key: cord- -pjv wy authors: hong, xiaoyun; wei, liangming; wu, fei; wu, zaozhan; chen, lizhu; liu, zhenguo; yuan, weien title: dissolving and biodegradable microneedle technologies for transdermal sustained delivery of drug and vaccine date: - - journal: drug des devel ther doi: . /dddt.s sha: doc_id: cord_uid: pjv wy microneedles were first conceptualized for drug delivery many decades ago, overcoming the shortages and preserving the advantages of hypodermic needle and conventional transdermal drug-delivery systems to some extent. dissolving and biodegradable microneedle technologies have been used for transdermal sustained deliveries of different drugs and vaccines. this review describes microneedle geometry and the representative dissolving and biodegradable microneedle delivery methods via the skin, followed by the fabricating methods. finally, this review puts forward some perspectives that require further investigation. hypodermic needles are used for most biotherapeutic and vaccine injections, providing a low-cost, rapid, and direct way to deliver almost any type of molecule into the body. however, hypodermic needles are utilized primarily in the clinic or at home by patients who have received special training, and safe needle disposal and other issues such as transportation are problems. conventional transdermal drug delivery offers many important advantages, such as accessibility, safety, painless drug administration, potential for self-administration, and avoidance of enzymatic degradation in the gastrointestinal tract or liver, but shows low bioavailability and poor permeability of drugs because of the physical barrier of the stratum corneum. , for parenteral controlled-release formulations, in situ-forming implants are also attractive alternatives to preformed implants and microparticles, avoiding the use of large needles or microsurgery and with relatively low cost of manufacture. such a system stays in a solid state before administration and becomes a hydrogel in situ after injection into the body. however, key issues remain to be solved, including variability of the implant shape and structure, avoidance of burst release during implant formation, and toxicity issues, thus these are far from practical use. recently, peng et al combined nanoparticle and thermosensitive hydrogel technologies in an appropriate manner, which would be a promising system for longer sustained and controlled drug delivery. however, compared to microneedle technologies, the promising system might have lower patient compliance and transportation convenience. deadman et al reported more recently that an in situ-forming drug depot, eligard, was approved by the us food and drug administration. upon administration, the water-miscible organic solvent dissipates into the surrounding tissue, which leads to polymer precipitation into a depot at the site of injection, entrapping the drug. the obvious disadvantages are the fact that an submit your manuscript | www.dovepress.com hong et al organic solvent is administered (which can lead to toxicity) and the variable shape and size of the implant formed in vivo, which leads to variable rates of drug release. microneedle technologies, which were first conceptualized for drug delivery many decades ago, overcome the shortages and preserve the advantages of hypodermic needles and conventional transdermal drug-delivery systems to some extent. they are also superior to in situ-forming implants and in situ-forming drug depot in some fields. microneedles are needle-like structures with diameters in the size order of microns and lengths up to µm. these structures are used to pierce the upper layer of the skin to enhance transdermal drug delivery by enabling the transport of a host of molecules that cannot be delivered across the skin by passive diffusion alone. there is no size limit for delivery of macromolecules through these microchannels, as the size of these microchannels is in microns and the maximum dimension of typical macromolecules administered into the body are in nanometers, thus microneedles can be used to deliver such macromolecules as insulin, growth hormones, immunobiologicals, proteins, and peptides. microneedle technologies can be divided into several categories: solid microneedles for skin pretreatment to increase skin permeability, microneedles coated with drugs, polymer microneedles that encapsulate drugs and fully or partially dissolve in the skin, and hollow microneedles for drug infusion into the skin. , microneedles are mostly applied for the transdermal delivery of drugs and vaccines that may require long exposure, among which the dissolving and biodegradable microneedle technologies are most commonly seen. compared to bolus delivery, the sustained delivery of drug and vaccine is more complicated and is a trend in microneedle technology, thus this article reviews this body of work. the stratum corneum is - µm thick and is indispensable as a barrier. the viable epidermis is located below the stratum corneum, and has a thickness of - µm. below the epidermis lies the dermis, which is about , µm thick and contains nerves, blood vessels, nociceptors, lymph vessels, hair follicles, and sweat glands ( figure ). the epidermal or epithelial layer is a covering carried in the superficial dermis or superficial lamina in the superficial plexus. in the case of skin as a model, the plexus is the intradermal plexus. there are some lymphatic vessels in that layer. in the deep dermal layer or deep lamina, the deep plexus is carried. in the case of skin, this is the subdermal plexus. in that layer, there are most of the lymphatic vessels, and the collagen content of that layer is much greater than in the superficial layer. microneedle technologies for sustained drug delivery has long been recognized as a highly immune reactive tissue containing an abundance of antigen-presenting cells and immunocompetent cells, especially within the epidermal and dermal skin layers. , skin microanatomy largely determines the geometry of microneedles. yana et al proved that microneedle arrays with longer needles (. µm) were more effective in creating pathways across skin and enhancing drug flux, and microneedle arrays with lower needle densities (, , needles/cm ) were more effective in enhancing drug flux if the microneedles had sufficient length (. µm). noh et al measured skin irritation using microneedles of µm depth, and concluded that there was little difference in the decrease of redness after microneedle application based on application time, but redness was generally maintained until minutes and rapidly decreased between minutes and hours. gomaa et al found that when µm-long microneedles were applied to the dermal side of skin, the transepidermal water-loss measurements remained at baseline levels. this is explainable by the fact that typically only %- % of the needle length actually penetrates tissue. gomaa et al also found that increasing microneedle density introduced complex effects. firstly, more skin channels may have been created, and these inhibited the long-term, tissue contraction-mediated partial recovery of the barrier. however, there was also some evidence that at high microneedle densities, individual needles may no longer effectively breach the barrier due to a "bed of nails" effect. leaving the array embedded in the skin seemed to diminish the magnitude of the initial postinsertion drop in barrier function. furthermore, lee et al concluded that pyramidal microneedles were stronger than conical ones, probably due to their larger cross-sectional area at the same base width/diameter. dissolving microneedles, mostly using different kinds of sugars as the matrix (figure ), usually release drugs or vaccines quickly in vivo, eg, ito et al reported that insulin released from microneedles very quickly, and almost all of the formulated insulin was released within hour when dextrin was used as matrix. nevertheless, sustained release of drugs or vaccines is also required under some circumstances. lee et al prepared microneedles with model drug encapsulated not within the microneedle tips but only in the backing layer, which served as a controlled-release reservoir that delivered molecules by a combination of swelling the backing with interstitial fluid drawn out of the skin and molecule diffusion into the skin via channels formed by dissolved microneedles. they revealed that sulforhodamine release from carboxymethyl cellulose microneedle patches exhibited an initial lag time of a few hours, followed by steady release for approximately day. similar behavior was seen for microneedle patches made of amylopectin, but with slower kinetics. in this case, lag time was longer and release took place over a few days. polymeric dissolving microneedles designed by donnelly et al have been produced from gantrez an- , and delivered % of the encapsulated theophylline into porcine skin within hours. kumar et al characterized and used maltose microneedles to microporate full-thickness pig-ear skin to evaluate drug delivery of model small (calcein) and large (human growth hormone) molecules. it was found that modulated transdermal delivery of small as well as large molecules is possible upon microporation of the skin in combination with iontophoresis (itp). the modulated itp protocol resulted in peaks in flux with application of current and gradual decrease with termination of current, and current density and time could be used appropriately to program a desired drug-delivery profile. garland et al studied the potential for itp to be combined with polymeric microneedle devices that remain in contact with the skin during the course of drug delivery procedure for the first time. furthermore, it has been shown that the application of an electric current enables the permeation of macromolecules from the entire microneedle-array matrix, and not just that which was contained within the microneedles alone. thus the application of an electric current significantly increased the extent of macromolecular delivery from the poly(methyl vinyl ether maleic anhydride) microneedle array, which is also promising for the sustained delivery of drugs and vaccines. wu et al reported a similar finding, wherein the transdermal permeation of high-molecular-weight compounds through microneedle-induced channels could be increased through the combination of itp. ito et al reported sustained-release self-dissolving micropiles (sdmps), in which porous silicon dioxide and porous calcium silicate were used as nanoporous microparticles to adsorb insulin, and the microparticleadsorbed insulin was molded to sdmps using chondroitin sulfate as a base. they concluded that long-acting sdmp preparation would be possible by means of porous silicate adsorbent-held insulin. biodegradable microneedles, mostly using different kinds of biodegradable polymers, including polylactic acid, chitosan, polyglycolic acid, or poly(lactide-co-glycolide) (plga) to form the matrix, degrade in the skin after application, whereby the release of incorporated drugs can be sustained for months by choosing the proper polymer. a previous study demonstrated the possibility of these biodegradable microneedles as a patient-friendly substitute for conventional sustained-delivery methods. however, these microneedles must be inserted and remain in the skin for several days to utilize the degradation property of biodegradable polymer effectively. kim et al demonstrated that microneedle separation into the skin was mediated by hydrogel swelling in response to contact with body fluid after the needles were inserted into the skin. the hydrogel particles absorbed water quickly, resulting in the cracking of the microneedles due to the difference in volume expansion between the needle-matrix polymer and the hydrogel particles. the swollen particles caused the microneedles to totally break down, leaving the microneedle tips in the porcine cadaver skin in vitro and in the hairless mouse skin in vivo (figure ). chu et al introduced separable arrowhead microneedles that featured micron-size sharp tips mounted on blunt shafts. upon insertion in the skin, the sharp-tipped polymer arrowheads encapsulating drug separate from their metal shafts and remain embedded in the skin for subsequent dissolution and drug release. the blunt metal shafts can then be discarded. due to rapid separation of the arrowhead tips from the shafts within seconds, administration using arrowhead microneedles can be carried out rapidly, while drug-release kinetics can be independently controlled, based on separable arrowhead formulation (figure ). park et al developed arrays of microneedles that were fabricated out of plga using a mold-based technique to encapsulate model drugs -calcein and bovine serum albumin -either as a single encapsulation within the needle matrix or as a double encapsulation, by first encapsulating the drug within carboxymethyl cellulose or poly-l-lactide microparticles and then encapsulating drug-loaded microparticles within needles. in vitro release of calcein and bovine serum albumin from three different encapsulation formulations was measured over time, and was shown to be controlled by the encapsulation method to achieve release kinetics ranging from hours to months. tsioris et al reported on a fabrication method to produce silk biopolymer microstructures with microneedle technologies for sustained drug delivery the high aspect ratios required to manufacture microneedle systems. room temperature and aqueous-based micromolding allow for the bulk loading of these microneedle structures with temperature-sensitive drugs such as peptides, antibiotics, and vaccines, or any temperature-labile therapeutic. controlled release of a model drug is achieved by adjusting the postprocessing conditions of the microneedle structures, mainly by controlling the silk protein secondary structure. ausiello et al released a method for extended and controlled delivery of parathyroid hormone to a patient in need, involving implanting a medical device into the patient, the medical device comprising a substrate, a plurality of reservoirs in the substrate, and a release system contained in each of the reservoirs, wherein the release system comprised parathyroid hormone, and controllably releasing a pharmaceutically effective amount of the parathyroid hormone from the reservoirs. microneedle delivery of nucleic acids, in particular plasmid dna (pdna), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunization. dna vaccines have many potential benefits, but have failed to generate robust immune responses in humans. demuth et al reported an approach for rapid implantation of vaccine-loaded polymer films carrying dna, immune-stimulatory rna, and biodegradable polycations using microneedles coated with releasable polyelectrolyte multilayers that promoted local transfection and controlled the persistence of dna and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. these "multilayer tattoo" dna vaccines induced immune responses against a model hiv antigen comparable to electroporation in mice, enhanced memory t-cell generation, and elicited -fold higher gene expression in nonhuman primate skin than intradermal dna injection, indicating the potential of this strategy for enhancing dna vaccination ( figure ). marc pearton et al proved that the pdna-coated microneedles facilitated reporter-gene expression in viable human skin, whilst the efficiency of gene expression from coated microneedles will depend upon suitable dna loading, efficient and reproducible skin puncture, and rapid in situ dissolution of the plasmid at the site of delivery. fabrication of microneedles usually follows the steps of molding, preparation of microneedle matrix, casting, removal, and drying. the concrete methods are determined by the desired property of the microneedles. however, harsh fabrication methods (ie, using high temperature or organic solvent) may damage temperature-sensitive drugs, particularly peptides and proteins, so this review mainly focuses on mild fabrication methods. vacuum and centrifugation are often applied in casting the microneedle matrix into the molds. whilst a polydimethylsiloxane micromold can be created with precise morphological fidelity to master microneedle structures, sugar solutions did not completely fill the micromold invaginations, due to the high surface tension of the solution. martin et al revealed a simple and novel low-temperature vacuum-deposition micromolding methodology for biodegradable sugar-glass microneedle fabrication. in the original vacuum-oven method, vacuum pressure could not be applied before the sugar solution was placed on the mold surface. subsequently, when the droplet of sugar solution was applied, air was entrapped within the micromold invaginations. an optimized method was developed whereby a vacuum was produced within an enclosed chamber before the sugar solution was applied to the mold surface. demuth et al developed microneedle-fabrication methods featuring drug-loaded plga microparticles or solid plga tips. plga microparticles were applied to the surface of the mold in an aqueous suspension and compacted into the mold cavities through centrifugation. excess hong et al microparticles were then removed, and the microparticleloaded mold was allowed to dry. then, a concentrated aqueous solution of poly(acrylic acid) was added to the mold surface and infiltrated into the packed plga particle bed via centrifugation. after being dried under vacuum, a solid plga-poly(acrylic acid) microparticle matrix was obtained. if the microparticle-loaded polydimethylsiloxane molds were dried and incubated under vacuum to fuse the embedded plga particles, solidified plga tips were obtained. demuth et al demonstrated a new approach for dna vaccination via multilayer "tattooing," using microneedles employing a ph-responsive release layer to implant biodegradable vaccine-loaded polymer films rapidly into the skin. mefti et al released a patent concerning the administration of drugs with microneedles. more specifically, it concerned microneedles comprising a part that dissolves by hydrolysis once they have penetrated the skin. it can also be concluded that enhancing methods such as itp, as well as with different drug carriers (eg, microand nanoparticles) will be applied to the fabrication of microneedles. microneedles effectively circumvent the skin barrier to offer this route as a potential alternative to oral and parenteral delivery of therapeutics, and relatively large doses can be administered due to bulk loading of dissolvable or biodegradable systems. however, there are still several problems that remain to be solved. for example, the barrier function of the skin changes from one site to another on the same person, from person to person, and with age. it is important to ensure that patients obtain the same and required dose during each microneedle administration, since the difference in individual skin and the penetration depth of microneedles relate to the stress upon the skin, thus an applicator may be required in order to obtain a reproducible penetration depth. besides, the sustained release of drugs or vaccines is more complicated than bolus release, and the kinetics should be further investigated and ensured. it is also necessary to investigate whether dissolved or degraded matrices have side effects if microneedles are used frequently. although these materials are commonly seen excipients in drugs, the accumulation levels and their influences should be noted for prudential reasons. in addition, in some microneedle-fabrication cases, the polymer-melting temperatures were above °c and vacuum necessary for processing, and these conditions could be detrimental to various temperature-sensitive drugs, particularly peptides and proteins. polymer microneedles for controlled-release delivery are also constrained by needle mechanical properties when the main matrix is plga or carboxymethyl cellulose. park et al found that microneedles with % drug loading retained sufficient mechanical strength, but needles with % loading did not. although the maximum dose constrains applications, controlled-release delivery of up to mg has a number of candidate drugs on the market, with more likely to be approved in the future. dna vaccines have been intensively studied because of potential advantages, such as ease of good-manufacturingpractice production, lack of antivector immunity, and the capability to promote both cellular and humoral immune responses. , since the discovery that genetically engineered dna can be delivered in vaccine form and elicit an immune response, there has been much progress in understanding the basic biology of this platform. , a large amount of data has been generated in preclinical model systems, , and more sustained cellular responses and more consistent antibody responses are being observed in the clinic. , microneedle application is promising in promoting local transfection and controlling the persistence of dna and adjuvants in the skin from days to weeks to function as an optimal strategy for safe, reproducible, and pain-free dna vaccination. for therapeutic proteins, preservation of protein structure during manufacture, storage, and use is considered even more important than for vaccines, and unwanted immunogenicity can lead to total loss of the therapeutic effect of a protein by neutralizing antibodies, and may even lead to depletion of endogenous proteins or breaking the immune tolerance to self-antigens. for dissolving microneedles, future studies will partially focus upon the incorporation of other bioactivity-enhancing methods including itp components into polymeric microneedle devices and investigation of their potential for efficient, electrically controlled pulsatile delivery of macromolecules from drug-loaded dissolving polymeric microneedle arrays. , altogether, dissolving and biodegradable microneedle technologies have a bright future for transdermal sustained delivery of drug and vaccine, and require further studies. pain following controlled cutaneous insertion of needles with different diameters intradermal injections: traditional bevel up versus bevel down needle-free vaccine delivery micro-scale devices for transdermal drug delivery transdermal drug delivery in situ forming implants -an attractive formulation principle for parenteral depot formulations injectable and biodegradable thermosensitive hydrogels loaded with phbhhx nanoparticles for the sustained and controlled release of insulin an investigation into the influence of drug lipophilicity on the in vivo absorption profiles from subcutaneous microspheres and in situ forming depots optical coherence tomography is a valuable tool in the study of the effects of microneedle geometry on skin penetration characteristics and in-skin dissolution development of a codrug approach for sustained drug delivery across microneedle-treated skin transdermal and intradermal delivery of therapeutic agents application of physical technologies microneedles: an emerging transdermal drug delivery system infusion pressure and pain during microneedle injection into skin of human subjects transcutaneous vaccines -current and emerging strategies microneedle technologies for (trans)dermal drug and vaccine delivery techniques of tissue handling and transfer intradermal vaccine delivery: will new delivery systems transform vaccine administration? vaccine intradermal, epidermal and transcutaneous vaccination: from immunology to clinical practice evaluation needle length and density of microneedle arrays in the pretreatment of skin for transdermal drug delivery in vitro characterization of the invasiveness of polymer microneedle against skin effects of microneedle length, density, insertion time and multiple applications on human skin barrier function: assessments by transepidermal water loss mechanism of fluid infusion during microneedle insertion and retraction dissolving microneedles for transdermal drug delivery laser-engineered dissolving microneedle arrays for transdermal macromolecular drug delivery feasibility of microneedles for percutaneous absorption of insulin design, optimization and characterisation of polymeric microneedle arrays prepared by a novel laser-based micromoulding technique modulated iontophoretic delivery of small and large molecules through microchannels dissolving polymeric microneedle arrays for electrically assisted transdermal drug delivery enhancement of skin permeation of high molecular compounds by a combination of microneedle pretreatment and iontophoresis sustained-release self-dissolving micropiles for percutaneous absorption of insulin in mice polymer microneedles for controlled-release drug delivery hydrogel swelling as a trigger to release biodegradable polymer microneedles in skin separable arrowhead microneedles fabrication of silk microneedles for controlled-release drug delivery method and device for the controlled delivery of parathyroid hormone. united states patent us cutaneous vaccination using microneedles coated with hepatitis c dna vaccine polymer multilayer tattooing for enhanced dna vaccination microneedle delivery of plasmid dna to living human skin: formulation coating, skin insertion and gene expression drug delivery using microneedles low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications composite dissolving microneedles for coordinated control of antigen and adjuvant delivery kinetics in transcutaneous vaccination soluble microneedle. world intellectual property organization patent wo/ / microneedles as transdermal delivery systems: combination with other enhancing strategies drug design, development and therapy poly vinyl alcohol re-usable masters for microneedle replication transdermal drug delivery system: a review enhancement of dna vaccine potency by linkage of antigen gene to an hsp gene a dna vaccine induces sars coronavirus neutralization and protective immunity in mice genetic immunization is a simple method for eliciting an immune response gene-based vaccines: recent technical and clinical advances studies of a prophylactic hiv- vaccine candidate based on modified vaccinia virus ankara (mva) with and without dna priming: effects of dosage and route on safety and immunogenicity electroporation as a "prime/boost" strategy for naked dna vaccination against a tumor antigen vaxfectin-formulated influenza dna vaccines encoding np and m viral proteins protect mice against lethal viral challenge dna vaccines: ready for prime time? needle-free epidermal powder immunization iontophoresis-driven penetration of nanovesicles through microneedle-induced skin microchannels for enhancing transdermal delivery of insulin the study was supported by the projects of national science foundation of china ( , and ), and projects of the shanghai committee of science and technology, people's republic of china ( nm , , and submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -iaocovf authors: schreiber, j. title: medikamenteninduzierte parenchymatöse lungenerkrankungen date: - - journal: pneumologe (berl) doi: . /s - - - sha: doc_id: cord_uid: iaocovf drug-induced alveolitis/pneumonitis and lung fibrosis are comparatively frequent manifestations of drug-induced damage to the respiratory system. these side effects rarely have pathognomonic features. therefore, they are relevant differential diagnoses of naturally occurring pulmonary diseases. side effects of drug therapy may mimic much of the clinical-radiological-pathological pattern of interstitial lung diseases; however, precise figures on the frequency of medicamentous induction of interstitial lung disorders are lacking. the diagnostics are based mainly on verifying a compatible disease pattern, excluding differential diagnoses, and assessing the temporal relationship and the consequences of drug abstention. reexposure is rarely indicated. strict elimination of the responsible drugs is the most important therapeutic measure. additional drug therapy, mostly with glucocorticosteroids, may be indicated. this article summarizes the spectrum of drug-induced disorders of the lung parenchyma. medikamenteninduzierte erkrankungen können sich in allen kompartimenten der atmungsorgane manifestieren. in diesem beitrag wird eine Übersicht über medikamentös induzierte erkrankungen des lungenparenchyms, aber auch reaktionsmuster wie das nichtkardiogene lungenödem, die diffuse alveoläre hämorrhagie, das medikamenteninduzierte ards ("acute respiratory distress syndrome") und eosinophile lungenparenchymerkrankungen gegeben. Überschneidungen zwischen diesen reaktionsmustern sind möglich. so können beispielsweise charakteristika eines diffusen alveolarschadens vor dem hintergrund eines nsip-musters ("non-specific interstitial pneumonia") auftreten. interstitielle lungenerkrankungen -alveolitiden und lungen-fibrosen -sind die häufigste form von arzneimittelnebenwirkungen am lungenparenchym. meist ist die lunge isoliert betroffen. eine gleichzeitig auftretende hepatotoxizität wurde z. b. für amiodaron oder nitrofurantoin beschrieben. dagegen sind medikamentös induzierte systemische krankheitsbilder mit pulmonaler beteiligung selten. es gibt medikamente, die im falle von nebenwirkungen ein uniformes reaktionsmuster an den atmungsorganen induzieren, einzelne medikamente können jedoch auch in abhängigkeit von der individuellen prädisposition verschiedenartige krankheitsbilder hervorrufen. andererseits können identische schädigungsmuster des lungenparenchyms durch strukturell und pharmakologisch unterschiedliche medikamente verursacht werden. für eine kaum noch überschaubare vielzahl von pharmakologisch und strukturell verschiedenartigen medikamenten wurden nebenwirkungen am lungenparenchym beschrieben. die am häufigsten ursächlichen medikamente bzw. medikamentengruppen sind zytostatika (bleomycin, busulfan, chlorambuzil, gemcitabin, nitrosoharnstoffe wie carmustin und lomustin, u.v.a.), amiodaron, antibiotika und biologische medikamente (wachstumsfaktoren, interferone, zytokine, antikörper wie rituximab, trastuzumab). die exzellente datenbank pneumotox-online [ ] enthält aktuell über medika-mente, für die bronchopulmonale nebenwirkungen beschrieben wurden. die relativen häufigkeiten verändern sich mit den sich ändernden modalitäten der pharmakotherapie. so ist beispielsweise die früher häufige nitrofurantoinlunge heute selten geworden, eine früher ebenfalls häufige goldlunge als folge einer therapie einer rheumatoiden arthritis mit goldpräparaten dürfte heute nicht mehr auftreten. für neuere substanzen, wie tyrosinkinaseinhibitoren (gefitinib, erlotinib), rapamycinderivate (sirolimus, everolimus, temserolimus), imatinib, interferone und anti-tnfα-antagonisten (eternacept, infliximab, adalimumab) wurden pneumotoxische nebenwirkungen nachgewiesen. die pathomechanismen der medikamenteninduzierten lungenerkrankungen sind vielfältig und je nach medikament und reaktionsmuster unterschiedlich, oft auch nicht im detail geklärt. sie reichen von einer direkten toxizität des medikaments oder seiner metaboliten über hypererge reaktionen bis hin zur induktion von autoimmunen oder allergischen reaktionen [ ] . die lunge ist ein stoffwechselaktives organ mit einer breiten enzymausstattung. die gesamtkonzentration der enzyme der cytochrom-p -superfamilie beträgt in der lunge etwa % der konzentration in der leber. auf zellulärer basis ist die konzentration dieser enzyme in den pulmonalen clara-zellen und den typ-ii-pneumozyten höher als in hepato- in abhängigkeit von den ergebnissen dieser diagnostik wird empfohlen, die wahrscheinlichkeit des kausalzusammenhangs einzuteilen in [ ] : f bewiesen ("causative"), f wahrscheinlich ("probable"), f möglich ("possible"). eine schädigung des lungenparenchyms durch medikamente manifestiert sich meist, jedoch nicht immer in form einer alveolitis oder lungenfibrose und kann durch eine vielzahl von arzneimitteln induziert werden (. infobox , . infobox , [ , , ] akute, subakute und chronische verläufe sind möglich. f bleomycin f busulfan f mitomycin f carmustin u. a. zytostatika f amiodaron f nitrofurantoin f amphotericin b f sulfasalazin f hydrochlorothiazid f goldsalze f bromocriptin f interstitielle lungenerkrankung · hämorrhagie · allergische reaktion · toxizität · medikamentenkarenz druginduceddisordersofthelungparenchyma abstract drug-induced alveolitis/pneumonitis and lung fibrosis are comparatively frequent manifestations of drug-induced damage to the respiratory system. these side effects rarely have pathognomonic features. therefore, they are relevant differential diagnoses of naturally occurring pulmonary diseases. side effects of drug therapy may mimic much of the clinical-radiological-pathological pattern of interstitial lung diseases; however, precise figures on the frequency of medicamentous induction of interstitial lung disorders are lacking. the diagnostics are based mainly on verifying a compatible disease pat-tern, excluding differential diagnoses, and assessing the temporal relationship and the consequences of drug abstention. reexposure is rarely indicated. strict elimination of the responsible drugs is the most important therapeutic measure. additional drug therapy, mostly with glucocorticosteroids, may be indicated. this article summarizes the spectrum of drug-induced disorders of the lung parenchyma. interstitial lung disease · hemorrhage · allergic reaction · toxicity · drug cessation bei begründetem verdacht ist eine medikamentenkarenz und in abhängigkeit vom schweregrad der reaktion eine therapie mit glukokortikosteroiden indiziert. eine granulomatöse entzündung ist nicht typisch für eine medikamentös induzierte schädigung des lungenparenchyms. einige medikamente können jedoch mehr oder weniger gut abgrenzbare pulmonale granulome induzieren: methotrexat, bleomycin, phenylbutazon, phenytoin, sirolimus, eternacept und leflunomid. die induktion einer sarkoidose kann die therapie mit interferon-α und -β komplizieren. ein diffuser alveolarschaden (dad) manifestiert sich klinisch oft als akutes lungenversagen ("acute respiratory distress syndrome", ards). das histomorpho-abb. [ ] . im folgenden werden einige medikamenten-und substanzgruppen wegen ihres besonderen nebenwirkungsspektrums oder ihres breiten einsatzes im detail dargestellt. eine antiarrhythmische therapie mit amiodaron wird relativ häufig durch pulmonale nebenwirkungen kompliziert. dieses krankheitsbild ist heutzutage seltener geworden, weil das medikament niedriger dosiert wird als früher. die häufigkeit einer apt ist dosisabhängig und beträgt , - , % bei einer dosis von mg/tag, wie sie in deutschland meist üblich ist, von - % bei einer dosierung bis mg/tag und reicht bis zu % bei tagesdosen von mg. ein risikofaktor ist eine hohe initialdosis. weitere triggerfaktoren sind thoraxchirurgische eingriffe, eine sauerstofftherapie und vermutlich auch infektionen. eine seltene, mit einer erheblichen letalität behaftete komplikation einer amiodarontherapie ist das akute lungenversa gen (ards), das nahezu ausschließlich nach thoraxchirurgischen eingriffen, einschließlich minimal-invasiver eingriffe auftritt [ , ] . für zahlreiche zytostatika sind nebenwirkungen an den atmungsorganen bekannt. auch die neueren chemotherapeutika und immunsuppressiven medi-kamente (fludarabin, gemcitabin, gefitinib, imatinib, irinotecan, rituximab, trastuzumab und taxane) wurden pneumotoxische nebenwirkungen beschrieben. durch den vermehrten einsatz von biologischen substanzen (wachstumsfaktoren, zytokine, antikörper) sind hinsichtlich der pulmonalen toxizität zusätzliche probleme entstanden. die pulmonale toxizität von zytostatika wird in eine früh, während der therapie einsetzende ("early onset") form (. abb. ) und eine monate bis jahre nach der therapie einsetzende ("late onset") form unterschieden. die früh einsetzende form manifestiert sich meist als alveolitis, toxisches lungenödem, ards, diffuse alveoläre hämorrhagie (dah) oder bronchiolitis obliterans, die spät einsetzende form meist als lungenfibrose. risikofaktoren für toxische nebenwirkungen von zytostatika an den lungen sind die kumulative medikamentendosis, eine kombinationstherapie mit anderen zytostatika, insbesondere eine second-line-oder third-line-therapie, eine zusätzliche behandlung mit hämatopoetischen wachstumsfaktoren, ein höheres alter der patienten (> jahre), eine simultane oder sequentielle strahlentherapie, eine sauerstofftherapie (f i o > , ) und thoraxchirurgische eingriffe. patienten mit vorbestehenden lungenparenchymerkrankungen. abb. drug-induced eosinophilic lung disease druginduced and iatrogenic infiltrative lung disease drug-induced respiratory disease in patients with hematological diseases interstitial lung disease induced by drugs and radiation amiodarone pulmonary toxicity methotrexate pulmonary toxicity druginduced lung disease: high-resolution ct and histological findings bronchoalveolar lavage in drug-induced lung disease pulmonary toxicity from novel antineoplastic agents drug-induced lung disease: high-resolution ct findings drug induces bronchiolitis obliterans organizing pneumonia pathologic characteristics of drug-induced lung disease pneumotox online. the drug-induced lung diseases understanding the mechanisms of drug-associated interstitial lung disease pulmonary toxicity of cardiac drugs drug-induced pulmonary edema and acute respiratory distress syndrome infiltrative lung disease due to non-toxic agents pulmonary toxicity of chemotherapy imaging features of drug-induced lung diseases drug-induced pulmonary damage pulmonary adverse events of anti-tumor necrosis factor alpha antibody therapy a method for estimating the probability of adverse drug reactions drug-induces lupus drug-related pulmonary problems in patients with rheumatoid arthritis drug-induced diffuse alveolar hemorrhage syndromes and vasculitis drug-induced eosinophilic pneumonia: high-resolution ct findings in patients bei der häufig notwendigen polychemotherapie kann es bei einer toxischen lungenschädigung unmöglich sein, eine einzelne substanz als ursache zu benennen. diese situation wird durch den terminus "chemotherapy lung" (chemotherapielunge) widergespiegelt (. abb. ; [ , ] ). in den letzten jahren haben sich die möglichkeiten der therapie onkologischer erkrankungen durch den einsatz von sog. "small molecules", wie thyrosinkinase (tk)-inhibitoren oder proteasomeninhibitoren, erweitert. auch für diese substanzen wurden schwere, teilweise letal verlaufende pulmonale nebenwirkungen beschrieben. so kann die therapie mit tk-inhibitoren wie gefitinib durch fibrosierende alveolitiden, die applikation von proteasomeninhibitoren (bortezomib) durch schwere alveoläre hämorrhagien kompliziert werden (. abb. ; [ ] ). mtx wird neben seinem einsatz in der onkologie in niedriger dosis zur behandlung von rheumatischen und entzündlichen erkrankungen eingesetzt. pulmonale nebenwirkungen sind auch bei niedrig dosierter mtx-therapie möglich und können zu jedem zeitpunkt der therapie auftreten. risikofaktoren sind ein höheres patientenalter, ein begleitender diabetes mellitus, pleuropulmonale manifestation einer rheumatoiden arthritis, eine vorangegangene therapie mit dmard ("disease modifying anti-rheumatic drugs") und eine hypalbuminämie. eine kombinationstherapie mit tnf-antagonisten kann die mtx-toxizität triggern.diagnostische kriterien einer mtx-alveolitis sind ein subakuter beginn innerhalb weniger tage mit husten, dyspnoe, fieber (> °c), beidseitigen pulmonalen infiltraten, leukozytose über g/l bei negativen blut-, sputum und bal-kulturen, eine restriktive ventilationsstörung mit dlco-und p a o -abfall. in der bal ist eine lymphozytose (cd + oder cd +) und im lungenparenchym sind histologisch eine alveolitis, hyperplasie der typ-ii-pneumozyten und granulomatöse entzündung sowie eine geringe, nicht obligate gewebseosinophilie nachweisbar. die wichtigste differenzialdiagnosen sind infektionen, einschließlich opportunistischer erreger (pneumocystis jirovecii, cytomegalie-virus, cryptococcus spp., herpesviren, nocardia spp.), die unter einer therapie mit mtx beschrieben wurden.die mtx-induzierte pulmonale toxizität kann einen perakuten verlauf zeigen und sich als diffuser alveolarschaden oder als diffuse alveoläre hämorrhagie manifestieren.die therapie besteht in einer medikamentenkarenz und der applikation von glukokortikosteroiden. eine reexposition ist kontraindiziert [ , , ] . eine potenzielle nebenwirkung an den atmungsorganen ist eine alveolitis, die sich histologisch als nsip manifestiert. auch können eosinophile infiltrate und pleuraergüsse auftreten. als nebenwirkung dieser medikamente wurde ein "acute druginduced chest syndrome" beschrieben, das wechselnde, subpleurale infiltrate mit starken, atemabhängigen pleuritischen schmerzen und dyspnoe umfasst, die nach karenz rückläufig sind und nach reexposition erneut auftreten. es ist eine abgrenzung von rezidivierenden lungenembolien erforderlich [ ] . die häufigsten und bekanntesten nebenwirkungen der ace-hemmer sind unproduktiver husten und ein potenziell vital bedrohliches glottis-oder larynxödem. weiterhin können diese substanzen eosinophile infiltrate oder eine subakute interstitielle pneumonie induzieren [ ] . in den letzten jahren wird nitrofurantoin als preisgünstiges medikament wieder vermehrt in der therapie von harnwegs-abb. röntgenuntersuchung der thoraxorgane einer -jährigen patientin mit einer alveolitis infolge einer nitrofurantointherapie (nitrofurantoinlunge): gering ausgeprägte interstitielle zeichnungsvermehrung. es lag eine schwere einschränkung der diffusionskapazität vor. die bal zeigte eine massive erhöhung der gesamtzellzahl mit vermehrung der lymphozyten und der eosinophilen granulozyten key: cord- -qdld hdc authors: fan, yue-nong; xiao, xuan; min, jian-liang; chou, kuo-chen title: inr-drug: predicting the interaction of drugs with nuclear receptors in cellular networking date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: qdld hdc nuclear receptors (nrs) are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. therefore, nrs have become a frequent target for drug development. during the process of developing drugs against these diseases by targeting nrs, we are often facing a problem: given a nr and chemical compound, can we identify whether they are really in interaction with each other in a cell? to address this problem, a predictor called “inr-drug” was developed. in the predictor, the drug compound concerned was formulated by a -d (dimensional) vector derived from its molecular fingerprint, and the nr by a -d vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the svm (support vector machine) algorithm. compared with the existing prediction methods in this area, inr-drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at http://www.jci-bioinfo.cn/inr-drug/, which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. it is anticipated that the inr-drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. with the ability to directly bind to dna ( figure ) and regulate the expression of adjacent genes, nuclear receptors (nrs) are a class of ligand-inducible transcription factors. they regulate various biological processes, such as homeostasis, differentiation, embryonic development, and organ physiology [ ] [ ] [ ] . the nr superfamily has been classified into seven families: nr (knirps or dax like) [ , ] ; nr (thyroid hormone like), nr (hnf -like), nr (estrogen like), nr (nerve growth factor ib-like), nr (fushi tarazu-f like), and nr (germ cell nuclear factor like). since they are involved in almost all aspects of human physiology and are implicated in many major diseases such as cancer, diabetes and osteoporosis, nuclear receptors have become major drug targets [ , ] , along with g protein-coupled receptors (gpcrs) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ion channels [ ] [ ] [ ] , and kinase proteins [ ] [ ] [ ] [ ] . identification of drug-target interactions is one of the most important steps for the new medicine development [ , ] . the method usually adopted in this step is molecular docking simulation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, to make molecular docking study feasible, a reliable d (three dimensional) structure of the target protein is the prerequisite condition. although x-ray crystallography is a powerful tool in determining protein d structures, it is time-consuming and expensive. particularly, not all proteins can be successfully crystallized. for example, membrane proteins are very difficult to crystallize and most of them will not dissolve in normal solvents. therefore, so far very few membrane protein d structures have been determined. although nmr (nuclear magnetic resonance) is indeed a very powerful tool in determining the d structures of membrane proteins as indicated by a series of recent publications (see, e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and a review article [ ] ), it is also time-consuming and costly. to acquire the d structural information in a timely manner, one has to resort to various structural bioinformatics tools (see, e.g., [ ] ), particularly the homologous modeling approach as utilized for a series of protein receptors urgently needed during the process of drug development [ , [ ] [ ] [ ] [ ] [ ] [ ] . unfortunately, the number of dependable templates for developing high quality d structures by means of homology modeling is very limited [ ] . to overcome the aforementioned problems, it would be of help to develop a computational method for predicting the interactions of drugs with nuclear receptors in cellular networking based on the sequences information of the latter. the results thus obtained can be used to pre-exclude the compounds identified not in interaction with the nuclear receptors, so as to timely stop wasting time and money on those unpromising compounds [ ] . actually, based on the functional groups and biological features, a powerful method was developed recently [ ] for this purpose. however, further development in this regard is definitely needed due to the following reasons. (a) he et al. [ ] did not provide a publicly accessible web-server for their method, and hence its practical application value is quite limited, particularly for the broad experimental scientists; (b) the prediction quality can be further enhanced by incorporating some key features into the formulation of nr-drug (nuclear receptor and drug) samples via the general form of pseudo amino acid composition [ ] . the present study was initiated with an attempt to develop a new method for predicting the interaction of drugs with nuclear receptors by addressing the two points. as demonstrated by a series of recent publications [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and summarized in a comprehensive review [ ] , to establish a really effective statistical predictor for a biomedical system, we need to consider the following steps: (a) select or construct a valid benchmark dataset to train and test the predictor; (b) represent the statistical samples with an effective formulation that can truly reflect their intrinsic correlation with the object to be predicted; (c) introduce or develop a powerful algorithm or engine to operate the prediction; (d) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; (e) establish a user-friendly web-server for the predictor that is accessible to the public. below, let us elaborate how to deal with these steps. the data used in the current study were collected from kegg (kyoto encyclopedia of genes and genomes) [ ] at http://www.kegg.jp/kegg/. kegg is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. here, the benchmark dataset can be formulated as where is the positive subset that consists of the interactive drug-nr pairs only, while the negative subset that contains of the non-interactive drug-nr pairs only, and the symbol represents the union in the set theory. the so-called "interactive" pair here means the pair whose two counterparts are interacting with each other in the drug-target networks as defined in the kegg database [ ] ; while the "non-interactive" pair means that its two counterparts are not interacting with each other in the drug-target networks. the positive dataset contains drug-nr pairs, which were taken from he et al. [ ] . the negative dataset contains non-interactive drug-nr pairs, which were derived according to the following procedures: (a) separating each of the pairs in into single drug and nr; (b) re-coupling each of the single drugs with each of the single nrs into pairs in a way that none of them occurred in ; (c) randomly picking the pairs thus formed until reaching the number two times as many as the pairs in . the interactive drug-nr pairs and non-interactive drug-nr pairs are given in supplementary information s , from which we can see that the + = pairs in the current benchmark dataset are actually formed by different nrs and different compounds. since each of the samples in the current network system contains a drug (compound) and a nr (protein), the following procedures were taken to represent the drug-nr pair sample. first, for the drug part in the current benchmark dataset, we can use a -d vector to formulate it as given by where d represents the vector for a drug compound, and d i its i-th (i = , , , ) component that can be derived by following the " d molecular fingerprint procedure" as elaborated in [ ] . the molecular fingerprint vectors thus obtained for the drugs in are, respectively, given in supplementary information s . the protein sequences of the different nrs in are listed in supplementary information s . suppose the sequence of a nuclear receptor protein p with l residues is generally expressed by where r represents the st residue of the protein sequence p , r the nd residue, and so forth. now the problem is how to effectively represent the sequence of equation ( ) with a non-sequential or discrete model [ ] . this is because all the existing operation engines, such as covariance discriminant (cd) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , neural network [ ] [ ] [ ] , support vector machine (svm) [ ] [ ] [ ] ] , random forest [ , ] , conditional random field [ ] , nearest neighbor (nn) [ , ] ; k-nearest neighbor (knn) [ ] [ ] [ ] , oet-knn [ ] [ ] [ ] [ ] , and fuzzy k-nearest neighbor [ , , , , ] , can only handle vector but not sequence samples. however, a vector defined in a discrete model may completely lose all the sequence-order information and hence limit the quality of prediction. facing such a dilemma, can we find an approach to partially incorporate the sequence-order effects? actually, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or a vector, yet still keep considerable sequence order information. to avoid completely losing the sequence-order information for proteins, the pseudo amino acid composition [ , ] or chou's pseaac [ ] was proposed. ever since the concept of pseaac was proposed in [ ] , it has penetrated into almost all the areas of computational proteomics, such as predicting anticancer peptides [ ] , predicting protein subcellular location [ ] [ ] [ ] [ ] [ ] [ ] [ ] , predicting membrane protein types [ , ] , predicting protein submitochondria locations [ ] [ ] [ ] [ ] , predicting gaba(a) receptor proteins [ ] , predicting enzyme subfamily classes [ ] , predicting antibacterial peptides [ ] , predicting supersecondary structure [ ] , predicting bacterial virulent proteins [ ] , predicting protein structural class [ ] , predicting the cofactors of oxidoreductases [ ] , predicting metalloproteinase family [ ] , identifying cysteine s-nitrosylation sites in proteins [ ] , identifying bacterial secreted proteins [ ] , identifying antibacterial peptides [ ] , identifying allergenic proteins [ ] , identifying protein quaternary structural attributes [ , ] , identifying risk type of human papillomaviruses [ ] , identifying cyclin proteins [ ] , identifying gpcrs and their types [ , ] , discriminating outer membrane proteins [ ] , classifying amino acids [ ] , detecting remote homologous proteins [ ] , among many others (see a long list of papers cited in the references section of [ ] ). moreover, the concept of pseaac was further extended to represent the feature vectors of nucleotides [ ] , as well as other biological samples (see, e.g., [ ] [ ] [ ] ). because it has been widely and increasingly used, recently two powerful soft-wares, called "pseaac-builder" [ ] and "propy" [ ] , were established for generating various special chou's pseudo-amino acid compositions, in addition to the web-server "pseaac" [ ] built in . according to a comprehensive review [ ] , the general form of pseaac for a protein sequence p is formulated by where the subscript  is an integer, and its value as well as the components ( , , , ) u u   will depend on how to extract the desired information from the amino acid sequence of p (cf. equation ( )). below, let us describe how to extract useful information to define the components of pseaac for the nr samples concerned. first, many earlier studies (see, e.g., [ ] [ ] [ ] [ ] [ ] [ ] ) have indicated that the amino acid composition (aac) of a protein plays an important role in determining its attributes. the aac contains components with each representing the occurrence frequency of one of the native amino acids in the protein concerned. thus, such aac components were used here to define the first elements in equation ( ); i.e., ( ) ( , , , ) ii fi   ( ) where f i ( ) is the normalized occurrence frequency of the i-th type native amino acid in the nuclear receptor concerned. since aac did not contain any sequence order information, the following steps were taken to make up this shortcoming. to avoid completely losing the local or short-range sequence order information, we considered the approach of dipeptide composition. it contained × = components [ ] . such components were used to define the next elements in equation ( ); i.e., ( ) ( , , , ) jj fj where ( ) j f is the normalized occurrence frequency of the j-th dipeptides in the nuclear receptor concerned. to incorporate the global or long-range sequence order information, let us consider the following approach. according to molecular evolution, all biological sequences have developed starting out from a very limited number of ancestral samples. driven by various evolutionary forces such as mutation, recombination, gene conversion, genetic drift, and selection, they have undergone many changes including changes of single residues, insertions and deletions of several residues [ ] , gene doubling, and gene fusion. with the accumulation of these changes over a long period of time, many original similarities between initial and resultant amino acid sequences are gradually faded out, but the corresponding proteins may still share many common attributes [ ] , such as having basically the same biological function and residing at a same subcellular location [ , ] . to extract the sequential evolution information and use it to define the components of equation ( ), the pssm (position specific scoring matrix) was used as described below. according to schaffer [ ] , the sequence evolution information of a nuclear receptor protein p with l amino acid residues can be expressed by a l matrix, as given by where ( ) were generated by using psi-blast [ ] to search the uniprotkb/swiss-prot database (the universal protein resource (uniprot); http://www.uniprot.org/) through three iterations with . as the e-value cutoff for multiple sequence alignment against the sequence of the nuclear receptor concerned. in order to make every element in equation ( ) be scaled from their original score ranges into the region of [ , ], we performed a conversion through the standard sigmoid function to make it become now we extract the useful information from equation ( ) moreover, we used the grey system model approach as elaborated in [ ] to further define the next components of equation ( ) ( , , , ) in the above equation, w , w , and w are weight factors, which were all set to in the current study; f j ( ) has the same meaning as in equation ( ) where   and combining equations ( ), ( ), ( ) and ( ), we found that the total number of the components obtained via the current approach for the pseaac of equation ( ) and each of the components is given by ( ) ( since the elements in equations ( ) and ( ) are well defined, we can now formulate the drug-nr pair by combining the two equations as given by   ( ) where g represents the drug-nr pair, Å the orthogonal sum, and the + = components are defined by equations ( ) and ( ) . for the sake of convenience, let us use x i (i = , , , ) to represent the components in equation ( ); i.e., ( ) to optimize the prediction quality with a time-saving approach, similar to the treatment [ ] [ ] [ ] , let us convert equation ( ) to where the symbol means taking the average of the quantity therein, and sd means the corresponding standard derivation. in this study, the svm (support vector machine) was used as the operation engine. svm has been widely used in the realm of bioinformatics (see, e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). the basic idea of svm is to transform the data into a high dimensional feature space, and then determine the optimal separating hyperplane using a kernel function. for a brief formulation of svm and how it works, see the papers [ , ] ; for more details about svm, see a monograph [ ] . in this study, the libsvm package [ ] was used as an implementation of svm, which can be downloaded from http://www.csie.ntu.edu.tw/~cjlin/libsvm/, the popular radial basis function (rbf) was taken as the kernel function. for the current svm classifier, there were two uncertain parameters: penalty parameter c and kernel parameter  . the method of how to determine the two parameters will be given later. the predictor obtained via the aforementioned procedure is called inr-drug, where "i" means identify, and "nr-drug" means the interaction between nuclear receptor and drug compound. to provide an intuitive overall picture, a flowchart is provided in figure to show the process of how the predictor works in identifying the interactions between nuclear receptors and drug compounds. to provide a more intuitive and easier-to-understand method to measure the prediction quality, the following set of metrics based on the formulation used by chou [ ] [ ] [ ] in predicting signal peptides was adopted. according to chou's formulation, the sensitivity, specificity, overall accuracy, and matthew's correlation coefficient can be respectively expressed as [ , [ ] [ ] [ ] sn where n  is the total number of the interactive nr-drug pairs investigated while n   the number of the interactive nr-drug pairs incorrectly predicted as the non-interactive nr-drug pairs; n  the total number of the non-interactive nr-drug pairs investigated while n   the number of the non-interactive nr-drug pairs incorrectly predicted as the interactive nr-drug pairs. according to equation ( ) we can easily see the following. when n    meaning none of the interactive nr-drug pairs was mispredicted to be a non-interactive nr-drug pair, we have the sensitivity sn = ; while nn    meaning that all the interactive nr-drug pairs were mispredicted to be the non-interactive nr-drug pairs, we have the sensitivity sn = . likewise, when n    meaning none of the non-interactive nr-drug pairs was mispredicted, we have the specificity sp we have mcc = meaning total disagreement between prediction and observation. as we can see from the above discussion, it is much more intuitive and easier to understand when using equation ( ) to examine a predictor for its four metrics, particularly for its mathew's correlation coefficient. it is instructive to point out that the metrics as defined in equation ( ) are valid for single label systems; for multi-label systems, a set of more complicated metrics should be used as given in [ ] . how to properly test a predictor for its anticipated success rates is very important for its development as well as its potential application value. generally speaking, the following three cross-validation methods are often used to examine the quality of a predictor and its effectiveness in practical application: independent dataset test, subsampling or k-fold (such as five-fold, seven-fold, or -fold) crossover test and jackknife test [ ] . however, as elaborated by a penetrating analysis in [ ] , considerable arbitrariness exists in the independent dataset test. also, as demonstrated in [ ] , the subsampling (or k-fold crossover validation) test cannot avoid arbitrariness either. only the jackknife test is the least arbitrary that can always yield a unique result for a given benchmark dataset [ , , , [ ] [ ] [ ] . therefore, the jackknife test has been widely recognized and increasingly utilized by investigators to examine the quality of various predictors (see, e.g., [ , , , , , , , , ] ). accordingly, in this study the jackknife test was also adopted to evaluate the accuracy of the current predictor. as mentioned above, the svm operation engine contains two uncertain parameters c and  . to find their optimal values, a -d grid search was conducted by the jackknife test on the benchmark dataset . the results thus obtained are shown in figure , from which it can be seen that the inr-drug predictor reaches its optimal status when c = and    . the corresponding rates for the four metrics (cf. equation ( )) are given in table , where for facilitating comparison, the overall accuracy acc reported by he et al. [ ] on the same benchmark dataset is also given although no results were reported by them for sn, sp and mcc. it can be observed from the table that the overall accuracy obtained by inr-drug is remarkably higher that of he et al. [ ] , and that the rates achieved by inr-drug for the other three metrics are also quite higher. these facts indicate that the current predictor not only can yield higher overall prediction accuracy but also is quite stable with low false prediction rates. as mentioned above (section . ), the jackknife test is the most objective method for examining the quality of a predictor. however, as a demonstration to show how to practically use the current predictor, we took nr-drug pairs from the study by yamanishi et al. [ ] that had been confirmed by experiments as interactive pairs. for such an independent dataset, were correctly identified by inr-drug as interactive pairs, i.e., sn = / = . %, which is quite consistent with the rate of . % achieved by the predictor on the benchmark dataset via the jackknife test as reported in table . it is anticipated that the inr-drug predictor developed in this paper may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well. since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful predictors [ , ] , a publicly accessible web-server for inr-drug was established. for the convenience of the vast majority of biologists and pharmaceutical scientists, here let us provide a step-by-step guide to show how the users can easily get the desired result by using inr-drug web-server without the need to follow the complicated mathematical equations presented in this paper for the process of developing the predictor and its integrity. step . open the web server at the site http://www.jci-bioinfo.cn/inr-drug/ and you will see the top page of the predictor on your computer screen, as shown in figure . click on the read me button to see a brief introduction about inr-drug predictor and the caveat when using it. step . either type or copy/paste the query nr-drug pairs into the input box at the center of figure . each query pair consists of two parts: one is for the nuclear receptor sequence, and the other for the drug. the nr sequence should be in fasta format, while the drug in the kegg code beginning with the symbol #. examples for the query pairs input and the corresponding output can be seen by clicking on the example button right above the input box. step . click on the submit button to see the predicted result. for example, if you use the three query pairs in the example window as the input, after clicking the submit button, you will see on your screen that the "hsa: " nr and the "d " drug are an interactive pair, and that the "hsa: " nr and the "d " drug are also an interactive pair, but that the "hsa: " nr and the "d " drug are not an interactive pair. all these results are fully consistent with the experimental observations. it takes about minutes before each of these results is shown on the screen; of course, the more query pairs there is, the more time that is usually needed. step . click on the citation button to find the relevant paper that documents the detailed development and algorithm of inr-durg. step . click on the data button to download the benchmark dataset used to train and test the inr-durg predictor. step . the program code is also available by clicking the button download on the lower panel of figure . nuclear receptors in cell life and death nuclear receptors nuclear receptors: current concepts and future challenges the nuclear receptor superfamily non-steroid nuclear receptors: what are genetic studies telling us their role in renal life? nuclear receptor drug discovery nuclear receptors and drug disposition gene regulation a web-server for identifying g-protein coupled receptors and their families with grey incidence analysis bioinformatical analysis of g-protein-coupled receptors igpcr-drug: a web server for predicting interaction between gpcrs and drugs in cellular networking a cellular automaton image approach for predicting g-protein-coupled receptor functional classes gpcr- l: predicting g protein-coupled receptors and their types by hybridizing two different modes of pseudo amino acid compositions prediction of g-protein-coupled receptor classes in low homology using chou's pseudo amino acid composition with approximate entropy and hydrophobicity patterns prediction of g-protein-coupled receptor classes based on the concept of chou's pseudo amino acid composition: an approach from discrete wavelet transform using ensemble svm to identify human gpcrs n-linked glycosylation sites based on the general form of chou's pseaac identifying gpcrs and their types with chou's pseudo amino acid composition: an approach from multi-scale energy representation and position specific scoring matrix prediction of g-protein-coupled receptor classes identify the channel-drug interaction in cellular networking with pseaac and molecular fingerprints insights from modelling three-dimensional structures of the human potassium and sodium channels influenza m proton channels a model of the complex between cyclin-dependent kinase (cdk ) and the activation domain of neuronal cdk activator rapid and accurate structure determination of coiled-coil domains using nmr dipolar couplings: application to cgmp-dependent protein kinase ialpha the three-dimensional structure of the cgmp-dependent protein kinase i-α leucine zipper domain and its interaction with the myosin binding subunit determination of the packing mode of the coiled-coil domain of cgmp-dependent protein kinase ialpha in solution using charge-predicted dipolar couplings a guide to drug discovery: target selection in drug discovery target discovery a fast flexible docking method using an incremental construction algorithm binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars nmr studies on how the binding complex of polyisoprenol recognition sequence peptides and polyisoprenols can modulate membrane structure review: progress in computational approach to drug development against sars molecular modelling and chemical modification for finding peptide inhibitor against sars cov mpro an in-depth analysis of the biological functional studies based on the nmr m channel structure of influenza a virus energetic analysis of the two controversial drug binding sites of the m proton channel in influenza a virus investigation into adamantane-based m inhibitors with fb-qsar designing inhibitors of m proton channel against h n swine influenza virus insights from investigating the interaction of oseltamivir (tamiflu) with neuraminidase of the h n swine flu virus review: structural bioinformatics and its impact to biomedical science identification of proteins interacting with human sp during the process of viral infections docking and molecular dynamics study on the inhibitory activity of novel inhibitors on epidermal growth factor receptor (egfr) novel inhibitor design for hemagglutinin against h n influenza virus by core hopping method design novel dual agonists for treating type- diabetes by targeting peroxisome proliferator-activated receptors with core hopping approach insights from modeling the d structure of new delhi metallo-betalactamase and its binding interactions with antibiotic drugs insights into the mutation-induced hhh syndrome from modeling human mitochondrial ornithine transporter- mitochondrial uncoupling protein structure determined by nmr molecular fragment searching structure and mechanism of the m proton channel of influenza a virus unusual architecture of the p channel from hepatitis c virus the structure of phospholamban pentamer reveals a channel-like architecture in membranes the structural basis for intramembrane assembly of an activating immunoreceptor complex solution nmr structure of the v a drug resistant mutant of influenza a m channel mechanism of drug inhibition and drug resistance of influenza a m channel solution structure and functional analysis of the influenza b proton channel prediction of the tertiary structure and substrate binding site of caspase- prediction of the tertiary structure of a caspase- /inhibitor complex prediction of the tertiary structure of the beta-secretase zymogen coupling interaction between thromboxane a receptor and alpha- subunit of guanine nucleotide-binding protein insights from modeling the d structure of dna-cbf b complex modeling the tertiary structure of human cathepsin-e assessment of chemical libraries for their druggability predicting drug-target interaction networks based on functional groups and biological features some remarks on protein attribute prediction and pseudo amino acid composition ( th anniversary year review) identify recombination spots with trinucleotide composition and pseudo amino acid components identify recombination spots with pseudo dinucleotide composition identifying the heat shock protein families using pseudo reduced amino acid alphabet composition combining evolutionary information extracted from frequency profiles with sequence-based kernels for protein remote homology detection inuc-physchem: a sequence-based predictor for identifying nucleosomes via physicochemical properties predict cysteine s-nitrosylation sites in proteins by incorporating position specific amino acid propensity into pseudo amino acid composition incorporating amino acid pairwise coupling into pseaac for predicting cysteine s-nitrosylation sites in proteins iezy-drug: a web server for identifying the interaction between enzymes and drugs in cellular networking iamp- l: a two-level multi-label classifier for identifying antimicrobial peptides and their functional types a sequence-based predictor for predicting nucleosome positioning in genomes with pseudo k-tuple nucleotide composition the kegg databases and tools facilitating omics analysis: latest developments involving human diseases and pharmaceuticals review: recent progresses in protein subcellular location prediction an intriguing controversy over protein structural class prediction some insights into protein structural class prediction prediction of enzyme family classes slle for predicting membrane protein types predicting protein structural classes with pseudo amino acid composition: an approach using geometric moments of cellular automaton image a novel approach to predicting protein structural classes in a ( - )-d amino acid composition space subcellular location prediction of apoptosis proteins boosting classifier for predicting protein domain structural class artificial neural network for predicting alpha-turn types neural network prediction of the hiv- protease cleavage sites a sequence-based predictor for identifying nuclear receptors and their subfamilies via physical-chemical property matrix idna-prot: identification of dna binding proteins using random forest with grey model afp-pred: a random forest approach for predicting antifreeze proteins from sequence-derived properties predicting subcellular localization of proteins in a hybridization space prediction of protease types in a hybridization space predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic k-nearest neighbor classifiers hum-ploc: a novel ensemble classifier for predicting human protein subcellular localization large-scale predictions of gram-negative bacterial protein subcellular locations euk-mploc: a fusion classifier for large-scale eukaryotic protein subcellular location prediction by incorporating multiple sites signal-cf: a subsite-coupled and window-fusing approach for predicting signal peptides using optimized evidence-theoretic k-nearest neighbor classifier and pseudo amino acid composition to predict membrane protein types a top-down approach to enhance the power of predicting human protein subcellular localization: hum-mploc . fuzzy knn for predicting membrane protein types from pseudo amino acid composition prediction of protein cellular attributes using pseudo amino acid composition using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes theoretical and experimental biology in one predicting anticancer peptides with chou's pseudo amino acid composition and investigating their mutagenicity via ames test predicting plant protein subcellular multi-localization by chou's pseaac formulation based multi-label homolog knowledge transfer learning euloc: a web-server for accurately predict protein subcellular localization in eukaryotes by incorporating various features of sequence segments into the general form of chou's pseaac predict mycobacterial proteins subcellular locations by incorporating pseudo-average chemical shift into the general form of chou's pseudo amino acid composition using radial basis function on the general form of chou's pseudo amino acid composition and pssm to predict subcellular locations of proteins with both single and multiple sites prediction of subcellular localization of apoptosis protein using chou's pseudo amino acid composition goasvm: a subcellular location predictor by incorporating term-frequency gene ontology into the general form of chou's pseudo-amino acid composition predicting protein subchloroplast locations with both single and multiple sites via three different modes of chou's pseudo amino acid compositions predicting membrane protein types by incorporating protein topology, domains, signal peptides, and physicochemical properties into the general form of chou's pseudo amino acid composition a multilabel model based on chou's pseudo-amino acid composition for identifying membrane proteins with both single and multiple functional types genetic programming for creating chou's pseudo amino acid based features for submitochondria localization predicting protein submitochondria locations by combining different descriptors into the general form of chou's pseudo amino acid composition multi-kernel transfer learning based on chou's pseaac formulation for protein submitochondria localization using the augmented chou's pseudo amino acid composition for predicting protein submitochondria locations based on auto covariance approach prediction of gaba(a) receptor proteins using the concept of chou's pseudo-amino acid composition and support vector machine using chou's amphiphilic pseudo-amino acid composition and support vector machine for prediction of enzyme subfamily classes predicting antibacterial peptides by the concept of chou;s pseudo-amino acid composition and machine learning methods supersecondary structure prediction using chou's pseudo amino acid composition identifying bacterial virulent proteins by fusing a set of classifiers based on variants of chou's pseudo amino acid composition and on evolutionary information a novel feature representation method based on chou's pseudo amino acid composition for protein structural class prediction predicting the cofactors of oxidoreductases based on amino acid composition distribution and chou's amphiphilic pseudo amino acid composition prediction of metalloproteinase family based on the concept of chou's pseudo amino acid composition using a machine learning approach secretp: identifying bacterial secreted proteins by fusing new features into chou's pseudo-amino acid composition prediction of allergenic proteins by means of the concept of chou's pseudo amino acid composition and a machine learning approach using chou's pseudo amino acid composition to predict protein quaternary structure: a sequence-segmented pseaac approach identifying protein quaternary structural attributes by incorporating physicochemical properties into the general form of chou's pseaac via discrete wavelet transform using the concept of chou's pseudo amino acid composition for risk type prediction of human papillomaviruses prediction of cyclin proteins using chou's pseudo amino acid composition discriminating outer membrane proteins with fuzzy k-nearest neighbor algorithms based on the general form of chou's pseaac use of fuzzy clustering technique and matrices to classify amino acids and its impact to chou's pseudo amino acid composition protein remote homology detection by combining chou's pseudo amino acid composition and profile-based protein representation identification of colorectal cancer related genes with mrmr and shortest path in protein-protein interaction network hepatitis c virus network based classification of hepatocellular cirrhosis and carcinoma signal propagation in protein interaction network during colorectal cancer progression pseaac-builder: a cross-platform stand-alone program for generating various special chou's pseudo-amino acid compositions propy: a tool to generate various modes of chou's pseaac pseaac: a flexible web-server for generating various kinds of protein pseudo amino acid composition the folding type of a protein is relevant to the amino acid composition an optimization approach to predicting protein structural class from amino acid composition monte carlo simulation studies on the prediction of protein folding types from amino acid composition predicting protein folding types by distance functions that make allowances for amino acid interactions monte carlo simulation studies on the prediction of protein folding types from amino acid composition. ii. correlative effect does the folding type of a protein depend on its amino acid composition? protein secondary structural content prediction the convergence-divergence duality in lectin domains of the selectin family and its implications a multi-label classifier for predicting the subcellular localization of singleplex and multiplex eukaryotic proteins using accumulation-label scale to predict subcellular locations of human proteins with both single and multiple sites improving the accuracy of psi-blast protein database searches with composition-based statistics and other refinements gapped blast and psi-blast: a new generation of protein database search programs a comparison of normalization methods for high density oligonucleotide array data based on variance and bias feature extraction and normalization algorithms for high-density oligonucleotide gene expression array data prediction of protein subcellular localization by support vector machines using multi-scale energy and pseudo amino acid composition low-frequency fourier spectrum for predicting membrane protein types using stacked generalization to predict membrane protein types based on pseudo amino acid composition prediction of linear b-cell epitopes using amino acid pair antigenicity scale predicting secretory proteins of malaria parasite by incorporating sequence evolution information into pseudo amino acid composition via grey system model using functional domain composition and support vector machines for prediction of protein subcellular location support vector machines for predicting membrane protein types by using functional domain composition an introduction of support vector machines and other kernel-based learning methodds libsvm: a library for support vector machines prediction of protein signal sequences and their cleavage sites using subsite coupling to predict signal peptides prediction of signal peptides using scaled window some remarks on predicting multi-label attributes in molecular biosystems review: prediction of protein structural classes cell-ploc: a package of web servers for predicting subcellular localization of proteins in various organisms cell-ploc . : an improved package of web-servers for predicting subcellular localization of proteins in various organisms predicting enzyme family classes by hybridizing gene product composition and pseudo-amino acid composition identify catalytic triads of serine hydrolases by support vector machines using pseudo amino acid composition to predict protein subcellular location: approached with amino acid composition distribution discriminating bioluminescent proteins by incorporating average chemical shift and evolutionary information into the general form of chou's pseudo amino acid composition a multi-layer classifier for predicting the subcellular localization of singleplex and multiplex gram-positive bacterial proteins drug-target interaction prediction from chemical, genomic and pharmacological data in an integrated framework review: recent advances in developing web-servers for predicting protein attributes the authors would like to express their gratitude to the three anonymous reviewers, whose constructive comments are very helpful for strengthening the presentation of the paper. the authors declare no conflict of interest. key: cord- -xhzxhwgo authors: nan title: book reviews date: journal: pharm res doi: . /b:pham. . .f sha: doc_id: cord_uid: xhzxhwgo nan the last few years have been a difficult time for the gene therapy field. a great deal of negative publicity was evoked by the occurrence of serious adverse consequences, including deaths, during clinical trials involving viral based gene delivery vectors. this has caused gene therapists to re-examine some of the basic premises in their field. in many ways, the volume from curiel and douglas is an outgrowth of that process. in their insightful preface, drs. curiel and douglas discuss the need for greater selectivity of gene therapy strategies, as well as the challenges in attaining that aim. in this broadranging volume they have included chapters on many aspects of selective targeting for both viral and non-viral gene delivery systems. the book is divided into five sub-sections. the first two sections cover current strategies for improving cell type selective delivery for viral and non-viral vectors. a third section deals with approaching selectivity by regulation at the transcriptional level. section four presents interesting discussions of how to find appropriate markers on target cells using various combinatorial library strategies. finally a last chapter deals with the in vivo evaluation of gene therapy using imaging technologies. to this reviewer, the first set of chapters, dealing with liposomes and other non-viral vectors, was only moderately interesting. many of the concepts and strategies discussed represented only modest refinements of ideas about cell targeting that have been around for a long time. one exception is the concept of using anionic liposomes (rather than the usually cationic variety) to convey dna into cells; thus the formation and characteristics of dna/poly-lysine/anionic liposome complexes is described in a chapter from l. huang and colleagues. the second section, dealing with the design of improved viral vectors, was quite fascinating. among the chief problems of adenovirus based gene therapy are ( ) the viral receptor, although widely expressed, is not present in certain cell types of key therapeutic interest, and ( ) humans have neutralizing antibodies to common adenoviral pseudotypes. investigators are addressing these problems in several ways. for example, molecular re-engineering of the virus fiber protein (which binds the cellular receptor) can change the tropism of the virus, suppressing binding to its usual receptor, and permitting binding to new cell-type specific targets. further, use of rarer viral serotypes as building blocks for gene delivery vectors may avoid the problem of wide spread pre-existing immunity to the more common serotypes. a chapter by t. wickham was particularly illuminating in terms of presenting an overall strategy for improved adenoviral vector design. some of the same, or similar, strategies for enhancing selectivity are also being applied to other viral vectors including adenoassociated virus, bacteriophages, sindbis virus and retroviruses. a third major section of the volume deals with regulation of the expression of the delivered gene. tissue specific or disease specific expression is often a keenly desired goal in gene therapy. although a number of tissue/disease specific promoters are known, often these fail to provide sufficiently high levels of gene expression or sufficiently "tight" control of expression to be of practical use. thus gene therapists have intensively investigated the optimization of promoter/ enhancer elements for various vector systems and therapeutic targets. r. muller and colleagues give a good account of this issue in a chapter. seeking cell-type specific or disease-specific targets is a key part of improving vector selectivity. in section four of the volume, various technologies for finding such targets are discussed. often this is done by sifting through large peptide libraries to find sequences that bind to specific cell surface determinants. in addition to the well-known phage display strategy, the volume provides descriptions of novel approaches, including retroviral display libraries, and bacterial display libraries. overall this volume provides a very comprehensive picture of the current state of selective targeting of gene therapy vectors. most of the contributors are well known in their respective areas, the chapters are timely, well written, and informative, and the editors provide the "big picture" to draw it all together. thus the volume should be of value both to investigators within the gene therapy field and to other scientists who may be seeking an introduction to this exciting area of research. chapters) focuses on the synthesis of small dna fragments and pna. the chapter by richard pon offers an excellent summary on chemical synthesis of oligonucleotides with a refreshing emphasis on various synthetic strategies for smallor large-scale production. salvatore marras provides an indepth analysis on both the theoretical and practical aspects of probe design for dna or rna hybridization. the synthesis, structural, and chemical properties of pna are the major focus of the chapter by beck and nielsen. this is a very informative chapter for those wishing to learn the basics of pna. lisa kelly's summary of methods for amplifying dna from dna or rna is particularly useful for pcr-lovers, because so many systems are available for pcr-based dna amplification. this chapter explains how relationships between primer design and the pcr products can have different biological applications. the histological aspects of pcr discovery described should be particularly interesting to young researchers who have not followed the history of science discovery closely. the second area ( chapters) focuses on dna manipulation. for a touch of genome structure, the chapter by lisa ganova-raeva gives an overview of genomic organization of various genetic elements. the inclusion of the methods and strategies utilized in the structural analysis of the genome is an additional feature of this chapter. in their chapter, glass and heinz summarize many new developments in site-specific mutagenesis, a powerful tool for structure-function analysis of gene products. a comprehensive review of the pros and cons of various methods for introducing mutations into a dna sequence is extremely useful for those who are interested in this technique. the last chapter in the second focused area is by pumpens and grens, who provide an in-depth discussion of the use of viral vectors for delivering a desirable gene into target cells for protein production. the discussion of the viral proteins involved and the summary of many of the wellstudied viruses will be extremely useful to those interested in developing viral vectors for gene delivery. for those interested in dna applications in immunology, the final four chapters should be especially helpful. renu tuteja's overview on minotopes summarizes various types of immunologically important epitopes. application of phage display technology to vaccine development is an excellent example of the application of artificial dna. the chapter by han et al describes more recent developments toward the use of dna to stimulate immune responses. the field of dna vaccines has attracted much attention in the scientific community because of its simplicity and safety features. the last two chapters by joy chang, and montano and pujol illustrate the direct application of phage display technology to antigen and antibody discovery. this technology may offer a practical and efficient means to obtain antigens and antibodies without using animals. overall, this is an excellent book presenting a panorama of the achievements and perspectives for further exploration from the perspective of recognized experts and leaders in the field. it covers a broad spectrum of topics ranging from how to make dna and its analogues to how to use them and where to look for new applications and improvement. it is a book that will meet the needs not only of those at entry level but also those experienced researchers who wish to refresh their memory of the past and glimpse the future of the field. as more knowledge of cellular components and their interactions is compiled, it is only natural that efforts are made to look at this information in novel ways that could provide new avenues of therapeutic intervention. with each layer of composition and interactions involving cellular function in healthy and disease states comes another new term. first, there was a focus on the genome that has culminated with a nearly complete sequence map for each of the human chromosomes. as information about the genome was gathered a wide range of important observations came to light related to functions of non-transcribed regions, microsequences, and how rna structures can act to inhibit the function of selected genes. next was the proteome. this effort initially provided information about the translation of proteins and how sets of proteins can be related to a particular cell function. with time it became apparent that critical information about protein function could be gathered from studying how proteins interacted with each other through specific domains that associate with great selectivity. the idea of scaffolding proteins that organize a set of functionally related proteins has now become a standard in assessing proteinrelated events. proteins, either alone or in these complexes, can perform many cellular functions. one set of these functions involves intracellular transmission of external stimuli. modulation of phosphorylation state of a large number of proteins (through the actions of kinases and phosphatases) is the basis for these signal transduction events. this has led to yet another new term-the kinome. where does this lead us? well, many places actually, with one of the most important ones being discoveries that show a critical role for some of these kinases and phosphatases in a variety of disease states. the book edited by finkel and gutkind provides a sweeping look at current information derived from assessing the role of some of these kinases and phosphatases on human conditions associated with disease. the book contains thoughtful, wellplanned chapters on topics such as cancer, cardiovascular disease, asthma, diabetes, infection, immunity, and neurodegenerative diseases. each chapter focuses on a particular topic in a well-organized manner with extensive citations and excellent artwork that describes relationships of that domain of the human kinome identified to be involved in that particular disease or condition. after reading the chapters in this book the reader comes away with an appreciation of how the kinome is both redundant and specific. the same kinases and phosphatases appear to be players in many of the diseases discussed, only in each case they perform slightly different functions or regulate dif-ferent events based upon the cell type or tissue in which they function. this provides the reader with an overall picture of how the kinome might be manipulated in a cell-or tissuespecific fashion to address the various disease states described. here is where i feel this text becomes extremely valuable to most of us reading it-getting a sense for where and how logical therapeutic interventions might be identified. from this, one gets a sense for the strategy of targeting components of the kinome. in certain situations, increased production of antibodies against body's own cell or tissue components might cause an autoaggressive disease. despite extensive research over the past decades, exact mechanisms of the phenomenon of "autoimmunity" are still unknown. this book is dedicated to the relationship between autoimmunity and apoptosis. the book represents a collection of papers contributed by leading scientists in this area from all over the world. it is organized in six parts. part is dedicated to general features of apoptosis and relations between apoptosis and autoimmunity. it describes the main mechanisms and immune functions of apoptotic cell death with emphasis on the role of caspases in the programmed cell death. this part discusses two general types of autoimmune diseases associated respectively with enhanced cell growth and survivals or abnormal processing in dying cells. part describes mechanisms of the clearance of apoptotic cells, anti-inflammatory and immunoregulatory effects of apoptotic cells. it discusses the role of complement, pentraxins, collectins, autoantibodies, atp-binding cassette transporters and cd in the clearance of apoptotic cells. part analyzes autoimmunity caused by defective execution of apoptosis or defective clearance of apoptotic cells. this part is mainly concentrated with the autoimmune lymphoproliferative syndrome (alps), systemic lupus erythematosus and rheumatoid arthritis. part deals with immunogenecity of apoptotic cells. it focuses on the dendritic cells pulsed with apoptotic tumor cells as vaccines and the immune response against apoptotic cells. part discusses autoantigens as substrates for apoptotic proteases and their implication for the pathogenesis of systemic autoimmune disease, the role of cleavage products of autoantigens, transglutaminases, and modifications of rna in autoimmunity. part analyzes the role of dna binding proteins in systemic autoimmunity, focuses specifically on nucleosomes and anti-nucleosome autoantibodies as mediators of glomerular pathology in systemic lupus erythematosus. the book is illustrated by original figures, schemas, and tables and contains interesting experimental data that support author's hypotheses. it is well written and organized and includes glossary of terms and subject index that significantly help readers to understand such complicated subject of modern biomedical science as the relationship between apoptosis and autoimmunity. when i was given the opportunity to review this book i was really excited. every month gibaldi writes an interesting column for aaps news and there is probably not a kineticist in the world who doesn't own a copy of the book he helped co-write on pharmacokinetics. so when i heard that he had co-written a book on biotech drugs i was filled with anticipation. then i received the book and was at first disappointed because i was expecting something more rigorous and formal, something more academic. after i started to read it i was pleasantly surprised because hidden beneath the glossy, csilike cover lies a wealth of knowledge. the chapters don't cover material in great depth (and if they did, this book will be many times larger) but they provide enough information to cover what someone new to the area would need to know to get started. it also provides a handy reference for properties of already marketed drugs. this book consists of three parts. part deals with the development aspects of biotechnology products: big pharma vs. biotech, small molecules vs. macromolecules, biotechnology processes, and pharmacologic principles of macromolecules. part deals with therapeutic classes: proteins (growth factors, hormones, and enzymes), monoclonal antibodies, and vaccines. each chapter in this section presents the basic pharmacology of the drug class and other relevant issues. each chapter usually concludes with drug monographs for all the approved drugs in that class providing the drug name, trade name, approval date, type of submission, and then a brief summary of the package insert for that drug. part deals with the future, where the authors see biotechnology heading. these chapters cover genomics and proteomics, gene therapy, individualized therapy based on pharmacogenetics, and advances in drug delivery. the appendices of the book present a tabular summary of the doses, dosage forms, and pharmacokinetics for each drug similar to the pharmacokinetics appendix in goodman and gilman's the pharmacological basis of therapeutics. this will be a useful book for someone wishing an introduction to drug development of macromolecules and for someone who needs a convenient reference for the properties and pharmacokinetics of approved biotechnology products. the chapters are short, filled with useful illustrations, and the tables are of good quality. what i found especially interesting were the "boxes" within each chapter. some of these are interesting anecdotes, such as how genentech was formed, while others are side-lines of particular note that do not quite fit in elsewhere within the chapter, such as how market exclusivity can become nonexclusive for drugs with orphan drug status. as a point of reference, this book is less like goodman and gilman's text and more like melmon and morelli's clinical pharmacology text. in summary, this will be a useful book for an upper-level undergraduate or graduate level class in the pharmacology and pharmacokinetics of biotechnologyderived products, as well, as a good introductory book for people new to the biotechnology industry. there has been an explosion of activity in the area of bioinformatics. the method of computational analysis allows great deal of progress to work in the sea of biological data. the book "introduction to bioinformatics. a theoretical and practical approach" is a good starting point for people stepping into the bioinformatics field for the first time. it starts with an explanation about the cell. parts i and ii provide a nice summary to bioinformatists with a computer science background to understand biological data they will handle. parts iii and iv focus on the computer applications used in the field. they cover the basic knowledge about unix operating system (chapter ) for novices in computer science, and describe the bioinformatics software (chapters , , and ) interesting to biologists for their research. the last section contains the overview of microarrays (chapter and ) which have got very popular for gene expression analysis these days. one more great thing about this book is that each chapter has a glossary at the end making all the abbreviations clear. also, the included cd-rom is a good add up, particularly with several software programs which can be installed on the computer to give readers the chance to play around. the book "structural bioinformatics" is more advanced than the other as can be noted by the title. this book is a good source to read if you are working on structural bioinformatics field. all the sections are carefully edited. the first section describes fundamentals on biological materials (for example, protein structure) and bioinformatics tools (for example, electron microscopy). then it spreads its touch into the detail aspects such as data representation (for example, databases) and structure prediction (for example, modeling). each section provides an own insight into the specific part of structural bioinformatics. membrane transporters are going to be the major role players in the rational design of next generation drugs such as peptides, nucleosides, carbohydrates, and complex lipids. in recent years, membrane transporters have been an intensive area of research because of their key roles in drug absorption, distribution, and efficacy. this book pulled together pharmacogenomics, bioinformatics, microarray technology, and upto-date methodology for studying membrane transporters. chapters and of this book give a general overview on pharmacogenomics and classification of membrane transporters. the first two chapters would be useful to someone who wants to have a general understanding of membrane transporters. chapter describes methodology and protocols for using bioinformatics database and computational tools in membrane transporters. authors provide examples on how to use bioinformatics protocols and address most frequently encountered problems in using bioinformatics tools in membrane transporters. chapters and describe hybridization protocols, problems associated with hybridization and computational method for microarray data analysis, respectively. the book also contains several chapters on the use of instrumental techniques such as fluorescence, laser capture, electrophysiological, x-ray scattering, nmr imaging and molecular modeling in studying and characterization of membrane transporters. chapter and gives a step-by-step approach to the reconstitution and quantitation of membrane transporters. overall, all chapters of the book contain step-by-step methodology for quantitation, characterization, and computational analysis for studying membrane transporters. however, this book could be a bit difficult to follow for scientists trained in drug delivery research. a brief introduction on the basic principles of the techniques would probably increase the readership of the book. further, the sequence of the chapters is somewhat inconsistent. for example, all instrumental techniques could be put in one part of the book, protocols and data analysis could be in another section. i strongly recommend this book to labs that are working on membrane transporter based drug delivery, design, and discovery. this book could also be a very useful reference for molecular biology labs that are working on characterization, quantitation, and reconstitution of membrane transporters. protocols described in this book can easily be followed by both experienced researchers and lab technicians with little training in the field. in the field of pharmaceutical sciences, membrane transporters are now accepted as the important determinant of drug absorption, distribution, and elimination. furthermore, coordinated reactions of transporters with metabolic enzymes for the detoxification of xenobiotics are now going to be clarified. so, it is essential to introduce the concept of transporters in the processes of membrane permeation for drug development and clinical drug therapy. however, membrane transporters are essential biological molecules for the normal cell functions and the predicted numbers of transporters in human genome is close to a thousand. from the presence of such a plenty of transporter molecules, it is not difficult to imagine that transporters have crucial roles in regulating the movements of physiologically important compounds across the cell membranes. accordingly, the deep understanding of membrane transporters should help to get new ideas for new drug targets, for the mechanisms of the toxicity induced by the drugs, for the mechanisms of drug absorption and disposition, and for the novel drug delivery. "transmembrane transporters" will give us the present status of membrane transporters, including classification, methods to study functionality and structure of transporters, and physiological roles. this book consists of chapters and the contents may not be directly related to drug absorption, distribution or disposition, but they show various and new technical approaches that can be applied to any transporters. the transporter molecules described in this book are for hexose, glutamic acid, neurotransmitters such as serotonin, dopamine, and gaba, citrate, and others, but each chapter intended not only to explain the characteristics of each transporter, but also to demonstrate the usefulness of the method used by including the brief protocols for the experimental procedures used in most of chapters. the techniques described are, for example, ( ) genetically modified yeast s. cerevisiae to isolate and characterize sugar transporters from various organisms such as humans, ( ) gene-knockout mice for neurotransmitter transporter to clarify the in vivo roles, ( ) the methods to identify the genetic variations such as snps and vntr polymorphisms in serotonin transporters, ( ) the usefulness of nonviral gene transfer into brain synaptic cells, and ( ) the methods to study trafficking of transporters by identifying the subcellular localization. other topics include structural analyses of membrane transporters by cysteine-scanning mutagenesis, photoaffinity labeling and subsequent proteolysis to identify the ligandprotein interacting sites, and basis and application of mass spectrometry. in addition, this book includes the functional analyses of transporters by voltage clamp fluorometry, electrophysiological assay, and imaging of neurotransmitter transporters by pet and spect using tracer-imaging agents. because all of the chapters have a list of related references, readers can deepen their understanding easily. as described above, this book does not intend to summarize transporters related to drugs rather tried to introduce many technologies to be used in future studies for molecular cloning, structure, functionality, regulation, and sorting in various point of views by using typical experimental results on physiologically important transporter molecules. accordingly, people, who are interested in transporter research, can get new idea for the next research from this book. with the latest incidence of the sars virus, i view the need of the advancement of the technologies for drug discovery, design and screening is to save lives more than anything else. the book gives a comprehensive overview of the latest tools used for drug discovery from the various disciplines such as chemistry, biology, and computational sciences. the book is divided into three sections. the first section focuses on the target identification and validation, the second section describes the tools for high throughput screening of new bioactive molecules, and the last section contains methodologies used for drug molecule synthesis and preparation. the technologies reported in the book are complex. the authors, however, were capable of delivering the information in a very clear and concise way. in many instances, explanations were built on the fundamentals and supporting materials. the current status of the technology was described with many practical examples and protocols. future progress and application of the technologies were also discussed. a well written chapter such as "adme-tox screening in drug discovery" discussed both pros and cons of the different methodologies. the author also pointed out the aspects (e.g. test models, assays validation) which still require improvement and further development to enhance the ability to select drug candidates with the best probability for further clinical development. this text is a valuable addition to the reference shelf for scientists who have a reasonable amount of knowledge in drug discovery. because readers may be interested in taking up the opportunity to learn about the latest technologies from this book, researchers may also consider a possible collaboration with scientists in their particular fields. because many of these technologies are rather sophisticated, establishment of these techniques or instruments in a laboratory could cost significant amounts of resources. the transfer of samples from one laboratory to another would seem to be a lot sim-pler and more feasible. collaborations can often be more fruitful and enjoyable than working alone. over the past years, high-performance liquid chromatography (hplc), sometimes called high-pressure liquid chromatography, has become a major assay technique due to endless advances in hplc system components such as detectors and hplc columns, along with computerization of the system. in addition, the united states pharmacopeia has played a critical role in expanding the application areas of hplc technique to a broad range of analytes. hplc technique becomes even more important especially when it comes to drug stability studies since it can overcome analytical hurdles such as interference from degradation products and excipients. the second edition of stability-indicating hplc methods for drug analysis is a unique, comprehensive, and condensed collection of previously published stability-indicating hplc methods and the examples of their use in evaluating drug stability in various pharmaceutical dosage forms. this book lists alphabetically monographs on different drug compounds of which are new to this edition. each monograph is clearly divided into two main components, basic information and hplc method summary with references. the basic information part, obtained from standard reference works including the merck index and ahfs drug information, presents chemical name(s), other name(s), form(s) of the drug molecule available in drug products, molecular formula, molecular weight, cas number, appearance and description, solubilities, and pk a values. this is followed by text format summaries of previously published stability-indicating hplc methods. the summaries of hplc methods are highly well-structured and subdivided into different sections. the first part of the method summary describes mainly information about hplc conditions whereas the second section is focused on sample preparation procedures containing dilution, extraction, and derivatization. in the third section, the stability-indicating nature of the method is presented by describing, for instance, decomposition techniques, absence of interference of degradants with intact drug, and retention times corresponding to intact drug and its decomposition products. lastly, the fourth section details the information on standard curve, intra-and inter-day variation, and limits of detection and quantitation. due to the fact that most of the efforts of analytical method development within the pharmaceutical sciences go into establishing and validating a stability-indicating hplc method, the book will save analytical chemists time and efforts in identifying a suitable stability-indicating hplc method available in the published literature. indeed, the usefulness of this book comes from the authors' effort to exclude general hplc methods primarily used for the measurement of drug concentrations in biological matrices, so that only stability-indicating methods are compiled in the book. for readers not only wishing to develop hplc methods as a means of studying drug stability but also carrying out research in the field of dosage form development, quality control, and drug regulation, this book will be an indispensable one. many people are uncomfortable speaking in public. i remember in graduate school we were required to give a seminar once a year. the semester we were to present, students started panicking and by the day of the talk the tension was unbearable. the reason for our panic was that despite getting a quality scientific education, we never received any training on how to speak in public or write effectively. i believe this is one of the great failures in science education in this country today. a scientist that cannot communicate ef-fectively cannot be effective. one way to become a good speaker is to either take a class or to read one or more of the many books on this topic and then practice. the emphasis on practice cannot be overstated because no matter how many books you read on the subject it is not until you have spoken in public many times that you really begin to understand what works and what doesn't. one of the latest books on public speaking is by the husband and wife team of peter and cheryl reimgold: the short road to great presentations. having read a few of these type of books, the first thing that struck me about this book was the title. this is by far the largest book i have seen on the subject, yet apparently it is the shortest road. all kidding aside, this is a pretty good book. the writing is easy, the chapters are well organized, and the figures are easy to digest. clearly this book was written to support their seminars on great presentations with the material written to support their own powerpoint presentation. what sets this book aside from others are the exercises at the end of the chapters, which i thought were helpful in reinforcing the material presented. this book is broken down into three sections. section introduces the concept of speaking with the audience in mind. poor speakers speak without giving any consideration to the audience. great speakers present as if they were sitting in the back row and speak with an understanding of what the audience wants from a talk. every book on effective communication has a mnemonic on how to present and this book's is: ramp: establish rapport, get the audience's attention, state your main message, and give the plan of your talk. the body of the talk is then designed to meet the talk's objective taking into consideration what the audience wants. the second section of the book is on delivery: preparation, connecting with the audience, attitude, and handling questions and surprises. the last section is on giving electronic presentations, such as using powerpoint and giving webcasts. the ironic thing about this book is that it is published by the institute for electrical and electronic engineers, but was not written for that audience. this is not a book on how to give great scientific presentations. i would say that the target audience is business professionals. there is an overemphasis on using clip-art to enhance a presentation with little material on what constitutes a useful scientific graphic. still, before one can give a great scientific lecture one must understand what makes a great public presentation and that is what this book has in mind. all books on effective presentations present roughly the same material and ideas. the difference is in the presentation. some books use a more literary approach with few figures and diagrams. this book takes a more casual approach with a powerpoint style. think of it as an executive summary approach to the problem. the chapters are easy to read and i believe the tips that are presented throughout are really useful. all in all, this is one of the top two or three books i have seen on effective communicating. this book is a useful place to start if someone wants some general guidance on how to speak and give presentations in public. do not expect much in the way of information about enhancing the technical side of a presentation. over the past several decades, polymer chemistry has made great impact on our daily lives and polymers have become truly indispensable materials to mankind. numerous polymers with different properties have been synthesized. polymer synthesis has been done mainly by step and chain polymerizations. step and chain polymerizations are also called condensation and addition polymerizations, respectively. this book focuses on the fundamental understanding of step-growth polymers in a structured and informative way and attempts to show the link between experiment and theory. this book is divided into chapters, covering fundamental concepts in step-growth polymerization processes and experimental methodologies. chapter provides a general introduction to step-growth polymers and their synthesis. chapters - deals with reviews of classical polymers, such as polyesters, polyamides, polyurethanes, polyureas, polyimides, and others. chapters and include nontraditional stepgrowth polymerizations, such as acyclic diene methathesis and transition metal coupling. these polymerization methods have been perceived as a versatile route for synthesis of a wide range of functionalized polymers or different types of polymers that cannot be easily accessible or are impossible to make by using traditional synthetic methods. the final chapter describes depolymerization and recycling process of each step-growth polymer, which seems highly useful for recent interests in polymer recycling and environmental concerns. this book has several strong points. although there exist many textbooks dealing with polymer science, few attempts have been made to bring together general knowledge, various synthetic methods, structure-property relationships, and detailed experimental methodologies of individual polymers in one volume. this book is well organized and provides comprehensive information and a lot of examples for practical applications. each chapter provides a polymer-based step-bystep description of not only basic information including various classification, application, and structure-property relationships, but also very practical descriptions of synthetic and analytic techniques that are believed to be good references in research laboratories. each technique is described in detail and potential applications are also highlighted well. the book is aimed at researchers and graduate students with a fair knowledge of polymer chemistry and engineering, whereas less devotion to basic principal concepts and theories may make this book undesirable as a textbook for the beginners or undergraduate students in polymer science. this book, however, contains vast amounts of experimental techniques highly useful for polymer chemists or other researchers who intend to prepare and use these polymers for their final products. this book may well serve as a first line of reference source for all polymer and material scientists. akina, inc. cumberland ave., #e west lafayette, in - khuh@akinainc.com since when the book "principles of polymer chemistry" by paul flory was in print, numerous books have been published on polymer chemistry, science, and technology. advances in polymer science and technology through the years resulted in emergence of new topics that include polymers with biologic activities, biodegradable polymers, smart polymers, high performance polymers, and electrically conductive polymers, to name a few. as the list of new topics grows, the magnitude of information to be digested also grows exponentially. even in the age of computerized information technology, the volume of information on polymer in general simply becomes too large for anyone to absorb. the goal of encyclopedia of polymer science and technology is to present comprehensive, authoritative, and lucid articles on topics that reflect the progress and evolution of polymer science and technology. the topics in the encyclopedia were chosen to present balanced account of all facets of polymer science and technology. each volume starts with the same ( pages) introduction, conversion factors, abbreviations, and unit symbols, so that the readers do not have to go back to other volumes for the information. general index ( pages the list of topics in volumes - is obviously not exhaustive, and additional topics starting from a to z (e.g., anionic polymerization, biodegradable polymers, electrically active polymers, molecularly imprinted polymers, superabsorbent polymers, and x-ray microscopy) will be covered in volumes - (part of the series). each topic in the current volumes was indeed dealt with in great detail to present authoritative information. reading an article provides thorough understanding on each topic, as an encyclopedia is supposed to do. i was in need of reviewing polymer coating for application on controlled drug delivery, and when i opened the index page i found items under coating. the articles on "coating methods, survey" and "coatings" were very informative. the coating methods described in the article are not specifically for controlled drug delivery applications, but understanding coating machines, coating processes, multilayer methods, discrete surface-coating methods, patch coating, and drying and solidification makes me appreciate what can be possible and what are not in coating for drug delivery. the article on coatings explains film formation, flow properties that control application of appearance of films, mechanical properties, exterior durability, adhesion of films to substrates, resins and cross-linkers, solvents, color and appearance, pigment dispersion (which is not any different from drug particle dispersion), and film defects. again, information in the article is not specifically for drug delivery, but it is highly relevant to provide new ideas on polymer coating for drug delivery. considering the breadth and depth of articles in the first four volumes of the series, i am anxious to examine parts and that will be published soon. the encyclopedia is definitely something that should be placed on a bookshelf of every library. it would be ideal if it can be available to all researchers dealing with polymers at a reasonable price. for the past decade, the microdrop technology, which is already an indispensable part of inkjet printing, has been adopted by a wide range of scientific disciplines for precision dispensing of liquid reagents and theoretical research of fluid dynamics. it is also not an exotic topic to many biologists, because the microdrop generator is the central element of a flow cytometer. more recently, the microdrop technology has gained sizable attention from pharmaceutical research by providing an excellent opportunity to produce microparticles with a narrow size distribution. it is not hard to imagine that more and more researchers outside the world of microdrops will be interested in this technology and benefit from the unique qualities of microdrops. the problem is, however, that there has been virtually no easy way for those who are about to venture into the field to approach the vast amount of information that has been rapidly growing on its own. for this reason, it is exciting to have a book that elegantly combines both the theoretical and practical aspects of the microdrop technology. written by a seasoned expert, the microdrop generation provides a comprehensive introduction to the theory and technology of microdrop generation with up-to-date references and a number of examples from the author's more than years of experience. the first chapter introduces how and why microdrop technology has been used in pure and applied scientific disciplines in addition to inkjet printing. the next two chapters continue to provide an introductory overview concerning various existing techniques of monodisperse microdrop generation and the basic theory behind the fluid dynamics of the ejected microdrops. throughout chapters , , , , and , the book covers the details of microdrop technology, including engineering requirements for drop generation; various ways to modify the surface charge of the microdrops, which makes the microdrops even more versatile; imaging microdrops; electronic elements used to drive piezoelectric transducers; and pressure control systems for reliable drop generation. chapters , , and describe the fluid systems compatible with the microdrop generators, which play a critical role in drop generation as much as the mechanical components of the drop ejectors. the book also serves as a practical lab manual via chapters and , which describe fabrication of the drop generator hardware. the author does not save pages in providing useful hints and stepby-step procedures, and successfully explains how one can build small aperture nozzles and piezoelectric drop ejectors in the laboratory. chapter deals with troubleshooting practical problems, such as clogging or nozzle malfunction, in a way that is only possible for those who are thoroughly experienced in the field. this -page book embraces so much useful information and at the same time does not lose the reader's interest in any of the chapters. each chapter begins with an insightful introduction that guides the readers to the details of the subjects. a number of useful photos and diagrams aid in the understanding of even complicated concepts, which could have been otherwise hard to grasp for those with no prior experience. i should not say this is an easy book that readers at any level can breeze through; however, this book will surely stimulate those who have considered the use of microdrops in their field and guide them into this fascinating world of microdrops. school of pharmacy west lafayette, in yeo @purdue.edu the analysis of controlled substances surface and thin film analysis spie-the society of photo-optical instrumentation engineers, bellingham, wa. www.spie.org. . pp. $ . . handbook of infrared spectroscopy of ultrathin films (preface) "starting from molecules, the smallest entities of matter that have distinct shapes and properties, chemists have developed a 'bottom-up' approach to the construction of molecular-level devices and machines of nanometer size.chemists are trying to construct much simpler molecular level devices and machines (than those present in nature), without mimicking the complexity of biological structures. throughout the book emphasis is placed on concepts that are then illustrated with examples of the various kinds of artificial device or machine, taken from recent literature. selected examples of natural and biomimetic molecularlevel systems are also presented to give the reader a flavor of the beauty and complexity of the chemical mechanisms responsible for the material aspects of life." although several new scientific encyclopedias or handbooks have appeared in the last few years, their usefulness has been marginal as they have been put together as "afterthoughts" to capture the growing market of the "one-stopshopping" attitude of researchers who do not have the time to consult the original sources. with the exception of very few (e.g., edith mathiowitz' brilliant "encyclopedia of controlled drug delivery," wiley, ), i have found most of these volumes ill-conceived and poorly prepared compilations of irrelevant "review" articles. not so here! the new encyclopedic reference on materials characterization reviewed here is a much needed addition to the vast literature on materials science. pharmaceutical scientists will find it a most welcome addition to their library. written by several outstanding contributors to the field, mostly physicists and materials scientists, the book is well integrated, complete and especially balanced.the two volumes cover computational and theoretical methods, mechanical testing, thermal analysis, electrical and electronic measurements, magnetic measurements, electrochemical techniques, optical imaging, resource methods, xray techniques, electron techniques, ion-beam techniques, and neutron techniques. pharmaceutical scientists will find most of these sections of importance to their work as they address positions of characterization of excipients and carriers for drug delivery.highly (preface) "this book attempts to describe the state of the art in measurement of ionization constants (pk a ), oil-water partition coefficients (log p/log d), solubility, and permeability (artificial phospholipid membrane barriers)." key: cord- -hn o authors: pivette, mathilde; mueller, judith e; crépey, pascal; bar-hen, avner title: drug sales data analysis for outbreak detection of infectious diseases: a systematic literature review date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: hn o background: this systematic literature review aimed to summarize evidence for the added value of drug sales data analysis for the surveillance of infectious diseases. methods: a search for relevant publications was conducted in pubmed, embase, scopus, cochrane library, african index medicus and lilacs databases. retrieved studies were evaluated in terms of objectives, diseases studied, data sources, methodologies and performance for real-time surveillance. most studies compared drug sales data to reference surveillance data using correlation measurements or indicators of outbreak detection performance (sensitivity, specificity, timeliness of the detection). results: we screened articles and included in the review. most studies focused on acute respiratory and gastroenteritis infections. nineteen studies retrospectively compared drug sales data to reference clinical data, and significant correlations were observed in of them. four studies found that over-the-counter drug sales preceded clinical data in terms of incidence increase. five studies developed and evaluated statistical algorithms for selecting drug groups to monitor specific diseases. another three studies developed models to predict incidence increase from drug sales. conclusions: drug sales data analyses appear to be a useful tool for surveillance of gastrointestinal and respiratory disease, and otc drugs have the potential for early outbreak detection. their utility remains to be investigated for other diseases, in particular those poorly surveyed. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. since the mid- s and the raise of concerns about bioterrorism and emerging diseases, non-diagnosis-based data have increasingly been used for routine disease surveillance and outbreak detection [ ] . the cdc defined "syndromic surveillance" as an investigational approach where health department staff, assisted by automated data acquisition and generation of statistical alerts, monitor disease indicators in real-time or near real-time to detect outbreaks of disease earlier than would otherwise be possible with traditional public health methods [ ] . in such efforts, different registries have served as data sources for public health surveillance [ , ] , including data on absenteeism at work or school [ ] , calls to health helplines [ , ] , emergency department consultations [ , ] , ambulance dispatching [ ] , or drug sales. although unspecific, such data sources can have the advantage over diagnosis-based surveillance of providing information within short delays since the event and in readily available electronic form for relatively low-cost, while capturing large parts of the population. drug sales data analysis may overcome the limitation of poor specificity when groups of drugs are exclusively used for the disease or disease syndrome of interest. furthermore, drug sales data may earlier capture changing population health status, as over-the-counter (otc) sales and a dense network of pharmacies in most developed countries make drugs easily accessible to patients at the earliest appearance of their symptoms. despite this potential interest, no state of the art of drug sales based surveillance is available to date. the present systematic literature review therefore summarized the evidence for an added value of drug sales data for infectious disease surveillance. we limited the scope of the review to infectious diseases, as they represent a public health problem for which early and valid signal detection is of particular concern, in light of potentially rapid emergence and opportunity for control interventions. we conducted a literature search from up to june to identify relevant peer-reviewed articles regarding surveillance of infectious diseases based on drug sales data. prisma guidelines were followed in the reporting of the review [ ] . published articles were searched for on electronic databases (pubmed, embase, scopus, lilacs, african index medicus, cochrane library), using combinations of the following key words: ("surveillance" or outbreak detection or warning system) and (overthe-counter or "prescription drugs" or pharmacy or (pharmaceutical or drug or medication) sales). the search was limited to articles in english or french. there were no limitations on study settings. to be included in the review, articles had to describe, test, or review an infectious disease surveillance based on drug sales data; and be original research that presented new data and results. we excluded studies that monitored chronic diseases, as well as prevalence studies whose purpose was not epidemic detection. one reviewer screened and evaluated the titles and abstracts. articles were widely included in a first stage. the full-text review and the final selection of the articles were made by two reviewers. we reviewed and described the articles in terms of objectives, diseases studied, data sources, methodologies, and performance for real time surveillance. to describe methods and results, we separated the articles into three groups based on their main objective: descriptive retrospective studies, drug selection studies, and prediction studies. outcomes selected to compare drug sales data to reference surveillance data of the corresponding disease were correlation measurements (strength and timeliness of the correlation) and indicators of outbreak detection performance (sensitivity, i.e. ability to identify true outbreaks; specificity, i.e. ability to identify true negative and timeliness of the detection). we screened a total of articles, of which were included in the final review. the search and selection process is presented in figure . articles excluded based on fulltext review (no drug sales data, no infectious disease, no outbreak detection) n= figure flow chart of study selection process in a systematic review of drug sales data analysis for syndromic surveillance of infectious diseases. three types of studies were defined: retrospective descriptive studies, drug selection studies and prediction studies. nineteen of the studies were descriptive retrospective studies assessing the strength of the correlation between drug sales and reference surveillance data of the corresponding disease or evaluating outbreak-detection performance [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . five studies used statistical algorithms to select groups of drugs that were closely associated with clinical surveillance data of a given disease and that would be most appropriate for future drugsales-based surveillance [ , [ ] [ ] [ ] [ ] . in a third group of three studies, the authors developed and evaluated statistical models to predict clinical surveillance data based on drug sales data [ ] [ ] [ ] . table summarizes the studies in terms of their general characteristics. most of the studies focused on respiratory illnesses ( studies) [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or gastrointestinal illnesses ( studies) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . only two other studies evaluated surveillance of pertussis [ ] and syphilis [ ] . most of the studies were set in the united states (n = studies, %), followed by canada (n = ), france (n = ), japan (n = ), the netherlands (n = ) and england (n = ). only one study was conducted in more than one country [ ] . in most retrospective studies, data were collected specifically for the purpose of the study from a sample of pharmacies [ , [ ] [ ] [ ] or from retailers [ , , , ] . for example, in a canadian study [ ] , electronic data were provided by one major retailer for all of their pharmacies in the study area. automatically compiled data sources were used in all the drug selection and prediction studies and in some retrospective studies. drug sales data were routinely collected in samples of a city's or country's pharmacies. such routine data collection systems were mainly implemented by research or public health groups, such as the johns hopkins applied physics laboratory [ , , , ] , the new york city department of health [ ] , the national institute of infectious diseases in japan [ ] , or the real-time outbreak and disease surveillance laboratory at the university of pittsburg [ ] . data are available the day after the day's sales in those systems. in eight other studies, private marketing companies had automatically aggregated and made available drug sales data from a sample ( - %) of pharmacies in a given city or country [ , , , [ ] [ ] [ ] [ ] ] . nineteen studies retrospectively compared drug sales data to gold standard reference data of the disease. details are given in table . reference data of the disease included medical case reports [ ] [ ] [ ] , diagnostic registries of microbiological laboratories [ , , , ] , hospital admission or discharge data [ ] [ ] [ ] [ ] [ ] ] , or clinical emergency department data [ ] [ ] [ ] . the selection of indicator drugs in these studies was based on the literature or expert opinion. for example, edge et al. [ ] selected all anti-nauseant and antidiarrheal otc drugs for gastrointestinal surveillance. in stirling et al. [ ] , pharmacists determined which common antidiarrheal drugs they would report. two methods were commonly used to compare drugsale and diagnostic data time series: correlation analysis and signal detection comparison ( table ). ten studies used cross-correlation function to measure the similarity of two curves and to determine the time lag at which the correlation between the datasets is maximized. cross-correlation is a standard method to determine the time delay between two signals. in three studies, only correlation between the time series was examined without analyzing time-lagged relationship. six studies used aberration detection methods to evaluate whether and by how long the date of signal detection by drug sales precedes the signal based on diagnostic data. the signal definition for aberration detection was based on either a simple threshold to define alerts [ ] or more complex algorithms such as the serfling method [ ] , arima models [ ] , the simple moving average method (ma), the cumulative sum method (cusum) [ , ] , or the exponentially weighted moving average (ewma) [ ] . these studies assessed the performance in terms of sensitivity, specificity and timeliness of disease outbreak detection. five other studies [ , [ ] [ ] [ ] ] only evaluated whether drug sales showed a significant increase during a known epidemic period. twelve of studies evaluating otc sales retrospectively found significant correlations or a significant increase in drug sales [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . only two studies didn't found any consistent correlation. for example, das et al. [ ] found a poor correlation between otc antidiarrheal drug sales and emergency department visits for diarrhea in new york city, with an r of . . they found however an increase in sales during a known outbreak of norovirus. otc drug sales preceded clinical data in three of eight studies that analyzed timeline correlations [ , , ] . for example, in hogan et al. [ ] , the correlation coefficient between electrolyte sales and hospital diagnoses of respiratory and diarrheal illness was . ( % ci, . - . ) when drug sales were assumed to precede clinical diagnosis data by . weeks. outbreaks were detected with % sensitivity and specificity in of studies that analyzed signal detection [ , , ] . drug sales data provided an earlier outbreak signal in two of them [ , ] . in davies et al. [ ] , the rate of cough/cold sales exceeded a threshold of units per week two weeks before the peak in emergency department admissions during three consecutive winters. in hogan et al. [ ] , detection from electrolytes sales occurred an average , weeks earlier than detection from hospital diagnoses of respiratory and diarrheal diseases. six of the seven studies that focused on prescribed drugs found strong correlations (r = . - . ) with clinical reference data or a significant increase in drug sales, without lead time however. the other study [ ] showed that the cusum signal generated for prescriptions for macrolide antibiotics was linked to a pertussis outbreak in a county of new york state. no association was observed between the type of reference data and the time lags observed. an important challenge for drug-sales-based surveillance is identifying relevant indicator drug groups to monitor diseases. five retrieved articles addressed this question. characteristics of the studies are described in table . two studies [ , ] developed methods to find homogeneous groups of otc products. the authors used unsupervised clustering algorithms for aggregating otc products in groups sharing similar sales histories. for example, magruder et al. [ ] first assigned otc products for respiratory diseases to subgroups qualitatively based on indication, dose form, and age group. a stepwise hierarchical clustering algorithm was then used to form categories sharing a similar sales history, leading to a set of product categories. in two studies [ , ] , the authors developed procedures to identify the drugs correlating with disease incidence. clusters were formed specifically for a particular disease. in pelat et al. [ ] , a hierarchical clustering procedure was applied to the time series of all therapeutic classes and the acute diarrhea incidence rate reported by a network of general practitioners. four therapeutic classes were found to cluster with diarrhea incidence and an algorithm based on the selected drugs allowed the detection of epidemics with a sensibility of %, a specificity of % and a timeliness of . weeks before official alerts. in three studies [ ] [ ] [ ] , the authors developed models to predict clinical data based on drug sales data. vergu et al. [ ] used a poisson regression model on selected otc sales to forecast influenza-like illness (ili) incidence as recorded by a sentinel network of general practitioners. the forecast at the national level - weeks ahead showed a strong correlation with observed ili incidence (r = . - . ). najmi et al. [ ] used least mean square filtering methods to estimate the incidence of emergency room consultations for respiratory conditions from past and present sales of groups of cold-remedy sales. in a later article [ ] , they succeeded in extending the estimation algorithm for predicting increases in clinical data several days in advance. the evidence gathered in this systematic literature review suggests that drug sales data analysis can be a useful tool for surveillance of acute respiratory and gastrointestinal infections. as could be expected, prescribed drug sales data were strongly correlated with clinical case reporting. no lead time was observed, which is consistent with the fact that patients purchase drugs after seeing a healthcare professional. analysis of prescribed drug sales data may nevertheless have an additional utility for epidemic detection, as these data might be available with a shorter delay than clinical surveillance data [ ] . a high correlation between otc drug sales data and reference surveillance data were found in almost all the retrospective studies. several studies also showed that otc drug sales can serve as an early indicator of disease epidemics. patients may buy nonprescription drugs during the early phase of illness when they become symptomatic, before consulting a health practitioner [ ] . a surveillance system based on drug data should ideally detect all the outbreaks, rapidly, with a low false alert rate. however, few studies in the review determined the sensitivity and specificity of the outbreak detection and those aspects should be analyzed in more details in future studies. surveillance based on otc drug sales could be particularly relevant for diseases whose prodromal phase persists for several days before the onset of more severe symptoms. for example, the early stages of dengue fever symptoms are nonspecific (fever, headache, myalgia, arthralgia, etc.) [ ] . the occurrence of grouped cases could trigger an excess of nonspecific drug sales over baseline levels, which in turn could provide an early warning of outbreak in an endemic area. results from drug selection studies showed that it is possible to identify groups of products strongly associated with incidence data, which can then be used to predict future trends in clinical data and help public health authorities to prepare response planning. such product selection procedures, however, depend on the existence of large clinical surveillance databases of the diseases concerned. similarly, the validity of drug sales data analysis has been evaluated mainly for two disease groups, respiratory and gastrointestinal illness, for which clinical reference data, used as the gold standard, are readily available. pertussis and syphilis have been evaluated in only one study each, and still require further confirmation. the concept of drug-based surveillance therefore needs to be validated for other infectious diseases. all the studies were conducted in developed countries or area. surveillance based on drug sales data requires electronic information systems for routine data analysis. besides, its implementation requires that the population has access to the health care system and mainly buy drugs in pharmacies. this could limit the use of drug based surveillance systems in developing countries. by improving the timeliness of epidemic detection compared to clinical data and giving information from a larger part of the population, drug sales data can be an additional source of information for already monitored diseases. besides, drug sales data analysis could have its greatest value in the surveillance of diseases for which clinical surveillance is cumbersome and costly, or where substantial under-reporting is suspected. to confirm the selected drug group as a valid proxy of disease, clinical surveillance may be conducted for a defined period in a representative population. examples of diseases for which this would be useful are typically varicella, urinary infections, allergies/asthma, and parasitic diseases. ideally, the drugs to be monitored should be specific to the disease and widely used to treat it in order to maximize the sensitivity of the signal. for example, benzylpenicillin benzathine . mui is the quasi exclusive treatment for syphilis infection [ ] and is a good candidate. in contrast, the treatment of measles is mostly symptomatic without a specific drug, which makes this disease unattractive for this approach. another limitation applies to diseases that are usually treated in hospitals or specialized centers, such as tuberculosis. surveillance based on drug sales, may not be appropriate to accurately estimate incidence of diseases, as the source population size is not precisely known. moreover, it may be difficult to link the number of drug packages sold to the number of patients with disease. however, the method is very efficient to determine temporal dynamics of a situation and to detect abnormal phenomena. surveillance based on drug sales is therefore well adapted to diseases with seasonal variations such as norovirus gastroenteritis, influenza and other infectious respiratory agents, or community outbreaks (foodborne illnesses, waterborne illnesses, hepatitis a, etc.). drug sales can be influenced by store promotions, sales period (holidays, weekends), and the media. also, we do not know whether people buy medications to treat a disease they currently have or a disease they fear they may have in the near future. for example, during the media coverage of avian influenza a (h n ) in the us, an increase in antiviral medications sales was observed [ ] , which corresponded to stockpiling behavior of the population. health-seeking behaviors also vary by demographic, social, cultural, and economic factors. a survey [ ] in canada analyzed the healthcare-seeking behaviors of patients with acute gastroenteritis. they found significant differences (patient age and sex) between the patients who used otc drugs and those who did not. consequently, factors that prompt self-medication should also be taken into account. the usefulness of drug sales based surveillance is also dependent on the available resources and the organization of the health care system. otc drug sales surveillance is for example less relevant in countries where reimbursement rate are high and patients mainly get prescribed drugs. population mobility, particularly in tourist areas, may lead to an increase in remedy sales, which could wrongly be interpreted as a disease outbreak. inversely, patients with high geographical mobility may not be included in the region of study and lead to an underestimation of the magnitude of an epidemic. despite some limitations, routine collection and analysis of drug sales data are likely to be developed in the coming years. many automated surveillance systems that collect drug data the day after the sales have been implemented in the last decade [ , , , ] . they allow a rapid assessment of the public health situation. early detection of outbreaks allows public health authorities to set up epidemic investigations and control measures sooner. most studies included in this review were published after the year , with their number increasing recently. they illustrate the need for improved surveillance systems, evidenced by recent public health crises (e.g., anthrax in , the sars outbreak in , the a/h n influenza pandemic in , etc.). drug sales data present indeed many advantages in terms of public health surveillance. data can be obtained in a real-time manner and usually cover a large portion of the population. data collection may be exhaustive, without selection of specific sales, and allows the simultaneous monitoring of a large number of diseases, especially new or emerging diseases. although non-specific, drug sales data are directly linked to patients' health conditions. drug sales data are therefore more specific than other syndromic surveillance data, such as tracking search patterns on the web and are likely to reflect more accurately disease activity in the population. moreover, it should be noted that alternative sources of data for disease surveillance are currently under development. healthcare management databases that can provide exhaustive information on drug consumption and diagnosis, as the dossier médical personnel [ ] in france, are promising tools for disease surveillance. our review may be affected by a publication bias since studies unable to show correlations between drug sales and reference data may have been less published. in addition, selections bias may have occurred in the studies. indeed, some studies in the review were based on a limited number of pharmacies and/or a limited study period (e.g. less than one year). language bias may exist as we were not able to identify studies published in languages other than english and french. the review focused on the temporal dynamics of infectious disease; consequently, further analyses are required to determine the capacity of these systems to efficiently monitor other aspects of infectious diseases such as spatial spreading. this review suggests that the analysis of drug sales data is a promising method for surveillance and outbreak detection of infectious diseases. it has the potential to trigger an outbreak alert earlier than most surveillance systems. however, the main challenges consist in the appropriate selection of indicator drug groups and the validation of this approach for diseases for which no or poor quality clinical surveillance data exists. the usefulness of the approach also depends on the available resources and the organization of the health care system. drug sales databases with real-time or near real-time data transmission are available in several countries; future studies should be encouraged to expand their use on other infectious diseases. what is syndromic surveillance? mmwr morb mortal wkly rep cdc: framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations from the cec working group review of syndromic surveillance: implications for waterborne disease detection absenteeism in schools during the influenza a(h n ) pandemic: a useful tool for early detection of influenza activity in the community? using ontario's "telehealth" health telephone helpline as an early-warning system: a study protocol using nurse hot line calls for disease surveillance disease outbreak detection system using syndromic data in the greater washington dc area assessment of a syndromic surveillance system based on morbidity data: results from the oscour network during a heat wave use of ambulance dispatch data as an early warning system for communitywide influenzalike illness preferred reporting items for systematic reviews and meta-analyses: the prisma statement use of medicaid prescription data for syndromic surveillance-new york a practical method for surveillance of novel h n influenza using automated hospital data syphilis surveillance in france monitoring over-the-counter medication sales for early detection of disease outbreaks sales of over-the-counter remedies as an early warning system for winter bed crises syndromic surveillance of gastrointestinal illness using pharmacy over-the-counter sales. a retrospective study of waterborne outbreaks in saskatchewan and ontario syndromic surveillance of norovirus using over-the-counter sales of medications related to gastrointestinal illness detection of pediatric respiratory and diarrheal outbreaks from sales of over-the-counter electrolyte products prediction of gastrointestinal disease with over-the-counter diarrheal remedy sales records in the san francisco bay area evaluation of over-the-counter pharmaceutical sales as a possible early warning indicator of human disease progress in understanding and using over-the-counter pharmaceuticals for syndromic surveillance experimental surveillance using data on sales of over-the-counter medications-japan using oral vancomycin prescriptions as a proxy measure for clostridium difficile infections: a spatial and time series analysis surveillance data for waterborne illness detection: an assessment following a massive waterborne outbreak of cryptosporidium infection pharmaceutical sales; a method for disease surveillance? waterborne cryptosporidiosis outbreak real-time prescription surveillance and its application to monitoring seasonal influenza activity in japan validation of syndromic surveillance for respiratory pathogen activity sales of nonprescription cold remedies: a unique method of influenza surveillance seasonal influenza surveillance using prescription data for anti-influenza medications mining aggregates of over-the-counter products for syndromic surveillance a multivariate procedure for identifying correlations between diagnoses and over-the-counter products from historical datasets a method for selecting and monitoring medication sales for surveillance of gastroenteritis unsupervised clustering of over-the-counter healthcare products into product categories estimation of hospital emergency room data using otc pharmaceutical sales and least mean square filters an adaptive prediction and detection algorithm for multistream syndromic surveillance medication sales and syndromic surveillance implementing syndromic surveillance : a practical guide informed by the early experience value of syndromic surveillance within the armed forces for early warning during a dengue fever outbreak in french guiana in increased antiviral medication sales before the - influenza season factors associated with the use of over-the-counter medications in cases of acute gastroenteritis in hamilton drug sales data analysis for outbreak detection of infectious diseases: a systematic literature review this research was funded by celtipharm (vannes, france) a company specialized in the real time collection and statistical processing of healthcare data (www.celtipharm.orgwww.openhealth.fr), through a doctoral thesis contract for mathilde pivette. mathilde pivette prepares a doctoral thesis under the french framework "cifre" (industrial contract for training through research; www.anrt.asso.fr), in partnership with the company celtipharm (www.celtipharm.org). the other authors declare they have no competing interests. all authors contributed to the study's design. mp and jm carried out the literature search and reviewed articles. mp drafted the manuscript. all authors interpreted the results, revised and approved the final manuscript. key: cord- -fn zlutj authors: nan title: abstracts of the th annual meeting of the german society of clinical pharmacology and therapy: hannover, – september date: journal: eur j clin pharmacol doi: . /bf sha: doc_id: cord_uid: fn zlutj nan grapefruit juice may considerably increase the systemic bioavailability of drugs as felodipine and nifedipine. this food-drug interaction has potential practical importance because citrus juices are often consumed at breakfasttime when drugs are often taken. it is likely that a plant flavonoid in grapefruit juice, naringenin, is responsible for this effect (inhibition of cytochrome p- enzymes in the liver or in the small intestinal wall). ethinylestradiol (ee ), the estrogen of oral contraceptive steroides, shows a high first-pass-metabolism in vivo. therefore, the purpose of this study is to test the interaction between grapefi-uite juice and ee , the area under the serum concentration-time curve (auc _ h) ofee was determined in a group of young healthy women (n = ) on day + ofmenstruale cycle. to compare intraindividually, the volunteers were randomly allocated to two test days. the female volunteers took lag ee together with either ml of herb tea or with the same amount of grapefruit juice (content of naringenin mg/ ). furthermore, on the day of testing the women drank times ml of the corresponding fluid every three hours up to four times. the auc . h of ee amounts to . + . pg x mi- x h after the administration of the drug with grapefruit juice; that means % higher in comparison to + . pg x m - x h after concomitant intake of tea. also, the mean cmax-value increases to %, p _< . ( . + . pg x m - and . + . pg x m - , respectively). this result shows that the systemic bioavailability ofee increases after intake of the drug with grapefruit juice. the extent of this effect is lower than the extent of known interindividual variability. procarbazine is a tumourstafic agent widely used m hodgin's disease, non-hodgldn's lymphomas and mmours of brain and lung. procarbazine is an inactive prodrug which is converted by a cytochrome p mediated reaction to its active metabolites, in the first step to azoprocarbazine. the kinetics of both procarbazine and azoprocarbazine is not described in humans up to now. on turnout patients we have investigated the plasma kinetics of both procarbazine and azoprocarbazine after oral adminislxation of mg procarbazine in form of capsules and drink solution, respectively. a hplc method with uv-detection ( nrn) and detection limits of and ng/ml was developed for procarbazine and azoprocarbazine respectively. after both the capsules and drink solution the parent drug could be detected in plasma only for h. in contrast the tl/ of terminal elimination of azoprocarbazine was estimated in the range of , to , h with a mean of , h - + , h. the auc of procarbazine was less than % of that of azoprocarbazine. cma x values of azoprocarbazine were determined in the range of , to ,l gg/ml. in comparison to the drink solution we determined on the basis of the plasma levels of azoprocarbazine a bioavailability of the therapeutic used procarbazine capsules of , + , %. prostaglandin e (pge ) is used for the treatment of patients with peripheral arterial disease, and probably effective due to its vasodilator and antiplatelet effects. l-arginine is the precursor of endogenously synthesized nitric oxide (no). in healthy human subjects, larginine also induces peripheral vasodi]ation and inhibits platelet aggregation due to an increased no production. in the present study the influence of a single intravenous dose of l-arginine ( g, min) or pge ( p.g, min) on blood pressure, peripheral hemodynamics (femoral artery duplex sonography), and urinary no -and cgmp excretion rates was assessed in ten patients with peripheral arterial disease (fontaine iii -iv). blood flow in the femoral artery was significantly increased by l-arginine by % (p < . ), and by pge by % (p < . ). l-arginine more strongly decreased systolic and diastolic blood pressure than pge . plasma arginine concentration was increased -fold by l-arginine, but unaffected by pge . urinary excretion of no -increased by % after l-arginine (p < . ), and by % after pge (p = n.s.). urinary cgmp excretion increased by % after l-arginine and by % after pgei (each p = n.s.). we conclude that intravenous l-arginine decreases peripheral arterial resistance, resulting in enhanced blood flow and decreased blood pressure in patients with peripheral arterial disease. these effects were paralleled by increased urinary no -excretion, indicating that systemic no production was enhanced by the infusion. increased no -excretion may be a sum effect of no synthase substrate provision (l-arginine) and increased shear stress (pge and l-arginine). it is weli established that the endothelial edrf/no-mediated relaxing mechanism is impaired in atherosclerotic and in hypertensive arteries. recently it was suggested that primary pulmonary hypertension might be another disease in which the endothelial edrf/no pathway is disturbed. we tested the hypothesis that intravenous administration of l-arginine (l-arg), the physiological precursor of edrf/no, stimulates the production of no, subsequently increasing plasma cgmp levels and reducing systemic and / or pulmonary vasular resistance, in patients with coronary heart disease (chd; n = ) and with primary pulmonary hypertension (pph; n = ). l-arg ( g, min) or placebo (nac ) was infused in chd patients, and l-arg was infused in pph patients undergoing cardiac catheterization. mean aortic (pao) and pulmonary (ppul) arterial pressures were continuously monitored. cardiac output (co; by thermodilution), and total peripheral resistance (tpr) were measured before and during the infusions. plasma cgmp was determined by ria. in chd patients, pao decreased from . + . to . + . mm hg during l-arg (p< . ), whereas ppul was unchanged. tpr decreased from . -+ . to . + . dyne sec cm - during l-arg administration (p< . ). co significantly increased during l-arg (from . + . to . + . /min, p< . ). placebo did not significantiy influence any of the haemodynamic parameters, cgmp slightly increased by . + . % during l-arg, but slightly decreased during placebo (- . + . %)(p < . for l-arg vs. placebo). in pph patients, l-arg induced no significant change in pao, tpr, and co. mean ppul was . + . mm hg at the beginning of the study, but was only slightly reduced by l-arg to . + , mm hg (p = n.s.). plasma cgmp was not affected by l-arg in these patients. we conclude that l-arg stimulates no production and induces vasorelaxation in chd patients, but not in patients with primary pulmonary hypertension. thus, the molecular defects underlying the impaired no foimation may be different m both diseases. institutes of clinical pharmacology, *cardiology, and **pneumology, medical school, hannover, germany. the influence of submaximal exercise on the urinary excretion of , -dinor-pgflc, (the major urinary prostacyclin metabolite), , dinor-txb (the major urinary thromboxane a metabolite), and pge (originating from the kidney), and on platelet aggregation was assessed in untrained and endurance-trained male subjects before and after days of rag/day of aspirin. urinary , -dinor-txb excretion was significantly higher in the athletes at rest (p < . ). submaximal exercise increased urinary , -dinor- -keto-pgfl~ excretion without affecting , -dinor-txb or pge excretion or platelet aggregation. aspirin treatment induced an - % inhibition of platelet aggregation and , -dinor-txb excretion in both groups. however, urinary , -dinor- -keto-pgfl~ was inhibited by only % in the untrained, but by % in the trained group (p < . ). urinary pge was unaffected by aspirin in both groups, indicating that cyclooxygenase activity was not impaired by a systemic aspirin effect. after low dose aspirin administration, the same selective stimulatory effect of submaximal exercise on urinary , -dinor- -keto-pgfl~ excretion was noted in both groups as before. the ratio of , -dinor- -keto-pgfld , -dinor-txb was increased by exercise; this effect was potentiated by aspirin (p < . ). our results suggest that the stimulatory effect of submaximal exercise on prostacyclin production is not due to an enhanced prostacyclin endoperoxide shift from activated platelets to the endothelium, but rather the result of endothelial prostacyclin synthesis activation from endogenous precursors. mg/day of aspirin potentiates the favorable effect of submaximal exercise on endothelial prostacyclin production by selectively blocking platelet cyclooxygenase activity. institute of clinical pharmacology, medical school, hannover, germany. soluble guanylyl cyclases (gc-s) are heterodimeric hemeproteins consisting of two protein subunits ( kda, kda). the enzyme is activated by nitric oxide (no) and catalyzes the formation of the signal molecule "cgmp" (cyclic guanosine- 's'-monophosphate) from gtp. numerous physiological effects of cgmp are already very well characterized. however, detailed insights in the no-activation mechanism of this enzyme have been described to date only in a hypothetical model ( ). recently, this concept was supported by experimental data using sitedirected mutagenesis to create a no-insensitive soluble guanylyl cyclase mutant ( ). it is generally accepted that the prostethic heine-group plays a crucial role in the activation mechanism of this protein. nonetheless, some interesting questions with regard to structure and regulation of soluble guanylyl cyclases still need to be uncovered (e.g. activation with other free radicals, such as carbon monoxide). since this kind of studies is limited so far by isolating large quantities of a biologically active enzyme with conventional purification techniques, the recombinant protein was expressed in the baculovirus / insect cell system. we describe here the construction and characterization of recombinant baculoviruses, harboring the genes that encode both protein subunits of the soluble guanylyl cyclase. insect cells infected with these recombinant baculoviruses produce between - % (as related to total cell protein) of functional soluble guanylyl cyclase. positive infection was monitored as a change in morphology of the cells and by production of the respective recombinant viruses detected by polymerase-chain-reaction (pcr). so far examined, the recombinant enzyme exhibits similar physicochemical characteristics as the "natural" protein. exogenous addition of several heme analogues to the infected cells is able to either stimulate or inhibit the enzymatic activity of gc-s. we are confident to purify milligram amounts of the recombinant protein in the near future. pet studies of myocardial pharmacology have principally concerned the sympathetic nervous system and u'acers have been developed to probe the integrity of both pre-and post-synaptic sites. the sympathetic nervous system plays a crucial role in the control of heart rate and myocardial contractility as well in the conlrol of the coronary circulation. alterations of this system have been implicated in the pathophysiology of a number of .cardiac disorders, in particular, heart failure, ventricular arrhythmogenesis, coronary artery disease, idiopathic dilated and hypertrophic cardiomyopathy. several beta blockers have been labelled with carbon-ll for imaging by pet. the most promising of these is cgp which is a non-selective beta adrenoceptor anatagonist particularly suited for pet studies due to its high affinity and low lipophilicity, thus enabling the functional receptor pool on the cell surface to be studied. studies in our institution in a group of young healthy subjects have yielded bmax values of . _+ . pmol/g myocardium. these data are consistent with literature values of bmax for beta adrenoceptors in human ventricular myocardium determined by a variety of in vitro assays. a recent study in patients with hypertrophic cardiomyopathy has shown that myocardial beta adrenoceptor density is decreased by approximately - % relative to values in normal subjects. the decrease in receptor density occurs in both hypertrophied and nonhypertrophied portions of the left ventricle. these data are consistent with the hypothesis that sympathetic overdrive might be involved in the phenotypic expression of hypertrophic cardiomyopathy. a further decrease of myocardial beta adrenoceptor density (to levels well below _ - . pmol/g) has been observed in those patients with hypertrophic cardiomyopathy who procede to ventricular dilatation and heart failure. cyp a hydroxylates polycyclic aromatic hydrocarbons such as benzo(a)pyrene occurring e.g. in cigarette smoke. two hereditary mutations are discovered: ml, a t to c transition , bp downstream of exon ; m , located at position , in exon representing an a to g transition resulting an isoleucine to valine substitution in the heme-binding region. recently we could demonstrate in caucasians that carriers of the m -mutation possess an increased risk towards lung cancer (drakoulis et al clin.lnvestig. : , ) , whereas the ml-mutation shows no such association. the phasg-ii enzyme gstm catalyses the conjugation of glutathione to electrophilic compounds such as products of cyp ai. gstm is absent in . % of the caucasian population due to base deletions in exon and of the gene. we found no contrariety in the gstm distribution, including frequencies of type a (p.) and type b (v) among lung cancer patients (odds ratio = . , n = ; cancer res. : res. : , . lung cancer patients and reference patients were investigated for mutations of cypia and gstm by allele-specific pcr and rflp. a statistically significant higher risk for lung cancer among carriers of the m trait was found (odds ratio = . , p = . ). interestingly, amid lung cancer, m -alleles were less often linked to ml than in controls (odds ratio = . , %-confidence limits = . - . , p = . ). however, the frequency of cypia mutations did not differ among active and defective gstm types. consequently, we could not confirm in the caucasian population the synergistic effects of cypia mutations (especially m ) and deficient gstm as combined susceptibility factors for lung cancer as described among the japanese (cancer res. : , in healthy subjects the effect of gastrointestinal hormones like somatostatin and glucagon on splanchnic hemodynamics is not well defined due to the invasiveness of the direct measurement of e.g. portal vein (pv) wedged pressure. methods : now, we applied duplex sonography ( . m~z) and color coded flow mapping to compare the effects of ocreotide (i ~g sc), a long acting somatostatin agonist, and glucagon (i mg iv) on the hemodynamics of the pv, superior mesenteric artery (sma) and common hepatic artery (ha) in healthy volunteers ( g,i q; ± y; x ± sem). basal values of pv flow ( . ± . cm/s), pv flow volume ( ± ml/min), sma systolic (sf: ± cm/s) and diastolic flow (df: ± cm/s), sma pourcelot index (pi) ( . ± . ), ha sf ( ± cm/s) and df ( ± cm/s) and ha pi ( . ± . ) well agreed with previously reported results. within min ocreotide resulted in a decrease of sma sf (- ± %) sma df (- ± %), ha sf (- ± %) and ha df (- ± %). maximum drop of pv flow (- ± %) and flow volume (- ± %) occurred at min. all effects diminished at min. no significant change of vessel diameter and pi was seen. min following its application glucagon caused a highly variable, only short lasting increase of pv flow volume (+ ± %) and sma df (+ ± %). ha fd (+ ± %) showed a tendency to rise (ns). we conclude that in clinical pharmacology duplex sonography is a valuable aid for measuring effects of hormones and drugs on splanchnic hemodynamics. pectanginal pain and signs of silent myocardial ischemia frequently occur in hypertensives, even in the absence of coronary artery disease (cad) and/or left ventricular hypertrophy, probably due to a reduced coronary flow reserve. since the oxygen extraction of the heart is nearly maximal during rest, increases of oxygen demand cannot be balanced by increases of myocardial perfusion: to assess the frequency of ischemic type st-segment depressions in this patients and to determine the influence of heart rate (hr) and blood pressure (bp), simultaneous h hoher-and h ambulatory bp monitoring were performed in hypertensives (age - years, f, m) without cad before and after four weeks on therapy with the -blocker betaxolol. episodes of significant st-segment depressions (> . mv,> min) of a total length of min could be demonsu'ated in / patients ( %) without antihypertensive therapy_ systolic bp significantly increased from + . mmhg (mean + sd, p < . ) min before to a maximum of + . mmhg during the ischemic episodes, hr and rate-pressure product (rpp) increased from + . min -t and . + . mmhg x rain -t x to _+ . min-: and . + . mmhg x min - x (p < . ). the extent of st-segment depressions significantly correlated with hr and rpp (p < . ). drug therapy with - mg/d betaxolol for weeks significantly decreased mean hr, systolic' and diastolic bp (p < . ). ischemic episodes of a total length of min were recorded only in of hypertensives ( . %; p < . ; x -test). in conclusion, increases of hr and systolic bp seem to be the most important factors which induce myocardial ischemia in hypertensives without cad. as silent ischemia is a independent risk factor for sudden cardiac death and other cardiac events, specific antihypertensive therapy should not only be aimed to normalize blood pressure, but should also address reduction of ischemic episodes as demonstrated here. phosphodiesterase inhibitors exert their positive inotropic effects by inhibiting camp degradation and increasing the intracellular calcium concentration in cardiomyocytes. an identical phosphodiesterase type i[ has been demonstrated in platelets and vascular smooth muscle cells. we studied the influence ofpiroximone on platelet function in vitro and ex vivo and the hemodynaimc effects of a bolus application of piroximone in patients with severe heart failure (nyha iii-iv) using a swan -ganz-catheter. in order to study the influence ofpiroximone on platelet function in vitro, platelet rich plasma from healthy volunteers was incubated with piroximone ( - ~tmol/l) from minute to hottrs and aggregation was induced by addition of adp. for the ex vivo experiments platelet rich plasma was obtained from patients, who received piroximone in doses of . , . , . or . mg/kg bw. blood samples were drawn immediately before and , , , and minutes after bolus application. the adp-induced platelet aggregation was inhibited time-and dosedependently. the ic value for piroximoue in vitro amounted to + omol/ . in the ex vivo experiments the maximal inhibition of adp-induced aggregation was obtained in prp from patients who had received mg/kg bw piroximune minutes before. the admitdstration ofpiroximone resulted in a marked hemodynamic improvement with a dose-dependent increase in cardiac index and decreases in pulmonary artery pressure and resistance. to treat conditions associated with acute and chronic multiorgan dysfunction. studies indicate patients receive approximately ten drugs, on average during their icu stay, from several drug classes. commonly prescribed drugs include narcotics, sedatives, antibiotics, antiarrhythmics, antihypertensives, drugs for stress ulcer prophylaxis, diuretics, vasopressors, and inotropes. reports suggest surgical icu patients cost the hospital an average of $ , /patient in un-reimbursed costs under fixed-price reimbursement. furthermore, patients with the greatest drain in revenue received catecholamines, triple antibiotics, or antifungal agents. thrombolytics, antibiotics, plasma expanders, and benzodiazepines account for nearly twothirds of the cost of drugs prescribed in medical and surgical icus. agents with considerable economic impact include biotechnology drugs for sepsis. pharmacoeconomic data in icu patients suggest increased attention should be directed towards several areas, including patients with pneumonia, intraabdominal sepsis, nosocomial bloodstream infections, optimizing sedation and analgesic therapy, preventing persistent paralysis from neuromuscular blockers, preventing stress ulcers, treating hypotension, and providing optimal nutritional support. studies are needed to assess the impact of strategies to improve icu drug prescribing on length of stay and quality of life. if expensive drugs are shown to decrease the length of icu stay, then their added costs can have positive economic benefits to the health care system. the responses to min iv. infusions of the -and -adrenoceptor agonist isoprenalin (iso) and the -(and c~-) adrenoceptor agonist adrenalin (adr) at constant rates of ijg/min were evaluated noninvasively after pretreatment (pre-tr) with placebo (pl), mg of the -selective adrenoceptor antagonist talinolol (tal) and mg of the non-selective antagonist propranolol (pro) in healthy subjects. the following were analysed: heart rate (hr, bpm), pre-ejection time (pep, ms), ejection time (vet, ms), hr-corrected electromechanical systole (qs c, ms), impedance-cardiographic estimates of stroke volume (sv, ml), cardiac output (co, i/min) and peripheral resistance (tpr, dyn.s.cm - ) calculated from co and mean blood pressure (sbp and dbp according to auscultatory korotkoff-i and -iv sounds this indicates that ) about half the rise of hr and co and half the shortening of pep is -respectively ~ -determined, ) that predominant -adrenergic responses, whilst not affecting vet, take optimal benefit from the inodilatory enhancement of pump performance, ) that an additional -adrenergic stimulation is proportionally less efficient, as vet is dramatically shortened, thus blunting the gain in sv so that the rise in co relies substantially on the amplified increase of hr and ), vet is more sensitive than qs c in expressing additional -adrenoceptor agonism and ) prime systolic time intervals provide a less speculative and physiologically more meaningful represenation of cardiac pump dynamics than hr-corrected ones. zentrum flit kardiovaskul~re pharmakologie, mathildenstral e , mainz, brd a regression between blunting of ergometric rise of heart rate and l~ladrenoceptor occupancies in healthy man c. de mey, d. palm, k. breithaupt-grsgler, g.g. belz the hr-responses to supine bicycle ergometry ( min at appr. watt) were investigated at several time points after the administration of propranolol (pro: , , mg), carvedilol (car: . , , , mg), talinolol (tal: , , , mg), metoprolol (met: mg) and celipro-iol (cel: mg) to healthy man. the effects of the agents (= difference of the ergometric response for active drug and placebo) were analysed for both the end values (end) and the increments (inc) from resting values immediately before ergometry up to end. the effects were correlated with the %-~l-adrenoceptor occupancies estimated using a standard emax-model (sigmoidicity=l) from the concentrations of active substrate in plasma determined by i~l-adrenoceptor specific radioreceptor assay. the respective intercepts (i), slopes (s) and correlation coefficients (r) are detailed here below : inhibition of leukotrienes is a promising approach to the treatmer~t of several diseases because excess formation of these lipid mediators has been shown to play an important role in a wide range of pathophysiological conditions. since until recently we were not able to obtain specific drugs suppressing leukotriene biosynthesis or action for clinical practice, we started investigating the effects of putative natural modulators of leukotriene biosynthesis such as fish oil. healthy male volunteers were supplemented for days with fish oil providing mg eicosapentaenoic and docosahexaenoic acid per kg body weight and day. the urinary concentration of leukotriene e plus n-acetyl leukotriene e served as a measure for the endogenous leukotriene production, treatment resulted in a significant increase in the eicosapentaenoate concentration in red blood cell membranes. fish oil reduced the endogenous leukotriene generation in of the volunteers. the effect was associated with a decrease in urinary prostaglandin metabolites, determined as tetranorprostanedioic acid. in contrast to what was expected from published in vitro and ex vivo experiments, no endogenously generated cysteinyl leukotrienes of the series could be identified. the inhibitory effect of fish oil on the endogenous leukotriene generation was not synergistic to the effect of vitamin e, which also exhibited some suppressive activity. early clinical data on the effects of fish oil on teukotriene production in patients with allergy or rheumatoid arthritis are not yet conclusive. we conclude that fish oil exhibits some inhibitory activity on leukotriene production in vivo. the effectivity of fish oil may be attenuated by concomitant modulation of other mediator systems e.g. up-regulation of tumor necrosis factor production. • the number and affinity of platelet thromboxane (txa ) and prostacyclin (pgi )-receptors are regulated by several factors. we studied the influence of oral intake of acetylsalieylic acid (asa) on ex-vivo binding studies with human platelet membranes on the binding of the specific thromboxane a antagonist h-sq- and the pgi agunist h-l]oprost. the number of receptors (bmm) and the binding affinity (kd) were calculated using scatchard's plot analysis. in healthy male volunteers o significant difference was seen following intake of mg/d of asa for days (mean -+ sem): the potency of meloxicam (mel), a new anti-inflammatory drug (nsaid), in the rat is higher than that of well-known nsaids, in adjuvant arthrtitis rats, mel is a potent inhibitor of the local and the systemic signs of the disease. mel is also a potent inhibitor of pg-biosynthesis by leukocytes found in pleuritic exudate in rats. conversely, the effect of mel on pg-biosynthesis in isotated enzyme preparations from bull seminal vesicle in vitro, the effect on intragastric and intrarenal pg-biosynthesis and the influence on the txb - evel in rat serum is weak. in spite of the high antiinflammatory potency in the rat, mel shows a low gastrointestinal toxicity and nephrotoxicity in rats. -cyclooxygenase- (cox- ) has been recently identified as a isoenzyme of cyclooxygenase. nsaids are anti-inflammatory through inhibition of pg-biosynthesis by inducible cox- and are ulcerogenic and nephrotoxic through inhibition of the constitutive cox- . we have investigated the effects of mel and other nsaids on cox- of non stimulated and on cox- of lps-stimulated guine pig peritoneal macrophages. cells were cultured with and without lps for hrs together with the nsaid. arachidonic acid was then added for further mins, the medium removed and pge measured by ria. bimakalim, emd , is a new investigational k+-channel activator with vasod[lating properties. single pereral doses of . mg bimakalim, mg diltlazem, either alone or in combination, were investigated in healthy male supine volunteers ( to years of age) [n a placebo-controlled, periodbalanced, randemised, double-blind, way cross-over design. point estimates of the global effects of bimakalim [k] , di]tiazem [d] and their interaction [kxd, = in case of mere additivity] incl. % confidence intervals (ci) were analysed for systolic and diastolic blood pressure (sbp, dbp; mmhg), heart rate (hr; bpm), pq (ms), systolic time intervals (pep, qs c, lvetc; ms), cardiac output (co; i.min- ), total peripheral resistance (tpr; dyn.s.cm- ), heather index (hi; q.s- ); , h after dosing, *statistically significant at a= . : - to - - to - to - to . to . - . to , . to . * - . to , - . to - . * - . tol, - . to , - . to . - . to . - . to . - . to . - . to . - to - to -& to . - . to . afterload reduction and a drop in dbp occurred with bimakalim associated with a rise in hr and mild increase in cardiac performance, diltiazem (slightly) decreased afterload and bp with little (reflectory) accompanying changes and had a negative dromotropic effect. the combination caused additive effects. center for cardiovascular pharmacology, zekapha gmbh, mathildenstr. , mainz, germany. rheumatoid arthritis (ra) is characterized by an immunological mediated inflammatory reaction in affected joints. infiltration of granulocytes and monocytes is the pathophysiological hallmark within the initial phase of inflammation. these cells are able to synthesize leukotrienes. ltb is a potent chemotactic factor and therefore could be responsible for the influx of granulocytes from the circulation. cysteinyl leukotrienes ltc , d and e augment vascular permeability and are potent vasoconstrictors. ltb and cysteinyl leukotrienes have been detected in synovial fluid of patients with ra. however, these results are difficult to interprete, because the procedure is invasive and artificial synthesis cannot be excluded. we used a different, noninvasive approach by assessing the excretion of lte into urine. studies with hltc have demonstrated that lte is unchanged excreted into urine and is the major udnary metabolite of cysteinyl leukotrienes in man. udnary lte was isolated from an aliquot of a hour urine collection by solid phase extraction followed by hplc and quantitated by ria. nine patients were enrolled in the present study. all met the american college of rheumatology criteria for ra. patients were treated with nonsteroidal inflammatory drugs and disease modifying drugs. therapy with prednisolon was started after collection of the initial hour urine sample. disease activity was assessed by crp (mean + mg/l) and esr (mean _+ mm/hour platelet aggregation is mediated by the binding of an adhesive protein, fibrinogen, to a surface receptor, the platelet glycoprotein lib/ilia. gpiib/llla is one of a family of adhesion receptors, integrins, which consist of a ca++-dependent complex of two distinct protein subunits. under resting conditions, gpiib/llla has a low affinity for fibrinogen in solution. however, activation of platelets by most agonists, including thrombin, adp and thromboxane results in a conformational change in the receptor and the expression of a high affinity site for fibrinogen. binding of fibrinogen to platelets is a common end-point for all agonists and therefore is a potential target for the development of antiplatelet drugs. these have included chimeric, partially humanised antibodies ( e ), peptides and peptidomimetics that bind to the receptor and prevent fibrinogen binding. the peptides often include the sequence rgd, a sequence that is present in fibrinogen and is one of the ligand's binding sites. when administered in vivo, antagonists of gpiib/llla markedly suppress platelet aggregation in response to all known agonists, without altering platelet shape change, a marker of platelet activation. they also prolong the bleeding time in a dose and perhaps drug dependent manner, often to more than rain. in experimental models of arterial thrombosis, gpllb/llla antagonists have proved highly effective and are more potent than aspirin. studies in man have focused on coronary angioplasty, unstable angina and coronary thrombolysis and have given promising results. e given as a bolus and infusion combined with aspirin and heparin reduced the need for urgent revascularisation in patients undergoing high-risk angioplasty, although bleeding was more common. some compounds have shown oral bioavailability raising the possibility that these agents could be administered chronically. antagonists of the platelet gpiib/llla provide a novel and potent approach to antithrombotic therapy. drug databases on computers are commonly textfiles or consist of tables of generic-names or prices for example. until now pharmacokinetic data are not easily available for regular use, because searching parameters in a textfile is time consuming and personal intensive. on the other hand these pharmacokinetic data are the fundamental background of every dosage regimen and individual dosage adjustment. for many drugs elimination is dependent on the patients renal function. renal failure leads to accumulation, possibly up to toxic plasma concentrations. therefore, the decision was to build up a pharmacokinetic database. the aim is to achieve simplicity and effectiveness by using the basic rules. only three parameters are needed to describe the pharmacokinetics: clearance (ci), volume of distribution (vd) and half-life (t~). moreover, with two parameters the third can be calculated ancl'controlled by the equation: cl = vd * , / t½ according to the dettli-equation and the bayes' theorem estimation of individual pharmacokinetic parameters will be done by a computer program. the advantage is that the impact of therapeutic drug monitoring can be increased. using the population data and the bayesian approach, only one measurement of serum drug concentrations might be enough to achieve an individual dosage regimens (el desoky et al., ther drug monitor , : ) higher therapeutic security for the patient can be achieved. there is also a major pharmacoeconomic aspect: adapting drug dosage reduces costs (susanka et al., am j hosp pharm , : ) the basic database for future pharmacokinetic clinical desicions is going to be built up. the pharmacokinetic interactions with grape#uit juice reported for many drugs are attributed to the inhibition of cytochrome p enzymes by nanngenin, which is the aglycene of the bitter juice component nadngin. however, only circumstantial evidence exist that naringenin is indeed formed when grapefruit juice is ingested, and the lack of drug interaction when naringin solution is given instead of the juice is still unexplained. we investigated the pharmacokinetics of naringin, naringenin and its conjugated metabolites following ingestion of ml grapefruit juice per kg body weight, containing ijm naringin, in male and female healthy adults. urine was collected - , - , - , - , - , - , - and - hours alter juice intake. naringin and naringenin concentrations were measured by reversed phase hplc following extraction using ethyl acetate, with a limit of quantitation of nm. conjugated metabolites in urine were transformed by incubation with glucuronidase ( u/ml) / sulfatase ( u/ml) from abalone entrails for h at ph . and determined as parent compounds. additionally, naringin and naringenin concentrations were measured in plasma samples from grapefl'uit juice interaction studies conducted previously. neither naringin nor its conjugated products were detected in any of the samples. naringenin was not found in plasma. small amounts of nanngenin appeared in urine alter a median lag time of hours and reached up to . % of the dose (measured as nanngin). after treatment with glucuronidase / sulfatase, up to % of the dose was recovered in urine: the absence of naringin and its conjugates and the lag time observed for naringenin to appear in urine suggests that cleavage of the sugar moeity may be required before the flavonoid can be absorbed as the aglycone. naringenin itself undergoes rapid phase ii metabolism. whether the conjugated metabolite is a potent cytochrome p inhibitor is unknown but not probable. the pronounced variability of naringenin excretion provides a possible explanation for apparently contradictory results in grapefruit and/or naringin interaction studies. grapefruit juice increases the oral bioavailablity of almost any dihydropyridine tested, presumably due to inhibition of first-pass metabolism mediated by the cytochrome p isoform cyp a / . the mean extent of increase was up to threefold, observed for felodipine, and more pronounced drug effects were also reported. thus, a such interaction may be of considerable clinical relevance. no data are yet available for nimodipine. we conducted a randomized cross-over interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of nimodipine and its metabolites m (pyridine analogue), m (demethylated) and m (pyridine analogue, demethylated). healthy young men ( smokers / nonsmokers) were included into the investigation. nimodipine was given as a single mg tablet (nimotop e) with either ml of water or ml of grapefruit juice (d~hler gmbh, darmstadt, mg/i naringin). concentrations ef nimodipine and its metabolites in plasma withdrawn up to hours p.ostdose were measured by gc-ecd, and model independent pharmacokinetic parameters were estimated. the study was handled as an equivalence problem, and anova based % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios. the absence of a relevant interaction was assumed if the ci were within the . to . range: grapefruit juice was reported to inhibit the metabolism of a variety of drugs, including dihydropyridines, verapamil, terfenadine, cyclosporine, and caffeine. these drugs are metabolized mainly by the cytochrome p isoforms cyp a (caffeine and, in part, verapamil) and cyp a (others). theophylline has a therapeutic range of - mg/i and is also in part metabolized by cyp a . therefore, we conducted a randomized changeover interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of theophylline. healthy young male nonsmokers were included. theophylline was given as a single dose of mg in solution (euphyllin e ), diluted by either ml of water or ml of grapefruit juice (d hler gmbhi darmstadt, mg/i nadngin). subsequently, additional fractionated . i of either juice or water were administered until hours postdose. theophylline concentrations in plasma withdrawn up to hours postdose were measured by hplc, and pharmacokinetics were estimated using compartment model independent methods. the study was handeled as an equivalence problem, and anova based % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios (trnax: differences thus, no inhibitory effect of grapefruit juice on theophylline pharmacokinetics was observed. the lower contribution of cyp a to primary theophylline metabolism or differences in naringin and/or naringenin kinetics are possible explanations for the apparent contradiction between the effects of grapefruit juice on caffeine and on theophylline metabolism. the physical stability of erythromycin stearate film tablets was studied according to a factorial design with experimental variables temperature, relative humidity, and storage time. after one half year of storage at oc and % relative humidity, the fraction of dose released within min in a usp xxl paddle apparatus under standard conditions decreased from % for the reference stored at ambient temperature in intact blister packages to % for the stress-tested specimens. chemical degradation of the active ingredient did not become apparent before months of storage. under all other storage conditions, no effects of physical aging upon drug release were found. the bioequivalence of reference and stress-tested samples was studied in six healthy volunteers. the extent of relative bioavailability of the test product was markedly reduced (mean: . %, range: - %), mean absorption times of the test product were significantly prolonged. the results indicate that the product tested can undergo physical alterations upon storage under unfavourable conditions, and lose its therapeutic efficacy. it can be expected that this phenomenon is reduced by suitable packaging, but the magnitude of deterioration may cause concern. on the other hand, incomplete drug release is in this case easily detected by dissolution testing. whether similar correlations exist for other erythromycin formulations remains to be demonstrated. the efficacy of a drug therapy is considerably influenced by patient compliance. within clinical trials the effects of poor compliance on the interpretation of study results frequently leads to underestimating the efficacy of the treatment. in the evaluation of the "lipid research clinics primary coronary prevention trial" and the "helsinki heart study" special attention was focused on compliance with medication. the strong influence of compliance on clinical outcome and the dilutional effect of poor compliance on the efficacy of the respective drugs occured in both these trials. there are indirect (e.g. pill-count, patient interview) and direct methods (e.g. measurement of drugs, metabolites or chemical markers in body fluids) used to assess compliance with drug therapy. the indirect methods mentioned are commonly considered as unreliable. the direct methods can prove dose ingestion a short time before the sample is taken, however, they cannot show the time history of the drug use. an advanced method of measuring compliance is to use electronic devices. the integration of time/date-recording microcirculty into pharmaceutical packaging, so as to compile a time history of package use, provides real-time data as indicative of the time when dosing occurred. this method supports a precise, quantitative definition of "patient compliance" as: the extent to which the actual time history of dosing corresponds to the prescribed drug regimen. by taking real-time compliance data into account the results from clinical trials show not only clearer evaluations of drug efficacy and dese-reponse-relationship but also a better understanding of dose dependant adverse drug reactions. in the present study, we examined the usefulness of eroderm- and eroderm- . seventy five impotent men, to years old, participated in the present trial. the patients were classified into groups, patients each. the first group was treated by cream containing only co-dergocrine mesilate (eroderm- ), the second received a cream containing isosorbide dinitrate, isoxsuprine hcl and co-dergocrine mesilate (eroderm- ), while the third used a cream containing placebo. the cream was applied to penile shaft and gland / - hr before sexual stimulation and intercourse. the patients were asked to report their experience via questi'onnaire after one week. the results of treatment are as follows: seven patients ( %) who applied eroderm- indicated a full erection and successful intercourse. the use of eroderm- restored potency in patients ( %) of the second group. three men ( %) of psychogenic type reported overall satisfaction with placebo cream. treatment of impotence with eroderm cream was most successful in patients with psychogenic disorders which are often coincident with minor vascular or neurological disorders. fair results were reported by patients afflicted by moderate neurological disorders. except for one case of drug allergy following the use of eroderm- , no side effects were reported. we believe that eroderm cream has obvious advantages and may be a suitable treatment before the use of non-safe method as intracavernous medication. a new type of topically applied drugs (eroderm creams) for impotence is presented. eroderm creams contain vasoactive drugs. these drugs have ability to penetrate the penile cutaneous tissue and facilitate erection. in the present study, we examine the usefulness of eroderm- in the treatment of erectile dysfunction. eroderm- contains tiemonium methylsulfate, a.f. piperazine and jsosorbide dinitrate. a randomized, double blinded control trial on patients was performed. the etiology of impotence was investigated. all patients received eroderm- and placebo cream. the patients randomized into groups of . the first group received eroderm- on day and placebo cream on day , however, group two received placebo on day . the patients were advised to apply the cream on the penile shaft / - hr, before sexual stimulation and intercourse. the patients reported their experience via questionnaire. overall percent of patients demonstrated a response with eroderm- . the other responders reported a partial erection and tumescenous. three men ( %) reported a full crection and satisfied intercourse with either cream. these patients were psychogenic impotence. neither eroderm- nor placebo cream produced marked response in patients. four patients were venous leakage which were advised to use tourniquet at the base of penis after / hr. of cream application. only one of them indicated a good response. the highest activity proved to occur in psychogenic impotence. less rate of success was observed in patients with minor to moderate neurological and/or arterial disorders. no marked side effects were recorded. for these reasons eroderm- may be proposed as first line therapy of erectile dysfunction. control of cell proliferation is a basic homeostatic function in multicellular organisms. we studied the effects of some prostaglandins and leukotrienes and of their pharmacological inhibitors on cell proliferation in murine mast cells and mast cell lines, in a human promyelocytic cell line (hl- cells) and in burkitt's lymphoma cell lines. in addition, prostaglandin and leukotriene production was investigated in mast cells, representing putative endogenous sources of these lipid mediators. murine mast cells were derived from bone marrow of balb/c mice. proliferation of cells was estimated using a colorimetric assay (mtt-test). production of prostaglandin d (pgd ), pgj , delta- -pgj , leukotriene c (ltc ) and ltb by mast cells was determined by combined use of high performanceliquid chromatography and radioimmunoassay. pgd and its metabolites pgj and delta- -pgj exhibited significant antiproliferative effects in the micromolar range in mast cells, mast cell lines, hl- and burkitt's lymphoma cell lines whereas inhibition of cyclooxygenase by indomethacin was without major effects. ltc and ltb had a small stimulatory effect on cell proliferation in hl- cells. degradation and possibly induction of cell differentiation may have attenuated the actions of leukotrienes. the leukotriene biosynthesis inhibitors aa- and mk- reduced proliferation of hl- and lymphoma cells significantly but had no major effects on mast cell growth. on the other hand, mast cells stimulated with calcium ionophore produced pgd and its metabolites, as well as ltb and ltc in significant amounts. from our data we conclude that prostaglandins and leukotrienes may play an important role in the control of cell proliferation. we compared the pattern of drug expenditures of several hospitals in (size: to beds). a, b are university hospitals in the ,,old"and c,d,e are university hospitals in the ,,new" german countries, f is a community based institution in an ,,old" german country. main data source were lists comprising all drags according to their expenditures in a rank order up to %. items were classified into i) pharmaceutical products including immunoglobulines, ii) blood and -derived products (cell concentrates, human albumin, clotting factors) and iii) contrast media (x-ray). with regard to group i) the highest expenditures nccured in hospitals a and b whereas drug costs in c -e were / less and came to only % in hospital f. the main groups of drugs which together account for > % of these expenditures are shown in the table. ) products were about % up to % of group i and highest in hospitals a, b and e, but about / lower in hospitals c and d. these results suggest meaningful differences in the drug utilization between the old and new countries as well as betv,,een university institutions and community based hospitals. however, although all hospitals provide oncology and traumatology services and all university hospitals offer ntx, differences in other subspecialities e.g bone marrow and liver transplantation and treatment of patients with haemophilia must be considered, too. dr.medsebastian harder, dept c]inicai pharmacology, university hospital frankfurt, theodor stern kai , frankfurt/main frg m. hgnicka, r. spahr, m. feelisch, and r. gerzer organic nitrates like glyceryl trinitrate (gtn) act as prodrugs and release nitric oxide (no), which corresponds to the endogenously produced endothelium-derived relaxing factor. in the vascular tissue, no induces relaxation of smooth muscle cells, whereas in platelets it shows an antiaggregatory effect. both activities are mainly mediated via stimulation of soluble guanylyl cyclase (sgc) by no. in contrast to compounds which release no spontaneously, a membrane-associated biotransformation step is thought to be required for no release from organic nitrates. glutathione-s-transferasea and cytochrome p- enzymes have been shown to metabolize organic nitrates in the liver, but little is known as to whether these enzymes are involved in the metabolic conversion of organic nitrates in the vasculature. furthermore, it is still unclear whether or not platelets are capable of metabolizing organic nitrates to no. we isolated the microsomal fraction of bovine aorta in order to characterize the activities towards organic nitrates using the guanylyl cyclase reaction as an indirect and the oxyhemoglobin-technique as a direct measure for no liberation. gtn was metabolized to no by the microsomal fraction under aerobic conditions already in the absence of added cofactors. this activity was not influenced by the cytochrome p- inhibitors cimetidine and metyrapone. in contrast, the glutathione s-transferase substrate -chloro- , -dinitrobenzene and the glutathione s-transferase inhibitors sulfobromophthalein and ethacrynic acid did not affect no release, but potently inhibited sgc activity. blocking of microsomal thiol-groups resulted in a decreased no release from gtn. homogenates of human plateles isolated by thrombapheresis and stabilized by addition of mm n-acetylcysteine did not show no-release from gtn as determined by the stimulation of the platelet sgc even after addition of the possible cosubstrates glutathione and nadph. these data demonstrate ( ) that bovine aortic microsomes exhibit an organic nitrate metabolizing and no-releasing activity whose properties are clearly different from classical cytochrome p- enzymes and from glutathione s-transferases, and ( ) that human platelets itself are not capable of bioactivating organic nitrates and therefore require organic nitrate metabolism in the vessel wall for antiaggregation to occur. bioavailability of acesal ®, acesal ® extra, micristin ® (all mg acetylsalicylic acid -asa), and miniasal ® ( mg asa), opw oranienbufg, relative to respective listed references was studied in female and male healthy volunteers (age - y, weight - kg, height - cm). asa and salicylic acid (sa) were measured using an hplc method validated from ng/ml to pg/ml. extent of absorption was assessed by auc (bioequivalence range . - . ), rate by cr~/auc (bioequivalence range . - . ). geometric means and %-confidence limits of the ratios test/reference (multiplicative model) are shown in the acesal ® and micdstin ® were bioequivalent in rate and extent of absorption with the reference formulations. the fast liberating acesal ® extra was bioequivalent with respect to extent only. asa from miniasal ® was absorbed more slowly than from an asa solution (cm= ( %-range): - ng/ml and - ng/ml; t~ (min-max): . - . h and . - . h). asa from micdstin ® and the corresponding reference was absorbed more slowly than from acesal ® and acesal ® extra. this was accompanied by decreased aucasa (increase of first pass metabolism) and increased apparent trrz (absorption being rate limiting). all ratios of aucsa/aucasa after administration of mg asa were markedly higher than after mg asa. thus, the formation of salicyludc acid from sa might be capacity limited at doses of mg asa. in the study >>physicians' assessment of internal practice-conditions and regional health-services-conditions in accordance with ambulatory patient-management<< a sampie of primary care physicians -comprising gps and internists -provide data for continuons analyses of arnbulatory health care quality and structure. focussing on the physicians' drug prescription, the impacts of reform law (gesundheitsstralcturgesctz, gsg) upon primary care providers and their therapeutic decisions were examined in . four different surveys were carded out during the year, dealing with frequent patients' reasons for encounter in gps' offices. after a pretest was carried out, physicians reported on patient-physician-encounters, basing on mailed questionnaires. for every therapeutic change patients received, the reasons for the change were recorded (e.g, reform law, medical indication) and above the physicians' expectations towards three criteria to measure the quality: ) physicians' assessment of the patients' satisfaction, ) adverse drug effects, ) therapeutic benefit. according to therapeutic changes due to reform law (drag budgets, blacklist) it can be stated: ) therapeutic changes due to reform law were carried out with relevant frequency. ) the reform law was of different concern regarding the different reasons for encounter we investigate& ) the impacts' strangth of the legal control mechanisms differed among several groups of physicians: those who already have been liable to recourse before more often carried out therapeutic changes according to fixed drug budget. different multivariate logistic regression-models yield an estimation of the odds-ratio of about . ) therapeutic changes in accordance with the reform law having been carried out at the beginning of the year more often suffered from negative expectations towards the therapeutic quality then changes during the actual encounter, e.g. >>joint pains<c % decrease of the therapeutic benefit in contrast to %. during the year a decrease of negative expectations is noticable. ) patients overestimating the severity of their own health problem in comparison to the physician more often suffered from negative expectations towards the therapeutic change than other patients, e.g. concerning the therapeutic benefit ( % in contrast to %). generally speaking, firstly the reform law led to insecurities -particularly in the introduction phase -among susceptible subgroups of physicians and patients, regarding the four investigated reasons for encounter. secondly, there is evidence of a loss of quality in ambulatory health care -predominandy in terms of subjective criteria. these impacts considerably subsided within the field phase duration from may to november, . the presented analyses give evidence of the practicability of studies assessing effects of legally-initiated mechanisms to control health costs upon primary health care quality. postansehrifi: iseg, bissendorfer sm , d- hannover the cellular and biochemical mechanisms that lead to the longerm complicatiorts of diabetes mellitus are poorly understood. glycation is a multireaetion beginning with a condensation reaction between reducing sugars and reactive amino groups of proteins. the accumulation of the glycated products of various proteins may contribute to the development of the complications of diabetes and aging. in order to find suitable non-toxic glycation inhibitors we used sulfur deriva-tives (penicillamine, eaptopril and alpha-lipoic acid) and amino derivatives (aminoguani-dine and aminoguanidine propionic acid) and postulated the mechanisms involved.the inhibitou effect of the drags on the early (amadori product) and advanced glycation products (age). determined by affinity chromatography, deglycation method. i-iplc and fluorescence spectroscopy, differs markedly depending on the drag used and is greatest (up to %) with aminoguauidine and penicillamine ( mm) and smallest with alpha-lipoic acid. aminoguanidine propionic acid had no inhibitory effect on the glycation process. the ability of penicillamine to inhibit both the formation of amadori products and age was demonstrated by isolation of the adducts of penicillamine with ribose, glucose, glucosa-mine and p-tolylglucosamine, a standard for glycated protein. their structures were characterized using ir-and h-nmr and c-nmr spectroscopy. the oxidative deg-radation of glycated human serum albun{in in the presence of copper (ii), forming the carbox jmethyllysine, was inltibited by the sulfur derivatives. the in vitro formation of pentosidine, a crossliuking product was influenced by all drugs used however captopril, alpha-lipoic acid and aminoguanidine propionic acid do not condense with sugar derivatives.the mechanism by which captopril and alpha-lipoic acid inhibit the formation of age may be involve radical formation, where a spontaneus thiol/disul-fide interchange between drug and protein takes place. complications can appear at any time in diabetic patients.the cause of these complica-lions in some cases may be due glycation and/or oxidation. the clinical applications of substances which are able to prevent these events may have advantages. these results and the mechanism proposed show that the amino and sulfl~ydryl groups of drags reduce glycation and postglycation of proteins and thus have potential value in this regard. non-enzymatic glycation starts by covalent binding of single sugars to o~ or e amino groups of proteins. in subsequent reactions a number of early glycation products like schiff bases or amadori products, and of advanced glycation endproducts (ages) is formed. glycation is increased in a variety of proteins in diabetic patients [schleicher e ( ) diabetes und stoffwechsel : ] including human serum albumin (hsa), the red cell membrane, hemoglobin, collagen, laminin, immunoglobulin g, low-and high-density lipoproteins. the process is supposed to be a key mechanism in the pathogenesis of diabetic complications and in tisstie ageing [vlassara het al. ( ) lab invest : ] . inhibition of glycation may therefore be important for the prevention of late diabetic complications. we investigated the in vitro glycation of human serum albumin (hsa) and its inhibition by aminoguanidine, penicillamine, captopril and c~-lipoic acid ( ram). hsa ( g/l) was glycated by incubation with mm glucose in . m phosphate buffered saline, ph . , at °c for days under sterile conditions in the presence of . % natrium azide. the glycation rate was determined by two different methods: deglyeation -measures amadori products based on the colorimetry of -ketoglucose which is released from the glycated protein (ketoamine) the effect of aniseylated #asminogen streptokinase activator complex (apsac) on left ventricuiar (ll r) function ;,.,as investigated in ! patien~ with acute myocardial infarction (am!). a dose of q units apsac was given intraveneusiy to patien~ with ami within hours after onset of pain, patients underwent cardiac angiegraphy at minutes and at day . wail motion ,.~s measured by the global ejection fraction and by the centefline method in :'=he infarct and neninfar~ regions and expressed in units of sf~ndard deviations beiew normai, as defined in normai patient. group a with successfui ',th, rombo!ysis (timi and ) was compared with group b without successfui thrombolysis (timi and !). at minutes there vras no significant difference in giebal ejection fraction between both groups, by using the regiona! wa[i motion by the centerline method group a showed a significant improvement of lv function (p ~ . ). the benefit of successeli thrombeiysis was more marked in the peripheral infarct region than in the centrai infarct region. thus early repeffusion is an important factor in maintaining good lv function after ami. the re=gionai wai! motion in the infarct region seems to be more sensmve man me g!oba! eiection fraction to show ear!y differences in lv function after ami. the quinoline derivative bay x ((r)- -[ -(quinoliu- -yl-metho.,qc)phenyl]- cyclopentyl acetic acid) is a selective inhibitor of ltb -biosynthesis in various in vitro and in vivo models. it acts via binding to the integral nuclear membrane protein five-lipoxygenase activating protein (flap), which has been shown to be an essential component of the cellular leukotriene synthesis machinary. the function of flap is only poorly understood, but recently it has been shown that a radiolabeled photoaffinity-analog of arachidonic acid binds specifically to flap. consequently, a role of flap in the substrate transfer to -lipoxygenase ( -lox) has been suggested. using intact and fractionated human polymorphonuclear leukocytes (pmnl), we present data showin~ that exogenously added arachidonie acid diminishes dose-dependently binding of [ c]-bay x to its target. this effect correlates with the reduction of bay x mediated inhibition of ltb synthesis after stimulation of intact pmnl with tile calcium ionophore a in the presence of increasing extracellular arachidonic acid concentrations in addition, enhancing the release of intracellular arachidonic acid from phospholipids after activation of phospholipase a (pla ) with increasing a concentrations or after prolonged incubation of the cells with the ionophore results in a loss of bay x potency. to further study" the effect of arachidonic acid on the bay x mediated ltb synthesis inhibition, human pmnl were stimulated with paf, c a or fmlp in the presence of exogenous arachidouic acid. under such conditions, ltb synthesis is significantly enhanced in response to paf, c a or fmlp. which are only weak activators of cell-associated pea the effect of extracellular arachidonic acid is dosedependently inhibited by bay x . coincubation of the cells with arachidonic acid and paf, c a or fmlp, respectively, enhances synergistically the translocation of -lox from the cnosol to the membrane fraction. again, this effect is inhibited by bay x . in summary, using bay x , we provide evidence, that flap is involved in the utilization of the -lox substrate araehidouic acid from exogenous or endogenous sources for the agonist-induced leukotrieue biosynthesis. switzerland, but their effect on the expression and function of the multiple drug resistance gene product has not been studied in detail. we have therefore examined the effect of gm-csf and g-csf on the uptake and toxicty of vincristine with and without the chemosensitizers r-verapamil and norverapamil in the pglycoprotein-containing vincristine-resistant cell-line, hl- /vi,o. following a h pre-incubation with ng/ml g-csf or gm-csf, cells were washed and reincubated in rpmi medium with hvincristine and the modulators for hours. the uptake of hvincristine was increased up to -fold and this effect was equally marked when the vincristine-uptake was stimulated with the chemosensitizers in the range of . pg/ml to pg/ml. although cytokine-pretreatment increased the accumulation of vincristine, we could not show an increase in vincristine toxicity over h with the same concentrations of vincristine and chemosensitizers. it is concluded that the effects of cytokines on vincristine accumulation are compensated by a positive effect on cell-survival or cell-growth. department of clinical pharmacology, university clinic, frankfurt, theodor stern kai , frankfurt, germany. from the economic point of view people often tend only to regard the price of a product. savings for the health care system as a whole which could be gained by use of these drugs and which could more than compensate the high costs of medication are not considered. the cost-benefit-analysis enables a comprehensive analysis of the economic effectiveness of a pharmaceutical. the relevant cost factors such as the costs for in-and out-patient treatment depending on the therapeutic success and the inability to work are compared to the costs of medications. using the example of stroke prevention, it was possible to show that prophylaxis with an expensive but effective pharmaceutical is economically better than standard medication. according to the tass study ticlopidine can prevent at least more strokes per i treated patients than ass. on the basis of this study cost savings of up to dm , per i treated patients can be reached including higher costs of ticlopidine. projected onto i , patients up to dm , per year can be saved for the german health care system by the use of ticlopidine in secondary prophylaxis after tia. the naphthodianthrones hypericin (h) and pseudohypericin (ph) were proved to be elfectire in mild depressive disorders. recently they were discovered to exhibit high in vitro activity against enveloped viruses fostering clinical trials in infections with hiv or herpes viruses, for studying the pharmacokinetics of h and ph a solidified extract from st. john's wort (hypericum pefforatum l) was orally administered to healthy male volunteers in three single doses of , , and ~g of h and , , and p.g of ph, followed by a -days course of multiple dosing of p.g h and pg of ph tds. plasma concentrations were determined by hplc, the quantification limit was . #g/i (staffeldl et al., nervenheilkunde ( ) : ). single,dose kinetics, h showed a median lagtime of . (range: , - . ) h, which was significantly longer compared to , (range: . - , ) h of ph. h was eliminated with a median half-life of (range: - ) h, and in case of ph the hail-life ranged between . and (median: , ) h. median crux levels were . , . , . ~g/i for h and . , . , . pg/i for ph for the three single-dose levels given above, respectively. lowest dose crre, x and auc of h was lower then expected from medium and highesl dose, which was statistically significantly for crnax (p= . , anova; p= . , onferroni/dunn), indicating nonproportionality of pharmacokinetic response may exist in the lower dosing range. mul!iple-dose kinetics. during long-term dosing steady-state trough levels (ctreugh) of . (range: . - . ) ~g/i for h and . (range: -to. ) ~g/i for ph were reached after days for both. cmax after days of treatment were . (range: . c. kori-lindner, r. eberhardt the healthcare restructure act ( ) changed the evaluation of drugs from efficacy to cost effectiveness: therapeutic necessity, utility, purpose; medical progress and economic utility have to be considered. criterias and suroundings of pharmacooeconomic evaluations and studies according to the bealthcare restructure act are demonstrated by an example: "costs of nsaid-induced gastro-duodenal ulcers and prevention with miso~rostol". methods: cost-utility, cost-benefit, legal requirements, decision-model and decision -tree, basic calculation and the transfer to the socialsystem of the frg. premissions: rate of ulcers without prevention , %, with prevention , %; rate of compliance %, rate of hospitalisation % and duration of hospitalisation , days. rheumatic disorders have become a frequent reason for services m ambulatory care. therefore the use of drugs which are administered in rheumatic complaints is widespread. data of the sientific institute of statutory health insurances (arzneimittelindex ) demonstrate that the group of antirheumatics/analgetics held the first rank due to the number of prescriptions. with regard to the costs thts drug class were amongst the top three. apart from anurheumatics and analgetics different other drug classes are used for the treatment of rheumatic disorders for example corticoids. to estimate the total expenditure for drug utilization in out-patients suffer from musculoskeletal disorders data of the statutory health insurance dortmund (aok dortmund) were investigated. a % random sample of insurees (n= ) was analysed for diagnoses and drug therapy. the data were collected in a individual-related way for an observation period of one year ( ). the results demonstrate the high frequency of rheumatic disorders among out-patients: the -month prevalence of musculoskeletal disorders was about % in the studied population of whom one half suffered from back pain. approximately % of the patients were prescribed any drugs for treatment of rheumatic disorders. as might be expected nonsteroidal antiinflammatory drugs were used most frequently. the extensive use of topical products was a considerable factor of costs. more than one half of the patients received drugs for topical application. further details will be presented. diachronous data of statutory health insurences that have been gathered in a person-related way permit the estimation of drug expenditure in outpatients with regard to the several musculoskeletal disorders. misleading results of endocrine tests in patients suffering from liver diseases promted us to investigate the influence of liver function on the pharmacokinetics of dexamethasone (dex). it was the aim of this study to find out whether routine parameters of liver function may be usefull in roughly predicting the clearance of dexamethasone. methods : in patients liver function was rated on the basis of their plasma cholinesterase activity (che) and prothrombin time (pt). blood samples were withdrawn , , , , , , , and min following an iv bolus injection of mg of dexamethasone- -dihydrogenphosphate corresponding to , mg of dex for the determination of dex pharmacokinetics by ria. results : computer assisted best fit analysis ( compartment model) of the plasma dex decay did not show any influence of liver function on the distribution half-life (t½-a) and on the initial volume of distribution of dex, but a % increase in the terminal phase, "metabolic" half-life (t½-b) with decreasing liver function. while the t½-b increased from ± min in patients with a che activity > . ku/l to ± min in those with a che s . ku/l, the metabolic clearance rate (mcr) decreased from ± ml/min to iii ± ml/min. in patients on phenytoin the t½-b was reduced to <i min. che activity (r=- . ) and pt (r=- . ) were significantly (p< . ) correlated with the t½-b of dex. we conclude that the dosage of dexamethasone given for diagnostic or therapeutic purpose needs to be adjusted in patients on phenytoin therapy and in patients with liver disease causing a che s ku/l. etofenamate is one of the most frequently applied topical nsaids in germany. it can be regarded as a lipophilic prodrug of flufenamic acid, which is liberated after transdermal absorption and cleavage of the ester. we compared several topical nsaids in patients suffering from osteoarthrosis or rheumatoid arthritis with concomitant knee joint effusions. the products contained between and % of etofenamate and were administered according to the recommended dosage schedule. following multiple applications of defined doses we obtained synovial fluid and plasma specimen in order to measure the concentrations and to compare the bioavailabilily of several products. the analysis of synovial fluid and plasma samples was achieved by hplc. following the addition of other n-phenylanthranilic acid derivatives as internal standards the samples were extracted twice with a -fold volume of acetonitrile/methanol ( : ). the diluted extracts were concentrated with the use of preconditioned ods-columns and subsequently eluted with methanol. the separation of the products was perlormed on a reversed phase column with an elution medium of methanol/water/acetic acid ( : . : . ). the highest synovial fluid and plasma concentrations were measured after hours following the final administration of etofenamate. the concentrations reached average maximum values of . ng/ml of flufenanamic acid in plasma and . ng/ml in synovial fluid. the bioavailability could be estimated from the area under the concentration time interval between . and hours, during which samples were obtained. they served for the comparison of the topical products. from in vitro data of cyclooxygenase or lipoxygenase inhibition with n-phenylanthranilic acid derivatives it can be concluded, that the synovial fluid levels of etofenamate and its metabolites are unsufficient to produce effects relying on this mechanism. it can not be excluded, however, that other effects contribute to the clinical improvements observed by other investigators. in animal studies it has been shown to have excellent anti-arthritic efficacy and an improved tolerability profile over other nsaids. early preclinical studies have shown that mel preferentially inhibits cyclooxygenase- (cox- ). the aims of this three-week, parallel, multicentre trial were to assess the efficacy anct safety of two different doses of mel in comparison to placebo (pla) in patients with active ra. the specific question was, whether cox- inhibition relates to therapeutic efficacy and safety in the clinical'setting. methods: patients ( f, m, mean age years) were randomised (mel . mg= , mel mg = , pla= ) and analysed by an intention-to-treat analysis. all trial drugs were given orally once daily. results: patients ( %) in the pla group, patients ( %) in the . mg mel group and patients ( %) in the mg mel group discontinued the trial prematurely due to lack of efficacy or adverse events. mg mel was significantly superior (p< . ) to pla in of the primary endpoints: disease activity assessed by the investigator and by the patient and reduction of the tender/painful joint count. the difference between . mg mel and pla reached statistical significance in of the primary endpoints: disease activity assessed by the patient and reduction of the tender/painful joint count. in none of the primary endpoints a statistically significant difference between mg and . mg mel occured. global tolerance assessed by the patient and investigator at the last trial visit was similar in all three treatment groups. adverse events were slightly more frequent with meloxicam than with placebo. in none of the patients gastric or duodenal ulcers, perforations or bleeds were observed. conclusion: the efficacy of meloxicam . mg and mg per day was significantly superior to placebo in the treatment of patients with ra. there were no relevant differences regarding safety parameters between the meloxicam doses and placebo. the preferential cox- inhibition may be the reason for the favorable efficacy and safety profile of meloxicam. the major metabolites of morphine are morphine - -b-d-glucuronide (m g) and morphine - - -d-glucuronide (m g). in humans plasma aucs of m g and m g exceed those of morphine depending on form and route of application. m g and m g penetrate the blood-brain-barrier due to an unexpectedly high lipophilicity, m g is an antagonist able to provoke with&awl symptoms even in opioid naive animals. m g is an agonist markedly more potent than morphine. methods: pig brains were homogenized and the homogenate centrifuged to give a microsomal preparation free of nuclei and coarse debris. aliquots were incubated at °c in buffer (ph . ) containing . nm [ h]damgo. results: the ic for morphine, m g and m g were . nm, nm and . pm, respectively. m g and m g, respectively, shifted the ic for morphine markedly to the right. discussion: our results on the single substances confirm data from the literature indicating that m g and m g inhibit binding to the ~t-or less potent than morphine. from the fact that m g and m g shift the ic for morphine to the right we infer binding of m g and m g to the morphine binding site at the ~t-or. bypassing activity and producing cost of dm . ,-was not included in the above shown results. • blood products, coagulation factors and anttmierobial agents are the most expensive substances, however their use could be more restricted. • chemotherapy represents also a high-cost factor, but alternatives do not exist. [ntensiv care units are the most expensive wards consuming - % of the hospital budget, drug use reviews on sicu's of the university hospitals frankfurt ( beds) and jena ( beds) were compared after recording the indication related administration of pharmaceuticals and blood products over a months investigation period using notebook-pc's. all substances were included, wich produced % and % of total pharmacentical cost in the year before, respectively. in frankfurt and jena and patients were registered, causing total cost of dm . ,-and dm . . ,-and causing cost per patient and day of average dm ,and dm ,-, respectively. in jena most expensive diagnoses were severe trauma ( % of total cost; n= ), gastre-intestinal bleeding ( . %; n= ) and malignant growth ( . %; n= ). in frankfiart nigh cost diagnoses were liver transplantation ( % of total cost; n= l), severe trauma ( , %; n= ) and coronary bypass craring ( . %" n= ). although the patient populations were different, the percentage cost of the substance groups on total drug cost were the same in both sicu's: blced-products ± %, antibiotic and chemotherapeutics io, %, immuna-ghibulines , :t , % anyway, inside these groups drug usage patterns differed distinctly. for example, in jena the nigh-cost antibiotics were tobramycin, cefuroxim, amoxicillin and ambisome ® causing . %, . %, . % and % of total antibiotic cost, respectively. corresponding data for franlffurt are ambisome* . %, imipedem . %, cefotaxim , % and cefamandol . %. severe sepsis was the most abundant complication occuring in beth sicu's in patients and producing cost of dm . ,-in frankfurt and dm . ,-in jena. in sepsis blood products were the most expensive substances with . % (frankfurt) and . % (jena) followed by antibiotics with . % and . % and immuneghibulines with . % and . %, respectively conclusion: * recording the indication related drug administration drug usage patterns may be identified and targets for economical intervention may be found. • blood and blood-products must be included because they produce about % of total cost. • complications, especially sepsis, produce nigh cost, which should be included in the discussion with the health assurances concerning the payment system using diagnosis related groups. in a drug-utilisafion-review-group (durg) was founded by clinical pharmacologists. the task of this group is to identify drug usage patterns, to record indication related drug administration including cost and to analyse these data. as a result recommendations concerning rational and economical use of pharmaceuticals and blood-products were given (e.g. ht -receptor-antagonists, human albumin, g-csf the most expensive substances are blood-products ( %), antimicrobial agents ( %) and immunoglobulines ( %). in conclusion, at the universitiy hospital frankfurvmain the activities of the drug-utilisation-review-group seem to be successful and are at least inpart responsible for the reduction of drug-cost of more than dm mio last year. the aim of this study was to determine the extent and the time course of fenoterol induced tachycardia and hypokalemia during and after a continuous intravenous infusion with three different rates within the therapeutic range for tocolysis. furthermore we compared the point estimators and precision of parameters obtained with a standard two step approach and a population model. according to a randomized double blind multiple cross over design, healthy females received an intravenous infusion of either placebo or fenoterol ( . ijg/min, ijg/min, ljg/min) over hours. the plasma concentration of fenoterol, heart rate, blood pressure, plasma potassium and plasma glucose were monitored during and up to hours after the infusion. the plasma concentrations of fenoterol and potassium and the heart rate were fitted to an integrated pkpd model. both individual parameter estimates and population estimates were obtained with nonmem. both effects correlated best with the concentration in the central compartment. the relationship between heart rate and concentration was adequately described by an emax model. modelling of the dose dependent hypokalemic effect of fenoterol required an negative sigmoid emax model with an additional term allowing for passive diffusion of potassium from the intra-to the extracellular compartment. during the observation period, the concentration response relationship remained stable for both effects. as to be expected, the estimates of the population parameters were much more stable than those of the individual patients. it is concluded that the population method might be superior to the sts approach even in a data rich situation. volunteers received different controlled release formulations (a, b, c, containing mg is- -m ", ) in a randomised cross-over design. plasma samples were taken from to hours after application. one week wash-out phase was kept between investigations. is- -mn were determined in plasma using a gc-method. in vitro dissolution of a, ] , c was investigated by using the rotating bottle method according nf xiv. mean absorption time (mat) was calculated as the difference of mrt and ke - , the mean dissolution time (mtdiss) was calculated as proposed by brockmeier ( ). additionally half value duration (hvd) and plateau time opt) are reported. the difference of the dissolution profiles between the product is not reflected in the parameters mat, hvd and pt. although preparation c has the longest mtdiss it has the largest cmax value and shortest hvd. the origin of this apparent discrepancy between in vivo/in vitro profiles deserves further elucidation ( ) acta pharm. technol. ( ) calciumantagonists (ca) are lipophilic molecules which, however, have no common structural similarities. nevertheless, ca are characterized by the ability to attach to various receptors and binding places in cardiac and smooth muscle preventing calcium overload, necrosis and cellular death. in order to study the physiological effects of ca on cerebral cortex of normal and vit d calcified rats, i.v. infusion of ca have been applied. cerebral blood flow (via hydrogen clearance) and oxygen supply (via surface oxygen tension) have been measured in normal and sclerotic albino rats in order to differentiate cerebral and systemic effects. miniaturized surface oxygen and hydrogen electrodes were placed on top of the exposed cerebral cortex of ketamine~xylazine anesthetized rats. nifedipen, vcrapamil, diltiazem and flunarizine induced hypotension in normal and monckebcrg type sclerotic rats. nifedipen and verapamil showed comparable improvements in cbf and surface po , the initial hypotension recovered completely under nifedipen but not under verapamil. compared with vcrapamil, the hypotension was even more pronounced under flunarizine ( : %). nevertheless, in response to both ca cbf was improved to about %. however, diltiazem did not increase cbf due to hypotension. sclerotic rats showed a map-decrease from to mm hg, the po histogram was left-shifted. diltiazem did not improve the cortical supply of sclerotic animals. the influence of intracoronary enoximone at a dose of . mg/kg/lo min on global and regional wall motion, myocardial perfusion (contrast echo) and diastolic lv function during pacing-induced ischemia was investigated in pts. with significant lad stenoses. results: enoximone did not change either heart rate at "rest ( ± vs ± min -i) or blood pressure (lvsp: ± vs ± mmhg) . in the postpacing ischemic period, lvedp fell from a mean of . to . mmhg after enoximone (p< . ), dp/dtmax increased from to mmhg/s (p< . ) and regional ef from % to % (p< . ) with global ef remaining unchanged ( % vs %). st segment depression was reduced significantly from . to . mm (p< . ). enoximone induced an increase in myocardial perfusion by % (p< . ) in the stenosis-depending myocardial areas with shortening of the wash-out half-time of the echo contrast medium from a mean of to s (p< . ) . the isovolumetric relaxation (echo) was shortened by % (p< . ), the e wave by %, and dp/dtmin was increased by % (p< . treated with pivaloyl-pivaloyl-substituted - actamantibiotics belong to a new group of prodrugs with an improved bioavailability following oral administration. after metabolic cleavage the pivalic acid and the free - actam are eliminated separately. pivalic acid is subjected to enzymatic coupling with endogenous camitine leading to carnitinepivaloylesler, which can be found in large amounts in the urine. free camitine is reduced under the treatment which may lead to neurological side effects in patients with metabolic predisposition. we showed, that the pattern of other acylcarnitineesters excreted with the urine was not changed significantly after administration of a pivaloyl.substituted cephalosporine in healthy subjects. in order to compare the pattern of excreted carnitineesters we analyzed urine samples of pediatric patients treated with cefetamet-pivoxil. the analysis of carnitineesters in the urine was achieved by hplc following precolumn derivatization. as an internal standrad we used undecanoyl-carnitine aster, which does not occur in human plasma or urine. after the extraction using ion exchange columns the samples were incubated with p-bromo-phenacyl reagent. the mixture of derivatives was separated on a reversed phase column applying a gradient of different buffers. with this method the whole spectrum of carnitineesters can be quantitatively analyzed. the percentage of the pivalic acid ester eliminated with the urine in pediatric patients did not differ from that observed in healthy subjects, therefore an increased risk in this patient group cannot be inferred. institut for klinische pharmakologie, universit~.tsklinikum charit& humboldt-universit&t zu berlin, schumannstral~e , d- berlin; pharmacokinetics and bioavailability of bupranolol-l"rs and oral bupranolol in patients with impaired liver function compared to healthy volunteers k.-h. molz*, w. cawello**, g. haug-pihale*", r. bonn** w. hammes**, a. weil*** and g. popescu*** bupranolol is nearly completely absorbed after oral administration, but more than % of the parent compound are metabolized by first pass through the liver. it can be expected that transdermal application of the substance will bypass the high first pass metabolism resulting in higher plasma concentrations or lower therapeutic doses, respectively. a reduced capacity of metabolizing liver enzymes might mimic this effect. the study was performed to investigate if metabolisation by hepatic enzyme systems results in liver function dependent pharmacokinetics. special interest was to be given to possible differences between oral and transdermal application. in an open sequential single dose-study including patients with impaired liver function and age/sex/weight-matched healthy volunteers a bupranolol transdermal therapeutic system (containing mg/ h) and bupranolol tablets ( mg) were administered to each participant separated by a washout period of a week. phenotyping for mephenytoin and sparteine was done to exclude poor metabolizers. blood samples for the quantification of bupranolol plasma concentrations were drawn before and , , , , , , , , (before patch removal), , , , , , and hours after patch application as well as before tablet administration and . , , . , , , , , , , , , , , , and hours post dose. urine samples were collected in ('its) and (tablets) intervals after dosing. blood pressure and heart rate were measured before and , , , and hours after dosing, ecg's were recorded before and , and hours after dosing. the pharmacokinetic results indicate only minor differences between patients and healthy controls after patch applications. for example the main parameter auc( -~) were . ± . ng/ml.h (mean±sd) in healthy controls and . ± . ng/ml.h in patients. completely other situation was given in comparison of data from oral administration: auc( -=)decreased dramatically to . ± . ng/ml-h in healthy controls and increased to . ± . ng/ml.h in patients. vascular complications are a common fmding in patients with diabetes mellitus. it has been reported that elevated glucose impairs acetylcholine-but not nitruprusside-mediated relaxation of rabbit aorta in vitro suggesting an abnormal endothelium-mediated vasodilator mechanism (cohen, circulation ; (suppl. v): ). the purpose of the study was to investigate whether this phenomenon . implies general impairment of the nitric oxidemediated relaxation (or only specific interference with muscarinic receptors), and . can be also demonstrated for coronary arteries. porcine coronary arteries were cut in rings and were set up in organ baths for isometric tension recordings. rings were randomized and incubated with different glucose concentrations ( . , , , or . mm) for hours according to cohen and coworkers. rings were preconstricted with prostaglandin f ~t ( . - ~m); the nitric oxidemediated relaxation was induced by either substance p ( . - nm), bradykinin ( . - nm), or calciumionophor a ( - nm), the endothelium-independent relaxation by nitroglycerin ( - nm). in addition, the effects of glucose were studied on acetylcholine-( . - ~tm) and serotonin-( . - ~m) induced vascular responses. results: surprisingly, elevated glucose concentrations ( and mm) had neither an effect on the potency nor the efficacy of any of the agonists tested. it is concluded that this previously reported impairment of endothelium-mediated vasodilation of rabbit aorta by elevated glucose concentrations may not be important in other species or types of arteries. nitric oxide production may be induced by cytokine-mediated immune responses. we tested the hypothesis that graft rejection in cardiac transplanted patients is associated with an increased production of nitric oxide. the study group consisted of patients in whom one or several myocardial biopsies (total biopsies) were obtained to assess for graft rejection. at the day of biopsy, urine was collected for a time period of at least hours ( % isopropanol was added for antimicrobial activity). nitrate was measuerd in urine samples by gaschromatography. nitrate excretion was expressed as the quotient of nitrate and creatinine content (gmol nitrate/mmol creatinine). since nitric oxide is oxidized in biologic systems to nitrite and finally nitrate, it was assumed that nitrate excretion reflects total nitric oxide release. graft rejection was classified according to the hannover classification system (a /i= no/minor rejection, a -a =moderate/severe rejection). results: nitrate excretion showed a large interindividual variation ( to i.tmol nitrate/mmol creatinine). /£ tough not significant, the nitrate excretion tended to increase with the degree of rejection (a : + ; a : + ; a : + ; a / : + ; mean + se). in a subgroup of patients (n= ) who underwent several biopsies with and without rejection, moderate/severe rejection was significantly associated with increased nitrate excretion (a /ai: + vs. a -a : + , p< . ). conclusion: the urinary nitrate excretion is increased in patients with moderate/severe graft rejection. because of large variation in excretion, this marker may be not suitable for rejection diagnosis. the combination of thremboxane a~/prostaglandin endoperoxide (epo) receptor antagonism with thromboxane synthetase inhibition (trasi) is designed to both abolish the epos' prothrombotic role and direct epos from activated platelets to adjacent cells for the generation of antithrombotic prostaglandins. thus, we have investigated the effect of the potent trasi dt-tx on thrombus formation in models of vascular injury (of the media versus selective deendothelialization) in man ex vivo. healthy volunteers were treated with an oral dose of vehicle (placebo; n= ) or , , , mg; n= per dose). blood was taken before and , , and h after the ingestion, anticoagulated and allowed to flow over a thrombogenic cell-free subendothelial matrix (cfm) or a layer of human venous smooth muscle cells (smc) for min. the "en face" area of platelets/thrombi deposited was determined by microscopic morphometry. in the absence of cells of the vessel wall (cfm) the thrombus formation was inhibited by + % (+sd) after mg, +_ % after mg, + % after mg and +p % after mg dt-tx versus + % after placebo. the subclass of the large thrombi (= % of all platelets/thrombi but incorporating > % of the platelet mass) was much stronger affected by the dt-tx treatment: the mean area was reduced by +p % after rag, + % after mg, _+ % after mg, + % after mg and _+ % after mg dt-tx versus - + % after placebo. in the presence of cells of the vessel wall (smc) the overall thrombus formation was reduced by up to + % after only mg, + % after mg, +_ % after mg, _+ % after rag and -+ % after mg dt-tx versus +_ % after placebo. dt-tx , a molecule combining potent and specific th romboxane synthetase inhibition with prostaglandin endoperoxide/thromboxane a receptor antagonism, has been examined in healthy male subjects. collagen-induced platelet aggregation in platelet rich plasma prepared from venous blood was measu red photometrically before and up to hours after a single oral dose of , , , or mg dt-tx in a placebo-controlled, double-blind study. platelet aggregation was induced in the ex vivo samples by collagen in concentrations between . and p.g/ml to evaluate platelet aggregation in relation to the strength of the proaggregatory stimulus. the ecs , i.e. the concentration of collagen required for a half-maximal aggregatory response (defined as the maximal change of the optical density), was determined. in the placebo-treated control group, the mean ecso was + ng/ml collagen (+ se; n= ) before treatment. it then varied between + and +_ ng/ml collagen after treatment. the'ratio of the post-to the individual pre-treatment ecso values was . _+ . (n= ) at . h, . _+ . at lh, . _+ . at h, . - . at h, . + . at h and . + . at h. this indicates that the sensitivity of the platelets to collagen was not affected by the placebo treatment. oral treatment with dt-tx , however, strongly inhibited the aggregatory response of the platelets to collagen stimulation. the ecs -ratio was increased to a maximum of . the detection of endogenous opioids suggested the opinion that in case of the presence in the organism of a receptor for an exogenous substance there is probably a similar endogenous substance.the occurrence in the blood of persons, who were not treated with cardiac glycosides, of endogenous digoxin-like or ouabain-like [actors confirms that opinion. in our study we took up the research of other drug-like [actors in the blood serum of healthy people. in two hundered and twenty-five healthy volunteers (llom,ll f) non-smokers not receiving any treatment before or during the test and aged between ib and y(mean age y) the occurrence of drug-like [actors in blood serum was studied.the examinations were carried out with the use of the fluorescence-polarization-immunoassay (fpia)-tdabbott. th e presence of the following endogenous drug-like foctors in the blood serum was evaluated: quinidine,phenytoin, earbamazepine,theophylline, cyclosporineand gentamicin. the presence of endogenous phenytoin-like, theophyllinelike and cyclosporine-like [actors has been demonstrated. the drug-like [actors were not found in the case of quinidine ,carbamazepine and gentamicin. the phenytoin-like factor was found in , ~, theophylline-like [actor , ~ and cyclosporine-like [actor in , ~ of examined volunteers.the mean value of the drug-like [actors were as follow : phenytoin , ~ , pg/ml,theophylline , ~ o,ll pg/ml and cyclosporine , z , ng/ml. the supposition may be proponued that organism produces drug-like substances according to its needs. the acetylation and oxidation phenotypes were studied in healthy volunteers ( m, [) aged between ib and years (mean y) in the wielkopolska region in poland. the acetylation phenotype was studied with the use of sulphadimidine which was given in a dose of mg/kg b.w. per os.sulphadimidine was determined by a spectrophotometric method.the border value of m.r. was ~ in urine. the oxidation phenotype was studied with the use of sparteine which was given in a dose of , mg/kg b.w.per de. sparteine was determined by the gas chromatographic method in urine. if m~ was <hr these are values for intensive metabolism, if mr was , <mr( these are values for poor metabolism. the determinated phenotypes were bimodally distributed. our studies have short that healthy volunteers ( . ~) were slow and ( , ~) were fast metabolizers. intensive oxidation phenotype could be ascertained in volunteers ( , ~) and poor oxidation phenotype in volunteers ( , ~). these findings are comparable with the values typical for the caucasian population. the genes coding for rapid acety!ation were present in . % of the cases, genes coding for slow acetylation were detected in . %. the frequency of specific nat alleles were: r , . %; r , . %; sla, . %; slb, . %; slc, . %; s , . %; s , . %; and s , . %. there was no mutation found at nt. in cases ( . %) we have stated nat genoand phenotype concordance, but in cases ( . %), genotype and phenotype deviated from each other. seven individuals were genotyped as slas , one as slasle, and one as slas but were phenotypically rapid acetylators. one child was genotyped as r s a but proved a slow acetylator. we can confirm that in about % nat genotyping allows to properly predict nat phenotype. discrepancies may derive, i. a., from yet unknown mutations which we try to detect by dna-sequencing. several studies demonstrated that cardiac glycosides reduce diameter of epicardial coronary arteries. in patients (ffs) with coronary artery disease (cad) this vasoconstrictive side effect of glycosides may cause ischemic complications in the presence ot high-grade stenoses. this study evaluated quantitatively the effect of an i.v. injection of digoxin on diameter of epicardial coronary arteries in pts with cad. eleven grs (group ) were treated with aspirin mg/d, nine frs (group ) were treated with aspirin and rag nisoldipin hours prior to angiography. coronary angiograms were taken in identical projections before ( ) and , , and minutes after i.v. injection of . mg digoxin, at rain .ling of nitroglycerin (ntg) was given. the mean diameters of normal segments and the minimum diameters of stenoses were analysed with cms (medis, leiden). a total of normal and stenotic segments were analysed. figure shows the change of coronary diameter (% predrug-vahie; mean + se) for group i (left) and ii (right): ( -( -cyclohexylureido-phenoxy)- -hydroxy- -tert.-butylaminopropan; awd, dresden) is frequently used as a cardioselective g-adrenoceptor antagonist. talinolol metabolites in urine were detected by hplc and gc/ms with previously characterized reference compounds and were quantified by hplc with a normal phase silica column (oertel et al., br. j. clin. pharmacol. ( ) ) . less than one per cent of administered dose was recovered in urine as bydroxylated talinolol. other metabolites could be excluded. in serum after therapeutic doses talinolol metabolites were not traceable. however, in a suicidal patient with a parent compound serum concentration of ng/ml (manifold higher as after usual therapeutic dose) two hydroxy metabolites at concentrations near the detection limit ( ng/ml) were found. obviously only a small amount of the talinolol dose is metabolized in human. in this study the relation between structure, polarity and urinary recovery of the metabolites was examined. four different isomers of mono-hydroxylated talinolol were identified in urine of volunteers after a single dose of talinolol. eight isomers are possible: six hydroxy-cyclohexyl-compounds and two hydroxy-phenoxy-compounds. there was a low degree of specificity of biotransformation seen with respect to the point of attack on the cyelohexyl ring. hydroxylation of the phenyl ring could be excluded in human. using hplc methods very small quantities of reference compounds are sufficient to estimate and compare the polarity and hydrophily of similar compounds. long retention times in a normal phase system and short retention times in a reversed phase (rp) system are found for polar and hydropbilic compounds. the shortest retention times of all hydroxylated talinolol compounds in the normal phase system as well as the longest retention times in the rp system were found for the main metabolites -trans-and -cis-hydroxycyclohexyl-talinolol. accordingly, the main metabolites are the most polar and hydrophilic mono-hydroxylated talinolol isomers. lysine clonixinate is a non-steroidal antiinflarnmatory drug with marked analgesic effects. it is used for treatment of acute pain such as postoperative, dental or menstrual pain. we have determined the biosynthesis of thromboxane (tx)b z and prostaglandin (pg)f ~ in clotting whole blood ex vivo as well as collagen-induced platelet aggregation before and . , . (only txb and pgf ,~ biosynthesis), , . , , and hours after oral intake of mg lysine clonixinate. the results were compared with data obtained after oral intake of mg acetylsalicylic acid (asa). while both txb and pgf ,~ biosynthesis measured radioimmunologically were inhibited significantly (p< , and p< . , respectively) . hours, but not hours after lysine clonixinate, inhibition by asa was much greater and still highly significant (p< , ) after hours. the mean concentrations of lysine clonixinate and of asa inhibiting txb biosynthesis in vitro by % (ecs ) were . + . ~tm and . _+ . gm, respectively. aggregation of . ml aliquots of platelet-rich plasma induced by gg/ml collagen was inhibited by lysine clonixinate significantly (p< . ), but not completely for up to hours, while aggregation induced by gg/ml collagen was inhibited for up to hours only (p< . ). platelet aggregation induced by both collagen concentrations was inhibited by asa almost completely (p< . ) for at least hours. the ecs for in vitro inhibition of submaximal collageff-induced platelet aggregation was + btm for lysine clonixinate and + lam for asa. it remains to be investigated, whether other mechanisms than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate. the profile of side effects including only moderate inhibition of platelet function by lysine clonixinate as compared to asa might be an advantage with regard to its use as an analgesic. increased cell proliferation induced by ras-oncogenes is mediated by growth factors. growth factors bind to cell membrane surface receptors and activate tyrosine kinases leading to protein phosphorylation. this triggers posttranslational modification of the function of low molecular mass gtp binding ras proteins, e.g. p ras, including heterotrimetric g proteins. th~se proteins carry a c-terminal amino acid sequence, caax where c is cysteine, which is the target of isoprenylation by famesyl protein transferases (fpptases) and geranyl-geranyl protein transferases (ggptases). the amino acids of this sequence determine whether the ras protein is substrate for fptases or ggptases. this biochemical pathway is related to mevalonate metabolism, therefore, cholesterol biosynthesis shares several steps with isoprenylation. the active compounds include a series of isoprenoid inter-mediates, such as geranyl pyrophosphate (c- ), farnesyl pyrophosphate (fpp, c- ) and geranyl-geranyl pyrophosphate (ggpp, which are critical for cell proliferation. inhibitors of isoprenylation may (i) compete with fpp to its binding site on the alpha subunit of the enzyme, (ii) inhibit the zn protease of fptase beta subunit, and (iii) interfere with the binding of the caax peptide to its binding site on the beta subunit. the inhibitors include microbial products like manumycin, gliotoxin, acetylgliotoxin and pepticin-namins, fpp analogues, tetrapeptides and related peptidomimetics that may compete for the caax sequence, products of plant origin like limonene as well as synthetic compounds. all these compounds have been known to retard cell proliferation, and their effect can be abolished by mevalonic acid. inhibitors of hmg-coa reductase, such as lovastatin, which have been developed as antihypeflipidemic drugs, may also effective as anticancer agents. the recognition of the importance of mevalonate metabolism in cancerogenesis provides possibility to test simple chemical comp unds against tumor growth, and gives promise for pharmacological exploitation of inhibitors of isoprenylation and hmg-coa reductase inhibitors for cancer chemotherapy. dopamine (da) and epinine (epi, the active metabolite of ibopamine) are da-and ~-and ~-adrenergic receptor (r) agonists. to study whether da-r mediated effects can be separated from ~-and ~-ar effects we assessed in male volunteers effects of i.v. da and epi ( . ; . ; . and . ~g/kg/min for min each) on changes in serum prolactin (prl, da-r mediated), blood pressure (p) and heart rate (hr, ~-and ~-ar mediated), natriuresis (urinary excretion of sodium), diuresis (urine flow rate) and plasma noradrenaline (na) levels. in the . and . ~g doses da and epi did not affect psyst, pdiast and hr, but significantly decreased prl (da: from . ± . to . ± . ng/ml; epi: from . ±i.i to . ± . ng/ml, p<o. ). in the ~g dose da and epi increased psyst by mmhg, pdiast by and mmhg, and hr by and beats/min but further decreased prl. in addition, both da and epi significantiy increased diuresis and natriuresis; in contrast, only da, but not epi, dose-dependently increased plasma na ievels, an effect that was not inhibited by ~-ar antagonists. we conclude that in . and . ~g doses (plasma levels of - nmol/ ) oa and epl act only at da-r. in higher doses, however, da and epi can activate ~-and ~-ar whereby epi has a stronger ~-ar component than da. moreover, part of da-effects are indirect via release of endogenous na whereas epl-effects do obviously not include an indirect component. j. raderrnacher, r.mliller-bartels, f. barrels, h. baurngarten, k. thon, d. tsikas and j.c. fr ich, patients with essential hypertension were ireated with cicletanin ( rag/d) for weeks. compliance was verified by measuring urinary cicletanin excretion. before entering the study anti_hypertensive medication had been withdrawn for at least weeks (wash out phase). the study was preceeded by a placabo phase of week duration. blood pressure measurements and urine collections for measurements of , dinor- -keto-prostaglandin f a (a stable prostacyclin metabolite) were performed at the start and end of placebo phase, atter week of treatment with cicletanin and in monthly intervalls thereafter. cicletanin decreased systolic and diastolic blood pressure, the effect becoming significant at weeks. urinary excretion of the prostacyclin metabolite , dinor- keto pgfla.increased by %. hypertensive patients and control subjects (n= ; , dinor- -keto-pgfla: + pg/rnl] did not differ significantly in urinary excretion of , dinor- keto pgf ct. neither systolic nor diastolic blood pressure showed a significant correlation with , dinor- keto-pgf alpha excretion (systolic bp: r-- , , p-- , l; diastolic bp: r= , , p=l, ). these data show that cicletanin is effective in reducing blood pressure. increased prostacyclin production is probably not responsible for the observed reduction of blood pressure. in the past the realization of trials on the pharmacokinetics of nicergoline in human beings break down according to analytical difficulties. from animal studies with labelled compounds it is known, that nicergoline is rapidly absorbed from the gastrointestinal tract and two main metabolites (mdl, i-mmdl) can be detected.the mean elimination half-life for nicergoline is given with , h, for mdl - h and for i-mmdl with - h. the compound is excreted mainly by the kidney. due to known problems of the application of labelled drugs in human beings there are two approaches for the determination of these compounds: first the detection by monoclonal antibodies by using a ria-method, secondly a hplc-analysis. although there are promising in-vitro results of the immunological method, quantitative analyses of serum samples show deceptive results. the hplcassay reveales that in human serum samples after oral administration of nicergoline only the metabolites mdl and i-mmdl can be detected. further analytical development lower the limit of detection which should be i/i of c~x. with an improved hplc-method it will be demonstrated that only the measurement of the metabolite mdl is suitable to give sufficient results in pharmacokinetic trials and in studies on bioavailability with formulations of nicergoline. antiplatelet treatment with aspirin is well established as secondary prophylaxis after a transient ischemic attack or minor ischemic stroke, but the effect of aspirin treatment on the course of carotid atheroselerosis is unknown. we tried to investigate the effect of aspirin on the initial stages of carotid atherosc}erosis. patients were recruited from a prospective, randomized, double blind clinical trial to compare two doses of aspirin ( mg vs mg claity) with respect to restenoses after lower limb angjoplasty. of the patients admitted to the angioplasty trial, patients showing small carotid atheroma (< % lumen narrowing) were examined at entry and after year of aspirin treatment using a high-resolution ultrasound duplex system. disease progression and regression were defined by a change of maximal plaque area (as measured by longitudinal ultrasound sections) of more than standard deviations of the method. the change in plaque area was significantly different for the treatment groups: average plaque size remained'unchanged after treatment with mg aspirin daily but increased markedly after treatment with mg aspirin daily (p = . ). there were significantly more lesions in the -mg group showing progression than in the -mg group ( plaques [ %] vs plaques [ %], p = . ). ultrasonic disappearance of a lesion was observed only in the -mg group in cases ( soft plaques, ulcerative plaques, p = . ). the patients on mg aspirin who continued smoking during the study showed significantly more progression as compared to the non-smokers in the -mg group ( plaques [ %] vs plaques [ %], p = . ). the results of our study indicate that aspirin treatment slows carotid plaque growth in a dose-dependent fashion, with a dose of mg daily more efficient than mg a day. gentamicin is frequently applied in neonatal patients for the treatment of severe infections. the use is associated with high incidences of oto-and nephrotoxicity. ototoxicity occurs in - . % of the treated patients. it can be avoided, if the trough levels remain under p.g/ml between the administered doses. if the nephrotoxic effect, which occurs in - % of the treated patients, can also be reduced under these circumstances is not yet clear. recently it has been suggested to administer gentamicin only once daily in order reach the desired trough levels. this applies especially to children, in which the terminal half-life can be prolonged to approximately hours. this dosage regimen assures a sufficient effectivity due to the marked postantibiotic effect, but should reduce the incidence of the toxic effects. in order to test this hypothesis we perform a case-control study with neonatal children treated with gentamicin. the cases of nephrotoxicity during the previous months are identified from the hospital records. for these patients the plasma level time curves are obtained with the use of a population based pharmacokinetics programme, which considers the measured plasma concentrations and the administered doses. plasma concentrations were measured routinely in the process of therapeutic drug monitoring. from these curves the duration of times with concentrations lower than p.g/ml during the last days prior to the diagnosis are estimated. for the cases matched controls were identified according to age, age of gestation and other factors affecting the renal function e.g. comedication. different analyses are carried out according to the definition of exposure. the results wiij be discussed with regards to the modification of the dosage schedule for gentamicin and the application of population kinetics in order to reduce the adverse effects. economic evaluation of drug therapy is an important task of clinical pharmacology. drug costs amount to a relatively small percentage of total health care costs. some drugs may help to avoid more expensive therapeutic procedures. drugs with small therapeutic ratio need e therapeutic drug monitoring (tdm) to optimize therapeutic results and to avoid side effects. tdm may enhance the costs of drug therapy and the question arises, whether the additional costs are justified from the economic point of view. the aim of the study was to investigate the effectiveness of theophylline tdm in chronic obstructive pulmonary disease. methods: outpatients suffering from chronic obstructive pu[monary disease and receiving theophylline as a basic medication were included in this study. during the one-year observation period the parameters of pulmonary function were examined four times ( st, rd, th and th month) by the same physician and theophylline trough levels were estimated to individualize the dosage. the clinical outcome was assessed by intraindividual comparison. results: impaired parameters of pulmonary function were improved. hospital admissions were reduced. during the one-year observation period out of patients had hospital admissions with totally days. under the conditions of theophylline therapy without tdm during the year before of them had hospital admissions with days of stay in hospital. discussion: the results agree with a previous study on inpatients. there it was observed, that parameters of pulmonary function significantly were improved due to tdm-supported theophy[line therapy in comparison to a group receiving theophylline without tdm. despite the additional costs thaopylline tdm contributes not only to improved clinical outcome but also to decreased costs for the care of patients with chronic obstructive pulmonary disease. in cancer treatment arterial blood flow reduction by embolisation followed by intra-artedal chemotherapy may be advantageous by achieving high and prolonged drug concentrations in the tumor and lower systemic drug exposure. pharmacokinetics of mitomycin-c (mmc) were investigated in colorectal cancer patients (body weight: - kg) with liver metastases undergoing chemoembolisation. after hepatic artery branch catheterization and microspheres injection (polyvinylalcobel, mg mmc dissolved in ml physiological naci solution were infused in - rain, followed by ethiblock application. serum mmc-concentrations were determined from peripheral venous blood samples by reverse-phase hplcsystem with ultraviolet detection (c -column, elution: phosphate-buffer ( . m, ph: . )) : methanol, v:v= : , nm). pharmacokinetic parameters were computed using non-compartmental or two-compartment model analysis assuming linear kinetics (top fit . software). results: on the average, maximum serum concentrations were observed at tmax = . rain following the beginning of mmc-infusion and amounted to cmax = ng/ml (range: - ng/ml). mean steady state distribution volume was . i/kg with a central compartment volume of . i/kg. concentration-time curves could be descnbed by a distributional phase (t / = . min) followed by a terminal elimination phase (]' / = . min). mean total clearance amounted to . ml/min.kg. the area under the concentration time curve was pg.m - .min. conclusion: pharmacokinetic parameters for mmc-disposition in this study do not substantially differ from those reported for similar mmc-amounts administered as intravenous bolus. systemic mmc exposure does not seem to be reduced following the embolisation procedure used compared to intravenous application. pseudocholinesterase-activity (pche) is an important determinant of the elimination kinetics of drugs like mivacudum. therefore, the time course of pche was investigated in patients undergoing cardiosurgery with cardiopulmonary bypass (cpb) in normo-or hypothermia. anesthesia was induced and maintained with midazolam, propofol and fentanyl using mivacurium as a muscle relaxant. by starting cpb, i of pdming fluid were added to circulation volume. from collected blood samples, pche (u/i) was determined photometrically according to eiiman ( )(butyrylthiocholine, dithiobisnitrobenzoate, nm, ph= . , oc) . total plasma protein concentration (g%) was measured according to lowry, and the specific pche was calculated as the quotient of pche and protein concentration (u/g). serum hepadn-activity was determined in patients (berichreme heparin test kit). moreover, the influence of hepadn and aprotinin, both contained in the cpb pdming solution, on pche in vitro was examined using fresh serum from healthy volunteers (n= ). results: the induction of anesthesia had no significant influence on pche or protein concentration (p> . ). cpb induced a significant decrease of pche (- %)(p< . ) and protein concentration (- %)(p< . ) and a less pronounced numedcal reduction the specific pche (- %)(p> . ). the reduction of pche and protein concentration was not significantly affected by ending cpb (p> . ), and the values remained low over the remaining operation time. there was no significant difference in pche, measured at °c in vitro, or protein concentration between the normothermic and hypothermic group (p> . ). furthermore, there was no correlation between serum hepadn-activity and pche reduction. pche in the plasma of healthy volunteers was not significantly affected by either hepadn up to u/ml or apretinin up to u/ml (p> . ). conclusion: ( ) the concentration of the antitumor antibiotic mitomycin c (mmc), used in ophtalmic surgery for its antiproliferative effects, was measured in the aqueous humor of glaucoma patients undergoing trabeculectomy. sponges soaked with mmc-solution ( ul of mmc-solution . mg/ml: rag) were applied intraoperatively under the scleral flap for rain. to ul of aqueous humor were drawn with a needle min following the end of topical mmc-treatment. samples were assayed for mmc using a reverse-phase hplc-system with ultraviolet detection (c -column, elution: phosphate-buffer ( . m, ph: . ):methanol, v:v = : , nm). swabs were extracted in phosphatebuffer ( . m, ph: . ) before hplc-analysis. external calibration was used for mmc quantitetion. quantitation limit was ng/ml. in all aqueous humor samples mmc-concentration was below ng/ml. mmc in the swabs amounted to % of the mmc amount applied. conclusion: after intraoperetive topical application, mmc concentration in the aqueous humor of patients is very low. the substantial loss of mmc from the swabs used for the topical mmc-treatment suggests ( ) rapid systemic absorption of mmc and/or ( ) a loss through irngation of the operative field following topical mmc-application. institut fur pharmakologie und * klinik for augenheilkunde, universitcit k n, gleuelerstrasse , k n al a due to runaway costs of the national health service which are reflected as well in growing expenditures for drugs at the university hospital of jena investigation of indication related drug administration patterns becomes more and more interesting. this holds especially true for intensive care units (itu's) which are determined by similar high costs for technical equipment as for drugs ( ) although any economical considerations seem to be questionable due to ethical reasons ( ). over a month period indication related drug administrations of surgical itu's of the university hospital jena have been recorded and analyzed by using a pc-notebook. total expenditures for all included patients add up to dm . . regarding these drugs and blood products which caused % of total costs in . the leading substances ( antithrombin ill, human albumin %, prothrembine complex, ...) represent % of total costs including blood products, antibiotics and ig m endched intravenous immunglobine. therefore the indication of particulary these drugs became mere interesting for further investigations. already during the study actual discussions with the treating medical staff have been made leading to new developed therapy recommendations. providing same high standard of medical treatment a remarkable cost saving of some drugs by more cdtical and purposeful use could already be achieved as a first result. however, the results of the study underline impressivly the benefit of such investigations for improvement of drug treatment. the simple replacement of expensive drugs ( e.g. prothrembine complex ) by higher quantities of cheaper ones of the same indication group ( e.g. fresh frozen plasma ( )) does not necessarily mean less expenditures in all cases but may cause unsiderable side effects. ( ketokonazole is known to decrease pituitary acth secretion in vitro and inhibits adrenal ll-hydroxylase activity. to work out the clinical significance of both effects analysis of episodic secretion of acth, cortisol (f) and ll-deoxycortisol (df) was performed in patients with cushing's syndrome (cs) requiring adrenostatic therapy. methods : ketokonazole was started in ii patients with cs ( acth-secreting pituitary adenomas [cd], adrenal adenoma [aa] ). in of them ( cd, aa) blood samples were obtained for hours at i min intervals ( samples/patient) before and again under treatment (mean dose i mg/d, > weeks). hormone levels were measured by ria and secretion patterns analysed by means of pulsar, cluster and desade. patients were investigated only once because treatment was stopped due to side effects. results : the we conclude that the observed % increase of plasma acth and the % decrease of f/df ratio demonstrate that inhibition of adrenal li -hydroxylase activity is the primary mode of action of ketoconzole in vivo. even at high doses acth and f secretion patterns could not be normalized. the improvement of pain and swelling conditions by means of drugs is an important method of achieving an enhanced perioperative quality of life in cases of dentoalveolar surgery. in prospective, randomised, double-blind studies the influence of various concentrations of local anaesthetics and accompanying analgesic and antioedematons drugs was investigated in the case of osteotoimes. all of the studies were carded out according to a standardised study procedure. a comparison of the local anaesthetics articaine % mad articaine % (study ) demonstrated the superior effect of articaiue % with respect to onset relief on pain, period of effectiveness and ischaemia. recordings of the cheek swelling in the remaining studies were made both sonographically and with tape measurement, while the documentation of the pain was carried out by means of visual analogue scales on the day of operation and on the first and third post-operative days. tile perioperative, exculsive administration of x mg dexamethasone (study ) resulted in a significant reduction in the swelling ( %) while the exclusive administration of x mg lbuprofen (study ) was accompained by a marked decrease in pain ( %) but no significant reduction of swelling in comparison to the placebo group. the combination of x mg ibuprofen und mg methylprednisolone (study ) yielded a decrease in pain of . % and a reduction in swelling of %. a cdmparison between a mono-drug ibuprofen and a combination drug ass/paracetamol (study ) resulted in no significant difference in the reduction of swelling and pain and therefore highlighted no advantages for the combined drug. a mono-drug should therefore be given priority as an analgesic. the combinatton of ibuprofen und methylprednisolone offers the greatest reduction in pain and swelling. using the results of the randomised studies, a phased plan for a patietu-orietued, anti-inflammatory therapy to accompany dento-alveolar surgery is presented. in a placebo controlled study patients with congestive heart failure (nyka class ii) were treated orally for seven days with i mg ibopamine t.i.d, i subjects had a normal renal function (mean inulin clearance (gfr) ± , ml/min), i patients suffered from chronic renal insufficiency (gfr ± , ml/min; x ± sem). pharmacokinetic parameters of epinine, the maximum plasma concentration, the time to reach maximum plasma concentration and the area under the curve from to hours were unaltered in impaired renal function when measured on the first or on the seventh treatment day. however plasma concentrations in both groups were significantly higher on the first treatment day than after one week of ibopamine administration. in this context antipyrine clearance as a parameter of oxidative liver metabolism which might have been induced by ibopamine revealed no differences between placebo and ibopamine values. in conclusion kinetic and dynamic behaviour of ibopamine was not altered by impaired renal function. human protein c (hpc) is a vitamin k-dependent in the liver produced glycoprotein with anticoagulant properties. when active protein c splits the coagulation factors va and vuia by means of limited proteolysis (kisiel et al ) . its concentration in normal plasma is - lag/m[ i-ipc's biological importance became evident when a congenital protein c deficiency, which results in difficult recurrent thromboembolic diseases was discovered (griffin eta/ ) . the recognition of a congenital hpc deficiency, as wall as the connection between acquired protein c deficiency and the appearance of thromboembolic complications by means of highly accurate and sensitive ascertained methods is therefore of great practical importance for the clinic. murine monoclonal antibodies (moabs) against hpc were formed. antibody producing hybridomas were tested by an ,,indirect elisa" against soluble antigens. the plates were coated with purified hpc up to ng/ al. the peroxydase-system was used to identify antibodies the antibodies were tested with the remaining vitamin k-dependent proteins for cross-reactivity, as well as with hpc deficiency plasma for disturbances by other plasma proteins. the above described experiment represents a sensitive and specific method for measuring the hpc concentration with moabs. assessment of local drug absorption differences ("absorption window") in the human gastrointestinal tract is relevant for the development of prolonged release preparations and for the prediction of possible absorption changes by modification of gastrointestinal motility. current methods are either invasive and expensive (catheterization of the intestinum, hf-capsule method) or do not deliver the drug to a precisely defined localization. we evaluated the delay of drug release from tablets coated with methacrylic acid copolymer dissolving at different ph values as an alternative method. three coated preparations of caffeine tablets (onset of drug release in in vitro tests at ph . , . and . ) and an uncoated tablet (control) were given to six healthy male volunteers in a randomized order. caffeine was used because of its rapid and complete absorption and good tolerability. blood samples were drawn up to h postdose (coating ph . up to h postdose), and caffeine concentrations were measured by hplc. auc, time to reach measurable caffeine concentrations (tia~), tr, ax, cmax and mean absorption time (mat) values for coated preparations were compared to the reference tablet (mean + sd of n= ): the relative bioavailibility for the coated preparations did not differ from the reference, suggesting complete release of caffeine. all coatings delayed caffeine absorption onset. the tlag for the ph . preparation suggests that release started immediately after the tablet had left the stomach. the mean delay of . h for the ph . coating was highly reproducible and should reflect small intestine release. the ph . coating delayed absorption to the highest extent, however the drug was probably released before the colon was reached. there is evidence that nitric oxide (no) plays a role in cardiovascular disease like hypertension, myocardial ischemia and septic cardiomyopath.y. no stimulates the guanylyl cyclase leading to an increase m cgmp content we investigated by immunoblotting the expression of the inducible nitric oxide synthase (inos) in left ventricular myocardium from failing human hearts due to idiopathic dilative cardiomyopathy (idc, n= ), ischemic cardiomyopathy (icm, n= ), beeker muscular dystrophy (n= ) and sepsis (sh, n= ) compared to non-failing human hearts (nf, n= ). cytokine-stimulated mouse macrophages were used as positive controls sds-polyacrylamide gel electrophoresis ( . %) was perfomed with homogenates of left ventricular myocardium and mouse macrophages respectively. proteins were detected by enhanced chemiluminescence using a mouse monoclnal antibody raised against inos. furthermore, we measured the cgmp content in these hearts by radioimmunoassy. a band at about kda was observed in two out of three hearts from patients with sepsis and in stimulated mouse macrophage~ no inos-protein expression was detected in either non-failing human hearts (n= ) or failing human hearts due to idc, ihd or bmd. in ventricular tissue from patients with sepsis cgmp content was increased to % ( + fmol/mg ww, n= ) compared to non-failing hearts ( % or + . fmol/mg ww, n= ). in left ventricular tissue tissue from patients with heart failure due to idc, ihd and bmd cgmp content did not differ from that in non-failing hearts. it is concluded that an enhanced inos protein expression may play a role in endotoxin shock, but is unlikely to be involved in the pathophysiology of end-stage heart failure due to idc, ihd and bmd. (supported by the dfg.) nitric oxide (no) has been shown to be a major messenger molecule regulating blood vessel dilatation, platelet aggregation and serving as central and peripheral neurotransmitter; furthermore no is a crucial mediator of macrophage cytotoxicity. no production can be assessed reliably by determination of its main metabolites nitrite and nitrate in serum, reflecting no synthesis at the time of sampling, or in h urine, reflecting daily no synthesis. farrell et ai. (ann rheum dis ; : ) recently reported elevated serum levels of nitrite in patients with rheumatoid arthritis (ra). we report here total body nitrate production and the effect of prednisolone in patients with ra. nitrate excretion in h urines of patients with ra as defined by the revised criteria of the american rheumatism association was measured by gas chromatography at times: first before start of a antiinflammatory therapy with prednisolone, when the patients had high inflammatory activity as indicated by mean crp serum concentrations of + sd mg/i and elevated esr with a mean of ]: after hour. secondly - weeks after start of prednisolone therapy in a dosage of . mg/kg body weight, when the patients showed clinical and biochemical improvement (crp + mg/i, p< . , esg + , p< . , two-tailed, paired t-test). for comparison h urines from healthy volunteers were obtained. before start of predniselone therapy the urinary nitrate excretion in patients with ra (mean + sd p.mol/mmol creatinine) was more than twofold higher (p< . , twoaailed unpaired t-test) than in healthy volunteers ( + ~tmol/mmol creatinine). the urinary nitrate excretion decreased significantly (p< . , two-tailed, paired t-test) to + i.tmol/mmol creatinine under therapy with prednisolone, when inflammatory activity was reduced considerably. despite the decrease the urinary nitrate excretion was still twc, fold higher (p< . , two-tailed, unpaired t-test) in patients with ra than in the control group. our data suggest that the endogenous no production is enhanced in patients with ra. furthermore the results indicate that this elevated no synthesis could be reduced in accordance with suppression of systemic inflammation by prednisolone therapy. but now as ever the physicians are entitled to prescribe drugs which have to prepare in a pharmacy for a particular patient. little information is available on the frequency and patterns of these prescriptions. we had occasion to analyse the prescriptions of drugs which were prepared in pharmacies in north thuringia (east germany) from october to december at the expense of a large health insurance company (allgemeine ortskrankenkasse). the selected pharmacies are loealised in cities. we found prescriptions of drugs made up in pharmacies among a total number of reviewed drug prescriptions. this is . % of the total. most of these prescriptions were performed by dermatologists ( . %), general practitioners ( . %), paediatrists ( . %) and otolaryngologists ( . %). according to this, the most frequently prescribed groups of drugs were dermatics enteric eoated tablets with nag and nag acetylsalicylic acid (asa) have been developed wluch should avoid the known gastrointestinal adverse events by a controlled drug release mainly in the duodenum after having passed the stomach. a -way cross-over study in healthy male subjects, aged from - years, was conducted to investigate the pharmacokinetics, bioavailability, safety, and tolerance of asa and its metabolites salicylic acid and salicylurie acid following enteric coated tablets in comparison with plain tablets. asa and its metabolites were determined by a sensitive, specific, and validated hplc method. pharmacokinetic parameters were determined by non-compartreental analysis. bioequivalence was assessed by % confidence intervals. following the admimstration of enteric coated tablets, a delayed absorption can be observed for both the mg dose and the rag dose. this is likely due to a delayed release of the active substance from the enteric-coated tablets in the small intestine arer gastric passage. considering the mean residence times (mrt), there is a difference of at least . h following the enteric coated tablets compared to the plain tablets for asa and the two metabolites measured• this difference represents the sum of residence time in the stomach plus the time needed to destroy the coating of the tablet when it left the stomach• in general, the maximum observed concentrations of both enteric coated formulations occurred - h post dose. the pharmacokinetics of a novel immunoglobulin g (lgg) preparation (bt , biotest, dreieich, frg) have been determined in healthy, male anti-hbs-negative volunteers. for this preparation only plasma from hiv-, hbv-and hcv-negative donors was used, the quality control for the product was in accordance with the ec-guideline for virus removal and inactivation procedures. each volunteer received a single, intravenous infusion of ml bt containing g igg and anti-hbs > , iu. anti-hbs was used as a simply measurable and representative marker for the igg. blood samples for determination of anti-hbs (ausab eia, abbott, frg) were drawn before and directly after the infusion, after , , , and hours, on day , , , , , , , , , and . additionally, total protein, igg, iga, igm and c /c complement were measured and blood hematology and clinical chemistry parameters determined. the phar~gacokinetic parameters of anti-hbs were calculated using the topfit ~" pc program assuming a -compartment model. pharmacoeconomic evaluations (pe) describe the relationship between a certain health care input (costs) for a defined treatment and the clinical outcome of patients measured in common natural units (e.g. blood pressure reduction in mmhg), quality of life (qol) gained, lifes saved or even in money saved due to the improvement in patients functional status. this implies that the efficacy of a treatment has been measured and proven in clinical trials. in addition, in order to transfer data obtained in clinical trials to the clinical setting, an epidemiological database for diseases and eventually drug utiiization may be required. the evaluation of the efficacy depends on the disease to be treated or prevented and the mode of treatment. for acute, e.g. infectious diseases, the endpoint can be defined easily by the cure rate, but for pe the time (length of hospital stay) and other factors (e.g. no. of dally drug administrations) have to be considered. in the case of chronic diseases, e.g. hypertension or hypercholesterolaemia, surrogate endpoints (blood pressure or serum cholesterol reduction) and information on side effects may be acceptable for the approval, but cannot be used for a meaningful pe. the latter should include the endpoints of the disease, i.e. cardiovascular events (requiring hospitalisation and additional treatment) and mortality. furthermore, the qol has to be measured and considered for chronic treatment. several questionaires have been developed to measure the overall qol or the health related qol. especially the latter may be a more useful tool to detect mad quantify the impact of a treatment on qol. combining the clinical endpoint mortality and qol by using qalys (quality-adjusted lifeyears) may be a useful tool to determine the value and costs of a given drug treatment but cannot be applied to all treatments under all circumstances. sorbitol was used as a model substance to investigate the dynamics of the initial distribution process following bolus intravenous injection of drugs. to avoid a priori assumptions on the existence of well-mixed compartments data analysis was based upon the concept of residence time density in a recirculatory system regarding the pulmonary and systemic circulation as subsystems. the inverse gaussian distribution was used as an empirical model for the transit time distribution of sorbitol across the subsystems, distribution kinetics was evaluated by the relative dispersion of transit (circulation) times. the distribution volumes calculated from the mean transit times were compared with the modelindependent estimate of the steady-state volume of distribution. kinetic data and estimates of cardiac output were obtained from patients after percutaneous transluminal coronary angioplasty. each received a single . g iv bolus dose of sorbitol. arterial blood samples were collected over hours. while the disposition curve could be well fitted by a tri-exponential function the results indicate that distribution kinetics is also influenced by the transit time through the lungs, in contrast to the assumption of a wellmixed plasma pool underlying compartmental modelling. a karit@ "bu£ter" is used traditionally in west afr%can manding colture as a cosmetic to protect the skin against the.sun. gas chromatography was used to analyze the ingredients of karit@ butter from guinea. we found % palmitic acid, % stearic acid, % oleic acid and % linoleic acid and . % of other fatty acids with higher chain lengths like arachidonio acid. some of these are essential fatty aclds (vitamine f). furthermore karit@ contains vitamine a and d as well as triterpene alcohols and phytosterines. an original extract was used to prepare a skin cream. this preparation was tested in volunteers ( women, men; age - y.). the cream contained at least % karit@, glycerol, emulsifiers and no preservative agent except for sorbic acid. of the volunteers very well tolerated the cream and thought it effective. the skin became more tender and elastic. good results were obtained when the volunteers suffered from very dry skin. two of them who were known to be allergic against the most available skin creams had no problems in using our karit cream. pure karit@ butter was used for four months to treat an african infant with neurodermitis. after this time the symptoms had markedly improved whereas previous therapy trials with other usual topical medicaments had been unsuccessful. these pre-studies had shown that dermatologic preparations containing karit# may be a good alternative in the treatment of therapyreslstent skin diseases and may in some cases be able to replace eorticoid treatment. ) and a low molecular weight heparin preparation (fragmin ~, iu/kg bodyweight s.c.) on coagulation and platelet activation in vivo by measuring specific coagulation activation peptides [prothrombin fragment + (f + ), thrombin antithrombin iii complex (tat), -thromboglobulin (~-tg)] in bleeding time blood (activated state) and in venous blood (basal state). in bleeding time blood, r-hirudin and the heparin preparations significantly inhibited the formation of both tat and f + . however, the inhibitory effect of r-hirudin on f + generation was short-lived and weaker compared to ufh and lmwh and the tat/f + ratio was significantly lower after r-hirudin than both ufh and lmwh. thus, in vivo when the coagulation system is in an activated state r-hirudin exerts its anticoagulant effects predominantly by inhibiting thrombin (lla), whereas ufh and lmwh are directed against both xa and ila. a different mode of action of ufh and lmwh was not detectable. in venous blood, r-hirudin caused a moderate reduction of tat formation and an increase (at hour) rather than decrease of f + generation. formation of tat and f + was suppressed at various time points following both ufh and lmwh. there was no difference in the tat/f + ratio after r-h[rudin and heparin. thus, a predominant effect of rhirudin on ila (as found in bleeding time blood) was not detectable in venous blood. in bleeding time blood, r-hirudin (but neither ufh nor lmwh) significantly inhibited ~-tg release. in contrast, both ufh and lmwh caused an increase of ~-tg hours after hepadn application. our observation of reduction of platelet function after r-hirudin compared to delayed platelet activation following ufh and lmwh suggests an advantage of r-h[rudin over heparin, especially in those clinical situations (such as arterial thromboembolism) where enhanced platelet activity has been shown to be of particular importance. the human cytochrome p isoform cyp a determines the level of a variety of drugs metabolized by the enzyme, including caffeine (ca) and theophylline (th). more than compounds are potential or proven inhibitors of this enzyme. some of them were reported to be substrates or inhibitors to cyp a in vitro, ethers caused pharmacokinetic interactions with drugs metabolised by cyp a . we characterized a series of these compounds with.respect to their effect on cyp a in human liver microsomes in relation to-published pharmacokinetic interactions in vivo. cyp a activity in vitro was measured as ca -demethylation at the high affinity site in human liver microsomes, using rain incubation at °c with - jm caffeine, an nadph generating system, and inhibitor concentrations covering . orders of magnitude. apparent kr values were estimated using nonlinear regression analysis. for inhibitory effects on cyp a activity in vivo, the absorbed oral dose causing % reduction in ca or th clearance (edso) was estimated from all published interaction studies using the emax model. %)i followed by disinfectants ( . %)r ointments ( . %) and solutions ( . %) were the most frequent drug forms %) or german ( . %). our results show that even now drugs prepared trend analysis of the expenses at the various departments may be a basis for a ratio-hal and economic use of the drug budget. total drug expenses amounted to mill. dm in . s milldm ( %) were used in surgical departments with intensive care units (icu) (general surgery, kardiovascular surgery, neurosurgery, gynecology, anaesthesiology) of wtfich % are needed by the icu and % in the operating rooms. surgical departments without scu but similar patient numbers (ophthalmology, ent, orthopedics and urology) get only % of the budget ( % needed for the operating rooms). the medical departments spent s mill.dm of which icu needs only % whereas the oncology (oncu) and antiinfective units uses more than %• similar relation could be seen in the child hospital ( . milldm, %) where % were spent for icu and % for oncu. the departments of dermatology and neurology get %, the depart-merits of radiology, nuclear medicine and radiation therapy only % of the budget. antiinfective drugs (antibiotics, antimycotics, virustatics) are most expensive ( % of budget) followed by drugs used for radiological procedures ( %) sncreasing the knowledge about the costs of medical items and the rational and economical use may stop the overproportional increase of the drug budget the mostly used : ) and a -fold higher efficiency than the r-form the elimination of the talinolol enantiomers was studied in healthy volunteers (age: - years, body weight: - kg) given a single oral dose ( mg) or an intravenous infusion ( rag) of the racemi c drug. three volunteers were phenotypically poor metabolisers and nine were extensive metabolisers of the debrisoquine-type of hydroxylation. the r-and senantiomers of talinolol were analysed in urine by a hplc method after enantioselective derivatisation. the concentrations of the enantiomers within every sampling period as well as the amounts of s-and r-enantiomer this corresponds to a s/r-ratio of , + , . the mean total amount (= s-+ r-enantiomer) eliminated was on average % &the administered dose. after oral administration _+ % of the dose were eliminated within h. the amounts of talinolol enantiomers recovered were equally (senantiomer: _+ gg the ratios of s-to r-concentrations at every sampling interval and of every volunteer were assessed between , and , (mean: , after infusion and , after oral administration, respectively) medizinische fakult~t carl gustav cams, teelmische university, t, fiedlerstr nitric oxide (no), synthesized by the inducib]e form of no synthase, has been implicated as an important mediator of-specific and non-specific immune response, little is known about the in vivo synthesis or no in inflammatory joint diseases. therefore we have studied the excretion of the major urinary metabolite of no, nitrate, in rats with adjuvant arthritis, a well established model of polyarthritis in addition we assessed the urinary excretion of cyclic gmp, which is known to serve as second messenger for the vascular effects of no, synthesized by the constitutive form of no synthase, affecting blood vessels, plate]et aggregation and neurotransmission, in h urines of male sprague daw]ey rats at day after induction of adjuvant arthritis we measured nitrate excretion by gas chromatography and cyclic gmp by radioimmunoassay. for contro] we determined the same parameters in h urines of non-arthritic rats of the same strain and age, we found a significant (p < , two-tailed, unpaired t-test), more than -fo]d increase of urinary nitrate excretion in arthritic rats (mean ± sd pmo]/mmol creatinine) as compared to non arthritic rats ( _+ izmot/mmo] creatinine). urinary cyclic gmp excretion was slightly, but not significant]y lower in arthritic rats ( ± nmol/mmol creatinine) than in controls ( ± nmo]/mmo] creatinine).there were no major differences in food or water intake which cou]d account for these results. the increased urinary nitrate excretion accompanied by normal cyclic gmp excretion suggests that no production by the inducible form of no synthase is enhanced in rats with adjuvant arthritis institute of c]inica] pharmacology~ hannover medical school, d- hannover, germany and *research center gr@nentha] gmbh, zieg]erstr , d- aachen, germany background: pge has been shown to be efficacious in the treatment of critical leg ischemia. despite of an almost complete first pass metabolism in the lung the clinical effects of intraarterial and intravenous pge do not differ significantly. in addition, it is not fully understood which of the various pharmacological actions of pge is the main factor; by most authors, however, it is thought to be the increase of cutaneous and muscular blood flow. by means of [ - ]-h -pet, we studied muscular blood flow (mbf) of the leg in patients with peripheral arterial disease comparing intraarterial and intravenous pge . patients and methods: patients ( f, m; mean age y) with pad were studied, ( atherosclerosis, thromboangiitis obliterans). at the first day, pg pge were infused intraarterially within minutes; pet scanning of the lower leg was performed at minutes , und . at the following day, pg pge were infused intravenously within hours; pet scanning was performed at minutes , , and . results: in the infused leg the increase of mbf caused by intraarterial pge averaged + % at minute and _ % at minute ; in the not infused leg there was no effect. the increase rate in the infused leg was highly variable but did not correlate with sex, age, disease or clinical outcome. for intravenous pge the change of mbf at any time averaged almost %. conclusion: unlike intraarterial pge , intravenous pge does not increase the muscular blood flow of the leg. a comparable clinical effect provided, increase of muscular blood flow may not be considered the main way of action of pge in critical leg ischemia. eslrogen(er) and progesterone(pr) receptor status as well as lymph node involvement are important factors in predicting prognosis and sensitivity to hormone and chemotherapy in patients with breast cancer. prognostic relevance of ps -protein, egfr and cathepsin d is currently under debate. especially ps and egfr expression appears to provide additional information regarding the responsiveness of the tumour tissue to tamoxifen. the aim of the present study was to investigate the relationships between these parameters and established prognostic factors in breast cancer. in a prospective study ps and cathepsin d were assayed immunoradiometricauy in the tumour cytosol of patients, egfr was measured by elisa. relating the level of these factors to the lymph node involvement, menopausal status as well as turnout size, no significant association could be established. jn our findings er and pr are significantly correlated with the expression of ps but none is correlated with the cathepsin d status. egfr was shown to be inversely correlated with the content of er. a significant association between cathepsin d and ps could be established in patients with early recurrence. at a median follow-up of - months, recurrence was more common in patients with tumours having negative status for ps , independent of receptor status. in conclusion, because of the relative independence on the er and pr status and other prognostic factors and the influence on the recurrence behaviour, demonslrated here, and their role in promoting tumour dissemination and changing hormone therapy sensitivity, all three factors represent markers of prognostic relevance.deparlancnts of clinical pharmacology l, nuclear medicine and surgery ,pharmacoeconomic studies, conducted either separately from or together with clinical trials are increasing in both number and meaning. in a period of limited health care budgets, political and medical decision makers alike run the risk of accepting the results of such studies without critical reflection. careful evaluation of those studies by state-of-the-art methods is one way out of the trap. another could be to refer to ethical considerations. the problem in this context is, that the discussion concerning ethical aspects of pharmacoeconomic research, at least in europe, is just in its beginning. therefore, no widely accepted standards are available. but they are essential to answer four main questions: . who should perfom a pharmacoeconomic study? . which objectives should be considered? . what kind of study should be performed (e. g. cost-effectiveness, cost-utility, cost-benefit analysis)? . which consequences will be drawn from the results?based on the case study-orientated "moral cost-benefit model" (r. wilson, sci. tech. human values : - , ) , a three-step decision and evaluation model is proposed to handle bioethical problems in pharmacoeconomic studies: . moral risk analysis . moral risk assessment . moral risk management. possible practical consequences for decision making in research policy, study design and assessment of results are discussed. hirudin is the most potent known natural inhibitor of thrombin and is presently gaining popularity as an anticoagulant since recombinant forms have become available. the aim of the present study was to compare platelet aggregation, sensitivity to prostaglandin e (pge ) and thromboxane a (txa ) release in r-hirudinized and heparinized blood. platelet aggregation was measured turbidimetrically using a dual channel aggregometer (labor, germany) in blood samples of healthy volunteers anticoagulated with r-hirndin w (behring) and hepatin ( gg/mi blood each). aggregation was induced by arachidonic acid (aa; . , . and . ram) and adp ( . lam). pge in concentrations , and ng/ml was used. plasma txb content was measured by gas chromatography/mass spectrometry. this study showed a significantly lower a.a-induced platelet aggregation in r-hirudinized plasma. three minutes after the aggregation induction by . mm aa the plasma txb concentration was ng/ml in blood anticoagulated with rhimdin and . ng/ml in heparin-anticoagulated blood. the extent of the adp-induced aggregation was nearly the same in rhimdinized and heparinized plasma. platelet sensitivity to pge was significantly higher in r-hirudinized blood. thus, aa-induced platelet aggregation is significantly lower and sensitivity to pgei higher in r-himdin-anticoagulated blood in comparison with beparin-anticoagulated blood.university of tartu, puusepa str. , tartu ee , estonia anaemia has been reported in renal transplant (ntx) recipients treated with azathioprine (aza) and angiotensin converting enzyme-inhibitors (ace-i). an abnormal aza metabolism with increased -thioguanine nucleotide (tgn) levels in erythrocytes is a possible cause of severe megaloblastic anaemia (lennard et al, br j clin pharmaco ). methods: ntx patients receiving aza ( , _+ , mg/kg/d), prednisolone ( , + , mg/kg/d) and enalapril (ena) ( , + , mg/kg/d) for more than months were studied prospectively. blood samples were taken before and h after administration of aza on visits during ena treatment and weeks after ena had been replaced by other antihypertensives (x). tgn in erythrocytes, -mercaptopurin (mp) and -thiouric acid (tua) in h post dose plasma (p.) und h urine (u.) samples were analyzed by hplc using a mercurial cellulose resin for selective absorption of the thiol compounds. pharmacodynamic variables were hemoglobin (hb), erythropoietin (epo) and creatinine clearance (ci ace~,lcholine plays an important role in regulating various functions in the airway's. in human lung less is known about regional differences in cholinergic innervation and about receptor-mediated r%m.flation of acetylcholine release. in the present study the tissue content of endogenous acetylcholine and the release of newly-synthesized [~h]acetylcholine were measured in human lung human tissue was obtained at thoracotomy from patients with lung cancer moreover, in isolated rat tracheae with intact extrinsic vagal innervation possible effects of g__-adrenoceptor agonists on evoked ph]acctylcholine release were studied. endogenous acetylcholine was measured by hplc with ec-detection; evoked ph]acetylcholme release was measured after a preceding incubation of the tissue with [~h]choline. huma n large (main bronchi) and small (subsegmental bronchi) airways contained similar amounts of acetylcholine ( pmol/ mg), whereas significantly less acetylcholine was found in lung parenchym ( pmol/ mg). release of [ h]acetylcholine ,,,,'as evoked in human bronchi by transmural electrical stimulation (four s trains at hz). oxotremorine, an agonist at muscarine receptors, inhibited evoked [~hiacetylcholine release indicating the existence of neuronal inhibitor ' receptors on pulmona~ parasympathetic neurones. scopolamine shifted the oxotremorine curve to the right suggesting a competitive interaction (pa value: : slope &the schild plot not different from unity) however, a rather sluggish schdd plot was obtained for pirenzepine. scopolamine but not pirenzepine enhanced evoked [ h]acetylcholine release. the present experiments indicate a dense cholinergic innervation in human bronchi; release of aceu, lcholine appears to be controlled by facilitatory and inhibitou' nmscarinc receptors. in isolated, mucosa-intact rat tracheae isoprenaline ( nm) inhibited [~h]acetylcholine release evoked by preganglionic nerve stimulation isoprenaline was ineffective in mucosa-denuded tracheae or in the presence of indomethacin thus, adrenoceptor agonists appear to inhibit acetylcholine release in the airways by the liberation of inhibitoiy prostanoids from the mucosa. the occurrence of the non-enzymatic reactions between glucose and structural proteins is well known (vlassara h et al. ( ) lab invest : - ) . the reaction between proteins and fructose (i.e. fmctation), however, can also occur. like glucose-protein adducts the fructose analognes are able to form so-called advanced glycation endproducts (age). the inhibition of early and advanced products of fmctation may be ilnportant for the prevention of diabetic late complications (mcpherson jd et al. ( ) biochemistry : - . we investigated the in vitro fmctation of human serum albumin (hsa) and its inhibition by selected drugs. hsa was fmctated by incubation with mmol/ fructose in . i mol/l phosphate buffer, ph= . .,at ° c for days. the rate of fmctation was measured by the following methods: -a colorimetric method based on deglycatien of glycated, proteins by hydrazine (kobayashi k et ai.( ) bioi pharm bull : - ), -affinity chromatography with aminophenyl-boronate-agarose, -fluorescence measurement for the delermination of age we used aminoguanidine, pcnicillamine, captopril and alpha-lipoic acid( mmol/ ) to study the inhibition of hsa fmctation. after three weeks incubation the formation of early glycation products was inhibited by aminogalanidine ( %) and captopril ( %) whereas penicillamine and alpha-lipoic acid showed minimal inhibition. aminognanidine inhibited the formation of age by %, penicillamine by %, alpha-lipoic acid by % and captopril by %. these results may suggest a potential use of the investigated drags in the prevention of the formation of protein-fructose addncts. key: cord- -mdej nhj authors: kumar de, amit; datta, sriparna; mukherjee, arup title: application of an amine functionalized biopolymer in the colonic delivery of glycyrrhizin: a design and in vivo efficacy study date: - - journal: sci pharm doi: . /scipharm. - sha: doc_id: cord_uid: mdej nhj in our current study, a newer amine functionalized guar gum derivative was studied for its efficacy in colonic drug delivery. glycyrrhizic acid mono-ammonium salt was used as the model drug. drug-loaded microparticles were formulated by ionic crosslinking using sodium tripolyphosphate. the scanning electron microscopic study revealed spherical particles of sizes from . ± . μm to . ± . μm. the ft-ir studies presented a possible interaction between the drug and the polymer. the drug was encapsulated in amorphous form as observed from the powder x-ray diffraction studies. a cumulative drug release study was carried out in simulated gastric, intestinal, and colonic fluids. the cumulative drug release studies presented a burst release followed by a sustained release of the drug in simulated colonic fluid containing rat cecal contents. the drug-polymer ratio was optimised using a ( ) factorial design by taking the amounts of glycyrrhizic acid (x( )) and guar gum alkyl amine (x( )) as the independant variables. the percent cumulative drug release at mins (q( )), mins (q( )), and at , mins (q( )) were considered as the dependant variables. the efficacy of the optimized formulation was studied in a , , -trinitrobenzene sulfonic acid-induced rat colitis model. the tissue’s nitric oxide, malondialdehyde, and myeloperoxidase activities were found to be much lower in the microparticle-treated group compared to free drug-treated group. the histology of the colonic tissue from the treated group of animals revealed almost no infiltration of inflammatory cells in the tissue for the microparticle-treated group of animals. the synthesized amine derivative of guar gum was found to be better in vitro with a better in vivo efficacy in the colonic delivery of glycyrrhizic acid monoammonium salt and can be considered as a newer modified biopolymer for colonic drug delivery. glycyrrhizic acid monoammonium salt and can be considered as a newer modified biopolymer for colonic drug delivery. inflammatory bowel disease is a common disease among urban populations [ ] [ ] [ ] [ ] . drugs prescribed for the treatment of this disease manifest a large number of side effects. a localised delivery is therefore suitable for the treatment of this disease. the development of polymer-based microspheres, microcapsules, and microbeads are currently the focused area of research for intestinally targeted drug delivery [ ] . this also prevents a sudden unwanted higher concentration of the drug in the tissue and maintains controlled release of the drug at the target site. polysaccharides are mainly used in the design of oral drug delivery devices. the increased adhesion of particles in the thick mucosal zones of inflammation or ulceration is an effective physiological parameter that can be exploited for targeted delivery of the drug encapsulated in the microcarriers [ , ] . in inflammatory bowel disease, microparticulate drug carriers are expected to accumulate at the sites of inflammation in the colon. due to effective adhesion to colonic mucosa, these carriers will have enough time to deliver their drug load to the sites of inflammation in the colon. guar gum obtained from guar flour is a polysaccharide commonly used for designing colontargeted drug delivery devices [ , ] . guar gum is metabolised by the colonic microflora that releases its contents specifically to the colon. a newer amine derivative of guar gum has been synthesised from commercially available guar gum. the derivative retains the biological properties of the mother polysaccharide and shows additional properties of the amine group. this makes the polymer positively charged. in our study, this newly synthesised derivative was utilized for the preparation of microparticles. preparation of guar gum microparticles by the ionic gelation technique using metal ions and glutaraldehyde has been reported earlier [ ] [ ] [ ] . in our current study, we used sodium tripolyphosphate as the crosslinker with guar gum alkyl amine. glycyrrhizic acid is a triterpene saponin found from the root extract of glycyrrhiza glabra [ ] . glycyrrhizin is found to have anti-inflammatory, antiulcer, anti-allergic, immunemodulating, and antiviral properties [ ] [ ] [ ] [ ] [ ] . colonic delivery of microencapsulated glycyrrhizic acid can be used to ameliorate the severity of colitis due to its anti-inflammatory [ , ] properties and its efficacy in the amelioration of peptic ulcer and inflammatory bowel disease [ , ] . in our study, glycyrrhizic acid monoammonium salt (ga) was used as the model drug. the prepared microparticles were characterised using the scanning electron microscopy (sem), fourier transform infrared spectroscopy (ft-ir), and powder x-ray diffraction (pxrd) studies. the entrapment efficiency and in vitro drug release were studied in simulated gastric, intestinal, and colonic fluids containing rat cecal contents. the drug-polymer ratio was optimized using a factorial design model. the in vivo efficacy of the microparticles was studied in a , , -trinitrobenzene sulfonic acid-induced rat model of colitis. the efficacy of the formulations was determined following a clinical score system [ ] . an increase in the number of neutrophils, natural killer cells, mast cells, and regulatory t-cells has been reported to play an important role in the pathophysiology of inflammatory bowel disease [ ] . myeloperoxidase (mpo) is an important constituent of inflammatory cells, mainly the neutrophils [ ] . the estimation of tissue mpo-content could be an efficient tool to measure the extent of colonic inflammation. in tnbs-induced colitis, the tissue no was found to increase [ , ] . malondialdehyde (mda), a secondary product of lipid peroxidation, was considered as an important parameter to study the extent of tissue damage [ ] . analysis of all three inflammatory parameters was carried out to study the efficacy of the ga microparticles in the amelioration of colonic ulcer. the results were corroborated by the histological studies of the colon tissue of the normal, control, and treated groups of animals. glycyrrhizic acid monoammonium salt (purity = . %) (glycyrrhizin; ga), sodium tripolyphosphate (tpp), and dialysis tubing d (mw cut off , kd) were purchased from sigma aldrich india ltd. guar gum (mannose to galactose ratio : ) and all other reagents used for the synthesis of the alkyl amine derivatives were purchased from merck india ltd. phosphoric acid of hplc quality and the solvents used for chromatography of hplc grade were purchased from spectrochem india limited. male sprague-dawley rats ( weeks old, - g) were purchased from the central ayurvedic research institute (kolkata, india) and kept on a normal diet with water ad libitum. the animals were housed in polymer cages at ± ºc temperature, ± % relative humidity, and a -hour light-dark cycle. they were acclimatized for seven days before any treatment. guar gum alkyl amine (ggaa) was synthesised from guar gum following a single-step method mentioned elsewhere [ ] . the product was washed several times with an isopropanol-water mixture in order to remove unwanted chemicals and reagents. the product was air-dried. the final product was purified by dialysis against millipore water for hours. an aliquot of . % ggaa solution was taken in a glass homogeniser and homogenised at rpm on an ice bath. a . % ga solution was added dropwise to the ggaa solution under homogenisation in an ice bath followed by . % tpp solution under similar conditions. the homogenisation was further continued for minutes in an ice bath and thereafter tween ( . % solution) solution was added dropwise and homogenised. the product was stirred overnight using a magnetic stirrer. the final product was harvested by centrifugation at , rpm for minutes at °c, washed three times with hplc grade water, dried for days in a vacuum desiccator, and preserved at °c in tightly closed vials till further use. the gams were characterized by their shape and particle size. a scanning electron microscope (sem) (ion sputter s- n hitachi, japan) was used for the physical characterization of the gams. the sample was analysed at kv at . k se magnification. the particle size was determined by measuring the diameters of particles chosen at random. the particle shape and surface were examined using the sem photomicrographs. the ft-ir spectra for ga, ggaa, and the gams were recorded in spectrum one ft-ir spectrometer (perkin elmer, usa) in pressed potassium bromide pellets. the spectra were stacked for the determination of any drug-polymer interactions. the x-ray diffraction patterns were determined with the x-ray powder diffractometer (xpert-pro, panalytical) using cukα radiation at a scan rate of . °/ . sec over the ɵ range of - °. diffraction patterns of the pure drug, polymers, physical mixture, and the prepared microparticles were compared. a validated reversed-phase chromatographic method was used throughout. the chromatographic determinations were carried out on a waters (waters, usa) binary gradient system equipped with a waters pump (two numbers), a manual rheodyne injector port attached with a µl loop, and a waters pda detector. the system control and data acquisition were carried out using empower software (waters, usa). the separation was carried out in a reversed-phase kromasil c column ( mm x . mm, µ; akzonobel, usa). the standard, sample, and mobile phase (mixture of % acetonitrile and % phosphate buffer (ph = . ), prepared by dissolving potassium phosphate monobasic . g and potassium phosphate dibasic . g in ml water, adjusted to ph= . using orthophosphoric acid) was filtered through a . µm membrane filter (pall life science, india). the flow rate was . ml/minute and the column was maintained at ambient temperature. the column effluent was monitored at nm with pda detection. the retention time of ga was . ± . minutes (± s.d.; n= ). a peak area (along y axis) vs. concentration in µg/ml (along x axis) graph for ga was first prepared (y = x + , r² = . ) and used to estimate ga concentrations throughout. the mass of ga in solution before and after the microparticulation in supernatant was determined by the hplc experiments for the calculation of entrapment efficiencies as presented in equation . ga % encapsulation = �mass of ga originally taken -mass of ga in supernatant� �mass of ga originally taken� × male sprague-dawley (sd) rats weighing between to gm were used in the test. the protocol was approved by the institutional animal ethics committee (iaec), university of calcutta, department of chemical technology with cpcsea registration number / /a/cpcsea. the rats were asphyxiated by excessive inhalation of carbon dioxide (co ) [ ] . the abdomens were opened and the cecum were traced, legated at both ends, dissected, and immediately transferred to the simulated colonic fluid [ ] previously bubbled with co . the cecums were opened, weighed separately, pooled, and then a % solution was prepared. the solution was then centrifuged at rpm for minutes. the supernatant was used as rat cecal content medium in the dissolution studies. the in vitro drug release study was carried out in simulated gastric fluid of ph . , simulated intestinal fluid of ph . , and simulated colonic fluid of ph . containing rat cecal contents under anaerobic conditions. gams equivalent to about mg of the drug ga were dispersed in . ml of the simulated biological fluids placed in dialysis bags and both ends were sealed with a clean thread. the bag was suspended in ml of the respective simulated fluid taken in a tube and maintained at °c in a beaker under constant stirring at rpm (fig. ). at predetermined time intervals, the release medium from the tube was taken out and replaced with fresh dissolution medium. the amount of ga released at regular intervals was estimated by the hplc method mentioned under the drug entrapment study. a (three level two factors) full-fledged factorial design [ ] was applied to optimize the two independent variables, namely the amount of ga (x ) and the amount of ggaa (x ). the factorial design layout for the nine different batches are presented in tab. a and b. the in vitro drug release in the simulated colonic fluid was monitored first at mins and then every hour. the release at mins, mins, and , mins were analyzed as response parameters in the factorial design studies. results are expressed as the second order polynomial equation . eq. . where b denotes the arithmetic mean response for nine runs, b i (i = , ) denotes the estimated coefficient for the factors and x i (i = , ) denotes the effect of changing one factor at a time from its lowest to highest level. the interaction terms x x denotes the effect when both the factors were changed simultaneously. the polynomial terms x i (i = , ) was used to explain non-linearity [ ] . y was the measured response parameter in each experiment and was taken as a dependant variable. the coefficients corresponding to the linear effects, b and b , interactions, b , and the quadratic effects, b and b , were determined from the experimental results. all animal experiments were performed in accordance with the recommendations of the institutional animal ethics committee, dept. of chemical technology, university of calcutta, india with registration number / /a cpcsea. the tnbs rat model is a recognized model for the induction of experimental colitis with a very low mortality rate [ ] . the inflammation was induced in male sd rats by instilling . ml of the tnbsethanol mixture ( . ml of % w/v tnbs in . ml of % ethanol) into the lumen of the colon using a baby feeding tube, followed up by flushing the tube with . ml of air. the treatment schedule is presented in the figure below (fig. ) . programme schedule for the development of experimental colitis and subsequent drug treatment. the animals in the treatment group received either . ml of the suspension of gam in saline or gam at a dose equivalent to mg of gam per kg body weight once daily. the tnbs control group received only saline and the void control group received the void microparticle suspension. the animals were sacrificed hours after administration of the last drug/gam dose. their abdomens were cut open and the colon was legated at both ends. finally, the colon was resected, cleaned, and preserved till further use. the degree of severity of colitis and inflammation was analyzed using a clinical activity index like weight loss, stool consistency, and rectal bleeding as described earlier [ ] . the resected colon tissue was opened longitudinally and cleaned thoroughly with ice cold phosphate buffer saline to remove its luminal contents. the colons were examined for the visual severity of the colitis. the colon weight and length were examined. they were expressed as a ratio with the respective body weights. the measurement of nitric oxide and myeloperoxidase activity in the inflamed tissue was performed to study the extent of the severity of the colitis. the level of malondialdehyde in the colonic tissue was determined as an indicator of lipid peroxidation [ , , ] . the other part of the colon tissue was preserved in formalin buffer solution for histological examination. the results were presented as the mean ± s.d. values. for analysis of statistical significance, student's-t test and anova were applied. furthermore, a p< . was considered to be significant. ggaa was synthesised, purified, and subjected to ir spectroscopic analysis for the presence of an amine group. the dried purified product was used for the preparation of gams. the gams were prepared by ionic crosslinking by using sodium tripolyphosphate. the microparticles were almost spherical with an average particle size varying from . ± . µm to . ± . µm (tab. a) as revealed from scanning electron photomicrograph (fig. ) . studies by lampretch et al. and his colleagues have shown that the bioadhesion of the microparticle carriers to the inflamed colonic mucosa depends on the size of the microparticle [ ] . also, particle sizes smaller than µm were found to show prolonged passage time. this indicates a longer residence time for the prepared microparticles in the colon for the delivery of the total drug load into the colonic mucosa. the prepared microparticles also had smooth surfaces with very few aggregations. representative scanning electron photomicrograph of prepared microparticles the ft-ir spectroscopy (fig. ) of the optimized formulation f was carried out to observe any drug-polymer interaction. ga showed a sharp peak at . cm − which showed no shift in the gam formulations. however, a sharp shift was observed from . cm − in the ga spectrum to cm − in the formulation. the peak at . cm − in the ga spectrum might be due to a primary alcohol and its shift to cm − was due to a hydrogen bonding interaction between ga and ggaa [ ] . this might be the reason for satisfactory entrapment of ga within the gams. the x-ray diffractograms (fig. ) manifested two distinct peaks at ɵ: . ° and . ° for intact ga and a clear peak at . º for ggaa. the diffraction pattern of the physical mixtures presented the lowering in peak intensities of the intact drug due to the dilution effect of the polymer and no qualitative differences in the diffraction pattern of the drug. gams prepared with ggaa were characterized by the absence of the distinct diffraction peaks of ga and a peak for the polymer at . °, signifying the entrapment of the drug in amorphous form or its solvation in the polymeric carrier (fig. ) . therefore, the pxrd studies verified the decrease in crystallinity of the drug in the microparticles and the effective entrapment of ga in amorphous form within the polymer matrix. the drug entrapment within the gams was found to be on the higher side for all the nine formulations f to f . the average drug entrapment varied from . ± . % to . ± . % of the total drug used for each gam formulation (tab. a, b). the drug release studies from the microparticles were carried out in the simulated gastric fluid of ph . , simulated intestinal fluid of ph . , and simulated colonic fluid of ph . containing rat cecal contents [ , ] . altogether, nine formulations f to f were prepared with varying proportions of the polymer and the amount of drug. fig. , , and demonstrate ga release profiles from gams in three different mediums of ph . , . , and . respectively. in the simulated gastric and intestinal fluid, minimum drug release was observed and the rate of drug release was also very slow. in the simulated gastric fluid for formulations f , f , and f , a maximum of only % cumulative drug release was observed, whereas for the others it was only about %. in the simulated intestinal fluid, only about % of the drug was released in the first mins of the study for all nine formulations. in the simulated colonic fluid containing rat cecal contents, the drug release was a bit more augmented with an initial burst release common for each of the designed the results predict the drug-polymer ratio to be the key factor for controlling the release. however, a complex interaction between the drug and polymer molecules or the simple adsorption of the drug molecules on the surface of the polymeric mps due to electrostatic adhesions may be responsible for the successful entrapment of the drug within the polymer matrix. a minimum drug release in the simulated gastric and intestinal fluid implies a minimal chance of the drug molecule getting adsorbed to the surface of the microparticle, as adsorption of ga on the surface of the microparticle would result in a substantial drug release earlier in the simulated gastric and intestinal fluid. thus, a proper entrapment of ga might be predicted from the study. the increase in drug release in the simulated colonic fluid was facilitated by the enzymatic degradation of the polymer and by the colonic microflora present in the rat cecal contents. the nature of the release profile predicts the dependence of the cumulative drug release on the drug-polymer ratio. therefore, the formulation was optimized on the basis of the drug-polymer ratio using a factorial design model. the response obtained from in vitro release studies was evaluated using a statistical model which involved a number of polynomial terms as explained earlier. the percent cumulative drug release values at three different time intervals of all nine formulations were fitted in the quadratic equation . the coefficients were determined for responses q , q and q and the respective equations , and were obtained. the data indicated that the release profile of the drug was strongly dependant on the selected independent variables i.e the drug-polymer ratio. the data fitted in equation for the full model relating to responses q , q and q and the transformed factors have been presented in tab a positive value indicates a synergistic effect that favours optimization, while a negative sign represents an antagonistic effect or an inverse effect of the factor on the selected response. it was observed that for all the three dependant variables q , q and q , the linear contribution of both coded factors shows an antagonistic effect (p < . , tab. ). for response q the linear contribution of x , x and the contribution of the interaction term x x were significant with p < . respectively. at mins (q ) a significant contribution of the polymer in drug release from the formulations (p < . ) was observed. at minutes (q ) the individual contribution of drug and polymer became significant. in order to determine the significance of each factor, analysis of variance was performed. the results have been presented in tab. . the high values of correlation coefficients for q , q and q indicated a good fit. the response surface regression analysis was performed using coded values of factor levels (− , , + ) for each factor to understand the contribution of each independent variable. the response surface plots were presented in fig. , and corresponding to release at minutes, minutes and minutes respectively. the three dimensional plots gives us an idea about the change in the response surface. the formulation was optimized on the basis of observed and predicted values of the responses [ , ] . the observed values for the formulation f were very close to the predicted value. this implies that the cumulative drug release from formulation f followed the required pattern of drug release to the colon. the in vivo efficacy study was carried out with the optimized formulation f having drug/polymer ratio : . the therapeutic efficacy of this optimized gam formulation was studied on tnbs induced rat model of colitis. the animals under study were divided into six groups -normal control, solvent control, tnbs control, void control, ga and gam groups. the normal control group was kept to study the normal condition of colon. the solvent control group was kept to study whether the solvent used had any adverse effect. the void control group was kept in order to study the possible effect of the delivery device itself. the ameliorative efficacy of the gam was studied by comparing the results with the ga treated groups. intra-colonic administration of tnbs in % ethanol resulted in an inflammatory response characterized by extensive mucosal disruption, linear and deep ulcers, haemorrhage, and sub-mucosal oedema (fig. ). diarrhoea and rectal bleeding were evident in all the rats with colitis which were without any treatment. the results of the control groups were compared with the treated groups i.e. ga and gam treated groups. both the diarrhoea and the rectal bleeding was reduced significantly in both the drug treated groups but optimized formulation f showed higher efficacy in the healing process than that of ga and the studied parameters for gam treated group was close to the healthy control group [ ] . the length of the colon from appendix to anus was measured and was found to be . cm for the normal group, . cm for the tnbs control group, . cm for ga group and . cm for gam group. thus both ga and gam recovered the colon length from shortening which was more significant for gam. the colon to body weight ratio for gam group was . and was much lower than the tnbs control group . (tab ). the tissue biochemical parameters namely tissue nitric oxide, myeloperoxidase and malondialdehyde presented a much lower value for those treated with ga and gam compared to the tnbs control group, gam treated group showing the lowest value for all the three studied parameters (p< . ) indicating the best healing effect shown by gam formulation (tab. ) [ , ] . thus encapsulation of ga within the microparticles in formulation f showed greater efficacy in the amelioration of colitis. in vivo efficacy study histological examination presented extensive ulcer formation and infiltration of inflammatory cells mostly neutrophils and florid ulcer formation with inflammation and necrosis among the tnbs control group. the ga treated group showed presence of inflammatory cells and moderate improvement with granulated tissue formation (fig. ) . only in the case of gam group mild ulcer and minimum inflammation was observed with very low infiltration of the inflammatory cells. the colon to body weight ratio (tab. ) and the colon length (fig. ) for the gam treated group were close to healthy control group after treatment. the studied biological parameters (tab. ) present enhanced efficacy of gams over free ga [ ] . the visual severity of colitis presented minimum damage of colonic mucosa in case of gam treated animals compared with ga treated group (fig. ) . excessive accumulation of inflammatory cells mainly neutrophils was observed at the sites of inflammation in tnbs control group and an increased tissue myeloperoxidase [ ] was thus observed. in case of gam group, the mpo activity in the colonic tissue was found to be the minimum, indicating lesser inflammation and better healing of colitis [ , ] . the histological examination of the colonic tissue ( fig. ) presented least accumulation of inflammatory cells in colonic tissue of gam treated animals [ , ] . the higher localized delivery of the drug to the affected colonic tissue may have reduced the extent of ulcer formation compared to the ga treated group of animals where much of the drug may have been absorbed at normal tissues of intestine and colon. the monoammonium glycyrrhizinate-loaded microparticles prepared through the ionic crosslinking of a newer biopolymer guar gum alkyl amine. this formulation was found to deliver the optimal drug load to the colon. the ft-ir studies presented a possible interaction of the drug with the polymer and retention of the drug within the polymer matrix. the drug-polymer ratio was found to be : on the optimization using a factorial design model. the release profile of the optimised formulation and in vivo efficacy studies on the basis of physiological and biochemical parameters presented the successful application of this newer biopolymer in the preparation of colonic delivery devices. inflammatory bowel disease in india -changing paradigms inflammatory bowel disease in the rural indian subcontinent: a survey of patients attending mission hospitals incidence and prevalence of ulcerative colitis in punjab an update on the epidemiology of inflammatory bowel disease in asia size-dependant bioadhesion of micro-and nanoparticulate carriers to the inflamed colonic mucosa multiparticulate formulation approach to colon specific drug delivery: current perspectives studies on guar gum compression-coated -aminosalicylic acid tablets for colon-specific drug delivery guar gum as a carrier for colon specific delivery; influence of metronidazole and tinidazole on in vitro release of albendazole from guar gum matrix tablet cross-liked guar gum microspheres: a visible approach for improved delivery of anticancer drugs for the treatment of colorectal cancer controlled release of tamoxifen citrate encapsulated in cross-linked guar gum nanoparticles preparation of cross-linked guar gum nanospheres containing tamoxifen citrate by single step emulsion in situ polymer cross-linking method glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus glycyrrhizic acid and β-glycyrrhetinic acid inhibit inflammation via pi k/akt/gsk β signaling and glucocorticoid receptor activation affinity of liquorice derivatives for mineralocorticoid and glucocorticoid receptors inhibition of phospholipase a and platelet aggregation by glycyrrhizin, an antiinflammation drug glycyrrhizin inhibits prostaglandin e production by activated peritoneal macrophages from rats mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin glycyrrhizic acid and β-glycyrrhetinicacid inhibit inflammation via pi k/akt/gsk β signaling and glucocorticoid receptor activation glycyrrhizic acid suppresses cox- -mediated anti-inflammatory responses during leishmaniadonovani infection chinese prescription, and its active ingredient glycyrrhizin ameliorate experimental colitis through regulating cytokine balance some properties of glycyrrhizic acid efficacy and toxicity of eudragit-coated chitosan-succinyl-prednisolone conjugate microspheres using rats with , , -trinitrobenzenesulfonic acid-induced colitis nanoparticles enhance therapeutic efficacy by selectively increased local drug dose in experimental colitis in rats measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker intracolonic release of nitric oxide during trinitrobenzene sulfonic acid rat colitis change of nitric oxide in experimental colitis and its inhibition by melatonin in vivo and in vitro -amino salicylic acid bound nanoparticles for therapy of inflammatory bowel disease new water resistant biomaterial biocide film based on guar gum development of polysaccharide based colon targeted drug delivery systems for the treatment of amoebiasis pharmaceutical experimental design and interpretation factorial experiments. statistical methods hapten-induced model of chronic inflammation and ulceration in the rat colon mucosal surface ph of the large intestine of rat and of normal and inflamed large intestine of man transit of pharmaceutical dosage forms through small intestine myeloperoxidase immunohistochemistry as a measure of disease activity in ulcerative colitis: association with ulcerative colitis-colorectal cancer, tumor necrosis factor polymorphism and runx methylation critical role of il- receptor signaling in acute tnbs-induced colitis colonic insufflation with carbon monoxide gas inhibits the development of intestinal inflammationin rats infliximab modifies mesenteric adipose tissue alterations and intestinal inflammation in rats with tnbs the authors acknowledge the generous guidance and support of scientific and clinical research laboratory, kolkata, west bengal, india for preparation of tissue sections and carrying out the histological analysis of the tissue samples. the authors declare no conflict of interest. all animal experiments were performed in accordance with the recommendations of the institutional animal ethics committee, dept. of chemical technology, university of calcutta, india with registration number / /a cpcsea. key: cord- - mw okmt authors: bule, mohammed; khan, fazlullah; niaz, kamal title: antivirals: past, present and future date: - - journal: recent advances in animal virology doi: . / - - - - _ sha: doc_id: cord_uid: mw okmt the uses of antiviral agents are increasing in the new era along with the development of vaccines for the effective control of viral diseases. the main aims of antiviral agents are to minimize harm to the host system and eradicate deadly viral diseases. however, the replications of viruses in host system represent a massive therapeutic challenge than bacteria and fungi. antiviral drugs not just penetrate to disrupt the virus’ cellular divisions but also have a negative impact on normal physiological pathways in the host. due to these issues, antiviral agents have a narrow therapeutic index than antibacterial drugs. nephrotoxicity is the main adverse reaction of antiviral drugs in human and animals. in this chapter, we summarize the antiviral agents’ past, present and future perspectives with the main focus on the brief history of antiviral in animals, miscellaneous drugs, natural products, herbal and repurposing drugs. vaccine development has long been the first and foremost approach in the control of viral diseases (saminathan et al. ) . however, with the development of the first human antiviral drug, idoxuridine, and its later approval in , a new era of antiviral treatment development began (de clercq and li ) . because of viral replication cycle, which involves usage of host cell biochemical machinery, antiviral drugs can also affect the function of the host's pathways resulting in the great risk of toxicity. therefore, the major concern in antiviral drug development is the identification of specific targets with increased selectivity and reduced side effects, which limit the therapeutic use of antiviral drugs in comparison to antibacterial agents (dal pozzo and thiry ) . in the early s, state-of-the-art review articles on antiviral chemotherapy in veterinary medicine listed several drawbacks of low antiviral drugs use in veterinary medicine. those included usage restricted to a single virus and specific animal species, problems with high spectrum activity and low cytotoxicity, high costs of development of new chemical compounds and absence of rapid diagnostic techniques allowing prompt use of a specific antiviral agent in the course of an acute infection (rollinson a, b) . most of the antiviral drugs used in animal medicine have been originally developed against human viral infections and their clinical use in veterinary medicine is not widespread and common. nevertheless, several licensed human antiviral agents are being used with cascade principle for treatment of animal diseases (e.g. acyclovir, idoxuridine and trifluridine against feline herpesvirus- ocular infection in cats) (thiry et al. ). currently, the only licensed antiviral drug in veterinary medicine is feline interferonomega (ifn-ω), whose mechanism of action involves a combination of antiviral and immunostimulatory activity (de clercq and li ; bracklein et al. ) . most antiviral agents interfere with the synthesis or regulation of viral nucleic acids ( fig. . ) and act by nucleoside analogues that block elongation of newly synthetized dna or rna chain. other antiviral agents used in veterinary medicine act as neuraminidase (oseltamivir) or amino acid (l-lysine) inhibitors, while novel treatment options such as small inhibitory rnas are also under investigation (dal pozzo and thiry ; sykes ) . in recent years, the use of antiviral agents in veterinary medicine has become more favourable with growing interest in its research. this is partially due to successful outcomes of antiviral therapy in some human diseases and partially due to advances in internal veterinary medicine with the development of novel and sophisticated diagnostic and treatment protocols. in addition to that, current measures for control of viral infections such as vaccination or removal of infected animals from breeding stock by culling have many limitations. therefore, antiviral agents represent a promising alternative for the treatment of viral diseases in veterinary medicine (dal pozzo and thiry ) . one of the most common approaches in antiviral drug discovery is rational drug design, which is based on the understanding of the structures and functions of target molecules. it comprises three steps of drug design: ( ) identification of the receptor or enzyme relevant for the disease that the drug is being developed for, ( ) discovery of the structure and function of the receptor or enzyme of interest and ( ) use of the information from step in order to design a drug molecule that would interact with the receptor or enzyme in a therapeutically beneficial way. the best known example of this approach is azidothymidine (azt), used in the treatment of human immunodeficiency virus (hiv), which acts by inhibiting hiv reverse transcription. interestingly, it was originally developed to target reverse transcription of avian retroviruses that may cause cancer and was later successfully applied to hiv as well (olivero ) . the other widely used approach is high-throughput screening (hts) methods, which enable validation of a number of biological modulators against a chosen set of defined targets. they yield rich data sets over a short span of time by combining expertise in liquid handling and robotic automation, multiplatform plate reading and high-content imaging. the number of thus emerged "active hits" is normally around % of the total number of potential biological modulators screened. steps in hts pipeline can be summarized as follows: ( ) sample preparation, ( ) sample handling and ( ) readouts and data acquisition. the most common hts methods are targeted/selected screens, diversity and high-content screens and rnai screens (szymański et al. ) . targeted or selected screening is based on identification of compounds that can selectively inhibit or bind to a specific protein of interest. if the crystal structure of the protein of interest is known, it is usually done by in silico three-dimensional ( -d) modelling, while if the ligand for the protein of interest is known, software can search libraries for the other compounds with similar characteristics and binding properties. examples of such approach include identification of compounds against hiv, filoviruses, poxviruses, arenaviruses, etc. (marriott et al. diversity screening, based on identification of compounds that inhibit viral replication or pathogenesis at any level. this approach involves a much broader target base, instead of focusing the screening against one specific protein of interest. it has been applied in the identification of candidate small-molecule inhibitors against dengue virus, yellow fever virus and new world arenaviruses (valler and green ) . high-content screening (hcs) is a subclass of diversity screening method developed upon automation of cellular imaging and analysis techniques. it allows imaging multiple cells at the same time and measurement of multiple parameters, such as shape, texture, staining localization and intensity, total number of cells, size of the nucleus and percentage of virus-positive cells (brodin and christophe ) . another type of screening for antiviral drug discovery is rnai screens. sirnas incorporate into the rna-induced silencing complex and bind to the target mrna, thereby inducing degradation of the mrna and thus preventing its translation into a protein. shrna, on the other hand, silences protein by forming a "short hairpin" loop through folding back upon itself. genome-wide rnai screens have been used to study the pathogenesis of hiv, influenza virus, west nile virus, ebola virus, etc. (hirsch ) . in addition to the above-mentioned methods, recent advances in genomics, bioinformatics and associated technologies offer new opportunities in antiviral drug discovery. computational methods enabled construction of databases that contain information related to biological function, chemical structure, biologic activity and many other properties of potential antiviral compounds that can all contribute to identification of new lead bioactive species (prichard ) . vidarabine ( -d-arabinofuranosyl adenine) was the first antiviral agent licensed for systemic treatment of herpes viral infections in humans (fenner et al. ) . it is an adenosine analogue that is converted by cellular enzymes to its active intracellular derivate, vidarabine triphosphate. obtained triphosphate form further acts as competitive inhibitor of both viral and host dna polymerase, where viral enzymes are much more susceptible to the drug than that of the host cell (sykes ; schaechter ) . however, independence on viral thymidine kinase-mediated phosphorylation results in greater host cell toxicity (sykes ) . it is used as a topical treatment for feline herpes keratitis, albeit in vitro studies have shown it to be less potent against feline herpesviruses than trifluridine and idoxuridine (nasisse ). in vitro activity has also been demonstrated against feline end equine rhinopneumonitis (ayisi et al. ). five to six times daily administration as a % ophthalmic ointment was reported to be well tolerated by cats and effective in the treatment of feline keratoconjunctivitis sicca. it has also been reported to be effective against idoxuridine-resistant strains (sykes ; stiles ) . acyclovir (acycloguanosine) is an acyclic analogue of the purine nucleoside deoxyguanosine that has been widely used to treat herpesvirus family infections (sykes and papich ; perazella and shirali ) . the activation involves phosphorylation of the drug firstly by virus-encoded thymidine kinase enzymes into monophosphate form, followed by further phosphorylation to triphosphates by host cell enzymes (sykes and papich ) . acyclovir triphosphate is a better substrate to viral than host dna polymerase, resulting in its concentration in infected cells. due to the lack of ′-hydroxyl group, the drug inhibits viral dna polymerase enzyme, as upon its incorporation further dna chain elongation is disabled (sykes and papich ; salvaggio and gnann ) . hence, as a therapeutic, it is mostly used to treat dna virus infections, in particular herpes simplex virus types and and varicella-zoster virus (salvaggio and gnann ) . in animal medicine, it has been primarily used against feline herpesvirus- (fhv- ) infections, but not as efficiently as against the same human virus in vitro, which is related to its low oral bioavailability in cats (maggs and clarke ; gaskell et al. ; nasisse et al. ) . the acyclovir prodrug valacyclovir shows better pharmacokinetic properties in terms of its enhanced bioavailability, resulting in faster absorption after oral administration upon which it gets rapidly metabolized to acyclovir. acyclovir and entecavir had the ability to block nucleic acid synthesis ( fig. . ). however, administration of valacyclovir as well as subsequent increased plasma acyclovir concentrations has been associated with adverse effects, such as nephrotoxicity, myelosuppression and renal and hepatic necrosis, and yet was not effective against fhv- infection (nasisse et al. ). these findings suggest systemic administration of neither acyclovir nor valacyclovir is recommended for treatment of herpesvirus infections in cats. ganciclovir, another purine nucleoside analogue that resembles acyclovir and is widely used to treat cytomegalovirus infections in human medicine, has been shown to be more effective against fhv- in vitro, but unfortunately there is lack of data on its efficacy and safety in animals (sykes ). on the other hand, topical acyclovir treatment was shown to be effective against fhv- conjunctivitis and keratitis when applied at least times a day and did not produce toxic effects (williams et al. ) . apart from cats, the existing studies provide data on acyclovir treatment in horses, where intravenous administration resulted in , -hour halflife, contrary to very low oral absorption (< %) (williams et al. ; riviere and papich ) . it has therefore been suggested that iv treatment could be administered twice daily for equine herpesvirus- (ehv- ) (riviere and papich ) . in birds, oral treatment with mg/kg of acyclovir every h has been shown as the minimum dose necessary to maintain concentrations that exhibit antiviral effect in pheasants (rush et al. ) . studies in dogs report oral absorption of acyclovir of - %, but it becomes saturated at high doses (de miranda et al. ). penciclovir [ -( -hydroxy- -hydroxymethylbut- -yl)] is guanosine analogue that resembles acyclovir in structure, mechanism of action and antiviral activity spectrum. comparing to acyclovir, penciclovir-triphosphate accumulates in virusinfected cells in much higher concentrations and for longer half-life ( - times longer than acyclovir) (salvaggio and gnann ; gill and wood ) . however, it is less potent than acyclovir triphosphate as it exhibits lower affinity for viral dna polymerase enzyme, which would allow lower and less frequent dosage in clinical use. in vitro studies have proven its efficacy against ffhv- virus and hepatitis b virus (dannaoui et al. ; shaw et al. ; korba and boyd ) . because of penciclovir's poor oral bioavailability (< %), famciclovir was developed as the oral formulation (salvaggio and gnann ) . the use of penciclovir will be further described along with famciclovir. famciclovir (the diacetyl ester of -deoxy-penciclovir) is the oral prodrug of acyclovir with the improved bioavailability which, following oral administration, gets rapidly converted to its active metabolite penciclovir by di-deacetylation and oxidation. however, the pharmacokinetics of penciclovir and famciclovir in cats appears to be nonlinear (saturable) and absorption variable compared to other species. this is supported by the observation that administration of the same doses of famciclovir to cats and other species resulted in much lower plasma concentrations and longer time required to reach peak plasma concentrations in cats (thomasy et al. ). thus, limited famciclovir metabolism stems from deficiency of hepatic aldehyde oxidase enzyme in cats, which converts famciclovir to its active form (dick et al. ) . even though famciclovir has to be administered in high oral doses to develop adequate plasma concentrations in cats, it seems to be well tolerated and successful in the treatment of fhv- -associated conjunctivitis (thomasy et al. ; malik et al. ; thomasy et al. ). due to saturable metabolism, oral administration of both and mg famciclovir/kg to cats resulted in equivalent serum and tear penciclovir concentrations, implying that mg/kg is equally effective against fhv- as the higher dose (thomasy et al. ) . in rats and dogs, famciclovir absorption and metabolism appear to be similar to those previously reported in people, despite the observed slower conversion of famciclovir to penciclovir in both species (filer et al. ). as first described by witkowski et al. in , ribavirin ( -β-d-ribofuranosyl- , , triazole- -carboxamide) is a triazole nucleoside analogue that inhibits replication of both dna and rna viruses by interfering with viral mrna synthesis (witkowski et al. ) . it is active against a wide range of viruses including adenoviruses, arenaviruses, bunyaviruses, herpesviruses, orthomyxoviruses, paramyxoviruses, picornaviruses, poxviruses, retroviruses, rhabdoviruses and rotaviruses, but the strongest effect exhibits against influenza viruses and, in combination with interferon, against hepatitis c virus (dolin ; gustafson ; te et al. ). it has several possible mechanisms of action. firstly, as ribavirin monophosphate, generated by adenosine kinase-mediated phosphorylation, it can indirectly inhibit the synthesis of guanine nucleotides. further, the phosphorylated triphosphate form competitively inhibits binding of atp and gtp to rna polymerase (riviere and papich ) . orally administered ribavirin has been shown to worsen the condition of cats experimentally infected with calicivirus. toxic effects mainly resulted from drug-induced thrombocytopenia and include depression of red and white blood cells, increased alanine aminotransferase activity, icterus and body weight loss. however, observed clinical symptoms withdrew within one week after treatment discontinuation (riviere and papich ; povey ) . interestingly, these side effects were not observed in dogs treated for weeks with mg/kg of the drug (canonico ) . in kittens experimentally infected with feline infectious peritonitis virus (fipv), treatment with neither free nor liposomal ribavirin improved survival rate and, similarly to animals infected with calicivirus, resulted in intrinsic toxicity (riviere and papich ; weiss et al. ). activity of ribavirin has also been demonstrated against bovine viral diarrhoea virus, bovine herpes virus- and parrot bornavirus in cell culture models (glotov et al. ; musser et al. ) . the antiviral activity of benzimidazole nucleosides was first reported by tamm, folker and co-workers in . they designed , -dichloro- -(β-d-ribofuranosyl) benzimidazole (drb), which had various biological activities including antiviral activity against rna and dna viruses. the antiviral activity of drb is via inhibiting cellular rna polymerase ii thus inhibiting viral and cellular rna synthesis (migawa et al. ; porcari et al. ; chen et al. ; townsend et al. ) . in pharmaceutical chemistry, heterocyclic compounds particularly the benzene-fused are of great importance. in the class of benzene-fused compounds, benzimidazole and its derivatives are known for their wide variety of biological activities. biologically active compounds such as vitamin b , albendazole, mebendazole and thiabendazole contain a benzimidazole nucleus in their structure ( fig. . ) (shaharyar et al. ). structure-activity relationship (sar) studies show that due to a change in the group on the basic structure, benzimidazoles display a wide array of biological activities including analgesic, antibacterial, antifungal, anticancer and antiviral (alaqeel ) . moreover, a range of structurally varied nonnucleoside inhibitors (nni) of the hcv polymerases sharing the benzimidazole pharmacophore has been reported. among these classes of compounds, jtk- , which is an orally active benzimidazole derivative, is in its phase i and ii clinical trial stage in japan (tan et al. ; tomei et al. ) . currently, a number of benzimidazole derivatives are available in the market such as omeprazole and rabeprazole used for gastric ulcers, telmisartan and candesartan for hypertension, astemizole and mizolastine for allergic rhinitis, and albendazole, oxibendazole and mebendazole for parasitosis (wang et al. ) . benzimidazoles readily interact with the biopolymers of the living system due to the fact that they are bioisosteres of cellular nucleotides (starčević et al. ). arildone is an antiviral drug of the -[ -( -chloro- -methoxy)phenoxyl]hexyl- , heptanedione class, which is active against both dna and rna viruses (kuhrt et al. ) . it is primarily suggested to be used as a broad-spectrum antiviral agent because it is relatively a less toxic drug and inhibits viral replication at lower concentration (kim et al. ) . sar studies demonstrated that omission of the lipophilic substituents of arildone diminished the antiviral activity (mcsharry et al. ) . arildone (fig. . ) inhibits replication of enterovirus, particularly poliovirus, via interaction with the viral capsid and hence blocking viral uncoating (nikolaeva-glomb and galabov ) . further in vitro studies provided evidence that a direct interaction of arildone with the poliovirus capsid stabilizes the virion against heat and alkaline treatment, resulting in loss of the vp capsid polypeptide and blocked release of viral rna (fox et al. ). the uncoating inhibition action of arildone at lower dose can block replication of herpes virus at an earlier stage than the polymerase. arildone is administered as a solution in dimethyl sulphoxide (dmso) due to its poor solubility in water, and the solvent properties of dmso may have augmented its antiviral action (hutchinson ) . in an animal model experiment, the ability of arildone to block the virion uncoating property to prevent paralysis and death was studied in mice intracerebrally infected with a higher dose of poliovirus type- (strain mef). moreover, ip administration of arildone suspended in gum tragacanth successfully protected the animals from paralysis and death in a dose-dependent fashion (minimal inhibitory dose = mg/kg, x/day) (mckinlay et al. ) . arildone is the first of the capsid inhibitors that demonstrated in vitro inhibition of poliovirus replication and prevented paralysis and death in poliovirus-infected mice. such compounds with better bioactivity showed potency orally in the mouse model, even when administered days after intracerebral infection (mckinlay et al. ). compounds consisting the carbon-phosphorous bond are rare in nature and were considered non-existent till recently. it was in that phosphonoacetic acid (paa) was first synthesized and its antiviral activity was discovered almost years later in (shipkowitz et al. ). the discovery of paa (fig. . ) as an antiviral drug gave rise to intense research on its biological activities, which demonstrated paa and its derivatives' ability to inhibit the replication of a number of viruses such as immunodeficiency, hepatitis and herpes viruses. in animal studies it was shown that paa is active against herpes keratitis in rabbits and herpes dermatitis in mice. paa and its derivatives being analogues of antimetabolites of pyrophosphates have their action against herpes viruses, especially in epstein-barr virus, cmv and hsv, through inhibiting dna polymerase, which is important in herpes virus replication (alimbarova et al. ; overby et al. ). in addition, it was depicted in a study that polymerase activity was inhibited in lysed cultures of infected cells by paa without affecting the enzyme in normal cells. polymerases from both normal and infected cells were highly purified and investigated to verify their differential sensitivity towards paa (mao et al. ). amycolatopsis rifamycinica is the first soil bacteria that provided the rifamycins in . for a while, it was considered the only bacterial source of rifamycins till their discovery in salinispora group. although there are several rifamycins isolated from bacteria, the most widely used derivative of rifampicin (rifampin) is a semisynthetic rifamycin. rifamycins are preferable as they can cross mammalian tissue and cell membrane easily (bhattacharjee ) . as a result, rifamycin-sv and its derivatives are deemed first line in the treatment of intracellular pathogens and demonstrated inhibitory action in various biological systems. among the antibacterial agents of these derivatives, some act by inhibiting the bacterial dna-dependent rna polymerase. furthermore, rifampin inhibits poxvirus replication in vitro via a mechanism other than inhibiting dna-dependent rna polymerase. in vitro screening for selective inhibition of rna-dependent dna polymerase (reverse transcriptase) on a number of derivatives revealed that certain derivatives prevented focus formation by rna tumour viruses (szabo et al. ). rifamycin derivatives were also found to act against type ii dna topoisomerases. besides, phylogenetic studies showed that viral type ii dna topoisomerase and their bacterial counterparts have similarities indicating that the antibacterial topoisomerase inhibitors can act against african swine fever (asfv) replication. in fact, fluoroquinolones, a class of synthetic antibacterial drugs, were shown to inhibit the asfv replication by interacting with type ii topoisomerase (zakaryan and revilla ) . rifampicin, rifapentine and rifabutin ( fig. . ) are semisynthetic and water-soluble derivatives of -formylrifamycin sv, used in therapies against different gram-positive and gram-negative bacterial strains including methicillin-resistant staphylococcus aureus (mrsa), mycobacteria (mycobacterium bovis or mycobacterium tuberculosis) and leprosy, legionella. they are also able to prevent viral infections (e.g. influenza) (czerwonka et al. ) . the antiviral potential of antibacterial drugs has been studied on various drugs. minocycline is among the well investigated for its actions against a number of ailments. it is a synthetic second-generation tetracycline derivative with immunomodulatory and anti-inflammatory action and widely used for the treatment of acne, rheumatoid arthritis and utis. potential antiviral action of minocycline against human immunodeficiency virus, japanese encephalitis virus and west nile virus has been reported. it was also found promising in reducing dengue virus infection, with a prompt action against all the four serotypes of the virus. minocycline generally diminished viral rna synthesis, intracellular viral protein synthesis and thus infectious virus production. it was also found to decrease erk / phosphorylation, which is associated with intensifying pathogenesis and organ damage in dengue virus infection (leela et al. ) . furthermore, the quinolones have showed an antiviral activity towards hiv and hepatitis c virus (hcv) in addition to their antibacterial and anticancer activity. particularly the antimalarial drugs chloroquine and amodiaquine displayed activity against viruses like dengue virus, west nile virus and ebola virus by interfering with viral entry and replication (savoia ). on the other hand, the compound teicoplanin isolated from an actinobacteria member, actinoplanes teichomyceticus, is a fermentation product that exerts bactericidal action through inhibiting bacterial cell wall biosynthesis. this semisynthetic glycopeptide teicoplanin showed a significant inhibitory activity against ebola envelope pseudotyped viruses in vero cells when used in the clinic. besides, teicoplanin and other glycopeptide antibiotics, including dalbavancin, oritavancin and telavancin, but not vancomycin, had inhibitory action against the entry of ebola virus, sars-cov and mers-cov transcription and replication-competent virus-like particles. with regard to teicoplanin's antiviral activity, various studies have reported about its action against hiv, hepatitis c virus, flaviviruses, coronaviruses, respiratory syncytial virus and influenza virus (colson and raoult ) . the world has benefited from the phenomenal discovery of penicillin by alexander fleming in and its development in the s by chain, florey, heatley and abraham at oxford. similarly, the s invention of important streptomycete products by waksman, woodruff, schatz and lechevalier at rutgers university has resulted in the selective action of antibiotics against pathogenic bacteria and fungi. ever since the invention of penicillin, microbes have played a very significant role in the discovery of newer natural product-based drugs. currently, over , active compounds of microbial origin including antimicrobials, antivirals and cytotoxic and immunosuppressive compounds, of which % are made by fungi and % by filamentous bacteria, the actinomycetes, are available (demain ) . cyclosporin , which is a fungus-derived potent immunosuppressant that acts through inhibition of cyclophilin, is found to have antiviral activity. nevertheless, its immunosuppressive and calcineurin-related side effects have made it impossible for use as an antiviral agent. therefore, continued search for structurally related cyclosporin analogues with minimal immunosuppressive activity and strong cyclophilin inhibitory action resulted in its derivative nim . on the contrary, nim had times less immunosuppressive activities than cyclosporin with a lesser toxicity profile and has demonstrated to possess anti-hiv and hcv activity. nim has passed evaluation in phase i trial for the treatment of hcv (butler ) . even though majority of natural products have been produced from terrestrial environments, marine organisms have also contributed quite a large number of bioactive compounds. between and , about bioactive compounds have been isolated from marine microbes only. various compounds with anticancer, antibacterial, antiviral, immunomodulatory and protease-inhibition activities have been isolated from marine cyanobacteria. marketed marine products include cytarabine (cytosar) for non-hodgkin's lymphoma, the antiviral vidarabine (vira-a), ziconotide (prialt) and trabectedin (yondelis) (demain ). the history of herbal drug use is widespread in both developed and developing countries, and they are still utilized because of several reasons such as fewer side effects, relatively less expensive, patient tolerance and acceptance due to long history of use (vermani and garg ) . veregen (polyphenon e ointment), which is a green tea leaf extract and a mixture of catechins, was the first herbal remedy to obtain fda approval in for treating genital warts. additionally, a perennial herb glycyrrhiza glabra has been in use for over years in japan for treatment of hepatitis. its dried and processed root licorice has a unique odour and sweet taste. various studies have investigated the pharmacological activity of licorice against viral hepatitis. a randomized controlled trial conducted on glycyrrhiza glabra derived compound glycyrrhizin and its derivatives demonstrated diminished hepatocellular damage in chronic hepatitis b and c (fiore et al. , indicating that it can be a potential antibiotic. furthermore, rutin has also been stated to inhibit replication of parasites, bacteria, fungi and viruses (rotavirus and hsv) (chiang et al. ) . on the other hand, in china and taiwan, ocimum basilicum is widely used traditionally against a number of infections. a number of compounds have been reportedly found from ocimum basilicum including monoterpenoids (carvone, cineole, fenchone, geraniol, linalool, myrcene and thujone), sesquiterpenoids (caryophyllene and farnesol), triterpenoid (ursolic acid) and flavonoid (apigenin). in particular, ursolic acid was shown to have inhibitory activity against herpes simplex virus (hsv)- and human immunodeficiency virus (hiv), as well as tumour growth (chiang et al. ). drug repurposing (or drug repositioning) is the method of assigning a new medical indication for an existing drug. the repositioned drug might be currently on the market for other medication, withdrawn due to adverse effects or proved to be less efficacious. as a matter of fact, most of the drug repositioning emerged as a result of beneficial side effects (by serendipity); however, current efforts to attain repurposing are accomplished in a more systematic way (naveja et al. ) . nowadays, the problem of antimicrobial drug resistances poses a growing threat to global public health and demands newer or repositioned drugs. with regard to utilizing already fda-approved drugs for another indication, the entities can be used for treating the new indication without any further structural modification of the compound at hand (though dosing and formulation could be modified) (savoia ; klug et al. ). the case of ebola virus outbreak in west africa that reached to a scale not ever seen in history was the greatest public health emergency. antibody-based therapy was proved effective in a macaque model and had been used to treat few patients; however, the supply of the drug was quite limited. therefore, drug repurposing was the best option to come up with an old drug with new indication to speed up the discovery and development of anti-ebola virus drugs for the treatment of patients with ebola virus infection. as a result, initial drug repurposing screen subsequently provided approved drugs with ebola virus-like particle entry-blocking activity including the macrolide antibiotics azithromycin and clarithromycin, which block bacterial protein synthesis (kouznetsova et al. ). finally, six antibiotics which inhibit ebola virus infection (azithromycin, erythromycin, spiramycin, dirithromycin, maduramicin, clarithromycin) were selected for anti-ebola activity out of fda-approved drugs (veljkovic et al. ) . there was no herbal therapy for zika virus infection; however recently, two antiviral agents have been approved by fda for zika virus infection (cheng et al. ) . it is important to design or develop a therapeutic approach to overcome zika virus infection with a special focus on drugs targeting the virus helicase protein, nucleosides, inhibitors of ns protein, small molecules, methyltransferase inhibitor and repurposed drugs. repurposed drugs such as chloroquine, azithromycin and niclosamide are used for the treatment of zika virus infection (munjal et al. ) . new studies revealed that alzheimer's drugs may moderate zika virus-mediated neuronal damage. so, alzheimer's disease drugs which overstimulate n-methyl-d-aspartate receptors (nmdars) lead to damage neuronal death interlinked with zika virus infection. therefore, blocking of nmdar channels with memantine and/or other antagonists helps to lessen the neuronal damage associated with zika virus infection, which act as a pre-approved drug from the food and drug authority (fda) which need more clinical trials (sirohi and kuhn ) . modification of specific or non-specific immune responses is a promising intervention for ongoing viral infections. the most suitable for immunotherapy are chronic viral infections (hegde et al. ). the first example is monoclonal antibodies, which are specific for one to one antigen or one epitope. in fibroblasts and neuroblastoma cells, monoclonal antibodies specific for nucleoprotein and nonstructural protein of the nucleocapsid have been shown to inhibit rabies virus, in a dose-dependent way, by impairing transcription of the genome or neutralizing newly translated proteins (lafon and lafage ) . in addition, another study found that monospecific antibody against rabies virus nucleoprotein recognizes lyssavirusspecific antigen (inoue et al. ) . monospecific antibodies have shown to be effective against non-capsid proteins of poliovirus (pasamontes et al. ) and various livestock diseases as rotaviral diarrhoea, bluetongue, classical swine fever, hendra and nipah viral infections (deb et al. ) . neutralization by antibody can be mediated by different mechanisms such as destabilization of the virion structure, aggregation of virions, inhibition of virion attachment to target cells, inhibition of the virion lipid membrane fusion with the membrane of the host cell, inhibition of the entry of the genome of non-enveloped viruses into the cell cytoplasm and inhibition of a function of the virion core through a signal transduced by an antibody (reading and dimmock ) . another therapeutic strategy for infectious viral diseases are recombinant antibodies, which, unlike monoclonal antibodies, do not need hybridomas and animals in the production process, but only synthetic antibody coding genes, and are delivered in high reproducibility, specificity and scalability (echko and dozier ) . examples include avian antibody against vp of infectious bursal disease virus protecting against viral infection in chicken (zhang et al. ) , porcine circovirus type (yang et al. ) , the e protein of classical swine fever virus (chen et al. ) and capsid protein of bovine immunodeficiency virus (bhatia et al. ) . antiviral drugs have been tested for various viral diseases of animals. antiviral therapy has been developed against a number of rna viral infections in livestock. first among them, against foot-and-mouth disease (fmd), involves vaccine that contains an inactivated whole-virus antigen. however, since vaccinated animals cannot be differentiated from the infected ones, the vaccine is not useful in eliminating fmd outbreaks from previously disease-free countries. hence, interferons (ifns) have emerged as another treatment agent, including ifn-α, ifn-β and ifn-γ, which are used both individually and synergistically. ifn-γ has been described to have several targets that possess antiviral properties, such as indoleamine , -dioxygenase and inducible nitric oxide synthase (moraes et al. ). one of the most widespread diseases in domestic livestock is caused by another rna viral infection, bluetongue virus (btv). an aminothiophenecarboxylic acid derivative named compound (c ) and its derivative compound (c ) have been identified as virostatic molecules against btv. they exert their effect by inhibiting btv-induced apoptosis via inhibition of caspase- /caspase- activation and inhibition of host autophagy activation (gu et al. ) . furthermore, feline herpes virus type (fhv- ) is a common cause of various diseases in cats, such as ocular surface disease, respiratory disease, dermatitis and potentially intraocular disease. a number of antiviral agents have been described against the virus, but the most effective antiviral therapies are the ones that target viral proteins involved in dna synthesis, many of which have been used against closely related human herpes simplex virus type . for example, nucleoside and nucleotide analogues have been reported for topical administration, such as vidarabine that affects dna polymerase and subsequently disrupts dna synthesis, trifluridine which acts as a fluorinated nucleoside analogue of thymidine and cidofovir which is a cytosine analogue acting on two host-mediated phosphorylation steps (thomasy and maggs ) . purine analogues and their oral prodrugs have also been described as well as other antiviral drugs, such as foscarnet that inhibits pyrophosphate binding site on viral dna polymerases, while numerous novel compounds have been investigated against fhv- including sirnas which target the fhv- glycoprotein d (gd) alone or jointly with dna polymerase genes (wilkes and kania ) . to conclude with, equine herpesvirus type (ehv- ) infection causes outbreak of respiratory and various neurological diseases in horses, against which acyclovir and valacyclovir are the most common drugs, but also ifn targeting ifngr complex as a key mediator of virus-specific cellular immunity (poelaert et al. ). feline immunodeficiency virus (fiv) is a complex lentivirus causing immunodeficiency disease in cats, manifested as the body's inability to develop normal immune response. as a retrovirus, it inserts copies of its genetic material into the dna of a host cell, where it can replicate. the most commonly used antiretroviral drugs are reverse transcriptase inhibitors (rtis), in particular the ones acting as nucleoside analogues, which are similar in structure to intrinsic nucleosides and can therefore block enzymatic activity by binding to the active centre of the enzyme (hartmann et al. ) . the first among them, zidovudine (azt), has been reported to improve the immunologic and clinical status of fiv-infected cats, increase quality of life as well as prolong life expectancy. it has been shown to increase the cd / cd ratio in naturally fiv-infected cats and acts by inhibiting rt but also cellular polymerases, which can lead to bone marrow suppression (hartmann ) . another drug acting as rti is stavudine that has been shown to be active against fiv in vitro, however with many resistant strains arisen. similarly, didanosine and lamivudine have shown potency against fiv in in vitro conditions (schwartz et al. ). additionally, a combination of zidovudine and lamivudine has been investigated, resulting in synergistic anti-fiv effects in cell cultures. also, a high-dose zidovudine/lamivudine combination was shown to protect from infection when treatment was initiated before virus inoculation (arai et al. ) . moreover, all of the above-mentioned antiviral agents are also effective against hiv infection. interferons (ifns) are a multigene family of inducible cytokines that possess antiviral activity. the ifn system comprises the cells synthesizing ifn in response to an external stimulus, such as viral infection, and cells that respond to ifn by establishing the antiviral state. they represent an early host defence, the one that occurs prior to the immune response onset. ifns are classified as ifn-α and ifn-β, which are produced by the cell in response to virus infection, and ifn-γ, synthetized upon antigen or mitogen stimulation (samuel ) . their activity has been reported against a number of feline viruses, including feline calicivirus (fcv), where they act by stimulating downstream genes such as ′- ′oligoadenylatedependent ribonuclease l (rnase l), which degrades single-stranded viral rnas. also, feline irf- , shown to be reduced upon fcv infection, has been reported to positively regulate ifn signalling by triggering the production of endogenous ifn and the expression of downstream targeted genes (liu et al. ) . as mentioned before, another feline virus reported for ifn therapeutic solutions is fiv. the best known among them, recombinant rfeifn-ω, is the first interferon compound that has been licensed for use in veterinary medicine, shown to significantly increase levels of acute phase proteins (apps) (doménech et al. ) . rfeifn-ω has also been reported for anti-inflammatory properties, exerted via interleukin- (il- ) (leal et al. ) . recombinant human interferon alpha- b (rhuifn-alpha b) and recombinant feline interferon omega (rfeifn-omega) have also exhibited an antiviral effect against feline herpesvirus (fhv)- in in vitro settings, as evidenced by significant reduction in plaque size (siebeck et al. ) . interferons also showed therapeutic potential against feline leukaemia virus (felv), with recombinant feline interferon rfeifn-omega, resulting in improvement of clinical signs and survival of infected cats (de mari et al. ) . idoxuridine and trifluridine are structurally similar thymidine analogues that inhibit synthesis of dna. they have been applied in the treatment of feline herpesvirus- (fhv- ), significantly reducing the number of viral plaques in vitro (nasisse et al. ). idoxuridine has also exhibited therapeutic potential against equine herpesvirus type (ehv- ) by alleviating the ocular symptoms caused by the infection (collinson et al. ) . it has also been reported that idoxuridine in the concentration of . % and . % trifluridine can limit the viral replication but do not kill the virus (plummer et al. ) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. however, they mostly selectively target only a single virus, known as a "one drug-one bug" principle. on the contrary, broad-spectrum antivirals (bsas) cover multiple viruses and genotypes, therefore reducing the likelihood of resistance development. they can, hence, reduce the complexity of the treatment, ensuring management of new or drug-resistant viral strains, first-line treatment or prophylaxis of acute infections, as well as co-infections (zhu et al. ) . against some viruses, such as hepatitis c, a direct-acting antiviral agents (daas) have been developed in the past few years. they act on ns / a protease inhibitors, ns a inhibitors or ns b inhibitors and ensure efficient, tolerable, safe and interferon-free oral therapies (das and pandya ) . furthermore, further development of antiviral agents will not only focus on viral factors as the potential targets for inhibition but also on the host factors as well, such as cellular receptors, adhesion molecules, cyclophilins and micrornas. therefore, an effort will be put on the combination of viral and host inhibitors, eventually leading to interferon-free therapies for consistent clearing of infection (bryan-marrugo et al. ) . perspectives for use of antiviral drugs in livestock animals are envisaged as the mass treatment for the control of the disease (on a large scale), whereas treatment in companion animals favours an individual approach. the main prerequisite for successful veterinary antiviral chemotherapy is a better understanding of the viral infection pathogenesis as well as development of sophisticated means for drug delivery. these will mainly focus on targeted approaches that aim specific molecular targets with a narrow niche, allowing for better specificity and less side effects of antiviral agents. advances in the field of molecular biology, in particular computational approaches, would contribute to development of a new generation of antiviral therapy, which would be of importance in the control of various kinds of animal diseases. the use of animal models for viruses of human and veterinary importance is still abundantly used to develop therapeutic agents. however, the current interest of these various viruses leads to multiple drug resistance due to the use of higherdosage therapies. the approaches are different for companion animals as a single method is preferred, while for large-scale livestock, mass treatment therapy is used; that is why antiviral drugs and other natural as well as herbal products are characterized through a novel and optimistic approach. still it is worthy to notice that these therapies lead to multiple drug resistance which should be overcome in the future. synthetic approaches to benzimidazoles from o-phenylenediamine: a literature review composition on the basis of phosphonoacetic acid. synthesis and antiviral activity is azt/ tc therapy effective against fiv infection or immunopathogenesis? combination chemotherapy: interaction of -methoxymethyldeoxyuridine with adenine arabinoside, -ethyldeoxyuridine, -iododeoxyuridine, and phosphonoacetic acid against herpes simplex virus types and single-chain fragment variable antibody against the capsid protein of bovine immunodeficiency virus and its use in elisa cham bracklein t et al ( ) activity of feline interferon-omega after ocular or oral administration in cats as indicated by mx protein expression in conjunctival and white blood cells high-content screening in infectious diseases history and progress of antiviral drugs: from acyclovir to directacting antiviral agents (daas) for hepatitis c natural products to drugs: natural product-derived compounds in clinical trials efficacy, toxicology and clinical applications of ribavirin against virulent rna viral infections synthesis and antiviral evaluation of trisubstituted indole n-nucleosides as analogues of , , -trichloro- -(β-d-ribofuranosyl) benzimidazole (tcrb) expression and characterization of a recombinant porcinized antibody against the e protein of classical swine fever virus drug repurposing: new treatments for zika virus infection? in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids antiviral activities of extracts and selected pure constituents of ocimum basilicum isolation of equine herpesvirus type (equine gammaherpesvirus ) from foals with keratoconjunctivitis fighting viruses with antibiotics: an overlooked path structure-activity relationship studies of new rifamycins containing l-amino acid esters as inhibitors of bacterial rna polymerases antiviral chemotherapy in veterinary medicine: current applications and perspectives inhibitory effect of penciclovir-triphosphate on duck hepatitis b virus reverse transcription recent advancement of direct-acting antiviral agents (daas) in hepatitis c therapy approved antiviral drugs over the past years therapeutic effects of recombinant feline interferon-co on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats the disposition of acyclovir in different species monoclonal antibody and its use in the diagnosis of livestock diseases importance of microbial natural products and the need to revitalize their discovery identification of aldehyde oxidase as the neonicotinoid nitroreductase antiviral chemotherapy and chemoprophylaxis use of recombinant interferon omega in feline retrovirosis: from theory to practice recombinant antibody technology for the production of antibodies without the use of animals veterinary virology metabolic and pharmacokinetic studies following oral administration of c-famciclovir to healthy subjects antiviral effects of glycyrrhiza species prevention of rhinovirus and poliovirus uncoating by win , a new antiviral drug feline herpesvirus the clinical pharmacokinetics of famciclovir study of antiviral activity of different drugs against bovine herpes virus and pestivirus novel virostatic agents against bluetongue virus antiviral therapy feline immunodeficiency virus infection: an overview efficacy of antiviral drugs against feline immunodeficiency virus the use of rnai-based screens to identify host proteins involved in viral replication metal chelators as potential antiviral agents cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues antiviral activity of arildone on deoxyribonucleic acid and ribonucleic acid viruses repurposing strategies for tropical disease drug discovery penciclovir is a selective inhibitor of hepatitis b virus replication in cultured human hepatoblastoma cells identification of compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs preliminary studies of the mode of action of arildone, a novel antiviral agent antiviral activity of monoclonal antibodies specific for the internal proteins n and ns of rabies virus evaluation of viremia, proviral load and cytokine profile in naturally feline immunodeficiency virus infected cats treated with two different protocols of recombinant feline interferon omega drug repurposing of minocycline against dengue virus infection identification of feline interferon regulatory factor as an efficient antiviral factor against the replication of feline calicivirus and other feline viruses in vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type- treatment of feline herpesvirus- associated disease in cats with famciclovir and related drugs inhibition of dna polymerase from herpes simplex virus-infected wi- cells by phosphonoacetic acid lead generation using pharmacophore mapping and three-dimensional database searching: application to muscarinic m receptor antagonists prevention of human poliovirus-induced paralysis and death in mice by the novel antiviral agent arildone progress in the development of poliovirus antiviral agents and their essential role in reducing risks that threaten eradication inhibition of uncoating of poliovirus by arildone, a new antiviral drug design, synthesis, and antiviral activity of α-nucleosides: d-and l-isomers of lyxofuranosyl-and ( -deoxylyxofuranosyl) benzimidazoles enhanced antiviral activity against foot-and-mouth disease virus by a combination of type i and ii porcine interferons advances in developing therapies to combat zika virus: current knowledge and future perspectives ribavirin inhibits parrot bornavirus replication in cell culture feline herpesvirus ocular disease in vitro susceptibility of feline herpesvirus- to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine drug repurposing for epigenetic targets guided by computational methods synergistic drug combinations against the in vitro replication of coxsackie b virus antiretroviral drugs, genotoxicity, and carcinogenesis. in: carcinogens, dna damage and cancer risk: mechanisms of chemical carcinogenesis inhibition of herpes simplex virus replication by phosphonoacetic acid production of monoclonal and monospecific antibodies against non-capsid proteins of poliovirus kidney disease caused by therapeutic agents ocular infections abortigenic but not neurotropic equine herpes virus modulates the interferon antiviral defense design, synthesis, and antiviral evaluations of -(substituted benzyl)- -substituted- , -dichlorobenzimidazoles as nonnucleoside analogues of , , -trichloro- -(β-d-ribofuranosyl) benzimidazole effect of orally administered ribavirin on experimental feline calicivirus infection in cats new approaches to antiviral drug discovery (genomics/proteomics) hoboken rollinson e ( a) prospects for antiviral chemotherapy in veterinary medicine: . feline virus diseases prospects for antiviral chemotherapy in veterinary medicine: . avian, piscine, canine, porcine, bovine and equine virus diseases pharmacokinetics of acyclovir in tragopans (tragopan sp.) drugs for herpesvirus infections prevalence, diagnosis, management and control of important diseases of ruminants with special reference to indian scenario antiviral actions of interferons new antimicrobial approaches: reuse of old drugs desk encyclopedia of microbiology. academic, amsterdam schwartz am et al ( ) antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells synthesis, characterization and pharmacological screening of novel benzimidazole derivatives in vitro antiviral activity of penciclovir, a novel purine nucleoside, against duck hepatitis b virus suppression of herpes simplex virus infection by phosphonoacetic acid effects of human recombinant alpha- b interferon and feline recombinant omega interferon on in vitro replication of feline herpesvirus- can an fda-approved alzheimer's drug be repurposed for alleviating neuronal symptoms of zika virus synthesis, antiviral and antitumor activity of -substituted- -amidinobenzimidazoles treatment of cats with ocular disease attributable to herpesvirus infection: cases ( - ) canine and feline infectious diseases-e-book antiviral and immunomodulatory drugs. canine and feline infectious diseases inhibition of infectious rous sarcoma virus production by rifamycin derivative adaptation of high-throughput screening in drug discovery-toxicological screening tests hepatitis c therapeutics: current status and emerging strategies mechanism of action of ribavirin in the treatment of chronic hepatitis c feline herpesvirus infection. abcd guidelines on prevention and management a review of antiviral drugs and other compounds with activity against feline herpesvirus type pharmacokinetics and safety of penciclovir following oral administration of famciclovir to cats pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study mechanism of action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis c virus rna-dependent rna polymerase design, synthesis, and antiviral activity of certain , , -trihalo- -(. beta.-d-ribofuranosyl) benzimidazoles diversity screening versus focussed screening in drug discovery virtual screen for repurposing approved and experimental drugs for candidate inhibitors of ebola virus infection herbal medicines for sexually transmitted diseases and aids new substituted benzimidazole derivatives: a patent review evaluation of free or liposome-encapsulated ribavirin for antiviral therapy of experimentally induced feline infectious peritonitis evaluation of the effects of small interfering rnas on in vitro replication of feline herpesvirus- synthesis and antiviral activity of , , -triazole- -thiocarboxamide and , , -triazole- -carboxamidine ribonucleosides selection and identification of single-domain antibody fragment against capsid protein of porcine circovirus type (pcv ) from c. bactrianus african swine fever virus: current state and future perspectives in vaccine and antiviral research a recombinant avian antibody against vp of infectious bursal disease virus protects chicken from viral infection broad-spectrum antiviral agents acknowledgements all the authors of the manuscript thank and acknowledge their respective universities and institutes. there is no conflict of interest. key: cord- - ta fg authors: van norman, gail a. title: expanding patient access to investigational new drugs: overview of intermediate and widespread treatment investigational new drugs, and emergency authorization in public health emergencies date: - - journal: jacc basic transl sci doi: . /j.jacbts. . . sha: doc_id: cord_uid: ta fg individual patients with life-threatening or severely debilitating diseases can petition the u.s. food and drug administration (fda) through their physicians to have expanded access (ea) to drugs that are in clinical trials but have not reached full fda approval (the “single-patient” investigational new drug [ind] application). additionally, recent state and federal laws—so-called “right to try legislation”—allow patients to approach drug companies directly for access prior to fda approval. while these pathways provide potential access for individual patients to investigational drugs, different ea pathways permit entire groups of certain patients to access investigational drugs prior to fda approval. this review focuses on special categories of ea inds intended for multiple patients—the intermediate-group ind and the widespread-treatment ind—as well as emergency authorization for use of investigational drugs and biological products (e.g., vaccines) in public health emergencies. for patients with life-threatening or severely debilitating disease, the wait for approval is simply too long, and can both abolish hope for those who diseases will be quickly fatal, and lead to sustained or even permanent disability for those whose diseases linger but are without effective proven therapies. spurred by patient advocacy during the early days of the acquired immunodeficiency syndrome (aids) epidemic in the late s, and facilitated by subsequent legislative efforts over the next years, regulatory initiatives permit the fda to release drugs for use in individual patients through expanded access (ea) inds ( , ) , in many cases allowing emergency treatment with nonapproved drugs within hours of application, and nonemergency treatment within an average of days ( ) . further, most states have enacted so-called "right to try" legislation, permitting "compassionate use" of investigational drugs by individual patients through applications directly to the manufacturer ( ) . it should be noted that although the terms "compassionate use" or "preapproval access" are often used informally to refer to the use of an investigational drug to treat a patient outside of a clinical trial, these terms are not defined or described in fda regulations, which simply refer to expanded access to investigational drugs. the call for ea is not limited to individual patients. advocacy organizations have pressed for groups of patients with rare and/or "orphan" diseases, for example, to be able to access promising new therapies prior to their approval. indeed, social media is increasingly becoming a consumer/patient advocacy tool for implementing fda regulatory changes and promoting access to investigational therapeutics ( ) . in addition, once a drug has completed phase testing and is awaiting approval, patients who have benefited from in-trial treatments may want continued therapy, and such use requires some form of "bridging approval" from the fda to allow potentially large groups of patients to continue treatment while final fda approval is pending. a previous review discussed individual patient emergency and nonemergency access to investigational drugs ( ) . this review will focus on fda ea for intermediate-sized groups of patients (the "intermediate-sized ind") and ea for entire classes of patients (the "widespread treatment use" ind), as well as emergency release of investigational drugs and biologics for use in public health emergencies. releasing investigational new drugs to individual patients who are facing certain death or disability seems to be a relatively uncomplicated decision, but allowing ea to entire groups of patients for treatment with an investigational new drug presents more complex regulatory, logistical, and ethical challenges for scientists, commercial entities, and the fda. the current regulatory process from ind filing to drug approval has evolved and includes not only the fda's historical primary mission of ensuring patient safety, but also, since the latter half of the th century, the newer mission of ensuring that marketed drugs are actually efficacious for their advertised/approved use. ea for a single patient may not present much of a challenge to the assertion that a drug's benefits outweigh the risks, because as presumably the patient requesting compassionate use faces an otherwise dismal clinical future, taking even significant risks with a new drug still presents potential benefits to a patient without other options. early in a drug's regulatory pathway, however, it is not usually possible to ensure that a drug has a reasonable risk/benefit ratio for all patients, including those in the early stages of disease. drug companies face bigger issues when the seeker of ea is a group of patients or an entire class of patients. before marketing, manufacture of the drug for clinical studies is nearly an "all cost" proposition for the commercial entity; the drug cannot be marketed to cover its costs. thus, companies generally only manufacture sufficient quantities (plus a small margin) to cover the requirements of clinical studies, rather than devote resources to manufacturing large quantities of a drug which has a < % chance of ever making it to market ( , ) . the fda approval process begins with the filing of an investigational new drug (ind). making the drug available to groups or classes of patients who might then deplete the supply of drug for clinical studies could compromise the very research that would more completely disclose a drug's risks and benefits; thus, it could possibly impede full market approval that would make the drug more widely available. companies have also expressed concern about how data from such "compassionate use" may be applied in the approval process. for cmv infection ( ) . later studies also showed that ganciclovir patients were living longer ( ). the fda refused approval of ganciclovir for treatment of cmv retinitis, because they had no animal studies for that use, nor significant human placebocontrolled trials on which to base a marketing application. many questioned whether the use of ganciclovir was wise, or safe ( , ) . but, because of ganciclovir's known efficacy, it became paradoxically impossible to carry out human controlled trials, because such trials are only ethically justifiable if investigators are honestly uncertain about whether net positive benefits over placebo exists ( ) . furthermore, neither patients nor doctors were willing to risk assignment to placebo and further loss of eyesight after the results were published. syntex then sought approval to study ganciclovir for treatment of cmv colitis, knowing that once marketing approval of the drug was obtained, fda rules would allow "off-label" use for retinitis ( ) . at this time, ea of nonapproved drugs for treatment of more than patient at a time is achievable only through the fda, in contrast with access for individual patients, which technically can be legally obtained without the fda by applying to the manufacturer directly ( ) . as with individual ea inds, specific conditions for group patient access apply: ) the patients must have a serious or immediately life-threatening disease or condition with no comparable therapy or satisfactory alternative therapy; ) the potential benefit must justify the potential risks of the treatment; and ) providing the treatment must not compromise or interfere with the ongoing fda drug development program, such as by critically depleting a limited supply of investigational drug that is also needed for an ongoing study or a future study that is in the planning stages ( ). an "immediately life-threatening condition or disease" is defined by the fda as "a stage of disease in which there is reasonable likelihood that death will occur within a matter of months or in which premature death is likely without early treatment." a serious disease or condition is defined as being "associated with morbidity that has substantial impact on day-to-day functioning." furthermore, while short-lived or self-limited morbidity will usually not be a sufficient qualifying condition, the morbidity "need not be irreversible, provided it is persistent or recurrent." the fda states that whether a condition is serious or not "is a matter of clinical judgment, based on its impact on such factors as survival, day-to-day functioning, or the likelihood that the disease, if left untreated, will progress from a less severe condition to a more serious one" ( ) . and other animal species such as cats, armadillos, guinea pigs, swine and ferrets in which thalidomide had been tested, teratogenic effects had been induced only occasionally" ( ) . in fact, when human birth defects began to appear in the offspring of women who had ingested thalidomide during pregnancy as a sedative and to treat nausea, researchers pointed out that thalidomide had failed to demonstrate teratogenicity in rats, and at first insisted that thalidomide could not be the culprit. in germany, where the drug was first developed, thalidomide was held to be so safe that no prescription was required for its use, it was advertised for use in pregnant women ( ) , and the drug company distributed free samples to its factory employees ( , ) . given the most severe rating for drugs that contribute to fetal deformities, and for drugs whose risks prioritizing those who should receive treatment, such as women and children ( ) . the fact that the treatment was made available to u.s. citizens and not to africans, who comprised most of its victims, engendered anger over the social justice of such decisions, providing, as enserink ( ) points out, a tragic validation to the satirical yet somewhat prophetic paper that had appeared in the onion only weeks before titled "experts: ebola vaccine at least white people away" ( ) . as of now, products have been approved under the animal rule, of which were issued quickly after the guidance was published ( table there is a reasonably well-understood pathophysiological mechanism of the toxicity of the toxic substance and its prevention or substantial reduction by the product. the effect is demonstrated in more than animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans. the animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity. the data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans. drugs, devices, and the fda: part understanding fda regulatory requirements for investigational new drug applications for sponsor-investigators policy and procedures: office of new drugs: ind clinical holds expanded access (compassionate use) early access programs: benefits, challenges and key considerations for successful implementation expanding patient access to investigational drugs: single patient inds and the "right to try going "social" to access experimental and potentially life-saving treatment: an assessment of the policy and on-line patient advocacy environment for expanded access compassionate use: a story of ethics and science in the development of a new drug -propoxymethyl)guanine in patients with aids and other immunodeficiencies oral ganciclovir for the prevention of cytomegalovirus disease in persons with aids blinded by science: dhpg and the conflict between "clean data" and humane health care (abstract th.g.p. ). paper presented at: int conf aids medicine: drugs from the underground the question of clinical equipoise and patients' best interests off-label" and investigational use of marketed drugs, biologicals and medical devices-information sheet expanded access to investigational drugs for treatment usequestions and answers; guidance for industry food and drug administration. food and drug administration amendments act (faaa) of administrationamendmentsactof /default.htm fda basics webinar: a brief overview of risk evaluation and mitigation strategies (rems) drugs as teratogens the death and afterlife of thalidomide. retro report. the new york times changing the face of medicine use of thalidomide in leprosy years after thalidomide: why regulation matters kefauver-harris amendments revolutionized drug development thalidomide approved to treat leprosy, with other uses seen. the new york times a comprehensive program for controlling and monitoring access to thalidomide the embryo project encyclopedia. us regulatory response to thalidomide project bioshield act of project bioshield: what is it, why is it needed, and its accomplishments so far regulatory information: emergency use authorization of medical products summary of process for eua issuance considerations for use of investigational drugs in public health emergencies food and drug administration how two u.s. patients changed the debate about using untested ebola drugs world health organization. ethical considerations for use of unregistered interventions for ebola virus diseases: report of an advisory panel to who. who, geneva switzerland _eng.pdf?ua¼ . accessed experts: ebola vaccine at least white people away fda product development under the animal rule: guidance for industry working with the u.s. food and drug administration to obtain approval of products under the animal rule food and drug administration ebola virus disease survivors: clinical and immunologic follow-up prevail iv: double-blind, randomized two-phase, placebo-controlled phase ii trial of gs- to assess the antiviral activity, longer-term clearance of ebola virus and safety in male ebola survivors with evidence of ebola virus persistence expanded access (compassionate use): ind submissions medicine, university of washington, th avenue west, seattle, washington . e-mail: lbsparrow@ yahoo.com. key: cord- -q sx dm authors: cacabelos, ramón title: pharmacogenomic biomarkers in neuropsychiatry: the path to personalized medicine in mental disorders date: journal: the handbook of neuropsychiatric biomarkers, endophenotypes and genes doi: . / - - - - _ sha: doc_id: cord_uid: q sx dm neuropsychiatric disorders and dementia represent a major cause of disability and high cost in developed societies. most disorders of the central nervous system (cns) share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways, poor therapeutic outcomes, and chronic disability. recent advances in genomic medicine can contribute to accelerate our understanding on the pathogenesis of cns disorders, improve diagnostic accuracy with the introduction of novel biomarkers, and personalize therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. the pharmacological treatment of cns disorders, in general, accounts for – % of direct costs, and less than – % of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). pharmacogenetic and pharmacogenomic factors may account for – % of drug variability in drug disposition and pharmacodynamics. approximately – % of cns drugs are metabolized via enzymes of the cyp gene superfamily; % of neuroleptics are major substrates of cyp a enzymes, % of cyp d , and % of cyp a ; % of antidepressants are major substrates of cyp a enzymes, % of cyp b , % of cyp c , % of cyp d , and % of cyp a ; % of benzodiazepines are major substrates of cyp c enzymes, % of cyp d , and % of cyp a . about – % of caucasians are carriers of defective cyp d polymorphic variants that alter the metabolism of many psychotropic agents. other genes participate in the efficacy and safety of psychotropic drugs. the incorporation of pharmacogenetic/ pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety. to achieve this goal several measures have to be taken, including: (a) educate physicians and the public on the use of genetic/ genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. central nervous system (cns) disorders are the third problem of health in developed countries, representing - % of deaths, after cardiovascular disorders ( - %) and cancer ( - %) . approximately, million europeans suffer brain disorders. the total annual cost of brain disorders in europe is about € billion, with € billion of direct medical expenditures (€ billion, inpatients; € billion, outpatients; € billion, pharmacological treatment), € billion of indirect costs (lost workdays, productivity loss, permanent disability), and € billion of direct non-medical costs. mental disorders represent € billion ( % of the total cost, excluding dementia), followed by neurological diseases (€ billion, %). senile dementia is becoming a major problem of health in developed countries, and the primary cause of disability in the elderly. alzheimer's disease (ad) is the most frequent form of dementia ( - %), followed by vascular dementia ( - %) , and mixed dementia ( - %) . these prevalent forms of agerelated neurodegeneration affect more than million people at present, and probably more than million people will be at risk in the next - years worldwide. the prevalence of dementia increases exponentially from approximately % at - years of age to more than - % in people older than years. it is very likely that in those patients older than - years of age most cases of dementia are mixed in nature (degenerative + vascular), whereas pure ad cases are very rare after years of age. the average annual cost per person with dementia ranges from € , to , , depending upon disease stage and country, with a lifetime cost per patient of more than € , . in some countries, approximately % of the global costs of dementia (direct + indirect costs) are assumed by the patients and/or their families. about - % of the costs in dementia are attributed to pharmacological treatment, including anti-dementia drugs, psychotropics (antidepressants, neuroleptics, anxiolytics), and other drugs currently prescribed in the elderly (antiparkinsonians, anticonvulsants, vasoactive compounds, antiinfl ammatory drugs, etc). in addition, during the past years more than drugs have been partially or totally developed for ad, with the subsequent costs for the pharmaceutical industry, and only drugs with moderate-to-poor effi cacy and questionable cost-effectiveness have been approved in developed countries. [ ] [ ] [ ] the lack of accurate diagnostic markers for early prediction and an effective therapy of cns disorders are the two most important problems to effi ciently diagnose and halt disease progression. the pharmacological treatment of cns disorders, in general, accounts for - % of direct costs, and less than - % of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). in the case of dementia, less than % of the patients can benefi t from current drugs (donepezil, rivastigmine, galantamine, memantine), with doubtful cost-effectiveness. the pathogenic mechanisms of most cns disorders (e.g., psychosis, depression, anxiety, alzheimer's disease, parkinson's disease, huntington's disease, multiple sclerosis, etc) are poorly understood. this circumstance makes it diffi cult the implantation of a molecular intervention to neutralize causative factors. in fact, more than % of the , genes integrating the human genome are expressed in the cns at different periods of the life span, and only a few neurotransmitters (e.g., noradrenaline, dopamine, acetylcholine, gaba, histamine, and less than ten neuropeptides) are the actual targets of conventional psychopharmacology. common features in cns disorders include the following: (a) polygenic/ complex disorders in which genomic and environmental factors are involved; (b) deterioration of higher activities of the cns; (c) multifactorial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegeneration. for instance, the neuropathological hallmark of alzheimer's disease (ad) (amyloid deposition in senile plaques, neurofi brillary tangle formation, and neuronal loss) is but the phenotypic expression of a pathogenic process in which more than genes and their products are potentially involved. drug metabolism, and the mechanisms underlying drug effi cacy and safety, are also genetically regulated complex traits in which hundreds of genes cooperatively participate. structural and functional genomics studies demonstrate that genomic factors, probably induced by environmental factors, cerebrovascular dysfunction, and epigenetic phenomena, might be responsible for pathogenic events leading to premature neuronal dysfunction and/or death. pharmacogenetic and pharmacogenomic factors may account for - % of drug variability in drug disposition and pharmacodynamics. about - % of caucasians are carriers of defective cyp d polymorphic variants that alter the metabolism of many psychotropic agents. the incorporation of pharmacogenetic/pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug effi cacy and safety. [ ] [ ] [ ] extensive molecular genetics studies carried out in the past decades have demonstrated that most cns disorders are multifactorial, polygenic/complex disorders in which hundreds of genes distributed across the human genome might be involved (tables . - . ). , for example, genes have been associated with dementia (table . ), with schizophrenia (table . ), with depression (table . ), with anxiety, with stroke, with different types of ataxia, with epilepsiy, with meningioma, with glioblastoma, with astrocytoma, with parkinson's disease, and more than genes with cerebrovascular disorders. , many of these genetic associations could not be replicated in different settings and different populations due to many complex (methodological, technological) factors. , , furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an apparently common phenotype, this increasing the complexity of genomic studies (e.g., patient recruitment, pure controls, concomitant pathology, epigenetic factors, environmental factors). several candidate genes for schizophrenia may also be associated with bipolar disorder, including g , disc , nrg , rgs , ncam , dao, grm , grm , grin b, mlc , syngr , and slc a . genes associated with bipolar disorder include trpm ( q . ), gpr (xq ), citron ( q ), chp . ( p . ), gchi ( q - ), mlc ( q ), gabra ( q -q ), bcr ( q ), cux , flj ( q -q ), and napg ( p ). another paradigmatic example of heterogeneity and complexity is dementia, one of the most heterogeneous disorders of the cns. the genetic defects identifi ed in ad during the past years can be classifi ed into three main categories: (a) mendelian or mutational defects in genes directly linked to ad, including (i) mutations in the amyloid beta (aβ)(abp) precursor protein (app) gene ( q ); (ii) mutations in the presenilin (ps ) gene ( q . ); and (iii) mutations in the presenilin (ps ) gene ( q -q ) , , (table . ). (b) multiple polymorphic variants of risk characterized in more than different genes distributed across the human genome can increase neuronal vulnerability to premature death (table . ). among these genes of susceptibility, the apolipoprotein e (apoe) gene ( q . ) is the most prevalent as a risk factor for ad, especially in those subjects harbouring the apoe- allele, whereas carriers of the apoe- allele might be protected against dementia. apoe-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of ad. (c) diverse mutations located in mitochondrial dna (mtdna) through heteroplasmic transmission can infl uence aging and oxidative stress conditions, conferring phenotypic heterogeneity. , , it is also likely that defective functions of genes associated with longevity may infl uence premature neuronal survival, since neurons are potential pacemakers defi ning life span in mammals. all these genetic factors may interact in still unknown genetic networks leading to a cascade of pathogenic events characterized by abnormal protein processing and misfolding with subsequent accumulation of abnormal proteins (conformational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport, and chaperone misoperation , - ( fig. . ). these pathogenic events may exert an additive effect, converging in fi nal pathways leading to premature neuronal death. some of these mechanisms are common to several neurodegenerative disorders which differ depending upon the gene(s) affected and the involvement of specifi c genetic networks, together with cerebrovascular factors, epigenetic factors (dna methylation) and environmental conditions (nutrition, toxicity, social factors, etc). , [ ] [ ] [ ] [ ] [ ] [ ] [ ] the higher the number of genes involved in ad pathogenesis, the earlier the onset of the disease, the faster its clinical course, and the poorer its therapeutic outcome. , [ ] [ ] [ ] [ ] [ ] high throughput microarray gene expression profi ling is an effective approach for the identifi cation of candidate genes and associated molecular pathways implicated in a wide variety of biological processes or disease states. the cellular complexity of the cns (with different cell types) and synapses (with each of the neurons in the brain having around - synapses with a complex multiprotein structure integrated by different proteins) requires a very powerful technology for gene expression profi ling, which is still in the very early stages and is not devoid of technical obstacles and limitations. transcripts of , genes have been measured in different cns regions. each region possess its own unique transcriptome fi ngerprint that is independent of age, gender and energy intake. less than % of genes are affected by age, diet or gender, with most of these changes occurring between middle and old age. gender and energy restriction have robust infl uences on the hippocampal transcriptome of middle-aged animals. prominent functional groups of age-and energy-sensitive genes are those encoding proteins involved in dna damage responses, mitochondrial and proteasome functions, cell fate determination and synaptic vesicle traffi cking. the systematic transcriptome dataset provides a window into mechanisms of neuropathogenesis and cns vulnerability. with the advent of modern genomic technologies, new loci have been associated with different neuropsychiatric disorders, and novel pathogenic mechanisms have been postulated. cryptic chromosome imbalances are increasingly acknowledged as a cause for mental retardation and learning disability. with subtelomeric screening, nine chromosomal anomalies and submicroscopic deletions of pter, qter, pter, qter and qter have been identifi ed in patients with mental retardation. increased dna fragmentation was observed in non-gabaergic neurons in bipolar disorder, suggesting that non-gabaergic cell may be selectively vulnerable to oxidative stress and apoptosis in patients with bipolar disorder. [ ] [ ] [ ] [ ] with laser microdissection, rna amplifi cation, and array hybridization, expression of more than , genes was detected in ca and ca hippocampal neurons under normoxic conditions. the comparison of each region under normoxic and ischemic conditions revealed more than , ischemia-regulated genes for each individual cell type. microarray technology has helped to elucidate gene expression profi les and potential pathogenic mechanisms in many other cns disorders including schizophrenia and bipolar disorder, [ ] [ ] [ ] speech and language disorders, parkinson's disease, , huntington's disease, prion disease, drug addiction, , alcoholism, brain trauma, epilepsy, [ ] [ ] [ ] cockayne syndrome, rett syndrome, friedreich ataxia, neuronal ceroid lipofuscinosis, multiple sclerosis, amyotrophic lateral esclerosis, acute pneumococcal meningitis, and the role of lipids in brain injury, psychiatric disorders, and neurodegenerative diseases. [ ] [ ] [ ] interactions between genomic factors and environmental factors have been proposed as important contributors for brain neuropathology. in schizophrenia, neurodevelopmental disturbances, neurotoxins and perinatal infections, myelin-and olygodendrocytes abnormalities and synaptic dysfunctions have been suggested as pathophysiological factors. individual genotoxicants can induce distinct gene expression signatures. exposure of the brain to environmental agents during critical periods of neuronal development can alter neuronal viability and differentiation, global gene expression, stress and immune response, and signal transduction. the binomial genome-neurotoxicants effect can be documented in cases of drug abuse or alcohol dependence. functional gene expression differences between inbred alcohol-preferring and nonpreferring rats suggest the presence of powerful genomic infl uences on alcohol dependence. alcohol dependence and associated cognitive impairment may result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. it has been suggested that cycles of alcohol intoxication/withdrawal, which may initially activate nuclear factor-kappa b (nf-κb), when repeated over years downregulate p (rela) mrna expression and nf-κb and p homodimer dna-binding. downregulation of the dominant p homodimer, a potent inhibitor of gene transcription apparently results in depression of κb regulated genes. alterations in expression of p homodimer/nf-κb regulated genes may contribute to neuroplastic adaptation underlying alcoholism. gene expression profi ling of the nucleus accumbens of cocaine abusers suggests a dysregulation of myelin. humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and golgi/er function. another important issue in the pathogenesis and therapeutics of cns disorders is the role of micror-nas (mirnas). mirnas are small ( nucleotide), endogenous noncoding rna molecules that posttranscriptionally regulate expression of protein-coding genes. computational predictions estimate that the vertebrate genomes may contain up to , mirna genes. mirnas are generated from long primary transcripts that are processed in multiple steps to cytoplasmic nucleotide mature mirnas. the mature mirna is incorporated into the mirna-induced silencing complex (mirisc), which guides it to target sequences located in ′ utrs where by incomplete base-pairing induce mrna destabilization or translational repression of the target genes. an inventory of mirna expression profi les from regions of the mouse cns has been reported. this inventory of cns mirna profi les provides an important step toward further elucidation of mirna function and mirna-related gene regulatory networks in the mammalian cns. the introduction of novel procedures into an integral genomic medicine protocol for cns disorders is an imperative requirement in drug development and in the clinical practice to improve diagnostic accuracy and to optimize therapeutics. this kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) cardiovascular evaluation, (v) conventional x-ray technology, (vi) structural neuroimaging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenomics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artifi cial intelligence procedures for diagnostic assignments and probabilistic therapeutic options (table . ). , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , all these procedures, under personalized strategies adapted to the complexity of each case, are essential to depict a clinical profi le based on specifi c biomarkers correlating with individual genomic profi les. functional genomics studies have demonstrated the infl uence of many genes on cns pathogenesis and phenotype expression (tables . - . ). taking ad as an example, it has been demonstrated that mutations in the app, ps , ps , and mapt genes give rise to wellcharacterized differential neuropathological and clinical phenotypes of dementia. the analysis of genotypephenotype correlations has also revealed that the presence of the apoe- allele in ad, in conjunction with other genes, infl uences disease onset, brain atrophy, cerebrovascular perfusion, blood pressure, β-amyloid deposition, apoe secretion, lipid metabolism, brain bioelectrical activity, cognition, apoptosis, and treatment outcome. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] the characterization of phenotypic profi les according to age, cognitive performance (mmse and adas-cog score), serum apoe levels, serum lipid levels including cholesterol (cho), hdl-cho, ldl-cho, vldl-cho, and triglyceride (tg) levels, as well as serum nitric oxide (no), β-amyloid, and histamine levels, reveals sex-related differences in % of the biological parameters and almost no differences ( . %) when patients are classifi ed as apoe- (−) and apoe- (+) carriers, probably indicating that genderrelated factors may infl uence these parametric variables more powerfully than the presence or absence of the apoe- allele; in contrast, when patients are classifi ed according to their apoe genotype, dramatic differences emerge among apoe genotypes (> %), with a clear biological disadvantage in apoe- / carriers who exhibit (i) earlier age of onset, (ii) low apoe levels, (iii) high cho and ldl-cho levels, and (iv) low no, β-amyloid, and histamine levels in blood. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] these phenotypic differences are less pronounced when ad patients are classifi ed according to their ps ( . %) or ace genotypes ( . %), refl ecting a weak impact of ps -and ace-related genotypes on the phenotypic expression of biological markers in ad. ps related genotypes appear to infl uence age of onset, blood histamine levels and cerebrovascular hemodynamics, as refl ected by signifi cant changes in systolic (sv), diastolic (dv), and mean velocities (mv) in the left middle cerebral arteries (mca). ace-related phenotypes seem to be more infl uential than ps genotypes in defi ning biological phenotypes, such as age of onset, cognitive performance, hdl-cho levels, ace and no levels, and brain blood fl ow mv in mca. however, when apoe and ps genotypes are integrated in bigenic clusters and the resulting bigenic genotypes are differentiated according to their corresponding phenotypes, an almost logarithmic increased expression of differential phenotypes is observed ( . % variation), indicating the existence of a synergistic effect of the bigenic (apoe + ps ) cluster on the expression of biological markers, apparently unrelated to app/ps mutations, since none of the patients included in the sample were carriers of either app or ps mutations. , , these examples illustrate the potential additive effects of ad-related genes on the phenotypic expression of biological markers. furthermore, the analysis of genotype-phenotype correlations with a monogenic or bigenic approach documents a modest genotype-related variation in serum amyloid-β (abp) levels, suggesting that peripheral levels of abp are of relative value as predictors of disease-stage or as markers of disease progression and/ or treatment-related disease-modifying effects. , , the peripheral levels of abp in serum exhibit an apoe-dependent pattern according to which both apoe- (+) and apoe- (+) carriers tend to show higher abp levels than apoe- (−) or apoe- carriers , - ( fig. est concentration of serum histamine is systematically present in apoe- (+) and apoe- (+) carriers, and the highest levels of histamine are seen in apoe- (+) carriers ( fig. . ). central and peripheral histaminergic mechanisms may regulate cerebrovascular function in ad, which is signifi cantly altered in apoe- / carriers. , [ ] [ ] [ ] [ ] [ ] [ ] these observations can lead to the conclusion that the simple quantifi cation of biochemical markers in fl uids or tissues of ad patients with the aim of identifying pathogenic mechanisms and/or monitoring therapeutic effects, when they are not accompanied by differential genotyping for sample homogenization, are of very poor value. differential patterns of apoe-, ps -, ps -, and trigenic (apoe + ps + ps ) cluster-related lymphocyte apoptosis have been detected in ad. fas receptor expression is signifi cantly increased in ad, especially in apoe- carriers where lymphocyte apoptosis is more relevant. , it has been demonstrated that brain activity slowing correlates with progressive gds staging in dementia , , [ ] [ ] [ ] (fig. . ). in the general population subjects harbouring the apoe- / genotype exhibit a premature slowing in brain mapping activity represented by increased slow delta and theta activities as compared with other apoe genotypes. in patients with ad, slow activity predominates in apoe- carriers with similar gds stage , , - ( fig. . ) . ad patients harbouring the apoe- / genotype also exhibit a dramatically different brain optical topography map refl ecting a genotype-specifi c differential pattern of neocortical oxygenation as well as a poorer activation of cortical neurons in response to somatosensory stimuli ( fig. . ). all these examples of genotype-phenotype correlations, as a gross approach to functional genomics, illustrate the importance of genotype-related differences in ad and their impact on phenotype expression. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , similar protocols are applied to schizophrenia, depression, anxiety and other neuropsychiatric disorders. most biological parameters, potentially modifi able by monogenic genotypes and/or polygenic cluster profi les, can be used in clinical trials for monitoring effi cacy outcomes. these parametric variables also show a genotypedependent profi le in different types of dementia (e.g., ad vs. vascular dementia). for instance, striking differences have been found between ad and vascular dementia in structural and functional genomics studies. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , our understanding of the pathophysiology of cns disorders has advanced dramatically in the last years, especially in terms of their molecular pathogenesis and genetics. drug treatment of cns disorders has also made remarkable strides, with the introduction of many new drugs for the treatment of schizophrenia, depression, anxiety, epilepsy, parkinson's disease, and alzheimer's disease, among many other quantitatively and qualita-tively important neuropsychiatric disorders. improvement in terms of clinical outcome, however, has fallen short of expectations, with up to one third of the patients continuing to experience clinical relapse or unacceptable medication-related side effects in spite of efforts to identify optimal treatment regimes with one or more drugs. potential reasons to explain this historical setback might be that: (a) the molecular pathology of most cns disorders is still poorly understood; (b) drug targets are inappropriate, not fi tting into the real etiology of the disease; (c) most treatments are symptomatic, but not anti-pathogenic; (d) the genetic component of most cns disorders is poorly defi ned; and (e) the understanding of genome-drug interactions is very limited. with the advent of recent knowledge on the human genome , and the identifi cation and characterization of many genes associated with cns disorders, , as well as novel data regarding cyp family genes and other genes whose enzymatic products are responsible for drug metabolism in the liver (e.g., nats, abcbs/ mdrs, tpmt), it has been convincingly postulated that the incorporation of pharmacogenetic and pharmacogenomic procedures ( fig. . ) in drug development might bring about substantial benefi ts in terms of therapeutics optimization in cns disorders and in many other complex disorders, assuming that genetic factors are determinant for both neuronal dysregulation (and/or neuronal death) , - and drug metabolism. [ ] [ ] [ ] fig. . effi cacy and safety issues associated with pharmacogenetics and pharmacogenomics (adapted from r. cacabelos , ) however, this fi eld is still in its infancy; and the incorporation of pharmacogenomic strategies to drug development and pharmacological screening in cns disorders is not an easy task. the natural course of technical events to achieve effi cient goals in pharmacogenetics and pharmacogenomics include the following steps: (a) genetic testing of mutant genes and/or polymorphic variants of risk; (b) genomic screening, and understanding of transcriptomic, proteomic, and metabolomic networks; (c) functional genomics studies and genotype-phenotype correlation analysis; and (d) pharmacogenetics and pharmacogenomics developments, addressing drug safety and effi cacy, respectively. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] with pharmacogenetics we can understand how genomic factors associated with genes encoding enzymes responsible for drug metabolism regulate pharmacokinetics and pharmacodynamics (mostly safety issues). [ ] [ ] [ ] with pharmacogenomics we can differentiate the specifi c disease-modifying effects of drugs (effi cacy issues) acting on pathogenic mechanisms directly linked to genes whose mutations determine the disease phenotype. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the capacity of drugs to reverse the effects of the activation of pathogenic cascades (phenotype expression) regulated by networking genes basically deals with effi cacy issues. at present, the terms pharmacogenetics and pharmacogenomics are often used interchangeably to refer to studies of the contribution of inheritance to variation in the drug response phenotype ; however, from historical and didactic reasons (until a more suitable and universal defi nition can be established) it would be preferable to maintain the term of pharmacogenetics for the discipline dealing with genetic factors associated with drug metabolism and safety issues, whereas pharmacogenomics would refer to the reciprocal infl uence of drugs and genomic factors on pathogenetic cascades and disease-associated gene expression (effi cacy issues). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the application of these procedures to cns disorders is a very diffi cult task, since most neuropsychiatric diseases are complex disorders in which hundreds of genes might be involved , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (tables . - . ). in addition, it is very unlikely that a single drug be able to reverse the multifactorial mechanisms associated with neuronal dysfunction in most cns processes with a complex phenotype affecting mood, personality, behaviour, cognition, and functioning. this heterogeneous clinical picture usually requires the utilization of different drugs administered simultaneously. this is particularly important in the elderly population. in fact, the average number of drugs taken by patients with dementia ranges from six to more than ten per day depending upon their physical and mental conditions. nursing home residents receive, on average, seven to eight medications each month, and more than % of residents have monthly drug regimes of nine or more medications, including (in descending order) analgesics, antipyretics, gastrointestinal agents, electrolytic and caloric preparations, central nervous system (cns) agents, anti-infective agents, and cardiovascular agents. in population-based studies more than % of patients older than years are moderate or chronic antidepressant users. polypharmacy, drug-drug interactions, adverse reactions, and non-compliance are substantial therapeutic problems in the pharmacological management of elderly patients, adding further complications and costs to the patients and their caregivers. in - , . - . % of elderly individuals received at least of potentially inappropriate medications in ten health maintenance organizations (hmos) of the usa. although drug effect is a complex phenotype that depends on many factors, it is estimated that genetics accounts for - % of variability in drug disposition and pharmacodynamics. under these circumstances, therapeutics optimization is a major goal in neuropsychiatric disorders and in the elderly population, and novel pharmacogenetic and pharmacogenomic procedures may help in this endeavour. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the pharmacogenomic outcome depends upon many different determinant factors including (i) genomic profi le (family history, ethnic background, disease-related genotype, pharmacogenetic genotype, pharmacogenomic genotype, nutrigenetic genotype, nutrigenomic genotype), (ii) disease phenotype (age at onset, disease severity, clinical symptoms), (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gender, (vii) pharmacological profi le of the drugs, (viii) drug-drug interactions, (ix) gene expression profi le, (x) transcriptomic cascade, (xi) proteomic profi le, and (xii) metabolomic networking ( fig. . ) . the dissection and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic outcome in terms of safety and effi cacy (figs. . and . ). more than % of psychotropic drugs (table . ) are metabolized by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: n-acetyltransferase, sulfotransferase, thiol methyltransferase, thiopurine methyltransferase, catechol-o-methyltransferase, glutathione-s-transferases, udp-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) reductases: nadph: quinine oxidoreductase, glucose- -phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase b, catalase, superoxide dismutase, trimethylamine n-oxidase, dihydropyrimidine dehydrogenase; and (e) cytochrome p enzymes, such as cyp a , cyp a , cyp c , cyp c , cyp c , cyp d , cyp e , cyp a (table . ) and many others. , polymorphic variants in these genes can induce alterations in drug metabolism modifying the effi cacy and safety of the prescribed drugs. drug metabolism includes phase i reactions (i.e., oxidation, reduction, hydrolysis) and phase ii conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). the principal enzymes with polymorphic variants involved in phase i reactions are the following: cyp a / / , cyp e , cyp d , cyp c , cyp c , cyp c , cyp b , cyp a , cyp b , cyp a / , epoxide hydrolase, esterases, nqo (nadph-quinone oxidoreductase), dpd (dihydropyrimidine dehydrogenase), adh (alcohol dehydrogenase), and aldh (aldehyde dehydrogenase). major enzymes involved in phase ii reactions include the following: ugts (uridine ′-triphosphate glucuronosyl transferases), tpmt (thiopurine methyltransferase), comt (catechol-o-methyltransferase), hmt (histamine methyl-transferase), sts (sulfotransferases), gst-a (glutathion s-transferase a), gst-p, gst-t, gst-m, nat (n-acetyl transferase), nat , and others. polymorphisms in genes associated with phase ii metabolism enzymes, such as gstm , gstt , nat and tpmt are well understood, and information is also emerging on other gst polymorphisms and on polymorphisms in the udp-glucuronosyltransferases and sulfotransferases. the typical paradigm for the pharmacogenetics of phase i drug metabolism is represented by the cytochrome p- enzymes, a superfamily of microsomal nonsteroidal anti-infl ammatory drug drug-metabolizing enzymes. p enzymes comprise a superfamily of heme-thiolate proteins widely distributed in bacteria, fungi, plants and animals. the p enzymes are encoded in genes of the cyp superfamily (table . ) and act as terminal oxidases in multicomponent electron transfer chains which are called p containing monooxigenase systems. some of the enzymatic products of the cyp gene superfamily can share substrates, inhibitors and inducers whereas others are quite specifi c for their substrates and interacting drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are more than p genes identifi ed in different species. saito et al provided a catalogue of variants among eight cyp genes, nine esterase genes, and two other genes in the japanese population. the microsomal, membrane-associated, p isoforms cyp a , cyp d , cyp c , cyp c , cyp e , and cyp a are responsible for the oxidative metabolism of more than % of marketed drugs. about - % of the psychotropic agents currently used for the treatment of neuropsychiatric disorders are metabolized via enzymes of the cyp family, especially cyp a , cyp b , cyp c / , cyp c , cyp d and cyp a (table . ). cyp a metabolizes more drug molecules than all other isoforms together. most of these polymorphisms exhibit geographic and ethnic differences. [ ] [ ] [ ] [ ] [ ] [ ] [ ] these differences infl uence drug metabolism in different ethnic groups in which drug dosage should be adjusted according to their enzymatic capacity, differentiating normal or extensive metabolizers (ems), poor metabolizers (pms) and ultrarapid metabolizers (ums). most drugs act as substrates, inhibitors or inducers of cyp enzymes. enzyme induction enables some xenobiotics to accelerate their own biotransformation (auto-induction) or the biotransformation and elimination of other drugs. a number of p enzymes in human liver are inducible. induction of the majority of p enzymes occurs by increase in the rate of gene transcription and involves ligand-activated transcription factors, aryl hydrocarbon receptor, constitutive androstane receptor (car), and pregnane x receptor (pxr). , in general, binding of the appropriate ligand to the receptor initiates the induction process that cascades through a dimerization of the receptors, their translocation to the nucleus and binding to specifi c regions in the promoters of cyps. cyps are also expressed in the cns, and a complete characterization of constitutive and induced cyps in brain is essential for understanding the role of these enzymes in neurobiological functions and in age-related and xenobiotic-induced neurotoxicity. assuming that the human genome contains about , - , genes, at the present time only . % of commercial drugs have been assigned to corresponding genes whose gene products might be involved in pharmacokinetic and pharmacodynamic activities of a given drug; and only % of the human genes have been assigned to a particular drug metabolic pathway. supposing a theoretical number of , chemicals in current use worldwide, and assuming that practically all human genes can interact with drugs taken by human beings, each gene in the human genome should be involved in the metabolism and/or biopharmacological effect of - drugs; however, assuming that most xenobiotic substances in contact with our organism can infl uence genomic function, it might be possible that for , , xenobiotics in daily contact with humans, an average of - xenobiotics have to be assigned to each one of the genes potentially involved in drug metabolism and/or xenobiotics processing. to fulfi l this task a single gene has to possess the capacity of metabolizing many different xenobiotic substances and at the same time many different genes have to cooperate in orchestrated networks to metabolize a particular drug or xenobiotic under sequential biotransformation steps (figs. . and . ). numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cyps, transferases and transporters. many natural and artifi cial substances induce the hepatic cyp subfamilies in humans, and these inductions might lead to clinically important drug-drug interactions. some of the key cellular receptors that mediate such inductions have been recently identifi ed, including nuclear receptors, such as the constitutive androstane receptor (car, nr i ), the retinoid x receptor (rxr, nr b ), the pregnane x receptor (pxr, nr i ), and the vitamin d receptor (vdr, nr i ) and steroid receptors such as the glucocorticoid receptor (gr, nr c ). there is a wide promiscuity of these receptors in the induction of cyps in response to xenobiotics. indeed, this adaptive system acts as an effective network where receptors share partners, ligands, dna response elements and target genes, infl uencing their mutual relative expression. , the most important enzymes of the p cytochrome family in drug metabolism by decreasing order are cyp a , cyp d , cyp c , cyp c , and cyp a . [ ] [ ] [ ] , , the predominant allelic variants in the cyp a gene are cyp a * (leu his) and cyp a del. the cyp a * mutation inactivates the enzyme and is present in - % of caucasians. the cyp a del mutation results in no enzyme activity and is present in % of caucasians and % of asians. [ ] [ ] [ ] the most frequent mutations in the cyp c gene are cyp c * (arg cys), with reduced affi nity for p in - % of caucasians, and cyp c * (ile leu), with alterations in the specifi city for the substrate in - % of caucasians and - % of asians. [ ] [ ] [ ] the most prevalent polymorphic variants in the cyp c gene are cyp c * , with an aberrant splicing site resulting in enzyme inactivation in % of caucasians, - % of asians, % of africans, and - % of ethiopians and saoudians, and cyp c * , a premature stop codon resulting in an inactive enzyme present in - % of asians, and almost absent in caucasians. [ ] [ ] [ ] , the most important mutations in the cyp d gene are the following: cyp d * xn, cyp d * , cyp d * , cyp d * and cyp d * . [ ] [ ] [ ] , the cyp d * xn mutation gives rise to a gene duplication or multiplication resulting in an increased enzyme activity which appears in - % of the caucasian population, - % of asians, % of africans, and - % of ethiopians. the defective splicing caused by the cyp d * mutation inactivates the enzyme and is present in - % of caucasians. the deletion in cyp d * abolishes enzyme activity and shows a frequency of - % in caucasians, % in asians, % in africans, and - % in ethiopians. the polymorphism cyp d * causes pro ser and ser thr mutations with unstable enzyme activity in - % of caucasians, % of asians, % of africans, and - % of ethiopians. the cyp d * variant causes thr ile and arg cys substitutions which produce a reduced affi nity for substrates in % of asians, % of africans, and - % of ethiopians, and is practically absent in caucasians. [ ] [ ] [ ] , , the cyp d enzyme, encoded by a gene that maps on q . - . , catalyses the oxidative metabolism of more than clinically important and commonly prescribed drugs such as cholinesterase inhibitors, antidepressants, neuroleptics, opioids, some β-blockers, class i antiarrhythmics, analgesics and many other drug categories, acting as substrates, inhibitors or inducers with which most psychotropics may potentially interact (table . ), this leading to the outcome of adrs. [ ] [ ] [ ] , , the cyp d locus is highly polymorphic, with more than different cyp d alleles identifi ed in the general population showing defi cient (poor metabolizers, pm), normal (extensive metabolizers, em) or increased enzymatic activity (ultra-rapid metabolizers, um). , most individuals (> %) are ems; however, remarkable interethnic differences exist in the frequency of the pm and um phenotypes among different societies all over the world. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] on the average, approximately . % of the world population belongs to the pm category. europeans ( . %), polynesians ( . %), and africans ( . %) exhibit the highest rate of pms, whereas orientals ( . %) show the lowest rate. the frequency of pms among middle eastern populations, asians, and americans is in the range of - %. [ ] [ ] [ ] [ ] [ ] cyp d gene duplications are relatively infrequent among northern europeans, but in east africa the frequency of alleles with duplication of cyp d is as high as %. the most frequent cyp d alleles in the european population are the following: cyp d * (wild-type) (normal), cyp d * ( c > t)(normal), cyp d * ( a > del)(inactive), cyp d * ( g > a)(inactive), cyp d * (gene deletion)(inactive), cyp d * ( t > del)(inactive), cyp d * ( a > c)(inac-tive), cyp d * ( g > t)(inactive), cyp d * ( - delaga)(partially active), cyp d * ( c > t)(partially active), cyp d * ( g > c) (inactive), cyp d * ( g > a)(inactive), cyp d * ( c > t)(partially active), and cyp d gene duplications (with increased or decreased enzymatic activity depending upon the alleles involved). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the spanish population, where the mixture of ancestral cultures has occurred for centuries, the distribution of the cyp d genotypes differentiates major categories of cyp d -related metabolizer types: (i) extensive metabolizers (em)( * / * , * / * ); (ii) intermediate metabolizers (im)( * / * , * / * , * / * , * / * , * / * , * / * , * / * , * / * , * / * ); (iii) poor metabolizers (pm)( * / * , * / * ); and (iv) ultra-rapid metabolizers (um)( * xn/ * , * xn/ * , dupl). in this sample we have found . % ems, . % ims, . % pms, and . % ums. , [ ] [ ] [ ] [ ] the distribution of all major genotypes is the following: * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * xn/ * , . %; * xn/ * , . %; and dupl, . %. , [ ] [ ] [ ] [ ] in some instances, there is association of cyp d variants of risk with genes potentially involved in the pathogenesis of specifi c cns disorders. when comparing ad cases with controls, we observed that ems are more prevalent in ad ( * / * , . %; * / * , . %)(total ad-ems: . %) than in controls ( * / * , . %; * / * , %)(total c-ems: . %). in contrast, ims are more frequent in controls ( . %) than in ad ( . %), especially the * / * (c: . %; ad: . %) and * / * genotypes (c: . %; ad: . %). the frequency of pms was similar in ad ( . %) and controls ( . %), and ums were more frequent among ad cases ( . %) than in controls ( . %). , , , we have also investigated the association of cyp d genotypes with ad-related genes, such as app, mapt, apoe, ps , ps , a m, ace, agt, fos, and prnp variants. , , , no app or mapt mutations have been found in ad cases. homozygous apoe- / ( . %) and apoe- / ( . %) accumulate in ums, and apoe- / cases were also more frequent in pms ( . %) than in ems ( . %) or ims ( %). ps - / genotypes were more frequent in ems ( %), whereas ps- / genotypes were over-represented in ims ( . %) and ums ( %). the presence of the ps - / genotype was especially high in pms ( . %) and ums ( %). a mutation in the ps gene exon (ps e +) was markedly present in ums ( . %). about % of ums were a m-v i-a/a, and the a m-v i-g/g genotype was absent in pms and ums. the a m-i/i genotype was absent in ums, and % of ums were a m-i/d and ace-d/d. homozygous mutations in the fos gene (b/b) were only present in ums, as well. agt-t t cases were absent in pms, and the agt-m m genotype appeared in % of pms. likewise, the prnp-m m variant was present in % of pms and ums. , , , these association studies clearly show that in pms and ums there is an accumulation of ad-related polymorphic variants of risk which might be responsible for the defective therapeutic responses currently seen in these ad clusters. , [ ] [ ] [ ] [ ] it appears that different cyp d variants, expressing ems, ims, pms, and ums, infl uence to some extent several biochemical parameters, liver function, and vascular hemodynamic parameters which might affect drug effi cacy and safety. blood glucose levels are found elevated in ems ( * / * vs. * / * , p < . ) and in some ims ( * / * vs. * xn/ * , p < . ), whereas other ims ( * / * vs. * / * , p < . ) tend to show lower levels of glucose compared with pms ( * / * ) or ums ( * xn/ * ) (table . ). the highest levels of total-cholesterol are detected in the ems with the cyp d * / * genotype (vs. * / * , * / * and * xn/ * , p < . ). the same pattern has been observed with regard to ldlcholesterol levels, which are signifi cantly higher in the em-* / * . in general, both total cholesterol levels and ldl-cholesterol levels are higher in ems (with a signifi cant difference between * / * and * / * ), intermediate levels are seen in ims, and much lower levels in pms and ums; and the opposite occurs with hdlcholesterol levels, which on average appear much lower in ems than in ims, pms, and ums, with the highest levels detected in * / * and * xn/ * (table . ). the levels of triglycerides are very variable among different cyp d polymorphisms, with the highest levels present in ims ( * / * vs. * / * and * xn/ * , p < . ). these data clearly indicate that lipid metabolism can be infl uenced by cyp d variants or that specifi c phenotypes determined by multiple lipid-related genomic clusters are necessary to confer the character of ems and ims. other possibility might be that some lipid metabolism genotypes interact with cyp d -related enzyme products leading to defi ne the pheno-genotype of pms and ums. no signifi cant changes in blood pressure values have been found among cyp d genotypes; however, important differences became apparent in brain cerebrovascular hemodynamics (table . ). in general terms, the best ( ) . ± . ( ) . ± . ( ldl-cholesterol levels, than in ims ( * / * , p < . ); and diastolic velocities (dv) also tend to be much lower in * / * and especially in pms ( * / * ) and ums ( * xn/ * ), whereas the best dv is measured in * / * ims. more striking are the results of both the pulsatility index (pi = (sv-dv)/mv) and resistance index (ri = (sv-dv)/sv), which are worse in ims and pms than in ems and ums (table . ). these data taken together seem to indicate that cyp d -related ad pms exhibit a poorer cerebrovascular function which might affect drug penetration in the brain with the consequent therapeutic implications. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] some conventional anti-dementia drugs (tacrine, donepezil, galantamine) are metabolized via cyp-related enzymes, especially cyp d , cyp a , and cyp a , and polymorphic variants of the cyp d gene can affect the liver metabolism, safety and effi cacy of some cholinesterase inhibitors. , in order to elucidate whether or not cyp d -related variants may infl uence transaminase activity, we have studied the association of got, gpt, and ggt activity with the most prevalent cyp d genotypes in ad (table . ). globally, ums and pms tend to show the highest got activity and ims the lowest. signifi cant differences appear among different im-related genotypes. the * / * genotype exhibited the lowest got activity with marked differences as compared to ums (p < . vs. * xn/ * ; p < . vs. * xn/ * ). gpt activity was signifi cantly higher in pms ( * / * ) than in ems ( * / * , p < . ) or ims ( * / * , * / * , p < . ). the lowest gpt activity was found in ems and ims. striking differences have been found in ggt activity between pms ( * / * ), which showed the highest levels, and ems ( * / * , p < . ; * / * , p < . ), ims ( * / * , p < . ), or ums ( * xn/ * , p < . ) ) ( table . ). interesting enough, the * / * genotype, with the lowest values of got and gpt, exhibited the second highest levels of ggt after * / * , probably indicating that cyp d -related enzymes differentially regulate drug metabolism and transaminase activity in the liver. these results are also clear in demonstrating the direct effect of cyp d variants on transaminase activity , , (table . ). ( ) . ± . ( ) . ± . ( ) intermediate metabolizers * / * . ± . ( , ) . ± . . ± . * / * . ± . ( , ) . ± . . ± . * / * . ± . ( , ) . ± . ( , ) . ± . ( no clinical trials have been performed to date to elucidate the infl uence of cyp d variants on the therapeutic outcome in ad in response to cholinesterase inhibitors or other anti-dementia drugs. to overcome this lack of pharmacogenetic information, we have performed the fi rst prospective study in ad patients who received a combination therapy with (a) an endogenous nucleotide and choline donor, cdp-choline ( mg/day), (b) a nootropic substance, piracetam ( , mg/day), (c) a vasoactive compound, , dimethyl β-( bromonicotinoyl-oxymethyl)- α-methoxyergoline (nicergoline) ( (fig. . ). among ems, ad patients harbouring the * / * genotype responded better than patients with the * / * genotype. the best responders among ims were the * / * , * / * and * / * genotypes, whereas the * / * , * / * , and * / * genotypes were poor responders. among pms and ums, the poorest responders were carriers of the * / * and * xn/ * genotypes, respectively. , , from all these data we can conclude the following: (i) the most frequent cyp d variants in the spanish population are the * / * ( . %), * / * ( . %), * / * ( . %), * / * ( . %) and * xn/ * ( . %), accounting for more than % of the population; (ii) the frequency of ems, ims, pms, and ums is about . %, . %, . %, and . %, respectively; (iii) ems are more prevalent in ad ( . %) than in controls ( . %); ims are more frequent in controls ( . %) fig. . cyp d -related therapeutic response to a multifactorial treatment in alzheimer's disease over a -year period (adapted from r. cacabelos , ).patients received a combina-tion therapy for year, and cognitive function (mmse score) was assessed at baseline (b) and after , , , , and months of treatment. than in ad ( . %), especially the * / * (c: . %; ad: . %) and * / * genotypes (c: . %; ad: . %); the frequency of pms is similar in ad ( . %) and controls ( . %); and ums are more frequent among ad cases ( . %) than in controls ( . %); (iv) there is an accumulation of ad-related genes of risk in pms and ums; (v) pms and ums tend to show higher transaminase activities than ems and ims; (vi) ems and ims are the best responders, and pms and ums are the worst responders to a combination therapy with cholinesterase inhibitors, neuroprotectants, and vasoactive substances; and (vii) the pharmacogenetic response in ad appears to be dependent upon the networking activity of genes involved in drug metabolism and genes involved in ad pathogenesis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , taking into consideration the available data, it might be inferred that at least % of the ad population may exhibit an abnormal metabolism of cholinesterase inhibitors and/or other drugs which undergo oxidation via cyp d -related enzymes. approximately % of this population cluster would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors to reach a therapeutic threshold, whereas the other % of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. if we take into account that approximately - % of therapeutic outcomes depend upon pharmacogenomic criteria (e.g., pathogenic mechanisms associated with ad-related genes), it can be postulated that pharmacogenetic and pharmacogenomic factors are responsible for - % of the therapeutic response (effi cacy) in ad patients treated with conventional drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , of particular interest are the potential interactions of cholinesterase inhibitors with other drugs of current use in patients with ad, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolized by the cytochrome p cyp d enzyme. although most studies predict the safety of donepezil and galantamine, as the two principal cholinesterase inhibitors metabolized by cyp d -related enzymes, , no pharmacogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling together pharmacological and clinical information obtained with routine procedures. , [ ] [ ] [ ] in certain cases, genetic polymorphism in the expression of cyp d is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major meta-bolic pathways are glucuronidation, o-demethylation, n-demethylation, n-oxidation, and epimerization. however, excretion rates are substantially different in ems and pms. for instance, in ems, urinary metabolites resulting from o-demethylation of galantamine represent . % of the dose compared with . % in pms, which show correspondingly higher urinary excretion of unchanged galantamine and its n-oxide. therefore, still there are many unanswered questions regarding the metabolism of cholinesterase inhibitors and their interaction with other drugs (potentially leading to adrs) which require pharmacogenetic elucidation. it is also worth to mention that dose titration (a common practice in ad patients treated with cholinesterase inhibitors; e.g., tacrine, donepezil) is an unwise strategy, since approximately - % of drug failure or lack of therapeutic effi cacy (and/or adr manifestation) is not a matter of drug dosage but a problem of poor metabolizing capacity in pms. additionally, inappropriate drug use is one of the risk factors for adverse drug reactions (adrs) in the elderly. the prevalence of use of potentially inappropriate medications in patients older than years of age admitted to a general medical or geriatric ward ranges from % to %, and these numbers may double in ambulatory patients. overall, the most prevalent inappropriate drugs currently prescribed to the elderly are amiodarone, long-acting benzodiazepines and anticholinergic antispasmodics; however, the list of drugs with potential risk also include antidepressant, antihistaminics, nsaids, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics, most of which are processed via cyp d and cyp a enzymes. therefore, pre-treatment cyp screening might be of great help to rationalize and optimize therapeutics in the elderly, by avoiding medications of risk in pms and ums. there are substantial differences between individuals in the effects of psychotropic drugs in the treatment of neuropsychiatric disorders. pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specifi c candidate genes and the positive and adverse effects of drug treatment. more than different genes are potentially involved in the metabolism of psychotropic drugs infl uencing pharmacokinetics and pharmacodynamics. of all genes affecting drug metabolism, effi cacy and safety, the cyp gene family is the most relevant since more than % of cns drugs are metabolized by cytochrome p enzymes. [ ] [ ] [ ] approximately, % of neuroleptics are major substrates of cyp a enzymes, % of cyp d , and % of cyp a ; % of antidepressants are major substrates of cyp a enzymes, % of cyp b , % of cyp c , % of cyp d , and % of cyp a ; % of benzodiazepines are major substrates of cyp c enzymes, % of cyp d , and % of cyp a (table . ). approximately, % of patients with resistant depression, % of patients non-responsive to neuroleptics, and - % of patients with paradoxical responses to benzodiazepines are carriers of mutant variants of the cyp d , cyp c and cyp a genes, falling within the categories of poor or ultra-rapid metabolizers. other genes infl uencing psychotropic drug activity include the following: abcb ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table . ) . historically, the vast majority of pharmacogenetic studies of cns disorders have been addressed to evaluate the impact of cytochrome p enzymes on drug metabolism. [ ] [ ] [ ] furthermore, conventional targets for psychotropic drugs were the neurotransmitters dopamine, serotonin, noradrenaline, gaba, ion channels, acetylcholine and their respective biosynthetic and catalyzing enzymes, receptors and transporters ; however, in the past few years many different genes have been associated with both pathogenesis and pharmacogenomics of neuropsychiatric disorders. some of these genes and their products constitute potential targets for future treatments. new developments in genomics, including whole genome genotyping approaches and comprehensive information on genomic variation across populations, coupled with large-scale clinical trials in which dna collection is routine, now provide the impetus for a next generation of pharmacogenetic studies and identifi cation of novel candidate drugs. [ ] [ ] [ ] cyclic nucleotide phosphodiesterases (pdes) are a family of enzymes that degrade camp and cgmp. intracellular cyclic nucleotide levels increase in response to neurotransmitters and are down-regulated through hydrolysis catalyzed by pdes, which are therefore candidate therapeutic targets. camp is a second messenger involved in learning, memory, and mood, and cgmp modulates brain processes that are controlled by the nitric oxide (no)/cgmp pathway. the analysis of snps in genes of this superfamily revealed that polymorphisms in pde a and pde a are associated with major depressive disorder. in addition, remission on antidepressants was associated with polymorphisms in pde a and pde a. according to these results, it has been postulated that pde a (haplotype gaacc) has a role in the pathogenesis of major depression. another example is the purinergic receptor gene p rx( ), located in a major linkage hotspot for schizophrenia and bipolar disorder ( q - ), which has been associated with bipolar disorder, but nine functionally characterized variants of p rx( ) did not show association with schizophrenia. the possible role of a tag snp (the g/a polymorphism) of the gene encoding the cannabinoid receptor type (cnr ) has been investigated in schizophrenics treated with atypical antipsychotics. no difference in g/a polymorphism was observed between patients and control subjects, and no relation-ships were noted between this polymorphism and any clinical parameter considered as potential intermediate factor; however, the g allele was signifi cantly higher among non-responders vs. responsive patients, suggesting that the g allele of the cnr gene could be a pharmacogenetic rather than a vulnerability factor for schizophrenics. synaptic dysfunction is a potential pathogenic factor in schizophrenia. cholesterol is an essential component of myelin and has proved important for synapse formation and lipid raft function. it has been demonstrated that the antipsychotic drugs clozapine and haloperidol stimulate lipogenic gene expression in glioma cells in culture through activation of the sterol regulatory element-binding protein (srebp) transcription factors. recently, the action of chlorpromazine, haloperidol, clozapine, olanzapine, risperidone and ziprasidone on srebp and srebp-controlled gene expression (acetyl-coa acetyltransferase , acetoacetyl-coa thiolase, acat ; -hydroxy- -methylglutaryl-coa reductase, hmgcr; -hydroxy- -methylglutaryl-coa synthase , hmgcs ; fdps; sterol-c -desaturase like, sc dl; -dehydrocholesterol reductase, dhcr ; low density lipoprotein receptor, ldlr; fatty acid synthase; farsenyl diphosphate synthase, fasn; stearoyl-coa desaturase, delta- -desaturase, scd ) has been investigated in different cns human cell lines, demonstrating that antipsychotic-induced activation of lipogenesis is most prominent in glial cells and that this mechanism could be relevant for the therapeutic efficacy of some antipsychotic drugs. rgs (regulator of g-protein signaling ) modulates dopamine receptor signal transduction. functional variants of this gene (rgs -rs c/g) may infl uence susceptibility to extrapyramidal symptoms induced by antipsychotic drugs. this snp is located in the ′-regulatory region of the gene, and is known to infl uence rgs mrna levels and protein expression. furthermore, rgs (regulator of g protein signaling ) genotypes predict both the severity at baseline symptoms and relative responsiveness to antipsychotic medication. tardive dyskinesia is characterized by involuntary movements predominantly in the orofacial region and develops in approximately % of patients during long-term treatment with typical antipsychotics. polymorphic variants of cyp a , cyp d , and drd genes have been associated with tardive dyskinesia in schizophrenics. , in contrast, the haplotype t- b-glu of the endothelial nitric oxide synthase (nos ) gene (- t > c in the promoter region, -bp variable number of tandem repeats in intron , glu asp in exon ) might represent a protective haplotype against tardive dyskinesia after long-term antipsychotic treatment. the t c variant in the serotonin a receptor (htr a) and the ser gly variant in the dopamine d receptor (drd ) were associated with a risperidone response to exacerbated schizophrenia. the patients with t/t in the htr a gene show less clinical improvement than do those with t/c or c/c. the c allele is more frequent in responders. when combinations of both polymorphisms are considered, patients who have t/t in the htr a gene and encode ser/ser or ser/gly from drd gene have a higher propensity to non-responsiveness compared to other subjects, suggesting that the htr a t c variant could be a potential indicator of clinical improvement after risperidone treatment. there is a signifi cant relationship between a promoter region polymorphism in the serotonin transporter gene and antidepressant response, as well as for associations between candidate neurotransmitter receptor genes and second generation antipsychotic drug response. polymorphic variants of several serotonin receptor subtypes seem to be involved in the efficacy and symptomatic response of schizophrenic patients to atypical antipsychotics. for instance, the − c/g polymorphism of the htr a receptor gene is associated with negative symptom response to risperidone in schizophrenics. interaction between comt and notch genotypes may also predict the treatment response to typical neuroleptics in patients with schizophrenia. the effi cacy of iloperidone in patients with schizophrenia has been associated with the homozygous condition for the rs g/g genotype of the ciliary neurotrophic factor (cntf) gene. dopamine receptor interacting proteins (drips) are pivotally involved in regulating dopamine receptor signal transduction. two snps in the dopamine receptor interacting protein gene, nef , which encodes the drip, neurofi lament-medium (nf-m), were associated with early response (rs , rs ). a snp haplotype spanning nef was over-represented in early responders. since nef is primarily associated with dopamine d receptor function, it is likely that both genes cooperate in eliciting genotype-specifi c antipsychotic response. the improvement in the positive and negative syndrome scale (panss) positive subscore was found signifi cantly greater in patients homozygous for the a allele of the slc a (solute carrier family (noradrenaline transporter), member ) gene, and smaller in patients homozygous for the c- allele of the slc a gene, suggesting that these polymorphisms of the noradrenaline transporter gene are specifi cally involved in the variation of positive symptoms in schizophrenia. weight gain is a problem commonly found in patients treated with neuroleptics, tricyclic antidepressants, and some antiepileptics (e.g., valproic acid). the adipocyte-derived hormone, leptin, has been associated with body weight and energy homeostasis, and abnormal regulation of leptin could play a role in weight gain induced by antipsychotics. the leptin gene promoter variant g a was associated with clozapine-induced weight in chinese patients with chronic schizophrenia. likewise, studies in caucasians suggest that genetic vulnerability in the leptin gene (− g/a) and leptin receptor (q r) may predispose some individuals to excessive weight gain from increased exposure to olanzapine. , the development of selective type metabotropic glutamate receptor (mglu ) antagonists, such as -methyl- -(phenylethynyl)-pyridine (mpep) and -[( -methyl- , -thiazol- -yl)ethynyl]-pyridine (mtep), has demonstrated the potential involvement of these receptors in several cns disorders including depression, anxiety, epilepsy, parkinson's disease, drug addiction, and alcoholism. treatment with mpep and mtep can induce gene expression related to atp synthesis, hydrolase activity, and signaling pathways associated with mitogen-activated protein kinase (mapk) in the frontal cortex, this constituting another potential therapeutic target in some neuropsychiatric disorders. a new marker (rs ) in the grik gene, which codes for the kainic acid-type glutamate receptor ka , has been associated with response to the antidepressant citalopram, suggesting that the glutamate system plays a role in modulating response to selective serotonin reuptake inhibitors (ssris). glycogen synthase kinase- β (gsk b) activity is increased in the brain of patients with major depressive disorders. inhibition of gsk b is thought to be a key feature in the therapeutic mechanism of antidepressants. four polymorphisms of the gsk b gene [rs (− t > c); rs (ivs + a > g); rs (ivs + g > t); rs (ivs + t > a)] have been genotyped in chinese patients with major depression. gsk b tagt carriers showed poorer response to antidepressants. lithium has been used for over years as an effective prophylactic agent in bipolar disorder. response to lithium treatment seems to be, at least in part, genetically determined. it has been suggested that lithium exerts an effect on signal transduction pathways, such as the cyclic adenosine monophosphate (camp) pathway. association studies in patients with bipolar disorders revealed that creb - h snp (g/a change at q . -q ) and creb - h (t/c change) may be associated with bipolar disorder and lithium response. dna oligonucleotide microarrays have been used to evaluate gene expression in the substantia nigra of patients with parkinson's disease (pd). sporadic pd is characterized by progressive death of dopaminergic neurons within the substantia nigra, where cell death is not uniform. the lateral tier of the substantia nigra (snl) degenerates earlier and more severely than the more medial nigral component (snm). genes expressed more highly in the pd snl included the cell death gene, p effector related to pmp , the tnfr gene, tnfr superfamily, member , and the mitochondrial complex i gene, nadh dehydrogenase (ubiquinone) -beta subcomplex, , kda (ndufβ ). genes that were more highly expressed in pd snm included the dopamine cell signaling gene, cyclic adenosine monophosphate-regulated phosphoprotein, kda, the activated macrophage gene, stabilin , and two glutathione peroxidase (gpx) genes, gpx and gpx . this gene expression profi le reveals that there is increased expression of genes encoding pro-infl ammatory cytokines and subunits of the mitochondrial electron transport chain in glial cells, and that there is a decreased expression of several glutathione-related genes in the gnl, suggesting a molecular basis for pathoclisis. these fi ndings may contribute to open new therapeutic avenues in pd, where glial cells might represent potential targets to halt disease progression. pharmacological inhibition of cyclic-dependent kinase (cdk ) protects neurons under distinct stressful conditions. in ad and amyotrophic lateral sclerosis deregulation of cdk causes hyperphosphorylation of tau and neurofi lament proteins, respectively, leading to neuronal cell death. by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of fl ight (maldi-tof)-mass spectrometry, several phosphoproteins that are modulated by cdk inhibitors have been identifi ed. these phosphoproteins include syndapin i which is involved in vesicle recycling, and dynein light intermediate chain which represents a regulatory subunit of the dynein protein complex, confi rming the role of cdk in synaptic signaling and axonal transport. other phosphoproteins detected are cofi lin and collapsing response mediator protein, involved in neuronal survival and/or neurite outgrowth. selective cdk inhibitors can also block mitochondrial translocation of pro-apoptotic cofi lin. phosphoproteome and transcriptome analysis of neurons indicate that cdk inhibitors promote both neuronal survival and neurite outgrowth. these compounds might represent novel therapeutic alternatives in neurodegenerative disorders. despite the promising results obtained with structural and functional genomic procedures to identify associations with disease pathogenesis and potential drug targets in cns disorders, it must be kept in mind that allelic mrna expression is affected by genetic and epigenetic events, both with the potential to modulate neurotransmitter tone in the cns. epigenetics is the study of how the environment can affect the genome of the individual during its development as well as the development of its descendants, all without changing the dna sequence, but inducing modifi cations in gene expression through dna methylation-demethylation or through modifi cation of histones by processes of methylation, deacetylation, and phosphorylation. cumulative experiences throughout life history interact with genetic predispositions to shape the individual's behaviour. epigenetic phenomena can not be neglected in the pathogenesis and pharmacogenomics of cns disorders. studies in cancer research have demonstrated the antineoplastic effects of the dna methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid, of current use in epilepsy. novel effects of some pleiotropic drugs with activity on the cns have to be explored to understand in full their mechanisms of action and adjust their dosages for new indications. both hyper-and hypo-dna methylation changes of the regulatory regions play critical roles in defi ning the altered functionality of genes (mb-comt, maoa, dat , th, drd , drd , reln, bdnf) in major psychiatric disorders, such as schizophrenia and bipolar disorder. this complexity requires a multifactorial approach to overcome the hurdles that cns drug development faces at the present time. polymorphic variants in the apoe gene ( q . ) are associated with risk (apoe- allele) or protection (apoe- allele) for ad. , [ ] [ ] [ ] for many years, alterations in apoe and defects in the apoe gene have been associated with dysfunctions in lipid metabolism, cardiovascular disease, and atherosclerosis. during the past years an enormous amount of studies clearly documented the role of apoe- as a risk factor for ad, an the accumulation of the apoe- allele has been reported as a risk factor for other forms of dementia and cns disorders. , [ ] [ ] [ ] apoe- may infl uence ad pathology interacting with app metabolism and abp accumulation, enhancing hyperphosphorylation of tau protein and nft formation, reducing choline acetyltransferase activity, increasing oxidative processes, modifying infl ammation-related neuroimmunotrophic activity and glial activation, altering lipid metabolism, lipid transport and membrane biosynthesis in sprouting and synaptic remodelling, and inducing neuronal apoptosis. , [ ] [ ] [ ] different apoe genotypes confer specifi c phenotypic profi les to ad patients. some of these profi les may add risk or benefi t when the patients are treated with conventional drugs, and in many instances the clinical phenotype demands the administration of additional drugs which increase the complexity of therapeutic protocols. from studies designed to defi ne apoe-related ad phenotypes, , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , several confi rmed conclusions can be drawn: (i) the ageat-onset is - years earlier in approximately % of ad cases harbouring the apoe- / genotype; (ii) the serum levels of apoe are the lowest in apoe- / , intermediate in apoe- / and apoe- / , and highest in apoe- / and apoe- / ; (iii) serum cholesterol levels are higher in apoe- / than in the other genotypes; (iv) hdl-cholesterol levels tend to be lower in apoe- homozygotes than in apoe- allele carriers; (v) ldl-cholesterol levels are systematically higher in apoe- / than in any other genotype; (vi) triglyceride levels are signifi cantly lower in apoe- / ; (vii) nitric oxide levels are slightly lower in apoe- / ; (viii) serum abp levels do not differ between apoe- / and the other most frequent genotypes (apoe- / , apoe- / ); (ix) blood histamine levels are dramatically reduced in apoe- / as compared with the other genotypes; (x) brain atrophy is markedly increased in apoe- / > apoe- / > apoe- / ; (xi) brain mapping activity shows a signifi cant increase in slow wave activity in apoe- / from early stages of the disease (fig. . ) ; (xii) brain hemodynamics, as refl ected by reduced brain blood fl ow velocity and increase pulsatility and resistance indices, is signifi cantly worst in apoe- / (and in apoe- carriers, in general, as compared with apoe- carriers); (xiii) lymphocyte apoptosis is markedly enhanced in apoe- carriers; (xiv) cognitive deterioration is faster in apoe- / patients than in carriers of any other apoe genotype; (xv) occasionally, in approximately - % of the ad cases, the presence of some dementia-related metabolic dysfunctions (e.g., iron, folic acid, vitamin b defi ciencies) accumulate in apoe- carriers more than in apoe- carriers; (xvi) some behavioral disturbances (bizarre behaviors, psychotic symptoms), alterations in circadian rhythm patterns (e.g., sleep disorders), and mood disorders (anxiety, depression) are slightly more frequent in apoe- carriers; (xvii) aortic and systemic atherosclerosis is also more frequent in apoe- carriers; (xviii) liver metabolism and transaminase activity also differ in apoe- / with respect to other genotypes; (xix) blood pressure (hypertension) and other cardiovascular risk factors also accumulate in apoe- ; and (xx) apoe- / are the poorest responders to conventional drugs ( fig. . ). these major phenotypic features clearly illustrate the biological disadvantage of apoe- homozygotes and the potential consequences that these patients may experience when they receive pharmacological treatment. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , fig. . apoe-related cognitive performance in patients with alzheimer's disease treated with a combination therapy for year (adapted from r. cacabelos , ). patients received a combination therapy for year, and cognitive function (mmse score) was assessed at baseline (b) and after , , , , and months of treatment. several studies indicate that the presence of the apoe- allele differentially affects the quality and size of drug responsiveness in ad patients treated with cholinergic enhancers, neuroprotective compounds or combination therapies; however, controversial results are frequently found due to methodological problems, study design, and patients recruitment in clinical trials. from these studies we can conclude the following: (i) multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and nmda antagonists associated with metabolic supplementation on an individual basis adapted to the phenotype of the patient may be useful to improve cognition and slow-down disease progression in ad. (ii) in our personal experience the best results have been obtained combining (a) cdp-choline with piracetam and metabolic supplementation, (b) cdp-choline with piracetam and anapsos, (c) cdp-choline with piracetam and cholinesterase inhibitors (donepezil, rivastigmine), (d) cdp-choline with memantine, and (e) cdpcholine, piracetam and nicergoline. (iii) some of these combination therapies have proven to be effective, improving cognition during the fi rst months of treatment, and not showing apparent side-effects. (iv) the therapeutic response in ad seems to be genotypespecifi c under different pharmacogenomic conditions. (v) in monogenic-related studies, patients with the apoe- / and apoe- / genotypes are the best responders, and apoe- / carriers are the worst responders ( fig. . ). (vi) ps -and ps -related genotypes do not appear to infl uence the therapeutic response in ad as independent genomic entities; however, app, ps , and ps mutations may drastically modify the therapeutic response to conventional drugs. (vii) in trigenic-related studies the best responders are those patients carrying the -, -, -, and -genomic clusters. (viii) a genetic defect in the exon of the ps gene seems to exert a negative effect on cognition conferring ps + carriers in trigenic clusters the condition of poor responders to combination therapy. (ix) the worst responders in all genomic clusters are patients with the + genotype. (x) the apoe- / genotype seems to accelerate neurodegeneration anticipating the onset of the disease by - years; and, in general, apoe- / carriers show a faster disease progression and a poorer therapeutic response to all available treatments than any other polymorphic variant. (xi) pharmacogenomic studies using trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity are very useful to widen the therapeutic scope of limited pharmacological resources. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , apoe infl uences liver function and cyp d -related enzymes probably via regulation of hepatic lipid metabolism. , , [ ] [ ] [ ] [ ] it has been observed that apoe may infl uence liver function and drug metabolism by modifying hepatic steatosis and transaminase activity. there is a clear correlation between apoe-related tg levels and got, gpt, and ggt activities in ad. , [ ] [ ] [ ] [ ] both plasma tg levels and transaminase activity are signifi cantly lower in ad patients harbouring the apoe- / genotype, probably indicating (a) that low tg levels protect against liver steatosis, and (b) that the presence of the apoe- allele infl uences tg levels, liver steatosis, and transaminase activity. consequently, it is very likely that apoe infl uences drug metabolism in the liver through different mechanisms, including interactions with enzymes such as transaminases and/ or cytochrome p -related enzymes encoded in genes of the cyp superfamily. , [ ] [ ] [ ] [ ] , when apoe and cyp d genotypes are integrated in bigenic clusters and the apoe + cyp d -related therapeutic response to a combination therapy is analyzed in ad patients after year of treatment, it becomes clear that the presence of the apoe- / genotype is able to convert pure cyp d * / * ems into full pms (fig. . ) , indicating the existence of a powerful infl uence of the apoe- homozygous genotype on the drug metabolizing capacity of pure cyp d -ems. , , , behavioral disturbances and mood disorders are intrinsic components of dementia associated with memory disorders. , [ ] [ ] [ ] the appearance of anxiety, depression, psychotic symptoms, verbal and physical aggressiveness, agitation, wandering and sleep disorders complicate the clinical picture of dementia and add important problems to the therapeutics of ad and the daily management of patients as well. under these conditions, psychotropic drugs (antidepressants, anxyolitics, hypnotics, and neuroleptics) are required, and most of these substances contribute to deteriorate cognition and psychomotor functions. apoe-related polymorphic variants have been associated with mood disorders , and panic disorder. gender, age, dementia severity, apoe- , and general medical health appear to infl uence the occurrence of individual neuropsychiatric symptoms in dementia, and medical comorbidity increases the risk of agitation, irritability, disinhibition, and aberrant motor behavior. a positive association between apoe- and neuropsychiatric symptoms and depressive symptoms in ad has been reported, especially in women. in other studies, no association of apoe- with behavioral dyscontrol (euphoria, disinhibition, aberrant motor behavior, and sleep and appetite disturbances), psychosis (delusions and hallucinations), mood (depression, anxiety, and apathy), and agitation (aggression and irritability) could be found. some authors did not fi nd association of apoe- with major depression in ad , or in patients with major depression in a community of older adults, but an apparent protective effect of apoe- on depressive symptoms was detected. others, in contrast, found that apoe- was associated with an earlier age-of-onset, but not cognitive functioning, in late-life depression. apoe−/− mice without human apoe or with apoe- , but not apoe- , show increased measures of anxiety. differences in anxiety-related behavior have been observed between apoe-defi cient c bl/ and wild type c bl/ mice, suggesting that apoe variants may affect emotional state. histamine h autoreceptor antagonists increase anxiety measures in wildtype, but not apoe−/−, mice, and apoe defi cient mice show higher sensitivity to the anxiety-reducing effects of the h receptor antagonist mepyramine than wildtype mice, suggesting a role of h -autoreceptormediated signaling in anxiety-like symptoms in this ad-related animal model. in humans, apoe- carriers with deep white matter hyperintensities in mri show association with depressive symptoms and vascular depression. . reduced caudate nucleus volumes and genetic determinants of homocysteine metabolism accumulate in patients with psychomotor slowing and cognitive defi cits, and older depressed subjects have persisting cognitive impairments associated with hippocampal volume reduction. , depressive symptoms are also associated with stroke and atherogenic lipid profi le. some multifactorial treatments addressing neuroprotection have shown to be effective in reducing anxiety progressively from the fi rst month to the month of treatment. the anxiety rate was declining from a baseline hrs-a score of . ± . to . ± . (p < . ) at month, . ± . (p < . ) at months, . ± . (p < . ) at months, . ± . (p < . ) at months, and . ± . (p < . ) at months of treatment (r = − . , a coef.: . , b coef.: − . ). similar striking results were found in depression, suggesting that improvement in mood conditions can contribute to stabilize cognitive function or that neuroprotection (with the consequent stabilization or improvement in mental performance) can enhance emotional equilibrium. , , , at baseline, all apoe variants showed similar anxiety and depression rates, except the apoe- / carriers who differed from the rest in a signifi cantly lower rates of anxiety and depression (figs. . and . ). remarkable changes in anxiety were found among different apoe genotypes (fig. . ) . practically, all apoe variants responded with a signifi cant diminution of anxiogenic symptoms, except patients with the apoe- / genotype who only showed a slight improvement. the best responders were apoe- / > apoe- / > apoe- / > apoe- / carriers (fig. . ) . the modest anxiolytic effect seen in apoe- / patients might be due to the very low anxiety rate observed at baseline. concerning depression, all apoe genotypes improved their depressive symptoms with treatment except those with the apoe- / genotype which worsen along the treatment period, especially after months (fig. . ). the best responders were patients with apoe- / > apoe- / > apoe- / > apoe- / , and the worst responders were patients harbouring the apoe- / genotype , , , (fig. . ) . the optimization of cns therapeutics requires the establishment of new postulates regarding (a) the costs of medicines, (b) the assessment of protocols for multifactorial treatment in chronic disorders, (c) the implementation of novel therapeutics addressing causative factors, and (d) the setting-up of pharmacogenetic/ pharmacogenomic strategies for drug development. , [ ] [ ] [ ] [ ] the cost of medicines is a very important issue in many countries because of (i) the growing of the aging population (> % disability), (ii) neuropsychiatric and demented patients (> % of the population) belong to an unproductive sector with low income, and (iii) the high cost of health care systems and new health technologies in developed countries. despite the effort of the pharmaceutical industry to demonstrate the benefi ts and cost-effectiveness of available drugs, the general impression in the medical community and in some governments is that some psychotropics and most anti-dementia drugs present in the market are not costeffective. , [ ] [ ] [ ] [ ] conventional drugs for neuropsychi-atric disorders are relatively simple compounds with unreasonable prices. some new products are not superior to conventional antidepressants, neuroleptics, and anxiolytics. there is an urgent need to assess the costs of new trials with pharmacogenetics and pharmacogenomics strategies, and to implement pharmacogenetic procedures to predict drug-related adverse events. , , , cost-effectiveness analysis has been the most commonly applied framework for evaluating pharmacogenetics. pharmacogenetic testing is potentially relevant to large populations that incur in high costs. for instance, the most commonly drugs metabolized by cyp d account for million prescriptions and us$ . billion annually in expenditures in the us, which represent - % of total utilization and expenditures for outpatient prescription drugs. pharmacogenomics offer great potential to improve patients' health in a cost-effective manner; however, pharmacogenetics/pharmacogenomics will not be applied to all drugs available in the market, and careful evaluations should be done on a case-by-case basis prior to investing resources in r&d of pharmacogenomic-based therapeutics and making reimbursement decisions. in performing pharmacogenomic studies in cns disorders, it is necessary to rethink the therapeutic expectations of novel drugs, redesign the protocols for drug clinical trials, and incorporate biological markers as assessable parameters of effi cacy and prevention. in addition to the characterization of genomic profi les, phenotypic profi ling of responders and non-responders to conventional drugs is also important (and currently neglected). brain imaging techniques, computerized electrophysiology, and optical topography in combination with genotyping of polygenic clusters can help in the differentiation of responders and non-responders. the early identifi cation of predictive risks requires genomic screening and molecular diagnosis, and individualized preventive programs will only be achieved when pharmacogenomic/pharmacogenetic protocols are incorporated to the clinical armamentarium with powerful bioinformatics support. - , , . an important issue in ad therapeutics is that antidementia drugs should be effective in covering the clinical spectrum of dementia symptoms represented by memory defi cits, behavioural changes, and functional decline. it is diffi cult (or impossible) that a single drug be able to fulfi l this criteria. a potential solution to this problem is the implementation of cost-effective, multifactorial (combination) treatments integrating several drugs, taking into consideration that traditional neuroleptics and novel antipsychotics (and many other psychotropics) deteriorate both cognitive and psychomotor functions in the elderly and may also increase the risk of stroke. few studies with combination treatments have been reported and most of them are poorly designed. we have also to realize that the vast majority of dementia cases in people older than - % are of a mixed type, in which the cerebrovascular component associated with neurodegeneration can not be therapeutically neglected. in most cases of dementia, the multifactorial (combination) therapy appears to be the most effective strategy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the combination of several drugs (neuroprotectants, vasoactive substances, acheis, metabolic supplementation) increases the direct costs (e.g., medication) by - %, but in turn, annual global costs are reduced by approximately - % and the average survival rate increases about % (from to years post-diagnosis). there are major concerns regarding the validity of clinical trials in patients with severe dementia. despite the questionable experience with memantine, simi-lar strategies have been used to demonstrate the utility of donepezil in severe ad. this kind of studies bears some important pitfalls, including (a) short duration (< year), (b) institutionalized patients, (c) patients receiving many different types of drugs, (d) non-evaluated drug-drug interactions, (e) side-effects (e.g., hallucinations, gastrointestinal disorders) that may require the administration of additional medication, (f) lack of biological parameters demonstrating actual benefi ts, and (e) no cost-effectiveness assessment, among many other possibilities of technical criticism. [ ] [ ] [ ] , some of these methodological (and costly) problems might be overcome with the introduction of pharmacogenetic/ pharmacogenomic strategies to identify good responders who might obtain some benefi t by taking expensive medications. major impact factors associated with drug effi cacy and safety include the following: (i) the mechanisms of action of drugs, (ii) drug-specifi c adverse reactions, (iii) drug-drug interactions, (iv) nutritional factors, (v) vascular factors, (vi) social factors, and (vii) genomic factors (nutrigenetics, nutrigenomics, pharmacogenetics, pharmacogenomics). among genomic factors, nutrigenetics/nutrigenomics and pharmacogenetics/pharmacogenomics account for more than % of effi cacy-safety outcomes in current therapeutics. [ ] [ ] [ ] , , , some authors consider that priority areas for pharmacogenetic research are to predict serious adverse reactions (adrs) and to establish variation in effi cacy. both requirements are necessary in cns disorders to cope with effi cacy and safety issues associated with either current cns drugs and new drugs. , since drug response is a complex trait, genome-wide approaches (oligonucleotide microarrays, proteomic profi ling) may provide new insights into drug metabolism and drug response. genome-wide family-based association studies, using single snps or haplotypes, can identify associations with genome-wide signifi cance. , to achieve a mature discipline of pharmacogenetics and pharmacogenomics in cns disorders and dementia it would be convenient to accelerate the following processes: (a) educate physicians and the public on the use of genetic/genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , costs of disorders of the brain in europe. executive summary a conceptual introduction to geriatric neuroscience the clinical and costeffectiveness of donepezil, rivastigmine, galantamine and memantine for alzheimer's disease pharmacological treatment of alzheimer disease: from phychotropic drugs and cholinesterase inhibitors to pharmacogenomics pharmacogenomics in alzheimer's disease pharmacogenomics for the treatment of dementia pharmacogenetics and drug development: the path to safer and more effective drugs molecular genetics of alzheimer's disease and aging molecular genetics of bipolar disorder and depression confounding in genetic association studies and its solutions molecular genetics of schizophrenia: a critical review lifespan and mitochondrial control of neurodegeneration high aggregate burden of somatic mtdna point mutations in aging and alzheimer's disease brain the application of functional genomics to alzheimer's disease pharmacogenomics and therapeutic prospects in alzheimer's disease pharmacogenomics, nutrigenomics and therapeutic optimization in alzheimer's disease pharmacogenomics, nutrigenomics and future therapeutics in alzheimer's disease pharmacogenomics in alzheimer's disease cerebrovascular risk factors in alzheimer's disease: brain hemodynamics and pharmacogenomic implications phenotypic profi les and functional genomics in dementia with a vascular component high throughput protein expression screening in the nervous system -needs and limitations gene expression atlas of the mouse central nervous system: impact and interactions of age, energy intake and gender subtelomeric study of patients with mental retardation reveals chromosomal anomalies and contributes to the delineation of submicroscopic deletions of pter, qter, pter, qter and qter dna fragmentation is increased in non-gabaergic neurons in bipolar disorder but not in schizophrenia the functional genome of ca and ca neurons under native conditions and in response to ischemia fibroblast and lymphoblast gene expression profi les in schizophrenia : are non-neural cells informative ? runs of homozygosity reveal highly penetrant recessive loci in schizophrenia the use of microarrays to characterize neuropsychiatric disorders: post-mortem studies of substance abuse and schizophrenia high-throughput analysis of promoter occupancy reveals direct neuronal targets of foxp , a gene mutated in speech and language disorders microarray analysis of oxidative stress regulated genes in mesencephalic dopaminergic neuronal cells: relevance to oxidative damage in parkinson's disease towards a pathway defi nition of parkinson's disease: a complex disorder with links to cancer, diabetes and infl ammation analysis of potential transcriptomic biomarkers for huntington's disease in peripheral blood comprehensive transcriptional profi ling of prion infection in mouse models reveals networks of responsive genes transcriptional profi ling in the human prefrontal cortex : evidence for two activational states with cocaine abuse gene expression profi ling in the brains of human cocaine abusers ethanol and brain damage genomic responses in rat cerebral cortex after traumatic injury transcriptional profi ling in human epilepsy: expression array and single cell real-time qrt-pcr analysis reveal distinct cellular gene regulation molecular profi ling of temporal lobe epilepsy: comparison of data from human tissue and animal models the antiepileptic drug levetiracetam selectively modifi es kindling-induced alterations in gene expression in the temporal lobe of rats increased apoptosis, p up-regulation, and cerebellar neuronal degeneration in repair-defi cient cockayne syndrome mice inhibitors of differentiation (id , id , id and id ) genes are neuronal targets of mecp that are elevated in rett síndrome hdac inhibitors correct frataxin defi ciency in a friedreich ataxia mouse model gene expression profi ling in a mouse model of infantile neuronal lipofuscinosis reveals upregulation of immediate early genes and mediators of the infl ammatory response multiple sclerosis as a generalized cns disease -comparative microarray analysis of normal appearing white matter and lesions in secondary progressive ms pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis gene expression in cortex and hippocampus during acute pneumococcal meningitis role of lipids in brain injury and diseases expression profi le analysis of neurodegenerative disease: advances in specifi city and resolution methodological considerations regarding single-cell gene expression profi ling for brain injury genotoxicants target distinct molecular networks in neonatal neurons functional gene expression differences between inbred alcohol-preferring and -non-prerats in fi ve brain regions neuroadaptations in human chronic alcoholics: tion of the nf-κb system gene expression profi le of the nucleus accumbens of human cocaine abusers : evidence for dysregulation of myelin transcriptional changes common to human cocaine, cannabis and phencyclidine abuse microrna expression in the adult mouse central nervous system a pharmacogenomic approach to alzheimer's disease clinical psychiatry. jobe th genomics and phenotypic profi les in dementia: implications for pharmacological treatment a functional genomics approach to the analysis of biological markers in alzheimer disease genomic characterization of alzheimer's disease and genotype-related phenotypic analysis of biological markers in dementia the histamine-cytokine network in alzheimer disease: etiopathogenic and pharmacogenomic implications histamine function in brain disorders histamine in alzheimer's disease pathogenesis: biochemistry and functional genomics characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and alzheimer's disease individuals international human genome sequencing consortium. finishing the euchromatic sequence of the human genome implications of the human genome for understanding human biology and medicine pharmacogenetics and pharmacogenomics. in: emery and rimoin's principles and practice of medical genetics. th edn pharmacogenomics: the inherited basis for interindividual differences in drug response pharmacogenetics and pharmacogenomics: development, science, and translation pharmacogenomics and therapeutic prospect in dementia pharmacogenetic basis for therapeutic optimization in alzheimer's disease infl uence of pharmacogenetic factors on alzheimer's disease therapeutics pharmacogenetic aspects of therapy with cholinesterase inhibitors: the role of cyp d in alzheimer's disease pharmacogenetics from pharmacogenetics and ecogenetics to pharmacogenomics inheritance and drug response pharmacogenomics-drug disposition, drug targets, and side effects national estimates of medication use in nursing homes medicare current benefi ciary survey and the medical expenditure survey antidepressant drugs prescribing among elderly subjects: a population-based study potentially inappropriate medication use among elderly home care patients in europe potentially inappropriate medication use by elderly persons in u.s. health maintenance organizations the metabolic & molecular bases of inherited disease catalog of variants among eight cytochrome p (cyp) genes: nine esterase genes, and two other genes in the japanese population dna sequence variations in a . -kb noncoding sequence -prime of the cyp a gene: implications for human population history and natural selection pm frequencies of major cyps in asians and caucasians polymorphisms of drug-metabolizing enzymes cyp c , cyp c , cyp d , cyp a , nat and of p-glycoprotein in a russian population cyp c allelic variants: ethnic distribution and functional signifi cance identifi cation and functional characterization of a new cyp c variant (cyp c * ) expressed among african americans molecular basis of ethnic differences in drug disposition and response isolation, sequence and genotyping of the drug metabolizer cyp d gene in the colombian population effects of prototypical microsomal enzyme inducers on cytochrome p expression in cultured human hepatocytes cytochrome p in the brain: a review the expression of cyp b , cyp c and cyp a genes: a tangle of networks of nuclear and steroid receptors regulation of cytochrome p (cyp) genes by nuclear receptors the cyp c enzyme polymorphism cytochrome p d variants in a caucasian population: allele frequencies and phenotypic consequences clinically signifi cant drug interactions with cholinesterase inhibitors: a guide for neurologists assessment of the predictive power of genotypes for the in-vivo catalytic function of cyp d in a german population ten percent of north spanish individuals carry duplicated or triplicated cyp d genes associated with ultrarapid metabolism of debrisoquine clinical pharmacokinetics of galantamine impact of the cyp d polymorphism on steady-state plasma concentrations and clinical outcome of donepezil in alzheimer's disease patients molecular pathology and pharmacogenomics in alzheimer's disease: polygenic-related effects of multifactorial treatments on cognition, anxiety, and depression pharmacogenomic studies with a combination therapy in alzheimer's disease interethnic differences in genetic polymorphisms of cyp d in the u.s. population: clinical implications donepezil use in alzheimer disease effects of cholinergic markers in rat brain and blood after short and prolonged administration of donepezil the o-demethylation of the antidementia drug galantamine is catalyzed by cytochrome p d cholinesterase inhibitors in the treatment of alzheimer's disease: a comparison of tolerability and pharmacology galantamine pharmacokinetics, safety, and tolerability profi les are similar in healthy caucasian and japanese subjects pharmacokinetics and drug interactions of cholinesterase inhibitors administered in alzheimer's disease the metabolism and excretion of galantamine in rats, dogs, and humans prevalence of potentially inappropriate medication use in elderly patients. comparison between general medicine and geriatric wards phargkb update: ii. cyp a , cytochrome p , family , subfamily a, polypeptide genomics and the future of pharmacotherapy in psychiatry cyp d polymorphism: implications for antipsychotic drug response, schizophrenia and personality traits cytochrome p polymorphisms and response to antipsychotic therapy infl uence of cytochrome p polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects pharmaco-genomics handbook. nd edn. lexi-comp drug information handbook with international trade names index. th edn. lexi-comp drug information handbook for psychiatry. th edn. lexi-comp pharmacogenomics in schizophrenia: the quest for individualized therapy the role of -ht c receptor polymorphisms in the pharmacogenetics of antipsychotic drug treatment dopamine d receptor gene exon iii polymorphism and interindividual variation in response to clozapine association between multidrug resistance (mdr ) gene polymorphism and therapeutic response to bromperidol in schizophrenic patients: a preliminary study genetic susceptibility to tardive dyskinesia among schizophrenia subjects: iv. role of dopaminergic pathway gene polymorphisms drd promoter region variation as a predictor of sustained response to antipsychotic medication in fi rst-episode schizophrenic patients the relationship between p-glycoprotein (pgp) polymorphisms and response to olanzapine treatment in schizophrenia polymorphisms of the abcb gene are associated with the therapeutic response to risperidone in chinese schizophrenia patients systematic investigation of genetic variability in human genes -implications for studying variable drug response pharmacogenetics of psychotropic drug response individualizing antipsychotic drug therapy in schizophrenia: the promise of pharmacogenetics pharmacogenetics and schizophrenia pharmacogenetics and pharmacogenomics of schizophrenia: a review of last decade of research phosphodiesterase genes are associated with susceptibility to major depression and antidepressant treatment response variation in the purinergic p rx( ) receptor gene and schizophrenia the cnr gene as a pharmacogenetic factor for antipsychotics rather than a susceptibility gene for schizophrenia druginduced activation of srep-controlled lipogenic gene expression in cns-related cell lines: marked differences between various antipsychotic drugs further evidence for association of the rgs gene with antipsychoticinduced parkinsonism: protective role of a functional polymorphism in the ′-untranslated region ethnic stratifi cation of the association of rgs variants with antipsychotic treatment response in schizophrenia pharmacogenetic assessment of antipsychotic-induced movement disorders: contribution of the dopamine d receptor and cytochrome p a genes association between cyp d genotype and tardive dyskinesia in korean schizophrenics haplotype analysis of endothelial nitric oxide synthase (nos ) genetic variants and tardive dyskinesia in patients with schizophrenia could htr a t c and drd ser gly predict clinical improvement in patients with acutely exacerbated schizophrenia? results from treatment responses to risperidone in a naturalistic setting the - c/g polymorphism of the -ht a receptor gene is associated with negative symptom response to risperidone treatment in schizophrenia patients interaction between notch and catechol-o-methyltransferase genotypes in schizophrenia patients with poor response to typical neuroleptics effect of a ciliary neurotrophic factor polymorphism on schizophrenia symptom improvement in an iloperidone clinical trial association of the dopamine receptor interacting protein gene, nef , with early response to antipsychotic medication pharmacogenetic study of atypical antipsychotic drug response : involvement of the norepinephrine transporter gene association of clozapineinduced weight gain with a polymorphism in the leptin promoter region in patients with chronic schizophrenia in a chinese population leptin and leptin receptor gene polymorphisms and increases in body mass index (bmi) from olanzapine treatment in persons with schizophrenia polymorphisms of the -ht c receptor and leptin genes are associated with antipsychotic drug-induced weight gain in caucasian subjects with a fi rst-episode psicosis transcriptional profi ling of the rat frontal cortex following administration of the mglu antagonists mpep and mtep association of grik with outcome of antidepressant treatment in the star * d cohort glycogen synthase kinase- beta gene is associated with antidepressant treatment response in chinese major depressive disorder lithium response and genetic variation in the creb family of genes the medial and lateral substantia nigra in parkinson's disease: mrna profi les associated with higher brain tissue vulnerability phosphoproteome and transcriptome analysis of the neuronal response to a cdk inhibitor allelic mrna expression of x-linked monoamine oxidase a (maoa) in human brain : dissection of epigenetic and genetic factors epigenetics and its implications for behavioural neuroendocrinology antineoplastic effects of the dna methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in cancer cell lines epigenetic alterations of the dopaminergic system in major psychiatric disorders central nervous system drug development: an integrative biomarker approach toward individualized medicine pleiotropic effects of apoe in dementia: infl uence on functional genomics and pharmacogenetics. in: advances in alzheimer's and parkinson's disease. insights, progress, and perspectives apoe-related dementia symptoms: frequency and progression apoe-related frequency of cognitive and noncognitive symptoms in dementia behavioral changes associated with different apolipoprotein e genotypes in dementia polymorphisms in the angiotensin-converting enzyme gene are associated with unipolar depression, ace activity and hypercortisolism apolipoprotein e gene polymorphism in early and late onset bipolar patients angiotensinrelated genes in patients with panic disorder risk factors for neuropsychiatric symptoms in dementia : the cache county study apolipoprotein e genotype infl uences presence and severity of delusions and aggressive behavior in alzheimer disease alzheimer genes and proteins, and measures of cognition and depression in older men depression in alzheimer's disease might be associated with apolipoprotein e epsilon allele frequency in women but not in men four components describe behavioral symptoms in , individuals with late-onset alzheimer's disease association analysis of apolipoprotein e genotype and risk of depressive symptoms in alzheimer's disease behavioural pathology in alzheimer's disease with special reference to apolipoprotein e genotype apolipoprotein e genotype and major depression in a community of older adults. the cache county study protective effect of the apoε allele in major depressive disorder in taiwanese apoe is associated with age-of-onset, but not cognitive functioning, in late-life depression apoe isoforms and measures of anxiety in probable ad patients and apoe-/-mice differences in anxietyrelated behaviour between apolipoprotein e-defi cient c bl/ and wild type c bl/ mice role of h -receptor-mediated signaling in anxiety and cognition in wild-type and apoe-/-mice relationship of deep white matter hyperintensities and apolipoprotein e genotype to depressive symptoms in older adults without clinical depression caudate nucleus volumes and genetic determinants of homocysteine metabolism in the prediction of psychomotor speed in older persons with depression a longitudinal study of hippocampal volume, cortisol levels, and cognition in older depressed subjects reduced hippocapal volumes and memory loss in patients with early-and late-onset depression vascular/risk and late-life depression in a korean community population measuring the value of pharmacogenomics assessing the cost-effectiveness of pharmacogenomics pharmacological treatment of neuropsychiatric symptoms of dementia. a review of the evidence memantine in moderate-to-severe alzheimer's disease donepezil in patients with severe alzheimer's disease: double-blind, parallel-group, placebo-controlled study donepezil for severe alzheimer's disease priorities and standards in pharmacogenetic research genomic screening and replication using the same data set in familybased association testing pharm acogenomics and individualized drug therapy ethical considerations in the use of dna for the diagnosis of disease key: cord- -hd p authors: wu, han-chung; chang, de-kuan title: peptide-mediated liposomal drug delivery system targeting tumor blood vessels in anticancer therapy date: - - journal: j oncol doi: . / / sha: doc_id: cord_uid: hd p solid tumors are known to recruit new blood vessels to support their growth. therefore, unique molecules expressed on tumor endothelial cells can function as targets for the antiangiogenic therapy of cancer. current efforts are focusing on developing therapeutic agents capable of specifically targeting cancer cells and tumor-associated microenvironments including tumor blood vessels. these therapies hold the promise of high efficacy and low toxicity. one recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics is to encapsulate anticancer drugs into targeting liposomes that bind to the cell surface receptors expressed on tumor-associated endothelial cells. these anti-angiogenic drug delivery systems could be used to target both tumor blood vessels as well as the tumor cells, themselves. this article reviews the mechanisms and advantages of various present and potential methods using peptide-conjugated liposomes to specifically destroy tumor blood vessels in anticancer therapy. one of the primary goals of a successful cancer treatment regimen is to deliver sufficient amounts of drug to tumors while minimizing damage to normal tissues. most chemotherapeutic agents enter normal tissues in the body with indiscriminate cytotoxicity and do not preferentially accumulate at tumor sites. at times the dose reaching the tumor may be as little as % to % of the doses accumulating in normal organs [ , ] . one reason for the inability for drugs to accumulate at target sites is that the interstitial fluid pressure (ifp) in solid tumors is higher than in normal tissues, that blocking transcapillary transport of chemotherapeutic drugs or antibodies [ ] [ ] [ ] . in this way, the anticancer effect is decreased and toxic effect to normal cells is increased. fear of severely harming the patients often limits the dose of anticancer drugs that can be given to a patient. these lower than optimal doses elicit incomplete tumor responses which leads to disease relapse and drug resistance. therefore, most cancer drugs fail in clinical studies not because they are ineffective in killing cancer cells but because they cannot be administered in doses high enough to eradicate the tumor without severely harming the patient. several approaches have been developed to improve the ability of anticancer drug to more specifically target tumors and avoid normal organs. one of the most effective strategies is to encapsulate drugs in particles that deliver them preferentially to tumor sites. for example, liposome particles have been found able to deliver radionuclides, genes, and chemotherapeutic agents to tumor sites. [ ] [ ] [ ] [ ] [ ] . another promising strategy is to encapsulate anticancer drugs in liposomes conjugated with moieties, such as antibodies and peptides, that target particular types of target tumor cells or tumor vasculatures [ ] [ ] [ ] . use of internalizing ligands for targeting liposomes conjugated with such moieties makes it possible to deliver the chemotherapeutic drugs encapsulated within them to the cytosol through the receptor-mediated endocytosis [ ] [ ] [ ] [ ] . this article reviews the current research in developing liposomal drug delivery systems that use peptide ligands to target blood vessels in solid tumors. we discuss the identification of peptides that can target tumor blood vessels and the use of targeting and nontargeting ggt gga ggt tcg- figure : selection of peptides that target tumor blood vessels using in vivo phage display. peptide or antibody libraries are expressed as fusion proteins with a coat protein (piii) of a bacteriophage, and the fused proteins are displayed on the surface of the virion. a phagedisplayed peptide library was injected through the tail vein of tumor-bearing mice. eight minutes after injection, the mice were perfused through the heart. phage recovered from the tumor was amplified and reinjected in mice for another four rounds. tumor-targeting phages were further identified by in vivo tumor-homing assay, synthetic peptide binding and competition assay, and immunohistochemical staining. the identified peptides can be used as ligands to recognize cell surface markers or tumor antigens to develop targeted therapy. scid mice bearing human cancer xenografts were successfully treated with ligand-conjugated antiangiogenic targeting liposomes. liposomes to encapsulate and deliver chemotherapeutic drugs to tumor sites. virtually every conventional cytotoxic drug has been found to be antiangiogenic in in vitro and in vivo models [ ] . one treatment approach known as metronomic therapy uses frequent administrations of low-dose antiangiogenic agents to destroy vessels in tumors while decreasing the toxicity to normal tissues [ ] [ ] [ ] . for example, it has been found in mice that frequent administration of relatively low, noncytotoxic doses of liposome-encapsulated doxorubicin can shrink various solid tumor xenografts [ , ] . the antiangiogenic agent bevacizumab (avastin), a humanized monoclonal antibody against vascular endothelial growth factor (vegf), has been used with some success to treat advanced colon cancer. one study compared the effect using three chemotherapeutic agents alone to treat advanced colon cancer with using the three agents combined with bevacizumab [ ] . they found that the combined use of chemotherapeutic agents and bevacizumab extended overall survival by approximately . months compared to the use of chemotherapeutic agents alone [ ] . other angiogenesis inhibitors, including sunitinib and sorafenib, have also been found to improve clinical outcomes when used to treat various cancer types [ , ] . the targeting of proliferating endothelial cells in the blood vessels of tumors has several advantages. first, endothelial cells in malignant tumors are genetically stable, nonmalignant, and rarely drug resistant, compared to the cancer cells [ , ] . however, some recent studies show that tumor-associated endothelial cells can acquire cytogenetic abnormalities while they are in the tumor microenvironment [ , ] . second, the destruction of endothelial cells using this method amplifies the drugs antitumor effect. it has been reported that the elimination of one endothelial cell can inhibit the growth of as many as a hundred tumor cells [ , ] . third, antiangiogenic therapy decreases ifp within the tumor allowing better penetration by chemotherapeutic agents [ ] [ ] [ ] [ ] . for example, jain found that bevacizumab could decrease ifp by normalizing tumor vasculature and decreasing vascular leakage [ , ] . fourth, antiangiogenic therapy is known to inhibit the growth of both primary and metastatic solid tumors. finally, intravenously injected angiogenesis inhibitors can directly reach endothelial cells. in addition, we can take advantage of the differences between endothelial cell plasma membrane proteins (i.e., vascular zip codes) to develop drug delivery systems capable of guiding therapeutic or imaging agents to a particular [ , , ] . the reaction was completed and confirmed by quantifying the remaining amino groups using tnbs (trinitrobenzenesulfonate) reagent [ ] . peptidyl-peg-dspe was transferred to preformed liposomes after coincubation at a temperature above the transition temperature of the lipid bilayer [ ] . there were peptide molecules per liposome [ ] . the mean diameter of the targeting liposome is approximately nm [ , ] . organ or tumor [ , ] . endothelial cells of blood vessels within solid tumors express certain molecular structures that are absent or minimally detectable in normal blood vessels [ , , ] . these structures can be used as molecular targets for antitumor treatment. the key to delivering drugs specifically to these targets is to identify and use ligands that specifically bind to and that can be internalized by endothelial cells in tumors. combinatorial peptide libraries displayed on microorganisms have become a research tool for identifying cell surface-binding peptides that can become targets for antitumor treatment. of the many molecular display techniques, phage display has been the most popular approach. phage display is a selection technique in which a peptide or protein is fused with a coat protein of bacteriophage and displayed on the surface of the virion. phage-displayed random peptide libraries have helped researchers map b-cell epitopes [ ] [ ] [ ] , discover protein-protein contacts [ , ] , and identify bioactive peptides bound to receptors [ , ] or proteins [ , ] . peptide libraries can be used to find disease-specific antigens [ , ] and cell- [ , ] and organ-specific peptides [ , , ] . recently, using affinity selection (biopanning) of phagedisplayed peptide libraries, researchers have discovered molecules that are expressed on tumor blood vessels exclusively [ , [ ] [ ] [ ] . the strategy for identifying tumortargeting ligands and developing ligand-mediated targeted therapy is shown in figure . researchers have used in vivo affinity selection of phage libraries to identify peptides that interact with the molecules found on endothelia in tumors [ , ] . the ngr peptide motif targets angiogenic blood vessels [ ] and the tumor-homing property of ngr motif relies on recognition of a cd isoform selectively expressed within tumor blood vessels [ ] . compared with the nontargeting liposomal doxorubicin (caelyx), ngr peptide-conjugated caelyx significant improvements in survival was seen in clinically relevant animal models of neuroblastoma, ovarian, and lung cancers [ ] . another peptide, sp - , has been found to recognize blood vessels created in tumors but not normal blood vessels in severe combined immunodeficiency (scid) mice bearing solid tumors. several selected phage clones display pro-ser-pro, a motif crucial to peptide binding to tumor neovasculature [ ] . several tumor homing peptides have been found to bind to blood vessels in surgical specimens of human cancer and they have also been found to home to tumor tissues of different human tumor xenografts as confirmed by in vivo homing assays [ ] . these studies found a greater correlation between increased tumoral accumulation of the targeting liposomes and antitumor efficacy than the accumulation of free drugs or drugs formulated in the nontargeting liposomes [ , , ]. most of the drug delivery systems approved for marketing are liposomal-or lipid-based formulations or therapeutic molecules linked to polyethylene glycol (peg) [ , , , ] . one such product is pegylated liposomal doxorubicin, figure : diagram of the molecular mechanism of peptide-conjugated liposomes on cancer therapy. these liposomes prolong circulation time in blood and improve pharmacokinetic and biodistribution of their encapsulated drugs. after intravenous administration, liposomes are large enough to be excluded from normal endothelium. in solid tumors, the angiogenic tumor vasculature becomes leakiness that particulate liposomes can extravasate and localize in the tissue interstitial space making it possible for more drug delivering liposomes to accumulate within the tumor by epr effect. coupling liposomes with peptides targeted to tumor cells or tumor vasculature further enhances the specificity and accumulation of liposomes in the tumor. on arrival in the tumor tissues, the liposomes are bound and internalized by tumor cells or tumor-associated endothelial cells through receptor-mediated endocytosis, fused with the low ph compartments of the endosomes, and subsequently broken down the liposomes and to release encapsulated drugs into the intracellular space of the cells. which is known as doxil in the us and caelyx in europe [ ] . it is currently approved for the treatment of aidsrelated kaposi's sarcoma and recurrent ovarian cancer in north america, europe, and other countries, and for metastatic breast cancer in europe. liposome-encapsulated doxorubicin has been found to significantly improve the therapeutic index of doxorubicin both in preclinical [ ] [ ] [ ] and in clinical studies [ ] [ ] [ ] [ ] . an important advantage of pegylated liposomal doxorubicin is that the heart muscle uptakes much less of it than free doxorubicin [ , ] . one study found no cardiotoxicity in patients receiving cumulative doses of - mg/m of doxorubicin [ ] . free doxorubicin, on the other hand, is limited to a maximum recommended cumulative dose of - mg/m . colbern et al. found that the activity of pegylated liposomal doxorubicin - mg/kg was almost equivalent to that of free doxorubicin mg/kg in mouse lewis lung carcinoma [ ] . one clinical study reported that most (> %) of the drug circulating in the blood stream remains in encapsulated in liposomes [ ] , suggesting that little of the liposomal drugs will be leaked to the circulation system during its journey to the tumor tissues. the hyperpermeability of tumor vasculature is a key factor for the success of liposome-delivered chemotherapy agents. the "leakiness" of the angiogenic tumor vasculature is estimated to have an average pore size of - nm [ ] . these pores are significantly larger than the gap junction found in normal endothelium, which are typically < nm wide [ ] . liposomes with diameters of approximately - nm [ , , ] are small enough to passively infiltrate tumor endothelium but large enough to be excluded from normal endothelium. hence, they selectively extravasate into the tumor interstitial space. in the tissue of solid tumors, vasculature becomes so permeable that particulate liposomes can extravasate and localize in the tissue interstitial space [ , ] . in addition, tumor tissues frequently lack effective lymphatic drainage [ ] , which means that the liposomes can be retained longer. together, these factors increase the journal of oncology accumulation of the drug within the tumor, which has been referred to as the "enhanced permeability and retention (epr) effect" by maeda et al. [ , ] . epr-mediated passive tumor targeting by liposomes can increase the concentration of drugs in solid tumors by as much as ten times, compared to free drugs [ ] . passively targeted liposomal drug delivery systems have some disadvantages. normal organ uptake of liposomes leads to accumulation of the encapsulated drug in mononuclear phagocytic system cells in the liver, spleen, and bone marrow [ ] , which may present hazards to these tissues. for example, with increased circulation time of these drugs may come increased toxicity inducing such problems as handfoot syndrome, mucositis, and hematological toxicities such as neutropenia, thrombocytopenia, and leucopenia [ ] [ ] [ ] [ ] . therefore, ongoing research aims at enhancing the tumor site-specific action of the liposomes by attaching ligands to surface molecules of tumor cells and tumor vasculature, a process called active or ligand-mediated targeting liposomes [ , , , ] . the disadvantage of the passive pegylated liposomes can be overcome by creating ligand-mediated targeting liposomes with more selective anticancer activity. the activity of anticancer drugs can be enhanced by coupling targeting moieties to the surface of liposomes to promote selective binding to tumor-associated antigens and facilitate the delivery of drug-containing liposomes to the intended cellular sites. this drug delivery system has a higher drug-to-carrier ratio than immunoconjugates and multivalent presentation of ligands, which increases their binding avidity [ ] . antibodies that bind to tumor-specific antigens have so far yielded little success as a drug delivery system for solid tumors, which make up more than % of all cancers in humans. although monoclonal antibodies have shown clinical potential as tumor targeting agents, they are limited by their large molecular size and poor tumor penetration [ ] , by the immunogenicity associated with immunoliposomes, and by their toxicity to liver and bone marrow from nonspecific antibody uptake. these limitations can be overcome by using peptide ligands, which are smaller, less immunogenic molecules, and easier to produce and manipulate. furthermore, peptide ligands have moderate affinity to antigens, which is beneficial because extremely high affinity of antibody-binding can impair tumor penetration [ ] . compared with antibody ligands, peptide ligands can improve tumor penetration and decrease mps clearance of conjugated liposomes [ , ] . the increasing use of peptides as targeting ligands has been aided by the use of phage display to identify novel ligands (figure ). researchers have already produced liposomes conjugated with ligands that specifically target tumor cells or tumor vasculature [ , , ] . peptide-conjugated liposomes have three main components: anticancer drug, a liposome carrier, and targeting peptide ( figure ). remote loading methods such as the ammonium sulfate method [ , ] and the ph gradient method [ ] can encapsulate weak bases such as doxorubicin or vinorelbine into the liposomes with more than % efficiency. schedule-dependent drugs such as vinca alkaloids, topotecan, and -fluorouracil are also potential candidates for liposomal delivery because they can extend the time when cancer cells are exposed to therapeutic levels of the drug. the bioavailability and pharmacodynamics of liposomeencapsulated chemotherapeutic drugs must be considered in developing these delivery systems. to take advantage of the epr effect, liposomes need to have long half-lives so that the drug stays within the carrier as long as possible in blood circulation until it accumulates in diseased tissues [ ] . once liposomes are localized to a solid tumor, the drug they contain must be released and become bioavailable at a rate remains therapeutically effective for a period of time. the rate of active drug's release into tumor cells, not the total drug concentration in the tumor tissues, is critical for measuring the actual bioavailability of the liposomal drug [ ] . some targeting liposomes have not been found to have greater therapeutic efficacy than passive liposomal drugs, possibly because the lack of internalizing ligands does not give the drug greater access inside tumor cells [ , ] . drug delivery can be further enhanced if the liposome-attached ligands bind selectively to internalizing antigens which would help increase the concentration of drugs inside tumor or tumor-associated endothelial cells resulting in higher drug concentration inside the cells [ , , , ] . this binding to internalizing antigens by ligands can induce receptor-mediated endocytosis of liposomes into endosome compartments with low ph, where the liposomes break down and release the encapsulated drug into the intracellular space ( figure ). these steps lead to higher intracellular drug concentration and greater destruction or inhibition of tumor cells. studies have confirmed greater cytotoxic effects produced by liposomes with peptides that target internalizing antigens through enhanced specificity and improved drug bioavailability [ , ] . the use of drug-encapsulated liposomes with ligands to target tumor blood vessels allows us to destroy both tumor blood vessels and tumor cells. in mice bearing human cancer xenograft, low dose of peptide-conjugated liposomal doxorubicin has been found to markedly inhibit vascularization and reduce total volume and weight of tumors [ , , ] . the immunofluorescent analysis of the tumors in several studies has revealed an association between significant decreases in microvessel density and increases in the apoptosis of tumor cells and tumor-associated endothelial cells. the severe damage to tumor vasculature caused by peptide-conjugated liposomal doxorubicin throughout the tumors suggested an improvement in chemotherapeutic efficacy over nontargeting liposomes and conventional drugs [ , , ] . this dual action may produce a greater, more durable anticancer effect than is found with the use of simple antiangiogenic therapy. one peptide-conjugated liposome can deliver over ten thousand anticancer drug molecules directly into target tumor cells efficiently and effectively. the targeted and sustained release of the drug molecules can increase the maximum tolerated dose (mtd) of the cytotoxic drugs journal of oncology and dramatically lower dose-limiting toxicities, and in turn prevent treatment delay or discontinuation. the affinity of targeting ligands may allow the liposomes to move past the high ifp barrier within tumors [ , , , ] . advances in nanotechnology and molecular biology are moving us closer to developing an ideal "multifunctional smart nanodrug delivery system" using various types of ligands and drugs based on the kinds of diagnosis, imaging, or therapy needed. such smart nanodrug delivery systems will allow accurate, specific, and noninvasive disease treatment, early diagnosis, and monitoring. in the future, combining ligands that specifically bind to cancer cells (including cancer stem cells) and tumor blood vessels with multifunctional liposomal drug delivery systems may help improve the effectiveness of cancer treatment and minimize the side effects traditionally associated with chemotherapy. the development of highly selective anticancer drugs that can discriminate between tumor cells and normal cells is the most important goal of current oncology research. the potential use of ligand-conjugated liposome-encapsulated drugs to target tumor cells and vasculature is very promising. peptides that specifically bind to tumor targets can be coupled to the peg terminus of sterically stabilized liposomes and subsequently precisely deliver chemotherapeutic agents to tumor cells or blood vessels. peptide-mediated liposomes that target vasculature are a new generation of chemotherapy delivery systems with superior pharmacokinetics, controlled biodistribution, efficacy, and safety profiles. elucidation of the mechanism enabling tumor selective prodrug monotherapy a novel peptide enhances therapeutic efficacy of liposomal anti-cancer drugs in mice models of human lung cancer transport of molecules in the tumor interstitium: a review high interstitial fluid pressure-an obstacle in cancer therapy targeted-therapy for cancer drug delivery systems: entering the mainstream detection of tumor angiogenesis in vivo by alphavbeta-targeted magnetic resonance imaging a dna vaccine against vegf receptor prevents effective angiogenesis and inhibits tumor growth tumor regression by targeted gene delivery to the neovasculature therapeutic nanoparticles for drug delivery in cancer ligand-targeted therapeutics in anticancer therapy vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy peptide-mediated targeting to tumor blood vessels of lung cancer for drug delivery a novel peptide specifically binding to nasopharyngeal carcinoma for targeted drug delivery internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs antiangiogenic targeting liposomes increase therapeutic efficacy for solid tumors enhanced antitumor efficacy of clinical-grade vasculature-targeted liposomal doxorubicin protracted low-dose effects on human endothelial cell proliferation and survival in vitro reveal a selective antiangiogenic window for various chemotherapeutic drugs antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer less is, more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice continuous lowdose therapy with vinblastine and vegf receptor- antibody induces sustained tumor regression without overt toxicity bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer sunitinib versus interferon alfa in metastatic renal-cell carcinoma phase ii placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma cytogenetic abnormalities of tumor-associated endothelial cells in human malignant tumors tumor-associated endothelial cells with cytogenetic abnormalities angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy proteins expressed on tumor endothelial cells as potential targets for anti-angiogenic therapy normalization of tumor vasculature: an emerging concept in antiangiogenic therapy inhibition of pdgf receptor signaling in tumor stroma enhances antitumor effect of chemotherapy effect of antivascular endothelial growth factor treatment on the intratumoral uptake of cpt- direct evidence that the vegf-specific antibody bevacizumab has antivascular effects in human rectal cancer antiangiogenic therapy for cancer: current and emerging concepts steps toward mapping the human vasculature by phage display novel challenges in exploring peptide ligands and corresponding tissue-specific endothelial receptors cancer treatment by targeted drug delivery to tumor vasculature in a mouse model aminopeptidase n is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis identification of b-cell epitope of dengue virus type and its application in diagnosis of patients identification of a dengue virus type (den- ) serotype-specific b-cell epitope and detection of den- -immunized animal serum samples using an epitope-based peptide antigen generation and characterization of monoclonal antibodies against dengue virus type for epitope mapping and serological detection by epitope-based peptide antigens structural plasticity in a remodeled protein-protein interface binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain minimization of a polypeptide hormone tissuepenetrating delivery of compounds and nanoparticles into tumors peptide binding and presentation by mouse cd a synthetic peptide mediated active targeting of cisplatin liposomes to tie expressing cells a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera disease-specific b cell epitopes for serum antibodies from patients with severe acute respiratory syndrome (sars) and serologic detection of sars antibodies by epitope-based peptide antigens cell-specific peptide binding by human neutrophils hepatocellular carcinoma cell-specific peptide ligand for targeted drug delivery determination of free amino groups in proteins by trinitrobenzenesulfonic acid poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains sterically stabilized anti-her immunoliposomes: design and targeting to human breast cancer cells in vitro differential binding of drugs containing the ngr motif to cd isoforms in tumor vessels, epithelia, and myeloid cells nanoparticle and targeted systems for cancer therapy polymer conjugates as anticancer nanomedicines phase iii data on caelyx in ovarian cancer sterically stabilized liposomes: improvements in pharmacokinetics and journal of oncology antitumor therapeutic efficacy significant increase in antitumor potency of doxorubicin hcl by its encapsulation in pegylated liposomes sequential administration with oxaliplatin-containing pegcoated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor randomized phase iii study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression pegylated liposomal doxorubicin (doxil): reduced clinical cardiotoxicity in patients reaching or exceeding cumulative doses of mg/m effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes dose escalation and pharmacokinetics of pegylated liposomal doxorubicin (doxil) in children with solid tumors: a pediatric oncology group study cardiac safety of liposomal anthracyclines openings between defective endothelial cells explain tumor vessel leakiness optimizing liposomes for delivery of chemotherapeutic agents to solid tumors a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs tumor vascular permeability and the epr effect in macromolecular therapeutics: a review doxorubicin encapsulated in liposomes containing surface-bound polyethylene glycol: pharmacokinetics, tumor localization, and safety in patients with aids-related kaposi's sarcoma liposomal doxorubicin: antitumor activity and unique toxicities during two complementary phase i studies hand-foot syndrome associated with liposome-encapsulated doxorubicin therapy phase i and pharmacokinetic study of mcc- , a doxorubicin (dxr) encapsulated in peg immunoliposome, in patients with metastatic stomach cancer the clinical benefit of pegylated liposomal doxorubicin in patients with metastatic breast cancer previously treated with conventional anthracyclines: a multicentre phase ii trial targeting liposomal chemotherapy via both tumor cell-specific and tumor vasculature-specific ligands potentiates therapeutic efficacy penetration of tumor tissue by antibodies and other immunoproteins high affinity restricts the localization and tumor penetration of singlechain fv antibody molecules cancer-specific ligands identified from screening of peptide-display libraries ammonium sulfate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandoliposomes liposomal vincristine which exhibits increased drug retention and increased circulation longevity cures mice bearing p tumors pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies immunoliposome-mediated targeting of doxorubicin to human ovarian carcinoma in vitro and in vivo nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump antibody targeting of doxorubicin-loaded liposomes suppresses the growth and metastatic spread of established human lung tumor xenografts in severe combined immunodeficient mice anti-her immunoliposomes: enhanced efficacy attributable to targeted delivery acknowledgment h-c. wu is supported by the funding from academia sinica and national science council, taiwan. key: cord- - fexcpt authors: reddy, mynampati akshitha; pradhan, bikash kumar; qureshi, dilshad; pal, sumit kumar; pal, kunal title: internet-of-things-enabled dual-channel iontophoretic drug delivery system for elderly patient medication management date: - - journal: j med device doi: . / . sha: doc_id: cord_uid: fexcpt wireless controllers have found its application in the supervision of the patients in the hospitals. it is not only a valid issue for the developing countries but also for the developed countries. for this reason, scientists are working on the advancement of medical devices that are capable of decreasing the workload of health caregivers. in this study, the development of an iontophoretic drug delivery device that could be controlled using a mobile is described. for the purpose, hardware and a software module were developed. the hardware module consisted of a two-channel voltage-controlled constant current sources that were used for driving the iontophoretic device. a mobile app was developed to control the two-channel iontophoretic device and to monitor the loose lead of the active and the passive patches. in the case of detection of the loose lead, the specific iontophoretic channel was stopped. further, the audio-visual indicator was developed for the detection of the detachment of the patches (loose lead). the device was tested in vitro by performing the drug release study using drug-loaded emulsion gels that were formulated. medication management for elderly patients is a critical component in the efficacy of the treatment modality. the process is complicated when a number of medicines are given, and the elderly patients have to manage the task of remembering the doses, which is quite taxing. this is further complicated if the patient is suffering from physical, visual, or cognitive impairments. furthermore, it has been observed that even the elderly patients are advised oral drug delivery systems, which they have to swallow. unfortunately, many of the patients (especially having age > years) suffer from dysphagia and face a tremendous obstacle in taking the medication. regular use of the injectables in these patients is also not feasible. due to the aforesaid reasons, the help of caregivers has been proposed. nevertheless, this is associated with a high recurring cost. many researchers have proposed to deliver drugs by applying the drug formulations over the skin surface [ ] . the iontophoretic drug delivery system is one such method that has gained much importance in the last few decades. the main advantage of iontophoretic drug delivery is its ability to deliver drugs within the deeper layer of the skin tissue and/or systemic circulation in a noninvasive manner [ ] . with the advent of technological advancements, it is quite feasible to develop electronically-controlled drug delivery systems, including iontophoretic drug delivery systems, at a cheap price. the use of the internet for manipulating various machines, devices, and objects is known as the internet-of-things (iot). due to the significant advancements made in the field of internet technology, iot has been able to revolutionize the current day technology system. in recent times, the term medical-iot has been gaining importance. medical-iot is a field of study that involves controlling medical devices from a remote location using the internet technology. the application of iot technology in medicine can significantly improve the quality and efficiency of the healthcare service, especially for elderly patients, patients with chronic conditions, and for the people who need continuous monitoring [ ] . this technology has also been explored for iontophoretic drug delivery applications. this type of device can help in monitoring and delivering the drug from a remote location (like office and car) by the relatives. sayeed et al. developed an iotbased responsive drug delivery system that can inject the drug into the epileptogenic zone on seizure detection [ ] . in another study, junginger et al. have designed a chip-controlled iontophoretic system that can deliver apomorphine to the patients suffering from parkinson's disease [ ] . in the proposed method, the amount of the drug delivered could be controlled by controlling the current density. conformal contact between the device and the skin must exist to increase the device efficacy [ ] . any loss in the connection between the skin and the patch(es) results in the underdosing of the drugs to the patients and can cause morbidity. however, to the best of our knowledge, no arrangement was made by any existing literature to detect the loss of connection between the skin and the patches. another critical issue found in every drug delivery system was its ability to deliver only one drug at a time. the report says elderly people are the largest per capita consumer of drugs and are prescribed a higher number of medications [ ] . in such patients, polypharmacy (multiple drug use) is mainly related to chronic diseases like cardiovascular diseases, diabetes, and memory-related diseases (e.g., dementia and alzheimer's disease). polypharmacy is also found in low-income countries where disease like tuberculosis is among the most common chronic illness [ ] . hence, a single-channel system may not be sufficient, and the requirement of a multichannel iontophoretic drug delivery system becomes essential. taking the cue from the above discussion, a smartphone-based remote-controlled iontophoretic drug delivery device has been developed in this study. provisions have been made in the device to deliver multiple drugs. the program of the device can be regulated to tailor the duration of the drug delivery, and hence, the amount of the drug to be delivered into a patient as per the choice of the programmer. further, an in-built program to detect and alert the healthcare professionals or relatives on the detachment of the drug delivery patch(es) from the site of the application is also proposed. materials. esp e nodemcu module (espressif systems, china), arduino due (arduino, italy), arduino mega adk (arduino, italy), resistances ( kx, kx, and kx), capacitors ( lf, lf, and lf), diode, light emitting diodes (leds) (green, orange), v batteries (mercury-cadmium type), and groundnut oil (farm naturelle, india) were bought from the local market. an operational amplifier (ua cn), ic lm , and ic lm voltage regulator were purchased from texas instrumentation. the free version of the eagle printed circuit board (pcb) design software (autodesk, san rafael, ca) was used for designing the pcb of the developed circuit. sorbitan development of the signal generator. in this study, a programmable signal generator was developed [ ] . for the purpose, the arduino due microcontroller board was used. a look-up table (two-dimensional array) was used to generate a sinusoidal signal (appendix). the signal was generated using points from the two-dimensional array. the sample index point was incremented until the points within a fixed time of ms. this resulted in the generation of a sinusoidal signal having a frequency of khz and a peak-to-peak amplitude of . v. the signal generator was programmed to generate independent analog signals, which were presented at the dac and dac terminals of the microcontroller board. the signal generator was programmed to generate signals for a period of h. designing of the iontophoretic device. the circuit for the iontophoretic drug delivery system was developed as per the previously reported literature [ ] . some modifications were made in the circuits to suit our needs. the sinusoidal signal generator, which was developed using the arduino due, was used as the voltage signal generator. the output of the signal generator was processed using a passive high-pass filter (f c ¼ hz). the output signal from the high-pass filter was then used as the input for the operational amplifier (op-amp) based voltage buffer circuit. the output of the buffer was later converted into a current signal using a voltage-to-current converter. the arrangements were made so that the generated current was then injected through the fig. flow chart of the working principle for a loose-lead monitoring system iontophoresis cell. an led-based visual indicator circuit was also incorporated to indicate the working status of the device. all the op-amp were powered using a v battery-powered power supply (see fig. s available in the supplemental materials on the asme digital collection). the circuit diagram of the developed iontophoretic circuit has been shown in fig. . testing of the iontophoretic circuit. the characteristic of the high-pass filter was tested using a variable function generator. the function generator was manually controlled to generate a sinusoidal signal of . vpp signal. the frequency of the signal was varied from hz to khz. subsequently, the frequency versus amplitude response was plotted. further, the testing of the iontophoresis circuit was carried out by analyzing the signal at different stages of the circuit. designing of the loose-lead monitoring system. the signal at the output of the voltage-to-current converter was used as the input for the buffer amplifier being powered with v power supply. the output of the buffer amplifier was then served as the input to the inverting terminal of an op-amp based comparator circuit. the noninverting terminal of the comparator was supplied with a þ . v dc voltage. the output of the comparator was then digitized. the digitized values were then passed through a moving average [ ] having a window of points. the average values were compared with a constant value of . a value of more than was considered as good connectivity between the patches and the human body. on the contrary, a value of less than was considered improper connectivity between the patches and the human body. in case any loose-lead was detected, a high output was generated and sent to the designed website through the wifi module. the circuit diagram of the developed loose-lead monitoring system has been shown in fig. . the processed flowchart of the loose-lead monitoring system has been shown in fig. . pcb designing of the iontophoretic device with loose-lead monitoring. eagle pcb design software was used for the designing of the pcb for the developed circuit of the iontophoretic device with loose-lead monitoring capability (see fig. s available in the supplemental materials on the asme digital collection). a layout of the circuit of the device was made (see fig. s available in the supplemental materials on the asme digital collection). the layout was then printed on a glossy paper using a laserjet printer. the printed layout, consisting of the carbon particles, was then transferred to copper-clad laminates. the copper-clad laminates with the transferred carbon were then kept for etching in ferric chloride (fecl ) solution for removing the exposed copper area. necessary holes were made at the designated places. subsequently, the components were placed at the specified slots and then soldered. the connectivity of the tracks was tested using a multimeter. finally, the working of the developed circuit was tested using a khz sinusoidal signal ( . vpp), and the output was monitored in a dso (digital storage oscilloscope). with the iontophoretic device. a webpage was designed using html protocol [ ] to communicate with the iontophoretic device. the webpage was hosted using a hypertext transfer protocol (http)based web server, which was created by programming an esp e nodemcu module (using arduino ide) in station mode. the webpage consisted of two sets of control buttons for switching "on" and switching "off" of each of the signal generators. once the switches were switched on, they were expected to trigger the signal generators that would continue for h. provisions were also made to switch off either or both of the signal generators before h in case of any need. further, two virtual leds were incorporated to indicate the status of the connections of the drug reservoir(s) to the human body. finally, a mobile app was designed using mit app inventor for this webpage. integration of the mobile app and the hardware. the integration of the mobile app and the iontophoretic drug delivery system was achieved by interfacing the iontophoretic drug delivery system with the capability to monitor the loose-lead monitoring and the mobile app. the same was achieved by hosting the webpage of the mobile app (containing the control buttons for the fig. flow diagram of the functioning of the loose-lead monitoring system signal generators, developed using mit app inventor) using the webserver of the esp- e wifi module [ ] and establishing the communication between them using http communication protocol [ ] . the schematic representation of the proposed drug delivery system has been provided in fig. . the proposed flowchart of the integrated device has been given in fig. . drug release study. a drug release study was conducted using the developed iontophoretic drug delivery system. for the purpose, two emulsion gel formulations were made ( table ). the formulations were prepared by the hot emulsification method. in gist, groundnut oil, emulsifier (sorbitan monopalmitate (smp)), and water were mixed in different proportions. smp was dissolved in groundnut oil ( c, rpm). to this hot solution, water ( c) was added dropwise and subsequently homogenized for min at rpm. the model hydrophilic drug (metronidazole, . g) was mixed to the homogeneous mixture. the hot emulsion was then cooled down to room temperature in order to induce gelation [ ] . the developed drug-loaded emulsion gels were used to perform the drug release study using the designed iontophoretic circuit. both the iontophoretic channels were switched on using the mobile app. the study was conducted for h (as was set in the iontophoretic device). channel- reservoir of the iontophoretic device consisted of smp -m, while the smp -m was loaded in the channel- reservoir. samples were withdrawn at regular intervals and tested for the amount of the released drug. similarly, another set of drug release experiment was carried out using the same setup without activating the iontophoresis device. the tests were conducted in triplicates. the schematic diagram of the setup is provided in fig. . development of the signal generator. arduino due board houses an atmel sam x e arm cortex-m microcontroller. the board has an in-built two-channel digital-to-analog converter (dac). the microcontroller was programmed so that the arduino due board could synthesize sinusoidal signals independently from either or both the dac channels. the synthesized sinusoidal signal has been shown in figs. s (a) and s (b) that are available in the supplemental materials on the asme digital collection. a visual inspection of the signal suggested the presence of a dc offset ( . v, . v) in the generated signals. the peak-to-peak amplitude was found to be . vpp and . v. the signals were designing and testing of the iontophoretic device. the analysis of the synthesized signals suggested the presence of an offset voltage of . v. so, to eliminate the same, a passive high-pass filter having a cut-off frequency of hz was employed. the gain response of the filter was determined (fig. (a) ). it was found that the gain at khz frequency was db. this suggested that the designed filter would not affect the nature of the khz signal. however, it would be capable of eliminating the dc component from the synthesized signal. hence, the designed filter was used to process the synthesized signals. the output of the high-pass filter has been given in fig. (b) . it could be seen here that the processed signal did not have any offset. further, there was no alteration in the peak-to-peak amplitude of the processed signals as compared to the synthesized signal. the processed signals were then presented to a buffer amplifier. a buffer amplifier provides a gain of . hence, there is no magnification of the signal component (fig. (c) ). in our study, we also found that the input signal from the high-pass filter and the output of the buffer amplifier were the same. further, the buffer amplifier imparts an electrical impedance transformation while connecting two circuits. this helps to prevent the signal source from being affected by the load of the sink circuit. the output of the buffer amplifier was then given to the voltage-controlled constant current source that was operating in the inverting amplifier mode. the input resistance ( kx) of the constant current source governs the current injected into the human body through the patches. this resulted in the generation of a sinusoidal current having a peak-to-peak current amplitude of . la (ipp). hence, the positive and negative peak currents were . la (ip). the current was then injected into the iontophoresis cells, which were used as a simulated human body. since the system was operating in the inverting amplifier mode, there was a deg phase shift of the voltage signal across the iontophoresis cells (fig. (d) ). designing of the loose-lead monitoring system. continuous monitoring of the impedance between the active and the passive patches was employed for the loose-lead monitoring. the passive patch was virtually grounded as it was connected with the inverting terminal of the constant current source, while the noninverting terminal was directly grounded to the ground of the electrical circuit. hence, the current is injected into the iontophoresis cell through the active patch. accordingly, the voltage measured at the active patch against the ground would be directly proportional to the impedance across the active and the passive patches. this means, when the patches are properly connected with the iontophoresis cell, we will get a voltage value that is proportional to the total impedance of the active patch, passive patch, and the iontophoresis cell. since the input signal to the iontophoresis cell is sinusoidal, the output would also be sinusoidal (fig. (a) ). however, if either of the patches or any one of the patches gets disconnected, the circuit becomes an open circuit, and the impedance would be infinite. here, we would get a square signal. the positive peak would go to positive saturation, while the negative peak would go to the negative saturation ( fig. (b) ). subsequently, the output of the iontophoresis cell is given to the buffer amplifier. the output signal was then used as the input to the inverting terminal of a voltage comparator circuit that was being operated using a v power supply. a constant dc voltage of . v was used for the comparison, which was connected to the noninverting terminal of the comparator. the comparator was operated using v power supply so as to prevent damage to the microcontroller board due to the input of the voltage that was higher than þ v. the microcontroller board rejects the negative voltage as it functions in the range of v and v. the output of the comparator during the open and the close cases has been given in figs. (c) and (d) . during the open case, a square wave of . vpp was obtained. on the contrary, during the close case, a dc voltage of . v was obtained. these signals were then pcb designing of the iontophoretic device with loose-lead monitoring. the layout of the circuit made during the developmental stage ( fig. (a) ) was designed using the eagle pcb design software. the layout was printed on a glossy paper ( fig. (b) ), which was then transferred over the copper-clad laminates ( fig. (c) ). the excess copper was removed from the laminates ( fig. (d) ), drilled, and the components were placed and consequently soldered. the components were placed as per the requirements and then soldering was performed. the developed pcb board has been shown in fig. (e). two numbers of pcbs were fabricated for implementing the dual-channel device. the connectivity of the tracks was then tested using the multimeter. finally, the testing of the circuit was performed using a khz sinusoidal signal. the output of the signal at each step was observed in a dso (digital oscilloscope) and compared with the observations made during the development stage (see fig. s available in the supplemental materials on the asme digital collection). the observations made during the developmental stage and after the pcb fabrication were found to be similar. this suggested that the designed pcbs were working fine. then, the pcbs were integrated with the microcontroller boards. the complete system has been shown in fig. . development of mobile app for communicating with the iontophoretic device. a mobile app was developed. when the app is opened, it shows the control system for channel- and channel- ( fig. (a) ). as the iontophoresis device is put on, the app showed two activated virtual green leds with a note "electrode connected" (fig. (b) ). the loose-lead was artificially generated by intentionally detaching any or both of the patches from the iontophoresis cell. during this case, the green virtual led changed color to red. further, the note electrode connected was changed to "loose-lead detected." (fig. (c) ). fig. testing of the iontophoretic device. (a) gain response of the designed high pass filter, (b) voltage signal at the output of the high pass filters (same phase of the input and the output signals), (c) voltage signal at the output of the buffer amplifier (same phase of the input and the output signals), and (d) differential voltage signal across active electrode-formulation-diffusion media-electrode arrangement ( deg phase-difference among the input and the output signals) (note: the upper waveform represents output from the signal generator; the lower waveform represents the output at various stages of the circuit). fig. testing of the loose-lead monitoring system. (a) differential voltage signal across active electrode-formulationdiffusion media-electrode arrangement when the electrodes were properly connected, (b) differential voltage signal across active electrode-formulation-diffusion media-electrode arrangement when one of the electrodes was displaced, (c) output of the comparator circuit when the electrodes were properly connected, (d) output of the comparator circuit when one of the electrodes was displaced, (e) the serial monitor reading when the electrodes were properly connected, and (f) the serial monitor reading when one of the electrodes was displaced (note: the upper waveform represents the output from the signal generator; the lower waveform represents the output at various stages of the circuit). drug release study. the drug release study was performed using the prepared emulgels. the emulgels contained metronidazole as the model drug. the drug release studies were conducted in both passive and active modes. in the passive mode, the iontophoretic device was not put on. while doing the drug release study in the active mode, the iontophoretic device was put on using the developed mobile app. the release of the drug was monitored using a uv-vis spectrophotometer at a different time interval. the test was carried out for h. the cumulative drug release (cpdr) profiles were determined (figs. (a) and (b) ) [ ] . it was observed that the release of the drug was higher during iontophoresis as compared to the passive study. it is important to note that there was not much difference between the release profiles. this can be accounted for the use of the dialysis membrane, which does not exert sufficient hindrance to the drug transport as that of the skin tissue. the release profiles were modeled using the korsmeyer-peppas model (eq. ( ); figs. (c) and (d)) [ ] . it was found that the diffusion of the drug was higher in both the samples when the active experiment was carried out (table ) . further, the diffusion exponent value "n" was < . during the active experiment using smp -m. this suggested that fickian diffusion played an essential role during the said experiment. in all other experiments, n value was > . . this is suggestive of non-fickian diffusion of the drug molecules during the experiment where m represents the solute fraction released, t is the sampling time, k is constant representing rate of drug release from the polymeric matrix, and n is the diffusion coefficient. iontophoresis is a noninvasive method of drug transport that is used to transfer drug molecules into the tissues or systemic circulation by the application of an electric field [ ] . yan et al. [ ] , in their study, have compared the constant current and alternating current iontophoretic drug delivery and suggested that a constant ac current iontophoresis can provide fluxes that are comparable to the . ma of constant current dc. in another study, it has been reported that the modulated ac current of a duration of h at a frequency of khz was found to be comparable to a dc current of h [ ] . hence, for increased efficiency and simple design circuitry, fig. pcb designing and testing of the iontophoretic device with loose-lead monitoring: (a) breadboard design of the prototype iontophoretic drug delivery-cum-loose-lead monitoring system, (b) the layout of the pcb printed on to the glossy paper, (c) carbon transfer of the pcb layout to the copper-clad laminate, (d) pcb layout after etching and washing of the copper-clad laminate, and (e) the designed pcb board for the iontophoretic drug delivery-cum-loose-lead monitoring system a sinusoidal current was used for developing this drug delivery system. the biological tissues, such as skin tissues, act as a capacitor due to their ability to store electrons. the skin contact impedance represents the total electrical opposition of the iontophoretic circuit to the passage of the current through it [ ] . in the proposed system, this impedance was used as a measure to design a looselead monitoring system. in the case of detachment of the patch(es), the skin contact impedance will become infinite, and the voltage pattern across the patches (active and the passive) changes. this change in the voltage pattern was used for recognizing the loose-lead. subsequently, this information was sent to the user via the app and stops the waveform generation for the specific channel. noncompliance with medication is a major problem in old age, and the factors associated with these conditions are visual and cognitive dysfunction, forgetfulness, increased physical disability with increased age, complicated drug regime, multimorbidity, and its associated high rate of medications, [ , ] . more than million people are suffering from dementia worldwide [ ] . it has been reported that over three out of four alzheimer's patients need help in managing their daily medication. again, it is not possible for the members of the patient's family to continuously monitor them. a survey conducted by the national alliance of caregiving, showed that approximately % of the caregivers leave the patients alone at least for h and monitor them only from a distance [ , ] . the current study uses an iot-based drug delivery system that can be controlled using a mobile application. prior reports inferred . % of the people aged above , % of the people aged between and years, and % of people under the age of experience morbidity [ ] , and are prescribed, a higher number of medications. the proposed system can be operated in a dual-channel mode, i.e., two different types of therapeutic agents can be delivered to the patient's body simultaneously or individually one after another according to the user's requirement. this can fulfill the need for multiple drug usage or polypharmacy. in the current study, a dual-channel iontophoretic drug delivery system has been proposed. a programmable dual-channel sinusoidal signal generator was designed to provide alternating current signals for the proposed drug delivery system. the alternating current signal was meant for carrying out the iontophoresis-based drug delivery through the skin surface. an android app was designed, and an esp e nodemcu was used to enable wireless control of the drug delivery device. the drug delivery device can deliver single or multiple drugs simultaneously for the desired time duration. it is also capable of detecting the loose attachment of the drug patches with the human skin surface and generates a visual alert on detecting the loose contact. the components of the proposed device, namely, the signal generator, the iontophoretic setup, the loose-lead monitoring system, and the android app based wireless control system, were tested for their proper functionality. finally, an in vitro drug release study (both in active and passive modes) was performed, which suggested non-fickian diffusion of the drug molecules and a relatively higher drug release was observed during the iontophoretic drug delivery as compared to the passive drug delivery. the current system is a prototype device for a dual-channel iontophoretic drug delivery system. however, before practical implementation, the miniaturization of the device is essential using different advanced device development techniques, such as microfabrication, nanofabrication, and mems-based technologies. this would allow us to develop a wearable system. we have conducted the in vitro drug release study of the proposed system. there is a need to conduct in vivo studies, including animal and human tests, to validate the functioning of the proposed device. the number of drug delivery channels can also be increased so as to avoid the complications of polypharmacy. an alarm system can also be integrated in the future that can give a gentle reminder regarding the time and dosage of medication, thereby, improving the patient-medication compliance. polypharmacy in elderly patients iontophoretic delivery of drugs: fundamentals, developments and biomedical applications development of the elderly healthcare monitoring system with iot an iot-based drug delivery system for refractory epilepsy iontophoretic delivery of apomorphine: from in-vitro modelling to the parkinson patient device-assisted transdermal drug delivery drug identification and interaction checker based on iot to minimize adverse drug reactions and improve drug compliance designing of a variable frequency standalone impedance analyzer for in vitro biological applications development of a wireless controlled iontophoretic drug delivery system variance of errors and elimination of outliers in the least squares analysis of impedance spectra the proportional integral derivative control system on the mini conveyor with message queuing telemetry transport protocol based on the internet of things groundnut oil based emulsion gels for passive and iontophoretic delivery of therapeutics effect of sorbitan monostearate concentration on the thermal, mechanical and drug release properties of oleogels effect of span on the microstructure, crystallization kinetics, and mechanical properties of stearic acid oleogels: an in-depth analysis recent progress in ocular drug delivery for posterior segment disease: emphasis on transscleral iontophoresis evaluation of constant current alternating current iontophoresis for transdermal drug delivery effect of modulated alternating and direct current iontophoresis on transdermal delivery of lidocaine hydrochloride electrical characteristics of the skin: the impedance of the surface sheath and deep tissues elderly patients' problems with medication compliance with prescribed medication by elderly patients family caregivers of people with dementia the role of human factors in home health care: workshop summary public health for an aging society methods to reduce prescribing errors in elderly patients with multimorbidity the authors would like to thank dr. sumit chakravarty, assistant professor of electrical engineering at kennesaw state university, u.s., for his support, guidance, and checking of the paper. look up table for sine wave generation using arduino due microcontroller board. f x ff, x a, x f, x a , xadd, xba , xbff, xcb , xdf , xe , xeb , xf f, xf a, xfb , xfe , xfff, xfe , xfb , xf a, xf f, xeb , xe , xdf , xcb , xbff, xba , xadd, x a , x f, x a, x ff, x , x bf, x ed, x , x d, x a , x c, x , x , x , xdf, x , x c, x , x , x , x c, x , xdf, x , x , x , x c, x a , x d, x , x ed, x bf, x , g key: cord- - ht cqhu authors: smith, silas w. title: drugs and pharmaceuticals: management of intoxication and antidotes date: - - journal: molecular, clinical and environmental toxicology doi: . / - - - - _ sha: doc_id: cord_uid: ht cqhu the treatment of patients poisoned with drugs and pharmaceuticals can be quite challenging. diverse exposure circumstances, varied clinical presentations, unique patient-specific factors, and inconsistent diagnostic and therapeutic infrastructure support, coupled with relatively few definitive antidotes, may complicate evaluation and management. the historical approach to poisoned patients (patient arousal, toxin elimination, and toxin identification) has given way to rigorous attention to the fundamental aspects of basic life suppport — airway management, oxygenation and ventilation, circulatory competence, thermoregulation, and substrate availability. selected patients may benefit from methods to alter toxin pharmacokinetics to minimize systemic, target organ, or tissue compartment exposure (either by decreasing absorption or increasing elimination). these may include syrup of ipecac, orogastric lavage, activated single- or multi-dose charcoal, whole bowel irrigation, endoscopy and surgery, urinary alkalinization, saline diuresis, or extracorporeal methods (hemodialysis, charcoal hemoperfusion, continuous venovenous hemofiltration, and exchange transfusion). pharmaceutical adjuncts and antidotes may be useful in toxicant-induced hyperthermias. in the context of analgesic, anti-inflammatory, anticholinergic, anticonvulsant, antihyperglycemic, antimicrobial, antineoplastic, cardiovascular, opioid, or sedative-hypnotic agents overdose, n-acetylcysteine, physostigmine, l-carnitine, dextrose, octreotide, pyridoxine, dexrazoxane, leucovorin, glucarpidase, atropine, calcium, digoxin-specific antibody fragments, glucagon, high-dose insulin euglycemia therapy, lipid emulsion, magnesium, sodium bicarbonate, naloxone, and flumazenil are specifically reviewed. in summary, patients generally benefit from aggressive support of vital functions, careful history and physical examination, specific laboratory analyses, a thoughtful consideration of the risks and benefits of decontamination and enhanced elimination, and the use of specific antidotes where warranted. data supporting antidotes effectiveness vary considerably. clinicians are encouraged to utilize consultation with regional poison centers or those with toxicology training to assist with diagnosis, management, and administration of antidotes, particularly in unfamiliar cases. the challenges to effective evaluation and management of a patient poisoned by drugs and pharmaceuticals are diverse. the circumstances surrounding exposure are often incompletely accessible. poisoning signs or symptoms may be subtle or delayed. patient-specific factors -pharmacogenetics and unique susceptibilities, drug-drug interactions, cultural or geographic practices, and underlying comorbidities -may complicate presentation, response to treatment, and outcome. polypharmacy or mixed exposures may confuse the clinical presentation. compared to the near-inexhaustible list of products and possible combinations, few specific antidotes exist. the toxicological profiles of newly intro-organ injury. specific management of toxicant-induced hyperthermias follows later. hypothermia may require active or passive rewarming techniques. clinical hypoglycemia, which implies neuroglycopenia, must be rapidly reversed with administration of . - . g/kg of age-appropriate dextrose-containing solutions (d in adults, d in children, and d in neonates). benzodiazepines (e.g., diazepam, midazolam, and lorazepam) are generally well tolerated and are first line agents for drug-and withdrawal-induced seizures and agitation. persistent or refractory seizures should prompt consideration of empiric administration of pyridoxine and barbiturates (phenobarbital, pentobarbital), propofol, or ultimately, general anesthesia. coincident endotracheal intubation may be required. phenytoin and non-barbiturate anticonvulsants are typically ineffective or harmful in toxin-induced seizures [ , ] . altered mental status should also prompt parenteral administration of mg thiamine hydrochloride. alcohol-dependent patients without clinically apparent wernicke's encephalopathy may require at least mg of parenteral thiamine to improve neurological symptoms; overt wernicke's encephalopathy necessitates a minimum of mg thiamine hydrochloride three times daily for - days [ ] . naloxone use is considered in a separate section. toxidromes (toxic syndromes) are characteristic signs and symptoms that correlate with exposure to certain xenobiotics. identifying toxidromes suggests the etiology of the patient's condition and helps guide management. "classic" class-effect toxidromes include anticholinergic, cholinergic, sedative-hypnotic, sedative-hypnotic withdrawal, opioid, and opioid withdrawal. these should be actively sought and managed if identified. while the patient is being stabilized, diagnostic investigations including a complete and thorough history and physical examination, laboratory analyses, and radiological studies may be undertaken to further characterize the exposure and effect. for significantly compromised patients, a typical "chemistry panel" (providing electrolytes, blood urea nitrogen, creatinine, and indirectly the anion gap), a complete blood count, arterial (or venous) blood gas, and lactate are reasonable studies. urine or serum ketones may be required to determine the etiology of acidemia. female patients benefit from an assessment of pregnancy status. it is useful to determine a serum acetaminophen concentration in suicidal patients or those with altered consciousness, as patients with significant acetaminophen poisoning may present without a toxidrome. serum acetaminophen is detectable in - % of patients without a reported history of ingestion; treatable concentrations are found slightly less frequently [ , ] . toxin-specific studies and other serum determinations are often not rapidly returned and should be obtained only if suggested by the history, physical examination, or bedside testing. urine drug screening (uds) is of minimal use in the acute management of intoxication. results are not typically returned for hours; a reported "positive" substance may not be the proximate cause of the presenting condition (as the measured metabolites may persist in urine for days to weeks); and the uds lacks sensitivity and specificity (particularly for opioids, benzodiazepines and other sedative-hypnotics, and amphetamines). selected patients may benefit from methods to alter toxin pharmacokinetics -limiting exposure. a discussion of these modalities and their risks and benefits occurs in the following section. ultimately, patients will require disposition depending of severity of presentation and anticipated sequelae, which may range from admission to intensive care units, cardiac monitoring (telemetry) units, ward beds, continued emergency department evaluation, to discharge. a psychiatric assessment and social assessment, when appropriate, should precede release from medical care. appropriate and early consultation with medical toxicologists or regional poison centers may also assist with diagnosis and management. in the u.s., this has been simplified by a uniform telephone number ( . . . ) for regional poison center consultation. the international programme on chemical safety (ipcs) maintains a world directory of poison centers (http://www.who.int/ipcs/poisons/centre/directory/en/). adjuncts to alter toxicant pharmacokinetics aim to minimize systemic exposure (either by decreasing absorption or increasing elimination) or to minimize exposure of a target organ or tissue compartment. in practice, this is achieved by expulsion or removal from the upper gastrointestinal tract (induced emesis, gastric lavage, or endoscopy); intraluminal binding to adsorptive materials (activated charcoal); or increasing intestinal transit time (cathartics and whole bowel irrigation). endogenous elimination may be improved by more effective urinary clearance (urinary alkalinization and forced diuresis), improved hepatobiliary clearance, or "gut dialysis" with multiple-dose activated charcoal. rarely, hepatic metabolism is altered to preclude ultimate toxicant formation (e.g., cimetidine to mitigate production of dapsone's methemoglobinemia inducing metabolite). exogenous clearance utilizes hemodialysis, charcoal hemoperfusion, continuous renal replacement therapies, and exchange transfusion. all the adjuncts attempt to shift where a patient lies upon a particular dose-response curve (fig. ) . drug recovery following gastrointestinal emptying techniques has been inconsistent; human studies attempting to demonstrate a survival benefit of any decontamination modality are inconclusive. randomized trials in which a control group might not receive any decontamination could be considered unethical; volunteer studies using sublethal doses of xenobiotic cannot show mortality benefit. as might be anticipated from the fact that supportive care suffices for the majority of poisoned patients, a typical study of routine administration of charcoal following oral overdose of primarily benzodiazepines, acetaminophen, and selective serotonin reuptake inhibitors could not demonstrate benefit [ , , ] . past studies have suffered from significant exclusions. recommendations are based both on theoretical grounds (animal and in vitro studies demonstrating lower peak serum concentration or faster serum drugs and pharmaceuticals: management of intoxication and antidotes clearance) and human studies with surrogate endpoints such as marker studies or area under the curve of plasma concentration versus time (auc) improvement. aggressive detoxification may be required for certain lethal toxins for which few antidotal options exist. most gastric emptying techniques are thought to be relatively ineffective beyond hour. these constraints diminish possible benefit. for example, the median time from ingestion to arrival at a health care facility is on the order of hours, and only about % of patients can be lavaged within the idealized -hour time frame [ ] . although in ideal situations (patients presenting early to experienced health care providers with readily available ipecac syrup) pill retrieval averages - %, ipecac's benefits can be completely negated when administration is delayed as briefly as min [ ] [ ] [ ] [ ] . when orogastric lavage is performed by experienced providers within min of ingestion, clinical manifestations of ingested xenobiotics have been prevented [ ] . practically, efficacy of tablet retrieval rates reduces to % in some cases and improvements in auc vary from zero to % (averaging ~ %) [ , [ ] [ ] [ ] . similarly, restricting activated charcoal (ac) administration to patients presenting to health care within the first hour post ingestion would exclude up to % of poisoned patients from the potential benefits of ac when administered beyond an hour [ , ] . earlier administration of ac is more efficacious [ ] . however, home and prehospital use of ac decreases the time to treatment, but has not improved clinical outcomes [ ] . drugs with opioid or anticholinergic properties that decrease peristalsis or particularly large ingestions, which independently decrease intestinal motility, may modify decision making in delayed presentations [ , ] . figure . adjuncts to alter toxicant pharmacokinetics attempt to shift where a patient lies upon a particular (idealized) dose-response curve. risk will likely outweigh benefit if the patient begins at point a (negligible morbidity and mortality) and systemic exposure is reduced to b. this is the case for many drug poisonings which are managed effectively by supportive care alone. decontamination might provide significant benefit if the patient lies upon the steep aspect of the curve [reduction from c to d -the same fixed amount as from a to b (although a percentage reduction could also be envisaged)]. with overwhelming overdose (point e), despite decontamination, benefit would be unlikely (point f). independent of side effects, the efficacy of one modality over another or combination therapy is debated. some studies rate ipecac syrup more efficacious than orogastric lavage, but most studies have found little or no difference, and neither has been shown to be more effective than spontaneous emesis [ , , , ] . ac has demonstrated ~ % better reductions in auc than ipecac, which may improve or worsen its efficacy [ , , ] . gastric lavage adds no benefit to ac, except for the most critically ill patients [ , , ] . compared directly, ac has better impact than lavage on auc and clinical effect [ , , ] . data are equivocal regarding whole bowel irrigation's ability to function similar to multiple-dose ac (mdac) as a medium for "gut dialysis" [ , ] . syrup of ipecac is obtained from a root extract of the amazonian flowering plant psychotria ipecacuanha [ ] . its active alkaloid components, cephaeline and emetine, induce emesis via local irritation and central stimulation of -hydroxytryptamine (serotonin) -ht receptors [ ] . following appropriate dose ( ml for infants, - ml for children under , and ml otherwise), roughly % of patients have a first episode of emesis within min [ , ] . patients average three episodes in min [ ] . however, since ipecac's removal from most homes, the median time to administration in the acute care setting is delayed on the order of an hour, with only one-third of patients successfully vomiting within the first hour post ingestion [ ] . indications for ipecac are limited. a routinely cited example is a patient known to have taken multiple lithium tablets, which do not bind ac and may not fit through a lavage tube, who presents early to health care [ ] . the american academy of pediatrics no longer recommends ipecac syrup for home use; ipecac use does not impact outcomes or decrease utilization of emergency services [ , ] . ipecac may or may not have a role in other rare ingestions that mandate gastrointestinal decontamination, but are not amenable to orogastric lavage, ac, whole bowel irrigation, or an antidote; the patient must present alert and early (< min post ingestion) to medical care [ ] . unsurprisingly, ipecac's most common side effect is persistent emesis. as many as eight emetic episodes occurring more than min after ipecac administration have been reported [ ] . this impairs administration of oral therapeutic agents, as induced emesis can last up to several hours [ ] . prolonged vomiting associated with induced sedation or absent airway reflexes increases the risk of aspiration bronchospasm, pneumonitis, and pneumonia [ , ] . other life-threatening side effects have been reported, including bradycardia, cns depression, mallory-weiss esophageal tears, pneumomediastinum, pneumoretroperitoneum, and intracranial hemorrhage [ ] . emesis of caustics reexposes damaged esophageal mucosa to the caustic agent. analogous pulmonary aspiration concerns accompany induced emesis of hydrocarbons. orogastric lavage is performed via a large bore orogastric tube (adults, [ ] [ ] [ ] [ ] [ ] french; children, - french) with fenestrae large enough to accommodate whole tablets [ ] . serial -ml aliquots ( - ml in pediatric patients) of normal saline or lactated ringer's solution are administered and suctioned until retrieved liquid is clear. orogastric lavage can be expected to have its best risk-to-benefit ratio when patients present early enough to have a significant gastric burden, and when severe toxicological effects are manifest or expected to become manifest [ , ] . because advancement of stomach contents does occur despite proper left lateral decubitus positioning [ ] , ac (see below) is sometimes provided prior to crystalline lavage [ , ] . introduction of a large, relatively rigid tube requires a cooperative patient with a protected airway (typically an endotracheal tube if the patient is ill enough to warrant gastric lavage). orogastric lavage risks hypoxia, dysrhythmia, laryngospasm, hypothermia, gastrointestinal or pharyngeal traumatic laceration or perforation, fluid and electrolyte abnormalities, and vomiting with subsequent aspiration pneumonia [ , , ] . ac is a convoluted macromolecule created via pyrolysis of carbonaceous material and subsequently "activated" with steam to further increase surface area [ ] . the multiple pores of various size on the surface of each macromolecule of ac account for its high adsorptive affinity for a multitude of xenobiotics -particularly chemical species that are nonionized, aromatic, and/or branched [ , , ] . maximal xenobiotic binding occurs in - min [ ] . ac decreases auc by as much as %, seems to improve clinical outcomes for critically ill patients, and may benefit in certain poisonings such as acetaminophen [ , , , ] . it also increases the rate of endogenous clearance of drugs with long half-lives and some degree of entero-enteric or enterohepatic circulation [ , , ] . those findings suggested the use of mdac as a "gut dialysis" for toxins with slow pharmacokinetics [ , ] . a meta-analysis of volunteer studies demonstrated increased clearance of xenobiotics with longer half-lives, but not necessarily improved clinical outcome [ , ] . mdac has enhanced amitriptyline, carbamazepine, dapsone, dextromethorphan, phenobarbital, phenytoin, quinine, and theophylline elimination, although without definitive clinical benefit in controlled trials [ , , ] . two studies provided conflicting results for survival benefit of mdac for yellow oleander poisoning [ , ] . the fraction of unbound xenobiotic decreases as the charcoal-to-toxin ratio increases from . : up to : , although the yield curve levels off near : [ , ] . in theory, the dose of ac administered to a poisoned patient would be ten times the mass of ingested xenobiotic, but those values are unknown in most clinical situations [ ] . ac is practically dosed based on the patient's weight ( g/kg), which can be divided into multiple smaller doses to be administered every - hours [ ] . although optimum dosing is unclear, mdac is administered hourly, every hours, or every hours at a dose equivalent to . g/hour [ ] . pediatric charcoal doses are lower due to generally smaller ingestions and gut capacity. the total dose administered is the major determinant of efficacy particularly for larger overdoses, and can be administered continuously [ ] . emesis occurs in up to % of patients receiving ac; patients receiving ac via nasogastric tube or who vomited previously are at greater risk for emesis [ , ] . rarer complications include aspiration and intestinal obstruction or perforation [ , , , ] . aspirated ac may produce bronchiolitis obliterans, acute respiratory distress syndrome (ards), and death [ ] . ac adheres to mucosa and obscures endoscopy; mineral acids and bases will not adhere to charcoal. ac poorly adsorbs short chain alcohols and metals such as iron, lead, and lithium [ ] . ac administration requires an intact mental status or protected airway. flavoring agents increase the palatability of ac for volunteers, but poisoned patients do not show increased compliance/tolerance with flavored ac [ ] . cathartics induce watery evacuation of bowel within a few hours. hyperosmotic cathartic agents such as sorbitol are non-absorbed, osmotically active substances that draw water into the lumen, where increased intestinal volume and pressure promote peristalsis. so-called "saline" cathartic agents such as magnesium salts also directly stimulate smooth muscle to induce peristalsis [ ] . cathartics alone are not recommended for ingested poisons [ ] . cathartics have many adverse effects, including volume depletion, hypernatremia, hypermagnesemia, hyperphosphatemia, hypocalcemia, metabolic alkalosis, pain, nausea, emesis, and flatus [ , ] . sorbitol or laxatives are sometimes used in conjunction with the first dose of ac. while theoretically beneficial -minimizing the possible constipation of ac or promptly delivering ac to the duodenum, they do not increase the efficacy of ac [ , , ] . sorbitol is implicated in the fluid/electrolyte changes that occur with mdac: hypermagnesemia, hypernatremia, and volume depletion [ , , ] . repetitive cathartic doses have been associated with rectal prolapse and death [ , ] . whole bowel irrigation (wbi) employs polyethylene glycol (peg), a large, non-absorbable organic polymer and an electrolyte lavage solution (els) is-drugs and pharmaceuticals: management of intoxication and antidotes osmotic to serum. large peg-els volumes are introduced into the alimentary canal with less risk for fluid and electrolyte shifts caused by traditional cathartics. peg-els provides non-viscous bulk for rapid transit of material in a normally functioning gastrointestinal tract. wbi should induce evacuation within min, but requires hours on average for complete effect. reported improvements in auc are modest given the more rapid absorption time for most pharmaceuticals [ ] . however, reduction in auc can be as high as % with poorly absorbed products or modified release preparations [ ] . wbi might be considered for slowly absorbed significant ingestions such as iron, lead, and lithium, as well as modified-release preparations of β-adrenergic antagonists, bupropion, calcium-channel antagonists, carbamazepine, and theophylline [ ] [ ] [ ] [ ] . wbi is also employed to rid patients of enterally transported illicit substances which produce toxicity upon packet rupture or leakage (e.g., cocaine, heroin, and methamphetamine) [ ] . standard dosing protocols are . - l/h ( ml/kg per h) enterally until rectal effluent is clear [ ] . at this point, intestinal contents are assumed to have been displaced, although this is not always true [ , ] . nasogastric tube placement is generally required to sustain compliance with the large volume requirements, and pretreatment with an antiemetic is prudent [ ] . wbi may produce nausea, vomiting, cramping, and flatus. peg-els for colonoscopy has precipitated colonic perforation [ ] . unintentional bronchial administration of peg-els can produce acute lung injury [ ] . ileus, obstruction, perforation or threatened perforation should preclude wbi; a protected airway is required. desorption of toxins from ac by peg has been demonstrated in vitro and in vivo [ , ] . support for endoscopic therapy consists of limited case reports of retrieval in ingestions of cocaine packets, lead pellets, and medication such as sustained release calcium channel antagonists, clomipramine, iron, and meprobamate [ ] [ ] [ ] [ ] [ ] . the procedure might be warranted for certain ingestions or cases of pharmacobezoar formation of toxic substances. complications include perforation, aspiration, hemorrhage, and anesthetic-associated hemodynamic changes. when endoscopy fails, surgery may be required for definitive removal [ , ] . surgery may be required in patients with enterally transported illicit substances either due to failure of passage (with or without wbi), obstruction, or severe toxicity upon packet rupture or leakage [ , ] . weak acids in an alkaline environment exist predominantly in ionized form. biological membranes are relatively impermeable to these charged molecules. alkaline serum thus inhibits the diffusion of acidic toxins (low pk a ) across cellular membranes. similarly, an alkaline urinary ph promotes renal sequestration (or "ion-trapping") of acidic species from the systemic circulation. the relative intolerance of biological systems to acidosis limits the effectiveness of converse urinary acidification (via ascorbic acid or diluted hcl solutions) for renal sequestration of weak bases. critically ill patients may have reduced drug clearances due to decreased hepatic and renal perfusion, and thus interventions that increase clearance/ elimination have the potential to significantly reduce toxicity [ ] . alkalinization improves renal elimination of chlorpropamide, , -dichlorophenoxyacetic acid, diflunisal, fluoride, mecoprop, methotrexate, phenobarbital, and salicylate [ ] . urine alkalinization is considered first line therapy in patients with moderate salicylism who do not meet hemodialysis indications. dosing of - meq/kg of . - . % bicarbonate provided over - min is followed by "normal" bicarbonate infused at double the standard rate of i.v. fluid maintenance. the "normal" bicarbonate solution is prepared by adding three ampules of sodium bicarbonate (totaling - meq) in l % dextrose in water (d w). the rate is titrated to maintain an alkaline urinary ph, without exceeding a serum ph of . [ ] . alkalemia decreases ionized calcium. volume overload may occur, particularly in patients with congestive heart failure, acute renal failure, or end-stage liver disease. bicarbonate treatment induces hypokalemia. as the proximal renal tubular cells conserve serum potassium by exchanging protons for urinary potassium, this defeats urinary alkalinization. therefore, maintaining a normal serum potassium, with frequent monitoring and supplemental administration and/or inclusion in the bicarbonate solution, are important components of urine alkalinization. saline diuresis is utilized to improve excretion and minimize toxicity of overdose of ions such as magnesium, calcium, and lithium in patients who do not meet hemodialysis indications [ ] [ ] [ ] . hypermagnesemia may occur with excessive antacid use, gargling or ingesting magnesium sulfate compounds, and iatrogenic error [ , ] . hypercalcemia can result from excess calcium (in antacid tablets) or vitamin d ingestion or parenteral administration [ , ] . renal lithium toxicity presumably results from cytotoxic accumulation of lithium entering via the apical epithelial sodium channel [ ] . ensuing nephrogenic diabetes insipidus, characterized by increased water and sodium diuresis, can result in dehydration, hyperchloremic metabolic acidosis, and renal tubular acidosis. in volume depletion, activation of the reninangiotensin-aldosterone axis leads to active resorption of sodium, and thus lithium, from the distal convoluted tubules. therefore, adequate volume repletion with saline is prerequisite for effective renal elimination of lithium. boluses of . % sodium chloride are administered until the patient is clinically euvolemic. saline infusion is then provided at . - times a standard maintenance rate. throughout treatment renal function, urine output, and electrolytes are monitored. congestive heart failure, renal failure, or end-stage liver disease moderate volume administration and make saline diuresis less attractive than hemodialysis in significant ingestions. loop diuretics such as furosemide inhibit sodium resorption in the proximal convoluted tubules, and would theoretically promote elimination of lithium as natriuretics. however, these effects are countered by the action of the renin-angiotensin-aldosterone axis on the distal convoluted tubules, and diuretics do not seem to improve outcomes in lithium overdose or radiographic contrast exposure [ , ] . in hemodialysis (hd) the patient's blood is pumped through a circuit that includes a cartridge consisting of thousands of semi-permeable, membranelined capillary tubes. the blood traverses the cartridge counter-current to a circulating buffered salt solution (a.k.a. dialysate) before returning to the patient's venous circulation. diffusible molecules flow down their electrochemical gradient from the serum to the dialysate. hemoperfusion (hp) employs a similar circuit, but the cartridge is enveloped with ac (rather than a circulating dialysate) to adsorb xenobiotics regardless of plasma protein binding, and leave serum electrolytes largely unchanged. continuous arteriovenous or venovenous hemofiltration (cavh or cvvh) employ lower pressures and flow rates than hd over longer sessions for patients unable to tolerate hd or to remove xenobiotics with slow tissue redistribution [ , ] . peritoneal dialysis (pd) is ineffective in poisoning management, given its inherently slow kinetics and the availability of hd [ ] . extracorporeal therapies may be warranted when criteria are met for both the xenobiotic and the patient [ ] . favorable dialyzable toxin properties include low volume of distribution (v d ), relatively low molecular weight, and poor serum protein binding (or binding that worsens in overdose, as is the case for salicylate and valproate) [ ] . patient characteristics suggesting extracorporeal therapy include signs or symptoms of significant end organ toxicity; impaired elimination secondary to baseline comorbidities or critical illness-induced hypoperfusion; inability to tolerate or refractory to antidotal strategies (such as bicarbonate or saline); inadequate response to supportive care measures; concurrent electrolyte derangements (e.g., metformin-associated lactic acidosis); or serum drug concentrations historically associated with severe outcome [ ] . traditionally, charcoal hp was used for xenobiotics significantly bound to plasma proteins, but its use is declining while (high-flux membrane) hd increases. methanol, ethylene glycol, salicylates, lithium, halides, theophylline, and metformin-associated lactic acidosis are commonly treated with dialysis [ ] . hd is used for valproate and carbamazepine poisoning; however, in the absence of high-flux dialysis membranes, the characteristics of charcoal hp may more appropriately address the larger v d and protein binding [ ] . common side effects of extracorporeal elimination include hypotension, bleeding, and infection. enhanced clearance of therapeutic medications and antidotes (e.g., antibiotics, fomepizole, n-acetylcysteine, water-soluble vitamins) may occur. the need for dialysis must be anticipated early; several hours of preparation time may be required to secure vascular access, equipment, and personnel. exchange transfusion is a total blood volume exchange administered in small aliquots. serial frequent phlebotomy of a small amount of circulating blood occurs with simultaneous transfusion of equivalent donor blood. this process is repeated until two to four vascular volumes have been exchanged. while the procedure is very rarely used for toxin removal, exchange transfusion is more familiar to clinicians treating severe hemolytic diseases of the newborn, hyperbilirubinemia without hemolysis, and sickle cell crisis. exchange transfusion removes xenobiotics that are large or bound to plasma proteins, such as thyroxine, iron, or theophylline [ , ] . for lifethreatening ingestions, exchange transfusion is a viable option for neonates and infants whose immature vasculature cannot tolerate extracorporeal elimination modalities or in institutions lacking pediatric dialysis capacity. exchange transfusion has been successfully employed in pediatric iron, isoniazid, phenobarbital, salicylate, theophylline, and vincristine overdose [ ] [ ] [ ] [ ] [ ] [ ] . it has also been suggested for refractory drug-induced methemoglobinemia [ ] . whole blood exchange was utilized in an adult with a fold cyclosporine dosing error [ ] . anticipated complications arise from vascular access, bleeding, hypoglycemia, hypotension, and blood product administration (immune-mediated reactions, blood incompatibility, and infections). several hyperthermic syndromes are caused by xenobiotics. these are generally spectrum disorders, whose features may overlap with other conditions such as cns infection, agitated delirium, and sepsis. malignant hyperthermia (mh) occurs in patients with an autosomal-dominant defect in genes encoding the skeletal muscle ryanodine receptor (ryr- ) or the voltage-gated calcium channel (cav . ) who are exposed to volatile anesthetics or depolarizing muscle relaxants (succinylcholine) [ ] . hypomagnesemia may increase the probability and possibly severity of an mh event [ ] . the subsequent rapid increase in myoplasmic calcium concentration increases muscle metabolism and heat production and produces muscle contractures and hyperthermia. neuroleptic malignant syndrome (nms) is characterized by high fever, autonomic instability, altered mental status, and muscle rigidity. potent antipsychotics (neuroleptics) such as haloperidol and other medications (metoclopramide, droperidol, and promethazine) with significant dopamine antagonism, as well as abrupt cessation of dopaminergic agents such as those used in parkinsonism, can precipitate this life-threatening syndrome [ ] . nms typically develops over several days and is characterized by ''lead-pipe'' rigidity [ ] . drugs that impair serotonin breakdown or re-uptake, those that act as serotonin precursors or enhance its release, or those that are serotonin agonists may lead to serotonin syndrome. like nms, serotonin syndrome is a spectrum disorder for which various signs and symptoms have been proposed to establish diagnosis (e.g., sternbach and hunter criteria) [ , ] . in its most severe form it consists of high fever, autonomic instability, altered mental status, and may have associated diaphoresis, shivering, tremor, diarrhea, or spontaneous clonus. in serotonin syndrome, onset of symptoms is usually rapid, with % of patients with the serotonin syndrome presenting within hours of drug exposure, and tremor and hyperreflexia predominant in the lower extremities may be a prominent feature [ ] . sympathomimetic-associated hyperthermia, seen with acute intoxication with cocaine, amphetamines, substituted amphetamines, and phencyclidine, may be clinically indistinguishable from serotonin syndrome [ ] . additionally, the agitated delirium engendered by these agents may be difficult to distinguish from that induced by hyperthermia itself. patients with anticholinergic-associated hyperthermia will generally present with a compatible "toxidrome" -agitation; mydriasis; dry, hot, and erythematous skin; hypoactive bowel sounds; and urinary retention. while rare, thyrotoxicosis factitia, the ingestion of excess thyroid hormones due to inadvertent intake (pharmaceutical or food contamination), misuse (dieting), or significant intentional ingestion may produce hyperthermia [ , ] . hyperthermia may accompany toxicity with agents that uncouple oxidative phosphorylation (e.g., salicylates, dinitrophenol, pentachlorophenol) [ ] . multiple medications can also complicate or contribute to environmental hyperthermia. several reviews and epidemiological data from major heat waves have demonstrated that anticholinergics, antiepileptics, antihistamines, antihypertensives in general and diuretics in particular, antipsychotics, and others contribute to excess morbidity and mortality [ , ] . conversely, exogenous heat stress can increase mortality from specific xenobiotics. in an urban setting at ambient temperatures above . °c, the mean daily number of fatal cocaine overdoses increased markedly [ ] . regardless of the cause for the hyperthermic syndrome, cessation of any possible offending or contributing agents and rapid cooling is critical. the degree of hyperthermia produced correlates with death and neurotoxicity in animal models, and temperature normalizing intervention is critically impor-tant in attenuating cns injury and mortality [ ] . studies from the chicago and france heat waves show that this is rarely done in a timely manner (if at all) in cases of environmental hyperthermia, with devastating results [ , ] . the benefits of rapid cooling by ice water immersion were demonstrated over years ago [ ] . a large review concluded that cooling methods based on evaporative heat loss are less efficient than immersion in ice water in dissipating heat [ ] . additional studies demonstrate that cooling rates of up to . - . °c/min can be achieved with immersion, two to three times that of evaporation [ , ] . regardless of the method used, effectiveness should be repeatedly assessed. sedation with benzodiazepines and rigorous supportive care are necessary adjuncts in significant cases. this is primarily accomplished with titrated doses of benzodiazepines to inhibit muscle rigidity and control agitation. animal models have demonstrated the benefit of benzodiazepines in prolonging survival, preventing seizure, and attenuating agitation in the toxicological hyperthermias [ , ] . phenytoin is ineffective in animal models [ ] . phenothiazines and butyrophenones, while reported, may have delayed onset and compromise mental status, lower seizure threshold, impair heat dissipation, and worsen hypotension [ ] . neuromuscular paralysis may be required to limit further heat generation in cases of nms, serotonin syndrome, and sympathomimetic-associated hyperthermia. as the pathophysiology of mh is beyond the neuromuscular junction, paralytics are unlikely to provide benefit. rapid i.v. administration of dantrolene, a direct-acting skeletal muscle relaxant, is the only drug proven effective for prevention and treatment of mh. dantrolene disrupts the pathogenic excitation-contraction coupling by acting at ryr- to suppress depolarizationinduced sarcoplasmic reticulum calcium release and normalize the voltage dependence of contractile activation [ ] . reversal of increased myotube sensitivity may also play a role [ ] . intravenous - mg/kg dantrolene is repeated until symptoms are controlled or mg/kg (or more) has been administered. following initial treatment, - mg/kg i.v. or per os is given every hours for - hours to prevent recurrence. dantrolene is packaged in vials containing mg dantrolene sodium; thus, multiple vials are needed for treatment of adult patients. a large review of nms cases did not suggest a beneficial role for dantrolene, although one case-controlled analysis found benefit [ , ] . bromocriptine, a dopamine agonist, has been used (off-label) to treat nms at doses ranging from to mg every hours [ ] . common side effects include hypotension, dyskinesia, erythromelalgia, and hallucinations. cyproheptadine, developed as an antihistamine, additionally antagonizes -ht receptors. cyproheptadine for serotonin syndrome (off-label) is initially used in a dose range of - mg, followed by mg every hours for persistent symptoms; upon symptom control, mg maintenance dosing is provided every hours [ ] . the tablet form necessitates administration orally or crushed via nasogastric tube. n-acetylcysteine (nac) provides an effective means of prevention and treatment of acetaminophen (n-acetyl-p-aminophenol, apap; paracetamol)induced hepatotoxicity. nac is also employed to preclude radiographic contrast-induced nephropathy [ ] . the ultimate toxicant of apap, n-acetyl-pbenzoquinone imine (napqi) generated primarily by cyp e and cyp a , depletes glutathione (gsh), binds intracellular components, and, through an incompletely understood process, produces hepatic injury, centrilobular necrosis, or hepatic failure [ , ] . nac works by multiple mechanisms. it augments apap sulfation to a nontoxic metabolite, it acts as a glutathione precursor or glutathione substitute to detoxify napqi, and possibly reverses napqi oxidation [ , ] . nac provides substantial benefit even in cases of delayed presentation following overdose [ ] . extra-hepatic benefits of nac include improving cardiac index and systemic mean oxygen delivery despite decreasing systemic vascular resistance [ ] . in a range of hepatic disorders, nac improved baseline oxygen delivery, oxygen consumption, and dye clearance in a majority of patients [ ] . liver blood flow and cardiac index improved in septic shock patients provided nac [ ] . only l-nac is beneficial. animal experiments demonstrate that the l-isomer, derived from physiological l-cysteine, prevents hepatotoxicity and provides prolonged elevations of hepatic glutathione [ ] . nonphysiological d-nac cannot increase glutathione stores or prevent hepatotoxicity, despite increasing acetaminophen sulfation [ ] . according to rumack [ ] , the oral nac dosing strategy was reached by estimating the absorption and turnover rate of glutathione at mg/kg per h and an fda safety factor of , to yield mg/kg every hours [ (mg/kg per h) × (h) × (safety factor) = ≈ mg/kg every h]. there were several assumptions as to "normal" hepatic glutathione levels and apap to napqi conversion. a mg/kg loading dose was added to provide an early high hepatic dose. the -hour duration of oral therapy was based on previous observations of multiple patients with prolonged apap half-lives and a desire to implement a protocol that would accommodate those with half-lives longer than hours (anticipating disappearance after five half-lives). while many have suggested that the -hour oral course is excessive, particularly after apap has disappeared from the serum, the optimal duration of therapy is unclear. studies assessing a shortened or "patient-tailored" approach have been small or methodologically limited [ , ] . the rumack-matthew nomogram guides initiation of nac therapy in single acute ingestions. the "treatment line" is anchored at an apap serum concentration of either μg/ml (" line") or μg/ml (" line") at hours post ingestion and decreased by % every hours. the slope of the treatment line does not reflect apap kinetics. the " line" is utilized in all patients in the u.s. and australia; in the u.k. and elsewhere the " line" is employed, with a " line" modification for an array of individuals deemed at "high-risk": ethanol tolerant, those at risk for glutathione depletion (malnutrition, hiv, eating disorders, cystic fibrosis), pregnancy, and those prescribed enzyme-inducing drugs (carbamazepine, phenytoin, phenobarbitone rifampacin, isoniazid, etc.) [ , ] . the u.s. multicenter study substantiated the safety and efficacy of its approach [ ] . proponents of the " line" point to the fact that . - . % of patients above the " line" but below the " line" developed biochemical hepatotoxicity (aspartate aminotransferase, ast > iu/l at any time during their course) in the u.s. multicenter trial and that patient deaths have occurred in untreated patients "between the lines" [ , ] . in patients presenting near hours after ingestion, or if a level is not available before hours post ingestion, nac is begun while awaiting apap results and then continued or stopped once the results are available and have been plotted on the nomogram. if the time of ingestion is unknown or more than hours has passed, nac is administered. when apap concentration and transaminase results are obtained, if transaminases are elevated or if measurable apap exists, a full course of treatment is provided. with normal aminotransferases and without detectable apap, treatment is not required. concentrations obtained less than hours post ingestion are not useful except to completely exclude ingestion (i.e., it is useful only if the apap concentration is undetectable). ongoing absorption may place individuals above the line at hours, or metabolism or charcoal administration may result in a patient falling below the nomogram at hours. in cases of chronic ingestion (> . g/day in adult), laboratory evaluation and treatment are provided as for an unknown time of ingestion. with elevated transaminases or measurable apap, nac is provided. oral nac is cheap and familiar to clinicians. it has minimal side effects (other than vomiting and odor) and is preferred in patients with bronchospastic disease. its use can become problematic in cases where oral delivery is compromised, e.g., in patients with depressed mental status, significant vomiting, or impaired gastric motility. use of an anti-emetic is encouraged. intravenous nac appears to be similarly efficacious to oral nac and eliminates many delivery issues. it has a much shorter therapy course ( hours), expediting medical and psychiatric disposition. it avoids first pass metabolism in cases where the liver is not the only target or interest, such as those with cerebral edema or pregnancy. while i.v. nac is slightly more expensive, total hospital charges may be less due to decreased treatment time. histamine-mediated anaphylactoid reactions are more commonly seen with rapid i.v. loading and in patients with lower apap levels [ ] . mild reactions have been treated by slowing the infusion rate and providing i.v. diphenhydramine, although this might alter nac and apap kinetics. dosing complexity - mg/kg in ml of % dextrose over hour, followed by mg/kg in ml of % dextrose over hours ( . mg/kg per h), and then mg/kg in ml of % dextrose over hours ( . mg/kg per h) -yields frequent administration errors [ ] . the supplied % solution was too concentrated for children, and dilution according to adult guidelines resulted in excess free water, and cases of hyponatremia and seizures [ ] . the current u.s. package prescribing information (http://www.acetadote.net/pi_acetadote_revised_apr .pdf) and dosage calculator website (http://www.acetadote.net/dosecalc.shtml) provide dosing and administration guidelines in patients of less than kg. in a study limited by different comparison groups, data acquisition methodology, treatment location and several other factors, -hour only i.v. nac was favored in patients with early presentation (< hours), whereas late presentation favored oral -hour nac [ ] . however, continuous i.v. infusion in delayed presentations with apap-induced fulminant hepatic failure showed clear benefit in a prospective study [ ] . whatever the route, prior to cessation of nac therapy, negative apap concentrations and normal transaminases must be ensured, particularly in cases of massive ingestion; hepatotoxicity may follow premature cessation of therapy [ , ] . the -hour maintenance dose is continued in patients receiving i.v. nac until apap is undetectable and transaminases are normal (or at baseline). experimental evidence and human case reports demonstrate both delayed absorption, delayed increase following initial decline, and "crossing the nomogram" with extended-relief, opioid-or anticholinergic-containing apap products, or co-ingestants [ , ] . in cases of hepatic failure, i.v. nac is continued until resolution, transplant, or death. historically, physostigmine (eserine), a reversible carbamate inhibitor derived from the seed (calabar bean) of the vine physostigma venenosum balfour, was used in the ancient trial by ordeal [ ] . medicinal use of physostigmine was first reported in to reverse severe atropine poisoning [ ] . naturally available (-)-physostigmine is over times more effective in inhibiting acetylcholinesterase and butylcholinesterase in tissue, erythrocytes, and serum in humans and animal models than its stereoisomer [ , ] . this activity depends upon interactions within the hydrophobic pocket of the acetylcholinesterase active center, which is distinct from the catalytic site [ ] . additionally, physostigmine binds nicotinic receptors close to, but distinct from, the acetylcholine binding site on the α-subunit [ ] . at low doses, physostigmine functions as an ineffective nicotinic receptor agonist, while at higher doses it produces marked channel blockade. physostigmine's nonspecific analeptic properties [ ] are no longer considered useful in overdose, given the clear benefits of supportive care. indiscriminate use of physostigmine and an incomplete understanding of the pathophysiology of tricyclic antidepressant (tca) poisoning was associated with bradydysrhythmias including asystole, seizure, and several deaths [ , ] . in animal models, physostigmine is ineffective in attenuating tcainduced seizures [ ] . it failed to abolish dysrhythmias, decreased blood pressure, and at high doses enhanced tca toxicity [ ] . physostigmine is currently recommended as a diagnostic and therapeutic agent for antimuscarinic poisoning [ ] . patients should have clear peripheral or central manifestations of the anticholinergic toxidrome. as a tertiary amine, physostigmine can cross the blood-brain barrier to reverse the central effects. an ecg should exclude sodium or potassium channel blockade (qrs or qt prolongation). excessive physostigmine will produce a cholinergic syndrome, with muscarinic and nicotinic effects. as the adverse effects of bradycardia and bronchorrhea can produce significant morbidity, continuous cardiac monitoring and immediate access to atropine are recommended during physostigmine administration. physostigmine, - mg in adults and . mg/kg (maximum . mg) in children is infused slowly over at least min [ ] . repeat doses every - min can be provided if an adequate response does not occur and adverse effects are absent. re-bolusing may be required in the setting of antimuscarinics with a prolonged duration of action. the anticonvulsants include carbamazepine, ethosuximide, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, tiagabine, topiramate, valproic acid (vpa), vigabatrin, and zonisamide. these drugs enjoy widespread approved and offlabel use for additional conditions, e.g., fibromyalgia (pregabalin); neuropathy and neuropathic pain (carbamazepine, gabapentin, lamotrigine, levetiracetam, and pregabalin); panic disorder (tiagabine); migraine prophylaxis and treatment of obesity, ethanol dependence, and depression (topiramate); and bipolar disorder (carbamazepine, lamotrigine, and vpa). treatment of anticonvulsant overdose is largely supportive, with particular attention to the cns-depressant and cardiovascular effects of some of these agents. l-(r)-carnitine exists as the sole specific antidote in this class for significant vpa (di-n-dipropylacetic acid, -propylpentanoic acid) poisoning. patients with drug-associated mitochondrial toxicity (particularly from nucleoside analogs) and anthracycline cardiotoxicity might also benefit from its administration [ , ] . the anticonvulsant properties of vpa derive from its ability to increase γ-aminobutyric acid (gaba) availability via inhibition of gaba transaminase and succinic semialdehyde dehydrogenase, to attenuate n-methyl-daspartate (nmda)-type glutamate receptor excitatory effects, and to slow the rate of recovery from sodium channel inactivation [ ] [ ] [ ] . additionally, vpa appears to affect inositol levels similar to lithium. therapeutic concen-trations are - mg/l. potentially toxic concentrations are greater than mg/l. oral absorption of vpa is excellent [ ] . peak plasma concentrations are generally seen in - hours, although this may be markedly delayed by overdose, enteric coating, or meals [ ] . manifestations of significant vpa toxicity include cns effects (lethargy, seizure, coma, cerebral edema), respiratory depression, metabolic derangement (hypernatremia, hyperammonemia, hypocalcemia, metabolic acidosis, carnitine deficiency), gastrointestinal effects (nausea, vomiting, and abdominal pain), pancytopenia, pancreatitis, and hepatotoxicity [ , ] . valproate toxicity is seen both in intentional acute overdose and in those on chronic therapy, either without adequate carnitine supplementation or on complex regimes. cells attempt to metabolize the vpa that is not directly excreted or glucuronidated in a manner similar to other fatty acids (fig. ) . thus, vpa is conjugated with coenzyme a (coa). carnitine enters via an atp-dependent transporter. vpa is then transferred to carnitine, the normal mechanism for fatty acids entry into the mitochondrion. however, vpa-carnitine both inhibits the carnitine transporter and also diffuses out of the cell to be lost via renal excretion [ ] . renal resorption of carnitine is also impaired [ ] . these factors contribute to intracellular carnitine depletion. once vpa-carnitine is shuttled into the mitochondrion, it is reattached to coa. it then undergoes β-oxidation, in an attempt to generate -carbon molecules for entry into the krebs cycle. the -en-vpa-coa product is neurotoxic with a prolonged half-life. the terminal -keto-vpa product traps coa, leading to its mitochondrial depletion. decreased mitochondrial coa yields decreased atp production, diminishing usable cellular energy currency and further limiting carnitine entry into the cell (via an atp-dependent carnitine transporter). once carnitine is depleted, normal fatty acid metabolism cannot occur [ ] . fatty acid build up is thought to underlie the reye's-like steatohepatitis, which can be seen in toxicity [ ] . coa is also needed to make n-acetylglutamate, an activator of carbamoylphosphate synthetase i (cps i), a critical enzyme in the urea cycle. when its effectiveness is limited due to inadequate activator, ammonia cannot be incorporated, and consequently, its concentrations increase. furthermore, as coa is depleted, β-oxidation shifts to omega (ω), or terminal carbon oxidation. this creates (among others) the hepatotoxic -en-vpa product. -en-vpa additionally inhibits cps i, further preventing nitrogen elimination and contributing to hyperammonemia. l-carnitine (levocarnitine) supplementation has been recommended to reverse the adverse metabolic effects of vpa in cases of vpa-induced hepatotoxicity, vpa overdose, and primary carnitine-transporter defects [ , ] . hyperammonemia and serum and muscle carnitine deficiency are well described in patients chronically taking vpa [ ] [ ] [ ] . several studies and case reports demonstrate that carnitine supplementation reverses clinical symptoms, hypocarnitinemia, hyperammonemia, and vpa half-life prolongation in patients with toxicity due to chronic administration [ ] [ ] [ ] . in patients with acute vpa overdose, limited clinical and laboratory data derived from case reports also suggest that reversal of metabolic derangements and improvement in clinical symptoms occurs when carnitine is provided [ ] [ ] [ ] . a single large retrospective analysis showed a significant survival benefit with i.v. carnitine supplementation (with vpa cessation) in patients with valproate-induced hepatotoxicity [ ] . l-carnitine dosing for cases of overdose is not currently evidence based. an oral or i.v. dose of mg/kg per day, divided and given every hours (maximum daily dose g), is provided to those patients with acute overdose and [ , , , , ] . asymptomatic hyperammonemia or hepatotoxicity in the absence of cns depression or metabolic derangement [ ] . symptomatic patients with hyperammonemia or symptomatic hepatotoxicity should receive mg/kg l-carnitine i.v. over min (maximum g), followed by mg/kg every hours over - min until clinical improvement occurs [ , ] . others have supplemented at the higher dosing strategy when vpa concentrations exceed mg/l [ ] . in addition, given the decrease in protein binding that occurs, hemodialysis or hemoperfusion is recommended for patients with vpa concentrations exceeding - mg/l or with severe clinical symptoms [ ] . l-carnitine is generally well tolerated. side effects associated with carnitine supplementation are nausea, abdominal discomfort, dose-related diarrhea, and fishy body odor [ ] . a small retrospective chart review found no adverse effects or allergic reactions in vpa overdose patients administered carnitine [ ] . the current l-carnitine package inserts have no warnings or contraindications, but note that seizures have been reported to occur in patients, with or without pre-existing seizure activity, who received either oral or i.v. l-carnitine [ ] . up to mg/kg per day for days has been provided without complications [ ] . the d-isomer and the racemate (d,l-carnitine) are contraindicated. historic use of racemic d,l-carnitine was associated with myasthenialike syndromes and cardiac dysrhythmias, which disappeared after l-carnitine administration [ ] . d-carnitine also competitively depletes cardiac and skeletal muscles and kidneys of l-carnitine [ ] . dextrose dextrose (d-glucose) is indicated to rapidly reverse organic or toxin-induced hypoglycemia (e.g., from sulfonylureas, insulin, ethanol, salicylates, β-adrenergic antagonists, quinolines, pentamidine, ritodrine, and disopyramide) [ , ] . hypoglycemia onset may be significantly delayed with certain agents (e.g., long-acting insulin or sulfonylureas). limited cns glycogen stores (in astrocytes) and the inability to acutely use free fatty acids make the cns particularly vulnerable to hypoglycemia [ ] . patients (and providers) may be unaware of hypoglycemia in the absence of objective testing; both the counter-regulatory autonomic response and overt neurological deficit may be absent [ , ] . additionally, significant neuroglycopenia and hypoglycemia-associated delirium (particularly in salicylism) may occur despite a "normal" peripheral blood glucose [ ] . a wide range of clinical presentations have been described, including diaphoresis, nausea, tachycardia, tremor, hypothermia, focal neurological deficits, and cns agitation, confusion, or depression. these are generally reversible upon prompt treatment. untreated hypoglycemia may result in seizure, coma, and death. hypoglycemic seizures increase cerebral metabolic rate, contribute to atp depletion, and produce irre-versible brain damage [ , ] . for these reasons, when bedside testing is unavailable, a risk-benefit calculation has generally favored empiric dextrose administration in the absence of a very clear alternative history or explanation for altered mental status. following a determination of absolute or relative hypoglycemia, . - . g/kg i.v of age-appropriate dextrose containing solutions should be provided immediately -d w ( g/ ml) in adults, d w ( g/ ml) in children, and d w ( g/ ml) in neonates. frequent re-evaluation of response to therapy is required. glucose uptake and distribution, hyperglycemia-induced insulin secretion in those with a competent pancreas, and ongoing toxin exposure may cause recurrent hypoglycemia and necessitate repeat dosing. feeding, which provides significantly more calories than each ml ampule of d w ( kcal according to one manufacturer [ ] ), should be commenced as soon as practicable. while d w "maintenance" solutions may be subsequently required, at an infusion rate of ml/h, this concentration only provides kcal per hour. continuous infusion of more concentrated solutions (e.g., d w) requires a central venous catheter for administration. only the d-isomer is clinically useful. most glucose transporters (gluts) and the specific transporter required for facilitated diffusion of glucose across the blood-brain barrier, glut (slc a ), have a high affinity for d-glucose and negligible affinity for l-glucose [ , ] . d-glucose is also generally favored over other d-glucose epimers such as d-mannose or d-galactose. d w is hypertonic and may cause phlebitis or thrombosis at the site of injection. extravasations of solutions containing as low as % dextrose have caused significant tissue injury and necrosis, particularly in young children [ ] . pseudoagglutination of red blood cells may occur if concentrated dextrose solutions without electrolytes are administered simultaneously with blood through the same infusion set [ ] . hypertonic dextrose administration may also induce generally clinically irrelevant hypophosphatemia [ ] . octreotide acetate, a synthetic somatostatin analogue, is now favored in cases of refractory hypoglycemia due to sulfonylureas or quinine. it is fda approved for treatment of acromegaly, carcinoid tumors, and vasoactive intestinal peptide tumors [ ] . it is a more potent inhibitor of insulin secretion than the natural hormone [ ] . in pancreatic β-islet cells, atp generated from glucose uptake and subsequent metabolism normally induces closure of the atp-dependent potassium channel by binding to its pore subunit (fig. ) . sulfonylureas similarly induce channel closure after binding to a regulatory (sur ) subunit. increased intracellular potassium triggers calcium entry through voltage-dependent calcium channels, leading to increased cytosolic calcium and insulin exocytosis [ , ] . additionally, atp contributes to insulin vesicles movement and provides a substrate for protein kinase a (pka)-mediated phosphorylation. octreotide binds to the somatostatin receptor (primarily sstr ) [ ] . the subsequent effects continue to be explored and include inhibitory calcium channel effects, inhibition of adenylyl cyclase, and dephosphorylation of specific proteins required for movement and/or docking of vesicles [ , , ] . octreotide effectively suppresses endogenous insulin release in controlled studies in diabetics and in cases of sulfonylurea overdose, but does not (and would not be expected to have) an effect on exogenously administered insulin [ ] [ ] [ ] . several factors support octreotide usage following failure of initial dextrose administration and feeding. bolused dextrose may produce hyperglycemia and thus subsequently stimulate an exaggerated insulin response, particularly when figure . pancreatic β-islet cell mechanisms of insulin release and octreotide action (see text for details). enzymes and substances: ac, adenylyl cyclase; atp, adenosine triphosphate; camp, cyclic adenosine monophosphate; glut , glucose transporter ; pka, protein kinase a; sfu, sulfonylurea; sstr, somatostatin receptor. symbols: ⊕, agonism or co-factor; ⊗, antagonism. data used can be found in [ ] [ ] [ ] [ ] . sulfonylureas persist. this contributes to recurrent (sometimes more significant) hypoglycemia. a vicious cycle of serum glucose concentrations is described in case reports and controlled trials following dextrose administration after sulfonylurea exposure [ ] [ ] [ ] . additionally, as has been demonstrated, classic neuroglycopenic symptoms may not be present, and patients may need to be admitted during periods when circadian sleep patterns would complicate assessment. octreotide administration also obviates the concern of excess water administration in pediatric patients receiving i.v. dextrose solutions. relatively few trials are available to judge the efficacy of octreotide for sulfonylurea-induced hypoglycemia. in one study, glipizide was used to induce induced hypoglycemia ( mg/dl) in eight healthy volunteers, who were then resuscitated with dextrose infusion, diazoxide, or octreotide [ ] . dextrose requirements were markedly less in patients provided octreotide and hypoglycemic events were markedly attenuated after all therapies were stopped. one retrospective chart review of nine patients demonstrated that octreotide significantly reduced the number of recurrent hypoglycemic events and dextrose requirement [ ] . one prospective randomized controlled trial in poisoned patients, despite a failure to control for carbohydrate intake and having an unusual dosing strategy (a single octreotide μg dose subcutaneously), demonstrated consistently higher glucose values for the duration for which octreotide would be expected to be effective ( - hours) [ ] . controlled animal studies with - μg octreotide demonstrated a similar decrease in hypoglycemic events [ ] . the remainder of human clinical experience of the effectiveness of octreotide in sulfonylurea overdose comes from abstracts, case reports, and case series (e.g., [ , , [ ] [ ] [ ] ). pediatric experience in sulfonylurea overdose comes only in the form of limited abstracts and case reports in children aged months to years [ , [ ] [ ] [ ] . however, octreotide has been used for prolonged periods to treat persistent hyperinsulinemic hypoglycemia of infancy [ , ] . two human studies examined the effectiveness of octreotide in quinineinduced hypoglycemia. in one study of nine healthy volunteers, μg/hour octreotide as a continuous i.v. infusion abolished quinine-induced insulin release [ ] . the authors reported resolution of hypoglycemia in an additional patient being treated with quinine for plasmodium falciparum malaria. a subsequent study in eight patients with p. falciparum malaria confirmed octreotide suppression of quinine-induced hyperinsulinemia [ ] . optimal dosing of octreotide has not been definitively determined. initial doses of - μg subcutaneously in adults have been reported, although μg every - hours is commonly provided [ , ] . in children, an initial dose of . - . μg/kg is used, although up to . μg/kg (or more) has been reported [ , ] . peak serum concentrations are achieved within min after subcutaneous administration and within min after a short ( min) i.v. infusion [ ] . the elimination half-life (by either route of administration) is approximately . hours. in patients with severe renal impairment (which may have contributed to sulfonylurea-induced hypoglycemia in the first place), the plasma clearance is reduced by % [ ] . the subcutaneous route is recommended due to longer duration of effect, as i.v. administration has resulted in treatment failure [ ] . side effects are generally minimal. octreotide does inhibit gallbladder contractility and decreases bile secretion in normal volunteers [ ] . when octreotide has been used to reverse sulfonylurea-induced hypoglycemia, bradycardia, hypokalemia, anaphylactoid reaction, and hypertension and apnea have been reported [ , ] . other adverse events include nausea, abdominal cramps, diarrhea, fat malabsorption and flatulence [ ] . octreotide also suppresses glucagon release, although hypoglycemia has been a concern only in patients on long-term therapy for organic hyperinsulinemia [ ] . glucagon is not generally recommended to correct hypoglycemia. glycogen stores are frequently depleted by the time toxin-induced hypoglycemia manifests; glucagon's half-life (less than min) is inadequate given the prolonged duration of the effect of sulfonylureas; and glucagon may exacerbate hyperinsulinemia [ ] . diazoxide, an antihypertensive agent, which reduces insulin release by opening the atp-dependent potassium channel, is now of historical interest due to associated hypotension, reflex tachycardia, nausea and vomiting, and fluid retention [ , ] . since its introduction in , isoniazid (inh, isonicotinic hydrazide, pyridine- -carbohydrazide) has remained a mainstay for treatment and prophylaxis of mycobacterial infections [ ] . the adult single tablet, mg daily dose ( . mg/kg in a kg individual) targets a peak plasma concentration of - μg/ml [ ] . acute inh toxicity may occur following ingestion of mg/kg inh; it is common above - mg/kg [ ] . the relatively narrow therapeutic window poses a significant risk for those with suicidal intent and for those who ingest extra pills to "catch up" after a brief period of incomplete compliance [ ] . historically, death rates of % were reported [ ] . seizures refractory to typical therapy, severe metabolic lactic acidosis, and coma may occur as early as min post ingestion due to the rapid and nearly complete absorption of inh from the gastrointestinal tract. seizures may occur at lower doses in those with pre-existing susceptibility. associated respiratory failure, hypotension, and rhabdomyolysis may ensue. in patients provided . - . g ( - mg/kg) inh due to medication error, all experienced nausea or vomiting, vertigo, and coma within min to hours after ingestion [ ] . abnormal generalized discharges as sharp and slow waves were seen on eeg in all patients. chronic inh toxicity may present with nausea, vomiting, hepatitis, hemolytic anemia, and neurological findings (restlessness, neuropathy, cerebellar findings, and psychosis). the acute clinical effects are a product of the multiple biochemical actions of inh, which lead to pyridoxine depletion and subsequent neuronal hyperexcitability (fig. ) [ , [ ] [ ] [ ] . inh hydrazones inhibit pyridoxine phosphokinase, which activates pyridoxine. inh hydrazines and hydrazides inactivate active pyridoxal -phosphate. inh metabolites also complex with pyridoxal -phosphate, leading to increased urinary elimination. glutamic acid decarboxylase (gad) and gaba transaminase (gaba-t) both require pyridoxal -phosphate as a co-factor. inhibition of gad exceeds that of gaba-t [ ] . the resulting gaba depletion and loss of neuronal inhibition is thought to underlie seizure activity. metabolic acidosis may be profound -survival has been reported with a ph of . [ ] . seizure-associated lactate generation is substantial; inh-induced metabolic acidosis does not develop in paralyzed dogs (despite eeg evidence of seizure) [ ] . importantly, merely correcting the acidosis (e.g., by bicarbonate) does not prevent additional seizures or terminate inh toxicity [ , ] . inh also impairs lactate conversion to pyruvate (fig. ) . increased metabolism of fatty acids due to impaired glucose metabolism with hyperglycemia and ketonuria has been reported [ , ] . inh also impairs cellular reduction-oxidation capacity via competitive inhibition of nad [ , ] . pyridoxine deficiency also appears to play a role in inh-induced mental status changes (coma and lethargy) [ , , ] . appropriately dosed pyridoxine (vitamin b ) has been the mainstay of antidotal therapy for inh intoxication since the early reports of benefit versus his- figure . mechanisms of isoniazid (inh) toxicity (see text for details). enzymes (italicized): gaba-t, gaba transaminase; gad, glutamic acid decarboxylase; got, glutamic-oxaloacetic transaminase; ldh, lactate dehydrogenase; ppk, pyridoxine phosphokinase; and sr, serine racemase. substances: gaba, γ-aminobutyric acid; and ssa, succinic semi-aldehyde. symbols: ⊕, agonism or co-factor; ⊗, antagonism. data used can be located in [ , [ ] [ ] [ ] ] . drugs and pharmaceuticals: management of intoxication and antidotes torical controls [ ] . exogenous vitamin b provides the necessary precursor for the co-factor for gaba regeneration. clinical experience with pyridoxine comes from case series, case reports, and animal data [ , , , , [ ] [ ] [ ] . clinical trials are absent due to ethical considerations. vitamin b (as pyridoxine hydrochloride) is provided on a gram per gram basis for each gram of inh ingested, to a maximum of g or mg/kg (the empiric dose in ingestions of unknown quantity) [ , , ] . a repeat dose can be provided if necessary. due to the large amount of pyridoxine required, inadequate stocking and depletion of institutional and entire regional supplies have been widely reported [ , , ] . in the convulsing patient, pyridoxine is administered i.v. at . g/min ( g maximum) until seizure termination, with the remainder over - hours. pediatric dosing should not exceed mg/kg ( g maximum). large doses of pyridoxine have been safely administered; however, sensory neuropathy may occur with massive acute doses (> g) or chronic large daily doses [ ] . co-administration of benzodiazepines is synergistic in controlling seizures [ , ] . massive inh ingestion may require additional sedative hypnotics or anesthetic agents to suppress seizures [ ] . inh is dialyzable, and hemodialysis has been used successfully in cases refractory to antidotal treatment, in those with extremely high plasma inh concentrations, and in patients with renal failure [ , ] . pyridoxine also appears to rapidly reverse the impaired consciousness seen in inh overdose [ , ] . the cns excitatory neurotransmitters include glutamate and d-serine, which with glutamate is a co-agonist of the nmda receptor [ ] . examination of the metabolic pathways affected by pyridoxal -phosphate depletion (fig. ) suggests that inadequate stores of these neurotransmitters (due to inadequate co-factors for glutamic-oxaloacetic transaminase and serine racemase) might be contributory, in addition to general substrate or catecholamine deficiency. pyridoxine therapy is also recommended for poisoning through other hydrazines or hydrazine precursors (e.g., gyrometra mushrooms, monomethylhydrazine, and unsymmetrical dimethylhydrazine fuel). pyridoxine is effective in treating the chronic inh-associated neuropathy, particularly in patients with renal failure. doses of - mg pyridoxine/day have typically been used in the chronic setting [ ] . pyridoxine has no effect in prevention or treatment of inh-associated hepatic injury. antineoplastic agents are used for the treatment of a variety of benign and malignant neoplasms. some antineoplastic agents (such as the antifolates) have an expanded spectrum that includes use in rheumatology, dermatology, and obstetrics and gynecology. toxicity may be due to the agent itself or delivery of the agent to an unintended target (e.g., extravasation). several antidotes are used in a prophylactic fashion or on chronic basis. amifostine (wr- ) -which is dephosphorylated by alkaline phosphatase to an activated, protective thiol form -is approved to decrease toxicity associated with radiotherapy and renal injury associated with cisplatin [ ] . it has also been used to reduce chemotherapy-induced neutropenia; genitourinary injury associated with cyclophosphamide; and transfusion requirements, gastrointestinal and hepatic toxicity in pediatric patients [ , ] . cyclophosphamide and ifosfamide induce bladder toxicity (hemorrhagic cystitis) via their metabolite acrolein. mesna ( -mercaptoethane sulfonate), a thiol agent that complexes with and inactivates acrolein, is provided orally or i.v. as prophylaxis [ ] . diethyldithiocarbamate (ddtc), the major metabolite of disulfiram, is an investigational agent for prevention of neuropathy from cisplatin and its analogs; it increased nephrotoxicity in one study [ ] . granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage colony-stimulating factor (gm-csf), erythropoietin (hemopoietin) and its derivatives, oprelvekin (recombinant interleukin- ), and other stimulating factors are employed as adjuvants to reconstitute various hematopoietic lines damaged by chemotherapy and radiation [ , ] . palifermin (recombinant truncated human keratinocyte growth factor) is used to prevent severe mucositis in patients receiving stem-cell transplantation with a total body irradiation conditioning regimen [ ] . the remaining section focuses on antineoplastic antidotes used in the acute setting. a dreaded complication of administration of vesicant chemotherapeutic agents is extravasation. risk factors for extravasation include small, fragile, or sclerosed veins, obesity, comorbid conditions (diabetes, circulatory disorders, impaired sensory perception), use of rigid i.v. catheters, and clinicians' lack of knowledge and skills [ ] . redness, burning pain, and swelling may portend later blistering, ulceration, and necrosis. dexrazoxane is u.s. fda approved for treatment of extravasation resulting from i.v. anthracycline chemotherapy, to diminish tissue damage and the need for surgical excision of necrotic tissue [ ] . clinical efficacy data comes from two simultaneously reported openlabel, single-arm, prospective multicenter studies in which only out of patients with biopsy-proven extravasation required surgical debridement [ ] . additional instances of successful dexrazoxane treatment of anthracycline extravasation are provided as case reports ( [ ] and others). dexrazoxane is provided once daily for consecutive days, with the first infusion initiated as soon as possible. daily doses are as follows: day , mg/m (maximum mg); day , mg/m (maximum mg); day , mg/m (maximum mg) [ ] . the dose is reduced by % in patients with creatinine clearance of less than ml/min. in mice, efficacy rapidly decreased when dexrazoxane was provided beyond hours after extravasation [ ] . dexrazoxane's mechanism of action appears to involve reversible inhibition of topoisomerase ii and inhibition by its metabolite, an ethylenediamintetraacetic acid (edta) analogue, of free radical formation via iron removal from the iron-doxorubicin complex [ ] . topoisomerase ii-independent effects have also been described [ ] . in contrast, some authors have encouraged the nonconcurrent, off-label use of topical dimethyl sulfoxide (dmso) for anthracycline extravasation because of the risk of infection, neutropenia, and thrombocytopenia associated with dexrazoxane [ ] . dexrazoxane is also used prophylactically to limit anthracycline-associated cardiomyopathy [ ] . in , methotrexate (mtx) joined the oncological armamentarium for leukemia [ ] . mtx treatment of solid cancers was reported in , and it gained fda approval for psoriasis in [ , ] . mtx is now used intramuscularly, intrathecally, i.v., and orally for a range of dermatological, rheumatological, obstetric, and gynecological conditions. the dose ranges from . - mg orally once weekly for psoriasis or rheumatoid arthritis to - g/m or more for osteosarcoma, leukemia, and lymphoma [ ] [ ] [ ] . mtx poisoning may result from intentional overdose; unintentional ingestion, prescription, dispensing, administration, and patient errors; or renal insufficiency leading to persistent mtx in patients receiving high-dose chemotherapy regimens [ , ] . mtx antagonizes folate metabolism (and rapidly proliferating cells) via multiple mechanisms. dihydrofolate reductase inhibition by mtx and its polyglutamated metabolites ensures that neither dihydrofolate nor active tetrahydrofolate can be generated from folate, nor can existing dihydrofolate be recycled. thymidylate synthase inhibition compromises thymidine synthesis. purine ring synthesis is impaired by inhibition of the participating enzymes amidophospho-ribosyltransferase (ppat) and -aminoimidazole- -carboxamide ribonucleotide transformylase (aicart) [ , ] . maintenance of brisk urinary elimination with i.v. hydration and urinary alkalinization are standard therapies for patients receiving mtx. mtx is ten times more soluble in alkalinized urine (i.e., ph . ) than at ph . [ ] . folate (folic acid) is an ineffective therapy for mtx poisoning. while folate will inhibit renal resorption of mtx, persistent dihydrofolate reductase inhibition by mtx inhibits folate's activation. leucovorin (folinic acid, -formyltetrahydrofolic acid, citrovorum factor) sustains the folate cycle by bypassing the blocked dihydrofolate reductase pathways. addition of leucovorin "rescue" permitted the administration of very-high-dose mtx chemotherapy [ ] . however, in patients receiving mtx chemotherapy, -hour mtx concentrations greater than × - m ( μmol/l), -hour concentrations greater than × - m ( μmol/l), or -hour concentrations greater than × - m ( . μmol/l, nm), or those with evidence of renal dysfunction are considered at high risk for toxicity [ ] . in the setting of mtx persistence or toxicity, leucovorin i.v. doses are increased to mg/m or mg/m every hours according to established nomograms; doses and as high as g/day have been used [ , ] . leucovorin therapy continues until mtx concentration are less than . × - - . × - m ( . - . μmol/l, - nm) [ ] . however, adequate leucovorin concentrations cannot be achieved for competitive reversal of mtx toxicity when mtx concentrations are persistently above - μmol/l; other antidotal strategies are then considered [ ] . treatment of patients ingesting mtx should not be delayed pending mtx concentrations. inhibition of dna synthesis is nearly complete when mtx plasma concentrations are greater than × - m ( . μmol/l, nmol/l) [ ] . therefore, leucovorin is provided until mtx concentrations are less than × - m in patients receiving mtx for non-oncological indications or in patients not receiving mtx therapeutically [ ] . only leucovorin's s-form [levoleucovorin, ( s)-leucovorin] is active and rapidly metabolized to usable, reduced folates; the inactive isomer is slowly eliminated by renal excretion during i.v. administration [ ] . leucovorin was available in the u.s. only as a racemate until , when levoleucovorin received fda approval. levoleucovorin at one-half of the usual racemic dose (as it is entirely active) appears to provide similar rescue therapy in high-dose mtx chemotherapy [ ] . oral rescue is not routinely recommended as leucovorin's bioavailability is poor above mg due to saturation of active intestinal transport [ ] . the calcium content of leucovorin ( . meq calcium/mg leucovorin) mandates that infusion should not exceed mg/min. intrathecal administration of leucovorin is contraindicated, as death may result [ ] . glucarpidase (carboxypeptidase g , cpdg ) is undergoing evaluation as an additional antidote for mtx toxicity. u.s. or european marketing approval for glucarpidase has not been granted at the time of writing. competitive and complete reversal of mtx toxicity by leucovorin may not be possible at mtx concentrations above μmol/l (and perhaps even lower) [ , , ] . patients with systemic mtx toxicity (significant mucositis, gastrointestinal distress, myelosuppression, hepatitis, or neurotoxicity), persistent serum mtx, and renal impairment following high-dose mtx have been considered for glucarpidase therapy in addition to leucovorin. recommendations for glucarpidase above certain mtx concentrations have varied by malignancy, degree of renal impairment, initial mtx dose, and serum mtx concentration (e.g., clinical trials nct , nct , and [ , [ ] [ ] [ ] ). purification of "carboxypeptidase g", a pseudomonad zinc-dependent enzyme capable of mtx cleavage, was reported in [ ] . its antidotal potential was suggested in . in mice injected with lethal mtx doses, carboxypeptidase g rapidly decreased mtx concentrations and improved survival [ ] . cpdg selectively eliminated systemic mtx in patients treated with high dosages targeting cns malignancy, and rescued a patient receiving mtx with renal failure in [ , ] . after the original enzyme source of cpdg was lost, a revived recombinant cpdg product demonstrated success in both i.v. and intrathecal rescue of mtx overdose in non-human primates [ ] [ ] [ ] . successful use in multiple case reports and human trials in adult and pediatric patients with i.v. and intrathecal mtx overdose emerged [ , , , , [ ] [ ] [ ] . glucarpidase is a dimerized protein with two domains -a zinc-dependent catalytic domain that removes c-terminal glutamate residues of folate and folate analogues and a β-sheet interaction site [ ] . glucarpidase splits mtx and its -hydroxy-mtx metabolite into inactive -{[ , -diamino- -(pteridinyl)methyl]-methylamino}-benzoic acid (dampa) and hydroxy-dampa plus glutamate [ , ] . mtx serum concentrations decline by - % within minutes after glucarpidase [ , , , , , ] . intracellular, intraluminal (gastrointestinal tract) and intracerebral mtx is unaffected, creating the potential for rebound concentrations and persistent cytotoxicity [ , , , [ ] [ ] [ ] . leucovorin therapy must continue after carboxypeptidase administration. dampa's poor urinary solubility also requires ongoing alkalinization and saline diuresis to prevent renal precipitation [ , ] . anti-glucarpidase antibodies have been detected in patients receiving glucarpidase, although patients have been successfully treated with additional doses of glucarpidase for persistently elevated mtx concentrations [ , , , , , , ] . hplc must be used to determine actual mtx concentrations after glucarpidase as both mtx metabolites, -hydroxy-mtx and dampa, interfere with immunoassay techniques [ ] . glucarpidase has an affinity for mtx approximately -to -fold higher than it does for leucovorin; however, its affinity for folate and -methyltetrahydrofolate are similar [ , ] . glucarpidase eliminates active levo-( s)-leucovorin about % faster than nonphysiological dextro-( r)-leucovorin [ ] . a study to address the clinical consequence is ongoing. because of the stereoselective destruction of active leucovorin and its metabolites, many protocols attempt to separate leucovorin administration from glucarpidase administration by - hours. administration of glucarpidase more proximate to leucovorin administration, and which antidote to provide should glucarpidase become available at a leucovorin dosing interval, requires a thoughtful benefit-risk assessment. countryspecific information on obtaining glucarpidase, institutional review board protocol, and consent issues have been made available online (www.btgplc.com/ btgpipeline/ /voraxaze.html; and www.fda.gov/cder/cancer/singleind. htm). cardiovascular pharmaceuticals comprise a wide variety of agents including anti-dysrhythmics, β-adrenergic antagonists (β-blockers, bbs), angiotensin antagonists, calcium channel antagonists (ccbs), cardioactive glycosides, and imidazoline derivatives. overdose of these agents alone or in combination can generate potentially lethal combinations of impaired conduction, dysrhythmia, vasodilatation, and negative inotropy. management of severe cases may necessitate diagnostic adjuncts such as echocardiography and right heart catheterization (swan-ganz measurements). in cases refractory to routine supportive care, vigorous gastrointestinal decontamination, and pharmacological intervention, aggressive measures including cardiac pacing, intra-aortic balloon counter-pulsation, or extracorporeal circulation (cardiopulmonary bypass) may be required until toxin elimination can be achieved [ ] . cardiac pacing may improve heart rate without increasing cardiac output if inotropy is compromised. use of naloxone in the management of overdose of clonidine and angiotensin receptor antagonists and angiotensin converting enzyme inhibitors is provided in the opioid antagonists section. strategies to mitigate the anticoagulant toxicity of vitamin k antagonists (i.e., coumadin) including exogenous oral or i.v. vitamin k, fresh frozen plasma, prothrombin concentrates, and recombinant factor vii are detailed in the american college of chest physicians evidence-based clinical practice guidelines [ ] . the guidelines also address protamine sulfate for reversal of heparin anticoagulation and use of nonheparin anticoagulants for treatment and prevention of heparininduced thrombocytopenia [ , ] . atropine (d,l-hyoscyamine) is familiar to clinicians due to its use in several advanced cardiac life support (acls) algorithms [ ] . atropine is a centralacting, competitive antagonist of muscarinic acetylcholine receptors (m -m ) [ ] . it is used to counteract bradycardia from bbs, ccbs, cardioactive glycosides, and clonidine. atropine increases basal heart rate; it does not affect the basal force of contraction [ ] . positive chronotropy alone may not produce systemic benefit in severe poisoning, and conduction system poisoning may limit responsiveness to atropine [ ] . for symptomatic bradycardia, atropine . - . mg (pediatric dose: . mg/kg) i.v. is provided every - min to a maximum dose of mg. paradoxical parasympathetic response may occur during slow infusions or doses less then . mg ( . mg minimum in children) [ ] . in slowing gastrointestinal motility, atropine may impair decontamination with wbi or ac. ccbs antagonize l-type calcium channels, slowing entry of calcium ions during myocyte depolarization; however, intracellular calcium release is not directly affected. this disrupts calcium-mediated excitation-contraction coupling, action potential generation and conduction, and vascular smooth muscle tone [ ] . exogenous i.v. calcium is indicated in cases of ccb and bb toxi-city [ ] . in animal models, calcium salts reverse ccb-induced deficits in contractility, blood pressure, and cardiac output [ ] . multiple uncontrolled cases reports document the effectiveness of calcium salts; however, interpretation of effectiveness is complicated by the co-administration of other modalities. some authors advocate aggressive high-dose calcium therapy, providing large amounts of calcium without apparent ill effect [ ] . this approach does carry a risk of death from hypercalcemia [reported concentration, . mg/dl ( . mmol/l) after g calcium] [ ] . others recommend a bolus dose followed by continuous infusion to maintain physiological calcium levels [ ] . peripheral administration as calcium gluconate decreases the risk of extravasation and tissue necrosis. a standard container of ml of % calcium gluconate provide . meq ( mg) elemental ca + ; ml of % calcium chloride ( g total cacl ) yields . meq elemental ca + [ ] . a suggested approach is to initially administer a . ml/kg ( . meq/kg) bolus of % calcium gluconate ( . ml/kg % cacl ) over - min [ , ] . empirically, this is roughly one vial ( g) of % cacl or three vials ( g) of % calcium gluconate i.v. the bolus may be repeated several times. due to bolus dissipation, most patients are placed on an infusion of % calcium gluconate at . - . ml/kg per hour ( . - . meq/kg per hour) or . - . ml/kg per hour [ , ] . serum phosphate, calcium, and hydration status should be closely monitored. calcium administration for hyperkalemia has been generally contraindicated in cases of cardioactive glycoside toxicity, out of concern for dysrhythmias or systolic arrest (also known as "stone heart") [ ] . while more recent studies have challenged this assertion, it is advisable to withhold calcium until the definitive cardiac glycoside antidote, digoxinspecific fab fragments, has been provided [ ] . digoxin and cardioactive glycosides inhibit the cardiac sodium-potassium atpase. the subsequent accumulation of sodium in the cytoplasm dissipates the driving force for calcium expulsion via the sodium-calcium exchanger. increased intracellular calcium enhances actin-myosin coupling, myocyte contraction, and inotropy. in overdose, the excess calcium may result in membrane hyperexcitability and delayed after-depolarizations. increased vagal tone decreases conduction through the av node. the combination of increased automaticity and vagotonicity may yield lethal ventricular escape rhythms. digoxin-specific antibody fragments bind free digoxin in serum to decrease digoxin serum concentrations to undetectable levels within minutes [ ] . successful reversal of digoxin toxicity with digoxin-specific fab was first reported in [ ] . the results of a prospective multicenter study demonstrated significant effectiveness in reversing life-threatening digitalis toxicity, and more recent studies confirm ongoing fab fragment utility [ , ] . digoxin-specific fab were also shown to be effective in children [ ] . digoxin-specific fab are produced from purified ovine-derived immunoglobulin g. cleaving the fc antibody portion significantly improves renal excretion of the complex, decreases immunogenicity, and facilitates diffusion of remaining free fab into tissue [ ] . reflecting digoxin redistribution from target organs of toxicity, the initial response to digoxin-specific fab was min ( - min), and complete reversal of systemic toxicity occurred on average by min ( - min) [ ] . indications for therapy include life-threatening or progressive dysrhythmia or shock; potassium greater than . meq/l (acute poisoning); chronic poisoning with other end-organ manifestations such as altered mental status, significant gastrointestinal symptoms or renal impairment; or serum digoxin concentration > ng/ml or greater than ng/ml beyond hours after ingestion. hyperkalemia is rapidly reversed by digoxin-specific fab [ ] . one vial neutralizes approximately . mg of digoxin (or digitoxin). dosing is based either on amount ingested [number of vials = amount ingested (in mg) × . (oral bioavailability) / . ], or a serum concentration [number of vials = serum digoxin concentration (ng/ml) × patient weight (kg) / ]. the number of vials is rounded up and administered i.v. over min. empiric therapy is - vials for adult or pediatric patients in acute poisoning or - vials ( - vials in children) in chronic poisoning. partial reversal is recommended by some authors [ ] , but is not common u.s. practice due in part to concern for recrudescent toxicity with inadequate therapy [ ] . following treatment, free digoxin concentrations may rebound upwards within - hours, most likely reflecting tissue redistribution into the vascular space [ ] . this provides a measure of protection against development of significant congestive heart failure (chf) in patients dependent upon digoxin for inotropy, although exacerbation of chf may occur [ ] . clinically significant late rebound of digoxin concentrations and toxicity have occurred in patients with marked renal dysfunction [ ] . immunogenicity from repeat digoxin-specific fab has generally not been significant, although allergic reactions have been infrequently reported with administration [ ] . digoxin-specific fab has been used clinically or experimentally to treat poisoning by other cardiac glycosides -yellow oleander (thevetia peruviana), nerium oleander, chan su and "love stone" (extract of the bufo bufo gargarizans toad) [ , ] . higher dosing may be required due to poor binding affinity. bbs competitively antagonize catecholamine effects at cardiac β-receptors, leading to decreased inotropy and slowed conduction through the av node. bradycardia, conduction delay, hypotension, and decreased cardiac output may accompany significant poisoning. bb interference with gluconeogenesis and glycogenolysis may lead to hypoglycemia, as well as blunt the catecholamine response that is important in its recognition. glucagon, a -amino acid peptide hormone secreted by pancreatic α-cells, counteracts hypoglycemia and the actions of insulin; regulates gastrointestinal motility; and mediates the rate of renal filtration, urea excretion, and water resorption [ ] . the current glucagon product is now produced in non-pathogenic e. coli by recombinant techniques [ ] . myocardial binding occurs at a distinct glucagon receptor (gcgr) coupled with the β-agonist binding site. antidotal (off-label) use of glucagon thus bypasses β-receptor blockade to directly induce g-protein-mediated stimulation of adenylate cyclase to convert atp to camp [ ] . camp, in turn, activates protein kinase a (pka), which promotes the phosphorylation and opening of dormant l-type calcium channels to improve calcium-dependent excitation-contraction coupling [ ] . another proposed mechanism is c-terminal cleavage of glucagon to miniglucagon, which has a direct effect on sarcoplasmic reticulum calcium stores via arachidonic acid [ ] . in human volunteers evaluated by cardiac catheterization, glucagon increased heart rate, cardiac index, and mean atrial pressure, but not left ventricular end-diastolic pressure (edp) or systemic vascular resistance (svr) [ ] . clinical experience in overdose consists primarily of case reports [ , ] . due to the complex nature of overdose, glucagon is often used in combination with other agents in severe bb overdose. additionally, several ex vivo experiments, controlled animal studies, and uncontrolled case reports have demonstrated that glucagon can be beneficial in ccb exposure [ ] [ ] [ ] . the recommended initial bolus dose of glucagon is - μg/kg, which may be repeated after - min [ ] . a continuous infusion corresponding to the total effective bolus reversal dose is then provided per hour (e.g., if clinical response was observed following administration of mg, mg, and finally mg, the hourly infusion would be mg/hour). the effects of glucagon administered i.v. begin within - min, peak at - min and last for approximately min [ ] . nausea and vomiting are common and should be anticipated. this may complicate management of patients with depressed mental status or airway concerns. flushing, transient hyperglycemia, and smooth muscle relaxation, and ileus may also occur. since ccbs antagonize the l-type calcium channel in pancreatic islet cells, a subsequent decreased insulin production can produce hyperglycemia [ ] . animals poisoned by ccbs have impaired myocardial fatty acid uptake (leaving them dependent upon carbohydrate metabolism), impaired uptake of glucose, and myocardial insulin resistance [ , ] . in humans, intracoronary verapamil increased glucose release and altered myocardial lactate use from consumption to release [ ] . decades ago, glucose-insulin-potassium (gik) was proposed as adjuvant therapy for acute myocardial infarction, with the intent of suppressing uptake of free fatty acids, improve myocardial energy production, and stabilize intracellular potassium [ ] . randomized trials of gik therapy in patients with acute myocardial infarction (ami) have not shown benefit, although the insulin doses tend to be low (in general, ≤ . u/kg) [ ] . experience in the surgical literature in cases where much higher insulin doses have been used has been somewhat different [ ] . patients undergoing aortic valve replacement and coronary artery bypass who received high-dose insulin at unit/kg per hour demonstrated more rapid lactate clearance, lower glucose, lower dobutamine requirements, a trend for improved cardiac indices, and potential anti-inflammatory benefit (lower c-reactive protein and free fatty acid levels) [ ] . insulin doses of . units/kg were tolerated without excess increase of insulin-induced potassium elimination [ ] . in combination with dopamine, insulin units/kg was used to significantly augment cardiac output and decrease systemic vascular resistance in post-coronary artery bypass graft (cabg) patients without generating excess in oxygen demand [ ] . additional benefits of high-dose insulin included overcoming insulin resistance, increased expression of glucose transporters, and improved turnover of sodium-potassium-atpases [ ] . the basis for high-dose insulin euglycemia therapy (hiet) (off-label) in overdose has been explored in a series of animal models of ccb and bb toxicity [ , , [ ] [ ] [ ] . hiet increased myocardial lactate uptake and improved systolic and diastolic heart function. insulin outperformed epinephrine and glucagon [ ] [ ] [ ] . multiple human cases of successful management of ccb overdose with hiet have been described [ , ] . because the beneficial cardiovascular effects of hiet are not seen for - min after initiation, it must be considered early, before patients become unsalvageable [ ] . a proposed dosing scheme includes a bolus dose of regular insulin of . units/kg, followed by an infusion of . - . units/kg per hour, titrated upwards as necessary [ ] . a dextrose bolus is also provided unless significant hyperglycemia exists, followed by an infusion of . - . g/kg per hour to maintain blood glucose between and mg/dl. persistent physician reticence to utilizing the high-dose insulin out of concern for excess hypoglycemia presents an obstacle for implementation of adequate hiet [ ] . this ignores a body of physiological data that demonstrate that the insulin transport follows saturation kinetics [ , ] . alternatively, it has also been demonstrated that insulin-stimulated glucose clearance reaches a maximum in both lean and obese subjects [ ] . taken together, this suggests that, from a therapeutic standpoint, since insulin effect via insulin receptors appears saturable, additional mechanisms must be at work. the effects of hiet may include counteracting ccb-mediated insulin impairment or shockinduced hyperglycemia, improving myocardial substrate utilization, and improving myocardial metabolism [ ] . from an adverse-effects standpoint, once adequate and ongoing glucose has been provided, hypoglycemia should not present an excessive risk [ ] , although frequent serum glucose and potassium evaluation are obvious components of hiet therapy. due to the high dosing, insulin may persist after the infusion cessation and necessitate ongoing supplemental dextrose beyond insulin infusion. as hypokalemia is an intracellular result of shift, it is supplemented cautiously. during administration of local anesthetics, severe toxicity may result from systemic absorption or unintended intravascular administration. loss of consciousness, dysrhythmia, cardiovascular collapse, seizures, and lactate-associated acidemia may rapidly ensue [ ] . furthermore, in animal models, for some of the local anesthetics (bupivacaine, levobupivacaine, and ropivacaine), treatment with "standard" advanced cardiac life support (acls) drugs such as epinephrine may precipitate ventricular dysrhythmia [ ] . following a serendipitous observation that pretreatment with a lipid emulsion altered the dose-response to bupivacaine-induced asystole, murine and canine studies provided evidence of survival benefit with lipids in bupivacaine toxicity [ , ] . case reports of successful resuscitation of patients severely affected by bupivacaine, levobupivacaine, mepivacaine, prilocaine and ropivacaine (alone or in combination) followed [ , [ ] [ ] [ ] . pediatric experience is limited to a case of successful resuscitation following lidocaine/ropivacaine toxicity from a posterior lumbar plexus block [ ] . lipid therapy has been successfully applied in human bupropion toxicity and combined quetiapine and sertraline overdose [ , ] . animal models have suggested a possible benefit in clomipramine, propranolol, thiopentone, and verapamil poisoning [ ] [ ] [ ] [ ] . an understanding of lipid's mechanism of action is incomplete. it may act as a "circulating lipid sink" in which excess lipophilic drug may dissolve; modulate intracellular processes; or provide an alternative myocardial energy supply [ ] . presumably due to central sympathetic activation, human volunteers given a -hour lipid emulsion ( %) infusion had increased systemic vascular resistance, blood pressure, muscle sympathetic nerve activity, and concentrations of insulin and aldosterone, without increased cardiac output [ ] . lipid emulsion increased inotropy in both spontaneously beating and paced isolated rat hearts poisoned with levobupivacaine [ ] . dosing guidelines for the off-label use of lipid emulsion in resuscitation are provisional, as optimal bolus and continuous infusion therapy and timing are still being explored. the association of anaesthetists of great britain and ireland recommends an i.v. bolus of . ml/kg intralipid ® ( %) over min, which may be repeated twice at -min intervals if an adequate circulation has not been restored [ ] . following the initial bolus, an infusion is commenced at . ml/kg per min (which may be increased to . ml/kg per min in inadequate circulation). propofol is an inadequate substitute [ , ] . ongoing lipid therapy may be required as recrudescence may occur [ ] . hyperamylasemia may be anticipated. additional concerns include pancreatitis, allergic reactions, acute myocardial infarction, fat embolism, and altered coagulation [ ] . in lapine and porcine models of asphyxial cardiac collapse (pulseless electrical activity or arrest), lipid emulsion was markedly ineffective [ , ] . in vitro, lipid affinity for both bupivacaine and ropivacaine is also adversely affected by low ph (by a factor of . in a ph drop from . to . ) [ ] . these data suggest that ventilatory status must be aggressively addressed early in toxicity. due to their physicochemical characteristics and structure, many non-antiarrhythmic drugs are able to antagonize or alter expression of the myocardial potassium delayed rectifier channel (herg, kcnh , lqt ). with channel block, potassium efflux is compromised, and the repolarizing cardiac i kr current is impaired. the surface ecg reflects this as qt prolongation. age, female gender, comorbidities such as structural heart disease, electrolyte disturbances such as hypokalemia, and heart rate (bradycardia) may provide additional risk. certain antibiotics, antihistamines, antipsychotics, antidepressants, and methadone are prone to induce qt prolongation. qt prolongation is associated with torsade de pointes, a polymorphic ventricular arrhythmia that can degenerate into ventricular fibrillation, cardiac arrest and sudden death [ ] . if significant qt prolongation (qtc > ms) is detected, administration of - g magnesium sulfate i.v. (pediatric dose, - mg/kg) over to min (depending on urgency of presentation), followed by an infusion of - mg/min is suggested [ ] . rapid infusion may cause hypotension, and magnesium should be administered cautiously in renal failure. a second bolus can be provided - min later [ ] . magnesium sulfate i.v. is effective in arrhythmias occurring due to early or delayed depolarization-induced triggered activity [ ] . acceleration of the heart rate with isoproterenol or transvenous pacing (overdrive pacing) may be needed to preclude recurrence of torsade de pointes while correction of underlying risk factors (hypokalemia and hypocalcemia) ensues. immediate non-synchronized defibrillation is required for unstable polymorphic ventricular tachycardia or ventricular fibrillation. severe cardiovascular toxicity may result from blockade of cardiac sodium channels by tricyclic antidepressants (tcas) -leading to conduction delays, dysrhythmias, and myocardial depression. tcas adversely affect maximum upstroke velocity (v max ), which approximates the magnitude of sodium entry [ ] . the sodium channel blockade displays rate dependence. at slow rates the tca has time to disassociate, allowing channel recovery. at faster rates, block progressively worsens. given the anticholinergic effects of tcas that speed the heart rate, this is a significant concern. however, attempts to decrease heart rate with propranolol produced hypotension and lethality in canine studies [ , ] . with progressive sodium channel block, ventricular impulse propagation becomes delayed. sodium channel blockade manifests on the surface ecg as qrs widening. a qrs equal or greater than ms is a significant predictor of seizure; a qrs ≥ ms predicts ventricular dysrhythmia [ ] . the right bundle branch has a relatively longer refractory period, and it is affected disproportionately by impaired intraventricular conduction delay. rightward terminal axis shift or outright bundle branch block may be present [ ] . these rightward terminal forces may also produce terminal r waves in leftwarddirected leads [ ] . acidemia secondary to hypoperfusion or seizure may generate progressively worsened block. in an acidemic environment, free tca concentrations increase as binding to α- acid glycoprotein decreases, the tca ionized fraction increases, and sodium channel blockade worsens [ ] . seizures are severe and consequential, leading to qrs widening and hypotension [ ] . administration of sodium bicarbonate improves v max and action potential amplitude by increasing extracellular ph and sodium concentration [ ] . consequentially, compromised myocardial inotropy, conduction aberrations, and dysrhythmia are reversed. several animal studies have demonstrated these beneficial effects [ , ] . both the sodium and alkalemia induced by sodium bicarbonate improve cardiac performance [ ] . the enhanced inotropy with sodium bicarbonate is independent of and additive to vasopressor treatment [ ] . hyperventilation-induced alkalinization similarly narrows the qrs [ ] . sodium bicarbonate outperformed hyperventilation in a swine model, although hypertonic saline was superior to both [ ] . this approach has been reported clinically [ ] . while sodium bicarbonate is recommended for qrs widening in tca evidence-based consensus guidelines for out-ofhospital management, actual human studies are not as extensive as one might suspect [ , ] . initially, hypertonic sodium bicarbonate - meq/kg i.v. is provided, preferably with continued ecg monitoring of the qrs. institutions usually stock either an . % solution ( meq/ml sodium and bicarbonate ions) or a . % solution ( . meq/ml sodium and bicarbonate ions). rarely, a % solution may be encountered ( . meq/ml). a "standard" -ml ampule of . % or . % solutions would deliver or . meq of nahco . several boluses may be required, either initially or as the bolus effect declines due to redistribution [ ] . ongoing alkalinization should be provided as discussed previously, with a goal of serum ph . . if sodium bicarbonate administration is problematic due to fluid load, hyperventilation and/or hypertonic saline may be required [ , ] . due to mechanistic similarities, sodium bicarbonate has been recommended for qrs widening seen in poisoning by vaughn-williams class ia and ic antidysrhythmics, cocaine, diphenhydramine, carbamazepine, and propoxy-phene [ ] [ ] [ ] [ ] . treatment of bupropion-induced qrs widening with sodium bicarbonate has met with both success and failure [ ] . sodium bicarbonate has also been suggested to treat qrs widening from venlafaxine; similar effects seen with lamotrigine might also be amenable [ , ] . sodium bicarbonate therapy may have a role in taxus species (yew berry) toxicity [ ] . treatment of amantadine-induced qrs widening with sodium bicarbonate may be complicated by concurrent profound hypokalemia [ ] . naloxone is a competitive opioid antagonist at all receptor subtypes [ ] . it can prevent or reverse the effects of opioids, notably cns and respiratory depression. massive doses of naloxone ( . mg/kg with . mg/kg per hour infusion) have been administered safely in non-opioid tolerant individuals suffering from spinal cord injury [ ] . however, indiscriminate use of naloxone in opioid-tolerant individuals can precipitate acute opioid withdrawal, with attendant acute lung injury, seizure, hypertension, or cardiac dysrhythmia [ ] . these are likely associated with the abrupt, significant, and sustained increases in plasma catecholamine concentrations (epinephrine and norepinephrine) that accompany narcotic reversal, particularly in the setting of hypercapnia [ ] . withdrawal-induced vomiting may compromise the airway in patients with concomitant sedative-hypnotic ingestion. precipitated withdrawal-associated agitation and violent behavior may require chemical restraint, leading to a vicious cycle of compromised cns and cardiopulmonary function as naloxone wears off. self-release and relapse following naloxone administration is also a concern in opioids with prolonged duration of effect (methadone, controlled-release oxycodone hydrochloride, etc.). naloxone is no longer recommended as the initial resuscitation of newborns with respiratory depression in the delivery room; precipitation of acute neonatal opioid withdrawal may produce severe consequences [ ] . sudden cardiac arrest has occurred in preterm neonates given naloxone to reverse opioid overdose [ ] . naloxone is utilized in those individuals with clear evidence of the opioid toxidrome. those with a respiratory rate ≤ or hypopnea are likely to benefit [ ] . the goal of therapy is titration to adequate ventilatory status without withdrawal. after normocapnia is achieved by supported ventilation, this can be done with i.v. administration of . - . mg initially (e.g., ml of . mg naloxone in ml diluent or mg naloxone in ml diluent). due to rapid onset, effectiveness can be assessed, and if required, the dose can be titrated upwards incrementally to . mg, mg, or even mg. patients without response to mg naloxone are unlikely to have opioid-induced respiratory depression. nonopioid-dependent adults are administered . - mg i.v. pediatric dosing for infants and children from birth to years of age or less than kg body weight is . mg/kg; children older than years of age or weighing more than kg are provided mg [ ] . for longer acting opioids, following adequate initial opioid antagonism, two-thirds of the initial naloxone reversal bolus is provided as a continuous i.v. infusion [ ] . naloxone can successfully antagonize buprenorphine overdose in children, although prolonged therapy and monitoring may be required [ ] . higher doses may be required due to reverse buprenorphine effects because of its high affinity for opioid receptors [ ] . naloxone has also been used to reverse clonidine toxicity, although this is not always the case [ ] . it has been postulated that patients with higher hyperadrenergic tone (who have higher concentrations of endogenous opioids) or those in whom clonidine induces more endogenous opioid release may respond best to naloxone [ ] . mental status, blood pressure, and heart rate may respond differently. naloxone has been employed in angiotensin converting enzyme inhibitor overdose. one author reported that a . mg bolus of naloxone followed by repeat mg bolus reversed hypotension due to overdose with mg captopril [ ] . naloxone has been ineffective in reversing hypotension in other cases complicated by co-ingestants [ ] . the mechanism may relate to antagonism of endogenous opioids [ ] . co-administration of . mg/kg naloxone mitigated captopril-related decreases in systolic and diastolic blood pressure in healthy volunteers [ ] . a placebo-controlled study of healthy men found that naloxone pretreatment with mg followed by . mg/hour infusion precluded systolic blood pressure decrease induced by captopril ( mg) [ ] . under different experimental conditions [naloxone, . mg bolus and a -hour continuous infusion ( . mg/hour), and captopril ( mg)], no difference was observed [ ] . analogous to naloxone antagonism at opioid receptors, flumazenil competitively antagonizes benzodiazepine receptors -allosteric sites located at the macromolecular gaba a receptor complex, which regulate chloride ion flux within the associated ion channel [ ] . flumazenil reverses the sedative, psychomotor, and amnestic effects of benzodiazepines [ ] . flumazenil's effectiveness depends upon the number of benzodiazepine receptors that can be occupied according to the mass-action law, the affinity of a particular benzodiazepine for the receptor, and the free benzodiazepine concentration near the receptor [ ] . in contrast, antagonism of benzodiazepine-induced respiratory depression is inconsistent, and acute tolerance may develop to large doses [ ] [ ] [ ] . flumazenil administration can reverse bispectral index (bis) depression and permit earlier emergence from anesthesia in patients provided non-benzodiazepine anesthesia (propofol/remifentanil) [ ] . postulated mechanisms included intrinsic cns stimulant activity or antagonism of endogenous benzodiazepine-like ligands (endozepines). under certain experimental conditions, flumazenil may also demonstrate partial agonist or even inverse agonist activity [ , ] . the appropriate utilization of flumazenil as an antidote in patients with benzodiazepine overdose is still a matter of debate. patients who ingest benzodiazepines alone or in combination generally have acceptable outcomes with supportive care alone. proponents argue that awakening is therapeutic and diagnostic, obviates requirements for investigatory procedures, and limits complications of sedation. opponents point to the low risk of mortality with benzodiazepine ingestion, frequent co-ingestants for which flumazenil is ineffective or contraindicated, relapse, and risks of reversal. while flumazenil can be administered safely, indiscriminate flumazenil administration may produce an acute withdrawal syndrome in benzodiazepine-dependent patients, seizures, dysrhythmias, vomiting, and agitation [ ] [ ] [ ] [ ] . flumazenil is not recommended in cases complicated by co-ingestants capable of inducing seizures or dysrhythmias (e.g., bupropion, carbamazepine, chloral hydrate, chlorinated hydrocarbons, chloroquine, cocaine, cyclic antidepressants, cyclosporine, isoniazid, lithium, methylxanthines, monoamine oxidase inhibitors, phenothiazines, and propoxyphene) [ , , ] . as might be anticipated with an antidote of lesser half-life than many benzodiazepines, clinical condition may deteriorate following initial improvement, mandating ongoing monitoring. in one study, patients with primarily benzodiazepines ingestion remained awake for ± min following flumazenil; this was markedly decreased to ± min with co-ingestants [ ] . this may be problematic in patients who, once aroused, demand release from medical care. after excluding co-ingestants of concern, vital sign abnormalities, and an aberrant ecg, and considering the risk-benefit ratio, flumazenil is administered slowly i.v., titrated to clinical effect ( . mg/min, max ≤ mg) [ ] . offlabel continuous infusions of . - . mg/hour have been provided to preclude relapse. patients poisoned by pharmaceuticals present many challenges to the treating clinicians. they generally benefit from aggressive support of vital functions, a careful history and physical examination, specific laboratory analyses, and a thoughtful consideration of the risks and benefits of decontamination and enhanced elimination. data on the effectiveness of certain antidotes ranges from isolated case reports to robust clinical trials. clinicians are encouraged to liberally utilize consultation with regional poison centers or those with toxicology training to assist with diagnosis, management, and administration of antidotes, particularly in unfamiliar cases. no outside funding or support was received. the author has no financial interest in any products mentioned or the companies that produce them. use of trade names is for identification purposes only and does not constitute endorsement by the author, the nyu school of medicine, the new york city poison control center, or the new york city department of health and mental hygiene. within the medical literature, pharmaceuticals, pharmaceutical combinations, and other products are used offlabel as antidotal therapies; off-label uses are referred to in this review. this is for discussion purposes only and does not constitute endorsement of off-label use by the author, the nyu school of medicine, the new york city poison control center, or the new york city department of health and mental hygiene. as medicine is an ever-changing science, readers are encouraged to confirm the information contained in this review -by consulting product and safety information sheets, regional poison centers, those with toxicological expertise, and other resources -particularly in the case of new or infrequently used drugs. analeptic use in clinical toxicology: a historical appraisal treatment of choice in barbiturate poisoning; series of twenty-nine cases of barbiturate poisoning treated with pentylenetetrazole (metrazol) and supportive therapy a study of the anti-barbiturate effects of bemegride respiratory stimulant effects of ethamivan and picrotoxin the case against the use of respiratory stimulants a double blind comparison of doxapram, ethamivan and methylphenidate nagd regimen for the coma of drug-related overdose physostigmine in coma due to drug overdose physostigmine for γ-hydroxybutyrate coma: inefficacy, adverse events, and review the poisoned patient with altered consciousness. controversies in the use of a 'coma cocktail anonymous ( ) practical pharmacology. xxiv management of oil of citronella poisoning emetics, cathartics, and gastric lavage fullers earth; its absorptive power, and its antidotal value for alkaloids management of acute childhood poisoning therapeutic trends in the treatment of barbiturate poisoning. the scandinavian method patients with acute poisoning seen in a general medical unit ( - ) antagonism of imipramine poisoning by anticonvulsants in the rat phenytoin prophylaxis of cardiotoxicity in experimental amitriptyline poisoning wernicke's encephalopathy: new clinical settings and recent advances in diagnosis and management acetaminophen and salicylate serum levels in patients with suicidal ingestion or altered mental status value of rapid screening for acetaminophen in all patients with intentional drug overdose a randomized clinical trial of activated charcoal for the routine management of oral drug overdose how feasible is it to conform to the european guidelines on administration of activated charcoal within one hour of an overdose? position statement: ipecac syrup gastric emptying procedures in the self-poisoned patient: are we forcing gastric content beyond the pylorus? residual gastric content after gastric lavage and ipecacuanhainduced emesis in self-poisoned patients: an endoscopic study efficacy of ipecac during the first hour after drug ingestion in human volunteers gastric decontamination performed min after the ingestion of temazepam, verapamil and moclobemide: charcoal is superior to lavage gastric lavage for liquid poisons efficacy of ipecac-induced emesis, orogastric lavage, and activated charcoal for acute drug overdose position paper: gastric lavage efficacy of superactivated charcoal administered late ( hours) after acetaminophen overdose activated charcoal alone or after gastric lavage: a simulated large paracetamol intoxication out-of-hospital administration of activated charcoal by emergency medical services prolonged gastric emptying half-time and gastric hypomotility after drug overdose effect of anticholinergic drugs on the efficacy of activated charcoal efficacy of gastric emptying: gastric lavage versus emesis induced with ipecac superiority of activated charcoal alone compared with ipecac and activated charcoal in the treatment of acute toxic ingestions management of acutely poisoned patients without gastric emptying assessment of the efficacy of activated charcoal following gastric lavage in acute drug emergencies gastric emptying in acute overdose: a prospective randomised controlled trial comparison of three methods of gut decontamination in tricyclic antidepressant overdose whole-bowel irrigation versus activated charcoal in sorbitol for the ingestion of modified-release pharmaceuticals multiple-dose charcoal and whole-bowel irrigation do drugs and pharmaceuticals: management of intoxication and antidotes not increase clearance of absorbed salicylate ipecacuanha: the south american vomiting root toxicology of ipecac: a review effectiveness of -ml versus -ml doses of syrup of ipecac in children urinary excretion of ipecac alkaloids in human volunteers position paper: ipecac syrup evaluation of the time frame for home ipecac syrup use when not kept in the home poison treatment in the home home syrup of ipecac use does not reduce emergency department use or improve outcome a comparison of ipecac syrup and apomorphine in the immediate treatment of ingestion of poisons the frequency of complications associated with the use of multiple-dose activated charcoal esophageal laceration and charcoal mediastinum complicating gastric lavage severe hypernatremia secondary to gastric lavage advantage of high-surface-area charcoal for gastrointestinal decontamination in a human acetaminophen ingestion model position paper: single-dose activated charcoal clinical pharmacokinetics of oral activated charcoal in acute intoxications efficacy of activated charcoal administered more than four hours after acetaminophen overdose activated charcoal reduces the need for n-acetylcysteine treatment after acetaminophen (paracetamol) overdose acceleration of the body clearance of phenobarbital by oral activated charcoal enhancement of theophylline clearance by oral activated charcoal gastrointestinal clearance of drugs with activated charcoal european association of poisons centres and clinical toxicologists ( ) position statement and practice guidelines on the use of multi-dose activated charcoal in the treatment of acute poisoning physicochemical characteristics of drugs and response to repeatdose activated charcoal randomized study of the treatment of phenobarbital overdose with repeated doses of activated charcoal the treatment of tricyclic antidepressant overdose with repeated charcoal multiple-dose activated charcoal for treatment of yellow oleander poisoning: a single-blind, randomised, placebo-controlled trial multiple-dose activated charcoal in acute self-poisoning: a randomised controlled trial effect of charcoal-drug ratio on antidotal efficacy of oral activated charcoal in man the effect of food and ice cream on the adsorption capacity of paracetamol to high surface activated charcoal: in vitro studies role of repeated doses of oral activated charcoal in the treatment of acute intoxications aspiration of activated charcoal and gastric contents risk factors for emesis after therapeutic use of activated charcoal in acutely poisoned children gastrointestinal tract perforation with charcoal peritoneum complicating orogastric intubation and lavage gastrointestinal obstruction associated with multiple-dose activated charcoal fatal pulmonary aspiration of oral activated charcoal activated charcoal and the absorption of ferrous sulfate in rats treatment of acetaminophen ingestion with a superactivated charcoal-cola mixture effects of emetic and cathartic agents on the gastrointestinal tract and the treatment of toxic ingestion position statement: cathartics severe hypermagnesemia due to multiple-dose cathartic therapy electrolyte disorders following oral sodium phosphate administration for bowel cleansing in elderly patients sorbitol catharsis does not enhance efficacy of charcoal in a simulated acetaminophen overdose the effects of charcoal and sorbitol (alone and in combination) on plasma theophylline concentrations after a sustained-release formulation hypernatremia due to repeated doses of charcoal-sorbitol fatal poisoning from sodium phosphate enema. case report and experimental study rectal prolapse after oral cathartics effect of whole bowel irrigation on the pharmacokinetics of an acetaminophen formulation and progression of radiopaque markers through the gastrointestinal tract position statement: whole bowel irrigation activated charcoal alone and followed by whole-bowel irrigation in preventing the absorption of sustained-release drugs use of whole bowel irrigation in an infant following iron overdose whole-bowel irrigation as a treatment for acute lithium overdose whole bowel irrigation in an acute oral lead intoxication parachuting" meth: a novel delivery method for methamphetamine and delayed-onset toxicity from "body stuffing drugs and pharmaceuticals: management of intoxication and antidotes efficiency of whole bowel irrigation with and without metoclopramide pretreatment colonic perforation with volume laxatives life-threatening respiratory failure following accidental infusion of polyethylene glycol electrolyte solution into the lung theophylline desorption from activated charcoal caused by whole bowel irrigation solution endoscopic removal of iron bezoar following acute overdose endoscopic removal of slow release clomipramine bezoars in two cases of acute poisoning bezoar formation requiring endoscopic removal after intentional overdose of extended-release nifedipine endoscopic removal of a cocaine packet from the stomach acute pediatric lead poisoning: combined whole bowel irrigation, succimer therapy, and endoscopic removal of ingested lead pellets gastrotomy -a surgical approach to iron overdose radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of cases prognosis of cocaine body-packers surgical treatment in cocaine body packers and body pushers the altered pharmacokinetics and pharmacodynamics of drugs commonly used in critically ill patients position paper on urine alkalinization acute hypermagnesemia: a rare complication of antacid administration after bone marrow transplantation methods used to decrease lithium absorption or enhance elimination hypercalcemic crisis in pregnancy associated with excessive ingestion of calcium carbonate antacid (milk-alkali syndrome): successful treatment with hemodialysis hypermagnesemia-induced fatality following epsom salt gargles excessive calcium ingestion leading to milk-alkali syndrome pamidronate treatment of hypercalcemia caused by vitamin d toxicity lithium nephrotoxicity revisited forced euvolemic ciuresis with mannitol and furosemide for prevention of contrast-induced nephropathy in patients with ckd undergoing coronary angiography: a randomized controlled trial lithium poisoning: pharmacokinetics and clearance during different therapeutic measures treatment of life-threatening lithium toxicity with high-volume continuous venovenous hemofiltration clinical pharmacokinetics during continuous haemofiltration treatment of acute leukemia with amethopterin ( -amino, -methyl pteroyl glutamic acid) principles and techniques applied to enhance elimination successful treatment of valproic acid overdose with hemodialysis hemodialysis in the treatment of lactic acidosis in diabetics treated by metformin: a study of metformin elimination hemodialysis is as effective as hemoperfusion for drug removal in carbamazepine poisoning severe iron intoxication treated with exchange transfusion exchange transfusion in the treatment of severe theophylline poisoning exchange transfusion in severe infant salicylism acute poisoning with isoniazid treated by exchange transfusion exchange transfusion treatment in a newborn with phenobarbital intoxication vincristine overdose: experience with patients drug-induced methaemoglobinaemia successful whole blood exchange by apheresis in a patient with acute cyclosporine intoxication without long-term sequelae therapeutic approaches to ion channel diseases halothane modulation of skeletal muscle ryanodine receptors: dependence on ca + , mg + , and atp medical complications of psychiatric treatment the hunter serotonin toxicity criteria: simple and accurate diagnostic decision rules for serotonin toxicity the serotonin syndrome the serotonin syndrome toxin-induced hyperthermic syndromes a thyrotoxicosis outbreak due to dietary pills in paris thyrotoxicosis factitia in a post-aortocoronary bypass patient pediatric fatality following ingestion of dinitrophenol: postmortem identification of a "dietary supplement shortand long-term outcomes of heatstroke following the heat wave in lyon, france near-fatal heat stroke during the heat wave in chicago ambient temperature and mortality from unintentional cocaine overdose further studies of the role of hyperthermia in methamphetamine neurotoxicity heat stroke: clinical and chemical observations on cases cooling and hemodynamic management in heatstroke: practical recommendations whole-body cooling of hyperthermic runners: comparison of two field therapies emergency treatment of exertional heatstroke and comparison of whole body cooling techniques diazepam and chlormethiazole attenuate the development of hyperthermia in an animal model of the serotonin syndrome antagonism of cocaine, amphetamine, and methamphetamine toxicity effects of diltiazem on hyperthermia induced seizures in the rat pup dantrolene stabilizes domain interactions within the ryanodine receptor enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation r c is attenuated by dantrolene managing an effective treatment for neuroleptic malignant syndrome drug treatment of the neuroleptic malignant syndrome contrast medium-induced nephropathy: strategies for prevention acetaminophen hepatotoxicity: the first years acetaminophen bioactivation by human cytochrome p enzymes and animal microsomes acetylcysteine for acetaminophen poisoning mechanism of action of n-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo intravenous acetylcysteine in paracetamol induced fulminant hepatic failure: a prospective controlled trial improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure n-acetylcysteine improves indocyanine green extraction and oxygen transport during hepatic dysfunction n-acetylcysteine increases liver blood flow and improves liver function in septic shock patients: results of a prospective, randomized, double-blind study selective effects of n-acetylcysteine stereoisomers on hepatic glutathione and plasma sulfate in mice dissociation of increased sulfation from sulfate replenishment and hepatoprotection in acetaminophen-poisoned mice by n-acetylcysteine stereoisomers a patient-tailored n-acetylcysteine protocol for acute acetaminophen intoxication a prospective evaluation of shortened course oral n-acetylcysteine for the treatment of acute acetaminophen poisoning guidelines for the management of paracetamol poisoning in australia and new zealand -explanation and elaboration efficacy of oral n-acetylcysteine in the treatment of acetaminophen overdose. analysis of the national multicenter study ( to ) deaths from low dose paracetamol poisoning acetaminophen misconceptions risk factors and mechanisms of anaphylactoid reactions to acetylcysteine in acetaminophen overdose frequency of medication errors with intravenous acetylcysteine for acetaminophen overdose dilution of intravenous n-acetylcysteine as a cause of hyponatremia comparison of the -hour intravenous and -hour oral acetylcysteine protocols for the treatment of acute acetaminophen poisoning hepatotoxicity despite early administration of intravenous n-acetylcysteine for acute acetaminophen overdose acetaminophen overdose with altered acetaminophen pharmacokinetics and hepatotoxicity associated with premature cessation of intravenous n-acetylcysteine therapy pharmacokinetic effects of diphenhydramine or oxycodone in simulated acetaminophen overdose tylenol extended relief overdose on the characters, actions, and therapeutic uses of the bean of calabar the first use of physostigmine in the treatment of atropine poisoning. a translation of kleinwachter's paper entitled 'observations on the effect of calabar bean extract as an antidote to atropine poisoning comparative inhibitory effects of various physostigmine analogs against acetyl-and butyrylcholinesterases inhibition of acetylcholinesterase from electric eel by (-)-and (+)-physostigmine and related compounds accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions unconventional ligands and modulators of nicotinic receptors asystole complicating physostigmine treatment of tricyclic antidepressant overdose assessing physostigmine's contraindication in cyclic antidepressant ingestions mechanism of imipramine-induced seizures in amygdala-kindled rats failure of physostigmine in intoxications with tricyclic antidepres-drugs and pharmaceuticals: management of intoxication and antidotes sants in rats a comparison of physostigmine and benzodiazepines for the treatment of anticholinergic poisoning physostigmine: is there a role for this antidote in pediatric poisonings? detecting life-threatening lactic acidosis related to nucleosideanalog treatment of human immunodeficiency virus-infected patients, and treatment with l-carnitine supplementation with l-carnitine does not reduce the efficacy of epirubicin treatment in breast cancer cells science review: carnitine in the treatment of valproic acid-induced toxicity -what is the evidence? case files of the children's hospital of michigan regional poison control center: the use of carnitine for the management of acute valproic acid toxicity basic pharmacology of valproate: a review after years of clinical use for the treatment of epilepsy valproic acid metabolism and its effects on mitochondrial fatty acid oxidation: a review delayed valproic acid toxicity: a retrospective case series acute valproate poisoning: pharmacokinetics, alteration in fatty acid metabolism, and changes during therapy valproic acid toxicity: overview and management carnitine disposition before and during valproate therapy in patients with epilepsy involvement of recognition and interaction of carnitine transporter in the decrease of l-carnitine concentration induced by pivalic acid and valproic acid acute valproate-associated microvesicular steatosis: could the [ c]methionine breath test be useful to assess liver mitochondrial function? carnitine as an antidote for acute valproate toxicity in children l-carnitine supplementation in childhood epilepsy: current perspectives serum and muscle carnitine levels in epileptic children receiving sodium valproate vigabatrin, lamotrigine, topiramate and serum carnitine levels carnitine deficiency and hyperammonemia in children receiving valproic acid with and without other anticonvulsant drugs the effect of carnitine supplementation in valproateinduced hyperammonaemia diet-and valproate-induced transient hyperammonemia: effect of l-carnitine carnitine deficiency during valproic acid treatment valproic acid overdose and l-carnitine therapy a case of valproateassociated hepatotoxicity treated with l-carnitine valproate-associated hyperammonemic encephalopathy effect of l-carnitine treatment for valproate-induced hepatotoxicity the role of carnitine supplementation during valproic acid therapy valproic acid overdoses: a retrospective study comparing serum drug levels and the incidence of adverse outcomes l-carnitine was safely administered in the setting of valproate toxicity carni-tor ® (levocarnitine) oral solution ( g per ml multidose). carnitor ® sf (levocarnitine) sugar-free oral solution ( g per ml multidose) a case of hemoperfusion and l-carnitine management in valproic acid overdose emg changes in chronically dialyzed uraemic subjects undergoing d,l-carnitine treatment enhancement of activities relative to fatty acid oxidation in the liver of rats depleted of l-carnitine by d-carnitine and a γ-butyrobetaine hydroxylase inhibitor drug-induced hypoglycemia. a review of cases drug-induced disorders of glucose metabolism. mechanisms and management astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter brain glucose uptake and unawareness of hypoglycemia in patients with insulin-dependent diabetes mellitus neuroglycopenia and adrenergic responses to hypoglycaemia: insights from a local epidemic of serendipitous massive overdose of glibenclamide reversal of salicylate-induced euglycemic delirium with dextrose hypoglycemic seizures during transient hypoglycemia exacerbate hippocampal dysfunction the distribution of hypoglycemic brain damage concentrated dextrose for intravenous administration docking studies show that d-glucose and quercetin slide through the transporter glut peripheral intravenous infiltration necrosis the effect of grams i.v. glucose on serum inorganic phosphate levels sandostatin ® octreotide acetate injection prescribing information pharmacological agents that directly modulate insulin secretion oral hypoglycemic agents drugs and pharmaceuticals: management of intoxication and antidotes inhibition of insulin secretion as a new drug target in the treatment of metabolic disorders molecular signaling of somatostatin receptors effect of octreotide on insulin requirement, hepatic glucose production, growth hormone, glucagon and c-peptide levels in type diabetic patients with chronic renal failure or normal renal function octreotide for pediatric sulfonylurea poisoning a diabetic woman with worsening heart failure, hunger, and tremor effectiveness of octreotide in a case of refractory sulfonylureainduced hypoglycemia octreotide reverses hyperinsulinemia and prevents hypoglycemia induced by sulfonylurea overdoses octreotide: an antidote for sulfonylureainduced hypoglycemia comparison of octreotide and standard therapy versus standard therapy alone for the treatment of sulfonylurea-induced hypoglycemia the effectiveness of various doses of octreotide for sulfonylurea-induced hypoglycemia after overdose successful treatment of sulfonylureainduced prolonged hypoglycemia with use of octreotide octreotide for sulfonylurea-induced hypoglycemia following overdose anaphylactoid reaction to octreotide case files of the medical toxicology fellowship training program at the children's hospital of philadelphia: a pediatric exploratory sulfonylurea ingestion bradycardia and hyperkalemia associated with octreotide administration octreotide in children with hypoglycaemia due to sulfonylurea ingestion laparoscopic pancreatectomy for persistent hyperinsulinemic hypoglycemia of infancy octreotide therapy for persistent hyperinsulinemic hypoglycemia of infancy effectiveness of sms - , a synthetic, long-acting somatostatin analogue, in treatment of quinine-induced hyperinsulinaemia hypoglycaemia and counterregulatory hormone responses in severe falciparum malaria: treatment with sandostatin bench-to-bedside review: antidotal treatment of sulfonylurea-induced hypoglycaemia with octreotide somatostatin and somatostatin analogues: pharmacokinetics and pharmacodynamic effects glyburide sold as "street steroid" causes hypoglycemia complicated by inappropriate iv administration of octreotide long-term non-surgical therapy of severe persistent congenital hyperinsulinism with glucagon isoniazid-associated hepatitis. report of an outbreak therapeutic drug monitoring in the treatment of tuberculosis pyridoxine in clinical toxicology: a review acute isoniazid poisoning accidental isoniazid poisoning -a report the origin and turnover of d-serine in brain glutamatergic regulation of serine racemase via reversal of pip inhibition the effect on gaba metabolism in brain of isonicotinic acid hydrazide and pyridoxine as a fuction of time after administration profound acidosis caused by isoniazid ingestion convulsions as the etiology of lactic acidosis in acute isoniazid toxicity in dogs single high-dose pyridoxine treatment for isoniazid overdose isoniazid self-poisoning fitzpatrick's dermatology in general medicine acute isoniazid poisoning in childhood reversal of prolonged isoniazid-induced coma by pyridoxine acute isoniazid neurotoxicity in an urban hospital isoniazid overdose: four case reports and review of the literature evaluation of diazepam and pyridoxine as antidotes to isoniazid intoxication in rats and dogs acute isoniazid toxicity and the need for adequate pyridoxine supplies isoniazid toxicity: a survey of pyridoxine availability sensory neuropathy from pyridoxine abuse. a new megavitamin syndrome treatment of refractory seizures in massive isoniazid overdose clinical practice guideline update: use of chemotherapy and radiation therapy protectants cytoprotective effects of amifostine in the treatment of childhood malignancies amifostine and glutathione prevent ifosfamide-and acrolein-induced hemorrhagic cystitis drugs and pharmaceuticals: management of intoxication and antidotes randomized placebo-controlled multicenter evaluation of diethyldithiocarbamate for chemoprotection against cisplatin-induced toxicities myeloid growth factors neumega ® [nu-meg<a] (oprelvekin) european oncology nursing society extravasation guidelines distributed by integrated commercialization solutions. manufactured by ben venue laboratories inc., and hameln pharmaceuticals gmbh for topotarget a/s. marketed by treatment of anthracycline extravasation with savene (dexrazoxane): results from two prospective clinical multicentre studies treatment of anthracycline extravasation with dexrazoxane -clinical experience dexrazoxane (totect): fda review and approval for the treatment of accidental extravasation following intravenous anthracycline chemotherapy management of anthracycline extravasation injuries topoisomerase iiα-dependent and -independent apoptotic effects of dexrazoxane and doxorubicin anthracycline extravasation: continue using dimethyl sulfoxide the response of acute leukemia to the administration of the folic acid antagonists, aminopterin and a-methopterin effect of methotrexate therapy upon choriocarcinoma and chorioadenoma immunosuppressant and cytotoxic drugs: unapproved uses or indications methotrexate in psoriasis: consensus conference the use of methotrexate in rheumatoid arthritis carboxypeptidase g rescue in patients with methotrexate intoxication and renal failure iatrogenically-related, fatal methotrexate intoxication: a series of four cases high-dose methotrexate-induced nephrotoxicity in patients with osteosarcoma polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis mechanisms of action of methotrexate theoretically required urinary flow during high-dose methotrexate infusion methotrexate: clinical pharmacology, current status and therapeutic guidelines high-dose methotrexate with citrovorum factor rescue: predictive value of serum methotrexate concentrations and corrective measures to avert toxicity high-dose leucovorin as sole therapy for methotrexate toxicity threshold methotrexate concentration for in vivo inhibition of dna synthesis in normal and tumorous target tissues clinical pharmacokinetics of high-dose leucovorin calcium after intravenous and oral administration safety and efficacy of l-leucovorin rescue following high-dose methotrexate for osteosarcoma intrathecal leucovorin after intrathecal methotrexate overdose carboxypeptidase-g rescue in cancer patients with delayed methotrexate elimination after high-dose methotrexate therapy the reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides glucarpidase (carboxypeptidase g ) intervention in adult and elderly cancer patients with renal dysfunction and delayed methotrexate elimination after high-dose methotrexate therapy treatment of accidental intrathecal methotrexate overdose with intrathecal carboxypeptidase g understanding and managing methotrexate nephrotoxicity carboxypeptidase g: purification and properties enzymatic cleavage of methotrexate provides a method for prevention of drug toxicity comparative effects of citrovorum factor and carboxypeptidase g on cerebrospinal fluid-methotrexate pharmacokinetics hemodialysis and enzymatic cleavage of methotrexate in man methotrexate pharmacokinetics following administration of recombinant carboxypeptidase-g in rhesus monkeys rescue of experimental intrathecal methotrexate overdose with carboxypeptidase-g successful carboxypeptidase g rescue in delayed methotrexate elimination due to renal failure successful treatment of intrathecal methotrexate overdose by using ventriculolumbar perfusion and intrathecal instillation of carboxypeptidase g optimal management of methotrexate intoxication in a child with osteosarcoma drugs and pharmaceuticals: management of intoxication and antidotes crystal structure of carboxypeptidase g , a bacterial enzyme with applications in cancer therapy pharmacokinetics and metabolism of the methotrexate metabolite , -diamino-n -methylpteroic acid carboxypeptidase-g rescue in a patient with high dose methotrexate-induced nephrotoxicity carboxypeptidase-g , thymidine, and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction favorable outcome in excessive methotrexate (mtx) intoxication after high-dose (hd) mtx therapy by early use of carboxypeptidase g (cpg ) carboxypeptidase g rescue after high-dose methotrexate comparative effects of citrovorum factor and carboxypeptidase g on cerebrospinal fluid-methotrexate pharmacokinetics treatment of accidental intrathecal methotrexate overdose with intrathecal carboxypeptidase g interactions of carboxypeptidase g with s-leucovorin and r-leucovorin in vitro: implications for the application in case of methotrexate intoxications interference of -hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays pre-authorisation evaluation of medicines for human use. withdrawal assessment report for voraxaze. emea/chmp/ / purification and properties of carboxypeptidase g from pseudomonas sp. strain rs- . use of a novel triazine dye affinity method tox-acls: toxicologic-oriented advanced cardiac life support pharmacology and management of the vitamin k antagonists: american college of chest physicians evidence-based clinical practice guidelines treatment and prevention of heparininduced thrombocytopenia: american college of chest physicians evidence-based clinical practice guidelines pharmacotherapy considerations in advanced cardiac life support muscarinic receptors in the mammalian heart calcium channel blocking drug overdose: an australian series management of β-adrenergic blocker and calcium channel antagonist toxicity a fatal case of iatrogenic hypercalcemia after calcium channel blocker overdose management of calcium channel antagonist overdose calcium gluconate injection, usp % assessment of the synergistic relationship between serum calcium and digitalis case files of the medical toxicology fellowship of the california poison control system-san francisco: calcium plus digoxin-more taboo than toxic treatment of severely digitalis-toxic patients with digoxin-specific antibody fragments reversal of advanced digoxin intoxication with fab fragments of digoxin-specific antibodies treatment of cases of life-threatening digitalis intoxication with digoxin-specific fab antibody fragments. final report of a multicenter study digoxin-specific fab fragments as single first-line therapy in digitalis poisoning the use of digoxin-specific fab fragments for severe digitalis intoxication in children review of clinical experience with digoxin immune fab (ovine) digoxin-specific antibody fragments: how much and when? pharmacokinetic aspects of digoxin-specific fab therapy in the management of digitalis toxicity late rebound digoxin toxicity after digoxin-specific antibody fab fragments therapy in anuric patient recurrent digoxin overdose and treatment with digoxin-specific fab antibody fragments successful treatment of oleander intoxication (cardiac glycosides) with digoxin-specific fab antibody fragments in a -year-old child: case report and review of literature efficacy of digoxin specific fab fragments (digibind) in the treatment of toad venom poisoning benefits and limitations of reducing glucagon action for the treatment of type diabetes information for the physician. glucagon for injection (rdna origin) treatment of poisoning caused by β-adrenergic and calcium-channel blockers arachidonic acid drives mini-glucagon action in cardiac cells cardiovascular effects of glucagon in man towards evidence based emergency medicine: best bets from the manchester royal infirmary. glucagon for the treatment of symptomatic β blocker overdose glucagon use in symptomatic β blocker overdose treatment of verapamil overdose with glucagon in dogs amelioration of nifedipine poisoning associated with glucagon therapy glucagon antagonism of calcium channel blockerinduced myocardial dysfunction myocardial metabolism dur-drugs and pharmaceuticals: management of intoxication and antidotes ing graded intraportal verapamil infusion in awake dogs beneficial myocardial metabolic effects of insulin during verapamil toxicity in the anesthetized canine myocardial lactate release after intracoronary verapamil application in humans: acute effects of intracoronary verapamil on systemic and coronary hemodynamics, myocardial metabolism, and norepinephrine levels effect of glucose-insulinpotassium infusion on mortality in critical care settings: a systematic review and meta-analysis forty years of glucose-insulin-potassium (gik) in cardiac surgery: a review of randomized, controlled trials anti-inflammatory effect of high-dose insulin treatment after urgent coronary revascularization surgery therapy with insulin in cardiac surgery: controversies and possible solutions high-dose insulin improves the efficacy of dopamine early after cardiac surgery. a study of myocardial performance and oxygen consumption insulin improves heart function and metabolism during non-ischemic cardiogenic shock in awake canines insulin improves survival in a canine model of acute β-blocker toxicity insulin versus vasopressin and epinephrine to treat β-blocker toxicity relative safety of hyperinsulinaemia/euglycaemia therapy in the management of calcium channel blocker overdose: a prospective observational study tarka(r) (trandolapril/verapamil hydrochloride extended-release) overdose the effect of insulin dose on the measurement of insulin sensitivity by the minimal model technique. evidence for saturable insulin transport in humans interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals dose-response characteristics of insulin action on glucose metabolism: a non-steady-state approach levobupivacaine-induced seizures and cardiovascular collapse treated with intralipid cardiac resuscitation after incremental overdosage with lidocaine, bupivacaine, levobupivacaine, and ropivacaine in anesthetized dogs lipid emulsion infusion rescues dogs from bupivacaine-induced cardiac toxicity resuscitation with lipid versus epinephrine in a rat model of bupivacaine overdose intravenous lipid infusion in the successful resuscitation of local anesthetic-induced cardiovascular collapse after supraclavicular brachial plexus block successful resuscitation of a patient with ropivacaineinduced asystole after axillary plexus block using lipid infusion reversal of central nervous system and cardiac toxicity after local anesthetic intoxication by lipid emulsion injection successful resuscitation after ropivacaine and lidocaine-induced ventricular arrhythmia following posterior lumbar plexus block in a child early treatment of a quetiapine and sertraline overdose with intralipid use of lipid emulsion in the resuscitation of a patient with prolonged cardiovascular collapse after overdose of bupropion and lamotrigine intralipid outperforms sodium bicarbonate in a rabbit model of clomipramine toxicity intralipid infusion ameliorates propranolol-induced hypotension in rabbits hemodynamic effects of intravenous fat emulsion in an animal model of severe verapamil toxicity resuscitated with atropine, calcium, and saline intralipid ameliorates thiopentone induced respiratory depression in rats: investigative pilot study non-esterified fatty acids increase arterial pressure via central sympathetic activation in humans the effects of lipid infusion on myocardial function and bioenergetics in l-bupivacaine toxicity in the isolated rat heart the association of anaesthetists of great britain & ireland ( ) guidelines for the management of severe local anaesthetic toxicity intravenous lipid emulsion for local anesthetic toxicity: a review of the literature recurrence of cardiotoxicity after lipid rescue from bupivacaine-induced cardiac arrest intralipid infusion diminishes return of spontaneous circulation after hypoxic cardiac arrest in rabbits a comparison of the combination of epinephrine and vasopressin with lipid emulsion in a porcine model of asphyxial cardiac arrest after intravenous injection of bupivacaine binding of long-lasting local anesthetics to lipid emulsions in-hospital cardiac arrest is associated with use of non-antiarrhythmic qtc-prolonging drugs current concepts in the mechanisms and management of drug-induced qt prolongation and torsade de pointes novel therapeutics for treatment of long-qt syndrome and torsade de pointes mechanism of reversal of toxic effects of amitriptyline on cardiac purkinje fibers by sodium bicarbonate experimental amitriptyline intoxication: treatment of cardiac toxicity with sodium bicarbonate tricyclic antidepressant overdosage: experimental studies on the management of circulatory complications value of the qrs duration versus the serum drug level in predicting seizures and ventricular arrhythmias after an acute overdose of tricyclic antidepressants electrocardiographic criteria for tricyclic antidepressant cardiotoxicity drugs and pharmaceuticals: management of intoxication and antidotes ecg lead avr versus qrs interval in predicting seizures and arrhythmias in acute tricyclic antidepressant toxicity effects of high dose α- -acid glycoprotein on desipramine toxicity in rats cardiovascular repercussions of seizures during cyclic antidepressant poisoning epinephrine and sodium bicarbonate independently and additively increase survival in experimental amitriptyline poisoning improvement of cardiac conduction after hyperventilation in tricyclic antidepressant overdose experimental tricyclic antidepressant toxicity: a randomized, controlled comparison of hypertonic saline solution, sodium bicarbonate, and hyperventilation reversal of severe tricyclic antidepressant-induced cardiotoxicity with intravenous hypertonic saline solution sodium bicarbonate treatment for tricyclic antidepressant arrhythmias in children tricyclic antidepressant poisoning: an evidence-based consensus guideline for out-of-hospital management diphenhydramine-induced wide complex dysrhythmia responds to treatment with sodium bicarbonate propoxyphene-induced wide qrs complex dysrhythmia responsive to sodium bicarbonate -a case report case files of the medical toxicology fellowship at the toxikon consortium in chicago: cocaine-associated wide-complex dysrhythmias and cardiac arrest -treatment nuances and controversies acute carbamazepine toxicity associated with a widened qrs interval treated with intravenous sodium bicarbonate bupropion-associated qrs prolongation unresponsive to sodium bicarbonate therapy a fatal case of venlafaxine overdose self-poisoning with lamotrigine management of isolated yew berry toxicity with sodium bicarbonate: a case report in treatment efficacy cardiotoxicity after massive amantadine overdose naloxone in opioid poisoning: walking the tightrope a randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. results of the second national acute spinal cord injury study adverse events after naloxone treatment of episodes of suspected acute opioid overdose narcotic reversal in hypercapnic dogs: comparison of naloxone and nalbuphine the ilcor consensus on science with treatment recommendations for pediatric and neonatal patients: neonatal resuscitation cardiac arrest following naloxone in an extremely preterm neonate the empiric use of naloxone in patients with altered mental status: a reappraisal a dosing nomogram for continuous infusion intravenous naloxone adverse effects in children after unintentional buprenorphine exposure naloxone reversal of buprenorphine-induced respiratory depression accidental clonidine patch ingestion in a child clonidine toxicity revisited naloxone reversal of hypotension due to captopril overdose profound prolonged hypotension following captopril overdose attenuation of the antihypertensive effect of captopril by the opioid receptor antagonist naloxone effect of naloxone on the actions of captopril naloxone does not modify the antihypertensive effect of captopril in essential hypertensive patients molecular structure and stereoelectronic properties of sarmazenil -a weak inverse agonist at the omega modulatory sites (benzodiazepine receptors): comparison with bretazenil and flumazenil flumazenil: us clinical pharmacology studies clinical pharmacology of flumazenil sedative and ventilatory effects of midazolam infusion: effect of flumazenil reversal the effects of large-dose flumazenil on midazolam-induced ventilatory depression effect of flumazenil on benzodiazepineinduced respiratory depression effect of flumazenil on bispectral index monitoring in unpremedicated patients electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action characterization of the analgesic effects of the benzodiazepine antagonist, ro - empiric use of flumazenil in comatose patients: limited applicability of criteria to define low risk use of flumazenil in the treatment of drug overdose: a double-blind and open clinical study in patients flumazenil and seizures: analysis of cases treatment of benzodiazepine overdose with flumazenil. the flumazenil in benzodiazepine intoxication multicenter study group a risk-benefit assessment of flumazenil in the management of benzodiazepine overdose valproic acid and metabolites: pharmacological and toxicological studies the author is indebted to lewis s. nelson, md, facep, facmt for manuscript review. key: cord- -ni iyzdn authors: he, zhisong; zhang, jian; shi, xiao-he; hu, le-le; kong, xiangyin; cai, yu-dong; chou, kuo-chen title: predicting drug-target interaction networks based on functional groups and biological features date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ni iyzdn background: study of drug-target interaction networks is an important topic for drug development. it is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. as a complement, the in silico prediction methods can provide us with very useful information in a timely manner. methods/principal findings: to realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. the optimal feature selection procedures are adopted by means of the mrmr (maximum relevance minimum redundancy) method. instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, g-protein- coupled receptors and nuclear receptors. thus, four independent predictors are established using the nearest neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. as a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are . %, . %, . %, and . %, respectively. conclusion/significance: our results indicate that the network prediction system thus established is quite promising and encouraging. identification of drug-target interaction networks is an essential step in the drug discovery pipeline [ ] . the emergence of molecular medicine and the completion of the human genome project provide more opportunity to discover unknown target proteins of drugs. many efforts have been made to discover new drugs in the past few years. however, the number of new drug approvals remains quite low (around only per year). this is partially because many compounds or drug candidates have to be withdrawn owing to unacceptable toxicity. such failures have wasted a lot of money. it would be beneficial to develop computational methods for predicting the sensitivity and toxicity before a drug candidate was synthesized [ , , ] . however, a number of problems need to be overcome in order to find out the exact effects of a drug. firstly, drugs could have numerous effects including positive and negative effects, and it is hard to find out and elucidate the possible effects; secondly, different people would have completely different responses to a drug even though the same gene products are only slightly different [ , , , ] ; thirdly, it is very hard to trace the drug effects since the biological interaction pathways are extremely complicated in human beings. therefore, it would be very helpful for drug development if the interactions between drugs and target proteins could be predicted more accurately and the underlying mechanisms could be better understood. several computational approaches have been developed for analyzing and predicting drug-protein interactions. the most commonly used are docking simulations [ , , , ] , literature text mining [ ] , and combining chemical structure, genomic sequence, and d structure information [ ] , among others (see, e.g., [ , , ] ). machine learning and data mining methods have been widely used in the computational biology and bioinformatics area. many researchers have made lots of efforts to develop useful algorithms and softwares to investigate various drug-related biological problems, such as hiv protease cleavage site prediction [ , ] , identification of gpcr (g protein-coupled receptors) type [ , ] , protein signal peptide prediction [ ] , protein subcellular location prediction [ , , ] , analysis of specificity of galnac-transferase protein [ ] , identification of protease type [ , ] , membrane protein type prediction [ , , , ] , and a series of relevant webserver predictors as summarized in a recent review [ ] . here we propose a predictor for drug-target interactions based on the nearest neighbor algorithm [ ] . since biochemical and physicochemical features [ ] are important for characterizing proteins, in this study they are used to represent proteins as done by many previous investigators (see, e.g., [ , , ] . to improve the predictor's performance, minimum redundancy maximum relevance (mrmr) algorithm [ ] is used to rank the features. meanwhile, the incremental feature selection and forward feature selection are applied for feature selection. the protein targets for drugs are divided into enzymes, ion channels [ , , , ] , gpcrs [ , ] , and nuclear receptors [ ] in this study. finally, four predictors for predicting the interactions of drugs with each of the four protein families are developed in hopes that they can help provide useful information for drug design. in addition to the dataset used by yamanishi et al. [ ] , information about drug compounds and genes can be obtained from kegg [ , ] by the ftp operations: ftp://ftp.genome.jp/ pub/kegg/ligand/drug/drug for the drugs, and ftp://ftp.genome. jp/pub/kegg/genes/fasta/gene.pep for the genes. after excluding the drug-target pairs that lack experimental information, we finally obtained a total of , drug-target pairs, of which , for enzymes, , for ion channels, for gpcrs, and for nuclear receptors. all these datasets were used as the positive datasets in the current study. the corresponding negative datasets were derived from the above positive datasets via the following steps: ( ) separate the pairs in the above positive dataset into single drugs and proteins; ( ) re-couple these singles into pairs in a way that none of them occurs in the corresponding positive dataset; ( ) randomly picked the negative pairs thus formed until they reached the number two times as many as the positive pairs. the drug-target benchmark datasets thus obtained for enzymes, ion-channels, gpcrs, and nuclear receptors are given in online supporting information s , s , s , and s , respectively. representing drugs with chemical functional groups composition. the number of drugs is extremely large. however, most of them are small organic molecules and are composed of some fixed small structures, called functional groups. since functional groups usually represent the characteristics of a compound as well as its reaction mechanism with other molecules, features derived from its functional groups could be very effective in characterizing a drug. moreover, the number of common functional groups is quite small, and hence it is possible to use the functional group composition to uniquely represent a drug [ ] . a number of functional groups are available in nature, and we selected the following common groups for the current study: ( ) alcohol, ( ) aldehyde, ( ) amide, ( ) amine, ( ) hydroxamic acid, ( ) phosphorus, ( ) carboxylate, ( ) methyl, ( ) ester, ( ) ether, ( ) imine, ( ) ketone, ( ) nitro, ( ) halogen, ( ) thiol, ( ) sulfonic acid, ( ) sulfone, ( ) sulfonamide, ( ) sulfoxide, ( ) sulfide, ( ) a_ c_ring, ( ) ar_ c_ring, ( ) non_ar_ c_ring, ( ) non_ar_ c_ring, ( ) hetero ar_ _ring, ( ) hetero non_ar_ _ring, ( ) hetero non_ar_ _ring, and ( ) hetero ar_ _ring. thus, following the same treatment as in [ ] , a drug compound can now be formulated as a -d (dimensional) vector given below: where g i (i~ , , Á Á Á , ) is the occurrence frequency of the i-th functional group in the drug d, and t the matrix transpose operator. representing target proteins with pseudo amino acid composition by incorporating biochemical and physicochemical features. now the problem is how to effectively represent a target protein. two kinds of representations are generally used in this regard: the sequential representation and the non-sequential representation. the most typical sequential representation for a protein sample is its entire amino acid sequence, which can contain the most complete information of a protein. to deal with this model, the sequence-similarity-searchbased tools, such as blast [ ] , are usually used to find the desired results. unfortunately, this kind of approach failed to work when the query protein did not have significant homology to the proteins in the training dataset. thus, various non-sequential representations or discrete models were proposed. the simplest discrete model was based on the amino acid composition (aac) (see, e.g., [ ] ). however, if using the aac model to represent a protein, all its sequence-order information will be lost. to avoid completely losing the sequence-order information, the pseudo amino acid composition (pse-aac) was proposed [ ] to represent the sample of a protein. the pseaac can be used to represent a protein sequence with a discrete model yet without completely losing its sequence-order information. for further information about pseaac, see the web-page by clicking the link http://en. wikipedia.org/wiki/pseudo_amino_acid_composition. ever since the concept of pseaac was introduced, it has been widely used to study various problems in proteins and protein-related systems (see, e.g., [ , , , , , , , , , , , , , , , , ] ). meanwhile, many different forms of discrete models were also proposed (see, e.g., [ , , , , , , , , , , , , , , , , , , , ] ). however, regardless of how much different these models are, they just belong to different forms of pseaac, as elucidated in a recent comprehensive review [ ] . here, we are to propose a different pseaac to represent drug-targeted proteins in terms of their biochemical and physicochemical features [ ] . six different types of features were considered: ( ) hydrophobicity, ( ) polarizability, ( ) polarity, ( ) secondary structure, ( ) normalized van der waals volume, and ( ) solvent accessibility. each amino acid residue in a protein sequence can be represented by a set of different states according to its features. for instance, its hydrophobicity feature can be marked by one of the following three states: ''polar'', ''neutral'', or ''hydrophobic'' [ ] ; its solvent accessibility feature by one of the two: ''buried'' or ''exposed to solvent'', as predicted by predacc [ ] ; its secondary structure feature by one of the three: ''helix'', ''sheet'', or ''coil'', as predicted by the method in [ ] ; and so forth. thus, a protein sequence can be translated to a series of codes according to the biochemical and physicochemical properties of its constituent amino acid residues. for example, if using ''p'', ''n'' and ''h'' to represent the three states of hydrophobicity: ''polar'', ''neutral'', and ''hydrophobic'', the protein sequence ''dmaeimsdkp-qagml'' can be translated to ''phnphhnppnpnnhh'' according to the codes of the hydrophobic property feature. the encoded sequences thus obtained would have different length for proteins of different sizes, which will make the prediction engine difficult to handle. to make the feature-encoded sequence to be a vector with a fixed number of dimensions, three properties of a sequence was used: composition (c), transition (t), and distribution (d). c represents the global composition of each letter in the sequence; t, the frequency of a code letter changing from one to another; d, the distribution pattern of the code letters along the sequence, measuring the percentage of the sequence length within which the first, %, %, %, and % of the amino acids of each code letter is located. take the above hydrophobic property sequence as an example: its c feature is / = . % for all of p, h, and n, while the t feature is / = %, / = % and / = % for the changes between h and p, n and h, n and p, respectively. the measurement of feature d is a little more complicated. for the letter h, the first, %, %, % and % of hs in the sequence is located at the position of , , , , and . thus its d feature is ( , with a total of components. likewise, for the sequences encoded by the other four biochemical properties, each is also corresponding to components. but for the sequence encoded by the solvent accessibility with only two states (''buried'' or ''exposed to solvent''), the encoded sequence is corresponding to only components. finally, by adding the components of aac [ ] into the vector concerned, the total number of components thus obtained for a given protein is | z z ~ ; i.e., the protein can be formulated as a -d vector given by where p i (i~ , , Á Á Á , ) is the i-th component of the protein p. of the components, are derived according to the codes of the above six biochemical and physicochemical features, and are the aac components of p. with all samples represented by a feature vector, now it is possible for us to construct our predictor using the machine learning approach. the nn (nearest neighbor) algorithm is quite popular in pattern recognition community owing to its good performance and simple-to-use feature. according to the nn rule [ ] , the query sample should be assigned to the subset represented by its nearest neighbor. in this study, if the drug-target pair with the shortest distance is a positive sample, meaning that they can interact with each other, the sample for test is seen as a positive drug-target pair. otherwise, the test sample is seen as a negative one. there are many different definitions to measure the ''nearness'' for the nn algorithm, such as euclidean distance, hamming distance [ ] , and mahalanobis distance [ , , ] . in the current study, the following equation was adopted to measure the nearness between samples v x and v y where v x : v y is the dot product of the two vectors, and v x k k and v y their modulus, respectively. when v x :v y we have d(v x ,v y )~ , indicating the ''distance'' between these two sample vectors is zero and hence they have perfect or % similarity. after constructing the drug-target interaction predictor, we have to evaluate its performance. in statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (k-fold cross-validation) test, and jackknife test [ ] . however, as elucidated by [ ] and demonstrated by eq. in [ ] , among the three cross-validation methods, the jackknife test is deemed the most objective that can always yield a unique result for a given benchmark dataset, and hence has been increasingly used and widely recognized by investigators to examine the accuracy of various predictors (see, e.g. [ , , , , , , , , , , , , ] ).'' accordingly, in this study the jackknife cross-validation was adopted to calculate the success prediction rates as well. although we've constructed the drug-target predictor based on the original feature set described above, it is possible to improve its performance with a better feature set. apparently, not every feature in the feature set is equally relevant to the drug-target interaction. what's more, features may not be independent with each other. the ''bad'' will have negative impact on the accuracy and efficiency of the predictor, so it is possible to do the feature selection process to construct a more compact and effective feature set. the first step is using maximum relevance minimum redundancy (mrmr) [ ] to do feature evaluation. maximum relevance minimum redundancy (mrmr) [ ] was firstly developed for analysis of microarray data. it ranks each feature according to its relevance to the target and redundancy to other features. the better a feature is deemed to be, the higher the rank it will be assigned to. mutual information (mi), denoted by i to indicate the dependence of two features used to quantify the relevance and redundancy. mi is defined as following: based on mi, we can quantify relevance (d) and redundancy (r) as: where f candidate is the feature to be calculated, and c is the target variable. by combining the above two equations to maximize relevance and minimize redundancy, the following mrmr function is constructed: where v s and v t are the already-selected feature set and to-beselected feature set, respectively, and m and n are the sizes of these two feature sets, respectively. the earlier a feature is selected, the better it would be though of. finally, we can get an ordered feature list with a rank for every feature to indicate its importance in the feature set. in our study, the mrmr program is obtained from: http://research. janelia.org/peng/proj/mrmr/index.htm. to calculate mi, the joint probabilistic density and the marginal probabilistic densities of the two vectors were used. a parameter t is introduced here to deal with these variables. suppose mean to be the average value of one feature in all samples, and std to be the standard deviation, the feature of each sample would be classified into one of the three groups according to the boundaries: mean+(t : std). in our study, t was assigned to be . as mentioned above, the importance of each feature is rated according to its rank in the mrmr analysis. the next step is to determine which features should be selected as the optimal feature set for our drug-target predictor. here the ifs (incremental feature selection) procedure is used to solve the problem. each feature in the mrmr feature list was added one by one, and n different feature sets are obtained if the total feature number is n, while the i-th feature set is: based on each of the n feature sets, an nn algorithm predictor was constructed and tested with the jackknife cross-validation test. with all the n overall accurate rates calculated, we could draw an ifs curve with the index i to be the x-axis and the corresponding overall accurate rate to be the y-axis. thus, s opt~f f , f , :::, f n g is regarded as the optimal feature set if the curve reach its peak where the value of its x-axis is nƒn. because four independent predictors are needed for the four different classes of drug-target pairs, the ifs analysis procedure will be processed four times with each for a specific predictor. to refine feature selection, the ffs (forward feature selection) procedure based on the result of ifs was used. ffs is a feature selection method based on ifs results which tries every feature in the candidate feature set and adds the feature that achieves the highest prediction accuracy into the already-selected feature set in each goes. suppose the ifs curve reaches its peak with apex as its x-axis, the initial ffs-selected feature set was constructed as: more features in ffs-to-be-selected feature set would be added into the ffs-selected feature set one by one. the ffs-to-beselected feature set with m features covers the features with mrmr ranks between k+ and k+ +m, where m is a user-defined positive integer smaller than n{k with n to be the size of the original feature set. in each round of ffs, each feature in ffs-tobe-selected feature set would be taken out and added to the ffsselected feature set. each predictor based on each new ffsselected feature set would be tested, and the feature set obtained the highest overall accurate rate would be used as the new ffsselected feature set. this process would be run for m times, until the ffs-to-be-selected feature set becomes a null set. an ffs curve similar to the ifs curve could be drawn with x-axis as the index and y-axis as the overall accurate rate. in this study, ffs was run for each of the four benchmark datasets based on the corresponding ifs result. m for all these processes was set to , while k for each ffs was set to be the index of the point with the first maximum value (i.e. the maximum point with the smallest index) in the corresponding ifs curve. to improve performance of the predictor of drug-target interaction, feature selection process was carried out. the first step of feature selection is feature evaluation. in this study, mrmr was used to evaluate every feature in original feature set. listed in online supporting information s are two kinds of outputs: the first one is the maxrel list which shows ranks of features for their relevance to the target; the second is mrmr list showing the mrmr ranks according to the feature order satisfying eq. . in this study, only the mrmr list was used as the results of feature evaluation. since there are four groups of samples, mrmr was run four times with each for one of them. with the four mrmr lists, ifs was processed for each of the four sample groups, generating four ifs curves. based on these results, we set k in ffs to be , , and for the data of enzymes, ion channels, gpcrs and nuclear receptors, respectively. each of these figures is the index of the point of the first maximum value in the corresponding ifs curve. shown in fig. are the four ifs curves with their corresponding ffs curves. the peaks of the four ffs curves finally reach the overall success rates of . % with features, . % with features, . % with features, and . % with features for enzyme group, ion channel group, gpcr group and nuclear receptor groups, respectively. features selected by mrmr+ffs for the four different groups are quite different from each other, showing the intrinsic differences between them. although there are more features for target than those for drug in the original feature set, more drug features were selected, showing the important role of drugs. many of the selected target features are for protein secondary structure, especially for enzyme group (half of selected target features are for this). all types of features are selected in at least one group, showing that all biochemical and physicochemical features have their irreplaceable positions in drug-target interaction process. for the details of the optimal feature-set outputs by ffs for the four benchmark datasets, see the online supporting information s . for the specificity and promiscuity, we divided the drug-protein interactions into four groups according to the targets of drugs: enzymes, ion channels, gpcrs, and nuclear receptors. we used all the known drugs and target proteins in the gold standard data as training data to predict the potential interactions between all human proteins annotated as members of the four classes in kegg genes and all compounds in kegg ligands. enzyme recognition is the primary event involved in the interaction of proteins with other proteins and with small molecules such as metabolites and therapeutics. predicting drugenzyme interactions has direct application for completing genome annotations, finding enzymes for synthetic chemistry, and predicting drug specificity, promiscuity and pharmacology. it is suggested that the secondary structure information plays the major role in determining the drug-enzyme interactions activity. for example, cytochrome p (cyp) induction-mediated interaction is one of the major concerns in clinical practice and for the pharmaceutical industry [ ] . induction of cyp a enzymes with a specific structure-stable state may activate some xenobiotics to their reactive metabolites, leading to toxicity [ , ] . amino acid composition and hydrophobicity also contribute considerably to these interactions. an insertion/deletion (i/d) polymorphism of the angiotensin i-converting enzyme (ace) have an influence on the antihypertensive response, particularly when using ace inhibitors (acei) [ ] , mirroring that the amino acid composition did contribute to the interactions. hydrophobicity plays a role in determining the coefficients of drug-enzyme interaction energy with the application to drug screening as well as in silico target protein screening [ , ] . the g-protein coupled receptor (gpcr) superfamily, which is comprised of estimated - , members, is another largest known class of molecular targets with varieties of physiological activities and proven therapeutic value [ ] . they are integral membrane proteins sharing a common global topology that consists of seven transmembrane alpha helices, intracellular cterminal, an extracellular n-terminal, three intracellular loops and three extracellular loops [ , ] . it is suggested that secondary structure and polarity would play a major role in determining the drug-gpcrs interactions activity. small secondary structures such as helices and loops are identified as entities potentially involved in stabilizing interactions with ligands [ ] . these motifs were situated mainly in the apical region of transmembrane segments and included a few extracellular residues [ ] . crystal structures of engineered human beta -adrenergic receptors (ars) in complex with an inverse agonist ligand, carazolol, provide threedimensional snapshots of an important g protein-coupled receptor (gpcr) with a beta-sheet structure and forms part of the chromophore-binding site [ ] . glida provides interaction data between gpcrs and their ligands, along with chemical information on the ligands, as well as biological information regarding gpcrs [ ] . some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane, the formation of extensive networks of interhelical h-bonds and sulfur-aromatic clusters that are spatially organized as ''polarity'', the close packing of side-chains throughout the transmembrane domain. when more experimental d structures become available for gpcrs in the future, this will help building reliable models for a wider range of gpcrs that would be suitable for docking studies. joint use of ligand-based chemogenomic and docking would certainly improve the prediction. ion channels are a large superfamily of membrane proteins that pass ions across membranes and are critical to diverse physiological functions in both excitable and nonexcitable cells and underlie many diseases. as a result, they are an important target class which is proven to be highly ''druggable''. according to our analysis, secondary structure and polarity play the major role in determining the drug-ion channels interactions activity. secondary structure controls the membrane potential and interrogates ion channels in different conformational states. the drug-ion channels interaction needs gated state where they can switch conformation between a closed and an open state [ , ] . simulations on model nanopores reveal that a narrow hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small increase in pore radius or an increase in polarity [ , ] . nowadays, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction [ ] . nuclear receptors (nr) are ligand-activated transcription factors that regulate the activation of a variety of important target genes, which are the most important drug targets in terms of potential therapeutic application. according to our results, secondary structure and polarizability play the major role in determining the drug-nrs interactions. the conservative motif of the nr is typically described as three stacked alpha-helical sheets. the helices that make up the ''front'' and ''back'' sheets are aligned parallel to one another. the helices in the middle sheet run across the two outer sheets and only occupy the space in the upper portion of the domain. the space in the lower part of the domain is relatively void of protein, and for most nrs, this creates an internal cavity for small-molecule ligands [ ] . hydrogen bonds with polarizability activity play a crucial role in protein-drug interactions (see, e.g., [ ] ). our approaches and the results thus obtained could be used to demonstrate how nuclear hormone receptors form a network of direct interactions. and this network increases in complexity to describe the interactions with target genes as well as small molecules known to bind a receptor, enzyme, or transporter. a comprehensive drug-target interaction network system has been established that contains four classifiers for predicting the drugable interaction of compounds with enzymes, ion-channels, gpcrs, and nuclear receptors, respectively. it is anticipated that the network predictor system may become a very useful tool for drug development. particularly it may help us find new or potential drug-target interactions. online supporting information s the benchmark dataset for the drug-target enzyme interaction system. it contains , genedrug pair samples, of which , are positive and , negative. the st column of the table indicates the nature of samples with for positive and for negative; the nd column shows the code of target gene; and the rd column shows the code of drug. all the detailed information for the genes and drugs listed here can be found in a guide to drug discovery: target selection in drug discovery predicting human safety: screening and computational approaches assessment of chemical libraries for their druggability review: progress in computational approach to drug development against sars d structure modeling of cytochrome p c and its implication for personalized drug design molecular modeling of two cyp c snps and its implications for personalized drug design review: pharmacogenomics and personalized use of drugs review: structure of cytochrome p s and personalized drug structure-based maximal affinity model predicts small-molecule druggability a fast flexible docking method using an incremental construction algorithm review: structural bioinformatics and its impact to biomedical science binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars a probabilistic model for mining implicit 'chemical compound-gene' relations from literature prediction of drug-target interaction networks from the integration of chemical and genomic spaces statistical prediction of protein chemical interactions based on chemical structure and mass spectrometry data integrating statistical predictions and experimental verifications for enhancing protein-chemical interaction predictions in virtual screening alignment-free prediction of a drug-target complex network based on parameters of drug connectivity and protein sequence of receptors a vectorized sequence-coupling model for predicting hiv protease cleavage sites in proteins review: prediction of hiv protease cleavage sites in proteins gpcr-ca: a cellular automaton image approach for predicting g-protein-coupled receptor functional classes gpcr-gia: a web-server for identifying gprotein coupled receptors and their families with grey incidence analysis signal-cf: a subsite-coupled and window-fusing approach for predicting signal peptides using functional domain composition and support vector machines for prediction of protein subcellular location cell-ploc: a package of web-servers for predicting subcellular localization of proteins in various organisms euk-mploc: a fusion classifier for large-scale eukaryotic protein subcellular location prediction by incorporating multiple sites a sequence-coupled vector-projection model for predicting the specificity of galnac-transferase protident: a web server for identifying proteases and their types by fusing functional domain and sequential evolution information prediction of protease types in a hybridization space prediction of membrane protein types and subcellular locations low-frequency fourier spectrum for predicting membrane protein types memtype- l: a web server for predicting membrane proteins and their types by incorporating evolution information through pse-pssm support vector machines for predicting membrane protein types by using functional domain composition review: recent advances in developing web-servers for predicting protein attributes a k-nearest neighbor classification rule based on dempster-shafer theory predacc: prediction of solvent accessibility prediction of protein cellular attributes using pseudo amino acid composition using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes digital coding of amino acids based on hydrophobic index feature selection based on mutual information: criteria of max-dependency, max-relevance, and min-redundancy insights from modelling three-dimensional structures of the human potassium and sodium channels the structure of phospholamban pentamer reveals a channel-like architecture in membranes mechanism of drug inhibition and drug resistance of influenza a m channel structure and mechanism of the m proton channel of influenza a virus prediction of g-protein-coupled receptor classes coupling interaction between thromboxane a receptor and alpha- subunit of guanine nucleotide-binding protein ligand: chemical database for enzyme reactions from genomics to chemical genomics: new developments in kegg predicting networking couples for metabolic pathways of evaluating the statistical significance of multiple distinct local alignments a novel approach to predicting protein structural classes in a ( - )-d amino acid composition space prediction of protein secondary structure content by using the concept of chou's pseudo amino acid composition and support vector machine use of fuzzy clustering technique and matrices to classify amino acids and its impact to chou's pseudo amino acid composition using the concept of chou's pseudo amino acid composition to predict apoptosis proteins subcellular location: an approach by approximate entropy predicting protein subcellular location using chou's pseudo amino acid composition and improved hybrid approach the modified mahalanobis discriminant for predicting outer membrane proteins by using chou's pseudo amino acid composition predicting subcellular localization of mycobacterial proteins by using chou's pseudo amino acid composition prediction of subcellular localization of apoptosis protein using chou's pseudo amino acid composition prediction of g-protein-coupled receptor classes based on the concept of chou's pseudo amino acid composition: an approach from discrete wavelet transform using the augmented chou's pseudo amino acid composition for predicting protein submitochondria locations based on auto covariance approach predicting the cofactors of oxidoreductases based on amino acid composition distribution and chou's amphiphilic pseudo amino acid composition predicting lipase types by improved chou's pseudo-amino acid composition using chou's amphiphilic pseudoamino acid composition and support vector machine for prediction of enzyme subfamily classes using chou's pseudo amino acid composition to predict subcellular localization of apoptosis proteins: an approach with immune genetic algorithm-based ensemble classifier prediction of cell wall lytic enzymes using chou's amphiphilic pseudo amino acid composition medicinal chemistry and bioinformatics -current trends in drugs discovery with networks topological indices proteomics, networks, and connectivity indices application of pseudo amino acid composition for predicting protein subcellular location: stochastic signal processing approach weighted-support vector machines for predicting membrane protein types based on pseudo amino acid composition slle for predicting membrane protein types using pseudo amino acid composition to predict protein structural classes: approached with complexity measure factor using pseudo amino acid composition to predict protein subcellular location: approached with lyapunov index, bessel function, and chebyshev filter using cellular automata to generate image representation for biological sequences using cellular automata images and pseudo amino acid composition to predict protein subcellular location using pseudo amino acid composition to predict transmembrane regions in protein: cellular automata and lempel-ziv complexity using pseudo amino acid composition to predict protein structural class: approached by incorporating dipeptide components predicting protein structural classes with pseudo amino acid composition: an approach using geometric moments of cellular automaton image using grey dynamic modeling and pseudo amino acid composition to predict protein structural classes predicting membrane protein type by functional domain composition and pseudo amino acid composition hum-ploc: a novel ensemble classifier for predicting human protein subcellular localization large-scale plant protein subcellular location prediction predicting membrane protein types by the llda algorithm predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic k-nearest neighbor classifiers pseudo amino acid composition and its applications in bioinformatics, proteomics and system biology recognition of a protein fold in the context of the structural classification of proteins (scop) classification the classification and origins of protein folding patterns seventy-five percent accuracy in protein secondary structure prediction predicting protein folding types by distance functions that make allowances for amino acid interactions a fuzzy k-nearest neighbours algorithm discriminant analysis; chapter multivariate analysis of variance on the generalized distance in statistics this reference also presents a brief biography of mahalanobis who was a man of great originality and who made considerable contributions to statistics review: prediction of protein structural classes review: recent progresses in protein subcellular location prediction an intriguing controversy over protein structural class prediction some insights into protein structural class prediction subcellular location prediction of apoptosis proteins cyp induction-mediated drug interactions: in vitro assessment and clinical implications cyp a : friend or foe? inhibition and induction of human cytochrome p enzymes: current status angiotensin i-converting enzyme gene polymorphism and drug response genome scale enzymemetabolite and drug-target interaction predictions using the signature molecular descriptor svm-prot: web-based support vector machine software for functional classification of a protein from its primary sequence molecular tinkering of g protein-coupled receptors: an evolutionary success critical role for the second extracellular loop in the binding of both orthosteric and allosteric g protein-coupled receptor ligands structural basis for ligand binding and specificity in adrenergic receptors: implications for gpcr-targeted drug discovery glida: gpcr-ligand database for chemical genomics drug discovery-database and tools update investigation into adamantane-based m inhibitors with fb-qsar an in-depth analysis of the biological functional studies based on the nmr m channel structure of influenza a virus ion channel pharmacology the nuclear receptor superfamily and drug discovery key: cord- -x tleomb authors: dodiuk-gad, roni p.; chung, wen-hung; shear, neil h. title: adverse medication reactions date: - - journal: clinical and basic immunodermatology doi: . / - - - - _ sha: doc_id: cord_uid: x tleomb cutaneous adverse drug reactions (adrs) are among the most frequent adverse reactions in patients receiving drug therapy. they have a broad spectrum of clinical manifestations, are caused by various drugs, and result from different pathophysiological mechanisms. hence, their diagnosis and management is challenging. severe cutaneous adrs comprise a group of diseases with major morbidity and mortality, reaching % mortality rate in cases of toxic epidermal necrolysis. this chapter covers the terminology, epidemiology, pathogenesis and classification of cutaneous adr, describes the severe cutaneous adrs and the clinical and laboratory approach to the patient with cutaneous adr and presents the translation of laboratory-based discoveries on the genetic predisposition and pathogenesis of cutaneous adrs to clinical management guidelines. the world health organization defined an adverse drug reaction (adr) in as "a response to a drug that is noxious and unintended and occurs at doses normally used in man" [ ] . edwards and aronson [ ] proposed a different definition in : "an appreciably harmful or unpleasant reaction, resulting from an intervention related to the use of a medicinal product, which predicts hazard from future administration and warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal of the product." the terms 'adverse reaction' and 'adverse effect' are interchangeable, except that an adverse reaction is seen from the point of view of the patient and adverse effect is seen from the point of view of the drug. however, both terms must be distinguished from 'adverse event'. an adverse event is an adverse outcome that occurs while a patient is taking a drug, but is not or not necessarily attributable to it [ ] . differentiating between serious adr and severe adr is imperative. serious adr is a legal term applied to any untoward medical occurrence that at any dose results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect [ ] . conversely, the term 'severe' is a clinical term used to describe the intensity (severity) of a medical event, as in the grading 'mild', 'moderate', and 'severe'; thus, a severe skin reaction need not be serious [ ] . adrs are associated with significant morbidity and mortality and have considerable economic implications. clinical manifestations of an adr are variable and may include cutaneous and or systemic features [ ] . when analyzing the type of adrs most encountered, two major groups emerge; common-mild reactions and raresevere reactions. common-severe reactions are not approved for clinical usage and rare-mild reactions are usually not noticed or reported. cutaneous adrs are among the most frequent adverse reactions in patients receiving drug therapy [ ] . they accounted for % of all reported adrs in a -year retrospective study in taiwan [ ] . the prevalence and incidence of cutaneous adrs vary greatly among different populations [ ] [ ] [ ] [ ] [ ] . in the usa, a -year prospective study found that the prevalence of cutaneous adrs was . % in hospitalized patients [ ] ; and an -year retrospective study found the annual incidence of cutaneous adrs to be . per , persons [ ] . in denmark, in a -year cross-sectional study the prevalence of cutaneous adrs was . % in in-patients and . % in outpatients [ ] . in southern china, in an -year retrospective study, the prevalence of cutaneous adrs was . % in hospitalized patients [ ] . in india, in a -month prospective study, the primary incidence of cutaneous adrs was . per , persons [ ] . the need to survey adrs in clinical practice is universally recognized. various methods may be employed: spontaneous surveillance, prescription-event monitoring (pem), linkage analysis, case-control surveillance and cohort studies [ ] . in , the th world health assembly reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medications. this affirmation led to the creation of the world health organization (who) programme for international drug monitoring, under whose auspices systems have been created in member states for the collection and evaluation of individual case safety reports (icsrs) [ ] . in the who set up its international drug monitoring programme in sweden at the uppsala monitoring centre (umc) http://www.who-umc. org. the us food and drug administration (fda) provides several options for reporting adverse events. one such option is medwatch, the fda safety information and adverse event reporting program http://www.fda.gov/safety/ medwatch/default.htm, founded in as a system for both consumers and healthcare professionals to report adverse events. medwatch is intended to detect safety hazard signals for medical products; in the event a signal is detected, the fda can issue medical product safety alerts or order product recalls, withdrawals, or labelling changes to protect the public health [ ] . a number of international research groups are investigating severe cutaneous adrs (scars): the regiscar network, an international registry of scar established in , the japanese research committee, j-scar, the asian scar consisting of japan and taiwan scar groups (j-scar and t-scar) established in , and the southeast asia network, sea-scar, with ten member countries: brunei, cambodia, indonesia, laos, malaysia, myanmar, philippines, thailand, singapore, and vietnam. the international serious adverse event consortium (isaec), a non-profit organization formed in , is a pharmaceutical industry-and fda-led international consortium that focuses on identifying and validating dna variants useful in predicting the risk of rare drug-induced serious adverse events [ ] . mechanisms of adverse drug reactions (adrs) can be classified into immunologic and non-immunologic etiologies. there are two common types of immune-mediated drug reactions: immediate-type hypersensitivity (type i hypersensitivity) and delayed-type hypersensitivity (type iv hypersensitivity). . immediate-type drug hypersensitivity: immediate-type drug hypersensitivity reactions usually occur minutes to hours after drug exposure, with clinical manifestations including pruritus, urticaria, angioedema, and bronchospasm to anaphylaxis. the reaction is mediated mainly by drug-specific ige, the most common causative agents being penicillins, cephalosporins and neuromuscular blocking agents. ige-mediated reactions to drugs are usually thought to be an immune response to a hapten/carrier complex. in the primary drug sensitization, drug-specific ige is formed when plasma cells transformed from activated b cells interact with t cells. in an allergic reaction, drug allergens bind to mast cells with high-affinity fc receptor, to which drug-specific ige is bound, causing mast cells to release mediators, such as histamine, leukotrienes, prostaglandins and cytokines [ ] . . delayed-type drug hypersensitivity: delayed-type drug hypersensitivity reactions usually take several days to weeks following drug exposure, with variable clinical presentations that may include maculopapular eruption (mpe), fixed drug eruption (fde), acute generalized exanthematous pustulosis (agep), stevens-johnson syndrome (sjs), toxic epidermal necrolysis (ten) and drug reaction with eosinophilia and systemic symptoms (dress). t cell receptor (tcr), cd + and cd + t cells are involved in different delayed-type drug hypersensitivity reactions [ ] . drugs are low molecular weight and usually considered not able to bind to tcrs to activate adaptive immunity. in the case of drug allergy, drug interactions with tcrs may involve a drug-peptide complex presented by human leucocyte antigen (hla) molecules of antigen-presenting cells (apcs). this process is known as the hapten concept; an example is β-lactams that covalently bind to lysine residues [ ] . drugs can also interact directly with tcrs without binding to the peptide/hla of the apc in what is known as the p-i concept (pharmacological interaction of drugs with immune receptors) [ ] . for example, carbamazepine is not able to bind covalently to peptides or proteins, but can associate with low affinity to tcrs and provoke t cell activation [ ] . the immunohistologic characteristics of delayed type drug hypersensitivity are summarized in table . . the skin of mpe is infiltrated by numerous mononuclear cells (cd and cd t cells, monocyte/macrophages) and some eosinophils. typically, interface dermatitis is seen with a predominance of cd + t cells. these cells are located mainly in the perivascular dermis, and both cd + and cd + t cells are located at the dermoepidermal junction [ ] . skin manifestations of dress may vary from mpe-like to exfoliative dermatitis and are characterized by a heavy infiltration of cd + and cd + t cells, monocyte/macro-phages and eosinophils [ ] . mpe and dress share many pathological features, but dress exhibits more severe dyskeratosis (keratinocyte death in epidermis) and a greater extent of systemic involvement and eosinophilia [ ] . immunohistology of skin lesions in agep reveals intraepidermal pustules with infiltration of neutrophils surrounded by il- producing t cells [ ] . despite very diverse clinical presentations, constant features of delayed-type drug hypersensitivity are the presence of high numbers of drug-specific cd + cytotoxic t cells and low numbers of innate nk lymphocytes [ , , ] . cd + t cells of cutaneous adrs have classic cytotoxic functions: lysis of autologous lymphocytes or keratinocytes in an mhc class i-restricted and drug-dependent manner [ ] . cytotoxic immune cells in sjs/ten drug-induced sjs and ten are severe cutaneous adrs in which cytotoxic t lymphocytes (ctls) and natural killer (nk) cells are activated, and subsequently carry out the cellular immune reactions directed at keratinocytes in a major histocompatibility class (mhc) i-restricted manner. upon activation of these immunocytes, various cytotoxic signals, including granulysin, perforin/granzyme b, fas/fas ligand, and cytokines/chemokines, are relayed to the skin lesions to mediate the disseminated keratinocyte death [ ] [ ] [ ] . it is noteworthy that the number of granulysin-positive cells in fixed drug eruptions was found to be similar to that observed in sjs/ten [ ] . non-immune-mediated hypersensitivity is commonly referred to as pseudoallergic reactions because they do not involve a specific immune mechanism -neither ige-mediated (type i) nor delayed (type iv) hypersensitivity. clinical manifestations, which range from milder erythematous to urticarial reactions to severe lethal anaphylaxis, may be indistinguishable from immune system-mediated hypersensitivity reactions. common non-immune-mediated hypersensitivity can be caused by contrast media, vancomycin, non-steroidal anti-inflammatory drugs (nsaids), opiates, plasma expanders, and drugs used in general anesthesia [ ] . nsaids-induced pseudoallergic reactions have been attributed to cyclooxygenase- inhibition and overproduction of leukotrienes, and may require higher drug doses than are needed for true ige-mediated reactions [ ] . mast cell drug-specific t cells mediate skin inflammation in variable clinical presentations of delayed-type drug hypersensitivity through the release and induction of different cytokines and chemokines (table . ) [ ] . the heterogeneous cytokines include th cytokines (interferon-γ) and th cytokines (il- , il- ) [ ] . increased expression of il- , which is a key cytokine for activation of eosinophils, is commonly seen in delayed-type drug hypersensitivity [ ] . the activation of eosinophils can be further enhanced by the chemokines eotaxin and rantes [ ] . thymus and activation-regulated chemokine (tarc/ccl ) has been reported to be a dress specific cytokine [ ] . in addition to th and th cytokines, a recent study demonstrated the involvement of il- aproducing th in dress and sjs/ten [ ] . elevated expression of the neutrophil-attracting il- has been known to be the key cytokine involved in agep. there are several cytokines involved in sjs/ ten. numerous studies have shown tumor necrosis factor alpha (tnf-α) strongly expressed in sjs/ten lesions and correlated with disease severity [ , , ] . tnf-α is a potent cytokine that induces cell apoptosis, cell activation, differentiation, and inflammatory processes [ , ] . interferon gamma (ifn-γ) is a common cytokine involved in delayed-type drug hypersensitivity, including sjs/ ten. ifn-γ was intensely expressed in the superficial dermis and epidermis of sjs/ten lesions [ , ] . ifn-γ is also known to promote antigen presentation and thus stimulate the cell-mediated immunity by upregulation of mhc molecules [ ] [ ] [ ] . in addition to tnf-α and ifn-γ, several cytokines and chemokine receptors that are responsible for trafficking, proliferation, and activation of t-cells and other immune cells have been found elevated in the skin lesions, blister fluids, blister cells, pbmcs, or plasma of sjs/ten patients . these cytokines/chemokines include il- , il- , il- , il- , il- , il- , il- , il- , ccr , cxcr , cxcr , and ccr [ , , , [ ] [ ] [ ] . the central hypothesis proposed to explain the severe mucocutaneous lesions of sjs/ten is the cd + cytotoxic t cell and natural killer (nk) cell-mediated cytotoxic immune reactions. three major cytotoxic signals from cytotoxic cells are reported to be involved in the extensive skin necrosis of sjs/ten, including the fas-fasl interaction, perforin/granzyme b, and granulysin, which can induce keratinocyte apoptosis [ , , ] . granulysin is not only a cytotoxic protein; it is also a chemoattractant and proinflammatory activator that can promote monocyte expression of ccl [ ] , and is capable of promoting antigen-presenting (dendritic) cells and leukocyte recruitment, and activating specific immune responses, such as il- b,il- , il- , tnf-a [ ] . reports on the familial occurrence of severe drug hypersensitivity and cases occurring in identical twins suggest genetic links [ ] [ ] [ ] [ ] . the hla genes show strong association with drug hypersensitivity. examples of strong associations of hla alleles with specific drug-induced hypersensitivity reactions include abacavir, nevirapine, carbamazepine, and allopurinol (table . ). the view that hla alleles are the main genetic determinants of sjs/ten was first proposed by roujeau et al. [ ] , who reported the weak associations of hla-a , b , and mpe maculopapular drug eruption, dress drug reaction with eosinophilia and systemic symptoms, sjs/ten stevens-johnson syndrome/toxic epidermal necrolysis dr in sulfonamide-related ten, and hla-a , b in oxicam-related ten in europeans [ ] . following the immunological hypothesis, the most striking evidence of genetic susceptibility to sjs/ten was provided by the findings that hla-b* : is strongly associated with carbamazepine-induced sjs/ten [ ] , hla-b* : with allopurinol-induced sjs/ten or dress [ ] , and hla-b* with abacavir hypersensitivity [ ] . the hla association to specific drug-induced hypersensitivity can be ethnic and phenotype-specific. the strength of hla associations with specific drug-induced hypersensitivity in different populations has been found related to the prevalence of the susceptibility allele in the ethnic population. the association of hla-b* : with carbamazepineinduced sjs/ten was replicated in other asian countries, including thailand, hong kong, malaysia, china, vietnam, cambodia, reunion, philippines and indian ethnicities, which carry high hla-b* : allele frequency, but not in europeans, which carry low hla-b* : allele frequency (< %) [ ] . in contrast, the strong association of hla-b* : with allopurinol-induced sjs/ten is more universal, being found in han chinese in china, thai populations, korean, japanese, and european populations; hla-b* : is the allele common to all these populations [ ] . the phenotype-specific characteristics are exemplified by carbamazepine hypersensitivity. while hla-b* : is strongly associated with carbamazepine-induced sjs/ten, it is not associated with carbamazepine-induced dress; in an international study, hla-a* : was strongly associated with carbamazepine-induced dress, but not with carbamazepineinduced sjs/ten [ ] . phenytoin -an aromatic antiepileptic drug structurally related to carbamazepine -also frequently causes sjs/ten and dress [ , ] . hla-b* : has been associated with phenytoin-related sjs/ten in asians, although the association is much weaker than that found for carbamazepine-related sjs/ten [ ] . a recent genome-wide association study by chung wh et al. turned up cytochrome (cyp) cvariants, including cyp c * , that showed a strong association with phenytoin-related scar. the significant association between cyp c * and phenytoin-related severe cutaneous ards was replicated in different asian populations [ ] . similar to delayed-type drug hypersensitivity, genetic predisposing factors have been reported in immediate-type drug hypersensitivity. β-lactam allergy was reported associated with gene variants of il , il , and il ra [ ] [ ] [ ] [ ] . several genetic predisposing factors, including gene polymorphisms in cysteinyl leukotriene receptor type (cysltr ) and leukotriene c synthase (ltc s) [ ] and high-affinity ige receptor (fcepsilonr ) [ ] , were associated with aspirin. cutaneous adrs may be classified in terms of their presumed mechanism, severity of the reaction, histological findings, and cutaneous morphological manifestations. the modern pharmacological classification of adrs differentiates two basic types of reactions; type a, predictable reactions, and type b, unpredictable or idiosyncratic reactions. type a reactions ('augmented') are dose-dependent, common and predictable based on the pharmacology of the drug; about % of all adrs are type a. type b reactions ('bizarre') do not occur at any dose in most patients, but may be dose dependent in susceptible individuals. they are uncommon, affecting a small number of patients based on an individual predisposition that depends on both genetic and environmental factors [ , ] . the pathogenesis of type a reaction was described in the sixteenth century by paracelsus, the swiss german renaissance physician who founded the discipline of toxicology: "all things are poison, and nothing is without poison; only the dose permits something not to be poisonous" [ ] . the pathogenesis of type b reaction was designated in the first century bc didactic poem, de rerum natura (on the nature of things), by the roman poet and philosopher lucretius: "one man's meat is another man's poison" [ ] . type b reactions can be categorized into different subtypes according to gell and coombs' classification system [ ] . the effector phase of the allergic reaction is classified into four types: type i mediated by drug-specific ige antibodies, types ii and iii mediated by drug specific igg or igm or iga antibodies, and type iv induced by drug-specifc t lymphocytes [ ] . this classification system may be helpful in daily clinical practice as a guide to diagnostic and therapeutic decisions. in addition to the basic classification of type a and b reactions, further types of reactions were subsequently added; type c-dose and time-related, 'chronic'; type dtime-related. 'delayed'; type e-withdrawal effects, 'end of use'; and type f-unexpected failure of therapy, 'failure' [ ] . the diagnosis of a cutaneous adr must be followed by differentiation between a simple reaction involving only the skin and a complex reaction that includes systemic involvement of organs in addition to the skin [ ] . systemic involvement should be explored even in a mild cutaneous eruption due to a drug since the severity of skin manifestation does not necessarily mirror the severity of the systemic involvement. systemic involvement is evaluated by assessing the patient's symptoms, including fever, facial edema, malaise, chills, dyspnea, cough, palpitations, nausea, vomiting, diarrhea, sore throat and arthralgia. further investigation is based on the patient's symptoms. basic laboratory screen, conducted in cases of suspected systemic involvement, includes a full blood count, liver and renal function tests, and urine analysis [ ] . skin biopsy is an invaluable diagnostic modality in the assessment of drug eruptions. histologically, drug eruptions can elicit a variety of inflammatory disease patterns in the skin and panniculus, and overlapping reaction patterns. ackerman et al.'s basic patterns of inflammatory skin diseases [ ] (table . ) are a helpful guide. the most common pattern of drug eruptions is the perivascular type, while psoriasiform and granulomatous patterns are rarely reported [ ] . drug eruptions may also mimic specific skin diseases such as lupus, lichen planus or lymphoma [ ] . a single drug may cause a wide range of reaction patterns and no reaction pattern is specific for a particular drug [ ] . while the histological changes are not distinctive in many cases of drug eruption, a few important histopathological clues may aid in the diagnosis: ( ) overlapping histological patterns in one specimen (e.g., lichenoid and spongiotic). ( ) presence of eosinophils (although not mandatory); although eosinophils are an important tell-tale sign of a drug-induced reaction, they may also be conspicuous in skin rashes devoid of a drug association and sparse or absent in some drug exanthems. ( ) apoptotic keratinocytes. ( ) mismatch between clinical and histomorphological features [ , , ] . in a study assessing the histological pattern of cases of diagnosed drug eruption during a -year period in one institution [ ] , the majority of the cases ( %) were morbilliform-type rashes. the most common histological pattern was superficial perivascular and interstitial with interface changes. eosinophils were present in only % of cases, and approximately half ( %) of the cases exhibited epidermal-dermal interface changes [ ] . in view of the large diversity of cutaneous drug reactions, it is helpful to approach them as clinicopathologic entities and to base the diagnosis on a combination of clinical, histo- logical and disease course data [ ] . heightened awareness of the possible mimicry of other skin diseases and of the suspicious histopathological clues pointing to drug etiology are key elements to the appropriate histological diagnosis of drug reactions in the skin [ , , ] . a widely accepted approach to diagnosing the type of drug eruption is a simplified method based on the morphology of the primary lesions. the four main categories are maculopapular, urticarial, pustular and blistering [ ] . the diagnosis of the drug eruption can be challenging since the same cutaneous morphology can be manifested in a simple reaction involving only the skin and in a complex reaction including systemic involvement in addition to the skin. therefore, there are two major steps in diagnosing drug eruptions: determine the morphology and assess systemic involvement [ ] . terminology the term 'maculopapular' is descriptive. morbilliform means measles-like, the rash of measles consisting of macules and papules that tends to confluence. the etymon of 'exanthema' is the greek 'exanthema', which means 'a breaking out'. thus exanthema merely means 'rash', and 'exanthematous rash' literally means 'rash-like rash'. therefore, the terminology is redundant [ ] . polymorphous pink-to-red macules and or papules usually in a symmetric distribution that may coalesce to form plaques ( fig. . ) [ ] . the eruption begins on the trunk and upper extremities and progressively becomes confluent. in addition, purpuric lesions may appear on the ankles and feet [ ] . the drug eruption can also manifest in a scarlatiniform pattern of pinpoint-sized pink-red papules coalescing and giving the skin the texture of sandpaper [ ] . frequency the most common drug-induced eruptions, occurring in - % of first-time users of most drugs [ ] . lag period - days [ ] . symptoms pruritus and low-grade fever are common [ ] . the eruption usually begins on the trunk and becomes generalized. palms and soles are often involved; mucous membranes are usually spared [ ] . histology nonspecific changes consisting of mostly superficial but also deep perivascular and interstitial infiltrate of lymphocytes. eosinophils and epidermal-dermal interface changes appear in approximately half the cases [ ] . differential diagnosis viral exanthems, scarlet fever, toxic shock syndrome, acute graft versus host disease (gvhd), kawasaki disease, juvenile idiopathic arthritis [ ] . treatment identifying and discontinuing the causative drug are the most important steps in management. symptomatic treatment with antipruritic agents and potent topical glucocorticoids may be helpful [ ] . a decision can be made to continue the drug and offer symptomatic treatment if the drug is of paramount importance, but the risk: benefit ratio of this option has to be carefully weighed, and the evolution of the eruption must be meticulously monitored [ ] . prognosis the eruption often fades within - days of discontinuation of the offending drug and scaling and desquamation may follow. re-challenge may lead to reappearance of the reaction within a few days [ ] . offending drugs the most common classes of drugs implicated are penicillins, sulfonamides, cephalosporins, and antiepileptics [ ] . terminology the term 'urticaria', first introduced by william cullen in the eighteenth century, is derived from urtica urens (common european stinging nettle). one of the earliest descriptions of urticaria comes from china, and is more than erythematous macules and papules coalescent into illdefined plaques on the trunk -maculopapular morphology of cutaneous adr , years old. in the huangdi neijing, written around bc, urticaria is referred to as feng yin zheng ('wind type concealed rash'). in ancient latin medical literature, urticaria was called 'uredo' (urere means 'to burn'), and in the old persian medical texts, 'essera' (meaning 'elevation') [ ] . skin signs urticaria is induced by superficial dermal swelling due to plasma leakage and vasodilation triggered by activation of mast cells. the skin manifestations of this process include erythematous and edematous papules and plaques (wheals) of various sizes that may coalesce to form large plaques [ ] . wheals may be characterized by pink or pale center and assume a figurate or polycyclic configuration. linear lesions can be seen with dermatographism [ , ] . frequency drug-induced urticarial eruptions are the second most common type of cutaneous drug eruption and account for approximately % of all cutaneous drug eruptions [ ] . lag period urticaria occurs within minutes to days of drug administration [ ] . symptoms a major clinical feature is pruritus, the lack of which should put the diagnosis in doubt. the lesions can also be painful if they occur on the soles, over joints, or in areas where the skin is tightly adhered to subcutaneous tissue [ ] . a single lesion lasts less than h and upon resolution leaves normal skin. however, new lesions may continue to arise for various periods of time. acute urticaria is defined when a bout of hives lasts less than weeks; when it lasts longer, it is defined as chronic urticaria [ ] . urticaria may be associated with angioedema [ ] . angioedema is defined as a deep, dermal, subcutaneous and/or mucous swelling that may involve the intestinal lining and the upper respiratory tract. symptoms include slight heat, burning, pain and sensation of pressure or tightness. however, pruritus is minimal or absent. swelling of gastrointestinal tract mucosa can induce abdominal pain, vomiting and diarrhea. edema of the respiratory tract may induce various symptoms including life-threatening asphyxia. drug-induced angioedema is associated with urticaria in approximately % of cases. some drugs may induce angioedema without urticaria [ ] . common sites of involvement lesions of urticaria can appear anywhere on the skin, including the palms, soles and scalp, but not on mucosal surfaces [ ] . angioedema most commonly occurs in the head, neck and hands, but can occur anywhere and frequently involves mucosal tissue. swelling may be more prominent in areas of looser skin, such as the scrotum, labia, lips, and eyelids [ ] . histology urticarial drug reactions are characterised by dermal edema and a superficial and deep perivascular and interstitial dermatitis. the mixed inflammatory infiltrate comprises lymphocytes, histiocytes, mast cells, eosinophils and neutrophils. the presence of neutrophils and deep vascular plexus involvement may be a clue to the drug-induced nature of the urticaria [ ] . the wheals with central red halo of urticaria may resemble the target lesions of erythema multiforme. four clinical signs of urticaria can help distinguish it from erythema multiforme: ( ) the central zone consists of normal skin, whereas in erythema multiforme, skin is dusty, bullous or crusted. ( ) each lesion is transient, lasting less than h, whereas erythema multiforme lesions are 'fixed' for a few days. ( ) new lesions appear daily and in erythema multiforme all lesions appear within the first h. ( ) there may be associated swelling of face, hands and feet and in erythema multiforme there is no edema [ ] . differential diagnosis of urticaria includes also bullous pemphigoid, urticarial vasculitis and serum sickness-like reaction (sslr). drug-induced urticaria needs to be differentiated from cases of urticaria induced by other etiologies, such as food, environmental allergens, insects, systemic illness, physical stimuli, genetic and idiopathic [ ] . urticaria and angioedema are the most common symptoms of anaphylaxis ( % of cases), and are one of the clinical criteria of the national institute of allergy and infectious disease (niaid) and the food allergy and anaphylaxis network (faan) for the diagnosis of anaphylaxis [ ] . therefore, all cases of sudden acute urticaria and angioedema should be evaluated for indications of the anaphylactic type of reaction: presence of respiratory compromise, decreased blood pressure, and end-organ dysfunction (collapse, syncope, incontinence) [ ] . treatment the most important step in the management of drug induced urticaria with or without angioedema is withdrawal of the causative agent. in most cases of acute urticaria, when the trigger is removed the rash quickly resolves. h -receptor blockers are the mainstay of treatment for patients with only cutaneous symptoms. systemic glucocorticoids are indicated in all cases with upper airway edema and should be considered in cases with extensive cutaneous involvement. epinephrine is reserved for angioedema with upper airway involvement [ ] . the presence or absence of any airway involvement should be specifically investigated. prognosis both urticaria and angioedema fade without visible sequelae. following resolution, there should be no residual pigmentary changes unless excoriated [ ] . offending drugs many drugs can induce acute urticaria, and do so by both immunologic and non-immunolgic mechanisms. the major drugs responsible for immunologically based urticaria are antibiotics, especially penicillins and cephalosporins [ ] . the major drugs triggering mast cell release (non-immunolgic mechanisms) are aspirin, nonsteroidal anti-inflammatory drugs (nsaids), opioids and radiocontrast media [ ] . viral infections or connective tissue diseases may induce or augment urticarial drug reactions [ ] . the national institute of allergy and infectious diseases (niaid) and the food allergy and anaphylaxis network (faan) defined anaphylaxis as a systemic reaction resulting from the sudden release of multiple mediators from mast cells and basophils, often life threatening, and usually unexpected. the world allergy organization (wao) has divided anaphylaxis into immunologic (further divided into immunoglobulin e [ige]-mediated and non-ige-mediated), non-immunologic, and idiopathic causes. drugs are the second most common cause of anaphylaxis after food, which constitutes % of triggers [ ] . common medications associated with anaphylaxis include penicillins, nsaids, and biologic response modifiers [ ] . the niaid/ faan definition of anaphylaxis has been translated into clinical diagnostic criteria that include an acute onset of illness (minutes to hours) and involvement of the dermatologic, respiratory, cardiovascular, or gastrointestinal systems [ ] . epinephrine is the only first-line treatment for anaphylaxis and is the sole effective treatment for an acute reaction. delays in administration have been associated with fatalities. supportive treatment with oxygen, fluids and additional drugs are also necessary according to the cardiopulmonary resuscitation (cpr) anaphylaxis algorithm [ ] . • serum sickness-like reaction (sslr) -see severe cutaneous adverse drug reactions. terminology the term pustule originates in classical latin in which pustule means a blister [ ] . skin signs pustular drug eruptions are characterized by monomorphic eruption consisting of erythematous papules (mostly follicular) and pustules at the same location lacking comedones. acneiform drug eruptions (acne medicamentosa) the term acneiform is applied to eruptions that resemble acne vulgaris. frequency varies, depending on the drug. the highest incidence involves epidermal growth factor receptor inhibitors (egfris), affecting - % of patients [ ] . lag period the eruption begins after a variable delay; corticosteroids may induce an acneiform eruption from shortly after their introduction ( - weeks) to several months [ ] . acneiform eruptions induced by egfris usually appear after - weeks of treatment but can also occur after only a few days [ ] . symptoms pruritus, tenderness and pain may occur. in cases of chemotherapy-related side effects, their appearance and severity are part of the criteria used for the classification of the adr [ ] . common sites of involvement lesions may be located in and beyond the seborrheic areas, such as the arms, trunk, lower back and genitalia [ ] . histology drug-induced acneiform eruptions show histopathologic features similar to acne vulgaris. early lesions most commonly have a corneocytic plug within a widened infundibulum, accompanied by infundibular spongiosis, perifollicular edema, with sparse perivascular and periinfundibular infiltrates of neutrophils and lymphocytes. larger older lesions show similar findings but the infiltrate is denser, with more neutrophils around the involved follicles, and infundibular rupture [ , ] . in a review of the histological findings of acneiform eruptions induced by egfris [ ] , all ten cases showed a superficial, predominantly neutrophilic suppurative folliculitis with ectatic infundibula and a rupture of the epithelial lining. differential diagnosis the main differential diagnosis is acne. the following clinical characteristics of acneiform drug eruptions may aid in differentiating between the two entities: ( ) clinical presentation: monomorphic pattern, lack of comedones and cysts and localization on areas beyond the seborrheic area. ( ) patient characteristics: age of onset before or after the teens, and absence of past history of acne. ( ) resistance to conventional acne therapy. ( ) time relationship: onset after recent drug introduction, improvement after drug withdrawal, and recurrence after drug reintroduction [ ] . the differential diagnosis also includes folliculitis, rosacea, perioral dermatitis, demodicosis, acne cosmetic, acne mechanica, chloracne, acne necrotica and acneiform presentation of cutaneous lymphomas [ ] . treatment the main treatment is withdrawal of the offending drug and the application of topical treatments as needed (benzoyl peroxide topical antibiotics and topical retinoids) [ ] . the management of acneiform eruptions associated with chemotherapy differs from all other types of acneiform drug euptions, as acneiform eruption is an expected outcome and discontinuation of the medication is not an option in a patient who is responding to therapy [ , , , ] . in fact, continuation of egfri therapy in these patients may be especially favourable in view of studies that have shown an increased survival with increasing severity of rash [ ] . the cutaneous reaction serves as an important clinical tool for determining tumor response and survival [ ] . the national cancer institute developed a scale for defining the degree of rash and laid down management guidelines for each stage [ ] . other management protocols were suggested by bachet et al. [ ] , who recommended that unless contraindicated, a tetracycline should be routinely prescribed for the prevention of acneiform eruption in patients treated with an egfri for more than weeks. chiang et al. [ ] reported successful treatment with isotretinoin for high grade and refractory cases. prognosis in most patients with acneiform drug eruption, the rash resolves upon discontinuation of the offending drug and the use of topical treatment. in egfri-induced acneiform eruption, prophylactic administration of a tetracycline was associated with significantly lower incidence of grade - folliculitis and improved quality of life of patients [ ] . few cases of drug-induced eosinophilic pustular folliculitis have been reported [ , [ ] [ ] [ ] [ ] . drugs reported include chemotherapy (cyclophosphamide, methotrexate, and -fluorouracil) [ ] , minocycline [ ] , carbamazepine [ ] , and allopurinol with timedium bromide [ ] . clinical presentation includes pruritic follicular papules and pustules on the face, scalp, trunk and arms [ ] . histological findings include spongiosis of the follicular epithelium, and an intraand perifollicular lymphohistiocytic infiltrate with numerous eosinophils that form microabscesses within the follicular epithelium [ ] . topical steroids are the first line of treatment [ ] . acute generalized exanthematous pustulosis (agep) -see severe cutaneous adverse drug reactions. terminology the term pseudoporphyria was coined in by korting to describe patients with chronic renal failure and a bullous disease resembling porphyria cutanea tarda (pct) [ ] . frequency the incidence of pseudoporphyria is unknown. however, in a -month prospective study, % ( / ) of children taking naproxen for juvenile idiopathic arthritis developed pseudoporphyria [ ] . lag period the skin lesions appear following drug intake combined with exposure to light. various time durations were reported, weeks to months [ ] [ ] [ ] . the clinical features of pseudoporphyria may be identical to those of pct; both exhibit vesicles, bullae, milia, and scarring on sun-exposed skin. in contrast to pct, however, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are rarely reported in pseudoporphyria [ ] . often, fragility and bruising may be the only clinical signs [ ] . in children, facial scarring resembling erythropoietic protoporphyria (epp) may be found [ ] . symptoms skin fragility and photosensitivity [ ] . the lesions appear on sunexposed skin, particularly the hands and feet, but also on the face and extensor surfaces of legs [ ] . histology the histological features are identical to those seen in pct. the blisters are subepidermal and the floor of the blister is typically lined by well-preserved dermal papillae (festooning). there is usually no significant inflammatory component although a light perivascular lymphocytic infiltrate may occasionally be seen in the superficial dermis. thickening of the superficial vessels (highlighted by a pas stain) and dermal sclerosis with elastosis may be apparent. in both pseudoporphyria and pct, direct immunofluorescence reveals granular deposits of igg and c at the basement membrane zone and in the perivascular region [ ] . differential diagnosis while pseudoporphyria and pct share clinical and histologic features, they can be differentiated by several features. most important, by definition, biochemical porphyrin abnormalities are absent in pseudoporphyria. epidemiologically, pseudoporphyria affects mainly women while there is a male predilection in pct. clinically, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are frequently evident in pct and conspicuously absent in pseudoporphyria [ ] . the differential diagnosis also includes other types of cutaneous porphyria that manifest with blistering, epidermolysis bullosa acquisita, polymorphous light eruption, and other photosensitive dermatosis [ ] . treatment treatment entails discontinuation of suspected agents and sun protection, especially against uva wavelengths, for several months following withdrawal of the drug [ ] . prognosis blisters may continue to appear for weeksmonths after discontinuation of the offending drug [ ] . offending drugs the most common group of drugs causing pseudoporphyria are nsaids [ ] . other groups are antibiotics, diuretics and retinoids. additional culprits are hemodialysis, renal failure, tanning beds and excessive sun exposure [ ] . terminology fixed drug eruption (fde) was first reported by boums in [ ] , and the term was coined by brocq in [ ] . frequency the incidence is not known, but is suspected to vary greatly by geographic region [ ] . lag period after initial use of the offending agent, a variable refractory period of weeks, months or years may pass before the lesions first appear on the skin of a sensitized individual [ ] . repeated exposure to the agent typically results in acute lesions within min to h. a refractory phase may occur following an acute flare in which exposure to the offending drug will not exacerbate the lesion for weeks to months [ ] . in its classical form, fde typically presents round or oval, sharply demarcated, red to livid, slightly elevated plaques ranging from several millimeters to over cm in diameter. vesicles or even blisters can develop [ ] . usually only a single lesion appears. sometimes, multiple lesions are present and even lead to generalized fde characterized by multiple, sharply defined, deep red macules distributed bilaterally and often symmetrically. generalized bullous fde is characterized by flaccid blisters arising on these macules. mucosal lesions are usually bullous and may appear with or without involvement of other areas of the skin [ ] . symptoms patients often complain of burning and itching in the lesions. general symptoms such as fever, nausea, dysuria, abdominal cramps and diarrhea are rare [ ] . pruritus and burning may be the only manifestations of reactivation in a postinflammatory hyperpigmentation lesion [ ] . common sites of involvement the eruption can occur anywhere on the body, but the lips, palms, soles, genitalia (especially male genitalia), groin and occasionally oral mucosa are favored sites [ ] . the diagnostic hallmark of fde is the reappearance of the lesions precisely over the previously affected sites. studies investigating the predilection areas indicate that some specific kind of drugs cause fde predominantly at specific sites: examples are tetracycline and location on the male genital area, and naproxen and fde on the lips [ ] . in rare cases, fde manifests in old trauma sites such as bcg vaccination, burn scar, venipuncture site or insect bite. with each recurrence, additional sites may be affected. the presence of numerous lesions is referred to as generalized fde [ ] . histology histologically, the acute phase is characterized by marked basal cell hydropic degeneration, with lymphocyte tagging along the dermoepidermal junction and individual keratinocyte necrosis. marked pigmentary incontinence is typical, and may be the sole histological finding in late lesions [ ] . differential diagnosis skin lesions can imitate various dermatoses, including lichen planus, erythema multiforme, erythema annulare centrifugum, and pityriasis rosea. in generalized fde, residual pigmentation in healed lesions may be reminiscent of erythema dyschromicum perstans. involvement of oral and genital mucosa raises the possibility of herpes simplex, pemphigus vulgaris, aphthous stomatitis, behçet syndrome, and erosive lichen planus [ ] . generalized bullous fde may resemble sjs/ten. the following typical clinical features of generalized bullous fde may aid in differentiating between conditions: ( ) blistering usually affects only a small percentage of body surface area, and between the large blisters there are sizable areas of intact skin. ( ) erosive mucosal involvement is rare, and when it does occur is rather mild. ( ) patients usually do not feel sick or have fever, and generally are in much better overall health than those with sjs/ten. ( ) most patients report a history of a similar, often local reaction [ ] . treatment for mild lesions, topical corticosteroids usually suffice. in severe involvement, especially generalized bullous fde, systemic corticosteroids may be indicated. strict avoidance of the causative drug and cross-reacting substances is essential for prophylaxis. successful desensitization was reported [ ] . prognosis the prognosis of localized fde is good and the lesions fade within a few days to leave a post-inflammatory brown pigmentation [ ] . generalized bullous fde does not have this benign nature and the mortality rate was % in a recent case control study of patients [ ] . offending drugs the most common groups of drugs implicated are antibiotics, analgesics, antiphlogistics and hypnotics [ ] . there is usually only one causative drug (monosensitivity), but sometimes several drugs can induce fde in the same patient (multisensitivity). it has also been claimed that recurrences of fde can be induced in nonspecific fashion by mast cell degranulators such as food, acetylsalicylic acid, bacterial toxins, or physical stimuli [ ] . • drug-induced/triggered autoimmune blistering dermatosis (pemphigus, bullous pemphigoid (bp)) and linear iga bullous dermatosis (labd) [ ] . cases of autoimmune blistering dermatosis resulting from exposure to drugs present clinical, histologic and immunopathologic features identical or very similar to those seen in idiopathic disease, but are induced by systemic ingestion or local use of certain drugs. there appear to be two main types: drug-induced autoimmune blistering dermatosis proper, the acute and self-limiting type with rapid resolution after withdrawal of the offending agent; and drug-triggered autoimmune blistering dermatosis in which the role played by the drug is only secondary to hereditary and immunologic factors. the drug stimulates a predisposition (hidden susceptibility) to develop the disease and is considered the chronic type in which the disease persists despite withdrawal of the offending agent [ , ] . frequency unknown. [ , , ] . of note, drug-induced labd patients tend to be older than idopathic type patients [ , ] . the polymorphic nature of the eruption may mimic other bullous diseases and or drug-induced bullous diseases such as sjs, ten, and fde [ ] . treatment treatment consists of discontinuing the offending agent, and, depending on the severity of the disease, systemic immunosuppressive treatment [ ] . prognosis drug-induced autoimmune blistering dermatosis remits after the offending drug is withdrawn, while drugtriggered autoimmune blistering dermatosis may persist despite withdrawal of the offending agent and chronic immunosuppressive treatment may be required [ , ] . two major groups of chemical structures were found in the drugs or their metabolites implicated in pemphigus: sulfhydryl radical drugs (thiol drugs or sh drugs) such as penicillamine, and phenol drugs such as aspirin [ , , ] . bp many drugs were reported [ , , ] , the most frequent being nsaids, cardiovascular agents and penicillin-derived antibiotics [ ] . in addition, external use of skin and mucous membrane preparations has been documented to provoke cases of either bp or cicatricial pemphigoid [ ] . labd of the various drugs reported, vancomycin is the most common [ , , ] . reactions. the incidence of dress remains to be determined because of variable presentations and lack of universally accepted diagnostic criteria [ ] . the estimated risk at first or second prescription of an aromatic antiepileptic drug was - . in , [ ] . a slight female predominance was found in the regiscar study (male/female . ) [ ] . the drugs most commonly inducing dress are anti-convulsants (mainly aromatic anti-convulsants such as carbamazepine), allopurinol, sulfonamides (the anti-infective sulfamethaxazole-trimethoprim, and the anti-inflammatory sulfasalazine), and antibiotics (such as vancomycin and minocycline) [ ] . numerous other drugs have been reported [ , , ] . the role of human herpesvirus (hhv) reactivation in the development of this adverse drug reaction is well recognized, especially hhv- [ ] . hhv- reactivation is among the diagnostic criteria of the japanese consensus group for dress/drug-induced hypersensitivity syndrome [ ] . the reactivation of other herpesviruses, including hhv- , cytomegalovirus (cmv), epstein-barr virus (ebv), and human herpes simplex virus was also reported [ ] . dress is considered to result from complex interactions between genetic predisposition, exposure to drug and viral reactivation [ ] . delayed onset of - weeks after drug administration followed by a stepwise development of manifestations. rechallenge can result in a reaction within hours to days [ ] . the lag period differs between drugs; carbamazepine tended to show a longer latency (median days) than allopurinol (median days) in the regiscar study [ ] . dress has multi-organ involvement with cutaneous, mucosal, hematological and solid organ manifestations. the cutaneous involvement in dress is typically extensive and symptomatic (pruritus, burning and pain) [ , ] . various dermatological features were reported. walsh et al. [ ] proposed a classification system based on four distinct patterns: ( ) urticated papular exanthema, the most common, ( ) morbilliform erythema, ( ) exfoliative erythroderma, and ( ) erythema multiforme-like (em-like), which was prognostic of more severe hepatic involvement. the extent of skin involvement varies between studies: it exceeded % of the body surface area in most of the patients ( %) according to the regiscar study [ ] ; head and neck edema observed in most patients [ , ] ; and pustules reported in various studies, predominantly in a facial distribution of the edema [ , ] . [ ] . most frequent were oral lesions including lips, oral cavity and throat [ ] . the manifestations of oral lesions in dress include cheilitis, erosions and dysphagia that may appear before skin lesions, and oropharaynx is considered the first site of herpesvirus reactivation in dress [ ] . involvement of eyes and genitalia were also reported in the regiscar study [ ] . multi-organ involvement is common in dress and may include a wide variety of systems. highgrade fever ( - °c) is a typical early manifestation that may last for several weeks; it often precedes the cutaneous eruption by several days [ ] . lymphadenopathy is common and has two distinct types: a benign pattern of lymphoid hyperplasia and a pseudolymphoma pattern [ ] . hematologic abnormalities are frequent and diverse, the most common being marked leukocytosis, eosinophilia and atypical lymphocytes [ ] . however, neutrophilia, monocytosis, thrombocytopenia, anemia, pancytopenia and hemophagocytic syndrome were also reported [ , , , ] . hypereosinophilia and activated neutrophils, if persistent, can contribute to organ damage [ ] . the liver is the most frequently affected visceral organ in dress; hepatitis with isolated elevation of liver enzymes is common and usually anicteric and without cholangitis. however, severe acute hepatitis with liver failure may result and is the primary cause of mortality in dress [ ] . renal involvement is common [ ] . involvement of the following organs was also reported: lungs, muscle, heart, pancreas, colon, thyroid, joints, parotid gland and brain [ ] . the type of organs involved was found to be related to the eliciting drug [ ] . the most common pathological changes found in a study of patients with dress were basket-weave hyperkeratosis ( %), dyskeratosis ( %), lymphocytic exocytosis ( %), spongiosis ( %), papillary edema ( %), perivascular lymphocytic infiltration ( %), eosinophilic infiltration ( %), and interface vacuolization in the dermoepidermal junction ( %) [ ] . the presence of severe dyskeratosis was correlated with a greater extent of systemic involvement [ ] . in a different study assessing the histological findings of cases with dress [ ] , the predominant pathological pattern was spongiotic dermatitis with superficial lymphocytic infiltrate ( %); necrotic keratinocytes were noted in % of cases, and were associated with a worse hepatic involvement [ ] . the diverse presentations in dress have hampered efforts to define diagnostic criteria. three diagnostic criteria have been proposed: bacquet et al. [ ] , the japanese study group of severe cutaneous adverse reactions to drugs (j-scar) [ ] , and the regiscar network [ ] . the first step in the management is immediate withdrawal of the culprit drug. the treatment is tailored according to the severity and extent of systemic involvement, and the diagnosis of viral reactivation of herpesviruses (mostly hhv- ) [ , , ] . management protocol for dress based on the consensus of experts was designed by the french society of dermatology [ ] , and includes four visceral involvement severity categories and corresponding treatment: counselling both the patient and his family members about drug avoidance is necessary. first-degree relatives have a higher risk of developing the same drug reactions [ ] . increased knowledge of hla susceptibility genes enables screening patients with dress for several high risk drugs [ , ] . symptoms are usually present for several weeks even after discontinuation of the offending agent and appropriate treatment [ ] . late complications include the appearance of autoimmune diseases such as lupus erythematosus and autoimmune thyroiditis, with laboratory evidence of autoantibodies [ ] . systemic corticosteroids were found beneficial in the prevention of autoimmune disease. however, this effect needs to be counterbalanced against the higher risk of viral reactivation and infection. [ ] . in a -year follow-up study of affected patients with dress in taiwan, the overall cumulative incidence of long-term sequelae was . %; four developed autoimmune diseases (graves disease, type diabetes mellitus and autoimmune hemolytic anemia); and the other two developed renal failure and required lifelong hemodialysis. the author concluded that the sequelae of dress can be divided into two major types that appear in different age groups: young patients tend to develop autoimmune diseases; elderly patients are more vulnerable to end-organ failure [ ] . mortality in dress has been estimated at %, with most patients dying from liver failure [ ] . pancytopenia, leukocytosis, tachycardia, tachypnea, coagulopathy, gastrointestinal bleeding and systemic inflammatory response syndrome were associated with a poor outcome in dress patients [ , ] . the incidence of sslr is unknown. epidemiology studies in children suggest that the overall frequency induced by cefaclor is . - . % per course of the drug [ ] . most reactions were reported in children under years old, mainly during the second and third courses of therapy [ ] . cefaclor is the most common cause of sslr in children, inducing . % of cases [ ] . other drugs implicated include other cephalosporins, [ ] penicillins, [ ] minocycline, [ ] insulin, [ ] and infliximab [ ] . usually - days (range - days) [ , ] . skin the skin is the most frequent finding in sslr, including erythema that progresses to urticarial lesions (pruritic and migratory), urticarial wheals with dusty to purple centers ('purple urticaria') that morphologically resemble erythema multiforme (em) [ ] and other cutaneous manifestations including morbilliform or scarlatiniform eruptions [ ] . mucous membranes are not involved [ ] . systemic involvement joint involvement may be prominent, presenting with edema, decreased range of motion, warmth, pain, and difficulty walking. polyarticular involvement is often observed, with involvement mainly of the wrists, ankles, hips and knees [ ] . some authors suggested that joint involvement may be related in part to increased fluid in the skin around affected joints due to urticarial eruption rather than arthritis [ ] . fever, malaise, myalgia and lymphadenopathy were also reported. neurologic involvement, gastrointestinal symptoms and renal complications were rarely documented [ ] . notable laboratory abnormalities include elevated erythrocyte sedimentation rate (esr), c-reactive protein (crp) and leukocytosis [ , ] . the histological findings of sslr appear to be in the spectrum of urticaria with no vasculitis [ ] . histology can be helpful in differentiating sslr from acute hemorrhagic edema of infancy, which is characterized by vasculitis [ ] . there are no diagnostic criteria. the diagnosis is based on clinical findings [ ] . withdrawal of the offending agent and symptomatic treatment with oral antihistamines and topical corticosteroids are usually sufficient. a short course of oral corticosteroids may be required in patients with severe symptoms [ ] . the disease course is benign and resolves in a few days. however, a few cases lasting several weeks have been described [ ] . no long-term morbidity has been reported [ ] . the estimated incidence of agep is - cases per million per year [ ] . female predominance was reported in several studies [ ] [ ] [ ] . the majority of cases appear to be related to drugs (> %), mainly antibacterials [ ] . in a large multinational casecontrol study (the euroscar study), the following agents were highly suspected drugs for agep: prestinomycin, ampicillin/amoxicillin, quinolones, (hydroxy)chloroquine, anti-infective sulfonamides, terbinafine and diltiazem [ ] . latent periods fall into two categories, according to the offending drug: median duration of day, associated with antibiotics (including sulphonamides), and median duration of days for all other associated drugs [ ] . longer periods of months were reported in a few agep cases with an underlying malignancy [ ] . agep is a severe acute pustular cutaneous reaction characterized by a rapid clinical course [ ] . skin the typical morphology of agep is an acute edematous erythema with burning and or itching sensation, followed by dozens to hundreds of small (pinhead sized) non-follicular sterile pustules with a predilection for the big folds, or with widespread distribution (fig. . ) . sometimes confluence of pustules may mimic a positive nikolsky's sign [ , ] . additional cutaneous manifestations include marked edema of the face, purpura, blisters and target-like lesions [ , , ] , all of which overlap with manifestation of agep and ten [ , ] , and acute localized exanthematous pustulosis (alep) [ , ] . mild, nonerosive mucous membrane involvement of one location (mostly oral) occurs in about % of cases [ ] . systemic involvement fever (above °c) and leukocytosis with neutrophilia are almost always apparent. lymphadenopathy, myalgia, headache, mild eosinophilia, elevated crp, slight reduction of creatinine clearance, and mild elevation of aminotransferases were also reported [ , ] . a -year retrospective review of patients with agep [ ] turned up patients ( %) with at least one systemic involvement in the acute phase, with abnormal hepatic function test, with renal insufficiency, two with acute respiratory distress and one patient with agranulocytosis. mean peripheral neutrophil counts and mean c-reactive protein levels were elevated significantly in patients with systemic involvement [ ] . biopsy specimen should be obtained from an early pustular lesion [ ] . a histopathological study of agep cases [ ] found the following histopathological features: ( ) all cases demonstrated pustules (sub/intracorneal and or intraepidermal). the agep validation score developed by the euro-scar study group is a standardized scoring system made up of data related to clinical features (morphology and clinical course) and histopathology. based on this score, agep cases can be categorized as no agep, possible agep, probable agep, and definite agep [ ] . treatment consists of discontinuation of the causative drug and supportive treatment. although, specific treatment is generally unnecessary, topical and systemic steroids were reported [ , ] . the treatment of overlapping agep and ten cases is not yet established [ ] , although successful treatment with infliximab was documented [ ] . after elimination of the causative drug, pustules usually spontaneously disappear in a few days with desquamation, and the reaction fully resolves within days [ ] . the overall prognosis is good, although high fever or superinfection of skin lesions can sometimes lead to life-threatening situations in patients of old age or poor general condition [ ] . the mortality rate is about % [ ] . the annual incidence of sjs and ten is . - and . - . per million individuals, respectively [ , ] . the annual incidence of sjs and/or ten in hiv patients is estimated at - per individuals, approximately -fold higher than that of the general population [ ] . the incidence of sjs/ten increases with age; children less than years of age account for only % of the samples in most studies [ ] . women are two times more likely to be affected by sjs/ten than men in the adult population, while the male to female ratio is about equal in children [ ] . drug exposure is the most common cause of sjs/ten [ ] , with more than drugs identified [ ] . the groups of medications associated with high risk of inducing sjs/ fig. . multiple, pin-head sized, non-follicular pustules on erythematous skin on the trunk in a patient with agep ten vary according to the population. in the general population in europe, high risk drugs for sjs/ten include allopurinol, carbamazepine, cotrimoxazole and other anti-infective sulfonamides, lamotrigine, nevirapine, oxicam-nsaids, phenytoin, phenobarbital and sulfasalazine [ ] . in the pediatric population in europe, they include anti-infective sulfonamides, phenobarbital, carbamazepine and lamotrogone [ ] . in africa, they include antibacterial sulfonamides, nevirapine, tuberculosis drugs, nsaids, antiepileptics, aminopenicillin, analgesics and allopurinol [ ] . non-medication triggers, implicated mainly in sjs, include infections, contrast media and vaccinations [ ] [ ] [ ] . alden is an algorithm for the assessment of drug causality in sjs/ten developed by the regiscar study group and consists of parameters according to which the drug causality is classified as very unlikely, unlikely, possible, probable and very probable [ ] . usually - days. the median latency was longer (above weeks) for drugs with no associated risk [ ] . sjs and ten represent different degrees of a severe, acute and life-threatening mucocutaneous reaction. we will refer to this disease spectrum as a single entity, namely sjs/ ten. the classification of sjs/ten, defined by bastuji-garin et al. [ ] , is based on the extent of epidermal detachment and the findings of characteristic skin lesions (table . ). it should be emphasized that only necrotic skin, which is already detached (e.g., blisters, erosions), or detach-able skin (positive nikolsky sign whereby slight rubbing of the skin results in exfoliation of the outermost layer) should be included in the evaluation of the extent of epidermal detachment [ ] . the characteristic skin morphology of sjs/ten consists of 'flat, atypical target lesions' and 'spots/macules', which are defined as follows. flat, atypical target lesions are round lesions, with only two zones and/or a poorly defined border, nonpalpable with the exception of potential central blister. 'spots/macules' are nonpalpable, erythematous or purpuric macules with irregular shape and size, often confluent [ ] . epidermal necrosis, the hallmark process of sjs/ten, induces flaccid blisters with positive asboe-hansen sign (lateral extension of bullae with pressure), erosions, positive nikolsky sign, and in severe cases extensive skin sloughing [ ] . at least % of epidermal detachment is required for the diagnosis of sjs/ten [ ] . in rare instances, extensive epidermal necrosis occurs with only widespread erythema and no evidence of 'flat, atypical target lesions' or 'spots/macules'; these cases were classified as 'ten without spots' (table . ). a characteristic sign of sjs/ten is severe pain and tenderness of the skin [ ] . mucosal involvement is evident in most of the cases with erythema, erosions and ulceration, due to necrosis of the epithelial lining [ ] . sjs/ten involve more than mucosal sites in - % of cases [ ] . most common sites are oral (fig. . ) , ocular and genital mucous membranes, although any mucous membrane may be involved, such as respiratory, gastrointestinal and urethral [ ] . fuchs syndrome is a unique type of sjs that involves the mucosa without skin lesions and was reported to be associated with mycoplasma pneumoniae, mostly in children and adolescents [ ] . table . classification of erythema multiforme major (emm), stevens-johnson syndrome (sjs) and toxic epidermal necrolysis (ten) according to bastuji-garin et al. [ ] emm sjs a sjs-ten overlap ten ten with spots ten without spots/ten with widespread erythema systemic involvement systemic findings in sjs/ten include: ( ) flu-like symptoms (malaise, fever, anorexia) that are usually the initial signs of the disease in the prodromal phase prior to the cutaneous involvement. ( ) epidermal barrier breakdown-related symptoms including hypothermia, dehydration and sepsis. ( ) organ involvement induced by necrosis of epithelial lining, including respiratory distress syndrome, colitis, hepatitis and nephritis [ ] . characteristic histologic features include extensive keratinocyte destruction via apoptosis with separation of the epidermis from the dermis at the dermoepidermal junction. a paucicellular, dermal mononuclear infiltrate has been commonly described. lymphocytes cross the dermoepidermal junction with moderate infiltration of the epidermis. em and sjs often demonstrate less keratinocyte destruction on a background of extensive dermal mononuclear inflammation [ ] . in a retrospective analysis of the clinical records and histologic material of patients with ten, the histologic spectrum ranged from sparse to extensive dermal mononuclear inflammation, the extent of which predicts clinical outcome approximately as well as scorten. increased inflammation correlated with a worse prognosis; a mean cell count of dermal mononuclear > cells per high-power field predicted a worse prognosis ( %) vs % mortality in those with < cells in patients with % or more total body surface area sloughing [ ] . however, in a retrospective study analyzing clinical records and skin biopsy of patients with sjs, sjs/ten overlap and ten, dermal infiltrate severity was not associated with day- scorten or hospital death, but full-thickness epidermal necrosis was associated with mortality [ ] . diagnostic criteria based on integration of the major clinical characteristics of skin and mucous membrane findings, pathology assessment, lag period and systemic signs remain to be defined. the management of sjs/ten consists of a multidisciplinary approach that includes the following important aspects: . identification and withdrawal of the culprit drug: documenting the medication history during the previous months and withdrawal of all suspected and unessential medications [ ] . . transfer of the patient to intensive care, burn unit or other specialty unit: supportive care including thermoregulation, fluid replacement, nutritional support, monitoring for infection, sedation and pain management, and psychological support [ ] . . assessment of skin, mucous membranes and systemic involvement and the scorten score: type of lesions in the skin, extent of epidermal detachment, and mucous membranes and systemic involvement. all patients should be evaluated by an ophthalmologist promptly following the diagnosis and at regular follow-up intervals to minimize potential long-term ocular sequelae [ ] . possible acute manifestations include the eyelids, conjunctiva and cornea, and result in the classification of ocular involvement as mild, moderate or severe [ ] . bringing other specialists in on the patient's care is decided in accordance with the relevant findings. the scorten system, a severity-of-illness score for toxic epidermal necrolysis, developed to stratify severity of illness and predict mortality in patients with ten, includes seven independent risk factors: age, malignancy, tachycardia, initial body surface area of epidermal detachment, serum urea, serum glucose, and bicarbonate [ ] . . skin treatment: there are no clinical guidelines for the skin care of patients with sjs/ten. debridement of the necrotic epidermis was recommended in past publications [ , ] . recent publications advise avoiding debridement, which may cause hypertrophic scars, and recommend considering the detached epidermis as a natural biological dressing that favors reepithelialization [ , , ] . various topical treatments reported include bioactive skin substitutes, semi-synthetic and synthetic dressings, and topical antimicrobials [ , ] . a recent report on the management of sjs/ten in an experienced french referral center described the following treatment; wound care once a day with minimal manipulation to prevent skin detachment, including a bath containing a solution of chlorhexidine / (morphine is given prior to the bath and/or equimolar mix of oxygen and nitrogen monoxide during the bath); if bathing is not possible, the chlorhexidine solution is sprayed - times daily on the skin, blister fluid is aspirated while maintaining the blister roof, vaseline is systemically applied over all detached skin areas, topical sulfa-containing medications are avoided, and hydrocellular or absorbent nonadhesive dressings are applied at least once daily to cover pressure points [ ] . . mucous membranes treatment: specialized care is essential to prevent lifelong complications [ ] . although there is no standardized care for ocular management, the following supportive local treatment is advised: tear replacement solutions, removal of pseudomembranes, lysis of symblepharon, debridement of loosened epithelium, topical antibiotics to prevent secondary infection, topical corticosteroid to prevent scar formation, and cycloplegic drops to relieve pain, photophobia and ciliary spasm [ ] . amniotic membrane transplantation was found effective in the acute and chronic stages of sjs/ten [ , ] . a 'triple-ten' protocol for severe ocular cases was recently reported [ ] , comprised of the following: ( ) subconjunctival triamcinolone (kenalog mg) administered into each of the fornices to curb the local inflammatory response without compromising systemic immunity. ( ) placement of amniotic membrane tissue mounted on a polycarbonate skirt (prokera) over the corneal and limbal regions to facilitate reepithelialization of the ocular surface. ( ) insertion of a steeply curved acrylic scleral shell spacer (technovent, sc ) to vault the lids away from the globe and provide a barrier to symblephara formation. this treatment offers an effective therapeutic option, without the need for microsurgical equipment, microscope, or sutures in the critical care setting. oral-the mouth should be rinsed several times a day with an antiseptic or anifungal solution and the lips lubricated with an ointment such as dexpanthenol [ ] . genital-wet dressings or sitz baths and lubrication with emollient are recommended to avoid adhesions and strictures of genital erosions in females [ , ] . a specialist is required in case of involvement of other mucous membranes: respiratory, gastrointestinal and/or urethral. . systemic immunomodulatory treatment: the optimal therapeutic regimen has yet to be established, but according to recent publications, the following conclusions can be drawn: the use of ivig does not yield survival benefits in sjs/ten [ ] ; cyclosporine decreased the death rate and the progression of detachment (dosage of mg/kg/ day for days) [ ] ; systemic corticosteroids were associated with clinical benefit according to the euroscarstudy [ ] and were reported to be the most common treatment for sjs/ten in a recent survey of drug hypersensitivity experts from countries [ ] . one of the suggested protocols is iv dexamethasone . mg/kg pulse therapy (given for - min) for consecutive days [ ] . treatment with anti-tnf biologic treatment was reported to be beneficial [ ] [ ] [ ] . a prospective, randomized, open-label trial currently underway in taiwan [ ] comparing etanercept versus systemic corticosteroids in patients with sjs/ten, reported that the average duration to reach maximal skin detachment and complete skin healing was shorter in the etanercept group. in vitro investigations demonstrated that etanercept, steroids or thalidomide significantly decreased granulysin expression of blister cells. etanercept did not, however, increase the cytotoxic effect to keratinocytes found with thalidomide [ ] . . causality assessment and communication with the patient and his/her family, health-care providers and regulatory agencies: recent discoveries of specific hlas that predict genetic susceptibility to sjs/ten offer a simple, fast, safe and reliable method for establishing clear causality between a drug and a disease [ ] . the hlas are specific to a drug and an ethnic background [ ] . since these tests are available only for certain drugs and a negative test does not exclude the drug as the offending agent, additional clinical and laboratory methods are available for assessing causality. the mortality rates of sjs/ten are variable. that of ten may approach % [ ] , and that of children with sjs/ten is approximately - . % [ ] . in a large-scale, populationbased, -year follow-up study of sjs/ten patients, the -week in-hospital mortality rate was %, and the death rate from weeks to year was % [ ] . the mortality rate at year in this study was % for sjs, % for sjs and ten overlap, and % for ten. several factors were found to affect mortality: age, severity of reaction, recent malignancy, preexisting severe kidney or liver disorder, and recent infection. the last two factors were recognized for the first time in this study as being independent risk factors for death. all other factors are part of the scorten [ ] . the severity of the reaction was a major risk factor for death in the first few weeks, and severe co-morbidities and older age had major impact on mortality after weeks [ ] . early and late physical complications are common among patients who survive sjs/ten [ ] , with some % experiencing long-term sequelae [ ] . complications may affect multiple organ systems including skin, nails, hair, oral and genital mucosal sufaces, eyes, kidneys, gastrointestinal tract, and respiratory system [ ] . ocular complications, which can lead to blindness, are the major long-term morbidity [ ] . a few studies have dealt with the quality of life of patients surviving sjs/ten [ ] [ ] [ ] , which was found to be lower in every domain from before hospitalization to follow-up and a low rate of return to previous employment was documented [ ] . patients reported concerns about social interactions, fear of taking medications, and fear of contracting an illness necessitating medication [ ] . insufficient information and support for patients surviving sjs/ ten was also documented [ ] [ ] [ ] . unfortunately, because of the rarity of sjs/ten, most physicians are not aware of the long-term complications of the diseases [ ] . there are several methods to approach a patient with a cutaneous adr. the following is the authors' protocol: clinical assessment of drug-induced skin injury: ds by dr. shear a cutaneous eruption in a patient taking a medication should immediately raise the suspicion of a cutaneous adr. the physician must then determine whether the patient's clinical symptoms are signs of a cutaneous adr or of another skin disease not related to a drug. the diagnosis of a cutaneous adr is based on three key clinical elements (fig. . ) : ( ) appearancethe morphology of the cutaneous eruption according to four main categories of the primary lesion: maculopapular, urticarial, bullous and pustular (see section "morphological classification of cutaneous adrs"). ( ) systemicextra-cutaneous signs (fever, dyspnea, lymphadenopathy, etc.) that distinguish between a simple reaction involving only the skin and a complex reaction that includes systemic involvement in addition to the skin (see section "severity of cutaneous adrs: skin only (simple) versus skin and systemic involvement (complex)") and ( ) histologyhistopathology and direct immunofluorescence studies of skin biopsies to confirm the clinical impression and to distinguish between a drug-induced eruption and other skin diseases (see section "histological classification of cutaneous adrs"). establishing a differential diagnosis that takes into account all possible diagnoses is essential. ranking the approximate likelihood of each condition is encouraged. all medications, regardless of route of administration, must be considered, especially new drugs taken in the weeks prior to the skin reaction. drugs taken intermittently, such as vitamins, sedatives, pain relievers, laxatives and natural products, must also be considered. assessment of the lag period -the time between initiation of the drug and onset of the cutaneous reaction -is crucial in view of the different lag times for different cutaneous drug reactions. a recommended method for drug exposure analysis is to chart a timeline in order to visualize the chronology and facilitate comprehension of the event. the timeline includes the relevant information (starting day, dosage, and discontinuing day) for each drug and the signs and symptoms throughout the period in question [ ] . the most important challenge in assessing drug-induced skin injury is establishing whether there is a causal relationship between the suspected drug and the untoward clinical event. the following methods are helpful: ( ) patient history: the patient should be questioned about previous cutaneous reactions to drugs, and whether rechallenge with the drug improved the eruption [ ] . these data should also be part of the above timeline. ( ) good communication strategies will aid in the interactions with the patient and family following a cutaneous adr and decrease the likelihood of lawsuits, especially in cases of severe reactions such as sjs/ten. physicians are advised to follow these steps: ( ) express empathy and say "sorry" according to the "apology laws" in an honest and respectful fashion and in a way that protects the physician from having an apology used against him in case of legal action. http:// www.sorryworks.net/. ( ) provide disclosure in a "disclosure meeting" planned according to the acronym cones: context -arrange the setting for a quiet, uninterrupted meeting and decide on the participants; opening shot -the first sentence in the meeting explains the aim of the conversation; narrative -lay out the facts; it is advised to avoid using the words "error" and "mistake" since the adr is a result of multiple factors, particularly when the facts are not completely known; emotions -provide an empathic environment; summary ( ) provide the patient with clear information on his cutaneous adr, the name of the offending drug, potential cross-reacting drugs, and drugs which can be safely taken as an alternative to the offending drug. in addition, advise the patient to wear a medic-alert bracelet. ( ) family counselling is part of the management plan since the predisposition to some cutaneous adrs may be genetic [ ] . information on the adverse event must be provided to the family physician and entered in the patient's records. report the cutaneous adr to the manufacturer and regulatory agencies [ ] . the strong associations found between hla alleles and specific drug-induced hypersensitivity reactions have fostered pharmacogenetic testing to prevent the development of lifethreatening drug-induced hypersensitivity reactions, such as sjs/ten and dress. the usefulness of such testing is dependent on a number of factors, including the incidence and severity of the adverse event, the sensitivity and specificity of the predictive markers, and the availability of equally effective, alternative medications for individuals who test positive. although the incidence of sjs/ten is relatively low, it is life-threatening and many patients who survive have longterm sequelae, such as ocular complications. hla-b* is a useful and strong predictive marker with high sensitivity and specificity for carbamazepine-induced sjs/ten in asian populations. this genetic association is strong enough that it prompted the usfda and many countries to relabel the genetic information for carbamazepine, and to recommend screening for hla-b* before prescribing the drug for subjects of asian descent. the hla-b* test for carbamazepine-induced sjs/ten has very high sensitivity (near %) and specificity ( %). with the . % prevalence rate of carbamazepine-induced sjs/ten among chinese, the hla-b* test has a . % positive predictive value and % negative predictive value for detecting [ ] . in view of the serious consequences of sjs/ten and the availability of alternative drugs, withholding carbamazepine from screened patients who test positive for hla-b* and switching to alternative antiepileptic drugs is reasonable and feasible in the high risk populations, including chinese and south-east asians. abacavir is used in the treatment of hiv infection, and has been associated with drug hypersensitivity syndrome in % of patients [ ] . hla-b* is a strong and useful predictive marker with high sensitivity and specificity for abacavir hypersensitivity in caucasians, prompting the usfda and many other countries to recommend screening for it before prescribing the drug. the hla-b* test for immunologically-mediated abacavir hypersensitivity has very high sensitivity ( %) and specificity ( . %) as well as positive predictive value ( %) and negative predictive value ( %) [ ] . hla-b* is a potentially useful predictive marker for allopurinol-induced sjs/ten or dress, with % positive predictive value and almost % negative predictive value for detecting allopurinol-induced sjs/ten or dress in chinese (table . ). this association was significant in caucasian and other asian populations as well. the recent american college of rheumatology guidelines for the management of gout recommend hla-b* screening for populations with high frequency of the allele [ ] . other recently discovered hla alleles related to drug hypersensitivity of potential usefullness in clinic practice are hla-b* for dapsone hypersensitivity [ ] , hla-a* for carbamazepine-related dress [ ] , and cyp c * for phenytoin hypersensitivity [ ] . the lymphocyte transformation test (ltt) is a widely used in vitro assay for the diagnosis and identification of offending drugs with t cell-mediated drug hypersensitivity [ ] . ltt is based on the activation and proliferation of t cells from pbmc obtained from drug-sensitized patients after stimulation, and incubation with the culprit drug in vitro [ ] . following in vitro stimulation by specific drugs, drug-specific t cells are activated and release several cytokines that promote proliferation of t cells. this in vitro proliferation of specific drug-activated t cells can be detected by the incorporation of h-thymidine during dna synthesis after days of culture. the results of ltt are expressed as the stimulation index (si): the relationship between the h-thymidine uptake in cells (counts per minute (c.p.m.)) with and without the drug antigen [ ] . the general sensitivity of the ltt is - %, varying with different drugs and different phenotypes of delayed-type hypersensitivity reactions; thus, a negative result does not exclude the possibility of drug hypersensitivity. extensive studies on ltt for beta-lactam drugs report even higher sensitivity [ ] [ ] [ ] [ ] [ ] . the specificity of the ltt is - % in different studies [ ] [ ] [ ] ] . ltt for the diagnosis of drug hypersensitivity has limitations. because it is measured by radioisotopes, the sensitivity can be very low and negative results are commonly observed for specific drugs (e.g., allopurinol, lamotrigine) and specific phenotypes (e.g., sjs/ten) [ , ] . several nonradioactive methods have been developed for measuring lymphocyte proliferation or activation in in vitro tests for diagnosis of delayed-type drug hypersensitivity, including the use of carboxyfluorescein succinimidyl ester (cfse) cell staining dye [ , ] , and measuring cytokines or cytotoxic proteins expression, such as inf-γ, il- , il- , il- , il- , granzyme-b, and macrophage migration inhibitory factor [ ] [ ] [ ] [ ] [ ] . flow cytometry-assisted basophil activation test (bat), which measures specific cell makers such as cd or cd c to quantify basophil activation after antigenspecific stimulation, has been widely used in the diagnosis of immediate-type drug hypersensitivity [ ] . bat directly measures basophil responses instead of ige sensitization. it has been applied to the diagnosis of different drugs implicated in immediate-type hypersensitivity, including beta-lactam antibiotics, neuromuscular blocking agents, aspirin, nsaids and radiocontrast media [ ] [ ] [ ] . the sensitivity of bat varies in different types of drugs: that for beta-lactam antibiotics ranged from . to % [ , ] ; that for nsaids ranged from to % [ , ] . recent data have shown that the unique interaction between drug, t-cell receptor and hla molecule is a key factor in the development of immune-mediated adverse reactions to drugs. the discovery of strong association of specific hla alleles with specific drug-induced hypersensitivity (e.g., hla-b* to carbamazepine-sjs/ten, hla-b* to allopurinol-sjs/ten/dress, and hla-b* to abacavir hypersensitivity), and studies of the functional role of hla-b* allele (e.g., hla-b* ) directly interacting with a specific drug (e.g., carbamazepine) and unique t-cell receptor support the hypotheses of the 'pharmacological interaction with immune receptors' (p-i) [ , , ] . in recent years, bioinformatics and computer modeling have been applied to elucidate how drug molecules interact with specific hla in drug hypersensitivity. hla alleles have been associated with liver injury induced by different drugs (such as flucloxacillin). using silico strategies to examine hla haplotype relationships, and bioinformatics tools, alfirevic et al. [ ] demonstrated a connection between the different hla alleles associated with drug-induced liver injury caused by therapeutically and structurally different drugs, suggesting a mechanism of peptide binding of one of the associated hla alleles [ ] . computer modeling of the molecular interaction between hla-b* and carbamazepine predicted a favorable drug-binding position in the b pocket of the hla-b* protein, where the side chain of arg could form a hydrogen bond with the ketone group of -carboxamide of carbamazepine ( fig. . ) [ ] . cutaneous adrs have a wide spectrum of clinical manifestations that may be caused by multiple drugs and different mechanisms. in this decade, our understanding of the pathogenesis of cutaneous adrs had progressed greatly. understanding how a drug can possibly cause reactions in the skin has led to an understanding of the cellular immunology, cytokines and immunogenetics. these key insights can help mitigate the risk of reactions by testing for genetic factors, and to understand the treatment of drug reactions by better understanding the pathways involved. the future will depend on better genetic screening and directed approved therapies. internationl drug monitoring: the role of national centres adverse drug reactions: definitions, diagnosis, and management what is a serious adverse event? epidemiology of severe drug hypersensitivity epidemiology of cutaneous drug-induced reactions a comprehensive -year survey of adverse drug reactions using a network-based hospital system drug-induced cutaneous reactions. a report from the boston collaborative drug surveillance program on , consecutive inpatients the prevalence of acute cutaneous drug reactions in a scandinavian university hospital cutaneous adverse drug reactions: an -year retrospective study on hospitalized patients in southern china cutaneous adverse drug reaction profile in a tertiary care out patient setting in eastern india skin manifestations of outpatient adverse drug events in the united states: a national analysis who strategy for collecting safety data in public health programmes: complementing spontaneous reporting systems medical devices, the fda, and the home healthcare clinician the th international congress on cutaneous adverse drug reactions, taiwan, : focus on severe cutaneous adverse reactions mechanisms of drug-induced allergy the immunological and clinical spectrum of delayed drug-induced exanthems new aspects in betalactam recognition pharmacological interaction of drugs with antigenspecific immune receptors: the p-i concept shared and restricted t-cell receptor use is crucial for carbamazepine-induced stevens-johnson syndrome infiltration of cytotoxic t cells in drug-induced cutaneous eruptions the dress syndrome: a literature review delayed drug hypersensitivity reactions granulysin is a key mediator for disseminated keratinocyte death in stevens-johnson syndrome and toxic epidermal necrolysis delayed reactions to drugs show levels of perforin, granzyme b, and fas-l to be related to disease severity toxic epidermal necrolysis: effector cells are drugspecific cytotoxic t cells histopathological analysis and clinical correlation of drug reaction with eosinophilia and systemic symptoms (dress) nkp + cells express granulysin in multiple cutaneous adverse drug reactions drug specific cytotoxic t-cells in the skin lesions of a patient with toxic epidermal necrolysis nonallergic drug hypersensitivity reactions nonallergic hypersensitivity to nonsteroidal antiinflammatory drugs, angiotensinconverting enzyme inhibitors, radiocontrast media, local anesthetics, volume substitutes and medications used in general anesthesia immunohistology of drug-induced exanthema: clues to pathogenesis high il- production by human drug-specific t cell clones functional expression of the eotaxin receptor ccr in t lymphocytes co-localizing with eosinophils identification of thymus and activation-regulated chemokine (tarc/ccl ) as a potential marker for early indication of disease and prediction of disease activity in drug-induced hypersensitivity syndrome (dihs)/drug rash with eosinophilia and systemic symptoms (dress) increased frequencies of th cells in drug eruptions evaluation of the potential role of cytokines in toxic epidermal necrolysis expression of cytokines and chemokine receptors in the cutaneous lesions of erythema multiforme and stevens-johnson syndrome/toxic epidermal necrolysis molecular mechanism of tnf signaling and beyond cell death mechanisms induced by cytotoxic lymphocytes interferon-gamma: an overview of signals, mechanisms and functions regulation of mhc class ii expression by interferon-gamma mediated by the transactivator gene ciita antigen presentation by mhc class i and its regulation by interferon gamma immunoregulatory effector cells in drug-induced toxic epidermal necrolysis increased interleukin , tumor necrosis factor alpha, and interleukin levels in blister fluid of toxic epidermal necrolysis involvement of ccl -ccr interactions in drug-induced cutaneous reactions inhibition of toxic epidermal necrolysis by blockade of cd with human intravenous immunoglobulin expression and purification of kda granulysin utilizing an insect cell secretion system granulysin activates antigen-presenting cells through tlr and acts as an immune alarmin familial occurrence of stevens-johnson syndrome severe drug rashes in three siblings simultaneously stevens-johnson syndrome occurring in identical twins with apparent response to terramycin and aureomycin concordance of primary generalised epilepsy and carbamazepine hypersensitivity in monozygotic twins medical genetics: a marker for stevens-johnson syndrome hla-b* allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol hla-b* screening for hypersensitivity to abacavir hla-b* genotype is a major determinant of drug-induced liver injury due to flucloxacillin a genome-wide study identifies hla alleles associated with lumiracoxib-related liver injury hla-b* : and the dapsone hypersensitivity syndrome predisposition to nevirapine hypersensitivity associated with hla-drb * and abrogated by low cd t-cell counts hla-b* is strongly associated with methazolamide-induced stevens-johnson syndrome/toxic epidermal necrolysis genetic susceptibility to toxic epidermal necrolysis association between presence of hla-b* , hla-dr , and hla-dq and hypersensitivity to hiv- reversetranscriptase inhibitor abacavir recent advances in the genetics and immunology of stevens-johnson syndrome and toxic epidermal necrosis clinical pharmacogenetics implementation consortium guidelines for human leukocyte antigen-b genotype and allopurinol dosing hla-a* : and different types of carbamazepineinduced severe cutaneous adverse reactions: an international study and meta-analysis risk of stevens-johnson syndrome and toxic epidermal necrolysis in new users of antiepileptics severe cutaneous adverse reactions to antiepileptic drugs in asians common risk allele in aromatic antiepileptic-drug induced stevens-johnson syndrome and toxic epidermal necrolysis in han chinese genetic variants associated with phenytoinrelated severe cutaneous adverse reactions clinical and genetic risk factors of self-reported penicillin allergy gene-gene interactions of il and il ra variants in immediate allergic reactions to betalactam antibiotics relationships between specific serum ige, cytokines and polymorphisms in the il- , il- ralpha in patients with penicillins allergy polymorphisms of il- and il- -il- -snps in patients with penicillin allergies differential contribution of the cysltr gene in patients with aspirin hypersensitivity analysis of high-affinity ige receptor (fcepsilonr ) polymorphisms in patients with aspirin-intolerant chronic urticaria idiosyncratic drug reactions: the reactive metabolite syndromes drug hypersensitivity reactions involving skin the dose makes the poison on the nature of things classification, diagnosis, and therapy of immunological aspects of disease according to reaction types severe cutaneous adverse reactions: impact of immunology, genetics, and pharmacology drug eruptions: approaching the diagnosis of drug-induced skin diseases phenotype standardization for immunemediated drug-induced skin injury histologic diagnosis of inflammatory skin diseases: an algorithmic method based on pattern analysis pattern analysis of drug-induced skin diseases drug-induced cutaneous pathology granulomatous drug eruptions histopathological patterns indicative of distinct adverse drug reactions cutaneous drug eruptions: a -year experience cutaneous drug reactions in children: an update clinical practice. exanthematous drug eruptions recognition and management of severe cutaneous drug reactions urticaria and angioedema revisions to the international guidelines on the diagnosis and therapy of chronic urticaria recent advances in drug-induced angioedema erythema multiforme recognition and first-line treatment of anaphylaxis anaphylaxis in the young adult population webster's new world college dictionary drug-induced acneiform eruption acneiform eruptions associated with epidermal growth factor receptor-targeted chemotherapy semiology of skin toxicity associated with epidermal growth factor receptor (egfr) inhibitors. support care cancer acneiform eruptions histopathology of acneiform eruptions in patients treated with epidermal growth factor receptor inhibitors isotretinoin for high-grade or refractory epidermal growth factor receptor inhibitor-related acneiform papulopustular eruptions folliculitis induced by egfr inhibitors, preventive and curative efficacy of tetracyclines in the management and incidence rates according to the type of egfr inhibitor administered: a systematic literature review eosinophilic pustular folliculitis induced by chemotherapy eosinophilic cellulitis and eosinophilic pustular folliculitis eosinophilic pustular folliculitis induced by carbamazepine eosinophilic pustular folliculitis induced by allopurinol and timepidium bromide porphyria cutanea tarda-like aspects in two prolonged hemodialysis patients (author's transl) naproxen-induced pseudoporphyria in patients with juvenile rheumatoid arthritis pseudoporphyria induced by voriconazole pseudoporphyria induced by propionic acid derivatives diclofenac-induced pseudoporphyria; an under-recognized condition? unusual effects of antipyrine Éruption érythemato-pigmentée fixe due a l'antipyrine prognosis of generalized bullous fixed drug eruption: comparison with stevens-johnson syndrome and toxic epidermal necrolysis fixed drug eruptions: a case report and review of the literature fixed drug eruption: state of the art stevens-johnson syndrome and toxic epidermal necrolysis: clinical patterns, diagnostic considerations, etiology, and therapeutic management trials of penicillin therapy in massive doses in some dermatoses of unknown etiology pemphigus in a patient treated with penicillamine for wilson's disease bullous pemphigoid in an -year-old boy vancomycin-induced linear iga bullous dermatosis drug-induced pemphigus. i. a survey furosemide-induced bullous pemphigoid: case report and review of literature angiotensin-converting enzyme inhibitor-induced pemphigus: three case reports and literature review childhood pemphigus associated with montelukast administration bullous pemphigoid-an adverse effect of ampicillin bullous pemphigoid mimicking bullous erythema multiforme: an untoward side effect of penicillins drug-induced linear iga bullous dermatosis vancomycin-induced linear iga bullous disease presenting as toxic epidermal necrolysis drug-induced pemphigoid: bullous and cicatricial drug-induced pemphigus drug-induced pemphigus. ii. pathomechanisms and experimental investigations vancomycin-induced linear iga bullous dermatosis: morphology is a key to diagnosis drug-reaction eosinophilia and systemic symptoms and drug-induced hypersensitivity syndrome risk of serious cutaneous disorders after initiation of use of phenytoin, carbamazepine, or sodium valproate: a record linkage study drug reaction with eosinophilia and systemic symptoms (dress): an original multisystem adverse drug reaction. results from the prospective regiscar study drug reaction with eosinophilia and systemic symptoms: is cutaneous phenotype a prognostic marker for outcome? a review of clinicopathological features of cases short-and long-term outcomes of patients with drug-induced hypersensitivity syndrome in a single institution saliva polymerase chain reaction assay for detection and follow-up of herpesvirus reactivation in patients with drug reaction with eosinophilia and systemic symptoms (dress) drug-induced hypersensitivity syndrome (dihs): a reaction induced by a complex interplay among herpesviruses and antiviral and antidrug immune responses reactivation of human herpesvirus (hhv) family members other than hhv- in drug-induced hypersensitivity syndrome fever, rash, and systemic symptoms: understanding the role of virus and hla in severe cutaneous drug allergy drug reaction with eosinophilia and systemic symptoms (dress) syndrome and dysphagia: a noteworthy association drug reaction with eosinophilia and systemic symptoms (dress)/drug-induced hypersensitivity syndrome (dihs): a review of current concepts ceftazidime-induced drug reaction with eosinophilia and systemic symptoms (dress) complicated by hemophagocytic lymphohistiocytosis the variable clinical picture of drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms in relation to the eliciting drug drug-induced pseudolymphoma and drug hypersensitivity syndrome (drug rash with eosinophilia and systemic symptoms: dress) variability in the clinical pattern of cutaneous side-effects of drugs with systemic symptoms: does a dress syndrome really exist? dress syndrome: part ii. management and therapeutics management of drug reaction with eosinophilia and systemic symptoms (dress) recommendations for hla-b* : and hla-a* : genetic testing to reduce the risk of carbamazepineinduced hypersensitivity reactions long-term sequelae of drug reaction with eosinophilia and systemic symptoms: a retrospective cohort study from taiwan drug reaction with eosinophilia and systemic symptoms: a retrospective study of cases identifying prognostic factors for drug rash with eosinophilia and systemic symptoms (dress) serum sickness-like reactions from cefaclor in children intestinal mucosal permeability of children with cefaclor-associated serum sickness-like reactions serum sickness-like reaction in children due to cefditoren severe serum sickness-like reaction to oral penicillin drugs: three case reports minocycline-induced serum sickness-like reaction severe serum sickness-like type iii reaction to insulin detemir serum sickness-like reactions in patients receiving intravenous infliximab serious dermatologic reactions in children serum sickness-like reaction to cefuroxime: a case report and review of the literature serum sickness-like reactions serum sickness-like reaction: histopathology and case report cefaclor-a cluster of adverse reactions acute generalized exanthematous pustulosis (agep)-a clinical reaction pattern profile of acute generalized exanthematous pustulosis in israel during - : results of the regiscar study clinicopathologic manifestations of korean patients with acute generalized exanthematous pustulosis: a case series and review of the literature risk factors for acute generalized exanthematous pustulosis (agep)-results of a multinational case-control study (euroscar) acute generalized exanthematous pustulosis caused by sennoside in a patient with multiple myeloma acute generalized exanthematous pustulosis acute localized exanthematous pustulosis (alep) caused by amoxicillin-clavulanic acid acute generalized exanthematous pustulosis simulating toxic epidermal necrolysis: a case report and review of the literature overlap of acute generalized exanthematous pustulosis and toxic epidermal necrolysis: response to antitumour necrosis factor-alpha antibody infliximab: report of three cases acute localized exanthematous pustulosis (alep) caused by ibuprofen. a case report acute generalized exanthematous pustulosis: report of five cases and systematic review of clinical and histopathological findings systemic involvement of acute generalized exanthematous pustulosis: a retrospective study on patients the spectrum of histopathological features in acute generalized exanthematous pustulosis: a study of cases erythema multiforme, stevens-johnson syndrome and toxic epidermal necrolysis in children: a review of years' experience topical treatment options for drug-induced toxic epidermal necrolysis (ten) incidence of toxic epidermal necrolysis and stevens-johnson syndrome in an hiv cohort: an observational, retrospective case series study medications as risk factors of stevens-johnson syndrome and toxic epidermal necrolysis in children: a pooled analysis toxic epidermal necrolysis and stevens-johnson syndrome clinical risk management of stevens-johnson syndrome/toxic epidermal necrolysis spectrum stevens-johnson syndrome and toxic epidermal necrolysis: assessment of medication risks with emphasis on recently marketed drugs. the euroscar-study stevens-johnson syndrome and toxic epidermal necrolysis in sub-saharan africa: a multicentric study in four countries toxic epidermal necrolysis associated with mycoplasma pneumoniae infection case of fatal toxic epidermal necrolysis due to cardiac catheterization dye stevens-johnson syndrome and toxic epidermal necrolysis after vaccination: reports to the vaccine adverse event reporting system alden, an algorithm for assessment of drug causality in stevens-johnson syndrome and toxic epidermal necrolysis: comparison with case-control analysis clinical classification of cases of toxic epidermal necrolysis, stevens-johnson syndrome, and erythema multiforme toxic epidermal necrolysis: part i. introduction, history, classification, clinical features, systemic manifestations, etiology, and immunopathogenesis toxic epidermal necrolysis: part ii: prognosis, sequelae, diagnosis, differential diagnosis, prevention, and treatment stevens-johnson syndrome without skin lesions (fuchs syndrome): a literature review of adult cases with mycoplasma cause uncovering histologic criteria with prognostic significance in toxic epidermal necrolysis prognostic value of histologic features of toxic epidermal necrolysis toxic epidermal necrolysis clinical guidelines french referral center management of stevens-johnson syndrome/toxic epidermal necrolysis the ophthalmologist's role in the management of acute stevens-johnson syndrome and toxic epidermal necrolysis scorten: a severity-of-illness score for toxic epidermal necrolysis the current understanding of stevens-johnson syndrome and toxic epidermal necrolysis amniotic membrane transplantation as a new therapy for the acute ocular manifestations of stevens-johnson syndrome and toxic epidermal necrolysis indications and outcomes of amniotic membrane transplantation in the management of acute stevens-johnson syndrome and toxic epidermal necrolysis: a case-control study triple-ten" in the treatment of acute ocular complications from toxic epidermal necrolysis the role of intravenous immunoglobulin in toxic epidermal necrolysis: a retrospective analysis of patients managed in a specialized centre open trial of ciclosporin treatment for stevens-johnson syndrome and toxic epidermal necrolysis effects of treatments on the mortality of stevens-johnson syndrome and toxic epidermal necrolysis: a retrospective study on patients included in the prospective euroscar study dexamethasone pulse therapy for stevens-johnson syndrome/toxic epidermal necrolysis toxic epidermal necrolysis in a child successfully treated with infliximab toxic epidermal necrolysis successfully treated with etanercept etanercept therapy for toxic epidermal necrolysis comprehensive survival analysis of a cohort of patients with stevens-johnson syndrome and toxic epidermal necrolysis stevens-johnson syndrome and toxic epidermal necrolysis: improving the support to victims late outcomes in adult survivors of toxic epidermal necrolysis after treatment in a burn center internet accounts of serious adverse drug reactions: a study of experiences of stevens-johnson syndrome and toxic epidermal necrolysis patient experiences of serious adverse drug reactions and their attitudes to medicines: a qualitative study of survivors of stevens-johnson syndrome and toxic epidermal necrolysis in the uk a method for estimating the probability of adverse drug reactions evaluation of the extent of under-reporting of serious adverse drug reactions: the case of toxic epidermal necrolysis human leukocyte antigens and drug hypersensitivity hypersensitivity reactions during therapy with the nucleoside reverse transcriptase inhibitor abacavir american college of rheumatology guidelines for management of gout. part : systematic nonpharmacologic and pharmacologic therapeutic approaches to hyperuricemia the lymphocyte transformation test in the diagnosis of drug hypersensitivity the lymphocyte transformation test for the diagnosis of drug allergy: sensitivity and specificity skin and laboratory tests in amoxicillinand penicillin-induced morbilliform skin eruption in vitro t-cell responses to beta-lactam drugs in immediate and nonimmediate allergic reactions t cell involvement in cutaneous drug eruptions t-cell reactions to drugs in distinct clinical manifestations of drug allergy characterization of drug-specific t cells in lamotrigine hypersensitivity poor relevance of a lymphocyte proliferation assay in lamotrigine-induced stevens-johnson syndrome or toxic epidermal necrolysis in vitro drug causality assessment in stevens-johnson syndrome -alternatives for lymphocyte transformation test allergen-specific t-cell response in patients with phenytoin hypersensitivity; simultaneous analysis of proliferation and cytokine production by carboxyfluorescein succinimidyl ester (cfse) dilution assay long-lasting reactivity and high frequency of drug-specific t cells after severe systemic drug hypersensitivity reactions in vitro detection of cytotoxic t and nk cells in peripheral blood of patients with various drug-induced skin diseases drug-specific in vitro release of il- , il- , il- and ifn-gamma in patients with delayed-type drug hypersensitivity detection and quantification of drug-specific t cells in penicillin allergy the appearance of macrophage migration-inhibition factor in drug reactions macrophage migration inhibition factor (mif) in drug eruption predicting drug hypersensitivity by in vitro tests recent applications of basophil activation tests in the diagnosis of drug hypersensitivity a new basophil activation test using cd and ccr in allergy to antibiotics usefulness of the basophil activation test (bat) in the diagnosis of life-threatening drug anaphylaxis basophil activation and sulfidoleukotriene production in patients with immediate allergy to betalactam antibiotics and negative skin tests diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study the flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal antiinflammatory drugs (nsaids) is useful for in vitro diagnosis of the nsaid hypersensitivity syndrome basophil activation tests in the diagnosis of drug reactions direct interaction between hla-b and carbamazepine activates t cells in patients with stevens-johnson syndrome in silico analysis of hla associations with drug-induced liver injury: use of a hla-genotyped dna archive from healthy volunteers key: cord- - d authn authors: gil, jp; gil berglund, e title: cyp c and antimalaria drug efficacy date: - - journal: pharmacogenomics doi: . / . . . sha: doc_id: cord_uid: d authn malaria is a major infectious disease. in the last years it has killed more than million people, mainly small children in africa. the highly efficacious artemisinine combination therapy is being launched globally, constituting the main hope for fighting the disease. amodiaquine is a main partner in these combinations. amodiaquine is almost entirely metabolized by the polymorphic cytochrome p (cyp) isoform c to the pharmacologically active desethylamodiaquine. the question remains whether the efficacy of amodiaquine is affected by the gene polymorphism. genotype-inferred low metabolizers are found in – % of african populations, which corresponds to millions of expected exposures to the drug. in vivo pharmacokinetic data on amodiaquine is limited. by combining it with published in vitro pharmacodynamic and drug metabolism information, we review and predict the possible relevance, or lack of, of cyp c polymorphisms in the present and future efficacy of amodiaquine. chloroquine and dapsone, both substrates of cyp c , are also discussed in the same context. malaria is a major infectious disease. in the last years it has killed more than million people, mainly small children in africa. the highly efficacious artemisinine combination therapy is being launched globally, constituting the main hope for fighting the disease. amodiaquine is a main partner in these combinations. amodiaquine is almost entirely metabolized by the polymorphic cytochrome p (cyp) isoform c to the pharmacologically active desethylamodiaquine. the question remains whether the efficacy of amodiaquine is affected by the gene polymorphism. genotype-inferred low metabolizers are found in - % of african populations, which corresponds to millions of expected exposures to the drug. in vivo pharmacokinetic data on amodiaquine is limited. by combining it with published in vitro pharmacodynamic and drug metabolism information, we review and predict the possible relevance, or lack of, of cyp c polymorphisms in the present and future efficacy of amodiaquine. chloroquine and dapsone, both substrates of cyp c , are also discussed in the same context. malaria, aids and tuberculosis (tb) are the three top killing communicable diseases. malaria alone claims at least one life every s. the overall burden of malaria can reach million casualties/year, mostly among children aged under years and pregnant women in sub-saharan africa [ , ] . the human malaria agents are parasites belonging to the genus plasmodia. the clinically most important among them are p. vivax and p. falciparum, the latter being responsible for the mortality associated with the disease. even after decades of research, the development of a vaccine with significant impact in the endemic populations is still many years away [ ] . this leaves drug therapy as the main tool for the global treatment and control of the disease, presently in the successful shape of the highly effective combination of artemisinine (art) derivatives with longer half-life partners [ ] . amodiaquine (aq), a -aminoquinoline, is one of the most important drugs for malaria control in the african continent, being the basis of the first-line treatment in countries as an art combination therapy (act) drug partner (aq-artesunate). aq is a very specific substrate of cytochrome p (cyp) c [ ] , to the extent of constituting a potential in vivo probe drug for this enzyme [ ] . cyp c has been also demonstrated in vitro to metabolize the presently less relevant antimalarial drugs chloroquine (cq) and dapsone [ , ] . compared with their importance, the body of knowledge concerning the enzymes involved in the metabolism of these antimalarials is disproportionately small [ ] . accordingly, in vivo data regarding the relation of the efficacy of these drugs with pharmacogenetics is generally not available. the objective of this review is to contribute to filling this gap by inferring from the available data the relevance -or lack of it -of the cyp c gene and its polymorphisms in the presently applied chemotherapies, with particular focus on aq. this will be described in the context of the current knowledge concerning parasite multidrug resistance mechanisms and the specific scenario of malaria as a major infectious disease. unless otherwise stated, this review focuses on p. falciparum malaria. the cyp c genes are located in a cluster at chromosome q , organized as cent-cyp c -cyp c -cyp c -cyp c -tel [ ] . the cyp c gene spans approximately kb, including nine exons (figure ) [ ] . this open reading frame codes for a amino acid protein, which quantitatively represents approximately % of the total liver cyp enzymes and approximately % of the hepatic cyp c subfamily content. among the major members of the cyp c subfamily ( c , c and c ), cyp c is the most divergent in terms of protein sequence: while cyp c and cyp c share over % of homology, cyp c is only % similar to each one of these enzymes. notably, the cyp c active site has recently been shown to be significantly different from the other members, in size ( Å ) and shape [ ] . the volume of the active pocket allows the accommodation of relatively large molecules, probably contributing for the differences in ranges of substrates as compared with c and c [ ] . cyp c transcription activity has been identified in extrahepatic tissues, including the kidneys, adrenal gland, uterus, brain [ , ] , mammary glands, ovaries, duodenum [ ] , small intestine, lung, prostate, testis [ ] and the heart [ ] . the actual cyp c protein has been immunologicaly identified in the kidneys, gut (small and large intestine), salivary glands, adrenal cortical cells and tonsils [ ] . several nonhomologous single nucleotide polymorphisms (snps) have been identified in the cyp c open reading frame, giving rise to at least ten formally distinct alleles [ ] . besides the alleles found in the original communications on cyp c biodiversity (* ,* ,* ,* ) [ , ] , the remaining were discovered in surveys conducted in japan (* -* ) [ ] [ ] [ ] . cyp c null homozygous have not been described, pointing to the probable physiological importance of this gene. cyp c * and * are the most frequent alleles found worldwide. the former is characteristic of populations of african origin (table ) , while * is essentially present among caucasians. orientals seem to have very high prevalence of wildtype alleles, although more extensive studies are warranted (e.g., in the large populations of china and india). both alleles code for enzymes with decreased performance: while the cyp c * allele was observed to cause a twofold increase on the k m for paclitaxel transformation [ ] ; * was documented to be associated with an arachidonic acid hydroxylation turnover of only % as compared with the wild-type allele (* ) [ ] . in figure , the structure of the cyp c gene is presented, as well as a compilation of snps published or readily accessible in several internet-accessible databases. in table , the available data on the frequencies of the main studied alleles (* , * and * ) in malaria endemic regions is presented. only the single nucleotide polymorphisms present in the coding region of the gene are represented. ºalleles coding for documented or predicted reduced enzyme activity; * : two-fold higher k m for paclitaxel transformation [ ] ; * : % of * -associated turnover number [ ] ; * : expected to be an inactive gene [ ] ; * : no in vitro detectable activity [ ] ; * : in vitro enzyme activity % of the wild-type [ ] ; p a: reduced protein expression leading to decreased v max /k m [ ] . malaria infection starts with the injection of malaria sporozoites during the blood meal of an infected female mosquito (anopheles spp.). in min these forms will invade the liver. in - days, liver schizontes will mature, each one giving rise to tens of thousands of merozoites. these are released in the blood stream, where they promptly invade red blood cells, initiating the intraerythrocytic cycle. asexual multiplication progresses in this cycle, with invading merozoites growing into trophozoites that will mature towards multinuclear intraerythrocytic schizonts. each of these will provide - new merozoites, which are released at the burst of the erythrocytes. these will attack new red blood cells, re-initiating the typical (p. falciparum, and p. vivax) hour intraerythrocyte cycle. during this process some merozoites will develop into gametocytes, sexual forms that, if ingested by the mosquito during the blood meal, will progress into fertilization and production of new sporozoites, ready for re-invading the human host and completing the overall cycle. in the case of p. vivax, the parasites can stay dormant in the liver for years before developing blood-stage infections. all drugs herein discussed act in the intraerythrocyte cycle. act is presently being adopted by most malaria-endemic countries for the control of the disease. the combination has its basis on the observation that art represents the most powerful class of antimalarials in use in terms of parasite reduction ratio (prr) [ ] , that is, the ratio of the parasitemia before the initiation of the treatment with the value registered h later (one p. falciparum intraerythrocytic cycle). this has been shown to be approximately (reduction to . % of the initial parasite load), which is significantly different from, for example, the - value characteristic of mefloquine. although rapid killers, art have short half-lives ( . - h), implicating a characteristically long monotherapy treatment course for any coadministrated antimalarial. in act, art is combined with a long halflife antimalarial expected to have a different mechanism of action (e.g., aq, of importance in this review). the basic rationale (figure ) is [ ] : • art acts very fast, significantly reducing the parasite load in the first hours of treatment; • the slowly eliminated partner drug (e.g., a quinoline antimalarial) is left to face a much decreased number of parasites; • as the mechanisms of action of art are expected to be different from the partner drug, the latter also acts over a particularly sensitive surviving population of parasites potentially selected by art; • the effect is further enhanced by the frequent pharmacodynamic synergism between its components (e.g., artesunate and mefloquine), which constitutes a frequent further advantage of act. act is an actual combination for a relatively short period of time due to the short half-life of art. some hours after the administration of act, the partner drug is essentially alone in circulation, that is, act becomes a monotherapy. in regions of low malaria transmission (e.g., most of south east asia) the risk * = . ; * = . ; * = . ; * = . ; * = . [ ] . cyp: cytochrome p ; lm: low-metabolizer, inferred from the frequency of subjects homozygous for the mutant alleles. future science group future science group of re-infection is low, so the long half-life partner drug will not interact with new parasites until it is totally eliminated. the situation in high transmission zones is different -a cured patient can be re-infected in just some days, at a point when the plasma concentrations of the long half-life partner drug are at subtherapeutic levels. the new incoming parasites will be exposed to low drug concentrations that prompt the selection of low-resistance ones ('tolerant parasites') [ ] . progressively, the population is expectedly stepwise selected to ever higher levels of tolerance until it reaches the previously established therapeutic window, generating clinical failure (in vivo resistance). amodiaquine: a main partner in artemisinine combination therapy aq was synthesized for the first time over years ago. the drug is, in general, clinically effective against chloroquine-resistant strains, although cross-resistance with this drug has been documented for at least years [ ] . aq is a major combination therapy partner, being mainly employed as act with the artemisinine derivative artesunate, being a main first-line treatment in west african countries in particular [ ] . aq is usually given in combination with artesunate as oral doses of mg/kg aq and mg/kg artesunate once daily for days. the two drugs are presently supplied separately. a future convenient aq-artesunate fixed combination (coarsucam™) is being developed by sanofi-aventis in collaboration with medicine for malaria venture [ ] and is presently under efficacy trials [ ] . its expected low price -less than us$ . for a children treatment coursewill probably command an increase of its use in african populations. an alternative combination, aq-sulfadoxine-pyrimethamine, has also been advocated, particularly for countries not ready to afford the rapid implementation of act [ ] . this combination is currently being employed under official malaria control programs in papua new guinea and colombia. artemisinine combination therapy is the main hope for the global control of malaria. the strategy is based on an elegant and simple concept, herein depicted from the example of the artesunate-mefloquine combination in current use in thailand. the very fast parasite reduction rate (prr) of artesunate ( / , ) [ ] rapidly reduces the parasite biomass, allowing the partner drug (mefloquine [mq], with a substantial slower prr, / ) to face a number of parasites several orders of magnitude smaller. no parasites remain when mq serum concentrations reached subtherapeutic levels where selection of tolerants might ocour. the expected different mechanisms of action of these structurally different drugs, associated with their documented in vitro pharmacodynamic synergy, further contributes to protect the combination from resistance. figure adapted from [ ] and [ ] . p. falciparum image courtesy of b schmidt, department of medicine, karolinska institute, sweden. although the main target patient population is pediatric, aq pharmacokinetics has mainly been studied in adults. this should be kept in mind when extrapolating the pharmacokinetic data to the clinical situation as the children may have higher or lower clearance/kg body weight, depending on their age and stage of maturation. the plasma concentration time course is biphasic with a fast distribution phase and a slower elimination phase. after an intravenous -h infusion of mg base/kg to ten adult patients with p. falciparum malaria, the half-life of the first disposition phase was min (geometric mean; range - min) and the halflife of the terminal phase h (geometric mean; range - h). clearance was l/h/kg (range - ) [ ] . after an intravenous injection of mg base/kg to six healthy volunteers, similar characteristics were observed but clearance was higher (geometric mean l/h/kg; range - ) and the terminal half-life shorter (geometric mean h, range . - h) than in patients. these differences in observed clearance may be due to a reduced hepatic bloodflow or reduced enzyme activity in acute malaria. it is possible that there is another disposition phase of aq with a longer half-life. this is indicated by the detection of aq in urine for longer time than expected by winstanley and colleagues [ ] . the authors only present long-term urinary data for one subject, but in this subject . µg/ml aq was observed months after a mg oral single dose. aq pharmacokinetics is independent of dose in the dose-range - mg administered as single doses to healthy volunteers. the drug appears to be eliminated mainly through metabolism with some contribution of renal excretion [ ] . the main metabolite observed is the pharmacologicaly active desethylamodiaquine (deaq) (figure ) , which is found in large amounts in the circulation. two other metabolites, -hydroxy-deaq and n-bisdesenthyl-aq, have also been found in the circulation in similar or higher concentrations than aq [ , ] . after oral administration, the exposure of deaq is many-fold higher than the exposure of aq. after a mg oral dose of aq to healthy volunteers, the deaq:aq area under the plasma-time curve (auc) -∞ ratio was approximately (the estimate is rough as both auc -∞ values included a large area extrapolated from the last data point). deaq accumulates in the red blood cells, reaching a red blood cells/plasma ratio of [ ] . both aq and deaq are highly (> %) protein-bound [ ] . deaq is assumed to be the main entity responsible for the therapeutic effect of aq treatment. the disposition of deaq in healthy volunteers after a mg/kg oral dose of aq base has been observed to have three phases; two initial disposition phases with half-lives of - h and - h and a terminal phase with a half-life of - days in plasma and - days in whole blood [ ] . in vitro metabolism studies with enzyme expression systems, human liver microsomes, enzyme activity correlation analysis and enzyme docking analysis, strongly indicate that cyp c is the only hepatic cyp isoform involved in the formation of deaq [ ] , and the main hepatic enzyme responsible for aq metabolism. deaq appears to be eliminated through renal excretion as well as further metabolized by other, probably extrahepatic, cyp enzymes, namely cyp a [ ] (i cavaco, university of algarve, faro, portugal. unpublished data). cyp c polymorphisms probably influence amodiaquine metabolism there is, as yet, no direct demonstration of the influence of the cyp c inhibition or polymorphism on the pharmacokinetics, efficacy and safety of aq in malaria settings. an expected importance of the polymorphism stems from the following observations: • cyp c is essentially the sole hepatic cyp enzyme catalyzing the formation in vitro of deaq [ ]; • significant variability in the pharmacokinetics of aq has been documented [ , ] ; • the main studied cyp c polymorphisms are associated with significant alterations in in vitro enzyme activity [ , , ] . the relation between these alleles and in vivo pharmacokinetics (pk) parameters is less clear, partly because of the fact that the tested drugs are also metabolized by other cyps. in fact, although some reports did find significant difference in the pk of the drugs under study [ ] [ ] [ ] , others did not observe it, albeit with other cyp c substrates [ ] [ ] [ ] . it is important to emphasize that aq is actually one of the best candidates as an in vivo cyp c probe substrate [ ] . from this scenario, it is presently only possible to predict the importance of cyp c for the efficacy of aq. for this purpose it is important to also take into account some characteristics of the variable response of the pathogen to aq and deaq. pharmacogenomics ( ) ( ) future science group future science group p. falciparum amodiaquine/ desethylamodiaquine resistance malaria therapy with aq is in most cases effective against cq resistance p. falciparum. nevertheless, events of the parasite being simultaneously refractory in vivo to both drugs are not rare, pointing for a certain degree of cross-resistance [ ] . in vivo p. falciparum resistance to aq regimens seems to be associated with mutations in the parasite p. falciparum cq resistance transporter (pfcrt) gene [ ] , coherent with the occasional observation of cq/aq clinical cross-resistance. pfcrt codes for a transmembrane protein located in the parasite food vacuole, the central organelle in the metabolism of hemoglobin, an essential requirement for the progression of the parasite cycle. during the metabolism of hemoglobin the toxic heme is liberated from its molecular scaffold. cq (and possibly aq or deaq) binds heme, disturbing its inactivation by the parasite [ ] . pfcrt snps, in particular a k t variation, render the parasite resistant to the drug, probably through the efflux of cq from the organelle. cq-resistance is reversible in vitro by the action of the calcium blocker verapamil (vp), known to interfere specifically with k t harboring pfcrt proteins. interestingly, although deaq sensitivity is reversible with vp, aq response is not significantly affected [ ] . this data points for pfcrt k t to be associated mainly with deaq response, partially explaining previously observed in vivo cq/aq cross-resistance in vivo, which should maybe be better interpreted as cq/deaq. furthermore, due to the aforementioned 'tail effect', the slowly eliminated deaq is predicted to be the weak link promoting resistance in the aq-art combination. can cyp c polymorphisms influence amodiaquine regimens efficacy & safety? the effect of cyp c polymorphism on deaq auc is dependent on the contribution of this pathway to aq total clearance. in vitro, cyp c was found to be the main hepatic cyp involved in the metabolism of this drug. however, some contribution of the extrahepatic enzymes cyp a and cyp b was also observed [ ] . no in vivo data on the contribution of different elimination pathways has been documented. if the extrahepatic pathways are considered likely to be of minor importance for total clearance, deaq auc will not be significantly affected by cyp c polymorphism. the deaq formation will be slowly giving rise to some decrease in maximum plasma concentration (c max ), with the auc not expected to be affected. aq is a more active antimalarial then deaq, with half-maximal inhibitory concentration (ic ) values - -fold lower [ , , ] . the little information available shows that the c max of aq is relatively low -approximately nm ( mg dose) - [ ] , falling anyway inside the in vitro ic values of a significant fraction of field analyzed aq sensitive parasites [ , [ ] [ ] [ ] [ ] [ ] . this suggests that aq might give a small but non-negligible contribution for the success of the therapy, particularly among a predicted cyp c 'low-metabolizer' genotype carrier associated with an increased drug exposure for the parasite. the positive effect of an increased aq exposure might not be evident in these early days of high efficacy act, as observed in a small trial of patients in papua new guinea [ ] . in this study, all patients were determined as cyp c wild type and were successfully treated, the authors concluding that "the aq regimen seems pharmacokinetically adequate in the absence of cyp c polymorphism" [ ] . nevertheless, with the potential development of p. falciparum resistance to deaq, the benefit of a high aq exposure might increase. this contribution is probably more important through another venue: in vitro experiments have consistently shown that aq is capable of marked synergism with artemsinine derivatives [ , ] and, most notably, with its own deaq metabolite [ ] . again, the presence of cyp c alleles predisposing for a higher aq exposure will be expected to prolong the synergistic influence over deaq. results in vitro show that aq is able to significantly enhance the action of its metabolite towards extraordinary low aq:deaq ratios, beyond : , . this observation implicates that although aq serum levels generally drops below the present level of detection (approximately nm) h after administration, very low concentrations -down to the picomolar range -may significantly aid deaq antiparasitic action for several days. again, low-metabolizer patients are potentially associated with an enhanced performance of the aq chemotherapy, possibly even in areas where deaq resistance might be rising, as this synergistic effect seemed to be independent at least of the pfcrt k t status of the tested parasites [ ] . in the s, reports documented fatal adverse drug reactions among travelers under aq-based prophylaxis (incidence : ), mainly through agranulocytosis and hepatic toxicity [ ] . this led to a who recommendation towards the withdrawal of aq from malaria control programs, a decision later considered 'more prudent than practical' [ ] . in vitro studies have shown that when compared with deaq, aq is more frequently transformed to the highly reactive quinoneimine species proposed as responsible for adverse events associated with aq profilaxis [ ] [ ] [ ] . hence, being a low-metabolizer potentially elevates the risk of aq associated serious side effects [ ] . treatment with aq has not been documented to be associated with severe adverse effects, showing to be not more toxic then cq [ , ] . however, more time and further pharmacovigilance efforts are needed for reliable conclusions to be drawn [ ] , in particular in a scenario of possible increase in aq doses following a significant decrease in parasite susceptibility to this chemotherapy. these can be aided by the identification of carriers of cyp c alleles predicted to be associated with aq low metabolism. in the african continent, the analysis of the * allele would be sufficient to identify most of these. * /* carrying patients could be selected for enhanced surveillance. cyp c has been shown to be readily inducible in vitro via nuclear receptors pregnane x receptor (pxr), constitutive androstane receptor (car) and glucocorticoid receptor (gr) activators [ ] . recently, it has been demonstrated that artemisinine, artemether and artether derivatives are potent pxr/car agonists, leading to the induction of the cyp b , cyp a and mdr in primary hepatocytes [ ] , as well as to increased cyp b and cyp a enzyme activities in vivo in a rodent model [ ] . although the authors did not test the induction potency of artesunate, neither the induction of cyp c , the possibility remains that when used in act, aq might be metabolized at a higher rate during act due to artesunate-based activation of pxr and/or car [ ] . furthermore, it is also possible that other commonly co-administered pxr activators, such as the antituberculosis agent rifampicin and some of the hiv drugs, may further induce the cyp c expression. such interactions are likely to reduce aq exposure. in this scenario, the relative positive impact of low-metabolizer status in the efficacy of aq might still be present, as a less active enzyme will be induced as compared with an induced fast-metabolizer. the importance of cyp c for drug metabolism has not been acknowledged until recently; hence few drugs are known to markedly inhibit cyp c in vivo. a number of drugs have been identified as potent cyp c inhibitors in vitro, with predicted risks of drug-drug interactions with substrates of this enzyme having been calculated (inhibitor concentration/inhibition constant values) [ ] . some cyp c inhibitors are under therapeutic use in areas where aq is also being administered for malaria treatment, including ritonavir (anti-hiv drug), ketoconazole (antifungal) and the antibacterial thrimethoprim. pharmacogenomics ( ) ( ) future science group future science group aq low-metabolizers can possibly be at additional risk due to their lower enzyme activity during cotreatment with cyp c inhibitors. due to the development of parasite resistance, cq is presently being officially phased out in most endemic countries. it is still part of the national malaria control strategy in several countries, for example, congo brazzaville and togo [ ] . in other regions, although other drugs have been adopted, their implementation will still take significant time extending the use of this drug. cq is still a main drug of choice at a global level for the treatment of p. vivax malaria. chloroquine pharmacokinetics cq is well absorbed after oral administration and has a bioavailability of approximately % [ ] . cq is eliminated through metabolism, as well as renal excretion, the latter pathway accounting for approximately % of total drug clearance [ ] . the main metabolite of cq is n-desethylchloroquine [ ] . other metabolites, such as bisdesenthyl chloroquine and -chloro- aminoquinolone, are also formed. bisdesenthyl chloroquine is present in concentrations approximately % of those of cq. the metabolite is pharmacologically active and has been proposed to be further metabolized by the enzyme also catalyzing its formation [ ] . cq exhibits a very long terminal half-life (up to days). the length of the half-life is mainly caused by its very large volume of distribution (up to l/kg), as the clearance of the drug is relatively rapid (up to l/h/kg). cq is metabolized in vitro by cyp c and cyp a , with a non-negligible contribution of cyp d , particularly at drug concentrations below µm. [ ] . the quantitative contributions of these enzymes in vivo are not clear. due to the large contribution of renal excretion and the multiple enzymes involved, at least in vitro, the effect of cyp c polymorphism is not expected to be marked in patients with a normal renal function. it is to note that patients with malaria have been observed to have a minor impairment of renal function [ ] . however, the impairment may be too small to significantly affect cq excretion. dapsone is, with chlorproguanil, a part of the antifolate lapdap™ combination, developed as a future replacement of sulfadoxine-pyrimethamine, as it is active against parasites resistant to this drug [ ] . resistance is expected to develop slower than with sulfadoxine-pyrimethamine, as both dapsone and chlorproguanil have significantly shorter half-lives, hence avoiding the 'tail effect' [ ] . unfortunately, the mechanism of resistance to these drugs is probably similar to the associated sulfadoxine-pyrimethamine (snps in p. falciparum dihydrofolate reductase and dihydropteroate synthase coding genes), well known to be particularly fast to rise [ ] . dapsone is reversibly metabolized to monoacetyldapsone, but the major pathway appears to be the formation of dapsone hydroxylamine, which has been associated with toxicity. the latter is cyp-catalyzed [ ] . in vitro studies performed at clinically relevant concentrations of dapsone ( µm) have shown that the drug is mainly metabolized by cyp c , with an expected minor cyp c contribution [ ] . this data, although not yet confirmed in vivo, points for a minor contribution of genetic cyp c variability for the pharmacokinetics, efficacy and safety of the drug. antimalarials are administered in high numbers in the developing world, in many settings as a default for most fever events without confirmation of the presence of the parasite. in a time when large changes in the national drug policies are underway, a more detailed knowledge on host factors influencing the efficacy and safety of the act partner drugs should be mandatory. this includes the understanding of the pharmacogenetic characteristics of the target populations. cyp c appears to be the only hepatic cyp enzyme metabolizing aq, suggesting that polymorphisms in this gene can be useful tools for predicting individual exposure to the drug. unfortunately, the importance of its polymorphism in the individual exposure to aq is still to be experimentally established. furthermore, the bulk of data concerning the pharmacokinetics of aq is almost two decades old, involving only small studies, mainly with adult subjects. new investigations adapted to the present clinical scenario of the use of aq in africa are as urgent as fundamental. in case of its development, such molecular tools might be used as a convenient way for sampling populations under aq-based first-line chemotherapies, determining the likelihood of a patient to come under higher-than-average exposure. this can be of importance in eventual changes in dosing, for example, due to increased future science group future science group www.futuremedicine.com p. falciparum deaq tolerance. this would also be valuable information for the future introduction of other drugs also metabolized by cyp c . nevertheless, it is to note that in this moment, when p. falciparum resistance to the newly employed drugs is still not maturated, the influence of cyp c pharmacogenetics in the efficacy of aq-based regimens is less likely to be important. however, the increased exposure to aq in poor-metabolizers may predispose to adverse events. finally, the influence of cyp c polymorphism in the therapeutic efficacy of cq and dapsone is not expected to be significant. p. falciparum has shown in the last decade to be a resilient pathogen, able to timely develop resistance to every applied antimalarial. in the long term, it is not unexpected that the same will happen with the presently used chemotherapies. in the specific case of aq, a shrinking of the therapeutic window in the next years, particularly through falling efficacy of the long half-life deaq, might highlight the importance of interindividual differences in aq exposure. future research will clarify the influence of cyp c polymorphisms in the modulation of aq exposure and the associated clinical efficacy, and potential risk of adverse reactions. in the next years, this might permit the development of cyp c allele analysis as one of a set of convenient molecular tools aiding pharmacovigilance programs of massive employed drugs in the developing world. in the case of africa, where most of the aq is presently being used, the main nonwild-type allele present is cyp c * , leading to the notion that the sole analysis of the i f snp will cover most of the predicted low-metabolizer individuals. this simplified approach can facilitate the logistics of the future implementation of this type of tool, potentially important in the definition of groups with an expected different therapeutic window from the * /* carrier majority. adjustments of the drug dosage might be of interest for exploiting in the best way the pharmacokinetic characteristics of this minority group. • cytochrome p (cyp) c is essentially the sole cyp enzyme involved in in vitro amodiaquine (aq) metabolism. • the cyp c gene shows polymorphism. null alleles are known, but are very rare. over % of the mutant allele frequency is explained in most populations by the presence of three variants (* , * and * ). these are expected to influence aq exposure, although the present data indicates that the exposure to desethylamodiaquine (deaq) will be approximately the same. studies formally confirming this hypothesis are still missing, not currently allowing a precise picture to be formed. • the efficacy of aq seems to be presently satisfactory in those homozygous for the cyp c * allele, although more data needs to be gathered, particularly concerning the aq-artesunate combination. clinical outcome is more certain to be influenced by parasite characteristics and other factors. however, variable cyp c activity can become important in the future event of increased p. falciparum insensitivity to deaq: low-metabolizers will have more contribution of aq in the elimination of deaq-tolerant parasites, particularly through the marked synergy of aq with deaq and artesunate. • on the other hand, aq poor-metabolizers are probably at higher risk of adverse events related to this drug. this may prove to be even more important if the aq dose is increased in order to circumvent deaq tolerance. • cyp c is probably of low relevance in the performance of chloroquine and dapsone. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers. n-hydroxylation at clinical concentrations of dapsone impact of pharmacogenomics on neglected diseases of the developing world a . -megabase physical map spanning the cyp c gene cluster on chromosome q gene structure of cyp c and extrahepatic distribution of the human cyp cs structural diversity of human xenobiotic-metabolizing cytochrome p monooxygenases cytochrome p c : substrates, inhibitors, pharmacogenetics and clinical relevance • excellent review on cyp c , being a gateway for other aspects of this gene and enzyme evaluation of gene induction of drug-metabolizing enzymes and transporters in primary culture of human hepatocytes using high-sensitivity real-time reverse transcription pcr gene expression in distinct regions of the heart distribution of soluble epoxide hydrolase and of cytochrome p c , c , and j in human tissues polymorphisms in human cyp c decrease metabolism of the anticancer drug paclitaxel and arachidonic acid •• discovery of the main cyp c alleles cyp c polymorphisms in caucasians and their relationship with paclitaxel α-hydroxylase activity in human liver microsomes non-synonymous single nucleotide alterations found in the cyp c gene result in reduced in vitro paclitaxel metabolism five novel single nucleotide polymorphisms in the cyp c gene, one of which induces a frame-shift functional characterization of five novel cyp c variants, g s, r x, r g, k r, and k n, found in a japanese population assessment of the pharmacodynamic properties of antimalarial drugs in vivo • valuable review on the pharmacodynamics of antimalaria drug action coartem (artemether-lumefantrine) in africa: the beginning of the end? the epidemiology of drug-resistant malaria chloroquine or amodiaquine combined with sulfadoxinepyrimethamine for uncomplicated malaria: a systematic review the disposition of amodiaquine in man after oral administration amodiaquine as a prodrug: importance of metabolite(s) in the antimalarial effect of amodiaquine in humans sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection antimalarial -aminoquinolines: mode of action and pharmacokinetics disposition of monodesethylamodiaquine after a single oral dose of amodiaquine and three regimens for prophylaxis against plasmodium falciparum malaria pharmacokinetics of intravenous amodiaquine • study supporting interindividual variability on aq metabolism amiodarone n-deethylation by cyp c and its variants, cyp c * polymorphism in cyp c is associated with reduced plasma concentrations of repaglinide interindividual variability in ibuprofen pharmacokinetics is related to interaction of cytochrome p c and c amino acid polymorphisms the effect of the cytochrome p cyp c polymorphism on the disposition of (r)-ibuprofen enantiomer in healthy subjects the impact of cyp c polymorphism and grapefruit juice on the pharmacokinetics of repaglinide association of cyp c , cyp a , cyp a , and abcb polymorphisms with the pharmacokinetics of paclitaxel the effects of human cyp c genotype and fluvoxamine on the pharmacokinetics of rosiglitazone in healthy subjects amodiaquine resistant plasmodium falciparum malaria in vivo is associated with selection of pfcrt t and pfmdr y an integrated model of chloroquine action physicochemical properties correlated with drug resistance and the reversal of drug resistance in plasmodium falciparum susceptibility of colombian plasmodium falciparum isolates to -aminoquinolines and the definition of amodiaquine resistance in vitro synergism between amodiaquine and its major metabolite, desethylamodiaquine, against plasmodium falciparum in vitro in vitro demonstration of the synergy between aq and its main metabolite, desethylamodiaquine high-performance liquid chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples in vitro susceptibility of gabonese wild isolates of plasmodium falciparum to artemether, and comparison with chloroquine, quinine, halofantrine and amodiaquine in vitro activity of pyronaridine and amodiaquine against african isolates (senegal) of plasmodium falciparum in comparison with standard antimalarial agents evaluation under field conditions of the colourimetric deli-microtest for the assessment of plasmodium falciparum drug resistance in vitro activities of ferrochloroquine against senegalese isolates of plasmodium falciparum in comparison with those of standard antimalarial drugs in vitro sensitivity of plasmodium falciparum to amodiaquine compared with other major antimalarials in madagascar the disposition of oral amodiaquine in papua new guinean children with falciparum malaria plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine pharmacodynamic interactions of amodiaquine and its major metabolite desethylamodiaquine with artemisinin, quinine and atovaquone in plasmodium falciparum in vitro amodiaquine induced agranulocytosis and liver damage systematic review of amodiaquine treatment in uncomplicated malaria • review on the use of aq for treatment detection of antidrug igg antibodies in patients with adverse drug reaction to amodiaquine role of hepatic metabolism in the bioactivation and detoxification of amodiaquine the bioactivation of amodiaquine by human polymorphonuclear leucocytes in vitro: chemical mechanisms and the effects of fluorine substitution amodiaquine for treating malaria • authorative review of the use of aq to treat malaria artemisinin-based combination therapies (acts), best hope for malaria treatment but inaccessible to the needy! induction of cyp c genes in human hepatocytes in primary culture • seminal paper on in vitro cyp c induction driven by pregnane x receptor (pxr) and constitutive androstane receptor (car) ligands antimalarial artemisinin drugs induce cytochrome p and mdr expression by activation of xenosensors pregnane x receptor and constitutive androstane receptor • artemisinin and its derivatives are in vitro pxr and car ligands in vivo and mechanistic evidence of nuclear receptor car induction by artemisinin examination of drugs for inhibition of cytochrome p c on the question of dose-dependent chloroquine elimination of a single oral dose single dose pharmacokinetics of chloroquine and its main metabolite in healthy volunteers pharmacokinetics of chloroquine and some of its metabolites in healthy volunteers: a single dose study renal dysfunction in children with uncomplicated, plasmodium falciparum malaria in comparison of chlorproguanil-dapsone with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in young african children: double-blind randomised controlled trial chlorproguanil/dapsone for uncomplicated plasmodium falciparum malaria in young children: pharmacokinetics and therapeutic range mutations in dhfr in plasmodium falciparum infections selected by chlorproguanildapsone treatment development of dapsone toxicity in patients with inflammatory dermatoses: activity of acetylation and hydroxylation of dapsone as risk factors cyp c polymorphism frequencies among malaria patients in zanzibar • first account of the frequencies of the main cyp c alleles in an african native population high prevalence of the cytochrome p c * mutation in northern ghana genetic polymorphism of cyp c in three malaysian ethnics: cyp c * and cyp c * are found in malaysian indians genetic variation in eleven phase i drug metabolism genes in an ethnically diverse population the prevalence of cyp c , c , j , and soluble epoxide hydrolase polymorphisms in african americans with hypertension single nucleotide polymorphisms in the cyp j and cyp c genes and the risk of hypertension cyp c polymorphism among the portuguese genetic polymorphisms of cyp c in japanese population key: cord- -b yywn f authors: hasan, ashfaq; praveen, sai haranath; tarke, chandrakant; abdullah, fahad title: clinical aspects and principles of management of tuberculosis date: - - journal: mycobacterium tuberculosis: molecular infection biology, pathogenesis, diagnostics and new interventions doi: . / - - - - _ sha: doc_id: cord_uid: b yywn f tuberculosis over the ages, has killed more people than any other infection has. notwithstanding the advances in modern science, clinical diagnosis sometimes remains elusive, owing principally to the frequent paucibacillary occurrence of the disease and the slow doubling time of the organism; empiric treatment is often fraught with risks in the era of increasing drug resistance. this chapter attempts to provide an overview of the disease, beginning with the pathogenesis and its protean clinical presentations. it also discusses the recent evolution of molecular methods that have lately provided an impetus to early diagnosis with a clear opportunity to unmask drug resistance before initiating “blind”, potentially ineffective, and sometimes harmful treatment with standard therapy. the chapter also provides insight into tuberculosis in special situations, and discusses briefly the treatments in uncomplicated cases as well as in special situations, and in instances of drug resistance. preventive methods including current and upcoming vaccines are mentioned. finally, a short discussion of the sequelae of tuberculosis—which have the potential to be confused with active disease—is presented. tb can involve all organs except the nails, hair and teeth [ekaterina kulchavenya. ther adv infect dis. apr; ( ): - ]. pulmonary tb is the most common form of tb. the symptomatology of tb has to do with its pathogenesis, the specific organ system involved and the duration of the disease. primary tb, which occurs in the mycobacterium-naive individual, usually presents with a frequently occult primary focus in the peripheral lung parenchyma (stead's focus) accompanied by a pleural effusion; or by a primary focus-frequently inapparent-in the lower part of the upper lobe or the upper part of the lower lobe (ghon's focus) accompanied by lymphadenopathy. other manifestations of primary tb such as erythema nodosum or phlyctenular conjunctivitis may be present. in young children with immature cellular immunity, or in immunocompromised individuals, the process may evolve to progressive primary disease. symptoms and signs relate to the compressive effect of enlarged lymph nodes on adjacent structures (collapse or hyperinflation of a lobe or segment), or rupture of caseating lymph nodes into a bronchus, with bronchiectasis later supervening in such a lobe. post-primary disease, the typical adult form of tb, usually occurs by reactivation of a dormant focus-or less frequently, by reinfection; the better aerated lung apices are preferentially involved. the classical triad of the constitutional symptoms of tb (fever, night sweats, weight loss)-most usually of several weeks' duration-is present in a high proportion of patients (heemskerk et al. ) . the extent of disease at the time of first diagnosis may be variable, from patchy consolidation to a large cavity. this not only reflects the protean pulmonary forms of the process but also has to do with host immunity, the virulence of the strain and, most of all, with the time elapsed between disease onset and diagnosis. cavitation is an important milestone in the timeline of the post-primary pulmonary tuberculosis. with ingress of o -rich air into a fresh cavity (caused by the coughing out of a caseous collection), the aerobic mycobacterium is presented with an opportunity to multiply manifold in an aerobic environment: the infectiousness of the subject (and the possibility of a positive diagnosis on sputum testing) commensurately increases (golub et al. ) . cavitation also increases the propensity to haemoptysis (which may sometimes be massive) and that of cavitary colonization with a fungal ball (mycetoma) later on in healed disease. one third of all individuals affected with post-primary tb die within a few months ("galloping consumption") (reported tuberculosis in the united states, ). the remainder either go on to develop a chronic pattern with a more gradual progressive decline or undergo spontaneous remission of the process and restoration to health. with chronicity, fibrosis is common. disseminated tb, which often casts "miliary" shadows in the lungs, occurs due to haematogenous dissemination following either primary or post-primary tb. the name derives from the resemblance of the widespread granulomatous lesions to millet seeds (seen in pathological specimens as - mm yellow granules). symptoms may be nonspecific and depend on the site and degree of organ involvement. hepatosplenomegaly, lymphadenopathy and pleural effusions may be present. fundus examination, often neglected, will frequently show choroid tubercles, which are virtually diagnostic of the condition in a likely setting. diagnosis often hinges on microbiology from broncho-alveolar lavage (bal) or on biopsy of involved organs (lewinsohn et al. ) though it may occasionally be made on sputum (sponaneously expectorated or induced). disseminated tb disseminated tb should always raise suspicion for an accompanying immune deficiency such as hiv disease. sometimes an occult disseminated form of tb ("cryptic miliary tb") presents as a "fever of unknown origin", usually in the elderly or immunocompromised patients. in difficult cases (as with the classic miliary form), bone marrow or liver biopsy with mycobacterial cultures may be diagnostic. unlike the post-primary form of disease, untreated miliary tb is predictably fatal. extra-pulmonary tuberculosis has been increasingly seen since the advent of the hiv epidemic. tb can involve any organ of the body (other than nails, hair and teeth). organ systems typically involved include pleura (pleural effusions), lymph nodes (generally cervical or mediastinal), airway (endobronchial tuberculosis), skeletal (joint or vertebral involvement, sometimes with a psoas abscess), gastrointestinal, genitourinary, meningeal and pericardial involvement. symptoms pertain to the system involved, but diagnosis may be difficult unless a high degree of suspicion is maintained. tissue or fluid samples from the involved organs with staining, mycobacterial cultures or molecular probe assays are often diagnostic although radiological patterns in specific cases may be very suggestive of the process (e.g. vertebral involvement with a paraspinal abscess) (lewinsohn et al. ) . the diagnosis of tuberculosis, especially in the developed nations, rests on the keystone of suspicion. physical examination is generally unrewarding. auscultation of the lung typically underestimates the lung problem relative to that seen on imaging. with a pleural effusion, breath entry is typically decreased, with a flat note over the effusion. cavernous bronchial breathing may be detected over a cavity, and tubular bronchial breathing over an area of consolidation or dense fibrosis. the radiologic features in the various forms of pulmonary tb have already been discussed above under the relevant heads. microbiological isolation of the mycobacterium tuberculosis on culture has been the only definitive test for tb thus far, though it could be argued that molecular probes for specific mycobacterial genes and other technologies (discussed below and also elsewhere in this book) offer viable and more attractive options for diagnosis. presumptive diagnosis is typically achieved by microscopy (usually of sputum, but also of fluid obtained by bal and other means) (pai et al. ). mycobacteria, not being stainable by "conventional" stains, require a modified acid stain (the ziehl-neelsen method) in order to be visualized; led microscopy though relatively expensive, by making the bacilli easy to see under low power, enables more fields to be scanned in a shorter period of time, and thus increases the sensitivity of microscopy several-fold (bhalla et al. ) . conventional culture methods are slow ( - weeks, less by radiometric assays), and frequently there is a compelling need to start appropriate anti-tubercular therapy as early as possible. rapid culture techniques are now in universal use. they use liquid rather than solid media. the mycobacteria growth indicator tube (mgit) method takes days rather than weeks and facilitates drug sensitivity testing. since relapses (see below) occur in approximately % of patients after months of standard therapy, it is a moot point whether establishment of minimum inhibitory concentrations (mics) in the presence of a "critical concentration" of drug is relevant in selected cases (colangeli et al. ) . automated nucleic acid amplification technology (naat) has the potential of amplifying even a fragment of mycobacterial dna rapidly (in h) and has revolutionized tb diagnosis. in its currently available form, the genexpert test is intended specifically to diagnose mycobacterium tuberculosis (and thereby differentiate it from other mycobacteria such as environmental mycobacteria), and also presumptively diagnose rifampicin resistance. though not invariable, rifampicin resistance also implies inh resistance. together, rifampicin and inh resistance fulfil the diagnosis of multiple drugresistant tb (mdr-tb). thus a positive genexpert test makes a presumptive diagnosis of mdr-tb (steingart et al. ). the test is more sensitive than direct microscopy and extremely specific (steingart et al. ). the test is not infallible, however, and any result that is discordant with the clinical picture should either be repeated or further information sought using another modality. false-positive naat results can occur due to laboratory contamination. importantly, naat can detect nucleic acid from dead non-viable bacilli, and so a positive test after completion of the course of treatment has no relevance in that situation (boyles et al. ) nor is it of relevance in monitoring response to treatment (friedrich et al. ) . it is important to realize that naat is not a substitute for afb smear and culture testing; culture is essential for species identification and for drug susceptibility testing (cheng et al. ) . a more sensitive version of xpert has been developed. the line probe assays (lpa) can permit rapid identification of specific gene markers associated with rifampicin resistance alone or in combination with isoniazid, and provide clinically relevant information about the level of inh resistance (low level associated with the inh-a gene; versus high level associated with the kat-g gene) (who treatment guidance for drug resistant tuberculosis ). definitive diagnosis has traditionally hinged on the culture characteristics of mtb: species discrimination can be achieved on the basis of colony morphology as well as on the basis of biochemical tests performed on the culture. conventional culture (lowenstein-jensen medium and its variants) is capable of detecting as few as ten bacteria per millilitre with the sensitivity and specificity of sputum culture approximating and %, respectively (morgan et al. ) . following initial culture, species identification is done by any of the following techniques: biochemical methods (testing, nucleic acid hybridization probes, high-pressure liquid chromatography or mass spectrophotometry). drug sensitivity testing is subsequently carried out, usually at a reference laboratory (shinnick and good ) to first-line anti-tubercular agents, but sometimes to second-line agents as well when resistance to components of first-line anti-tubercular agents has been documented. in individuals living with hiv who have cd counts < cells/mm , or those who are immunocompromised, mycobacterial cultures of blood and urine should complement conventional testing. point-of-care urinary detection of lipoarabinomannan, a component of the mycobacterial cell wall (urine lam test) may be useful for hiv-infected individuals, and its use may possibly confer a mortality benefit (reported tuberculosis in the united states, ). the future of point-of-care devices is next-generation sequencing, and, perhaps, innovations like detection of volatile organic compounds in exhaled breath (garcia-basteiro et al. ) serological tests (tests for antibodies against tb) have poor sensitivity and specificity; false-positive tests are ubiquitous, and serologic testing for the diagnosis of tb is discouraged by the who. a pleural fluid adenosine deaminase (ada) > u/l has been reported to be % sensitive and % specific for the diagnosis of pleural tb in lymphocytic exudates (garcia-zamalloa and taboada-gomez ). tb infection can rapidly progress to disease in infants and young children (progressive primary tb). mortality is high in early childhood because of the severe forms of tb (such as miliary tb and meningeal tb) that occur more frequently in young children. progressive primary tb is a serious consequence of primary tuberculosis. consolidation or bronchopneumonia is more common than cavitary disease in children. haematogenous dissemination leading to miliary tb is also more common in infants and young children. tubercular pleural effusion, however, is less common in children below years of age. central nervous tb is the most serious form of childhood tb (prevention ). the presentation is usually with nonspecific symptoms such as fever, headache, drowsiness and irritability, but in advanced cases, vomiting, neck rigidity, seizures, focal neurologic deficit and coma may supervene. since sputum samples for diagnosis are difficult to obtain in young children (children tend to swallow sputum rather than expectorate), gastric washings can rather be sent for microbiological examination. a ppd test that is greater than mm in bcg-unvaccinated children and > mm in bcg-vaccinated children is considered positive. (targeted tuberculin testing and treatment of latent tuberculosis infection. this official statement of the american thoracic society was adopted by the ats board of directors, july . this is a joint statement of the american thoracic society (ats) and the centers for disease control and prevention (cdc). this statement was endorsed by the council of the infectious diseases society of america. (idsa), september , and the sections of this statement .) the principles of treatment of tuberculosis in children are similar to that of adults. paediatric tb patients should be monitored carefully for ethambutol-induced ocular toxicity, owing to the fact that children are less likely to report visual impairment than adults. in a prospective study of children on multidrug-resistant tuberculosis, children had lower serum concentrations in spite of higher dosing of moxifloxacin, a fact that was ascribed to increased drug elimination in children. quinolones have also been known to rarely cause arthropathy and tendon rupture. more recent studies have shown that most quinolones are quite well tolerated but very specific and narrow indications are present for quinolone use in children (patel and goldman ) . tuberculosis in pregnancy has important implications for the mother and child (loto and awowole ) . the diagnosis of tuberculosis in pregnancy can be challenging, since many of the symptoms of early tb may be confounded by pregnancy; for instance, the normal weight gain in pregnancy may temporarily mask the associated weight loss. chest radiograph is not an absolute contraindication and when necessary may be carried out with "abdominal shielding" of the fetus. the complications of tb in pregnancy include early spontaneous abortion, intrauterine growth retardation, preterm delivery, low birth weight and increased neonatal mortality. congenital tb is rare, but can be associated with high perinatal mortality. first-line oral drugs, i.e. isoniazid, rifampicin, ethambutol and pyrazinamide, can all be used during pregnancy safely. streptomycin and other aminoglycosides have been proven to be potentially teratogenic in pregnancy. they are also capable of causing eighth nerve paralysis, with deficits ranging from mild hearing loss to bilateral deafness (loto and awowole ) . concerns have been raised about the use of fluoroquinolones in pregnancy even though the weight of the evidence seems to point toward safety (acar et al. ) tb is considered an aids-defining disease [centers for disease control and prevention. appendix a: aids-defining conditions. mmwr. ; (rr ): ]. the risk of developing tb is estimated to be between and times greater in people living with hiv than among those without hiv infection. hiv and tb have a salutary impact upon each other and expedite each other's progression. the risk of progressing from latent to active tb is estimated to be between and times greater in people living with hiv than among those without hiv infection (global tb report ). against the backdrop of hiv infection, the rate of progression of tb from the acquisition of infection to full-blown disease may take weeks, rather than years-as is the case in hiv-negative individuals (mayer and dukes hamilton ) . it is important to realize that tb can occur at all "stages" of hiv disease. the clinical presentation of hiv patients who have relatively high cd counts is no different to that which occurs in individuals without hiv. such individuals manifest with the classical manifestations of post-primary tuberculosis such as upper lobe infiltrates. on the other hand, lower lobe involvement, pleural effusions and intrathoracic lymphadenopathy are common in individuals with low cd counts (typically < /μl). indeed the chest x-ray may sometimes only show vague interstitial infiltrates or even be normal (mayer and dukes hamilton ) . taken together, the fact that in advanced hiv disease (a) the sputum is less likely to be positive for afb (b) the ppd is usually non-reactive (c) the x-ray abnormalities are likely to represent other opportunistic infections or even non-infective conditions associated with hiv disease and (d) histopathology of involved tissues is characterized by lack of typical granulomata; these considerations make the diagnosis extremely challenging in this setting. in addition, there is increased frequency of extra-pulmonary tb among hiv-positive individuals. frequently, a positive naat (genexpert) test is the justification for starting treatment. however it must be cautioned that even a negative naat test cannot convincingly rule out disease, and, given that delays in treatment may be fatal, the decision to initiate treatment is sometimes taken on clinical grounds. the treatment of tuberculosis differs from the treatment of most other infections in certain respects. multiple drugs are required in a regimen since spontaneous mutations can lead to resistance to the action of one or more drugs. also, because the bacterium is relatively slow growing, it takes longer to achieve bacteriologic sterility. multidrug therapy, the norm for tb, has been very effective, with a modest rate of relapse (colangeli et al. ) . the standard four-drug regime ("short course") comprises a -month intensive phase with the four front-line drugs (rifampicin, isoniazid, ethambutol and pyrazinamide), followed by a continuation phase with the drugs (minus pyrazinamide and possibly ethambutol) for a further months. the four front-line drugs have been chosen based on their efficacy in rapidly reducing the number of organisms initially (during the initial phase), thus reducing infectivity; and on their sterilizing potential (the ability to eradicate viable bacteria completely and so prevent relapses); and their relative lack of side effects (see table . ). these drugs are combined into regimens. the six classes of second-line agents (fluoroquinolones, the aminoglycosides, capreomycin, ethionamide and prothionamide, para-amino salicylic acid (pas), cycloserine and terizidone) have a rather lower efficacy and a higher propensity for side effect and drug intolerance. several other antibiotics of uncertain efficacy have also been categorized as anti-tubercular agents and have a role in treating multidrug-resistant tb (mdr-tb) (see table . ). two new agents, bedaquiline (a diaryl-quinolone) and delamanid (a nitroimidazole), are now approved for the treatment of mdr-tb. the who recommends using a daily regimen and fixed-dose combinations (guidelines for treatment of drug-susceptible tuberculosis and patient care ). administration of the drugs on a daily basis both during the intensive and during the continuation phase is preferable although daily administration during the initial phase and intermittent (thrice weekly) administration during the continuation phase is also acceptable (guidelines for treatment of drug-susceptible tuberculosis and patient care ). intermittent administration of drugs (especially in the initial phase) is not offered in the presence of hiv disease. if at the end of the intensive phase the bacteria are sensitive to the three most important agents (isoniazid, rifampicin and pyrazinamide), ethambutol may be discontinued (nahid et al. ). however, drug testing for three of the four frontline drugs for each patient at the end of the intensive phase is impractical, and limited testing (genexpert) is usually performed initially in those patients whose sputum remains positive. there is evidence to show that extending the continuation phase by months (making up a total duration of treatment of months) helps in preventing relapse in those patients who have cavitation on initial chest x-rays; and also in those who show positive cultures at the end of the initial phase of treatment (benator et al. ) ; as well as in those whose medication given during the initial phase did not include pyrazinamide. anti-tubercular drugs require to be supplemented with carefully monitored steroid therapy in two circumstances: tubercular meningitis (a short course of dexamethasone or prednisolone is typically given, tapered over to weeks) and tuberculous pericarditis (guidelines for treatment of drug-susceptible tuberculosis and patient care ). since many patients cannot be relied upon to regularly take their drugs, and irregular intake of drugs has the potential to result in drug resistance (see treatment failure and relapse; and drug resistant tb, below), it is imperative to ensure adherence to the regimen. directly observed therapy, short-course (dots) is the direct observation by another person, of the patient taking the medication (chaudhuri ) . the use of dots has improved adherence in subgroups of patients such as tb with hiv or if dots was at the site of dots centre or provider location. self-administered therapy (sat) modified with some form of monitoring incorporated into the process shows some promise. although dots could be administered by a family member, the preferred person is a trained lay provider or healthcare worker (guidelines for treatment of drug-susceptible tuberculosis and patient care ). the treatment of tb in hiv disease merits special consideration. antiretroviral therapy (art) should be started irrespective of the clinical stage of hiv disease and cd count. anti-tubercular therapy (att) should precede the commencement of art. art should then be commenced as soon as it is evident that att is being tolerated (typically between weeks and months). the immune reconstitution inflammatory syndrome (iris) is sometimes seen with the returning immune competence that follows art; an increased immune response to tubercle bacilli or antigens occurs. iris usually occurs between and months of the commencement of art and is associated with a decrease in viral load and an increase in cd + t cell count. there is a worsening in the clinical picture weeks or months into treatment, with an increase in the size of lymph nodes, development of pleural effusions and features of noncommunicating hydrocephalus in a patient with tb meningitis. although most patients with iris have mild to moderate symptoms, iris, especially when related to tb meningitis, can be life-threatening. an "unmasking iris" may occur in patients with unsuspected tb (raviglione ) . treatment of iris includes addition of steroids (which probably work by decreasing the levels of pro-inflammatory cytokines) and the continuation of att and art. with both att and art on board, drug-drug interactions especially involving rifampicin (by induction of the cytochrome p system) may occur. rifamycins induce the metabolism of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. rifampicin is a more potent inducer of the cytochrome p system than rifapentine, which in turn is more potent than rifabutin. despite potential drug interactions, rifamycins should nevertheless be included in tb regimens, with dosage adjustment if necessary. rifabutin is the preferred rifamycin in hiv-positive individuals. host-directed therapy to modulate the immune response to tb is an active area of research (kaufmann et al. ) . the use of an anti-diabetic drug-metformin, for example-is being investigated. also, studies of autologous mesenchymal stromal cell infusions is being investigated in mdr and xdr-tb (skrahin et al. ). an approach using high-altitude sanatoria was also explored (murray ) in a throwback to the pre-anti-tubercular chemotherapy era. study using atypical mycobacterial injection in tuberculous did not show any major outcome differences from placebo although steroids did decrease the subsequent development of constrictive pericarditis (mayosi et al. ) . given the role that vitamin d plays at the level of the antigen-presenting cell, the role of vitamin d deficiency as a possible player in tb predisposition has recently come into focus; indeed a recent meta-analysis has revealed that vitamin d supplementation in fact did not affect time to sputum culture conversion overall, though it did accelerate sputum culture conversion in patients with multidrug-resistant pulmonary tuberculosis (jolliffe et al. ). patients with tb may be admitted to an icu for a variety of reasons, including respiratory failure, multiorgan failure, decreased consciousness associated with tubercular meningitis, pneumothorax, cardiogenic shock (due to massive pericardial effusion) and liver or kidney failure due to a drug reaction induced by att. confluent tuberculous bronchopneumonia and tb ards are less common causes of respiratory failure (hagan and nathani ) . tuberculosis treatment is especially challenging in critically ill patients due to potential for poor gastric absorption and the high rates of organ dysfunction and drug toxicity in this subset of patients. deciding the optimal regimen and dosing becomes challenging in the presence of concomitant hepatic and renal failure. in a recent study done on tb patients requiring icu admission, mortality was . % (tatar et al. ). treatment failure is suspected when the sputum remains positive for afb at the end of the first months of treatment. it occurs due to factors that may involve incorrect regimens or dosing or indeed due to malabsorption or poor-quality drugs (lambregts-van weezenbeek and veen ) . patients are regarded as treatment failures when a microbiologic indicator like sputum smear/culture for tb remains positive after - months of starting treatment or comes back positive after a period of remaining negative (fall and rise phenomenon) (prasad et al. ) . patients may respond for a short time after starting treatment or do not respond to treatment from the inception. in any case the implication of treatment failure is drug-resistant tb (see below), and strenuous efforts must be made to look for microbiological resistance to first-and, in the proper context, to second-line agents-by the available tools. relapse, on the other hand, is the recurrence of tb after completion of treatment that has been successful, with documented culture negativity. relapse may be due either to a reinfection or reactivation of the dormant bacillus. drugresistant tb is to be considered in these settings. the implications of relapse are not as grave, the isolates generally being drug sensitive. in either case, the exact regimen chosen should be based upon the drug sensitivity pattern. specific recommendations upon the construction of such a regimen are available at who treatment guidelines for drug-resistant tuberculosis update (http://www.who.int). drug-resistant tb is a major global problem and is a threat to the success of the end tb strategy. mdr-tb or multidrug-resistant tb (mdr-tb) is defined by resistance of m. tuberculosis against to at least rifampicin and isoniazid. since these two drugs are the most effective drugs available against tb, resistance to both considerably shortens the odds of complete cure. xdr-tb or extensively drug-resistant tb is defined as resistance to rifampicin and inh plus resistance to at least one fluoroquinolone and one second-line injectable agent (amikacin, kanamycin or capreomycin). some of the reasons that lead to drug resistance have been mentioned in the section above. however the most important of these are failure to comply with the treatment regimen on the part of the patient and the addition of a single drug ("monotherapy") to a failing regimen; or a situation of unrecognized primary (that which is present at the start of therapy) or secondary (that which develops during treatment) drug resistance. inh mono-resistance of itself probably does not impact the outcome of therapy provided that ethambutol and possibly pyrazinamide are used for the entire duration of therapy. consideration should be given in this case to extending the duration of treatment to months from the standard (sadanand ). for reasons already discussed, rifampicin-resistant individuals should be managed as mdr-tb patients. in such patients it is not unusual to encounter resistance to additional drugs (e.g. ethambutol). it has been shown that clinical cure is possible in patients with mdr and even xdr-tb (who drug resistant tb ). however, owing to a variety of reasons not the least of which is drug intolerance, only about half of mdr-tb and a third of xdr-tb patients ever complete therapy. the who estimates there are over , mdr-tb or rifampicin-resistant cases annually (global tb report, who update ; wwww.who.int). the treatment of mdr-tb and xdr-tb is complex and requires specialized knowledge and close monitoring to ensure rational and safe therapy (lange et al. ) . the choice of drugs for treatment of mdr-tb is frequently difficult. formulation of drugs into regimens is facilitated by the organization of drugs into groups: group a ¼ levofloxacin/moxifloxacin, bedaquiline, linezolid. group b ¼ clofazimine, cycloserine/terizidone. group c ¼ ethambutol, delamanid, pyrazinamide, imipenem-cilastatin, meropenem, amikacin (streptomycin) , ethionamide/prothionamide, p-aminosalicylic acid. the who makes specific recommendations for short and long mdr-tb regimens. in the who's update (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis ), for those mdr-tb patients "who have not been previously treated for more than one month with second-line medicines used in the shorter mdr regimen or in whom resistance to fluoroquinolones and second-line injectable agents has been excluded," the who permits a shorter mdr-tb regimen of - months. for mdr/rr (rr¼ rifampicin resistant) tb patients on longer regimens, the who recommends that "all three group-a agents and at least one group-b agent (should) be included to ensure that treatment starts with at least tb agents likely to be effective, and that at least three agents (are) included for the rest of treatment after bedaquiline is stopped. if only one or two group a agents are used, both group b agents are to be included. if the regimen cannot be composed with agents from groups a and b alone, group c agents are added to complete it. kanamycin and capreomycin are not to be included in the treatment of mdr/rr-tb patients on longer regimens" (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis ). a detailed discussion about the treatment of mdr-tb is available at the foregoing resource. novel anti-tubercular agents (e.g. bedaquiline and delamanid) have been discussed elsewhere in this text. the remodelling of the lung that follows tb can significantly contribute to morbidity and mortality. haemoptysis is frequently one of the remote sequelae and can on occasion be life-threatening. an unresolved cavity is frequently the cause, and bleeding from an unsupported blood vessel in the cavity wall or in a post tubercular bronchiectatic segment can be a source of troublesome bleed. cavities colonized by fungi (aspergillomas), broncholiths eroding into the bronchial wall and rarely expansion and subsequent rupture of an unsupported blood vessel in the cavity wall (rasmussen's aneurysm) can cause massive haemoptysis (van den heuvel and van rensburg ) . a scar carcinoma sometimes develops in an area of fibrosis and may sometimes be difficult to distinguish from a dense fibrotic infiltrate (gao et al. ) . post-tubercular bronchiectasis is predisposed to when caseation necrosis and inflammation with retention of secretions leads to destruction of bronchial walls with ensuing permanent dilatation. sometimes compression of bronchial lumen by enlarged lymph node can produce bronchiectasis by a different mechanism. postprimary tb involves the upper lobes of the lung, and so the gravitationally advantaged upper lobes do not usually undergo a process of chronic and recurrent infection unlike the lower lobes that are involved by bronchiectasis of other aetiologies. mycetoma (fungal ball) is a mass of fungal hyphal material that grows within a cavity. aspergillus fumigatus is the most common aetiological agent, but other fungi such as mucor or fusarium can also cause a fungal ball to develop (rippon ) . such a mycetoma is usually asymptomatic but can sometimes lead to haemoptysis. no treatment required in asymptomatic cases, but surgical treatment needs to be considered in patients with massive or recurrent haemoptysis. a list of adverse effects associated with anti-tubercular agents appears in table . . side effects are possible with any of the anti-tubercular agents, and on rare occasions, they may be serious and even fatal. the risk of liver injury with anti-tb therapy is always a concern. among the four first-line agents inh, rifampicin and pyrazinamide are all capable of causing hepatotoxicity. isoniazid is capable of causing asymptomatic mild elevation in transaminases in - % as well as fatal severe acute hepatitis in . - %. jaundice with liver injury can occur in . - %. peripheral neuropathy due to pyridoxine deficiency can occur with inh therapy in up to . % of elderly individuals (ruan et al. ); pyridoxine supplementation is required for all patients taking inh (john ). factors increasing the chances of inh hepatotoxicity include age, female gender, alcohol use, prior hepatitis, concurrent use of cytochrome-inducing agents and slow acetylator status (sharma et al. ) . acute inh toxicity can present as seizures due to decreased gaba levels. inh is metabolized to several metabolites including the hepatotoxic metabolite hydrazine, toxic-free radicals and mono-methylhydrazine. degradation by acetylation occurs via n-acetyltransferase- gene (nat ); a deficiency of the nat enzyme can lead to accumulation of hepatotoxic metabolite. ten percent of patients with anti-tubercular drug-induced hepatotoxicity show progression to acute liver failure; the rest are self-limited (badrinath and john ) . if the serum bilirubin rises to ! mg/dl or serum transaminases exceed more than five times the upper limit of normal (or exceed three times the upper limit of normal in a symptomatic patient), all drugs should be stopped (nahid et al. ). the decision on what regimen to use on restarting should be individualized. the regimen, even if retailed, might then still include one or more potentially hepatotoxic drugs, and these should be restarted one at a time, only when liver function tests return to their baseline (e.g. the transaminases fall to less than twice normal). in general, a cholestatic type of derangement would prompt addition of inh first; with a non-cholestatic picture, rifampicin could first be added (sterling) . needless to say, careful monitoring with frequent testing of serum liver function is required. most physicians would omit pyrazinamide when restarting att except in the mildest of cases or with other compelling reasons (sterling) . patients receiving ethambutol (emb) should be questioned regarding their vision at each monthly visit. monthly visual acuity and colour vision discrimination testing is recommended in those patients in whom emb doses of greater than - mg/kg are being used, those who have been taking ethambutol for longer than months and those with renal insufficiency in whom the levels of drug may be expected to rise (blumberg et al. ) . for patients who require a regimen with no hepatotoxic agents, potential agents include ethambutol, levofloxacin or moxifloxacin, an injectable agent and other second-line oral drugs. the optimal choice of agents and duration of treatment (at least - months) is uncertain. (see "antituberculous drugs: an overview", section on "second-line agents".) in the end, efforts to finally eradicate tb will need to hinge upon preventative aspects as much as upon its treatment. these strategies would include currently established practices, as well as novel strategies. the former include the following. tuberculosis is now the primary cause of death from infectious diseases exceeding hiv, aids and malaria. the who estimates . million new cases and . million deaths every year (who global tb report ). of these, . million are hiv patients who die mostly in lmic (low-and middle-income countries) (who global tb report ; www.who.int). within the next years, the goal is to reduce tb incidence by % but the progress has been painfully slow. the most effective way to reduce the burden of tb in the community, to date, has been to identify patients affected with tb as early as possible and to treat them effectively with antitubercular therapy. the role of vaccines in this regard, though potentially vital, has been disappointing. of all vaccine tested to date, the bacillus calmette-guerin (bcg)-a live attenuated vaccine that was made in after an incredible years and sub-cultures (hasan )-is still the most effective vaccine we have today. yet its effect has been inconsistent, ranging from % to zero protection; several factors including geographical differences appear to play a role (british adolescents had better responses than south indian participants perhaps due to background exposure to mtb or atypical mycobacteria). the efficacy of bcg has been most manifested in its protection of infants and young children from the dangerous forms of tb (such as disseminated "miliary" tb and tb meningitis). also, its effects appear to wane with time (hasan ) . the bcg is not a single vaccine. several strains-all derived from the original bgc strain-are in use, which also partly explains its variable efficacy. in , the sanger centre in cambridge, britain and paris's pasteur institute in france cracked the genetic code for the old h rv strain of tubercle bacillus, and then, the genome of highly virulent cdc (the "oshkosh" strain) was mapped at the tigr (fine ). these developments have led to the possibility of exciting new candidate vaccines, several of which are in fact in the pipeline. it has been estimated that one-third of the world has been infected with tb bacteria. a relatively small number of immunocompetent individuals affected with ltbi will ever progress to active disease. reactivation of tb occurs when the immune system is compromised by external factors such as steroids or other immunosuppressants or by immune deficiency syndromes, which enables the latent (dormant) bacteria to "awaken" causing active disease. there is unfortunately no test that can currently predict which of these individuals will progress to active disease immunocompromised individuals have a high possibility of doing so. given that perspective, treatment for latent tb infection is most relevant for children under years, the elderly and individuals with hiv infection. to eliminate tb from the planet, a multipronged strategy incorporating early and effective treatment, vaccination and elimination of dormant reservoir of latent tb will be essential. ltbi treatment with inh ("chemoprophylaxis") is still the mainstay for treating latent tb infection but given that treatment extends to months, and that there is one death from hepatotoxicity for every , - , cases with such treatment, alternate strategies including rifamycin-based regimens are being explored (rangaka et al. ) . pregnancy outcomes following quinolone and fluoroquinolone exposure during pregnancy: a systematic review and meta-analysis rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in hiv-negative patients: a randomised clinical trial performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis infectious diseases society of america: treatment of tuberculosis false-positive xpert (r) mtb/rif assays in previously treated patients: need for caution in interpreting results recent changes in technical and operational guidelines for tuberculosis control programme in india - : a paradigm shift in tuberculosis control molecular diagnostics in tuberculosis bacterial factors that predict relapse after tuberculosis therapy the bcg story: lessons from the past and implications for the future five-year follow-up of a controlled trial of five -month regimens of chemotherapy for pulmonary tuberculosis assessment of the sensitivity and specificity of xpert mtb/rif assay as an early sputum biomarker of response to tuberculosis treatment ct features of lung scar cancer point of care diagnostics for tuberculosis diagnostic accuracy of adenosine deaminase and lymphocyte proportion in pleural fluid for tuberculous pleurisy in different prevalence scenarios delayed tuberculosis diagnosis and tuberculosis transmission guidelines for treatment of drug-susceptible tuberculosis and patient care ( ) world health organization clinical review: tuberculosis on the intensive care unit adjunctive vitamin d in tuberculosis treatment: meta-analysis of individual participant data progress in tuberculosis vaccine development and host-directed therapies -a state of the art review control of drug-resistant tuberculosis drug-resistant tuberculosis: an update on disease burden, diagnosis and treatment infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children tuberculosis in pregnancy: a review synergistic pandemics: confronting the global hiv and tuberculosis epidemics prednisolone and mycobacterium indicus pranii in tuberculous pericarditis comparison of a radiometric method (bactec) and conventional culture media for recovery of mycobacteria from smearnegative specimens worth a try in extensively drug-resistant tuberculosis? official american thoracic society/ centers for disease control and prevention/infectious diseases society of america clinical practice guidelines: treatment of drug-susceptible tuberculosis tuberculosis diagnostics: state of the art and future directions safety concerns surrounding quinolone use in children multidrug-resistant tuberculosis/rifampicin-resistant tuberculosis: principles of management reported tuberculosis in the united states controlling the seedbeds of tuberculosis: diagnosis and treatment of tuberculosis infection (ed) harrison's infectious diseases, th edn. mcgraw-hill education medical mycology: the pathogenic fungi and the pathogenic actinomycetes isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by ( )h nmr based metabolomics approach harrison's infectious diseases miliary tuberculosis: a new look at an old foe diagnostic mycobacteriology laboratory practices autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase safety trial xpert(r) mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults treatment of drug-susceptible pulmonary tuberculosis in hiv-uninfected adults this official statement of the american thoracic society was adopted by the ats board of directors contributing factors to mortality rates of pulmonary tuberculosis in intensive care units who treatment guidance for drug resistant tuberculosis ( ) who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis fluoroquinolones for treating tuberculosis key: cord- - mbsib authors: devarajan, padma v.; dawre, shilpa m.; dutta, rinku title: infectious diseases: need for targeted drug delivery date: - - journal: targeted drug delivery : concepts and design doi: . / - - - - _ sha: doc_id: cord_uid: mbsib infectious diseases are a leading cause of death worldwide, with the constant fear of global epidemics. it is indeed an irony that the reticuloendothelial system (res), the body’s major defence system, is the primary site for intracellular infections which are more difficult to treat. pro-inflammatory m macrophages play an important role in defence. however, ingenious pathogen survival mechanisms including phagolysosome destruction enable their persistence. microbial biofilms present additional challenges. low intracellular drug concentrations, drug efflux by efflux pumps and/or enzymatic degradation, emergence of multi-drug resistance (mdr), are serious limitations of conventional therapy. targeted delivery using nanocarriers, and passive and active targeting strategies could provide quantum increase in intracellular drug concentration. receptor mediated endocytosis using appropriate ligands is a viable approach. liposomes and polymeric/lipidic nanoparticles, dendrimers micelles and micro/nanoemulsions could all be relied upon. specialised targeting approaches are demonstrated for important diseases like tuberculosis, hiv and malaria. application of targeted delivery in the treatment of veterinary infections is exemplified and future possibilities indicated. the chapter thus provides an overview on important aspects of infectious diseases and the challenges therein, while stressing on the promise of targeted drug delivery in augmenting therapy of infectious diseases. of the microorganism. intracellular infections result when the organisms cleverly evade destruction following phagocytosis. the intracellular location of these microorganisms protects them from the host defence mechanisms, such as antibodies or complement, and from the action of drugs that are unable to penetrate the cell effi ciently. hence, while adequate drug concentrations are readily achieved at extracellular infection sites to enable effi cient therapy, intracellular infections are more diffi cult to treat. some common intracellular and extracellular infectious diseases and their causative organisms are listed in table . . the res also known as the mononuclear phagocytic system (mps)/macrophage system is the primary defence mechanism of the human body and hence the site of intracellular infections. the macrophages constitute the major defence cells of the res. derived from the bone marrow the res also contributes to both non-specifi c and specifi c immunity. recognition by the res is facilitated by opsonins, with the step of opsonisation being a precursor to phagocytosis. opsonisation is the process by which bacteria are altered by opsonins so as to become more readily and effi ciently engulfed by phagocytosis. opsonisation is mediated by the complement system: c b, c b, and ic b, antibodies igg and igm and mannose-binding lectin. mannose binding lectin initiates the formation of c b. opsonisation of particles enables recognition by the fc receptors, complement receptors or specifi c receptors for phagocytosis. opsonins are generally proteins which can bind to pattern-recognition receptors (prrs) or other specifi c receptors expressed on the surface of macrophages. pentraxins [c-reactive protein and serum amyloid p] [ ] , mindin, collectins [ ] and fi colins [ ] are such opsonins. the function of pattern-recognition receptors (prrs) is to recognise and enhance phagocytosis of pathogen-associated molecular patterns (pamps), specifi c patterns present on microbial pathogens like lipopolysaccharide (lps) in gram-negative bacteria, lipotechoic acid (lta) in gram-positive bacteria and mannans in yeast. toll-like receptors (tlrs) are prrs essential for recognition of microbial components such as tlr (lps) [ - ] , tlr [double-stranded rna] [ ] , tlr [mycoplasmal macrophage-activating lipopeptide- kda] [ ] , tlr [cpg bacterial dna] [ ] , tlr [bacterial fl agellin] [ ] , and tlr [peptidoglycan] . however, the exact mechanisms of tlr recognition of microbial components remain unclear. opsonisation facilitates adherence of pathogens to macrophages, and is facilitated by integrins. adherence induces membrane protrusions, called pseudopodia, to extend around the attached material. following fusion with the macrophage, the pseudopodia forms a phagosome that encloses the pathogen within a membrane, which then enters the endocytic process. phagosomes coalesce with intracellular organelles to mature into phagolysosomes, which have an acidic environment with many digestive proteins which fi nally degrades the internalised material. phagocytised material is eliminated by exocytosis. the process of phagocytosis is mediated by several proteins such as actin, dynamin and cortactin. while actin is connected to the lipidic membrane and responsible for invagination of the membrane to form the endosome, cortactin is an actin-binding protein which stimulates its polymerisation. dynamin hydrolyses guanidine triphosphate and uses the resulting energy for the contraction of actin and formation of endosome. particulates that cannot be digested remain sequestered in residual bodies within the cell. other cells such as fi broblast, endothelial and epithelial cells also exhibit phagocytic activity and can engulf microbes like shigella, listeria and yersinia [ ] . such phagocytosis is mediated by laminin and fi bronectin receptors/heparan sulfate present on the membrane surface [ ] . however, the major cells responsible for phagocytosis are macrophages. macrophages (greek: makros means large and phagein means eat) are cells formed by the differentiation of monocytes in tissues. macrophages play an important role in both innate and adaptive immunity in vertebrates. these specialised phagocytic cells engulf and destroy infectious microbes, foreign particles and cancer cells [ ] . the macrophages also regulate lymphocyte, granulocyte populations and important tumor growth modulators [ ] . macrophages act by both oxygen-dependent killing and oxygen independent killing mechanisms. the mediators for oxygen-dependent killing are reactive oxygen intermediates (rois) (superoxide anion, hydroxyl radicals, hydrogen peroxide and hypochlorite anion), reactive nitrogen intermediates (rnis) (nitric oxide, nitrogen dioxide and nitrous acid) and monochloramine, while the mediators for oxygen independent killing are defensins, tumor necrosis factor (macrophage only), lysozyme and hydrolytic enzymes. floating macrophages predominate in the vascular system, while tissue macrophages are localised in specifi c tissues. based on the tissue of residence they have specifi c nomenclature ( fig. . ). macrophages can be classifi ed mainly into two groups: ( ) pro-infl ammatory or classically activated macrophages (m ) and ( ) anti-infl ammatory or alternatively activated macrophages (m ). m macrophages are immune effector cells that aggressively work against microbes and cause their destruction much more readily. m is mainly associated with gastrointestinal infections (e.g. typhoid fever and helicobacter pylori gastritis) and active tuberculosis. m macrophages are stimulated by interferon (ifn)-g or lipopolysaccharide (lps) to release nitric oxide (no), important for killing intracellular pathogens. activated macrophages are characterised by expression of major histocompatibility molecule like mhc class ii and cd and their ability to secrete proinfl ammatory cytokines such as tumor necrosis factor (tnf)-a, il- b, il- , il- and the chemokines ccl , ccl , cxcl - and cxcl [ ] . activated m macrophages facilitate killing of microorganisms by endocytosis, synthesising reactive oxygen intermediates (roi), limiting the uptake of nutrients and iron essential for the growth of bacteria and replication of viruses, or production of nitric oxide facilitated by ifn-g-inducible no synthase (inos). m macrophages are important for killing extracellular parasites, wound healing, tissue repair, and to turn-off immune system activation. m macrophages are activated by interleukin (il)- or il- (m a) to produce il- , transforming growth factor (tgf)-b and arginase- (arg ), to enable this function [ ] . m macrophages are mostly observed in lepromatous leprosy, whipple's disease and localised infections (keratitis, chronic rhinosinusitis). a number of infectious organisms which manage to overcome the res defence develop unique adaptive mechanisms which enable them to survive within the cell for prolonged periods of time. eradication of such intracellular organisms poses immense challenges. many pathogens have an innate ability to develop adaptive mechanisms under stress conditions to fi ght for their survival. such adaptive mechanisms or protective strategies, enables them to exhibit greater defence to the host and there by prolong survival. the different adaptive mechanisms employed by pathogens are discussed below. strategies adopted by microorganisms to inhibit phagolysosome formation include interference with the transformation of primary endosomes into late endosome, fusion with lysosomes and or phagosome acidifi cation. this delays the fusion of endosomes with lysosomes [ ] or blocks the same [ ] . the strategies to inhibit phagolysosome formation and the pathogens which exhibit the same [ ] are summarised in table . . pathogens which exhibit this adaptation survive and multiply in vesicles formed by fusion of endosomes with cell organelles other than the lysosome, such as the rough endoplasmic reticulum, ribosome or mitochondria [ ] and thus avoid phagolysosome formation. they thereby bypass destruction due to the enzymatic activity in the lysosome [ ] . escape from endocytosis is a crucial step for intramacrophagic survival. pathogens from this category contain lytic enzymes which enable them to break the endosomes membrane and disrupt membrane of the vacuole [ ] , and hence evade degradation in the phagolysosome, and enter the cytosol rich in nutrients [ ] . specifi c enzymes are produced by the microorganisms for instance, l. monocytogenes pujol et al. [ ] disturbs the formation of lipid rafts by producing beta- , glucans brucella spp. brucellosis roy [ ] alteration of host cell signaling by dephosphorylation of signal regulated kinase leishmania spp. leishmaniasis ghosh et al. [ ] produces listeriolysin o (llo) [ ] and haemolysin c [ ] while phospholipases are produced by the rickettsia spp. [ ] . the microbes in this category exhibit virulence factors which allow them to survive in lytic enzymes, acidic conditions and oxidants, the harsh conditions in the phagolysosome environment. intramacrophagic resistance employing multiple virulence factors enables alternative pathways for survival and multiplication [ ] . pathogens are internalised into macrophages by alternate routes. they traverse inside the cell by receptor mediated pathways like clathrin [ ] and lipid rafts [ ] . formation of vesicles with new properties after fusion between the pathogen and membrane of the cell, like the parasitophorous vacuole formed by toxoplasma gondii [ ] also provides protection. in certain infections successful fusion of microorganisms with the macrophage is followed by secretion of antiapoptotic molecules (e.g. bcl ). this results in impairment of apoptosis of the infected cells. in addition to the adaptive mechanisms certain microbes employ highly specifi c strategies for persistence inside the cell. such strategies are discussed with reference to some important diseases. the adaptive mechanisms of mycobacterium tuberculosis to survive inside the macrophages are prevention of fusion of the phagosome with lysosomes by producing tryptophan-aspartate-containing coat protein (taco). transformation of primary endosomes into phagolysosomes is prevented by a number of actions that occur simultaneously. these include reduced levels of proton atpase inside the endosomes [ ] and scavenger receptors [ , ] have also been implicated in mycobacterial uptake. uptake of mycobacteria by the complement receptor pathway protects it from the aggressive lysosomal compartment ensuring relatively hospitable conditions. salmonella specifi cally forms a glycolipid capsule or biofi lm. biofi lm formation in salmonella is related to the multicellular and aggregative response of rdar [ ] , rugose [ ] , or lacy [ ] . this multicellular behavior is a property of salmonellae [ ] and is responsible for elaboration of thin fi mbriae like tafi , curli [ ] , cellulose [ ] , and other uncharacterised extracellular polysaccharides. together, these components form the extracellular matrix that confers resistance to acid and bleach and facilitates environmental persistence [ , , - ] . pathogens which cause fungal infections adapt various mechanisms to increase their pathogenesis and survive inside macrophages. c. albicans contains superoxide dismutases (sod) and catalase enzymes which are able to convert o into molecular oxygen and hydrogen peroxide, thereby decreasing the scavenging and toxic effects of o and h o levels by certain reactions [ ] . further, c. neoformans evade phagocytic uptake by phenotypic switching. this mechanism is observed in yeast cells that express glucuronoxylomannan mucoid capsule that resist phagocytic uptake and cause high lethality in mice [ ] . in case of aspergillus conidia infection collectins, pentraxin proteins are essential for opsonisation, but their defi ciency is responsible for high susceptibility to infection in immunocompetent mice. furthermore, several enzymes such as elastases and proteases released by the fungus enable conidia to escape from phagocytic uptake by alveolar macrophages. in hiv- -infected macrophages, the viral envelope protein induces macrophage colony-stimulating factor (m-csf). this pro-survival cytokine down regulates the trail (tumor necrosis factor-related apoptosis-inducing ligands) receptor and up regulates the anti-apoptotic genes bfl - and mcl- enabling hiv to survive inside the macrophages. hiv invades the macrophage through ccr a chemokine receptor and through binding of gp to cd [ ] . macropinocytosis as a route of entry of hiv- into macrophages [ ] also enables intracellular protection. leshmania prevent activation of macrophages by inhibiting secretion of cytokines such as the infl ammatory response il- and tumor necrosis factor beta (tnf-beta) or t-lymphocyte activation (il- ) and produce various immunosuppressive signaling molecules, such as arachidonic acid metabolites and the cytokines tnfbeta and il- . l. chagasi induces tnf-beta production in the immediate environment of the infected human macrophage, and this may lead to inhibition of immune responses [ ] . further, this pathogen induces alteration of host cell signaling. macrophages infected with l. donovani or l. mexicana have shown altered ca + dependent responses, such as chemotaxis and production of roi [ , ] . based on the adaptive mechanisms microorganisms reside in different cells and at different locations in the cells. treating diseases therefore, necessitates an understanding of both the resident cells and target organelles. illustrative examples of microorganism and their cellular/organelles targets are listed out in table . . the granulocytes are classifi ed as neutrophils, eosinophils, or basophils on the basis of cellular morphology. neutrophils play the major role in the body's defence. neutrophils are produced in the bone marrow by hematopoiesis. they are released into blood where they circulate for - h and migrate into tissues where they have a life span of a few days. during infection the bone marrow releases more than usual tularemia -cytosol al-khodor [ ] number of neutrophils, which migrate to the site of the infection. they act by both oxygen-dependent and oxygen-independent pathways to kill microbes. neutrophils exhibit a larger respiratory burst than macrophages and consequently are able to generate more reactive oxygen intermediates and reactive nitrogen intermediates. in addition, neutrophils express higher levels of defensins than macrophages do. hence, neutrophils are more active than macrophages in killing ingested microorganisms. dendritic cells are antigen-presenting cells and constitute . - % of the leukocyte population in the peripheral blood mononuclear cells. they are found mostly in nonlymphoid tissues and organs such as skin, heart, liver, lungs, and mucosal surfaces. the function of these cells is to initiate, stimulate and regulate a t cell response which includes antigen-specifi c t lymphocytes, th /th modulation, regulatory t cell induction and peripheral t cell deletion. there are four types of dendritic cells, i.e. langerhans cells, myeloid dendritic cells, plasmacytoid dendritic cells and infi ltrating infl ammatory dendritic epidermal cells. cd b, cd a, cd b and cd c, the thrombospondin receptor (cd ), and the mannose receptor (cd ), present on infl ammatory dendritic epidermal cells, are known to be involved in the uptake of bacterial components. in case of mycobacterium tuberculosis infection, alveolar macrophages (dust cells), along with dendritic cells engulf bacteria and exhibit innate as well as an adaptive immune response. combined efforts by macrophages and dendritic cells establish protective immunity in % of infected individuals. natural killer cells (nkc) are non-phagocytic cells present mostly in mammalian and avian species [ ] . nkc express surface receptors for the fc portion of igg and their function is to mediate antibody-dependent cytotoxicity against tumor target cells [ ] . it is also suggested that nkc play a role in resistance against some microbial infections. nkc also play a role in natural genetic resistance to infections caused by cytomegalovirus and herpes simplex type i [ , ] . however, there is also evidence against the role of nkc in resistance to some other viruses [ ] . lymphocytes are cells present % in the lymph and constitute - % of the body's white blood cells. there are approximately ~ - lymphocytes in the human body, and this can vary with body weight and age. they circulate in the lymph and blood, and can migrate into tissue spaces and lymphoid organs, enabling integration with the immune system. the two main categories of lymphoid cells that can recognise and react against a wide range of specifi c antigens are b lymphocytes or b cells and t lymphocytes or t cells. the main function of b cells is to produce antibodies against antigens [ ] . each of the approximately . natural t lymphocytes mature in the thymus region and survive in the periphery. the chief function of t cells is to respond to signals associated with tissue destruction and to minimise the collateral tissue damage they cause [ ] . t cells express t-cell receptors (tcr) which are a composite of polypeptides including cd and either of one of the two membrane molecules, cd and cd . tcr recognises virus infected cells and cancer cells. however, unlike b cells, tcr does not recognise free antigen, unless it is bound to mhc molecules on the membrane of antigen presenting cells. the main function of t cells is to induce death of virus infected cells by secretion of cytotoxins and cytokines which activates b cells, macrophages and cytotoxic t cells. t cells also play role in infectious diseases such as leishmaniasis [ ] , infection by hepatitis c virus (hcv), etc. their ability to confi ne exuberant immune reactivity, associated with many chronic infections is benefi cial the host due to limited tissue damage [ ] . infectious diseases are also located in cells other than cells of the res. such cells include hepatocytes, epithelial cells and erythrocytes. hepatocytes are located in the liver and are major site for infections such as hepatitis b/c and malaria. the hepatocytes are discussed in greater detail in chapter of this book. epithelial cells bind together to form the epithelial tissue which is held together by adherens, tight junctions, gap junctions and desmosomes. the functions of epithelial cells are boundary and protection of vital organs, transportation, absorption, secretion, lubrication and movement. these epithelial cells can be readily attacked by microbes such as hiv virus, infl uenza, herpes simplex virus (hsv- ) and cause infections. furthermore, erythrocytes are infected and act as hosts for plasmodium causing malaria, one of the current fatal infections posing serious challenges. the introduction of antimicrobial agents such as penicillin resulted in a major breakthrough to decrease morbidity and mortality caused by infectious diseases. antibiotics represented one of the greatest discoveries. this euphoria was short lived due to adverse effects and the emergence of drug resistance. conventional therapy when associated with side effects or necessitates long term treatment, results in low patient compliance. further inadequate drug concentration within cells is a major barrier for effective treatment of intracellular diseases. increasing the dose, however, resulted in enhanced toxicity. mono-drug therapy evolved into multi-drug therapy, and enabled a good degree of success and continues to form standard therapy, even today. classic examples include the multi-drug combination for tuberculosis akt , akt , akt comprising , or drugs, respectively. the haart combination for aids and two drug combinations for malaria are also examples of successful therapy. nevertheless, the alarming rate at which drug resistance is occurring, and more so the emergence of multi-drug resistance are a matter of great concern. tuberculosis is one such major disease which has evolved from resistant to multi-drug resistant(mdr) to total drug resistant (tdr), the latest being extremely drug resistant tuberculosis (xxdr), wherein, resistance is seen to almost all known antitubercular drugs. the emergence of multi-drug resistance is attributed to a number of factors. pathogens resort to different mechanisms to avoid intracellular killing. some pathogens secrete exotoxins which destroy phagocytes and prevent phagocytosis. bacteria with pore forming cytolysins avoid the phagosome and also escape lysosomal destruction [ - ] . certain bacteria interfere with the production of cytotoxic metabolites of phagocytes or contain the antioxidant proteins, thereby overcoming the effects of rnis or rois and cause obstruction in phagocytosis [ , ] . bacteria adhere to surfaces, aggregate and form a hydrated polymeric matrix comprising of exopolysaccharide known as biofi lms [ ] . biofi lms are developed by various bacteria such as salmonella , streptococcus, vibrio cholerae, klebsiella pneumonia and haemophilus infl uenzae. further some cells in the biofi lm experience nutrient limitation and therefore survive in the starved state. such cells are slow growing cells and less susceptible to antimicrobial agents [ ] . certain cells in a biofi lm adapt a different and protected phenotype. biofi lms are resistant to antibodies, phagocytes, and antibiotics . although p hagocytes reach the biofi lms, they become frustrated and release their enzymes, which cause damage to the tissue around the biofi lm. release of bacteria through the damaged biofi lm results in dissemination of the infection, leading to acute infection in the surrounding tissues [ , ] . effl ux pump genes and proteins are present in almost all organisms. effl ux pumps thwart the entry of an antibiotic in the bacterial cell and export an antibiotic from the cell. as effl ux pumps can be specifi c for one substrate or for drugs of dissimilar structure, they can be associated with multi-drug resistance. multi-drug-resistance effl ux pumps are a known cause for the development of bacterial resistance against antibiotics. bacterial effl ux-pump proteins related with mdr are divided into fi ve families namely the atp binding cassette (abc) superfamily, the major facilitator superfamily (mfs), the multi-drug and toxic-compound extrusion (mate) family, the small multidrug resistance (smr) family and the resistance nodulation division (rnd) family [ ] . multi-drug resistance occurs, when effl ux proteins are overexpressed on the cell, and easily identify and effi ciently expel a broad range of antibiotics from the cells [ ] . gram-negative bacteria express several families of transporters which cause resistance [ ] . gram-positive bacteria mainly staphylococcus aureus and streptococcus pneumoniae express mdr effl ux pumps. s. aureus (responsible for skin and soft-tissue infections) overexpress mfs effl ux pump nora which enables resistance to chloramphenicol and fl uoroquinolones. the s. pneumoniae mfs effl ux pumppmra exports the fl uoroquinolones ciprofl oxacin, norfl oxacin, and also expels the dyes acrifl avine and ethidium bromide [ - ] escherichia coli emre express a member of the small multi-drug resistance (smr) superfamily and acrab-tolc, a member of the resistance-nodulation-cell division (rnd) superfamily. vibrio parahaemolyticus overexpress norm, a member of the multi-drug and toxic compound extrusion (mate) superfamily. multi-drug-resistant tuberculosis (mdr-tb) is appearing as a ghost among the mdr bacteria because tb patients are at high risk of death due to failure of treatment. it is evident that mdr exhibits p effl ux pumps which play a crucial role in the pathogenicity of the microorganisms, and is responsible for the effl ux of tetracycline and aminoglycosides. this has opened a vast array for research in identifying mutants which are responsible for overexpressing these protein pumps in cases of elevated virulence [ ] . chemical modifi cation of antibiotics resulting in their inactivation and hence, ineffective dug concentration can be a cause of bacterial resistance. the inactivation reactions include hydrolysis, redox, and group transfer. hydrolysis is the major cause of degradation of beta lactam antibiotics. the group transfer approach is the most varied and includes modifi cation by thiol transfer, glycosylation, acyl transfer, ribosylation, nucleotidylation and phosphorylation transfer. drugs which are degraded by group transfer are aminoglycoside, chloramphenicol, rifamycin, macrolides, etc. [ ] . one important strategy to overcome the limitation of conventional drug delivery is to deliver high therapeutic payloads intracellularly. this could ensure high efficacy, coupled with low toxicity to provide major advantages. targeted nanocarriers provide high promise as potential drug delivery systems with the capacity to address this specifi c challenge. targeted nanocarriers could therefore prove to be the magic wand. passive and active targeting approaches could be relied on to achieve organ based targeting (fi rst order), specifi c cell based targeting in an organ (second order) and cell organelle based targeting (third order) [ ] . a major requirement, however, besides reaching the targeting site is to ensure adequate concentration and adequate retention at the site. passive targeting can be described as deposition of drug or drug-carrier systems at a particular location due to pharmacological or physicochemical factors [ ] . passive targeting can be achieved by exploiting pathophysiological and anatomical opportunities. introduction of drugs directly into various anatomical sites for example lungs and the eye by using non-invasive or invasive methods such as catheters or direct injections can enable local targeting. these site specifi c drug delivery methods limit systemic toxicity of the drug thus reducing adverse effects of drugs in the nontarget tissues [ ] . exploiting altered pathological conditions in diseased tissues are strategies that can be adopted for passive targeting for example chemotactic factors released in infected or infl amed tissues increased permeability of vascular tissues, decreased ph and/or increased temperature [ , ] . increased vascular permeability specifi cally in cancers has enabled passive targeting of nanocarriers and is cited as the enhanced permeation and retention effect (epr) effect [ ] . surface properties such as particle size, shape, hydrophobicity and surface charge have great impact on macrophage activation and phagocytosis. particle size plays essential role in distribution and elimination of nanocarriers [ ] . particles size can infl uence attachment, adhesion, phagocytosis, distribution, circulation half-life and endocytic pathways [ , ] . the opsonisation and phagocytosis of particles is strongly affected by size of nanocarriers. although macrophages engulfed . versus μm igg-coated spherical particles by different mechanisms, they followed similar kinetics [clathrin endocytosis versus fc-receptor mediated phagocytosis]. phagocytic uptake is generally observed with polymeric particles and liposomes with high particle size [> -microns] [ ] . table . highlights the size of a number of nanocarriers evaluated for targeted delivery in infectious diseases. a broad range of non-spherical shaped particles studied including cylinders, cubes, hemispheres, ellipsoids, cones and complex shapes like fi lamentous, biconcave discoid showed varying effects on phagocytosis [ ] . non-spherical shaped particles bypassed phagocytosis due to incomplete actin structure formation. particle shape affected attachment and internalisation during phagocytosis [ ] . for instance oblate ellipsoids show best attachment and internalisation by phagocytosis, while prolate ellipsoids showed good attachment but poor internalisation. champion et al. reported that worm-like particles showed low phagocytosis as compared to spherical particles of the same volume [ ] . asymmetric polymer lipid nanostructures (lipomer) of doxycycline hydrochloride (dh) in the range of ( - nm) [ ] revealed enhanced splenic delivery. the irregular shape of the lipomer coupled with rigidity resulted in fi ltration and non-phagocytic accumulation to reveal splenotropy in sinusoidal spleen models, rat, rabbit and dog. a high spleen liver ratio of . : . was seen in the dog model (fig. . ) [ ] . surface properties like hydrophobicity and surface charge also impact opsonisation, phagocytosis and biodistribution of nanoparticles [ ] . hydrophobic nanocarriers are readily coated by complement proteins, albumin, and immunoglobulin and scavenged by res [ ] . surface charge of particles also infl uences interaction and stability with cells [ ] . reports suggest that positively charged particles showed high phagocytic uptake over negatively charged particles probably due to better interaction with the negatively charged cell membrane. cationic and neutral nanocarriers are less taken up by res as compare to negatively charge [ - ] . however, negatively charged nanoparticles can potentially attach to cationic sites on the macrophages namely the scavenger receptors, which facilitate their uptake by res [ ] . for details on infl uence of particle size, shape and charge readers are directed to the following reviews [ , ] . active targeting, defi ned as specifi c targeting of drugs or drug containing nanocarriers by anchoring active agents or ligands, provides selectivity, recognisability and potential to interact with specifi c cells and tissues in the body [ ] . targeting by attaching ligands has been investigated as an additional strategy to enhance translocation of antimicrobials inside cells. attaching ligands facilitates greater uptake and can be mediated by various mechanisms. the membrane of macrophages expresses various receptors to facilitate the internalisation of cargoes inside the cell and their degradation. receptor mediated endocytosis (rme) permits the rapid internalisation of ligand attached particles as compared to untargeted particles [ ] . the common rme mechanisms are macropinocytosis, clathrin dependent endocytosis (cde), caveolae-mediated endocytosis and clathrin independent endocytosis (cie). each approach exhibits different binding and internalisation mechanisms. further, the predominant uptake mechanism is often dictated by the nature of the ligand. receptor mediated processes are relatively slower than phagocytic processes, with the ligand playing an important role. the sizes, geometry, charge and density of the ligand signifi cantly infl uences receptor mediated endocytosis [ ] . for more references readers can refer to [ , , ] . table . lists the endocytic pathways, endosome morphology and the proteins involved in the endocytic pathways. macrophages possess large number of surface receptors which help in the process of recognition and endocytosis of engineered particulate carriers. infection of macrophages leads to changes in the expression pattern of the concerned receptors, which can be exploited for targeted drug delivery employing nanocarriers. table . is a summary of the important receptors on macrophages and illustrative examples of ligands for the same that could play a role in designing targeted nanocarriers for infectious disease therapy. cd [ ] , decay accelerating factor (cd ), endo [ ] are also receptors which could be targeted. nevertheless, ligands for the same need to be explored. all known nanocarriers can be effectively employed for targeted delivery in intracellular infections. both passive and active targeting approaches have been evaluated. the following tables . and . illustrate examples of nanocarriers, limited to major anti-infective agents for active and passive targeting, respectively. size being a major parameter infl uencing targeting to res. table . also highlights the size of nanocarriers, which is a primary factor in passive targeting. tuberculosis is persistent and deadly infectious disease, caused mycobacterium tuberculosis which is non-specifi cally phagocytosed by alveolar macrophages. malaria is a complex disease caused by plasmodium and majorly resides in non-res cells like red blood cells (rbcs) and hepatocytes. entry of the parasite into the brain causes cerebral malaria. malaria can be targeted at the exoerythrocytic stage by targeting rbcs, or targeting the hypnozoites to tackle malarial relapse and further in case of cerebral malaria targeting the brain. increased permeability of infected rbcs is seen after - h of plasmodium invasion through formation of channels. these channels are "new permeability pathways" (npps) which allow entry of molecules such as dextran, protein a and igg a antibody thereby differentiating the non-infected and infected rbcs. such pathways could be targeted to enable high drug loading in the erythrocytes specifi cally through design of nanocarriers of < nm [ ] . this could be supported through design of stealth nanocarriers which could enable long circulation, using various stealth agents like poly(ethyleneglycol) (peg), pluronic, etc. [ ] . chloroquine liposomes anchored with anti-erythrocyte f (ab′) were studied for targeting to erythrocytes [ ] . hepatocytes the residence of hypnozoites expresses the asialoglycoprotein receptor (asgpr), which is overexpressed in infections. targeting this receptor using nanocarriers anchored with asgpr ligands is a strategy for hepatocyte targeting. joshi et al. prepared in situ primaquine nanocarboplex of primaquine phosphate anchored with pullulan as the asgpr ligand for specifi c targeting to hepatocytes. signifi cantly, enhanced hepatic accumulation with preferential accumulation in the hepatocytes and a high hepatocytes/nonparenchymal cells ratio of : confi rmed hepatocyte targeting [ ] . transferrin (tf)-anchored solid lipid nanoparticles (slns) were intravenously administered for targeting quinine dihydrochloride to the brain, in cerebral malaria. compared to conventional slns or drug solution the tf-slns signifi cantly enhanced the brain uptake of quinine [ ] . a major feature of hiv that complicates therapy is the existence of hiv in multiple reservoirs, which include various cellular and anatomical sites [ ] . the typical reservoirs are the liver, spleen, lungs, git and genital tract with the brain and bone marrow representing remote sites [ ] . targeted delivery for hiv therefore needs to address delivery to maximum sites simultaneously to achieve remission. one strategy that we propose is a combination of nanocarriers of size < nm to target remote sites and size > nm target major res organs (unpublished data). viral replication is inhibited by the antioxidant glutathione. erythrocytes containing glutathione (gsh) in combination with azidothymidine (azt) and didanosine (ddi) showed higher reduction in viral dna in bone marrow and brain as compared to ddi + gsh alone [ ] . immunoliposomes containing sirna for targeting the lymphocyte function-associated antigen- (lfa- ) integrin, which is expressed on all leukocytes, was selectively taken up by t cells and macrophages, the primary site of hiv. further, in vivo administration of anti-ccr sirna resulted in leukocytespecifi c gene silencing that was sustained for days [ ] . nanogels comprising non-reverse transcriptase inhibitors (nrits) decorated with a peptide for brain specifi c apolipoprotein e (apoe) receptors, showed tenfold suppression of retroviral activity and decrease infl ammation in humanised mouse model of hiv- infection in the brain [ ] . targeted drug delivery for the therapy of veterinary infections assumes immense importance not only for improved animal health but due to the challenges posed by zoonotic diseases. about zoonotic diseases including brucellosis, tuberculosis, trypanosomiasis, cysticercosis and others are related to . billion cases of infection in humans and over two million deaths annually [ , ] . such infections exist both in domestic animals and wild life. the close proximity of humans especially with such domestic animals is a cause of global concern. the who policy of "cull and kill" results not only in the loss of lives but also heavy monetary losses to the farmer. targeted treatment strategies using nanodrug delivery systems could provide a revolutionary strategy to benefi t both the animals and man. the benefi ts of targeted nanomedicine strategies are slowly gaining recognition as evident from a number of reported studies. liposomes have been used by many researchers for treating various veterinary diseases such as leishmaniasis [ , ] , brucellosis [ ] , blastomycosis [ ] , babesiosis [ ] , etc. patil et al. [ ] developed an asymmetric lipomer. this is a combination of polymer-lipid containing doxycycline which could have application in the treatment of intracellular infections that are primarily resident in the spleen like brucellosis, ehrlichiosis, etc. a number of studies are reported on horses infected with babesiosis, streptococcus equi, t. gondii and strongylus vulgaris infections using liposomes [ ] , polymeric nanospheres [ ] , dendrimers [ ] and micelles [ ] respectively. a recent study revealed the improved therapy of theileiriosis in cattle with solid lipid nanoparticles (sln) of buparvaquone [ ] . sln revealed comparable effect with the intramuscular injection at signifi cantly lower doses. nanodrug delivery systems have also been evaluated in dogs, sheep and pigs. for details on nanodrug delivery applications in targeted delivery in veterinary infections, readers are directed to the following reference [ ] . targeted delivery for infectious diseases has immense scope. tackling infections using nanodrug delivery systems could provide a practical alternative as a short term strategy. a rate-limiting factor however would be the serious concerns of toxicity. nanodrug delivery systems due to their high intracellular delivery could precipitate new and unknown toxicities. evolving strategies to predict the same is an important path forward. while vaccines could probably provide the ultimate cure and control, vaccine development is a complex process and not yet easily attained as evident from the limited success stories. however, designing nano-vaccines targeted to exhibit greater cellular response is also a near future prospect. pentraxins: structure, function, and role in infl ammation collectins -soluble proteins containing collagenous regions and lectin domains -and their roles in innate immunity ficolins and infectious diseases defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene cutting edge: toll-like receptor (tlr )-defi cient mice are hyporesponsive to lipopolysaccharide: evidence for tlr as the lps gene product endotoxin-tolerant mice have mutations in toll-like receptor (tlr ) recognition of double-stranded rna and activation of nf-kappab by toll-like receptor differential roles of tlr and tlr in recognition of gram-negative and gram-positive bacterial cell wall components a toll-like receptor recognizes bacterial dna the innate immune response to bacterial fl agellin is mediated by tolllike receptor professional and non-professional phagocytes: an introduction cell size of alveolar macrophages: an interspecies comparison distinct role of macrophages in different tumor microenvironments exploring the full spectrum of macrophage activation a microbial strategy to multiply in macrophages: the pregnant pause mechanism of phagolysosome biogenesis block by viable mycobacterium tuberculosis yersinia pestis can reside in autophagosomes and avoid xenophagy in murine macrophages by preventing vacuole acidifi cation lack of acidifi cation in mycobacterium phagosomes produced by exclusion of the vesicular proton-atpase mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event internalized listeria monocytogenes modulates intracellular traffi cking and delays maturation of the phagosome inhibition of macrophage phagosome-lysosome fusion by salmonella typhimurium inhibition of phagolysosomal biogenesis by the leishmania lipophosphoglycan avoidance of innate immune mechanisms by the protozoan parasite, leishmania spp in protozoans in macrophages phagosome acidifi cation blocked by intracellular toxoplasma gondii helicobacter pylori phagosome maturation in primary human macrophages characterization of the catalytic subunit of trypanosoma cruzi cyclic amp-dependent protein kinase trimming the fat: a brucella abortus survival strategy interaction of leishmania parasites with dendritic cells and its functional consequences association of legionella pneumophila with the macrophage endoplasmic reticulum two distinct defects in intracellular growth complemented by a single genetic locus in legionella pneumophila life on the inside: the intracellular lifestyle of cytosolic bacteria survival of intracellular pathogens within macrophages listeriolysin o: a genuine cytolysin optimized for an intracellular parasite expression of the rickettsia prowazekii pld or tlyc gene in salmonella enterica serovar typhimurium mediates phagosomal escape distinct isoforms of phospholipase a mediate the ability of salmonella enterica serotype typhimurium and shigella fl exneri to induce the transepithelial migration of neutrophils a potential new pathway for staphylococcus aureus dissemination: the silent survival of s. aureus phagocytosed by human monocyte-derived macrophages selective receptor blockade during phagocytosis does not alter the survival and growth of mycobacterium tuberculosis in human macrophages macrophage receptors for mycobacterium tuberculosis multicellular and aggregative behavior of salmonella typhimurium strains is controlled by mutations in the agfd promoter salmonella enterica serovar typhimurium dt displays a rugose phenotype a novel relationship between o-antigen variation, matrix formation, and invasiveness of salmonella enteritidis thin aggregative fi mbriae and cellulose enhance long-term survival and persistence of salmonella thin, aggregative fi mbriae mediate binding of salmonella enteritidis to fi bronectin the multicellular morphotypes of salmonella typhimurium and escherichia coli produce cellulose as the second component of the extracellular matrix biofi lm formation by escherichia coli o :h on stainless steel: effect of exopolysaccharide and curli production on its resistance to chlorine effect of heat, acidifi cation, and chlorination on salmonella enterica serovar typhimurium cells in a biofi lm formed at the air-liquid interface genetic analysis of salmonella enteritidis biofi lm formation: critical role of cellulose salmonella produces an o-antigen capsule regulated by agfd and important for environmental persistence lower intracellular hydrogen peroxide levels in cells overexpressing cuzn-superoxide dismutase phenotypic switching of cryptococcus neoformans occurs in vivo and infl uences the outcome of infection hiv- and the macrophages human immunodefi ciency virus type entry into macrophages mediated by macropinocytosis activation of tgf-beta by leishmania chagasi: importance for parasite survival in macrophages the effect of parasitization by leishmania mexicana on macrophage function in vitro subversion mechanisms by which immune response: a signaling point of leishmania parasites can escape the host optimal cytoplasmic transport in viral infections adaptation of the brucellae to their intracellular niche brucella evades macrophage killing via virb-dependent sustained interactions with the endoplasmic reticulum differing infl uences of virus burden and immune activation on disease severity in secondary dengue- virus infections membrane association of hepatitis c virus nonstructural proteins and identifi cation of the membrane alteration that harbors the viral replication complex change of processing and nucleocytoplasmic transport of mrna in hsv- -infected cells transgenic mice with intracellular immunity to infl uenza virus how the parasitic bacterium legionella pneumophila modifi es its phagosome and transforms it into rough er: implications for conversion of plasma membrane to the er membrane an amphipathic sulphated glycoconjugate of leishmania: characterization with monoclonal antibodies assessing the contributions of the lias histidine kinase to the innate resistance of listeria monocytogenes to nisin, cephalosporins, and disinfectants the biochemistry of intracellular parasitism innate immune response in the gut against salmonella -review adaptive changes in gene expression of mycobacterium tuberculosis during the development of the infection triggering ras signalling by intracellular francisella tularensis through recruitment of pkca and bi to the sos /grb complex is essential for bacterial proliferation in the cytosol natural killer cells: their role in defenses against disease natural killer cells may be the only cells in normal mouse lymphoid cell populations endowed with cytolytic ability for antibody-coated tumour target cells genetic control of natural resistance to infection and malignancy genetic infl uences on the augmentation of natural killer (nk) cells during murine cytomegalovirus infection: correlation with patterns of resistance natural cell-mediated immunity during viral infections the cellular basis of the immune response. an approach to immunobiology regulating the regulators regulatory t cells: friend or foe in immunity to infection? natural regulatory t cells in infectious disease fusion of azurophil granules with phagosomes and activation of the tyrosin ekinase hck are specifi cally inhibited during phagocytosis of mycobacteria by human neutrophils legionella pneumophila dota protein is required for early phagosome traffi cking decisions that occur within minutes of bacterial uptake apoptosis in macrophages and alveolar epithelial cells during early stages of infection by legionella pneumophila and its role in cytopathogenicity legionella pneumophila utilizes the same genes to multiply within acanthamoeba castellanii and human macrophages survival of mycobacterium avium and mycobacterium tuberculosis in acidifi ed vacuoles of murine macrophages interactions between professional phagocytes and brucella spp nitric oxide-mediated crosstalk between helicobacter and gastric mucosa bacterial biofi lms: a common cause of persistent infections growth rate control of adherent bacterial populations the diffusion characteristics of antibiotics in mucus glycoprotein gels a review of experimental measurements of effective diffusive permeabilities and effective diffusion coeffi cients in biofi lms multidrug-resistance effl ux pumps not just for resistance multidrug effl ux pumps and resistance: regulation and evolution antibiotic effl ux mechanisms the multidrug effl ux transporter of bacillus subtilis is a structural and functional homolog of the staphylococcus nora protein nucleotide sequence and characterization of the staphylococcus aureus nor a gene, which confers resistance to quinolones effl ux-mediated fl uoroquinolone resistance in staphylococcus aureus bacterial resistance to antibiotics: enzymatic degradation and modifi cation drug targeting targeted drug conjugates: principles and progress nanosystems in drug targeting, opportunities and challenges targeted drug delivery to enhance effi cacy and shorten treatment duration in disseminated mycobacterium avium infection in mi host factors infl uencing the preferential localization of sterically stabilized liposomes in klebsiella pneumoniae-infected rat lung tissue neutrophil transepithelial migration: evidence for sequential, contact-dependent signaling events and enhanced paracellular permeability independent of transjunctional migration tumor vascular permeability and the epr effect in macromolecular therapeutics: a review characterization of the size, shape, and state of dispersion of nanoparticles for toxicological studies the effect of particle design on cellular internalization pathways nanocarriers entry into the cell: relevance to drug delivery antimicrobial effectiveness of liposomal polymyxin b against resistant gram-negative bacterial strains vitro activities of free and liposomal drugs against mycobacterium avium-m. intracellular complex and m. tuberculosis biological evaluation of pyrazinamide liposomes for treatment of mycobacterium tuberculosis rifabutin encapsulated in liposomes exhibits increased therapeutic activity in a model of disseminated tuberculosis liposome-entrapped ampicillin in the treatment of experimental murine listeriosis and salmonellosis treatment of brucella canis and brucella abortus in vitro and in vivo by stable plurilamellar vesicle-encapsulated aminoglycosides liposome-incorporated ciprofl oxacin in treatment of murine salmonellosis parasitic diseases: liposomes and polymeric nanoparticles versus lipid nanoparticles liposomes as carriers of the antiretroviral agent dideoxycytidine- %-triphosphate gelatin nanocarriers as potential vectors for effective management of tuberculosis development and characterization of guar gum nanoparticles for oral immunization against tuberculosis in vivo/in vitro pharmacokinetic and pharmacodynamic study of spray-dried poly-(dl-lactic-co-glycolic) acid nanoparticles encapsulating rifampicin and isoniazid preparation and antibacterial activity evaluation of rifampicin-loaded poly lactide-co-glycolide nanoparticles macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroaids targeted delivery of antibiotics to intracellular chlamydial infections using plga nanoparticles targeted delivery of arjunglucoside i using surface hydrophilic and hydrophobic nanocarriers to combat experimental leishmaniasis increase in gentamicin uptake by cultured mouse peritoneal macrophages and rat hepatocytes by its binding to polybutylcyanoacrylate nanoparticles targeted delivery of nanoparticles for the treatment of lung diseases encapsulation of moxifl oxacin within poly(butyl cyanoacrylate) nanoparticles enhances effi cacy against intracellular mycobacterium tuberculosis nanoencapsulation increases quinine antimalarial effi cacy against plasmodium berghei in vivo in vitro antileishmanial activity of amphotericin b loaded in poly(ε-caprolactone) nanospheres inhalable microparticles containing isoniazid and rifabutin target macrophages and "stimulate the phagocyte" to achieve high effi cacy microparticle-based lung delivery of inh decreases inh metabolism and targets alveolar macrophages lopinavir loaded solid lipid nanoparticles (sln) for intestinal lymphatic targeting transmucosal transport of tobramycin incorporated in solid lipid nanoparticles after duodenal administration to rats. part ii tissue distribution solid lipid nanoparticles as drug carriers incorporation and retention of the lipophilic prodrug ´-azido- ´-deoxythymidine palmitate solid lipid nanoparticles enhance the delivery of the hiv protease inhibitor, atazanavir, by a human brain endothelial cell line pharmacokinetics, tissue distribution and relative bioavailability of isoniazid-solid lipid nanoparticles targeted intracellular delivery of antituberculosis drugs to mycobacterium tuberculosis-infected macrophages via functionalized mesoporous silica nanoparticles niosomal delivery of isoniazid development and characterization formulation, antimalarial activity and biodistribution of oral lipid nanoemulsion of primaquine a novel injectable water-soluble amphotericin b-arabinogalactan conjugate targeting potential and anti-hiv activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer glycodendrimeric nanoparticulate carriers of primaquine phosphate for liver targeting targeted killing of leishmania donovani in vivo and in vitro with amphotericin b attached to functionalized carbon nanotubes characterisation of copper oxide nanoparticles for antimicrobial applications investigations into the antibacterial behavior of copper nanoparticles against escherichia coli global trends in emerging infectious diseases antibacterial activity of zno nanoparticle suspensions on a broad spectrum of microorganisms effect of iron oxide and gold nanoparticles on bacterial growth leading towards biological application role of target geometry in phagocytosis polymer particle shape independently infl uences binding and internalization by macrophages role of lipids in enhancing splenic uptake of polymer-lipid (lipomer) nanoparticles particle shape: a new design parameter for passive targeting in splenotropic drug delivery particle size, surface coating, and pegylation infl uence the biodistribution of quantum dots in living mice plga nanoparticles of different surface properties: preparation and evaluation of their body distribution effect of surface properties on nanoparticle-cell interactions surface-modifi ed biodegradable albumin nano-and microspheres. ii: effect of surface charges on in vitro phagocytosis and biodistribution in rats drug targeting by surface cationization the effect of surface charge on in vivo biodistribution of peg-oligocholic acid based micellar nanoparticles the class b scavenger receptors sr-bi and cd are receptors for anionic phospholipids targeting to macrophages: role of physicochemical properties of particulate carriers liposomes and microspheres on the phagocytosis by macrophages lymphatic targeting with nanoparticulate system endocytic mechanisms for targeted drug delivery the effect of nanoparticle size, shape, and surface chemistry on biological systems mechanisms of endocytosis endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies simultaneous binding of ptdins p and clathrin by ap in the nucleation of clathrin lattices on membranes yolk protein uptake in the oocyte of the mosquito aedes aegypti the multiple faces of caveolae caveolin, a protein component of caveolae membrane coats caveolins: structure and function in signal transduction flotillin- defi nes a clathrin-independent endocytic pathway in mammalian cells coassembly of fl otillins induces formation of membrane microdomains, membrane curvature, and vesicle budding arf family gtp loading is activated by, and generates, positive membrane curvature characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements clathrin-independent endocytosis: a unique platform for cell signaling and pm remodelling clathrin-independent endocytosis: new insights into caveolae and noncaveolar lipid raft carriers quantitative microscopy reveals d organization and kinetics of endocytosis in rat hepatocytes a dynamin-cortactin-arp / complex mediates actin reorganization in growth factor-stimulated cells clathrinindependent endocytosis used by the il- receptor is regulated by rac , pak and pak interleukin receptors and detergent-resistant membrane domains defi ne a clathrin-independent endocytic pathway uptake of pneumocystis carinii mediated by the macrophage mannose receptor tuftsin-bearing liposomes in treatment of macrophage-based infections tuftsin (an ig-associated tetrapeptide) triggers the immunogenic function of macrophages: implications for activation of programmed cells microglial scavenger receptors and their roles in the pathogenesis of alzheimer's disease targeting the hemoglobin scavenger receptor cd in macrophages highly increases the anti-infl ammatory potency of dexamethasone the function of fcγ receptors in dendritic cells and macrophages primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains folate receptor β is expressed by tumor-associated macrophages and constitutes a marker for m anti infl ammatory/regulatory macrophages folate receptor β as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients targeted drug delivery via the transferrin receptor-mediated endocytosis pathway the role of pattern-recognition receptors in innate immunity: update on toll-like receptors macrophage complement receptors and pathogen clearance ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex toll receptors, cd , and macrophage activation and deactivation by lps drug delivery system: targeting of pentamidines to specifi c sites using sugar grafted liposomes effi cient drug delivery to alveolar macrophages and lung epithelial lining fl uid following pulmonary administration of liposomal ciprofl oxacin in rats with pneumonia and estimation of its antibacterial effects hyaluronan-modifi ed and regular multilamellar liposomes provide sub-cellular targeting to macrophages, without eliciting a pro-infl ammatory response the role of apolipoprotein e in alzheimer's disease targeting leishmania (l.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine therapeutic effi cacies of isoniazid and rifampin encapsulated in lung-specifi c stealth liposomes against mycobacterium tuberculosis infection induced in mice aerosolized liposome-based delivery of amphotericin b to alveolar macrophages uptake and processing of immunoglobulin-coated liposomes by subpopulations of rat liver macrophages sterically stabilized liposomes bearing anti-hla-dr antibodies for targeting the primary cellular reservoirs of hiv- targeted delivery of indinavir to hiv- primary reservoirs with immunoliposomes development and optimization of curcumin-loaded mannosylated chitosan nanoparticles using response surface methodology in the treatment of visceral leishmaniasis ionic complexation as a noncovalent approach for the design of folate anchored rifampicin gantrez nanoparticles surfacemodifi ed cobalt oxide nanoparticles: new opportunities for anti-cancer drug development tat-conjugated nanoparticles for the cns delivery of anti-hiv drugs targeted brain delivery of azt via transferrin anchored pegylated albumin nanoparticles transferrin-and transferrin-receptor-antibody-modifi ed nanoparticles enable drug delivery across the blood-brain barrier (bbb) mannan-coated gelatin nanoparticles for sustained and targeted delivery of didanosine: in vitro and in vivo evaluation mannan-modifi ed solid lipid nanoparticles for targeted gene delivery to alveolar macrophages mannosylated nanoparticulate carriers of rifabutin for alveolar targeting transferrin-conjugated solid lipid nanoparticles for enhanced delivery of quinine dihydrochloride to the brain polyamidoamine dendrimers-capped carbon dots/au nanocrystal nanocomposites and its application for electrochemical immunosensor toxicological investigation of surface engineered fi fth generation poly (propyleneimine) dendrimers in vivo targeting of efavirenz loaded tuftsin conjugated poly (propyleneimine) dendrimers to hiv infected macrophages in vitro intracellular macrophage uptake of rifampicin loaded mannosylated dendrimers macrophages targeting of amphotericin b through mannosylated multiwalled carbon nanotubes inhalable microparticles containing nitric oxide donors: saying no to intracellular mycobacterium tuberculosis protein transport across the parasitophorous vacuole of plasmodium falciparum: into the great wide open design aspects of poly(alkylcyanoacrylate) nanoparticles for drug delivery functional drug targeting to erythrocytes in vivo using antibody bearing liposomes as drug vehicles receptor-mediated hepatocyte-targeted delivery of primaquine phosphate nanocarboplex using a carbohydrate ligand the challenge of viral reservoirs in hiv- infection hiv disease management in the highly active antiretroviral therapy (haart) era new drug combinations for the treatment of murine aids and macrophage protection rnai-mediated ccr silencing by lfa- -targeted nanoparticles prevents hiv infection in blt mice nano-nrtis demonstrate low neurotoxicity and high antiviral activity against hiv infection in the brain effi cacy of the liposome trifl uralin in the treatment of experimental canine leishmaniasis reduced tissue parasitic load and infectivity to sand fl ies in dogs naturally infected by leishmania (leishmania) chagasi following treatment with a liposome formulation of meglumine antimoniate effi cacy of various treatment regimens, using liposomal streptomycin in cows with brucellosis use of an amphotericin b lipid complex for treatment of blastomycosis in dogs liposomal diamidine (imidocarb), preparation and animal studies enhanced mucosal immunoglobulin a response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide evaluation of immune responses in sheep induced by dna immunization with genes encoding gra , gra , gra and gra antigens of toxoplasma gondii effi cacy of ivermectin in injectable and oral paste formulations against eight-week-old strongylus vulgaris larvae in ponies buparvaquone loaded solid lipid nanoparticles for targeted delivery in theleriosis targeted nanomedicine strategies for livestock infections. nanotechnology for animal health and production key: cord- -e vcf un authors: nan title: forum date: - - journal: pharmaceut med doi: . /s - - -z sha: doc_id: cord_uid: e vcf un nan the us food and drug administration (fda) is introducing electronic submission of safety reports for investigational new drug (ind) applications through the fda's adverse event reporting system (faers). the fda has released new draft guidance on the process as well as supporting technical specification documents. the changes will enable the fda to review pre-and post-marketing safety data within in the same system, with the same data standard. publication of the draft guidance and technical documents should assist drug manufacturers in beginning preparations for when final draft guidance is issued and becomes effective. ind safety reports will be submitted in a reporting format that is consistent with international council for harmonisation of technical requirements for pharmaceuticals for human use (ich) e b guidelines. the fda will shortly announce when sponsors can begin voluntary submissions of ind safety reports to faers. "the fda highly encourages sponsors of all inds, both commercial and noncommercial, to begin submitting ind safety reports to faers voluntarily as soon as the new submission process is available," said dr. janet woodcock, director of the fda's center for drug evaluation and research (cder). ind safety reporting via faers will be voluntary until years after the final guidance has been issued, after which it will become mandatory for commercial inds. the fda has created a separate submission path to faers for ind safety report submissions; they will remain designated as investigational and will not be publicly available. us food and drug administration. digital submission of adverse event reports for investigational new drug applications reflects fda's ongoing modernization efforts. oct . https ://www.fda.gov/news-event s/press -annou nceme nts/digit al-submi ssion -adver se-event -repor ts-inves tigat ional -new-drug-appli catio ns-refle cts-fdas-ongoi ng. accessed dec . the us institute for safe medication practices (ismp) and the ecri institute have announced that they have formed an agreement to work together to enhance patient safety with regard to medicines, medical devices and healthcare practices. ismp will become a subsidiary of the ecri institute on january , and together the two non-profit organisations will create one of the largest patient safety entities in the world. approximately % of us hospitals rely on data and recommendations from the ecri institute to protect patients from unsafe practices and ineffective products, while the ismp's efforts to improve safety in patients have resulted in changes to clinical practice and public policy, including improvements in drug labelling, packaging, preparation and administration. "this agreement will strengthen our critical contributions to medication safety," said ismp president michael cohen, "it allows both organizations to retain their core missions while immediately extending our ability to share lifesaving information and further a vision where safe, high-quality healthcare is more readily available". "for both organizations, this agreement furthers the mission, deepens expertise, and broadens relationships. it's a good move for both of us and for all of the organizations we serve, and ultimately for the patients worldwide," commented ecri institute president and ceo marcus schabacker. institute for safe medication practices. ecri institute and institute for safe medication practices join forces to enhance patient safety. nov . https ://www.ismp.org/news/ ecri-insti tute-and-insti tute-safe-medic ation -pract ices-joinforce s-enhan ce-patie nt-safet y. accessed dec . the us fda has published a draft document on best postmarketing drug safety surveillance practices, says dr. janet woodcock, director of the cder, in an fda statement. the fda evaluates over two million adverse event reports annually that are submitted to faers through the med-watch program, and to the vaccine adverse event reporting system (vaers) by patients or their families or healthcare providers, as well as adverse event reports submitted by pharmaceutical companies, and this information is used by the fda's office of surveillance and epidemiology, and the center for biologics evaluation and research's office of biostatistics and epidemiology, to identify safety concerns and recommend actions to improve safety. the cures act was introduced in to amend the federal food, drug, and cosmetic act by eliminating the requirement for the fda to prepare a summary analysis of reports of adverse drug reactions (adrs) received up to months after drug approval or after use of the drug by , patients. a new requirement of the cures act was for the fda to make its best practices for drug safety surveillance publicly available on the web. the fda has now announced the availability of a draft document entitled "best practices in drug and biological product postmarket safety surveillance for fda staff", which outlines the agency's approach to timely postmarketing analyses of drugs and biologics, and "includes a high-level overview of tools, methods, and signal detection and evaluation activities, using varied data sources, for drug safety surveillance to provide a broader context and a general overview of our overarching effort and commitment in this area", says woodcock. the fda is constantly seeking new methods for improving its surveillance practices, and is inviting the public to comment on its draft document on postmarketing surveillance. us food and drug administration. statement on the agency's efforts to protect patients through postmarket drug safety surveillance practices. nov . https ://www.fda. gov/news-event s/press -annou nceme nts/state ment-agenc ys-effor ts-prote ct-patie nts-throu gh-postm arket -drug-safet y-surve illan ce-pract ices. accessed dec . a number of improvements are being introduced to make medicine labels clearer in an effort to reduce medication errors in australia, says the therapeutic goods administration (tga). the regulatory agency has developed a set of posters targeted at both healthcare professionals and consumers, which will be released over a -year transition period. one of these posters is a quick reference guide for healthcare professionals entitled "your medicine, your knowledge-improved medicine labels", which highlights "some of the key medicine labelling changes that are important for consumers", noted the tga. in addition, another three posters have been developed for consumers to raise their awareness of the labelling improvements, and are intended to be displayed in waiting rooms or public areas. the tga noted that "medicine labels are already starting to change" and that some of the key information that consumers should be aware of will include the following: • prominence of active ingredients; • clearer medicine information, which may include a "critical health information table" that will provide information in a consistent order and will be easy to recognise; • information on declarable substances (e.g. allergens) will now be required on the medicine label; • easier dispensing of medicines from pharmacies; • updated names for australian medicine ingredients to align with names used internationally. the tga highlighted that medicine sponsors will have years (i.e. until august ) to implement these changes and fully comply with the new labelling rules. during this transition period, the agency will release targeted communications to consumers regarding the labelling changes. therapeutic goods administration. labelling changes: information for health professionals. oct . https :// www.tga.gov.au/label ling-chang es-infor matio n-healt h-profe ssion als. accessed dec . experts speaking at the european public health alliance's universal access and affordable medicines forum in november urged that drug approval in the eu should require the pharmaceutical company's agreement on a fair price, said elisabeth mahase in the bmj. at the forum, experts across the eu discussed the need for improved transparency from drug companies to enable fairer drug pricing. "we need a change in law. after european medicines agency (ema) registration, we need to identify what is a fair price as a basis for negotiation", commented carin uyl-de groot, director of the institute for medical technology assessment at erasmus university, rotterdam, the netherlands. she said that the marketing authorisation process should include assessments of cost effectiveness and fair pricing by the ema or another organisation after drug effectiveness and safety have been reviewed, and countries could use the fair pricing assessment as a basis for price negotiations with drug companies. experts also discussed the need for more research on cancer drugs before their approval, to ensure their effectiveness and to identify the specific patient population which will benefit from treatment with them. "only - % of cancer drugs actually have an impact. we make drugs available at a very high speed, without actually knowing how to use them", said denis lacombe, director general at the european organisation for research and treatment of cancer. he called for more research into the optimal way to use cancer drugs, and said that they should be less expensive if this information is not available. mahase e. eu drug approval should include price evaluation, says expert panel. bmj. ; :i . in response to increasing overseas enquiries, the national institute for health and care excellence (nice) is relaunching its international division to advise organisations, ministries and government agencies from outside the uk on health related, evidence-based decision making. the not-for-profit international division, operating on a fee for service basis, draws on the expertise of internal staff, academic partners and world-wide experts to provide support with cost effective, transparent resource allocation, improved quality of care and equitable access. clients can attend knowledge transfer seminars in the uk, overseas or via web conference, or employ the consultancy service for more intensive support with technical and institutional training including implementing new methods and programmes, and adapting nice guidelines to local healthcare settings. chief executive at nice, andrew dillon said "we're delighted to be relaunching nice international so that we can share what we've learnt over the last years to help other international health and care systems optimise their use of evidence-based practice". national institute for health and care excellence. nice international returns to deal with growing overseas enquiries. nov . https ://www.nice.org.uk/news/artic le/ nice-inter natio nal-retur ns-to-deal-with-growi ng-overs easenqui ries. accessed dec . market exclusivity extensions to promote antibiotic drug development may lead to billions of dollars in additional societal spending on prescription drugs, according to a study conducted by researchers from harvard medical school in boston, us. the researchers estimated the economic impact of the re-valuing anti-microbial products (revamp) act introduced in the us house of representatives in june with the goal to promote research and development of novel antibiotic drugs, specifically those targeting multi-drug resistant pathogens. the act offers manufacturers that gain fda approval for specific novel antibiotics a -month transferable market exclusivity extension voucher that could be applied to an existing brandname drug or sold to another manufacturer. the analysis identified antibiotics approved by the fda from through that would likely have qualified for an exclusivity voucher, and each antibiotic was matched to the fast-track drug with the highest revenue losing exclusivity within years of the antimicrobial's approval date. the median annual revenue of these antimicrobials prior to generic entry was estimated at $us million. accounting for a % spending reduction after generic entry, the median spending associated with an exclusivity voucher was $ million. total spending associated with a -year exclusivity extension for all drugs was estimated at $ . billion. "our results raise important concerns about the overall cost of transferable exclusivity vouchers", conclude the researchers. rome bn, kesselheim as. transferrable market exclusivity extensions to promote antibiotic development: an economic analysis. clin infect dis. epub oct . http:// doi.org/ . /cid/ciz . accessed dec . the newly developed technical verification (tech-ver) checklist for validating health economic decision-analytical models can help identify causes of model implementation errors and be used as a training and quality control tool for the development of models, say authors of a report published in pharmacoeconomics. embase and medline databases were searched up to may for studies on the credibility, validation and verification of models for health technology assessment (hta) or cost-effectiveness analysis. a draft checklist was developed based on findings from studies identified in the literature review, and applied to health economic models which varied with respect to stakeholder developer, purpose and the maturity of clinical evidence. iterative revision of the checklist was performed and checked after implementation. the checklist then underwent further revision after discussions with other health economists, until the final version of tech-ver was created. the tech-ver requires a model reviewer, a transparent model, input sources, and detailed documentation reporting the concept, implementation, model inputs and results. it includes five domains: input calculations, event-state calculations, result calculations, uncertainty analysis calculations, and overall checks. the reviewer should assess the justifications of methods used in calculations. verification tests conducted to check the correctness of implementation of these calculations should include (in consecutive order): black-box tests to check that model calculations align with a priori expectations; white-box testing of program code line by line, or cell by cell for crucial calculations if black-box test results are unexpected; and model replication/parallel programming if issues related to unexpected results from black-box tests cannot be resolved through white-box testing. "the tech-ver checklist is a comprehensive checklist for the technical verification of decision analytical models, aiming to help identify model implementation errors and their root causes while improving the transparency and efficiency of the verification efforts", said the authors. "it is the authors' aim that the tech-ver checklist transforms itself to an open-source living document, with possible future versions, or 'bolt-on' extensions for specific applications with additional 'fit-for-purpose' tests, as well as 'tips and tricks' and some demonstrative error examples," they commented. buyukkaramikli nc, rutten-van mölken mpmh, severens jl, et al. tech-ver: a verification checklist to reduce errors in models and improve their credibility. pharmacoeconomics. epub nov . https ://doi.org/ . /s - - -y. accessed dec . in most cases, dosage adjustments in patients with renal impairment can be effectively determined using their estimated glomerular filtration rate (egfr). however, the uk's medicines and healthcare products regulatory agency (mhra) notes in a drug safety update that "in some circumstances, the cockcroft-gault formula should be used to calculate creatinine clearance (crcl)". the mhra has received queries about choosing which renal function measurement to use. in some patient groups or clinical situations, egfr can overestimate renal function, and can lead to prescribing higher than recommended medication doses. the agency has received reports of adrs when egfr was used to determine dosage adjustments in patients with renal impairment. one yellow card report involved an elderly patient treated with a direct-acting anticoagulant (doac) who developed a significant bleeding event; a review revealed that the dose initiated "was too high for the patient". a cross-sectional study of drugs in general practices from the uk revealed that in elderly patients with reduced renal function, there was widespread prescribing outside recommendations; using egfr overestimated renal function for up to % of patients. using crcl is recommended for patients in the following situations: ≥ years of age; at extremes of muscle mass; taking doacs; taking nephrotoxic drugs; or taking drugs which are largely renally excreted and have a narrow therapeutic index. healthcare professionals are requested to report suspected adrs via the yellow card scheme. medicines and healthcare products regulatory agency. prescribing medicines in renal impairment: using the appropriate estimate of renal function to avoid the risk of adverse drug reactions. drug safety update. ; ( ): - . gifts from pharmaceutical companies to general practitioners (gps) in france appears to influence primary care prescribing, according to findings of a retrospective study published in the bmj. data from the national health data system database managed by the french national health insurance (nhi) system, and from the french transparency in healthcare database, were used to investigate the association between the monetary value of drug company gifts to gps and prescribing patterns of , gps who worked in the private sector in france in . the amount per visit reimbursed by the nhi was significantly lower in gps for whom no gifts were reported in the transparency in healthcare database than in those who received at least one drug company gift valued at ≥ € in (− € . ; p < . ). gps who received no gifts more frequently prescribed generic antibacterials, antihypertensives and hmg-coa reductase inhibitors (statins) than gps who received gifts valued at ≥ € (all p < . ). they significantly less frequently prescribed vasodilators but significantly more often prescribed ace inhibitors versus ace inhibitors plus angiotensin-ii receptor antagonists (sartans) compared with gps who received gifts valued at ≥ € . there were no significant differences in prescribing of aspirin or generic antidepressants or proton pump inhibitors between gps with and without gifts from drug companies. "our study shows that gifts to gps are common and associated with less rational drug prescriptions for patients and more expenses for the national health insurance", said the authors. the findings "suggest that french gps who do not receive gifts from pharmaceutical companies have better drug prescription efficiency indicators (as defined by france's national healthcare insurance) and less costly drug prescriptions than those who receive gifts", they concluded. in australia, adverse events in vaccinated patients are monitored by the ausvaxsafety system, notes the department of health, and a report of safety during indicated that vaccines in the national immunisation program (nip) are very safe. vaccine recipients or their carer are sent an sms message by the participating immunisation clinics, asking whether the recipient had any postvaccination adverse events. responses include yes, no, or stop (to opt out). during , > , sms messages were sent, with > , responses. a short survey is sent to yes responders, requesting a description of the adverse event. report of a visit to the emergency department (ed) or a physician is flagged with the provider, who follows up with the recipient and notifies the tga division if required. responses are closely monitored by ausvaxsafety, enabling potential problems to be detected and acted on. children are vaccinated against serious diseases at the schedule points of , , , and months of age and at years. nip modifications from july include the third dose of pneumococcal vaccine at months rather than months of age, and hib vaccine at months of age and a quadrivalent meningococcal vaccine at months of age rather than a combined hib-meningococcal vaccine at months of age. an adverse event was reported by %, %, %, %, % and % of patients at the , , , and months and years schedule points, respectively. an ed or physician visit was reported by . %, . %, . %, . %, . % and % of patients. the most frequently reported adverse events were fever, irritability, and injection site pain, swelling or redness, which are similar to reports in previous years. the proportion of serious adverse events (saes) was unaltered, and the overall type and proportion of adverse events was similar before and after modification of the nip schedule. adolescents are vaccinated against human papillomavirus and diphtheria/tetanus/whooping cough. adverse events were reported by % of patients, with . % requiring an ed or physician visit. the most commonly reported events were tiredness and injection site pain. pregnant women are vaccinated against influenza and diphtheria/tetanus/whooping cough. adverse events were reported by % of patients, with . % requiring an ed or physician visit. the most commonly reported events were injection site pain, swelling or redness. department of health-australia. vaccine safety in australia. ausvaxsafety summary report . nov . https ://www.healt h.gov.au/sites /defau lt/files /docum ents/ / /vacci ne-safet y-in-austr alia-ausva xsafe tysumma ry-repor t- .pdf. accessed dec . results of a preliminary study, reported in the journal of oncology practice, show that use of the decision aid developed by the american society of clinical oncology (asco) improves the accuracy of reporters in the attribution of saes to a drug, but has no apparent effect on determining seriousness. under us fda regulations, sponsors of clinical trials relating to ind applications are required to reports saes that are unexpected and suspected to be drug-related. based on the fda's final rule and related guidances, asco developed the one-page decision aid stepwise flowchart. the crossover study included physician-investigators or research staff from community or academic research sites who are involved in sae reporting decisions at their site. ten clinical case studies were evaluated, with five assessed with the assistance of the decision aid tool. compared with an unassisted assessment, the decision aid tool did not significantly improve the accuracy of determining serious for the case studies (odds ratio [or] . ; . , . ). however, the accuracy of attributing an sae to the drug was significantly increased (or . ; . , . ). most participants assessed the tool as being helpful ( %) and allowing improved decision-making time ( %) and confidence in reporting ( %), and indicated that they would use the tool in practice ( %). the preferred delivery method was by mobile application, followed by hard copy/ paper or via the asco web site. potential barriers to tool use included contract/protocol expectations or restrictions, and variability of the sae review and decision-making process among sites. "the decision aid shows promise as a method to improve the quality of sae attribution", note the authors, "which may improve the detection of valid safety signals and reduce the administrative burden of uninformative investigational new drug safety reports". they add that "study of the decision aid in a larger sample with analysis stratified by participant role and sae reporting experience would further assess the tool's impact". the accuracy of forecasting cancer drug costs can be improved by using a hybrid approach combining automated time-series forecasting and expert customisation, report researchers from canada. the researchers developed a forecasting framework from both a technical and a policy perspective to provide a flexible tool that can improve the accuracy of forecasts while incorporating multiple forecasts for the canadian province of ontario. the optimal forecasts for the top nine drug policies that made up the first % of the total budget in the previous months ('big budget drivers' [bbds]) funded by the provincial drug reimbursement programs resulted in errors within ± %. it was estimated that the forecast error from the automated approach for these nine bbd policies would have been approximately $can . million, compared with approximately $can . million using a manual approach. this corresponds to savings of $can million for these policies. "the flexibility provided by the hybrid approach should ultimately lead to improved accuracy, which will help achieve our policy-related goal: to help the programme make better decisions regarding to ability to fund drugs in the oncology pipeline", note the researchers. this approach to drug budget forecasting was implemented in ontario for the first time in the / fiscal year. the forecasts resulted in a % error for the overall budget, corresponding to an overestimate of expenditures by $can million. although the high-cost car-t cell therapies tisagenlecleucel (kymriah) and axicabtagene ciloleucel ( while icer considered evidence provided certainty of the cost effectiveness of yescarta and kymriah, nice and the tlv granted access to the drugs despite a high level of uncertainty in their cost effectiveness. gaps in clinical evidence were identified for both drugs in all five countries, primarily with regard to the lack of long-term data on overall survival and progression-free survival, and a lack of comparative data. nice, g-ba, tlv and the tc required further evidence to demonstrate the long-term clinical benefit and cost-effectiveness of the drugs. strategies have been implemented to mitigate the risk of introducing these high-cost drugs. "the fact that both yescarta and kymriah have widespread access, despite significant uncertainty in clinical evidence and cost-effectiveness (where relevant), is largely reflective of the high unmet need in dlbcl, the willingness of payers to access car-t therapies and the high level of innovation perceived", said the authors. "for both drugs, hta bodies are recommending that re-assessment occurs when more clinical and economic data (from clinical trials, country-specific real world evidence and indirect comparisons) becomes available", they noted. many drugs used in dermatology appear to be associated with idiopathic intracranial hypertension (iih), leading to the suggestion that the term drug-induced intracranial hypertension (diih) should be introduced, say authors of a systematic review published in the american journal of clinical dermatology. investigators searched medline, embase, and cochrane review databases for all articles reporting potentially drug-related cases of iih up until june . in total, articles were considered to be relevant. for all cases which met modified dandy criteria for diagnosis of iih, the koh algorithm for adverse drug reactions (adr) was used to assess the likelihood of iih being drug-related. overall, cases of diih were verifiable. vitamin a (retinol) and its derivatives (isotretinoin and tretinoin) were most frequently associated with diih ( cases). other drugs or drug classes most strongly associated with diih (≥ cases) included recombinant growth hormone and tetracyclines (including doxycycline, minocycline and tetracycline). lithium was also strongly associated with diih ( - cases) while corticosteroids were determined to be moderately associated with diih ( - cases). drugs found to be weakly associated with diih included amiodarone, ciclosporin, combined oral contraceptives, danazol, fluoroquinolones (including ciprofloxacin, levofloxacin, nalidixic acid and ofloxacin), gonadotropin-releasing hormones, luteinising hormone stimulants, subcutaneous implantable progestogen-only contraceptives (nexaplanon and norplant), stanozolol, sulfasalazine, sulfenazone, ustekinumab and valproate semisodium (divalproic acid). "we suggest using the term 'drug-induced intracranial hypertension' (diih) and propose a set of diagnostic criteria for diih… this may ultimately assist physicians in counselling patients about the risk of diih when prescribing medications and recognizing this uncommon yet sightthreatening condition", said the authors. "if diih is suspected by the physician, a timely referral for co-evaluation and co-management by a neurologist and an ophthalmologist is recommended", they commented. tan mg, worley b, kim wb, et al. drug-induced intracranial hypertension: a systematic review and critical assessment of drug-induced causes. am j clin dermatol. epub nov . http://doi.org/ . /s - - -z. accessed dec . despite premarketing efforts to reduce the risk of medication errors (mes) and a mandatory eu risk management plan for all newly licensed medicinal products, over a quarter of centrally authorised products in the european economic area (eea) are associated with me safety concerns, according to findings of a study published in drug safety. data from the european public assessment report registry were used to investigate me safety concerns included in risk management plans of originator centrally authorised products which were authorised between and in the eea. for each product, safety concerns, categories for the summary of safety concerns, and how mes were addressed, were assessed. in total, centrally authorised products were approved during the study period, and ( %) of the products had a total of me safety concerns. the proportion of products with me safety concerns ranged from . % in to . % in . the most frequent types of me were drug administration error (n = ), product dosage form confusion (n = ) and product preparation error (n = ). additional risk minimisation measures (armms) were required to address me safety concerns in of the products, and included educational material for healthcare professionals ( . %) and/or patients ( . %). studies evaluating the effectiveness of the additional measures were agreed upon for . % of products with armms for mes. "the high number of products with me safety concerns and the high proportion of me safety concerns with armms suggest awareness regarding mes at the level of the pharmaceutical industry and regulators. there is limited knowledge regarding the effectiveness of the measures available to prevent mes. therefore, studies are necessary to evaluate the suitability of the current risk minimisation framework for mes", said the authors. in patients hospitalised for an exacerbation of chronic obstructive pulmonary disease (copd), there is a high prevalence of drug-related problems, according to study results reported in drug safety. the study was conducted in an academic teaching hospital in china. drug-related problems and interventions were analysed based on the pharmaceutical care network europe (pcne)-drp v . classification, where one problem (p) may have multiple causes (c) and lead to more than one intervention (i) but lead to only one outcome (o). there were drug-related problems in patients, or an average of . per patient, which had corresponding causes. at least one problem occurred in patients ( . %). the major type of problem identified was "treatment safety p " ( . %), followed by "treatment effectiveness p " ( . %). the most frequently identified cause of the problem was "drug selection c " ( . %), followed by "dose selection c " ( . %) and "treatment duration c " ( . %). the three major cause subcategories were drug dose too high, duration of treatment too long, and patient administers/uses the drug in a wrong way. "this indicates that the pharmacist should provide necessary patient education on the correct use of drugs when providing medication review", note the authors. most patients ( . %) were receiving polypharmacy (≥ medications), with an average of . medications per patient. the five most frequently used medication classes were antibiotics, antihypertensives, bronchodilators, corticosteroids, and expectorants. the drug class most frequently involved in drug-related problems was antibiotics ( . %), followed by corticosteroids ( . %) and proton pump inhibitors ( . %). in multivariate logistic regression analysis, there was a significant association with drug-related problems in patients with renal dysfunction, those taking ≥ drugs, and those who are hospitalised for ≥ days, and an association in patients with ≥ comorbidities. pharmacists proposed interventions to solve the drug-related problems, an average of . per problem identified. these were mostly made at the "drug level i " ( . %), followed by at the "prescriber level i " ( . %) and at the "patient level i " ( . %). overall, . % of interventions were accepted, and . % were fully implemented; . % of problems were solved. "pharmacists can have an important role in addressing the problems and optimizing the safety and effectiveness of therapies for hospitalized copd patients", note the authors. ranitidine products containing n-nitrosodimethylamine (ndma) levels above acceptable limits will be recalled in the us while investigations are ongoing into the contamination, says cder director dr. janet woodcock. a number of us-approved medicines containing ranitidine (commonly known as zantac) have been tested over the past few months, and a summary of current investigation results was recently presented by dr. woodcock. she noted that the cder had "conducted tests that simulate what happens to ranitidine after it has been exposed to acid in the stomach with a normal diet and results of these tests indicate that ndma is not formed through this process". ndma was also not formed when ranitidine was exposed to a simulated small intestine environment, but dr. woodcock noted that human trials still needed to be carried out to fully understand if ranitidine forms ndma. although many ndma levels observed in us fda testing to date "are much lower than the levels some third-party scientists first claimed, some levels still exceed what the fda considers acceptable for these medicines", highlighted dr. woodcock. if the fda or manufacturers find ndma levels that are above acceptable limits (i.e. ng/day, or . parts per million), then companies are being asked to voluntarily recall their ranitidine product(s). in addition, manufacturers are being asked to voluntarily recall products containing nizatidine (commonly known as axid, and chemically similar to ranitidine) if ndma levels are found to be above the acceptable daily intake level. manufacturers are also requested to continue conducting their own laboratory testing to assess ndma levels in products containing ranitidine or nizatidine, and samples should also be sent to the fda so that the agency could conduct its own tests. to date, fda testing of a ranitidine syrup commonly used in paediatric patients has revealed some samples with ndma levels above the acceptable limits, which are now being recalled. testing of a ranitidine injection is also ongoing. woodcock j. statement on new testing results, including low levels of impurities in ranitidine drugs. nov . https ://www.fda.gov/news-event s/press -annou nceme nts/state ment-new-testi ng-resul ts-inclu ding-low-level s-impur ities -ranit idine -drugs . accessed dec . attention-deficit hyperactivity disorder (adhd) medication does not appear to be associated with serious cardiovascular (scv) events in children or adolescents with adhd or autism spectrum disorder (asd), according to findings of a f. hoffmann-la roche-funded study published in cns drugs. commercial claims data ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and medicaid claims data ( - ) from the us truven health marketscan database were used to conduct nested case-control studies in paediatric patients - years of age with adhd (n = , , ) or asd ( , ). each case with the composite outcome of stroke, myocardial infarction (mi) or serious arrhythmias was matched with ten controls based on age, sex and insurance type. conditional logistic regression analysis was used to evaluate the associations between scv events and current adhd medication use including cns stimulants such as methylphenidate or non-stimulants such as atomoxetine. the risk of scv events was not significantly increased in patients with adhd or asd; in the adhd cohort . % of cases and . % of controls were exposed to adhd medication (or . ; % ci . , . ), while in the asd cohort . % of cases and . % of controls were exposed to adhd drugs (or . ; % ci . , . ). there was also no increased risk of scv events after adjustment for covariates, or for individual scv outcomes. "in conclusion, in a large, contemporary insurance database, we found low rates of scv events in children and adolescents with adhd ( . / , person years) and asd ( . / , person years). furthermore, we found no evidence of an increased scv risk when exposed to adhd medications," said the authors. magnesium sulphate is the drug most frequently associated with adrs in women with high-risk pregnancy in brazil, according to findings of a study published in the european journal of clinical pharmacy. adrs were investigated in women with highrisk pregnancies admitted to an obstetric intensive care unit (icu) in brazil between june and december . patients were excluded if they were admitted to the icu for non-obstetric conditions, stayed in the icu for less than h, or were readmitted to the icu. the overall incidence of adrs was . %. no severe adrs were reported but . % of adrs were of moderate severity. the incidence of adrs was highest in patients receiving magnesium sulphate ( . %), and was much lower for all other drugs, including vancomycin ( . %), meropenem ( . %), cefalexin ( . %), azithromycin ( . %), methyldopa ( . %), mineral oil ( . %), insulin ( . %), ferrous sulphate ( . %) and captopril ( . %). adrs reported in patients receiving magnesium sulphate included somnolence ( . %), absent patellar reflex ( . %) and hypotension ( . %). all four cases of methyldopa-related adrs were somnolence. moderate hypoglycaemia was reported in one patient receiving insulin, and mild diarrhoea was reported in patients receiving azithromycin, cefalexin, meropenem, ferrous sulphate and vancomycin (one case each). multivariate analysis found that higher bp (adjusted odds ratio [aor] . ), higher haemoglobin level (aor . ) and lower body temperature (aor . ) significantly increased the risk of adrs. considering its therapeutic importance in preeclampsia/ eclampsia and for foetal neuroprotection, the benefits of magnesium sulphate to the mother and foetus outweigh its risks, even though its use is associated with significant adr, said the investigators. xavier da costa t, de almeida pimenta cunha md, do vale bezerra pk, et al. incidence of adverse drug reactions in high-risk pregnancy: a prospective cohort study in obstetric intensive care. eur j clin pharmacol. epub nov . https ://doi.org/ . /s - - - . accessed dec . the risk of interstitial lung disease will be added to the package inserts for two prostate cancer medicines-apalutamide and enzalutamide, according to japan's pharmaceuticals and medical devices agency (pmda). apalutamide (erleada tablets) is indicated for treatment of castration-resistant prostate cancer without remote metastasis. since the launch of this product in japan in may , there have been a total of four cases involving interstitial lung disease (including two cases for which a causal relationship to the drug could not be ruled out). in addition, there has been one patient death reported to date, but again, a causal relationship between the drug and death subsequent to the patient's interstitial lung disease could not be ruled out [ ] . enzalutamide (xtandi capsules, xtandi tablets) is indicated for the treatment of castration-resistant prostate cancer. over the past fiscal years in japan, a total of cases involving interstitial lung disease have been reported to date (including five cases for which a causal relationship to the drug could not be ruled out). furthermore, three patient deaths have been reported, but a causal relationship between the drug and deaths could not be established in any of these cases [ ] . based on its investigation of currently available evidence and consultation with expert advisors, the mhlw/pmda concluded that the following package insert revisions are necessary for both apalutamide and enzalutamide: reports) and fluticasone-associated adrenal cortical hypofunction (prr . ; % ci . , . ; reports) . all ten signals with the highest prrs were already known. there were signals classified as new. new signals unmasked by calculating the prr by therapeutic area included herpes virus infections and bacterial infections associated with omalizumab, and herpes virus infections associated with budesonide. other new signals included hypertrichoses with budesonide and encephalopathy with theophylline. overall, % of the signals in paediatric patients did not appear in the total patient population. "although % of the statistical signals were already known, we observed new signals, especially for omalizumab. calculation of the prr by therapeutic area, age and sex revealed additional new signals, pointing to masking due to confounding by indication or effect modifi-cation… safety signals of asthma drugs were mainly related to psychiatric disorders, especially in combination with the use of montelukast'', said the authors. baan ej, de smet va, hoeve ce, et al. exploratory study of signals for asthma drugs in children, using the eudravigilance database of spontaneous reports. drug saf. epub patients with interstitial lung disease or a history of the disease" should be added to the "careful administration" section • a cautionary statement for interstitial lung disease should be added to the "important precautions" section interstitial lung disease" should be added to the "clinically significant adverse reactions" section pharmaceuticals and medical devices agency of japan. summary of investigation results -apalutamide usa) notes that development of antiviral vaccines began when peyton rous postulated in the early s that sarcoma transmission between hens may be virus-related, followed by identification in of the first human dna tumour virus, the epstein-barr virus. papillomavirus dna was isolated in , with > types of hpv subsequently classified; the first hpv vaccine was licensed in the usa in safety of the -valent human papillomavirus vaccine near real-time surveillance to assess the safety of the -valent human papillomavirus vaccine from peyton rous to the hpv vaccine: a journey of discovery and progress according to findings of a study published in drug safety that was based on faers reports. faers data from , , reports over a -year period ( - ) were used to determine reporting odds ratios (rors) for associations between antibacterials (intramuscular, intravenous, subcutaneous or parenteral) and akis. there were , reports of aki to faers during the study period cotrimoxazole (trimethoprim/sulfamethoxazole % ci . , . ), and fluoroquinolones this study found classes of antibiotics having significant reporting associations with aki. among the antibiotics evaluated in this study, colistin had the highest aki ror and moxifloxacin had the lowest comparing acute kidney injury reports among antibiotics: a pharmacovigilance study of the fda adverse event reporting system (faers) angiotensin receptor antagonists increase risk of suicide treatment with statins does not appear to be associated with memory or cognitive disorders in elderly patients, according to findings of the sydney memory and aging study published in the journal of the american college of cardiology (jacc) [ ] . this prospective study investigated the effects of statins (atorvastatin, pravastatin or simvastatin) on memory and global cognition over a -year period in community-dwelling patients in australia who were - years of age, and the effects of statins on brain volume over a -year period in a subgroup of patients. five memory tests and cognitive tests were used. brain volume was assessed using magnetic resonance imaging.rates of memory decline and global cognitive decline did not differ significantly between statin users and patients who never used statins.overall, initiation of statins was associated with a reduced rate of memory decline. statin use was associated with attenuated decline in memory test performance in patients with heart disorders and those with the apolipoprotein eε (apoeε ) genotype.changes in brain volume did not differ significantly between statin users and non-users."statin use in the elderly population was not associated with any acceleration in decline in memory, global cognition, or brain volumes… protective associations were found for some aspects of memory testing in those with dementia risk factors such as heart disease and apoeε gene carriage", concluded the investigators."contrary to popular concern, statin use was not associated with cognitive decline in this observational study", said drs. constantino iadecola and neal parikh from weill cornell medicine, new york, usa, in an accompanying editorial published in the jacc [ ] . "these data support the view that worries about cognitive impairment should not limit statin use and raise the possibility that statins may favorably alter cognitive trajectories in a group of elderly subjects at high risk of alzheimer disease", they commented. the low uptake of human papillomavirus (hpv) vaccines in the usa may be due to concerns about vaccine safety. two studies reported in pediatrics investigated the safety of the -valent hpv vaccine [ vhpv vaccine; gardasil ]. the first study investigated postlicensure surveillance reports to the us fda's vaccine adverse event reporting system (vaers) from december until december [ ] . most reports were submitted by the vaccine manufacturer ( . %), with . % of reports submitted by healthcare providers. disproportional reporting was evaluated using empirical bayesian data mining. most reports ( . %) were not classified as serious, and vhpv vaccine was given alone in . % of reports. two deaths were reported, but "no information in autopsy reports or death certificates suggested a causal relationship with vaccination", note the authors. the most commonly reported events were dizziness ( . %), syncope ( . %), headache ( . %), and injection site pain ( . %) or erythema ( . %).as about million doses of vhpv vaccine were distributed during the study period, the crude adverse event reporting rate was per million doses overall and per million doses for serious events. the rate for syncope was per million doses, which did exceed the threshold for disproportional reporting, but is a known adverse event for injectable vaccines. rates for all other events were < per million doses.analysis of prespecified conditions revealed reports of anaphylaxis, reports of guillain-barré syndrome, reports of postural orthostatic tachycardia syndrome, reports of primary ovarian insufficiency, report of complex regional pain syndrome, and reports of acute disseminated encephalomyelitis. however the authors note that "most reported events did not meet diagnostic criteria or did not contain sufficient information to make a determination on the diagnosis".the authors conclude that "no new or unexpected safety concerns or reporting patterns of vhpv with clinically important aes were detected". the second study investigated reports to the vaccine safety datalink (vsd) between october and october in patients - years of age, when , vaccine doses were administered [ ] . rapid cycle analysis (rsa) methodology was used to compare adverse events in recently vaccinated and unvaccinated groups, with near real-time data. relative risks were investigated using maximised sequential probability ratio test (maxsprt), conditional maxsprt (cmaxsprt) and exact sequential analysis (esa) analyses.unexpected statistical signals were detected for the following events: pancreatitis in men - years of age after any dose; appendicitis after dose in boys - years of age; allergic reactions after dose in women - years of age; and allergic reactions in girls - years of age after any dose. further investigation did not confirm signal generation, with results classified as false-positives. signals were also detected for syncope, injection site reactions, and nonspecific reactions, but no further investigations were conducted because they were "expected based on clinical trials of vhpv", note the authors, "and because the diagnoses were unlikely to indicate a serious adverse event". they add that "with this large observational study, we contribute reassuring postlicensure data that will help bolster the safety profile of vhpv".angiotensin receptor blockers (arbs) appear to increase the risk of suicide in elderly patients compared with ace inhibitors (aceis), according to findings of a canadian case-control study published in jama network open [ ] .data from administrative claims databases in ontario were used to investigate death by suicide during treatment with arbs or aceis in patients years of age and over. conditional logistic regression analysis was used to assess the association between suicide and exposure to arbs versus aceis in patients who died by suicide within days of receiving an acei or arb (cases) compared with acei-or arb-treated patients matched by age, sex and comorbid hypertension and diabetes mellitus who did not die by suicide (controls). of patients exposed to arbs (most frequently candesartan, telmisartan or valsartan), . % were cases and . % were controls, while of patients exposed to aceis (most frequently enalapril or ramipril), . % were cases and . % were controls.treatment with arbs significantly increased the risk of death by suicide compared with treatment with aceis (aor . ; % ci . , . ). sensitivity analysis showed similar findings after exclusion of patients with a history of self-harm (aor . ; % ci . , . ). "our findings suggest a possible increased risk of suicide associated with the use of arbs compared with aceis among adults aged years and older. given their high prevalence of use, the severity of the outcome, and the similar efficacy of these drug classes in treating the same conditions, clinicians may opt for preferential use of aceis over arbs where possible", concluded the authors."the report by mamdani et al. that arbs are associated with increases in the risk of suicide in a study using real-world data requires conceptual replication. the strength of the methods, the importance of preventing suicide, and the number of people exposed to arbs all support the need to encourage additional studies and to translate the combined findings into guidance about prescribing", said dr. ira katz from the office of mental health and suicide prevention, department of veterans affairs, philadelphia, pennsylvania, usa, in an accompanying invited commentary published in jama network open [ ] . analysis of spontaneous adr reports to the european medicine agency's eudravigilance database has identified new safety signals for asthma drugs in paediatric patients, say authors of a study published in drug safety. a proportional reporting ratio (prr) was calculated for each asthma drug-event combination (dec) reported to eudravigilance between and . signals in paediatric patients up to years of age were compared with those in the total patient population.among the , reports in pediatric patients, there were , antiasthmatic-related decs associated with single drug or fixed-dose combinations.there were unique decs in paediatric patients, of which met the criteria for a safety signal. the signals were most frequently for psychiatric disorders (n = ), respiratory, thoracic and mediastinal disorders ( ) and nervous system disorders ( ). there were also signals related to injury, poisoning and procedural complications ( ) after off-label use or accidental exposure to an antiasthmatic.the highest prrs in paediatric patients were for montelukast-associated vasculitides (prr . ; % ci . , . ; key: cord- -tpqsjjet authors: nan title: section ii: poster sessions date: - - journal: j urban health doi: . /jurban/jti sha: doc_id: cord_uid: tpqsjjet nan food and nutrition programs in large urban areas have not traditionally followed a systems approach towards mitigating food related health issues, and instead have relied upon specific issue interventions char deal with downstream indicators of illness and disease. in june of , the san francisco food alliance, a group of city agencies, community based organizations and residents, initiated a collahorarive indicator project called rhe san francisco food and agriculture assessment. in order to attend to root causes of food related illnesses and diseases, the purpose of the assessment is to provide a holistic, systemic view of san francisco\'s food system with a focus on three main areas that have a profound affect on urban public health: food assistance, urban agriculture, and food retailing. using participatory, consensus methods, the san francisco food alliance jointly developed a sec of indicators to assess the state of the local food system and co set benchmarks for future analysis. members collected data from various city and stare departments as well as community based organizations. through the use of geographic information systems software, a series of maps were created to illustrate the assets and limitations within the food system in different neighborhoods and throughout the city as a whole. this participatory assessment process illustrates how to more effectively attend to structural food systems issues in large urban areas by ( t) focusing on prevention rather than crisis management, ( ) emphasizing collaboration to ensure institutional and structural changes, and ( ) aptly translating data into meaningful community driven prevention activities. to ~xplore the strategies to overcome barriers to population sample, we examined the data from three rapid surveys conducted at los angeles county (lac). the surveys were community-based partic· patory surveys utilizing a modified two-stage cluster survey method. the field modifications of the method resulted in better design effect than conventional cluster sample survey (design effect dose to that if the survey was done as simple random sample survey of the same size). the surveys were con· ducte~ among parents of hispanic and african american children in lac. geographic area was selected and d .v ded int.o small c~usters. in the first stage, clusters were selected with probability proportionate to estimated size of children from the census data. these clusters were enumerated to identify and develop a list of households with eligible children from where a random sample was withdrawn. data collectmn for consented respondents involved - minutes in-home interview and abstraction of infor· ma~ion from vaccine record card. the survey staff had implemented community outreach activities designed to fost~r an~ maintain community trust and cooperation. the successful strategies included: developing re.lat on .w. th local community organizations; recruitment of community personnel and pro· vide them with training to conduct the enumeration and interview; teaming the trained community introduction: though much research has been done on the health and social benefits of pet ownership for many groups, there have been no explorations of what pet ownership can mean to adults who are marginalized, living on fixed incomes or on the street in canada. we are a community group of researchers from downtown toronto. made up of front line staff and community members, we believe that community research is important so that our concerns, visions, views and values are presented by us. we also believe that research can and should lead to social change. method: using qualitative and exploratory methods, we have investigated how pet ownership enriched and challenged the lives of homeless and transitionally housed people. our research team photographed and conducted one-on-one interviews with pet owners who have experienced home· lessncss and live on fixed incomes. we had community participation in the research through a partnership with the fred vicror centre camera club. many of the fred victor centre camera club members have experienced homelessness and being marginalized because of poverty. the members of the dub took the photos and assisted in developing the photos. they also participated in the presenta· tion of our project. results: we found that pet ownership brings important health and social benefits to our partici· pants. in one of the most poignant statements, one participant said that pet ownership " ... stops you from being invisible." another commented that "well, he taught me to slow down, cut down the heavy drugs .. " we also found that pet ownership brings challenges that can at times be difficult when one is liv· ing on a fixed income. we found that the most difficult thing for most of the pet owners was finding affordable vet care for their animals. conclusion: as a group, we decided that research should only be done if we try to make some cha.nges about what we have learned. we continue the project through exploring means of affecting social change--for example, ~eti.tions and informing others about the result of our project. we would like to present our ~mdmgs and experience with community-based participatory action resea.rch m an oral. presentarton at yo~r conference in october. our presentation will include com· mumty representation ~f. both front-hne staff and people with lived experience of marginalization and homdessness. if this is not accepted as an oral presentation, we are willing to present the project m poster format. introduction the concept of a healthy city was adopted by the world health organization some time ago and it includes strong support for local involvement in problem solving and implementation of solutions. while aimed at improving social, economic or environmental conditions in a given community, more significantly the process is considered to be a building block for poliq reform and larger scale 'hange, i.e. "acting locally while thinking globally." neighbourhood planning can he the entry point for citizens to hegin engaging with neighbours on issues of the greater common good. methods: this presentation will outline how two community driven projects have unfolded to address air pollution. the first was an uphill push to create bike lanes where car lanes previously existed and the second is an ongoing, multi-sectoral round table focused on pollution and planning. both dt•monstrate the importance of having support with the process and a health focus. borrowing from traditions of "technical aid"• and community development the health promoter /planner has incorporated a range of "determinants of health" into neighbourhood planning discussions. as in most urban conditions the physical environment is linked to a range of health stressors such as social isolation, crowding, noise, lack of open space /recreation, mobility and safety. however typical planning processes do not hring in a health perspective. health as a focus for neighbourhood planning is a powerful starti_ng point when discussing transportation planning or changing land-uses. by raising awareness on determmants of health, citizens can begin to better understand how to engage in a process and affect change. often local level politics are involved and citizens witness policy change in action. the environmental liaison committee and the dundas east hike lanes project resulted from local level initiatives aimed at finding solutions to air pollution -a priority identified hy the community. srchc supported the process with facilitation and technical aid. _the processs had tangible results that ultimately improve living conditions and health. •tn the united kmgdom plannm in the 's established "technical aid" offices much like our present day legal aid system to provide professional support and advocacy for communities undergoing change. p - (c) integrating community based research: the experience of street health, a community service agency i.aura cowan and jacqueline wood street health began offering services to homeless men and women in east downtown ~oronto in . nursing stations at drop-in centres and shelters were fo~lowed by hiv/aids prevent ~, harm reduction and mental health outreach, hepatitis c support, sleeping bag exchange, and personal tdennfication replacement and storage programs. as street health's progi;ams expanded, so to~ did the agency:s recognition that more nee~ed t~ be done to. address the underl~ing causes of, th~ soct~l and economic exclusion experienced by its clients. knowing t.h~t. a~voca~y ts. helped by . evtd~nce , street he.alt~ embarked on a community-based research (cbr) initiative to dent fy commumty-dnven research priorities within the homeless and underhoused population. methods: five focus groups were conducted with homeless people, asking participants to identify positive and negative forces in their lives, and which topics were important to take action on and learn more about. findings were validated through a validation meeting with participants. results: participants identified several important positive and negative forces in their lives. key positive forces included caring and respectful service delivery, hopefulness and peer networks. key negative forces included lack of access to adequate housing and income security, poor service delivery and negative perceptions of homeless people. five topics for future research emerged from the process, focusing on funding to address homelessness and housing; use of community services for homeless people; the daily survival needs of homeless people and barriers to transitioning out of homelessness; new approaches to service delivery that foster empowerment; and policy makers' understanding of poverty and homelessness. conclusions: although participants expressed numerous issues and provided much valuable insight, definitive research ideas and action areas were not clearly identified by participants. however, engagement in a cbr process led to some important lessons and benefits for street health. we learned that the community involvement of homeless people and front-line staff is critical to ensuring relevance and validity for a research project; that existing strong relationships with community parmers are essential to the successful implementation of a project involving marginalized groups; and that an action approach focusing on positive change can make research relevant to directly affected people and community agency staff. street health benefited from using a cbr approach, as the research process facilitated capacity building among staff and within the organization as a whole. p - (c) a collaborative process to achieve access to primary health care for black women and women of colour: a model of community based participartory research notisha massaquoi, charmaine williams, amoaba gooden, and tulika agerwal in the current healthcare environment, a significant number of black women and women of color face barriers to accessing effective, high quality services. research has identified several issues that contribute to decreased access to primary health care for this population however racism has emerged as an overarching determinant of health and healthcare access. this is further amplified by simultaneous membership in multiple groups that experience discrimination and barriers to healthcare for example those affected by sexism, homelessness, poverty, homophobia and heterosexism, disability and hiv infection. the collaborative process to achieve access to primary health care for black women and women of colour project was developed with the university of toronto faculty of social work and five community partners using a collaborative methodology to address a pressing need within the community ro increase access to primary health care for black women and women of colour. women's health in women's hands community health centre, sistering, parkdale community health centre, rexdale community health centre and planed parenthood of toronto developed this community-based participatory-action research project to collaboratively barriers affecting these women, and to develop a model of care that will increase their access to health services. this framework was developed using a process which ensured that community members from the target population and service providers working in multiculrural clinical settings, were a part of the research process. they were given the opportunity to shape the course of action, from the design of the project to the evaluation and dissemination phase. empowerment is a goal of the participatory action process, therefore, the research process has deliberately prioritized _ro enabling women to increase control over their health and well-being. in this session, the presenters will explore community-based participatory research and how such a model can be useful for understanding and contextualizing the experiences of black women and women of colour. they will address. the development and use of community parmerships, design and implementation of the research prorect, challenges encountered, lessons learnt and action outcomes. they will examine how the results from a collaborative community-based research project can be used as an action strategy to poster sessions v address che social determinants of women health. finally the session will provide tools for service providers and researchers to explore ways to increase partnerships and to integrate strategies to meet the needs of che target population who face multiple barriers to accessing services. lynn scruby and rachel rapaport beck the purpose of this project was ro bring traditionally disenfranchised winnipeg and surrounding area women into decision-making roles. the researchers have built upon the relationships and information gachered from a pilot project and enhanced the role of input from participants on their policy prioriries. the project is guided by an advisory committee consisting of program providers and community representatives, as well as the researchers. participants included program users at four family resource cencres, two in winnipeg and two located rurally, where they participated in focus groups. the participants answered a series of questions relating to their contact with government services and then provided inpuc as to their perceptions for needed changes within government policy. following data analysis, the researchers will return to the four centres to share the information and continue che discussion on methods for advocating for change. recommendations for program planning and policy development and implementation will be discussed and have relevance to all participants in the research program. women's health vera lefranc, louise hara, denise darrell, sonya boyce, and colleen reid women's experiences with paid and unpaid work, and with the formal and informal economies, have shifted over the last years. in british columbia, women's employability is affected by government legislation, federal and provincial policy changes, and local practices. two years ago we formed the coalition ior women's economic advancement to explore ways of dealing with women's worsening economic situations. since the formation of the coalition we have discussed the need for research into women's employabilicy and how women were coping and surviving. we also identified how the need to document the nature of women's employability and reliance on the informal economy bore significanc mechodological and ethical challenges. inherent in our approach is a social model of women's health that recognizes health as containing social, economic, and environmental determinants. we aim to examine the social contexc of women's healch by exploring and legitimizing women's own experiences, challenging medical dominance in understandings of health, and explaining women's health in terms of their subordination and marginalizacion. through using a feminist action research (far) methodology we will explore the relationship between women's employability and health in communities that represent bricish columbia's social, economic, cultural/ethnic, and geographic diversities: skidegate, fort st . .john, lumhy, and surrey. over the course of our year project, in each community we will establish and work with advisory committees, hire and train local researchers, conduct far (including a range of qualitative methods), and support action and advocacy. since the selected communities are diverse, the ways that the research unfolds will ·ary between communities. expected outcomes, such as the provision of a written report and resources, the establishment of a website for networking among the communities, and a video do.:umentary, are aimed at supporting the research participants, coalition members, and advisory conuniuces in their action efforts. p t (c) health & housing: assessing the impact of transitional housing for people living with hiv i aids currently, there is a dearth of available literature which examines supporrive housing for phas in the canadian context. using qualitative, one-on-one interviews we investigace the impact of transitional housing for phaswho have lived in the up to nine month long hastings program. our post<'r pr<·senta-t on will highlight research findings, as well as an examination of transitional housing and th<· imp;kt it has on the everyday lives of phas in canada. this research is one of two ground breaking undertakings within the province of ontario in which fife house is involved. p - (c) eating our way to justice: widening grassroots approaches to food security, the stop community food centre as a working model charles l.evkoe food hanks in north america have come co play a central role as the widespread response to growing rates of hunger. originally thought to be a short term-solution, over the last years, they have v poster sessions be · · · · d wi'thi'n society by filling the gaps in the social safety net while relieving govemcome mst tut ona ze . . . t f the ir responsibilities. dependent on corporate donations and sngmauzmg to users, food banks men so th' . · i i . are incapable of addressing the structural cause~ of ~u~ger. s pres~ntation w e~~ ore a ternanve approaches to addressing urban food security while bmldmg more sustamabl.e c~mmumt es. i:nrough the f t h st p community food centre, a toronto-based grassroots orgamzanon, a model is presented case e h'l k' b 'id · b that both responds to the emergency food needs of communities w e wor mg to. u ~ sustama le and just food system. termed, the community food centre model. (cfc), ~he s~op is worki?g to widen its approach to issues of food insecurity by combining respectful ~ rect service wit~ com~~mty ~evelop ment, social justice and environmental sustainability. through this approach, various critical discourses around hunger converge with different strategic and varied implications for a~ion. as a plac~-based organization, the stop is rooted within a geographical space and connected directly to a neighbourhood. through working to increase access to healthy food, it is active in maintaining people's dignity, building a strong and democratic community and educating for social change. connected to coalitions and alliances, the stop is also active in organizing across scales in connection with the global food justice movement. inner city shelter vicky stergiopoulos, carolyn dewa, katherine rouleau, shawn yoder, and lorne tugg introduction: in the city of toronto there are more than , hostel users each year, many with mental health and addiction issues. although shelters have responded in various ways to the health needs of their clients, evidence on the effectiveness of programs delivering mental health services to the home· less in canada has been scant. the objective of this community based research was to provide a forma· tive evaluation of a multi-agency collaborative care team providing comprehensive care for high needs clients at toronto's largest shelter for homeless men. methods: a logic model provided the framework for analysis. a chart review of clients referred over a nine month period was completed. demographic data were collected, and process and outcome indicators were identified for which data was obtained and analyzed. the two main outcome measures were mental status and housing status months after referral to the program. improvement or lack of improvement in mental status was established by chart review and team consensus. housing outcomes were determined by chart review and the hostel databases. results: of the clients referred % were single and % were unemployed. forty four percent had a psychiatric hospitalization within the previous two years. the prevalence of severe and persistent men· tal illness, alcohol and substance use disorders were %, % and % respectively. six months after referral to the program % of clients had improved mental status and % were housed. logistic regression controlling for the number of general practitioner and psychiatrist visits, presence of person· ality or substance use disorder and treatment non adherence identified two variables significantly associ· ated mental status improvement: the number of psychiatric visits (or, . ; % ci, . - . ) and treatment non adherence (or, . ; % ci, . - . ). the same two variables were associated with housing outcomes. history of forensic involvement, the presence of a personality or substance use disorder and the number of visits with a family physician were not significantly associated with either outcome. conclusions: despite the limitations in sample sire and study design, this study can yield useful informa· tion to program planners. our results suggest that strategies to improve treatment adherence and access to mental health specialists can improve outcomes for this population. although within primary care teams the appropriate collaborative care model for this population remains to be established, access to psychiatric follow up, in addition to psychiatric assessment services, may be an important component of a successful program. mount sinai hospital (msh) has become one of the pre-eminent hospitals in the world by contributing to the development of innovative approaches to effective health care and disease prevention. recently, the hospital has dedicated resources towards the development of a strategy aimed at enhancing the hospital's integration with its community partners. this approach will better serve the hospital in the current health care environment where local health integration networks have been struck to enhance and support local capacity to plan, coordinate and integrate service delivery. msh has had early success with developing partnerships. these alliances have been linked to programs serving key target populations with _estabhshe~. points of access to msh. recognizing the need to build upon these achievements to remain compe~mve, the hospital has developed a community integration strategy. at the forefront of this strategy is c.a.r.e (community advisory reference engine): the hospital's compendium of poster sessions v community partners. as a single point of access to community partner information, c.a.r.e. is more than a database. c.a.r.e. serves as the foundation for community-focused forecasting and a vehicle for inter and intra-organizational knowledge transfer. information gleaned from the catalog of community parmers can be used to prepare strategic, long-term partnership plans aimed at ensuring that a comprehensive array of services can be provided to the hospital community. c.a.r.e. also houses a permanent record of the hospital's alliances. this prevents administrative duplication and facilitates the formation of new alliances that best serve both the patient and the hospital. c.a.r.e. is not a stand-alone tool and is most powerful when combined with other aspects of the hospital's community integration strategy. it iscxpected that data from the hospital's community advisory committees and performance measurement department will also be stored alongside stakeholder details. this information can then be used to drive discussions at senior management and the board, ensuring congruence between stakeholder, patient and hospital objectives. the patient stands to benefit from this strategy. the unique, distinct point of reference to a wide array of community services provides case managers and discharge planners with the information they need to connect patients with appropriate community services. creating these linkages enhances the patient's capacity to convalesce in their homes or places of residence and fosters long-term connections to neighborhood supports. these connections can be used to assist with identifying patients' ongoing health care needs and potentially prevent readmission to hospital. introduction: recruiting high-risk drug users and sex workers for hiv-prevention research has often been hampered by limited access to hard to reach, socially stigmatized individuals. our recruitment effom have deployed ethnographic methodology to identify and target risk pockets. in particular, ethnographers have modeled their research on a street-outreach model, walking around with hiv-prevention materials and engaging in informal and structured conversations with local residents, and service providers, as well as self-identified drug users and sex workers. while such a methodology identifies people who feel comfortable engaging with outreach workers, it risks missing key connections with those who occupy the margins of even this marginal culture. methods: ethnographers formed a women's laundry group at a laundromat that had a central role as community switchboard and had previously functioned as a party location for the target population. the new manager helped the ethnographers invite women at high risk for hiv back into the space, this time as customers. during weekly laundry sessions, women initiated discussions about hiv-prevention, sexual health, and eventually, the vaccine research for which the center would be recruiting women. ra.its: the benefits of the group included reintroducing women to a familiar locale, this time as customers rather than unwelcome intruders; creating a span of time (wash and dry) to discuss issues important to me women and to gather data for future recruitment efforts; creating a location to meet women encountered during more traditional outreach research; establishing the site as a place for potential retention efforts; and supporting a local business. women who participated in the group completed a necessary household task while learning information that they could then bring back to the community, empowering them to be experts on hiv-prevention and vaccine research. some of these women now assist recruitment efforts. the challenges included keeping the group women-only, especially after lunch was provided, keeping the membership of the group focused on women at risk for hiv, and keeping the women in the group while they did their laundry. conclusion: public health educators and researchers can benefit from identifying alternate congregation sites within risk pockets to provide a comfortable space to discuss hiv prevention issues with high-risk community members. in our presentation, we will describe the context necessary for similar research, document the method's pitfalls and successes, and argue that the laundry group constitutes an ethical, respectful, community-based method for recruitment in an hiv-prevention vaccine trial. p - t (c) upgrading inner city infrastructure and services for improved environmental hygiene and health: a case of mirzapur in u.p. india madhusree mazumdar in urgency for agricultural and industrial progress to promote economic d.evelopment follo_wing independence, the government of india had neglected health promotion and given less emphasis on infrastructure to promote public health for enhanced human pro uct v_ity. ong wit r~p m astrucrure development, which has become essential if citie~ are to. act ~s harbmger.s of econ~nuc ~owth, especially after the adoption of the economic liberalization policy, importance _is a_lso ~emg g ve.n to foster environmental hygiene for preventive healthcare. the world health orga~ sat ~ is also trj:' ! g to help the government to build a lobby at the local level for the purpose by off~rmg to mrroduce_ its heal.thy city concept to improve public health conditions, so as to reduce th_e disease burden. this pape~ s a report of the efforts being made towards such a goal: the paper descr~bes ~ c~se study ?f ~ small city of india called mirzapur, located on the banks of the nver ganga, a ma or lifeline of india, m the eastern part of the state of uttar pradesh, where action for improvement began by building better sanitation and environmental infrastructure as per the ganga action plan, but continued with an effort to promote pre· ventive healthcare for overall social development through community participation in and around the city. asthma physician visits in toronto, canada tara burra, rahim moineddin, mohammad agha, and richard glazier introduction: air pollution and socio-economic status are both known to be associated with asthma in concentrated urban settings but little is known about the relationship between these factors. this study investigates socio-economic variation in ambulatory physician consultations for asthma and assesses possible effect modification of socio-economic status on the association between physician visits and ambient air pollution levels for children aged to and adults aged to in toronto, canada between and . methods: generalized additive models were used to estimate the adjusted relative risk of asthma physician visits associated with an interquartile range increase in sulphur dioxide, nitrogen dioxide, pm . , and ozone, respectively. results: a consistent socio-economic gradient in the number of physician visits was observed among children and adults and both sexes. positive associations between ambient concentrations of sul· phur dioxide, nitrogen dioxide and pm . and physician visits were observed across age and sex strata, whereas the associations with ozone were negative. the relative risk estimates for the low socio-«onomic group were not significantly greater than those for the high socio-economic group. conclusions: these findings suggest that increased ambulatory physician visits represent another component of the public health impact of exposure to urban air pollution. further, these results did not identify an age, sex, or socio-economic subgroup in which the association between physician visits and air pollution was significantly stronger than in any other population subgroup. eco-life-center (ela) in albania supports a holistic approach to justice, recognizing the environ· mental justice, social justice and economic justice depend upon and support each other. low income cit· izens and minorities suffer disproportionately from environmental hazards in the workplace, at home, and in their communities. inadequate laws, lax enforcement of existing environmental regulations, and ~ea.k penalties for infractions undermine environmental protection. in the last decade, the environmental ust ce m~ve~ent in tirana metropolis has provided a framework for identifying and exposing the links ~tween irrational development practices, disproportionate siting of toxic facilities, economic depres· s on, and a diminished quality of life in low-income communities and communities of color. the envi· ~onmental justice agenda has always been rooted in economic, racial, and social justice. tirana and the issues su.rroun~ing brow~fields redevelopment are crucial points of advocacy and activism for creating ~ubstantia~ social chan~~ m low-income communities and communities of color. we engaging intensively m prevcnnng co'.' mumnes, especially low income or minority communities, from being coerced by gov· ernmenta~ age_nc es or companies into siting hazardous materials, or accepting environmentally hazard· ous_ practices m order to create jobs. although environmental regulations do now exist to address the environmental, health, and social impacts of undesirable land uses, these regulations are difficult to poster sessions v enforce because many of these sites have been toxic-ridden for many years and investigation and cleanup of these sites can be expensive. removing health risks must be the main priority of all brown fields action plans. environmental health hazards are disproportionately concentrated in low-income communities of color. policy requirements and enforcement mechanisms to safeguard environmental health should be strengthened for all brownfields projects located in these communities. if sites are potentially endangering the health of the community, all efforts should be made for site remediation to be carried out to the highest cleanup standards possible towards the removal of this risk. the assurance of the health of the community should take precedence over any other benefits, economic or otherwise, expected to result from brownfields redevelopment. it's important to require from companies to observe a "good neighbor" policy that includes on-site visitations by a community watchdog committee, and the appointment of a neighborhood environmentalist to their board of directors in accordance with the environmental principles. vancouver - michael buzzelli, jason su, and nhu le this is the second paper of research programme concerned with the geographical patterning of environmental and population health at the urban neighbourhood scale. based on the vancouver metropolitan region, the aim is to better understand the role of neighbourhoods as epidemiological spaces where environmental and social characteristics combine as health processes and outcomes at the community and individual levels. this paper builds a cohort of commensurate neighbourhoods across all six censuses periods from to , assembles neighbourhood air pollution data (several criterion/health effects pollutants), and providing an analysis to demonstrate how air pollution systematically and consistently maps onto neighbourhood socioeconomic markers, in this case low education and lone-parent families. we conclude with a discussion of how the neighbourhood cohort can be further developed to address emergent priorities in the population and environmental health literatures, namely the need for temporally matched data, a lifecourse approach, and analyses that control for spatial scale effects. solid waste management and environment in mumbai (india) by uttam jakoji sonkamble and bairam paswan abstract: mumbi is the individual financial capital of india. the population of greater mumbai is , , and sq. km. area. the density of population , per sq. km. the dayto-day administration and rendering of public services within gr. mumbai is provided by the brihan mumbai mahanagar palika (mumbai corporation of gr. mumbai) that is a body of elected councilors on a -year team. mumcial corporation provides varies conservancy services such as street sweeping, collection of solid waste, removal and transportation, disposal of solid waste, disposal of dead bodies of animals, construction, maintance and cleaning of urinals and public sanitary conveniences. the solid waste becoming complicated due to increase in unplanned urbanization and industrialization, the environment has deteriorated significantly due to inter, intra and international migration stream to mumbai. the volume of inter state migration to mumbai is considerably high i.e. . lakh and international migrant . lakh have migrated to mumbai. present paper gives the view on solid waste management and its implications to environment and health. pollution from a wide varity of emission, such as from automobiles and industrial activities, has reached critical level in mumbai, causing respiratory, ocular, water born diseases and other health problems. sources of generation of waste are -household waste, commercial waste, institutional waste, street sweeping, silt removed from drain/nallah/cleanings. disposal of solid waste in gr. mumbai done under incineration . processing to produce organic manure. . vermi-composting . landfill the study shows that the quantity of waste disposal of through processing and conversion to organic ~anure is about - m.t. per day. the processing is done by a private agency m/s excel industries ltd. who had set up a plant at the chincholi dumping ground in western mumbai for this purpose. the corporation is also disposal a plant of its waste mainly market waste through the environment friendly, natural pro-ces~ known as vermi-composing about m.t. of market waste is disposed of in this manner at the various sites. there are four land fill sites are available and percent of the waste matter generated m mumbai is disposed of through landfill. continuous flow of migrant and increa~e in slum population is a complex barrier in the solid waste management whenever community pamc pat on work strongly than only we can achieved eco-friendly environment in mumbai. persons exposed to residential craffic have elevated races of respiratory morbidity an~ ~ortality. since poverty is an important determinant of ill-health, some h~ve argued that t~es~ assoc at ons may relate to che lower socioeconomic status of those living along ma or roads. our ob ect ve was to evaluate the association between traffic intensity at home and hospital admissions for respiratory diagnoses among montreal residents older than years. morning peak traffic estimates from the emmej montreal traffic model (motrem ) were used as an indicator of exposure to road traffic outside the homes of those hospitalised. the influence of socioeconomic status on the relationship between traffic intensity and hospital admissions for respiratory diagnoses was explored through assessment of confounding by lodging value, expressed as the dollar average over road segments. this indicator of socioeconomic status, as calculated from the montreal property assessment database, is available at a finer geographic scale than socioeconomic information accessible from the canadian census. there was an inverse relationship between traffic intensity estimates and lodging values for those hospitalised (rho - . , p vehicles during che hour morning peak), even after adjustment for lodging value (crude or . , cl % . - . ; adjusted or . , cl % . - . ). in montreal, elderly persons living along major roads are at higher risk of being hospitalised for respiratory illnesses, which appears not simply to reflect the fact that those living along major roads are at relative economic disadvantage. the paper argues that human beings ought to be at the centre of the concern for sustainable development. while acknowledging the importance of protecting natural resources and the ecosystem in order to secure long term global sustainability, the paper maintain that the proper starting point in the quest for urban sustainability in africa is the 'brown agenda' to improve che living and working environment of che people, especially che urban poor who face a more immediate environmental threat to their health and well-being. as the un-habitat has rightly observed, it is absolutely essential "to ensure that all people have a sufficient stake in the present to motivate them to take part in the struggle to secure the future for humanity.~ the human development approach calls for rethinking and broadening the narrow technical focus of conventional town planning and urban management in order to incorporate the emerging new ideas and principles of urban health and sustainability. i will examine how cities in sub-saharan africa have developed over the last fifty years; the extent to which government policies and programmes have facilitated or constrained urban growth, and the strategies needed to achieve better functioning, safer and more inclusive cities. in this regard i will explore insights from the united nations conferences of the s, especially local agenda of the rio summit, and the istanbul declaration/habitat agenda, paying particular attention to the principles of enablement, decentralization and partnership canvassed by these movements. also, i will consider the contributions of the various global initiatives especially the cities alliance for cities without slums sponsored by the world bank and other partners; che sustainable cities programme, the global campaigns for good governance and for secure tenure canvassed by unhabit at, the healthy cities programme promoted by who, and so on. the concluding section will reflect on the future of the african city; what form it will take, and how to bring about the changes needed to make the cities healthier, more productive and equitable, and better able to meet people's needs. heather jones-otazo, john clarke, donald cole, and miriam diamond urban areas, as centers of population and resource consumption, have elevated emissions and concentrations of a wide range of chemical contaminants. we have developed a modeling framework in which we first ~stimate the emissions and transport of contaminants in a city and second, use these estimates along with measured contaminant concentrations in food, to estimate the potential health risk posed by these che.micals. the latter is accomplished using risk assessment. we applied our modeling framework to consider two groups of chemical contaminants, polycyclic aromatic hydrocarbons (pah) a.nd the flame re~ardants polybrominated diphenyl ethers (pbde). pah originate from vehicles and stationary combustion sources. ~veral pah are potent carcinogens and some compounds also cause noncancer effects. pbdes are additive flame retardants used in polyurethane foams (e.g., car seats, furniture) fer sessions v and cl~ equipm~nt (e.g., compute~~· televisio~s). two out of three pbdes formulations are being voluntarily phased by mdustry due to rmng levels m human tissues and their world-wide distribution. pbdes have been .related to adv.erse neurological, developmental and reproductive effects in laboratory ijlimals. we apphed our modelmg framework to the city of toronto where we considered the southcattral area of by km that has a population of . million. for pah, local vehicle traffic and area sources contribute at least half of total pah in toronto. local contributions to pbdes range from - %, depending on the assumptions made. air concentrations of both compounds are about times higher downtown than km north of toronto. although measured pah concentrations in food date to the s, we estimate that the greatest exposure and contribution to lifetime cancer risk comes from ingestion of infant formula, which is consistent with toxicological evidence. the next greatest exposure and cancer risk are attributable to eating animal products (e.g. milk, eggs, fish). breathing downtown air contributes an additional percent to one's lifetime cancer risk. eating vegetables from a home garden localed downtown contributes negligibly to exposure and risk. for pbdes, the greatest lifetime exposure comes through breast milk (we did not have data for infant formula), followed by ingestion of dust by the toddler and infant. these results suggest strategies to mitigate exposure and health risk. p - (a) immigration and socioeconomic inequalities in cervical cancer screening in toronto, canada aisha lofters, rahim moineddin, maria creatore, mohammad agha, and richard glazier llltroduction: pap smears are recommended for cervical cancer screening from the onset of sexual activity to age . socioeconomic and ethnoracial gradients in self-reported cervical cancer screening have been documented in north america but there have been few direct measures of pap smear use among immigrants or other socially disadvantaged groups. our purpose was to investigate whether immigration and socioeconomic factors are related to cervical cancer screening in toronto, canada. methods: pap smears were identified using fee codes and laboratory codes in ontario physician service claims (ohip) for three years starting in for women age - and - . all women with any health system contact during the three years were used as the denominator. social and economic factors were derived from the canadian census for census tracts and divided into quintiles of roughly equal population. recent registrants, over % of whom are expected to be recent immigrants to canada, were identified as women who first registered for health coverage in ontario after january , . results: among , women age - and , women age - , . % and . %, rtspcctively, had pap smears within three years. low income, low education, recent immigration, visible minority and non-english language were all associated with lower rates (least advantaged quintile:most advantaged quintile rate ratios were . , . , . , . , . , respectively, p < . for all). similar gradients were found in both age groups. recent registrants comprised . % of women and had mm;h lower pap smear rates than non-recent registrants ( . % versus . % for women age - and . % versus . % for women age - ). conclnsions: pap smear rates in toronto fall well below those dictated by evidence-based practice. at the area level, immigration, visible minority, language and socioeconomic characteristics are associated with pap smear rates. recent registrants, representing a largely immigrant group, have particularly low rates. efforts to improve coverage of cervical cancer screening need to be directed to all ~omen, their providers and the health system but with special emphasis on women who recently arrived m ontario and those with social and economic disadvantage. challeges faced: a) most of the resources are now being ~pent in ~reventing the sprea.d of hiv/ aids and maintaining the lives of those already affected. b) skilled medical ~rs~nal are dymg under· mining the capacity to provide the required health care services. ~) th.e comphcat o~s of hiv/aids has complicated the treatment of other diseases e.g. tbs d) the ep dem c has led. to mcrease number of h n requiring care and support. this has further stretched the resources available for health care. orp a s d db . . i methods used on our research: . a simple community survey con ucte y our orgamzat on vo · unteers in three urban centres members of the community, workers and health care prov~ders were interviewed ... . meeting/discussions were organized in hospitals, commun.ity centre a~d with government officials ... . written questionnaires to health workers, doctors and pohcy makers m th.e health sectors. lessors learning: • the biggest-health bigger-go towards hiv/aids prevention • aids are spreading faster in those families which are poor and without education. •women are the most affected. •all health facilities are usually overcrowded with hiv/aids patients. actions needed:• community education oh how to prevent the spread of hiv/aids • hiv/aids testing need to be encouraged to detect early infections for proper medical cover. • people to eat healthy • people should avoid drugs. implications of our research: community members and civic society-introduction of home based care programs to take care of the sick who cannot get a space in the overcrowded public hospitals. prl-v a te sector private sector has established programs to support and care for the staff already affected. government provision of support to care-givers, in terms of resources and finances. training more health workers. introduction: australian prisons contain in excess of , prisoners. as in most other western countries, reliance on 'deprivation of liberty' is increasing. prisoner numbers are increasing at % per annum; incarceration of women has doubled in the last ten years. the impacts on the community are great - % of children have a parent in custody before their th birthday. for aboriginal communities, the harm is greater -aboriginal and/or torres strait islanders are incarcerated at a rate ten times higher than other australians. % of their children have a parent in custody before their th birthday. australian prisons operate under state and territory jurisdictions, there being no federal prison system. eight independent health systems, supporting the eight custodial systems, have evolved. this variability provides an unique opportunity to assess the capacity of these health providers in addressing the very high service needs of prisoners. results: five models of health service provision are identified -four of which operate in one form or another in australia: • provided by the custodial authority (queensland and western australia)• pro· vided by the health ministry through a secondary agent (south australia, the australian capital territory and tasmania) • provided through tendered contract by a private organization (victoria and northern territory) • provided by an independent health authority (new south wales) • (provided by medics as an integral component of the custodial enterprise) since the model of the independent health authority has developed in new south wales. the health needs of the prisoner population have been quantified, and attempts are being made to quantify specific health risks /benefits of incarceration. specific enquiry has been conducted into prisoner attitudes to their health care, including issues such as client information confidentiality and access to health services. specific reference will be made to: • two inmate health surveys • two inmate access surveys, and • two service demand studies. conclusions: the model of care provision, with legislative, ethical, funding and operational independence would seem provide the best opportunity to define and then respond to the health needs of prisoners. this model is being adopted in the united kingdom. better health outcomes in this high-risk group, could translate into healthier families and their communities. p - (a) lnregrated ethnic-specific health care systems: their development and role in increasing access to and quality of care for marginalized ethnic minorities joshua yang introduction: changing demographics in urban areas globally have resulted in urban health systems that are racially and ethnically homogenous relative to the patient populations they aim to serve. the resultant disparities in access to and quality of health care experienced by ethnic minority groups have been addressed by short-term, instirutional level strategies. noticeably absent, however, have been structural approaches to reducing culturally-rooted disparities in health care. the development of ethnic-specific h~alth car~ systems i~ a structural, long-term approach to reducing barriers to quality health care for eth· me mmonty populations. methods: this work is based on a qualitative study on the health care experiences of san francisco chinatown in the united states, an ethnic community with a model ethnic-specific health care infrastrucrure. using snowball sampling, interviews were conducted with key stakeholders and archival research was conducted to trace and model the developmental process that led to the current ethnic-specific health care system available to the chinese in san francisco. grounded theory was the methodology ijltd to analysis of qualitative data. the result of the study is four-stage developmental model of ethnic-specific health infrastrueture development that emerged from the data. the first stage of development is the creation of the human capital resources needed for an ethnic-specific health infrastructure, with emphasis on a bilingual and bicultural health care workforce. the second stage is the effective organization of health care resources for maximal access by constituents. the third is the strengthening and stability of those institutional forms through increased organizational capacity. integration of the ethnic-specific health care system into the mainstream health care infrastructure is the final stage of development for an ethnic-specific infrastructure. conclusion: integrated ethnic-specific health care systems are an effective, long-term strategy to address the linguistic and cultural barriers that are being faced by the spectrum of ethnic populations in urban areas, acting as culturally appropriate points of access to the mainstream health care system. the model presented is a roadmap to empower ethnic communities to act on the constraints of their health and political environments to improve their health care experiences. at a policy level, ethnic-specific health care organizations are an effective long-term strategy to increase access to care and improve qualiiy of care for marginalized ethnic groups. each stage of the model serves as a target area for policy interventions to address the access and care issues faced by culturally and linguistically diverse populations. users in baltimore md: - noya galai, gregory lucas, peter o'driscoll, david celentano, david vlahov, gregory kirk, and shruti mehta introduction: frequent use of emergency rooms (er) and hospitalizations among injection drug users (idus) has been reported and has often been attributed to lack of access to primary health care. however, there is little longitudinal data which examine health care utilization over individual drug use careers. we examined factors associated with hospitalizations, er and outpatient (op) visits among idus over years of follow-up. methods: idus were recruited through community outreach into the aids link to lntravenous experience (alive) study and followed semi-annually. , who had at least follow-up visits were included in this analysis. outcomes were self-reported episodes of hospitalizations and er/op visits in the prior six months. poisson regression was used accounting for intra-person correlation with generalized estimation equations. hits: at enrollment, % were male, % were african-american, % were hiv positive, median age was years, and median duration of drug use was years. over a total of , visits, mean individual rates of utilization were per person years (py) for hospitalizations and per py for er/op visits. adjusting for age and duration of drug use, factors significantly associated with higher rates of hospitalization included hiv infection (relative incidence [ri(, . ), female gender (ri, . ), homelessness (ri, . ), as well as not being employed, injecting at least daily, snorting heroin, havmg a regular source of health care, having health insurance and being in methadone mainte.nance treatment (mmt). similar associations were observed for er/op visits except for mmt which was not associated with er/op visits. additional factors associated with lower er/op visits were use of alcohol, crack, injecting at least daily and trading sex for drugs. % of the cohort accounted for % of total er/op visits, while % of the cohort never reported an op visit during follow-up. . . . lgbt) populations. we hypothesized that prov dmg .appomtments .for p~t ~nts w thm hours would ensure timely care, increase patient satisfaction, and improve practice eff c ency. further, we anticipated that the greatest change would occur amongst our homeless patients.. . methods: we tested an experimental introduction of advanced access scheduling (usmg a hour rule) in the primary care medical clinic. we tracked variables inclu~ing waiting ti~e fo~ next available appointment; number of patients seen; and no-show rates, for an eight week penod pnor to and post introduction of the new scheduling system. both patient and provider satisfaction were assessed using a brief survey ( questions rated on a -pt scale). results and conclusion: preliminary analyses demonstrated shorter waiting times for appointments across the clinic, decreased no-show rates, and increased clinic capacity. introduction of the advanced access scheduling also increased both patient and provider satisfaction. the new scheduling was initiated in july . quantitative analyses to measure initial and sustained changes, and to look at differential responses across populations within our clinic, are currently underway. introduction: there are three recognized approaches to linking socio-economic factors and health: use of census data, gis-based measures of accessibility/availability, and resident self-reported opinion on neighborhood conditions. this research project is primarily concerned with residents' views about their neighborhoods, identifying problems, and proposing policy changes to address them. the other two techniques will be used in future research to build a more comprehensive image of neighborhood depri· vation and health. methods: a telephone survey of london, ontario residents is currently being conducted to assess: a) community resource availability, quality, access and use, b) participation in neighborhood activities, c) perceived quality of neighborhood, d) neighborhood problems, and e) neighborhood cohesion. the survey instrument is composed of indices and scales previously validated and adapted to reflect london specifically. thirty city planning districts are used to define neighborhoods. the sample size for each neighborhood reflects the size of the planning district. responses will be compared within and across neighborhoods. data will be linked with census information to study variation across socio-eco· nomic and demographic groups. linear and gis-based methods will be used for analysis. preliminary results: the survey follows a qualitative study providing a first look at how experts involved in community resource planning and administration and city residents perceive the availability, accessibility, and quality of community resources linked to neighborhood health and wellbeing, and what are the most immediate needs that should be addressed. key-informant interviews and focus groups were used. the survey was pre-tested to ensure that the language and content reflects real experiences of city residents. the qualitative research confirmed our hypothesis that planning districts are an acceptable surrogate for neighborhood, and that the language and content of the survey is appropriate for imple· mentation in london. scales and indices showed good to excellent reliability and validity during the pre· test (cronbach's alpha from . - . ). preliminary results of the survey will be detailed at the conference. conclusions: this study will help assess where community resources are lacking or need improve· ment, thus contributing to a more effective allocation of public funds. it is also hypothesized that engaged neighborhoods with a well-developed sense of community are more likely to respond to health programs and interventions. it is hoped that this study will allow london residents to better understand the needs and problems of their neighborhoods and provide a research foundation to support local understandmg of community improvement with the goal of promoting healthy neighborhoods. p - (a) hiv positive in new york city and no outpatient care: who and why? hannah wolfe and victoria sharp introduction: there are approximately million hiv positive individuals living in the united sta!es. about. % of these know their hiy status and are enrolled in outpatient care. of the remaining yo, approx~mately half do not know their status; the other group frequently know their status but are not enrolled m any .sys~em of outpatient care. this group primarily accesses care through emergency departments. when md cated, they are admitted to hospitals, receive acute care services and then, upon poster sessions v di 'harge, disappear from the health care system until a new crisis occurs, when they return to the emergency department. as a large urban hiv center, caring for over individuals with hiv we have an active inpatient service ".'ith appr~xi~.ately discharges annually. we decided to survey our inpatients to better charactenze those md v duals who were not enrolled in any system of outpatient care. results: % of inpatients were not enrolled in regular outpatient care: % at roosevelt hospital and % at st.luke\'s hospital. substance abuse and homelessness were highly prevalent in the cohort of patients not enrolled in regular outpatient care. % of patients not in care (vs. % of those in care) were deemed in need of substance use treatment by the inpatient social worker. % of those not in care were homeless (vs. % of those in care.) patients not in care did not differ significantly from those in me in terms of age, race, or gender. patients not in care were asked "why not:" the two most frequent responses were: "i haven't really been sick before" and "i'd rather not think about my health. conclusions: this study suggests that there is an opportunity to engage these patients during their stay on the inpatient units and attempt to enroll them in outpatient care. simple referral to an hiv clinic is insufficient, particularly given the burden of homelessness and substance use in this population. efforts are currently underway to design an intervention to focus efforts on this group of patients. p .q (a) healthcare availability and accessibility in an urban area: the case of ibadan city, nigeria in oder to cater for the healthcare need of the populace, for many years after nigeria's politicl independence, empphasis was laid on the construction of teaching, general, and specialist hospital all of which were located in the urban centres. the realisation of the inadequacies of this approach in adequately meeting the healthcare needs of the people made the country to change and adopt the primary health care (phc) system in . the primary health care system which is in line with the alma ata declaration of of , wsa aimed at making health care available to as many people as possible on the basis of of equity and social justice. thus, close to two decades, nigeria has operated primary health care system as a strategy for providing health care for rural and urban dwellers. this study focusing on urban area, examimes the availabilty and accessibility of health care in one of nigeria's urban centre, ibadan city to be specific. this is done within the contest of the country's national heath policy of which pimary health care is the main thrust. the study also offers necessary suggestion for policy consideration. in spite of the accessibility to services provided by educated and trained midwifes in many parts of fars province (iran) there are still some deliveries conducted by untrained traditional birth attendants in rural parts of the province. as a result, a considerable proportion of deliveries are conducted under a higher risk due to unauthorised and uneducated attendants. this study has conducted to reveal the pro· portion of deliveries with un-authorized attendants and some spatial and social factors affecting the selection of delivery attendants. method: this study using a case control design compared some potentially effective parameters indud· ing: spatial, social and educational factors of mothers with deliveries attended by traditional midwifes (n= ) with those assisted by educated and trained midwifes (n= ). the mothers interviewed in our study were selected from rural areas using a cluster sampling method considering each village as a cluster. results: more than % of deliveries in the rural area were assisted by traditional midwifes. there are significant direct relationship between asking a traditional birth attendant for delivery and mother age, the number of previous deliveries and distance to a health facility provided for delivery. significant inverse relationships were found between mother's education and ability to use a vehicle to get to the facilities. conclusion: despite the accessibility of mothers to educated birth attendants and health facilities (according to the government health standards), some mothers still tend to ask traditional birth attendants for help. this is partly because of unrealistic definition of accessibility. the other considerable point is the preference of the traditional attendants for older and less educated mothers showing the necessity of changing theirs knowledge and attitude to understand the risks of deliveries attended by traditional and un-educated midwifes. p - (a) identification and optimization of service patterns provided by assertive community treatment teams in a major urban setting: preliminary findings &om toronto, canada jonathan weyman, peter gozdyra, margaret gehrs, daniela sota, and richard glazier objective: assertive community treatment (act) teams are financed by the ontario ministry of health and long-term care (mohltc) and are mandated to provide treatment, rehabilitation and support services in the community to people with severe and persistent mental illness. there are such teams located in various regions across the city of toronto conducting home visits - times per week to each of their approximately respective clients. each team consists of multidisciplinary health professionals who assist clients to identify their needs, establish goals and work toward them. due to complex referral patterns, the need for service continuity and the locations of supportive housing, clients of any one team are often found scattered across the city which increases home visit travel times and decreases efficiency of service provision. this project examines the locations of clients in relation to the home bases of all act teams and identifies options for overcoming the geographical challenges which arise in a large urban setting. methods: using geographic information systems (gis) we geocoded all client and act agency addresses and depicted them on location maps. at a later stage using spatial methods of network analysis we plan to calculate average travel rimes for each act team, propose optimization of catchment areas and assess potential travel time savings. resnlts: initial results show a substantial scattering of clients from several act teams and substan· rial overlap of visit travel routes for most teams. conclusions: reallocation of catchment areas and optimization of act teams' travel patterns should lead to substantial savings in travel times, increased service efficiency and better utilization of resourc_~· ~e l'<!tenri~i modifications to catchment areas must be conducted with appropriate attention to specific clients servtce needs, service continuity and applicable regulations by the mohl tc. venice lido is a popular island within and outside italy because it hosts the international cinema festival yearly. only few people remember the island for its hospital, which, in the fifties, was one of the most important hospitals in italy and in europe, being a modern center for wind-, sun-and thermal-therapy. since the sixties, its fame became to drop away, since its structure was no longer adequate for the evolving medicine science. at the end of the seventies all the wards were moved into a new big building in the same area. at present the hospital is made up by about forty 'pavilions' (mostly partially used or empty) and a big building, disseminated in a hectares area, on the sea front, on the m long beach. the aim of my research is to suggest a realistic solution, sustainable on the functional use of the area according to basic local needs and economically realizable. i used a philological and multi-disciplinary method, more in detail: -from historical documents, i found what was the very first call of the property and how it adapted to the needs of the demographic development and the sanitary supply over time; -by contacting some operators from different fields, i realized which functions were suitable to the buildings in existence, to the population's needs and to the present sanitary supply. moreover the planning idea considers: -the debate going on between local people who strongly want to preserve the property, and die ones who want to renew it by different new opportunities; and -the regional sanitary politics, aiming at rationalizing the regional hospitals dislocation, and the budget needs. this plan analyzes and proposes some new alternative solutions to satisfy different needs. the different activities will be dealt out and the environmental fall will be limited as much as possible. after having analyzed its basic call, considered its environmental peculiarities and its position, verified the emerging needs of the local population, the plan proposes different activities: the hospital; the social-rehabilitation center; the elderly house; the thermal center; the residential center; and other activities. such a plan wants to share in the solution of a basic problem by giving architectonic and functional dignity to an historical completely degraded area and by starting again a social-economical broken off process, by bringing in venice lido some fresh necessary elements for its so wished new throw. p - (c) dilemma of free health care in spokane, wa david bunting introduction: the paper reports on the activities of project access spokane [pas], a physician sponsored initiative to provide uncompensated health care to low income, uninsured residents of spokane county wa. program providers included all county hospitals, over physicians, other medical professionals, and specialized clinics. each prescription requires only a $ co-payment. sponsored and administered by the county medical society, pas is funded by local and national foundations, private corporations, municipalities, civic organizations and individuals. method: the paper is based on a first year assessment report funded by pas, using hcfa [health care financing administration] insurance claim data documenting utilization rates, disease categories and donated charge information and patient cares [centralized applications, referrals and enrollment status) data. results: while essentially free for enrollees, the program involved real costs. an administrative organization to refer over patients to any of over providers had to be developed and staffed, using both paid and unpaid personnel. methods to identify potential patients and veri.fy eligibility. had to ~devised. patient appointments and transportation had to be scheduled and monitored. pu~hc relanons, provider solicitations and fundraising activities were continuous. information requirements regarding program operation were also significant. data reporting the volume. ~nd value of dona~ed medical services were necessary to demonstrate accomplishments and attr~ct addmon~l support. providers required assurances their contributions were both significant and eqmtable. funding sources sought evidence that their support had important consequences. however, generating this ~~ta proved difficult. many providers failed to submit appropriate insurance claims forms because .of a~dmonal donated effort and administrative cost, thereby not only reducing their own apparent conmbut ons but also the overall significance of the program. conclusions: during its initial year, the estimated cost to deliver free health care was ~bout per enrollee. the lesson is that without an elaborate administrative structure and record keeping s.yst~~· efforts to provide free health care probably will fail. eligible enrollees c~n not be ~parated from in~hgi ble ones, care will not be accessed or delivered in an efficient manner, neither fundmg nor ~upport "". be offered without evidence of tangible benefits, and providers will withdraw if they perceive mequ table tteannent. v p - s (c) mobility in prostitution and the impact of health therese van der helm and henk sulman poster sessions introduction: many of the estimated . commer~ial ~ex wor~ers (c~w)in a'.' ~terdam ~~me from developing countries and eastern europe. to gain ins~ght mto their workmg and_ hvmg condinons the intermediary project (ip) at the municipal health serv ~e _contacts these women via ?utreach work on regular basis. since the new brothel law in october s ~troduc~d, ~sw ~rom outside the eu and who have no dutch residence permit are not allowed to work m prost tut n. smee then, many of these csw therefore have gone "underground." the consequences of the new law in the netherlands will be discussed with regard to the accessibility of csw and their risk for stl/hiv acquisition and unplanned pregnancies. methods: topics during outreach work are the interventions to increase the knowledge of safe sex and birth control for csw. written information is handed out in relevant languages and with addresses of health and social services. in conversations with the women, it appeared that many are not aware of these services. to provide easier access to health care for women, staff of the ip carries out free sti control csw working places, in windows brothels and sex houses. also, women are offered vaccination against hepatitis b (hbv). results: from january till july we approached csw for first contact. one third was dutch; others were from developing countries, other eu countries and eastern europe. sti consul· rations were done among non-iv drug using sex workers, of whom % was dutch. of these, % was diagnosed with syphilis, % with gonorrhea, and % with chlamydia. of women tested for hiv, none were infected. due to the high turnover of csw in .brothels, most wnmen were tested only once. among csw tested for hbv -of whom one third is dutch - % had antibodies for hbv, were carrier of hbv, most of whom came from hbv endemic areas. conclusions: sti control and hbv vaccination in brothels is an important support in health care. our findings suggest that csw do not play an important role in the transmission of sti. many csw are highly mobile and often not aware of the existing health and social services. continuous outreach is important to remain in contact with this mobile population. regulations in the new brothel law should not hamper contacts with (illegal) csw. heart disease (cho) is projected to become the leading cause of death. studies conducted in europe and the united states have proven a higher rate of cho in south asians who have migrated from south asia, most notably india, pakistan and bangladesh. approximately % of all heart attacks among south asian men occur under the age of ; % of them occur under the age of -unheard of in any other population. associated risk factors such as diabetes, high blood pressure and cholesterol are also com· mon in this group. the population of south asians residing in nyc has more than doubled over the past decade to over , , the majority being young, working-class and with limited health care access. many south asian men are employed as taxi drivers ( out of nyc taxi drivers are from south asial, working daily hour plus shifts. methods: acknowledging that this vulnerable, at-risk group was unavailable for outreach through conventional venues, three of the hospitals of the new york city health and hospital corporation, (elmhurst, queens and bellevue) conducted a one day outreach effort at nyc's jfk international air· ~rt's taxi holdin~ ar~a, ~here over one thousand taxis regularly assemble. bringing an outreach event directly to the taxi dnvers workplace, a tent was set up where cardiovascular-related health screenings, in~luding bl~ pressure, sugar, cholesterol, body mass index (bmi), were provided. results were shared with and explamed to the drivers and each participant received a south asian health education brochure. a total of taxi drivers who identified as south asian were involved. rau/ts: participants were all male, ranging from to years; mean age, . . screenings revealed % hypertensive; % had cholesterols over ; % blood glucose over ; % had a bmi greater than . conc~on: a unique outreach activity, adapting to logistical, occupational limitations, is pre· se~ted. on-site f~back and explanation of results, reiterating and reinforcing the concern that south asia~. men are at mcreased danger for early coronary heart disease, and the dissemination of a culturally sensmve and r~levant brochure, may heighten awareness of prevailing cardiac risk factors in this immi· grant community. p - (c) the community-hospital integration program framework: community-hospital panoenhips to improve the population's health john stevenson, richard blickstead, ann-marie marcolin, and sandi kendal v introduction: st. josephs health centre is a catholic community teaching hospital located in the heart of southwest toronto. st. joseph\'s was founded from a community-expressed need for hospital services, and since then has stayed committed to fostering a healthy community through collaboration and parmership. st. joseph's has developed an innovative model, the community-hospital integration program (chip), to strengthen st. joseph's partnerships with community stakeholders, and facilitates the building of links between community needs, service provision, and public policy outcomes to improve the health and wellness of the diverse neighbourhoods they together serve. methods: development of the chip framework st. joseph's health centre developed the chip framework through a multidisciplinary approach that included extensive international literature reviews, consultations, and key informant interviews with community service agencies and academics. to ensure active community voice, st. joseph's has hosted a number of community consultations including a community outreach forum attended by over ninety representatives from community partners. results: the chip framework the chip framework aims to improve the health and wellness of the urban communities served by st. josephs health centre through four intersecting pillars: • raising community voices provides an infrastructure and process that supports community stakeholder input into health care service planning, decision-making, and delivery by the hospital and across the continuum of care; • sharing reciprocal capacity promotes healthy communities through the sharing of our intellectual and physical capacity with our community partners; • cultivating integration initiatives facilitates vertical, horizontal, and intersectoral integration initiatives in support of community-identified needs and gaps; and • facilitating healthy exchange develops best practices in community integration through community-based research, and facilitates community voice in informing public policy. the chip framework drives the complex inter-relationships between community-hospital engagement, reciprocal capacity-building, integration initiatives, and community-based research and evaluation, to create an interconnected network of health care services. the fluidity and simplicity of this framework, and its dedication to balancing community needs and hospital mandate, posits the st. josephs health centre's chip model as an integral component in building healthy and thriving communities. p - (c) home based care promotion: improving acess to quality services and livelihood in the face of aids home based care is a service provided to persons affected/living with hiv/aids, a menu of care/support services at home/community. it is a prominent alternative approach. in uganda, hospital bed ocupacny is high, patient health care worker ratio deteriorating. empowering communities offers solutions to the problems; adressing cost, effectiveness of care. hiv/aids demands changes in attitudes, behaviour, approaches which is enhanced in home/community settings. for example art requires high levels of adherecnce, which medical workers have vety little opportunity to enforce. it also demands other support mechanisims in terms of nutrition, personal displine, etc which work best in stigma free environments. hence the relevance of home based care. this paper highlights the gaps that call for increased action in terms of home based care, examples of whre it has worked key challenges and recommendations for the future. p - (c) health care for one ... health care for alli katharina kovacs burns introduction: at the surface, it appears that every person in canada _has. ~ccess to a publicly funded health care system. those living in urban centers should have the best ava l~b hty, chmce, and access to a variety of health care services because of the distribution of health care services, fac lmes, and health professionals in concentrated in urban centers. this is not true for everyone -people with low income or who are homeless experience the challenges of social exclusion and being marginalized: there are many factors at play making it difficult for the latter group of people to access health care. service they need,.when they need them. health care is not as accessible or inclusive as intended. the quesuon to be explore.cl ~: if health care is available and accessible to one person, what makes it not available to all people? the ~ gmfic~nce of having marginalized people accessing health care and other services includes e~hanced quality of hfe and decreased risks associated with being homeless, and cost savings in ion~ ter~ w rh acute health care. methodology: community-based participatory research is applied m a case study on health care access and challenges experienced by low income and homeless people in one urban centre, edmonton, edmonton, and their challenges. the data is being gathered and ana_lyzed usmg basic m x~d methods. results: the results will focus on how services can be more mtegrated and accessible to all people with low income and who are homeless. there will also be discussion about specific perceptions of not only the service providers but also of people with low income an~ who are homeless~ and vario~s decision makers. the results will be compared to the literature regardmg health care services access mother urban centers in canada and elsewhere. conclusions: recommendations will need to address social exclusion and other factors which can make health care and other services more available to all people in the community. the implications for health care will also be discussed. additional information will be gathered in the next phases of the study to develop a community services access model. p - (c) availability and access exemplified: a case study beth hayhoe and ruth ewert introduction: access to health care in canada requires either the correct documentation or money. street youth have neither. in addition, health services in canada are designed by adults for adults. youth in general are often wary of having trust in adults with respect to their health concerns and frequently feel misunderstood. street youth are even more mistrustful of adults and public organizations, making their contact with health care professionals minimal. this combined with their risky behaviours and dangerous environment creates a population at risk for numerous health issues but with no place to have them investi· gated. at our non-government, not for profit health service designed specifically to provide a broad range of health care to street youth, professional services are provided almost entirely by volunteers. methods: using a retrospective analysis of the years of data gathered from this organization and reviewing the initial reasons gathered from youth about their needs, the success of the health centre was examined. results: all the things youth had listed as being important in a health centre have been implemented, and even more things have been added to improve the breadth and quality of services offered. the use of the health centre has increased by more than six times since its opening in . in addition, tens of thousands of visits have been paid to the health centre, costing the government and taxpayers nothing. as far as we know there is nothing else in canada that offers so many diverse and innovative services to youth in need. conclusion: it is clear from this case, that health care targeted at the needs of specific disadvan· taged populations can successfully provide them with appropriate health care. a model of accessible health care for marginalized populations can easily be developed for high impact and low cost from this successful case. toronto is one of the most ethno-racially diverse cities in the world of the estimated , , toronto residents, % ( , ) live in scarborough population growth in scarborough is faster than toronto. scarborough's population increased by % from to while toronto increased only by %. toronto's population is projected to increase by % ( ) in while scarbor· ough's will increase by %. (census canada ) low income, pove;ty, seniors, hidden homeless, new~omers to canada, the uninsured, are just a few of the issues concerning health care in our community. health care needs in scarborough are greatly impacted by diversity of ethnic and racial groups, poverty and settlement issues. this paper will share how the scarborough hospital (tsh) an urb~n communir_y hospital cares f~~ it~ community. tsh recognizes: •the changing community• b_arriers t~ accessing care • lnequalmes m health care the hospital in caring for its community pro· v ~es services that '!'e~e cu~turally, racially, and linguistically sensitive to our community. the paper will share the hospitals unique program on access and equity. the paper will share the needs in scar· borough and tsh's response to these needs: working in partnership with the local health committee, i:ne scarborough homeless co.mminee, the scarborough network of immigrant services organiza· nons a~d others. :' e paper will also share the many initiatives this urban community hospital has taken viz. community and home programs in service areas such as mental health, haemodialysis day care, t~~ home oxyg~n program, the palliative at-home program, and above all support for the volunteer clinic for the uninsured. j"'" tllu:lion: in canada's universal health care system it is generally assumed that everyone has equal access to health care. however, some immigrant groups in canada have historically been uninswed. these groups include failed refugee claimants, those overstaying their visas, and new immigrants in transition who are waiting to become eligible for health insurance. some estimates have suggested that at the present time there may be as many as one hundred thousand undocumented and thus uninsured immigrants in toronto alone. understandably, information on the characteristics of this marginalized population is very limited, since undocumented immigrants tend to have little contact with formal institutions. knowledge of the demographic characteristics of this population may aid in the development of health and social programs to better identify and meet the needs of undocumented immigrants in toronto. we conducted a review of all undocumented, uninsured patients, years of age or older at access alliance multicultural community health centre (aamchc) between and . demographic information such as age, gender, preferred language, length of time living in canada, selfreported education level and household income were recorded. rmdts: % of all adult patients at access alliance were undocumented and uninsured. % were under years of age (with a median age of years), % were women and only % spoke english. this group lived in canada for an average period of . years before they were first seen at aamchc. % of undocumented clients reported having completed at least a secondary or post-secondary education. % had self-reported household incomes of less than , $/year, while % of households reported earning less than $ , /year. the mean income per person in an average household was $/month. conchuions: uninsured individuals at aamchc were predominantly young females with limited knowledge of the english language. despite having attained a high level of education most were living well below the poverty line. recognition of these characteristics may assist in the development of health and social programs that are better adapted to meet the needs of undocumented immigrants. p - (c) sharing expertise: a role for the hospital lactation consultant in the community. badrgrmuul: st. michael's hospital is a tertiary care hospital that serves a large inner city population in downtown toronto. in order to provide care to this population, the hospital has developed a number of unique outreach roles. one such role was developed to work with pregnant and breastfeeding women living in regent park, a social housing complex located near the hospital. the population of regent park includes new immigrants, refugees and the socially disadvantaged. approach: the outreach lactation consultant provides care, as part of the canada prenatal nutrition program, in the community prenatally, in the hospital during their inpatient stay and postnatally when they return to the community. the continuity of care enhances the probability of long term successful breastfeeding. aside from the health benefits of breastmilk, breastfeeding is especially important for women who are financially needy and must depend on food banks for formula. . . lason learned: aher two years of partnership, the relationship between the an-hospital ~stpar tum experience for breastfeeding mothers and the community postpartum support, the breastfeeding rate ~thin this community is high. the professional support and visibility of the : ctation consultant, both m the hospital and in the community, contributes to the support of breastfee~m~.. . in light of ongoing funding constraints, this ha son between hospital and community could be at risk and discontinued. in light of the benefits to the mother/baby dyad, the lactation consultant as a community partner in the canada prenatal nutrition program should be safeguarded. . lldpodiu:tion: although the importance of adequate health care in pregna~cy is well recognised, ethnic disparities in utilization of prenatal care still exist in many western coun~~es. a fun~amental factor in these disparities may be language proficiency, as it influences women's ab hty. to ob~m and understand health information, to find the way in the health care system and to communicate with health care v poster sessions providers. we investigated the role of language proficiency in use and knowledge of folic acid as aspect of prenatal care in an urban multi-ethnic pregnancy c?hort. . . design: prospective cohort study. setting and parnc pants: amsterdam ~regnant women attending obstetric care providers for their first antenatal visit (n= ). country of birth defined ethnicity: the netherlands, surinam, antilles, turkey, morocco, ghana, other non-~est~m (nw) and other western (w) country. main outcome measures: knowledge about and use of.fohc a~d (fa) supplements in pregnancy, and determinants of these in diffe~~t ethnic ~~ups. deterrrunants mcl~ded age, ed~ tion, parity, pregnancy intention and dutch prof c ency. stansncs: ~ to co~p~re e~c groups, stranfied logistic regression (forward stepwise method) to explore determmants "'.'thm ethmc groups .. use of fa supplements was significantly lower among ghanaian, moroccan, turkish and other nw women ( %to %) than among dutch ( %) or other w women ( %). use amongsurinames and antillean women was intermediate ( %and %). ethnic differences in fa knowledge were similar. knowledge was the strongest determinant of use in all ethnic groups, with odds ratios (ors) ranging from . to . . language proficiency was the strongest determi~a~t of kno~ledge in ethnic groups with a mother tongue different from dutch (ors good vs. low proficiency ranging from . ro . ). educational level had a modifying role, as shown by an interaction effect between education and language proficiency in the nw group. here, the odds of having fa knowledge given a high education and good dutch proficiency was times the odds given a good proficiency but low education. conclusions: appropriate periconceptional use of folic acid supplements in non-western ethnic groups is low, reflecting an absence of knowledge that is largely determined by the inability to speak and understand the language of the habitual country. tailored interventions using communication channels most likely to address (pregnant) women from ethnic minority groups are necessary. apan from specific interventions, our results are in support of strong incentives on language education despite current political debate. introduction: recent research suggests living in an economically disadvantaged neighborhood is asso· ciated with decreased likelihood of undergoing mammography and increased risk of late-stage breast can· cer diagnosis. long distances and travel times to facilities offering low-or no-cost mammography may be imponant barriers to adherence to mammography screening recommendations for residents of economically disadvantaged neighborhoods. the purpose of this study was to examine whether facilities providing low-and no-cost screening mammography were less spatially accessible in low-income neighborhoods in chicago, and the extent to which the relationship between neighborhood income and the spatial accessibility of facilities varied by the proponion of african-american residents in the neighborhood. methods: the sample consisted of chicago neighborhoods. for each neighborhood, we con· structed three measures of the spatial accessibility of facilities: street network distance to the nearest facility, public transportation travel time to the nearest facility, and shortest automobile travel time to a facility. using decennial census data, we characterized the neighborhoods according to proportion of residents with incomes below the poverty line, proportion of residents in each of four raciavethnic groups (african-american, latino, white, and other), and population density. we used ordinary least squares (ols) and spatial exogenous lag regression to examine relationships. model estimated the relationship between neighborhood poverty and the spatial accessibility of facilities, adjusting for raciaveth· nic composition and population density. model added a multiplicative interaction term between neighborhood poverty and african-american. restllts: we identified deven facilities that provided low-or no-cost screening mammography to chicago residents in . we found that the distance and travel times via automobile and public trans· portation to facilities generally decreased as neighborhood poveny increased. however, we also found that the ~egative associations between neighborhood poverty and two of the spatial accessibility mea· sures -distance and public transportation travel time -were less strong in neighborhoods with the highest proponions of african-american residents. among neighborhoods in the highest tertile for poveny, th~ mean distance and mean public transportation travel time to facilities were over twice as long in neighborhoods in the highest tertile than in those in the lowest tenile of african-american residents. . ~ persistent socioeconomic and racial/ethnic disparities in breast cancer stage at diagno-s ~nd su~ val suggest that ensuring an equitable distribution of affordable mammography is a worth-w~e policy .goal. the ~~dy sugges~ that ~ture investigations should consider both neighborhood soc oecono d c charactensbes and rac avethmc composition when examining the spatial distribution of health resources. , ) . epidemiological data suggest tha~ hiv prevalence among msm is over % in some metropolitan cities, such .as beijing (ibid.). due to widespread homophobia in chinese society, however, this population remains invisible within current health/social services. lack of research on sexuality, specifically homosexuality, has impaired our understanding of health and health practices of chinese msm. focusing on the illness experiences of chinese msm with hiv/aids, this paper explores the socio-cultural impacts of hiv/ aids on this group. the data used for this paper were collected through semi-constructed in-depth face-toiace interviews with adult plwhas (including msm) in beijing, china. with the permission of the participants, the interviews were audio-taped or recorded in notes. the transcribed interviews and interview notes were analyzed by using n-vivo, a software program for qualitative data analysis. this phenomenological study aimed to understand the experiences chinese plwhas (including msm) from their own perspectives. results: it is found that the illness experiences of chinese msm with hiv/aids are profoundly shaped by the socio-cultural meanings of homosexuality, which are further complicated by the dominant discourses on hiv/aids in china. at a macro level, homophobia in the larger society has decreased the capability of this group to respond to this epidemic in a more efficient way. at a meso level, aids-phobia within the chinese msm circuits has prevented this group from openly confronting this disease and offering support to those who are already affected by it. at a micro level, however, these hiv-infected/ affected msm have tried their best to locate spaces to live and to construct hope for their future lives despite various barriers within family, community, and the larger society. this study suggests that facilitating chinese msm's response to the aids crisis urgently requires bridging awareness, commitment, knowledge and resources both within and without this group. it also illustrates the importance of understanding homosexuality in the local context of hiv i aids, which will be helpful for developing culturally sensitive hiv/aids programs for this population on the community, national and international levels. introduction: having a regular family doctor (rfd) is known to be positively correlated with a good medical follow up, including access to preventive and/or chronic care. inversely, a lack of primary care may lead to high rates of avoidable hospitalizations, especially among poor people and/or in underserved neighbourhoods. in such a matter, a recent comparative study showed that paris was better ranged than other cities, such as nyc, tokyo or london. nevertheless, despite a quasi universal health insurance and a high density of general practitioners, a noticeable fraction of the paris population does not have any regular physician and we aimed to understand who they are and for what reasons rhey don't have any rfd. mdbods: cross sectional population survey among a random sample of households in ~nderpriv ileged neighbourhoods in paris inner city, performed in , using a face-to-face quest nna re collecr- ng more than social and health characteristics. ruults: a quarter ( . %) of rhe study popularion did not have any rfd. this proporrion was iignificantly higher among male (or= . , %ci = [ . - . ), younger (e.g. or - /> .s l _= ._oo, " .ci = ( . - . )), and/or unemployed (or = . , %ci = ij . - . _ people. in multtvanate analysis, after a full adjustment on gender, age, health status, health insura~ce, income, occupat n and tducation level, we observed significant associations between having no rfd and: ~arrtal and_ pare~t hood status (e.g. or single no kids/in couple+kids = . , %ci = ( . - . ()~ quality of relattonsh ps with neighbours (or bad/good= . , %ci = [ . - . )), and length of residence m the neighbourhood (with a dose/effect statistical relationship). . co clusion: gender, age, employment status, mariral and parenthood stat~s as well as ~e gh bourhood anchorage seem to be major predictors of having a rfd, even when um.versa! health i~sur ance has reduced most of financial barriers. in urban contexts, where residential migrattons and single lift (or family ruptures) are frequent, specific information may be conducted to encourage people to ket rfd. :tu~y tries to assess the health effects and costs and also analyse the availability and accessibility to health care for poor. . methods: data for this study was collected by a survey on households of the local community living near the factories and households where radiation hazard w~s n?~ present. ~~art from mor· bidity status and health expenditure, data was collected ~n access, a~ail~b .hty and eff c ency of healrh care. a discriminant analysis was done to identify the vanables that d scnmmate between the study and control group households in terms of health care pattern. a contingent valuation survey was also undertaken among the study group to find out the factors affecting their willingness to pay for health insurance and was analysed using logit model. results: the health costs and indebtedness in families of the study group was high as compared to control group households and this was mainly due to high health expenditure. the discriminant analysis showed that expenditure incurred by private hospital inpatient and outpatient expenditure were significant variables, which discriminated between the two types of households. the logit analysis showed !hat variables like indebtedness of households, better health care and presence of radiation induced illnesses were significant factors influencing willingness to pay for health insurance. the study showed that study group households were dependent on private sector to get better health care and there were problems with access and availability at the public sector. conclusion: the study found out that the quality of life of the local community is poor due to health effects of radiation and the burden of radiation induced illnesses are so high for them. there is an urgent need for government intervention in this matter. there is also a need for the public sector to be efficient to cater to the needs of the poor. a health insurance or other forms of support to these households will improve the quality for health care services, better and fast access to health care facilities and reduces the financial burden of the local fishing community. the prevalence of substance abuse is an increasing problem among low-income urban women in puerto rico. latina access to treatment may play an important role in remission from substance abuse. little is known, however, about latinas' access to drug treatment. further, the role of social capital in substance abuse treatment utilization is unknown. this study examines the relative roles of social capital and other factors in obtaining substance abuse treatment, in a three-wave longitudinal study of women ages - living in high-risk urban areas of puerto rico, the inner city latina drug using study (icldus). social capital is measured at the individual level and includes variables from social support and networks, familism, physical environment, and religion instruments of the icdus. the study also elucidates the role of treatment received during the study in bringing about changes in social capital. the theoretical framework used in exploring the utilization of substance abuse treatment is the social support approach to social capital. the research addresses three main questions: ( t) does social capital predict parti~ipating in treatment programs? ( ) does participation in drug treatment programs increase social capital?, and ( ) is there a significant difference among treatment modalities in affecting change in ~ial capital? the findings revealed no significant association between levels of social capital and gettmg treatment. also, women who received drug treatment did not increase their levels of social capital. the findings, however, revealed a number of significant predictors of social capital and receiving drug ~buse treatment. predictors of social capital at wave iii include employment status, total monthly mcoi:rie, and baseline social capital. predictors of receiving drug abuse treatment include perception of physical health and total amount of money spent on drugs. other different variables were associated to treatment receipt prior to the icldus study. no significant difference in changes of social capital was found among users of different treatment modalities. this research represents an initial attempt to elucidate the two-way relationship between social capital and substance abuse treatment. more work is necessary to unden~nd. ~e role of political forces that promote social inequalities in creating drug abuse problems and ava lab hty of treatment; the relationship between the benefits provided by current treatment poster sessions v sctrings and treatment-seeking behaviors; the paths of recovery; and the efficacy and effectiveness of the trtaanent. and alejandro jadad health professionals in urban centres must meet the challenge of providing equitable care to a population with diverse needs and abilities to access and use available services. within the canadian health care system, providers are time-pressured and ill-equipped to deal with patients who face barriers of poverty, literacy, language, culture and social isolation. directing patients to needed supportive care services is even more difficult than providing them with appropriate technical care. a large proportion of the population do not have equitable access to services and face major problems navigating complex systems. new approaches are needed to bridge across diverse populations and reach out to underserved patients most in need. the objective of this project was to develop an innovative program to help underserved cancer patients access, understand and use needed health and social services. it implemented and evaluated, a pilot intervention employing trained 'personal health coaches' to assist underserved patients from a variety of ethno-linguistic, socio economic and educational backgrounds to meet their supportive cancer care needs. the intervention was tested with a group of underserved cancer patients at the princess margaret hospital, toronto. personal coaches helped patients identify needs, access information, and use supportive care services. triangulation was used to compare and contrast multiple sources of quantitative and qualitative evaluation data provided by patients, personal health coaches, and health care providers to assess needs, barriers and the effectiveness of the coach program. many patients faced multiple barriers and had complex unmet needs. barriers of poverty and language were the easiest to detect. a formal, systematic method to identify and meet supportive care needs was not in place at the hospital. however, when patients were referred to the program, an overwhelming majority of participants were highly satisfied with the intervention. the service also appeared to have important implications for improved technical health care by ensuring attendance at appointments, arranging transportation and translation services, encouraging adherence to therapy and mitigating financial hardship -using existing community services. this intervention identified a new approach that was effective in helping very needy patients navigate health and social services systems. such programs hold potential to improve both emotional and physical health out· comes. since assistance from a coach at the right time can prevent crises, it can create efficiencies in the health system. the successful use of individuals who were not licensed health professionals for this purpose has implications for health manpower planning. needle exchange programs (neps) have been distributing harm reduction materials in toronto since . counterfit harm reduction program is a small project operated out of a community health centre in south-east toronto. the project is operated by a single full-time coordinator, one pan-rime mobile outreach worker and two peers who work a few hours each week. all of counterfit's staff, peers, and volunteers identify themselves as active illicit drug users. yet the program dis~rib utes more needles and safer crack using kits and serves more illicit drug u~rs t~an the comb ~e~ number of all neps in toronto. this presentation will discuss the reasons behind this success, .s~ f cally the extended hours of operation, delivery models, and the inclusion of an. extremely marg ~ahzed community in all aspects of program design, implementation and eva.luat ?n. ~ounterfit was recently evaluated by drs. peggy milson and carol strike, two leading ep dem olog st and researchers in the hiv and nep fields in toronto and below are some of their findings: "the program has experienced considerable success in delivering a high quality, accessible and well-used program .... the pro· gram has allowed (service users) to become active participants in providing. services to others and has resulted in true community development in the best sense. " ... counterf t has ~~n verr succe~sful attracting and retaining clients, developing an effective peer-based model an.d assisting chen~s ~ th a vast range of issues .... the program has become a model for harm red~ctmn progr~ms withm the province of ontario and beyond." in june , the association of ?ntano co~mumty heal~~ <:en· ires recognized counterfit's acheivements with the excellence m community health initiatives award. in kenya, health outcomes and the performance of government health service~ have det~riorated since the late s, trends which coincide with a period of severe resource constramts necessitated by macro-economic stabilization measures after the extreme neo-liberalism of the s. when the govern· ment withdrew from direct service provision as reform trends and donor advocacy suggested, how does it perform its new indirect role of managing relations with new direct health services providers in terms of regulating, enabling, and managing relations with these health services providers? in this paper therefore, we seek to investigate how healthcare access and availability in the slums of nairobi has been impacted upon by the government's withdrawal from direct health care provision. the methodology involved col· leering primary data by conducting field visits to health institutions located in the slum areas of kibera and korogocho in nairobi. purposive random sampling was utilized in this study because this sampling technique allowed the researcher(s) to select those health care seekers and providers who had the required information with respect to the objectives of the study. in-depth interviews using a semi-structured ques· tionnaire were administered ro key informants in health care institutions. this sought to explore ways in which the government and the private sector had responded and addressed in practical terms to new demands of health care provision following the structural adjustment programmes of the s. this was complemented by secondary literature review of publications and records of key governmental, bilateral and multilateral development partners in nairobi. the study notes a number of weaknesses especially of kenya's ministry of health to perform its expected roles such as managing user fee revenue and financial sustainability of health insurance systems. this changing face of health services provision in kenya there· fore creates a complex situation, which demands greater understanding of the roles of competition and choice, regulatory structures and models of financing in shaving the evolution of health services. we rec· ommend that the introduction of user fees, decentralization of service provision and contracting-out of non-clinical to private and voluntary agencies require a new management culture, and new and clear insri· tutional relationships. experience with private sector involvement in health projects underlines the need not only for innovative financial structures to deal with a multitude of contractual, political, market and risks, but also building credible structures to ensure that health services projects are environmentally responsive, socially sensitive, economically viable, and politically feasible. purpose: the purpose of this study is to examine the status of mammography screening utilization and its predictors among muslim women living in southern california. methods: we conducted a cross-sectional study that included women aged ::!: years. we col· leered data using a questionnaire in the primary language of the subjects. the questionnaire included questions on demography; practices of breast self-examination (bse) and clinical breast examination (cbe); utilization of mammography; and family history of breast cancer. bivariate and multiple logistic regression analyses were performed to estimate the odds ratios of mammography use as a function of demographic and other predictor variables. . results: among the women, % were married, % were - years old, and % had family h story of breast cancer. thirty-two percent of the participating women never practiced bse and % had not undergone cbe during the past two years. the data indicated that % of the women did not have mammography in the last two years. logistic regression analysis showed that age ( r= . , % confi· dcnc~ interval (cl)=l. - . ), having clinical breast examination ( r= . , % cl= . - . ), and practtce of self-breast examination ( r= . , % cl= . - . ), were strong predictors of mammography use . . conclusions: the data point to the need for intervention targeting muslim women to inform and motivate th.cm a~ut practices for early detection of breast cancer and screening. further studies are needed to investigate the factors associated with low utilization of mammography among muslim women population in california. we conducted a review of the scientific literature and° government documents to describe ditnational health care program "barrio adentro" (inside the neighborhood). we also conducted qualiurivt interviews with members of the local health committees in urban settings to descrihe the comm unity participation component of the program. rtsmlts: until recently, the venezuelan public health system was characterized by a lack or limited access w health care ( % of the population) and long waiting lists that amounted to denial of service. moit than half of the mds worked in the five wealthiest metropolitan areas of the country. jn the spring oi , a pilot program hired cuban mds to live in the slums of caracas to provide health care to piople who had previously been marginalized from social programs. the program underwent a massive expansion and in only two years , cuban and , venezuelan health care providers were working acmss the country. they provide a daily average of - medical consultations and home visits, c lly out neighborhood rounds, and deliver health prevention initiatives, including immunization programs. they also provide generic medicines at no cost to patients, which treat % of presenting ill-ij!m, barrio adentro aims to build , clinics (primary care), , diagnostic and rehabilitation ctnrres (secondary care), and upgrade the current hospital infrastructure (tertiary care). local health committees survey the community to identify needs and organize a variety of lobby groups to improve dit material conditions of the community. last year, barrio adentro conducted . times the medical visits conducted by the ministry of health. the philosophy of care follows an integrated approach where btalrh is related to housing, education, employment, sports, environment, and food security. conclusions: barrio adentro is a unique collaboration between low-middle income countries to provide health care to people who have been traditionally excluded from social programs. this program shows that it is possible to develop an effective international collaboration based on participatory democracy. low-income americans are at the greatest risk of being uninsured and often face multiple health concerns. this evaluation of the neighborhood health initiative (nh!), an organization which uses multiple programmatic approaches to meet the multiple health needs of clients, reflected the program's many activities and the clients' many service needs. nh! serves low-income, underserved, and hard-to-reach residents in the des moines enterprise community. multiple approaches (fourth-generation evaluation, grounded theory, strengths-and needs-based) and methods (staff and client interviews, concept mapping, observations, qualitative and quantitative analysis) were used to achieve that reflection. results indicate good targeting of residents in the zip code and positive findings in the way of health insurance coverage and reported unmet health needs of clients. program activities were found to match client nttds, validating the organization\'s assessment of clients. important components of nhi were the staff composition and that the organization had become part of both the formal and informal networks. nhi positioned as a link between the target population and local health and social sc:rvice agencies, working to connect residents with services and information as well as aid local agencies in reaching this underserved population. p - (c) welfare: definition by new york city maribeth gregory for an individual who resides in new york city, to obtain health insurance under the medicaid policy one must fall under certain criteria .. (new york city's welfare programs ) if the individual _is on ssi or earns equal to or less than $ per month, he is entitled to receive no more than $ , m resources. a family the size of two would need to earn less than $ per month to qualify for no greater than ss, worth of medicaid benefits. a family of three would qualify for $ , is they earned less than $ per month and so on. introduction: the vancouver gay communiry has a significant number of asian descendan!l. because of their double minority status of being gay and asian, many asian men who have sex with men (msm) are struggling with unique issues. dealing with racism in both mainstream society and the gay communiry, cultural differences, traditional family relations, and language challenges can be some of their everyday srruggles. however, culturally, sexually, and linguistically specific services for asian msm are very limited. a lack of availability and accessibiliry of culturally appropriate sexual health services isolates asian msm from mainstream society, the gay community, and their own cultural communities, deprives them of self-esteem, and endangers their sexual well-being. this research focuses on the qualita· tive narrative voices of asian msm who express their issues related to their sexualiry and the challenges of asking for help. by listening to their voices, practitioners can get ideas of what we are missing and how we need to intervene in order to reach asian msm and ensure their sexual health. methods: since many asian msm are very discreet, it is crucial to build up trust relationships between the researcher and asian msm in order to collect qualitative data. for this reason, a community based participatory research model was adopted by forming a six week discussion group for asian msm. in each group session, the researcher tape recorded the discussion, observed interactions among the participants, and analyzed the data by focusing on participants' personal thoughts, experiences, and emotions for given discussion topics. ra lts: many asian msm share challenges such as coping with a language barrier, cultural differ· ences for interpreting issues and problems, and westerncentrism when they approach existing sexual health services. moreover, because of their fear of being disclosed in their small ethnic communities, a lot of asian msm feel insecure about seeking sexual health services when their issues are related to their sexual orientation. conclflsion: sexual health services should contain multilingual and multicultural capacities to meet minority clients' needs. for asian msm, outreach may be a more effective way to provide them with accessible sexual health services since many asian msm are closeted and are therefore reluctant to approach the services. building a communiry for asian msm is also a significant step toward including them in healthcare services. a communiry-based panicipatory approach can help to build a community for asian msm since it creates a rrust relationship between a worker and clients. p - (c) identifying key techniques to sustain interpretation services for assisting newcomers isolated by linguistic and cultural barriers from accessing health services s. gopi krishna lntrodaetion: the greater toronto area (gta) is home to many newcomer immigrants and other vulnerable groups who can't access health resources due to linguistic, cultural and systemic barriers. linguistic and cultural issues are of special concern to suburbs like scarborough, which is home to thousands of newcomer immigrants and refugees lacking fluency in english. multilingual community ~nterpreter. service~ (mcis) is a non-profit social service organization mandated to provide high quality mterpretanon services. to help newcomers access health services, mcis partnered with the scarborough network of immigrant serving organizations (sniso) to recruit and train volunteer interpreters to accompany clienrs lacking fluency in english and interpret for them to access health services at various locati?ns, incl~~ing communiry ~c:-lth centres/social service agencies and hospitals. the model envisioned agencies recruin~ and mcis ~.mm.g and creating an online database of pooled interpreter resources. this da.tabase, acces& bl~ to all pama~~g ?rganization is to be maintained through administrative/member · ship fees to. be ~ d by each parnapanng organization. this paper analyzes the results of the project, defines and identifies suc:cases before providing a detailed analysis for the reasons for the success . . methods:. this ~per~ q~ntitative (i.e. client numben) and qualitative analysis (i.e. results of key •~ormant m~rv ews with semce ~sers and interpreters) to analyze the project development, training and mplementanon phases of the project. it then identifies the successes and failures through the afore· mentioned analysis. poster sessions vss resljts: the results of the analysis can be summarized as: • the program saw modest success both ia l?lllls of numbers of clients served as well as sustainability at various locations, except in the hospital iririog. o the success of the program rests strongly on the commitment of not just the volunteer interprmr, but on service users acknowledgments through providing transponation allowance, small honororia, letter of reference etc. • the hospital sustained the program better at the hospital due to the iolume and nature of the need, as well as innate capacity for managing and acknowledging volunceers. collc/llsion: it is possible to facilitate and sustain vulnerable newcomer immigrants access to health !ul'ices through the training and commitment of an interpreter volunteer core. acknowledging volunteer commitment is key to the sustenance of the project. this finding is important to immigration and health policy given the significant numbers of newcomer immigrants arriving in canada's urban communities. nity program was established in to provide support to people dying at home, especially those who were waiting for admission to the resi<lential hospice. with the advent of haart and corresponding challenges for people living with hiv/aids in the community, the community program has developed a unique case management approach. this model features an interdisciplinary team providing a clientdrivcn service. the case manager provides continuity of care to clients in a variety of settings both within and outside of the health care system. a case study is used to demonstrate how formal and informal networks of support facilitate efficient and appropriate resource utilization to promote client health. this case study demonstrates the value of consistent coordination with a client who has complex physical, spiritual, substance use and mental health needs. the authors will show how they liaised with the housing authority, police, social services as well as the family physician, clinic staff and a hospital emergtncy room to ensure this client did not fall between the cracks. tarek hussain recognition of the importance of community involvement and sectoral cooperation in health and !ocial services, formalized in alma-ata declaration in , was reinforced at riga meeting held at the mid·point between alma-ata and the year . then at the th anniversary of alma-ata declaration, ir was declared that 'primary health care is everybody's business'. health does not exist in isolation. health cannot be defined only as an outcome of 'medical care' but also as one of 'social action'; the responsibility of disease prevention and health promotion rests not only on governments, but also with don·govemmentorgan izations, the community, and individuals. the major accomplishment to date may be that rhere is growing realization in the health sector that community involvement is more than a political right-it is absolute necessary. lessons have learned during the implementation of disease-specific l'cltical programmes, such as cdd, ari, and epi; first, integrated and holistic approaches to childcare art needed, and second, the need for community involvement has become evident. imc! is a broad inte-gra ed strategy with an overall objective contributing to reducing child morbidity and mortality in developing countries. and one of the imci components, 'community !mci', which is now broadening ~s an entry point for child-focused community development initiatives. community jmcl/c~ ld health is ~n mtcgrated approach to the promotion of key family and community practices that have impact on: child growth and development, disease prevention, home care for sick, malnourished child~en, a.nd care-seekmg behavior and compliance with advice and treatment. the presentation addres~es, m brief, the ~evel opinent of community !mci, operational aspect of community/im cl, key strategies and tools ava la~le for implementation of c/imci. the paper draws some successful programme examples on working logether for imc! with the community in various countries which had substantial benefns on programme outcomes. p - t (c) healthy child screening: an innovative service initiative ann-marie marcolin and joyce allen . introduction: with mounting attention for creative and integrated models of c~re .that are populallon and community focused, the healthy child screening initiative achiev.es t~ese pnncipl~s and more! . • parkdale high park rotary (sponsorship) the heal~h ~h ld scree? ?g is a ~opl~-centred model of care. agency and professional sector-specific silos are ehmmated, ~rov dmg f~~ hes wit~ one-sto~ access to primary prevention services in a local school gym! children at nsk are rece vmg early mtervent n and appropriate referrals to the right care provider, at the right time a~d in t~e right place. further, with a complimentary focus on prevention, the program serves to keep at nsk children healthy. healthy child screening results: the service model is developed around four distinct phases: ) planned outreach; ) coordinated intake and registration; ) interdisciplinary screening; and ) targeted referral. since the fall and through six collaborative healthy child screenings children ranging in age from to have benefited from this unique service model of care. conclusion: the presentation will provide practical information on the development and implementation of the model. there will also be a focus on lessons learned in building community service provider-hospital relationships. according to statistics canada ( ) % of the toronto population was born outside of canada and % were new immigrants. immigrants often arrive in canada in superior health compared to the canadian born population, but they lose this health advantage over time. changes in immigrant health status over time may be attributed to a variety of factors including barriers to accessing and receiving care as well as limitations in the mainstream health service organizations and their approaches to health care delivery (hyman, ) . for example, immigrant women are less likely to receive pap tests, which places them at a greater risk for developing cervical cancer. similarly, immigrant women receive less mammography screening than their canadian counterparts. the immigrant women's health centre mobile health unit (mhu) was created as an alternative care delivery model to address the need for increased accessibility as well as culturally and linguistically appropriate reproductive health care. toronto western hospital and the immigrant women's health centre have formed a collaborative partnership which incorporates a primary care nurse practitioner and lay ethnocultural counselors providing care on the mhu. currently, a qualitative ethnonursing study is underway to understand the unique experiences of women using the mhu for their reproductive health care as well as the perspectives of the mhu health care providers. based on a literature review and preliminary findings from provider and participant interviews, the proposed presentation will address the themes of health status for immigrant and refugee women, accessibility issues as well strategies to improve their reproductive health care. we will discuss benefits as well as limitations to health care provision using this alternative service delivery model. michael carden, brian edlin, andrew talal, elizabeth getter, and marla shu lntrod.ction: to date most active drug users have not had access to treatment for hepatitis c virus (hcv) infection and substantial barriers continue to exist that interfere with treatment availability for this population. methods: active illicit drug users interested in pursuing hcv care and treatment were recruited in partnership w~th co~munity-based organizations (cbo's) in new york city. baseline data were collected on quahty of hfe, substance use patterns, depression, health care utilization, hcv health beliefs and other relev.ant variables. medical evaluations were conducted, including liver biopsy when indicated, an~ treatment ts ~ffered when no contraindications are present. participants also receive psychiatric evaluahons to determme the appropriateness of interferon treatment. renlts: fifteen individuals have been recruited to date and received an initial medical evaluation. the mean age was years and _the average age of initial heroin or cocaine use was . . eight ( %) ha~ been homeless at least once m the last months and s ( %) were homeless for most or all of this penod. fourteen ( %) had a history of injection drug use with the mean age at first injection being s frsewons v ,an. elrven persc.!ns ( %) reported inje~~ng .heroin or cocaine within the last days while the .wing four ( l'o) reported regular non-m ect on use of cocaine and street-obtained benzodiazepines. sijiy seven percent of the sample (n= ) reported ever being referred to hcv treatment while ( t % ) ftponeddiattreann~nt had been offered by~ ~e.dical provider. none had ever received a liver biopsy or araanent. most believed that hcv was a s gmf cant threat to their health ( %) and that there was a pm)dchance they would eventually die if their hepatitis c was not treated ( %). though individuals % believed that there was no cure for hcv, ( %) reported that they would initiate treatment if i was recommended by their doctor. thirteen ( %) had a current psychiatric diagnosis including «pmsion, anxiety disorder, schizophrenia, schizoaffective disorder, and borderline and antisocial persooaliry disorders. among eight individuals who had the beck depression inventory administered, the mean score was . . attendance rates were % ( / ) at scheduled medical and psychiatric ppointments and % ( / ) at liver biopsy. <andtuions: active drug users, despite experiencing multiple psycho-social problems, can be mgaged in hcv care using a multidisciplinary approach, but require intensive follow-up support. hcv aunnent of active drug users should not be dismissed. n- (c) antiretroviral therapy in mv infected infants: when to initiate therapy an african experience kingsley okonkwo, ademola adeyemo, israel sokeye, and nwaife umeike /""°""ction: as technology for early diagnosis of human immunodeficiency virus [hiv] improves, m to commence highly active antiretroviral therapy [haarn is yet to be fully evaluated. this prospective study describes our initial experience with early haart in hiv infected nigerian infants and rqions the outcome. we also analyzed the socioeconomic implications of early haart therapy in infants in the african setting. method: hiv infected infants were started on haart before months and followed up at mkly intervals.we monitored baseline and monthly cd . conclusion: initial results suggest early haart before months resulted. in improv~d out~ome in african children. treatment appears to be as effective as in developed counmes. in. a~nca as. m most developing countries with limited resources it is possible and effective to use haart m ~~ants if p~oper llloniroring facilities are available. however the occurrence of long-term drug related tox c ty and mk of emergence of resistant viral strains create need for further evaluation of haart in infants. background: most risk factors for cardiovascular disease (cvd) can be mitigated through ~th clinical interventions and lifestyle change. we used data from a telephone survey of new york city (nyc) adults to characterize health care access and healthy behavior among those with three or more cvd risk factors. methods: the study population included respondents to the nyc community health survey (n= , ) self-reporting~ of the following: smoking, diabetes, high cholesterol level, high blood pres· sure, body mass index > , and age > (males) or > (females) (n= , ). results: based on self-report, an estimated . , ( %) of nyc adults have~ or more cvd risk factors. this population is % male, % white, % black, and % with s years of education. most report good access to health care, indicated by having health insurance ( %), regular doctor ( %), their blood pressure checked within last months ( %), and their choles· terol checked within the past year ( % ). only % reported getting at least minutes of exercise ~ times per week and only % eating ~ servings of fruits and vegetables the previous day. among current smokers, % attempted to quit in past months, but only % used medication or counseling. implications: these data suggest that most nyc adults known to be at high risk for cvd have access to regular health care, but most do not engage in healthy lifestyle or, if they smoke, attempt effective quit strategies. more clinic-based and population-level interventions are needed to support lifestyle change among those at high risk of cvd. introduction: recently, much interest has been directed at "obesogenic" (obesity-promoting) (swinburn, egger & raza, ) built environments, and at geographic information systems (gis) as a tool for their exploration. a major geographical concept is accessibility, or the ease of moving from an origin to a destination point, which has been recently explored in several health promotion-related stud· ies. there are several methods of calculating accessibility to an urban feature, each with its own strengths, drawbacks and level of precision that can be applied to various health promotion research issues. the purpose of this paper is to describe, compare and contrast four common methods of calculating accessibility to urban amenities in terms of their utility to obesity-related health promotion research. practical and conceptual issues surrounding these methods are introduced and discussed with the intent of providing health promotion researchers with information useful for selecting the most appropria e accessibility method for their research goal~ ~ethod: this paper describes methodological insights from two studies, both of which assessed the neighbourhood-level accessibility of fast-food establishments in edmonton, canada -one which used a relatively simple coverage method and one which used a more complex minimum cos method. res.its: both methods of calculating accessibility revealed similar patterns of high and low access to fast-food outlets. however, a major drawback of both methods is that they assume the characteristics of the a~e~ities and of the populations using them are all the same, and are static. the gravity potential method is introduced as an alternative, since it is ·capable of factoring in measures of quality and choice. a n~mber of conceptual and pr~ctical iss~es, illustrated by the example of situational influences on food choice, make the use of the gravity potential model unwieldy for health promotion research into sociallydetermined conditions such as obesity. co.nclusions: i~ ~ommended that geographical approaches be used in partnership with, or as a foun~ation for, ~admonal exploratory methodologies such as group interviews or other forms of commumty consultation that are more inclusive and representative of the populations of interest. qilhl in los angeles county ,,..ia shaheen, richard casey, fernando cardenas, holman arthurs, and richard baker ~the retinomax autorefractor has been used for vision screening of preschool age childien. ir bas been suggested to be used and test school age children but not been validated in this age poup. ob;taiw: to compare the results of retinomax autorefractor with findings from a comprehensive i!' examination using wet retinoscopy for refractive error. mllhods: children - years old recruited from elementary schools at los angeles county were iaml with snellen's chart and the retinomax autorefractor and bad comprehensive eye examination with dilation. the proportion of children with abnormal eye examination as well as diesensitiviry and specificity of the screening tools using retinomax autorefractor alone and in combinalion wirh snellen's chart. results of the children enrolled in the study (average age= . ± . years; age range, - years), ?% had abnormal eye examination using retinoscopy with dilation. for the lerinomax, the sensitivity was % ( % confidence interval [ci] %- %), and the specificity was % ( % ci, %- o/o). simultaneous testing using snellen's chart and retinomax resulted in gain in sitiviry ( %, % cl= , ), and loss in specificity ( %, % cl= %- %). the study showed that screening school age children with retinomax autorefractor could identify most cases with abnormal vision but would be associated with many false-positive results. simuhaneous resting using snellen's chart and retinomax maximize the case finding but with very low specificiry. mdhotjs: a language-stratified, random sample of members of the college of family physicians of canada received a confidential survey. the questionnaire collected data on socio-demographic characteristics, medical training, practice type, setting and hcv-related care practices. the self-adminisratd questionnaire was also made available to participants for completion on the internet. batdti: response proportion was %. median age was years ( % female) and the proporlionoffrench questionnaires was %. approximately % had completed family medicine residency lllining in canada; median year of training completion was . sixty-seven percent, % and % work in private offices/clinics, community hospitals and emergency departments, respectively. regarding ~practices, % had ever requested a hcv test and % of physicians had screened for hcv iafrction in rhe past months· median number of tests was . while % reported having no hcv-uaed patients in their practic~, % had - hcv-infected patients. regarding the level of hcv care provided, . % provide ongoing advanced hcv care including treatment and dose monitoring for ctmduions: in this sample of canadian family physicians, most had pro~ided hcv screening. to •least one patient in the past year. less than half had - hcv-infected patients and % provide ~:relared care the role of socio-demographic factors, medical training as wel_i as hcv ca~e percep-lldas rhe provision of appropriate hcv screening will be examined and described at the time of the canference. ' - (c) healthcare services: the context of nepal meen poudyal chhetri """ tl.ction healthcare service is related with the human rights and fundamental righ~ of the ci~ ciaaiuntry. however, the growing demand foi health care services, quality heal~care service, accessib b~ id die mass population and paucity of funds are the different but interrelated issues to .be ~ddressed. m nepat. n view of this context, public health sector in nepal is among other sectors, which is struggling -.i for scarce resources. . . . nepal, the problems in the field of healthcare servic~s do not bnut ~o the. paucity of faads and resources only, but there are other problems like: rural -urban imbalance, regional unbalance, poster sessio~ f the ll ·m ·ted resources poor healthcare services, inequity and inaccessibility of the poor management o , . poor people of the rural, remote and hilly areas for the healthcare services and so on.. . . . · . i f ct the best resource allocation is the one that max m zes t e sum o m ivi ua s u · ea t services. n a , · h d' ·b · · · h . ·t effi.ciency and efficient management are correlated. it might be t e re istn utmn of mes. ence, equi y, . . . . . income or redistribution of services. moreover, maximizanon of available resources, qua tty healthcare services and efficient management of them are the very important and necessary tools and techmques to meet the growing demand and quality healthcare services in nepal. p - (a) an jn-depth analysis of medical detox clients to assist in evidence based decision making xin li, huiying sun, ajay puri, david marsh, and aslam anis introduction: problematic substance use represents an ever-increasing public health challenge. in the vancouver coastal health (vch) region, there are more than , individuals having some probability of drug or alcohol dependence. to accommodate this potential demand for addiction related services, vch provides various services and treatment, including four levels of withdrawal management services (wms). clients seeking wms are screened and referred to appropriate services through a central telephone intake service (access i). the present study seeks to rigorously evaluate one of the services, vancouver detox, a medically monitored -bed residential detox facility, and its clients. doing so will allow decision makers to utilize evidence based decision-making in order to improve the accessibility and efficiency of wms, and therefore, the health of these clients. methods: we extract one-year data (october , -september , from an efficient and comprehensive database. the occupancy rate of the detox centre along with the clients' wait time for service and length of stay (los) are calculated. in addition, the effect of seasonality on these variables and the impact of the once per month welfare check issuance on the occupancy rate are also evaluated. results: among the clients (median age , % male) who were referred by access! to vancouver detox over the one-year period, were admitted. the majority ( %) of those who are not admitted are either lost to follow up (i.e., clients not having a fixed address or telephone) or declined service at time of callback. the median wait time was day [q -ql: - ], the median los was days iq -qt: - ], and the average bed occupancy rate was %. however, during the threeday welfare check issue period the occupancy rate was lower compared to the other days of the year % vs. %, p conclusion: our analysis indicates that there was a relatively short wait time at vancouver detox, however % of the potential clients were not served. in addition, the occupancy rate declined during the welfare check issuance period and during the summer. this suggests that accessibility and efficiency at vancouver detox could be improved by specifically addressing these factors. background: intimate partner violence (ipv) is associated with acute and chronic physical and men· tal health outcomes for women resulting in greater use of health services. yet, a vast literature attests to cultural variations in perceptions of health and help-seeking behaviour. fewer studies have examined differences in perceptions of ipv among women from ethnocultural communities. the recognition, definition, and understanding of ipv, as well as the language used to describe these experiences, may be different in these communities. as such, a woman's response, including whether or not to disclose or seek help, may vary according to her understanding of the problem. methods: this pilot study explores the influence of cultural factors on perceptions of and responses to ipv among canadian born and immigrant young women. in-depth focus group interviews were con· ducted with women, aged to years, living in toronto. open-ended and semi-structured interview questions were designed to elicit information regarding how young women socially construct jpv and where they would go to receive help. interviews were transcribed, then read and independently coded by the research team. codes were compared and disagreements resolved. qualitative software qsr n was used to assist with data management. . ruu~ts_: res~nses_abo~t what constitutes ipv were similar across the study groups. when considering specific ab.us ve ~ tuanons and types of relationships, participants held fairly relativistic views about ipv, especially with regard to help-seeking behaviour. cultural differences in beliefs about normaive m;ile/femal~ relations. familial.roles, and customs governing acceptable behaviours influenced partictpants perceptions about what n ght be helpful to abused women. interview data highlight the social l ter srnfons v suucrural _impact these factors ha:e on you?g women and provide details regarding the dynamics of cibnocultur~ m~uences on help-~eekmg behav ur: t~e ro~e of such factors such as gender inequality within rtlaoo?sh ps and t_he ~erce ved degree of ~oc al solat on and support nerworks are highlighted. collc~ the~ findmgs unde~score the _ mporta_nc_e of understanding cultural variations in percrprions of ipv ~ relanon to ~elp-seekmg beha~ ':'ur. th s_mformation is critical for health professionals iodiey may connnue developmg culturally sensmve practices, including screening guidelines and protorol s. ln addition, _this study demonstr~tes that focus group interviews are valuable for engaging young romen in discussions about ipv, helpmg them to 'name' their experiences, and consider sources of help when warranted. p -s (a) health problems and health care use of young drug users in amsterdam .wieke krol, evelien van geffen, angela buchholz, esther welp, erik van ameijden, and maria prins / trod ction: recent advances in health care and drug treatment have improved the health of populations with special social and health care needs, such as drug users. however, still a substantial number dots not have access to the type of services required to improve their health status. in the netherlands, tspccially young adult drug users (yad) whose primary drug is cocaine might have limited access to drugrreatment services. in this study we examined the history and current use of (drug associated) treatmmt services, the determinants for loss of contact, and the current health care needs in the young drug mm amsterdam study (yodam). methods: yodam started in and is embedded in the amsterdam cohort study among drug mm. data were derived from y ad aged < years who had used cocaine, heroin, ampheramines and i or methadone at least days a week during the months prior to enrolment. res lts:of yao, median age was years (range: - years), % was male and % had dutch nationality at enrolment. nearly all participants ( %) reported a history of contact with drug llt.lnnent services (methadone maintenance, rehabilitation clinics and judicial treatment), mental health car? (ambulant mental care and psychiatric hospital) or general treatment services (day-care, night-care, hdp for living arrangements, work and finance). however, only % reported contact in the past six l!xlllths. this figure was similar in the first and second follow-up visit. among y ad who reported no current contact with the health care system, % would like to have contact with general treatment serl' icts. among participants who have never had contact with drug treatment services, % used primarily cocaine compared with % and % among those who reported past or current contact, respectively. saied on the addiction severity index, % reported at least one mental health problem in the past days, but only % had current contact with mental health services. concl sion: results from this study among young adult drug users show that despite a high contact rm with health care providers, the health care system seems to lose contact with yao. since % indicatt the need of general treatment services, especially for arranging house and living conditions, health m services that effectively integrate general health care with drug treatment services and mental health care might be more successful to keep contact with young cocaine users. mtthods: respondents included adults aged and over who met dsm-iv diagn?snc criteria for an anxiety or depressive disorder in the past months. we performed two sets of logisnc regressmns. thtdichotomous dependent variables for each of the regressions indicated whether rhe respondenr_vis-ud a psychiatrist, psychologist, family physician or social worker in the _past_ months. no relationship for income. there was no significant interaction between educatmn an mco~:· r: ::or respondents with at least a high school education to seek help ~rom any of the four servic p were almost twice that for respondents who had not completed high school. th . d ec of analyses found che associacion becween educacion and use of md-provided care e secon s · · be d · · ·f· ly ·n che low income group for non-md care, the assoc anon cween e ucatlon and was s gm icant on -· . . . . use of social workers was significant in both income groups, but significant only for use of psychologists in che high-income group. . . . conclusion: we found differences in healch service use by education level. ind v duals who have nor compleced high school appeared co use less mental he~lt~ servi~es provided ~y psyc~iatrists, psycholo· gists, family physicians and social workers. we found limited e.v dence _suggesting the influence of educa· tion on service use varies according to income and type of service provider. results suggesc there may be a need to develop and evaluate progr~ms.designe~ to deliver targeted services to consumers who have noc completed high school. further quahtanve studies about the expen· ence of individuals with low education are needed to clarify whether education's relationship with ser· vice use is provider or consumer driven, and to disentangle the interrelated influences of income and education. system for homeless, hiv-infected patients in nyc? nancy sahler, chinazo cunningham, and kathryn anastos introduction: racial/ethnic disparities in access to health care have been consistently documented. one potential reason for disparities is that the cultural distance between minority patients and their providers discourage chese patients from seeking and continuing care. many institucions have incorporated cultural compecency craining and culturally sensicive models of health care delivery, hoping co encourage better relacionships becween patients and providers, more posicive views about the health care system, and, ulcimacely, improved health outcomes for minority patients. the current scudy tests whether cultural distance between physicians and patients, measured by racial discordance, predicts poorer patient attitudes about their providers and the health care system in a severely disadvantaged hiv-infected population in new york city that typically reports inconsistent patterns of health care. methods: we collected data from unscably housed black and latino/a people with hiv who reported having a regular health care provider. we asked them to report on their attitudes about their provider and the health care system using validated instruments. subjects were categorized as being racially "concordant" or "discordant" with their providers, and attitudes of these two groups were compared. results: the sample consisted of ( %) black and ( %) latino/a people, who reported having ( %) black physicians, ( %) latino/a physicians, ( %) white physicians, and ( %) physicians of another/unknown race/ethnicity. overall, ( %) subjects had physicians of a different race/ethnicity than their own. racial discordance did not predict negative attitudes about rela· tionship with providers: the mean rating of a i-item trust in provider scale (lo=high and o=low) was . for both concordant and discordant groups, and the mean score in -icem relationship with provider scale ( =high and !=low) was . for both groups. however discordance was significantly associated with distrust in che health care syscem: che mean score on a -icem scale ( =high discrust and l=low distrust) was . for discordant group and . for che concordant group (t= . , p= . ). we further explored these patterns separacely in black and lacino/a subgroups, and using different strategies ro conceptualize racial/ethic discordance. conclusions: in this sample of unscably housed black and latino/a people who receive hiv care in new york city, having a physician from the same racial/ethnic background may be less important for developing a positive doctor-patient relationship than for helping the patients to dispel fear and distrust about the health care system as a whole. we discuss the policy implications of these findings. ilene hyman and samuel noh . .abstract objectiw: this study examines patterns of mental healthcare utilization among ethiopian mm grants living in toronto. methods: a probability sample of ethiopian adults ( years and older) completed structured face-to-face interviews. variables ... define, especially who are non-health care providers. plan of analysis. results: approximately % of respondents received memal health services from mainstream healthcare providers and % consulted non-healthcare professionals. of those who sought mental health services from mainstream healthcare providers, . % saw family physicians, . % visited a psychiatrist. and . % consulted other healthcare providers. compared with males, a significantly higher proportion gsfer sessions v ri ftlnales consulted non-healthcare_ professionals for emotional or mental health problems (p< . ). tlbile ethiopian's overall use of mamstream healthcare services for emotional problems ( %) did not prlydiffer from the rate ( %) of the general population of ontario, only a small proportion ( . %) rjerhiopians with mental health needs used services from mainstream healthcare providers. of these, !oj% received family physicians' services, . % visited a psychiatrist, and . % consulted other healthll/c providers. our data also suggested that ethiopian immigrants were more likely to consult tradioooal healers than health professionals for emotional or mental health problems ( . % vs. . % ). our bivariate analyses found the number of somatic symptoms and stressful life events to be associated with an increased use of medical services and the presence of a mental disorder to be associated with a dfcreased use of medical services for emotional problems. however, using multivariate methods, only die number of somatic symptoms remained significantly associated with use of medical services for emooonal problems. diu#ssion: study findings suggest that there is a need for ethnic-specific and culturally-appropriate mrcrvention programs to help ethiopian immigrants and refugees with mental health needs. since there ~a strong association between somatic symptoms and the use family physicians' services, there appears robe a critical role for community-based family physicians to detect potential mental health problems among their ethiopian patients, and to provide appropriate treatment and/or referral. the authors acknowledge the centre of excellence for research in immigration and settlement (ceris) in toronto and canadian heritage who provided funding for the study. we also acknowledge linn clark whose editorial work has improved significantly the quality of this manuscript. we want to thank all the participants of the study, and the ethiopian community leaders without whose honest contributions the present study would have not been possible. this paper addresses the impact of the rationalization of health-care services on the clinical decision-making of emergency physicians in two urban hospital emergency departments in atlantic canada. using the combined strategies of observational analysis and in-depth interviewing, this study provides a qualitative understanding of how physicians and, by extension, patients are impacred by the increasing ancmpts to make health-care both more efficient and cost-effective. such attempts have resulted in significantly compromised access to primary care within the community. as a consequence, patients are, out of necessity, inappropriately relying upon emergency departments for primary care services as well as access to specialty services. within the hospital, rationalization has resulted in bed closures and severely rmricted access to in-patient services. emergency physicians and their patients are in a tenuous position having many needs but few resources. furthermore, in response to demands for greater accountability, physicians have also adopted rationality in the form of evidence-based medicine. ultimately, ho~ever, rationality whether imposed upon, or adopted by, the profession significantly undermines physu.: ans' ability to make decisions in the best interests of their patients. johnjasek, gretchen van wye, and bonnie kerker introduction: hispanics comprise an increasing proportion of th.e new york city (nyc) populanon !currently about %). like males in the general population, h spamc males (hm) have a lower prrval,nce of healthcare utilization than females. however, they face additional access barriers such as bnguage differences and high rates of uninsurance. they also bear a heavy burden of health problems lllehasobesity and hiv/aids. this paper examines patterns of healthcare access and ut hzat on by hm compared to other nyc adults and identifies key areas for intervention. . . . and older are significantly lower than the nhm popu anon . v. . , p<. ), though hi\' screening and immunizations are comparable between the two groups. conclusion: findings suggest that hm have less access t? healthcare than hf or nhm. hown r, hm ble to obtain certain discrete medical services as easily as other groups, perhapsdueto!rtor are a hm. i i . subsidized programs. for other services, utilization among s ower. mprovmg acc~tocareinthis group will help ensure routine, quality care, which can lead to a greater use of prevennve services iii! thus bener health outcomes. introduction: cancer registry is considered as one of the most important issues in cancer epidemiology and prevention. bias or under-reporting of cancer cases can affect the accuracy of the results of epidemiological studies and control programs. the aim of this study was to assess the reliability of the regional cancer report in a relatively small province (yasuj) with almost all facilities needed for c llcll diagnosis and treatment. methods: finding the total number of cancer cases we reviewed records of all patients diagnoicd with cancer (icd - ) and registered in any hospital or pathology centre from until i n yasuj and all ( ) surrounding provinces. results: of patients who were originally residents of yasui province, . % wereaccoulll!d for yasuj province. the proportion varies according to the type of cancer, for exarnplecancetsofdiglstive system, skin and breast were more frequently reported by yasuj's health facilities whereas cancmoi blood, brain and bone were mostly reported by neighbouring provinces. the remaining cases ( . % were diagnosed, treated and recorded by neighbouring provinces as their incident cases. this is partly because of the fact that patients seek medical services from other provinces as they believed that the facil. ities are offered by more experienced and higher quality stuffs and their relative's or temporary acooiii' modation addresses were reported as their place of residence. conclusion: measuring the spatial incidence of cancer according to the location of report ortht current address affected the spatial statistics of cancer. to correct this problem recording the permanm! address of diagnosed cases is important. p - (c). providing primary healthcare to a disadvantaged population at a university-run commumty healthcare facility tracey rickards the. c:ommuni~y .h~alth ~linic (chc) is a university sponsored nurse-managed primary bealthwt (p~c:l clime. the clm c is an innovative model of healthcare delivery in canada that has integrated tht principles of phc ser · · h' . vices wit ma community development framework. it serves to provide access to phc services for members of th · · illi · dru is ii be . . e community, particularly the poor and those who use or gs, we mg a service-learning facil'ty f d · · · · · · d rionll h . . .,m.:. · t · . meet c ient nee s. chmc nursing and social work staff and srudents r·--· ipa em various phc activities and h .l.hont" less i . f . outreac services in the local shelters and on the streels to'"" popu auon o fredericton as well th chc · model iii fosterin an on oi : . • e promotes and supports a harm reduction . · local d!or an~ h ng ~art:ersh p with aids new brunswick and their needle exchange program, w tha ing condoms and :xu:t h:~~~e e~aint~nance therapy clients, and with the clie~ts themselves ~_r; benefits of receiving health f ucation, a place to shower, and a small clothing and food oai~· care rom a nurse p · · d d · --""~'i"· are evidenced in th r research that involved needsaans mvo ves clients, staff, and students. to date the chc has unacn- · sessment/enviro i . d ; •• '"""" ll eva uanon. the clinic has also e . d nmenta scan, cost-benefit analysis, an on-go...,, "".'i'~ facility and compassionate lea x~mme the model of care delivery' focusing on nursing roles wi~ cj rmng among students. finally, the clinic strives to share the resu•p v . -arch with the community in which it provides service by distributing a bi-monthly newsletter, and plllicipating in in-services and educational sessions in a variety of situations. the plan for the future is coolinued research and the use of evidence-based practice in order to guide the staff in choosing how much n~ primary healthcare services to marginalized populations will be provided. n- (c) tuming up the volume: marginalized women's health concerns tckla hendrickson and betty jane richmond bdrotbu:tion: the marginalization of urban women due to socio-economic status and other determinants negatively affects their health and that of their families. this undermines the overall vitaliry of urban communities. for example, regarding access to primary health care, women of lower economic surus and education levels are less likely to be screened for breast and cervical cancer. what is not as widely reported is how marginalized urban women in ontario understand and articulate their lack of access to health care, how they attribute this, and the solutions that they offer. this paper reports on the rnults of the ontario women's health network (owhn) focus group project highlighting urban women's concerns and suggestions regarding access to health care. it also raises larger issues about urban health, dual-purpose focus group design, community-based research and health planning processes. mdhods: focus group methodology was used to facilitate a total of discussions with urban and rural women across ontario from to . the women were invited to participate by local women's and health agencies and represented a range of ages, incomes, and access issues. discussions focussed on women's current health concerns, access to health care, and information needs. results were analyzed using grounded theory. the focus groups departed from traditional focus group research goals and had two purposes: ) data collection and dissemination (representation of women's voices), and ) fostering closer social ties between women, local agencies, and owhn. the paper provides a discussion and rationale for a dual approach. rax/ts: the results confirm current research on women's health access in women's own voices: urban women report difficulty finding responsive doctors, accessing helpful information such as visual aids in doctors' offices, and prohibitive prescription costs, in contrast with rural women's key concern of finding a family doctor. the research suggests that women's health focus groups can address access issues by helping women to network and initiate collective solutions. the study shows that marginalized urban women are articulate about their health conctrns and those of their families, often understanding them in larger socio-economic frameworks; howtver, women need greater access to primary care and women-friendly information in more languages and in places that they go for other purposes. it is crucial that urban health planning processes consult directly with women as key health care managers, and turn up the volume on marginalized women's voices. women: an evaluation of awareness, attitudes and beliefs introduction: nigeria has one of the highest rates of human immunodeficiency virus ihivi seroprrvalence in the world. as in most developing countries vertical transmission from mother to child account for most hiv infection in nigerian children. the purpose of this study was ro. determine the awareness, attitudes and beliefs of pregnant nigerian women towards voluntary counseling and testing ivct! for hiv. mnbod: a pre-tested questionnaire was used to survey a cross section '.>f. pregnant women ~t (lrlleral antenatal clinics in awka, nigeria. data was reviewed based on willingness to ~c~ept or re ect vct and the reasons for disapproval. knowledge of hiv infection, routes of hiv transm ssmn and ant rnroviral therapy iart) was evaluated. hsults: % of the women had good knowledge of hiv, i % had fair knowledge while . % had poor knowledge of hiv infection. % of the women were not aware of the association of hreast milk feeding and transmission of hiv to their babies. majority of the women % approved v~t while % disapproved vct, % of those who approved said it was because vct could ~educe risk of rransmission of hiv to their babies. all respondents, % who accepted vc.i ~ere willing to be tnted if results are kept confidential only % accepted to be tested if vc.t results w. be s~ared w .th pinner and relatives % attributed their refusal to the effect it may have on their marriage whale '-gave the social 'and cultural stigmatization associated with hiv infection for their r~fusal.s % wall accept vct if they will be tested at the same time with their partners. ~ of ~omen wall pref~r to breast feed even if they tested positive to hiv. women with a higher education diploma were times v more likely to accept vct. knowledge of art for hiv infected pregnant women as a means of pre. vention of maternal to child transmission [pmtct) was generally poor, % of respondents wm aware of art in pregnancy. conclusion: the acceptance of vct by pregnant women seems to depend on their understanding that vct has proven benefits for their unborn child. socio-cultur al factors such as stigmatizationof hiv positive individuals appears to be the maj_or impedi~ent towards widespread acceptanee of ycr in nigeria. involvemen t of male partners may mpro~e attitudes t~wa~ds vct:the developmentofm novative health education strategies is essential to provide women with mformanon as regards the benefits of vct and other means of pmtct. p - (c) ethnic health care advisors in information centers on health care and welfare in four districts of amsterdam arlette hesselink, karien stronks, and arnoud verhoeff introduction : in amsterdam, migrants report a "worse actual health and a lower use of health care services than the native dutch population. this difference might be partly caused by problems migrants have with the dutch language and health care and welfare system. to support migrants finding their way through this system, in four districts in amsterdam information centers on health care and welfare were developed in which ethnic health care advisors were employed. their main task is to provide infor· mation to individuals or groups in order to bridge the gap between migrants and health care providers. methods: the implementat ion of the centers is evaluated using a process evaluation in order to give inside in the factors hampering and promoting the implementat ion. information is gathered using reports, attending meetings of local steering groups, and by semi-structu red interviews with persons (in)directly involved in the implementat ion of the centers. in addition, all individual and groupcontaets of the health care advisors are registered extensively. results: since four information centers, employing ethnic health care advisors, are implemented. the ethnicity of the health care advisors corresponds to the main migrant groups in the different districts (e.g. moroccan, turkeys, surinamese and african). depending on the local steering groups, the focus of the activities of the health care advisors in the centers varies. in total, around individual and group educational sessions have been registered since the start. most participants were positive about the individual and group sessions. the number of clients and type of questions asked depend highly on the location of the centers (e.g. as part of a welfare centre or as part of a housing corporation). in all districts implementa tion was hampered by lack of ongoing commitment of parties involved (e.g. health care providers, migrant organization s) and lack of integration with existing health care and welfare facilities. discussion: the migrant health advisors seem to have an important role in providing information on health and welfare to migrant clients, and therefore contribute in bridging the gap between migrants and professionals in health care and welfare. however, the lack of integration of the centers with the existing health care and welfare facilities in the different districts hampers further implementation . therefore, in most districts the information centres will be closed down as independent facilicities in the near future, and efforts are made to better connect the position of migrant health advisor in existing facilities. the who report ranks the philippines as ninth among countries with a high tb prevalence. about a fourth of the country's population is infected, with majority of cases coming from the lower socioeconomic segments of the community. metro manila is not only the economic and political capital of the philippines but also the site of major universities and educational institutions. initial interviews with the school's clinicians have established the need to come up with treatment guidelines and protocols for students and personnel when tb is diagnosed. these cases are often identified during annual physical examinations as part of the school's requirements. in many instances, students and personnel diagnosed with tb are referred to private physicians where they are often lost to follow-up and may have failure of treatment due to un monitored self-administered therapy. this practice ignores the school clinic's great potential as a tb treatment partner. through its single practice network (spn) initiative, the philippine tuberculosis initiatives for the private sector (philippine tips), has established a model wherein school clinics serve as satellite treatment partners of larger clinics in the delivery of the directly observed treatment, short course (dots) protocol. this "treatment at the source" allows school-based patients to get their free government-suppl ied tb medicines from the clinic each day. it also cancels out the difficulty in accessing medicines through the old model where the patient has to go to the larger clinic outside his/her school to get treatment. the model also enables the clinic to monitor the treatment progress of the student and assumes more responsibility over their health. this experience illustrates how social justice in health could be achieved from means other than fund generation. the harnessing of existing health service providers in urban communities through standardized models of treatment delivery increases the probability of treatment success, not only for tb but for other conditions as well. p - (c) voices for vulnerable populations: communalities across cbpr using qualitative methods martha ann carey, aja lesh, jo-ellen asbury, and mickey smith introduction: providing an opportunity to include, in all stages of health studies, the perspectives and experiences of vulnerable and marginalized populations is increasingly being recognized as a necessary component in uncovering new solutions to issues in health care. qualitative methods, especially focus groups, have been used to understand the perspectives and needs of community members and clinical staff in the development of program theory, process evaluation and refinement of interventions, and for understanding and interpreting results. however, little guidance is available for the optimal use of such information. methods: this presentation will draw on diverse experiences with children and their families in an asthma program in california, a preschool latino population in southern california, a small city afterschool prevention program for children in ohio, hiv/aids military personnel across all branches of the service in the united states, and methadone clinic clients in the south bronx in new york city. focus groups were used to elicit information from community members who would not usually have input into problem definitions and solutions. using a fairly common approach, thematic analysis as adapted from grounded theory, was used to identify concerns in each study. next we looked across these studies, in a meta-synthesis approach, to examine communalities in what was learned and in how information was used in program development and refinement. results: while the purposes and populations were diverse, and the type of concerns and the reporting of results varied, the conceptual framework that guided the planning and implementation of each study was similar, which led to a similar data analysis approach. we will briefly present the results of each study, and in more depth we will describe the communalities and how they were generated. conclusions: while some useful guidance for planning future studies of community based research was gained by looking across these diverse studies, it would be useful to pursue a broader examination of the range of populations and purposes to more fully develop guidance. background: the majority of studies examining the relationship between residential environments and cardiovascular disease have used census derived measures of neighborhood ses. there is a need to identify specific features of neighborhoods relevant to cardiovascular disease risk. we aim to ) develop methods· data on neighborhood conditions were collected from a telephone survey of s, fesi· dents in balth:.ore, md; forsyth county, nc; and new york, ny. a sample of of the i.ni~~l l'elpondents was re-interviewed - weeks after the initial interview t~ measure the tes~-~etest rebab ~ ty of ~e neighborhood scales. information was collected across seven ~e ghborho~ cond ~ons (aesth~~ ~uah~, walking environment, availability of healthy foods, safety, violence, social cohesion, and acnvmes with neighbors). neighborhoods were defined as census tracts or homogen~us census tra~ clusters. ~sycho metric properties.of the neighborhood scales were accessed by ca~cu~~.ng chronba~h s alpha~ (mtemal consistency) and intraclass correlation coefficients (test-r~test reliabilmes) .. pear~n s .corre~anons were calculated to test for associations between indicators of neighborhood ses (tncludmg d mens ons of race/ ethnic composition, family structure, housing, area crowding, residential stability, education, employment, occupation, and income/wealth) and our seven neighborhood scales. . chronbach's alphas ranged from . (walking environment) to . (violence). intraclass correlations ranged from . (waling environment) to . (safety) and wer~ high~~~ . ~ for ~urout of the seven neighborhood dimensions. our neighborhood scales (excluding achv hes with neighbors) were consistently correlated with commonly used census derived indicators of neighborhood ses. the results suggest that neighborhood attributes can be reliably measured. further development of such scales will improve our understanding of neighborhood conditions and their importance to health. childhood to young adulthood in a national u.s. sample jen jen chang lntrodfldion: prior studies indicate higher risk of substance use in children of depressed mothers, but no prior studies have followed up the offspring from childhood into adulthood to obtain more precise estimates of risk. this study aimed to examine the association between early exposure to maternal depl'elsive symptoms (mds) and offspring substance use across time in childhood, adolescence, and young adulthood. methods: data were obtained from the national longitudinal survey of youth. the study sample includes , mother-child/young adult dyads interviewed biennially between and with children aged to years old at baseline. data were gathered using a computer-assisted personal interview method. mds were measured in using the center for epidemiologic studies depression scale. offspring substance use was assessed biennially between and . logistic and passion regression models with generalized estimation equation approach was used for parameter estimates to account for possible correlations among repeated measures in a longitudinal study. rnlllta: most mothers in the study sample were whites ( %), urban residents ( %), had a mean age of years with at least a high school degree ( %). the mean child age at baseline was years old. offspring cigarette and alcohol use increased monotonically across childhood, adolescence, and young adulthood. differential risk of substance use by gender was observed. early exposure to mds was associated with increased risk of cigarette (adjusted odds ratio (aor) = . , % confidence interval ( ): . , . ) and marijuana use (aor = . , % ci: . , . ), but not with alcohol use across childhood, adolescence, and young adulthood, controlling for a child's characteristics, socioeconomic status, ~ligiosity, maternal drug use, and father's involvement. among the covariates, higher levels of father's mvolvement condluion: results from this study confirm previous suggestions that maternal depressive symptoms are associated with adverse child development. findings from the present study on early life experi-e~ce have the potential to inform valuable prevention programs for problem substance use before disturbances become severe and therefore, typically, much more difficult to ameliorate effectively. the ~act (~r-city men~ health study predicting filv/aids, club and other drug transi-b~) study a multi-level study aimed at determining the association between features of the urban enyjronment mental health, drug use, and risky sexual behaviors. the study is randomly sampling foster sessions v neighborhood residents and assessing the relations between characteristics of ethnographically defined urban neighborhoods and the health outcomes of interest. a limitation of existing systematic methods for evaluating the physical and social environments of urban neighborhoods is that they are expensive and time-consuming, therefore limiting the number of times such assessments can be conducted. this is particularly problematic for multi-year studies, where neighborhoods may change as a result of seasonality, gentrification, municipal projects, immigration and the like. therefore, we developed a simpler neighborhood assessment scale that systematically assessed the physical and social environment of urban neighborhoods. the impact neighborhood evaluation scale was developed based on existing and validated instruments, including the new york city housing and vacancy survey which is performed by the u.s. census bureau, and the nyc mayor's office of operations scorecard cleanliness program, and modified through pilot testing and cognitive testing with neighborhood residents. aspects of the physical environment assessed in the scale included physical decay, vacancy and construction, municipal investment and green space. aspects of the social environment measured include social disorder, social trust, affluence and formal and informal street economy. the scale assesses features of the neighborhood environment that are determined by personal (e.g., presence of dog feces), community (e.g., presence of a community garden), and municipal (e.g., street cleanliness) factors. the scale is administered systematically block-by-block in a neighborhood. trained research staff start at the northeast corner of an intersection and walk around the blocks in a clockwise direction. staff complete the scale for each street of the block, only evaluating the right side of the street. thus for each block, three or more assessments are completed. we are in the process of assessing psychometric properties of the instrument, including inter-rater reliability and internal consistency, and determining the minimum number of blocks or street segments that need to be assessed in order to provide an accurate estimate of the neighborhood environment. these data will be presented at the conference. obj«tive: to describe and analyze the perceptions of longterm injection drug users (idus) about their initiation into injecting. toronto. purposive sampling was used to seek out an ethnoculturally diverse sample of idus of both genders and from all areas of the city, through recruitment from harm reduction services and from referral by other study participants. interviews asked about drug use history including first use and first injecting, as well as questions about health issues, service utilization and needs. thematic analysis was used to examine initiation of drug use and of injection. results: two conditions appeared necessary for initiation of injection. one was a developed conception of drugs and their (desirable) effects, as suggested by the work of becker for marijuana. thus virtually all panicipants had used drugs by other routes prior to injecting, and had developed expectations about effects they considered pleasureable or beneficial. the second condition was a group and social context in which such use arose. no participants perceived their initiation to injecting as involving peer pressure. rather they suggested that they sought out peers with a similar social situation and interest in using drugs. observing injection by others often served as a means to initiate injection. injection served symbolic purposes for some participants, enhancing their status in their group and marking a transition to a different social world. concl ion: better understanding of social and contextual factors motivating drug users who initiate injection can assist in prevention efforts. ma!onty of them had higher educational level ( %-highschool or higher).about . yo adffiltted to have history of alcohol & another . % had history of smoking. only . % people were on hrt & . % were receiving steroid. majority of them ( . ) did not have history of osteoporosis. . % have difficulty in ambulating. only . % had family history of osteoporosis. bmd measurements as me~sured by dual xray absorptiometry (dexa) were used for the analysis. bmd results were compare~ w ~ rbc folate & serum vitamin b levels. no statistical significance found between bmd & serum v taffiln b level but high levels of folate level is associated with normal bmd in bivariate and multivariate analysis. conclusion: in the studied elderly population, there was no relationship between bmd and vitamin b ; but there was a significant association between folate levels & bmd. introduction: adolescence is a critical period for identity formation. western studies have investigated the relationship of identity to adolescent well-being. special emphasis has been placed on the influence of ethnic identity on health, especially among forced migrants in different foreign countries. methodology: this study asses by the means of an open ended question identity categorization among youth in three economically disadvantaged urban communities in beirut, the capital of lebanon. these three communities have different histories of displacement and different socio-demographic makeup. however, they share a history of displacement due to war. results and conclusion: the results indicated that nationality was the major category of identification in all three communities followed by origin and religion. however, the percentages that self-identify by particular identity categories were significantly different among youth in the three communities, perhaps reflecting different context in which they have grown up. mechanical heart valve replacement amanda hu, chi-ming chow, diem dao, lee errett, and mary keith introduction: patients with mechanical heart valves must follow lifelong warfarin therapy. war· farin, however, is a difficult drug to take because it has a narrow therapeutic window with potential seri· ous side effects. successful anticoagulation therapy is dependent upon the patient's knowledge of this drug; however, little is known regarding the determinants of such knowledge. the purpose of this study was to determine the influence of socioeconomic status on patients' knowledge of warfarin therapy. methods: a telephone survey was conducted among patients to months following mechan· ical heart valve replacement. a previously validated -item questionnaire was used to measure the patient's knowledge of warfarin, its side effects, and vitamin k food sources. demographic information, socioeconomic status data, and medical education information were also collected. results: sixty-one percent of participants had scores indicative of insufficient knowledge of warfarin therapy (score :s; %). age was negatively related to warfarin knowledge scores (r= . , p = . ). in univariate analysis, patients with family incomes greater than $ , , who had greater. than a grade education and who were employed or self employed had significantly higher warfarm knowledge scores (p= . , p= . and p= . respectively). gender, ethnicity, and warfar~n therapy prior to surgery were not related to warfarin knowledge scores. furthermore, none of t~e. m-hospital tea~hing practices significantly influenced warfarin knowledge scores. however, panic ~ants who _rece v~d post discharge co~unity counseling had significantly higher knowledge scores tn comp~r son with those who did not (p= . ). multivariate regression analysis revealed that und~r~tandmg the ~oncept of ?ternational normalized ratio (inr), knowing the acronym, age and receiving ~ommum !' counseling after discharge were the strongest predictors of warfarin kn~wledge. s~ oeconom c status was not an important predictor of knowledge scores on the multivanate analysis. poster sessions v ~the majority of patients at our institution have insufficient knowledge of warfarin therapy.post-discharge counseling, not socioeconomic status, was found to be an important predictor of warfarin knowledge. since improved knowledge has been associated with improved compliance and control, our findings support the need to develop a comprehensive post-discharge education program or, at least, to ensure that patients have access to a community counselor to compliment the in-hospital educatiop program. brenda stade, tony barozzino, lorna bartholomew, and michael sgro lnttotl#ction: due to the paucity of prospective studies conducted and the inconsistency of results, the effects of prenatal cocaine exposure on functional abilities during childhood remain unclear. unlike the diagnosis of fetal alcohol spectrum disorder, a presentation of prenatal cocaine exposure and developmental and cognitive disabilities does not meet the criteria for specialized services. implications for public policy and services are substantial. objective: to describe the characteristics of children exposed to cocaine during gestation who present to an inner city specialty clinic. mnbods: prospective cohort research design. sample and setting: children ages to years old, referred to an inner city prenatal substance exposure clinic since november, . data collection: data on consecutive children seen in the clinic were collected over an month period. instrument: a thirteen ( ) page intake and diagnostic form, and a detailed physical examination were used to collect data on prenatal substance history, school history, behavioral problems, neuro-psychological profile, growth and physical health of each of the participants. data analysis: content analysis of the data obtained was conducted. results: twenty children aged to years (mean= . years) participated in the study. all participants had a significant history of cocaine exposure and none had maternal history or laboratory (urine, meconium or hair) exposure to alcohol or other substances. none met the criteria of fetal alcohol spectrum disorder. all were greater than the tenth percentile on height, weight, and head circumference, and were physically healthy. twelve of the children had iqs at the th percentile or less. for all of the children, keeping up with age appropriate peers was an ongoing challenge because of problems in attention, motivation, motor control, sensory integration and expressive language. seventy-four percent of participants had significant behavioral and/or psychological problems including aggressiveness, hyperactivity, lying, poor peer relationships, extreme anxiety, phobias, and poor self-esteem. conclusion: pilot study results demonstrated that children prenatally exposed to cocaine have significant learning, behavioural, and social problems. further research focusing on the characteristics of children prenatally exposed to cocaine has the potential for changing policy and improving services for this population. methods: trained interviewers conducted anonymous quantitative surveys with a random sample (n= ) of female detainees upon providing informed consent. the survey focused on: sociodemographic background; health status; housing and neighborhood stability and social resource availability upon release. results: participants were % african-american, % white, % mixed race and % native american. participants' median age was , the reported median income was <s , year, and % reported receiving a high school diploma or equivalent. women reported a number of health conditions rypical of underserved populations, including asthma ( %), sexually transmitted infections ( %), high blood pressure ( %), hcv ( %), diabetes ( %) and hiv ( %). nearly half ( %) did not anticipate having a place to stay for at least days upon release. factors significantly associated housing stability upon release were: high family support score (adjusted odds ratio [aor) . ; % confidence interval ( % cl) . , . ), high neighborhood stability score (aor . ; % cl . , . ), wanting a commercial sex worker (csw) support group (aor . ; % cl . , . ); and identifying as bisexual (aor . ; % cl . , . ). women desired employment ( %), housing ( %), education ( %), drug treattnent ( %), help with custody of children ( %), childcare ( %), and domestic violence suppon ( %) services. conclusions: female detainees represent one of urban centers' most marginalized pcjpwatioos. short periods of detention represent a unique opportunity for _interven?~ns bri~ co~s and public health institutions. service providers should collaborate ~ th local ~ails to .p~de a con uwnof care for female detainees upon release that includes transportation , housing, residential drug treallllcnt, employment, education, family reunification, childcare and domestic viol~nce su~rt. ~pecial attenlion is warranted to lesbian and bisexual women and csws, who may be especially margmalized &om family and service-based support networks. introduction: "health care and ethnic minority immigrants" examines how health care policy dil· ferences between canada and the united states impact the health-related hardships, access and use of preventative care, and health outcomes of urban ethnic minority immigrants in both countries. methods: the findings of this paper build on the results of my qualitative comparative study of hotel workers in vancouver, bc and seattle, wa that revealed greater health related hardships for similarly matched employees in seattle compared to vancouver. in this paper, i examine the generalizability of these findings at the cross-national level for similar ethnic minority immigrant groups. i compare the impact of health care policy differences on specific groups that have emigrated to cities in both canada and the united states. these include immigrants from china, viemam, philippines, west indies, africa, sri lanka, pakistan, and latin america. comparative statistical analysis -utilizing rel· evant data from statistics canada and the u.s. census -reveal the extent to which health policy differ· ences help explain how current health policy in the united states disadvantages urban ethnic minority immigrants. results: many ethnic minority immigrants are a part of the working poor, a group which disproportionately lacks health insurance coverage in the united states. their position in the labour market makes them most vulnerable to exclusion from health care services. the data reveals how current u.s. health policy creates barriers for recently arrived ethnic minority immigrants to access health insurance and care. it remains difficult to ascribe health outcome differences directly to policy differences because of the challenge in disentangling the complex interacting interventing variables, including income inequality, that determine health outcomes. yer suggestive evidence suggests that health policy in the united states is harming the health of urban ethnic minority immigrants. the current health policy regime in the united states harms the health access, care, and outcomes of urban ethnic minority immigrants. comparing the fortunes of similar immigrants in cana· dian and u.s. cities reveals these patterns of disadvantage and some of their consequences. these barriers are an understudied factor in the sociological literature on "segmented assimilation" and social exclu· sion. the paper ends with policy recommendatio ns to improve access to health care among ethnic minor· ity immigrants in both countries, with the goal of reducing health related hardships, and improving health outcomes. mass index deyu pan, richard baker, and keith norris badtgrou~ over the past decades, there has been dramatic increase in prevalence of obesity in the us population. however, the relationship between obesity and the prevalence of cardiovascular dis· ease (cv~) risk factors in different race/ethnic groups over time is not well known. . ik~rgn: _we use cross-sectional , nationally representative surveys: national health and nutri· t nal examination survey (nhanes iii - (n= ) and nhanes nhanes - , including white, black, and hispa~ic races who were no~preg~ant, aged to years. res lt : the preval~nce of high cholesterol level (~ mg/dl), untreated high blood pressure ( : l / mm hg) an~ diabetes were calculated according to bmi group (lean, < ; overweight, - ; and obese, : ) for different race/ethnicity groups. over the approximately years period (from l ~ ~o [ ] [ ] [ ] [ ] , reducrion in cvd risk factors in non-hispanic white has been observed. for mmonty race/~hnicity groups, black and hispanic, there had been increases in prevalence in high blood fa pressure and diabetes. the lean group experienced larger increase in prevalence of those cvd risk ctors. . fo~~ the prevalence in cvd risk factors over time had reduced in most of the bmi ca egoes r t e w ~~e population. however, for black and hispanic, prevalence of of cvd risk factors ave generauy uncreased, especially in overweight and obese groups. methods: strategies identified to achieve these goals include: developing meaningful neighbourhood and district boundaries for comparing health information, providing free access to neighbourhood health profiles (including relevant data at the smallest level for which valid rates could be calculated); mapping relationships of inequality at a variety of nested levels; providing a range of formats (maps, tables) to meet the needs of users; providing technical support; seeking user input to advance and improve the site; and conducting workshops to foster access and use of data for decision-making , advocacy and collaboration. an evaluation strategy is being developed to assess the efforts and impact of these strategies. a preliminary assessment of the results of the first six months of activity will be completed in october . results: significant differences in health are shown at all the geographic levels indicating success in developing the boundaries. results tabulated for the first six months use of the site include: site visits; media use; reference/acknow ledgement of the site in other documents; number of contacts through presentations and workshops; feedback from users identifying strengths and limitations; and, response from health decision makers. members of the partnership are identifying additional tools and services to further support actions that reduce health inequalities. the assessment will be used to develop more specific goals, targets and monitoring mechanisms. conclusions: our experience with paper-based health profiles indicates that they have been used extensively for program planning and advocacy. the greater availability of information in a publiclyavailable web-based format is expected to translate into even greater usefulness and wider application. the web site has had considerable usage to date, extensive media coverage. site users and workshop participants have provided positive feedback and suggestions for next steps. the six month review will be available in october . in india, more than and a half people are hiv-positive and half a million have aids. india currently has the largest number of positive people in the world, oumumbering south africa and accounting for nearly one tenth of the global hiv/aids prevalence. the world bank predicts that by , there will be million hiv/aids cases in india. the central challenge facing hiv prevention efforts in south asia today is learning how to respond to the societal determinants of vulnerability to hiv. hiv/aids is an issue largely engulfed by social stigma. stigma and discrimination are often considered the foremost barriers to effective poster sess ns v prevention and care initiatives. stigma can make a person afraid of disclosing their status~ ~yone el se, and may make them feel depressed or alone. stigma can .also le~d to poor adherence to medicanon, ~ likelihood to access health and social services, and less desire to hve. hiv+ men and women may beafraidtotd! their co-workers that they have hiv for fear of their reactions or for fear of losing their jobs. in a study conducted of people living with hiv/aids in delhi, india ov~r. -~, many respo~ts shared their experiences of discrimination in their families, in their communmes, an~ m health ~e settmgs. these findings will be shared in the workshop, and will be compa~ed to m~ ex~nences workmg as an ~ co~l~ at the center for aids prevention studies in san francisco, cahforma and congreso de lannos unidos m philadelphia, pennsylvania. the workshop will include an overview of hiv/aids in urban contexts in tht united states (los angeles, san francisco, new york, philadelphia) and south asia (delhi, hyderabad, chennai). differences in methods for outreach, prevention and treatment efforts between devdoped world and developing world contexts will be discussed. a large portion of the workshop will be centered on discussion. participants will engage in an exercise to categorize their ideas of stigma. the workshop will also include a powerpoint presentation on the quantitative data gathered from the survey. the number of homeless and runaway youth is increasing in toronto, which is currently home to out of youth in the gt a who live alone, and it is the preferred locale for the street-involved and homeless youth. the city's youth population is expected to grow by nearly % by , the school dropout rate is rising, and there are is a paucity of empirical data on what works with these at-risk youth. over a two-year period, ( ) ( ) ( ) , youthlink-inner city, conducted an evaluation of the impact of a harm reduction program aimed at reducing street youth's risk of contracting/infecting others with the hep c virus, which has both short-term and long-term deleterious effects. demographic data from street-youth, along with resources/services used, employment methods, type and frequency of drug use, health status, police contacts, and their views of program impact were analyzed using a standardized sur· vey. additionally, focus groups with youth, agency staff and community key informants were conducted, as well as in-depth interviews with three youth, over a sixth-month period. this paper details study results, which both inform both practice and future study about what works, why it works, and how best to work with these vulnerable youth. sho~ed .that the wasteland in this area was contaminated by as cd zn pb cu simultaneously. the con-ta~matton of as ~d was quit significant. the contamination problems and environmental issues in this region are very. serious. the awareness on environmental and health issues was investigated by spot survey and analysis. overall awareness among the residents in the region is very poor. the measures must be taken to assu~e t~e .sustainable economy development by local and central gorvernments. keywords nandan guangx ; mmmg area; environmental awareness. concern like, epileptic fits, vision problems, earache, cough of more than weeks and urinary problems. ninety-five subjects did not take bath regularly and were exposed to sex. the health problems identified are only of a particular point of time and it may be difficult to interpret the depth of "iceberg" of the street children's world also draw the health-illness continuum. periodic interaction and follow-up is very difficult as they generally disappear from the research setting due to their frequent mobility for survival measures. as per the prediction of the iceberg theory, only some problems may have been identified, larger problems in respect to their health may not have been identified. in spite of various limitations, this attempt had proven that, it is possible to enter the street children world, explore the iceberg of illnesses, and establish data of health-illness continuum through an effective nursing agency. it is recommended to deworm these children, provide nutritional education, establish "condom corners" and "street children help line" in the city and urban slum areas and undertake action research on their health. malnutrition is a serious problem in developing countries like bangladesh. malnutrition causes a great deal of child suffering. malnutrition is one of the major causes of morbidity and mortality among the child. the aim of present study was to investigate the nutritional status and its relation to socioeconomic conditions of urban disadvantaged child of under five years age. nutritional status was determined by anthropoemetric measurement(heig ht, weight, mid-arm circumference etc.). determination of total protein and serum albumin were done for biochemical examination. haematological examination was done by blood haemoglobin profile estimation by cynmethhaemoglob in method and determination of haematocrit value or packed cell volume (pcv). stool examination for ova of hook worm, round worm, trichuris species and other species were also done. relevant information on socioeconomic characteristics was recorded by interviewing the house hold head using pretested questionnaire. our study demonstrated that nutritional status of the children are positively correlated with family income, parents level of formal education and negatively related to the family size. the effect of family income on the nutritional status, haematological and biochemical indices of urban disadvantaged child under five will be discussed in detail which will help us to determine proper way of utilization of health care facilities exists in developing countries. introduction: with the increase in morbidity and reduction in mortality and with the introduction of highly active antiretroviral therapy (haart), there is increasing focus on the determinants of healthrelated quality of life (hrqol) in hiv-infection. the focus on the present study is to examine the relationship between social support, depression, and hiv disease severity, and the extent to which these variables impact on hrqol in adult men with hiv-infection. methods: as part of a larger ongoing natural history study focusing on the neurobehavioural complications of hiv and aids, we administered questionnaires to assess depression (beck depression inventory), social support, and hrqol (mos-hiv) to adult men with hiv-infection. a structured interview was used to collect health information and data on hiv disease severity. physical and mental health summary scores (phs and mhs) were derived through principal components analysis. participants were grouped according to level of social support: "well supported" (n= ), "moderately supported" (n= ), "little or ineffective support" (n= ). analysis of variance was conducted to assess the effects of three levels of perceived social support, depression status (present vs absent), and hiv disease severity (aids vs non-aids) on mental and physical health dimensions of hrqol. potential interaction effects were also evaluated. results: social support was found to have a significant effect on both mhs and phs. presence of depression was found to have a significant effect on mhs but not phs. hiv disease severity (presence of aids diagnosis) was found to have a significant effect on phs but not mhs. manov a analyses revealed no significant interactions effects. conclusions and implications: level of social support, presence of depression and hiv disease severity were shown to have independent effects on hrqol. feeling "well supported" appears to have significant benefits for mental and physical health. biological indicators of hiv disease appear to have a selective impact on physical health while presence of depression is related to significant reductions in mental health. the development and evaluation of behavioural interventions to improve perceived social support network and depression will likely result in significant improvements in health outcomes of adults with hiv and aids. introduction: ongoing medical advances have greatly enhanced the longevity of penons living with aids (plwa). however, for certain subgroups living with hiv/aids, including, members of minority groups, women and the economically underprivilege d, aids outcomes have not changed so favorably. one obvious factor to consider when explaining disparities in aids outcomes is the environ· ment within which an individual resides. few studies of aids outcomes have focused on how commu· nity setting influences the effectiveness of care delivery. the principle research questions were: ) what is the relationship between the locations of areas with concentrations of pl wa and the distribution of both primary and ancillary service providers? ) how has the widespread availability of haart (after ) impacted aids outcomes at the community level? ) how do aids mortality and fatality rates vary when related community characteristics, such as household income, ethnic mix, and geographic access to primary and ancillary hiv/aids services are considered? we examine this issue across residential communities in the los angeles area. methods: los angeles county's comprehensive aids service providers database (compiled and maintained by aids project los angeles) was geo-referenced and distances between weighted mean population centers and the nearest hiv/aids service providers were calculated to provide a measure of access which was then merged with aids diagnosis and mortality data along with relevant socioeconomic and population indicators and for analysis using bivariate and multivariate statistics at the zip code level. results: there is a growing disparity in hiv/aids outcomes between low-income minority areas and affluent non-minority areas in the post-haart era (p = . ). ancillary aids services such as case management and counseling and testing are located near the places where low income hiv/aids service consumers are likely to live (p =. ). in spite of having more access to ancillary services low income areas continue to be associated with smaller decreases post-haart aids mortality rates (p= . ). conclusions: given the increasing disparities in aids outcomes between lower and upper income communities in the post -haart era, more specific research into the capacities of aids services providers and the efficiency of their interventions is required. although it is clear that ancillary services are operating in the areas of greatest need, their effectiveness remains limited. understanding the reasons for t~is, be they lack of adequate resources for inner city service providers, failure to reach target popula· tmns or other causes, requires additional research. in ? , y.out~ who were able to speak either french or english and had been absent from their parent's/ c~regivers ~es. dence for at least three consecutive nights took part in the survey which consisted of inter· viewer-adm ms tered question~aires. p.arti~i~an~s were recruited from drop in centres in cities across <?-nada.y~uth self-report as either using in ect on or non-injection drugs. statistical analyses were car· r ed out using sas version . the 'home' is a contested site for many women living in urban areas. women living in poverty and using illicit drugs are largely excluded from participation in dominant 'home-base' gender roles of wife and mother because of economic disadvantage. they thus 'make home' in ways that are specific to their everyday experiences. while research has established links between housing, health and drug use, this literature does not tend to consider the meanings that home may have within the lives of women who lack access to adequate tenured living spaces. this presentation reports the findings of my master's thesis work. using grounded theory methodology within a poststructuralist feminist conceptual framework, i conducted in-depth, open-ended interviews with women living in the toronto area. the women were recruited from within the community through posters at different social service agencies (e.g., drop-in centres, community health centres). they were asked to describe their housing experiences and what home meant to them over the course of their lives. through the data analysis process, a substantive theory of 'making home' for women living in poverty and using illicit drugs emerged. the central concept of making home is a continual process of learning about and interacting with the environment. the process of making home entails different subprocesses: finding home (knowing what is available and accessible, and accessing home), adaptation, and leaving home. the different meanings of home that women developed from their experiences and knowledge emerged as critical components for the process of making home. the women i interviewed were very mobile and experienced constant change with regards to home. home was not considered only in relation to physical living space. home could be made within a relationship or with regards to a possession. while dominant meanings of home relation to tenured living spaces are generally positive, my research indicated that home could also be a negative context. the presentation will describe my research process and findings as well as the theoretical and policy implications of my results. in the western democracies, social citizenship bundles the right to social provision (defined as a share of the social good, not necessarily proportionate to the market value of a citizen's contribution) with participation in the paid labour force. for this reason, social citizenship rights, which usually involve some kind of redistributive measures, are a key mechanism by which states ensure economic equality, or at least, economic equality of opportunity, but are also a mechanism of social exclusion. social rights are inscribed around a single kind of relationship, that which exists between a male family breadwinner and the paid labor market; those who make economic contributions that fall outside this relationship -such as unpaid care-work, or, work that occurs in shadow or underground economiesare to a greater or lesser degree barred from accessing citizen-based social provisions. furthermore, social provision that occurs outside of the breadwinner/market-eco nomy dyad is very often meanstested and is accompanied by regulatory state apparatuses (this includes a range of social services, from welfare payments, to child-care subsidies, to disability pensions). social citizenship is thus stratified; the legal relationship that is at the heart of social citizenship acts as a form of social closure against claims for a share of the social good made by those who are 'lesser' citizens than others. of particular interest in this paper is how citizenship inequalities translate into inequalities in the social provisions that emerge from economic participation: the right to safety on the job, protection from employer harassment, protection from the vagaries of the market as well the right to adequate, comprehensive, and appropriate health services. drawing on mixed methods, longitudinal data from adult sex workers (n= ) located in two research sites with different social welfare regimes -one in canada and one in the u.s. -we examine the consequences of working in a "shadow" or "underground" economy on various facets of health and access to health services, taking into consideration the mediating role that citizenship constructs and social welfare regimes play in accessing other key social and economic determinants of health. methods: the study population included methadone patients (re)admitted at the mhs in the period from / / to / / , born in the netherlands, morocco, surinam, dutch antilles or turkye, and officially registered in the population register of amsterdam. the population register was used to ascer· rain the vital status. causes of death were provided by the central bureau of statistics. mortality was categorised as hiv related, directly drug related (overdose) and other.mortality. results: the methadone patients had the following characteristics: years of age at entry; % male; % ethnic minority; % ever injected. in total, personyear (py) of observation time and deaths ( % hiv related, % overdose; % other) were observed. hence, the crude mortality rate was / py ( % ci - / py). the percentage of (ever) injectors was higher among the native dutch than among the ethnic minorities, % and % respectively. higher rates were observed among native dutch drug users compared to those belonging to an ethnic minority (age adjusted hazard ratio (hr) . ( % ci . - . ). the age adjusted hr of (ever) injecting drug users versus non injecting users was . ( % cl . - . ). after additional adjustment for route of administration a non significant difference between the dutch and ethnic minorities remained (hr . , % cl . - . , p-value . . conclusion: the mortality rate among the heroin users in amsterdam is strongly affected by the high prevalence of non-injecting drug use. moreover, this study confirms that differences in mortality between the native dutch and the ethnic minorities is related to differences in route of administration. the ongoing reduction of injecting drug use (due tot selctieve mortality, and switching route ofadministration and popularity of crack cocaine) may lead to a further reduction of mortality rates among heroin users in amsterdam. interventions preventing the initiation of injecting should be encouraged. . introduction: food insecurity is an inequality that is central in the lives of many low-income cana· d ans. people lack food security when regular access to nutritious food is limited or variable due to high prices'. low income, lack of transportation or inadequate food distribution, or they become disadvan· taged m other w~ys through the acquisition of food. a group comprising academics, health profession· als, and commumty workers who have experience in food insecurity, developed a pilot study in ottawa that sought to und~rstand the lived experience of food insecurity. the study also used the united states department of agriculture (usda) community food security module. methods: a series of group meetings determined the best research approaches; questionnaires and consent forms were developed by a working group. twenty-three self-identified food insecure respon· dents were interviewed. the data were analysed using frequency distributions· the food security module was coded according to the usda coding guide, and open-ended responses w~re coded using a grounded approach. renlts: preliminary results from our pilot study have given the research team cause for considerable concern. out of households without children, experienced food insecurity with hunger; ~ere at the severe l~vel. out of households with children, experienced food insecurity with hunger. ~•rst person reflecnons commonly featured feelings of depression low self-esteem and despair. participants' account tha d . an cu ty gettmg to the store, severely limited putting knowledge and skills mto practice. l'oster sessions v conclusion: based on these early findings, our research team is looking to challenge what appear to be assumptions about poverty and food insecurity and for best ways to use this information. some policy approaches and interventions that encourage people to eat better and exercise more, may be missing the mark for low-income individuals, and may serve to maintain food insecurity. future research will build on this pilot. with methods augmentation, and a comprehensive knowledge dissemination plan, we hope co contribute to research and policy frameworks at all levels. ps- (a) mental health and the corrections system: population-based analyses in urban, semi-urban, and rural settings julian somers, robert watts, and michelle patterson introduction: according to recent epidemiological research, rates of mental disorders vary considerably across countries and across regions within countries. nevertheless, rates of mental disorders (including substance use disorders) are consistently higher in populations involved in the corrections sys- em. courts have long recognized the heavy burden of mental illness within their purview, and have developed innovative programs to better manage the needs of such individuals. the majority of these innovations (e.g., specialized courts) exist in urban settings. however, few studies have examined the degree of variability in rates of different mental disorders between courts in urban and other settings (e.g., semi-urban, rural). variability between courts in different settings, if present, holds implications for needs-based resource planning. the present study was conducted as part of a long-term inter-ministerial collaboration in british columbia (bc). analyses were conducted on linked administrative data regarding population health and correctional services. a database was constructed including all individuals sentenced in bc in a single year (n= , ). corrections records were matched to records of health services utilization (hospital and outpatient services) for mental disorders and substance use disorders in the years preceding sentencing. services for mental health and substance-related problems were aggregated into three groups: severe mental illness (smi); less-severe mental illness (lsmi); alcohol/other drug (ad). courts were grouped, based on their annual volumes, inro three categories: "urban"; "semi-urban" and "rural." rates of service use for mental disorders were compared between groups for -year and -years preceding sentencing. results: there were significant differences in the rates of mental disorders in courts of different size (urban, semi-urban, rural) . however, differences between the rates of disorders within each of these groups exceeded the discrepancies between groups. comparatively high and low rates of disorders were observed in courts of each size. moreover, the courts with the highest rates of certain disorders (e.g., ad) did not have the highest rates of other types of mental disorders (e.g., smi). conclusions: rates of mental disorders differed significantly among the annual populations sentenced across courts in bc. urban courts (which process comparatively large numbers of people) have implemented services to address the needs of the mentally ill. the present analyses strongly suggest that program development should be closely linked to the demonstration of need. the results caution that a uniform approach to court programs for the mentally ill is likely to be inefficient, over-supplying services in some settings and underestimating need in others. results: public settings used for injection are characterized by highly unhygienic conditions, the presence of unsafely disposed syringes, limited availability of sterile syringes and a lack of sterile water for injecting. a number of unsafe injection practices observed were common among public injectors. the presence of police officers nearby and the potential of assault by street predators fostered rushed injecting among those consuming drugs in public spaces. the ecological features of these environments encourage unhygienic and unsafe injecting practices, heighten risk for overdose and the transmission of blood borne viruses. introduction: approximately % of women experience depression in the first weeks or months after giving birth. although it has been argued that postpartum depression (ppd) is a culture-bound p~ nomenon, experienced only in western, industrialized countries, recent international research studies have confirmed that the prevalence of ppd is similar around the world. however, little is known about variables that may influence the experience of ppd in women from diverse cultures and immigrant women. research is needed to identify the role culture may play in the presentation of ppd (i.e., types of symptoms most commonly reported), how women from various racial, ethnic and cultural backgrounds perceive and attribute their symptoms of ppd, and finally, how culture can be appropriately addressed in ppd treatment programs. method: to examine the role of culture in ppd, semi-structured interviews and self-report question· naires were administered to clients of the women's health centre of st. joseph's health centre, tor· onto. participants were identified as either first generation canadians (relative newcomen) (n= ) or second generation canadian (parents were immigrants to canada) (n= ). data were collected qd these two groups of women based on: severity of depression, symptom presentation, perceived role of social supports and culturally-specific traditions, and barriers and responses to treatment. the interview data were analyzed using qualitative methods (thematic content analysis), and the following themes were identified: ) stress about passing on their culture to their children, ) lack of social support and feelings of isolation, ) perceived importance or lack of importance of cultural based tra· ditions, ) conflicts with family members. about cultural traditions and beliefs, ) mental health stigma within ethnic communities and beyond, ) race-and sex-based discrimination, and ) language barriers. conclrllion: identifying the role culture plays in the presentation of ppd, and how women from diverse backgrounds perceive and attribute their symptoms of ppd, is critical in order to establishcultur· ally appropriate guidelines for treatment options. furthermore, information gathered from this study can be used to develop policies and resources for delivering culturally competent care for newcomer wo~ who are dealing with ppd as well as the issues around acculturation (i.e., language barriers, racism). llus is of particular importance for urban health centers which provide postpartum care to diverse groups of women, and particularly recent immigrants or newcomers. ps-~ (al. contaminated 'therapeutic landscape': perceptions of the aamjiwnaang fint nation keyln smith and isaac luginaah . in~: this. paper pres;ents the findings of an ongoing study among the aamjiwnaang f!rst nanon. aam wnaang is located m the heart of samia's 'chemical valley', surrounded by cherrucal ~(ants such as esso, imperial oil, shell, suncor energy, and canada's largest hazardous waste disposal sate. safety kleen. this fint nation community is located within the st. clair river •area of concem'. as destgna~ by health canada for the population of samia and the surrounding region. although this comm_umty, by way of. its proximity to the numerous chemical plants is already exposed to high levels pollu~on, recent che?ucal dumping in ~mjiwnaang, as well as a failed proposal for another ethanol plant m the community, only helped to mtensify community concerns about potential health impacts of exposure. research on the cultural beliefs and traditions of first nation communities has established that th~ ~isting rel~tionship between first nations people and 'mother earth' is not only physical, but also_ spmtual. in this study we explore how the complex relationship between the aamjiwnaang first naaon and 'mother earth' be ha · · · i .. may c ngmg a contammated 'therapeutic' landscape. the study a so ~lores h?w aam wnaang residents are coping with these changes and with the probable conramina· boa of their community. ra!jts: aamjiwnaang residents acknowledge that they are living in a contaminated environment and are being impacted by emissions from the surrounding 'chemical valley' especially for future generations, and have expressed concerns that members of the community and mother earth are 'sick' as a result of this exposure. while moving from that 'place' has been discussed, residents have strong personal and generational connections to the land and insist however, that aamjiwnaang is their 'home' and will remain that way despite growing community concerns about the impact from industry. the contribution of this study lies in the direct involvement of community leadership in designing and conducting this research and distributing the results to further local policy development regarding community concerns on the reserve. background: truck driver are at very high risk for drug abuse because the factor contributing to drug abuse among driver are multiple. it is important to gain an understanding of the key factors that contributing to drug abuse among truck drivers. methods: seventy-truck driver aged - years were conveniently selected from the population. all the participants were male. an interview schedule was developed in the light of literature data and data were collected by personal interview with informed consent in selected urban area of eastern part of nepal. results: a considerable percentage of truck driver have less knowledge about drug abuse, its effect on body and their complication. majority of the participants ( %) explained that lack of education, financial crisis were leading factors of drug abuse. the average numbers of respondents were believed that lack of meaning in life, marital disharmony. friendship with drug abuser, stress, tiredness, modernization of life pattern and freedom from the home pressure were the influencing risk factors to drug abuse. conchuion: this study reveals those truck drivers are at risk in urban area for developing drugs abuse, mostly influenced by social and environmental factors. hence, these groups should undergo certain educational programme to prevent not only drug abuse, but also prevent transmission of infectious disease among these groups. introduction: food insecurity is an inequality that is central in the lives of many low-income canadians. our group of university researchers, health professionals, and community developers conducted a study in ottawa with community workers who work with people identified as food insecure. the purpose of the study was to understand the nature of their work, and their perspectives, perceptions and experiences of the causes and consequences of food insecurity. (a parallel study was conducted with food insecure participants). methods: sampling was through local knowledge of organizations involved in the provision of food to community members. written permission for the researchers to contact workers in confidence was obtained from each organization. thirteen in-depth interviews were conducted. the interviews consisted of a number of open-ended questions. the data were collated, and analysed using a grounded approach. results: workers interviewed represented organizations that offered diverse programming and methods of food distribution intended to address the needs of the community. the provision of food was seen as a necessary response to address hunger that arose for the majority from poverty caused by low income, or levels of government income support and safety nets that were insufficient to support food requirements. workers reported that food requirements varied according to different. ethnic backgrounds, ages of recipients and family configurations, and with food preferences, and d'.etary (health) requirements. workers' observations of the effects of insufficient food on adults and children such as depression, low energy and non-participation or social disengagement matched those of food insecure respondents. food insecurity was cited as "another srressor among the mountain of problems." overall, workers presented a picture of a silent, stigmatized, poor population including children, and a lack of advocacy to address the issues. be tha th · ·ty the results suggest that workers and organizations, like the commuruty mem rs t ey msecun • · · · · f ilab'l' f food · t d ·n thei·r ab 'l 'ty to address food insecurity. l m tanons m terms o ava ty o , serve, are restnc e . . funding, and donations appeared to set the para? eters of pr~g:am r.espon~ to commuruty food msecurity. workers were concerned with program des g": and a~m mstranon that m general addr~d hunger on an emergency short-term basis, although food msecunty was long-ter_m for ~any. sustainable solutions that include knowledge translation strategies, and health and social pohcy responses were suggested and will be explored in future research that hopes to build on this pilot. background: socioeconomic deprivation as well as low levels of soc!al s.uppoi:r have .been associated with poor outcomes following cardiac surgery. pre-operative depression m ~anents with coron~ry artery disease awaiting cabg ranges from - % and is an independent predictor of post-operanve mortality. since st. michael's hospital has a large inner city population, this study was designed to determine the both the prevalence of depressive symptomatology (ds) in patients awaiting cabg as well as its relationship with socioeconomic status (ses) and social support. methods: consecutive patients referred for isolated cabg were invited to complete the centre for epidemiological studies depression scale (ces-d) as well as a social support scale and a life stressors inventory. a ces-d score of : represents mild ds and a score of : represents moderate to severe ds. participants also recorded information on functional status, perceived progression of heart failure, demographic and ses variables. all participants with ces-d scores : were considered to have ds. results: of the patients ( %) who returned the survey, ( . %) of participants had a ces-d score : with ( . %) of those having scores suggestive of severe depression. females and those with ongoing medical concerns tended to have more ds (p= . and p= . respectively). age was not related to ds. perceived worsening of symptoms and increased ccs angina class were significantly related to the presence of ds (p= . and p= . respectively) but not the number of diseased heart vessels. satisfaction with personal relationships including having someone to confide in (p= . ), feeling lonely (p= . ) and not having a partner (p= . ) were significantly related to ds. being very upset by a recent breakup in the immediate family was also significantly related to ds (p= . ). having greater than a grade education was the only ses variable related to ds (p= . ). multivariate logistic regression suggested that not being partnered and having low education were the most significant predictors of ds. conclnsion: pre-operative depression is present in at least one quarter of patients referred for cabg. since ds is related to poor outcomes following cabg, single patients with few social supports and low education should be considered at high risk for ds. these findings support the need for the development of supportive interventions in patients at risk in order to decrease post-operative morbidity. this study examines the relationship between childhood sexual abuse, sexual assault during adolescence, and hiv-risk-related behaviours in a sample of homeless youths. over the past decade, there has been increasing research devoted to the impact of childhood sexual abuse. some studies have suggested that the experience of childhood sexual abuse is associated with substance use and abuse, at· risk sexual behaviour and subsequent higher rates of hiv infection (ompad et al., ; cinq-mars et al., ; brown et al., ) . other studies have found that childhood abuse, especially sexual abuse, is c_o~n among stree~ youth (noell et al. ). this study compares sexually abused males and females livtng on the street ~th non-sexually abused street youth, with regards to unsafe sexual behaviours and e~ures to potmnal t:dv sources (e.g., tattooing, body piercing and injection drug use). the hypothe-sis oft~ ~nt study is that the prevalence of hn-risk-related behaviours will be higher among street youth vtctuns of sexual abuse than those who have not been sexually abused. the sample includes youths aged to who met specific criteria's for itinerancy ( male and female, with a mean ~of . y· and a standard deviation of . ). they were recruited through five organisations working ~th~ you~ and were~ of an ongoing cohort study. all youth completed a to minute ques· ~onnaue on their sexual behaviours, use of drugs and alcohol and other hiv risk behaviours. oral spec· unens for hiv testing were. also collected. a total of ( . %) youth reported at least one incident of ~i •~use/assault ( ~ girls ( . % of all the girls) and boys ( . % of all the boys]). preliminary descnpnve analyses pomt to sexually abused/assaulted youth showing the highest prevalence of sexual poster sessions v at-risk behaviours. for example, . % of sexual abuse/assault victims have engaged in prostitution in their lives compared with . % in the non-sexually abused/assaulted group. in addition, . % of sexual abuse/assault victims reported having injected drugs, compared with . % in the non-sexually abused/assaulted group. more statistical analyses will bring other significant factors to light, especially when we analyze separately those victim of childhood sexual abuse as compare to those victim of sexual assault after childhood. elucidating the risk factors for getting involved in hiv-risk-related behaviours is important for the development of comprehensive and appropriate prevention and treatment intervention strategies. introduction: new immigrants and refugees to canada frequently emigrate from regions of the world where intestinal parasitic infections with worms are endemic. of note, some of these parasites area capable of surviving for years to decades within a given host and can have life threatening consequences many years after initial infection. thus, early diagnosis and treatment of intestinal parasitic worms is considered important in high risk immigrant populations, however there remains a lack of consensus regarding the best method of accomplishing this. diagnosing worm infections through stool examinations is costly and has limited sensitivity, while presumptive treatment of all immigrants, although advocated by some, is expensive and results in healthy individuals being unnecessarily treated. herein, we examine the utility of a hematologic marker (i.e. peripheral blood eosinophilia) as a screening instrument to identify parasitic worm infections in new immigrants to toronto. we identified adults, years of age or older, who were screened for intestinal parasitic infections by stool examination as part of a recently developed screening protocol at a downtown toronto community health centre. we examined the prevalence of infections with worms and subsequently evaluated the impact of several demographic factors on the presence of worm infections. we also determined the sensitivity and specificity of peripheral blood eosinophilia (defined as greater than eosinophils per ml) as a marker for intestinal parasitic worm infections. results: overall, % of the immigrants tested had evidence of at least one worm infection on a single stool examination, while % of such individuals bad infections with multiple worms concurrently. age did not appear to be associated with the presence of worms, however % of males had evidence of worm infections compared with % of females (p= . ). immigrants from asia, sub-saharan africa, and central america had the highest burden of overall infection. the sensitivity and specificity of peripheral blood eosinophilia for worm infections was % and % respectively. conclusions: one in five recent immigrants presenting to a community health centre in downtown toronto had evidence of at least one intestinal parasitic worm infection. the highest burden of illness was in men and in those emigrating from less developed regions of the world. peripheral blood eosinophilia is a poor screening test for worm infections however its presence is highly suggestive of the existence of worms in new immigrants. more than a decade ago, the journal of american college health recommended that a national study be conducted to measure the health status of african american college students, 'both health disparities and [their] positive healthful behaviors' (fennell, ) . it was later decided that when exploring the level of health risk behaviors for african american students, gender is an important variable and should be included in the research (fennell, ) . at historically black college and universities (hbcus) college health researchers must tackle gender disparities through highly-effective health promotion and disease prevention programs. the purpose of this review is to examine the literature that addresses the overarching health behaviors (i.e. risky sexual behavior, poor diet, smoking, physical inactivity, etc.) of african american men who attend hbcus. despite the various social, cultural, and academic benefits of african american students who attend hbcus, the number of research studies that adequately address their health and health behaviors is limited. studies indicate that african american men who attend hbcus are actively engaged in poor health behaviors. compared to females at hbcus, african american men are more likely to behave in ways that could result in injuries and the use tobacco, alcohol, and ~ther dru~. the city of toronto has recently proposed to conduct a street count of the homeless. like proposals that have come before, this most recent proposal is controversial among many homeless advocates and service providers because it is perceived as intrusive, likely to undercount the target population, and unnecessary for addressing the problem. advocates of the count view it as necessary to gauge the scope of the problem and advise city leaders about how to most effectively direct resources ro.the various homeless services. this paper first reviews the history of efforts to count homeless populat ns m toronto, describing the motivations and arguments of people on both sides of the issue. second, the paper reviews the methodological complexities of counting homeless populations, describing approaches that use shelter-counts, administrative data and street observations. those interested in counts for toronto and elsewhere can benefit from this review of approaches that have been proposed and carried out in other municipalities. third, the paper proposes a new community-base d strategy that joins the skills of methodologists, local experts, and service providers to ensure a fair and accurate count. the method of systematic social observation avoids some of the problems of early counts by utilizing a repeated identification approach to minimize undercount bias. the method also has the advantage of involving and compensating homeless individuals for assisting with the street count estimates. the estimation approach can be implemented on a rolling basis throughout the year. drug transitions) is a multi-level study aimed at examining the associations between features of the urban environment and mental health, drug use, and risky sexual behaviors. research participants are being recruited in new york city (nyc) neighborhoods. an essential first step in the implementation of this study was to delineate boundaries for each target neighborhood that could then be used to establish sampling frames and as key units of analytic interest. although many neighborhood studies rely on pre-established definitions (e.g. those used for the census or administrative purposes), such definitions do not always reflect contemporary settlement patterns. we felt that street level observations were a neces· sary component of the definition process. methods: the procedures used to delineate neighborhoods were: ( ) development of census block maps of targeted areas; ( ) review of land use maps and census tract data to ensure, prior to going into the field, tha.t particular blocks are residential and likely to have sufficient population for recruitment purposes; and ( ) field vislts and observation in each of the targeted areas. field observations focused on: housing characteristics, including housing type and condition; characteristics of commercial areas, such as likely customer base (e.g. local or no~, socio-economic status and ethnicity of customers); pedestrian volume (including con· gregauons of people m parks and outside stores or residences); obstructions to pedestrian traffic (e.g. ma or thoroughfares, multi-block industry or institutions); and maintenance and use of open spaces. results: in making the final decisions regarding neighborhood boundaries and the inclusion or exclusion of particular. blocks, we considered a broad range of factors including evidence of homogeneity an~ heterogeneity (socmeconomic, ethnic, housing type, environmental) across blocks within and blocks ad acent to a nei.ghborhood, and across the sample neighborhoods; the potential for efficient recruit· ment of appropriate particip.ants (i.e. sufficient local pedestrian traffic); and boundaries used in reporting census data (s? that population data can be used in our analysis). . altho~gh utilization of field observations for neighborhood definitions is relatively time consuming (ap~~ox mately hours for a block area, with more diverse neighborhoods taking even longer), we annc p~t~ that it will substantially improve the quality and consistency of our data gath· ~rmg ~nd analyse~. spec f c e?'amples from neighborhoods defined through this process will be given dur· m~ this presentatmn. we will also discuss the merits and limitations of characterizing neighborhoods usmg street level observations. has been no consideration for how social networks are associated with sse. the objective of this study was to identify personal and social network characteristics associated with being a recipient of sse. in this cross-sectional study, idus who injected in the past months were recruited from syringe exchange programs in montreal, canada, between april and january . information on each participant and on the persons with whom contact had occurred in the past month were elicited using a structured, interviewer-administe red questionnaire. participants provided detailed demographic and drug use information on up to idu members with whom drugs or injecting materials were shared. using logistic regression, personal and network characteristics were examined in relation to receipt of sterile syringes. res.its: of idus, % reported receiving sterile syringes in the past month from another idu. the sample mean age was years (range - ), % male, % caucasian, and % cocaine injectors. in multivariate analyses adjusted for age and gender, recipients of sterile syringes were more likely than non-recipients to share drugs after preparation ( r= . ( . - . )), to require help or to have helped inject another idu (or= . ( . - . )), to have asked someone to get sterile syringes from a syringe exchange program (or= . [ . - . )), to lend syringes (or= . [ . - . ) ) and to use drug preparation water that was previously used by another iou ( r= . ( . - . ) ) during the past months. recipients also had a higher rate of change (p= . ) of idus in their drug injecting network during the past month. with respect to social networks, % ( / ) of idu network members were providers of sterile syringes and were more likely than non-providers to inject everyday (or= . ( . - . )). when stratified by their role as a sexual partner or as someone who provides support (e.g. friend, family member), further risk behaviours were observed in relation to sharing injecting materials with the participant. conclusion: recipients of sterile syringes and their idu network members had several high-risk behaviours for infection with bloodborne diseases. sse may afford an opportunity for the dissemination of other salutary behaviours such as information sharing on safe injecting practices between recipients and distributors. furthermore, drug injecting networks may be an avenue to reach idus who may not otherwise be exposed to preventive measures. there are numerous sources of stigma and labelling of mental and physical illness. first, the sociocultural dimension to stigma and labelling clearly influences patients, families, health care professionals and society's perception and treatment of illness. this creates multiple challenges related to illness identification and recovery, particularly in culturally diverse societies. second, the media is a major source of stigma and stereotyping with regard to a variety of mental and physical illnesses. despite increased public knowledge of many illnesses, studies have shown that stigma has intensified over the years in certain cases. a third element of stigma and labels appears with the use of diagnosis as a solution to human problems with the result that difficult to treat patients are repudiated and social issues are unaddressed. this presentation will serve to outline the development of stigma, various expressions of stigma, and consequences of stigma. it will provide some practical recommendations for the reduction of stigma related to mental and physical illness. purpose: to assess the knowledge, attitude, and practice about immunization for parent of - years old hispanic and african american children in los angeles county (lac). methods: we conducted two cluster surveys in lac. the sample was selected with probability proportionate to estimated size at the first stage and simple random sample of children at the second stage. data were collected through interview. we absuacted vaccine coverage data from immunization record card (irc) at home or from clinic records. the participation was % in hispanics and % in african americans populations. irc was not available at home for % of african american and % of hispanic children. results: all parents perceived their children got all necessary vaccine~. fa~orable attitudes to.w~rd immunization were reported by % of african american and % of h s?amc parents. '.he m~ ority of the parents ( % african american and % hispanic) agreed that vaccines prevent senous d se~ses among children. regarding vaccine safety, significantly more hispanic parents ( %) than african american parents ( %) mentioned that vaccines are safe. introduction: cervical cancer has been shown to be preventable by papanicolau tests in heterosexual women; however, the utility of papanicolau test in lesbians is controversial. cervical cancer is caused by the human papillomavirus (hpv) which is transmitted by sexual _intercourse; howev_er, its' transmission by homosexual intercourse is also controversial. the goal of this study was to review the evidence for papanicolau tests in lesbians. methods: a pub med, ovid ( ovid ( to , and cochrane library search was performed using the mesh headings "homosexuality , female," "vaginal smears," and "papillomavirus, human." in pub med, the clinical queries feature was used with "diagnosis" and "broad, sensitive" filters. a google advanced search of the internet was conducted with the terms "lesbian," "cervical cancer," and "pap rest." the url was limited to ".ca," ".edu," or ".gov." results: the search yielded original articles and reviews, but no cochrane reviews. none of the publications were canadian. website hits were found and selected websites were reviewed. studies showed that lesbians have fewer papanicolau tests than heterosexual women; however, many reported risk factors for cervical cancer, including multiple past or current sexual partners (male and female), early age of first coitus, history of sexually transmitted diseases, and cigarette smoking. hpv has been detected in - % of lesbians and - % of lesbians who have never had sex with men. case studies of abnormal papanicolau tests in lesbians who have never had sex with men have also been reponed. sexual transmission of hpv among lesbians can be explained by several factors: ) - % of lesbians have had sex with men and - % of lesbians continue to have sex with men. ) hpv may be transmitted by sexual behaviours among women, like digital-vaginal sex, digital-anal sex, oral sex, and the use of insertive sex toys. ) hpv transmission requires only skin-to-skin contact. genital hpv has been identified on human fingers. some professional organizations, like the society of obstetricians and gynecologists of canada and the american cancer society, recognize the need for papanicolau tests in lesbians. other organizations, like the canadian cancer society, do not have official policy statements. the current ontario cervical screening program does not specifically address lesbians. conclusion: although the data was limited, most studies recommended that lesbians should receive papanicolau tests. the frequency of these tests was controversial. policy makers and professional organizations should address the needs of this special population and make specific recommendations . monique chaaya, ahia sibai, hiam chemaitelly, and zana el roueiheb . literature concerning the mental and physical effect of work at an old age is contradictory. in addinon, urban environments are physically and socially harsh with reduced potential for income generation and employment and increased potential for depression. the present paper is an attempt to study how do work, or lack of work, link to the experience of depression among older adults in underprivileged lebanese urban communities. the data comes from a cross-sectional survey, where elderly residing in underprivileged communities in beirut, including a palestinian refugee camp, were successfully inter-v ewed through face-to-face interviews. logistic regression was performed to assess the effect of work on depression ad_justing for many other covariates. data showed that . % of people aged years and more were still workmg. there was a highly protective effect of work on depression in elderly males (or= . , % cl= . - . ). the current study is the first of its kind in the arab region and more work shou_ld be _don_e in this area. it is important for the lebanese government to consider elderly rights for social mclus n m terms of employment opportunities. amam nuru-jeter, nancy adler, and burton singer . l~u~on:_ the socioeconomic gradient is perhaps the most persistent and significant finding in social epidem ological research. the insistent nature of the ses effect is evidenced by its significance after acc?untmg for ~umerous confounds. the significance and magnitude of the effect, however, varies by ::•al group. evidence_ o_f the relative effect of race and ses is inconclusive. further, less attention has f ~ ptid to the specific interactions of race and ses than to examinations of which is a better predictor d ~ th. ~sues are further complicated by the explanatory complexity of the various measures of ses anl . owht ey relate to the health of various social groups. understanding the specific nature of these re a ons ips s necessary for guiding he ith d · i · · h a an soc a po mes and programs aimed at improving t e poster sessions v health of our most disadvantaged groups, and therefore population health, overall. this study examines the synergistic effects of ses with both race and gender in predicting group differences in mortality. methods: analyses use data from the national longitudinal mortality survey (n= , ) for the years , , and - ; and were linked to the national death index for deaths occurring through . results were confirmed using data from the national mortality followback survey for people who died in (n= , ) and the national health interview survey (denominator). we used correlation analysis and the standardized mortality ratio to examine ses (education and income), race, and gender as predictors of group differences in changes in mortality. results: for the total population, analyses support the income gradient showing significant declines in mortality ratios from low to high income along the income strata. education shows a significant drop in mortality with completion of high school and completion of college, exhibiting a threshold rather than a gradient effect. compared to whites, blacks receive diminishing health returns for each subsequent level of income and education; with whites closely mirroring the total population in ses thresholds. similarly, compared to men, women do not experience the same health gains at comparable ses levels. for education, black men and women only show significant mortality declines upon completion of high school. the ses gradient operates differently among race and gender subgroups. threshold effects suggest resource gains in life opportunities at particular milestones along the ses gradient. further, ses matters less for blacks and women compared to whites and men suggesting differing implications for health and social policy aimed at reducing disparities. objective: we assessed the impact of diabetes in a large puerto rican community of chicago by measuring the prevalence of diagnosed diabetes and calculating the diabetes mortality rate. methods: we analyzed data from a comprehensive health survey conducted in randomly selected households in community areas. questions on diagnosed diabetes and selected risk factors were asked. in addition, mortality data were analyzed in order to calculate the age-adjusted diabetes mortality rate. when possible, rates were compared to those found in other studies. results: the diabetes prevalence located in this community ( . %: % cl= . %- . %) is one of the highest ever reported for puerto ricans. for instance, it is more than three times higher than the prevalence for the us ( . %) and twice the prevalence for puerto ricans in new york ( . %) and puerto rico ( . %- . %). diagnosed diabetes was found to be significantly associated with obesity (p= . ). the prevalence was particularly high among older people, females, those horn in the us, and those with a family history. the diabetes mortality rate ( . per , population) was more than twice the rate for all of chicago ( . ) and the us ( . ). conclusions: understanding why the diabetes and mortality rates for puerto ricans in this commu· nity are so much higher than those of other communities is imperative. collaboration between researchers, service providers and community members can help address the issues of diabetes education, early screening and diagnosis and effective treatment needed in this community. introduction: sars is a respiratory illness that is spread from person to person through dose contact. this infectious disease outbreak was unusual due to the high rate of infection among health care workers. the aim of the study is to explore the demographic and attitudinal determinants of psychological distress due to sars among health care workers of a toronto hospital. methods: a total of adult participants completed questionnaires co assess psychological distress (impact of event scale -jes) and attitudes to sars. of these, participants completed the questionnaire during the sars i outbreak, and participants completed the questionnaire during the sars ii outbreak. participants also provided information on demographic variables, including occupation and exposure to sars. principal components analysis was used to derive eight attitudinal scales: fear: system, protection, prevention, job stress, stigma, instrumental coping, and psychological copm~. l ear regression analyses were applied to evaluate the associations of the demographic and amtudmal variables, as well as time, with psychological distress. . . regression analyses showed that time and higher scores on fear, ob stress, stigma'. and instrumental coping were associated with a higher total ies score: (r =. ). contrary to expectations, none of the demographic variables was associated with total ies score. conclusion: the presence of psychological distress in health care workers is indicative of intrusive or avoidant responses to thoughts, feelings, and memories associated with the sars outbreak. this study shows that ( ) fear for one's health and the he~lth of.others, ( ) ex~erien~in~ job stress, ( )_tbe per· ception of stigma, ( ) usage of instrumental copmg skills, ~n~ ( ) t m_e s grufi~an~ly c~nmb~te to increased psychological distress in health care workers. potential mterventmns during mfecttous diseaie outbreaks in health care settings should be targeted to minimize the impact of these factors. purpose: the purpose of this study was to expand knowledge regarding feeding practices, know!· edge and nutritional beliefs of mothers currently residing in the dominican republic (dr). the rising incidence of obesity in children is a major national health concern and obesity in first and second-generation immigrant children also continues to rise. we know that parents (particularly mothers) shape children's early diet patterns and that immigrant mothers experience additional challenges related to acculturation and assimilation into a new culture, however there is a paucity of literature concerning the effect of immigrant adaptation to american culture on the nutrition of children. in addition, new immigrant knowledge about the causes of obesity and the resulting health implications for childhood obesity has not been assessed and reported. this qualitative study used focus group methodology to discuss views and practices related to the introduction of food for infants and feeding practices for young children, along with a discussion on knowledge and beliefs related to the causes and health implications of child· hood obesity. ten dominican mothers' of young children (birth to years old) who reside in a small town in the western frontier area of the dr were participants in the focus group. results from this srudy will be compared to results from an identical study that will be conducted in fall with new immi· grant mothers from the dr to the boston area. we will compare feeding practices, beliefs and knowledge about nutrition for young children between these two groups. methods: using participatory principles, a focus group design was used to interview a group of mothers in the dr. the investigator along with a bilingual translator conducted the focus group. the session was tape recorded and is currently in the process of being transcribed and translated. transcribed data will be systematically analyzed themes will be identified. a qualitative data analysis software will be used to organize and code the data according to the specific aims of the study. supporting quotations will be selected to substrate each theme. lmpli~tions for urban health practice: an ultimate goal of this study is to lay a foundation for understanding the process of acculturation on feeding practices of mothers when they immigrate to the us. understanding beliefs, knowledge and feeding practices that mothers use with young children will help m ~he ~evelopment of culturally and linguistically appropriate educational strategies and materials for use m primary prevention of pediatric obesity. in canada more than seventy-five percent of immigrants choose to stay in three major urban centers of toronto, montreal and vancouver. approximately fifty percent of the immigrants eventually set· tie m ~~e greater tor<_>nto area. there is mounting evidence that the increasing immigrant population has a_ sigmfic~nt health disadvantage over canadian-born residents. this health disadvantage manifests particularly m the ma "ority of "mm "gr t h h d be · · h . . . . an s w o a en m canada for longer than ten years. this group as ~n associ~te~ with higher risk of chronic disease such as cardiovascular diseases. this disparity twccb n ma onty of the immigrant population and the canadian-born population is of great importance to ur an health providers d" · i i · b as isproporttonate y arge immigrant population has settled in the ma or ur an centers. generally the health stat f · · · · · · h h been . us most mm grants s dynamic. recent mm grants w o av_e ant •;ffca~ada _for less ~han ~en years are known to have a health advantage known as 'healthy imm • ~ants r::r · ~:s eff~ ~ defined by the observed superior health of both male and female recent immi- immigrant participation in canadian society particularly the labour market. a new explanation of the loss of 'healthy immigrant effect' is given with the help of additional factors. lt appears that the effects of social exclusion from the labour market leading to social inequalities first experienced by recent immigrant has been responsible for the loss of healthy immigrant effect. this loss results in the subsequent health disadvantage observed in the older immigrant population. a study on patients perspectives regarding tuberculosis treatment by s.j.chander, community health cell, bangalore, india. introduction: the national tuberculosis control programme was in place over three decades; still tuberculosis control remains a challenge unmet. every day about people die of tuberculosis in india. tuberculosis affects the poor more and the poor seek help from more than one place due to various reasons. this adversely affects the treatment outcome and the patient's pocket. many tuberculosis patients become non-adherence to treatment due to many reasons. the goal of the study was to understand the patient's perspective regarding tuberculois treatment provided by the bangalore city corporation. (bmc) under the rntcp (revised national tuberculosis control programme) using dots (directly observed treatment, short course) approach. bmc were identified. the information was collected using an in-depth interview technique. they were both male and female aged between - years suffering from pulmonary and extra pulmonary tuberculosis. all patients were from the poor socio economic background. results: most patients who first sought help from private practitioners were not diagnosed and treated correctly. they sought help form them as they were easily accessible and available but they. most patients sought help later than four weeks as they lacked awareness. a few of patients sought help from traditional healers and magicians, as it did not help they turned to allopathic practitioners. the patients interviewed were inadequately informed about various aspect of the disease due to fear of stigma. the patient's family members were generally supportive during the treatment period there was no report of negative attitude of neighbours who were aware of tuberculosis patients instead sympathetic attitude was reported. there exists many myth and misconception associated with marriage and sexual relationship while one suffers from tuberculosis. patients who visited referral hospitals reported that money was demanded for providing services. most patients had to borrow money for treatment. patients want health centres to be clean and be opened on time. they don't like the staff shouting at them to cover their mouth while coughing. conclusion: community education would lead to seek help early and to take preventive measures. adequate patient education would remove all myth and conception and help the patients adhere to treatment. since tb thrives among the poor, poverty eradiation measures need to be given more emphasis. mere treatment approach would not help control tuberculosis. lntrod#ction: the main causative factor in cervical cancer is the presence of oncogenic human papillomavitus (hpv). several factors have been identified in the acquisition of hpv infection and cervical cancer and include early coitarche, large number of lifetime sexual partners, tobacco smoking, poor diet, and concomitant sexually transmitted diseases. it is known that street youth are at much higher risk for these factors and are therefore at higher risk of acquiring hpv infection and cervical cancer. thus, we endeavoured to determine the prevalence of oncogenic hpv infection, and pap test abnormalities, in street youth. ~tbods: this quantitative study uses data collected from a non governmental, not for profit dropin centre for street youth in canada. over one hundred females between the ages of sixteen and twentyfour were enrolled in the study. of these females, all underwent pap testing about those with a previous history of an abnormal pap test, or an abnormal-appearing cervix on clinical examination, underwent hpv-deoxyribonucleic (dna) testing with the digene hybrid capture ii. results: data analysis is underway. the following results will be presented: ) number of positive hpv-dna results, ) pap test results in this group, ) recommended follow-up. . the results of this study will provide information about the prevalence of oncogemc hpv-dna infection and pap test abnormalities in a population of street youth. the practice implic~ tions related to our research include the potential for improved gynecologic care of street youth. in addition, our recommendations on the usefulness of hpv testing in this population will be addressed. methods: a health promotion and disease prevention tool was developed over a period of several years to meet the health needs of recent immigrants and refugees seen at access alliance multicultural community health centre (aamchc), an inner city community health centre in downtown toronto. this instrument was derived from the anecdotal experience of health care providers, a review of medical literature, and con· sultations with experts in migration health. herein we present the individual components of this instrument, aimed at promoting health and preventing disease in new immigrants and refugees to toronto. results: the health promotion and disease prevention tool for immigrants focuses on three primary health related areas: ) globally important infectious diseases including tuberculosis (tb), hiv/aids, syphilis, viral hepatitis, intestinal parasites, and vaccine preventable diseases (vpd), ) cancers caused by infectious diseases or those endemic to developing regions of the world, and ) mental illnesses includiog those developing among survivors of torture. the health needs of new immigrants and refugees are complex, heterogeneous, and ohen reflect conditions found in the immigrant's country of origin. ideally, the management of all new immigrants should be adapted to their experiences prior to migration, however the scale and complexity of this strategy prohibits its general use by healthcare providers in industrialized countries. an immigrant specific disease prevention instrument could help quickly identify and potentially prevent the spread of dangerous infectious diseases, detect cancers at earlier stages of development, and inform health care providers and decision makers about the most effective and efficient strategies to prevent serious illness in new immigrants and refugees. lntrodmction: as poverty continues to grip pakistan, the number of urban street children grows and has now reached alarming proportions, demanding far greater action than presently offered. urbanization, natural catastrophe, drought, disease, war and internal conflict, economic breakdown causing unemployment, and homelessness have forced families and children in search of a "better life," often putting children at risk of abuse and exploitation. objectives: to reduce drug use on the streets in particular injectable drug use and to prevent the transmission of stds/hiv/aids among vulnerable youth. methodology: baseline study and situation assessment of health problems particularly hiv and stds among street children of quetta, pakistan. the program launched a peer education program, including: awareness o_f self and body protection focusing on child sexual abuse, stds/hiv/aids , life skills, gender and sexual rights awareness, preventive health measures, and care at work. it also opened care and counseling center for these working and street children ar.d handed these centers over to local communities. relationships among aids-related knowledge and bt:liefs and sexual behavior of young adults were determined. rea.sons for unsafe sex included: misconception about disease etiology, conflicting cultural values, risk demal, partner pressur~, trust and partner significance, accusation of promiscuity, lack of community endorsement of protecnve measures, and barriers to condom access. in addition socio-economic pressure, physiological issues, poor community participation and anitudes and low ~ducation level limited the effectiveness of existing aids prevention education. according to 'the baseline study the male children are ex~ to ~owledge of safe sex through peers, hakims, and blue films. working children found sexual mfor~anon through older children and their teachers (ustad). recommendation s: it was found that working children are highly vulnerable to stds/hiv/aids, as they lack protective meas":res in sexual abuse and are unaware of safe sexual practices. conclusion: non-fatal overdose was a common occurrence for idu in vancouver, and was associated with several factors considered including crystal methamphetamine use. these findings indicate a need for structural interventions that seek to modify the social and contextual risks for overdose, increased access to treatment programs, and trials of novel interventions such as take-home naloxone programs. background: injection drug users (idus) are at elevated risk for involvement in the criminal justice system due to possession of illicit drugs and participation in drug sales or markets. the criminalization of drug use may produce significant social, economic and health consequences for urban poor drug users. injection-related risks have also been associated with criminal justice involvement or risk of such involvement. previous research has identified racial differences in drug-related arrests and incarceration in the general population. we assess whether criminal justice system involvement differs by race/ethnicity among a community sample of idus. we analyzed data collected from idus (n = , ) who were recruited in san francisco, and interviewed and tested for hiv. criminal justice system involvement was measured by arrest, incarceration, drug felony, and loss/denial of social services associated with the possession of a drug felony. multivariate analyses compared measures of criminal justice involvement and race/ethnicity after adjusting for socio-demographic and drug-use behaviors including drug preference, years of injection drug use, injection frequency, age, housing status, and gender. the six-month prevalence of arrest was highest for whites ( %), compared to african americans ( %) and latinos ( % ), in addition to the mean number of weeks spent in jail in the past months ( . vs. . and . weeks). these differences did not remain statistically significant in multivariate analyses. latinos reported the highest prevalence of a lifetime drug felony conviction ( %) and mean years of lifetime incarceration in prison ( . years), compared to african americans ( %, . years) and whites ( %, . years). being african american was independently associated with having a felony conviction and years of incarceration in prison as compared to whites. the history of involvement in the criminal justice system is widespread in this sample. when looking at racial/ethnic differences over a lifetime including total years of incarceration and drug felony conviction, the involvement of african americans in the criminal justice system is higher as compared to whites. more rigorous examination of these data and others on how criminal justice involvement varies by race, as well as the implications for the health and well-being of idus, is warranted. homelessness is a major social concern that has great im~act on th~se living.in urban commu?ities. metro manila, the capital of the philippines is a highly urbanized ar~ w. t~ the h gh~st concentration of urban poor population-an estimated , families or , , md v duals. this exploratory study v is the first definitive study done in manila that explores the needs and concerns of street dwdlent\omc. less. it aims to establish the demographic profile, lifestyle patterns and needs of the streetdwdlersindit six districts city of manila to establish a database for planning health and other related interventions. based on protocol-guid ed field interviews of street dwellers, the data is useful as a template for ref!!. ence in analyzing urban homelessness in asian developing country contexts. results of the study show that generally, the state of homelessness reflects a feeling of discontent, disenfranchisem ent and pow!!· lessness that contribute to their difficulty in getting out of the streets. the perceived problems andlar dangers in living on the streets are generally associated with their exposure to extreme weather condirioll! and their status of being vagrants making them prone to harassment by the police. the health needs of the street dweller respondents established in this study indicate that the existing health related servias for the homeless poor is ineffective. the street dweller respondents have little or no access to social and health services, if any. some respondents claimed that although they were able to get service from heallh centers or government hospitals, the medicines required for treatment are not usually free and are beyond their means. this group of homeless people needs well-planned interventions to hdp them improve their current situations and support their daily living. the expressed social needs of the sucet dweller respondents were significantly concentrated on the economic aspect, which is, having a perma· nent source of income to afford food, shelter, clothing and education. these reflect the street dweller' s need for personal upliftment and safety. in short, most of their expressed need is a combination of socioeconomic resources that would provide long-term options that are better than the choice of living on the streets. the suggested interventions based on the findings will be discussed. . methods: idu~ aged i and older who injected drugs within the prior month were recruited in usmg rds which relies on referral networks to generate unbiased prevalence estimates. a diverse and mon· vated g~o~p of idu "seeds." were given three uniquely coded coupons and encouraged to refer up to three other ehgibl~ idu~, for which they received $ usd per recruit. all subjects provided informed consent, an anonymous ~t erv ew and a venous blood sample for serologic testing of hiv, hcv and syphilis anti~!· results. a total of idus were recruited in tijuana and in juarez, of whom the maion!)' were .male < .l. % and . %) and median age was . melhotls: using the data from a multi-site survey on health and well being of a random sample of older chinese in seven canadian cities, this paper examined the effects of size of the chinese community and the health status of the aging chinese. the sample (n= , ) consisted of aging chinese aged years and older. physical and mental status of the participants was measured by a chinese version medical outcome study short form sf- . one-way analysis of variance and post-hoc scheffe test were used to test the differences in health status between the participants residing in cities representing three different sizes of the chinese community. regression analysis was also used to examine the contribution of size of the chinese community to physical and mental health status. rmdts: in general, aging chinese who resided in cities with a smaller chinese population were healthier than those who resided in cities with a larger chinese population. the size of the chinese community was significant in predicting both physical and mental health status of the participants. the findings also indicated the potential underlying effects of the variations in country of origin, access barriers, and socio-economic status of the aging chinese in communities with different chinese population size. the study concluded that size of an ethnic community affected the health status of the aging population from the same ethnic community. the intra-group diversity within the aging chinese identified in this study helped to demonstrate the different socio-cultural and structural challenges facing the aging population in different urban settings. urban health and demographic surveillance system, which is implemented by the african population & health research center (aphrc) in two slum settlements of nairobi city. this study focuses on common child illnesses including diarrhea, fever, cough, common cold and malaria, as well as on curative health care service utilization. measures of ses were created using information collected at the household level. other variables of interest included are maternal demographic and cultural factors, and child characteristics. statistical methods appropriate for clustered data were used to identify correlates of child morbidity. preliminary ratdts: morbidity was reported for , ( . %) out of , children accounting for a total of , illness episodes. cough, diarrhoea, runny nose/common cold, abdominal pains, malaria and fever made up the top six forms of morbidity. the only factors that had a significant associ· ation with morbidity were the child's age, ethnicity and type of toilet facility. however, all measures of socioeconomic status (mother's education, socioeconomic status, and mother's work status) had a significant effect on seeking outside care. age of child, severity of illness, type of illness and survival of father and mother were also significantly associated with seeking health care outside home. the results of this study have highlighted the need to address environmental conditions, basic amenities, and livelihood circumstances to improve child health in poor communities. the fact that socioeconomic indicators did not have a significant effect on prevalence of morbidity but were significant for health seeking behavior, indicate that while economic resources may have limited effect in preventing child illnesses when children are living in poor environmental conditions, being enlightened and having greater economic resources would mitigate the impact of the poor environmental conditions and reduce child mortality through better treatment of sick children. inequality in human life chances is about the most visible character of the third world urban space. f.conomic variability and social efficiency have often been fingered to justify such inequalities. within this separation households exist that share similar characteristics and are found to inhabit a given spatial unit of the 'city. the residential geography of cities in the third world is thus characterized by native areas whose core is made up of deteriorated slum property, poor living conditions and a decayed environment; features which personify deprivation in its unimaginable ma~t~de. there are .eviden~es that these conditions are manifested through disturbingly high levels of morbidity and mortality. ban · h h d-and a host of other factors (corrupt n, msens t ve leaders p, poor ur ty on t e one an , . · f · · · th t ) that suggest cracks in the levels and adherence to the prmc p es o socta usnce. ese governance, e c . . . . . ps £factors combine to reinforce the impacts of depnvat n and perpetuate these unpacts. by den· grou o . · "id . . bothh tifying health problems that are caused or driven by either matena _or soc a e~nvanon or , t e paper concludes that deprivation need not be accepted as a way. of hfe a~d a deliberate effon must be made to stem the tide of the on going levels of abject poverty m the third world. to the extent that income related poverty is about the most important of all ramifications of po~erty, efforts n_iu_st include fiscal empowerment of the poor in deprived areas like the inner c~ty. this will ~p~ove ~he willingness of such people to use facilities of care because they are able to effectively demand t, smce m real sense there is no such thing as free medical services. ). there were men with hiv-infection included in the present study (mean age and education of . (sd= . ) and . (sd= . ), respectively). a series of multiple regressions were used to examine the unique contributions of symptom burden (depression, cognitive, pain, fatigue), neuropsychologic al impairment (psychomotor efficiency), demographics (age and education) and hiv disease (cdc- staging) on iirs total score and jirs subscores: ( ) activities of daily living (work, recreation, diet, health, finances); ( ) psychosocial functioning (e.g., self-expression, community involvement); and ( ) intimacy (sex life and relationship with partner). resnlts: total iirs score (r " . ) was associated with aids diagnosis (ii= . , p < . ) and symptoms of pain (ii= - . , p < . ), fatigue (ji = - . , p < . ) and cognitive difficulties (p = . , p < . ). for the three dimensions of the iirs, multiple regression results revealed: ( ) activities of daily living (r = . ) were associated with aids diagnosis (ii = . , p < . ) and symptoms of pain <p =- j , p < . ), fatigue (ji = - . , p < . ) and cognitive difficulties (ji = . , p < . ); ( ) psychosocial functioning (r = . ) was associated with was associated with symptoms of fatigue (ii= - . , p < . ) and cognitive difficulties <ii = . , p < . ); and ( ) intimacy (r = . ) was related to was associated with symptoms of fatigue (ij = - . , p < . ) and cognitive difficulties (ii= . , p < . ). methods: the sif evaluation methodology involves a comprehensive database located at the sif, a randomly selected p~o.spective coh~rt of sif users, and two pre-existing external control cohorts. . . ~ts: in addmon to reporting process data and descriptions of the sif users, we report on data indicating that the establishment of the sif has been independently associated with reductions in public ~':°g ~ (p <?. ), and syringe sharing (aor = . [ %ci: . _ . ); p = . ) and other unsafe m ecnon pract _ces_ (p ~ . ), and increased uptake of detox services (log-rank p = . ). as well, we report on da~ md cat ng that the sif has not prompted adverse changes in community drug use patterns. the vancouver sif has been well accepted by the target population, and while adve~ events s~c~ as overdoses have occurred, these events have been successfully managed. externally compiled data indicate that the sif has been associated with substantial declines in public disorder !'oster sessions v associated with injection drug use, syringe sharing, and increased uptake of detox services. as well, the establishment of the sif has not exacerbated community drug use patterns. ongoing evaluation activities wiu involve assessing the impact of the sif on a variety of outcomes, including infectious disease transmission, fatal overdose, and utilization of health and social services. city, brazil, - maria cristina almeida, waleska caiaffa, renato assum;iio, and fernando proietti dengue cases were described according to time, space, intensity, age, gender, onset of symptoms, residence address and census tract. spatial distribution of two groups (children and women over years old and men aged - ) were compared, under assumption that they have distinct behaviors regarding their displacement around the city. analysis included local moran index, ripley\\'s k function and recurrence of census areas over time. about , cases were identified in seven epidemic waves. concentration of cases in small areas, followed by a spread either in space or time suggested endemic patterns. regarding age-gender groups, distinct patterns suggested that residence might not be a good proxy in determining the local of infection for men aged - . dengue is shifting to endemic pattern in this city and transmission may not be sole related to home environment, suggesting that additional spatial information must be couected aiming efficient control measures. murukan kandamuthan and subodh kandamuthan lnl uduction: illness or disablement of a child may affect the economic functioning of a family as it alters the employment pattern and earnings of parents this paper tries to assess the health status and the quality of life of disabled children, and how generally, severe disablement in a child affected their fami-lies\' finances, and to quantify any such effects the problems faced by children in their home and to provide information relevant to the formation of criteria for new or increased cash support. methods: a case-control study was done in the thiruvananthapuram district, capital city of kerala by selecting a random sample of families with severely disabled children below years and a random sample of normal children matched for age and occupation of the parents. comparative and subjective data were collected. money spent on children\'s medical care, loss of earnings of the parents, and other economic burden to the family due to the disability of the child in the family. a discriminant analysis along with univariate analysis was done to assess the factors that mostly differentiate the disabled and normal children. the mean expenditure of the families with a disabled child was rs. /-per month, which is significantly higher than the corresponding expenditure of rs. /-per month of families with normal child, (t= . , p <. ). of the disabled children, % were not getting any social security payments and % had no special concessions for medical and other educational purposes. the analysis of income and expenditure pattern indicated that financial impact of disablement in a child on the family is significant. the discriminant model results revealed that medical expenditure was a significant variable that differentiated the disabled and normal child. the study found that the health status and quality of life of the disabled children were far from satisfactory. this in fact affected the economic status of the disabled children. in addition to reduced earnings, there are extra costs for disabled children for travel, domestic help, medical care, and health care expenditures (hospital care, physician services, dentistry, drugs and others) for disabled individuals. a strong case can be made for their long-term care by improving the financial support to such families possibly through the introduction of an allowance specially to compensate for the earnings lost by parents due to the disability of their children. widows in india are considered disadvantage group of population. because they are characterized by pangs of separation from spouse, and consequently they go through mental agony, social ~s?lation, and economic dislocation. in addition to these, poverty, landlessness, homelessness, malnutr non etc. endure serious deterioration of their health. according to census , there are million women are widowed and one fourth live in urban areas. there is a noticeable increase of urban widows from the previous census (almost half). the paper examines the type of disability and chronic ailment borne among elderly widows ( years and above). fifty second round of national sample survey aske~ the individuals three sets of questions regarding their health: their status of health, whether they are physically poster sessions v immobile, and whether or not they have had any specific chronic illnesses. although health infrastruc- · ble ·n urban setting in india as compared to rural counterpart, prevalence of tures are arge y ava a . . . . . h · · · h h"gher "n urban areas analysis shows omt problems m old age qmte common c romc ness is muc • . . and nearly percent elderly widows are suffering from this ail'. ent in urban areas. one-fifth of widows are suffering from high/low bp. heart disease, diabetes and urinary problems are much ~ore pre~alent in urban areas though disabilities is comparatively lower in urban areas. however, the d~fference is not statistically significant. disability with respect to vision is more pronounced and one third_ of the total respondents have problems regarding eyesight. one fifths ofdderly widows are _ha.rd of hearing. though disability increases with age, similar trend is not observed with respect to chr~mc illness. the percepnon of self-health among elderly becomes poorer as their age increases. the perceived health status need not be always corrected to objective indicators of health. it is noted that around per~en~ of those perceived their health as "excellenr" or "very good" are found to be suffering from chrome disease of omts and percent have visual disability. to conclude an elderly widow living in urban areas have similar health problems as she jives in rural areas. this can be easily explained, as they were not made aware to seek treatment from health facility. in addition, the study shows that the economic conditions are deterrent factors for their illness. it is essential that intervention should be taken up improving their economic conditions so that wellbeing of this most vulnerable group of the society can be taken care of. heart failure is a disease that affects nearly million people world wide. the united states alone accounts for one third of this population. blacks are stricken with heart failure twice as often as whites. men are more likely to develop chf than women, however since the majority of the chf population are seniors, there are actually more women than men. congestive heart failure (chf) has become a pandemic. as the baby boomer generation ages, the number of chf cases will rise dramatically. (young, j. & mills, r., ). after the age of , each proceeding decade doubles the incidence level of chf. the average person has a in chance of developing heart failure within their lifespan according to the framingham study (nih.gov). individuals aging or older compose over half of the entire documented heart failure population. in this age group, chf is the leading cause of hospitalization. many patients are continuously readmitted. it is estimated that the chf population will increase by one million within the next years. mortality was at % for a two year period and % for five years (young, j. & mills, r., ). data from: aha as technology evolves, the understanding of the nature of heart failure has become clearer. in the beginning of the th century heart failure was diagnosed as a condition that presented with edema (swelling) in the extremities caused by water retention. treatment in the beginning of the past cenrury was limited to abdominal drainage and diuretics. the discovery of the heart's insufficient pumping ability in heart failure patients lead to new surgeries and devices such as heart transplants and ventricular assist devices (bridge until transplant is available). ventricular hypertrophy often occurs before the development of heart failure. hypertrophy is the term given for cardiac remodeling. cardiac remodeling also includes chamber dilation (young, j. & mills, r., ) . chf financial burden the us healthcare financing administration has ranked chf as the highest cost illness in the diagnose-related area. direct costs related to chf in the us for were nearly $ billion. these costs included hospital admissions, physician fees, home health aids and medications. indirect costs were over $ billion in . loss of jobs or death due to chf was the criteria for this category (young, j. & mills, r., ). . introduction: the injection drug user quality of life scale (iduqol) is a relatively new scale ~es gned to capture the unique and individual circumstances that determine quality of life among injection drug users (idus). the life areas comprising the instrument were based on the research literature an~ ~ocus group discussions with idus. the purpose of the present study was to evaluate the content validity of t.h~ iduqol using judgmental methods based on subject matter experts' (smes) ratings. content vah~ ty refers to the de~ee to which elements of an assessment tool are representative of the construct of interest and appropnate for a given population. methods: data were obtained from six smes, from the field of idu research and practice, who ~ere. asked t.o evaluate various aspects of the iduqol, including the title of the instrument, administraon instruchons, clarity of the names and descriptions of each life area, response format, scoring instruc-no~s and examples, record form and whether each life area should be included in the instrument. sme ratings were made using either or point scales. sm es were also given the opportunity to comment on poster sessions v each section and to suggest whether important life areas had been overlooked, were redundant, not relevant, or in need of revision. (cvi) and the ~v.erage ~eviation index (ad) were used to evaluate smes' agreement on ratings of the content vahd ty vanables. both measures provided support for the content validity of various aspects of the iduqol, although results from the stricter ad index noted some areas of disagreement, including the description of particular life areas (e.g., being useful, drugs, sex), the inclusion of some life areas (e.g., drug treatment, education, independence and free choice), and the ease of the scoring instructions. the findings from this study provide (a) content validity evidence to support the use of the iduqol as well as (b) recommendations to suengthen both the instrument (e.g., improved descriptions for some items) and the accompanying administration and scoring manual (e.g., an easier scoring template). changes made to the instrument and its manual make the iduqol even easier for researchers, practitioners, and program evaluators to use as a way of assessing, and tracking changes over time or as a result of interventions in, quality of life in injection drug users. introduction: strict adherence to prescribed regimens is required to derive maximal benefit from highly active antireuoviral therapy (haart) in persons living with hiv/aids (phas). adherence is a key determinant of the degree and durability of viral suppression. adherence is also essential in reducing the spread of hiv and ensuring that today's treatments remain effective for as long as possible. the objective of this study is to conduct a systematic review of the research literature on the effectiveness of education and patient support strategies for improving adherence to haart. methods: a systematic search of the core databases was performed from january until may . randomized controlled trials examining the effectiveness of education and patient support interventions to improve the adherence to haart were considered for inclusion. only those studies that measured adherence at a minimum of six weeks were included. study selection, quality assessments, and data abstraction were performed independently by two reviewers. results: study heterogeneity with respect to differing populations, interventions, outcomes, and length of follow-up did not allow for meta-analysis. we included studies involving , ph as. sample sizes ranged from to . study duration ranged from a single session to a variable number of sessions delivered over one year. many of the interventions involved the provision of an educational component to improve adherence and often addressed strategies to overcome barriers to adherence and the development of problem-solving skills. they ranged from simple interventions (e.g., the provision of reminder devices) to complex interventions (e.g., cognitive-behavioural therapy delivered by licensed psychologists). eleven of the studies demonstrated a statistically significant advantage associated with the described adherence intervention. the advantage associated with adherence outcomes does not seem to translate into the more clinically relevant outcome of viral suppression. only out of studies that reported virological or immunological results found a significant effect associated with the intervention. the studies had several methodological shortcomings. conclusions: there is a need for standardization and methodological rigour in the conduct of adherence trials. some interventions may have a significant impact on improving adherence. further research is required before any specific adherence improving strategy can confidently be incorporated into standard clinical practice. focus groups were used to collect data from a purposive sample of treatmentexperienced participants. focus group data were analyzed using a grou~ded t~eory appr~ach. i:ne themes identified from the interviews were used to identify the effects of ant rerrov rals on quality of hfe. the content of the mos-hiv was appraised against the themes identified from our analysis. participants also completed the mos-hiv survey and were asked whether the survey covered all important aspects of quality of life on antiretroviral medications. results: five key informant interviews and five focus groups with participants were used to identify effects on quality of life that are not directly ~aptu~ed by_ the mos-hiv health survey. our~~ showed that our participants described quality of hfe as mvol~mg_ a set of ~rsonal trad~ffs ~stay alive. in most cases, worsened quality of life was traded-off for gams ~ longevlty from ~~canon use. these eff~'ts or tradeoffs included: ( ) downstream consequences of side effects and toxlanes; ( ) loss of lrufe. pende~t decision-making privileges, ( ) having to choose drugs over ~areer; (~)burden of medication-taking responsibilities; and ( ) living life under a pretense and havmg to hide-the net effect of the tradeoffs, or "prices" to pay led to what was described as "longevity with hn without hope and future~. conclusion: our study highlights five major themes summarizing the impact of haart on quality on life, and suggests that currently used scales for measuring quality of life do not a~atd~ cap~e these findings and the associated consequences. the conceptual framework for measuring quality of bfe in hiv should be reconsidered in order to better capture the important effects of the medications on quality of life. this may involve widening the boundaries of the definition of health-related quality oflife to include aspects such as finances, employment, and housing. we should further consider the development of a haart adverse effect specific instrument, using a broad definition of adverse effect in order to capture all types of adverse impacts of hiv treatments on quality of life. introduction: measles (rubeola) is the most contagious vaccine preventable disease of humans. while cases of measles are uncommon in canada today, the disease continues to be a leading cause of morbidity and death in the developing world. as a result of human migration, measles cases still occur in canada, primari~ among immigrants, returning travelers, and other susceptible groups. canada's national advisory council on immunizations (naci) recommends that immigrants without records of measles immunization be considered for vac:cination since these individuals may not have been appropriately vaccinated in their native country. on rhe other hand, natural immunity to measles in immigrants is likely to be high given the high incidence of disease in developing parts of rhe world. thus it remains unclear if the most efficient strategy to achieve high levels of measles immunity in immigrants should entail initial serologic screening followed by vaccination of susceptible individuals or preemptive vaccination of all persons lacking immunization records. understanding the epidemiology of measles immunity in immigrant populations could help guide such decisions. . methods: we identified adults, years of age or older, who were screened for serologtc immunity to measles as part of a recently developed screening protocol at a downtown toronto community health centre. we subsequently determined if these individuals had any immunization records from their native country or from within canada. we then examined the relationship between several demographic factors and immunity to measles. results: % of immigrants had no prior immunization records. overall, % of the immigrants rested for immunity to measles were found to be immune; % of those under the age of , % of those between and years of age, % of those between and years of age, and % of those years of age or older. gender, education status, household income, and immigration status were nor ~ssoc ated wirh immunity to measles. immunity to measles was lowest among immigrants from eastern europe ( ~%), whil~ immigrants from other regions of the globe had greater than % immunity. c~lusrons: immigrants and refugees appear to have reasonably high levels of immunity to mea· sics, possibly related to natural infection wirh the measles virus. immigrants from eastern europe appear to have slightly. lower l~vel~ of immunity to the measles virus. universal screening for immunity to meales or preemptive vac:cmauon may both be inefficient public health strategies, however further research is needed to corroborate these findings. nancy r~ss, stephane tremblay, saeeda khan, daniel crouse, mark tremblay, and jean-marie berrhelor o~ve: the speed of the rise in obesity in places like canada suggests that rather than a shift in the gcncnc_ com~sirion of the population, the root of the obesity pandemic is an environment that suprrs ~besity. this paper examines the influence of neighbourhood and metropolitan characteristics. bour~lts:. while accounting. for individual socio-demographics and behaviours, residents of neighs with a large proportmn of poorly educated individuals had higher bmis than those living in poster sessions v neighbourhoods with more highly educated individuals (p <. ). residing in a neighbourhood with a high proportion of recent immigrants was associated with lower bmi for men (p<. ), but not women. neighbourhood dwelling density, a proxy for walkability, was not associated with bm! for either sex. metropolitan sprawl was associated with higher male bmi (p=. ) but the effect was negligible for women (p=. ). bmi was significantly lower for women (p<. ) living in a city in the province of quebec, even after accounting for the influence of individual and neighbourhood covariates. conclusions: bm! was strongly patterned by individual-level social position in urban canada. the incremental influence on bmi of neighbourhood related to the neighbourhood's social conditions and not to their physical form. metropolitan sprawl was associated with higher bm! for men, a group with longer car-dependent commutes than women. the findings suggest that both individuals and their environments (both social and physical) influence the distribution of bmi in urban populations. introduction: urban environments have been linked to a range of human health issues. in singapore, prevalence of end stage renal disease (esrd) is predicted to rise ( in to in , kidney international, vol. , . the clinical and economic burden of esrd has promoted development of strategies aimed at preventing the development and progression of chronic kidney disease. these include population based screening programs such as the national kidney foundation, sin-gapore\'s (nkfs) "partnership for prevention."the program, aims to reduce incidence of esrd through comprehensive intervention and screening for early detection of urinary abnormalities, blood pressure (bp), and random blood glucose (rbg). objective of this study is to examine gender, race and age wise prevalence of elevated bp, rbg and proteinuria. methodology: this current analysis includes , subjects, age to years, who underwent screening during september and december . participants were asked to give demographic information and the history of diseases. tests were given to get clinical information such as proteinuria, blood glucose, sbp, and dbp. blood pressure abnormalities were defined according to ]nc vi criteria. proteinuria was defined as the presence of plus or greater protein (equivalent to> mg/di) on dipstick analysis. results: there were , ( . %) males. racial distribution was chinese ( . % ), malay ( . % ), indians ( . %) and others ( . % ).among participants, who were apparently "healthy" (asymptomatic and without history of dm, ht, or kd), gender and race wise % prevalence of elevated (bp> / ), rbg (> mg/di) and positive urine dipstick for protein was as follows male: ( . ; . ; . ) female:( . ; . ; . ) chinese:( . ; . ; ) malay: ( . ; . ; . ) indian:( . ; . ; . ) others: ( . ; . ; . ) total:(l . , . , . ). percentage of participants with more than one abnormality were as follows. those with bp> / mmhg, % also had rbg> mg/dl and . % had proteinuria> i. those with rbg> mgldl, % also had proteinuria> and % had bp> / mmhg. those with proteinuria> , % also had rbg> mg/dl, and % had bp> / mmhg. conclusion: we conclude that sub clinical abnormalities in urinalysis, bp and rbg readings are prevalent across all genders and racial groups in the adult population. the overlap of abnormalities, point towards the high risk for esrd as well as cardiovascular disease. this indicates the urgent need for population based programs aimed at creating awareness, and initiatives to control and retard progression of disease. introduction: various theories have been proposed that link differential psychological vulnerability to health outcomes, including developmental theories about attachment, separation, and the formation of psychopathology. research in the area of psychosomatic medicine suggests an association between attachment style and physical illness, with stress as a mediator. there are two main hypotheses explored in the present study: ( t) that individuals living with hiv who were upsychologically vulne~able" at study entry would be more likely to experience symptoms of depression, anxiety and phys ca! illness over. the course of the -month study period; and ( ) life stressors and social support would mediate the relat nship between psychological vulnerability and the psychological ~nd physical outcomes. . (rsles), state-trait anxiety inventory (stai), beck depr~ssi~n lnvento~ (bdi), and~ _ -item pbys~i symptoms inventory. we characterized participants as havmg psychological vulnerability and low resilience" as scoring above on the raas (insecure attachment) or above on the das (negative expectations about oneself). . . . . . " . . ,, . results: at baseline, % of parnc pants were classified as havmg low resilience. focusmg on anxiety, the average cumulative stai score of the low-resilience group was significandy hi~e~ than that of the high-resilience group ( . sd= . versus . sd= . ; f(l, )= . , p <. ). similar results were obtained for bdi and physical symptoms (f( , )= . , p<. and f( , )= . , p<. , respec· tively). after controlling for resilience, the effects of variance in life stres".°rs averaged over time wa~ a_sig· nificant predictor of depressive and physical symptoms, but not of anxiety. ho~e_ver, these assooan~s became non-significant when four participants with high values were removed. s id larly, after controlling for resilience, the effects of variance in social support averaged over time became insignificant. conclusion: not only did "low resilience" predict poor psychological and physical outcomes, it was also predictive of life events and social support; that is, individuals who were low in resilience were more likely to experience more life events and poorer social support than individuals who were resilient. for individuals with vulnerability to physical, psychological, and social outcomes, there is need to develop and test interventions to improve health outcomes in this group. rajat kapoor, ruby gupta, and jugal kishore introduction: young people in india represent almost one-fourth of the total population. they face significant risks related to sexual and reproductive health. many lack the information and skills neces· sary to make informed sexual and reproductive health choices. objective: to study the level of awareness about contraceptives among youth residing in urban and rural areas of delhi. method: a sample of youths was selected from barwala (rural; n= ) and balmiki basti (urban slums; n= ) the field practice areas of the department of community medicine, maulana azad medical college, in delhi. a pre-tested questionnaire was used to collect the information. when/(calen· dar time), by , fisher exact and t were appliedxwhom (authors?). statistical tests such as as appropriate. result: nearly out of ( . %) youth had heard of at least one type of contraceptive and majority ( . %) had heard about condoms. however, awareness regarding usage of contraceptives was as low as . % for terminal methods to . % for condom. condom was the best technique before and after marriage and also after childbirth. the difference in rural and urban groups was statistically signif· icant (p=. , give confidence interval too, if you provide the exact p value). youth knew that contra· ceptives were easily available ( %), mainly at dispensary ( . %) and chemist shops ( . %). only . % knew about emergency contraception. only advantage of contraceptives cited was population con· trol ( . %); however, . % believed that they could also control hiv transmission. awareness of side effects was poor among both the groups but the differences were statistically significant for pills (p= . ). media was the main source of information ( %). majority of youth was willing to discuss a~ut contraceptive with their spouse ( . %), but not with others. . % youth believed that people in their age group use contraceptives. % of youth accepted that they had used contraceptives at least once. % felt children in family is appropriate, but only . % believed in year spacing. . conclusion: awareness about contraceptives is vital for youth to protect their sexual and reproduc· tive health .. knowledge about terminal methods, emergency contraception, and side effects of various contraceptives need to be strengthened in mass media and contraceptive awareness campaigns. mdbot:ls: elderly aged + were interviewed in poor communities in beirut the capital of f:ebanon, ~e of which is a palestinia~. refugee camp. depression was assessed using the i -item geriat· nc depressi~n score (~l?s- ). specific q~estions relating to the aspects of religiosity were asked as well as questions perta rung to demographic, psychosocial and health-related variables. results: depression was prevalent in % of the interviewed elderly with the highest proportion being in the palestinian refugee camp ( %). mosque attendance significantly reduced the odds of being depressed only for the palestinian respondents. depression was further associated, in particular communities, with low satisfaction with income, functional disability, and illness during last year. condiuion: religious practice, which was only related to depression among the refugee population, is discussed as more of an indicator of social cohesion, solidarity than an aspect of religiosity. furthermore, it has been suggested that minority groups rely on religious stratagems to cope with their pain more than other groups. implications of findings are discussed with particular relevance to the populations studied. nearly thirty percent of india's population lives in urban areas. the outcome of urbanization has resulted in rapid growth of urban slums. in a mega-city chennai, the slum populations ( . percent) face greater health hazards due to overcrowding, poor sanitation, lack of access to safe drinking water and environmental pollution. amongst the slum population the health of women and children are most neglected, resulting in burden of both communicable and non-communicable diseases. the focus of the paper is to present the epidemiology profile of children (below years) in slums of chennai, their health status, hygiene and nutritional factors, the social response to health, the trends in child health and urbanization over a decade, the health accessibility factors, the role of gender in health care and assessment impact of health education to children. the available data prove that child health in slums is worse than rural areas. though the slum population is decreasing there is a need to explore the program intervention and carry out surveys for collecting data on some specific health implications of the slum children. objective: during the summer of there was a heat wave in central europe, producing an excess number of deaths in many countries including spain. the city of barcelona was one of the places in spain where temperatures often surpassed the excess heat threshold related with an increase in mortality. the objective of the study was to determine whether the excess of mortality which occurred in barcelona was dependent on age, gender or educational level, important but often neglected dimensions of heat wave-related studies. methods: barcelona, the second largest city in spain ( , , inhabitants in ) , is located on the north eastern coast. we included all deaths of residents of barcelona older than years that occurred in the city during the months of june, july and august of and also during the same months during the preceding years. all the analyses were performed for each sex separately. the daily number of deaths in the year was compared with the mean daily number of deaths for the period - for each educational level. poisson regression models were fitted to obtain the rr of death in with respect to the period - for each educational level and age group. results: the excess of mortality during that summer was more important for women than for men and among older ages. although the increase was observed in all educational groups, in some age-groups the increase was larger for people with less than primary education. for example, for women in the group aged - , the rr of dying for compared to - for women with no education was . ( %ci: . - - ) and for women with primary education or higher was . ( %ci: . - . ). when we consider the number of excess deaths, for total mortality (>= years) the excess numbers were higher for those with no education ( . for women and . for men) and those with less than primary education ( . for women and - for men) than those with more than primary edm:ation ( . for women and - . for men). conclusion: age, gender and educational level were important in the barcelona heat wave. it is necessary to implement response plans to reduce heat morbidity and mortality. policies should he addressed to all population but also focusing particularly to the oldest population of low educational level. introduction: recently there has been much public discourse on homelessness and its imp~ct on health. measures have intensified to get people off the street into permanent housing. for maximum v poster sessions success it is important to first determine the needs of those to be housed. their views on housing and support requirements have to be considered, as th~y ar~ the ones affected. as few res.earch studies mclude the perspectives of homeless people themselves, httle is known on ho~ they e~penence the mpacrs on their health and what kinds of supports they believe they need to obtain housing and stay housed. the purpose of this study was to add the perspectives of homeless people to the discourse, based in the assumption that they are the experts on their own situations and needs. housing is seen as a major deter· minant of health. the research questions were: what are the effects of homelessness on health? what kind of supports are needed for homeless people to get off the street? both questions sought the views of homeless individuals on these issues. methods: this study is qualitative, descriptive, exploratory. semi-structured interviews were conducted with homeless persons on street corners, in parks and drop-ins. subsequently a thematic analysis was carried out on the data. results: the findings show that individuals' experiences of homelessness deeply affect their health. apart from physical impacts all talked about how their emotional health and self-esteem are affected. the system itself, rather than being useful, was often perceived as disabling and dehumanizing, resulting in hopelessness and resignation to life on the street. neither welfare nor minimum wage jobs are sufficient to live and pay rent. educational upgrading and job training, rather than enforced idleness, are desired by most initially. in general, the longer persons were homeless, the more they fell into patterned cycles of shelter /street life, temporary employment /unemployment, sometimes addictions and often unsuccessful housing episodes. conclusions: participants believe that resources should be put into training and education for acquisition of job skills and confidence to avoid homelessness or minimize its duration. to afford housing low-income people and welfare recipients need subsidies. early interventions, 'housing first', more humane and efficient processes for negotiating the welfare system, respectful treatment by service providers and some extra financial support in crisis initially, were suggested as helpful for avoiding homelessness altogether or helping most homeless people to leave the street. this study is a national homelessness initiative funded analysis examining the experiences and perceptions of street youth vis-a-vis their health/wellness status. through in-depth interviews with street youth in halifax, montreal, toronto, calgary, ottawa and vancouver, this paper explores healthy and not-so healthy practices of young people living on the street. qualitative interviews with health/ social service providers complement the analysis. more specifically, the investigation uncovers how street youth understand health and wellness; how they define good and bad health; and their experiences in accessing diverse health services. findings suggest that living on the street impacts physical, emotional and spiritual well·being, leading to cycles of despair, anger and helplessness. the majority of street youth services act as "brokers" for young people who desire health care services yet refuse to approach formal heal~h care organizational structures. as such, this study also provides case examples of promising youth services across canada who are emerging as critical spaces for street youth to heal from the ravages of ~treet cultur~. as young people increasingly make up a substantial proportion of the homeless population in canada, it becomes urgent to explore the multiple ways in which we can support them to regain a sense of wellbeing and "citizenship." p - (c) health and livelihood implications of marginalization of slum dwellers in provision of water and sanitation services in nairobi city elizabeth kimani, eliya zulu, and chi-chi undie . ~ntrodfldion: un-habitat estimates that % of urban residents in kenya live in slums; yet due to their illegal status, they are not provided with basic services such as water sanitation and health care. ~nseque~tly, the services are provided by vendors who typically provide' poor services at exorbitant prices .. this paper investigates how the inequality in provision of basic services affects health and livelihood circumstances of the poor residents of nairobi slums . . methods: this study uses qualitative and quantitative data collected through the ongoing longitudmal .health and demographic study conducted by the african population and health research center m slum communities in n ·rob" w d · · · · ai . e use escnpnve analytical and qualitative techmques to assess h~w concerns relating to water supply and environmental sanitation services rank among the c~mmumty's general and health needs/concerns, and how this context affect their health and livelihood circumstances. results: water ( %) and sanitation ( %) were the most commonly reported health needs and also key among general needs (after unemployment) among slum dwellers. water and sanitation services are mainly provided by exploitative vendors who operate without any regulatory mechanism and charge exorbitantly for their poor services. for instance slum residents pay about times more for water than non-slum households. water supply is irregular and residents often go for a week without water; prices are hiked and hygiene is compromised during such shortages. most houses do not have toilets and residents have to use commercial toilets or adopt unorthodox means such as disposing of their excreta in the nearby bushes or plastic bags that they throw in the open. as a direct result of the poor environmental conditions and inaccessible health services, slum residents are not only sicker, they are also less likely to utilise health services and consequently, more likely to die than non-slum residents. for instance, the prevalence of diarrhoea among children in the slums was % compared to % in nairobi as a whole and % in rural areas, while under-five mortality rates were / , / and / respectively. the results demonstrate the need for change in governments' policies that deprive the rapidly expanding urban poor population of basic services and regulatory mechanisms that would protect them from exploitation. the poor environmental sanitation and lack of basic services compound slum residents' poverty since they pay much more for the relatively poor services than their non-slum counterparts, and also increase their vulnerability to infectious diseases and mortality. since iepas've been working in harm reduction becoming the pioneer in latin america that brought this methodology for brazil. nowadays the main goal is to expand this strategy in the region and strive to change the drug policy in brazil. in this way harm reduction: health and citizenship program work in two areas to promote the citizenship of !du and for people living with hiv/aids offering law assistance for this population and outreach work for needle exchange to reduce damages and dissemination of hiv/aids/hepatit is. the methodology used in outreach work is peer education, needle exchange, condoms and folders distribution to reduce damages and the dissemination of diseases like hiv/aids/hepatitis besides counseling to search for basic health and rights are activities in this program. law attendance for the target population at iepas headquarters every week in order to provide law assistance that includes only supply people with correct law information or file a lawsuit. presentations in harm reduction and drug policy to expand these subjects for police chiefs and governmental in the last year attended !du and nidu reached and . needles and syringes exchanged. in law assistance ( people living with aids, drug users, inject drug users, were not in profile) people attended. lawsuits filed lawsuits in current activity. broadcasting of the harm reduction strategies by the press helps to move the public opinion, gather supporters and diminish controversies regarding such actions. a majority number of police officer doesn't know the existence of this policy. it's still polemic discuss this subject in this part of population. women remain one of the most under seviced segments of the nigerian populationand a focus on their health and other needs is of special importance.the singular focus of the nigerian family welfare program is mostly on demographic targets by seeking to increase contraceptive prevalence.this has meant the neglect of many areas of of women's reproductive health. reproductive health is affected by a variety of socio-cultural and biological factors on on e hand and the quality of the service delivery system and its responsiveness on the other.a woman's based approach is one which responds to the needs of the adult woman and adolescent girls in a culturally sensitive manner.women's unequal access to resources including health care is well known in nigeria in which stark gender disparities are a reality .maternal health activities are unbalanced,focusi ng on immunisation and provision of iron and folic acid,rather than on sustained care of women or on the detection and referral of high risk cases. a cross-sectional study of a municipal government -owned hospitalfrom each of the geo-political regions in igeria was carried out (atotal of ce~ters) .. as _part ~f t~e re.search, the h~spital records were uesd as a background in addition to a -week mtens ve mvesuganon m the obstemc and gynecology departments. . . . : little is known for example of the extent of gynecological morbtdtty among women; the little known suggest that teh majority suffer from one or more reproductive tr~ct infect~ons. although abortion is widespread, it continues to be performed under ilegal and unsafe condmons. with the growing v poster sessions hiv pandemic, while high riskgroups such ascomn;iercial sex workers and their clients have been studied, little has been accomplished in the large populat ns, and particularly among women, regardmgstd an hiv education. . . conclusions: programs of various governmentalor non-governmental agen,c es to mvolve strategies to broaden the narrow focus of services, and more importan~, to put wo~en s reproducnve health services and information needs in the forefront are urgently required. there is a n~d to reonent commuication and education activities to incorprate a wider interpretation of reproducnve health, to focus on the varying information needs of women, men, and youth and to the media most suitable to convey information to these diverse groups on reproductive health. introduction: it is estimated that there are - youths living on the streets, on their own with the assistance of social services or in poverty with a parent in ottawa. this population is under-serviced in many areas including health care. many of these adolescents are uncomfortable or unable to access the health care system through conventional methods and have been treated in walk-in clinics and emergency rooms without ongoing follow up. in march , the ontario government provided the ct lamont institute with a grant to open an interdisciplinary and teaching medical/dental clinic for street youth in a drop-in center in downtown ottawa. bringing community organizations together to provide primary medical care and dental hygiene to the streetyouths of ottawa ages - , it is staffed by a family physician, family medicine residents, a nurse practitioner, public health nurses, a dental hygienist, dental hygiene students and a chiropodist who link to social services already provided at the centre including housing, life skills programs and counselling. project objectives: . to improve the health of high risk youth by providing accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. . to model and teach interdisciplinary adolescent care to undergraduate medical students, family medicine residents and dental hygiene students. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative:a) semi-structured interviews b) focus groups with youth quantitative:a) electronic medical records for months b) records (budget, photos, project information). results: in progress-results from first months available in august . early results suggest that locating the clinic in a safe and familiar environment is a key factor in attracting the over youths the clinic has seen to date. other findings include the prevalence of preventative interventions including vaccinations, std testing and prenatal care. the poster presentation will present these and other impacts that the clinic has had on the health of the youth in the first year of the study. conclusions: ) the clinic has improved the health of ottawa streetyouth and will continue beyond the initial pilot project phase. ) this project demonstrates that with strong community partnerships, it is possible meet make healthcare more accessible for urban youth. right to health care campaign by s.j.chander, community health cell, bangalore, india. introduction: the people's health movement in india launched a campaign known as 'right to health care' during the silver jubilee year of the alma ata declaration of 'health for all' by ad in collahoration with the national human rights commission (nhrc). the aim of the campaign was to establish the 'right to health care' as a basic human right and to address structural deficiencies in the pubic health care system and unregulated private sector . . methods: as part of the campaign a public hearing was organized in a slum in bangalore. former chairman of the nhrc chaired the hearing panel, consisting of a senior health official and other eminent people in the city. detailed documentation of individual case studies on 'denial of access to health care' in different parts of the city was carried out using a specific format. the focus was on cases where denial of health services has led to loss of life, physical damage or severe financial losses to the patient. results: _fourte_en people, except one who had accessed a private clinic, presented their testimonies of their experiences m accessing the public health care services in government health centres. all the people, e_xcept_ one person who spontaneously shared her testimony, were identified by the organizations worki_ng with the slum dwellers. corruption and ill treatment were the main issues of concern to the people. five of the fourteen testimonies presented resulted in death due to negligence. the public health cen· n:s not only demand money for the supposedly free services but also ill-treats them with verbal abuse. five of these fourteen case studies were presented before the national human right commission. the poster sessions v nhrc has asked the government health officials to look into the cases that were presented and to rectify the anomalies in the system. as a result of the public hearing held in the slum, the nhrc identified urban health as one of key areas for focus during the national public hearing. cond#sion: a campaign is necessary to check the corrupted public health care system and a covetous private health care system. it helps people to understand the structure and functioning of public health care system and to assert their right to assess heath care. the public hearings or people's tribunals held during the campaign are an instrument in making the public health system accountable. ps- (a) violence among women who inject drugs nadia fairbairn, jo-anne stoltz, evan wood, kathy li, julio montaner, and thomas kerr background/object ives: violence is a major cause of morbidity and mortality among women living in urban settings. though it is widely recognized that violence is endemic to inner-city illicit drug markets, little is known about violence experienced by women injection drug users (!du). therefore, the present analyses were conducted to evaluate the prevalence of, and characteristics associated with, experiencing violence among a cohort of female idu in vancouver. methods: we evaluated factors associated with violence among female participants enrolled in the vancouver injection drug user study (vidus) using univariate analyses. we also examined self-reported relationships with the perpetrator of the attack and the nature of the violent attack. results: of the active iou followed between december , and may , , ( . %) had experienced violence during the last six months. variables positively associated with experiencing violence included: homelessness (or= . , % ci: . - . , p < . ), public injecting (or= . , % ci: . - . , p < . ), frequent crack use (or= . , % ci: . - . , p < . ), recent incarceration (or = . , % cl: . - . , p < . ), receiving help injecting (or = . , % cl: . - . , p < . ), shooting gallery attendance (or = . , % ci: . - . , p < . ), sex trade work (or = . , % cl: . - . , p < . ), frequent heroin injection (or= . , % cl: . - . , p < . ), and residence in the downtown eastside (odds ratio [or] = . , % ci: . - . , p < . ). variables negatively associated with experiencing violence included: being married or common-law (or = . . % ci: . - . , p < . ) and being in methadone treatment (or = . , % ci: . - . , p < . ). the most common perpetrators of the attack were acquaintances ( . %), strangers ( . %), police ( . %), or dealers ( . %). attacks were most frequently in the form of beatings ( . %), robberies ( . %), and assault with a weapon ( . %). conclusion: violence was a common experience among women !du in this cohort. being the victim of violence was associated with various factors, including homelessness and public injecting. these findings indicate the need for targeted prevention and support services, such as supportive housing programs and safer injection facilities, for women iou. introduction: although research on determinants of tobacco use among arab youth has been carried out at several ecologic levels, such research has included conceptual models and has compared the two different types of tobacco that are most commonly used among the lebanese youth, namely cigarette and argileh. this study uses the ecological model to investigate differences between the genders as related to the determinants of both cigarette and argileh use among youth. methodology: quantitative data was collected from youth in economically disadvantaged urban communities in beirut, the capital of lebanon. results: the results indicated that there are differences by gender at a variety of ecological levels of influence on smoking behavior. for cigarettes, gender differences were found in knowledge, peer, family, and community influences. for argileh, gender differences were found at the peer, family, and community l.evels. the differential prevalence of cigarette and argileh smoking between boys and girls s therefore understandable and partially explained by the variation in the interpersonal and community envi.ronment which surrounds them. interventions therefore need to be tailored to the specific needs of boys and girls. introduction: the objective of this study was to assess the relationship between parents' employment status and children' health among professional immigrant families in vancouver. our target communmes v poster sessions included immigrants from five ethnicity groups: south korean, indian, chine~e, ~ussian, and irani~ with professional degrees (i.e., mds, lawyers, engineers, ma?~ger~, and uru~ers ty professors) w h no relevant job to their professions and those who had been hvmg m the studied area at least for months. methodology: the participants were recruited by collaboration from three local community agencies and were interviewed individually during the fall of . ra#lts: totally, complete interviews were analyzed: from south-east asia, from south asia, from russia and other eastern europe. overall, . % were employed, . % were underemployed, % indicated they were unemployed. overall, . % were not satisfied with their current job. russians and other eastern europeans were most likely satisfied with their current job, while south-east asians were most satisfied from their life in canada. about % indicated that their spouses were not satisfied with their life in canada, while % believed that their children are very satisfied from their life in canada. in addition, around % said they were not satisfied from their family relationship in canada. while most of the responders ranked their own and their spouses' health status as either poor or very poor, jut % indicated that their first child's health was very poor. in most cases they ranked their children's health as excellent or very good. the results of this pilot study show that there is a need to create culturally specific child health and behavioral scales when conducting research in immigrant communities. for instance, in many asian cultures, it is customary for a parent either to praise their children profusely, or to condemn them. this cultural practice, called "saving face," can affect research results, as it might have affected the present study. necessary steps, therefore, are needed to revise the current standard health and behavioral scales for further studies by developing a new scale that is more relevant and culturally sensitive to the targeted immigrant families. metboda: database: national health survey (ministry of health www.msc.es). two thousand interviews were performed among madrid population ( . % of the whole); corresponded to older adults ( . % of the . million aged years and over). study sample constitutes . % ( out of ) of those older adults, who live in urban areas. demographic structure (by age and gender) of this population in relation to health services use (medical consultations, dentist visits, emergence services, hospitalisation) was studied using general linear model univariate procedure. a p . ), while age was associated with emergence services use ( % of the population: %, % and % of each age group) and hos~italisation ( % .oft~~ population: %, % and %, of each age ~oup) (p . ) was fou~d with respect to dennst v s ts ( % vs %), medical consultations ( % vs %), and emergence services use ( % vs %), while an association (p= . ) was found according to hospitalisation ( % vs %). age. an~ g~der interaction effect on health services use was not found (p> . ), but a trend towards bosp tal sanon (p= . ) could be considered. concl.uions: demographic structure of urban older adults is associated with two of the four health se~ices use studi~. a relation.ship ber_ween age. and hospital services use (emergence units and hospitalisanon), but not with ~ut-hosp tal sei:vices (medical and dentist consultations), was found. in addition ro age, gender also contnbutes to explam hospitalisation. . sexua experiences. we exammed the prevalence expenences relation to ethnic origin and other sociodemographic variables as wc i as y j die relation between unwanted sexual experiences, depression and agreuion. we did so for boys and prts separately. mdhods: data on unwanted sexual expcric:nces, depressive symptoms (ce.s-d), aggrc:uion (bohi-di and sociodemographic facron were collected by self-report quescionnairc:s administettd to students in the: nd grade (aged - ) of secondary schools in amsterdam, the netherlands. data on the nature ol unwanted sexual experiences were collected during penonal interviews by trained schoolnursn. ltaijtj: overall prevalences of unwanted sexual experiences for boys and girls were . % and . % respectively. unwanted sexual experiences were more often ttported by turkish ( . %), moroc· an ( . %) and surinamese/anrillian boys ( . %) than by dutch boys ( . %). moroccan and turkish girls, however, reported fewer unwanted sexual experiences (respectively . and . %) than durch girls did ( . %). depressive symptoms(or= . , cl= . - . ) covert agression ( r• . , cl• . - . ) and cmrt aggression (or= . , cl• . - . ) were more common in girls with an unwanted sexual experi· met. boys with an unwanted sexual experience reported more depressive symptoms (or= . ; cl• i . .l· . ) and oven agression (or= . , cl= . - . ) . of the reported unwanted sexual experiences rnpec· timy . % and . % were confirmed by male and female adolescents during a personal interview. cond sion: we ..:an conclude that the prevalence of unwanted sexual experiences among turkish and moroccan boys is disturbing. it is possible that unwanted sexual experiences are more reported hy boys who belong to a religion or culture where the virginity of girls is a maner of family honour and talking about sexuality is taboo. more boys than girls did not confirm their initial disdosurc of an lllwalltc:d sexual experience. the low rates of disclosure among boys suggcsu a necd to educ.:atc hcahh care providen and others who work with migrant boys in the recognition and repomng of exu.il ... iction. viramin a aupplc:tmntation i at .h'yo, till far from tafl'eted %. feedinit pracn~:n panku· lerty for new born earn demand lot of educatton ernpha a• cxdu ve hrealt fecdtnit for dnared rcnoj of months was observtd in only .s% of childrrn thoulh colckturm w. givm n rn% of mwly horn ct.ildrm. the proportion of children hclow- waz (malnounshrdl .con" a• h!jh •• . % anj "rt'i· acimy tc.. compared to data. mother's ~alth: from all is womm in ttprod~uvr •ill' poup, % were married and among marned w~ .\ % only w\"rt' u mic wmr cnntr.-:cruve mt h· odl % were married bdorc thc •ar of yean and % had thnr ftnc prcicnancy hcftitt dlt' •icr nf yean. the lt'f'vicn are not uutfactory or they arc adequate but nae unh ed opumally. of thote' l'h mothen who had deliverrd in last one year, % had nailed ntmaral eum nat on ira" oncc, .~o-... bad matt rhan four ttmn and ma ortty had heir tetanus toxotd tnin,"t or"'" nlht "'"'"· ljn r ned rn· win ronductrd . % dchvcnn and % had home deh\'t'oc'i. ~md~: the tervtcn unbud or u led are !tu than dnarame. the wr· l'kft provided are inadequate and on dechm reprcwnttng a looun t ~p of h hnto good coytti\#' ol wr· ncn. l!.ckground chanpng pnoriry cannoc be ruled out u °"" of thc coatnbutory bc f. ps-ii ia) dcpn:wioa aad anuccy ia mip'mu ia awccr._ many de wn, witco tui~bmjer. jack dekker, aart·jan lttkman, wim gonmc:n. and amoud verhoeff ~ a dutch commumry-bucd icudy thawed -moarh•·prc:yalm«i al . ' . kw anx · ay daorden and . % foi' dqrasion m anmttdam. nm .. p tficantly hlllhn than dwwhrft .. dw ~thew diffamca m pttyalcnca att probably rdarcd to tlk' largr populanoa of napaan ..\mturdam. <turkish %, moroccan %, and surinam % . lndttd. ic'wt'll ltudla hatt ~ ~ high prevalena rata among migrant poupi in rbe ncdwrlands. howc'ytt au ttlluchn iuffenod from wry low raporne rara, or med screnung talel thac lack a ~y yabdarcd ~ in order to study the prevalence of depression and anxiety, and the (barriers to) use of mental health care among the different ethnic groups the following study was recently conducted: . the study consisted of a two-step approach. the first step was mcl~ded m the ~ h ith survey of (ahs), and consisted of three screening scales for depression and aruaety (klo, g::q- and mhl- ). in this survey all respondents were asked_ permission for~ second app~ :' t consisted of a structured interview containing the cidi for aruaety and depression, and questtollllalres on health care seeking, amongst others. all respondents who gave permission for a second approachwere invited by jetter for a suuctured interview with multilingual interviewers at home at a preset date and time in the following week. . in total, dutch, turkish, moroccan and surinam respondents took part in the ahs and gave permission for a second approach. in the second step, fo~ ~ tur~h, m~roc can surinamese and dutch respondents, information from the extensive interview was available (res~nse rates between and % ). since the data collection was completed only recently (june ), prevalence estimates can be presented at the conference for the first time. we have shown that with this approach it is feasible to achieve an acceptable response rate in a study on mental health among migrants. therefore we expect that this study will provide reli· able estimates of depression and anxiety in the turkish, moroccan and surinam migrants in amstw! ~· for the first time. in addition, insight into mechanisms underlying the differences between and within ethnic groups and into barriers to mental health care will provide pretexts for the improvement of pre· vention and mental health care for migrant groups. this study aimed to determine spatial patterns of mortality and morbidity of five major health problems in an urban environment: homicides, pregnancy among adolescents ((< years old), asthma hospitalization among young children(< years old) and two mosquito-borne diseasesdengue and visceral leishmaniasis, during - . all events were obtained through the city health database and, subsequently, geoproccsscd using the address of residence and the geographical and administrative division of the municipality, composed by unit of planning (up), which in tum were formed, each one, by census tract units. two research questions were investigated: are there spatial patterns to the events dis· tribution, and moreover, are these patterns overlapping across space? we use thematic maps, index of comparative mortality/morbidity by up and the overlapped rank of the th worse up rates for each event. a spatial pattern of high rates of homicides, proportion of young mothers and hospitalization of asthma were overlapping in areas social and economically disadvantaged. for mosquito-borne diseases, high rates with great dispersion were found in unprivileged areas in contrast with very low rares among privileged ones. these results pointed toward a coexistence of heavier burden of diseases for those living in areas of the city where misery, poverty, lack of political public health may be modulating social health problems. a possible environmental intervention in one mosquito•bome disease might be playing a role in the occurrence of other. although with limitations, this study may provide useful information for a joint urban planning under the public perspective, articulated for use in health impact assessment. jalil safaei bdrod#aion: the association of socioeconomic status (ses), usually measured by income, and health status is an established result in bcalth studies. the poor and low income individuals have lower health sta· tus then_ those with higher incomes. in general. such finding has been usually obtained from sample surveys on speafic populations. a problem with many sample survey$ is that their results are not comparable aaoss ~ndaries as they may use different sampling methods, ask different questions, categorize the answers differently, or define groups differently. moreover, the sample data need to be standardized using the demographic: katurea of a ~ population. this study, however, uses the national population health survey (nphs) cycle public use mictodata files which is based on a common survey template ·~~three~ area in canada, namely montreal, toronto, and vancouver. it also utili .es the built-m stanclatdizaaon of the nphs data which accounts for its complex survey design. . ~:the stu~ usc:s two well-known measures of health inequalitythe relative index of meqaality and die concentranon indexto capture the extent of income related health inequality among poster sessions v urban female and male populations in each of the three metropolitan areas. personal income is classified into ten groups by the nphs with "no income" as the first group and "more than $ , " as the last. health status is measured by three variables -having a chronic condition (chc), self assessed health (sah), and health utility index (hui) -using appropriate indices. alternative formulations are used to calculate the standard deviations of the estimated or calculated measures. the findings of the study suggest that the measured health inequality depends on the health measure used. for chc and hui the measured inequality indices do indicate poorer health for lower income individulas, however, they are not statistically insignificant. health inequalities are more pronounced when health is measured by sah. the results do not support any systematic ranking of the three metropolitan areas in terms of health inequalities. they do not reveal any systematic pattern of inequality between men and women, either. conclusions: despite the use of highly aggregated nphs data, income related health disparities are observed in the three metropolitan areas in canada. this is more the case with self perceived (sah) health. such disparities are preventable and call for broader health policies that address poverty and low income among other social determinants of health. introduction: the analysis of health indicators under the spatial perspective is configured as an important instrument in the detection of intra-urban differentials. this study aimed to examine the spatial distribution of the births occurred in belo horizonte, in , analyzing the presence of spatial clusters of health indicators for newborns (rn) and their mothers, using data from the information system on live birth database (sinasc). method: for each covered area of the basic units of health (ubs), we calculated the proportion of: adolescent mothers, mothers with less than years of schooling, first pregnancy, mothers with four or more pregnancies, dead babies born on previous pregnancies, stillbirths, cesarean section, less than four prenatal care attendance, moderate and severe hypoxemia in the st and th minutes of life, low or very low birth weight. we used empiric bayesian methods for smoothing the estimates. for spatial analysis, the indicators obtained from the global moran (i) index and local indicators of spatial association (lisa) were used. maps were built to allow the visualization of the spatial clusters. in all analysis, significance statistics was considered p~ . . results: a total of , births of residents in belo horizonte were registered in i. the estimated bayesian proportions for the entire municipality was close to the one observed for all variables. analysis using lisa showed the presence of relevant spatial clusters for adolescent and low educated mothers, dead babies form previous pregnancies, cesarean section, low attendance to the prenatal care especially in area with low socio-demographic characteristics. three areas presented consistent clusters, with important spatial auto-correlation for almost all studied indicators. conclusion: the used methodology was configured as a great instrument of detection risk areas where clustering occurs. it can easily be incorporated in any surveillance system as a mechanism for controlling events related to births in the municipality. moreover, it can be applied to discriminate target areas for prompt public health interventions, such as improving health services access and the consolidation of better obstetric practices. introduction: gentrification, the displacement of low income and non-majority people out of longtime neighborhoods and residences is a global phenomenon. it has been identified as occurring in both developed and less developed countries and as inequality persists or increases, many communities are vulnerable to disruption and loss. while there may be some benefits to gentrification, these benefits are less likely to impact those who have been forced to move out of a gentrifying community or those who remain but economically stressed. . . . . this paper lays out a theoretical framework for dent fymg and ~ddressm~. the_ health consequences of gentrification on residents living in a community prior .to o.r durmg gent~ ~ cat. n. by drawing on the literature of housing, sociology, health and urban plannmg, t places genmf catt~n and neighborhood change into the context of other forces affecting th~ .hea.lth of vulnerable populations. it then proposes solutions to prevent or mitigate the impacts of genmf cat on. results: four main categories of consequences potentiallr re~ult from ~entr~fica~~n: !'°°r housing· related issues, spatial effects, mental health impacts and contr bunons to racial ~spanttes m heal~. the housing issues include increased susceptibility to asthma, lead exposure, allergiesl~topy an~. acetdents/ injuries. spatial effects include reduced access to health ~are, reduce~ access to obslttadinonal food sources, decreased physical activity, and increased obesity. the __ pnmary mental health effects_ :ire increased stress increased risk of depression and disruption of trad t onal support networks. in addinon to the above he~lth issues, gentrification has the potential to increase racial disparities in health by reduc· ing access to long term and multi-culturally experienced health care provide~s. conclusions: public health practice provides a framework for addressmg the health consequences of gentrification. in this context primary prevention would involve preventing gentrification in the first place along with a renewed commitment to helping distressed communities a~~ im_proving the quality ~f life for long term residents. the next layer of meeting the challenges of gentrification would be to assjst long rime residents to stay in a community and thus potentially allowing them to participate in the bene~ts of gentrification (such as better public services). only if all these efforts fail -and these other prevennon strategies must be a priority, public policy should then work to help people and instirutions move to other communities through grants and the provision of alternative safe affordable housing and neighborhoods. sarah romans, marsha cohen, and tonia forte lnh'odlletion: there has been sustained interest in urban rural (ur) differences in mental health over the last years of epidemiological research, with most studies reporting higher urban rates of mor· bidity, particularly when psychotic disorders have been studied. most recently, in britain, urban rates of non-psychotic disorder were greater than rural and semi-rural rates, both before and after the more adverse circumstances of urban dwellers were considered (paykel et al ) . surprisingly, there were no ur differences in help seeking. in canada, wang ( ) found greater urban rates for major depression only after he controlled for the confounding effects of race, immigration, employment and marital status, a result reminiscent of work from the us (blazer et ) . rural participants were less likely to have sought help for their mental health problems. in this study, we sought to examine urban/rural differences in rates of depression and help seeking in canada. method; data came from the canadian community health survey . , a community survey conducted by statistics canada of people aged and older, with the total sample size of , respon· dents, % response. analyses used weighted data; differences were assessed by chi-squared. ra.its: bivariate cross-tabulations showed a modest increase in month depression rates for urban ( . %) over rural ( . %, p= . ) dwellers; there were no ur differences when the sample was examined separately by gender and age group ( - , - , - ) . in general, more urban ( . %) than rural people ( . %, p= . ) had sought mental health treatment in the past year. there was no ur difference in helping seeking amongst those with depression (u . %, r . % ns). amongst the whole population, helping seeking was gendered with more urban men accessing help ( . %) than rural men ( . %, p= . ); this did not apply for women ( . % vs . % p= . ). ~ons: the urban-rural demographic continues to generate intriguing findings, even recently when internal migration is common and people can seek out the environment most conducive to their health, ~th physical and mental. people with depression are a disadvantaged, frequently stigmatized group, with ~r quality of life a_nd often impaired function. unlike many other factors associated wi~h urban or rural hfe per se, depression can be treated. the low help seeking rates is a cause for concern, m both the ur~n and_ rural populations. it behooves researchers, policy makers and service planners to ensutt effective services are available for both humane and economic reasons. methods: using united states census income data, we assigned the manhattan community districts to two major socioeconomic groups, manhattan i and ii. manhattan i is composed of community districts with average household incomes above the median for new york city; manhattan ii is those below. with public new york city department of health death records and us census data, we calculated a "standard" new york city population and estimated mortality rate distributions for manhattan i and ii. we then compared these age-adjusted actual mortality distributions with a standard t-test. results: we explored premature deaths using several cut-off points (before the ages of , , and ), and with subcomparisons for males and females. every comparison showed the wealthier area, manhattan i, to have significantly lower premature mortality rates than the lower income manhattan ii. further, we compared age-adjusted mortality rates among the city's five boroughs, and found no significant differences based on local geography alone. conclusions: these findings suggest that in manhattan, characterized by its extremes of wealth and poverty, health status, as measured by premature mortality, does vary significantly with the local income level. in manhattan, the relative average income by community district varies as much as : . this ratio is greater than that found in the other cities we have studied: for example, the range among tokyo's kus is half that in manhattan, or about : . paris has shown the longest life expectancy and changing premature mortality rates. from our preliminary work, we expect to find less variation in premature mortality rates in comparable cities. this study will continue to explore the relationship of premature mortality rates and life expectancy to income levels and different health systems among wcp cities with a view to measuring health policy options. introduction: according to the world health organization (who), smoking-related illness is currently the world's second major cause of death, currently responsible for one out of ten adult deaths worldwide. the who's framework convention on tobacco control (fctc) aims to reduce tobaccorelated disease and deaths by changing national public policies. mexico, which, according to the who, had smoking prevalences of % among adults in and % among health care providers (hcps) in , ratified the fctc in . objective: to examine knowledge of and attitudes towards cigarette smoking and smoking cessation among hcps, and clinical practices regarding smoking cessation. methods: in june , a convenience sample of hcps from one public clinic in urban oaxaca, mexico, part of mexico's national network of public health care clinics, were interviewed in spanish using a verbally administered questionnaire that was standard among all participants. during primary care outpatient visits, hcps were observed treating patients to assess the relative importance of smoking cessation as a health care priority. results: of the hcps interviewed, including ten physicians, three nurses, and five medical students, % reported smoking regularly. all hcps reported assessing patients' history of smoking, knowledge of the health risks associated with smoking, and feeling qualified to discuss these risks with patients. all hcps reported counseling patients who currently smoked to quit. all hcps reported recommending nicotine replacement therapy (eg., patch and/or gum) and/or behavioral therapy (eg., psychologist, support groups) to patients who smoked. all hcps reported that there was no government funding for smoking cessation, and that all costs associated with smoking cessation were patients' out-of-pocket expenses. observation of hcp interaction with patients revealed that hcps always inquired about smoking history with new patients and rarely inquired about smoking history with returning patients. hcps were not observed counseling patients who reported current smoking on smoking cessation methods and its benefits. observation of the clinical environment suggested a focus on preventative child health (eg., immunization, water-borne illnesses), family planning, and chronic diseases (eg., diabetes, hypertension). conclusions: while the mexican government has made smoking cessation a public health priority and hcps report addressing smoking cessation, observation of hcps suggests: ) smoking status is assessed only in new patients; and ) smoking cessation methods are not addresse~. observ_atio~ of hcps and the clinical environment in a public clinic in oaxaca suggests that smoking cessation s of lower priority than other heath care issues. p - (a) urban agriculture and food and nutrition security in kampala, uganda fiona yeudall, renee sebastian, abdelrahman lubowa, selahadin ibrahim, and donald cole introduction: urban agriculture is an important livelihood strategy contributing to household food security by increasing access and availability to food in urban settings (koc et al., ) . the purpose of poster sessions v rhis study was to examine relationships between child nutritional securiry outcomes, household food security, and urban agriculture activiries in kampala, uga~da. . . questionnaires assessing socio-demographic, farmmg and household food secunry (hfsi characreristics were administered to households. food diversiry was ~lculate~ from ~e number of foods consumed over hours for one child ( - years) per household. heigh~ weight,. nud-upper·ann· circumference (muac) and tricep skinfolds (tsf) were measured for the mdex child. z-sc.ores for height-for·age (haz), weighr-for-age (waz) and body mass index (zbmi) were ~~ulated usmg cen· rres for disease control and prevenrion reference data. z-scores for body composition measures were calculated using nhanes i and ii data for african americans. the lms method was used to c~.t for skewed z·score indices. all dara was checked for normaliry and univariable and backward mulnvanablc linear regression analysis conducted. ruwlts: household food securiry was significantly associated with wealth (b= . , p< 'a acre were more dependenr on assets for hfs. although sex of head of household was ~ot related to j-:ifs, it modi· fied relationships in rhar female headed households had greater food securtry when fai:mmg less than , acre compared ro male headed households, and vice versa. hfs was significantly associated with food diversiry (s= . , p). urbanization, especially in developing countries, has substantially increased the vulnerability of rhe mass of low-income urban dwellers. the livelihoods and quality of life for many of the poor espe· cially in larin america, asia, and africa, have deteriorated significantly over the years. urban areas are increasingly unable to provide for their populations, resulting in poverry, massive unemploymenr, job cuts, poor housing, lack of or poor public services, and a compromised health status. botswana, a country rhat lies in the sourhern parr of africa, has had its share of urban problems such as rhe mush· rooming of squarters and low-income urban sertlements. despite efforts to address the substandard living conditions in low-income urban areas, these problems have continued to grow. these living con· diuons are porenrially stressful to rhe residenrs and likely affects their health. the primary aim of the udy wa to examine the complex relationship between communiry-level stressors and interveners and health-related quality of life among residents of low-income neighborhoods in francistown, botswana. u i"" a croa -icctional quantitative design (both descriptive and explanatory) and using primarily cloae-mded interview• with a random sample of residents, this study examined the role of chrome life trnson and environmental quality on overall health status qualiry of life) and the physical, psy· chological and level of independence domains of health. the major hypothesis of the study was that community-levrl stre sor would influence health-related quality of life and that social capital would moderate these relationships. findings indicate that neighborhood quality is a powerful predictor of healrh tatu rhan socioeconomic status and individual life stressors. social capital was also found to he a ificant positive predictor of healrh and also moderator of structural factors. social capital moderated the effects of low environmental quality on level of indrpendence and on physical health outcomn, but nor on psychological and global health outcomes. these findings suggest that as the environment get betttr. stresson are reduced, hence promoting berter health outcomes. the study ends with implication• for social justice, public health and social work practice, and research, focusing mostly on the ~ole of social capital and the environmental quality in predicting health outcomes. spt· cafically • anenhon should be focused on political and civic society's commitment for social justice and poveny alleviation, ~uction of the threat of insecuriry and violence; cultivating social capital and good governance; and improving the health and social environments, especially housing and environ· mental aiutanon to name a few. urban and other uprisings by non-elites that lead to transitions, can disrupt sexual and injection "risk" networks that transmit infectious diseases by changing mixing patterns. they can also weaken urban social networks, their associated protective norms and their informal social control mechanisms which can lead to increased sexual and drug risk behaviors and violence (fullilove ; wallace & wallace , aral , friedman & reid hankins et al ) . for example, the revolution and transition to theocratic rule, and subsequent urban and national conflicts, have been followed by many urban youth in iran engaging in clandestine high-risk sexual and drug behaviors. in palestine, conflicts and restrictions on movement have led to increased fatalities from chronic diseases due to limited access to hospitals and modern medical facilities (union of palestinian medical relief committees); and heroin use and violence-related blood exposure have also increased. social movements "from below" that are rooted both in urban social network dynamics and in underlying patterns of injustice can have both protective and risky effects. for example, the social movements that led to the collapse of the ussr and its dependent governments in other countries-with subsequent increases in sex trade, alcoholism, drug use, tuberculosis, hiv, stis, and mortality in many localities-were based originally on such city-based movements in eastern europe. their impacts on health were mediated by urban social dynamics and structures. some urban social movements have improved health by prevent· ing the spread of hiv, hepatitis and other diseases. examples include movements of (a) gays and lesbians and (b) drug users. these have been rooted in social networks that overlap with risk networks. theories of urban health should include social conflict as a core concept. mechanisms that generate conflicts and the pathways by which conflicts affect health should be a major part of urban health research agendas. p - (c) demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital (lasuth) chest clinic john bako, adewale akeredolu, and wale alabi tuberculosis has become a resurgent public health problem in recent times in the world. because resources are limited, control programmes frequently must target population at greatest risk. the purpose of this study was to study the demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital. a total of patients who have been both radiologically and bacteriologically confirmed tuberculosis patients were used for this study between january and march. ( %) were males, while ( %) were females. notable risk factors among patients with tuberculosis were overcrowding ( . % ), homelessness ( . %) cigarette smoking ( . % ), alcoholism ( . %), non vaccination ( %), secondary contact ( . %) and poor knowledge ( . l 'x,). man· agement history patterns among the patients were herbs ( . 'yo), orthodox medicine ( .h . .l'x.). intervention targeting early-identified groups may be an effective way to reduce the incidence of tuber· culosis. such intervention should focus on health education, modification of life style and improvement of standard of living. p - (c) profiles in urban health in cities of the americas in the countries of the americas, there is an increasing migration of national and international populations to urban centers. this presentation will focus on some of the issues created by this influx of populations, the ways that cities in countries are dealing with them, and the efforts to promote local participation and solutions in management and decision-making. the methodology used to collect this information has been to draw upon and analyze information produced by the mayor's and ministry of health and development offices in each of the cities under consideration. an analysis of the impact of urbanization on health and health determinants will be presented. the information covers issues such as health status by geographic areas within the cities, highlighting issues such as marginality, barriers and physical, economic and cultural constructs and inequities in the delivery of and access to essential services (such as health, education, water, and basic sanitation). issues of governance and citizen participation will be highlighted to provide insight in~o the balance of power and mechanisms for decision-making at the local level. part of the analysis will show the relationship between democratic processes and social participation. public ~olicies will be reviewed to indicate those that are most beneficial in promoting health and overcoming barriers to it, as well as ways of capitalizing on local assets and resources. final~y, evidence of effectiveness of various strategies will be indicated. the presentation will conclude wuh suggesuons for future strategic directions to improve the quality of life and the promotion of health in large urban centers of the americas. introduction: much of the illicit drug in mexico is concentrated in northern border areas. the mile border between the u.s. and mexico is also characterized by migration; the border crossing between tijuana, baja california, mexico and san diego, california, u.s.a. is rep~rt~dl~ the busiest land border crossing in the world. we attempt to describe the migration scene among m ect on drug users (idus) m tijuana and investigate service needs. methods: migration trends were investigated in a cross-sectional study conducted from february to june among idus in tijuana. enrollment criteria included informed consent, being years or older, and having injected drugs within the prior month. subjects were recruited by respondent-dnven sampling and were administered a quantitative survey and serology for hiv, hcv, and syphilis. logistic regression was used to compare idus who had migrated to the u.s. versus those who had not. results: of idus enrolled, % were male and median age and age at first injection were and , respectively. drug combinations injected most frequently in the past months were heroin ( % and crystal methamphetamine mixed with heroin ( %). of those enrolled, responded to quesnons on migration and drug treatment and were included in this analysis. although % had resided in tijuana for at least years, % of users were born outside of baja california. working outside of mexico was common, with % working abroad in the last years ( % in u.s.), and % in the past year. in the last months, % of idus had crossed the border to the u.s., with % crossing at least once per month. those working outside of mexico in the past year were less likely to have ever received substance abuse treatment, [ % vs. %, or . , ] and were marginally less likely to have received drug treatment in the past months [ % vs. %, or . , % ci ( . - . )]. drug use patterns, age and gender did not differ between migrants and non-migrants, although migrants were more likely to report being in need of substance abuse treatment ( % vs. %, respectively). conclusions: migration is common among idus in tijuana. maintaining drug treatment regimens and determining how to best target education and services to a highly mobile population pose challenges to officials on both sides of the border. these data underscore the need to develop coordinated binational prevention and treatment efforts. introduction: despite the expanding literature on the important role public policy plays in influencing the broader determinants of the public\'s health, profound differences exist among jurisdictions in rhe attention placed upon such activities. we take an international perspective on health by examining rhe d?minant public _health paradigms of canada, usa, uk, and sweden and exploring how these paradigms shape public health practice. _method: we carefully analyze governmental and public health agencies documents to discern ~he dommant para~igms driving public health approaches and practices. we also consider the unique paht · cal and economic contexts within each nation and consider how these contexts drive public health pahcy and approaches. . . ~u~: the canadian and usa public health communities -with some exceptions --focus upon m~ividuahzed approaches to risk management. in contrast, the uk and swedish public health scenes are oriented toward broader approaches to health determinants. we find that the extent to which govern· ments, public health agencies and public health workers concern themselves with public policy approaches £<_> ~ddress ~ro.ad~r .determinants of health depends upon the particular health parad'.~ adhered. ~o withm each urisd ctton. and whether a paradigm is adopted depends upon the ideologi~a and pol~ncal context of each nation. nations such as sweden that have a long tradition of public policies promonng social jus~ce an~ equity are naturally receptive to evolving population health concepts. '[he usa represen~ a ~bey en~ro~~t where such is~ues are clear!~ subordinate. ., our findings mdicate that there s a strong political component that influences pubh ~ealth a~proaches and practi~ within the jurisdictions examined. the implications are that those seek· m~ to raise the broader detennmants of the public's health should work in coalition to raise these issues with non-health organizations and age · ca d d th · - badrgrollnd: in developed countries, social inequalities in health have endured or even worsened comparatively throughout different social groups since the s. in france, a country where access to medical and surgical care is theoretically affordable for everyone, health inequalities are among the high· est in western europe. in developing countries, health and access to care have remained critical issues. in madagascar, poverty has even increased in recent years, since the country wenr through political crisis and structural adjustment policies. objectives. we aimed to estimate and compare the impact of socio· economic status but also psychosocial characteristics (social integration, health beliefs, expectations and representation, and psychological characteristics) on the risk of having forgone healthcare in these dif· fercnt contexts. methods: population surveys conducted among random samples of households in some under· served paris neighbourhoods (n= ) and in the whole antananarivo city (n= ) in , using a common individual questionnaire in french and malagasy. reslllts: as expected, the impact of socioeconomic status is stronger in antananarivo than in paris. but, after making adjustments for numerous individual socio-economic and health characteristics, we observed in both cities a higher (and statically significant) occurrence of reponed forgone healthcare among people who have experienced childhood and/or adulthood difficulties (with relative risks up to and .s respectively in paris and antananarivo) and who complained about unhealthy living conditions. in paris, it is also correlated with a lack of trust in health services. coneluions: aside from purely financial hurdles, other individual factors play a role in the non-use of healthcare services. health insurance or free healthcare seems to be necessary hut not sufficienr to achieve an equitable access to care. therefore, health policies must not only focus on the reduction of the financial barriers to healthcare, but also must be supplemented by programmes (e.g. outreach care ser· vices, health education, health promotion programmes) and discretionary local policies tailored to the needs of those with poor health concern .. acknowledgments. this project was supported by the mal>io project and the national institute of statistics (instat) in madagascar, and hy the development research institute (ird) and the avenir programme of the national institute of health and medical research (inserm) in france. for the cities of developing countries, poverty is often described in terms of the living standard~ of slum populations, and there is good reason to believe that the health risks facing these populations are even greater, in some instances, than those facing rural villagers. yet much remains to be learned ahour the connections between urban poverty and health. it is not known what percentage of all urban poor live in slums, that is, in communities of concentrated poverty; neither is it known what proportion of slum residents are, in fact, poor. funhermore, no quantitative accounting is yet available that would sep· arare the health risks of slum life into those due to a househoid•s own poverty and those stemminic from poveny in the surrounding neighborhood. if urban health interventions are to be effectively targeted in developing countries, substantial progress must be made in addressing these cenrral issues. this paper examines poverty and children's health and survival using two large surveys, one a demographic and health survey fielded in urban egypt (with an oversampling of slums) and the other a survey of the slums of allahabad, india. using multivariate statistical methods. we find, in both settings: ( substan· rial evidence of living standards heterogeneity within the slums; ( strong evidence indicating that household-level poverty is an imponant influence on health; and ( ) staristically significant (though less strong) evidence that with household living standards held constant, neighborhood levels of poverty adversely affect health. the paper doses with a discussion of the implications of these findings for the targeting of health and poverty program interventions. p - (a) urban environment and the changing epidemiological surfacr. the cardiovascular ~ &om dorin, nigeria the emergence of cardiovascular diseases had been explained through the concomitants o_f the demographic transition wherein the prevalent causes of morbidity and monality ~hangr pr~mmant infectious diseases to diseases of lifestyle or chronic disease (see deck, ) . a ma or frustration m the v poster sessions case of cvd is its multifactural nature. it is acknowledged that the environment, however defined is the d · f · t' b tween agents and hosts such that chronic disease pathogenesis also reqmre a me an o mterac ion e . spatio-temporal coincidence of these two parties. what is not clear is which among ~ever~( potennal fac· · h b pace exacerbate cvd risk more· and to what extent does the ep dem olog cal trans · tors m t e ur an s ' . . . . tion h othesis relevant in the explanation of urban disease outlook even the developmg cities like nigeri~: thesis paper explorer these within a traditional city in nigeria. . . . the data for the study were obtained from two tertiary level hospitals m the metropolis for years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the data contain reported cases of cvd in the two facilities for the period. adopting a series of parametric and non-parametric statistics, we draw inferences between the observed cases of cvds and various demographic and locational variables of the patients. findings: about % of rhe cases occurred in years ( ) ( ) ( ) coinciding with the last year of military rule with great instability. . % occurred among male. . % also occurred among people aged - years. these are groups who are also likely to engage in most stressful life patterns. ~e study also shows that % of all cases occurred in the frontier wards with minor city areas also havmg their •fair' share. our result conformed with many empirical observation on the elusive nature of causation of cvd. this multifactoral nature had precluded the production of a map of hypertension that would be consistent with ideas of spatial prediction. cvd -cardiovascular diseases. mumbai is the commercial capital of india. as the hub of a rapidly transiting economy, mumbai provides an interesting case study into the health of urban populations in a developing country. with high-rise multimillion-dollar construction projects and crowded slums next to each other, mumbai presents a con· trast in development. there are a host of hi-tech hospitals which provide high quality care to the many who can afford it (including many westerners eager to jump the queue in their healthcare systems-'medical tour· ism'), at the same time there is a overcrowded and strained public healthcare system for those who cannot afford to pay. voluntary organizations are engaged in service provision as well as advocacy. the paper will outline role of the voluntary sector in the context of the development of the healthcare system in mumbai. mumbai has distinct upper, middle and lower economic classes, and the health needs and problems of all three have similarities and differences. these will be showcased, and the response of the healthcare system to these will be documented. a rising hiv prevalence rate, among the highest in india, is a challenge to the mumbai public healthcare system. the role of the voluntary sector in service provision, advocacy, and empowerment of local populations with regards to urban health has been paramount. the emergence of the voluntary sector as a major player in the puzzle of urban mumbai health, and it being visualized as voices of civil society or communiry representatives has advantages as well as pitfalls. this paper will be a unique attempt at examining urban health in india as a complex web of players. the influence of everyday socio·polirical-cultural and economic reality of the urban mumbai population will be a cross cutting theme in the analysis. the paper will thus help in filling a critical void in this context. the paper will thus map out issues of social justice, gender, equiry, effect of environment, through the lens of the role of the voluntary sector to construct a quilt of the realiry of healthcare in mumbai. the successes and failures of a long tradi· tion of the active advocacy and participation of the voluntary sector in trying to achieve social justice in the urban mumbai community will be analyzed. this will help in a better understanding of global urban health, and m how the voluntary sector/ngos fir into the larger picture. ba~und: o~er. half _of n~irobi's . million inhabitants live in illegal informal settlements that compose yo of the city s res dent al land area. the majority of slum residents lack access to proper san· iranon and a clean and adequate water supply. this research was designed to gain a clearer understand· mg of what kappr · · · h f . . opnate samtanon means or the urban poor, to determine the linkages between gender, hvehhoods, and access to water and sanitation, and to assess the ability of community sanitation blocks to meet water and sanitation needs in urban areas. m~tbojs_: _a household survey, gender specific focus groups and key informant interviews were conducted m maih saba, a peri-urban informal settlement. qualitative and quantitative research tools were u~ to asses~ the impact and effectiveness of community sanitation blocks in two informal settlements. results ropna e samtarmn me u es not only safe and clean latrines, but also provision ° adequate drainage and access to water supply for cleaning of clothes and homes. safety and cleanliness poster sessions v were priorities for women in latrines. levels of poverty within the informal settlements were identified and access to water and sanitation services improved with increased income. environmental health problems related to inadequate water and sanitation remain a problem for all residents. community sanitation blocks have improved the overall local environment and usage is far greater than envisioned in the design phase. women and children use the blocks less than men. this is a result of financial, social, and safety constraints. the results highlight the importance a need to expand participatory approaches for the design of water and sanitation interventions for the urban poor. plans need to recognize "appropriate sanitation" goes beyond provision of latrines and gender and socioeconomic differences must be taken into account. lessons and resources from pilot projects must be learned from, shared and leveraged so that solutions can be scaled up. underlying all the challenges facing improving water and sanitation for the urban poor are issues of land tenure. p - (c) integrating tqm (total quality management), good governance and social mobilization principles in health promotion leadership training programmes for new urban settings in countries/ areas: the prolead experience susan mercado, faren abdelaziz, and dorjursen bayarsaikhan introduction: globalization and urbanization have resulted in "new urban settings" characterized by a radical process of change with positive and negative effects, increased inequities, greater environmental impacts, expanding metropolitan areas and fast-growing slums and vulnerable populations. the key role of municipal health governance in mitigating and modulating these processes cannot be overemphasized. new and more effective ways of working with a wide variety of stakeholders is an underpinning theme for good governance in new urban settings. in relation to this, organizing and sustaining infrastructure and financing to promote health in cities through better governance is of paramount importance. there is a wealth of information on how health promotion can be enhanced in cities. despite this, appropriate capacity building programmes to enable municipal players to effectively respond to the challenges and impacts on health of globalization, urbanization and increasing inequity in new urban settings are deficient. the who kobe centre, (funded by the kobe group( and in collaboration with regional offices (emro, searo, wpro) with initial support from the japan voluntary contribution, developed a health promotion leadership training programme called "prolead" that focuses on new and autonomous structures and sustainable financing for health promotion in the context of new urban settings. methodology: country and/or city-level teams from areas, (china, fiji, india, japan, lebanon, malaysia, mongolia, oman, philippines, republic of korea, tonga and viet nam) worked on projects to advance health promotion infrastructure and financing in their areas over a month period. tools were provided to integrate principles of total quality management, good governance and social mobili .ation. results: six countries/areas have commenced projects on earmarking of tobacco and alcohol taxes for health, moblization of sports and arts organizations, integration of health promotion and social health insurance, organizational reforms, training in advocacy and lobbying, private sector and corporate mobilization and community mobilization. results from the other six areas will be reported in ..;obcr. conclusions: total quality management, good governance and social mobilization principles and skills are useful and relevant for helping municipal teams focus on strategic interventions to address complex and overwhelming determinants of health at the municipal level. the prolead training programmes hopes to inform other processes for building health promotion leadership capacity for new urban settings. the impact of city living and urbanization on the health of citizens in developing countries has received increasing attention in recent years. urban areas contribute largely to national economies. however, rapid and unplanned urban growth is often associated with poverty, environmental degradation and population demands that outstrip service capacity which conditions place human health at risk. local and national governments as well as multi national organizations are all grappling with the challenges of urbanization. with limited data and information available, urban health characteristics, including the types, quantities, locations and sources in kampala, are largely unknown. moreover, there is n? basis for assessing the impact of the resultant initiatives to improve health ~onditions amo~g ~o": ": um ties settled in unplanned areas. since urban areas are more than the aggregation ?f ~?pie w~th md_ v dual risk factors and health care needs, this paper argues that factors beyond the md v dual, mcludmg the poster sessions v · i d h · i · ment and systems of health and social services are determinants of the health soc a an p ys ca environ . of urban populations. however, as part of an ongoing study? ~s pape~ .addresses the basic concerns of urban health in kampala city. while applying the "urban hvmg conditions and the urban heal~ pen· alty" frameworks, this paper use aggregated urban health d~ta t~ explore the role of place an~ st tu· tions in shaping health and well-being of the population m kampala by understanding how characteristics of the urban environment and specific features of the city are causally related to health of invisible and forgotten urban poor population: results i~dica~e that a .range o~ urb~n he~l~h hazards m the city of kampala include substandard housing, crowdmg, mdoor air poll.ut on, msuff c ent a~d con· taminated water, inadequate sanitation and solid waste management services, vector borne .diseases, industrial waste increased motor vehicle traffic among others. the impact of these on the envtronment and community.health are mutually reinforcing. arising out of the withdra"'.al of city pl~nning systems and service delivery systems or just planning failure, thousands of people part cularl~ low-mc~me groups have been pushed to the most undesirable sections of the city where they are faced with ~ va_r ety ~f envj· ronmental insults. the number of initiatives to improve urban health is, however, growing mvolvjng the interaction of many sectors (health, environment, housing, energy, transportation and urban planning) and stakeholders (local government, non governmental organizations, aid donors and local community groups). key words: urban health governance, health risks, kampala. introduction: the viability of urban communities is dependent upon reliable and affordable mass transit. in particular, subway systems play an especially important role in the mass transit network, since they provide service to vast numbers of ridersseven of the subway systems worldwide report over one billion passenger rides each year. surprisingly, given the large number of people potentially affected, very little is known about the health and safety hazards that could affect both passengers and transit workers; these include physical (e.g., noise, vibration, accidents, electrified sources, temperature extremes), biological (e.g., transmission of infectious diseases, either through person-to-person spread or vector-borne, for example, through rodents), chemical (e.g., exposure to toxic and irritant chemicals and metals, gas emissions, fumes), electro-magnetic radiation, and psychosocial (e.g., violence, workstress). more recently, we need to consider the threat of terrorism, which could take the form of a mass casualty event (e.g., resulting from conventional incendiary devices), radiological attack (e.g., "dirty bomb"), chemical terrorist attack (e.g., sarin gas), or bioterrorist attack (e.g., weapons grade anthrax). given the large number of riders and workers potentially at risk, the public health implications are considerable. methods: to assess the hazards associated with subways, a structured review of the (english) litera· ture was conducted. ruults: based on our review, non-violent crime, followed by accidents, and violent crimes are most prevalent. compared to all other forms of mass transit, subways present greater health and safety risks. however, the rate of subway associated fatalities is much lower than the fatality rate associated with automobile travel ( . vs. . per million passenger miles), and cities with high subway ridership rates have a % lower per capita rate of transportation related fatalities than low ridership cities ( . versus . annual deaths per , residents). available data also suggest that subway noise levels and levels of air pollutants may exceed recommended levels. . ~: there is a paucity of published research examining the health and safety hazards associated with subways. most of the available data came from government agencies, who rely on passively reported data. research is warranted on this topic for a number of reasons, not only to address important knowled~ gaps, but also because the population at potential risk is large. importantly, from an urban perspecnve, the benefits of mass transit are optimized by high ridership ratesand these could be adversely affu:ted by unsafe conditions and health and safety concerns. veena joshi, jeremy lim. and benjamin chua ~ ~rban health issues have moved beyond infectious diseases and now centre largely on chrome diseases. diabetes is one of the most prevalent non-communicable diseases globally. % of adult ¥ benefit in providing splash pads in more parks. given the high temperature and humidity of london summers, this is an important aspect and asset of parks. interviewed parents claimed to visit city parks anywhere between to days per week. corrduion: given that the vast majority of canadian children are insufficiently active to gain health benefits, identifying effective qualities of local parks, that may support and foster physical activity is essential. strategies to promote activity within children's environments are an important health initiative. the results from this study have implications for city planners and policy makers; parents' opinions of, and use of city parks provides feedback as to the state current local parks, and modifications that should be made for new ones being developed. this study may also provide important feedback for health promoters trying to advocate for physical activity among children. introdt clion: a rapidly increasing proportion of urban dwellers in africa live below the poverty line in overcrowded slums characterized by uncollected garbage, unsafe water, and deficient sanitation and overflowing sewers. this growth of urban poverty challenges the commonly held assumption that urban populations enjoy better health than their rural counterparts. the objectives of this study are (i) to compare the vaccination status, and morbidity and mortality outcomes among children in the slums of nairobi with rural kenya, and (ii) to examine the factors associated with poor child health in the slums. we use data from demographic and health survey representative of all slum settlements in nairobi city carried out in by the african population & health research center. a total of , women aged - from , households were interviewed. our sample consists of , children aged - months. the comparison data are from the kenya demographic and health survey. the outcomes of interest include child vaccination status, morbidity (diarrhea, fever and cough) and mortality, all dichotomized. socioeconomic, environmental, demographic, and behavioral factors, as well as child and mother characteristics, are included in the multivariate analyses. multilevel logistic regression models are used. l'nlimin ry rest lts: about % of children in the slums had diarrhea in the two weeks prior to the survey, compared to % of rural children. these disparities between the urban poor anj the rural residents are also observed for fever ( % against %), cough ( % versus %), infant mortality ( / against / ), and complete vaccination ( % against %). preliminary multivariate results indicate that health service utilization and maternal education have the strongest predictive power on child morbidity and mortality in the slums, and that household wealth has only minor, statistically insignificant effects. conclruion: the superiority of health of urban children, compared with their rural counterparts, masks significant disparities within urban areas. compared to rural residents, children of slum dwellers in nairobi are sicker, are less likely to utilize health services when sick, and stand greater risk to die. our results suggest policies and programs contributing to the attainment of the millennium development goal on child health should pay particular attention to the urban poor. the insignificance of socioeconomic status suggests that poor health outcomes in these communities are compounded by poor environmental sanitation and behavioral factors that could partly be improved through female education and behavior change communication. introduction: historic trade city surat with its industrial and political peace has remained a center of attraction for people from all the comers of india resulting in to pop.ulatio~ explosio~ a~d stressed social and service infrastructure. the topography,dimate and demographic profile of the city s threat to the healthy environment. aim of this analysis is to review the impact of managemt'nt reform on health indicators. method: this paper is an analysis of the changing profile of population, sanitary infr~s~rucrure, local self government management and public health service reform, secondary health stat st cs data, health indicator and process monitoring of years. . . health of entire city and challenge to the management system. plague outbr~ak ( ) was the turning point in the history of civic service management including p~blic ~e~lth service management. ~ocal self government management system was revitalized by reg~lar_ field v s ts o~ al~ cadre~, _decentraltzanon of power and responsibility, equity, regular vigilant momtormg, commumcanon facility, ream_approach and people participation. reform in public health service management was throu_gh stan~~rd zed intervention protocol, innovative intervention, public private partnership, community part c panon, academic and service institute collaboration and research. sanitation service coverage have reached nearer to universal. area covered by safe water supply reached to %( ) from % ( ) and underground drainage to % ( ) from % ( ) the overhauling of the system have reflected on health indicators of vector and water born disease. malaria spr declined to . ( ) from . 'yo(! ) and diarrhea case report declined to ( ) from ( ). except dengue fever in no major disease outbreaks are reported after . city is recipient of international/national awards/ranking for these achievements. the health department have developed an evidence and experience based intervention and monitoring system and protocol for routine as well as disaster situation. the health service and management structure of surat city have emerged as an urban health model for the country. introduction: the center for healthy communities (chc) in the department of family and com· munity medicine at the medical college of wisconsin developed a pilot project to: ) assess the know· ledge, attitudes, and behaviors of female milwaukee public housing residents related to breast cancer; develop culturally and literacy appropriate education and screening modules; ) implement the developed modules; ) evaluate the modules; and ) provide follow-up services. using a community-based participatory research model the chc worked collaboratively with on-site nurse case management to meet these objectives. methods: a "breast health kick off event" was held at four separate milwaukee public housing sites for elderly and disabled adults. female residents were invited to complete a -item breast health survey, designed to accommodate various literacy levels. responses were anonymous and voluntary. the survey asked women about their previous physical exams for breast health, and then presented a series of state· ments about breast cancer to determine any existing myths. the final part gathered information about personal risk for breast cancer, the highest level of education completed, and whether the respondents h;td ever used hormone replacement therapy and/or consumed alcohol. responses were collected for descriptive analysis. results: a total of surveys (representing % of the total female population in the four sites) were completed and analyzed. % reported that they had a physical exam in the previous rwo years. % of respondents indicated they never had been diagnosed with breast cancer. % reported having had a mammogram and % having had a clinical breast exam. those that never had a mammogram reported a fear of what the provider would discover or there were not any current breast problems ro warrant an exam. % agreed that finding breast cancer early could lower the chance of dying of cancer. over % reported that mammograms were helpful in finding cancer. however, % believed that hav· ing a mammogram actually prevents breast cancer. % indicated that mammograms actually cause cancer and % reported that a woman should get a mammogram only if there is breast cancer in her family. conclusion: this survey indicates that current information about the importance of mammograms and clinical breast exams is reaching traditionally underserved women. yet there are still critical oppor· tunities to provide valuable education on breast health. this pilot study can serve as a tool for shaping future studies of health education messages for underserved populations. located in a yourh serv· ~ng agency m downtow~ ottawa, the clinic brings together community partners to provide primary medical care. and dent~i hygiene t? the street youths of ottawa aged - . the primary goal of the project is to provide accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. these efforts respond to the pre-existing body of evidence suggesting that the principle barrier in accessing such care for these youths are feelings of intimidation and vulnerability in the face of a complex healthcare system. the bruyere fhn satellite clinic is located in the basement of a downtown drop-in and brings together a family medicine physician and her residents, a dental hygienist and her nd year students, a nurse practitioner, a chiropodist and public health nurses to provide primary care. the clinic has been extremely busy and well received by the youth. this workshop will demonstrate how five community organizations have come together to meet the needs of high risk youths in ottawa. this presentation will showcase the development of the clinic from its inception through its first year including reaction of the youths, partnerships and lessons learned. it will also focus on its sustainability without continued funding. we hope to have developed a model of service delivery that could be reproduced and sustained in other large cities with faculties of medicine. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative-a) semi structured interviews with providers & partners b)focus groups with youth quantitative a)electronic medical records for months records (budget, photos, project information). results: ) successfully built and opened a medicaudental clinic which will celebrate its year anniversary in august. ) over youths have been seen, and we have had over visits. conclusion: ) the clinic will continue to operate beyond the month project funding. ) the health of high risk youth in ottawa will continue to improve due to increased access to medical services. p - (a) health services -for the citizens of bangalore -past, present and future savita sathyagala, girish rao, thandavamurthy shetty, and subhash chandra bangalore city, the capital of karnataka with . million is the th most populous city in india; supporting % of the urban population of karnataka, it is considered as one of the fastest growing cities in india. known as the 'silicon valley of india', bangalore is nearly years old. bangalore city corporation (bmp), is a local self government and has the statutory commitment to provide to the citizens of bangalore: good roads, sanitation, street lighting, safe drinking water apart from other social obligations, cultural development and poverty alleviation activities. providing preventive and promotive heahh services is also a specific component. the objective of this study was to review the planning process with respect to health care services in the period since india independence; the specific research questions being what has been the strategies adopted by the city planners to address to the growing needs of the population amidst the background of the different strategies adopted by the country as a whole. three broad rime ranges have been considered for analysis: the s, s and the s. the salient results are: major area of focus has been on the maternal and child care with activities ranging from day-care to in-patient-care; though the number of institutions have grown from to the current day , their distribution has been far from satisfactory; obtaining support from the india population projects and major upgradarions have been undertaken in terms of infrastructure; over the years, in addition to the dispensaries of modern system of medicine, local traditional systems have also been initiated; the city has partnered with the healthy cities campaign with mixed success; disease surveillance, addressing the problems related to the emerging non-communicable diseases including mental health and road traffic injuries are still in its infancy. isolated attempts have been made to address the risks groups of elderly care and adolescent care. what stands out remarkably amongst the cities achievements is its ability to elicit participation from ngos, cbos and neighbourhood groups. however, the harnessing of this ability into the health sector cannot be said totally successful. the moot question in all the above observed development are: has the city rationally addressed it planning needs? the progress made so far can be considered as stuttered. the analysis and its presentation would identify the key posirive elements in the growth of banglore city and spell a framework for the new public health. introduction: anaemia associated with pregnancy is a major public health problem all over the world. different studies in different parts of india shown prevalence of anaemia between - %. anaemia remains a serious health problem in pregnancy despite of strong action taken by the government of india through national programmes. in the present study we identified th~ social beha~iors, responsible for low compliance of if a tablets consumption in pregnancy at community level and intervention was given with new modified behaviors on trial bases. . in vadodara urban. anganwadies out of were selected from the list by random sampling for tips (trials of improved practices) study. . . participants: pregnant women ( , intervention group+ , control. group) registered m the above anganwadies. study was conducted in to three phases: phase: . formative research and baseline survey (frbs). data was collected from all pregnant women to identify behaviors that are responsible for low compliance of ifa tablets. both qualitative and quantitative data were collected. haemoglobin was estimated of all pregnant women by haemo-cue. phase: . phase of tips. behaviors were identified both social & clinical for low compliance of ifa tablets consumption in pregnancy from frbs and against those, modified behaviors were proposed to pregnant women in the intervention group on trial bases by health education. trial period of weeks was given for trial of new behaviors to pregnant women in the interven· tion group. phase: . in this phase, feedbacks on behaviors tried or not tried were taken from pregnant women in intervention group. haemoglobin estimation was carried out again in all pregnant women. at the end of the study, messages were formulated on the bases of feedbacks from the pregnant women. results: all pregnant women in the intervention group had given positive feedback on new modified behaviors after intervention. mean haemoglobin concentration was higher in intervention group ( . ± . gm%) than control group ( . ± . gm%). ifa tablets compliance was improved in intervention group ( . %) than control group ( . %). conclusion: all pregnant women got benefits after trial of new modified behaviors in the intervention group. messages were formulated from the new modified behaviors, which can be used for longterm strategies for anaemia control in the community. introduction: in order to develop a comprehensive mch handbook for pregnant women and to assess its effect among them, a pilot study was carried out at the maternal and child health training institute (mchti), in dhaka, bangladesh. methods: from mchti a sample of pregnant women was selected and all subjects were women who were attending the first visit of their current pregnancy by using a random sampling method. of the subjects, women were given the mch handbook as case and women were not given the handbook as control. data on pre and post intervention of the handbook from the cases and controls were taken from data recording forms between the st of november and st of october, and data was analysed by using a multilevel analysis approach. this was a hospital-based action (case-control) research, and was applied in order to measure the outcome of pre and post intervention following the introduction of the handbook. data was used to assess the effects of utilisation of the handbook on women's knowledge, practice and utilisation of mch services. results: this study showed that the change of knowledge about antenatal care visits was . % among case mothers. knowledge of danger signs improved . %, breast feeding results . %, vaccination . % and family planning results improved . % among case. results showed some positive changes in women's attitudes among case mothers and study showed the change of practice in antenatal care visits was .u. % in the case. other notable changes were: change of practice in case mother's tetanus toxoid (ti), . %; and family planning . %. in addition, handbook assessment study indicated that most women brought the handbook on subsequent visits ( . %), the handbook was highly utilised (i.e. it was read by . %, filled-in by . %, and was used as a health education tool by . %). most women kept the handbook ( . %) and found it highly useful ( . %) with a high client satisfaction rate of . %. conclusion: pregnant women in the case group had higher knowledge, better practices, and higher utilisation of mch services than mothers in the control groups who used alternative health cards. if the handbook is developed with a focus on utilising a problem-oriented approach and involving the recomendations .of end~users, it is anticipated that the mch handbook will contribute significantly to ensuring the quahry of hfe of women and their children in bangladesh. after several meetmgs to identify the needs of the community, a faso clinic was opened at ncfs. health care professionals from smh joined with developmental and social service workers from ncfs to implement the faso diagnostic process and to provide culturally appropriate after-care. the clinic is unique in that its focus is the high risk urban aboriginal population of toronto. it accepts referrals of not only children and youth, but also of adults. lessons learned: response to the faso clinic at native child and family services has been overwhelming. aboriginal children with f asd are receiving timely diagnosis and interventions. aboriginal youth and adults who have been struggling with poveny, substance abuse, and homelessness are more willing to enter the ncfs centre for diagnosis and treatment. aboriginal infants prenatally exposed to alcohol born at st. michael's hospital or referred by other centres have access to the developmental programs located in both of the partnering agencies. the presentation will describe the clinic's development, and will detail the outcomes described, including interventions unique to the aboriginal culture. p - (c) seeds, soil, and stories: an exploration of community gardening in southeast toronto carolin taran, sarah wakefield, jennifer reynolds, and fiona yeudall introduction: community gardens are increasingly seen as a mechanism for improving nutrition and increasing food security in urban neighbourhoods, but the evidence available to support these claims is limited. in order to begin to address this gap in a way that is respectful of community knowledge and needs, the urban gardening research opportunities workgroup (ugrow) project explored the benefits and potential risks of community gardening in southeast toronto. the project used a community-based research (cbr) model to assess community gardens as a means of improving local health. the research process included interviews, focus groups, and participant observation (documented in field notes). we also directly engaged the community in the research process, through co-learning activities and community events which allowed participants to express their views and comment on emerging results. most of the research was conducted by a community-based research associate, herself a community gardener. key results were derived from these various sources through line-by-line coding of interview transcripts and field note review, an interactive and iterative process which involved both academic and community partners. results: these various data sources all suggest that enhanced health and access to fresh produce are important components of the gardening experience. they also highlight the central importance of empowering and community-building aspects of gardening to gardeners. community gardens were thought to play a role in developing friendships and social support, sharing food and other resources, appreciating cultural diversity, learning together, enhancing local place attachment and stewardship, and mobilizing to solve local problems (both inside and outside the garden). potential challenges to community gardens as a mechanism for communiry development include bureaucratic resistance to gardens, insecure land tenure and access, concerns about soil contamination, and a lack of awareness and under· standing by community members and decision-makers of all kinds. conclusion: the results highlight many health and broader social benefits experienced by commu· nity gardeners. they also point to the need for greater support for community gardening programs, par· ticularly ongoing the ongoing provision of resources and education programs to support gardens in their many roles. this research project is supported by the wellesley central health corporation and the centre for urban health initiatives, a cihr funded centre for research development hased at the univer· sity of toronto. p - (c) developing resiliency in children living in disadvantaged neighbourhoods sarah farrell, lorna weigand, and wayne hammond the traditional idea of targeting risk reduction by focusing on the development of eff~ctive coping strategies and educational programs has merit in light of the research reportmg_ that_ ~ lupl.e forms of problem behaviour consistently appear to be predicted by increasing exposure to den_uf able risk factors. as a result, many of the disadvantaged child and youth studies have focused on trymg to better _unde.r· stand the multiple risk factors that increase the likelihood of the development of at nsk behaviour m ch ldren/youth and the potential implications for prevention. this in turn has led t_o. the conclus on that community and health programs need to focus on risk reduction by helpm~ md v duals develop more effective coping strategies and a better understanding of the limitations of cenam pathologies, problematic v poster sessions coping behaviours and risk factors potentially inheren~ in high needs co~unities. ~owever, another ai:ea of research has proposed that preventative interventions should cons de~ .~rotecnve fa~ors alo~~ with reducing risk factors. as opposed to just emphasizing problems, vulnerab ht es, and deficits, a res liencybased perspective holds the belief that children, youth and their families. have strengths, reso~ce.s and the ability to cope with significant adversity in ways that are not only effective, but tend to result m mcreased ability to constructively respond to future adversity. with this in mind, a participatory research project sponsored by the united way of greater toronto was initiated to evaluate and determine the resiliency profiles of children - years (n = ) of recent immigrant families living in significantly disadvantaged communities in the toronto area. the presentation will provide an overview of the identified protective factors (both intrinsic and extrinsic) and resiliency profiles in an aggregated format as well as a summary of how the children and their parents interpreted and explained these strength-based results. as part of the focus groups, current community programs and services were examined by the participants as to what might be best practices for supporting the development and maintaining of resiliency in children, families and communities. it was proposed that the community model of assessing resiliency and protective factors as well as proposed best strength-based practice could serve as a guide for all in the community sector who provide services and programs to those in disadvantaged neighbourhoods. p - (c) naloxone by prescription in san francisco, ca and new york, ny emalie huriaux the harm reduction coalition's overdose project works to reduce the number of fatal overdoses to zero. located in new york, ny and san francisco, ca, the overdose project provides overdose education for social service providers, single-room occupancy hotel (sro) residents, and syringe exchange participants. the project also conducts an innovative naloxone prescription program, providing naloxone, an opiate antagonist traditionally administered by paramedics to temporarily reverse the effects of opiate overdose, to injection drug users (idus). we will describe how naloxone distribution became a reality in new york and san francisco, how the project works, and our results. the naloxone prescription program utilizes multiple models to reach idus, including sro-and street-based trainings, and office-based trainings at syringe exchange sites. trainings include information on overdose prevention, recognition, and response. a clinician conducts a medical intake with participants and provides them with pre-filled units of naloxone. in new york, funding was initially provided by tides foundation. new york city council provides current funding. new york department of mental health and hygiene provides program oversight. while the new york project was initiated in june , over half the trainings have been since march . in san francisco, california endowment, tides foundation, and san francisco department of public health (sfdph) provide funding. in addition, sfdph purchases naloxone and provides clinicians who conduct medical intakes with participants. trainings have been conducted since november . to date, nearly individuals have been trained and provided with naloxone. approximately of them have returned for refills and reported that they used naloxone to reverse an opiate-related overdose. limited episodes of adverse effects have been reported, including vomiting, seizure, and "loss of friendship." in new york, individuals have been trained and provided with naloxone. over overdose reversals have been reported. over half of the participants in new york have been trained in the south bronx, the area of new york with the highest rate of overdose fatalities. in san francisco, individuals have been trained and provided with naloxone. over overdose reversals have been reported. the majority of the participants in san francisco have been trained in the tenderloin, th street corridor, and mission, areas with the highest rates of overdose fatalities. the experience of the overdose project in both cities indicates that providing idus low-threshold access to naloxone and overdose information is a cost-effective, efficient, and safe intervention to prevent accidental death in this population. p - (c) successful strategies to regulate nuisance liquor stores using community mobilization, law enforcement, city council, merchants and researchers tahra goraya presenta~ion _will discuss ~uccessful environmental and public policy strategies employed in one southen: cahf?rmna commumty to remedy problems associated with nuisance liquor stores. participants ~ be given tools to understand the importance of utilizing various substance abuse prevention str~tegi~ to change local policies and the importance of involving various sectors in the community to a~_ st with and advocate for community-wide policy changes. recent policy successes from the commultles of pa~ad~na and altad~na will highlight the collaborative process by which the community mobilized resulnng m several ordmances, how local law enforcement was given more authority to monitor poster sessions v nonconforming liquor stores, how collaborative efforts with liquor store owners helped to remove high alcohol content alcohol products from their establishments and how a community-based organiz,uion worked with local legislators to introduce statewide legislation regarding the regulation of nuisance liquor outlets. p - (c) "dialogue on sex and life": a reliable health promotion tool among street-involved youth beth hayhoe and tracey methven introduction: street involved youth are a marginalized population that participate in extremely risky behaviours and have multiple health issues. unfortunately, because of previous abuses and negative experiences, they also have an extreme distrust of the adults who could help them. in , toronto public health granted funding to a non governmental, nor for profit drop-in centre for street youth aged - , to educate them about how to decrease rhe risk of acquiring hiv. since then the funding has been renewed yearly and the program has evolved as needed in order to target the maximum number of youth and provide them with vital information in a candid and enjoyable atmosphere. methods: using a retrospective analysis of the six years of data gathered from the "dialogue on sex and life" program, the researchers examined the number of youth involved, the kinds of things discussed, and the number of youth trained as peer leaders. also reviewed, was written feedback from the weekly logs, and anecdotal outcomes noted by the facilitators and other staff in the organization. results: over the five year period of this program, many of youth have participated in one hour sessions of candid discussion regarding a wide range of topics including sexual health, drug use, harm reduction, relationship issues, parenting, street culture, safety and life skills. many were new youth who had not participated in the program before and were often new to the street. some of the youth were given specific training regarding facilitation skills, sexual anatomy and physiology, birth control, sexually transmitted infections, hiv, substance use/abuse, harm reduction, relationships and discussion of their next steps/future plans following completion of the training. feedback has been overwhelmingly positive and stories of life changing decisions have been reported. conclusion: clearly, this program is a successful tool to reach street involved youth who may otherwise be wary of adults and their beliefs. based on data from the evaluation, recommendations have been made to public health to expand the funding and the training for peer leaders in order ro target between - new youth per year, increase the total numbers of youth reached and to increase the level of knowledge among the peer leaders. p - (c) access to identification and services jane kali replacing identification has become increasingly more complex as rhe government identification issuing offices introduce new requirements rhar create significant barriers for homeless people to replace their id. new forms of identification have also been introduced that art' not accessible to homekss peoplt-(e.g. the permanent resident card). ar rhe same time, many service providers continue to require identifi· cation ro access supports such as income, housing, food, health care, employment and employmt·nt training programs. street health, as well as a number of other agencies and community health centres, h, , been assisting with identification replacement for homeless peoplt· for a number of years. the rnrrt·nr challenges inherent within new replacement requirements, as well as the introduction of new forn ' of identification, have resulted in further barriers homeless people encounter when rrring to access t:ssential services. street health has been highlighting these issues to government identification issuing offices, as well as policy makers, in an effort to ensure rhar people who are homeless and marginalized have ac'ess to needed essential services. bandar is a somali word for •·a safe place." the bandar research project is the product of the regent park community health centre. the research looks ar the increasing number of somali and afri· can men in the homeless and precariously house population in the inner city core of down~own toronto. in the first phase of the pilot project, a needs assessment was conducted to dennfy barners and issues faced by rhe somali and other african men who are homeless and have add cr ns issues. th_e second phase of rhe research project was to identify long rerm resources and service delivery mechamsms that v poster sessions would enhance the abiity of this population to better access detox, treatment, and post treatment ser· vices. the final phase of the project was to facilitate the development of a conceptual model of seamless continual services and supports from the streets to detox to treatment to long term rehabilitation to housing. "between the pestle and mortar" -safe place. p - (c) successful methods for studying transient populations while improving public health beth hayhoe, ruth ewert, eileen mcmahon, and dan jang introduction: street youth are a group that do not regularly access healthcare because of their mis· trust of adults. when they do access health care, it is usually for issues severe enough for hospitalization or for episodic care in community clinics. health promotion and illness prevention is rarely a part of their thinking. thus, standard public health measures implemented in a more stable population do not work in this group. for example, pap tests, which have dearly been shown to decrease prevalence of cer· vical cancer, are rarely done and when they are, rarely followed up. methods to meet the health care needs and increase the health of this population are frequently being sought. methods: a drop-in centre for street youth in canada has participated in several studies investigating sexual health in both men and women. we required the sponsoring agencies to pay the youth for their rime, even though the testing they were undergoing was necessary according to public health stan· dards. we surmised that this would increase both initial participation and return. results: many results requiring intervention have been detected. given the transient nature of this population, return rates have been encouraging so far. conclusion: it seems evident that even a small incentive for this population increases participation in needed health examinations and studies. it is possible that matching the initial and follow-up incentives would increase the return rate even further. the fact that the youth were recruited on site, and not from any external advertising, indicates that studies done where youth trust the staff, are more likely to be successful. the presentation will share the results of the "empowering stroke prevention project" which incor· porated self-help mutual aids strategies as a health promotion methodology. the presentation will include project's theoretical basis, methodology, outcomes and evaluation results. self-help methodology has proven successful in consumer involvement and behaviour modification in "at risk," "marginalized" settings. self-help is a process of learning with and from each other which provides participants oppor· tunities for support in dealing with a problem, issue, condition or need. self-help groups are mechanisms for the participants to investigate existing solutions and discover alternatives, empowering themselves in this process. learning dynamic in self-help groups is similar to that of cooperative learning and peertraining, has proven successful, effective and efficient (haller et al, ) . the mutual support provided by participation in these groups is documented as contributory factor in the improved health of those involved. cognizant of the above theoretical basis, in the self-help resource centre initiated the "empowering stroke prevention project." the project was implemented after the input from health organizations, a scan of more than resources and an in-depth analysis of risk-factor-specific stroke prevention materials indicated the need for such a program. the project objectives were:• to develop a holistic and empowering health promotion model for stroke prevention that incorporates selfhelp and peer support strategies. • to develop educational materials that place modifiable risk factors and lifestyle information in a relevant context that validates project participants' life experiences and perspectives.• to educate members of at-risk communities about the modifiable risk factors associated with stroke, and promote healthy living. to achieve the above, a diverse group of community members were engaged as "co-editors" in the development of stroke prevention education materials which reflected and validated their life experiences. these community members received training to become lay health promoters (trained volunteer peer facilitators). in collaboration with local health organizations, these trained lay health promoters were then supported in organizing their own community-based stroke prevention activities. in addition, an educational booklet written in plain language, entitled healthy ways to prevent stroke: a guide for you, and a companion guide called healthy ways to pre· vent stroke: a facilitator's guide were produced. the presentation will include the results of a tw<>tiered evaluation of the program methodology, educational materials and the use of the materials beyond the life of the project. this poster presentation will focus on the development and structure of an innovative street outreach service that assists individuals who struggle mental illness/addictions and are experiencing homelessness. the mental health/outreach team at public health and community services (phcs) of hamilton, ontario assists individuals in reconnecting with health and social services. each worker brings to the ream his or her own skills-set, rendering it extremely effective at addressing the multidimensional and complex needs of clients. using a capacity building framework, each ream member is employed under a service contract between public health and community services and a local grassroots agency. there are public health nurses (phn), two of whom run a street health centre and one of canada's oldest and most successful needle exchange programs, mental health workers, housing specialists, a harm reduction worker, youth workers, and a united church minister, to name a few. a community advisory board, composed of consumers and professionals, advises the program quarterly. the program is featured on raising the roors 'shared learnings on homelessness' website at www.sharedlearnings.ca. through our poster presentation participants will learn how to create effective partnerships between government and grassroots agencies using a capacity building model that builds on existing programs. this study aims to assess the effects of broadcasting a series of documentary and drama videos, intended to provide information about the bc healthguide program in farsi, on the awareness about and the patterns of the service usage among farsi-speaking communities in the greater vancouver area. the major goals of the present study were twofold; ( ) to compare two methods of communications (direct vs. indirect messages) on the attitudes and perceptions of the viewers regarding the credibility of messengers and the relevance of the information provided in the videos, and ( ) to compare and contrast the impact of providing health information (i.e., the produced videos) via local tvs with the same materials when presented in group sessions (using vcr) on participants' attitudes and perceptions cowards the bc healrhguide services. results: through a telephone survey, farsi-speaking adults were interviewed in november and december . the preliminary findings show that % of the participants had seen the aired videos, from which, % watched at least one of the 'drama' clips, % watched only 'documentary' clip, and % watched both types of video. in addition, % of the respondents claimed that they were aware about the program before watching the aired videos, while % said they leaned about the services only after watching the videos. from this group, % said they called the bchg for their own or their "hildren's health problems in the past month. % also indicated that they would use the services in the future whenever it would be needed. % considered the videos as "very good" and thought they rnuld deliver relevant messages and % expressed their wish to increase the variety of subjects (produ\:e more videos) and increase the frequency of video dips. conclusion: the results of this study will assist public health specialists in bc who want to choose the best medium for disseminating information and apply communication interventions in multi\:ultural communities. introduction: many theorists and practitioners in community-based research (cbr) and knowledge transfer (kt) strongly advocate for involvement of potential users of research in the development of research projects, yet few examples of such involvement exist for urban workplace health interventions. we describe the process of developing a collaborative research program. methods: four different sets of stakeholders were identified as potential contributors to and users of the research: workplace health policy makers, employers, trade unions, and health and safety associations. representatives of these stakeholders formed an advisory committee which met quarterly. over the month research development period, an additional meetings were held between resc:ar~h~rs and stakeholders. in keeping with participant observation approaches, field notes of group and md v ~ ual meetings were kept by the two co-authors. emails and telephone calls were also documented. qu~h tative approaches to textual analysis were used, with particular attention paid to collaborattve v poster sessions relationships established (as per cbr), indicators of stakeholders' knowledge utilization (as per kt), and transformations of the proposed research (as per cbr). results: despite initial strong differences of opinion both among stakeho~ders .an~ between stakeholders and researchers, goodwill was noted among all involved. acts of rec~proc ty included mu.rual sharing of assessment tools, guidance on data utilization to stakeho~der orga~ zat ns, and suggestions on workplace recruitment to researchers. stakeholders demonstrated mcreases m concep~ual. un~erstand ing of workplace health e.g. they more commonly discussed more complex,. psychosocial md cators of organizational health. stakeholders made instrumental use of shared materials based on research e.g. adapting their consulting model to more sophisticated dat~ analysis. sta~ehol?~rs recogni_zed the strategic use of their alliance with researchers e.g., transformational leadership trainmg as a~ inducement to improve health and safety among small service franchises. stakeholders helped re-define the research questions, dramatically changed the method of recruitment from researcher cold call to stakeholderbased recruitment, and strongly influenced pilot research designs. owing a great deal to the elaborate joint development process, the four collaboratively developed pilot project submissions which were all successfully funded. conclusion: the intensive process of collaborative development of a research program among stakeholders and researchers was not a smooth process and was time consuming. nevertheless, the result of the collaborative process was a set of projects that were more responsive to stakeholder needs, more feasible for implementation, and more broadly applicable to relevant workplace health problems. introduction: environmental groups, municipal public health authorities and, increasingly, the general public are advocating for reductions in pesticide use in urban areas, primarily because of concern around potential adverse health impacts in vulnerable populations. however, limited evidence of the relative merits of different intervention strategies in different contexts exists. in a pilot research project, we sought to explore the options for evaluating pesticide reduction interventions across ontario municipalities. methods: the project team and a multi-stakeholder project advisory committee (pac), generated a list of potential key informants (kl) and an open ended interview guide. thirteen ki from municipal government, industry, health care, and environmental organizations completed face to face or telephone interviews lasting - minutes. in a parallel process, a workshop involving similar representatives and health researchers was held to discuss the role of pesticide exposure monitoring. minutes from pac meetings, field notes taken during ki interviews, and workshop proceedings were synthesized to generate potential evaluation methods and indicators. results: current evaluation activities were limited but all kls supported greater evaluation effons beginning with fuller indicator monitoring. indicators of education and outreach services were imponant for industry representatives changing applicator practices as well as most public health units and environmental organizations. lndictors based on bylaw enforcement were only applicable in the two cities with bylaws, though changing attitudes toward legal approaches were being assessed in many communities. the public health rapid risk factor surveillance system could use historical baseline data to assess changes in community behaviour through reported pesticide uses and practices, though it had limited penetration in immigrant communities not comfortable in english. pesticide sales (economic) data were only available in regional aggregates not useful for city specific change documentation. testing for watercourse or environmental contamination might be helpful, but it is sporadic and expensive. human exposure monitoring was fraught with ethical issues, floor effects from low levels of exposure, and prohibitive costs. clinical episodes of pesticide exposure reported to the regional poison centre (all ages) or the mother risk program (pregnant or breastfeeding women) are likely substantial underestimates that would be need to be supplemented with sentinel practice surveillance. focus on special clinical populations e.g., multiple chemical sensitivity would require additional data collection efforts . . conc~ons: broad support for evaluation and multiple indicators were proposed, though con-s~raints associate~ with access, coverage, sensitivity and feasibility were all raised, demonstrating the difficulty of evaluating such urban primary prevention initiatives. interventionists. an important aim of the youth monitor is to learn more about the health development of children and adolescents and the factors that can influence this development. special attention is paid to emo· tional and behavioural problems. the youth monitor identifies high-risk groups and factors that are associated with health problems. at various stages, the youth monitor chancrs the course of life of a child. the sources of informa· tion and methods of research are different for each age group. the results arc used to generate various kinds of repons: for children and young persons, parents, schools, neighbourhoods, boroughs and the municipality of rotterdam and its environs. any problems can be spotted early, at borough and neigh· bourhood level, based on the type of school or among the young persons and children themselves. together with schools, parents, youngsters and various organisations in the area, the municipal health service aims to really address these problems. on request, an overview is offered of potentially suitable interventions. the authors will present the philosophy, working method, preliminary effects and future developments of this instrument, which serves as the backbone for the rotterdam local youth policy. social workers to be leaders in response to aging urban populations: the practicum partnership program sarah sisco, alissa yarkony, and patricia volland "'" tliu:tion: across the us, . % of those over live in urban areas. these aging urban popu· lations, including the baby boomers, have already begun encounter a range of heahh and mental hcahh conditions. to compound these effects, health and social service delivery fluciuates in cities, whit:h arc increasingly diverse both in their recipients and their systems. common to other disciplines (medicine, nursing, psychology, etc.) the social work profession faces a shortage of workers who are well-equipped to navigate the many systems, services, and requisite care that this vast population requires. in the next two decades, it is projected that nearly , social workers will be required to provide suppon to our older urban populations. social workers must be prepared to be aging-savvy leaders in their field, whether they specialize in gerontology or work across the life span. mllhotu: in , a study conducted at the new york academy of medicine d<> :umcntcd the need for improved synchroniciry in two aspects of social work education, classroom instruction and the field experience. with suppon from the john a. hanford foundation, our team created a pilot proj~"t entitled the practicum pannership program (ppp) in master's level schools of social work, to improvt" aginr exposure in field and classroom content through use of the following: i) community-university partnrr· ships, ) increased, diverse student field rotations, ll infusion of competcn ."}'·drivm coursework, enhancement of field instructors' roles, and ) concentrated student recruitment. we conductt"d a prr· and post-test survey into students' knowledge, skills. and satisfaction. icarlja: surveys of over graduates and field inltnk."tors rcflected increased numlk-n of . rrm:y· univmity panncrships, as well as in students placed in aging agencin for field placements. there wa marked increase in student commitments to an aging specialization. onr year por.t·gradu:nion rcvealrd that % of those surveyed were gainfully employed, with % employed in the field of aginic. by com· bining curricular enhancement with real-world experiences the ppp instilled a broad exposurr for llu· dents who worked with aging populations in multiple urban settings. coltdtuion: increased exposure to a range of levels of practicr, including clinical, policy/ajvocaq, and community-based can potentially improve service delivery for older adulh who live in elfin, and potentially improve national policy. the hanford foundation has now elected to uppon cxpantion of the ppp to schools nationwide (urban and rural) to complement other domntic initiatives to cnhalk"c" holistic services for older adults across the aging spectrum. bodrgnn.ntl: we arc a team of rcscarcbcn and community panncn working tcj c(her to develop an in"itepth understanding of the mental health needs of homeless youth ~ages to ) (using qualiutivc and quantitative methods ' panicipatory rncarch methods). it is readily apparmt that '-neless youth cxpcricnce a range of mental health problems. for youth living on the street, menul illnew may be either a major risk factor for homelessnal or may frequently emcsge in response to coping with rhe multitudinous stressors associated with homclcslllcsi including exposure to violence, prasutt to pamaplte in v poster sessions survival sex and/or drug use. the most frequent psychiatric diagnoses amongst the homeless gencrally include: depression, anxiety and psychosis. . . . the ultimate ob ective of the pr~am of rei:e~ is to ~evelop a plan for intervention to meet the mental health needs of street youth. prior t_o pl~nnmg mtervenbons, .it is necessary to undertake a comprehensive assessment ~f mental health needs m this ~lnerable populanon. thus, the immediate objective of this research study is to undertake a comprehensive assessment of men· tal health needs. . . melbotlology: a mixed methodology triangulating qualitative, participatory acnon and quantitative methods will capture the data related to mental health needs of homeless youth. a purposive sample of approximately - subjecrs. ages to , is currently being ~ted ~participate from the commu.nity agencies covenant house, evergreen centre fo~ srrc;et youth, turning p? ?t and street ~ serv~. youth living on the street or in short -term residennal programs for a mmimum of month pnor to their participation; ages to and able to give infonned consent will be invited to participate in the study. o..tcomes: the expected outcome of this initial survey will be an increased understanding of mental health needs of street youth that will be used to develop effective interventions. it is anticipated that results from this study will contribute to the development of mental health policy, as well as future programs that are relevant to the mental health needs of street youth. note: it is anticipated that preliminary quantitative data ( subjects) and qualitative data will be available for the conference. the authors intend to present the identification of the research focus, the formation of our community-based team, relevance for policy, as well as preliminary results. p - (a) the need for developing a firm health policy for urban informal worken: the case of despite their critical role in producing food for urban in kenya, urban farmers have largely been ignored by government planners and policymakers. their activity is at best dismissed as peripheral eveo, inappropriate retention of peasant culture in cities and at worst illegal and often some-times criminal· ized. urban agriculture is also condemned for its presumed negative health impact. a myth that contin· ues despite proof to the contrary is that malarial mosquitoes breed in maize grown in east african towns. however, potential health risks are insignificant compared with the benefits of urban food production. recent studies too rightly do point to the commercial value of food produced in the urban area while underscoring the importance of urban farming as a survival strategy among the urban poor, especially women-headed households. since the millennium declaration, health has emerged as one of the most serious casualties consequent on the poverty, social exclusion, marginalisation and lack of sustain· able development in africa. hiv/aids epidemic poses an unprecedented challenge, while malaria, tuber· culosis, communicable diseases of childhood all add to the untenable burden. malnutrition underpins much ill-health and is linked to more than per cent of all childhood deaths. kenya's urban poor people ~ace ~ h~ge burde~ of preventable and treatable health problems, measured by any social and bi~ medical md cator, which not only cause unnecessary death and suffering, but also undermine econonuc development and damage the country's social fabric. the burden is in spite of the availability of suitable tools and re:c=hnology for prevention and treatment and is largely rooted in poverty and in weak healah •rstems. this pa~ therefore challenges development planners who perceive a dichotomy instead of con· tmuum between informal and formal urban wage earners in so far as access to health services is con· cemed. it i~ this gap that calls for a need to developing and building sustainable health systems among the urban mformal ~wellers. we recommend a focus on an urban health policy that can build and strengthen the capacity of urban dwellers to access health services that is cost-effective and sustainable. such ~ health poli<=>: must strive for equity for the urban poor, displaced or marginalized; mobilise and effect ~ely use sufficient sustainable resources in order to build secure health systems and services. special anenti_on. should ~ afforded hiv/aids in view of the unprecedented challenge that this epidemic poses to africa s economic and social development and to health services on the continent. methods: a review of the literature led us to construct three simple models and a composite model of exposure to traffic. the data were collected with the help of a daily diary of travel activities using a sample of cyclists who went to or come back from work or study. to calculate the distance, the length of journey, and the number of intersections crossed by a cyclist different geographic information systems (gis) were operated. statistical analysis was used to determine the significance between a measure of exposure on the one hand, and the sociodemographic characteristics of the panicipants or their geographic location on the other hand. restlltj: our results indicate that cyclists were significantly exposed to road accidents, no matter of where they live or what are their sociodemographic characteristics. we also stress the point that the fact of having been involved in a road accident was significantly related to the helmet use, but did not reduce the propensity of the cyclists to expose themselves to the road hazards. condlllion: the efforts of the various authorities as regards road safety should not be directed towards the reduction of the exposure of the vulnerable users, but rather towards the reduction of the dangers to which they could face. keywords: cyclist, daily diary of activities, measures of exposure to traffic, island of montreal. p - (a) intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods olumuyiwa akinbamijo intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods abstract urbanization panicularly in nigerian cities, ponends unprecedented crises of grave dimensions. from physical and demographic viewpoints, city growth rates are staggering coupled with gross inabilities to cope with the consequences. environmental and social ills associated with unguarded rapid urbanization characterize nigerian cities and threaten urban existence. this paper repons the findings of a recent study of the relationship between environmental health across inrraurban residential communities of akure, south west nigeria. it discuses the typical urbanization process of nigerian cities and its dynamic spatial-temporal characteristics. physical and socio-demographic attributes as well as the levels and effectiveness of urban infrastructural services are examined across the core residential districts and the elite residential layouts in the town. the incidence rate of cenain environmentally induced tropical diseases across residential neighborhoods and communes is examined. salient environmental variables that are germane to health procurement in the residential districts, incidence of diseases and diseases parasitology, diseases prevention and control were studied. field data were subjected to analysis ranging from the univariate and bivariate analysis. inferential statistics using the chi-square test were done to establish the truthfulness of the guiding hypothesis. given the above, the study affirms that there is strong independence in the studied communities, between the environment and incidence of diseases hence health of residents of the town. this assertion, tested statistically at the district levels revealed that residents of the core districts have very strong independence between the environment and incidences of diseases. the strength of this relationship however thins out towards the city peripheral districts. the study therefore concludes that since most of the city dwellers live in urban deprivation, urban health sensitive policies must be evolved. this is to cater for the urban dwellers who occupy fringe peripheral sites where the extension of facilities often times are illegally done. urban infrastructural facilities and services need be provided as a matter of public good for which there is no exclusive consumption or access even for the poorest of the urban poor. many suffer from low-self esteem, shame and guilt about their drug use. in addition, they often lack suppon or encounter opposition from their panners, family and friends in seeking treatment. these personal barriers are compounded by fragmented addiction, prenatal and social care services, inflexible intake systems and poor communication among sectors. the experience of accessing adequate care between services can be overwhelming and too demanding. the toronto centre for substance use in pregnancy (t-cup) is a unique program developed to minimize barriers by providing kone-stop" comprehensive healthcare. t-cup is a primary care based program located in the department of family medicine at st. joseph\'s health centre, a community teaching hospital in toronto. the interdisciplinary staff provides prenatal and addiction services, case management, as well as care of newborns affected by substance use. regular care plan meetings are held between t-cup, labour and delivery nurses and social workers in the y poster sessions maternity and child care program. t-cup also connects "'.omen with. inpatient treatment programs and community agencies such as breaking the cycle, an on-site counselmg group for pregnant substance users. · f · d d h ith method: retrospective chart review, qualitative patient ~ans action stu ~· an ea care provider surveys are used to determine outcomes. primary outcomes mclude changes m maternal su~tance use, psychosocial status and obstetrical complications (e.g. pre-rupture of membrane, pre-eclampsia, placen· ral abruption and hemorrhage). neonatal measures ~~nsisted of .bir~h pa_rame~ers, length of h~spital st.ay and complications (e.g. feral distress, meconium stammg, resuscitation, aund ce, hypoglycemia, seventy of withdrawal and treatment length). chart review consisted of all t-cup patients who met clinical cri· reria for alcohol or drug dependence and received prenatal and intra-partum care at st. joseph's from october to june . participants in the qualitative study included former and current t-cup patients. provider surveys were distributed on-site and to a local community hospital. raulb: preliminary evaluation has demonstrated positive results. treatment retention and satisfaction rates were high, maternal substance use was markedly reduced and neonatal outcomes have shown to be above those reported in literature. conclusion: this comprehensive, primary care model has shown to be optimal in the management of substance use in pregnancy and for improving neonatal outcomes. future research will focus on how this inexpensive program can be replicated in other health care settings. t-cup may prove to be the optimal model for providing care to pregnant substance users in canada. lntrod ction: cigarette smoking is one of the most serious health problems in taiwan. the prevalence of smoking in is . % in males . % in females aged years and older. although the government of taiwan passed a tobacco hazards control act in , it has not been strongly enforced in many places. therefore, community residents have often reported exposure of second hand smoke. the purpose of the study was to establish a device to build up more smoke-free environments in the city of tainan. methods: unique from traditional intervention studies, the study used a healthy city approach to help build up smoke-free environments. the major concept of the approach is to build up a healthy city platform, including organizing a steering committee, setting up policies and indicators, creating intersectoral collaboration, and increasing community participation. first, more than enthusiastic researchers, experts, governmental officers, city counselors and community leaders in tainan were invited in the healthy city committee. second, smoke-free policies, indicators for smoke-free environments, and mechanisms for inter-departmen· tal inspections were set up. third, community volunteers were recruited and trained for persuading related stakeholders. lastly, both penalties and rewards were used for help build up the environments. raults: aher two-year ( aher two-year ( - execution of the project, the results qualitatively showed that smoke-free environments in tainan were widely accepted and established, including smoke-free schools, smoke-free workpla~es, smoke-free households, smoke-free internet shops, and smoke-free restaurants. smoke~s were. effectively educated not to smoke in public places. community residents including adults and children m the smoke-free communities clearly understand the adverse effects of environmental tobacco smoke and actively participated anti-smoking activities. conclruions: healthy city platform is effective to conquer the barrier of limited anti-smoking rc:sources. nor. only can it enlar:ge community actions for anti-smoking campaigns, but also it can provide par_merships for collaboratjon. by establishing related policies and indicators the effects of smoke· free environments can be susta ·ned a d th · · · ' · n e progression can be monitored m a commuruty. these issues are used ~· oi::c it~ goals, weuha identifies issues that put people's health at risk. presently, team com~u:c: ran ee~tion !earns. (iats) that design integrative solutions ~tesj'°~ g om six to fifteen members. methods in order to establish wo-poster sessions v projects for weuha, the following approach was undertaken: i. a project-polling template was created and sent to all members of the alliance for their input. each member was asked to identify thdr top two population groups, and to suggest a project on which to focus over a - month period for each identified population. . there was a % response to the poll and the top three population groups were identified. data from the toronto community health profile database were utilized to contextualize the information supplied for these populations. a presentation was made to the steering committee and three population-based projects were selected, leaders identified and iats formed. three population-based projects: the population-based projects and health care issues identified are: newcomer prenatal uninsured women; this project will address the challenges faced by providers to a growing number of non-insured prenatal women seeking care. a service model where the barrier of "catchments" is removed to allow enhanced access and improved and co-ordinated service delivery will be pilot-tested. children/obesity/diab etes: using a health promotion model this team will focus on screening, intervention, and promoting healthy lifestyles (physical activity and nutrition) for families as well as for overweight and obese children. seniors health promotion and circle of discharge: this team will develop an early intervention model to assist seniors/family unit/caregivers in accessing information and receiving treatment/care in the community. the circle of discharge initiative will address ways of utilizing community supports to keep seniors in the community and minimize readmissions to acute care facilities. results/expected outcomes: coordinated and enhanced service delivery to identified populations, leading to improved access, improved quality of life, and health care for these targeted populations. introduction: basic human rights are often denied to high-risk populations and people living with hiv/aids. their rights to work and social security, health, privacy, non discrimination, liberty and freedom of movement, marriage and having a family have been compromised due to their sero-positive status and risk of being positive. the spread of hiv/aids has been accelerating due to the lack of general human rights among vulnerable groups. to formulate and implement effective responses needs dialogue and to prevent the epidemic to go underground barriers like stigma need to be overcome. objective: how to reduce the situation of stigma, discrimination and human rights violations experienced by people living with hiv/aids and those who are vulnerable to hiv/aids. methodology and findings: consultation meetings were strm.-rured around presentations, field visits, community meetings and group work to formulate recommendations on how govt and ngos/cbos should move forward based on objective. pakistan being a low prevalence country, the whole sense of compl;u:enc.:y that individuals are not subject to situations of vulnerable to hiv is the major threat to an explosion in th•· epidemic, therefore urgent measures are needed to integrate human rights issues from the very start of the response. the protection and promotion of human rights in an integral component of ;tll responses to the hiv/aids epidemic. it has been recognized that the response to hiv/aios must he multi sectoral and multi faceted, with each group contributing its particular expertise. for this to occur along with other knowlcdg<" more information is required in human rights abuses related to hiv/ aids in a particular scenario. the ~·on sultarion meetings on hiv/aids and human rights were an exemplary effort to achieve the same ohj<..:tivc. recommendations: the need for a comprehensive, integrated and a multi-sectoral appro;u.:h in addressing the issue of hiv/aids was highlighted. the need social, cultural and religious asp•·ct' to he: prominently addressed were identified. it was thought imperative measures even in low prevalence countries. education has a key role to play, there is a need for a code of ethics for media people and h<"alth care providers and violations should be closely monitored and follow up action taken. p - (c) how can community-based funding programs contribute to building community capacity and how can we measure this elusive goal? mary frances maclellan-wright, brenda cantin, mary jane buchanan, and tammy simpson community capacity building is recognized by the public health agency of canada (phac) as an important strategy for improving the overall health of communities by enabling communities to addre~s priority issues such as social and economic determinants of health. in / phac.:, alberta/nwf region's population health fund (phf) supported community-based projects to build community capacity on or across the determinants of health. specifically, this included creating accessible and sup· portive social and physical environments as well as creating tools and processes necessary for healthy policy development and implementation. the objective of this presentation is to highlight how the community capacity building tool, developed by phac ab/nwf region, can demonstrate gains in v poster sessions · · the course of a pror· ect and be used as a reflective tool for project planning and community capacity over . . . . i · a art of their reporting requirements, pro ect sites completed the community caparny eva uanon. s p . . th t i ii i'd d . building tool at the beginning and end of their ~ne-year prorect. e oo ~o ects va an reliable data in the context of community-based health prorects. developed through a vigorous ~nd collabora ve research process, the tool uses plain languag~ to expl~re nine key f~atures o~ commuruty cap~city with 't ch with a section for contextual information, of which also mdude a four-pomt raong ems, ea f fu d · scale. results show an increase in community capacity over the course o the nde prorects. pre and post aggregate data from the one-year projects measure~ statistic.ally si~n~ficant changes for of the scaled items. projects identified key areas of commumty capacity bmldmg that needed strengthemng, such as increasing participation, particularly among people with low incomes; engaging community members in identifying root causes; and linking with community groups. in completing the tool, projects examined root causes of the social and economic determinants of health, thereby exploring social justice issues related to the health of their community. results of the tool also served as a reflec· cion on the process of community capacity building; that is, how the project outcomes were achieved. projects also reported that the tool helped identify gaps and future directions, and was useful as a project planning, needs assessment and evaluation tool. community capacity building is a strategy that can be measured. the community capacity building tool provides a practical means to demonstrate gains in community capacity building. strengthening the elements of community capacity building through community-based funding can serve as building blocks for addressing other community issues. needs of marginalized crack users lorraine barnaby, victoria okazawa, barb panter, alan simpson, and bo yee thom background: the safer crack use coalition of toronto (scuc) was formed in in response to the growing concern for the health and well-being of marginalized crack users. a central concern was the alarm· ing hepatitis c rate ( %) amongst crack smokers and the lack of connection to prevention and health ser· vices. scuc is an innovative grassroots coalition comprised of front-line workers, crack users, researcher! and advocates. despite opposition and without funding, scuc has grown into the largest crack specific harm reduction coalition in canada and developed a nationally recognized sarer crack kit distribution program (involving community-based agencies that provide outreach to users). the success of our coalition derives from our dedication to the issue and from the involvement of those directly affected by crack use. setting: scuc's primary service region is greater toronto, a diverse, large urban centre. much ofour work is done in areas where homeless people, sex trade workers and drug users tend to congregate. recently, scuc has reached out to regional and national stakeholders to provide leadership and education. mandate: our mandate is to advocate for marginalized crack users and support the devdopmentof a com.p.rehensive harm reduction model that addresses the health and social needs facing crack users; and to fac htare the exchange of information between crack users, service providers, researchers, and policy developers across canada. owrview: the proposed workshop will provide participants with an overview of the devdopment of scuc, our current projects (including research, education, direct intervention and consultation), our challenge~ and s~ccesses and the role of community development and advocacy within the coalition. pre-senter~ will consist of community members who have personal crack use experience and front-line work· ers-, sc.uc conducted a community-based research project (toronto crack users perspectives, ) , in w~ich s focus groups with marginalized crack users across toronto were conducted. participants iden· t f ed health and social issues affecti h b · · · d " red . . ng t em, arrsers to needed services, personal strategies, an oue recommendations for improved services. presenters will share the methodology, results and recommen· datmns resulting from the research project. conc/usio": research, field observations and consultations with stakeholders have shown that cradck shmoke~s are at an. increased risk for sexually transmitted infections hiv/aids hepatitis c, tb an ot er serious health issues health · ff, · ' ' · · . · issues a ectmg crack users are due to high risk behavmurs, socio· economic factors, such as homeless d. · · · · d · . . ness, scrsmmat on, unemployment, violence incarceraoons, an soc a so at on, and a lack of comprehe · h i h · ' ns ve ea t and social services targeting crack users. · · sinct · s, owever arge remains a gross underesurnaoon. poster sessions v these are hospital-based reports and many known cases go unreported. however teh case, young age at first intercourse, inconsistent condom use and multiple partnersplace adolescents at high risks for a diverse array of stls, including hiv. about % of female nigerian secondary school students report initiating sexual intercourse before age years. % of nigerian female secondary school students report not using a condom the last time they had sexual intercourse. more than % of urban nigerian teens report inconsistent condom use. methods: adolescents were studied, ages to , from benin city in edo state. the models used were mother-daughter( ), mother -son( ), father -son ( ), and father-daughter( ). the effect of parent-child sexual communicationat baseline on child\'s report of sexual behavior, to months later were studied. greater amounts of sexual risk communication were asociated with markedly fewer episodes of unprotected sexual intercourse, reduced number of sexual partners and fewer episodes of unprotected sexual intercourse. results: this study proved that parents can exert more influence on the sexual knowledge attitudes and practise of their adolescent children through desired practises or rolemodeling, reiterating their values and appropriate monitoring of the adolescents\' behavior. they also stand to provide information about sexuality and various sexual topics. parental-child sexual communication has been found to be particularly influential and has been associated with later onset of sexual initiation among adolescents, less sexual activity, more responsible sexual attitudes including greater condom use, self efficacy and lower self -reported incidence of stis. conclusions: parents need to be trained to relate more effectively with their children/wards about issues related to sex and sexuality. family -based programs to reduce sexual risk-taking need to be developed. there is also the need to carry out cross-ethnicaland cross-cultural studies to identify how parent-child influences on adolescent sexual risk behavior may vary in different regions or countries, especially inthis era of the hiv pandemic. introduction: public health interventions to identify and eliminate health disparities require evidence-based policy and adequate model specification, which includes individuals within a socioecological context, and requires the integration of biosociomedical information. multiple public and private data sources need to be linked to apportion variation in health disparities ro individual risk factors, the health delivery system, and the geosocial environment. multilevel mapping of health disparities furthers the development of evidence-based interventions through the growth of the public health information network (phin-cdc) by linking clinical and population health data. clinical encounter data, administrative hospital data, population socioenvironmental data, and local health policy were examined in a three-level geocoded multilevel model to establish a tracking system for health disparities. nj has a long established political tradition of "home rule" based in elected municipal governments, which are responsible for the well-being of their populations. municipalities are contained within counties as defined by the us census, and health data are linked mostly at the municipality level. marika schwandt community organizers from the ontario coaliti~n again~t pove~, .along ":ith ~edical practitioners who have endorsed the campaign and have been mvolved m prescnbmg special diet needs for ow and odsp recipients, will discuss the raise the rates campaign. the organizati~n has used a special diet needs supplement as a political tool, meeting the urgent needs o.f .poor ~ople m toront~ while raising the issues of poverty as a primary determinant of health and nutrtnous diet as a preventative health mea· sure. health professionals carry the responsibility to ensure that they use all means available to them to improve the health of the individuals that they serve, and to prevent future disease and health conditions. most health practitioners know that those on social assistance are not able to afford nutritious foods or even sufficient amounts of food, but many are not aware of the extra dietary funds that are available aher consideration by a health practitioner. responsible nurse practitioners and physicians cannot, in good conscience, ignore the special needs diet supplement that is available to all recipients of welfare and disabiliry (ow and odsp). a number of toronto physicians have taken the position that all clients can justifiably benefit from vitamins, organic foods and high fiber diets as a preventative health measure. we know that income is one of the greatest predictors of poor health. the special needs diet is a health promotion intervention which will prevent numerous future health conditions, including chronic conditions such as cardiovascular disease, cancer, diabetes and osteoporosis. many communiry health centres and other providers have chosen to hold clinics to allow many patients to get signed up for the supplement at one time. initiated by the ontario coalition against poverty, these clinics have brought together commu· niry organizers, community health centers, health practitioners, and individuals, who believe that poverty is the primary determinant of poor health. we believe that rates must be increased to address the health problems of all people on social assistance, kids, elders, people with hiv/aids -everyone. even in the context of understaffing, it could be considered a priority activity that has potentially important health promotion benefits. many clients can be processed in a two hour clinic. most providers find it a very interesting, rewarding undertaking. in the ontario coalition for social justice found that a toronto family with two adults and two kids receives $ , . this is $ , below the poverty line. p - (c) the health of street youth compared to similar aged youth beth hayhoe and ruth ewert . lntrod~on: street youth are at an age normally associated with good health, but due to their risky ~hav ours and th~ conditions in which they live, they experience health conditions unlike their peer~ an more stable env r~nments. in addition, the majority of street youth have experienced significant physical, sexual ~nd em.ot onal abuse as younger children, directly impacting many of the choices they make around their physical and emotional health. we examined how different their health really is. . , methodl: using a retrospective analysis of the years of data gathered from yonge street mis· ~ • evergreen health centre, the top conditions of youth were examined and compared with national tren~s for similar aged youth. based on knowledge of the risk factors present in the group, rea· sons for the difference were examined. d' ~its: street youth experience more illness than other youth their age and their illnesses can bt . irect t ·~kc~ to the. conditions in which they live. long-term impacts of abus~ contribute to such signif· ~~nt t e t d~slpl air that youth may voluntarily engage in behaviours or lack of self care in the hope at t cir ve~ w perhaps come to a quicker end. concl non: although it has ion b k h th' dy clearly shows d'fi . h g ee~ no~n t at poverty negatively affects health, ~siu be used to make ; erence m t .e health of this particular marginalized population. the infonnanon can relates to th . ecommendatio.ns around public policy that affects children and youth, especially as it e r access to appropriate health care and follow up. p - (cl why do urban children · b gt . tarek hussain an adesh die: how to save our children? the traditional belief that urban child alid. a recent study (dhs d fr r~n are better off than rural children might be no longer v urban migrants are highata th om h c~untn~s i demonstrates that the child survival prospects of rural· er an t ose m their r j · · ·grants. in bangladesh, currently million ~r~ ~ gm and lower than those of urban non-idi million. health of the urban ~ p~e are hvmg m urban area and by the year , it would be so the popu at on s a key a eals that urban poor have the worse h h . concern. recent study on the urban poor rev ea t situation than the nation as a whole. this study shows that infant poster sessions v mortality among the urban poor as per thousand, which are above the rural and national level estimates. the mortality levels of the dhaka poor are well above those of the rest of the city's population but much of the difference in death rates is explained by the experience of children, especially infants. analyzing demographic surveillance data from a large zone of the city containing all sectors of the population, research showed that the one-fifth of the households with the least possessions exhibited u child mortality almost three times as high as that recorded by the rest of the population. why children die in bangladesh? because their parents are too poor to provide them with enough food, clean water and other basic needs to help them avoid infection and recover from illness. researchers believed that girls are more at risk than boys, as mothers regularly feed boys first. this reflects the different value placed on girls and boys, as well as resources which may not stretch far enough to provide for everyone. many studies show that housing conditions such as household construction materials and access to safe drinking water and hygienic toilet facilities are the most critical determinants of child survival in urban areas of developing countries. the present situation stressed on the need for renewed emphasis on maternal and child healthcare and child nutrition programs. mapping path for progress to save our children would need be done strategically. we have the policies on hand, we have the means, to change the world so that every child will survive and has the opportunity to develop himself fully as a healthy human being. we need the political will--courage and determination to make that a reality. p - (c) sherbourne health centre: innovation in healthcare for the transgendered community james read introduction: sherbourne health centre (shc), a primary health care centre located in downtown toronto, was established to address health service gaps in the local community. its mission is to reduce barriers to health by working with the people of its diverse urban communities to promote wellness and provide innovative primary health services. in addition to the local communities there are three populations of focus: the lesbian, gay, bisexual, transgendered and transexual communities (lgbtt); people who are homeless or underhoused; and newcomers to canada. shc is dedicated to providing health services in an interdisciplinary manner and its health providers include nurses, a nurse practitioner, mental health counsellors, health promoters, client-resource workers, and physicians. in january shc began offering medical care. among the challenges faced was how to provide responsive, respectful services to the trans community. providers had considerable expertise in the area of counselling and community work, but little in the area of hormone therapy -a key health service for those who want to transition from one gender to another. method: in preparing to offer community-based health care to the trans community it was clear that shc was being welcomed but also being watched with a critical eye. trans people have traditionally experienced significant barriers in accessing medical care. to respond to this challenge a working group of members of the trans community and health providers was created to develop an overall approach to care and specific protocols for hormone therapy. the group met over a one year period and their work culminated in the development of medical protocols for the provision of hormone therapy to trans individuals. results: shc is currently providing health care to registered clients who identify as trans individuals (march ) through primary care and mental health programs. in an audit of shc medical charts (january to september ) female-to-male (ftm) and male-to-female (mtf) clients were identified. less than half of the ftm group and just over two-thirds of the mtf group presented specifically for the provision of hormones. based on this chart audit and ongoing experience shc continues to update and refine these protocols to ensure delivery of quality care. conclusion: this program is an example of innovative community-based health delivery to a population who have traditionally faced barriers. shc services also include counselling, health promotion, outreach and education. p - (c) healthy cities for canadian women: a national consultation sandra kerr, kimberly walker, and gail lush on march , the national network on environment and women's health held a pan-canadian consultation to identify opportunities for health research, policy change, and action. this consultation also worked to facilitate information sharing and networking between canadian women working as urban planners, policy makers, researchers, and service workers on issues pertaining to the health of women living in canadian cities. methods: for this research project, participants included front-line service workers, policy workers, researchers, and advocates from coast to coast, including francophone women, women with disabilities, racialized women, and other marginalized groups. the following key areas were selected as topics for du.bnes i alto kading .:auk of end·sugr ieaal clileue ia singapore, accounting for more than so% of new can singapore (nkfs) to embark on a prevention program (pp) empo~r d ahc j u f dieir condition bttter, emphasizing education and disease sdf·managemen lkilla a. essennal camponenn of good glycaemic control. we sought explore the effects of a pecialijed edu.:a on pro· pun od glycacmic conuol, as indicated by, serum hba ic values budine serum hba ic values were determined before un<k-rgmng the education progr ;am, which couisted of comprehensive dietary advice, ideal weight goals, and improving lipid profiln. serum hbaic values were obtained ar , , months later to determine impact of the program. analy • wu done uling paired !·tests significant reduction in hba le levels from ro month• was observed in females, chinese race, older age group(> so yean). ohew-ibmi ~ .nwm , wai hip ratio> l),up to primary and above secondary level education and those having om urine iclt showed that increasing hbalc levels ( ) had increasing urmary protein ( .± ; . ±i ih so± ) and crearinine (s .s ± s ± ; ioi± s) levels fbg rnults showed that the management nf d abetn m the nkfs preven· tion programme is effec;rive. results also indicated har hba le leve have a linnr trend wnh unnary protein and creatinine which are imponant determinants of renal diseate tal family-focused cinical palbway promoce politivc outcollln for ua inner city canu allicy ipmai jerrnjm care llctivirits in preparation for an infanr'' dilchargr honlr, and art m endnl lo improve effi.:k'fl.:tn of c.are. lere i paucity of tttran:h, and inconsi trncy of rnulta on ht-•m!*- of f m ly·fc"-'uw d nm a: to determinr whrthrr implrmentation of family.focuted c:pt n ntnn.tt.tl unit w"n mg an inner city ;ommunity drcl't'aki leftarh of lf•y (i.osi and rromclll'i family uo•fkllon and rt. j nest for dikhargr. md odt: family-focuk"d cpi data wm coll«ted for all infant• horn btrwttn and wft"k• t"lal mi atr who wrtt . dm ed to the ntonatal unit lmgdl of -.y . n. . day'o p c o.osi ind pma . d•mr., ho.nr . t . n. . ± i. i wb, p < o.os) wett n« fiamly f.lfrt n the pre.(]' poup. ~ .fxtmon icofn for famihn wrre high. and families noctd thc:y wnr mott prepued to ah thrar t..lby "'-· thett was .a cosi uving of s , (cdn) per patient d teharpd home n the pmi-cp poap c.-pated the p"''lfoup· cortclaion· lmplrmrnr.rion of family·foanrd c:p. in a nrona . i umt tc"fyidi an nnn an com· muniry decre.ned length of'"'" mft with a high dcgrft of family uujamon, and wrre coll~nt at least % percent of the kathmandu population lives in slum like conditions with poor access to basic health services. in these disadvantaged areas, a large proportion of children do not receive treatment due to inaccessibility to medical services. in these areas, diarrhea, pneumonia, and measles, are the key determinants of infant mortality. protein energy malnutrition and vitamin a deficiency persists and communicable diseases are compounded by the emergence of diseases like hiv/aids. while the health challenges for disadvantaged populations in kathmandu are substantial, the city has also experienced various forms of innovative and effective community development health programs. for example, there are community primary health centers established by the kathmandu municipality to deliver essential health services to targeted communities. these centers not only provide equal access to health services to the people through an effective management system but also educate them hy organizing health related awareness programs. this program is considered one of the most effective urban health programs. the paper/presentation this paper will review large, innovative, and effective urhan health programs that are operating in kathmandu. most of these programs are currently run by international and national ngos a) early detection of emerging diseases in urban settings through syndromic surveillance: data pilot study kate bassil of community resources, and without adequate follow-up. in november shelter pr.oviders ~et with hospital social workers and ccac to strike a working group to address some of th~ issues by mcre.asing knowledge among hospital staff of issues surrounding homelessness, and to build a stro?g workmg relationship between both systems in hamilton. to date the hswg has conducted four w~lkmg to~ of downtown shelters for hospital staff and local politicians. recently the hswg launched its ·~ool.k t for staff working with patients who are homeless', which contains community resources and gu dehnes to help with effective discharge plans. a scpi proposal has been submitted to incre~se the capacity of the hswg to address education gaps and opportunities with both shelters and hospitals around homelessness and healthcare. the purpose of this poster presentation is to share hamilton's experience and learnings with communities who are experiencing similar issues. it will provide for intera~tion around shared experiences and a chance to network with practitioners across canada re: best practices. introduction and objectives: canadians view health as the biggest priority for the federal government, where health policies are often based on models that rely on abstract definitions of health that provide little assistance in the policy and analytical arena. the main objectives of this paper are to provide a functional definition of health, to create a didactic model for devising policies and determining forms of intervention, to aid health professionals and analysts to strategize and prioritize policy objectives via cost benefit analysis, and to prompt readers to view health in terms of capacity measures as opposed to status measures. this paper provides a different perspective on health, which can be applied to various applications of health such as strategies of aid and poverty reduction, and measuring the health of an individual/ community/country. this paper aims to discuss theoretical, conceptual, methodological, and applied implications associated with different health policies and strategies, which can be extended to urban communities. essentially, our paper touches on the following two main themes of this conference: •health status of disadvantaged populations; and •interventions to improve the health of urban communities.methodology: we initially surveyed other models on this topic, and extrapolated key aspects into our conceptual framework. we then devised a theoretical framework that parallels simple theories of physkal energy, where health is viewed in terms of personal/societal health capacities and effort components.after establishing a theoretical model, we constructed a graphical representation of our model using selfrated health status and life expectancy measures. ultimately, we formulated a new definition of health, and a rudimentary method of conducting cost benefit analysis on policy initiatives. we end the paper with an application example discussing the issues surrounding the introduction of a seniors program.results: this paper provides both a conceptual and theoretical model that outlines how one can go about conducting a cost-benefit analysis when implementing a program. it also devises a new definition and model for health barred on our concept of individual and societal capacities. by devising a definition for health that links with a conceptual and theoretical framework, strategies can be more logically constructed where the repercussions on the general population are minimized. equally important, our model also sets itself up nicely for future microsimulation modeling and analysis.implications: this research enhances one's ability to conduct community-based cost-benefit analysis, and acts as a pedagogical tool when identifying which strategies provide the best outcome. p - (a) good playgrounds are hard to find: parents' perceptions of neighbourhood parks patricia tucker, martin holmes, jennifer irwin, and jason gilliland introduction: neighbourhood opportunities, including public parks and physical activity or sports fields hav~ been. iden.tified as correlates to physical activity among youth. increasingly, physical activity among children s bemg acknowledged as a vital component of children's lives as it is a modifiable determinant of childh~d obesity. children's use of parks is mainly under the influence of parents; therefore, the purpose of this study was to assess parents' perspectives of city parks, using london ontario as a case study.m~~: this qualitative study targeted a heterogeneous sample of parents of children using local parks w thm london. parents with children using the parks were asked for minutes of their time and if willing, a s.hort interview was conducted. the interview guide asked parents for their opinion 'of city parks, particularly the one they were currently using. a sample size of parents is expected by the end of the summer.results: preliminary findings are identifying parents concern with the current jack of shade in local parks. most parents have identified this as a limitation of existing parks, and when asked what would make the parks better, parents agree that shade is vital. additionally, some parents are recognizing the v poster sessions focused discussions during the consultation: . women in _poverty . women with disability . immi· grant and racialized women . the built and _physica_l environment. . . . . r its· participants voiced the need for integration of the following issues withm the research and policy :::na; t) the intersectional nature of urban women's health i~sues wh~ch reflects the reality of women's complex lives ) the multisectional aspect of urban wo_m~n s health, ss~es, which reflects the diversity within women's lives ) the interse~roral _dynamics within _womens hves and urban health issues. these concepts span multiple sectors -mdudmg health, educat n, and economics -when leveraging community, research, and policy support, and engaging all levels of government.policy jmplicatiom: jn order to work towards health equity for women, plans for gender equity must be incorporated nationally and internationally within urban development initiatives: • reintroduce "women" and "gender" as distinct sectors for research, analysis, advocacy, and action. •integrate the multisectional, intersectional, and intersecroral aspects of women's lives within the framework of research and policy development, as well as in the development of action strategies. • develop a strategic framework to house the consultation priorities for future health research and policy development (for example, advocacy, relationship building, evidence-based policy-relevant research, priority initiatives}.note: research conducted by nnewh has been made possible through a financial contribution from health canada. the views expressed herein do not necessarily represent the views of health canada.p - (c) drugs, culture and disadvantaged populations leticia folgar and cecilia rado lntroducci n: a partir de un proyecto de reducci n de daiios en una comunidad urbana en situ· aci n de extrema vulnerahilidad surge la reflexion sobre el lugar prioritario de los elementos sociocuhurales en el acceso a los servicios de salud de diferentes colectivos urbanos. las "formas de hacer, pensar y sentir" orientan las acciones y delimitan las posibilidades que tienen los individuos de definir que algo es o no problema, asf como tambien los mecanismos de pedido de ayuda. el analisis permanenre del campo de "las culturas cotidianas" de los llamados "usuarios de drogas" aporta a la comprension de la complejidad del tema en sus escenarios reales, y colabora en los diseiios contextualizados de politicas y propuestas socio-sanitarias de intervenci n, tornandolas mas efectivas.mitodos: esta experiencia de investigaci n-acci n que utiliza el merodo emografico identifica elementos socio estructurales, patrones de consumo y profundiza en los elementos socio-simb icos que estructuran los discursos de los usuarios, caracterizandolos y diferenciandolos en tanto constitutivos de identidades socia les que condicionan la implementaci n del programas de reduccion de daiios.resultados: los resultados que presentaremos dan cuenta de las caracteristicas diferenciadas v relaciones particulares ~ntre los consumidores de drogas en este contexto espedfico. a partir de este e~tudio de caso se mtentara co ? enzar a responder preguntas que entendemos significativas a la hora de pensar intcrvcnciones a la med da de poblaciones que comparten ciertas caracteristicas socio-culturales. (cuales serian las .motivaciones para el cambio en estas comunidades?, cque elementos comunitarios nos ayudan a i:nnstnur dema~~a? • cque tenemos para aprender de las "soluciones" que ellos mismos encuenrran a los usos problemat cos? methods: our study was conducted by a team of two researchers at three different sites. the mapping consisted of filling in a chart of observable neighbourhood features such as graffiti, litter, and boarded housing, and the presence or absence of each feature was noted for each city block. qualitative observations were also recorded throughout the process. researchers analyzed the compiled quantitative and qualitative neighbourhood data and then analyzed the process of data collection itself.results: this study reveals the need for further research into the effects of physical environments on individual health and sense of well-being, and perception of investment in neighbourhoods. the process reveals that perceptions of health and safety are not easily quantified. we make specific recommendations about the mapping methodology including the importance of considering how factors such as researcher social location may impact the experience of neighbourhoods and how similar neighbourhood characteristics are experienced differently in various spaces. further, we discuss some of the practical considerations around the mapping exercise such as recording of findings, time of day, temperature, and researcher safety.conclusion: this study revealed the importance of exploring conceptions of health and well-being beyond basic physical wellness. it suggests the importance of considering one's environment and one's own perception of health, safety, and well-being in determining health. this conclusion suggests that attention needs to be paid to the connection between the workplace and the external environment it is situated in. the individual's workday experience does not start and stop at the front door of their workplace, but rather extends into the neighbourhood and environment around them. our procedural observations and recommendations will allow other researchers interested in the effect of urban environments on health to consider using this innovative methodology. introduction: responding ro protests against poor medical attention for sexually assaulted women and deplorable conviction rates for sex offenders, in the late s, the ontario government established what would become over hospital-based sexual assault care and treatment centres (sactcs) across the province. these centres, staffed around the clock with specially trained heath care providers, have become the centralized locations for the simultaneous health care treatment of and forensic evidence collection from sexually assaulted women for the purpose of facilitating positive social and legal outcomes. since the introduction of these centres, very little evaluative research has been conducted to determine the impact of this intervention. the purpose of our study was to investigate it from the perspectives of sexually assaulted women who have undergone forensic medical examinations at these centres.method: women were referred to our study by sactc coordinators across ontario. we developed an interview schedule composed of both closed and open-ended questions. twenty-two women were interviewed, face-to-face. these interviews were approximately one-to-two hours in length, and were transcribed verbatim. to date, have been analyzed for key themes.results: preliminary findings indicate that most women interviewed were canadian born ( 'yo), and ranged in age from to years. a substantial proportion self-identified as a visible minority ( 'x.). approximately half were single or never married ( %) and living with a spouse or family of origin ( %). most were either students or not employed ( %). two-thirds ( %) had completed high school and onethird ( %) was from a lower socio-economic stratum. almost two-fifths ( %) of women perceived the medical forensic examination as revictimizing citing, for example, the internal examination and having blood drawn. the other two-thirds ( %) indicated that it was an empowering experience, as it gave them a sense of control at a time when they described feeling otherwise powerless. most ( %) women stated that they had presented to a centre due to health care concerns and were very satisfied ( % ) with their experiences and interactions with staff. almost all ( %) women felt supported and understood.conclusions: this research has important implications for clinical practice and for appropriately addressing the needs of sexually assaulted women. what is apparent is that continued high-quality medical attention administered in the milieu of specialized hospital-based services is essential. at the same time, we would suggest that some forensic evidence collection procedures warrant reevaluation. the study will take an experiential, approach by chroruclmg the impa~ of the transition f m the streets to stabilization in a managed alcohol program through the techruque of narrative i~:uiry. in keeping with the shift in thinking in the mental health fie!~ ~his stu~y is based on a paradigm of recovery rather than one of pathology. the "inner views of part c pants hves as they portray their worlds, experiences and observations" will be presented (charm~z, , ~· ~)-"i?e p~ of the study is to: identify barriers to recovery. it will explore the exj?cnence of ~n~t zanon pnor to entry into the program; and following entry will: explore the meanmg ~nd defirutto~s of r~overy ~~d the impact of the new environment and highlight what supports were instrumental m movmg pan apants along the recovery paradigm.p -st (a) treating the "untreatable": the politics of public health in vancouver's inner city introdudion: this paper explores the everyday practices of therapeutic programs in the treadnent of hiv in vancouver's inner city. as anthropologists have shown elsewhere, therapeutic programs do not siinply treat physical ailments but they shape, regulate and manage social lives. in vancouver's inner city, there are few therapeutic options available for the treatment of -ilv. public health initiatives in the inner city have instead largely focused on prevention and harm reduction strategies such as needle exchange programs, safe injection sites, and safer-sex education. epidemiological reports suggest that less than a quarter of those living with hiv in the downtown eastside (dtes) are taking antiretroviral therapies raising critical questions regarding the therapeutic economy of antiretrovirals and rights to health care for the urban poor.methods: this paper is drawn from ethnographic fieldwork in vancouver's otes neighborhood focusing on therapeutic programs for hiv treatment among "hard-to-reach" populations. the research includes participant-observation at inner city health clinics specializing in the treatment of hiv; semi· structured interviews with hiv positive participants, health care professionals providing hiv treatment, and administraton working in the field of inner city public health; and, lastly, observation at public meetings and conferences surrounding hiv treatment.r.awlts: hiv prevention and treatment is a central concern in the lives of many residents living in the inner city -although it is just one of many health priorities afflicting the community. concerns about drug resistance, cost of antiretrovirals, and illicit drug use means that hiv therapy for most is characterized by the daily observation of their medicine ingestion at health clinics or pharmacies. daily observed treatment (dot) is increasingly being adopted as a strategy in the therapeutic management of "untreatable" populations. dot programs demand a particular type of subject -one who is "compliant" to the rules and regimes of public health. over emphasis on "risky practices," "chaotic lives," and "~dh~rence" preve~ts the public health system from meaningful engagement with the health of the marginalized who continue to suffer from multiple and serious health conditions and who continue to experience considerable disparities in health.~ the ~ffec~s of hiv in the inner city are compounded by poverty, laclc of safe and affordable houamg, vanous llegal underground economies increased rates of violence and outbreaks of ~~~·~ly tr~nsmitted infections, hepatitis, and tuberculosi: but this research suggests 'that public health uunauves aimed at reducing health disparities may be failing the most vulnerable and marginal of citiztl s. margaret malone ~ vi~lence that occurs in families and in intimate relationships is a significant urban, ~unity, and pu~hc health problem. it has major consequences and far-reaching effects for women, ~~--renho, you~ sen on, and families. violence also has significant effects for those who provide and ukllc w receive health care violence · · i · · . all lasses, · is a soc a act mvolvmg a senous abuse of power. it crosses : ' : ' ~ s;nden, ag~ ~ti~, cultures, sexualities, abilities, and religions. societal responseshali ra y oc:used on identificatton, crisis intervention and services for families and individuajs.promoten are only "-"--:-g to add h · ' · i in intimate relationshi with"-~"'.". ress t e issues of violence against women and vjoence lenga to consider i~ m families. in thi_s p~per, i analyze issues, propose strategies, and note c~· cannot be full -...l'-~ whork towards erad canng violence, while arguing that social justice and equity y -. ucvcu w en thett are people wh mnhod: critical social theory, an analysis that addresses culturally and ethnically diverse communities, together with a population health promotion perspective frame this analysis. social determinants of health are used to highlight the extent of the problem of violence and the social and health care costs.the ottawa charter is integrated to focus on strategies for developing personal skills, strengthening community action, creating supportive environments, devdoping healthy public policies, and re-orientating health and social services. attention is directed to approaches for working with individuals, families, groups, communities, populations, and society.ratdts: this analysis demonstrates that a comprehensive interdisciplinary, multisectoral, and multifaceted approach within an overall health promoting perspective helps to focus on the relevant issues, aitical analysis, and strategies required for action. it also illuminates a number of interacting, intersecting, and interconnecting factors related to violence. attention, which is often focused on individuals who are blamed for the problem of violence, is redirected to the expertise of non-health professionals and to community-based solutions. the challenge for health promoters working in the area of violence in families and in intimate relationships is to work to empower ourselves and the communities with whom we work to create health-promoting urban environments. social justice, equiry, and emancipatory possibilities are positioned in relation to recommendations for future community-based participatory research, pedagogical practices for health care practitioners, and policy development in relation to violence and urban health. the mid-main community health center, located in vancouver british columbia (bc), has a diverse patient base reflecting various cultures, languages, abilities, and socio-economic statuses. due to these differences, some mid-main patients experience greater digital divide barriers in accessing computers and reputable, government produced consumer health information (chi) websites, such as the bc healthguide and canadian health network. inequitable access is problematic because patient empowerment is the basis of many government produced chi websites. an internet terminal was introduced at mid-main in the summer of , as part of an action research project to attempt to bridge the digital divide and make government produced chi resources useful to a broad array of patients. multi-level interventions in co-operation with patients, with the clinic and eventually government ministries were envisioned to meet this goal. the idea of implementing multi-level interventions was adopted to counter the tendency in interactive design to implement a universal solution for the 'ideal' end-user [ ), which discounts diversity. to design and execute the interventions, various action-oriented and ethnographic methods were employed before and during the implementation of the internet terminal. upon the introduction of the internet terminal, participant observation and interviews were conducted using a motion capture software program to record a digital video and audio track of patients' internet sessions. this research provided insight into the spectrum of patients' capacities to use technology to fulfil their health information needs and become empowered. at the mid-main clinic it is noteworthy that the most significant intervention to enhance the usefulness of chi websites for patients appeared to be a human rather than a technological presence. as demonstrated in other ethnographic research of community internet access, technical support and capacity building is a significant component of empowerment ( ). the mid-main wired waiting room project indicates that medical practitioners, medical administrators, and human intermediaries remain integral to making chi websites useful to patients and their potential empowerment. ( ) over the past years the environmental yo~th alliance has been of~ering a.youth as~t. mappin~ program which trains young people in community research and evaluation. wh ~st the positive expenenc~ and relationships that have developed over this time attest to the success of this program, no evaluations has yet been undertaken to find out what works for t.he youth, what ~ould be changed, and what long term outcomes this approach offers for the youth, their local community, and urban governance. these topics will be shared and discussed to help other community disorganizing and uncials governments build better, youth-driven structures in the places they live.p - (a) the world trade center health registry: a unique resource for urban health researchers deborah walker, lorna thorpe, mark farfel, erin gregg, and robert brackbill introduction: the world trade center health registry (wfchr) was developed as a public health response to document and evaluate the long-term physical and mental health effects of the / disaster on a large, diverse population. over , people completed a wfchr enrollment baseline survey, creating the largest u.s. health registry. while studies have begun to characterize / bealth impacts, questions on long-term impacts remain that require additional studies involving carefully selected populations, long-term follow-up and appropriate physical exams and laboratory tests. wtchr provides an exposed population directory valuable for such studies with features that make ita unique resource: (a) a large diverse population of residents, school children/staff, people in lower man· hattan on / including occupants of damaged/destroyed buildings, and rescue/recovery/cleanup work· ers; (b) consent by % of enrollees to receive information about / -related health studies; (c) represenration of many groups not well-studied by other researchers; (c) email addresses of % of enrollees; (d) % of enrollees recruited from lists with denominator estimates; and (e) available com· parison data for nyc residents. wfchr strives to maintain up-to-date contact information for all enrollees, an interested pool of potential study participants. follow-up surveys are planned.methods: to promote the wtchr as a public health resource, guidelines for external researcher.; were developed and posted on (www.wtcregistry.org) which include a short application form, a twopage proposal and documentation of irb approval. proposals are limited to medical, public health, or other scientific research. researchers can request de-identified baseline data or have dohmh send information about their studies to selected wfchr enrollees via mail or email. applications are scored by the wtchr review committee, comprised of representatives from dohmh, the agency for toxic subst~nces and disease registry, and wtchr's scientific, community and labor advisory committees. a data file users manual will be available in early fall .~suits: three external applications have been approved in , including one &om a non-u.s. ~esearcher, all requesting information to be sent to selected wtchr enrollees. the one completed mail· mg~~ wtchr enrollee~ (o , wfc tower evacuees) generated a positive survey response rate. three additional researchers mtend to submit applications in . wfchr encourages collaborations between researchers and labor and community leaders.conclusion: studies involving wtchr enrollees will provide vital information about the long· term health consequences of / . wtchr-related research can inform communities, researcher.;, policy makers, health care providers and public health officials examining and reacting to and other disasters. t .,. dp'"f'osed: thi is presentation will discuss the findings of attitudes toward the repeat male client iden· ie as su e a and substance us'n p · · · · i · 'd . . - g. articipants will learn about some identified effective strategies or service prov ers to assist this group of i · f men are oft · d bl men. n emergency care settings, studies show that this group en viewe as pro emaric patient d i r for mental health p bl h h an are more ikely to be discharged without an assessmen !) ea rofr ems t. an or er, more cooperative patients (forster and wu · hickey er al., · r y resu ts om this study suggest th · · ' ' l · d tel' mining how best to h . d at negative amtudes towards patients, difficu nes e · as well pathways l_e_ p patientsblan ~ck of conrinuity of care influence pathways to mental health care. • uc\:ome pro emat c when p ti k · che system. m a ems present repeatedly and become "get stuc id methods: semi-structured intervie d . · (n= ), ed nurses (n= ) other ed ;s were con ucted with male ed patients (n= ), ed phys oans ' sta (n= ) and family physicians (n= ). patients also completed a poster sessions v diagnostic interview. interviews were tape-recorded, transcribed verbatim and managed using n . transcripts were coded using an iterative process and memos prepared capture emergent themes. ethics approval was obtained and all participants signed a detailed informed consent form.introduction: urban settings are particularly susceptible to the emergence and rapid spread of nt•w or rare diseases. the emergence of infectious diseases such as sars and increasing concerns over the next influenza pandemic has heightened interest in developing and using a surveillance systt·m which detects emerging public health problems early. syndromic surveillance systems, which use data b, scd on symptoms rather than disease, offer substantial potential for this by providing near-real-rime data which are linked to an automated warning system. in toronto, we are piloting syndromic data from the · emergency medical services (ems) database to examine how this information can be used on an ongoing basis for the early detection of syndromes including heat-related illness (hri), and influenza-like-illness (ill). this presentation will provide an outline of the planned desi_gn of this system and proposed evaluation. for one year, call codes which reflect heat-related illness or influenza-like-illness will be selected and searched for daily using software with a multifactorial algorithm. calls will he stratified by call code, extracted from the -ems database and transferred electronically to toronto public health. the data will be analyzed for clusters and aberrations from the expected with the realtime outbreak and disease surveillance (rods) system, a computer-based public health tool for the early detection of disease outbreaks. this -ems surveillance system will be assessed in terms of its specificity and sensitivity through comparisons with the well-established tracking systems already in place for hr! and ill. others sources of data including paramedic ambulance call reports of signs and . this study will introduce complementary data sources t~ the ed ch e~ complamt an~ o~~rthe-counter pharmacy sales syndromic surveillance data currently bemg evaluated m ~ther ontar~o cltles. . syndromic surveillance is a unique approach to proactive(~ dete~tmg early c.hangesm the health status of urban communities. the proposed study aims to provide evidence of differential effectiveness through investigating the use of -ems call data as a source of syndromic surveillance information for hr! and ili in toronto. introduction: there is strong evidence that primary care interventions, including screening, brief advi<:e, treatment referral and pharmacotherapy are effective in reducing morbidity and mortality caused by substance abuse. yet physicians are poor at intervening with substance users, in part because of lack of time, training and support. this study examines the hypothesis that shared care in addictions will result in decreased substance use and improved health status of patients, as well as increased use of primary care interventions by primary care practitioners (pcps). methods: the addiction medicine service (ams) at st. joseph's health centre's family medicine department is in the process of being transformed from its current structure as a traditional consult service into a shared care model called addiction shared care (asc). the program will have three components: education, office systems and clinical shared care. as opposed to a traditional consult service, the patient will be booked with both a primary care liaison worker (pcl) and addictions physician. patients referred from community physicians, the emergency department and inpatient medical and psychiatric wards will be recruited for the study as well as pcps from the surrounding community. the target sample size is - physicians and a similar number of patients. after initial consult, patient will be recruited into the study with their consent. the shared-case model underlines the interaction and collaboration with the patient's main pcp. asc will provide them with telephone consults, advice, support and re-assessment for their patients, as well as educational sessions and materials such as newsletters and informational kits.results: the impact of this transition on our patient care and on pcp's satisfaction with the asc model is currently being evaluated through a grant provided by the ministry of health & long term care. a retrospective chart review will be conducted using information on the patient's substance use, er/clinic visits, and their health/mood status. pcp satisfaction with the program will be measured through surveys and focus groups. our cost-effectiveness analysis will calculate the overall cost of the program per patient..conclusion: this low-cost service holds promise to serve as an optimal model and strategy to improve outcomes and reduce health care utilization in addict patients. the inner city public health project introduction the inner city public health project (icphp) was desi.gned to explore new an~ innovative ways to reach marginalized inner city populations that par-t c pate m high health-nsk beha~ ors. much of this population struggles with poverty, addictions, mental illness and homelessness, creatmg barriers to accessing health services and receiving follow-up. this pro ect was de~igned to evaluat~ .~e success of offering clinics in the community for testing and followup of communicable diseases uuhzmg an aboriginal outreach worker to build relationships with individuals and agencies. v n(demographics~ history ~f testi~g ~nd immunization and participation in various health-risk behaviors), records of tesnng and mmumzat ons, and mterviews with partner agency and project staff after one year.. results: t~e chr ~as i~strumental in building relationships with individuals and partner agencies ' .° the c~mmun_ ~ re_sultmg m req~ests for on-site outreach clinics from many of the agencies. the increase m parnc pat n, the chr mvolvement in the community, and the positive feedback from the agen? staff de~onstrated that.the project was successfully creating partnerships and becoming increasingly integrated m the community. data collected from clients at the initial visit indicated that the project was reaching its target populations and highlighted the unique health needs of clients, the large unmet need for health services and the barriers that exist to accessing those services. ~usion: the outreach clinics were successful at providing services to target populations of high health-nsk groups and had great support from the community agencies. the role of the chr was critical to the success of the project and proved the value of this category of health care worker in an urban aboriginal population. the unmet health needs of this disadvantaged population support the need for more dedicated resources with an emphasis on reducing access barriers. building a caring community old strathcona's whyte avenue, a district in edmonton, brought concern about increases in the population of panhandlers, street people and homeless persons to the attention of all levels of government. the issue was not only the problems of homelessness and related issues, but feelings of insecurity and disempowerment by the neighbourhood residents and businesses. their concerns were acknowledged, and civic support was offered, but it was up to the community itself to solve the problem. within a year of those meetings, an adult outreach worker program was created. the outreach worker, meets people in their own environments, including river valley camps. she provides wrap-around services rooted in harm reduction and health promotion principles. her relationship-based practice establishes the trust for helping clients with appropriate housing, physical and/or mental health issues, who have little or no income and family support to transition from homelessness. the program is an excellent example of collaboration that has been established with the businesses, community residents, community associations, churches, municiple services, and inner-city agencies such as boyle street community services. statistics are tracked using the canadian outcomes research institute homes database, and feedback from participants, including people who are street involved. this includes an annual general meeting for community and people who are homeless. the program's holistic approach to serving the homeless population has been integral, both in creating community awareness and equipping residents and businesses to effectively interact with people who are homeless. through this community development work, the outreach worker engages old strathcona in meeting the financial and material needs of the marginalized community. the success of this program has been surprising -the fact is that homeless people's lives are being changed; one person at a time and the community has been changed in how they view and treat those without homes. over two years, the program has successfully connected with approximately seventy-five individuals who call old strathcona home, but are homeless. thirty-six individuals are now in homes, while numerous others have been assisted in obtaining a healthier and safer lifestyles by becoming connected with other social/health agencies. the program highlights the roots of homelessness, barriers to change and requirements for success. it has been a thriving program and a model that works by showing how a caring community has rallied together to achieve prosperous outcomes. the spn has created models of tb service delivery to be used m part~ers~ p with phannaceunca compa-. · · -. t' ns cooperatives and health maintenance orgamzanons (hmos). for example, the mes, c v c orgamza , . · b tb d' · spn has established a system with pharmaceutical companies that help patients to uy me cmes at a special discounted rate. this scheme also allows patients to get a free one-_month's worth of~ dru_g supply if they purchase the first months of their regimen. the sy_s~e~s were ~es gned to be cm~pattble with existing policies for recording and documentation of the ph hppme national tuberculosis program (ntp). aside from that, stakeholders were also encouraged to be dots-enabled through the use of m~nual~ and on-line training courses. the spn initiative offers an alternative in easing the burden of tb sc:rv ce delivery from rhe public sector through the harnessing of existing private-sector (dsos). the learnmgs from the spn experience would benefit groups from other locales that _work no~ only on ~ but other health concerns as well. the spn experience showcases how well-coordinated private sector involvements help promote social justice in health delivery in urban communities.p - (c) young people in control; doing it safe. the safe sex comedy juan walter and pepijn v. empelen introduction: high prevalence of chlamydia and gonorrhoea have been reported among migrants youth in amsterdam, originating from the dutch antilles, suriname and sub-sahara africa. in addition, these groups also have high rates of teenage-pregnancy (stuart, ) and abortions (rademakers ), indicating unsafe sexual behaviour of these young people. young people (aged - ) from the so· called urban scene (young trendsetters in r&b/hip hop music and lifestyle) in amsterdam have been approached by the municipal health service (mhs) to collaborate on a safe sex project. their input was to use comedy as vehicle to get the message a cross. for the mhs this collaboration was a valuable opportunity to reach a hard-to-reach group.mdhods: first we conducted a need assessment by means of a online survey to assess basic knowledge and to similtaneously examine issues of interest concerning sex, sexuality, safer sex and the opposite sex. second, a small literature study was conducted about elements and essential conditions for succesful entertainment & education (e&e) (bouman ), with as most important condition to ensure that the message is realistic (buckingham & bragg, ) . third a program plan was developed aiming at enhancing the stl/hiv and sexuality knowledge of the young people and addressing communication and educational skills, by means of drama. subsequently a safe sex comedy show was developed, with as main topics: being in love, sexuality, empowerment, stigma, sti, hivand safer sex. the messages where carried by a mix of video presentation, stand up comedy, spoken word, rap and dance.results: there have been two safe sex comedy shows. the attendance was good; the group was divers' with an age range between and year, with the majority being younger than year. more women than men attended the show. the story lines were considered realistic and most of the audients recognised the situations displayed. eighty percent of the audients found the show entertaining and % found it edm:arional. from this %, one third considers the information as new. almost all respondents pointed our that they would promote this show to their friends.con.clusion: the s.h<_>w reached the hard-to-reach group of young people out of the urban scene and was cons d_ered entert~mmg, educational and realistic. in addition, the program was able in addressing important issues, and impacted on the percieved personal risk of acquiring an sti when not using condoms, as well as on basic knowledge about stl's. introduction: modernity has contributed mightily to the marginality of adults who live with mental illnesses and the subsequent denial of opportunities to them. limited access to social, vocational, educational, and residential opportunities exacerbates their disenfranchisement, strengthening the stigma that has been associated with mental illness in western society, and resulting in the denial of their basic human rights and their exclusion from active participation in civil society. the clubhouse approach tn recovery has led to the reduction of both marginality and stigma in every locale in which it has been implemented judiciously. its elucidation via the prism of social justice principles will lead to a deeper appreciation of its efficacy and relevance to an array of settings. methods: a review of the literature on social justice and mental health was conducted to determine core principles and relationships between the concepts. in particular, fondacaro and weinberg's ( ) conceptualization of social justice in community psychology suggests the desirability of the clubhouse approach in community mental health practice. a review of clubhouse philosophy and practice has led to the inescapable conclusion that there is a strong connection between clubhouse philosophy-which represents a unique approach co recovery--and social justice principles. placing this highly effective model of community mental health practice within the context of these principles is long overdue. via textual analysis, we will glean the principles of social justice inherent in the rich trove of clubhouse literature, particularly the international standards of clubhouse development.results: fondacarao and weinberg highlight three primary social justice themes within their community psychology framework: prevention and health promotion; empowerment, and a critical pnsp<"<·tive. utilizing the prescriptive principles that inform every detail of clubhouse development and th<" movement toward recovery for individuals at a fully-realized clubhouse, this presentation asserts that both clubhouse philosophy and practice embody these social justice themes, promote human rights, and empower clubhouse members, individuals who live with mental illnesses, to achieve a level of wdl-heing and productivity previously unimagined.conclusion: a social justice framework is critical to and enhances an understanding of the clubhouse model. this model creates inclusive communities that lead to opportunities for full partic pil!ion civil society of a previously marginalized group. the implication is that clubhouses that an· based on the international standards for clubhouse programs offer an effective intervention strategy to guarantee the human rights of a sizable, worthy, and earnest group of citizens. to a drastic increase in school enrollment from . million in to . million in .s. however, while gross enrollment rates increased to °/., in the whole country after the introduction of fpe, it remained conspicuously low at % in the capital city, nairobi. nairobi city's enrollment rate is lower .than thatof all regions in the country except the nomadic north-eastern province. !h.e.d sadvantage of children bas_ed in the capital city was also noted in uganda after the introduction of fpe m the late s_-many_ education experts in kenya attribute the city's poor performance to the high propornon of children hvmg m slums, which are grossly underserved as far as social services are concerned. this paper ~xammes the impact of fpe and explores reasons for poor enrollment in informal settlements m na rob city. methods: the study utilizes quantitative and qualitative schooling data from the longitudinal health and demographic study being implemented by the african population and health research center in two informal settlements in nairobi. descriptive statistics are used to depict trends in enrollment rates for children aged - years in slum settlements for the period - . results: the results show that school enrollment has surprisingly steadily declined for children aged - while it increased marginally for those aged - . the number of new enrollments (among those aged years) did not change much between and while it declined consistently among those aged - since . these results show that the underlying reasons for poor school attendance in poverty-stricken populations go far beyond the lack of school fees. indeed, the results show that lack of finances (for uniform, transportation, and scholastic materials) has continued to be a key barrier to schooling for many children in slums. furthermore, slum children have not benefited from fpe because they mostly attend informal schools since they do not have access to government schools where the policy is being implemented.conclusion: the results show the need for equity considerations in the design and implementation of the fpe program in kenya. without paying particular attention to the schooling needs of the urban poor children, the millennium development goal aimed at achieving universal primary education will remain but a pipe dream for the rapidly increasing number of children living in poor urban neighborhoods.ps- (c) programing for hiv/aids in the urban workplace: issues and insights joseph kamoga hiv/aids has had a major effect on the workforce. according to !lo million persons who are engaged in some form of production are affectefd by hiv/aids. the working class mises out on programs that take place in communities, yet in a number of jobs, there are high risks to hiv infection. working persons sopend much of their active life time in workplaces and that is where they start getting involved in risky behaviour putting entire families at risk. and when they are infected with hiv, working people face high levels of seclusion, stigmatisation and some miss out on benefits especially in countries where there are no strong workplace programs. adressing hiv/aids in the workplace is key for sucessfull responses. this paper presents a case for workplace programing; the needs, issues and recommendations especially for urban places in developing countries where the private sector workers face major challenges. key: cord- - yniw t authors: ben-chetrit, eldad title: colchicine date: - - journal: textbook of autoinflammation doi: . / - - - - _ sha: doc_id: cord_uid: yniw t colchicine is an alkaloid which was originally extracted from bulbs of a plant called colchicum autumnale (meadow saffron). its active pharmacological component was isolated in and in the active ingredient was purified and named colchicine. it consists of three hexameric rings termed a, b, and c. it was first recommended for the treatment of gout by alexander of tralles in the sixth century ad. later it has been employed for suggested and approved indications including primary biliary cirrhosis (pbc), alcohol induced hepatitis, psoriasis, behçet disease, sweet syndrome, scleroderma, sarcoidosis and amyloidosis. perhaps the most effective results have been obtained in the prophylaxis of familial mediterranean fever (fmf). colchicine is absorbed in the jejunum and ileum and is trapped in the body tissues. it is metabolized in the liver and the intestine by cytochrome p (cyp) a and p-glycoprotein (pgy) . colchicine is excreted mainly by the biliary system, intestines and the kidneys. it has a narrow therapeutic range, but with normal liver and kidney functions is relatively safe and can be used during pregnancy, nursing and in infants. the main mechanism of action of colchicine is probably through interaction with microtubules affecting leukocyte chemotaxis, thereby suppressing inflammation. the blood level of colchicine may be affected by concomitant drug administration and therefore, caution should be exercised when such medications are added. colchicine is an alkaloid which has been used for centuries for treating gout [ ] . the source of its name is colchis, a kingdom on the black sea in western georgia. its history is related to medea who was the daughter of aeetes, ruler of colchis. aeetes kept the "golden fleece" under heavy guard. his daughter, medea used to "harvest grasses and to extract harmful juices squeezed from twisted roots" [ ] . among the most potent products squeezed from these bulb-corms was the juice of colchicum autumnale, the yellow crocus of colchis. on his quest to fetch the "golden fleece" from colchis, jason met medea and fell in love with her. the princess used her potions (most of which consisted of concentrated colchicine) to help jason poison the warriors and dragons that stood guard over the fleece. the use of the bulb-like corms of colchicum for gout traces back to a.d., as the "hermodactyl (iris)" recommended by the byzantine physician, alexander of tralles (today-aydin in turkey) [ ] . while the greeks and romans knew about the use of colchicine for gout, the drug wasn't available in pure form until the late nineteenth century. the active pharmacological component of the plant, colchicum, was isolated in and in , p.l. geiger purified the active ingredient, which he named colchicine [ ] . in the nineteenth century, alfred garrod, dyce duckworth, and many others, reached a consensus that colchicine was relatively specific for the treatment of gout [ ] . pernice, an italian pathologist, found that when therapeutic doses of colchicine were given to experimental animals, lesions were produced in the nuclei of gastric and intestinal cells as these cells were arrested in metaphase [ ] . in , ed taylor and gary borisy used tritiated colchicine to identify the target of colchicine in dividing and non-dividing cells [ ] . the protein they identified was the dimeric building block of microtubules, subsequently given the name "tubulin" by mohri [ ] . we now know that the traffic of intracellular materials is carried over tracks formed by microtubules [ ] . in the last years, colchicine has been employed for an increasing number of diseases, in addition to gout, including familial mediterranean fever (fmf), idiopathic recurrent pericarditis, behçet disease, sweet syndrome, systemic sclerosis, amyloidosis and hepatic cirrhosis [ ] (table . ). in acute gout, colchicine is effective in alleviating the acute attack and as a prophylactic medication. in behçet disease, colchicine is effective mainly in the treatment of mucosal ulcers, especially in female genitalia. in systemic sclerosis, colchicine may decrease the stiffness of the skin whereas in amyloidosis it may result in regression of amyloid deposition loci where serum amyloid a (saa) fibers are deposited [ , ] . since , colchicine has gained popularity as being the main effective remedy for fmf [ ] . in this disease, colchicine prevents the occurrence of the acute inflammatory episodes and the development of amyloidosis. however, colchicine is not effective in controlling acute attacks when administered once they occur. • colchicine is an alkaloid which is absorbed in the jejunum and ileum and is trapped in the body tissues • colchicine is metabolized in the liver and the intestine by cytochrome p (cyp) a and p-glycoprotein (pgy) • colchicine is excreted mainly by the biliary system, intestine and the kidneys • the half-life of colchicine is between and h following oral ingestion • caution should be exercised in patients with liver or kidney disease or damage colchicine is a tricyclic, lipid-soluble alkaloid with the chemical formula; n- ( , , , , tetrahydro-l, , , , tetramethoxy- oxobenzo[a] hep-tain- -yl) acetamide n ( fig. . ). pharmacokinetic studies of colchicine are limited, and their results somewhat contradictory because of the different methods used for its measurement. previously, most studies employed thin-layer chromatography, whereas recently a more sensitive radioimmunoassay and high performance liquid chromatography with mass spectrometry (hplc-ms) has been used [ ] . colchicine can be administered by mouth as . , . and in some localities mg tablets. until recently an intravenous (iv) formula was also available. however, in , the united states food and drug administration (fda) withdrew approval following several cases of fatal toxicity [ ] . formerly, it was thought that colchicine is almost completely absorbed after oral administration. however, recent studies have shown that its bioavailability ranged from to % [ ] . the exact site of absorption is unknown, but the drug seems to reach the jejunum and ileum because dysfunction of these bowel regions is common in cases of chronic colchicine overdose [ ] . the plasma membrane-localized multidrug transporter p-glycoprotein (pgy ) [also known as atp-binding cassette subfamily b member (abcb ), multiple drug resistant (mdr ), and cd ] transports multiple classes of substrates across cell membranes [ , ] . altered intestinal pgy expression can change the absorption of drugs transported by this peptide, including colchicine. therefore, pgy- is critical in the regulation of its bioavailability and plasma concentrations, which can lead to sub-optimal therapeutic effects or, alternatively, to drug toxicity [ , ] . also, pgy clearly participates in the removal of drug metabolites through bile, the intestinal lumen, and urine [ ] [ ] [ ] [ ] . colchicine is predominantly eliminated by biliary excretion and through the stool, with gastrointestinal tract lining cell turnover playing a variable role in this process [ , , ] . normally, a lesser, but significant role in colchicine metabolism (~ %) is played by enteric and hepatic cytochrome p a (cyp a ), which catalyzes the demethylation of colchicine to and demethtylcolchicine, which are inactive metabolites [ ] . renal elimination has been estimated to be responsible for - % of drug disposition in normal subjects. however, cyp a and renal disposition of colchicine can be significantly impacted by certain drug-drug interactions that affect pgy- (abcb ), as well as hepatobiliary dysfunction and aging [ , ] . the bioavailability of oral colchicine tablets is comparable in the young and elderly, but colchicine pharmacokinetics differ markedly, with volume of distribution at steady state (vss) and total body clearance significantly reduced in the elderly, and the plasma cmax significantly higher at equivalent colchicine doses [ ] . over the last decade, several single nucleotide polymorphisms of pgy (abcb ) were identified with the potential to influence expression and quantitative transporter function [ ] . for instance, - % of patients diagnosed with fmf do not respond to treatment with colchicine. compared with non-responders (who usually have more severe disease), treatment responders had a twofold greater concentration of colchicine in mononuclear cells, which was attributed to a potential genetic effect independent of mefv mutations [ , ] . specifically, the distribution of abcb c and t-genotypes was significantly different between colchicine-responders and non-responders, with the c allele significantly increasing the risk of resistance to colchicine, whereas in patients with the t genotype a decreased colchicine dose was needed to obtain an adequate response [ ] . wallace and ertel [ ] were pioneers in studying the pharmacokinetics of colchicine. they found that after iv administration of mg of the drug to healthy volunteers, the peak plasma concentration averaged . + . μg/ ml. after iv administration of . mg of colchicine, a rapid drop of plasma levels occurred during the first min, followed by a logarithmic decline [ ] . the rapidity of colchicine disappearance from the plasma and its persistent excretion days after drug ingestion suggest that it is trapped in body tissues for a prolonged period. indeed, the [ ] . an example for such binding is the detection of colchicine in leukocytes days after a single iv dose [ ] . furthermore, it was shown that in plasma % of the colchicine is bound to albumin [ ] . after oral administration of colchicine, the tmax (the time needed to reach peak plasma levels) is - h [ ] . however, maximal anti-inflammatory effects develop over - h based upon intra-leukocytes accumulation of the drug [ ] . initial studies reported terminal elimination half-life time (t / ) from min (after iv administration) to h (after oral ingestion) [ ] . in an additional study, the mean t / was . h, similar to findings in patients with fmf without renal or liver disease (t / of about ± h) [ , ] . in a study in which we evaluated the pharmacokinetics of colchicine in patients with fmf with and without renal or liver disease, we found that colchicine clearance was significantly impaired in those with kidney or liver failure [ ] . the t / of colchicine in patients with severe renal failure was two-to threefold longer and in a patient with both renal failure and cirrhosis tenfold longer than in patients with normal renal and liver function. leighton et al. [ ] reported similar results in patients with liver cirrhosis. these findings suggest that patients with either liver or renal disease should be closely monitored even when treated with conventional doses of colchicine. • colchicine binds non-polymerized tubulin forming a tubulin-colchicine complex • heterodimers of αand β-tubulin form microtubules that can elongate and contract as filaments • colchicine, can bind the tubulin molecule and inhibit its polymerization into microtubules in vitro • since microtubules are involved in cell division, in signal transduction, regulation of older studies claimed that most of the effects of colchicine at the cellular level are attributed to its interaction with tubulin, the main building block of microtubules [ ] . colchicine binds in an equimolar and poorly reversible manner to soluble non-polymerized tubulin with high activation energy, forming a tubulin-colchicine complex [ , , ] . heterodimers of α-and β-tubulin form dynamic polymers termed microtubules that can elongate and contract as filaments, to change the structure and function of the cytoskeleton, exemplified by the interphase microtubule network and the mitotic spindle. microtubules are involved not only in cell division, but also in signal transduction, regulation of gene expression, migration, and secretion [ ] . microtubules are widely distributed in organelles present in nerve cells, ciliated cells, leukocytes, and sperm tails. it was shown that tropolone methyl ester, which is a precise analog of the ring c of colchicine, can bind the tubulin molecule and inhibit its polymerization into microtubules in vitro. furthermore, mescaline, which is an analog of the methoxyphenyl moiety of ring a of colchicine, also may inhibit microtubular assembly [ ] . these data suggest that the action of colchicine is dependent on its two rings which bind microtubules, inhibiting the movement of intracellular granules, thereby disturbing the secretion of various components to the cell exterior. colchicine has an inhibitory effect on several leukocyte functions such as adhesiveness, ameboid motility, mobilization and degranulation of lysosomes [ ] . however, the most potent effect of colchicine is on leukocyte chemotaxis [ ] . of all the effects of colchicine, only the inhibition of chemotaxis was shown to occur at concentrations as low as × − mol/l. a higher dose is necessary to inhibit other effects. it was suggested that the primary anti-inflammatory effect of colchicine is derived from its potent inhibitory effect on leukocytes chemotaxis [ ] . however, additional studies have shown that colchicine decreases the expression of adhesion molecules on neutrophil membranes, leading to significant inhibition in migration and interaction with endothelial cells [ ] . other investigators have shown that colchicine may modulate cytokine production by polymorphonuclear cells. several studies have shown a relatively high concentration of colchicine in leukocytes explaining its potential effect on these cells [ , ] . however, the cause for the special "affinity" of colchicine to leukocytes was unclear. we and other investigators, have shown that granulocytes have low activity of the pgy efflux pump and therefore when colchicine enters these cells it accumulates in their cytoplasm [ , ] . conversely, lymphocytes and mononuclear cells have a higher activity level of pgy . therefore, some of the colchicine entering these cells is effluxed, explaining why the level of colchicine in granulocytes exceeds that of lymphocytes and monocytes. mechanisms of colchicine several mechanisms have been ascribed to the therapeutic action of colchicine (table . ). bessis and gorius discovered that colchicine disrupts microtubules in a dose-dependent fashion [ ] . colchicine does not enhance microtubule dissolution but abrogates the process of microtu-bule self-assembly by forming tubulin-colchicine complexes [ , ] . colchicine reduces the generation of tumor necrosis factor (tnf)-α by macrophages and its receptors on endothelial cells [ , ] . colchicine also has been shown to interfere with the interaction of neutrophils and the vascular endothelium by abrogating their binding to adhesion molecules. colchicine abrogates the e-selectin-mediated adhesiveness of the cytokinestimulated vascular endothelium to neutrophils. it also alters the distribution of the adhesion molecules on the surface of endothelial cells and neutrophils, significantly reducing their interaction [ ] . in addition, at high concentrations colchicine suppresses phospholipase a activation, lysosomal enzyme release and phagocytosis [ ] . conversely, colchicine does not exert its anti-inflammatory effect through inhibition of cyclooxygenases [ ] . in , the gene likely to cause fmf was isolated and cloned [ , ] . following the isolation of the mefv gene and the finding that it is fully expressed in neutrophils, a question was raised regarding the possibility of a direct effect of col- abrogates the microtubule self-assembly by forming complex with tubulin reduces the generation of tumor necrosis factor (tnf)-α by macrophages abrogates e-selectin-mediated adhesiveness of endothelium to neutrophils alters distribution of adhesion molecules on endothelial cells may suppress phospholipase a activation (at high concentrations) may inhibit lysosomal enzyme release, and phagocytosis raises mefv gene expression in a cell line of peritoneal fibroblasts may modulate the expression of numerous genes in endothelial cells suppresses the activation of caspase- may inhibit superoxide production by neutrophils leads to release of guanine nucleotide exchange factor (gef)-h factor thereby activating rat sarcoma homolog gene family, member a (rhoa) reduces neutrophils elasticity and relaxation inhibiting chemotaxis inhibits liver fibrosis by inducing stellate cell apoptosis inhibits anti-transforming growth factor (tgf)-β activity chicine on the gene or its protein. in a study where we tested this hypothesis, we showed that colchicine did not up-regulate the expression of mefv gene in neutrophils [ ] . however, colchicine increased the mefv gene expression in a primary cell line of peritoneal fibroblasts. the exact significance of this finding is unknown. nevertheless, it should be borne in mind that peritoneal cells comprise an important target in the acute attack of fmf (peritonitis), suggesting a potential local effect of colchicine. a few studies suggested that colchicine modulation of pyrin expression and interaction with pyrin in the cytosol contributed to its efficacy in fmf [ , ] . at relatively high concentrations, colchicine also modulates the expression of numerous genes in cultured endothelial cells, with a significant delay in the onset of action [ ] . this may explain the observation that initiation of colchicine treatment during acute attacks of fmf does not effectively terminate them. colchicine has recently been shown to suppress the activation of caspase- , the enzymatic component of the nucleotide-binding oligomerization domain (nod) receptor family pyrin (nlrp ) inflammasome. caspase- suppression blocks conversion of pro-interleukin (il)- β to active il- β, leading to secondary reduction in cytokines such as tnf-α and il- . the effect of colchicine on this process may be upstream of the inflammasome, rather than a direct effect [ ] . to date, inflammasome inhibition has been assessed at colchicine concentrations to -fold higher than that achieved in the serum. whether colchicine inhibits caspase- at physiologic concentrations, or whether colchicine accumulation in leukocytes is sufficient to block the inflammasome, remains to be determined. another mechanism by which colchicine may suppress inflammation is by inhibition of superoxide production by neutrophils. chia et al. demonstrated that colchicine inhibits monosodium urate (msu)-induced superoxide production by murine peritoneal macrophages in vivo at doses times lower than that required to inhibit neutrophil infiltration [ ] . this suggests that superoxide anion production is more sensitive to suppression by colchicine than microtubule formation involved in cell migration. attacks of fmf? • rat sarcoma homolog gene family, member a (rho a) protein is a peptide which controls the action of gtpases thereby affects tubulin dynamics • pyrin is a specific immune sensor (pattern recognition receptor-prr) for bacterial modifications of rho and gtpases • activation of rhoa inhibits pyrin activity while inactivation of rhoa causes over activation of pyrin resulting in increased production of interleukin (il)- , thereby enhancing inflammation • colchicine may activate rhoa by guanine nucleotide exchange factor (gef)-h , thereby suppressing pyrin activity and inflammation • colchicine also disrupts microtubules structure reducing neutrophils membrane elasticity and relaxation, thereby preventing their extravasation from the blood vessels to the inflammatory site to understand this process, we first need to explain the cellular role of pyrin. pyrin is the protein encoded by the mefv gene, which is mutated in fmf (see chap. ). rat sarcoma homolog gene family, member a (rho a) protein is an intra-cellular peptide which controls the action of gtpases. gtpases are important enzymes in regulation of actin and tubulin dynamics. actintubulin interaction has a major role in the motility and chemotaxis of neutrophils [ , [ ] [ ] [ ] . bacterial toxins such as that of clostridium may modify the effect of rho on gtpases, thereby inhibiting actin activity and leukocytes chemotaxis. xu et al. found that pyrin is a specific immune sensor (pattern recognition receptor-prr) for bacterial modifications of rho and gtpases [ ] . pyrin does not directly recognize rho modification, but probably senses events downstream of rho modification. xu et al. showed that activation of rhoa inhibits pyrin activity while inactivation of rhoa causes over activation of pyrin resulting in increased production of il- , thereby enhancing inflammation. clostridium toxin inactivates rhoa thereby leading to less production or recruitment of protein kinases (pkns) and thereby less phosphorylation of pyrin [ ] . ** peptides (**the name reflect the migration pattern on d deae gel of these acidic proteins family) are group of proteins which interact with phosphorylated pyrin [ ] . decreased phosphorylation of pyrin leads to less interaction and reduced binding by peptides. this increases the amount of free pyrin which can recruit apoptosis-associated speck-like protein containing a card (asc) and procaspase − to construct the pyrin inflammasome, leading to increased secretion of il- β and an enhanced inflammatory process (fig. . ) . colchicine may also suppress inflammation in fmf by the following mechanism. it binds to tubulin and depolymerizes microtubules, resulting in the release of the rhoa activator, guanine nucleotide exchange factor (gef)-h , which is inactive when bound to microtubules [ , ] . thus, colchicine indirectly activates rhoa. activation of this protein results in enhancement of pyrin phosphorylation, thereby higher binding by the pep-tides and hence, less free pyrin is available for constructing the inflammasome, required for secreting inflammatory cytokines, including il- . in a recent study, we looked more closely to the mechanistic features of the effect of colchicine on the neutrophil membrane [ ] . we found that colchicine disrupts the microtubules structure and reduces neutrophil elasticity and relaxation, thereby preventing their extravasation from the blood vessels to the site of inflammation. this may be the final and most effective step in inhibiting chemotaxis. colchicine has anti-fibrotic effects. in a rat model of hypertensive chronic kidney disease, colchicine inhibited renal fibrosis via inhibition of rhoa signaling and infiltration of inflammatory cells [ ] . in another rat model, colchicine inhibited liver fibrosis by inhibiting the activation of hepatic stellate cells and inducing stellate cell apoptosis [ ] . in an encapsulating peritoneal sclerosis model, colchicine inhibited anti-transforming growth factor (tgf)-β activity [ ] . • colchicine is a relatively safe medication but has a narrow therapeutic window • the metabolism of colchicine is affected by the functions of cytochrome p (cyp) a , p-glycoprotein (pgy) , the liver and the kidneys and by additional medications taken concomitantly colchicine is a relatively safe medication but has a narrow therapeutic window [ , ] . colchicine is well tolerated when taken in age dependent doses that are less than mg/day in children (up to year old) and mg/day in adults with normal liver and kidney function, when not taking concomitant drugs that may affect its pharmacokinetics. nevertheless, therapeutic oral doses of colchicine ( - mg/day), may cause cramping, abdominal pain, hyperperistalsis, diarrhea and vomiting. these effects are usually mild and transient. when abdominal cramps persist, lowering colchicine dose may be effective. when diarrhea persists dividing the dose of colchicine (twice daily) may be helpful. in resistant cases, a short course of anti-diarrhea medications such as loperamide may be beneficial [ ] . decreasing use of lactose containing products or using lactase prior to their consumption may also reduce symptoms. leukopenia is a very rare adverse event in therapeutic dose and neuromuscular complications may occur when renal functions are impaired or with use of concomitant drugs such as clarithromycin. colchicine overdose may lead to a cholera-like syndrome associated with dehydration, shock, multiple organ failure, alopecia, disseminated intravascular coagulation (dic), seizures, coma and death [ ] . colchicine doses of . - . mg/kg are highly toxic, and doses of more than . mg/kg are typically lethal [ ] . cumulative doses of colchicine causing toxicity when administered intravenously were mg given over days, mg in days and even mg given within days. the lowest reported oral dose causing lethal colchicine toxicity was mg given over days to a -year old male [ ] . the course of colchicine intoxication can be divided into three stages, with overlap between the stages. in the first stage, gastrointestinal symptoms dominate. there may be excessive fluid loss through diarrhea, leading to volume depletion and dehydration. this stage develops within - h following ingestion of the drug. the second stage is dominated by multi-organ failure which may include: bone marrow failure, renal insufficiency, adult respiratory distress syndrome (ards), arrhythmias, disseminated intravascular coagulation (dic), neuromuscular disturbances and alopecia. this stage develops over - days. patients surviving this stage may enter the third stage which is characterized by bone marrow recovery and rebound leukocytosis, resolution of organ failure and regrowth of hair. clinical management of colchicine intoxication is basically supportive. in a single case, treatment with f(ab) fragments of anti-colchicine antibodies was successful [ ] . unfortunately, these antibodies, raised in goats, are not currently available. one of the problems of managing colchicine overdose is that it is not dialyzable using regular dialysis membranes. recently, new high flux polysulfone membranes have been introduced to improve dialysis [ ] . many medications and substrates which were non-dialyzable with older membranes are now dialyzable. colchicine is one of the medications of which steady state levels were reduced when given to patients with fmf on high flux dialysis [ ] . however, our study showed that the rate of excretion of colchicine by these membranes is far less than the rate needed for effective treatment of colchicine intoxication. drug-drug interactions have increasingly become apparent as a cause of colchicine toxicity in patients treated with "standard" daily prophylactic regimens. abcb (pgy ) can undergo conformational changes with expression modulation thereby promoting clinically significant colchicine drug-drug interactions [ , , , ] . cyclosporine is a prime example of a drug that can inhibit or modulate the abcb transporter [ ] . potentiation of colchicine neuromyopathy can occur within weeks of commencement of cyclosporine therapy. this complication is often associated with cyclosporine nephropathy and a decline in the glomerular filtration rate [ , ] . notably, cyclosporine was found to delay colchicine-induced diarrhea in an animal model system, likely due to modulation of intestinal abcb . hence, it is suspected that cyclosporine could mask the gastrointestinal side effects of colchicine in humans that might otherwise be a clue to the development of systemic colchicine toxicity. cyp a is the most abundant of the human p enzymes and metabolizes multiple structural classes of drugs (e.g. cyclosporine, quinidine, testosterone, nifedipine, etc) [ , ] . cyp a is a focal point of many drug-drug interactions and dietary and herbal interactions. cyp a may be stimulated by its substrates ("normotropic cooperativity") or its effectors ("heterotropic cooperativity"), which renders prediction of drug-drug interactions difficult [ , ] . these substances may be divided into three groups. the first group contains drugs, such as cimetidine, which have an inhibitory effect on the entire cytochrome system. indeed, in animal studies, it was shown that concomitant administration of cimetidine and colchicine resulted in a significant rise in serum colchicine concentration. the second group contains substances that have a specific inhibitory effect on the isoform cyp a which metabolizes colchicine. these include erythromycin, ketoconazole and other medications. the third group includes drugs that are also metabolized by cyp a , such as cyclosporine and nifedipine, and may compete with colchicine for binding to the enzyme. the interaction in these cases is dictated by the affinity of each medication for the enzyme. thus, coadministration of medications and substances metabolized by the same cytochrome system may lead, in principle, to an increase of one or more of the drugs, exposing the patient to a higher risk of toxicity. certain drugs increase the potential for colchicine toxicity via dual modulation of abcb and cyp a (table . ). these include the macrolide antibiotics erythromycin and clarithromycin, and the statins, e.g. lovastatin, simvastatin and atorvastatin [ ] . clarithromycin is a particularly striking case in point and has been associated with at least fatalities with concomitant colchicine use [ ] . however, azithromycin (a weak cyp a inhibitor) had minimal effects on colchicine concentration and terminal elimination half-life, and decreased total apparent oral clearance by only %. azithromycin should be recommended as a safer alternative to clarithromycin in patients taking colchicine [ ] . mutual potentiation by colchicine and statins of myopathy (sometimes including rhabdomyolysis) is a notable concern [ , ] . significantly, case studies have reported acute myopathy after concurrent use of colchicine with a statin that was either not metabolized or minimally metabolized by the cyp a isoenzyme [ , ] . in this context, fluvastatin can disrupt the integrity of the cytoskeleton and is linked with vacuolization and other pathology in muscle, which is pertinent given the capacity of colchicine to promote vacuolization in muscle by disruption of the microtubule network. a new set of evidence-based guidelines provides an algorithm for reducing colchicine doses to prevent toxicity in patients who are concomitantly taking other drugs [ ] . the researchers conducted a series of studies designed to show the effects of a single-dose of colchicine given with known inhibitors of cyp a or pgy . among the drugs tested were: cyclosporine, ketoconazole, ritonavir, clarithromycin, azithromycin, verapamil extended release (er), and diltiazem er. it was shown that the mean maxi-mum concentration of colchicine was % higher when colchicine was co-administered with ketoconazole compared with colchicine alone. the mean maximum concentration of colchicine was % higher and the mean total colchicine exposure was % higher when colchicine was combined with ritonavir as compared with colchicine alone [ ] . colchicine treatment can alter absorption of other compounds or medications from the intestines. it may induce malabsorption of vitamin b by reducing the number of b intrinsic factor receptors as shown in the intestinal mucosa of guinea pigs [ ] . colchicine-induced lactose intolerance occurs in a significantly higher per- centage of patients with fmf treated with oral colchicine compared with non-treated patients [ ] . reduction in iron absorption was also observed among patients with fmf taking colchicine. • colchicine rarely causes oligo/azoospermia • colchicine does not affect sperm motility • it is safe to take colchicine during pregnancy and nursing provided the liver and kidney functions are normal • colchicine is relatively safe in treating children and toddlers and does not affect their growth colchicine is a drug which may affect the function of microtubules in various cells. in high concentrations, it may inhibit mitosis within the process of cell division. therefore, concern was raised as to the effect of colchicine on sperm proliferation and motility in patients taking colchicine. a case report by merlin described a patient with gout who developed azoospermia following treatment with colchicine [ ] . re-challenge again induced azoospermia. because patients with fmf receiving colchicine are often in their child-bearing years, the concern about fertility is pertinent. indeed, rabbits treated with a relatively high dose of colchicine showed various degenerative changes of the testes, including loss of differentiation from spermatogonia to spermatozoa [ ] . however, cohen et al. performed cytogenetic evaluation in patients with fmf receiving long-term colchicine. mitotic rates, percentage of tetraploidy, and chromosomal breakage rates were determined in lymphocytes [ ] . no significant differences were found between the patients and controls. in a study by levy et al., patients receiving long term colchicine therapy were evaluated. no effect on fertility was noted and levels of spermatocytes, testosterone stimulating hormone, luteinizing hormone and prolactin were all within normal limits [ ] . another study showed that four out of males with fmf receiving colchicine suffered from infertility [ ] . one had azoospermia and the others had a normal spermatogram, but the sperms could not penetrate the ova normally. since sperm motility and hence ovum penetration depends upon microtubule function, we hypothesized that colchicine may affect the movement of sperm. accordingly, we studied the effect of colchicine on sperm motility in an invitro system employing the "swim up" technique for sperm selection [ ] . sperm motility was inhibited significantly only after an incubation period of at least h, with a minimal colchicine concentration of μg/ml. because plasma colchicine concentration under therapeutic dose is about - ŋg/ml, the amount of colchicine needed for affecting sperm motility in vitro is -fold higher. thus, it seems unlikely that standard colchicine treatment would inhibit sperm motility unless the drug has a very high and special affinity to the testes. the frequency of oligo or azoospermia with colchicine depends on the underlying disease. bremner and paulson failed to show any effect on spermatogenesis in six healthy volunteers who received commonly used doses of colchicine for - months [ ] . conversely, in a study of turkish men treated with colchicine for behçet disease, oligospermia was evident in ( %) patients and azoospermia in two patients [ ] . if corroborated, these findings suggest that infertility and disturbed spermatogenesis result not only from colchicine use but also may depend on other factors such as genetic background or underlying disease. the vasculitis associated with behçet disease may further contribute to this complication by adding local ischemia to the potential toxicity of colchicine. based upon the above observations, it is tempting to ascribe the development of azoospermia in patients with fmf to colchicine. however, in three cases of azoospermia we performed a testicular biopsy which demonstrated amyloido-sis of the testes [ ] . thus, amyloidosis of the testes should also be considered in patients with fmf presenting with azoospermia (see chaps. and ). another concern related to male fertility is the question on the outcome of pregnancies induced by male patients with fmf. in a study by zemer et al. of patients with fmf, females conceived while their male partners were treated with colchicine [ ] . there was no mention concerning fertility or delivery problems. due to limited data on this issue, some physicians in the past used to advise to discontinue colchicine months before attempting to conceive. in a prospective study, we followed the outcome of pregnancies and deliveries of female partners of males with fmf, were on colchicine when their partners conceived [ ] . as a control group, we followed the outcome of pregnancies and deliveries in healthy women married to healthy men. our findings revealed no difference regarding the rate of early or late abortions, or of congenital malformations. therefore, it seems that there is no need for males with fmf to discontinue colchicine prior to planning conception. the potential effects of colchicine on microtubules, cell division and growth raise a serious concern as to the female reproduction system. fmf attacks may be triggered in some patients by their menstrual period [ ] . the association of fmf attacks and menstruation raises the possibility of hormonal influences (see chap. ). indeed, it was shown that estrogen significantly decreases intercellular adhesion molecules [ ] . furthermore, it was demonstrated that estrogens inhibit tubulin assembly by interacting directly with tubulin s sites analogous to colchicine sites [ ] . moreover, estrogen is metabolized by the a liver cyp- complex and competes in binding it with colchicine. thus, it is tempting to speculate that estrogens mimic the effect of colchicine on tubules and adhesion molecules, thereby enhancing the effect of colchicine. during menstruation, there is a sharp decrease in estrogens, thus their accumulative suppressive effect on inflammation is suddenly diminished. in addition, the reduction in estrogen, allows for a more effective metabolism of colchicine by the a cytochrome (less inhibitory competition by estrogen) so that the effective level of colchicine may be further reduced. this situation may lead to menstruation associated fmf attack. to control these attacks, it is recommended to increase the dose of colchicine (by . mg) for days prior to the onset of the menstrual period and for two more days after its onset (see chap. ). theoretically, colchicine may affect female fertility by affecting the ovaries via its potential effect on cell division. however, this has not been shown. serious concern was raised regarding a teratogenic effect of colchicine. therefore, in the s physicians advised their patients to discontinue colchicine months before planning conception and during pregnancy. sporadic reports claimed that colchicine was safe during pregnancy. furthermore, in a study which followed pregnant women with fmf treated with colchicine, the outcome of the newborns was the same as in an untreated control group [ ] . in another study, all women with fmf, who had pregnancies and were on colchicine, gave birth to normal children [ ] . however, rabinovitch et al. reported that newborns out of ( : ) deliveries of fmf patients were born with trisomy , twice the expected rate of a comparable normal population [ ] . it was not clear whether colchicine therapy itself plays a role with this increment outcome. recently, diav-citrin et al. examined the safety of fetuses by following mothers exposed to colchicine during pregnancy. in a prospective observational comparative cohort study between and they found that colchicine did not appear to be a major human teratogen, and, probably, has no cytogenetic effect [ ] . we followed the outcome of pregnancy in a group of patients with fmf who took colchicine during pregnancy and compared them with a group of patients with fmf who were not treated with colchicine and with a group of healthy pregnant individuals [ ] . we showed that colchicine was not associated with a higher rate of miscarriage or stillbirth. there was no reduction of the duration of pregnancy or the birth weight of the babies. based upon these results, we do not recommend amniocentesis for patients with fmf solely because of treatment with colchicine during pregnancy. leaflets of pharmaceutical companies and textbooks of pharmacology warn women not to nurse their babies while treated with colchicine. milunsky and milunsky found that colchicine was present in the breast milk of patients taking the drug [ ] . we also measured the levels of colchicine in sera and milk of women with fmf at various time points after drug ingestion [ ] . colchicine was detected in all samples of sera and milk, with similar concentrations. however, the estimated daily amount of colchicine ingestion by the nursing babies was less than one tenth the therapeutic dose (per kilogram) given to adult patients with fmf. this rough estimation was concordant with our favorable clinical experience in following more than children of mothers who continued to breastfeed while taking colchicine. therefore, we suggest that breast feeding is safe while taking colchicine. since growth depends upon cell division, the potential effect of colchicine on child growth and development may raise concerns. the diagnosis of fmf can be made as early as several months of age. initially, we were reluctant to start treatment with colchicine before the age of years. during this period, children continued to suffer from recurrent attacks of fmf and were also at risk of developing amyloidosis. furthermore, they exhibited growth delay when compared with healthy children of the same age. following the start of colchicine treatment and control of fmf attacks, their appetites improved and a marked growth spurt was evident. we followed seven children since the age of - years and measured their height and weight every months for a period of years [ ] . their growth while treated with colchicine was within the normal expected percentile range. similar results were observed in larger and more recent studies [ ] . savgan-gurol et al. evaluated the growth process and insulin like growth factor- (igf- ) levels in children with fmf [ ] . they found that igf- levels of children with fmf did not differ from their healthy peers. however, there was a positive correlation between the rate of growth and the cumulative colchicine dose. therefore, they suggested that colchicine enhances the growth of children with fmf by suppressing disease activity and inflammation. the literature suggests that the minimal daily dose for preventing the development of amyloidosis in adult fmf patients is mg/day, even if attacks can be suppressed with a lower dose [ ] . nevertheless, several series from japan, reported that their adult patients with fmf were controlled by low-dose colchicine ( . mg daily). it is hypothesized that the reason for that is their carriage of mutations (in exon and ) which are known to cause a milder disease [ ] . the dose of colchicine should not exceed the maximal tolerated dose and should not be more than mg per day in adults without comorbidity and mg per day in prepubertal children [ ] . it is of paramount importance to avoid toxicity due to concomitant administration of cyp a or pgy inhibitors (table . ). drug-drug interactions need to be considered and the dose of colchicine should be appropriately adjusted. it is necessary to closely monitor the kidney and liver function of these patients (see sect. . for details). we recommend taking the entire colchicine dose at once in order to improve compliance. if the patient develops diarrhea due to ingestion of a relatively high single dose of colchicine the dose should be divided to twice per day without affecting its effectiveness [ ] . although colchicine has been used to treat gout for centuries, relatively few controlled trials have assessed its efficacy. the most recent major trial, acute gout flare receiving colchicine evaluation (agree), randomized patients with acute gout to receive a lower-dose colchicine regimen ( . mg dose followed by one . mg dose h later), a traditional higher dose regimen ( . mg dose followed by . mg every hour for a maximum of . mg) or placebo within h of the onset of attack [ ] . the lower-and higher-dose regimens demonstrated similar efficacy ( . % vs. . % achieving ≥ % improvement within h), but adverse events in the lower-dose regimen were similar to placebo. accordingly, the lower-dose regimen was approved by the fda. american college of rheumatology guidelines recommend the lowerdose colchicine regimen as a first-line therapy option for acute gouty attacks [ ] . in addition to its role in acute gout, colchicine is used prophylactically to reduce gout flare frequency, particularly when patients are initiating urate-lowering therapy. an analysis of three randomized controlled trials found that colchicine use for up to months during initial urate lowering provided greater prophylaxis of flares than its use for only weeks [ ] . there is only limited evidence for the use of colchicine in prophylaxis of pseudogout, although this is recommended practice [ ] . the rationale is the shared nlrp -inflammasome and il- driven inflammatory pathogenesis of urate and calcium pyrophosphate crystal deposition. recent european league against rheumatism (eular) guidelines recommend giving colchicine for acute attacks at a dose of . mg three times daily with or without a mg loading dose and for prophylaxis . mg per day. these recommendations are based largely on expert opinion and a single uncontrolled trial [ , ] . colchicine is recommended in the management of behçet disease, particularly for the mucocutaneous manifestations (oral and genital ulcers) and joint symptoms, based on controlled trials performed to date [ , ] . over the years colchicine has been studied for disorders of hepatic fibrosis. primary biliary cirrhosis is a rational potential indication, given the intense concentration of colchicine in bile and the evolving understanding of the capacity of colchicine to modulate bile composition [ ] . however, a meta-analysis combining the results of randomized controlled trials encompassing subjects with alcoholic or non-alcoholic liver cirrhosis, demonstrated no significant effects of colchicine on mortality, liver related mortality, complications and other outcomes [ ] . in a recent double blind, randomized controlled trial in subjects with chronic liver cirrhosis who could not be treated with interferon-α, muntoni et al. demonstrated that colchicine at a dose of mg per day significantly increased survival ( . % vs. . % p = . ), and decreased serum procollagen iii (a biomarker of liver fibrosis) over a follow up period of . years [ ] .this suggests that there may be a beneficial effect of colchicine for selected subjects with liver cirrhosis. additional research has assessed whether colchicine can delay the development of hepatic cell carcinoma (hcc) in patients diagnosed with hepatitis virus-related liver cirrhosis [ ] . while the effect of colchicine on the progression of cirrhosis was disappointing, colchicine suppressed the development of hcc. nine percent of patients treated with colchicine developed hcc versus % of untreated patients (p − . ), and the time to development of hcc was longer in the colchicine-treated group. colchicine is beneficial in neutrophilic skin conditions. sweet syndrome typically occurs in women aged - years and is characterized by fever, neutrophilia, arthralgia, tender erythematous skin lesions, and neutrophilic infiltrates in the upper dermis. maillard et al. gave colchicine to patients with sweet syndrome ( . mg three times daily for - days). they reported that patients experienced resolution of fever, skin lesions, arthralgia and neutrophilia within days [ ] . interestingly, in pyrin associated autoinflammatory disease with neutrophilic dermatosis (paand) colchicine is not effective and anti-il agents are required for disease control [ ] . the accumulating understanding of the role of inflammation in cardiovascular diseases has been accompanied by recognition of the potential anti-inflammatory benefit of colchicine in these settings. practice guidelines from the european society of cardiology advocate that colchicine appears to be effective when added to a nonsteroidal antiinflammatory drug regimen or as monotherapy to treat an initial attack or to prevent recurrence of pericarditis [ ] . these recommendations have subsequently been supported by randomized trials [ , ] . colchicine was as effective at reducing multiple recurrences of pericarditis as it was for first recurrences. [ , ] similar benefits were observed for treatment of an initial attack of acute pericarditis [ , ] . c-reactive protein (crp) is a biomarker of inflammation and infection. elevated highsensitivity (hs) crp is both a predictor and a pathogenic factor in vascular events such as coronary artery disease [ ] . a recent pilot study evaluated whether low-dose colchicine as an anti-inflammatory treatment could lower hs-crp levels in patients with stable coronary artery disease whose crp remained elevated despite aspirin and high-dose atorvastatin therapy [ ] . low-dose oral colchicine ( . mg twice daily) was associated with decreased hs-crp levels by %. however, in a separate, randomized study of patients with acute coronary syndrome or stroke, raju et al. found no significant association between colchicine use ( mg daily) and crp reduction, suggesting either that the dose studied was insufficient in the acute setting, or that colchicine may be more effective when administered as a prophylactic rather than as a treatment agent [ ] . more recently, nidorf et al. evaluated the effect of colchicine ( . mg daily) on secondary cardiovascular events in patients with stable coronary artery disease already on aspirin and/or clopidogrel and statin therapy [ ] . patients were followed for a median of years. the primary outcome,-the composite incidence of acute syndromes, out-ofhospital cardiac arrest, and non-cardio-embolic ischemic stroke-occurred in . % of patients treated with colchicine versus % of patients receiving placebo. these results warrant further study of the potential benefit of colchicine in the prevention of acute coronary syndrome. there is growing evidence that inflammation plays a role in the re-stenosis process. therefore, one study randomized patients with diabetes mellitus and coronary artery disease who underwent percutaneous coronary intervention with bare-metal stent placement to receive either colchicine . mg or placebo twice daily for months [ ] . subsequent angiography indicated that the rate of angiographic in-stent restenosis was % in the colchicine group versus % in the placebo group (p = . ). post-operative atrial fibrillation is a common occurrence after cardiac surgery and is presumed to be driven by surgery-related inflammation. the colchicine for the prevention of the postpericardiotomy syndrome (copps) atrial fibrillation substudy demonstrated that post-operative administration of colchicine was associated with a % reduction in the incidence of post-operative atrial fibrillation [ ] . however, a second multicenter, randomized, double-blind, placebocontrolled trial (copps- ) found no reduction in post-operative atrial fibrillation among patients receiving colchicine [ ] . colchicine may also reduce the risk of recurrence of atrial fibrillation after ablation therapy. in a randomized, double-blind, placebocontrolled study by deftereos et al., months of colchicine monotherapy was associated with a % reduction in the incidence of atrial fibrillation after pulmonary vein isolation with radiofrequency ablation [ ] . the colchicine group also experienced a significant decrease in il- and crp levels. colchicine update- indianapolis: hackett colchicine: new uses of an old, old drug ueber einige neue giftige organische alkalien (on some new poisonous organic alkalis) medea and the microtubule: research has been translational ever since colchis sulla cariocinesi delle cellule epiteliale e dell endotelio dei vasi della mucosa dello stomato e dell' intestino, nello studio gastroenterite sperimentale (nell avvelenamento per colcico) binding of colchicine- h to cellular protein amino-acid composition of "tubulin" constituting microtubules of sperm flagella the role of microtubule movement in bidirectional organelle transport colchicine in dermatology colchicine in the prevention and treatment of the amyloidosis of familial mediterranean fever colchicine for familial mediterranean fever pharmacokinetics of colchicine in pediatric and adult patients with familial mediterranean fever pharmacokinetic bioavailability of colchicine in healthy male volunteers improvement of colchicine oral bioavailability by incorporating eugenol in the nanoemulsion as an oil excipient and enhancer genuine functions of p-glycoprotein (abcb) concise review: clinical relevance of drugdrug and herb-drug interactions mediated by the abc transporter abcb substrateinduced conformational changes in the transmembrane segment of human p-glycoprotein p-glycoprotein: so many ways to turn it on colchicine today pharmacokinetics and absolute bioavailability of colchicine after i.v. and oral administration in healthy human volunteers and elderly subjects association of drug transporter gene abcb (mdr ) c to t polymorphism with colchicine response in familial mediterranean fever colchicine nonresponsiveness in familial mediterranean fever: genetic, pharmacokinetic, and socioeconomic characterization plasma levels of colchicine after administration of a single dose colchicine plasma levels: implications as to pharmacology and mechanism of action colchicine disposition in patients with familial mediterranean fever with renal impairment measurement of colchicine in urine and peripheral leukocytes (abstract) binding of drugs by albumin and plasma protein colchicine disposition in human leukocytes after single and multiple oral administration colchicine: a state-ofthe-art review bioavailability and pharmacokinetics of two formulations in volunteers colchicine clearance is impaired in alcoholic cirrhosis interaction of tubulin with single ring analogue of colchicine effect of colchicine on lymphocytes and monocytes function and its relation to fibroblast proliferation in primary biliary cirrhosis appearance of chemotactic activity following intracellular injection of monosodium urate crystals: effect of colchicine colchicine inhibition of chemotaxis a new mode of action for an old drug: colchicine decreases surface expression of adhesion molecules on both neutrophils (pmns) and endothelium (abstract) p-glycoprotein expression and function in circulating blood cells from normal volunteers does the lack of the p-glycoprotein efflux pump in neutrophils explain the efficacy of colchicine in fmf and other inflammatory diseases rapports entre noyau et centrioles dans les granulocytes étalalés. rúle des microtubules molecular mechanism of colchicine action: induced local unfolding of b-tubulin response of microtubules to the addition of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with microtubules inhibition of lpsinduced tumor necrosis factor-α production by colchicine and other microtubules disrupting drugs downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by mictotubule depolarizing agents colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils involvement of secretory phospholipase a activity in the zymosan air pouch model of inflammation the effects of colchicine and hydroxychloroquine on the cyclo-oxygenases cox- and cox- the french consortium. a candidate gene for familial mediterranean fever ancient missense mutations in a new member of the roret gene family are likely to cause familial mediterranean fever effect of colchicine and cytokines on mefv expression and c a inhibitor activity in human primary fibroblast culture the familial mediterranean fever protein, pyrin, associates with microtubules and colocalizes with actin filaments the pharmacologic basis of treatment with colchicine in children with familial mediterranean fever mechanism of the anti-inflammatory effect of colchicine in rheumatic diseases: a possible new outlook through microarray analysis gout-associated uric acid crystals activate the nalp inflammasome colchicine suppresses neutrophil superoxide production in a murine model of gouty arthritis: a rationale for use of low-dose colchicine innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome interaction of pyrin with . . in an isoform-specific and phosphorylation-dependent manner regulates its translocation to the nucleus nucleotide exchange factor gef-h mediates cross-talk between microtubules and the actin cytoskeleton pyrin inflammasome activation and rhoa signaling in the autoinflammatory diseases fmf and hids technical advance: inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments colchicine attenuates renal injury in a model of hypertensive chronic kidney disease curcumin prevents liver fibrosis by inducing apoptosis and suppressing activation of hepatic stellate cells the effects of colchicine on the progression and regression of encapsulating peritoneal sclerosis colchicine use in children and adolescents with familial mediterranean fever: literature review and consensus statement colchicine is a safe drug in children with familial mediterranean fever current trends in colchicine treatment in familial mediterranean fever colchicine intoxication clinical pharmacology, risk factors, features and management colchicine: old and new estimation of colchicine in a poisoned patient by using high performance liquid chromatography treatment of severe colchicine overdose with colchicine specific fab fragments vos fr high flux dialysis membranes improve lipid profile in chronic hemodialysis patients colchicine clearance by high-flux polysulfone dialyzers abc drug transporters as molecular targets for the prevention of multidrug resistance and drug-drug interactions colchicine myotoxicity: case reports and literature review colchicine use in cyclosporine treated transplant recipients: how little is too much? mdr-and cyp a -mediated drug-drug interactions influence of cytochrome p polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects multiple sequential steps involved in the binding of inhibitors to cytochrome p a heterotropic cooperativity of cytochrome p a and potential drug-drug interactions acute myopathy in a patient with concomitant use of pravastatin and colchicine fatal interaction between clarithromycin and colchicine in patients with renal insufficiency: a retrospective study cytoskeletal myotoxicity from simvastatin and colchicine rhabdomyolysis in a patient treated with colchicine and atorvastin possible colchicine rhabdomyolysis in a fluvastatin-treated patient novel evidence-based colchicine dosereduction algorithm to predict and prevent colchicine toxicity in the presence of cytochrome p effect of colchicine on guinea pig intrinsic factor-vitamin b receptor colchicineinduced lactose malabsorption in patients with familial mediterranean fever azoospermia caused by colchicine-a case report the effect of colchicine on the spermatogenesis of rabbits a cytogenetic evaluation of long term colchicines therapy in the treatment of familial mediterranean fever (fmf) testicular function in patients with familial mediterranean fever, long-term colchicine treatment fertility and obstetric history in patients with familial mediterranean fever on long term colchicine therapy the effect of colchicine on human spermatozoal motility in vitro colchicine and testicular function in man urological evaluation of behçet patients and the effect of colchicine on fertility azoospermia in fmf patients-the role of colchicine and amyloidosis daily prophylactic colchicines in familial mediterranean fever the effect of fmf and colchicine on pregnancies outcome of wives of patients with fmf familial mediterranean fever and menstruation effects of hormone therapy on inflammatory cell adhesion molecules in postmenopausal healthy women qualitative study of the interaction mechanism of estrogenic drugs with tubulin safety of colchicine therapy during pregnancy familial mediterranean fever and its implications for fertility and pregnancy colchicine treatment in conception and pregnancy: two hundred and thirty one pregnancies in patients with familial mediterranean fever pregnancy outcome after in utero exposure to colchicine outcome of pregnancies in fmf women with colchicine breast feeding during colchicine therapy for familial mediterranean fever colchicine in breast-milk of patients with fmf colchicine treatment in familial mediterranean fever (fmf)-reappraisal after years colchicine therapy of children with fmf (abstract) growth and igf- levels of children with familial mediterranean fever on colchicine treatment eular recommendations for the management of familial mediterranean fever familial mediterranean fever is no longer a rare disease in japan evidencebased recommendations for the practical management of familial mediterranean fever comparison of the efficacy of once-and twice-daily colchicine dosage in pediatric patients with familial mediterranean fever-a randomized controlled non-inferiority trial high versus low dosing of oral colchicine for early acute gout flare: twenty-four-hour outcome of the first multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-comparison colchicine study american college of rheumatology guidelines for management of gout. part : therapy and antiinflammatory prophylaxis of acute gouty arthritis effect of prophylaxis on gout flares after the initiation of urate-lowering therapy: analysis of data from three phase iii trials eular recommendations for calcium pyrophosphate deposition. part ii: management intravenous colchicine in the treatment of acute pseudogout a double-blind trial of colchicine in behçet's syndrome eular recommendations for the management of behcet disease mechanisms by which camp increases bile acid secretion in rat liver and canalicular membrane vesicles colchicine for alcoholic and non-alcoholic liver fibrosis and cirrhosis colchicine reduces procollagen iii and increases pseudocholinesterase in chronic liver disease colchicine delays the development of hepatocellular carcinoma in patients with hepatitis virusrelated liver cirrhosis colchicine for sweet's syndrome. a study of cases familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation guidelines on the diagnosis and management of pericardial diseases executive summary; the task force on the diagnosis and management of pericardial diseases of the european society of cardiology colchicine as first-choice therapy for recurrent pericarditis: results of the core (colchicine for recurrent pericarditis) trial colchicine for recurrent pericarditis (corp): a randomized trial efficacy and safety of colchicine for treatment of multiple recurrences of pericarditis (corp- ): a multicentre, double-blind, placebo-controlled, randomised trial colchicine for treatment of acute or recurrent pericarditis colchicine in addition to conventional therapy for acute pericarditis: results of the colchicine for acute pericarditis (cope) trial a randomized trial of colchicine for acute pericarditis c-reactive protein and atherogenesis: from fatty streak to clinical event effect of colchicine ( . mg twice daily) on high-sensitivity c-reactive protein independent of aspirin and atorvastatin in patients with stable coronary artery disease effect of colchicine compared with placebo on high sensitivity c-reactive protein in patients with acute coronary syndrome or acute stroke: a pilot randomized controlled trial low-dose colchicine for secondary prevention of cardiovascular disease colchicine treatment for the prevention of baremetal stent restenosis in diabetic patients colchicine reduces postoperative atrial fibrillation: results of the colchicine for the prevention of the postpericardiotomy syndrome (copps) atrial fibrillation substudy colchicine for prevention of postpericardiotomy syndrome and postoperative atrial fibrillation: the copps- randomized clinical trial colchicine for prevention of early atrial fibrillation recurrence after pulmonary vein isolation: a randomized controlled study key: cord- -jy fdsp authors: orhobor, oghenejokpeme i.; french, joseph; soldatova, larisa n.; king, ross d. title: generating explainable and effective data descriptors using relational learning: application to cancer biology date: - - journal: discovery science doi: . / - - - - _ sha: doc_id: cord_uid: jy fdsp the key to success in machine learning is the use of effective data representations. the success of deep neural networks (dnns) is based on their ability to utilize multiple neural network layers, and big data, to learn how to convert simple input representations into richer internal representations that are effective for learning. however, these internal representations are sub-symbolic and difficult to explain. in many scientific problems explainable models are required, and the input data is semantically complex and unsuitable for dnns. this is true in the fundamental problem of understanding the mechanism of cancer drugs, which requires complex background knowledge about the functions of genes/proteins, their cells, and the molecular structure of the drugs. this background knowledge cannot be compactly expressed propositionally, and requires at least the expressive power of datalog. here we demonstrate the use of relational learning to generate new data descriptors in such semantically complex background knowledge. these new descriptors are effective: adding them to standard propositional learning methods significantly improves prediction accuracy. they are also explainable, and add to our understanding of cancer. our approach can readily be expanded to include other complex forms of background knowledge, and combines the generality of relational learning with the efficiency of standard propositional learning. descriptors, i.e. the data can be put into a single table, where the descriptors (attributes) are the columns, and the examples are rows. descriptors are properties of the examples that are believed to be important: for example if one wishes to classify pictures of animals then image pixel values are useful descriptors. such tuple-based representations are essentially based on propositional logic [ ] . the effectiveness of the propositional descriptors used for learning can vary greatly, and traditionally, most of the effort in ml went into hand-crafting effective descriptors. this has changed with the success of deep neural-networks (dnns), which has been based on their capacity to utilize multiple neural network layers, and large amounts of data, to learn how to convert raw propositional descriptors (e.g., image pixel values) into richer internal representations that are effective for learning. thanks to this ability dnns have succeeded in domains that had previously proved recalcitrant to ml, such as face recognition and learning to play go. the archetypal success is face recognition, which was once considered to be intractable, but can now be solved with super-human ability on certain limited problems [ ] . therefore, a key lesson of the success of dnns is: use ml to learn better data representations for ml. for many problems the standard propositional representation of data is problematic, as such a representation cannot efficiently express all the known relational structure (background knowledge) in the data. in some cases this structure can be encoded for using special purpose methods. for example convolutional neural networks encode relational information about the position of descriptors in the structure of the net. similarly, recurrent neural networks encode information about temporal structure in the net, graph neural networks encode graphical information, etc. in many cases such special purpose methods can work very well. however, these methods must be redesigned for each new type of problem, and the structure encoded in the learning process is not explicit. it would be more beneficial (and elegant) if the learning biases in dnns were explicit, and not inherent in the structure of the network. a more general approach to encoding known structure in data is to use logic programs [ ] to represent the data -relational learning (rl) [ ] . such programs can express spatial, temporal, graphical structure, etc. using a single formalism, and, crucially, this structure is explicit instead of being implicit (e.g. in the connection of neurons). logic programs provide a unified way of representing the relations between objects. they also promote explainable ml, as it is usually straightforward to translate logic programs into a series of easily understandable sentences that can be interpreted by domain experts. more formally, logic programs are a subset of st-order predicate logic, and therefore more general that propositional representations. the main disadvantages of using a relational representation compared to a standard propositional one are that rl is more computationally expensive and difficult, as the search space of possible models is much larger, and that rl technology is much less developed. this suggests a hybrid strategy where rl is used to learn effective descriptors, and then standard ml is used to learn the final model [ ] . this hybrid approach is particularly suited to problems where the data is semantically complicated, and where symbolic explainable models are required. in such problems rl has the potential to effectively learn new descriptors that are understandable to domain experts. many biomedical ml problems are potentially suitable for a hybrid rl approach, such as understanding the mechanism of cancer drugs. in this problem one needs to encode background knowledge (problem structure) about gene/protein function, associated pathways, known drug targets, cancer cell type, the molecular structure of drugs, etc. we took data on this problem from the library of integrated networkbased cellular signatures [ ] (lincs). specifically, we used the phase ii data, which consists of gene expression levels for landmark human genes under perturbation conditions, making this a regression problem. the perturbation conditions consist of a cancer drug added to a cancer cell line, and it is worth noting here that only the response gene expression values are provided, and one would need to independently construct the input variables from the provided metadata. we hypothesized that we could improve both ml model explainability, and predictive accuracy, by including additional background knowledge in the learning process using a hybrid rl approach. a key source of this background knowledge was the stanford biomedical network dataset collection [ ] (sbnd). using rl we mined frequent patterns about each drug found in relation to additional background knowledge. these patterns are expressed in datalog [ ] and are explainable to domain experts. they can also be used as binary descriptors in standard ml methods. it is worth noting that ml model explainability heavily depends on the learning algorithm, as some learning algorithms are more interpretable than others. however, we argue that the descriptors generated using the hybrid rl approach will generally be more interpretable than their propositional counterparts. we evaluated the predictive performance of the newly learnt rl descriptors versus the standard descriptors, both when used by standard ml in isolation, and in combination. we compared two approaches to combining sets of descriptors: one in which the features from both representations are concatenated to form a single dataset, and another where predictions are stacked [ ] . we found that the standard descriptors generally outperform the rl descriptors when used in isolation. however, the rl descriptors significantly improve predictive performance when used in combination. moreover, these new effective rl descriptors are understandable by domain experts. the main contributions of the paper are as follows: . demonstration of the effectiveness of hybrid rl learning on an important real-world problem. . learnt explainable patterns underlying common cancer drugs. . a fully integrated biomedical knowledge base in datalog. the problem of building models to predict drug effects has been widely studied, from potential adverse effects [ ] and drug-drug interactions [ ] to cancer cell sensitivity [ ] . one such task is the learning of quantitative structure activity relationships (qsars), where one is interested in predicting the effect of a drug or chemical compound from its molecular structure [ ] . molecular structure is usually represented using molecular fingerprints, which are tuples of boolean descriptors [ ] . however, several other approaches also exist. for example, some authors have used the -dimensional structure of chemicals [ ] , while others have extracted molecular vector embeddings using graph neural networks [ ] . in our evaluation, we used the most widely adopted molecular fingerprint representation as the propositional approach. the lincs data has been used in several studies, e.g. for the task of predicting gene expression levels using perturbation conditions [ ] . in contrast to our evaluation, the authors do not utilise background knowledge in the learning process. several techniques have been applied to interconnected knowledge bases for various problems in biology [ , ] . rl in particular has been used in problems such as predicting gene function [ ] , gene regulation [ ] and qsar-related problems [ ] . rl algorithms such as warmr, which we use in our evaluation, have been shown to be successful in identifying relationships in linked data [ ] . however, there are other algorithms like amie [ ] which have also been shown to perform remarkably well. furthermore, there exist several other approaches for learning representations from graph or inherently relational data [ , ] with varying levels of predictive performance and interpretability. we argue that our decision to use warmr in our evaluation offers a good foundation from which all of these other methods can be explored in tackling the stated problem as part of future work. one can think of the boolean molecular fingerprint and rl representations of the drugs in the stated problem as views in multi-view learning, as both of these representations offer different perspectives in what constitutes the known properties of a drug. in a standard multi-view learning problem the views are typically distinct, meaning that special consideration is made as to the learning algorithm used in building a model for a particular view. multiple kernel learning [ ] , which is essentially a form of stacking, has been proposed for such a scenario, where a kernel that is best suited for a particular view is used and the predictions from all views are then combined to form the final prediction. this is in contrast to how we perform our evaluation, because though we considered multiple learning algorithms, a specific learning algorithm is not used for a particular representation. the lincs phase ii dataset with accession code gse provides the expression levels for landmark human genes for , perturbation conditions. in the metadata, the perturbation conditions are described by their cell line, cell site, drug dosage, drug timepoint, and the applied drug. the broad institute identifiers along with their canonical smiles are also provided for the drugs. we were able to map , of the applied drugs to their drugbank [ ] and chembl [ ] identifiers. this is relevant because the sbnd knowledge graph uses drug-bank identifiers. the drugs we could map across these databases were applied to only , of the , perturbation conditions. for all the aforementioned perturbation condition properties but the drugs, we engineered features for the perturbation conditions using one-hot encoding, and treat them as base features. for the propositional representation of the drugs, we converted them into molecular fingerprints using rdkit [ ] , with bits, a radius of , and usefeatures set to true. for the rl representation of the drugs, we formalised the following relations from the sbnd: drug-drug (chch-miner), drug-gene (chg-miner), genefunction (gf-miner), disease-drug (dch-miner), and disease-function (df-miner). additionally, we included relationships between functions from gene ontology [ ] , such as is a and part of. furthermore, we included the chemical properties of each drug, such as the presence of rings. in total, this datalog knowledge base contains , drugs, , genes, , functions and , diseases. it is available for download at https://github.com/oghenejokpeme/ rlcbkb. the hypothesis language we used is given in fig. . drug_drug(+drug, -drug) drug_disease(+drug, -disease) disease_function(+disease, -function) drug_gene(+drug, -gene) gene_function(+gene, -function) has_functional_group(+drug, #group) has_ring(+drug, #ringtype) has_group_count(+drug, #group, #count) has_ring_count(+drug, #ringtype, #count) has_group_ring_attachment(+drug, #group, #ringtype) is_a(+function, #function) part_of(+function, #function) has_part(+function, #function) regulates(+function, #function) neg_regulates(+function, #function) pos_regulates(+function, #function) using this knowledge base, we learned , frequent patterns using the warmr algorithm in the aleph inductive logic programming engine [ ] for each drug, which we then use as binary features. warmr is a levelwise rl algorithm based on a breath-first search of the input knowledge base, as relations are structured as a lattice. it allows for the learning of frequent patterns present in a knowledge base within pre-specified constraints, such as the proportion of the sample space a learned pattern must cover [ ] . in aleph, we used a minimum cover of %, a maximum clause length of , and nodes. we should note that there is evidence that suggests that beyond a certain point, increasing the number of learned features leads to no performance gains [ ] . with the base features, we then created three data descriptors for the perturbation conditions. one with the base and rl features, another with the base and fingerprint features, and finally, one with the base, rl and fingerprint features. we refer to these datasets as rl, fp, and rl+fp for the remainder of this paper, all of which at this point, have , samples. we used a train-test split in our evaluation and selected only a subset of the samples due to computational complexity. the selection procedure entailed an initial random split of the , samples into training and testing buckets, %- %. we then randomly selected , samples from the training bucket for the training set and , samples from the testing bucket for the test set. this was performed exactly once, and the dataset is available here: http://dx.doi.org/ . / mgyb dyxv. . one might argue that this is a paired-input problem, as we are predicting gene expression on pairs of drug perturbation conditions and cancer cell lines. therefore, we should extend our evaluation to take this into account. we expect that the naive train-test split evaluation approach we have taken will produce more optimistic results than an evaluation procedure for which each entity in a pair present in the test set is also not present in the training set [ ] . however, we argue that for the purposes of evaluating standard propositional and rl data descriptors, such an evaluation setting will suffice. as we mentioned previously, the lincs dataset contains the gene expression levels of genes. we selected genes that are dissimilar from one another by associated function using gene ontology associations at a tree depth of , where are the base nodes. this process selected genes, which we used in our experiments. we did this to reduce computational complexity and to select genes that are uncorrelated on a functional level in order to get performance estimates that are generally representative of the complete set of genes. for learning algorithms, we used the least absolute shrinkage and selection operator (lasso) [ ] , ridge regression (rr) [ ] , and random forests (rf) in our evaluation. for lasso and rr the regularization parameter was chosen using internal -fold crossvalidation, and the rf models were built with trees and default settings. the performance metric reported is the coefficient of determination (r ), as we are most interested in the amount of variance explained by the built models. apart from the standard regression experiments using all three datasets, we also evaluated integrating the predictions made by the rl and fp representations using simple averaging, which is a form of stacking [ ] . in this case, we averaged the predictions made using the rl and fp representations. we refer to these results as avg in the discussion of the evaluation results. all code used for this experiment is available at https://github.com/oghenejokpeme/rlcbexp. we observed that on average the rl representation consistently performs worse than all the other representations (see table ). for the approaches which combine the rl and fp representations, we found that rl+fp consistently outperforms rl and does not strictly outperform fp on any of the learners. however, rl+fp and fp perform equally well on lasso. like rl+fp, avg also consistently outperforms rl, but is outperformed by fp on both lasso and rr, but not on rf. these results suggests that the effect the rl representations have when used to augment fp representations depends on two things; the choice of learning algorithm and how the representations are combined. from the mean performance results in table , one might argue that overall, the performance of the representations is generally low. while this is true, we would argue that this is to be expected, as we are attempting to recreate laboratory conditions in silico, and predict the expression of genes which often vary in concert and not in isolation of each other. furthermore, it is worth pointing out that the representations perform reasonably well on some genes, with a maximum r of . when the rl and fp predictions for rf are averaged (table ) . table . the predictive performance (r ) of the engineered datasets (rl, fp and rl+fp) and the aggregation by mean of the predictions made by rl and fp (avg) on the learning algorithms. we show the mean with the minimum and maximum performance for the considered genes. the best performing descriptor for each learner is in boldface. given the difference in performance between the different methods, we tested for statistical significance using sign tests and paired t-tests. for lasso, table shows that rl+fp underperforms when compared to fp, with a ratio of - of the considered genes and a . % average performance decrease from fp to rl+fp. however, this difference in performance is not statistically significant for both sign test and paired t-test. when avg is compared to fp, we found that fp performed better on more genes, with a ratio of - . however, we found that when compared to fp, avg achieves a . % average percentage performance increase over fp, with statistical significance according to the paired t-tests. further investigation showed that although fp outperformed avg on table . performance (r ) comparisons between the different datasets for the learning algorithms we considered. the comparisons are structured as approach a/b. for each compared pair, the number of genes for which one strictly outperforms the other is given. additionally, an asterisk ( * ) and a dagger ( †) are used to indicate a statistically significant difference in performance with a significance level of . for a sign test and a paired t-test respectively. lastly, the average percentage performance increase or decrease when approach a is compared to b is given. it is worth noting that this average percentage performance is calculated by taking the mean percentage difference in performance of genes between a and b, and not simply the percentage difference in mean performance given in table rl enables the introduction of additional background knowledge to the model building process, and it can improve both understandability and predictive performance. in our experiments we were interested in learning frequent patterns present in the knowledge base for the considered drugs. we learned such rules as: the former rule can be interpreted as a frequent pattern is for a drug to have an oxide group and two benzene rings. note that the standard fingerprint representation of molecules cannot express the simple concept of a molecule having two benzene rings unless a special descriptor 'two benzene rings' is pregenerated. nor can it express the concept of a drug having an oxide group and two benzene rings unless it is pre-generated. to pre-generate all possible descriptors would produce an exponential number of descriptors. the latter rule can be interpreted as a frequent pattern is for a drug to target a gene that positively regulates a metabolic process. note that is a second-order pattern, the drug targets a gene that in turn regulates metabolism. most drugs inhibit their targets, and in this pattern the overall result is likely to be decrease in a metabolic process, which is generally desirable in cancer therapy. these examples show that rules are easily understandable by a human reader. one can conjecture that if feature selection is performed when such rules are used as features in a predictive problem, the why of the observed variance in the target could be explained easier. however, it is beyond the scope of this work. the great success of dnns is based on their ability to learn how to transform a simple input data representation into an effective internal representation. the limitations of the dnn approach are that it requires a large amount of data, the internal representation is obscure, and there is not a general way to encode known problem structure and background knowledge. in many biomedical problems, such as understanding the effect of anti-cancer drugs, it is required to encode a large amount of background knowledge. in this paper we have shown that a hybrid rl approach can learn new descriptors that are effective and explainable. the limitations of the hybrid rl approach are that it is a two stage approach rather than end-to-end learning (it is computationally efficient to learn frequent patterns, but they are not necessarily effective), and that the learning model is not differentiable, which makes it more difficult to find model improvements. the main criticism of rl in the past was that it was too inefficient to be applied. however, now, given the vast resources used to train dnns this no longer applies. it is therefore interesting to consider whether there is a more general way of learning how to improve data representations that combines the advantages of dnns and the hybrid rl approach, as is the case with deep relational machines [ ] . furthermore, it is worth noting that though the rl representations might be explainable, the interpretability of the models built using them will vary based on the learning algorithm. for example, one might conceivably inspect the important variables of a random forest model, but will find this far more challenging in a deep attention neural network. in this paper we report the use of rl representations to enhance the predictive accuracy of traditional propositional data representations for a relevant problem in cancer biology. apart from improved predictive accuracy, we also learnt explainable patterns underlying common anti-cancer drugs, and built a fully integrated biomedical knowledge base in datalog which is now publicly available. we intend to investigate other forms of rl as part of future work. prediction of new associations between ncrnas and diseases exploiting multi-type hierarchical clustering stacked regressions spatial associative classification: propositional vs structural approach gene expression inference with deep learning qsar modeling: where have you been? where are you going to? machine learning of functional class from phenotype data large-scale assessment of deep relational machines discovery of frequent datalog patterns inductive logic programming for gene regulation prediction amie: association rule mining under incomplete evidence in ontological knowledge bases chembl: a large-scale bioactivity database for drug discovery the gene ontology (go) database and informatics resource representation learning on graphs: methods and applications ridge regression: biased estimation for nonorthogonal problems fp vec: a new molecular featurizer for learning molecular properties applying inductive logic programming to predicting gene function warmr: a data mining tool for chemical data data portal for the library of integrated networkbased cellular signatures (lincs) program: integrated access to diverse large-scale cellular perturbation response data rdkit: open-source cheminformatics deep learning foundations of logic programming biosnap datasets: stanford biomedical network dataset collection machine learning prediction of cancer cell sensitivity to drugs based on genomic and chemical properties ilp turns meta-qsar: a large-scale application of meta-learning to drug design and discovery a general framework for building accurate and understandable genomic models: a study in rice (oryza sativa) flaws in evaluation schemes for pair-input computational predictions artificial intelligence: a modern approach large scale multiple kernel learning the aleph manual quantitative pharmacophore models with inductive logic programming predicting drug-drug interactions through drug structural similarities and interaction networks incorporating pharmacokinetics and pharmacodynamics knowledge data-driven prediction of drug effects and interactions regression shrinkage and selection via the lasso d-qsar in drug design-a review drugbank: a comprehensive resource for in silico drug discovery and exploration modeling polypharmacy side effects with graph convolutional networks open access this chapter is licensed under the terms of the creative commons key: cord- - p rwp authors: nan title: escp th european symposium on clinical pharmacy ‘implementing clinical pharmacy in community and hospital settings: sharing the experience’, istanbul, turkey – october ; abstracts date: - - journal: pharm world sci doi: . /s - - - sha: doc_id: cord_uid: p rwp nan pharmacy, groupe hospitalier pitié salpétrière, paris, france background and objective: strongyloides stercoralis infects each year millions of persons worldwide. in immunocompromised patients, this intestinal nematode can disseminate and cause a fulminant fatal illness: hyperinfection syndrome. oral ivermectin is the principal treatment. since one of the features of s. stercoralis hyperinfection is the development of an ileus and small bowel obstruction, the drug absorption is impaired and thus a reduced efficacy is noted. no parenteral antihelminthic drug is licensed for human use, but parenteral ivermectin is commonly used in veterinary medicine. we report two cases of s. stercoralis hyperinfection syndrome that were refractory to oral drugs and, as a life saving therapy, were treated with a veterinary formulation of parenteral ivermectin (ivomec Ò , merial) after agreement from the french drug administration (afssaps). design: case report. setting: pneumology and neurochirurgical intensive care units, university hospital, paris, france. main outcome measures: case report. results: patient a -year-old african man has been hospitalized in august for a degradation of his condition in neurosarcoidosis with hydrocephalus, associated with enterococcus faecalis meningitis. he underwent ventriculo-peritoneal shunt and was treated with corticosteroids (prednisone mg/day). his condition worsened on september , with the diagnosis of a disseminated strongyloidiasis with paralytic ileus. he was initially treated with ivermectin ( mg bid) via the nasogastric tube. antibiotics were added on day to control the sepsis. on day albendazole ( mg/day) was added. subcutaneous ivermectin was then obtained and administered on day ( lg/kg) in association with ivermectin via nasogastric tube while albendazole was discontinued. the patient's condition improved during the following days. he completed days of ivomec Ò and days of oral ivermectin. he returned home months later. patient a -year-old african man was hospitalized in november because of a kg weight loss. he was diagnosed with hiv, meningeal tuberculosis, urinary and pulmonary infections and s. stercoralis hyperinfection treated with oral ivermectin. on january he was admitted in pneumology intensive care unit with melena on severe immunodepression. on february he developed a septic shock with ards on an important bowel obstruction. therefore, among other antiinfectious therapies, a veterinary formulation of subcutaneous ivermectin was administered ( lg/kg/day). the sepsis was controlled, but on february he died of an acute haematological deterioration. conclusions: as the occurrence of malabsorption is a frequent complication of disseminated strongyloidiasis, a parenteral formulation of ivermectin would be really helpful, especially as the efficacy of the subcutaneous form has been proved in the literature. keywords: strongyloides stercoralis, hyperinfection, parenteral ivermectin • each pharmacy received three covert sp visits over six weeks. verbal and written feedback was provided to the pharmacy staff after each sp visit. no pharmacy was visited by the same sp more than once. each pharmacy was visited by the same pe throughout the study. background and objective: heart failure (hf) is a common disease with an estimated prevalence of . to % in europe. patients with hf have frequent episodes of exacerbation. non-compliance to medical and dietary advice is a significant clinical problem as is suboptimal treatment. one example of factors influencing the ability to comply with a treatment plan is impaired comprehension. the objectives were to construct a medication assessment tool and to establish face validity for its use in this project, to construct an interview schedule in order to identify non-compliance, poor patient comprehension and suboptimal treatment, to conduct a survey and to report the findings to the clinic. design: a cross-sectional study performed during april -may . setting: the emergency department and medical wards at malmö university hospital. main outcome measures: comparison of compliance, comprehension and optimal treatment on a population basis between men and women, younger (\ years) and elderly ([ years) patients, and patients in different new york heart association (nyha) classes, in order to assess if exacerbation could have been caused by any of these factors. results: of the patients included, % reported high compliance. in the subgroup analysis, women and elderly patients reported a significant higher compliance than men and younger patients. comprehension on self-care was poor. only % weighed themselves regularly and % did not limit the amount of fluids. no more than % reported they would contact a health care provider in case of experiencing more symptoms. suboptimal treatment was also found to be a great concern with only % being treated with angiotensinconverting enzyme inhibitors (acei) or angiotensin ii receptor blocker (arb), % with beta blockers, and % with aldosterone receptor antagonists, but no consideration to other co-morbidities has been taken into account. the majority treated with recommended agents had not achieved target dose as recommended in guidelines. conclusions: poor patient compliance and comprehension as well as suboptimal treatment could contribute to hf exacerbation and efforts should be made to improve these factors in order to reduce hf exacerbation. keywords: heart failure, compliance, sub-optimal treatment background and objective: adherence with inhaled corticosteroids has repeatedly been reported to be poor. poor adherence could lead to inadequate control of asthma complaints. monitoring of repeat prescriptions by a pharmacist could offer an opportunity to reach concordance with the patient and improve adherence. the objective of this study is was to improve asthma control by optimizing use of asthma medicines. design: retrospective follow up study. all pharmacy dispensing records concerning respiratory medication (r ) from st october to th september were collected. between st october to th september monitoring of repeat medication was conducted by a pharmacist. pharmacists discussed asthma complaints and use of asthma medicines with all patients calling for repeat prescriptions. when indicated the pharmacist proposed adjustments of asthma medicines to the gp after this telephone consultation. setting: community pharmacy and one gp practice ( gp's) in leiden, the netherlands, serving a community of . patients. main outcome measures: self-reported use of short-acting betaagonists (saba) by intervention patients. defined daily doses (ddds) of short-acting beta-agonists (saba), long-acting beta-agonists (laba) and inhaled corticosteroids (ics). results: consultations were registered for intervention patients. for patients more than consultation was registered. at the first consultation only of patients ( %) reported use of pharmaceutical sciences, strathclyde institute of pharmacy and biomedical sciences, university of strathclyde, glasgow, united kingdom background and objective: to test a method to quantify adherence of medication use to clinical guideline recommendations in a primary care setting. design: retrospective survey to field-test a -item instrument (mat-cvd) based on earlier studies of quality of medication use in cardiovascular disease (cvd) . setting: a database of patients [ % male, mean (sd) aged ( ) years] coded with circulatory system disease (read code 'g*') was drawn from computerised records of all patients receiving care from a single community pharmacist and general medical practitioner (gp) collaboration (n = , ). the pharmacist worked as a supplementary prescriber and had remote access to the electronic records of the gp. patients had diagnoses of diabetes (n = ), hypertension (htn; n = ), ischaemic heart disease (ihd; n = ), other ischaemic vascular disease (cerebrovascular n = ; peripheral vascular n = ), heart failure (hf; n = ), atrial fibrillation (af; n = ), were anticoagulated (warfarin, n = ) or otherwise identified as potential candidates for primary prevention of cvd (n = ). main outcome measures: adherence (%) to criteria based on guideline recommendations on primary and secondary prevention of cvd, treatment of htn, ihd, hf, af and warfarin therapy; overall applicability of criteria and quantification of insufficient data; interrater agreement of application of individual mat-cvd criteria and of the overall tool (cohen's r) results: a total of criteria were applicable and for ( %) of these there was insufficient data to apply the standard. the guideline adherence ( % ci) overall was ( - )%. highest adherence was to 'primary/secondary prevention of cvd' [ ( - )% adherence, n = criteria]. lowest adherence was to 'treatment of af' [ ( - )% adherence, n = criteria]. non-adherences were found to at least one criterion in ( %) and to c criteria in ( %) patients. inter-rater agreement was assessed on the application of the tool to all patients by two independent raters. all six sections and the overall tool were found to have inter-rater agreement r [ . and a percentage agreement [ %. among the ( %) of individual mat criteria that were applicable to c patients showed r [ . . in two of the remaining seven criteria the base-rate problem was responsible for r \ . and when taken into account the number of individual criteria with acceptable inter-rater agreement was ( %). conclusions: the application of the mat-cvd to routine primary care records in a scottish primary care setting is feasible and reliable; the tool has potential use in continuous quality improvement of prescribing in primary care. pc- self-management of complications in diabetic patients: a pharmaceutical care program in community pharmacies pharmacy, general medicine unit, university medical outpatient clinic, lausanne, switzerland background and objective: seamless care refers to continuity of patient care in the health system across caregivers. the objectives of the present study were ( ) to identify barriers to seamless information between a given hospital and an outpatient clinic in switzerland and ( ) to propose tools for improving pharmaceutical seamless care. design: this is a retrospective study with a convenient sample of patients for mapping the information flow network. the inclusion criteria were: ( ) at least one stay lasting more than hours in the hospital in , ( ) regular checkups with a gp in the outpatient clinic and ( ) medication delivered by the community pharmacy of the outpatient clinic months prior to months after the hospitalization. setting: both hospital and outpatient clinic are independent and run their own pharmacy. the hospital pharmacy is implied in drug production and distribution without generalized pharmaceutical care activities, and the community pharmacy delivers rx or otc medication for outpatients. geographic and computer proximity between both entities constitutes an ideal setting for seamless care projects. main outcome measures: ( ) to map medical or administrative information between community pharmacy, gp and hospital and ( ) to find opportunities to improve pharmaceutical seamless care. results: sixteen patients met inclusion criteria ( women, men, average years, mean visits/patient/year with gp: , and with community pharmacist: , i.e. -time more with pharmacist than gp). we observed that administrative information is computerized on a common database for both hospital and outpatient clinic. in contrast, clinical information is mainly handwritten and difficult to share between hospital and outpatient clinic caregivers. patient medication database is managed by a community pharmacy software not linked to medical information. however administrative information flows in one direction from the administrative to the community pharmacy database. we identified potential tools easily available to the community pharmacy to improve pharmaceutical seamless care in the center: ( ) an alarm through the administrative database connection if a patient is hospitalized to allow pharmacist to contact hospital physician for medication history and ( ) an access to patient discharge letter and lab results to improve rx validation process. conclusions: clinical information is not easily shared between caregivers of the hospital and the outpatient clinic. if global seamless care still remains a long term goal, initial actual steps promoted by community pharmacists can be easily implemented. keywords: seamless, information, community pk- implementation of a protocol for pharmacokinetic monitoring of high-dose methotrexate background and objective: to quantify the impact of the implementation of a protocol for the pharmacokinetic monitoring of patients receiving high-dose methotrexate. design: prospective experimental study, in which the hospital pharmacy department designed a specific protocol for the pharmacokinetics follow-up of these patients and for gathering the data required for a correct rescue. results were compared between three months before and three months after implementation of this new protocol. setting: -hr infusions of methotrexate at a dose of c g/m were evaluated in adult patients admitted to the oncohaematological area of a tertiary level hospital. main outcome measures: number of infusions started at the correct time, number of missed blood extractions, number of missed leucovorin doses, calculation of the elimination half-life, and measurement of the urinary ph (dichotomous variables). degree of compliance with the leucovorin rescue dosage protocol was measured on a scale of - points, with all items carrying the same score (correct loading dose, dosage as function of body surface area, and dosage as function of the concentrations of methotrexate obtained). results: the number of infusions started at the correct time increased from % to %. the number of missed blood extractions fell from . to . extractions per course; and missed leucovorin doses dropped from . to per course. the elimination half-life could be calculated in only % of courses in the first study period versus % of courses after protocol implementation. urinary ph changed from not being measured in any cycle to being measured in % of cycles. compliance with rescue dosage protocol was scored with . points before versus . points after implementation. background and objective: to quantitatively evaluate the safety of the current injectables medication process, from prescription to administration, in the paediatric and neonatal intensive care units. to compare the potential impact of safety measures on the risk. to classify these measures from a pharmacoeconomic point of view. design: assessment by a prospective risk analysis according to the failure modes, effects and criticality analysis (fmeca) method [ ] by a multidisciplinary team: one physician, two nurses, three pharmacists. three drugs chosen as models (gentamicin; morphine; dopamine). failure modes (fm) defined during brainstorming and criticality indexes calculated on the basis of their likelihood of occurrence, potential severity for the patients and detectability. impact of ten safety measures on the criticality indexes of the selected three drugs, extrapolation to all drugs injected daily and calculation for each measure of the investment in euros per year to improve the safety by quali (- point of criticality) per day. setting: university hospital, fifteen nicu beds and ten picu beds. main outcome measures: mean criticality indexes; gain in qualies per day; cost-efficacy ratios for each safety measure. results: in the current situation, the sum of mean criticality indexes of thirty-one identified fm was , for the selected three drugs. we gain , qualies ( , by extrapolation to all drugs injected daily) with civas (centralized intravenous additives services), , ( , ) with a clinical pharmacist, ( , ) with double check by nurses, ( , ) with cpoe (computerized physician order entry), ( , ) with in-line filters, ( , ) with vial of dilution, ( , ) with horizontal laminar airflow hood, ( , ) with intermediate dilution, ( , ) with simple additional measures of asepsis and ( ) with a drug planer. the best cost-efficacy ratios were obtained by a clinical pharmacist ( quali = . euros) or by double check by nurses ( quali = . euros) or by civas ( quali = . euros). the highest ratio was obtained with cpoe, due to the very high costs investment ( quali = . euros). conclusions: the use of a prospective risk analysis allowed us to quantitatively evaluate the relationship between the medication process of injectables and the paediatric patient safety and to build a strategy for continuous quality improvement, by selecting the most appropriate evolutions. based on the results of the pharmacoeconomic analysis, development of clinical pharmacy and civas for some drugs will be discussed with the paediatric department background and objective: studies show that up to % of patients starting treatment with antidepressants fill only a single prescription at the pharmacy, apparently not accepting treatment. the aim of this study was to determine characteristics and reasons associated with non-acceptance of ssri treatment. design: retrospective questionnaire study. patients presenting a gp prescription for a newly started ssri treatment to a community pharmacy were selected. 'non-accepters' were defined as those patients filling only a single ssri prescription, and patients who received at least three prescriptions were defined as 'accepters'. setting: community pharmacies in the netherlands. main outcome measures: characteristics evaluated included sociodemographic (e.g. level of education), disease (e.g. reason for use) and treatment (e.g. type of ssri) characteristics. 'non-accepters' were also asked for the reason not filling a second prescription. results: 'non-accepters' and 'accepters' were included in the analysis. 'non-acceptance' was more common among patients with a low level of education (or . ; ci . - . ) and in patients who reported aspecific symptoms like fatigue, stress and restlessness as the reason for ssri use (or . ; ci . - . ). in addition, there was a trend that 'non-acceptance' was more common among patients over years of age (or . ; ci . - . ) . of all 'non-accepters', . % (n = ) did not start ssri use, while . % (n = ) discontinued ssri use within two weeks. fear of side effects and the actual occurrence of side effects are main reasons for not accepting ssri treatment. in addition, a considerable number of 'non-accepters' indicated that they felt an aversion towards medicine use, were feeling better meanwhile or disagreed the gp's diagnosis. of the 'nonaccepters', . % discontinued treatment without informing the gp. conclusions: acceptance of ssri treatment is a decisive moment in compliance to treatment initiated by gps, and deserves more attention. gps and pharmacists should address issues related to the use of ssris especially in groups who are at risk for non-acceptance. keywords: antidepressants, discontinuation, nonadherence edu- pharmaceutical interventions by pharmacists working within surgery and medicine departments julie prince , stephanie diallo , eric grandsire , anne lecoeur , caroline fijalkowski , michelle lebas-certain , franck le mercier pharmacy, ambroise pare hospital ap-hp, boulogne-billancourt, france background and objective: since , our pharmacy department set up a nominative daily drug distribution system without computarization. in each pharmaceutical unit localized in clinical departments, the prescriptions are screened daily by a pharmacist, then the drugs are delivered by a technician. our objective was to compare the frequency and content of pharmaceutical interventions in surgery and medicine departments. pharmacy, haematology, nurses' management, cancer centre henri becquerel, rouen, france background and objective: while several studies have evaluated the frequency and the consequences of medication errors, few have explored their causes. in particular, knowledge of nurses regarding treatment of their patients has been scarcely studied. this survey has been carried out to determine how nurses master medications prescribed to the patients they care for, and how often they access drugs database. design: this work is a prospective study carried out from february to april . we have decided to focus on the clinical audit method, following french health authorities recommendations. a questionnaire has been elaborated and submitted to nurses during semistructured interviews. setting: french cancer centre: nurses from an oncology department and from a haematology department. main outcome measures: data collected were: nurses' profile (age, length of service, competencies' self-assessment), knowledge on drugs prescribed to their patients (usage, administration, side-effects, drug interactions…), use of existing tools (i.e. drugs database) and possible tools to be developed by the pharmacy ward to help them in their daily practice. results: twenty out of twenty six nurses (mean age: , mean length of service: years) consider their medical knowledge as intermediate level. % of pharmaceutical classes are quite well known ( % of the indications are known). only % of drugs' inn are given and more than half of the generic drugs' names are not mastered. administration conditions and conservation are known for respectively % and % of the products. however, side-effects ( %), contraindications ( %) and drug-drug interactions ( %) are not acquired. in their daily routine, nurses face problems mainly related to: drug administration ( %), drug conservation ( %), and dealing with generic drugs and therapeutic equivalence ( %). % of nurses refer to a drug database several times a week when only % more than once a day. pharmacy ward is considered to give information on drugs on a 'regular' basis. three tools have been identified for their potential to help nurses: summarized data on drugs (card format), drugs administration and conservation tables. conclusions: this study has helped to define nurses' difficulties regarding patients' treatment, and their needs for information on drugs. it is also useful for the pharmacy ward to improve its relationships with clinical wards and feedback on treatments. training sessions will shortly be organised to improve the above results. results: the aim of the vita (vienna transdanubia aging) study is the early detection of alzheimer dementia and the discovery of its risk factors. at basic examination, dementia was diagnosed in out of patients ( %) at an age of years. in % of these cases dementia was classified as alzheimer disease. in addition, a clinically relevant depression was diagnosed in % of patients at basic examination, but only % of them were treated accordingly. the first re-examination after . years included those patients, who showed no or only mild signs of cognitive disorders at basic examination. % of these patients developed dementia within the period of . years. the first reexamination also revealed a rapid increase of patients with depression ( % vs. %). the incidence for the development of dementia was % in patients, who have never suffered from depression. however, in patients with the diagnosis depression at basic examination, the risk for dementia was doubled. we aimed to prove whether there is a statistically significant correlation between long-term medication with selected drugs and the development of depression. eleven classes of drugs were investigated, including calcium channel blockers, beta-and alpha-blockers, corticoids, statines, non-steroidal anti-inflammatory drugs, h -blockers, neuroleptic drugs, benzodiazepines, levodopa and opiates. medication was documented from those patients pharm world sci ( ) : - ( male, female) without dementia and depression at basic examination, and without dementia at first re-examination. at first reexamination of them were depressive ( % male, % female), and had no depression (control group). for each class of drug, patients were divided into groups according to gender and duration of medication. a statistically significant (p \ . ) correlation was found between the treatment with benzodiazepines (c months) as well as beta-blockers (c months) and the development of depression in both male and female. conclusions: in elderly long-term therapy with benzodiazepines and beta-blockers can aggravate the development of depression. background and objective: pharmacogenomic studies aim to elucidate the genetic bases for interindividual differences and use such genetic information to predict the efficacy, response rate and safety of a selected drug. to date, the prognostic value of fccr polymorphisms as markers to predict treatment outcome in nhl is still being studied. our goal was to determine whether there is any correlation between fccriia polymorphisms and clinical response to rituximab in patients with nhl. design: in the present study we analysed fccriia polymorphisms in the genomic dna isolated from peripheral blood of patients with nhl who have undergone immunotherapy with rituximab. genotype analysis was based on a polymerase chain reaction (pcr) method followed by a restriction fragment length polymorphism (rflp) study. data were analysed using the computer software spss for windows (version . ) and treatment outcomes of the patients were compared using chi-square or fisher's exact test. a cut-off p-value of . was adopted for all the statistical analysis. survival estimates were calculated using the kaplan-meier method. the curves were examined by the log-rank test. setting: unit of molecular oncology of instituto português de oncologia, porto, portugal. main outcome measures: the response to therapeutics with rituximab was evaluated according to physical examination and computed tomography images. responses were scored according to international working group consensus. overall response rate (orr) was considered as complete response (cr), unconfirmed complete response (cru) and partial response (pr). overall survival (os) duration was defined as the period of time between st treatment with rituximab and either death or the last clinical evaluation of the patient. event-free survival (efs) was defined as the time interval between st treatment with rituximab and the occurrence of an event (recurrence or death) or the time of the last clinical evaluation of the patient. results: the orr for hh genotype was % and for r allele was % (p = . ). however, our results demonstrate that all patients carrying the hh genotype had complete responses to rituximab therapy. complete response rate for hh genotype was % and for r allele was % (p = . ). when comparing the fccriia genotypes, hh genotype or r allele does not have a significant impact on os at -year (p = . ) or on efs at -year (p = . ). conclusions: this study demonstrates that fccriia polymorphism is predictive of complete response to regimens containing rituximab in nhl patients, but is not predictive of overall or event-free survival. based on the current observation, rituximab has in some way an fccriia-dependent mechanism of action which is ameliorated in patients with hh genotype. we hypothesize that hh genotype increases affinity of fccriia receptor not only for naturally occurring igg , via antibody-dependent cellular cytotoxicity but also ameliorate connection with chimeric igg rituximab. keywords: non-hodgkin lymphoma, rituximab, pharmacogenomics pt- platinum salts, cancer and renal insufficiency. sub-group analysis of the irma study xavier pourrat , nicolas janus , stéphane oudard , isabelle ray-coquard , jean-philippe spano , jospeh gligorov , jean-françois morere , philippe beuzeboc , gilbert deray , vincent launay-vacher pharmacy, hôpital trousseau, tours, nephrology, gh pitié-salpêtrière, medical oncology, hôpital européen georges pompidou, paris, medical oncology, centre léon bérard, lyon, medical oncology, gh pitié-salpêtrière, medical oncology, hôpital tenon, paris, medical oncology, hôpital avicenne, bobigny, medical oncology, institut curie, paris, france background and objective: the irma study reported the high prevalence of renal insufficiency (ri) in solid tumour patients: mean age . , mean weight . kg ( . % between and kg), glomerular filtration rate (gfr) \ ml/min for - % [ ] . we present the results for irma patients who received a platinum salt (ps) as part of their chemotherapy. design: data were retrospectively collected for in and outpatients with cancer presenting over two periods in (february st- th and october st- th). setting: anticancer centers in france. main outcome measures: subgroup analysis of irma patients who received ps. data collected: sex, age, weight, serum creatinine (scr), type of tumor and anticancer drugs. the prevalence of scr [ lmol/l was assessed. gfr was estimated with cockcroft-gault (cg) [ ] and amdrd [ ] formulae. chi-square test was used to compare the prevalence of ri between patients who received ps and patients who did not. results: patients were included: mean age . and weight kg, men. the prevalence of scr [ lmol/l was . %. gfr \ ml/min was . % with cg and % with amdrd. the prevalence of ri was significantly higher in patients who received ps as compared to patients who did not receive ps (p = . ). there were prescriptions: . % carboplatin, . % cisplatin and . % oxaliplatin. . % of patients received carboplatin or cisplatin, the two drugs of this class needing dosage adjustment and being nephrotoxic. conclusions: ri is highly frequent in cancer patients receiving ps. appropriate evaluation of renal function necessitates cg or amdrd calculation. in addition, two third of those patients with pre-existing ri are at risk for iatrogenic acute renal failure still receive nephrotoxic ps. consequently, appropriate methods for the nephrotoxicity prevention of those drugs should be used as recommended for cisplatin by the escp special interest group on cancer care [ ] . [ ] prevalence of renal insufficiency in cancer patients and implications for anticancer drugs management: the irma study. cancer (in press). [ ] prediction of creatinine clearance from serum creatinine. nephron, ; : - . [ ] a simplified equation to predict glomerular filtration rate from serum creatinine [abstract] . j am soc nephrol ; : - . pharmacy, hospital la candelaria, tenerife, spain background and objective: to describe an quantify the pharmaceutical interventions in patients on total parenteral nutrition (tpn)and the drug related problems (drp)in patients on this type of nutritional support. to know the acceptance degree of the interventions and its relevance on patients' care and quality of life. design: prospective longitudinal study for four months (from january to april ). all patients on tpn were included. the registered data were: patients number, hospital departments, type of interventions and modifications on the tpn. setting: the pharmaceutical interventions were classified in: indication, effectiveness, safety and adherence according to the cipolle and cols methodology in order to identify de drp related to the tpn and/ or to the drugs. main outcome measures: all interventions were recorded both in the patient medical record and in a excel database in the pharmacy department. results: patients were evaluated and interventions were recorded. that means an average of interventions per patient and a duration average per nutrition of . ± . days. the drp were: indication . %, safety . %, effectiveness % and adherence . %, being the drp the most representative. the drp were listed in: nutritional assessment ( . %), monitoring ( . %) and individualized tpn ( . %). a total of patients ( . %) was favoured through some type of pharmaceutical intervention, being the most implicated hospital departments the neonatology and digestive surgery departments. a % of the interventions were focus on monitoring and optimization of nutritional support and % on drugs (diuretics, insulin, digoxine, enalaprile, and propofol). the acceptance degree of the interventions was %. conclusions: the individual monitoring of the patients with tpn represents an improvement of their clinical outcome and a lower incidence of drp. therefore, with this method we contribute to a lower hospital stay and it also may prevent the appearance of new adverse effects. main outcome measures: the number of interventions carried out and accepted, items concerned: regulation problems, nitrogenous and calorie intake, electrolytic intake, prescription omissions and eventually other fields. results: altogether, parenteral nutrition prescriptions were analyzed for one month in five pediatric units. the pharmaceutical analysis, which consumes hours a day for pharmacists, generated interventions for prescribers: % concerned electrolytic intake, more than half of which concerned potassium and sodium, the main dangerous electrolytes; % were prescriptions omissions; % about nitrogenous and calorie intake; % about other fields (weight error, incompatibility of lipids with divalent ions). of these interventions, which concern exactly prescriptions, were accepted by prescribers, that is %, leading to prescription modifications. conclusions: putting in place a systematic pharmaceutical analysis of parenteral nutrition prescriptions has ensured the detection and the correction of prescription errors. these errors concern mainly non adjustment of electrolytes to the biological results of the child. pharmaceutical interventions are important for safety of the patient and represent a privileged way to communicate with prescribers and their acceptance is, on the whole, satisfactory. background and objective: the number of elderly intensively increases and the fact that they consume a great amount of pom and otc drugs makes them a significant group of patients that need pharmacy care. unfortunately pharmacists often find their interaction with elderly clients very difficult and determined by many factors such as the sensory and physical limitations that accompany the aging process. to test the readiness of the elderly patients to communicate with the pharmacist, to assess the barriers that hinder the proper communication process and to provide a communication skills training in order to be improved the communication process. design: an experimental design involving two stages -assessment and education. setting: setting: the elderly patient center (hospice) and private community pharmacies both situated in the city of sofia, bulgaria. participants: patients aged + ( community pharmacy patients and patients from the elderly center). main outcome measures: an initial interview with the patients and questionnaire with the selected pharmacists to assess the level of communication and to clarify the hinders. communication skills training leaflets provided to the pharmacists. test of the newly received skills. final interview with the patients to be assessed the level of their satisfaction. results: the trained pharmacists that have passed the education process are more facilitated in providing pharmaceutical care that leads to the elderly patients' satisfaction (about %). additionally, the elderly patients obtained significantly more information from their pharmacists that leads to better care and avoidance of nearly half of the drug-related problems (drps) for this age. conclusions: pharmacist communication skills' training appears to be an effective means of enhancing the communication process in the pharmacy. background and objective: as the hospital has no pharmacists working in the multidisciplinary teams on the wards, we wanted to introduce and evaluate a clinical pharmacy service. design: a month prospective pilot study with three aims: . identify drug related problems (drps) (data collecting period of eight months). . design drug related information sheets and teach nurses. . evaluate the service by questionnaire. suggest cost-effective measures. setting: paediatric ward with beds, national university hospital. main outcome measures: the acceptance rate of the drps identified and suggested by the pharmacist, the number of drug information sheets introduced and lectures given to nurses. physicians' and nurses' views on the service. results: the pharmacist identified drps in ( %) of the charts that was screened. immediate action was taken in ( %) of the cases, the physician considered ( %) of the suggestion rational but no immediate action was taken due to various reasons, and ( %) of the suggestions were not approved by the physician. the most commented drp was ''dosage'' ( %), which included too low or too high dose, non-optimal administration time or inappropriate formulation. the pharmacist designed six drug information sheets and gave five lectures. cost-effective measures were suggested for drug handling and specific drugs. seven out of eight physicians and all nurses (n = ) considered the pharmacist a natural participant in the multidisciplinary team. conclusions: quality assurance of drug treatment may be performed by a clinical pharmacist, not only by the traditional way of identifying drps, but also by designing drug information sheets and teaching. the clinical pharmacist is also capable of suggesting cost-effective measures. physicians and nurses considered the clinical pharmacist a natural participant in the multidisciplinary team. as a result of this project, the clinical service will continue and also be introduced to one of the other paediatric wards. keywords: drp, paediatrics, quality assurance pc- iatrogeny and drug dispensations for outpatients: implication of a hospital pharmacy emilie degris , isabelle peyranne , anne laure sarda , nadine malric , brigitte bellon pharmacie, hôpital paule de viguier, chu toulouse, toulouse cedex , france background and objective: in france, some drugs are not available in community and outpatients have to go to hospital to obtain their treatment. our objective was to assess the role of the pharmacist in prevention of iatrogeny when dispensing drugs, in particular medication errors at high risk for the patient. design: a month retrospective study, from december to may setting: pharmacy of paule de viguier, teaching hospital of toulouse, france main outcome measures: each error encountered was recorded and analysed. first, we determined the number of errors avoided and the number of errors effective (divided into groups: non avoided and created by pharmacy). then, we quantified the frequency ( = once, = from twice to ten times, = more than ten times) and the severity ( = no risk, = weak, = moderate, = high) of each error. the multiplication of those two parameters gave us the level of the risk of error for the patient ( = no risk, to = weak risk, and = moderate risk, = high risk). finally, for each type of error we noted the actors. results: we made dispensations during the period of the study. we recorded errors ( . %): ( for dispensations) were avoided by the evaluation of the pharmacist, were not avoided ( for dispensations) and were created ( for dispensations). among the avoided errors, ( . for ) were at high risk ( ), ( for ) at moderate risk ( or ), ( . for ) at weak risk ( or ). the actor of of them was the prescriber (mainly lack of information on the prescription like no dosage). among the effective errors, ( . for ) were at moderate risk ( or ), ( . for ) at weak risk ( to ), ( . for ) had no risk ( ). the actor of of them was the pharmacy. conclusions: the errors for the activity ''retrocession'' are not numerous. the majority of them are stopped by the evaluation of the pharmacist, in particular those at high risk for the patient. we implemented curative and preventive measures to decrease the number of errors made both by prescribers and pharmacy. background and objective: despite improved treatments and guidelines, asthma control remains suboptimal. in a recent observational study, we described the asthma control test Ò (act) as an easy tool to measure asthma control of patients presenting at community pharmacies ( ) . the present randomised controlled trial was set up to study the hypothesis that a pharmacist intervention, focused on optimal use of asthma medication and tailor-made to the patient's current asthma control, would result in improved asthma control in adult patients. design: a -month randomised controlled trial in asthma patients: patients in the control group (c) and patients in the intervention group (i). patients in the control group received usual care. patients in the intervention group received a protocol defined pharmacist intervention, mainly focusing on inhaler technique and adherence to controller medication. setting: randomly selected community pharmacies in flanders (the dutch speaking part of belgium). main outcome measures: primary outcome was the level of asthma control, as measured by the asthma control test Ò . secondary outcomes included rescue medication use, night-time awakenings due to asthma, patients' peak expiratory flow, inhalation technique, adherence to controller medication, quality of life, knowledge on asthma and smoking behaviour. results: mean act scores did not change from baseline for both study groups (act at baseline for c: . , i: . -act at months for c: . , i: . ). however, a predefined subgroup analysis of patients having insufficiently controlled asthma at baseline showed that the intervention significantly increased act scores during the course of the study compared with usual care (p = . ). the intervention also significantly reduced reliever medication use (p = . ) and the frequency of night-time awakenings due to asthma (p = . ). inhalation technique (p = . ) and adherence to controller medication (p = . ) were significantly better in the intervention group. these findings suggest that the more effective use of asthma medication is responsible for the improvements in symptom control. there were no differences between control and intervention group in peak expiratory flow, quality of life, knowledge on asthma and smoking behaviour. conclusions: a pharmacist intervention can significantly improve outcomes for asthma patients (clinicaltrials.gov number nct ). background and objective: patients admitted to surgery departments receive multiple drugs before, during and after surgical procedures. drug-related problems (drp) are the most common cause of injury to hospitalized patients. in pharmacotherapeutic follow-up (ptf) a pharmacist is responsible for drug-related patient needs by detecting, preventing and solving drug-related problems (drp) aiming at specific results to improve patient quality of life. drp are pharmacotherapy negative outcomes leading to failed therapeutic goals or undesirable events. when a drp appears, it affects not only older hip fracture patient health, but also the effectiveness of hospital health care. the general objective of this study was to demonstrate that ptf improves the hip fracture assistential process quality, comparing some quality indicators of this process between patients in study group (sg) and control group (cg). design: cuasi-experimental study with control group. ptf was the intervention. setting: two traumatology wards in a large teaching hospital, ''hospital san cecilio'', granada, spain. the period of study was from january to july (sg) and the same period but in (cg). main outcome measures: incidence and types of drp; drp solved in sg; differences in lengh of stay, six-months mortality and threemonths readmissions between study and control groups. results: the incidence of drp was % in sg (n = ) and . % in cg (n = ). in sg, more than % of drp were resolved. in sg and cg the % and % of drp were related to medication need, % and % to effectiveness, and % and % to safety, respectively. mean length of stay was days in sg and . in cg. in general, patients with drp had a significative longer length of stay ( . d) than those without drp ( d); but in sg, patients in which drp were solved had the same length of stay than those without drp. sixmonths mortality was . % in sg and . % in cg, and readmissions was . % and . % respectively. . % (n = ) of patients reported an aspirin allergy that triggers their asthma attacks. . % (n = ) had never used aspirin before and did not know whether they had any sensitivity to aspirin. . % (n = ) of subjects could correctly describe and distinguish between preventor and releiver drugs. . % (n = ) confused the terms, while . % (n = ) had no idea about these terms. % (n = ) of asthmatics had received previous inhaler usage education from a specialist (doctor, nurse or pharmacist). of these patients had ineffective inhalator usage although they had ostensibly received education. the inhaler technique of the remaining who had been previously educated was accepted as successful. % (n = ) of the patients had never received inhaler usage education before a specialist. of these patients demonstrated successful technique but of failed. conclusions: the results of this pilot study indicate that some asthmatics are ignorant of their condition. in addition most of them seem to have no comprehension of the concepts of preventor or reliever therapy. despite prior education about half (n = ) were unable to demonstrate successful technique. furthermore cigarette smoking may be a detremental factor to the lives of asthmatic patients. this results of this study suggest the potential benefit of an innovative pharmacist led patient education service among asthmatic patients in turkey. background and objective: according to a recent meta-analysis, drug-related morbidity leads to . % of preventable hospital admissions causing enormous expenditures. in austria, there are only data on the incidence of adverse drug reactions (adrs) of psychiatric drugs. clinical pharmacy is not widely practised at hospital ward level. with this study, we aim to evaluate and document adrs leading to or occurring during hospital admissions. to improve the co-operation of doctors and pharmacists in an austrian hospital, to enhance doctors sensitivity in detecting drug-related morbidity, to increase patient safety and lower costs by reducing hospital admissions. design: two study nurses especially instructed about typical symptoms of adrs identify and document these cases prospectively in cooperation with doctors on selected internal wards for a period of three months. these cases are evaluated by a clinical pharmacist by means of a computer tool and data-base specialised on detecting causality and severity of adrs. results and outcomes form the basis for structured feedback to doctors. setting: university teaching hospital. main outcome measures: quantity and quality of adrs connected with hospital admission. results: during the first six weeks, patients were screened. sixty three adrs ( female) were identified ( . % of admissions). more than % of adrs occurred in patients more than years old. reasons: polypharmacy (mean number of drugs on admission . ) and reduced renal function (mean creatinine clearance . ml/min). diuretics, oral anticoagulants, nsaids, digoxin and antibiotics were most frequently associated with drug-related problems. water-electrolyte imbalance, overantigoagulation with or without bleeding, gastrointestinal problems and bradycardia are some of the most common problems. results concerning the severity of adrs will be available in september . conclusions: adrs are frequent in austria. incidences are comparable to numbers given in the literature. mainly older patients are affected. the impact on clinical practice is yet unknown. background and objective: adherence to cardiovascular treatment, particularly in the first year, is low and can result in serious complications. depression is associated with a fold increased risk of nonadherence with medical treatment. therefore, our aim was to investigate whether illness and treatment perceptions were associated to depressive symptoms in patients starting treatment for cardiovascular diseases. design: cross-sectional study with mailed questionnaire. setting: patients, who were dispensed at least a first prescription for a cardiovascular disease (anti-thrombotics excluded), were selected from pharmacies in the netherlands. main outcome measures: the questionnaire comprised the illness perception questionnaire-brief (ipq-b), beliefs about medicines questionnaire (bmq), the medication adherence report scale (mars) and the centre for epidemiological studies depression scale (ces-d). descriptive statistics and associations between depressive symptoms and the other study variables were assessed by bivariate correlations. results: sixty two ( . %) of eligible patients returned our questionnaire. the mean age was . yr ± . (range - ) and . % was female. patients reported to have hypertension ( . %), cardiac arrhythmia ( . %) and hypercholestereamia ( . %). the mean score on ces-d was . ± . and median self-reported adherence (mars) was . reports of depressive symptoms increased with emotional response (ipq-b emotional response, r = . ), the perceived consequences (ipq-b consequences, r = . ) and increased experience of symptoms (ipq-b identity, r = . ) attributed to their cardiovascular disease. depressive symptoms correlated with concerns about medication (bmq, r = . ), but not with self-reported adherence. adherence was relatively high, as . % of the sample had the maximum mars score of . conclusions: in patients who started cardiovascular treatment, perceptions about cardiovascular disease and concerns about medication are associated with report of depressive symptoms. depressive symptoms did not correlate with self-reported adherence. the majority of patients reported excellent medication taking behaviour, which might reflect their awareness of the importance of adherence or reluctance to report deviant behaviour rather than their actual behaviour. further research is needed to clarify this finding. results: fifteen nurses completed the questionnaire. % of the nurses were aware of the purpose of controlled release formulations. pharmaceutical codes added to brand names such as uno, zok, la and ocas related to prolonged activity were not recognised in % of cases. in contrast, retard and cr were linked to slow release by % of the responders. the purpose of enteric coated (ec) drugs was only known by %. in general, the nursing staff did not pay a lot of attention towards the prevention of drug-nutrient and/or drug-tube interactions. the recommended time interval between administration of enteral feeding and drugs was not respected. % of the responders would crush drugs together (in the same mortar) when multiple drugs are prescribed. based on the results of the survey, an intervention plan has been developed. this consisted of information rounds, a poster related to the topic and implementation of the use of a website dedicated to crushing medication developed by the flemish association of hospital pharmacists. background and objective: the detection and solution of drugrelated problems is an important activity within pharmaceutical care. this study focused on drug-related problems (drps) detected during dispensing of new prescriptions in community pharmacies and aimed to explore frequency as well as nature and the pharmacist's management of them. design: during their pharmacy internships fifth-year pharmacy students collected consecutively hospital discharge and primary care prescriptions. after training, they documented drps and interventions on an adapted pcne classification form. inclusion criteria were: age over , at least one new medication, at least prescribed drugs. setting: swiss community pharmacies affording the opportunity of internships for fifth year pharmacy students. main outcome measures: prevalence, nature and management of drps in community pharmacies assessed with an adapted pcne classification form. results: the patient's median age was years (iqr ) and they received a median of (iqr ; range - ) different drugs. prescriptions of patients ( ( . %) discharged from hospital) were analysed. in ( . %) of all prescriptions at least one drp was detected. the most frequent drps were potential interactions ( . %), wrong/improper application or time of drug intake ( . %), inappropriate drug ( . %) or inappropriate drug form for indication ( . %), no clear indication for drug use ( . %) and too high or too low dosage ( . %). these drps led to a total of interventions (multiple answers): patient counselling ( ); request of information from prescriber ( ); change of drug ( ; there from after consultation with physician), drug form ( ), dosage ( ), instruction for application ( ) or deliverable drug amount ( ); drug stopped ( ); start with new drug ( ); referral to a physician ( ); others ( ). out of all interventions . % could be managed by the pharmacist without any contact to the prescriber. there were no differences between hospital discharge and primary care prescriptions. conclusions: in the delivery process of new prescribed drugs drps are frequently observed prompting many interventions. most drps can be managed by the pharmacy. further studies are needed to analyse relevance of the problems and impact of according interventions. the main selection criteria were clinical relevancy (patient centred initiatives) reproducibility of clinical and economical outcome, outcome indicators and multidisciplinary approach. an approval by the hospital board and medical council must underline the willingness to integrate the clinical pharmacy in the patient care team. results: projects has submitted (on a total of hospitals). a total number of hospitals were selected to receive funding for clinical pharmacy activities. projects were quoted for a full time equivalent and projects for a half time clinical pharmacist. the projects described different fields or a combination of different aspects of pharmaceutical care like e.g. the transfer of information on medication use on admission and discharge conclusions: the funding of the belgian health authorities triggered a very high response rate, which proves the increasing attitude from the belgian hospitals to the positive impact of clinical pharmacy. the funding was complementary to other national projects to improve overall safety of medical treatment. also, many hospital administrators took the opportunity to enhance more economical and rational use of drugs. financial support by the belgian authorities of clinical pharmacy and the results of the projects could trigger a further integration of the hospital pharmacies into a patient care team. background and objective: the task of assisting patients in selfmedication practice is an important component of pharmaceutical care in spain. in order to provide appropriate self-medication counselling pharmacist should be able to distinguish between a minor ailment and one that it is not, and should, consequently, refer patients as necessary to gps. nevertheless, there are no criteria for referral to gp in spain. the objectives were:( )to identify the most relevant minor ailments, agreeing on the specific criteria for referral to the gp.( )to select the non-prescription drugs, with evidence of safety and effectiveness, for the treatment of the identified minor ailments design: qualitative study with an expert panel which was made up of primary care physician from semfyc and six community pharmacists (two members of sefac and four members of giaf-ugr). the expert panel held two meetings, of five hours each. it was established which minor ailments were considered most relevant within the framework of community pharmacy in spain. subsequently, the expert panel, reach an agreement on the general content that should be included in the protocols for the management of each selected minor ailment. finally, a working team composed of gps and three community pharmacists prepared the protocols, which were compiled into a guide for self-medication counselling. setting: university of granada, spain during . main outcome measures: identified minor ailments, content of the protocols for each minor ailment, non prescription drugs selected. results: it was selected minor ailments, allocated as follows; respiratory (rhinitis, cough, cold, flu), pain (period pains, sore throat, headache, backache, toothache), gastrointestinal (heartburn, diarrhea, constipation, vomiting, hemorrhoids), skin and mucous membrane (aphthae, acne vulgaris, cutaneous wounds, burns, stings, urticaria, herpes labialis, eczema lesions) and others (vaginitis, varicose veins, fever, conjunctivitis, insomnia). the following sections were specified in each protocol: banal and serious reasons or conditions that can lead to the symptom (including drugs); referral criteria according to the duration of the symptom and associated signs; drug treatment and non-pharmacologic therapies. it was selected a total of different non prescription drugs. conclusions: a total of minor ailments were identified as the most frequently demanded in community pharmacies in spain. referral criteria were based mainly in the duration of the symptom and other associated symptoms that are indicative of illness. for the treatment of these minor ailments, different non prescription drugs were selected. keywords: non-prescription drugs, minor ailment, community pharmacy services mareike kunkel , matthias ganso , irene kraemer pharmacy, johannes-gutenberg-university hospital, mainz, germany background and objective: in clinical pharmacy service was implemented in three surgical clinics (inclusive icu). drug related problems (drp) were identified by medication review and discussed with the physicians. from january to june all pharmaceutical interventions (pi) from pharmacists ( fte) were recorded (paper based) and classified according to drp (with the pi-doc Ò -system, which was modified to comply the requirements for hospital use ), intervention type, outcome and clinical relevance. the pis were documented and evaluated with an access Ò database. design: retrospective study of pis in surgical patients, identification of drp by medication review. setting: departments of neurosurgery, accident surgery and general/abdominal surgery ( , and beds, respectively), university hospital. main outcome measures: drp, intervention type, outcome, clinical relevance, drugs and admission diagnoses being at risk for drps. results: within six month patients were admitted. drps were identified in % (n = ) of the patients. patients with drps were older (mean = y sd ± vs. y sd ± ) and had an increased length of stay (mean = d sd ± vs. d sd ± ). pis were made. the acceptance by the physicians was . %. pis were classified to the outcome subcategory patient safety and clinical relevance was estimated as major (n = ) or moderate (n = ) by pharmacists. further data are based on these pis. the most often addressed drps categories were overdose ( %), no or insufficient drug monitoring, when necessary ( %), untreated indication ( %) and increased risk of an adverse drug reaction ( %). the type of recommended intervention varied: change dose/time of application ( %), stop drug ( %) and conduct drug monitoring ( %). drps related with the outcome patient safety and at least moderate clinical relevance were caused by drugs ( %). the most affected drugs were vancomycin ( %), diclofenac ( %), potassium ( %), acetaminophen ( %), digitoxin ( %), phenytoin ( %) and theophylline ( %). the incidence of the most frequently admission diagnoses of patients with relevant drp differed from the incidence of diagnoses of all admitted patients (incidence icd- (cost-)effectiveness has started to emerge ( ) . a literature review was carried out that a) summarized the findings of pharmaco-economic studies; b) evaluated the methodology employed by studies; and c) suggested how future research has to be designed to meet the requirements of a pharmaco-economic analysis. design: studies to be included are identified by searching electronic databases. due to limited relevance of older studies, the scope is limited to studies published between and . mainly three techniques can be used to conduct an economic evaluation: costeffectiveness analysis, cost-utility analysis and cost-benefit analysis. ( ) all studies are reviewed regarding results and methodological quality. nineteen out of studies met our eligible criteria. setting: clinical pharmacy services provided in a hospital setting. main outcome measures: results were analyzed in terms of number of preventable adverse drug events (ade), length of stay (los) and financial savings. methodological quality was assessed with respect to perspective, scope and measurement of costs and consequences, sources of data on costs and consequences, and application of an incremental analysis. results: a) nearly all studies conclude a financial benefit based on direct cost saving and estimated cost avoidance as a measure of prevented ade or shortened los. b) methodologically there are a number of shortcomings: e.g. not including the wage of the personnel, lack of control groups, use of expert panels to estimate savings and costs, possible selection bias, no valorization of health effects. c) a methodology for conducting a prospective economic evaluation of an observational study is proposed. conclusions: it is not obvious to calculate the net savings of a clinical pharmacy program or to compare different programs because there are no common guidelines for this type of assessment. the ideal protocol is hard to achieve, so best practice will be more realistic. addition of direct cost saving, labor cost and economic value of prevented ade and shorter los results in a lucrative service. these savings are higher for specific inverventions (like preventing ade, switch therapy) or disciplines (e.g. intensive care unit versus geriatrics background and objective: the contribution of pharmacists to the delivery of public health in scotland is recognised in national pol-icy , . the new community pharmacy contract with its emphasis on public health will provide a new framework in which the contribution of community pharmacy to improving health in scotland can be delivered. the objective was to define the core public health competencies applicable to community pharmacy practise, using the 'skills for health public health practice competency framework' . design: a web based delphi methodology was used to achieve consensus on which competencies, from the 'skills for health public health practice competency framework', should be met or aspired to by practising community pharmacists using a multidisciplinary group of expert stakeholders. two rounds took place. setting: primary and secondary healthcare and academia. main outcome measures: panel members rated their extent of agreement/disagreement that each community pharmacist should achieve or be striving to achieve that particular competency. consensus was defined as c % rating a competency as strongly agree/ agree. results: ten organisations ( % of those invited) and organisation members ( % of those invited) agreed to participate. responses were received from ( %) individuals in round and ( %) in round . consensus was achieved for / ( %) competencies in round and a further / ( %) in round . competencies achieving consensus predominantly focused on health improvement activities at individual and local community levels and ethical management of self, rather than those relating to surveillance and assessment, strategic leadership or research and development. conclusions: this research has identified that many of the competencies in the 'skills for health' document can be applied to community pharmacy. research has since been carried out, using focus group and questionnaire methodology, to investigate the views of practising community pharmacists. background and objective: in spain, off-label drug utilization (nonapproved indications, patient population, doses, administration route, association), must be derived to compassionate use, which requires a prior national health authorities (nha) approval and a monitoring plan and follow up information provided to them. request to nha includes circumstances of case and patient protection measures, including: physician assessment, informed consent and institutional clearance. the objective of this study is to analyse the strength of recommendation, strength of evidence and clinical efficacy of drugs prescribed outside the terms of product licence (off-label) in paediatric patients of our hospital. design: literature review to evaluate the evidence level: micromedex healthcare series, cochrane library, pub-med, embase, expert opinion or consensus. sample: % off-label drugs used in at least paediatric patients (prior spanish nha treatment approval required for every patient). years, retrospective observational study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . setting: paediatric hospital ( beds) and pharmacy department ( pharmacist) in a large general teaching hospital ( beds, pharmacists). main outcome measures: categorisation of evidence-based medicine according to thomson ratings of recommendation (class i-iii), evidence (category a-c) and efficacy (class i-iii). results: paediatric patients ( % total patients: adults and paediatrics) used off-label drugs ( % total drugs: adults and paediatrics). out of this off-label drugs, % ( / ) only approved for adults, % ( / ) outside of license in terms of indication for adults and paediatric patients and % ( / ) both causes. off-label drugs for indications were used in at least paediatric patients: % anti-infective agents, % haematopoietic growth factors, % cytokines, % hormone therapy, % antiarrhythmics, % other therapeutic groups. the categorisation according to evidence-based medicine was: a) strength of recommendation class: % i, % iia; % iib, % iii and % indeterminant; b) strength of evidence category: % a, % b, % c and % no evidence; c) clinical efficacy class: % i, % iia, % iib and % indeterminant. conclusions: a higher proportion of off-label prescriptions is observed among paediatric patients, most of them related to nonapproved indications in this population. there is a broad range of therapeutic groups involved. the evidence of most off-label therapies are based on meta-analyses of randomized controlled trials with conflicting conclusions, small numbers of patients or significant methodological flaws or nonrandomized studies and, although the weight of evidence favors efficacy of the treatment for a specific indication, the therapy may be useful and indicated in some, but not most, cases. keywords: medicine-based evidence, off-label, paediatrics background and objective: there is an increasing demand towards the involvement of the community pharmacists in health promotion. it has been reported that community pharmacists have a successful role in providing services, which help to improve and promote health with regard to smoking cessation, coronary heart disease, skin cancer prevention, drug misuse, sexual health, immunization, mental health, diabetes, nutrition and physical activity [ ] . the aims of this study were to describe the current practice of community pharmacists with regard to their provision of health promotion activities, identify their willingness to participate in health promotion and identify the barriers that may limit their participation. design: a descriptive cross sectional study, which included community pharmacies that selected via stratified and systematic random sampling. data were collected via face-to-face structured interview of the respondents using a pre-tested questionnaire. setting: community pharmacies in khartoum state. main outcome measures: the extent of the pharmacists' involvement in counselling patients about health promotion topics, their preparation to counsel patients in health promotion topics, and their success in changing the patients' health behaviour. results: the response rate was . %. seventy five ( . %) of the study participants were strongly involved in counselling patients on health promotion related to medications, but less involved in counseling them on the other personal health behaviours such as tobacco use ( . %), alcohol use ( . %) and exercise habits ( . %). seventy two ( . %) of the respondents perceived themselves as very prepared to counsel patients on taking drugs and less very prepared to counsel them on other personal health behaviours. fifty two ( . %) claimed a high level of success in helping patients to change their behaviour with regard to medications, but not in relation to other personal health behaviours. ninety eight percent of respondents indicated their willingness to participate in continuing education programs to gain more knowledge and skills about health education and promotion. the main barriers facing the community pharmacists' participation in health promotion as perceived by respondents were lack of information and/or training ( . %) and lack of pharmacists' time ( . %). conclusions: community pharmacists reported to achieve considerable success in helping patients to change their behaviours in relation to medications, but were less successful of their ability to change personal health behaviours. the majority of the respondents have the interest and willingness to be a prime source of advice and support on health promotion. results: during the first study period, inpatients were exposed to major or moderate pddis. ( %) of these pddis were judged clinically relevant by the pharmacist. recommendations including pddi information, and simply information leaflets were handed out to the physicians. % ( of ) of the recommendations were accepted. at hospital discharge, in % ( of reviewed instances, which were accepted) the drug changes due to the recommendations were implemented. during the second study period, patients at hospital discharge were exposed to major and moderate pddis. ( %) pddis were assessed as clinically relevant by the pharmacist. recommendations including pddi information, and simply information leaflets were sent to the physicians. % ( of ) of the recommendations were accepted. one year after hospital discharge, of drug changes due to recommendations were still existent. overall, in % and %, respectively, of all major and moderate pddis detected by pharmavista, clinical management was adapted accordingly. conclusions: the management of clinically relevant pddis can be improved by physicians' advice of clinical pharmacists. changes in medication due to pddis were found to persist up to one year after hospital discharge. background and objective: current treatment options for metastatic renal cell carcinoma (mrcc) are limited and there is a need to identify novel and effective therapies. sunitinib is an oral multitargeted tyrosine kinasa inhibitor, which has shown activity in cytokinerefractory metastatic rcc patients. this agent inhibits vascular endothelial grown factor receptor and platelet derived growth factor receptor. the purpose of this study is to analyse the efficacy and safety profile of this agent in patients with mrcc. design: retrospective assessment in seven patients treated with sunitinib as second-line treatment in mrcc. data were obtained from clinical histories and informatic records from the oncology pharmacy department. setting: oncology and pharmacy department. la paz university hospital. madrid. spain. main outcome measures: assessment of clinical response and adverse events. results: seven patients were evaluated ( men, women), median age was ( - ). six of them presented bone, lung, brain or liver metastases, all patients were treated with vinblastine and ifn-alpha as first-line therapy. patients received sunitinib at a starting dose of mg per day in repeated -week cycles for consecutive weeks followed by weeks off treatment. they started therapy with sunitinib because of progressive disease in patients and adverse events in patients on previous therapy. sunitinib was discontinued in four of them, causes were: adverse events ( patient), volunteered dropout ( patient) and progressive disease ( patients). the median progression-free survival was . months. the median number cycles received was six and of the patients are still in treatment at the time of data analysis. dosage was reduced mg daily because of unacceptable toxicity: hand-foot syndrome( patient) and hypothyroidism ( patient). the most common adverse events experienced were: asthenia ( patients), diarrhea ( patients), damaged nails( patients), insomnia ( patients), dermatitis ( patients)and dehydration ( patients). conclusions: in our experience, sunitinib has demonstrated an acceptable efficacy and safety profile as a single agent in second-line therapy for patients with mrcc. (ii) validated questionnaire to guide discussion; (iii) fostering group interaction to generate data; (iv) post-interview analysis of verbatim transcripts with specialized software (qsr nvivo . for windows Ò ), based on the grounded theory approach (classification of emerging themes). setting: groups: prescribing physicians ( ), nurses ( ) , and laboratory technicians ( ) , all involved in antibiotic tdm as performed in orthopaedic surgery, general surgery, neurosurgery, vascular surgery, haematology, and pulmonary wards in a beds teaching hospital. main outcome measures: (i) issues causing poor antibiotic overall tdm performance, (ii) approaches for optimizing tdm performance supported by group consensus results: key identified issues: (i) nursing work overload; (ii) insufficient education to pharmacokinetics and lack of specific training; (iii) insufficient information communication and lack of coordination and involvement of all stakeholders; (iv) conflicting guidelines; (v) lack of perception of positive benefit/risk ratio. approaches for optimization (consensus): (i) continuous education of all stakeholders; (ii) daily multidisciplinary collaboration with infectious disease physicians and clinical pharmacists; (iii) simplification and uniformization of guidelines and procedures; (iv) implementation of a simpler administration scheme (v) increased staffing. conclusions: correct performance of tdm and its implementation in routine clinical care needs to be critically assessed and appears to be mainly dependent on non laboratory-related parameters. background and objective: new data published at the end of and the beginings of the , suggest not to exceed a haemoglobin level of g/dl to avoid cardiovascular morbility-mortality in patients with anaemia and chronic kidney disease (ckd) treated with recombinant human erythropoietin (rhuepo). before these evidences the optimal target haemoglobin levels was greater than g/dl. our aim is to evalue if these new published evidences have changed the clinical practice in pre-dialysis ckd patients. design: retrospective observational study. all the pre-dialysis patients who received rhuepo were including. in order to evaluate the possible changes in clinical practice, we measured the levels of haemoglobin prior to the publication of evidences (march-may of : group ) and after the publication of these evidences (march-may of : group ). we made a descriptive analysis of independent data. setting: department of hospital pharmacy. main outcome measures: the main outcome measures were: age, sex, mean glomerular filtration rate (gfr), mean haemoglobin level, mean haematocrit and type of rhuepo used. results: we studied patients ( in group and in group ). patients age ranging between to years (median = years). the proportion of women was . %. mean gfr for both years located around ml/min and the most frequent stages of renal injury were and . the most rhuepo used was darbepoetin alfa ( . % of patients). mean haemoglobin level for group was . g/dl (sd = . ) and . g/dl (sd = . ) for group . mean haematocrit was . % (sd = . ) and . % (sd = . ) for group and , respectively. conclusions: our nephrologist are cautious about of prescribing rhuepo, not only after the publication of the new scientific evidences on this subject, but before this too. it s worth questioning if clinical practice in ours hospital is different from the published evidences. background and objective: tacrolimus (tac)-based immunosuppression is effective in adult renal transplant patients with acute or chronic rejection or cyclosporin (cya)-related toxicity. the conversion from cya to tac resulted in improved cardiovascular risk profile and increased prevalence of post-transplant diabetes mellitus (ptdm)compared with treatment with cya. the aim of this study was to review clinical documents for renal transplant patients and assess patients' outcomes. design: a retrospective review of clinical data. excluded from the study were patients converted to tac less than months posttransplantation. statistical analysis (one sample paired -tailed t test) was performed using microsoft office excel . the graft survival was analysed with kaplan-maier survival curve using xl stat software. the study was approved by the northern ethics committee, auckland, new zealand. setting: tertiary care setting. main outcome measures: mean serum creatinine, incidence of ptdm, mean total cholesterol, hdl cholesterol, ldl cholesterol, total/hdl cholesterol ratio, mean blood pressure and antihypertensive scores, graft survival censored for death. results: forty-four patients were converted to tac more than months post-transplantation from to june . mean serum creatinine (scr) increased in the months prior to conversion from lmol/l ( % ci - ) to lmol/l ( % ci - ) at months post conversion to tac (p-value = . ). thirty-four patients were taking cya for more than months. the mean scr increased from lmol/l ( %ci - ) at the months prior to conversion to lmol/l ( %ci - ) at months post conversion (p-value . ). if scr for seven patients who had an acute rejection episode were excluded, the mean scr did not show any change in slope after conversion and showed a tendency to gradually increase from lmol/l ( %ci - ) to lmol/l ( %ci - ) months post conversion (p-value . ) eleven out of patients were affected by diabetes mellitus. six patients were diabetic pre transplantation and remained diabetic post transplantation and post conversion to tac. two patients developed new onset ptdm post transplantation and two became glucose intolerant. after conversion to tac, glucose intolerance resolved in one patient and one patient ( %) developed new onset ptdm. in patients converted to tac more than months post transplantation, mean total cholesterol was reduced from . to . mmol/l (p-value . ) and mean ldl cholesterol from . to . mmol/l (p-value . ). in june , / patients ( . %) were taking tac with a mean scr of +- lmol/l. four patients ( %) lost their grafts. mean graft survival time was . months. -year graft survival was . %. conclusions: conversion from cya to tac was beneficial with respect to renal function and cardiovascular risk profile. the conversion had no added benefit on renal function in patients with stable renal function taking cya more than months post transplantation. the reported incidence of ptdm was found to be low ( %). background and objective: most of the cancer patients suffer from severe pain especially during the terminal phase of the disease. it is essential to monitor these patients to achieve adequate and successive pain management, not just because of the importance of the effects, side effects and overdose problems; but also to improve quality of life. the aim of the study was to evaluate oncology pharmacist interventions on pain management. design: numeric pain scales was conducted prospectively among the cancer patient who were over years old and were selected randomly. patients were separated into two groups: of the patients was control group, pharmacist had been effectively included to the rest patient's pain management strategies, which was pharmacist intervention group. all of the patients had been evaluated by numeric pain scales (time ). the patients who were on pharmacist intervention group were monitored by pharmacist on treatment effectiveness and side effect profile every three days during the study. after one month, numeric scales were repeated (time ). the interventions that pharmacist done were pain evaluation, suggestion on appropriate pain reliever, dose management, patient education and patient monitoring. our therapy recommendations were made on the basis of the world health organization's analgesic ladder following the results of assessments. setting: oncology outpatient unit of a university hospital main outcome measures: the demographic and diagnostic information of the patients were collected. the results of the evaluations via numeric pain scales were calculated by using arithmetic mean value. results: the mean of pain intensity was significantly decreased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). the mean of pain's effect on daily activity was significantly decreased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). the mean of drug effectiveness was significantly increased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). conclusions: the harmonious working of the pharmacist with the other health care staff working in oncology unit, helped patients to achieve more effective pain management. in this study; pain intensity was decreased, pain interfered less with daily activities was, and the reported effectiveness of drugs was increased in the pharmacist intervention group compared to the control group. all these outcomes show that the clinical pharmacists have an important role in oncology services, especially pain management. background and objective: to investigate and compare the frequency and nature of prescrbing errors requiring contact with the prescriber at community pharmacies in norway, estonia and sweden. design: a protocol, based on a scheme originally presented by rupp ( ), revised and developed by kennedy ( ) and translated and transformed to the nordic context by haavik ( ), was used in all three settings. in norway the protocol was self-completed by the pharmacists; in sweden and estonia observers (trained students) recorded and classified the interventions. setting: norway - community pharmacies in southern and western norway; estonia - community pharmacies in three cities; sweden - community pharmacies in swedish cities and public pharmacies at hospitals in sweden. main outcome measures: prescriptions with errors or ambiguities where the pharmacist decided to contact the prescriber to correct, clarify or complete the information on the prescription. results: the total numbers of dispensed prescriptions were: norway , , estonia , , sweden , (community pharmacies) and , (public pharmacies at hospitals). the proportion of handwritten prescriptions and prescriptions where pharmacists contacted the prescriber was higher in estonia than the other countries. administrative problems -reimbursement issues; prescriber data and distribution and licensing issues -were the reason for more than one third ( - %) of all contacts with the prescribers in all settings. however, the patterns of prescription problems with potential clinical hazards varied -in estonia and norway, errors concerning strength, administration form and number of doses were the most common errors and constituted and % of the problems. in sweden, errors concerning the prescribed dosage were the most common reasons. conclusions: the proportion of problem prescriptions requiring a clarifying contact with the prescriber was higher in estonia compared to norway and sweden. the main reason may be that most prescriptions in estonia were handwritten. administrative problems (reimbursement and availability of prescribed products) constituted a similar large portion in the three countries. however, prescription problems with potential clinical consequences for the patients, varied. , presenting with salmonella bacteremia and neurological deterioration due to cerebral toxoplasmosis was admitted to the intensive care unit. he was immediately intubated. to treat toxoplasmosis, cotrimoxazole was started in a dose of mg smx/ mg tmp qd. days later the patient developed leucopenia (absolute wbc count: . /l, neutrophils: . /l). folinic acid mg od was associated to restore white blood cell count. neutrophils further dropped to attain its nadir ( . /l) on day of cotrimoxazole therapy. cotrimoxazole was stopped and clindamycin mg td was used instead. neutrophil count restored, normalizing on day after stopping cotrimoxazole. this event was attributed to the administration of cotrimoxazole. the time relation between the administration of cotrimoxazole and the onset of neutropenia as well as the normalisation of neutrophils was clear. other explanations, such as the contribution of concomitant medication (ranitidin, ceftriaxon, ethambutol, isoniazid, rifampicin, aciclovir, amphotericin b, enoxaparin)could be ruled out. the naranjo score, which estimates the probability of adverse drug reactions, is . the use of folinic acid as rescue therapy in association with cotrimoxazole is controversial, as it can theoretically antagonise the anti-infective action of cotrimoxazole. therapeutic failure in aids patients, receiving this combination for pneumocystis jiroveci pneumonia, has been reported. nevertheless, we decided to start folinic acid to further prevent nosocomial infections in this severe immunocompromised host. we don't know whether folinic acid contributed to quick recovery of neutrophil count in our patient. further studies are necessary to clarify its role as rescue agent during treatment with folic acid antagonists. conclusions: this case report illustrates that cotrimoxazole, frequently used in opportunistic infections, can be associated with agranulocytosis. this dangerous complication in immunocompromised patients with severe infections must be prevented, although the effectiveness of folinic acid as rescue therapy is still a matter of debate. background and objective: intravenous immunoglobulin (ivig) therapy is increasingly used in inflammatory and autoimmune disorders, because of its therapeutic benefit and its good safety profile. cutaneous adverse events are rare and include prurit, rash, alopecia and eczema. in the literature, about cases of eczematous skin reactions have been reported. most of the cases were treated for a neurological or neuromuscular disease. erythematous eruptions on hands and feet have been notably reported after high-dose infusion. in most of the cases, the eruption was progressively extending to involve the entire body. when ivig were readministered, eruptions were more rapid and more intensive. we report an eczematous skin reaction of the palms after ivig infusion without extensive eruption, in spite of three administrations. design: case report. setting: neurology ward, university hospital, grenoble, france. main outcome measures: a -year-old man was treated with ivig (tegeline Ò , lfb, france) for an inclusion body myositis. he developed a skin reaction, days after the end of a days ivig infusion (dose of . g/kg was given daily for consecutive days). the eruption was a non-pruriginous erythematous maculopapulovesicular rash located on the palms. this reaction occurred th day after completing the second therapy and did not extend. the lesions regressed progressively with topical application of fatty ointment. three days after the third infusion, the same lesions reappeared, and regressed the same way. results: clinical pharmacist with the help of pharmacovigilance experts and doctor worked collaboratively: because of the chronology of exposure to other treatments, intrinsic imputability and recurrence on reintroduction, we ruled out an adverse drug reaction to any other medication. we decided not to interrupt the infusion. we advised the patient to continue fatty ointment application and to tell the healthcare team if the reaction became more serious. conclusions: dermatologic adverse reactions such as eczematous skin reactions are rare and usually mild. there is no reason to limit the use of ivig in a case like this one, as long as the treatment is effective. however, a narrow clinical and biological follow-up is required. if necessary, this adverse effect can be prevented by antihistamines or even steroids. anticoagulants must be monitored closely by physicians, because this products have a narrow therapeutic index. numerous interactions with herbs are documented, either increasing or decreasing the anticoagulant effect. our main objective is to identify this interactions in our surgery and if they are clinically significant. design: six months observational study; interviewing patients with their inr alterated about herbs that they were taking at that moment. literature review. setting: the anticoagulant oral treatment surgery. main outcome measures: the two oral anticoagulant drugs available in spain are acenocoumarol and warfarin. the international normalized ratio (inr) is the laboratory test used to measure therapeutic efficacy and safety of vitamin-k antagonists. a control test is done every four weeks and if necessary it can be done earlier. results: among patients with inr [ , six of them were taking herbs at the same time, and we could relate the increase of the effect of oral anticoagulants to those products. one of this patients who was taking dandelion (tarxacum officinale) had a inr. an other one who was taking chamomille (matricaria capensis) and passion flower (passiflora incarnata) had a . inr. all of these products have coumarins compounds. two patients who were taking equinacea (equinacea purpurea, equinacea angustifolia) also had their inr test altered: one had a . inr and the other one . . an other one was taking bilberry (vaccinium myrtillus) and had a . inr. both, equinacea and bilberry inhibit different isoenzimes of cytochrome p . the last patient was taking garlic oil (allium sativum) and had a inr. garlic increases the anticoagulant effect. conclusions: it is commonly believed that herbal products are inofensive, that is the reason why mainly of the patients do not take medical advise before starting a treatment with them. however, there can appear interactions with the usual treatment. if we fix on the vitamin-k antagonists the risk resides on the hemorragic or strokes events. in conclusion, we believe that patients should be educated about the potential risk of using herbal products while being treated with vitamin-k antagonists. a year-old man, treated with clopidogrel after coronary stenting, is hospitalized for aa (neutrophils: g/l ( . - g/l); haemoglobin: . g/dl ( - g/dl); platelets: g/l ( - g/l)). his permanent medications were insulin, perindopril, omeprazole, atorvastatine, bisoprolol, and acetylsalicylic acid. clopidogrel ( mg/d) was prescribed weeks before aa occurence. clopidogrel is withdrawn and aa therapy is started, consisting in the sequential association of anti-thymocyte globulin therapy ( mg/kg) and ciclosporin ( mg/ kg/d) in a filtered-air room. but the severe co-morbidities lead to early stop ciclosporin, then relayed by androgen therapy (norethandrolone). finally, at weeks from the diagnosis, the evolution ends to a resolution of aa, but with platelet-transfusion dependance. after the elimination of the other aetiologies, iatrogenic cause is envisaged. to blame clopidogrel is difficult with regard to the other drugs, especially perindopril and omeprazole known to induce bone marrow failures. four arguments lead to target clopidogrel: (i) the length of treatment by perindopril and omeprazole without complications, (ii) the timing between the onset of aa and the addition of clopidogrel to treatment, (iii) the resolution of aa whereas neither perindopril nor omeprazole were withdrawn, and (iv) the support of the literature. conclusions: clopidogrel seems to be responsible of this side effect. we unfortunately lack in specific biological tests to prove it. keywords: clopidogrel, side-effects, pharmacovigilance background and objective: informing patients on their medicines is a patient right. what does current information provision on antidepressants to patients with a depression admitted to a psychiatric hospital look like? what is the current practice of health care professionals? what are the experiences of patients? this study aims to explore current practice on drug information provision in psychiatric hospitals. design: a qualitative study consisting of semi-structured interviews with separate interview guides for health care professionals and for patients. interviews were tape recorded, verbatim transcribed and analyzed using nvivo software. setting: eight flemish psychiatric hospitals. main outcome measures: identification and evaluation of current approaches to drug information provision on antidepressants from the point of view of health care professionals as well as patients. results: patients get information on antidepressants, firstly, through psychiatrists and, secondly, through nurses. hospital pharmacists have a supporting role. the approach in giving information depends on patient characteristics and his/her mental state. information is provided mainly orally. leaflets are not frequently distributed to patients. patients also get information on antidepressants during psycho-educational sessions. on request, patients can read a package insert under supervision of a health care professional. health care professionals consider non-verbal cues of patients to verify if information has been understood. information is repeated when the first instruction was not clear for patients. there are no systematic interdisciplinary contacts on information interventions. patients as well as health care professionals are satisfied with current practice on information provision. health care professionals reported lack of time and lack of interdisciplinary contacts as negative aspects. patients indicated that health care professionals take too little initiative to give information about medicines. positive aspects reported by health care professionals are the hospitals' openness and the opportunity for patients to ask their questions to psychiatrists as well as nurses. suggestions for improving practice are: providing more medication information to patients, in particular on side-effects; enhancing the availability of easy readable information; and organizing continuing education for nurses on medicines. patients are informed about their antidepressants through various pathways. however, there seems to be room for improvement as a number of suggestions were formulated to support these pathways of drug information. keywords: medication information, antidepressant, psychiatry claire chapuis , christine chevallier , céline villier , pierrick bedouch , benoît allenet , jean calop , gérard besson pharmacy, pharmacovigilance, neurology, university hospital, grenoble, france background and objective: the use of intravenous immunoglobulin (ivig) is expanding. the risk for adverse effects can be minimized by taking some precautions. there are yet no standardized practice guidelines for prevention and management of adverse effects occurring during the infusion, and there is a need for it. the purpose was to conduct a study of their knowledge among the nurse community and to make a review of the best practices. design: we search in medline and carried out a questionnaire for nurses. we evaluated knowledge and practices of nurses in neurology, pneumology and haematology units, experienced in intravenous immunoglobulins administration, in order to define their role and the prescribers role in ensuring patients safety during therapy. we used all information and synthesized it in a table. for every type of adverse reaction, we indicated mechanism, frequency, seriousness, risk factors, practical guidelines of management and prevention and actor. the guidelines were reviewed by experts (pharmacist in charge of human derivative products, pharmacovigilance experts and neurologists). setting: university hospital, grenoble, france. main outcome measures: formalisation of practical guidelines on prevention and management of intravenous immunoglobulin adverse effects, for prescribers and nurses in the local hospital network and possibly other hospitals. results: nurses always checked the doses before administration ( / ), always prepared the product aseptically ( / ), warmed up the product until it reached room temperature ( / ), but only few recorded the patients tolerability during the infusion ( / ), and very few knew that most adverse events could be minimized first by slowing down the rate of infusion ( / ). all nurses called a doctor as an adverse effect appeared. conclusions: nurses must be involved in the management of adverse effects, even if the prescriber remains the one who makes the prevention by evaluating risk factors, co-medications, dosing and frequency of treatment and the one who makes the decision to interrupt the treatment if an adverse effect occurs. the clinical pharmacist in care units works collaboratively with both prescriber and nurse. he plays a central role for preventing drugs' adverse effects while counselling every member of the healthcare team background and objective: fludrocortisone tablets - lg (f) is mainly used in the treatment of adrenocortical insufficiency. it may also be used in treatment of orthostatic hypotension. f is manufactured by ageps (public special-order manufacturer) and dispensed to outpatients by hospital pharmacies, as a ''hospital preparation''. in order to follow gmp guidelines, a patient information leaflet for f was elaborated in our pharmacy outpatient unit. the leaflet was approved by our regulation unit. to evaluate the usefulness of this leaflet and to improve its quality, we performed a patient satisfaction survey. design: during days, for every dispensation of f, a leaflet was presented to the patient and an anonymous satisfaction survey was performed. setting: pharmacy outpatient unit, agence générale des equipements et produits de santé (ageps) (ap-hp), paris, france. main outcome measures: the questionnaire consisted of three items: general information about patient and its treatment; patient's knowledge of f before reading the leaflet (uses, precautions, adverse events, storage); patient satisfaction of leaflet (general presentation, language simplicity, information volume, utility). results: patients answered the questionnaire. the mean age was years ( - years). mean f treatment duration was years ( months- years). % of patients were already informed about f: % by physicians and % by associations and internet. % knew the indication ( % adrenocortical insufficiency and % orthostatic hypotension). % knew about precautions, % knew about side effects, and % knew about storage conditions. % of patients did not read the leaflet and had no opinion about satisfaction items. % were satisfied of general presentation. language was understandable for %, and non understandable for %. information volume was sufficient for %, insufficient for %, and too large for %. leaflet was useful for % of pts. patients who found the information insufficient suggested the following items: results of clinical trials, management of acute situations due to disease or f, and contacts of qualified centres in case of serious events. conclusions: nearly half of the patients were informed about f by their physicians, but information is communicated orally without written support. the majority of patients treated by f knew about precautions, side effects, and storage conditions. however, patients were satisfied of our information leaflet and find it useful. leaflet appears to be a good tool to communicate information from the pharmacist to the patient when not available in the packaging. of these recommendations taken into account ( %), within a period of days for most (n = ), involved the intervention of a pharmacy student, were given only via the computer software, and via a telephone call. conclusions: computerisation of prescriptions is an indispensable tool in order to make the pharmaceutical distribution circuit safer. however, its use as a vehicle for pharmaceutical interventions is limited, as shown by this study. it is impossible to analyse a nonresponse of our recommendations: is our advice even red, is it considered as inadequate? a discussion with the clinician (via pharmacy students or on the telephone) allowing a constructive exchange of knowledge, leads to a better transmission of recommendations. background and objective: in hospital setting, employees may be exposed to hazardous drugs. risks and protective measures needed when handling parenteral cytotoxic drugs are well described, whereas information related to drugs like monoclonal antibodies or antivirals are lacking. we developed a standardized method to evaluate drugs potential toxicity and occupational risks taking into account the pharmaceutical forms of the drugs to balance the risks. design: development of an algorithm for toxicity evaluation using material safety data sheet (risk and safety phrases), international agency for cancer research (iarc) classification and official manufacturers' data. . evaluation of chronic toxicity (mutagenicity, carcinogenicity), acute toxicity (sensitisation or irritation in contact with skin, eyes or by inhalation) and toxicity to reproduction . balancing of toxicity according to the pharmaceutical forms . assessment of protective measures (centralization of drug preparation in the pharmacy, wearing of mask, gloves and/or glasses) centralization of drug preparation in the pharmacy is recommended only in case of documented mutagenicity and carcinogenicity and when there is a risk of respiratory or cutaneous exposure related to the pharmaceutical form. setting: university hospital ( beds). main outcome measures: • chronic (r , r , r or iarc group , a or b), acute toxicity (r - , - , - ; s - , - ) and toxicity to reproduction (r - ; cat.d,x) • pharmaceutical forms associated with a risk of respiratory (e.g. tablets crushing), cutaneous (e.g. drug in solution) or ocular contact (e.g. inhalation) results: occupational risks of parenteral monoclonal antibodies, oral and parenteral antivirals, oral cytotoxics and other drugs forms were analysed. according to our algorithm, crushing of % of the tablets forms should be done in the pharmacy (e.g. valganciclovir). only parenteral antiviral should be reconstituted at the pharmacy (ganciclovir). monoclonal antibodies were found not to be at risks of mutagenicity or carcinogenicity and only gloves will be recommended for their manipulation. no ''class-effect'' has been pointed out (e.g. only a few antivirals were found to be hazardous). products were at risks for pregnant women. protective measures to be taken by pregnant nurses or those wishing to have a baby will be discussed institutionally. conclusions: toxicity evaluation of hazardous drugs handling in hospital should take the pharmaceutical forms into account as some toxic drugs may not be associated with occupational risks (e.g. coated tablets). our method allows a standardized way to evaluate whether a drug should be treated as hazardous or not. results will be discussed institutionally in order to implement applicable policies and procedures. results: lidocaine % injectable solution is indicated for tracheobronchial anesthesia, via bolus of mg in adults, and to mg in children. the maximal dose is restricted to mg/kg in adults and - mg/kg in children. epinephrine is indicated in hemorrhagic complications occurring during interventions; the solution is diluted to mg/ ml with nacl . %; a lg bolus is injected and can be repeated up to mg. terlipressine can be used in heavy bleedings; the powder should be reconstituted with nacl . %, at mg/ ml; mg is injected, repeated if necessary. mesna is used to dissolve mucous plugs, especially in patients with cystic fibrosis. the solution is diluted to mg/ml with nacl . %; bolus of ml are injected to allow visibility in the lower airways. there are no data available on the maximal dose. all preparations have to be compounded aseptically and extemporaneously by the nurse when requested by the physician, because of the lack of stability studies in those ranges of concentrations. conclusions: these guidelines are intended to standardize flexible bronchoscopy procedures between the different units, in order to minimize the occurrence of medication errors. almost half of the enquiries ( . %) were about stability and incompatibility in intravenous (iv) admixtures. the frequency of these calls was significantly higher than the calls came from other health professionals (p \ . ). the following types of enquiries were about dosage ( . %) and availability of pharmaceutical products ( . %). while . % of enquiries were answered by hizbim in less than minutes, in % of all calls took more than day to gather and tailor which requires more literature search. the mostly ( . %) used references in retrieving information was general references followed by micromedex ccis inc. with . %. • hizbim has been consulted by nurses in a rising pattern. this means that this service is of importance for them because of its rapidity, accuracy and currency • the preparing iv admixtures that has been left to nurses' responsibilities in practice in our country can be reconsidered by pharmacists since this service has been fulfilled by ''drug experts'', namely pharmacists in many countries. • drug information centers can provide not only emergent information for nurses but also education such as seminars and conferences in some topics they need. the establishment of drug information centers in hospital settings will be of use to stimulate the frequency of consultations by nurses and to improve patient care provided by them. background and objective: the concept of pharmaceutical care (pc) is capturing the attention of a growing number of pharmacists. the strategy followed by us to spread out the implementation of pc in pharmacies involves the utilization of internet tool as an innovation of traditional education. design: to do so, we designed an on line pc course with interactive cases. the specific goals of the course are: • the enhancement of the pharmacy practice. • the promotion of practice research. • the personal and professional development of pharmacists. the objective was to analyse the evolution of the program, whose pedagogical design was based in a process of teaching-learning in the field of the clinical pharmacy and pharmaceutical care that permit the training of the future pharmacists in the use of medicines. design: all the classes were designed with practical-theoretical modality including workshops, reading and analysis of the bibliography, discussion of cases and utilization of virtual simulators. setting: clinical pharmacy. department of biological sciences main outcome measures: students evaluation was carried out by tests of the general contents, degree of active participation during the workshops, resolution exposition and discussion of the cases. results: the development of clinical pharmacy education began in . the students appreciated this experience and the % of them are satisfied with the process of teaching-learning employed. this teaching system improved them the adequate and rational use of the medicines. in these two years the % of the students has approved the examination. the students also realized two poster presentations during this period, namely: . ''comparative study of the information supplies in the leaflet of tablets of omeprazol mg'', in and . ''study of the information supplied by the pharmaceutical laboratories in the television media'' in . conclusions: we consider the importance of the incorporation of clinical pharmacy in the new pharmacy curricula, because the students have major task about the practical education involvement patients and related to their professional future. background and objective: clinical trials' managing and dispensation is one of the obligatory missions of a hospital pharmacy. the respect of good clinical practices is necessary to ensure proper trial performance and to be aware of errors in prescription and in drugs dispensation. the aim of our study was to analyse the nonconformities in the steps of prescription and dispensation and to evaluate their severity. design: the pharmacy department manages clinical trials for all clinical wards with a pharmacist and a technician who change every four months. there is no specific computerized system but an excel software for data filing. we realized a retrospective study of ongoing clinical trials. all the prescriptions were reviewed and we focussed on all the items required for the prescription (e.g., identification of the protocol, the patient, the investigator, the dosage) and for the dispensation (e.g., identification of the drug, the quantity dispensed, batch number, expiry date) setting: results: gps participated to the survey. they agreed (median = , iqr = - ) that the following concepts are crucial in a patientcentred approach: the need of a specific training in counselling in under and post graduate education; the necessity of working with patients to develop mutually agreed-upon goals; the role of information in the decision making process, the ability to understand patients readiness to make change, and to identify barriers to change, the importance to recognize that patients are the experts when it comes to their own behaviour related issues. for only one item (time dedicated to the consultation) iqr changed (iqr = - ) indicating some difficulty in implementing this aspect in practice. conclusions: for benevento community gps, a patient-centred approach is a useful way to help change and promote behaviour. knowing how to support it is an important skill for all care professionals, but education is needed to shift from theory to practice. type ii and gestationnal diabetes. the average age was years and for the half of these patients, the pathology developed since more than years. patients were painful and patients presented a positive score to dn questionnaire. symptoms frequently observed were: pins and needles, prickles, and numbness. among these patients, were treated by antidepressants (amytriptiline) or antiepileptics (clonazepam, gabapentine, tegretol, carbamazepine or pregabaline) or both. the average number of molecules received by patients was . . the average number of lines of treatment was: . . first intention treatment insisted of an anti-epileptic monotherapy in patients. second line treatment involved the introduction of another anti-epileptic or an anti-depressant drug. the drugs have been well tolerated except a respiratory depression under clonazepam. among the not-treated patients, only one benefitted from a treatment initiation. conclusions: patients are treated by ''old'' molecules with a large prescription of clonazepam but their efficacy is very variable. the physicians have been sensibilised to dn questionnaire and they concluded that it is easy to use. however there is a lack of physician's informations about the management of neuropathic pain; besides they are hesitant to initiate a treatment. the relationship between pharmacists and physicians and the development of clinical pharmacy seems important to optimize the management of this pain. background and objective: pharmacy education in the faculty of pharmacy and biochemistry of the university of buenos aires is taught as a product-oriented profession with a focus on the basic sciences. however in pharmaceutical care and clinical pharmacy was integrated as an optional course into the pharmacy curriculum by resolution cd / . the object of the emphasis was on the students' ability to provide clinical pharmacy and pharmaceutical care upon graduation. hence, therapeutic plays a significant role in building students' knowledge and skills in preparation for clinical practice. design: theoretical education and program for students and collaborative implication in the hospital activity or in the community pharmacy. the development of the program is carried out in two phases. in the first phase the clinical activities of the pharmacists, the unidose drug distribution, the role of the drug information centers, pharmacoepidemiology and surveillance studies are explained. in the second phase the concept of pharmaceutical care is introduced and its implementation in different pathologies is developed. an active approach of the patient and contact with the treating physician was considered as tool in a strong learning environment. setting: undergraduate pharmacy students at university of buenos aires. main outcome measures: the students find them abilities to identify drug related problems and to assess patient care and follow-up. results: more than students have attended pharmaceutical care and clinical pharmacy to: % passed and % failed. students option was that the strongest aspect are the case discussion and the weakest the very few number of hours not enough to discuss other important illnesses. they also say that many topics should be taught sooner in the career and it was not considered an emphasis an the clinical and patient oriented-aspects of the profession. conclusions: an approach to clinical pharmacy education in which the integration of teaching and learning are collaborative creates an atmosphere that is conducive to effective student learning. moreover the clinical pharmacy is a valued and important tool of the general practice team regarding quality improvement in drug therapy. all of the most popular cpd linked pharmacy activities were from the competency-'participation as a member of the multidisciplinary team'. telephone interview findings showed pharmacists cited 'priority for their service development' as the principle reason for cpd identification. no pharmacist identified a cpd need for: 'supporting patient/family motivation'; 'ecording problems in blood glucose control requiring balancing food intake and insulin dose'; 'sharing reflections of where your performance leaves room for improvement within a pharmacists group' and 'taking part in a local multi-disciplinary mentoring group.' conclusions: currently cpd workbooks appear not to be widely used within the pharmacy profession in the uk although there are examples of successful use of reflective portfolio. , almost half of the participants chose the workbook as a means of support leading to a substantial number of identified cpd issues. for community pharmacists to deliver high quality care for diabetes, more attention is required to forms of training and to both uniprofessional and multiprofessional peer support. setting: we retrospectively reviewed all prescriptions of ivig issued from three infectious disease departments ( beds) in a french bed university hospital. main outcome measures: for each ivig prescription, the following data were collected: patient identification, name of ivig product, quantity of ivig issued, date of ivig release, indication for treatment and level of relevance. these levels are determined as follows. level : indications approved by health authorities or for which comprehensive guidelines have been published (high level of proof). level : relevant indications based on scientific publications. level : off-label indications more difficult to prove on a scientific basis (few or no high quality randomized controlled clinical trials). results: during the studied period, grams of ivig were administered for a total of patients and prescriptions. . % ivig used saccharose as a stabilizing agent, other products contain either glucose ( . %), or maltose ( . %). the purpose of this study is to produce an evaluation focus on the risk profile and counselling activities concerning therapeutic and lifestyle change. design: descriptive study. individual measurements were recorded by pre graduation students on a standardized datasheet. blood pressure was measured using a digital wrist device. blood glucose monitoring systems of two different brands were used. setting: the screening was made in the street using passer-by volunteers. in the course of a single day in may , a total of subjects ( . % males and . % females, mean age . years, sd = . ) had glycaemia and blood pressure measured. main outcome measures: age, gender, medication taken, cigarette smoking, body mass index (bmi), blood pressure, capillary blood glucose results: . % of the sample presented bmi [ kg/m. . % had elevated systolic blood pressure values and . % elevated diastolic blood pressure values. . % had elevated occasional blood glucose. . % are cigarette smokers. systolic (t = - . ; p = . ) and diastolic (t = - . ; p = . ) blood pressure values were significantly higher in smokers than in non-smokers. concerning patients taking diabetes medication, fewer patients with blood glucose controled and more patients blood glucose uncontrolled were found than those expected, suggesting either low compliance or lack of efficiency of medication (v = . ; p \ . ). concerning the hypertension medication, similar results were found. more patients under therapy with blood pressure uncontrolled were found than expected. the concordance within the two measures of the blood glucose with different monitorizing system was found to be strong and significant (k = . ; p \ . ). positive and significant correlation between bmi and diastolic blood pressure was found. however, no significant correlation between bmi and systolic blood pressure was found. conclusions: events such as this screening improves the quality of education, as well as develops the interests and opinions of students. as well as it shows face to face were can be apply their knowledge of clinical pharmacy furthermore, events such as these are found by the students to be invaluable in acquiring training in similar-to-professional setting and expertise in field work. background and objective: nhs education for scotland (nes) has worked with the scottish executive health department (sehd) to develop training packages to support the use of validated needs assessment tools (nat) for several longterm conditions. consequently a specific training package was then developed to support and standardise needs assessment and pharmaceutical care delivery by palliative care pharmacists in the local networks set up in scotland. additional support materials (trainers package) was developed for representatives from the scottish palliative care pharmacists' association to deliver the training to their local network pharmacists in a consistent manner. design: development and evaluation of a training package and course for palliative care pharmacists. evaluation of the outcomes from using a nat for delivery of care. identification of issues %; relevance of questions %; time to complete nat - mins; barrier -time; benefits -care issues identified by nat; care issues -counselling and compliance issues, side-effects identified (nausea, dry mouth, constipation), pain relief not adequate. conclusions: nes are proactively supporting national policy and practice through a process of identifying and meeting the educational needs through direct and self-directed learning for continuing professional development (cpd). the needs of palliative care patients are seen as an appropriate target group for pharmaceutical care. the evaluation and feedback from the courses, training pack and outcomes from the use of the nat in practice have been very positive and amendments will be made for further implementation in scotland. background and objective: pharmacy students represent a broad spectrum of learning preferences and styles. this diversity presents a responsibility for the lecturers and instructors to meet the educational needs of all students. in order to develop appropriate learning approaches the instructors need to know the students learning preferences. therefore, the aim of this study was to identify the learning preferences of pharmacy students. design: the visual, auditory, reading/writing, kinesthetic (vark) questionnaire identifies student's preferences for particular modes of information presentation. the vark questionnaire is freeware that can be completed online. however, we administered the vark questionnaire as a hard copy at the end of the 'clinical pharmacy practices' final exam to the fourth-grade pharmacy students. setting: marmara university -faculty of pharmacy. main outcome measures: the frequency of students' preference for modes of information presentation. results: we administered the vark questionnaire to students and ( %) returned the completed questionnaire. almost half of the students ( . %) preferred a single mode of information presentation. among these students, % preferred visual (learning from graphs, charts, and flow diagrams), . % preferred auditory (learning from speech), and . % preferred printed words (learning from reading and writing), and % preferred using all their senses (kinesthetics: learning from touch, hearing, smell, taste, and sight). the other half ( . %) preferred multiple modes [ modes ( . %), modes ( . %), or modes ( . %)] of information presentation. a total of ( . %) students preferred 'kinesthetic' learning solely or in a multimodal combination. conclusions: the students represented a variety of learning styles. student motivation and performance improves when instruction is adapted to student learning preferences and styles; so, it is the responsibility of the instructor to address this diversity of learning styles and develop optimum learning approaches. escitalopram, fluoxetine and mirtazapine were rarely prescribed. the posology and taking plan were generally respected. however, some improvements in terms of treatment optimization could have been brought. they could have lead to actions of clinical pharmacy within the framework of prospective study: posology optimizations and taking plan optimizations. nineteen patients ( %) had an adjustment in their antidepressant treatment: nature and dosage. for all the antidepressants, a sufficient duration was respected before increasing the doses. an average number of interactions by prescription were . but none was clinically significant. conclusions: therapeutic strategies corresponded to guidelines recommendations. the dosage adjustments and duration before increasing the doses respected the indication of antidepressants. in the future, to optimize the medicinal treatment, decision-making tools carried out could facilitate psychiatrists' prescriptions. the pharmaceutical validation of prescriptions will be facilitated by complying with them within the framework of clinical pharmacy activity. background and objective: al-amal hospital is a bed oncology/ hematology hospital. al-amal hospital is now the first hospital in qatar to be accredited by the joint commission international (jci), the worldwide leader in improving the quality of healthcare. the objective is to implement clinical pharmacy services in al-amal hospital in the state of qatar by training the pharmacists about clinical pharmacy services. design: a pharmacist designed the training program and took the initiative and responsibility for training other pharmacists in aah about clinical pharmacy. the department of medical oncology/ hematology, the hospital administration and the pharmacy department agreed that the pharmacists should have central responsibility for antineoplastic agents and other drugs related problems. pharmacists for the program were selected from the existing staff. the healthcare team is consisting of two pharmacists rotating every months. each pharmacist join teams consisting of a pharmacists, a consultant, a specialist, a resident, a rotating resident, nurses, a dietitian, physiotherapist, social worker and a psychologist. we used to have oncology teams and hematology team. both pharmacists participate in the medical rounds and morning report days per week. the pharmacists provide clinical pharmacy services including chart review, pharmacy patient profile review, laboratory tests, therapeutic drug monitoring, antibiotics monitoring, interviews with patients and/or relatives. drug related problems were identified, resulting in interventions. setting: in patient wards, al-amal hospital, qatar. main outcome measures: to identify drug related problems, which well result in interventions and to help the medical team and the patients to reach their treatment goals. patient outcomes were evaluated by follow up with the medical team or by patient interview. we refer patients to the dietitian, physicians, the clinical psychologist as needed. results: more time is needed to evaluate the clinical pharmacy services provided by the pharmacists as the program was just started. patient and physicians were satisfied by starting the training program. conclusions: hematology/oncology setting provides an excellent opportunity to involve pharmacists. a. leads to encephalopathy and progressive neurodegeneration in the infant who is not treated. early diagnosis and dietary management can prevent complications and may allow for normal intellectual development. however, neurologic function may deteriorate rapidly at any age because of acute metabolic decompensation. these severe episodes are caused by catabolism of endogenous protein, which may be provoked by physiological stress (infections, post-surgery). during these crisis, the patient must have immediately intravenous glucose infusion and enteral nutrition free of b.c.a.a. design: case report. setting: department of pharmacy, hospital charles nicolle, rouen. main outcome measures: case report results: one patient with classical m.s.u.d. is followed in our establishment since many years. his disease has been diagnosed in neonatal period. a diet free of b.c.a.a. has been instaured. this diet is successful, now this patient is years old and had a normal development except myalgia and hypoesthesy of the left leg. however, when the diet is not well followed or when he's infected, acute episode occurs (on average or times per years). as the crises starts, the patient is sleepy and confused. in order to be able to treat him very quickly, the medical staff decided to set up an emergency protocol, which include an adapted enteral nutrition formulation. the pharmacy is implicated in this protocol to prepare the mixture. the formula includes: m.s.u.d mix, dextrin maltose, oligoelements, ions, lipids (sunflower oil), vitamins and water. the pharmacy must be able to carried out the preparation at any time and the components must be always available. conclusions: because m.s.u.d is an unherited disease, published report of treatment are rare and they are no consensus for the treatment of acute decompensation. since years, this protocol is successful: b.c.a.a. levels decrease between to days after the setting-up and the patient always recovered rapidly. this formula is administrated by nasogastric tube and avoid the use of hemodialysis which is the last solution to remove b.c.a.a. this is an example that a personnal follow-up program (with plan for clinical and metabolic evaluations) during common intercurrent illnesses can have optimal outcomes. keywords: leucinosis, metabolic decompensation, adapted enteral nutrition nutr- ensuring phosphorus adequacy of human-milk-fed preterm babies canadell laura , cañete carmen , pardo rocio , albujar mar , valldeperez cinta , carretero juan , gallart m jesus , closa ricardo pharmacy service, neonatology, hospital universitari joan xxiii, tarragona, spain background and objective: human milk is the feed of choice for preterm infants both for nutritional and non-nutritional reasons. phosphorus levels in human milk are insufficient for most premature infants. this deficit is the major cause of osteopenia in prematurity. fortification with a commercial multinutrient product should only be considered after weeks of mother's milk feeding, however, phosphorus supplement must be given initially. to describe a standardized scheme for early nutritional support with phosphorus of very preterm infants (\ - g)and describe the phosphorous oral solution we use as a supplement is the aim of this study. design: clinically relevant reports were reviewed to establish a standardized scheme for early nutritional support with phosphorus of the very preterm infants. a standardized formula of oral phosphorous was established to diminish the medical errors when the addition of this mineral is required. setting: pharmacy service and neonatology unit of a third level hospital. main outcome measures: to describe the scheme of adding phosphorous to human milk as well as the standardized formula we use, ''phosphorous oral solution'' ( mg p/ ml). results: phosphorous oral solution procedure: composition, stability and the scheme of addition to human milk to ensure the requirements for bone substrate needs in preterm infants to avoid osteopenia of prematurity. conclusions: various methods have been tested to decide when additional supplements must be given. individual adjustment is not possible due to the delay of laboratory results on milk analysis and the fast changes in infants' requirements. therefore, it is necessary to make a standard adjustment scheme on the dose of the fortifier that needs to be added. shortly to the pancreaticduodenectomy, total parenteral nutrition (tpn) was started ( kcal in progress until reaching his energy requirements). in addition to parenteral nutrition, supplementation enteral nutrition was delivered via jejunostomy along four postoperative days. on post day , transition to a complete enteral formula was achieved (standard formula, ml = kcal: kcal/d). on day , patient complained of colic pain in upper hemiabdomen. an emergency tc revealed presence of liquid in the abdominal cavity from anastomosis pancreatogastric. with the suspect of a leak from jejunostomy, the catheter was removed. tpn was reintroduced and kept as the only way of nutrition until later when oral tolerance was started. during hospital stay ( days) periodic blood controls were performed. main methabolic complication was high blood sugar, needing the administration of insuline. from day to , mean plasma levels of albumin ( . vs . g/dl), total proteins ( . vs . g/dl), total serum cholesterol ( vs mg/dl), total lymphocite count ( . vs . %) and prealbumin ( . vs . mg/dl) increased significantly conclusions: the leak of artificial nutrition to the abdominal space in patients with jejunal feeding is a frequent complication of ne. its incidence is probably related to the length of the tube inserted into the lumen. protocols are need to prevent complications like tube displacement and to encourage early enteral nutrition. increase in plasma concentrations of nutritional parameters suggests effective uptake admixtures might be either prescribed and made ''a la carte'' according to the newborn's needs or provided by pharmaceutical companies as standard formulations. the aim of the study is to review individual pn prescriptions in a neonatology care unit in order to assess the potential for using standardized pn instead. design: prospective study one day per week during weeks. setting: neonatal intensive care unit, strasbourg university hospital. main outcome measures: the major criteria for the comparison are carbohydrate concentration and then amino acid intake. results: prescriptions were analysed and compared with a standardized formulation, pediaven Ò (fresenius kabi). the first point of comparison based on carbohydrate concentration resulted in an exclusion of % ( / ) of the total prescriptions because their carbohydrate concentration was less than g/ ml or more than g/ ml (pediaven Ò glucose concentration, g/ ml). among the prescriptions retained, only prescriptions whose amino acid concentration was less than . g/ ml were included (pediaven Ò : . g/ ml background and objective: it is known that propofol protect myocardial tissue against global myocardial ischemic-reperfusion injury in the isolated rat heart model. the aim of this study was to investigate whether propofol, at a clinically relevant concentration infused during both preischemia and reperfusion (peri-ischemic) period, also provide protective effect against regional myocardial ischemic-reperfusion injury in vivo. design: mail sd rats weighing between and g were anesthetized with mg/kg of ketamine and mg/kg of xylazine. a haparinized g catheter was placed in the left femoral vein. the trachea was intubated and then mechanically ventilated with room air using a volume-controlled rodent ventilator. a left thoracotomy was performed, and the pericardium was opened. for the ischemia-reperfusion experiments, a snare was passed around a left anterior coronary artery territory to induce regional myocardial ischemia. coronary occlusion was produced by pulling the snare and clamping it with a mosquito hemostat. reperfusion was produced by releasing the clamp. setting: rats were subjected to minutes of coronary artery occlusion followed by hours of reperfusion. propofol or intralipid was administrated during minutes starting minutes before the onset of ischemia until minutes after the onset of reperfusion. main outcome measures: the micro-manometer catheter was advanced into the left ventricle via right internal carotid artery and hemodynamic function was checked after hours of reperfusion. infarct size was determined by triphenyltetrazolium staining after hours of reperfusion. results: propofol administration during both preischemia and reperfusion (peri-ischemic) period showed protective effects on myocardial function and infarct reduction. in the control group, the peak rate of ventricular pressure rise (+dp/dtmax) and the peak rate of intraventricular pressure decline (-dp/dtmin) significantly decreased than sham group. in the propofol group, the +dp/dtmax and -dp/dtmin significantly improved than conrol group. infarct size was . % of the area at risk in control group, and was reduced markedly by administration of propofol during peri-ischemic period to . % in the propofol group (p \ . ). infarct size of intralipid group was . % of the area at risk, intralipid had no effect on infarct size compared with the control group. conclusions: propofol, at a clinically relevant concentration infused during peri-ischemic period, provided protective effect after regional myocardial ischemic-reperfusion injury at in vivo rat heart model. the results showed hemoglobin level of less than g/dl in . % of the subjects, transferrin saturation (tsat) of less than % in . % of the hd patients, tsat \ % and ferritin \ ng/ml in . % of the patients, serum alb level of less than g/dl in % of the patients, serum p level of more than . mg/dl in . % of the subjects, ca p product of more than in % of the patients, parathyroid hormone (pth) \ pg/ml (adynamic bone disease) in . % of the subjects and serum pth concentration of more than pg/ml (uncontrolled secondary hyperparathyroidism) in . % of the subjects. the results showed that more than half of the hd patients need erythropoietin and ferrous dose adjustment or follow up for resistant anemia, more than half of the subjects need phosphate binders dose adjustment or replacement and about % of he patients need rocaltrol dose adjustment. we are planning to compare these results with the findings following the participation of a clinical pharmacist in this hd center rounds and monitoring of hd patients. since enough management of complications of crd patients and their drugs monitoring are necessary to improve quality of life of hd patients, clinical pharmacist may have a major role in hd centers. background and objective: computerization of our drug circuit has been deployed gradually to every hospitalisation units of our hospital since . pharmacists coordinated the extension, the installation and the support for starting. they were the first interlocutors to analyze dysfunctions and to help solving them. difficulties encountered by nurses were often notified to head nurses and transmitted to pharmacists. the objective was to study as a whole difficulties of users and to bring a workable solution to their problems. design: a working subgroup depending on the drug commission was created. it was composed with nurses from different departments (intensive care, infectious, pediatric and rehabilitation departments), head nurses and pharmacists. several meetings, organized between october and december , made it possible to the participants to announce their difficulties. reports were written and diffused for validation. to evaluate the pharmacist -patient communication and the level of counseling for otc and prescription drugs dispense; to improve the professional relationship between the patient and the community pharmacist; to assess the effect of clinical pharmacist intervention over those parameters. design: interventional study (visits done by clinical pharmacists, especially employed for), repeated after weeks and again after months. setting: chain pharmacies from bucharest, romania. main outcome measures: the investigation was conducted using a multiple sections protocol. the assessed parameters were: pharmacist's attitude toward the patients, his/her availability to communication and the level of counseling when otc or prescription medication is released. results: during the first visit, in sixteen pharmacies only ( %) the pharmacists greet the patients. two weeks later after the intervention, this number increased to ( %), although after six moths it decreased to pharmacies ( %). in more than % of the chain pharmacies, the professionals had a positive attitude toward the patients. as an example, an empathic approach has been encountered initially in pharmacies ( %), then in pharmacies ( %) and finally raised up to pharmacies ( %) after months. although the clinical pharmacist's intervention (therapeutic counseling) had positive impact, the extent of minimal counseling at otc or prescription drugs dispensing was found to be low at the first visit, since it increased from to pharmacies ( to %) only, during the study period. the processed data showed a very low level of minimal counseling ( % at the end of surveillance period). by considering the patients benefits (quality of life, better control and management of chronic diseases, reduction of medication costs), the pharmacist interventions are imperatively needed in bucharest chain community pharmacies. background and objective: to compare the level of minimal consultation services in chain community pharmacies located in city center or district of bucharest (capital city) vs. independent pharmacy in a country town (cluj-napoca). design: prospective, months, multicenter study. setting: pharmacies accepted to fill in the study protocols. both shifts were covered, monday to friday (week-end days not included). main outcome measures: the investigation was conducted using a multiple sections protocol. the assessed parameters were: the level of minimal consultation when otc and prescription medications are dispensed and the extent of chronic medication release without a medical prescription. at the end of trial period, the results were centralized on weekly and monthly protocols. the interpretation of the collected data was done using percentile calculations. results: to evaluate the minimal consultation, the percentage of counseled patients from the monthly total was calculated, separately for otc and for prescription drugs dispense. the period of the study (from june to january ) was divided in various slices of consecutive months, when certain pharmacies were compared. the level of otc medication counseling in the five studied pharmacies is different and varies from to % (maximum level reached in the country town pharmacy). the counseling level for medication on prescription varied from to %. by counting separately, the percentage of patients who requested chronic medication without presenting a prescription is as high as up to %. conclusions: the level of counseling, especially for otc drugs (recommended or auto-medication), was generally low in the studied pharmacies and may threat the health state of the patients, due to improper administration. as a third of patients come in pharmacy and request chronic medication without a physician's prescription, this commonly leads to complications which aren't discovered and treated in time. background and objective: the quality of medication use in nursing homes (nhs) is subject to growing concern. focus should not only be on appropriateness of prescribing, but also on correct pharm world sci ( ) : - administration of the medication. the aim of this study was to investigate ) the type and frequency of medication administration errors in nhs, ) their clinical relevance and ) whether a training session by a pharmacist on good medication administration practices can contribute to the prevention of the detected errors. design: the study had a pre-post design. during the first phase (pre), medication administration was observed during days per ward by pharmacists (barker method). phase (intervention) consisted of a general information session on good medication administration principles provided by the pharmacists to the nursing staff. moreover, the observed errors were discussed with the head nurse of each ward. phase (post) took part one month after the intervention and consisted again of a -day observation on each ward. finally, in the last phase (phase ), the clinical relevance of the detected errors was scored by an expert panel (geriatrician and clinical pharmacist). setting: volunteering nhs with different medication distribution systems. in total, medication administration was observed for residents. results: the number of detected errors was considerably lower in nh than in nh . however, the type of errors did not differ. besides the unnecessary or forgotten preparation of medication, most problems occurred during the administration stage. . % of crushed medications indeed were not suitable for crushing. the same applied to . % of the opened capsules. moreover, the crushing hygiene was problematic: all medications for one resident were crushed together and the crushing device was not cleaned between different residents. inhalation techniques were inadequate in almost all cases (insufficient inhalation by the resident, coordination problems or expiration in the device). furthermore, specific administration moments were not taken into account. for example, the administration of alendronate (fosamax Ò ) was observed in a horizontal position after breakfast, while it should be administered minutes prior to breakfast in a vertical position. the nursing staff experienced the training course by the pharmacists as very interesting. . % of the attendants found that the discussed topics were not sufficiently covered during their education. background and objective: the off-label use of intravitreal injection of bevacizumab (iib) for the treatment of macular edema (me) requires the approval of its use by health authorities. pharmacy department (pd) participates in that process, assessing each treatment request and preparing a sterile syringe for intravitreal administration. our aim is to evaluate the short term anatomic and visual acuity (va) response after iib in patients affected of me due to diabetic retinopathy or retinal vein thrombosis. the aim is to give an answer within hours. setting: all three pharmacists are working in the virga jesse hospital, a large peripheral hospital of beds in belgium. main outcome measures: implementation of the clinical helpline in the entire hospital. to make sure clinical pharmacy services are known by every physician and nurse and are easy to contact. results: we made an e-mail address and a schedule, so every day another clinical pharmacist is responsible for answering the questions. to let the physicians and nurses on the ward know we exist, we made flyers with the address and the explanation of the service. on a patient safety congress in the hospital, the clinical pharmacists presented a lecture concerning the advantages of clinical pharmacy services. the main aim is to explore other ways of delivering clinical pharmacy services. in belgium, the hospitals don't have a tradition of clinical pharmacy and there is no governmental support for this pharmaceutical function. with the clinical helpline we try to spread our services without having a clinical pharmacist on every ward. in the pharmacy we prepare the question thoroughly on paper. the clinical pharmacist has computerized access to all necessary medical information and pharmaceutical data. afterwards the pharmacist goes to the ward, to see the patient and to have a discussion with the physician. the physician can decide if he agrees with the given pharmaceutical advice or not. the clinical pharmacist has only an advisory function and doesn't do any therapeutic changes in the prescription. by collaboration of several caregivers, the patient receives a more complete and optimal therapy in our hospital. conclusions: by implementation of a clinical helpline, by an e-mail address, it is possible to spread our clinical pharmacy services over the entire hospital, without having a clinical pharmacist on every ward. the aim is to make an advice and go to the ward to discuss it with the doctor. not only physicians can use this e-mail address, also nurses can ask their questions. in this way we reach every caregiver. % of these pi was followed by modification of the prescription few days later. this study showed that % of the opinions were related to psychotropic drug overdose (often confirmed by psychiatrists after pi), especially neuroleptics, the most prescribed therapeutic class on the establishment. . % of the pi was related to inappropriateness to available guidelines. we note an important proportion of no respect of correct use recommendations of long-acting rispéridone injection: no respect of posological equivalence, patients not stabilized by oral way, insufficient period of co-administration oral/im during the initialization of treatment… . % of pi was guiding to drug management and to clinical and biological monitoring. drug related problems still under estimated without clinical and biological data accessible to pharmacists. however, pi may identify the risks related to therapeutic, to prevent potential problems, to reinforce the clinical and biological monitoring. comparison with a similar retrospectif study in shows that the number of prescriptions was increased ( in vs ), and number of pi doubled. however, nature and type of pi are virtually the same. conclusions: in order to reduce drp of overdose (the most frequent problem), an information strategy targeted to psychiatrists was developed and a updated list of maximal psychotrops posology was diffused and put on line. our study doesn't include problem of second -generation atypical antipsychotics association, this association still increasing despite fewer evidence and lucid guidelines; a second study will be soon conducted to identify pi having a significant clinical impact. conclusions: this study showed that clinical practices don't change even after reminding guidelines. information by fact sheet is not the best tool to spread guidelines. study's results will be submitted to an interactive presentation in medical staff, and clinical trials with iva are discussed and changed with acetaminophen oral route. the proportion of gp's that received, reviewed and returned the patient selection to the pharmacist, and the proportion of long-term users that received the informative letter. results: substantially more pharmacists in the intervention ( %) than in the control group ( %) handed over the patient selection to their gp's. % resp. % of the gp's received (n.s.), and % resp. % of the gp's reviewed and returned the list (n.s.). substantially more pharmacies in the intervention group got back any lists ( % vs %) and sent any letters ( % vs %). % and % of all longterm users received the informative letter in the intervention resp. control groep (n.s.). conclusions: the maximal implementation strategy was effective in getting the pharmacists started. the main outcome measures were not significantly different in both groups, though the realized effect on a large scale was relevant in practice. of the participants, total of . % has heard of the term 'pharmacovigilance' in the period- , which is entirely the academicians, while it is increased to . % in the period- (chi-square test with yates correction, p \ . ), which are mainly expressed by the hospital ( . %) and academician ( . %) pharmacists. during the period- , only % of the participants know where to report any adrs ( . % academicians and . % hospital pharmacists), whereas during the period- , this figure is increased to % ( . % academicians, % hospital and . % community pharmacists) (p [ . ). the participants preferred to report any adrs mainly by the internet ( . % vs %) and by the telephone ( % vs . %) at the period- and the period- , respectively (p [ . ). in terms of having reported any adrs among the participants, it is indicated that none of the pharmacists reported adrs at the period- , but only three hospital pharmacists reported an adr at the period- . conclusions: by the national regulations for pharmacovigilance, the pharmacists are entitled to report any adrs to the turkish pharmacovigilance centre. although this study is limited by the small number of pharmacists and location, it shows that there is an increased awareness and knowledge about pharmacovigilance. by the provision of pharmaceutical care, pharmacists' involvement in detecting and reporting adrs will improve, mainly in hospital and in community settings. background and objective: in france, global drug dispensing (gdd) is the common way to dispense drug inpatient. after an experience of implementation of individual drug dispensing (idd) the objective of the department of pharmacy was to prove the benefit offered by this system in comparing the clinical impact of the intercepted dispensing errors of both dispensing system. design: thirty days prospective study. setting: orthopaedic surgical care unit; department of pharmacy. main outcome measures: data related to drug preparation were collected by two pharm d students over a days period and analysed. identified preparation errors were classified in four groups: discordance between prescription and drug administration, exceeding or missing treatment and, unidentified delivered drugs. then errors were classified in potential or effective errors. at last, pharmacist and prescriber have quote this errors. preparation errors were classified according to their clinical impact from (no clinical impact) to (life threatening). results: drugs units were prepared with gdd vs with idd. eighty effectives errors ( . %) were observed with gdd and ( . %) with idd. clinical impact were ( %), ( %), ( %) for gdd and ( %), ( %) and ( %) for idd. a khi test highlight a difference between the dispensing system for clinical impact and (p \ . ). moreover, the mean value of weighting was about . with gdd vs . with idd (p \ . ). conclusions: this study shows the major benefit offered by idd versus gdd. errors weighting represent a relevant parameter for physicians. the results provided by this study highlight the role of the pharmacy staff, to reduce the incidence of medication errors and to promote a rational use of medicines. this work was the second step of our quality medication process; the next step will be the development of idd in several care unit by use and test automated process for preparing doses. for each prescription we collected information about the patient, the prescribed drug, the steps involved in its preparation and its administration. results: we collected prescriptions ( different drugs) concerning children (average age: years). sixty-two percent of medications were oral forms: % liquids, % tablets, % capsules. thirty-two percent of the tablets were cut; only % of those were authorized. due to the age, % of the tablets were crushed to facilitate administration whereas grinding was allowed in only % of the cases. most of the capsules were opened ( %) and % fractionated for an adapted dose. opening of capsules was possible in % of the cases. intravenous drugs represented %, the average injection was . % of the vial; in %, less than a quarter was given. conclusions: about % of pediatric preparations were inappropriate. the results of this study highlight the need to provide drugs adapted to pediatric care, which is one of the main tasks of the pharmacy. we are thus developing more appropriate pharmaceutical formulations for children, such as oral suspensions. a comparative statement of solubilization rates in liquid of crushed tablets or capsules after opening has to be established. moreover if a pharmacist is present in the care units, most medication error will be avoided. background and objective: the world's population is aging. the elderly have many chronic disorders and consequently use more drugs than any other age group. safe, effective pharmacotherapy is one of the results: fifty patients received antibiotics according to several schemes. nineteen patients received prophylactic treatments during . days on average. the employed molecules were mostly part of beta-lactams' family ( . %) including . % of co-amoxiclav. the prophylactic treatment seemed to be effective in . % of case because it wasn't followed by other treatment. thirty-one patients received probabilistic treatment: . % of them received quadruple therapies, . % received triple therapies, . % bitherapies, and . % monotherapies (only beta-lactams). the average duration of the probabilistic treatment was days. twenty-three patients got curative treatment. compared with the probabilistic treatment or with the treatment at the entry, the curative treatment corresponded to a reduction of the spectrum in % of cases with mainly an arrest of the vancomycin ( %). monotherapy was the most prescribed ( . %) and especially beta-lactams ( . %). then came bitherapies ( . %) and triple therapies ( . %). the most frequent isolated germs were escherichia coli ( cases), staphylococcus aureus ( cases), pseudomonas aeruginosa ( cases) and pneumococcus sp. ( cases) alone or associated, including . % of multiple-drugsresistant bacterias. the monitoring of aminosids and vancomycin was globally well carried out. only . % of vancomycin's dosages and . % of aminosides' dosages were not done. thirty-two patients increased their transaminases and/or their creatininemy. these increases were often physiopathological, related to multiviscérales failures or to septic shocks. the antibiotics could be clearly blamed in only one case. conclusions: in the surgical intensive care unit, antibioprophylaxy is longer than in recommendations. the probabilistic treatments used often associate active molecules on multiple-drugs-resistant bacterias. this can be explained by the gravity of infections, and the increased risk of bacteriologic resistances due to former treatments. moreover, bacteriological tests are systematically done, so antibiotics can quickly be adapt to the germs' resistances. background and objective: most of proton pump inhibitors (ppis) do not have legal mention for a paediatric use. however these drugs are largely prescribed to children. one disadvantage resides in the absence of liquid form which causes problems for their administration in nasogastric tubes. indeed, the absence of use recommendations involves many misuses responsible for inefficiency and/or tube obstruction. we tried to evaluate if ppis can be administered through paediatric nasogastric tubes. design: to quantify the transit of different ppis through paediatric nasogastric tubes and to optimise their modes of administration. setting: laboratory of clinical pharmacy and biotechnics. faculty of pharmacy. main outcome measures: we administered four ppis (mopral Ò , ogast Ò , inexium Ò , ogastoro Ò ) through nasogastric tubes by respecting their positioning in a child in a °elevation. for each ppi a study plan was drawn up to assess the influence of different variables: the volume of water to dissolve or put in suspension the ppis ( or ml), the rinse volume ( . or ml), the length ( or cm) and the diameter ( or french) of the polyurethane tubes. for every tests (n = ) we carried out an analysis of each active ingredient at the tube outlet by uv spectrometry. results: all f tubes were obstructed by ppis. through f tubes, we observed a mean recovery of active ingredient of % for ogastoro Ò , . % for inexium Ò but only . % for ogast Ò and . % for mopral Ò . the length of the tubes had no significant influence on the loss of ppi at the outlet of the tube. a water volume of ml instead of ml increased only the final concentration of inexium Ò (+ %). a rinse volume of ml improved significantly the transit of mopral Ò , ogast Ò and ogastoro Ò (+ . %, + . %, + . % respectively). this rinse volume allowed to obtain a . % recovery of lanzoprazole for ogastoro Ò whatever the water volume employed for its administration. conclusions: the most satisfactory results were obtained with ogastoro Ò : an administration volume of ml and a rinse volume of ml allowed a near-complete transit of lanzoprazole. under these conditions only % of inexium Ò was recovered. it is disadvised using mopral Ò and ogast Ò through f nasogastric tubes because no condition ensure the transit of an efficient concentration of active ingredient. keywords: nasogastric tubes, proton pump inhibitors, children background and objective: feeding tube occlusion is a frequent problem. practices to make the clogging off are very varied and are not the subject of any consensus. no study have assessed the impact of the different products on the inner surface of the tubes. in this context, it seams to be important to evaluate if these products are safe in order to rationalize the practices. design: to study the inner surface of nasogastric feeding tubes after contact with various products used to unblock them. setting: laboratory of clinical pharmacy and biotechnics. faculty of pharmacy. main outcome measures: we have put in contact f nasogastric tubes made of silicone or polyurethane with the following products: water, . % sodium bicarbonate, orange juice, pineapple juice, cola, papain syrup, pancreatic enzymes. an analysis of the inner surface of the tubes was carried out after , and days by scanning electron microscopy (sem). photos of unexposed tubes were used as negative controls. photos of tubes exposed to heat, ether or sodium hydroxide were used as positive controls. results: the analysis by sem shows that the silicone tubes are not altered by the different products tested. on the other hand, the surface of polyurethane tubes is modified in the presence of . % sodium bicarbonate and pancreatic enzymes. the papain syrup seems to settle on the surface of the tubes without altering it. water, fruit juices and cola do not modify the biomaterial whatever the exposure time. conclusions: . % sodium bicarbonate, pancreatic enzymes and even papain syrup should not be used in practice to unblock the feeding tubes. the orange and pineapple juices as well as cola can be recommended because of their harmlessness with biomaterials. keywords: nasogastric feeding tubes, occlusion, scanning electron microscopy pc- assessment of administration practices of extemporaneous formulation pediatric capsule preparations helene richard , anne jalabert , sylvie hansel-esteller pharmacy unit, lapeyronie and arnaud de villeuneuve hospitals, montpellier, france background and objective: to assess administration practices of pediatric capsules made as an extemporaneous formulation preparation by the laboratory of the pharmacy unit, in pediatric units. design: a questionnaire was designed and filled in by asking questions directly to nurses about their administration practices, from february to april . setting: altogether, six pediatric units were consulted, corresponding to the units for which the laboratory carries out the most pediatric extemporaneous formulation preparations. main outcome measures: the questionnaire concerned the ten most made up pediatric preparations, which are: amiodarone, warfarine, captopril, propranolol, ursodesoxycholic acid, omeprazole, fludrocortisone, spironolactone, hydrocortisone and calcium carbonate. items filled in were: hygiene rules, the existence of administration procedures in units, preparation conservation, and practical administration details. results: in months, questionnaires were filled in. concerning hygiene rules, nurses wash their hands before every manipulation, % wear gloves, % wear a mask or a mobcap. no administration procedures were available in units and more than % of nurses would like to have one. in every unit, preparations were conserved at room temperature, in a dry place, and % in a light-free place. for amiodarone, propranolol, fludrocortisone, spironolactone and hydrocortisone capsules, nurses use sweetened water (g %) to dilute capsule contents, sometimes milk (especially in newborns units). for the other drugs, the vehicules the most used were: sweetened water ( %), solid vehicules like yogurt, apple sauce, jam ( %), fruit juice or syrup ( %), milk ( %), and coca-cola ( %). the vehicule volume used fluctuates between ml and cl. nurses administer capsules by syringue into the mouth ( %), per os ( %), or by enteral nutrition ( %). conclusions: most of the administration practices of pediatric preparations are homogeneous in the different units (except the vehicule volume used). in the absence of administration procedures, the administration of preparations is more adapted to the child than to the drug. the aim of this study is to provide pediatric units with guidelines about good use of extemporaneous formulation pediatric preparations. services compared to a pharmaceutical care model. items pertaining to pharmacists' communication with the patient and the physician as well as the pharmacists potential to manage drug therapy were assessed. results: of the patients approached, ( %) response rate was achieved. cronbach's alpha = . (expectations) and . (satisfaction). females were ( %), married ( %) and % had post-secondary education. some % expect accurate dispensing of their medications, % expect pharmacists to simplify their medications and % expect the pharmacist to spend as much time as possible with them. expectations were resolved into components: humaneness, friendly attitude and professional competence. only % were satisfied with the professional appearance of the pharmacy, the rest of the items received dissatisfaction rating. two principal components of satisfaction were identified as humaneness and professional competence. marital status and level of education were associated with satisfaction scores. conclusions: patients' expectations of pharmaceutical care services were high but the satisfaction with current services compared to pharmaceutical care was below average. professional competence and humaneness were important dimensions of both patient expectations and satisfaction. there is a need to introduce pharmaceutical care. results: fourteen criteria were defined. a yes, no or not applicable answer has been given to each criterion according to the guidelines. drawing lots have been realised to set up a representative sample of twenty patients among those included in the study: six males, fourteen females, average age: . years old. two patients are below the average. the average mark of the patients is . %. hemoglobin level of % patients is inferior to the targeted hemoglobin level ( g/dl) at the end of the study. six criteria obtain a score superior or equal to %. five criteria obtain a score under %. the three criteria which obtain the best results concern the monitoring of the hemoglobin levels during correction phase and maintenance phase, and the maximum weekly dose of erythropoetin. the three criteria which obtain the lowest results concern the assessment of iron status before erythropoetin therapy starts ( %), the rate of increase in hemoglobin levels which should be - g/dl per month ( %) and the adjustment of total weekly erythropoetin dose. conclusions: the average grade obtained by the patients highlights the deviation from the guidelines. this indicates the needs of improving the anemia management in our haemodialysis unit. so it is necessary to make another time the team of doctors and nurses aware of the problem and to reinforce the pharmaceutic implication into the haemodialysis unit. a re-evaluation of practices (in the guise of clinical audit) must be planned in order to estimate the impact of the setting up of correctional measures. inclusion criteria were: oral and injectable drugs' prescriptions concerning patients who were present in the ward since more than hours. results: medical records were analyzed. patient age varied from to years old and the average duration of the hospitalisation from . to days. omitted variables were: patient's weight in all cases, prescriber's quality in % of cases, prescription's hour in % and prescriber's signature in . %. for each patient, all drugs prescribed since the beginning of the hospitalization in the unit was studied i.e. lines of drugs. we have noted the inn was absent in % of the cases, pharmaceutical form in % and administration route in %. only one prescription had all variables. thus, the global rate of prescription conformity was %. in the wards, nurses write the original prescriptions on a card-index (ci). then, drugs administrations are written on a temperature chart (tc) and on pharmacy's book (pb) for drugs order. we have compared documents one by one, for each drug. conformity rates are . % (prescription/ci), . % (ci/ tc) and % (ci/pb). the noted differences are multiple(modification of the administration or hour route, increase or dicrease of dosage…). the clinical relevance of non conformity was not studied. conclusions: from this study, it can be concluded that many prescriptions do not comply to the regulation and many retranscriptions are not identical to original prescriptions. it constitutes of course a potential iatrogenic impact. the development of computerized physician order entry (cpoe) in our hospital should be a corrective measure. the next stage will consist of realizing this study with cpoe and compared the studies. anne-claire buire , emilie prevost , françois lebargy , bertrand gourdier pharmacy, pneumology, reims teaching hospital, reims, france background and objective: assessment of antibacterial prescriptions regarding community-acquired lower respiratory tract infections and comparison with national guidelines. design: patients treated with antibiotics were included each day, during the analysis of the computerized prescriptions in the pharmacy. then, a report sheet was indicated in the unit with the prescribers for each patients with pneumonia or exacerbation of chronic obstructive lung disease. one month prospective study (in april ) included all patients hospitalised for pneumonia or exacerbation of chronic obstructive pulmonary disease (ecopd) in a unit of the pneumology department and treated with antibiotics. setting: pneumology department -reims university hospital. main outcome measures: for each patient the following data were assessed: age, hospitalization duration, diagnosis, severity factors, comorbidities and antibiotic prescriptions were analyzed. treatments were thought consistent when following the national guidelines [ ; ] . results: new patients were hospitalized during this month. ( %) were treated with antibiotics: with no pulmonary pathology and with pulmonary infection ( patients with pneumonia, with ecopd, with pleurisy, with exacerbation of asthma, with chest secondary infection, with acute respiratory infection, with thoracic pain and with cough). then, the study focused on patients: pneumonia and ecopd (m/f = / , mean age: years old). hospitalization duration was about days. patients ( %) had at least one co-morbidities factor (the majority: with pulmonary antecedents) and ( %) a severity factor (the majority: with an attack of the vital functions). antibiotic treatments were initiated in the unit and out ( in emergency department and by general practitioner). all treatments initiated in the unit were consistent with the recommendations and revaluated with the bacterial results (except one treatment) or changed because of bad tolerability. bacteriological documentation was always researched and the results were significant for patients ( %): streptococcus pneumoniae, pseudomonas aeruginosa, proteus mirabilis with morganella morganii and haemophilus influenzae. when it was possible, per os relay was realized in the at hours, except for patients (relay in the days). conclusions: in this survey, the management of pneumonia and ecopd was globally consistent with the national guidelines. nevertheless, the bacterial documentation is poor and the antibiotics prescriptions for bronchopulmonary infections are difficult and need using molecules with large spectrum. collaborate with all of the health care professionals and patients and to identify causes of errors. the unique position and possibilities of a pharmacist enable him to follow up the errors, from a fact that he is an expert on medication properties and is basically the last in a row who deals with drugs before their administration to the patients. the aim of this study was to describe and evaluate the role of pharmacist in identification and dealing with medication errors in czech pharmacy. design: pharmacists identified and recorded all medication errors over a period of six months of pharmaceutical care. basic characteristic of pharmacists: mean age . years; mean length of pharmaceutical practice . years; of them worked in the hospital pharmacy. of them got the first grade of attestation in pharmacy (by years of working experience in pharmacy and by passing exams). pharmacists collected the following data: types and causes or errors, their interventions, drugs and time concerning errors, subjects making errors and patient's characteristics as age, gender, other drugs used and co-morbidities. all the data were processed by descriptive statistics. background and objective: there is no standard of care to prevent oral mucositis for patients with cancer treated by chemotherapy. most common treatment is local (mouthwashes). no commercial mouthwash solution is available. special preparations are compounded by clinical units. a new . % sodium bicarbonate solution presentation was recently proposed for local treatment. our hospital drug committee decided to evaluate the clinical practices of oral mucositis prevention related to anti-neoplasic agents. design: an observational study conducted in pitié-salpétrière hospital. interview of nurses and physicians by pharmacy students about mouthwashes prescription and practice. data recorded on pre-established questionnaire and analyzed in pharmacy department using microsoft excel Ò . setting: study performed in oncology, hematology and radiotherapy clinical departments and in clinical units with oncologic activity (gastroenterology…). during one year, more than patients received anticancer chemotherapy. main outcome measures: questionnaire items were: use of a specific mouthwash procedure within the service, use of a single agent or in combination. data collected from the nurse point of view were: preparation and administration practices; from the physicians' perspective: circumstances of prescription and duration of the treatment. results: questionnaires were analyzed (nurses: , physicians: ) from clinical units. there was a standard written procedure in only one clinical unit. several formulations were used: each physician proposed his own, including antifungal prophylaxis (amphotericin b, nystatin), antimicrobial agents (povidone, chlorhexidine), mucosal surface protectant (sucralfate), alkalin solution (sodium bicarbonate), anti-inflammatory agent (aspirin), anaesthetic drugs (lidocaïn) and other agent (glycothymoline). preparations were not systematically labelled with patients name, formulation, date of preparation and stability duration. these were administered from to times daily regardless the stability compounding. for physicians, prescriptions of mouthwashes were done for patients with specific toxic anticancer drugs ( -fu, anthracyclin, capecitabin and sunitinib), were concomitant with chemotherapy and systematic after radiotherapy. conclusions: there are variations among clinical units in terms of mouth care regimen used. treatment efficacy was never evaluated. drug committee worked on guidelines in order to prescribe antifungal therapy only for curative aim or avoid anaesthetic drugs (swallowing difficulties). good practices included, before chemotherapy, dental hygiene. maintenance is realized by the patient himself (mouthrinses with alkalin solution or chlorhexidine). analgesics can be taken orally in case of mouth pain. design: the open, randomized, single-blind two-sequence, two-period crossover study design was performed. setting: under fasting conditions, each subject received a single oral dose of mg olanzapine tablet as a test or reference formulation on treatment days. the treatment periods were separated by a oneweek washout period. main outcome measures: the plasma concentrations of drug were analyzed by a rapid and sensitive hplc method with uv detection. results: the pharmacokinetic parameters included auc - h, auc -infinity, cmax, t / , and ke. the mean auc -infinity of olanzapine was . and . ng h/ml for the test and reference formulation, respectively. the maximum plasma concentration (cmax) of olanzapine was on average . ng/ml for the test and . ng/ml for the reference product. no statistical differences were observed for cmax and the area under the plasma concentration-time curve for test and reference tablets. % confidence limits calculated for cmax and auc -infinity of cefixime were included in the bioequivalence range ( . - . %). conclusions: therefore, the two tablet formulations were considered to be bioequivalent. conclusions: clinical pharmacists effectively validate % of these prescriptions but one third of prescriptions will inevitably remain validated by pharmacist residents during night and week-ends. performance criteria must be proposed to measure impact of pharmaceutical improvement initiatives (validation procedures writing, continuous education for example). we suggest to test clinical pharmacy activities related to economic indicators and to medication events reduction. background and objective: education of patients with type diabetes is a key point of non-medical management of that disease. thus objective was evaluation of patients' knowledge about their disease and its management for further development of pharmaceutical care protocols. design: prospective cohort control study. total patients casually were divided in two equivalent groups: study group (n = ), age . ± . , diabetes duration . ± . who were questioned by face-to-face technique and received pharmaceutical care during the interview; control group (n = ), age . ± . , diabetes duration . ± . and filled in questionnaires without clinical pharmacist. inclusive criteria were: presence of type diabetes mellitus, duration of the disease was not considered as criteria. patients were inquired once following standardized questionnaire. all included patients could read and write. two groups controlled their diabetes mostly by oral antidiabetics, only n = in study group and n = in control used insulin. setting: out-patient setting of lviv clinical hospital no , endocrinologist's office. main outcome measures: assessment of patients' knowledge about diabetes and self-monitoring in study and control groups. results: only . % and . % of inquired patients in both groups respectively stated that they possess good knowledge about diabetes. but as it was shown by evaluation of their level of knowledge through assessment of keeping to diet, regular physical activity, self monitoring it didn't conform completely. diet was implemented by almost % of patients in two groups, while regular physical activity was declared only by . % in study and % in control groups. smoking was reported by . % and . % respectively. next step was evaluation of self-monitoring. it has been revealed that only . % and . % of patients in two groups performed blood glucose monitoring at home; body weight was controlled by . % and . % respectively; blood pressure by . % and . % as well. target level of blood pressure was achieved by . % of subjects in study group and in . % -in control. in study group foot examination was performed everyday by . % of patients when in control only by . %. no one from both groups evaluated glycated haemoglobin regularly as it is recommended by american diabetes association, those they couldn't state their present or previous parameter. conclusions: it was estimated that knowledge about necessity of diet following, regular physical activity, and particularly -self-monitoring was very poor in diabetic patients, no regarding compliance which possibly also will be low. even if patients stated good knowledge about the disease they had problems with self-monitoring. it is obvious that patients of this out-patient setting require adequate education, which should be a component of pharmaceutical care program implemented by the clinical pharmacist. protocols of pharmaceutical care for these patients must be developed and include standard procedure of type diabetes patients' education. pharmacy department, hospital universitari vall d'hebron, pharmacy department, pharmacy department, hospital universitari vall hebron, barcelona, spain background and objective: patients who are admitted for a programmed orthopedic surgery in a tertiary hospital are usually elderly and present high co-morbidity which makes therapy complexity increase. the objective is to evaluate whether a pharmaceutical care is necessary before patients hospital admission. design: we analysed the domiciliary treatment in patients who attended to a pre-surgery visit. a form designed by the pharmacy department and validated by anaesthesiology unit was delivered to the patient to fill in with domiciliary therapy. the average of prescribed drugs was . per patient, being this reduced to . drugs in patients younger than and increased to . in patients older than . over one third of patients ( / ) were taken orally nonsteroidal anti-inflammatory drugs (nsaids). from ai, would need a pharmacological evaluation in the preoperative. those were included in drugs, meaning the . % of the different drugs prescriptions. this percentage belonged to patients. conclusions: the fact that a high percentage of patients over with an orthopedic pathology take a considerable amount of drugs at home makes it necessary to monitories the therapy in order to minimise the iatrogenic problems during the admission. we have seen that this situation is so frequently ( %) in our study, so the pharmacy department has established a collaborative job with anaesthesiologist unit to reduce as much as possible therapy problems. levation. through each tube, a ml nutritive solution was delivered on h each day during one week. after dissolution in ml water the ppis were administered once a day after stopping of the enteral nutrition and rinsing of the tube with ml of water. the tubes were then rinsed with ml of water and the nutrition was started again. during each administration of ppi the suspension was collected at the tube exit in order to quantify the ppi by uv-spectrometry. results: no tube was obstructed. the enteral nutrition mixture did not adversely affect the transit of lansoprazole through f nasogastric tubes. the transit of lansoprazole through the tube was complete and regular during the days of the study ( . ± . ). for esomeprazole the mean recovery of active ingredient was of . ± . (coefficient of variation: %) this variability can be explained by the incomplete and inconstant dissolution of esomeprazole because of the low volume of water usable in paediatry. conclusions: orally disintegrating tablet of lansoprazole can be administered through f nasogastric tubes in a concomitant way to an enteral nutrition mixture. for esomeprazole there is a variability of the administered active ingredient. however enteral nutrition doesn't seem to affect the esomeprazole transit. low volumes of water used in paediatry seem to be responsible for the variability. results: we analyzed pharmacological treatment in patients, with a mean age of years old ( - ). the average of drugs per patients was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . after checking the sources we detected possible pi ( severe, moderate and slight), observed times. pi were detected in of patients and mean per patient was . ( - ) . the following pi were described: increase of sedation ( patients), risk of bleeding ( patients), serum potassium levels alteration ( patients), hypotension ( patients), risk of hepatotoxicity ( patients), increase of creatin kinase levels and risk of myopathy ( patients). from all these, only two were observed: sedation which was observed in all patients and hypotension which permitted the reduction of patient antihypertensive treatment. conclusions: the most frequent pi was sedation which is considered beneficial in order to reduce anxiety in sci patients. arterial tension, electrolytic balance, hepatic and renal function, as well as risk of bleeding, are regularly controlled in the sci unit. this way, if one of these parameters was altered it would be easily detected. to conclude, it would be important to carry out regular checking to detect pi, especially for those drugs that are not usual and for those symptoms that are not controlled regularly in the sci unit. background and objective: pharmacists' individualized counselling of patients has positive impacts on the management of hyperlipidaemia, including improved compliance and better treatment endpoints. the objective was to evaluate patient knowledge on hypercholesterolemia and its treatment and to assess impact of pharmacist intervention at the lipid clinic. design: one hundred and fifty statin-treated patients were recruited by convenience sampling. following completion of a scored pre-intervention questionnaire, the pharmacist provided education on hypercholesterolaemia and the use of statins. a leaflet was prepared, evaluated and distributed to the patients. the patients completed again the same questionnaire after the intervention (post-intervention). results: patient demographics: % ( ) were males, % ( ) were females, mean age was years (range - years). a response rate of % was achieved with patients completing both questionnaires. following the educational intervention by the pharmacist, knowledge regarding the correct action to be taken if muscle pain or tenderness occur during statin therapy increased by % (p = ). the awareness regarding the normal total blood cholesterol level increased by % (p = . ) and the knowledge regarding the need for low-fat diet consumption during statin therapy increased by % (p = ). face and content validity of the patients' leaflet were strong. the average gunning fog index obtained for the leaflet was . indicating good readability for individuals. the leaflet was endorsed by the local health promotion department and copies were printed and distributed to patients. background and objective: in order to assess the performance of bayesian individualization of busulfan (bu) dosage regimens, venoocclusive disease (vod) rate was monitored for paediatric patients undergoing allogeneic bone marrow transplantation (bmt). design: consecutive patients undergoing allogeneic bmt with bu as conditioning regimen during five years period (january to febuary ) were retrospectively reviewed ( patients). setting: vod was major outcome variable. preconditioning risk of vod was estimated for each patient using a scoring system that included type of transplant, recipient cmv-positive status and total parenteral nutrition provided pretransplantation. a risk-adjusted cumulative sum method was used to compare observed versus predicted outcome by assigning a risk score, based on log-likelihood ratios, to each patient. main outcome measures: the cumulative scores were sequentially plotted with preset control limits for ''signalling'' where results were substantially different than expected (doubling or halving of odds ratio). results: sixty-six children received bmt after oral busulfan-based conditioning regimen with median age . years, . % of male. median preconditioning risk of vod was . range ( . - . ). observed vod rate was . % (n = ) which was . % ( patients) fewer than the expected number estimated by the risk score. the resulting risk-adjusted score for each patient was plotted sequentially. this plot adopted early a negative slope, crossing the lower control limit twice, after and patients, indicating improved results compared to those expected. background and objective: geriatric patients use numerous drugs; because they have several concurrent diseases. % of those years old and over have at least one chronic disease, % have or more chronic diseases ( ) . the purpose of this study is to evaluate data on the geriatrics' drug usage, assess the appropriateness of their drug treatment and identify their pharmaceutical care requirements. design: patients, who were years old and over and live in a nursing home in the anatolian part of istanbul, were included in our study. by interviewing the patients, individualized information was obtained regarding the drugs they used, dose and frequency of drugs, the purposes of medication use and side effects, and who suggested or prescribed the drug. the patients at risk of drug induced problems were defined and a risk map was developed. patients who have or more risk factors were accepted as being in a high risk category. setting: a nursing home. main outcome measures: the demographic, clinical and drug data of the patients were recorded. the pills count that patients used daily and totally; side effects of the drugs; knowledge of patient's diseases and drugs; risk category of patients were assessed. results: polypharmacy was identified in % of those included in the study (total of patients who take drug therapy). generally it was observed that the drugs were prescribed at an appropriate dosage and time; however % of the patients didn't know for what they were taking drugs. just ( . %) of the patients were aware of the conditions under which they should take drugs. of the patients ( . %) were not aware on how to and when they should receive their drugs. patients ( . %) were using their drugs by self-administration. the drugs of patients ( . %) were administered by their nurse. . % of the patients were receiving no medication. mean of number of drug used by patients was . ± . . . % of patients were in a high risk category. conclusions: as a result, drug effects alter due to polypharmacy, physiological and psychological changes. drug treatment should be individualized and monitored in geriatrics. according to our results, patients have lack of knowledge on drug use and they have never been educated in this matter. it is necessary to begin meeting their factor analytical technique can be either exploratory or confirmatory. exploratory factor analysis (efa) remains one of the standard and most widely used methods to demonstrate construct validity of new instruments. it is used to help development of the instrument by revealing items that may be made redundant from the questionnaire because they contribute little to the presumed construct. the seip consists of items in five domains and is scored on a four-to-fivepoint likert scale. the aim of the study was to assess further psychometric property of the seip using principal component factor analysis (pca). the strength of the inter-correlations among the items will be assessed by the presence of coefficients greater than . in the correlation matrix. if few correlations above this level are found, then factor analysis may not be appropriate. issues were identified including related to inr controls not performed and due to a poor/no adjustement of the oat dosage. % of the issues resolved following the pharmacist recommandations conclusions: computerized follow-up system allows to check inrs at the right date and to reduce the loss of results. pharmacists' recommendations were well accepted by physicians. the impact of the follow-up on the reduction of the overdose incidence will have to be evaluated. indications were hip (n = ) and knee (n = ) total replacement, other orthopaedic surgery of lower limbs (n = ) and pulmonary embolism treatment (n = ). indications were ''off label'' for patients with allergic reaction to heparine and were not documented. at least one of the haemorrhagic risks factors was identified in patients ( %). conclusions: fondaparinux is mainly used in approved indications. within the patients with haemorrhagic risks factors, no haemorrhagic accident was notified in the pharmacovigilance records of our hospital. nevertheless, fondaparinux has to be administred cautiously in this population of patients for whom the . mg dose will be necessary and hopefully available in a few month. diagnose group a streptococcal (gas) acute pharyngitis. this study aimed to evaluate the service; examine whether current clinical records were adequate; and test hypotheses investigating the association between clinical prediction rules (center criteria, cc), rapid antigen detection tests (radt) and antibiotic prescription. design: initially, a semi-structured interview was conducted to gain an insight into the service. data was retrospectively obtained from a standardised template (apef), completed by a pharmacist at the time of each patient visit. patient demographics and symptoms, radt results, cc scores and follow up rates were analysed. chi squared analyses were performed to investigate the aforementioned hypotheses. setting: jones pharmacy, spokane, washington dc. main outcome measures: establishing adequacy and effectiveness of clinical records and relationships between clinical prediction rules, radt and the antibiotic prescription. results: out of a total of patients (mean age = ; male = ) there were children (mean age = . ; male ) and adults (mean age = ; male = ). ten ( . %) of the negative radt results amongst children were referred to a primary healthcare provider. out of patients, ( . %) needed a radt to be performed. only ( . %) out of patients were followed up. significant association existed between radt outcomes and the prescription of antibiotics (p = . , p \ . ). conclusions: the findings from this study indicate that pharmacists need to be educated on the importance of a comprehensive clinical record. inconsistent practices occur amongst pharmacists due to the conflict between us guidelines, particularly relating to the referral of children with negative radt results to a primary care provider. the template was an efficient tool for data collection. jones did not tailor their data collection for the purpose of the study: with improved data collection, jones can yield information demonstrating the value of this service in future studies. only % of the rhophylac prescriptions. lg of anti d ig were administered when natead was available, whereas lg with rhophylac excepted times where , or lg were administered. times the pharmacist has induced a modification of the posology. however, none prescription was made according to the afssaps recommendations. prescriptions were made for children in hematology unit concerning boys and girls. among prescriptions of lg natead, % were correct and % for the rhophylac. the dose administered was not standard ( , or lg) and none prescription was conformed to the recommendations. conclusions: this study shows that since rhophylac was commercialized (more expensive than natead), the doses administered have doubled and the national recommendations (existent for adults only) are not followed. the reason is that hematologic patients may have a lot of transfusions so they want to protect them from any immunization, despite they are immunocompromised patients. new recommendations are needed, more applied to the practice and precising doses for children. the role of the pharmacist is then to remind physicians good practices of prescription. pharmacy, chu bicêtre, le kremlin bicêtre, france background and objective: computerized prescriptions are settled in clinical wards of our hospital. as pharmacist we have to validate those prescriptions and make recommendations to change part of the prescription such as dose, drug drug interactions. the objective of this study is to evaluate what kind of pharmaceutical interventions physicians really pay attention to. design: the prescriptions had been analysed for three months in three medical care units by two pharmacists. one of them take part in the physician round in order to integrate particular medical practices. pharmacist's interventions were recorded and categorized. the ratio of accepted interventions by the physicians was assessed. setting: three medical care units: internal medicine services (acute care unit: beds and long term hospitalisation: beds) and acute geriatric unit ( beds) in a -bed french university hospital. main outcome measures: description and analysis of pharmacist's interventions in clinical wards. results: we analysed prescriptions. interventions were performed and categorized. it should be noticed that % of pharmaceutical interventions were for misused of the new software: wrong selection of unit ( %) and redundant order ( %). the other % interventions were related to the prescription, our suggestions were as follow: time of administration ( %), adequate drug formulation ( %), dose adjustment ( %), therapeutic drug monitoring or biologic follow up ( %), to stop treatment ( %), route of administration ( %). among these recommendations, % were the consequence of drug-drug interaction. % of the interventions led to change in the prescription. % of the physicians maintained their prescription despite the recommendations, mainly for staggered administration to avoid drug interaction. background and objective: sliding scale insulin therapy (ssi) is a commonly used method of adjusting insulin in an attempt to control a patient's blood glucose levels. previous research investigating ssi use in the hospital setting has determined that ssi therapy leads to poor glycaemic control and poor patient outcomes. therefore, a corrective schedule for ssi therapy is recommended by the american diabetes association (ada). the aims of this study were therefore to determine, for the first time whether ssi recipients experience problems in the home care setting and whether ada guidelines on ssi use are being adhered to. design: eligible patients were identified through electronic records of patients admitted from st january to st december . the list was utilised to obtain medical charts of the patients. relevant information including patient demographics, blood glucose readings and documented problems were recorded using a standardised data collection form. chi-squared statistical analysis was determined using spss version . . setting: the visiting nurses association (vna) home care agency, spokane, washington state, usa. main outcome measures: use of 'traditional ssi therapy' versus 'corrective' version recommended by ada. results: of the total (male = , female = ; mean age = . , age range = - ) patient medical records examined in this study, . % (n = ) had at least one problem documented with their insulin regimen. the most common problem that affected over a quarter of the population, . % (n = ) was 'lack of control' which included any hyper-and hypoglycemic events. more than a third of sliding scale recipients, . % (n = ) had preprandial blood glucose levels above mg/dl. in total . % (n = ) of patients were prescribed the non-recommended traditional ssi therapy and only . % (n = ) were using the recommended corrective ssi therapy. conclusions: the findings of this study support previous concerns that ssi use is prone to problems and poor glycaemic control. furthermore, this study has established the lack of adherence to the ada recommended use of the corrective ssi schedule in the home care setting. it is hoped that this study will influence use of ssi in hospital and home care agencies as well as national and international guidelines. background and objective: prolonged (more than hours) mechanical ventilation (mv) is the most important factor associated with nosocomial pneumonia ( ) . nosocomial pneumonia (np) is differentiated in to ventilator-associated pneumonia (vap) if the process arose after the patient has been receiving at least h of mv ( ) . vap is defined as an inflamation of the lung parenchima caused by infectious agents not present or incubating at time mv was started ( ). design: longitudinal, prospective and observational study. setting: we made a prospective evaluation of the clinical files of a patient population of cases with vap diagnosed between april and december , who were assisted in hospital garcia de orta. a total of patients, % male and % female, aged ± years were diagnosed with vap. vap was defined as new positive respiratory culture after at least hours of mv. main outcome measures: these data sugest that the increase of pcr for documented vap caused by pseudomonas aeruginosa ocurred more frequently with ceftazidim than other antibiotics. results: in all vap episodes, an aetiologic microrganism, was isolated from hemocultures and bronchic secretions. the gramnegative bacteria were the most commonly isolated microorganisms ( %). we collected pcr and leucocytes data of seven documented schedules (n = ) for erradication of pseudomonas aeruginosa: ( ) ceftazidime ( g q h) + gentamicin ( mg/kg qd), ( ) ceftazidime ( g q h) + ciprofloxacin ( mg q h), ( ) piperacillin-tazobactam ( g/ mg q h) + aztreonam ( g q h), ( ) piperacillintazobactam ( g/ mg q h) + gentamicin ( mg/kg qd), ( ) piperacillin-tazobactam ( g/ mg q h) + amikacin ( mg/kg qd), ( ) imipenem-cilastatine ( mg q h) + gentamicin ( mg/kg qd), ( ) meropenem ( g q h) + gentamicin ( mg/kg qd). the mean duration of antibiotic therapy was days. there was an increase of the pcr values of the patients who were scheduled with ceftazidim but in the other groups there was a decrease of this parameter. the number of leucocytes didn't have any impact of the pcr variation (p [ . , t-test). considering the group of the patients, % ( ) of the positive cultures for pseudomonas aeruginosa which were sensitive to either ceftazidim or a carbapenem or piperacillin-tazobactam were treated with ceftazidim instead. conclusions: this approach provides useful information on the relation of host defenses and the clinical outcome. it is also useful to study the prevalence of acquired resistance to several antibiotics that may be used in documented antibiotherapy for pseudomonas aeruginosa. background and objective: rfviia is increasingly used as rescue therapy in uncontrolled bleeding, however little information is available regarding its safety and efficacy in this indication. -indication for use: massive bleeding when first-line treatment (surgical control of bleeding, use of blood products) has failed; -to achieve the correction of factors that may interfere with coagulation (hypothermia, severe acidosis, hypocalcemia); -before administration of rfviia the patient or his family should be informed about the treatment; -the prescription of fviia should be initialized by a referent physician -to conform the dose of mu/kg in cardiac surgery patient. conclusions: several solutions have been proposed to the physicians to improve their professional practices: to develop an algorithm for use of rfviia, to fill in a checklist before administration of rfviia to be sure to follow the guidelines, … this study will be extended in others departments using rfviia as gastroenterology and traumatology. pharmacy, administração regional de saúde de lisboa, pharmacy, administração regional de saúde de lisboa, lisboa, portugal background and objective: the pharmacist can contribute to the maintenance and recovery of population health conditions participating in patient house visit s health teams. pharmaceutical care at this level may also have a real impact on the health system costs. the aim of this project is to establish a pathway at this care level in which all aspects of pharmaceutical care are explained and defined. design: program description. participation of the pharmacist in health care teams. definition of field areas, between the pharmaceutical hospital care and the community pharmacy and health care centers. articulation between the hospital pharmacist, the health care center pharmacist and this new ''home pharmacist''. definition of the different types of pharmaceutical care to be implemented in this setting. background and objective: hip fracture is a major public health problem with a high incidence and prevalence in people aged years and older. changes in body composition and organ function, drug-drug interactions, and co-morbidities should be taken into account in the pharmaceutical care of this group of patients. the aim of this study is to analyse pharmacological treatment of elderly patients ongoing hip fracture in order to improve pharmaceutical care in this group of patients. design: a prospective pilot-study was performed during one month, (may-june ) in patients admitted in a tertiary hospital ongoing hip fracture. these variables were recorded for each patient: sex, age, body mass index (bmi), diseases antecedents, serum creatinine and creatinine clearance estimated by cockroft-gault formula, serum albumin levels, lymphocytes count, sodium and potassium levels. drug treatment was recorded from pharmacy database. setting: patients with hip fracture admitted in a tertiary hospital. main outcome measures: prescription profile in elderly patients with hip fracture. results: of patients, were female. mean age was . years old ( - ). mean bmi was . (n = , range - ). albumin levels were lower than . g/dl in patients. sodium levels were out of the normal range in patients. five patients had creatinine clearance lower than ml/min, of them less than ml/min. we analysed prescriptions which included drugs. they were classified in categories: not adjustment required ( drugs), adjustment required ( ) , inappropriate based in beer's criteria ( ), precaution in elderly people ( ) and not enough information available in geriatric population ( ) . mean number of drugs per patient was ( - ). of prescriptions revised, required adjustment and of them were correctly adjusted. of prescriptions in precaution group were correctly prescribed. conclusions: dosage adjustment or precaution was required in % of prescriptions. of these, % ( / ) needed dosage adjustment according to renal function. besides, % of patients had renal function alteration. thus, it is important to improve pharmaceutical care in this group of patients specially for those drugs that need dosage adjustment in renal failure. keywords: pharmaceutical care, hip fracture, elderly patients pc- hospital pharmacists and community pharmacists: an experiment of pharmaceutical information transmission carried out in an anticancer center anne lebreton , erwin raingeard , christelle audeval , sophie rochard anticancer center, centre rené gauducheau, nantes, france background and objective: to evaluate a programme of pharmaceutical information transmission from hospital pharmacists to the community pharmacists about drugs, particularly anticancer drugs, dispensed until then by hospital pharmacy and now distributed by them. design: while doing the last dispensation by the hospital pharmacy, an informative fax was sent to the community pharmacists indicating the name of the patient, prescribed drug and its posology, date and quantity dispensed, approximate date of the next dispensation and general information about the delivered drug. one month later a questionnaire was sent to the pharmacists to get their appreciation about this document. in the case of having no answer from them, the same questionnaire was re-sent. setting: pharmacy of french anticancer center, centre rené gauducheau, nantes. -appropriateness of the way of transmission -pertinence of information sent -efficiency of the programme results: out of patients treated ( vinorelbine oral, erlotinib, sorafenib, sunitinib), ( . %) were registered in this study, involving pharmacists. six pharmacists ( . %) answered after the first sending of the questionnaire and ( . %) answered after they received the remainder. fourteen pharmacists ( . %) did not answer. all the pharmacists were satisfied with the way of transmission. however, one of them suggested having the information sent by e-mail. seventeen professionals ( %) thought information was useful and ( %) thought that it was sufficient for their practice. only pharmacists ( %) encouraged us to continue the programme; the others did not express any opinion about its efficiency. moreover, none of them called us even though we suggested so. conclusions: this experiment seems to be interesting and to correspond to the needs of the pharmacists. it would also be an easy way to make dispensation safe. nevertheless, many problems appeared: much time spent and the difficulty making an exhaustive follow-up of all the patients thus limiting the application of this kind of programme to larger cohort. background and objective: to make a proposal of software that facilitates the analysis of clinical relevance of antiretroviral drug interactions, in the medical prescription, dispensation and dader methodology of pharmaceutical care study phase. design: pubmed and other databases evaluate revision. antiretroviral drug interactions were classified in four levels according to probability and severity of the interaction. the probability was grouped in categories: defined, probable and possible. so, severity was grouped in categories: serious, moderate, and slight. the levels are: level (serious and defined or probable); level (serious and possible, moderate and defined or probable); level (moderate and possible, slight and defined or probable) and level (slight and possible). we used the pubmed and database review for identified and organized the information to software elaboration. were by the enzymatic inhibition. antiarrhythmics, antihistaminics, ergot alkaloids, prokinetics, benzodiazepins, statines, calcium channels antagonists, phosphodiesterase inhibitors, azoles antifungics, selective serotonin reuptake inhibitors, opioid analgesic, immunosuppressants, macrolides, classic anticonvulsivants, and riphamicins were the most common drug therapeutic groups with anti-retroviral drug interactions. conclusions: interactions were classified, which % were drug-drug interactions; as well, . % were pharmacokinetics interactions and in their majority ( . %) were mediated by the enzymatic inhibition. about . % of interactions were level and , this levels of greater clinical relevance and whose the alert generated by software are contributions that help to analyze and take decisions with respect to the handling from the same ones. outcomes (e.g. mortality, morbidity, adverse drug reactions.); a profile of patients and clinical activities; an update of task description based on evidences and context. results: studies demonstrate that pharmacists have an impact on selected clinical outcomes following clinical pharmacy activities (e.g. drug therapy monitoring, pharmacokinetics and education for medical and paramedical professional). patients admitted in the intensive care unit have a higher level of complexity of care than average patients in the hospital, but a similar length of stay. for pharmacy services, their drug cost per admission is higher ( $cad vs $cad), as the number of pharmacist paid hours per admission ( . vs . ) and the number of pharmaceutical interventions per admission ( . vs . ). the approach helped us to identify solutions to problems like the nonparticipation to the cardiology patient's round, the absence of a medication reconciliation process or the inconstant documentation of interventions. a revised task description will be tested by both clinicians. conclusions: this study illustrates an approach for the evaluation of a pharmaceutical care model in a pediatric intensive care unit. background and objective: from , an increase in the consumption of biological and synthetic glues was noted in the cardiac service of surgery. these are drugs and expensive medical devices, which represent an expenditure of more than k€. the impact of their use was evaluated on the post-operative bleedings, the duration and the cost of stay by the way of a economic medical study. design: an observational exploratory study was carried out between january and march . all the operated patients were included. setting: the computerized consultation of the patient files made it possible to identify the following criteria of judgement: indication, surgeon, type of glue used, catch of platelet aggregation inhibitors or oral anticoagulants, volume of drainage, number blood transfused, duration of hospitalization. the statistical analysis related to two groups of patients, treated or not by glues, with a stratification on the hemorrhagic factors of risk. main outcome measures: the comparison of the averages of the various parameters was carried out by tests of student, for the large samples presenting comparable variables, and of fisher in the other cases. the cost of each hospitalization was calculated from the numbers of stays. results: during the three months of study, patients, whom average age was years [ - ], were operated. the two principal indications were the valvular replacement ( . %) and aorto-coronary bridging ( %). on all six surgeons, % of the interventions were dealt with by three. on the whole, patients receive a glue ( %), of biological type with tissucol Ò ( . %), and synthetic with bioglue Ò ( %), arista Ò ( %) and the grf Ò ( %). some patients received two types of glues ( . %). no significant difference between the two groups appeared in the total analysis. among the patients who received a pre-operative anticoagulant treatment ( %), only % were treated by a glue, for which the number of transfused globular bases and the duration of stay in reanimation were significantly lower (p \ . ; p \ . ), compared to untreated patients. conclusions: in general, the use of glues in cardiac surgery does not decrease the post-operative bleedings, but increases the cost of the stay. it however finds its utility with patients at the hemorrhagic risk. a standardization of the local practices of the surgeons is in progress because there is not any national consensus. these results should be confirmed by randomized studies on a large scale. although za is times more expensive than pa, it seems more effective with a shortened administration time ( minutes vs - hours). in respect of the contract of good use (cbu), our objective was to evaluate the real cost of a daily hospital (hdj) session per patient treated with bp, and to compare it with the one obtained from the national study of costs (enc) in order to determine if whether our hospital gained or loosed benefit from this new refunding status. design: retrospective study. setting: conception hospital, bd baille, marseille ce-dex , france. main outcome measures: for every hdj session, we accounted pharmacy direct expenditures (including implants, medical devices or drugs), medical technical acts, and structure and logistic supports costs. moreover, we evaluated matching of prescriptions with the cbu criteria. we analyzed data together with the public health and medical information ward and the department of management control. results: real costs of hdj session amounted to € without bp, and increased to € and € with pa and za administration, respectively. according to enc results, each session including intravenous bp was refunded for . €. in , on a total of hdj sessions ( patients), sessions had bp administration ( . %) and concerned patients ( . %). real costs of hdj were € ( € and €, for and hdj sessions with pa and za, respectively). if bp administrations were integrated in the ghs at this time, the sessions would have been refunded for € ( € and € with pa and za, respectively) resulting in a profit of € for the hospital. among bp prescriptions, % matched approved indications, . % were part of temporary protocol of use and . % were medical publication-based. internal medicine ward initiated % of the prescriptions. conclusions: using pamidronic acid compensates the deficit generated by zoledronic acid use. in respect with the cbu, zoledronic acid use will be restrained to approved indications in outpatients and pamidronic acid will be preferred in all others indications. keywords: biphosphanate, refund, economy pec- evaluation of the implementation of automated medication-dispensing system in an intensive care ward gregory gaudillot , marie antignac , fabien heck , nadine casimir , robert farinotti pharmacy, groupe hospitalier pitié-salpétrière, paris, france background and objective: the development of the implementation of automated medication-dispensing system in french hospitals is the main aim, defined in the ''good use'' contract voted in . the purpose is to improve the safety around the drug dispensing by the nurses. the objective of this work was to evaluate this implementation in thrusts: safety, economic and organization. design: comparison between two different organization ways: before the implementation (pharmacy order by nurses chief) and months afterwards (automated order and arrangement by a chemist assistant). setting: surgery intensive care unit ( beds) in the pitie-salpétrière hospital (ghps), a large teaching hospital. main outcome measures: study ''before and after'': follow up of prescribing and dispensing matches, analysis of time repartition between nurses and chemist assistants, follow up of line of emergency order, analysis of the results of a nurses satisfaction survey and costs study of drug consumption and drug storage. results: this study, performed over days, showed very small differences between prescribing and administered medications ( % before versus % afterwards = no significant difference). organization: implementation of automated device allowed a better division of activities, the management by nurses chief of the pharmacy order decreased (from % to % of their weekly working time), because this part was attributed to the chemist assistant ( % of their weekly working time). satisfaction: % of nurses preferred the automated system and especially because they find that this system was safe. in the same time, % of them appreciate with the new relationship with the pharmacy department. costs study: a huge decrease of the storage: - % of cost ( , € versus , €) and - % of references number ( versus ). in the same time, over months, the drug consumption of the unit has been reduced by % (- , €). conclusions: even if the study did not demonstrate a decrease of the number of medication errors (due to the tiny number of them as much before than afterwards the implementation), this automated system allowed a safety access to the drugs (biometric system) and contributed to reduce the risk of medication errors. the other great interest is the real involvement of the pharmacy in the clinical wards. the presence of the chemist assistant ensures a better management of drug storage (decrease of emergency order), a direct follow up and allowed a contact with nurses. results: in in france, intracranial stents with various technical characteristics are available (steel or nitinol, learning curve: to procedures, follow-up of patients is from months to years). stents with a hight radial force are used for intracranial artery angioplasty. more precisely, those devices are implanted in patients with recidive stroke, with an intracranial stenosis c % and who had failed medical therapy. two stents are identified in this therapeutic use: wingspan Ò (boston) and pharos Ò (micrus). about implantations are estimated for in france, whom implantations (cost = , €) in paris hospitals. the need is not actually clearly identified: the number of eligible patients is not already known and not systematically searched by neuroradiologists. so number of implantations will evolve. intracranial stents are used in combination with detachable coils embolization in patients with wide-necked cerebral aneurysms: léo Ò (balt), neuroform Ò (boston), entreprise Ò (cordis) and pharos Ò (micrus, double therapeutic use). for , about to implantations are estimated in france ( to % of endovascular treatment of intracranial aneurysm), whom implantations (cost = €) in paris hospitals. those data might increase. indeed, there is evidence of the effectiveness of stent implantation in intracranial aneurysm which generates lower rate of aneurysm recanalisation. conclusions: intracranial stents has been identified in precise therapeutic uses. however, an exact quantitative assessment can not be realised: those medical devices are innovating and are actually changing the management of patients concerned. those quantitative results might evolve. further similar studies will be necessary in order to follow up those innovating therapeutic uses in neuroradiology departments and to estimate their cost impact, actually negligible. keywords: stent, intracranial angioplasty, intracranial aneurysm, implantable medical device, cost evaluation pec- feasibility of economical impact of management of cytotoxic remainders in a centralized cytotoxic unit hélène corneau , déborah schlecht , sébastien bauer , sylvie froger , jacqueline grassin background and objective: the french law forecasts to reimburse the most expensive cytotoxic drugs to the real quantity administered to the patient. so to set up a secured procedure to use remainders of cytotoxic drugs in a centralized cytotoxic preparation unit in order to conform to the french law. design: prospective study. setting: clinic unit of oncologic pharmacy. main outcome measures: this procedure was tested during ten weeks and had concerned the three most expensive cytotoxic drugs used in digestive cancers. generated remaining quantities are conditioned inside isolator in radio-sterilized bags, and identified with the number of register of prescriptions, the drug's name, the remaining quantity, the conservation' conditions, the opening date, the duration of physico-chemical stability according to the data of the literature. bags are joined when they are gone out of the isolator. a theoretical differential is daily established between the doses which are prepared with or without management of remainders. results: the management of remainders involves that a rigorous manipulation during the conditioning and a daily management of the out-of-dates. the average of realized savings € a week, that represents , € extrapolated to one year and a decrease of . % of expenses for these three drugs. pharm world sci ( ) : - our software of the preparations of drugs is adapted for the use of the remainders. but the invoice-software allows only one invoice for one flask, which involves with the management of the remainders an unequal invoicing system for the patients conclusions: the management of remainders generates a real financial profit. but the really administered quantity cannot be imputed to the patient because the software of inventory control and invoicing does not manage the fraction of flasks. nevertheless, according to the french law we must express under fractional shape the quantities of cytotoxics, which are administered by stay to the patients for the most expensive drugs. background and objective: since july , financing of drugs in belgian hospitals is based on a lump-sump system. this decision favours efforts leading to more rational use of medication like for example the sequential treatment. efforts like posters, recommendation letters and information rounds were part of the strategy. since - - a clinical pharmacist puts also attention on this subject by contacting physicians and nurses regarding individual drug therapies. documenting the results of interventions suggested by a clinical pharmacist is often quite difficult. the number of ddd of intravenous administered drug versus the total amount of ddd [oral + iv] of the particular drug administered has been described. this parameter can be disturbed by the use of the oral form over a long period of time or by the early discharge of patients combined with a continuation of the therapy at home. to follow up the sequential treatment on the emergency department, this parameter seems to be accurate by the fact that the stay of the patient here is between and days. cost savings by sequential treatment of paracetamol and levofloxacin were euro and euro, calculated from the start of the activities the clinical pharmacist (for months). old habits are difficult to change among them the administration of drugs by intravenous route. almost every medical and surgical patient admitted to the emergency department receives an intravenous line, so the threshold for intravenous administration is low. furthermore a wide variety in patients' medical conditions added to a rapidly changing timetable for technical examinations and surgery necessitates an individual approach of the most suitable route of drug administration. official letters and posters may be useful. however this report confirms the importance of a clinical pharmacists' permanent presence on the ward for maintaining awareness of sequential treatment. results: applications for compassionate use treatment were processed ( . processings/ inhabitants). the higher number of applications has corresponded to gynecology unit, processings of misoprostol to use in delayed curettage. secondly, botulinum toxin was processed in cases, . % of them corresponding to anaesthesic and reanimation unit for miofascial pain. active substance that has caused a higher impact on the hospital s budget was inhalated tobramicin, processed for a total of patients with bronchiectasias colonized by pseudomonas aeruginosa. the expenses for this indication has added up to , € during . secondly, as regards to economic impact we found botulinum toxin which raised up to , €, expenses that were attributed in % to anaesthesic and reanimation unit. the highest cost/treatment by patient during corresponded to inhalated tobramicin that has increased up to , €, followed by infliximab approved for hydrosadenitis treatment which accounted for , €. total cost of treatments for compassionate use has involved approximately . % of total consumption for drugs during . conclusions: the higher number of processings in corresponds to misoprostol requested by gynecology unit although its economic impact on the consumption of medicinal products for compassionate use is very low. inhalated tobramicin utilization for colonized bronchiectasias involves the highest global cost and the highest cost/treatment on total medicinal products for compassionate use. the percentage accounted for medicinal products for compassionate use on total consumption of medicinal products is low. it would be interesting to perform a multicenter study in order to value economic impact in last years of medicinal products for compassionate use. the vacuum-assisted closure (vac) therapy: a -months medico-economic retrospective study stéphanie roche , nathalie herment , sandrine havet , amélie pruvost , willemin jean-claude , frances carole pharmacie, reims teaching hospital, reims, france, background and objective: in the management of wounds care, spectacular results have been achieved through the application of negative pressure wound therapy. this approach known as vacuum-assisted closure (vac) involves the use of a defined controlled negative pressure (delivered by an ambulatory motor) over a polyurethane or polyvinyl sponge (which are considered as consumables) placed in the wound. in our hospital, this therapy was first introduced for acute traumatic wounds. in june , the interdisciplinary wound and cicatrisation group decided to extend indications to chronic wounds. to improve management regarding this larger and complex use through all units, a specific prescription form associated with recommendations was set up. this document is available as well as paper or electronic form. the purpose of this study was to evaluate conditions of use and global costs of vac therapy. design: a -months retrospective study was based on the analysis of nominative prescriptions of vac consumables (canisters with gel, small, medium or large foam dressing kits and y connectors . the cost of consumables was estimated to . euros per day per patient. total cost over the study period amounted to euros, including the hiring of the vac system ( . euros per day). these data indicate that the use seems to be appropriate and optimized without overuse. conclusions: facing the high cost of this technical therapy, its use must be closely managed. this study suggests that the multidisciplinary collaboration in our hospital between medical staff and pharmacist unit contributes to guarantee the optimal use of this specific therapy. to analyze the effectiveness of an educational programme to increase the adrs reports during this period. design: clinically relevant events possibly caused by exposure to drugs have been analysed in a retrospective study for a period of five months. we analysed the prevalence of adrs reported, the medical conditions of the patients, the relationship between adr and the suspected drug using karch-lasagne algorithm and the severity of the reaction using who criteria. during this period, we implemented a pharmacovigilance programme in order to increase the reports and to estimate the prevalence of adr in this unit. setting: pharmacy service and critical care service of a general hospital main outcome measures: to evaluate prevalence, characteristics, nature and severity of adrs in uci. to measure the effectiveness of an educational programme to improve the awareness and detection of adrs. results: a total of patients were hospitalized during this period, adrs were detected ( . %). severe sepsis and cardiac arrest were the most frequent diagnosis in this group of patients. . % males and . % females, . years in average. dermatologic effects as urticaria rashes and haematological effects as pancytopenia were the most frequently noted, with more than % each. therapies most often associated with the reported events were antibiotics (piperacillin/tazobactam, ertapenem, azitromicin) in . % of cases and nitroglycerin in . % of them. the adr reported were classified as low severity in . % of cases, medium in . % and high severity in % of them. level of causality more frequent was ''probable'' in a . % of the reports followed by ''possible'' in . % of them. since the implementation of the educational programme the number of reports of adr have increased from . % to . %. the high level severity of adrs reported has increased in this period from % to %. conclusions: communication and educational programmes should be implemented to promote detection, identification, reporting and evaluation of adrs. the analysis to determine the probability, causality and severity of adr is necessary to establish the measures needed to improve the security and the quality of health attention. background and objective: ace inhibitors are used for controlling blood pressure, treating heart failure and preventing kidney damage in people with hypertension or diabetes. although ace inhibitors are generally well-tolerated by the most individuals, they are not free of side effects. dry cough is one of the most common side effects seen in patients during ace inhibor therapy. in this study, we have evaluated the incidence of dry cough that appears during the ace inhibitors therapy and relationships with the other coughing factors and the other side effects that may appear. design: the questionnaire was applied on ambulatory patients ( m/ f) who have used or been using ace inhibitors and hypertension patients as control group who use also anti-hypertensive drugs except ace inhibitors. ace inhibitors, and the reactions against the dry cough were determined by pharmacist's questionnaire on the patients who come to the community pharmacy. results: dry cough was observed on the patients out of , during their using of ace inhibitors ( %). % of male patients and % of female patients were having a cough. the patients out of ( %) from control group were having a cough. the incidence of dry cough that appears on the patients who use ace inhibitors were; silazopril %, ramipril %, lisinopril %, fosinopril %, qinapril %, perindopril %, enalapril % and trandolapril %. the treatments of patients out of who complain from coughing during the therapy of ace inhibitors were changed with angiotensin receptor antagonists and calcium antagonists by their physician. treatment changes were resulted in increasing in the cost by . ytl monthly if it was calculated on the base of generics. conclusions: as a conclusion, the incidence of dry cough from ace inhibitors was found to be % in the blacksea region of turkey. the pharmacist can play an important role in determining side effects such as dry cough and refer the these patients to physician. patient counselling and drug therapy monitoring in the community pharmacies will increase the compliance and provide better outcomes in many chronic diseases. setting: community in _ istanbul main outcome measures: visual analouge scale (vas) was used for assessment of pain. the universe of the study consisted of randomly selected women and men (n = ). a pilot study was run on individuals from different occupation groups to determine the validity and intelligibility of the questionnaire. results: the data were analyzed using a spss . program. the level of significance was accepted as p \ . . the results show that women experience more intense pain than men; their mean vas score is higher than men ( vs ). headache is the most common type of pain ( . %) and also its vas scores reached the highest level ( ) ( ) . this pain is especially caused by migraine and hypertension. in addition, % of questionees prefer to take an analgesic drug in order to manage their pain problem. it was recorded that especially non-steroidal antiinflammatory drugs (nsaids) and preparations containing paracetamol are the first choice in pain management. most respondees took analgesics when pain begun ( . %), when pain increased ( . %) or when was intolerable ( . %). conclusions: the majority of those who participated in the study took their analgesic medication without consulting a health professional. this indicates a high level of self medication in our population. this gives rise to a number of potential drug problems such as nsaids usage in gastrointestinal disorders, hypertension and renal failure. although many participants used their drugs in a rondom and improper way, unfortunately the pharmacist was rated second to last as a drug consultant. background and objective: many patients visit the pharmacy for their oral problems like toothache and ask for appropriate pain relievers. the purpose of this study is to examine the attitude and role of pharmacists, dentists and non-health workers towards solving of dental and oral health (like management of toothache). design: different questionnaires were applied randomly on pharmacists, dentists and non-health workers. setting: canakkale -turkey. main outcome measures: dentists' and pharmacists' approach to patients with toothache, drug usage evaluation in dental problems, the type of information given by pharmacists, patient behaviors. results: % of non-health workers (n = ) indicated that if they complain about toothache they choose their pharmacist as their consultant. % of non-health workers (n = ) indicated that they use various medications without consulting a dentist ( % naproxen sodium, % paracetamol, % methimazole, % flurbiprofen, % amoxicillin, % carnation essence). the most common suggested antibiotics was amoxicillin ( %) by pharmacists and amoxicillinclavulanate ( %) by dentists. the most common suggested pain relievers were naproxen sodium by both pharmacists ( %) and dentists ( %). only % of dentists declared that they consult with a pharmacist about drug usage. % of dentists indicated the importance of consultation of patients by pharmacists. conclusions: according to our results; pharmacists must take an important role in prevention and management of oral and dental health problems including informing the patients about convenient drug usage. also collaboration between dental staff and pharmacists need to be improved. background and objective: in hibbard and smithells suggested a link between inadequate maternal intake of folic acid and neural tube defects in their offspring ( ) . consequently, it has been recommended that all women planning to become pregnant should consume additional folic acid before conception and during the first weeks of pregnancy. despite these recommendations, periconceptional intake of additional folic acid is still low in many developed countries and a substantial percentage of women are not aware of its benefits ( ) . design: a questionnaire was used in a face-to-face encounter. setting: postnatal wards of a teaching and a private hospital in iran. main outcome measures: awareness of the effects of folic acid on the fetus was evaluated among women. the questionnaire included questions about demographic information, folic acid supplementation before and during pregnancy, its effects and the most susceptible periods in pregnancy and the source of information regarding the drug's effects during pregnancy. results: the mean age of women was . (± . ) years old. the majority of the subjects had more than high-school education ( % vs. %). out of subjects, ( . %) took folic acid supplement before and during pregnancy. only . % believed that its usage was unnecessary, . % believed in its positive effects. in the subjects' opinion, the most important time for taking this supplement was the first trimester ( . %), then prior to pregnancy ( . %). the second and third trimester were noted important by . %. . % believed in the importance of this supplement during all nine months. the advisor for taking this supplement was doctors ( . %), health visitor ( . %), self-medication ( . %), tv and radio ( . %), family members and friends ( . %) and pharmacist ( %). conclusions: awareness of the value of periconceptional folic acid was high among women of iranian nationality compare to similar studies ( ) . the majority of the participants believed in the positive effects of folic acid. the advices provided by doctors and pharmacists had the greatest and least effect on the use of this medication. regarding the best time of usage of this supplement, the most emphasis was on the first trimester and next on prior to pregnancy. background and objective: diabetes mellitus type-ii is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. before type ii diabetes stage, people almost always have ''pre-diabetes''; in which blood glucose levels are higher than normal but not yet high enough to be diagnosed as diabetes. while some people with type-ii diabetes have symptoms, the majority may go - years without apparent symptoms. because some of the symptoms for diabetes mimic other diseases or conditions, makes it harder to predict an precise diagnosis without any additional information. the purpose of this study is early diagnosis of pre-diabetes, prevention or delay of the complications with a collaboration of community pharmacists and patients. design: pharmacists used a structured questionnaire containing questions concerning demographic data and informations which indicate prediabetes symptoms. setting: four community pharmacies in istanbul. main outcome measures: data: age, gender, body mass index (bmi), genetic predisposition, blood pressure, pysical activity and hypoglycemia symptoms. results: one hundred people were screened for undiagnosed diabetes. and the risk for pre-diabetes is evaluated according fasting plasma glucose (fpg) and total oral glucose tolerance (ogtt) test results. blood glucose level between and mg/dl with the fpg test and and mg/dl with ogtt test are considered as pre-diabetes. conclusions: diagnosis of pre-diabetes can prevent the development of type ii diabetes and making changes in nutrition and increasing the level physical activity may even be able to return the elevated blood glucose levels to the normal levels. pepi- awareness among pregnant women of the effects of drugs on the fetus and mother maryam dilmaghanizadeh , simin mashayekhi , masoud naghizadeh , zahra fardiazar , roghaiieh bamdad moghaddam pharmaceutics department, the faculty of medicine, tabriz university of medical sciences, tabriz, iran (islamic republic of) background and objective: since the talidomide catastrophy [ ] concern about the safety of drugs in pregnancy has been increasingly evident. studies have revealed that pregnant women continue to take considerable quantities of drugs. however, all pregnant women worry about whether to take any medications. because of an estimated % of birth defects resulted from maternal drug exposure [ ] , this fear is well justified. little is known about the knowledge of pregnant women regarding the safety of medications during pregnancy. to our knowledge the present study is the first performed in iran. design: a questionnaire was used in a face-to-face encounter. setting: postnatal wards of a teaching and a private hospital in iran. main outcome measures: awareness of the safety of drug use during pregnancy among women. the questionnaire included questions about demographic information, drugs use before and during pregnancy, information regarding the safety of drugs during pregnancy and the most susceptible periods in pregnancy, the source of information regarding drugs' safety during pregnancy. results: the mean age of women was . years old. only an . % and . % used conventional medications and herbal remedies during pregnancy, respectively. a great percentages ( . %) believed in harmfulness of drugs during pregnancy, but only a . % believed in harmfulness of herbal remedies. the first trimester and the second trimester were believed to be the most and the least susceptible period, respectively ( . % vs. . %). the sources of information for the subjects regarding the safety of medications was specialist doctors ( . %), general practitioner ( . %), pharmacist ( . %), midwives ( . %), health center ( . %), media and books ( . %) and friends and family member ( %). discussion: a common concern about the care of pregnant women involves the medications, which led us to establish a special initiative to review available knowledge among our general population. the present study highlights weakness of the role the pharmacists play in providing the information to this vulnerable and eligible group of people, who nourishing our next generation. conclusions: a common concern about the care of pregnant women involves the medications, which led us to establish a special initiative to review available knowledge among our general population. the present study highlights weakness of the role the pharmacists play in providing the information to this vulnerable and eligible group of people, who nourishing our next generation. ayce celiker , nergiz nemutlu , gulru ozkaya hacettepe drug and poison information center, hacettepe university, ankara, turkey background and objective: poisoning casualties require be approached with utmost attention due to their ''medico-social emergency'' nature. children are more vulnerable because of their inherent interests for knowing the environment besides ignorance and carelessness of adults. drugs are among the leading offending agents in children poisonings. the services of drug and/or poison information centers have been regarded as one of the main challenge areas of clinical pharmacy practice. being a pioneer in turkey, hacettepe drug and poison information center (hizbim), has been run for years in working hours basis. objective: the objective is to evaluate the demographic and epidemiological characteristics of drug poisonings in children, thus, to contribute to clarify the actual ''intoxication profile'' and identify necessary measures for the children in turkey. design: the data of childhood (aged \ years) poisoning enquiries received by hizbim between january , and december , were collected retrospectively and analyzed with spss . Ò setting: hizbim is affiliated to hacettepe university, faculty of pharmacy main outcome measures: categorization and comparison of data of children intoxicated with drugs during ten years results: children were involved in % of all poisoning cases and % of those cases were due to drugs. there was no gender difference among very young children, however, girls dominated as the age increases (p \ . ). % of the cases were accidental, and analgesics were involved in % of the accidental poisonings (p \ . ). while the most offending agent group was analgesics in children younger than years (p \ . ), multiple drug ingestions were the main causes of the cases involved older children (p \ . ). multiple drugs were mostly encountered ( %) in suicidal attempts where the dominant gender was girls ( %) (p \ . ). the most frequently reported symptoms indicated central nervous system involvement almost in all intoxication cases and in all age groups. conclusions: pediatric poisonings are rather high in turkey like many other countries and drugs are accounted for mostly in those injuries whether accidental or suicidal exposures. some regulations, the attitudes of physicians, pharmacists, and parents and other care givers make contributions to that outcome. in the prevention of childhood drug intoxications it is essential to make cooperation between drug manufacturers, regulatory authorities, health professionals, and families, besides increasing social awareness. drug and/or poison information centers stimulate rational drug use through providing accurate and rapid information to health care providers, educating people directly, documenting current epidemiological data. recently, we noticed a great interest of pharmaceutical companies in drugs such as monoclonal antibodies or in diseases such as inflammatory rheumatism. the aim of this study is to put in evidence the orientations of biomedical research. we particularly analysed the pathologies concerned, the type and the target of the experimental drugs, and the aim of the biomedical researches. design: analysis of the protocols and of the investigator's brochures of the ongoing clinical trials in january . setting: clinical trials sector of lapeyronie-arnaud de villeneuve hospital, montpellier. main outcome measures: methodological aspects were first analysed: aim of the study, phase, type of sponsor, type of binding, inclusion rate. then, we focused on the experimental drugs involved: type of drug, target, therapeutic area concerned. results: % of clinical trials are promoted by pharmaceutical companies. % are phase trials, % are open-label trials. objectives can be found: testing a new molecule on a new target ( %), testing a new molecule in an known pharmacological group ( %), testing a new formulation ( %), testing an known molecule in a new indication ( %), evaluating a therapeutic strategy ( %). % concern rheumatology, % pneumology, % infectiology, % haematology, and % nephrology. the most frequent pathologies concerned are inflamatory rheumatism ( %), malignancies ( %), asthma/cobp ( %), hiv infection ( %), and post renal transplantation immunosuppression ( %). the percentage of patients included (number of patients in the trials concerned/total number of included patients) and the inclusion rate (real number of inclusions/ number of expected inclusions)are respectively % and % in rheumatology, % and % in pneumology, % and % in haematology, % and % in infectiology and % and % in nephrology. % of experimental drugs are injectable. % are little molecules obtained by chemical synthesis, and % are issued from biotechnologies: monoclonal antibodies ( %), peptide ( %), and gene therapy ( %). the new targets ( % of clinical trials) are receptors ( %), enzymes ( %), gene transcription ( %), cytokines ( %). conclusions: this analysis gives an idea of the future commercialized drugs. it puts in evidence the development of drugs in therapeutic areas which concern a lot of patients and whose financial rentability is high. furthermore, only % of clinical trials concern new drugs and new targets. development of me-too and optimization of therapeutic strategies are the most frequent clinical trials. background and objective: the increasing use of performanceenhancing substances and methods in sport threatens not only the meaning and the ethical value of the sport itself, but also the health of the athletes. this study aims to assess the drug utilization profiles of the amateur football players, as well as their attitudes and knowledge on ''performance enhancing drugs''. design: this study was conducted on male players of the amateur football league. the players were asked to fill in a standard questionnaire, where information about their drug utilization profiles as well as their attitudes and knowledge on ''performance enhancing drugs'' were sought through various questions. setting: various amateur football clubs in turkey. main outcome measures: drug consumption rates of the players. answers to the pre-prepared questions. results: the age of the players ranged between and years. forty-four ( %) players thought that drugs have a positive impact on sports performance; while % did not share this idea. the resource of this idea was a team-member for . %, the trainer for . %, sports magazines for . % and a family-member for . %. the players were asked to name the drugs that could be used for performance enhancement and . % replied as vitamins, while . % replied as central nervous system (cns) stimulants and . % replied as anabolic steroids. thirty-six players ( . %) reported that they use various drugs with the aim of performance enhancement. the drug utilization profile was as follows: vitamins were consumed by . % of the players; where cns stimulants, anabolic steroids, diuretics and growth hormone were consumed by . %, . %, . % and . % of the players, respectively. the players reported the reasons referring them to drug-use as follows: . drugs always have a positive impact on sports performance ( . % of the drug-users); . drugs are necessary in case of inadequate training ( . % of the drug-users); . performance enhancement leads to individual success in the team and this brings prices and rewards in return ( . % of the drug-users). about % of the players thought that cns stimulants have the main effects of increasing the heart rate, endurance and strength. the main potential adverse effects of the cns stimulants were reported as gastrointestinal problems, dependency and tachycardia. about % of the players thought that anabolic steroids increase muscle volume, endurance and strength. the main potential adverse effects of the anabolic steroids were reported as hypertension, hepatotoxicity and dependency. conclusions: the results of the questionnaire suggests that drug-use with the aim of performance-enhancement was common among the amateur football players; and the players were not adequately and properly informed on the effects and adverse effects of the (so called performance-enhancing) drugs. this reality yields new responsibilities in this challenging area of practice, for the clinical pharmacist. background and objective: psychiatric disorders and opiate misuse are associated with chronic pain syndromes, but their incidence in fibromyalgia (fms) is unknown. the aim of this study was to identify if the incidence of affective disorders, and opioid misuse, was more common in patients with fms than those with other forms of nonmalignant chronic pain. design: a prospective, cohort study was carried out involving patients, who were internally referred to a chronic pain management program. subjects with a working diagnosis of fms were matched by age (mean = ) and sex ( % female) and compared to a control group of patients with alternative forms of non-malignant chronic pain. individuals were compared using urine toxicological screens, drugrelated criminal convictions, diagnoses of affective disorders, and responses to the following inventories: screener and opioid assessment for patients in pain, the pain disability index, the personal health questionnaire and the fibromyalgia impact questionnaire. patients also underwent a standardised physical examination using american college of rheumatology (acr) guidelines to diagnose fms. setting: this was a quantitative, hypothesis-testing cohort study, conducted in an academic general internal medicine practice. main outcome measures: diagnosis of fibromyalgia, clinician's awareness of acr guidelines, pain intensity and impact of fibromyalgia on physical and psychosocial activity. results: response rate was % (n = ; mean age = , median = , age range = - ; male = ); the most common background and objective: the increase in the frequency of the metabolic syndrome and its implication, in the development of ischemic cardiovascular disease and type ii diabetes mellitus, represent a real public health problem and of much interest in the medical field. otherwise, the cardiovascular pathologies are twice more frequent among chronics psychotics patients, for that reason we were interested in the prevention of these pathologys. this phenomena have been accentuated with the arrival of second generation antipsychotic drugs which were associated with weight gain, disorders of glycemia and lipidemia. in the aim to elaborate recommendations, at first we proceeded to the assessment of biological and clinical follow-up of the patients hospitalized in the saint-egreves hospital. design: literature review, months prospective study. setting: we asked doctors of each unit ( ) to answer questionaries corresponding to patient files. we worked out a general questionary to avoid revealing our objective. main outcome measures: blood pressure, body weight, height, abdominal perimeter, glycemia, total cholesterol, triglyceridemia, cholesterol's fractions(hdl and ldl). results: our results showed a good clinical follow-up but the frequency of biological control was not sufficient. the blood pressure and weight were evaluated respectively in and % of the patients, the total cholesterol, glycemia and triglyceridemia were in % of patients. on the other hand, cholesterol's fractions (hdl and ldl) were rarely evaluated. as for the abdominal perimeter, where the increase is predictive of cardiovascular disease, is never measured. conclusions: it seems difficult to evaluate the risk of either cardiovascular disease or metabolic syndrome of these patients or to determine if there is any possible relationship between the nombre of cardiovascular risk factors and the apparition of this syndrome. these findings imply to identify these high risk subjects and to define the optimal preventive, or curative, management strategy of metabolic syndrome and this, through simple measurements to realize in clinical practice. background and objective: availability of a medicine in western markets can be delayed either due to differential submission strategies of companies or differences in review process between countries. the aim of this study was to examine delays in patient access to medicines for compounds approved by two or more authorities (us fda, eu emea, australian tga, health canada and swissmedic), by characterising potential drivers for new active substances (nas) approved between and , from both a company and regulatory agency perspective. design: nass approved by fda since were compared to nass approved by the other agencies. this data was analysed comparing the difference between submission and approval dates and characterised by; type of approval route, company size, and therapy area. setting: data on nas's approved by the authorities was collected from agencies and from public domain sources. main outcome measures: the difference in patient access to new medicines in different countries and factors influencing such differences. results: nass have been approved by fda and one or more of the agencies studied. the median time ranged between submissions to fda and another authority from days at emea to days at tga. the difference between approval dates ranged from a median of days at emea to at health canada. however these differ depending on company size, therapy area and approval route. conclusions: availability of a new medicine is a mixture of company submission strategies and approval process, although therapeutic profile of submissions and company size are also influencing factors. in europe the main driver to patient access is review timelines rather than delay in submission by companies. main outcome measures: data on sex, age, origin of ppi prescription and indication were collected by a standardized questionnaire and were retrospectively analysed. results: men and women were reviewed. the median age was years (range, - ). % of patients received ppi therapy by pantoprazole (available in our hospital) when hospitalized. % of patients received daily mg of pantoprazole and % received mg a day. % of the prescriptions were validated. the main off-label indications were prevention of hemorrhagic risk of antiplatelet agent ( %), hemoglobin decrease( %), anticoagulant co-prescription( %), steroids co-prescription( %). conclusions: this prospective study confirms the large prescription of ppi therapy in a department of internal medicine. nevertheless, this study highlights the difficulties to interrupt this well tolerated therapy after the first prescription by family physicians. clinical pharmacist interventions in the department consist of explaining the difference of indications between pantoprazole mg and pantoprazole mg, he makes physicians aware of prescribing ppi therapy with a cautious reweighted cost/benefit consideration. background and objective: to develop and validate a system for regulatory authorities to provide feedback to companies on the quality of their submissions while companies report to authorities on the quality of the review. a standardised report format will allow performance comparisons within and across companies and agencies. design: draft scorecards were tested in a study on the same four compounds, each reviewed recently by the agencies in australia, canada and switzerland. the agencies provided feedback on the quality of submissions and sponsors, astrazeneca, gsk, novo nordisk and pfizer gave views on the conduct of reviews. background and objective: adherence with chronic medication such as inhaled corticosteroids (ics) has repeatedly been reported to be low. non-adherence could be related to inadequate knowledge of ics' actions and lack of ics' instructions on the use of inhalers. this has not been reported previously to our knowledge among new users of ics who discontinued ics treatment early. the aim of the study is therefore to describe, among new users of ics that discontinued, their knowledge of ics' actions and whether they were instructed on the use of their inhaler. design: a cross-sectional study among new users of ics that discontinued use. patients were interviewed by telephone and their gp received a mailed questionnaire. automated dispensing records of all patients were retrieved. setting: community pharmacies in the netherlands. main outcome measures: by use of conditional logistic regression the association between knowledge and study variables was assessed. results: from eligible patients, ( . %) were interviewed. the majority ( . %) of these new users of ics who discontinued ics early was not aware of the anti-inflammatory actions of ics. most patients ( . %) were instructed on the use of their inhaler, predominantly by the gp ( . %). after adjusting for symptom experience by acq, asthma diagnosis, having persistend asthma or use of medication only age (or . % ci . - . ) and male gender (or . % ci . - . ) were associated with unawareness of anti-inflammatory actions. conclusions: this study shows that a substantial number of new patients that did not refill their ics prescriptions, were unaware of ics' inflammatory actions. surprisingly only age and gender seemed associated with awareness of ics actions. most patients were instructed on the use on their inhaler by a health care provider. physicians and pharmacists could cooperate in identifying and motivating these patients to continue ics use. background and objective: to assess the quality of antibiotic therapeutic drug monitoring (tdm) in routine hospital practice and establish baseline status for rationally defining future actions aimed at improving it (by implementation of clinical pharmacy services). design: months prospective observational study with validated data collection form using predefined criteria for tdm quality assessment. descriptive statistics performed with spss . for windows Ò . setting: orthopaedic surgery, general surgery, neurosurgery, vascular surgery, haematology and pulmonary wards of a beds teaching hospital, using vancomycin twice daily and amikacin once daily administration schemes. main outcome measures: adherence to predefined criteria for sample timing, information transmission, and follow up of dose adjustment recommendations. criteria: (i) sampling time: less than ± min (amikacin) and ± min (vancomycin) deviation from preset time for peak levels; less than ± min for trough levels; (ii) information transmission: patient's full name, dose, schedule of administration, time of previous and current dose, actual time of peak and trough level sampling; (iii) quality of the analysis [internal and external controls]; (iv) acceptance of dose adjustment (more than %) recommendations. results: inclusion: patients ( vancomycin and amikacin courses). correct sampling times: (i) peak levels: % (n = ) for vancomycin and % (n = ) for amikacin, (ii) trough levels: % (n = ) for both antibiotics. correct information transmission: % (n = ). no issue noted for the quality of the laboratory analyses. implementation of recommendations: % (n = ) for vancomycin and % (n = ) for amikacin. conclusions: incorrect sampling times and deficiencies in communication between the ward and the laboratory are key factors affecting the quality of tdm, leading to dosage adjustment recommendations that are only infrequently implemented. the companion abstract examines the underlying reasons for such poor performance of the tdm process using a qualitative approach. corresponding values of d were: background and objective: to evaluate the relation between vancomycin and amikacin pharmacokinetic (pk) parameters in an intensive care unit population. design: data from intensive care unit patients were collected over a -month period, through a retrospective review of medical records and therapeutic drug monitoring (tdm) reports. patients were included only if at least two blood samples, at steady state conditions, had been drawn. data were first evaluated for completeness and consistency of recorded sampling and dosing times. individual pk parameters were estimated (bayesian analysis) using a one-compartmental pk model for amikacin and a two-compartmental pk model for vancomycin (pks Ò abbot). phase i of the study determined relationships between vancomycin and amikacin pharmacokinetic parameters, mainly clearance and volume of distribution. for that purpose, linear regression analysis of data from out of patients (analysis dataset) was performed. phase ii tested the predictability of the developed equations in an additional sample of patients (validation dataset) by comparing predicted pk parameters from equations (predeq) to those estimated by bayesian analysis (predbay) . t-test between predeq and predbay from each antimicrobial was performed. bias and precision were evaluated calculating the mean prediction error (mpe) and mean absolute error (mape), respectively (s-plus ). setting: intensive care units. tertiary university hospital. main outcome measures: patients demographics, clinical and tmd records, creatinine clearance by cockcroft-gault, vancomycin and amikacin blood levels and pk parameters. results: eighty-three critically ill patients ( females, males)were recruited for the study (mean values: age . yr, weight: . kg, cr: . mg/dl). a correlation between vancomycin and amikacin regarding their cl was found (clvancomycin = . clamikacin + . ; r = . ). however, no correlation was observed for vd (r = . ). concerning phase ii, differences in demographic data from both datasets were not statistically significant. no significant differences were observed when performing predeq versus predbay t-test. nevertheless, boxplot graphs for predeq and predbay residuals showed a wide variability of the values distribution and a lack of precision for both antimicrobials. conclusions: in our patient population this studied approach reveals an existing relation between amikacin and vancomycin pk parameters (or vice versa). however, the poor precision and large bias of residual values prevents us from recommending the use of these equations as pk parameters predictors (or regimen dose predictors) in intensive care patients. further studies with larger samples are definitely required in such an heterogeneous population. they were asked to identify which items of the ashp guidelines and gedefo they considered that must be filled in, in a prescription or in an identification label. consensus was defined as an agreement rate c %. prescriptions/labels evaluation: all breast or colon intravenous chemotherapy prescriptions, from a central hospital, have been evaluated from january to december (n = ), based on the parameters identified in the consensus document. a two month analysis of identification labels was performed. results: consensus document: a total of hospital pharmacists ( . %) completed the rounds of the delphi. consensus was obtained for . % of the prescription items and for . % of the labels items. prescriptions/labels evaluation: more than / of the analysed prescriptions were for breast cancer ( %) and the rest for colon. none of the analysed prescriptions had all the consensus items filledin. information that allowed the validation of the prescription by the pharmacist (ex: height, weight, body surface or number of cycle) was present in less then % of the prescriptions. no one had the prescriptor telephone, or the justification for dose reduction (when appropriate). only . % ( / ) of the labels mentioned the full identification of the solvent ( % miss the concentration) used and none of them stressed out the need for filter use when applicable. conclusions: consensus was obtained about a large number of items, which may constitute a difficulty in daily practice the evaluation of prescriptions highlights the lack of information that could allow confirmation by the pharmacist. labels do not seem to alert about special administration conditions. background and objective: background: the ministerial advisor on hepatitis c in catalonia has established a series of recommendations concerning hepatitis c treatment distinguishing between two groups: viral genotypes and , and viral genotypes and . in genotypes and it is necessary to evaluate treatment continuity after and weeks, depending on viral load and it is also necessary to prolong the treatment to weeks if there is viral response. in genotypes and it has not to be longer than weeks regardless of viral load. objective: to evaluate the adequacy of hepatitis c treatment and analytic monitoring, following the recommendations of the catalonian ministerial advisor. design: observational and retrospective study. setting: patients that started treatment with peg-interferon plus ribavirine in this hospital during . the information obtained was: viral genotype, the beginning and the end of treatment, quantitative basal rna (in all genotypes), quantitative rna ( weeks) and qualitative rna ( weeks) in genotypes and and at the end of the treatment in all genotypes. main outcome measures: the application of global advices ranges from % to % according to viral genotype and established recommendation. results: the compliance degree in genotypes and : % application of quantitative basal rna and % after weeks. % of treatments were discontinued with quantitative rna positive after weeks, % with qualitative rna positive after weeks and % the treatment was continued longer than weeks. in genotypes and : % application of quantitative basal rna, % application of qualitative rna after weeks and % the treatment was continued longer than weeks. conclusions: following the recommendations on viral response evaluation after and weeks allows the early suspension of therapy in non-responsive patients. this leads to and improvement in patients' quality of life, a reduction in adverse side-effects and savings in medical care costs. treatment monitoring by hospital pharmacist provides medical decision support. in consequence these patients constitute a target group to establish pharmacy care programs focused on hospital outpatients. topical application of mitomycin . % during two minutes and postoperative stenting for a period of four-ten weeks were used, no second application of mitomycin was used. the follow-up was years in bilateral case and six months for the unilateral case. setting: pharmacy and otorhinolaryngology service. hospital universitari joan xxiii de tarragona. spain. main outcome measures: the success of the repair was measured according to the following items: endoscopic evaluation of the patency of the choanae, respiratory distress and nasal drainage. results: bilateral membranous choanal atresia was surgical repaired five days after birth, using transnasal endoscopic approach and topical mitomycin . % during de proceeding. no operative complications occurred and stents were removed four weeks after repair. the choanae was inspected endoscopically to asses healing and no presence of re-estenosis was found. no clinical symptomatology. unilateral mixted atresia on the left side was diagnostic at six years old and surgical repaired because of association of respiratory distress and nasal mucus drainage symptomatology. no restenosis has appeared and syntomatology has improved. nome patient required surgical revision. conclusions: although, the exact role of the topical application of mitomycin . % must to be further investigated, the use of this drug as an adjunct to the surgical repair of choanal atresia may offer decreased need for revision surgery due to re-estenosis. keywords: mitomycin . %, choanal atresia pt- adherence to clinical guidelines for upper respiratory and ear infections in out-of-hours primary carea retrospective study sara claesson biofarmaci, uppsala university, täby, sweden background and objective: infections in the upper airways and ears are a frequently occurring reason for patients to visit primary care settings. prescribing adherence to local guidelines for handling infections of ears and upper airways and antibiotic prescribing is of both local and national concern. increasing antibiotic resistance is one reason, cost and patient quality of care are others. the objective of this study was to investigate physician adherence to clinical guidelines at the out-of-hours primary care clinic in täby. design: a retrospective study. clinical case notes were scrutinised for all patients seeking care for problems with ear, nose, throat, fever, cough or cold between january and march . data was analysed for patients who were diagnosed with specified ear or upper respiratory infections. laboratory tests taken and antibiotic prescriptions were anonymously documented. antibiotic prescription filling dates were investigated. setting: husläkarjouren, the out-of-hours primary care clinic in täby. main outcome measures: adherence to and fulfillment of local therapeutic guidelines and professional quality criteria was defined with respect to immediate, delayed or no prescription, drug choice, dose and duration and the use of diagnostic tests. adherence was defined as complete or not, and deviations from the guidelines were separately analysed. prescription filling was analysed with respect to time from clinic visit to pharmacy visit. results: data from patient visits were analysed. adherence to local guidelines was disappointingly low. general treatment of infections was only according to guidelines in % of the cases and only laboratory testing met the quality criteria. adherence to antibiotic prescribing guidelines was even lower, only % of antibiotic prescriptions were completely according to local guidelines. % of all antibiotic prescriptions were filled within one day from the visit to the clinic. conclusions: communicating guidelines to prescribers and continuous follow up of prescribing behaviour is essential for improving patient care and decreasing the risk of antibiotic resistance in the community. this study exposes gaps in the quality of care that may not be picked up by traditional follow up measurments. studies with a wider scope and in depth analysis of reasons for nonadherence to guidelines are warranted if antibiotic use is to be improved. . lines after other therapies. before bortezomib one patient received therapeutic with thalidomide/dexametasone, one vad, one cyclophosphamide (ctx), three followed by vad, one mp followed vad and followed ctx, and another with vad followed by ctx. the therapeutic selection followed the nccn guidelines in all patients. the average number of cycles with bortezomib has . . from the patients, stopped, one had a generalized face oedema bortezomib related, although the disease was in remission, and another died by sepsis not related with this drug. in relation to bortezomib's effectiveness, ( %) patients had a very good response, since the immunoglobulin decreased and three ( %) of these patients are actually at consolidation cycles ( th and th). conclusions: the use of bortezomib in our hospital was according the nccn guidelines and the experience was very positive. nevertheless, and considering also the high cost of this therapeutic, we consider very important to continue to follow up this group in order to evaluate the rate of response to bortezomib during time. pt- evaluation of the time interval between admission on the emergency department and administration of the first dose of antibiotics background and objective: when patients are admitted with a proven or suspected infection on the emergency department, adequate antibiotic treatment must be started as soon as possible. literature reveals that the time between admission and administration of the first dose of antibiotics can reach about hours and this can influence the prognosis of the patient. international guidelines for community acquired pneumonia (cap) and bacterial meningitis define this time interval of hours( ) and less than hours( ) respectively. to evaluate the practice on the emergency department of our hospital, with an average daily admission of patients, a study on this interval was carried out. . % of all administered antibiotics is in adherence to the local guidelines; however this was only evaluated by the clinical pharmacist. a possible explanation for the relatively short time intervals could be that the antibiotics which are frequently used are available at the emergency department and that the guidelines are at all time available on our hospital intranet. the time intervals of cap and bacterial meningitis are shorter than the defined international guidelines.( , ) conclusions: the time interval, found in our study, between admission on the emergency department and administration of the first dose of antibiotics is short, compared with similar conducted studies.( , ) pharmacy, otolaryngology, de octubre university hospital, madrid, spain background and objective: a woman diagnosed with laryngeal papillomatosis. treatment consisted of laser excision of the granulomatous lesions, followed by mitomycin c instillation over the surgical bed. description of the preparation and utilization of mitomycin c instillation over the surgical bed for the treatment of laryngeal papillomatosis. design: request by the ent service for compassionate use of mitomycin c. following authorization from the health authorities, a literature search was made. after determining the dose/day for the patient, elaboration was carried out according to the literature references. setting: vials of mg of mitomycin c were used as starting material, with -ml syringes to facilitate instillation in the operating room. dilution was made to double the standard in our centre ( ml). main outcome measures: two -ml syringes were prepared per cycle, with a mitomycin c solution of . mg/ml for instillation, though finally in all cases only one of them was used. stability was established as hours at room temperature, without special considerations regarding light exposure, as specified by the literature. the entire procedure was carried out in a laminar flow chamber, in compliance with the specifications for manipulating cytostatic agents. results: the patient received a total of three cycles of mitomycin c, the last two being maintenance cycles. the patient has experienced no papilloma relapse, and only right vocal cord hyperaemia is observed, probably secondary to surgery. conclusions: collaboration between the service of pharmacy and the ent service allowed adequate treatment and recovery of the patient, without apparent adverse effects. swollen and tender joints over joints evaluated, and the value of erythrocyte sedimentation rate, comparing the das from one patient on two different time points; eular response, that classifies patients in groups according to treatment response, and decrease in mhaq (modified health assessment questionnaire), that indicates patient's awareness of disability. data were recollected at beginning and at weeks of initiating treatment with rituximab. differences between b-lymphocyte count at beginning and at weeks was of initiating treatment was used as a secondary variable. results: we collected data of patients, all being females, was rf positive and were anti-ccp positive. at women) a . % of patients received adjusted prophylaxis to their degree of risk ( . % with heparin prescription and . % without it) from all . % of inadequate prescription ( % of the patients with lmwh prescription); patients lost follow up. patients submitted to cancer surgery ( surgeries, men, women), % received apropiate prophylaxis ( . % with lmwh and . % without it). same percentage of patients ( %) was not adapted to correct prophylaxis ( % patients with lmwh); patients lost follow up. conclusions: there is a greater percentage of choledoco surgery patients with lmhw prescription in compliance with published guidelines than oncology surgery patients, however it will be necessary carry out a better implementation among healthy professionals in order to increase this percentage of patientes. results: prescriptions of these broad-spectrum antibiotics were collected and analysed. % of the prescriptions were initiated by residents, % by senior physicians. vancomycin was the most prescribed antibiotic ( %), mainly in the orthopaedic surgery unit. the indications of antibiotics were osteomyelitis ( %), septic arthritis ( %), prosthetic joint infections ( %) and pneumonia ( %). only % of antibiotic doses were not correctly adapted to creatinine clearances or to plasmatic vancomycin rates. the initial choice of antibiotic was considered appropriate in % of cases. regarding bacteriological results (bacteria, antibiogram), the continuation of the treatement was acceptable in % of cases. however in % of prescriptions, an adjustment of therapy with a more narrow spectrum antibiotic could have been done. conclusions: these results have to be extended with further investigation (at least months). were systematically reviewed to retrieve prospective trials describing esa doses in dialysis pts who converted from rhuepo to da. search words included: ''epoetin, darbepoetin, esrd, ckd, and dialysis.'' the inclusion criteria required a study to have dose data available during the evaluation period for both rhuepo and da. study selection and data extraction were performed by independent reviewers and verified by a rd. relative doses and dose changes after conversion from rhuepo to da were estimated using an initial rhuepo:da : conversion ratio (aranesp Ò eu label). study quality assessment was performed using the downs-black checklist, a standard method used to assess the quality of a study using ebm principles. setting: meta-analysis. main outcome measures: dose efficiency. results: the search yielded studies meeting the inclusion criteria. upon further review, studies were excluded ( had unextractable data, were retrospective analyses, and had predialysis pts). the remaining studies were analyzed: rcts with parallel control groups, cross-over trials, and observational conversion studies (table) . the studies yielded data on , rhuepo and , da pts, with a mean treatment duration being weeks. we found the average study quality was %, with rcts (n = ) having a higher quality score ( %) than crossover ( %; n = ) or observational ( %; n = ) studies. there was a notable dose efficiency observed when pts were converted to da from rhuepo. this effect was greater in the rcts ( . %) than in crossover ( . %) or observational ( . %) studies. conclusions: we found a notable da dose efficiency (up to %) in pts who were converted from rhuepo to da using a : conversion ratio. additionally, studies with the highest quality scores (eg rcts) had the greatest observed dose efficiency while non controlled studies scored lowest in both quality and dose efficiency. background and objective: the purpose of this study is to evaluate the frequency of otitis media and the assessment of its relationship to patients' socio-demographic characteristics and determination of systemic and topical drug profiles in otitis media. design: this retrospective study included the assessment of patients diagnosed with otitis media, who admitted to the study hospital during a months period (march-may ). demographic, clinical and prescription data of these patients were collected and analyzed. setting: the ear-nose-throat out-patient clinic of a states hospital. main outcome measures: the socio-demographic data of patients; the frequency of otitis media; type and percentage of prescribed drugs; the most used treatment regimens. results: patients ( women and men), who were diagnosed with otitis media, were included in our study. patients were diagnosed as chronic otitis media or serous otitis media or acute otitis media or external otitis media at rates of . %, . %, . %, and . %; respectively. in children, the acute and serous otitis media were seen more often than in adults (p \ . ). however, chronic otitis media were seen more frequently in adults (p \ . ). all patients were administered drug therapy for their diseases. it was observed that antibiotics (oral and/or topical), analgesics, decongestants, and topical corticosteroids were prescribed at rates of . %, . %, . %, and . %; respectively. prescribed oral antibiotics were cephalosporins [cefixime ( . %), cefuroxime axetil ( . %) and cefaclor ( . %)], amoxicillin-clavulanate, and floroquinolones [levofloxacin ( . %) and ciprofloxacin ( . %)] at rates of . %, %, and . % respectively. rifampin and ciprofloxacin were prescribed as topical antibiotics at rates of . %, . % respectively. when the number of drug used by the patients was evaluated, were on quadri-therapy, on tri-therapy, on dualtherapy and on mono-therapy. patients were treated with the combination of antibiotic-analgesic-decongestants. conclusions: our study indicated that the most frequently prescribed drugs were antibiotics in otitis media. clinical pharmacists have a potential role in rational antibiotic use by providing clinical pharmacy services such as antibiotic selection, drug monitoring and patient education; so that they would reduce both antibiotic resistance and treatment costs. background and objective: the sampling method is crucial for the physical and chemical quality control of antineoplastic chemotherapies. this step acts upon the dosage correctness and may lead to risk of needle-stick or cytotoxic drug projection during its achievement. beyond, the sampling time must be as short as possible and the sample be directly placed on the analysis machine. this work evaluates a new and improved sampling method, specially worked out for this application. design: this study was designed to ensure the vial airtightness and the volume sampling. a cost and time study was also performed. setting: the vial is an hplc type, chromacol Ò ml, mm dp = hpa, (interchim Ò , montluçon, ). the vial is airtight thanks to a ptfe silicon septum set with an aluminium collar. the vials are vacuum-packed in a pvc bag (didop Ò , compiègne, ). a void test was led on unpacked vials up to months, to ensure a maximum conservation, and showed a filling volume of ± ll, very close of the ll target. main outcome measures: one week samples have been weighed to calculate the filling volume in real conditions of use. this volume is ± ll ranging from to ll. since this sampling method has been set up, the percentage of refusal for insufficient volume is lower than . %. this technique was compared to the previous method in terms of cost: vial, sampling adjuncts, handlingtime and waste. the vials are filled with a secured double-needle eclipse Ò , mm, / (becton-dickinson Ò , le-pont-de-claix, ). results: about , chemotherapies controls are made each year in the hospital. the vial and the sampling device costs are higher than the previous ( € versus . €, and . € versus . €). on the contrary, the handling-time for sampling was estimated minute lower (which corresponds to . € less per sample). furthermore, the waste weight is gram lighter with the new devices, which costs . € less in the waste disposal. the total cost difference is . € higher per sample. an estimation of a needle-stick accident has been carried out, with the human cost (pharmacist technician's compensation and medical consulting) and the equipment including gloves, sterilization devices and post-sterilization check; the lowest estimation cost of an accident is . €. conclusions: the secured needle makes the sampling operation easier for the workers and it lowers the risk of needle-stick. besides, this closed system avoids completely the antineoplastic contact for the manipulators during the confection and the control. moreover, this system allows to secure the sample library. background and objective: the prevalence of diabetes mellitus in kuwait ranks amongst the highest in world at about %. diabetes is a well recognized independent risk factor for cardiovascular disease (cvd). the increased prevalence of several other known risk factors for cvd in kuwaiti diabetics further increases the risk. since cvd is the leading cause of death in kuwait, the high incidence of diabetes has major social and economic impact. diabetics aged years or older have, in general, an increased -year risk of developing cvd compared to younger patients but there have been no audits involving this group of patients in kuwait. the objective of this study was therefore to audit achievement of cvd risk factor goals according to pt- pharmacotherapy of first-episode psychosis in the psychiatry clinics of the north estonia regional hospital (nerh) and the tartu university hospital (tuh) jana lass , agnes männik , simon j. bell pharmacy department, north estonia regional hospital, tallinn, institute of pharmacy, university of tartu, tartu, estonia, faculty of pharmacy, university of helsinki, helsinki, finland background and objective: treatment guidelines provide recommendations for the evidence-based treatment of schizophrenia. adherence to these guidelines is often sub-optimal. our aim was to compare and contrast the pharmacotherapy of first-episode psychosis at the nerh and tuhs, with respect to both treatment location and evidence-based guidelines. design: retrospective study. case notes for consecutive patients with schizophrenia, schizotypal or delusional disorders (icd- ) admitted to the nerh and tuh between september and september were retrospectively reviewed. setting: psychiatry clinics of two tertiary care hospitals -nerh and tuh. main outcome measures: outcome measures included the choice and daily dose of antipsychotics, incidence of antipsychotic polypharmacy and reasons for changes in therapy plan. results: patients form nerh and from tuh were included in the final analyses. median age (sd) of the patients was ( . ) in the nerh and ( . ) in the tuh patients were hospitalised for longer in the nerh than in the tuh, ( . ) vs. ( . ). the most frequently prescribed antipsychotic was risperidone at both study locations - % of prescriptions in the nerh and % in the tuh. conventional antipsychotics were administered twice often in the nerh than in the tuh. in the tuh olanzapine was administered in higher prescribed daily doses than in the nerh. the number of antipsychotics prescribed per patient was higher in the nerh than in the tuh - . vs . . the prevalence of antipsychotic polypharmacy was . % among the patients in the nerh, whereas only one patient was treated with antipsychotic polypharmacy in the tuh. conclusions: analyses revealed significant differences in the pharmacotherapy of first episode psychosis at the nerh and the tuh. mechanisms to facilitate improved adherence to the evidence-based treatment guidelines should be investigated. pharmacy, hospital universitario de getafe, getafe, spain background and objective: to analyze the use of tnf-alfa inhibitors in rheumatoid arthritis diagnosed patients in a bed hospital. design: retrospective study of patients with anti-tnf-alfa during year . the following data were compiled: age, sex and anti-tnfalfa prescription including dosage and duration of treatment. setting: hospital universitario de getafe. main outcome measures: dosage and duration of treatment. results: a total of patients were included. patients ( . %) received just one anti-tnf-alfa drug, ( . %) needed two lines of treatment and ( . %) of them needed three. patients who were treated with just one drug had a median age of . years and those who required two lines of treatment had a median of . years. the first line drug was etanercept in . %, infliximab in . % and adalimumab in . % of patients. second option was etanercept in . %, adalimumab in . % and infliximab in . % of patients. the average duration of treatment with etanercept as forward edge was days. the treatment was suspended in . % of patients. when infliximab was used as first line, average duration was days, and treatment was interrupted in . %. with adalimumab, average duration was days and treatment was interrupted in % of patients. conclusions: those of our patients who need an only one treatment line are younger than those who need or , due to the chronic and progressive course of the disease. etanercept is used as much in first option (followed by infliximab) as in second one (followed of adalimumab), although these differences are not statistically significant and it would be necessary to make a study including more patients. the duration of treatment with infliximab is the longest, as this was the first drug available. regarding treatment failure, etanercept shows the greatest percentage. this should be taken into account when establishing first line treatment. background and objective: plantar warts are hyperkeratotic lesions on the plantar surface caused by infection with human papillomavirus. lesions caused by warts are commonly refractory to therapy and may become large and painful in immunodeficient patients. cidofovir is a cytidine analogue with activity against a broad spectrum of dna viruses. it is indicated for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome and without renal dysfunction. we describe a case of plantar warts that was treated with topical cidofovir in a highly immunodeficient patient. design: case report, evaluation and discussion based in clinical chart and literature review. setting: pharmacy department, general teaching hospital. main outcome measures plantar warts regression, which was evaluated on the basis of change in overall surface area of the treated lesions compared with baseline. to evaluate the organoleptics properties of the galenic formulation. results: a years old woman, who received kidney transplant in , presenting plantar warts refractory to conventional therapy since last four years. she was treated with topical % cidofovir cream twice daily. the treatment was authorised as compassionate use by the national regulatory agency on drugs. the glomerular filtration rate (gfr) was monitored in order to detect nephrotoxicity due to cidofovir. the % cidofovir ointment was compounded as follows: -cidofovir mg/ml ml vial .…….. ml -anhydrous lanolin ……………………….. g -beeler base …..sufficient to produce g it was packaged and labelled in a light-resistant containers and we assumed an expiration date of months based on the duration of treatment and published studies. the quality controls of organoléptics properties were made according to the good manufacturing practice (gmp) after weeks of therapy the patient did not show any improvement and developed severe local erosion, so treatment with cidofovir was withdrawn. two weeks later this local erosion disappeared spontaneously. no systemic side effects were observed. the colour, texture and smell organoleptics characters were complied with gmps. conclusions: there are not formal studies of optimal formulations or treatment regimens and further studies are needed to elucidate the role of cidofovir in treatment of plantar warts. the immunodeficiency of the patient and the large wart area could be related with the failure to the treatment. background and objective: hypersensitivity reactions (hr) to platinum salts can be serious and should lead to interrupt chemotherapy regimen. skin tests may be used to confirm diagnosis of hr. usually, these tests are prick tests and intradermal tests performed with diluted solutions of platinum salts. there is no standard solutions for these drugs, which local toxicity depends on concentration. the main objective of this study was to assess the safety of platinum salts skin tests performed in our hospital. the secondary objective was to assess their efficacy to verify the allergic nature of the reactions observed. design: pilot study of skin tests preparations between january and june . these preparations were dilutions ( / , / , / ) of a primary platinum salt solution, which concentration was roughly the one used in the chemotherapy regimen of most patients. the compatibility of platinum salts with dilution solvent was checked, and all solutions were prepared extemporaneously in a centralised cytotoxic drug preparation unit, in order to protect handlers. setting: allergology department and pharmacy department in a university hospital. main outcome measures: skin tests results: positive if a papule appeared, negative if there was no reaction, local toxicity if an irritative reaction happened. results: patients with clinical symptoms of hr with a chemotherapy regimen containing platinum salts were explored by skin tests. drugs assessed were: oxaliplatin ( patients), cisplatin ( ), and carboplatin ( ) . no patient developed local toxicity. tests results were positive in cases ( oxaliplatin and cisplatin), and negative in cases. patients received both cisplatin and oxaliplatin skin tests: patients had a single positive reaction with no cross reaction between the two drugs, and the third had no reaction. in cases, tests results and clinical history of hypersensitivity mismatched. conclusions: this study shows that these skin test solutions were safe. their efficacy was judged correct: positive reactions confirmed the diagnosis of hypersensibility for patients. the main limit of the results is the absence of control subjects. these tests allowed to explore patients' hr, and to help oncologists to choose the more appropriate treatment for them. stability studies are still needed to assess the pharmaceutical quality of these diluted solutions. these preparations have now been standardized in our hospital. background and objective: guidelines regarding appropriate use of prophylactic antibiotics have been implemented at university hospitals leuven. however, the degree of compliance with these guidelines is unknown. the aim of this study is to develop a method to quantify compliance with antibiotic prophylaxis guidelines and to apply this method to the clinical areas of appendectomy and heart valve surgery. design: a retrospective case series was carried out of all prophylaxis episodes related to appendectomy and heart valve surgery at university hospitals leuven between august and february . four grades of compliance with antibiotic prophylaxis guidelines were identified: grade compliance, reflecting administration of the antibiotic proposed by the guidelines in a dosage within - % of the recommended dosage; grade compliance, defined as the administration of the antibiotic proposed by the guidelines in a dosage outside - % of the recommended dosage; grade compliance, referring to the administration of an antibiotic equivalent to the antibiotic proposed, but not mentioned by the guidelines; and grade compliance, representing any other antibiotic prophylaxis scheme. setting: divisions of abdominal and cardiac surgery, university hospitals leuven. main outcome measures: the percentage of prophylaxis episodes that satisfy each grade of compliance with antibiotic guidelines. results: prophylaxis guidelines relating to appendectomy ( , episodes) recommend administration of three times cefazolin g and a single dose of metronidazol . g. the proportion of episodes that satisfied grade , , and of compliance with guidelines amounted to %, %, %, and %, respectively. cefazolin g and metronidazol . g was used in episodes. prophylaxis guidelines applying to heart valve surgery ( , episodes) recommend administration of cefazolin g. the proportion of episodes that satisfied grade , and of compliance amounted to %, % and %, respectively. grade does not apply to heart valve surgery as no equivalent antibiotics were identified. the difference in compliance with prophylaxis guidelines between both surgical procedures could be explained by differences in infectious pathology, the peri-operative adaptation of the antibiotic regimen by the abdominal surgeon, and the use of a second regimen related to the severity of the appendicitis. a case can be made for combining grade - compliance with respect to appendectomy, resulting in a higher compliance rate. conclusions: our proposed method to measure compliance needs to be validated by future research. the method can be applied to different surgical procedures, thereby stimulating surgeons to explain differences in compliance between procedures and promoting the development of instruments to enhance compliance. closer interaction with surgeons is required to further develop the measurement of compliance with antibiotic prophylaxis. keywords: antibiotic prophylaxis, compliance, guidelines pt- non-specific immunoglobulins for immune neonatal thrombopenia nuria ibañ ez , maria del pilar m. p. bautista , ana maria a. m. iglesias , roberto r. ortiz , javier j. sanchez-rubio , maria del carmen m. c. giron , marta m. arteta pharmacy, pediatry, hospital universitario de getafe, madrid, spain background and objective: non-specific human immunoglobulins are being used at the moment in neonatal population for treatment of immune thrombopenia. the dosis commonly used varies between mg/kg and g/kg from one to five days. corticoids and platelet transfusions can be used jointly. to study the effectiveness and safety of non-specific human immunoglobulins in a neonatal unit for treatment of immune thrombopenia. design: retrospective study of neonatal patients diagnosed with immune thrombopenia during and treated with non-specific human immunoglobulins. a revision of clinical histories is made and following data are collected: sex, gestational age, born weight, age at the moment of infusion, administered dose and duration of treatment, use of corticoids and platelet transfusions, number of platelets/ll before infusion, at , at hours of initiate the treatment and at discharge. possible adverse reactions is also considered. setting: hospital universitario de getafe. main outcome measures: the effectiveness and safety of nonspecific human immunoglobulins for treatment of immune neonatal thrombopenia. results: three children were included in the study, two of them were males. thrombopenia was diagnosed from probable alloimmune origin, including positive confirmation study in one of the cases. gestational ages ranged from + to + weeks. born weight ranged between . kg and . kg. immunoglobulin treatment was initiated between first and sixth day of life. administered dose varies between mg/kg/day and g/kg/day from two to five days. all children needed platelet transfusions, while only one of them was treated with corticoids. the number of platelets/ll before infusion of immunoglobulins, at hours, at hours and at discharge was: children : , , , , , and , platelets/ll. children : , , , , , and , . children : , and , platelets/ll hours after initiation of treatment, there were no analytical data at hours, but number of platelets at discharge was , . no adverse effects were observed in any children. conclusions: although eventually the three children recovered the number of platelets, it can not be concluded that this was due to immunoglobulin treatment, because it is overlapped with administration of platelet transfusions and corticoids. a higher number of patients is required to evaluate efficacy and safety of non-specific human immunoglobulins in treatment of neonatal thrombopenia. background and objective: pulmonary hypertension (ph) is one of the most difficult childhood disease to treat. in spain, oral sildenafil has recently been approved in adults to treat ph, but it s an off-label drug for children (its utilization must be derived to ''compassionate use'', which requires a prior national health authorities approval for every children), and an oral suspension must be formulated at the pharmacy department for them. the objective of this study is to analyse the use of oral sildenafil for ph in paediatric patients. design: years retrospective study. % paediatrics patients with oral sildenafil for ph. clinical data review. setting: paediatric cardiology unit and pharmacy department ( pharmacist) in a paediatric hospital ( beds), in a large general teaching hospital ( beds, pharmacists). main outcome measures: patient data (diagnosis, age, weight). treatment description (dose, length of treatment). treatment effectiveness: peripheral arterial oxygen saturation and six-minute walk test. treatment security: side effects registered. results: children ( girls). age: months to years, median . years. diagnosis: / ph secondary to surgery due to congenital heart disease and / primary ph. sildenafil doses ranged from . mg/kg/ h to mg/ h; median length of treatment was . months ( month- . years). children have used the oral suspension formulated and monthly dispensed at the pharmacy department. other treatments: spironolactone ( ), furosemide ( ) , captopril ( ), acenocoumarol ( ), aspirin ( ), ranitidine ( ) and propranolol ( ). patients have experimented clinical improvement and are on treatment. sildenafil was withdrawn in patients because it was indicated to ameliorate the effects of inhaled nitric oxide withdrawn. patients died. no data available in patient. only patient experimented occasional headache. mensual treatment cost range from - €/patient. conclusions: oral sildenafilo seems to be a safe and effective therapy for paediatric patients with pulmonary hypertensión. due to the lack of an oral formulation for paediatrics patients, it should be elaborated at the pharmacy department. background and objective: we will describe the case of a bi-pulmonary transplant women who developed an invasive aspergillosis located in the lungs and the brain. she received intravenous voriconazole during days. she was then diagnosed with an aspergillus endophthalmitis. even though a dual therapy consisting of caspofungin and posaconazole was initiated, the patient underwent a partial vitrectomy. this therapeutic failure could be explained by a late diagnosis and insufficient vitreous and aqueous humor penetration of the systemic drugs. design: a retrospective analysis of an endophthalmitis management. setting: clinical unit in a french teaching hospital main outcome measures: to secure a high ocular concentration, the ophtalmologist recommended voriconazole intravitreal injections. his prescription was based on several case reports. results: we found articles dealing with animal testing: one concluded that voriconazole was a safe intravitreal agent which may be injected in human eye. another study described the successfull use of intra-ocular voriconazole to treat a fungal endophthalmitis: it allowed a significant improvement in visual acuity and the patient's recovery. however, further studies are needed to assess the optimal dosage and frequency of administration. we prepared voriconazole syringes under a horizontal laminar air flow hood, as follows: -preparation of a mg/ml solution with ml of water for injection and dilution in ml of water for injection, to obtain a mg/ml solution pharm world sci ( ) : - -we sampled . ml of this solution in a ml syringe, which was closed with an occluder, labelled and refrigerated. since we had no data regarding stability, it was administrated extemporaneously. conclusions: intravitreal injections failed to prevent deterioration. had they been introduced precociously, they might have been more efficient. an early diagnosis and prompt management might improve the extremely poor visual prognosis of this devastating condition. were are currently studying the preparation stability. pharmacy service, hospital universitario de getafe, getafe, spain background and objective: in professionals were alerted about an elevated frequency of early virological failure in patients treated with tenofovir (tdf) and didanosine (ddi) associated to lamivudine. in similar results related to the administration of tdf and ddi, in association with a non-nucleoside reverse transcriptase inhibitor (nnrti) were notified. therefore similar events can be observed when tdf and ddi are co-administered in combination with other antiretroviral classes, such as protease inhibitors (pi). subsequent pharmacokinetic studies have shown that tdf when co-administered with ddi increases ddi plasma concentrations leves by up to - %, with a higher risk of didanosine-related adverse events, like pancreatitis and lactic acidosis. the administration of a reduced dose of didanosine ( mg) to avoid over-exposure to didanosine may also contribute to a higher rate of virological failure and emergence of resistance at early stage. the objective of the study is to asses the rate of virological failure in patients treated with tdf and ddi associated to a pi. results: during the study period patients had prescription for drotrecogin alfa for sepsis syndrome; . % were male and the mean age was . years (range - years). all patients had proven infection: . % had pneumonia (n = ), . % pyelonephritis(n = ), . % soft tissue infection(n = ) and . % abdominal infection(n = ). the main isolated micro-organisms were klebsiella pneumonia(n = ), escherichia coli(n = ), pseudomonas aeruginosa(n = ), enterococcus faecium(n = ), staphylococcus aureus(n = ), legionella pneumophila(n = ), proteus vulgaris(n = ), enterococcus faecium(n = ), klebsiella oxytoca(n = ). all patients started treatment within h of the onset of severe sepsis. the treatment was not completed in one patient due to adverse events. contraindications were present in patients: platelet count \ . /l (n = ), age under years (n = ) and major surgery (n = ). the mean organ failures was . (range - organs). adverse reactions were present in patients: thrombocytopenia (n = ), pancytopenia (n = ), bleeding (n = ) and elevation of activated partial thromboplastin time (n = ). mortality at days was found to be % (n = ). conclusions: despite the presence of some contraindications, in most patients drotrecogin alfa was used according to current guidelines. nevertheless, since apache ii score was not determined, the real risk of death is unknown and there can be no extrapolation to literature results. upon these findings, a systematic evaluation of apache ii score must be implemented in order to optimize patient selection and the risk-benefit ratio, improving the use of drotrecogin alfa. pharmacy, oncology, hôpital tenon aphp, paris, france background and objective: it is necessary to focus on side effects for pharmaceutical analysis. dosage reductions are commonly used in cancer chemotherapy. however, little is known concerning the way these reductions are performed in clinical practice. the objectives were to evaluate the incidence, the reason and the percentage of dosage reduction. design: prospective four-week study during which we analysed prescriptions with dosage reductions. setting: pharmacy and clinical oncology department in a paris university hospital. main outcome measures: we focused on prescriptions with dosage reduction and we recorded : -patient information -cancer localisation -chemotherapy regimen -dosage reduction characteristics (date, reduction percentage, reason) the toxicities were classified according to the nci-ctc criteria (grade to ). individual interviews were performed in order to assess how physicians decided the reduction ratio. results: patients ( % women; mean age years) have been treated during that period. diagnosis majority were breast ( %), colorectal ( %) and lung ( %) cancer. patients required a dosage reduction (incidence %). hematological toxicities were the main cause of reductions ( %). the hematological toxicities observed were thrombopenia ( %), neutropenia ( %) and neutropenia-thrombopenia associations ( %). the toxicities observed were grade ( %) or ( %). the other major causes of reductions were neurological ( %) and gastrointestinal ( %). the average percentage of reductions was between % and %. the individual interviews have shown that physicians didn't base the dosage reductions on literature results (established criteria) but on their own clinical practice (experience). conclusions: % of the prescriptions showed a decrease of the regimen. even if there is few literature, clinical trials recommend a decrease of % of the usual dosage of the drugs. the percentage in practice is lower than the one defined by clinical trials. the choices of reduction percentage were not standardized. recommendations for dosage reductions are still needed. keywords: dosage reduction, anticancer chemotherapy, toxicity pt- interdisciplinary approach to dose adjustment in patients with renal impairment in secondary care liekweg a, hinnerkort a, ebeling g, braband s, dreischulte t, heilenkötter k, sander s, schiffmann s, schrimpff u, siems m, wagner k, weiland t, zeigermann g, melzer s hospital pharmacy of the asklepios kliniken hamburg gmbh background and objective: as part of a unit dose dispensing system, patient medication profiles are routinely entered in an electronic database. medication profile and laboratory data are accessible online by clinical pharmacists. the project was conducted in order to optimise pharmacotherapy in patients with renal impairment and to integrate the clinical pharmacist in the therapeutic team. setting: unit-dose supplied wards (n = ) in four asklepios hopitals in hamburg with approximately patients per day. the project was conducted in cooperation with clinical pharmacists, physicians and the laboratory department over a period of . months ( / - / ). programm description: clinical pharmacists receive a list of all patients with an estimated glomerular filtration rate (egfr) \ ml/ min/ , m (mdrd) from the laboratory department on a daily basis. they screen medication profiles daily with regard to apparently inappropriate dosing of renally excreted drugs (q o \ . ). critical cases are reported to physicians by phone or entry in medical case notes. following an interdisciplinary discussion with the physician the drug dose or dosing interval is either adjusted, the medication is stopped or paused or an alternative is started. during the pilot phase the number of altered medications as a result of pharmacists' recommendations was documented. results: a prevalence of % of patients with a egfr \ ml/min/ . m was found in the examined setting. during the pilot phase of prescribed drugs ( %) were renally excreted or considered nephrotoxic. antibiotics ( %), antidiabetics ( %), diuretics ( %) or nsais ( %) were predominantly involved. overall, of pharmaceutical recommendations ( %) were accepted and acted upon by physicians. conclusion: the number of recommendations demonstrates the importance of this service in optimising pharmacotherapy. clinical pharmacists' contributions in matters of dose adjustment in patients with renal impairment is well received by physicians especially in non-nephrologic departments. the new service was found to be feasible in daily practice and has become part of the clinical routine. background and objective: the sources and availability of drug information for patients are growing, e.g. through the internet and official patient information leaflets (pils). however, the quality of the information on the internet might be questioned. furthermore, pils are not standardized, the layout is not reader friendly and the information covers all approved indications for the drug, some of them not relevant for rheumatic patients. also, over the years various information leaflets for drugs have been developed in the departments of rheumatology in norway. these are not standardized and the accessibility is limited. the objective was to develop a system for producing and maintaining reader friendly patient information leaflets about antirheumatic drugs, which takes the quality assurance aspect into account, and is easily accessible for the users. design: development project, consensus method. a national multidisciplinary project group was set up in december , with members from the social leagues (two members), pharmacists' organization (four) and rheumatologists' organization (two). mandate and regulations were approved by the organizations, as well as a legal disclaimer. the pharmacists make a draft for each drug which is e-mailed to all the members of the group. based on the comments a revision is made followed by another hearing until consensus is reached. the rheumatologists approve the leaflet. setting: national multidisciplinary consensus including patients associations main outcome measures: establishment of a dedicated website. number of leaflets published. results: a web address for publication of the leaflets is set up on the home page of the norwegian society for rheumatology: www.legeforeningen.no/nrf. there is a link to this address on the home pages of the social leagues and the norwegian association of hospital pharmacists. during the first year different drug leaflets have been developed and published on the web site. it is possible to search by trade name, generic name and groups of drugs such as ''antiinflammatory drugs'', so the numbers of hits adding up to . conclusions: this national multidisciplinary approach has made it possible to develop a system for making patient information leaflets about anti-rheumatic drugs, which are standardized and easily accessible. keywords: patient information leaflets, drugs in rheumatic diseases, multidisciplinary the need for pharmaceutical care in the prevention of coronary heart disease; an exploratory study in acute myocardial infarction patients the right medicine: a strategy for pharmaceutical care in scotland pharmacy for health: the way forward for pharmaceutical public health in scotland. glasgow: phis . . the sector skills council for health. skills for health: public health practice competences qualitative research: consensus methods for medical and health services research prescribing problems and pharmacist intervention in community practice a dictation system for reporting prescribing errors in community pharmacies a competency framework for the care of a person with diabetes pharmaceutical care of the patient with type diabetes mellitus: a consensus model for delivery of structured pharmaceutical care by community pharmacists in scotland development of a leadership course tailored for pharmacists in scotland pharmacy students attitudes and views about portfolio-based learning: a questionnaire survey using portfolios to learn about prescribing: qualitative insights into students experiences implementing clinical pharmacy services in an outpatient oncology clinic an evaluation of pharmacist contribution in oncology ward in a swedish hospital evaluation of clinical pharmacy services in a hematology/oncology outpatient setting rheumatic illness (ri): ( . %), multiple sclerosis (ms): ( . %), hepatitis c (hc): ( . %). : cfk: ( . %), others: ( . %), ri: ( . %), hiv: ( . %), ms: ( . %), hc: ( . %). : cfk: ( . %) total economic impact (tei): , € valorisation of clinical pharmacy activities: validation of a standard tool for routine interventions quotation in french hospitals keywords: drug related problems hiv-infected patients' perceived satisfaction with an outpatient pharmaceutical care unit (opcu) quality perceived by outpatients at the pharmaceutical care clinic antibiothérapie par voie générale en pratique courante au cours des infectons respiratoires basses de l'adulte et de l'enfant éme conférence de consensus en thérapeutique anti-references simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching comparative study and optimisation of the administration mode of three proton pump inhibitors by nasogastric tube is the administration of esomeprazole through a nasogastric tube modified by concomitant delivery of a nutrition mixture? gerontology and geriatric medicine the clinical implications of platelet transfusions associated with abo or rh(d) incompatibility platelet transfusion: products, indications anti-d ig pc- pharmacist's interventions in three medical care units: analysis and impact on the physicians' prescriptions ventilator-associated pneumonia epidemiology and outcomes of health-care-associated pneumonia keywords: pseudomonas aeruginosa, antibiotherapy, vap (ventilator-associated pneumonia) folic acid metabolism and human embryopathy folic acid in general medicine and dermatology folic acid awareness and intake survey in the united arab emirates keywords: pregnancy, folic acid, neural tube defects pepi- pre-diabetes screening program: a proactive study in istanbul community pharmacies www.diabetes.org keywords: type-ii diabetes years later…where do we stand? over-the-counter medications in pregnancy north american association for the study of obesity. consensus development conference on antipsychotic drugs and obesity and diabetes second-generation (atypical) antipsychotics and metabolic effects. a comprehensive literature review keywords: metabolic syndrome, second-generation antipsychotic, biological and clinical follow-up references analysis of taurine in blood plasma of epileptic patients using an improved isocratic hplc method for amino acids cardiovascular actions of taurine pharmacokinetics and pharmacodynamics of the effects of taurine on human blood pressure and heart rate references criteris d'indicació en el tractament de les hepatitis víriques. direcció general de recursos sanitaris peginterferon-alpha a and ribavirin combination therapy in chronic hepatitis c prevalence, incidence and predictors of severe anaemia with zidovudine-containing regimens in african adults with hiv infection within the dart trial the anemia prevalence study group. prevalence of anemia and correlation with biomarkers and specific antiretroviral regimens in human-immunodeficiency-virus-infected patients: findings of the anemia prevalence study clinical pharmacy and medication safety keywords: clinical pharmacist, chronic kidney disease, treatment safety reference nccn clinical practice guidelines in oncoloy for multiple myeloma a phase study of bortezomib in relapsed keywords: antibiotics, emergency department the use of mitomycin-c for respiratory papillomas: clinical, histologic and biochemical correlation mitomycin c: prevention and treatment of anterior glottic synechia preliminary results of intraoperative mitomycin-c in the treatment and prevention of glottic and subglottic stenosis airway complications from topical mitomycin c. otolaryngol head neck surg keywords: mitomycin c, laryngeal papillomatosis, papillomas pt- effectiveness of rituximab in rheumatoid arthritis background and objective: to assess the response to rituximab in patients with rheumatoid arthritis (ra) who were refractory to anti-tnf treatment. design: observational, cross-sectional study. performed on patients diagnosed of ra according to acr criteria the main variables used to assess clinical evolution were: decrease in das -esr, considering the number of references noss eh et al. rituximab as therapy for refractory polymyositis and dermatomyositis treatment of early and refractory dermatomyositis with infliximab: a report of two cases keywords: dermatomyositis, rituximab, infliximab were included in the study. main outcome measures: percentage of patients achieving optimum and minimum audit standards for serum total cholesterol, ldl-c, glycosylated haemoglobin (hba c), bp and take up of aspirin of these, optimum treatment standards for total cholesterol (\ mmol/l) and ldl-c (\ mmol/l) were achieved by . % (n = ) and . % (n = ), respectively. patients within the minimum audit goal for total cholesterol (\ mmol/l) and ldl-c (\ mmol/l) were joint british societies' guidelines on prevention of cardiovascular disease in clinical practice pt- pediatric use of infliximab: retrospective study vanida brunie reference french guidelines for assumption of responsibility of candida sp. and aspergillus sp. invasive infections. french society of anesthesia and reanimation keywords: assessment, antifungal agents comparisons of psychotropic drug prescribing patterns in acute psychiatric wards across europe to score personal risks factors ( to : standard risk, to : high risk) design of a therapeutic scheme following asco's guidelines, as follow. day : aprepitant mg per os h before chemotherapy ondansetron mg iv min before chemotherapy methylprednisolone (mp) mg iv min before chemotherapy days and : aprepitant per os mg methylprednisolone per os mg b.i.d. for the beam strategy, this treatment is given on day (carmustine) and on day (melphalan). the course of corticosteroid was reduced on purpose for clinical oncology guideline for antiemetics in oncology: update . kris and coll keywords: aprepitant, emesis, hematology pt- use of anti-tnf-alfa in rheumatoid arthritis intravitreal voriconazole for the treatment of endogenous aspergillus endophthalmitis intravitreal voriconazole for drug-resistant fungal endophthalmitis: case series fungal endophthalmitis caused by aspergillus ustus in a patient following cataract surgery intravitreal voriconazole: an electroretinographic and histopathologic study management of endogenous fungal endophthalmitis with voriconazole andcaspofungin histological examination of an eye with endogenous aspergillus endophthalmitis treated with oral voriconazole: a case report aspergillus endophthalmitis: an unusual complication of disseminated infection in renal transplant patients maria eugenia martínez nú ñ ez , javier sánchez-rubio ferrández , noelia garrido peño , carolina apezteguía fernández background and objective: zidovudine (zdv) is the first drug that approved for treatment of hiv infected patients and now has wide use in haart regimens. this drug can cause hypoproliferative anemia bone marrow toxicity. the object of this study is evaluation of incidence of anemia in iranian hiv positive patients that received zdv in haart regimens. design: in a prospective study, hiv positive patients were referred to iranian hiv research center that start zdv in haart combination were entered the study and have followed for at least one year. baseline and monthly hematological parameters were recorded. setting: iranian hiv research center. main outcome measures: patients demographic parameters, route of infection exposure, stage of disease, cd counts, cbc, and hematological parameters. results: twenty nine ( ) patients were excluded from the study because of impossible follow-up. from patients, of them have anemia (hemoglobin less than g/dl for female and less than g/dl for male). thirty there ( ) patients have anemia before starting haart. thirty four ( ) patients have showed anemia following received zdv. twenty ( ) patients have improved anemia after were changed zdv to stavudine. conclusions: about % of hiv positive patients that were received zdv have experienced anemia.background and objective: in june , the use of infliximab has been approved by emea for the treatment of severe active crohn's disease in pediatric patients aged to years old, who have not responded to conventional therapy (corticosteroid, immunomodulator and nutrition therapy). however, pediatricians were already using infliximab for patients with inflammatory bowel syndrome (ibd) such as crohn's disease (cd), ulcerative colitis (uc) and indeterminate colitis (ic). the goal of the study was to analyze infliximab prescriptions for children and to evaluate changes in prescriptions of corticosteroid due to the introduction of infliximab. design: retrospective study in ibd patients in a pediatric teaching hospital. setting: gastroenterology unit and pharmacy department. main outcome measures: indications, infliximab dosage, anterior treatments, reason of therapeutic change (non-tolerance or inefficiency of anterior treatment and/or cortico-dependance), evolution of corticosteroid dosage and months after the introduction of infliximab. results: thirty-three children were treated by infliximab: cd, uc and ic. age for diagnosis was an average of years old ( . - . ) and . years old ( - ) for the beginning of infliximab. previous treatment to infliximab was immunomodulators, single therapy for patients (azathioprine n = , mercaptopurine n = , methotrexate n = ) or dual therapy (n = azathioprine + methotrexate), with corticosteroids (n = ) and/or mesalazine (n = ). various etiologies justified infliximab administration: corticodependance (n = ), corticoresistance (n = ), non compliance to corticotherapy (n = ), insufficient efficacy of previous treatment (n = ), non tolerance to previous treatment (n = ). at the beginning, dosage of infliximab was mg/kg. dosages were increased ( mg/kg) for patients due to insufficient clinical results. one patient also had to be switched for adalimumab because he developed human antichimeric antibody (haca). among corticodependant patients ( ), corticosteroids have been stopped after or months, ( %) and patients ( %) respectively. for patients, corticosteroids were continued without reduction of dosages, six months after the introduction of infliximab. conclusions: infliximab is the only therapeutic alternative for children who are non tolerant or non respondent to conventional treatment. moreover, this treatment permits the use of decreased dosage of corticosteroids, limiting their side effects, especially on children growth. however, haca occurrence could limit its use in a long-term disease. keywords: infliximab, inflammatory bowel syndrome, pediatrics chemotherapy indication was autologous bone marrow transplant (bmt) ( %), leukaemia chemotherapy induction or consolidation ( %), leukaemia intensive chemotherapy ( %), myeloblastic allogeneic bmt ( %) and mini allogeneic bmt ( %).treatments were prophylactic ( %), empirical ( %) or curative ( % for aspergillus sp but no for candida sp infections); % of the prescriptions related to local candidosis and % remained unknown.although % of prescriptions were in accordance with internal guidelines concerned antifungal drug indication, % had wrong dosages e.g. no loading dose for voriconazole. moreover, only % of the prescriptions were in accordance with french recommendations: neither voriconazole is approved in prophylaxis of aspergillosis in patients with autologous bmt nor antifungal drugs associations (ten prescriptions). nevertheless, it may be a good way of medical management as hopeful patients outcomes have been obtained.conclusions: hematology department guidelines should be reviewed in accordance with french recommendations, department's ecology and the state-of-the-art about treatment of fungal infections in patients with haematological malignancies. the accordance to further recommendations should be regularly assessed as well as resistance emergence. background and objective: to evaluate the efficacy and safety of rituximab (rtx) for the treatment of refractory autoimmune cytopenia, including autoimmune hemolytic anemia (aha) and immunemediated thrombocytopenia (idiopathic thrombocytopenic purpura itp and thrombotic thrombocytopenic purpura ttp) design: descriptive, retrospective study based on rituximab prescriptions analysis. patients were identified through medical reports delivered by compassionate use program. data collection was made through the pharmaco-therapeutic profile and medical chart review setting: general teaching hospital ( beds) main outcome measures: patients who received any course of rtx for refractory immune cytopenia from january to may were evaluated. data recorded included patients details, diagnosis, previous treatment, rtx schedule, number of courses and baseline hemoglobin (hb) and platelet count (pq) values. effectiveness and tolerance were also considered. response was evaluated according to criteria found in the literature: clinical symptoms resolution and a normal pq count of . /mm for itp/ttp or an hb level [ g/ dl achieved and maintained for at least months for aha. additional response criteria for aha was an hb increase [ . g/dl month after the last dose of rtx. results: patients ( men), doses rtx; average age years (range - ). diagnosis: aha ( cases cold agglutinin disease), itp and ttp. in patients cytopenia (aha) was associated with chronic lymphocytic leukemia. all patients had been previously treated with steroids and had received or more other treatment modalities ( splenectomy, immunosupressive agents, intravenous immunoglobulin). patients received - rtx infusions at a standard dose of mg/m once per week, in combination with steroids therapy in cases. no serious infusion-related effects occurred, but patients reported hematologic toxicity (fever and infection). all patients with aha ( / ) and patients with itp ( / ) responded to the first course of rtx. one patient aha had relapse after months and responded to retreatment. itp responders achieved durable response ( and months) and were offered second course of rtx after relapse ( patients did not respond to retreatment). after months follow-up, patient with ttp remained with acceptable pq counts. hb levels increased by a median of . g/dl (range - , ) among the aha responders. itp + ttp responders achieved a median increase in pq count of . /mm (range - ). only responders who reached a months follow-up were considered for response duration assessment: aha, ttp, itp ( retreatment). median response duration was months (range - ) for aha and months (range - ) for itp + ttp conclusions: most of the literature findings for rtx in this setting were related to small series or isolated case descriptions. despite the common limitation of the number of patients, our results showed that rtx appears to be a promising agent for the treatment of refractory autoimmune cytopenia. key: cord- -dkw sugl authors: singh, indu; swami, rajan; khan, wahid; sistla, ramakrishna title: delivery systems for lymphatic targeting date: - - journal: focal controlled drug delivery doi: . / - - - - _ sha: doc_id: cord_uid: dkw sugl the lymphatic system has a critical role in the immune system’s recognition and response to disease, and it is an additional circulatory system throughout the entire body. most solid cancers primarily spread from the main site via the tumour’s surrounding lymphatics before haematological dissemination. targeting drugs to lymphatic system is quite complicated because of its intricate physiology. therefore, it tends to be an important target for developing novel therapeutics. currently, nanocarriers have encouraged the lymphatic targeting, but still there are challenges of locating drugs and bioactives to specific sites, maintaining desired action and crossing all the physiological barriers. lymphatic therapy using drug-encapsulated colloidal carriers especially liposomes and solid lipid nanoparticles emerges as a new technology to provide better penetration into the lymphatics where residual disease exists. optimising the proper procedure, selecting the proper delivery route and target area and making use of surface engineering tool, better carrier for lymphotropic system can be achieved. thus, new methods of delivering drugs and other carriers to lymph nodes are currently under investigation. the lymphatic system was fi rst recognised by gaspare aselli in , and the anatomy of the lymphatic system was almost completely characterised by the early nineteenth century. however, knowledge of the blood circulation continued to grow rapidly in the last century [ ] . two different theories are proposed which are in favour of origin of the lymphatic vessels. firstly, centrifugal theory of embryologic origin of the lymphatics was described in the early twentieth century by sabin and later by lewis, postulating that lymphatic endothelial cells (lecs) are derived from the venous endothelium. later the centripetal theory of lymphatic development was proposed by huntington and mcclure in which describes the development of the lymphatic system beginning with lymphangioblasts, mesenchymal progenitor cells, arising independently of veins. the venous connection to the lymphatic system then happens later in development [ ] . the lymphatic vessels in the embryo are originated at mid-gestation and are developed after the cardiovascular system is fully established and functional [ ] . a dual origin of lymphatic vessels from embryonic veins and mesenchymal lymphangioblasts is also proposed [ ] . recent studies provide strong support of the venous origin of lymphatic vessels [ - ] . the recent discovery of various molecular markers has allowed for more in-depth research of the lymphatic system and its role in health and disease. the lymphatic system has recently been elucidated as playing an active role in cancer metastasis. the knowledge of the active processes involved in lymphatic metastasis provides novel treatment targets for various malignancies. the lymphatic system consists of the lymphatic vessels, lymph nodes, spleen, thymus, peyer's patches and tonsils, which play important roles in immune surveillance and response. the lymphatic system serves as the body's second vascular system in vertebrates and functions co-dependently with the cardiovascular system [ , ] . the lymphatic system comprises a single irreversible, open-ended transit network without a principal driving force [ ] . it consists of fi ve main types of conduits including the capillaries, collecting vessels, lymph nodes, trunks and ducts. the lymphatic system originates in the dermis with initial lymphatic vessels and blind-ended lymphatic capillaries that are nearly equivalent in size to but less abundant than regular capillaries [ , ] . lymphatic capillaries consist of a single layer of thin-walled, non-fenestrated lymphatic endothelial cells (lecs), alike to blood capillaries. the lecs, on the contrary to blood vessels, have poorly developed basement membrane and lack tight junctions and adherent junctions too. these very porous capillaries act as gateway for large particles, cells and interstitial fl uid. particles as large as nm in diameter can extravasate into the interstitial space, get phagocytosed by macrophages and are ultimately passed on to lymph nodes [ - ] . lymphatic capillary endothelial cells are affi xed to the extracellular matrix by elastic anchoring fi laments, which check vessel collapse under high interstitial pressure. these initial lymphatics, under a positive pressure gradient, distend and create an opening between loosely anchored endothelial cells letting for the entry of lymph, a protein-rich exudate from the blood capillaries [ , , ] . in initial lymphatic vessels, overlying endothelial cell-cell contacts prevent fl uid refl ux back into the interstitial space [ , ] . after the collection of lymph by the lymphatic capillaries, it is transported through a system of converging lymphatic vessels of progressively larger size, is fi ltered through lymph nodes where bacteria and particulate matter are removed and fi nally goes back to the blood circulation. lymph is received from the initial capillary lymphatic by deeper collecting vessels that contain valves to maintain unidirectional fl ow of lymph. these collecting vessels have basement membranes and are surrounded by smooth muscle cells with intrinsic contractile activity that in combination with contraction of surrounding skeletal muscles and arterial pulsations propels the lymph to lymph nodes [ - ] . the collecting lymphatic vessels unite into lymphatic trunks, and the lymph is fi nally returned to the venous circulation via the thoracic duct into the left subclavian vein [ , ] . the fl ow of lymph toward the circulatory system is supported by increases in interstitial pressure as well as contractions of the lymphatic vessels themselves. roughly l of lymphatic fl uid enters the cardiovascular system each day [ ] . the key functions of the lymphatic system are maintenance of normal tissue fl uid balance, absorption of lipids and fat-soluble vitamins from the intestine and magnetism and transport of immune cells. lymphatics transport the antigen-presenting cells as well as antigens from the interstitium of peripheral tissues to the draining lymph nodes where they initiate immune responses via b-and t-cells in the lymph nodes [ , , , ] . tissue fl uid balance is maintained by restoring interstitial fl uid to the cardiovascular system [ ] . although capillaries have very low permeability to proteins, these molecules as well as other macromolecules and bacteria accumulate in the interstitium. due to the accumulation of these large molecules in the interstitium, signifi cant tissue oedema would result. the lymphatic system offers the mechanism by which these large molecules re-enter the blood circulation [ ]. the lymphatic system is the site of many diseases such as metastitial tuberculosis (tb), cancer and fi lariasis [ ] . due to the peculiar nature and anatomy of the lymphatic system, localisation of drugs in the lymphatics has been particularly diffi cult to achieve. the lymphatic system has an active role in cancer metastasis. although many cancers may be treated with surgical resection, microscopic disease may remain and lead to locoregional recurrence. conventional systemic chemotherapy cannot prove effective for delivering drugs to the lymphatic system without dose-limiting toxicities [ ]. lymphatic system functions in the clearance of particulate matter from the interstitium following presentation to lymph nodes have created interest in developing microparticulate systems to target regional lymph nodes. molecule's composition is important in determining uptake into the lymphatics and retention within the lymph nodes. colloidal materials, for example, liposomes, activated carbon particles, emulsions, lipids and polymeric particulates, are highly taken up by the lymphatics; that's why nowadays these substances are emerging as potential carriers for lymphatic drug targeting [ ] . the vast majority of drugs following oral administration are absorbed directly into portal blood, but a number of lipophilic molecules may get access to the systemic circulation via the lymphatic pathway [ , ] . intestinal lymphatic transport of lipophilic molecules is signifi cant and presents benefi ts in a number of situations: the lymphatic system also acts as the primary systemic transport pathway for b-and t-lymphocytes as well as the main route of metastatic spread of a number of solid tumours [ , ] . therefore, lymphatic absorption of the immunomodulatory and anticancer compounds may be more effective [ , ] . the presence of wide amounts of hiv-susceptible immune cells in the lymphoid organs makes antiretroviral drug targeting to these sites of tremendous interest in hiv therapy. this strategy comprises once again targeting nanosystems to immune cell populations, particularly macrophages. also evidence further suggests that lymph and lymphoid tissue, and in particular gut-associated lymphoid tissue, play a major role in the development of hiv and antivirals which target acquired immunodefi ciency syndrome (aids) may therefore be more effective when absorbed via the intestinal lymphatics [ , ] targeting drugs to lymphatic system is a tough and challenging task, and it totally depends upon the intricate physiology of the lymphatic system. targeting facilitates direct contact of drug with the specifi c site, decreasing the dose of the drugs and minimising the side effects caused by them. currently, nanocarriers have encouraged the lymphatic targeting, but still there are challenges of locating drugs and bioactives to specifi c sites, maintaining desired action and crossing all the physiological barriers. these hurdles could be overcome by the use of modifi ed nanosystems achieved by the surface engineering phenomena. from the growing awareness of the importance of lymph nodes in cancer prognosis, their signifi cance for vaccine immune stimulation and the comprehension that the lymph nodes harbour hiv as well as other infectious diseases stems the development of new methods of lymph node drug delivery [ - ] . new methods of delivering drugs and other carriers to lymph nodes are currently under investigation. lymph node dissemination is the primary cause of the spread of majority of solid cancers [ ] . in regard to cancer metastasis, the status of the lymph node is a major determinant of the patient's diagnosis. the most important factor that determines the appropriate care of the patient is correct lymph node staging [ ] . but patient survivals have been shown to improve by the therapeutic interventions that treat metastatic cancer in lymph nodes with either surgery or local radiation therapy [ ] . viraemia is an early indication of primary infection with hiv followed by a specifi c hiv immune response and a dramatic decline of virus in the plasma [ ] . long after the hiv virus can be found in the blood, hiv can be found in high levels in mononuclear cells located in lymph nodes. viral replication in these lymph nodes has been reported to be about -to -fold higher than in the peripheral blood mononuclear cells [ ] . standard oral or intravenous drug delivery to these lymph node mononuclear cells is diffi cult [ ] . even if highly active antiretroviral therapy (haart) can reduce plasma viral loads in hiv-infected patients by %, active virus can still be isolated from lymph nodes even after months of haart therapy. lymph nodes are the key element of the life cycle of several parasite organisms, including fi laria. lymphatic vessels and lymph nodes of infected patients can carry adult worms. this adult fi laria obstructs the lymphatic drainage that results into swelling of extremities that are distal to the infected lymph node. these very symptoms of swollen limbs in patients with fi larial disease have been termed elephantiasis. the eradication of adult worms in lymph nodes is not frequently possible, and commonly a much extended course of medical therapy is required for it to be successful [ ] . new methods of curing anthrax have become a burning interest following the recent outburst of anthrax infections and deaths in the usa as a result of terrorism. in anthrax infection, endospores from bacillus anthracis that gain access into the body are phagocytosed by macrophages and carried to regional lymph nodes where the endospores germinate inside the macrophages and become vegetative bacteria [ ] . according to one literature, computed tomography of the chest was performed on eight patients infected with inhalational anthrax. mediastinal lymphadenopathy was found in seven of the eight patients [ ] . in another case report of a patient, the anthrax bacillus was shown to be rapidly sterilised within the blood stream after initiation of antibiotic therapy. however, viable anthrax bacteria were still present in postmortem mediastinal lymph node specimens [ ] . treatment and control of these diseases are hard to accomplish because of the limited access of drugs to mediastinal nodes using common pathways of drug delivery. also, the anatomical location of mediastinal nodes represents a diffi cult target for external beam irradiation. newer methods to target antituberculosis drugs to these lymph nodes could possibly decrease the amount of time of drug therapy. tb requires lengthy treatment minimum of approximately months probably because of its diffi culty in delivering drugs into the tubercular lesions. the tb infection is caused by mycobacteria that invade and grow chiefl y in phagocytic cells. lymph node tb is the most common form of extrapulmonary tb rating approximately as . %. this is frequently found to spread from the lungs to lymph nodes. in one study, total tb lymph node involvement was found as % of the intrathoracic lymph nodes, % of the cervical lymph nodes and % of the axillary lymph nodes [ ] . targeted delivery of drugs can be achieved utilising carriers with a specifi ed affi nity to the target tissue. there are two approaches for the targeting, i.e. passive and active. in passive targeting, most of the carriers accumulate to the target site during continuous systemic circulation to deliver the drug substance, the behaviour of which depends highly upon the physicochemical characteristics of the carriers. whereas much effort has been concentrated on active targeting, this involves delivering drugs more actively to the target site. passive targeting involves the transport of carriers through leaky tumour vasculature into the tumour interstitium and cells by convection or passive diffusion. further, nanocarriers and drug then accumulate at the target site by the enhanced permeation and retention (epr) effect [ ] . the epr effect is most prominent mainly in cancer targeting. moreover, the epr effect is pertinent for about all fast-growing solid tumours [ ] . the epr effect will be most positive if nanocarriers can escape immune surveillance and circulate for a long period. very high local concentrations of drug-loaded nanocarriers can be attained at the target site, for example, about -to -fold higher than in normal tissue within - days [ ] . however, there exist some limitations for passively targeting the tumour; fi rst is the degree of tumour vascularisation and angiogenesis which is important for passive targeting of nanocarriers [ ] . and, second, due to the poor lymphatic drainage in tumours, the interstitial fl uid pressure increases which correlates nanocarrier size relationship with the epr effect: larger and long-circulating nanocarriers ( nm) are more retained in the tumour, whereas smaller molecules easily diffuse [ ] . active targeting is based upon the attachment of targeting ligands on the surface of the nanocarrier for appropriate receptor binding that are expressed at the target site. the ligand particularly binds to a receptor overexpressed in particular diseased cells or tumour vasculature and not expressed by normal cells. in addition, targeted receptors should be present uniformly on all targeted cells. targeting ligands are either monoclonal antibodies (mabs) and antibody fragments or non-antibody ligands (peptidic or not). these can also be termed as ligand-targeted therapeutics [ , ] . targeting approaches for lymphatic targeting are shown in fig. . . current research is focussed on two types of carriers, namely, colloidal carriers and polymeric carriers. targeting strategies for lymphatics are shown in fig. much effort has been concentrated to achieve lymphatic targeting of drugs using colloidal carriers. the physicochemical nature of the colloid itself has been shown to be of particular relevance, with the main considerations being size of colloid and hydrophobicity. the major purpose of lymphatic targeting is to provide an effective anticancer chemotherapy to prevent the metastasis of cancer cells by accumulating the drug in the regional lymph node. emulsions are probably well-known particulate carriers with comparative histories of research and have been widely used as a carrier for lymph targeting. hashida et al. demonstrated that injection of water-in-oil (w/o) or oil-in-water (o/w) emulsions favoured lymphatic transport of mitomycin c via the intraperitoneal and intramuscular routes and uptake into the regional lymphatics was reported in the order of o/w > w/o > aqueous solution. the nanoparticle-in-oil emulsion system, containing anti-fi larial drug in gelatin nanoparticles, was studied for enhancing lymphatic targeting [ ] . pirarubicin and lipiodol emulsion formulation was developed for treating gastric cancer and metastatic lymph nodes [ , ] . after endoscopic injection of the pirarubicin-lipiodol emulsion, the drug retained over days at the injection site and in the regional lymph node. hauss et al. in their study have explored the lymphotropic potential of emulsions and self-emulsifying drug delivery systems (sedds). they investigated the effects of a range of lipid-based formulations on the bioavailability and lymphatic transport of ontazolast following oral administration to conscious rats and found that all the lipid formulations increased the bioavailability of ontazolast comparative to the control suspension, the sedds promoted more rapid absorption and maximum lymphatic transport is found with the emulsion [ , ] . lymphatic delivery of drug-encapsulated liposomal formulations has been investigated extensively in the past decade. liposomes possess ideal features for delivering therapeutic agents to the lymph nodes which are based on their size, which prevents their direct absorption into the blood; the large amount of drugs and other therapeutic agents that liposomes can carry; and their biocompatibility. the utility of liposomes as a carrier for lymphatic delivery was fi rst investigated by segal et al. in [ ] . orally administered drug-incorporated liposomes enter the systemic circulation via the portal vein and intestinal lymphatics. drugs entering the intestinal lymphatic through the intestinal lumen avoid liver and fi rst-pass metabolism as they fi rst migrate to lymphatic vessels and draining lymph nodes before entering systemic circulation. lymphatic uptake of carriers via the intestinal route increases bioavailability of a number of drugs. for oral delivery of drug-encapsulated liposomal formulations, intestinal absorbability and stability are the primary formulation concerns. ling et al. evaluated oral delivery of a poorly bioavailable hydrophilic drug, cefotaxime, in three different forms: liposomal formulation, aqueous-free drug and a physical mixture of the drug and empty liposomes [ ] . the liposomal formulation of the drug turned out to exhibit a . -fold increase in its oral bioavailability compared to the aqueous dosage and a . -fold increase for the physical mixture. they also accounted that the liposomal formulation leads to a signifi cant enhancement of the lymphatic localisation of the drug relative to the other two formulations. as a result, liposome systems emerged as useful carriers for poorly bioavailable hydrophilic drugs, promoting their lymphatic transport in the intestinal lymph as well as their systemic bioavailability. conventional liposomal formulations contain anticancer drugs incorporated in them for intravenous infusion in treating various types of cancers. doxil, a chemotherapeutic formulation of pegylated liposomes of doxorubicin, is widely used as fi rst-line therapy of aids-related kaposi's sarcoma, breast cancer, ovarian cancer and other solid tumours [ - ] . liposomal delivery of anticancer drug actinomycin d via intratesticular injection has shown greater concentration of the drug in the local lymph nodes. furthermore, a study by hirnle et al. found liposomes as a better carrier for intralymphatically delivered drugs contrasted with bleomycin emulsions [ ] . systemic liposomal chemotherapy is preferred mainly because of its reduced side effects compared to the standard therapy and improved inhibition of the anticancer drugs from enzymatic digestion in the systemic circulation. effective chemotherapy by pulmonary route could overcome various lacunas associated with systemic chemotherapy like serious non-targeted toxicities, poor drug penetration into the lymphatic vessels and surrounding lymph node and fi rst-pass clearance concentrating drugs in the lungs and draining lymphatics in the case of oral delivery. latimer et al. developed liposomes of paclitaxel and a vitamin e analogue α-tocopheryloxy acetic acid (α-tea) in an aerosol formulation for treating murine mammary tumours and metastases [ ] . similarly, lawson et al. performed a comparative study for the anti-proliferative effi cacy of a -nitro-camptothecin ( -nc)-encapsulated dilauroylphosphatidylcholine liposomal delivery, α-tea and a combination therapy of -nc and α-tea, in a metastatic murine mammary tumour model. liposome-encapsulated individual as well as combination treatment was delivered via an aerosol for curing metastases of lungs and of the surrounding lymph node. the animals treated with the combination therapy were found to have less proliferative cells compared to the animals treated with -nc alone when immunostained with ki- . the in vivo anticancer effi cacy studies demonstrated that the combination treatment greatly hindered the tumour progression compared to each treatment alone, leading to the prolonged survival rate [ ] . high levels of drugs could be targeted to lymph nodes containing tb using liposomal antituberculosis drug therapy [ ] . deep lung lymphatic drainage could also be visualised using mtc radioactive marker-incorporated liposomes. in addition, botelho et al. delivered aerosolised nanoradioliposomal formulation to wild boars and observed their deep lung lymphatic network and surrounding lymph nodes [ ] . also, this technique has offered new information of the complicated structure of lymphatic network and has emerged as a new and non-invasive molecular imaging technique for the diagnosis of early dissemination of lung cancers as compared to the conventional computed tomography. solid lipid nanoparticles (sln) could be a good formulation strategy for incorporating drugs with poor oral bioavailability due to low solubility in gi tract or pre-systemic hepatic metabolism (fi rst-pass effect) permitting transportation into the systemic circulation through the intestinal lymphatics. bargoni et al. have performed various studies on absorption and distribution of sln after duodenal administration [ - ] . in one study, i- -iodoheptadecanoic acid-labelled drug-free sln were delivered into the duodenal lumen of fed rats, and transmission electron microscopy and photon correlation spectroscopy results of the lymph and blood samples verifi ed the transmucosal transport of sln [ ] . in a later study of tobramycin-loaded sln after duodenal administration, the improvement of drug absorption and bioavailability was ascribed mostly to the favoured transmucosal transport of sln to the lymph compared to the blood [ ] . the same group conducted a study using idarubicin-loaded sln, administered via the duodenal route rather than intravenous route, and observed enhancement in drug bioavailability [ ] . reddy et al. prepared etoposide-loaded tripalmitin (etpl) sln radiolabelled with mtc and administered the etpl nanoparticles subcutaneously, intraperitoneally and intravenously, to mice bearing dalton's lymphoma tumours, and h after subcutaneous administration, gamma scintigraphy and the radioactivity measurements showed that the etpl sln revealed a clearly higher degree of tumour uptake given via subcutaneous route ( -and -fold higher than that of the intraperitoneal and intravenous routes, respectively) and reduced accumulation in reticuloendothelial system organs [ ] . targeting therapies are of great potential in small cell lung cancer considering intrathoracic lymph node metastasis occurring in approximately % of the limited stage patients and to nearly % of the extensive stage patients [ ] . considering the case of non-small cell lung cancer, extensive rate of metastasis of lymphatics is seen in greater than % of stage iv patients [ ] . videira et al. compared the biodistribution of inhaled mtc-d,l -hexamethylpropyleneamine oxime (hmpao)radiolabelled sln with that of the free tracer administered through the same route, and gamma scintigraphic results specifi ed that the radiolabelled sln were primarily cleared from lungs via the lymphatics [ , ] . nanocapsules tend to be the most promising approach for lymphatic targeting because of their possibility of attaining distinct qualities with an easy manufacturing process. nanocapsules coated with hydrophobic polymers could be easily captured by lymphatic cells in the body, when administered, because the hydrophobic particle is generally recognised as a foreign substance. the lymphatic targeting ability of poly(isobutylcyanoacrylate) nanocapsules encapsulating -( -anthroxy) stearic acid upon intramuscular administration was evaluated and compared with three conventional colloidal carriers [ ] . in vivo study in rats proved that poly(isobutylcyanoacrylate) nanocapsules retained in the right iliac regional lymph nodes in comparison with other colloidal carriers following intramuscular administration. for effective targeted and sustained delivery of drugs to lymph, several polymeric particles have been designed and studied. the polymers are categorised in two types based on their origin either natural polymers like dextran, alginate, chitosan, gelatin, pullulan and hyaluronan or synthetic polymers like plga, pla and pmma. dextran a natural polysaccharide has been used as a carrier for a range of drug molecules due to its outstanding biocompatibility. bhatnagar et al. synthesised cyclosporine a-loaded dextran acetate particles labelled with mtc. these particles gradually distributed cyclosporine a all through the lymph nodes following subcutaneous injection into the footpad of rats [ ] . dextran (average molecular weights of , and kda)-conjugated lymphotropic delivery system of mitomycin c has been studied and it was reported that after intramuscular injection in mice, this mitomycin c-dextran conjugates retained for a longer period in regional lymph nodes for nearly h while the free mitomycin was quickly cleared. hyaluronan, also called as hyaluronic acid, is a natural biocompatible polymer that follows lymphatic drainage from the interstitial spaces. cai et al. demonstrated a novel intralymphatic drug delivery method synthesising a cisplatin-hyaluronic acid conjugate for breast cancer treatment. following subcutaneous injection into the upper mammary fat pad of female rats, most of the carrier localised in the regional nodal tissue compared to the standard cisplatin formulation [ ] . poly(lactide-co-glycolide) as synthetic polymer that is used to prepare biodegradable nanospheres has been accounted to deliver drugs and diagnostic agents to the lymphatic system. similarly, nanospheres coated with block copolymers of poloxamers and poloxamines with radiolabelled in-oxine are used to trace the nanoparticles in vivo. upon s.c. injection, the regional lymph node showed a maximum uptake of % of the administered dose [ ] . dunne et al. synthesised a conjugate of block copolymer cis-diamminedichloroplatinum(ii) (cddp) and poly(ethylene oxide)-block-poly(lysine) (peo-b-plys) for treating lymph node metastasis. one animal treatment with wt.% cddp-polymer resulted into limited tumour growth in the draining lymph nodes and prevention of systemic metastasis [ ] . johnston and coworkers designed a biodegradable intrapleural (ipl) implant of paclitaxel consisting gelatin sponge impregnated with poly(lactide-co-glycolide) (plga-ptx) for targeting thoracic lymphatics. in rat model, this system exhibited lymphatic targeting capability and showed sustained drug release properties [ ] . kumanohoso et al. designed a new drug delivery system for bleomycin by loading it into a small cylinder of biodegradable polylactic acid to target lesions. this system showed signifi cantly higher antitumour effect compared to bleomycin solution and no treatment [ ] . to treat lesions, a new biodegradable colloidal particulatebased nanocarrier system was designed to target thoracic lymphatics and lymph nodes. various nano-and microparticles of charcoal, polystyrene and poly(lactideco-glycolide) were studied for the lymphatic distribution after intrapleural implantation in rats, and after h of intrapleural injection, the lymphatic uptake was observed [ ] . kobayashi et al. utilised dendrimer-based contrast agents for dynamic magnetic resonance lymphangiography [ ] . gadolinium (gd)-containing dendrimers of different sizes and molecular structures (pamam-g , pamam-g and dab-g ) (pamam, polyamidoamine; dab, diaminobutyl) are used as contrast agents. size and molecular structure play a great role in distribution and pharmacokinetics of dendrimers. for example, pamam-g when injected intravenously had a comparatively long life in the circulatory system with minimum leakage out of the vessels, whereas pamam-g cleared rapidly from the systemic circulation due to rapid renal clearance but had immediate survival in lymphatic circulation. the smaller-sized dab-g showed greater accumulation and retention in lymph nodes useful for lymph node imaging using mr-lg. gadomer- and gd-dtpadimeglumine (magnevist) were evaluated as controls. imaging experiments revealed that all of the reagents are able to visualise the deep lymphatic system except gd-dtpa-dimeglumine. to visualise the lymphatic vessels and lymph nodes, pamam-g and dab-g were used, respectively. while pamam-g provided good contrast of both the nodes and connecting vessels, gadomer- was able to visualise lymph nodes, but not as clear as gd-based dendrimers. kobayashi also delivered various gd-pamam (pamam-g , pamam-g , pamam-g , pamam-g ) and dab-g dendrimers to the sentinel lymph nodes and evaluated its visualisation with other nodes. the g dendrimer provided excellent opacifi cation of sentinel lymph nodes and was able to be absorbed and retained in the lymphatic system [ ] . using a combination of mri and fl uorescence with pamam-g -gd-cy, the sentinel nodes were more clearly observed signifying the potential of the dendrimers as platform for dual imaging. kobayashi et al. further overcame the sensitivity limitation and depth limitations of each individual method by the simultaneous use of two modalities (radionuclide and optical imaging). making use of pamam-g dendrimers conjugated with near-infrared (nir) dyes and an in radionuclide probe, multimodal nanoprobes were developed for radionuclide and multicolour optical lymphatic imaging [ , ] . later kobayashi also proposed the use of quantum dots for labelling cancer cells and dendrimer-based optical agents for visualising lymphatic drainage and identifying sentinel lymph nodes [ ] . polylysine dendrimers have been best used for targeting the lymphatic system and lymph nodes. carbon nanotubes (cnt) possess various mechanochemical properties like high surface area, mechanical strength and thermal and chemical stability which cause them to be versatile carriers for drugs, proteins, radiologicals and peptides to target tumour tissues. hydrophilic multiwalled carbon nanotubes (mwnts) coated with magnetic nanoparticles (mn-mwnts) have emerged as an effective delivery system for lymphatic targeting following subcutaneous injection of these particles into the left footpad of sprague dawley rats; the left popliteal lymph nodes were dyed black. mn-mwnts were favourably absorbed by lymphatic vessels following their transfer into lymph nodes and no uptake was seen in chief internal organs such as the liver, spleen, kidney, heart and lungs. gemcitabine loaded in these particles was evaluated for its lymphatic delivery effi ciency and mn-mwnts-gemcitabine displayed the maximum concentration of gemcitabine in the lymph nodes [ ] . mcdevitt et al. synthesised tumour-targeting water-soluble cnt constructs by covalent attachment of monoclonal antibodies like rituximab and lintuzumab using , , , -tetraazacyclododecane- , , , -tetraacetic acid (dota) as a metal ion chelator while the fl uorescent probe was fl uorescein. cnt-([ in] dota) (rituximab) explicitly targeted a disseminated human lymphoma in vivo trials compared to the controls cnt-([ in] dota) (lintuzumab) and [ in]rituximab [ ] . tsuchida and coworkers evaluated the drug delivery effi ciency of water-dispersed carbon nanohorns in a non-small cell lung cancer model. polyethylene glycol (peg)-doxorubicin conjugate bound oxidised single-wall carbon nanohorns (oxswnhs) injected intratumourally into mice bearing human non-small cell lung cancer (nci-h ) caused a signifi cant retardation of tumour growth. histological analyses showed (probably by means of interstitial lymphatic fl uid transport), migration of oxswnhs to the axillary lymph node occurred which is a major site of breast cancer metastasis near the tumour [ ] . shimada et al. described a silica particle-based lymphatic drug delivery system of bleomycin and compared its therapeutic effi cacy to that of free bleomycin solution in a transplanted tumour model in animals. silica particle-adsorbed bleomycin showed considerable inhibitory effect on tumour growth and lymph node metastasis compared to free bleomycin solution [ ] . activated carbon particles of aclarubicin are used for adsorption and sustained release into lymph nodes. upon subcutaneous administration into the fore foot-pads of rats these particles showed signifi cantly elevated distribution of aclarubicin to the auxiliary lymph nodes compared to aqueous solution of the drug [ ] . activated carbon particles of aclacinomycin a, adriamycin, mitomycin c and pepleomycin have also been used by another group for adsorption. higher level of drug concentration was maintained in the new dosage form than in the solution form [ ] . antibody-drug conjugates enhance the cytotoxic activity of anticancer drugs by conjugating them with antibodies. antibodies conjugated with cytostatic drugs such as calicheamicin have been used for the treatment of various lymphomas, including non-hodgkin b-cell lymphoma (nhl), follicular lymphoma (fl) and diffuse large b-cell lymphoma (dlbcl) [ - ] . cd b-cell marker is expressed on the surface membrane of pre-b-lymphocytes and mature b-lymphocytes. the anti-cd mab rituximab (rituxan) is now the most potential antibody for the treatment of non-hodgkin b-cell lymphomas (b-nhl) [ ] . rituximab-conjugated calicheamicin elevated the antitumour activity of rituximab against human b-cell lymphoma (bcl) xenografts in preclinical models [ ] . cd is a b-lymphoid lineage-specifi c differentiation antigen expressed on the surface of both normal and malignant b-cells. hence, the cd -specifi c antibody could be effective in delivering chemotherapeutic drugs to malignant b-cells. also, cd (siglec- ) antibodies targeting to cd are suited for a trojan horse strategy. thus, antibody-conjugated therapeutic agents bind to the siglec and are carried effi ciently into the cell [ ] . a lot of interest has been seen in clinical progress of the conjugated anti-cd antibodies, especially inotuzumab ozogamicin (cmc- ) [ ] . cd is expressed in the malignant hodgkin and reed-sternberg cells of classical hodgkin lymphoma (hl) and anaplastic large-cell lymphoma. younes and bartlett reported an ongoing phase i dose-escalation trial in relapsed and refractory hl patients with seattle genetics (sgn- ) , a novel anti-cd -antibody-monomethylauristatin e conjugate. sgn- was stable in the blood and released the conjugate only upon internalisation into cd -expressing tumour cells [ ] . huang et al. constructed (anti-her / neu -igg -ifn α ), another antibody-drug conjugate, and examined its effect on a murine b-cell lymphoma, c , expressing human her / neu , and this signifi cantly inhibited c /her tumour growth in vivo [ ] . hybrid systems use combination of two or more delivery forms for effective targeting. khatri et al. prepared and investigated the in vivo effi cacy of plasmid dna-loaded chitosan nanoparticles for nasal mucosal immunisation against hepatitis b. chitosan-dna nanoparticles prepared by the coacervation process adhered to the nasal or gastrointestinal epithelia and are easily transported to the nasal-associated lymphoid tissue (nalt) and peyer's patches of the gut-associated lymphoid tissue (galt) both as iga inductive site [ ] , in which chitosan-dna might be taken in by m cell, and transported across the mucosal boundary and thereby transfect immune cells within nalt or galt [ ] . a work demonstrates targeting of three peptides containing sequences that bind to cell markers expressed in the tumour vasculature (p -nrp- and p -flt- ) [ , ] and tumour lymphatics (p -lyp- ) [ ] and were tested for their ability to target (nitrilotriacetic acid)-ditetradecylamine (nta -dtda) containing liposomes to subcutaneous b -f tumours. signifi cantly, a potential antitumour effect was seen after administration of doxorubicin-loaded peg liposomes engrafted with p -nrp- . hybrid liposomes composed of l -α-dimyristoylphosphatidylcholine and polyoxyethylene ( ) dodecyl ether prepared by sonication showed remarkable reduction of tumour volume in model mice of acute lymphatic leukaemia (all) treated intravenously with hl- without drugs after the subcutaneous inoculation of human all (molt- ) cells was verifi ed in vivo. prolonged survival (> %) was noted in model mice of all after the treatment with hl- without drugs [ ] . in a report, lyp- peptide-conjugated pegylated liposomes loaded with fl uorescein or doxorubicin were prepared for targeting and treating lymphatic metastatic tumours. the in vitro cellular uptake and in vivo near-infrared fl uorescence imaging results confi rmed that lyp- -modifi ed liposome increased uptake by tumour cells and metastatic lymph nodes. in another study, in vitro cellular uptake of peg-plga nanoparticle (lyp- -nps) was about four times that of peg-plga nanoparticles without lyp- (nps). in vivo study, about eight times lymph node uptake of lyp- -nps was seen in metastasis than that of nps, indicated lyp- -np as a promising carrier for targetspecifi c drug delivery to lymphatic metastatic tumours [ ] . currently, surgery, radiation therapy and chemotherapy are the principal methods for cancer treatment. gene therapies may act synergistically or additively with them. for example, another case demonstrated that replacement of the p (protein ) gene in p -defi cient cancer cell lines enhanced the sensitivity of these cells to ad-p (adenovirus-expressed protein ) and cisplatin (cddp) and resulted into greater tumour cell death [ ] . later, son and huang [ ] stated that treatment of cddp-resistant tumour cells with cddp increased the sensitivity of these cells to transduction by dna-carrying liposomes. also, chen et al. [ ] described that to improve tumour killing, herpes simplex virus thymidine kinase (hsv-tk) and interleukin (il) expression can be combined. on the whole, greater therapeutic effect can be achieved by effectively combining conventional cancer treatments and gene therapy together. mainly colloidal carriers have emerged as potential targeting agents to lymphatic system. physicochemical properties affect the effi ciency of colloid uptake into the lymphatic system [ ]. these properties include size, number of particles, surface charge, molecular weight and colloid lipophilicity. physicochemical properties are altered by adsorption of group of hydrophilic polymers like poloxamers and poloxamines to the particle surface. these properties modifi ed the biodistribution of particles in vivo, particularly the avoidance of the reticuloendothelial system (res) upon intravenous administration [ , ] . in one study, it was opined that opsonisation may cause alteration of the particle surface in vivo [ ] . size could be important factor in defi ning the behaviour of particulates after subcutaneous injection. small particles with diameter less than a few nanometres generally exchanged through the blood capillaries, whereas larger particles of diameters up to a few tens of nanometres absorbed into the lymph capillaries. but particles over a size of few hundred nanometres remain trapped in the interstitial space for a long time [ ] . christy et al. have shown a relationship between colloid size and ease of injection site drainage using model polystyrene nanospheres after subcutaneous administration to the rat [ ] . results showed distribution of polystyrene nanospheres in the size range - nm h after administration and - % of the recovered dose retained at the administration site, and as particle diameter increased, drainage became slower. it has been proposed earlier that the optimum colloid size range for lymphoscintigraphic agents is l - nm [ ] . size has less importance when colloids are administered intraperitoneally (i.p.) within the nanometre size range, as drainage is only from a cavity into the initial lymphatics; hence, no diffusion is required through the interstitial space [ ]. the size limit of the open junctions of the initial lymphatic wall is the only barrier to uptake from the peritoneal cavity into the lymphatics [ ] . more number of particles at the injection site decreases their rate of drainage, owing to increased obstruction of their diffusion through the interstitial space [ , ] . scientists at nottingham university investigated this effect using polystyrene nanospheres of nm. following administration to the rat, the concentration range of nanospheres was approximately . - . mg/ml. lower lymphatic uptake was seen on increasing the concentration of nanospheres in the injection volume due to slower drainage from the injection site. injecting oily vehicles intramuscularly to the rat, the effect of injection volume has been studied. increasing volume of sesame oil accelerated oil transport into the lymphatic system. upon s.c. administration, volumes of aqueous polystyrene particle suspensions have been investigated in the range - μl [ ]. surface charge studies have been done utilising liposome as colloidal carrier. the surface charge of liposomes affected their lymphatic uptake from s.c. and i.p. injection sites. negatively charged liposomes showed faster drainage than that for positive liposomes after i.p. administration [ ] . patel et al. also indicated that liposome localisation in the lymph nodes followed a particular order negative > positive > neutral [ ] . macromolecule having high molecular weight has a decreased ability for exchange across blood capillaries and lymphatic drainage becomes the route of drainage from the injection site which shows a linear relationship between the molecular weight of macromolecules and the proportion of the dose absorbed by the lymphatics. for a compound to be absorbed by the lymphatics, the molecular weight should range between , and , [ , ] . the effect of molecular weight becomes negligible when targeting carriers to the lymphatic system as the molecular weight of a colloidal carrier is generally less than , da. the most important determinant of the phagocytic response and so lymphatic uptake is the lipophilicity of a colloid [ ] . opsonins generally unite with lipophilic rather than hydrophilic surfaces; hence, the hydrophilic particles show reduced phagocytosis [ ] . hydrophobic polystyrene nanospheres adsorbed with hydrophilic block copolymers showed drastic reduction in phagocytosis prior to i.v. administration [ ] . in the case of polystyrene nanospheres of -nm diameter, peo chains of the poloxamers and poloxamines adsorbed onto the surface of the particle described the relationship between interstitial injection site drainage and lymph node uptake in rat [ ] . uncoated nanospheres of this diameter showed reduced drainage from the injection site with % of the administered dose remaining after h. the adsorption of block copolymers can enhance the drainage from the injection site such that levels remaining at the injection site may be as little as % after h, with very hydrophilic polymers such as poloxamine . uptake of nanospheres into the regional lymph nodes may also be improved by the adsorption of block copolymers with intermediate lengths of polyoxyethylene, such as poloxamine . this polymer may sequester up to % of the given dose by the lymph nodes after h [ ] . surface modifi cation could prove as an effective strategy for potential targeting to lymphatic system. the infl uence can be quoted in following ways. coating of a carrier with hydrophilic and sterically stabilised peg layer can successfully enhance lymphatic absorption, reducing specifi c interaction of particle with the interstitial surrounding, and inhibit the formation of too large particle structure [ ] . surface modifi cation of liposomes with peg also does not have a significant effect on lymph node uptake. small liposomes coated with peg showed greatest clearance from the s.c. injection site with small -nm peg-coated liposomes having < % remaining at the injection site at h. larger neutral and negatively charged liposomes had a clearance > % remaining at the initial s.c. injection site. however, this smaller amount of large liposomes that were cleared from the injection site was compensated by better retention in the lymph node [ ] . oussoren et al. reported that the amount of liposomes cleared from the injection site was somewhat greater with the peg-coated liposomes [ ] . this improved clearance did not result in improved lymph node retention because the fraction of peg liposomes retained by the lymph node is decreased. phillips et al. also studied the slightly improved clearance of peg-coated liposomes from the s.c. injection site [ ] . porter and coworkers demonstrated that pegylation of poly-l -lysine dendrimers resulted into better absorption from s.c. injection sites and stated that the extent of lymphatic transport may be improved by increasing the size of the pegylated dendrimer complex. they estimated the lymphatic uptake and lymph node retention properties of several generation four dendrimers coated with peg or -benzene sulphonate after subcutaneous administration in rats. for this surface modifi cation study, three types of pegs with molecular weights of , or , da were taken. peg -derived dendrimers showed rapid and complete absorption into the blood when injected subcutaneously, and only % of the total given dose was found in the pooled thoracic lymph over h, whereas peg -and peg derived dendrimers showed lesser absorption, and a higher amount was recovered in lymphatics ( %) over h. however, the benzene sulphonate-capped dendrimer was not well absorbed either in the blood or in lymph following subcutaneous injection [ ] . carriers capped with nonspecifi c human antibodies as ligands showed greater lymphatic uptake and lymph node retention compared to uncoated one at the s.c. site. liposomes coated with the antibody, igg, have been shown to increase lymph node localisation of liposomes to . % of the injected dose at h, but this level decreased to % by h [ ] . in a study, the liposomes containing positively charged lipids had approximately - times the lymph node localisation (up to . % of the injected dose) than liposomes containing neutral or negatively charged lipids ( . % of the injected dose) [ ] . attachment of mannose to the surface of a liposome increased lymph node uptake by threefold compared to control liposomes [ ] . another study demonstrated hbsag entrapped dried liposomes with their surfaces modifi ed with galactose. pharmacokinetic study in rats showed that galactosylated liposomes delivered higher amounts of hbsag to the regional lymph nodes than other ungalactosylated formulations [ ] . lectin is another ligand that can be attached to the carriers for improved targeting to intestinal lymphatics. bovine serum albumin containing acid phosphatase model protein and polystyrene microspheres conjugated with mouse m-cell-specifi c ulex europaeus lectin. ex vivo results showed that there was favoured binding of the lectin-conjugated microspheres to the follicle-associated epithelium. final results indicated that coupling of ligands such as lectin specifi c to cells of the follicleassociated epithelium can improve the targeting of encapsulated candidate antigens for delivery to the peyer's patches of the intestine for better oral delivery [ ] . to improve carrier retention in lymph nodes, a new method of increasing lymphatic uptake of subcutaneously injected liposome utilises the high-affi nity ligands biotin and avidin. biotin is a naturally occurring cofactor and avidin is a protein derived from eggs. avidin and biotin are having extremely high affi nity for each other. for instance, upon injection, the avidin and the biotin liposomes move into the lymphatic vessels. biotin liposomes that migrate through the lymphatic vessels meet the avidin resulting in an aggregate that becomes trapped in the lymph nodes [ , ] . the biotin liposome/avidin system has promising potential as therapeutic agent for delivery to lymph nodes. it can be applied not only to s.c. targeting of lymph nodes but also to intracavitary lymph node targeting [ ] . different ligands with their application in lymphatic targeting are represented in table . . the lymphatics have the potential to play a major role in anticancer treatment as lymphatic spread is recognised to precede haematological spread in many cancers including melanoma, breast, colon, lung and prostate cancers. currently, the focus is on the development of drug carriers that can localise chemotherapy to the lymphatic system, thus improving the treatment of localised disease while minimising the exposure of healthy organs to cytotoxic drugs. the delivery of novel carriers to lymph nodes for therapeutic purposes has much promise. giving importance to the lymphatic route in metastasis, this delivery system may have great potential for targeted delivery of various therapeutic agents to tumours and their metastatic lymph nodes. various delivery systems have been discussed here but colloidal carriers, especially, liposomes have been the carrier of choice to date. the purpose of this review is to provide an improved and effective lymphotropic system with a satisfactory quality for clinical use and to establish a preparation method applicable for industrial production. surface-engineered lymphotropic systems may prove as an effective carrier for anti-hiv, anticancer and oral vaccine delivery in near future. . delivery of antigens to gut-associated lymphoid tissue (galt) intestinal delivery [ ] . microparticles active targeting of peripheral lymph nodes doppler ultrasonography contrast agent [ ] . lymph vaccine delivery [ - ] . . block copolymer of poloxamine and poloxamer nanospheres regional lymph nodes - [ ] . lyp- nanoparticles, liposomes targeted to lymphatic vessels and also in tumour cells within hypoxic area antitumour [ ] . liposomes targeting to lymph node mediastinal lymph node targeting [ ] . liposome targeting to lymph node increased lymph node retention [ , ] the physiology of the lymphatic system the anatomy and development of the jugular lymph sacs in the domestic cat (felis domestica) on the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig dual origin of avian lymphatics lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature an essential role for prox in the induction of the lymphatic endothelial cell phenotype prox function is required for the development of the murine lymphatic system live imaging of lymphatic development in the zebrafi sh developmental and pathological lymphangiogenesis: from models to human disease tumor lymphangiogenesis and melanoma metastasis cardiovascular physiology new insights into the molecular control of the lymphatic vascular system and its role in disease advanced colloid-based systems for effi cient delivery of drugs and diagnostic agents to the lymphatic tissues the structure of lymphatic capillaries in lymph formation specifi c adhesion molecules bind anchoring fi laments and endothelial cells in human skin initial lymphatics focal adhesion molecules expression and fi brillin deposition by lymphatic and blood vessel endothelial cells in culture the second valve system in lymphatics evidence for a second valve system in lymphatics: endothelial microvalves ultrastructural studies on the lymphatic anchoring fi laments new horizons for imaging lymphatic function lymphatic smooth muscle: the motor unit of lymph drainage the fi ne structure and functioning of tissue channels and lymphatics clinically oriented anatomy lymphangiogenesis in development and human disease acyclic nucleoside phosphonate analogs delivered in ph-sensitive liposomes liposomes for drug targeting in the lymphatic system liposomes to target the lymphatics by subcutaneous administration novel method of greatly enhanced delivery of liposomes to lymph nodes current concepts in lymph node imaging old friends, new ways: revisiting extended lymphadenectomy and neoadjuvant chemotherapy to improve outcomes targeted delivery of indinavir to hiv- primary reservoirs with immunoliposomes studies on lymphoid tissue from hivinfected individuals: implications for the design of therapeutic strategies lymphoid tissue targeting of anti-hiv drugs using liposomes a randomized clinical trial comparing single-and multi-dose combination therapy with diethylcarbamazine and albendazole for treatment of bancroftian fi lariasis infección bacteriana por ántrax bioterrorism-related inhalational anthrax: the fi rst cases reported in the united states fatal inhalational anthrax in a -year-old connecticut woman extrapulmonary tuberculosis: clinical and epidemiologic spectrum of cases nanoparticles for drug delivery in cancer treatment polymeric drugs for effi cient tumor-targeted drug delivery based on epr-effect exploiting the enhanced permeability and retention effect for tumor targeting drug targeting and tumor heterogeneity does a targeting ligand infl uence nanoparticle tumor localization or uptake? high affi nity restricts the localization and tumor penetration of single-chain fv antibody molecules vcam- directed immunoliposomes selectively target tumor vasculature in vivo lymphatic targeting with nanoparticulate system a lymphotropic colloidal carrier system for diethylcarbamazine: preparation and performance evaluation evaluation of endoscopic pirarubicin-lipiodol emulsion injection therapy for gastric cancer targeted lymphatic transport and modifi ed systemic distribution of ci- , a lipophilic lipid-regulator drug, via a formulation approach self-emulsifying drug delivery systems (sedds) of coenzyme q : formulation development and bioavailability assessment liposomes as vehicles for the local release of drugs enhanced oral bioavailability and intestinal lymphatic transport of a hydrophilic drug using liposomes delivery of liposomal doxorubicin (doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure reduced cardiotoxicity and comparable effi cacy in a phase iii trial of pegylated liposomal doxorubicin hcl (caelyx™/doxil®) versus conventional doxorubicin for fi rst-line treatment of metastatic breast cancer doxil offers hope to ks sufferers liposomal doxorubicin (doxil): in vitro stability, pharmacokinetics, imaging and biodistribution in a head and neck squamous cell carcinoma xenograft model caelyx/doxil for the treatment of metastatic ovarian and breast cancer patent blue v encapsulation in liposomes: potential applicability to endolympatic therapy and preoperative chromolymphography aerosol delivery of liposomal formulated paclitaxel and vitamin e analog reduces murine mammary tumor burden and metastases novel vitamin e analogue and -nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis use of liposome preparation to treat mycobacterial infections nanoradioliposomes molecularly modulated to study the lung deep lymphatic drainage solid lipid nanoparticles in lymph and plasma after duodenal administration to rats duodenal administration of solid lipid nanoparticles loaded with different percentages of tobramycin transmucosal transport of tobramycin incorporated in sln after duodenal administration to rats. part i-a pharmacokinetic study pharmacokinetics and tissue distribution of idarubicin-loaded solid lipid nanoparticles after duodenal administration to rats infl uence of administration route on tumor uptake and biodistribution of etoposide loaded solid lipid nanoparticles in dalton's lymphoma tumor bearing mice metastatic patterns in small-cell lung cancer: correlation of autopsy fi ndings with clinical parameters in patients metastatic pattern in non-resectable non-small cell lung cancer solid lipid nanoparticles (sln) for controlled drug delivery-a review of the state of the art lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles infl ammation imaging using tc- m dextran intralymphatic chemotherapy using a hyaluronan-cisplatin conjugate lymph node localisation of biodegradable nanospheres surface modifi ed with poloxamer and poloxamine block co-polymers block copolymer carrier systems for translymphatic chemotherapy of lymph node metastases translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer enhancement of therapeutic effi cacy of bleomycin by incorporation into biodegradable poly-d, l-lactic acid targeting colloidal particulates to thoracic lymph nodes comparison of dendrimer-based macromolecular contrast agents for dynamic micro-magnetic resonance lymphangiography delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging multimodal nanoprobes for radionuclide and fi ve-color near-infrared optical lymphatic imaging a dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fl uorescence imaging to localize the sentinel lymph node in mice multicolor imaging of lymphatic function with two nanomaterials: quantum dot-labeled cancer cells and dendrimerbased optical agents hydrophilic multi-walled carbon nanotubes decorated with magnetite nanoparticles as lymphatic targeted drug delivery vehicles tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes waterdispersed single-wall carbon nanohorns as drug carriers for local cancer chemotherapy enhanced effi cacy of bleomycin adsorbed on silica particles against lymph node metastasis derived from a transplanted tumor selective distribution of aclarubicin to regional lymph nodes with a new dosage form: aclarubicin adsorbed on activated carbon particles carbon dye as an adjunct to isosulfan blue dye for sentinel lymph node dissection safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of b-cell non-hodgkin's lymphoma: results of a phase i study therapeutic potential of cd -specifi c antibody-targeted chemotherapy using inotuzumab ozogamicin (cmc- ) for the treatment of acute lymphoblastic leukemia antibody-targeted chemotherapy with cmc- : a cd -targeted immunoconjugate of calicheamicin for the treatment of b-lymphoid malignancies preclinical anti-tumor activity of antibody-targeted chemotherapy with cmc- (inotuzumab ozogamicin), a cd -specifi c immunoconjugate of calicheamicin, compared with non-targeted combination chemotherapy with cvp or chop rituximab (rituxan®/mabthera®): the fi rst decade cd -specifi c antibody-targeted chemotherapy of non-hodgkin's b-cell lymphoma using calicheamicin-conjugated rituximab siglecs as targets for therapy in immune-cell-mediated disease clinical activity of the immunoconjugate cmc- in b-cell malignancies: preliminary report of the expanded maximum tolerated dose (mtd) cohort of a phase study objective responses in a phase i dose-escalation study of sgn- , a novel antibody-drug conjugate (adc) targeting cd , in patients with relapsed or refractory hodgkin lymphoma targeted delivery of interferonalpha via fusion to anti-cd results in potent antitumor activity against b-cell lymphoma chitosan for mucosal vaccination polysaccharide colloidal particles as delivery systems for macromolecules suppression of tumor growth and metastasis by a vegfr- antagonizing peptide identifi ed from a phage display library antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin- a tumor-homing peptide with a targeting specifi city related to lymphatic vessels chemotherapy with hybrid liposomes for acute lymphatic leukemia leading to apoptosis in vivo lyp- -conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector exposure of human ovarian carcinoma to cisplatin transiently sensitizes the tumor cells for liposome-mediated gene transfer combination gene therapy for liver metastasis of colon carcinoma in vivo polymeric microspheres as drug carriers the organ distribution and circulation time of intravenously injected colloidal carriers sterically stabilized with a blockcopolymerpoloxamine fate of liposomes in vivo: a brief introductory review the characterisation of radio colloids used for administration to the lymphatic system effect of size on the lymphatic uptake of a model colloid system radiolabeled colloids and macromolecules in the lymphatic system electron microscopic studies on the peritoneal resorption of intraperitoneally injected latex particles via the diaphragmatic lymphatics lymphatic transport of liposomeencapsulated drugs following intraperitoneal administration-effect of lipid composition assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of -technetium marker to represent intact liposomes in lymph nodes effect of molecular weight on the lymphatic absorption of water-soluble compounds following subcutaneous administration surface engineered nanospheres with enhanced drainage into lymphatics and uptake by macrophages of the regional lymph nodes serum opsonins and liposomes: their interaction and opsonophagocytosis physicochemical principles of pharmacy targeting of colloids to lymph nodes: infl uence of lymphatic physiology and colloidal characteristics evaluation of [( m) tc] liposomes as lymphoscintigraphic agents: comparison with [( m) tc] sulfur colloid and [( m) tc] human serum albumin lymphatic uptake and biodistribution of liposomes after subcutaneous injection: iii. infl uence of surface modifi cation with poly(ethyleneglycol) pegylation of polylysine dendrimers improves absorption and lymphatic targeting following sc administration in rats lymph node localization of non-specifi c antibody-coated liposomes modifi ed in vivo behavior of liposomes containing synthetic glycolipids enhanced lymph node delivery and immunogenicity of hepatitis b surface antigen entrapped in galactosylated liposomes targeted delivery of antigens to the gut-associated lymphoid tissues: . ex vivo evaluation of lectinlabelled albumin microspheres for targeted delivery of antigens to the m-cells of the peyer's patches avidin/biotin-liposome system injected in the pleural space for drug delivery to mediastinal lymph nodes pharmacokinetics and biodistribution of in avidin and tc biotin-liposomes injected in the pleural space for the targeting of mediastinal nodes folate-peg-ckk -dtpa, a potential carrier for lymph-metastasized tumor targeting nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery molecular targeting of lymph nodes with l-selectin ligand-specifi c us contrast agent: a feasibility study in mice and dogs hyaluronan in drug delivery lymphatic targeting of zidovudine using surfaceengineered liposomes alginate/chitosan microparticles for tamoxifen delivery to the lymphatic system homing of negatively charged albumins to the lymphatic system: general implications for drug targeting to peripheral tissues and viral reservoirs key: cord- - rblzyry authors: hill, andrew; wang, junzheng; levi, jacob; heath, katie; fortunak, joseph title: minimum costs to manufacture new treatments for covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: rblzyry introduction: ‘repurposing’ existing drugs to treat covid- is vital to reducing mortality and controlling the pandemic. several promising drugs have been identified and are in various stages of clinical trials globally. if efficacy of these drugs is demonstrated, rapid, mass availability at an affordable cost would be essential to ensuring equity and access especially amongst low- and middle-income economies. methods: minimum costs of production were estimated from the costs of active pharmaceutical ingredients using established methodology, which had good predictive accuracy for medicines for hepatitis c and hiv amongst others. data were extracted from global export shipment records or analysis of the route of chemical synthesis. the estimated costs were compared with list prices from a range of countries where pricing data were available. results: minimum estimated costs of production were us $ . /day for remdesivir, $ . /day for favipiravir, $ . /day for hydroxychloroquine, $ . /day for chloroquine, $ . /day for azithromycin, $ . /day for lopinavir/ritonavir, $ . /day for sofosbuvir/daclatasvir and $ . /day for pirfenidone. costs of production ranged between $ . and $ per treatment course ( – days). current prices of these drugs were far higher than the costs of production, particularly in the us. conclusions: should repurposed drugs demonstrate efficacy against covid- , they could be manufactured profitably at very low costs, for much less than current list prices. estimations for the minimum production costs can strengthen price negotiations and help ensure affordable access to vital treatment for covid- at low prices globally. introduction as the sars-cov pandemic continues to grow, researchers worldwide are urgently looking for new treatments to prevent new infections, cure those already infected or lessen the severity of disease. an effective vaccine may not be widely available until late , even if trials are successful [ ] . there are clinical trials in progress to 'repurpose' drugs, normally indicated for other diseases, to treat covid- [ , ] . the shortened development timeline and reduced costs to this approach [ ] of using already existing compounds is particularly advantageous compared with new drug discovery in a pandemic situation, where time is of the essence. antiviral drugs include nucleotide analogue remdesivir, which was previously used experimentally but unsuccessfully against ebola [ ] [ ] [ ] [ ] , favipiravir, used to treat influenza [ ] , the hiv protease inhibitor lopinavir/ritonavir (lpv/r) [ , ] , the antimalarials chloroquine and hydroxychloroquine [ ] [ ] [ ] , and the directacting antivirals sofosbuvir and daclatasvir [ ] , which are all potential candidates. additionally, treatments to improve lung function and reduce inflammation, such as pirfenidone [ , ] and tocilizumab [ , ] , are being evaluated in clinical trials. most of the clinical trials reported so far are small pilot studies, often non-randomised, making interpretation of current evidence difficult. however, results from randomised trials of these repurposed treatments should become available from may onwards. if favourable results are shown from these new trials, there is the potential to rapidly upscale production of the most promising drugs. the safety profiles of these drugs have already been established from clinical trials for other diseases, so they could be rapidly deployed to treat covid- before vaccines become available. low-and middle-income countries will need access to these treatments at minimum prices to ensure all those in need can be treated. even in high-income countries, the burden of disease could be so great that access to drugs at minimum costs could also be necessary. the hiv epidemic has been controlled by mass treatment with antiretroviral drugs worldwide, at very low unit costs. large donor organisations such as the global fund for aids, tb and malaria (gfatm) and the president's emergency plan for aids relief (pepfar) order drugs to treat > million people with hiv, at prices close to the cost of production [ , ] . this system allows low-and middle-income countries to access high-quality drugs at affordable prices. the minimum costs of drug production can be estimated by calculating the cost of active pharmaceutical ingredients (api), which is combined with costs of excipients, formulation, packaging and a profit margin, to estimate the price of 'final finished product' (ffp) -the drug ready for use. there are established methods for these calculations, which have reliably predicted the minimum costs of drugs to cure hepatitis c [ , ] and other diseases [ , ] . the purpose of this analysis was to apply the same calculations to the new candidate treatments for covid- . the leading candidate drugs to treat covid- were selected based on recent reviews and analysis of ongoing clinical trials [ , , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the treatments selected were remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, chloroquine, azithromycin, pirfenidone, tocilizumab and sofosbuvir/daclatasvir. all are currently being evaluated in randomised trials, with results expected between may and september . the methods used to estimate minimum costs of production have been described previously [ , ] . briefly, we analysed the costs of exports of api from india using the online tracking database panjiva [ ] , which shows details of all shipments of api with quantities and costs per kilogram. we used all available costing data for each drug api found on panjiva, excluding shipments < kg in size, alongside the lowest and highest % of results based on prices per kg. a % api loss during tableting process was factored into our calculations, and a conversion cost of us $ . per tablet was used. a multiplier based on api mass was applied to account for the price of excipients, which are additional substances needed to convert api into fpp. our estimated api costs presume that production is carried out by a generic provider of apis, where associated costs of capital investment, overhead and labour are not as high as for production by originator companies. this method was applied to small-molecule drugs only. in addition, the drug remdesivir, which is administered by iv infusion, was considered separately when estimating formulation costs. a profit margin of % and indian taxation of % on profit was added. these results were cross-checked with a second api database, pharmacompass [ ] , which displayed records up to only. panjiva was selected to provide real time, up-to-date shipment and cost data. the estimates assume a volume of kg of api for each compound. three drugs did not have available data on api production: remdesivir and favipiravir and tocilizumab. for the first two, api production costs were estimated based on published routes of chemical synthesis [ , , , ] . since remdesivir is administered by iv infusion, the costs of production were adjusted to include the cost of injections, according to an established method used for the world health organization (who) essential medicines list [ ] . it was not possible to track the cost of api for the monoclonal antibody tocilizumab; therefore, we tracked list prices in a range of countries, in particular developing economies as a proxy for minimum costs. the costs of regulatory filings and approvals are often significant add-ons to the initial use of drugs in any specific country. all drugs analysed in this study, except for remdesivir have been approved for treatment for some indications in all countries, but few are approved for treatment of covid- . remdesivir is an investigational drug without any prior regulatory approvals, but it has a known favourable safety profile after clinical trials against ebola. favipiravir has been approved for the treatment of influenza in japan since [ ] , and in china since march . it has also been approved for investigational use (china and italy) against covid- in march . we have not included the cost or timing associated with regulatory approvals for the use of these drugs. we are assuming in our analysis that the who and other influential regulatory agencies will cooperate to define a pathway for use of these drugs which does not include additional financial outlays or filing for marketing approvals. the minimum costs of production were then compared with published list prices for each drug in a range of countries -usa, uk, france, sweden, south africa, india, bangladesh, malaysia, brazil, turkey, pakistan, egypt and china [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] -to give a representative sample of prices in countries with different levels of economic development. for consistency, we selected a single data source per country to be used for all searches of drug prices within that country, based on the organisation of data and perceived reliability. not all drugs analysed in this study were available in our selected countries, and in some countries, online pharmacy sites were used because national databases were not available. where several prices were available in the same database, the lowest price was selected. available clinical trial data for each drug were collected from literature searches, clinicaltrials.gov and the chinese clinical trials registry. results from pilot studies were included, together with the planned clinical trial programmes and expected completion dates. the predicted costs of production, and the highest/lowest available list prices of all drugs analysed are shown in table , and chemical structures for all drugs are shown in figure . remdesivir remdesivir (formerly gs- ) has been evaluated for treatment of sars and ebola [ , ] . there are five randomised trials of remdesivir for sars-cov , with first results expected at the end of april . there is a -day course of treatment under evaluation. the dose of remdesivir is mg on the first day with mg per day thereafter. remdesivir is administered by iv infusion. one metric ton of remdesivir is sufficient api to manufacture , courses of treatment, without allowance for any losses during formulation. based on analysis of the published second-generation route of chemical synthesis and assumed overheads such as occupancy rate per hour and labour of an india-based generic producer, the cost of api was estimated to be $ /kg [ , ] for -kg batches produced without capital investment in dedicated remdesivir production facilities. the -day course of treatment would therefore cost $ . per person. after adjustment for the cost of formulation (and % losses projected during formulation), cost of vials, profit margin and tax, the estimated cost per treatment would be approximately $ per person as shown in figure a . daily cost is estimated to be $ . . as an iv infusion, there would be an additional cost for saline, estimated at $ per infusion [ ] , equipment such as syringe, sterile water for reconstitution and iv lines, as well as staff-time cost associated with the healthcare professional administrating the infusion. these additional non-drug components are likely to be more expensive than the estimated cost of the drug itself and are not included in our cost estimate. the main randomised trial of favipiravir evaluated up to days of treatment, vs another influenza drug, umifenovir, in patients. after days of treatment, the clinical recovery rate was % for favipiravir vs % for umifenovir (p< . ). recovery from fever was also faster for people treated with favipiravir (p< . ), but there was no difference between arms in auxiliary oxygen therapy or non-invasive mechanical ventilation rates [ ] . a second, non-randomised study in china evaluated days of treatment with either favipiravir or lpv/r. the median time to virus clearance was significantly shorter in the favipiravir group ( days) vs the lpv/r group ( days, p< . ) [ ] . favipiravir is dosed mg twice daily. a metric ton of favipiravir, therefore, would provide approximately , courses of treatment. the cost of api was estimated at about $ /kg, based [ , ] . this is a simple molecule to synthesise: several very basic steps involved are more akin to processes for manufacturing fine chemicals rather than pharmaceuticals. the structure is a -fluoro substituted -hydroxypyrazine- -carboxamide. based on a -day course of treatment, the cost of the api would be $ . . after adjustment for loss, formulation, packaging and profit margins, the estimated cost of production is $ per -day treatment cost (figure b ), or $ . per day. in late february , favipiravir was launched for sale in china for $ per treatment course, in an announcement by the shandong provincial public resource trading centre [ ] . the standard dose of lpv/r is / mg twice daily. lpv/r has been evaluated in two published studies of sars-cov infection [ ] . in these studies, there has been no difference in measures of efficacy between lpv/r (given for days) and control treatment. a systematic review by the who showed no clear evidence for the efficacy of lpv/r. however, there were very few clinical trials available for this evaluation at the time of the review [ ] . it is not clear whether this hiv protease inhibitor will work in the earlier stage of sars-cov infection, or if used in combination with other drugs. results from other clinical trials are expected between june and august . the estimated cost of production is $ . per treatment course, based on api cost of $ /kg for lopinavir and $ /kg for ritonavir. after adjustment for loss, formulation, packaging and profit margins, the estimated cost of production of the combined drug is $ per -day course, or $ . per day. searches in selected national databases and online pharmacies yielded a range of list prices between $ in the us, to $ in south africa per -day dose, as shown in figure a . additionally, lpv/r is also available through the global fund for a range of low-and middle-income countries, with a median cost of $ per -day course [ ] . in vitro, hydroxychloroquine is predicted to show superior activity against covid- compared with chloroquine [ ] . in a recent parallel-group, a randomised chinese study in wuhan of patients found treatment with hydroxychloroquine mg per day led to faster symptomatic improvement times in the treatment arm (n = ) after days in terms of temperature normalisation and cough remission, and a greater proportion of patients ( . % vs . %) with improved pneumonia [ ] . similarly, another chinese randomised trial in shanghai [ ] of patients also used mg per day in the treatment arm but found no statistically significant difference in clinical findings, symptomatic improvements or radiological improvements between the arms by day . the authors also highlighted the need for much larger, better powered trials to reach reliable conclusions. journal of virus eradication ; : - minimum costs to manufacture new treatments for covid- different dosing protocols are being used for hydroxychloroquine, including mg daily in the small, open-label, non-randomised french study by gautret et al. (n = ) that has suggested improved efficacy for hydroxychloroquine [ ] . for our analysis, we have therefore chosen mg daily, which was the most commonly used dosage. this is also the upper recommended dose by the british national formulary for existing indications [ ] . api cost-per-kilogram of hydroxychloroquine is $ /kg, with a -day dose-equivalent of api costing $ . . after adjustment for the cost of formulation, packaging and profit margin, the final cost would be $ per -day treatment course ($ . per day). globally, list prices range between $ per -day course in china and only $ per -day course in india ( figure b ). for chloroquine, the cost of api is $ /kg from panjiva data. a -day course equivalent of api would therefore cost $ . based on the equivalent dose of mg per day of chloroquine base. after adjustment for the cost of loss, formulation, packaging and profit margin, the final cost would be $ . per -day treatment course of chloroquine, equivalent to $ . per day. available list prices vary from $ in the us, to $ . per -day course in bangladesh, which is less than our estimated generic treatment cost. it is worth noting that the us price for chloroquine may be considered an outlier, given the next most expensive list price, found in the uk, was only $ per -day course (figure c ). azithromycin this macrolide antibiotic has been used as an adjunctive treatment for six patients in the small french pilot study of hydroxychloroquine by gautret et al. to prevent bacterial superinfection, with all six patients virologically cured by day six [ ] . however, this finding is contradicted by a small, open-label study (n = ) in paris by molina et al. [ ] , who found no strong viral clearance effect associated with hydroxychloroquine/azithromycin combination therapy. the cost of api derived from panjiva shipment data for azithromycin is $ /kg. a -day course equivalent of api at a dose of mg per day would therefore cost $ . . after adjustment for the cost of loss, formulation, packaging and profit margin, the final cost would be $ . per day or $ . per -day treatment course. list prices for azithromycin range between $ per -day course in the us, and $ per -day course in india and bangladesh (figure d ). sofosbuvir/daclatasvir combination treatment with sofosbuvir/daclatasvir, direct-acting antivirals normally used to treat hepatitis c, is being evaluated in iran for covid- patients with moderate to severe symptoms [ ] . the dosage of sofosbuvir/daclatasvir is / mg daily. api per kilogram was $ for sofosbuvir and $ for daclatasvir. fourteen-day dose-equivalent of api for the combined drug therefore costs $ . . after adjusting for the cost of loss, formulation, packaging and profit margin, the final cost would be $ per -day treatment course, or $ . per day. the cost of sofosbuvir/daclatasvir api has been falling significantly in recent years; earlier estimates in for a -week course of treatment were $ per patient, or $ per days [ ] and in , $ per -week course, or $ . per days [ ] . therefore, our new estimates represent a . -fold reduction in the minimum cost of production since . globally, -day course list prices range between $ , in the us and $ in india, or $ in neighbouring pakistan, as shown in figure e [ ] . there is a randomised trial of pirfenidone vs placebo in progress [ ] . there are patients with severe or critical sars-cov infection being evaluated in this clinical trial, with results expected in may . the dose being evaluated is mg three times daily for weeks. the cost of api from the panjiva database was $ /kg, representing a -week api cost of $ . after adjustment for costs of loss, formulation, packaging and profit margins, the minimum cost of treatment would be $ per person, or $ . per day. there is again a large variation between individual countries' list prices. pirfenidone is available in the us for $ for a -week course, but only $ in bangladesh and $ in india for a generic version (figure f) . however, even at $ per month, this is still higher than our api cost-based estimate. there are several large clinical trials of this monoclonal antibody in progress, for patients with late-stage disease [ , ] . as an iv infusion, doses are based on bodyweight ( mg/kg) with a maximum single dose of mg every hours. we therefore made the assumption of average bodyweight being kg, with a single dose of mg. no api data were available for tocilizumab; therefore, we were unable to estimate the minimum cost of production. list prices per mg single dose varied from $ in the us to $ in pakistan (figure g ). across several developing economies with available list prices -india, bangladesh, turkey, south africa, egypt and pakistan -the median was $ per dose. several tocilizumab biosimilars are currently under development [ , ] ; however, none has yet been approved and launched. the general experience so far of biosimilars has been that they offer health care systems the potential to lower costs significantly [ ] , with the uk alone expected to save up to gbp -gbp million per year through increased uptake of better-value biological medicines [ ] . this analysis shows that drugs to treat covid- could be manufactured for very low prices, between $ and $ per course. many of these drugs are already available as generics, at prices close to the cost of manufacture, in low-and middle-income countries. we do not yet know which of these drugs will show significant benefits. however, if promising results emerge from pivotal clinical trials, there is the potential to upscale generic production and provide treatment for millions of people at very low unit prices. there is an established mechanism to do this: donor organisations such as gfatm and pepfar already provide mass treatment of hiv, tb and malaria worldwide at prices close to the costs of production. the drugs in this analysis were not designed against the sars-cov virus; they were developed to treat other viruses or diseases. some, such as chloroquine, were developed in the s. most of the clinical trials of treatments have been funded by national health authorities and donor agencies rather than pharmaceutical companies. patients with covid- have risked their own health in these clinical trials, often with no clear benefits. companies should be encouraged to continue their research, with costs of clinical trials supported by public funding. since the start of the pandemic, the money spent by pharmaceutical companies on research and development of these drugs will be minimal, relative to funding from national health authorities. where pharmaceutical companies have donated drugs for clinical trials, there are already tax rebate systems in place that will recover the costs of the donated drugs. for mass production of these drugs, our analysis assumed a profit margin of % to companies manufacturing the drugs. this is similar to the pricing structure for hiv, tb and malaria, where generic drug companies still earn acceptable profits while mass producing these drugs at prices close to production costs. large-volume orders are needed to incentivise generic companies to manufacture drugs at low prices. other mechanisms exist to optimise drug manufacture. with pooled procurement, a set of countries can order drug supplies together, to take advantage of economies of scale. there can be volume-price guarantees to procure large amounts of drugs at fixed prices for a set number of years. prequalification of key companies by the who can be recognised by any country as an indicator of drug quality, including adherence to good manufacturing practice and the stability, or viability of the drug over its stated shelf life, alongside the bioequivalence of generic drugs vs the original branded versions. additionally, the costs of treatment may be higher if combinations of two or three drugs are needed. other infectious diseases such as hiv, hepatitis c or tb are best treated with two/threedrug combination treatments. drugs which have not shown efficacy against covid- as monotherapy should not necessarily be discarded: they might still contribute to the efficacy of two-or three-drug combination treatments. drugs that are not curative but lessen disease severity are also needed. these treatments could lessen the burden on healthcare systems, which could otherwise be overwhelmed by a lack of ventilators or other supportive services. when these drugs are repurposed to treat covid- , we will need to ensure a constant supply of drugs for the original indications, for example, pirfenidone for people with pulmonary fibrosis, or hydroxychloroquine for people with rheumatoid arthritis and systemic lupus erythematosus. the costs of these treatments will be higher if used for longerterm prevention, for example, in healthcare workers. randomised trials of chloroquine and hydroxychloroquine for prevention of sars-cov infection are in progress [ , ] and other candidate drugs could emerge for use as prophylactics. we highlight four limitations of our study, for consideration in future work. first, this analysis does not include all candidate drugs for covid- . there are drugs at earlier stages of development; a wide range of candidate drugs have been identified by machine learning models [ ] . these drugs may need further in vitro testing before being introduced into human studies. second, treatments like lpv/r and sofosbuvir/daclatasvir have only a small chance of showing significant benefits to patients in ongoing trials, given current evidence. third, for newer drugs such as remdesivir, favipiravir and pirfenidone, costs of production could continue to fall over time through economies of scale. this trend has been seen for drugs to treat hiv and hepatitis c. the cost of api for the hepatitis c drug daclatasvir fell by % in the years after initial launch, as more generic companies upscaled synthesis of the api with greater competition in the market. fourth, many drugs may have been given discounts from the list prices that we have identified for comparison in this analysis following in-country negotiations. even so, list prices can be over -fold higher than the predicted costs of production in some cases. we propose four main recommendations to ensure that any patient with covid- , in any country, can access the drugs they need: . treatments showing efficacy in well-powered clinical trials should be made available worldwide at prices close to the cost of manufacture. all the treatments being evaluated in clinical trials are very cheap to manufacture. clearly, the mass production of these drugs will need to be economically sustainable. treatments for hiv, tb and malaria are distributed worldwide by gfatm and pepfar, to treat millions of people at prices close the cost of manufacture. the prices paid allow generic companies to make acceptable profits. we should adopt a similar model of drug distribution for covid- . . there should be parallel manufacture by at least three different companies for each product, sourcing their api from different countries. in the early stages of the sars-cov epidemic, api production in china was severely disrupted because of quarantine of key workers and delays in transporting key raw materials between factories [ ] . india has suspended export of several key drugs because of anticipated local shortages. production of drugs in a range of countries will protect us from disruption or shortages in individual countries. . there should be no intellectual property barriers preventing mass production of these treatments worldwide. we need open 'technology transfer' so that the methods used to manufacture the key drugs can be shared with any country deciding to produce the drugs locally. . results and databases from all covid- clinical trials should be fully accessible so others can learn from them. to speed up access to these drugs, countries could rely on recognition of the review and approval of key treatments by regulatory authorities in the us or europe, or other stringent regulatory authorities. there may not be time for the normal times of regulatory review by all individual countries. in summary, repurposed drugs may be our only option to treat covid- for the next - months, until effective vaccines can be developed and manufactured at scale. if repurposed drugs do show efficacy against covid- , they could be manufactured at very low unit prices, in the range of $ to $ per treatment course. the system of mass production and distribution of drugs to treat hiv, tb and malaria via gfatm and pepfar could act as a blueprint for the treatment of sars-cov , to ensure access to effective drugs at low prices worldwide. responding to covid- -a once-in-a-century pandemic? therapeutic options for the novel coronavirus ( -ncov) predicting commercially available antiviral drugs that may act on the novel coronavirus ( -ncov), wuhan, china through a drug-target interaction deep learning model drug repurposing: progress, challenges and recommendations broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses methods for treating filoviridae. pubchem database: patent=us . available at: pubchem.ncbi.nlm.nih.gov/ patent/us discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[ , -f][triazine- -amino] adenine c-nucleoside (gs- ) for the treatment of ebola and emerging viruses clinicaltrials.gov. severe -ncov remdesivir rct: identifier: nct . bethesda (md): us national library of medicine influenza virus polymerase inhibitors in clinical development systematic review of the efficacy and safety of antiretroviral drugs against sars, mers, or covid- : initial assessment a trial of lopinavir-ritonavir in adults hospitalized with severe covid- in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) drugs for treating protozoan infections a to ipca laboratories kumar a, vyas kd, singh d, nandavadekar s, bhise s, jadhav a. available at: patentscope.wipo.int/search/en/detail.jsf?docid=wo &tab=pctbiblio identifier: irct n . a prospective randomized controlled clinical trial comparing the therapeutic efficacy of sovodak (sofosbuvir/daclatasvir) with standard care in patients with moderate to severe coronavirus (covid- ) virus bollu rb, mandadapu vpk, indukuri vsk, chava s. . available at: patentscope.wipo.int/search/en/detail.jsf?docid=wo clinicaltrials.gov. a study to evaluate the efficacy and safety of pirfenidone with novel coronavirus infection: identifier: nct . bethesda (md): us national library of medicine favipiravir combined with tocilizumab for the treatment of coronavirus disease : identifier: nct . bethesda (md): us national library of medicine tocilizumab in covid- pneumonia (tocivid- ): identifier: nct . bethesda (md): us national library of medicine global fund for aids, tb and malaria. results report the united states president's emergency plan for aids relief. annual report to congress minimum costs for producing hepatitis c directacting antivirals for use in large-scale treatment access programs in developing countries rapid reductions in prices for generic sofosbuvir and daclatasvir to treat hepatitis c estimation of cost-based prices for injectable medicines in the who essential medicines list estimated costs of production and potential prices for the who essential medicines list efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) hydroxychloroquine and azithromycin as a treatment for covid- : results of an open-label, non-randomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies favipiravir versus arbidol for covid- : a randomized clinical trial favipiravir treatment of covid- pneumonia initial clinical results released hydroxychloroquine post exposure prophylaxis for coronavirus disease (covid- ): identifier: nct . bethesda (md): us national library of medicine panjiva supply chain intelligence. available at: panjiva.com pharmacompass import-export database. available at: www.pharmacompass.com/ manufacturers-suppliers-exporters xiang h et al. a practical and step-economic route to favipiravir the complete synthesis of favipiravir from -aminopyrazine british national formulary. london: nice, . available at: bnf.nice.org.uk drug price information. available at: www.drugs.com/price-guide office of procurement, acquisition and logistics: pharmaceutical prices medicine price registry. south africa. available at: openup.org.za/tools/ mpr.html drug price lists. available at: portal.anvisa. gov.br/listas-de-precos french ministry of solidarity and health. base de données publique des médicaments. available at: base-donnees-publique.medicaments.gouv.fr online medicine index of bangladesh. available at: medex. com.bd pharmaceutical services programme. consumer price guide. available at: www.pharmacy.gov.my/v /en/apps/drug-price drug price of all the brand names. available at: www.medindia.net/ drug-price/index ilacabak turkey. available at: ilacabak.com egyption drug store. actemra mg/ ml (tocilizumab) vial for iv infusion. available at: egyptiandrugstore.com/index.php?route=product/product&product_ id= impact of fluid choice in systemic inflammatory response syndrome patients on hospital cost savings pharmaceutical favipiravir price announced: cny/box [chinese]. febuary. available at: med.sina.com/article_detail_ _ _ . html the global fund. price reference report - -present. available at: public. tableau.com/profile/the.global.fund#!/vizhome/pqrpricelist_english/pricelist treatment advocate tactics to expand access to antiviral therapy for hiv and viral hepatitis c in low-to high-income settings: making sure no one is left behind the road to elimination of hepatitis c: costs per cure fall to us$ per person. american association for the study of liver disease: the liver meeting mycenax biotech inc. lusinex (tocilizumab, mycenax in-house). available at: www.mycenax.com.tw/en/product.php?act=view&id= clinicaltrials.gov. comparative study of bat to roactemra® in rheumatoid arthritis patients with inadequate response to methotrexate: identifier: nct . bethesda (md): us national library of medicine of biosimilars in rheumatology: reshaping the healthcare environment commissioning framework for biological medicines (including biosimilar medicines indian pharma threatened by covid- shutdowns in china acknowledgements conflicts of interest ah received a consultancy payment from merck for a clinical trial review that is not connected with this project.funding ah received funding from the international treatment preparedness coalition (itpc) as part of the unitaid-supported project 'affordable medicines for developing countries'. journal of virus eradication ; : - minimum costs to manufacture new treatments for covid- key: cord- -d at y authors: ghasemiyeh, parisa; mohammadi-samani, soliman title: covid- outbreak: challenges in pharmacotherapy based on pharmacokinetic and pharmacodynamic aspects of drug therapy in patients with moderate to severe infection date: - - journal: heart lung doi: . /j.hrtlng. . . sha: doc_id: cord_uid: d at y the new coronavirus (covid- ) was first detected in wuhan city of china in december . most patients infected with covid- had clinical presentations of dry cough, fever, dyspnea, chest pain, fatigue and malaise, pneumonia, and bilateral infiltration in chest ct. soon covid- was spread around the world and became a pandemic. now many patients around the world are suffering from this disease. patients with predisposing diseases are highly prone to covid- and manifesting severe infection especially with organ function damage such as acute respiratory distress syndrome, acute kidney injury, septic shock, ventilator-associated pneumonia, and death. till now many drugs have been considered in the treatment of covid- pneumonia, but pharmacotherapy in elderly patients and patients with pre-existing comorbidities is highly challenging. in this review, different potential drugs which have been considered in covid- treatment have been discussed in detail. also, challenges in the pharmacotherapy of covid- pneumonia in patients with the underlying disease have been considered based on pharmacokinetic and pharmacodynamic aspects of these drugs. wuhan city of china. the most common clinical signs and symptoms of these patients were dry coughs, fever, dyspnea, and bilateral infiltration in chest ct. all these patients were associated with wuhan's huanan seafood wholesale market which sells fish and other live animals such as bats, poultry, snakes, etc. the causative agent, new coronavirus, was first detected through a swab sample which was drawn from the throat of these patients . this new coronavirus was subsequently named severe acute respiratory syndrome coronavirus (sars-cov- ). soon this disease, which called coronavirus disease (covid- ) by world health organization (who), promptly spreads around the world , and to date over . million cases have been diagnosed with covid- and this disease became a pandemic. on the late january covid- chinese outbreak, were introduced as a public health emergency of international concern . so although previously coronaviruses were considered as a potential cause of the common cold now we know that they are more than just the common cold! most of the covid- infected patients have an average age of s, it is slightly more predominant in the male sex, approximately % of infected patients involved with severe disease were required to intensive care unit services and % of them were required to mechanical ventilation . a published report from italian patients revealed that covid- was predominant in men ( . % in male and . % in female), most of the patients (about %) were over years old, approximately % of all confirmed patients had mild disease, % had severe disease, % were in a critical situation, and rest of the patients showed few symptoms, unspecified symptoms or were completely asymptomatic . according to recently published researches, the most common clinical presentations in covid- patients were fever in % to % of patients, dry cough in to %, and fatigue or myalgia in to % of them. other signs and symptoms which have been reported include sore throat, headache, confusion, rhinorrhea, sneezing, ageusia, anosmia, chest pain, hypoxemia, pneumonia, hemoptysis, acute cardiac injury, neurologic complications , , and gastrointestinal presentations such as nausea, vomiting, diarrhea and abdominal pain , - . patients with underlying diseases are highly prone to present with severe infection especially with organ function damage such as acute respiratory distress syndrome (ards), acute kidney injury (aki), septic shock, and ventilator-associated pneumonia (vap) , . severe covid- could cause death due to huge alveolar damage and highly progressive respiratory failure . covid- particles could spread through the respiratory mucosa and fecal-oral route . the nucleic acid of the virus was detected in stool, saliva, and respiratory specimens . this virus could be transmitted between humans during the epidemic and then pandemic of covid- . human-to-human transmission could highly accelerate the spread of this virus around the world. this type of transmission among humans is restricted to close contact and through sneezing or coughing of the infected patients who are capable to spread the respiratory droplets. then these respiratory droplets could settle in oral mucosa and lung of the people who inhaled the contaminated air near (about feet) to the infected patients , . although some researches have been focused on the airborne transmission of this virus but this route of transmission has not been approved yet and further studies are required. researches revealed that covid- could also be transmitted through asymptomatic carriers with an incubation period of to days . in order to prevent spreading of this new virus: hands should be washed frequently, the face should not be touched with unwashed hands, regular surface disinfecting is required, social distancing from people with respiratory symptoms is essential, sneezing or coughing should be done into the elbow or soft tissue if available . based on the published reports, in most of the patients with covid- , the absolute value of lymphocytes was reduced, which indicated that this novel coronavirus (covid- ) acts more on lymphocytes especially t lymphocytes, just similar to sars coronavirus . it seems that covid- could induce a cytokine storm and activate immune responses which could be appeared as changes in the number of white blood cells and immune cells especially lymphocytes, the clinical outcome of such events would be respiratory distress syndrome, septic shock and finally end-organ damage . covid- could also affect the liver which could be presented as hypoproteinemia, elevated aminotransferases, and prolonged prothrombin time. hepatotoxicity could be attributed to the higher expression of angiotensin converting enzyme ii (ace ) in cholangiocytes, ace could act as an entry receptor for covid- . so it seems that this new virus can directly damage the intrahepatic bile ducts . pathological findings of a liver biopsy from a patient with covid- showed moderate micro-vesicular steatosis and also a mild portal and lobular activity which could be a result of direct sars-cov- liver damage or antiviral drug-induced hepatotoxicity . almost all covid- patients had abnormal lung ct when diagnosed. according to the recently published article, an average of . ± . segments were involved in patients and the number of involved lung segments was significantly higher in symptomatic patients group in comparison to asymptomatic ones. ct findings revealed that affected covid- patients could present as bilateral lung involvement, peripheral distribution, or diffuse distribution. the most common presentation in chest ct was ground-glass opacity pattern, consolidation, and ill-defined margins , . laboratory confirmation of steatosis could be performed by real-time reverse-transcription polymerase chain reaction (rrt-pcr) , . according to who approved laboratory testing for covid- diagnosis is based on nucleic acid amplification test (naat) such as rrt-pcr which could detect the sequence of the rna of covid- . governments need to appreciate people to obey social distancing and isolation. in some situations, quarantine of major cities is also suggestive. global health governance should apply the least restrictive measures for people according to the international health regulations (ihr) , . scientists around the world are looking for drugs that could be beneficial in covid- treatment. many drugs have been studied that are listed in table with the usual dosage ranges in adults and pediatrics. the latest guidelines for the prevention, diagnosis, and treatment of novel coronavirus-induced pneumonia, have been suggested antiviral agents containing: interferon alpha (ifn-α), lopinavir/ritonavir, chloroquine phosphate, ribavirin, and arbidol as potential options in covid- treatment . drugs that have been considered in covid- management have been classified as investigational drugs, drugs under clinical trials, and drugs that have received u.s. food and drug administration (fda) as shown in table . . although many previous studies emphasized the potential therapeutic effects of these drugs in covid- management, unfortunately some recent publications reported that the efficacy of chloroquine/hydroxychloroquine in covid- management is not consistent. also, their safety is still remaining a major concern for physicians and pharmacists, since chloroquine/hydroxychloroquine could cause qt prolongation and arrhythmia. since the possible risks of these drugs could overweigh their potential benefits and efficacy, united states food and drug administration (fda) no longer recommended these two drugs as potential options for covid- management . results of a systematic review on the efficacy of hydroxychloroquine or chloroquine on the prevention or treatment of covid- revealed that the available evidences on their benefits and risks are weak and controversial . results of another systematic review and meta-analysis on randomized clinical trials on administration of hydroxychloroquine in covid- management revealed that hydroxychloroquine administration (case group) was significantly associated with higher incidence of total adverse effects in comparison to placebo or no treatment (control group) in overall population of patients with covid- . so, the recruitment of chloroquine/hydroxychloroquine in covid- management is still controversial and further larger multi-center randomized clinical trials are required to evaluate their efficacy, safety, risk-benefit ratio, dose and duration of individualized pharmacotherapy. also, close patient monitoring, especially cardiac, ocular, and neurotoxicity assessments, are required and strongly recommended during drug administration . recently published studies revealed that chloroquine could highly reduce covid- replication . chloroquine is a weak base that could be entrapped in organelles that are membrane-enclosed and have low-ph, so interfering with their acidification process. therefore chloroquine could inhibit ph-dependent viral fusion and replication. also, it might inhibit viral assembly in endoplasmic reticulum-golgi intermediate like structures . another possible antiviral mechanism of chloroquine is its immunomodulatory effect through cell signaling pathways and regulating the action of proinflammatory cytokines that can enhance its antiviral effect synergistically , , . chloroquine/hydroxychloroquine could prevent from covid- -induced ards by attenuating the pro-inflammatory cytokines and receptors . hydroxychloroquine can enhance intracellular ph and avoid lysosomal activity in antigen presenting cells containing b cells, also they can avoid antigen processing and mhc-ii presentation to t cells. so, t cell activation could be reduced by the action of hydroxychloroquine. it can suppress the cytokine release syndrome (crs), which is a result of immune system over-activation, caused by covid- . according to this mechanism, hydroxychloroquine could alleviate symptoms of mild to severe covid- pneumonia . chloroquine has different adverse reactions such as cardiovascular adverse reactions . also the results of a systematic review on dermatologic adverse effects of hydroxychloroquine emphasized that the most common dermatologic reactions due to hydroxychloroquine administration were rash, sjs, toxic epidermal necrolysis (ten), pruritus, hyperpigmentation, and hair loss. these dermatologic reactions were mostly occurred after cumulative dosages of hydroxychloroquine . in overall, since hydroxychloroquine has lower tissue accumulation potential in comparison with chloroquine, it has fewer adverse drug reactions and would be better choice . contraindications in chloroquine use contains hypersensitivity to chloroquine ( -aminoquinolone compounds) and the presence of retinal or visual field changes . chloroquine and hydroxychloroquine have a narrow therapeutic index and poisoning could be occurred with cardiovascular features so it should be used with caution in patients with predisposing cardiovascular disease . long-term exposure to these drugs could induce cardiomyopathy . chloroquine in patients consuming heparin, prone the patients to risk of bleeding. also, chloroquine in patients with digitalization (using digoxin) could cause cardiac block . there is no dosage adjustment available for chloroquine or hydroxychloroquine in patients with hepatic failure but it should be used with caution . there is no dosage adjustments available for chloroquine in patients with renal failure from the manufacture's labeling but according to uptodate some clinicians use the following guideline : a) patients with gfr ≥ ml/min: no dosage adjustment is required. b) patients with gfr < ml/min: dosage should be reduced to %. c) patients with peritoneal-or hemodialysis: dosage should be reduced to %. required. there is no dosage adjustments available for hydroxychloroquine in renal failure but it should be used with caution . although some studies showed a low risk of congenital abnormalities in patients receiving chloroquine during pregnancy because of the lack of a pattern in these congenital defects, the possible association is unlikely and it seems that the benefits of its use are higher than risks . hydroxychloroquine use during pregnancy could not be accompanied by risks for fetuses, especially in low doses. but patient monitoring during pregnancy is required . in general, since chloroquine may induce severe side effects during fetal development, so hydroxychloroquine would be a better option in pregnant women with covid- infection because of its safety profile during pregnancy . according to the american academy of pediatrics, chloroquine is compatible with breastfeeding. although it could be excreted into the milk, this amount was not considered harmful for nursing infants . according to the american academy of pediatrics, hydroxychloroquine is compatible with breastfeeding. small amounts of hydroxychloroquine could be excreted to the milk, but because of the slow elimination rate and the possibility of drug accumulation and toxicity, breastfeeding during hydroxychloroquine therapy should be done with caution . umifenovir is a broad-spectrum antiviral agent which is effective against enveloped and nonenveloped rna or dna viruses especially against influenza virus type a and b, respiratory syncytial virus, sars-cov, adenovirus, hepatitis c virus (hcv), etc. umifenovir was first developed in russia and now its usage is more common in russia and china and is less common in western countries. its possible antiviral mechanism is the inhibition of viral fusion with targeted membrane and preventing from the viral entrance to targeted cells . umifenovir has a dual pharmacologic action: first is its beneficial effect on respiratory viruses such as the covid- virus and the second is its immune-stimulating function which can activate serum interferon and phagocytes. since , umifenovir was patented for its beneficial effect in the treatment of severe acute respiratory distress (sars) coronavirus-induced atypical pneumonia . results revealed that umifenovir can induce direct viricidal effect so it would be a promising direct-acting antiviral (daa) agent. umifenovir could affect critical stages of viral life cycles such as cell attachment, cell internalization, viral replication, assembly, and budding so it also would be a promising host targeting agent (hta). its dual pharmacologic function is related to its potential interaction with both cell membranes and with cellular and viral lipids and proteins . the most important adverse reactions associated with umifenovir are diarrhea, nausea, vomiting, dizziness, confusion, and elevated liver enzymes (serum aminotransferases) . umifenovir is an indole derivative with poor water solubility which could affect its bioavailability and pharmacokinetics. after oral administration of umifenovir, it could rapidly distribute to organs and tissues, maximum plasma concentration (c max ) was achieved after to . hours. in the russian population, it had elimination half-life (t ½) of to hours, but t ½ was shorter in the chinese population. after multiple-dose administration of umifenovir, little drug accumulation could be predictable. the main site of drug metabolization is the liver. umifenovir could undergo several metabolism pathways such as oxidation at the s site, ndemethylation, glucuronidation, and conjugation at -hydroxy moiety. the potential antiviral effects of umifenovir metabolites are unknown until now . since the major site of umifenovir metabolization is in the liver, so it should be used with caution in patients with predisposing liver diseases. animal data revealed that umifenovir therapy couldn't induce embryo-toxic effects during pregnancy. according to these results umifenovir would be a promising safe and well-tolerated antiviral agent in pregnancy with a wide therapeutic index in administration for a few days up to one month . ribavirin is a nucleoside antihepaciviral agent (anti-hcv) which has been suggested for covid- treatment. ribavirin is a direct-acting antiviral (daa) agent . ribavirin is a nucleoside analog that has antiviral action against a variety of rna and dna viruses. the potential antiviral activity of ribavirin is inhibition of inosine monophosphate dehydrogenase (impdh) cellular protein and therefore intracellular gtp would be diminished which inhibits rna replication of viral genomes, so viral growth might be stopped. another possible antiviral activity of ribavirin is its immunomodulatory effects by suppression of il- . ribavirin also could inhibit rna polymerase activity and therefore inhibition of rna fragments' initiation and elongation, so viral protein synthesis could be inhibited. the ribavirin is contraindicated in patients with hypersensitivity to ribavirin, pregnant women and their partner, patients with severe renal failure, patients with severe hepatic failure, and patients with major hemoglobinopathies such as sickle cell anemia and major thalassemia . ribavirin distribution could significantly prolonged in erythrocytes for about to days, which is responsible for ribavirin-induced anemia . ribavirin has hepatic metabolism. its oral bioavailability (f) is about %. ribavirin elimination half-life (t ½) in the normal population is hours but in patients with pre-existing chronic hepatitis c infection, half-life could be increased to hours. so because of its prolonged half-life and potential overdose toxicity, ribavirin is contraindicated in patients with hepatic failure (child-pugh class b and c). time to peak level (t max ) after oral administration is between to hours. ribavirin excretion could take place through both urine and feces routes. because of its renal elimination, dose adjustment in patients with underlying kidney disease is highly essential. according to the previous pharmacokinetic/pharmacodynamic study, bayesian therapeutic drug monitoring would be a suitable approach to control ribavirin-induced anemia [ ] . one of the most important side effects of ribavirin is hemolytic anemia which could worsen cardiac disease in patients with underlying cardiac diseases and it could induce fatal or non-fatal myocardial infarction in them. so ribavirin should be avoided in patients with a history of unstable or severe cardiac diseases. ribavirin is contraindicated in patients with hepatic decompensation (child-pugh class b and c). ribavirin dosage adjustment in patients with renal failure highly depends on different formulations which are available. these data are shown in table . in children, if serum creatinine level rises over mg/dl during administration, ribavirin should be discontinued promptly . ribavirin has teratogenic and mutagenic effects. so it is a high-risk drug in pregnancy according to animal data. also because of its half-life of hours in multiple-dose drug therapy and the possibility of drug accumulation in tissue compartments for up to months, ribavirin administration is contraindicated in pregnant women and also in men who are pregnant women's partner. it was suggested that pregnancy should be avoided during ribavirin therapy and at least months after completion of therapy in women or men . ribavirin because of the prolonged plasma elimination half-life and molecular weight of da, potentially would have toxicity in nursing infants but there are no human data available . ribavirin may precipitate hematologic adverse effects of organ transplantation regimen such as immunosuppressive agents (mycophenolate mofetil, azathioprine, mtor inhibitors), trimethoprim/sulfamethoxazole, and valganciclovir. these hematologic adverse reactions would also worsen hematologic reactions related to covid- . so close patient monitoring is essential. . also, lopinavir would be a promising drug of choice in children with covid- . lopinavir/ritonavir are protease inhibitor, anti-retroviral agents. the potential antiviral mechanism of lopinavir/ritonavir is inhibition of viral protease, which is a critical enzyme in viral maturation and infectivity. low dose ritonavir in combination with lopinavir act as a pharmacokinetic enhancer by inhibition of lopinavir inactivation metabolism . the since lopinavir undergoes hepatic metabolism through cyp a enzyme, potential drug-drug interactions could occur with all drugs that are strong inhibitors or inducers of cyp a enzyme and p-glycoprotein inhibitors. also, ritonavir has hepatic metabolism via cyp a and cyp d . ritonavir has serious and life-threatening drug interactions with sedative-hypnotic agents, antiarrhythmic drugs, and ergot alkaloid agents because of the effect of ritonavir on their hepatic metabolism through cyp a and cyp d . concurrent use of these agents with ritonavir is absolutely contraindicated and should be avoided . lopinavir/ritonavir is contraindicated in patients with a history of hypersensitivity reactions such as toxic epidermal necrolysis (ten), stevens-johnson syndrome, angioedema, etc. to lopinavir/ritonavir or its components . absorption and oral bioavailability are highly affected by fasting or fed state, its absorption, bioavailability, and peak level (c max ) could be significantly increased with food. but food can delay t max from hours in fasting state to hours in the fed state. ritonavir also has small renal elimination so dosage adjustment is not required in patients with underlying kidney disease . lopinavir/ritonavir have major interactions with drugs used in cardiovascular diseases such as anti-coagulating agents (anti-factor xa inhibitors), none dihydropyridine calcium channel blockers, digoxin, antiarrhythmic agents such as amiodarone, etc. so close patient monitoring is required in covid- patients with pre-existing cardiovascular disease who are planned to treat with lopinavir/ritonavir and sometimes alternative drugs might be considered . in patients with mild to severe hepatic failure, there is no dosage adjustments available but lopinavir has primary liver metabolism and its auc will be increase by about %, so it should be used with caution . there is no dosage adjustments based on renal function according to the manufacture's labeling . results revealed that embryo-fetal risk of lopinavir/ritonavir is low, so it is compatible with pregnancy and should not be stopped during pregnancy . lopinavir and ritonavir with a molecular weight of and da respectively and their lipid solubility nature, are good candidates for excretion to milk during lactation period but their high plasma protein binding could limit this excretion. a comprehensive data is not available yet. it has been recommended that breastfeeding during lopinavir/ritonavir therapy is better to be avoided especially in developed countries . immunosuppressive agents are critical drugs in patients undergone solid organ transplantation. lopinavir/ritonavir cannot be administered in combination with immunosuppressive agents because of the occurrence of strong drug interactions. lopinavir/ritonavir can enhance the plasma level of immunosuppressive agents such as calcineurin inhibitors and mtor inhibitors. so if co-administration is essential, immunosuppressive agents dose reduction and therapeutic drug monitoring, to maintain optimum immunosuppressive plasma level, is highly recommended. it has been suggested that during covid- treatment, calcineurin inhibitors (ex. cyclosporine and tacrolimus) and mtor inhibitors (ex. sirolimus and everolimus) could be discontinued and replaced with lopinavir/ritonavir but it seems that the benefit of this drug discontinuation could not overlay the risk of allograft transplant rejection . tocilizumab is an interleukin- (il- ) inhibitor which is a disease modifying anti-rheumatic agent . a small retrospective observational study on covid- pneumonia patients receiving tocilizumab revealed that this drug would have potential benefits in these patients such as since an active immune response against respiratory viruses such as covid- is highly dependent on cytotoxic t cells' action, so in patients with total t cell count of fewer than cells/µl, aggressive intervention is essential. one of the possible approaches in enhancing t cell count in these patients is the administration of tocilizomab, because there is a reverse relationship between t cell count and the number of cytokines such as il- . ifn-α is a broad-spectrum antiviral agent that is commonly used in hepatitis management. after corticosteroids are a double-edged sword, they can inhibit our immune response and so the clearance of covid- could be delayed, but on the other hand, they can suppress our inflammatory response which is highly responsive to the lung damage and ards during viral convalescent plasma or immunoglobulins would be a promising therapy in covid- patients. previous results revealed that convalescent plasma therapy during viral infection outbreaks, it has been recommended that patients with covid- who are suffering from refractory hypoxemia should be managed with extracorporeal membrane oxygenation (ecmo in some countries such as switzerland, tocilizumab has been considered in patients with multiorgan failure and inotropic support . ace has a critical role in cardiac and immune systems. ace is related to heart function and it could be a potential cause of hypertension and diabetes mellitus development . since ace is a functional receptor for covid- , in patients with underlying cardiovascular diseases, clinical symptoms of covid- are more severe and fatal than the general population because, in cardiovascular diseases, ace secretion might be enhanced. administration of reninangiotensin-aldosterone system inhibitors, such as angiotensin converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs) could enhance ace level. thiazolidinediones and ibuprofen might also enhance the ace level pneumonia but arbs, through blockade of angiotensin receptors, may have beneficial effects in these patients. most of the hepatic metabolites of drugs considered in covid- treatment such as chloroquine, hydroxychloroquine, and lopinavir/ritonavir would be found in urine due to renal elimination. so in patients with chronic kidney disease (ckd), the accumulation of drug metabolites would be expected if administered in routinely recommended doses for the normal population. therefore, individualized dose adjustment based on kidney function is required for each drug as mentioned above . a small study on covid- patients with end-stage renal disease (esrd) who undergone hemodialysis, revealed that the number of total t cells (cytotoxic and helper t cells), natural killer (nk) cells, and inflammatory cytokines were significantly lower than these levels in non-hemodialysis patients with covid- . this study revealed that esrd patients with hemodialysis who infected with covid- had a good prognosis and they had mild symptoms of pneumonia, it might be related to the fewer number of inflammatory cytokines and reduced immune function which can avoid crs but further studies are required to confirm this hypothesis . according to the recent studies, liver abnormalities (such as elevated ast and alt serum levels) have been occurred after and during infection with covid- . these abnormalities would be related to viral infection pathogenesis and direct liver injury or it may be drug-induced . almost all of the potential drugs in covid- treatment containing chloroquine, hydroxychloroquine, ribavirin, and lopinavir/ritonavir have hepatic metabolism. so injury to the liver because of pre-existing liver disease or acute hepatic failure would impair drug metabolism and therefore drug accumulation and enhancement in plasma level, which can lead to drug toxicity. in these patients, frequent liver function monitoring is essential to achieve an optimal serum drug level . also, dosage adjustment for each drug should be done individually according to the patients' liver function as mentioned above. the impact of chronic liver diseases such as chronic viral hepatitis, alcoholic and non-alcoholic liver diseases on occurrence of liver injury related to covid- infection, still is not clear. it seems that in patients with underlying liver disease, with the immunocompromised condition, who infected with covid- , more intensive and individualized pharmacotherapy is required. further studies would be also helpful to explain the exact role of pre-existing liver diseases in covid- prognosis . the new coronavirus (covid- ) was first detected in wuhan city of china in december . soon this coronavirus disease (covid- ) spreads around the world and became a pandemic. now many patients around the world are suffering from this disease. patients with underlying diseases are highly prone to severe covid- pneumonia. till now many drugs have been considered in the treatment of covid- pneumonia, but pharmacotherapy in patients with pre-existing comorbidities is highly challenging. in this review, different potential drugs which have been considered in covid- treatment have been discussed in detail. also, challenges in the pharmacotherapy of covid- pneumonia in patients with underlying disease especially heart diseases have been considered based on pharmacokinetic and pharmacodynamic aspects of drugs. patients with covid- who have cardiac diseases such as coronary heart disease and those who are aceis consumers should be highly considered in treatment options. also, patients with liver and kidney disease and those with organ transplants should be considered to avoid the occurrence of drug overdose toxicities and potential drug-drug interactions. funding: this work was not funded. coronavirus infections-more than just the common cold the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status covid- -new insights on a rapidly changing epidemic the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention world health organization declares global emergency: a review of the coronavirus disease (covid- ) in italy neurological complications of coronavirus disease (covid- ): encephalopathy. cureus major neurologic adverse drug reactions, potential drug-drug interactions and pharmacokinetic aspects of drugs used in covid- patients with stroke: a narrative review epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study covid- : gastrointestinal manifestations and potential fecal-oral transmission the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak comorbidities and multi-organ injuries in the treatment of covid- pathological findings of covid- associated with acute respiratory distress syndrome. the lancet respiratory medicine novel coronavirus (covid- ) outbreak: a review of the current literature stopping the spread of covid- presumed asymptomatic carrier transmission of covid- radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study. the lancet infectious diseases chest ct findings in coronavirus disease- (covid- ): relationship to duration of infection clinical characteristics of coronavirus disease in china positive rt-pcr test results in patients recovered from covid- laboratory testing for coronavirus disease (covid- ) in suspected human cases: interim guidance novel coronavirus-important information for clinicians discovering drugs to treat coronavirus disease (covid- ) a brief review of antiviral drugs evaluated in registered clinical trials for covid- . medrxiv pharmaceutical care recommendations for antiviral treatments in children with coronavirus disease remdesivir as a possible therapeutic option for the covid- fact sheet for health care providers: emergency use authorization (eua) of remdesivir (gs- ™). silver spring, md, us food and drug administration the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality ongoing clinical trials for the management of the covid- pandemic use of iron chelators to reduce the severity of covid- iron chelating agents: promising supportive therapies in severe cases of covid- ? etoposid-based therapy for severe forms of covid- of chloroquine and covid- covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression chloroquine and hydroxychloroquine as available weapons to fight covid- new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial chloroquine or hydroxychloroquine for management of coronavirus disease : friend or foe? hydroxychloroquine or chloroquine for treatment or prophylaxis of covid- : a living systematic review safety of hydroxychloroquine in covid- and other diseases: a systematic review and meta-analysis of randomized trials hydroxychloroquine and chloroquine: a potential and controversial treatment for covid- insights from nanomedicine into chloroquine efficacy against covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine-induced stevens-johnson syndrome in covid- : a rare case report characterizing the adverse dermatologic effects of hydroxychloroquine: a systematic review american pharmaceutical association drugs in pregnancy and lactation: a reference guide to fetal and neonatal risk arbidol: a broad-spectrum antiviral compound that blocks viral fusion arbidol as a broad-spectrum antiviral: an update anti-hcv, nucleotide inhibitors, repurposing against covid- ribavirin's antiviral mechanism of action: lethal mutagenesis? journal of molecular medicine pharmacokinetic/pharmacodynamic and time-to-event models of ribavirin-induced anaemia in chronic hepatitis c in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds case report combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome a trial of lopinavir-ritonavir in adults hospitalized with severe covid- case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr patients of covid- may benefit from sustained lopinavir-combined regimen and the increase of eosinophil may predict the outcome of covid- progression simultaneous population pharmacokinetic model for lopinavir and ritonavir in hiv-infected adults should covid- concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade the rheumatologist's role in covid- covid- infection and rheumatoid arthritis: faraway, so close! autoimmun rev effective treatment of severe covid- patients with tocilizumab. chinaxiv preprint reduction and functional exhaustion of t cells in patients with coronavirus disease therapeutic options for the novel coronavirus ( -ncov) covid- ) update: fda issues emergency use authorization for potential covid- treatment remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial. the lancet compassionate use of remdesivir for patients with severe covid- covid- : low dose steroid cuts death in ventilated patients by one third, trial finds convalescent plasma as a potential therapy for covid the feasibility of convalescent plasma therapy in severe covid- patients: a pilot study high-dose intravenous vitamin c treatment for covid- a review of the novel coronavirus (covid- ) based on current evidence preparing for the most critically ill patients with covid- : the potential role of extracorporeal membrane oxygenation are patients with hypertension and diabetes mellitus at increased risk for covid- infection? coronavirus disease (covid- ) and cardiovascular disease clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor liver injury in covid- : management and challenges. the lancet gastroenterology & hepatology ethical approval: not required. p.g. and s.m. were contributed equally in data gathering, writing-original draft, reviewing, and revising the final version of this manuscript. key: cord- - gacqii authors: murthy, sreekant; papazoglou, elisabeth; kanagarajan, nandhakumar; murthy, narasim s. title: nanotechnology: towards the detection and treatment of inflammatory diseases date: journal: in vivo models of inflammation doi: . / - - - - _ sha: doc_id: cord_uid: gacqii biological systems operate at the nanoscale. nanomedicine is the application of nanotechnology to monitor and treat biological systems in health and disease. this is accomplished by real time monitoring of molecular signaling at the cellular and tissue level. during the past decade, there has been an explosion in this field, resulting in revolutionary advances in determining the microstructure and function of living systems. these discoveries have led to the development of powerful tools for fundamental biological and medical research. nanotechnology has been applied to targeted drug delivery to minimize side effects, creating implantable materials as scaffolds for tissue engineering, creating implantable devices, surgical aids and nanorobotics, as well as throughput drug screening and medical diagnostic imaging. the nanoinitiatives are funded by governments and private sources throughout the world to develop or further refine the technology to provide the beyond-imaginable, most sophisticated tools to a physician and scientists to inflammatory diseases. no doubt, there will be many technical, regulatory and legal challenges in the deployment of these technologies. unquestionably, there is enough desire and commitment to meet these challenges for the good of society and betterment of the quality of life. accomplishments, combining them with available new technologies to identify the earliest signatures of inflammation and cancer. such developments will allow us to provide immediate and specific intervention and monitor the progress before it cascades into chronic inflammation and malignancy. to fulfill this objective, it requires the development of technologies of - nm size, which display unique mechanical, electrical, chemical, and optical properties and assist in visualizing or interacting with receptors, cytoskeleton, specific organelles and nuclear components within the cells. it will be very rewarding when many of these technologies can migrate into monitoring the disease condition through non-invasive methods in vivo in a physically undisturbed state, thus minimizing the influence of artifacts induced by physical methods while securing biological samples. the integration of nanotechnology with biology and medicine has given birth to a new field of science called "nanomedicine". the ultimate goal of nanomedicine is to develop well-engineered nanotools for the prevention, diagnosis and treatment of many diseases. in the past decade, extraordinary growth in nanotechnology has brought us closer to being able to vividly visualize molecular and cellular structures. these technologies are beginning to assist us in our ability to differentiate between normal and abnormal cells and to detect and quantify minute amounts of signature molecules produced by these cells. most of these represent real time measurements, relating to the dynamic relationship among structures in the damaged area and also to repair of damaged tissues. novel pharmaceutical preparations have been developed to fabricate nanovehicles to deliver drugs, proteins and genes, contrast enhancement agents for imaging, and hyperthermia agents to kill cancer cells. several of these inventions have already transitioned into basic medical research and clinical applications. because of this, some social, ethical, legal and environmental issues have emerged. thus, regulatory and educational strategy needs to be developed for the society to gain benefit from these discoveries. the focus of this chapter is to provide an overview of the state-of-the-art in nanotechnology with particular reference to detecting and treating inflammation and cancer at the earliest settings. nanotechnology encompasses multiple scientific disciplines, which exploit materials and devices with functional co-assembled molecules or sensors that has been engineered at the nanometer scale typically ranging from . to nm [ ] . in medical fields, it offers a wide range of tools that can be used as drug delivery platforms [ ] , better contrast agents in imaging [ ] , chip-based bio-laboratories [ ] and nanoscale probes [ ] that are able to track cell movements and manipulate molecules. multifunctional nanostructures can combine diagnostic and therapeutic modalities and target cellular events. the concept of molecular medicine to develop personalized treatments can be made possible with the information available with the developed nanotools. these devices, systems and functionalized structures contain unique properties as a result of their nanosize. for example, gold nanoparticles and carbon nanotubes possess different properties [ ] at the micron scale. semiconductor particles exemplified by quantum dots exhibit quantum confinement effects, hence they fluoresce at various wavelengths compared to the semiconductor particles at micron size which do not exhibit the same optical or magnetic properties [ ] . macromolecular structures such as dendrimers and liposomes at the nanoscale are also considered valid nanotools [ ] , while biological molecules of nanometer size in their native state such as dna and monoclonal antibodies are not examples of "nanotools". thus, nanometer size is of critical importance to the cell and the living organisms. interestingly, fabricated nanoscale devices are of the same size as subcellular organelles. some nanoscale structures are of the order of enzymes, receptors and key molecules within the cell membrane or cytoplasm. for example, the lipid bilayer surrounding cells is nm and hemoglobin is nm in size. by modifying the surface chemistry of these nanostructures, which permits covalent or ligand-receptor (lock-key-type) or electrostatic interaction with key molecules, we can identify biomolecules on the cell surface and in the cytoplasm. an advantage of this will be our ability to map the transport of those molecules from the cytoplasm across the cell membrane so we can understand the cellular behavior in health and disease. nanoparticles smaller than nm can pass through blood vessel walls [ ] , which opens opportunities of diagnostic imaging and targeted delivery of drugs when non-toxic nanoparticles are used. what is critical for scientists engaged in inflammation science and engineering is that these technologies should be applicable to detect or monitor: -host biochemical and immune responses -bacterial and viral pathogens interacting within the local immune system -effect of noxious chemicals and pharmaceutical agents -thrombosis -neurogenic inflammation -wound healing and remodeling -imaging -diagnosing and treating vulnerable plaques -drug delivery and therapeutics for local delivery and retention nanotechnology in the medical field offers a wide range of tools that can be used as drug delivery platforms, better contrast agents in imaging, chip-based biolabs and nanoscale probes able to track cell movements and manipulate molecules [ ] . combination of these multifunctional nanostructures through cross-disciplinary interactions may further enhance our diagnostic and therapeutic capabilities and to monitor intra-and extracellular cellular events in inflammation and cancer. existing and emerging technologies, which may impact on early detection of inflammation, prevention and early detection of cancer, include several diverse technological innovations. they are bio-mimicry self-assembling peptide systems, which serve as building blocks to produce nucleotides, peptides and phospholipids, which support cell proliferation and differentiation and give insights into protein-protein interactions [ ] . microchip drug release systems, micromachining hollow needles and two-dimensional needle arrays from single crystal silicon for painless drug infusion, intracellular injections, microsurgeries and needle-stick blood diagnosis form another group of tools [ , ] . all of these inventions could one day lead to develop personalized treatments [ ] . the creation, control and use of structures, devices and systems with a length scale of - nm is the domain of nanotechnology. macromolecular structures such as dendrimers and liposomes at the nanoscale are also considered valid nanotools [ , ] . the application of various nanotools in various areas of medicine is depicted in figure . this list in the figure is by no means exhaustive as nanotechnology is continuing to grow with new technologies emerging each day. biomolecular nanopore detector technology was first developed to rapidly discriminate between nearly identical strands of dna thereby replacing the tedious process of running billions of copies of dna through sequencing machines and minimizing errors and saving time [ ] . in this technology single molecules of dna are drawn through pores that are - nm in size and serve as a sensitive detector. the detection system through its electronic signature process can sequence more than one base pair per millisecond. this technology has the potential to detect dna polyploidy, and dna mutations. this field includes biomimetics encouraged to mimic nature and create biomolecular nanomachines to handle various biological problems. many biological systems use self assembly to assemble various complex molecules and structures [ ] . numerous man-made self-assembling systems that mimic natural self assembly of molecules are created to snap together fundamental building blocks of complex polymer molecules structured easily, and inexpensively, on beads, tubes, wires, and flat supports, and in suspensions and liposomes. these assemblies can have geneti-cally introduced bifunctionality so that nonspecific molecules are repelled from fusing with the cell membrane fusion layers. dna, lipid bilayers, atp synthase, peptides and protein foldings are target candidates for self assembly. liposomes are an example of a human-made supramolecular structure. nanoscale cantilevers are about -mm-wide flexible diving board-like beams that can be coated with antibodies and dna complementary to a specific protein or a gene. when molecules come in contact with these substrates coated on the surface of cantilevers, they bind to the substrate and make the cantilevers resonate or bend as a result of this binding event [ ] . this bending deflection is proportional to the quantity of binding, thus making it a quantitative technique. multiple cantilevers can be used simultaneously to differentiate between bound and unbound molecules. likewise, multiple antibodies can be used in the same reaction set up to quantify several markers at a time. an important advantage of this technique is that there is no need to add fluorescent tags to detect and quantify the molecule. any biological sample containing biomolecules of interest can be tested. nanoscale cantilevers, constructed as part of a larger diagnostic device, can provide rapid and sensitive detection of inflammation and cancer-related molecules and to evaluate how various drugs bind to their targets at a concentration times lower than clinical threshold. carbon nanotubes, also known as "bucky tube" and "buckyballs" are a member of fullerene structural family potentially useful in a number of biological applications. they could be cylindrical (nanotubes), spherical (buckyballs) or branched (fullerenes). nanotubes could be single-walled nanotubes (swnt) or multi-walled nanotubes (mwnt). the usefulness of nanotubes in drug delivery and cancer therapy is accomplished through the transporting capabilities of carbon nanotubes via suitable functionalization chemistry and their intrinsic optical properties. proper surface functionalization is necessary to make carbon nanotubes biocompatible. in their most recent applications, swnts have been used to transport dna inside living cells [ ] . intracellular protein transport has also been accomplished [ ] , although they are suspected to cause severe immune responses. most swnts have diameters close to nm, with a tube length that can be many thousands of times larger. swnts with lengths up to the order of centimeters have been produced [ ] . swnts are a very important class of carbon nanotubes because they exhibit important electrical properties not shared by the mwnt variants. on the other hand, mwnts are fabricated as multiple concentric nanotubes precisely nested within one another for perfect linear or rotational bearing. the technology has now advanced into merging these mwnts with magnetic nanomaterials like magnetite, which can be functionalized. gadofullerenes offer the ability to concentrate more gadolinium at the site of disease, than traditional gd-dtpa. this is the result of the shielding that the carbon structure provides and its ability to link more gadolinium per conjugate. gadofullerenes also take advantage of the gadolinium-water interactions as the gadolinium is brought along the periphery of the structure and can maintain its interaction with water, which is the basis of traditional proton density magnetic resonance imagine (mri). these properties lead to a greater signal, which can increase sensitivity to small lesions. recently, the technology has been further improved by developing smart bionanotubes that could be manipulated to produce open or closed end nanotubes to encapsulate drugs or genes to deliver them in a particular location [ ] . thus, possibilities exist for using nanotubes to improve gene sensing, gene separation, drug delivery and detection of biomarkers to improve health care, protection against bioterrorism and other areas of molecular sensing. they are mostly spherical particles with specific properties that allow their detection, analysis and quantification. fundamentally, they are nanoprobes where these particles are complexed with biomolecules, drugs and other reagents. they exhibit various physical and optical properties. for example, iron oxide nanoparticles exhibit super paramagnetic properties [ ] , and gold nanoparticles specific optical absorption properties depending on their size [ ] . it is important to note that particles made from the same materials but of micron dimensions do not exhibit such unique optical or magnetic properties [ ] . one can thus combine the immense surface to volume ratio of these nanostructures to deliver higher loads of compounds encapsulated or linked to their surface, while their presence can be measured due to their characteristic magnetic or optical properties. quantum dots (qds) are tiny light-emitting particles on the nanometer scale. they are emerging as a new class of biological probes that could replace traditional organic dyes and fluorescent proteins. the fundamental benefit of using qds is the high quantum yield and strong emission intensity. the emission spectrum of qds is a function of the particle size, and hence by varying particle size qds can emit from visible to infrared wavelengths [ ] . they can be excited by uv light. their broad excitation spectrum and narrow emission spectrum with little or no spectral overlap makes them attractive for imaging and resolving multiple species at the same time without complex optics and data acquisition systems. qds offer higher signal to noise ratios compared to traditional fluorochromes. their high sensitivity allows accurate detection even in the presence of strong autofluorescence signals encountered during in vivo imaging [ ] . their excellent resistance to photobleaching is particularly useful for long-term monitoring of biological phenomena, critical in live cell imaging and thick tissue specimens. already qds are finding increasing use in live cell imaging, by themselves or as fluorescence resonance energy transfer (fret) donors combined with traditional fluorchromes, and in in vitro assays and live animal imaging for cancer and tumor diagnostics. limitations of qds can arise from the stability of the core shell structure. most commercially available materials comprise a core of cdse and a shell of zns. to render this inorganic structure hydrophilic, amphiphilic polymers are used to cap the shell layer and provide reactive sites for further linking to proteins. it is, therefore, the stability of this layer that controls the aggregation of qds, as well as possible release of core materials (cd ions) to their surroundings, which may result in toxicity [ ] . this has limited the immediate clinical use of qds, but has focused applications to animal testing and in vitro assay developments. another reported problem is the blinking property of the qds. qds tends to blink at the single dot level and hence present some limitation in absolute fluorescence quantification. however, when used for imaging of biomarkers, this property does not have much of an effect as there are hundreds or thousands of qds in a sample to allow proper averaging. paramagnetic iron oxide nanoparticles are a new class of contrast agents that are finding increasing applications in the field of diagnostics and molecular imaging based on magnetic resonance (mr) [ ] . traditional mri agents rely on the interaction of the proton density, i.e., water molecules and the magnetic properties of the tissue. these paramagnetic agents accelerate the rate of relaxation of protons in the longitudinal direction, resulting in bright images, and hence are highly dependent on water molecules. however, the super paramagnetic iron oxide nanoparticles, by the virtue of their nanoscale properties, disturb the magnetic field independently of their environment, and hence are not dependent on presence of water molecules. they are also called negative enhancers as they act as negative contrast agents and appear dark where they are sequestered. the traditional mr agents such as gadolinium-diethylenetriamine penta-acetic acid (dtpa) enhance the signal from the vascular compartments and are nonspecific, whereas the nanoparticle-based contrast agents impact the mr signal from tissues and cells. iron oxide nanoparticles are classified into two types depending on their size: ( ) superparamagnetic iron oxides (spios) ( - nm), and ( ) ultra-small super paramagnetic iron oxide (< nm). the advantage of these contrast agents lies in their ability to get sequestered anywhere within a support matrix and still generate a contrast, whereas the traditional mr agents need water in their vicinity of generate contrast. these nanoparticles can be used for both passive and active targeting. because of the small size of these particles, tissue macrophages readily take up these agents, and hence it is possible to image liver, spleen, lymph nodes, and lungs. in addition, it is also possible to functionalize these nanoparticles using a wide variety of ligands, antibodies, peptides, aptamers, drugs, etc., to achieve site-specific or biomarker-specific targeting. this is an added advantage since traditional paramagnetic formulations are difficult to conjugate to antibodies, and even when conjugated, owing to the small number of cellular receptors, the signal intensity is not sufficient for accu-rate imaging. thus, as a result of their superparamagnetic properties, iron oxide nanoparticles have been used as contrast agent for imaging of cancer, brain inflammation, arthritis, and atherosclerotic plaques. because of the small size, these iron oxide particles have been able to distinguish between the normal and tumor-bearing lymphatic nodes [ ] . these nanoparticles may also distinguish very small metastases (less than mm in diameter) within normal lymph nodes, a size well below the detection limit of the most sensitive imaging techniques such as positron-emission tomography (pet) available today. using cells loaded with iron oxide nanoparticles, it has been shown that these particles are non toxic and are cleared from the cell after five to eight divisions. lewin et al. [ ] labeled stem cells with iron oxide particles using hiv tat peptide and injected them systemically. the labeled stem cells homed on to the bone marrow, and the labeled stem cells did not cause any impairment. however, due to the small size of these particles, a long time is required (up to h) to clear them from the organs and blood to reduce background signals. thus, mri using spios may result in improved sensitivity and selectivity, and may assist diagnosis of tumors at the earliest stages of malignancy or metastasis. dendrimers are a new class of hyper-branched polymer macromolecules that radiate from a central core with structural symmetry. they can vary in shape, size, surface, flexibility and topography, enabling fabrication of functional nanoscale materials that would have unique properties [ , ] . they may be useful for developing antiviral drugs, tissue repair scaffolds, and targeted carriers of chemotherapeutics. certain dendrimers are now being used commercially as immuno-diagnostic agents and gene transfection vectors. dendrimers complexed with gadolinium (iii) ions (gadomer- ) are being tested (phase i clinical trial) for mri angiography [ ] . it is anticipated that many exciting developments will emerge from the use of dendrimers in the near future. these are made up of phospholipid bilayers exhibiting multifunctioning characteristics. they facilitate encapsulating of various classes of drugs and diagnostic agents for their controlled delivery into cellular and therapeutic targets. important drug delivery strategies utilizing these agents include polymersomes, hydrogel matrices, nanovesicles/nanofiber mats and biodegradables [ ] . both small and large molecules can be used. biodegradeable polymersomes based on polyethylene oxide have been synthesized, and they may be used as a surface to anchor antibodies or other targeting molecules. quite recently fluorescent materials have been embedded into these cell-like vesicles [ ] to produce near-infrared emissive polymersomes that could be used to locate areas of inflammation and deliver a load of drug to inflammation sites. interestingly, inflammation sites deeper than cm could be imaged with this technique. efforts are being made to target dna complexes into hepatocytes and macrophages with the idea of enabling gene therapy and delivering genetically derived vaccines in a safe and efficacious manner. polymeric micelles are useful as developing agents for -scintigraphy, mri, and computed tomography (ct) [ ] . liposomes can be injected intravenously and when they are modified with lipids that render their surface more hydrophilic, their circulation time in the bloodstream can be increased significantly. another class of polymersomes, called polymer nanotubes, has been synthesized by directly pulling on the membrane of polymersomes using either optical tweezers or a micropipette [ ] . these polymersomes are composed of amphiphilic diblock copolymers consisting of an aqueous core connected to the aqueous interior of the polymersome, which are less than nm in diameter. they are unusually long (about cm) and are stable enough to maintain their shape indefinitely. the pulled nanotubes are stabilized by subsequent chemical cross-linking. the aqueous core of the polymer nanotubes together with their robust character offer opportunities for nanofluidics and other applications in biotechnology, especially in the development of nanohyperdermic syringes [ ] . development and use of analytical tools in diagnostic area possibly presents immediate benefits to the user. many diagnostic tools have been developed to improve human health. the diagnostic detection methods involve measuring antibody or antigen-based complexes, enzyme-based reaction rates, and polymerase chain reactions using micro-electro-mechanical systems (mems) [ ] . other methods include whole-cell bacterial sensors and biosensors which utilize aptamers, which are biomimetic synthetic bioreceptors that can complex with proteins, nucleic acids and drugs. the signal processing in these systems may be optical, electrochemical, or mass-related, creating resonance and thermal detection. some diagnostic methods utilize nanoparticles as nanoprobes where nanoparticles are interfaced with biological molecules such as antigens, antibodies or chemicals. the nanoparticles used in diagnostics include qds, nanobarcodes, metallic nanobeads, silica, magnetic beads, carbon nanotubes, optical fibers and nanopores [ ] . in antibody/antigen-based detection methods, for example, - -nm-wide, boron-doped silicon wires laid down on a silicon grid can be coated with antigens to provide real time detection of antibodies. antibody binding to immobilized antigen gives a measurable conductance change at antibody concentrations less than nm. detection of single copies of multiple viruses has been accomplished via antibody-conjugated nanowire field effect transistors [ ] . the dream of optical biopsy is closer to reality with antibody-functionalized semiconductor nanoparticles (qds) detected by fluorescence microscopy. multiplexed assays can be developed since the fluorescence emission of qds is tunable by changing their size. outstanding detection sensitivity of antibodies in whole blood (picogram per ml) has been obtained using gold nanoparticle conjugates [ ] . for detection purposes classical tools can be used as well as nanobased methods, such as atomic force microscopy (afm) and near-field scanning optical microscopy (nsom), methods where quantum tunneling plays a key role in amplifying detection capability. again, such phenomena are related to the nanometer distance between the instrument probe and the surface/specimen under examination. afm is used to elucidate structures of biomolecules under physiological conditions [ ] , to determine antibody/antigen binding properties [ ] , to image the topology of viruses [ ] , and to image pathologies at the molecular scale [ ] . researchers in academia and the pharmaceutical industries traditionally use bioassays, which are often cumbersome and riddled with errors. the recent explosive development in the field of microfluidics, biotechnology and functional genomics has resulted in the miniaturization of these bioanalytical assays to micron scales for routine and throughput screening [ ] . these assays have been used for genomic and proteomics analysis, though their application to proteomics still requires refinement since replication of proteins as opposed to dna is yet to be fully realized. efforts are being made to improve miniature microarrays, which are still used for analyzing proteins. these include fabrication of afm-based dip-pen nanolithography (dpn), which can probe complex mixtures of proteins, reactions involving the protein features and antigens in complex solutions, and can aid the study of cellular adhesion at the submicrometer scale. protein nanoarrays generated by dip-pen nanolithography are emerging [ ] . with further advances in miniaturization techniques like dpn, it will be possible to design nanoarrays that can detect biological entities on a single particle level in a time-and cost-efficient manner and also profiling of new diagnostic biomarkers at a detection level beyond our imagination. the pharmaceutical industry, physicians and patients have long desired better pharmaceutical formulations to improve and extend the economic life of proprietary drugs, to reduce the costs of preparation and treatment, and reduce toxicity or even death. nanotechnology has already made significant inroads into the problems of improving delivery of injectibles, oral formulations, drug device implants, and topical and transdermal delivery of drugs. more is expected from nanotechnology in improving the delivery of drugs to the brain, as many of the formulations aimed at treating diseases of the brain fail to cross the blood-brain barrier. various methods have been tested for drug delivery. for example, carbon-based materials, nanostructures, silicone-based materials, polymers and liposomes, which are capable of delivering drug molecules directly into cells, tumors and sites of inflammation either actively or passively. there is no question that there are many limitations such as opsonization, problems with encapsulation and leakiness of drug that needs to be tackled. some of these obstacles have been overcome by the development of agents like "stealth liposomes", which escape attack by the immune system. thus, nanotechnology is expanding our capabilities through promising approaches for delivery of therapeutic agents. nanosystems and nanoparticles have opened up hitherto unforeseen avenues in diagnostics and therapeutics in medicine, especially in the fields of inflammation and cancer. the previous treatment strategies in the fields of autoimmune diseases and cancer involved non-targeted treatment options with extensive "collateral damage". nanodelivery of drugs is envisioned to reduce this collateral damage, extend a drug's availability and effectiveness at the site, and reduce toxicity, cost and storage. the focus of this section is to highlight several nanomedicine applications that have made an immediate major impact in these fields. biological nanostructures used in drug delivery systems include lipid-, silica-, polymer-, fullerene (carbonbased buckyballs, bucky tubes)-based nanostructures such as liposomes, micelles and nanoparticle systems. liposomes have been widely used as drug delivery systems, but current knowledge extends the use of any nanoparticle as an efficient carrier with necessary modifications. liposomes are vesicles with phospholipid membranes that contain hydrophilic substances in their core. the properties vary widely based on the size, lipid composition, surface charge and method of preparation [ ] . liposomal formulations have been used as anti-cancer and anti-fungal drugs, and have helped reduce the adverse effects of these drugs, while improving the efficacy and pharmacokinetics. conventional liposomes are short-lived in vivo, and are rapidly cleared by the reticuloendothelial system (res). a novel liposomal formulation with a polyethylene glycol (peg) coating avoids res-mediated clearing, and is called a stealth liposome. these stealth liposomes have favorable properties like long circulation half-life and targeted accumulation in tumor tissues [ ] . liposomes have been extensively used in cancer therapy. some of the major classes of anti-cancer drugs in liposomal formulations that are currently available or in late stages of development include anthracyclines, camptothecins, platinum derivatives, anti-metabolites and cell-cycle-specific drugs like vincristine and doxorubicin. liposomal formulations have been shown by clinical trials to decrease cardiotoxicity as compared to conventional formulations [ ] . current liposomal formulations include pegylated liposomal doxorubicin (doxil ® orthobiotech, caelyx ® schering-plough) non-pegylated doxorubicin (myocet ® elan pharma) and liposomal daunorubicin (daunoxome ® , gilead sciences). this protective strategy to limit toxicity has aided in limiting the cumulative dose of anthracyclines and administration of dexrazoxane, a highly effective cardioprotective agent, prior to anthracycline administration [ ] . liposomal platinum derivatives like cisplatin and carboplatin are used in the treatment of head and neck cancers, testicular cancer, lung cancer and many other malignancies. they have shown significantly reduced toxicity and better pharmacokinetic profiles compared to conventional formulations [ ] . some formulations have not yielded the best results. for example, spi- , a stealth liposomal cisplatin showed low clinical efficacy in phase i/ii clinical trials, possibly secondary to inadequate release of drug from liposomes [ , ] . lipoplatin, which has shown lipid bilayer fusing properties [ ] , has shown significant nephrotoxicity, but further clinical research is awaited to see if this translates into improved clinical efficacy. the use of liposomes has also been extended for enhancing immunotherapeutic effects. it is now known that liposomal targeting can be achieved by passive targeting or active targeting. passive targeting is achieved in both inflammatory and cancerous conditions taking advantage of the leakiness caused by many vascular factors that enhance permeability. this opens up a window of opportunity to increase the drug delivery, with accumulation of drug at higher concentration at the targeted site by extravasation, thereby reducing toxicity and collateral damage. on the other hand, active targeting depends on certain unique properties and molecular strategies involving monoclonal antibody-liposomal conjugates (immunoliposomes), which enables specific tumor cell targeting by antigen identification and drug delivery by internalization of the liposome by tumor cells [ ] . promising examples are the enhanced anti-tumor activity of anti-her immunoliposomes containing doxorubicin [ ] , and the use of anti-epidermal growth factor receptor (anti-egfr) immunoliposome, which showed increased cytotoxic effect in vitro against tumor cells overexpressing egfr and enhanced efficacy in vivo in xenograft models [ ] . the recent advent of the widespread use of monoclonal antibodies in cancer therapy promises more such targeted therapeutic agents. liposomal preparations have also been studied in various autoimmune and chronic inflammatory diseases. targeted delivery of anti-inflammatory agents to inflamed tissue is a promising approach limiting the adverse impact of these agents on healthy tissues. in animal colitis models, liposomal formulations of -asa achieved significantly higher local concentrations of -asa in inflamed colonic tissues compared to current treatment methods. on the other hand, liposomal preparation of mercaptopurine ( -mp) failed to improve local delivery [ ] because the drug is metabolized before it reaches the inflammation site. when peg-liposomes containing glucocorticoids were injected in mouse collagen arthritis models, longlasting reduction in joint inflammation was achieved with a single dose, while regular steroids needed multiple injections [ ] . weekly inhaled liposomal budesonide was as effective as daily inhaled budesonide in a mouse model of asthma [ ] . if this result can be replicated in clinical trials, it can greatly enhance patient compliance. this is particularly important since treatment of chronic inflammatory diseases is hampered by patient non-compliance. similarly, it is reported that liposomal preparations of anti-oxidants can also be used in diseases like adult respiratory distress syndrome (ards), sepsis, radiation lung injury and emphysema [ ] . liposomes have also been shown to be effective in diverse clinical applications such as enhanced drug delivery systems for analgesics [ ] [ ] [ ] . the carbon nanostructures that have gained most attention have been fullerene nanotubes and the geodesic dome-shaped c fullerenes. they have wide ranging applications as drug carriers and can also be used as vaccine delivery tools enhancing the immune response [ ] . they have demonstrated neuroprotective properties in cortical cell cultures and have potential therapeutic applications in neuronal-inflammation and neurodegenerative disorders like parkinson's disease, amyotropic lateral sclerosis (als) and cerebral ischemia [ ] . carbon nanotubes can cross the cell membrane without causing damage and they can act as "nanoneedles" [ ] . tectodendrimers are multicomponent dendrimers capable of multiple functions like identifying defective cells, delivering imaging and therapeutic agents to the cell and reporting the response to therapy. they can be individualized for each specific disease state and can be mass produced. baker and colleagues [ ] designed dendrimers with folic acid, fluorescein and methotrexate, and showed a -fold increase in the cytotoxic response of cells to methotrexate. in some cases, nanoparticles also aided in avoiding harmful adverse effects of drug vehicles, as in the case of abraxane ® (american bioscience), nanospheres of albumin-bound paclitaxel thus avoiding the need for toxic solvents like cremophor [ ] . recently, kriz et al. [ ] described a new sensing technology platform integrating a magnetic permeability detection and a two-site heterogeneous immunoassay using monoclonal anti-crp-conjugated superparamagnetic nanoparticles and solid-phase polyclonal anti-crp-conjugated silica microparticles to assay crp in blood samples. the results were comparable to assays performed in a clinical laboratory. the methodology seems applicable for rapid screening of biomarkers and drugs in a rapid and cost-effective manner using whole blood samples. the field of molecular imaging has exploded in recent times. significant advances have been made in real-time cellular imaging and for detecting cellular pathophysiology. over the last few years, varieties of nanostructures containing novel contrast agents and nanomaterials such as qds, gold nanoparticles or nanoshells, supramagnetic nanoparticles complexed with biological agents that can specifically bind molecular signatures of inflammation and cancer have been described [ , ] . qds have enabled in vivo live imaging, down to the level of a single qd inside a cell. qds provide several advantages over organic fluorochromes since they are photostable permitting imaging over extended periods of time, avoid interference with cellular autofluorescence, permit tracking of multiple processes simultaneously in the cells and are less toxic than organic dyes [ ] . although there is a possibility of cellular toxicity from the metallic components of qds, no cellular toxicity was seen, even under selection pressure, when qds were used to track metastatic tumor cell extravasations in an animal model [ ] . however, toxicity to humans is still being debated. qds have multimodal applications as contrast agents in bioimaging, microarrays, and facs analysis, in monitoring pharmacokinetics of therapeutic agents, and in multicolor optical coding for high throughput screening [ ] . qds have been successfully been used for sentinel lymph node sampling in gastrointestinal tract in pig models. qds can have immediate applications in oncological surgery if the safety profile can be established for humans [ ] . there are several potential pitfalls, including lack of convincing evidence for absence of cytotoxicity. further research is needed before we move forward towards widespread use of qds in biological systems [ ] . qds can also potentially replace conventional fluorochromes in complex fluoroimaging techniques like fret and fluorescence lifetime imaging microscopy, but intensive research is needed before that can happen. spio crystal core nanoparticles have magnetic properties that can be used to enhance current mri techniques due to their selective activity during t relaxation times. they can also act as 'negative enhancers' [ ] . utilizing the lymphotropic properties of these nanoparticles, weissleder and colleagues [ ] showed that superparamagnetic nanoparticle enhanced high-resolution mri. it was far superior to conventional high-resolution mri in detecting clinically occult prostatic cancer metastasis to lymph nodes. the combined use of qds with superoxide paramag-netic crystals may provide additional information by targeting specific molecular targets for imaging [ ] . a particularly interesting application is the use of spio particles for the study of nucleic acid sequences and surface topography of subcellular organelles. this is achieved by a modified afm with a nanoneedle mounted on a cantilever beam that deflects when it comes in contact with a paramagnetic nanoparticle. this response can be quantified and mapped [ ] . metal nanoshells are nanoparticles that can serve as strong near infrared absorbers. this property has been exploited to provide targeted thermal therapy selective to tumor cells without damaging normal tissue using gold nanoshells [ ] . gadolinium neutron capture therapy has several advantages, including more efficient tumor killing effects and the potential for simultaneous mri to assess response. fukumori and colleagues [ ] utilized cationic polymer chitosan nanoparticles that incorporated gadolinium for efficient cellular uptake, and demonstrated significant in vitro tumoricidal effect at relatively low concentrations. recently, bankiewicz and colleagues [ ] described an integrated strategy to delivery drugs to the brain. the combined technology involved conventionenhanced delivery (ced) to deliver liposomes containing gadoteridol, with dil-ds and mri to obtain detailed images of drugs moving through a living primate brain following ced for imaging, and to induce better clinical efficacy. molecular imaging has now crossed-over into medical imaging through the use of smart imaging agents for in vivo molecular imaging and imaging of animal models [ ] [ ] [ ] . a recent study showed that magnetic nanoparticle conjugated with anti-vcam- antibodies can detect vcam- expression through fluorescence and magnetic resonance on endothelial cells in vivo and in vitro [ ] . this is an important step, opening up opportunities to use many specific markers specific for inflammation and cancer to diagnose and monitor many inflammatory diseases and cancers. biological systems operate at the nanoscale. nanomedicine is the application of nanotechnology to monitor and treat biological systems in health and disease. this is accomplished by real time monitoring of molecular signaling at the cellular and tissue level. during the past decade, there has been an explosion in this field, resulting in revolutionary advances in determining the microstructure and function of living systems. these discoveries have led to the development of powerful tools for fundamental biological and medical research. nanotechnology has been applied to targeted drug delivery to minimize side effects, creating implantable materials as scaffolds for tissue engineering, creating implantable devices, surgical aids and nanorobotics, as well as throughput drug screening and medical diagnostic imaging. the nanoinitiatives are funded by governments and private sources throughout the world to develop or further refine the technology to provide the beyond-imag- inable, most sophisticated tools to a physician and scientists to inflammatory diseases. no doubt, there will be many technical, regulatory and legal challenges in the deployment of these technologies. unquestionably, there is enough desire and commitment to meet these challenges for the good of society and betterment of the quality of life. intestinal inflammation: a complex interplay of immune and nonimmune cell interactions nanoparticle and targeted systems for cancer therapy surfactant stabilized contrast agent on the nanoscale for diagnostic ultrasound imaging nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent sypro ruby dye plasmonics-based nanostructures for surfaceenhanced raman scattering bioanalysis engineered materials for biophotonics applications: improving sensing, and therapeutics biological applications of dendrimers nanomedicine: current status and future prospects emerging biological materials through molecular self-assembly micromachined needles and lancets with design adjustable bevel angles microfabricated microneedles for gene and drug delivery role of nanobiotechnology in developing personalized medicine for cancer genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery rapid discrimination among individual dna hairpin molecules at singlenucleotide resolution using an ion channel molecular self-assembly bioassays based on molecular nanomechanics carbon nanotubes as intracellular transporters for proteins and dna: an investigation of the uptake mechanism and pathway carbon nanotubes as intracellular protein transporters: generality and biological functionality direct synthesis of long single-walled carbon nanotubes strands cationic liposome-microtubule complexes: pathways to the formation of two-state lipid-protein nanotubes with open or closed ends accumulation of ultra-small super paramagnetic particles of iron oxide in human atherosclerotic plaques can be detected by in vivo magnetic resonance imaging applications of nanotechnology to biotechnology quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules quantum dot bioconjugates for imaging, labeling and sensing differences in subcellular distribution and toxicity of green and red emitting cdte quantum dots superparamagnetic iron oxide nanoparticles: nodal metastases and beyond tat peptidederivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells dendrimers for pharmaceutical and biomedical applications designing dendrimers for biological applications synthesis and evaluation of gadolinium complexes based on pamam as mri contrast agents biodegradable polymersomes as a basis for artificial cells: encapsulation, release and targeting near-infrared-emissive polymersomes: self-assembled soft matter for in vivo optical imaging recent advances with liposomes as pharmaceutical carriers stable and robust polymer nanotubes stretched from polymersomes nanotechnology on duty in medical applications electrical detection of single viruses whole-blood immunoassay facilitated by gold nanoshell-conjugate antibodies biological cryo atomic force microscopy: a brief review adhesion forces between individual ligand-receptor pairs atomic force microscopy imaging of retroviruses: human immunodeficiency virus and murine leukemia virus adhesion at calcium oxalate crystal surfaces and the effect of urinary constituents functional protein nanoarrays for biomarker profiling protein nanoarrays generated by dip-pen nanolithography self-assembled lipid superstructures: beyond vesicles and liposomes from conventional to stealth liposomes: a new frontier in cancer chemotherapy reduced cardiotoxicity and comparable efficacy in a phase iii trial of pegylated liposomal doxorubicin hcl (caelyx/doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer the effect of dexrazoxane on myocardial injury in doxorubicin-treated children with acute lymphoblastic leukemia comparative intravenous toxicity of cisplatin solution and cisplatin encapsulated in long-circulating, pegylated liposomes in cynomolgus monkeys phase i-ii study of pegylated liposomal cisplatin (spi- ) in patients with inoperable head and neck cancer phase i and pharmacokinetic study of spi- , a liposomal encapsulated dosage form of cisplatin pharmacokinetics and adverse reactions of a new liposomal cisplatin (lipoplatin): phase i study immunoliposomes for the targeted delivery of antitumor drugs anti-her immunoliposomes: enhanced efficacy attributable to targeted delivery epidermal growth factor receptor (egfr)-targeted immunoliposomes mediate specific and efficient drug delivery to egfr-and egfrviii-overexpressing tumor cells liposomal formulations of inflammatory bowel disease drugs: local versus systemic drug delivery in a rat model liposomal targeting of glucocorticoids to synovial lining cells strongly increases therapeutic benefit in collagen type ii arthritis efficacy of liposomal budesonide in experimental asthma antioxidant strategies in respiratory medicine transdermal delivery of an analgesic agent using elastic liposomes: preparation, characterization and performance evaluation preparation oral liposome-encapsulated recombinant helicobacter pylori heat shock protein vaccine for prevention of hp infection topical liposomal gel of idoxuridine for the treatment of herpes simplex: pharmaceutical and clinical implications immunization with peptide-functionalized carbon nanotubes enhances virus-specific neutralizing antibody responses fullerenebased antioxidants and neurodegenerative disorders carbon nanotubes for the delivery of therapeutic molecules design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor phase i and pharmacokinetic study of abi- , a cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel detection of c-reactive protein utilizing magnetic permeability detection based immunoassays nanocrystal imaging in vivo in vivo cancer targeting and imaging with semiconductor quantum dots turning the spotlight on cellular imaging tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy quantum dots for live cells, in vivo imaging, and diagnostics sentinel lymph node mapping of the gastrointestinal tract by using invisible light potentials and pitfalls of fluorescent quantum dots for biological imaging use of magnetic nanoparticles as nanosensors to probe for molecular interactions noninvasive detection of clinically occult lymph-node metastases in prostate cancer oligomerization of paramagnetic substrates result in signal amplification and can be used for mr imaging of molecular targets novel nanosensors for rapid analysis of telomerase activity nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance in vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain pet scanners dedicated to molecular imaging of small animal models in vivo molecular and genomic imaging: new challenges for imaging physics current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research in vivo imaging of activated endothelium using an anti-vcam- magnetooptical probe key: cord- - rg qtdq authors: watkins, laura c.; degrado, william f.; voth, gregory a. title: influenza a m inhibitor binding understood through mechanisms of excess proton stabilization and channel dynamics date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: rg qtdq prevalent resistance to inhibitors that target the influenza a m proton channel has necessitated a continued drug design effort, supported by a sustained study of the mechanism of channel function and inhibition. recent high-resolution x-ray crystal structures present the first opportunity to see how the adamantyl-amine class of inhibitors bind to m and disrupt and interact with the channel’s water network, providing insight into the critical properties that enable their effective inhibition in wildtype m . in this work, we test the hypothesis that these drugs act primarily as mechanism-based inhibitors by comparing hydrated excess proton stabilization during proton transport in m with the interactions revealed in the crystal structures, using the multiscale reactive molecular dynamics (ms-rmd) methodology. ms-rmd, unlike classical molecular dynamics, models the hydrated proton (hydronium-like cation) as a dynamic excess charge defect and allows bonds to break and form, capturing the intricate interactions between the hydrated excess proton, protein atoms, and water. through this, we show that the ammonium group of the inhibitors is effectively positioned to take advantage of the channel’s natural ability to stabilize an excess protonic charge and is thus acting as a hydronium-mimic. additionally, we show that the channel is especially stable in the drug binding region, highlighting the importance of this property for binding the adamantane group. finally, we characterize an additional hinge point near val , which dynamically responds to charge and inhibitor binding. altogether, this work further illuminates a dynamic understanding of the mechanism of drug inhibition in m , grounded in the fundamental properties that enable the channel to transport and stabilize excess protons, with critical implications for future drug design efforts. toc graphic proton transport (pt) across cellular membranes is a critical component of many biomolecular systems, necessary, for example, to maintain ph gradients, , to drive atp synthesis, and to facilitate the co-or anti-transport of other small molecules. [ ] [ ] [ ] because of their essential role in such systems, channels and transporters with pt functionality are often targets for drug design to inhibit or control pt-in the case of viruses and bacteria, to slow or prevent infection, but there are myriad other disease applications. [ ] [ ] [ ] drug design is notoriously challenging, as both thermodynamic and kinetic factors must be considered but are difficult to predict and control, and its success depends on high quality structures, an understanding of structural dynamics, and a knowledge of the protein's function and its mechanism. thus, beyond elucidating mechanisms of pt in order to understand how a specific channel or transporter works, studying the detailed interactions that facilitate pt can provide valuable insight to help guide drug design efforts. the influenza virus kills up to , people each year, and the impact of the recent global coronavirus pandemic emphasizes how critical it is to maintain our focus on understanding and treating viral infections. the influenza a virus matrix (m ) proton channel is a homo-tetrameric protein responsible for the acidification of the viral interior, a critical step in the influenza infection process. [ ] [ ] [ ] it is the target of two of the three currently available oral antivirals, amantadine and rimantadine. , while these are effective at blocking pt in wildtype m , drug-resistant mutants have become the predominate strains, rendering these drugs ineffective and thus requiring a continued drug design effort informed by a deeper understanding of the pt and drug inhibition mechanisms. additionally, m is considered an archetype for the viroporin family, a class of viral channels considered ideal drug targets. the sars-cov- virus responsible for the covid- pandemic contains two viroporins, protein e and . [ ] [ ] [ ] thus, viroporins are a critical class of proteins to study as potential therapeutic targets. m is located in the viral capsid and is acid-activated: as the ph of the endosome encapsulating the virus is lowered, the m channel becomes activated and facilitates unidirectional proton flow to the viral interior, allowing the virus to escape the endosome and infect the cell. the key residue that controls activation is his , - which can bind one additional proton and take on a + charge. one histidine from each helix forms the his tetrad, which can collectively hold a + to + excess charge, dependent on ph. the channel becomes activated and the c-terminal portion opens (adopting the inwardopen conformation) upon reaching the + state, and pt occurs as the channel cycles through a transporter-like mechanism. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] amantadine and rimantadine belong to the adamantyl-amine class of inhibitors, binding in the upper-middle portion of the channel. these drugs were the predecessors of many related adamantane-based compounds featuring a relatively rigid, apolar group and an attached charged group. [ ] [ ] [ ] [ ] [ ] [ ] [ ] recently, thomaston et al. published several high-resolution x-ray crystal structures of m with amantadine, rimantadine, and a novel spiro-adamantyl amine bound. these structures provided the first opportunity to see the specific interactions that facilitate stable inhibitor binding and the disruption of the hydrogen-bonded water network otherwise present. along with an earlier qualitative md simulation study that guided the design of the spiro-adamantyl amine inhibitors, the crystallographic analysis provided potential insights into the mechanism of inhibition, suggesting that the backbone carbonyls of pore-lining residues act as physiochemical chameleons, able to engage in both hydrophobic and hydrophilic interactions, and that the drug is tilted off the channel's axis and interacts with waters in the ala layer. taken together, it is hypothesized that amantadine acts as a mechanism-based inhibitor, with the ammonium group functioning as a hydronium mimic. computational studies to date have primarily focused on the means of entry into the channel and location of binding, - but have not deduced specific interactions between the drug, channel, and channel water involved in binding as they relate specifically to similar interactions seen in the pt mechanism. proton transport is an inherently quantum mechanical process, as the hydrated proton structure (hydronium-like) exists in a complex hydrogen-bonded network that rearranges dynamically as bonds break and form according to the grotthuss shuttling mechanism. - thus, classical molecular dynamics (md) with fixed bonding topology cannot be used to study pt; moreover, ab initio methods are not efficient enough to reach the many nanosecond timescales necessary to obtain sufficient sampling in biomolecular systems that may have important degrees of freedom several orders of magnitude slower than proton shuttling. multiscale reactive molecular dynamics (ms-rmd) [ ] [ ] [ ] [ ] (and multistate empirical valence bond, ms-evb, before it) was developed to efficiently and accurately capture the solvation and delocalization of an excess proton in water, such that the quantum-chemical nature of the hydrated proton can be studied in the context of membrane proteins over the long timescales needed for accurate simulation of such systems. ms-rmd has been successfully applied in several protein systems to predict and explain mechanisms of pt. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in previous work, , quantum mechanics/molecular mechanics (qm/mm) and ms-rmd was used to calculate potentials of mean force (pmfs, i.e., free energy profiles) of pt through the m channel in the + - states, providing critical insight into the ph-dependent activation behavior and the role of the his tetrad in pt. most recently, we further analyzed the ms-rmd simulations to explore the detailed interactions between the hydrated excess proton and the channel and found that the proton dynamically, as a function of its position, alters several properties of the protein and pore waters, including the hydrogen-bonding network and the protein structure. this latter work illustrates how ms-rmd can be used successfully to investigate explicit, dynamic interactions between a hydrated proton and its immediate environment, as well as its indirect effects on other parts of the system. here, we employ a similar approach as in this previous work to focus specifically on properties related to drug binding and how the position of the bound drug relates to the overall pt mechanism. through this analysis, we examine the hypothesis that the adamantyl-amine drugs act as mechanism-based inhibitors. our results indicate that the ammonium group of amantadine and rimantadine are aptly positioned to take advantage of the channel's ability to stabilize an excess proton. additionally, by examining conformational fluctuations, we show that the drug binding pocket is an especially stable and symmetrical portion of the channel, conducive to binding a roughly spherical drug, and we reveal an additional minor hinge point towards the top of the channel which may be a relevant feature for future drug design efforts. simulations for calculating properties as the proton moves through the top of the channel were run as follows. starting structures were taken from previous simulations, which were initiated from a crystal structure of the transmembrane portion of the m channel (this construct is referred to as m tm) resolved at room-temperature and high ph (pdb: qkl ) embedded in a -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) bilayer solvated with water. m tm is the minimum construct necessary to retain proton conduction similar to full-length m , and it has been shown that the presence of amphipathic helices, included in the full-length m protein, do not significantly influence the pt mechanism. the collective variable (cv) defined for umbrella sampling (us) is the z-coordinate of the vector between the excess proton center of excess charge (cec, see below) and the center of mass of the four gly alpha carbons, as in our previous work, such that the cv has negative values at the top (n-terminal end) of the protein and progresses to positive values at the bottom (cterminal end). the excess proton cec is defined as: is the center-of-charge of the diabatic ms-rmd state, and is the amplitude of that state. the sum is over all states. ms-rmd simulations were run with the excess proton at every . Å along the cv coordinate between - . and . , generating windows. to ensure that the proton remained in the channel, a cylindrical restraint was added at Å with a force constant of kcal/mol·Å using the opensource, community-developed plumed library. , the ms-evb version . parameters were used to describe the hydrated excess proton. after a ps ms-rmd equilibration, the replica exchange umbrella sampling technique was used to facilitate convergence. production simulations were run for ~ - ns with frames saved every ps. for calculating hydrogen bond residence times, longer independent trajectories were run with the cec restrained in different positions using us as described above. each hydrogens on the ammonium group of amantadine were added. right, a snapshot from an ms-rmd trajectory with the most hydroniumlike water indicated in green. in both, two opposing chains of m are shown in silver. the ser , his sidechains and the gly , ala backbone carbonyls are shown. the z-coordinate for the system is included on the left, where z = Å is defined as the center-of-mass of the gly alpha-carbons. trajectory was run for . - ps with frames saved every fs. simulation frames were binned by excess proton cec value for subsequent analyses, which were performed in python using the scipy, numpy, and pandas libraries. for hydrogen bond analysis, values were averaged over the four helices. hydrogen bonds were defined by the following criteria: the donor-acceptor distance must be less than . Å, and the donor-hydrogen-acceptor angle must be greater than °. several hydrogen bond definitions were tested and did not affect the conclusions (not shown). for calculating residence times, a hydrogen bond was considered in place as long as the particular water molecule remained the closest water to the protein atom and the hydrogen bond criteria were met. images of molecular structures were rendered in visual molecular dynamics (vmd), while other figures were generated using matplotlib. if the adamantyl-amine drugs are acting as mechanism-based inhibitors as hypothesized, we would expect to see specific aspects of the pt mechanism taken advantage of or replicated by the drug upon drug binding. to test this, we performed ms-rmd simulations of m in the + his charge state with an explicit excess proton to evaluate the hydrogen-bonding networks, pore shape, and protein fluctuations throughout pt that relate to drug binding. by focusing on pt in the + state, we are studying the process of proton entry and diffusion to his in the first key step of channel activation, paralleling inhibitor entry into the channel. we additionally expect this to be a prevalent charge state in drug-bound structures due to the lowered his pkas. replica exchange umbrella sampling was used to obtain sufficient sampling of proton positions throughout the top portion of the channel, with windows from cecz = - . to . Å where the coordinate origin is defined as center of mass of the gly alpha carbons. the channel is aligned along the z-axis for all subsequent analyses. to understand how properties of pt may provide insight into drug binding, we primarily examine variations dependent on proton position. we compared the values of each property when the proton is at the drugs' ammonium group positions versus other parts of the channel to determine if the drugs could be taking advantage of the channel's natural ability to stabilize a proton. this idea is highlighted in figure , which shows both the drug-bound crystal structure and a snapshot of a hydrated excess proton in the channel from our simulations. we refer to the drugs' ammonium nitrogen position along the z-axis in the crystal structure as ammnz. this value is - . and - . Å for the the inwardclosed amantadine and rimantadine bound structures, respectively (averaged over the two tetramers in each crystal structure). flexible hydrogen bonds stabilize the excess proton near ammnz. it has been shown in our previous work that hydrogen bonds within the channel, including those between water and protein atoms, help facilitate proton transport by altering their direction and frequency of interaction as the proton moves through the channel. here, we focus specifically on water interactions that may help account for excess charge stabilization near ammnz. in figure , we calculate the occupancy of three different hydrogen bonds between protein atoms and water as a function of the excess proton position in the channel. while the ala hydrogen bond occupancy is consistent as the excess proton enters and moves through the top of the channel, as it approaches the ala carbonyls, the occupancy decreases ~ %. this dip indicates the ala hydrogen-bonded waters can flexibly reduce their interaction with the protein as a result of an excess charge in their vicinity. additionally, this dip is centered at - . Å, near ammnz. at this point, the role of the waters near ala carbonyls in hydrating the proton is maximized. this supports the hypothesis that amantadine and rimantadine are mechanism-based inhibitors and take advantage of the channel's natural ability to stabilize a hydrated excess proton in order to stabilize the drug's ammonium group. the hydrogen bond occupancy of waters with the gly carbonyls increases once the excess proton passes through the val gate and remains fairly consistent across proton posi tions thereafter, exhibiting little dependence on the hydrated proton position once it is in the channel. the ser sidechain water occupancies are shown for comparison, which do not show a noticeable trend based on proton position. thus, this change in interactions is not a universal effect throughout the channel, but the ala waters seem to be uniquely flexible in this manner. these differences are consistent with drug design studies -while compounds such as spiro-adamantyl amine have been able to displace the water in the ala layer, no designed inhibitors have displaced the water around gly . to further understand how the dynamics of the hydrogenbond network may show how these drugs benefit from the channel's inherent excess-charge stabilization used in proton transport, we examined the average residence times of hydrogen bonds between water and several important protein atoms. to do this, independent trajectories were run for five different excess proton positions, including two trajectories with the proton completely outside the channel (cecz = - . , . Å) and three when the proton is near ammnz. these results are shown in figure . the ala water residence times slightly increase when the excess proton is near ammnz, the ser water residence times do not show any significant difference between the proton outside the channel and at ammnz, and those of gly waters decrease at ammnz. the waters hydrogen bonded to his imidazole nitrogens show the greatest change in residence times and are shown to highlight the ability of this method to describe such differences. we note that classical md does not wholly capture charge transfer in hydrogen bonds, resulting in an overall weaker interaction. thus, while these simulations provide valuable insight into these interactions and their trends, we expect that they are stronger in the real system and any differences will be more prominent. with the above results for ala , this may indicate that several waters remain tightly hydrogen bonded to the ala backbone carbonyls, while one or more are bonded less frequently. while the gly hydrogen bonds do not form less frequently (as indicated in figure ) with an excess charge in this region, they do exhibit greater dynamics and flexibility. this change indicates an increase in water dynamics when the excess proton is near, which could help stabilize and solvate the excess charge in the ala water layer. taken together, these results further support the hypothesis that amantadine and rimantadine act as mechanism-based inhibitors: the channel acts as a scaffold to facilitate pt by harboring flexible protein-water interactions that can adapt and respond to a positive excess charge, with specific ability to stabilize an excess proton near ammnz that the charged drugs can take advantage of. drug tilt positions ammonium group in highest cec density. one prominent characteristic of the amantadine and rimantadine bound structures is the drug's tilt within the pore. this tilted conformation is also seen in solid-state nmr studies. given the drug's three-fold symmetry in a four-fold symmetric channel, the ammonium group cannot form hydrogen bonds with all waters hydrogen-bonded to ala , leading in part to this tilt. based on our previous work examining the proton's path through the channel, we used a similar analysis to examine the density of cec positions when the excess proton is near the ammonium position in drug-bound structures. figure a the excess proton prefers to be near the edge of the pore, unlike the predominate preference for the center of the pore figure b shows the radial density of the cec in this same region of the channel. possible positions of amantadine's ammonium group nitrogens were calculated based on the drug's position and tilt in the crystal structure, and their radii are included as dashed lines (these positions were also used to generate the image in figure ). these hydrogens can extend to a radius ~ . Å in this static crystal structure, which indicates that the slightly off-centered ammonium group directly positions - of its hydrogens in the region of the cec's highest density. the cec's propensity for the edge of the pore indicates that the drug's tilt in the channel may not only be a necessary component of its binding, but also a thermodynamic advantage. this tilt further allows the drug to act as a hydronium mimic, as the hydrogens of the ammonium group are in the favorable positions of the solvated excess proton. the analysis of hydrogen bonding changes and proton densities indicates how the ammonium group is a functional addition to the adamantane scaffold, as the charged group is positioned in a region where the channel is especially adept to stabilize an excess charge. this stabilization relies on flexible water structures and hydrogen-bond interactions that can undergo minor changes to accommodate the proton, suggesting that the adamantyl-amine inhibitors are acting as hydronium mimics -they take advantage of these inherent features to help solvate the charged ammonium group. the identification of other regions of the channel with increased ability to stabilizing an excess charge, such as areas of increased proton density or significantly flexible water interactions, could help provide new targets for drugs to act as hydronium-mimics. pore shape and stability near ser are ideal for adamantane binding. another hypothesis about the adamantyl-amine class of inhibitors is that adamantane is effectively spherical and can freely rotate within the channel, but has no rotatable bonds, which minimizes the entropy lost upon binding. this rapid rotation can be seen on the nmr time scale and is consistent with the recent thomaston et al. crystallographic studies, in which the motion was indirectly inferred. nevertheless, its significance depends on the dynamic nature of the channel -if the protein exhibits great structural fluctuations in the region where the drug binds, then drug binding may induce changes that greatly decrease the entropy and this hypothesis would not fully explain the drugs' efficacy. to better understand how the channel's natural dynamics may lend itself to favorable drug binding, we examined the pore shape throughout our trajectories. as an estimate of the asymmetry of the channel, we calculated the eccentricity, which is essentially a measure of how "circular" a given oval is. the eccentricity is defined as: where a and b are the semi-major and semi-minor axes, respectively, which we approximate by the distance between alpha-carbons on opposing helices. a schematic of this is shown in si figure . eccentricity can have values between and , with indicating a circle and indicating a parabola. these results are shown in figure . eccentricity maximum, minimum, and rmsd values are calculated in si table . the pore-lining residues in the bottom half of the channel, gly , his , and trp , all show a greater degree of asymmetry and a wider range of eccentricity values, dependent on the excess proton position, than the pore-lining residues in the top part of the channel. interestingly, proton entry at cecz = - Å has a pronounced effect on the channel near trp , greater than that when the proton nears the center of the channel. ser , however, has overall the smallest average eccentricity and the lowest minimum value than the other pore-lining residues during pt in this portion of the channel. additionally, ser and val have smaller proton position dependent changes in eccentricity than the pore-lining residues in the bottom half of the channel. this result indicates that the ser region is the most symmetrical and stable in the channel. while analyzing the alpha-carbon distances and eccentricity, we also examined the correlation between these alpha-carbons distances on opposing helices, shown in figure , to gain further insight into protein motion and conformational fluctuations on the nanosecond timescale. these motions captured here are equilibrium fluctuations in the + , inwardclosed state, not necessarily motions driving the transition between inwardopen and inwardclosed. the calculated correlations indicate that the channel's equilibrium structural fluctuations are dominated by alternating inward-outward motions of opposing helices. at each pore-lining residue, the distances are negatively correlated-that is, when helices a and c move farther apart, helices b and d move closer together, and vice-versa. gly is known to be the hinge point whose kinking controls the large structural change between inward-open and inwardclosed conformations, which may falsely lead to the conclusion that the conformational fluctuations at equilibrium above and below gly are decorrelated, with a stable core centered at gly . interestingly, however, the motions at gly are strongly and similarly correlated with the motions at both ser and his . this correlation indicates that in this fixed charge state, the gly kink is relatively rigid. instead, there is a noticeable lack of correlation between val and the other porelining residues, suggesting that there is a secondary, minor "hinge" between val and ser that decorrelate the inwardoutward motions between the helices above and below this point. this natural hinge observed near val furthers our understanding of val acting as a secondary gate that opens to allow proton and water entry into the channel. , in our simulations, this valve can readily hydrate, particularly in the presence of a nearby excess proton. moreover, it is frequently closed, which may make passage of a hydrated sodium or chloride ion more difficult. this aspect of the val gate and its relevance for pt and proton selectivity is likely an important feature of the m channel and could be further explored in future work. given that the adamantane group of the drug is centered in the ser tetrad plane, we hypothesize that these facets of the channel's dynamics are critical for fully explaining the drugs' favorable binding. because of the more circular shape of the pore at the ser tetrad, the spherical adamantane group can fit snugly under the hydrophobic val cleft and block pt. additionally, the relative stability of the pore in the region of drug binding helps explain why drug binding is thermodynamically favorable. because the channel exhibits smaller structural fluctuations here than in other regions of the channel, the adamantane based drugs are able to bind with minimal loss of entropy as the channel does not need to lose flexibility to create a stable drug-binding interface. this has figure . pearson correlation coefficients of the distances between alpha-carbons on opposing helices, for all pore-lining residues, when excess proton cecz=- . Å. each row and column correspond to a specific residue and distance, as labeled, where 'a-c' is the distance between the alpha-carbons of helices and and , and 'b-d' is that of helices and . only those values with a pvalue < . are shown, any other values are set to . . important implications for designing drugs that use scaffolds different from the adamantane group, , - or that interact with drug-resistant mutants such as s n. while drug binding to more flexible regions of the channel is possible, only modest changes in potency are often observed despite large changes in the size of the drugs. this is likely because of the need to counter the greater loss of entropy resultant from structural changes and reduced fluctuations. altogether, we have shown how the adamantyl-amine inhibitors of m are suited to exploit various inherent features of the m channel that naturally facilitate proton transport, further supporting the claim that they function as mechanismbased inhibitors. the ability of hydrogen bond interactions to flexibly respond to the hydrated excess proton cec, measured in both hydrogen bond occupancy and residence times, indicate how the channel is suited to stabilizing an excess charge near ammnz. thus, the ammonium group of these inhibitors can act as a hydronium-mimic by binding in this region. we also analyzed the pore shape throughout the channel by calculating the eccentricity of the pore based on alpha-carbon distances. the results from these calculations indicate that the drug binding pocket is an especially stable and symmetrical portion of the channel, conducive to binding a roughly spherical drug. finally, by examining the correlations between these distances, we found an additional minor hinge point towards the top of the channel which may be a relevant feature for future drug design efforts. understanding these features as they relate to drug binding gives further insight into the specific interactions that stabilize drug binding, and could help inform drug design efforts by highlighting other aspects of the m channel and proton transport mechanism that a drug could take advantage of. through this understanding, we hope future drug-design efforts can take advantage of this approach to methodically create new inhibitors for the more prevalent mutant strains. with the recent publication of high resolution influenza b m (bm ) structures, we hope similar studies to elucidate the detailed pt mechanism in bm will be conducted to guide drug design in this functionally similar protein. this work also shows how similar analyses to understand the details of explicit proton transport mechanisms (not those inferred by water structures alone) could be used in other systems, and extended to ion transporters such as the sars-cov- viroporins, to help inform mechanism-based inhibitor design. elucidating the inherent features of drug-targetable proton transporters, such as flexible water and hydrogen bonding interactions, preferred proton positions, dynamic pore shapes, and structural fluctuations, can help guide the design of drug scaffolds and added substituents. the ms-rmd simulation methodology utilized in this work has also made these studies possible both for m and other important drug targets. supporting information. a table of average, maximum, minimum, and rmsd for eccentricity is included in the si. this material is available free of charge via the internet at http://pubs.acs.org. corresponding author * gavoth@uchicago.edu the authors declare no competing financial interest. voltage-gated proton channels and other proton transfer pathways a protonmotive force drives atp synthesis in bacteria coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism chapters and chance and design--proton transfer in water, channels and bioenergetic proteins importance of ph homeostasis in metabolic health and diseases: crucial role of membrane proton transport lysosomal storage disease upon disruption of the neuronal chloride transport protein clc- (seasonal) (accessed march , ). . world health organization the m proton channels of influenza a and b viruses selective proton permeability and ph regulation of the influenza virus m channel expressed in mouse erythroleukaemia cells ion channel activity of influenza a virus m protein: characterization of the amantadine block viroporins: structure, function and potential as antiviral targets sars coronavirus e protein forms cation-selective ion channels severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release activation of the m ion channel of influenza virus: a role for the transmembrane domain histidine residue histidines, heart of the hydrogen ion channel from influenza a virus: toward an understanding of conductance and proton selectivity structure and mechanism of the m proton channel of influenza a virus mechanisms of proton conduction and gating in influenza m proton channels from solid-state nmr effect of cytosolic ph on inward currents reveals structural characteristics of the proton transport cycle in the influenza a protein m in cell-free membrane patches of xenopus oocytes acid activation mechanism of the influenza a m proton channel put a cork in it: plugging the m viral ion channel to sink influenza in vitro pharmacokinetic optimizations of am -s n channel blockers led to the discovery of slow-binding inhibitors with potent antiviral activity against drug-resistant influenza a viruses exploring the requirements for the hydrophobic scaffold and polar amine in inhibitors of m from influenza a virus identification of hits as matrix- protein inhibitors through the focused screening of a small primary amine library interpreting thermodynamic profiles of aminoadamantane compounds inhibiting the m proton channel of influenza a by free energy calculations hydrogen-bonded water molecules in the m channel of the influenza a virus guide the binding preferences of ammonium-based inhibitors computationally efficient multiconfigurational reactive molecular dynamics computationally efficient multiscale reactive molecular dynamics to describe amino acid deprotonation in proteins understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase proton movement and coupling in the pot family of peptide transporters modulating the chemical transport properties of a transmembrane antiporter via alternative anion flux exchange pathways in clc-ec antiporter proton-induced conformational and hydration dynamics in the influenza a m channel high-resolution structures of the m channel from influenza a virus reveal dynamic pathways for proton stabilization and transduction a second generation multistate empirical valence bond model for proton transport in aqueous systems a guide to numpy {vmd} --{v}isual {m}olecular {d}ynamics matplotlib: a d graphics environment the chemical and dynamical influence of the anti-viral drug amantadine on the m proton channel transmembrane domain computational study of drug binding to the membrane-bound tetrameric m peptide bundle from influenza a virus exploring the size limit of templates for inhibitors of the m ion channel of influenza a virus undecane derivatives: from wild-type inhibitors of the m ion channel of influenza a virus to derivatives with potent activity against the v a mutant expeditious lead optimization of isoxazole-containing influenza a virus m -s n inhibitors using the suzuki-miyaura cross-coupling reaction key: cord- -i z ju authors: criado, paulo ricardo title: adverse drug reactions date: - - journal: dermatology in public health environments doi: . / - - - - _ sha: doc_id: cord_uid: i z ju adverse events and adverse drug reactions are common in clinical practice. side effects range from the common to the rare and may be confused with other mucocutaneous manifestations resulting from several medications to treat infections, other medical conditions, and in the clinical setting of oncologic treatment. the objective of this chapter to review current data on adverse drug reactions, here classified as (i) severe adverse drug reactions, (ii) uncomplicated cutaneous adverse drug reactions, and (iii) adverse drug reactions caused by chemotherapy drugs, particularly those cases whereby the dermatologist is requested to issue a report and asked to comment on the safety and viability of readministration of a specific drug. we describe aspects associated with these events, presenting a detailed analysis of each of them. • if possible identify the physiopathologic mechanism involved in the reaction; • identify as rapidly as possible the drug inducing the reaction and always opt for its withdrawal; in some circumstances the choice is difficult as there is no alternative drug and its use is essential for the maintenance of life; • a careful and intensive observation is recommended for the occurrence of warning signs regarding the appearance of a potentially severe adverse drug reaction, especially in relation to mucous, oral, ocular, and genital involvement and progression of any present cutaneous eruption; • it is imperative that the drug responsible may be withdrawn on a permanent basis together with chemically related com-pounds, and this advice is also valid for first-degree relatives who can present the same type of reaction. function. the aim of this study is to describe these reactions in order to facilitate recognition and treatment. this group of drug reactions includes anaphylaxis, stevens-johnson syndrome (sjs), toxic epidermal necrolysis (ten), and, depending on the systemic involvement, erythroderma. in this chapter we approach the characteristics and treatment of some adverse reactions to drugs, including anaphylaxis, erythroderma, sjs, and ten. the prevalence of scards is estimated at in , hospitalized patients. sjs and ten are particularly severe [ ] . in general, fatal cutaneous drug-induced reactions occur in . % of clinical patients and . % of surgery patients [ ] . scards may be defined as usually requiring hospitalization, at times in intensive therapy or burn care units for close observation of vital signs and visceral function. this group of drug reactions includes anaphylaxis, sjs, ten, drug hypersensitivity, and, depending on the systemic involvement, erythroderma, acute generalized exanthematous pustulosis (agep), cutaneous necrosis induced by anticoagulants, druginduced vasculitis, and reactions such as serum disease [ ] . quick differentiation between a scard and a less severe eruption may be difficult, although essential. withdrawal of the suspected drug is the surest way of intervening to reduce mortality [ ] . most cutaneous reactions to drugs are usually observed as a morbilliform or maculopapulous exanthema [ , , ] . unfortunately, erythema morbilliform ( fig. . ) most often characterizes the appearance at onset in the severest of cases, including ten, serum disease, and drug hypersensitivity syndrome [ ] . djien et al. [ ] , studying patients with reactions to drugs clinically presenting with erythematous cutaneous eruptions (morbilliform and scarlatiniform exanthema, maculopapulous, and small isolated papules), concluded that three types of severe clinical markers exist with respect to this kind of reaction: fever, lymphadenopathy, and extensive cutaneous affection. the authors excluded specific forms from the study, such as sjs, ten, fixed drug eruption (fde), agep, phototoxicity, and vasculitis. this suggests that in cases of drug-induced reactions with extensive cutaneous affection, with or without lymphadenopathy, a laboratory investigation is required with a complete hemogram and hepatic function test. in , roujeau and stern [ ] put forth clinical and laboratory criteria leading to the suspicion that a reaction to drugs could develop into more severe behavior (chart . ). we next discuss the following reactions: anaphylaxis and anaphylactoid reactions, erythroderma, and the clinical spectrum of sjs and ten (lyell's disease). anaphylaxis is a quick systemic reaction usually presenting a risk to life and resulting in immediate hypersensibility mediated by immunoglobulin e (ige). anaphylactoid reactions mimic anaphylaxis, although they are not related to immunologic mechanisms [ , ] . these reactions lead to a powerful activation of mastocytes, with a massive release of mediators [ , ] . drugs are not the more important cause of anaphylaxis, as they are responsible for merely - % of cases [ ] .drugs that do cause anaphylactic reactions include β-lactam antibiotics (responsible for % of fatal anaphylactic reactions in the united states), cephalosporin, sulfonamides, hemoderivatives, enzymes (trypsin, chymopapain, and streptokinase), insulin (very rare nowadays, owing to use of recombinant human insulin), vaccines (due to preservatives, proteic components, and gelatin; some reports of patients show sensitivity to eggs and allergic reactions to vaccines), allergenic extracts, protamine, and progesterone [ , ] . anaphylactoid reactions may occur with acetylsalicylic acid, nonhormonal anti-inflammatory drugs, iodide contrasts, angiotensin-converting enzyme (ace) inhibitors, and fluorescein [ ] . during general anesthesia, anaphylactic and anaphylactoid reactions may occur. these are difficult to differentiate because of the large amount of medications used, such as anesthetics, muscular relaxants, analgesics, nonhormonal anti-inflammatories, and antibiotics [ ] . their clinical emergence tends to occur suddenly, within -min to -h intervals after contact with the precipitating factor, although delayed reactions are rarer. they show an appearance of pruritus, urticaria ( fig. . ), rhi- lymphocytosis with atypia abnormalities of liver enzymes or function noconjunctival symptoms, angioedema symptoms (especially laryngitis), hypotension, and lung sounds [ ] . the following ailments may be observed: abdominal pain, diarrhea, vomiting, uterine contraction, and cardiac arrhythmia. after a few hours symptoms may reappear during a late phase, although this is by no means automatic [ , ] . patients with anaphylaxis must be identified as fast as possible, and treatment must be initiated immediately [ ] . this reduces the risk of fatal reactions [ ] . the following are signs of anaphylaxis that pose a risk to life: presence of stridor, edema of the glottis, intense dyspnea, lung sounds, hypotension, cardiac arrhythmia, shock, convulsions, and loss of consciousness [ , ] . in patients using β-blockers, anaphylaxis is often severe and may be resistant to conventional treatment [ ] . various conditions must be considered in the differential diagnosis when suspecting anaphylaxis [ ] : cardiac arrhythmias, acute myocardial infarction, food aspiration, convulsive disease, reaction to insulin, pulmonary embolism, syndrome etiology (e.g., the presence of carcinoid tumors or reaction to alcohol and chlorpropamide), hysterical behavior, vasovagal reactions, and fictitious allergic reactions. vasovagal reactions are most often confused with anaphylaxis [ ] . in general they are consequences of procedures such as injections, which present as a clinical condition consisting of facial paleness, nausea, profuse sweating, and syncope, with symptoms improving without treatment to min later [ ] . absence of pruritus in the presence of a slow pulse and normal blood pressure distinguish vasovagal reactions from anaphylaxis [ ] . the treatment of anaphylaxis consists of short-and long-term measures [ ] . the immediate goal is to maintain the permeability of the airways and blood pressure, in addition to administering oxygen in more severe cases [ ] . epinephrine must be administered as soon as possible, with a standard dose of . mg/kg of a : , solution, up to a maximum of . - . ml, subcutaneously every - min until the patient is stabilized. this is a condition characterized by a state of generalized erythema and scaling (exfoliative dermatitis) of the skin. it has the morphologic appearance of various cutaneous diseases such as psoriasis, atopic dermatitis, t-cell cutaneous lymphoma, and reactions to drugs [ ] . the dissemination of a maculopapular condition caused by medication may lead to the emergence of an erythrodermic syndrome. various types of drug-induced cutaneous reactions (including contact dermatitis, photosensitivity, and maculopapulous reactions) would be responsible for roughly . % of erythroderma cases [ ] .the secondary drug-induced erythroderma conditions, as opposed to erythrodermas resulting from other etiologies, most often set in quickly and also tend to regress quickly after withdrawal of the medication being used [ ] . pruritus arises - weeks after starting drug use, in association with diffuse erythema covering roughly % of the body surface, followed by lymphadenopathy and scaling. when acute, large amounts of epidermis are exfoliated; when chronic, it produces small elements [ ] ( fig. . ). pruritus and a sensation of diffuse burning occur [ ] . exfoliative dermatitis leads to systemic complications such as hydroelectrolytic and thermoregulatory disturbances, high cardiac insufficiency, tachycardia, capillary leak syndrome, and infection [ ] [ ] [ ] . the effect of exfoliative dermatitis on the organism depends on the intensity and duration of the process [ ] . common laboratory findings in the erythrodermic state include light anemia, leukocytosis with eosinophil, high ige, an increase of the hemosedimentation process, a drop in serum albumin, and a rise in uric acid [ , ] . increased ige and eosinophil is a nonspecific finding and is found only in secondary drug-induced erythrodermas, although it might also be due to atopic dermatitis [ , ] . multiple cutaneous biopsies performed simultaneously on distinct points of the skin might increase the accuracy of the diagnosis of the base disease [ ] . in drug reactions vacuolar alterations may be observed on the epidermis, as well as necrotic keratinocytes [ ] . the initial treatment of drug reaction in an erythrodermic patient is identical to that for erythrodermas of other causes [ , ] . suspending the drug is the quickest way to improve the patient's condition. one ought to consider the nutritional state and hydroelectrolytic replacement, as well as administering local measures such as antiseptic baths, humid compresses on the crusts, application of soft emollients, and low-strength corticosteroids [ ] . classic oral antihistamines may be prescribed to alleviate the pruritus and anxiety. they provide the patient with a warm and humid environment so as to prevent hypothermia and improve cutaneous hydration [ , ] . symptoms and signs of cardiac and respiratory insufficiency may require emergency assistance and hospitalization [ ] . the most aggressive and debilitating erythrodermic states may require care similar to that offered to sjs or ten patients. the differential diagnosis must be performed with other types of secondary erythrodermas to cutaneous diseases, such as psoriasis, contact dermatitis, seborrheic dermatitis, lichen planus, bullous pemphigoid, and pemphigus foliaceus, as well as systemic diseases such as leukemias, t-cell cutaneous lymphoma, and hodgkin's lymphoma, in addition to erythrodermic states secondary to internal cancer [ , ] . what currently exists is a combination of concepts according to which spectrum of erythema multiforme (em), including em minor and em major (emm), is separated from another spectrum of reactions, which includes sjs and ten (lyell's syndrome), referred to here as the sjs/ ten spectrum [ ] [ ] [ ] [ ] . however, according to assier et al. [ ] , it seems possible to separate emm patients from true sjs patients based on clinical symptoms and disease origin. these authors define the emm pattern as consisting of characteristic mucous erosions and cutaneous lesions (typical targets, with or without blisters), symmetrically distributed and commonly acral. sjs would be represented by mucous erosions and disseminated cutaneous purpuric macules that are frequently confluent, with a positive nikolsky sign and epidermal scaling limited to less than % of the body surface [ , ] . em would include recurrent, postinfectious cases (especially related to herpes simplex and mycoplasma), or eventually related to exposure to medication, with a low mortality rate and without lethality. on the other hand, sjs would comprise a severe adr with high mortality rates and a reserved prognosis for many cases [ , , ] . in , bastuji-garin et al. [ ] put forward a clinical classification of the spectrum that included me bullosa up to ten. to better understand this classification [ ] , we note the characteristics of the dermatologic lesions of which the group consists, defined as follows: • epidermal detachment: refers to epidermal loss, which at times occurs in flaps ( fig. . ). • typical targets: lesions less than cm in diameter, in disc shape, with well-defined borders, and exhibiting at least three distinct zones, namely two concentric halos around a central disc ( fig. . ). • atypical flat targets: lesions that are not raised, but are round or disc shaped, with two zones and/or borders that are not well defined. • atypical raised targets: round or disc-shaped lesions, palpable or raised, but without the two zones and/or well-defined borders. • macules/spots: erythematous or purpuric stains, irregularly shaped or confluent, with or without blisters (fig. . ). insofar as the area of epidermal necrolysis makes up one of the two main factors of prognosis, a consensus was reached on classifying the spectrum [ , ] : (i) sjs in cases with mucous erosions and disseminated purpuric macules and scaling of the epidermis below %; (ii) sjs/ten superposition or transition in cases with epidermal scaling between % and % of the body surface; and (iii) ten in cases with disseminated purpuric macules and epidermal scaling above %; or (iv) in rare cases with disseminated necrolysis (over % scaling) without any of the lesions described above. sjs is an entity characterized by the presence of lesions similar to those of em, but with purpuric macula and widely distributed blisters or even lesions in atypical targets dispersed over the dorsal aspect of the hands, palms, soles of the feet, extensor region of the extremities, neck, face, ears, and perineum; the face ( fig. . ) and trunk ( fig. . ) are prominently involved [ ] . incidence of sjs is estimated at roughly one in three cases per million residents yearly [ ] [ ] [ ] . sjs may be preceded by a discrete maculopapulous eruption similar to exanthema morbilliform [ ] . blister formations are possible, though usually not determined by an epidermal detachment of over % of the body surface [ , , ] . mucous involvement occurs in roughly % of cases, generally on two distinct mucous surfaces; this may precede or follow cutaneous involvement [ , , ] . onset begins with enanthema and edema, which give rise to erosions and pseudomembranous formations on the eyes, mouth, genitals, pharynx, and upper airways [ ] . some - % of cases occur with fever and lesions in the gastrointestinal and respiratory tracts [ ] . its prognosis seems to not be affected by the type and dose of the drug responsible, nor by human immunodeficiency virus (hiv) infection [ ] . the therapeutic options for sjs are limited and controversial [ , , ] . corticosteroids are frequently used [ ] , although some cases have not shown a satisfactory response [ ] . in agreement with most authors, the use of systemic corticosteroids on the initial sjs and ten forms do not currently demonstrate any proven benefits. the advanced forms of this spectrum of relations have clearly deleterious effects on the patient [ ] . the treatment and prognosis of sjs are tackled in combination with that of ten. ten is an entity characterized by extensive scaling of the epidermis in the wake of necrosis (epidermal necrosis) [ , , ] . the term "toxic epidermal necrosis" was introduced by lyell in [ ] . fortunately, it consists of a very rare adverse reaction to drugs. in europe, its incidence is estimated to be at . [ ] cases per million residents yearly [ ] . in aids patients, however, the risk of this reaction does rise, estimated at one case in every , patients yearly [ ] . in general, there is a slight predominance among women ( . - cases in females for every male case). indeed, the disease's occurrence in aids patients ends up balancing out the incidence rate between the sexes [ ] . the initial characteristics of ten are nonspecific influenza-like symptoms, such as fever, sore throat, coughing, and burning eyes. these are considered prodromic manifestations preceding a cutaneous and mucous affection by - days [ ] . an erythematous eruption emerges symmetrically on the face ( fig. . ) and the upper part of the trunk, extending to the craniocaudal region and provoking symptoms of burning or painful skin [ , ] . the individual cutaneous lesions are, for the most part, characterized by erythematous macules with poorly defined contours and a purple center. they progressively spread over the anterior thorax and back [ , ] . less commonly, the initial eruption may consist of an extended scarlatiniform exanthema. in roughly - days or, at times, within a few hours, or more seldom in about a week, complete extension of the cutaneous condition occurs [ ] . at first, some cases may present lesions persisting in sun-exposed areas of the skin [ ] . the apex of the process consists of characteristic denuding of the necrotic epidermis, standing out as veritable red strips or flaps on the areas affected by the base erythema ( fig. . ) [ , ] . the epidermis is raised by the serum content of flaccid blisters, which are progressively confluent and provoke rupture of the blisters and detachment of the skin. this causes an aspect of severe burns on the patient's skin, with the skin denuded, bleeding, and with an erythematouspurple color, as well as continued elimination of serosity, which contributes to hydroelectrolytic unbalance and accentuated protein loss [ , ] . the nikolsky sign is positive over widespread areas of the skin [ , ] . the areas of the skin subjected to pressure, such as the lower shoulders, back, and buttocks, are the first to release epidermal flaps [ , ] . cutaneous extensor affection might determine a state of acute cutaneous failure [ , ] . the cutaneous surface can virtually be % affected, although scalp affection is exceptional [ ] . the mucous membranes are affected in - % of patients, commonly preceding skin involvement by a day or two [ ] . in the order of frequency, the disease afflicts the oropharynx, eyes, genitalia, and anus [ ] . extensive and painful erosions lead to labial crusts, salivation, feeding obstruction, photophobia, and painful urination and evacuation [ ] . severe eye sequelae, with the formation of synechiae between the eyelids and conjunctiva by pseudomembranous conjunctival erosions, and blindness may occur [ , ] . ceratitis and corneal erosions have been reported, as well as a secondary sicca-like syndrome [ ] . high fever or hypothermia may occur because of a thermoregulatory imbalance until complete healing, even in the absence of concomitant infections [ ] . the abrupt drop in temperature is more indicative of sepsis than of fever itself [ ] . psychomotor agitation and mental confusion are not uncommon, and usually indicative of hemodynamic complications and sepsis [ ] . many internal organs are affected by the same pathologic process that involves the skin and determines a spectrum of systemic manifestations [ , ] . systemic involvement occurs, causing erosion in the esophagus and gastrointestinal tract, which may progress to esophageal constrictions, transaminase increases in % of cases (hepatitis in %), pseudomembranous colitis, and pancreatitis [ ] . in the respiratory tract tracheobronchial erosions and secondary pulmonary interstitial edema, with the correction of hypovolemia, can be found [ ] . anemia can be constantly observed, as well as lymphopenia in up to % of patients [ ] . thrombocytopenia is found in % of patients; neutropenia occurs in % of cases, and when present indicates a worse prognosis [ , ] . the medications most commonly causing ten are sulfas, phenobarbital, carbamazepine, dipyrone, piroxicam, phenylbutazone, aminopenicillin, allopurinol, and nevirapine. however, it is necessary to consider that new drugs are continually being reported as triggering ten [ , , , ] . the exact mechanism by which sjs and ten develop is not well defined. some authors have suggested the participation of the altered metabolism of drugs with the predominance of a slow acetylator genotype in sjs and ten patients, and a deficiency in the mechanisms involved in detoxification of reactive intermediary metabolites [ ] [ ] [ ] . in addition to the metabolic mechanisms, there is evidence to suggest that, especially in ten, the epidermal necrosis is mediated immunologically [ , , ] . it is known today that sjs and ten are disturbances mediated by t cells, similarly to acute graft-versus-host disease (gvhd), with cytotoxic t cells being responsible for the epidermal necrosis through an apoptosis in keratinocytes [ , ] . posadas et al. [ ] have shown the association of high tumor necrosis factor α (tnf-α) levels with the severity of the reaction. this cytokine has been related to an induction in the adhesion and activation of t cells and monocytes. it also participates in apoptosis, irrespective of the action of perforins [ ] . it has been demonstrated also that apart from tnf-α, the perforins granzyme b (grb) and fas ligand (fasl) are found to be high in the initial stages of a drug reaction, particularly in sjs and ten. this reinforces the hypothesis of the participation of cytotoxic mechanisms [ ] . nowadays, cytotoxic reaction caused by granulosin liberation from t cells is the major mediator involved in cell apoptosis, and the spectrum of sjs/ten is grouped in type ivc of immune reactions, as classified by pichler. correia et al. [ ] have observed a similar seric cytokine profile between ten and acute gvhd. these authors showed a significantly high serum level of interleukin (il)- and il- in ten and acute gvhd patients as opposed to normal blood donors [ ] . il- is a multifunctional proinflammatory cytokine produced by various cells, including keratinocytes. it consists of a main circulating endogenous pyrogen [ ] . this explains the presence of fever that is unrelated to the infection in the first days of ten and gvhd [ ] . in turn, il- is an endogenous antipyrogen agent. it is produced by keratinocytes with the purpose of blocking inflammatory cytokines such as il- , il- , and tnf-α, in addition to being a powerful suppressant of macrophage, t-cell, and natural killer cell functions [ ] . by contrast, as il- recruits cd + lymphocytes from the peripheral blood, its increase in blister fluid explains the high number of these cells in patients' epidermis [ ] .the elevation of il- creates a natural mechanism against excessive tissue inflammatory reaction [ ] . chosidow et al. [ ] have suggested that the cellular cytotoxic targets are viral antigens with a potential to alter immune responses resulting from exposure to medications. treatment for sjs and ten patients is similar to that for patients who have suffered extensive burns, with a number of rare exceptions [ ] . all patients have to submit to cutaneous biopsy to confirm the diagnosis [ ] . the patient must be observed in an intensive therapy unit, in an isolated and heated environment so as to avoid any cutaneous trauma [ , , ] . the treatment must proceed by suspending any drug that is not essential to the patient's life and begin replacement of intravenous fluid, mainly when an oral mucous lesion obstructs liquids from being ingested [ , , ] . isolation and feeding must be carried out through the nasogastric probe because the patient shows calorie and protein loss [ , , ] . corticosteroids should only be administered within h of the condition's onset. it has not proved to be beneficial after this period because of its delaying epithelialization and increasing protein catabolism, in addition to increasing the risk of infection [ , ] . antibiotic therapy has to be administered to cases whereby a sudden drop in temperature occurs and with a concomitant drop in the general status or increase of cultivated bacteria on the skin with a predominance of a single strain [ , ] . it must be emphasized that during the first days, the most common infections are by staphylococcus aureus and later by gramnegatives (pseudomonas aeruginosa) or candida albicans [ ] . noncontrolled reports and studies on the treatment of ten exist, reporting the use of intravenous immunoglobulin, cyclosporine, cyclophosphamide, plasmapheresis, and anticytokine monoclonal antibodies, among others, in an attempt to curb the process of epidermal necrosis. the value of these studies has been questioned, however, particularly owing to the fact that in most patients who are hospitalized the phenomenon of necrosis virtually comes to a halt [ ] . prins et al. [ ] published a multicenter, retrospective study on intravenous immunoglobulin use in treating ten patients, which obtained excellent results. a -patient cohort, average age years (± ) and consisting of women and men, with a - % variation of epidermal detachment of the total body surface area, was treated. mucous membrane was affected in . % of these patients. the patients received intravenous infusion of gammaglobulins begun on average days after onset of ten (with a variation of - days). it was administered over a period of - days, in doses varying from . to . g/kg (mean total dose of . g/kg). an objective positive response to treatment occurred with a break in the progression of ten, observed in ( %) of the patients. in all there were six deaths. the authors concluded that early use of intravenous gammaglobulin is safe, with a recommended dose of g/kg daily for days in a row. in contrast to the studies of prins et al. [ ] , a french group (bachot, revuz, and roujeau) led a noncomparative prospective study of patients diagnosed with sjs (nine patients), sjs/ten overlapping (five patients), and ten ( patients). they concluded that intravenous gammaglobulin in a -g/kg daily dose, administered for days in a row, did not reduce patient mortality [ ] . until such discrepancies in the results have been cleared up, intravenous gammaglobulin use in treating ten will remain controversial [ ] . however, as the volume of data encourages its application and effective alternative therapies remain lacking, it seems difficult to not suggest a high dose of intravenous gammaglobulin, especially as a way of intervening early in quickly progressing ten cases. whereas mortality rate is low for emm (< %) and sjs (roughly %), it is above % for ten patients with macules [ ] . the mortality rate rises with age range and increased surface area of the epidermal scaling [ ] . we reiterate the classification methodology adopted by multicenter studies, prospectively named scard (severe cutaneous adverse reactions). the results of the latter were recently published based on the analysis of patients and , controls [ ] . this classification system (named scorten) is summarized in chart . . despite the large range and amount of drugs that may pose a great risk of contracting sjs and ten, an annual risk rate of five cases per year among medication users has not been exceeded [ , ] . [ ] [ ] [ ] dress syndrome is an acronym derived from the term "drug rash with eosinophilia and systemic symptoms" coined by bocquet et al. also known as drug-induced hypersensitivity syndrome (dihs), it was first recognized in by chaiken in a patient using an anticonvulsant. there are many synonyms used, most of them referring to the origin of the drugs involved in the drug reaction, such as dapsone syndrome, allopurinol hypersensitivity syndrome or the anticonvulsant hypersensitivity syndrome. although a dermatosis is usual in dress, the extent of skin involvement is variable and therefore the "r" in dress was subsequently changed from "rash" to "reaction." clinically, in its complete form, this syndrome includes an extensive mucocutaneous rash, fever, lymphadenopathy, hepatitis, and hematologic abnormalities with eosinophilia and atypical lymphocytes, and may involve other organs with eosinophilic infiltration, producing damage in several systems, especially in kidney, heart, lungs, and pancreas. this multivisceral involvement differentiates dress from other common skin reactions to drugs. another unique feature of this syndrome is its late onset in relation to the period of introduction of the causative drug, i.e., at around weeks to months, and its possible persistence or worsening despite the withdrawal of the offending drug. the incidence of this syndrome is estimated to vary from one case among , - , drug exposures. adults are more affected than children, and although the precise incidence of drug reaction has not yet been determined, it is much more common than sjs, which has an incidence of . - cases per million person-years, and most cases are sporadic, with no gender predilection. recognition of this syndrome is of paramount importance, since the mortality rate is about - % and a specific therapy may be necessary. the exact mechanism of dress/dihs remains to be determined but, in cases related to anticonvulsant drugs, three components are considered: (i) deficiency or abnormality of the epoxide hydroxylase enzyme that detoxifies the metabolites of aromatic amine anticonvulsants (metabolic pathway); (ii) associated sequential reactivation of herpesvirus family; and (iii) ethnic predisposition with certain human leukocyte antigen (hla) alleles (immune response). this type of reaction is most commonly seen using seven different drug groups: (i) anticonvulsants, such as the aromatic anticonvulsants (phenytoin, carbamazepine, phenobarbital, primidone), mexiletine, lamotrigine, valproate, ethosuximide, and zonisamide; (ii) antidepressants (desipramine, amitriptyline, fluoxetine); (iii) sulfonamides and sulfones (dapsone, sulfasalazine, trimethoprim-sulfamethoxazole, salazosulfopyridine); (iv) anti-inflammatory drugs (piroxicam, naproxen, diclofenac, sundilac, phenylbutazone, ibuprofen); (v) anti-infectives (abacavir, cidofovir, terbinafine, nevirapine, minocycline, linezolid, doxycycline, telaprevir, nitrofurantoin, zalcitabine, spiramycin, metronidazole, piperacillin-tazobactam, ceftriaxone); (vi) angiotensin-converting enzyme inhibitors (captopril, enalapril); and (vii) β-blockers (atenolol, celiprolol). cases have been reported with allopurinol, gold salts, thalidomide, calcium channel blockers (diltiazem), ranitidine, sorbinil, azathioprine, dobutamine, methimazole, propylthiouracil, and efamizulab. the cases more consistent with dress/dihs were caused by aromatic anticonvulsants, dapsone, salazosulfopyridine, allopurinol, and minocycline. other drugs causing less typical cases are reported in the literature, but less frequently. aromatic anticonvulsants have an estimated occurrence of dress/dihs of one case for every , people exposed to the drug, and the reaction is especially common among black patients. the aromatic anticonvulsant drugs that have been associated most frequently with drss/dihs are phenytoin, phenobarbital, and carbamazepine. however, newer anticonvulsant medications also containing aromatic structure (felbamate, oxcarbazepine, zonisamide, and lamotrigine) can also be involved, and the cross-reactivity between the various aromatic anticonvulsant drugs is well documented, varying between % and %. the pathogenic mechanism of idiosyncratic reactions to drugs, such as dress/dihs, has not been fully elucidated. sullivan and shear proposed a multifactorial model for the pathogenesis of dress/dihs. its occurrence would be determined by the combination of exposure to a drug capable of causing adverse reaction given in sufficient dosage and period of use to a susceptible patient. a certain group of drugs associated with dress/dihs, including the aromatic anticonvulsants, is metabolized to reactive oxygen intermediates that appear to be inefficiently detoxified in patients with acquired or pharmacogenetic variations in the metabolism of these drugs. aromatic anticonvulsants such as carbamazepine, phenytoin, and phenobarbital are metabolized by the hepatic cytochrome p enzymes and undergo oxidation by aromatic hydroxylation, with subsequent formation of arene oxides. arene oxides are toxic reactive intermediates that are normally enzymatically converted to nontoxic metabolites by epoxide hydroxylase or glutathione transferase. in addition, spontaneous conversion to nontoxic phenol derivatives can occur. in cases of defective or deficient epoxide hydroxylase, arene oxides can accumulate and cause direct cellular toxicity or immune response ( fig. . ). drug interactions can be important in this syndrome. concomitant use of lamotrigine and valproic acid increases the occurrence of the syndrome. it is thought that the mechanism for this drug interaction is the competition between valproic acid and lamotrigine for hepatic metabolism by glucuronidation, which doubles the halflife of lamotrigine and predictably would increase the possibility of adverse effects. positive patch tests and testing of blast transformation of lymphocytes indicate the presence of an immune reaction in which t cells participate in specific core function. clones of drugspecific t cells have been isolated from patients sensitive to carbamazepine and lamotrigine. several clinical similarities that could be observed between dress/dihs and infectious mononucleosis (im) have led researchers to implicate a possible range of viruses as triggers for this syndrome. in addition, unique features of this syndrome are its late onset in relation to the period of introduction of the causative medication and frequent clinical and laboratory deterioration, as well as episodes of exacerbation despite the withdrawal of the offending drug, so that these characteristics are not necessarily typical of a reaction of specific drug etiology. although there are conflicting views on the pathogenesis of dress/dihs in different parts of the world, recent studies have suggested a close relationship between human herpesvirus (hhv- ) and the development of dress/dihs. sporadic reports have shown that not only hhv- , but also other herpesviruses such as hhv- , epstein-barr virus (ebv), and cytomegalovirus (cmv), can be reactivated during the course of the dress/dihs. results obtained with analysis by polymerase chain reaction showed that various herpesviruses are sequentially reactivated during the course of dress/dihs in most patients. the cascade of viral reactivation is initiated by ebv or hhv- and extends over a period to hhv- and eventually to cmv [ ] . in some patients, the clinical manifestations of this syndrome persist despite discontinuation of the drug involved, coinciding with the reactivation of herpesvirus, as shown in fig. . . the reactivation of hhv- is evidenced by increases in the titers of igg anti-hhv- dna levels, and hhv- is commonly found in the second or third week after the onset of rash, despite the high variability of clinical manifestations among patients with this drug reaction. since the reactivation of hhv- can be detected only in patients with dress/dihs, but not other adrs, in japan this diagnostic test has become sensitive and specific for the diagnosis of all patients with dress/dihs [ ] . the detection of hhv- reactivation seems to be the gold-standard diagnostic test for dress/dihs in japan, with other asian countries and europe helping to confirm the identification of this condition. however, it is still unknown how detection of the viral genome in peripheral blood reflects the true status of viral reactivation in progress in many different organs and systems. specifically, it is possible that in different compartments and organs such as spleen and lymph nodes, different herpesviruses can reactivate in sequential order completely independent of what occurs in the blood, which would explain why blood samples negative for the viral genome are obtained during the clinical activity of dress/dihs. what remains unclear is the role of herpesvirus in early dress/dihs. there are two possibilities: (i) dress/dihs began as an "allergic" immune reaction to a particular drug, which seems to possess an innate ability to stimulate t cells. ( ) in genetically predisposed individuals or by additional factors, an impaired detoxification and accumulation of these metabolites occur ( ), which can cause cellular damage generating danger signs that can stimulate resting t cells, inducing costimulatory pathways ( ). in addition, ethnic predisposition to certain hla types may contribute to the formation of neoantigens from the combination of these intermediary reactive metabolites with tissue macromolecules and formation of haptens ( a), which can be presented via the human histocompatibility complex class i (hla-dr) or class ii (hla-a, -b or -c), to cd or cd t cells ( ) . it was demonstrated that carbamazepine, valproic acid, and amoxicillin are able to exert immunomodulatory actions by inhibiting histone decarboxylase on b lymphocytes, producing a hypogammaglobulinemia that precedes the clinical onset of dress/dihs. the clonal expansion of t cells requires sequential reactivation of latent herpesvirus, and at the same time cd + cla + t cells are produced, which are directed toward skin, cd + ccr + t cells addressed to the lungs ( b), and cd + il- , il- producer and il- cd th + producer that cause tissue and peripheral eosinophilia in the context of t-cell activation is a massive activation of herpesvirus housed in these cells, since the stimulation of t cells by the drug may reactivate the viral genome into the cell. thus, the drug in turn can activate a specific cellular and humoral immune response to herpesvirus. this could explain why different herpesviruses are activated and because in another intense immune process, so-called gvhd, a similar reactivation can be observed. (ii) the viral reactivation can occur but is initially clinically unapparent. however, t cells stimulated by virus present significant crossreactivity with certain drugs, and exposure to these drugs leads to an expansion of t cells specific to the drug (and viruses), which persists even after drug withdrawal due to persistence of viral antigens. the simultaneous appearance of multiple concurrent viral reactivation could be explained by the ability of hhv- and hhv- counterparts to reactivate virus. thus, if the symptoms of dress/ dihs are mediated by both the various gene products and herpesvirus immune responses to viral replication, the frequent deterioration or the several exacerbations that occur despite withdrawal of the offending drug could derive, at least in part, from the sequential activation of this herpesvirus. the viral reactivation may provide a "danger signal" (danger sign) that stimulates massive clonal expansion of both cd + and cd + nonspecific t cells and causes the complete development of the syndrome. shiohara et al. proposed the possibility that the clinical symptoms during the course of evolutionary dress/dihs do not seem to be only mediated by oligoclonal expansion of drug-specific t cells, but also by antiviral t cells that cross-react with drugs. how is hhv- acquired? hhv- infects almost all humans around years of age. most infections arise through the exchange of infected saliva during the first year of life, although perinatal transmission can occur. it was demonstrated that the dna of hhv- can be integrated into the host dna, and once part of the human dna, congenital transmission can occur [ ] . this was also demonstrated in the course of the dress/dihs. the temporal relationship between onset of drug use and the onset of dress/dihs ( weeks to months) suggests that viruses have no primary function in the syndrome, favoring primary pathogenesis related to drug allergy. patients with dress/dihs have decreased total serum igg, iga, igm, and b-lymphocyte count at onset, while there is an expansion of memory t cells that cross-react with both drug and virus. it is noteworthy that the lymphocyte transformation test is negative in the first week of illness and remains negative in % of patients weeks after the onset of symptoms, becoming positive only - weeks after the initial drug reaction. this could be due to the expansion of regulatory t cells (which suppress the proliferation of memory t cells) in the early stages of the disease and its subsequent reduction by apoptosis. several cytokines are increased during dress/dihs. in particular, levels of tnf-α and il- , which are typically proinflammatory cytokines, are elevated in this syndrome before the reactivation of hhv- . interestingly, il- becomes undetectable during viral replication and increases again after the infection in most patients. dress/dihs is an entity distinct from other serious adrs because of the dynamic changes in the immune response observed during the course of the disease. the phenotype of circulating cd + t cells is changed to cd + phenotype at the time of viral reactivation. regulatory t cells are initially increased in number in the circulation and skin, but decrease in parallel the function of the different organs or systems. the reactivation of hhv- is considered a condition requiring immunosuppression, demonstrated on several immune abnormalities in the early syndrome: marked decrease of serum immunoglobulins, the number of circulating b cells, and regulatory t-cell dysfunction. moreover, the participation of skin inflammation may be involved in the induction of immunosuppressive conditions. sugita et al. demonstrated a reduction in the number of plasmacytoid dendritic cells (pdc) in peripheral blood of patients, but an increase in the expression of these cells in skin affected by the rash. the pdc human leukocyte subtypes are capable of producing large amounts of interferon-α (ifn-α), which induces the maturation of b cells in order to produce igg and plays a critical role in antiviral defense. the pdc from circulation may accumulate in the skin and thus reduce the number of pdc in the circulation. therefore, antiviral responses may be reduced, facilitating viral reactivation in peripheral blood and tissues other than the skin. although the terms dress and dihs are often and mistakenly used interchangeably, there is currently a tendency to believe that the dihs represents the most severe cases of dress, with reactivation of hhv- detected in a large majority of patients and only in a limited number of patients with dress [ ] . the most popular hypothesis to explain the immunoallergic reactions to drugs is the theory of hapten/pro-hapten: according to this hypothesis, the drug (or metabolite) is processed by antigen-presenting cells (apcs) and expressed in the cell membrane in the context of hla-a, -b, or -c type i (mhci) or hla-d type ii (mhcii). the complex hla drug (hapten) is presented to native t cells (naive) via their t-cell receptor (tcr), which initiates different types of immune responses, depending on the hla expressed on the apc and the cytokine environment. the story of "hla-drug" correlation truly began in the twenty-first century with abacavir. in , two independent groups observed the abacavir hypersensitivity syndrome and that this was restricted to the allele hla-b* , which conferred an elevated odds ratio (> ). glaxosmithkline (london, uk) led the largest international randomized pharmacogenetic clinical trial to date, which demonstrated the correlation between abacavir hypersensitivity reactions and patients with this allele, and proved that the exclusion of abacavir introduction to the patients with this allele resulted in the disappearance of the syndrome, which was first seen in % of patients overall who received the drug during the first weeks of antiretroviral treatment. this allele test is now routinely used before the introduction of abacavir in several countries. the hla alleles have a high negative predictive value but low positive predictive value in relation to adrs, indicating that these biogenetic markers are necessary but not sufficient to trigger the allergic immune reactions. according to the theory of hla-drug (hapten), the complex hapten only triggers an immune-allergic reaction in the presence of a specific hla allele. thus, prospective hla screening should prevent some patients from having serious idiosyncratic reactions such as dress/dihs, sjs, and ten if they have a specific risk allele by not receiving the drug related to it. hla pharmacogenomics is a recent field of study that has been rapidly developed and implemented into clinical practice and has improved drug prescription, which is likely to become more and more important in coming years. besides causing sjs and ten, carbamazepine also induces other types of adrs, including maculopapular exanthema (mpe) and dress/ dihs. the association between hla-b* and carbamazepine-induced mpe was not detected in populations of ethnic han chinese and hong kong or thai populations. studies involving han chinese residents in taiwan and caucasians showed no association between cases of dress/dihs caused by carbamazepine and hla-b* . these data indicate that the association between hla-b* and cutaneous adrs induced by carbamazepine are specific to sjs/ten. kano et al. showed that in four of their japanese patients ( . %) with dress/dihs in whom reactivation of hhv- was proved, the syndrome was triggered by aromatic anticonvulsants (carbamazepine in ten, phenobarbital in two, and phenytoin in one) had hla-b* . the frequency of this allele was much higher than in the japanese population ( . %). although this difference was not statistically significant after correction for multiple comparisons, the authors proposed that the presence of certain alleles of hla-b on the reactivation of the virus contributed, at least in part, to the association of hla-b allele with dress/dihs. kashiwagi et al. demonstrated a significant association between adverse skin reactions to carbamazepine and hla-a* among japanese patients, including erythema multiforme, erythroderma, dress/dihs, ssj, and other drug reactions. eleven of these patients ( %), including two patients with sjs and others, were carriers of hla-a* and allele frequency was much higher in these patients ( %) than in the japanese population ( . %) (p = × - , odds ratio (or) = . ). in a case-control study in a han-chinese population a strong association between the presence of hla-b* and sjs/ten, or dress/dihs triggered by allopurinol among patients ( %) was found, compared with out of ( %) allopurinol-tolerant patients and out of controls ( %) (p (pc value . × (- ), or = ). japanese patients with different clinical types of cutaneous adrs caused by allopurinol, including sjs, ten, and dress/dihs, had the same hla-b* allele. pirmohamed et al. found an increased frequency of hla-dr and hla-dq in a group of patients with carbamazepine-induced dress/ dihs (respectively p = . , or = . ; p = . , or = . ). it was demonstrated that activation of cd + t cells with il- is essential for the spread of hhv- in vitro. genotyping of patients revealed that they had positive hla-dr (drb * ) and hla-dq (dqb * ). thus, in recent years increased attention has been given to genetic factors as a cause of variation in both the interpersonal effectiveness and adverse effects of medicines. idiosyncratic reactions are often mediated through immune, usually severe, and unpredictable course. the main region of human dna with genetic variations that predispose to drug hypersensitivity reactions is the region hla. this region harbors the gene locus of most diseases and contains many genes associated with immune functions. although strong associations have been demonstrated between certain hla alleles and some types of adverse skin reaction to drugs, there is no definitive evidence or published data concerning the functions involved in these alleles. the activation of t cells restricted to hla is necessary for the induction of immune reactions and, moreover, there is the possibility that some hla proteins have high binding affinity combined with other drugs or a metabolite of the drug through covalent and noncovalent mechanisms. on the other hand, a protective effect of hla has also been suggested. alfirevic et al. reported a potential protective effect of hla-b* against severe adverse skin reactions induced by carbamazepine in caucasian patients. the implications of pharmacogenomics are varied; one example is the recommendation of the us food and drug administration (fda), which currently recommends genetic testing for users of more than ten drugs currently marketed in that country. histopathology of the skin shows a diffuse, dense superficial and/or perivascular lymphocytic infiltrate. eosinophils in the dermis or swelling may or may not be present ( fig. . a ). on some occasions there is a band-like infiltrate with atypical lymphocytes simulating epidermotropism such as mycosis fungoides. fernando et al. described a patient with dress/dihs triggered by carbamazepine whose rash biopsy presented an unusual form of superficial perivascular inflammatory infiltrate, in which tiny granulomas along with a moderate number of lymphocytes were found. the authors speculated that granuloma formation may be due to a sustained exposure to the drug, even after the onset of dress/dihs. the expansion of cd + t cells producing ifn and other cytokines results in recruitment of macrophages which, as a result of maintained exposure to the drug and persistence of cytokine release, promote differentiation into epithelioid cells, which then secrete tnf to promote fusion of these cells into multinucleated giant cells. thus, biopsies of organs involved in dress/ dihs, such as skin and liver, on a significant number of patients may demonstrate the true frequency of granulomatous infiltration in the disease and assist in understanding the pathogenesis of the reaction. the syndrome usually develops within months after drug introduction, more often in weeks to months of the introduction of the drug, or earlier if constituting readministration. fever, often high ( °- °c), which is the most common symptom (seen in - % of cases), and rash ( % of cases) are the first signs, especially when related to antiepileptic drugs. the cutaneous eruption consists of a morbilliform rash, which is indistinguishable from the rash of other less severe reactions ( fig. . b, c) . the face, upper trunk, and upper extremities are initially affected, with subsequent progression to the lower extremities occurring in about % of cases, which later spreads to the legs and the development of erythrodermic rash. the maculopapular eruption later becomes infiltrated with edematous follicular accentuation. swelling of the face, with marked periorbital involvement, is a warning for the diagnosis, occurring in about % of patients, and can be so intense that the patient becomes disfigured. vesicles may arise, and fine bubbles caused by edema of the dermis can be present. no necrosis of the epidermis such as ten occurs, except in rare cases of overlapping dress/dihs and ten. small sterile perifollicular pustules and nonfollicular pustules may appear, which are different from acute generalized exanthematous pustulosis and do not predominate on the main ridges of the skin. often atypical targets may arise. over time the rash becomes purplish, with sharp definition on lower limbs and the resolution of scaling another form of presentation is a picture of exfoliative dermatitis, which may be associated with mucosal involvement, such as cheilitis, erosions, pharyngitis, and enanthematous enlarged tonsils. bilateral edema and infiltration of the salivary glands with xerostomia has been frequently reported. lymphadenopathy is common ( - % of cases), limited to the lymph nodes or generalized, and painful, gradually resolving with the withdrawal of the drug. the lymph nodes may reveal two distinct types of involvement: a benign pattern of lymphoid hyperplasia with maintenance of normal lymph node architecture, and another a b c various hematologic abnormalities are observed, which consist of marked leukocytosis, eosinophilia ( % of cases), and atypical lymphocytes similar to mononucleosis. these findings guide the diagnosis toward dress, but can sometimes be difficult to distinguish from viral infections such as infection by ebv or hematologic diseases. lymphopenia, leukopenia, or leukocytosis usually precedes it, although they often are not detected because they occur several days before establishment of the clinical syndrome. leukocytosis may be high, up to , leukocytes/mm , and eosinophilia reaches values higher than , /mm . the eosinophilia may determine the involvement of internal organs with pulmonary infiltrates. in general, eosinophilia may be observed about - weeks after the onset of the syndrome, or may even occur after the increase in liver enzymes has normalized. hemophagocytic syndrome (hps) can rarely be observed in the course of dress/dihs. hps is associated with and triggered by various conditions, including viral infections, particularly ebv, malignant tumors, or autoimmune diseases. when involved with the course of dress/dihs, hps usually occurs weeks after the onset of drug eruption. there is a decrease in white blood cells and platelets that are detected simultaneously with elevation of lactate dehydrogenase (ldh). bone marrow aspirate reveals hemophagocytosis in an increased number of macrophages. multiorgan involvement may include a wide variety of organs and systems with myocarditis/ myositis, pericarditis, interstitial nephritis ( % of cases), necrotizing granulomatous vasculitis in kidney, brain involvement (encephalitis or meningitis), colitis, and thyroiditis. this potentially fatal visceral involvement form may be symptomatic or not, and begins - weeks after the onset of rash. we observed a patient who developed acute pancreatitis that evolved into a lethal course. there are reports of shock and respiratory distress syndrome with hypotension, pyrexia, hepatitis, and renal failure related to a hydantoin reaction. arthritis or arthralgia may occur in the context of this syndrome, including myositis. liver involvement is the most common visceral manifestation ( - % of patients) after lymphadenopathy. hepatomegaly may constitute a finding on physical examination. hepatitis with isolated elevation of liver transaminases is common ( % of cases), usually anicteric, but liver failure is a leading contributory factor to mortality. liver biopsy shows central lobular necrosis and dense inflammatory infiltrate of lymphocytes and eosinophils or granulomas. the reaction is accompanied by cholestasis and hepatocyte necrosis. in more severe cases, widespread or focal hepatic necrosis may be present. the presence of an active coinfection with hepatitis viruses b and/or c often determines deterioration in liver function and prolonged liver dysfunction. there are few cases reported in the literature of dress/dihs with severe acute hepatitis (defined by the presence of alanine aminotransferase (alt) to more than × upper limit of normal and/or acute liver failure, such as coagulopathy and encephalopathy), mostly observed in women between the second and fourth decade of life, especially in relation to the use of sulfasalazine. about % result in death or liver transplantation, and the course of the disease is apparently unchanged by the use of immunosuppressants. the rapid recognition of the syndrome and prompt withdrawal of the drug can limit the liver damage, although this may be possibly even worse for several weeks and take months to resolve. renal involvement occurs in about % of cases, being particularly evident in cases arising from the use of allopurinol, whereby there was an increase in serum creatinine and urea and decreased creatinine clearance. in urine tests, increased content of eosinophils can be observed. although pulmonary involvement is rarely reported in dress/dihs, interstitial pneumonia with eosinophilia is often observed among patients whose syndrome was triggered by minocycline. possibly the cases with lower intensity of pulmonary manifestations are less reported, leading to a bias in the published literature. pulmonary complications include acute interstitial pneumonitis, lymphocytic interstitial pneumonia, and adult respiratory distress syndrome (ards). myocarditis may develop at the beginning of the syndrome or up to days after establishment. symptoms include heart failure, chest pain, sudden tachycardia, dyspnea, and hypotension in early dress/dihs, but some patients are asymptomatic. the echocardiogram shows a reduction in ejection fraction, chest x-ray demonstrates cardiomegaly, and the electrocardiogram shows nonspecific changes in the st-t segment. there is an increase in enzymes such as cpk and ck-mb, but no apparent changes in levels of troponin- . neurologic complications include meningitis and encephalitis. meningoencephalitis occurs about - weeks after initiation of drug reaction, and may lead to coma, seizures, headaches, disorders of speech, and paresis and paralysis of the cranial nerve. gastrointestinal bleeding may be an abrupt complication caused by ulcers derived from cmv. endoscopic examination reveals arterial bleeding from punched-out gastric ulcerations. kennebeck compiled the frequency of clinical manifestations and laboratory data of the anticonvulsant hypersensitivity syndrome: fever ( - %), cutaneous eruption ( - %), lymphadenopathy ( %), hepatitis ( - %), hematologic abnormalities ( - %), periorbital and orofacial edema ( %), myalgia and arthritis ( %), nephritis ( %), pharyngitis ( %), and pulmonary manifestation ( %). the visceral involvement in acute dress/ dihs until resolution of clinical disease is, therefore, extensive and varied, some of these events being closely related to hhv reactivation: enterocolitis and intestinal bleeding, hemophagocytic syndrome (hps), hepatitis, limbic encephalitis, myocarditis, nephritis, mumps, pneumonia, pleurisy, and the syndrome of inappropriate antidiuretic hormone (siadh). the exclusion of other serious infections, particularly bacteremia, neoplastic diseases (lymphoma, leukemia, hypereosinophilic syndrome, paraneoplastic syndrome), and autoimmune or connective tissue conditions (adult-onset still's disease, lupus erythematosus, vasculitis) is necessary for an accurate diagnosis of dress/ dihs. complications are rare and include limbic encephalitis, thyroid disease, renal failure, splenic rupture, eosinophilic colitis, eosinophilic esophagitis, enterocolitis, and fatal cmv. the mortality rate can reach %, especially in cases related to advanced age, renal impairment, jaundice, and hepatitis with reactivation of cmv. by contrast, cases where there is a reactivation of ebv seem to have a less severe course, but are more likely to later (usually after several years) develop autoimmune diseases such as diabetes mellitus type and autoimmune hypothyroidism. several authors have reported the occurrence of autoimmune diseases and/or the production of autoantibodies after the resolution of dress/ dihs, in a period ranging from several months or years after the resolution of the syndrome, and some are similar to those seen after bone marrow transplant. the related conditions include diabetes mellitus type , lupus erythematosus, hashimoto's thyroiditis, enteropathy, sclerodermiform lesions, gvhd, and bullous pemphigoid. the diagnosis is difficult since there are incomplete or less characteristic clinical features, for example, hepatitis without rash, or merely pulmonary infiltrate with eosinophilia. bocquet, bagot, and roujeau were the first authors who proposed criteria for dress diagnosis. according to these authors the diagnosis is established if there are at least three criteria present: there is still no international consensus on the best criteria for the definition of dress/dihs diagnosis. bocquet et al. and southeimer and houpt have proposed different definitions and nosology for dress/dihs in order to clarify clinical and pathologic characteristics of this syndrome. the japanese study group for severe cutaneous adverse reactions to drugs (scar-j) has adopted other criteria, as presented on chart . . however, the universal adoption of these criteria may be impaired, because one of the criteria is viral replication during the course of infection, and some tests, such as measurement of igg titer anti-hhv- , are not yet routinely available in all hospitals or laboratories. in our view, the criteria adopted by the european group regiscar, published by kardaun et al. in , is the best to meet the needs in the diagnosis of dress/dihs. here the use of a system score for the diagnosis of dress/ dihs was suggested, based on the presence of symptoms and clinical and laboratory signs, as displayed in table . . given the suspicion of the syndrome relevant examinations should be performed, keeping in mind that this syndrome has evolutionary behavior. the initial tests are oriented to verify the data and research into hematological visceral involvement, as proposed by descamps et al. at admission: complete blood count, alt, aspartate aminotransferase (ast), total bilirubin, γ-glutamyl transferase, alkaline phosphatase, sodium, potassium, creatinine and creatinine clearance, -h urine protein and urinary eosinophil count, cpk, ldh, ferritin, triglycerides, calcium and parathyroid hormone, blood glucose, prothrombin time and activated partial thromboplastin time, lipase, protein electrophoresis, c-reactive protein, quantitative pcr for hhv- , - , ebv, and cmv, blood culture, and antinuclear factor. follow-up (two times per week): complete blood count, alt, ast, creatinine, ldh, and other laboratory tests according to changes found on admission tests. evolutive follow-up: quantitative pcr for hhv- , - , ebv, and cmv, complete blood count, alt, ast, alkaline phosphatase, creatinine, ldh, ferritin, and triglycerides. the early recognition of adrs and withdrawal of the offending drug is the most important and essential steps toward clinical improvement. empiric treatment with antibiotics or antiinflammatory drugs should not be administered during the acute disease, since they may confuse or worsen the clinical picture of patients because of an unexplained cross-reactivity between drugs. prognosis is generally worse in the elderly while the recovery is usually faster and usually complete in children. for many years, the treatment of dress has been based on the use of systemic corticoste- the diagnosis is confirmed by the presence of the seven criteria (typical dihs) or of the first five criteria (atypical dihs) a this can be replaced by other organ involvement such as renal involvement b reactivation is detected from the second to third week after symptom onset, through igg anti-hhv- titer elevation roids (dose equal to or greater than - . mg/ kg/day of prednisone or equivalent) with marked improvement of symptoms and laboratory parameters only several days after the start of treatment. systemic corticosteroids should have their dose reduced, after clinical and laboratory control of the disease, slowly over - weeks to prevent recurrence of the symptoms of disease. abrupt deterioration of various symptoms is observed when the withdrawal is accidental or by rapid reduction of the dose of corticosteroids. shiohara et al. recommend that all patients should be hospitalized even when the initial presentation is mild. if symptoms worsen despite the use of oral corticosteroids, other options used in case series are the use of pulsed methylprednisolone ( mg/kg intravenously for days), intravenous immunoglobulin (ivig), and plasmapheresis, or a combination of these therapies. it should be remembered that the immunosuppressive therapies may increase the risk of infectious complications and sepsis. mild cases can recover simply by drug withdrawal and supportive treatment after a few weeks, even without the use of corticosteroids. however, even in mild cases, the monitoring of liver function tests should be conducted and appropriate tests ordered to rule out the involvement of other organs such as lungs, thyroid, and heart. special attention should be given to possible reactivation of cmv, especially in patients with severe dress/dihs. physicians should also pay attention to a proper balance between the needs of corticosteroids for relief of symptoms and been shown that dihs/dress is a manifestation of newly observed immune reconstitution syndrome (irs), and herpes zoster is observed as the most common manifestation of irs after highly active antiretroviral therapy for aids. relevant clues related to dress syndrome include: • dress/dihs is an adr caused by an apparent group of drugs, and one-third of cases are related to anticonvulsants, in addition to sulfonamides and allopurinol, which can cause - % mortality. • the syndrome is characterized by a latency period ranging between weeks and months after the introduction of the offending drug, and its course is marked by apparent sequential reactivation of hhv and subsequent development of autoimmune diseases, providing an opportunity to establish a connection between viral infections and the emergence of autoimmune diseases. • in early dress/dihs hypogammaglobulinemia and reduced peripheral b cells are found, and cd + cd + foxp + (regulatory t cells) levels are high at the beginning of the syndrome, regardless of whether or not patients are treated with corticosteroids. this clonal expansion of regulatory t cells appears to prevent activation of antiviral t cells in an appropriate manner and sequential reactivation of virus is presented in the syndrome. these regulatory t cells have the phenotype ccr + and cla + , which address the skin. in the last stage of the syndrome's activity, phenotype of cytotoxic t cells becomes prominent and cd + lymphocytes are intensely diminished. these cells are depleted over time, suffering apoptosis and becoming reduced after the resolution of the syndrome, which could be a predisposing factor for the development of autoimmunity. pustulosis [ ] agep is a clinical entity that appears in the intertriginous areas or on the face as a diffuse erythema (scarlatiniform) with acute presentation. patients report pruritus or local burning sensation. after this appearance, the erythema is replaced by hundreds of nonfollicular sterile small pustules (< mm in diameter) (figs. . and . ). these pustules may sometimes converge and mimic nikolsky's sign, leading to misdiagnosis as ten. intense edema of the face may occur, with purpuric lesions mainly on the legs and the onset of lesions similar to em of the legs. there may be mucous involvement in about % of the patients, although it is usually mild and self-limited, occurring in just one location. the cutaneous symptoms are almost always accompanied by fever of > °c. frequently there is leukocytosis in the blood count, and eosinophilia may also occur in one-third of the patients. usually this eruption regresses within - days after withdrawal of the drug and in typical cases leaves a lamellar or punctiform desquamation. disease prognosis worsens when there is hyperthermia or infection of the lesions, and when it affects elderly individuals, who should be hospitalized. the drugs described as a cause of agep are most frequently β-lactams (penicillin, cephalosporins), macrolides (azithromycin, erythromycin), cyclines (doxycycline), sulfonamides (trimethoprim, sulfasalazine), chloramphenicol, isoniazid, streptomycin, vancomycin, quinolones (ciprofloxacin, norfloxacin), itraconazole, terbinafine, allopurinol, carbamazepine, phenytoin, diltiazem, nifedipine, chromium picolinate, diclofenac, enalapril, disulfiram, furosemide, hydroxychloroquine, paracetamol, mercury, thalidomide, protease inhibitors, and bamifylline. sidoroff and et al. proposed some characteristics that might aid in the differentiation between pustular psoriasis and agep. in the latter, a history of psoriasis is rare, the lesions are most frequent in the cutaneous folds, the duration of the fever and the pustules is short, and there is usually a history of recent exposure to the drug; arthritis is rare. histopathology may show subcorneal and/or intraepidermal spongiform pustules, edema of the papillary dermis, vasculitis, exocytosis of eosinophils, and focal necrosis of keratinocytes (fig. . ). on the other hand, in pustular psoriasis a history of psoriasis is common, the involvement is generalized, the duration of the fever and the pustules is longer, history of drug exposure is less frequent, arthritis occurs in about % of the patients, and histopathologic recently, britschgi and colleagues demonstrated high expression of il- in these patients. it is known that il- is a chemokine with potent activity in the recruitment of neutrophils, which is produced by the keratinocytes and mononuclear cells of the cutaneous inflammatory infiltration. these authors concluded that agep might be the expression of a reaction whereby a cell bound to the drug triggers a drug-specific cd + and cd + immune response, which results in high expression of il- (type vid in the pichler classification). [ ] in , von piquet and shick described serum sickness in children treated with horse serum containing diphtheria antitoxin. more recently serum sickness has been observed in patients treated with horse antithymocyte globulin or vaccines of rabbit antihuman diploid cells. this constitutes a type iii hypersensitivity reaction, mediated by immunocomplexes deposited on the walls of the vessels, activation of the complement, and recruitment of granulocytes. it presents particular cutaneous manifestations: typically there is erythema in the lateral portion of the fingers and toes that precedes a more disseminated eruption (occurring in % of cases), which frequently is morbilliform (twothirds of the patients) and sometimes urticariform. the presence of urticaria, leukocytoclastic vasculitis, and multiform erythema is rarely observed. in half of the cases there is visceral involvement [ ] . the following clinical findings are common: fever, cutaneous eruption, constitutional symptoms, arthritis, and arthralgia. the disease begins about - days after the initial exposure to the foreign protein. the drugs related with this type of manifestation are the heterologous sera and vaccines. serum sicknesslike reactions can also be caused by penicillin, cephalosporin, minocycline, propranolol, streptokinase, and nonhormonal anti-inflammatories. there are no data on the prevalence of this disease in brazil, although reports of cases of this disease are not infrequent in the medical literature. fractions c and c of the complement are strongly decreased in serum sickness while they are usually normal in serum sickness-like reactions. treatment of the disease constitutes withdrawal of the drug allied to the use of systemic corticosteroids, in addition to antihistamines for symptomatic relief of pruritus when present. careful observation of the clinical course of the patient's systemic involvement is imperative. [ , ] several medications can induce a cutaneous vasculitis-type response, the histopathologic definition of which is the presence of inflammation and necrosis in the wall of the cutaneous blood vessels. clinically it presents as tangible purpura or maculopapular purpuric eruption. this disease can also occur in the form of hemorrhagic blisters, urticaria, ulceration, nodules, raynaud's disease, and digital necrosis. the same vasculitis the disease develops about - days after initiating the drug; however there can be a longer time interval, and any medication instituted within the months prior to the presentation should be considered suspect. given the absence of confirmatory tests for this entity, one should value anamnesis and the correlation with drug exposure, which in general occurs - weeks before onset of the cutaneous picture. however, the exposure can have occurred in periods as disparate as days to years. withdrawal of the drug leads to a rapid resolution of the picture, and systemic corticosteroids can benefit some patients. the process is usually solved without sequels. the clinical, epidemic, and pathologic characteristics of drug-induced vasculitis have been little reported in the medical literature, since there is no consensus in the definition of this disease, with various revisions using different criteria for inclusion of cases. vasculitis attributed to exposure to medicines is rare, but seemingly account for about - % of dermal vasculitis cases. it is difficult to quantify the frequency with which drug-induced vasculitis is strictly cutaneous. clinical experience suggests that most of the cases are confined to the skin and have a selflimited course, although it can be associated with varied degrees of systemic symptoms including arthralgia, indisposition, and fever. visceral involvement is well described and pathologically heterogeneous. glomerulonephritis and interstitial renal disease, varied degrees of hepatocellular damage, and formation of granulomas in the liver have been described, besides involvement of the heart, lungs, and central nervous system. furthermore, there are rare cases of drug-induced vasculitis with renal and hepatic involvement in the absence of cutaneous disease. the drugs most frequently referred to in the literature, in the form of case reports or series studies, as causative of vasculitis are propylthiouracil, hydralazine, granulocyte colony-stimulating factor (g-csf), cefaclor, minocycline, allopurinol, d-penicillamine, phenytoin, isotretinoin, and methotrexate. as many of the cases of drug-induced vasculitis are not reported in the literature, other drugs are also possible important causative agents of this reaction type. other drugs have been reported less often as causal agents of vasculitis: several antibiotics, etretinate, didanosine, zidovudine, acebutolol, atenolol, sotalol, propranolol, chlorothiazide, furosemide, diltiazem, nifedipine, methyldopa, captopril, enalapril, lisinopril, losartan, procainamide, quinidine, antithyroid medications, painkillers and antipyretics, levamisole, tamoxifen, arabinoside c, interferon, interleukin- , sulfasalazine, etanercept, gold, carbamazepine, antidepressants, zafirlukast, chromalin, cimetidine, ranitidine, l-tryptophan, radiocontrast, streptokinase, heparin, coumarin, chlorpromazine, metformin, pimagedine, and diphenhydramine. drugs that induce vasculitis associated with antineutrophil cytoplasmic antibodies (anca) include hydralazine, propylthiouracil, minocycline, and anti-tnfα biological agents. about % of the patients who use propylthiouracil develop anca, a fact that is related to a higher risk of glomerulonephritis. a particularly relevant form among the drug-induced vasculites is propylthiouracil hypersensitivity vasculitis. there are cases with other antithyroid compounds, such as methimazole, thiamazole/ methylthiouracil, and carbimazole, which, similarly to propylthiouracil, contain a thioamide group and cause allergic cross-reactions. although uncommon, nowadays a larger number of case reports of this entity are observed, suggesting that cases were previously not reported or were included among other nosologic entities, since propylthiouracil is a drug classically dedicated to the treatment of hyperthyroidism. the clinical symptoms and signs begin after initiating propylthiouracil. although the duration of drug use is extremely variable, from week to years, it appears under a classic tetrad of symptoms that include fever, sore throat, arthralgia and cutaneous eruption; there can also be myalgia, fatigue, weight loss, conjunctivitis, rhinitis, and hemoptysis. the disease course is that of a systemic vasculitis. there can be a lupus-like syndrome, wegener-like granulomatosis, or nodular-like polyarthritis with multiple involvement of organs, such as kidneys, joints, lungs, and others associated with cutaneous lesions. the cutaneous lesions usually consist of plaques or acral purpuric nodules arranged in a livedoid pattern, with a preference for the extremities (fig. . ) , face, breasts, and characteristically the lobes and helices of the ears, mimicking the leprosy type reaction of lucio's phenomenon. hemorrhagic blisters appear on these lesions that progress to central necrosis of the skin, which can be so extensive that it simulates the clinical presentation of purpura fulminans observed in septic infectious states with disseminated intravascular coagulation. laboratory tests reveal anemia, leukopenia, and platelet depletion in the blood count; increased erythrocyte sedimentation rate, urea, creatinine, transaminases, and bilirubin; hypoalbuminemia; alterations in the coagulation time, prothrombin time, and partial activated thromboplastin time; and immunological abnormalities such as positive anca, rheumatoid factor, and hypergammaglobulinemia can be found. positivity can also be present in anti-ssa, antidouble-stranded dna, anticardiolipin, antismooth muscle antibodies, antimitochondrial, parietal, and antiadrenergic antibodies, besides hypocomplementemia, cryoglobulinemia, and elevation of c-reactive protein. histopathologic study demonstrates a leukocytoclastic vasculitis of the superficial and profound vessels of the dermis. the finding of immunocomplexes deposited in the vascular wall is uncommon, such that some authors have named them pauci-immune anca-positive vasculitis. most of the patients recover completely following withdrawal of propylthiouracil, although some develop impairment of the kidneys or other internal organs, or skin, requiring high doses of prednisone for several months. the dermatologic findings in patients with drug-induced vasculitis associated with anca include plaques and purpuric acral nodules, which appear more commonly on the extremities, face, breasts, and ears. in addition, the patients report the same signs and symptoms as found in other small-vessel vasculites associated with anca (wegener's granulomatosis, churg-strauss syndrome), including glomerulonephritis, pulmonary hemorrhage, and digital gangrene. besides withdrawal of the offending drug, it is generally necessary to use corticosteroids in high doses or in pulse therapy, plasmapheresis, and immunosuppressants for several months. the mortality rate is approximately %. necrosis [ ] this is a rare and severe adverse effect from treatment with warfarin (anti-vitamin k agents), occurring with cutaneous necrosis secondary to occlusive thrombosis in the vessels of the skin and subcutaneous cellular tissue. it usually presents - days after use of the drug, as painful (fig. . ) , with hemorrhagic blisters or necrotic scars in the areas rich in subcutaneous tissue, such as buttocks, breasts, and hip. the risk of this disease increases in patients who are female, obese, and users of high doses of the medication [ ] . the necrotic tissue requires debridement and grafts. this type of reaction has also been described with the use of heparin. this kind of adr is represented by several conditions related to drug exposure, which do not represent life-threatening conditions to the patients except discomfort. there is no severe temporary or permanent remaining lesions or long-term internal organ sequelae and, in most of cases, no mortality or severe impact on patients' health. in contrast to severe adrs, in the clinical setting of uncomplicated cadrs admission to the intensive care or burn unit is usually not necessary for the majority of patients. for this reason, the physician must be able to identify the signs and symptoms that indicate severe cadr [ ] . in particular, dermatologists must pay attention to identifying these reactions, since the skin is among the most common organs or systems of clinical manifestation of adrs and concurs with at least % of adrs [ ] . the most common forms of cadrs are urticarial and exanthematous eruptions, which together constitute - % of all cards [ ] . these two types carry few to no long-term consequences [ ] . the severe adrs (sadrs), described earlier in this chapter, probably represent around % of all adrs [ ] . sadrs often are associated with high levels of morbidity and mortality, and therefore a prompt recognition of the reaction, withdrawal of all possible offending agents, and appropriate triage, hospital admittance, workup, and specific treatment are critical [ ] . regarding studies of severe versus uncomplicated adrs, swanson and colven [ ] proposed a staged patient evaluation as shown in fig. . . signs and symptoms severe adr are listed in fig. . , and in this clinical scenario the physician should have a low threshold to admit the patient to hospital, perform a complete workup including evaluation of other medical specialties, withdraw suspected medications, and initiate adequate therapy when indicated [ ] . another relevant aspect in recognizing the type of adr is the time from medication introduction to the onset of a cutaneous reaction, since this is related to the subtype of adr, as proposed in fig. . [ ] . often patients have been exposed to several medications in the same period, and creating a "drug list" that details the dates of all medications taken is helpful in narrowing down the most probable culprits [ ] . physicians should pay attention to prodromal symptoms (skin pain, fever, malaise, throat pain or discomfort, arthralgia, etc.) that can to precede the cutaneous eruption, and associated internal symptoms (abdominal pain, ocular discomfort, dysuria, respiratory distress, etc.), and proceed to complete physical examination including full skin examination of groin, genitalia, eyes, oropharynx, thorax auscultation, abdomen, and lymph node palpation [ ] . the physician needs remember that several risk factors for the development of more severe cutaneous adrs have been identified, including female gender, older age, viral infections (herpesvirus family or hiv), genetic susceptibility (specific single-nucleotide polymorphisms in the hla region), iatrogenic immunosuppression, underlying immune-mediated diseases, and cancer [ ] . [ , ] exanthematous or maculopapular drug eruptions, sometimes inappropriately designated "drug rashes" or "drug eruptions" by some generalists, are the most common adrs in the skin. the eruption usually occurs between and days after the initiation of a new medication or chemical substance, although it can develop sooner, exanthematous eruptions generally are composed of erythematous macules and/or papules and more rarely by vesicles or pustules, usually with a pattern of symmetric distribution on skin. the eruption often begins on the trunk followed by centrifugal dissemination to the proximal limbs. skin lesions progressively become confluent and may cover large areas of the body (fig. . ). pruritus and/or low-grade fever are often associated with the exanthema. in some patients the exanthema may progress to erythroderma or more severe reactions such as sjs/ten or dress syndrome after some days or weeks. under histopathology examination this type of adr demonstrates interface dermatitis with vacuolar changes in keratinocytes at the basal layer of the epidermis, and upper dermal mononuclear cells infiltrate with some eosinophils. the pathogenesis involves the overexpression of several cytokines of th pattern, such as il- and il- , causing epidermal damage by uncomplicated exanthematous drug eruptions can occur with almost any medication, but the following drugs have higher risks (more than % of patients): allopurinol, aminopenicillins, cephalosporins, antiepileptic drugs, and antibacterial sulfonamides. viral infections may increase the incidence of morbilliform drug eruptions, as seen in the setting of mononucleosis infection under treatment with ampicillin, or in severe exanthema with internal damage as in dress syndrome related to the hhv family (ebv, cytomegalovirus, hhv- and - ). morbilliform reaction is the most common presentation of exanthematous drug eruption. morbilliform is defined as a rash resembling measles and is clinically depicted by erythematous macules and/or papules, often coalescing into larger plaques. many studies have shown that cutaneous biopsy alone cannot distinguish with certainly that a reaction is due to a drug. there are some clues that suggest the diagnosis: (i) epidermis (mild spongiosis is the most consistent feature, with occasional hyperplasia of the epidermis. few lymphocytes are commonly present in the epidermis. in % of biopsies, vacuolization was found in the dermoepidermal junction); (ii) dermis (perivascular infiltrate is virtually always present, composed of lymphocytes and in % of cases scattered eosinophils); (iii) papillary dermal edema; (iv) dilated lymph and blood vessels. the primary differential diagnosis for morbilliform eruptions includes viral exanthemas (e.g., ebv, hhv- , and cmv), bacterial toxin resection (streptococcal or staphylococcal), kawasaki syndrome, and others such as secondary syphilis, scarlet fever, acute hiv, or acute gvhd. the treatment is supportive. the first measure is the withdrawal the causative agent. topical corticosteroids and systemic antihistamines can be administered in the first step. if necessary, this can be combined with a short cycle of systemic corticosteroids (oral prednisone, . mg/kg/day, with progressively tapering dosages over several days). antihistamines are indicated as adjuvant therapy in cases of itching. [ ] [ ] [ ] drug-induced urticaria is the second most common form of cutaneous drug reaction after exanthematous reactions. urticarial eruption can be broken down into simple acute urticarial eruptions, those involving angioedema or anaphylaxis, and serum sicknesslike reactions as previously described in this chapter. simple urticarial reactions caused by drugs consist of erythematous and edematous lesions, which have central clearing with a red border. the lesions can be located anywhere on the body and wax and wane over hours to days. pruritus is an associated symptom. this type of drug reaction takes place minutes to days after exposure to the offending drug. common drugs responsible for urticarial reactions include antibiotics, such as penicillins, cephalosporins, sulfonamides, and tetracyclines, generally due to ige-mediated hypersensitivity reaction. another common class of drugs related to urticarial eruptions is nonsteroidal anti-inflammatory drugs (nsaids). nsaids cause most frequently non-ige-mediated urticaria and angioedema because of their pharmacologic activity of cyclooxygenase- enzyme inhibition, particularly of prostaglandin e , and results in the generation of leukotriene c and activation of inflammatory cells. urticaria and angioedema are associated in about % of cases. regarding ace inhibitors, angioedema is described in . % of patients treated with this class of drugs, and often without urticarial lesions. rarely, angiotensin ii receptor blockers result in the same complication. oral or injectable antihistamines and systemic corticosteroids are sometimes needed for severe acute urticaria and intramuscular epinephrine for angioedema. in cases of isolated angioedema caused by ace inhibitors, epinephrine will not control the symptoms and it is necessary to use the selective bradykinin b receptor antagonist icatibant in this clinical setting. [ , ] this entity is defined as recurrent lesions that, upon repeated uptake of the causative drug, always appear at the same skin or mucosal sites. fdes present as well as circumscribed, single or multiple, often pruritic or burning erythematous, dusky patches (fig. . ) , ranging from several millimeters to over cm in diameter. vesicles or even blisters can develop. as the lesions resolve, they leave residual hyperpigmentation. the hallmark of fdes is geographic memory. if a reaction recurs, it tends to recur in the same location as previously (although a new location can also be involved). lips, hands, genitalia (especially male genitalia), and occasionally oral mucosa are favored sites of fde occurrence, although the lesions can be found anywhere on the skin and mucous membranes. after intake of the offending drug, fde appears within minutes up to several hours (about min to h). the cutaneous lesions can be accompanied by general symptoms such as fever, nausea, dysuria, abdominal cramps, and diarrhea, though rarely. on occasion the disease presents in an atypical form with blunt-margined, non-pigmented, giant (> cm in diameter), urticarial, purpuric, targetoid, linear, reticular, and butterflylike lesions. the histopathologic hallmark is brisk interface dermatitis with varying amounts of epidermal necrosis as well as melanophages and eosinophils in the upper dermis. fdes reveal a reaction pattern with lichenoid or erythema multiforme-like changes. atypical histopathologic reaction patterns such as leukocytoclastic vasculitis, neutrophilic reaction, and a predominantly dermal reaction without pigment incontinence in what is termed nonpigmented fde have been reported. the pathogenesis of fde is based on the new subclassification of delayed type iv immune reactions (werner pichler), a type ivc reaction. in this kind of immune response cytotoxic t cells play a predominant function, whereby autoaggressive αβ + cd + memory t cells persist intraepidermally in previous fde sites and play a central role in new flare-ups during drug recall. under drug exposition, keratinocytes are stimulated to participate in immune response through tnf-α and a rapid expression of icam- molecule, and then stimulate cd + t cells to produce ifn-γ and many drugs have been found to cause fde, with common offenders including sulfonamides, nsaids (e.g., ibuprofen), allopurinol, barbiturates, hydroxyzine, laxatives, tetracycline, phenolphthalein, and feprazone. usually there is only one causative drug (monosensitivity), although sometimes several drugs can induce fdes in the same patient (multisensitivity). the most common multisensitivity is the cross-reaction between chemically related drugs such as tetracyclines. less frequently, multisensitivity can occur because of polysensitivity, whereby two or more chemically unrelated drugs either induce the identical fde lesion or each drug determine flare-ups in separate lesions. treatment is mainly symptomatic with discontinuation of offending agent, topical corticosteroids, and antihistamines. [ , ] this kind of adr produces lesions resembling acne vulgaris, but unlike acne vulgaris, druginduced acneiform eruptions typically are not associated with the presence of comedones (blackheads and whiteheads). acneiform drug eruptions appear as erythematous papules or erythematous pustules on the face and trunk (fig. . ) and proximal extremities, but sometimes can be present on the forearms and legs, an unusual site in acne vulgaris. the most relevant hallmark is the monomorphous pattern of this eruption and the resolution without scarring. drug-induced acneiform eruptions represent only % of drug eruptions. several medications are related to flare-ups of drug-induced acneiform eruptions, the most strongly associated being lithium, androgens, oral contraceptives, corticosteroids, vitamin b complex, and nowadays epidermal growth receptor (egfr) inhibitors for chemotherapy. iodine, bromide, isoniazid, actinomycin d, and phenytoin have also been associated. in the last decade, the use of supplementary complexes by bodybuilders, such as milk and whey proteinbased products, have been reported as being involved in acneiform eruptions. this is an effect caused by elevations of postprandial insulin and basal insulin-like growth factor i plasma levels. treatment involves discontinuing the use of the offending drug, except in the case of egfr inhibitors, when discontinuation may not be possible. benzoyl peroxide, topical retinoids, and topical or oral antibiotics, such as doxycycline, can be used to treat the reaction, similar to the treatment of acne vulgaris. lichenoid eruptions [ , ] drug-induced lichenoid eruptions are uncommon adrs that appear similar or even identical to lichen planus, with shiny violaceous polygonal papules and plaques (fig. . ) . drug-induced lichenoid eruptions can present virtually anywhere on the body surface, but certain clues in the distribution can help suggest drug eruption over lichen planus. drug-induced lichenoid eruptions tend to be absent from the flexor surface of the wrists, genitals, and mucous membranes, whereas these locations are often involved in common lichen planus. lichen planus drug eruptions also often favor sun-exposed areas of the body. several drugs have been reported to be related to drug-induced lichenoid eruptions: gold salts, antimalarials, methyldopa, nsaids, penicillamines, lithium, sulfonylureas, phenylenediamine derivatives, thiazide diuretics, β-blockers, omeprazole, and pantoprazole. the time from initiation of the drug to onset of lichenoid drug eruption varies greatly depen ding on the causative medication. reactions caused by naproxen, for example, tend to occur approximately days after administration. by contrast, certain drugs such as lithium, methyldopa, and acebutolol can develop lichenoid eruptions several years later. hiv infection can contribute to lichenoid drug eruptions on photoexposed areas of the body. treatment typically is symptomatic, with topical corticosteroids a mainstay. once discontinuation of the medication has been accomplished, the eruption resolves spontaneously after a period of a few weeks or months. [ ] [ ] [ ] acute photosensitivity ranges from common polymorphous light eruptions to phototoxicity, or rare photoallergies. photosensitivity refers to reactions that occur when a photosensitizing agent (chromophore substance) in or on the skin reacts with ultraviolet (uv) radiation, often in doses smaller than those associated with sunburn. up to % of cutaneous drug reactions are photosensitivity eruptions. typically, a photosensitivity reaction occurs within hours to days of exposure to sunlight and may last for up to week or more. more frequent reactions are named "phototoxic reactions," in which skin signs resemble moderate to severe sunburn, with erythema, blistering, weeping, and desquamation. photoallergic reactions resemble eczematous lesions, often in subacute or chronic presentation. phototoxic and photoallergic reactions occur in sun-exposed areas of the skin; however, widespread eruptions can occur, which may suggest a systemic photosensitizing agent (photoallergy). these reactions are dose related and are most commonly seen in patients who have been exposed to high doses of both the drug and uv radiation. one's susceptibility to this type of syndrome is variable and likely based on drug absorption and metabolism, as well as the amount of melanin in the skin. piroxicam, fluoroquinolone antibiotics, tricyclic antidepressants, and nsaids are classes of drugs that have been reported to be frequent photosensitizers, with fluoroquinolones being the most potent. other antibiotics, such as tmp-smx and tetracyclines, have also been implicated. recently voriconazole, a third generation of azole antifungal agents, has been reported as a photosensitivity agent, especially in phototoxic reactions (fig. . ), in % of outpatients treated, besides increasing the potential of nonmelanoma skin cancer (particularly squamous cell carcinomas) arising from potential photocarcinogenesis related to voriconazole. phototoxic reactions are the most common dermatologic adverse effect of amiodarone therapy, affecting - % of patients on long-term treatment. photoallergy is considerably less likely to occur, but the risk also increases with prolongation of the therapy. skin changes usually occur after at least months of therapy and with the minimal cumulative dose, which is g. management of photosensitivity reactions includes limiting exposure to sunlight, using potent sunscreen, and wearing protective clothing. oral and topical corticosteroids agents may be employed in the treatment. [ , ] coma blisters are uncommon skin eruptions seen in patients with impaired consciousness. the original case was described in a patient who was heavily sedated because of barbiturate intoxication. subsequently anticonvulsants have been reported, including certain benzodiazepines such as clobazam (fig. . ) , and valproic acid and amitriptyline overdose. there were a few reports of coma blisters and peripheral neuropathy caused by amitriptyline overdose. these blisters are most often seen in pressure areas, particularly over bony prominences in contact with hospital beds. the hallmark histologic feature that defines coma blisters is eccrine gland necrosis in the skin. differential diagnosis with bullous pemphigoid is obtained with a negative direct immunofluorescence biopsy of the skin. until recently, coma blisters were thought to be a self-limiting process that did not require withdrawal of the offending agent. however, in some patients the eruption resolves only upon withdrawal of the drugs. nodosum [ ] erythema nodosum is a skin reaction manifested by tender or painful erythematous subcutaneous nodules, located usually on the extensor aspects of the lower extremities. histologically it is a septal panniculitis without vasculitis. several conditions can be induce and act as an antigenic stimuli, including drugs, benign and malignant systemic diseases, leprosy, and bacterial (e.g., tuberculosis) and fungal infections. frequently the cause is unknown. drugs that may cause erythema nodosum are antimicrobial agents (amoxicillin, penicillin, sulfonamides), bromide, iodine, gold salts, analgesics and antipyretics (including paracetamol), carbimazole, isotretinoin, azathioprine, vemurafenib, gm-csf, oral contraceptives (estrogens/ progesterones), and estrogens. erythema nodosum disappears within a couple of weeks after withdrawal of the causative drug. psoriasis [ , ] drug-induced psoriasis is well documented. such eruptions may occur in patients with pre-existing psoriasis (exacerbation phenomenon) or those without a personal or family history. lesions typically improve with drug withdrawal, although persistent disease is possible. more frequent drugs involved are β-blockers, lithium, antimalarials, nsaids, anti-tnfα agents (fig. . ) , and bupropion. exacerbation of psoriasis caused by the following medications has also been observed: adrenergic antagonists, ifn, gemfibrozil, iodine, digoxin, and clonidine. exanthema [ ] in individuals previously sensitized to an allergen through contact, systemic exposure results in the development of a condition classically termed systemic contact dermatitis. one of the most common manifestations of this condition is socalled baboon syndrome (bs). a subsequent study by hausermann et al. [ ] examined a series of cases of bs and found that about half of the patients exhibited no evidence of prior skin sensitization. for that group the authors proposed the term "symmetric drugrelated intertriginous and flexural exanthema" (sdrife) to describe a peculiar form of drug rash with symptoms similar to those of true bs. bs is historically often equated with a mercuryinduced exanthem in patients with previous contact sensitization. sdrife specifically refers to the typical clinical pattern of this drug eruption, and the following diagnostic criteria are proposed [ ] : ( ) exposure to a systemically administered drug either at the first or repeated dose (excluding contact allergens); ( ) sharply demarcated erythema of the gluteal/perianal area and/or v-shaped erythema of the inguinal/perigenital area; ( ) involvement of at least one other intertriginous/flexural localization (fig. . ) ; ( ) symmetry of affected areas; and ( ) absence of systemic symptoms and signs. several drugs are reported to induce sdrife [ ] : (i) β-lactam antibiotics (amoxicillin, ampicillin, amoxicillin/clavulanic acid, pivampicillin, penicillin v, ceftriaxone, cefuroxime, cephalexin) and non-β-lactam antibiotics (including clindamycin, roxithromycin); (ii) corticosteroids: deflazacort; (iii) radiocontrast barium; (iv) other drugs: sulfate iomeprol, iopromide, monoclonal antibodies (cetuximab, glembatumumab), vedotin (cr , vcmmae), psychopharmaceuticals (risperidone loflazepate ethyl), allopurinol, cimetidine hydroxyurea, heparin (intravenous), ivig, mitomycin c, naproxen, oxycodone, pseudoephedrine, salsalate, terbinafine, and valacyclovir. [ , ] in , freudenthal described full-thickness dermal necrosis associated with intramuscular injection of oily bismuth suspension, which was used to treat syphilis at that time. he described the histologic appearance of these suspended particles deep within the cutaneous arteries, distant from the injection site. this condition was also described by nicolau the following year, and the syndrome more often bears his name despite freudenthal's precedence in the literature. nicolau syndrome is an iatrogenic syndrome caused by intramuscular injection leading to variable degrees of tissue necrosis, with variable severity, including the skin and deeper tissues. intense pain in the immediate postinjection period and purplish discoloration of the overlying skin, with or without a reticulate pattern (livedo racemosa-like pattern), is highly characteristic of this syndrome. intramuscular, subcutaneous, intravenous, and intra-articular injections have been reported to produce this syndrome. the skin necrosis resolves with severe and disfiguring scarring. it is therefore important that dermatologists and cutaneous surgeons are aware of this agonizing and deforming iatrogenic complication of injections. discoloration of the skin may result in necrosis and ulceration, which might involve the subcutaneous tissue and the muscular layer. paralysis of the lower extremities has been reported and attributed to embolization of the medication, mainly resulting from the force of injection from the gluteal vessels into the internal iliac arteries, and ischemia of sciatic nerve. application of cold devices or compress tends to aggravate the tissue injury and necrosis. several drugs are related to ecm: (i) intramuscular injections (vitamin k, nsaids, hydroxyzine, vaccination, bismuth, benzathine penicillin, penicillin g); (ii) intravenous injections (polidocanol %); (iii) subacromial injection (triamcinolone acetate); (iv) subcutaneous injection: pegylated ifn-α, glatiramer acetate; and (v) intra-articular: glucocorticoid. aspirating just before injecting has been suggested as a method of preventing nicolau syndrome, as it is thought to help prevent embolism caused by intra-arterial deposition of medication. however, it is doubtful as to whether nicolau syndrome can be prevented by this method, as the spasm of the vessel or vasocompressive effect in nicolau syndrome is usually difficult to recognize. the essential difference between those cases of ecm and the pathophysiology seen with vascular obstruction by dermal fillers (hyaluronic acid, polymethylmethacrylate microspheres) is that the former often involves inflammatory pathways being activated by the injected material, whereas the latter typically involves a more purely mechanical vascular obstruction (although some dermal fillers may promote blood clotting, hyaluronic acid-based dermal fillers by design are minimally reactive in tissues). the phenomena are similar in that the inciting event is accidental intravascular injection, followed by some degree of intravascular transport, finally resulting in distal vascular obstruction, ischemia, and so forth, such that the ultimate clinical presentation is the same. diagnosis is mainly clinical; cutaneous biopsy reveals necrotic changes caused by ischemia. ultrasonography study of the skin and magnetic resonance imaging help in delineating the extent of damage. prompt treatment has been reported to avert necrosis of the skin. in the immediate post-event period, treatment is based on various approaches to improve blood supply such as pentoxyphylline, hyperbaric oxygen, intravenous alprostadil, and thrombolysis with heparin. intralesional corticosteroid has also been used to reduce inflammation. surgical debridement of the necrotic scar is of utmost importance as it reduces infection and enhances wound healing. [ , ] the autoimmune form. the most commonly used medication associated with this kind of adr is vancomycin; however, other drugs include amiodarone, atorvastatin, captopril, ceftriaxone, diclofenac, furosemide, lithium, metronidazole, penicillin, phenytoin, piroxicam, rifampin, and trimethoprim-sulfamethoxazole. treatment of drug-induced labd includes discontinuation of the causative agent and treatment with topical or systemic steroids, dapsone, and/or nonsteroidal systemic immunosuppressive agents. pemphigoid [ ] this entity is very similar to the autoimmune form. multiple tense bullous lesions appear on the skin and pruritus is a common symptom. often the medications associated with druginduced bullous pemphigoid include furosemide, ace inhibitors (especially captopril and enalapril), penicillin, ampicillin, chloroquine, psoralen-uva treatment, and sulfasalazine. treatment is aimed at discontinuation of the offending drug as well as topical or systemic corticosteroids and steroid-sparing immunosuppressive drugs as indicated. [ ] similar other drug-induced reactions related to counterpart autoimmune conditions, druginduced pemphigus most closely resembles pemphigus foliaceus, with flaccid vesicles or bullae that rupture, creating crusted or desquamated erosions, with mucous membranes often spared. the histologic hallmark of this drug-induced eruption is the acantholysis of epidermal cells, but this phenomenon is not a pathognomonic sign of this type of adr. both autoimmune (idiopathic) and drug-induced pemphigus have a positive nikolsky sign, as observed in the sjs/ten spectrum, although sjs/ten does not demonstrate acantholysis in the skin biopsy. drugs containing thiol molecules (penicillamine, thiopurine, pyritinol, gold sodium thiomalate, captopril) are responsible for % of the cases, and other drugs implicated include levodopa, penicillin, phenobarbital, piroxicam, propranolol, and rifampicin. drug-induced pemphigus can occur any time within the first year of initiation of one of the offending drugs. treatment generally consists of withdrawal of the drug and use of systemic corticosteroids. [ , [ ] [ ] [ ] [ ] the skin, mucous membranes, annexes (sebaceous and sudoriferous glands), and the phaneros (hair and nails) are tissues with rapid cellular proliferation and are thus susceptible to adverse reactions (toxic or hypersensitive) resulting from systemic chemotherapeutic treatment. antineoplastic agents are defined as substances that inhibit or prevent the proliferation of neoplasms. because of their high metabolic rate, the skin, mucous membranes, and annexes are the most important target organs of the toxicity associated with chemotherapy. reactions can present with disseminated exanthematous eruptions, nonspecifically, or as distinct cutaneous lesions. some drugs can trigger localized reactions caused by extravasation to tissues adjacent to the areas of application. exanthematous reactions, such as nonspecific erythema multiforme, are more common, and many of them are attributed to hypersensitivity mechanisms. certain local toxicity, such as alopecia, mucositis, nail alterations, or hand-foot syndrome, is more specific and less common, frequently associated with particular drugs or groups of drugs. the identification of the reaction pattern associated with the trigger drug and of the possible dose-limiting toxicity is of extreme importance to the physician, as is the differential diagnosis with infectious processes and specific manifestations of the neoplasm. [ , [ ] [ ] [ ] [ ] alopecia [ , [ ] [ ] [ ] [ ] alopecia is the most common adverse skin manifestation of chemotherapeutic treatment. there are two types of drug-induced alopecia: the anagen effluvium and the telogen effluvium. in the anagen effluvium hair loss occurs because of the sudden interruption of the mitotic activity of the hair matrix, - weeks after the start of chemotherapy, leading to lack of hair production or its thinning (pohl-pinkus constrictions). the weakening of the hair shaft in this context predisposes the hair to breakage and shedding during the act of combing. they involve the hair, eyebrows, beard, axillary hair, and pubic hair. it is dose-dependent and reversible. new hairs often grow back with a different color and texture. in the telogen effluvium, hairs move prematurely to a resting phase with subsequent loss of normal hair. the antineoplastic agents that most frequently cause the anagen effluvium lead to diffuse hair loss, of sudden onset, from to days after the start of chemotherapy. hair loss becomes more pronounced about - months after the start of treatment. even though hair loss is intense, about % of the pilous follicles are usually in a resting phase at the time of the administration of the drug, and this determines incomplete hair loss. with repeated treatment cycles, alopecia totalis may occur. this type of effluvium is generally reversible when treatment is suspended, and occasionally permanent with the use of cyclophosphamide and busulfan. hair grows around cm per month, possibly showing new texture and color. the chemotherapeutic drugs more often associated with alopecia when used in isolation are: (i) complete alopecia (cyclophosphamide at high dose, doxorubicin, docetaxel, dactinomycin, irinotecan, topotecan, bleomycin, paclitaxel); (ii) incomplete alopecia (etoposide, ifosfamide, mitomycin c, -fluorouracil, melphalan, mitoxantrone, gemcitabine, vinca alkaloids). most reactions can be reversed by dose reduction or by increasing the interval between doses. some toxic effects can be successfully treated or prevented. medication administered before the chemotherapeutic treatment can prevent hypersensitivity reactions. the use of oral antiseptic solutions is useful in the control of mucositis. some dermatologic reactions to new antineoplastic agents, such as egfr inhibitors, have been associated with anticancer efficacy. other adverse effects may be mistaken for reactions to chemotherapeutic drugs and include infections resulting from immunosuppression, paraneoplastic syndromes, gvhd, nutritional deficiencies, development of skin malignancies, and metastatic primitive tumor. there are several classifications of reactions to antineoplastic drugs. the lack of a systematized multidisciplinary approach does not provide all the microscopic data and physiopathogenic mechanisms that delineate the lesions. therefore, the classification adopted didactically groups with the eruptions based on the target cells and mechanism of action of the drugs. preventive measures to limit hair loss have had limited success. hypothermia of the hair scalp or tourniquets applied in this region may reduce the perfusion of the drug in the pilous follicles and delay the start of or minimize hair loss. this procedure is contraindicated for patients with hematologic neoplasms such as leukemias, lymphomas, and other potentially metastatic tumors of the hair scalp. topical minoxidil is not effective in the prevention of drug-induced alopecia, but may shorten its duration. [ , [ ] [ ] [ ] [ ] hair alterations with acceleration of growth and shaft changes are observed with the use egfr inhibitors (fig. . ) . alterations [ , [ ] [ ] [ ] [ ] nail alterations can present with a reduction of the nail growth speed, fragility, lines of discoloration (mees' lines), transversal depressions (beau's lines), hyperpigmentation, onycholysis with subungual aseptic abscesses, photo-onycholysis, paronychia, and pyogenic granulomas of the periungual folds. nearly all antineoplastic agents can lead to reduction of growth speed, nail fragility, mees' lines, and beau's lines. hyperpigmentation can occur after the use of cyclophosphamide, hydroxyurea, fluoropyrimidines such as -fluorouracil, and especially anthracyclines such as doxorubicin and daunorubicin. painful onychomycosis and subungual abscesses are due to the use of taxanes (docetaxel/ paclitaxel) and anthracyclines (doxorubicin). ingrown nails, paronychia, and pyogenic granuloma are associated with the use of tyrosine kinase inhibitors of egfr (fig. . ), such as erlotinib and gefitinib. the fenestration or avulsion of the lamina should be considered when abscesses that involve more than % of the nail bed are present. in these more severe cases, the temporary suspension of treatment, longer intervals between cycles, and dose reduction should be considered. [ , [ ] [ ] [ ] [ ] this rare, nonspecific disease often occurs when chemotherapeutic drugs are used in combination, making it difficult to know which drugs are responsible for causing the disease. cytarabine is the most commonly cited drug; however, others are also implicated, such as bleomycin, chlorambucil, cyclophosphamide, cytarabine, doxorubicin, lomustine, mitoxantrone, busulfan, carmustine, cisplatin, cyclophosphamide, etoposide, -fluorouracil, methotrexate, and thiotepa. some authors consider neutrophilic eccrine hidradenitis (neh) as a paraneoplastic phenomenon, since it has been found in an early case of acute myeloid leukemia not yet treated. it has been associated with hiv infection, nocardia, serratia, enterobacter, staphylococcus, and with patients receiving gm-csf. the mechanism is unknown, but may be due to the excretion of the chemotherapeutic drug by the eccrine glands and its direct toxic effect on the eccrine epithelium. the clinical condition may be preceded by fever and unspecific clinical signs. skin eruptions are distributed in the head, neck, trunk, and extremities, with lesions that vary from erythema, papules, nodules, and pustules to papular plaques. lesions may be purpuric or hyperchromic, single or multiple. they appear between days and weeks from the start of treatment, regressing spontaneously without scarring or sequelae - weeks after the suspension of the drug. the differential diagnosis is vast and includes sepsis, septic embolism in a postchemotherapeutic neutropenic patient, vasculitis, leukemia cutis, hypersensitivity reaction, urticaria, polymorphous erythema, and neutrophilic dermatoses such as sweet's syndrome, bullous pyoderma gangrenosum, and atypical pyoderma gangrenosum. owing to the unspecific clinical presentation of the disease and the great number of differential diagnoses, some authors suggest that neh be included in the diagnostic hypotheses of any ingrown nail and pyogenic granuloma in a patient using erlotinib eruption that may occur in patients undergoing chemotherapy, and its final diagnosis is established by histopathology. therefore, histopathology is essential for conclusive diagnosis. it is constituted by a dense neutrophilic infiltrate, inside and around the eccrine glands, with necrosis of the eccrine epithelium cells. involvement of the apocrine glands has been reported. occasionally, squamous syringometaplasia, hemorrhage and edema of the dermis, spongiosis and/or vacuolization of the basal layer of the epidermis, necrosis of keratinocytes, and mucin deposits inside and around the eccrine glands may occur. in patients with severe neutropenia, the neutrophilic infiltrate may be absent; however, necrosis of the eccrine epithelium is typical. neh is a self-limiting adverse reaction. frequently the process resolves within a month, without treatment. in other chemotherapy cycles, % of the patients may relapse. the efficacy of the prophylactic or therapeutic use of systemic corticosteroids, dapsone, or nonhormonal antiinflammatories is still questionable. [ , [ ] [ ] [ ] [ ] eccrine squamous syringometaplasia is an unusual adverse reaction to chemotherapeutic drugs. it can also be found in association with chronic ulcerations, skin tumors, exposure to toxic agents, and several inflammatory processes. therefore, it is not a histopathologic reaction exclusive to the use of chemotherapeutic drugs. the mechanism of neutrophilic eccrine hidradenitis is unknown, but it can be the result of the excretion of the drug by the eccrine glands and its direct toxic effect on the eccrine epithelium. it is postulated that eccrine squamous syringometaplasia represents the final noninflammatory spectrum of adverse reactions to chemotherapeutic drugs in the eccrine glands. similarly to neh, eccrine squamous syringometaplasia also has an unspecific clinical presentation, constituted by erythematous maculae, papules, and papular plaques or vesicles, localized or disseminated. lesions develop between and days after the start of chemotherapy and improve spontaneously after weeks. the diagnosis is histopathologic, characterized by the presence of squamous metaplasia of the eccrine glands in the papillary dermis. minimal and focal necrosis of the eccrine gland epithelium, fibroblastic proliferation, and edema of the periductal stroma may occur. contrary to neh, the neutrophilic infiltrate is minimal or absent. squamous eccrine syringometaplasia has been described as an accidental histologic finding in other conditions not associated with chemotherapy. eccrine squamous syringometaplasia does not appear to be associated with a specific chemotherapy agent or malignancy. numerous drugs have been related such as cytarabine, mitoxantrone, daunorubicin, cisplatin, -fluorouracil, doxorubicin, cyclophosphamide, etoposide, methotrexate, busulfan, melphalan, and carmustine. eccrine squamous syringometaplasia has been observed in association with palmoplantar erythrodysesthesia syndrome, in radiation-induced memory reactions, and in patients who underwent bone marrow transplantation and received high doses of chemotherapeutic drugs. the condition often spontaneously resolves. [ , [ ] [ ] [ ] [ ] first described in , this syndrome is also known as burgdorf's syndrome, palmoplantar erythema, hand-foot syndrome, and toxic erythema of the palms and soles. it occurs more frequently in patients treated with cytarabine and, fluoropyrimidines, especially capecitabine, which is the oral -fluorouracil prodrug. after alopecia and mucositis, it is the most common adverse reaction to chemotherapy. other agents less frequently associated with palmoplantar erythrodysesthesia syndrome are cisplatin, cyclophosphamide, cytarabine, doxorubicin, daunorubicin, doxifluridine, etoposide, floxuridine, hydroxyurea, mercaptopurine, methotrexate, mitotane, paclitaxel, docetaxel, and vinorelbine. it is estimated that this adverse reaction occurs in - % of the patients treated with different chemotherapeutic regimens. most patients show a prodrome of dysesthesia, with a tingling (pins and needles) sensation on the palms and soles. within a few days the reaction evolves to a feeling of pain and burning with a well-demarcated edema and erythema. the erythema is symmetric and sometimes more pronounced on the soft parts of the distal phalanges. hands are often more affected than feet (fig. . a) . some patients show light scaling with or without erythema. a bullous variant has been described (fig. . b) , representing a more severe form of the reaction, specifically associated with cytarabine and methotrexate. lesions are aggravated if the treatment is not suspended, and the associated pain and edema may limit the movement of fingers. when the drug is suspended, the reaction progressively improves within weeks. in some patients, when treatment is maintained despite the development of erythrodysesthesia syndrome, palmoplantar keratodermia may occur. the reaction occurs more frequently in patients who undergo oral or continuous infusional therapy with fluoropyrimidines ( - %), as compared with those submitted to bolus therapy ( . - %). it is thought that in the pathogenesis of the process the local accumulation of the drug leads to degeneration with necrosis of the sweat glands, because its microscopic aspects are similar to those of eccrine squamous syringometaplasia and neutrophilic eccrine hidradenitis. in the differential diagnosis the following should be considered: polymorphous erythema, erythromelalgia, eccrine squamous syringometaplasia, and neutrophilic eccrine hidradenitis. the most relevant differential diagnosis is acute gvhd. the fundamental difference is that acute gvhd occurs in patients who have received a bone marrow transplant, in addition to extracutaneous involvement with gastrointestinal alterations (abdominal pain and diarrhea, elevation of hepatic enzymes). in cases of acute gvhd without extracutaneous manifestations, differentiation may be difficult. nevertheless, acute gvhd presents with diffuse erythema and can form papules, whereas palmoplantar erythrodysesthesia syndrome shows a well-demarcated erythema and edema. there are no relevant histopathologic differences between them, except for necrosis of the satellite cell in all layers of the epidermis (apoptotic keratinocytes adjacent to lymphocytes) in acute gvhd and sometimes presence of squamous syringometaplasia in palmoplantar erythrodysesthesia syndrome. the differentiation between these two disorders is essential because the use of cyclosporine is necessary to treat acute gvhd, but worsens the patient's pain if used in the treatment of palmoplantar erythrodysesthesia. apart from dose reduction, longer intervals between the cycles of chemotherapy and, as a last resort, the suspension of the drug, there is no specific treatment for palmoplantar erythrodysesthesia syndrome that has proved to be effective in a large series of cases. some treatments have been suggested for small series of patients or case reports. general measures should be taken, such as reduction or suspension of the drug, longer intervals between chemotherapy cycles, dressings, elevation of the extremity, cold compresses, analgesic medication, and emollients. as a specific treatment, pyridoxine can be used if -fluorouracil, liposomal doxorubicin, doxorubicin, docetaxel, and etoposide have been administered; hand cooling (docetaxel); oral corticosteroids (doxorubicin, -fluorouracil); strong topical corticosteroids (liposomal doxorubicin, cisplatin, and -fluorouracil); and topical dimethyl sulfoxide (dmso) at % (liposomal doxorubicin). symptoms can be relieved with lesion care to prevent infection and elevation of the limb to reduce the edema. cooling of hands and feet during treatment reduces the blood flow in these areas and may decrease the severity of the reaction. strong topical corticosteroids have been used with mixed results when associated with emollients. systemic corticosteroids are useful in some situations. pyridoxine (vitamin b ) in doses of - mg/ day can be useful to treat and prevent this reaction, except when cytarabine or vincristine is used. topical dmso at % four times a day for days has cured some cases of palmoplantar erythrodysesthesia syndrome induced by pegylated liposomal doxorubicin. [ , [ ] [ ] [ ] [ ] some authors prefer to associate toxic erythema caused by chemotherapy with clinical lesions that present with painful erythema, with or without edema, often affecting the hands and feet, intertriginous areas such as the axillary and inguinal regions, and less frequently the elbows, knees, and auricular pavilion. these eruptions may have a bullous component, are self-limited, and generally evolve with resolution and scaling associated with postinflammatory hyperpigmentation. many denominations used refer to histopathologic findings or those given by various authors on different occasions. disorders such as eccrine squamous syringometaplasia, neh, acral erythema, and palmoplantar erythrodysesthesia syndrome would be, according to these authors, grouped under toxic erythema caused by chemotherapeutic drugs. the objective to group many disorders under the same denomination seeks to emphasize the superposition of clinical characteristics and promote an easy dialogue between medical specialties and with the patient. the clinical characteristics of the toxic erythema associated with chemotherapy are: ( ) maculae or erythematous and/or edematous plaques on the hands and feet, intertriginous areas, and less frequently on the elbows, knees, and auricular pavilions, often appearing - days after the administration of the drug; ( ) associated symptoms of pain (that may be debilitating), burning, paresthesia, pruritus, and/or hypersensitivity; ( ) pale color, petechiae, and/or sterile blisters, followed by erosion in areas of intense erythema; ( ) scaling and spontaneous resolution without specific treatment; and ( ) chance of relapse if an equal or higher dose is administered. isolated papules may be found in the periphery of plaques. papules and plaques may also be found in the head, cervical region, trunk, and extremities. onset of lesions after - months can be observed. the histologic characteristics observed are atypia (larger cells and nuclei and nuclear pleomorphism), apoptosis of keratinocytes, mitotic figures and bizarre mitotic configurations (astral mitosis), loss of polarity of the epidermal cells and apoptosis of keratinocytes, vacuolar degeneration of the basal layer of the epidermis, dermal edema, and eccrine squamous syringometaplasia. moreover, necrosis of the upper epidermis, similar to the alterations observed in pellagra, may also occur. the inflammatory infiltrates are usually minimal despite their abundant clinical profile. from these observations, it has been suggested that erythema is secondary and results from damage to keratinocytes, leading to the release of cytokines and vasodilation. [ , [ ] [ ] [ ] [ ] acneiform eruption is the adverse effect more often associated with the use of egfr inhibitors. onset occurs week after the start of treatment with the egfr inhibitor as a self-limiting eruption, dose-related, that affects the face, central region of the thorax, upper dorsum and, more rarely, limbs. it presents with follicular erythematous papules, pustules with or without comedones, and scaling of the interfollicular skin (fig. . ) . often an association with the following conditions is observed: acral asteatosis, paronychia with pyogenic granuloma, oral and nasal aphthous ulcerations, and hair alterations. palms and soles are often free of lesions. excessive follicular hyperkeratosis leading to the obstruction of the ostium with formation of a follicular corneal plug, rupture of the glandular wall, and consequent inflammatory process are suggested as pathogenic mechanisms. in the histopathologic examination a prominent corneal plug, with dilated infundibulum, with or without neutrophilic folliculitis, is observed. there is a positive correlation between the severity of the eruption and the tumoral response and survival. we emphasize the need for attention to the eruption to improve adherence to the chemotherapeutic treatment. the use of topical anti-acne agents and oral tetracyclines improve the condition. topical emollients are indicated to treat xerosis. [ , [ ] [ ] [ ] [ ] stomatitis [ , [ ] [ ] [ ] [ ] oral mucositis is the main dose-limiting reaction of most chemotherapeutic drugs. about % of the patients being treated show some type of oral complication. these complications are often associated with drugs that affect the synthesis of dna. the main causative agents are antimetabolic drugs and antitumoral antibiotics. the drugs more frequently associated with stomatitis are bleomycin, dactinomycin, methotrexate, topotecan, and -fluorouracil. unusually, the stomatitis caused by -fluorouracil is related to its continuous infusional administration or to the use of its oral prodrug, capecitabine, and is less frequently observed when -fluorouracil is administrated in bolus. the main mechanism is the direct toxicity of the drug, but it can result secondarily from the indirect effects of the drug on the bone marrow. in patients with head and neck tumors, cisplatin used during radiotherapy acts as a strong radiosensitizer. in these cases there is more tumoral control but also greater severity of stomatitis caused by a boost in the direct effect of radiotherapy. since oral epithelium cells have a high mitotic index (renewal every - days), they become susceptible to the toxic effects of chemotherapeutic drugs. moreover, there is atrophy of the oral mucosa, causing odynophagia, burning, xerostomia, and mucous membrane ulcerations. ulcerations may be initially focal and then become diffuse and confluent, with occasional vesicles and blisters. these alterations are more common in the nonkeratinized mucosa and appear - days after use of the drug. resolution of lesions may occur after treatment is suspended, often within - weeks. spontaneous or induced hemorrhage, especially gingival, may occur when the platelet count is below , /mm . patients at a higher risk of developing stomatitis are those with hematologic neoplasms, those who are under years old (high mitotic activity of the epithelium), and patients with pre-existing oral disease and poor mouth hygiene. preventive measures include proper maintenance of oral hygiene by washing the mouth with water, saline solution, sodium bicarbonate, or hydrogen peroxide. the use of cold water to prevent mucositis induced by -fluorouracil and melphalan in high doses appears to be helpful. other alternative clinical procedures, still not fully proven, consist in the use of chlorhexidine gluconate, β-carotene, and benzydamine chlorhydrate or sucralfate. treatment essentially consists of support with oral care, using agents such as magnesium or aluminum hydroxide and vitamin e. in addition, pain-relief drugs such as paracetamol and opioids (codeine and morphine) may be necessary when the use of topical anesthetics such as benzocaine and lidocaine are not effective. additional complications occur because of secondary bacterial, viral, or fungal infections that may become systemic. palifermin, when used prophylactically, reduces the occurrence and duration of severe stomatitis in patients with hematologic tumors and submitted to bone marrow transplantation. palifermin is a human recombinant factor of keratinocyte growth and protects various epithelial tissues. it acts not only on stomatitis but also on mucositis in general. a possible tumoral stimulating factor still limits its use in patients with epithelial tumors. hyperthermic chemotherapy with mitomycin c [ ] [ ] [ ] peritoneal carcinomatosis frequently signals the terminal stage in some cancers of gastrointestinal and gynecologic origins. cytoreductive surgery and intraperitoneal hyperthermic chemotherapy was introduced in the early s and has become the mainstay of treatment in particular clinical settings for patients with peritoneal carcinomatoses, especially in pseudomyxoma peritonei, significantly improving overall survival rates. there are four case reports of this recently described entity using a new procedure to treat peritoneal carcinomatosis after intraperitoneal hyperthermic chemotherapy with mitomycin c. one of them was described [ ] in a male patient after days of the procedure. the patient developed pain and scrotal necrosis on the anterior aspect of the scrotum (fig. . ) . two possible causes of the scrotal ulcers were proposed. (i) a patent processus vaginalis, allowing mitomycin c to become sequestered in the scrotum, inducing an inflammatory reaction, resulting in scrotal wall inflammation and subsequent ulceration (fig. . ). this was proposed since previous studies have shown that intradermal administration of mitomycin c inhibits wound healing and induces skin necrosis. (ii) local spillage of mitomycin c onto the scrotal skin, with resulting inflammation and ulceration. previous studies with patients on continuous ambulatory peritoneal dialysis (capd) have shown that % of capd patients developed genital swelling [ ] . a possible cause of the scrotal swelling is a patent processus vaginalis causing a communicating hydrocele, which can be found in - % of adult men and may not be clinically evident until capd has begun. patients may develop this complication months after the surgery; hence, any complaint of scrotal pain or discomfort in these patients should warrant immediate investigation and attention, even if the complaints present much later. our group suggests that an image investigation, such as computed tomographic peritoneography, should be considered for male patients prior to intraperitoneal chemotherapy, since this complication is potentially serious for the patient. vasomotor changes [ ] various vascular alterations have been described, probably as a result of a direct effect on arterial smooth muscle fibers or by acting on the autonomic nervous system. manifestations may include blood vessel spasms with livedo, raynaud's phenomenon, and distal necroses, which may be triggered by bleomycin and cisplatin. vasodilatation with erythema and flushing may result from the use of bleomycin, cisplatin, asparaginase, dacarbazine, taxanes, -fluorouracil, doxorubicin, cyclophosphamide, gefitinib, and carmustine. flushing [ ] flushing consists of a temporary erythema of the face, neck, upper chest, ears, or upper abdomen. the mechanism responsible for flushing is a transitory vasodilation mediated by the autonomic nervous system or by the direct effect of circulating substances that act on the musculature of the vessel walls. the nerves of the autonomic nervous system also control the sweat glands so that flushing mediated by these nerves is known as "wet flushing," whereas when the substance acts directly on the vascular wall muscles it is known as "dry flushing." derivatives of biological agents probable mechanism of mitomycin c deposition in a patent processus vaginalis into the scrotum such as l-asparaginase and bleomycin are notorious for causing flushing, which occurs soon after infusion. irinotecan, a topoisomerase i inhibitor, causes dysautonomia, the symptoms of which include diarrhea, bradycardia, and flushing. hormonal agents such as antiestrogens (tamoxifen, anastrozole), lhrh analogs (leuprolide), and antiandrogens (flutamide and diethylstilbestrol) may result in flushing. other agents that also deserve mention include -fluorouracil, carboplatin, cisplatin, cyclophosphamide, dacarbazine, doxorubicin, etoposide, and methotrexate. interaction with uv light [ ] eruptions resulting from photosensitivity are caused by various agents, principally following exposure to uv radiation. phototoxicity caused by dacarbazine, fluoropyrimidines (systemic -fluorouracil, topical -fluorouracil, tegafur, and capecitabine) and vinblastine has been well documented. phototoxicity caused by dactinomycin, doxorubicin, hydroxyurea, procarbazine, brequinar sodium, mitomycin, -thioguanine, and flutamide, as well as by the porphyrin compounds that are used in photodynamic therapy, is uncommon. reactivation of sunburn is a well-documented adverse effect following the use of methotrexate. it occurs when the drug is administered - days after exposure to uv radiation, when the erythema from the previous exposure has been in the process of disappearing. leucovorin does not prevent this reaction. phototoxic reactions resemble intense sunburn in areas of the skin that are exposed to light, with erythema, edema, pain, or pruritus. blisters may be present and desquamation may occur in severe cases. residual hyperpigmentation may persist for months. hydroxyurea has been described as being associated with development of dermatomyositislike eruption due to photosensitivity (fig. . ) [ , ] . hydroxyurea is an anticancer agent that inhibits dna synthesis through its action on the enzyme ribonucleotide reductase [ ] . it is used in chronic myeloproliferative diseases such as polycythemia vera, chronic myeloid leukemia, and essential thrombocytosis, although it has also been prescribed to patients with refractory psoriasis [ ] . patients on long-term therapy with hydroxyurea can develop various side effects, including a wide variety of mucocutaneous manifestations, which appear in - % of patients [ ] . the most common skin changes are facial erythema, hyperpigmentation, xerosis, alopecia, skin atrophy, melanonychia, and ulcers on the [ ] . other less frequent adverse effects are dermatomyositis-like rash and nonmelanoma skin cancer (fig. . ) [ ] . dermatomyositis-like rash resembles true dermatomyositis both clinically and histologically [ ] . it presents as desquamating erythematous papules or plaques on the dorsum of the hands, typically associated with facial erythema and pronounced xerosis of the skin. patients rarely report other accompanying symptoms and there are usually no significant alterations of laboratory tests [ ] . histologically, a lichenoid inflammatory infiltrate is found at the dermoepidermal interface, with vacuolization of the basal layer, dyskeratosis, and, rarely, mucin deposition ( fig. . ) [ ] . diagnosis is made according to the distribution of the lesions and by the temporal relationship between chemotherapy and light exposure. treatment includes discontinuation of the agent and protection from the sun for at least weeks. physical sunscreens are recommended. cold compresses, systemic antihistamines, and topical or oral corticosteroids are used as associated symptomatic treatment. [ ] this is a phenomenon whereby the chemotherapeutic agent induces an inflammatory reaction in an area previously exposed to radiation. these reactions are predominantly cutaneous; however, they may affect internal organs such as the lungs, heart, bladder mucosa, esophagus, oral or bowel mucosa, and supraglottic larynx. it occurs more often with the use of doxorubicin, dactinomycin, and gemcitabine and is less common with bleomycin, etoposide, hydroxyurea, methotrexate, trimetrexate, vinblastine, -fluorouracil, lomustine, daunorubicin, melphalan, cyclophosphamide, cytarabine, docetaxel, edatrexate, idarubicin, paclitaxel, tamoxifen, and vinblastine. the mechanism of radiation recall is unknown but it is probably related to dna repair. relapsing dermatitis or radiation recall may occur between and days following radiotherapy and generally appears hours to days after administration of the chemotherapeutic agent. clinically, the patient may or may not experience a painful erythema with or without vesiculation, edema, desquamation, and pruritus. the borders of the lesion are well defined and correspond to the exact site at which the radiation was applied. in severe cases, necrosis and ulceration may occur. the severity appears to directly reflect the brevity between radiation and chemotherapy as well as the doses of both radiation and chemotherapy. the reaction improves spontaneously within hours or weeks following cessation of chemotherapy, treatment being symptomatic. the use of systemic corticosteroids associated with the discontinuation of chemotherapy generally results in a marked improvement and may permit reintroduction of the treatment. [ ] this occurs when a chemotherapeutic agent increases the toxicity of radiotherapy. this phenomenon may occur in virtually all the organs of the body including the skin, mucosa, esophagus, lungs, heart, digestive tract, kidneys, liver, brain, bladder, and eyes. the agents most associated with exacerbation of radiation are bleomycin, gemcitabine, dactinomycin, doxorubicin, -fluorouracil, hydroxyurea, -mercaptopurine, oxaliplatin, and methotrexate. clinically, the reaction resembles residual dermatitis secondary to acute dermatitis from radiation, with erythema, edema, vesiculation, blisters, or erosions. the reaction generally appears at the site of radiation; however, the area affected may be more extensive. severe mucositis may occur. the reaction is associated with the dose, the type of drug used, and the sequence of time between radiation and the use of chemotherapy. toxicity may be additive or supra-additive (synergic). in supra-additive toxicity, the reaction is greater than that of the sum of each one of the types of treatment alone. treatment is symptomatic: applying cold compresses, taking precautions at the site to prevent infection and avoiding trauma, heat, and uv light. sequelae such as fibrosis, skin atrophy, and telangiectasia-related disorders may occur. [ ] in theory, all chemotherapeutic agents may trigger hypersensitivity eruptions. with certain drugs derived from biological agents such as l-asparaginase, mitomycin c, and bleomycin in addition to paclitaxel, the incidence of hypersensitivity reactions is high. in the case of paclitaxel, this is due to the fact that it is dissolved in cremophor el castor oil. according to the classification system defined by gell and coombs, the majority of hypersensitivity reactions are type i, i.e., ige-mediated. they present as urticaria, pruritus, angioedema, and anaphylaxis. they generally occur within the first hour after use of the drug, but onset may be delayed until up to h after using the medication. type iii reactions occur because of formation of circulating immunocomplexes, and cause eruptions such as polymorphous erythema and vasculitis. nonetheless, l-asparaginase and procarbazine cause urticarial reactions via type iii reactions. allergic contact dermatitis, a type iv reaction, may occur, principally as a consequence of the topical use of nitrogen mustard (mechlorethamine). other severe reactions may occur, such as sjs and ten, as well as exanthematous eruptions, all currently classified as type iv reactions according to the extended gell and coombs classification, i.e., sjs and ten, respectively (type ivc, mediated by fas, granzymes, and perforin) and exanthematous eruptions (type ivb, mediated by t cells with il- production, with chemotaxis of eosinophils). agents [ ] local toxicity [ ] antineoplastic drugs may be classified according to their potential aggressiveness toward blood vessels and adjacent tissues. they may be nonirritating, irritating, or vesicant, causing effects that range from mere local discomfort to tissue necrosis. nonirritating drugs (thioguanine, asparaginase, bleomycin, cyclophosphamide, chlorambucil, methotrexate, hydroxyurea) provoke an edema that is indicative of a site of extravasation; however, they do not cause necrosis or tissue irritation. irritating drugs ( -fluorouracil, carmustine, docetaxel, and etoposide) cause tissue damage that does not progress to necrosis. they trigger erythema, pain, inflammation at the puncture site and along the venous pathway, burning, and local edema, without blistering. the vesicant drugs (dactinomycin, doxorubicin, melphalan, vincristine, vinblastine, and dacarbazine) cause severe skin irritation with pain, erythema, edema, blistering, and necrosis with functional and aesthetic damage. [ ] this is defined as the leakage of a chemotherapeutic drug from the vessel bed to the surrounding tissues, either as a result of vascular rupture or by direct infiltration. the frequency of this event in adults is estimated at . - % and it is more common among children. severe sequelae are rare. the severity of tissue damage is related to the type of chemotherapeutic agent used and the quantity and concentration of the drug administered. cytotoxic agents are classified as irritants or vesicants as a function of their potential for local toxicity. an irritant is defined as an agent that causes an inflammatory reaction, paresthesia, pain, or phlebitis at the puncture site or along the venous pathway. clinical signs include sclerosis and hyperchromia along the passage, as well as burning, increased temperature at the site, discomfort, erythema, and pain at the area of extravasation. necrosis does not occur with this condition. the symptoms are generally short-lived and leave no sequelae. the drugs most associated with this complication are -fluorouracil, carboplatin, cisplatin, bleomycin, mitomycin, dactinomycin, idarubicin, daunorubicin, dacarbazine, ifosfamide, cyclophosphamide, mechlorethamine, carmustine, mitoxantrone, paclitaxel, docetaxel, streptozotocin, vinblastine, vinorelbine, and etoposide. the vesicant agents (melphalan, bleomycin, mechlorethamine, carmustine, mitomycin, mitoxantrone, cisplatin, paclitaxel, dacarbazine, dactinomycin, daunorubicin, streptozotocin, doxorubicin, epirubicin, vinblastine, vincristine, etoposide, vindesine, and vinorelbine) have the potential to cause more severe and long-lasting tissue damage, including necrosis of the affected area. the initial manifestations are often subclinical and may appear immediately following extravasation or after several days or weeks. the initial signs include local burning or paresthesia at the site of infusion, mild erythema, pruritus, and edema. a change in the infusion rate or the absence of venous return in the aspirate may indicate the occurrence of extravasation. after - days, erythema increases and there is pain, a brownish discoloration, induration, dry desquamation, or the appearance of blisters. if the amount extravasated was small, the signs and symptoms may disappear in the following weeks. if a significant amount was extravasated, the following symptoms may appear in the coming weeks: necrosis, formation of eschar and painful, necrotic ulceration with raised, erythematous borders and a yellowish base. there is generally no granulation tissue with these ulcerations. they may resolve slowly or persist, increasing gradually in area. involvement of the tendons, nerves, and vessels may occur if appropriate treatment is not given, leading to severe sequelae such as nerve compression syndrome, a reduction in joint mobility, contractures, neural deficits, and reflex sympathetic dystrophy. cellulitis and the formation of abscesses are rare events. the interval between detecting the condition and adopting the appropriate measures should be short as possible. the nursing team should be trained in this respect. preventive measures should be adopted such as avoiding puncturing emaciated limbs, lower limbs, limbs with multiple punctures, limbs with phlebitis or those that have been subjected to radiation, the ipsilateral limb to a mastectomy, in vena cava syndrome, and in veins that protect joints, nerves, and tendons. it is important to evaluate the venous conditions of the patient and, if necessary, to use an indwelling catheter. the use of common needles for venous access should be avoided. adequate fixation should be performed and blood reflux should be tested, with an infusion of . % saline solution or % glucose-saline solution used for every ml of the chemotherapeutic agent. after administration of all the drugs, ml of saline or glucose-saline solution should be infused to reduce any possibly toxic residues. vesicant drugs should always be given first. in prolonged sessions of chemotherapy (those lasting over an hour) with vesicant drugs, central venous access should be used. always listen to the patient's complaints. if extravasation occurs, stop the infusion immediately. remove the puncture device and elevate the affected limb. in the case of extravasation of drugs such as etoposide, paclitaxel, vinblastine, vincristine, and vinorelbine, apply local heat (leading to vasodilation and dilution of the drug) for min and ice (venous constriction and greater degradation of the toxic metabolites in addition to alleviating pain and inflammation) every min, six times a day in the first h. for the other drugs, apply ice every min, six times a day. when indicated, the specific antidote for the drug in question should be used. the use of intralesional corticosteroid and sodium bicarbonate should be avoided. ulcers that fail to heal may require debridement and grafting. in case of persistent edema and erythema and pain without ulceration that persists despite conservative therapy or in the presence of extensive areas of necrotic tissue or skin ulceration, surgery may be indicated. periorbital edema [ ] edema of the eyelids has been described with the use of gemcitabine. recovery [ ] cutaneous eruption of lymphocyte recovery (elr) is observed in leukemia patients who receive bone marrow ablation. in general, it appears between the th and st days after chemotherapy. this point corresponds to the beginning of the recovery of peripheral lymphocytes following the nadir of leukocyte count induced by chemotherapy. although the exact mechanism has yet to be clarified, it is believed that the eruption is caused by the return of immunocompetent lymphocytes to peripheral circulation with cutaneous cytotoxicity. t lymphocytes and langerhans cells are found on histopathologic evaluation of these reaction sites. clinically, the condition consists of pruriginous, erythematous macules, papules, or papulous plaques that become confluent. erythrodermia may occur. in addition, this condition is associated with an elevation in body temperature that occurs together with the appearance of the eruption. the temperature falls in the following - days and the skin eruption tends to diminish after several days, progressing with desquamation and mild residual hyperchromia. the drugs most associated with these reactions are cytarabine, daunorubicin, amsacrine, etoposide, cyclophosphamide, and vincristine. differential diagnosis should be made with sepsis, viral exanthems, gvdh, leukemia or lymphoma cutis, and drug hypersensitivity or toxicity. histopathology is nonspecific. the most characteristic findings are superficial perivascular mononuclear cell infiltrate, mild epidermal alterations such as spongiosis, vacuolar alteration of the basal cell layer, and loss of keratinocyte maturation secondary to chemotherapy. dyskeratotic keratinocytes are rare and eosinophils are absent. on occasions when the patient was treated with gm-csf associated with il- , atypical lymphocytes with large pleomorphic and hyperchromatic nuclei were found at histopathology. differentiation may be difficult between elr and gvhd. receptor tyrosine kinase inhibitors [ ] anti-egfrs currently consist of panitumumab, cetuximab, erlotinib, and gefitinib. skin toxicity with anti-egfrs is actually more of a pharmacologic effect than a hypersensitivity reaction, since this is a clinical marker of the efficacy of the inhibiting effect of these drugs on the tumor, with the severity of the eruption corresponding to tumor response. the skin effects observed with the anti-egfr are alterations in capillary growth and in the texture of the hair, paronychia with or without secondary infection, or the formation of pyogenic granuloma, generalized asteatosis, skin desquamation, and blepharitis. the most characteristic and intense manifestation is a papulopustular, follicular, comedone, or non-comedo (acneiform eruption) that occurs on the head, neck, and the central portion of the chest and back, which later disseminates. there may be pruritus, which differentiates this reaction from the acneiform eruptions caused by corticosteroids, antiepileptic drugs, and vitamins b and b . acneiform eruptions occur in more than % of patients with use of cetuximab, and this percentage may reach as high as - %. the manifestations generally occur in the first weeks ( days to weeks) after the beginning of treatment (cetuximab and panitumumab). the eruption is dose dependent; however, the duration of the condition does not correlate with the duration of treatment. the acneiform eruptions induced by monoclonal antibodies are more severe and extensive than those resulting from the use of tyrosine kinase inhibitors. blepharitis caused by anti-egfrs may range from mild to intense. histopathology of the papulopustular lesions shows no increase in sebaceous gland activity, comedones, or follicular rupture that would explain the inflammation, differentiating it from acne vulgaris. the follicles are rather wide and at times obstructed by an excess of keratinocytes. in the dermis, neutrophilic infiltrate may be found, particularly involving the follicular infundibulum. intraepidermal acantholysis may be present in association with the eccrine gland ducts. in the lesions of patients using gefitinib, there is an expressive thinning of the stratum corneum layer with loss of the normal basket-weave pattern. paronychia occurs in around - % of patients who are using cetuximab and gefitinib, appearing at - weeks of treatment or sometimes after months. it affects various fingers and the first toes. treatment consists of potent topical corticosteroids. in case of onychocryptosis, anti-egfr may be temporarily interrupted and canthotomy performed. asteatosis occurs in around % of patients, particularly with the use of gefitinib. there is a predilection for the areas previously or simultaneously affected by acneiform eruption. some patients have xerosis of the vaginal mucosa, with dysuria. xerosis may progress to chronic asteatotic eczema (fig. . ) , with a greater susceptibility to staphylococcus aureus infection or hhv- . emollients and topical corticosteroids should be used for the eczema. fissures can be treated with a solution of % propylene glycol under plastic occlusion or a hydrocolloid dressing. adverse drug reactions (adrs) include all unintended pharmacologic effects of a drug except therapeutic failures, intentional overdosage, abuse of the drug, or errors in administration. they can be classified as predictable (type a - % of the adrs) or unpredictable (type b). anaphylaxis an immediate systemic reaction that occurs when a previously sensitized individual is re-exposed to an allergen. it is caused by rapid ige-mediated immune release of vasoactive mediators from tissue mast cells and blood basophils with a potential late component. this is a systemic severe adr affecting skin, mucous membranes, gastrointestinal tract, respiratory tract, and cardiovascular system. drug allergy an immunologically mediated response to a pharmaceutical and/or formulation (excipient) agent in a previous sensitized patient. drug idiosyncrasy an abnormal and unexpected effect that is unrelated to the intended pharmacologic action of a drug and has an unknown mechanism. it is not mediated by a humoral or cellular immune response but is reproducible on readministration. it may be due to underlying abnormalities of metabolism, excretion, or bioavailability. drug intolerance an undesirable pharmacologic effect that may occur at low or conventional doses of the drug without underlying abnormalities of metabolism, excretion, or bioavailability of the drug. humoral or cellular immune mechanisms are not thought to be involved, and a definitive mechanism for such exaggerated responses has not been established (e.g., acetylsalicylic acid-induced tinnitus at low doses). immediate systemic reactions that mimic anaphylaxis but are caused by non-ige-mediated release of mediators from mast cells and basophils. often caused by radiocontrast agents. severe adrs include all adverse effects which are unpredictable life-threatening adrs and need prompt recognition to reduce integumentary and internal organ damage and, thus, morbidity and mortality. uncomplicated adrs include mild or moderate adverse drug effects on healthy patients, not involving life-threatening situations. diagnostic testing for drug hypersensitivity drug-induced reactions: a report from the boston collaborative drug surveillance program on . consecutive inpatients sémilogie et marqueurs de sévérité des toxidermies érythémateuses severe adverse cutaneous reaction to drugs the nature of adverse events in hospitalized patients: results of the harvard medical practice study ii cutaneous drug reactions: clinical types and causative agents: a five-fig. . intense xerosis caused by erlotinib year survey of in-patients ( - ) joint task force on practice parameters of american academy of allergy, asthma and immunology, american college of allergy, asthma and immunology, joint concil of allergy, asthma and immunology anaphylaxis and anaphylactoid reactions. a guide to prevention, recognition and emergent treatment erythroderma: analysis of cases dermatitis exfoliativa: cases admitted in the decade - to the dermatological clinic, karolinska sjukhuset exfoliative dermatitis: a prospective study of patients dermatology in general medicine advances in toxic epidermal necrolysis toxic epidermal necrolysis erythema multiforme: a critical review of characteristics, diagnostic criteria, and causes acute disseminated epidermal necrosis types , , and : study of sixty cases erythema multiforme major and stevens-johnson syndrome are clinically different disorders with distinct etiologies clinical classification of cases of toxic epidermal necrolysis, stevens-johnson syndrome and erythema multiforme epidemiology of erythema exsudativum multiforme majus, stevens-jonhson syndrome, and toxic epidermal necrolysis in germany ( - ): structure and results of a populationbased registry toxic epidermal necrolysis (lyell syndrome). incidence and drug etiology in france the incidence of erythema multiforme, stevens-johnson syndrome, and toxic epidermal necrolysis. a population-based study with particular reference to reactions caused by drugs among outpatients toxic epidermal necrolysis (lyell syndrome) intravenous immunoglobulin therapy for stevens-johnson syndrome effectiveness of early therapy with corticosteroids in stevens-johnson syndrome: experience with cases and a hypothesis regarding pathogenesis treatment of severe drug reactions: stevens-johnson syndrome, toxic epidermal necrolysis and hypersensitivity syndrome o espectro do eritema multiforme (eritema multiforme minor e major) e o espectro da síndrome de stevens-johnson e da necrólise epidérmica tóxica (síndrome de lyell) low n-acetylating capacity in patients with stevens-johnson syndrome and toxic epidermal necrolysis a slow acetylator genotype is a risk factor for sulphonamide-indiced toxic epidermal necrolysis and stevens-johnson syndrome cutaneous adverse drug reactions. stevens-johnson syndrome and toxic epidermal necrolysis delayed reactions to drugs show levels of perforin, granzyme b, and fas-l to be related to disease severity increased interleukin , tumor necrosis factor a, and interleukin levels in blister fluid of toxic epidermal necrolysis drug rashes. what are the targets of cell-mediated cytotoxicity? treatment of toxic epidermal necrolysis with high-dose intravenous immunoglobulins intravenous immunoglobulin treatment for stevens-johnson syndrome and toxic epidermal necrolysis treatment of toxic epidermal necrolysis epidemiology of druginduced severe skin reactions correlations between clinical patterns and causes of erythema multiforme majus, stevens-johnson syndrome, and toxic epidermal necrolysis medication use and the risk of stevens-johnson syndrome or toxic epidermal necrolysis severe cutaneous adverse reactions to drugs -relevant aspects to diagnosis and treatment -part i: anaphylaxis and anaphylactoid reactions, erythroderma and the clinical spectrum of stevens-johnson syndrome and toxic epidermal necrolysis (lyell's disease) drug reaction with eosinophilia and systemic symptoms (dress): a complex interaction of drugs, viruses and the immune system drug reaction with eosinophilia and systemic symptoms (dress)/drug-induced hypersensitivity syndrome (dihs): a review of current concepts drug reaction with eosinophilia and systemic symptoms/drug-inducedhypersensitivity syndrome: clinical features of patients adverse mucocutaneous reactions to chemotherapeutic agents: part i severe cutaneous adverse drug reactions: relevant aspects to diagnosis and treatmentpart ii propylthiouracil-induced vasculitis with antineutrophil cytoplasmic antibody approach to the patient with a suspected cutaneous adverse drug reaction adverse drug reactions and organ damage: the skin a review of cutaneous drug eruptions dermal dendrocytes fxiiia+ phagocytizing extruded mast cell granules in drug-induced acute urticaria fixed drug eruption: state of the art evidence for acne-promoting effects of milk and other insulinotropic dairy products lichenoid drug eruption with proton pump inhibitors getting under the skin of adverse drug reactions cutaneous adverse reactions of amiodarone phototoxicity and photocarcinogenesis associated with voriconazole an update on pediatric cutaneous drug eruptions coma blisters, peripheral neuropathy, amitriptyline overdose: a brief report in vitro interferon-gamma release test in the diagnosis of druginduced erythema nodosum drug induced psoriasis advances in the diagnosis of drug eruptions baboon syndrome resulting from systemic drugs: is there strife between sdrife and allergic contact dermatitis syndrome? contact dermatitis a new proposal for a clinical-oriented subclassification of baboon syndrome and a review of baboon syndrome nicolau syndrome: an iatrogenic cutaneous necrosis complications of the injectable fillers, part : vascular complications cutaneous drug reactions in the pediatric population chemotherapeutic agents and the skin: an update mucocutaneous reactions to chemotherapy cutaneous reactionsto chemotherapy drugs: the art of consultation reações cutâneas desencadeadas por drogas scrotal ulcer developed after intraperitoneal hyperthermic chemotherapy with mitomycin-c scrotal ulcer after intraperitoneal hyperthermic chemotherapy scrotal pain and ulceration post hipec: a case report adverse mucocutaneous reactions related to chemotherapeutic agents: part ii dermatomyositis-like eruption associated with hydroxyurea therapy: a premalignant condition key: cord- -i hbx pz authors: matthews, abigail a.; ee, pui lai rachel; ge, ruowen title: developing inhaled protein therapeutics for lung diseases date: - - journal: mol biomed doi: . /s - - -z sha: doc_id: cord_uid: i hbx pz biologic therapeutics such as protein/polypeptide drugs are conventionally administered systemically via intravenous injection for the treatment of diseases including lung diseases, although this approach leads to low target site accumulation and the potential risk for systemic side effects. in comparison, topical delivery of protein drugs to the lung via inhalation is deemed to be a more effective approach for lung diseases, as proteins would directly reach the target in the lung while exhibiting poor diffusion into the systemic circulation, leading to higher lung drug retention and efficacy while minimising toxicity to other organs. this review examines the important considerations and challenges in designing an inhaled protein therapeutics for local lung delivery: the choice of inhalation device, structural changes affecting drug deposition in diseased lungs, clearance mechanisms affecting an inhaled protein drug’s lung accumulation, protein stability, and immunogenicity. possible approaches to overcoming these issues will also be discussed. biological drugs are revolutionising the treatment and management of many serious illnesses including cancer, autoimmune disorders, and rare genetic diseases, with about a third of all new drug approvals by the food and drug administration (fda) consisting of biological drugs [ ] . however, over the past decades, the development of inhaled therapeutics for the treatment of respiratory diseases has largely been focused on small molecules (corticosteroids, β agonists, and muscarinic antagonists), with only one inhaled protein biologic drug pulmozyme® being approved by the fda to date [ ] . in the treatment of lung diseases via inhalation therapy, biological drugs such as proteins/polypeptides offer many advantages over small molecule drugs. proteins delivered via the pulmonary route could accumulate in the lungs while having a poor ability to traverse the airblood barrier due to their large molecular weight. this would result in higher target site accumulation (airway epithelial cells, alveolar macrophages, neutrophils etc) and minimise systemic toxicity, as compared to small molecule drugs that would pass easily into the systemic circulation after reaching the lungs [ ] [ ] [ ] . in addition, protein therapeutics display higher potencies (picomolar to femtomolar range) than small molecules (nanomolar range), as well as highly specific receptor binding to reduce off-target effects [ ] . notably, although peptide drugs (< kda or < - amino acids in length) share some of the characteristics with protein/polypeptide drugs such as both are composed of amino acids linked via peptide bond and both having high target specificity, most of peptide drugs are much smaller in sizes, conferring them with some distinct differences including less enzyme stability, higher tissue penetration ability etc. in addition, many peptide drugs harbour chemical modifications in the form of peptidomimetics and/or cyclization, and some have direct cell membrane penetrating ability. these peptide drugs are not covered in this review. protein therapeutics are conventionally administered via the systemic route, although this has proven to be an inefficient approach for drug delivery to the lung, not mentioning the additional danger of exposing the rest of the body vulnerable to toxicity [ , ] . for instance, monoclonal antibodies (mabs) are found at higher levels ( - , times more) in serum than in bronchoalveolar lavage (bal) fluid following intravenous administration, a trend that has been demonstrated in all species [ ] . by the same token, protein therapeutics delivered via the airways also pass poorly from the lungs into the systemic circulation. for example, only low amounts of anti-vegf-a g - mab ( . %) and cetuximab ( %), an anti-egfr mab, were present in the serum after aerosol delivery in mouse models of lung cancer [ , ] . this means that high concentrations of the protein drug can be attained in the lung via pulmonary delivery, suggesting that lower doses of inhaled protein can have an equivalent or even superior therapeutic effect for lung diseases when compared to the higher doses that would be needed from systemic administration [ ] . indeed, it was reported that the nebulised effective dose of avidinox-anchored biotinylated cetuximab was / , of the intravenous effective dose in a mouse model of advanced metastatic lung cancer [ ] . although higher pulmonary levels of protein therapeutics can be achieved through inhalation compared to systemic administration, this could be offset by the short residence time of proteins in the lung compared to plasma. proteins and antibodies are mostly cleared from the lungs within h, while plasma half-lives of full-length antibodies following intravenous injection can reach weeks and more [ ] . nevertheless, there are strategies that can be employed to increase the local residence time of protein therapeutics in the lungs, and these will be discussed in detail later on in this review. besides improving pharmacokinetic and toxicity profiles of protein therapeutics, the inhalation route is non-invasive and allows for self-administration, which could improve patient compliance [ , , ] . in , pulmozyme® (dornase alfa/deoxyribonuclease i), was introduced for the treatment of cystic fibrosis [ ] . it has been almost three decades since then, and no other inhaled protein therapeutics for topical treatment of a lung disease has reached the market, despite the aforementioned advantages that inhaled proteins possess. presently, and to the best of our knowledge, ten inhaled protein therapeutics are being assessed in clinical trials for the treatment of a range of lung diseases including asthma, cystic fibrosis, lung cancer, copd and covid- (table ) [ ] [ ] [ ] [ ] [ ] [ ] . there are also protein therapeutics such as mabs that are in the preclinical stages [ , ] . in order to drive more of these therapies into clinical development and eventually to the market, it is crucial to take into account the challenges unique to the development of these agents into potential treatments so that they may be utilised successfully for pulmonary delivery. in this review, we will discuss the challenges in developing an inhaled protein therapeutic for lung diseases, as well as approaches that could help to circumvent these issues. the focus of this review is on the pulmonary delivery of protein drugs for local action in the lung, however, examples of inhaled proteins for systemic action will be mentioned wherever relevant. challenges and considerations in the development of protein therapeutics for local lung delivery choice and limitations of drug delivery device there are mainly three classes of inhalation devices namely dry powder inhalers (dpis), nebulisers, and metered dose inhalers (mdis). dpis deliver drug as a solid aerosol, and powder formulations possess inherent stability and shelf life benefits [ , ] . however, the temperature and shear stress during the manufacturing processes needed to produce powders (e.g. freeze drying, spray drying) could lead to protein degradation [ ] . dpis have been used for the marketed inhalable insulin formulations exubera® (approved in , but was discontinued after year due to large device size, high pricing, and safety concerns) and afrezza® (still commercially available) [ ] . moreover, dpis have shown promising results in studies assessing their use for inhaled protein formulations. for example, weers et al. ( ) showed that dry powder formulations of csj (antithymic stromal lymphopoietin (tslp) mab fragment) could achieve a total lung dose (tld) of about % of the delivered dose (dd) with the use of particle engineering techniques such as via the introduction of surface corrugation through the addition of trileucine [ ] . nebulisers (jet, ultrasonic, and mesh) generate aerosol droplets from a liquid solution of the drug [ ] . the first and only inhaled protein formulation approved for pulmonary delivery to date, pulmozyme®, is administered via jet nebuliser. nebulised formulations are less expensive to produce and test, because the manufacturing process for these formulations does not include extra drying steps. nevertheless, prolonged storage of proteins in liquid solutions can lead to protein instability through degradation pathways (i.e. deamidation and hydrolysis), temperature and ph changes, and aggregation (through agitation of the aqueous carrier) [ ] . furthermore, across all nebuliser types, the process of nebulisation exposes the protein to physical stresses such as shearing forces and heat, as well as the large air-liquid interface (ali) that could alter protein conformation and/or structure through denaturation, chemical modifications (oxidation, deamidation), and aggregation [ , ] . device-specific limitations such as the shear forces generated by jet nebulisers, and the temperature increases that occur in ultrasonic nebulisers, can also lead to protein degradation. jet and ultrasonic nebulisers actually recycle % of the primary aerosol, and a molecule would typically be subjected to - cycles of nebulisation before leaving the nebuliser as a secondary aerosol [ ] . this subjects the molecules to high shear stress in these devices, resulting in the denaturation of proteins, with the extent of protein denaturation and degradation varies depending on the characteristics of the individual protein [ ] . for example, for jet nebulizer delivery of ldh and urease, there is a log-linear degradation with a fraction of protein degraded with every recirculation [ , ] . in contrast, igg and g-csf has a rapid initial decline in native proteins in the first - min [ , ] . for ultrasonic nebulizers, heating resulting from ultrasonic radiation in addition to aerosol recirculation generated various protein denaturation and degradation in different proteins [ ] . for example, the degradation of ldh with ultrasonic nebulizer presented a sigmoidal progression instead, indicating different denaturation process and factors involved from jet nebulizers [ ] . by comparison, vibrating mesh nebulisers employ single-pass technology, ensuring that there is no recirculation of droplets into the reservoir; they do not alter solution temperature, and produce less shear forces inside the drug reservoir during nebulisation, making them more suitable for the delivery of protein therapeutics [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, several studies have reported that jet and ultrasonic nebulisers produce lower levels of activity, lower amounts of protein monomers (because of partial degradation), and more aggregates (with or without excipients). on the other hand, mesh nebulisers appear to maintain protein integrity to a greater extent than other nebulisers [ ] . safe aerosolisation with mesh nebulisers has already been demonstrated in several studies of labile drugs including proteins and mabs [ ] [ ] [ ] [ ] [ ] [ ] . the detailed designs and comparisons of various types of nebulizers have been reviewed previously and we will not elaborate further here [ , [ ] [ ] [ ] ] . recently, nanoengineered particles using metal-phenolic networks (mpns) with highly defined physical properties have been used to encapsulate both small molecule and macromolecules including proteins for pulmonary delivery via nebulisation. intratracheal nebulization delivery of fitc-labelled bovine serum albumin (bsa, kda) in mice demonstrated that these capsules are biocompatible and biodegradable, showing > % of the capsules in the lung after h, while only < % remaining after days without causing obvious lung inflammation or toxicity. although still in early stage of development, these mpn particles may revolutionize the nebulization delivery of protein drugs and provide a more protected environment for effective pulmonary delivery [ ] . moreover, new generation nebulisers are being developed such as surface acoustic wave (saw) nebulisers and the more recent hydra (hybrid resonant acoustics) nebulisers that provide new platforms for inhaled drug delivery. saw use surface waves to generate aerosols which can preserve macromolecule integrity that has been shown to be efficient in aerosolising proteins [ ] . hydra uses a hybrid combination of surface and bulk sound waves to generate the aerosol droplets, and can overcome the low nebulisation rate of saw nebulisers and conventional nebulisers, while also avoiding the potential damage to proteins due to high shear (jet nebulisation) or cavitation (ultrasonic nebulisation). the first human lung deposition study using a prototype hydra nebuliser has been reported recently, indicating successful lung deposition of a radiolabelled small molecule [ ] . it is probable that hydra nebulisers may be developed for pulmonary protein drug delivery. mdis deliver drug through an aerosol burst, and allow for the controlled delivery of specific amounts of drug to the lungs [ , ] . however, as with nebulisers, the use of aqueous solutions is not ideal for protein storage [ ] . there are also concerns that the hydrofluoroalkane (hfa) propellants used in mdis could denature proteins [ , ] . despite this concern, there are examples of studies showing that proteins can remain stable in hfa-containing mdi formulations. quinn et al. ( ) utilised raman spectroscopy to analyse the secondary conformations of lysozyme in the hfa propellants tetrafluoroethane (hfa a) and heptafluoropropane (hfa ), demonstrating that structural integrity of lysozyme was preserved in both hfas, and that there is potential for proteins to be developed as mdi formulations without compromising their conformational stability [ ] . moreover, liao et al. ( ) demonstrated that spray-dried lysozyme and catalase that were stabilised with excipients (sugars and/or % polyvinyl alcohol) and then stored in hfa a at room temperature for months showed retention of biological activity [ ] . when choosing an inhalation device, it is important to be cognisant of the fact that not all devices in the same category are equivalent. for instance, although it is generally accepted that there is minimal heating of the drug reservoir in vibrating mesh nebulisers, and that heating occurs to a lesser degree than in ultrasonic nebulisers, considerable temperature increases have been reported in some brands of vibrating mesh nebulisers (pari eflow®, akita apixneb®, and aeroneb go), with temperatures of up to °c being reached towards the end of nebulisation [ ] . therefore, it is essential to choose the inhalation device carefully, bearing in mind that the best device type is the one that confers the most stability to the protein drug formulation. soft mist inhaler (smi), which also generate aerosols from liquid, is the newest type of inhaler which does not use any propellent. as only one medicine respimat uses smi, its suitability for protein drug delivery is not clear. another factor to consider is the aerodynamic diameter of aerosol particles, which is critical to control where the particles will be deposited in the respiratory track after inhalation. to be therapeutically effective, the drug containing particles need to be deposited into the correct location within the respiratory track. for example, for therapeutics for copd, drugs need to be delivered to the deep lung (the alveolar space) for which it requires the aerodynamic diameter of the particles to be between and μm. larger size particles will generally be deposited in the oropharyngeal region and be ingested, while small particles < μm may be exhaled during the next breathing cycle. thus, suitable aerosol particle sizes need to be selected for precise drug delivery into the lung to enhance drug efficacy while simultaneously reducing harmful side effects [ ] . the respiratory tract comprises of a series of branching airways, which can be categorised into two parts: the conducting zone and the respiratory zone. the conducting zone consists of the trachea, bronchi, bronchioles, and terminal bronchioles. the airway wall in the conducting zone is too thick for diffusion and this region does not contain alveoli. as such, no gas exchange takes place here, and the purposes of the conducting zone include transmitting air to the respiratory zone, as well as to warm, moisture and cleaning the inspired air. the respiratory zone consists of respiratory bronchioles, alveolar ducts, and alveolar sacs, and facilitates gas exchange between the air and the bloodstream. alveoli can occasionally be found in the walls of the respiratory bronchioles, and are abundant in the alveolar ducts and alveolar sacs [ , ] . given the branched structure of the lungs, it is not only important to achieve high deposition rates, but also to obtain an appropriate deposition pattern for the respiratory disease in question i.e. the protein therapeutic would not only need to reach the lung, but would also need to reach the correct target site within the lung. for instance, a therapeutic for asthma would have to reach the large airway, as asthma mainly affects the bronchi, while a drug for emphysema in copd would need to go deeper and reach the small airways of the lung because emphysema affects the alveolar region [ ] . the amount and pattern of lung deposition is not only affected by the device and the characteristics of the inhaled drug (particle size and physicochemical properties of the formulation), but also by factors that are influenced by the specific disease state, including breathing patterns, lung geometry (i.e. airway diameter, number of alveoli) and structure, and nasal, oral, and pharyngeal anatomy [ ] . these factors need to be considered, and if possible, alterations to the drug formulation can be made to address these issues. for example, in certain lung pathologies (for instance cystic fibrosis, copd, and chronic sinusitis), the airway mucus becomes thicker. it has been reported that the thickness of the mucus layer ranges from to μm in normal lungs to more than μm in cystic fibrosis and other obstructive airway diseases [ ] . this presents a physical barrier that the protein drug would need to penetrate to reach its target site in the lung and exert its effects. the addition of anti-adhesive molecules (e.g. polyethylene glycol, peg) in the formulation may help to promote the translocation of the protein drug through the thickened mucus, although it should be noted that the adhesive properties of peg depend on peg molecular weight (mw) [ ] . while high mw pegs display mucoadhesive properties, low mw pegs are able to prevent mucoadhesion, with pegs of mw up to kda able to provide effective mucus penetration [ ] [ ] [ ] . notably, as macromolecules, proteins have a relatively poor ability to penetrate the epithelial layer to reach the deep parenchyma lung. however, depending on the molecular weight and aerosol characteristics, a portion of the proteins would be able to reach the abluminal side of the epithelium or the air-blood interface in the thin alveolar wall, triggering local or even systemic immune signalling that may provide beneficial therapeutic effect in some cases [ ] . a good lung deposition pattern would be worthless if the protein therapeutic cannot withstand the lung's clearance mechanisms. inhaled proteins would be subjected to clearance by three mechanisms. the first clearance mechanism is mucociliary clearance (mcc), which is the coordinated beating of cilia lining the nasal cavity, trachea, and bronchi, in order to move the mucus towards the larynx/pharynx, thereby pushing dust, microorganisms, and insoluble particles that are trapped in the mucus out of the lungs and into the upper airways to eventually be swallowed [ ] . the surface lining of the airways in normal lungs consists of an aqueous layer adjacent to the epithelium and a surfactant containing film layer at the air-liquid interface. the peri-ciliary aqueous layer has a relatively low viscosity, while the surfactant film layer is more viscous. the surfactant film plays an important role in the displacement of airway particles towards the epithelium where they will be immersed and retained. the extent of particle immersion depends on the surface tension of the film. the lower the surface tension, the greater the immersion of particles into the aqueous layer adjacent to the epithelium [ , ] . it is possible that some protein monomers could quickly reach the stagnant aqueous layer and not be subjected to mcc. on the other hand, some protein monomers would become aggregated during the inhalation delivery process and the aggregates may stay with the surfactant film layer at the air-liquid interface for some time for mcc to take effect. anti-adhesive formulations (achieved by using lower mw pegs for example) could be used to circumvent mcc clearance of inhaled therapeutics, thus increasing their lung accumulation. the mucus-penetrating ability of such pegs has already been demonstrated in multiple studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the second clearance mechanism is macrophage uptake, which is the primary clearance mechanism in the alveoli. proteins are taken up by alveolar macrophages in the deep lung via pinocytosis, and the uptake of particles is size dependent [ , , ] . large proteins (≥ kda) would have more time to be engulfed by alveolar macrophages by virtue of their slower transport and absorption across the alveolo-capillary barrier, while small proteins and peptides (≤ kda) are absorbed rapidly from the airspaces and thus, may not be impacted by alveolar macrophage uptake as much. in essence, pinocytosis by alveolar macrophages could become significant for macromolecules with mw > kda [ ] . the use of excipients can help to reduce clearance of large proteins by alveolar macrophages. for example, koussoroplis et al. ( ) showed that pegylation conferred increased residence time to antibody fragments anti-il- a f (ab ′) and anti-il- fab′ (unconjugated f (ab′) was kda and unconjugated fab′ was kda), and that the effect was due to mucoadhesion as well as evasion of alveolar macrophage uptake [ ] . protein pegylation may potentially also alter its deposition pattern in the respiratory track, due to changes in molecular weight, hydrophilicity etc. however, systemic analyses of the effect of pegylation on protein deposition pattern in the respiratory track are still needed in order to know if a consistent deposition pattern and behaviour can be reached based on how pegylation is achieved. the third clearance mechanism is absorption into the systemic circulation. after deposition in the alveolar region, aerosol drug particles may dissolve in pulmonary epithelial lining fluid if the drug is water-soluble, and become available for systemic absorption and clearance [ ] . for the purpose of topical lung treatment, the goal would be to minimise systemic absorption, which is greatly influenced by protein mw. the bioavailability of a protein after absorption from the lung decreases as protein mw increases. small peptides are absorbed rapidly from the lungs with - % of the bioavailability for subcutaneous injection [ ] . proteins with mw of - kda exhibit moderate absorption, with bioavailability ranging from to %, although it should be noted that pulmonary absorption studies in animals may lead to an overestimation of bioavailability. for example, systemic bioavailability after aerosol administration in animals for growth hormone (gh) and interferon α (ifnα) was % and % respectively, compared to only - % in humans [ , ] . large mw antibodies (~ kda) are not significantly absorbed across the lung, and bioavailability is negligible (<< %) unless an active transport system is included [ ] . apart from high protein mw, the presence of obstructive lung diseases (e.g. asthma, copd, cystic fibrosis) can also reduce systemic absorption and bioavailability of proteins and other drugs [ ] . for example, henry et al. ( ) reported that healthy subjects had significantly higher area under the curve (auc) and mean maximum concentration (c max ) after insulin inhalation than asthma patients, indicating that less insulin was absorbed into the systemic circulation in asthma patients [ ] . in addition, diderichsen et al. ( ) reported that the c max of an inhaled long acting β agonist (pf- ) was found to be reduced by % and % for copd and asthma patients respectively, compared to healthy volunteers [ ] . additional possibility for the lower systemic absorption of drugs in lung disease patients could be due to altered drug deposition pattern in the diseased lung, for which further studies are needed. regardless the underlying mechanisms, effective local lung retention of protein drugs may not be a major issue for the successful inhalation treatment of obstructive lung diseases, since the protein can be relatively well retained in the lungs. inhaled protein therapeutics may undergo various degradation mechanisms during production, processing and/or storage. these degradation pathways may be physical (denaturation and non-covalent aggregation) or chemical (mainly covalent aggregation, deamidation, oxidation and/or glycation). denaturation is the result of physical stresses including low/high temperatures, high salt concentrations, organic solvents, and air/water or ice/water interfaces. removal of the stressor may be spontaneously reversible (for some single domain proteins), but is usually irreversible for most of the larger multi-domain proteins [ ] . surface-induced aggregation is one of the common mechanisms of non-covalent aggregation, and one example of when it occurs is during the process of nebulisation [ , ] . as most proteins are amphiphilic and surface active, they have a tendency towards adsorption at the ali. upon adsorption, conformation changes may occur, exposing hydrophobic residues to the interface to avoid contact with water, thus leading to aggregation and unfolding, which are the main factors contributing to protein instability [ , ] . chemical degradation of proteins (deamidation, oxidation, glycation) may also cause aggregation (either covalent or non-covalent) [ ] . aggregation has been extensively studied but chemical modifications have not, despite having implications on biological activity and immunogenicity [ , ] . aggregation can result from both physical and chemical pathways; therefore, it is useful to also evaluate chemical changes in inhaled proteins. most studies assessing stability of inhaled protein formulations focus on formation of aggregates, while studies that also examine chemical changes are few and far between. one study did, however, consider chemical changes when evaluating the technical feasibility of delivering dornase alfa using perforated vibrating membrane devices for nebulisation. in this study, besides detecting protein aggregates, stability was also evaluated by measuring the percent deamidation of dornase alfa at asn (the main chemical change for the protein), which was shown to be inversely proportional to dornase alfa potency [ ] . another study looked at methionine oxidation [met(o)] of nebulised insulin-like growth factor- (igf-i), and how that correlated with aggregate formation and bioactivity. highly aggregated samples displayed a complete loss of bioactivity, while samples with complete oxidation but minimal aggregation showed partial retention of bioactivity. limited met(o) formation and no aggregation was observed following delivery with air-jet or vibrating mesh nebulisers [ ] . bandi et al. ( ) conducted a study to compare the effects of deamidation and oxidation on interferon alpha- a (ifna a), as deamidation of asparagine and glutamine residues, and oxidation of methionine residues are two of the most common chemical alterations that occur in pharmaceutical proteins that could compromise their efficacy and safety [ ] . these findings revealed that deamidation destabilised ifna a and enhanced its tendency to aggregate under stressful conditions, and reduced its function to a greater extent than oxidation. this is the first study that quantitatively compared the effects between deamidation and oxidation of a therapeutic protein [ ] . it would be a good strategy to conduct such studies early on in the development of therapeutic protein candidates in order to identify the chemical modifications that a particular protein would be susceptible to, and to test out various excipients that could resolve specific stability issues. the protein therapeutic also needs to remain stable after reaching the lung, which can be challenging due to the high numbers of serine proteases and aminopeptidases present in the lung mucosa [ ] [ ] [ ] [ ] . these proteases could degrade protein drugs even before they reach their target sites within the lung. the use of appropriate excipients in the formulation such as peg, could help to enhance protein resistance to proteolysis by these lung proteases. for example, zhang et al. ( ) evaluated the stability of fibronectin (fn) preferentially pegylated at lysine residues using different mw pegs [ kda (peg ), kda (peg ) or kda (peg )] against the protease α-chymotrypsin. they showed that pegylation protected fn from proteolysis and that peg mw positively correlated with proteolytic stability (i.e. after min of proteolysis, %, %, % and % of the starting amounts of native fn, fn-peg , fn-peg and fn-peg respectively were remaining) [ ] . one must also be aware of the possibility of protein aggregates forming in the lungs. lasagna-reeves et al. [ ] demonstrated that mice exposed to inhaled insulin (exubera®) in a chamber twice daily for week developed amyloid aggregates of insulin in both the proximal and distal airways, as well as the lung parenchyma (epithelium and muscle layer of the bronchi, bronchioles, and in the alveolar lining cells). the formation of insulin aggregates coincided with a significant decrease in respiratory flow rates, and also with caspase- activation. previous studies investigating the link between changes in pulmonary function and inhaled insulin use focused on formation of anti-insulin antibodies, or pulmonary inflammation and subsequent airway remodelling, but none of the published works before this looked at insulin aggregation in the lungs as a contributor to pulmonary dysfunction after inhaled insulin use [ ] . indeed, exu-bera® was reported to cause cough, dyspnea, increased sputum and epistaxis [ ] . this example highlights the possibility of inhaled proteins forming aggregates in the lungs, and thus the need for toxicity testing and safety studies examining this possibility to be done early on in the development of an inhaled protein candidate, during preclinical studies. in addition, proteins and other macromolecules have the potential to induce immunogenicity, with the production of anti-drug antibodies (adas) as the main immune response [ ] . the development of adas in patients can alter pharmacokinetics, drastically reduce efficacy, and can also lead to severe adverse events or even lethal consequences [ ] . immunogenicity is also linked to protein stability, as the presence of aggregates can render the protein immunogenic. as aggregates are typically composed of denatured molecules, they would exhibit no or decreased activity, but at the same time, aggregates are usually immunogenic leading to adas with important clinical implications [ ] . aggregates are believed to be recognised and processed via non-specific uptake by antigen presenting cells and specific uptake by b cells. they may unmask neo−/cryptic/repetitive epitopes, and these differences may influence the mechanism by which they activate the immune system [ ] . currently, only a few excipients have been approved by the fda for inhalation due to a dearth of toxicological studies for inhaled excipients [ , ] . there is also very limited number of excipients that are approved by fda for biologics, rendering formulators limited choices to improve protein formulations when excipients are searched on the fda's inactive ingredient database guidance. furthermore, very few novel excipients have been investigated for biologic products; most are cyclodextrin-based excipients [ ] . as such, there is a need for more extensive toxicity testing to identify novel excipients for pulmonary delivery. for excipients already known to increase protein stability, a trial and error approach needs to be taken in determining their suitability for a particular protein formulation, as an excipient may work for one protein but not for another for various reasons including sequence differences [ ] . excipients that are commonly used in liquid formulations (nebulisers and mdis) include buffering or ph adjusting agents, and surfactants, and those that are commonly used in dry powder formulations (dpis) include sugars, polyols, and amino acids [ , ] . buffering or ph adjusting agents such as sodium chloride, sodium citrate, hydrochloric acid, sodium hydroxide, and citric acid, are added to maintain the ph of the formulation. it is important to choose the right buffering agent at an appropriate concentration, as most proteins in solution only remain stable within a narrow ph range. different buffer systems and concentrations can also affect the aggregation pattern of proteins [ ] . kim et al. [ ] analysed the stability of a fusion protein, etanercept (marketed enbrel®), with changing ph and buffer concentrations. increasing the ph of etanercept from ph . to . resulted in a decrease in protein size and increase in aggregation. under high buffer concentrations ( mm tris buffer), changes in protein size was reduced and irreversible aggregation was not observed, while in lower buffer concentrations ( mm tris buffer), larger aggregates (~ μm) were observed across the ph range [ ] . surfactants (polysorbates, sorbitan esters, oleic acid, and soy lecithin) are frequently used to prevent aggregate formation, and they work by displacing protein molecules from the ali [ , ] . polysorbates are the most commonly used surfactants, and are already being used to preclude aggregation in formulations of intravenously administered antibodies [ ] . polysorbate has been reported to lead to stabilisation in various inhaled protein formulations including those for granulocyte-colony stimulating factor (g-csf), lactate dehydrogenase (ldh), tissue plasminogen activator (t-pa) and aviscumine (recombinant mistletoe lectin) [ ] . the ability of polysorbates and other surfactants to stabilise a protein and hinder aggregate formation is contingent on the protein-to-surfactant ratio. respaud et al. [ ] examined the effects of various antibody and surfactant (polysorbate ) concentrations to optimise the protein-to-surfactant ratio for a nebulised antibody formulation. the authors determined that high concentrations of either surfactant or protein could minimise the formation of medium and large-sized aggregates, without significantly affecting the volume mean diameter (vmd) of the aerosol cloud, ensuring suitability for inhalation. therefore, including surfactants and raising protein concentration to enhance the stability of inhaled protein formulations is a viable strategy, although it should be noted that this approach needs to be evaluated and optimised for each drug and device pairing being developed into an inhaled protein formulation [ ] . sugars (sucrose, trehalose, raffinose and lactose) and polyols (mannitol) stabilise proteins through the preferential hydration of proteins via steric repulsion of sugar/ polyol molecules from the native protein [ , ] . lactose is often used as a drug carrier in dpis, however, it may not be suitable for proteins because it is a reducing sugar, and it could interact with amino groups in proteins (maillard reaction) [ ] . on the other hand, non-reducing sugars such as sucrose, trehalose and raffinose would not undergo the maillard reaction with proteins, and thus could be used as alternatives to lactose [ ] . sellers et al. [ ] demonstrated that sucrose could help to improve the stability of a dry powder formulation of ldh. supercritical fluid (scf) drying of ldh without excipients lead to irreversible loss of activity (only % recovered after rehydration). inclusion of % (w/w) sucrose during dehydration lead to an increase in activity recovered (to~ %), and there was almost complete retention of activity when polysorbate was added in addition to sucrose [ ] . trehalose and raffinose are currently not approved for any administration routes, but have been evaluated in experimental studies with promising results. for instance, Ógáin et al. [ ] incorporated lysozyme into nanoporous microparticles of trehalose and raffinose. lysozyme showed good retention of specific activity after storage for weeks at either °c ( . ± . % for lysozyme:trehalose and . ± . % for lyzosyme:raffinose) or °c ( . ± . % for lysozyme:trehalose and . ± . % for lyzosyme:raffinose) [ ] . mannitol was used as an excipient in the formulation of exubera® (table ) [ ] . small amino acids (histidine, arginine, alanine, glycine, lysine, isoleucine) are also used as stabilisers, and they work by the "water substitution mechanism" in which the amino acids hydrogen bond with the protein during drying to preserve the native protein structure in the dried state [ , ] . ajmera and scherlieβ [ ] screened different amino acids and their combinations for their ability to stabilize catalase during spray drying. when various ratios of arginine, glycine and histidine were mixed with catalase, some formulations were able to maintain close to % catalase activity [ ] . despite encouraging results in studies such as this one, there is a lack of data on the local toxicity of the various amino acids following inhalation, which could limit their use. however, as they are endogenous substances, they may not present major safety issues for local lung delivery [ , ] . the polyol, peg, could be used for both liquid and powder formulations of inhaled proteins. small mw pegs (< kda) are often used as excipients in oral, intravenous and nasal formulations. larger pegs (up to kda) may be used in pegylated biopharmaceuticals, and safety testing for these formulations are done during development on a case-by-case basis [ ] . pegylation is a commonly used method to enhance solubility and stability, as well as to decrease immunogenicity of bioactive drugs including but not limited to proteins, peptides, antibody fragments, and enzymes, and is achieved by the covalent or noncovalent conjugation of peg to the biomolecule [ , ] . pegylation can also help to reduce clearance and increase lung accumulation and residence time of inhaled protein therapeutics. for instance, conjugation of a peg chain to two antibody fragments (anti-il- a f (ab′) and anti-il- fab′) increased their levels in mouse lungs following intranasal administration. fortyeight hours post-administration, levels of unconjugated antibody fragments in the lungs had dropped to % and % of the original deposited dose of f (ab′) and fab′ respectively, while this value was % for both pegylated fragments [ ] . furthermore, conjugation of a peg chain to an anti-il- a fab' antibody fragment increased pulmonary retention in all three species tested (mice, rats, and rabbits) following intratracheal administration. unconjugated fragments were cleared from the lungs within h while large amounts of pegylated fragments still remained for up to h [ ] . the two biggest challenges in developing particle systems for pulmonary drug delivery are to maintain colloidal stability during aerosolisation and to achieve high delivery efficacy. encapsulation of proteins in carriers could provide multiple benefits such as protection from enzymatic degradation and specific targeting to the site of action through targeting ligands [ ] . furthermore, carriers may also be used to provide sustained drug release, accumulating in the lungs and releasing therapeutic levels of the protein drug over extended periods of time. this would enhance efficacy while averting peaks in local drug concentrations that could cause pulmonary toxicity [ ] . proteins, including insulin, calcitonin, and igg, have already been loaded into various carriers such as microparticles, liposomes, and solid lipid nanoparticles [ , ] . indeed, afrezza® uses techno-sphere® technology, in which fumaryl diketopiperazine (fdkp), an excipient added into the formulation, selfassembles into microspheres, entrapping the insulin. upon reaching the alveolar zone of the lung, the tech-nosphere® particles rapidly dissolve in the ph-neutral environment and release the insulin for systemic absorption [ ] . although this approach has not been extensively explored for topical lung delivery of proteins, and more work needs to be done on the use of carriers for the purpose of systemic delivery of proteins through the lungs, some promising results have been reported that support further development of this approach. tawfeek et al. [ ] encapsulated a model mucinolytic enzyme, αchymotrypsin (which is very sensitive to unfolding and formulation conditions), in a novel biodegradable pegco-polyester microparticle carrier. the encapsulated αchymotrypsin exhibited retention of enzymatic activity and the results indicated suitability of the carrier for potential use in the delivery of macromolecules as dpi formulations for the treatment of lung diseases [ ] . in another study by osman et al. [ ] , various surface modifications were made to dnase i loaded microparticles using different excipients in order to provide higher lung deposition, enzyme stability and biological activity. surface modifying the microparticles with polyglutamic acid (pga) or dextran was found to provide high inhalation indices (emitted fraction (ef), respirable particle fraction (rp), and effective inhalation index (ei)) and increased mucolytic activity in cystic fibrosis sputum. this could be explained by the resulting surfaces of the particles after modification with pga (rough dented surfaces) or dextran (dimpled surfaces). compared to spherical particles with similar physical properties, corrugated particles have surface asperities that could reduce the true contact area between particles, decreasing powder cohesiveness and enhancing aerosol performance [ ] . advancements in drug-loaded capsules for pulmonary delivery have been made in both inhalable dry powder or liquid drug formulations [ ] [ ] [ ] . for dry powder drug particles, precise control of the particle size has been reported using the particle replication in nonwetting templates (print) technology [ , ] . for control of aerodynamic particle size in liquid aerosols such as in nebulized liquid formulation, the recently reported mpns have presented promising possibilities. mpnbased drug-loaded capsules with highly defined physical properties can be generated for both macromolecular protein drugs and small molecule chemical drugs [ ] . these new developments may transform inhalation drug delivery in the near future. one drawback with the use of carriers is their rapid uptake by alveolar macrophages [ , ] . phagocytosis of carriers by alveolar macrophages can result in fast clearance and reduced residence time, limiting the therapeutic efficacy of the carrier-associated drug. this would be an issue for the treatment of chronic lung diseases such as asthma and copd, where the goal of using a carrier system would be to achieve controlled and continuous drug release over an extended period of time. however, various formulation design strategies may be employed to reduce the uptake of particulate carriers by alveolar macrophages including modulation of particle size, shape, surface charge and surface coating [ ] . studies on the use of various polymer coatings demonstrate reduced alveolar macrophage uptake of coated carriers. for example, jones et al. [ ] showed that respirable microspheres coated with dipalmitoyl phosphatidylcholine (dppc; a major component of lung surfactant) were able to significantly reduce phagocytic uptake by nr in cultured alveolar macrophages compared to uncoated microspheres. the uptake of dppc coated microspheres was found to be only . ± . %, . ± . % or . ± . %, of the uptake of uncoated microspheres for ratios of , or excess microspheres per nr cell respectively [ ] . furthermore, shen et al. [ ] demonstrated that surface coating of hydrogel nano-and microparticles with peg showed significantly reduced uptake by alveolar macrophages both in vitro (in mh-s cells) and in vivo (in mice) compared to unpegylated particles of the respective size. at h post-dose, the fold difference between pegylated and unpegylated × nm, . μm, and μm particles in bronchioalveolar lavage fluid (balf), was . , . and . respectively [ ] . on the other hand, drug-loaded particles may be advantageous for anti-tuberculosis drugs as efficient uptake of drugs into alveolar macrophages could potentially enhance the drug's efficacy to kill the parasitic mycobacterium tuberculosis that hide inside the cells [ ] . if the usage of a carrier is to be included in the protein formulation, it should be noted that the formulation (i.e. combination of protein and carrier) would need to be optimised together with the choice of device, as the chosen carrier may not work well with all inhalation device types. for instance, liposomes may be delivered to the lungs either by dry powder inhalation or nebulisation of a liposome suspension. however, nebulised solutions of liposomes may cause instability as nebulisation has been reported to disrupt liposomal structure, leading to the release of loaded drug. these issues can be avoided with the use of dry powders of liposomes instead [ ] . hence, although this review has presented a general overview for the various aspects of protein formulation design (such as choice of device, excipients), it is important to test out the formulation and device together to determine which combination works best. this review analyses the various obstacles that an inhaled protein drug would need to overcome in order to reach the lungs and exert its therapeutic effects. these obstacles include the physical and chemical stresses experienced by the protein during production/storage/ aerosolisation, the need to overcome mucociliary clearance and physical barriers arising from disease conditions in order to reach target sites within the lung, and the need to remain stable in spite of the presence of abundant proteases, and to evade clearance by alveolar macrophages after reaching the lungs (fig. ) . all of these threats to the integrity of the protein need to be carefully considered, so that pre-emptive measures can be taken while designing the protein formulation to ensure its therapeutic efficacy. nevertheless, although the information provided here may serve as general considerations in developing pulmonary protein therapeutics, empirical testing of the formulation together with the device should still be performed to determine the best combination for a particular protein. several key areas will require further investigation in order to support the development of more successful inhaled protein therapies, and maintaining the stability of the inhaled protein is of paramount importance. firstly, more studies could look at other instability issues beyond protein aggregation. in depth studies on the specific chemical modifications that a protein would be susceptible to, such as the one conducted by bandi et al. ( ) , could be carried out on therapeutic protein candidates so that they may be developed into stable and effective treatments [ ] . moreover, the scarcity of fdaapproved excipients for inhaled therapeutics further limits drug developers, and expanding this list through increased toxicological testing of new excipients would provide more options for formulation design. finally, innovative approaches such as the use of novel carrier systems should be employed for the purpose of topical lung delivery, as carrier systems could greatly enhance the stability and pharmacokinetic profile of proteins. these approaches would greatly benefit the field of pulmonary drug delivery, and will ultimately allow more inhaled protein therapeutics to reach the clinic. funding support for this work is provided by the grant moe -t - - awarded to ruowen ge from singapore ministry of education, republic of singapore. capturing the benefits of competition for patients current approaches to the discovery of novel inhaled medicines inhaled protein/peptide-based therapies for respiratory disease nebulization as a delivery method for mabs in respiratory diseases carriers for the targeted delivery of aerosolized macromolecules for pulmonary pathologies inhalation of immuno-therapeutics/ −prophylactics to fight respiratory tract infections: an appropriate drug at the right place! front immunol vegf neutralizing aerosol therapy in primary pulmonary adenocarcinoma with kras activating-mutations the airways, a novel route for delivering monoclonal antibodies to treat lung tumors inhaled therapy in respiratory disease: the complex interplay of pulmonary kinetic processes efficacy of aerosol therapy of lung cancer correlates with egfr paralysis induced by avidinox-anchored biotinylated cetuximab pegylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract repurposing of gamma interferon via inhalation delivery phase ii clinical trial results of alidornase alfa for the treatment of cystic fibrosis protalix biotherapeutics announces phase ii clinical trial results for alidornase alfa in cystic fibrosis presented at the th european cystic fibrosis society conference. protalix biotherapeutics designing inhaled protein therapeutics for topical lung delivery: what are the next steps? inhaled gm-csf in a pulmonary alveolar proteinosis patient refractory to plasmapheresis combined with multiple whole lung lavages synairgen doses first patient in covid- trial ansun biopharma enrolls first patient in proof of concept trial of das for the treatment of covid- direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies in a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration challenges and future prospects for the delivery of biologics: oral mucosal, pulmonary, and transdermal routes non-invasive delivery strategies for biologics idealhalers versus realhalers: is it possible to bypass deposition in the upper respiratory tract? that's cool! -nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer protein stability in pulmonary drug delivery via nebulization protein nebulization: i. stability of lactate dehydrogenase and recombinant granulocyte-colony stimulating factor to air-jet nebulization stability of urease during aerosolization protein nebulization some factors associated with the ultrasonic nebulization of proteins nebulizers for drug delivery to the lungs the function and performance of aqueous aerosol devices for inhalation therapy devices for improved delivery of nebulized pharmaceutical aerosols to the lungs vibrating mesh nebulisation of pro-antimicrobial peptides for use in cystic fibrosis effect of formulation on the stability and aerosol performance of a nebulized antibody a technical feasibility study of dornase alfa delivery with eflow® vibrating membrane nebulizers: aerosol characteristics and physicochemical stability insulin-like growth factor-i aerosol formulations for pulmonary delivery development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin effective nebulization of interferon-γ using a novel vibrating mesh factors to consider when selecting a nebulizer for a new inhaled drug product development program engineering of nebulized metal-phenolic capsules for controlled pulmonary deposition pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform in vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (hydra) technology prospects of formulating proteins/peptides as aerosols for pulmonary drug delivery protein conformational stability in the hydrofluoroalkane propellants tetrafluoroethane and heptafluoropropane analysed by fourier transform raman spectroscopy computational modeling of lung deposition of inhaled particles in chronic obstructive pulmonary disease (copd) patients: identification of gaps in knowledge and data the impact of pulmonary diseases on the fate of inhaled medicines-a review drug delivery for traditional and emerging airway models. organs-on-a-chip nanodelivery in airway diseases: challenges and therapeutic applications addressing the peg mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier nanoparticles coated with high molecular weight peg penetrate mucus and provide uniform vaginal and colorectal distribution in vivo surfactant displaces particles toward the epithelium in airways and alveoli pulmonary surfactant: surface properties and function of alveolar and airway surfactant transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy protein nanocages that penetrate airway mucus and tumor tissue use of single-site-functionalized peg dendrons to prepare gene vectors that penetrate human mucus barriers rapid transport of large polymeric nanoparticles in fresh undiluted human mucus highly compacted biodegradable dna nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy pegylated enhanced cell penetrating peptide nanoparticles for lung gene therapy lung gene therapy with highly compacted dna nanoparticles that overcome the mucus barrier the penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles preclinical models for pulmonary drug delivery formulation technology to repurpose drugs for inhalation delivery will pulmonary drug delivery for systemic application ever fulfill its rich promise? inhaled insulin using the aerx insulin diabetes management system in healthy and asthmatic subjects characterizing systemic exposure of inhaled drugs: application to the long-acting β -agonist pf- inhaled proteins: challenges and perspectives d nmr analysis of the effect of asparagine deamidation versus methionine oxidation on the structure, stability, aggregation, and function of a therapeutic protein respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants role of proteases in lung disease: a brief overview role of proteases in chronic obstructive pulmonary disease proteases and antiproteases in chronic neutrophilic lung disease -relevance to drug discovery pegylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity inhaled insulin forms toxic pulmonary amyloid aggregates technosphere®: an inhalation system for pulmonary delivery of biopharmaceuticals immunogenicity of different stressed igg monoclonal antibody formulations in immune tolerant transgenic mice the effects of substituted cyclodextrins on the colloidal and conformational stability of selected proteins formulation strategy and use of excipients in pulmonary drug delivery effects of ph and buffer concentration on the thermal stability of etanercept using dsc and dls practical, regulatory and clinical considerations for development of inhalation drug products particle engineering of materials for oral inhalation by dry powder inhalers. i-particles of sugar excipients (trehalose and raffinose) for protein delivery dry powders of stable protein formulations from aqueous solutions prepared using supercritical co -assisted aerosolization mechanisms of protein stabilization in the solid state stabilisation of proteins via mixtures of amino acids during spray drying amorphous powders for inhalation drug delivery pegylation, an approach for improving the pulmonary delivery of biopharmaceuticals what is the future of pegylated therapies? from synthesis to characterization of site-selective pegylated proteins pegylation prolongs the pulmonary retention of an anti-il- a fab' antibody fragment after pulmonary delivery in three different species delivery strategies for sustained drug release in the lungs multifunctional nanocarriers for lung drug delivery dry powder inhalation of macromolecules using novel peg-copolyester microparticle carriers inhalable dnase i microparticles engineered with biologically active excipients inhaled nano-and microparticles for drug delivery nanoparticles for drug delivery to the lungs advanced therapeutic strategies for chronic lung disease using nanoparticle-based drug delivery formulation of high-performance dry powder aerosols for pulmonary protein delivery microfabricated engineered particle systems for respiratory drug delivery and other pharmaceutical applications polymeric nanoparticles in development for treatment of pulmonary infectious diseases update on macrophage clearance of inhaled micro-and nanoparticles particle engineering to enhance or lessen particle uptake by alveolar macrophages and to influence the therapeutic outcome the inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages distribution and cellular uptake of pegylated polymeric particles in the lung towards cell-specific targeted delivery drug delivery for tuberculosis: is inhaled therapy the key to success? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -jejgkcql authors: jain, k.k. title: synthetic biology and personalized medicine date: - - journal: med princ pract doi: . / sha: doc_id: cord_uid: jejgkcql synthetic biology, application of synthetic chemistry to biology, is a broad term that covers the engineering of biological systems with structures and functions not found in nature to process information, manipulate chemicals, produce energy, maintain cell environment and enhance human health. synthetic biology devices contribute not only to improve our understanding of disease mechanisms, but also provide novel diagnostic tools. methods based on synthetic biology enable the design of novel strategies for the treatment of cancer, immune diseases metabolic disorders and infectious diseases as well as the production of cheap drugs. the potential of synthetic genome, using an expanded genetic code that is designed for specific drug synthesis as well as delivery and activation of the drug in vivo by a pathological signal, was already pointed out during a lecture delivered at kuwait university in . of two approaches to synthetic biology, top-down and bottom-up, the latter is more relevant to the development of personalized medicines as it provides more flexibility in constructing a partially synthetic cell from basic building blocks for a desired task. synthetic biology, application of synthetic chemistry to biology, is a broad term that covers the engineering of biological systems with structures and functions not found in nature to process information, manipulate chemicals, produce energy, maintain cell environment and enhance human health [ ] . synthetic biology includes technologies for dna synthesis and assembly of fragments of dna for gene synthesis, sometimes referred to as synthetic genomics. craig venter, a pioneer in this area, has described synthetic biology in a video (http://www.youtube.com/ watch?v=dvbv qnszwo). genome engineering includes approaches to construct synthetic chromosomes and goes beyond traditional genetic engineering. metabolic engineering can be used to manipulate metabolic pathways for correction of metabolic disorders and can also be applied to microorganisms to produce chemicals more efficiently than by genetic engineering. synthetic biology devices contribute not only to improve our understanding of disease mechanisms, but also provide novel diagnostic tools. methods based on synthetic biology enable the design of novel strategies for the treatment of cancer, immune diseases metabolic disorders and infectious diseases as well as the production of cheap drugs [ ] . the potential of synthetic genome, using an expanded genetic code that is designed for specific drug synthesis as well as delivery and activation of the drug in vivo by a pathological signal has been reported [ ] . personalized medicine simply means the prescription of specific therapeutics best suited to an individual. it is usually based on pharmacogenetics, pharmacogenomics, transcriptomics, pharmacoproteomics and pharmacometabolomic information. other individual variations in patients and environmental factors are also taken into consideration [ ] . personalized medicine means improving healthcare by incorporating early detection of disease, preventive medicine, rational drug discovery and development, and monitoring of therapy. the broad scope and interrelationships of personalized medicine are shown in figure . the concept of personalized medicine as systems medicine is the best way of integrating new technologies and translating them into clinical application for improving healthcare. nanobiotechnology has already helped in the development of personalized medicine [ ] . biological therapies are making significant contribution to personalized medicine [ ] . initially pharmacogenomics and pharmacogenetics were used to select drugs from those already available, but sequencing information now provides the opportunity to design and develop new personalized medicines as shown in figure . sequencing of the genomes of various organisms has provided the basis for development of synthetic biology. the genome sequence is an organism's blueprint: the set of instructions dictating its biological traits. resequencing, using next generation technologies, means determination of variations of dna sequence in an organism where the nominal sequence is already known, and it is more relevant for synthetic biology as well as translation into diagnostics and clinical applications. the j. craig venter institute has reported the design, synthesis and assembly of the genome starting from digitized genome sequence information and its transplantation into a recipient cell to create new bacterial cells that are controlled only by the synthetic dna [ ] . the researchers built up the synthetic genome of mycoplasma mycoides , a fast-growing bacterium with a million-base genome, by stitching together shorter stretches of dna, each about , bases. they then transferred the completed genome into the shell of another bacterium, mycoplasma capricolum , whose own dna had been removed. the transplanted genome 'booted up' the host cell and took over its biological machinery. after cell divisions, there were billions of synthetic bacteria in the laboratory dishes -all of them making exclusively the biological molecules associated with m. mycoides . the only dna in the cells is the designed synthetic dna sequence, including 'watermark' sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. the new cells have expected phenotypic properties and are capable of continuous selfreplication. the synthetic bacteria have 'watermark sequences' attached to their genome -inert stretches of dna added to distinguish them from their natural counterparts. they behaved and divided in laboratory dishes like natural bacteria. to apply this method to other types of cells, researchers will have to design and synthesize the dna of choice and develop a system to boot up the genome. these two tasks will have to be accomplished in parallel for every new species. accuracy in the genetic code is important for engineering of synthetic genes. the j. craig venter institute is currently working on a project with the national institutes of health of the usa and novartis on vaccine production using this technology. synthetic genes are mostly assembled by using ssdna, and those oligonucleotides (oligos) tend to contain sequence errors. purification of the source oligos can improve the purity of the pool, but at the cost of discarding ϳ % of the oligos. errase, an enzyme technology, facilitates gene assembly at lower costs because it reduces input expenses through use of unpurified oligos [ ] . it reduces errors in synthetic gene sequences by detecting and correcting mismatched based pairs that can be introduced during the procedure. novici biotech/life technologies are developing errase technology commercially for synthetic biology. biosensing is an important activity due to myriads of cell signaling pathways and circuits in cells. it is an important component of two-way communication between implanted engineered cells and natural cells of the body. examples of synthetic biosensing are [ ] : ( ) transcriptional biosensors are built by linking environment-responsive promoters to engineered gene circuits for programmed transcription. ( ) translational biosensors are typically built by linking rna aptamer domains to rna regulatory domains. ( ) posttranslational biosensors consist of membrane-bound protein receptors that trigger signal transduction cascades. in conventional one-way biosensing, a therapeutic substance is released from an implanted device or a genetically engineered cell in response to a programmed mechanism, e.g. release of insulin according to rise of blood glucose level. a two-way communication between the host, enabled by construction of synthetic circuits, can enable more personalized control of adjustment of therapy from implanted cells. the ability to control expression in target areas within a genome is important for synthetic biologists who are developing chemical-producing organisms for commercial applications. in transcription activator-like (tal) effector technology, two hypervariable amino acid residues in each repeat recognize bp in the target dna [ ] . recognition sequences of tal effectors are predictable. the tal code is similar to a navigation system for the genome, allowing pinpoint delivery of functional control elements to any specified sequence. the modular protein architecture has enabled the construction of artificial effectors with new specificities. a central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. as an initial step toward achieving this goal, synthetic proteins have been created that can function in escherichia coli . using a so-called binary code method that relies on strategic placement of polar and non-polar residues, a team of scientists made more than a million stably folded strings of amino acids from genetic sequences distinct from those known to occur naturally [ ] . they then screened these synthetic proteins in dozens of e. coli strains missing essential genes, identifying artificial proteins that could substitute for the organism's own proteins. they are molecular machines that function quite well within a living organism even though they were designed from scratch and expressed from artificial genes. they found that of the artificial proteins could rescue the growth of e. coli mutant strains missing the essential enzyme-coding genes serb, glta, ilva or fes. the information encoded in these artificial genes does not come from and is not significantly related to information encoded by natural genes, and yet the end result is a living, functional microbe. the synthetic proteins are substantially less active and may function by different mechanisms than the natural proteins they replace. nonetheless, co-expression of several novel proteins rescues a strain in which multiple natural genes were deleted simultaneously. this work suggests that the construction of artificial genomes capable of sustaining cell life is feasible. synthesis of therapeutic proteins by this approach has not been explored as yet. complex synthetic biological networks that operate inside living cells in a desired manner usually have controllable genetic switches that are capable of processing therapeutic signals by sensing and responding to the environment. for biomedical applications, these engineered cells need to be sealed in order to protect them from the host immune system and enable their removal after completion of therapy. microencapsulation of cells to be transplanted is an established technology. microencapsulation of defined cells into a semi-permeable and biocompatible microcapsule shields them from the external environment but allows exchange to occur on a molecular basis. powerful combination of synthetic biology and microencapsulation has opened the door to novel and innovative cell-based biomedical applications, such as smart implantable drug delivery systems [ ] . whereas traditional genetic engineering is a haphazard affair, synthetic biology imposes a sense of order to the process. knowledge of how pieces of dna can be put together can facilitate computer-aided design. tools developed for synthetic biology will enable arrangement of atoms in making a desired chemical. it could enable harnessing of vast amounts of genomic information to create microbes with new metabolic pathways, a sort of mix and match approach. whereas genetic engineering focuses on individual genes, synthetic biology strings together a series of molecular components, such as dna, rna, proteins and cells, into circuits and networks. synthetic biology is more versatile than genetic engineering for the development of biopharmaceuticals such as oral vaccines and engineered stem cells or bacteria to detect and treat tumors. synthetic biology enables better quantitative control over molecular engineering techniques with higher throughput and predictable properties. since several complex diseases are caused by disorders of human metabolism, elucidation of the underlying molecular mechanisms is important for developing an effective and personalized approach for their treatment. systems biology can facilitate the analysis of complex metabolic diseases by use of computational approaches. a systems medicine approach can utilize such data about metabolic processes to reconstruct genome-scale metabolic models for study of the function of specific enzymes and pathways in the context of the complete metabolic network. reconstruction of genome-scale models in systems medicine may contribute towards the development of personalized medicine for human metabolic disorders [ ] . engineering of complex biological pathways provides efficient routes for manufacture of natural products with desirable pharmaceutical properties, e.g. artemisinin, which is a chinese herb derived from the plant artemisia annua for the treatment of malaria. this is accomplished through channeling the flux of the isoprenoid pathway to the specific genes involved in artemisinin biosynthesis [ ] . biosynthesis of artemisinic acid through the synthetic biology approach has led to manufacture and commercialization of the drug at low cost for developing countries. by the same metabolic engineering approach, efficient coupling of the isoprenoid pathway also leads to the construction of an e. coli strain that produces a high titer of taxadiene, which is the first committed intermediate for biosynthesis of the anticancer drug taxol. next generation sequencing is expected to advance our understanding of the molecular basis of common diseases and pharmacophore response by improving the compilation of a reference catalog of genes, transcripts, nucleotide variants and structural variants. information gathered from these efforts can be used in synthetic biology-based drug discovery, e.g. translation of signals identified in genome-wide association studies into drug targets. next generation sequencing is also being used directly in drug discovery to identify variants associated with common traits by non-hypothesis directed sequencing of many human genomes. synthetic biology provides the following advantages over conventional techniques for drug discovery and development: ( ) synthetic biology can enable the design of cells to screen drug molecules and reduce drug discovery time/expense. ( ) metabolic pathways can be more precisely regulated in synthetic organisms and manipulated by molecular tools. ( ) parallel to recombinant e. colibased biotechnology drug production, synthetic biology enables redesigning of cells to produce desirable molecules with higher efficacy and lower toxicity. ( ) data accumulated with advances in sequencing can be used for combining synthetic biology with personalized medicine. ( ) reduction of cost of biologicals, particularly therapeutic proteins. ( ) potential for production of synthetic therapeutic proteins. synthetic biology has contributed to the development of personalized medicine from several approaches. the most important of these are by enabling the design and discovery of personalized therapies. the impact of synthetic biology is also discussed on various components of personalized medicine. pharmacogenetics, a term recognized in pharmacology in the pre-genomic era, is the study of influence of genetic factors on action of drugs as opposed to genetic causes of disease. genetic factors may have a major impact on the pharmacokinetics and pharmacodynamics of a particular drug and thereby influence the sensitivity to such a drug in an individual patient with a certain genotype. pharmacogenetics has a three-fold role in the pharmaceutical industry, which is relevant to the development of personalized medicines: ( ) study of the drug metabolism and pharmacological effects, ( ) prediction of genetically determined adverse reactions, and ( ) drug discovery and development. with the synthetic biology approach, pharmacogenetic knowledge can be used to design drugs with less adverse effects and improved efficacy. pharmacogenomics implies the use of genetic sequence and genomics information of the host (normal or diseased) or of the pathogen in patient management to enable therapy decisions. pharmacogenomics, an important part of personalized medicine, can have an impact on all phases of drug development, from drug discovery to clinical trials. this also applies to a wide range of therapeutic products including bioengineered proteins, cell therapy, antisense therapy and gene therapy. synthetic biology can be used for pathway modeling to improve the understanding of the mechanisms of cellular signaling, and for discovering new therapeutic targets for the treatment of various diseases. an important goal of synthetic biology is to devise molecules that can regulate genes and networks in a programmable manner. to achieve these goals, it is necessary to chart the sequence specificity of natural and engineered dna-binding molecules [ ] . synthetic gene network design and prototype therapeutic circuits will have an impact on future gene-and cell-based therapies and usher a new era of drug discovery that may enable treatment of complex diseases in a personalized manner. encyclopedic information that is available on gene function in the postgenomic era provides correlations, and systems biology is now delivering comprehensive details on the dynamics of biochemical reaction networks in living organisms. these catalogued items can be reassembled to design new biological devices and systems with novel and useful functions in a rational and systematic manner [ ] . transgene control systems and networks can be used for gene therapy to treat single-gene defects by triggering inducible expression of complementary transgenes. they can also be used for genetic modification of bacteria to enable them to search and destroy cancer cells. tailored synthetic gene networks can be uploaded into cells to therapeutically target the body's endogenous networks, causing a transition from disease to healthy state [ ] . synthetic mammalian circuits may be able to function as cell implants that could be programmed to sense diseaseassociated metabolites and produce an appropriately adjusted therapeutic response. transgenic cell implants containing prosthetic networks would be more effective than transplants of genetically modified cells in preventing or reversing disease development in a self-sufficient manner without repetition of treatment. a synthetic multi-enzyme glyoxylate shunt has been used to protect mice on a high-fat diet from diet-induced obesity. this shunt prevents the complete oxidation of fatty acids in plants and bacteria, but when introduced in mice, it increases fatty acid oxidation [ ] . aptamers are single-stranded oligonucleotides that can bind to a given ligand with high affinity and specificity due to their particular d structure. aptamer-binding proteins are versatile and powerful building blocks for the construction of artificial genetic switches. an aptamer-based construct regulating the tet off system in a tetracycline-independent manner has been shown to achieve control of transgene expression [ ] . tetr protein-inhibiting aptamer enables the rna-responsive control of the tetracycline-dependent transactivator (tta). by attaching the theophylline aptamer as a sensor, the inhibitory tetr aptamer and thus tta activity became dependent on the ligand of the sensor aptamer. aptamer-based control of the widely used tet system introduces a new layer of regulation to facilitate the construction of more complex gene networks. it could also be used as a safeguard for regulation of gene expression in microencapsulated cell implants. reconstruction of pathogens by dna synthesis can be used to produce diagnostic high-density antigen arrays such as those used to profile post-lyme disease syndrome [ ] . synthesis and analysis of chimeric viruses have also made a substantial contribution to the understanding of viruses that were responsible for the severe acute respiratory syndrome pandemic of . use of transgenic viral strains that harbor lethal synthetic circuitry may control the insect vector populations and suppress transmission of malarial parasites and dengue viruses [ ] . similarly, a synthetic homing endonuclease-based gene drive system can be used to spread genetic modifications, e.g. as resistance against malaria from engineered mosquitoes to the field population. advances in sequencing are facilitating the characterization of pathways of antibiotic biosynthesis. new systematic methods for de novo biosynthetic pathway prediction are enabling the exploration of the metabolic chemical space beyond metabolic engineering. computer-assisted design of modular assembly lines in polyketide synthases and non-ribosomal peptide synthases may enable the development of tailor-made antibiotics. production of novel antibiotics may be transferred into any chosen chassis by optimizing a host factory through specific strain modifications. advances in metabolic engineering and synthetic biology provide novel strategies for engineering antimicrobial agents with desired specificities [ ] . sequencing of tumors provides considerable data on mutations and structural variations, which can be used to predict individual tumor progression and response to treatment. however, prediction of the functional consequences of these mutations, e.g. phenotypes of cancer, is limited. information about the genetic makeup of cancer cells has been combined with functional genomics approaches for identification of novel targets, and exploration of rewiring of cellular pathways [ ] . this is a key step toward personalized cancer diagnostics and therapy. applications of interactive gene networks include synthetic biology-based approach for future gene therapy, as well as the utilization of synthetic gene circuits as blueprints for the design of stimuli-responsive biohybrid materials. the recent progress in synthetic biology, including the rewiring of biosensing devices with the body's endogenous network as well as novel therapeutic approaches originating from interdisciplinary work, generates numerous opportunities for future biomedical applications [ ] . cell therapy, where the patient's own cells are used for treatment, is one of the earlier forms of personalized medicine. different types of cells including embryonic stem cells are now being employed for therapy, and many new technologies are used to modify as well as to synthesize cells. research is progressing on the construction of minimal synthetic or semi-synthetic cells by a bottom-up approach by encapsulating different macromolecules into a lipid vesicle. in situ generation of enzymes and proteins is a prerequisite for constructing functional cells that can replicate their genetic material or respond to chemical messengers by means of surface-mediated receptors [ ] . liposomes have been studied as the most likely precursors of biological cells, and complex biochemical reactions have been described inside liposomes, up to the expression of proteins. the cell membrane between the inner and outer environment of the compartment should have specific properties such as semi-permeability to enable cell-to-cell communication and molecular transport [ ] . such membranes can be built from natural constituents or from synthetic polymers. synthetic cells could include essential components for a personalized therapy and would be a cheaper and more effective tool for treatment. controlling gene expression is the key to stem cell differentiation for applications such as tissue regeneration and cancer therapy. stem cells can be developed into specialized cells, e.g. cardiomyocytes, by insertion of specific sequences. a simple, non-integrating strategy has been described for induced pluripotent stem cells (ipscs) based on administration of synthetic mrna that function as mrna transcripts for the four key genes [ ] . the transcripts are translated into proteins that induce pluripotency without the integration of extra genes into the genomes, which can also be used to efficiently direct the differentiation of rna-ipscs into terminally differentiated myogenic cells. this strategy for somatic cell reprogramming and directing cell fate can be applied for basic research and regenerative medicine. silenced developmental regulators of stem cells can be reactivated by a synthetic transcription factor that interacts with chromatin rather than dna [ ] . potential applications of stem cells modified with synthetic biology include the following: ( ) drug screening for personalized therapies. ( ) engineering of stem cells, to achieve new functions not present in our body, and to introduce them back into the donor, e.g. cells involved in immune response can be programmed to recognize specific microorganisms and target them in a more efficient way than our own immune system can. ( ) to maintain a population level of ␤ cells in diabetics using auto-regulated differentiation of embryonic stem cells that counter-balances the auto-immune attenuation. ( ) tissue engineering with specific desirable properties. the function of synthetic microenvironments is to act as a physical substrate for stem cell attachment and migration, similar to the natural extracellular matrix. nanofabrication technologies were shown to enable the design of synthetic microenvironments that offer new ways to control stem cell fate, e.g. differentiation of mesenchymal stem cells into tissues that most closely match the mechanical properties of the polyacrylamide substrate upon which they are cultured [ ] . mesenchymal stem cells cultured on medium stiffness gels differentiated into muscle cells, whereas those cultured on compliant gels differentiated into neural cells and those cultured on stiff (bone-like) gels differentiated into osteoblasts. by directing the development of stem cells into the desired cell type, synthetic microenvironments facilitate their use for personalized tissue regeneration. conventional vaccine strategies mainly focus on liveattenuated vaccines, inactivated microorganisms, and subunits thereof comprising purified components or recombinant proteins formulated with adjuvants. the development of new vaccines is limited by several drawbacks, including risks associated with the use of attenuated pathogens, along with difficulties altering vaccine target specificity. synthetic biology-based vaccines aim to overcome some of these drawbacks and enable economic and rapid chemical synthesis of dna encoding the immunogens designed in silico as well as their efficient assembly with delivery systems to obtain vectored vaccines [ ] . altogether, synthetic biology can help develop improved vaccine candidates in considerably less time compared to conventional approaches. some examples are given in the following sections. a biocompatible as well as biodegradable nanoparticle has been designed by computer modeling, which self-assembles from single polypeptide chains to produce a structure with isohedral symmetry and a diameter of ϳ nm [ ] . these peptide nanoparticles are multifunctional with high binding affinity and specificity. they can be customized to a high functional density. this platform was used to design and produce a prototypic ma-laria vaccine that can repetitively display a tandem repeat of the b cell immunodominant repeat epitope of the circumsporozoite protein of rodent malaria parasite plasmodium berghei [ ] . administered without an adjuvant, this vaccine conferred a long-lasting antibody response against b cell epitope and protected mice against malarial parasite for up to months. the use of liposomes has been proposed as artificial microbes for vaccination as they can be genetically programmed to produce specific antigens at will [ ] . studies in mice with such vaccines showed that antigen-expressing immunostimulatory liposomes (anexils) elicited higher specific humoral immune responses against the produced antigens than control vaccines. anexils can be used as a synthetic biology platform to construct dnabased vaccines, which combines antigen production, adjuvants and delivery in one system, offering several advantages over existing vaccine formulations. this system can be easily altered for other antigens by simply changing the dna template and carries no risk of infection by attenuated pathogens. availability of complete genome sequences, high throughput technologies and synthetic biology has enabled reverse vaccinology (rv). availability of sequence data from different specimens of the same species of a pathogen provides an opportunity to select novel vaccine candidates. thus the empiric approach to vaccine development is being replaced by vaccine design. the rv approach is one of the most powerful examples of biotechnology applied to the field of vaccinology for identifying new protein-based vaccines. rv combines the availability of genomic data, the analyzing capabilities of new bioinformatic tools and the application of high throughput expression. purification systems can be combined with serological screening assays for a coordinated screening process of the entire genomic repertoire of bacterial, viral or parasitic pathogens. the application of rv to neisseria meningitidis serogroup b represents the first success of this novel approach. this approach can be easily applied to any pathogen [ ] . vaxign is the first web-based vaccine design system that predicts vaccine targets based on genome sequences using the strategy of rv. predicted features in the vaxign pipeline include protein subcellular location, transmembrane helices, adhesin probability, conservation to human and/ or mouse proteins, sequence exclusion from genome(s) of nonpathogenic strain(s) and epitope binding to mhc class i and class ii. the precomputed vaxign database contains prediction of vaccine targets for genomes. vaxign also performs dynamic vaccine target prediction based on input sequences. vaccine candidates against e. coli were predicted using vaxign and results indicate that vaxign is an accurate and efficient vaccine design program [ ] . most of the current treatments of cancer do not discriminate between cancer and normal tissues. besides individual variations, personalized therapy takes into consideration the fact that cancer varies both genetically and phenotypically among patients who may have identical type and stage of cancer. personalized therapy aims to deliver therapy to the malignant tumors while sparing normal tissues. synthetic biology provides many opportunities for design of personalized therapies for cancer. discoveries made through application of human genome sequencing have already an impact on practice of oncology and have influenced the design of clinical trials for new cancer therapies. in the future, research into cancer will expand to generate full genome sequences of various cancers, yielding complete catalogues of somatic mutations in each one. these studies will reveal essentially the full repertoire of mutated cancer genes, enabling us to determine how many and what combinations of mutated cancer genes are necessary to generate an individual cancer. sequencing is evolving from a research tool to applications for cancer diagnostics. analyses of the cancer genome as well as the transcriptome and their applications into clinical trials in order to exploit the full clinical potential of information within the cancer genome are generating new predictors of drug responsiveness and prognosis, which will enable personalized management of cancer [ ] . bacteria can be synthetically engineered to target, invade and destroy cancer cells selectively, or knock down a specific, endogenous cancer-related network of genes. an example of the potential application of this approach is brain cancer (glioblastoma multiforme), which is one of the most challenging cancers, and efforts to cure it have failed in over years of history of modern neurosurgery. the challenge is due to complete eradication required for cure as even a few residual cells multiply rapidly with recurrence of the tumor that kills the patient. response to conventional treatments such as surgical resection and chemotherapy varies according to the characteristics of individual tumors, but none are curative. one of the innovations is the use of genetically modified bacteria to selectively destroy the tumor without invading the surrounding normal brain [ ] . genetic modification of bacteria is complicated as it requires selection of aggressive invasive species and extensive modifications, i.e. excision of harmful genes and insertion of genes for selective destruction of the tumor. synthetic bacteria may be easier to construct and designed according to the characteristics of an individual tumor and the required tasks with better prospects of cure. there are ethical concerns because of the perception that techniques of synthetic biology can be used to create life. in response to this, us president barack obama asked the presidential commission for the study of bioethical issues to review the developing field of synthetic biology and identify appropriate ethical boundaries to maximize public benefits and minimize risks. the findings of this commission were published in december . 'the recommendations detailed in this report provide a publicly accountable basis for ensuring that the field of synthetic biology advances to improve human health and public welfare with processes in place to identify, assess, monitor, and mitigate risks on an ongoing basis as the field matures. risk assessment should precede field release of the products of synthetic biology. ongoing assessment and review is required in several areas to avoid unnecessary limits on science and social progress, and to ensure appropriate restrictions to protect individual safety and our shared environment. ongoing dialogue about concerns regarding the implications of synthetic biology for humans, other species, nature, and the environment should continue as synthetic biology develops from its infancy to a fully mature field of scientific inquiry and innovation' [ ] . synthetic biology is a further stage in the evolution of the biotechnology industry that has been taking place for more than years and has resulted in the development of commercial products. safety issues of synthetic biology are covered by existing regulations for biotechnology drugs. synthetic biology is already being used by biotechnology companies and has the potential to substantially reduce research and development time of products towards commercialization [ ] . in the future, as synthetic biology products enter the market and if scientific evidence warrants it, there may be need for special regulatory oversight beyond that for biotechnology products in general. in may , the us department of defense granted usd . million for synthetic biology research at several universities and institutes with the aim of speeding up bioengineering production. there are eight projects being funded through the defense advanced research projects agency under this new initiative. research conducted under the program will seek to create the basic production methods and tools that will be required to make bioengineering swifter and more accurate, and to design the blueprints for synthetic biology factories. these projects will pursue the initiative's aims of developing new tools, technologies and methods to enable the rapid development of bioengineered products, such as new materials and medicines. also in may , the uk's engineering and physical sciences research council (http://www.epsrc.ac.uk) awarded a gbp million (usd . million) five-year grant to a consortium of universities to develop a webbased synthetic biology information system called syn-bis that is currently in beta trials. synbis will host the software platform biocad and modeling tools, opening up the possibility of undertaking high-level software design of bioparts and devices. at the same time, the uk government plans to provide a boost to the nation's synthetic biology sector under a new initiative that will provide nearly gbp . million (usd . million) in research funding. applicants for the funding also will be expected to consider any ethical, social and regulatory implications related to the use of their technologies. funding of research in synthetic biology by government agencies in the usa and the uk is encouraging. acceptance of personalized medicine by the pharmaceutical industry and healthcare professionals is increasing. currently research on synthetic biology is being conducted at over places in the usa and europe [ ] . these include companies ( in the usa and in europe) and universities ( in the usa and in europe); the rest are research institutes and laboratories. this number has been increasing over the past few years and is expected to continue increasing. the synthetic biology research market, which was estimated to be worth usd million in , is estimated to grow to usd billion during the next decade [ ] . among new technologies, synthetic biology will contribute by the introduction of therapeutic systems based on a synthetic genome, using an expanded genetic code, and designed for specific personalized drug synthesis as well as delivery and activation by a pathological signal. improvements in the speed and cost of dna synthesis will enable scientists to design modified bacterial chromosomes that can be used in the production of pharmaceutical intermediates and healthcare products. synthetic mammalian gene network-based design of biohybrid materials will be useful for applications in personalized medicine because of its interactive functionality [ ] . synthetic biology may provide the tools to devise tailored treatments for some currently incurable conditions. synthetic biology and personalized medicine will provide commercial opportunities for the biopharmaceutical industry by discovery of personalized drugs that will be cheaper to manufacture and have a shorter and less expensive development path with lower failure rate compared to traditional drugs. although there are a number of ethical issues they are being addressed. as further research is carried out, funding covers the investigation of relevant new ethical issues that may arise. synthetic biology remains a promising technology for advancing personalized medicine. engineering life through synthetic biology emerging biomedical applications of synthetic biology drug discovery and delivery in the st century textbook of personalized medicine the role of nanobiotechnology in the development of personalized medicine role of biological therapies in the development of personalized medicine creation of a bacterial cell controlled by a chemically synthesized genome the changing economics of dna synthesis synthetic biology: applications come of age breaking the code of dna binding specificity of tal-type iii effectors de novo designed proteins from a library of artificial sequences function in escherichia coli and enable cell growth smart medication through combination of synthetic biology and cell microencapsulation systems medicine and metabolic modelling metabolic engineering for the production of clinically important molecules: omega- fatty acids, artemisinin, and taxol sequence-specificity and energy landscapes of dna-binding molecules mammalian synthetic biology -from tools to therapies synthetic biology moving into the clinic resistance to dietinduced obesity in mice with synthetic glyoxylate shunt rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells anti-borrelia burgdorferi antibody profile in post-lyme disease syndrome black wc th: genetic elimination of dengue vector mosquitoes engineering antibiotic production and overcoming bacterial resistance screens, maps & networks: from genome sequences to personalized medicine therapeutic synthetic gene networks compartmentalized reactions as a case of softmatter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles synthetic cells and organelles: compartmentalization strategies highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna synthetic reversal of epigenetic silencing bioinspired materials for controlling stem cell fate synthetic biology: impact on the design of innovative vaccines structure-based design of peptides that self-assemble into regular polyhedral nanoparticles a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine antigen identification starting from the genome: a 'reverse vaccinology' approach applied to menb vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development genomics and the continuum of cancer care future prospects for the cure of brain cancer presidential commission for the study of bioethical issues: the ethics of synthetic biology and emerging technologies. washington, dc synthetic biology: regulating industry uses of new biotechnologies synthetic biology project: mapping the emerging synthetic biology landscape synthetic biology: technologies, markets and companies. basel synthetic mammalian gene networks as a blueprint for the design of interactive biohybrid materials key: cord- -v ye authors: ganguly, nirmal kumar; saha, gautam kumar title: pharmacogenomics and personalized medicine for infectious diseases date: - - journal: omics for personalized medicine doi: . / - - - - _ sha: doc_id: cord_uid: v ye humans have been plagued by the scourge of invasion by pathogens leading to infectious diseases from the time in memoriam and are still the cause of morbidity and mortality among millions of individuals. trying to understand the disease mechanisms and finding the remedial measures have been the quest of humankind. the susceptibility to disease of an individual in a given population is determined by ones genetic buildup. response to treatment and the disease prognosis also depends upon individual’s genetic predisposition. the environmental stress induces mutations and is leading to the emergence of ever-increasing more dreaded infectious pathogens, and now we are in the era of increasing antibiotic resistance that has thrown up a challenge to find new treatment regimes. discoveries in the science of high-throughput sequencing and array technologies have shown new hope and are bringing a revolution in human health. the information gained from sequencing of both human and pathogen genomes is a way forward in deciphering host-pathogen interactions. deciphering the pathogen virulence factors, host susceptibility genes, and the molecular programs involved in the pathogenesis of disease has paved the way for discovery of new molecular targets for drugs, diagnostic markers, and vaccines. the genomic diversity in the human population leads to differences in host responses to drugs and vaccines and is the cause of poor response to treatment as well as adverse reactions. the study of pharmacogenomics of infectious diseases is still at an early stage of development, and many intricacies of the host-pathogen interaction are yet to be understood in full measure. however, progress has been made over the decades of research in some of the important infectious diseases revealing how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the efficacy and toxicology of the drugs used for treatment. further, the field of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of “omics” technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. over the millennia with the progression of human civilization, the condition of human health has changed considerably. the lifespan of the human has increased considerably with the advent of vaccines against several diseases which has eradicated small pox, and now we are embarking on the global campaign to eradicate poliomyelitis and have controlled the disease in most parts of the world. the treatment of infectious diseases got a boost with the discovery of penicillin by alexander fleming in the earlier part of the twentieth century. as a range of antibiotics were later discovered, infectious diseases such as meningitis, bacterial pneumonia, sepsis, and other lifethreatening bacterial infection were treatable. also the survivability of patients undergoing operative procedures and aggressive chemotherapy was feasible and their recovery high. the last years of the twentieth century have been eventful with the discovery of antimicrobials that had given us the hope that we shall eradicate all infectious diseases. despite all the efforts and progress we have made in medical science, infectious disease remained a major health problem. but the golden era of the antibiotics will not be long if we go on unregulated administering and promoting rampant use of antibiotics, as is evident from the rise of antibiotic resistance (caramia and ruffi ni ) , and the new emerging diseases have posed major challenge (table . ). in the last few decades with the advent of fi eld of genomics, there is a new hope in prevention, diagnostics, and treatment of infectious diseases. we will explore in this chapter the table highlights the organisms that are of public health importance and their year of discovery source : world health organization: newly discovered organisms of public health importance: page . from who regional offi ce east-asia: combating emerging infectious diseases in south east asia region ( ) how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the effi cacy and toxicology of the drugs used for treatment. further, the fi eld of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of "omics" technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. how the advent of new technologies is bringing about a change in medical treatment of the infectious diseases. for the use of "omics" technology to be successful requires considerable information of pathogen genome as well as genome information of the host. the pathogen genomic and proteomic information helps to identify antigens that can give us information necessary for making a diagnostic tool and vaccine design. the pathogen genome on one hand gives us the information about the important genes conferring disease pathogenesis as well as drug resistance, while the genome of the host on the other hand will reveal the susceptibility genes, and the further knowledge of polymorphisms in genes of the host metabolic and immune system will lead to the new vaccine strategies, drugs targets, and also their treatment outcomes. rapid advances of biotechnological and informatics tools in the past few decades mainly in fi elds of genetics, genomics, and proteomics are leading the way in identifying treating and thus improving the health of human beings. the effective treatment in a patient can only be achieved by fi rst rapid diagnosis of the disease and also identifying its causative agent that is particularly important in the cases of infectious diseases. new insights gained by the analysis of genome and structural feature of pathogen macromolecules have brought about new hope in the treatment of the dreaded diseases. the knowledge of system biology in respect to the microbial infections is still in development, and data available is mostly for few human infections. the rapid development of new generation sequencing technologies have led to generation of new knowledge base and with more advancement of such technologies in the coming years has brought in a hope that all diseases will conquered. in the near future, we will have complete sequences of the total transcriptomes, like genome sequences a decade earlier, and proteomic technologies will attain the throughput and sensitivity of microarrays. other technologies like metabolomics, glycomics, lipidomics, and phosphoproteomics when referred to in the context of infectious diseases are still in various stages of development, but we are taking the right steps in the direction of development of such technologies (antony et al. ). the technologies of transcriptomics and functional genomics are transforming our understanding of microbial infections and helping us decipher the reason of infections susceptibility in the humans. transcriptomics have been developed and used by scientists to broaden our understanding of infectious diseases. to elucidate the hostpathogen interaction, cdna microarrays have been widely used. the studies have focused on how the pathogens effect the host cell gene expression. the wild-type virulent strains and isogenic mutants have been used to gauge the responses of the host cell. the major fi ndings of these studies have shown how the pathogen virulence factors modify host cell factor expression (roy and mocarski ) . the role of pathogen recognition receptors (prr) affecting hostpathogen interaction has been studied. these studies have shown that the host cell responses have an alarm signal (jenner and young ) . studies have also shown that gene cluster signals are responsible for generating the alarm signals that are the target of attack by the invading pathogens (hamon and cossart ) . array technologies are being used with molecular probes of host human (also animals/plants) and microbial genes to monitor and at the same time point the expression of genes from host cells and those of the pathogens to better understand the full complexity of host-pathogen interaction. functional genomics have also led to the development of tools to decipher infectious diseases by manipulating the cellular mechanisms. the technology of the rna interference (rnai) has undergone tremendous development in the last decade which has led to the large-scale reverse genetic screens in human cells and model organisms (boutros and ahringer ) . rnai technology uses double-stranded ribonucleic acid (dsrna) with having complementary sequence to the target mrna sequence and is used to silence or downregulate the gene expression of its target. long dsrna induces interferons or other unspecifi c responses in mammalian cells which is avoided by the use of small interfering dsrna (sirna) directly or small hairpin rna (shrna). rnai screening using rna probes, which induces loss of function of host genes, leads to discovery of host resistance factors (hrf). it is made possible when silencing this restrictive factors leads to invading pathogen replication enhancement and may also identify host susceptibility factors (hsf) and also identify permissive factors, that when silenced will decrease the pathogen replication. the rnai screens still have some limitations due to offtarget sirna effect (echeverri et al. ) . thus, the primary screening validation is made by using additional sirna screens. from the several rnai screenings in human and in fruit fl y cells, only host factors were validated from about , and , targets identifi ed in the initial screen (agaisse et al. ; brass et al. ; konig et al. ; krishnan et al. ; zhou et al. ) . to make the system biological tools like rnai more effective in fi nding mechanisms in host-pathogen interaction and thus fi nding cure for the microbial infections, there is a need for integrations of all data obtained from the omics technologies. in addition several rounds of biological experimentations are required by using mutant pathogens, cellular rnai knockdowns, or humanized animal models using mice or primate infection model. the resulting inferences from the validated data would help us build predictive models which could lead us to the better understanding of pathogen interactions with the host. it is evident from human history of infectious diseases that not everybody in a given population is affected by an infective disease. for an infective organism to cause an infection, both the virulence of the pathogen and the host susceptibility are important. the identifi cation of genetic factors of host innate and adaptive immunity that determine the protection from pathogen is an important endeavor of scientists. animal models of infectious diseases especially mouse models have been used to fi nd the genetic factors and biochemical mechanism of disease susceptibility (marquet and schurr ) . the identifi cation of candidate genes responsible for disease susceptibility or resistance and the occurrence of genetic polymorphism in them give us the best possible biological scenario of the disease. researchers have found that in bacterial diseases, tuberculosis and leprosy seem to have similar genetic susceptibility determinants in the host as exemplifi ed from the fi nding that higher incidence of these diseases was found in the monozygotic twins than in dizygotic twins and siblings (abel et al. ; vidal et al. ) . mouse model of infections has revealed that gene encoding the natural resistance-associated macrophage protein ( nramp ) confers natural resistance to infections caused by mycobacterium , salmonella , and leishmania . nramp is found in the membrane of the phagosome of the macrophages where it seems to be probably affecting the replication of the infecting intracellular bacterium (gruenheid et al. ) . the genomic analysis in humans has found a similar gene to that of mice which is also having similar pattern of expression. hence, it has been inferred that humans too carry similar susceptibility gene. there have been further studies which have shown that polymorphisms found in the nramp gene are related to the infectivity of leprosy and tuberculosis (abel et al. ). in one of the studies, it was found that persons who carry an nramp heterozygous variant will be four times more likely to be infected by tuberculosis than persons who are carrying more common variants of the nramp (bellamy et al. ). cell-mediated immunity plays an important role in the context of tuberculosis and is well studied. research has further gone ahead to fi nd links between tuberculosis susceptibility and polymorphism in the gene coding for receptors of interferon-γ and interleukin- , which are cytokines belonging to t-helper cell type (th ). the absence of functional copies of either of these genes in families and of isolated patients leads to high susceptibility to m. tuberculosis infection (jouanguy et al. (jouanguy et al. , newport et al. ; altare et al. ) . the highly polymorphic human leukocyte antigen (hla) system is the name of the major histocompatibility complex (mhc) in humans. the hla class i glycoproteins are highly expressed on the surface of every nucleated human cell, and they present endogenous peptides derived from the cell to the cytotoxic t cells. hla class i glycoprotein plays a major role during the viral infection as it presents intracellular viral peptides on the surface which leads to cellmediated immune response, which further leads to the destruction of virus-infected cells. hla class ii glycoproteins which are present on the surface of antigen-presenting cells (apcs) on the other hand present - amino acids long peptides that are derived from the engulfed pathogen and displayed on the surface which then are recognized by t cell as foreign antigens, and it will elicit an immune response to the antigen. the length of antigen as well as composition are important in deciding if the antigenic peptide will bind to the antigenic peptide-binding cleft. polymorphisms occur almost solely in the peptide-binding cleft of hla class i and ii in the glycoproteins. the diversity of hla-binding region ensures that some pathogenic peptides will be preferentially presented compared to the others. thus, in a given population, the hla diversity ensures the advantage that some of the hla glycoprotein peptide-binding clefts will be able to bind and present the pathogenic antigen peptide which will lead to an immune response to any invading pathogen. this ensures the survivability of the species against an infection. thus, genomic studies have focused on the identifi cation of susceptibility genes which would lead to the better management of infectious diseases in the population. the tuberculosis susceptibility has been associated with hla class ii genetics. the association is evident from the studies that have been found between pulmonary tuberculosis and class ii hla antigens in several populations (marquet and schurr ) . as is now clear, the knowledge of the mechanism of action of the pathogen and the identifi cation of the susceptibility genes goes a long way in the management of the disease in context of a public health perspective to prevent, diagnose, identify, and target the vulnerable populations against a given infectious disease. the requirement of the genomic information of both the host and pathogen is important to fully carry out infectious disease management. the fi rst sequence map of human genome being completed in june (lander et al. ; venter et al. ) , followed by discovery of genome-wide single-nucleotide polymorphisms (sachidanandam et al. ) and further genomic sequencing of several pathogens by institutes like the j. craig venter institute ( www.jcvi.org ) gradually opened the pathway to create new treatment and disease management methods. the ultimate aim of genomic technologies to bring personalized medicine for every infectious disease scenario is still decades away, but here we will focus only on important breakthrough which has shown us the way forward in respect to infectious disease identifi cation and treatment. neisseria meningitidis is a bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a lifethreaten-ing sepsis ( www.cdc.gov/meningococcal/ ). n. meningitidis is a major cause of morbidity and mortality during childhood in industrialized countries and has been responsible for epidemics in africa and in asia . neisseria meningitidis serogroup b is responsible for causing about one third of infection. the genome sequence of n. meningitidis has opened a new way for disease management (pizza et al. ; tettelin et al. ) . the vaccine that was used only contained capsular polysaccharides from the serogroups a, c, y, and w only. serogroup b polysaccharide contained elements that resemble human polysialic acid and hence is poorly immunogenic and might generate autoantibodies (hayrinen et al. ) . in this scenario the n. meningitidis serogroup b was sequenced, and potential antigens from the serogroup b were expressed in escherichia coli to fi nd the potential vaccine candidate. the expressed proteins are injected into mice to fi nd immunogenic antigens that can developed into a vaccine. similar strategies are being used to fi nd vaccine candidates for other serotypes of neisseria species and for other pathogenic organisms. pathogen genomic information is also being used to fi nd the immunologically important peptides for cytotoxic t lymphocytes (ctls) epitopes. the response of ctls is to seek out virally infected cells by recognizing the peptides presented by human leukocyte antigen (hla) glycoproteins on the cell surface and killing the infected cells. ctl epitopes are the viral peptide that is presented by the hla and recognized by ctls. the peptides are of the length of amino acids, and genome sequence is used to fi nd out and synthesize these peptides for immunogenic evaluation. amino acids are divided into segments of peptide, measuring amino acids in length and overlapping the previous peptides amino acids, for example, west nile virus genome translates into , amino acids which can be segmented into , peptides that are amino acids in length. immunoinformatics, a fi eld of bioinformatics, is speeding up the fi nding of ctl epitopes for the scientist working in the fi eld. algorithms on computer softwares are being used to match the viral peptides with the hla glycoproteins in silico for binding based on previously known results and are being tested (de groot et al. ). informatics based algorithms help eliminate % of the peptides that would not be used in the experimental screens. thus, the time and effort to screen for the ctl epitopes have been reduced drastically. ctl epitopes may be used for making subunit-based vaccines and diagnostic tests. virusspecifi c antibody found using ctl epitopes can be used in enzyme-linked immunosorbent assay. it may be even possible to use ctl epitopes to test for the antigen itself. basically as of now only four types of molecular diagnostic tests are carried out to detect infection in laboratory setup. first is by direct detection where the pathogens can be detected directly by imaging technologies of microscopy and cell culture. second method of diagnosis is by the detection of proteins produced by pathogens by the use of specifi c antibodies, like that used in enzymelinked immunosorbent assay (elisa). third method is by detection of the specifi c antibodies iga, igm, and igg against the pathogens and the changes in their titers using antibody capture assay. fourth method uses detection of nucleic acid of the pathogens and amplifying their signal using techniques like polymerase chain reaction. latest diagnostic technologies have been developed on these basic four biotechnological technologies (speers ) . pathogen genome can also be used to identify the infecting organism itself. microbial dna in the clinical specimen can be used to identify the disease-causing pathogens. human immunodeficiency virus (hiv), hepatitis virus, borrelia burgdorferi (causative agent of lyme disease), and mycobacteria are few examples of pathogens that can be identifi ed by their genomic sequences. mycobacterium tuberculosis antimicrobial resistance strain-caused infections are becoming quite common, and genomic information has deciphered few potential candidates like katg gene mutations in resistant strains (siqueira et al. a , b ; marahatta et al. ) . the traditional culture test for mycobacterium which is time-consuming and less sensitive is giving way to restriction fragment length polymorphism (rflp), a specifi c technique used in dna fi ngerprinting (van soolingen ). the technology uses restriction enzymes that cut dna at the places having certain particular nucleotide sequences. the nucleotide pattern that is obtained is then compared to the previously identifi ed specifi c nucleotide pattern of the genome of the pathogen dna. dna patterns can be separated on the basis of length, and the pattern of dna fragments in the dna fi ngerprint is characteristic of particular isolate, and each particular pathogen has a unique pattern. dna fi ngerprint technology is faster and reliable than culturing of the mycobacteria, ideal for discovering new drug-resistant strains from unique genomic sequences of each mycobacterium. the technology is very useful for identification of strains during the time of outbreaks and further epidemiological studies. the knowledge of viral load in patients is also important for dosage determination in drug therapy; hence, detection of the viral pathogenic dna and rna in clinical specimens is of paramount importance. treatment of viral diseases like hiv, chronic hepatitis b, and hepatitis c often depends on the knowledge of viral load (revets et al. ) . for example, hiv viral loads are detected by enzymatic amplifi cation of the viral nucleic acid and detection of the signal from the labeled probes that hybridizes with them. the signal usually is either a color signal conjugated to the probe or a chemiluminescent probe, and the intensity of the signal corresponds to the number of copies of the nucleic acid rna. capillary electrophoresis detects hybridized probes at a very high sensitivity with detecting as low as , copies of hiv rna in milliliters of plasma (kolesar et al. ). the rampant uncontrolled use of antimicrobials has led to increased number of antibiotic bacterial strains. genomic mutations allow the certain bacterial strains to overcome the effects of antimicrobials and are able to propagate in spite of the presence of antibiotics. fluoroquinolones are drugs that act on the bacterial dna replication by binding to bacterial enzymes involved in bacterial dna replication, that is, dna gyrase and topoisomerase. the bacterial resistance to quinolone occurs due to mutation in the quinolone-binding site in the enzymes mentioned above. the mutation leads to change in the amino acid at the site of binding of fl uoroquinolones to the enzymes. if both the bacterial enzymes are mutated, then high-level resistance occurs to the quinolone drug affecting the treatment of infection as compared to when either of the enzymes is mutated (hooper ) . now genetic test is available to detect antimicrobial resistance in the infecting pathogens. the information is important because it would lead to a better treatment management of the infection. the methicillin-resistant staphylococcus aureus phenotype is detected when cultured in the presence of oxacillin after a period of h. before the era of omics technology, the only means of resistance detection was by culture test which is a very time-consuming test. methicillin resistance in s . aureus is controlled by alternations of penicillin-binding protein pbp a. gene meca controls the production of pbp a. polymerase chain reaction test is used to detect the presence of meca in reference laboratories, while commercially developed kit can detect the same using a fl uorescein-labeled meca probe. both dna probe and pcr technology when used for analysis can detect meca -resistant gene in a given sample in less than h. the rapid detection of antimicrobial resistance in pathogens helps patients in providing adequate treatment opportunities (louie et al. ) . the study of the host genome becomes important to fully understand the drug effects and as such design more effective methods of treatments. although the ultimate goal is to decipher the system biological effect, the trend of single gene effects is also very important. cytokines play a very important role in human immunity (paul and seder ) . in hepatitis c infection, interferon alpha is used to stimulate cell-mediated immunity against the viral infection and is the primary treatment. however, studies have shown that response to interferon-alpha treatment is only % in some cases even when combined with other antiviral treatment (manns et al. ) . further studies have shown that if chronic hepatitis c patients have il- polymorphism variant, it leads to the reduction in expression of il- itself, and they will have fi ve times more chance of effective treatment with interferon alpha than those who do not carry the polymorphism (edwards-smith et al. ) . interleukin- (il- ) is a polymorphic cytokine and is a t-helper cell type ii (th ) cytokine that is associated with the induction of the production of large amount of antibodies in body's immune response. th cytokines which promote cell-mediated immunity inhibit th response and vice versa. thus, people with high-expressing il- genotype if infected and suffering with chronic hepatitis c infection are less likely to respond to interferon-alpha treatment. new treatment regimes have to be developed for patients suffering from chronic hepatitis c infection and carrying il- polymorphism associated with high cytokine expression. vaccine responses can be used as a system of gauging the state of immune system ( poland a , b ) . vaccines are administered to large number of population as an integral part of public health system. vaccines are used to mimic the infective disease conditions that induce immunological memory to protect the individual against subsequent exposure to the pathogen and lead to prevention of disease. the phenomenon to gain protective immunity against a pathogen upon being vaccinated for the particular pathogen depends on individual genetic build. as studies have shown, not all healthy individuals are able to generate a protective immune response upon vaccination. it has been observed in the case of measles vaccination that only % of the population was seronegative and clustered in family (poland a , b ; poland et al. c ). both hla i and hla ii class alleles have been responsible for the measles vaccine response, while hla-b , hla-b , hla-drb * , and hla-dqa * are associated with positive measles vaccine response, and hla-b, hla-dr, and hla-dqa have been responsible for the vaccine being noneffective (hayney et al. (hayney et al. , poland et al. ). drugs used for targeting any pathogenic infection can only be successful if we are aware how it is affecting the host and pathogen at genomic level and hence are able to explain the host effi cacy and toxicity. we look at few important infectious diseases where pharmacogenomic research has been bringing a landscape change in the disease treatment. leishmaniasis is a very complex major tropical infection transmitted by the vector sand fl y is all right. the infection is caused by intracellular protozoan parasites of leishmania genus. there are more than species of leishmania . the type of infective species, virulence factors, and host immune responses and depending on the clinical symptoms, the disease is categorized into cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). vl is also known as kala-azar; the origin of the name is from the eastern and northeastern part of the indian subcontinent where the disease is endemic. depending upon the place where one has acquired the infection, cl is further categorized into "new world" from central america and south america and "old world" if from asia, middle east, africa, or southern europe. more than - . million cases of leishmaniasis occur worldwide (about countries are affected) with major countries being the developing nations of asia, africa, and latin america ( www.who.int/topics/leishmaniasis/en/ ) . species of leishmania are several causing different clinical manifestations of the infectious disease. l . donovani produces primary cutaneous disease as well as gives rise to visceral leishmaniasis (vl) and also post-kala-azar dermal leishmaniasis (pkdl) that is manifest after the treatment of the initial visceral disease. visceral leishmaniasis main causative pathogen is the l. donovani complex with old world vl disease being caused by the species l. donovani and l. infantum, and new world disease is mainly caused by different species of l. chagasi in the cases of infection caused by l. braziliensis complex, there is always a chance that the infection dissemination to mucosal region can occur to give rise to mucocutaneous leishmaniasis (mcl) (herwaldt ) . the complex disease is manifested due to multiple factors ranging from environmental factors such as time and number of exposure with infected vector sand fl ies, species of the infecting leishmania pathogen, to host genetic factors that include immune status of both innate and adoptive immune systems that determine the clinical outcome of the disease. other reasons for leishmania disease susceptibility are malnutrition, immunodefi ciency with hiv coinfection, and young age. the protection against invading pathogenic leishmania protozoa and even the curative resolution of the disease is provided by th cytokine response involving cytokine interferon gamma (ifn-γ), interleukin (il)- , and tumor necrosis factor alpha (tnf-α), whereas th response cytokines il- , transforming (tgf)-β, and il- have been implicated in increasing susceptibility to the disease in the experimental animals (reed and scott ; sacks and noben-trauth ) . nonhealing lesions and diffused lesions in cl have been implicated to th response, while self-healing lesion has been associated with th response (melby et al. ) . however, in some situation il- (a th cytokine) has been implicated to induce il- production and lead to th cytokine response, and it has also been found in some cases that th response occurs independent of il- (alexander and bryson ; mansueto et al. ). leishmania infection is a complex infection depending on host factors as well as strain polymorphism. leishmania mexicana cysteine proteases which target il- that prevents th protective response (buxbaum et al. ) , while the leishmania analogue of activated c kinase (lack) from the leishmania major induces th response that leads to host parasitization (kelly et al. ) . polymorphism of l. braziliensis also affects disease outcomes (cupolillo et al. ; schriefer et al. ). pkdl is a complication arising after treatment of vl, affecting % of vl patients in sudan (study carried out in united sudan) and also - % patients in india. pkdl has been found to be associated with increased levels of il- (zijlstra et al. ; ganguly et al. ). the major treatment regime of cl which has propensity of dissemination towards vl and mcl is with parenteral antimonials like sodium stibogluconate or meglumine antimoniate, pentamidine, and oral miltefosine (olliaro et al. ; ameen ; amato et al. ) , whereas cl with low risk of spread is treated with local and physical therapies such as intralesional antimonials, topical paromomycin, cryotherapy, and thermotherapy or by oral azoles. however, when the disease progresses to mcl, treatment is prolonged, and toxicity from such long-duration drug use is a common occurrence (marsden ; amato et al. ) . vaccine is still elusive in the case of leishmania. some trials with dna vaccines do have shown a way forward. these vaccines have shown the promise to be effective as they have been able to induce l- production, which was in response from the persistent antigen exposure from the dna vaccine (requena et al. ). in venezuela killed leishmania promastigotes along with bacillus calmette-guerin (bcg) used as immunotherapy have shown results with a high cure rate in clinical trials by inducing th response (convit et al. ) . l. major vaccine trial with bcg and parenteral antimony combined have been successfully used for treatment of pkdl (mansueto et al. ). the search for effective vaccine for leishmania had got a boost with knowledge from genome sequence data of several leishmania strains. more vaccine candidate genes will be evaluated in the future (stober et al. ). in the absence of an effective vaccine with recurring infection such as pkdl, dissemination infection to mucosa leads to aggravating of the disease. prolonged treatment with parenteral antimonials that give rise to high-level risk of toxicity with high morbidity and mortality from the disease is a problem of concern (convit et al. ; muse et al. (samaranayake et al. ) . l. donovani though normally associated with causing vl is shown in few places, kenya, yemen, cyprus, and the himalayan region of north india, and is the main causative pathogen of cl (mebrahtu et al. ; pratlong et al. ; sharma et al. ; antoniou et al. ) . to deduce the genetic susceptibility of the leishmania disease, experimental murine animal models along with clonal parasite line (to control environmental variable) have been used to fi nd the genes responsible for disease progression along with their human homologues of disease susceptibility (handman et al. ) . first genes that were used to deduce from such analysis in murine model were nramp and the h- locus had been implicated in l. donovani infection (blackwell et al. ) . hla class ii antigen hla-pq is found to be associated with cl in venezuela (lara et al. ) and mcl in brazil caused by l. braziliensis (petzl-erler et al. ) . pcr genotyping studies in mexico on leishmania patients has found an association with hla class ii genes with cutaneous leishmaniasis (cl) (olivo-diaz et al. ) . high blood tnf has been found to be associated with mcl (castes et al. ) and acute vl (barral-netto et al. a , b ) . a venezuelan study has implicated that allele of tnf-β polymorphism with high risk of developing mcl caused l. braziliensis and higher frequency of allele of tnf-α polymorphism was also associated with mcl (cabrera et al. ) . in brazil by using family-based disequilibrium test analysis (tdt), investigation has shown that tnf polymorphism has been linked to l. chagasi infection (karplus et al. ) . in asymptomatic patients having positive skin test, l. chagasi has been associated with tnf- allele of tnf-α gene, while in case of symptomatic l. chagasi vl patients, tnf- allele is implicated. due to parasite heterogeneity, this tnf polymorphism association has not been correlated to infection by other leishmania species such as in l. infantum vl (meddeb-garnaoui et al. ) and l. major cl (kamali-sarvestani et al. ) . variation in promoter of il- and ifn-γ gene polymorphism has been found to be linked to l. major cl disease susceptibility and progression respectively in an iranian study, while in a sudanese vl patient study, il- polymorphism has been shown to increase disease susceptibility (mohamed et al. ) . polymorphism in promoter region of il- gene leads to higher il- production which has been shown to increase the risk of having skin lesions during an infection of l. braziliensis (salhi et al. ). il- can diminish the high th proinfl ammatory response that occurs when l. braziliensis cl progresses to mcl (hatzigeorgiou et al. ; bacellar et al. ) . il- polymorphism plays an important role in the progression of l. braziliensis cl to mcl, and this fi nding is important since their genetic markers have high prognostic value (castellucci et al. ) . genome-wide linkage have been performed for l. donovani-infected vl patients in artinga ethnic group in sudan to help identify loci on chromosome q and is associated with disease susceptibility genes (bucheton et al. a , b ) . il- receptor β chain (il rb) gene is present in the highly susceptible loci on chromosome q that was identifi ed from this study. il- receptor has been detected in high levels during vl infection and plays a critical role in t cell genetic responses (barral-netto b ) . further studies have shown il rb polymorphism in association with l. donovani vl (bucheton et al. ) . another candidate gene found is slc a (formerly nrampi) on chromosome q , an innate resistance gene that regulates macrophage activation and contributes to increased vl risk in sudanese population (bucheton et al. a , b ; mohamed et al. ) as well as increased susceptibility to several intracellular pathogens (blackwell et al. ) . other studies have shown that genotypes having signifi cantly high level of mannan binding lecithin occur more prominently in patients with clinical vl. an opsonin, mannanbinding protein, is known to enhance pathogen infection. polymorphism in mannan-binding gene has been shown to increase risk of developing l. chagasi vl in brazilian study population (alonso et al. ) . in pkdl there is elevated level of ifn-γ. polymorphism of ifn-γ receptor from study in sudan has been implicated in pkdl (salih et al. ). the ifn receptor expression is important for the activation of macrophages via ifn-γ. drug treatments are not very effi cient in the treatment of leishmaniasis disease; more effective treatment regimes can be developed by thoroughly knowing the genetic factors that lead to disease progression. thus, unnecessary drug use and adverse reaction can be avoided. as various genetic susceptibility studies have shown, cytokine response determines the disease progression in leishmaniasis. role of il- in pathogenesis of leishmaniasis is known and is well established, and il- polymorphisms have shown to increase risk of lesions in l. braziliensis infection. in a study with l. guyanensis infected cl patients from french guiana, high level of mrna il- within lesions leads to poor chemotherapy response and treatment failure (bourreau et al. ) . it is hypothesized that il- might be regulating the response to chemotherapy by blocking the th response. the increased level of il- has been linked to the active vl (nylen and sacks ) and pkdl (saha et al. ) and also associated with persistent cl infection occurring from l. major (melby et al. ) and l. mexicana (louzir et al. ). success of vl treatment with amphotericin b and the complete elimination of il- are associated with one another (saha et al. ). on the other hand, mcl is associated with low il- receptor expression and low il- secretion that decrease the ability for modulation of proinfl ammatory response (faria et al. ) . progress has been made to fi nd the susceptibility genes and will provide further insight into disease pathogenesis and will lead to progress in the fi eld of diagnostic markers, drug targets, and vaccine development to control, treat, and eradicate leishmaniasis. improving the treatment of malaria by pharmacogenomics malaria is vector-borne (mosquito) disease that has been one of the top causes of mortality in the world for generations especially in tropical countries of asia and africa. even after renewed global efforts, still there is high infectivity and mortality. three billion people are at risk with - million deaths attributed to malaria each year ( www.who.int/topics/malaria/en/ ). four species of protozoan parasite are involved from genus plasmodium, i.e., p. falciparum , p. vivax , p. malariae, and p. ovale . these malaria-causing combination parasitic species occur in human population and occur in infected individuals (gurarie et al. ) . in respect to prevalence, virulence, and multidrug resistance, p. falciparum has been a major cause of mortality and morbidity. p. falciparum accounts for about % cases of malaria in africa (roca-feltrer et al. ). next to it is p. vivax which causes - million cases annually (price et al. ). the most commonly used drugs are chloroquine (cq) and sulfadoxine-pyrimethamine (s-p fansidar ® ) that are becoming less effective due to the development of resistance in malaria parasite by p. falciparum, and the species has become predominant and become a threat to travelers and people alike (schlagenhauf and petersen ) . in the absence of vaccine and in addition, development of resistance even in the mosquito vector control against chemical methods using insecticides has thrown new challenges for the researchers (greenwood et al. ) (fig. . a ). some of the recent developments in malarial treatment using pharmacogenomics are bringing about improvements in the effi cacy of treatment regime of malaria. current treatment regimes have recommended artemisinin combination treatments (acts) in cases of uncomplicated falciparum malaria in nonpregnant adults (lin et al. ) . the drug regime is highly effi cacious and has reduced development to resistance. in cases of uncomplicated malaria, the act is being used in countries by . in the coming years, a number of patients including women and children will be brought under act therapy regime as per world health organization. like the treatment of hiv and tuberculosis, combination therapy is now being used for malaria treatment too, which reduces resistance among the highly effi cacious drug the artemisinins which rapidly eliminate the parasite from blood and thus limit the number of parasites so that the other more bioavailable drugs given in combination act on the parasite. unrelated mode of action of two or more combination drugs also reduces the chances of resistance (yeung et al. ). among many other factors which contribute to drug effectiveness, malarial drug bioavailability and tolerability are depended upon the host metabolic mechanisms. the severe drug reaction to primaquine in the s used in antimalarial treatment was instrumental in the discovery of glucose- -phosphate dehydrogenase (g pd) defi ciency in ; thus, importance of the use of pharmacogenetics in malarial treatment was realized (alving et al. ). the polymorphism leading to variation g pd or even its defi ciency is a grave problem in designing the effective drugs. even now during malarial terminal prophylaxis to decrease transmission, primaquines are administered. thus, the g pd status of patient becomes quite important (luzzatto ) . knowledge of both the host and parasitic genetics is necessary to designing drugs and dosage for effective treatment regimes. parasitic genetics helps us in deciphering the modes of resistance, and host genetics help us in giving the information about host drug bioavailability and explain adverse reaction to the drugs. g pd polymorphisms and genetic variation in cyp c can play pivotal role in point of care diagnostics, but these genetic testings will have to be incorporated into the laboratories and national health programs. the knowledge of this important genetic variations in population would ultimately reduce cost and make the treatment regime more effective and with lesser adverse reaction and ultimately reduce the suffering of the patients. the pharmacogenetic drug policy in context of malaria is slowly becoming a reality as per efforts of the who and other agencies. genetics is becoming a guide to new drug policy. amodiaquine was generally known to be tolerated in malarial treatment, but later when it was found in the caucasian population during the decades of and to be responsible to cause agranulocytosis with fatalities and also cause hepatotoxicity (hatton et al. ; raymond et al. ; phillips-howard and west ) , the drug was fi rst removed from the list of essential drugs against malaria but then had to be added back to the list as alternate drugs started showing resistance. amodiaquine induced adverse reaction in individuals was attributed to genetic make up of the individual. the genotypes of individuals harboring cyp c , cypia , and cyp b have been reported in studies to show immunogenic adverse reaction to amodiaquine (li et al. ; kerb et al. ). some population in africa has shown hepatotoxicity and leucopenia with only two doses given weeks apart (orrell et al. ) . amodiaquine when administered to an individual with reduced cyp c activity impairs the metabolism of the drug and hence leads to the cause of hepatotoxicity and agranulocytosis. other common variants of the enzymes cyp c * and cyp c * have been associated with decrease in the metabolizing activity of cyp c enzyme as is evident from studies with anticancer drugs (dai et al. ) . individuals with cyp c * genotype have no cyp c enzymatic activity in vitro (parikh et al. ). in a study from burkina faso, patients carrying cyp c * genotype showed common adverse effects to amodiaquine and in addition patients have also reported to experience more abdominal pain when compared to healthy individuals. the study from burkina faso and ghana could not clearly establish the relation between drug efficacy and cyp c genotype (adjei et al. ) . though the inactivated gene of cyp c is not very high in population, estimates have shown that cyp c * and cyp c * occur in about . % of the population in zanzibar, united republic of tanzania, which was about , patients of the total malarial patients ~ , (cavaco et al. ) . in ghana it was found that . % of the population has been estimated to have metabolic variants of cyp c . hence, due to high disease burden, the study of pharmacogenomics for drug metabolism was carried out in large patient samples from the population to get a clear correlation between genotype and effi cacy of drug treatment as well as adverse reaction. major active antimalaria metabolite of artemisinin is dihydroartemisinin (dha) (ilett et al. ) . artesunate is rapidly converted to its active metabolite catalyzed via cyp a which is a major enzyme; conversion to dha also includes minor enzymes cyp b , cyp a , and cyp a (li et al. ) . cyp a has about variant forms of which at least have been implicated as slow metabolizing enzymes, and have been reported to show no activity in vitro (di et al. ) . hence, lower level of cypba enzymes in patients will have reduced bioavailability of dha the major antimalarial metabolite and hence have lower antimalarial activity. major endemic areas of malaria like sub-saharan africa, ghana, sabah region of malaysia have been evaluated for the presence of cyp a genotype. among these population of ghana has high wild-type cyp a along with % alleles being cyp a * a (gyamfi et al. ) , whereas malaysian population has an allele cyp a * a frequency of % with only % wild-type enzyme (yusof and gan ). other asian populations have been reported to carry several other alleles of cyp a with even alleles that do not show any cyp a enzyme activity at all. no activity variant of cyp a is found about . - . % in japanese, chinese, and thai populations (gyamfi et al. ) . hence, artesunate is expected to be more effective in population of ghana. in some parts of thailand, about % of patients have shown resistance to artemisinins (white ) . though it has been found by study that about % frequency of cypz alleles have no activity in the thai population and the antibiotic resistance is indicative to be related to cyp a activity and ability to convert artesunate to dha (noedl et al. ), more studies require to be done to clearly establish the relation between the genotype and resistance to artemisinin-based therapy. several mutations in gene targeted by antimalarial drug have been identifi ed which led to the resistance in vivo of act drug partners such as mefl oquine, lumefantrine, amodiaquine, and chlorproguanil (kerb et al. ; mehlotra et al. ). identifi cation of genes and mechanism is important for controlling the infection. research has yielded the information regarding the gene responsible and underlying mechanism of action resistance of some drugs against p. falciparum and p. vivax . chloroquine resistance (cqr) in p. falciparum has been linked to point mutation cq resistance transporter gene (pfcrt chromosome ). the mutation pfcrt -k t is a reliable marker for cqr. while cq-sensitive strain carries wild-type allele cvmnk , the variant cqr alleles are s agt vmnt (asia, south america, africa), s tct vmnt (south america), cvmnt (south america, philippines), cviet (southeast asia, africa), and cvmet (colombia). another multidrug resistance gene ( pfmdr chromosome ) is a parasite transporter gene. polymorphism, point mutation, and copy number variation have been implicated in multi drug resistance. in different geographic regions, the pfmdr two mutant alleles have been reported, namely, y_ y_ s_ n_ d found mostly in asia and africa and n_ f_ c_ d_ y predominantly from south america (valderramos and fidock ) . the pfcrt- and pfmdr - y mutations have been related jointly to contribute in giving rise to cqr phenotype in addition to other likely parasite genes (hayton and su ) . p . falciparum dhps enzyme ( pf-dhps , chromosome ) has been linked to resistance to the sulfa class of antimalarial drugs, while mutations in dhfr ( pf-dhfr , chromosome ) domain have been linked to high level of pyrimethamine resistance. combination of sulfadoxine-pyrimethamine (s-p) treatment failure has been found to be associated with pf-dhps double mutant ( g with either e or g), combined with the pf-dhfr triple mutant ( n_ _ r) (hayton and su ; hyde ) . point mutations in p. vivax ortholog of pfcrt (pcvg ) are associated with clinical cqr. pfmdr p. vivax ortholog that is pvmdr has been proven and has also been identifi ed. y cf point mutation of pvmdr has been linked to cqr. pv-dhs and pv-dhr gene point mutations have been identifi ed and are suspected to link to clinical resistance in antimalarial s-p treatment (hayton and su ) . more data is required for new mutations in the parasite genes, and in addition more data is needed for therapy of other act drug partners like sulfadoxine-pyrimethamine and lumefantrine. a new rejuvenation is taking place in pharmacology and pharmacokinetics development of databases of antimalarial pharmacogenetics. worldwide antimalarial resistance network ( http://www.wwarn.org/ ) has set up a module together with high-quality pharmacological research data for optimum drug dosage in light drug resistance information and adverse event reporting. the aim to achieve global cooperation will go a long way to personalized malarial treatment as per population needs. during the last half a century (for about years), the most effective treatment regimes have been the combination therapies of drugs that was because a single drug treatment was found to be in invariantly leading to resistance for the drug, leading to much more severity and complications (crofton ) . due to rampant and unregulated use of tuberculosis drugs, however, this has led to emergence of multidrug resistant tuberculosis (mdr) (fig. . b ) . now the treatment course is usually for months with the combination of isoniazid, rifampicin, pyrazinamide, and ethambutol for the fi rst months. this has to be followed up by the next months with isoniazid and rifampicin treatment. if the treatment is taken up with diligence and patient completes the whole drug course, then it has been reported that effi ciency of the treatment is very high with more than > % patients getting cured and relapse is in less than % of patients (menzies et al. ). another advantage of multidrug treatment is that the treatment regime helps in treating different population of tubercle bacilli. for the last years, knowledge from the fi eld of genetic molecular basis of drug treatment outcomes has helped us in the better management of and understanding of treatment effi ciency and of drug. the difference in drug response is found among different individuals of the population. the individual person tends to show similar type of response to tuberculosis drugs that do not change over time. thus, in light of above observations, we say that there is a huge variation in drug response among individuals due to variation in genes involved in drug metabolism, drug transporters, and drug targets compared to minimal within-subject variation as found from studies. further studies on drug response revealed that - % of variation in drug pharmacokinetics is due to genetic factors (kalow et al. ). the sequence variation in drug-metabolizing enzymes, drug transporters, or drug targets leads to the variation in drug response among individuals (evans and relling ; evans and johnson ) . some nongenetic factors such as nutrition organ function, age and other concomitant therapies, nature of disease, and drug interaction can also effect in drug response, but genetic determinant remains constant throughout the lifetime of the individual. pharmacogenomics have played an important role in deciphering therapeutic effi cacy of drug metabolism and occurrence of adverse events. though research is still being pursued to decipher the intricacies of how genetic differences play an important role in regard to clinical application of the drug however, through research we have gained information on the role of genetic polymorphism with respect to drug effi cacy for the treatment of tuberculosis. in this section we will discuss the knowledge we have gained through newer technologies in regard to different drugs being used for tuberculosis. since isoniazid has been in use for antituberculosis treatment since , it is the most well studied of the lot (ellard and gammon ) . this drug has been found to be tuberculosis specifi c in its action against tubercle bacilli and has relatively minimal toxicity. now pharmacogenomics is playing a very important role in making isoniazid the fi rst-line treatment drug. acetylation of isoniazid takes place mainly in the liver and gut mucosa. for any drug ingested in the body, it is absorbed and metabolized and then its soluble by-products are released or excreted out of the body. the drugs have specifi c retention and metabolizing rates depending upon their chemical composition and the genetic polymorphisms of the metabolizing enzymes. the activation of isoniazid is catalyzed by highly polymorphic enzyme n-acetyltransferase (nat ) and leads to formation of acetyl isoniazid. this is formed by the transfer of acetyl group from the acetyl coenzyme a to acceptor amine leading to formation of an amide. acetyl isoniazid combines with several other cellular compounds to give a variety of metabolites which do not have any antituberculosis activity. the level of acetylating isoniazid that will be subjected to during metabolism in the body determines the disease outcome. the level of bioavailability of the drugs determines whether the drug would be effective for elimination of the invading pathogen or toxic to the human body. acetylation of isoniazid varies from individual to individual depending as per his or her genetic predisposition. genetics determines the amount of active nat enzyme that an individual expresses. the metabolism of isoniazid is catalyzed by nat enzyme which takes place in liver or gut mucosa. thus, the level of nat gene expression is controlled by the type of polymorphism in nat that particular individual carries. thus, for the pharmacogenomic and personal medicine in effect to succeed, the dosage for the drugs that are metabolized by nat should be tailor made as per the enzymatic activity depending upon the polymorphic variant (roy et al. ) . the enzymatic activity being highly variable cascorbi and roots ( ) has been studied over the years in human subjects who have been categorized as slow or rapid inactivators ( http://www. brti.co.zw ). the categorization has been based on the measure of capacity of nat enzyme to acetylate isoniazid to acetyl isoniazid thus inactivating it. here the rapid inactivators are those who have more concentration of active nat enzymes than slow inactivators. based on the new technologies, genotypic studies have led to further classifi cation depending upon enzymatic activity nat variant as rapid acetylators, that is, the wild type gene which codes for the completely active enzyme. rapid acetylators are highly active forms of the enzyme denoted by nat * allele. patients harboring these alleles can tolerate conventional dosage of drug that is rapidly metabolized by nat enzyme. individuals who carry nat heterozygous alleles where only one of the allele is active/functional should be administered lower than average drug (those are nat metabolized) dosage to get an optimum effective drug response without adverse drug response. mutations in nat enzyme in human individuals designated as nat * a , nat * b , nat * a, and several others which lead to rendering the nat gene activity are termed as slow acetylators which can lead to diminished drug clearance and toxicity. the variation of frequency of slow acetylator gene is depended on the race, population type, and the ethnicity from one country to the next. it is found in a study that % of middle eastern, % in south indian population, caucasian and negroid, and % of the us population harbor slow acetylator gene. in mongoloid populations like the eskimos, japanese, and the chinese, slow acetylators are found in only % of study subjects. in another study carried out in a population of healthy caucasian, there is variability in isoniazid clearance. while isoniazid preparation is responsible for only % variation and body weight accounted for only % variation in isoniazid clearance, the majority variation of % in isoniazid clearance was due to nat genotypes (kinzig-schippers et al. ) . highactivity nat allele-carrying individuals have higher isoniazid clearance. other studies have shown that - times more isoniazid concentration in individual is carrying slow acetylator nat genotype (parkin et al. ) . a study estimating the comparison of urinary isoniazid excretion in japanese patients to normal, healthy individuals showed that persons with higher number of active nat alleles had higher level of isoniazid acetylation (kita et al. ) . the relation between isoniazid concentration in blood with drug effi cacy and toxicity knowledge is important. peak isoniazid concentration to minimum inhibitory concentration ratio has been proposed to serve as a means to outcome tuberculosis treatment (mitchison ) . mean early bactericidal activity of isoniazid depends on its level in the plasma which in turn depends upon the variant nat genotype carried by an individual. comparatively the mean bacterial activity is lower in rapid acetylators than in the slow acetylators (donald et al. ) . therapeutic failure or relapse of infection is thus attributed to the lower plasma level due to rapid metabolism of isoniazid in rapid acetylator genotypes, while on other hand high level of isoniazid in slow acetylators may lead to the high level of toxicity (weiner et al. ) . nat allele genotyping of tuberculosis patients prior to the treatment with isoniazid is the way forward. dosage adjustment of isoniazid could be carried out depending upon if the patient is harboring none, one, or two alleles nat rapid acetylators. thus, isoniazid would be more pharmaceutically viable for treatment of tuberculosis. in pulmonary tuberculosis patients with known acetylator state, the response to isoniazid treatment analysis was carried out when it is administered alone or in combination with p-aminosalicylic acid. the study compared isoniazid response between nat slow and fast acetylators, and the study revealed that there is association with treatment response and bacteriological negativity (selkon et al. ) . tuberculosis treatment trials used dosage regimes of daily, twice weekly (tuberculosis research centre madras, study , , or three times weekly drug regimes (ellard and gammon ) . by means of controlled clinical trials, it was observed that using once a week uptake of isoniazid showed better clinical response to treatment compared to rapid acetylators, with cure rate of - %. it was postulated that metabolic status of isoniazid may have lesser clinical signifi cance for daily isoniazid treatment regime as compared to thrice weekly or twice weekly treatments. in slow acetylator individuals, the peak concentration of isoniazid was higher than rapid acetylators, and the level of isoniazid decreased more gradually. the effectiveness of a drug in tuberculosis treatment is determined in terms of coverage and the exposure. coverage has been defi ned as the number of hours for which bacteriostatic concentration of isoniazid is ( . µg/ml) maintained in the blood, while exposure is defi ned as area under concentration time curve. both parameters have been found to be signifi cantly greater in slow acetylators. hence, in rapid acetylator individuals, there is a suboptimal concentration of isoniazid which leads to failure of once-weekly regime of isoniazid (sarma et al. ) . other studies using onceweekly isoniazid-rifapentine were compared with twice-weekly isoniazid-rifampicin; treatment also showed that in case of once-weekly treatment regimes, treatment outcome was poor and was associated with isoniazid acetylator status of the patients (weiner et al. ) . the clinical studies have shown that rapid acetylators having infected from combined tuberculosis and hiv infection are at a further disadvantage since it has been found that antituberculosis drug bioavailability becomes suboptimal in those individuals (gurumurthy et al. ) . tuberculosis patients having chronic renal failure are also at a risk from adverse drug reactions if they also happen to harbor slow acetylator genotypes of nat . studies have shown that slow acetylators have higher peak isoniazid concentration, exposure, and half-life compared to rapid acetylators and healthy subjects ( gurumurthy et al. ) . hence, in the case of pulmonary tuberculosis patient also suffering from chronic renal failure, the isoniazid dosage should be administered based on their nat genotypes status. in adult pulmonary patients, studies were carried out to determine correlation between isoniazid dosage and nat genotypic and phenotypic status. determination of isoniazid therapeutic dosage has shown that the fast acetylators need higher drug dosage to have an optimum positive treatment response. fast acetylators tuberculosis patients when administered with mg/kg isoniazid had similar exposure level as mg/kg isoniazid administered to slow acetylators does (donald et al. ). in a further study in a population of south african tuberculosis patients, it was found that current treatment regimes were causing suboptimal exposure of isoniazid in patients having rapid acetylator status (wilkins et al. ) . several fi eld studies have further suggested that there is a need for calibration of isoniazid dosage as per the individual tuberculosis patient's age, acetylator status, and disease process for an effective antimicrobial outcome of drug treatment (jeena et al. ) . in children affected with tuberculosis, it was shown through several studies that the exposure of isoniazid was reduced in the rapid acetylators when compared to the slow acetylators and thus likely to affect the outcome of the treatment of tuberculosis (cranswick and mulholland ; schaaf et al. ; mcilleron et al. ). though isoniazid has been found to be nontoxic during conventional regimes, two types of adverse reactions to isoniazid have been reported. the most common isoniazid toxicity reported is hepatotoxicity which affects - % of the patients (tostmann et al. ) . another isoniazid-associated adverse event is peripheral neuropathy. neuropathy usually occurs in slow acetylators due to administration of high doses of isoniazid (devadatta et al. ) . hepatotoxicity is the major adverse reaction of isoniazid, and the factors that are responsible are nat acetylation, oxidation by cytochrome p s oxidation (cyp) ei, and detoxifi cation by glutathione s-transferase (gst) enzyme activity (roy et al. ) . accumulation of acetyl hydrazine, a toxic metabolite of isoniazid, has been implicated in peripheral neuropathy, and the condition in humans is reversible by concomitant administration of pyridoxine (zilber et al. ) . further, it has also been deduced that hepatotoxicity occurs due to hydrazine metabolites of isoniazid. rifampicin also causes induction of isoniazid metabolism and inducing isoniazid hydrolase to produce isonicotinic acid and hydrazine. the rifampicin induction is more pronounced in slow acetylators compared to rapid acetylators (sarma et al. ) . in some populations studies have established association with nat acetylator status and isoniazid-induced hepatotoxicity, while in other studies it has not. studies in japanese and taiwanese populations have shown that the acetylator status of nat increased the risk factor for hepatotoxicity by -fold isoniazidinduced hepatotoxicity (ohno et al. ; huang et al. ) . in another study on the korean population, the nat slow acetylator status has been implicated to increase isoniazidinduced hepatotoxicity by two-to eightfold, and hence the nat acetylator genotype could serve as predictor of hepatotoxicity (cho et al. ) . nat * / b , nat * a/ a , it/t, and a/a diplotypes have been indicated and could be used as biomarkers for prediction of antituberculosis drug-induced toxicity (ben mahmoud et al. ) . slow acetylator nat alleles have been attributed to increase - -fold to -fold higher risk in isoniazid-induced hepatotoxicity. but other studies in tuberculosis patients have not been able to fi nd any association of nat acetylator status and the drug-induced hepatotoxicity. case studies of caucasian origin patients with tuberculosis (leiro-fernandez et al. ) , genotyping in an indian population (roy et al. ) , and study on heterogeneous population of hispanics, africans, caucasian, south american, and asian have not reported any linkage between nat acetylator status and isoniazid-induced hepatotoxicity polymorphisms (vuilleumier et al. ) . cytochrome p e is one of the enzymes of the hepatic microsomal enzyme system. cyp e gene encodes a member of the cytochrome p superfamily of enzymes. the cytochrome p proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids. cyp e is an enzyme which brings about conversion of acetyl hydrazine to hepatotoxins, such as acetyl diazone and ketene, and brings about conversion of acetylonium ion (nelson et al. ) . polymorphism in cyp e has been linked with increasing the risk factor associated with isoniazid-induced liver injury (lee et al. ) . the enzyme relocates to the endoplasmic reticulum and can be induced by isoniazid or its metabolite hydrazine. in animal model studies using rat, it has been found that cy pe activity is linked to blood isoniazid levels (yue et al. ). in the presence of variant genotype of cyp e , isoniazid could on the other hand inhibit the activity of the cytochrome p e enzyme. enhanced cytochrome p e activity leads to the increased production of hepatotoxins and hence causing hepatotoxicity. both nat and cyp e polymorphisms have been shown to be associated with susceptibility of fi rst-line druginduced hepatitis. cyp e polymorphisms have been related to increase in risk of antituberculosis drug-induced liver toxicity. the common * a/* a genotype of cyp e , in tuberculosis patients from taiwan, has been linked to increase in the liver damage risk by . times. presence of both slow nat acetylator status and the * a/* a genotype further increases risk of hepatotoxicity when compared to presence of either of the single polymorphism (huang et al. ) . the cyple * and * a* * d haplotypes in indian pediatric patients have been shown in sep-arate study and have shown to increase the liver toxicity. further, the common * a allele at cyp e has been implicated to hepatotoxicity in various heterogeneous population comprising of asians, africans, caucasians, hispanics, and south americans (leiro-fernandez et al. ), but study done on a korean population on the other hand could not fi nd any association between cyp e polymorphism and liver toxicity (huang et al. ) . the glutathione s-transferases are class of two distinct supergene families of proteins located in cytosolic and membrane-bound forms. glutathione s-transferases are a class of enzymes that are responsible for detoxifi cation of therapeutic medication, carcinogens, therapeutic medication, and toxic chemicals that are mostly electrophilic in nature. gsts are present both in eukaryotes and prokaryotes. at present, eight distinct classes of the soluble and cytoplasmic mammalian glutathione s-transferases have been identifi ed: alpha, kappa, mu, omega, pi, sigma, theta, and zeta. the cytosolic gst enzymes are encoded by at least fi ve different loci coding for gst enzymes, distantly related gene families (designated class alpha, mu, pi, sigma, and theta gst), whereas the membrane-bound enzymes, microsomal gst, and leukotriene c synthetase are encoded by single genes and both have arisen separately from the soluble gst (simon et al. ; strange et al. ) . glutathione s-transferase catalyzed elimination of toxic chemicals from the human body is carried out by making the toxic chemical soluble by conjugation with glutathione. in context of isoniazid-related hepatotoxicity, studies have indicated that deletions of gst mu ( gstm ) and gst theta ( gstt ) are associated with liver damage (cho et al. ; huang et al. ) . gst enzymes play an important role in removing the harmful metabolites of isoniazid from the body. the toxic metabolites generated by isoniazid metabolisms are from intracellular free radicals that are scavenged by conjugation with glutathione in reactions catalyzed by gst enzymes. now, studies in indian patients suffering from tuberculosis show that those harboring homozygous gstm mutations have higher risk of hepatotoxicity. it was also found in a study on taiwanese tuberculosis that patients have twice the risk of isoniazid-induced hepatotoxicity if they have homozygous gstm deletion. thus, it can be inferred from similar studies that identifi cation of gstm deletion in patients will lead to the better management of isoniazidinduced hepatotoxicity. reactive oxygen species as we have been aware is a causative agent for damage to hepatic tissue. it has been deduced that level of mitochondrial oxygen species is reduced by the action of manganese superoxide dismutase (msd). as the name suggests, msd catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide and is the fi rst line of defense against reactive oxygen species. polymorphism in the msd enzyme has been found in a study in the population in taiwan, where genotypes having t > c polymorphism in codon lead to variant amino acid valine in place of alanine which increases in risk associated with antituberculosis druginduced hepatotoxicity (huang et al. ) . the presence of valine at codon causes the increased activity in the enzyme manganese superoxide dismutase which leads to the accumulation of the toxic byproduct hydrogen peroxide which can cause hepatotoxicity. rifampicin has been proven to show concentration-dependent activity against m. tuberculosis and is a very important fi rst-line drug against tuberculosis (ji et al. ; jayaram et al. ) . drug transporters p-glycoprotein and oatp b transporters uptake rifampicin as a substrate and hence play an important role in distributing the drug throughout the body. the drug transporters are transcriptionally regulated by the nuclear receptors, i.e., pregnane x receptor and constitutive androstane receptor. the phenomenon variation in bioavailability of rifampicin among individuals in a population on administration of standard dosage has been subjected to investigation by the scientists. the pharmacokinetics of rifampicin depends upon the uptake of machinery of the cells in the body. it has been found that there is a relation between pharmacokinetics and polymorphisms of genes that is responsible for drug effl ux and infl ux. a study group of individuals suffering from tuberculosis who were categorize as per place of origin africans versus non-africans, it was observed that in drug transporter gene, slco b c > a polymorphism leads to reduced rifampicin exposure and bioavailability (weiner et al. ) . the people of african origin (black subjects) that carry slco b c > a gene polymorphisms have been associated with more pronounced reduced rifampicin exposure compared to people from other races. the study thus showed for the very fi rst time that marked interindividual variation in rifampicin exposure can be attributed to slco b polymorphism. another study in south africa has also highlighted that the variant allele of slco b rs polymorphism reduced the bioavailability of rifampicin in the body of the patients when present both in the homozygous and heterozygous states (chigutsa et al. ). this fi nding has been attributed to the observation that there is about % variability in drug clearance among the patients. polymorphisms in the abcb , pxr, and car genes have not been found to affect the pharmacokinetics of rifampicin in the patients in any signifi cant manner. researchers have further predicted by means of stimulation that increasing the dosage of rifampicin in patients carrying slco b rs would increase the plasma availability of the drug and thus would have a positive impact on the treatment outcome. however, more studies needed to be carried out to know the exact association of slco b gene polymorphisms between rifampicin bioavailability to provide an effective treatment regime. as has been already mentioned, the variation in human leukocyte antigens also is known cause of disease susceptibility and the response to treatment has also in indian patients the lack of human allele hla dqai* , while the presence of dqb * has been reported to be associated with antituberculosis-induced hepatotoxicity (sharma et al. ) . aminoglycosides are antibiotics which are molecules that consist of amino-modifi ed sugars, and some of the drugs of this class have been used for treatment of tuberculosis. aminoglycosides such as kanamycin, streptomycin, and amikacin have been used to treat tuberculosis. aminoglycosides have known to cause ototoxicity. ototoxicity is term used when there is damage to the ear (oto-), specifi cally the cochlea or auditory nerve and sometimes the vestibular system due to toxins. the association between aminoglycosideinduced ototoxicity and mitochondrial mutations has been found in a study in chinese family. the deafness phenotype was found to be associated with c > t s rrna gene polymorphisms which could be induced with the administration of aminoglycosides or even get more aggravated (zhao et al. ). there is still no clear relationship of ethnicity and genetic background and response to antituberculosis treatment, and no single variant of nat and cyp e genes is associated with signifi cant liver damage (yamada et al. ). more extensive pharmacogenomic research is still needed for realization of robust personalized medicine for tuberculosis. the antifungal medicine amphotericin b has been found to be effective and well quite toxic. further investigations revealed that the immunomodulatory role of amphotericin b also involves the induction of production of proinfl ammatory cytokine. in human cell the amphotericin-induced higher mrna expression and cytokine production have been detected in studies (rogers et al. ) . the discovery of induction of proinfl ammatory cytokine production was able to explain the infusion-related toxicity effect like nausea, fever, chills, and hypotension that are characteristics of this cytokine release. it was also able to explain the mechanism of action of amphotericin b since the proinfl ammatory cytokines are responsible for the activation of monocytes, macrophages, and promote chemotaxis that led to enhanced immune response to the infection. since the advent of the era of omics technology, the number of drugs that have been discovered have not delivered as was fi rst predicted especially in the case of infectious disease. some of the shortcoming and the remedial measures have already been discovered in the previous sections. it has been found that even with high-throughput screening of number of drug, candidates have not been successful always (payne et al. ). the fi eld of scientifi c research which has become all encompassing and interdisciplinary has added strength along the way and opened new avenues. the fi eld of biology is intertwined with structural biology and chemistry has given rise to the fi eld of medical structural genomics. the exact causes of failure of high-throughput screens have not been well defi ned. the fi eld of structure-based drug discovery has tried to overcome these limitations in the availability of chemical libraries and absence of structural information of many of the targets. the fi eld of structure-based drug discovery has its origin from the fi eld of x-ray crystallography and nuclear magnetic resonance (nmr) technology. with the availability of human genome sequences and pathogen genome sequence databases, the fi eld of structural genomics has gained importance, and hence over the past decade, more than such projects have been taken up. the fi eld of structural biology has got a boost with the coming together of robotics and informatics in the biological research sphere (haquin et al. ) . for the synthesis of an effective drug, by means of medical structural genomics, the protein which drug will affect should be well defi ned experimentally in both structure and functional aspects as the potential target. the protein should not only be well characterized structurally but also should be well defi ned as essential for the survival of the pathogen. once drug and its protein target in a microorganism is identifi ed, the fi eld of medical structural genomics provides rapid mechanisms using high-throughput x-ray crystallography and nmr assay system to fi nd the ligand-bound structures. to identify such drug targets, it is very essential to know the complex host and pathogen interaction. the mode the pathogen uses to cause the infections is very diffi cult to elucidate and is a long process. the technologies of rna interference and other gene knockout techniques should be complimented with experimental chemical biology approaches as microorganisms adopt multiple mechanisms for survival. this has been emphasized for the fact examples of efforts of scientist for targeting the fatty acid biosynthesis pathways of bacteria. at fi rst drugs were found to have high bioavailability and are potent against the bacterial replication in vitro. these compounds were subjected to be tested in animals and have been found to be not effective, the reason being that the bacterium utilizes the fatty acids present in their host vertebrates (brinster et al. ). hence, this study proves that there is need for more effective screening using the services of scientist from several spectra of biology like microbiologist, biochemists along with structural biologist, and chemical biologists to fi nd effective molecules and compounds which can eliminate the pathogen under proper infective conditions (hoon et al. ) . in pharmaceutical research scenario, it might also be possible that the drug target for a cell active compound is not known and then medical structural genomics provides a number of purifi ed protein targets which can be assayed for binding interaction with bioactive compound by means of number of biophysical techniques like thermal stability (ericsson et al. ). such efforts have already been carried out in the fi eld of protozoan pathogens. the program of medical structural genomics of protozoan pathogens ( http://msgpp.org/description.shtml ) has been initiated to screen for drugs for ten protozoan diseases. the initiative has screening of thousands of potential antimalarial drugs against about putative plasmodium falciparum protein targets by expressing them in bacterial expression system in the laboratory and deciphering their d structures. further, the com-pounds are assayed for their effectiveness in live organisms and further validated in appropriate disease model. the terms chemical validation and drugability are often used in conjunction in such cases. drugability is meant to be used how tractable a given drug target is for the development of a drug candidate, while chemical validation means that drugs have been found to be active in live organism. drugs which fulfi ll the abovementioned criteria are worth the effort, time, and resources. in the future more collaborative efforts between medical structural genomic centers and the chemical biology institutes would be possible with the availability of collection of phenotypically defi ned compound that would have proven anti-pathogen activity resulting in the synergistic target validation and hit to lead development using structure-based drug design. pharmaceutical industry has now taken fragment-based drug discovery methodology as an alternatively less expensive and at times more effective than high throughput screening. variety of methods like x-ray crystallography, nmr, surface fl uorescence polarization, plasmon resonance yield, and differential thermal denaturation have been used to obtain macromolecular structure to screen libraries of small fragment that are obtained from compounds that are building blocks of drugs and hence can be more drug like. the fragment-based drug discovery is based on the screening libraries of small molecules on the rule of three which has the molecular weight < da, the calculated log of octanol/water coeffi cient (clog p) < , and ≤ rotatable bonds and hydrogen bonds (rees et al. ; congreve et al. ) . protein-protein interactions are important for all biological processes. metabolic activities in the biological system are catalyzed by proteinbased enzyme where in certain cases their activities are regulated by modulation of an equilibrium of an alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) that have now been described as mode allostery. the oligomerization from the protein-protein interaction need not lead to gain in free energy, and it has been found that small molecules can block or disrupt any protein-protein interaction that is necessary for biological systems, for example, being in the development of potent peptidomimetic inhibitor of hsv ribonucleotide reductase with antiviral activity (liuzzi et al. ). the discoveries have opened avenues where structure-based information can be used to develop small novel antimicrobial molecules that can be made which can target protein-protein interfaces (wells and mcclendon ) . an example of this technology has been used to fi nd small-molecule species-specifi c allosteric drugs for porphobilinogen synthase (pbgs). the oligomeric equilibrium for porphobilinogen synthase (pbgs) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. in silico docking analysis from a small molecule library helped in selecting suitable compounds and molecules that had more affi nity for docking pbgs allosteric site and thus were subjected to testing in vitro . in one compound whose inhibition mechanism is species specifi c, conversion of pbgs octamers to hexamers was thus identifi ed (lawrence et al. ) . the above fi ndings have led the way of targeting of oligomeric enzymes in pathogenic organism bacteria. prime example is bacterial inorganic pyrophosphatases, which function as hexamer (kankare et al. ) . on the other hand, the eukaryotic, cytosolic, and mitochondrial pyrophosphatases function as homodimers (oksanen et al. ) and hence have different interfaces than its bacterial counterparts as evident from the study of evolutionary aspects of inorganic phosphatases. in this context the strategy has been to target the oligomeric state of the bacterial inorganic pyrophosphatase enzymes to inhibit their activity rather than their conserved active site (sivula et al. ) . the technology has opened a novel pathway where more antibiotics can be developed. the amount of knowledge of protein structures being generated is enormous; the need of the hour is dissemination of the knowledge databases among scientists and academic researchers on a worldwide scale. the protein structural know-how should be in the public domain without any constrains and copyright restrictions; also in addition the databases should be available free of any monetary charge. structural genomic projects the world over have solved the structures of many proteins and have made the knowledge available for world community by submitting the structures to protein data bank {pdb; http://www.wwpdb.org/ }. worldwide protein data bank is the site whose mission is to maintain a single protein structural public database which can be accessed by the global community (berman et al. ) . there is lot of structural data of protein-ligand complexes that is in private pharmaceutical industries not in the public domain. the economic incentives of drug discovery are driving force for this secrecy, but in this process there are a lot of valuable data that are duplicated and lots of valuable resources and energy efforts. the learning process from failures and successes in pharmaceutical corporate sectors is never known to the scientifi c community, and a major loss is of most valuable time. hence, as we can see, the drug discovery resources are not being adequately utilized across the academia and industry, so there is suggestion to have open-access industry-academia partnerships as possible mechanisms to overcome the problem. a frame work is need where both fi nancial and intellectual properties of the innovators are safeguarded when there structural data are deposited in the databases like pdb. a simple proposition would delay the release date of such structural data so that protection of intellectual property is feasible. policies which can bring into the public domain structural data from the corporate world could only be possible by the concerted efforts of all stakeholders from industry, national, and international research funding agencies from all nations (edwards et al. ). apart from easier dissemination of structural information related to infectious diseases and collaboration of structural biologist with medical chemist and molecular biologist, there is need for development of automation in several technologies to bring about unprecedented growth in the new drug discovery. fragment-based drug design needs the support of high throughput technologies such that along with structural genomics, there will be more success in the determining protein-ligand structure determination. decades of experience have shown that the infectious diseases would emerge with more vigor and virulence. when the diseases are not controlled, then it would take a considerable toll of human health both in terms of mortality and morbidity. the life would be affected by emerging microbial disease-causing pathogen whatever the region, ethnicity, lifestyle, socioeconomic status, and ethnic background. hence, the threat from infectious diseases is real and the situation is overtly challenging. the great advances in the genetics, genomics, and proteomics have the potential to take up the challenge in the coming decades. it is evident that these technologies have the potential to change the fi eld of diagnostics, treatment, and discovery of drugs and vaccines. the need of the hour is to strengthen the public health programs at both the national and the international levels to strengthen research in the fi eld of omics to fully realize the potential of scientifi c technologies that would usher in the era of pharmacogenomicbased personalized medicine. complex segregation analysis of leprosy in southern vietnam susceptibility to leprosy is linked to the human nramp gene effect of concomitant artesunate administration and cytochrome p c polymorphisms on the pharmacokinetics of amodiaquine in ghanaian children with uncomplicated malaria genome-wide rnai screen for host factors required for intracellular bacterial infection t helper (h) /th and leishmania: paradox rather than paradigm genotypes of the mannanbinding lectin gene and susceptibility to visceral leishmaniasis and clinical complications impairment of mycobacterial immunity in human interleukin- receptor defi ciency enzymatic defi ciency in primaquine-sensitive erythrocytes mucosal leishmaniasis: current scenario and prospects for treatment cutaneous leishmaniasis: therapeutic strategies and future directions leishmania donovani leishmaniasis in cyprus from systems biology to systems biomedicine up-regulation of th -type responses in mucosal leishmaniasis patients tumor necrosis factor (cachectin) in human visceral leishmaniasis soluble il- receptor as an agent of serum-mediated suppression in human visceral leishmaniasis variations in the nramp gene and susceptibility to tuberculosis in west africans polymorphism of the n-acetyltransferase gene as a susceptibility risk factor for antituberculosis druginduced hepatotoxicity in tunisian patients with tuberculosis the worldwide protein data bank (wwpdb): ensuring a single, uniform archive of pdb data infl uence of h- complex on acquired resistance to leishmania donovani infection in mice slc a (formerly nramp ) and disease resistance high intralesional interleukin- messenger rna expression in localized cutaneous leishmaniasis is associated with unresponsiveness to treatment the art and design of genetic screens: rna interference identifi cation of host proteins required for hiv infection through a functional genomic screen type ii fatty acid synthesis is not a suitable antibiotic target for gram-positive pathogens a major susceptibility locus on chromosome q plays a critical role in the control of kala-azar genetic control of visceral leishmaniasis in a sudanese population: candidate gene testing indicates a linkage to the nramp region identifi cation of a novel g r polymorphism in the il- receptor beta membrane proximal domain associated with human visceral leishmaniasis cysteine protease b of leishmania mexicana inhibits host th responses and protective immunity polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis proper antibiotic therapy. from penicillin to pharmacogenomic pitfalls in n-acetyltransferase genotyping promoter polymorphism infl uences susceptibility to mucosal but not localized cutaneous leishmaniasis in brazil serum levels of tumor necrosis factor in patients with american cutaneous leishmaniasis cyp c polymorphism frequencies among malaria patients in zanzibar the yellow book, cdc health information for international travel the slco b rs polymorphism is highly prevalent in south africans and is associated with reduced rifampin concentrations: dosing implications genetic polymorphisms of nat and cyp e associated with antituberculosis druginduced hepatotoxicity in korean patients with pulmonary tuberculosis recent developments in fragment-based drug discovery immunotherapy of american cutaneous leishmaniasis in venezuela during the period - isoniazid treatment of children: can genetics help guide treatment? global challenge of tuberculosis genetic polymorphism and molecular epidemiology of leishmania (viannia) braziliensis from different hosts and geographic areas in brazil polymorphisms in human cyp c decrease metabolism of the anticancer drug paclitaxel and arachidonic acid rapid determination of hla b* ligands from the west nile virus ny genome peripheral neuritis due to isoniazid structure, function, regulation and polymorphism of human cytochrome p a the infl uence of human n-acetyltransferase genotype on the early bactericidal activity of isoniazid the infl uence of dose and n-acetyltransferase- (nat ) genotype and phenotype on the pharmacokinetics and pharmacodynamics of isoniazid minimizing the risk of reporting false positives in large-scale rnai screens open access chemical and clinical probes to support drug discovery interleukin- promoter polymorphism predicts initial response of chronic hepatitis c to interferon alfa pharmacokinetics of isoniazid metabolism in man thermofl uor-based highthroughput stability optimization of proteins for structural studies pharmacogenomics: the inherited basis for interindividual differences in drug response pharmacogenomics: translating functional genomics into rational therapeutics decreased in situ expression of interleukin- receptor is correlated with the exacerbated infl ammatory and cytotoxic responses observed in mucosal leishmaniasis increased levels of interleukin- and igg are hallmarks of indian post-kalaazar dermal leishmaniasis malaria: progress, perils, and prospects for eradication natural resistance to infection with intracellular pathogens: the nramp protein is recruited to the membrane of the phagosome dynamic regulation of single-and mixed-species malaria infection: insights to specifi c and non-specifi c mechanisms of control single-dose pharmacokinetics of isoniazid and rifampicin in patients with chronic renal failure decreased bioavailability of rifampin and other antituberculosis drugs in patients with advanced human immunodefi ciency virus disease high prevalence of cytochrome p a * a alleles in a black african population of ghana histone modifi cations and chromatin remodeling during bacterial infections genes and susceptibility to leishmaniasis data management in structural genomics: an overview frequency of severe neutropenia associated with amodiaquine prophylaxis against malaria il- down-modulates the cytokineenhanced antileishmanial activity in human macrophages the infl uence of the hla-drb * allele on measles vaccine response relationship of hla-dqa alleles and humoral antibody following measles vaccination antibodies to polysialic acid and its n-propyl derivative: binding properties and interaction with human embryonal brain glycopeptides genetic and biochemical aspects of drug resistance in malaria parasites an integrated platform of genomic assays reveals small-molecule bioactivities mechanisms of action of antimicrobials: focus on fl uoroquinolones polymorphism of the n-acetyltransferase gene as a susceptibility risk factor for antituberculosis drug-induced hepatitis cytochrome p e genotype and the susceptibility to antituberculosis drug-induced hepatitis genetic polymorphisms of manganese superoxide dismutase, nad(p)h: quinone oxidoreductase, glutathione s-transferase m and t , and the susceptibility to drug-induced liver injury drug-resistant malaria -an insight glucuronidation of dihydroartemisinin in vivo and by human liver microsomes and expressed udp-glucuronosyltransferases pharmacokineticspharmacodynamics of rifampin in an aerosol infection model of tuberculosis in silico children and the glass mouse model: clinical trial simulations to identify and individualize optimal isoniazid doses in children with tuberculosis insights into host responses against pathogens from transcriptional profi ling effectiveness of rifampin, rifabutin, and rifapentine for preventive therapy of tuberculosis in mice interferon-gammareceptor defi ciency in an infant with fatal bacille calmette-guerin infection partial interferon-gamma receptor defi ciency in a child with tuberculoid bacillus calmette-guerin infection and a sibling with clinical tuberculosis hypothesis: comparisons of inter-and intra-individual variations can substitute for twin studies in drug research cytokine gene polymorphisms and susceptibility to cutaneous leishmaniasis in iranian patients structure of escherichia coli inorganic pyrophosphatase at . a resolution association between the tumor necrosis factor locus and the clinical outcome of leishmania chagasi infection leishmania major lack antigen is required for effi cient vertebrate parasitization pharmacogenetics of antimalarial drugs: effect on metabolism and transport should we use n-acetyltransferase type genotyping to personalize isoniazid doses? n-acetyltransferase genotype correlated with isoniazid acetylation in japanese tuberculous patients direct quantification of hiv- rna by capillary electrophoresis with laser-induced fl uorescence global analysis of host-pathogen interactions that regulate early-stage hiv- replication rna interference screen for human genes associated with west nile virus infection initial sequencing and analysis of the human genome immunogenetics of human american cutaneous leishmaniasis: study of hla haplotypes in families from venezuela shape shifting leads to small-molecule allosteric drug discovery nat and cyp e polymorphisms and susceptibility to fi rst-line anti-tuberculosis druginduced hepatitis n-acetyltransferase polymorphisms and risk of anti-tuberculosis drug-induced hepatotoxicity in caucasians amodiaquine clearance and its metabolism to n-desethylamodiaquine is mediated by cyp c : a new high affi nity and turnover enzyme-specifi c probe substrate identifi cation of human cytochrome p( )s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data drugresistant malaria: the era of act a potent peptidomimetic inhibitor of hsv ribonucleotide reductase with antiviral activity in vivo evaluation of three rapid methods for detection of methicillin resistance in staphylococcus aureus immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to leishmania major the rise and fall of the antimalarial lapdap: a lesson in pharmacogenetics peginterferon alfa- b plus ribavirin compared with interferon alfa- b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial immunopathology of leishmaniasis: an update katg (ser thr) gene mutation in isoniazid resistant mycobacterium tuberculosis genetics of susceptibility to infectious diseases: tuberculosis and leprosy as examples mucosal leishmaniasis (espundia escomel isoniazid plasma concentrations in a cohort of south african children with tuberculosis: implications for international pediatric dosing guidelines human cutaneous leishmaniasis caused by leishmania donovani s.l. in kenya association analysis of hla-class ii and class iii gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis application of pharmacogenomics to malaria: a holistic approach for successful chemotherapy increased expression of proinfl ammatory cytokines in chronic lesions of human cutaneous leishmaniasis in situ expression of interleukin- and interleukin- in active human cutaneous leishmaniasis effect of duration and intermittency of rifampin on tuberculosis treatment outcomes: a systematic review and meta-analysis drug resistance in mycobacteria genetic susceptibility to visceral leishmaniasis in the sudan: linkage and association with il and ifngr slc a (formerly nramp ) and susceptibility to visceral leishmaniasis in the sudan immunochemotherapy of persistent post-kala-azar dermal leishmaniasis: a novel approach to treatment isoniazid and iproniazid: activation of metabolites to toxic intermediates in man and rat a mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection artemisininresistant malaria in asia interleukin- and the pathogenesis of human visceral leishmaniasis slow n-acetyltransferase genotype affects the incidence of isoniazid and rifampicin-induced hepatotoxicity a complete structural description of the catalytic cycle of yeast pyrophosphatase role of hla class ii alleles in susceptibility to and protection from localized cutaneous leishmaniasis treatment options for visceral leishmaniasis: a systematic review of clinical studies done in india pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers amodiaquine metabolism is impaired by common polymorphisms in cyp c : implications for malaria treatment in africa trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis lymphocyte responses and cytokines drugs for bad bugs: confronting the challenges of antibacterial discovery association of mucosal leishmaniasis with hla serious adverse drug reactions to pyrimethamine-sulphadoxine, pyrimethamine-dapsone and to amodiaquine in britain identifi cation of vaccine candidates against serogroup b meningococcus by whole-genome sequencing current paradoxes and changing paradigms in vaccinology immunogenetic mechanisms of antibody response to measles vaccine: the role of the hla genes the association between hla class i alleles and measles vaccine-induced antibody response: evidence of a signifi cant association measles antibody seroprevalence rates among immunized inuit, innu and caucasian subjects human cutaneous leishmaniasis caused by leishmania donovani sensu stricto in yemen targeted profi ling of oral bacteria in human saliva and in vitro biofi lms with quantitative real-time pcr fatal acute hepatitis due to amodiaquine t-cell and cytokine responses in leishmaniasis fragment-based lead discovery recent advances in vaccines for leishmaniasis comparative evaluation of nasba hiv- rna qt, amplicor-hiv monitor, and quantiplex hiv rna assay, three methods for quantifi cation of human immunodefi ciency virus type rna in plasma estimates of the burden of malaria morbidity in africa in children under the age of years bulletin of world health organization: can pharmacogenomics improve malaria drug policy? amphotericin b induces expression of genes encoding chemokines and cell adhesion molecules in the human monocytic cell line thp- pathogen subversion of cell-intrinsic innate immunity increased risk of antituberculosis drug-induced hepatotoxicity in individuals with glutathione s-transferase m 'null' mutation pharmacogenomics of anti-tb drugs-related hepatotoxicity a map of human genome sequence variation containing . million single nucleotide polymorphisms the immunology of susceptibility and resistance to leishmania major in mice il- -and tgfbeta-mediated susceptibility in kala-azar and postkala-azar dermal leishmaniasis: the signifi cance of amphotericin b in the control of leishmania donovani infection in india immunological and genetic evidence for a crucial role of il- in cutaneous lesions in humans infected with leishmania braziliensis ifng and ifngr gene polymorphisms and susceptibility to post-kala-azar dermal leishmaniasis in sudan clinical manifestations of cutaneous leishmaniasis in sri lanka -possible evidence for genetic susceptibility among the sinhalese studies of serial plasma isoniazid concentrations with different doses of a slow-release preparation of isoniazid rifampin-induced release of hydrazine from isoniazid. a possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin isoniazid pharmacokinetics in children treated for respiratory tuberculosis malaria chemoprophylaxis: strategies for risk groups multiclonal leishmania braziliensis population structure and its clinical implication in a region of endemicity for american tegumentary leishmaniasis rate of inactivation of isoniazid in south indian patients with pulmonary tuberculosis. . clinical implications in the treatment of pulmonary tuberculosis with isoniazid either alone or in combination with pas evaluation of clinical and immunogenetic risk factors for the development of hepatotoxicity during antituberculosis treatment localized cutaneous leishmaniasis due to leishmania donovani and leishmania tropica: preliminary fi ndings of the study of new cases from a new endemic focus in himachal pradesh combined glutathione-s-transferase m and t genetic polymorphism and tacrine hepatotoxicity clinical evolution of a group of patients with multidrug-resistant tb treated at a referral center in the city of rio de janeiro, brazil isoniazid-resistant mycobacterium tuberculosis strains arising from mutations in two different regions of the katg gene evolutionary aspects of inorganic pyrophosphatase clinical applications of molecular biology for infectious diseases from genome to vaccines for leishmaniasis: screening novel vaccine candidates against murine leishmania major infection glutathione s-transferase: genetics and role in toxicology complete genome sequence of neisseria meningitidis serogroup b strain mc antituberculosis drug-induced hepatotoxicity: concise up-to-date review tuberculosis research centre madras, study ( ) a controlled comparison of a twice-weekly and three once-weekly regimens in the initial treatment of pulmonary tuberculosis tuberculosis research centre madras, study ( ) controlled comparison of oral twice-weekly and oral daily isoniazid plus pas in newly diagnosed pulmonary tuberculosis transporters involved in resistance to antimalarial drugs molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements the sequence of the human genome the ity/lsh/bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the nramp gene cyp e genotype and isoniazidinduced hepatotoxicity in patients treated for latent tuberculosis low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine effects of tuberculosis, race, and human gene slco b polymorphisms on rifampin concentrations reaching for highhanging fruit in drug discovery at protein-protein interfaces qinghaosu (artemisinin): the price of success variability in the population pharmacokinetics of isoniazid in south african tuberculosis patients genetic variations of nat and cyp e and isoniazid hepatotoxicity in a diverse population antimalarial drug resistance, artemisinin-based combination therapy, and the contribution of modeling to elucidating policy choices cyp e mediated isoniazid-induced hepatotoxicity in rats high prevalence of cyp a * and cyp a * alleles detected among a malaysian population maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel c t mutation in the mitochondrial s rrna gene in a large chinese family genome-scale rnai screen for host factors required for hiv replication post-kala-azar dermal leishmaniasis the prevention and treatment of isoniazid toxicity in the therapy of pulmonary tuberculosis. . an assessment of the prophylactic effect of pyridoxine in low dosage key: cord- - nnqx g authors: canturk, semih; singh, aman; st-amant, patrick; behrmann, jason title: machine-learning driven drug repurposing for covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: nnqx g the integration of machine learning methods into bioinformatics provides particular benefits in identifying how therapeutics effective in one context might have utility in an unknown clinical context or against a novel pathology. we aim to discover the underlying associations between viral proteins and antiviral therapeutics that are effective against them by employing neural network models. using the national center for biotechnology information virus protein database and the drugvirus database, which provides a comprehensive report of broad-spectrum antiviral agents (bsaas) and viruses they inhibit, we trained ann models with virus protein sequences as inputs and antiviral agents deemed safe-in-humans as outputs. model training excluded sars-cov- proteins and included only phases ii, iii, iv and approved level drugs. using sequences for sars-cov- (the coronavirus that causes covid- ) as inputs to the trained models produces outputs of tentative safe-in-human antiviral candidates for treating covid- . our results suggest multiple drug candidates, some of which complement recent findings from noteworthy clinical studies. our in-silico approach to drug repurposing has promise in identifying new drug candidates and treatments for other viruses. artificial intelligence (ai) technology is a recent addition to bioinformatics that shows much promise in streamlining the discovery of pharmacologically active compounds [ ] . machine learning (ml) provides particular benefits in identifying how drugs effective in one context might have utility in an unknown clinical context or against a novel pathology [ ] . the application of ml in biomedical research provides new means to conduct exploratory studies and high-throughput analyses using information already available. in addition to deriving more value from past research, researchers can develop ml tools in relatively short periods of time. past research now provides a sizable bank of information concerning drug-biomolecule interactions. using drug repurposing as an example, we can now train predictive algorithms to identify patterns in how antiviral compounds bind to proteins from diverse virus species. we aim to train an ml model so that when presented with the proteome of a novel virus, it will suggest antivirals based on the protein segments present in the proteome. the final output from the model is a best-fit prediction as to which known antivirals are likely to associate with those familiar protein segments. these benefits are of particular interest for the current covid- health crisis. the novelty of sars-cov- requires that we execute health interventions based on past observations. grappling with an unforeseen pandemic with no known treatments or vaccines, the potential for rapid innovation from ml is of utmost significance. the ability to conduct complex analyses with ml enables us to research insights quickly that can help steer us in the right direction for future studies likely to produce fruitful results. we present here multiple models that produced a number of antiviral candidates for treating covid- . out of our top predicted drugs, of them have shown positive results in recent findings based on cell culture results and clinical trials. these promising antivirals are lopinavir, ritonavir, ribavirin [ ] , cyclosporine, [ ] , rapamycin [ ] , and nitazoxanide [ ] . for the other predicted drugs, further research is needed to evaluate their effectiveness against sars-cov- . we used two main data sources for this study. the first database was the drugvirus database [ ] ; drugvirus provides a database of broad-spectrum antiviral agents (bsaas) and the associated viruses they inhibit. the database covers viruses and compounds, and provides information on the status as antiviral of each compound-virus pair. these statuses fall into eight categories representing the progressive drug trial phases: cell cultures/co-cultures, primary cells/ organoids, animal model, phases i-iv and approved. see appendix a for a more intuitive pivot table view of the database. the second database is the national center for biotechnology information (ncbi) virus portal [ ] ; as of april , this database provides approximately . million amino-acid and million nucleotide sequences from viruses with humans as hosts. each row of this database contains an amino acid sequence specimen from a study, as well as metadata that includes the associated virus species. in our work, we considered sequences only from the virus species in the drugvirus database or their subspecies in order to be able to merge the two data sources successfully. we also constrained ourselves to amino-acid sequences only in the current iteration. the main reasons for this are two-fold: . amino-acid sequences are essentially derived from the dna sequences, which may encode overlapping information on different levels. in somewhat simplified terms, amino-acid sequences are the outputs of a layer of preprocessing on genetic material (in the form of dna/rna). . nucleotide triplets (codons) map to amino-acids, making amino-acid sequences much shorter and easier to extract features both in preprocessing and in the machine learning methods themselves. shorter sequences also mean the ml pipeline will be more resource-efficient, i.e. easier to train. the amino-acids were downloaded as three datasets: hiv types & ( , , sequences), influenza types a, b & c ( , sequences), and the "main" dataset for all other types including sars-cov- ( , sequences). each dataset came with two components. the "sequence" component is composed on accession ids and the amino-acid sequence itself, while the "metadata" component includes all other data (e.g. virus species, date specimen was taken, an identifier of the related study) as well as the accession id to enable merging the two components. the amount of research with a focus on influenza and hiv naturally lead to these viruses comprising most of the samples. in our experiments, we have excluded these viruses, and have worked only with dataset # , though the other datasets can be integrated into the main one during the class balancing process, an idea we will discuss in section , future work. the first step of the preparation phase was to merge the "sequence" and "metadata" components into a single ncbi dataset based on sequence ids. afterwards, we mapped the "species" column in this main dataset to the virus name column in the drugvirus database. this step was required as these two columns that denote the virus species in the respective datasets did not match due to subspecies present in the sequence dataset and alternative naming of some viruses. afterwards, we processed the drugvirus dataset to a format suitable for merging with the ncbi data frame. every row of the drugvirus dataset consists of a single drug-virus pairing and their respective interaction/drug trial phase, meaning any given drug and virus appeared in multiple rows of the dataset. we derived a new drugvirus dataset that functioned as a dictionary where each unique virus was a key, and the interactions with antivirals encoded as a multi-label binary vector ( if viable antiviral according to the original dataset, if not) of length (the number of antivirals) which corresponded to the value. we came up with three "versions" depending on how we decided an antiviral was a viable candidate to inhibit a virus. the criteria depended on drug trial phases: . in the first version, any interaction between a drug-virus pair is designated by a . this means drugs that did not go past cell cultures/co-cultures or primary cells/organoids testing are still considered viable candidates. . this second version expands upon the first stemming from our discovery that an attained trial phase in the database does not necessarily mean previous phases were also listed in the database. for example, we found that for a given virus, a given drug had undergone phase iii testing, designated by a , but phase i & ii were listed as s. this undermined our assumption that drug trials are hierarchical; though, in reality this is usually the case. this can be caused by missing data reporting or possibly skipped phases. we proceeded with the hierarchy assumption, and extended the database in ( ) to account for the previous phases. this meant that in this second version, an approved drug will have all phases designated with s, for example. keeping track of the phases meant that the size of the database also grew by . . in the third version, we considered a drug-virus pair as viable only if it has attained phase ii or further drug trials, signifying some success with human trials have been observed. in the results presented in section , our training database was based on this third version of the drugvirus database. the full dataset was then generated by merging this "new" version of the drugvirus dataset with the ncbi dataset. we then generated two versions of this full dataset: one that consists of all sars-cov- sequences and one that consists of all other viruses available. this enabled us to compare how successful our models are in a case when they have not been trained on the virus species at all and have to detect peptide substructures in the sequences to suggest antivirals. a sample of this final database (with some columns excluded for brevity) is available in appendix b. upon inspection of the data, we found that there were replete of duplicate or extremely similar virus sequences. to reduce this exploitability and pose a more challenging problem, we removed the duplicate sequences that belonged to the same species and had the exact same length. this reduced the size of the dataset by approximately %. the counts for each virus species before and after dropping duplicate viruses is available in appendix c and c . our main database also contained a class imbalance in the number of times certain virus species appeared in the database. we oversampled rare viruses (e.g., west nile virus: sequences) and excluded the very rare species which compose less than . % of the available unique samples in the dataset (e.g., andes virus: sequences), and undersampled the common viruses (e.g., hepatitis c: , sequences). this produced a more modest database of , amino acid sequences, with each virus having samples in the - range (see appendix c ). we kept the size of the dataset small both to enable easier model training and validation in early iterations and to handle data imbalance more smoothly. the class imbalance problem also presented itself in the antiviral compounds. even with balanced virus classes, the number of times each drug occurred within the dataset varied, simply because some drugs apply to more viruses than others. to alleviate this, we computed class weights for each drug, which we then provided to the models in training. this enabled a fairer assessment and a more varied distribution of antivirals in predicted outputs. the final step of data processing involved generating the training and validation sets. we split the data in two different ways, resulting in two different experiments (see section . , experiment setup for the full experiment pipeline). experiment i is based on a standard, randomized an % training/ % validation split on the main dataset. for experiment ii, we split the data on virus species, meaning the models were forced to predict drugs for a species that it was not trained on, and have to detect peptide substructures in the amino-acid sequences to suggest drugs. in this setup we also guaranteed that the sars-cov- sequences were always in the test set, in addition to three other viruses randomly picked from the dataset. we used a variant of this setup that trains on all virus sequences except sars-cov- and is validated on sars-cov- only to generate the results presented in section . a growing number of studies demonstrate the success of using artificial neural networks (ann) in evaluating biological sequences in drug repositioning and repurposing [ ] [ ] . previous work on training neural networks on nucleotide or amino-acid sequences have been successful with recurrent models such as gated recurrent units (gru), long short-term memory networks (lstm) and bidirectional lstms (bilstm), as well as d convolutions and d convolutional neural networks (cnn) [ ] [ ] . we have therefore focused on these network architectures, and conducted our experiments with an lstm with d convolutions and bidirectional layers as well as a cnn. the network architectures are explained briefly below. lstm and d convolutions for the lstm, a character-level tokenizer was used to encode the fasta sequences into vectors consumable by the network. the sequences were then padded with zeros or cut off to a fixed length to maintain a fixed input size. the network architecture consisted of an embedding layer, followed by d convolution and bidirectional lstm layers (each followed by maxpooling), and two fully connected layers. a more detailed architecture diagram is available in appendix d. convolutional neural network (cnn) for the cnn, the input features were one-hot encoded based on the fasta alphabet/charset, which assisted in interpretability when examining the d input arrays as images. the inputs are also fixed at a length of , resulting in x images, where is the number of elements in the fasta charset. the network architecture consists of four d convolutions with filter sizes of x , x , x and x respectively, which are maxpooled, concatenated and passed through a fully connected layer. a more detailed architecture diagram is available in appendix e. the experiments were run on a computer with an . ghz intel broadwell cpu ( gb ram) and nvidia k gpu ( gb) . both models completed a -epoch experiment in - minutes. one to three training and evaluation runs were made for each setup during model and hyperparameter selections, and ten training and evaluation runs were done to produce the average metrics in section . the experiments start by determining the model to use and apply the appropriate preprocessing steps mentioned in section . . we then proceed with determining the dataset to train and validate on. this part of the experiment setup is more extensively covered in section . . , train/test splitting. we used binary cross entropy (bce) loss, adam optimizer, precision, recall and f -score as metrics since accuracy tends to be an unreliable metric given the class imbalance and the sparse nature of our outputs. after training and validation, predictions were done on the validation set and the results were post-processed for interpretability. in post-processing, we applied a threshold to the sigmoid function outputs of the neural network, where we assigned each drug a probability of being a potential antiviral for a given amino acid sequence. after experimenting with different values, we settled on a threshold value of . . postprocessing outputs a list of drugs that were selected along with the respective probabilities for the drugs being "effective" against the virus with the given amino acid sequence. for other hyperparameters involved as well as information on hyperparameter tuning, see appendix f. here we present the results for the two experiments described in section . . , train/test splitting. the figures and tables presented in this section are based on the lstm and cnn architectures described in section . , which were trained on batch size and . and . learning rates respectively for epochs with an adam optimizer. in the regular setup, we performed an %/ % train-test split on our data of , sequences. the metrics for the best set of hyperparameters (based on validation set f -score) for both the cnn and lstm architectures respectively are presented in table . similarly, plots for the same set of models and hyperparameters over epochs are presented in figures and . our models handled the task successfully, achieving . f -score in a multi-label multi-class problem setting. this means that the models were able to match the virus species with the sequence substructures and appropriately assign the inhibiting antivirals with accuracy. these satisfactory results led to us implementing experiment ii. in experiment ii, the models predicted antiviral drugs for virus species they haven't been trained on. this meant the models were not able to recommend drugs by "recognizing" the virus from the sequence and therefore had to rely only on peptide substructures in the sequences to assign drugs. in the results presented below, the test set consists of sars-cov- , herpes simplex virus , human astrovirus and ebola virus, whose sequences were removed from the training set. we see here that the cnn (and the lstm) had issues with convergence, and the accuracies are clearly below their counterparts in the regular setup, though this is certainly expected. we now turn to the actual predictions on the sequences and attempt to interpret them. upon examination of drug predictions for herpes simplex virus (hsv- ), however, we see that our cnn was in fact quite successful. in table and table , count represents how many times each drug was flagged as potentially effective for hsv- sequences, and mean probability denotes the average confidence predicted over all instances of the drug. a sample of the outputs where these metrics are derived from is available in appendix g. antivirals used for phase ii and further trials for hsv- are highlighted in bold, meaning all six drugs in the database that are used for phase ii and further trials are predicted by our model. three of the top five predictions are approved antivirals for hsv- and the only remaining one is predicted th among antivirals. this high level of accuracy is remarkable given that our model has not been trained on hsv- sequences. predictions for sars-cov- with some variation between the two, both the lstm (table a ) and the cnn (table b) seem to converge on a number of drugs: ritonavir, lopinavir (both phase iii for mers-cov), tilorone (approved for mers-cov) and brincidofovir are in the top five candidates in both, while valacyclovir, ganciclovir, rapamycin and cidofovir rank high up in both lists. most of the remaining drugs are present in both lists as well. the lstm is more conservative in its predictions than the cnn, and the overall counts for sars-cov- are significantly lower than for herpes simplex virus for both, pointing a comparable lack of confidence on the models' part in predicting sars-cov- sequences. a further step we took for the sars-cov- sequences was visualizing the layer activations in the zetane engine to validate that the model was processing the data at a fine-grained level. this was done in similar fashion to a study where integrated gradients were used to generate attributions on a neural network performing molecule classification [ ] . the layer activations in both models showed that different antivirals activated different subsequences of a given sequence at the amino acid level, thus validating our approach. the filter activations are available in appendix h. the preliminary results of our experiments show promise and merit further investigation. we note that our ml models predict that some antivirals that show promise as treatments against mers-cov may also be effective against sars-cov- . these include the broad-spectrum antiviral tilorone [ ] and the drug lopinavir [ ] , the latter of which is now in phase iv clinical trials to determine its efficacy against covid- [ ] . such observations suggest with confidence that our models can recognize reliable patterns between particular antivirals and species of viruses containing homologous amino acid sequences in their proteome. additional observations that support our findings have come to light from a study in the lancet published shortly before this article [ ] . this open-label, randomized, phase ii trial observed that the combined administration of the drugs interferon beta- b, lopinavir, ritonavir and ribavirin provides an effective treatment of covid- in patients with mild to moderate symptoms. both of our models flagged three of the drugs in that trial (note that interferon was not part of our datasets). in terms of number of occurrences aka count, ritonavir, lopinavir and ribavirin were ranked th, th and th by the lstm, while the cnn model ranked them rd, th and th, respectively. other studies have also focused on the treatment of sars-cov- by drugs predicted in our experiments. wang et al. discovered that nitazoxanide (lstm rank th, cnn rank th) inhibited sars-cov- at a low-micromolar concentration [ ] . gordon th) is known to be effective against diverse coronaviruses [ ] . such observations are encouraging. they demonstrate that predictive models may have value in identifying potential therapeutics that merit priority for advanced clinical trials. they also add to growing observations that support using ml to streamline drug discovery. from that perspective, our models suggest that the broad spectrum antiviral tilorone, for instance, may be a top candidate for covid- clinical trials in the near future. other candidates highlighted by our results and may merit further studies are brincidofovir, foscarnet, artesunate, cidofovir, valacyclovir and ganciclovir. the antivirals identified here have some discrepancies with emerging research findings as well. for instance, our models did not highlight the widely available anti-parasitic ivermectin. one research study observed that ivermectin could inhibit the replication of sars-cov- in vitro [ ] . another large-scale drug repositioning survey screened a library of nearly , drugs and identified six candidate antivirals for sars-cov- : pikfyve kinase inhibitor apilimod, cysteine protease inhibitors mdl- , z lvg chn , vby- , and ono , and the ccr antagonist mln- [ ] . it comes as no surprise that our models did not identify these compounds as our data sources did not contain them. future efforts to strengthen our ml models will thus require us to integrate a growing bank of novel data from emerging research findings into our ml pipeline. in terms of our machine learning models, better feature extraction can improve predictions drastically. this step involves improvements through better data engineering and working with domain experts who are familiar with applied bioinformatics to better understand the nature of our data and find ways to improve our data processing pipeline. some proposals for future work that could strengthen the performance of our machine learning process are as follows: . deeper interaction with domain experts and further lab testing would lead to a better understanding of the antivirals and the amino-acid sequences they target, leading to building better ml pipelines for drug repurposing. . better handling of duplicates can improve the quality of data available. the current approach (which is based on species and sequence length) can be improved through using string similarity measures such as dice coefficient, cosine similarity, levenshtein distance etc. . influenza and hiv datasets should be integrated into the data generation and processing pipeline to enhance available data. . vectorizers can be used to extract features as n-grams (small sequences of chars), which has attained success in similar problems [ ] . other unsupervised learning methods such as singular value decomposition also may be applicable to our study [ ] . we hope that the machine learning approaches and pipelines developed here may provide longterm benefit to public health. the fact that our results show much promise in streamlining drug discovery for sars-cov- motivates us to adapt our current models so we can conduct identical drug repurposing assessments for other known viruses. moreover, experimental data suggests that our approaches are generalizable to other viruses (see the hsv- example in section . , experiment ii) -we are therefore confident that we could adapt our models to conduct equivalent studies during the next outbreak of a novel virus. this also means our methods can be used to repurpose existing drugs in order to find more potent treatments for known viruses. the direct beneficiaries of our findings are members of the clinical research community. using relatively few resources, ml-guided drug repurposing technology can help prioritize clinical investigations and streamline drug discovery. in addition to reducing costs and expediting clinical innovation, such efficiency gains may reduce the number of clinical trials -and thus human subjects used in risky research -needed to find effective treatments (this pertains to the ethical imperative to avoid harm when possible). also of importance is that in-silico analyses using machine learning provide yet another means to employ past research findings in new investigations. ml-guided drug repurposing thus provides means to obtain further value from knowledge on-hand; maximizing value in this case is laudable on many fronts, especially in terms of providing maximum benefit from publicly-funded research. the negative consequences that could arise should our models fail appear limited but are noteworthy nonetheless. note that our models aim to only indicate possible therapeutics that merit further clinical investigation in order to prove any antiviral activity against sars-cov- . should our models fail by recommending spurious treatments, these incorrect predictions may divert limited time and resources towards frivolous investigations. it should also be noted that our methods aim to primarily work as guidance for medical experts, and not as a be-all-end-all solution. and any incorrect inferences made by our models are bound to be detected early by medical experts. communicating any machine-learning predictions of tentative antiviral drugs from this study requires much caution. the current pandemic continues to demonstrate how fear, misinformation and a lack of knowledge about a novel communicable disease can encourage counterproductive health-seeking behaviour amongst the public. soon after the coronavirus became a widely understood threat, the internet was awash in false -sometimes downright harmful -information about preventing and treating covid- . included within this misleading health information were premature claims by some prominent government officials that therapeutics like chloroquine and hydroxychloroquine might hold promise as a repurposed drug for covid- . such unfounded advice caused avoidable poisonings from people self-medicating with chloroquine. subsequent clinical investigations demonstrated no notable benefit and potential adverse reactions to chloroquine when used to treat covid- . such unfortunate events remind us that preliminary findings may be misinterpreted as conclusive treatments or as evidence to support inconclusive health claims. the hyperparameters tested in our experiments are presented in section f it is certainly possible to improve the accuracies of our experiments by conducting a vaster coverage of the loss landscape through more extensive training (e.g. running longer experiments with smaller learning rates on more complex network architectures), especially for results in experiment ii. however, due to performance constraints, the scope of hyperparameter tuning as well as the ann architectures experimented on are relatively constrained as we focused on the methodology as opposed to optimal performance in this study. it should be noted that much improvement is possible in this front, as pointed out in discussion and future work. additional notes regarding our observations during hyperparameter tuning are presented below. • for the threshold, we wanted to predict eagerly, i.e. we considered false negatives more costly errors than false positives. a high threshold would mean the outputs would be composed only of the antivirals our models are very confident about per amino acid sequence. this we deem undesirable, as while we do hope these outputs narrow the scope of antivirals to focus on, over-restricting could prevent antivirals that are predicted frequently yet with low probability be detected. a low threshold such as . filtered the number of antivirals sufficiently, but also left enough breathing room for the domain experts to draw their own conclusions on a per-drug basis. • while a larger sequence length cutoff was possible and not detrimental to the results, we deemed a suitable trade-off in terms of performance versus accuracy, as many sequences do not reach lengths in the thousands to begin with. • as mentioned, the number of epochs trained could be increased, as we did not see dramatic signs of overfitting at epochs or further. however, a flattening of the metrics were evident around epochs with the hyperparameters listed, which therefore was selected a suitable stopping point. table : a section of sample outputs for amino acid sequences and their associated antivirals. post-processing outputs a list of drugs that were selected along with the respective probabilities of the drugs being "effective" against the virus with the given amino acid sequence. survey of machine learning techniques in drug discovery drug repositioning: a machine-learning approach through data integration triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial cyclosporin a inhibits the replication of diverse coronaviruses a sars-cov- protein interaction map reveals targets for drug repurposing remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro discovery and development of safe-in-man broad-spectrum antiviral agents ncbi viral genomes resource drug repurposing using deep embeddings of gene expression profiles deepdr: a network-based deep learning approach to in silico drug repositioning protein family classification with neural networks deepsf: deep convolutional neural network for mapping protein sequences to folds using attribution to decode binding mechanism in neural network models for chemistry tilorone: a broad-spectrum antiviral invented in the usa and commercialized in russia and beyond a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option corona virus drugs -a brief overview of past, present and future the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro a large-scale drug repositioning survey for sars-cov- antivirals near perfect protein multi-label classification with deep neural networks neural networks for full-scale protein sequence classification: sequence encoding with singular value decomposition we would like to thank the administrators of the drugvirus and the ncbi virus portal for providing the datasets that are central to this study. we appreciate comments on preliminary drafts of this manuscript from dr tariq daouda from the massachusetts general hospital, broad institute, harvard medical school.the authors declare they will not obtain any direct financial benefit from investigating and reporting on any given pharmaceutical compound. the following study is funded by the authors' employer, zetane systems, which produces software for ai technologies implemented in industrial and enterprise contexts. c database profile c. virus counts before dropping duplicate sequences key: cord- -qah pk authors: ekins, s; mestres, j; testa, b title: in silico pharmacology for drug discovery: applications to targets and beyond date: - - journal: british journal of pharmacology doi: . /sj.bjp. sha: doc_id: cord_uid: qah pk computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. these in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. the first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. the aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. we will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediating the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets. the first part of this review (ekins et al., ) has briefly described the history and development of a field that can be globally referred to as in silico pharmacology. this included the development of methods and databases, quantitative structure-activity relationships (qsars), similarity searching, pharmacophores, homology models and other molecular modelling, machine learning, data mining, network analysis and data analysis tools that all use a computer. we have also previously introduced how some of these methods can be used for virtual ligand-and target-based screening and virtual affinity profiling. in this second part of the review, we will greatly expand on the applications of these methods to many different target proteins and complex properties, and discuss the pharmacological space covered by some of these in silico efforts. in the process, we will detail the success of in silico methods at identifying new pharmacologically active molecules for many targets and highlight the resulting enrichment factors when screening active druglike databases. we will finally discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. the applicability of computational approaches to ligand and target space in which a lead molecule against one gene family member is used for another similar target (termed chemogenomics) (morphy et al., ; sharom et al., ) , will be discussed thoroughly in an upcoming review in this journal from didier rognan (personal communication) and will be only briefly addressed here. however, there have been several attempts to establish relationships between molecular structure and broad biological activity and effects that should be considered (see also section . . in ekins et al. ( ) ) (kauvar et al., (kauvar et al., , b kauvar and laborde, a) . for example, the work of fliri et al. ( b) presented the biological spectra for a cross-section of the proteome. using hierarchical clustering of the spectra similarity enabled a relationship between structure and bioactivity to be constructed. this work was extended to identify agonist and antagonist profiles at various receptors, correctly classifying similar functional activity in the absence of drug target information (fliri et al., c) . interestingly, using ic data as affinity fingerprints did not identify functional activity similarities between molecules as this approach was suggested to introduce a pharmacophoric bias (fliri et al., c) . a similar probabilistic approach has also been applied by the same authors to link adverse effects for drugs (obtained from the drug labelling information) with biological spectra. for instance, clustering molecules by side effect profile showed that similar molecules had overlapping profiles, in the same way that they had similar biological spectra, linking preclinical with clinical effects (fliri et al., a) . this work offers the intriguing possibility of predicting a biospectra profile, possible functional activity and a side effect profile for a new molecule based on similarity alone. however, confidence in this approach would be greatly enhanced by further prospective testing with a large test set of drug-like molecules not used to generate the underlying signature database. a second group also from pfizer presented a global mapping of pharmacological space and in particular focused on a polypharmacology network of molecules with activity against multiple proteins (paolini et al., ) . they have additionally generated bayesian binary models (for molecules active at o mm or inactive) for targets using over molecules with biological data (from their in-house collection and the literature), suggesting that they would be useful for predicting primary pharmacology. assessment of approved oral drugs in two-dimensional ( d) molecular property space (molecular weight versus clogp) showed that many of them had clogp and mw . in spite of this, their associated targets were potentially druggable but had yet to realize their potential (paolini et al., ) . perhaps this work needs to be combined with that of fliri and others for its true potential to be realized, to enable simultaneous understanding and prediction of target, proteomic, functional activity and side effects. a recent analysis using molecular d descriptors followed by principal component (pca) of over anticancer molecules representing cancer medicinal chemistry space, showed that they populated a different space broader than hit-like space and orally available drug-like space. this would indicate that in order to find molecules for anticancer targets in commercially available databases, different rules are required other than those widely used for drug-likeness, as they may unfortunately filter out possible clinical candidates (lloyd et al., ) . methods to predict the potential biological targets for molecules from just chemical structure have been attempted by using different approaches to those already described above. for example, one study used probabilistic neural networks with atom-type descriptors to classify molecules from the mdl drug data reports (mddr) database with activity against one of the seven targets (g protein-coupled receptors (gpcrs), kinases, enzymes, nuclear hormone receptors and zinc peptidases) with excellent training, testing and prediction statistics (niwa, ) . twenty-one targets related to depression were selected and molecules from the mddr database were used to create support vector machine (svm) classification models from atom-type descriptors (lepp et al., ) . these models had satisfactory predictions and recall values between and %, the molecules recovered being on average of low molecular weight (o ) and some were active against more than one model. it was suggested that general svm filters would be useful for virtual screening owing to their speed. others have used similarity searching of the mddr database against small numbers of reference inhibitors for several different targets and were able to show variable enrichment factors that were greater than random (hert et al., ) . the structure-based alternative to understanding small molecule-protein interactions is to flexibly dock molecules into multiple proteins. a representative of this inverse docking approach is invdock, which was recently applied for identifying potential adverse reactions using a database of proteins related to toxicities (dart). this method has been recently demonstrated with marketed anti-hiv drugs resulting in reasonable accuracy against the dna polymerase beta and dna topoisomerase i (ji et al., ) . the public availability of data on drugs and drug-like molecules may make the analyses described above possible for scientists outside the private sector. for example, chemical repositories such as drugbank (http://redpoll. pharmacy.ualberta.ca/drugbank/) (wishart et al., ) , pubchem (http://pubchem.ncbi.nlm.nih.gov/), kidb (http:// kidb.bioc.cwru.edu/) (roth et al., ; strachan et al., ) and others consist of a wealth of target and small molecule data that can be mined and used for computational pharmacology approaches. although much of the in silico pharmacology research to date has been focused on human targets, many of these databases contain data from other species that would also be useful for understanding species differences and promoting discovery of molecules for animal healthcare as well as assisting in understanding the significance of toxicological findings for chemicals released into the environment. to exhaustively describe all of the proteins that have been computationally modelled under the auspices of in silico pharmacology would be impossible in the confines of this review. therefore, we will briefly overview the types of proteins that have been modelled and the methods used (see below and table ). in addition, we will focus on and describe particular pharmacological applications with regard to virtual screening where novel ligands have been identified. the reader is highly encouraged to study an extensive review of success stories in computer-aided design, which covers a large number of proteins that have been targets for all manner of in silico methods (kubinyi, ) , as well as other reviews that have dealt with the successes of individual methods (fujita, ; kurogi and guner, a; guner et al., ) . as described previously, computational approaches for drug discovery and development may have more impact if integrated (swaan and ekins, ) and we have previously attempted to show that computational methods have been broadly applied to virtually all important proteins in absorption, distribution, metabolism, excretion and toxicity (adme/tox) (ekins and swaan, b) . the qaim of this paper is to provide an up-to-date review of all proteins and protein families addressed through current state-of-the-art in silico pharmacology methods. drug target examples. enzymes: the ubiquitin regulatory pathway, in which ubiquitin is conjugated and deconjugated with substrate proteins, represents a source of many potential targets for modulation of cancer and other diseases santo et al. ( ) abbreviations: ampa, a-amino- -hydroxy- -methyl- -isoxazole propionate; cox, cyclooxygenase; cyp, cytochrome p ; hiv- , human immunodeficiency virus; lox, lipoxygenase. in silico pharmacology for drug discovery s ekins et al (wong et al., ) . the recent crystal structure of a mammalian de-ubiquitinating enzyme hausp, which specifically de-ubiquitinates the ubiquitinated p protein, may also assist in drug development despite the peptidic nature of its substrate (hu et al., ) . novel non-peptidic inhibitors of the protease ubiquitin isopeptidase, which not only de-ubiquitinates p but other general ubiquitinated proteins as well, were discovered recently using a simple pharmacophore-based search of the national cancer institute (nci) database (mullally et al., ; mullally and fitzpatrick, ) . these inhibitors had ic values in the low micromolar range and caused cell death independent of the tumour suppressor p , which is mutated in greater than % of all cancers (hence, p inhibition per se may not represent an optimal target for modulation). the ubiquitin isopeptidase inhibitors shikoccin, dibenzylideneacetone, curcumin and the more recently described punaglandins from coral indicate that a sterically accessible a,b-unsaturated ketone is essential for bioactivity (verbitski et al., ) . all these molecules represent valuable leads for further chemical optimization. aromatase (cytochrome p (cyp) ) is a validated target for breast cancer. a ligand-based pharmacophore was generated with three non-steroidal inhibitors. this model could recognize known inhibitors from an in-house library and was further refined by the addition of molecular shape. the model was further used to search the nci database and molecules were scored with a quantitative catalyst hypo-refine (accelrys inc., san diego, ca, usa) model generated with molecules. the hits were also filtered with other pharmacophores for toxicity-related proteins, before testing. two out of the three compounds were ultimately found to be micromolar inhibitors (schuster et al., ) . a structure-based catalyst pharmacophore was developed for acetylcholine esterase, which was subsequently used to search a natural product database. the strategy identified scopoletin and scopolin as hits and were later shown to have moderate in vivo activity (rollinger et al., ) . the same database was also screened against cyclooxygenase (cox)- and cox- structure-based pharmacophores, leading to the identification of known cox inhibitors. these represent examples where a combination of ethnopharmacological and computational approaches may aid drug discovery (rollinger et al., ) . a combined ligand-based and structure-based approach was taken to gaining structural insights into the human -lipoxygenase (lox). a catalyst qualitative hiphop model was created with different molecules that resulted in a five-feature pharmacophore. a homology model of the enzyme was based on two soybean lox enzymes and one rabbit lox enzyme. molecular docking was then used to update and refine the pharmacophore to a four-feature model that could also be visualized in the homology model of -lox. as a result of these models, amino-acid residues in the binding site were suggested as targets for site-directed mutagenesis while virtual screening with the pharmacophore had suggested compounds with a phenylthiourea or pyrimidine- -carboxylate group for testing (charlier et al., ) . homology models for the human -lox and -lox have also been used with the flexible ligand docking programme glide (schrödinger inc.) to perform virtual screening of compounds. out of compounds tested, had inhibitory activity and several were in the low micromolar range (kenyon et al., ) . more than years of research on renin have not been enough to deliver a marketed drug that inhibits this enzyme. in spite of this, renin remains an attractive yet elusive target for hypertension (fisher and hollenberg, ; stanton, ) . in this respect, application of structure-based design leads to the identification of new non-peptidic inhibitors of human renin. these molecules include aliskiren (rahuel et al., ; torres et al., ) , piperidines, including ro- oefner et al., ; vieira et al., ) , and related , -disubstituted piperidines (marki et al., ) . interestingly, these piperidines bind to and stabilize a different conformer of the protein termed 'open renin' (bursavich and rich, ) , whereas aliskiren binds to 'closed renin'. since these latter structure-based design efforts, there have been remarkably very few published attempts at computer-aided design of novel renin inhibitors. a single early qsar was derived for a series of chainmodified peptide analogues of angiotensinogen. the activity of these molecules was found to correlate with kier's firstorder molecular connectivity index descriptor and molecular weight but not with lipophilicity as measured by logp (khadikar et al., ) . another computational method for renin drug discovery used the de novo design software growmol, which could apparently regenerate , -disubstituted piperidines in % of the grown structures (bursavich and rich, ). an attempt to use a catalyst pharmacophore to discover new renin inhibitors was described in the early s (van drie, ) . several novel molecules from the pomona database (an early three-dimensional ( d) molecule database) were found that mapped to a renin pharmacophore but apparently were not tested in vitro. more recently, a ligandfit docking study with a crystal structure of the 'open renin' form was able to detect known inhibitors seeded in a library of compounds within the top . % when using a consensus scoring function. four examples of high-scoring compounds that were not tested as inhibitors fulfilled the pharmacophore derived from the x-ray data, consisting of four hydrophobes, a hydrogen bond donor or positive ionizable feature as well as excluded volumes (krovat and langer, ) . another study has used similarity searching of the mddr database (for over compounds) using renin inhibitors and was able to produce enrichment factors that were -fold greater than random (hert et al., ) . genetic algorithms have also been used for class discrimination between renin inhibitors and noninhibitors in a subset of the mddr using a small number of interpretable descriptors. among them, amide bond count, molecular weight and hydrogen bond donor counts were found to be much higher in renin inhibitors (ganguly et al., ) . the recent publications on novel renin inhibitors represent a considerable amount of new information that could be used for further qsar model development and database searching efforts in order to derive novel starting scaffolds for optimization. cathepsin d is an aspartic protease found mainly in lysosomes, which may have a role in b-amyloid precursor protein release and hence may well be a target for in silico pharmacology for drug discovery s ekins et al alzheimer's disease. cathepsin d may also be elevated in breast cancer and ovarian cancer hence a means to modulate this activity could be beneficial in these diseases. there has been a brief overview of cathepsin d in a comprehensive review of protease inhibitors (leung et al., ) . a combination of a structure-based design algorithm and combinatorial chemistry has been successfully applied to finding novel molecules for cathepsin d in the nanomolar range (kick et al., ) . structures based on pepstatin (a . pm inhibitor (baldwin et al., ) ) yielded a - % hit rate. these molecules were tested in vitro using hippocampal slices and were shown to block the formation of hyperphosphorylated tau fragments (bi et al., ) . there have been relatively few computational studies to date on cathepsin d and other related aspartic proteases such as renin and b-secretase. one study has used molecular dynamics and free energy analyses (mm-pbsa) of cathepsin d inhibitor interactions to suggest new substitutions that may improve binding (huo et al., ) . a genetic algorithmbased de novo design tool, adapt has also been used to rediscover active cathepsin d molecules, by placing key fragments in the correct positions (pegg et al., ) . computational models may aid in the selection of novel ligands for protease inhibition that are non-peptidic and selective. using the structural features of eight published inhibitors for cathepsin d (huo et al., ) , a five-feature pharmacophore was derived consisting of three hydrophobes and two hydrogen bond acceptors (r ¼ . ). this pharmacophore was used to search a molecule database and selected molecules out of present. in contrast, a similarity search at the % level using chemfinder (cambridgesoft, cambridge, ma, usa) suggested different molecules. all of these were selected for testing in vitro. the pharmacophore produced four hits ( % hit rate) and the similarity search generated five hits ( % hit rate), where at least one replicate showed greater than % inhibition (ekins et al., a) . in silico evaluation of the adme properties for all active compounds estimated that the molecules would be well absorbed, although some were predicted to have solubility and cyp d inhibition problems. pharmacophore-and structure-based approaches have been used to optimize an acyl urea hit for human glycogen phosphorylase. a catalyst hypogen five-feature pharmacophore was developed and used to guide further analogue synthesis. these compounds showed a good correlation with prediction (r ¼ . ). an x-ray structure for one molecule was used to confirm the predicted binding conformation. ultimately, a comparative molecular field analysis (comfa) model was generated with all molecules synthesized and was found to be complementary to the x-ray structure. the outcome of this study was a molecule with good cellular activity that could inhibit blood glucose levels in vivo in rat (klabunde et al., ) . the human sirtuin type , a target for controlling aging and some cancers, deacetylates a-tubulin and has been crystallized at high resolution. this structure has been used for docking the maybridge database and returned a small hit list from which compounds were tested and showed activity at the micromolar level (tervo et al., ) . catechol o-methyltransferase is a target for parkinson's disease and there is currently a crystal structure of the enzyme that has been used to generate a homology model of the human enzyme. this model was used to dock with flexx software several catechins from tea and understand the structure-activity relationship (sar) for these molecules and their metabolites, which had been tested in vitro. ultimately, the combination of in vitro and computational work indicated that the galloyl group on catechins, the distance between lys on the enzyme, and the reacting catecholic hydroxy group were important for inhibition . kinases: the kinases represent an attractive family of over targets for the pharmaceutical industry, with several drugs approved recently. kinase space has been mapped using selectivity data for small molecules to create a chemogenomic dendrogram for kinases that showed the highly homologous kinases to be inhibited similarly by small molecules (vieth et al., ) . virtual screening methods have been applied quite widely for kinases to date (fischer, ) . the structure-based design method has produced new potent inhibitors of cdk starting from the highly similar apo cdk and the positioning of olomoucine. a few aminoacid residues were mutated to conform to the cdk sequence. macromodel was used to energy minimize molecules in the atp pocket and visual inspection suggested points for molecular modification on the ligand. very quickly, design efforts guided ligand optimization to improve activity from . mm to nm (furet et al., ) . a more recent cdk /cyclin b homology model was also used to manually dock ligands, which enabled progression from alsterpaullone with an ic of nm to a derivative with an ic of . nm (kunick et al., ) . a structure-based in silico screening method was pursued for the syk c-terminal sh domain using dock to find low molecular weight fragments for each binding site with millimolar binding affinity. the fragments were then linked to result in molecules in the - mm range, which is a starting point for further lead optimization (niimi et al., ) . a pseudoreceptor model was built with a set of epidermal growth factor receptor (egfr) tyrosine kinase inhibitors with the flexible atom receptor model method. the top models created had high r and q and were also validated with a six-molecule test set. the pseudoreceptor was also in accord with a crystal structure of cdk (peng et al., ) . virtual screening using dock with the crystal structure of the lck sh domain was used to screen two million commercially available molecules. extensive filtering was required to result in a manageable hit list using molecular weight and diversity. out of compounds tested in vitro, were inhibitory at mm, while had activities of and mm. fluorescence titrations of some of these compounds suggested the k d values were in the low micromolar range (huang et al., ) . the same group also took a similar approach to discover inhibitors of erk by screening compounds computationally and testing in vitro of them (hancock et al., ) . five of these molecules inhibited cell in silico pharmacology for drug discovery s ekins et al proliferation and two were shown by fluorescence titration to bind erk with k d values, which were in the low micromolar range. in both cases, docking of the active molecules suggested orientations for verification by x-ray crystallography (hancock et al., ) . the ligand scout method was used with bcr-abl tyrosine kinase to find sti- (imatinib, gleevec) in a single and multiple conformation database (wolber and langer, ) . a structurally related three-substituted benzamidine derivative of sti- was suggested by structure-based design and when manually docked into the binding site and energy minimized, it was shown to form favourable interactions with a hydrophobic pocket. ck and pkd are part of the cop signalosome and can control stability of p and c-jun, which are important for tumour development. curcumin, besides being an inhibitor of ubiquitin isopeptidase (mullally et al., ; mullally and fitzpatrick, ) and activator protein- (tsuchida et al., ) , also inhibits ck and pkd. using curcumin and emodin as reference structures against which a database of over a million molecules was screened by means of d and d similarity searches retrieved molecules. among them, seven possessed inhibitory activity. for example, piceatannol was more potent than curcumin against both ck and pkd, with ic values of . and . mm, respectively (fullbeck et al., ) . obviously, these examples suggest there has been some success in finding active molecules for kinases, but interestingly in few of these studies is selectivity toward other kinases accounted for. ultimately, for therapeutic success activity toward several kinases (but selectivity toward others) may be required. drug-metabolizing enzymes and transporters: mathematical models describing quantitative structure-metabolism relationships were pioneered by hansch et al. ( ) using small sets of similar molecules and a few molecular descriptors. later, lewis and co-workers provided many qsar and homology models for the individual human cyps (lewis, ) . as more sophisticated computational modelling tools became available, we have seen a growth in the number of available models (de groot and ekins, b; de graaf et al., ; de groot, ) and the size of the data sets they encompass. some more recent methods are also incorporating water molecules into the binding sites when docking molecules into these enzymes and these may be important as hydrogen bond mediators with the binding site amino acids (lill et al., ) . docking methods can also be useful for suggesting novel metabolites for drugs. a recent example used a homology model of cyp d and docked metoclopramide as well as other drugs to show a good correlation between ic and docking score r ¼ . . a novel aromatic n-hydroxy metabolite was suggested as the major metabolite and confirmed in vitro. now that several crystal structures of the mammalian cyps are available, they have been found to compare quite favourably to the prior computational models (rowland et al., ) . however, for some enzymes like cyp a , where there is both ligand and protein promiscuity, there may be difficulty in making reliable predictions with some computational approaches such as docking with the available crystal structures (ekroos and sjogren, ) . hence, multiple pharmacophores or models may be necessary for this and other enzymes (ekins et al., a, b) , as it has been indicated by others more recently (mao et al., ) . the udp-glucuronosyltransferases are a class of versatile enzymes involved in the elimination of drugs by catalysing the conjugation of glucuronic acid to substrates bearing a suitable functional group, so called phase ii enzymes. there have been numerous qsar and pharmacophore models that have been generated with relatively small data sets for rat and human enzymes. the pharmacophores for the human ugt a , ugt a and ugt a all have in common two hydrophobes and a glucuronidation feature, while ugt a has an additional hydrogen bond acceptor feature sorich et al., ) . sulfotransferases, a second class of conjugating enzymes, have been crystallized (dajani et al., ; gamage et al., ) and a qsar method has also been used to predict substrate affinity to sult a (dajani et al., ) . to the best of our knowledge, computational models for other isozymes have not been developed. in general, conjugating enzymes have generally been infrequently targeted for in silico models. perhaps because of a paucity of in vitro data and limited diversity of molecules tested, they have been less widely applied in industry. the computational modelling of drug transporters has been thoroughly reviewed by numerous groups (zhang et al., a, b; chang and swaan, ) and will not be addressed here in detail. various transporter models have also been applied to database searching to discover substrates and inhibitors pleban et al., ; chang et al., b) and increase the efficiency of in vitro screening (chang et al., a) or enrichment over random screening. a pharmacophore model of the na þ /d-glucose co-transporter found in renal proximal tubules was derived indirectly using phlorizin analogues with the disco programme to superpose molecules. this enabled an estimate of the size of the binding site to be obtained. in contrast to more recent studies with transporter pharmacophores, this model was not tested or used for database searching (wielert-badt et al., ) . receptors: there are more than different families of receptors that are present in the plasma membrane, altogether representing over proteins of the receptorome (strachan et al., ) . receptors have been widely used as drug targets and they have a wide array of potential ligands. however, it should be noted that to date we have only characterized and found agonists and antagonists for a small percentage of the receptorome. the a-amino- -hydroxy- -methyl- -isoxazole propionate receptor is central to many central nervous system (cns) pathologies and ligands have been synthesized as anticonvulsants and neuroprotectants. there is currently no d structure information and therefore a four-point catalyst hiphop pharmacophore was developed with antagonists. this was then used to search the maybridge database and select eight compounds for testing of which six of these were found to be active in vivo as anticonvulsants (barreca et al., ) . serotonin plays a role in many physiological systems, from the cns to the intestinal wall. along with its many receptors, it has a major developmental function regulating cardiovascular morphogenesis. the -ht receptor family are g protein-coupled -transmembrane spanning receptors with -ht b expressed in cardiovascular, gut, brain tissues, as well as human carcinoid tumors (nebigil et al., ) . in recent years, this receptor has been implicated in the valvular heart disease defects caused by the now banned 'fen-phen' treatment of patients. the primary metabolite, norfenfluramine, potently stimulates -ht b (fitzgerald et al., ; rothman et al., ) . computational modelling of this receptor has been limited to date. a traditional qsar study used a small number of tetrahydro-b-carboline derivatives as antagonists of the rat -ht b contractile receptor in the rat stomach fundus (singh and kumar, ) . a d-qsar with grid-golpe using (aminoalkyl)benzo and heterocycloalkanones as antagonists of the human receptor resulted in very poor model statistics, possibly owing to the limited range of activity measured and the fact that the data corresponded to a functional response that is likely more complex (brea et al., ) . neither of these models was validated with external predictions. on the basis of bacteriorhodopsin and rhodopsin, homology models for the mouse and human -ht b receptor have been combined with site-directed mutagenesis. the bacteriorhodopsin structure provided more reliable models, which confirmed an aromatic box hypothesis for ligand interaction along transmembrane domains , , with serotonin (manivet et al., ) . a more recent -ht b homology model based on the rhodopsin-based model of the rat -ht a was used to determine the sites of interaction for norfenfluramine following molecular dynamics simulations. site-directed mutagenesis showed that val . was implicated in highaffinity binding through van der waals interactions and the ligand methyl groups (setola et al., ) . there is certainly an opportunity to develop further qsar models for this receptor in order to rapidly screen libraries of molecules to identify undesirable potent inhibitors. the serotonin -ht a receptor has been frequently modelled. for example, a conformational study of four ligands defined a pharmacophore of the antagonist site using sybyl (hibert et al., ) . the model resulting from such an active analogue approach was used in molecule design and predicted molecule stereospecificity. more recently, a series of over homology models were iteratively created based on the crystal structure of the bovine rhodopsin that were in turn tuned by flexx docking of known ligands. the final model was used in a virtual screening simulation that was enriched with inhibitors, compared with random selection and from this the authors suggested its utility for a real virtual screen (nowak et al., ) . a homology model of the -ht a receptor has also been used with dock to screen a library of compounds seeded with -ht a ligands. ninety percent of these active compounds were ranked in the top compounds (becker et al., ) , representing a significant enrichment. the same model was used to screen a library of vendor compounds and select for testing, of which had activities below mm, one possessing nm affinity. structure-based in silico optimization was then performed to improve selectivity with other gpcrs and optimize the pharmacokinetic (pk) profile. however, as this proceeded, the molecules were found to have affinity for the human ether a-go-go-related gene (herg), and this was subsequently computationally assessed using a homology model that pointed to adjusting the hydrophobicity. the resulting clinical candidate had good target and antitarget selectivity and backup compounds were selected in the same way (becker et al., ) . another early computer-aided pharmacophore generated with sybyl using a set of selective and non-selective analogues was used to design agonists for -ht d as antimigraine agents with selectivity against -ht a (linked to undesirable changes in blood pressure) (glen et al., ) . a range of typical and atypical antipsychotics bind to the -ht receptor. based on the structure of bovine rhodopsin, homology models of the human and rodent -ht receptors were constructed and used to dock ligands that were known to exhibit species differences in binding (hirst et al., ) . following sequence alignment, amino-acid residues were identified for mutation and the rationalization of these mutations and their effects on ligand binding were obtained from the docking studies. the models generated were in good agreement with the in vitro data and could be used for further molecule design. this study was a good example where computational, molecular biology and traditional pharmacology methods were combined (hirst et al., ) . the na þ , k þ -atpase is a receptor for cardiotonic steroids, which in turn inhibit the atpase and cation transport and have ionotropic actions. although the effects of digitalis have been known for hundreds of years, a molecular understanding has remained absent until recently. a homology model was generated with the serca a crystal structure and tested with nine cardiac glycosides (keenan et al., ) . the model was also mutated to mimic the rat receptor and showed how oubain would orient differently in these models, perhaps explaining the species difference in affinity. these models also suggested amino acids that could be experimentally mutated to validate the hypothesis for the binding site identification, although this has yet to be tested. the dopamine receptors have been implicated in parkinson's disease and schizophrenia. unfortunately, no crystal structure is currently available and thus the search for new antagonists has used qsar models. a set of compounds was used with four different qsar methods (comfa, simulated annealing-partial least square (pls), k-nearest neighbours (knn) and svm), and training as well as testing statistics were generated. svm and knn models were also used to mine compound databases of over molecules that resulted in consensus hits. five of these hits were known to bind the receptor and were not in the training set, while other suggested hits did not contain the catechol group normally seen in most dopamine inhibitors (oloff et al., ) . the a a receptor is a target for controlling vascular tone and therefore useful for antihypertensive agents. a novel approach for ligand-based screening called multiple feature tree (mtree) describes the training set molecules as a feature tree descriptor derived from a topological molecular graph that is then aligned in a pairwise fashion (hessler et al., ) . a set of six antagonists was used to derive a model with this method and was compared with a catalyst pharmacophore model. both approaches identified a central positive ionizable feature flanked by hydrophobic regions at either end. these two methods were compared for their ability to rank a database of over molecules. within the top % of the database, mtree had an enrichment factor that was over twice that obtained with catalyst (hessler et al., ) . nuclear receptors: nuclear receptors constitute a family of ligand-activated transcription factors of paramount importance for the pharmaceutical industry since many of its members are often considered as double-edged swords (shi, ) . on the one hand, because of their important regulatory role in a variety of biological processes, mutations in nuclear receptors are associated with many common human diseases such as cancer, diabetes and osteoporosis and thus, they are also considered highly relevant therapeutic targets. on the other hand, nuclear receptors act also as regulators of some the cyp enzymes responsible for the metabolism of pharmaceutically relevant molecules, as well as transporters that can mediate drug efflux, and thus they are also regarded as potential therapeutic antitargets (off-targets). examples of the use of target-based virtual screening to identify novel small molecule modulators of nuclear receptors have been recently reported. using the available structure of the oestrogen receptor subtype a (era) in its antagonist conformation, a homology model of the retinoic acid receptor a (rara) was constructed (schapira et al., ) . using this homology model, virtual screening of a compound library lead to the identification of two novel rara antagonists in the micromolar range. the same approach was later applied to discover novel and diverse micromolar antagonists of the thyroid hormone receptor (schapira et al., ) . by means of a procedure designed particularly to select compounds fitting onto the lxxll peptide-binding surface of the oestrogen receptor, novel era antagonists were identified (shao et al., ) . since poor displacement of b-estradiol was observed in the er-ligand competition assay, these compounds may represent new classes of era antagonists, with the potential to provide an alternative to current anti-oestrogen therapies. the discovery of three low micromolar hits for erb displaying over -fold binding selectivity with respect to era was also recently reported using database screening (zhao and brinton, ) . a final example reports the identification and optimization of a novel family of peroxisome proliferator-activated receptors-g partial agonists based upon pyrazol- -ylbenzenesulfonamide after employing structure-based virtual screening, with good selectivity profile against the other subtypes of the same nuclear receptor group . ion channels: therapeutically important channels include voltage-gated ion channels for potassium, sodium and calcium that are present in the outer membrane of many different cells such as those responsible for the electrical excitability and signalling in nerve and muscle cells (terlau and stuhmer, ) . these represent validated therapeutic targets for anaesthesia, cns and cardiovascular diseases (kang et al., ) . a recent review has discussed the various qsar methods such as pharmacophores, comfa, svm, d-qsar, genetic programming, self organizing maps and recursive partitioning that have been applied to most ion channels (aronov et al., ) in the absence of crystal structures. to date l-type calcium channels and herg appear to have been the most extensively studied channels in this regard. in contrast, there are far fewer examples of computational models for the sodium channel. these three classes of ion channels have been studied as they represent either therapeutic targets or antitargets to be avoided. for example, one of many models for the herg potassium channel has compared three different methods with the same set of molecules for training and a test set. recursive partitioning, sammon maps and kohonen maps were used with atom path lengths . the average classification quality was high for both training and test selections. the sammon mapping technique outperformed the kohonen maps in classification of compounds from the external test set. the quantitative predictions for recursive partitioning could be filtered using a tanimoto similarity to remove molecules that were markedly different to the training set (willett, ) . the path length descriptors can also be used to visualize the similarity of the molecules in the whole training set (figure a ). in addition, a subset of molecules can also be compared, with those highlighted in blue representing close neighbours and those in red being more distant (figure b) . transcription factors: a cyclic decapeptide with activity against the ap- transcription factor was used to derive a d pharmacophore to which low energy conformations of non-peptidic compounds were compared. new -thia- azaspiro [ , ] decane and benzophenone derivatives with activity in binding and cell-based assays were discovered as ap- inhibitors in a lead hopping approach (tsuchida et al., ) . antibacterials: twenty deoxythymidine monophosphate analogues were used along with docking to generate a pharmacophore for mycobacterium tuberculosis thymidine monophosphosphate kinase inhibitors with the catalyst software. a final model was used to screen a large database spiked with known inhibitors. the model was suggested to have an enrichment factor of , which is highly significant. in addition, the model was used to rapidly screen half a million compounds in an effort to discover new inhibitors (gopalakrishnan et al., ) . antivirals: neuroamidase is a major surface protein in influenza virus. a structure-based approach was used to generate catalyst pharmacophores and these in turn were used for a database search and aided the discovery of known inhibitors. the hit lists were also very selective (steindl and langer, ) . human rhinovirus c protease is an antirhinitis target. a structure-based pharmacophore was developed initially around ag but this proved too restrictive. a second pharmacophore was developed from seven peptidic inhibitors using the catalyst hiphop method. this hypothesis was useful in searching the world drug index database to retrieve in silico pharmacology for drug discovery s ekins et al compounds with known antiviral activity and several novel compounds were selected from other databases with good fits to the pharmacophore, indicative that they would be worth testing although these ultimate testing validation data were not presented (steindl et al., a) . human rhinovirus coat protein is another target for antirhinitis. a combined pharmacophore, docking approach and pca-based clustering was used. a pharmacophore was generated from the structure and shape of a known inhibitor and tested for its ability to find known inhibitors in a database. ultimately, after screening the maybridge database, compounds were suggested that were then docked and scored. six compounds were tested and found to inhibit viral growth. however, the majority of them were found to be cytotoxic or had poor solubility (steindl et al., b) . the ligand scout approach was tested on the rhinovirus serotype and was able to find known inhibitors in the pdb (wolber and langer, ) . the sars coronavirus c-like proteinase has been addressed as a potential drug design target. a homology model was built and chemical databases were docked into it. a pharmacophore model and drug-like rules were used to narrow the hit list. forty compounds were tested and three were found with micromolar activity, the best being calmidazolium at mm (liu et al., ) , perhaps a starting point for further optimization. a pharmacophore has also been developed to predict the hepatitis c virus rna-dependent rna polymerase inhibition of diketo acid derivatives. a catalyst hypogen model was figure (a) a distance matrix plot of the molecule herg training set showing in general that the molecules are globally dissimilar as the plot is primarily red . (b) a distance matrix plot of a subset of the training set to show molecules similar to astemizole. blue represents close molecules and red represents distant molecules based on the chemtree pathlength descriptors (see colour scale). in silico pharmacology for drug discovery s ekins et al derived with molecules with activities over three log orders to result in a five-feature pharmacophore model. this was in turn tested with compounds from the same data set as well as nine diketo acid derivatives, for which the predicted and experimental data were in good agreement (di santo et al., ) . other therapeutic targets: the integrin vla- (a b ) is a target for autoimmune and inflammatory diseases such as asthma and rheumatoid arthritis. the search for antagonists has included using a catalyst pharmacophore derived from the x-ray crystal structure of a peptidic inhibitor (singh et al., b) . this was used to search a virtual database of compounds that could be made with reagents from the available chemicals directory. twelve compounds were then selected and synthesized, with resulting activities in the range between . nm and mm. hence, a peptide was used to derive non-peptide inhibitors that were active in vivo. a second study by the same group used comfa with a set of antagonists with activity from to nm to generate a model with good internal validation statistics that was subsequently used to indicate favourable regions for molecule substituent changes (singh et al., a) . it is unclear whether the comfa model was also successful for design of further molecules. it is possible to use approved drugs as a starting point for drug discovery for other diseases. for example, the list of world health organization essential drugs has been searched to try to find leads for prion diseases using d tanimoto similarity or d searching with known inhibitors. this work to date has suggested compounds, yet they appear not to have been tested, so the approach has not been completely validated (lorenzen et al., ) . protein-protein interactions are key components of cellular signalling cascades, the selective interruption of which would represent a sought after therapeutic mechanism to modulate various diseases (tesmer, ) . however, such pharmacological targets have been difficult for in silico methods to derive small molecule inhibitors owing to generally quite shallow binding sites. the g-protein gbg complex can regulate a number of signalling proteins via protein-protein interactions. the search for small molecules to interfere with the gbg-protein-protein interaction has been targeted using flexx docking and consensus scoring of molecules from the nci diversity set database (bonacci et al., ) . after testing compounds as inhibitors of the gb g -sirk peptide, nine compounds were identified with ic values from nm to mm. further substructure searching was used to identify similar compounds to one of the most potent inhibitors to build a sar. these efforts may eventually lead to more potent lead compounds. up to this point, we have generally considered in silico pharmacology models that essentially relate to a single target protein and either the discovery of molecules as agonists, antagonists or with other biological activity after database searching and in vitro testing or following searching of databases seeded with molecules of known activity for the target. however, there are many complex properties that have been modelled in silico and these will be briefly discussed here. it should also be pointed out that while several physicochemical properties such as clogp and water solubility have been extensively studied, the training sets for these models are in the s or tens of thousands of molecules, while other complex properties have generally used much smaller training sets in the range of hundreds of molecules. for example, a measure of molecule clearance would be indicative of elimination half-life that would naturally be of value for selecting candidates. the intrinsic clearance has therefore been used as a measure of the enzyme activity toward a compound and this may involve multiple enzymes. some of the earliest models for this property includes a comfa model of the cyp-mediated metabolism of chlorinated volatile organic compounds, likely representative of cyp e (waller et al., ) . a more generic set of molecules with clearance data derived from human hepatocytes has been used to predict human in vivo clearance using multiple linear regression, pca, pls, neural networks with leave-oneout cross-validation (schneider et al., ) . microsomal and hepatocyte clearance data sets have also been used separately to generate catalyst pharmacophores, which were then tested by predicting the opposing data set. this method assumes there are some pharmacophore features intrinsic to the molecules that dictate intrinsic clearance (ekins and obach, ) . a second complex property is the volume of distribution that is a function of the extent of drug partitioning into tissue versus plasma and there have been several attempts at modelling this property (lombardo et al., (lombardo et al., , . this property, along with the plasma half-life, determines the appropriate dose of a drug. for example, diverse drugs from the literature were used with eight molecular descriptors with sammon and kohonen mapping methods. these models appeared to classify correctly % of the compounds (balakin et al., ) . recently, a set of drugs with literature volume of distribution at steady-state data was used with a mixture discriminant analysis-random forest method and molecular descriptors to generate a predictive model. this model was tested with molecules, resulting in a geometric mean fold error of . , which was comparable to the values for other predictions for this property from animal, in vitro, or other methods (lombardo et al., ) . a third property, the plasma half-life determined by numerous adme properties has also been modelled with sammon and kohonen maps using data for drugs from the literature and four molecular descriptors. like the previously described volume of distribution models, these models appeared to classify correctly % of the compounds (balakin et al., ) . a fourth complex property is renal clearance, which assumes the excretion of the unchanged drug that takes place only by this route, hence this represents a method of monitoring the proportion of drug metabolized. in one set of published qsar models, molecules were used with volsurf or molconn-z descriptors. the models were tested with molecules and one using soft independent modelling of class analogies and molconn-z descriptors obtained % correct classification between the two classes ( - and - %) (doddareddy et al., ) . a fifth example of a complex property is the proteinligand interaction and appropriate scoring functions for which several methods have been developed such as force fields, empirical and knowledge-based approaches (see also ekins et al., ) . these are important in computational structure-based design methods for assessing virtual candidate molecules to select those that are likely to bind a protein with highest affinity (shimada, ) . recently, a kernel partial least squares (k-plss) qsar approach has been used along with a genetic algorithm feature selection method for the distance-dependent atom pair descriptors from the or small molecule training sets with binding affinity data and the proteins they bind to. bootstrapping, scrambling the data and external test sets were used to test the models (deng et al., ) . in essence, such k-pls qsar models across many proteins perhaps isolate the key molecular descriptors that relate to the highest affinity interactions. it will be interesting to see whether such models can continue to be generated with the much larger binding affinity data sets that are now available. a final example of a complex property is the v max of an enzyme that has been modelled on a few occasions (hirashima et al., ; mager et al., ; ghafourian and rashidi, ; sipila and taskinen, ) . this value will depend on the properties of the compound in question and will be influenced by the steric properties of the active site as well as the ease of expulsion of the leaving group from the active site. balakin et al. ( ) , have recently used neural network methods to model the v max data for n-dealkylation mediated by cyp d and cyp a , using whole molecules, centroid of the reaction and leaving group-related descriptors. these models were also used to predict small sets of molecules not included in training. ultimately, many other reactions and the evaluation of other enzymes will be necessary. similarly, larger test sets are required for all the above complex property models to provide further confidence in the models in terms of their utility and applicability. uses of in silico pharmacology we propose a general schema for in silico pharmacology, which is shown in figure . this demonstrates some of the key roles of the computational technologies that can assist pharmacology. these roles include finding new antagonists or agonists for a target using an array of methods either in the absence or presence of a structure for the target. computational methods may also aid in understanding the underlying biology using network/pathways based on annotated data (signalling cascades), determining the connectivity of drug as a network with targets to understand selectivity, integration with other models for pk/pd (pharmacodynamic) and ultimately the emergence of systems in silico pharmacology. obviously, we have taken more of a pharmaceutical bias in this review but we would argue these methods are equally amenable and should be considered to discover new chemical probes for the academic pharmacologist as opposed to lead molecules for optimization to become drugs. some of the advantages of in silico pharmacology and in silico methods in general are the reduction in the number of molecules made and tested through database searching to find inhibitors or substrates, increased speed of experiments through reliable prediction of most pharmaceutical properties from molecule structure alone and ultimately reductions in animal and reagent use. we must however consider the multiple optimization of numerous predicted properties, possibly either weighting in silico pharmacology models by importance (or confidence in the model and or data), as well as data set size and diversity. similarly, we should consider the disadvantages of in silico pharmacology methods as protein flexibility, molecule conformation and promiscuity all hinder accurate predictions. for example, even with the recent availability of crystal structures for several mammalian drug-metabolizing enzymes, there is still considerable difficulty in reliable metabolism predictions. our focus thus far has been on the creation of many in silico pharmacology models for human properties, yet as pharmacology uses animals for much in vivo testing and subcellular preparations from several species for in vitro experiments, we need models from other species both to understand differences as well as enable better scaling between them. a widely discussed disadvantage of in silico methods is the applicability of the model, which will now be discussed further. defining in silico model applicability domain some of the in silico pharmacology methods that can be used have similar limitations to models used in other areas, such as those for predicting physicochemical and adme/tox properties. for example, models may be generated with a narrow homologous series of pharmacologically relevant molecules (local model) or a structurally diverse range of molecules (global model). these two approaches have their pros and cons, respectively. the applicability domain of the local model may be much narrower than for the global model such that changing to a new chemical series will result in prediction failure. however, global models may also fail if the predicted molecule falls far enough away from representative molecules in the training set. these limitations are particularly specific to qsar models. from many of the in silico pharmacology model examples described above, the qsar models are generally local in nature and this will limit lead hopping to new structural series, whereas global models may be more useful for this feature. several papers have described the applicability domain of models and methods in considerable detail (dimitrov et al., ; tetko et al., ) to calculate this property. molecular similarity to training set compounds may be a reliable measure for prediction quality (sheridan et al., ) as demonstrated for a herg model . to our knowledge, there has not been a specific analysis of the applicability domain specifically for in silico pharmacology models (other than for those examples described above) to the same degree as there has been for physicochemical properties like solubility and logp. the applicability domain of pharmacophore models have not been addressed either as the focus has primarily been on statistical qsar methods. as we shift toward hybrid or meta-computational methods (that integrate several modelling approaches and algorithms) for predicting from molecular structure the possible physicochemical and pharmacological properties, then these could be used to provide prediction confidence by consensus. the docking methods with homology models for certain proteins of pharmacological interest could be used alongside qsar or pharmacophore models if these are also available. there have been numerous occasions in the study of drug-metabolizing enzymes were qsar and homology models have been combined or used to validate each other (de groot et al., a; de graaf et al., ; de groot, ) . drug metabolism is a good example as several simultaneous outcomes (for example, metabolites) often occur, a condition not normally found in other pharmacological assays where a single set of conditions yields a single outcome. it is here that the classification into specific ('local') and comprehensive ('global') methods finds its clearest use (see figure ) , with local methods being applicable to simple biological systems such as a single enzyme or a single enzymatic activity (testa and krämer, ) . the production of regioselective metabolites (for example, hydroxylation to a phenol and an alcohol) is usually predictable from such methods, but that of different routes (for example, oxidation versus glucuronidation) is not. this is where global algorithms (that is, applicable to versatile biological systems) are most useful in their potential capacity to encompass all or most metabolic reactions and offer predictions, which are much closer to the in vivo situation. it is readily apparent that in a minority of papers we have found that computational approaches have resulted in predicted lead compounds for testing without the authors providing further experimental verification of biological activity (krovat and langer, ; langer et al., ; steindl and langer, ; gopalakrishnan et al., ; lorenzen et al., ; steindl et al., a; amin and welsh, ) . this is an interesting observation as for many years computational studies were generally performed after synthesis of molecules, and essentially provided illustrative pictures and explanation of the data. now it appears we are seeing a shift in the other direction as predictions are published for pharmacological activity without apparently requiring in vitro or in vivo experimental verification, as long as the models themselves are validated in some manner. as the models may only have a limited prediction domain so perhaps in future we will see some discussion of the predicted molecules and their distance from the training set or some other measure of how far the predictions can be extended. many of the molecules identified by virtual screening techniques have not been tested in vitro to ensure that they are not false positives that may actually be involved in molecule aggregation. these types of molecules have been termed so-called 'promiscuous inhibitors', occurring as micromolar inhibitors of several proteins mcgovern and shoichet, ; seidler et al., ) . a preliminary computational model was developed to help identify these potential promiscuous inhibitors (seidler et al., ) . from reviewing the literature, we suggest it would be worth researchers either implementing filters for 'promiscuous inhibitors' or performing rigorous experimental verification of their predicted bioactive molecules to rule out this possibility. . it would certainly be very useful to know the existence of difficult targets for modelling with different methods, as this apparently is a process of trial and error for each investigator currently. in summary, in this and the accompanying review (ekins et al., ) , we have presented our interpretation of in silico pharmacology and described how the field has developed so far and is used for: discovery of molecules that bind to many different targets and display bioactivity, prediction of complex properties and the understanding of the underlying metabolic and network interactions. while we have not explicitly discussed pk/pd, whole organ, cell or disease simulations in this review, we recognize they too are an important component of the computer-aided drug design approach (noble and colatsky, ; gomeni et al., ; kansal, ) and may be more widely integrated with other in silico pharmacology methods described previously . the brief history of in silico pharmacology has taken perhaps a rather predictable route with computational models applied to many of the most important biological targets where they have the capacity to be used to search large databases and quickly suggest molecules for testing. many of the examples we have presented have demonstrated significant enrichments over random selection of molecules and so far these have been the most plentiful types of metrics that are routinely used to validate in silico models. the future of in silico pharmacology may be somewhat difficult to predict. while we are seeing a closer interaction between computational and in vitro approaches to date, will we see a similar relationship with in vivo studies in the future? more broadly, will in silico pharmacology ever be able to replace entirely experimental approaches in vitro and even in vivo, as some animal rights activists want us to believe? the answer here can only be a clear and resounding 'no' (at least in the near future), for two irrefutable reasons. first, biological entities are nonlinear systems showing 'chaotic behaviour'. as such, there is no relation between the magnitude of the input and the magnitude of the output, with even the most minuscule differences between initial conditions rapidly translating into major differences in the output. and second, no computer programme, however 'complex and systemslike', will ever be able to fully model the complexity of biological systems. indeed, and in the formulation of the mathematician gregory chaitin, biological systems are algorithmically incompressible, meaning that they cannot be modelled fully by an algorithm shorter than themselves. in the meantime, in silico pharmacology will likely become more complex requiring some degree of integration of models, as we are seeing in the combined metabolism modelling approaches (figure ) . ultimately, to have a much broader impact, the in silico tools will need to become a part of every pharmacologist's tool kit and this will require training in modelling and informatics, alongside the in vivo, in vitro and molecular skills. this should provide a realistic appreciation of what the different in silico methods can and cannot be expected to do with regard to the pharmacologists aim of discovering new therapeutics. in silico pharmacology for drug discovery s ekins et al a preliminary in silico lead series of -phthalimidinoglutaric acid analogues designed as mmp- inhibitors applications of qsar methods to ion channels. in: ekins s (ed) computational toxicology: risk assessment for pharmaceutical and environmental chemicals quantitative structure-metabolism relationship modeling of the metabolic n-dealkylation rates comprehensive computational assessment of adme properties using mapping techniques crystal structures of native and inhibited forms of human cathepsin d: implications for lysosomal targeting and drug design pharmacophore modeling as an efficient tool in the discovery of novel noncompetitive ampa receptor antagonists an integrated in silico d model-driven discovery of a novel, potent, and selective amidosulfonamide -ht a agonist (prx- ) for the treatment of anxiety and depression novel cathepsin d inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus differential targeting of gbetagamma-subunit signaling with small molecules new serotonin -ht( a), -ht( b), and -ht( c) receptor antagonists: synthesis, pharmacology, d-qsar, and molecular modeling of (aminoalkyl)benzo and heterocycloalkanones designing non-peptide peptidomimetics in the st century: inhibitors targeting conformational ensembles developing a dynamic pharmacophore model for hiv- integrase rapid identification of p-glycoprotein substrates and inhibitors pharmacophorebased discovery of ligands for drug transporters computational approaches to modeling drug transporters structural insights into human -lipoxygenase inhibition: combined ligand-based and target-based approach inhibition of human liver catechol-o-methyltransferase by tea catechins and their metabolites: structure-activity relationship and molecular-modeling studies x-ray crystal structure of human dopamine sulfotransferase, sult a cytochrome p in silico: an integrative modeling approach designing better drugs: predicting cytochrome p metabolism development of a combined protein and pharmacophore model for cytochrome p c pharmacophore modeling of cytochromes p generation of predictive pharmacophore models for ccr antagonists: study with piperidine-and piperazine-based compounds as a new class of hiv- entry inhibitors predicting proteinligand binding affinities using novel geometrical descriptors and machine-learning methods simple but highly effective three-dimensional chemical-feature-based pharmacophore model for diketo acid derivatives as hepatitis c virus rna-dependent rna polymerase inhibitors a stepwise approach for defining the applicability domain of sar and qsar models in silico renal clearance model using classical volsurf approach molecular docking and highthroughput screening for novel inhibitors of protein tyrosine phosphatase- b insights for human ether-a-go-go-related gene potassium channel inhibition using recursive partitioning, kohonen and sammon mapping techniques applying computational and in vitro approaches to lead selection three and four dimensional-quantitative structure activity relationship analyses of cyp a inhibitors three dimensional quantitative structure activity relationship ( d-qsar) analysis of cyp a substrates in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling techniques: application of systems biology to absorption, distribution, metabolism, excretion, and toxicity three dimensional-quantitative structure activity relationship computational approaches of prediction of human in vitro intrinsic clearance development of computational models for enzymes, transporters, channels and receptors relevant to adme/tox structural basis for ligand promiscuity in cytochrome p a the design of drug candidate molecules as selective inhibitors of therapeutically relevant protein kinases is there a future for renin inhibitors possible role of valvular serotonin -ht( b) receptors in the cardiopathy associated with fenfluramine analysis of drug-induced effect patterns to link structure and side effects of medicines biological spectra analysis: linking biological activity profiles to molecular structure biospectra analysis: model proteome characterizations for linking molecular structure and biological response identification of nonpeptidic urotensin ii receptor antagonists by virtual screening based on a pharmacophore model derived from structure-activity relationships and nuclear magnetic resonance studies on urotensin ii recent success stories leading to commercializable bioactive compounds with the aid of traditional qsar procedures novel curcumin-and emodin-related compounds identified by in silico d/ d conformer screening induce apoptosis in tumor cells structure-based design of potent cdk inhibitors derived from olomoucine structure of a human carcinogenconverting enzyme, sult a introducing the consensus modeling concept in genetic algorithms: application to interpretable discriminant analysis quantitative study of the structural requirements of phthalazine/quinazoline derivatives for interaction with human liver aldehyde oxidase computer-aided design and synthesis of -substituted tryptamines and their pharmacology at the -ht d receptor: discovery of compounds with potential anti-migraine properties computerassisted drug development (cadd): an emerging technology for designing first-time-in-man and proof-of-concept studies from preclinical experiments a virtual screening approach for thymidine monophosphate kinase inhibitors as antitubercular agents based on docking and pharmacophore models combining structurebased drug design and pharmacophores piperidine-renin inhibitors compounds with improved physicochemical properties pharmacophore modeling and three dimensional database searching for drug design using catalyst: recent advances identification of novel extracellular signal-regulated kinase docking domain inhibitors structure-activity correlations in the metabolism of drugs comparison of fingerprint-based methods for virtual screening using multiple bioactive reference structures multiple-ligand-based virtual screening: methods and applications of the mtree approach graphics computer-aided receptor mapping as a predictive tool for drug design: development of potent, selective, and stereospecific ligands for the -ht a receptor quantitative structure-activity studies of octopaminergic -(arylimino)thiazolidines and oxazolidines against the nervous system of periplaneta americana l differences in the central nervous system distribution and pharmacology of the mouse -hydroxytryptamine- receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p lck sh domain via in silico screening against the py þ binding site molecular dynamics and free energy analyses of cathepsin d-inhibitor interactions: insight into structure-based ligand design in silico search of putative adverse drug reaction related proteins as a potential tool for facilitating drug adverse effect prediction identification of novel farnesyl protein transferase inhibitors using three-dimensional searching methods interactions of a series of fluoroquinolone antibacterial drugs with the human cardiac k þ channel herg modeling approaches to type diabetes predicting ligand binding to proteins by affinity fingerprinting the diversity challenge in combinatorial chemistry protein affinity map of chemical space elucidation of the na þ , k þ -atpase digitalis binding site novel human lipoxygenase inhibitors discovered using virtual screening with homology models qsar studies on , -dithiole- -thiones: modeling of lipophilicity, quinone reductase specific activity, and production of growth hormone structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin d acyl ureas as human liver glycogen phosphorylase inhibitors for the treatment of type diabetes development of novel edg antagonists using a d database search and their structure-activity relationships impact of scoring functions on enrichment in docking-based virtual screening: an application study on renin inhibitors computer applications in pharmaceutical research and development structure-aided optimization of kinase inhibitors derived from alsterpaullone pharmacophore modeling and threedimensional database searching for drug design using catalyst discovery of novel mesangial cell proliferation three-dimensional database searching method lead identification for modulators of multidrug resistance based on in silico screening with a pharmacophoric feature model screening for new antidepressant leads of multiple activities by support vector machines protease inhibitors: current status and future prospects on the recognition of mammalian microsomal cytochrome p substrates and their characteristics prediction of small-molecule binding to cytochrome p a : flexible docking combined with multidimensional qsar virtual screening of novel noncovalent inhibitors for sars-cov c-like proteinase oncology exploration: chartering cancer medicinal chemistry space a hybrid mixture discriminant analysis-random forest computational model for the prediction of volume of distribution of drugs in human prediction of human volume of distribution values for neutral and basic drugs. . extended data set and leave-class-out statistics prediction of volume of distribution values in humans for neutral and basic drugs using physicochemical measurements and plasma protein binding in silico screening of drug databases for tse inhibitors structure-based drug design of a novel family of ppargamma partial agonists: virtual screening, x-ray crystallography, and in vitro/in vivo biological activities judging models in qsar-and lfe-like studies if there are no replications: correlation of dipeptidyl peptidase iv hydrolytic activities of l-alanyl-l-alanine phenylamides the serotonin binding site of human and murine -ht b receptors: molecular modeling and site-directed mutagenesis qsar modeling of in vitro inhibition of cytochrome p a piperidine renin inhibitors: from leads to drugs a common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening kinase inhibitors: not just for kinases anymore from magic bullets to designed multiple ligands pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p -independent cell death cyclopentenone prostaglandins of the j series inhibit the ubiquitin isopeptidase activity of the proteasome pathway serotonin b receptor is required for heart development hiv- integrase pharmacophore: discovery of inhibitors through three-dimensional database searching design and synthesis of non-peptidic inhibitors for the syk c-terminal sh domain based on structure-based in-silico screening prediction of biological targets using probabilistic neural networks and atom-type descriptors a return to rational drug discovery: computer-based models of cells, organs and systems in drug target identification homology modeling of the serotonin -ht a receptor using automated docking of bioactive compounds with defined geometry computational tools for the analysis and visualization of multiple protein-ligand complexes renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases? application of validated qsar models of d dopaminergic antagonists for database mining global mapping of pharmacological space a genetic algorithm for structure-based de novo design d-qsar and receptor modeling of tyrosine kinase inhibitors with flexible atom receptor model (flarm) targeting drug-efflux pumps -a pharmacoinformatic approach structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin discovering cox-inhibiting constituents of morus root bark: activity-guided versus computer-aided methods acetylcholinesterase inhibitory activity of scopolin and scopoletin discovered by virtual screening of natural products screening the receptorome to discover the molecular targets for plant-derived psychoactive compounds: a novel approach for cns drug discovery evidence for possible involvement of -ht( b) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications crystal structure of human cytochrome p d rational discovery of novel nuclear hormone receptor antagonists combining in vitro and in vivo pharmacokinetic data for prediction of hepatic drug clearance in humans by artificial neural networks and multivariate statistical techniques pharmacophore modeling and in silico screening for new p (aromatase) inhibitors in silico pharmacology for drug discovery s ekins et al identification and prediction of promiscuous aggregating inhibitors among known drugs molecular determinants for the interaction of the valvulopathic anorexigen norfenfluramine with the -ht b receptor identification of novel estrogen receptor alpha antagonists from large networks to small molecules similarity to molecules in the training set is a good discriminator for prediction accuracy in qsar orphan nuclear receptors, excellent targets of drug discovery the challenges of making useful protein-ligand free energy predictions for drug discovery. in: ekins s (ed) computer applications in pharmaceutical research and development d qsar (comfa) of a series of potent and highly selective vla- antagonists identification of potent and novel alpha beta antagonists using in silico screening quantitative structure-activity relationship study on tetrahydro-beta-carboline antagonists of the serotonin b ( ht b) contractile receptor in the rat stomach fundus comfa modeling of human catechol o-methyltransferase enzyme kinetics development of biologically active compounds by combining d qsar and structure-based design methods towards integrated adme prediction: past, present and future directions for modelling metabolism by udp-glucuronosyltransferases multiple pharmacophores for the investigation of human udp-glucuronosyltransferase isoform substrate selectivity evaluation of a novel shape-based computational filter for lead evolution: application to thrombin inhibitors potential of renin inhibition in cardiovascular disease human rhinovirus c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening influenza virus neuraminidase inhibitors: generation and comparison of structure-based and common feature pharmacophore hypotheses and their application in virtual screening pharmacophore modeling, docking, and principal component analysis based clustering: combined computer-assisted approaches to identify new inhibitors of the human rhinovirus coat protein screening the receptorome: an efficient approach for drug discovery and target validation reengineering the pharmaceutical industry by crash-testing molecules structure and function of voltage-gated ion channels an in silico approach to discovering novel inhibitors of human sirtuin type hitting the hot spots of cell signaling cascades the biochemistry of drug metabolism -an introduction. part : principles and overview can we estimate the accuracy of adme-tox predictions? solution structure of cnerg (ergtoxin), a herg specific scorpion toxin discovery of nonpeptidic small-molecule ap- inhibitors: lead hopping based on a three-dimensional pharmacophore model protein-structure-based drug discovery of renin inhibitors punaglandins, chlorinated prostaglandins, function as potent michael receptors to inhibit ubiquitin isopeptidase activity substituted piperidines-highly potent renin inhibitors due to induced fit adaption of the active site kinomics-structural biology and chemogenomics of kinase inhibitors and targets modeling the cytochrome p -mediated metabolism of chlorinated volatile organic compounds the discovery of novel, structurally diverse protein kinase c agonists through computer d-database pharmacophore search. molecular modeling studies probing the conformation of the sugar transport inhibitor phlorizin by d-nmr, molecular dynamics studies, and pharmacophore analysis similarity-based approaches to virtual screening drugbank: a comprehensive resource for in silico drug discovery and exploration ligandscout: -d pharmacophores derived from protein-bound ligands and their use as virtual screening filters drug discovery in the ubiquitin regulatory pathway in silico prediction of drug binding to cyp d : identification of a new metabolite of metoclopramide structural biology and function of solute transporters: implications for identifying and designing substrates modeling of active transport systems structure-based virtual screening for plant-based erbeta-selective ligands as potential preventative therapy against age-related neurodegenerative diseases acknowledgements se gratefully acknowledges dr cheng chang and dr peter w swaan (university of maryland), dr konstantin v balakin (chemical diversity inc.) for in silico pharmacology collaborations over the past several years and dr hugo kubinyi for his insightful efforts in tabulating the successful applications of in silico approaches, which was inspirational. gold-enhelix inc. graciously provided chemtree. se kindly acknowledges dr maggie az hupcey for her support. jm acknowledges the research funding provided by the spanish ministerio de educació n y ciencia (project reference bio - ) and the instituto de salud carlos iii. owing to limited space, it was not possible to cite all in silico pharmacology-related papers, our sincere apologies to those omitted. the authors state no conflict of interest. key: cord- - rfc qb authors: karabasz, alicja; bzowska, monika; szczepanowicz, krzysztof title: biomedical applications of multifunctional polymeric nanocarriers: a review of current literature date: - - journal: int j nanomedicine doi: . /ijn.s sha: doc_id: cord_uid: rfc qb polymeric nanomaterials have become a prominent area of research in the field of drug delivery. their application in nanomedicine can improve bioavailability, pharmacokinetics, and, therefore, the effectiveness of various therapeutics or contrast agents. there are many studies for developing new polymeric nanocarriers; however, their clinical application is somewhat limited. in this review, we present new complex and multifunctional polymeric nanocarriers as promising and innovative diagnostic or therapeutic systems. their multifunctionality, resulting from the unique chemical and biological properties of the polymers used, ensures better delivery, and a controlled, sequential release of many different therapeutics to the diseased tissue. we present a brief introduction of the classical formulation techniques and describe examples of multifunctional nanocarriers, whose biological assessment has been carried out at least in vitro. most of them, however, also underwent evaluation in vivo on animal models. selected polymeric nanocarriers were grouped depending on their medical application: anti-cancer drug nanocarriers, nanomaterials delivering compounds for cancer immunotherapy or regenerative medicine, components of vaccines nanomaterials used for topical application, and lifestyle diseases, ie, diabetes. medical application of nanomaterials is becoming increasingly crucial in diagnostics (delivery of contrast agents for imaging) as well as in prophylaxis (vaccines) and therapy of various diseases (drug delivery systems). nanocarriers employed in medicine improve the bioavailability, pharmacokinetics, and effectiveness of various therapeutics or contrast agents, ensuring: increased hydrophilicity, reduced interaction with plasma and cellular proteins, and better accumulation in target tissues. the most promising purpose of exploiting nanoparticles in medicine is their potential to localize (or be targeted) in a specific manner to the site of disease and reduce or eliminate the possible side effects. targeting can be broadly classified into three regimes; passive, active, andphysical. passive is based on accumulating nanoparticulate systems containing drugs through leaky vasculature of a diseased area. it was found in tumors, infarcts, and inflammation regions. in such regions with increased vascular permeability, nanoparticulate systems can selectively accumulate and release a drug that exerts a therapeuticeffect. this phenomenon allows the delivery of drugs to the tumors, for which "enhanced permeability and retention" (epr) effect is observed. , , the epr effect is predominantly described for solid tumors where fenestrations between epithelial cells, disorders in the pericyte coverage, and the lack of a smooth muscle layer of new blood vessels are sufficient for nanomedicines accumulation. additionally, impaired lymphatic drainage in this tissue is responsible for nanocarriers retention. another approach, active targeting, is based on the specific recognition of targeted diseased region/cells' ligands. finally, physical targeting includes ph-sensitive, temperature-sensitive, redox potential sensitive, ultrasound sensitive, and magnetic sensitive systems. as a result of nanomaterials' research, over nanoformulations for oral, intravenous, subcutaneous, intrabronchial, topical, or intramuscular administration have already been approved by the american food and drug administration (fda) or the european medicines agency (ema) for human use. , the most clinically approved nanomedicines belong to liposomes and polymeric nanoparticles; however, the number of nanomaterials accepted by the fda for medical applications is still low. an example of a polymeric nanomedicine is apealea/paclical containing micellar paclitaxel (paclitaxel formulated with the surfactant xr ) used to treat epithelial ovarian cancer, primary peritoneal cancer, and fallopian tube cancer. the new polymer nanomaterials' safety and therapeutic efficacy recently verified several clinical studies that mostly concerned nanocarriers of anti-cancer drugs. phase three of clinical trials have already been completed for yet another paclitaxel formulation -nk , a "core-shell-type" polymeric micellar nanoparticle formulation used in patients with metastatic or recurrent breast cancer. still, other studies concerned nanomedicines such as cyclodextrin-based polymer-camptothecin crlx or conjugated docetaxel crlx- . soon, the first phase of clinical trials will begin on polymeric nanoparticles loaded with monoclonal antibody cetuximab and decorated with somatostatin analog for the targeting of colon cancer (data from clinicaltrials.gov). in connection with the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic, at least several different nano-based-tools have been developed that could be useful in the treatment of patients with the coronavirus disease- . , clinical trials have recently started for a full-length recombinant sars-cov- glycoprotein nanoparticle vaccine adjuvanted with the saponin-based matrix m (nvx-cov , developed by novavax, nct ) or inhaled nanoparticle formulation of remdesivir (developed by neuroactiva, nct , data from clinicaltrials.gov). nanocarriers were initially developed to improve the pharmacokinetics of drugs, but they unexpectedly received much attention from researchers and have become the center of this promising trend of multifunctional nanomedicine. unlike molecular drugs or imaging agents, nanoparticles can easily integrate more than one kind of contrast or therapeutic agent, making them potential multifunctional nanoplatforms. the concept of multifunctional nanocarriers also refers to the precise delivery of more than one drug simultaneously, which increases the effectiveness of therapy or overcome resistance to a given compound. delivery of more than one agent also allows the use of various therapeutic methods (for example, chemotherapy and immunotherapy, chemotherapy and photodynamic therapy, induction of the immune response to the selected antigen, and enhancement of this response). there are constant enormous demands on new, advanced, multifunctional nanomaterials to be used in the future of medicine. first of all, they should be biodegradable and biocompatible. they are also supposed to be very stable in the body, have optimal pharmacokinetics and biodistribution, ensure the protection of healthy tissues, and accumulate drugs in diseased tissue at the right time of the disease process. additionally, they should deliver chemically diverse compounds for including small drugs (antibiotics, cytostatics), nucleic acids, peptides, proteins, and magnetic nanoparticles. appropriate synthesis methods should allow the biological activity of transported drugs to be preserved. it is especially important when providing active therapeutic proteins (enzymes, cytokines) or antigens that are the essential components of vaccines. the purpose of this work is to present the latest scientific reports on polymeric nanocarriers for medical applications. we selected papers that were published in - . we describe nanomaterials whose biological assessment has been carried out at least in vitro. most of them have also been tested in vivo. a literature search (pubmed) revealed that polymeric nanocarriers are most often used as drug delivery systems. since effective cancer treatment still seems to be the most significant medical challenge, most of the new nanomaterials that have recently been obtained and characterized are primarily anti-cancer drug carriers ( figure ). in this work, we also present less numerous polymer nanomaterials to treat other diseases such as diabetes, atherosclerosis, or diseases based on chronic inflammation. we focused primarily on these polymeric nanocarriers, which in our opinion, can be described as multifunctional, with high capacity as innovative diagnostic and therapeutic systems for the clinical field in the treatment of various diseases, and with the considerable potential for improving human health. nanocarriers are generally considered as particulate structures with a diameter smaller than nm. depends on the morphology, nanoparticles, nanospheres, and nanocapsules can be defined as presented in figure . nanoparticles/nanospheres are generally homogenous matrix systems in which the drug is dispersed in the material, forming them or can be adsorbed in their pores or at their surface, or the drug can be conjugated to them. on the other hand, nanocapsules' structure can be divided into two parts, the core and the shell. the core material can be solid, liquid, or gas, depending on the type of application, while the shell is usually formed from the polymeric material. generally, the drug is located in the core part, while the shell protects them from the external environment. however, incorporation in shell material and adsorption at their surface is also possible, allowing simultaneous delivery of one or several drugs in different nanocapsule compartments. the shell may be made permeable, semi-permeable, or impermeable, depending on the application. permeable or semi-permeable shells are used for controlled release applications. the release rate from capsules is governed mainly by the shell's thickness, its pore's size, and the core's material. there are three main classical polymeric nanocarriers preparations: layer-by-layer method, nanoprecipitation method, and emulsion templated method. emulsion templated methods mainly include emulsification-diffusion, emulsification-evaporation, emulsification-coacervation, multiple emulsification, and emulsion/polymer coating. [ ] [ ] [ ] [ ] [ ] nevertheless, other methods have been used, such as methodologies for preparing polymer-coated liposomes, polymersomes, dendrimers, or self-assembled polymeric micelles inorganic the main biomedical application of polymeric nanocapsules/nanocarriers. notes: pubmed resources searched in all fields using the term "polymeric nanocapsules" filtered on "in the last five years" ( - ). seven hundred seventy results were obtained (on march , ), and the authors selected publications in which nanocapsules were used in the biomedical field (excluding diagnostic application and review papers). (a) polymer nanocapsules were divided into three categories depending on their application in medicine. (b) since the vast majority were nanocapsules used as a drug delivery system ( from publications), the authors present the types of transported drugs, w/o drugs stand for nanocapsules which were obtained empty and have potential as drug transporter. nanoparticles modified with polymeric materials as well as protein/peptide self-assembled nanocarriers. - sketches of selected polymeric nanocarriers are presented in figure . on the other hand, the mentioned methods of preparation of polymeric nanocarriers could be divided mainly by two approaches: (i) dispersion of the preformed polymers; and (ii) polymerization of monomers. the commonly used polymers are: arabian gum, gelatin, ethylcellulose, hydroxypropylmethylcellulose phthalate, poly-e-caprolactone (pcl), poly(lactide) (pla), poly(lactide-co-glycolide) (plga), poly(alkyl cyanoacrylate) (paca), while the polyelectrolytes are: polyethyleneimine (pei), polyacrylic acid (paa,), poly(diallyldimethylammonium, chloride) (pdadmac), poly(sodium -styrene sulfonate) (pss), and poly(allylamine hydrochloride) (pah), dextran sulfate (ds), poly(glutamic acid) (pga), alginates and alginic acid (alg), hyaluronic acid (hyl), chitosan (chit), poly(lysine) (pll). synthetic polymers have higher purity and better reproducibility than natural ones, while natural ones possess better biocompatibility. , , another class of polymeric nanoparticles is based on proteins or peptides. they are biological macromolecules composed of amino acids, with proved biocompatibility and bioavailability. protein/peptide nanoparticles are widely used to enhance the bioavailability of drugs. surface modification of nanocarriers for the preparation of multifunctional drug delivery systems eg for passive, active, and physical targeting, are performed. , , polymeric nanocarriers have been increasing in interest and applied in the pharmaceutical field, especially as drug delivery systems ( figure ). since several specialized reviews have already discussed nanotechnology and polymeric materials for the formulation of nanocarriers, [ ] [ ] [ ] , in this review, we present a brief introduction of the classical formulation techniques. the sequential adsorption of oppositely charged nanoobjects (layer-by-layer (lbl) method) is a powerful approach for fabricating multilayer thin films. , the origin of multilayered adsorption can be traced back to the mid- s when iler proposed deposition of microparticles onto solid substrates in the lbl manner. in the s, decher and hong expended the lbl by using a combination of linear polycations and polyanions. the layer-by-layer adsorption of charged nanoobjects like polyelectrolytes, nanoparticles, proteins, organic molecules, etc., is considered as a convenient method to obtain multilayer "nanocarriers" shells on colloidal cores (figure ) , and those types of capsules have been the subject of intensive research since when sukhoukovr proposed the method of forming polyelectrolyte multilayers shell on solid microparticles. furthermore, the layer-by-layer method was applied for liquid and gas cores. , the layer-by-layer (lbl) method's main advantage are the ease of manipulation and the multifunctionality that comes from the possibility of modifying the polyelectrolyte shell by organic molecules, polymers, inorganic nanoparticles, carbon nanotubes, antibodies, lipids, or nanoparticles. [ ] [ ] [ ] that multifunctionality is of fundamental importance for the preparation of multifunctional drug delivery systems. for passive targeting, the polyelectrolyte shell is modified by the adsorption of so-called "stealth" polymers like poly(ethylene glycol) (peg), poly-(acrylamide), poly(vinylpyrrolidone), polysaccharides, or dextrans. [ ] [ ] [ ] that modification prolongs circulation time and allows nanocarriers' accumulation in the targeted area through an epr effect. , , for active targeting, polyelectrolyte shells can be functionalized by adsorption/incorporation of targeting ligands like antibodies or folic acids present on the surface of the targeted cells; for physical targeting, by the incorporation of stimuli-responsive building blocks in multilayered shell eg ph-responsive polymers, magnetic nanoparticles, etc. for example, the incorporation of magnetic nanoparticles that allows controlling magnetically responsive nanocarriers using an external magnetic field could be indicated. [ ] [ ] [ ] that incorporation allows the application of such nanocarriers as a tool for cell-biology research to separate and purify cell populations, tissue repair, magnetic drug delivery, magnetic resonance imaging (mri), magnetic hyperthermia, etc. [ ] [ ] [ ] [ ] [ ] [ ] excellent books and comprehensive reviews of lbl nanocarriers have been written. [ ] [ ] [ ] [ ] the nanoprecipitation method the nanoprecipitation method is also known as solventdisplacement, or interfacial deposition method, or solvent shifting method. according to fessi et al, the nanocarrier synthesis needs both solvent and non-solvent phases (usually, the solvent and non-solvent phases are called organic and aqueous phases, respectively). a polymer and active substance solution in a miscible organic solvent or a mixture of solvents (ie ethanol, acetone, hexane, methylene chloride, or dioxane) is added to a nonsolvent or mixture of nonsolvents supplemented with one or more surfactants. it can be performed either by dialysis or by the dropping technique ( figure ). after adding the organic phase to the water phase, the polymer diffuses with the organic solvent and precipitates at the interface between oil and water, forming nanocarriers. the principle of nanocarriers formation by nanoprecipitation is a fluctuation of polymer concentration at the interface between solvent and precipitant during the solvent to nonsolvent exchange. the initial supersaturation at the interface layer occurs, followed by the interfacial deposition of the polymer. the nanocarriers' characteristics can be mainly influenced by polymer concentration, injection method of the organic phase (dialysis or dropping), the volume ratios components, and the nature of materials. the interfacial deposition method has been used intensively in the last decade, due to the simple operation without applied external high energy force and extensive applicability for various payloads. the studies of polymeric nanocarriers formulation by the nanoprecipitation method are excellently summarized in the following review. [ ] [ ] [ ] [ ] the nanoemulsion templated methods nanoemulsions are thermodynamically unstable but kinetically stabilized systems. , for nanoemulsion preparation, the organic or aqueous phase is emulsified in the aqueous or organic phase in the presence of a surfactant (stabilizing agents). during the nanoemulsion formation, surfactants are self-assembled at the interface between two immiscible phases to reduce the interfacial tension, thereby achieving a stable state. the various methods of formation of nanoemulsion including a high energy ones (eg, ultrasonication, high mechanical shear forces) and low energy ones (eg, phase inversion composition, phase inversion temperature, membrane emulsification, selfemulsification) have been developed. , the size of the droplets of nanoemulsion is in the range of about - nm with a low polydispersity index. nanodroplets found application as a template for polymeric nanocarrier synthesis both from preformed polymers and monomers in a polymerization process. the second approach is generally restricted to synthetic polymer materials, in which case one of the phases usually contains monomers, which in the polymerization process form polymeric nanocarriers. here four main methods ( figure ): emulsification-diffusion, emulsification-evaporation, emulsification-coacervation, multiple emulsification will be described; nevertheless, other nanoemulsions templating methods have been used such as nanoemulsion/polymer coating or nanoemulsion polymerization. , [ ] [ ] [ ] in this method, the polymer and active substance containing the oil phase are emulsified into an aqueous solution in the presence of surfactants to make oil in water nanoemulsion. after the formation of nanoemulsion, the organic solvent is evaporated either by increasing the temperature/under pressure or by continuous stirring results in polymeric nanocarriers' formation. nanocarriers are formed by polymer precipitation during an evaporation procedure. the influence of process parameters on the properties of prepared polymeric nanocarriers has been discussed. , , , , in this method, the oil phase contains polymers and active substances dissolved in the organic solvent (eg dichloromethane or chloroform) and another solvent that is miscible with water (eg acetone, ethanol, or ethyl acetate), thereby removing them by diffusion into water. , , , , such an oil phase is emulsified into the aqueous solution in the presence of surfactants to make oil in water nanoemulsion; subsequently, due to the spontaneous diffusion of water-miscible solvent, nanocarriers are formed by a combination of polymer precipitation and interfacial phenomena. in the emulsification-coacervation method, polymeric nanocarriers are formed and stabilized by physical coacervation or chemical crosslinking at the interface. this method is principally used for materials (monomers or polymers) possessing crosslinking function groups. physical coacervation (eg based on electrostatic interaction), as well as chemical ones (eg by chemical crosslinking or interfacial polymerization), have been proposed. , , , , multiple emulsions are heterodisperse systems called "emulsions of emulsions" formed when oil droplets in a continuous water phase contain dispersed water droplets (w/o/w). a hydrophilic active substance is dissolved in water, that is dispersed into the oil phase containing a dissolved polymer in the presence of a stabilizer. this primary/internal emulsion containing hydrophilic active substance is emulsified in an outer water phase again in the presence of a stabilizer. the procedure for obtaining the polymeric nanocarriers is similar to the single nanoemulsion technique for solvent removal, ie, diffusion, evaporation, coacervation, or a combination of these approaches. a reverse system w/o/w may also be formed ( figure ) . , chemotherapy remains one of the most important treatments for all cancer types, but its efficacy is lower than expected. the right approach to improve it seems to be developing better formulations, including nanoformulations of already approved drugs. however, the clinic's nanoformulations are less toxic than conventional therapy; their therapeutic efficiency remains unchanged. therefore, even better nanoformulations of anti-cancer drugs are continually being sought. various polymeric nanocarriers have recently been successfully obtained and characterized in vivo and seem promising as the oral or intravenous transporters of the following chemotherapeutic agents: gemcitabine, diphenyl diselenide, etoposide, thiazolidinediones, imiquimod, methotrexate, or tamoxifen (table ) . very complex nanocarriers have been developed to deliver some drugs, eg, doxorubicin, to simultaneously increase drug delivery and precisely control drug release in the tumor. the free doxorubicin may cause cardiotoxicity in some patients leading to the discontinuation of therapy. wang et al proposed the use of polymer nanocarriers made of reduction-responsive arginine-tethered amphiphilic copolymers and microwave absorbing ammonium bicarbonate for doxorubicin encapsulation. these nanocarriers can easily penetrate the tumor and are efficiently taken up by cancer cells. after irradiation by the pulsed microwave in the cell, in nanocarriers, ammonium bicarbonate converts the adsorbed energy to kinetic energy, which may generate thermocavitation and production of gas bubbles in cells. consequently, intracellular structures are destroyed, and the doxorubicin releases from nanocarriers immediately target tumor cells. still, other polymeric nanocarriers have been designed to release doxorubicin under the influence of reactive oxygen species (ros). because the oxidative environment is characteristic of tumors or tissues affected by chronic inflammation, the release of drugs can be most active there. it has been demonstrated for nanocarriers made of a biocompatible copolymer, namely phenylboronic acid pinacol ester-functionalized poly (ethylene glycol)-blockpoly (phthalic anhydride-alter-glycidyl propargyl ether). due to the phenylboronic acid ester's presence, this nanocarrier was sensitive to oxidative stress and demonstrated an improved anti-cancer effect in a breast cancer mouse model. another exciting approach is multidrug therapy using optimally selected agents, simultaneously or sequentially administered, to achieve a synergistic therapeutic effect. attempts are being made to develop various nanomaterials, including polymeric nanocarriers, that would allow more than one drug to be delivered efficiently. zhou et al proposed an attractive approach for sequential co-delivery of gefitinib (a small-molecule inhibitor of the epidermal growth factor receptor -egfr) and doxorubicin. they obtained nanocarriers based on drug-polymer conjugation and ion pairing. gefitinib was complexed with dioleoyl phosphatidic acid (dopa) and encapsulated by coprecipitation with doxorubicin-conjugated poly (l-lactide)-block-polyethylene glycol (dox-pla -b-peg). the biological activity of these nanocarriers was compared in vitro with doxil (pegylated liposome with doxorubicin -fda approved doxorubicin nanoformulation), demonstrating a better anti-cancer effect against a (human non-small cell lung cancer) and mda-mb - (human mammary adenocarcinoma, triple-negative breast cancer cells). moreover, the analysis of pharmacokinetic and biodistribution in the mouse model confirmed the high nanocapsule accumulation inside the breast tumors. however, the authors have not yet conducted studies on the developed nanocarriers' anti-tumor activity in vivo. there are many examples of polymeric nanocarriers equipped with more than one chemotherapeutic. nanoparticles with popular drugs, eg, doxorubicin, paclitaxel, curcumin, -fluorouracil, were proposed. in all cases, more than one anti-cancer agent's encapsulation allows a better therapeutic effect than one drug (summarized in table ) . a more complex multifunctional nanocarrier system, namely rgd-ps-peg calcium phosphate nanoparticles, was obtained and characterized previously by the team of dong et al. they used the polymer rgd-ps-peg (rgd corresponds to peptide h-(d-val) -arg-gly-asp-glu-oh; ps-peg is organic phosphatidylserine-polyethylene glycol) self-assembling into micelles to encapsulate the anti-cancer drug novantrone (mitoxantrone hydrochloride). moreover, calcium phosphate that adsorbs verapamil was used to form an inorganic shell on the organic core (rgd-ps-peg with novantrol) to stabilize these nanoparticles. because verapamil is a p-gp inhibitor, these nanocarriers co-deliver a chemotherapeutic and anti-multidrug resistance agent. it is worth emphasizing that, in this case, the carrier was additionally actively targeted by the covalent coupling of ps-peg with the rgd peptide. rgd is a cell adhesion motif displayed on extracellular matrix and is a ligand, for example, for αvβ or α β integrins. the expression of these integrins is high on the tumor endothelium cells. therefore rgdfunctionalized nanocarriers may increase drug delivery to the tumor. in vivo studies performed after intravenous administration of rgd-ps-peg calcium phosphate nanoparticles to animal bearing mcf /mdr tumors (multidrug-resistant human breast cancer cells mc ) indeed revealed the increased concentration of encapsulated drugs in tumors, improved therapeutic effect, and submit your manuscript | www.dovepress.com international journal of nanomedicine : showed the beneficial effect of functionalization on drug accumulation, resulting in better therapeutic efficacy and lower toxicity to healthy tissues. , there are also attempts at dual encapsulation of chemotherapy and inorganic nanoparticles. jeon et al obtained paclitaxel loaded nanocarriers coated with crosslinked gold nanoparticles and poly(amidoamine) dendrimers. the therapeutic efficacy of this combination was confirmed in a mouse tumor model. besides, superparamagnetic iron oxide nanoparticles (spions) can be used for magnetic targeting. plga-peg nanocarriers with spion and docetaxel showed faster and stronger accumulation, followed by better tumor growth inhibition after local exposure to the magnetic field. because encapsulated inorganic compounds are predominantly applied as contrast agents, plga-peg nanocarriers were also used as a magnetically-targeted triple-modal imaging system. this time spions and indocyanine green (fluorescence near-infrared dye) were encapsulated in the hydrophobic core, and diethylene triamine penta acetic acid was conjugated to plga-peg in order to indium- (radioisotope) chelation. this approach was tested on a mice model after intravenous injection and allowed for successful tumor visualization via fluorescence, mri, and nuclear imaging. furthermore, the accumulation of nanocarriers, hence the signal intensity, was more significant after adding an external magnetic field around the tumor. other examples of mri contrast agents are semicrystalline polyurea nanocarriers with gadobutrol (commercial contrast agent of gadolinium(iii) complex), which poses enhanced tissue contrast compared to the free agent. also, pegylated nanocarriers with cubic cobalt ferrite nanocubes were detectable via mri in the tumor tissue after its intratumoral injection. an exciting example is actively targeted perfluoropropane filled covered with gold nanoshelled plga nanocarriers functionalized with anti-vegfr (vascular endothelial growth factor receptor type overexpressed on tumor surrounding vascular endothelial cells) and anti-p antibodies for precise ultrasound molecular imaging of breast cancer. the authors have shown that dual-targeted nanocarriers accumulate in the tumor (orthotopic mice breast cancer) about two times more than nontargeted, ensuring accurate breast cancer detection. interestingly, regardless of double targeting, the amount of the injected dosage detected in the tumor was %, which is still lower than expected. the easily modifiable thickness of the polymeric shell allows, for example, to obtain thin-shelled nanocarriers to deliver perfluorooctyl bromide, which finds application in ultrasonography as a contrasting agent. the liquid core of nanocarriers and the appropriately thin shell made of plga-peg ensure very good echogenicity and acoustic response. besides, nanocarriers' surface functionalization with a monoclonal therapeutic antibody specific for egfr (cetuximab) improves the accumulation of capsules within tumors that overexpress this receptor. anti-cancer theranostics uses advanced nanomaterials to simultaneously and accurately detect disease processes in the patient's body and deliver drugs directly to areas affected by the disease. an interesting example is amphiphilic nanocarriers synthesized using block copolymer, succinic anhydride functionalized poly ( -diisopropylaminoethyl methacrylate) -block poly ( -aminoethyl methacrylate hydrochloride) with hydrophobic ruthenium entrapped in the core. the acidic tumor environment allows this compound's active release, a known inhibitor of dna replication with fluorescent properties. exciting examples of anti-cancer theranostics are multifunctional polymeric plga nanocarriers designed for the high-intensity focused ultrasound (hifu) combined with transarterial chemoembolization treatment of hepatocellular carcinoma model in rabbits. the nanocapsule shell contained magnetic fe o as a contrast compound for mri, and hydrophobic perfluorohexane was encapsulated into the core. hifu is a method for local cancer treatment based on high temperatures leading to tissue destruction. because perfluorohexane may increase energy deposition in cancer tissue, it is an enhancing agent for hifu. simultaneously, the embolization of blood vessels in the tumor allows retaining more nanocarriers within a tumor and improving therapy's efficacy. multifunctional, intelligent, and dual-stimulus responsive nanocarriers, functionalized with folate for active delivery of doxorubicin and indocyanine green (icg), have also been obtained using plga -polyethylene glycol -poly (n-isopropyl acrylamide). icg is known as a near-infrared fluorescence (nir) imaging agent, as well as the photosensitizer in photodynamic therapy (pdt), or photothermal agent in photothermal therapy (ptt). at the same time, fe/feo nanocrystals were tethered with plga polymer, finally forming the shell of multifunctional nanocarriers. fe/feo nanocrystals are mri contrasting agents, and they may provide additional, precise diagnostic information about the exact tumor location and size. after irradiation with nir laser, nanocarriers shrink and degrade, releasing doxorubicin and icg while fe/feo simultaneously degrades in the acidic tumor environment, leading to the overproduction of ros as a result of the fenton reaction. results obtained in mouse tumor models suggest that it may help overcome hypoxia-related tumor resistance to pdt. wang et al showed that the problem of insufficient oxygen in the tumor significantly limited the effectiveness of pdt, which can be solved by the simultaneous delivery of a photosensitizer, in this case, meso-tetra (p-hydroxyphenyl) porphine and catalase, an enzyme that decomposes endogenous tumor h o into water and oxygen. this approach was possible due to the use of unique polymeric nanocarriers obtained due to the covalent coupling of catalase, meso-tetra (p-hydroxyphenyl) porphine, and peg, and additionally labeled with in vivo single-photon emission computed tomography imaging. icg converts nir light energy into local heat leading to cancer tissue ablation. however, cellular stress-induced during this therapy may activate autophagy in cancer cells, which is a complex process that eliminates damaged cellular structures. it can promote tumor cell survival and further tumor progression. that is why wu et al have proposed yet another multifunctional system for the delivery of icg and improve ptt's effectiveness. it uses nanocarriers made of malpeg-b-poly (l-phenylalanine-co-l-aspartic acid), loaded with icg and primaquine -autophagy inhibitor. the nanocarriers' surface has been modified by covalently attaching to the polymer the so-called cell-penetrating peptide (cpp, cys-krptmrfrytwnpmk), allowing shielding of the entire capsule as a result of hyaluronic acid (ha) binding. ha actively directs nanocarriers to the tumor due to interaction with cd (ha receptor). then, ha corona is decomposed in the tumor's acidic environment, exposing cpp that facilitates nanocarriers uptake. the anti-cancer effects of these nanocarriers have already been confirmed by in vivo studies. the intelligent, pegylated bismuth selenide nanocarriers were designed to deliver doxorubicin and yet another agent used in pdt/ptt, namely chlorin (ce ). the entrapment of small drugs in bismuth selenide is simple because of their mesoporous and hollow structure (sponge-like structure). it is worth emphasizing that because previous studies confirmed the usefulness of bismuth selenide as a computed tomography contrasting agent, these nanocarriers enabled bimodal imaging of tumors, fluorescence, and ct. fluorescence imaging confirmed efficient tumor accumulation of nanocarriers and remarkable tumor eradication. it resulted from the synergistic effect of doxorubicin and ptt/pdt-induced tumor ablation. at the same time, toxicity analysis revealed, the nanocarriers were not harmful to healthy tissues. the high therapy specificity resulted from the selective drug release primarily in the tumor. protonation of the amino group of dox under acidic tumor conditions altered, leading to increased drug hydrophilicity and its release from nanocarriers. additionally, upon nir laser irradiation, there was also a release of drugs from the nanocarriers in the tumor. pegylated, biodegradable hollow mesoporous organosilica nanocarriers were obtained to deliver up to different anti-cancer compounds. in addition to icg, they contained gemcitabine and aag, the heat-shock protein inhibitors. the use of aag allows to overcome tumor thermoresistance, the phenomenon regulated by heat shock proteins that are responsible for the proper functioning of selected cancer defense mechanisms, that affect anti-cancer effects of ptt. a unique example of theranostic nanocarriers can be peptide nanoparticles that possess fluorescent properties. the team of fan et al obtained self-assembling nanoparticles based on octa-peptide cyclo -[(d-ala-l-glu-d-ala-l-trp) -] and zn + ions and used them to deliver a chemotherapeutic compound, epirubicin. studies performed on the mouse esophagus cancer model demonstrated that zn + -coordinated cyclic peptides forming these nanoparticles emit nir fluorescence due to quantum confinement within the nanostructures allowing tumors to be imaged without the use of any other imaging compounds. the presence of rgd on their surface ensured sufficient accumulation of nanocapsules in cancer. it is worth asking why so few polymeric nanocarriers intended for the transport of cancer drugs are functionalized with specific ligands (ie anti-cd or anti-her /erbb monoclonal antibodies for active targeting of nanocarriers to cancer cells overexpressing these antigens). it is believed that actively targeted nanoparticles more effectively affect cancer. however, retention of even modified nanomaterials in tumors depends on epr. since most studies on the epr effect have been conducted on rodent models, it is doubtful whether this effect occurs in humans and whether its functioning depends on the type of cancer. , also, in the case of human-emerging tumors, significant tumor volumes are elements of the so-called tumor microenvironment such as tumor-associated fibroblasts, immune cells, and extracellular matrix, which impede the nanoformulation of penetration into the tumor. additionally, intratumor heterogeneity makes it difficult to select the appropriate molecular target and hampers actively targeted nanoparticles. it should also be mentioned that the vast majority of ligands for actively targeted nanodrugs only affect cancer cells without considering other tumor microenvironment forming cells. the problem is also the lack of efficient and cheap methods ensuring the attachment of ligands, especially those with a complex structure than rgd or folate (eg antibodies) while maintaining their biological activity. this makes the production of targeted nanodrugs expensive and costinefficient. these factors probably contribute to the phenomenon observed in the clinic, indicating the lack of submit your manuscript | www.dovepress.com international journal of nanomedicine : improvement in the effectiveness of many drugs given in nanoformulation compared to their free forms. the application of nanomaterials in anti-cancer therapies is not limited to the transport of chemotherapeutics. numerous studies indicate that nanomaterials, including polymeric nanocarriers, may also be used in cancer immunotherapy. briefly, immunotherapy's essential goal is to stimulate immune cells, primarily cytotoxic t lymphocytes (ctl), capable of eliminating cancer cells. since strict ctl activation depends on their cooperation with antigen-presenting cells such as dendritic cells (dc) and macrophages, it is crucial to properly stimulate these cells' activity, eg, by specific adjuvants. an interesting approach was using intradermally administered nanocarriers made of plga for providing low-molecular agonists (imidazoquinoline-based esters) of endosomal pathogen recognizing toll-like receptor / (tlr / ). due to encapsulation, these compounds underwent effective delivery and uptake by dc, enhancing on their surface the expression of ctl co-stimulatory molecules (cd , cd , cd ) and inducing the expression of selected cytokines. this activation of dendritic cells led to ctl stimulation and a better anti-tumor immune response. in turn, the use of poly (β-amino ester)s copolymers assembling into nanocarriers with encapsulated il- were efficiently delivered to the tumor after intravenous administration. this approach provides reversion of the tumor infiltrated macrophages from m to m phenotype, which resulted in a more effective anti-tumor response. it is worth emphasizing that the administration of il- in nanocarriers allowed for a significant reduction of side effects resulting from this proinflammatory cytokine's systemic activity. with the help of polymeric nanocarriers, cancer cells' surface can also undergo modification by exposing them to specialized immune cells such as natural killer cells (nk cells). this approach effectively stimulates immune cells, but limits their activity only to the tumor, bypassing other tissues. zheng et al received very complex polymeric nanocarriers, in which the first layer was made of bovine serum albumin, with a covalently attached antibody and phenylboronic acid. the outer layer was glucose-modified poly ( -methacryloyloxyethyl phosphorylcholine) -b-poly (n-( -aminopropyl) -methacrylamine) associated with the albumin layer due to the interaction between phenylboronic acid and glucose. because this interaction is fragile in the acid environment of the tumor, it is possible to disassemble the outer layer easily. it results in simultaneous exposure of phenylboronic acid capable of binding sialic acids present on the surface of the cancer cells. then, nanocarriers bind to the surface of cancer cells, and due to the presence of an antibody to lure nk cells. these cells may employ a direct killing mechanism involving antibody-dependent cell-mediated cytotoxicity. as demonstrated recently, an interesting and even more comprehensive approach to overcoming tumor tolerance may be possible using nanocarriers whose shell is made of antigen (e.g, protein, overexpressed on tumor cells). simultaneously, inside the carriers, there are two different adjuvants (resiquimod and muramyl dipeptide) stimulating dc. such complex nanocarriers are, therefore, a therapeutic anti-cancer nano-vaccine. however, the immunostimulatory activity of these nanocarriers still requires verification in vivo. nanoparticles indeed offer many advantages as modern therapeutic and preventive vaccines for tumor, viral, and bacterial antigens (e.g, recombinant proteins and nucleic acid) delivering. they might help effectively protect the integrity of the antigen, increase its immunogenicity, and, as a consequence, improve vaccine efficacy. for example, incorporating the antigen into the nanoparticles allows for more effective delivery primarily from the injection site (subcutaneous or intramuscular injection) via the lymphatic system to secondary lymphoid organs where adaptive immune response develops. however, not without significance is the fact that nanomaterials with antigen, due to their size, shape, density, surface charge, and chemical complexity, can mimic the pathogen. thus nano-vaccines more effectively stimulate immune cells (b cells and t cells), leading to efficient antibody synthesis and long-term immunity. this approach has already been successfully implemented to prepare clinically approved the influenza vaccine inflexal/crucell, where virus haemagglutinin was incorporated into liposomes. currently, there are various polymeric nanocarriers in development for the delivery of vaccine components. in that case, an antigen is displayed on the nanocarrier surface. it associates with shell-forming polymers already after the synthesis. the effectiveness of international journal of nanomedicine : submit your manuscript | www.dovepress.com dovepress this process is monitored by analyzing changes in the zeta potential values. the research carried out on the mouse model revealed that the polymers that build nanocarrier shells are essential for the antigen's immunogenicity embedded in/on the surface. peleteiro et al confirmed the higher efficacy of protamine nanocarriers over polyarginine ones in the induction of an immune response against the recombinant hepatitis b antigen. vaccination of animals with protamine nanocarriers resulted in the higher production of antigen-specific antibodies and selected cytokines, including il- a and il- β. these cytokines, due to their proinflammatory activity, play a fundamental role in creating the right environment for the developing immune response. an effective, but the slightly different solution is the use of two-layer nanocarriers, in which the antigen is trapped between two polymeric layers with opposite charges. thus, an antigen is not exposed on the surface of the nanocarriers. crecente-canto analyzed in vivo on the mice model two different nanocapsule types: nanocarriers made of chitosan associated with bacterial antigen and nanocarriers in which the antigen bound to the chitosan layer was covered with a second layer of dextran sulfate. he concluded that coating the antigen with an additional layer of polymer may induce a better immune response. it is probably due to the increased stability of the delivered antigen, which is longer available for antigenpresentingcells. it is worth emphasizing that the effectiveness of many vaccines based on the polymeric nanocarriers, although satisfactory, still seems lower than vaccines based on the antigen administered together with the standard adjuvant alum. many researchers claim the effectiveness of nanovaccines can be significantly increased by additional encapsulation molecules that modulate the immune response, such as imiquimod, cpg or polyi: c. , interestingly, fichter et al proposed the use of nanocarriers built exclusively from hepatitis c virus nonstructural protein a and functionalized with adjuvant monophosphoryl lipid a (mpla) as a vaccine against the hepatitis c virus. studies conducted on a mouse model showed that after intravenous administration, these nanocarriers efficiently and preferentially accumulated primarily in the liver, and thanks to mpla, which is a ligand for tlr , were detectable in liver-resident antigen-presenting cells such as kupffer cells (specialized macrophages located in the liver) or dcs leading to induction of intrahepatic immune response specific to the viral antigen. the authors also emphasized that an essential advantage of using nanocarriers made solely of antigen is the lack of induction of an immune response directed against the additional material present in nanocarriers providing viral antigen. finally, polymeric nanocarriers can be useful in immunotherapy, aiming to induce immune tolerance to allergens (antigens). briefly, this therapy involves supplying the body with a small amount of antigen to inhibit the synthesis of the underlying allergy molecules, namely ige class antibodies and cytokines such as il- , il- , and il- . in vivo tests carried out by smarr et al have shown that an effective and safe approach for the abrogation of allergen-specific response and inhibition of allergic inflammation can be the use of antigen-conjugated biodegradable plga, and antigen-encapsulated plga. it is worth emphasizing that, in this case, the antigen is encapsulated or covalently bound to the nanocapsule-building polymer. nanomaterials have also found a broad application in regenerative medicine, which aims to develop and apply therapies that support the healing of tissue injuries resulting from diseases or mechanical damage. encapsulation of phenytoin (stimulate fibroblast for cytokine and growth factor production) into pcl nanocarriers significantly reduced drug skin permeabilization compared to a free drug when supplied with chitosan hydrogel. another example of polymeric nanocarriers embedded in a hydrogel was utilized as local anesthesia of the intraoral mucosa. the standard application of emla (a mixture of lidocaine and prilocaine) is limited because it inconveniences the patient (eg bitter taste and burning). muniz et al proposed pcl nanocarriers for double encapsulation of lidocaine and prilocaine trapped in carbopol (acrylic acid copolymer matrix) hydrogel. studies in a rat model have shown that nanoformulation provides a better anesthesia duration compared to commercially available emla and free drugs embedded in the hydrogel. at the same time, tissue regeneration is a multistep process, and the use of more than one active agent and long-term sequential release of the drug at the appropriate stage of the healing process is required. an interesting example of a nanosystem used for wound healing is dual encapsulation of bromelain and salvianolic acid b into an ultrafine core-shell nanofiber built from pcl, poly (vinyl alcohol), and gelatin. the nanofibers were designed to submit your manuscript | www.dovepress.com international journal of nanomedicine : release bromelain sequentially at an early stage, and salvianolic acid b at a later stage of tissue healing. it is essential because bromelain poses anti-inflammatory, antimicrobial, and anticoagulant properties necessary at the initial inflammation phase. salvianolic acid b promotes wound healing by stimulating angiogenesis, reepithelialization, and cell proliferation at proliferative, maturation, and remodeling phases of skin regeneration. during the in vitro evaluation, the authors proved biocompatibility, antibacterial, and proangiogenic properties of tested nanofibers. besides, they confirmed that the subsequent sequential release of two compounds provides accelerated wound healing in rats compared to saline control and unloaded nanofibers. because in the late phase of bone healing, the surrounding environment becomes more alkaline tian et al proposed ph-dependent nanocarriers with growth factorbone morphogenic protein- (nbmp- ). the authors used free-radical polymerization of n-( -aminopropyl) methacrylamide, acrylamide, and glycerol dimethacrylate (degradable cross-linker) to synthesize alkalinedegradable polymeric shells. this solution provides a sustained release of bmp- at the therapeutic window. during research on the posterolateral spinal fusion in rats, nbmp- poses superior characteristics upon free bmp- and provides a better quality of newly formed bone and reduced undesirable inflammatory edema. additionally, proteins like growth factors are sensitive to tissue proteases, which provide their short half-life in the body. thus, the nanocarriers application also reduces the risk of transported cargo proteolysis. due to their pleiotropic properties, polyphenols were proposed for osteoarthritis treatment. to dual transport resveratrol and curcumin, self-assembling lipid-core nanocarriers with grape seed oil cores and pcl shells were proposed. because of differences in the polyphenols solubility in oil, these drugs pose subsequent sequential releases from nanocarriers. the results indicate that this combination of polyphenols administered in nanocarriers protects chondrocytes against oxidative stress and shows better therapeutic efficacy against rat arthritis without undesired toxicity. , there are also examples of the use of gene therapy in the treatment of osteoporosis. sezlev bilecen et al successfully proposed plga nanocarriers with sirna inhibiting rank expression (receptor activator of nuclear factor κ b) protein, essential in the activation of osteoclasts. furthermore, nanocarriers built of amphiphilic gelatin with encapsulated tgf-β (transforming growth factor-β ) and magnetic nanoparticles were proposed for cartilage regeneration. this approach combines magnetic guidance with chondrocyte stimulation via growth factor. the mentioned nanoformulation still requires evaluation of toxicity as well as therapeutic efficiency in vivo. an interesting example is drug delivery as eye drops, which is limited due to the rapid dilution and clearance of the drug that follows tear production and blink reflex. the effectiveness of nanoparticles in this area is due to their improved corneal interaction. reimondez-troitiño et al presented oil nanodroplets surrounded by cell-penetrating cationic polypeptides -protamine or polyarginine for corneal wound healing. even empty nanocarriers administered in eye drops were able to accelerate wound healing in vivo process similarly to commercially available cacicol ® . the use of protamine nanocarriers resulted from a decrease degree of corneal haze following tissue healing compared to polyarginine. this effect was probably due to the antifibrotic activity of protamine. however, additional encapsulation of cyclosporin a or vitamin a did not improve the therapy's effectiveness compared to empty nanocarriers. all nanoformulations were non-toxic in a mouse ocular tolerance test. recently published papers indicate that polymeric nanocarriers could be used in the future for the oral delivery of drugs, in particular biological drugs such as peptides and proteins. oral administration requires developing a formulation that will protect them from degradation and loss of biological activity in the gastrointestinal tract and allow their effective absorption. lifestyle diseases are a combination of diet, physical inactivity, and consumption of stimulants. they emerge more often each year, and polymeric nanocarriers have already been proposed as a drug delivery system for venous thromboembolism, hypertension, peripheral arterial disease, and inflammatory bowel disease (summaries in table ). among these diseases, diabetes requires a new drug formulation improvement of patient safety. many studies involve the development of an oral insulin formulation. this peptide hormone is widely used in treating type i diabetes and advanced type ii diabetes when patients have inefficient insulin synthesis resulting from the destruction of the pancreatic beta-cells caused by prolonged hyperglycemia or dyslipidemia. because insulin is a drug taken only by subcutaneous injection, developing international journal of nanomedicine : submit your manuscript | www.dovepress.com an oral formulation would significantly improve patients' comfort of life and reduce therapy costs. lee et al received insulin loaded pluronic based nanocarriers. interestingly, to ensure the nanocarriers' best absorption in the small intestine, they armed them with two different ligands: chitosan and hexamer peptide at- . due to its interaction with the intestinal mucosa, chitosan allows nanocarriers to adhere to epithelial cells and improves interstitial permeability. its activity is synergistic with the activity of at- . at- is a peptide derived from zonula occludens toxin (zot), a virulence factor of vibrio cholera, a pathogen responsible for the diarrhoeal disease cholera. because at- , like zot reversibly and transiently enhances the intestinal permeability by rearranging the intestinal cell cytoskeleton strategically located to modulate tight intercellular junctions, ensuring effective transport of nanocarriers across the epithelium to the blood. in vivo studies conducted in diabetic rats confirmed that the nanocarriers prepared in this way were effectively absorbed and that the encapsulated insulin retained biological activity. the slow insulin release profile from nanocarriers allowed normal blood glucose levels to be maintained up to hours after oral administration. later, sun et al proposed a unique synthesis method termed flash nanocomplexation to obtain polyelectrolyte nanocomplexes to deliver insulin. first, insulin-loaded n-( -hydroxy) -propyl- -trimethylammonium- -chloride modified chitosan/sodium tripolyphosphate nanocomplexes were produced as a nanocore. in the next step, they encapsulated nanocores into enteric eudragit l - polymer (polymer with mucoadhesive properties). its task is to protect nanocomplexes with the drug against degradation in the digestive system and achieve a specific release of nanocomplexes in the ileum. importantly, the authors claimed that nanocarrier synthesis's optimized process is a scalable manner allowing easy clinical translation. the effectiveness of the obtained nanocarriers revealed studies on diabetic rats. nasal polymeric oil-core nanocarriers with shells covered with eudragit rs (poly (ethyl acrylate-co-methyl methacrylate-co-trimethylamine methacrylate chloride) and with encapsulated steroid triamcinolone acetonide revealed excellent efficacy for the treatment of allergic rhinitis. unique mucoadhesive properties of eudragit polymers enabled excellent retention of nanocarriers in the nasal cavity, high local absorption of the drug, which limited its systemic distribution, and the resulting side effects. as other numerous studies have shown, polymeric nanocarriers could be used to formulate and deliver a wide variety of anti-inflammatory drugs (table ) . gene therapy is a sophisticated approach to the treatment of different diseases based on nucleic acid delivery. a new method of crispr gene editing was developed. chen et al utilized polymeric nanocarriers to transport the preassembled complex of cas nuclease and a singleguide rna (cas rnp), which provided safe and effective crispr-mediated therapeutic gene editing. because cas rnp was surrounded by oppositely charged polymers, imidazole-containing monomer, covalently crosslinked glutathione, and acrylate methoxy poly(ethylene glycol), nanocarriers underwent endosomal escape followed by cytoplasmatic degradations. subsequently, cas rnp was able to enter the nucleus. moreover, for in vivo experiments, nanocarriers were decorated with all-trans retinoic acid, which binds to the interphotoreceptor retinoidbinding protein. results obtained for either subretinal or intramuscular injection have shown more effective genome editing compared to unencapsulated cas rnp. one of the biggest challenges of modern medicine is undoubtedly treating central nervous system diseases such as neurodegenerative disease and cancer. the fundamental problem is a weak blood-brain barrier (bbb) permeability for small molecule drugs and therapeutic proteins. hence, many studies on developing systems that allow the effective transport of therapeutics through bbb-forming endothelial cells. one of the more interesting approaches is the use of nanocarriers obtained from the degradable crosslinker, poly (lactide) -b-poly (ethylene glycol) -b-poly (lactide) -diacrylate triblock copolymer and -methacryloyloxyethyl phosphorylcholine. the latter compound is made of choline and acetylcholine analogs. receptors for these neurotransmitters are commonly found on cells located within the central nervous system and on endothelial barrier cells of the brain; hence they can facilitate transcytosis of functionalized nanocarriers carrying therapeutic proteins (monoclonal antibodies or growth factors supporting the regeneration of damaged brain areas). , other examples of polymeric nanocarriers transporting drugs for neurological disorders are summarized in table . furthermore, polymer nanocarriers have been used to treat various infectious diseases as transporters of antibacterial, antifungal, and anti-parasitic compounds (summarized in table ). the requirements that medicine places in modern therapies, especially those useful in treating such complex diseases as cancer, chronic inflammatory diseases, or autoimmune diseases, lead scientists to seek more and more advanced therapeutics. modern therapies are simultaneously based on many drugs, often biochemically different, but their administration is expected to be safe and as low as possible for the patient. hence the necessity to design biocompatible drug nanocarriers efficiently delivering drugs to the diseased tissue and ensuring their specific release. recently performed studies indicate that polymeric nanomaterials could help in developing such nanocarriers and therapies of the future. the biocompatible polymers used to synthesize polymeric nanomaterials ensure low toxicity and allow obtaining complex and various nanocarriers. polymeric nanocarriers are flexible in size, efficiently transport, and rapidly release cargo in the target tissue dependent on ph or temperature stimuli. however, these nanocarriers are often so complicated that it is difficult to predict the cost of mass production and how stable in vivo they would ultimately be. the usefulness of many polymeric nanomaterials has not even been studied in biological systems. therefore, our knowledge about numerous advanced and complex nanosystems is still low. it is necessary to develop optimal schemes allowing for a detailed characterization of toxicity and verifying these nanomaterials' effectiveness in vivo. nanostructured porous silicon in preclinical imaging: moving from bench to bedside key targeting approaches for pharmaceutical drug delivery vascular permeability in cancer and infection as related to macromolecular drug delivery, with emphasis on the epr effect for tumor-selective drug targeting vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs factors and mechanism of "epr" effect and the enhanced antitumor effects of macromolecular drugs including smancs nanoparticles in the clinic nanoparticles in the clinic: an update pharmacokinetics of total and unbound paclitaxel after administration of paclitaxel micellar or nab-paclitaxel: an open, randomized, cross-over, explorative study in breast cancer patients a multi-national, randomised, open-label, parallel, phase iii non-inferiority study comparing nk and paclitaxel in metastatic or recurrent breast cancer patients toward nanotechnology-enabled approaches against the covid- pandemic immunemediated approaches against covid- phase - trial of a sars-cov - recombinant spike protein nanoparticle vaccine multifunctional nanoparticles for multimodal imaging and theragnosis nanocapsules: the weapons for novel drug delivery systems nanocapsule technology: a review polymeric nanocapsules as nanotechnological alternative for drug delivery system: current status, challenges and opportunities microparticulate drug delivery systems: release kinetic models. microspheres microcapsules liposomes polymer-based nanocapsules for drug delivery submit your manuscript | www.dovepress.com dovepress . ottenbrite rm, kim sw. polymeric drugs and drug delivery systems polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy new developments in liposomal drug delivery injectable lipid-based depot formulations: where do we stand? nanoencapsulation i. methods for preparation of drug-loaded polymeric nanoparticles nanoencapsulation for drug delivery biodegradable polymeric nanoparticles as drug delivery devices the application of biomacromolecules to improve oral absorption by enhanced intestinal permeability: a mini-review self-assembled fluorescent tripeptide nanoparticles for bioimaging and drug delivery applications near infrared fluorescent peptide nanoparticles for enhancing esophageal cancer therapeutic efficacy an investigation into the role of surfactants in controlling particle size of polymeric nanocapsules containing penicillin-g in double emulsion multifunctional nanocarriers nanoparticle-based targeted drug delivery buildup of ultrathin multilayer films by a self-assembly process: iii. consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces fuzzy nanoassemblies: toward layered polymeric multicomposites multilayers of colloidal particles layer-by-layer self assembly of polyelectrolytes on colloidal particles biocompatible long-sustained release oil-core polyelectrolyte nanocarriers: from controlling physical state and stability to biological impact encapsulation of liquid cores by layer-by-layer adsorption of polyelectrolytes nanoparticle synthesis in engineered organic nanoscale reactors monodisperse polyelectrolyte-supported asymmetric lipid-bilayer vesicles bioinspired colloidal systems via layer-by-layer assembly which polymers can make nanoparticulate drug carriers long-circulating? long-circulating and target-specific nanoparticles: theory to practice surfaces that resist bioadhesion the epr effect and polymeric drugs: a paradigm shift for cancer chemotherapy in the st century stimuli-sensitive nanoparticulate systems for drug delivery magnetic targeting for site-specific drug delivery: applications and clinical potential drug targeting scientific and clinical applications of magnetic carriers applications of magnetic nanoparticles in biomedicine development and experimental use of receptor-specific mr contrast media characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular mr imaging synthesis and surface engineering of iron oxide nanoparticles for biomedical applications polymer-coated iron oxide nanoparticles for medical imaging. massachusetts institute of technology multilayered materials based on biopolymers as drug delivery systems engineering nanolayered particles for modular drug delivery layer-by-layer films for biomedical applications layer-by-layer engineered capsules and their applications process for preparing a colloidal and disperse system in the shape of nanocapsules nanoprecipitation and nanoformulation of polymers: from history to powerful possibilities beyond poly(lactic acid) nanoparticles based on hydrophobic polysaccharide derivatives-formation principles, characterization techniques, and biomedical applications physicochemical parameters associated with nanoparticle formation in the salting-out, emulsification-diffusion, and nanoprecipitation methods synthetic polymeric nanoparticles by nanoprecipitation nanocapsule formation by interfacial polymer deposition following solvent displacement an overview of nanoemulsion: concepts of development and cosmeceutical applications nanoemulsions: formation, properties and applications preparation of polymeric nanocapsules by miniemulsion polymerization encapsulation through (mini)emulsion polymerization nanoparticles: properties, applications and toxicities mono-and multilayer covered drops as carriers doxorubicininduced cardiotoxicity: from mechanisms to development of efficient therapy explosible nanocapsules excited by pulsed microwaves for efficient thermoacoustic-chemo combination therapy a pegylated alternating copolymer with oxidation-sensitive phenylboronic ester pendants for anticancer drug delivery addition of vandetanib to pegylated liposomal doxorubicin (pld) in patients with recurrent ovarian cancer. a randomized phase i/ii study of the ago study group (ago-ovar . ) delayed sequential co-delivery of gefitinib and doxorubicin for targeted combination chemotherapy incorporation of drug efflux inhibitor and chemotherapeutic agent into an inorganic/organic platform for the effective treatment of multidrug resistant breast cancer the functions and applications of rgd in tumor therapy and tissue engineering folate receptor-targeted hybrid lipid-core nanocapsules for sequential delivery of doxorubicin and tanespimycin synthesis of size-tunable hollow polypyrrole nanostructures and their assembly into folate-targeting and ph-responsive anticancer drug-delivery agents nanoparticle-dendrimer hybrid nanocapsules for therapeutic delivery magnetic drug targeting: preclinical in vivo studies, mathematical modeling, and extrapolation to humans triple-modal imaging of magnetically-targeted nanocapsules in solid tumours in vivo highly loaded semipermeable nanocapsules for magnetic resonance imaging oil core-peg shell nanocarriers for in vivo mri imaging ultrasound molecular imaging of breast cancer in mcf- orthotopic mice using gold nanoshelled poly(lactic-co-glycolic acid) nanocapsules: a novel dual-targeted ultrasound contrast agent thin-shelled pegylated perfluorooctyl bromide nanocapsules for tumor-targeted ultrasound contrast agent a ph-sensitive nanocarrier for tumor targeting: delivery of ruthenium complex for tumor theranostic by ph-sensitive nanocapsule nanoparticle-enhanced synergistic hifu ablation and transarterial chemoembolization for efficient cancer therapy near-infrared light and tumor microenvironment dual responsive size-switchable nanocapsules for multimodal tumor theranostics photosensitizer-crosslinked in-situ polymerization on catalase for tumor hypoxia modulation & enhanced photodynamic therapy a cascade-targeting nanocapsule for enhanced photothermal tumor therapy with aid of autophagy inhibition stimuli responsive pegylated bismuth selenide hollow nanocapsules for fluorescence/ct imaging and light-driven multimodal tumor therapy chemodrug-gated biodegradable hollow mesoporous organosilica nanotheranostics for multimodal imaging-guided low-temperature photothermal therapy/chemotherapy of cancer review on targeting nanoparticles for breast cancer submit your manuscript | www in vivo distribution of polymeric nanoparticles at the whole-body, tumor, and cellular levels challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology nanodrug delivery: is the enhanced permeability and retention effect sufficient for curing cancer? stromal biology and therapy in pancreatic cancer intratumor heterogeneity and branched evolution revealed by multiregion sequencing early returns from the era of precision medicine polymeric nanoparticles encapsulating novel tlr / agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy polymeric nanoparticles enable reversing macrophage in tumor microenvironment for immunotherapy in situ modification of the tumor cell surface with immunomodulating nanoparticles for effective suppression of tumor growth in mice vaccine delivery: a matter of size, geometry, kinetics and molecular patterns polymeric nanocapsules for vaccine delivery: influence of the polymeric shell on the interaction with the immune system bilayer polymeric nanocapsules: a formulation approach for a thermostable and adjuvanted e. coli antigen vaccine multi-enveloping of particulated antigens with biopolymers and immunostimulant polynucleotides co-delivery of viral proteins and a tlr agonist from polysaccharide nanocapsules: a needle-free vaccination strategy polymeric hepatitis c virus non-structural protein a nanocapsules induce intrahepatic antigen-specific immune responses biodegradable antigen-associated plg nanoparticles tolerize th -mediated allergic airway inflammation pre-and postsensitization chitosan hydrogels containing nanoencapsulated phenytoin for cutaneous use: skin permeation/penetration and efficacy in wound healing hybrid hydrogel composed of polymeric nanocapsules co-loading lidocaine and prilocaine for topical intraoral anesthesia fabrication of core-shell nanofibers for controlled delivery of bromelain and salvianolic acid b for skin regeneration in wound therapeutics growth-factor nanocapsules that enable tunable controlled release for bone regeneration intracellular delivery of poorly soluble polyphenols: elucidating the interplay of self-assembling nanocarriers and human chondrocytes a novel approach to arthritis treatment based on resveratrol and curcumin co-encapsulated in lipid-core nanocapsules: in vivo studies development of pei-rank sirna complex loaded plga nanocapsules for the treatment of osteoporosis using the interplay of magnetic guidance and controlled tgf-β release from protein-based nanocapsules to stimulate chondrogenesis polymeric nanocapsules: a potential new therapy for corneal wound healing zot-derived peptide and chitosan functionalized nanocarrier for oral delivery of protein drug zonula occludin toxin, a microtubule binding protein scalable manufacturing of enteric encapsulation systems for site-specific oral insulin delivery rationally designed nanocarriers for intranasal therapy of allergic rhinitis: influence of carrier type on in vivo nasal deposition a biodegradable nanocapsule delivers a cas ribonucleoprotein complex for in vivo genome editing a bioinspired platform for effective delivery of protein therapeutics to the central nervous system efficient delivery of nerve growth factors to the central nervous system for neural regeneration -( -methoxyphenyl)- -((piperidin- -yl)ethyl)thiazolidin- -oneloaded polymeric nanocapsules: in vitro antiglioma activity and in vivo toxicity evaluation antitumor action of diphenyl diselenide nanocapsules: in vitro assessments and preclinical evidence in an animal model of glioblastoma multiforme mitochondria targeting non-isocyanate-based polyurethane nanocapsules for enzyme-triggered drug release near-infrared triggered decomposition of nanocapsules with high tumor accumulation and stimuli responsive fast elimination polyarginine nanocapsules as a potential oral peptide delivery carrier pegylated polypeptide lipid nanocapsules to enhance the anticancer efficacy of erlotinib in non-small cell lung cancer preparation and evaluation of etoposide-loaded lipid-based nanosuspensions for high-dose treatment of lymphoma novel therapeutic mechanisms determine the effectiveness of lipid-core nanocapsules on melanoma models pegylated squalenoyl-gemcitabine nanoparticles for the treatment of glioblastoma polymeric glabrescione b nanocapsules for passive targeting of hedgehog-dependent tumor therapy mechanisms of the effectiveness of poly(ε-caprolactone) lipid-core nanocapsules loaded with methotrexate on glioblastoma multiforme treatment nanoencapsulation of psoralidin via chitosan and eudragit s for enhancement of oral bioavailability resveratrol-loaded nanocapsules inhibit murine melanoma tumor growth tamoxifen citrate-loaded poly(d,l) lactic acid nanoparticles: evaluation for their anticancer activity in vitro and in vivo new insight into polydopamine@zif- nanohybrids: a zinc-releasing container for potential anticancer activity nano-co-delivery of berberine and anticancer drug using plga nanoparticles: exploration of better anticancer activity and in vivo kinetics selol nanocapsules with a poly(methyl vinyl ether-co-maleic anhydride) shell conjugated to doxorubicin for combinatorial chemotherapy against murine breast adenocarcinoma in vivo preparation of biodegradable polymeric nanocapsules for treatment of malignant tumor using coaxial capillary microfluidic device pegylated lipid bilayer coated mesoporous silica nanoparticles co-delivery of paclitaxel and curcumin leads to increased tumor site drug accumulation and reduced tumor burden paclitaxel-loaded pegylated nanocapsules of perfluorooctyl bromide as theranostic agents ultrasound-induced mild hyperthermia improves the anticancer efficacy of both taxol ® and paclitaxel-loaded nanocapsules design of a liquid nano-sized drug delivery system with enhanced solubility of rivaroxaban for venous thromboembolism management in paediatric patients and emergency cases antidiabetic activity enhancement in streptozotocin + nicotinamide-induced diabetic rats through combinational polymeric nanoformulation studies on core-shell nanocapsules of felodipine: in vitro-in vivo evaluations in vivo evaluation of enhanced drug carrier efficiency and cardiac anti-hypertrophy therapeutic potential of nano-curcumin encapsulated photo-plasmonic nanoparticles combined polymerized nano-vesicles: a novel strategy longlasting anti-platelet activity of cilostazol from poly(εcaprolactone)-poly(ethylene glycol) blend nanocapsules budesonideloaded eudragit s nanocapsules for the treatment of acetic acid-induced colitis in animal model curcumin entrapped hyaluronan containing niosomes: preparation, characterisation and in vitro/in vivo evaluation starch nanocapsules containing a novel neutrophil elastase inhibitor with improved pharmaceutical performance anti-inflammatory effect of nano-encapsulated nerolidol on zymosan-induced arthritis in mice orally delivered resveratrol-loaded lipid-core nanocapsules ameliorate lps-induced acute lung injury via the erk and pi k/akt pathways pegylated meloxicam-loaded nanocapsules reverse in vitro damage on caspase activity and do not induce toxicity in cultured human lymphocytes and mice enhanced pharmacological actions of p,p'-methoxyl-diphenyl diselenide-loaded polymeric nanocapsules in a mouse model of neuropathic pain: behavioral and molecular insights submit your manuscript | www a surface modification of clozapine-loaded nanocapsules improves their efficacy: a study of formulation development and biological assessment nfl-lipid nanocapsules for brain neural stem cell targeting in vitro and in vivo triphenyl phosphonium coated nano-quercetin for oral delivery: neuroprotective effects in attenuating age related global moderate cerebral ischemia reperfusion injury in rats the nasal delivery of nanoencapsulated statins -an approach for brain delivery effect of free and nano-encapsulated curcumin on treatment and energetic metabolism of gerbils infected by listeria monocytogenes design and evaluation of ph-dependent nanosystems based on cellulose acetate phthalate, nanoparticles loaded with chlorhexidine for periodontal treatment in vivo vaginal fungal load reduction after treatment with itraconazole-loaded polycaprolactone-nanoparticles lychnopholide in poly(d,l-lactide)-block-polyethylene glycol nanocapsules cures infection with a drug-resistant trypanosoma cruzi strain at acute and chronic phases biodegradable polymeric nanocapsules prevent cardiotoxicity of anti-trypanosomal lychnopholide efficacy of lychnopholide polymeric nanocapsules after oral and intravenous administration in murine experimental chagas disease lipid-core nanocapsules increase the oral efficacy of quercetin in cutaneous leishmaniasis effects of surface characteristics of polymeric nanocapsules on the pharmacokinetics and efficacy of antimalarial quinine artesunate-heparin conjugate based nanocapsules with improved pharmacokinetics to combat malaria conanoencapsulation of antimalarial drugs increases their in vitro efficacy against plasmodium falciparum and decreases their toxicity to caenorhabditis elegans optimization of curcuma oil/quinine-loaded nanocapsules for malaria treatment reduced cardiotoxicity and increased oral efficacy of artemether polymeric nanocapsules in plasmodium berghei-infected mice ksz acknowledges financial assistance from the statutory research fund of icsc pas. the authors report no conflicts of interest in this work. key: cord- -guiy x authors: cojocaru, florina-daniela; botezat, doru; gardikiotis, ioannis; uritu, cristina-mariana; dodi, gianina; trandafir, laura; rezus, ciprian; rezus, elena; tamba, bogdan-ionel; mihai, cosmin-teodor title: nanomaterials designed for antiviral drug delivery transport across biological barriers date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: guiy x viral infections are a major global health problem, representing a significant cause of mortality with an unfavorable continuously amplified socio-economic impact. the increased drug resistance and constant viral replication have been the trigger for important studies regarding the use of nanotechnology in antiviral therapies. nanomaterials offer unique physico-chemical properties that have linked benefits for drug delivery as ideal tools for viral treatment. currently, different types of nanomaterials namely nanoparticles, liposomes, nanospheres, nanogels, nanosuspensions and nanoemulsions were studied either in vitro or in vivo for drug delivery of antiviral agents with prospects to be translated in clinical practice. this review highlights the drug delivery nanosystems incorporating the major antiviral classes and their transport across specific barriers at cellular and intracellular level. important reflections on nanomedicines currently approved or undergoing investigations for the treatment of viral infections are also discussed. finally, the authors present an overview on the requirements for the design of antiviral nanotherapeutics. today, we are living through a so-called fourth great transitional period, after the other three waves of epidemiological transitions, namely early agrarian-based settlements, early eurasian civilizations and european expansionism (more details can be found in mcmichael's description [ ] ). the current configuration and variety of infectious diseases closely followed the combined evolutions in demography, environment, technology, social change and behaviours. medicine itself has created new opportunities for microbes either through blood transfusions, organ transplants, the use of hypodermic syringes or the excessive use of antibiotics thus contributing to the induction of iatrogenic effects in some treatments for infections such as hepatitis c, hiv and others [ ] . in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [ ] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [ ] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [ ] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure presents statistical facts related to the most "burden-generator" viral infection diseases [ ] [ ] [ ] [ ] [ ] . pharmaceutics , , x for peer review of in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [ ] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [ ] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [ ] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure presents statistical facts related to the most "burden-generator" viral infection diseases [ ] [ ] [ ] [ ] [ ] . hiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide long- hiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide long-term care and support for the ill person, demanding a higher level of treatment costs for the hiv-affected households. consequently, hiv/aids causes depletion of savings and productive assets, and increases the indebtedness of the hiv-affected households [ , ] . moreover, the higher health care expenditure of the households reduces investment for nutritional food for the family members, investment for farming or business, and the children education. death during the working age of the patient is a major factor in the economic impact of hiv/aids. the household level impact of hiv/aids includes direct costs, including medical and non-medical costs, and productivity costs such as loss of labour time, as a result of the morbidity of hiv positive household members, as well as time spent by others caring for them [ ] . this evidence suggests that hiv/aids places significant economic pressure on households trying to pay for health care costs, and trying to make up for lost income. the difficulty in accurately quantifying and explaining the morbidity and mortality related to viral hepatitis stems from the fact that hepatitis deaths are caused by five distinct viruses (hepatitis a-e) with different routes of transmission, or from the fact that death occurs decades after infection, and that when people die with hepatitis-related liver cancer and cirrhosis, these deaths are not always linked to the underlying infection. although antiviral therapy appears to be expensive (for example, average cost for a treated hcv case ranges from $ , to $ , [ ] ), is considered to be also cost effective when compared with other well-accepted medical interventions [ ] due to sustained viral response to therapy, the cost savings and quality-of-life improvement and prolongation of life expectancy from the prevention of hcv complications. in the era of new daas, the statement "provide treatment to hcv-patients" generates savings compared to not provide it. low and middle-income countries may consider hcv-treatment as a cost-saving intervention for the health system, not only in a long-term horizon, but in - years [ ] . economic impact of no treatment is higher than treatment costs itself. but still, the enthusiasm for daa therapies, however, has been tempered by two major concerns: the price, still very high, of these medications, and, the challenges patients and clinicians face with respect to drug access in many countries. hsv and herpes zoster (varicella-zosterian virus-vzv reactivation) are some of the most common infections in humans, with no effective treatment available at this time. the impact on the health degree varies from a very small impairment to severe, disabling forms, in most cases limiting methods are applied to local infection and reducing side effects (pain, manifestations dermis etc.). also, specific psychological issues are related with this disease, such as negative feelings correlated to the condition following diagnosis, in particular if they have acquired the genital form of the disease. feelings can include depression, fear of rejection, feelings of isolation, fear of being found out, and self-destructive feelings [ ] . as mentioned above, all the available reports present crude estimates rather than precise measures of the economic costs of the illness, because most of the costs are calculated directly on interventions or on medical budgetary chapters, without taking into account the societal losses. moreover, different approaches are discrepant (can be explained, at least in part, with the influence of compliance to treatment and possible under sampling of subpopulations in the data set) [ ] . also, limitations must be placed on the ability to generalize the results beyond the sample. moreover, not only the cost matters. the costs of an intervention have to be compared with the results of interventions because the effectiveness of treatments and the efficiency can produce societal gains that must be offset by losses [ ] . the usual most accurate methodology to estimate a burden (associated with a disease) is the so-called "cost-of-illness" [ ] , and measures all the costs of a particular disease, including the direct, indirect, and intangible dimensions. it is widely accepted that estimating the total social cost of a disease is useful in establishing policy decisions [ ] . but there are also other methods as the cross-sectional surveys of samples of primary and secondary care physicians, analyzing health care resource utilization or approaches based on the analysis of a large administrative data sets, such as values of spending or drugs consuming lists [ ] . other approaches are used to estimate the number of patients seeking medical treatment, the average medical expenditures (as health inputs employed per unit multiplied by number of units) and estimated national costs. these comprehensive studies can often be advantageous in allocating total national expenditures among the major diagnostic categories [ ] . however, regardless of the method, such analyzes are not possible otherwise than inside countries (where the impact determined by cultural and social aspects can vary substantially). but even in the absence of global data of this nature, we still can extract from the information presented the relevant issue for the topic of this paper: the current arrangements in the management of viral infections (treatments, prevention and limitations of spread) are costly and less effective, unaffordable in some cases and burdensome for medical systems. for example, according to the current analysis of globe newswire reports and data [ ], the global antiviral drugs market was valued at $ . billion in and is expected to reach $ . billion by year . sales of antivirals increased by approximately % each two years. moreover, thanks to better diagnostics, innovative drugs and new therapeutics, the market is likely to witness even further future growth. however, the list of viral diseases for which antiviral therapies are available is still relatively short [ ] . there are several factors that hinder the development of antiviral drugs: • dependence of viruses replication on host cell biosynthetic machinery [ ] , that leads to a limited number of virus-specific metabolic functions can be targeted by antiviral drugs without any damage to the host; • the viruses' functions are specific to each virus, preventing the development of a broad-spectrum antivirals fighting against different viruses that cause similar symptoms. antivirals developed for some viruses (as hsv and hiv) can treat the acute illness, but do not cure the latent infection. this leads to recurrent or chronic diseases that require treatment for longer periods of time [ ] . all these limitations prompted the need for a paradigm shift. the great challenge of antiviral therapies is to move on to developing new drug formulas. this involves changing the physico-chemical and bio-pharmaceutical properties of antiviral molecules using new scientific strategies during the preparation or in dosage configuration. viruses are sub-microscopic intracellular parasitic particles of genetic material contained in a protein coat, totally dependent by host for cell replication, showing both living and non-living characteristics [ ] . living characteristics of the viruses are represented by the high rate of multiplication (only in living host cells) and by the ability to mutate. the non-living characteristics for viruses consist in acellularity (lack of cytoplasm and organelles), the replication only by using host cell's metabolic machinery and the composition with dna or rna [ ] . in humans, viral infections are responsible for different diseases as briefly presented in table . according to international committee on taxonomy of viruses (ictv) there are phylum, subphylia, six classes, orders, seven suborders, families, subfamilies, genera, subgenera and species [ ] . currently, viruses are classified based on their type of nucleic acid (dna, rna, single-stranded, double-stranded) and their way of replication, known as baltimore classification [ ] , divided as seven baltimore classes: • i-dsdna viruses (e.g., adenoviruses, herpesviruses, poxviruses): enter to the host nucleus and are dependent by host cell polymerases to replicate viral genome. the virus may induce the cell to forcefully undergo cell division, which may lead to transformation of the cell and, ultimately, to cancer. • ii-ssdna viruses (+ strand or "sense") dna (e.g., parvoviruses), consists of viruses that have a single-stranded dna genome of the same polarity as the mrna. excepting parvoviruses, most of them have circular genomes and are replicating within nucleus. • iii-dsrna viruses (e.g., reoviruses): not dependent by host replication polymerases and their replication (monocistronic) is realized into capsid (in cytoplasm). • iv-(+)ssrna viruses (+ strand or sense) rna (e.g., picornaviruses, togaviruses): the rna can be directly accessed by ribosomes of the host to form proteins, and use a simple reproduction pathway (viruses with polycistronic mrna) or a more complex transcription pathway (for which subgenomic mrnas, ribosomal frameshifting, and proteolytic processing of polyproteins may be used). [ ] [ ] [ ] [ ] [ ] gastroenteritis adenoviruses, rotaviruses, noroviruses, astroviruses, coronaviruses [ ] [ ] [ ] sexually transmitted diseases herpes simplex type , human papillomavirus hiv [ ] there are distinct stages of viral replication (cell entry, uncoating, transcription of viral genome, translation of viral proteins, post-translational modifications and assembly of virion components) and the classes of antiviral agents that can act at each stage, the correspondence between stage of replication and classes of selective inhibitors being described in detail in reference [ ] and their pharmacological properties as it follows in the next section. according to our knowledge (based on found studies) only the antivirals summarized in figure were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals pharmaceutics , , of can be found in described in detail in references [ ] [ ] [ ] [ ] [ ] . briefly, acv pharmacology can be explained as follows: • the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv; • then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [ ] [ ] [ ] ; • occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [ ] . according to our knowledge (based on found studies) only the antivirals summarized in figure were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals can be found in described in detail in references [ ] [ ] [ ] [ ] [ ] . briefly, acv pharmacology can be explained as follows:  the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv;  then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [ ] [ ] [ ] ;  occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [ ] . the mechanism of gcv inhibits viral dna synthesis [ ] as briefly explained below:  it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; the mechanism of gcv inhibits viral dna synthesis [ ] as briefly explained below: • it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; • cmv can become resistant to gcv by mutations in the viral phosphotransferase encoded by the ul gene and by mutations in viral dna polymerase [ ] . the conventional treatment (prophylaxis or therapy) of an influenza virus infection, as a major public health concern worldwide, is designed to target viral proteins and could be used, either alone or in combination [ ] . these include also amantadine and neuraminidase inhibitors (zanamivir and oseltamivir), that have been encapsulated into nanoparticles as specified in table . potent vacuolar atpase (v-atpase) inhibitors, namely diphyllin and bafilomycin, previously shown to have broad-spectrum antiviral activity represent another possibility against influenza virus infection [ ] [ ] [ ] . briefly, the antiviral mechanism of amantadine is based on nterference with the viral protein, m (an ion channel), the protein needed for the viral particle to become uncoated once it is taken inside the cell by endocytosis [ ] . also, oseltamivir carboxylate mechanism implies a selective inhibition of influenza virus neuraminidase enzymes, which are glycoproteins found on the virion surface, very important for viral entry into uninfected cells, for the release of recently formed virus particles from infected cells, and for the further spread of the infectious virus in the body [ , ] . hepatitis viruses have been the subject of intense study in the last years, with a special attention on therapy. as mentioned in the first section, hepatitis treatment depends upon the type of hepatitis, therefore different antivirals are considered and summarized in detail in references [ ] [ ] [ ] [ ] [ ] [ ] . currently, interferons (ifns) α, β, and γ have antiviral activity, the first two being produced by nearly all cells as response to viral infections, while the third is restricted to t-lymphocytes and nk cells. ifn-induced proteins include - -oligoadenylate [ - (a)] synthetase and a protein kinase, either of which can inhibit protein synthesis in the presence of double-stranded rna. the - (a) synthetase produces adenylate oligomers that activate a latent cellular endoribonuclease (rnase l) to cleave both cellular and viral single-stranded rnas. antiretroviral therapy-art-refers to the treatment with hiv medicines. according to the last updated list approved by food and drug administration (fda) these drugs can be classified as can be seen in figure [ , ] . in the following paragraph the authors describe the pharmacological mechanism of the medicines for hiv treatment that were included/encapsulated/incorporated into nanomaterials. the representative nucleoside reverse transcriptase inhibitor (nrti) zidovudine (azt) is phosphorylated intracellular by kinases specific to azt -triphosphate, a metabolite responsible for termination in elongation of proviral dna because it is incorporated by reverse transcriptase into nascent dna but lacks a -hydroxyl group. resistance to azt is associated with mutations at reverse transcriptase codons , , , , , , and [ ] . also, lamivudine (lam), another nrti agent, enters cells by passive diffusion, and then is converted to the monophosphate by deoxycytidine kinase, and undergoes further phosphorylation by deoxycytidine monophosphate kinase and nucleoside diphosphate kinase to yield lamivudine -triphosphate, which is the active anabolite [ ] . tenofovir disoproxil is a derivative of adenosine -monophosphate lacking a complete ribose ring, and it is the only nucleotide analogue currently marketed for the treatment of hiv infection, being active against hiv- , hiv- and hbv. after a rapid hydrolysis, tenofovir is formed, being then phosphorylated by cellular kinases to its active metabolite, tenofovir diphosphate which is a competitive inhibitor of viral reverse transcriptases and is incorporated into hiv dna, causing chain termination [ , ] . non-nucleoside reverse transcriptase inhibitors (nnrtis) include a variety of chemical substrates that bind to a hydrophobic pocket in the p subunit of the hiv- reverse transcriptase and induce a conformational change in the d structure of the enzyme that greatly reduces its activity, and thus they act as non-competitive inhibitors. unlike nucleoside and nucleotide reverse transcriptase inhibitors, these compounds do not require intracellular phosphorylation to attain activity [ ] . also, the binding site for nnrtis is virus-strain-specific and the approved agents are active against hiv- but not hiv- or other retroviruses and should not be used to treat hiv- infection. hiv protease inhibitors (pis) are peptide-like chemicals that competitively inhibit the action of the virus aspartyl protease (a homodimer consisting of two -amino acid monomers). the preferred cleavage site for this enzyme is the n-terminal side of proline residues, especially between phenylalanine and proline. these drugs prevent proteolytic cleavage of hiv gag and pol precursor polypeptides that include essential structural (p , p , p , and p ) and enzymatic (reverse transcriptase, protease, and integrase) components of the virus, preventing the metamorphosis of hiv virus particles into their mature infectious form [ , ] . only sqv, indinavir, atv, rtv, nfv and lopinavir are currently employed in nanotechnology research. there are two drugs available in the entry inhibitors class: enfuvirtide-enf and maraviroc-mcv, with different mechanisms of action, both incorporated into nanoparticles as specified in table . enf inhibits fusion of the viral and cell membranes mediated by gp and cd interactions, while mcv is a chemokine receptor antagonist and binds to the host cell ccr receptor to block binding of viral gp . as such, mcv is the only approved antiretroviral drug that targets a host protein [ ] [ ] [ ] . raltegravir (ral), the first approved hiv integrase inhibitor, has potent activity against both hiv- and hiv- , and also retains activity against viruses that have become resistant to antiretroviral agents of other classes because of its unique mechanism of action [ ] [ ] [ ] [ ] . ral was encapsulated into a polymeric plga nano-formulation and gold nanoparticles (see table ). the first line of defence [ ] that any substance encounters is the biological barriers penetration into the organism. the "security" system includes physiological barriers, such as blood-brain barrier (bbb), epithelium, stratum corneum, air-blood lung barrier [ ] , reproductive system barrier, etc. all of which control the extracellular and intracellular access and trafficking of foreign substances such as bacteria, viruses, fungi, and chemicals [ ] but also provide selective access to "suitable candidates" such as nutrition and/or therapy molecules. it is well established that more than one mechanism may be involved in intracellular drug delivery. the mechanisms involved in nano-based intracellular drug delivery include passive diffusion of free drug, non-specific phagocytosis of the nanocarrier, nanocarrier uptake by pinocytosis, and receptor-mediated endocytosis [ ] . in this section, we will discuss in particular how to overcome biological barriers such as mucus, skin, cell membrane and bbb in antiviral therapy. the gastrointestinal tract, respiratory system, the urogenital cavities, eyes and mouth, are all covered with mucosal membranes. the highly adhesive mucus acts as protective layer as well as for lubrication purposes. although large molecules cannot pass, small ones along with viruses can easily penetrate. these are also the reasons that drug delivery is so challenging. mucus contains % of water along with mucin fibres, lipids, salts, cholesterol and proteins. it is continuously produced but the thickness, ph and amount differ by its position. two strategies have to be followed for passing through mucus, mainly depending how fast the turnover is: fast mucosal penetration or highly adhesive particles (slow turnover) to increase the drug's residence on the targeted mucosa. mucoadhesion can be the resultant of interactions like hydrophobic, hydrogen bonding, ionic bonding or van der waals ones. other possible attractive interactions can be covalent bond formation between catechols, maleimides, thiols, and acrylates with domains of mucin glycoproteins rich in cysteine. chitosan, alginate, pectin or cellulose polymers are mostly used for achieving adhesion on mucosa. furthermore, it is known that thiolation of the mentioned polymers develops high mucoadhesive properties. mucopenetration is the second strategy found to permeate the mucus layers by two potential mechanisms: active strategy characterized by the interaction with the mucus and chemically shifting the features of the mucus or their own structures and passive strategy that uses hydrophilicity enhancers to penetrate the mucus [ ] . the classical hsv therapy includes daily dosing of orally administered acv [ ] that is effective in most cases, and of course problematic in other cases, for example, in long-term use of acv patients report resistance against the drug followed by renal injury [ , ] . another issue produced by standard hsv treatment in this case for the topical form of trifluridine (tft) and ganciclovir (acv analog) gels, is denoted by low retention time on vaginal and corneal mucus followed by multiple doses up to times [ ] . the nanotechnology field offers a great deal of drug delivery modalities in order to overpass the biological barriers, deliver efficiently the incorporated active principle in a controlled and targeted pharmaceutics , , of manner, reduce circulating drug levels and attenuate the renal damage. a recent review points out the nanogels based on the above-mentioned materials capabilities as an adequate example to pass through these types of biological barriers [ ] . for example, the synthesized nano-drug delivery micelles based on chitosan-g-oligo (nipaam) copolymers stabilised by ionotropic crosslinking by raskin et al. [ ] , gave good results for delivering antiretroviral drugs (efv) through mucosa. more studies on how acv penetrates different barriers can be found in table and detailed in section . it is important to mention a "special barrier" namely the ocular mucus that changes completely in to min, making the drug absorption unfavourable. in addition, the eye is protected by blood-anterior chamber and blood-retinal barriers. in case of drug administration, a combination of penetration and adhesion of the substances is necessary. polymers like phosphotyrosine could be the solution. it was demonstrated that if intestinal alkaline phosphatase is present, polymers can manifest a zeta potential change, thus causing their immobilization after penetration. another kind of construct for combining adhesion and penetration is through thiolated systems with mucolytic enzymes, ph dependent. therefore, at acid ph, the absence of disulphide bonds formation with cysteine-rich domains in mucins does not manifest mucosal adhesion unless they are near the epithelium [ ] . skin, as the largest organ of our body, protects us from microorganisms and chemicals, regulates our body temperature and maintains hydroelectrolytic balance. the two layers of the skin are the epidermis and the dermis. the first one is avascular and is composed of stratified, keratinized squamous epithelium, in four layers from bottom up: basale, spinosum, granulosum and corneum. thick skin (palms and soles) has a fifth layer (under the most superficial corneum) called lucidum. the dermis consists of two layers (reticular and the more superficial papillary) of connective tissue of elastin and collagenous fibres and has in its component blood and lymphatic vessels network, nerves, touch receptors (meissner corpuscles), adipocytes, phagocytes, hair follicles and sweat glands [ ] . topical, through skin drug delivery, has a local effect, requiring less drug for the targeted outcome. transdermal therapy results in fewer side effects with no need of regular treatment but systemic distribution of the drug. both methods of treatment have a common blockage, stratum corneum. to pass through this barrier, different approaches were developed based mostly on disrupting this structure chemically with substances like surfactants, alcohols, esters, amines, terpenes, alkanes phospholipids, or mechanically by using ultrasounds, micro needling, magnetophoresis, iontophoresis, electroporation or lasers. excessive use, though, can damage the skin. analysing the literature data, we can suggest that there are different processes and mechanisms that govern the penetration of small/large molecules through skin barrier. according to schneider et al. [ ] review there are two general pathways for skin absorption: through skin appendages or through the stratum corneum and the underlying layers. the lipophilic statum corneum medium determines the first mechanism of skin penetration, namely absorption of lipophilic compounds [ ] . the three transport routes of substances across stratum corneum can be classified in transcellular, intercellular and trans-appendageal pathways as defined by liang et al. [ ] . more examples are available but the conclusion is the same: "the full understanding of the penetration or absorption processes is still under evaluation" due to the challenges associated with delivering complex burdens through the skin barrie [ ] . as specified in section , the current antiviral therapy for hsv infection includes topical formulations of acv that is unable permeate stratum corneum and target the virus site at the basal epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [ ] . nanotechnology strategies [ ] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nano-platforms as presented in figure . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the abovementioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [ ] . nanotechnology strategies [ ] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nanoplatforms as presented in figure . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the above-mentioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [ ] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [ ] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nanoplatforms can grow the adhesion to the membrane and the amount of drug entered in the cell [ ] . thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [ ] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [ ] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nano-platforms can grow the adhesion to the membrane and the amount of drug entered in the cell [ ] . the bbb is a highly selective semipermeable structure composed of five parts: the basement membrane, the astrocytes, the immune cells, the pericytes and an endothelial cell layer of capillaries. the area between basement membrane and the neurons is called virchow-robin space. in this region there is interstitial fluid in which reside microglia. all the above components are called neurovascular unit. the kinetics of this unit is crucial to the role of bbb and its states of illness. the capillaries of bbb compose a layer of squamous epithelial cells that fold to form a circular vessel. these cells are linked with strong connections for blocking entrance or exit of materials through central nervous system. protein transportation facilitates the selective flow of molecules through vessel lumens, essential biomolecules being in higher concentrations, like glucose and at the same time eliminating toxins. the bbb is a physical and metabolic "obstacle", physiologically important and active, which survey blood-brain traffic and control it, restricting the paracellular diffusion between the endothelial cells (microvessels) and the efflux pumps activity that quickly expel back into the capillary lumen a wide variety of xenobiotics. bbb integrity and function is critically influenced by what is now referred to as "the extended neurovascular unit" that incorporates not only microvascular endothelial cells and adjacent pericytes, astrocytes and neurons, but also neighbouring smooth muscle cells and microglia in the brain, and blood cells in the capillary lumen such as polymorphonuclear cells, lymphocytes and monocytes [ ] . transport at the bbb level is assured by numerous transport mechanisms that provides to the brain the necessary nutrients and also protects from the toxic xenobiotics. the main transport mechanisms are represented by free diffusion of small lipophilic substances or by active transport (carrier mediated, receptor mediated and active efflux transport). active efflux transport is assured by two major types of transporters that extrude metabolic waste, xenobiotics and a large number of drugs from the brain back into the blood. the first superfamily of bbb efflux transporters is the solute carrier proteins (slc) superfamily, being represented at the level of bbb by slc and slco (slc ) efflux transporters. the second is the atp-binding cassette (abc) efflux transporter family represented by permeability glycoprotein (p-gp), breast cancer resistance protein (bcrp) and the multidrug resistance associated proteins (mrps) [ ] [ ] [ ] . based on their localisation, the abc efflux transporters prevent lipophilic and amphiphilic environmental toxic compounds or drugs, including anti-inflammatory, immunosuppressive, anti-infectious, antineoplastic drugs, some antiepileptic, antidepressant and psychotropic agents, and drug conjugates by an energy-dependent, unidirectional direct transport mechanism, from entering specific substrates [ ] . the bbb's efflux machinery does an excellent job of recognizing xenobiotics, but a poor job on distinguishing between toxicants and therapeutic drugs, creating an important obstacle to treatment of brain cancer, epilepsy and neuro aids [ ] . the penetration of the bbb for drug delivery, although challenging, captivates the interest of numerous researchers in antiviral therapy since the mechanisms by which for example hsv- penetrates the cns remain unclear. the most likely routes include retrograde transport via the olfactory or trigeminal nerve fibres, occasionally leading to herpes simplex encephalitis (hse) caused by hsv- [ ] . another studied virus that is involved in encephalitis and bbb disruption is hiv, known to cause severe neurological disorders and leading to hiv-related encephalitis [ ] since bbb is impermeable to % of antiretroviral drugs [ ] . the possible mechanism responsible for bbb disruption in hiv- encephalitis is considered a "trojan horse" mechanism, where hiv infects specific t-lymphocytes and circulating monocytes, then entering to cns through bbb gaps and followed by inflammatory reactions [ ] , but, in the last years nanotechnology has been intensely explored and several experimental attempts have been carried out in order to enhance the bbb permeability toward antiretroviral drugs, briefly described below based on the nano-based formulation composition, since it is well known that the size and surface functionalization influence transport properties within tissues: • polymeric polybutylcyanoacrylate (pbca) nanoparticles with two incorporeated antiretroviral drugs (azt and lamivudine) showed a - and - fold increase in bbb permeation, by three possible mechanisms as presented by the authors: prolonged interaction interval between drug-loaded nanoparticles and brain-microvascular endothelial cells elevated the concentration gradient between blood and the brain, polysorbate covering on the periphery of nanoparticles was able to be absorbed and degraded nanoparticles improved drug absorption [ ] ; • spherical transferrin coated-pegylated albumin nanoparticles encapsulating azt prepared by ultra-emulsification method using chemical cross-linking by glutaraldehyde gained an access across the bbb through the transferrin receptor mediated endocytosis on the membrane [ ] ; • transferrin-conjugated quantum rod nanoparticles conjugated with saquinavir crossed an in vitro bbb model by exploiting a receptor-mediated transport [ ] ; • magnetic liposomal nanoformulations of azidothymidine -triphosphate (the active form of azidothymidine) migrate across bbb in vitro, either directly or by a monocyte-mediated transport, under the influence of an external magnetic field [ ] ; • novel nanodrug consisting of an iron oxide nanoparticle coated with pma amphiphilic polymer and functionalized with the antiretroviral peptide enfuvirtide crossed the bbb by a passive diffusion, probably mediated by the absorption of the amphiphilic coating on the cell membrane [ ] . as briefly presented the preliminary results are more than encouraging. certainly, future investigations on the mechanisms about bbb disruption are needed along with novel, innovative, safe and efficacious therapeutic approaches. up to now we have concluded that current antiviral therapy has not yet achieved the ideal shape and efficiency and also that the complex biological barriers are major obstacles, but can we critically say that nanotechnology could be the identified solution? search engine queries on "nanotechnology" generate more than , items on specialized platforms (science direct, for example) that represents potential and challenges in different fields from biosensors and industry-related applications up to nanomedicine and biomaterials. when the search keywords are "nanotechnology as antiviral therapy" the same engine only returns results starting from . it is a given fact that nanotechnology is defined as the application of materials on the nanometer scale. according to the literature data results, namomaterials designed with different shapes and morphologies display numerous advantages for use in antiviral therapy, namely: nanometric size that permits drug delivery through impermeable barriers [ ] , large surface area to volume ratios for large drug payloads incorporation [ ] and improved efficacy, surface modification and/or backbone functionalization versatility that facilitates cellular membranes passage [ ] or enhancing stability and bioavailability [ ] , virucidal activity against a series of viruses (hiv, hsv, hbv, etc.) due to biomimetic properties [ ] , increased specificity, improved antiviral delivery and controlled drug release to the target [ ] through engineered moieties, decrease the emergence of drug resistance, personalized therapy possibility, protection of the drugs and low adverse drug side effects mainly due to the composition. the mechanisms of nanomaterial-mediated drug delivery are determined by the chemistry, the architecture and the specific properties of each nanosystem (as presented in the schematic representation in figure ). the design of new drug delivery systems for the antiviral therapy is focused on manipulating these features that are relevant in viral diseases where high drug doses are compulsory, implies high costs and the patient is depended on the administration protocol. lembo and cavalli [ ] present the current status up to in the nanoparticulate delivery systems in antiviral therapy area, highlighting their perspective on the challenges that must be tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. in , liu and chen [ ] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in , milovanovic et al. [ ] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table highlights part of their summary and described based on virus classification in section . in , cao and woodrow [ ] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section . nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table and described in section . in , liu and chen [ ] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in , milovanovic et al. [ ] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table highlights part of their summary and described based on virus classification in section . in , cao and woodrow [ ] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section . nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table and described in section . recently, arca-lafuente et al. [ ] overviewed nanotechnology-based systems as reliable alternative diagnostic tools for hcv infectious disease. even if our review does not cover screening, it is important to mention that new diagnostic methods are required in order to overcome current drawbacks of hcv under-diagnosed infection as highlighted in the above-mentioned review. the nanotechnology-based tools described in the review seem to fulfil the necessary features for hcv elimination. with the aim of developing new personalized diagnostic tools, farzin et al. [ ] summarized current strategies and under-development tools for early diagnosis of hiv. their review combines the use of nanomaterials such as carbon nanostructures, nanoclusters, quantum dots, metallic and metal oxide nps as advanced structures for hiv detection with possible biosensing strategies targeting to offer innovative outlooks for developing intelligent, sensitive and specific nano-objects for in situ and real-time detection of hiv. in this section the authors point out the suitability of nanomaterials (recent data) for antiviral therapy, highlighting the enhanced features pursued to overcome the identified issues as related above. donalisio et al. [ ] have reported the preparation by a modified nano-emulsion method of chitosan nanospheres (ns) loaded with . % acv as a topical formulation against both hsv- and hsv- herpes virus strains. the main component, chitosan, a natural polycationic polysaccharide, was selected as a material for acv release, due to its distinctive properties: hydrophilic character, in situ gelling, mucoadhesion, permeation enhancing, in addition to a low cytotoxicity, biocompatibility and bioresorbability features [ ] . the obtained gel formulation based on acv-loaded ns proved an enhanced ability to penetrate porcine skin to about % (at h) greater than the commercial cream product ( %). ic values against hsv- and hsv- were also determined on vero cell cultures infected with above-mentioned strains, displaying significant reduced values of . µm and . µm, respectively, when using the ns formulation as compare to . µm and . µm for free acyclovir. this nano-technological approach attests the higher efficacy of the described formulation and with promising expectations for further preclinical and clinical experiments. yadavalli et al. [ ] have explored the potential of highly porous activated carbon (hpac) particles as a model for restricting hsv- and hsv- from entering target cells. they have considered this material due to the charcoal surface-active that could provide antiviral effects through virion sequestration approach. furthermore, acv molecules adsorbed or encapsulated inside the hpac pores revealed sustained drug release acting in a synergistic manner to obtain an enhanced therapeutic effect. the hpac compound prove d a to % reduction in hsv- and hsv- entry for concentrations as low as mg/ml. the ic value of hpac corresponding to hsv- and hsv- infection in prophylactic administration was found of . and mg/ml, respectively, significantly lower than clinically accepted tc value (half maximal toxicity concentration) of hpac. following the promising outcomes from in vitro tests, further determinations of antiviral efficacy on in vivo studies using a murine model of ocular (hsv- ) and genital (hsv- ) infection were performed. as a result, the acv loaded hpac acts by capturing the virus and releasing the encapsulated drug, hindering inflammation and immune cell infiltration in targeted tissue. the strong antiviral activity of this product was assigned to the charged surface of its pores which may interact with the cell's surface, stimulating an active exchange of ions (na + , k + , ca + , cl − , and oh − ), when sustained or slow release of acv has been acquired. moreover, these particles exhibited both prophylactic and therapeutic effects against hsv- /hsv- cells, unlike the free drug that did not demonstrate a prophylactically antiviral response. gold and silver nps using seaweed sargassum wightii with anti-herpetic activity biogenic gold (au) and silver (ag) nps were prepared using the seaweed sargassum wightii (sw) and investigated for their antiviral activity against hsv- and hsv- strains [ ] . the nps synthesis resided in an eco-friendly method, previously described in the literature [ ] , replacing the use of different reducing agents. the obtained nps, sw-au and sw-ag, were evaluated concerning both cytotoxic and antiviral effect, using mtt and cpe (cytopathic effect) assays on vero cells. the results showed that cell viability ranged from % to % when the concentration ranged between . and µl per sample in sw-au, and from % to . % for concentrations of . and µl per sample in sw-ag. the antiviral assay have shown a % decrease of cpe on both hsv- and hsv- when vero cells were treated with and µl sw-au, whereas sw-ag exhibit similar reduction of cpe at a concentration of only . µl per sample. higher concentrations of sw-ag are not accepted due to an increased cytotoxic effect. the authors claimed that the obtained results are in agreement with other published research and they inferred that functionalized metallic nps act as antiviral agents by blocking the virus attachments and cell access, depending on particle size. cagno et al. [ ] conducted a research study concerning broad-spectrum antiviral products, which usually mimic heparan sulfate proteoglycans (hspg), as well-preserved target of "viral attachment ligands" (vals). the antiviral effect relies on the binding mechanism of the nanoparticles to the virus surface, thus preventing virus-cell attachment. in most cases, the reversibility of these bonds is reported [ ] , so that by increasing the dilution viral inhibition is lost, causing those compounds not to be considered antiviral drugs. the aforementioned authors have designed antiviral nanoparticles of virucidal effect based on long and flexible linkers simulating hspg, leading to irreversible viral deformation. of the synthesized compounds, the most notable virucidal effect was found in the aunps coated with a : mixture of decanesulfonic acid (mus) and -octanethiol (ot). mus allows a multivalent binding [ ] as a consequence of its structure comprising a long hydrophobic chain, sulfonic acid terminated. the enhanced activity of mus:ot-nps was assigned to the new construct using mus linker that caused local distortions and then a global virus deformation, leading to irreversible loss of infectivity. the mus:ot-nps exhibited efficient virucidal effect against hsv- and hsv- , human papilloma virus (hpv- ), respiratory syncytial virus (rsv), dengue and lenti virus. the in vivo testing on balb/c mice infected with rsv reveals the efficacy of mus:ot-nps treatment that prevented the pulmonary dissemination of the infection. these results are in agreement with previous published data [ ] , which assessed the relationship between the surface structure of nano-objects and their ability to cross cell membranes. both in vivo and in vitro tests on cell cultures have proven the lack of toxicity of mus:ot-nps. coumestrol is an isoflavonoid-like compound having the ability to inhibit the replication of hsv- (both acyclovir sensitive and resistant strains) and also some strains of hsv- [ ] . argenta et al. [ ] have designed a formulation in an effort to obtain a topical product for coumestrol delivery at the level of mucosa. in this approach, the bioactive compound was entrapped by fluid or rigid phospholipid nanoemulsions (dioleylphosphocholine, dopc and distearoylphosphocholine, dspc, respectively) dispersed in a hydroxyethylcellulose gel. the effectiveness of the proposed antiviral agents was tested regarding permeation and retention ability on intact and damaged porcine esophageal mucosae and for antiherpes activity on cell culture assays using vero and gmk ah cell lines. the greatest performance of both coumestrol-loaded nanoemulsions ne-cou/dopc and the same product thickened with hydroxyethylcellulose, hne-cou/dopc, as compared to those based on dspc, relies largely on the physico-chemical properties of the nanoemulsion. the positively charged nanoemulsion showing highest values of ζ-potential may interact with negatively charged surface of mucosa membrane, with beneficial consequences relating to transmucosal delivery of coumestrol [ ] . the length of phospholipids alkyl chain, the number of unsaturations, the lipophilic/hydrophilic balance of the active principle also contributed to the global effect, so that the fluid-state of hydrocarbon chain induced by dopc explained the interaction between the oil-water interface and mucosa, increasing coumestrol permeation and retention. the low ic values proved an enhanced antiviral activity against hsv- and hsv- after coumestrol formulation using nanoemulsions based on dopc, which could be considered for advanced studies in order to be introduced in therapy. the huge socio-economic impact of hiv, as mentioned in section , determined a continuously increased trend of studies related with finding an almost perfect treatment. since the seven classes of antiretroviral drugs defined by fda contain a large number of active principles, plenty of studies regarding their incorporation in different types of nanosystems can be found in the literature. several examples are presented below. rtis classes, nrtis and nnrtis, include some of the drugs often used in hiv treatment plans. recently, grande et al. [ ] published a complex review on rtis nanosystems for controlled drug delivery and our review complements part of the data presented in this article, emphasizing the penetration of biological barriers in vitro or in vivo by nanosystems containing rtis. azt, a high bioavailable drug, has serious side effects, the most common being bone marrow suppression, toxicity for some organs, neutropenia and anaemia. specific target drug delivery using different nanosystems is a promising solution [ ] . atz has been incorporated in hybrid nps based on alginate and stearic acid-poly ethylene glycol. c glioma, neuro brain and hela cells have been used to study the cellular uptake and the cytotoxicity of the nps in vitro. the results proved that these nanosystems are nontoxic and have significant brain cellular uptake, suggesting that they can be used for more complex internalization in brain cells studies [ ] . in another study, sol-oil chemistry has been used to prepare small nps lactoferrin loaded with azt ( - nm in size), stable in biological simulated fluids (gastric and intestinal). antiviral activity of nps has been analysed using supt cells infected with hiv- in virus and the results suggested that the encapsulation of azt in lactoferrin does not influence the drug activity. the nps loaded with azt and the drug alone have been orally administrated to wistar rats of both genders, the performed assays (bone marrow micronucleus, histopathological and biochemical analysis) showing that azt loaded in nps is more efficient and less toxic, compared with the soluble form [ ] . lamivudine-lam, a water-soluble drug with two main drawbacks: its half-life is only h and has a deficient bioavailability, especially in paediatric patients ( %) [ ] . lam has been included in nps based on poly(ε-caprolactone)-pcl [ , ] , poly lactic-co-glycolic acid-plga [ ] , chitosan and sodium alginate [ ] , eudragit e [ ] . the physico-chemical characterization of the obtained nps has shown an adequate size of the nps and a good stability. in vitro drug release tests indicate that nps can support the drug delivery for h, indicating a less frequent administration [ , ] . sneba et al. [ ] have reported a more complex study, where lam-polymeric non-cytotoxic nps have been included in films for drug delivery through the buccal mucosa barrier. four mucoadhesive polymers: polyvinyl alcohol-pva, polyvinyl pyrrolidone-pvp, sodium carboxymethylcellulose-scm, hydroxypropyl methylcellulose-hpmc have been used to prepare the films. moreover, ozturk et al. [ ] , obtained plga nps loaded with lam and proved that are physicochemical stable and slowly released the drug, a great property attributed to ester end-group of plga. because these nps were intended for oral administration, the authors evaluated the gastrointestinal stability of the nps in vitro, using different fluids of biological interest with ph in the range . - . phosphate buffer solution, intestinal fluid phosphate buffer solution, physiological serum and distilled water; the tests have been developed at • c for h. the results indicated that plga nps are promising intestinal targeted drug delivery system for lma, being stable in tested media. in clinical practice, lam is frequently administrated together with azt, therefore this combination of drugs is being studied for target delivery using different types of nps. sankar et al. [ ] used plga, methylmethacrylate-sulfopropylmethacrylate-mma-spm, poly lactic acid-pla, and poly methyl methacrylate-pmma to prepare different types of nps by emulsion polymerization, as drug delivery nanosystems for atz ( %) and lma ( %). in vivo acute toxicity has been studied in mice; the results proving the fact that the drug doses loaded in the nps are not toxic. atz-lam plga nps seemed to be the most promising nanosystems. efavirenz-efv, one of the most used nnrtis in clinical practice, is a poorly water-soluble drug, and the incorporation in different drug delivery nanosystems being a solution for this drawback. patel et al. [ ] obtained nanosuspensions-ns based on povidone polymer-polyvinylpyrrolidone (pvp) k , poloxamers steric stabilizer ( and ) and an anionic electrostatic stabilizer (sodium lauryl sulphate, a steric stabilizer-sls). compared with the drug alone, an important improvement of saturation solubility has been noticed for the ns with efv. the incorneporation of efv in ns increased the absorption of the drug in rat intestine in situ, and very important the oral bioavailability in the studies on albino rabbits. lactoferin used to prepare nps loaded with atz [ ] , as described above, has been used also to encapsulate efv [ ] , based on the same preparation technique: sol-oil chemistry. compared with free efv, the encapsulation of the drug in nps showed a reduced toxicity to peripheral blood mononuclear cells, jurkat t cells and b -f cells, an increased anti-hiv- activity and improved oral bioavailability and pharmacokinetic profile in studies on rats. atv. low brain permeability and antiretroviral drug resistance are two of the most important disadvantages of atv. this pis drug has been encapsulated in slns and studied as nanosystems for brain delivery using hcmec/d as a blood-brain barrier in vitro model, hcmec/d being human brain microvessel endothelial cell line. average atv had an important increase regarding the cellular uptake once delivered through average nanosystems (around nm) [ ] . sqv, another anti-hiv pis, has been incorporated in poly(ethylene oxide)-modified poly (ε-caprolactone) (peo-pcl) nps [ ] by a solvent displacement process. human monocyte/ macrophage (mo/mac) cell line-thp- has been used for in vitro cellular uptake assay. the drug has been successively released intracellular and a meaningful uptake of the sqv-peo-pcl has been noticed. nfv, used in hiv- and hiv- treatment as pi, is a promising drug that can be used also for other grave medical disorders like cancer [ ] . some studies have showed the ability of different types of nps loaded with nfv to activate latent hiv and to restrict viral spread in vitro. kovochich et al. [ ] showed that lipid nps-lnps incorporated with bryostatin- , a protein kinase c activator (lnp-bry), can be loaded with nfv (lnp-bry-nfv), and proved the above mentioned abilities on j-lat full length cells ( . ). tang et al. [ ] have prepared nps based on poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers and anti-cd ro antibody conjugated with suberoylanilide hydroxamic acid (saha) and nfv and tested theirs in vitro properties on ach- cells. more complex studies have been performed by venkatesh et al. [ ] plga nps loaded with nfv have significantly enhanced the oral bioavailability of the drug studied in vivo in new zealand rabbits, a reduced frequency of dosing being needed in this case. the literature data reports also several studies where combinations of pis drugs have been incorporated in nanosystems and pre-clinically evaluated. duan et al. [ ] have included separately atv and drv in lnps, but only atv-lnps proved to form stable drug-lipid concentrations. based on these results, the authors have developed lnps containing atv and rtv and also lnps containing atv + rtv + tenofovir (tfv-an hiv nrtis), the last ones being prepared in a large volume for a preliminary primate pharmacokinetic study. after lnps subcutaneously administration, the three drugs have been detected in plasma for seven days. enf is a fusion inhibitor that is incapable to cross the cerebrospinal fluid. fiandra et al. [ ] proved that by including it into a nanosystem composed from magnetic nanoparticles (mnp) synthesized by solvothermal decomposition in organic solvent followed by fluorescent labelled pma coating could solve enf drawback. in vitro model (co-colture of rbmvecs and astrocytes) and in vivo model (balb/c mice) studies proved that nanoconjugated enf could penetrate bbb. mcv, an entry inhibitor acting as a ccr co-receptor antagonist, has been also included in some nanosystems in order to increase its oral bioavailability. solid drug nanoparticles-sdns, containing wt % mvc and % some polymer/surfactant excipients have been prepared using the emulsion-template freeze drying technique. monolayers of caco- have been used as a human gut in vitro model in order to study the absorption behaviour of mvc sdns and in vivo oral pharmacokinetics of the mvc solid drug nanoparticles (sdns) has been analysed on a rat model. both studies indicated an advanced permeability of the mcv nps (based on pva and sodium , -bis( ethylhexoxy)- , -dioxobutane- -sulfonate (aot) excipients) correlated with the normal drug [ ] . bowman et al. [ ] proved that small organic monovalent molecules conjugated to aunps acts as fusion inhibitors in vitro, while vijayakumar et al. [ ] proved that aunps alone acts as entry inhibitors. moreover, integrase inhibitor, ral, has been functionalized with a thiol group in order to link au-nps. in vitro cellular uptake has been tested on macrophages, human brain microendothelial cells and primary peripheral blood mononuclear cells, the results suggesting that ral-pmba-au-nps penetrate the cells and also can exhibit antiviral activity. in vivo studies performed by injection of ral-pmba-au-nps in female adult balb mice tail proved that the studied nps could cross the bbb [ ] . nanomedicine represents a fast-revolutionizing field that faces rapidly and constantly progress assessed by the numerous nanodrugs that have entered clinical practice and also by even more being investigated in clinical trials. table presents the approved antiviral nanomedicines, from which half are vaccines. [ ] according to singh et al. [ ] review from and also to available information on the respective websites there are still several nanomedicines under evaluation, namely: • fluquit (stp ) from sirnaomics inc. currently under preclinical evaluation, a polymer-based nanotherapeutic that incorporates sirna and targeting the h n (avian flu), h n (swine flu) influenza, and newly emerging h n ; and cervisil (stp ), a nanobased drug candidate, which incorporates sirna for the treatment of hpv and hpv ; • dermavir from genetic immunity, a synthetic pathogen-like nanomedicine that incorporates single plasmid dna expressing hiv antigens that assemble to hiv-like particles; dermavir vaccine completed phase i/ii randomized, placebo-controlled, dose-finding, double-blinded, multicenter study to assess the safety, tolerability and immune response in hiv- -infected adults who are currently receiving anti-hiv treatment (number nct ) [ ] ; • doravirine (mk- ), from merck, a novel, next generation nnrti described as solid drug nanoparticle formulation tested for hiv; currently doravirine completed the pharmacokinetic trial of the bioavailability of four mk- nano formulations in healthy adults (number nct ) [ ] ; • lipid nanoparticles of arb- tkm-hbv containing three rnai therapeutics for hbv genome targeting from arbutus biopharma; in the company completed the phase a, single blind, randomized, placebo controlled, study evaluating the safety, anti-viral activity, and pharmacokinetics (pk) following multiple doses of intravenous arb- (number nct ) [ ] . as discussed above, nanotechnology started to be a critical player in the antiviral therapy. as mentioned by ross et al. [ ] , nanotechnology frees the current therapy payloads in terms of delivery across biological complex barriers, and could resolve the low bioavailability drawback as already stated in section . nanomaterials impart many physical, chemical and biological advantages [ , ] such as: ( ) small particle size in order to facilitate drug delivery through biological barriers, ( ) large surface area to volume ratios to ensure large drug payloads, ( ) tunable surface charge to facilitate cellular entry across the negatively charged cellular membrane, ( ) biomimetic properties which result in intrinsic antiviral assets, ( ) ability to anchor targeting moieties to increase specificity to desired cell types, tissue or other compartments, ( ) improved solubility and pharmacokinetic and/or pharmacodynamics properties translated in longer time to allow greater accumulation, controlled and sustained release, ( ) enhanced efficiency gained either by drug molecules entrapment to protect them from physiologically hostile media, or by using surface conjugation to target drugs to specific tissues, ( ) reduced toxicity and ( ) multifunctionality by combining several beneficial features in a stable construct, designed to simultaneously stimulate the replication of latent virus and deliver an antiviral to the activated cell [ ] . several limitations were also acknowledged such as: ( ) degradation, for example nanoparticles are degraded in the gut following oral administration, or fail to penetrate the mucus barrier and are thus minimally absorbed [ ] , ( ) undesired interactions with biological molecules that leads to opsonization, uptake by macrophages and reduced plasma half-life [ ] , ( ) non-specifically absorption that may induce apoptosis and disrupt cell membrane and adverse immunological responses [ ] , ( ) large dimension for renal clearance therefore cannot be degraded within the body, and are accumulated, leading to toxicity [ ] , and ( ) scaling up issues and high costs. in our perspective, the "ideal" nanocarrier for efficient antiviral delivery must take into considerations several key factors namely: clinical outcome, since patients need safe, effective, targeted, available and affordable therapy, as they are our inspiration; • from the clinical perspective, the future antiviral candidates should improve the efficacy of the fused/encapsulated drug, reduce the intake frequency and time, restrict adverse side effects and reduce therapy costs; • design consideration for the nanoplatforms that will allow targeted delivery of the drugs in sustained released manner and improves efficacy, safety and patient convenience; therefore, from a chemist point of view, hybrid nanosystems can gather all the necessary features in terms of composition, shape and size by overcoming limitations of individual systems and offers greater advantages. starting with the composition, the chosen materials should be biodegradable, biocompatible, and non-toxic, for example polymers are very attractive since they offer the possibility for chemical modifications over the surface or backbone. in addition to these advantages, the second component from the hybrid architecture (in the shape of potential liposomes) should offer besides advanced barrier penetration, higher encapsulation efficiency for the intended drug, which in combination with the polymeric piece will be able to modulate the release kinetics, the stability and prolong drug release. when thinking about the shape, we have in mind targeting capabilities as impact. as we already know, the shape is linked with size and surface charge and density, therefore a complex puzzle that must be solved. the surface charge and density should be carefully chosen during the nanoplatforms design through the surface modification possibility. the ideal candidate here from our perspective is peg due to its versatility to exhibit various charges, shapes and sizes but also to enhance tolerability, reduce clearance, and lengthen circulation time. the size influences the biodistribution and the uptake rate therefore the "nominee" has to be in the submicron size range, recommended to be under nm. taking into consideration the performance indicators of nanomedicine, we claim that the development a personalized nanomedicine is possible via a synergistically approach. since the development of "best" viral carriers involves a multidisciplinary team, virologists should be directly implicated in the development, offering specialized support on the following matters: identification of differentially expressed moieties virus cells for targeted delivery, elucidation of the type of desired targeted and the response from the host cells to nanodelivery platforms. therefore, multidisciplinary research-oriented efforts have to be related also to system biology by exploring machine learning for process optimization and pharmacology in order to introduce best appropriate combination of therapeutic agents. in , an interdisciplinary team of virologists and biochemists, which developed low-cost and "cell-friendly" nanogels that can efficiently prevent viral infections, addressed these challenges [ ] . here, the flexible, nontoxic and broad-spectrum nanogels based on dendritic polyglycerol sulfate mimic cellular surface receptors where several viral families bind. the designed nanogels can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection since they act as robust inhibitors for these viruses. when thinking about the translation into the clinical practice, the nano-based future antiviral therapy must follow a specific flow-chart, starting with the optimization and scale-up practices according to the good manufacturing practice, the elaboration of suitable regulatory guidelines and finishing with the development of cost-effective and high quality formulations available worldwide. taken into consideration all these enhanced features, the road to clinical practice still has many addressed issues in order to provide effective and safe antiviral nano-formulations to patients. treating or improving treatment success rate for viral diseases are fundamental responsibilities. the established potential and boosted progress of nanotechnology in antiviral therapy development generates great expectations for new therapeutic innovative strategies for attacking or eradicating viral disorders. at present, studies explored numerous and diverse nano-platforms including nanoparticles, liposomes, micelles, with different compositions, size, with single or combined entrapped drugs that may serve as potential antiviral drug delivery transporters. these nano-based systems have exhibited versatile features to improve the identified current therapy drawback. however, the clinical use of a nano-based antiviral formulation to date based on our knowledge has turned out just a few approved or under clinical trials nanoformulations, mainly vaccines, despite more than years of constant efforts. it is expected in the upcoming years that part of this "success preclinical story" to be scaled-up, translated and applied for better outcome, convenience and access for patients. environmental and social influences on emerging infectious diseases: past, present and future triage nurse application of the ottawa knee rule the joint united nations programme on hiv/aids. available online who. herpes simplex virus key facts the estimated economic burden of genital herpes in the united states. an analysis using two costing approaches socio-economic impact of antiretroviral treatment in hiv patients. an economic review of cost savings after introduction of haart the evolution of three decades of antiretroviral therapy: challenges, triumphs and the promise of the future: three decades of antiretroviral therapy socio-economic impact of antiviral intervention cost and access to direct-acting antiviral agents costs, and affordability of new medicines for hepatitis c in countries: an economic analysis evidence from cost-effectiveness research economic note: cost of illness studies methods for the economic evaluation of health care programmes cost-of-illness studies: concepts, scopes, and methods nanoparticulate delivery systems for antiviral drugs how viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication the common cold herpetic eye disease study: lessons learned adenoviral keratoconjunctivitis viral pneumonia la revue de médecine interne viral myocarditis viral infections in type diabetes mellitus-why the β cells? hepatitis a and b infections hepatitis d virus: introduction and epidemiology. cold spring harb hepatitis e: discovery, global impact, control and cure clinical features of varicella-zoster virus infection molecular mechanisms of varicella zoster virus pathogenesis viral skin diseases viral gastroenteritis in the adult population viral gastroenteritis in adults epidemiology of gastroenteritis viruses in japan: prevalence, seasonality, and outbreak: epidemiology of gastroenteritis viruses in japan sexually transmitted and anorectal infectious diseases expression of animal virus genomes the pharmacological basis of therapeutics a new agent with potent anti-herpesvirus activity long-term foscarnet therapy remodels thymidine analogue mutations and alters resistance to zidovudine and lamivudine in hiv- docosanol: a topical antiviral for herpes labialis phosphorylation of acyclovir diphosphate by cellular enzymes history, pharmacokinetics, and pharmacology of acyclovir causes of severe pneumonia requiring hospital admission in children without hiv infection from africa and asia: the perch multi-country case-control study antiviral drug resistance: mechanisms and clinical implications an update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients antiviral treatment of cytomegalovirus infection and resistant strains antiviral agents active against influenza a viruses inhibitory and combinatorial effect of diphyllin, a v-atpase blocker, on influenza viruses inhibitors of v-atpases: old and new players suppression of influenza a virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin a antiviral treatments. clin neuraminidase inhibitors for preventing and treating influenza in adults and children oseltamivir resistance during treatment of influenza a (h n ) infection mechanism of action of ribavirin in the treatment of chronic hepatitis c amino acid ester prodrugs of nucleoside and nucleotide antivirals clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of dna virus and retrovirus infections an evaluation of entecavir for the treatment of chronic hepatitis b infection in adults telbivudine for the management of chronic hepatitis b virus infection telbivudine: a new treatment for chronic hepatitis b antiretroviral drugs used in the treatment of hiv infection nucleoside and nucleotide analogue reverse transcriptase inhibitors: a clinical review of antiretroviral resistance lamivudine: a review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in the management of hiv infection a review of the toxicity of hiv medications efficacy of tenofovir % vaginal gel in reducing the risk of hiv- and hsv- infection conformational changes in hiv- reverse transcriptase induced by nonnucleoside reverse transcriptase inhibitor binding hiv protease inhibitors: a review of molecular selectivity and toxicity pharmacotherapy of hiv- infection: focus on ccr antagonist maraviroc hiv entry inhibitors and their potential in hiv therapy hiv entry inhibitors: progress in development and application progress in hiv- integrase inhibitors: a review of their chemical structure diversity raltegravir with optimized background therapy for resistant hiv- infection the first hiv integrase inhibitor the first hiv type integrase inhibitor canton, i. inspired by nature: fundamentals in nanotechnology design to overcome biological barriers air-blood barrier translocation of tracheally instilled gold nanoparticles inversely depends on particle size walking the line: the fate of nanomaterials at biological barriers anti-hiv- nanotherapeutics: promises and challenges for the future approaches in polymeric nanoparticles for vaginal drug delivery: a review of the state of the herpetic eye disease study group. oral acyclovir for herpes simplex virus eye disease: effect on prevention of epithelial keratitis and stromal keratitis drug delivery approaches of an antiviral drug: a comprehensive review acyclovir: a decade later comparison of famciclovir, valaciclovir, and brivudine treatments in adult immunocompetent patients with herpes zoster crossing biological barriers with nanogels to improve drug delivery performance mucoadhesive nanogels by ionotropic crosslinking of chitosan-goligo(nipaam) polymeric micelles as novel drug nanocarriers layers of the skin nanoparticles and their interactions with the dermal barrier penetration of nanoparticles into human skin nano-enabled delivery of diverse payloads across complex biological barriers nanocarriers for effective topical delivery of anti-infectives the cell membrane physiology and pathophysiology of the blood-brain barrier: p-glycoprotein and occludin trafficking as therapeutic targets to optimize central nervous system drug delivery efflux mechanisms at the developing brain barriers: abc-transporters in the fetal and postnatal rat multidrug resistance in cancer: role of atp-dependent transporters abc transporters in drug-resistant epilepsy: mechanisms of upregulation and therapeutic approaches abc transporters at the blood-brain barrier regulation of abc transporters at the blood-brain barrier mechanisms of blood-brain barrier disruption in herpes simplex encephalitis disruption of the blood brain barrier is vital property of neurotropic viral infection of the central nervous system towards nanomedicines for neuroaids: nanomedicines for neuroaids analysis of human immunodeficiency virus type gp sequences from a patient with hiv dementia: evidence for monocyte trafficking into brain effect of nanoparticulate polybutylcyanoacrylate and methylmethacrylatesulfopropylmethacrylate on the permeability of zidovudine and lamivudine across the in vitro blood-brain barrier targeted brain delivery of azt via transferrin anchored pegylated albumin nanoparticles blood-brain barrier transport of therapeutics via receptor-mediation magnetic nanoformulation of azidothymidine -triphosphate for targeted delivery across the blood & ndash nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice unique benefits of nanotechnology to drug delivery and diagnostics. in characterization of nanoparticles intended for drug delivery strategies in the design of nanoparticles for therapeutic applications nanostructured materials for applications in drug delivery and tissue engineering biomimetic and bioinspired nanoparticles for targeted drug delivery targeted nanomedicines: effective treatment modalities for cancer, aids and brain disorders role of nanotechnology in hiv/aids vaccine development nanotechnology approaches to eradicating hiv reservoirs preparation and ocular pharmacokinetics of ganciclovir liposomes recombinant high-density lipoprotein complex as a targeting system of nosiheptide to liver cells liver targeting and anti-hbv activity of reconstituted hdl-acyclovir palmitate complex targeted delivery of sirna against hepatitis c virus by apolipoprotein a-i-bound cationic liposomes rnai-mediated ccr silencing by lfa- -targeted nanoparticles prevents hiv infection in blt mice butyldeoxynojirimycin is a broadly effective anti-hiv therapy significantly enhanced by targeted liposome delivery targeted delivery of indinavir to hiv- primary reservoirs with immunoliposomes sustained and specific in vitro inhibition of hiv- replication by a protease inhibitor encapsulated in gp -targeted liposomes drug-encapsulated carbon (decon): a novel platform for enhanced drug delivery nanoparticle-based topical ophthalmic formulation for sustained release of stereoisomeric dipeptide prodrugs of ganciclovir inhibition of h n influenza virus-induced apoptosis by functionalized selenium nanoparticles with amantadine through ros-mediated akt signaling pathways inhibitory activity of selenium nanoparticles functionalized with oseltamivir on h n influenza virus reversal of h n influenza virus-induced apoptosis by silver nanoparticles functionalized with amantadine antiviral efficacy of nanoparticulate vacuolar atpase inhibitors against influenza virus infection bioerodible polymeric nanoparticles for targeted delivery of proteic drugs improved safety, bioavailability and pharmacokinetics of zidovudine through lactoferrin nanoparticles during oral administration in rats preparation and in vitro characterization of lamivudine loaded nanoparticles prepared by acid and/or ester terminated plga for effective oral anti-retroviral therapy formulation and in-vitro evaluation of zidovudine-lamivudine nanoparticles intracellular delivery of saquinavir in biodegradable polymeric nanoparticles for hiv/aids plga-peg nanoparticles coated with anti-cd ro and loaded with hdac plus protease inhibitors activate latent hiv and inhibit viral spread fabrication and in vivo evaluation of nelfinavir loaded plga nanoparticles for enhancing oral bioavailability and therapeutic effect an oral formulation of efavirenz-loaded lactoferrin nanoparticles with improved biodistribution and pharmacokinetic profile the mixed lineage kinase- inhibitor urmc- improves therapeutic outcomes for long-acting antiretroviral therapy improving maraviroc oral bioavailability by formation of solid drug nanoparticles polymeric nanoparticles containing combination antiretroviral drugs for hiv type treatment pharmacokinetic and tissue distribution profile of long acting tenofovir alafenamide and elvitegravir loaded nanoparticles in humanized mice model triple drug combination of zidovudine, efavirenz and lamivudine loaded lactoferrin nanoparticles: an effective nano first-line regimen for hiv therapy multivalent peptide-nanoparticle conjugates for influenza-virus inhibition amantadine surface-modified silver nanorods improves immunotherapy of hiv vaccine against hiv-infected cells enhancing the delivery of anti retroviral drug "saquinavir"; across the blood brain barrier using nanoparticles acyclovir-loaded chitosan nanospheres from nano-emulsion templating for the topical treatment of herpesviruses infections nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation assessment of ocular pharmacokinetics and safety of ganciclovir loaded nanoformulations tissue distribution of borneol-modified ganciclovir-loaded solid lipid nanoparticles in mice after intravenous administration solid lipid nanoparticles enhance the delivery of the hiv protease inhibitor, atazanavir, by a human brain endothelial cell line activation of latent hiv using drug-loaded nanoparticles evaluation of atazanavir and darunavir interactions with lipids for developing ph-responsive anti-hiv drug combination nanoparticles anti-hiv drug-combination nanoparticles enhance plasma drug exposure duration as well as triple-drug combination levels in cells within lymph nodes and blood in primates nanotechnology: a reality for diagnosis of hcv infectious disease hiv biosensors for early diagnosis of infection: the intertwine of nanotechnology with sensing strategies chitosan as a bioactive polymer: processing, properties and applications anti-herpes simplex virus (hsv- and hsv- ) activity of biogenic gold and silver nanoparticles using seaweed sargassum wightii a novel extracellular synthesis of monodisperse gold nanoparticles using marine alga, sargassum wightii greville broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism inhibition of human metapneumovirus binding to heparan sulfate blocks infection in human lung cells and airway tissues pathogen inhibition by multivalent ligand architectures surface-structureregulated cell-membrane penetration by monolayer-protected nanoparticles antiherpes evaluation of soybean isoflavonoids topical delivery of coumestrol from lipid nanoemulsions thickened with hydroxyethylcellulose for antiherpes treatment lecithin based nanoemulsions: a comparative study of the influence of non-ionic surfactants and the cationic phytosphingosine on physicochemical behaviour and skin permeation reverse transcriptase inhibitors nanosystems designed for drug stability and controlled delivery novel dendritic structure of alginate hybrid nanoparticles for effective anti-viral drug delivery design of antiretroviral drug-polymeric nanoparticles laden buccal films for chronic hiv therapy in paediatrics influence of solvent evaporation technique parameters on diameter of submicron lamivudine-poly-ε-caprolactone conjugate particles nanoencapsulation of water-soluble drug, lamivudine, using a double emulsion spray-drying technique for improving hiv treatment formulation and characterisation of chitosan based lamivudine nanoparticles the antiretroviral agent nelfinavir mesylate: a potential therapy for systemic sclerosis inhibition of hiv fusion with multivalent gold nanoparticles gold nanoparticles as an hiv entry inhibitor gold nanoparticles to improve hiv drug delivery the role of nanotechnology in the treatment of viral infections epaxal ® : a virosomal vaccine to prevent hepatitis a infection pharmacokinetics and pharmacodynamics modeling and simulation systems to support the development and regulation of liposomal drugs eleven years of inflexal ® v-a virosomal adjuvanted influenza vaccine package insert peg-introntm (peginterferon alfa- b) powder for injection, schering corporation progress in nanomedicine: approved and investigational nanodrugs tolerability and immune response to lc , an experimental therapeutic vaccine bioavailability of mk- experimental nano formulations in healthy adults (mk- - )-clinicaltrials.gov identifier: nct . available online subjects with chronic hbv infection receiving nucleos(t)ide analogue therapy-clinicaltrials.gov identifier: nct . available online therapeutic nanomedicine surmounts the limitations of pharmacotherapy nanotechnology and the treatment of hiv infection physiologically based pharmacokinetic modeling of nanoparticles multifunctional nanoparticles-properties and prospects for their use in human medicine state of the art of nanocrystals-special features, production, nanotoxicology aspects and intracellular delivery nanotechnology and drug delivery part : nanostructures for drug delivery multivalent flexible nanogels exhibit broad-spectrum antiviral activity by blocking virus entry this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -zult f authors: nguyen, thin; le, hang; quinn, thomas p.; nguyen, tri; le, thuc duy; venkatesh, svetha title: graphdta: predicting drug–target binding affinity with graph neural networks date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: zult f the development of new drugs is costly, time consuming, and often accompanied with safety issues. drug repurposing can avoid the expensive and lengthy process of drug development by finding new uses for already approved drugs. in order to repurpose drugs effectively, it is useful to know which proteins are targeted by which drugs. computational models that estimate the interaction strength of new drug--target pairs have the potential to expedite drug repurposing. several models have been proposed for this task. however, these models represent the drugs as strings, which is not a natural way to represent molecules. we propose a new model called graphdta that represents drugs as graphs and uses graph neural networks to predict drug--target affinity. we show that graph neural networks not only predict drug--target affinity better than non-deep learning models, but also outperform competing deep learning methods. our results confirm that deep learning models are appropriate for drug--target binding affinity prediction, and that representing drugs as graphs can lead to further improvements. availability of data and materials the proposed models are implemented in python. related data, pre-trained models, and source code are publicly available at https://github.com/thinng/graphdta. all scripts and data needed to reproduce the post-hoc statistical analysis are available from https://doi.org/ . /zenodo. . contact thin.nguyen@deakin.edu.au it costs about . billion us dollars to develop a new drug [ ] , and can take up to years for fda approval [ , ] . finding new uses for already approved drugs avoids the expensive and lengthy process of drug development [ , ] . for example, nearly existing fda-approved drugs are currently being investigated to see if they can be repurposed to treat covid- [ ] . in order to repurpose drugs effectively, it is useful to know which proteins are targeted by which drugs. high-throughput screening experiments are used to examine the affinity of a drug toward its targets; however, these experiments are costly and time-consuming [ , ] , and an exhaustive search is infeasible because there are millions of drug-like compounds [ ] and hundreds of potential targets [ , ] . as such, there is a strong motivation to build computational models that can estimate the interaction strength of new drug-target pairs based on previous drug-target experiments. several computational approaches have been proposed for drug-target affinity (dta) prediction [ , , ] . one approach is molecular docking, which predicts the stable d structure of a drug-target complex via a scoring function [ ] . even though the molecular docking approach is potentially more informative, it require knowledge about the crystallized structure of proteins which may not be available. another approach uses collaborative filtering. for example, the simboost model uses the affinity similarities among drugs and among targets to build new features. these features are then used as input in a gradient boosting machine to predict the binding affinity for unknown drug-target pairs [ ] . alternatively, the similarities could come from others sources (rather than the training data affinities). for example, kernel-based methods use kernels built from molecular descriptors of the drugs and targets within a regularized least squares regression (rls) framework [ , ] . to speed up model training, the kronrls model computes a pairwise kernel k from the kronecker product of the drug-by-drug and protein-by-protein kernels [ , ] (for which any similarity measure can be used). dta prediction may also benefit from adopting methods for predicting drug-target interactions (dti). approaches in this line of work include dti-cdf [ ] , a cascade deep forest model, or dti-mlcd [ ] , a multi-label learning supported with community detection. another approach uses neural networks trained on d representations of the drug and protein sequences. for example, the deepdta model uses d representations and layers of d convolutions (with pooling) to capture predictive patterns within the data [ ] . the final convolution layers are then concatenated, passed through a number of hidden layers, and regressed with the drug-target affinity scores. the widedta model is an extension of deep-dta in which the sequences of the drugs and proteins are first summarized as higher-order features [ ] . for example, the drugs are represented by the most common sub-structures (the ligand maximum common substructures (lmcs) [ ] ), while the proteins are represented by the most conserved sub-sequences (the protein domain profiles or motifs (pdm) from prosite [ ] ). while widedta [ ] and deepdta [ ] learn a latent feature vector for each protein, the padme model [ ] uses fixed-rule descriptors to represent proteins, and performs similarly to deepdta [ ] . the deep learning models are among the best performers in dta prediction [ ] . however, these models represent the drugs as strings, which are not a natural way to represent molecules. when using strings, the structural information of the molecule is lost, which could impair the predictive power of a model as well as the functional relevance of the learned latent space. already, graph convolutional networks have been used in computational drug discovery, including interaction prediction, synthesis prediction, de novo molecular design, and quantitative structure prediction [ , , , , , , ] . however, graph neural networks have not been used for dta prediction. of these, [ , , ] are closest to our work, but look at binary prediction, while our model looks to predict a continuous value of binding affinity. also, in [ ] , the input is a drug descriptor (single input), while our model takes as input both a drug descriptor and a sequence (dual input). in this article, we propose graphdta, a new neural network architecture capable of directly modelling drugs as molecular graphs, and show that this approach outperforms state-of-the-art deep learning models on two drug-target affinity prediction benchmarks. the approach is based on the solution we submitted to the idg-dream drug-kinase binding prediction challenge , where we were among the top ten performers from registered participants . in order to better understand how our graph-based model works, we performed a multivariable statistical analysis of the model's latent space. we identified correlations between hidden node activations and domain-specific drug annotations, such as the number of aliphatic oh groups, which suggests that our graph neural network can automatically assign importance to well-defined chemical features without any prior knowledge. we also examine the model's performance and find that a handful of drugs contribute disproportionately to the total prediction error, and that these drugs are inliers (i.e., not outliers) in an ordination of the model's latent space. taken together, our results suggest that graph neural networks are highly accurate, abstract meaningful concepts, and yet fail in predictable ways. we conclude with a discussion about how these insights can feedback into the research cycle. we propose a novel deep learning model called graphdta for drug-target affinity (dta) prediction. we frame the dta prediction problem as a regression task where the input is a drug-target pair and the output is a continuous measurement of binding affinity for that pair. existing methods represent the input drugs and proteins as d sequences. our approach is different; we represent the drugs as molecular graphs so that the model can directly capture the bonds among atoms. smiles (simplified molecular input line entry system) was invented to represent molecules to be readable by computers [ ] , enabling several efficient figure : this figure shows the graphdta architecture. it takes a drug-target pair as the input data, and the pair's affinity as the output data. it works in stages. first, the smiles code of a drug is converted into a molecular graph, and a deep learning algorithm learns a graph representation. meanwhile, the protein sequence is encoded and embedded, and several d convolutional layers learn a sequence representation. finally, the two representation vectors are concatenated and passed through several fully connected layers to estimate the output drug-target affinity value. applications, including fast retrieval and substructure searching. from the smiles code, drug descriptors like the number of heavy atoms or valence electrons can be inferred and readily used as features for affinity prediction. one could also view the smiles code as a string. then, one could featurize the strings with natural language processing (nlp) techniques, or use them directly in a convolutional neural network (cnn). instead, we view drug compounds as a graph of the interactions between atoms, and build our model around this conceptualization. to describe a node in the graph, we use a set of atomic features adapted from deepchem [ ] . here, each node is a multi-dimensional binary feature vector expressing five pieces of information: the atom symbol, the number of adjacent atoms, the number of adjacent hydrogens, the implicit value of the atom, and whether the atom is in an aromatic structure [ ] . we convert the smiles code to its corresponding molecular graph and extract atomic features using the open-source chemical informatics software rdkit [ ]. one-hot encoding has been used in previous works to represent both drugs and proteins, as well as other biological sequences like dna and rna. this paper tests the hypothesis that a graph structure could yield a better representation for drugs, and so only drugs were represented as a graph. although one could also represent proteins as graphs, doing so is more difficult because the tertiary structure is not always available in a reliable form. as such, we elected to use the popular one-hot encoding representation of proteins instead. for each target in the experimented datasets, a protein sequence is obtained from the uniprot database using the target's gene name. the sequence is a string of ascii characters which represent amino acids. each amino acid type is encoded with an integer based on its associated alphabetical symbol (e.g., alanine (a) is , cystine (c) is , aspartic acid (d) is , and so on), allowing the protein to be represented as an integer sequence. to make it convenient for training, the sequence is cut or padded to a fixed length sequence of residues. in case a sequence is shorter, it is padded with zero values. these integer sequences are used as input to the embedding layers which return a -dimensional vector representation. next, three d convolutional layers are used to learn different levels of abstract features from the input. finally, a max pooling layer is applied to get a representation vector of the input protein sequence. having the drug compounds represented as graphs, the task now is to design an algorithm that learns effectively from graphical data. the recent success of cnn in computer vision, speech recognition, and natural language processing has encouraged research into graph convolution. a number of works have been proposed to handle two main challenges in generalizing cnn to graphs: ( ) the formation of receptive fields in graphs whose data points are not arranged as euclidean grids, and ( ) the pooling operation to down-sample a graph. these new models are called graph neural networks. in this work, we propose a new dta prediction model based on a combination of graph neural networks and conventional cnn. figure shows a schematic of the model. for the proteins, we use a string of ascii characters and apply several d cnn layers over the text to learn a sequence representation vector. specifically, the protein sequence is first categorically encoded, then an embedding layer is added to the sequence where each (encoded) character is represented by a -dimensional vector. next, three d convolutional layers are used to learn different levels of abstract features from the input. finally, a max pooling layer is applied to get a representation vector of the input protein sequence. this approach is similar to the existing baseline models. for the drugs, we use the molecular graphs and trial graph neural network variants, including gcn [ ] , gat [ ] , gin [ ] , and a combined gat-gcn architecture, all of which we describe below. in this work, we focus on predicting a continuous value indicating the level of interaction of a drug and a protein sequence. each drug is encoded as a graph and each protein is represented as a string of characters. to this aim, we make use of gcn model [ ] for learning on graph representation of drugs. note that, however, the original gcn is designed for semi-supervised node classification problem, i.e., the model learns the node-level feature vectors. for our goal, to estimate the drug-protein interaction, a graph-level representation of each drug is required. common techniques to aggregate the whole graph feature from learned node features include sum, average, and max pooling. in our experiments, the use of max pooling layer in gcn-based graphdta usually results in better performance compared to that of the remaining. formally, denote a graph for a given drug as g = (v, e), where v is the set of n nodes each is represented by a c-dimensional vector and e is the set of edges represented as an adjacency matrix a. a multi-layer graph convolutional network (gcn) takes as input a node feature matrix x ∈ r n ×c (n = |v |, c: the number of features per node) and an adjacency matrix a ∈ r n ×n ; then produces a node-level output z ∈ r n ×f (f : the number of output features per node). a propagation rule can be written in the normalized form for stability, as in [ ] : whereà = a + i n is the adjacency matrix of the undirected graph with added self-connections,d ii = ià ii ; h (l) ∈ r n ×c is the matrix of activation in the l th layer, h ( ) = x, σ is an activation function, and w is learnable parameters. a layer-wise convolution operation can be approximated, as in [ ] : where Θ ∈ r c×f (f : the number of filters or feature maps) is the matrix of filter parameters. note that, however, the gcn model learns node-level outputs z ∈ r n ×f . to make the gcn applicable to the task of learning a representation vector of the whole graph, we add a global max pooling layer right after the last gcn layer. in our gcn-based model, we use three consecutive gcn layers, each activated by a relu function. then a global max pooling layer is added to obtain the graph representation vector. unlike graph convolution, the graph attention network (gat) [ ] proposes an attention-based architecture to learn hidden representations of nodes in a graph by applying a self-attention mechanism. the building block of a gat architecture is a graph attention layer. the gat layer takes the set of nodes of a graph as input, and applies a linear transformation to every node by a weigh matrix w. for each input node i in the graph, the attention coefficients between i and its first-order neighbors are computed as this value indicates the importance of node j to node i. these attention coefficients are then normalized by applying a soft-max function, then used to compute the output features for nodes as σ( where σ(.) is a non-linear activation function and α ij are the normalized attention coefficients. in our model, the gat-based graph learning architecture includes two gat layers, activated by a relu function, then followed a global max pooling layer to obtain the graph representation vector. for the first gat layer, multi-headattentions are applied with the number of heads set to , and the number of output features set to the number of input features. the number of output features of the second gat is set to . the graph isomorphism network (gin) [ ] is newer method that supposedly achieves maximum discriminative power among graph neural networks. specifically, gin [ , ] . we compare graph neural network variants: gin [ ] , gat [ ] , gcn [ ] , and combined gat-gcn [ , ] . italics: best for baseline models, bold: better than baselines. where is either a learnable parameter or a fixed scalar, x is the node feature vector, and b(i) is the set of nodes neighboring i. in our model, the gin-based graph neural net consists of five gin layers, each followed by a batch normalization layer. finally, a global max pooling layer is added to obtain the graph representation vector. we also investigate a combined gat-gcn model. here, the graph neural network begins with a gat layer that takes the graph as input, then passes a convolved feature matrix to the subsequent gcn layer. each layer is activated by a relu function. the final graph representation vector is then computed by concatenating the global max pooling and global mean pooling layers from the gcn layer output. to compare our model with the state-of-the-art deepdta [ ] and widedta [ ] models, we use the same datasets from the [ , ] benchmarks: davis contains the binding affinities for all pairs of drugs and targets, measured as k d constants and ranging from . to . [ ] . [ , ] . we compare graph neural network variants: gin [ ] , gat [ ] , gcn [ ] , and combined gat-gcn [ , ] . italics: best for baseline models, bold: better than baselines. kiba contains the binding affinities for , drugs and targets, measured as kiba scores and ranging from . to . [ ] . to make the comparison as fair as possible, we use the same set of training and testing examples from [ , ] , as well as the same performance metrics: mean square error (mse, the smaller the better) and concordance index (ci, the larger the better). for all baseline methods, we report the performance metrics as originally published in [ , ] . the hyper-parameters used for our experiments are summarized in table . the hyper-parameters were not tuned, but chosen a priori based on our past modelling experience. the activation of nodes within layers of a deep neural network are called latent variables, and can be analyzed directly to understand how a model's performance relates to domain knowledge [ ] . we obtained the latent variables from the graph neural network layer, and analyzed them directly through a redundancy analysis. this multivariable statistical method allows us to measure the percent of the total variance within the latent variables that can be explained by an external data source. in our case, the external data source is a matrix of molecular joelib features/descriptors [ ] for each drug (available from chemmine tools [ ] ). we also compare the value of the principal components from these latent variables with the per-drug test set error. here, the per-drug (or per-protein) error refers to the median of the absolute error between the predicted dta and the ground-truth dta for all test set pairs containing that drug (or that protein). for these analyses, we focus on the gin model [ ] (because of its superior performance) and the kiba dataset [ ] (because of its larger drug catalog). . graphical models outperform the state-of-the-art table compares the performance of variant graphdta models with the existing baseline models for the davis dataset. here, all variants had the lowest mse. the best variant had an mse of . which is . % lower than the best baseline of . . the improvement is less obvious according to the ci metric, where only of the variants had the highest ci. the best ci for a baseline model was . . by comparison, the gat and gin models achieved a ci of . and . , respectively. table compares the performance of the graphdta models with the existing baseline models for the kiba dataset. here, of the variants had the lowest mse and the highest ci, including gin, gcn, and gat-gcn. of note, the best mse here is . , which is . % lower than the best baseline of . . of all variants tested, gin is the only one that had the best performance for both datasets and for both performance measures. for this reason, we focus on the gin in all post-hoc statistical analyses. a graph neural network works by abstracting the molecular graph of each drug into a new feature vector of latent variables. in our model, there are latent variables which together characterise the structural properties of the drug. since the latent variables are learned during the dta prediction task, we assume that they represent graphical features that contribute meaningfully to dta. unfortunately, it is not straightforward to determine the molecular substructures to which each latent variable corresponds. however, we can regress [ ] . the blue dots represent drugs, the green dots represent latent variables (the furthest from origin are labelled), and the arrows represent molecular descriptors (the longest are labelled). the right panel of the figure shows the activation of two latent variables plotted against the number of aliphatic oh groups in that drug. these results suggest that the graph convolutional network can abstract known molecular descriptors without any prior knowledge. here, we see that the errors are not distributed evenly across the drugs. it is harder to predict the target affinities for some drugs than others. the learned latent space with a matrix of known molecular descriptors to look for overlap. figure shows a redundancy analysis of the latent variables regressed with molecular descriptors [ ] (available from chemmine tools [ ] ). from this, we find that . % of the latent space is explained by the known descriptors, with the "number of aliphatic oh groups" contributing most to the explained variance. indeed, two latent variables correlate strongly with this descriptor: hidden nodes v and v both tend to have high activation when the number of aliphatic oh groups is large. this finding provides some insight into how the graphical model might "see" the drugs as a set of molecular sub-structures, though most of the latent space is orthogonal to the known molecular descriptors. although the graphdta model outperforms its competitors, we wanted to know more about why its predictions sometimes failed. for this, we averaged the prediction error for each drug (and each protein), for both the davis and kiba test sets. figures and show the median of the absolute error (mae) for affinity prediction, sorted from smallest to largest. interestingly, we see that a handful of drugs (and a handful of proteins) contribute disproportionately to the overall error. of note, chembl (an alk inhibitor), chembl (a pdk inhibitor) and the protein csnk e all had an mae above . we examined the latent space with regard to the prediction error, but could here, we see that the errors are not distributed evenly across the proteins. it is harder to predict the target affinities for some proteins than others. not find any obvious pattern that separated hard-to-predict drugs from easy-topredict drugs. the only trend we could find is that the easy-to-predict drugs are more likely to appear as outliers in a pca of the latent space. supplemental figure (https://github.com/thinng/graphdta/blob/master/supplement.pdf) shows the median errors plotted against the first six principal components, where we see that the hard-to-predict drugs usually appear close to the origin. we interpret this to mean that drugs with unique molecular sub-structures are always easy to predict. on the other hand, the hard-to-predict drugs tend to lack unique structures, though this is apparently true for many easy-to-predict drugs too. knowing how a model works and when a model fails can feedback into the research cycle. in the post-hoc statistical analysis of our model, we find that a graph neural network can learn the importance of known molecular descriptors without any prior knowledge. however, most of the learned latent variables remain unexplained by the available descriptors. yet, the model's performance implies that these learned representations are useful in affinity prediction. this suggests that there are both similarities and differences in how machines "see" chemicals versus how human experts see them. understanding this distinction may further improve model performance or reveal new mechanisms behind drugtarget interactions. meanwhile, the distribution of the test set errors suggest that there are "problem drugs" (and "problem proteins") for which prediction is especially difficult. one could action this insight either by collecting more training data for these drugs (or proteins), or by using domain-knowledge to engineer features that complement the molecular graphs. indeed, knowing that the pca outliers are the easiest to predict suggests that some additional feature input may be needed to differentiate between drugs that lack distinct molecular sub-graphs. although d graphs contain more information than d strings, our model still neglects the stereochemistry of the molecules. future experiments could test whether representing drugs in d (or proteins in d) further improves model performance. interestingly, under-representation of proteins in the training set does seem to be the reason for the "problem proteins". the supplement shows an analysis of the effect of homologous proteins on test set performance. although we see that test set error varies across clustered protein groups, the training set represents all protein clusters equally well. this suggests that the variation in test set performance is not simply explained by asymmetrical representation of protein groups within the training set. we test graphdta with four graph neural network variants, including gcn, gat, gin, and a combined gat-gcn architecture, for the task of drug-affinity prediction. we benchmark the performance of these models on the davis and kiba datasets. we find graphdta performs well for two separate benchmark datasets and for two key performance metrics. in a post-hoc statistical analysis of our model, we find that graphdta can learn the importance of known molecular descriptors without any prior knowledge. we also examine the model's performance and find that a handful of drugs contribute disproportionately to the total prediction error. although we focus on drug-target affinity prediction, our graphdta model is a generic solution for any similar problem where either data input can be represented as a graph. it may be possible to improve performance further by representing proteins as graphs too, for example by a graph of their d structure. however, determining the d structure of a target protein is very challenging. we chose to use the primary protein sequence because it is readily available. the use of d sequences, instead of d structures, also reduces the number of parameters that we need to learn, making it less likely that we over-fit our model to the training data. still, for some problem applications, it may make sense to use structural information, as well as binding site, binding confirmation, and solution environment information, to augment the model. new drugs cost us $ . billion to develop drug repositioning: identifying and developing new uses for existing drugs pharmacogenetics in drug discovery and development: a translational perspective overcoming drug development bottlenecks with repurposing: old drugs learn new tricks a sars-cov- -human protein-protein interaction map reveals drug targets and potential drug-repurposing. biorxiv protein kinases-the major drug targets of the twenty-first century? protein kinase inhibitors: insights into drug design from structure frequent substructure-based approaches for classifying chemical compounds the protein kinase complement of the human genome maximizing diversity from a kinase screen: identification of novel and selective pan-trk inhibitors for chronic pain a machine learning-based method to improve docking scoring functions and its application to drug repurposing drug discovery in the age of systems biology: the rise of computational approaches for data integration the drug repurposing hub: a next-generation drug library and information resource an overview of scoring functions used for protein-ligand interactions in molecular docking simboost: a read-across approach for predicting drug-target binding affinities using gradient boosting machines computational-experimental approach to drug-target interaction mapping: a case study on kinase inhibitors learning with multiple pairwise kernels for drug bioactivity prediction dti-cdf: a cascade deep forest model towards the prediction of drugtarget interactions based on hybrid features predicting drug-target interactions using multi-label learning with community detection method (dti-mlcd). biorxiv deepdta: deep drugtarget binding affinity prediction widedta: prediction of drug-target binding affinity linguistic measures of chemical diversity and the 'keywords' of molecular collections prosite, a protein domain database for functional characterization and annotation padme: a deep learning-based framework for drug-target interaction prediction djork-arné clevert, and sepp hochreiter. large-scale comparison of machine learning methods for drug target prediction on chembl graph convolutional neural networks for predicting drug-target interactions chemi-net: a molecular graph convolutional network for accurate drug property prediction convolutional neural network based on smiles representation of compounds for detecting chemical motif molecular graph convolutions: moving beyond fingerprints graph convolutional networks for computational drug development and discovery large-scale learnable graph convolutional networks interpretable drug target prediction using deep neural representation smiles, a chemical language and information system. . introduction to methodology and encoding rules deep learning for the life sciences: applying deep learning to genomics rdkit: open-source cheminformatics semi-supervised classification with graph convolutional networks graph attention networks how powerful are graph neural networks? comprehensive analysis of kinase inhibitor selectivity making sense of large-scale kinase inhibitor bioactivity data sets: a comparative and integrative analysis deep in the bowel: highly interpretable neural encoder-decoder networks predict gut metabolites from gut microbiome feature selection for descriptor based classification models. . human intestinal absorption (hia) chemmine tools: an online service for analyzing and clustering small molecules key: cord- -qtmjgrml authors: mirjalili, mahtabalsadat; shafiekhani, mojtaba; vazin, afsaneh title: coronavirus disease (covid- ) and transplantation: pharmacotherapeutic management of immunosuppression regimen date: - - journal: ther clin risk manag doi: . /tcrm.s sha: doc_id: cord_uid: qtmjgrml the novel coronavirus disease (covid- ) was first detected in wuhan, hubei province, china, in late . since then, covid- has spread to more than countries in the world, and a global pandemic has been declared by the world health organization (who). at present, no vaccines or therapeutic regimens with proven efficacy are available for the management of covid- . hydroxychloroquine/chloroquine, lopinavir/ritonavir, ribavirin, interferons, umifenovir, remdesivir, and interleukin antagonists, such as tocilizumab, have been recommended as potential treatment options in covid- . transplant patients receiving immunosuppressant medications are at the highest risk of severe illness from covid- . at the same time, with regard to receiving polypharmacy and immunosuppressants, treatment options should be chosen with more attention in this population. considering drug–drug interactions and adverse effects of medications used for the treatment of covid- , such as qt prolongation, the dose reduction of some immunosuppressants or avoidance is recommended in transplant recipients with covid- . thus, this narrative review describes clinically important considerations about the treatment of covid- and immunosuppressive regimens regarding modifications, side effects, and interactions in adult kidney or liver allograft recipients. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel virus which was first detected in humans in late december its emergence was first reported in wuhan, hubei province, china, followed by a large outbreak in that country. , by january , a global public health emergency had been announced by the world health organization (who) and two months later, in march, the who declared the coronavirus outbreak a global pandemic. by may , , a total number of , , cases and , confirmed deaths caused by had been reported by the who. since then, covid- has continued to spread, and cases are currently reported in countries. transplant patients receiving immunosuppressive therapy are at the highest risk of severe illness from covid- . the prevalence of human coronavirus (hcov) was . % in immunocompetent vs . % in immunocompromised patients. in another study evaluating bronchoalveolar lavage (bal) samples from patients in a -month period, more than half of the patients diagnosed with hcov were solid organ recipients. studies have also been published in spain and italy, as active centers in solid organ transplantation in europe, evaluating transplanted patients with covid- . , so, a balance is needed between optimal and safe immunosuppression regimens to maintain graft function and the management of covid- . two of the most important challenges ahead are modifying immunosuppressive regimens and the management of drug interactions, as well as adverse events of treatment options for covid- in transplant patients. considering the novelty of covid- and the lack of valid randomized clinical trials regarding its treatment, particularly the management of transplant patients, this study aims to review the published articles and interim guidelines in this regard. because of the limited experience on covid- in transplant recipients, the following points are based on studies conducted so far on this disease and also previous articles regarding severe acute respiratory syndrome coronavirus (sars-cov- ) and middle east respiratory syndrome-related coronavirus (mers-cov). on the other hand, more attention should be paid to therapeutic interventions for these patients, particularly in the icu setting and ensuring safe medication use. thus, this review describes clinical important considerations about the treatment of covid- and immunosuppressive regimens, regarding modifications, side effects, and interactions in adult kidney or liver allograft recipients. table shows a summary of medications which are used or suggested for the management of covid- patients, according to recent studies. in stable patients who can be treated as outpatients, monotherapy with chloroquine/hydroxychloroquine (with doses mentioned in table ) or combination therapy with oseltamivir in high-risk areas for h n outbreaks is suggested. based on interim guidelines from china and belgium, in patients with unstable respiratory conditions (respiratory rate > breaths/min, pao < %, or extensive pulmonary infiltration) who need to be admitted to hospital because of sars, chloroquine/hydroxychloroquine in combination with lopinavir/ritonavir after the discontinuation of chloroquine/hydroxychloroquine, with or without ribavirin, or using remdesivir and interleukin (il) antagonists, such as tocilizumab, is suggested. , however, some guidelines may advise against these suggestions. the data regarding modification of the immunosuppressive regimen in transplant patients with covid- are limited; thus, we have to utilize the recommendations provided in transplant guidelines by scientific communities related to transplantation. for instance, the guideline provided by massachusetts general hospital has suggested that in non-critically ill kidney and liver transplant recipients, cyclosporine and tacrolimus doses should be reduced by % and their plasma concentrations should remain within - ng/ml and - ng/ml, respectively. also, it has been recommended that antimetabolites, such as mycophenolate mofetil or mycophenolatesodium (myfortic and cellcept) and azathioprine, should be stopped. at the same time, this guideline has recommended stopping all immunosuppressants in critically ill patients and administering prednisolone, but the dose has not been mentioned.- meanwhile, the american association for the study of liver diseases (aasld) has announced that there is no need to discontinue or decrease the dose of immunosuppressants in non-critically ill patients. in one case report regarding the successful treatment of a kidney transplant recipient with pneumonia caused by sars-cov- in china, all the immunosuppressants were stopped and the patient received g intravenous immunoglobulin (ivig) on the first day and then g/day for the next days, with mg/day methylprednisolone for days and million units/day interferon as atomization inhalation. however, using corticosteroids in the management of patients with acute respiratory distress syndrome (ards) or those affected with covid- is controversial. the who advises against the use of corticosteroids for the management of severe acute respiratory infection when sars-cov- is suspected, unless indicated for another reason, such as an asthma attack or reversible septic shock. , inhibition of the immune response, reduction in pathogen clearance and an increase in viral shedding have been observed following the use of corticosteroids. , considering that adverse clinical outcomes and increased mortality and morbidity following the administration of corticosteroids in patients with respiratory infections caused by respiratory syncytial virus (rsv), influenza, sars-cov- , or mers-cov may be due to an increased risk of secondary bacterial infections, their use for the prevention of disease progression or its treatment remains under discussion. in this regard, more precautions should be taken in transplant patients, because these agents are the main components of immunosuppression and their abrupt discontinuation may lead to an increased risk of acute rejection or flares in some liver transplant situations, such as autoimmune hepatitis. so, the aasld suggests avoiding high doses of corticosteroids in these patients, and whenever the dose reduction is unavoidable, a minimum dose of mg/day prednisone should be considered to avoid adrenal insufficiency. considering the pulmonary adverse effects of mammalian target of rapamycin (mtor) inhibitors (ie, sirolimus and everolimus), including drug-induced pneumonitis and interstitial lung disease, it is reasonable to stop mtor inhibitors and substitute them with calcineurin inhibitors (cnis). so far, no specific regimen has been suggested for acute rejection episodes in liver and kidney transplant patients suffering from covid- , but considering the positive role of ivig in the management of patients with covid- in some experiences, administering ivig as part of the treatment of antibody-mediated acute rejection in kidney transplant patients may be useful. , as mentioned earlier in this section, the decrease in immunosuppression in transplant recipients leads to an increase in rejection risk. thus, modification of the immunosuppression regimen should be rational and individualized. another important issue to be considered is that sars-cov- can present as renal or hepatic impairment and it is necessary to differentiate these clinical manifestations from rejection episodes in liver or kidney recipients. according to the affinity between sars-cov- and angiotensin-converting enzyme- (ace- ) receptors, which are enriched in the lung, liver, and biliary systems, an increase in liver enzymes, such as aspartate transaminase (ast), alanine transaminase (alt),alkaline phosphatase (alp), and gamma-glutamyl transferase (ggt), was seen in patients affected with covid- . , the incidence of elevated liver enzymes in these patients ranges from % to %. features of renal impairment, such as massive albuminuria, hematuria, and elevation in blood urea nitrogen or serum creatinine, are also present in these patients, with an incidence ranging between . % and %. therapeutic options for the management of liver or kidney injury following covid- include limiting the use of hepatotoxic medications such as acetaminophen for fever management. using compounds such as glycyrrhizic acid, owing to its steroid-like characteristics, could reduce hepatocyte inflammation to some extent. also, one stud suggested using ursodeoxycholic acid and s-adenosylmethionine for the improvement of cholestatic liver injury caused by covid- . the suggested drug regimens for the treatment of covid- have several drug-drug interactions and adverse drug reactions regarding immunosuppressive medications, which are mentioned in table . these points are discussed in more detail below. chloroquine phosphate or hydroxychloroquine, which has antimalarial characteristics, is one of the drugs used for the management of covid- owing to its inhibitory effect on viral replication and its appropriate lung permeability. , the suggested dose for chloroquine and hydroxychloroquine is mg twice daily for days and mg daily for - days, respectively. if hydroxychloroquine is used in combination with lopinavir/ritonavir, a loading dose of mg twice daily should be administered on the first day, then hydroxychloroquine should be discontinued and lopinavir/ritonavir initiated. an increase in liver enzymes, particularly when used concurrently with hepatotoxic drugs, gastrointestinal upset, and hemolytic anemia in patients with glucose- -phosphate dehydrogenase (g p) deficiency are important adverse effects of this drug. this drug can cause qt prolongation, too. so, prior to initiating this medication, the risk factors for qt prolongation, including hypomagnesemia, hypokalemia, and cardiomyopathy, should be evaluated and eliminated if possible. this adverse effect can also occur when chloroquine or hydroxychloroquine is used in combination with lopinavir/ritonavir, another drug used for the management of covid- , or some other qtc-prolonging medications, including antipsychotics (eg, pimozide) or cardiac medications (eg, digoxin). occasional headaches, dizziness, loss of appetite, and maculopathy (in long-term use) are other adverse effects of this drug. regarding the interactions between these drugs and immunosuppressants, it should be mentioned that chloroquine and hydroxychloroquine, as well as cnis and mtor inhibitors, can cause qt prolongation. furthermore, everolimus is a cyp a inhibitor and can theoretically increase hydroxychloroquine and chloroquine concentration, and potentiates the qt-prolonging effect of these drugs. cyclosporine concentration is also affected by hydroxychloroquine and chloroquine by inhibiting their metabolism through the inhibition of cyp a . therefore, the cyclosporine level should be monitored periodically and the dose may be decreased if necessary. , removal of the drug by hemodialysis is negligible. it is suggested that % of the usual dose should be administered in patients with a glomerular filtration rate (gfr) < ml/min, but % of the usual dose is recommended for patients on continuous renal replacement therapy (crrt). , lopinavir/ritonavir another drug of interest in the management of covid- is lopinavir/ritonavir. lopinavir is a protease inhibitor (pi) which has been used for years in the treatment of human immunodeficiency virus (hiv). ritonavir increases the plasma half-life of lopinavir due to its inhibition of cyp enzymes. some previous studies have shown positive results in using this drug against sars and mers. , thus, this drug has drawn medical attention for the management of covid- . although some studies have reported positive effects of this drug on covid- , , a recently published article questioned its efficacy. based on limited studies, using lopinavir as monotherapy or in combination with umifenovir (arbidol®), ribavirin, or interferon could help in the management of patients with covid- ; however, a clinical trial did not demonstrate any superiority of lopinavir monotherapy over standard care supportive therapy. therefore, further evaluations are required regarding its efficacy and safety. so far, few studies have been conducted regarding the use of this drug in liver and kidney transplant patients, but if it is administered to this population, its adverse effects and interactions with immunosuppressants and other medications used in transplant patients, such as fluoroquinolones for the treatment of gram-negative infections, should be considered. although gastrointestinal upset, including nausea and vomiting, is the most important adverse effect of this drug, qt prolongation in combination with other medications used in the covid- treatment regimen is also an issue of concern. thus, it should be prescribed cautiously in patients with significant risk factors, such as previous second-and third-degree heart block. a rise in liver enzymes, particularly in decompensated cirrhosis, and pancreatitis have also been reported with its use in hiv patients. ritonavir is an inhibitor of cyp a and to a lesser extent cyp d , as well as an inhibitor of intestinal glycoprotein p. the majority of immunosuppressants are metabolized by cyp isoenzymes and eliminated via glycoprotein p. for instance, tacrolimus and cyclosporine are metabolized by this system, while mycophenolic acid is metabolized by uridine diphosphate glucuronosyltransferase (ugt) and has fewer interactions with antiretroviral therapy. , several studies have reported a significant increase in the plasma concentration of tacrolimus ( - fold) when used with pis. , therefore, based on the available evidence on hiv-positive kidney and liver recipients, who received tacrolimus and lopinavir concurrently, the recommended dose of tacrolimus is . mg every - days and its plasma concentration should be maintained between and ng/ml. , if daily administration of tacrolimus is preferred, the dose of . - . mg daily or a reduction to one-twentieth to one-fiftieth of baseline daily dose is recommended. , another cni, cyclosporine, has less severe drug interactions with pis than tacrolimus, but an increase in its plasma concentration, particularly when used with hydroxychloroquine, is of great importance. thus, based on studies conducted on hiv-positive kidney and liver recipients who received cyclosporine and lopinavir/ritonavir concurrently, the cyclosporine dose should be reduced to one-fifth of the total daily dose to achieve - ng/ml as the target plasma concentration (eg, mg every - days), and daily plasma concentration measurement is also recommended. , as mentioned earlier (see "immunosuppressive medications", above), temporary discontinuation of mtor inhibitors in transplant patients with covid- at the discretion of the transplant team is recommended. however, if they are continued, the drug interaction with pis should be noted, and a - % reduction in dose of sirolimus and discontinuation of everolimus should be considered in concurrent use with pis. , ribavirin ribavirin is a nucleotide/nucleoside analogue which is effective against a wide range of dna and rna viruses. the oral dosage form in combination with interferon is used in the treatment of chronic hepatitis c and the inhalation form is administered in the treatment of rsv. it is considered as a promising agent in the management of covid- because of effective clinical experience with this agent in mers. , if this drug is administered in transplant patients with covid- , the risk of drug interactions and adverse effects should be noted. hemolytic anemia - weeks after the initiation of ribavirin, particularly in combination with interferons, is of great importance. patients with cardiovascular comorbidity need more attention regarding this adverse effect. the concurrent use of ribavirin with bone marrow suppressant drugs, such as antimetabolites, cotrimoxazole, ganciclovir, and valganciclovir, which may be used in transplant patients, causes thrombocytopenia and pancytopenia. considering renal elimination of ribavirin, its use is not recommended in patients with a gfr < ml/min because the risk of hemolytic anemia increases. remdesivir, which is an adenosine analogue and rna polymerase inhibitor, acts as an antiviral agent and has been successful in the treatment of mers-cov and ebola in clinical and animal models. , thus, this drug has been mentioned in some studies as one of the treatment options for covid- and its efficacy in covid- is under investigation in clinical trials. , currently, nine clinical trials are being conducted regarding the safety and efficacy of remdesivir in patients with covid- . at this time, there is no widespread access to this drug and its use is limited to clinical trials, emergency treatment, and compassionate use requests, but its manufacturer, gilead (foster city), is trying to expand its access programs during the covid- outbreak. , the suggested dose is mg stat and then mg daily for days. considering the limited studies on remdesivir, sufficient data regarding its side effects, drug interactions, and safety are not available and we should wait for further studies. no drug interactions are expected between remdesivir and immunosuppressants used in transplantation settings. however, considering the lack of knowledge regarding the safety of remdesivir coadministration with immunosuppressants, it is suggested that their blood concentrations should be closely monitored. the aasld has warned about its hepatotoxic effects in transplanted patients. umifenovir is another potential option for covid- treatment. it is a broad-spectrum antiviral agent used in the treatment and prevention of influenza and it has in vitro activity against sars-cov- . studies published after the emergence of sars-cov- indicate that it has positive effects on covid- , in combination with other therapeutic options. the suggested dose in one study is mg every hours for days. in a case report regarding two kidney transplant patients with covid- who were treated successfully, umifenovir was one of the therapeutic options. umifenovir is metabolized by the liver, particularly cyp a , so it should be used with caution in patients with liver failure. its protein binding is high; thus, it should be used cautiously in patients receiving other medications with high protein binding. so far, no data are available regarding its effects when used concurrently with immunosuppressants. ifn type i, such as ifn-α and ifn-β, and ifn type ii, such as inf-γ, are considered important components of the host immune response to viral infections. interferons have been used in the treatment of hepatitis c virus (hcv) in combination with ribavirin, owing to their positive effect on viral replication and immunomodulatory properties. after the emergence of sars-cov- in and mers-cov in , studies regarding the effects of ifns in their treatment have been conducted. , currently, ifns are being studied for the treatment of covid- . ifns have been used as nebulization or subcutaneous injections in these studies. , although it is too early to make judgments about the efficacy of ifns in the management of covid- , the following points should be considered in kidney or liver transplant recipients with covid- for whom infs are initiated. long-term use of ifns may cause bone marrow suppression, so it is recommended not to use these agents in severe neutropenia or thrombocytopenia. moreover, their use can lead to hepatotoxicity. there are several reports of hepatoencephalopathy, jaundice, and acute liver failure in patients receiving ifn for the treatment of hcv. at the same time, acute and chronic rejection, plasma cell hepatitis, and consequently graft failure have been reported in liver transplant recipients using pegylated interferon. , also, acute humoral rejection in kidney transplant recipients with hcv treated with ifn has been reported. therefore, ifns are not recommended in kidney and liver transplant recipients because of the risk of rejection. a hyperinflammatory syndrome, which presents with fulminant and fatal hypercytokinemia, is mentioned as covid- pathogenesis in the majority of studies conducted in this regard. some studies have mentioned positive effects of il- inhibitor (anakinra) and il- antagonist (tocilizumab) in covid- , and clinical trials are being conducted to evaluate the efficacy of these agents in the management of this disease. , thus, we should wait for the results of these studies. studies on the safety of tocilizumab in transplantation have so far been restricted to steroid-refractory acute graft-versus-host disease (gvhd), , and studies regarding the safety of this agent in kidney and liver transplantation are limited. although studies on the efficacy and safety of tocilizumab in transplantation are scarce, some case reports have indicated that the use of monotherapy or combination therapy with tocilizumab improved the status of transplant recipients with covid- . , it is worth mentioning that some aspects of tocilizumab administration, such as time of initiation (early vs late phase), route of administration (subcutaneous vs intravenous), and dose, are not clear yet, and we have to wait for the results of clinical trials. we found case reports mentioning liver damage and fulminant liver failure due to hepatitis b reactivation following tocilizumab therapy. , however, there are some studies reporting successful treatment by tocilizumab of chronic antibody-mediated rejection in kidney transplant patients. , considering the specific adverse effects of tocilizumab, including upper respiratory tract infections, cardiovascular complications, and hepatic failure, it seems that until an official announcement of the results of ongoing studies, its use in transplant patients with covid- is debatable. sometimes, medications other than antivirals are used for symptomatic therapy or supportive care in covid- patients. for the management of fever, myalgia, or headache caused by covid- or by medication such as hydroxychloroquine, acetaminophen (paracetamol) is recommended as the first line agent, but acetaminopheninduced hepatotoxicity should be noted and the dose should be no more than g/day. regarding the use of non-steroidal anti-inflammatory drugs (nsaids) in covid- patients, no conclusive evidence is available. the mechanism of action of nsaids includes inhibition of cyclooxygenase (cox) /cox . these enzymes are responsible for the production of prostaglandins (pgs), such as pge /pgd and pgi , which can promote and restrain inflammation. it is reported in one study that inflammation worsens following nsaid administration in covid- patients, particularly those receiving ibuprofen. in one study, ibuprofen induced overexpression of ace in diabetic rats. this effect can theoretically worsen the inflammatory course in patients suffering from covid- . however, some studies have concluded that owing to the anti-influenza properties of naproxen, , it may be effective in covid- patients. also, considering the antiviral activity of indomethacin on coronavirus replication in vitro, it may be effective in covid- patients. it is too early to make judgments about the effects of nsaids in the management of covid- patients, and particularly transplant recipients, and we should wait for the results of ongoing studies, but the following points should be considered in the management of covid- in kidney and liver transplant recipients. administration of nsaids in kidney transplant patients increases the risk of acute kidney injury (aki) owing to their mechanism of action (inhibition of pg synthesis) and decrease in renal blood flow. the risk of aki increases with the concurrent use of cnis as the basis of immunosuppression in kidney transplant recipients. also, concern exists regarding nsaid administration in liver transplant patients, particularly due to diclofenac liver injury. thus, caution should be applied when administering nsaids in transplant recipients. another complication in patients with covid- is nausea and vomiting (n/v). serotonin antagonists ( -ht receptor antagonists), dopamine antagonists, nk antagonists, and antihistamines are common drugs used for the management of n/v. considering polypharmacy in patients with covid- , special attention should be given to drug interactions between antiemetics and other administered medications when treating n/v in this population. for instance, the risk of qt prolongation is high in concurrent use of lopinavir/ritonavir, hydroxychloroquine, and serotonin antagonists, such as ondansetron and granisetron, or nk antagonists, such as aprepitant, and it is better to avoid their concurrent use. furthermore, serotonin syndrome is predictable in concurrent use of serotonin antagonists with some common medications in critically ill patients, such as linezolid and fentanyl. , therefore, considering the safety of antiemetics, it is recommended to use antihistamines, such as diphenhydramine or dimenhydrinate, in patients with covid- . also, it is advised to administer medications such as lopinavir/ritonavir after meals, and to insert an interval of at least hour between these agents and emetogenic drugs, such as oseltamivir. dry cough is the main complaint of patients with covid- . dextromethorphan, guaifenesin, codeine, and levocloperastine are safe in majority of patients and do not have any potential drug-drug interactions. , however, the risk of serotonin syndrome with high-dose administration or prolonged use of dextromethorphan or other agents that increase serotonin should be noted. in transplant patients with cardiovascular diseases and covid- , it is necessary to continue cardiovascular medications. based on animal laboratory studies, ace receptors in the lung are binding sites for sars-cov- . at first, the hypothesis was considered that taking ace inhibitors (aceis) or angiotensin ii receptor blockers (arbs) can worsen the pulmonary function of patients with covid- , but further studies demonstrated that aceis or arbs can be considered as potential therapeutic options for the management of patients with covid- . , , therefore, cardiovascular disease societies recommend that patients taking aceis or arbs continue receiving their treatment and avoid the abrupt discontinuation of these drugs due to adverse effects on the course of covid- disease. statins are another prescribed class of medications discussed in patients with covid- . considering their antiinflammatory properties, positive clinical experience with statins in the improvement of ards in patients with ebola and mers, and their effect in upregulation of the activity of the ace pathway, some reports have recommended their use in patients with covid- . , however, some studies do not recommend the initiation or routine use of statins in patients with covid- owing to the paradoxical effects of statins for improving lung function in patients with viral infections and their potential side effects. the american college of cardiology recommends statin therapy only for patients with cardiovascular adverse effects caused by covid- or patients with clinical atherosclerotic cardiovascular disease (ascvd) or diabetes, or those at high risk of ascvd. , , we should wait for further studies regarding the prescription of statins in kidney and liver transplant patients with covid- . also, the side effects of statins, such as a rise in liver enzymes, myalgia, or, in more severe cases, rhabdomyolysis, should be considered. considering drug interactions between immunosuppressants and statins, it seems that tacrolimus is a safer option than cyclosporine in concurrent use of atorvastatin. , it is worth mentioning that most statins are metabolized via cyt p isoenzymes, particularly a , as well as p-glycoproteins. the coadministration of pis, such as lopinavir and darunavir, and their pharmacokinetic enhancers, such as ritonavir and cobicistat, with statins leads to increased statin levels and their adverse effects. simvastatin or lovastatin should not be administered concomitantly with ritonavir/cobicistat-boosted pis. the maximum recommended daily dose of atorvastatin and simvastatin is and mg, respectively, when coadministered with ritonavir/cobicistat-boosted pis. mirjalili et al the maintenance of the transplanted graft and its proper function is of great importance in patients with covid- . with regard to receiving immunosuppressants and polypharmacy, treatment options should be chosen with prudence in these patients. it is suggested that mtor inhibitors be stopped throughout the course of covid- and substituted with cnis. also, close therapeutic drug monitoring of immunosuppressants should be conducted in this population. considering drug-drug interactions and adverse effects of medications used for the treatment of covid- , such as qt prolongation, the dose reduction of some immunosuppressants or avoidance of their administration should be considered in transplant recipients with covid- . qt prolongation should be considered when using hydroxychloroquine or lopinavir/ritonavir in combination with other qtc-prolonging drugs, such as antipsychotics and cardiac medications. we should wait for further clinical trials and multicenter studies to evaluate the safety and efficacy of treatment options for covid- in transplant patients. the authors report no conflicts of interest in this work. successful containment of covid- : the who-report on the covid- outbreak in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china who. coronavirus disease (covid- ) situation reports the challenge of respiratory virus infections in hematopoietic cell transplant recipients a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection covid- in solid organ transplant recipients: a single-center case series from spain the covid- outbreak in italy: initial implications for organ transplantation programs determining frequency of prescription, administration and transcription errors in internal intensive care unit of shahid faghihi hospital in shiraz with direct observation approach covid- : an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese-western medicine for the management of novel coronavirus disease interim clinical guidance for patients suspected of/confirmed with covid- in belgium massachusetts general hospital. department of medicine covid- treatment guidance clinical insights for hepatology and liver transplant providers during the covid- pandemic successful recovery of covid- pneumonia in a renal transplant recipient with long-term immunosuppression future drugs for treatment of acute respiratory distress syndrome pharmacologic treatment of transplant recipients infected with sars-cov- : considerations regarding therapeutic drug monitoring and drug-drug interactions clinical management of severe acute respiratory infection when novel coronavirus ( -ncov) infection is suspected: interim guidance risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease pneumonia in wuhan, china from emcrit project website clinical evidence does not support corticosteroid treatment for -ncov lung injury. the lancet sirolimus and mtor inhibitors: a review of side effects and specific management in solid organ transplantation patients of covid- may benefit from sustained lopinavir-combined regimen and the increase of eosinophil may predict the outcome of covid- progression high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease covid- and the liver: little cause for concern liver injury during highly pathogenic human coronavirus infections liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor liver injury in covid- : management and challenges the novel coronavirus epidemic and kidneys chinese medical association hepatology drug-induced liver disease study group. drug-induced liver injury diagnosis and treatment guidelines novel coronavirus pneumonia related liver injury: etiological analysis and treatment strategy a systematic review on the efficacy and safety of chloroquine for the treatment of covid- aminoquinolines against coronavirus disease (covid- ): chloroquine or hydroxychloroquine hydroxychloroquine: a multifaceted treatment in lupus life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review an open, randomized comparison study of cyclosporine a, cyclosporine a+ methotrexate and cyclosporine a+ hydroxychloroquine in the treatment of early severe rheumatoid arthritis long-term impact of different immunosuppressive drugs on qt and pr intervals in renal transplant patients hydroxychloroquine in patients with systemic lupus erythematosus with end-stage renal disease a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr a trial of lopinavir-ritonavir in adults hospitalized with severe covid- qt prolongation in hiv-positive patients pharmacologic issues of antiretroviral agents and immunosuppressive regimens in hivinfected solid organ transplant recipients drug-drug interactions between antiretroviral and immunosuppressive agents in hiv-infected patients after solid organ transplantation: a review daily dosing of tacrolimus in patients treated with hiv- therapy containing a ritonavir-boosted protease inhibitor or raltegravir tacrolimus and lopinavir/ ritonavir interaction in liver transplantation pharmacokinetics of tacrolimus and cyclosporine in liver transplant recipients receiving direct-acting antivirals as treatment for hepatitis c infection effect of coadministered hiv-protease inhibitors on tacrolimus and sirolimus blood concentrations in a kidney transplant recipients a review on therapeutic drug monitoring of the mtor class of immunosuppressants: everolimus and sirolimus severe covid- in a renal transplant recipient: a focus on pharmacokinetics ribavirin: pharmacology, multiple modes of action and possible future perspectives oral versus aerosolized ribavirin for the treatment of respiratory syncytial virus infections in hematopoietic cell transplant recipients ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study use of ribavirin in viruses other than hepatitis c. a review of the evidence mycophenolate mofetil/ribavirin interaction safety, tolerability, and pharmacokinetics of ribavirin in hepatitis c virus-infected patients with various degrees of renal impairment mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection therapeutic options for the novel coronavirus ( -ncov) institutes of health remdesivir for the treatment of covid- -preliminary report remdesivir as a possible therapeutic option for the covid- arbidol as a broad-spectrum antiviral: an update arbidol combined with lpv/r versus lpv/ r alone against corona virus disease : a retrospective cohort study novel coronavirus infection and acute kidney injury in two renal transplant recipients: case report pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans determination of plasma protein binding rate of arbidol interferon-beta and interferongamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) adherence to treatment of chronic hepatitis c: from interferon containing regimens to interferon and ribavirin free regimens role of interferons in the treatment of severe acute respiratory syndrome interferon and cytokine responses to sarscoronavirus infection treatment with interferon-α b and ribavirin improves outcome in mers-covinfected rhesus macaques discovering drugs to treat coronavirus disease (covid- ) treatment of covid- : old tricks for new challenges interferon-α induced severe thrombocytopenia: a case report and review of the literature presentation and outcomes with clinically apparent interferon beta hepatotoxicity risk for immune-mediated graft dysfunction in liver transplant recipients with recurrent hcv infection treated with pegylated interferon outcomes of acute rejection after interferon therapy in liver transplant recipients immune-mediated complications of the graft in interferon-treated hepatitis c positive liver transplant recipients interferon-alpha and its deleterious effects on kidney transplant: regarding one case covid- : consider cytokine storm syndromes and immunosuppression. the lancet effective treatment of severe covid- patients with tocilizumab covid- pneumonia in a kidney transplant recipient successfully treated with tocilizumab and hydroxychloroquine assessment of tocilizumab (anti-interleukin- receptor monoclonal) as a potential treatment for chronic antibody-mediated rejection and transplant glomerulopathy in hla-sensitized renal allograft recipients clinical course of covid- in a liver transplant recipient on hemodialysis and response to tocilizumab therapy: a case report tocilizumab in covid- pneumonia (tocivid- ) -clinicaltrials. gov toxic drug-induced liver failure during therapy of rheumatoid arthritis with tocilizumab subcutaneously: a case report case of severe liver damage after the induction of tocilizumab therapy for rheumatoid vasculitis fulminant liver failure due to hepatitis b reactivation during treatment with tocilizumab clinical safety of tocilizumab in rheumatoid arthritis misguided drug advice for covid- covid- : ibuprofen should not be used for managing symptoms, say doctors and scientists ibuprofen attenuates cardiac fibrosis in streptozotocin-induced diabetic rats covid- infection and rheumatoid arthritis: faraway, so close! autoimmun rev structure-based design of novel naproxen derivatives targeting monomeric nucleoprotein of influenza a virus structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza a virus naproxen exhibits broad anti-influenza virus activity in mice by impeding viral nucleoprotein nuclear export indomethacin has a potent antiviral activity against sars coronavirus a efficacy of addition of naproxen in the treatment of critically ill patients hospitalized for covid- infection (enacovid) longterm assessment of nsaid prescriptions and potential nephrotoxicity risk in adult kidney transplant recipients calcineurin inhibitor nephrotoxicity hepatotoxicity of nonsteroidal anti-inflammatory drugs: a systematic review of randomized controlled trials antiemetics: american society of clinical oncology clinical practice guideline update effect of intravenous ondansetron on the qt interval of patients' electrocardiograms serotonergic medications, herbal supplements, and perioperative serotonin syndrome. can j anesth/j canadien d'anesthésie psychotropic drug therapy in patients in the intensive care unit-usage, adverse effects, and drug interactions: a review efficacy and safety of levocloperastine in the treatment of dry cough: a prospective observational study clinical practice guidelines for diagnosis and management of cough-chinese thoracic society (cts) asthma consortium pharmacology of dextromethorphan: relevance to dextromethorphan/quinidine (nuedexta ® ) clinical use hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid- sars-cov : should inhibitors of the renin-angiotensin system be withdrawn in patients with covid- ? hiding in plain sight: an approach to treating patients with severe covid- infection treating ebola patients: a 'bottom up'approach using generic statins and angiotensin receptor blockers statins may decrease the fatality rate of middle east respiratory syndrome infection covid- clinical guidance for the cardiovascular team clinical controversy in transplantation: tacrolimus versus cyclosporine in statin drug interactions cyclosporine a, but not tacrolimus, shows relevant inhibition of organic anion-transporting protein b -mediated transport of atorvastatin considerations for statin therapy in patients with covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings remdesivir as a possible therapeutic option for the covid- addition of interleukin- inhibition with tocilizumab to standard graft-versushost disease prophylaxis after allogeneic stem-cell transplantation: a phase / trial impact of tocilizumab (anti-il- r) treatment on immunoglobulins and anti-hla antibodies in kidney transplant patients with chronic antibody-mediated rejection outcomes in patients with covid- infection taking acei/arb renin-angiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension statin potency and the risk of hospitalization for community-acquired pneumonia risk of pneumonia in patients taking statins: population-based nested case-control study therapeutics and clinical risk management is an international, peerreviewed journal of clinical therapeutics and risk management, focusing on concise rapid reporting of clinical studies in all therapeutic areas, outcomes, safety, and programs for the effective, safe, and sustained use of medicines key: cord- -yqf tyo authors: keshmiri neghab, hoda; azadeh, seyedeh sara; soheilifar, mohammad hasan; dashtestani, fariba title: nanoformulation-based antiviral combination therapy for treatment of covid- date: journal: avicenna j med biotechnol doi: nan sha: doc_id: cord_uid: yqf tyo nan in december , a novel coronavirus disease (covid- ) was detected in wuhan, china, which was accompanied by symptoms of fever, dry cough, weakness of immune system with reduction of the white blood cells. afterwards, coronavirus has speared and affected many countries in worldwide , . coronaviruses are subgroup of enveloped, singlestranded, positive-sense rna genome, phylogenetically associated with acute respiratory syndrome coronavirus (sars-cov- ) and middle east respiratory syndrome coronavirus (mers-cov) which are closely related to lung disease leading to acute respiratory distress syndrome (ards) . it has been shown that coronavirus spike protein (s protein) is a transmembrane glycoprotein responsible for cell entry into the target cells. it binds to the host cell receptors, angiotensin converting enzyme (ace- ), inducing conformational rearrangement that drives membrane fusion . since the primary goal of antiviral therapy is blocking virus entry, inhibition of ace- as a main entry key into cells for coronavirus is the most promising therapeutic approach . although ace- is widely distributed in the human body, and can affect a variety of organs and cells including the lung, heart, kidney, and small intestine, but promising and effective potential of ace- in blocking viral entry, inhibiting inflammation and reducing the damage of target organs cannot be ignored even after some adverse effects . dx is a novel, specific and potent peptide inhibitor of ace that can be used in antiviral treatment for covid- . in addition to ace- blocking strategy, preventing virus replication by inhibition of host inosine monophosphate dehydrogenase (impdh) enzyme as a crucial goal of antiviral therapy for virus infection could be a potential strategy in the battle against coronavirus. ribavirin is analogue of purine nucleoside that can inhibit the impdh enzyme to prevent replication of the genetic material (rna and dna) of viruses . it has been revealed that taking ribavirin by both intravenous and oral administration at different doses is the effective way for prevention and treatment of sars . but taking medication at high doses leads to adverse effects such as nausea, exacerbation of bronchospasm and dose-dependent anemia; even it may affect the embryo and cause a genetic mutation . it has been reported that combination antiretroviral therapy (cart) promotes the effectiveness of the treatment and decreases the risk of drug resistance . hence, administration of ribavirin and dx which are two antiviral agents would be more effective. moreover, nanomedicine with its rapid growth combines the nanotechnology with the biomedical and pharmaceutical sciences . the small particle size of nanoparticles (nps) creates dispersity in the stable nanostructure and enables easy entrance into cells; thereby, drug cost is reduced and the therapeutic efficacy will improve. moreover, the dose of drug to be administered can be reduced and concurrently the unwanted side effects are minimized. so, by including nanoparticles (nps) in drug formulations, the efficacy, safety, and dose of administered drug would be improved. the formidable barriers for gastrointestinal tract, skin and cell have limited the therapeutic effects of antiviral drugs. for example, functionalized single-walled carbon nanotubes were used as a nanodrug carrier for ribavirin for the treatment of viral diseases in fish. the results show that ribavirin intake was increased by nanocarrier and therapeutic dosage was significantly reduced . several researches have been conducted about antiviral drug delivery nanosystems and their transport across specific barriers at cellular and intracellular level . furthermore, improving the antiretroviral agents' delivery could overcome some probable limitations of current cart . thus, co-encapsulation of ribavirin and dx in nps would afford a novel means of drug delivery to sars-cov- in tissues. the studies in mice and in human cell lines revealed that dx is a potent ace inhibitor specific for only human ace . hence, by using dx in nano-formulation, the targeted drug delivery would be obtained. polylactic-co-glycolic acid (plga) and polyethylene glycol (peg) copolymer is a developed system with many advantages; it is biodegradable, biocompatible, easily synthesized by self-assembling into nanometric micelles, and it favors a time-dependent release manner, reduces blood clearance of nanocarriers and increases blood circulation time . consequently, co-encapsulation of ribavirin and dx- in plga-peg provides access to all good qualities of antiviral drugs and nanocarriers in one drug. schematic representation of using nanotechnology systems for delivery of antiretroviral drugs has been shown in figure . in the end, it is hypothesized that the use of plga-peg copolymer to deliver two or more antiretroviral drugs to suppress viral entry and viral replication by dx and ribavirin, respectively can be a promising tool in treatment of coronavirus. moreover, plga-peg copolymer has the potential to be applied as a nanocarrier agent for codelivery of any antiviral drugs to target tissue and consequently promotes the cart in living organisms. clinical features of patients infected with novel coronavirus in wuhan, china a new coronavirus associated with human respiratory disease in china the novel coronavirus: a bird's eye view angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target novel peptides inhibitors of angiotensin converting enzyme ribavirin in the treatment of sars: a new trick for an old drug? a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury recent developments of nanotherapeutics for targeted and long-acting, combination hiv chemotherapy a genosensor for detection of htlv-i based on photoluminescence quenching of fluorescent carbon dots in presence of iron magnetic nanoparticle-capped carbon nanotubebased nanocarrier loaded with ribavirin against grass carp reovirus nanomaterials designed for antiviral drug delivery transport across biological barriers progress in nanomedicine: approved and investigational nanodrugs lazartigues e. species-specific inhibitor sensitivity of angiotensin-converting enzyme (ace ) and its implication for ace activity assays biodegradable plgab-peg polymeric nanoparticles: synthesis, properties, and nanomedical applications as drug delivery system . department of medical laser key: cord- -vvb ffhv authors: mongia, aanchal; saha, sanjay kr.; chouzenoux, emilie; majumdar, angshul title: a computational approach to aid clinicians in selecting anti-viral drugs for covid- trials date: - - journal: nan doi: nan sha: doc_id: cord_uid: vvb ffhv covid- has fast-paced drug re-positioning for its treatment. this work builds computational models for the same. the aim is to assist clinicians with a tool for selecting prospective antiviral treatments. since the virus is known to mutate fast, the tool is likely to help clinicians in selecting the right set of antivirals for the mutated isolate. the main contribution of this work is a manually curated database publicly shared, comprising of existing associations between viruses and their corresponding antivirals. the database gathers similarity information using the chemical structure of drugs and the genomic structure of viruses. along with this database, we make available a set of state-of-the-art computational drug re-positioning tools based on matrix completion. the tools are first analysed on a standard set of experimental protocols for drug target interactions. the best performing ones are applied for the task of re-positioning antivirals for covid- . these tools select six drugs out of which four are currently under various stages of trial, namely remdesivir (as a cure), ribavarin (in combination with others for cure), umifenovir (as a prophylactic and cure) and sofosbuvir (as a cure). another unanimous prediction is tenofovir alafenamide, which is a novel tenofovir prodrug developed in order to improve renal safety when compared to the counterpart tenofovir disoproxil. both are under trail, the former as a cure and the latter as a prophylactic. these results establish that the computational methods are in sync with the state-of-practice. we also demonstrate how the selected drugs change as the sars-cov- mutates over time, suggesting the importance of such a tool in drug prediction. the dataset and software is available publicly at https://github.com/aanchalmongia/dva and the prediction tool with a user-friendly interface is available at http://dva.salsa.iiitd.edu.in. there has been an exponential rise in the total active cases and deaths due to covid- (corona virus disease- ) since the first case in wuhan, china in december, [ ] . the disease results in severe acute respiratory syndrome coronavirus (sars-cov- ), which is known to be highly transmittable and has spread across more than countries. this pandemic has wreaked havoc on people's social life, the global economy, and most importantly the health of the human race. the death numbers are frightening, confirming about k deaths worldwide till mid-june, [ ] . as medical professionals are striving to save lives, research scientists specialized in drug development, are racing against time to develop a vaccine against sars-cov- [ ] . the investigation involved for developing a vaccine (or even a new drug) is time consuming, requiring several phases of extensive trials. experts believe that it is highly unlikely that a vaccine will be ready before a year or more. in such circumstances the best bet may be to re-position existing drugs for treating covid- . this is a well known approach where existing drugs (which have already been approved for release in the market) are investigated for new disease/s [ ] . drug re-positioning is usually cost effective and fast (compared to developing a new drug / vaccine) since its effects are well studied. one classic example for drug re-positioning is chlorocyclizine , which was initially developed as an anti-allergic but later found to act against the hepatitis c virus [ ] . another example is imatinib mesylate (sold under the trade name gleevec), it was originally used as a treatment for leukemia but later was found to be effective against genes associated with gastrointestinal-stromal tumors [ , ] . given the relatively large drug-virus association space, manual investigation in wet-labs is not a scalable strategy. putting all the anti-virals in trials for treating corona is not very feasible either; especially because time is of essence. in such a scenario, computational approaches can help; they can be used to prune down the search space for the drugs to be investigated [ ] . practically, such approaches could also assist the clinicians to come up with treatments for rapidly mutating viruses by pruning the anti-viral drug space (see discussion section). specifically, a computational approach which takes into account the genomic structure of the latest viral isolate or its similarity with the previously occuring strains of viruses would be helpful in deciding the treatment. with this objective, we have manually curated a comprehensive database called dva (drug virus association), having the approved (antiviral) drug-virus associations in the literature along with the similarity measures associated with drugs (chemical structure similarity) and viruses (genome sequence similarity). to the best of our knowledge there is no existing database for drug virus association. the dva database we propose in this work lies the foundation for further computational studies on this topic. there can be various methodologies to predict drug virus association. the prediction problem can be approached via feature-based classification models, neighborhood models, matrix completion models, network diffusion models etc. a recent empirical study on well established drug-target interaction databases exhibit the best prediction performance by matrix completion models [ ] . in computer science, matrix completion is used routinely in recommendation systems. the general problem of drug-disease association can actually be thought of as a recommendation system, where drugs are being recommended for treating a disease. given the success of matrix completion techniques in drug target interaction, we deploy state-of-the-art matrix completion techniques on our curated dva database. we perform a thorough comparative analysis of those for predicting assessed drug-disease associations. then, we apply the methodology for pruning the search space of potential candidates for covid- trial drugs. finally, we show how the tool helps in selecting drugs as the virus mutates. our objective is to make our solution user friendly for clinicians and scientists. in pursuit of this goal, we have made the solution (dataset and algorithms) available as a webserver. the webserver can be used in two ways. first, given the genome of the virus, the webserver can predict (and rank) the probable antivirals. second, given a drug and a virus, it can output a normalized score depicting how effective the drug will be against the virus. we assess the performance of different matrix completion techniques in this section. the techniques have been described in the methods section. six matrix completion methods were used, which can be categorized into three families provided below. • basic frameworks (mf: matrix factorization [ ] and mc: matrix completion or nuclear norm minimization [ ] ) • deep frameworks (dmf: deep matrix factorization) [ ] , • graph regularized frameworks (grmf: graph regularized matrix factorization [ ] , grmc: graph regularized matrix completion [ ] , grbmc: graph regularized binary matrix completion [ ] ) matrix factorization (mf) is the traditional matrix completion method which factorizes the data matrix into two latent factor matrices (tall and short) and the algorithm recovers these factor matrices to recover the original matrix. since this problem is non-convex, it may not converge to a global minimum of the cost function. nuclear-norm minimization based matrix completion (mc) was proposed as a (mathematically) better alternative; it directly recovers the matrix by penalising its nuclear norm (convex surrogate of rank). deep matrix factorization (dmf) generalises mf to more than two factors. none of the techniques mentioned so far can take advantage of genomic structure of the viruses or chemical structure of the drugs. the said pieces of information can be incorporated into the graph regularized matrix completion techniques (grmf, grmc, grbmc). these techniques have been explained in detail in the methods section. the typical anti-viral drug discovery process involves genomic and biophysical understanding of the virus. it aims to target the enzymes or peptides involved in the viral replication cycle and takes years for successful clinical validation. other approaches involve screening all the broad-spectrum anti-viral drugs or chemical libraries comprising large numbers of existing compounds/databases (having information on transcriptional signatures in different cell lines) to be further evaluated by standard anti-viral assays [ ] . in view to assist acceleration of this process (by pruning down the search space), we create and share a publicly available dva database, along with a number of matrix completion techniques (mentioned above) for drug-virus association prediction. the originality of the proposed work lies in the formalization of the drug-virus association prediction as a matrix completion problem, without the need for any anti-viral assays. such a computational approach requires the chemical structure of the drugs and, in case of graph-regularized matrix completion techniques, the genome of the viruses, or existing associations otherwise. figure depicts the schematic flow of the proposed work involving data curation and implementation overview. in this sub-section, we carry out extensive experimental protocol to illustrate and compare the ability of the different methods to retrieve the drug-disease associations available in our curated dataset. the protocol dictates three variants of -fold cross-validation setting (cv). in the first setting cv (cross validation ), % of the associations selected at random are left out as testing set. this allows to assess each algorithm's ability to predict associations between existing drugs and viruses. to evaluate an algorithm for its ability to predict association for novel drugs and viruses i.e. those which have no association information, we use two other (more stringent) cv settings. in cv and cv , % of the complete virus and drug entities selected at random are left out as test set respectively. the standard metrics for evaluation are the auc (area under the receiver operating characteristic curve) and aupr (area under the precision-recall curve). auc is more common in machine learning literature, it assumes that the classes are evenly balanced. problems in drug-disease association have highly imbalanced classes, in such a scenario the aupr is a more appropriate metric for evaluation [ , ] . table shows how each of the tested algorithms performs in retrieving the associations. a clear observation from the experiments is that the graph regularized-based matrix completion algorithms that incorporate the similarity information associated with the drugs and viruses, perform fairly well giving an auc greater or equal than . in cv . the best performing algorithm (grbmc) exhibits an auc and aupr of . and . respectively. predicting the associations for novel drugs and viruses also have a reasonable performance with the best auc/aupr of . / . and . / . by grbmc and grmf. it can be noted that the standard matrix completion methods, which do not take into account the metadata, fail to learn from the association data giving a near-random performance as far as the prediction on novel viruses is concerned, depicting how very important the similarity information is. dva database and its associated computational tools can also be used on new drugs without any previously known virus association information. for evaluating this ability, we identified in our database all the drugs which are known to interact with only one virus (drugs associated with a single virus only) and hide that association to the methods. this allows us to assess the performance of the algorithms in predicting viruses associated with the new drugs in the database. we hide the only virus corresponding to each of the drugs (with only a single virus associated with it) and run matrix completion to predict candidate viruses for these drugs. the drugs for which the test virus associated with it is the top-ranked virus predicted by the algorithm would have the maximum precision value (mpv) of . the number and percentage of drugs with a maximum precision value of are reported in table . nearly % ( / ) of single association drugs with a maximum precision of were predicted using grmf. other graph regularized frameworks show comparable performance in terms of predicting drugs with mpv of . in this experiment, we add the sars-cov- sample in our database by providing its onf based d * similarity [ ] in the virus similarity matrix. we then apply the matrix completion algorithms to predict the associations and rank prediction scores corresponding to sars-cov- to predict the top recommended drugs. table : top- drugs predicted for sars-cov- by the dva computational methods. as can be seen from the results of section . (table ) , mc, mf and dmf often yield considerably worse results than their graph regularized counterparts (grmf, grmc and grbmc). such poor performance of non-graph regularized versions of matrix completion methods could be explained as they do not incorporate any knowledge about the genomic structure of the viruses and the chemical structure of the drugs. since the three graph-based methods perform reasonably well in the prediction task, we consider these techniques for the drug prediction on the novel coronavirus. the top- drugs they predicted have been reported in table (ranked by their predicted scores). drugs highlighted with blue text are unanimously predicted drugs by the three considered matrix completion techniques and those in red text are predicted by two methods. we also highlighted with yellow cells the drugs which are under trial/investigation as a potential cure/prophylactic against covid- . it can be seen that the three techniques have consistently and unanimously selected six drugs, namely remdesivir, ribavarin, sofosbuvir, taribavirin, tenofovir alafenamide and vidarabine. umifenovir has been recommended by two (grmf and grbmc) out of three techniques. amongst these recommendations, remdesivir [ ] , ribavarin [ , ] , sofosbuvir [ ] and umifenovir [ ] are under clinical trials. taribavirin is similar to ribavirin but it is not approved by the fda. tenofovir alafemanide (an antiretroviral for hiv- ) is on undergoing trial [ ] . grmf has additionally selected ibuprofen which is expected to be investigated in uk [ , ] . the fact that three techniques unanimously select the aforementioned drugs make us confident about these recommendation results. in the previous sub-section, we have established that the results from our models are mostly in sync with clinical practice. in this sub-section, we will demonstrate how our proposed approach can be of help to clinicians. all the results generated so far have been generated using the reference sequence of the sars-cov- strain (collected in december, in wuhan). the novel coronavirus is rapidly mutating [ ] . in such a scenario, it is necessary to select drugs that are effective against the mutated strain. while mutating, the virus isolates may develop resistance to previous drugs used for its treatment. our model may be of help to clinicians in this respect. before proposing a treatment regime (trial, for e.g.) for covid- treatment, the practitioner may use our approach to check the drugs selected for the particular isolate of novel coronavirus. in table , we have experimented with three isolates of the novel coronavirus (collected over an interval of months), in addition to the reference sequence (collected in december ). those three isolates have been collected in february (from usa), april (from australia) and june (from india). one can note from the table that the selected drugs change with mutations. baloxavir marboxil was not selected even once for the reference sequence from december , but has been selected by two methods for the february isolate. a recent pre-print [ ] reports the results of this antiviral on covid- patients. the drug ibuprofen, was selected by one of the methods for the december reference sequence, it was not selected for the february isolate, then it was selected by two methods for the april isolate and selected by all three for the june isolate. it may be worthy to note that lipid ibuprofen is being considered in a trial in uk from starting june, [ ] . similarly, pleconaril has been selected for by all three methods for the most recent (june) isolate, it was selected by only one of the techniques for the reference sequence (december) and was not selected for the february or june sequences. pleconaril, although developed for treating enterovirus and rhinovirus, is not fda approved. rilpivirine and etravirine are two antiretrovirals developed for treating hiv positive subjects. both of them have been predicted by all three methods in the latest isolate, but not in the previous isolates or in the reference sequence. to the best of our knowledge, this antiretroviral is not under study for covid- trials. note that vidarabine, which was getting predicted for the reference sequence (albeit wrongly) has not been predicted from the later ones. based on this discussion, we can see that how the mutations in genomic structure results in different predictions of drugs. since the novel coronavirus is mutating, it may be judicious to account for the structure of the latest isolate while deciding the treatments to be put in trial. in such a case, our model may be of help to clinicians. we recorded the time taken by each of the matrix completion algorithms for a single run (table ) , on a single core machine with a clock speed of . ghz, gb ram (intel(r) xeon(r) cpu e - v processor). all the methods have relatively low computational requirements. matrix factorization methods are faster than the nuclear norm minimization based techniques, with a difference of few seconds. such difference may not be practically significant, given the nature of our problem as an improved anti-viral prediction in pandemic is much more crucial than the running time in the order of seconds. we have collected a comprehensive dataset comprising of all the anti-viral drugs which act against viruses known to infect humans, along with the similarity information associated with the drugs and the viruses (see methods section). on this database, we deploy state-of-the-art drug target interaction techniques based on matrix completion. the drug-virus associations and the similarity information are assembled as three matrices: drug-virus association matrix (y ), drug similarity matrix (s d ) and virus similarity matrix (s v ). several matrix completion methods have then been implemented and compared. the matrix completion methods which are not designed to incorporate the similarity information take association matrix as input (assuming it to be a partially filled matrix from which the full low-rank association matrix would be recovered) along with the masking operator which stores information on the position of train and test indices. on the other hand, the graph regularized frameworks utilize similarity information and give an improved prediction performance in the cross-validation evaluation (best auc= . , aupr= . ). the graph regularized matrix completion methods are not only capable of predicting associations between existing drugs and viruses but can also take into account novel viruses and drugs for which no association information is known (as can be seen in the latter two cross-validation settings). the similarity information for such novel drugs/viruses (s v and s v ) can be added to the metadata using the chemical structure and sequence information of the drug and virus respectively. the validity of the proposed pipeline is illustrated by the fact that out of the drugs unanimously predicted in top- prediction by the graph regularized methods are already under trial for treating sars-cov- . from the prediction provided by graph regularized methods, we observe a consensus over the recommendation of six drugs, namely remdesivir, ribavarin, sofosbuvir, taribavirin, tenofovir alafenamide and vidarabine ( th common drug being umifenovir recommended by models). note that umifenovir and remdesivir are investigational as per fda, umifenovir is approved in russia and china and remdesivir has obtained approval for emergency use by fda. researchers working on ribavarin trials argue that since it is an established drug with ready availability and established supply chains, it is worth to investigate its potency against covid- . in a study published in april, , remdesivir was found to shorten the recovery time (median recovery time of days, % faster recovery time than patients who received placebo) in adults with covid- infection in clinical trials involving hospitalized patients in united states [ ] . it has received emergency use authorization (eua) by the us food and drug administration (fda) for patients hospitalized with severe disease [ ] . umifenovir (arbidol) is also being investigated as a potential prophylactic agent for the prevention of covid- [ ] . ribavirin [ , ] , in combination with other antiviral drugs has recently been studied for the effectiveness and safety of different antiviral regimens (combination therapies) for the treatment of covid- . sofosbuvir is used specifically for hepatitis c infection. currently it is under trial for treating covid- patients. this is because, superposition of the hepatitis c virus polymerase bound to sofosbuvir, with the sars-cov polymerase shows that the residues that bind to the drug are present in the latter [ ] . the clinical trial of tenofovir disoproxil as a prophylactic, is based on recent albeit sparse literature that shows that rna synthesis nucleos(t)ide analogue inhibitors, acting as viral rna chain terminators, like tenofovir disoproxil, abacavir or lamivudine, amongst others, could have an effect against sars-cov- [ ] . our algorithm selects tenofovir alafenamide, which is less harmful to the kidneys than tenofovir disoproxil. tenofovir alafenamide is known to have large antiviral efficacy at ten times lesser dose than tenofovir disoproxil. it is also under investigation as a combination therapy (emtricitabine/tenofovir-alafenamide and lopinavir/ritonavir) to treat covid- patients [ ] . in this sub-section, our objective is to establish that the drugs selected by our algorithms for sars-cov- are clinically sensible predictions, in the sense that they are known to be effective against a significant number of the covid- symptoms. the table shows that out of the top ten selections, six are common across all the three techniques and another (umifenovir) have been predicted by two out of three. although vidarabine has been selected by all three models, it is an antiviral effective against dna viruses. since the novel coronavirus is an rna virus this antiviral is not supposed to work. we have discussed the rest of the six antivirals in supplementary section . the descriptions have been primarily taken from drugbank.ca. furthermore, significance of such computational models for predicting anti-viral therapeutics would correlate with the rate at which a virus mutates. rna viruses (like hiv, flu virus) are known to mutate at a much faster rate than the dna viruses [ ] , helping them to evade the human immune system and develop drug resistance. sars-cov- is no exception and has been mutating over the past few months [ ] . clinically keeping up with the evolving viruses and drug resistance could be a major challenge for development of an anti-viral treatment [ ] . hence, artificial intelligence, and in particular the presented matrix completion techniques, could help the clinicians to prune down the drug space for viral strains which have been mutating rapidly and avoid unnecessary testing on drugs for the new viral strain/s. computational techniques have the inherent advantage of learning from the data (which can be huge) and scale to a large number of drugs and viruses and hence be of immense importance to the clinicians by narrowing down the search space for the clinical trials to be carried out. we would like to emphasize that the proposed dva database and methods are not particular to the novel coronavirus. such computational approaches have the general capability to help for identification of drugs which might be effective against a broad spectrum of viruses [ ] , or the viruses which can be targeted by multiple drugs (since many drugs could target specific elements of viral replication) [ ] . we believe that the proposed work will pave the way for more scientific ideas for anti-viral drug re-positioning and assist clinicians in the process. the proposed dva dataset aims at being exhaustive. it compiles various existing sources, housing together all the anti-viral drugs proved clinically to be effective against viruses infecting humans. we believe such resource would be highly useful for analysing and proposing anti-virals not only for the novel coronaviruses but other viruses too. along with that, it may also be used to computationally identify viruses that a newly discovered drug may target. the associated metadata (information about the drugs and viruses) may also help clinicians in manual analysis and having a deeper insight. all the associations corresponding to anti-viral drugs clinically shown to act against human host viruses have been assembled from standard drugbank database [ ] (https://www.drugbank.ca/categories/dbcat ). to ensure that the database is fully comprehensive, other literature works [ , , , , , , , , ] and resources such as vipr [ ] were also scanned for any additional drug-viral associations. vipr or niaid virus pathogen database and analysis resource (http://www.viprbrc.org/) is a repository of data and analysis tools for virology research [ ] capturing various types of information derived from comparative genomics analysis and visualization tools. it has antiviral drug information (for viral species) derived imported from drugbank (https://www.drugbank.ca/) [ ] . the drug-virus indications have been stored (see supplementary data section) and processed in a matrix form of size m × n (m being no of drugs in the database and n being the number of viruses) to be used as input for any of the matrix completion algorithms we made available in our repository. the drugbank identifier (drugbank id) of the anti-viral drugs involved is considered as the unique key for the drugs, obtained from drugbank vocabulary (https://www.drugbank.ca/releases/latest#open-data). along with the viral association information, we also store the target pathway and mechanism of action of each drug for quick reference in any further investigation. apart from this, each drug is mapped to its corresponding kegg identifier (kegg id) from the kegg compound/kegg drug database (https://www.genome.jp/kegg/drug/, https: //www.genome.jp/kegg/compound/) of the kegg (kyoto encyclopedia of genes and genomes) [ ] . the kegg ids were taken from the linking file provided at https://www.drugbank.ca/releases/latest#external-links [ ] or manually added in the case of drugs missing in the linking file. each virus is identified by an acronym assigned to it (in case of no acronym, full virus name is used). the viral family, genome type, transmission route and incubation period is also available in the virus metadata file along with the accession number of the complete genomic sequence of the viruses fetched from ncbi (national center for biotechnology information) viral genome browser https://www.ncbi.nlm.nih.gov/genomes/genomesgroup.cgi [ ]). to integrate the similarity information to the drug-virus associations, we have computed similarities between the drugs based on their chemical structures and between the viruses using their complete genomic sequences. • drug similarity: all the drugbank ids were mapped to kegg ids of the corresponding drug/compound in the kegg database [ ] . the chemical structure similarity was measured between the drugs by computing the simcomp score [ ] based on the maximum common substructures between the chemical structure of the compounds using the kegg api page at genomenet (https://www.genome.jp/tools/gn_tools_api. html). the drugs for which the simcomp score was less than the set cutoff ( . ) and the drugs with no kegg ids available were assigned a similarity score of to themselves and to other drugs in the dataset. • virus similarity: the d * distance based on onf (oligonucleotide frequency) measure between the dna sequences was shown to be the best amongst various other onf metrics with several k-mers length in host prediction accuracy at the genus level [ ] . hence, we compute d * dissimilarity/distance (at k= ) between the viral genome sequences obtained from ncbi [ ]. the reference sequences of viruses were saved in fasta format to be used by the distance computation software (https://github.com/jessieren/virhostmatcher) proposed by [ ] . the d * distance was subtracted from to obtain the similarity measure. for the viruses with segmented structure (influenza a virus, influenza b virus, influenza c virus, lassa mammarenavirus), the coding sequence in the nucleotide sequence of each genomic segment (taken in decreasing order of length was taken) was combined to form the complete viral sequence. in this subsection, we describe each of the matrix completion algorithms used (www.github.com/aanchalmongia/ dva), along with their mathematical formulations and resolution strategies. let x m×n be the complete drug-virus association matrix (with m drugs and n viruses) with binary entries ( denoting that the drug is known to act against the virus and denoting no association). here x is the matrix to be recovered from its sampled (partially known) entries in y . let m denote the masking operator (elementwise multiplied to x) having 's at positions where associations are known and otherwise. then, the matrix completion problem can be formulated as searching for x satisfying: under specific constraints. in particular, it is typically assumed that similar drugs act in a similar manner, hence x to be recovered (from y and m ) is of low-rank. the most straightforward technique of solving low-rank matrix completion is matrix factorization, where the data matrix x m×n is decomposed into two latent factor matrices u m×k and v k×n , where k denotes the number of latent (hidden) factors deciding if a drug is associated with a virus or not. x is recovered by solving for u and v in the following minimization problem: min the above problem is solved in an alternating manner, by first decoupling the mask using a majorization-minimization technique [ , ] and then using alternating least squares method [ ] to obtain u and v . the complete algorithm is described in [ ] . an extension of matrix factorization has been proposed motivated by the success of deep dictionary learning [ ] , where the data matrix x is decomposed into multiple factor matrices (analog to multiple layers) to capture more complex hidden features in the data. the formulation of the minimization problem in the case of -layer matrix factorization is given below: the above problem is solve alternatively. the minimization with respect to variables u and v , is done in a similar way to that of matrix factorization, while the update on u can be obtained as shown in [ ] . another variant of matrix factorization has been proposed to incorporate metadata associated with the row and column entities (drug and virus similarities in this case) [ ] . here, the drug and virus entities form the nodes of two separate graphs and the similarity between them is assumed to be the weights between the nodes. regularization is imposed by adding graph laplacian penalties to the cost function of matrix factorization as shown below: where µ > and µ > are coefficients penalizing the graph regularization laplacian terms and tr denotes the trace of the matrix. l d = d d − s d and l v = d v − s v are the graph laplacians [ ] for s d (row/drug similarity matrix) and s v (column/virus similarity matrix), respectively, and d ii d = Σ j s ij d and d ii v = Σ j s ij v are the associated degree matrices. a resolution technique for the above formulation has been shown in [ ] . matrix factorization based approach leads to a non-convex minimization problem and hence rarely benefits from global convergence guarantees. to limit the space of minimizers, it may be useful to impose a low-rank constraint on the solution x. since rank minimization is still an np-hard problem, it was proposed to relax the above constraint to its closest convex surrogate by making use of the nuclear norm penalty [ , ] . the formulation for the resulting nuclear norm minimization problem (referred to as matrix completion by the authors) is: the above problem can be solved alternatively, by invoking majorization-minimization arguments [ ] to deal with the mask operator m and by applying thresholding operations on the singular values to process the nuclear norm term [ ] . just like matrix factorization, nuclear norm minimization based matrix completion can also be graph regularized by incorporating graph laplacian penalties to take metadata/similarity information into account. the formulation for the minimization problem is given by: the above formulation can either be solved using admm (alternating direction method of multipliers) [ , ] as was done in [ ] (referred as grmc here) or by explicitly taking care of the constraint that the recovered values should be in the range [ , ]. if the latter range constraint is taken into account, we obtain then a new variant called graph regularized binary matrix completion. the minimization with respect to x can be solved by making use of the ppxa (parallel proximal algorithm) [ ] . such approach allows to decouple the constraints by introducing proxy variables and then solving each subproblem in a parallel fashion as shown in [ ] (referred as grbmc here). the stepsize, regularization parameters and latent factor dimensions, for the above techniques have been tuned using cross-validation on training set (after hiding % of the data) in each of the three cross-validation settings (see section . ). the parameters obtained after extensive cross-validation on the setting cv (randomly hiding the virus entities) have been further used in predicting drugs for sars-cov- and the corresponding isolates (see sections . and . ). similarly, the parameters selected for the setting cv (randomly hiding drug entities) have been used to evaluate the performance of the approaches in section . . a computational approach to aid clinicians in selecting anti-viral drugs for covid- trials description of drugs predicted by computational models • remdesivir (fda emergency use, investigational) -remdesivir was originally investigated as a treatment for ebola virus, but has potential to treat a variety of rna viruses. its activity against the coronavirus (cov) family of viruses, such as sars-cov and mers-cov, was shown in , and it is also being investigated as a potential treatment for covid- . • ribavirin (approved) -broad-spectrum activity against several rna and dna viruses. it is primarily indicated for use in treating hepatitis c and viral hemorrhagic fevers. it is reported that ribavirin might be only effective in early stages of viral hemorrhagic fevers including lasser fever, crimean-congo hemorrhagic fever, venezuelan hemorrhagic fever, and hantavirus infection. currently ribavirin is being used in combination with interferon beta- b, lopinavir-ritonavir in a trial for treating covid- . • umifenovir (investigational) -umifenovir is used for the treatment and prophylaxis of influenza and other respiratory infections. umifenovir's ability to exert antiviral effects through multiple pathways has resulted in considerable investigation into its use for a variety of enveloped and non-enveloped rna and dna viruses, including flavivirus / hemorrhagic fever, zika virus, lassa virus, ebola virus, chikungunya virus, hantaan virus, and coxsackie virus a and b. umifenovir is currently being investigated as a potential treatment and prophylactic agent for covid- . • sofosbuvir-sofosbuvir is a direct acting antiviral medication used as part of combination therapy to treat chronic hepatitis c. currently it is undergoing clinical trial for treating covid- . • taribavirin (similar to ribavirin) -the prodrug taribavirin is under development for the treatment of patients with chronic hepatitis c. taribavirin is metabolized by the liver and converted into its active metabolite, ribavirin. this pathway reduces exposure to red blood cells (rbcs) and increases exposure to the liver, the site of hcv replication. • tenofovir alafenamide -tenofovir alafenamide is a novel tenofovir prodrug developed in order to improve renal safety when compared to the counterpart tenofovir disoproxil. tenofovir alafenamide is indicated to treat chronic hepatitis b, treat hiv- and prevent hiv- infections. currently tenofovir disoproxil is undergoing trial as a prophylactic against covid- on healthcare workers. the study is supposed to be completed in july. • hemorrhagic fever symptoms (mayoclininc, clevelandclinic) -fever, fatigue, dizziness, body ache, headache, rashes, bleeding from the eyes, ears or mouth, difficulty breathing, internal bleeding, organ failure. drugs: ribavarin, umifenovir sars-cov symptoms (cdc, who) -fever, dry cough, sore throat, shortness of breath, headache, body ache, loss of appetite, malaise, night sweats, chills, confusion, rash and diarrhea. drugs: remdesivir mers-cov symptoms (cdc, who) -fever, chills, headache, body ache, malaise, shortness of breath, diarrhea, dry cough, sore throat, body ache and hypoxia. influenza virus (cdc) -fever, body ache, chills, sweats, headache, dry cough, fatigue, nasal congestion and sore throat. zika virus (cdc) -mild fever, rash, body ache, headache, red eyes and malaise. the web server (http://dva.salsa.iiitd.edu.in) has two tabs in the menu. the 'home' page provides reference to our work and the main functionalities of the web server. the 'about' page consists of a brief description of the dataset and the algorithms used. home -the 'home' page briefly discusses what this web server is about. it also includes reference to our pre-print. in the sub-section 'functionalities', there are two parts. the first one pertains to the prediction of drugs, given the genomic structure of the virus. the second takes in two inputs, a drug and a virus, and it returns a normalized score depicting the overall efficacy of the drug against the virus, as predicted by our computational model. figure shows the 'home' page of the server. figures , , , show how to use the web server to run an algorithm for the prediction of top- drugs for a virus already existing in the database (sample query : figures and ) or for a novel virus by uploading its genomic sequence in ".fasta" file format (sample query : figures and ) . figures and depict the usage of the software to predict the association score (normalized) between a drug and a virus predicted by the chosen algorithm (sample query : figures and ). about -this page contains of a brief description about how the dataset has been curated. it also gives short descriptions of the different algorithms used. the reference papers of the algorithms are also provided for interested new trial starts in uk to see if ibuprofen can help prevent severe breathing problems in covid- patients randomized clinical trial for the prevention of sars-cov- infection (covid- ) in healthcare personnel (epicos) coronavirus update (live)-worldometer alignment-free oligonucleotide frequency dissimilarity measure improves prediction of hosts from metagenomically-derived viral sequences drug repositioning: identifying and developing new uses for existing drugs remdesivir for the treatment of covid- -preliminary report distributed optimization and statistical learning via the alternating direction method of multipliers. foundations and trends in machine learning handling class imbalance in customer churn prediction exact matrix completion via convex optimization the power of convex relaxation: near-optimal matrix completion an overview of mutations occurring within the coronavirus- genome: mutations data reporting on sars-cov- . available at ssrn effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection a majorize-minimize subspace approach for l -l image regularization spectral graph theory. cbms regional conference series in mathematics heat shock protein positively regulates chikungunya virus replication by stabilizing viral non-structural protein nsp during infection approved antiviral drugs over the past years advance of promising targets and agents against -ncov in china computational prediction of drug-target interactions using chemogenomic approaches: an empirical survey drug-target interaction prediction with graph regularized matrix factorization fact sheet for health care providers: emergency use authorization (eua) of remdesivir (gs- tm ) drug discovery: playing dirty chikungunya virus: in vitro response to combination therapy with ribavirin and interferon alfa a coronavirus puts drug repurposing on the fast track matrix completion and low-rank svd via fast alternating least squares simcomp/subcomp: chemical structure search servers for network analyses repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis c virus infection prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial drug resistance in influenza a virus: the epidemiology and management structural basis for the inhibition of covid- virus main protease by carmofur, an antineoplastic drug. biorxiv from genomics to chemical genomics: new developments in kegg playing with duality: an overview of recent primal-dual approaches for solving large-scale optimization problems clinical outcomes and plasma concentrations of baloxavir marboxil and favipiravir in covid- patients: an exploratory randomized no current evidence supporting risk of using ibuprofen in patients with covid- imatinib binding and ckit inhibition is abrogated by the ckit kinase domain i missense mutation val ala computational prediction of drug-disease association based on graph-regularized one bit matrix completion. biorxiv deep matrix completion on graphs: application in drug target interaction prediction drug-target interaction prediction using multi graph regularized nuclear norm minimization deepmc: deep matrix completion for imputation of single-cell rna-seq data mcimpute: matrix completion based imputation for single cell rna-seq data emerging sars-cov- mutation hot spots include a novel rna-dependent-rna polymerase variant vipr: an open bioinformatics database and analysis resource for virology research parallel proximal algorithm for image restoration using hybrid regularization antiviral drugs for viruses other than human immunodeficiency virus sofosbuvir as a potential alternative to treat the sars-cov- epidemic viral mutation rates anti-hepatitis c virus drugs in development repurposing of the anti-malaria drug chloroquine for zika virus treatment and prophylaxis long-acting neuraminidase inhibitor laninamivir octanoate (cs- ) versus oseltamivir as treatment for children with influenza virus infection majorization-minimization algorithms in signal processing, communications, and machine learning deep dictionary learning clinical characteristics and therapeutic procedure for four cases with novel coronavirus pneumonia receiving combined chinese and western medicine treatment potential benefits of ibuprofen in the treatment of viral respiratory infections drugbank: a comprehensive resource for in silico drug discovery and exploration comparative effectiveness and safety of ribavirin plus interferon-alpha, lopinavir/ritonavir plus interferon-alpha, and ribavirin plus lopinavir/ritonavir plus interferon-alpha in patients with mild to moderate novel coronavirus disease : study protocol drugs: umifenovir, ribavarin ebola virus (cdc, webmd) -fever, body ache, headache, abdominal pain, rash, loss of appetite, fatigue, diarrhea, vomiting, unexplained hemorrhaging, bleeding or bruising. drugs: remdesivir, umifenovir chikungunya virus (cdc) -fever, body ache, headache, joint swelling and rash. drugs: umifenovir hantavirus (cdc) -fatigue, fever, body ache, headache, dizziness, chill, nausea, vomiting, diarrhea, abdominal pain, blurred vision, inflamed or red eyes, shortness of breath, rash, low blood pressure. drugs: umifenovir, ribavarin coxsackie virus a (webmd) -sore throat, blisters in mouth, and small tender lesions on the palms of their hands and bottom of their feet, inflammation of the spinal cord and brain. drugs: umifenovir coxsackie virus b (webmd) -fever, spasms of the abdominal and chest muscles, inflammation of the spinal cord and brain. drugs: umifenovir non-polio enterovirus (cdc) -fever, runny nose, sneezing, cough, skin rash, mouth blisters, body muscle aches. drugs: pleconaril rhinovirus (mayoclinic) -runny nose drugs: tenofovir alafenamide hiv- (acute phase) -fever, chills, rash, night sweats, body ache, sore throat, fatigue, swollen lymph nodes, mouth ulcers. drugs: tenofovir alafenamide figure : home page of the web-server readers. in the side menu, we also provide some important links key: cord- -tvqpv fp authors: corrin, bryan; nicholson, andrew g. title: occupational, environmental and iatrogenic lung disease date: - - journal: pathology of the lungs doi: . /b - - - - . - sha: doc_id: cord_uid: tvqpv fp nan in practice the term is confined to the effects of mineral dust on the lungs. diseases caused by organic dusts are not included among the pneumoconioses and, in medicolegal practice at least, the presence of dust alone is insufficient to indicate pneumoconiosis: for compensation to be considered, the mineral dust must alter the structure of the lung and cause disability. the british industrial injuries advisory council defined pneumoconiosis as 'permanent alteration of lung structure due to the inhalation of mineral dust and the tissue reactions of the lung to its presence, excluding bronchitis and emphysema' . parkes recommends that cancer and asthma caused by mineral dust should also be excluded from the definition, an opinion with which we concur. to reach the lung, dust particles have to be very small. particle density and shape also affect the aerodynamic properties of dust. host factors such as airflow characteristics, airway branching patterns and airway disease also affect dust deposition. three deposition mechanisms are recognised ( fig. . . ): . inertial impaction: when air streams change direction or velocity, the inertia of the entrained particles causes them to maintain their original direction for a distance that depends upon their density and the square of their diameter. the same rules govern a car approaching a bend too fast: the car crashes into the outside of the bend. . sedimentation (gravitational settlement): under the influence of gravity, particles settle with a speed that is proportional to their density and the square of their diameter. . diffusion: very small airborne particles acquire a random motion as a result of bombardment by the surrounding gas molecules. inhaled dust particles are liable to sediment out in the alveoli if they have a diameter in the range of - µm, are roughly spherical in shape, and in density approximate to that of water. larger or denser particles impact or precipitate on the walls of the conductive airways and are rapidly removed by ciliary action. smaller particles may reach the alveoli but do not sediment so readily and many are therefore exhaled. very small particles are deposited on the walls of alveoli by diffusion but because they are so small the total amount of dust deposited in this way is insignificant compared with that deposited by sedimentation ( fig. . . ). direct measurement shows that most lung dust ( %) has a particle diameter less than . µm. fibrous dust particles behave differently. fibres over µm in length may reach the alveoli if they are very thin and remain aligned with the air stream. fibre penetration is inversely related to path length and the number of bifurcations. tall people have longer conductive airways and experience more deposition in these sites than short people who have greater alveolar deposition for the same level of exposure. slightly more dust is deposited in the right lung than the left, probably because the right main bronchus is more in line with the trachea, and is broader and shorter than the left, and carries % of the inhaled air. , dust clearance from the lung inhaled dust that settles in the conductive airways is removed within a day or two by ciliary action. only dust that reaches the alveoli is liable to cause pneumoconiosis and much of this is also removed, but the clearance rate here is much slower: many coalminers continue to expectorate mine dust years after retirement. alveolar clearance is gravity largely effected by macrophages, principally via the airways to the pharynx but also via lymphatics to the regional lymph nodes. the airway and interstitial routes interconnect at the bronchiolar level where some dust-laden macrophages leave the interstitium for the air space. this interconnection is probably the route utilised by circulating macrophages clearing other parts of the body of endogenous or exogenous particulate matter via the lung. long asbestos fibres present a particular problem to macrophage clearance. some minerals, notably chrysotile asbestos, undergo slow physicochemical dissolution in the lungs. only a small fraction of the inhaled dust gains access to the interstitium, a necessary step if it is to cause pneumoconiosis. some free dust enters through the bronchus-associated lymphoid tissue , and some is taken up by, or pierces, the alveolar epithelium ( fig. . , p. ). [ ] [ ] [ ] some of this is transported within hours to the hilar lymph nodes. so rapid is this translocation that it is thought not to involve most, the lesions are more numerous and better developed in the upper lobes than the bases but the reverse is true of asbestosis. the reasons for this are complex but undoubtedly involve the dust deposition:clearance ratio for the effect of the dust will depend upon both its amount and the duration of its stay in the lungs. there are well-recognised regional differences in the distribution and clearance of inhaled material, which in turn are dependent upon man's upright posture, the consequent gravitational forces being maximal at the apices. when standing at rest, the apices of the lungs are hardly perfused, so that lymph formation and clearance are much better at the bases. [ ] [ ] [ ] similarly, the apices are relatively less well aerated; alveoli in the lower lobes receive more air than those in the upper lobes. , the greater respiratory excursions at the bases are thought to promote macrophage mobility there. it is to be expected therefore that the bases would both receive and clear more dust than the apices, rendering it difficult to predict on theoretical grounds which parts of the lungs carry the heaviest dust burden. in fact, more dust of all types is found in the upper lobes, the part most severely affected by every type of pneumoconiosis except asbestosis. , the predilection of asbestos to affect the periphery of the lower lobes is attributed to the dangerous long asbestos fibres preponderating there. , pulmonary reactions to mineral dust the main tissue reaction to mineral dust is fibrosis. silica is highly fibrogenic and is therefore very likely to cause pneumoconiosis. carbon is non-fibrogenic and therefore, unless there are complications, coal pneumoconiosis causes little disability. tin too is harmless, and stannosis therefore unimportant, although the chest radiograph is highly abnormal because tin is very radiopaque. stannosis is one of several terms that specify pneumoconiosis due to a particular mineral, the best known being silicosis, asbestosis and anthracosis. the blackness of carbon and red-brown colour of iron give ample evidence, both naked-eye and microscopically, of the type and amount of these dusts when they are present in the lung ( fig. . . ), but other inorganic dusts may be more difficult to identify. however, a flick-out substage condenser and polaroid filters to test for refractility and birefringence respectively are useful adjuncts that are too often neglected by the histopathologist. crystalline silica is traditionally regarded as being only weakly birefringent, in contrast to silicates which generally show up brightly with simple crossed polaroid filters. however, with modern microscope lamps, if the light source is set at high intensity when using polaroid filters, both silica and silicates are birefringent. mineralogists use polarising microscopy for analysis, but only by studying large polished crystals with controlled orientation of the light. the small dust particles found in tissue sections are too small to permit analysis by this technique but it is nevertheless very useful for detecting their presence ( fig. . . ) . particle shape gives a useful indication of mineral type but appearances are sometimes deceptive: the plate-like crystals of talc are seldom observed as such, usually being viewed edge-on, when they appear to be needle-shaped. occasionally, stains can be used to identify minerals, e.g. a modified perls' reaction for inhaled iron, and irwin's aluminon stain for aluminium, but these too have largely been replaced by modern analytical techniques. ultrafine dust particles are particularly liable to be transported across the alveolar epithelium. the integrity of the alveolar epithelium is very important to dust translocation from the air spaces to the interstitium. much more dust reaches the interstitium if the epithelium is damaged. , it is widely thought that macrophages that have left the interstitium for the alveolar space never return, , but this is probably untrue. heavily laden macrophages accumulate in alveoli bordering the terminal and respiratory bronchioles, eventually filling them completely. erosion of the alveolar epithelium permits re-entry of these macrophages into the interstitium, very close to foci of bronchial mucosaassociated lymphoid tissue (malt), which are found near the terminal bronchioles. these aggregates guard the mouths of lymphatics, which commence at this point; alveoli are devoid of lymphatics. dustladen interstitial macrophages accumulate in and around the bronchial malt, which macklin therefore referred to as dust sumps. most pneumoconiotic lesions are found in the region of the dust sumps and are therefore focal. asbestosis is diffuse rather than focal because the long asbestos fibres are not readily mobilised and cannot be concentrated in the centriacinar dust sumps. this is also seen on occasion with platy non-fibrous dusts such as talc, mica, kaolinite and feldspar. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] within the dust sumps the dust particles are not static. they are constantly being freed and reingested by interstitial macrophages and, because these cells are mobile, successively inhaled dusts soon become intimately mixed. macrophages play an important role in pneumoconiosis and if the dust is fibrogenic the repeated phagocytosis of indestructible mineral particles results in constant fibroblast stimulation. the zonal distribution of pneumoconiosis pneumoconiosis affects both lungs but seldom evenly and some pneumoconioses show characteristic patterns of lung involvement. in microincineration combined with dark-field microscopy can also be used to demonstrate small particles. incombustible mineral particles that cannot be seen with bright-field or polarising microscopy are rendered visible by this technique and their position on the slide can be compared with tissue reactions evident in a serial section that has not been incinerated. microincineration has, however, also been largely replaced by modern analytical techniques that will now be considered. analytical electron microscopy is very helpful in identifying minerals, whether applied to lung digests or tissue sections. [ ] [ ] [ ] [ ] scanning electron microscopy permits the examination of thicker sections than transmission electron microscopy but does not detect very small particles. however, scanning electron microscopy allows more tissue to be examined and avoids the difficulty of cutting mineral particles with an ultramicrotome. mineral particles in a -µm thick deparaffinised section can be recognised in a scanning electron microscope set to collect the back-scattered electrons. the instrument can then be focused on points of potential interest and switched to x-ray diffraction, which provides information on crystal structure (fig. . . ) . alternatively, elemental analysis may be undertaken with either energy-dispersive or wavelength-dispersive x-ray spectroscopy. with energy-dispersive x-ray spectroscopy, all elements of atomic number above are identified, whilst with wavelength-dispersive x-ray spectroscopy the section can be scanned for one particular element. with the former technique different elements are shown graphically as individual peaks, the heights of which are proportional to the amounts of the different elements within the particle studied, thereby giving information on probable molecular formula ( fig. . . ) . thus, different silicates can be distinguished from each other and also from silica, which registers as pure silicon, oxygen (atomic number ) not being detected. the fact that the elements of low atomic number that constitute organic chemicals are not detected means that any minerals present (except beryllium, atomic number ) can be recognised easily in tissue sections. only particles can be analysed however: elements present in only molecular amounts cannot be detected by x-ray analysis. the detection of trace amounts of substances such as beryllium requires bulk chemical analysis or techniques that are not widely available such as atomic absorption spectrometry, neutron activation analysis and microprobe mass spectrometry. , the last of these techniques can also provide molecular (as opposed to elemental) analysis of organic as well as inorganic particles. another analytical technique of interest is microscopic infrared spectroscopy which provides data on the compound nature of microscopic particles in tissue sections ( fig. . . ) . micro-raman spectroscopy is also useful in this respect. some metals cause hypersensitivity, which can be identified by exposing the patient's lymphocytes to metals and measuring their reaction in vitro. radiological grading of pneumoconiosis a scheme for grading pneumoconiosis radiologically by comparison with standard radiographs has been adopted by the international labour organisation (ilo) and is widely used. small opacities (up to cm diameter) are graded by their profusion, , and indicating increasing numbers, and by their size, increasing through p, q and r if rounded and s, t and u if irregular. type p opacities are described as punctiform and measure up to . mm in diameter; larger lesions up to mm in diameter (type q) are described as micronodular or miliary; and those over mm and up to cm in diameter (type r) are described as nodular. irregular opacities cannot be sized so accurately, s, t and u indicating fine, medium and coarse respectively. large opacities (over cm diameter) are graded by their combined size, increasing through a, an opacity measuring between and cm in diameter; b, one or more opacities whose combined area does not exceed the equivalent of one-third of the area of the right lung field (when they are regrouped in the mind's eye or measured with a transparent ruler); and c, one or more opacities whose combined area exceeds one-third of the area of the right lung field (when similarly regrouped). in coalworkers, small opacities (up to cm diameter) correspond to simple coalworker's pneumoconiosis and large opacities (over cm diameter) to complicated coalworker's pneumoconiosis, which is also known as progressive massive fibrosis. silicotic lesions have been identified in the lungs of egyptian mummies, and the injurious effects on the lungs of inhaling mine dust have been recognised for more than years. as long ago as the sixteenth century in joachimsthal, bohemia (now jachymov, czech republic), diseases of miners' lungs were attributed to the dust the miners breathed. silicosis, tuberculosis and lung cancer are all now known to have been prevalent among the miners in this region, the cancer being largely attributable to the high level of radioactivity in the mines. silicosis was recognised in the uk soon after the discovery in that the addition of calcined flint to the clay from which china is made produced a finer, whiter and tougher ware. the preparation and use of this flint powder were highly dangerous, causing the condition known as potter's rot, one of the first of the many trade names by which silicosis has since been known. aluminium oxide (alumina) now provides a safe, effective substitute for flint in this industry. in it was noted that sheffield fork grinders who used a dry grindstone died early, and amongst other preventive measures it was recommended that the occupation should be confined to criminals: fortunately for them, the substitution of carborundum (silicon carbide) for sandstone was effective enough. however, silicosis still occurs in some miners, tunnellers, quarrymen, stone dressers and metal workers. silica in one form or another is used in many trades -in the manufacture of glass and pottery, in the moulds used in iron foundries, as an abrasive in grinding and sandblasting, and as a furnace lining that is refractory to high temperatures. rocks such as granite and sandstone are siliceous and their dusts are encountered in many mining and quarrying operations. in coal mining in the uk the highest incidence of the disease was in pits where the thinness of the coal seams required the removal of a large amount of siliceous rock, a process known as 'hard heading' . in south africa, silicosis causes a high mortality among the gold miners on the witwatersrand, where the metallic ore is embedded in quartz. slate is a metamorphic rock that contains both silica and silicates, and slateworkers develop both silicosis and mixed-dust pneumoconiosis. , nor are rural industries immune from the disease, particularly if ventilation is inadequate, as it is in certain african huts where stone implements are used to pound meal and the occupants develop mixed-dust pneumoconiosis. silicosis and mixed-dust pneumoconiosis have also been reported in dental technicians. desert sand is practically pure silica but the particles are generally too large to reach the lungs. however, silicosis has been reported in inhabitants of the sahara, libyan and negev deserts and those living in windy valleys high in the himalayan mountains, [ ] [ ] [ ] [ ] [ ] [ ] [ ] whilst in california the inhalation of dust raised from earth has led to silicate pneumoconiosis in farm workers, horses and a variety of zoo animals. the silica in rocks such as granite, slate and sandstone is largely in the form of quartz and this is therefore the type of silica encountered in most of the industries considered above. cristobalite and tridymite, which are possibly even more fibrogenic than quartz, are more likely to be encountered in the ceramic, refractory and diatomaceous earth industries where processing involves high temperatures. many workers with silicosis are asymptomatic. as a general rule, exposure to silica dust extends over many years, often or more, before the symptoms of silicosis first appear: by the time the disease becomes overt clinically, much irreparable damage has been inflicted on the lungs. the initial symptoms are cough and breathlessness. from then onwards, respiratory disability progresses, even if the patient is no longer exposed to silica dust. ultimately, there may be distressing dyspnoea with even the slightest exercise. silicosis sometimes develops more rapidly, perhaps within a year or so of first exposure. such 'acute silicosis' was observed in the scouring powder industry in the s when these cleansing agents consisted of ground sandstone mixed with a little soap and washing soda. , the additives were considered to have rendered the silica in the sandstone more dangerous but it is possible that the rapidity of onset of the disease merely reflected the intensity of the dust cloud to which the packers were exposed. confusingly, the term 'acute silicosis' has since been applied to a further effect of heavy dust exposure in tunnellers, sand blasters and silica flour workers, namely pulmonary alveolar lipoproteinosis (see below), , whilst the terms 'accelerated silicosis' or 'cellular phase silicosis' have been substituted for 'acute silicosis' in referring to the rapid development of early cellular lesions. , the time from first exposure to the development of symptoms (the latency period) is inversely proportional to the exposure level. however, it is evident that a certain amount of silica can be tolerated in the lungs without fibrosis developing, indicating either a time factor in the pathogenetic process or a threshold dust load that has to be reached before fibrosis develops. silica particles that are roughly spherical in shape and of a diameter in the range of - µm sediment out in the alveoli and are concentrated within macrophages at macklin's dust sumps, as explained previously (see p. ). early lesions, as seen in so-called accelerated or cellular phase silicosis, consist of collections of macrophages separated by only an occasional wisp of collagen. the early lesions have been likened to granulomas and on occasion have been mistaken for langerhans cell histiocytosis or a storage disorder, but langerhans cells are scanty and the histiocytes contain dust particles rather than accumulated lipid or polysaccharide. the macrophages of the early lesion are gradually replaced by fibroblasts and collagen is laid down in a characteristic pattern. the mature silicotic nodule is largely acellular and consists of hyaline collagen arranged in a whorled pattern, the whole lesion being well demarcated ( fig. . . ) and sometimes calcified. small numbers of birefringent crystals are generally evident within the nodules when polarising filters are used, but these mainly represent silicates such as mica and talc, inhaled with the silica. silica particles are generally considered to be only weakly birefringent, but fairly strong birefringence is evident in strong light (see above). silicotic nodules develop first in the hilar lymph nodes and are generally better developed there than in the lungs. [ ] [ ] [ ] indeed, silicotic nodules are occasionally found in the hilar lymph nodes of persons who have no occupational history of exposure to silica and whose lungs are free of such lesions, the silica in the nodes being presumed to represent inhaled particles derived from quartz-rich soil. severely affected lymph nodes often calcify peripherally, giving a characteristic eggshell-like radiographic pattern. this is sometimes the only radiological abnormality. such enlarged lymph nodes may occasionally press upon and obstruct adjacent large bronchi or result in a left recurrent laryngeal nerve palsy, so simulating malignancy. sometimes the nodules develop within the walls of major bronchi, occasionally causing a middle-lobe syndrome (see p. ). silicotic nodules are also found along the lines of the pleural lymphatics , where they have been likened to drops of candle wax on the visceral pleura. very rarely, silica-induced fibrosis is more pronounced in the pleura than in the lungs. lung tissue between the nodules is often quite normal and not until the process is very advanced is there any disability ( fig. . . b). in severe cases large masses of fibrous tissue are formed, which may undergo central necrosis and cavitation ( fig. . . ). on close inspection it is evident that these consist of conglomerations of many silicotic nodules closely packed together. in such severe cases cor pulmonale develops. occasionally, silicotic nodules develop in the abdominal as well as the thoracic lymph nodes, and in the liver, spleen, peritoneum and bone marrow. [ ] [ ] [ ] [ ] [ ] in about % of cases, the typical pulmonary nodules that predominantly affect the upper lobes are accompanied by diffuse fibrosis that is maximal in the lower lobes. , , [ ] [ ] [ ] the latter may show 'honeycombing' and closely resemble idiopathic pulmonary fibrosis. the association is too common to be explained by chance and the diffuse fibrosis is therefore regarded as a further manifestation of the pneumoconiosis, possibly due to an interaction between the dust and the immunological factors discussed below. the pathogenesis of silicosis has excited much interest and many different theories have been advanced over the years. an early theory held that the hardness of the silica was responsible, but this was discounted by the observation that silicon carbide (carborundum) is harder than silica but is non-fibrogenic. theories based on the piezoelectric property and on the solubility of silica were successively abandoned although the latter had a long period of popularity. it gained support from kettle's experiments which showed that fibrosis developed about chambers placed in an animal's peritoneal cavity if the chambers contained silica powder sealed in by a collodion membrane through which solutes such as silicic acid could pass. however, it was later shown that the pores in a collodion membrane are quite irregular in size and when the experiments were repeated using chambers guarded by millipore membranes, no fibrosis developed, despite solutes being able to diffuse out. the solubility theory also fails to take account of the differing fibrogenicity of the various forms of silica despite them being of similar solubility. furthermore, if the outer, more soluble layer of the particles is removed by etching, fibrogenicity is increased although solubility is decreased. in line with this, freshly fractured crystalline silica is more pathogenic in every respect than its aged equivalent, which may partly explain the severity of silicosis in trades such as sandblasting. these observations suggest that the fibrogenicity of silica is connected with its surface configuration. it is now known that uptake of the silica by macrophages is necessary for silicosis to develop. if silica and macrophages are enclosed together in peritoneal millipore chambers, a soluble product of the macrophages diffuses out and causes fibrosis. this observation led to the realisation that the fibrogenicity of the various crystalline forms of silica correlated well with their toxicity to macrophages and for a time macrophage death was thought to be necessary. it is now considered that before the macrophages are killed by the ingested silica, they are stimulated to secrete factors that both damage other con stituents of the lung and promote fibrosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] transforming growth factor-β is one fibrogenic factor that has been implicated in the pathogenesis of silicosis. [ ] [ ] [ ] toxic damage to macrophages is due to silica particles injuring the phagolysosomal membranes, so releasing acid hydrolases into the cytoplasm. it is important in the pathogenesis of the disease indirectly because when the macrophage crumbles, the silica particles are taken up by fresh macrophages and the fibrogenic process continues. it has been suggested that early involvement of the hilar lymph nodes in the fibrogenic process promotes the development of the disease in the lung by delaying dust clearance. immunological factors have been implicated in the pathogenesis of silicosis because many patients with silicosis have polyclonal hypergammaglobulinaemia, rheumatoid factor or antinuclear antibodies, and because there is a well-recognised association between autoimmune diseases such as systemic sclerosis and rheumatoid disease and exposure to silica. , [ ] [ ] [ ] [ ] the relation of immunity to dust exposure appears to be a reciprocal one: on the one hand, the presence of dust results in rheumatoid lesions in the lungs being more florid (see caplan's syndrome, p. ), whilst on the other, non-specific immunisation of rabbits with horse serum results in experimental silicotic lesions being larger and more collagenous. it is doubtful whether pneumoconiosis and autoimmune disease play a causative role in each other but one seems to aggravate the other and may lead to its earlier development. one of the commonest and most feared complications of silicosis is chronic respiratory tuberculosis. once this infection has been added to the silicosis, the prognosis rapidly worsens. it is thought that in the presence of silica, the tubercle bacilli proliferate more rapidly because the ingested silica particles damage phagolysosomal membranes and thereby interfere with the defensive activity of the macrophages. the synergistic action of silica dust has long been held responsible for the inordinately high incidence of respiratory tuberculosis in mining communities. many former south african gold miners now have acquired immunodeficiency syndrome (aids) as well as silicosis and tuberculosis has consequently reached almost epidemic proportions amongst these men. phagocyte damage by ingested dust particles may also cause some cases of chronic necrotising aspergillosis complicating pneumoconiosis. a series of studies suggesting that there might be a link between silica inhalation and lung cancer was reviewed by the international agency for research on cancer in , leading to the conclusion that the evidence for carcinogenicity of crystalline silica in experimental animals was sufficient, while in humans it was limited. subsequent epidemiological publications were reviewed in , when it was concluded that the epidemiological evidence linking exposure to silica to the risk of lung cancer had become somewhat stronger but that in the absence of lung fibrosis remained scanty. the pathological evidence in humans is also weak in that premalignant changes around silicotic nodules are seldom evident. nevertheless, on this rather insubstantial evidence, lung cancer in the presence of silicosis (but not coal or mixed-dust pneumoconiosis) has been accepted as a prescribed industrial disease in the uk since . some subsequent studies have provided support for this decision. in contrast to the sparse data on classic silicosis, the evidence linking carcinoma of the lung to the rare diffuse pattern of fibrosis attributed to silica and mixed dusts is much stronger and appears incontrovertible. , alveolar lipoproteinosis in response to heavy dust exposure a further complication of exposure to silica is the development of alveolar lipoproteinosis (see p. ). , , , very heavy experimental exposure to silica, and indeed other dusts, stimulates hypersecretion of alveolar surfactant to such an extent that the normal clearance mechanism is overwhelmed. [ ] [ ] [ ] [ ] [ ] [ ] [ ] alveolar macrophages are enlarged by numerous phagolysosomes distended by lamellar bodies that represent ingested surfactant. the alveoli are filled by such cells and, having a foamy cytoplasm, they produce the appearances of endogenous lipid pneumonia, similar to that more usually encountered as part of an obstructive pneumonitis distal to a bronchial tumour. the macrophages gradually disintegrate and the free denatured surfactant slowly becomes compacted, during which time its staining with both eosin and the periodic acid-schiff reagents intensifies until the appearances are finally those of alveolar lipoproteinosis. this process prevents the aggregation and concentration of the dust in the usual foci and thereby hinders the development of silicosis. lipoproteinosis and silicosis may be seen in conjunction but, more often, different areas of the lung show one or the other. the lipoproteinosis has its own severe impact on lung function, but, unlike silicosis, is potentially reversible (by massive alveolar lavage). occasional patients exposed to silica develop renal disease. [ ] [ ] [ ] [ ] two mechanisms appear to operate. first, translocation of silica particles from the lungs leads to their deposition in the renal interstitium with resultant nephrotoxity. second, silica stimulates an autoimmune response characterised by the formation of various antibodies, notably rheumatoid factor and antinuclear antibodies, which leads to the development of immune complex-mediated glomerulonephritis. , amorphous silica manmade submicron forms of silica, variously known as amorphous, vitreous, colloidal, synthetic or precipitated silica, are widely used in industry. they consist of pure non-crystalline silicon dioxide. particle size ranges from to nm but aggregates of the particles measure from to µm. industrial surveys suggest that inhalation of such dust is harmless, observations that are in accord with the results of animal experiments. an amorphous silica is the principal component of the fossilised remains of diatoms that constitute the sedimentary rock, diatomite ( fig. . . ). this is generally obtained by open-cast mining, following which the rock is crushed and calcined. the calcined product is used in filters, insulation material and as a filler. being amorphous, the silica in diatomite is harmless, but calcining (> °c) results in its conversion to crystalline forms of silica. diatomaceous earth pneumoconiosis is unusual and its risk appears to be related to the amount of cristobalite and tridymite (two forms of crystalline silica) produced in the calcining process. the silicates are complex compounds in which silicon and oxygen form an anion combined with cations such as aluminium and magnesium: talc, for example, is a hydrated magnesium silicate with the formula mg si o (oh) . silicates include fibrous forms (asbestos and the zeolites), plate-like forms (talc and mica) and clays (kaolinite and fuller's earth). in histological sections, the platy talc and mica particles are generally cut tangentially and therefore appear needleshaped (see fig. . . ). they are strongly birefringent whereas the clays are only weakly so. talc particles in the lung exceeding µm in length should arouse suspicion of intravenous drug abuse. of the fibrous silicates, zeolite is used as a building material in certain communities, notably in central turkey. pneumoconiosis is not a problem but zeolites are of medical interest because, like asbestos, they present a mesothelioma risk. asbestos is dealt with separately (see below). pneumoconiosis has been described with various non-fibrous silicates, notably in the rubber industry, which uses talc and, less commonly, mica as lubricants. other occupations posing a risk include the extraction of kaolinite from china clay (kaolin), , , and in the open-cast and underground mining of fuller's earth (montmorillonite, bentonite and attapulgite clays, which were originally used in 'fulling' (degreasing) wool). , however, all these substances are commonly contaminated with silica, asbestos or both, and it has been questioned whether in pure state they are at all fibrogenic. the modifying effect of inert substances such as iron on that of silica is well known (see mixed-dust pneumoconiosis, below) and it has been suggested that talc, mica and fuller's earth act in a similar way in regard to their more fibrogenic contaminants, the pneumoconioses attributed to them in reality representing mixed-dust pneumoconiosis or asbestosis. contrary evidence comes from reports of pulmonary fibrosis in persons heavily exposed to pure talc, mica or kaolin. all these silicates are evident in the tissues as plate-like birefringent crystals which often provoke a foreign-body giant cell reaction (see fig. . . ) and may result in fibrotic nodules. large focal lesions resembling the progressive massive fibrosis of coalworkers may be produced, and also a diffuse 'asbestosis-like' form of pneumoconiosis, the latter attributed to poor macrophage mobilisation of the plate-like particles. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it would appear therefore that silicates are indeed fibrogenic if enough is inhaled; they appear to vary in fibrogenicity but in all cases they are less fibrogenic than silica. inert dusts are non-fibrogenic and therefore of little clinical consequence, although elements of high atomic number can give rise to a striking chest radiograph. it should be noted however that inert or lowly fibrogenic materials may be associated with substances of medical importance, for example, kaolin, bentonite and barytes (barite) may all be contaminated with silica , , and talc may be contaminated with asbestos. the best known of the inhaled inert mineral dusts is carbon while, of the remainder, iron is the most widespread. others include tin and barium. with all these dusts, particles retained in the lung are gathered at macklin's dust sumps by heavily laden macrophages which are lightly bound together there by a few reticulin fibres. collagen is not formed and the worker suffers no ill-effects. the lungs take on the colour of the dust and in siderosis assume a deep brick-red hue. carbon deposition is commonly found in the lungs, particularly those of city dwellers and tobacco smokers. it is also the principal constituent of coal, which is dealt with separately below, and large amounts of pure carbon may be inhaled by workers involved in the manufacture of carbon black, carbon electrodes and charcoal. [ ] [ ] [ ] [ ] although carbon is regarded as being non-fibrogenic, the very heavy lung burdens encountered in industries such as these may lead to the complicated form of pneumonconiosis known as progressive massive fibrosis that is more commonly encountered in coal workers (see p. ). heavy pure carbon deposition may also be acquired domestically when wood is burnt in buildings devoid of a chimney, so-called 'hut lung', a term that is also applied to the domestic acquisition of carbon mixed with silica or silicates, resulting in forms of mixeddust pneumoconiosis. , , anthracofibrosis is a term introduced by chinese bronchoscopists for bronchial stenosis or obliteration associated with carbon pigmentation of the mucosa. although the original description incriminated tuberculosis, mixtures of various mineral dusts acquired at work or domestically are a more likely cause. [ ] [ ] [ ] [ ] iron dust in the lungs was first described by zenker in , when he also introduced the terms siderosis and pneumonokoniosis. zenker was describing a woman who coloured paper with iron oxide powder ('rouge'), a substance which is still encountered by some workers engaged in polishing silver, glass, stone and cutlery. siderosis is also found in welders, iron foundry fettlers, steel workers, boiler scalers and haematite miners and crushers. iron dust particles are reddish-brown but in the lung may be masked by carbon : when evident, or revealed by microincineration, they resemble haemosiderin and generally give a positive perls' reaction, but particularly with haematite, heat ( - o c) and concentrated ( n) hydrochloric acid may be necessary. haematite miners in both the uk (cumbria) and france (lorraine) have an increased risk of bronchial carcinoma, but radon gas rather than haematite is the suspected carcinogen. radon is a decay product of uranium. minute amounts are present in all rocks but local concentrations occur and these are liable to build up in mines if ventilation is limited. silver, as well as iron, is found in the lungs of silver polishers, where it stains elastin in alveolar walls and pulmonary vessels grey. such argyrosiderosis is as harmless as siderosis. tin miners are subject to silicosis but not stannosis because the ore, which is found in association with siliceous rocks, contains only low concentrations of the metal. tin smelters, on the other hand, and factory workers exposed to high concentrations of tin dust or fume, are liable to inhale large amounts of this inert metal and develop the striking chest radiograph of stannosis. they remain in good health however for tin is completely non-fibrogenic. tin particles in the lung resemble carbon but are strongly birefringent and remain after microincineration: microprobe analysis provides positive identification. other inert dusts include barium, which also has a high atomic number and is therefore radiopaque, and minerals of low radiodensity such as limestone, marble and cement (all chiefly composed of calcium carbonate) and gypsum (hydrated calcium sulphate). however, the extraction of barium ore (almost entirely in the form of barium sulphate, which is known as barytes in europe and barite in the usa) may entail exposure to silica and silicates. pure baritosis resembles stannosis and siderosis. the term 'mixed-dust pneumoconiosis' refers to the changes brought about by inhaling a mixture of silica and some other less fibrogenic substance such as iron, carbon, kaolin or mica. , , [ ] [ ] [ ] the proportion of silica is usually less than %. typical occupations include foundry work and welding and the mining of coal, haematite, slate, shale and china clay. the action of the silica is modified and, although fibrotic nodules are formed, they lack the well-demarcated outline and concentric pattern of classic silicosis. the lesions are found in a centriacinar position and are stellate in outline with adjacent scar emphysema. they are firm and generally measure no more than mm in diameter. they closely resemble the fibrotic nodules of simple coal pneumoconiosis (see below). confluent lesions also occur on occasions. these resemble the progressive massive fibrosis of coalworkers and appear to represent a single large lesion rather than a conglomeration of individual nodules, as in advanced silicosis. abundant dust is generally evident in lesions of all sizes; this consists of black carbon or brown iron mixed with crystals of varying degrees of birefringence, silicates generally being strongly birefringent and silica weakly so. calcification is unusual. mixed-dust pneumoconiosis carries an increased risk of pulmonary tuberculosis, but not to the same degree as silicosis. in some cases the stellate nodules are accompanied by diffuse fibrosis, as in silicosis and again possibly involving interactions between the dust and immunological factors. involvement of the bronchi with consequent stenosis (so-called anthracofibrosis) is described above. the term 'anthracosis' was initially applied to changes observed in a coalminer's lung but is now often extended to include the common carbon pigmentation of city dwellers' lungs, and the term 'coal pneumoconiosis' is more appropriate to a special form of pneumoconiosis to which coalworkers are subject, particularly those who work underground. the principal constituent of coal, carbon, is non-fibrogenic, so suspicion has naturally fallen on the ash content of mine dust, some of which derives from the coal, some from adjacent rock strata and some from stone dust laid in the roadways to minimise the risk of coal dust explosions. coal itself appears to be the responsible agent because coal-trimmers, working in the docks and not exposed to rock dust, also develop the disease. coalminers encountering siliceous rock are, of course, also liable to develop silicosis like other underground workers. coal consists largely of elemental carbon, oxygen and hydrogen with traces of iron ore and clays such kaolinite, muscovite and illite, but no silica. the mineral content varies with the type and rank (calorific value) of the coal. all coal derives from peat, the youngest type being lignite and the oldest anthracite, with bituminous (house) coal in between. as it ages, the oxygen and mineral constituents diminish and the coal hardens. lignite is soft and said to be of low rank, anthracite hard and of high rank, with bituminous coal intermediate. although high-rank coal is of low mineral content, its dust is more toxic to macrophages in vitro and is cleared more slowly in vivo. this observation may explain why, in the uk, high-rank coal is associated with a higher prevalence of coal pneumoconiosis. the low mineral content of high-rank coal is reflected in the mineral content of the lungs of those who hew such coal in the uk, but in the ruhr, in germany, and in pennsylvania, in the usa, anthracite miners' lungs contain more silica than those who hew bituminous coal, the silica presumably deriving from other sources. not surprisingly, the presence of silica is reflected in the tissue reaction to the inhaled dust, resulting in a more fibrotic reaction very analogous to mixed-dust pneumoconiosis. a spectrum of changes is therefore encountered in coalminers' lungs, ranging from coal pneumoconiosis through mixeddust pneumoconiosis to silicosis; the findings in any individual depend upon the nature of the coal being mined and the type of work undertaken. in high-rank british collieries the development of coal pneumoconiosis appears to depend on the total mass of dust inhaled, whereas in low-rank british collieries the mineral content of the lung dust appears to be more important. this may explain apparently contrary data drawn from different coalfields -data based on coals of different composition that are not strictly comparable. some workers have stressed the importance of silica in the dust whereas others, particularly in the high-rank coalfields of south wales, have been unable to detect any association between silica and the level of pneumoconiosis. both findings may be correct, but only for the particular group of miners examined in each case. the lesions of coal pneumoconiosis are generally focal and fall into one or other of two major types, simple and complicated, depending upon whether the lesions measure up to or over cm; simple corresponds to categories - of the ilo grading system (see p. ) and complicated, which is also known as progressive massive fibrosis, to ilo categories a-c. more diffuse interstitial fibrosis has been reported in about % of welsh and west virginian coalminers, usually involving those carrying a particularly heavy dust burden; it runs a more benign course than non-occupational interstitial fibrosis (idiopathic pulmonary fibrosis). similar findings have been reported from france. simple coal pneumoconiosis consists of focal dust pigmentation of the lungs, which may be associated with a little fibrosis and varying degrees of emphysema. its clinical effects are relatively minor. some degree of black pigmentation (anthracosis) of the lungs is common in the general urban population, especially in industrial areas, but much denser pigmentation is seen in coalminers, whose lungs at necropsy are black or slate-grey. black pigment is evident in the visceral pleura along the lines of the lymphatics and on the cut surface where it outlines the interlobular septa and is concentrated in macklin's centriacinar dust sumps ( fig. . . ). the dust is generally more plentiful in the upper parts of the lungs and in the hilar lymph nodes, possibly due to poorer perfusion and consequently poorer lymphatic drainage there (see p. ). two forms of coal dust foci are recognised, macules and nodules, the former being soft and impalpable and the latter hard due to substantial amounts of collagen. both lesions are typically stellate but the more fibrotic the nodules, the more rounded they become, until it is difficult to distinguish them macroscopically from those of silicosis. in these circumstances reliance has to be placed on the whorled pattern of the collagen that is evident microscopically in silicosis. the stellate nodules are analogous to those seen in mixed-dust pneumoconiosis caused by mixtures of silica and inert dusts other than carbon (see above). with polarising filters, small numbers of birefringent crystals may be seen in both macules and nodules, usually representing mica or kaolinite derived from rock that bordered the coal. macules consist of closely packed dust particles, free or within heavily laden macrophages, so that the lesion appears black throughout ( fig. . . ). appropriate stains show that the dust-laden macrophages and free dust are lightly bound by reticulin. very little collagen is evident. although striking in their appearance, dust macules are thought to have little effect on lung function. nodules contain substantial amounts of collagen and are thought to have an adverse, but limited, effect on respiration. they vary from a heavily pigmented, stellate lesion, which apart from its collagen content resembles the dust macule ( fig. . . ), to one that is less pigmented and more circumscribed. the stellate, heavily pigmented type of nodule is seen in lungs that have a relatively low ash content whilst the more rounded and less pigmented nodule is seen in lungs with relatively high ash loads. radiologically (see p. ), p-type opacities correspond to macules, q-type opacities to the stellate nodules that resemble those of mixeddust pneumoconiosis and r-type opacities to the rounded nodules that resemble those of silicosis. , thus, the radiological changes of simple coalworker's pneumoconiosis are due to the dust and the small amount of collagen present and do not reflect any emphysema that may also be present. however, pulmonary dust foci are often associated with emphysema ( fig. . . ) and the severity of the emphysema appears to correlate with the dust load. the prevalence of chronic bronchitis and emphysema is high in the coal industry and it has long been debated whether occupation or cigarette smoking is the major factor contributing to emphysema in coalminers. [ ] [ ] [ ] [ ] as well as mineral dust, nitrous fumes from shot-firing form another occupational hazard of coal mining. heppleston made a special study of the emphysema found in coalminers, claiming that it differs from centriacinar emphysema, as seen in smokers in the general population, and attributing it to the dust. he introduced the term 'focal emphysema of coalworkers' to describe this special process. others find it very difficult to identify any convincing difference between the emphysema of coalworkers and that encountered outside the industry but heppleston based his claims on the study of serial sections. by this means he showed that, although both forms affect respiratory bronchioles, the focal emphysema of coalworkers affects more proximal orders of these airways and is not associated with the bronchiolitis seen with centriacinar emphysema. furthermore, focal emphysema is a dilatation lesion whereas coniosis, also known as progressive massive fibrosis, can have very serious consequences. particularly when the lesions are large, it is associated with productive cough, breathlessness, significant impairment of lung function and premature death. the major factor accounting for the development of progressive massive fibrosis appears to be the sheer bulk of coal dust in the lung, rather than coal rank or the silica content of the mine dust. progressive massive fibrosis has occasionally been recorded in dockers loading silica-free coal into the holds of ships and in workers exposed to pure carbon in the manufacture of carbon black and carbon electrodes. [ ] [ ] [ ] progressive massive fibrosis is characterised by large (over cm) black masses, situated anywhere in the lungs but most common in the upper lobes. the lesions may be solitary or multiple and very large, occupying most of the lobe and even crossing an interlobar fissure to involve an adjacent lobe (figs . . b, . . ). they cut fairly easily, often with the release from a central cavity of black fluid flecked by cholesterol crystals. for many years it was believed that the condition was the result of synergism between mycobacterial infection and dust but the failure of the attack rate to decrease as tuberculosis declined negated this view. today, more emphasis is placed on total dust load for the lesions tend to affect lungs that carry an unduly heavy dust burden. if the remainder of the lung shows little evidence of dust accumulation, the possibility of the masses representing caplantype lesions (see below) should be considered. centriacinar emphysema involves destruction of adjacent alveolar walls. by definition, therefore, focal emphysema is not a true emphysema at all (see p. ). however, it has been shown that mineral dusts cause elastin and collagen breakdown in the rat lung. focal emphysema may progress to the destructive centriacinar form and this has strengthened claims that mine dust plays a causal role in centriacinar emphysema. , [ ] [ ] [ ] [ ] [ ] [ ] in the uk, these claims have been accepted and chronic bronchitis and emphysema in coalminers and metal production workers have been accepted as prescribed industrial diseases since . in germany too, chronic obstructive pulmonary disease is now compensatable as an occupational disease. the conditions for compensation in the uk were initially: • underground coal mining for a minimum of years in aggregate • forced expiratory volume in second at least litre below that expected or less than litre in total • radiological category of at least / . however the last of these criteria has now been dropped. the inclusion of a time element and the omission of some estimate of dust load (such as radiological category) have been criticised, with some justification. as with lung cancer caused by chromates benefit is paid irrespective of smoking habits. whereas simple coal pneumoconiosis, particularly the macular variety, has little effect on lung function, complicated coal pneumo- microscopically, the lesions consist of dust and connective tissue intermixed in a random fashion. central necrosis and cavitation commonly occur. the necrosis is thought to be ischaemic. it is amorphous or finely granular, and eosinophilic apart from abundant dust particles and cholesterol crystal clefts. the fibrotic component in a complicated pneumoconiotic lesion is rich in fibronectin, with collagen only more abundant at the periphery. two types of progressive massive fibrosis are recognisable, corresponding to the two types of nodule described in simple coal pneumoconiosis. the first appears to have arisen by enlargement of a single nodule, whereas the second is a conglomeration of individual lesions, each of which corresponds to the more circumscribed type of nodule seen in simple coal pneumoconiosis. the ash content of the lungs bearing these two types of progressive massive fibrosis varies in the same way as with the two types of simple pneumoconiotic nodules, the enlarged single lesion being found in lungs with a relatively low ash content, and the conglomerate lesion in lungs with a relatively high ash content. the second type resembles the conglomerate nodules of large silicotic lesions but lacks the characteristic whorled pattern of the latter. the diffuse interstitial fibrosis found in a minority of coalworkers is associated with heavy dust deposition. it may progress to honeycombing but, as with the focal forms and unlike idiopathic interstitial fibrosis, it is better developed in the upper zones, the reasons for which are discussed above (see the zonal distribution of pneumoconiosis, p. ). the pathogenesis of coal pneumoconiosis has much in common with that of silicosis, and indeed many other pneumoconioses. it involves the promotion of fibrogenic factor synthesis and release by cells phagocytosing the inhaled dust. several such factors have now been identified, the degree of fibrosis produced varying with the amount of dust inhaled and the ability of its constituents to promote the production of the responsible cytokines. these include plateletderived growth factor, insulin-like growth factors and , transforming growth factor-β and tumour necrosis factor-α. , , as with other minerals, the indestructability of the dust perpetuates the process. as in silicosis, immunological factors appear to be involved, for there is an increased prevalence of rheumatoid arthritis and of circulating autoantibodies [ ] [ ] [ ] in miners with coal pneumoconiosis. rheumatoid factor has also been demonstrated within the lung lesions. these abnormalities are generally more pronounced in miners with complicated pneumoconiosis but are also found in those with the simple variety. it is also possibly pertinent to the immunological basis of coal pneumoconiosis that some of the pulmonary manifestations of rheumatoid disease are more pronounced in coalminers. this was first pointed out by caplan and will be considered next. caplan described distinctive radiographic opacities in the lungs of coalminers with rheumatoid disease, and it is now recognised that similar lesions may develop in rheumatoid patients exposed to siliceous dusts. the development of such rheumatoid pneumoconiosis does not correlate with the extrapulmonary or serological activity of the rheumatoid process. nor is there a strong relation to dust burden: caplan lesions are characteristically seen in chest radiographs that show little evidence of simple coal pneumoconiosis. pathologists recognise the lesions as particularly large necrobiotic nodules similar to those seen in rheumatoid patients who are not exposed to dust (fig. . . ) . however, because of their large size (up to cm diameter) they may be confused with progressive massive fibrosis undergoing central ischaemic necrosis (see above) or silicosis complicated by caseating tuberculosis. such errors will be less likely if the radiological evolution of the lesions is considered for they tend to cavitate and undergo rapid remission, only to be succeeded by others. they are also well demarcated radiologically. pathologically, they resemble rheumatoid nodules in showing peripheral palisading but differ in their large size and the presence of dust. the dust accumulates in circumferential bands or arcs within the necrotic centres of the lesion (fig. . . ), an arrangement that suggests periodic episodes of inflammatory activity. caplan lesions differ from tuberculosis in lacking satellite lesions and tubercle bacilli, and from progressive massive fibrosis in showing characteristic bands of dust pigmentation (table . asbestosis is defined as diffuse interstitial fibrosis of the lung caused by exposure to asbestos dust. , it does not cover asbestos-induced carcinoma of the lung or asbestos-induced pleural disease. the development of asbestosis depends on the presence of fairly large dust burdens: this is in contrast to mesothelioma and other forms of asbestos-induced pleural disease, which, although also dose-related, occur following the inhalation of far smaller amounts of asbestos dust. asbestos is a generic term for more than naturally occurring fibrous silicates, fibre being defined as an elongated particle with a length-tobreadth (aspect) ratio of at least . asbestos fibres have a high aspect ratio, generally over . based on their physical configuration they can be divided into two major groups, serpentine and amphibole. the physical dimensions and configuration of asbestos fibres are strongly linked to their pathogenicity. chrysotile (white asbestos) is the only important serpentine form. it accounts for most of the world production of asbestos of all types ( being a serpentine mineral, chrysotile consists of long, curly fibres that can be carded, spun and woven like cotton ( fig. . . ). the curly chrysotile fibres are carried into the lungs less readily than the straight amphibole asbestos fibres, and once there undergo physicochemical dissolution and are cleared more readily. they readily fragment into short particles that are easily ingested by macrophages and in the acidic environment of the macrophage phagolysosome they are particularly unstable. the half-life of chrysotile in the lungs is estimated to be in the order of only a few months. , not surprisingly therefore chrysotile is the least harmful type of asbestos in respect of all forms of asbestos-induced pleuropulmonary disease. [ ] [ ] [ ] it may nevertheless cause pulmonary fibrosis if sufficient is inhaled. , in contrast to chrysotile, amphibole forms of asbestos consist of straight rigid fibres that are stable within the lung. they do not fragment, they are insensitive to chemical attack and their clearance halflives are in the order of decades rather than months. the main amphibole forms of asbestos of commercial importance are crocidolite (blue asbestos) and amosite (brown asbestos). crocidolite, reputedly the most dangerous in regard to all forms of asbestos-related disease, was formerly mined in western australia (wittenoom) and south africa (cape province and the transvaal); it was the principal amphibole used in the uk. amosite, the name of which derives from the acronym for the former asbestos mines of south africa company in the transvaal, was the principal amphibole used in north america. amphiboles are no longer imported by the developed countries but much remains in old lagging and presents a considerable dust hazard when this is removed. tremolite, a further amphibole asbestos, contaminates quebec chrysotile deposits, montana vermiculite and many forms of commercial (non-cosmetic) talc and is responsible for much of the asbestos-related disease in chrysotile miners and millers. another amphibole asbestos, anthophyllite, was formerly mined in finland. it causes pleural plaques (see p. ) but not lung disease, possibly because its fibres are relatively thick ( fig. . . ) . erionite is a zeolite rather than a type of asbestos but is comparable in form to amphibole asbestos and is also biopersistent. it is found . these coated structures are termed 'asbestos bodies' . because other fibres may gain a similar coat, the non-specific term 'ferruginous body' has been advocated. however, coated carbon fibres (so-called coal bodies) are easily recognised as such by their black core. in practice, ferruginous bodies with the appearance of asbestos bodies almost always prove to have an asbestos core. , long fibres are more likely to be coated than short ones, which are cleared more quickly: in one study few fibres less than µm in length were coated and few fibres over µm in length were uncoated. amphiboles form bodies more readily than chrysotile. a comparison of light and electron microscopic fibre counts found that . % of chrysotile, % of crocidolite and . % of amosite formed bodies. nevertheless, sufficient chrysotile fibres are coated to permit recognition of asbestosis by standard histological criteria (diffuse fibrosis and asbestos bodies), even if chrysotile is the only asbestos present. despite the biodegradability of chrysotile, asbestos body numbers do not materially diminish with time. very occasionally however a patient with diffuse pulmonary fibrosis and a history of asbestos exposure has no evident asbestos bodies but analysis shows a fibre burden within the range found in asbestosis, justifying fibre analysis in such cases. a there is evidence that alveolar macrophages are involved in the coating of asbestos fibres to form asbestos bodies and that the bodies are less harmful to the macrophages than uncoated fibres. asbestos bodies give a prussian blue reaction for iron when stained by perls' method and their yellow-brown colour makes them easily recognisable in unstained films of sputum or in unstained histological sections. sections may be cut µm thick to increase the yield and help identify bodies that lie at an angle to the microtome blade. there is a good correlation between the numbers of asbestos bodies seen in lung sections and those in tissue digests. , the bodies may be found singly or in irregular clumps or stellate clusters. they are unevenly distributed but in well-established asbestosis they are easily found. if they are not evident, asbestos burden may be assessed quantitatively in tissue digests (see below). their presence in lung tissue, sputum or bronchoalveolar lavage fluid merely confirms exposure, not the presence of disease. however, the number of asbestos bodies in lavage fluid correlates well with lung asbestos burden , and the number in sputum correlates with the duration and intensity of exposure. [ ] [ ] [ ] fibre counts , , [ ] [ ] [ ] [ ] [ ] quantitation is desirable in certain circumstances (box . . ), in which case it is best effected on -cm blocks of fixed or fresh lung tissue obtained from three different sites, avoiding tumour and thickened pleura. the tissue blocks are digested with caustic soda or bleach, following which the fibres may be collected on a millipore membrane or viewed in suspension in a red blood cell-counting chamber. if phase contrast optics are used both coated and uncoated fibres can be assessed. alternatively, dark ground illumination can be used to demonstrate uncoated fibres. however, electron microscopy is to be preferred as it detects far more fibres than are visible by light microscopy and can also provide information on fibre type. it is important that the laboratory is well practised in fibre analysis and has established its own control range for the general population as well as asbestosis as most lungs contain some asbestos. ambient fibres are generally shorter than µm and some workers therefore confine their counts to fibres that are at least as long as this. justification for this comes from animal experiments demonstrating that long fibres cause more inflammation, chromosomal damage, fibrosis, lung tumours and mesotheliomas than short fibres, [ ] [ ] [ ] [ ] and from studies in humans suggesting that long fibres in parts of central turkey where it causes both mesothelioma and a pattern of interstitial pulmonary fibrosis that is comparable to asbestosis. , asbestos use and exposure exposure to asbestos occurs in countries where it is extracted ( asbestos is used particularly for fireproofing, in heat and sound insulation and for strengthening plastics and cement. thus, unless adequate precautions are taken, exposure is experienced by dockers unloading asbestos in the close confines of a ship's hold, by thermal insulation workers (laggers and strippers) in shipyards, power stations, train maintenance depots, factories and other large buildings, by construction workers such as carpenters cutting asbestos building panels, and by workers making asbestos products such as fireproof textiles, brake and clutch linings, and specialised cement. as well as such direct exposure, exposure may also be: • indirect, as experienced by the families of asbestos workers • paraoccupational, as experienced by those working alongside an asbestos worker • neighbourhood, as experienced by those living downwind of an asbestos works or mine • ambient, as experienced by those living or working in a building containing asbestos. exposure to asbestos incorporated in the structure of a building carries a negligible health risk if the asbestos material is well maintained to prevent shedding of dust. stripping asbestos out is more dangerous than maintaining it in situ, but maintenance is sometimes neglected. the near indestructibility of asbestos accentuates the health problems that its ubiquity poses. because of their aerodynamic properties, fibres of µm or more in length may reach the finer bronchioles and alveoli. once impacted, the sharp asbestos fibres become coated with a film of protein that is rich in iron. the coating is thickest at the ends of the fibres, giving a other human studies have shown that, although asbestos load is maximal in the upper lobes, more long fibres are found at the bases, where fibrosis is most marked. , a further reason for limiting attention to the longer fibres is that the shorter ones are cleared more easily and their number therefore varies with the time lapsed since last exposure. for these reasons asbestos regulations in many countries now limit attention to fibres that are over µm in length and have a length-to-diameter (aspect) ratio greater than : such fibres have become known as regulatory or world health organization (who) fibres. values are best expressed as fibres/g dry lung. by light microscopy, normal values range up to : over is seen with mesotheliomas, and over in asbestosis (table . . ). , , , however, compared with electron microscopy, light microscopy is relatively insensitive, showing only . % of the amosite, % of the crocidolite and . % of the chrysotile. light microscopic counts correlate poorly with severity of asbestosis and electron microscopy non-asbestos fibres commonly found in the lung include mullite, which derives from fly ash. this may constitute up to % of the total fibre burden (see table . . ) and is thought to be harmless. there is no firm evidence that manmade fibres present a health hazard but in certain localities natural non-asbestos mineral fibres, zeolites for example, are important causes of mesothelioma (see p. ) and also cause interstitial pulmonary fibrosis. in contrast to the first half of the twentieth century, much of the asbestosis encountered today is asymptomatic, identified radiologically or histologically in lungs resected for carcinoma or removed at autopsy. symptomatic cases are characterised by an insidious onset of breathlessness, a dry cough and crackles over the lower lung fields. finger clubbing is a variable feature. lung function tests show a restrictive respiratory defect. radiology initially shows small irregular basal opacities that gradually coalesce to become linear, coarsen and eventually progress to a honeycomb pattern of small cysts. the principal differential diagnosis, both clinically and pathologically, is from idiopathic pulmonary fibrosis. this is aided by the slow progression of asbestosis, which often extends over years, as opposed to an average course of - years from presentation to death for the idiopathic condition. most cases of asbestosis are diagnosed solely on the occupational history and these clinicoradiological features. recourse to histology is unusual but biopsy (preferably as a wedge of lung) may be undertaken if the clinical features are atypical. histology also arises when the pathologist samples lung parenchyma remote from a resected carcinoma (the universal importance of which cannot be overemphasised). asbestosis (established) over over the light microscopic counts include total fibres (coated and uncoated). the electron microscopic counts include only amphibole asbestos. results from different laboratories vary and these figures, derived from several sources, , , provide only a general guide. reliable results depend upon counts being made regularly and the normal range from that laboratory being ascertained. ratios of counts obtained by electron and light microscopy vary greatly but approximate to . is better in this respect. [ ] [ ] [ ] by transmission electron microscopy, values may range up to in controls, with asbestosis generally above and mesotheliomas found at any level down to , all these figures representing amphibole fibres/g dried lung (see table . . ). , , it should be noted that counts from different parts of the same lung may vary widely; , - caution should therefore be exercised in interpreting a count obtained on a single sample. there is also wide discrepancy between laboratories, even when analysing the same sample. results obtained in an individual case therefore have to be evaluated against a standard set of values unique to that laboratory. electron microscopy also provides valuable information on the type of fibre. chrysotile differs physically from the amphiboles in two respects: its fibres are both curved and hollow (figs . . and . . ). with an electron microscope equipped for microprobe analysis, the various forms of asbestos may also be distinguished from other fibres and from each other (box . . ), , an important point as the amphibole forms of asbestos are far more dangerous than chrysotile (table . . ). [ ] [ ] [ ] coroners require autopsy verification of the diagnosis in all suspected cases and this also necessitates hystology. when the lungs from a patient with asbestosis are seen at autopsy, pleural fibrosis is often found, and although this may also be attributable to asbestos exposure it is to be regarded as an independent process and not part of the asbestosis: it is dealt with separately on page . slicing the lung affected by asbestosis shows a fine subpleural fibrosis, especially of the lower lobes ( fig. . . ). in severe cases the fibrosis often extends upwards to involve the middle lobe and lingula, and sometimes the upper lobes also. microcystic change associated with the fibrosis develops in advanced cases and in severe disease there may be cysts over cm in diameter. however, these classic changes are seldom seen in developed countries today. following decades of dust suppression in asbestos factories, current patients have mild to moderate asbestosis and are dying of related cancer or of non-pulmonary disease. in some of these cases the asbestosis is only detectable microscopically. fixation of the lungs through the bronchi and the use of heard's barium sulphate impregnation technique facilitate demonstration of the fibrosis (see p. and fig. . . ). the mild degree of asbestosis currently encountered is of little functional significance but is often critical in determining whether an associated carcinoma of the lung should be attributed to asbestos exposure (see below). the histological diagnosis of asbestosis requires an appropriate pattern of interstitial fibrosis associated with the presence of asbestos bodies. both components must be present. the fibrosis is paucicellular, lacking any significant degree of inflammation and being collagenous rather than fibroblastic. it is generally considered that asbestosis begins about the respiratory bronchioles and alveolar ducts where most of the asbestos fibres impact. alveolar walls attached to these bronchioles show fine interstitial fibrosis. however, this early lesion has to be interpreted with caution because it is not specific to asbestos, being found with other inhaled mineral dusts , and even in many cigarette smokers who have not been so exposed. it more likely represents a non-specific reaction to a variety of inhaled particles. it may cause mild airflow obstruction but is not associated with the radiographic, clinical or restrictive changes of classic asbestosis. as the disease progresses, the focal changes join up so that the basal subpleural regions show widespread interstitial fibrosis and eventually complete destruction of the alveolar architecture. in severe cases there may be honeycombing and metaplastic changes in the alveolar and bronchiolar epithelium. apart from the presence of asbestos bodies the changes resemble those of non-specific interstitial pneumonia, or more rarely usual interstitial pneumonia. fibroblastic foci may be found but they are uncommon. there is often an increase in alveolar macrophages but the desquamative interstitial pneumonia that has been reported in association with asbestos , is not to be regarded as a variant of asbestosis ; concomitant smoking is a more likely cause. a variety of other non-specific inflammatory processes such as organising pneumonia have been reported in asbestos workers and if localised some have been suspected of representing malignancy until biopsied. several schemes have been proposed for grading the extent and severity of asbestosis. these are of value in epidemiological studies but should only be applied to cases meeting the histopathological criteria for a diagnosis of asbestosis. one such scheme is shown in box . . . , , in well-established asbestosis asbestos bodies are numerous and easy to find, aggregates of them sometimes forming clumps ( fig. . . ) . in earlier lesions a detailed search may be necessary, in which fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli b extension of fibrosis to involve alveolar ducts and/or two or more tiers of alveoli adjacent to the respiratory bronchiole, with sparing of at least some alveoli between adjacent bronchioles fibrotic thickening of the walls of all alveoli between at least two adjacent respiratory bronchioles honeycomb change a an average score is obtained for an individual case by adding the scores for each slide ( - ), then dividing by the number of slides examined b grade and, to a lesser extent, grade need to be distinguished from smoking-induced peribronchiolar fibrosis and mixed-dust pneumoconiosis. case the examination of unstained or perls-stained sections facilitates their identification. minimum criteria for the diagnosis of asbestosis require the identification of diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or other mass lesion and the presence of either two or more asbestos bodies in tissue with a section area of cm or a count of uncoated asbestos fibres that falls in the range recorded for asbestosis by the same laboratory. , there are marked variations in the concentration of asbestos fibres between samples from the same lung , and it is therefore recommended that at least three areas be sampled, the apices of the upper and lower lobes and the base of the lower lobe. the equivalent of mallory's alcoholic hyalin of the liver has been described in the lungs in asbestosis, , and subsequently in other [ ] [ ] [ ] [ ] it is seen as small eosinophilic cytoplasmic inclusions within hyperplastic type ii alveolar epithelial cells (fig. . . a ). electron microscopy shows that the inclusions consist of a tangle of tonofilaments ( fig. . . b ) and by immunocytochemistry a positive reaction is obtained with antibodies to cytokeratin, both these features being typical of mallory's hyalin in the liver. the inclusions also react for ubiquitin, the accumulation of which is indicative of cellular damage, in particular faulty proteinolysis. the differential diagnosis of asbestosis includes pulmonary fibrosis due to many other causes, any of which may of course affect an asbes-tos worker as much as members of the general population. the proportion of diffuse pulmonary fibrosis in asbestos workers that is not attributable to asbestos has been estimated to be as high as % and likely to rise as the risk of asbestosis diminishes with better industrial hygiene. the principal differential diagnosis of asbestosis is from idiopathic pulmonary fibrosis. both diseases affect the bases and periphery of the lungs predominantly. in the late stages, cystic change is more evident in idiopathic pulmonary fibrosis but this criterion is not totally reliable. nor is the presence of pleural fibrosis, although it is usually present in asbestosis and is seldom found in idiopathic pulmonary fibrosis. asbestosis seldom progresses or does so very slowly after exposure ceases , whereas idiopathic pulmonary fibrosis typically proves fatal within - years from onset. the fibrosis of asbestosis is generally paucicellular: inflammation is not a feature and the fibroblastic foci that characterise the usual interstitial pneumonia pattern of fibrosing alveolitis are seldom observed in asbestosis. very often the distinction from idiopathic pulmonary fibrosis has to be based on the amount of asbestos in the lung and, if asbestos bodies are not readily identifiable, this has to depend on fibre counts. errors are made both by overlooking substantial numbers of asbestos bodies completely and by ascribing undue importance to scanty bodies. if considering the possibility of minimal asbestosis (to justify attributing carcinoma of the lung to asbestos, for example) it should be remembered that a little peribronchiolar fibrosis is also characteristic of smokers' lungs, centriacinar emphysema and early mixeddust pneumoconiosis. [ ] [ ] [ ] as described above, at least two asbestos bodies/cm in the presence of interstitial fibrosis distant from any lung cancer or other mass lesion is required for a diagnosis of asbestosis. although the causes of asbestosis and idiopathic pulmonary fibrosis are very different, they resemble each other in several ways, suggesting that similar pathogenetic mechanisms may operate. , [ ] [ ] [ ] in both these diseases there is degeneration of the alveolar epithelium and capillary endothelium, with patchy loss of the former, and bronchoalveolar lavage shows an increase in macrophages that might perpetuate the damage by releasing lysosomal enzymes, nitric oxide and hydroxyl radicals. , [ ] [ ] [ ] both diseases are also characterised by an increased prevalence of circulating non-organ-specific autoantibodies. experimentally, asbestos exposure leads to the activation of a variety of fibrogenic cytokines at sites of lung injury. , [ ] [ ] [ ] [ ] [ ] [ ] inhaled asbestos activates a complement-dependent chemoattractant for macrophages and macrophage stimulation involves the secretion of fibroblast stimulating factors, [ ] [ ] [ ] asbestos being intermediate between haematite and silica in regard to macrophage-mediated fibrogenicity. the epithelial damage could be mediated directly by the needle-like asbestos fibres or indirectly through enhanced phagocyte generation of free radicals (which is much greater with amphibole asbestos than with either chrysotile or silica). , fibrogenic cytokines released by activated pulmonary phagocytes and regenerating alveolar epithelial cells in asbestosis include tumour necrosis factor-α and transforming growth factor-β, as in idiopathic pulmonary fibrosis. as a result of better industrial hygiene, asbestosis is less severe today than in earlier years when it followed much heavier exposure, with the consequence that death from respiratory failure and cor pulmonale is less common and sufferers are surviving longer. there is therefore now a greater risk of asbestos-related cancer eventually developing. asbestos exposure predisposes to two varieties of malignant neoplasm, carcinoma of the lung and mesothelioma of the pleura and peritoneum. the risk of malignancy increases with dose but the relative risk of carcinoma is much smaller than that of mesothelioma. for example, with heavy exposure, as in lagging, the risk of mesothelioma is increased -fold whereas it is increased only fivefold for lung cancer. hence, with light exposure there is a substantial risk of mesothelioma but only a small risk of lung cancer. asbestosis requires heavy exposure and in one group of patients with asbestosis, % died of pulmonary carcinoma, % of mesothelioma and % of other respiratory diseases. although there were many earlier reports, the link with carcinoma of the lung may be considered to have been firmly established by , that between crocidolite asbestos and mesothelioma by , and that between amosite asbestos and mesothelioma by . mesothelioma is considered on page . in regard to carcinoma of the lung, asbestos is not such a potent pulmonary carcinogen as cigarette smoke but together their effects are multiplicative rather than additive (table . . ). , however, the risk attributable to asbestos is the same regardless of smoking history, being increased fivefold in both smokers and non-smokers. there is usually a latent period in excess of years between first exposure to asbestos and the development of lung cancer and the risk increases the greater the cumulative exposure. the increased risk involves carcinomas of all the histological types encountered in the lung, although adenocarcinoma has been disproportionately overrepresented. , [ ] [ ] [ ] [ ] [ ] [ ] it is uncertain whether the increased risk of carcinoma is caused by the asbestos , [ ] [ ] [ ] [ ] [ ] [ ] or the asbestosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the latter view envisages the carcinoma arising in the foci of alveolar epithelial hyperplasia and dysplasia that commonly accompany any interstitial fibrosis (see carcinoma complicating idiopathic pulmonary fibrosis, p. ). however, most carcinomas complicating asbestosis arise in the bronchi rather than the alveolar tissue. on the other hand, more arise in the sites worst affected by asbestosis, the lower lobes and the periphery of the lung, than in the general population ( fig. . . ) . , , [ ] [ ] [ ] [ ] [ ] the majority view has been that asbestosis is a necessary precursor of the carcinoma but evidence to the contrary is finding increasing support (table . . ). in the uk, industrial compensation was formerly only awarded to an asbestos worker for carcinoma of the lung if there was also asbestosis or diffuse pleural fibrosis but new rules were introduced in . asbestosis remains a sufficient criterion but diffuse pleural thickening is not and asbestosis is no longer a necessary criterion: asbestos is deemed to have been responsible if the patient worked in asbestos textile manufacture, spraying, lagging or gas mask manufacture for at least years before or years after . the basis for these changes is the premise that heavy asbestos exposure is sufficient in itself to account for carcinoma of the lung. together, these factors have a multiplicative rather than additive effect mortality ratio non-smoking controls non-smoking asbestos workers cigarette-smoking controls cigarette-smoking asbestos workers asbestosis associated with carcinoma of the lung. the asbestosis has been highlighted by barium sulphate impregnation and is seen as a grey subpleural band to the right of the picture. although the carcinoma has arisen in the same lobe as the asbestosis it has not obviously arisen in an area affected by asbestosis. . asbestosis diagnosed clinically, radiologically or histologically or a minimum count of asbestos bodies per gram dry lung tissue (/g dry), or an uncoated asbestos fibre burden of million amphibole fibres more than µm in length/g dry, or million amphibole fibres more than µm in length/g dry or estimated cumulative exposure to asbestos of at least fibres/ ml-years or an occupational history of year of heavy exposure to asbestos (e.g. manufacture of asbestos products, asbestos spraying) or - years of moderate exposure (e.g. construction or ship-building) and . a minimum lag time of years lung fibre counts in the asbestosis range (see table . . ) provide valuable evidence of such exposure. compensation standards for asbestos-associated lung cancer in different countries are shown in box . . . , asbestos-induced airway disease although asbestosis causes a restrictive respiratory defect, airflow limitation is also seen in this disease. much of the airflow limitation is attributable to cigarette smoking but it is also seen in non-smoking asbestos workers and is worse in those with asbestosis. the pathological basis of this appears to be small-airways disease (see p. ). it is possibly a non-specific reaction to inhaled dust or cigarette smoke. because it is not established that this lesion progresses to interstitial alveolar fibrosis (asbestosis) the term 'asbestos airways disease' is suggested. fibrosis limited to the bronchioles is specifically excluded from the definition of asbestosis in the latest guidelines (although these retain grade for fibrosis limited to the bronchiolar walls). it should also be noted that, although emphysema is considered to be a destructive rather than fibrotic condition, a little focal the presence of asbestos-related bilateral pleural plaques or asbestos-related bilateral pleural thickening and occupational exposure and a lag time of at least years the presence of asbestosis or pleural plaques or diffuse pleural thickening or fibre-years of exposure only fibre-years of exposure are taken into account exposure, at least years' latency and asbestos-related pleural or parenchymal changes asbestosis is not required but smoking is taken into consideration attempts are made to quantify separately the attributability to asbestos, smoking and other factors (e.g. radon) fibrosis is generally evident in this common condition and does not necessarily indicate early asbestosis. aluminium has been implicated in the development of respiratory disease during the refining of its principal ore, bauxite, to yield various aluminium oxides (aluminas), in the preparation of the metal by smelting alumina, in the production of corundum abrasive and in the production of special aluminium powders used in explosives. bauxite is a mixture of various aluminium oxides, hydroxides and silicates, iron oxide and titanium dioxide. the oxides of aluminium are obtained by differential heating of the ore and the respiratory effects of this work appear to be no more than mild airway irritation. it is generally accepted that aluminium oxide is inert. aluminium is prepared by the electrolytic reduction of its oxide dissolved in sodium aluminium fluoride (cryolite), a process releasing a considerable amount of fluoride-rich effluent. exposed workers have complained of what is termed pot-room asthma. the pathology of this condition is not well described but the pathogenesis is thought to involve irritation rather than allergy. the abrasive corundum is formed from bauxite mixed with coke and iron heated in an electric arc furnace, a process in which workers may be exposed to the fumes of alumina and free silica. in the past some of these workers developed diffuse pulmonary fibrosis (shaver's disease) and, although this was initially attributed to the aluminium, it is now agreed that the free silica was the responsible agent. the exposure to free silica has been reduced and the disease is now regarded as historic. aluminium powder holds a paradoxical position in regard to lung disease. in certain industries it has caused very severe pulmonary fibrosis, yet in others it has proved harmless. indeed, at one time canadian miners breathed aluminium dust before work, in the belief that this would reduce the danger of silica in the mine dust and more recently silicosis has been treated by such means in france. it is questionable whether this practice is effective but it at least appears to cause no harm. the explanation for these contradictory observations probably lies in differing methods of manufacture of aluminium powder. aluminium metal appears to be an inert substance but this is only because it has a high affinity for oxygen and the surface layer of aluminium oxide so formed is firmly bound to the underlying metal, unlike ferric oxide which permits further rusting of iron. granular aluminium powders, produced in a ball mill or from a jet of molten aluminium, therefore acquire a protective coat of surface oxide and are inert. with stamped aluminium powders, however, surface oxidation is prevented by lubricants added to aid the separation of these flake-like particles. the usual lubricant (stearin) contains stearic acid and this polar compound combines with the underlying metal, which is thereby protected from both atmospheric oxidation and the action of body fluids when such dust is inhaled. in certain circumstances, however, non-polar lubricants in the form of mineral oils have been substituted for stearin. this happened in germany during the second world war when munition production was stepped up but stearin was difficult to obtain, , and in the uk in the s to make the powder darker for purely commercial reasons. in vitro, oil-coated stamped aluminium powder reacts with water to produce aluminium hydroxide, which affords the underlying metal no protection against further attack, so that aluminium hydroxide continues to be formed. this substance is a protein denaturant, once used in the tanning industry, and it is believed that this property underlies the very ex ceptional cases of severe pulmonary fibrosis that have occurred in connection with stamped aluminium powder produced with mineral oil rather than stearin. , the fibrosis has a very characteristic pattern, affecting the upper lobes and progressing rapidly, the interval from onset of symptoms to death being as short as years. there is marked shrinkage of the lungs with gross elevation of the diaphragm and buckling of the trachea (fig. . . ). the lungs are grey ( fig. . . ) and microscopically, numerous small black jagged particles are seen. these can be shown to contain aluminium with irwin's aluminon stain or by microprobe analysis. what appears to be a different pathological effect of aluminium dust on the lungs is the rare development of granulomatous disease resembling sarcoidosis and berylliosis. , this represents hypersensitivity to the metal, amenable to confirmation with a lymphocyte transformation test similar to that used to diagnosis berylliosis (see below). rare cases of desquamative interstitial pneumonia and pulmonary fibrosis have been reported in aluminium welders. , elements with atomic numbers from (lanthanum) to (lutetium) are known as the lanthanides or rare earth metals. they are used in many manufacturing processes, including the production of hightemperature ceramics and the grinding of optical lenses. carbon arc lamps used in reproduction photography emit appreciable quantities of oxidised lanthanides, particularly cerium oxide, and there are reports of pneumoconiosis in exposed individuals. the pathological changes reported have varied from granulomatous nodules to diffuse interstitial fibrosis indistinguishable from the idiopathic variety except for the presence of rare earth elements (usually cerium) detected by polarising light microscopy and electron microprobe analysis. hard metal is a tungsten alloy containing small amounts of cobalt, titanium, molybdenum and nickel. it is exceptionally tough and once formed can only be worked with diamond. it is used in the tips of drill bits, on abrasive wheels and discs, and in armaments. interstitial lung disease is liable to arise in its manufacture or in those using hard metal as an abrasive. experimental work suggests that cobalt is the dangerous constituent but this element is soluble and, unless industrial contact has been recent, analysis of lung tissue usually shows tungsten and titanium but no cobalt. the role of cobalt is also indicated by the development of similar interstitial lung disease in diamond polishers using high-speed polishing discs made with a diamond-cobalt surface that lacked tungsten carbide and the other constituents of hard metal. , hard-metal lung disease and cobalt lung take two forms, an industrial asthma and interstitial fibrosis. the latter has a diffuse lower zonal distribution and the appearances mimic idiopathic pulmonary fibrosis. however, an unusual feature is the presence of moderate, or perhaps only small numbers, of giant cells (fig. . . a, b) . , not only are there multinucleate alveolar macrophages but syncytial cell forms develop in the alveolar epithelium. electron microscopy confirms that these are multinucleate type ii pneumocytes (fig. . . c ). such epithelial changes are well known in measles pneumonia but the viral inclusion bodies that characterise this infection are not found in hard-metal pneumoconiosis. the changes are those initially described as a particular pattern of idiopathic interstitial pneumonia termed giant cell interstitial pneumonia or gip (see p. ). elemental analysis shows that many, but not all, cases of gip represent hardmetal disease. the exceptions seldom give a history of cobalt exposure and must be presumed to represent true idiopathic cases. conversely, epithelial giant cells are not always found in hard-metal pneumoconiosis and so their presence, although highly characteristic, is neither totally specific nor totally sensitive. beryllium is the lightest of metals. it has an atomic weight of and special properties that make it especially useful in many applications. it is more rigid than steel, has a high melting point and is an excellent conductor of heat and elecricity. unfortunately, the inhalation of beryllium dust or fume is exceedingly dangerous. , those who worked with beryllium compounds before precautionary measures were taken suffered a high morbidity and mortality. sometimes, the escape of dangerous fumes from the factories was on such a scale that people living in nearby houses, downwind from the places in which these materials were being worked, contracted and occasionally died from berylliosis ('neighbourhood cases'). alternatively, contamination of a beryllium worker's clothes might lead to berylliosis in a temperatures. the alloys of beryllium are also now widely used, especially those with copper, on which it confers elasticity and resistance to fatigue. alloy manufacture and the machining of beryllium alloys are therefore further activities that entail a risk of berylliosis, as is the recovery of the metal in the recycling of scrapped electronic and computer parts. seemingly innocuous occupations such as dental laboratory technician are not without risk of chronic berylliosis. there are good grounds for regarding chronic berylliosis as being an allergic condition. many of those affected react strongly to skin tests with dilute solutions of beryllium salts, although these must be undertaken with care: occasionally in a highly sensitised person even so small an exposure may evoke a systemic reaction. the skin reaction is of the delayed type, occurs in only % of exposed individuals, is not associated with a clear-cut dose-response curve and represents a granulomatous response. further evidence for the disease having an allergic basis derives from bronchoalveolar lavage, which demonstrates an excess of t-helper lymphocytes that proliferate in vitro on exposure to beryllium salts. a positive transformation test given by these lymphocytes is a more reliable indicator of disease than in vitro blood lymphocyte transformation testing, which is safe but not wholly reliable and indicates only sensitization, rather than berylliosis. susceptibility to berylliosis varies widely from person to person and it is notable that chronic pulmonary disease is strongly associated with the hla antigen dpβ and the glu gene. , the importance of genetic factors is supported by a report of the disease in identical twins. chronic berylliosis is thought to be initiated by the metal binding to tissue proteins and acting as a hapten to initiate a delayed hypersensitivity response characterised by a proliferation of t-helper lymphocytes. these sensitised cells in turn secrete a variety of cytokines (e.g. interleukin- , tumour necrosis factor-α and interferon-γ) that recruit and activate macrophages, which mature into epithelioid cells. the resultant epithelioid cell granulomas destroy the lung tissue and lead to pulmonary fibrosis. if beryllium enters the subcutaneous tissues through a cut or abrasion, as often happened in the earlier days of fluorescent lamp manufacture, a sarcoid-like granuloma soon appears at the site; in time, the overlying epidermis may break down to form an ulcer. even more serious are the lesions produced by the inhalation of beryllium. chronic pulmonary berylliosis takes the form of a widespread granulomatous pneumonia with a histological picture identical to that of sarcoidosis (fig. . . a ). both berylliosis and sarcoidosis affect the upper lobes more than the lower (fig. . . b ) and in both diseases the granulomas are preferentially distributed along lymphatics and may involve adjacent blood vessels. in neither condition is there widespread necrosis but in both diseases the granulomas occasionally display a little central necrosis or hyalinisation. as in sarcoidosis, the hilar lymph nodes may be involved but, unlike sarcoidosis, not in isolation. over a period of many years, the sarcoid-like granulomas gradually undergo progressive fibrosis, with consequent impairment of pulmonary function. in the later stages, when the disease has become chronic, dispersal of beryllium from its site of initial absorption may lead to generalisation of the disease and to the appearance of similar granulomas elsewhere, particularly in the liver, kidneys, spleen and skin, but this is unusual. relative. beryllium compounds may also cause contact dermatitis and conjunctivitis. beryllium is also classified as a probable pulmonary carcinogen, but this is controversial. two forms of berylliosis are recognised, acute and chronic. acute berylliosis was first reported in germany in and is now largely of historical interest, being only encountered as a result of rare accidental or unexpected exposure. it follows the inhalation of a soluble beryllium salt and represents chemical injury, the pathology being that of diffuse alveolar damage (see p. ). further consideration will be confined to chronic berylliosis, which is allergic in nature. chronic berylliosis was first reported in in the fluorescent lamp industry. beryllium has now been replaced in this application but it has since proved to be of great value in the nuclear, electronic, computer and aerospace industries and the production of refractory materials and crucibles that are to be subjected to particularly high and there is a lifelong risk of disease. progression often entails alternating exacerbations and remissions, long after exposure has ceased. in keeping with the view that berylliosis is a hypersensitivity reaction, very little beryllium is necessary to cause the disease. particulate beryllium is so scanty in the affected tissues and the atomic number of beryllium so low that electron microprobe analysis is generally unsuitable for its detection. furthermore conventional detectors are protected by a beryllium window. however, the substitution of a polymeric window has enabled beryllium to be detected by electron microprobe analysis, presumably in a patient with fairly heavy exposure. ion or laser microprobe mass spectroscopy can also detect very small amounts of beryllium in tissue sections but these techniques are not widely available. the differential diagnosis of chronic berylliosis is from sarcoidosis, to which it is identical morphologically. [ ] [ ] [ ] however, as noted above, it is unusual for berylliosis to cause significant hilar lymphadenopathy in the absence of pulmonary disease, which is a common feature of sarcoidosis. extrathoracic granulomas, erythema nodosum and uveitis, which are all common in sarcoidosis, are unusual in berylliosis. however, one group found that % of patients initially diagnosed as having sarcoidosis actually had chronic berylliosis. similar findings have been reported by others. , any patient thought to have sarcoidosis who has worked with or near metals should be offered a beryllium lymphocyte transformation test. a list of laboratories performing this test can be found at www.dimensional. com/~mhj/medical_testing.html. although polyvinyl is not a mineral and the reaction of the lungs to its presence is therefore not a true pneumoconiosis, it is generally so termed and is dealt with here for convenience. workers are exposed to polyvinyl chloride dust in the milling and bagging of this plastic and micronodular opacities may be detected in their lungs radiologically. however, the material is non-fibrogenic and histology merely shows a foreign-body reaction to the dust particles. the radiological opacities may abate when exposure ceases. nevertheless, one polyvinyl chloride worker developed systemic sclerosis, which is a recognised complication of silicosis (see p. ). polyvinyl chloride is produced from vinyl chloride monomer, which has a causal association with angiosarcoma of the liver and probably other forms of cancer, including carcinoma of the lung (see p. ). in the late s a characteristic lung disease was identified in workers at several factories producing plush material by spraying nylon flock on to an adhesive backing material. [ ] [ ] [ ] [ ] the flock fibres are too large to be inspired but may be mixed with smaller nylon shards of respirable size. the workers complained of cough and breathlessness and were found to have a restrictive ventilatory defect with interstitial markings on radiography. their symptoms improved on removal from the workplace but relapsed on return to work. pathologically, there was lymphocytic bronchiolitis and peribronchiolitis with widespread lymphoid hyperplasia represented by lymphoid aggregates. chronic berylliosis is characterised by the gradual onset of cough, shortness of breath, chest pain, night sweats and fatigue. these symptoms may develop within a few weeks of exposure or many years later. once the worker is exposed, the beryllium is retained in the tissues granulomas were not identified. the histological appearances suggest a severe immunological reaction and raise possibilities such as rheumatoid disease and sjögren's syndrome but consideration of the clinical and serological setting and the occupation should permit recognition of the cause. the industrial production of popcorn and other foodstuffs appears to carry a risk of obstructive airway disease. [ ] [ ] [ ] [ ] biopsy of affected workers has shown peribronchiolar fibrosis and granulomas and air sampling has identified many volatile organic compounds, of which the flavouring agent diacetyl ( , -butanedione) is suspected of being responsible for the bronchiolitis. it is difficult to continue paint spraying (air brushing, aerographics) without adequate respiratory protection but in the early s several small aerographic factories operated in the neighbourhood of alicante, southeastern spain without any concern for the workers' health. the workers were required to paint patterns on textiles using a hand-held spray gun. the atmospheric pollution was intense but complaints of respiratory difficulties were met with reassurances and the workers urged to continue. this they did because of the otherwise poor economy, often returning to work when disabling breathlessness had settled down. a change of paint (to acramin f) may have contributed because the worst-affected workers were employed at two plants that had made this switch. their illness has been described as the 'ardystil syndrome' after the name of one of these factories. some workers were left with permanent respiratory disability. one required a lung transplant and others died. [ ] [ ] [ ] [ ] transbronchial biopsy showed organising pneumonia, which in the fatal cases had progressed to irreversible interstitial fibrosis. a similar outbreak of respiratory disease was subsequently reported in algerian textile factories where acromin f was applied by the same technique. , acromin f is marketed as a paste and used as such without ill-effect. its use in heavy spray form appears to be responsible for the 'ardystil syndrome' . workers in engineering workshops may be exposed to the prolonged inhalation of fine sprays or mists of the longer-chain hydrocarbons that constitute many mineral oils. this may result in exogenous lipid pneumonia, which is described on page , or extrinsic allergic alveolitis. [ ] [ ] [ ] the vapour of shorter-chain hydrocarbons such as paraffin oil (kerosene: c - ) and petrol (gasoline: c - ) and gaseous hydrocarbons such as propane may act as acute asphyxiants or central nervous system depressants but have negligible pulmonary toxicity. however, if they are ingested or aspirated in their liquid form they are acutely toxic to the lungs, producing a chemical pneumonitis with the features of diffuse alveolar damage. ingestion may be accidental or deliberate (see fig. . , p. ) whereas aspiration is generally inadvertent, occurring in siphoning accidents, such as those experienced by fairground operatives who 'breath or eat fire' ('fire-eater's lung'). , animal experiments involving the intratracheal injection of kerosene resulted in acute pulmonary exudates, which cleared except for residual bronchiolitis. welder's pneumoconiosis, first recognised in , essentially represents the fairly harmless deposition of iron in the lungs (siderosis -see p. ). however, welders may suffer various ill-effects from the inhalation of substances other than iron (table . . ). some of these are para-occupational risks, that is, encountered by welders because they work near another process and are inadvertently exposed: thus, shipyard welders may be exposed to asbestos, and those in foundries to silica. welders may therefore develop a mixed-dust pneumoconiosis (see p. ), rather than just siderosis. however, one analytical investigation identified excess amounts of iron alone in association with pulmonary fibrosis; the silicon content did not differ from that in controls. more directly, welders may be exposed to asbestos insulation that they themselves use, while welders of special steel alloys run the risk of metal-induced asthma, metal fume fever, polymer fume fever and the consequences of toxic metal fume inhalation, all of which are described separately in this chapter, as is lung disease in aluminium welders. chronic bronchitis has been attributed to the inhalation of low concentrations of irritants such as ozone and nitrogen dioxide by welders but this risk is unproven and the subject of much controversy. welders may also inhale carcinogenic hexavalent chromium compounds in the course of their work and therefore develop lung cancer. the term 'welder's lung' is often applied indiscriminately to any of these diseases and, as it has no specific meaning, is best avoided. dust, fume and gas are some of the terms used to describe different physical forms of respirable agents. they are defined in table . . on the finely divided fume of several metals is highly toxic to the lungs and capable of producing severe acute and chronic damage to both the conductive airways and the alveoli, resulting in acute tracheobronchitis and bronchiolitis, diffuse alveolar damage, obliterative bronchiolitis and pulmonary fibrosis. important metal fumes in this respect include aluminium, which is released together with silica fume in bauxite smelting (see shaver's disease, above), cadmium from welding or cutting special steels, chromium from cutting its alloys or in the manufacture of chromates, cobalt released in the production and use of its alloys (see hard-metal disease, above), mercury released in various industries and in the home, nickel carbonyl released during the purification of metallic nickel or the manufacture of nickel alloys and beryllium (see above). many irritant gases cause severe acute and chronic damage to both the conductive airways and alveoli. the changes are non-specific and similar to those wrought by toxic metal fumes (see above) and viruses amongst other agents. they consist of acute tracheobronchitis and bronchiolitis, obliterative bronchiolitis, diffuse alveolar damage and pulmonary fibrosis. the gases liable to produce such damage include oxides of nitrogen, sulphur dioxide, ozone, phosgene, chlorine, ammonia and various constituents of smoke, notably acrolein. some of these are also touched upon in chapter . because they are of general as well as occupational importance, although there is no rigid difference between general and occupational pollution. ozone, sulphur dioxide and nitrogen dioxide are oxidising gases that may be found together as industrial atmospheric pollutants. each is capable of producing diffuse alveolar damage by means of its oxidising properties and the release of free active radicals. in addition, they cause damage to distal airways, particularly terminal and respiratory bronchioles, with resulting bronchiolitis. oxides of nitrogen may be encountered with fatal consequences by farmhands seeking to free a blockage in a silo when they encounter pockets of this gas that have accumulated on top of the fermenting silage: the term 'silo-filler's disease' is generally applied to the initial haemorrhagic oedema or the obliterative bronchiolitis that develops in those who survive the initial chemical injury. [ ] [ ] [ ] [ ] asphyxia due to the farmhand encountering pockets of carbon dioxide is a further hazard within agricultural silos. other farmhands have suffered from the inhalation of toxic gases or bacteria when handling liquid manure. [ ] [ ] [ ] [ ] welding, which is considered below, may also involve exposure to toxic gases such as oxides of nitrogen. ozone, the principal oxidant gas of photochemical smog, produces pulmonary changes at relatively low levels and may be encountered at higher concentrations in various industries. potentially dangerous levels of ozone are produced from atmospheric oxygen by ultraviolet radiation given off in welding while ozone is used in industry to sterilise water, bleach paper, flour and oils, and mask the odour of organic effluents. the damage wrought by ozone is predominantly centriacinar in distribution, affecting terminal and respiratory bronchiolar epithelium and proximal alveolar epithelium. [ ] [ ] [ ] there is loss of cilia and necrosis of centriacinar alveolar type i epithelial cells. the changes are dose-dependent and, in one study, the youngest animals were most sensitive. in long-term experiments, hyperplastic bronchiolar clara and ciliated cells extended peripherally to line alveolar ducts. the role of granulocytes is stressed in some experimental studies and it is notable that neutrophil migration is prominent when the human lungs are damaged by ozone. aldehydes such as acetaldehyde, formaldehyde and acrylic aldehyde (acrolein) are widely used in the plastics and chemical industries. the first is a liquid and the others are water-soluble gases. pathologists are of course familiar with formaldehyde solution from its use as a disinfectant and histological fixative. all these aldehydes are intensely irritant and their acute effects generally prevent prolonged exposure to high concentrations. chronic effects include skin sensitivity and asthma, and in rats nasal carcinoma. however, the doses to which these experimental animals were exposed far exceed any that are likely to be encountered by humans, in whom there is no convincing evidence of aldehyde-induced cancer. ammonia gas is extensively used in industry as a raw material, notably in the manufacture of nitrogenous products such as fertilisers and plastics. it is highly soluble and its acute irritative effects are mainly felt in the eyes, nose and throat, but high levels affect the major airways, possibly leading to them being blocked by exudates. survival usually brings full recovery but bronchiectasis and obliterative bronchiolitis have been described. chlorine gas is widely used in the chemical industry. it is transported and stored under pressure in liquid form. heavy exposure through its accidental release or use as a war gas has proved fatal through its acute toxicity causing exudative airway occlusion and pulmonary oedema. survivors usually recover completely but, as with nitrogen dioxide and ammonia, there is a risk of obliterative bronchiolitis. phosgene (carbonyl chloride, cocl ) is a poisonous, colourless gas that was responsible for thousands of deaths during world war i, when it was used in chemical warfare. it is used industrially in the preparation of some organic chemical compounds and is formed, perhaps inadvertently, by the combustion of methylene chloride in products such as paint strippers. phosgene causes injury to terminal bronchioles and alveoli, with resulting oedema and hyaline membrane formation. the mechanism of cell damage is uncertain but it may depend on inactivation of intracellular enzymes by the gas. longterm problems are rare but chronic bronchitis and emphysema have been described in survivors. mustard gas (bichloroethyl sulphide, c h cl s) is a further agent that has been used in chemical warfare. it is primarily a skin vesicant but when inhaled it results in widespread epithelial destruction and pulmonary oedema. survivors may be left with irritant-induced asthma (reactive airways dysfunction), chronic bronchitis, tracheobronchomalacia, bronchiectasis and bronchiolitis obliterans. [ ] [ ] [ ] thionyl chloride is used in the manufacture of lithium batteries where it is liable to result in the release of sulphur dioxide and hydrochloric acid fumes. workers in such factories have developed lung injury varying from mild, reversible interstitial disease to severe obliterative bronchiolitis. hydrogen sulphide is the principal chemical hazard of natural gas production. high levels of the gas also buid up in sheds housing large numbers of pigs, the source here being the pig manure. once inhaled the gas is rapidly absorbed into the blood stream. the effects are therefore widespread but include the usual respiratory effects of irritant gases, varying from sneezing to pulmonary oedema and acute respiratory distress, depending upon the exposure. in alberta cases were identified over a -year period. the overall mortality was %; % of victims were dead on arrival at hospital. most required admission to hospital but the survivors experienced no long-term adverse effects. a the danger of asphyxia from the inhalation of gases devoid of oxygen is fairly widespread in industry. it generally arises from the use of inert gases, which, being non-toxic, give a false sense of security. pockets of these gases tend to form in confined spaces. anoxic death from the accumulation of methane is well known in mines and has also occurred in slurry pits and sewers. anoxic asphyxia in diving (and anaesthesia) has resulted from the incorrect connection of gas cylinders or failure to notice that a mixed gas contains insufficient oxygen. deaths have occurred in welding when argon or carbon dioxide has been used to shield the weld and prevent oxidation of the metals at the high temperatures employed. deaths have also resulted from inadvertent entry to discharged oil tanks filled with nitrogen to reduce the risk of explosions, or from the formation of pockets of nitrogen gas applied in liquid form to freeze the contents of damaged pipes so that they can be repaired without the necessity to drain down. the respiration of a gas devoid of oxygen causes loss of consciousness within seconds because it not only fails to provide oxygen but removes that present in the pulmonary arterial blood. the changes at autopsy are those common to cellular hypoxia. they include cerebral and serosal petechiae and pulmonary congestion and haemorrhage but these features are not specific and are not always present. the cause of death can generally only be surmised from the circumstances surrounding the death. occupational asthma is the commonest cause of work-related respiratory disease in many western countries (table . . ). [ ] [ ] [ ] the reported incidence ranges from per million workers in south africa to per million workers in finland. , it occurs in many industries (table . . ) and occupational factors can be identified as contributing to asthma in about % of adult cases. over aetio- in the uk a third are organic, a third chemical, % metallic and the rest miscellaneous. the commonest, in descending order, are isocyanates, flour and grain, laboratory animals, glutaraldehyde, solder or colophony and hardening agents. atopy appears to predispose to occupational asthma when the allergen is of high molecular weight but not when it is of low molecular weight. for example, atopic individuals are particularly prone to develop asthma if employed in the manufacture of biological detergents, whereas atopy does not increase the risk of asthma from sensitisation to toluene di-isocyanate, which is a serious health problem in the manufacture of polyurethane. similarly, platinum salts are such potent sensitising agents that nearly all those exposed to them develop asthma. asthma-provoking metals other than platinum include chromium, cobalt, nickel and vanadium, all of which are used in steel alloys, and possibly aluminium (see pot-room asthma, p. ). other asthma-inducing factors encountered in industry include grain and flour dust, certain wood dusts, soldering fluxes containing colophony (pine resin), epoxy resin hardeners such as phthallic anhydride, isocyanate-containing foams and paints, formaldehyde and the excreta of laboratory animals. contaminated humidifiers may cause occupational asthma as well as humidifier fever and extrinsic allergic alveolitis. pathologically, occupational asthma is identical to nonoccupational asthma (see p. ). byssinosis is a further form of occupational asthma, one encountered in the cotton industry. the sensitising agent is a component of the cotton bract, which is the part of the cotton harvest other than the cotton fibre. bract consists of dried leaf, other plant debris and soil particles and contains a variety of fungal and bacterial residues, including lipopolysaccharide endotoxin, but the exact nature of the sensitising agent remains unknown. the endotoxin is unlikely to be responsible for byssinosis but may be the cause of so-called mill fever, a self-limiting illness characterised by malaise, fever and leukocytosis that is experienced by many people on first visiting a cotton mill. dust levels and the risk of byssinosis are particularly high in the carding rooms where the raw cotton is teased out before it is spun. affected workers are worse when they return to work after the weekend break, a feature attributed to antibody levels having built up during this brief respite from the cotton dust. there is no link with atopy and the fluctuating antibodies are precipitins of the immunoglobulin g class. complement activation by both arms of the complement cascade has been reported. , when the lancashire economy was largely cotton-based, necropsies on workers suffering from byssinosis generally showed gross emphysema, and this came to be accepted as evidence of byssinosis. however, it is now realised that in this heavily industrialised part of the uk, emphysema is as common in the general population as in cotton workers and it can no longer be considered a component of byssinosis. other findings in byssinosis are more commensurate with asthma, namely an increase in bronchial muscle and mucous cells. no granulomas or other evidence of extrinsic allergic alveolitis are found. fever may be the predominant feature in a variety of occupational illnesses and the unifying term 'inhalation fever' has been proposed. however, the individual occupations are of interest and these conditions will therefore be considered separately. mill fever has been mentioned above under byssinosis. humidifier fever is an acute illness characterised by malaise, fever, myalgia, cough, tightness in the chest and breathlessness, all of which are worse on monday mornings if the humidifier responsible is at work rather than home. the chest complaints, and their aggravation on return to work after the weekend, are features shared with byssinosis (see above) but the general complaints fit better with extrinsic allergic alveolitis (see p. ). humidifier fever develops in circum-stances that also lead to the development of a form of extrinsic allergic alveolitis, and not surprisingly the same name has been extended to this latter condition, with inevitable confusion. both diseases are caused by microbiological contamination of humidifiers or air conditioners so that a fine spray of microorganisms is emitted into the office, factory or home. investigations have generally shown the baffle plates of the air conditioner to be covered with a slime of bacteria, fungi or protozoa (mainly amoeba and ciliates), and extracts of this have been used to identify precipitins in the patient's sera, as in extrinsic allergic alveolitis. however, unlike extrinsic allergic alveolitis, humidifier fever resolves within a day and leaves no permanent injury. for this reason there is seldom the opportunity to study the tissue changes, and partly for this reason it remains unclear whether the disease is mediated by immune complexes, as in extrinsic allergic alveolitis, or by endotoxins derived from the contaminants. a febrile illness occurring in precipitin-negative farm-workers after heavy exposure to fungi in their silos was attributed to inhaled fungal toxins and named pulmonary mycotoxicosis. it is also known as precipitin test-negative farmer's lung and organic dust toxic syndrome. the condition is generally self-limiting and is seldom biopsied but desquamative interstitial pneumonia and diffuse alveolar damage have been reported. , metal fume fever this is a self-limiting acute illness characterised by fever, sweating, myalgia, chest pain, headache and nausea, that comes on monday mornings when occupational exposure is experienced after a weekend's respite, as with bysinnosis and humidifier fever; during the week tolerance develops. , the disease involves the release of cytokines such as tumour necrosis factor and is presumed to have an allergic basis. the metals involved are chiefly zinc, copper and magnesium, and, to a lesser extent, aluminium, antimony, iron, manganese and nickel. occupations at risk include any that generate such metal fumes, but particularly welding. it is most commonly associated with welding zinc-coated surfaces. if the symptoms persist, alternative diagnoses, such as acute cadmium poisoning and other specific toxic metal fume diseases, should be suspected: these are not self-limiting and may cause severe bronchiolitis or diffuse alveolar damage (see above). this illness resembles metal fume fever except that it occurs without regard to previous exposure: no tolerance develops and there is therefore no particular susceptibility on mondays. the polymers concerned are quite inert, except when heated to produce fume: polytetrafluorethylene (ptfe, teflon, fluon, halon) is a notable example. as with other self-limiting diseases, little is known of the tissue changes. environmental irradiation chiefly affects the skin but in some parts of the world rocks near the surface release significant amounts of radon gas. this carcinogen is liable to accumulate in buildings and be inhaled, so subjecting the occupants to an increased risk of lung cancer. the installation of underfloor ventilation is therefore advocated in such areas. this subject is explored more fully on page . the body is vulnerable to both increases and decreases in pressure and it is the lungs that often bear the brunt of the damage. increased pressure may result in blast injury or crushing of the chest while decreased pressure may result in the lungs literally bursting or dissolved gases being released within the blood (caisson disease), or the vascular alterations that underlie mountain sickness developing. some of these pressure changes entail a risk of pneumothorax and it is essential that this is properly investigated postmortem by the chest being opened under a water seal. loud music has been incriminated as a specific form of air pressure change causing pneumothorax and metereologists have shown that 'spontaneous' pneumothoraces tend to occur in clusters associated with natural drops in atmospheric pressure. , explosions may cause injury by the body being violently thrown against a less moveable object, by objects being thrown against the body or by the blast wave hitting the body. these mechanisms often act together but sometimes there is only blast injury, to which the lungs are particularly vulnerable. for a time it was considered that the damage was direct, the blast wave travelling down the airways to injure the lungs. however, at the start of the second world war, experiments conducted in the uk showed that the lungs were injured indirectly, the blast wave being transmitted to them through the chest wall: pulmonary blast injury is worst on the side of the body towards the explosion, and can be reduced by protective clothing. underwater explosions are particularly dangerous because water is incompressible. there may be severe internal injury but no external evidence of damage other than a trickle of blood from the mouth or nose. this is because the injury is rate-dependent. quite small thoracic deform-ation may produce severe pulmonary damage if peak compression is attained very quickly, typically in less than ms. conversely, severe chest wall distortion may produce only minor pulmonary contusion if this time is extended beyond ms. at necropsy, the lungs are contused, with blood evident in the airways and parenchyma. depending on the force of the blast, the haemorrhage may be pinpoint, patchy or confluent. it tends to follow the lines of the ribs and may be accompanied by pleuropulmonary lacerations having the same distribution. in this case there will also be haemothorax, pneumothorax and possibly air embolism. patchy pulmonary haemorrhages cuff the blood vessels. , in patients who survive for a few days, the lungs resemble the liver macroscopically and histologically show chronic interstitial inflammation and fibrosis as well as haemorrhage. other injuries are often present and fat embolism, aspiration pneumonia, fluid overload and infection may all be added to the effects of the blast wave. 'chest squeeze' is another form of barotrauma caused by high pressure but here the body is compressed rather than subject to a sudden wave of pressure as in blast injury. it is experienced by divers who descend very deeply, thereby subjecting their bodies to such high pressure that their chest walls are literally crushed, so that their ribs break and their lungs are severely compressed. more common mishaps experienced by divers include drowning and decompression sickness, both of which are dealt with below, and neurological syndromes such as nitrogen narcosis, which will not be considered further. 'burst lung' is the most acute form of decompression sickness. it is experienced by divers and submariners making rapid ascents from depth and by aviators who ascend too rapidly in unpressurised aeroplanes, experience failure of a plane's pressure system or have to eject at high altitudes. injury to the lung is caused by trapped alveolar gas expanding so rapidly that it exceeds total lung capacity before it can escape through the trachea. the lungs literally burst: the alveolar walls rupture and blood mixes directly with alveolar air. the victim experiences chest pain and there may be blood-stained froth at the mouth or frank haemoptysis. air may enter the alveolar walls to cause interstitial emphysema or air embolism. asthmatics may be at particular risk because of regional air-trapping. . diving mammals such as porpoises and whales are protected from such dangers of peripheral air-trapping by cartilage extending far out into the finest conductive airways so that these passages never close, even at the end of full expiration (fig. . . ) . , patients requiring positive-pressure artificial respiration are also at risk of burst lung, but the complications of the resultant interstitial emphysema differ from those experienced by divers. in divers, the chest wall is buttressed by the surrounding water and air in the interstitium is liable to track towards the hilum of the lungs and enter pulmonary veins, with resultant cerebral and coronary air embolism, either of which may prove fatal. iatrogenic burst lung, on the other hand, takes place in patients whose chest wall is not so buttressed, and then outward rupture of the interstitial air is more likely, resulting in pneumothorax. extension of the interstitial emphysema to the mediastinum, neck and chest wall is also more likely in such patients, resulting in surgical emphysema at these sites. however, there are exceptional cases marked by both cerebral embolism and extensive air tracking. the same circumstances that lead to burst lung may also cause decompression sickness, which is also known as caisson disease. in this condition there is a sudden release of nitrogen gas that has gone into solution in the lipids of adipose tissue and of myelinated nervous tissue at the higher pressure: the released nitrogen gains access to the blood stream in which it forms bubbles. doppler ultrasound techniques show that this is quite customary when divers ascend from depth, but the lungs generally provide an effective filter so that there are no untoward systemic effects, although there may be sudden chest pain on deep inspiration ('the chokes'). gradual decompression permits the nitrogen to diffuse across the alveolar membranes and be exhaled. if, however, substantial amounts of nitrogen are released from solution, sufficient pulmonary arteries may be blocked to cause pulmonary hypertension, with resultant opening of arteriovenous communications or a patent foraman ovale, so permitting the gas to enter the systemic circulation. this is often followed by limb pains ('the bends') and perhaps cerebral symptoms ('the staggers'). fatal cases are characterised by gas bubbles within blood vessels throughout the body and froth in the heart chambers. delayed effects include ischaemic necrosis of bones and other tissues. deep-diving mammals are protected by the same mechanism that prevents them suffering from burst lung. they exhale before diving and during the dive the chest is compressed to the extent that virtually all the gas in the lungs passes into the cartilage-buttressed nonrespiratory airways (see fig. . . ) , resulting in very little to be absorbed by the blood. the pulmonary collapse also serves to reduce buoyancy. the distribution of the little gas that is absorbed is minimised by bradycardia. many viscera experience anaerobic respiration but hypoxia is minimised in the heart and musculature by high levels of haemoglobin and myoglobin. the brain is further protected by the supplying arteries drawing on oxygen stored in an unusual spongelike cervical organ known as the rete mirabilis. mountain sickness is due to reduced atmospheric pressure brought about more slowly than that responsible for decompression sickness . , it may be acute or chronic. acute mountain sickness is likely to be experienced by anyone who ascends above - m without a period of acclimatisation at intermediate levels. symptoms are as liable to occur in people born at high altitude who return after a few weeks spent at sea level as in those who go to the mountains for the first time: acclimatisation is obviously short-lived and is therefore necessary whenever an ascent is to be made. the ill-effects are commonly precipitated by exercise. in the susceptible, acute mountain sickness commonly appears within days of ascent. the basis of acute mountain sickness is tissue hypoxia. it results in deteriorating intellectual and psychological function, headache, nausea, vomiting, and more rarely pulmonary and cerebral oedema. high-altitude pulmonary oedema is characterised by increasing dyspnoea, cyanosis and a dry cough, and later the production of copious, frothy sputum, which sometimes becomes blood-stained. the pulmonary artery pressure is markedly raised but wedge pressures are normal, indicating that the left side of the heart is unaffected and that pulmonary venous constriction is unlikely to be an important contributory factor. the pulmonary oedema fluid has a high protein content and the condition has been characterised as a non-cardiogenic high-permeability oedema associated with excessive pulmonary hypertension. , hypoxia is a well-known cause of pulmonary arteriolar constriction but in acute mountain sickness the vascular response appears to be exaggerated for the pulmonary artery pressure is considerably higher than is usual for the altitude. an association with certain hla complexes (hla-dr and hla-dq ) suggests that this has a genetic basis. although arteriolar constriction only tends to protect the pulmonary capillaries, it could explain the oedema if the process was patchy -as is the resultant oedema -for patchy arteriolar constriction would subject the rest of the lung to abnormally high pressures and lead to capillary stress failure in these areas (see pp. and ). , measurements of capillary pressure suggest that this is indeed the case. furthermore, vasodilators such as calcium channel blocking agents and inhaled nitric oxide gas , , have been used with success to counter acute mountain sickness, supporting the idea that hypoxic vasoconstriction plays a central role. autopsy shows the lungs to be heavy and firm. the cut surface weeps oedema fluid, which is often blood-stained, but a striking feature is the patchy distribution of the changes. areas of haemorrhagic oedema alternate with others that contain clear oedema fluid and others that are normal apart from overinflation. pulmonary arterial thrombi are commonly found. microscopy confirms the presence of haemorrhagic oedema and may show neutrophils and hyaline membranes in the alveoli. the alveolar capillaries are congested and may contain thrombi. there may also be an increase in mast cells and rarely pulmonary infarction. the right ventricle is commonly dilated whereas the left ventricle is normal. highlanders generally show right ventricular hypertrophy and increased muscle in their pulmonary arteriesm, changes that are not apparent in lowlanders. , chronic mountain sickness prolonged residence at high altitude leads to hypoxic pulmonary hypertension (see p. ), an increase in red cell mass and cor pulmonale. livestock taken from lowland plains to high-altitude pastures suffer similarly but the natural stock of the himalayas and ethiopian highlands are apparently immune. so too are other species long established at high altitude such as the llama and yak. these species are said to have adapted to their climate, that is, the forces of natural selection have bred out the pulmonary vasoconstrictive response to hypoxia. cattle of european origin and humans acclimatise to high altitude by processes such as increasing their red cell mass but generally they are not adapted like native species and suffer hypoxic pulmonary hypertension at altitudes in excess of m. certain himalayan highlanders may be an exception to this in that their small pulmonary arteries are reported not to show the muscularisation that characterises hypoxic pulmonary hypertension. in cattle of european origin, the dependent oedema of right-sided cardiac failure caused by hypoxic pulmonary hypertension affects the breast (brisket) particularly and in the rocky mountains of north america such cattle are said to have 'brisket disease' . a human counterpart of this has been described in children of chinese ancestry who have been taken to reside in tibet and who have developed a fatal form of subacute infantile mountain sickness. a small minority of permanent residents in the andes develop the changes of chronic mountain sickness to a marked degree and are said to suffer from monge's disease. the basis of this is alveolar hypoventilation, which leads to a progressive fall in systemic arterial oxygen saturation and elevation of haemoglobin concentration to an unusually severe degree. the latter averages about g/dl, which exceeds even the g/dl found in healthy high-altitude residents. patients with monge's disease are so deeply cyanosed that their lips are virtually black. their pulmonary artery resistance is also markedly raised. the cause of the alveolar hypoventilation is uncertain but the only cases of monge's disease that have come to necropsy had conditions such as kyphoscoliosis that predispose to alveolar hypoxia. drowning is defined as suffocation by submersion, and usually occurs in water. it is the commonest cause of accidental death among divers but % of drowning accidents do not involve deep descents. falling into quite shallow water is a particularly common cause of drowning in young children. in adults, men outnumber women by to . more die in fresh water than the sea, not because it is more hazardous to the lungs than sea water, but because unguarded inland waters and swimming pools are visited more frequently. alcohol consumption contributes to many deaths by drowning. drowning is not simply a matter of being unable to keep one's head above water. this may be merely a secondary event. for example, the entry dive may result in underwater head injury, or the exertion of swimming may precipitate a heart attack. furthermore, the struggling swimmer going down for the third time ('drowning not waving') is the exception: most drowning is characterised by the swimmer failing to surface or quietly dropping beneath the surface without anyone noticing. swimming underwater can be extremely hazardous if it is preceded by hyperventilation, a danger that needs to be more widely appreciated. hyperventilation results in undue loss of carbon dioxide so that instead of hypercapnia forcing the swimmer to surface to breathe, progress under water may be continued until hypoxia causes sudden loss of consciousness. panic contributes to many swimming accidents and is often precipitated by the inadvertent aspiration of just a little water. most people are naturally buoyant, but only slightly so. with the lungs fully expanded the average adult has a positive buoyancy of about . kg, which is sufficient to keep the head out of the water if the rest of the body is submerged. if an arm (weight about kg) is raised to wave for help, the head will go down. if the swimmer shouts, exhalation reduces buoyancy to neutral at normal end-expiration and to negative at residual volume. buoyancy cannot be regained when the head is submerged and unless able to swim to the surface, the person will continue to sink. autopsy generally shows that the lungs are full of water, but some victims die of 'dry drowning' due to laryngospasm. events may also be modified by the temperature of the water. sudden immersion in cold water may result in tachycardia, hypertension and hyperventilation, making it difficult for the victim to keep the airways free of water. it may also result in sudden death due to ventricular fibrillation. even a good swimmer loses consciousness within an hour of immersion in very cold water. drowning is then inevitable unless a correctly fitted life jacket is worn, in which case there is a danger of death from hypothermia. however, as in open heart surgery, cold prolongs the interval before there is irreversible brain damage. if the person is rescued, water in the lungs is quickly absorbed, even if it is saline, and therefore hyperosmolar:aspirated sea water is quickly equilibrated by pure water joining it from the blood but the alveolar epithelial barrier remains impermeable to protein and once osmotic equilibrium is reached, all is quickly reabsorbed. [ ] [ ] [ ] fresh water is absorbed even more quickly. it is unnecessary to tip the patient to hasten this process. any water recovered in this way comes from the stomach and time that should be devoted to mouth-to-mouth breathing and cardiac massage is lost. these resuscitative efforts may need to be prolonged as fresh water in particular inactivates alveolar surfactant, leading to alveolar collapse which persists until the surfactant is replenished. very few victims who are resuscitated on site fail to survive, and very few who cannot be resuscitated on site recover later. interchange of fluid between the blood and air spaces may cause major fluctuations in plasma volume with consequent changes in ionic concentrations and haemolysis. hypervolaemia may cause circulatory problems but hyperkalaemia consequent upon the haemolysis is not thought to be as important as was formerly believed: ventricular fibrillation following submersion is more likely to be a complication of hypothermia than of electrolyte imbalance. circulatory collapse may ensue shortly after rescue. this is due to loss of the circulatory support provided by the pressure the water exerts on the body, which results in a considerable increase in cardiac output while the body is immersed. on leaving the water the loss of this support results in a tendency to venous pooling. although this is countered by baroreceptor responses, these are reduced by prolonged immersion in cold water. circulatory collapse is believed to be the cause of death in many persons who perish within minutes of rescue. to counter this effect, patients should be lifted out of the water in the prone position. it can be seen that, in fatal cases, the pathologist is faced with several possibilities. thus, death may have been due to: • natural causes before the body entered the water • unnatural causes before entry, the body merely being disposed of in the water • natural causes in the water • injuries received in the water from impact with rocks, a boat or a ship's propeller, or in tropical waters from predators such as a crocodile or a shark (any of which may also be incurred after death, as may disfigurement by fish and rats) • 'dry drowning' • true drowning • hypothermia • circulatory failure after rescue. true drowning is indicated by froth in the airways and heavy waterfilled lungs. both fresh and salt water contain numerous microscopic algae known as diatoms and those representative of the water in which the drowning occurred are found in the lungs. unless death occurred before submersion, diatoms are also found in other viscera because these tiny life forms easily enter the circulation. thus, the presence of diatoms in digests of organs such as the kidneys, liver, brain and bone marrow suggests that death was due to drowning. because they have a siliceous capsule, diatoms are resistant to putrefaction as well as digestion and can be identified in the body long after death. however, a positive test is not always accepted as proof of drowning and a negative test does not exclude drowning. the various physical forms in which respirable environmental agents may be encountered are defined in table . . . some effects of inhalant lung injury are recognised as distinct disease entities and are dealt with elsewhere: for example, the pneumoconioses on page , extrinsic allergic alveolitis on page , chronic bronchitis on page and lung cancer on page . other respirable agents, such as lead fume and carbon monoxide gas, exert their harmful effects elsewhere in the body and will not be considered further. this section is concerned with toxic substances that may be inhaled by the general public. those that are more likely to be encountered in the workplace or in war zones are considered on page . the lungs have a rather stereotyped pattern of response to inhaled toxins, displaying degenerative changes and inflammation of varying degree, the former sometimes amounting to necrosis. in general, the site of maximal absorption or injury is related to solubility (for gases and vapours) and particle size (for aerosols such as dusts, fog, fumes, mists, smog and smoke): the less water-soluble and the smaller the particle size, the further down the respiratory tract the agent will penetrate ( fig. [ ] [ ] [ ] thus, ammonia produces intense congestion of the upper respiratory passages and laryngeal oedema whereas phosgene has little effect on these sites but causes pulmonary oedema. air pollution [ ] [ ] [ ] [ ] [ ] the toxic (as opposed to allergenic) air pollutants thought to pose the greatest threat to the lungs comprise smoke particles, sulphur dioxide, oxides of nitrogen, various aldehydes and ozone. smoke and sulphur dioxide derive particularly from the combustion of fossil fuels in domestic fires and power stations, nitrogen dioxide is an important car exhaust and domestic gas appliance pollutant and ozone is the principal photochemical product of smog. aldehydes such as formaldehyde and acrylic aldehyde (acrolein) also contribute to general air pollution because they are released in the combustion of diesel oil and petrol. collectively, these pollutants have been incriminated in the exacerbation (rather than causation) of asthma. they also predispose to respiratory infection and result in airway inflammation and hypersecretion. , their effect on children is of particular concern because development of the lungs is known to continue well into childhood and damage to the lungs before their growth is complete is likely to be irreparable. at the other extreme of life episodes of severe air pollution are known to hasten the deaths of many patients with chronic airway disease. particularly high concentrations of the agents responsible for air pollution may be encountered in industry and their effects are therefore also considered in chapter . , on occupational diseases of the lung. many of the polycyclic hydrocarbons found in polluted air are carcinogenic (see p. ) and it is therefore not surprising that urban air pollution has been found to be associated with excess mortality from lung cancer. domestic air pollution is rife in many of the poorer parts of the world due to the burning of biomass (wood, dried cow dung, bagasse, straw) in unventilated living rooms for heating and cooking. the women are particularly at risk of developing chronic bronchitis while their children have an increased incidence of acute respiratory infections. , , a volcanic ash (tephra) irritates the eyes, skin and respiratory tract and in some eruptions may contain much free silica (e.g. montserrat in and mount st helens, washington state, usa in ) or be associated with the release of radon gas (e.g. the azores in ). the destruction of the world trade center in caused massive air pollution of new york city that had lasting respiratory effects on survivors, rescue workers and local residents. [ ] [ ] [ ] at the time of the disaster there was much smoke from combustion of aeroplane fuel and flammable materials in the building while the collapse of the twin towers released dust from cement and dry-wall partitions that was highly alkaline. [ ] [ ] [ ] this caused considerable irritation of the eyes and the conductive airways. a year later many victims were still suffering from bronchial hyperreactivity and poor ventilatory function, in a so-called reactive airways dysfunction syndrome , and there was continuing spirometric decline years later. the respirable portion of the dust formed only a small fraction of the whole but given the level of exposure its future effects cannot be discounted, particularly as it contained substances such as asbestos. unusual effects attributed to the disaster include acute eosinophilic pneumonia and granulomatous pneumonitis. , allergenic air pollutants are dealt with in detail in the sections on asthma (see p. ) and extrinsic allergic alveolitis (see p. ). allergenic air pollution is generally occupational or domestic but periodic widespread air pollution was responsible for the epidemics of asthma seen in barcelona in the s, which were eventually traced to ships discharging cargoes of soya flour (see p. ). smoking-related diseases figure large throughout this book and in this section they are merely summarised collectively. of the greatest importance, both in the number of patients they affect and in their clinical effects on the individual, are the various forms of chronic obstructive lung disease and lung cancer, but there are many other respiratory diseases associated with smoking, and a few that are less common in smokers (box . . ). not surprisingly, these diseases are often encountered in combination and sometimes one may obscure another. for example, a cigarette smoker may have emphysema in the upper lobes and idiopathic pulmonary fibrosis in the lower lobes. , alternatively, langerhans cell histiocytosis and desquamative interstitial pneumonia may affect the same parts of the lungs, in which case the focal lesions of the former may be masked by the latter condition. the term 'smoking-related interstitial lung disease' has been introduced to cover a spectrum of interstitial diseases related to smoking , , as well as being used in a more restricted sense to describe a combination of air space enlargement and interstitial fibrosis predominantly affecting the lower lobes. , , a quite advanced interstitial fibrosis has been reported in smokers with no clinical evidence of interstitial lung disease. b early changes detectable in smokers include chronic bronchiolitis, fibrosis of the bronchiolar wall and mild peribronchiolar interstitial fibrosis. , even earlier changes are detectable at the molecular level: as many as smoking-responsive genes that are significantly up-regulated or down-regulated have been identified in normal cigarette smokers. there is marked individual variation, which may explain why many lifelong heavy smokers experience no respiratory problems. histological evidence that a patient smokes is provided by an increase in the number of alveolar macrophages and a characteristic brown discoloration of cytoplasm due to the phagocytosis of tar and other particulate matter derived from tobacco smoke (fig. . . ) . cigarette smokers are at greater risk of lung disease than cigar and pipe smokers, probably because they inhale more deeply. they do this because cigarette smoke is more acid than cigar and pipe smoke and its nicotine content is therefore absorbed more easily through the lungs than the buccal mucosa. smokers obviously put their own health at greatest risk but the lesser hazards of passive smoking are now well recognized (see p. ). passive smoking involves both the smoke exhaled by others and that coming from smouldering tobacco between puffs, the latter being known as sidestream smoke. the harmful effects of maternal smoking on the unborn child also come in this category. they include increased airway responsiveness and reduced lung function during the neonatal period and an increased risk of sudden infant death syndrome. reduced numbers of alveolar attachments to the bronchioles have been demonstrated in such infants. smoking is also associated with disease of other organs (e.g. carcinoma of the oesophagus and bladder) but these are outwith the remit of this text. tobacco smoking by waterpipe (shisha, hubble-bubble) is enjoying a rise in popularity, both in its heartland, the middle east, and western countries, and wherever it is practised it is widely perceived as being less dangerous than smoking cigarettes. this is probably a mis conception. what evidence there is suggests that waterpipe tobacco smoking is just as harmful as cigarette smoking, if not more so. the lungs may be injured in burned patients in many ways (box . . ) , but an important consideration when a body is recovered from a fire is whether death was due to the fire or took place beforehand, the latter raising the possibility of foul play. a vital reaction to the skin burns and the presence of soot in the lower airways provide evidence that death occurred in the fire but an absence of soot from the airways may be due to death occurring rapidly, from asphyxia or poisoning by gases released in the conflagration. soot is cleared rapidly and if the patient survives a few days an absence of soot from the airways is to be expected. lung injury may result directly from heat and smoke inhalation or indirectly from the release of mediators associated with blast injury or shock. although air temperature in a fire may reach very high temperatures thermal injury seldom extends beyond the carina but more extensive injury from heat alone was seen in men exposed to steam escaping from a fractured boiler pipe. those dying immediately showed coagulative necrosis of the respiratory mucosa down to the level of the alveolar ducts and alveolar congestion and oedema, while those surviving a little longer exhibited diffuse alveolar damage. the diffuse alveolar damage probably represented a manifestation of shock from their extensive cutaneous scalding whereas the mucosal necrosis is directly attributable to heat. diffuse alveolar damage is extrinsic allergic alveolitis sarcoidosis blast injury asphyxia poisoning by combustion products (e.g. carbon monoxide, cyanide) direct thermal injury (largely limited to the trachea) irritant smoke, fume and gas (e.g. oxides of nitrogen, ammonia, acrolein, sulphur dioxide) hypovolaemic shock secondary to skin loss septicaemic shock from: infected skin burns infected central lines secondary viral and bacterial pneumonia fluid overload tracheostomy complications, including tracheobronchitis, pneumonia and barotrauma oxygen toxicity absorption of toxic topical disinfectants thromboembolism uraemia usually part of systemic multiorgan failure in these patients, and is the leading cause of death in burns. the ubiquity of plastics today means that smoke contains numerous irritants, including isocyanates, aldehydes and fluorinated organic chemicals. irritant smoke products have two principal effects. firstly, they cause an immediate painful stimulation of the eyes and respiratory tract which at low concentrations may prevent escape and at high concentrations may cause laryngeal spasm and death. secondly, they cause bronchopulmonary injury some hours after exposure. burned patients dying within - days often show tracheobronchial necrosis and diffuse alveolar damage with prominent hyaline membranes. , , secondary herpesvirus infection is often present. , the respiratory changes caused by heat and smoke are non-specific and careful consideration of the many causes of lung injury in burned patients listed in box . . and of the clinical circumstances and management is generally required. often it will be concluded that the cause of the lung injury is multifactorial. long-term consequences of smoke inhalation include bronchiectasis and obliterative bronchiolitis. methyl isocyanate, the chemical released at bhopal the bhopal catastrophe of was caused by the accidental release of tons of methyl isocyanate gas (ch -n=c=o) from a pesticide plant. over people were exposed, of whom died, mostly within hours of exposure, and were seriously injured. the victims complained of intense ocular and respiratory irritation. some survivors were left with persistent respiratory impairment, which was thought to be due to obliterative bronchiolitis. , methyl isocyanate is an extremely potent respiratory irritant, destroying the epithelium throughout the conducting airways, with comparatively less parenchymal injury. in survivors, epithelial regeneration, often involving squamous metaplasia, quickly commences, but not before endobronchial granulation tissue projections have developed, resulting in obliterative bronchiolitis. tear gases are chemical irritants delivered as an aerosol for the purpose of riot control. they react with mucocutaneous sensory nerve receptors causing intense irritation of the eyes, mucous membranes and skin. the respiratory effects are mainly concentrated on the upper tract so that there is violent sneezing, severe rhinorrhoea and cough but there may also be tracheobronchitis and rarely pulmonary oedema. patients with pre-existent asthma or chronic obstructive pulmonary disease are most severely affected while others may be left with reactive airways dysfunction. toxins reaching the lungs via the blood stream may be drugs, food contaminants, metabolites produced elsewhere in the body, or chemicals ingested intentionally or accidentally, either in the home or the workplace. the lungs are selectively damaged by certain blood-borne toxins for a variety of reasons. for example, the herbicide paraquat is preferentially taken up by the lungs because of its molecular homology with certain endogenous substances. as detailed below, the type i alveolar epithelial cells are the cells that bear the brunt of the damage in paraquat poisoning. on the other hand, the alveolar capillary endothelium has its own selective uptake mechanisms (see metabolic functions of the pulmonary endothelium, p. ) which may be responsible for it being selectively damaged by other chemicals. the bronchiolar clara cells are selectively injured by some ingested chemicals because they are equipped to deal with inhaled xenobiotics, but occasionally this activity results in metabolites that are extremely toxic. an example of this from veterinary medicine is provided by the furan-derivative -ipomeanol, which is found in mouldy sweet potatoes and results in acute pulmonary oedema in cattle fed such a diet. when this chemical is injected into mice, the bronchioles are denuded of clara cells whereas the intervening ciliated cells are completely unaffected. the selective damage to the bronchiolar clara cells appears to stem from the oxidative efficiency of their p- cytochromes, which is much higher than those of the liver. chemicals having a similarly selective effect on bronchiolar clara cells include -methylfuran, carbon tetrachloride, naphthalene and , -dichloroethylene, the last of which is a volatile compound that is widely used in the plastics industry. procarcinogens may be activated in the airways by similar mechanisms. paraquat is a dipyridylium compound that is widely used in agriculture as a herbicide. it kills all green plants but is inactivated on contact with the soil. it is applied as a spray and if the manufacturer's instructions are followed there is no danger to health. most fatal cases of paraquat poisoning, both accidental and suicidal, have been due to ingestion of the % aqueous solution gramoxone. the less concentrated granular form weedol is unlikely to be ingested accidentally but may be taken suicidally. paraquat is not absorbed by the intact skin but repeated or prolonged application damages the epidermis so that absorption into the blood stream with consequent systemic effects is possible, but rare. although paraquat has toxic effects on the liver, kidneys and myocardium, these are transient and attention has centred on the pulmonary changes, which are usually fatal. following suicidal ingestion of large amounts of paraquat, death from multiorgan failure and pulmonary haemorrhage occurs within a few days, whereas most victims of accidental paraquat poisoning die from progressive pulmonary fibrosis between and days after ingestion. in those who survive longer, a honeycomb pattern of pulmonary fibrosis may be apparent. paraquat is a powerful oxidant and owes its toxicity to the production of active oxygen radicals. the lungs are particularly susceptible because paraquat is concentrated there by an active uptake mechanism in the alveolar epithelium. the inadvertent uptake of paraquat probably stems from a similarity between the molecular arrangement of its quaternary nitrogen atoms and the amine groups of endogenous oligoamines such as putrescine, spermidine and spermine, which are concerned in alveolar epithelial cell division and differentiation (fig. . . ) . this results in paraquat levels being - times higher in the lung than in the plasma. once taken up by the lung, paraquat is not metabolised but participates in redox cycling so that superoxide radicals are constantly produced. epithelial injury is proportional to the concentration of paraquat, while it is lessened by hypoxia and antioxidants such as superoxide dismutase, and potentiated by increased concentrations of oxygen. [ ] [ ] [ ] [ ] the high concentration of oxygen in the alveoli is a further reason why the lungs are particularly vulnerable to paraquat. knowledge of the toxic effects of paraquat comes from observations on autopsy series , , and from experimental studies that have enabled the sequence of pulmonary changes to be observed. [ ] [ ] [ ] [ ] in accordance with paraquat being taken up by the alveolar epithelium, electron microscopy shows that these cells suffer more profound damage than the endothelium. type i epithelial cells swell and undergo necrosis (fig. . . ), whilst type ii cells, although remaining capable of proliferation, show ultrastructural evidence of damage with derangement of cell organelles. , histological changes in the lungs follow the pattern of diffuse alveolar damage, with a characteristic feature of the early exudative phase being intense vascular congestion and alveolar haemorrhage. , , hyaline membranes are most clearly seen by about days (fig. . . ) and epithelial proliferation and fibrosis are conspicuous by about days. the pattern of pulmonary fibrosis in paraquat poisoning has been disputed. some authors have stressed its interstitial position, whereas others have clearly demonstrated that it is intra-alveolar. , , [ ] [ ] [ ] [ ] however, as described on page , it generally assumes an obliterative pattern of intra-alveolar fibrosis in which the lumina of several adjacent alveoli are totally effaced, rendering them completely airless (see fig. . , p. ). a new multisystem disease appeared abruptly in the environs of madrid in . [ ] [ ] [ ] over people were affected and about in died. the disease was initially thought to be mycoplasma pneumonia but was soon found to be associated with the use of adulterated oil sold illicitly by door-to-door salesmen. although it was sold for culinary purposes the oil had been produced for industrial use in steel manufacture. it consisted of rapeseed and olive oil mixed with liquified animal fat, aniline and other organic chemicals. it has not been possible to identify the exact chemical responsible for the disease or to reproduce the changes in other species but the later induction of similar pathological changes by another substance contaminated with an aniline derivative is possibly relevant (see l-tryptophan-induced eosinophilia-myalgia syndrome, p. ). some clinical and pathological features of the disease suggest that immune mechanisms may also be involved. the initial clinical features included fever, respiratory distress, cough, haemoptysis, skin eruptions and marked eosinophilia. radiographs suggested pulmonary oedema and sometimes showed pleural effusion. about % of patients died at this stage but most recovered quickly. however, within a few weeks many were readmitted to hospital with nausea, vomiting, diarrhoea and abdominal pain. about a quarter then proceeded to develop weakness, myalgia, weight loss, scleroderma-like skin signs and pulmonary hypertension. , many of these patients died after a long, wasting illness or are permanently disabled with neurological and hepatic disorders. in the early phase the lungs showed the most severe changes, which consisted of a combination of diffuse alveolar damage, eosinophilic infiltrates and arterial luminal narrowing by endothelial swelling and vacuolation, intimal foam cell infiltration and a non-necrotising vasculitis. , , there was also capillary thrombosis, which later extended into arteries and veins, culminating in fibrosing obliteration of these blood vessels. in some patients dying of haemoptysis, dilated thin-walled blood vessels were identified in the mucosa of major blood-filled airways. late features in the lungs included plexogenic arteriopathy (see p. ), possibly secondary to changes in the liver. similar inflammatory and vascular changes were seen in many other tissues. notable extrapulmonary features included fasciitis, vasculitis, neuronal degeneration, perineuritis, hepatic injury and tissue eosinophilia. sauropus androgynus is a vegetable that is widely cultivated for the table in many south-eastern asian countries. it is apparently harmless when cooked but recently there has been a vogue in taiwan for consuming large amounts of its unprocessed juice, blended with that of guavas or pineapple, because of its supposed efficacy as a slimming aid and in blood pressure control. coincident with this fad there has been an upsurge in patients with symptoms of obstructive lung disease. within a -month period more than such patients were seen at one hospital. [ ] [ ] [ ] they had four features in common: recent consumption of uncooked s. androgynus juice, fixed ventilatory obstruction, radiological evidence of bilateral bronchiectasis and an absence of any previous chronic respiratory disease. four patients agreed to undergo open-lung biopsy. this showed chronic bronchiolitis or obliterative bronchiolitis of constrictive pattern. the lymphocytes were mainly t cells but immunofluorescent and electron microscopy showed no evidence of an immune process. four patients underwent single-lung transplantation. the excised lungs showed sclerotic obliteration of bronchial arteries in the walls of bronchi - mm in diameter with segmental necrosis of bronchi - mm in diameter. the changes were considered to fit best with segmental ischaemic necrosis of bronchi at the watershed zone of the bronchial and pulmonary vasculature. further patients have required lung transplantation but public education of the dangers of this herbal medicine now appears to have been successful. alcohol and nicotine outstrip all other recreational drugs in popularity and their effects are of course well known. those of tobacco smoking are summarised above and dealt with in detail in the chapters on obstructive lung disease (chapter ) and carcinoma of the lung (chapter . ). less well known is the lung disease that results from smoking blackfat tobacco, a practice popular with guyanese indians. blackfat is the trade name of a type of tobacco that is flavoured with mineral oil, some of which vaporises and is inhaled when the tobacco is smoked, to cause exogenous lipid pneumonia (see p. [ch ] ). in recent years the smoking of two other substances, marijuana and cocaine, has gained in popularity. it would not be surprising if the long-term effects of smoking these substances were similar to those of cigarette smoking but as yet it is too early to judge. however, the short-term effects are similar to those of tobacco smoking and this bodes badly for their ultimate effects. marijuana consists of the dried leaves of the cannabis plant, also known as hemp, as opposed to hashish, which is the plant's resin, and a further extract known as 'weed oil' . all these substances are smoked because they contain cannabis alkaloids which have psychoactive effects. however, this habit also exposes the lungs to many of the same respiratory irritants that are found in tobacco smoke. initial exposure to marijuana smoke often results in coughing while habitual smokers produce black sputum. bronchial biopsy shows inflammation and squamous metaplasia and bronchoalveolar lavage demonstrates increased numbers of cells, which are predominantly macrophages but also include neutrophils. [ ] [ ] [ ] [ ] [ ] these changes are virtually identical to the short-term effects of tobacco smoke and are therefore likely to be similarly followed by the development of chronic obstructive lung disease and lung cancer. indeed, the dangers of smoking marijuana are probably greater than those of smoking tobacco as compared with tobacco smoking it is associated with a fivefold greater increase in blood carboxyhaemoglobin and a threefold increase in the amount of tar inhaled. it is estimated that three cannabis cigarettes result in the same degree of bronchial damage as tobacco cigarettes. there is also evidence that the effects of smoking marijuana and tobacco are additive. not surprisingly therefore, epidemiological studies report a doserelated impairment of large-airway function in marijuana smokers. there are also several reports attributing pneumothorax to marijuana smoking ( fig. . . ) . , the pneumothorax may be spontaneous or develop during the deep, sustained inspiratory effort involved in smoking marijuana (or cocaine), which may be enhanced by a partner applying positive ventilatory pressure by mouth-to-mouth contact. thoracoscopy in such cases has shown predominantly apical, irregular bullous emphysema, while lung biopsy has demonstrated widespread alveolar filling by heavily pigmented macrophages. , evidence is also beginning to accumulate that long-term cannabis use increases the risk of lung cancer. smoking cannabis in the form of weed oil is also reported to result in exogenous lipid pneumonia. cocaine cocaine hydrochloride is a fine white powder derived from the leaves of the plant erythroxolon coca by a complex chemical process. it is heat-labile and therefore cannot be smoked. users inject it intravenously or inhale it unheated through the nose, the latter practice being known as 'snorting' . however, a heat-stable free-base form that can be smoked is easily prepared from the hydrochloride with baking powder and a solvent such as ether. this process results in a crystalline deposit that is known as 'rock' because of its appearance or 'crack' because of the crackling sound it emits when heated. when smoked, the cocaine is readily absorbed and an intense surge of euphoria is experienced within seconds. the intravenous route takes twice as long and 'snorting' several minutes. the hard addict therefore prefers to smoke 'crack' . a variety of pulmonary complications of smoking free-base cocaine has been reported. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] acute effects include cough, shortness of breath, chest pain and haemoptysis. asthma may be aggravated, black sputum is produced, and pneumothorax and interstitial emphysema have resulted from valsalva manoeuvres undertaken in the belief that they promote even more rapid absorption. biopsy has shown pulmonary congestion and oedema, organising pneumonia, haemorrhage, haemosiderosis, diffuse alveolar damage and interstitial pneumonia or fibrosis. less common effects include eosinophilic pneumonia, extrapulmonary eosinophilic angiitis, medial thickening of pulmonary arteries and the barotrauma described above (see fig. . . ) . severe burning of the airways has also been seen due to 'crack' being smoked before all the ether used in its preparation has evaporated. 'snorting' unheated cocaine has its own complications: substances such as cellulose or talc with which the drug is 'cut' (mixed as a diluent) are liable to provoke a foreign-body giant cell reaction in the lungs (fig. . . ). however, particles of foreign material larger than those in the usual respirable range (allowing for the fibrous shape of substances such as cellulose) should suggest intravenous use (see 'filler embolism', below). heroin is usually injected, but it may be smoked, when, as with marijuana, it is liable to lead to a very pronounced macrophage response. intravenous heroin abuse sometimes causes the sudden onset of a potentially fatal high-permeability pulmonary oedema (fig. . . ). intravenous abuse of heroin and other drugs is also liable to cause 'filler embolism', which will now be considered. 'filler embolism' is the result of illicit drug usage in which compounds designed for oral use are injected intravenously to heighten their effects. oral preparations consist largely of fillers such as talc or starch and this insoluble particulate matter accumulates in the pulmonary capillaries. it provokes a foreign-body giant cell reaction, thrombosis and fibrosis and may cause pulmonary hypertension ( fig. this 'designer' drug, taken for its central stimulant activity (street names 'ice' or 'u- -e-uh', pronounced euphoria), is related to the appetite suppressor aminorex, discussed on page , and has similarly been associated with pulmonary hypertension. assessing whether a particular clinical manifestation represents an adverse drug reaction considers previous experience with the drug, alternative aetiological agents, the timing of events, drug levels, and the effect of withdrawing the drug and rechallenge with the drug. it is worth bearing in mind that: • one drug may cause several patterns of disease. • one pattern of disease may be produced by a variety of drugs. • a drug reaction may develop long after the drug has been withdrawn. • a drug reaction may develop suddenly even though the dose of the drug has not been altered. • drug effects may be augmented by factors such as age, previous radiotherapy and elevated oxygen levels. • drug reactions may be localised. • many drugs cross the placenta to affect the fetus. an alternative classification of adverse drug reactions, which is more appropriate to pathology practice and which will be followed here, is one based on the pattern of disease. some pathological patterns of drug-induced lung disease are shown in table central depression of respiration occurs as a side-effect of barbiturates, morphine and its derivatives, and even mild sedatives, and may be particularly troublesome in patients suffering from chronic obstructive lung disease. ventilation in such patients may be largely dependent on hypoxic respiratory drive and treatment with oxygen may therefore also have an adverse effect on respiration by lowering the degree of hypoxia and so diminishing the stimulation of the respiratory centre. peripheral impairment of the respiratory drive may be brought about by aminosides and other antibiotics, while corticosteroids may result in a myopathy affecting the respiratory muscles. other iatrogenic hazards affecting the peripheral nerves controlling respiration include nerve root disease complicating immunisation and surgical damage to the spinal and phrenic nerves. asthmatic patients are particularly susceptible to exacerbations of their disease by drugs (box . . ). this effect may occur either as a predictable pharmacological side-effect of the drug or as an idiosyncratic response. examples of the former include β-adrenergicic antagonists and cholinergic agents while examples of the latter include sensitivity to the colouring agent tartrazine, for which reason many manufacturers have eliminated tartrazine from their red, orange and yellow tablets. allergic bronchoconstriction also forms part of generalised anaphylactic reactions induced by vaccines and antisera and occurs as a localised response to penicillin, iodine-containing contrast media, iron dextran and other medicaments. bronchospasm may also be initiated by the non-specific irritant effect of inhaling nebulised drugs if they are prepared as a hypotonic solution, a side-effect that is prevented by using isotonic solutions. aspirin and other non-steroidal anti-inflammatory agents aspirin-induced asthma has been recognised for many years and more recently several of the newer anti-inflammatory drugs have been found to exacerbate asthma in certain sensitive individuals. the basis for this is uncertain but the likelihood of an individual antiinflammatory drug provoking an asthmatic response is related to its potency as an inhibitor of prostaglandin cyclooxygenase pathway, resulting in the production of leukotrienes. [ ] [ ] [ ] as well as asthma being exacerbated by drugs, the disease has been caused by occupational exposure in the pharmaceutical industry to certain drugs which can be inhaled during manufacture, notably penicillin, cephalosporin, methyldopa, cimetidine and piperazine. obliterative bronchiolitis of the constrictive type has been reported with penicillamine , and gold , but in many cases it is possibly the underlying condition rather than the drug that is res ponsible (see p. ). this is often rheumatoid disease, which is sometimes complicated by bronchiolitis obliterans whether the patient is under treatment or not. organising pneumonia extending into peripheral bronchioles (see p. ) may be seen with a variety of drugs but results in a restrictive rather than obstructive lung defect and is to be regarded as a cytotoxic effect of the drug acting primarily at the alveolar level (see below). raw sancropus androgyns taken as a slimming aid causes severe obliterative bronchiolitis (see p. ). the cytotoxic effects of drugs may be acute or chronic, leading to changes as varied as pulmonary oedema, diffuse alveolar damage, pulmonary haemorrhage and haemosiderosis, organising pneumonia, interstitial pneumonitis and interstitial fibrosis. , some of the most severe acute effects are seen with the chemotherapeutic agents used in malignant disease but they are also recorded with drugs that are not traditionally thought to be cytotoxic, e.g. desferrioxamine administered as a prolonged intravenous infusion in acute iron poisoning. pulmonary toxicity due to busulphan was first described in , and has been the subject of several subsequent studies. [ ] [ ] [ ] [ ] it remains the mainstay of treatment for chronic myeloid leukaemia. like other alkylating agents, it acts by cross-linking dna strands. clinical estimates of the incidence of pulmonary toxicity vary around % but subclinical damage is thought to be much more common. although not strictly dose-dependent, toxicity is rarely seen with a total cumulative dose of less than mg. synergy with radiation and other cytotoxic drugs occurs. similar effects have been reported for most cytotoxic agents, particularly bleomycin. pulmonary toxicity is seen less commonly with other alkylating agents, such as cyclophosphamide and melphalan. [ ] [ ] [ ] [ ] bleomycin is a cytotoxic antibiotic derived from streptomyces species. it is widely used in the treatment of neoplasms such as lymphomas and germ cell tumours, and is thought to produce its therapeutic and toxic effects by altering the normal balance between oxidants (active oxygen radicals) and antioxidant systems. bleomycin produces superoxide radicals when incubated with oxygen and iron in vitro. oxygen enhances its effects, a fact well known to anaesthetists who accordingly take care to limit concentrations of inspired oxygen to % in patients on bleomycin who are undergoing surgery. [ ] [ ] [ ] radiotherapy and cytotoxic agents such as bleomycin are also synergistic. bleomycin is preferentially concentrated in the lungs and pulmonary fibrosis can be produced in animals when it is administered intravenously, intraperitoneally or by intratracheal instillation. electron microscopy shows that the early changes consist of swelling and vesiculation of endothelial cells, interstitial oedema and type i epithelial cell necrosis. , the reported incidence of bleomycin toxicity varies from to % depending on the type of patient being treated and on dosage. in general, toxic effects increase with age and cumulative dose: above a total dose of about units they rise significantly. the acute morphological changes attributable to drugs include pulmonary oedema and diffuse alveolar damage. acute pulmonary oedema is seen in heroin addicts who die while injecting themselves intravenously but it is also seen in patients administered a variety of drugs therapeutically, for example hydrochlorothiazide, salicylate, opiates, vinorelbine,and desferrioxamine. the oedema is of the high permeability type (see p. ), rich in protein, and is occasionally haemorrhagic or accompanied by the hyaline membranes of diffuse alveolar damage. diffuse alveolar damage has alveolar epithelial necrosis as its basis (figs . . and . . ). however, the continuing action of many cytotoxic drugs affects the regeneration process so that atypical type ii epithelial cells develop, a characteristic feature that was first described with busulphan and subsequently with bleomycin. , these two drugs differ chemically but both act (by different mechanisms) on dna. the atypical cells have abundant deeply eosinophilic or amphophilic cytoplasm and large nuclei, which may be multiple but are usually single. the nuclei measure up to µm and are densely stained throughout or contain either large homogeneous deeply eosinophilic inclusions or clear vacuoles (fig. . . ) . electron microscopy distinguishes the inclusions from nucleoli and shows them to consist of tubular aggregates derived from the internal nuclear membrane. airway epithelium shows similar nuclear changes and often undergoes squamous metaplasia. the presence of such cells in sputum specimens submitted for cytology can lead to a misdiagnosis of malignancy. fibrosis may follow diffuse alveolar damage or develop insidiously, perhaps many years after drug therapy ceased (fig. . . ) . it may be both interstitial and intra-alveolar. the interstitial component is often accompanied by a non-specific chronic inflammatory infiltrate. the proportions of inflammation, which is potentially reversible, and fibrosis, which when collagenous is irreversible, obviously bear on the prognosis. however, most case reports antedate the recent classification of interstitial pneumonia described in chapter and it is uncertain how their pathological appearances would now be classified. the majority lack the classic features of usual interstitial pneumonia and fibrotic non-specific interstitial pneumonia. many show overlapping patterns of intersitital pneumonia and this alone should arouse suspicion that a drug may have been responsible. however, some cytotoxic drugs result in pulmonary changes by more than one mechanism: for example, methotrexate may produce hypersensitivity reactions with granuloma formation [ ] [ ] [ ] [ ] or pulmonary eosinophilia as well as diffuse alveolar damage. pulmonary toxicity is also occasionally seen in patients undergoing treatment with gold salts for rheumatoid disease: in addition to diffuse alveolar damage, there may be eosinophilia and dermatitis in these cases, again indicating possible hypersensitivity. nitrofurantoin is another example of a drug resulting in a variety of patterns of alveolar injury: diffuse alveolar damage, desquamative interstitial pneumonia, giant cell interstitial pneumonia, organising pneumonia and eosinophilic pneumonia have all been recorded in association with this drug. [ ] [ ] [ ] it should also be noted that in patients with neoplastic disease, clinical features suggestive of a pulmonary drug reaction may be due to factors other than drugs. in leukaemic patients, for example, these include direct infiltration of the lungs by leukaemic cells, opportunist infection and, if bone marrow transplantation has been undertaken, the effects of irradiation and possibly graft-versus-host disease. phospholipidosis is encountered with drugs such as the antidysrhythmic agent amiodarone, which block lysosomal enzymes involved in the breakdown of complex lipids. this leads to their accumulation throughout the body but the effect is most marked in tissues that take up the drug and contain cells rich in lysosomes. the lung fulfils both these requirements through its rich complement of alveolar macrophages. these cells accumulate the enzyme substrate (phospholipid) in their cytoplasm with the result that large foam cells fill the alveoli (fig. . . ). the appearances are those of endogenous lipid pneumonia, similar to that seen in obstructive pneumonitis. however, with amiodarone cytoplasmic vacuolation is also seen in epithelial and interstitial cells. the phospholipid inclusions contained within the vacuoles are particularly well seen in unstained frozen sections viewed by polarised light. identical changes to those induced by amiodarone were seen in the lungs of rats exposed to very high levels of the antidepressant drug iprindole and the anorectic drug chlorphentermine. these three compounds, iprindole, chlorphentermine and amiodarone, all belong to the amphiphilic group of drugs which block lysosomal phospholipase and sphingomyelinase. although their pharmacological actions are very different, a molecular homology is apparent (fig. . . ) . it is likely that all patients receiving substantial amounts of amiodarone develop phospholipidosis throughout the body, but this is generally well tolerated. only a minority experience respiratory impairment and in these there is also evidence of pulmonary inflammation and fibrosis, which is possibly mediated immunologically. these patients generally have a restrictive lung deficit, the onset of which may be acute or chronic. bronchoalveolar lavage shows foamy macrophages but these cells indicate exposure to the drug rather than drug toxicity; nor are they specific to amiodarone, being observed on occasion with other drugs. lymphocytes of suppressor type may also be detected on lavage. histologically, amiodarone toxicity is diagnosed on a combination of phospholipidosis and interstitial pneumonia and fibrosis. occasionally the hyaline membranes of diffuse alveolar damage are superimposed on the interstitial changes (see fig. . . ) . [ ] [ ] [ ] in some patients the fibrosis is intraalveolar rather than interstitial and the appearances are those of organising pneumonia. the process may be localised and mimic a neoplasm radiologically. , amiodarone toxicity is probably dose-dependent but there is considerable individual variation in the amount required, , which appears to be under genetic control. amiodarone toxicity is uncommon in patients taking daily doses of mg or less whereas the there are drugs that undoubtedly cause a usual interstitial pneumonia pattern, for example the chemotherapeutic agents and nitrofurantoin (fig. . . ), while others, for example the statins, are recorded as having induced a non-specific interstitial pneumonia pattern. a drug history is therefore imperative when assessing any patient with diffuse parenchymal lung disease. organising pneumonia similar to the cryptogenic condition described on page , and probably similarly reversible with steroids, has been encountered with a variety of drugs, including amiodarone, sulphasalazine and pencillamine. penicillamine has also been incriminated in the development of both diffuse alveolitis and bronchiolitis obliterans, but both these changes could well be due to the underlying rheumatoid disease for which the pencillamine is administered. in busulphan lung there may be an organising intraalveolar fibrinous exudate, which at its most extreme results in irreversible effacement of the alveolar architecture by sheets of loose connective tissue (see p. ). with continued experimental administration of the drug iprindole mentioned above, the phospholipidosis it produced gradually evolved into alveolar proteinosis (more properly called lipoproteinosis; see p. ), but this has not been reported as a drug effect in humans. alveolar proteinosis has however been recognised in a number of patients receiving chemotherapy for conditions such as leukaemia. the mechanism here is probably based on the cytotoxic action of the drug and the material filling the alveoli may represent the detritus of degenerate alveolar cells rather than excess pulmonary surfactant, as in the primary auto-immune form of alveolar proteinosis. eosinophilic pneumonia, the pathology of which is described on page , may be caused by several drugs, including nitrofurantoin, para-aminosalicylic acid, sulphasalazine, phenylbutazone, gold compounds, aspirin and penicillin (see box . , p. ). , it may also follow radiation to the chest. the tissue eosinophilia is generally accompanied by a rise in the number of eosinophils in the blood. the clinical picture varies from transient asymptomatic opacities on a chest radiograph to a life-threatening illness with severe respiratory distress and hypoxaemia, so-called acute eosinophilic pneumonia (see p. ). the reaction is often associated with a florid rash. withdrawal of the drug may be all that is required to effect resolution but corticosteroids are usually given as they produce a marked improvement. this syndrome of necrotising granulomatosis, vasculitis and eosinophilia in asthmatic patients, which is described more fully on page , has been reported when leukotriene receptor antagonists have been used to treat asthma. however, it is likely that the syndrome has been merely unmasked by the antileukotriene permitting a reduction in corticosteroid dose rather than representing a direct effect of the antileukotriene. , mesalazine has also been implicated in inducing a vasculitis during treatment for inflammatory bowel disease. the eosinophilia-myalgia syndrome was identified in the usa in and quickly identified as being due to the ingestion of ltryptophan from one particular japanese supplier. withdrawal of this substance led to the virtual elimination of the disease, but not before patients had been affected, in fatally. [ ] [ ] [ ] [ ] [ ] cases were subsequently described in europe where there were further fatalities. l-tryptophan is an essential amino acid that is freely available to the public: its purchase does not require a medical prescription. it has been promoted as a dietary supplement and as an agent against insomnia and premenstrual tension. women in the reproductive years preponderated in the patients affected by the resultant eosinophiliamyalgia. the clinicopathological features of the syndrome are similar to those of the spanish toxic oil syndrome (see p. ) and differ more in degree than type. the discovery of an aniline-derived contaminant in the tryptophan-induced condition is a further link connecting these two syndromes. an immune basis is suggested by the identification of t lymphocytes activated against fibroblasts in the eosinophilia-myalgia syndrome. the illness is a multisystem disorder and besides blood eosinophilia and myalgia there may be arthralgia, fever, rash and involvement of the lungs, liver and central nervous system. as in the toxic oil syndrome, there is fasciitis, wasting and muscle pain associated with blood and tissue eosinophilia. the lungs are affected in % of cases. pulmonary symptoms have included cough, dyspnoea and chest pain. radiographs have shown diffuse bilateral infiltrates and pulmonary hypertension has been documented in a few cases. histology of the lungs shows an oedematous myxoid intimal thickening affecting small pulmonary blood vessels and a diffuse interstitial lymphocytic and eosinophilic infiltrate. , , , , these cells may also be seen within the walls of the thickened blood vessels (fig. . . ) . , massive ingestion of l-tryptophan has resulted in the appearances of an organising pneumonia. as an adverse drug reaction, granulomatous alveolitis is best exemplified by the extrinsic allergic alveolitis of pituitary snuff-takers, but it is also encountered on rare occasions with cytotoxic and other drugs, including methotrexate, bacille calmette-guérin (bcg) immunisation, interferons, ciprofloxacin, antiviral therapy and tumour necrosis factor antagonists. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the histological appearances may suggest extrinsic allergic alveolitis or sarcoidosis but the centri-acinar or lymphangitic concentration of these conditions is usually lacking. however, unless an infective agent can be demonstrated the diagnosis generally requires consideration of the clinical and environmental details, including any drug regimen. exogenous lipid pneumonia may result from the unintentional aspiration of various fat-based medicaments such as liquid paraffin, oily nose drops and petroleum jelly or of fat-rich dietary supplements in the form of ghee. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the consumption of liquid paraffin as an aperient is common in some countries and may be taking place without the knowledge of the patient's medical practitioner. regurgitation and aspiration of ingested oil are especially likely to happen during sleep in the presence of a hiatus hernia or when the oesophagus fails to empty completely into the stomach because of achalasia of the cardia. the aspiration of vegetable oil occurred in the past from the use of menthol in olive oil for the treatment of tuberculous laryngitis, and occasionally from the use of iodinated vegetable oils for bronchography. [ ] [ ] [ ] [ ] more recently exogenous lipid pneumonia has developed from the constant sucking of lollipops formulated for the administration of the analgesic fentanyl but also containing a stearate component. the treatment of epistaxis by nasal packing with paraffin gauze has also led to exogenous lipid pneumonia. the pathology of exogenous lipid pneumonia is described on page . other medicines may also be aspirated unwittingly, for example a ferrous sulphate tablet may cause brown iron staining and necrosis of the bronchus at the point of impact, progressing to bronchial stenosis. [ ] [ ] [ ] distal infection is then likely, as with any foreign body. barium sulphate aspiration may complicate gastrointestinal radiography. large amounts may impair ventilation but being inert there is no permanent injury to the lungs, although the striking changes are evident on the chest radiograph. an outbreak of pulmonary hypertension affecting many swiss, austrian and german patients in the period - was probably due to the anorectic drug aminorex, which was accordingly withdrawn with regression in the number of new cases. the pathology in these patients was identical to that of primary pulmonary hypertension (see p. ) and it proved impossible to reproduce the condition in laboratory animals but the epidemiological evidence that aminorex was to blame is very strong. fenfluramine and phentermine, further anorectic drugs that are chemically similar to aminorex, have also been associated with such plexogenic pulmonary hypertension, [ ] [ ] [ ] [ ] [ ] and with fibroproliferative plaque on the tricuspid valve and pulmonary arteries. pulmonary hypertension due to pulmonary veno-occlusive disease has sometimes complicated the use of cytotoxic chemotherapeutic agents or followed bone marrow transplantation. non-steroidal anti-inflammatory agents such as indomethacin and diclofenac cross the placenta and, if given in late pregnancy, may cause premature closure of the ductus arteriosus, resulting in severe neonatal pulmonary hypertension. , pulmonary hypertension is a well-recognised association of human immunodeficiency virus (hiv) infection but until recently has been unexplained. now, however, evidence is emerging that the highly active antiretroviral therapy administered to hiv-positive patients might be responsible for the pulmonary hypertension. the older high-oestrogen contraceptive drugs carried a slight risk of thromboembolism but this is not seen with the newer preparations. pulmonary thromboembolism has also occurred with a drug-induced lupus syndrome associated with anticardiolipin antibodies. chemotherapeutic drugs such as mitomycin may cause widespread smallvessel thrombosis resulting in the haemolytic-uraemic (thrombotic microangiopathic) syndrome. there is prominent involvement of pulmonary vessels and patients often suffer from respiratory as well as renal insufficiency, and pulmonary hypertension. the syndrome can develop during treatment or up to several months after the drug has been withdrawn. pulmonary thromboembolism is also recorded as a complication of immunoglobulin infusion. non-traumatic fat embolism has resulted from the agglutination or 'creaming' of fat emulsions administered intravenously as a source of calories to debilitated patients. [ ] [ ] [ ] [ ] [ ] [ ] the agglutinated liposomes occlude fine blood vessels throughout the body, causing effects such as priapism, osteonecrosis and pancreatitis. they may be demonstrated in the pulmonary capillaries but the lungs have considerable vascular reserve and it is uncertain what effect the vascular occlusion has on pulmonary function. agglutination of these fat emulsions is particularly common in severely ill patients and this has been attributed to the elevated blood levels of acute-phase proteins, especially c-reactive protein, that are found in the very ill. the agglutination is also induced by calcium and may be brought about by administering calcium and other mineral supplements through the same venous line as the fat. once agglutinated, the fat is less soluble and may be demonstrated in paraffin sections. sudan black is especially useful for this purpose (fig. . . ). microvascular crystal embolism is a further risk of parenteral nutrition, the crystals representing various calcium salts that may precipitate in the circulation. transient diffusion abnormalities attributed to oil embolism are very common in patients undergoing lymphangiography but serious respiratory impairment is limited to those patients with pre-existing lung disease or in whom substantial amounts of contrast medium are injected rapidly. [ ] [ ] [ ] [ ] other emboli of an iatrogenic nature described in pulmonary arteries include the broken-off ends of intravenous catheters and cannulas, particles from dialysis tubing, prosthetic implants of substances such as teflon and silicone , [ ] [ ] [ ] [ ] and various materials injected to occlude abnormal blood vessels. , diffuse pulmonary haemorrhage diffuse pulmonary haemorrhage may result from interference with the clotting mechanism by anticoagulants or from widespread pulmonary capillaritis, the latter reported in leukaemic patients treated with retinoic acid. pulmonary haemorrhage has also been reported as an idiosyncratic reaction to lymphangiography media and as a complication of immunoglobulin infusion, while the development of anti-basement membrane antibodies resulting in goodpasture's syndrome has been attributed to penicillamine. a infection is a common pulmonary hazard in any patient receiving corticosteroids, chemotherapy or any other immunosuppressant drug. viral, bacterial, fungal and protozoal infections, often in combination, may all develop in the lungs of such patients and tissue reactions may be atypical. pneumocystis jiroveci, for example, may elicit a granulomatous reaction or cause diffuse alveolar damage rather than the usual foamy alveolar exudate (see p. ). metastatic calcification, described on page , may result from any drug causing hypercalcaemia, e.g. high doses of vitamin d, calcium and inorganic phosphate or excessive alkali intake in the treatment of peptic ulceration. carcinoma of the lung may be promoted by drugs. arsenicals cause squamous metaplasia of the bronchi and occasionally squamous carcinoma, while peripheral scar cancers, usually adenocarcinomas, have developed in lungs showing fibrosis due to drugs such as busulphan. drugs may result in a variety of pleural diseases. common examples include effusions, chronic inflammation and fibrosis. these are usually encountered in isolation but may be associated with chronic interstitial pneumonia or fibrosis. sometimes there is also serological evidence of systemic lupus erythematosus: many drugs, including hydantoin, practolol, procainamide, hydralazine and sulphonamides, are associated with the development of a syndrome resembling systemic lupus erythematosus that includes pleural disease. whether the drugs are directly responsible for the syndrome or merely promote the development of latent natural disease is uncertain. ergotamine derivatives such as methysergide and bromocriptine are notable for the production of pleural fibrosis, which is sometimes associated with mediastinal and retroperitoneal fibrosis large amounts or prolonged treatment are generally required to produce this effect. [ ] [ ] [ ] in patients given practolol, pleural thickening has become evident several years after the drug was discontinued. this shows the need for a careful drug history in any patient with unexplained pleural fibrosis. reports of radiation-induced lung damage began to appear soon after ionising radiation became widely used in the treatment of malignant disease. [ ] [ ] [ ] despite refinements in radiotherapy techniques it is often impossible to avoid irradiating small areas of lung when treating cancer of the lung, breast, spine, thymus and oesophagus. parts of the lungs are also included in 'mantle' irradiation of mediastinal lymph nodes affected by lymphoma. occasionally, the whole of both lungs is irradiated, as in the treatment of widespread pulmonary metastases or as part of whole-body irradiation prior to marrow transplantation for the treatment of leukaemia. radiation pneumonitis, usually localised, is estimated to affect about % of patients. therapeutic irradiation is given as divided doses over several weeks in order to minimise damage to adjacent tissue. the effects of such fractionated treatment are cumulative. in the lungs an early exudative phase soon passes and progressive damage becomes apparent only after months or even years. , the changes are generally confined to the area of lung that is irradiated but are widespread when the whole body is irradiated prior to bone marrow transplantation or there is accidental whole-body irradiation. however, localised irradiation of the lung has been followed by abnormalities in non-irradiated areas. these include bilateral alveolar exudates, migratory organising pneumonia affecting both lungs , and fulminant bilateral interstitial pneumonia. the likelihood of lung injury is increased by the simultaneous use of cytotoxic drugs and oxygen therapy. furthermore, chemotherapy following irradiation may result in exacerbation of the injury in areas previously irradiated, a phenomenon termed 'recall pneumonitis' . , in the long term, irradiation also results in an increased incidence of lung carcinoma. this was seen in patients given therapeutic irradiation to the spine for ankylosing spondylitis and is still encountered on occasion following irra diation for breast cancer. the pathogenesis of radiation injury is described on page . radiation damage to the lung is traditionally separated into fulminant acute injury coming on within days, subacute pneumonitis developing within several weeks (typically - months) and interstitial fibrosis slowly evolving from the subacute stage or making itself apparent years later. the migratory organising pneumonia referred to above is an unusual further effect, as is chronic eosinophilic pneumonia. in the pleura, radiation causes fibrinous effusions and adhesions. pleural effusion and pulmonary oedema may be augmented by the long-term effects of radiation on the heart. fulminant acute injury is an unusual and unexpected effect of therapeutic radiation but one that is likely to come to the attention of the pathologist as an autopsy is often requested. the clinical features are those of acute lung injury and the pathological changes are those of diffuse alveolar damage. the cause is likely to be accidental overdosage, augmentation of the radiation damage by accompanying oxygen therapy or treatment with cytotoxic drugs. occasionally however these factors can be excluded, in which case the damage has to be ascribed to 'hypersensitivity' . subacute radiation pneumonitis is encountered more commonly. after an interval of about - months the patient complains of shortness of breath and a non-productive cough. the chest radiograph shows hazy opacification proceeding to more dense consolidation. lung biopsy shows alveolar and interstitial oedema, possibly with residual hyaline membranes, proliferation of atypical alveolar epithelial cells and interstitial fibroblasts and organising thrombosis. later, as the process advances, there is widespread fibrosis comparable to that illustrated in figure . on page and ultimately dense scarring (fig. . . ) . tracheal and aortic injury may complicate radiation treatment of tracheal lesions, sometimes resulting in an aortotracheal fistula. patients requiring mechanical ventilation are liable to suffer lung injury in a number of ways. in addition to effects of barotrauma such as pneumothorax and surgical emphysema, they often develop diffuse alveolar damage. the high oxygen tension that is often combined with mechanical ventilation is a major factor - but mechanical forces other than the high pressures responsible for barotrauma can also contribute to this form of lung injury, notably by resulting in excessive end-expiratory stretch and repeated collapse/recruitment of the alveolar walls. , low tidal volume ventilation is therefore a fundamental part of the management of diffuse alveolar damage. although oxygen is necessary to life, it is cytotoxic in high concentrations. severe hyperoxia damages dna, inhibits cellular proliferation and ultimately kills cells. its toxicity is thought to be due to the intracellular production of active oxygen radicals, some of which derive from activated neutrophils attracted to the site of injury. [ ] [ ] [ ] [ ] under normal conditions most of the oxygen is reduced to water by cytochrome oxidase, and any active radicals produced are eliminated by superoxide dismutase, catalase and other antioxidants. however, these defence mechanisms may prove inadequate when active radicals are produced in excess. problems are likely to arise in clinical practice when lung disease necessitates the concentration of oxygen in the inspired air being raised in order to maintain normal blood levels of oxygen and prevent cerebral hypoxia. [ ] [ ] [ ] a 'safe' level for oxygen administration is not firmly established and, because of species differences in susceptibility to oxygen, caution is needed in extrapolating from animal studies. however, animal experiments have shown that previous damage to the lungs renders them unduly sensitive to oxygen , and conversely that prior exposure to high levels of oxygen confers some resistance to subsequent oxygen exposure. clinical studies suggest that less than % oxygen (at atmospheric pressure) can be tolerated for long periods without ill effect. little, if any, serious lung damage results from administration of % oxygen for up to hours but concentrations between % and % carry a risk of damage if this period is exceeded. , extracorporeal oxygenation of the blood circumvents the problem but if it is to be prolonged it becomes a major undertaking that poses its own hazards; it is therefore generally reserved for patients who remain hypoxaemic despite other measures. intravenous blood oxygenators are employed to minimise the supplementation of inspired oxygen and partial liquid ventilation utilising perfluorocarbon has also been used. experimentally, disruption of cd binding to reduce the release of proinflammatory cytokines has shown promising results in blunting oxygen-induced lung injury. none of the morphological changes attributable to oxygen toxicity is specific. the earliest ultrastructural change in experimental oxygen poisoning is swelling of endothelial cells, the cytoplasm of which becomes grossly oedematous and vacuolated. swelling and fragmentation of type i epithelial cells follow and these cells become separated from their basement membrane, which is then coated by thin strands of protein. this coating is replaced by proliferating type ii cells by the th day. with recovery in room air the lungs practically return to normal. the full clinical picture of oxygen poisoning is the acute respiratory distress syndrome and the corresponding pathological changes are those of diffuse alveolar damage, as described on page . patients with hypovolaemic shock or undergoing major surgery often require massive blood transfusions and this provides another possible cause of pulmonary damage. although hypervolaemia is the commonest cause of pulmonary oedema after blood transfusion, transfusion-related acute lung injury is more often fatal. platelet and white cell aggregates are known to develop in stored blood, but a relationship between the number of microaggregates transfused and the degree of respiratory impairment has not been convincingly demonstrated. leukocyte antibodies are a more likely cause of lung injury in these patients. such antibodies are often found in multiparous female donors as a result of sensitisation by fetal white cells during pregnancy. alternatively, the recipient may have developed them during pregnancy or as a result of previous blood transfusions. the implicated antibodies are thought to initiate alveolar capillary damage within hours of transfusion by stimulating granulocyte aggregation. , electron microscopy has shown capillary endothelial damage with activated granulocytes in contact with alveolar basement membranes. cardiopulmonary bypass entails oxygenation and circulation of the blood by extracorporeal devices, so permitting major heart surgery. in the early days of such surgery it was not unusual for patients to develop fatal respiratory insufficiency in the postoperative period. this led to the term 'postperfusion lung' . electron microscopic studies showed alveolar damage with degranulation of neutrophils in pulmonary capillaries. , the syndrome is now less common but infants remain susceptible. the most likely explanation is that the synthetic materials with which blood comes into contact during the bypass procedure are able to activate complement. this is mediated by hageman factor (factor xii) and the alternative pathway. aggregation of neutrophils leads to their sequestration in the lungs and damage results from their release of lysosomal enzymes and active radicals. [ ] [ ] [ ] the process is delayed by hypothermia. a postcardiac injury syndrome develops after a variety of myocardial or pericardial injuries: it has been described after cardiac surgery (postpericardiotomy syndrome), myocardial infarction (dressler's syndrome), blunt trauma to the chest, percutaneous puncture of the heart and implantation of a pacemaker. there is a delay of anything between a few days and a few months between the cardiac injury and the onset of symptoms, which comprise chest pain, breathlessness, dyspnoea and fever. examination usually reveals haemorrhagic pleural or pericardial effusions and pulmonary infiltrates. the syndrome usually resolves spontaneously and few pathological studies have therefore been conducted. however, the changes of diffuse alveolar damage have been reported, principally hyaline membrane formation and type ii pneumocyte hyperplasia. the pathogenesis is obscure. antibodies reacting with myocardial antigens often develop after cardiac surgery but there is no relationship between these and the development of the syndrome. [ ] [ ] [ ] this minimally invasive technique is used to destroy lesions as varied as pulmonary metastases and the connection between the left atrium and ectopic foci in the muscular sleeves that surround the terminations of the pulmonary veins (see p. ). the former may be complicated by pneumothorax and the latter by pulmonary vein stenosis. , central venous cannulation (synonym: catheterisation) is widely used in treating seriously ill patients and may give rise to serious complications. the commonest early complications related to the respiratory tract are caused by local trauma: they include pneumothorax, subcutaneous emphysema, haemothorax and air embolism. infection occurs later, causing endocarditis, septic emboli and lung abscesses. thrombosis is another common late complication: one autopsy study of patients with central venous lines showed that % had major pulmonary emboli and % had microscopic emboli in their pulmonary arteries. pulmonary artery cannulation, for example with a swan-ganz catheter, may result in pulmonary infarction or any of the traumatic complications of central venous catheterisation mentioned above. , tracheotomy entails a small immediate risk of haemorrhage from damaged subthyroidal arteries, while an endotracheal tube predisposes to infection, as with all foreign bodies. infection is also promoted by the filtering action of the upper respiratory air passages being bypassed. the latter factor also necessitates humidification of the inspired air and on occasion the humidifier or ventilator has become contaminated so that an aerosol of bacteria is introduced directly into the lower respiratory tract. high-pressure ventilation may also lead to interstitial emphysema, pneumothorax and surgical emphysema. asphyxia may follow an endotracheal tube becoming blocked by secretions or through it being badly positioned. secretions need to be constantly removed yet repeated suctioning to achieve this has led to cardiac dysrrythmia and even cardiac arrest. if the balloon on the endotracheal tube is too near the tracheostomy it may act as a fulcrum, causing the tip of the tube to press into the tracheal wall. pressure necrosis and perforation may follow, leading to mediastinitis, tracheo-oesophageal fistula or erosion of a large blood vessel. these are also complications of tracheobronchial laser therapy. pressure from the balloon may lead to a tracheal diverticulum and after the tube is withdrawn the trachea may become narrowed at either the site of the incision or further down where the balloon on the tracheal tube causes pressure. small, shallow ulcers generally heal quickly but deeper ulcers cause necrosis of the tracheal cartilage, and healing is then often accompanied by fibrous stenosis (fig. . . ) or web formation. this results in wheezing and dyspnoea but not before the trachea has narrowed to % of its original size, which may take months. earlier narrowing may be caused by oedema or a fibrinous pseudomembrane. , sometimes the stenosis takes the form of a large mass of granulation tissue at the tracheostomy site, a so-called granuloma ball. in children especially, intubation may lead to tracheomalacia so that after the tube is removed the airway collapses. necrotising sialometaplasia is a further complication of prolonged intubation. the incidence of such posttracheostomy complications can be minimised by careful placement of the stoma and tube, avoidance of large apertures and high cuff pressures, elimination of heavy connecting equipment and meticulous care of the tracheostomy. nasogastric feeding tubes may of course lead to aspiration lesions in the lungs and even fatal asphyxia if they are inadvertently allowed to enter the trachea rather than the oesophagus. bronchoscopy is generally a safe, almost routine procedure. a review of patients who underwent bronchoscopy identified severe complications in ( . %), of whom three died. the fatal cases comprised a -year-old with coronary heart disease who developed cardiac arrest and two patients who had had tracheal transplantation for oesophageal cancer and required bronchoscopic laser treatment but died of airway obstruction. the pleural cavity is intubated in the treatment of pneumothorax and pleural effusions the tube being placed anteriorly to drain air and posteriorly to drain fluid. complications include laceration of an intercostal artery or vein, the lung, the diaphragm and the heart. pneumonectomy has been practised since the s, since when the mortality associated with this operation has dropped from over % to near zero in the best hospitals. risk factors include underlying lung disease, other medical conditions and more extensive procedures such as pleuropneumonectomy and pneumonectomy combined with chest wall resection. the anatomical changes that take place soon after pneumonectomy have been extensively studied by radiologists who describe the air-filled postpneumonectomy space gradually filling with fluid and contracting as the mediastinum shifts and the ipsilateral dome of the diaphragm rises. much of the space is filled by fluid within weeks but complete opacification may take up to months. rapid filling in the immediate postoperative period suggests haemorrhage or chylothorax. however, fluid accumulation is normally rapid after pleuro-pneumonectomy and may compromise the function of the other lung. pathologists conducting autopsies long after the operation may find complete fibrous obliteration of the postpneumonectomy space, coupled with mediastinal shift and elevation of the hemidiaphragm, but often there is persistent brown fluid, which may be clear, cloudy or occasionally purulent. the remaining lung is generally enlarged, with its volume greater than predicted. animal studies have shown that if one lung is excised early in life the enlargement is partly due to enhanced growth but later it represents only dilatation of existing air spaces. hepatocyte growth factor is thought to be involved in the proliferation of residual lung cells following pneumonectomy. pulmonary complications include those typically seen after other thoracic procedures, such as haemorrhage and infection, and those unique to the postpneumonectomy state, namely anastomotic dehiscence and postpneumonectomy pulmonary oedema. the latter presents as the acute respiratory distress syndrome and represents the early stages of diffuse alveolar damage. it follows severe shift of the heart and mediastinum, which is commoner in children and young adults, in whom the tissues are more compliant. [ ] [ ] [ ] [ ] the condition complicates up to % of lung resections , and is commoner following excision of the right lung when severe herniation of the left lung into the postpneumonectomy space stretches the trachea and left main bronchus and the latter is compressed between the left pulmonary artery in front and the arch of the aorta behind. in the long term the compression can result in bronchomalacia and postobstructive bronchiectasis. if postpneumonectomy oedema develops the immediate postoperative mortality is high - % following pneumonectomy, % following lobectomy and % following sublobar resections. , the pathogenesis is probably multifactorial but apart from factors such as fluid overload and high inspired oxygen concentrations there is probably an element of alveolar wall injury, induced by oxidant generation secondary to lung stretching and general surgical trauma. , occupational dust exposure and chronic obstructive pulmonary disease -a systematic overview of the evidence occupational exposures and the risk of copd: dusty trades revisited the origin of the term 'pneumonokoniosis' industrial injuries advisory council. pneumoconiosis and byssinosis. london: hmso; aerosols: their deposition and clearance human lung parenchyma retains pm . airway branching patterns influence asbestos fiber location and the extent of tissue injury in the pulmonary parenchyma and lymph nodes in infants' lungs pulmonary sumps, dust accumulations, alveolar fluid and lymph vessels candida lung abscesses complicating parenteral nutrition a diffuse form of pulmonary silicosis in foundry workers inhalation of china stone and china clay dusts: relationship between mineralogy of dust retained in the lungs and pathological changes talc pneumoconiosis. a report of six patients with postmortem findings diffuse 'asbestosis-like' interstitial fibrosis of the lung pulmonary pathology in workers exposed to nonasbestiform talc talc pneumoconiosis -a pathologic and mineralogic study diffuse interstitial fibrosis in nonasbestos pneumoconiosis -a pathological study the disposal of coal and haematite dusts inhaled successively vertical gradient of alveolar size in lungs of dogs frozen intact apical localization of phthisis distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive co apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and progressive massive fibrosis of the lung effect of inspiratory flow rate on the regional distribution of inspired gas apical scars and their relationship to siliceous dust accumulation in non-silicotic lungs topographic distribution of asbestos fibres lung structure as a risk factor in adverse pulmonary responses to asbestos exposure. a case-referent study in quebec chrysotile miners and millers particle deposition in casts of the human upper tracheobronchial tree differences in particle deposition between the two lungs pulmonary retention of ultrafine and fine particles in rats experimental study of the dust-clearance mechanism of the lung the lung: an excretory route for macromolecules and particles phagocytic potential of pulmonary alveolar epithelium with particular reference to surfactant metabolism chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages the uptake of mineral particles by pulmonary epithelial cells translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships patterns of particle deposition and retention after instillation to mouse lung during acute injury and fibrotic repair silica deposition in the lung during epithelial injury potentiates fibrosis and increases particle translocation to lymph nodes the permeability of lung parenchyma to particulate matter the migration of bronchoalveolar macrophages into hilar lymph nodes etude au microscope electronique du granulome pulmonaire silicotique experimental the postnatal development of lymphoreticular aggregates in human lung in relation to occupational and non-occupational exposure the distribution of amosite asbestos in the periphery of the normal human lung diseases associated with exposure to silica and nonfibrous silicate minerals detection of silica particles in lung tissue by polarizing light microscopy a practical method for the identification of particulate and crystalline material in paraffin-embedded tissue specimens particles in paraffin sections demonstrated in the backscattered electron image quantitative analysis of inorganic particulate burden in situ in tissue sections microanalysis in histopathology ion microprobe mass analysis of beryllium in situ in human lung: preliminary results laser probe mass spectrometry (lamms) analysis of beryllium, sarcoidosis and other granulomatous diseases molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by fourier transform laser microprobe mass spectrometry value of beryllium lymphocyte transformation test in chronic beryllium disease and in potentially exposed workers radiographic and pathologic correlation of coal workers' pneumoconiosis slateworker's pneumoconiosis transkei silicosis three cases of dental technician's pneumoconiosis related to cobaltchromium-molybdenum dust exposure: diagnosis and follow-up deposition of silicosis dust in the lungs of the inhabitants of the sahara regions sulla possibilita e sulla frequenza della silicosi pulmonare tra gli abitanti del deserto libico simple siliceous pneumoconiosis of bedouin females in the negev desert simple siliceous pneumoconiosis in negev bedouins silicosis in a pakistani farmer silicosis in a himalayan village population -role of environmental dust non-occupational pneumoconiosis at high altitude villages in central ladakh silicate pneumoconiosis of farm workers silicate pneumoconiosis and pulmonary fibrosis in horses from the monterey-carmel peninsula comparative pathology of silicate pneumoconiosis two cases of acute silicosis -with a suggested theory of causation acute silicosis silicose aigue fatale par inhalation volontaire de poudre à recurer acute silicosis in tombstone sandblasters accelerated silicosis in scottish stonemasons the relation between fibrosis of hilar lymph glands and the development of parenchymal silicosis distribution of silicotic collagenization in relation to smoking habits silicosis presenting as bilateral hilar lymphadenopathy silicotic lymph node lesions in nonoccupationally exposed lung carcinoma patients obliterative central bronchitis due to mineral dust in patients with pneumoconiosis left recurrent laryngeal nerve palsy associated with silicosis anthracotic and anthracosilicotic spindle cell pseudotumors of mediastinal lymph nodes: report of five cases of a reactive lesion that simulates malignancy right middle lobe atelectasis associated with endobronchial silicotic lesions pleural pearls following silicosis: a histological and electronmicroscopic study silicainduced pleural disease: an unusual case mimicking malignant mesothelioma factors associated with massive fibrosis in silicosis silicosis of systemic distribution hepatosplenic silicosis hepatic silicosis silicotic lesions of the bone marrow: histopathology and microanalysis peritoneal silicosis pulmonary fibrosis in non-ferrous foundry workers irregular opacities in coalworkers' pneumoconiosis -correlation with pulmonary function and pathology pneumoconiosis-related lung cancers -preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis the effects of dusts on peritoneal cells within diffusion chambers freshly fractured quartz inhalation leads to enhanced lung injury and inflammation: potential role of free radicals an examination of the cytotoxic effects of silica on macrophages activity of macrophage factor in collagen formation by silica fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages effect of sio -liberated macrophage factor on protein synthesis in connective tissue in vitro the regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis the ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro cytokines and cytokine network in silicosis and coal workers' pneumoconiosis activation of murine macrophages by silica particles in vitro is a process independent of silica-induced cell death localization of type i procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis transforming growth factor-beta (tgfbeta) in silicosis mechanisms in the pathogenesis of asbestosis and silicosis changes in the composition of lung lipids and the 'turnover' of dipalmitoyl lecithin in experimental alveolar lipo-proteinosis induced by inhaled quartz case report: pulmonary alveolar proteinosis and aluminum dust exposure induction of surfactant protein (sp-a) biosynthesis and sp-a mrna in activated type ii cells during acute silicosis in rats silicon nephropathy: a possible occupational hazard rapidly progressive silicon nephropathy silica and glomerulonephritis: case report and review of the literature rapidly progressive crescenteric glomerulonephritis in a sandblaster with silicosis diatomaceous earth pneumoconiosis particle size for differentiation between inhalation and injection pulmonary talcosis kaolin pneumoconiosis. a case report kaolin pneumoconiosis. radiologic, pathologic and mineralogic findings silicosis in wyoming bentonite workers pneumoconiosis due to fuller's earth pulmonary disease caused by the inhalation of cosmetic talcum powder talc dust pneumoconiosis mica-associated pulmonary interstitial fibrosis baritosis: a benign pneumoconiosis silicosis in barium miners scleroderma in gold-miners on the witwatersrand with particular reference to pulmonary manifestations immunological factors in the pathogenesis of the hyaline tissue of silicosis mini-review -immunologic aspects of pneumoconiosis adverse effects of crystalline silica exposure chronic necrotizing pulmonary aspergillosis in pneumoconiosis -clinical and radiologic findings in patients silica and some silicates. monographs on the evaluation of the carcinogenic risk of chemicals to humans occupational lung disease: . exposure to crystalline silica and risk of lung cancer: the epidemiological evidence do silica and asbestos cause lung cancer? report by the industrial injuries advisory council in accordance with section of the social security administration act on the question whether lung cancer in relation to occupational exposure to silica should be prescribed. london: hmso; crystalline silica exposure, radiological silicosis, and lung cancer mortality in diatomaceous earth industry workers acute silicoproteinosis acute silicolipoproteinosis atypical reaction to inhaled silica alveolar proteinosis. its experimental production in rodents experimental endogenous lipid pneumonia and silicosis pathogenesis of experimental pulmonary alveolar proteinosis pneumoconiosis in carbon electrode makers graphite pneumoconiosis of electrotypers case report: a case of wood-smoke-related pulmonary disease hut lung. a domestically acquired particulate lung disease bronchial stenosis due to anthracofibrosis anthracofibrosis attributed to mixed mineral dust exposure: report of three cases anthracofibrosis, bronchial stenosis with overlying anthracotic mucosa: possibly a new occupational lung disorder occupational anthracofibrosis pneumoconiosis and other causes of death in iron and steel foundry workers histochemical study of certain iron ore dusts mixed-dust pneumoconiosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation chronic interstitial pneumonia in silicosis and mix-dust pneumoconiosis: its prevalence and comparison of ct findings with idiopathic pulmonary fibrosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation coal pneumoconiosis coal workers' pneumoconiosis pneumoconiosis in coal trimmers dust exposure, dust recovered from the lung, and associated pathology in a group of british coalminers the relationship between the mass and composition of coal mine dust and the development of pneumoconiosis interstitial fibrosis in coal miners -experience in wales and west virginia coal worker's pneumoconiosis: ct assessment in exposed workers and correlation with radiographic findings variations in the histological patterns of the lesions of coal workers' pneumoconiosis in britain and their relationship to lung dust content comparison of radiographic appearances with associated pathology and lung dust content in a group of coalworkers small airways disease and mineral dust exposure. prevalence, structure and function coal mining, emphysema, and compensation revisited coal mining, emphysema, and compensation revisited -reply coal mining and chronic obstructive pulmonary disease: a review of the evidence the pathogenesis of simple pneumokoniosis in coal workers mineral dusts cause elastin and collagen breakdown in the rat lung: a potential mechanism of dust-induced emphysema emphysema in coal workers' pneumoconiosis pulmonary disability in coal workers' pneumoconiosis quantified pathology of emphysema, pneumoconiosis, and chronic bronchitis in coal workers emphysema and dust exposure in a group of coal workers coal worker's lung: not only black, but also full of holes contributions of dust exposure and cigarette smoking to emphysema severity in coal miners in the united states report by the industrial injuries advisory council in accordance with section of the social security administration act on the question whether bronchitis and emphysema in coal miners and metal production workers should be prescribed. london: hmso; the new prescription: industrial injuries benefits for smokers? exposure to respirable coalmine dust and incidence of progressive massive fibrosis the attack rate of progressive massive fibrosis ischemic necrosis in anthracosilicosis the composition of massive lesions in coal miners mechanisms of fibrosis in coal workers' pneumoconiosis: increased production of platelet-derived growth factor, insulin-like growth factor type i, and transforming growth factor beta and relationship to disease severity secretion and mrna expression of tnf alpha and il- in the lungs of pneumoconiosis patients a broader concept of caplan's syndrome related to rheumatoid factors differential agglutination test in rheumatoid arthritis complicated by pneumoconiosis circulating antinuclear antibody and rheumatoid factor in coal pneumoconiosis serologic changes in pneumoconiosis and progressive massive fibrosis of coal workers immunological investigations of coalworkers' disease certain unusual radiological appearances in the chest of coal-miners suffering from rheumatoid arthritis pathological studies of modified pneumoconiosis in coal-miners with rheumatoid arthritis (caplan's syndrome) asbestos, asbestosis, and cancer: the helsinki criteria for diagnosis and attribution report of the asbestosis committee of the college of american pathologists and pulmonary pathology society the asbestos cancer epidemic rapid short-term clearance of chrysotile compared with amosite asbestos in the guinea pig persistence of natural mineral fibers in human lungs: an overview a pathological and mineralogical study of asbestos-related deaths in the united kingdom in correlation between fibre content of the lungs and disease in naval dockyard workers correlation between fibre content of the lung and disease in east london asbestos factory workers the effects of the inhalation of asbestos in rats mass and number of fibres in the pathogenesis of asbestos related lung disease in rats fiber burden and patterns of asbestos-related disease in chrysotile miners and millers carcinogenesis and mineral fibres lung diseases due to environmental exposures to erionite and asbestos in turkey prospective study of mesothelioma mortality in turkish villages with exposure to fibrous zeolite analysis of ferruginous bodies in bronchoalveolar lavage from foundry workers analysis of the cores of ferruginous (asbestos) bodies from the general population fiber counting and analysis in the diagnosis of asbestos-related disease concentrations and dimensions of coated and uncoated asbestos fibres in the human lung comparison of the results of asbestos fibre dust counts in lung tissue obtained by analytical electron microscopy and light microscopy asbestos bodies and the diagnosis of asbestosis in chrysotile workers asbestos fiber content of lungs with diffuse interstitial fibrosis: an analytical scanning electron microscopic analysis of cases comparison of phagocytosis of uncoated versus coated asbestos fibers by cultured human pulmonary alveolar macrophages numbers of asbestos bodies on iron-stained tissue sections in relation to asbestos body counts in lung tissue digests asbestos content of lung tissue in asbestos associated diseases: a study of cases analysis and interpretation of inorganic mineral particles in 'lung' tissues asbestos bodies in bronchoalveolar lavage in relation to asbestos bodies and asbestos fibres in lung parenchyma asbestos bodies in the sputum of asbestos workers: correlation with occupational exposure the optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to human pathology reaction assessment of mineral fibres from human lung tissue detection of asbestos fibres by dark ground microscopy analysis of lung asbestos content guidelines for mineral fibre analyses in biological samples: report of the ers working group relationship between occupations and asbestos-fibre content of the lungs in patients with pleural mesothelioma, lung cancer, and other diseases inflammation-generating potential of long and short-fibre amosite asbestos samples asbestos-stimulated tumour necrosis factor release from alveolar macrophages depends on fibre length and opsonization the pathogenicity of long versus short fibre amosite asbestos administered to rats by inhalation and intra-peritoneal injection cytogenetic and pathogenic effects of long and short amosite asbestos exposure and mineralogical correlates of pulmonary fibrosis in chrysotile asbestos workers quantitative assessment of inorganic fibrous particulates in dust samples with an analytical transmission electron microscope fibre distribution in the lungs and pleura of subjects with asbestos related diffuse pleural fibrosis malignant mesothelioma in women observations on the distribution of asbestos fibres in human lungs analysis of asbestos fibres and asbestos bodies in tissue samples from human lung: an international interlaboratory trial distribution and characteristics of amphibole asbestos fibres in the left lung of an insulation worker measured with the light microscope the identification of asbestos dust with an electron microprobe analyser symposium on man-made mineral fibres mortality in cases of asbestosis diagnosed by a pneumoconiosis medical panel small-airway lesions in patients exposed to nonasbestos mineral dusts small airways disease and mineral dust exposure relation of smoking and age to findings in lung parenchyma: a microscopic study electron microscopic studies in desquamative interstitial pneumonia associated with asbestos desquamative interstitial pneumonia associated with chrysotile asbestos fibres localized inflammatory pulmonary disease in subjects occupationally exposed to asbestos criteria for the diagnosis and grading of asbestosis report of the pneumoconiosis committee of the college of american pathologists and the national institute for occupational safety and health cytoplasmic hyalin in asbestosis further observations on cytoplasmic hyaline in the lung lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore lung pathology of fatal severe acute respiratory syndrome immunohistochemical detection of ubiquitin-positive intracytoplasmic eosinophilic inclusion bodies in diffuse alveolar damage idiopathic pulmonary fibrosis in asbestosexposed workers mortality of workers certified by pneumoconiosis medical panels as having asbestosis radiographic evidence of asbestos effects in american marine engineers chronic airflow obstruction in lung disease. philadelphia: saunders fine structural changes in cryptogenic fibrosing alveolitis and asbestosis pulmonary asbestosis and idiopathic pulmonary fibrosis: pathogenetic parallels crocidoliteinduced pulmonary fibrosis in mice hydroxyl radicals are formed in the rat lung after asbestos instillation invivo asbestos fibers and interferon-gamma up-regulate nitric oxide production in rat alveolar macrophages oxidant sand antioxidant mechanisms of lung disease caused by asbestos fibres antibodies in some chronic fibrosing lung diseases asbestos causes translocation of p protein and increases nf-kappa b dna binding activity in rat lung epithelial and pleural mesothelial cells rapid activation of pdgf-a and -b expression at sites of lung injury in asbestos-exposed rats upregulation of the pdgf-alpha receptor precedes asbestos-induced lung fibrosis in rats tnf-alpha receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers alveolar cell stretching in the presence of fibrous particles induces interleukin- responses the molecular basis of asbestos induced lung injury inhaled asbestos activates a complementdependent chemoattractant for macrophages alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis cellular and molecular basis of the asbestos-related diseases up-regulated expression of transforming growth factor-α in the bronchiolar-alveolar duct regions of asbestos-exposed rats a study of macrophage-mediated initiation of fibrosis by asbestos and silica using a diffusion chamber technique enhanced generation of free radicals from phagocytes induced by mineral dusts mortality from lung cancer in asbestos workers diffuse pleural mesotheliomas and asbestos exposure in northwestern cape province carcinogenicity of amosite asbestos asbestos exposure, cigarette smoking and death rates smoking, exposure to crocidolite, and the incidence of lung cancer and asbestosis pathology of carcinoma of the lung associated with asbestos exposure a study of the histological cell types of lung cancer in workers suffering from asbestosis in the united kingdom lung cancer cell type and asbestos exposure histological type of lung carcinoma in asbestos cement workers and matched controls incidence of lung cancer by histological type among asbestos cement workers in denmark lung cancer in asbestos cement workers in denmark asbestos and lung cancer in glasgow and the west of scotland exposure to crocidolite and the incidence of different histological types of lung cancer asbestos and lung cancer asbestos and lung cancer: is it attributable to asbestosis or to asbestos fibre burden? in: corrin b, editor. pathology of lung tumours asbestos fibreyears and lung cancer: a two phase case-control study with expert exposure assessment the quantitative risks of mesothelioma and lung cancer in relation to asbestos exposure is asbestos or asbestosis the cause of the increased risk of lung cancer in asbestos workers? relation between asbestosis and bronchial cancer in amphibole asbestos miners asbestosis as a precursor of asbestos related lung cancer -results of a prospective mortality study asbestos exposure, asbestosis, and asbestosattributable lung cancer asbestos and lung cancer -reply criteria for attributing lung cancer to asbestos exposure asbestosis: a marker for the increased risk of lung cancer among workers exposed to asbestos asbestos, asbestosis, and lung cancer: a critical assessment of the epidemiological evidence airway function in lifetime-nonsmoking older asbestos workers severe diffuse small airways abnormalities in long term chrysotile asbestos miners morphology of small-airway lesions in patients with asbestos exposure does aluminum smelting cause lung disease? the treatment of silicosis by aluminum powder silicose aigue. caractéristiques cliniques, radiologiques, fonctionnelles et cytologiques du liquide bronchoalveolaire. a propos de observations pathologie and pathogenesis der aluminiumlunge die aluminiumlunge -eine neue gewerbeerkrankung. z f d ges pulmonary fibrosis in workers exposed to finely powdered aluminium aluminium pneumoconiosis i. in vitro comparison of stamped aluminium powders containing different lubricating agents and a granular aluminium powder effect on the rat lung of intratracheal injections of stamped aluminium powders containing different lubricating agents and of a granular aluminium powder desquamative interstitial pneumonia in an aluminum welder sarcoidlike lung granulomatosis induced by aluminum dusts aluminum induced pulmonary granulomatosis aluminum welding fume-induced pneumoconiosis rare earth (cerium) pneumoconiosis rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review hard metal disease interstitial lung disease and asthma in hard-metal workers: bronchoalveolar lavage, ultrastructural, and analytical findings and results of bronchial provocation tests the biological action of tungsten carbide and cobalt: studies on experimental pulmonary histopathology cobalt lung in diamond polishers rapidly fatal progression of cobalt lung in a diamond polisher hard metal pneumoconiosis and the association of tumor necrosis factor-alpha the pulmonary toxicity of beryllium beryllium and lung disease recent chronic beryllium disease in residents surrounding a beryllium facility nonoccupational beryllium disease masquerading as sarcoidosis -identification by blood lymphocyte proliferative response to beryllium beryllium disease international agency for research on cancer. an evaluation of carcinogenic risk to humans. overall evaluation of carcinogenicity: an updating of iarc monographs vols - uber eine apparatur zur erzeugung niedriger staubkonzentrationen von grosser konstanz und eine methode zur mikrogravinctrischen staubbestemmung. anwendung bei der untersuchung von stauben aus der berylliumsgewinnung delayed chenical pneumonitis occurring in workers exposed to beryllium compounds chronic beryllium disease in a dental laboratory technician maintenance of alveolitis in patients with chronic beryllium disease by berylliumspecific helper t cells hla-dpb glutamate : a genetic marker of beryllium disease immunogenetic basis of environmental lung disease: lessons from the berylliosis model beryllium disease in identical twins beryllium detection in human lung tissue using electron probe x-ray microanalysis diagnostic criteria for chronic beryllium disease (cbd) based on the uk registry - united kingdom beryllium registry: mortality and autopsy study lung disease and the lightest of metals misdiagnosis of sarcoidosis in patients with chronic beryllium disease diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients the pathology of interstitial lung disease in nylon flock workers flock worker's lung: broadening the spectrum of clinicopathology, narrowing the spectrum of suspected etiologies polyethylene flock-associated interstitial lung disease in a spanish female popcorn workers' lung clinical bronchiolitis obliterans in workers at a microwave-popcorn plant clinical bronchiolitis obliterans in workers at a microwave-popcorn plant bronchiolitis obliterans syndrome in popcorn production plant workers popcorn worker's lung' in britain in a man making potato crisp flavouring toxicity in textile airbrushing in spain outbreak of organising pneumonia in textile printing sprayers epidemic outbreak of interstitial lung disease in aerographics textile workersthe 'ardystil syndrome': a first year follow up organizing pneumonia in textile printing workers: a clinical description outbreak of pulmonary disease in textile dye sprayers in algeria five-year follow-up of algerian victims of the ''ardystil syndrome mineral oils and petroleum . skorodin ms, chandrasekhar aj. an occupational cause of exogenous lipoid pneumonia machine operator's lung. a hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols metalworking fluid-associated hypersensitivity pneumonitis: a workshop summary an outbreak of hypersensitivity pneumonitis at a metalworking plant: a longitudinal assessment of intervention effectiveness stenius-aarniala b. fire-eater's lung fire-eater's lung: seventeen cases and a review of the literature pulmonary mechanics in dogs given different doses of kerosene intratracheally x-ray appearance of the lungs of electric arc welders the health of welders in naval dockyards: the risk of asbestosrelated diseases occurring in welders welders' pneumoconiosis: tissue elemental microanalysis by energy dispersive x ray analysis the respiratory health of welders toxic fumes and gases . nemery b. metal toxicity and the respiratory tract lung changes associated with the manufacture of alumina abrasives acute inorganic mercury vapor inhalation poisoning nickel poisoning ii. studies on patients suffering from acute exposure to vapors of nickel carbonyl silo-filler's disease -a syndrome caused by nitrogen dioxide silo-filler's disease: nitrogen dioxide-induced lung injury silo filler's disease silo-filler's disease in new york state dung lung: a report of toxic exposure to liquid manure death caused by fermenting manure acute toxic exposure to gases from liquid manure slurry lung': a report of three cases pulmonary lesions in rats exposed to ozone pathology of adult respiratory distress syndrome response of macaque bronchiolar epithelium to ambient concentrations of ozone ultrastructural alterations of alveolar tissue of mice differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for months ozone-induced acute tracheobronchial epithelial injury -relationship to granulocyte emigration in the lung ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy a cohort study of workers exposed to formaldehyde in the british chemical industry -an update pulmonary toxicity following exposure to methylene chloride and its combustion product, phosgene the diversity of the effects of sulfur mustard gas inhalation on respiratory system years after a single, heavy exposure: analysis of cases tracheobronchomalacia and air trapping after mustard gas exposure an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients thionyl-chloride-induced lung injury and bronchiolitis obliterans hydrogen sulfide poisoning: review of years' experience anoxic asphyxia anoxic asphyxia -a cause of industrial fatalities: a review sword ' : surveillance of work-related and occupational respiratory disease in the uk occupational asthma as identified by the surveillance of work-related and occupational respiratory diseases programme in south africa incidence of occupational asthma by occupation and industry in finland sword ' : surveillance of work-related and occupational respiratory disease in the uk american thoracic society statement: occupational contribution to the burden of airway disease aetiological agents in occupational asthma reported incidence of occupational asthma in the united kingdom, - occupational asthma in a factory with a contaminated humidifier the pathology of byssinosis byssinosis: a review in vitro alternative and classical activation of complement by extracts of cotton mill dust: a possible mechanism in the pathogenesis of byssinosis antibody independent complement activation by card-room cotton dust occupational fevers . raskandersen a, pratt ds. inhalation fever -a proposed unifying term for febrile reactions to inhalation of noxious substances humidifier fever pulmonary mycotoxicosis organic dust toxicity (pulmonary mycotoxicosis) associated with silo unloading desquamative interstitial pneumonitis and diffuse alveolar damage in textile workers: potential role of mycotoxins pulmonary mycotoxicosis: a clinicopathologic study of three cases pulmonary involvement in zinc fume fever cytokines in metal fume fever music: a new cause of primary spontaneous pneumothorax atmospheric pressure influences the risk of pneumothorax: beware of the storm experimental study of blast injuries to the lungs biophysics of impact injury to the chest and abdomen histologic, immunohistochemical, and ultrastructural findings in human blast lung injury histologic, immunohistochemical, and ultrastructural findings in human blast lung injury the ultrastructure of rat lung following acute primary blast injury disorders associated with diving decompression disorders in divers intrapulmonary air trapping in submarine escape training casualties airway structure and alveolar emptying in the lungs of sea lions and dogs the structure and function of the small airways in pinniped and sea otter lungs mechanisms of death in shallow-water scuba diving pulmonary barotrauma in submarine escape training the pathophysiology of decompression sickness hemodynamic changes induced by recreational scuba diving dysbaric osteonecrosis: aseptic necrosis of bone deep diving mammals: dive behavior and circulatory adjustments contribute to bends avoidance current concepts: high-altitude illness illnesses at high altitude high altitude pulmonary edema high altitude pulmonary oedema; characteristics of lung lavage fluid prevention of high-altitude pulmonary edema by nifedipine pulmonary circulation at high altitude association of high-altitude pulmonary edema with the major histocompatibility complex pathogenesis of high-altitude pulmonary oedema: direct evidence of stress failure of pulmonary capillaries high-altitude pulmonary edema: current concepts high-altitude pulmonary edema is initially caused by an increase in capillary pressure when lungs on mountains leak -studying pulmonary edema at high altitudes inhaled nitric oxide for high-altitude pulmonary edema lung pathology in high-altitude pulmonary edema pathological features of the lung in fatal high altitude pulmonary edema occurring at moderate altitude in japan lack of smooth muscle in the small pulmonary arteries of the native ladakhi -is the himalayan highlander adapted? aspects of hypoxia pulmonary blood vessels and endocrine cells in subacute infantile mountain sickness the human pulmonary circulation: its form and function in health and disease pulmonary edema associated with salt water near-drowning -new insights the effect of salt water on alveolar epithelial barrier function near-drowning: clinical course of lung injury in adults haggard hw. action of irritant gases upon the respiratory tract regional respiratory tract absorption of inhaled reactive gases respiratory irritants encountered at work health effects of outdoor air pollution committee of the environmental and occupational health assembly of the committee of the environmental and occupational health assembly of the fine particulate air pollution and mortality in us cities, - health effects of diesel exhaust emissions respiratory changes due to long-term exposure to urban levels of air pollution: a histopathologic study in humans chronic glandular bronchitis in young individuals residing in a metropolitan area an association between air pollution and mortality in united states cities chronic bronchitis in women using solid biomass fuel in rural peshawar indoor air pollution: a poverty-related cause of mortality among the children of the world risk of copd from exposure to biomass smoke human health impacts of volcanic activity persistent hyperreactivity and reactive airway dysfunction in firefighters at the world trade center the world trade center residents' respiratory health study: new-onset respiratory symptoms and pulmonary function pulmonary function after exposure to the world trade center collapse in the new york city fire department characterization of the dust/smoke aerosol that settled east of the world trade center (wtc) in lower manhattan after the collapse of the wtc chemical analysis of world trade center fine particulate matter for use in toxicologic assessment longitudinal assessment of spirometry in the world trade center medical monitoring program acute eosinophilic pneumonia in a new york city firefighter exposed to world trade center dust granulomatous pneumonitis following exposure to the world trade center collapse smoking-related interstitial lung diseases: a concise review concomitant upper-lobe bullous emphysema, lower-lobe interstitial fibrosis and pulmonary hypertension in heavy smokers: report of eight cases and review of the literature heterogeneity in combined pulmonary fibrosis and emphysema smoking-related interstitial lung diseases challenges in pulmonary fibrosis : smoking-induced diffuse interstitial lung diseases combined pulmonary fibrosis and emphysema: a distinct underrecognised entity smoking-related changes in the background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course clinical assembly contribution to the celebration of years of the ers. smoking-related lung diseases: a clinical perspective clinically occult interstitial fibrosis in smokers: classification and significance of a surprisingly common finding in lobectomy specimens bronchiolar inflammation and fibrosis associated with smoking -a morphologic cross-sectional population analysis relation of smoking and age to findings in lung parenchyma: a microscopic study variability in small airway epithelial gene expression among normal smokers airway alveolar attachment points and exposure to cigarette smoke in utero patterns of global tobacco use in young people and implications for future chronic disease burden in adults tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic pathology of the lung in fatally burned patients respiratory tract pathology in patients with severe burns heat injuries to the respiratory system death in the burn unit: sterile multiple organ failure pulmonary interstitial edema and hyaline membranes in adult burn patients herpesvirus infections in burn patients pulmonary herpes simplex in burns patients long-term course of bronchiectasis and bronchiolitis obliterans as late complication of smoke inhalation disaster at bhopal: the accident, early findings and respiratory health outlook in those injured chronic lung inflammation in victims of toxic gas leak at bhopal respiratory morbidity years after the union carbide gas leak at bhopal: a cross sectional survey management of the effects of exposure to tear gas ingested toxic agents . boyd mr. evidence for the clara cell as a site of cytochrome p -dependent mixed-function oxidase activity in lung pulmonary histological appearances in fatal paraquat poisoning pulmonary damage due to paraquat poisoning through skin absorption paraquat induced pulmonary fibrosis in three survivors kinetics and cellular localisation of putrescine uptake in human lung tissue enhancement of oxygen toxicity by the herbicide paraquat hypoxic protection in paraquat poisoning the effects of variable o tension and of exogenous superoxide dismutase on type ii pneumocytes exposed to paraquat paraquat-induced injury of type ii alveolar cells the changing pattern of paraquat poisoning in man remodeling of the alveolar structure in the paraquat lung of humans: a morphometric study experimental paraquat poisoning the pathogenesis and structure of paraquatinduced pulmonary fibrosis in rats pulmonary ultrastructure after oral and intravenous dosage of paraquat to rats patterns of pulmonary structural remodeling after experimental paraquat toxicity experimental models of interstitial pneumonia: paraquat, iprindole fine structure of the lung lesion in a case of paraquat poisoning fatal pulmonary intra-alveolar fibrosis after paraquat ingestion paraquat lung: a reappraisal an immunohistochemical study of the fibrosing process in paraquat lung injury toxic-allergic syndrome caused by ingestion of rapeseed oil denatured with aniline pathology of a new toxic syndrome caused by ingestion of adulterated oil in spain clinical epidemiology of toxic oil syndrome -(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome pulmonary hypertension due to toxic oil syndrome. a clinicopathologic study pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome pulmonary vascular lesions in the toxic oil syndrome in spain outbreak of bronchiolitis obliterans associated with consumption of sauropus androgynus in taiwan histopathological study of sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans sauropus androgynus-constrictive obliterative bronchitis/bronchiolitishistopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression segmental necrosis of small bronchi after prolonged intakes of sauropus androgynus in taiwan sauropus bronchiolitis -reply the lipoid pneumonia of blackfat tobacco smokers in guyana a morphometric analysis of the male and female tracheal epithelium after experimental exposure to marijuana smoke differential examination of bronchoalveolar lavage cells in tobacco cigarette and marijuana smokers tracheobronchial changes in habitual, heavy smokers of marijuana with and without tobacco airway inflammation in young marijuana and tobacco smokers an unusual cause of patchy ground-glass opacity pulmonary hazards of smoking marijuana as compared with tobacco british lung foundation. a smoking gun? tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco effects of cannabis on pulmonary structure, function and symptoms large lung bullae in marijuana smokers the pulmonary pathology of illicit drug and substance abuse bong lung: regular smokers of cannabis show relatively distinctive histologic changes that predispose to pneumothorax cannabis use and risk of lung cancer: a case-control study exogenous lipid pneumonia related to smoking weed oil following cadaveric renal transplantation a review of the respiratory effects of smoking cocaine medical complications of cocaine abuse blackened bronchoalveolar lavage fluid in crack smokers -a preliminary study pulmonary histopathology in cocaine abusers pulmonary complications of crack cocaine: a comprehensive review cocaine-induced churg-strauss vasculitis pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization crack lung: an acute pulmonary syndrome with a spectrum of clinical and histopathologic findings effects of 'crack' cocaine on pulmonary alveolar permeability pulmonary hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature cellulose granulomas in the lungs of a cocaine sniffer intravenous injection of talc-containing drugs intended for oral use. a cause of pulmonary granulomatosis and pulmonary hypertension autopsy findings in drug addicts pulmonary 'mainline' granulomatosis: talcosis of intravenous methadone abuse self induced pulmonary granulomatosis. a consequence of intravenous injection of drugs intended for oral use the pulmonary vascular lesions of intravenous drug abuse microcrystalline cellulose pulmonary embolism and granulomatosis talc granulomatosis: laboratory findings similar to sarcoidosis postmortem findings of pulmonary lesions of older datum in intravenous drug addicts. a forensic-pathologic study recreational use of aminorex and pulmonary hypertension fatal drug reactions among medical inpatients drug-related hospital admissions: a review of australian studies published - incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies (see comments) the quality in australian health care study adverse reactions to drugs drug-induced pulmonary disease -an update an algorithm for the operational assessment of adverse drug reactions. ii. demonstration of reproducibility and validity aspirin idiosyncrasy and tolerance aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks; diagnostic and physiopathological implications mechanism of aspirin sensitivity bronchiolitis and bronchitis in connective tissue disease. a possible relationship to the use of penicillamine bronchiolitis obliterans in a patient with localized scleroderma treated with d-penicillamine constrictive bronchiolitis obliterans following gold therapy for psoriatic arthritis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease progressive airway obliteration in adults and its association with rheumatoid disease drug-induced infiltrative lung disease drug-induced and iatrogenic infiltrative lung disease interstitial lung disease associated with drug therapy pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning interstitial pulmonary fibrosis following busulfan therapy busulphan lung report of a case and review of the literature busulfan lung: report of two cases and review of the literature a -year-old man with pulmonary infiltrates after a bone marrow transplantation -busulfan pneumonitis drug-induced pulmonary disease lung parenchymal injury induced by bleomycin pathology of high dose intermittent cyclophosphamide therapy cyclophosphamide pneumonitis pulmonary histopathologic changes associated with melphalan therapy melphalan-induced pulmonary interstitial fibrosis potentiation of bleomycin-induced lung injury by exposure to % oxygen factors influencing postoperative morbidity and mortality in patients treated with bleomycin bleomycin therapy and anaesthesia risk factors of anesthesia and surgery in bleomycin-treated patients the pathogenesis of bleomycin-induced pulmonary damage in mice experimentally induced bleomycin sulfate toxicity origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity late bcnu lung -a light and ultrastructural study on the delayed effect of bcnu on the lung parenchyma statin-induced fibrotic nonspecific interstitial pneumonia bronchiolitis obliterans organizing pneumonia during treatment with acebutolol and amiodarone methotrexate-induced pneumonitis (baltimore) bronchoalveolar lavage findings suggest an immunologic disorder pneumonitis complicating low-dose methotrexate therapy for rheumatoid arthritis -discrepancies between lung biopsy and bronchoalveolar lavage findings methotrexate pneumonitis: review of the literature and histopathological findings in nine patients the pulmonary toxicity of antineoplastic agents relationship of gold and penicillamine therapy to diffuse interstitial lung disease desquamative interstitial pneumonia following long-term nitrofurantoin therapy two unusual pathological reactions to nitrofurantoin: case reports bronchiolitis obliterans organising pneumonia associated with the use of nitrofurantoin amiodarone lung toxicity: a human and experimental study effect of amiodarone on the lung shown by polarized light microscopy fine structural alterations in the lungs of iprindole-treated rats effects of chlorphentermine on the rat lung amiodarone-induced hypersensitivity pneumonitis. evidence of an immunological cell-mediated mechanism amiodarone pneumonitis: three further cases with a review of published reports amiodarone-associated pulmonary toxicity. a clinical and pathologic study of eleven cases amiodarone lung: pathologic findings in clinically toxic patients amiodarone-induced bronchiolitis obliterans organizing pneumonia (boop) amiodarone-related pulmonary mass and unique membranous glomerulonephritis in a patient with valvular heart disease: diagnostic pitfall and new findings nodular amiodarone lung disease amiodarone pneumonitis: no safe dose amiodarone pneumonitis: no safe dose susceptibility to amiodarone-induced pulmonary toxicity: relationship to the uptake of amiodarone by isolated lung cells acute amiodarone-induced pulmonary toxicity following lung resection pulmonary alveolar proteinosis developing from desquamative interstitial pneumonia in long term toxicity studies of iprindole in the rat eosinophilic lung diseases mesalazine-induced eosinophilic pneumonia chronic eosinophilic pneumonia after radiation therapy for breast cancer churg-strauss syndrome in patients receiving montelukast as treatment for asthma inhaled corticosteroids and churg-strauss syndrome: a report of five cases pulmonary migratory infiltrates and pachypleuritis in a patient with crohn's disease immunodeficiency virus-infected patients receiving antiretroviral therapy ciprofloxacin-induced acute interstitial pneumonitis pulmonary granulomas after tumour necrosis factor alpha antagonist therapy pulmonary sarcoidosis developing during infliximab therapy sarcoid-like granulomatous disease following etanercept treatment for ra pneumonia due to liquid paraffin: with chemical analysis a case of chronic paraffin pneumonitis liquid paraffin pneumonia -with chemical analysis and electron microscopy paraffinoma confirmed by infrared spectrophotometry foreign body granulomata of the lungs due to liquid paraffin exogenous lipoid pneumonia due to nasal application of petroleum jelly reaction of human lungs to aspirated animal fat (ghee): a clinicopathological study clinical reactions following bronchography the reaction of pulmonary tissue to lipiodol experimental study of bronchographic media on lung a method for the identification of lipiodol in tissue sections of lungs, lipids, and lollipops bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration pulmonary disease associated with l-tryptophan-induced eosinophilic myalgia syndrome. clinical and pathologic features a case of the eosinophilia-myalgia syndrome associated with use of an l-tryptophan product an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use l-tryptophan and the eosinophiliamyalgia syndrome: pathologic findings in eight patients histopathologic features of the l-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome -(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome immunemediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by l-tryptophan pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome tryptophan-induced lung disease -an immunophenotypic, immunofluorescent, and electron microscopic study bronchiolitis obliterans organizing pneumonia associated with massive l-tryptophan ingestion pulmonary disease complicating intermittent therapy with methotrexate case records of the massachusetts general hospital. a -year-old man with increasing dyspnea, dry cough, and fever after chemotherapy for lymphoma interferon-alpha therapy associated with the development of sarcoidosis proliferation and differentiation in mammalian airway epithelium sarcoid-like pulmonary disorder in human review of the literature severe barium sulfate aspiration into the lung: clinical presentation, prognosis and therapy aminorex and the pulmonary circulation pulmonary hypertension and fenfluramine irreversible pulmonary hypertension after treatment with fenfluramine dietary pulmonary hypertension appetite-suppressant drugs and the risk of primary pulmonary hypertension fatal pulmonary hypertension associated with short-term use of fenfluramine and phentermine autopsy findings of heart and lungs in a patient with primary pulmonary hypertension associated with use of fenfluramine and phentermine pulmonary veno-occlusive disease following therapy for malignant neoplasms pulmonary veno-occlusive disease in an adult following bone marrow transplantation: case report and review of the literature indomethacin in the treatment of premature labor. effects on the fetal ductus arteriosus prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application roles and mechanisms of human immunodeficiency virus protease inhibitor ritonavir and other anti-human immunodeficiency virus drugs in endothelial dysfunction of porcine pulmonary arteries and human pulmonary artery endothelial cells pulmonary embolism after intravenous immunoglobulin fat embolism in infancy after intravenous fat infusions pulmonary fat accumulation after intralipid infusion in the preterm infant intralipid microemboli the pathogenesis of fat embolism pulmonary lipid emboli in association with long-term hyperalimentation the impact of intravenous fat emulsion administration in acute lung injury microvascular pulmonary emboli secondary to precipitated crystals in a patient receiving total parenteral nutrition -a case report and description of the high-resolution ct findings pulmonary complications following lymphangiography with a note on technique changes in pulmonary function due to lymphangiography pulmonary complications of lymphangiography respiratory distress syndrome from lymphangiography contrast medium spallation and migration of silicone from blood-pump tubing in patients on hemodialysis acute pneumonitis after subcutaneous injections of silicone in transsexual men pulmonary granulomas secondary to embolic prosthetic valve material pulmonary teflon granulomas following periurethral teflon injection for urinary incontinence acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis isobutyl- -cyanoacrylate pulmonary emboli associated with occlusive embolotherapy of cerebral arteriovenous malformations hemorragie alveolaire diffuse secondaire a l'utilisation d'anticoagulants oraux diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome intrapulmonary hemorrhage with anemia after lymphangiography alveolar hemorrhage as a complication of treatment with abciximab d-penicillamine induced goodpasture's syndrome in wilson's disease drugs and the pleura pleuropulmonary changes induced by ergoline drugs pleuropulmonary disease as a side-effect of treatment with bromcriptine pleuropulmonary disease due to pergolide use for restless legs syndrome fibrosis of the lung following roentgen-ray treatments for tumor radiation reaction in the lung radiation pneumonitis: experimental and pathologic observations radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors the pathogenesis of radiationinduced lung damage radiation pneumonitis: a review adult respiratory distress syndrome after limited thoracic radiotherapy migratory bronchiolitis obliterans organizing pneumonia after unilateral radiation therapy for breast carcinoma migratory organizing pneumonitis 'primed' by radiation therapy hamman-rich syndrome 'primed' by radiation? recall' pneumonitis: adriamycin potentiation of radiation pneumonitis in two children recall lung pneumonitis due to carmustine after radiotherapy pulmonary radiation injury mortality from cancer and other causes after radiotherapy for ankylosing spondylitis increased risk of lung cancer after breast cancer radiation therapy in cigarette smokers aortotracheal fistula secondary to bacterial aortitis respirator lung -a misnomer pathology of adult respiratory distress syndrome pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project lung injury caused by mechanical ventilation ventilator-induced lung injury oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes in vitro damage of rat lungs by oxygen metabolites intercellular adhesion molecule- contributes to pulmonary oxygen toxicity in mice -role of leukocytes revised intercellular adhesion molecule- expression on the alveolar epithelium and its modification by hyperoxia normobaric oxygen toxicity of the lung oxygen pneumonitis in man pathology of pulmonary oxygen toxicity diffuse alveolar damage -the role of oxygen, shock and related factors diffuse interstitial pulmonary fibrosis. pulmonary fibrosis in mice induced by treatment with butylated hydroxytoluene and oxygen potentiation of diffuse lung damage by oxygen: determining values resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung ultrastructural observations on the development of the alveolar lesions extracorporeal membrane oxygenation for adult respiratory failure pulmonary pathology of patients treated with partial liquid ventilation disruption of the cd -cd ligand system prevents an oxygen-induced respiratory distress syndrome pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. ii ultrastructural and morphometric studies diffuse alveolar damage, respiratory failure and blood transfusion pulmonary injury -secondary to extracorporeal circulation fine structural changes in the lungs following cardiopulmonary bypass complement and the damaging effects of cardiopulmonary bypass acute lung injury during cardiopulmonary bypass: are the neutrophils responsible? inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies the pleuropulmonary manifestations of the postcardiac injury syndrome the postmyocardial infarction syndrome. the nonspecificity of the pulmonary manifestations the postcardiac injury syndromes antiheart antibodies following open heart surgery: incidence and correlation with postpericardiotomy syndrome analysis of the factors associated with radiofrequency ablation-induced pneumothorax irreversible intrapulmonary vascular changes after pulmonary vein stenosis complicating catheter ablation for atrial fibrillation pulmonary venous stenosis after treatment for atrial fibrillation right sided infective endocarditis as a consequence of flow directed pulmonary artery catheterisation complications and consequences of endotracheal intubation and tracheotomy. a prospective study of critically ill adult patients pathologic changes of the trachea after percutaneous dilatational tracheotomy pseudomonas aeruginosa respiratory tract infections in patients receiving mechanical ventilation cardiac arrhythmias resulting from tracheal suctioning obstructive fibrinous tracheal pseudomembrane -a potentially fatal complication of tracheal intubation -h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial tracheobronchomalacia in children necrotizing sialometaplasia (adenometaplasia) of the trachea severe complications of bronchoscopy the postpneumonectomy state evaluation of post-pneumonectomy space by computed tomography the postpneumonectomy space: factors influencing its obliteration hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice severe airway obstruction caused by mediastinal displacement after right pneumonectomy in a child. a case report postpneumonectomy syndrome: diagnosis, management, and results treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis postpneumonectomy syndrome: another twist acute lung injury and acute respiratory distress syndrome after pulmonary resection the mortality from acute respiratory distress syndrome after pulmonary resection is reducing: a -year single institutional experience prevalence and mortality of acute lung injury and ards after lung resection lung injury following pulmonary resection in the isolated, blood-perfused rat lung the pathogenesis of lung injury following pulmonary resection it is estimated that % of all hospital admissions are due to effects of therapeutic drugs, that - % of inpatients experience a drug reaction and that % of deaths in hospital may be related to drug therapy. [ ] [ ] [ ] [ ] the lungs are often involved in these adverse reactions. the mechanism of an adverse drug reaction may be based on:• overdosage: toxicity linked to excess dose, or impaired excretion, one classification of adverse drug reactions is that based upon the type of drug (box . . ). this is not adopted here but in passing it is worth noting that pharmacists are generally very helpful in supplying details of adverse reactions to specific drugs. alternatively, information on the long list of potentially pneumotoxic drugs may be obtained at http://www.pneumotox.com. a useful scheme for chapter key: cord- -lrz bdx authors: nayyar, gaurvika m. l.; attaran, amir; clark, john p.; culzoni, m. julia; fernandez, facundo m.; herrington, james e.; kendall, megan; newton, paul n.; breman, joel g. title: responding to the pandemic of falsified medicines date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: lrz bdx over the past decade, the number of countries reporting falsified (fake, spurious/falsely labeled/counterfeit) medicines and the types and quantities of fraudulent drugs being distributed have increased greatly. the obstacles in combating falsified pharmaceuticals include ) lack of consensus on definitions, ) paucity of reliable and scalable technology to detect fakes before they reach patients, ) poor global and national leadership and accountability systems for combating this scourge, and ) deficient manufacturing and regulatory challenges, especially in china and india where fake products often originate. the major needs to improve the quality of the world's medicines fall into three main areas: ) research to develop and compare accurate and affordable tools to identify high-quality drugs at all levels of distribution; ) an international convention and national legislation to facilitate production and utilization of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; and ) a highly qualified, well-supported international science and public health organization that will establish standards, drug-quality surveillance, and training programs like the u.s. food and drug administration. such leadership would give authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines. the organization would also advocate strongly for including targets for achieving good quality medicines in the united nations millennium development goals and sustainable development goals. malaria is a devastating illness, particularly to young children and pregnant women in tropical countries. a recent review reported that the active pharmaceutical ingredient (api) was absent in over one-third of close to , antimalarial drug samples tested from pharmacies in seven southeast asian and sub-saharan african countries ; over % of the alleged artemisinin-containing drugs were falsified, outright fakes. a wide variety of falsified brand name and generic medicines and even falsified raw ingredients for several essential pharmaceuticals have been found in rich and poor countries. [ ] [ ] [ ] [ ] [ ] [ ] such drugs are often used for acutely ill patients, many of whom would die or suffer prolonged illness without proper treatment. in addition to patients' loss of confidence in the health-care delivery system, microbial resistance to the drug may develop and spread if medicines contain subtherapeutic doses or no api. the increasing global scientific and public awareness and epidemic proportions of the spreading problem are reflected in the number of articles on "fake drugs" cited in pubmed: until recently, there were a paucity of reports from pharmaceutical companies on the type and quantity of drugs that were fraudulently compounded or transferred by criminals. data are emerging from the pharmaceutical security institute (psi), a not-for-profit membership organization of pharmaceutical security directors, indicating that a large number of companies, products, and countries are targeted. for instance, since , pfizer pharmaceuticals (pfizer global security, new york, ny) has identified a rapidly increasing number of falsified products, countries reporting falsified drugs, and breaches of the legitimate supply chain national entry points (table ) ; the increases have been from % to over %. of pfizer products, those for erectile dysfunction are most frequently falsified ; other such products target patients with alzheimer's disease, cancer, high cholesterol, hypertension, malaria, and anxiety disorder. facilities where fake drugs were made or compounded were discovered with moldy walls, dirty equipment, and infested with rodents and insects ( figure ). falsifiers have created products that are visually indistinguishable from the genuine product, clearly demonstrating criminal intent to deceive. this increasingly recognized problem on virtually all continents is a pandemic defined as "an epidemic occurring over a very wide area, crossing international boundaries, and usually affecting a large number of people." definitions despite increasing awareness of the fraudulent drug epidemic, efforts to quantify and stop this peril have been stymied by multiple obstacles, not the least of which is agreement on definitions. , , poor quality drugs include substandard/ spurious/falsely labeled/falsified/counterfeit (ssffc) medical products. falsified (also commonly called fake or counterfeit products are intentionally and fraudulently produced and contain no api, the incorrect dose of the api, or the incorrect api. substandard medicines are caused by unintentional or negligent errors of manufacturing or by degradation after manufacturing resulting in insufficient api, poor dissolution properties, or degradation products. the nomenclature used by the world health organization (who), the world trade organization, the united nations (u.n.) office on drugs and crime, interpol, and others can be confusing; hence, we are using terms agreed upon by who. there are also properly manufactured medicines that are unlawful for reasons apart from their quality. these can be unregistered with company branded or generic medicines that, for reasons of theft or accidental or intentional diversion, do not have the legally required marketing authorization of the country's regulators to be imported or sold there, and medicines that infringe the trademark of a legal product. relatively little is known about medicines that have expired and are repackaged with a new date; these topics along with diverted products are beyond the realm of this article. this article focuses mainly on medical and public health considerations of falsified medicines that are particularly widespread in low-and middle-income countries. [ ] [ ] [ ] although there are increasing reports of detection of a variety of fake drugs from around the world, paradoxically, there are virtually no reports from middle-or high-income countries quantifying the state of poor quality medicines, only anecdotal case citations. governments have been hampered by a confusing array of expensive detection technologies. few functional national regulatory authorities exist in low-income nations that lack trained staff and suitably equipped laboratories to test drug quality centrally or in peripheral pharmacies or markets. , furthermore, the variability or absence of national and international criminal statutes, lack of an international agreement against trafficking of poor quality medicines, and inadequate punishments for convicted offenders reflect the weak legal framework for confronting drug fraud. one of the biggest obstacles in provision of quality-assured pharmaceuticals is the lack of effective manufacturing, regulatory, and quality processing in india and china. in , global public health agencies including providers, foundations, and research institutions contributed to developing an advocacy campaign to address falsified medicines, particularly in china. this campaign called fight the fakes is a step toward raising awareness about the problem, but legal action has to follow along with more public and political awareness. the u.s. institute of medicine (iom) has published a report "countering the problem of falsified and substandard drugs." the iom recommendations to "stem the global trade" in such products are laudable in advising that the u.s. food and drug administration (fda), the national institute of standards and technology, and other u.s. and international pharmaceutical and financing agencies be more actively involved in setting standards and financing improvements; yet this report falls far short of making a strong call for standardized, agreed-upon quality assessment technologies; an international law convention; and a more activist, internationally recognized lead organization, all three of which are essential for stopping the many health threats of fake drugs. global leadership to date has devolved in parts to the who, the u.n. office on drugs and crime, and interpol, each with diverse missions, responsibilities, limited authorities, and their own collaborations, funding networks, cultures, and languages. no organization is leading assertively. of the three areas listed, an international convention and improved national regulations are likely to have the most enduring value in concert with effective leadership and other innovations. the focus of all actions tied to drug quality must be on public and individual health, and strengthening national capacities to improve the health of their citizens. detection methods and technology. a major hindrance to understanding the types, names, extent, and amount of poor quality drugs nationally and globally has been ) the lack of agreed-upon field survey approaches and ) available lowcost tools to detect and classify bad drugs quickly at points of entry into countries, at public and private pharmacies, and in health units. in , the who published draft guidelines for surveys of medicine quality that are currently being revised. two or more levels of drug quality tests exist: ) methods useable in the field that are quick, inexpensive, and easy to use and teach; these methods are targeted mainly to examine packaging and detect drug contents and ) technologies requiring a laboratory equipped for exhaustive chemical analysis. these approaches are summarized in table , deriving from the iom report and a recent analysis by green and others from the centers for disease control and prevention reference laboratory. within each method there are numerous tools and prototypes being used and new ones tested. current technologies for field use rely on visual packaging inspection, lot number reporting via mobile phones, thin-layer chromatography, colorimetric tests, and simplified spectroscopic methods. gas and liquid chromatography and mass spectrometry are some of the more advanced and complex techniques for investigating drug quality in central laboratories. qualitative or semiquantitative tests for an api are not substitutes for proper manufacturing control, dissolution studies, pharmacokinetics equivalence, and supply chain integrity. a very promising recent development has been the u.s. pharmacopeia promoting the quality of medicines (pqm) program in several african, asian, and latin american countries using the "minilab" (global pharma health fund e.v., giessen, germany). [ ] [ ] [ ] this training program supported by the u.s. agency for international development (usaid) and the president's malaria initiative (pmi) has trained several hundred persons in rapid chemical analysis of drugs taken from public and private pharmacy stocks. a major reference training center has recently been opened in accra, ghana, with usaid and u.s. pharmacopeia support as a referral testing and regional training center. important also is the development of the counterfeit detection (cd)- (us food and drug administration, forensic chemistry center, cincinnati, oh), a promising handheld electronic device for peripheral use that detects fake packaging at point of sale with images and videos of the suspect samples. , the fda, skoll foundation, and other partners are supporting expansion of testing and use of this device. we recommend that a precertification of essential diagnostics, drugs, and vaccines should be required for specific regional and global control, elimination, and eradication programs and campaigns. more information is needed to confirm that precertification of products is occurring for the pmi and the global fund to fight aids, tuberculosis and malaria, and products purchased by u.n. international children's emergency fund (unicef) and who. essential drugs designated by who should also be targeted for special vigilance by quality assurance mechanisms. no independent agency has inventoried and performed comparative quality assessments of these packaging and drug-testing devices and made recommendations to countries for their use. objective comparisons are needed of the diversity of field methods in terms of accuracy, reliability, costs of equipment and supplies, level of training needed, ease of use, spare part availability, and maintenance requirements. simplified standard survey protocols and methods for sampling drugs at country entry points (seaports, airports, and roads); at major pharmacy depots; in health units (public and private hospitals and clinics); and at more peripheral distribution sites (district and village pharmacies and individual vendors) are also needed. low-cost, portable detection tools would empower pharmaceutical inspectors in numerous countries that have oversight of the medicine supply. results would be available promptly rather than delayed when samples are sent to national or international laboratories as occurs now; lamentably, intervals of several years have occurred from the time specimens were collected to the time the results were available to those needing to take action. ideally, central reference laboratories vetted by who, fda, or another agency would back up spot checks and random sampling of pharmaceuticals at the periphery. good quality medicines by law. falsified medicines are ultimately a problem that impacts public health. the solution needs to reflect various incentives, either via financial gain, avoidance of punishment or both. a multi-sectorial effort is essential for taking into account how this illegal market is interwoven with world trade agreements, business models, and associated legal ramifications. globalization has enlarged the international trade in medicines. for example, india exports over us$ . billion in pharmaceuticals, which are among their most important exports. as of , % of drugs and % of apis for drugs in the united states are imported from foreign countries. an international law convention against substandard and falsified medicines would address both regulatory and criminal international governance challenges simultaneously through technical, legal, and financial mechanisms. how would the convention work and what national benefits would it bring? a convention would provide four legal underpinnings that do not exist, that together would advance patient safety and access to quality medicines. first, a convention would define the various sorts of wrongful medicines accurately and thereby avoid misunderstandings caused by today's problematic or vague terminology (e.g., where countries seized good quality generic medicines as "counterfeit"). second, a convention would promote the requirement that signatory countries enact national laws to designate wrongful actssuch as the intentional manufacture, trafficking, or selling of falsified medicines-as criminal offences, with attendant obligations to alert health-care workers and to prosecute or extradite the offenders to justice promptly. third, a convention would provide the legal and institutional framework for participating countries to agree, implement, and evolve convergent standards of medicine regulation, so as to reduce poor quality medicines in international trade. fourth, and for lower income countries particularly, a convention would contain mechanisms for financial and technical assistance, and, to join local and regional networks. these actions would help build national and regional medicine regulatory authorities (mras) to a point where patients' access to quality medicines is protected. some have said that establishing recommended codes of practice that are nonbinding (soft law) are better than international norms and regulations that are binding (hard law). we disagree with soft law in regard to controlling the current fake-drug pandemic. there are precedents for using international law in this way. a treaty that internationally criminalizes counterfeit banknotes provides an analogy for falsified medicines. in the health field, there are treaties specifically addressing the illicit traffic of certain narcotic drugs and treaties to prevent harm-particularly, the framework convention on tobacco control and its associated protocols to stanch illicit trade. that convention has brought over us$ million new funding to global tobacco-control efforts, demonstrating that international law need not compete for resources, but can increase them. the u.n. office on drugs and crime has been developing "draft model legislative provisions on fraudulent medical products" for several years but there has been no agreement on final text; the focus appears to be on criminal and judicial issues. challenges ahead. information is accruing that large quantities of falsified drugs are being manufactured in asian countries. china and india are two of the largest producers of good quality drugs and vaccines, many of which are purchased or funded by the usaid; unicef; global fund to fight aids, tuberculosis and malaria; who; and other organizations, charities, and national agencies for global disease control and eradication programs. however, according to the world customs organization, in , % and % of unlawful drugs of all sorts confiscated worldwide were manufactured in india and china, respectively. the circuitous travel itineraries of fake medicines have been traced across continents, such that the unsuspecting recipient countries assume a bona fide origin. a particularly heinous example is that of multiples instances of the production, marketing, and international travel of falsified bevacizumab (avastin ), a cancer medicine; the fake drug closely matched the appearance of the real medicine, but tests indicated salt, starch, and various cleaning solvents instead of the active ingredient with resulting endophthalmitis. the internet has opened up an unregulated opportunity for criminals to promote and sell fake drugs to unsuspecting vulnerable populations, often the aged and others seeking convenience and low cost. a recent survey of over , online pharmacies found that % operated outside legal regulations and a large percentage closed operations within years of operation. , the fda and other organizations participate with interpol in annual international actions (operation pangea) to shutdown illegal pharmacy websites selling potentially counterfeit and illegal medical products. more than , such illegal websites were closed during one week in with seizure of us$ . million of pharmaceuticals worldwide. leadership, collaboration, and national strengthening. arguably, the major obstacle to solving the problem of poor quality medicines has been the lack of a clearly identified lead organization with a plan of action developed in concert with countries, pharmaceutical companies (multinational corporations and innovator/biotechnology enterprises), and national and international agencies-and a sense of urgency to implement the plan with resources and partners-including pharmaceutical companies in low-income countries. who has estimated that % of countries have inadequate medicines regulation authorities (mras) or none at all. moreover, who has found that % of african mras lack the capacity to undertake medicine regulatory functions and therefore cannot guarantee the quality, efficacy, and safety of medicines, , the new partnership for africa's development has found that there is either limited or declining government funding for mras in the east african community partner states. many have looked to who for this leadership, given its successful implementation of the public health treaty on tobacco control. however, some argue that the u.n. system, including the who, is poorly suited to be in a leadership role because of sparse technical expertise in products, manufacturing, and quality systems. u.n. agencies are beholden to member states and cannot regulate or enforce anything easily, especially, in india and china. in this regard, who could serve the role of a partner rather than a leader. the recently revitalized rapid alert program at who has begun to "track and trace" poor quality drugs as reported voluntarily by member countries. rapid alert notices are published periodically by who indicating the fake drug type, lot number, quantity of product, and place detected. strong action by countries can stem the tide as shown in rwanda and cambodia, although unique situations and major multi-sectorial engagements exist in these countries. one solution is creation of regional harmonization networks, addressing some elements of drug registration tied to regional economic communities; the african medicines registration harmonization initiative is one example of such a network. the u.n. office on drugs and crime (unodc) has also made recent attempts at facilitating international cooperation against falsified medicines. one proposal has been a trilateral coalition of the unodc, who, and interpol. still, active and transparent support from the fda, drug companies, individual countries, and other partners will be needed; the fda may be the most qualified as a leadership organization based on their technical expertise and global influence. mechanisms for training technical staff, regulating products, improving manufacturing practices, and stopping criminal production are needed to assure a good supply of medicines. given that the problem of substandard and falsified medicines should be approached primarily from a public health and equity perspective, it is important that the negotiations on the way forward be led by the ministries of health along with the ministers of finance and trade, while respecting legitimate intellectual property rights. could and should who be the lead organization in curbing the spreading epidemic of falsified pharmaceuticals? who's ability to take more assertive action is strengthened by the revised international health regulations. who's director general can convene emergency committees in response to public health emergencies as has been done recently for the influenza a (h n ) pandemic in , the middle east respiratory syndrome (mers-cov) in , the polio crises in , and the ebola epidemic in - . illicit drug trafficking is an emergency. the drug quality and security act, signed into law by president obama in , outlines steps to build an electronic system to identify and trace certain prescription drugs in the united states. the results of this system are awaited. finally, the millennium development goals (mdgs), under revision, should include measurable objectives for good quality drugs. this will encourage national establishment of baseline status and achievable targets, particularly for essential drugs. establishment of mdg targets and sustainable development goals (sdgs) will help greatly to solve the poor quality drug epidemic by application of available technology and good pharmaceutical vigilance and governance. one incentive that would transform the current system is applying a "universal quality standard" to drug products. for example, if india allows a substandard manufacturer to sell products in africa, the fda could ban import of products from india. although difficult to develop and implement, a combination of incentives and penalties driven at the political and economic levels is needed. the major urgent needs to improve the quality of the world's medicines fall into three main areas: ) research to develop and compare the most accurate and affordable tools to identify high-quality drugs at point of sale and deployment of the best methods; ) an international convention and national legislation to facilitate production and use of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; ) designation of a highly qualified, well-supported international organization, possibly the fda or who, that will establish standards, training programs, drug quality surveillance, and authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines. the organization would also advocate strongly for including targets for achieving good quality medicines in the mdgs and sdgs including certification of pharmaceutical products entering countries that request such services, and participate in global disease control and elimination programs. poorquality antimalarial drugs in southeast asia and sub-saharan africa substandard and counterfeit medicines: a systematic review of the literature the global counterfeit drug trade: patient safety and public health risks substandard/spurious/ falsely-labeled/falsified/counterfeit medical products analysis of counterfeit cialis tablets using raman microscopy and multivariate curve resolution confirmed glyburide poisoning from ingestion of "street valium characterization and identification of suspected counterfeit miltefosine capsules empirical analysis of counterfeit drug penetration in global legitimate medicine supply chains: a descriptive assessment internetordered viagra (slidenafil citrate) is rarely genuine a dictionary of epidemiology new definition for "substandard medicines how to achieve international action on falsified and substandard medicines impact of poorquality medicines in the 'developing' world united states pharmacopeia ten global health organizations united in a worldwide campaign to protect patients from fakes countering the problem of falsified and substandard drugs improving global health governance to combat counterfeit drugs: a proposal for a unodc-who-interpol trilateral mechanism guidelines for field surveys of the quality of medicines: a proposal recommendations on the content of a survey protocol for surveys of the quality of medicines integration of novel low-cost colorometric and visual fluorescent techniques for rapid identification of falsified artemether-lumefantrine and other drugs in resource poor areas survey of the quality of selected antimalarial medicines circulating in madagascar, senegal, and uganda the global pandemic of falsified medicines: laboratory and field innovations policies and perspectives evaluation of a new handheld instrument for the detection of counterfeit artesunate by visual florescence comparison fda facts: fda's counterfeit detection device cd- india aims to clock usd . bn pharma exports fy import of human drugs and human drug components global health and the law follow the money: how the billions of dollars that flow from smokers in poor nations to companies in rich nations greatly exceed funding for global tobacco control and what might be done about it counterfeiting, a global spread, a global threat counterfeit bevacizumab and endophthalmitis progress report for state and federal regulators medicines counterfeiting is a complex problem: a review of key challenges across the supply chain fda takes action against thousands of illegal internet pharmacies. food and drug administration news release effective medicines regulation: ensuring safety, efficacy and quality availability of drug regulatory and quality assurance elements in member states of the who african region the african medicines regulatory harmonization initiative: rationale and benefits international medical products anti-counterfeiting taskforce (impact) combating substandard and falsified medicines: a view from rwanda quality of antimalarials at the epicenter of antimalarial drug resistance: results from an overt and mystery client survey in cambodia why the mdgs need good governance in pharmaceutical systems to promote global health this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- - dq oe authors: greaves, peter title: respiratory tract date: - - journal: histopathology of preclinical toxicity studies doi: . /b - - / - sha: doc_id: cord_uid: dq oe the chapter describes different aspects of the respiratory tract. in preclinical safety studies, pathologies of the respiratory system can be a result of an intercurrent disease or can be induced by systemically administered drugs. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and technologies required to administer a drug. a complex technology is developed to support the assessment of adverse effects of inhaled substances in rodent and nonrodent species, and the extrapolation of experimental findings to humans. the nasal chambers are the structures that are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. in rodents, the relatively small size of the nose and nasal sinuses facilitates a histological examination. findings show that infectious agents cause inflammation in the nose and nasal sinuses, and this may be associated with inflammation in the conjunctiva, the middle ear, and the oral cavity. it has been observed that a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium, and to a lesser extent in respiratory and glandular epithelial cells. by far and away the most important pulmonary diseases in humans are related to the smoking of tobacco. however, occupational lung diseases caused by inhalation of industrial chemicals, particulate matter and antigens, are also important causes of morbidity and mortality. for this reason, considerable effort has been directed to the examination of airborne pollutants over recent years, including study of their effects in laboratory animals when administered by the inhalation route. extensive study has shown that a complex array of defensive mechanisms protects the lung against the adverse effects of airborne substances and pathogenic organisms. aerodynamic factors prevent access of particles larger than |im diameter for these are deposited on the walls of the nasal passages. particles measuring between and (im diameter tend to be trapped by the mucus-covered ciliated epithelium lining the bronchial tree and removed by mucociliary transport aided by the cough reflex. smaller particles may reach the alveoli where they are ingested and transported by pulmonary macrophages.^ considerations of airborne delivery to the lungs are also important in the development of therapies to be administered via the respiratory tract. whilst the inhalation route has been used for many years for volatile anaesthetic gases, the respiratory tract is increasingly being employed for delivery of therapy in not only for asthma and other lung diseases but also as a means of systemic delivery of polypeptides such as insulin. in contrast to the adverse pulmonary effects of cigarette smoke and industrial pollutants, therapeutic agents remain a relatively minor cause of pulmonary toxicity in humans although actual incidence is difficult to ascertain. however, drug-induced pulmonary disease appears to be an increasingly frequent clinical problem and the drugs associated with parenchymal pulmonary injury in humans continue to increase.^ although it is difficult to assess in the context of the underlying disease process, it has been suggested that about % of patients receiving wellestablished anticancer drugs develop various forms of pulmonary toxicity some novel antineoplastic therapies may have a similar liability.^ drug-induced toxicity usually occurs after exposure of lung tissue via the circulation to parent drug or metabolites, although increasingly the adverse effects of direct administration of drugs to the lungs needs consideration in preclinical studies. in patients a number of drugs have been associated with pulmonary toxicity which can occur through different mechanisms and take different forms.^"^ through their specific pharmacological action drugs can produce excessive effects on bronchial calibre or pulmonary function. drugs mediate allergic reactions in the bronchi or lungs. they may also produce a variety of obscure, diffuse pulmonary alveolar conditions including a pulmonary syndrome resembling systemic lupus er ^hematosis. as the respiratory tract is a major route by which microorganisms gain entry into the body, opportunistic pulmonary infections with bacteria, viruses, fungi or protozoa are consequences of immunosuppression or broad-spectrum antibacterial therapy. as in other organs, drugs that disturb coagulation may precipitate pulmonary thromboembolism or haemorrhage. localized lung lesions also result from accidental, diagnostic or therapeutic inhalation of xenobiotics. mucociliary clearance is also sensitive to therapeutic agents that affect the secretion of mucus and fluid, ciliary activity and transport.^ treatment with antacids or histamine h blockers can also increase the risk of pneumonia developing in patients in intensive care units through increasing gastric ph, which leads to an overgrowth of gram-negative bacteria in the stomach and retrograde pharyngeal colonization.^ in preclinical safety studies, pathology of the respiratory system can be the result of intercurrent disease or be induced by drugs administered systemically. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and the technologies required to administer drug. the different anatomical and physiological characteristics of the airways also influence drug toxicity, disposition and metabolism. the development of drugs to be administered by inhalation or intranasal routes is particularly difficult because of the perceived risks of high local drug concentration in respiratory tissues and their use in potentially vulnerable patients with pulmonary disease.^ a complex technology has been developed to support the assessment of the adverse effects of inhaled substances in rodent and non-rodent species and the extrapolation of the experimental findings to humans.^'^ in order to administer drugs by inhalation, it is necessary to generate aerosols (suspensions of particles in a gas) with a well-defined composition, particle size and shape. they must be delivered to the respiratory tract of laboratory animals in a way that parallels the likely human exposure. in case of therapeutic agents, this should avoid non-respiratory pathways through the skin and food. when aerosols are inhaled, various fractions of the particles are deposited at different locations in the respiratory tract. site of deposition depends primarily on particle size, but variability in the sites of deposition occurs among different laboratory animal species and humans by virtue of the differences in the size and shape of the respiratory passages as well as breathing patterns. ^^ in addition, there are different types of inhalers used in human therapy to consider in the assessment, which can deliver different materials to the lungs, for example nebulizers, propellant-driven metered dose inhalers and dry powder inhalers for asthma treatment. ^^ the subsequent fate of inhaled particles depends not only on their size but also their shape, chemical nature, and solubility in body fluids. soluble substances are absorbed into the blood stream and are removed by the pulmonary circulation. they may also undergo metabolism by enzymes present in the cell populations of the respiratory tract and reactive metabolites may cause local pulmonary damage. insoluble, inert particles are removed primarily by the mucociliary transport system of the trachea and bronchi or through phagocytosis by macrophages. overload of the lung by even relatively inert, nonfibrous particles such as titanium dioxide or carbon black may impair alveolar macrophage-mediated particle clearance. ^^ this may lead in turn to accumulation of dusts over time with eventual fibrotic and tumorigenic responses, particularly in rats.^^ measurements of respiration rate, tidal volume, airway resistance, pulmonary gas exchange and the disposition of the inhaled substances have an important place in the evaluation of chemically induced lung damage in laboratory animals.^"^'^^ however, the key component of the evaluation of the adverse effects of inhaled substances is careful morphological assessment of the fixed tissues. even though there are novel and very sensitive physiological methods for the characterization of oedema following lung injury in rodents, light and electron microscopy of lung tissue provides vital qualitative evidence of the nature of injury. ^^ the nasal chambers are the structures which are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. although these chambers are not usually examined in great detail in conventional toxicity studies in which substances are administered orally or by parenteral routes, they are carefully examined histologically when drugs are administered by inhalation. study of nasopharyngeal silicone rubber casts has shown considerable species differences in the anatomy of this part of the airway. ^^"^^ relative to total nasal length, the nasopharynx is longest in rats and shortest in humans with the dog in an intermediate position. maxilloturbinates are relatively simple structures in man and non-human primates but highly complex in dogs and rodents. as a consequence, regional nasal airflow and disposition patterns vary considerably and this influences the distribution of lesions produced by inhaled xenobiotics in the nasal cavity.^^ however, comparison of the nasal cavity of rhesus monkey and humans using magnetic resonance imaging and nasal casts have shown that many similarities in structure exist in these species. ^^ the anterior nares are lined by stratified squamous epithelium. in other zones the sinuses are covered either by respiratory or olfactory epithelium with a zone of transitional epithelium at the junction between the two types. respiratory epithelium is similar to that found elsewhere in the respiratory passages being composed of ciliated cells, serous and mucous cells, brush cells, intermediate cells and progenitor basal cells. it represents a cellular system engaged in mucociliary clearance carrying surface secretions to the nasopharynx to be cleared by swallowing. although this epithelium is similar to that lining the other large airways, key differences are the particularly rich complement of secretory cells and the complex vasculature of the nose which can modulate capillary, arterial and venous blood flow through the mucosa.^^ mucins may be particularly important. it has been postulated that they not only have a physical protective function but also possess antioxidant properties by virtue of the scavenging behaviour of their high proportion of sugar groups. ^^ the proportion of the nose lined by olfactory mucosa is variable between species, being disposed over a much larger area in dogs and rodents than in primates. ^^ however, it is structurally similar in humans and rodents. it is located in more dorsal or posterior regions of the nasal passages out of the direct line of airflow during normal respiration. olfactory mucosa is a pseudostratified columnar epithelium composed of basal cells, sustentacular cells and sensory cells with mucus-secreting bowman's glands situated in the lamina propria. basal cells are composed of two distinct types, light and dark cells. the light type represents the primitive, stem cell population. sustentacular or supporting cells are non-ciliated, columnar cells possessing microvilli that extend into the overlying layer of mucus. cell bodies of olfactory sensory neurons are situated in the middle layer of the epithelium between sustentacular and basal cells. their dendritic processes extend above the epithelial surface to end in a ciliated expansion referred to as the olfactory vesicle that is believed to be the receptor of odour perception. olfactory axons extend from the cell body, penetrate the basement membrane in bundles to become surrounded by schwann cells and eventually join with the olfactory bulb. the olfactory system is of importance in toxicology for it can be selectively damaged by xenobiotics, including therapeutic agents, presumably as a result of its high metabolizing capacity. the superficial location of neural cells in the olfactory epithelium also provides a model system for the study of the effects of xenobiotics on neural cells. submucosal mucous glands have been well characterized in the rat, hamster and dog, where they are divided into lateral nasal glands and maxillary recess glands. these are both situated in the posterior parts of the nasal cavity and composed of mucus-secreting cells.^^"^^ immunocytochemical study using antisera raised against the major isoenzymes of rat hepatic microsomal cytochromes p induced by ( -napthoflavone, -methylcholanthrene, phenobarbitone and pregnenolone- -a-carbonitrile as well as nadph-cytochrome p reductase, epoxide hydrolase and glutathione s-transferases b, c and e, has shown their presence in rat nasal mucosal cells.^^ cyp a enzymes appear to be expressed at high levels in the respiratory tract mucosa.^^"^^ this suggests that the nasal mucosa not only has a capacity for metabolizing and activating xenobiotics by oxidation, but also for hydration and inactivation of potentially toxic epoxides and conjugating electrophilic, reactive metabolites with reduced glutathione. it has been shown that the distribution of immune-reactive enzymes is different in olfactory and respiratory mucosa.^^ xenobiotics can be metabolized within both olfactory and respiratory mucosa but the olfactory regions appear to possess greatest capability for oxidative metabolism. consequently, regional differences in nasal toxicity and tumour formation from inhaled materials may not only be a response to different water solubility and deposition patterns but also differences in the formation of reactive metabolites.^'* another feature of this metabolizing activity is that it can be induced by systemically administered xenobiotics and this can alter the distribution of enzyme activity in the nasal mucosa.^^ studies of the mouse olfactory mucosa have shown that whilst typical hepatic inducers of cyp a do not significantly change its expression in the mucosa, olfactory toxicants can alter the pattern of enzyme distribution.^^'^^ like many other tissues exposed to external environmental agents, the nasal mucosa possesses aggregates of lymphoid tissue in the underlying lamina propria. in rats these areas, characterized by follicles containing both t and b cell areas, are located in the ventral aspects of the lateral walls of the nasal airways at the opening of the nasopharyngeal duct.^^'^^ like the gutassociated lymphoid tissue, these nasal follicles have been shown in the rat to be covered by specialized epithelium with islands of cells with microvilli, socalled m or membranous cells. little is known of any toxicity occurring in this tissue despite its strategic position in the respiratory tract.^^ in rodents, the relatively small size of the nose and nasal sinuses facilitates histological examination. usually this area is sectioned transversely into several standardized blocks following decalcification.^^ there have been a number of detailed publications describing the histological preparation and assessment and recording of pathology of the rodent nasal cavity.^^"^^ careful standardized histological sections, careful recording of lesions with the use of diagrams of the rodent nasal cavity are useful in the assessment of lesions in the nasal cavity found in inhalation studies."*^ in larger species, particularly dogs and primates, sectioning and blocking is more complex. although dissection is required, a similar procedure following decalcification can be adopted. careful examination of haematoxylin and eosin stained sections remains paramount in the assessment of the nasal cavity, although special stains may be helpful. examination of cytokeratin expression in the respiratory mucosa has been used as a marker of epithelial differentiation in the respiratory tract>^ a test system that relates to the innervation of the nasal mucosa is that proposed by alarie.^^ the trigeminal nerve endings in the nasal mucosa of mice mediate the response to sensory irritants and this can be measured by a decrease in respiratory rate. it has been shown that a good correlation exists between the decrease in respiration rate in mice exposed to airborne chemicals and the nasal irritancy potential of the chemicals in humans.^^ this enables the detection of airborne sensory irritants and the prediction acceptable levels of exposure to the upper respiratory tract in people. microbial pathogens infectious agents cause inflammation in the nose and nasal sinuses and this may be associated with inflammation in the conjunctiva, middle ear and oral cavity. murine pathogens may cause alterations in the respiratory tract that can confound the assessment of changes induced by xenobiotics.^^ in rats, microbiological agents implicated in the development of rhinitis and sinusitis include corynebacterium kutscheri (pseudotuberculosis), streptococcus pneumonia, pasteurella pneumotropica, klebsiella pneumoniae, mycoplasma pulmonis and the sialodacryoadenitis virus or rat corona virus.^^ rats infected with the sialodacryoadenitis virus show inflammation and necrosis of the upper respiratory epithelium as well as damage to salivary and lachrymal glands. the sendai virus, a paramyxovirus, also has marked tropism for the respiratory tract, including the nasal cavity, and is associated with systemic effects that can compromise studies in laboratory rodents. occasionally, fungal infections of the airways with. aspergillus fumigatus are reported.^^ a variable that has been shown to influence the severity of the rhinitis produced by mycoplasma pulmonis is the strain of rat. following housing of lewis and fischer strains together to eliminate microbial and environmental differences it was shown that the lewis strain developed a more severe rhinitis following inoculation with mycoplasma pulmonis than fischer rats, although the reason for the difference was unclear."^^ rats exposed to ammonia, a common pollutant of the air in laboratory animal cages, have also been shown to develop lesions of the dorsal meatus, dorsal nasal septum and prominence of the turbinates.^^ these lesions are characterized histologically by swelling or mild degeneration of the epithelium. it appeared that ammonia exposure potentiated the acute inflammatory response of the nasal cavity to microbiological pathogens. a microorganism reported in the nasal cavity of rhesus monkeys employed in inhalation studies is the nematode of the genus anatrichosoma.^^ sections of this nematode are found in the squamous epithelium of the nasal vestibule and are associated with acanthosis and hyperkeratosis of the epithehum and a multifocal or diffuse granulomatous inflammation in the submucosa. administration of toxic or irritant substances to laboratory animals by the inhalation route produces degenerative, inflammatory and reactive changes in the nasal mucosa. the range of histological features is similar to those found in other mucosal surfaces damaged by other exogenous agents. whilst therapeutic agents administered by the inhalation route do not usually produce a severe degenerative or inflammatory responses in the nasal mucosa, at least at therapeutic doses, the simple categories proposed by hardisty and colleagues in recording of degenerative and reactive lesions following exposure to volatile chemicals are useful.'^^ categories suggested are: degeneration, regeneration, atrophy (postdegenerative), respiratory metaplasia and basal cell hyperplasia and inflammation. degeneration is usually the earliest morphological change characterized by loss of sensory and sustentacular cells resulting in a thinner mucosa. bowman's glands and nerve bundles, individual cell necrosis may be seen in more severe cases. regeneration is characterized by proliferation of basal cells associated with an epithelium that loses its regular structural features. post-degenerative atrophy usually follows severe damage and is characterized by loss of sensory and sustentacular cells. respiratory metaplasia is a process whereby the normal olfactory mucosa is replaced by pseudostratified epithelium of respiratory type often with cilia. basal cell hyperplasia represents a longer term effect where the proliferating basal cells form a distinct layer of cells below the respiratory epithelium. an example of the type and distribution of the degenerative and inflammatory conditions which can be induced by inhaled irritants is provided by the study in which swiss-webster mice were given various irritants by inhalation for periods of hours per day for days at concentrations that produced a % decrease in respiratory rate (alarie test). although the degree of histological changes varied with different agents, the changes were broadly similar in type and distribution.^^ most agents examined produced little or no alteration in the squamous mucosa lining the anterior part of the nose apart from some mild increase in thickness of the squamous layers. principal sites of damage were shown to be the anterior respiratory epithelium adjacent to the vestibule and the olfactory epithelium of the dorsal meatus. there was a distinct decrease in severity in posterior regions. histologically, the lesions in respiratory epithelium ranged from mild loss of cilia and small areas of epithelial exfoliation to frank erosion, ulceration and necrosis of the epithelium and underlying tissues including bone. variable polymorphonuclear cell infiltration was also found. in some cases, early squamous metaplasia developed on the free margins or the naso-maxillo-turbinates. changes to the olfactory epithelium varied from focal to extensive loss of sensory cells associated with damage to sustentacular cells. in severe cases, complete loss of olfactory epithelium occurred. although the degree of histological change was shown to vary with different agents, lesions induced by the more water-soluble chemicals tended to remain localized in the anterior part of the nasal cavity whereas agents with relatively low water solubility produced lung lesions in addition. it was suggested that these findings demonstrated the powerful 'scrubbing' action of the nasal cavity for water soluble, airborne xenobiotics.^^ inflammatory alterations have been induced in the nasal cavity of rodents treated with therapeutic agents at high doses by inhalation. significantly irritant substances do not make viable therapies. however, the precise relevance of such changes for human therapy by the inhalation route are sometimes questionable when the nasal damage is limited to high doses and it is not associated with alterations in other parts of the respiratory tract. in the case of tulobuterol, a -adrenergic receptor agonist, it was argued that the nasal inflammation induced in rats in a one month inhalation toxicity study was the result of a particularly high exposure of the nasal epithelium to drug, not representative of the likely human exposure to tulobuterol by inhalation, where little or no nasal exposure would occur.^^ rp [ cyclopentyloxy)-ar-( , -dichloro- -pyridy)- -methoxybenzamide], a novel type iv phosphodiesterase inhibitor which was being developed for the treatment of asthma and rheumatoid arthritis, was also reported to produce degeneration of the olfactory epithelium in rats but neither dogs nor mice after single and repeated oral doses and by inhalation.^^ histologically, the olfactory epithelium showed necrosis of the superflcial epithelial layers including the sustentacular and sensory cells, with sparing of the basal cell layer. there was also damage to bowman's glands. the development of proliferative lesions and ultimately tumours of principally neuroectodermal origin followed chronic treatment. as rp was highly metabolized and the nasal lesions could be inhibited by treatment of rats with metyrapone, a non-speciflc inhibitor of cytochromes p , it was postulated that the changes were the result of p mediated activation in the olfactory tissues, not linked to its pharmacological phosphodiesterase activity.^^ nasal epithelial degeneration and necrosis has also been reported in both rats and dogs treated with another candidate anti-inflammatory drug ci- by the intranasal route. this agent affected olfactory epithelium more than respiratory mucosa, suggesting that metabolism was important in the generation of this toxicity^^ although the nasal cavity has not been often examined histologically in great detail in toxicity studies conducted on drugs administered orally or by parenteral routes, damage to the nasal mucosa can be induced by drugs administered by these routes. one example is methimazole, a thioureylene antithyroid drug used in clinical practice where oral doses of . - mg/kg/day are employed and abnormalities of taste and smell have been described.^^ administration of methimazole at relatively high doses to long-evans rats by single oral ( mg/kg) or intraperitoneal ( mg/kg) routes was shown to produce damage to the sustentacular and sensory cells with sparing of the basal cells and basement membrane.^^ bowman's glands were also involved. methimazole is metabolized by the flavin-containing monooxygenase system and it is employed as a model substrate for this enzyme in vitro. the presence of flavin-containing monooxygenase isoforms in olfactory mucosa of long-evans rats suggested that reactive intermediates may be responsible for the nasal toxicity ^^ similar changes have been reported in mice where depletion of glutathione in the olfactory mucosa has been demonstrated also suggesting formation of local reactive metabolites.^^ histological examination has also shown that intravenous administration of a single dose of vincristine to mice damages the olfactory epithelium.^^'^^ vincristine is a vinca alkaloid derivative used in cancer therapy which has antimitotic activity and binds to tubulin. cell death was noted in olfactory cells - days after dosing with a peak of cell proliferation at days and repair after about days. these features resemble those that can be seen in other proliferating tissues after single doses of antimitotic drugs. the risk of damage to human olfactory cells from agents with these effects in rat nasal mucosa often remains uncertain because an understanding of relative exposure and metabolism in different species and a better understanding of the metabolic potential of human olfactory mucosa is required. a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium and to a lesser extent in respiratory and glandular epithelial cells.^^'^^ these inclusions are pas-negative and ultrastructural examination shows that they are membrane-bound, ellipsoid bodies containing homogenous electron dense matrix. their significance remains uncertain. a consensus classification for the variety of proliferative, non-neoplastic changes and atypical epithelial lesions and neoplasms found in the rat nasal cavity has been defined by schwartz and colleagues.^^ the classification of the international agency for research on cancer provides a similar perspective for rats and mice.^^'^^ proliferative lesions may be occasionally seen in untreated rodents in carcinogenicity studies but are much more commonly induced by administration of xenobiotics in inhalation carcinogenicity studies. spontaneous nasal tumours are uncommon but most often squamous in type in rats whereas in mice spontaneous squamous tumours are extremely rare and haemangiomas and respiratory adenomas predominate.^^'^^ the generally agreed categories are described below: mucous (goblet) cell hypertrophy and hyperplasia affects the nasal respiratory epithelium and are characterized by the presence of enlarged mucus-filled goblet cells, some of which form clusters suggestive on intraepithial glands. squamous cell hyperplasia is seen in the stratified squamous epithelium of the nares and is characterized by a focal increase in the number of cell layers. cells may show atypia with irregular enlarged, pleomorphic nuclei and nucleoli. squamous metaplasia occurs to respiratory epithelium under conditions of chronic damage. it is characterized histologically by the presence of three or more layers of epithelial cells with eosinophilic cytoplasm and clear cell boundaries whereas advanced lesions show typical keratinization and formation of intercellular bridges. cellular atypia may also be seen and should be characterized when found. respiratory epithelial metaplasia (of the olfactory epithelium) represents atrophy and degeneration of the olfactory epithelium with loss of sensory cells and in advanced cases loss of sustentacular cells with replacement by ciliated and non-ciliated respiratory epithelium. it may be seen as a spontaneous focal lesion in aged rats. epithelial hyperplasia with cellular atypia (atypical hyperplasia, basal cell hyperplasia, dysplasia) is a term used to embrace proliferative lesions in the respiratory and olfactory mucosa in the nasal cavity in which there is varying degrees of altered differentiation and atypia. there is perturbation of the growth pattern of the epithelium such that the changes are not those found in the normal regenerative response to transient mucosal damage. adenomas (polypoid or villous adenoma, adenomatous or villous polyp) usually develop in the anterior part of the nasal cavity and are usually exophytic lesions that develop from respiratory epithelium or nasal glands. adenomas of respiratory epithelium may be papillary in form but are, by definition, well circumscribed with minimal cellular pleomorphism and atypia. they may very occasionally occur spontaneously in aged rats.^^ adenomas of nasal glands usually show an acinar pattern. squamous cell papillomas develop in the squamous epithelium of the nares or in areas of squamous metaplasia in respiratory or olfactory epithelium. they are exophytic lesions with limited connective tissue stroma. they may develop spontaneously in aged rats.^^ carcinomas of either squamous or glandular differentiation develop in the nasal mucosa. histologically, they have similar characteristics to those in other epithelial tissues. they are rare spontaneous lesions in aged laboratory rodents but may be induced by xenobiotics administered by inhalation, orally or by the parenteral route. squamous carcinomas have been reported to develop in a small number of untreated fischer rats used in carcinogenicity studies in association with point mutations in the c-h-ras and c-k-ras gene.^^ olfactory neuroblastoma (ethesioneuroblastoma, olfactory neuroepithelioma, olfactory neuroepithelial carcinoma) show olfactory differentiation and arise from olfactory epithelium. they do not seem to occur as spontaneous lesions in rats or mice and only rarely induced.^^'^^ cells are arranged in lobules or in solid sheets with scanty stroma. cells are relatively uniform with scanty cytoplasm with round or oval hyperchromatic nuclei. true rosettes with lumens or pseudorosettes are also seen. poorly differentiated tumours of this type may require ultrastructural study for diagnosis. olfactory neuroblastomas typically show the presence of electron-dense neurosecretory granules, neurofilaments or axons. as there is no detailed understanding of the biological behaviour of these neoplasms in laboratory rodents, the generic term olfactory neuroblastoma is usually preferred. they are almost always invasive tumours.^^ olfactory carcinomas forming glands, follicles and rosettes have been occasionally reported in aged syrian hamsters.^^'^^ mesenchymal neoplasms may be seen in the nasal cavity, particularly after exposure to potent carcinogens. their histological features are similar to those in the soft tissues and bone elsewhere in the body (see chapter ). the mucosa lining the larynx and trachea becomes involved as part of an upper or lower respiratory tract infection. for instance, in rats, an acute laryngitis or tracheitis has been shown to accompany experimental infection with mycoplasma pulmonis and the sialodycroadenitis virus."^^'^^ a spontaneous degenerative condition of tracheal and laryngeal cartilage of uncertain pathogenesis associated with granulomas has been reported in fischer rats.^^ the condition increases in severity and incidence with advancing age although it is seen in rats as young as weeks of age. tracheal cartilage rings may also show alterations in genetically engineered animals, such as the c bl/ j-tgn(c -l-tag)cjeg (tag) mice that have generalized defects in cartilage development.^^ the larynx of rodents is also susceptible to the effect of inhaled substances, notably tobacco smoke but also pharmaceutical agents and propellants.^^'^^ in view of the localized nature of induced lesions in the larynx, standardized histological sectioning techniques have been proposed for rats, mice and hamsters using anatomical landmarks.^^"^^ the target site is located on the ventral floor of the larynx near the base of the epiglottis cranial to the ventral laryngeal diverticulum. lesions tend to occur in the ventrolateral region, which is covered by respiratory epithelium and the inner aspect of the arytenoid processes which is lined by squamous mucosa. the larynx responds to inhaled irritants by inflammatory, degenerative and regenerative changes in a manner similar to other regions of the respiratory tract. these include disruption of the epithelial cells, inflammatory cell exudates and infiltration, goblet cell hyperplasia and squamous metaplasia.^^ however, these changes are not specific to inhaled irritants but also occur as a response to natural respiratory tract pathogens in conventionally housed rats.'^^ the pseudostratified ciliated and non-ciliated mucosa of the trachea may also show pathological alterations in inhalations studies, although sites at the bifurcation (carina) are those often first affected. consequently, the carina should be systematically included in examination of the respiratory tract for induced lesions.^^'^^ as in the nasal passages a range of proliferative lesions including squamous hyperplasia, mucous cell hyperplasia, as well as papilloma, carcinoma and mesenchymal tumours are occasionally reported in the airways in laboratory rodents. in humans and laboratory animals, the trachea terminates at the bifurcation giving rise to two main bronchi which serve left and right lungs. depending on species, the main bronchi subdivide into further branches that enter the different lobes. various forms of branching are recognized. bronchi may arise as side-branches from a parent or stem bronchus (monopodial). the parent bronchus can divide into two equal daughter bronchus (dichotomous) or several daughter bronchi ipolychotomous)j^ study of silicone rubber casts of the respiratory tract has shown that the bronchial trees of humans and non-human primates are essentially dichotomous, in contrast to the monopodial pattern of rodents.^^ the comparatively long trachea of the dog gives rise to dichotomous upper airways but monopodial branching develops peripherally within each lobe. the size of the lungs is generally dependent on size and weight of the different species. auometric studies have shown that lung volume, alveolar surface area and diffusing capacity increase proportionally with body weight across a broad range of mammalian species, although cell size and surface area appear to be more determined by cell function rather than species size.^^ dogs have comparatively smaller body mass and higher airway dimensions compared to humans.^^ the number of lobes is species-dependent. the human lung possesses an upper and lower left lobe and an upper, middle and lower right lobe. this contrasts with the upper, middle and lower left lobes and a fourth, azygos right lobe in rhesus monkeys and baboons.^^ the dog has three lobes on both right and left sides. rats, mice and hamsters show cranial, middle, caudal and postcaval right lobes with a single, left lobe in mice and rats and a superior and inferior lobe on the left side in hamsters. cell types lining the bronchi are generally similar between species.^ the majority of cells are the ciliated cells that are accompanied by variable but relatively smaller proportions of basal cells, intermediate cells, mucous or goblet cells, serous cells, neuroendocrine and brush cells. in addition, mucous cells line the adjacent bronchial glands.^^ unlike the tracheal mucosa, which is pseudostratified, the mucosa of intra-pulmonary bronchi is non-stratified. ciliated cells are tall, columnar cells attached to basal and intermediate cells by desmosomal junctions. tight junctions exist between adjacent specialized cells at the apex. each cell possesses or more cilia that are engaged in mucociliary clearance.^^ the superficial cell surface also shows a pronounced glycocalyx. the cytoplasm of ciliated cells contains scattered profiles of rough endoplasmic reticulum, a supranuclear golgi and numerous mitochondria particularly near the apex where a prominent cytoskeleton is also found. mucous or goblet cells are typical mucus-secreting cells representing about % of the bronchial mucosa cell population in man but less than % in pathogenfree rats.^ the serous cell is a cylindrical or pyramidal cell containing small, round, closely packed serous granules.^^ basal cells are compact, pyramidal cells resting on the basement membrane. they are believed to be progenitor stem cells with the intermediate cells representing an intermediate stage of cell differentiation. the mucus-secreting and ciliated cells form the cellular basis for the mucociliary clearance mechanism of the main conducting airways. the epithelium is covered by a mucous blanket that is fairly complete in humans and rabbits but patchier in rats.^ the mucous layer is segregated into an upper layer or gel phase separated from epithelial cells by a serous layer or sol phase. the complex carbohydrates of the glycocalyx and secreted mucosubstances show species-related differences in their sugar residues, which can be demonstrated histochemically by the use of labelled-lectins.^^ mucociliary clearance mechanisms are sensitive to the effects of many therapeutic agents, particularly those that alter mucins, fluid or electrolyte balance and ciliary activity. anaesthetic gases, barbiturates, narcotics and alcohol depress clearance function. by contrast, topical, oral or parenteral administration of ( -adrenergic agonists, isoprenaline and adrenalin, produce a dose-dependent stimulation of mucociliary transport by an effect on ciliary beat frequency, probably mediated by increasing levels of cyclic adenosine monophosphate in ciuated cells rather than through vascular changes. although basal mucociliary function is dependent on normal vagal tone, parasympathomimetic agents can affect mucociliary transport. acetylcholine and cholinergic agents stimulate ciliary activity whereas anticholinergic drugs, atropine and hyoscine, inhibit ciliary activity and mucociliary transport. these substances may alter deposition of inhaled particles in the lung.^ clara cells or non-ciliated bronchiolar cells located in the bronchiolar epithelium, first described by clara in , are small and cylindrical in shape with highly infolded nuclei, surface microvilli, well developed golgi, abundant endoplasmic reticulum and characteristic oval, homogeneous electron-dense granules in the apical cytoplasm. in rats, rabbits and humans the granules are pas-positive, although they are usually considered pas-negative in hamster and mouse.^^ clara cells have high metabolic activity. they contain cytochrome p -dependent enzymes and secrete a variety of proteins.^^"^^ clara cell secretory protein is the major component of their cytoplasmic granules and they have been shown to produce mucin following antigen challenge.^^ in most laboratory rodents, the conducting airways terminate abruptly at the non-cartilaginous terminal bronchiole that opens directly into an alveolar type airway, the alveolar duct which in turn communicates with the alveoli.^^ squamous epithelial or type i cells form only about % of all lung cells but they line over % of the alveolar surface, by virtue of extremely long cytoplasmic extensions. the principal gas exchange takes place across this cell. in the rat, the typical thickness of this barrier is nm for a cytoplasmic extension of a type i pneumocyte, nm for basal lamina and nm for an endothelial cell.^^ the type i cell contains juxtanuclear mitochondria and the long smooth cytoplasmic extensions contain many ribosomes and pinocytotic vesicles. the anatomical configuration and function of type i cells render them highly vulnerable to inhaled gases and particles. the other main alveolar lining cell is the granular pneumocyte or type ii cell which constitutes about % of all lung cells, but which covers only about % of the alveolar surface.^^ this cell does not possess the long cytoplasmic processes typical of type i cells and it shows many microvilli on its luminal surface. the cell cytoplasm contains rough endoplasmic reticulum, golgi apparatus, some mitochondria and characteristic oval, osmiophilic lamellar inclusions. surfactant, a microaggregate of phospholipid and protein which modifies alveolar surface tension at low inflation volumes, is secreted by type ii alveolar cells. ultrastructural immunocytochemistry has shown the presence of surfactant apoproteins in the synthetic organelles and in the lamellar bodies of these cells, in agreement with the concept that the surfactant apoproteins are synthesized in the rough endoplasmic reticulum, glycosylated in the golgi and are stored in lamellar bodies.^^ type ii cells are more resistant to the damaging effects of xenobiotics and unlike type i cells they retain the ability to undergo mitotic division. following damage to type i cells, increased numbers of mitoses are evident in type ii cells which results in the appearance of large undifferentiated epithelial cells which ultimately differentiate into type i and type ii cells.^^ the lung also contains a dense neural network and a population of endocrinelike cells believed to be important in lung function.^^ these neurosecretory cells (kultschitsky or apud cells) are scattered sparsely in the epithelial surface of the larynx, trachea bronchi, bronchioles and alveoli. these cells are oval or cuboidal with oval nuclei, and argyrophilic cytoplasm which electron microscopic examination shows to contain dense core granules. the role of neuroendocrine cells in the lung is uncertain but immunocytochemical study has shown them to contain a number of neuroendocrine substances including neurone-specific enolase, synaptophysin, chromogranin and a variety of other peptides similar to vasoactive intestinal peptide, bombesin, calcitonin, serotonin, leu-encephalin, p endorphin and acth.^^'^^ cells lining the bronchi, bronchioles and alveolar walls are capable of metabolizing xenobiotics. immunocytochemical study has shown the presence of immune-reactive cytochromes p , nadph cytochrome p reductase, epoxide hydroxylase and glutathione s-transferase in bronchial epithelial cells, ciliated bronchiolar cells, clara cells, type ii and possibly type i pneumocytes in the rat lung.^^ different cell populations contain different amounts of enzymes, clara cells containing the greatest concentrations of the phenobarbitoneinducible isoenzyme of cytochrome p , nadph-cytochrome p reductase and epoxide hydrolase. studies of microsomal enzyme activities suggest that lung tissue contains fewer p isoenzymes than liver, principally forms cyplal cyp b , cyp a and cyp bl^^ whereas p enzyme activity is highly concentrated in specific cell types, overall microsomal enzyme activity is low compared with liver on the basis of microsomal protein weight.^^ other important cells are the pulmonary alveolar macrophages and lymphocytes. lymphocytes are found in the epithelium of the airways, in the interstitium of alveoli and as part of follicles in bronchial walls. pulmonary macrophages form part of the specific immune defence system of the lung, involving, as elsewhere in the body, antigen presentation. in the rat and mouse, distinctive populations of pulmonary macrophages have been described based on enzyme activities and reactivity to monoclonal antibodies against monocyte and macrophages surface determinates.^^'^^ bronchus associated macrophages in rat and mouse have more acid phosphatase and less non-specific esterase activity than the populations found in the pulmonary alveoli and interstitial tissues. an important aspect of the immune system is the bronchus-associated lymphoid tissue or balt, which forms part of the mucosal lymphoid system found in other epithelia. the morphology of balt is a useful guide to the nature and degree of immune stimulus in the lung. balt is organized in a way that is characteristic of other peripheral lymphoid organs. it is structurally similar in the laboratory rat, mouse, rabbit, guinea pig as well as in man but its size and prominence is species and strain-dependent as well as a function of the degree of antigenic stimulus^"^"^^. in the rat, the balt is composed of lymphoid aggregates or fouicles located mostly between a bronchus and artery with a zone of lymphocytes situated immediately under the bronchial epithelium. as in other peripheral lymphoid tissue, balt is organized into b and t cell zones but in no predetermined manner. immunocytochemical staining has shown that b and t lymphocyte zones differ in location from one aggregate to another. there are about two t lymphoc ^es for every three b cells compared with a ratio of : in rat peyer's particles.^^ the ratios may be different in other species. quantitative observations of t cell subsets using monoclonal antibodies have also shown that rat balt normally contains twice as many t-helper as t-suppressor/cytotoxic lymphocytes.^^ the t cells are confined to one or two discrete zones with a light scattering of t cells within the b cell zones and immediately under the bronchial epithelium. in common with lymph nodes, interdigitating cells are also found. the epithelium overl dng balt shows anatomical modifications. it is composed of ciliated and non-ciliated cells covered by microvilli. in conventional, untreated laboratory rats, balt shows little activity and germinal centres are usually absent, although balt may be more prominent in some rat colonies in association with non-specific inflammatory lesions in lungs.^^'^^ in one colony of young wistar rats germinal centres were not seen in balt in untreated animals but they developed following the administration of a single intratracheal dose of lipopolysaccharide, a t cell-independent antigen.^^^ single intratracheal doses of t cell dependent antigens such as horseradish peroxidase, bovine serum albumin and bcg have been shown to produce only minor morphological changes which include expansion of the zone of lymphocytes immediately under the epithelium and infiltration of the bronchial epithelium overlying balt by lymphocytes.^^^ in addition, perivascular, peribronchial or alveolar infiltrates of small and large lymphocytes and macrophages were observed in the lungs of rats given bcg. immunoc ^ochemical study of the rat balt following intratracheal challenge with horseradish peroxidase showed that the majority of cells that infiltrated the bronchial epithelium were t helper (cd positive) lymphocytes. ^^^ furthermore, la antigen expression of the epithelial cells overlying the balt was shown to increase, associated with an increase in the number and size of microvilli, a more pronounced glycocalyx and a decrease in number and size of cilia. immunocytochemical study of the balt tissue in c b / mice using monoclonal antibodies to lymphoid and macrophage populations has demonstrated quite similar arrangements of cells to those in the rat with the majority of t cells belonging to the t helper (cd positive) class.^^ the pulmonary lymphatic system drains into mediastinal or cervical lymph nodes. although among rat strains differences in the location of lymph nodes and their drainage occur, tracer studies in the fischer rat using colloidal carbon have shown that the lung lymphatics drain mainly into posterior mediastinal lymph nodes and those in the tracheal wall drains primarily to the internal jugular and posterior cervical nodes.^^^ although a variety of fixation, embedding and staining procedures are available for light and electron microscopic examination of lung tissue, there is no substitute for initial, careful visual inspection of the lungs at autopsy. uneven collapse of lungs on opening the thoracic cavity, discoloration or alteration in texture of the pleural or cut surface, congestion and presence of fluid in the larger airways may indicate structural damage. in rodent lungs, small pulmonary adenomas may be detectable by inspection in good light. fresh lung weight is also a helpful measure in lung assessment, although passive vascular engorgement can significantly affect this value. nevertheless, studies in the normal fischer rat have shown that after exsanguination, wet lung weights show a close relationship to body weight and that dry weight of lungs consistently represents about % of the wet weights regardless of age or body weight. ^^^ an increase in wet weight over dry weight appears to be a good index of pulmonary oedema. ^^ various methods of fixation have been employed although simple immersion fixation in formalin for conventional light microscopy has the virtue of simplicity and it avoids the risk of translocation or removal of exudates from airways and alveoli. mixtures of formaldehyde, paraformaldehyde and glutaraldehyde are used in initial fixation for electron microscopy.^^ the best overall appreciation of lung architecture is achieved by instillation of fixative via the trachea under an appropriate constant pressure or by perfusion fixation of the pulmonary arteries that is less liable to dislodge intra-alveolar exudate. in a review of methods employed routinely in rodent toxicity studies, instillation of fixative via the trachea was the preferred method in most laboratories because its advantages were seen to outweigh its disadvantages.^^ the sampling procedure is an important aspect of histological examination of the bronchi and lungs, particularly those of large laboratory animals. the extent of histological sectioning in conventional toxicity studies should be modulated to take account of lesions found by macroscopic examination, the type of study and the nature of the test substance. the bronchi should be carefully sampled to allow assessment of any alterations in bronchial epithelium. morphometric analysis represents a sensitive tool of value in the evaluation of drug-induced lung changes, but it requires particularly rigorous sampling and evaluation procedures.^^^'^^^ a tiered, multiple stage or cascade sampling technique is normally considered the most appropriate for morphometric studies.^^'^ this involves dividing the lung into a series of homogeneous compartments or strata from which randomly selected samples can be examined by appropriate light or electron microscopic techniques. conventional special stains for reticulin and collagen as well as pas and alcian blue for mucins are helpful in the characterisation of lung damage and changes to the respiratory epithelium. immunocytochemistry and enzyme cytochemistry are also useful in the study of the heterogeneous cell population of the lung. xenobiotic metabolizing activity can be studied both by enzyme cytochemical methods as well as by immunocytochemical techniques using antisera specific for pulmonary monooxygenases and related enzymes.^^ important structural components, particularly collagen and laminin can be studied both at light and ultrastructural level with immunocytochemical methods.^^^ cytokeratin immunocytochemistry can be used as a method for the characterization of changes to epithelial cells.^^ clara cells can be localized by the presence of clara cell secretory protein and ciliated cells by the presence of tubulin.^^ endocrine cells are visualized by immunocytochemistry using antibodies to general neuroendocrine markers such as chromogranin and synaptophysin or regulatory peptides.^^ other useful antigens, which can be demonstrated in the lung, include surfactants, lysozyme, immunoglobulins and those of microorganisms that infect the lung.^^^ electron microscopy is particularly useful for the detailed characterization of injury to the cells of the alveolar epithelium and endothelium ( figure . ). pulmonary oedema is a component of many inflammatory conditions of the lung, including those induced by infectious agents. however, the term oedema is reserved for a poorly cellular exudate characterized by the presence of pale, homogenous eosinophilic material in the alveoli, sometimes associated with a similar exudate in the lung septae and perivascular connective tissue. it occurs in a number of spontaneous conditions such as in congestive cardiac failure, metastatic pulmonary neoplasms or as an agonal change in association with pulmonary congestion and haemorrhage. drugs may induce cardiogenic pulmonary oedema as a consequence of pulmonary hypertension or impaired ventricular contractility. cardiogenic oedema is often associated with vascular congestion and red blood cells and haemoglobin may leak into airspaces. this can give rise to the presence of haemoglobin crystals within the oedema fluid in formalin-fixed tissue sections. most importantly, pulmonary oedema may be a manifestation of acute lung injury. inhalation or systemic administration of toxic chemicals may produce acute pulmonary oedema (see figure . ). some substances such as phenylthiourea and a-naphlythiourea produce massive pulmonary oedema in laboratory animals when administered orally, principally as a result of damage to the endothelium of pulmonary capillaries and venules. ^^^ over drugs have been reported to produce non-cardiogenic pulmonary oedema in humans, either directly or through poorly understood immunogenic mechanisms.^ another form of pulmonary oedema involves the main airways. allergic reactions in sensitized airways of asthmatic individuals is believed to result from cross-linking of ige and activation of mast cells that degranulate and release inflammatory mediators. ^^^ this has been reproduced in the main airways of rats sensitized to ovalbumin and then challenged with ovalbumin by the intratracheal route. ^^^ this treatment leads to rapid accumulation of bronchial exudate, degranulation of mast cells and the development of mucosal oedema, most marked immediately below the respiratory epithelium. congestion and haemorrhage is a frequent finding in the lungs of laboratory animals, where it is usually related to certain modes of death. it can be associated with administration of drugs and chemicals that have adverse effects on cardiac function or on the coagulation system. administration of heparin to rats produces a characteristic extravasation of blood into the air spaces.^^^ lower respiratory tract infection is generally not a major health hazard among laboratory animals but it is nevertheless an ever-present threat that can cause overt respiratory disease within a colony or develop following administration of xenobiotics. subclinical pulmonary infections and infestations can also produce histological alterations in the bronchial airways or pulmonary parenchyma which mimic changes induced by inhaled irritants or systemically administered drugs.^^'^^ furthermore, some respiratory pathogens alter immune defences and exacerbate the effects of inhaled substances. ^^^ a range of bacterial and viral pathogens may produce inflammatory lung changes.^^ typically, bacterial pathogens such as steptococcus pneumoniae produce acute bronchitis associated with a variable degree of acute inflammation of the lung parenchyma (bronchopneumonia) or a confluent lobar pneumonia. viral agents are generally associated with histological features of bronchiolitis and interstitial pneumonia, characterized by an increase in mononuclear cells in the respiratory bronchioles and alveolar septa. the histological features are variable for they depend on the particular pathogen, species and strain, immune status, presence or absence of secondary infection and the particular stage at which the infection is examined. respiratory infections are frequently mixed. changes due to secondary bacterial infection are frequently superimposed on those induced by viruses. sequential histopathological examination of the lungs of laboratory animals following inoculation with respiratory tract pathogens has been able to characterize the evolution of pathological changes produced by individual organisms. for instance, following inoculation with mycoplasma pulmonis, one of the more important respiratory pathogens among laboratory rodents both lewis and fischer rats were shown to develop upper and lower respiratory tract inflammation. in the lewis strain this was characterized after days by a variable acute inflammatory exudate in bronchi and bronchioles with focal bronchiectasis, inflammation and hyperplasia of the epithelium with a predominantly macrophage infiltration of the alveoli and alveolar walls.^^'^^^ these changes were associated with marked hyperplasia of the bronchusassociated lymphoid tissue (balt), which extended down the airways and blood vessels towards the periphery of the lungs. although the lymphoid hyperplasia was also found in inoculated fischer rats, it was less marked and accompanied by little or no mucopurulent exudate or active inflammation of the bronchial walls. this disparity in response suggested that differences were related to the degree of lymphocyte activation in the two strains, an imbalance in regulation of lymphocyte proliferation in lewis rats, or both.^^^ other studies have been conducted in both rats and mice infected with another important respiratory pathogen of laboratory rodents, the sendai virus (parainfluenza type ). sequential studies showed that the initial damage to bronchial and bronchiolar epithelium is associated with polymorphonuclear and lymphocytic inflammation (bronchiolitis). immunocytochemical and ultrastructural studies revealed the presence of viral antigen in the mucosa.^^^ hyperplastic and multinucleated syncytial epithelial cells develop in the hyperplastic terminal bronchiolar epithelium and the inflammatory process extended to involve peribronchial or peribronchiolar parenchyma with infiltration of alveolar walls by mononuclear cells, macrophages and neutrophils. a similar cell population accompanied by cell debris and oedema fluid develops in air spaces. pulmonary arteries show only minor involvement with inflammatory cells and focal reactive hyperplasia of the endothelium. immunocytochemistry and ultrastructural examination suggested that virus replication takes place in alveolar type i and type ii epithelial cells and macrophages but not in endothelial or interstitial cells of the alveolar septae.^^^ it was shown that when repair occurs there may be residual distortion of bronchiolar and alveolar walls by collagen and hyperplastic cuboidal epithelium may line the thickened alveolar septa. air spaces may also contain enlarged macrophages with pale vacuolated cytoplasm.^^^ strain differences in susceptibility have also been demonstrated to this virus. there is differential pulmonary interleukin (il- ) gene expression between virus-susceptible brown norway rats and resistant fischer rats and il- treatment provides protection from virus-induced chronic airway inflammation and remodelling. moreover increased tumour necrosis factor a (tnfa) expression has been shown to be an important regulatory factor in the development of sendai virus-induced bronchiolar flbrosis in infected rats.^^^ virus-inoculated brown norway rats had increased tnfa pulmonary mrna levels and increased numbers of bronchiolar macrophages and fibroblasts expressing tnfa protein compared with virus-inoculated f rats.^^^ the corona virus, which causes sialodacryoadenitis in many rat colonies, also produces lower respiratory tract inflammation. this is characterized by acute bronchitis and bronchiolitis with focal extension into lung parenchyma. thickened oedematous, hypercellular alveolar walls infiltrated by monocytic cells are found.^^ immunocytochemistry has shown the presence of viral antigen in bronchial and bronchiolar epithelial cells. there is also peribronchial lymphocytic infiltration and increased prominence of balt. ultimately complete resolution occurs. viruses remain a potential source of spontaneous respiratory disease in laboratory dogs. canine adenovirus type , parainfiuenza sv , canine herpes virus, coronavirus and parvovirus have all been isolated from laboratory dogs developing respiratory disease.^^^ the syndrome of visceral larva migrans also incites focal inflammation, granulomas and fibrosis in the lungs of species such as dog and primate in which parasites are prevalent. the syndrome of visceral larva migrans is usually defined as that which results from the migration of nematode larvae into the viscera. it has been well described in the beagle dog lung where it results from the larvae of toxocara species or metastrongyloid nematodes.^^^'^^^ the precise identification of parasites is not always possible in tissue sections. histological appearances of infested lungs are highly variable. nematodes surrounded by granulomas and granulomatous inflammation, mostly in a subpleural location, may be visible in sections. in affected lungs there may be perivasculitis and active arteriolitis, bronchiolitis and peribronchiolitis. pleural involvement by the inflammatory process can be marked, particularly in regions overlying granulomas. scarring develops and pleural and sub-pleural fibrosis is frequently associated with epithelial hyperplasia and squamous metaplasia of the associated airways ( figure . ).^^^ the lesions may sufficiently severe to resemble those induced by high doses of anticancer drugs such as bleomycin (see below). pulmonary acariasis is a common infestation of many species of non-human primates caused by various species of the mite pneumonyssus. reproduction of the mites appears to take place in the terminal bronchioles. pneumonyssus simicola is the recognized form found in rhesus monkeys.^^^ although it is most prevalent in wild caught primates, the disease is not easily eliminated during breeding in captivity. ^^^ even when eliminated by ivemectin the lesions of chronic bronchiolitis, bronchiectasis and pigmentation may persist as an incidental finding. ^^^ as the mite can produce significant destructive pulmonary pathology and render animals susceptible to secondary pulmonary bacterial infections, it can disrupt or confound the interpretation of toxicity studies performed in primates. the lesions are located most frequently in cranial lobes and are characterized by the presence of bullae distending the pleural surface, parenchymal cysts, nodules and scar tissue.^^^'^^^ histologically, there is a wide range of inflammatory activity. fully developed lesions are characterized by granulomatous bronchiolitis and peribronchiolitis with involvement of immediately adjacent alveoli. cystic lesions involving the bronchiolar walls develop around the parasites giving rise to the appearance of walled-off cysts composed of highly cellular granulation tissue, associated with neutrophils, lymphocytes, macrophages, multinucleated giant cells and various pigments (see below). in less active lesions, dilated, cystic airways with walls composed of thick bands of smooth muscle and lined by squamous or cuboidal epithelium are found. pneumocystis carinii is an important cause of pneumonia in patients with the acquired immunodeficiency syndrome (aids) as well as in other immunocompromised patients, including those treated with immunosuppressive drugs.^^"^ the natural habitat of pneumocystis carinii is pulmonary alveoli and it is widely encountered in the human population without being associated with overt disease. both clinical and experimental evidence suggests that impaired cellular immunity is much more important as a predisposing factor than impaired humoral immunity. ^^^ as in humans, laboratory animals may have latent pneumocystis infection that becomes clinically evident following immunosuppression. it has been shown in the rat that chronic administration of various regimens of adenocorticosteroids, low protein diets, cyclophosphamide and other immunosuppressive drugs with concomitant antibiotic administration to prevent other infections gives rise to typical pneumocystis pneumonia. ^^^ rodents with genetically deficient cellular immunity also develop pneumocystis pneumonia. the importance of pneumocystis pneumonia in toxicology is that it can be considered as a sentinel of chronic immune depression. in haematoxylin and eosin stained sections, pneumocystis pneumonia is characterized in both humans and rodents by the presence of alveoli filled with foamy eosinophilic material containing a few macrophages and indistinct nuclei of pneumocystis (figure . fe). ovoid or crescent-shaped structures of the organisms become clearly visible with gomori methenamine silver or toluidine blue stains. ultrastructural study of rats with pneumocystis pneumonia shows that trophozoites attach themselves most frequently to type i pneumocytes by altering their morphology to the contours of the pneumocytes rather than by a process of invasion. ^^^ systemically administered therapeutic agents may produce histological changes within the lung parenchyma that mimic components of the normal response to respiratory pathogens. however, there is no sharp separation between agents that produce pulmonary oedema with those that are associated with acute inflammatory changes and histological features overlap because an acute inflammatory process is often accompanied by exudate within airspaces. an example of drug-induced pulmonary inflammation in laboratory animals and humans is reported following the administration of interleukin (il- ). il- is a glycoprotein lymphokine, molecular weight kda, which is normally produced by activated t cells and mediates immunoregulatory responses. it has been produced in large quantities by recombinant dna technology for use in tumour immunotherapy. however, high doses have been associated with a number of adverse effects, notably the 'vascular leak' syndrome, characterized clinically by pulmonary oedema, pleural effusions and ascites. ^^^ the vascular leak syndrome has been reported in laboratory animals given high doses of this agent. histological examination of the lungs of b d f mice developing this syndrome following administration of il- showed infiltration of the alveolar walls with large lymphocytes and intra-alveolar proteinaceous exudate containing large lymphocytes, macrophages and red blood cells.^^^'^^^ pulmonary venules and arterioles showed the presence of lymphocytes attached to or lying beneath the endothelium, infiltrating vessel walls or in a perivascular location where they were accompanied by oedema fluid or red blood cells. similar, but less severe changes have been demonstrated in rats given il- .^^^ in addition, treated rats showed an infiltration of pulmonary vasculature with eosinophils probably secondary to an eosinopoietic cytokine produced by il- stimulated lymphocytes. immunocytochemical evaluation of the lymphoid infiltrate in mice showed that most of the cells were thy . positive (cd ) lymphocytes. furthermore, co-administration of asialo gml (ganglio-n-tetrosyl-ceremide) with il- not only abrogated the clinical signs but also reduced the number of asialo gml-positive lymphocytes in the tissue sections. as lymphoid cells expressing lyt- (cds, suppressor/cytotoxic t cells) were unaffected by asialo gml treatment, it was postulated that the vascular leak syndrome (but not antitumour efficacy) in these mice was mediated by an endogeneous subset of il- stimulated lymphocytes or lymphokine-activated killer cells. ^^^ corresponding changes were also observed in liver and lymphoid tissue. immunocytochemical and detailed electron microscopic studies in rats have supported the concept that il- induces cytotoxic vascular damage that is mediated both directly by lymphokine-activated killer cells and cytotoxic t lymphocytes with secondary release of inflammatory cytokines.^^^ as in humans, severe chronic pulmonary inflammatory disease in laboratory animals may compromise pulmonary function and lead to secondary alterations in other organs. although the mechanisms were not explored in detail, a diffuse interstitial pulmonary inflammatory process with lung haemorrhage was induced in rats treated for two years with prizidilol (skandf -a ), an antihypertensive agent with both vasodilator and ( adrenoceptor blocking properties.^^^ affected animals developed dyspnoea associated with reduction in lung volume, deformity of the thoracic spinal column and marked cardiac hypertrophy. inflammation with granulomas develops in the lungs of laboratory animals under a variety of different circumstances, which have been alluded to above. a common cause in rodents is granulomatous pulmonary inflammation resulting from aspiration of stomach contents or food particles (aspiration pneumonia). this is sporadically observed in aged rodent where it is associated with general ill health, particularly resulting from pressure effects of large pituitary adenomas and subsequent disturbance of pharyngeal or laryngeal reflex mechanisms. ^^^ histologically, the lungs show peribronchial and peribronchiolar granulomatous inflammation with macrophages and foreign body cells associated with fragments of refractive vegetable matter. the associated bronchial mucosa may also show reactive changes including goblet cell hyperplasia in long-standing cases. as dogs and primates are more liable to be infested by parasites, granulomatous inflammation in response to pulmonary larvae is more common in these species. pulmonary tuberculosis represents a potential problem among non-human primate colonies in view of its insidious onset and its liability for transmission from monkeys to humans. ^^^ pathological findings are similar to those so well known in the human disease. the disease is characterized by the presence of granulomas in lung parenchyma and lymph nodes. in florid cases there may be caseation surrounded by epithelioid and multinucleated giant cells and variable numbers of lymphocytes, plasma cells and flbroblasts. diffuse granulomatous pneumonia as a result of tuberculosis is also reported in non-human primates. granulomatous pneumonitis is also produced in laboratory animals by the intravenous injection of bcg. twenty-eight days following intravenous injection of bacille calmette-guerin (bcg), the lungs of c b / mice contained numerous granulomas composed of histiocytes and round cells which were surrounded by alveoli with thickened walls and associated with mild interstitial pneumonitis. ^^^ these histological changes were associated with an increase in the number of thy . -positive (cd ) cells, especially lyt- (cds) positive lymphocytes. the histological changes were abrogated by treatment with cyclosporin a suggesting an important role for cd -positive lymphocytes in the development of the granulomas. discrete granulomas occur in the lungs of experimental animals in response to intra-tracheal or intravenous injection of certain relatively insoluble substances (figure . ) . intra-tracheal administration of insoluble polymerized dextran and latex micro-particles to mice showed that the morphology and the systemic effects of granulomas depends on the nature of the injected substances. it has been shown that large granulomas develop rapidly in the pulmonary parenchyma around dextran particles that subsequently regress quickly, whereas latex particles produce small, discrete stable granulomas. ^^^ although both forms of granulomas are of foreign body or non-immunological in type, those produced by dextran but not latex beads, are associated with anergy-like immunosuppression, probably caused by release of soluble factors from the granulomas. it has been reported that granuloma formation after instillation of sephadex beads is associated with increases in the interleukin -(il- ) like activity in the lung.^^^ studies comparing the effects of inhaled crystalline silica and titanium dioxide have shown a correlation between the release of the macrophage derived cytokine il- and granuloma formation, suggesting that il- might be a useful biomarker for granuloma formation. ^^^ localized, angiocentric granulomas of foreign body type, clustered around pulmonary arteries and arterioles and occasionally alveolar capillaries and venules also develop following intravenous injection of relatively insoluble polysaccharides or other polymers. ^^^ characteristic epithelioid and large, foreign body type giant cells efface the smaller vessels although overt necrosis is not usually observed (figure . ) . haemosiderin-laden macrophages accumulate in the alveou of laboratory animals in association with chronic pulmonary congestion and haemorrhage. similar changes occur in patients in congestive cardiac failure where the haemosiderin-laden macrophages are termed 'heart failure' cells. the lungs of non-human primates are especially liable to contain alveolar, perivascular and peribronchial aggregates of macrophages laden with various brown pigments. iron-containing pigments have been associated with the inflammatory changes produced by simian lung mites (pneumonyssus simicola) which are prevalent in many non-human primates. in addition, lungs from some primate colonies may show perivascular and peribronchial collections of brown-grey macrophages containing highly refractive spicules and plates composed of high concentrations of silica.^^^'^^^ it has been shown that in old world primates including rhesus and cynomolgus monkeys, this pigment contains fossil diatomaceous material, compatible with the concept that the animals inhale dusts containing diatoms and other silicon fragments to which they are exposed in their semi-arid, natural habitats.^^^ chronic lung injury from a variety of different causes is frequently associated with the development of pulmonary fibrosis characterized by the replacement of the normal pulmonary structure by a thickened collagenous matrix with consequent reduction in the capacity for gas exchange. regardless of the inciting agent, the fibrogenic process appears to be generally characterized by disruption of the normal alveolar-capillary structure, leakage of exudate from the vascular compartment into the airspaces, subsequent invasion by infiammatory cells and fibroblasts associated with excess matrix formation. studies in laboratory animals with different fibrogenic agents as well as in humans have suggested that central to pulmonary fibrogenesis is increased production of tnfa by macrophages.^^'^^^'^^^"^^^ this cytokine is a not only a mitogen for fibroblasts but also a potent activator and chemo-attractant for macrophages, capable of stimulating release of other cytokines and inducing expression of adhesion molecule expression on endothelial cells. moreover, it has been shown that tnfa receptor knockout mice appear protected from the fibroproliferative effects of inhaled asbestos. ^^^ pulmonary fibrosis is a common sequel of chronic lower respiratory tract inflammation. it may be associated with, or preceded by interstitial pneumonitis, characterized by infiltration of lymphocytes, plasma cells and macrophages with scattered polymorphonuclear cells. ^^^ focal pulmonary fibrosis occurs spontaneously in laboratory animals, although this is usually most prevalent in dogs and non-human primates as a response to chronic infestation by parasites, which are not easily eliminated during breeding. in humans, conditions leading to pulmonary fibrosis vary widely. they include infections, shock lung syndrome, ionizing radiation, inhalation of irritant particulate matter, exposure to antigens or excessive amounts of oxygen as well as the results of the toxicity of paraquat and a range of both cytotoxic and noncytotoxic therapeutic agents which cause pulmonary parenchymal injury.^'^^^ the principal therapeutic agents that produce pulmonary fibrosis in both humans and laboratory animals are anticancer drugs. bleomycin, a glycopeptide preparation derived from streptomyces verticillus, is the best known example but pulmonary fibrosis is also associated with the clinical use of a number of other anticancer agents, including l, -bis-( -chloroethyl)-l-nitrosourea (bcnu or carmustine), cyclophosphamide, busulphan, mitomycin c and methotrexate. ' , - the precise mechanisms involved in the induction of pulmonary fibrosis by antineoplastic drugs in humans are poorly understood. the true incidence for a particular drug is difficult to estimate because of confounding factors in cancer patients, such as concomitant administration of several drugs, radiation and oxygen therapy, diffuse pulmonary cancer and opportunistic infections. it is probable that drug-induced fibrosis is accentuated by concomitant administration of several antineoplastic agents, radiation therapy, hyperoxia, preexisting pulmonary damage and age of the patient. severity is often related to total dose of drug received.^^^ novel antineoplastic drugs may also produce lung toxicity.^ bleomycin is associated with the development of interstitial pneumonia and pulmonary fibrosis in clinical use and this can be reproduced in experimental animals. the histopathological appearances of bleomycin-induced pulmonary fibrosis in patients are in many instances different from those seen in laboratory animals because the lungs of patients treated with bleomycin are modified by the primary neoplastic disease, smoking, multiple drugs, radiation therapy and secondary pulmonary infections, interstitial pneumonitis and fibrosis.^^^ it has been postulated that tnfa is an important mediator in the development of bleomycin-induced fibrosis. ^'^ in the preclinical evaluation of bleomycin beagle dogs were given cycles of drug by the intravenous route for periods of up to weeks. ^^^ dogs developed anorexia, weight loss, a variety of epithelial lesions as well as focal interstitial pneumonia and fibrosis. the focal lung lesions were characterized by increased elastic fibres, reticulin, collagen and acid mucosubstances. the lesions were situated predominantly in the pleural and subpleural zones, suggestive of a potentiating effect of friction between the pleural surfaces. histologically the lesions resembled those produced by larvae migrans in the dog (see figure . a). similar histological changes have also been described in both rats and mice treated with bleomycin by both the intravenous and intratracheal route.^^^'^^^ as fibrosis is such a consistent change, bleomycin-treated rodents have been extensively employed as a model for pulmonary fibrosis. early changes include mild, diffuse increases in interstitial lymphocytes, macrophages, polymorphonuclear cells and perivascular or interstitial oedema. after about a week, interstitial infiltrates also comprise fibroblasts with early collagen deposition, associated with proliferation of macrophages and type ii pneumocytes.^^^'^^^ subsequently, the amount of interstitial collagen increases, with eventual scarring and collapse of lung tissue in proportion to the cumulative dose given. ^^^ immunohistochemical and ultrastructural study of rats and mice treated with bleomycin shows a large accumulation of immune-reactive laminin and reduplication of the basement lamina within the thickened alveolar walls. ^^^ in bleomycin-treated rats three-dimensional scanning electron microscopy shows drug-induced capillary remodelling comprising irregular alveolar and pleural capillaries with increased diameter and decreased branching. ^^^ certain strains of mice have been shown to possess greater sensitivity to bleomycin fibrogenesis. the c bl/ strain produces a greater fibroblastic response than dba/ and swiss mice and the balb/c strain demonstrates a particularly poor fibroblastic response. ^^^ therapeutic use of cyclophosphamide is also occasionally associated with the development of pulmonary interstitial fibrosis.^^^'^^^ it appears to be associated with two forms of pathology: an early-onset pneumonitis and a late onset progressive pulmonary fibrosis. ^^^ similar changes have been less easy to reproduce in laboratory animals. when mice were sequentially examined for periods of up to one year after a single intravenous dose of loomg/kg of cyclophosphamide, only slight pulmonary interstitial thickening and hypercellularity was observed in association with progressive multifocal accumulation of intra-alveolar macrophages. ^^^ however, these changes were also accompanied by a progressive increase in pulmonary hydroxyproline content and a decrease in pulmonary compliance with time in treated animals compared with controls. the changes were amplified by exposure to % ambient oxygen. the bronchiolitis, alveolar septal infiammation and fibrosis induced by gold therapy in patients with rheumatoid arthritis is probably immune-mediated. this condition is associated with peripheral eosinophilia and drug-induced alterations to the immune system.^^^ emphysema is characterized by abnormal, permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis. three principle types, centriacinar, panacinar and distal acinar emphysema, are recognized in humans. enlargement of air spaces as a result of congenital factors or fibrous scarring are grouped separately and not regarded as emphysema. ^^^ emphysema has been reported as an age-related spontaneous change in laboratory rats.^^^ however, several experimental rodent emphysema models have been developed, using intratracheal instillation of proteolytic enzymes papain, pancreatic and neutrophil elastase. this gives rise to histological appearances resembling panacinar emphysema in humans.^^^ irritant gases, notably oxides of nitrogen, are also capable of inducing changes in the lungs of laboratory rats and hamsters following long term exposure which resemble mild human, centrilobular emphysema.^^^'^^^ a variety of different names have been applied to membrane-bound, acid phosphatase-positive cytoplasmic inclusions with a lamella or crystalloid ultrastructural matrix. these include myeloid bodies, myelinoid bodies, myelin figures or myelinosomes. these lysosomal inclusions are seen in small numbers in a variety of normal cell types but they accumulate in various organs in laboratory animals following administration of a wide variety of drugs of diverse therapeutic classes.^'^^^"^^^ the generalized accumulation of these lysosomal cytoplasmic bodies is generally called phospholipidosis, a term coined to describe the tissue accumulation of phospholipids. ^^^ at the light microscopic level, phospholipids are characterized by the increase in the number of foam cells in the airspaces. examples of drug-induced phospholipidosis include the anorectic drug chlorphentermine, tricyclic antidepressants, inhibitors of cholesterol biosynthesis such as triparanol, the antihistamine chlorcyclizine and its analogues, the selective oestrogen receptor antagonist tamoxifen, chloroquine and the cardiovascular drugs amiodarone, , 'diethylamino-ethoxyhexestrol and perhexiline.^^^'^^^'^^^ many tissues and organs may develop the cytoplasmic inclusions, including lymphoid cells, liver, pancreas, endocrine tissue, nervous system, muscle cells, eyes and particularly lungs. aminoglycoside antibiotics may produce laminated phospholipid inclusions in the renal tubular cell and imidazol antifungals in hepatocytes (see under liver and kidney, chapters and ). many drugs that induce phospholipidosis usually share structural features, notably a hydrophilic cationic side chain, a primary, secondary or tertiary amine and a hydrophobic region that is usually an aromatic ring or ring system. as this structural pattern renders these molecules amphiphilic, these drugs probably bind with polar lipids by means of electrostatic and hydrophobic forces.^^"^ this leads to formation of drug-lipid complexes which are poorly degraded by lysosomal enzymes and which accumulate in the cell cytoplasm to form the inclusions described above. as the binding is not covalent, its reversibility depends on the dissociation rate constant under the particular intracellular conditions and drug concentration achieved. predictions of this activity based on molecular structure have shown reasonably good correlation with the ability of compounds to produce phospholipidosis in cultured rat peritoneal macrophages. more recently other cell based systems have been proposed for screening for phospholipidosis.^^^'^^^ however these perform less well in the prediction of in vivo potency, presumably because of differences in drug disposition in blood and tissues. it should be underlined that the accumulation of foamy macrophages in the alveolar spaces may also be a spontaneous change in laboratory animals. it has long been recognized as a spontaneous alteration in ageing rats.^^^ it may also be found in lung tissue distal to bronchial lesions that impede clearance mechanisms. in contrast to drug-induced changes, the spontaneous lipidosis characterized by accumulation of alveolar foam cells occurs sporadically in older rats and is observed in both controls and treated animals. drug-induced phospholipidosis occurs within a period of several months during which lungs of control animals remain fairly free of foam cell accumulation. the lungs appear especially vulnerable to drug-induced phospholipidosis, possibly because macrophages are in very close proximity to blood-borne agents. phospholipidosis is also more clearly visible microscopically in alveoli whereas it can be easily overlooked in other organs. the continuous uptake of phospholipid-rich surfactant material from the alveoli by macrophages leads to excessive accumulation of phospholipids when their catabolism is impaired.^^^'^^^ the fact that lungs are commonly affected is a potentially useful diagnostic feature because in many organs phospholipidosis can be extremely difficult to recognize in haematoxylin and eosin stained sections. although the changes in the lungs are not specific for drug-induced phospholipidosis, an increase in the number of lipid-containing lung macrophages in treated animals compared with controls is relatively easy to detect and provides a simple way for the pathologist to screen for this effect. in severe generalized phospholipidosis in rats, the lungs show irregular pale grey or yellowish patches of discoloration of the pleura and parenchyma. this is a result of patchy or confluent aggregates of large, pale, foamy macrophages. they may be free lying or packed in alveoli and accompanied by granular, extracellular material. their abundant cytoplasm shows a vacuolated appearance in which fine eosinophilic granules are sometimes visible. the nuclei are rounded and centrally located structures of variable size (figure . ) . multi-laminated cells are also occasionally seen, as are vacuolated cells firmly attached to alveolar walls, probably pneumocytes. these foamy cells stain typically for phospholipids (e.g. acid haematin), although neutral lipids may also be present and stain with oil red o. semi-thin plastic-embedded sections stained with toluidine blue allow better characterization of phospholipidosis in all organs, including the lungs. the macrophages in the air spaces contain unmistakable dense, dark round cytoplasmic inclusions of variable size, some over mm diameter. ^^^ plasticembedded sections also show the inclusions in other pulmonary cells including pneumocytes attached to the alveolar walls, from which they can be seen discharging into the alveolar spaces. as in other organs affected by phospholipidosis, ultrastructural examination reveals dense, multi-lamellar membranes and numerous heterogeneous dense bodies of lysosomal origin (figure . ) . these bodies need to be distinguished from membranous bodies that form as a result of fixation for ultrastructural study. lipids tend to leach out and become hydrated to form myelinoid membranes during glutaraldehyde fixation. these structures are subsequently fixed by osmium to give rise to electron-dense membranous figures both outside and inside cells, particularly in mitochondria where they may be mistaken for pathological lesions.^^^ the lamella patterns seen in phospholipidosis may be simple alternating dense and clear lines spaced at - nm, or more complex arrangements of clear and dense lines. the other typical crystalloid inclusions of hexagonal aggregates of tubular subunits seen in other organs are not usually found in the lungs. the significance of these various forms is uncertain but they probably represent the various phases in which phospholipids exist and are influenced by proportions of lipids present. electron microscopic examination reveals that not only are pulmonary macrophages affected by these changes but that inclusions may be present in pneumocytes types i and ii, pulmonary capillary endothelial cells, smooth muscle cells, bronchiolar epithelium and occasionally neutrophils.^^^"^^^ the changes are typically still visible several weeks after withdrawal from treatment with the offending agent. although the extent of pulmonary phospholipidosis in the lungs varies between dosage regimen and animal species, studies with chlorphentermine, , 'diethylaminoethoxyhexestrol and amiodarone indicate that similar cytological and ultrastructural changes occur in most laboratory animal species studied including rats, mice, hamsters, guinea pigs, rabbits and dog.^^^'^^^'^^^'^^^ safety assessment -amphiphilic drugs what are the imphcations for humans of drugs that induce phosphohpidosis in laboratory animals? novel compounds continue to be found that possess the property of producing phosphohpidosis in laboratory animals with varying degrees of severity.^^^'^^^ although not all drugs that produce phosphohpidosis in animals have been studied in humans, only very few drugs that produce phosphohpidosis in animals have been shown capable of inducing significant phosphohpidosis in human clinical practice.^ agents such as chloroquine, , 'diethylamindethoyhexestrol and amiodarone, which have been shown to produce phosphohpidosis in patients, can also induce cellular damage in the same organs. however the phenomenon of phosphohpidosis where phospholipids are packaged behind lysosomal membranes may not be causally related to cellular damage in humans. indeed the weight of evidence suggests that druginduced phosphohpidosis per se is an adaptive phenomenon and does not in itself have functional or deleterious consequences unless excessive. ^^^ hence, the finding of phosphohpidosis in animal studies with a novel drug requires careful assessment on a case by case basis with respect to its implications for the safety of humans. an example of this issue is the iodinated benzofuran derivative amiodarone, a potent antiarrhythmic drug effective against ventricular arrhythmia. lung toxicity continues to be a problem in patients treated for cardiac arrhythmias with this drug.^^"^ not only does phosphohpidosis occur in a wide variety of organs in laboratory animals treated with amiodarone,^^^'^^^ but also in liver, peripheral nerve cells, skin, lymphoid cells and lungs in patients at therapeutic doses.^^^'^^^'^^^ although pulmonary interstitial fibrosis occurs in association with phosphohpidosis in patients, amiodarone-induced phosphohpidosis in rodents is not associated with pulmonary fibrosis or significant functional alterations. several theories have been proposed for the pulmonary alveolitis and interstitial fibrosis in humans. the weight of evidence to date suggests that the accumulation of lipid-laden histiocytes is not causally related to the alveolitis or pulmonary fibrosis. ^^^ indeed, overall there is little evidence that the mere presence of phosphohpidosis is deleterious to the organism.^^^ cytotoxicity, possibly through the metabolite desethylamiodarone, has been proposed and an immune-mediated mechanism has been postulated, possibly favoured by the binding of drug to components of pulmonary tissue.^^^ it might also involve free radical formation or indirect influences on inflammatory mechanisms.^^^ it is also possible that pulmonary disease results from an interaction of several mechanisms and metabolic factors unique to particular patients. ^^^ despite undoubted differences in tissue and species sensitivity to development of phosphohpidosis, dose, drug disposition, metabolism and elimination and the degree of tissue exposure to drug are important considerations in safety assessment of drugs that produce phosphohpidosis in laboratory animals. although phosphohpidosis is more likely to occur at high doses employed in toxicity studies than at lower therapeutic doses used in patients, it has been suggested that this may be offset by faster ehmination of the drug, characteristic of small laboratory animals. ^^^ the potential for drugs to accumulate in critical tissues such as eye and heart is especially important when drugs are administered for long periods of time, particularly as tissue/plasma ratios of some amphiphilic drug may exceed , following repeated administration.^^^ consequently, although phospholipidosis may not have functional consequences, any implications for humans of drugs that induce phospholipidosis in laboratory animals can only be assessed on a case by case basis, with due consideration of mechanism, drug disposition and clinical risk-benefit analysis. it is important to underline that similar morphological changes due to the increased presence of phospholipids in lysosomes can also result from treatment with compounds that are not cationic amphiphilic structures. mechanisms include direct or indirect inhibition of lysosomal enzyme activity. this reenforces the need to understand the mechanism of any chemically induced increase of phospholipids in the lungs of laboratory animals. for example, it has been shown that when glycosaminoglycans accumulate in inherited human lysosomal disorders they inhibit other lysosomal enzymes, thereby inducing lysosomal phospholipid inclusions. ^^^ this is reflected by administration of high doses of the trypanocidal drug suramin to rats which induces intracellular storage of glycosaminoglycans associated with phospholipid inclusions in diverse organs including lungs. ^^^ although at light microscopy clear vacuoles are typically seen, electron microscopic examination shows the presence of both clear vacuoles containing glycosaminoglycans and lamellar phospholipid inclusions. a similar effect seems to have been produced in rats by elmiron®, a semi-synthetic heparin-like macromolecular carbohydrate derivative, chemically and structurally similar to glycosaminoglycans used clinically for anticoagulant effects and interstitial nephritis.^^^ another example is the induction of lysosomal inclusions in the lungs of rat and dogs by the macrolide antibiotic erythromycin.^^^ collections of foam cells were described in the lungs and lymphatic tissues of dogs and rats treated with high oral doses. foam cells in the lung showed a pattern of small whorls in vacuoles similar to that seen with other drugs that induce phospholipidosis. in vitro studies have suggested all macrolide antibiotics have the potential to cause phospholipidosis. biochemical studies suggest that drug binds to phosphatidylinositol-containing liposomes and inhibits activity of lysosomal phospholipase in close correlation with the number of cationic groups carried by each of the drugs. ^^^ hook reviewed other agents, such as oxidant gases and insoluble particles including silica, that can also increase phospholipid levels and histological appearances of phospholipidosis in the lungs.^^^ some of these agents inhibit phospholipid catabolism in the lungs giving rise to accumulation of surfactant protein a and surfactant lipoproteins and a clinico-pathological picture similar to pulmonary alveolar proteinosis in humans. studies from humans have shown that three chnically distinct forms of this condition occur: congenital, secondary or acquired. the congenital disease is caused by a diverse range of mutations in the genes encoding surfactant proteins or the ( c chain of the receptor of granulocyte-monocyte colony stimulating factor (gm-csf). the secondary form occurs in association with conditions where there is functional impairment or reduced numbers of alveolar macrophages, such as in haematological cancers, following immune suppression or inhalation of silica or toxic fumes. acquired or idiopathic alveolar proteinosis that accounts for over % of all cases ( . per persons) has been an enigma until recently. patients are at risk from infections, particularly nocardia, and the year survival rate appears to be about %. studies from transgenic mouse models and in humans have shown that autoantibodies against gm-csf are important in the development of the acquired form of the disease as this antibody causes a defect in macrophages function which impairs the catabolism of surfactant lipids and proteins. ^^^ in this context it is of interest to note that treatment with imatinib, a tyrosine kinase inhibitor of the bcr-abl tyrosine kinase constitutively expressed in philadelphia chromosome positive myeloid leukemia, has been associated with the accumulation of lamellar inclusions in pulmonary macrophages in a leukaemia patient, although this might have been a result of the primary disease. ^^^ studies in mice have suggested that imatinib mesylate actually inhibits the fibrogenic activity of transforming growth factor ( and prevents fibrosis induced by bleomycin.^^^ various forms of hyperplasia are found in the airways and lungs of laboratory animals. the mucosal surface of the bronchi may show hyperplasia of the goblet cells, squamous hyperplasia or metaplasia. the cells lining the terminal bronchiole and alveolus may also show hyperplasia and squamous metaplasia. standard classifications for the characterization of these changes in histological sections have been developed for use in rodent studies.^^"^^ goblet cell hyperplasia is a well-recognized response of the mucosa of conducting airways to chronic inflammation and inhalation of irritant substances such as cigarette smoke and sulphur dioxide.^^'^^'^^^'^^^ the degree of goblet cell hyperplasia is dictated by the severity and duration of the irritation or inflammatory process. florid cases of goblet cell hyperplasia are characterized by thickening and pseudostratification of the tracheal or bronchial mucosa by a population of tall, mucus secreting cells with abundant pale cytoplasm. in addition, goblet cells extend further down the airways than in normal animals and mucus may fill or distend the airways or impact in the alveoli. in less florid cases, a simple increase in the number of goblet cells may be found without other structural change.^^ goblet cell hyperplasia of the lining epithelium may be accompanied by an increase in size of the underlying submucosal glands. this has clearly been demonstrated in patients with chronic bronchitis and in rats where submucosal glands are normally quite prominent.^^^'^^^ species differences may exist because the airways of laboratory animals are variably endowed with goblet cells and submucosal mucous glands. the normal rat has more goblet cells lining the airways than either mouse or hamster. ^^^ the factors controlling these alterations are uncertain but is has been long suggested that increased mitotic activity as well as cell conversion, probably by metaplasia of serous or clara cells to mucous cells, is involved.^^^ it has more recently been shown in mice sensitized to ovalbumin and subject to a single antigen challenge by aerosol that clara cells in the proximal airways show great plasticity and become mucin-secreting cells.^^ pharmacological agents can induce goblet or mucous cell hyperplasia. rats given six or daily injections of isoprenaline, a non-selective ( receptor agonist showed a dose-and time-dependent increase in the number and size of alcian blue-positive goblet (mucous) cells as well as serous cells in the tracheal and bronchial mucosa. this was associated with an increase in length, width and depth of submucosal glands.^^^ similar changes were produced by pilocarpine, although both alcian blue-and pas-positive cells were increased in number following this agent, suggesting that pilocarpine induced both acid and neutral glycoprotein secretion. comparison of the distribution of these changes in the rat following isoprenaline, with those of salbutamol, pilocarpine and tobacco smoke, showed that there were regional differences in the distribution of these changes in the airways.^^'* isoprenaline produced a greater increase in secretory cells in peripheral airways than tobacco smoke, which itself produces a greater increase in mitotic activity. isoprenaline and pilocarpine produced a more diffuse change than the more selective ( agonist, salbutamol. the changes induced by these therapeutic agents are presumably the result of their pharmacological activity.^^'^ sturgess and reid showed that the changes in the rat were accompanied by hypertrophy of the pancreas, submaxillary and parotid salivary glands^^^ (see digestive system, chapter ). unlike the rat and mouse, the hamster appears predisposed to develop minor multifocal epithelial hyperplasia of the tracheal and bronchial mucosa spontaneously with advancing age. these changes are flat or polypoid in nature and are composed of clear cells and goblet cells.^^'^^ the epithelium of the bronchi shows squamous metaplasia in response to chronic irritation or injury. it is characterized by three or more layers of epithelial cells with abundant eosinophilic cytoplasm with prominent cell boundaries. it may be associated with degenerative alterations to the mucosa or goblet cell hyperplasia. squamous metaplasia can also develop in the alveolar parenchyma as a response to prolonged damage such as produced by large burden of inhaled irritant or insoluble dusts. the metaplasia is also characterized by the presence of several layers of flattened epithelial cells showing squamous differentiation. the term pulmonary keratinizing cyst has been recommended for pulmonary cystic lesions lined by non-neoplastic squamous epithelium without excessive proliferative change.^^^ hyperplasia, bronchiolo-alveolar (type ii cell hyperplasia) hyperplasia may involve the lining epithelium of the alveoli or bronchioli. this form of hyperplasia has been termed alveolar hyperplasia, adenomatosis, alveolar bronchiolization or epithelialization. it occurs spontaneously but can be induced by infections and administration of irritant xenobiotics in rats/^'^^^"^^^ j^j^g , ^^^ hamsters.^^^ histologically, the lesions consist of localized but unencapsulated foci of hyperchromatic regular, cuboidal or columnar cells investing airspaces without appreciable distortion of alveolar walls. neuroendocrine hyperplasia is well described in hamsters. although small aggregates of neuroendocrine cells (neuroepithelial bodies) are found at various levels of the bronchi and bronchioli in normal hamsters, administration of nitrosamines and -nitroquinoline -oxide produces neuroendocrine hyperplasia.^^^"^^^ hyperplastic lesions are recognizable as groups of non-ciliated cuboidal, oval or columnar cells located in the bronchial or bronchiolar epithelium. they contain argyrophilic granules that show immunoreactivity for corticotrophin (acth) and neurone-specific enolase. ultrastructural examination reveals the presence of dense-core cytoplasmic granules of apud type. proliferative changes have also been reported in other species, including rats and humans in hypoxic conditions, although it has been suggested that these changes might be a result of increased peptide content rather than cell proliferation.^^'^^^ the most frequently diagnosed neoplasm world wide is lung cancer, where it is usually caused by smoking tobacco.^^^ bronchogenic squamous carcinoma is generally the most common subtype but in north america the incidence of adenocarcinoma now exceeds that of squamous cell tumours for reasons not fully understood. some of the most aggressive subtypes are small and large cell neuroendocrine lung cancers, defined as small or large tumour cells with greater than ten mitoses per mm^.^^^ these seem to be seen almost exclusively in heavy cigarette smokers. in contrast to findings in people, squamous cell lung tumours are only occasionally seen arising spontaneously in laboratory animals. even laboratory animals -rats, mice, hamster, monkeys and dogs -exposed to tobacco smoke for long periods and at high doses fail to develop an increase in lung tumours.^^^'^^^ moreover, there appears to be no good experimental model for neuroendocrine lung cancer. thus, particular caution is merited if using animal models for prediction of lung tumorigenic potential of inhaled substances. by far the most common primary pulmonary neoplasms found in laboratory rats, mice and hamsters are adenomas and adenocarcinomas. these appear to develop from the bronchiolar or alveolar epithelium, although their precise histogenesis is somewhat disputed. although spontaneous squamous neoplasms are uncommon in rodents cystic keratinizing lesions can be induced in rats by high burdens of particulate material in the lungs.^^^ pleural mesotheliomas and mesenchymal neoplasms also occur in these species but are uncommon. they can be induced in rodents by mineral fibres.^^^ mesenchymal tumours have similar histological features to those in soft tissues and mesotheliomas may show either epithelial or mesenchymal differentiation or both. in most rat strains alveolar or bronchiolar neoplasms occur spontaneously in relatively small numbers, but morphologically identical neoplasia can be induced by administration of chemical carcinogens.^^^ the most common are classified as bronchiolo-alveolar adenoma (pulmonary adenoma) and bronchioloalveolar carcinoma. the national toxicology program database on control fischer rats used in carcinogenicity studies indicates an overall percentage of less than % of animals with bronchiolo-alveolar adenomas and less than % with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar adenomas in different studies was between and % in this series. histologically, bronchiolo-alveolar tumours are mostly small, discrete, rounded nodules located in the lung parenchyma and composed of fairly uniform cells with moderately hyperchromatic nuclei arranged in solid (alveolar), tubular, papillary or mixed growth patterns. they usually compress surrounding tissues without infiltration or metastatic spread (adenoma), although loss of differentiation, infiltration and spread to adjacent tissues can occur (adenocarcinoma). ultrastructural study of bronchiolar-alveolar neoplasia in fischer rats has shown the presence of osmiophilic, lamellated inclusion bodies similar to those found in alveolar type ii cells. therefore it has been suggested that the neoplasms are derived from this cell type.^^^ pulmonary squamous carcinoma occurs but is a very uncommon spontaneous neoplasm in the rat.^^ the large proliferative but benign cystic lesions found in the lungs of rats following accumulation of large amounts of particulate matter have been termed pulmonary cystic keratinizing epitheliomas for they have been regarded as benign neoplasms. when these lesions show evidence of tissue invasion they are regarded as pulmonary squamous cell carcinomas. similar lesions are very occasionally reported as spontaneous lesions.^^^ analogous neoplasms are found more commonly in most strains of laboratory mice used in carcinogenicity bioassays although considerable variation in incidence is reported. they are common in strain a mice where they are observed in low frequency at - months of age and incidences reach nearly % by months of age.^^^ fewer, but significant numbers are found in b c fi mice, although there is considerable inter-laboratory variation.^^^ the national toxicology program database on control b c fi mice used in carcinogenicity studies indicates an overall percentage of about % of males and % of females with bronchiolo-alveolar adenomas but only about % and . % respectively with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar tumour varied considerably between studies in this series. even in the same laboratory, mice housed under similar conditions show variation in incidence in these neoplasms with time. the incidence of lung adenomas and adenocarcinomas occurring in cd-i mice used as controls in -month carcinogenicity bioassays in the same laboratory under similar conditions for a period of years varied from between and % in males and to % in females.^^^ by contrast, some strains of mice such as the c / j strain show a very low predisposition to the development of lung adenomas.^^^ although these mouse pulmonary adenomas and adenocarcinomas do not resemble the common lung tumours in humans, strain differences have been exploited to study genetic susceptibility and resistance to pulmonary adenomas and carcinomas.^^^'^^^ histologically, pulmonary tumours of this type in mice are generally small, sharply circumscribed nodules composed of fairly uniform, closely packed columns of cuboidal or columnar cells arranged in tubular or papillary structures with scanty fibrovascular stroma ( figure . ). they may be less well differentiated, with cellular pleomorphism, and show intrabronchial growth, invade lung parenchyma and produce metastatic spread. the histogenesis of mouse pulmonary adenomas and adenocarcinomas is disputed. on the basis of sequential light and electron microscopic study of pulmonary adenomas induced in bagg-webster swiss mice by transplacental exposure to ethylnitrosourea, it has been suggested that they develop from either alveolar type ii cells or clara cells.^^^'^^^ careful, stepwise analysis using light microscopic and electron microscopic examination has suggested that adenomas can be divided into three principal groups. some are composed of solid growths of uniform cuboidal cells with expanding margins limited to alveolar septae (alveolar pattern). these cells contained concentrically arranged cytoplasmic lamellar bodies and abundant, large mitochondria similar to mitochondria found in alveolar type ii cells. tubular or papillary patterns are composed of cuboidal cells showing histological and ultrastructural features of clara cell differentiation.^^^ however, immunocytochemical studies of chemically induced and spontaneous pulmonary neoplasia in b c f , balb/c or a strain mice have shown that the majority of adenocarcinomas, including those showing papillary patterns, contain surfactant apoprotein, typical of type ii antigens, suggesting that most neoplasms show alveolar type ii differentiation.^^^ however, in view of the plasticity of clara cells, this does not exclude a clara cell origin of the tumours. immunocytochemistry of specific clara cell secretory protein expression in a transgenic mouse model of lung carcinomas developing from clara cells has shown that the protein is lost during tumour cell progression.^^^ it has also been shown in strain a mice that the proportion of tumours with papillary and solid/alveolar growth patterns varies with the inducing agent.^^^ this also suggests biological differences exist between histological subtypes. very few squamous carcinomas are reported in most series of mouse studies. a chemically induced mouse model of squamous cell carcinoma has been generated by administration of n-nitroso-tris-chloroethylurea. strain differences in susceptibility to squamous cancer development have been demonstrated in this model, with nih swiss, a/j and swr/j being highly susceptible, akr/j and c bl/ j being resistant and fvb/j and balb/cj mice showing an intermediate response to carcinogen.^^^ the high incidence and the inherent variabihty of pulmonary adenomas and adenocarcinomas in conventional mouse carcinogenicity bioassays sometimes gives rise to statistically significant differences between control and treatment groups. there is considerable risk in over-interpretation of such group differences in conventional mouse bioassays. in the analysis of group differences, consideration needs to be given to tissue sampling procedure, age-standardization, historical control incidence, effects on food intake as well as the results of mutagenicity studies and carcinogenicity bioassays in other rodent species. indeed, a considerable number of widely employed therapeutic agents of different classes have produced an increase in benign or malignant pulmonary tumours in carcinogenicity studies performed in mice without this proving of any significance to humans. davies and monro counted at least drugs of this type in the physicians' desk reference of the united states.^^^ for instance, in a carcinogenicity bioassay in which cfl mice were treated for weeks with the synthetic analgesic tilidine fumarate, a statistically significant difference (p < . ) was reported in the incidence of lung adenocarcinomas between the top dose female group ( %) and concurrent controls ( %).^^^ it was argued that group differences did not indicate tumorigenic potential of tilidine fumarate on the basis that the incidence in the high dose group was within the historical control range ( %) and that there was no tumorigenic effect in a parallel week rat carcinogenicity study. a more difficult evaluation concerned metronidazole, a nitroimidazole which is an important therapeutic agent active against anaerobic organisms and trichomonas species. administration of this compound led to an increased incidence of pulmonary adenomas and carcinomas in three separate mouse carcinogenicity bioassays.^^^'^^^ the analysis of these findings was somewhat complicated by evidence that metronidazole shows mutagenic activity in bacterial assays using some strains of salmonella typhimurium. it was argued that the risk to human patients was slight because the increase in prevalence in pulmonary tumours was likely to be a result of changes in nutritional status of the mice through the effect of metronidazole on gut fiora, as similar differences could occur between ad libitum fed mice and those fed the same but restricted diet.^^^ it was also postulated that the positive findings in bacterial mutagenesis assays were an inherent part of the antibacterial activity of metronidazole as a result of nitroreduction that does not occur in normal mammalian tissues. this conclusion was supported by negative effects in hamster carcinogenicity bioassays as well as lack of excess cancer risk in women followed up for years or more.^^^ the common occurrence of lung adenomas in strain a mice has been utilized in the development of a quantitative bioassay for carcinogenic activity. this followed the demonstration that administration of carcinogens such as -methylcholanthrene to this strain could significantly increase the incidence of pulmonary adenomas within periods of up to six months.^^^ over many years the strain a mouse pulmonary tumour assay has been used to test a large number of chemicals of different classes, including polycyclic hydrocarbons, nitrosamines, food additives, alkyl halides, metals and chemotherapeutic agents.^^^'^^^ however, as with many test systems, correlation of results in the strain a test with year carcinogenicity study data and genotoxicity results have been shown to be poor so prudence is needed in the use of this test.^^^ hamsters develop lung adenomas spontaneously in small numbers with advancing age. they are composed of uniform cylindrical cells similar to those found in bronchial epithelium or goblet cells showing distinct mucus production.^^'^^'^^^ an immunohistochemical study of similar pulmonary neoplasms induced in hamsters by n-nitrosodiethylamine showed the presence of clara cell antigen in early phase of development, but as the tumours developed they became more squamous in type and showed immunoreactivity for cytokeratins.^^^ a clara cell origin was suggested for most of these neoplasms. lung defenses against infection: a clinical correlation drug-induced lung toxicity pulmonary toxicity from novel antineoplastic agents drug-induced diseases. drug-induced respiratory disease mucociliary clearance and mucus secretion in the lung risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation considerations for toxicology studies of respiratory drug products overview of inhalation toxicology inhalation exposure technology, dosimetry, and regulatory issues pulmonary deposition: determinants and measurement techniques inhalation therapy: technological milestones in asthma treatment the relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report interpretation of new techniques used in determination of pulmonary function in rodents measurement of respiratory patterns in rodents using whole-body plethysmography and a pneumotachograph evaluation of lung injury in rats and mice comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants comparative aspects of nasal airway anatomy. relevance to inhalation toxicology comparative anatomy, physiology, and function of the upper respiratory tract comparative anatomy of mammalian respiratory tracts: the nasopharyngeal region and the tracheobronchial region airflow, gas deposition, and lesion distribution in the nasal passages a comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts the upper airways. . nasal physiology and defence of the lungs antioxidant protection: a function of tracheobronchial and gastrointestinal mucus the histochemical and microscopical differentiation of the respiratory glands around the maxillary sinus of the rat hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin d the lateral nasal gland of the dog, its structure and secretory content sites for xenobiotic activation and detoxication within the respiratory tract: implications for chemically induced toxicity cytochrome-p a of nasal epithelium -regulation and role in carcinogen metabolism purification and characterization of heterologously expressed mouse cyp a and cyp g : role in metabolic activation of acetaminophen and , -dichlorobenzonitrile in mouse olfactory mucosal microsomes differential xenobiotic induction of cyp a in mouse liver, kidney, lung, and olfactory mucosa nasal cytochrome p a: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen cell-specific expression of cyp a in the mouse respiratory tract: effects of olfactory toxicants biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach nasal lymphoid tissue in the rat toxicity to nasal associated lymphoid tissue histopathologic examination of the rat nasal cavity structural evaluation of the respiratory system normal histology of the nasal cavity and application of special techniques approaches to the identification and recording of nasal lesions in toxicology studies nasal diagrams -a tool for recording the distribution of nasal lesions in rats and mice histopathology of nasal olfactory mucosa front selected inhalation toxicity studies conducted with volatile chemicals cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. . determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung irritating properties of airborne materials to the upper respiratory tract bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man infectious diseases of the upper respiratory tract: implications for toxicology studies respiratory tract murine respiratory mycoplasmosis in lew and f rats: strain differences in lesion severity spontaneous proliferative lesions in the nasopharyngeal meatus of f rats spontaneous tumors and common diseases in three types of hamsters spontaneous tumors and common diseases in two colonies of syrian hamsters. ii respiratory tract and digestive system sialodacyoadenitis virus-associated lesions in the lower respiratory tract of rats oropharyngeal granulomas and tracheal cartilage degeneration in fischer- rats articular chondromatosis and chrondroid metaplasia in transgenic tag mice cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases histological sectioning of the rodent larynx for inhalation toxicity testing histologic methods and interspecies variations in the laryngeal histology of f /n rats and b c f mice interspecies variations in the histology of toxicologically important areas in the larynges of crl:cd rats and syrian golden hamsters revised guides for organ sampling and trimming in rats and mice. part : a joint publication of the rita and nacad groups a comparison of the pathology of the larynx from spf, germ-free, conventional, feral and myoplasma-infected rats modelling of biological tree structures allometric relationships of cell numbers and size in the mammalian lung the ultrastructure of various cell types in the lung of the rat length and distribution of cilia in human and canine airways lectin-binding affinities of the respiratory tract. a light microscopical study of ciliated epithelium in rat, guinea pig and hamster clara cell proteins identification, cellular-locahzation, isolation, and characterization of human clara cell-specific -kd protein mucin is produced by clara cells in the proximal airways of antigen-challenged mice morphology of the terminal bronchiolar region of common laboratory mammals morphological changes in the lung during the life span of fischer rats immunocytochemical localization of the major surfactant apoproteins in type ii cells, clara cells and alveolar macrophages of rat lung quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung what can the biology of small cell cancer of the lung teach us about the endocrine lung? the role of metabolism in chemical-induced pulmonary toxicity characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (balt) macrophages in the rat. an enzyme-cytochemical and immunocytochemical study subpopulations of lymphoid and non-lymphoid cells in bronchus-associated lymphoid tissue (balt) of the mouse bronchial lymphoid tissue. . morphologic characteristics the specificity of the high endothelial venule in bronchus-associated lymphoid tissue (balt) t cells and t cell subsets in rat bronchus associated lymphoid tissue (balt) in situ and in suspension have you seen this?' infiammatory lesions in the lungs of rats inflammatory lesions in the lungs of wistar rats histological changes in rat bronchus-associated lymphoid tissue after administration of five different antigens changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intracheal challenge with horseradish peroxidase patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat age-body weight relationships to lung growth in the f rat as indexed by lung weight measurements application of morphometric methods to study diffuse and focal injury in the lung caused by toxic agents morphometric assessment of pulmonary toxicity in the rodent lung the connective tissue of the rat lung: electron immunohistochemical studies immunohistochemical techniques and their applications in the histopathology of the respiratory system systemically applied chemicals that damage lung tissue bronchial mucosal mast cells and their implications in the pathogenesis of asthma morphological-changes in rat tracheal mucosa immediately after antigen challenge comparative studies of heparin and heparin fragments: distribution and toxicity in the rat interactions between sendai virus and bacterial pathogens in the murine lung: a review murine respiratory mycoplasmosis in f and lew rats -evolution of lesions and lung lymphoid-cell populations pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (sendai) virus respiratory tact lesions in weanling outbred rats infected with sendai virus increased tumor necrosis factor-alpha (tnf-alpha) gene expression in parainfluenza type (sendai) virus-induced bronchiolar fibrosis il- reduces the severity of sendai virus-induced bronchiolar inflammation and remodeling studies of respiratory disease in random source laboratory dogs: viral infections in unconditioned dogs visceral larva migrans in the dog lesions produced by a new lung worm in beagle dogs diagnostic exercise: macaque with dyspnoea treatment of pulmonary acariasis in rhesus macaques with ivermectin a primer of primate pathology: lesions and nonlesions attachment of microbes to host cells: relevance o^ pneumocystis carinii animal model: pneumocystis carinii pneumonia in the immunosuppressed rat attachment of pneumocystis carinii to rat pneumocytes a progress report on the treatment of patients with advanced cancer using lymphokine-activated killer cells and interleukin or high-dose interleukin alone toxicity of human recombinant interleukin- in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement toxicity of human recombinant interleukin- in the mouse is mediated by interleukin-activated lymphoc ^es. separation of efficacy and toxicity by selective lymphocyte subset depletion characterization of the pulmonary lesions induced in rats by human recombinant interleukin- dyspnoea and thoracic spinal deformation in rats after oral prizidilol (skandf -a ) diagnostic exercise: pneumonia in a rat multidrug chemotherapy of tuberculosis in rhesus monkeys granulomatous pneumonitis induced by bacille calmette-guerin in the mouse and its treatment with cyclosporin a anergy-like immunosuppression in mice bearing pulmonary foreign body granulomatous inflammation direct evidence for granulomainducing role of interleukin- cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity animal model: pulmonary granulomatous vasculitis induced in rats by treatment with glucan naturally occurring diatomaceous pneumoconiosis in subhuman primates ultrastructural and micropulse analysis of simian lung mite pigments lung c ^okine production in bleomycin-induced pulmonary fibrosis treatment of human recombinant soluble tnf receptor of pulmonary fibrosis induced by bleomycin or silica in mice the physiology of transforming growth factor-a tnf-a receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos extensive laminin and basement membrane accumulation occurs at the onset of bleomycin-induced rodent pulmonary fibrosis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease methotrexate pneumonitis induced by intrathecal methotrexate therapy. a case report with pharmacokinetic data cytotoxic drug-induced pulmonary disease: update s lung toxicity associated with cyclophosphamide use. two distinct patterns interstitial pneumonitis associated with bleomycin therapy bleomycin lung toxicity: who are the patients with increased risk? preclinical toxicologic evaluation of bleomycin (nsc ), a new antitumor antibiotic bleomycin-induced pulmonary toxicity in the rat intratracheal versus intravenous administration of bleomycin in mice: acute effects an investigation of possible models for the production of progressive pulmonary fibrosis in the rat. the effects of repeated intraatracheal instillation of bleomycin capillary remodeling in bleomycin-induced pulmonary fibrosis the role of strain variation in murine bleomycin-induced pulmonary fibrosis pulmonary toxicology of cyclophosphamide: a -year study animal models of emphysema lung scleroproteins in young and adult rats and in rats with spontaneous emphysema: comparative studies by biochemical and histochemical approach a quantitative study of stenosis in the respiratory bronchiole of the rat in n -induced emphysema long-term sequelae of bronchiolitis induced by nitrogen dioxide in hamsters cationic amphiphilic drug-induced phospholipidosis drug-induced phospholipidoses drug-induced lipidosis and the alveolar macrophage drug-induced lysosomal disorders in laboratory animals: new substances acting on lysosomes pulmonary and generalized lyosomal storage induced by amphiphilic drugs drug-induced generalized phospholipidosis tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses fine structural alterations in the lungs of iprindole-treated rats a cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development validation of an in vitro screen for phospholipidosis using a high-content biology platform multifocal histiocytosis in the lungs of rats the induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone collecting and processing tissues for diagnostic electron microscopy chlorphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species amiodarone lung toxicity: a human and experimental study generalized phospholipidosis induced by amphiphilic cationic psychotropic drug morphological and biochemical changes in the liver of various species in experimental phospholipidosis after diethylaminoethoxyhexestrol recovery from amiodarone-induced lipidosis in laboratory animals. a toxicological study epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine d selective antagonist pnu- myopathy related to administration of a cationic amphiphilic drug and the use of multidose drug distribution analysis to predict its occurrence drug-induced phospholipidosis: are there functional consequences? an -year-old man with dyspnea and pulmonary abnormalities amiodarone induced phospholipidosis. biochemical, morphological and functional changes in the lungs of rats chronically treated with amiodarone granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study amiodarone-associated pulmonary fibrosis. evidence of an immunologically mediated mechanism an evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity lipidosis induced by amphiphilic cationic drugs pathological discussion. case record of the massachusetts general hospital organomegaly and histopathology in an animal model of mucopolysaccharidosis induced by suramin lysosomal-storage disorder induced by elmiron following -days gavage administration in rats and mice foam cell response in the lung and lymphatic tissues during long-term high-level treatment with erythromycin interactions of macrolide antibiotics (erythromycin a, roxithromycin, erythromycylamine [dirithromycin], and azithromycin) with phospholipids: computer-aided conformational analysis and studies on acellular and cell culture models alveolar proteinosis and phospholipidosis of the lungs pulmonary alveolar proteinosis imatinib-associated pulmonary alveolar proteinosis imatinib mesylate inhibits the profibrogenic activity of tgf-( and prevents bleomycin-mediated lung fibrosis an experimental study of hypersecretion of mucus in the bronchial tree goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with or without the antiinflammatory agent phenylmethyloxadiazole measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis mitotic activity of airway epithelium after short exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole the effect of isoprenaline and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen experimental chronic bronchitis classification of cystic keratinising squamous lesions of the rat lung: report of a workshop pathological changes during aging in barrier-reared fischer male rats neoplastic and nonneoplastic lesions in aging f rats histopathological profile of a wistar rat stock including a survey of the literature naturally occurring sendai disease of mice immunohistochemical demonstration of clara cell antigen in lung tumors of bronchiolar origin induced by n-nitrosodiethylamine in syrian golden hamsters sequential morphologic alterations in the bronchial epithelium of syrian golden hamsters during n-nitrosomorpholine-induced pulmonary tumorigenesis lung endocrine-like cells in hamsters treated with diethylnitrosamine: alterations in vivo and in cell culture -nitroquinoline -oxide-induced pulmonary endocrine cell hyperplasia in s rrian golden hamsters increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine-cells of hypoxic rats the world health organization classification of lung tumors pleura, thymus and heart a review of chronic inhalation studies with mainstream cigarette smoke in rats and mice a review of chronic inhalation studies with mainstream cigarette smoke, in hamsters, dogs, and nonhuman primates animal models of mesothelioma induced by mineral fibers: implications for human risk assessment morphology of spontaneous and induced tumors in the bronchiolo-alveolar region of f rats spontaneous cystic keratinising epithelioma in the lung of a sprague-dawley rat strain a mouse lung tumor bioassay variability in the rates of some common naturally occurring tumors in fischer rats and (c bl/ nxc h/hen)fl (b c f ) mice a carcinogenicity study in mice of a ( -adrenegic antagonist, primidolol; increased total tumour incidence without tissue specificity effect of diet on spontaneous disease in the inbred mouse strain c b / j pol iota is a candidate for the mouse pulmonary adenoma resistance locus, a major modifier of chemically induced lung neoplasia cancer susceptibility in the mouse: genetics, biology and implications for human cancer histogenesis of the papillary clara cell adenoma histologic and ultrastructural features of the clara cell adenoma of the mouse lung immunocytochemical localization of the surfactant apoprotein and clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice immunohistochemical analysis of clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis strain a/j mouse lung adenoma patterns vary when induced by different carcinogens a chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility marketed human pharmaceuticals reported to be tumorigenic in rodents evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate induction of lung tumors and malignant lymphomas in mice by metronidazole toxicologic evaluation of metronidazole with particular reference to carcinogenic, mutagenic, and teratogenic potential induced pulmonary tumors in mice. ii: reaction of lungs of strain a mice to carcinogenic hydrocarbons. arc/iii;es of pathology strain a mouse pulmonary tumor test results for chemicals previously tested in the national cancer institute carcinogenicity tests animal model: spontaneous carcinoma of the lung in hamsters key: cord- - lmwnfda authors: ray, sumanta; lall, snehalika; mukhopadhyay, anirban; bandyopadhyay, sanghamitra; schonhuth, alexander title: predicting potential drug targets and repurposable drugs for covid- via a deep generative model for graphs date: - - journal: nan doi: nan sha: doc_id: cord_uid: lmwnfda coronavirus disease (covid- ) has been creating a worldwide pandemic situation. repurposing drugs, already shown to be free of harmful side effects, for the treatment of covid- patients is an important option in launching novel therapeutic strategies. therefore, reliable molecule interaction data are a crucial basis, where drug-/protein-protein interaction networks establish invaluable, year-long carefully curated data resources. however, these resources have not yet been systematically exploited using high-performance artificial intelligence approaches. here, we combine three networks, two of which are year-long curated, and one of which, on sars-cov- -human host-virus protein interactions, was published only most recently ( th of april ), raising a novel network that puts drugs, human and virus proteins into mutual context. we apply variational graph autoencoders (vgaes), representing most advanced deep learning based methodology for the analysis of data that are subject to network constraints. reliable simulations confirm that we operate at utmost accuracy in terms of predicting missing links. we then predict hitherto unknown links between drugs and human proteins against which virus proteins preferably bind. the corresponding therapeutic agents present splendid starting points for exploring novel host-directed therapy (hdt) options. the pandemic of covid- (coronavirus disease- ) has affected more than million people. so far, it has caused about . million deaths in over countries worldwide (https://coronavirus.jhu.edu/map.html), with numbers still increasing rapidly. covid- is an acute respiratory disease caused by a highly virulent and contagious novel coronavirus strain, sars-cov- , which is an enveloped, single-stranded rna virus . sensing the urgency, researchers have been relentlessly searching for possible therapeutic strategies in the last few weeks, so as to control the rapid spread. in their quest, drug repurposing establishes one of the most relevant options, where drugs that have been approved (at least preclinically) for fighting other diseases, are screened for their possible alternative use against the disease of interest, which is covid- here. because they were shown to lack severe side effects before, risks in the immediate application of repurposed drugs are limited. in comparison with de novo drug design, repurposing drugs offers various advantages. most importantly, the reduced time frame in development suits the urgency of the situation in general. furthermore, most recent, and most advanced artificial intelligence (ai) approaches have boosted drug repurposing in terms of throughput and accuracy enormously. finally, it is important to understand that the d structures of the majority of viral proteins have remained largely unknown, which raises the puts up the obstacles for direct approaches to work even higher. the foundation of ai based drug repurposing are molecule interaction data, optimally reflecting how drugs, viral and host proteins get into contact with each other. during the life cycle of a virus, the viral proteins interact with various human proteins in the infected cells. through these interactions, the virus hijacks the host cell machinery for replication, thereby affecting the normal function of the proteins it interacts with. to develop suitable therapeutic strategies and design antiviral drugs, a comprehensive understanding of the interactions between viral and human proteins is essential . when watching out for drugs that can be repurposed to fight the virus, one has to realize that targeting single virus proteins easily leads to the viruses escaping the (rather simpleminded) attack by raising resistance-inducing mutations. therefore, host-( ) we link existing high-quality, long-term curated and refined, large scale drug/protein -protein interaction data with ( ) molecular interaction data on sars-cov- itself, raised only a handful of weeks ago, ( ) exploit the resulting overarching network using most advanced, ai boosted techniques ( ) for repurposing drugs in the fight against sars-cov- ( ) in the frame of hdt based strategies. as for ( )-( ), we will highlight interactions between sars-cov- -host protein and human proteins important for the virus to persist using most advanced deep learning techniques that cater to exploiting network data. we are convinced that many of the fairly broad spectrum of drugs we raise will be amenable to developing successful hdt's against covid- . in the following, we will first describe the workflow of our analysis pipeline and the basic ideas that support it. we proceed by carrying out a simulation study that proves that our pipeline accurately predicts missing links in the encompassing drug -human protein -sars-cov- -protein network that we raise and analyze. namely we demonstrate that our (high-performance, ai supported) prediction pipeline accurately re-establishes links that had been explicitly removed before. this provides sound evidence that the interactions that we predict in the full network most likely reflect true interactions between molecular interfaces. subsequently, we continue with the core experiments. we predict links to be missing in the full (without artificially having removed links), encompassing drug -human protein -sars-cov- -protein network, raised by combining links from year-long curated resources on the one hand and most recently published covid- resources on the other hand. as per our simulation study, a large fraction, if not the vast majority of the predictions establish true, hence actionable interactions between drugs on the one hand and sars-cov- associated human proteins (hence of use in hdt) on the other hand. a b c d figure . overall workflow of the proposed method: the three networks sars-cov- -host ppi, human ppi, and drug-target network (panel-a) are mapped by their common interactors to form an integrated representation (panel-b). the neighborhood sampling strategy node vec converts the network into fixed-size low dimensional representations that perverse the properties of the nodes belonging to the three major components of the integrated network (panel-c). the resulting feature matrix (f) from the node embeddings and adjacency matrix (a) from the integrated network are used to train a vgae model, which is then used for prediction (panel-d). for the purposes of high-confidence validation, we carry out a literature study on the overall drugs we put forward. for this, we inspect the postulated mechanism-of-action of the drugs in the frame of several diseases, including sars-cov and mers-cov driven diseases in particular. see figure for the workflow of our analysis pipeline and the basic ideas that support it. we will describe all important steps in the paragraphs of this subsection. this reduces the training time compared to the general graph autoencoder model. we tested the model performance for a different number of sampled nodes, keeping track of the area under the roc curve (auc), average precision (ap) score, and model training time in the frame of a train-validation-test split at proportions : : . table shows the performance of the model for sampled sugraph sizes n s = , , , and . for sampled nodes, the model's performance is sufficiently good enough concerning its training time and validation-auc and -ap score. the average test roc-auc and ap score of the model for n s = are . ± . and . ± . . to know the efficacy of the model in discovering the existing edges between only cov-host and drug nodes, we train the model (with n s = ) on an incomplete version of the graph where the links between cov-host and drugs have been removed. we further compute the feature matrix f based on the incomplete graph, and use it. the test set consists of all the previously removed edges. the model performance is no doubt better for discovering those edges between cov-host and drug nodes (roc-auc: . ± . ap: . ± . for runs). the fastgae model is learned with the feature matrix (f) and adjacency matrix (a). the node feature matrix (f) is obtained from a using the node vec neighborhood sampling strategy. the model performance is evaluated with and without using f as feature matrix. figure shows the average performance of the model on validation sets with and without f as input for the different number of sampling nodes. we calculate average auc, and ap scores for complete runs of the model. from figure , it is evident that including f as feature matrix enhances the model's performance markedly. we use the node vec framework to learn low dimensional embeddings of each node in the compiled network. it uses the skipgram algorithm of the word vec model to learn the embeddings, which eventually groups nodes with a similar 'role' or having a similar 'connection pattern' within the graph. similar 'role' ensures that nodes within the sets/groups are structurally similar/equivalent than the other nodes outside the groups. two nodes are said to be structurally equivalent if they have identical connection patterns to the rest of the network . to explore this, we have analyzed the embedding results in two steps. first, we explore structurally equivalent nodes to identify 'roles' and similar connection patterns to the rest of the networks, and later use lovain clustering to examine the same within the groups/clusters. the most_similar function of the node vec inspects the structurally equivalent nodes within the network. we find out all the cov-host nodes which are most similar to the drug nodes. while it is expected to observe nodes of the same types within the neighborhood of a particular node, in some cases, we found some drugs are neighbors of cov-host proteins with high probability (pobs > . ). sars-cov- cl protease . some other drugs such as 'clenbuterol' and 'fenbendazole', the probable neighbor of ppp cb and eef a respectively, are used as bronchodilators in asthma. to explore the closely connected groups, we have constructed a neighborhood graph using the k-th nearest neighbor algorithm from the node embeddings and apply louvain clustering ( figure -panel-c). although there is a clear separation between host proteins (including cov-host) cluster and drug cluster, some of the louvain clusters contain both types of nodes. for example, louvain cluster- and - contain four and two drugs along with the other cov-host proteins, respectively. figure panel-d represents a network consisting of these six drugs and their most similar cov-host nodes. for drug-cov-host interaction prediction, we exploit variational graph autoencoder (vgae), an unsupervised graph neural network model, first introduced in to leverage the concept of variational autoencoder in graph-structured data. to make learning faster, we utilized the fastgae model to take advantage of the fast decoding phase. we have used two data matrices in the fastgae model for learning: one is the adjacency matrix, which represents the interaction information over all the nodes, and the other one is the feature matrix representing the low-dimensional embeddings of all the nodes in the network. we create a test set of 'non-edges' by removing all existing links between drugs and cov-host proteins from all possible combinations ( cov-host × drugs) of edges. the model is trained on the whole network with the adjacency matrix a and feature matrix f. the trained model is then applied to the test 'non-edges' to know the most probable links. we identified a total of most probable links with drugs and cov-host proteins with a probability threshold of . . the predicted cov-host proteins are involved in different crucial pathways of viral infection (table ). the p-values for pathway and go enrichment are calculated by using the hypergeometric test with . fdr corrections. figure , panel-a shows the heatmap of probability scores between predicted drugs and cov-host proteins. to get more details of the predicted bipartite graph, we figure . drug-cov-host predicted interaction: panel-a shows heatmap of probability scores between drugs and cov-host proteins. the four predicted bipartite modules are annotated as b , b , b and b within the heatmap. the drugs are colored based on their clinical phase (red-launched, preclinical-blue, phase /phase -green and phase- / phase- -black ). panel-b, c, d and e represents networks corresponding to b , b , b and b modules.the drugs are annotated using the disease area found in cmap database a b c d e figure . predicted interactions for probability threshold: . . panel-a shows the interaction graph between drugs and cov-host. drugs are annotated with their usage. panel-b, c, d and e represents quasi-bicliques for one, two, three and more than three drugs molecules respectively. use a weighted bipartite clustering algorithm proposed by j. beckett . this results in bipartite modules (panel-a figure ): b ( drugs, cov-host), b ( drugs, cov-host), b ( rugs and cov-host), and b ( drugs and cov-host). the other panels of the figure show the network diagram of four bipartite modules. b contains drugs, including some antibiotics (anisomycin, midecamycin), and anti-cancer drugs (doxorubicin, camptothecin). b also has some antibiotics such as puromycin, demeclocycline, dirithromycin, geldanamycin, and chlortetracycline, among them, the first three are widely used for bronchitis, pneumonia, and respiratory tract infections . some other drugs such as lobeline and ambroxol included in the b module have a variety of therapeutic uses, including respiratory disorders and bronchitis. the high confidence predicted interactions (with threshold . ) is shown in figure panel-a. to highlight some repurposable drug combination and their predicted cov-host target, we perform a weighted clustering (clusterone) on this network and found some quasy-bicluques (shown in panel-b-e) we matched our predicted drugs with the drug list recently published by zhou et al. and found six common drugs: mesalazine, vinblastine, menadione, medrysone, fulvestrant, and apigenin. among them, apigenin has a known effect in the antiviral activity together with quercetin, rutin, and other flavonoids . mesalazine is also proven to be extremely effective in the treatment of other viral diseases like influenza a/h n virus. . baclofen, a benzodiazepine receptor (gabaa-receptor) agonist, has a potential role in antiviral associated treatment . antiinflammatory antecedents fisetin is also tested for antiviral activity, such as for inhibition of dengue (denv) virus infection . it down-regulates the production of proinflammatory cytokines induced by a denv infection. both of the drugs are listed in the high confidence interaction set with the three cov-hosts: tapt (interacted with sars-cov- protein: orf c), slc a (interacted with sars-cov- protein: orf c), and trim (interacted with sars-cov- protein: orf a) ( figure -panel-c). topoisomerase inhibitors play an active role as antiviral agents by inhibiting the viral dna replication , . some topoisomerase inhibitors such as camptothecin, daunorubicin, doxorubicin, irinotecan and mitoxantrone are predicted to interact with several cov-host proteins. it has been demonstrated that the anticancer drug camptothecin (cpt) and its derivative irinotecan have a potential role in antiviral activity , . it inhibits host cell enzyme topoisomerase-i which is required for the initiation as well as completion of viral functions in host cell . daunorubicin (dnr) has also been demonstrated as an inhibitor of hiv- virus replication in human host cells . the conventional anticancer antibiotic doxorubicin was identified as a selective inhibitor of in vitro dengue and yellow fever virus replication . it is also reported that doxorubicin coupling with monoclonal antibody can create an immunoconjugate that can eliminate hiv- infection in mice cell . mitoxantrone shows antiviral activity against the human herpes simplex virus (hsv ) by reducing the transcription of viral genes in many human cells that are essential for dna synthesis . histone deacetylases inhibitors (hdaci) are generally used as latency-reversing agents for purging hiv- from the latent reservoir like cd memory cell . our predicted drug list (table ) contains two hdaci: scriptaid and vorinostat. vorinostrate can be used to achieve latency reversal in the hiv- virus safely and repeatedly . asymptomatic patients infected with sars-cov- are of significant concern as they are more vulnerable to infect large number of people than symptomatic patients. moreover, in most cases ( percentile), patients develop symptoms after an average of - days, which is longer than the incubation period of sars, mers, or other viruses . to this end, hdaci may serve as good candidates for recognizing and clearing the cells in which sars-cov- latency has been reversed. heat shock protein (hsp) is described as a crucial host factor in the life cycle of several viruses that includes an entry in the cell, nuclear import, transcription, and replication , . hsp is also shown to be an essential factor for sars-cov- envelop (e) protein . in , hsp is described as a promising target for antiviral drugs. the list of predicted drugs contains three hsp inhibitors: tanespimycin, geldanamycin, and its derivative alvespimycin. the first two have a substantial effect in inhibiting the replication of herpes simplex virus and human enterovirus (ev ), respectively. recently in , geldanamycin and its derivatives are proposed to be an effective drug in the treatment of covid- . inhibiting dna synthesis during viral replication is one of the critical steps in disrupting the viral infection. the list of predicted drugs contains six such small molecules/drugs, viz., niclosamide, azacitidine, anisomycin, novobiocin, primaquine, menadione, and metronidazole. dna synthesis inhibitor niclosamide has a great potential to treat a variety of viral infections, including sars-cov, mers-cov, and hcv virus and has recently been described as a potential candidate to fight the / sars-cov- virus . novobiocin, an aminocoumarin antibiotic, is also used in the treatment of zika virus (zikv) infections due to its protease inhibitory activity. in , chloroquine (cq) had been demonstrated as an effective drug against the spread of severe acute respiratory syndrome (sars) coronavirus (sars-cov). recently hydroxychloroquine (hcq) sulfate, a derivative of cq, has been evaluated to efficiently inhibit sars-cov- infection in vitro . therefore, another anti-malarial aminoquinolin drug primaquine may also contribute to the attenuation of the inflammatory response of covid- patients. primaquine is also established to be effective in the treatment of pneumocystis pneumonia (pcp) . cardiac glycosides have been shown to play a crucial role in antiviral drugs. these drugs target cell host proteins, which help reduce the resistance to antiviral treatments. the antiviral effects of cardiac glycosides have been described by inhibiting the pump function of na, k-atpase. this makes them essential drugs against human viral infections. the predicted list of drugs contains three cardiac glycosides atpase inhibitors: digoxin, digitoxigenin, and ouabain. these drugs have been reported to be effective against different viruses such as herpes simplex, influenza, chikungunya, coronavirus, and respiratory syncytial virus . mg , proteasomal inhibitor is established to be a strong inhibitor of sars-cov replication in early steps of the viral life cycle . mg inhibits the cysteine protease m-calpain, which results in a pronounced inhibition of sars-cov- replication in the host cell. in , resveratrol has been demonstrated to be a significant inhibitor mers-cov infection. resveratrol treatment decreases the expression of nucleocapsid (n) protein of mers-cov, which is essential for viral replication. as mg and resveratrol play a vital role in inhibiting the replication of other coronaviruses sars-cov and mers-cov, so they may be potential candidates for the prevention and treatment of sars-cov- . another drug captopril is known as angiotensin ii receptor blockers (arb), which directly inhibits the production of angiotensin ii. in , angiotensin-converting enzyme (ace ) is demonstrated as the binding site for sars-cov- . so angiotensin ii receptor blockers (arb) may be good candidates to use in the tentative treatment for sars-cov- infections . in summary, our proposed method predicts several drug targets and multiple repurposable drugs that have prominent literature evidence of uses as antiviral drugs, especially for two other coronavirus species sars-cov and mers-cov. some drugs are also directly associated with the treatment of sars-cov- identified by recent literature. however, further clinical trials and several preclinical experiments are required to validate the clinical benefits of these potential drugs and drug targets. in this work, we have successfully generated a list of high-confidence candidate drugs that can be repurposed to counteract sars-cov- infections. the novelties have been to integrate most recently published sars-cov- protein interaction data on the one hand, and to use most recent, most advanced ai (deep learning) based high-performance prediction machinery on the other hand, as the two major points. in experiments, we have validated that our prediction pipeline operates at utmost accuracy, confirming the quality of the predictions we have raised. the recent publication (april , ) of two novel sars-cov- -human protein interaction resources , has unlocked enormous possibilities in studying virulence and pathogenicity of sars-cov- , and the driving mechanisms behind it. only now, various experimental and computational approaches in the design of drugs against covid- have become conceivable, and only now such approaches can be exploited truly systematically, at both sufficiently high throughput and accuracy. here, to the best of our knowledge, we have done this for the first time. we have integrated the new sars-cov- protein interaction data with well established, long-term curated human protein and drug interaction data. these data capture hundreds of thousands approved interfaces between encompassing sets of molecules, either reflecting drugs or human proteins. as a result, we have obtained a comprehensive drug-human-virus interaction network that reflects the latest state of the art in terms of our knowledge about how sars-cov- and interacts with human proteins and repurposable drugs. for exploiting the new network-already establishing a new resource in its own right-we have opted for most recent and advanced deep learning based technology. a generic reason for this choice is the surge in advances and the resulting boost in operative prediction performance of related methods over the last - years. a particular reason is to make use of most advanced graph neural network based techniques, namely variational graph autoencoders as a deep generative model of utmost accuracy, the practical implementation of which was presented only a few months ago (just like the relevant network data). note that only this recent implementation enables to process networks of sizes in the range of common molecular interaction data. in essence, graph neural networks "learn" the structure of links in networks, and infer rules that underlie the interplay of links. based on the knowledge gained, they enable to predict links and output the corresponding links together with probabilities for them to indeed be missing. simulation experiments, reflecting scenarios where links known to exist in our network were re-established by prediction upon their removal, pointed out that our pipeline does indeed predict missing links at utmost accuracy. encouraged by these simulations, we proceeded by performing the core experiments, and predicted links to be missing without prior removal of links in our encompassing network. these core experiments revealed high confidence interactions relating to drugs. in our experiments, we focused on predicting links between drugs and human proteins that in turn are known to interact with sars-cov- proteins (sars-cov- associated host proteins). we have decidedly put the focus not on drug -sars-cov- -protein interactions, which would have reflected more direct therapy strategies against the virus. instead, we have focused on predicting drugs that serve the purposes of host-directed therapy (hdt) options, because hdt strategies have proven to be more sustainable with respect to mutations by which the virus escapes a response to the therapy applied. note that hdt strategies particularly cater to drug repurposing attempts, because repurposed drugs have already proven to lack severe side effects, because they are either already in use, or have successfully passed the preclinical trial stages. we further systematically categorized the repurposable drugs into categories based on their domains of application and molecular mechanism. according to this, we identified and highlighted several drugs that target host proteins that the virus needs to enter (and subsequently hijack) human cells. one such example is captopril, which directly inhibits the production of angiotensin-converting enzyme- (ace- ), in turn already known to be a crucial host factor for sars-cov- . further, we identified primaquine, as an antimalaria drug used to prevent the malaria and also pneumocystis pneumonia (pcp) relapses, because it interacts with the tim complex timm and alg . moreover, we have highlighted drugs that act as dna replication inhibitor (niclosamide, anisomycin), glucocorticoid receptor agonists (medrysone), atpase inhibitors (digitoxigenin, digoxin), topoisomerase inhibitors (camptothecin, irinotecan), and proteosomal inhibitors (mg- ). note that some drugs are known to have rather severe side effects from their original use (doxorubicin, vinblastine), but the disrupting effects of their short-term usage in severe covid- infections may mean sufficient compensation. in summary, we have compiled a list of drugs, which when repurposed are of great potential in the fight against the covid- pandemic, where therapy options are urgently needed. our list of predicted drugs suggests both options that had been identified and thoroughly discussed before and new opportunities that had not been pointed out earlier. the latter class of drugs may offer valuable chances for pursuing new therapy strategies against covid- . we have utilized three categories of interaction datasets: human protein-protein interactome data, sars-cov- -host protein interaction data, and drug-host interaction data. we have taken sars-cov- -host interaction information from two recent studies by gordon et al and dick et al , . in , high confidence interactions between sars-cov- and human proteins are predicted using using affinity-purification mass spectrometry (ap-ms). in , high confidence interactions are identified using sequence-based ppi predictors (pipe & sprint). the drug-target interaction information has been collected from five databases, viz., drugbank database (v . ) , chembl database, therapeutic target database (ttd) , pharmgkb database, and iuphar/bps guide to pharmacology . total number of drugs and drug-host interactions used in this study are and , respectively. we have built a comprehensive list of human ppis from two datasets: ( ) ccsb human interactome database consisting of , genes, and high-quality binary interactions - , ( ) the human protein reference database which consists of proteins and ppis. the summary of all the datasets is provided in table . cmap database is used to annotate the drugs with their usage different disease areas. we have utilized node vec , an algorithmic framework for learning continuous feature representations for nodes in networks. it maps the nodes to a low-dimensional feature space that maximizes the likelihood of preserving network neighborhoods. the principle of feature learning framework in a graph can be described as follows: let g = (v, e) be a given graph, where v represents a set of nodes, and e represents the set of edges. the feature representation of nodes (|v |) is given by a mapping function: f : v → r d , where d specify the feature dimension. the f may also be represented as a node feature matrix of dimension of |v | × d. for each node, v ∈ v , nn s (v) ⊂ v defines a network neighborhood of node v which is generated using a neighbourhood sampling strategy s. the sampling strategy can be described as an interpolation between breadth-first search and depth-first search technique . the objective function can be described as: this maximizes the likelihood of observing a network neighborhood nn s (v) for a node v given on its feature representation f . now the probability of observing a neighborhood node n i ∈ nn s (v) given the feature representation of the source node v is given as : where, n i is the i th neighbor of node v in neighborhood set nn s (v). the conditional likelihood of each source (v) and neighborhood node (n i ∈ nn s (v )) pair is represented as softmax of dot product of their features f (v) and f (n i ) as follows: variational graph autoencoder (vgae) is a framework for unsupervised learning on graph-structured data . this model uses latent variables and is effective in learning interpretable latent representations for undirected graphs. the graph autoencoder consists of two stacked models: ) encoder and ) decoder. first, an encoder based on graph convolution networks (gcn) maps the nodes into a low-dimensional embedding space. subsequently, a decoder attempts to reconstruct the original graph structure from the encoder representations. both models are jointly trained to optimize the quality of the reconstruction from the embedding space, in an unsupervised way. the functions of these two model can be described as follows: encoder: it uses graph convolution network (gcn) on adjacency matrix a and the feature representation matrix f. encoder generates a d -dimensional latent variable z i for each node i ∈ v , with |v | = n, that corresponds to each embedding node, with d ≤ n. the inference model of the encoder is given below: where, r(z i |a, f) corresponds to normal distribution, n ( z i µ i , σ i ), µ i and σ i are the gaussian mean and variance parameters. the actual embedding vectors z i are samples drawn from these distributions. decoder: it is a generative model that decodes the latent variables z i to reconstruct the matrix a using inner products with sigmoid activation from embedding vector, (z). where, a is the decoded adjacency matrix. the objective function of the variational graph autoencoder (vgae) can be written as: the objective function c v gae maximizes the likelihood of decoding the adjacency matrix w.r.t graph autoencoder weights using stochastic gradient decent. here, d kl (.||.) represents kullback-leibler divergence and p(z) is the prior distribution of latent variable. drug-sars-cov- link prediction . adjacency matrix preparation in this work, we consider an undirected graph g = (v, e) with |v | = n nodes and |e| = m edges. we denote a as the binary adjacency matrix of g. here v consists of sars-cov- proteins, cov-host proteins, drug-target proteins and drugs. the matrix (a) contains a total of n = nodes given as: where, n nc is the number of sars-cov- proteins. n dt is the number of drug targets, whereas n nt and n d represent the number of cov-host and drugs nodes, respectively. total number of edges is given by: where, e represents interactions between sars-cov- and human host proteins, e is the number of interactions among human proteins, and e represents the number of interactions between drugs and human host proteins. the neighborhood sampling strategy is used here to prepare a feature representation of all nodes. a flexible biased random walk procedure is employed to explore the neighborhood of each node. a random walk in a graph g can be described as the probability: where, π(v, x) is the transition probability between nodes v and x, where (v, x) ∈ e and a i is the i th node in the walk of length l. the transition probability is given by π(v, x) = c pq (t, x) * w vx , where t is the previous node of v n the walk, w vx is the static edge weights and p, q are the two parameters which guides the walk. the coefficient c pq (t, x) is given by where, distance(t, x) represents the shortest path distance between nodes t and node x. the process of feature matrix f n×d generation is governed by the node vec algorithm. it starts from every nodes and simulates r random walks of fixed length l. in every step of walk transition probability π(v, x) govern the sampling. the generated walk of each iteration is included to a walk-list. finally, the stochastic gradient descent is applied to optimize the list of walks and result is returned. . link prediction: scalable and fast variational graph autoencoder (fastvgae) is utilized in our proposed work to reduce the computational time of vgae in large network. the adjacency matrix a and the feature matrix f are given into the encoder of fastvgae. the encoder uses graph convolution neural network (gcn) on the entire graph to create the latent representation (z). the encoder works on full adjacency matrix a. after encoding, sampling is done and decoder works on the sampled sub graph. the mechanism of decoder of fastvgae is slightly different from traditional vgae. it regenerate the adjacency matrix a based on a subsample of graph nodes, v s . it uses a graph node sampling technique to randomly sample the reconstructed nodes at each iteration. each node is assigned with a probability p i and the selection of noes is based on the high score of p i . the probability p i is given by the following equation: where, f (i) is the degree of node i, and α is the sharpening parameter. we take α = in our study. the node selection process is repeated until |v s | = n s , where n s is the number of sampling nodes. the decoder reconstructs the smaller matrix, a s of dimension n s × n s instead of decoding the main adjacency matrix a. the decoder function follows the following equation: a s (i, j) = sigmoid(z t i .z j ), ∀(i, j) ∈ v s ×v s . at each training iteration different subgraph (g s ) is drawn using the sampling method. after the model is trained the drug-cov-host links are predicted using the following equation: where a i j represents the possible links between all combination of sars-cov- nodes and drug nodes. for each combination of nodes the model gives probability based on the logistic sigmoid function. a new coronavirus associated with human respiratory disease in china host-pathogen systems biology host-directed therapies for bacterial and viral infections network-based drug repositioning: approaches, resources, and research directions new horizons for antiviral drug discovery from virus-host protein interaction networks drug target prediction and repositioning using an integrated network-based approach mapping protein interactions between dengue virus and its human and insect hosts a review of in silico approaches for analysis and prediction of hiv- -human protein-protein interactions network-based study reveals potential infection pathways of hepatitis-c leading to various diseases prediction of the ebola virus infection related human genes using protein-protein interaction network a genome-wide positioning systems network algorithm for in silico drug repurposing deepdr: a network-based deep learning approach to in silico drug repositioning network-based drug repurposing for novel coronavirus -ncov/sars-cov- network bioinformatics analysis provides insight into drug repurposing for covid- a sars-cov- protein interaction map reveals targets for drug repurposing comprehensive prediction of the sars-cov- vs. human interactome using pipe , sprint, and pipe-sites scalable feature learning for networks fastgae: fast, scalable and effective graph autoencoders with stochastic subgraph decoding from community to role-based graph embeddings specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus a next generation connectivity map: l platform and the first , , profiles improved community detection in weighted bipartite networks drugbank: a comprehensive resource for in silico drug discovery and exploration detecting overlapping protein complexes in protein-protein interaction networks the therapeutic potential of apigenin delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus baclofen promotes alcohol abstinence in alcohol dependent cirrhotic patients with hepatitis c virus (hcv) infection antiviral and immunomodulatory effects of polyphenols on macrophages infected with dengue virus serotypes and enhanced or not with antibodies evaluation of topoisomerase inhibitors as potential antiviral agents potent antiviral activity of topoisomerase i and ii inhibitors against kaposi's sarcoma-associated herpesvirus antiviral action of camptothecin an analog of camptothecin inactive against topoisomerase i is broadly neutralizing of hiv- through inhibition of vif-dependent apobec g degradation water-insoluble camptothecin analogues as potential antiviral drugs inhibition of hiv- replication by daunorubicin a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro elimination of hiv- infection by treatment with a doxorubicin-conjugated anti-envelope antibody antiviral activity of mitoxantrone dihydrochloride against human herpes simplex virus mediated by suppression of the viral immediate early genes histone deacetylase inhibitors for purging hiv- from the latent reservoir interval dosing with the hdac inhibitor vorinostat effectively reverses hiv latency the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application synthesis and in vitro anti-hsv- activity of a novel hsp inhibitor bj-b heat shock protein facilitates formation of the hbv capsid via interacting with the hbv core protein dimers severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis hsp : a promising broad-spectrum antiviral drug target drug repositioning suggests a role for the heat shock protein inhibitor geldanamycin in treating covid- infection broad spectrum antiviral agent niclosamide and its therapeutic potential hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro pharmacokinetic optimisation in the treatment of pneumocystis carinii pneumonia the antiviral effects of na, k-atpase inhibition: a minireview severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain effective inhibition of mers-cov infection by resveratrol structural basis of receptor recognition by sars-cov- angiotensin receptor blockers as tentative sars-cov- therapeutics next-generation sequencing to generate interactome datasets development of human protein reference database as an initial platform for approaching systems biology in humans drugbank . : shedding new light on drug metabolism chembl: a large-scale bioactivity database for drug discovery therapeutic target database update : enriched resource for bench to clinical drug target and targeted pathway information the iuphar/bps guide to pharmacology: an expert-driven knowledgebase of drug targets and their ligands towards a proteome-scale map of the human protein-protein interaction network a proteome-scale map of the human interactome network a reference map of the human binary protein interactome stochastic backpropagation and approximate inference in deep generative models on information and sufficiency. the annals mathematical statistics key: cord- -j bmgmhz authors: parreiras martins, maria auxiliadora; fonseca de medeiros, amanda; dias carneiro de almeida, claudmeire; moreira reis, adriano max title: preparedness of pharmacists to respond to the emergency of the covid- pandemic in brazil: a comprehensive overview date: - - journal: drugs ther perspect doi: . /s - - - sha: doc_id: cord_uid: j bmgmhz the outbreak of covid- in low- and middle-income countries is worrisome due to the social inequalities in these countries, their limited health budgets and the significant burden of other acute and chronic diseases. the leap in the number of cases in brazil has imposed a huge strain on the healthcare system. we sought to provide a comprehensive overview of the challenges encountered by pharmacy services in responding to the covid- pandemic emergency in brazil and discuss the role of clinical pharmacists in this context. pharmaceutical services play a key role in the emergency response to the pandemic. the pharmacy workforce has been actively working to manage drug shortages, redesign workflow, and review drug formularies/protocols to improve safety for patients and healthcare professionals (hcps). covid- patients may present high risk in the use of medications and clinical pharmacists can contribute substantially as part of a multidisciplinary team to improve outcomes in drug therapy in severe and critical illness. the participation of pharmacists as members of antimicrobial stewardship programs should be enhanced to ensure appropriate and safe use of antibiotics in this context. hcps should be encouraged to seek improvements in the performance of pharmaceutical services and innovative practices to respond to the pandemic. further studies are needed to generate knowledge on covid- to improve patient care in vulnerable populations. coronavirus disease (covid- ) is an emerging infectious disease first described in wuhan, china, in december , caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] . the outbreak of covid- spread rapidly worldwide, being declared a pandemic on mar by the world health organization (who). human-to-human transmission occurs via droplets or contaminated hands or surfaces [ ] . various levels of classic public health strategies, such as social distancing, isolation, quarantine, and community containment, have been applied across countries to reduce the dissemination of the disease [ ] . by july , sars-cov- had infected > million people in countries, creating an enormous burden on healthcare systems and the economy [ ] . it reached the southern hemisphere later than the other continents and the progress of its transmission in the winter 'flu' season is unclear. the infection spread in low-and middle-income countries (lmics) is worrisome due to their inability to respond rapidly to a pandemic situation in a scenario of social inequalities, limited health budgets, and the significant burden of other acute and chronic diseases [ , ] . the clinical spectrum of covid- is heterogeneous, encompassing asymptomatic disease, mild upper respiratory tract infection, and severe viral pneumonia that may progress to critical illness and death [ , ] . common signs of the disease include fever, myalgia, cough, and dyspnea [ ] . around % of confirmed covid- cases develop severe or critical illness [ ] , representing the subgroup of patients who are challenging frontline healthcare professionals (hcps) in hospitals. prognosis is poor for older adults [ ] and in the presence of comorbidities, such as cardiovascular diseases [ , ] . knowledge regarding covid- is being acquired at the bedside during the accelerated race to find efficacious drug treatments and vaccines. worldwide, clinical trials have been designed to investigate the efficacy and safety of old and new drug candidates as treatments for covid- , but no guidelines have been established thus far [ ] . in the context of assistance to covid- patients, pharmacy services need to consider adjustments in drug supply, workflow, and pharmaceutical care. we sought to provide a comprehensive overview of the challenges encountered by pharmacy services to respond to the covid- pandemic emergency and to describe its role in the brazilian context. the first case of covid- in brazil was officially confirmed on feb , and the infection has spread rapidly, infecting , , individuals by july , with , deaths [ ] . brazil is in second place with regard to the number of cases and deaths, being declared by the who as the new epicenter of the coronavirus outbreak. due to the low testing capacity, testing of severe cases has been prioritized and the total number of cases is still underestimated. according to the brazilian ministry of health, the number of hospitalizations to manage acute respiratory distress syndrome (ards) was > % higher in than the same period in [ , ] . the transmission of the virus is not yet controlled and brazil has been reported to have the highest rate of transmission (r of . ) among analyzed countries [ ] . it is hypothesized that covid- was imported by wealthy brazilians who travelled abroad and then transmitted locally to poor communities, who mostly live in precarious conditions [ , ] . social isolation and containment measures, as well as hygiene recommendations, are almost impossible to follow in these environments. healthcare in brazil is provided by public and private health systems. the first suspected and confirmed cases of covid- were referred to private services, but later poor people began to seek assistance in the public system. brazil is a large south american country with states with diverse social, cultural, and economic backgrounds. hospital beds are unequally distributed across the country and most beds in intensive care units (icus) are concentrated in the southeast region. people requiring ventilation support may outnumber the available ventilators and icu beds [ ] . in this context, covid- remains unequally spread in a country with a political scenario of uncertainties [ ] , involving instability in the ministry of health and polarization in the political and ideological fields. to provide assistance of quality in the middle of a crisis is extremely challenging, especially if we consider the particularities of our population. prior to the emergence of covid- , brazil had undergone a transition in epidemiological age, related to decreasing mortality and increasing life expectancy [ ] , with a projected increase in the population aged > years of almost threefold by [ ] . the burden of chronic conditions, including diabetes mellitus, cardiovascular diseases (e.g., ischemic heart disease and stroke), cancer, and obesity is growing. additionally, the public healthcare system still needs to cope with chronic infectious diseases, including hiv/aids, tuberculosis, syphilis, and malaria, and also endemic and emerging vector-borne arboviruses, such as yellow fever, dengue, zika, and chikungunya [ ] . in late march , scheduled elective healthcare procedures and appointments started being gradually cancelled within brazilian states to prevent virus transmission. strategies have been implemented to reorganize the available beds to assist covid- patients and other emergency clinical and surgical patients, and increase the number of beds by building temporary field hospitals where necessary. healthcare settings have restructured workflow, adapting protocols to address new requirements of biosafety and providing training to staff. despite the lack of conclusive scientific evidence, protocols are being rapidly updated to improve safety for patients and hcps. pharmaceutical services have been encouraged to be actively involved in various steps of these processes. the early experience of pharmaceutical services during the outbreak of covid- in china highlighted the indispensable work of community [ , ] and hospital [ ] pharmacists in the emergency response to the pandemic [ ] . the international pharmaceutical federation (fip) released the "coronavirus -ncov outbreak: information and interim guidelines for pharmacists and the pharmacy workforce", drafted with the participation of the chinese pharmaceutical association (cpa), to provide reliable information on the management of pharmacy operations [ ] . the pharmacy emergency preparedness and response (pepr) recommendations were published to guide pharmacy endeavors towards full integration within public health and in recognition of pharmacists' skills, roles, and contributions as integral members of multidisciplinary healthcare teams [ ] . these references can be used to create regional guidance for pharmacists and adapted to local demands and resources. the brazilian society of hospital pharmacy released recommendations for pharmacists working in different scenarios to fight against the pandemic [ ] . an economic disruption without precedent has occurred together with a dramatic increase in the consumption of health products worldwide. global drug shortages were triggered by inadequate quantities of raw materials, procurement and manufacturing issues, and drug discontinuations [ ] . in brazil, drug shortages also involved importation difficulties, exorbitant costs, compulsory advance payments, and noncompliance with agreed quantities and delivery deadlines. the supply of essential drugs has been jeopardized, including sedatives (fentanyl and midazolam) and neuromuscular agents (suxamethonium, atracurium). commitment of pharmacists is required to manage drug shortages and minimize the impact on patient outcomes [ , , , ] . the brazilian population has been impacted by a flurry of fake news. the fear of catching covid- and free access to drugstores have caused a rush to buy medicines for selftreatment. data presented by the federal board of pharmacy revealed a substantial increase in drug sales involving ascorbic acid ( . %), acetaminophen ( . %) and hydroxychloroquine sulfate ( . %) from january to march relative to the same period in [ ] . the off-label use of chloroquine/hydroxychloroquine for preventing or treating covid- imposes safety issues and requires the implementation of prescription monitoring strategies [ ] . panic buying of alcohol-based formulations, face shields, n respirators and other personal protective equipment (ppe) has also occurred, leading to shortages in hospitals and other healthcare facilities. the supply has been partially restored after government interventions and the recommendation to the public to wear fabric face masks. population health interventions and the support of pharmacists for the rational use of drugs was shown to be of utter importance in this scenario. the brazilian branch of the institute for safe medication practices (ismp-brasil; https ://www.ismp-brasi l.org/ site/) has provided important contributions for this approach. another concern is the effect of covid- on increasing morbimortality of patients with chronic diseases, given their compromised access to healthcare and diagnostic tests. delays in assessment may worsen the prognosis of chronic diseases. kretchy et al. [ ] discussed the challenges of managing chronic diseases and promoting medication adherence in lmics. community pharmacists were identified as front-line personnel to promote healthy lifestyle adjustments, refill prescriptions, provide counseling on the management of chronic diseases, and minimize unsafe self-medication habits with over-the-counter medicines. pharmacists working through telehealth services could enhance outcomes in drug therapy and mitigate strategies to reduce the risks of covid- contagion, such as triaging people with flu-like symptoms and improving workflow for physical distancing and self-protection [ , , ] . strategies to support the public and hcps to cope with stress, anxiety, and poor sleep quality during the pandemic have been reported worldwide [ , , ] , and remote services have gradually started to operate with this aim in brazil. however, the number of people with access to remote assistance is still limited. pharmaceutical services have been adjusting to respond to the covid- outbreak at the level of public and private institutions, with attempts to meet the needs of patient care in each brazilian region, according to the available financial and human resources. hcps and students have been recruited by the ministry of health through the program "brasil conta comigo" ('brazil counts on me') as volunteers to face the pandemic. basheti et al. [ ] investigated the pharmacists' and pharmacy students' awareness of their role during this emergent situation in jordan and the source of their information on covid- . in brazil, the education and training of pharmacy professionals and students are under intense discussion in universities and healthcare settings. live broadcasts, remote trainings and recorded courses using the internet and other media are being used as important means of education. pharmacists have been working on laboratory testing for covid- , administration of pharmacy supplies and replenishment, and also patient care, replicating experiences in other countries [ , , , ] . attributes of pharmaceutical services in the community and hospital settings in brazil are summarized in fig. . in community pharmacies, actions are focused on patients, comprising provision of information on covid- , promotion of the safe use of medicines, and guidance on medication adherence in acute and chronic diseases. in hospital pharmacies, beyond interactions with patients, pharmacists develop integrated actions with multidisciplinary teams in different fields of expertise, such as emergency and intensive care. hospital pharmacists, in partnership with infection control committees, have produced remarkable results in the internal review of work processes, including rotation of working hours, home offices, distancing between workers, intensification of environmental control measures and self-protection of workers, new guidance on the use of sanitizers, and training on good practices for self-care at home, on the move, and at work. hospital pharmacies seek to optimize clinical, humanistic, and economical outcomes in drug therapy by performing medical history and drug therapy reviews, patient follow-up, and medication reconciliation to ensure safe transition of care, as well as participating in antimicrobial stewardship programs (asps). pharmacists in community and hospital settings should enhance medication safety strategies and precautions in special populations with the aim of reducing avoidable harm related to medications. the development and refinement of pharmacists' role in the community and hospital settings is heterogeneous across regions. however, establishing robust organization of pharmacy services with clear planning of structure, processes, and outcomes' assessment [ ] could contribute to ensuring the quality of the response to the pandemic. reimbursement issues need to be addressed and advanced in brazil. the pharmacy workforce need to understand the nature of covid- , how it is transmitted, and how to prevent its spread [ ] . clinical pharmacists should constantly seek updated knowledge on covid- to improve the monitoring of effectiveness and safety of drug therapy, and to promote productive interaction within the multidisciplinary team [ ] . however, at the time of writing, high-quality evidence to support pharmacologic treatments for covid- is still lacking [ ] . whilst waiting the results of ongoing clinical trials, experimental treatments are being used worldwide. various protocols have been proposed by different committees and institutions in brazil. on may, a consensus of the brazilian association of intensive medicine, brazilian society of infectology and brazilian society of pneumology and tisiology was published as a reference for the pharmacological treatment of covid- [ ] . off-label drugs currently used in brazil include hydroxychloroquine (chloroquine) solely or in combination with azithromycin, lopinavir/ritonavir/oseltamivir, tocilizumab, glucocorticoids and parenteral anticoagulation. due to conflicting scientific evidence, hcps should reach a consensus, with decisions on drug therapy being individualized, based on disease stage, risk/benefit assessment, and pharmacoeconomic aspects. pharmacovigilance is needed to monitor the safety of experimental treatments and provide up-to-date guidance to hcps. for instance, increasing numbers of patients with ards are receiving empirical treatment with oseltamivir due to limitations of covid- testing, which may increase the risk of drug toxicity. in brazil, the national health surveillance agency (agência nacional de vigilância sanitária; anvisa) is responsible for evaluating adverse drugs events (ade) notifications made through the vigimed ® system. clinical pharmacists play a key role in the identification and reporting of these ade, especially in the critically ill. individuals with severe or critical covid- should be considered high-risk patients with regard to the use of potentially beneficial therapies and should be closely monitored by clinical pharmacists. liver and kidney injuries in covid- may alter the pharmacokinetics of drugs currently prescribed to treat the disease. lopinavir/ritonavir/ oseltamivir, hydroxychloroquine and chloroquine are metabolized in the liver and most of their metabolites depend on renal excretion. therefore, injury to the liver and kidneys can hinder metabolism, excretion, and the achievement of desirable serum concentrations, increasing the risk of drug toxicity [ ] . pre-existent liver and kidney diseases, impairment of these organs in older adults, and the presence of comorbidities can elevate the risk of clinical complications in covid- [ , [ ] [ ] [ ] . other parameters to monitor effectiveness and safety of drug therapy should be included in clinical pharmacy protocols. thromboprophylaxis in covid- using non-fractionated heparin or low-molecular-weight heparins has been used to control the hypercoagulability state in hospitalized patients [ , ] , with multiple pharmacological mechanisms previously described [ , ] . risk-benefit assessments of anticoagulation therapy should consider individual contraindications, stratification of bleeding risk, and dosage adjustments in patients with renal impairment and obesity [ ] . the use of proarrhythmic medications should also be closely monitored. patients receiving hydroxychloroquine or chloroquine ± azithromycin should be frequently monitored for cardiac toxicity by checking their corrected qt (qtc) interval and for the use of other medications with a risk of qtc interval prolongation or torsade de pointes [ ] . additionally, the empiric use of antibiotics for suspected secondary bacterial infections needs to be optimized on initiation and rigorously de-escalated [ , ] . hospitals should not lose sight of the long-term threat of antimicrobial resistance, if their current structures and asps are disrupted during the pandemic [ ] . hence, the participation of clinical pharmacists with advanced infection disease training as members of asps is fundamental in ensuring appropriate and safe use of antibiotics and improvements in the quality of care. however, the involvement of asps in the covid- response is still incipient despite being recommended by experts in this field [ ] . the use of medications should take into account special populations, including pregnant [ ] , pediatric [ ] , breastfeeding [ ] , and cancer patients [ ] . the desirable attributes of clinical pharmacists to provide care for hospitalized covid- patients in brazil are summarized in table . due to the lack of definitive protocols, the management of covid- has been primarily supportive [ ] . the fasthug-maidens mnemonic (table ) is a standardized structured approach for pharmacists to identify drug-related problems in icus [ ] . several authors have described potential markers of critical illness, staging, or disease recovery in covid- (e.g., d-dimer, lactate dehydrogenase, interleukin- ) [ , , , [ ] [ ] [ ] [ ] . the course of an intense inflammatory process leads to alterations in many review of patients' medical history provision of real-time assessment and evidence-based (when possible) advice on drug therapy support on safe use of medications brought from home medication reconciliation at different levels of transition of care simplification of drug administration schedule to reduce the exposure of nurses to covid- patients monitoring of potential drug-drug, drug-food interactions and adverse drug reactions adjustments in dosing regimens according to liver and kidney functions prevention of medication errors optimization of drug therapy and electrolytes to minimize the risk of prolonged corrected qt intervals and torsade de pointes support on lung-protective ventilation and neuromuscular blocking agents to facilitate ventilator synchrony provision of conservative fluid strategies and monitoring of vasopressors use monitoring of empirical antibiotics for suspected bacterial co-infection with rigorous de-escalation employment of fasthug-maidens mnemonic to identify drug-related problems in intensive care units support on drug information to patients and multidisciplinary teams, following biosafety protocols considerations on special situations (pediatrics, older adults, people with chronic diseases, allergies) research and continuing education precise documentation of pharmaceutical interventions laboratory tests in patients with acute or severe/critical illness. specific guidance on the use of laboratory tests to predict critical illness in covid- is under investigation [ ] . thus, to date, laboratory tests need critical interpretation and hcps should make consensual decisions based on patients' clinical condition. laboratory tests with potential utility to monitor covid- in hospitals are listed in table . in brazil, the use of these tests is heterogeneous in healthcare settings, and may be limited due to cost issues, delayed release of results, and the lack of equipment or partnerships between institutions. there are many unanswered questions regarding covid- . thrombotic risk varies according to race and ethnicity [ ] ; however, the impact of the heterogeneous ancestral contributions from african, caucasian, and indigenous genomes in the brazilian populations [ ] are not yet clear. regarding the use of medications, the protective effect of chronic use of oral anticoagulants and the clinical impact of ace inhibitors and arbs commonly prescribed to treat hypertension on the risks of covid- are currently unclear [ , ] . furthermore, the association between the complications of severe covid- and chagas disease and rheumatic heart diseases, conditions that are still prevalent in brazil [ ] , is not established. we also do not know if the determinants of severity in brazil will be similar to those observed in the usa [ ] , china, and europe [ , ] , given other factors such as social inequality and endemic diseases. future studies in brazil should investigate clinical presentation, according to the features of inflammatory response, treatment outcomes, and the factors associated with critical illness. • pharmacists play a key role in the management of covid- in brazil by providing health interventions and specialized support to improve outcomes, especially in populations with underlying diseases and social inequalities. • pharmaceutical expertise has been relevant in managing drug shortages, redesigning workflow, and educating pharmacists in brazil. • pharmacists can contribute to multidisciplinary teams, including antimicrobial stewardship programs, to improve outcomes of drug therapy in severe and critically ill patients. • further studies are needed to improve covid- knowledge and patient care in low-and middle-income countries. world health organization. novel coronavirus-china. geneva: world health organization isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak world health organization. coronavirus disease (covid- ) managing covid- in lowand middle-income countries covid- in brazil the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) presumed asymptomatic carrier transmission of covid- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study estimates of the severity of coronavirus disease : a model-based analysis clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study race to find covid- treatments accelerates ministry of health of brazil, health surveillance department [ministério da saúde do brasil, secretaria de vigilância sanitária deep impact of covid- in the healthcare of latin america: the case of brazil coronavirus contagion rate in brazil is . , the highest among countries, points out imperial college routes for covid- importation in brazil covid- is deadlier for black brazilians, a legacy of structural racism that dates back to slavery so what burden of disease in brazil, - : a systematic subnational analysis for the global burden of disease study united nations department of economic and social affairs recommendations and guidance for providing pharmaceutical care services during covid- pandemic: a china perspective community pharmacist in public health emergencies: quick to action against the coronavirus -ncov outbreak hospital pharmacists' pharmaceutical care for hospitalized patients with covid- : recommendations and guidance from clinical experience coronavirus sars-cov- /covid- pandemic: information and interim guidelines for pharmacists and the pharmacy workforce pharmacy emergency preparedness and response (pepr): a proposed framework for expanding pharmacy professionals' roles and contributions to emergency preparedness and response during the covid- pandemic and beyond contingency plan in various pharmaceutical scenarios of the covid- pandemic global drug shortages due to covid- : impact on patient care and mitigation strategies drug shortage: a public health problem federal board of pharmacy -brazil [conselho federal de farmácia-brasil] survey shows how covid- 's fear impacted medicine sales chloroquine and hydroxychloroquine for the prevention or treatment of novel coronavirus disease (covid- ) in africa: caution for inappropriate off-label use in healthcare settings medication management and adherence during the covid- pandemic: perspectives and experiences from low-and middle-income countries necessity of pharmacistdriven non-prescription telehealth consult services in the era of covid- fighting against covid- : innovative strategies for clinical pharmacists clinical characteristics and drug therapies in patients with the common-type coronavirus disease in hunan, china pharmacists' readiness to deal with the coronavirus pandemic: assessing awareness and perception of roles providing pharmacy services at cabin hospitals at the coronavirus epicenter in china pharmacologic treatments for coronavirus disease (covid- ): a review guidelines for the pharmacological treatment of covid- . the task force/consensus guideline of the brazilian association of intensive care, the brazilian society of infectious diseases and the brazilian society of pulmonology and tisiology. rev bras ter intensiva liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor covid- and older adults: what we know new understanding of the damage of sars-cov- infection outside the respiratory system anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy a proposal for staging covid- coagulopathy heparin: effects upon the glycocalyx and endothelial cells the versatile heparin in covid- stratifying therapeutic enoxaparin dose in morbidly obese patients by bmi class: a retrospective cohort study guidance on minimizing risk of drug-induced ventricular arrhythmia during treatment of covid- : a statement from the canadian heart rhythm society antibiotic use in the intensive care unit: optimization and de-escalation covid- : the uninvited guest in the intensive care unit (icu) implications for pharmacotherapy covid- and the potential long-term impact on antimicrobial resistance involving antimicrobial stewardship programs in covid- response efforts: all hands on deck coronavirus disease (covid- ) and pregnancy: what obstetricians need to know hyperinflammatory shock in children during covid- pandemic breastfeeding and respiratory antivirals: coronavirus and influenza covid- and cancer: a comprehensive review a standardized, structured approach to identifying drug-related problems in the intensive care unit: fasthug-maidens abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia prediction models for diagnosis and prognosis of covid- infection: systematic review and critical appraisal the laboratory tests and host immunity of covid- patients with different severity of illness development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid- factors associated with hospital admission and critical illness among people with coronavirus disease covid coagulopathy in caucasian patients atrial fibrillation: prevalence in a large database of primary care patients in brazil acknowledgements this study was partially supported by the univer- key: cord- -afkeuwup authors: nan title: chapter emergency management of poisoning date: - - journal: haddad and winchester's clinical management of poisoning and drug overdose doi: . /b - - - - . - sha: doc_id: cord_uid: afkeuwup nan medical toxicology is one of the most important and dynamic fields in medicine today, since the practicing physician is continually faced with the management of poisoning, drug overdose, and adverse drug effects. the abuse of both prescription and illicit drugs in the united states continues unabated. because the process of drug approval is more rapid, it is often not until the agent has been in use for some time, during the postmarketing period, before its toxicity is fully appreciated. defining the incidence of human poisoning is not easy. there are multiple sources of data on drug overdose and substance abuse. the toxic exposure surveillance system (tess) of the american association of poison control centers tabulates referrals for human poisoning called into the nation's poison centers. in , it recorded , , exposures, with deaths; analgesics were the most common cause of a fatal outcome. the national institute of drug abuse surveys emergency department visits through its drug abuse warning network (dawn), and in reported that a total of deaths resulted from drug abuse, with cocaine being the most commonly implicated agent. , however, these sources vastly underestimate the number of toxic events in humans. for example, reports of intoxicated patients who die from trauma, drowning, and fires are not consistently included in any national data set, nor are those of patients with medical complications from therapy, such as chemotherapy or anesthetics. morbidity that results from chronic abuse (e.g., heart disease from cocaine or nicotine abuse and cirrhosis from alcohol abuse) or industrial exposures, and the long-term effects of environmental hazards, is not rigorously compiled and is probably impossible to quantify. the most common causes of poisoning-related death in the united states have been carbon monoxide poisoning, cocaine use, and tricyclic antidepressant overdose. poisoning with analgesics, aspirin, and acetaminophen also remains a leading cause of death. calcium channel blocker overdose has surpassed digitalis overdose as the most common cause of cardiovascular drug-related death. to poison means to injure or kill with a substance that is known or discovered to be harmful. thus, the term poisoning connotes clinical symptomatology. it also implies that the toxic exposure is unintentional (e.g., in the case of an elderly patient who misreads a drug label). in contrast, the term overdose implies intentional toxic exposure, either in the form of a suicide attempt or as inadvertent harm secondary to purposeful drug abuse. the terms poisoning and drug overdose often are used interchangeably, especially when prescription drugs are the agents, even though by definition a drug overdose does not produce poisoning unless it causes clinical symptoms. poisoning has a bimodal incidence, occurring most commonly in children who are to years of age and in the elderly. overdose, whether motivated by suicidal intent or the result of abuse, occurs through adulthood. toxic exposure in those between the ages of and years is uncommon; when it occurs, the patient must be assessed carefully to ensure that psychiatric follow-up is provided when indicated. the general approach to the poisoned patient can be divided into six phases: ( ) stabilization; ( ) laboratory assessment; ( ) decontamination of the gastrointestinal tract, skin, or eyes; ( ) administration of an antidote; ( ) elimination enhancement of the toxin; and ( ) observation and disposition. because overdose patients are often clinically unstable when discovered, resuscitation with establishment of the airway, adequate support of ventilation and perfusion, and maintenance of all vital signs (including temperature) must be accomplished first. continuous cardiac and pulse oximetry monitoring is essential. rapid-sequence intubation (rsi) may be indicated in patients with an airway in jeopardy. naloxone, mg intravenously (iv); thiamine, mg intravenously (iv); and % dextrose, ml iv (if patients are shown on dextrostix testing to be hypoglycemic) are generally given to all adults in coma, once an iv line has been established and appropriate blood studies have been performed. , maintenance of blood pressure and tissue perfusion may require the provision of volume, correction of acid-base disturbance, administration of pressor agents, and antidotal therapy. only for reversal of pure initial dose not to exceed mg benzodiazepine sedation β blockers glucagon adult: - mg (iv) initially stimulates camp synthesis, increasing child: - ug/kg (iv) initially myocardial contractility continuous infusion as needed calcium channel calcium chloride % g ( ml) (iv) over min as initial avoid extravasation; tissue destructive blockers dose; repeat as necessary in critical patients; doses up to g may be necessary to restore blood pressure insulin/glucose . - . u/kg initially then monitor serum potassium and glucose . - . u/kg/hr as needed to maintain systolic blood pressure therefore, this task requires a thorough understanding of advanced airway management principles and of their application in a manner that prevents worsening of the clinical situation. rsi is a method of rapidly obtaining airway control with minimal physiologic disturbance. the process of rsi involves a patterned sequence of preparation, drug administration, intubation, and postintubation management. [ ] [ ] [ ] in the emergency department, rsi has historically had its greatest role in the patient with severe head trauma in whom intubation could exacerbate already increased intracranial pressure. however, because it is designed to blunt or prevent all adverse responses associated with endotracheal intubation, rsi is the ideal method of intubation in the poisoned patient. with the use of drugs having a short duration of action, rsi also is advantageous because it is a measure that permits temporary airway control for the patient with mildly compromised airway reflexes who requires gastrointestinal decontamination (lavage followed by activated charcoal administration) but who does not require prolonged intubation. rsi requires several essential steps that include the use of pharmacologic agents (table a- ) . to be performed safely, rsi must occur in the following sequence. the clinician must first evaluate the patient's airway to determine the necessary equipment and the best technique for safe intubation. particular attention should be directed to abnormalities in the cervical spine and temporomandibular joint because these will significantly impede rapid and uncomplicated intubation. if there is any question about the stability of the cervical spine, immobilization must be maintained. the oral cavity should be closely examined for the presence of foreign bodies. in addition to basic airway management, many victims of poisoning require advanced management that includes endotracheal intubation. clinical situations in which endotracheal intubation may be necessary in poisoned patients are numerous (box a- ). intubation offers the advantages of complete airway control, protection from aspiration of gastric contents, provision of a route for suctioning of secretions, and a means of optimizing both oxygenation and ventilation. however, the process of intubating an awake patient is difficult and is associated with potential adverse effects, including coughing, gagging, vomiting, tachycardia or bradycardia, hypertension, hypoxia, and increased intracranial pressure. moreover, emergency intubation can be challenged by vocal cords that are obscured by secretions, unusual airway anatomy, a full stomach, or active vomiting. before intubation, all necessary equipment must be present so that serious delays or unforeseen complications can be prevented. an iv line should be established and the patient connected to a cardiac monitor and pulse oximeter. the equipment necessary for endotracheal intubation is outlined in box a- . the proper functioning of all equipment should be ensured before it is used. appropriate endotracheal tube size also should be determined (table a- ) . unanticipated difficulties with intubation are common; "difficult airway" equipment (e.g., illuminated or fiberoptic-directed endotracheal tubes) should be kept close at hand. oxygen should be administered for to minutes before intubation; this produces a washout of nitrogen from the lungs, replacing this gas with an oxygen reservoir. the oxygen reservoir allows several minutes of apnea during which intubation can be performed without the risk of producing hypoxia. assisted ventilation with bag-valve-mask apparatus should only be provided if the patient's own respiratory efforts are inadequate because it risks inflation of the stomach, which increases the likelihood of vomiting. patients who are breathing spontaneously should be given % oxygen by face mask for several minutes before intubation. pretreatment involves the administration of pharmacologic agents that prevent adverse physiologic changes that may occur during intubation. agents included in this category are lidocaine and atropine. iv administration of the anesthetic lidocaine appears to blunt the increase in intracranial pressure that concepts in medical toxicology anesthetics, ketamine can produce significant elevations in pulse, blood pressure, intracranial pressure, and myocardial oxygen consumption, and such an increase in any of these could worsen the patient's clinical condition. because ketamine has a potent bronchodilating effect, it retains its important role as an induction agent in the patient with severe bronchospasm. [ ] [ ] [ ] after administration of a sedative/anesthetic, skeletal muscle relaxation is performed. skeletal muscle relaxants, all of which interrupt acetylcholine function at the myoneural junction, are typically divided into depolarizing and nondepolarizing categories. depolarizing agents, of which succinylcholine is the model drug, produce muscle depolarization before paralysis; this results in initial generalized muscle fasciculation. nondepolarizing relaxants produce paralysis without initial depolarization. the nondepolarizing skeletal muscle relaxants include pancuronium, vecuronium, atracurium, and rocuronium. succinylcholine is the most popular muscle relaxant because it has several desirable properties, including a rapid onset of action (less than minute) and an extremely short duration of action. customary paralyzing doses of succinylcholine are to mg/kg iv. despite its efficacy and popularity, succinylcholine can produce several adverse effects. these include hyperkalemia, prolonged paralysis, malignant hyperthermia, and hemodynamic changes. hyperkalemia, which can be severe, has been most commonly associated with administration of succinylcholine to those with burns, crush injuries, select neuropathies (e.g., guillain-barré syndrome), and myopathies (e.g., childhood muscular dystrophies). prolonged paralysis can occur in those who have a genetic deficiency in serum cholinesterase, the enzyme that inactivates the drug. prolonged paralysis may also occur in patients with liver disease, the elderly, and those who have ingested anticholinesterase insecticides (carbamates or organophosphates). malignant hyperthermia is a syndrome characterized by muscle rigidity, hyperthermia, autonomic disturbances, acidosis, rhabdomyolysis, myoglobinuria, renal failure, and coagulopathy. occurring in genetically predisposed individuals, malignant hyperthermia may appear without warning in those who are given inhalation anesthetics or succinylcholine. the mortality rate associated with this syndrome is approximately % to %. a malignant hyperthermia-like picture can also occur in children with skeletal muscular disorders (e.g., muscular dystrophy) who are given succinylcholine. finally, succinylcholineinduced muscle depolarization can lead to transient increases in intracranial and intra-abdominal pressure, with accompanying changes in cardiac output. because of these potential adverse effects, nondepolarizing muscle relaxants are often recommended as adjuncts to or substitutes for succinylcholine use. as adjuncts, nondepolarizing agents, when given before succinylcholine, can prevent muscle fasciculation and its attendant physiologic effects. the so-called "defasciculating dose" of a nondepolarizing agent is approximately one tenth the full dose of that agent. for example, pancuronium can be given in a dose of . mg/kg iv before the accompanies intubation. although scientific proof of lidocaine's efficacy is sparse, it is appropriateparticularly in the patient with suspected intracranial hypertension-to administer lidocaine, . to . mg/kg iv, to minutes before intubation. [ ] [ ] [ ] bradycardia can accompany rsi in two circumstances. in young children, both posterior pharyngeal stimulation and administration of succinylcholine can result in severe bradycardia. therefore, in children younger than years, atropine should be administered before induction. the dose of atropine is . to . mg/kg (maximum, . mg). no less than . mg of atropine should be administered because smaller doses can produce paradoxical bradycardia. severe bradycardia can also occur in patients of any age who have been exposed to medications or toxins with negative chronotropic actions. for example, in patients who have ingested β antagonists (e.g., propranolol), calcium channel blockers, and digoxin, rsi can produce an abrupt decrease in heart rate or frank cardiac arrest. therefore, in patients who are undergoing rsi after exposure to these agents, atropine should either be administered prophylactically or kept immediately available should emergency administration become necessary. induction consists of two components: administration of a sedative/anesthetic agent to produce unconsciousness, and the subsequent administration of an agent that produces complete skeletal muscle relaxation (paralysis); both actions facilitate intubation. because administration of these drugs leads to apnea and paralysis, it is essential that induction proceed quickly and efficiently; this underscores the importance of having all intubation equipment immediately available and in working order. a number of medications of different pharmacologic classes are used to produce sedation before skeletal muscle relaxation (see table a - ). these drugs include benzodiazepines, opioids, barbiturates, propofol, etomidate, and ketamine. among the benzodiazepines, midazolam, when given in a dose of . mg/kg iv (up to a range of to mg in an adult), is ideal because its effects are rapid in onset and short in duration. the drug also offers the advantage of producing muscle relaxation and amnesia. opioids are another class of drugs that can be used; however, many opioids, such as morphine, may prompt histamine release, with resultant hemodynamic changes. fentanyl in a dose of to μg/kg is highly effective at producing rapid sedation and relaxation with minimal cardiovascular change. several barbiturates can produce rapid sedation and relaxation. the most popular of these is sodium thiopental (dose to mg/kg). equally effective but with a shorter duration of action are methohexital, propofol, and etomidate. finally, ketamine is a dissociative anesthetic that can produce rapid onset of a state in which the patient is insensitive to pain but maintains an awake appearance and continues to have protective airway reflexes. the typical iv induction dose of ketamine is to mg/kg. unlike other sedatives/ administration of succinylcholine to prevent fasciculation. nondepolarizing agents can also be used solely for skeletal muscle relaxation. however, they generally have a much slower onset of action (as long as to minutes) and produce a longer duration of paralysis. also, many nondepolarizing agents stimulate histamine release, producing significant hemodynamic changes. therefore, they are not ideal agents for rsi. rocuronium appears to have the most rapid onset of all nondepolarizing agents, approaching that of succinylcholine with regard to time to complete muscle relaxation in the less than ideal conditions generally found during emergency intubation. significant warnings to succinylcholine use in the pediatric population have been recently added, based on the possibility of life-threatening cardiac arrhythmias. according to these new warnings, children with undiagnosed myopathies (e.g., a muscular dystrophy) could develop hyperkalemia sufficient to produce a cardiac disturbance. intubation suction must be immediately available when intubation is performed. the patient undergoing emergency intubation often has a full stomach; the risk for vomiting and aspiration is therefore significant. this risk is minimized both by the rsi technique and by the direct application of pressure on the cricoid cartilage (sellick's maneuver), which occludes the esophagus. adequate preoxygenation and limiting the duration of the intubation attempt to less than to seconds should prevent significant hypoxia. the differences between the airway of the child and that of the adult have important implications for endotracheal intubation. . the child has a relatively large tongue; this makes direct visualization of the larynx difficult. . the child has larger tonsils, which also obscure visualization. . the infant's larynx is located more cephalad than that of the adult. as a result, the angle between the tongue and the glottis is more acute, and visualization of the larynx is impaired. . the subglottic area of the infant is the narrowest part of the larynx and may impede the passage of an endotracheal tube passed through the vocal cords. immediately after successful endotracheal intubation, placement of the endotracheal tube must be confirmed by detection of bilateral equal breath sounds on chest auscultation, end-tidal carbon dioxide monitoring, or chest radiography; of these, chest auscultation is the least sensitive method and should never be used in isolation to confirm endotracheal tube placement. after confirmation, the tube should be secured either with a strap or with benzoin and adhesive tape. inflation of the endotracheal tube cuff should be performed to minimize aspiration of gastric contents (although aspiration of activated charcoal around cuffed endotracheal tubes is a frequent occurrence). until recently, because the airway of the young child has an area of narrowing ("physiologic cuffing"), cuffed endotracheal tubes were not used in the pediatric patient. pediatric cuffed tubes are now available; their use is encouraged in most circumstances. if long-term intubation is necessary, sedatives/anesthetics and nondepolarizing muscle relaxants should continue to be administered. in unskilled or unprepared hands, emergency endotracheal intubation can have disastrous consequences. even when performed by the most experienced hands, this complex procedure can have complications that should be anticipated so that they can be quickly recognized and treated. these complications include: poisoned patients often present to the emergency department with hypotension or frank shock. provision of circulatory support through interventions that may include volume expansion, vasopressor therapy, antidote administration, and correction of electrolyte and acidbase disturbances is essential in initial management. many medications and toxins produce hypotension (box a- ). depending on the ingested substance, the low blood pressure may have a number of causes. for example, blood pressure depressions may occur from direct depression of myocardial contractility (e.g., quinidine), disturbances of central nervous system cardiorespiratory centers (e.g., clonidine), severe gastrointestinal fluid losses (e.g., acetaminophen, iron, arsenic, ricin, mushrooms), peripheral vasodilation (e.g., angiotensin-converting enzyme inhibitors), or a combination of these effects (e.g., theophylline, calcium channel blockers, tricyclic antidepressants). hypotension also can result from the secondary effects of toxins (e.g., cocaineinduced myocardial infarction). finally, blood pressure disturbances in the poisoned patient may represent accompanying trauma (e.g., severe spinal cord injury or internal hemorrhage). with the multitude of possible causes, the clinician, on the basis of the known pathophysiology of a particular drug and after having performed a thorough physical assessment, should determine, if at is mechanistically the source of intravascular volume loss, the use of colloid solutions may be preferred. whole blood is most valuable in situations in which there is frank blood loss. with severe hemolysis (e.g., after arsine or stibine exposure), exchange transfusion with whole blood may be necessary. usually, the adequacy of volume expansion is determined clinically by an increase in blood pressure. other clinical signs of improved cardiac output include resolution of cyanosis and normalization of capillary refill time. central venous pressure and swan-ganz catheter monitoring, although invasive, provide the best evidence of appropriate intravascular volume. fluid overload is a potential complication of volume expansion. this is most likely to occur in patients who receive excess fluids over a short period of time. also, after an overdose of a myocardial depressant such as tricyclic antidepressants or quinidine, a fluid bolus that could be tolerated by a healthy individual can produce pulmonary edema in the overdose patient. therefore, administration of modest boluses of fluid is generally recommended; if cardiac output remains inadequate after fluids have been given, vasopressor therapy should be initiated. in the patient with severe hypotension, vasopressor therapy is necessary if blood pressure is not satisfactorily improved after volume expansion. vasopressors are drugs that can be administered to maintain cardiac output. these agents have specific effects on the heart or blood vessels, augmenting myocardial function or increasing vasomotor tone, or both. with rare exception, vasopressors used in the acute management of hypotension are short-acting drugs that must be given by continuous iv infusion. vasopressors generally act at adrenergic (α and β), d (dopamine), or glucagon receptors (table a- ). the adrenergic system has been further defined with the recognition of two major α-adrenergic receptor subtypes (α and α ) and three β-adrenergic receptor subtypes (β , β , and β ). coupled with intracellular g proteins, these membrane-bound receptors effect an intracellular chain of events that includes changes in the activity of adenylate cyclase. this action goes on to modulate the level of intracellular cyclic adenosine monophosphate (camp), which in turn alters phospholipase activity or opens gated calcium channels. although the cellular mechanisms of this system have become much better defined, the general principles of vasopressor action remain unchanged. for example, α-adrenergic receptor agonists produce vascular smooth muscle contraction. β -adrenergic receptor agonists produce increased heart rate and contractility, whereas β -adrenergic receptor agonists promote generalized smooth muscle relaxation (including bronchial and vascular). vasopressor therapy is designed to improve cardiac output through manipulation of the specific receptor most appropriate for the clinical situation. a number of vasopressors can be used to provide blood pressure support (see table a - ) . the all possible, the probable cause of hypotension if he or she is to provide a specific intervention. appropriate cardiac output relies on the adequacy of intravascular volume. after poisoning, intravascular volume may decrease abruptly. this decrease can be absolute, occurring as a result of a direct loss of intravascular volume (e.g., pulmonary edema, gastrointestinal pooling), or relative, resulting from severe peripheral vasodilation (e.g., angiotensin-converting enzyme inhibitor or α-antagonist overdose). in either case, hypotension should first be treated with the administration of volumeexpanding agents. many fluids are acceptable for emergency volume expansion. normal saline and lactated ringer's solution are generally the most readily available isotonic agents. adults should receive up to -to -ml boluses of isotonic fluid while blood pressure is monitored; children should be given to ml/kg. after the administration of each bolus, the patient should be reassessed for improvements in cardiac output. alternative fluids that can be used for volume expansion in the poisoned patient include albumin and whole blood. each of these fluids has a role that is best determined by the pathophysiologic mechanism responsible for the hypotension. being colloid rather than crystalloid in nature, these fluids in theory maintain intravascular volume better than saline solutions do. in clinical situations in which a "leaky capillary syndrome" box a- ricin indications for the use of these drugs vary slightly, depending on the clinical circumstance. epinephrine elevates blood pressure primarily through its α-adrenergic-stimulating properties. this effect also is valuable in improving myocardial and cerebral blood flow. because it also has prominent β-adrenergic agonist effects, epinephrine is variably effective at producing marked increases in blood pressure. epinephrine therapy is initiated at a dose of . to . μg/kg/min. epinephrine is particularly effective in intoxications associated with hypotension and bronchospasm (e.g., hymenoptera envenomation and anaphylactic reactions). norepinephrine stimulates both αand β-adrenergic receptors, with slightly greater stimulation of α-adrenergic receptors. the effect is improved vasomotor tone in conjunction with increased myocardial chronotropy and inotropy. norepinephrine infusions are typically initiated in a dose of . to . μg/kg/min. dopamine is a precursor of norepinephrine. the most popular of vasopressors, dopamine appears to have at least three mechanisms of action: ( ) promotion of norepinephrine synthesis, ( ) a tyramine-like effect that stimulates release of preformed norepinephrine, and ( ) direct stimulation of vascular dopamine receptors. the cardiovascular effects of dopamine are variable, depending on the infusion rate. at relatively low doses ( to μg/kg/min), the drug dilates renal and mesenteric vessels without marked increases in heart rate or blood pressure. at doses of to μg/kg/min, β-adrenergic receptor stimulation predominates, producing significant increases in cardiac output. finally, at doses greater than μg/kg/min, α-adrenergic receptor stimulation is the primary action, resulting in marked peripheral vasoconstriction. the general dose range for dopamine infusion is to μg/kg/min. dopamine is safe and effective for any type of druginduced hypotension. in the past, there have been theoretic concerns that dopamine's β-adrenergic effect in the face of phenothiazine or tricyclic antidepressant intoxication would increase the peripheral vasodilatation associated with overdose, exacerbating hypotension. however, experimental data and clinical experience have failed to confirm this adverse effect from dopamine use. also, with hypotension after monoamine oxidase inhibitor overdose, dopamine's effects are somewhat unpredictable; it may be relatively ineffective (owing to the lack of preformed norepinephrine), or it can produce an exaggerated response (because of its tyramine-like action). dobutamine is a synthetic catecholamine with almost exclusive β-adrenergic receptor-stimulating effects. its primary mechanism of blood pressure improvement is direct myocardial inotropy; thus, reflex peripheral vasodilation may occur with its use. unlike dopamine, dobutamine does not release preformed norepinephrine. the usual dosage range for dobutamine is to μg/kg/min, although doses as high as μg/kg/min have been used. high-dose infusions often increase myocardial oxygen demands, which, if unmet, can result in myocardial ischemia. nonetheless, dobutamine is extremely effective in syndromes of heart failure. phenylephrine has both αand β-adrenergic receptorstimulating properties, although its α-adrenergic receptor actions predominate. phenylephrine is a potent stimulator of vasomotor tone; it is therefore very effective in patients in hypotensive states resulting from severe peripheral vasodilation (e.g., following overdose with an α-adrenergic antagonist, such as prazocin or a phenothiazine neuroleptic, e.g., chlorpromazine). phenylephrine centrations of extracellular calcium, particularly in the face of channel blockade (e.g., after overdose of calciumchannel blockers), sometimes improve contractility. administration of iv calcium chloride is indicated in the management of hypotension resulting from calcium channel blocker overdose (see table a - ), hyperkalemia, and hypocalcemia. a thorough history taking and physical examination are essential to the diagnosis of the toxic patient. poisoning should be suspected in any patient who presents with multisystem disturbance until proven otherwise. although the initial manifestations of poisoning are myriad, a patient with acute poisoning often presents with coma, cardiac arrhythmia, seizures, metabolic acidosis, or gastrointestinal disturbance, either together as symptom complexes or as isolated events. symptom complexes, or toxidromes (table a- ) , may give clues to an unknown poisoning. for example, a patient with a history of depression who presents with coma, seizures, a widened qrs complex or evidence of dysrhythmia on electrocardiography, and dilated pupils has likely taken a tricyclic antidepressant. hepatic, renal, respiratory, and hematologic disturbances are generally delayed manifestations of poisoning. the clinical evaluation, in addition to the history taking and physical examination, includes an assessment of major signs of toxicity presented by the patient and evaluation of the laboratory data. when one suspects poisoning or drug overdose, the primary goal of history taking is identification of the toxic agent. sometimes diagnosis is easy, as in the case of the toddler who ingests iron tablets in the mother's presence. sometimes it is difficult, as in the case of the patient who is hiding a history of drug abuse and passes out at work or who has an unexpected seizure. prior medical or psychiatric history, current medications, and allergies should be obtained from family or friends if the patient is unable to relate the information. the following questions may be revealing: what other medicines are in the house? what was the patient doing that day? does the patient live alone, did he or she just lose a job, or have there been recent emotionally traumatic events? is the patient eating a special diet or taking a new health food, alternative medication, or performance enhancer? could the patient inadvertently have taken too much of a prescribed medication? if it can be identified, is the substance nontoxic? (see box a- .) the physical examination can help in determining the extent of poisoning and may reveal the presence of a infusions are given in a typical dose range of . to . μg/kg/min. amrinone is a novel, nonadrenergic cardiac stimulant that improves myocardial contractility while inducing vasodilation. its mechanism of action appears to be direct inhibition of phosphodiesterase; the result of this is increased intracellular camp activity, an action that increases transmembrane calcium flux, potentiating cardiac chronotropy and inotropy. amrinone's effects have been compared with those of dobutamine and nitroprusside combination therapy. amrinone may be particularly valuable in the treatment of calcium channel blocker intoxication; its inhibition of camp breakdown results in greater phosphorylation of l-type calcium channels, potentially increasing their permeability. experimental data support its role in this specific poisoning. amrinone can be used to treat syndromes of left ventricular failure but should not be administered in the presence of myocardial ischemia; like dobutamine, it may increase myocardial demands, resulting in infarction. because of its potent vasodilating action, amrinone may cause a hypotensive response in those with low intravascular volume. the usual dosage range for this agent is to μg/kg/min; the total daily dose should not exceed mg/kg per day. glucagon is a single-chain pancreatic polypeptide that is an effective inotropic and chronotropic agent. its mechanism of action is direct stimulation of myocardial glucagon receptors; these receptors, when stimulated, increase the formation of myocardial camp. the resultant effect is positive inotropy and, to a lesser degree, positive chronotropy. glucagon is theoretically most effective after β blocker overdose, in which decreased β-adrenergic receptor activation leads to diminished camp production. the hormone may also provide therapeutic benefit in hypotension after calcium channel blocker overdose. glucagon is given in an initial dose of to mg ( to μg/kg in children). if effective in augmenting blood pressure, it can be given as a continuous infusion of to mg/hr ( μg/kg/hr in children). some preparations of glucagon are marketed as a lyophilized compound with a . % phenol-based diluent for reconstitution. while single doses of such a product can be given after standard reconstitution, glucagon for continuous infusion should be reconstituted with saline to prevent phenol toxicity. adverse effects from glucagon include hyperglycemia, nausea, vomiting, and ileus. calcium plays a key role in regulating cardiac inotropy through its binding to troponin c, an action that permits interaction between actin and myosin. although most of the calcium that produces this change resides in an intracellular calcium pool, extracellular calcium does diffuse into cells and contributes to increased contractility. although diffusion of calcium into the myocardium is "gated"-that is, it is tightly controlled-high con- hypoxia or prolonged pressure and are seen after sedativehypnotic overdoses (especially barbiturate overdose), carbon monoxide poisoning, and thermal burns. bullae may also follow rattlesnake envenomation. bullous lesions or soft tissue swelling should prompt evaluation for rhabdomyolysis, an occasional finding in patients following prolonged coma or severe hyperthermia, such as in cocaine abuse. it is important to smell the patient's breath. alcohol is the most common odor detected on the breath of an intoxicated patient in the emergency department. the accurate identification of other odors varies greatly among physicians. a fruity odor may be detectable in the patient with diabetic ketoacidosis. cyanide poisoning can be associated with the smell of almonds. the smell of cleaning fluid suggests carbon tetrachloride poisoning. gasoline, camphor, hydrogen sulfide, ether, turpentine, methyl salicylate, paraldehyde, phenol, and organophosphate insecticides all have characteristic odors. arsenic and tellurium intoxication is associated with the odor of garlic. a nasal examination may reveal chronic insufflation of cocaine. an edematous, often elongated uvula may be seen with marijuana use or exposure to corrosive agents. auscultation of the lungs may provide diagnostic clues. in narcotic or tricyclic antidepressant overdose, pulmonary edema may be a complication, leading to the appearance of adventitious noises. in all overdose patients, aspiration pneumonitis, the result of a depressed gag reflex, is a possibility. inhalation of toxic gases may produce wheezing and pulmonary compromise. pneumothorax may be detected in patients who smoke cocaine, methamphetamine, or any other heated, impure substance. mediastinal emphysema from marijuana or crack cocaine smoking also may be detected by auscultation. examination of the heart may reveal a new murmur, which in an intravenous drug abuser suggests endocarditis. bradycardia is common after the overdose of four classes of cardiac agents: calcium channel blocker, β blocking agents, digitalis preparations, and central α antagonists (e.g., clonidine or guanfacine). a ventricular arrhythmia on electrocardiography in a young patient suggests cocaine toxicity. an irregularly irregular heartbeat that is new in a patient on an alcoholic binge suggests atrial fibrillation-the so-called "holiday heart" syndrome. a boardlike abdomen in a patient with a history of spider bite is characteristic of black widow envenomation. examination of the abdomen in an overdose patient often reveals an adynamic ileus. in patients with abdominal pain, a surgical abdomen must be ruled toxic syndrome, of any underlying disease, or concomitant trauma. repeated assessment, especially of vital signs and of cardiac, pulmonary, and neurologic status, is critical to proper management of the toxicologic patient. the physical examination also can provide valuable clues as to the particular toxin involved (table a- ) . as part of the initial evaluation, complete determination of vital signs, including measurement of body temperature initially and throughout the emergency department assessment, is mandatory. obtaining a core body temperature measurement may be necessary. hyperthermia can occur with a number of ingestions and in infectious illness, but it is characteristic of poisoning with salicylates, anticholinergics, monoamine oxidase inhibitors, and dinitrophenol; it is occasionally also seen after intoxication with phencyclidine, lsd, or cocaine, especially following seizures. life-threatening malignant hyperthermia following drug overdose may occur. hypothermia is common and may occur because of exposure to cold, hypoglycemia, or overdose of a number of sedatives, especially barbiturates, ethanol, carbamazepine, narcotics, and phenothiazines. bradycardia can be seen with overdose of digitalis, cholinergic agents, β blockers, and calcium channel blockers, but it also may be seen with hypothermia or spinal cord trauma. hypertension is characteristic of intoxication with cocaine, amphetamines, phencyclidine, and sympathomimetics. the skin should be examined for needle tracks, burns, bruises, or lacerations. needle tracks may be confined to the groin or other areas that are not readily visible. a "boiled lobster" appearance suggests ingestion of a boric acid-containing roach powder insecticide. generalized flushing suggests an allergic reaction, niacin overdose, anticholinergic poisoning, scombroid fish poisoning, or an alcohol-disulfiram reaction. diaphoresis suggests hypoglycemia, salicylate or organophosphate poisoning, hyperthyroidism, drug or alcohol withdrawal, or shock from cardiac or other etiology. jaundice may follow overdose of acetaminophen, aspirin, iron, carbon tetrachloride, mushrooms, copper, or phosphorus. petechiae and ecchymoses suggest coumadin overdose. bullae may be secondary to skin out. hepatomegaly suggests liver congestion (e.g., with pyrrolizidine toxicity). all patients should undergo a careful neurologic examination. issues of major concern are concomitant head trauma and spinal cord trauma in comatose patients. serial neurologic examinations are key to proper assessment (see discussion of coma in section on level of consciousness). the extremities should be evaluated to detect thrombophlebitis, fracture or dislocation, or vascular insufficiency. rhabdomyolysis and the compartment syndrome are definite concerns in overdose patients, especially in those with prolonged coma or underlying trauma. the toxicologic patient presenting in the acute setting often exhibits the following, either alone or in combination: coma, cardiac arrhythmia, metabolic acidosis, gastrointestinal disturbance, and seizures. consciousness is defined as an awareness of self and the environment. coma is unarousable unresponsiveness. wakefulness implies the ability to be aroused. these three functional states are mediated by the ascending reticular activating system, a tract that courses through the diencephalon, midbrain, and pons. diseases produce coma either by diffusely affecting the brain or by encroaching upon the brainstem. coma may be produced by ( ) a supratentorial mass lesion, such as a subdural hematoma; ( ) a brainstem lesion (uncommon); or ( ) metabolic disorders that widely depress or interrupt brain function. one of the most common manifestations of acute poisoning is coma. the principles of coma management are relatively straightforward. patients in coma must be stabilized initially by establishment of an airway, proper oxygenation with continuous pulse oximetry, insertion of an iv line with normal saline, and resuscitation, if necessary (see earlier section on emergency management). the clinical evaluation of the comatose patient is invaluable not only in determining the depth of coma and assessing for trauma, but also in providing a baseline for repeated clinical assessment. coma can be assessed either using the simple avpu (alert, responsive only to verbal stimuli, responsive only to painful stimuli, unresponsive) or glascow coma scales. the major causes of coma in patients seen in the emergency department include poisoning (e.g., carbon monoxide poisoning), drug overdose, head trauma, cerebrovascular accident, anoxia, infection (e.g., meningitis), and diabetes and other systemic disorders such as renal failure, hepatic coma, and cardiac arrhythmia. the physician must rule out each condition before establishing the diagnosis of poisoning. supratentorial structural lesions are suggested by a rapid progression of signs, including changes in respiratory pattern, disconjugate gaze, lateralizing signs, or loss of doll's eyes movements. a metabolic cause of coma may be indicated by the persistence of the pupillary light reflex; a depression of respiration and consciousness more pronounced than other neurologic signs; preceding altered mental states; asterixis or fasciculations, or both; the presence of a ciliospinal reflex; and extracranial signs, such as jaundice. repeated assessment of the comatose patient is critical to proper management of poisoning. evaluation of the patient's pupils is most helpful. midpoint fixed pupils or a unilateral dilated pupil suggests a structural lesion. pinpoint pupils suggest overdose of opiates, clonidine, organophosphate insecticides, nerve agents (e.g., sarin), chloral hydrate, phenothiazines, or nicotine. dilated pupils are nonspecific. a disturbance of ocular movements (e.g., loss of doll's eyes movements) suggests a structural lesion. nystagmus suggests intoxication with phenytoin, phencyclidine, carbamazepine, and, occasionally, ethanol. it is important to note abnormal patterns of breathing. posthyperventilation apnea, cheyne-stokes respirations, and apneustic breathing strongly suggest that a structural lesion is the cause of the patient's coma. central neurogenic hyperventilation is a classic presentation of brainstem injury. kussmaul breathing can occur after salicylate or dinitrophenol poisoning. compensatory hyperventilation may accompany methanol or ethylene glycol poisoning or other toxin-producing metabolic acidosis. respiratory arrest is a common presentation in the patient who has taken a central nervous system depressant and may lead to multisystemic dysfunction resulting from severe hypoxic injury. decorticate and decerebrate posturing suggests a structural lesion. it is important to realize that patients with poisoning or drug overdose (e.g., tetrodotoxin intoxication) may appear brain dead; have fixed, dilated pupils; be in an unresponsive coma; and lack the cold caloric response, yet recover fully in time. a -lead electrocardiogram and continuous cardiac monitoring are essential for any patient with significant poisoning. evidence of an arrhythmia or other important diagnostic clues may be present on electrocardiography, such as a widened qrs complex in cyclic antidepressant overdose or a prolonged qt interval in trazadone or arsenic poisoning overdose. box a- lists common toxic causes of cardiac arrhythmia. the patient with life-threatening cardiac arrhythmia or cardiac arrest should be managed on the basis of the general principles of resuscitation and the american heart association's advanced cardiac life support causes of a high-anion gap metabolic acidosis are listed in box a- . the assessment of metabolic acidosis includes not only arterial (or, less ideally, venous) blood gas analysis, but also studies of serum sodium, potassium, chloride, carbon dioxide, blood urea nitrogen (bun), creatinine, glucose, acetone, serum osmolality, and urine ph, as well as urinalysis. determination of the anion gap is helpful in the diagnosis and management of poisoning. the clinician can measure serum osmolality either directly by determining the freezing point (osmometry) or by calculation. the formula for calculating osmolality is serum osmolality = × na + (meq/l) + bun (mg/dl)/ . + glucose (mg/dl)/ when laboratory data are expressed in international (si) units, the formula for calculation of serum osmolality simply equals × na + bun + glucose. the normal serum osmolality is to mosm/l. an osmometer measurement indicating a serum osmolality that is more than mosm/l greater than the calculated osmolality is termed an osmolar gap; it suggests the presence of an osmotically active substance that is not accounted for by the calculated osmolality. causes of an osmolar gap are listed in box a- . the most common cause of an osmolar gap is consumption of an alcohol. the osmolar gap can be used to estimate the serum concentration of an alcohol, based on that alcohol's molecular weight (table a- ) . a substance contributes to osmolality only if it achieves relatively high blood levels and has a low molecular weight. most drugs or intoxicants cannot be detected with use of the osmolar gap. the causes of toxic gastrointestinal disturbance are many. the patient with iron, arsenic, or ricin poisoning has severe, repeated episodes of vomiting and may develop gastrointestinal hemorrhage. theophylline overdose also causes persistent retching. acute lithium and arsenic poisoning characteristically produce massive diarrhea. patients with acute mercury poisoning have a mucous-type diarrhea, with the subsequent development (acls) guidelines. if cyclic antidepressant overdose is suspected, administration of iv sodium bicarbonate is indicated for correction of ventricular arrhythmia or conduction disturbances. sodium bicarbonate may also be effective in the treatment of other overdose by other agents associated with prolongation of the qrs interval, including diphenhydramine and cocaine. administration of iv calcium chloride is the primary therapeutic measure for calcium channel blocker overdose. use of digoxin antibody fragments is indicated for digitalis poisoning, and glucagon for β blocker overdose (see table a - ). in referred patients who have already been hospitalized elsewhere, ventricular arrhythmia may be due to hyperkalemia because renal failure may have ensued; in such patients, iv sodium bicarbonate, glucose/insulin, and, if necessary, calcium chloride administration may be warranted. magnesium has a singular role in the treatment of drug-induced prolongation of the qt interval, a conduction disturbance that is often the prelude to torsades de pointes and other lifethreatening disturbances. in all intoxicated patients, correction of hypoxia, metabolic acidosis, and fluid and electrolyte disturbance serves to reduce the incidence of cardiac arrhythmias. digoxin and other digitalis compounds lithium of hemorrhagic colitis. one of the most striking presentations is caused by phosphorus poisoning, which produces luminescent vomitus and flatus. the early presentation of organophosphate or nerve agent exposure is similar to that of acute gastroenteritis and is characterized by abdominal cramps, vomiting, and diarrhea, with subsequent development of neurologic signs. poisoning from mushrooms (see chapter ), toxic marine life (see chapter ), botulism, and food (see chapter ) should be included in the differential diagnosis. chemotherapeutic agents (see chapter ) are well-known causes of toxic gastroenteritis. the management of gastrointestinal disturbance in the toxic patient includes following the general principles of blood, fluid, and electrolyte resuscitation, when indicated; judicious use of parenteral antiemetics to control persistent vomiting; specific measures such as antidotal therapy (e.g., in iron or organophosphate poisoning); or interventional therapy, such as charcoal hemoperfusion (in theophylline overdose) or hemodialysis (in lithium overdose), when indicated. common agents that cause seizures are listed in box a- . almost any drug or toxin is capable of producing a seizure. delayed seizures occurring during a recovery period may be a sign of sedative-hypnotic or alcohol withdrawal. seizures should be managed first with establishment of an airway and oxygenation. patients with a simple isolated seizure may require only observation and supportive care, whereas repetitive seizures or status epilepticus, which can be life threatening, must be managed aggressively. some seizures are particularly difficult to control, such as those seen with theophylline or cocaine overdose. the standard regimen for seizure control in overdose of an unknown agent is use of the full therapeutic dosages of benzodiazepines (e.g., diazepam or lorazepam), followed by administration of phenytoin or a barbiturate (e.g., phenobarbital or pentobarbital). in patients with status epilepticus, rsi may be necessary and the use of thiopental is indicated, with electroencephalographic monitoring to ensure control of electrical seizure activity; the use of additional paralytics, such as pancuronium bromide, may be warranted. specific measures to control seizures may be indicated, such as administration of pyridoxine for isoniazidinduced seizures. box a- lists specific blood studies whose results may be used for diagnosis and to direct therapy of the overdose patient. in every significant poisoning, routine studies include a complete blood count; determination of serum electrolytes, glucose, bun, creatinine, and calcium; urinalysis; prothrombin time; pulse oximetry; end-tidal co monitoring, and -lead electrocardiography. arterial blood gas analysis is necessary for evaluating respiratory status and acid-base abnormalities, particularly in the comatose or seizure patient. the measurement of serum salicylate and acetaminophen levels is generally added in the case of the patient with overdose of an unknown substance, because these agents are often co-ingestants or are contained in combination drugs. measurement of hepatic enzymes is important in the evaluation of acetaminophen toxicity. the advantage of a toxicologic drug screen in initial management is equivocal. box a- gives a partial list of drugs and toxins not commonly ph monitoring is helpful in the management of salicylate overdose. urine is the best specimen to use for "drug screening" purposes. a urinalysis is also useful in the early identification of acute renal failure or rhabdomyolysis with myoglobinuria. the intravenous drug abuser requires special blood testing, such as evaluation for human immunodeficiency virus, a hepatitis profile, a blood culture to identify bacteremia, and evaluation for rhabdomyolysis. chest radiography is an aid for diagnosing aspiration pneumonia or pulmonary edema. box a- lists agents that are radiopaque on plain film radiography of the abdomen. computed tomography may be useful if underlying trauma is suspected. finally, lumbar puncture may be indicated for ruling out meningitis in a patient with fever and coma. ocular decontamination see chapter . being the largest and most superficial organ in the body, the skin is often subject to exposure to toxins and is affected in . % of reported cases. at least % of occupational illnesses involve the skin. the effects of these exposures can be local or systemic (box a- ) . the skin provides many barriers to the absorption of toxins. the stratum corneum forms an important first barrier and is highly effective when it is completely intact. however, when skin wounds are present, when the wounds are wet, and when exposure is to certain highly lipophilic substances (e.g., organophosphate insecticides), significant absorption of toxin through the skin can occur. the skin of infants is notable for being more permeable than that of adults to substances of all classes. detected with routine drug screening. further laboratory blood studies are tailored to assess the individual diagnostic and therapeutic needs of the patient. it is extremely important to remember to "treat the patient, not the lab." one should never withhold therapy while waiting for a confirmatory drug level in a critical patient, such as a patient with tricyclic antidepressant overdose who is exhibiting a widened qrs complex. in contrast, performing hemodialysis on a completely asymptomatic patient with lithium overdose on the basis of one test result indicating an elevated serum lithium concentration would be equally unwise. serial blood level determinations are often helpful in guiding therapy in patients undergoing hemodialysis; in patients in whom concretions have formed, such as those with barbiturate, iron, salicylates, or meprobamate intoxication; or in patients receiving antidotal therapy (e.g., serial measurement of lead level is useful in patients receiving iv cana edta for management of lead poisoning). a urinalysis is necessary. performing a urine pregnancy test is wise in all women of childbearing age. urine the range of dermal toxins is broad. most of these substances are corrosive agents capable of producing burns that may become full thickness (i.e., third degree). other types of agents are irritants, sensitizers (including photosensitizers), allergens, vesicants, and exfoliants. as with ocular exposures, the general principles of management after exposure to dermal toxins are many. as soon as a toxic dermal exposure is recognized, decontamination efforts should begin. if the victim is immersed in a toxic fluid, the first step in management is his or her extrication without injury to the assistant. the victim should disrobe him-or herself at the scene. if the victim requires assistance, undressing should be done as safely as possible. protective gear should be donned before assistance with decontamination is rendered. unless the agent is highly reactive (e.g., elemental sodium), it is appropriate to wash the victim thoroughly with water, preferably in a nearby decontamination shower. generally speaking, water should not be used to decontaminate skin in exposures to sodium, phosphorus, calcium oxide, chlorosulphonic acid, and titanium tetrachloride. when emergency medical personnel arrive to the scene, they should continue skin decontamination. again, if the agent is known to have significant dermal absorption, emergency medical personnel should provide themselves every available level of self-protection. certain toxins such as organophosphates can contaminate the air within the ambulance and produce ill effects among personnel if prehospital decontamination efforts are inadequate. upon arrival at a health care facility, the victim may require quarantine, depending on the nature of the agent. skin decontamination in a decontamination shower should continue. particular caution should be exercised in the decontamination of victims of organophosphate insecticide or organophosphate-based nerve gas exposure; health care personnel have been overcome secondarily by contaminants on victims when they assisted in their care without donning proper protective gear. water is the most commonly used skin decontaminant and is highly effective for most dermal exposures. in select cases, specific agents should be used to assist in management (table a- ) . without exception, toxin-induced skin burns should be treated according to existing burn management guidelines. these include wound débridement and dressing, monitoring for infection, fluid management, and surgical consultation when appropriate. after the ingestion of a toxic substance, with the exception of agents that have a direct toxic effect on the gastrointestinal tract (e.g., iron or corrosives), that substance must be systemically absorbed and circulated before it reaches a target organ and exerts clinical toxicity. preventing the absorption of toxin is therefore the foundation of treatment after ingestion of a toxin has occurred. the term gastrointestinal decontamination (gid) has been coined to describe those interventions that are useful in preventing toxin absorption. with the exception of rare interventions such as gastroscopy, gid is considered to have only three components: ( ) gastric evacuation, ( ) administration of adsorbent, and ( ) catharsis. gastric evacuation is accomplished through gastric lavage. syrup of ipecac, once used as an emetic for treatment of toxic ingestions, is no longer routinely recommended for this purpose. there are several agents that can adsorb toxic substances, reducing their systemic absorption and subsequent toxicity (table a- ) . of these, activated charcoal is the most important adsorbent; there are few substances that activated charcoal will not adsorb (box a- ). catharsis, once an integral part of management, also has a diminishing role in the treatment of poisoned patients. gastrointestinal decontamination is discussed in greater detail in chapter b. with the development of sophisticated new antidotes and the changing spectrum of clinical poisoning, the use of emergency antidotes is assuming an increasing role in clinical toxicology. however, antidotes are useful in only a fraction of poisonings. table a - lists the common emergency antidotes. in poisoning with a known substance, early antidote use is indicated for emergency stabilization, often within the first hour. ( ) urine alkalinization. potential roles for these interventions are found in table a- and box a- . further discussion of elimination of a substance that has already been absorbed is provided in chapter c. observation and supportive care are the mainstays of therapy for the poisoned patient. indiscriminate use of gastric lavage, antidotes, and drugs should be avoided. all too often, the toxic agent is unknown, multiple drugs have been taken, or the patient is too unstable to undergo an aggressive therapy such as hemodialysis. monitoring of vital signs, cardiac telemetry, and oxygen saturation is mandatory. hospitalization in an intensive care unit is generally indicated for the patient with serious poisoning. multisystem monitoring with blood studies and assessment of other parameters are indicated, and upon detection of any specific system disturbance, appropriate subspecialty consultation is warranted. some agents such as iron, mercury, acetaminophen, paraquat, carbon tetrachloride, and amanita phalloides toxin have a latent phase, in which the patient appears to recover from the initial insult, only to decompensate to hours postingestion. patients with overdose of sustained-release capsules, such as calcium channel blocker or theophylline preparations, also may have delayed manifestation of poisoning. rarely, the tricyclic antidepressants have been known to cause fatal arrhythmia up to days following ingestion. some effects are not seen until later, such as hypertension following phencyclidine ingestion, hemorrhagic colitis following mercury ingestion, and disseminated intravascular coagulation following snakebite. one must also watch for the delayed pulmonary (see chapter ), hepatic (see chapter ), renal (see chapter ) , and hematologic (see chapter ) manifestations of poisoning. hyperbaric oxygen can provide oxygen at pressures greater than normal atmospheric pressure, which is given as atmosphere (atm) or mm hg. three atmospheres is the maximal pressure humans can tolerate over a reasonable period of time; hyperbaric units generally do not exceed . to . atm. the use of hyperbaric oxygenation is becoming standard therapy for patients with significant carbon monoxide poisoning, and it is becoming more available (see chapter ) for carbon tetrachloride poisoning, and possibly for cyanide and hydrogen sulfide poisoning. admission to an intensive care unit following antidotal therapy for further management and observation is generally indicated. further discussion of each antidote and its use is provided in the chapter on the specific poison. patients may require observation because of an underlying disease that may be exacerbated because of the overdose, such as diabetes, congestive heart failure, cardiac rhythm disturbances, or chronic lung disease. observation may be necessary to evaluate or treat complications, such as in a patient with an overdose who fell and sustained trauma or in a patient who develops aspiration pneumonitis or interstitial pulmonary edema. the iv use of illicit drugs is associated with multiple complications; observation is especially indicated for patients experiencing these complications, which include bacterial endocarditis, rhabdomyolysis, and neurologic sequelae. the disposition of the patient with intoxication may involve medical and psychiatric care as well as social follow-up. all patients admitted to the hospital with intentional overdose warrant close observation and the institution of suicide precautions. these patients may need appropriate restraint or observation if further injury or additional overdose attempts are to be prevented. overt or subtle attempts or gestures indicate the need for psychiatric evaluation. often, outpatient follow-up is necessary; for example, a child with kerosene ingestion may require further examination and chest radiography, and a child who has ingested anticoagulant rat poison may require serial outpatient monitoring of prothrombin times. the issue of child abuse or neglect may need consideration whenever a pediatric patient is treated. finally, long-term follow-up may be indicated; for example, hepatitis and hiv testing may be needed in the iv drug abuser. concepts is very difficult in a single center or even multiple centers. confirmation of exposure by laboratory studies is often unavailable, requiring the clinician to depend on the history (which may be inaccurate) and physical findings (which may be nonspecific) in arriving at the diagnosis of poisoning. there are likewise problems with randomization schemes, and basic inclusion and exclusion criteria. in addition, there are problems with the process of evidence-based reviews themselves. language bias occurs in some evidence-based reviews, such that non-english language publications, potentially of good quality, are often excluded from consideration. in addition, the premises on which the evidence review is based may not be universally applicable. the conclusions reached, based on studies performed in urban tertiary care centers where hospitals are capable of providing state-ofthe-art intensive care, may not apply equally to a remote hospital in a rural area, or even less so to a clinic in a developing country. thus, while careful consideration should be given to position papers and consensus conference proceedings, individual judgment will necessarily enter into the decision to employ any decontamination method for a given case of exposure. unfortunately, an unintended consequence of the publication of position papers is that they may in fact squelch further research. a number of methods of decontamination exist and may be employed depending on the circumstances of exposure. decontamination of the skin and eyes, as well as the gastrointestinal tract, will be discussed. extracorporeal methods of purification (hemodialysis, charcoal filtration, etc.) are covered in chapter c. decontamination of the skin and eyes is employed to reduce local tissue injury (chemical burns or irritation) and/or absorption that may result in systemic consequences. the decision to perform skin and eye decontamination is often based on the presence of symptoms, such as burning or itching. this is an insensitive evaluation method; thus, decontamination of these organs should primarily depend on careful consideration of the circumstances of exposure and the physical and toxicologic properties of the compound. protection of personnel during eye and skin decontamination is important to avoid secondary contamination of health care providers. the choice of personal protective equipment is beyond the scope of this chapter. the reader is referred to chapter and to the recent occupational safety and healthy administration (osha) best practices document. in almost all cases, clothing, jewelry, and shoes should be rapidly and completely removed prior to washing. it has been suggested in studies of radionuclide contamination that this process alone can remove the majority of a contaminant. this will, of course, depend on the physical properties of the toxicant but is a logical first step. solids and dust should be gently brushed away before decontamination with a solution. in this way, the heat generated from water reactive compounds can be diminished, as is caking of solids. in some cases, water or other decontamination solutions may be unavailable or in short supply. in such cases, dry decontamination, using an absorbent material (charcoal, flour, earth) followed by brushing or wiping may be attempted. the selection of skin decontamination solutions has historically been a choice between water for polar (watersoluble) compounds and water plus a mild soap or detergent for nonpolar compounds. water alone is typically employed for initial eye decontamination. physiologic saline and other saline-based eye washes are often employed for eye decontamination in health care and industrial settings. the use of these solutions has been largely empiric and practical, based on widespread availability rather than on critical evaluation of their efficacy. yano and colleagues studied water irrigation of burns involving mol/l hcl in rats, measuring subcutaneous ph as a measure of penetration of the acid and efficacy of decontamination. these investigators found that maximal subcutaneous ph depression had occurred by minutes following application of the acid. animals undergoing water irrigation at or minutes postexposure demonstrated some benefit; however, animals irrigated at minutes had no appreciable improvement in ph, compared with control. these investigators had previously demonstrated lack of efficacy of water irrigation after minutes in a n naoh burn model. clearly time is of the essence in irrigation of corrosive exposures. with regard to eye exposures, kuckelkorn and colleagues pointed out that water is hypotonic to corneal stroma, allowing edema and increased penetration by chemicals. they recommend use of amphoteric solutions to avoid these problems. in recent years, a number of novel decontamination solutions have come to market. diphoterine (prevor laboratories, moulin de verville, france), an amphoteric solution has been proposed for use in both acid and alkali exposures of eyes and skin, with emphasis on immediate irrigation (at the scene of the incident) rather than for hospital treatment. a recent article that compared diphoterine to physiological saline in alkaline eye burns demonstrated more rapid healing of grade and burns with diphoterine than with saline. the study suffers from a number of deficiencies, including lack of randomization and significant delays and variability in initial irrigation (in the field) and secondary irrigation in hospital (with either diphoterine or saline). nonetheless, the time to corneal reepithelialization was approximately six times as long after saline for grade burns and almost twice as long for grade burns, compared with diphoterine-treated eyes. there were an insufficient number of grade burns to detect any significant difference between groups. despite its shortcomings, this study suggests the potential for improved healing using diphoterine in alkaline eye burns and warrants further investigation. the same study group had previously shown in a study of ammonia burns in new zealand albino rabbit eyes that early application (within minutes) of diphoterine rapidly corrected ph, whereas saline irrigation did not. furthermore, saline-treated eyes had stromal edema, whereas diphoterine-treated eyes did not. the need for early irrigation is emphasized by this experimental study; however, the cited clinical study demonstrates some benefit even with delayed treatment (mean . hours). cavallini and casati studied diphoterine in experimental skin burns in rats involving % hydrochloric acid. skin flushing with diphoterine reduced substance p release during the first hours after burn and was associated with better wound healing and higher concentrations of β-endorphin days later when compared with normal saline or % calcium gluconate. hall and colleagues most authors recommend against neutralization of acid and base burns due to the risk for exothermic reaction leading to thermal burns. simple dilution with water or milk after oral ingestion of corrosives is uncommon in europe, but the norm after ingestions in the united states. penner demonstrated in an ingestion model that dilution of concentrated sulfuric acid with an equivalent volume of water results in a temperature elevation of approximately °c. neutralization results in even greater heat production. he suggested that vigorous gastric aspiration (likewise considered controversial given the risk for esophageal or gastric perforation) prior to cold fluid lavage is the treatment of choice in patients treated immediately following acid ingestion. a recent experimental study involving irrigation of rat skin exposed to n naoh with % acetic acid suggests that neutralization may not always be contraindicated. the investigators demonstrated more rapid correction of ph, no difference in peak temperatures, and improved outcomes in animals treated with % acetic acid rather than water. these findings cannot inundated with contaminated casualties. decontamination protocols that are not deployable within minutes (preferably to minutes) after an incident may serve little purpose in protecting the facility and health care providers from contamination. serious consideration should be given to the complexity of the setup process, one that typically will not be employed frequently. one example of a locally developed immediate deployment sheltered outdoor decontamination unit is shown in figure b - . this unit can be deployed in less than minutes and provides for decontamination of both ambulatory and stretcher patients. it is common practice to utilize physicians, nurses, and other critical emergency department (ed) staff to do decontamination. this is probably not advisable for a number of reasons. first, decontamination does not require great technical skill, and very little stabilization be generalized, but indicate the need to readdress current dogma regarding neutralization. the ideal duration of eye and skin decontamination is yet to be determined. recommendations for copious irrigation are common, without further precision. fifteen minutes is probably the most commonly recommended duration for eye irrigation. kuckelkorn and colleagues have recommended a minimum of minutes for eye irrigation after chemical exposures. one retrospective study of eye burn victims suggested that outcomes were better among those who had prolonged ( to hours) irrigation. irrigating to a relatively neutral ph is often attempted. if this is employed, it is helpful to remember that the ph of saline for intravenous injection (often employed for eye irrigation) is appreciably acidic (approximately . ) so that one should wait a few minutes after irrigation to measure the ph in order to allow the patient's own tears to replace the saline irrigation fluid in the conjunctival sac. the appropriate temperature for decontamination fluids has also been poorly studied. while increasing temperature of decontamination liquids reduces the likelihood of hypothermia in inclement climes and improves water solubility, heat also dilates skin pores and blood vessels, which may lead to increased absorption. if excessive, it may aggravate chemical or thermal burns. osha's recently published best practices guide for first receivers recommends a -minute wash with tepid water, based on recommendations from the u.s. army for chemical decontamination. mcintyre and colleagues recommend "warm, but not excessively warm" water for decontamination. eye irrigation should be performed with room temperature solutions. careful thought should be given to environmental conditions and risk for exposure when decontamination must be done out of doors. the duration and type of skin and eye decontamination performed in mass casualty situations may vary from that in cases involving single patients based on triage considerations. management of mass casualties is covered in chapter . a great number of options have been developed in recent years for skin decontamination, due to the increased interest in hazardous materials and chemical terrorism issues. examples of decontamination stretchers and facilities are shown in figures b- to b- . many others exist. one of the overriding considerations in determining the kind of decontamination facilities and equipment to purchase should be their capacity for rapid deployment. the experience with the sarin terrorist attack in tokyo revealed that hospitals may be rapidly can be performed during the decontamination process. furthermore, if there are problems and decontaminating personnel are unable to continue, such a practice results in incapacitation of primary emergency care providers. neither should security personnel, in general, be tasked with this responsibility, since a mass casualty situation will require their services for security itself. a number of alternatives have been suggested. some hospitals train housekeeping staff to perform decontamination. others have nurses from other units (burn units have extensive experience in wound care and cleaning) don protective clothing and prepare for decontamination while the ed staff prepares the decontamination facility and the ed proper to receive casualties. decontamination of radionuclides from the skin may be performed in a manner analogous to chemical exposures. uranium hexafluoride exposures should be treated in a manner analogous to that for hydrofluoric acid burns. wounds heavily contaminated with radionuclides may require surgical débridement and should be covered after initial decontamination. see chapter regarding radiation emergency management for further information. hydrogen fluoride, ammonium biflouride, and other soluble fluorides may pose a unique case in terms of decontamination. while an initial quick flush with water is appropriate, the patient may benefit from rapid decontamination with a substance that can bind the fluoride. hexafluorine has been reported to prevent significant skin burns in both humans and experimental animals when applied immediately after exposure. , two randomized studies in rats found, however, that hexafluorine was no better than water in preventing electrolyte disturbances caused by fluoride , and perhaps less effective than water or water plus calcium gluconate in reducing burn injury. thus, hexafluorine's efficacy in fluoride injury remains controversial. researchers in hydrogen fluoride manufacturing facilities frequently recommend skin irrigation with benzalkonium chloride solution based on studies performed in pigs. , calcium gluconate irrigation of skin and eyes has also been recommended to bind fluoride and prevent further injury. other investigators have found calcium gluconate to be no more effective than water or saline and perhaps detrimental in eye irrigation. , in summary, the ideal decontamination of hydrogen fluoride burns to skin and eyes remains to be determined. phenol is unique in its capacity to cause nonpainful burns and systemic toxicity. water irrigation may increase phenol absorption. generally accepted skin irrigation therapy consists of isopropanol , or polyethylene glycol solutions. , white phosphorus is pyrophoric (i.e., it burns in the presence of air). it is thus indispensable to provide adequate copious irrigation with water. the application of copper sulfate has been recommended, but an experimental study in rats demonstrated increased lethality in animals receiving topical treatment with % copper sulfate. eldad and colleagues have evaluated various phosphorus burn treatment recommendations and have concluded that copious water irrigation is superior to other treatments. high-pressure injection injuries should be mentioned here due to their requirement for special care and high all eye and skin exposures resulting in violation of the epithelium should prompt consideration of the need for tetanus toxoid administration. the only emetic currently recommended for use in humans is syrup of ipecac. previously employed emetics continue to be responsible for significant pathology, however. the administration of table salt has long been condemned in the literature ; nonetheless, recent reports illustrate that its use has not been completely abandoned and that it remains potentially lethal. , liu reported that copper sulfate continues to be used in china for emetic purposes and has resulted in multiple fatalities in recent years. the dose of ipecac is to ml in children to months of age or ml in children to years of age. this should be followed by to ml water per kg body weight. older children and adults should receive ml ipecac followed by to ml of water. ipecac is contraindicated in the following situations: ingestion of petroleum distillates ingestion of strong acids or bases ingestion of strychnine or other proconvulsants unconsciousness or absence of gag reflex ipecac has a number of potential adverse effects, including lethargy, cramps, and diarrhea. when taken chronically, it may induce muscle cramps and both skeletal and cardiac myopathy. it is subject to frequent abuse by patients with eating disorders, a factor that led to a review of the product's safety by the fda in . the american academy of clinical toxicology (aact) and the european association of poisons centres and clinical toxicologists (eapcct) reviewed the medical literature regarding the use of ipecac in . this combined task force more recently examined their previous findings and literature that had appeared since their earlier review. in brief, they concluded that syrup of ipecac should not be administered routinely in the management of poisoned patients. they pointed out that in experimental studies the amount of marker removed by ipecac was highly variable and diminished with time. furthermore they concluded that there is no evidence from clinical studies that ipecac improves the outcome of poisoned patients and recommended that its routine administration in the ed be abandoned. finally, they signaled the absence of data to support or exclude ipecac administration soon after poison ingestion, the administration of ipecac potentially reducing the effectiveness of activated charcoal, oral antidotes, and whole bowel irrigation (wbi). there are indications that the use of syrup of ipecac has dramatically declined in recent years, and it is likely that this trend will continue. , bond examined the evolving use of ipecac in u.s. poison centers, comparing rates of referral to the ed and moderate or greater outcomes in patients younger than years with unintentional ingestions. this comparison was carried out according to the frequency with which centers recommended use of ipecac. overall, use of ipecac was extremely rare ( . %) and significant adverse outcomes even lower ( . %). comparing the two groups of centers each, there were no significant differences in referral rates or adverse outcomes. bond concluded that there was no reduction in resource utilization (ed referral) or improvement in patient outcome from the use of syrup of ipecac at home and that while the data could not exclude a benefit in a very limited set of poisonings, such a benefit remained to be proven. shannon, in an editorial in the same issue, sounded the demise of ipecac on the basis of lack of proven efficacy, changing patterns in poison center approaches to the management of pediatric ingestions (including preference for activated charcoal when decontamination is indicated), and the move by the fda to rescind ipecac's over-the-counter status. this prediction was prescient, since the american academy of pediatrics, based on bond's article and the factors cited by shannon, revised its position statement on ipecac use, calling for abandonment of the regular stocking of it in the home. more recently, a u.s. government-convened review panel of experts published their conclusions regarding ipecac use. the panel concluded that the use of ipecac syrup might have an acceptable benefit-to-risk ratio when: there is no contraindication to the use of ipecac syrup. there is substantial risk of serious toxicity to the victim. there is no alternative therapy available or effective to decrease gastrointestinal absorption (e.g., activated charcoal). there will be a delay of more than hour before the patient will arrive at an emergency medical facility. ipecac syrup can be administered within to minutes of the ingestion. ipecac syrup administration will not adversely affect more definitive treatment that might be provided at a hospital. given these restrictions, rural residents might consider keeping ipecac on hand for home use under poison center direction. otherwise, these recommendations sharply limit the applicability of the drug and, thus, the induction of vomiting in general. are many unknowns in any clinical trial, the most significant of these being the time between ingestion and treatment and the amount of toxicant ingested. it is fair to say that the evidence for efficacy is currently deficient, but that lack of efficacy has not been proven either. the use of endoscopy in the diagnosis of caustic injuries is addressed in chapter . its use in the retrieval of foreign objects, such as batteries, firearm cartridges, and various pill fragments [ ] [ ] [ ] [ ] [ ] [ ] has been reported in humans and studied in animals, but has not been systematically addressed in humans. faigel and colleagues report that endoscopic use of the roth net was most effective in removal of button batteries. like other forms of decontamination, endoscopy to retrieve tablet fragments has been associated with significant complications; thus, the decision to perform endoscopy in these circumstances should be carefully weighed against the risks. surgical gastrointestinal decontamination has been employed for button battery ingestions, - cocaine and narcotic drug packets, [ ] [ ] [ ] [ ] [ ] [ ] [ ] and bezoars of iron and theophylline, among others. batteries passing the esophagus usually are expelled in the feces and are generally believed to require no intervention, although recent reports of early battery leakage have called the "wait and see" approach into question. , button batteries, when impacted in the esophagus, should generally be removed by endoscopy unless perforation is suspected. , , the trend toward use of smaller batteries by manufacturers has decreased the incidence of this problem. in the case of cocaine and heroin bodypackers/stuffers, many cases can be managed conservatively with wbi or other purgatives. most researchers agree, however, that acute toxicity (drug leakage) and bowel obstruction are indications for immediate laparotomy. , , charcoal binds to diverse substances, rendering them less available for systemic absorption from the gastrointestinal tract. it is obtained as a product of pyrolysis of numerous organic compounds (petroleum, wood, peat) and "activated" by heating it to º f to º f, in the presence of steam, carbon dioxide, or air. this gives the product a small particle size and large surface area. while charcoal adheres to many substances, a significant number of compounds and classes of compounds are poorly absorbed by charcoal. these include metals (lithium, sodium, iron, potassium) and alcohols. thallium appears to be an exception, being relatively well absorbed by charcoal. the employment of gastric lavage is controversial and varies markedly depending on geographic area and the background and training of the practitioner. gastric lavage involves blind placement of a large-bore gastric tube into the stomach, in a patient who can either protect his or her own airway or in whom the airway has been protected by an endotracheal tube, with the goal of removing toxicant remaining in the stomach through a combination of instillation of water or physiological saline, followed by suction or gravity-induced drainage. this cycle of instillation/drainage is repeated until the effluent is clear or until several liters of water/saline have been passed through the tube. this procedure has been widely popular in the past and continues to be employed in many eds around the world. it is, however, a largely unproven therapy. the indications for gastric lavage are recent ingestion (generally less than hour, unless the ingestion involves agents that decrease gastric motility, such as anticholinergics) of a substance of sufficient quantity to be likely to cause serious harm in the absence of removal. the procedure should be given greater consideration in ingestions for which inadequate treatment modalities exist (paraquat) or in cases where delivery of proven effective therapy (antidotes or extracorporeal removal) is likely to be delayed. ingestion of low-viscosity petroleum products, corrosives (acids and alkalis) and inability to protect the airway (unless tracheal intubation has been performed beforehand) are contraindications to gastric lavage. serious adverse effects of gastric lavage are relatively rare but may be significant. the procedure may induce hypoxia, perforation of the gastrointestinal tract or pharynx, , fluid and electrolyte abnormalities, inadvertent tracheal intubation, as well as aspiration pneumonitis. tracheal intubation is not completely protective against aspiration. the aact/eapcct recently reviewed the animal and clinical literature regarding gastric lavage and published a position statement, which states in part that gastric lavage should not be employed routinely, if ever, in the management of poisoned patients. the study group pointed out that in experimental studies, the amount of marker removed by gastric lavage is highly variable and diminishes with time and that few clinical studies have demonstrated a beneficial effect on outcome. the quality of the gastric lavage literature is, for the most part, lacking. few studies have the power to detect significant differences in outcome for a single toxicant, and it seems unscientific to compare outcomes after ingestion of widely varying products. furthermore, there activated charcoal products containing sorbitol should be avoided where possible because ( ) the efficacy of cathartics is lacking (see below); ( ) sorbitol is emetogenic and can increase the risk for vomiting the charcoal; and ( ) sorbitol administration in infants is associated with dehydration and other life-threatening events. activated charcoal is administered as a slurry, either in water or sorbitol, orally or via a nasogastric tube. dose recommendations vary, but generally a larger dose is considered better, to assure that binding capacity exceeds the amount of toxicant present. the recommended dose is . to g/kg in children or to g in adults. in general, to be maximally effective, charcoal should be administered as soon as possible after ingestion of the toxicant, preferably within hour. green and colleagues studied this issue in healthy volunteers in a randomized crossover study. after giving g (the equivalent of eight extra-strength tablets) of acetaminophen to patients, then giving charcoal at , , or hours after ingestion, they found no differences in the area under the curve of plasma acetaminophen. the investigators stated that "data do not support the administration of activated charcoal as a gastrointestinal decontamination strategy beyond hour after drug overdose." while there are obvious problems in extrapolating toxicokinetic results from a study involving a nontoxic dose to all overdoses, the results do suggest that benefit clearly decreases over time. as for most decontamination measures, the indications for single dose activated charcoal are controversial. the recently released revision of the single-dose activated charcoal position statement of the aact/ eapcct states that single-dose activated charcoal should not be administered routinely in the management of poisoned patients, but that it may be considered if a patient has ingested a potentially toxic amount of a poison (which is known to be adsorbed to charcoal) up to hour previously. the researchers state that the potential for benefit after hour cannot be excluded. finally, they emphasize that there is no evidence that the administration of activated charcoal improves clinical outcome. charcoal administration is generally considered contraindicated in ingestions of caustics, since it is probably ineffective in reducing their potential for harm and furthermore makes endoscopy difficult. charcoal generally should not be administered when there is a high risk for gastrointestinal hemorrhage or perforation. charcoal is likewise contraindicated in any patient in whom the airway protection is not assured. it should not be administered in the presence of hydrocarbons with high aspiration potential. charcoal should not be administered in the case of ileus or mechanical bowel obstruction. one of the most common adverse events associated with charcoal administration is vomiting, which occurs in approximately % to % of patients. abdominal bloating is also quite common. both diarrhea and constipation may occur. complications have been described, including pulmonary aspiration and direct administration into the lungs via misplaced nasogastric tube. aspiration appears to be relatively rare, but may have serious consequences. , [ ] [ ] [ ] [ ] multiple-dose activated charcoal (mdac) has been proposed for use in the case of drugs that undergo extensive enterohepatic or enteroenteric circulation. drugs with small volumes of distribution are particularly susceptible to removal by adsorption to charcoal in the gut, which has sometimes been referred to as "gastrointestinal dialysis." although experimental and volunteer studies have demonstrated that mdac increased elimination of a number of compounds, there is little proof of clinical benefit. the aact/eapcct has concluded that although many studies in animal and volunteer studies have demonstrated mdac increases drug elimination significantly, there are no controlled studies in poisoned patients that demonstrate mdac reduces morbidity and mortality. pending further evidence of direct benefits, the study group recommended that mdac be considered only if a patient has ingested a life-threatening amount of carbamazepine, dapsone, phenobarbital, quinine, or theophylline. the contraindications for mdac are essentially those of single-dose activated charcoal. the admonition for use in intestinal obstruction is of even greater import in the case of mdac. the presence of decreased peristalsis (often associated with anticholinergic drugs and opiates) should provoke extreme caution in the administration of mdac. prussian blue is an effective absorbent for the management of thallium and cesium intoxications. see chapters and for further information. fuller's earth is often recommended for gastrointestinal decontamination of paraquat (see chapter ) . although effective for this purpose, this substance is found in few hospitals. activated charcoal is an effective absorbent of paraquat and should be employed when fuller's earth is not available. hypermagnesemia may result in cardiac dysrhythmias. elderly patients and those with renal dysfunction are at particular risk. [ ] [ ] [ ] massive doses of cathartics may result in cardiopulmonary arrest. wbi involves the administration, by mouth or nasogastric tube, of large amounts of an iso-osmotic polyethylene glycol electrolyte solution (go-lytely [braintree laboratories, braintree, ma], co-lyte [schwarz pharma, mequon, wi], and others) with the goal of removing unabsorbed toxicant from the gastrointestinal tract as rapidly as possible by rectal expulsion. one rationale for its use includes the fact that some compounds are poorly absorbed by charcoal, particularly iron and lithium. wbi may be of particular interest in the case of sustainedrelease or enteric-coated compounds and in the case of drug packets (body packers). the aact/eapcct's consensus panel concluded that wbi should not be used routinely and that there is no conclusive evidence that it improves the outcome of poisoned patients. based on evidence from volunteer studies, the group recommended that wbi be considered for potentially toxic ingestions of sustained-release or enteric-coated drugs, particularly in those patients who present more than hours after drug ingestion. they more strongly endorsed wbi for patients who have ingested substantial amounts of iron because the morbidity is high and there are no other effective options for gastrointestinal decontamination. another potential indication cited for the use of wbi is expulsion of ingested packets of illicit drugs. polyethylene glycol electrolyte solution (e.g., go-lytely, co-lyte, nulytely [braintree laboratories, braintree, ma]) is administered by mouth or nasogastric tube at to ml/kg/hr until the rectal effluent is clear or until the desired effect is otherwise demonstrated (e.g., passage of drug packets demonstrated by imaging studies). computed tomography (ct) with contrast has often been used to identify retained packets, but a recent case report demonstrated incomplete sensitivity of ct to detect all unexpelled drug packets. wbi is contraindicated in the presence of mechanical or functional (ileus) bowel obstruction or perforation and in the presence of significant gastrointestinal hemorrhage. it should likewise be avoided if the patient is hemodynamically unstable. nausea and vomiting are not uncommon. abdominal bloating and cramping may occur. vomiting in the case of an unprotected airway may result in pulmonary aspiration. cathartics comprise another group of compounds recommended since ancient times for the purpose of eliminating toxicants from the gastrointestinal tract. the two most common categories of cathartics are the magnesium salts (e.g., magnesium citrate, magnesium sulfate) and nondigestible carbohydrates (e.g., sorbitol). despite their long history of use, there is virtually no evidence of their efficacy. on the contrary, cathartics may induce significant harm in certain groups of patients, particularly children and those with renal disease. there are no evident indications for the use of a cathartic alone in the treatment of poisoning. an aact/eapcct position statement concluded that experimental data are conflicting regarding the use of cathartics in combination with activated charcoal. the study group found no published clinical studies that investigated the ability of a cathartic, with or without activated charcoal, to reduce the bioavailability of drugs or to improve the outcome of poisoned patients. they went on to say that based on available data, the routine use of a cathartic in combination with activated charcoal is not endorsed and that if a cathartic is used, it should be limited to a single dose in order to minimize adverse effects of the cathartic. the dose of sorbitol is approximately to g/kg. when given in combination with activated charcoal for single dose-activated charcoal therapy, the dose should be determined on the basis of charcoal dosing. if multiple doses of charcoal are to be administered, repeated use of sorbitol is not recommended. for magnesium citrate, the dose is to ml/kg in children and to ml in adults. cathartics are contraindicated in the presence of bowel obstruction, in the absence of bowel sounds, or in the case of recent bowel surgery or intestinal perforation. they should likewise not be employed in the case of corrosive ingestions or when significant electrolyte disturbances, dehydration, or hemodynamic instability are present. magnesium-containing cathartics must be avoided in patients with renal insufficiency and heart block. caution should be employed in patients at extremes of age. cathartics frequently cause cramping, nausea, and vomiting. significant dehydration may occur if catharsis is excessive, with resultant hypotension. cathartic-related the decision to employ decontamination methods in an individual case must be determined based on the factors present in that individual case. first and foremost in the decision to attempt decontamination is a determination of whether a significant exposure has occurred. this is critical for numerous reasons: ( ) needless decontamination procedures may delay other definitive therapy for systemic toxicity; ( ) conversely, failure to adequately decontaminate the skin may increase morbidity of contaminated patients and result in secondary contamination of health care providers and facilities; and ( ) decontamination, as discussed, is not without risks. it is vital to recall hippocrates' admonition: primum non nocerum. dose/ exposure assessment is extremely difficult on an acute basis due to the great number of unknowns. young children cannot recount the quantity or quality of what they have ingested. self-harm attempts are often accompanied by attempts to conceal or, conversely, to exaggerate the consumption of potentially toxic compounds. it is rare that contemporaneous exposure information (air concentrations or even product identification) is available after environmental exposures associated with hazardous materials releases or acts of terror. fear associated with these events may result in psychogenic illness at times indistinguishable from that of the toxic exposure. when doubt exists, it may be safer to decontaminate, but this should be a considered decision. once it is established that an exposure has occurred (or if exposure cannot be excluded) and some attempt has been made to determine the magnitude of the exposure, one must examine the toxic potential of the compound(s) in question, keeping in mind that the toxicity of combined substances is not always equal to the sum of their individual toxicities. approaches to the treatment of poisoning are deeply rooted in personal experience and colored by bias in the literature. recent studies have reexamined the need to decontaminate victims of certain exposures that were previously approached aggressively from a therapeutic standpoint. , decisions around decontamination must be individualized. a suggested approach is found in figure b - . this nonvalidated algorithm should simply be considered a pathway for considering options. there are numerous potential exceptions to the general suggestions in the figure. one might consider gastric lavage alone in the case of presentation of poisoning within hour of ingestion by a highly toxic compound that is not readily absorbed by charcoal. lithium is one such example. reiterating, gastric lavage is most likely to be effective in an early-presenting, potentially lethal ingestion. cyanide is an example of a highly toxic compound that might be removed by lavage, but is also readily absorbed by charcoal. yes. illicit drug packets containing cocaine or heroin may leak. a dose of activated charcoal given prophylactically (in the absence of symptoms) could theoretically absorb eventual leakage, while wbi accelerates passage of the packets. as a reminder, clinical evidence of toxicity (leakage) is an indication for laparotomy. similarly, charcoal followed by wbi may be indicated for ingestions of enteric coated tablets. in cases in which ingestion of a substance known to be effectively eliminated by wbi (such as lead, zinc, or iron) is not recent (and thus not likely to benefit from gastric lavage) and when the substance is not readily absorbed by charcoal, wbi alone may be indicated. it should be clear from the discussion that the previously common "reflex arc" of ingestion-decontamination should not apply. the decision to apply a particular procedure or combination may not be simple. for this reason, consultation with a regional poison center and/or medical toxicologist is strongly suggested in cases of uncertainty. . irritants, corrosives, and substances toxic by skin absorption should be removed. liquids with high volatility and potential for secondary contamination (organic solvents) should likewise be removed. when in doubt, decontamination is appropriate; however, life-saving interventions (abcs) take precedence over decontamination. personnel should wear protective garments appropriate to the hazard. . if the history is reliable and the ingestion is clearly nontoxic, no decontamination is required. the urge to "do something" should be weighed against the maxim to "first, do no harm." . single-dose activated charcoal (sdac) is considered most effective when administered less than hour after ingestion of a toxic substance. there is insufficient evidence to support or condemn its use with toxic ingestions presenting more than hour later. in general, metals (lithium, iron, but not thallium) and alcohols are poorly absorbed. . multiple-dose activated charcoal (mdac) should be considered primarily if a patient has ingested a life-threatening amount of carbamazepine, dapsone, phenobarbital, quinine, or theophylline. multiple-dose activated charcoal increases the elimination of amitriptyline, dextropropoxyphene, digitoxin, digoxin, disopyramide, nadolol, phenylbutazone, phenytoin, piroxicam, and sotalol, but evidence is insufficient to support its use in these ingestions. . whole bowel irrigation should not be performed in the presence of ileus or bowel obstruction. . if uncertainty exists about the need for decontamination procedures, contact the regional poison control center and/or a medical toxicologist. effective adsorbent ; it may even limit the adsorptive capacity of activated charcoal. two theories have been advanced to account for the observed acceleration of drug clearance associated with the use of mdac after the drug's absorption from the gastrointestinal tract. one theory explains this acceleration by charcoal's interruption of the enterohepatic recirculation of hepatically metabolized drugs. the other is aptly called "gastrointestinal dialysis," a term coined by levy in an editorial that accompanied the seminal work on this topic by berg and colleagues. , using an animal model of intravenous theophylline poisoning, kulig and colleagues measured bile theophylline concentrations and were able to demonstrate that the observed increase in theophylline clearance with mdac was not due to interruption of enterohepatic recirculation of the drug. arimori and colleagues demonstrated in an experimental model that the exsorption rate of theophylline into the intestinal lumen increased in proportion to the administered dose of theophylline, suggesting that the gastrointestinal dialysis effect may increase with escalating dose for some toxins. given that overall mortality from overdose is low, that the efficacy of gastrointestinal decontamination techniques declines significantly with advancing time after ingestion, and that significant delay to clinical presentation occurs in the majority of ingestions, elimination enhancement techniques, including mdac, have come under recent scrutiny. tenenbein cited case reports of complications from mdac with a lack of proven clinical benefit in arguing that its role in the treatment of poisoning required reassessment. these cautions notwithstanding, review of the available evidence suggests that mdac can accelerate drug clearance, achieving rates comparable to more invasive techniques such as hemodialysis. moreover, a recent study of complications associated with mdac use found that they occurred infrequently. there are also data suggesting that mdac improves outcome in selected poisoning cases. one report describes two presentations of the same patient with phenytoin toxicity, one in which mdac was not used and a second in which it was; the second hospitalization was days shorter despite the patient having a higher serum phenytoin level. a recent single-blind, randomized, placebo-controlled trial performed in sri lanka demonstrated a significant reduction in mortality from yellow oleander poisoning once a drug or toxin has been absorbed, a number of means exist to enhance its elimination. chapter d addresses extracorporeal techniques of toxin removal from the blood (e.g., hemodialysis and hemoperfusion), which, when performed on an emergency short-term basis such as for poisoning, are done intermittently using a large double-lumen catheter in a central vein with flow through the circuit driven by a pump. other types of dialysis exist that also usually do not require arterial access and are more versatile in that they do not pose as great a hemodynamic stress to the patient; however, they also are generally much slower in their rates of clearance. one of these alternatives, multiple-dose activated charcoal (mdac), involves use of the patient's own gastrointestinal mucosa as a dialyzer. all of these techniques share in common with dialysis the underlying principle of filtering a toxin from the blood using a semipermeable membrane in order to enhance its clearance. a number of substance-related factors affect the clearance rates that can be achieved by dialysis techniques. first, a toxin must distribute primarily into the intravascular compartment-that is, have a low volume of distribution, in order to be removed by a dialysis technique at a clinically significant rate. second, molecular size is important, with low molecular weight substances crossing the dialysis membrane from an area of high concentration (blood) to an area of low concentration (dialysate). even higher molecular weight substances can cross and thus be removed from the blood by convection when dialysis is supplemented with ultrafiltration, which relies on a membrane with a high permeability coefficient and a high transmembrane pressure. finally, high protein binding presents a large molecular size to the membrane (i.e., a protein-bound drug), thus limiting the rate of clearance, unless the toxin is adsorbed from the serum proteins such as with the use of a charcoal cartridge in hemoperfusion. delayed absorption may occur after toxic ingestion: ( ) with sustained-release preparations, ( ) if tablet conglomerates form in the gastrointestinal tract, ( ) if the substance delays gastrointestinal motility. or, ( ) if a poorly absorbed substance (e.g., phenytoin) is ingested. multiple doses of activated charcoal have been used in these situations to enhance preabsorptive elimination. whole bowel irrigation, the other treatment modality used to enhance preabsorptive elimination, appears to provide no additional benefit when administered to treat a drug overdose for which activated charcoal is an curtis p. snook, md ■ daniel a. handel, md, mph c with mdac therapy when compared with treatment with single-dose activated charcoal. further study is needed to define the circumstances in which the benefits of mdac appear to justify its risks. however, some predictions can be made based on available data. mdac has been demonstrated to accelerate the clearance of a number of toxins (box c- ). chyka and colleagues found in a porcine model that mdac enhanced elimination of acetaminophen, digoxin, and theophylline but not valproic acid. drugs with relatively lower intrinsic clearance (digoxin, theophylline) were cleared with mdac more rapidly than were drugs with higher intrinsic clearance (acetaminophen). interestingly, volume of distribution, half-life, and protein binding were not significantly correlated with mdac enhancement of clearance. however, therapeutic doses were administered in this study, limiting application of these results to the overdose setting. a volunteer study of dapsone ingestion using a randomized crossover design along with data from two overdose cases showed a doubling of elimination with mdac in healthy volunteers and a greater increase in the overdose patients, again suggesting that the effectiveness of mdac also may be a function of dose for a given toxin. significant adverse effects with mdac preclude its use in patients for which no significant clinical benefit is expected. there are some drugs or drug classes for which data are conflicting (box c- ). for example, the available studies suggest that mdac likely would not be beneficial in unselected cases of digoxin poisoning. however, one volunteer study of mdac for enhancement of digoxin clearance applied kinetic predictions to suggest that greater clearance with mdac would occur in patients with renal impairment ; animal data support this assertion. in a case of chronic digoxin poisoning, mdac effectively accelerated drug clearance when digoxin antibody fragments were unavailable and hemodialysis had been unsuccessful. box c- lists the drugs investigated for which mdac has not been shown to accelerate clearance. mdac often is poorly tolerated. virtually all patients in one study complained of poor palatability and bloating with mdac, with one patient withdrawing because of repeated vomiting. transient constipation may occur with mdac use in susceptible patients. protracted vomiting also has been reported to limit the usefulness of mdac in theophylline poisoning. aspiration of charcoal is another well-recognized complication of mdac. in one published case, an unintubated patient treated for phenobarbital and carbamazepine overdose with gastric lavage followed by mdac vomited and aspirated hours after therapy was initiated, eventually dying on hospital day . another reported patient with theophylline overdose was treated with ipecac followed by mdac. he went on to develop convulsions followed by aspiration, eventuating in his death. in the most sobering case, a patient aspirated activated charcoal despite airway control. this patient received mdac after intubation and gastric lavage for desipramine and thiothixene overdose. after extubation the next day, the patient vomited, aspirated charcoal, and had a cardiorespiratory arrest from which he could not be resuscitated. in a similar case, a patient who ingested thioridazine and imipramine underwent endotracheal intubation and gastric lavage after he had a seizure. he received g of activated charcoal by nasogastric tube followed by g and then g with sorbitol every hour for hours. when the nasogastric tube was removed, the patient vomited, became cyanotic and had a respiratory arrest. charcoal was suctioned from the patient's lungs until hospital day seven. rau mdac, multiple-dose activated charcoal. and colleagues described three deaths from charcoal aspiration among patients treated with charcoal for central nervous system-depressant overdose without airway protection. their subsequent six overdose patients were intubated prior to charcoal administration and no cases of aspiration occurred. in light of this experience, any patient at risk for aspiration (i.e., one in whom diminished consciousness, depressed airway reflexes, or seizures are present or anticipated), should have a firm indication for mdac and a protected airway before undertaking the procedure. equally important, there appears to be a ceiling effect for charcoal adsorption such that increasing dose or frequency beyond a certain point results in no additional therapeutic benefit. overdose victims with decreased gastrointestinal motility may develop bowel obstruction with use of mdac. a number of cases have reported mdac resulting in small bowel obstruction, abdominal distension, constipation, rectal bleeding, and even intestinal perforation when either the overdose itself or the therapy for the overdose involved anticholinergic drugs. [ ] [ ] [ ] [ ] [ ] [ ] appendicitis in association with mdac use has been reported. another patient with chronic theophylline toxicity treated with mdac developed small bowel obstruction and was found on laparotomy to have adhesions at the ileocecal valve secondary to a previous hysterectomy; multiple pieces of charcoal were found in the bowel at the site of obstruction measuring . × × cm in aggregate. cathartics are ineffective as a means of elimination enhancement after poisoning. however, to avoid constipation and charcoal inspissation, cathartics often are coadministered. sorbitol is the usual choice because of its rapid onset and palatability. sorbitol is typically marketed in a % concentration with activated charcoal because it is bacteriostatic at this concen-tration. , using excessive or multiple doses of sorbitol with activated charcoal in poisoning is associated with significant morbidity, including hypernatremia resulting in death, and should be avoided. magnesium citrate or sulfate occasionally are used instead of sorbitol. patients with premorbid magnesium abnormalities or those receiving excessive doses of magnesium cathartic can suffer serious morbidity, including hypermagnesemia, which can be fatal. the optimal dosing for activated charcoal in mdac is unknown. the commonly accepted dose is g/kg (maximum of g) of activated charcoal initially, followed by . g/kg (maximum g) every hours thereafter. others have suggested a dose of g/kg of activated charcoal in . ml/kg body weight of % sorbitol every hours until the first charcoal stool appears. in children, the same weight-based dose of activated charcoal is recommended. a third set of published guidelines for adults has suggested an initial dose of to g, with additional doses given at a rate of at least . g/hr until the patient is improving clinically and by relevant laboratory parameters. this latter set of recommendations includes lower dosing ( to g) for children younger than years of age, smaller doses more frequently along with antiemetic use in vomiting patients, and no use of cathartics, particularly in young children. mdac has been used safely in infants and neonates , ; it has even been used to treat neonatal hyperbilirubinemia. patients receiving mdac should be intubated, ideally with a cuffed endotracheal tube, if they are obtunded or have diminished airway reflexes or seizures. the charcoal should be diluted with at least ml of water per gram of charcoal. mdac should not be administered in the presence of ileus or bowel obstruction. coadministration of anticholinergic agents should be avoided during mdac. charcoal should be withheld hours prior to extubation. gastric contents should be aspirated with a nasogastric tube prior to extubation to avoid the complications previously discussed. magnesium cathartics should be used with caution, particularly in patients with decreased renal function, and are best avoided in such patients given that safer alternatives are available. it is a common practice to give the first dose of charcoal with a cathartic, usually sorbitol, and to give subsequent doses without cathartic. the availability of only sorbitol-charcoal preparations can be problematic in hospitals with such rigid stocking patterns. electrolytes should be closely monitored if more than one dose of cathartic is administered. box c- summarizes the indications, contraindications, and dosing guidelines for mdac use in poisoning. myoglobin in an animal study and in humans from drug-and exercise-induced rhabdomyolysis has been removed successfully by cvvh. [ ] [ ] [ ] severe lactic acidosis induced by propofol improved significantly with the use of cvvh. cvvh has been shown to be safe and effective in treating radiocontrast-induced acute renal failure after percu- continuous veno-venous hemofiltration (cvvh) and similar modalities used for what is termed "continuous renal replacement therapy" increasingly are being used in the therapy of critically ill patients, including those suffering from poisoning. cvvh offers a number of useful advantages over traditional dialysis techniques. first, it can be used in patients with renal failure who are hemodynamically unstable and require large volumes of parenteral nutrition. in cvvh isotonic fluid is removed from the femoral vein slowly and continuously rather than in -to -l increments over a -to -hour treatment session, the typical pattern for hemodialysis (hd). also, the diffusion clearance inherent to hd results in the return of a hypo-osmolar fluid to the intravascular space, resulting in a further loss of intravascular volume in comparison with cvvh. the latter restores volume losses with isotonic replacement fluid and fluid refilling from the overhydrated body parenchyma. second, trained nursing staff can administer cvvh, though it requires their continuous attention. finally, the typical rebound in serum drug levels from redistribution observed after hd are not seen with continuous modalities such as cvvh. the main disadvantage of cvvh in comparison with hd is the slower rate of toxin removal it can achieve. this disadvantage is significant if rapid toxin removal is critical to the patient's survival, which often is the case. thus, the use of cvvh for a toxin whose elimination is enhanced by dialysis techniques should be considered only when hemodynamic instability precludes the use of hd, when hd is not available, or if the inherently slower rate of toxin removal is clinically acceptable. cvvh does not require arterial access-a pump is used to provide the pressure gradient necessary for filtration. anticoagulation is required to optimize the lifespan of the apparatus (which requires replacement when it becomes clotted). however, the need for anticoagulation is less than for arteriovenous techniques because of the controlled blood flow provided by the pump, particularly in smaller pediatric circuits. animal data demonstrate that cvvh can achieve adequate flow rates with pediatric-sized filters and circuits. the blood is passed through a highly permeable hemofilter, forming an ultrafiltrate made up of plasma and filtered solutes. hemodiafiltration refers to the use of a dialysate fluid on the opposite side of the filter from the blood flowing in a counter current direction. box c- lists the toxins for which cvvh has been shown to speed elimination. toxin-related factors determining clearance by cvvh include the sieving coefficient (dependent upon molecular weight and protein binding) taneous coronary interventions. hypothermia is associated with poisoning, particularly when central nervous system depressants result in excessive exposures to cold ambient temperatures. cvvh has been used with success in rewarming a patient with severe accidental hypothermia. toxins for which cvvh has not been shown to speed elimination are listed in box c- . one patient treated with a lorazepam infusion developed toxicity from the propylene glycol diluent, despite receiving cvvh with dialysis (cvvhd), suggesting that the glycol was not effectively removed. care should be exercised in choosing the replacement fluid; lactate-buffered solutions have been reported to induce hyperlactatemia. since cvvh requires systemic anticoagulation, contraindications include recent surgery and gastrointestinal or intracranial hemorrhage. however, regional heparinization can be used with those patients for whom cvvh is necessary but who cannot tolerate systemic heparinization. trisodium citrate is a useful alternative to heparin as a regional anticoagulant with cvvhd to minimize the risk of hemorrhage and thrombocytopenia. however, caution should be exercised in patients with decreased hepatic function as use of trisodium citrate in this context is associated with severe hypercalcemia. a summary of the indications and contraindications for cvvh in poisoning are listed in box c- . though the first use of peritoneal dialysis pd dates back to , experiments with peritoneal lavage were carried out as early as . numerous advances have been made since this early work was done, including the addition of substrates to the dialysate to enhance the elimination of certain drugs. given the ease in most medical centers of obtaining hd, pd has fallen out of favor in most instances for the treatment of acute intoxication, although it continues to be used as a bridge when hd is not available or as an adjunct to enhance elimination. because of its "second-tier" status, recent literature and research on its use in poisoning are sparse. the peritoneal surface area of an adult has been estimated to be , cm , allowing this structure to serve as an excellent semipermeable membrane for dialysis. pd involves in principle the passive movement of drug or toxin down its concentration gradient from the intravascular space in capillaries dispersed throughout the lining of the peritoneum into the dialysis solution infused into this cavity. as with other dialysis techniques, pd is particularly effective in removing drugs with small volumes of distribution and low protein binding. the intermittent method of pd involves the use of one catheter to introduce the dialysate by gravity, removing it at a later time through the same access. this process also can be carried out using two catheters. two liters of fluid ( ml/m or ml/kg in children) are used per exchange, with the fluid typically left in the peritoneal cavity for to minutes. in contrast, during continuous ambulatory peritoneal dialysis (capd), dialysate is left in the peritoneum for much longer periods of time. extending this period beyond hours or leaving the catheter in place between courses of pd increases the risk of infection. various additives, such as glucagon, albumin, prostaglandin e (pge ), lipids, furosemide, streptokinase, and chelating agents, have been added to dialysis fluid in the attempt to enhance elimination with varying degrees of success. - a mildly hypo-osmolar solution is used to prevent water removal during dialysis, with electrolyte concentrations similar to that of extracellular fluid. when the osmolality of the dialysate is increased, increased solute removal occurs at the expense of a negative fluid balance with each exchange. whereas pd can be a continuous process, its elimination rate is significantly less than that of hd and other more invasive methods of extracorporeal therapy. blood flow via mesenteric circulation in pd cannot be adjusted as it can in hd, and the mechanics of instilling and draining in the pediatric population, pd is contraindicated when there is severe intraperitoneal hemorrhage that is sometimes seen in hemolytic-uremic syndrome with thrombocytopenia. the presence of focal peritonitis, a fecal fistula or colostomy, abdominal adhesions, and recent abdominal surgery with use of a prosthetic material, a major vascular anastomosis, or an open wound, are relative contraindications for pd. box c- lists the toxins for which peritoneal dialysis has not been found effective in accelerating clearance. a summary of the indications and contraindications for peritoneal dialysis in poisoning are listed in box c- . dialysate into and from the peritoneum limit achievable clearance rates. the time required to eliminate a given amount of toxin is three to six times greater with pd than with hd, depending on the properties of the agent. the elimination of small molecules that are highly membrane permeable is rate-limited by blood and dialysate flow rates, whereas the elimination rate of large molecules depends primarily on membrane surface area. in severe poisoning from a dialyzable toxin, the difference in achievable clearance rates between hd and pd can have enormous clinical significance, as one case series of methanol poisoning illustrates. thus, pd should not be substituted for hd in such cases unless the latter is absolutely inaccessible. if hd is not available in one treating facility but patient transfer is possible, the patient should be transferred. pd may be used as an adjunct during the transfer to speed toxin elimination. similar to other methods of extracorporeal elimination enhancement, pd can produce electrolyte shifts and loss of serum proteins. pulmonary complications, including bronchitis/pneumonia, pleural effusions, and atelectasis, also are known adverse effects. bowel perforation from catheter misplacement occurs with an incidence of . % according to one report. the other major complication of pd, especially capd, is peritonitis, which usually limits the long-term use of this technique. the dialysate additive icodextrin has been reported to cause a blistering skin reaction that resolves upon cessation of the use of the substance. pd can be used for toxins of up to , daltons in molecular weight and when a filtration rate of no greater than ml/min is required. it can be used when hd is not currently available and the patient's clinical status is critical. it can be used also when anticoagulation is contraindicated due to comorbidities. pd has been more widely used in the pediatric population, especially in the contexts of acute renal failure seen in hemolyticuremic syndrome, congestive heart failure, chronic renal failure, and hyperkalemia. , because of the difficulties of vascular access in small children as well as the difficulty in removing excess fluid gained between courses of dialysis without causing profound hypotension, pd offers distinct advantages in this population. the toxins for which pd hastens clearance and those for which its effects on clearance have been equivocal are listed in boxes c- and c- , respectively. note that there are toxins listed in box c- that are eliminated by the kidneys, such as baclofen, for which pd has been found useful in poisoning accompanied by renal failure even though pd effects on clearance are small when renal function is normal. also, pd has proven useful in treating toxin-induced renal failure apart from effects on toxin clearance. hypercalcemia from overadministration of vitamin d in an anephric child has been effectively box c- chapter c addresses the use of peritoneal dialysis, various forms of hemofiltration, and multiple-dose activated charcoal (mdac) as so-called "gastrointestinal dialysis." this chapter discusses the more classic methods for extracorporeal removal of toxicants, namely, hemodialysis (hd) and hemoperfusion (hp). in addition, we briefly discuss a newer methodology, the molecular adsorbents recirculating system (mars, also referred to as albumin dialysis), which was developed primarily for the treatment of fulminant liver failure (flf), but has recently been employed in the treatment of a variety of poisonings. as has been pointed out in previous chapters, extracorporeal methods for toxicant removal should be viewed as important adjuncts rather than as primary approaches to specific poisonings. the most effective method of removal of toxicants from the body in most cases is maintenance of properly functioning kidneys, liver, and lungs through excellent supportive care. the ultimate evaluation of efficacy of extracorporeal methods is in fact through direct comparison with spontaneous elimination by the body. in the absence of careful attention to resuscitation and supportive care, these adjuncts may be of little use; they may even be dangerous. further-more, while dozens of toxicants have been shown to be removed by hd or hp, evidence-based support for significant clinical improvement from these procedures is often lacking. as will be shown, each of these procedures has benefits and risks, which must be carefully weighed against other methods of treatment. it is difficult to ascertain the frequency with which hd and hp are employed in the treatment of poisoning. based on the most recent toxic exposure surveillance survey, it would appear that use of hd (n = ) largely exceeds that of hp (n = ). both procedures were far more commonly performed in adults older than years ( % of hd cases and % of hp cases) than in children. use of these extracorporeal removal techniques in children younger than years was rare (seven cases of hd, one case of hp). these figures are undoubtedly underestimates of real use, since reporting to poison centers by physicians is voluntary and incomplete. nonetheless, they suggest that extracorporeal purification therapies have a limited role in management of poisoning in the united states. hd requires the passage of blood from the body through an external circuit, in which it is anticoagulated and placed in sustained contact with a selectively permeable membrane prior to being returned to the patient. opposite the membrane and flowing in countercurrent direction is a heated dialysate, the composition of which can be varied according to the indications for dialysis. the variation of electrolyte concentrations, addition of buffers, and sometimes other additives allow the operator to manipulate the passage of these elements from one side of the membrane to the other. hd operates on the basis of three principles: diffusion, osmosis, and ultrafiltration. diffusion relies on concentration gradients, with solutes moving from an area of higher concentration to an area of lower concentration, ultimately resulting in equilibration. osmotic pressure results in the movement of water from one side of a semipermeable membrane to the other, dependent on a higher concentration of solutes on the other side. manipulation of plasma sodium concentrations (sodium profiling) during dialysis can induce a net flow of water from cells into the plasma, allowing for its ultimate removal from the plasma. ultrafiltration involves movement of fluid across the membrane due to the presence of a pressure gradient. this principle allows control of fluid loss during dialysis and can be manipulated via varying dialysate pressure (pressure control) or by variation of dialysate flow volumes (volumetric control). volumetric control is more common in modern dialysis machines. in the case of toxicants, diffusion into the dialysate is the primary end point, although amelioration of acid-base and electrolyte conditions is often an important additional goal. dialyzer specifications (blood flow rate, surface area, and membrane construction) determine, in part, the efficacy of drug or chemical removal from the blood. pharmacodynamic and pharmacokinetic factors (molecular weight, lipid solubility, protein binding, toxicant concentration gradient, and volume of distribution [vd] ) are equally critical factors which will be discussed in further detail below. hp implies the passage of blood through a device containing absorbent particles. it may be performed alone or in combination with hd and has been employed for a number of years in the management of poisonings. resin hp with variants of amberlite xad (rohm & haas, philadelphia, pa) was viewed as promising in the s and s. however, very few recent reports of resin hp have been published. [ ] [ ] [ ] [ ] [ ] [ ] this is likely in part due to unavailability of medical-grade resin cartridges in many geographic areas. activated charcoal appears to be the most commonly used absorbent material based on published reports, but hospital availability of even these cartridges appears to be limited. a recent survey of new york city -receiving hospitals revealed that only about one third of those surveyed had charcoal hemoperfusion (chp) cartridges readily available (only one hospital had pediatric cartridges). just of responding hospitals had used chp within the previous years. reasons cited by the authors for limited cartridge availability and use of chp were decreasing clinical use of phenobarbital and theophylline and improved efficacy of hd. chp irreversibly binds water-and lipid-soluble drugs and chemicals in the molecular weight range of to , da, a much larger range than that for hd (<~ da). plasma drug extraction ratios for hp are superior to those for hd for acetaminophen (paracetamol), digoxin, glutethimide, paraquat, phenobarbital, and theophylline. however, further examination of this list may reveal, in part, why hp is seldom employed in the united states. nacetylcysteine is effective for acetaminophen poisoning, widely available, and less invasive. digoxin fab fragments are far more effective than hp in binding and eliminating digitalis glycosides. glutethimide is rarely prescribed in the united states. paraquat poisonings are rather rare in the united states, and hp has not been shown to alter outcomes, despite of reduction of plasma concentrations. as mentioned previously, phenobarbital and theophylline have largely been replaced in clinical use by other agents, and the less invasive mdac has been shown to be effective in reducing plasma concentrations of these agents, although it has not been proven to alter outcomes. the molecular adsorbents recirculating system (mars) is a relatively new method of extracorporeal decontamination, which employs dialysis across a membrane impregnated with albumin and a % albumin dialysate, line has a molecular weight of . da, but is poorly removed by hd. this is due to its very large vd. the vd is the theoretical volume into which a drug distributes in the body and is generally expressed in liters or liters/kg body weight. substances with a small vd (< l/kg) reside to a greater extent in the bloodstream and can generally be effectively removed by using extracorporeal methods. the vd of a drug is the most important determinant of the efficacy of hp. even when extracorporeal methods are very efficient in removing toxicants from the blood, if the theoretical vd is high, the pharmodynamic effect achieved is likely to be minimal, as the concentration at the drug target tissue may be virtually unchanged. a third critical factor in predicting efficacy of extracorporeal methods is protein binding. hd is of limited value for substances that are highly protein bound. chp, on the other hand, remains effective in cases of drug overdose with substances of high protein binding capacity. a study conducted in patients treated with hp, with drug overdoses involving different drugs, showed that the efficacy of drug removal through absorption by activated charcoal was dependent on the binding affinity, which is related to the protein binding percentage. this study concluded that drugs that are protein bound at levels of up to % are effectively removed from the blood with hp. following hd or hp, substances with a higher vd and high protein binding percentage tend to redistribute from storage tissues to plasma. for this reason, a single extracorporeal substance removal session may be insufficient. water solubility is also an important factor in determining dialyzability. water-soluble substances like ethanol, methanol, ethylene glycol, salicylates, theophylline, lithium, and valproate tend to have limited distribution in adipose tissues, thus limiting their vd. this property makes them ideal for extracorporeal drug removal. poisoning by a substance susceptible to extracorporeal removal is not in itself an adequate justification for the procedure. in general, extracorporeal methods should be reserved for poisonings for which toxic metabolic activation is anticipated (e.g., methanol, ethylene glycol), for which blood concentrations or the amount of toxicant absorbed foretell very serious toxicity, and for patients who have not improved despite of appropriate supportive care (volume repletion, acid-base correction, use of vasopressors, ion-trapping diuresis, and administration of specific antidotes). winchester identifies six clinical considerations for hd or hp in poisoning: ( ) progressive deterioration; ( ) depression of midbrain function leading to hypoventilation, hypothermia, and hypotension; thus attracting highly protein-bound substances. in addition, charcoal and anion exchange resin cartridges are employed to filter the dialysate, regenerating it for continued use. mars may be of interest in the setting of poisons that have a predilection for liver toxicity, as the system is capable not only of removing certain hepatotoxins, but also reducing hyperbilirubinemia, restoring hemodynamics, diminishing hepatic encephalopathy, and improving renal function. mars has been used to maintain patients in liver failure during the peritransplant period. [ ] [ ] [ ] [ ] the existing data for mars in general are encouraging, but the evidence base is limited. this caveat is even truer in the setting of poisoning. a number of factors determine the suitability of use of hd or hp as extracorporeal methods of removal of a given toxicant. in the case of hd, chemical and drug removal are determined by factors as blood flow rate, dialysate flow rate, dialyzer surface area, and pore structure of the chosen membrane and by pharmacodynamic and pharmacokinetic factors such as molecular size (usually < da), lipid solubility, protein binding, vd, and concentration gradient. for hp, the selection of filter material has historically been important (charcoal vs. resin cartridges). with scarce availability of resin cartridges, the choice has essentially reverted to charcoal. hp is typically reserved for drugs with a low vd and high protein binding, but as mentioned previously, compounds of larger molecular weight may be removed. hd frequently causes hypotension, whereas this is reportedly less often a problem in hp. as such, in instances where hemodynamic instability after poisoning is an issue, hp may be preferable even if less efficacious for a particular toxicant. vasopressors such as dopamine and norepinephrine are absorbed by the cartridges and therefore should be administered in the circuit after the sorbent. a definite role for mars therapy in poisoning has not yet been established. in general, substances that are highly bound to albumin and poorly susceptible to other forms of treatment, including hd and hp, should prompt consideration of mars, particularly in the setting of liver failure. in order to be effectively hemodialyzed, toxicants should ideally be of low molecular weight (< da). a low molecular weight alone is not sufficient, however, to assure effective dialyzability. for example, nortripty-( ) development of, or predisposition to, complications of coma, such as pneumonia or septicemia; ( ) impairment of drug excretion; ( ) poisoning of substances possessing metabolic and/or delayed effects; and ( ) poisons extractable at a rate exceeding endogenous elimination. these considerations could be condensed to ( ) patients who are very sick or likely to become so and ( ) toxicants for which hd/hp are effective in removal and known to make a clinical difference in outcome. blood concentrations of certain toxicants (e.g., methanol and ethylene glycol) have been proposed as indicators for hd. historically, a plasma concentration of mg/dl has been used as a threshold for the need for dialysis in both ethylene glycol and methanol poisonings. the availability of fomepizole, a safe and effective inhibitor of alcohol dehydrogenase, has altered the indications for hd. [ ] [ ] [ ] [ ] while hd continues to be a useful and often necessary adjunct in the treatment of toxic alcohol poisonings, an elevated blood concentration of the alcohol alone is no longer considered sufficient to require hd. severe metabolic acidosis accompanying methanol and ethylene glycol, as well as other toxic alcohol poisonings, is a clear indication for hd, regardless of blood concentration. in these cases, hd not only effectively removes the toxicants and their acidic metabolites, but helps in directly correcting the acid-base balance of the patient. even when the toxicant's molecular weight, protein binding, or vd do not conform to the criteria necessary for dialyzability, hd should be considered in the setting of severe metabolic acidosis and/or renal failure. an example of this can be found in overdoses by metformin. metformin would be expected to be poorly dialyzable on the basis of its very large vd. however, metformin toxicity results in severe metabolic acidosis, which may be ameliorated by application of hd techniques. , both hd and hp may be performed in children, but both pose greater challenges than in adults. vascular access is obviously more difficult in small children. for this reason, peritoneal dialysis has been historically used more often in children. however, use of peritoneal dialysis for acute renal failure in children appears to be decreasing and use of hd increasing over time. as mentioned before, availability of chp cartridges suited to use in children may be extremely limited, even in major metropolitan areas. however, it appears that if antidotal therapy for ethylene glycol is administered before the onset of renal insufficiency, hd, even in children, may be safely avoided. given the long half-life of methanol, hd is still considered advisable in children after treatment with fomepizole. obviously, poisonings by substances normally eliminated by the kidneys may require hd or hp in patients with chronic renal failure. both hd and hp are associated with potential serious complications (box d- ). the most serious complications associated with hd include hypotension (which by extension may lead to myocardial or brain infarction) and bleeding associated with anticoagulation. an addi- concepts in medical toxicology box d- to the onset of acidosis and end-organ damage (renal failure or visual disturbances). while fomepizole is relatively expensive, a multiple-day icu admission and hd might exceed drug costs. in other situations, hd or hp may shorten icu or hospital stays and be cost conscious procedures. a formal cost-benefit analysis of these various treatment options would be useful. the evaluation of efficacy of extracorporeal methods of blood purification should ultimately rest on improvement in patient outcomes. however, a good outcome after hd or hp is not equivalent to established efficacy. indeed, the literature is replete with case reports of the "successful use" of hd and hp in cases of poisoning in which the procedure may have had little impact at all on outcome. as mentioned in the beginning, the efficacy of extracorporeal purification procedures must be compared with the efficacy of elimination of the substance in question by the kidneys, liver, and lungs. thus, one cannot estimate the utility by simply measuring blood concentrations before and after hd/hp. rather, the amount of extracted drug should be measured directly in the dialysate or via elution from the cartridge, or alternatively, indirectly from hourly differences in simultaneously arterial (inlet)-venous (outlet) concentrations multiplied by the purification system blood flow. these amounts can then be compared to the concentrations in urine (determination of spontaneous renal clearance) and to the quantity believed to have been absorbed to determine efficacy. before and after blood concentrations give some idea of the combined efficacy of corporeal and extracorporeal elimination, but the interpretation may be clouded by ongoing intestinal absorption or redistribution of the toxicant. elimination by other routes (pulmonary, sweat) should be likewise considered. the list of toxic substances that have been subjected to hd and/or hp is quite long. winchester provides a list of more than substances removed with dialysis and hp. as already mentioned, the ability to remove a toxic substance by hp or hd is not equivalent to an indication for these procedures. one must take into account the patient's underlying health (renal or hepatic insufficiency), the toxicity of the absorbed substance, the presence of or likelihood of advancing to severe illness, the availability of these procedures, and the availability of acceptable alternatives (good supportive care, antidotes). a list of a few substances that have frequently been subjected to extracorporeal removal follows (table d- ) . the list is not comprehensive and does not necessarily imply an indication for hd/hp. the reader is referred to individual chapters for detailed indications for these and other forms of therapy. tional significant consequence is reduction of therapeutic levels of drugs, which may result in unmasking of conditions protected by these agents. complications appear to be relatively fewer in hp, but may nonetheless be important. hp is associated with the destruction of platelets, such that significant thrombocytopenia and bleeding may occur. this complication has been reduced by coating of the sorbent particles with a polymer solution. hypocalcemia likewise may occur. rahman and colleagues recently reported on the case of a patient treated with chp for valproate poisoning. despite of the use of coated charcoal, the patient developed severe hemolysis, which the researchers attributed to mechanical damage to red cells by the high flow rate through the cartridge. the patient required packed red blood cells, platelets, and fibrin transfusions, developing oliguricanuric renal failure that required days of hd over a period of several weeks. she ultimately regained her normal renal function. transfer hd and hp are not universally available. thus, transfer to centers capable of performing these procedures may at times be required. the decision to transfer a patient for these therapies should rest on evaluation of several elements: ( ) is the patient stable for transport? ( ) will the patient's outcome likely be significantly better if the procedure is undertaken? ( ) are there acceptable alternatives to hd or hp? each of these issues should be studied prior to committing a patient to prolonged and expensive or potentially hazardous transport to another facility for extracorporeal decontamination procedures. as mentioned above, hd and hp have significant potential for complications. in certain poisonings, such as ethylene glycol and methanol intoxications, where hd was formerly routinely prescribed in the presence of high blood concentrations, recent improvements in specific antidotes have led to therapeutic alternatives. for example, if a patient with significant ethylene glycol poisoning arrives at the hospital before the onset of acidosis and renal failure, early treatment with fomepizole alone may preempt the need for hd. , , even in the case of methanol, hd may be avoided in selective cases after early treatment with fomepizole. methanol's elimination half-life is much greater than that of ethylene glycol, however, and may require prolonged antidotal treatment in the absence of hd. requirement for acute hd and/or hp generally implies costly intensive care unit (icu) admission. as mentioned above, early administration of fomepizole may forego the need for hd and icu admission in ethylene glycol and selected methanol poisonings, if administered prior the alcohol and glycols generally have toxicity that is inversely related to molecular weight. this, along with limited protein binding, small volumes of distribution, and relatively high water solubility, makes them particularly amenable to removal by hd. ethylene glycol (see chapter b) has a molecular weight of da, it has no significant protein binding, and it distributes primarily in total body water (vd = . - . l/kg), rendering it readily removable by hd. , additionally, glycolate, the toxic by-product of ethylene glycol responsible for acidosis, is effectively removed by hd. , the indications for hd in ethylene glycol poisoning have been reviewed. , , pizon and brooks have stated that an extremely high ethylene glycol level should be considered an indication for hd regardless of the patient's acid-base status or renal function due to hyperosmolarity. methanol (see chapter a) is likewise small ( da), with minimal protein binding and a vd of . l/kg. formic acid, the toxic by-product of methanol responsible for acidosis and retinal toxicity, is removed by hd. hovda and colleagues have suggested that hd may be performed on an "elective" basis in selected methanol poisonings if patients are rapidly treated with bicarbonate and fomepizole. , several researchers have recently reviewed the indications for hd after methanol poisoning. , , isopropanol (see chapter c) may also be removed by hd, although the indications for hd are limited, due to generally good outcomes with supportive care. , hd has been proven to be effective in removing both isopropanol and acetone from the plasma. lacouture and colleagues have recommended hd in cases where the blood isopropanol concentration exceeds mg/dl. salicylates (see chapter ) are compounds of low molecular weight. they are moderately to highly protein bound ( %- %), with very small volumes of distribution. although salicylates are highly protein bound at therapeutic concentrations, the fraction of unbound drug increases in the setting of overdose, rendering it amenable to removal by hd or hp. early hd is recommended due to the high propensity of salicylates to cause serious toxicity and death. the importance of concurrent alkalinization of the urine has been underscored by higgins and colleagues. because of acid-base and electrolyte abnormalities associated with salicylate poisoning, acute hd, rather than hp, is generally preferred. theophylline (see chapter ) has a low molecular weight and small vd with moderate protein binding. shannon addressed the use of hd and hp in theophylline poisoning in a -year prospective, observational study. the study included acute, chronic, and acute-ontherapeutic poisonings. the incidence of major toxicity was significantly greater in those undergoing hd. shannon concluded that while hp provides a higher theophylline clearance rate than hd, the latter appears to have comparable efficacy in reducing the morbidity of severe theophylline intoxication and is associated with a lower rate of procedural complications. lithium carbonate (see chapter ) remains a commonly used therapeutic agent in the treatment of bipolar disorder. while its vd is low, it concentrates in brain matter, rendering effective treatment more difficult. it has minimal protein binding and a very small molecular weight, and thus is amenable to treatment with hd. indications for dialysis remain controversial. recently, newer forms of extracorporeal purification for lithium poisoning have been proposed (see chapter c) which allow slower removal of lithium without rebound levels. annual report of the american association of poison control centers toxic exposure surveillance system management of intentional overdose in a&e departments diagnosis and management of the drug overdose patient pediatric rapid sequence intubation: a review rapid sequence intubation rapid-sequence intubation: a safe but ill-defined procedure rapid sequence induction medications: an update in patients with head injury undergoing rapid sequence intubation, does pretreatment with intravenous lignocaine/lidocaine lead to an improved neurological outcome? a review of the literature lidocaine and rapid sequence intubation acute asthma in emergency room does ketamine have a role in managing severe exacerbation of asthma in adults? emergency department use of ketamine in pediatric status asthmaticus in patients with head injuries who undergo rapid sequence intubation using succinylcholine, does pretreatment with a competitive neuromuscular blocking agent improve outcome? a literature review rocuronium versus succinylcholine for rapid sequence induction intubation pharmacologic management of the hypotensive patient management of calcium channel antagonist overdose drug-induced prolongation of the qt interval single-dose oral activated charcoal in the treatment of the self-poisoned patient: a prospective, randomized, controlled trial prospective evaluation of gastric emptying in the self-poisoned patient management of acutely poisoned patients without gastric emptying randomized study of the treatment of phenobarbital overdose with repeated doses of activated charcoal the revised position papers on gastric decontamination selecting the language of the publications included in a meta-analysis: is there a tower of babel bias? occupational health and safety administration: osha best practices for hospital-based first receivers of victims from mass casualty incidents involving the release of hazardous substances effects of washing acid injuries to the skin with water: an experimental study using rats experimental study on alkaline skin injuries-periodic changes in subcutaneous tissue ph and the effects exerted by washing emergency treatment of chemical and thermal eye burns alkali ocular burns in martinique (french west indies). evaluation of the use of an amphoteric solution as the rinsing product is there a delay in bathing the external eye in the treatment of ammonia eye burns? comparison of two ophthalmic solutions: physiological serum and diphoterine a prospective, randomized, blind comparison between saline, calcium gluconate and diphoterine for washing skin acid injuries in rats: effects on substance p and beta-endorphin release diphoterine for emergent eye/skin chemical splash decontamination: a review acid ingestion: toxicology and treatment the treatment of alkaline burns of the skin by neutralization poor prognosis of severe chemical and thermal eye burns: the need for adequate emergency care and primary prevention management of chemical eye injuries with prolonged irrigation army center for health promotion and preventive medicine (usachppm): personal protective equipment guide for military medical treatment facility personnel handling casualties from weapons of mass destruction and terrorism events weapons of mass destruction events with contaminated casualties: effective planning for health care facilities hexafluorine for emergent decontamination of hydrofluoric acid eye/skin splashes efficacy of hexafluorine for emergent decontamination of hydrofluoric acid eye and skin splashes hexafluorine vs. standard decontamination to reduce systemic toxicity after dermal exposure to hydrofluoric acid topical treatments for hydrofluoric acid burns: a blind controlled experimental study topical treatments for hydrofluoric acid burns: a blind controlled experimental study hydrofluoric acid dermal burns. an assessment of treatment efficacy using an experimental pig model topical treatments for hydrofluoric acid dermal burns. further assessment of efficacy using an experimental piq model comparison of topical magnesium and calcium treatment for dermal hydrofluoric acid burns the role of calcium gluconate in the treatment of hydrofluoric acid eye burn the efficacy of calcium gluconate in ocular hydrofluoric acid burns ocular hydrofluoric acid burns: animal model, mechanism of injury and therapy effects of isopropyl alcohol, ethanol, and polyethylene glycol/industrial methylated spirits in the treatment of acute phenol burns efficacy of topical phenol decontamination strategies on severity of acute phenol chemical burns and dermal absorption: in vitro and in vivo studies in pig skin chemical skin burns phosphorus burns: a practical approach to local treatment the phosphorous burn-a preliminary comparative experimental study of various forms of treatment phosphorous burns: evaluation of various modalities for primary treatment hand injuries due to highpressure injection devices for painting in shipyards: circumstances, management, and outcome in twelve patients high-pressure hand injection injuries caused by dry cleaning solvents: case reports, review of the literature, and treatment guidelines fatal hypernatremia from exogenous salt intake: report of a case and review of the literature fatal hypernatremia from saltwater used as an emetic fatal hypernatremia after using salt as an emetic-report of three autopsy cases death following cupric sulfate emesis lacomis d: case of the month rapidly reversible cardiomyopathy associated with chronic ipecac ingestion position statement: ipecac syrup. american academy of clinical toxicology; european association of poisons centres and clinical toxicologists conservative management of cocaine-packet ingestion: experience in milan, the main italian smuggling center of south american cocaine a massive, near-fatal cocaine intoxication in a body-stuffer. case report and review of the literature heroin body packers cocaine-condom ingestion. surgical treatment the cocaine 'body packer' syndrome. diagnosis and treatment surgical removal of iron tablets endoscopic removal of pharmacobezoar of slow release theophylline release of toxic metals from button batteries retained in the stomach: an in vitro study systemic absorption of lithium following ingestion of a lithium button battery button battery ingestion: an analysis of cases comparative efficacy of thallium adsorption by activated charcoal, prussian blue, and sodium polystyrene sulfonate how long after drug ingestion is activated charcoal still effective? position paper: singledose activated charcoal activated charcoal administration in a pediatric emergency department the frequency of complications associated with the use of multiple-dose activated charcoal chronic lung disease after activated charcoal aspiration incidence of aspiration pneumonia in intubated patients receiving activated charcoal american academy of clinical toxicology; european association of poisons centres and clinical toxicologists: position statement and practice guidelines on the use of multi-dose activated charcoal in the treatment of acute poisoning effects of fuller's earth and activated charcoal on oral absorption of paraquat in rabbits position paper: cathartics acute hypermagnesemia after laxative use hypermagnesemia induced by massive cathartic ingestion in an elderly woman without preexisting renal dysfunction severe hypermagnesemia due to multiple-dose cathartic therapy position paper: whole bowel irrigation a prospective study of acute, unintentional, pediatric superwarfarin ingestions managed without decontamination acute, unintentional pediatric brodifacoum ingestions position paper: ipecac syrup home syrup of ipecac use does not reduce emergency department use or improve outcome ipecac syrup-induced emesis . . . no evidence of benefit the demise of ipecac poison prevention: poison treatment in the home guideline on the use of ipecac syrup in the out-of-hospital management of ingested poisons changes in arterial oxygen tension after gastric lavage for drug overdose gastrointestinal tract perforation with charcoal peritoneum complicating orogastric intubation and lavage esophageal tear following forceful removal of an impacted oral-gastric lavage tube aspiration pneumonia following severe self-poisoning position paper: gastric lavage severe esophageal damage due to button battery ingestion: can it be prevented? bite the bullet: lead poisoning after ingestion of lead bullets successful treatment of theophylline toxicity by upper gastrointestinal endoscopy radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of cases urine sugar reagent tablet ingestion causing gastric and duodenal ulceration device choice and experience level in endoscopic foreign object retrieval: an in vivo study hazards of battery ingestion swallowed button batteries: is there a consensus on management? is surgical decontamination definitive treatment of "body-packers"? whole-bowel irrigation as adjunctive treatment for sustained-release theophylline overdose interaction between whole-bowel irrigation solution and activated charcoal: implications for the treatment of toxic ingestions acceleration of the body clearance of phenobarbital by oral activated charcoal gastrointestinal clearance of drugs with activated charcoal intravenous theophylline poisoning and multiple-dose charcoal in an animal model dose-dependency in the exsorption of theophylline and the intestinal dialysis of theophylline by oral activated charcoal in rats european association of poisons centres and clinical toxicologists: position statement and practice guidelines on the use of multi-dose activated charcoal in the treatment of acute poisoning multiple doses of activated charcoal: time for reappraisal? the frequency of complications associated with the use of multiple-dose activated charcoal use of multiple-dose activated charcoal in phenytoin toxicity multiple-dose activated charcoal for treatment of yellow oleander poisoning: a single-blind, randomized, placebo-controlled trial correlation of drug pharmacokinetics and effectiveness of multiple-dose activated charcoal therapy oral activated charcoal and dapsone elimination acceleration of digoxin clearance by activated charcoal digoxin toxicity in chronic renal failure: treatment by multiple dose activated charcoal intestinal dialysis characteristics of vomiting associated with acute sustained release theophylline poisoning: implications for management with oral activated charcoal fatal pulmonary aspiration of oral activated charcoal a fatality resulting from multiple dose activated charcoal therapy (abstract) aspiration of activated charcoal (letter) pulmonary aspiration of activated charcoal: a complication of its misuse in overdose management fatal pulmonary aspiration of oral activated charcoal (letter) activated charcoal surface area and its role in multiple-dose charcoal therapy gastrointestinal obstruction associated with multiple-dose activated charcoal charcoal bezoar. small bowel obstruction secondary to amitriptyline overdose therapy rectal ulcer with massive hemorrhage due to activated charcoal treatment in oral organophosphate poisoning intestinal pseudo-obstruction following the use of enteral charcoal and sorbitol and mechanical ventilation with papaveretum sedation for theophylline poisoning charcoal stercolith with intestinal perforation in a patient treated for amitriptyline ingestion treatment with activated charcoal complicated by gastrointestinal obstruction requiring surgery multiple doseactivated charcoal as a cause of acute appendicitis small-bowel obstruction secondary to activated charcoal and adhesions position statement: cathartics. american academy of clinical toxicology; european association of poisons centres and clinical toxicologists palatability of sucrose-, sorbitol-, and saccharinsweetened activated charcoal formulations dosage recommendations for activated charcoal-sorbitol treatment the genesis of the square meter-hour hypothesis methanol intoxication: comparison of peritoneal dialysis and hemodialysis treatment peritoneal dialysis in children: survey of its indications and applications allergic reactions to the polymeric glucose-based peritoneal dialysis fluid icodextrin in patients with renal failure pharmacokinetic evaluation of forced diuresis, dialysis, and hemoperfusion current concepts: peritoneal dialysis vitamin d intoxication in an anephric child acute digoxin overdose in a newborn with renal failure: use of digoxin immune fab and peritoneal dialysis carbamazepine overdosethe effects of multiple dose activated charcoal carbamazepine poisoning in adolescent suicide attempters. effectiveness of multiple-dose activated charcoal in enhancing carbamazepine elimination pediatric carbamazepine intoxication effect of oral charcoal and urine ph on dextropropoxyphene pharmacokinetics treatment of digitoxin overdose with oral activated charcoal the effects of activated charcoal on digoxin and digitoxin clearance enhancement of phenytoin elimination by multiple-dose activated charcoal treatment of phenytoin toxicity with repeated doses of activated charcoal multiple-dose activated charcoal in management of phenytoin overdose multiple-dose activated charcoal compared to urinary alkalinization for the enhancement of phenobarbital elimination pharmacokinetics of phenobarbital during certain enhanced elimination modalities to evaluate their clinical efficacy in management of drug overdose the effect of activated charcoal on the bioavailability of piroxicam in man ma: thallium and arsenic poisoning in a small midwestern town accelerated clearance of intravenously administered theophylline and phenobarbital by oral doses of activated charcoal in rats. a possibility of the intestinal dialysis effect of administration of activated charcoal and fibre on absorption, excretion and steady state blood levels of digoxin and digitoxin. evidence for intestinal excretion of the glycosides multiple doses of charcoal in digoxin poisoning activated charcoal increases digoxin elimination in patients misadventures with activated charcoal and recommendations for safe use treatment of phenobarbital poisoning with multiple dose activated charcoal in an infant multiple-dose activated charcoal in an accidental vancomycin overdose treatment of neonatal hyperbilirubinemia with repetitive oral activated charcoal as an adjunct to phototherapy prevalence of sorbitol in multiple-dose activated charcoal regimens in emergency departments continuous venovenous hemofiltration for the treatment of contrast-induced acute renal failure after percutaneous coronary interventions continuous arterial-venous diahemofiltration and continuous veno-venous diahemofiltration in infants and children functional characteristics of pediatric veno-venous hemofiltration rapid removal of vancomycin by continuous veno-venous hemofiltration is continuous veno-venous hemofiltration for acetaminophen-induced acute liver and renal failure worthwhile? hyperphosphatemia in tumor lysis syndrome: the role of hemodialysis and continuous veno-venous hemofiltration myoglobin clearance during continuous veno-venous hemofiltration with or without dialysis acute rhabdomyolysis after gemfibrozil therapy in a pregnant patient complicated with acute pancreatitis and hypertriglyceridemia while receiving continuous veno-venous hemofiltration therapy significant myoglobin removal during continuous veno-venous haemofiltration using f membrane (letter) lactic academia and bradyarrhythmia in a child sedated with propofol severe accidental hypothermia: rewarming with cvvhd propylene glycol toxicity associated with lorazepam infusion in a patient receiving continuous veno-venous hemofiltration with dialysis the acid-base effects of continuous hemofiltration with lactate or bicarbonate buffered replacement fluids profound hypercalcemia in continuous veno-venous hemofiltration dialysis with trisodium citrate anticoagulation and hepatic failure (letter) ueber die beseitgung giftiger stoffe aus blute durch dialyse peritoneal dialysis: a review fine rn: peritoneal dialysis update pharmacokinetics of drugs during various detoxification procedures for overdose and environmental exposure extracorporeal therapy in the treatment of intoxication interactions of drugs and peritoneal dialysis dialysis of poisons and drugs-annual review evaluation of the effects of multiple-dose activated charcoal on the absorption of orally administered salicylate in a simulated toxic ingestion model an evaluation of the effect of repeated doses of oral activated charcoal on salicylate elimination does multiple-dose charcoal therapy enhance salicylate excretion? multiple-dose charcoal and whole-bowel irrigation do not increase clearance of absorbed salicylate effect of multiple-dose activated charcoal on the clearance of high-dose intravenous aspirin in a porcine model effect of activated charcoal on absorption of nortriptyline effect of single and repeated doses of activated charcoal on the pharmacokinetics of doxepin lack of effect of oral activated charcoal on imipramine clearance pharmacokinetics of amitriptyline influenced by oral charcoal and urine ph effect of orally administered activated charcoal on vancomycin clearance failure of oral activated charcoal to accelerate the elimination of amiodarone and chloroquine the effect of activated charcoal on the absorption and elimination of astemizole effects of charcoal, sodium bicarbonate, and ammonium chloride on chlorpropamide kinetics tenenbein m: diltiazem overdose: pharmacokinetics of diltiazem and its metabolites and effect of multiple dose charcoal therapy effect of activated charcoal on the pharmacokinetics of pholcodine, with special reference to delayed charcoal ingestion the effect of repeateddose activated charcoal on the pharmacokinetics of sodium valproate in healthy volunteers effect of oral activated charcoal on tobramycin clearance elimination of amrinone during continuous veno-venous haemofiltration after cardiac surgery acute barium intoxication and hemodiafiltration pharmacokinetics of ceftriaxone in patients undergoing continuous veno-venous hemofiltration ethylene glycol antifreeze poisoning pharmacokinetics of imipenem-cilastatin in critically ill patients undergoing continuous venovenous hemofiltration continuous venovenous haemofiltration versus continuous veno-venous haemodialysis in severe lithium self-poisoning:a toxicokinetics study in an intensive care unit clearance of metformin by hemofiltration in overdose three case reports of the use of haemodiafiltration in the treatment of salicylate overdose vancomycin and tobramycin clearance in an infant during continuous hemofiltration amikacin pharmacokinetics during continuous veno-venous hemofiltration (letter) acute arsenic poisoning treated by intravenous dimercaptosuccinic acid (dmsa) and combined extrarenal epuration techniques hemodialysis versus continuous veno-venous hemodiafiltration in the management of severe valproate overdose peritoneal dialysis in acute poisoning: successful treatment of a -month-old child ingesting times the adult dose of achrocidin the early removal of amatoxins in the treatment of amanita phalloides poisoning (author's translation) management of amikacin overdose acute potassium dichromate poisoning: treated by peritoneal dialysis peritoneal dialysis for removal of copper role of albumin-enriched peritoneal dialysate in acute copper poisoning treatment of ethylene glycol poisoning with peritoneal dialysis intensified dialysis treatment of ethylene glycol intoxication treatment of glutethimide poisoning: a comparison of forced diuresis and dialysis peritoneal and hemodialysis for acute glutethimide treatment of glutethimide intoxication: an in vivo comparison of lipid, aqueous, and peritoneal dialysis with albumin peritoneal dialysis for isopropanol poisoning peritoneal dialysis for lithium poisoning lithium intoxication treated by peritoneal dialysis lithium intoxication with acute renal failure and death meprobamate poisoning treated by peritoneal dialysis meprobamate intoxication treated with peritoneal dialysis meprobamate poisoning: successful treatment with peritoneal dialysis amphetamine poisoning and peritoneal dialysis: a case report the use of peritoneal dialysis in acute methyl alcohol poisoning methanol poisoning in an infant: successful treatment with peritoneal dialysis acute mercury poisoning by intentional ingestion of mercuric chloride peritoneal dialysis for severe methyprylon intoxication peritoneal dialysis for severe methyprylon intoxication (letter) propoxyphene hydrochloride poisoning: report of the first fatality propoxyphene hydrochloride poisoning: report of a case treated with peritoneal dialysis acute propoxyphene hydrochloride intoxication peritoneal dialysis in quinine sulfate intoxication peritoneal dialysis in quinine intoxication quinine extraction during peritoneal dialysis: the role of nonionic diffusion hemodialysis, peritoneal dialysis, plasmapheresis and forced diuresis for the treatment of quinine overdose amitriptyline hydrochloride poisoning: unsuccessful treatment by peritoneal dialysis fatal chloroquine poisoning in a child: experience with peritoneal dialysis chlorpropamide intoxication-treatment with peritoneal dialysis accidental poisoning with isoniazid and rifampicin in an infant: role of peritoneal dialysis peritoneal dialysis and haemodialysis in methaqualone (mandrax) poisoning peritoneal dialysis in methaqualone overdose peritoneal dialysis and lysol poisoning thallium ingestion with survival: ineffectiveness of peritoneal dialysis and potassium chloride diuresis valacyclovir neurotoxicity in a patient with end-stage renal disease treated with continuous ambulatory peritoneal dialysis comparison of peritoneal and hemodialysis: treatment of methanol intoxication peritoneal dialysis for severe salicylism: an evaluation of indications and results rapid correction of acute salt poisoning by peritoneal dialysis toxicologic and pharmacokinetic evaluation of a case of vancomycin intoxication during continuous ambulatory peritoneal dialysis use of peritoneal dialysis in experimental amphetamine poisoning baclofen neurotxicity in uremic patients: is continuous ambulatory peritoneal dialysis less effective than intermittent hemodialysis? peritoneal dialysis in the treatment of boric acid poisoning acute boric acid poisoning: report of an infant successfully treated by peritoneal dialysis asymptomatic boric intoxication: value of peritoneal dialysis ingestion of boric acid by infants bromate poisoning treatment of bromate poisoning (letter) near-fatal caffeine intoxication treated with peritoneal dialysis dialysis treatment of acute chromium intoxication and comparative efficacy of peritoneal versus hemodialysis in chromium removal chromic acid burns: early aggressive excision is the best method to prevent systemic toxicity brief clinical and laboratory observations: management of neonatal gentamicin overdosage removal of mercury by peritoneal dialysis chapter emergency management of poisoning winchester jf: dialysis and hemoperfusion in poisoning annual report of the american association of poison control centers' national poisoning and exposure database toltec international inc: how hemodialysis (dialysis) works combined hemoperfusion and hemodialysis treatment of poisoning with cholinesterase inhibitors acute poisoning with bromofosmethyl (bromophos) use of hemoperfusion and cholinesterase in acute poisoning with organophosphate cholinesterase inhibitors-clinical analysis of patients the availability and use of charcoal hemoperfusion in the treatment of poisoned patients elimination of paraquat multiple-dose activated charcoal: a review of relevant clinical studies the role of the molecular adsorbents recirculating system (mars) in the management of liver failure preconditioning by extracorporeal liver support (mars) of patients with cirrhosis and severe liver failure evaluated for living donor liver transplantation-a pilot study application of mars artificial liver support as bridging therapy before split liver retransplantation in a -month-old child application of molecular adsorbent recirculating system in patients with severe liver failure after hepatic resection or transplantation: initial single-centre experiences experiences with mars liver support therapy in liver failure: analysis of patients of the international mars registry mars preconditioning for living donor liver transplantation: panacea or placebo? how tightly can a drug be bound to a protein and still be removable by charcoal hemoperfusion in overdose cases? extracorporeal therapies for acute intoxications quantitative pharmacogenetics of nortriptyline: a novel approach fomepizole for the treatment of ethylene glycol poisoning fomepizole for the treatment of methanol poisoning fomepizole in treatment of uncomplicated ethylene glycol poisoning current management of ethylene glycol poisoning references . bismuth c: biological valuation of extra-corporeal techniques in acute poisoning fomepizole may change indication for hemodialysis in methanol poisoning: prospective study in seven cases current recommendations for treatment of severe toxic alcohol poisonings severe lactic acidosis treated with prolonged hemodialysis: recovery after massive overdoses of metformin combination of intermittent haemodialysis and highvolume continuous haemofiltration for the treatment of severe metformin-induced lactic acidosis acute kidney failure: a pediatric experience over years treatment of severe pediatric ethylene glycol intoxication without hemodialysis childhood methanol ingestion treated with fomepizole and hemodialysis chronic renal disease patients with severe star fruit poisoning: hemoperfusion may be an effective alternative therapy extracorporeal techniques in the treatment of poisoned patients acute hemolysis with acute renal failure in a patient with valproic acid poisoning treated with charcoal hemoperfusion hemodialysis complications intradialytic complications during hemodialysis high-flux hemodialysis-an effective alternative to hemoperfusion in the treatment of carbamazepine intoxication high-efficiency dialysis for carbamazepine overdose high-flux haemodialysis treatment as treatment for carbamazepine intoxication management of carbamazepine overdose efficacy of charcoal hemoperfusion in massive carbamazepine poisoning repeat charcoal hemoperfusion treatments in life threatening carbamazepine overdose osmol gap as a surrogate marker for serum propylene glycol concentrations in patients receiving lorazepam for sedation use of the osmolal gap to guide the start and duration of dialysis in methanol poisoning removal of propylene glycol and correction of increased osmolar gap by hemodialysis in a patient on high dose lorazepam infusion therapy unusual d-lactic acid acidosis from propylene glycol metabolism in overdose treatment of the alcohol intoxications: ethylene glycol, methanol and isopropanol life-threatening isopropyl alcohol intoxication: is hemodialysis really necessary? extracorporeal management of valproic acid toxicity: a case report and review of the literature acute valproate poisoning: pharmacokinetics, alteration in fatty acid metabolism, and changes during therapy successful use of hemodialysis in acute valproic acid intoxication successful treatment of valproic acid overdose with hemodialysis high-flux hemodialysis without hemoperfusion is effective in acute valproic acid overdose efficiency of highflux hemodialysis in the treatment of valproic acid intoxication extracorporeal management of valproic acid overdose: a large regional experience valproic acid toxicity: overview and management glycolate kinetics and hemodialysis clearance in ethylene glycol poisoning. meta study group glycolate causes the acidosis in ethylene glycol poisoning and is effectively removed by hemodialysis formate kinetics in methanol poisoning an evidence based flowchart to guide the management of acute salicylate (aspirin) overdose alkalinization and hemodialysis in severe salicylate poisoning: comparison of elimination techniques in the same patient concepts and controversies in salicylate toxicity severe theophylline poisoning: charcoal haemoperfusion or haemodialysis comparative efficacy of hemodialysis and hemoperfusion in severe theophylline intoxication phosphorus-enriched hemodialysis for the treatment of patients with severe methanol intoxication validation of a method to predict required dialysis time for cases of methanol and ethylene glycol poisoning acute phenobarbital intoxication effectiveness of hemodialysis in the extracorporeal therapy of phenobarbital overdose conventional haemodialysis significantly lowers toxic levels of phenobarbital hemodialysis following butoxyethanol ingestion repeated ingestion of -butoxyethanol: case report and literature review the treatment of severe drug intoxication with charcoal hemoperfusion in series with hemodialysis critical, acutely poisoned patients treated with continuous arteriovenous hemoperfusion in the emergency department when should dialysis be performed in lithium poisoning? a kinetic study in cases of lithium poisoning hemodialysis followed by continuous hemofiltration for treatment of lithium intoxication in children hyperosmolality: another indication for hemodialysis following acute ethylene glycol poisoning ethylene glycol toxicity: the role of serum glycolic acid in hemodialysis hemodialysis as a treatment of severe ethanol poisoning intravenous -methylpyrazole as an antidote for diethylene glycol and triethylene glycol poisoning: a case report successful treatment of a child with fulminant liver failure and coma caused by amanita phalloides intoxication with albumin dialysis without liver transplantation molecular adsorbent recirculating system in dealing with maternal amanita poisoning during the second pregnancy trimester: a case report successful use of molecular absorbent regenerating system (mars) dialysis for the treatment of fulminant hepatic failure in children accidentally poisoned by toxic mushroom ingestion mars: optimistic therapy method in fulminant hepatic failure secondary to cytotoxic mushroom poisoning-a case report phenytoin intoxication in critically ill patients experience with albumin dialysis in five patients with severe overdoses of paracetamol mars procedure as a bridge to combined liver-kidney transplantation in severe chromium-copper acute intoxication: a paediatric case report childhood diethylene glycol poisoning treated with alcohol dehydrogenase inhibitor fomepizole and hemodialysis the role of continuous renal replacement therapy in the treatment of poisoning methanol and formate kinetics during treatment with fomepizole rethinking the toxic methanol level isopropyl alcohol intoxication: a diagnostic challenge acute isopropyl alcohol intoxication. diagnosis and management methods used to decrease lithium absorption or enhance elimination treatment of lithium intoxication with continuous venovenous hemodiafiltration successful treatment by direct hemoperfusion of coma possibly resulting from mitochondrial dysfunction in acute valproate intoxication treatment of severe theophylline poisoning with the molecular adsorbent recirculating system (mars) molecular adsorbent recirculating system-mars as a bridge to liver transplantation in amanita phalloides intoxication successful treatment of an adult with amanita phalloides-induced fulminant liver failure with molecular adsorbent recirculating system (mars) carbamazepine has a relatively low molecular weight ( da), but a fairly large vd ( - l/kg) and is about % protein bound at therapeutic levels. its active metabolite , -epoxide (cbze), is approximately % protein bound. , because of the high protein binding, activated charcoal and chp have generally been favored in treatment; however, high-efficiency dialysis may also be effective. valproic acid (see chapter ) is a low molecular weight ( da) anticonvulsant with a small vd and saturable protein binding. at therapeutic concentrations, % to % of valproic acid is protein bound, but in overdose the degree of protein binding is relatively less (i.e., there is more unbound drug available for extracorporeal purification). saturation of protein binding sites occurs at levels greater than μg/ml, at which % to % of the drug is protein bound. at a level of μg/ml, % of valproic acid is protein bound, , , rendering it amenable to extracorporeal drug removal techniques. , both hp and hd, alone and in combination, have been used in cases of valproate toxicity. valproic acid elimination has been shown to be enhanced about tenfold by the use of extracorporeal methods. while the half-life of the drug is effectively diminished, the precise role of these procedures in valproate toxicity remains to be established. a nonexhaustive list of substances for which mars has been used in poisoning includes theophylline, cytotoxic mushrooms, - phenytoin, acetaminophen (paracetamol), and a copper-chromium-containing solution. mars appears to improve liver failure from multiple causes, but further prospective studies are needed to determine the role this technique should play in patient care. in conclusion, hd and hp remain important, but secondary methods of treatment in poisoning by specific substances. these modalities cannot be substituted for excellent supportive care, and in some cases the need for them has been supplanted by effective antidotes. both hd and hp are associated with complications and are not universally available. careful evaluation of individual cases should guide the decision to use extracorporeal circulation. mars or albumin dialysis may be of clinical benefit in selected poisonings. key: cord- -ekgdsv f authors: mehta, meenu; deeksha,; tewari, devesh; gupta, gaurav; awasthi, rajendra; singh, harjeet; pandey, parijat; chellappan, dinesh kumar; wadhwa, ridhima; collet, trudi; hansbro, philip m.; kumar, s rajesh; thangavelu, lakshmi; negi, poonam; dua, kamal; satija, saurabh title: oligonucleotide therapy: an emerging focus area for drug delivery in chronic inflammatory respiratory diseases date: - - journal: chemico-biological interactions doi: . /j.cbi. . . sha: doc_id: cord_uid: ekgdsv f abstract oligonucleotide-based therapies are advanced novel interventions used in the management of various respiratory diseases such as asthma and chronic obstructive pulmonary disease (copd). these agents primarily act by gene silencing or rna interference. better methodologies and techniques are the need of the hour that can deliver these agents to tissues and cells in a target specific manner by which their maximum potential can be reached in the management of chronic inflammatory diseases. nanoparticles play an important role in the target-specific delivery of drugs. in addition, oligonucleotides also are extensively used for gene transfer in the form of polymeric, liposomal and inorganic carrier materials. therefore, the current review focuses on various novel dosage forms like nanoparticles, liposomes that can be used efficiently for the delivery of various oligonucleotides such as sirna and mirna. we also discuss the future perspectives and targets for oligonucleotides in the management of respiratory diseases. respiratory diseases like lung cancer, inflammatory diseases with chronic obstructive pulmonary disease (copd), asthma, respiratory infections and pulmonary fibrosis are some of the major causes of death globally [ ] [ ] [ ] . currently available treatment options for these diseases have limited efficacy [ ] . the approval of inhaled corticosteroids in the early s opened a major breakthrough and the path for the treatment of airway diseases. even after years, these are still the mainstay of respiratory disease therapy. however, there are several limitations when it comes to the management of chronic disease conditions [ ] . along these lines of research, double-stranded rnas (dsrnas) were discovered, which regulates gene function by rna interference (rnai). silencing mechanism. the mechanism represents a new and powerful therapeutic approach for the treatment and prevention of respiratory diseases by altering gene expression. different rna molecules can mediate rnai, such as short interfering rna (sirna), long dsrna, microrna (mirna) and short hairpin rna (shrna) [ ] . among these molecules and approaches, oligonucleotide therapies are emerging as a newer and effective class. oligonucleotides are defined as polynucleic acid chains that may be modified or unmodified and consists of various functional groups based on their use and source. the nucleotides contain five base pairs, two of which are purine derivatives (adenine and guanine) and the rest are pyrimidine derivatives (cytosine, thymine, and uracil). this class of substances function based on several approaches, namely, the rnai (mirna and sirna), the antisense, the aptamer, the immunomodulatory and the decoy approaches. most of these approaches are in early phases of development. however, there are several limitations with the current state of small molecule therapies. thus, the use of oligonucleotides has emerged as an advancement in the treatment of respiratory diseases, as these cover a wide range of targets [ , ] . some of the applications of oligonucleotides are listed below: • pcr (polymerase chain reaction) primer • rna, sirna and antisense studies • melting point optimization of oligonucleotides • molecular diagnostics • gene therapy • microarrays • fluorescence in situ hybridization (fish) • fluorescence resonance energy transfer (fret) [ ] . these are short, single stranded oligodeoxynucleotides that have the capability to modify rna and can alter the protein expression [ ] . based upon their mechanism of action, these are further categorized into two classes: a) rnase h-dependent oligonucleotides: these stimulate mrna degradation. most of the antisense drugs act through rnase h-dependent mechanism. the enzyme rnase h causes hydrolysis of the rna strand of rna/dna duplex as shown in fig. . their efficiency is - % in down-regulating protein and mrna expression and target any site of mrna. b) steric-blocker oligonucleotides: these hamper the process of splicing or translation. these oligonucleotides are effective only when they target a specific codon i.e. ′ or aug initiation codon [ ] . as these are capable of targeting the cause of development of the disease, asos have the potential of being used as a successful therapy as compared to the conventional therapies [ ] . these are non-coding rnas that have a distinctive role in gene regulation and are also very specific, as they act only on one mrna target. sirnas are responsible for gene silencing at the post transcriptional level by causing rna interference (rnai). rnai is a natural process that causes gene silencing through mrna degradation. a conventional sirna contains - nucleotides along with two nucleotides overhanging at the ' end, mostly tt and uu. their potency can be enhanced by elongating the length of double stranded rna (dsrna). although, these have the potential of being therapeutically utilized, there are yet certain challenges for their utilization into clinical practice such as, reduced stability and poor delivery [ ] . mirna is a single strand rna that consists of - nucleotides and are produced from primary mirna via the action of two rnase-iii type proteins-drosha (in nucleus) and dicer (in cytoplasm) [ , ] . these hinder the translation process through accumulation of mrna in processing bodies (p-bodies). these play a vital role in various processes such as cell division, cell death, breakdown of fats, neuronal pattering, hematopoietic differentiation and immunity [ ] . mirna acts on multiple targets and causes mrna degradation. they also face limitations in terms of instability and poor delivery. in addition, they also have the potential of being used in various complex disorders such as different types of cancers and neurodegenerative disorders [ ] . a comparison between mirna and sirna is shown in table . these are single stranded dna or rna molecules that can bind strongly to definite targets. these are less immunogenic, physically stable, and can be subjected to large scale production at relatively reasonable cost. these molecules are mostly used as therapeutic or diagnostic agents or as biosens [ ] . both rna and dna aptamers differ in their sequence and folding pattern, even though these act on similar targets [ ] . these are short, single stranded oligodeoxynucleotides that includes unmethylated cpg dinucleotides at a particular region. these have been divided into four classes depending upon their structural variations and the type of immune reaction they stimulate: a) k-type/b-type: these have - cpg dinucleotides on a phosphorothioate backbone that increase their resistance to nuclease digestion leading to an enhancement of half-life. they stimulate b-cells to produce igm. b) d-type/a-type: these molecules consist of a phosphodiester centre surrounded by phosphorothioate terminal nucleotides. they cause maturation of plasmacytoid dendritic cells (pdc) and stimulates the production of interferon α (ifnα). c) c-type: these substances enhance the production of il- by b-cells and ifnα by pdc. d) p-type: these are substances that consist double palindromes that make hairpin like structures at the gc rich ′-ends and stimulate the secretion of type i-ifn [ ] . a brief classification of oligonucleotides is shown in table . oligonucleotides have been utilized from the last two decades for their therapeutic properties. majorly these are used either for inhibition of genes or protein expression. following are few areas in which these can be used: • neurodegenerative disorders: oligonucleotides can be used as an effective therapy for the treatment of huntington's disease (hd) because it is an autosomal disease caused by mutation on single allele. oligonucleotides target the altered messenger rna (mrna) and decrease the synthesis of the causative protein-huntingtin [ ] . aso can also be used as a therapy for the treatment of spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als) and spinocerebellar ataxias [ ] . • respiratory disorders: oligonucleotides can be administered as an inhalation for the treatment of asthma and copd. they have fewer side effects as these molecules are directly targeted to the lungs. in addition, their uptake is usually enhanced at the target site which leads to their prolonged duration of action [ ] . • cancer: antisense oligonucleotides have emerged as a new therapeutical approach for the treatment of various types of cancers whereby, they attach with mrna and inhibit gene translation [ ] . however, non-specific protein binding and efficient delivery appear to be the major hurdles for their use in cancer treatment [ ] . • diabetic retinopathy: antisense oligonucleotides (e.g., ico- ) are currently under trials for the treatment of diabetic retinopathy. these act by down regulating the signaling pathway of multiple growth factors that are involved in the ocular angiogenesis and vascular leakage. they provide several advantages namely, increased half-life, lesser degradation and improved safety profile [ ] . it is interesting to note that the only oligonucleotide currently approved is vitravene ® (novartis, new york, ny, usa), which is used for cytomegalovirus retinitis, where the drug is directly administered to the site of disease (intravitreal). dna antisense oligonucleotide (aso) molecules, act by modulating the expression of a target gene by binding to its mrna, thereby prevent translation. the concept was first proposed by zamecnik and stephenson in [ ] . this was the first evidence reported in the literature, suggesting that oligonucleotides might be used for therapeutic intervention and since then aon technology has been developed for a wide array of therapeutic purposes. the growing interest of such approaches has risen from the fact that several copies of a protein can be produced by each mrna molecule. d'anjou and team had patented a formulation of antisense oligonucleotides targeted against genes coding for phosphodiesterase. these can be used either as analytical agents or for therapy in cases of asthma, copd, bronchitis, pulmonary fibrosis and leads to rise in cyclic amp and reduction in pde level [ ] . another patent involves an oligonucleotide that has nucleotide sequence corresponding to the respiratory syncytial virus nucleotide sequence number - or - of the respiratory syncytial virus antigenome. the invention also provides a composition including oligonucleotide and a physiologically acceptable carrier [ ] . it is, therefore, potentially a more efficient approach to target the mrna rather than the protein itself [ ] . furthermore, cpg oligonucleotides attach to toll-like receptor (tlr- ) which causes an immune response by stimulation of intracellular signaling and causes the stimulation of pro-inflammatory mediators such as nf-κb. aptamers bound tightly to the protein, targets to hinder their response. sirna acts on argonaute and rna-induced silencing complex. this complex has exonuclease and endonuclease activities that cause degradation of cellular mrna, thus inhibiting translation. it also acts on syk kinase, a signaling molecule that is responsible for inflammation [ ] . kinman and yamada had patented a method of administering oligodeoxynucleotides for the treatment of inflammatory lung diseases in . these were able to suppress immune response to cpg oligodeoxynucleotides for the management of inflammatory lung disorders [ ] . another invention involves a method for activation of immune response by using combination of immunostimulatory cpg oligonucleotides with immunopotentiating cytokines. these were administered as such or with nucleic acid delivery complex. these causes antigen specific immune response in both humans and animals [ ] . in order to attain the full potential of oligonucleotides as treatment option, better methodologies must be developed to deliver the agents to tissues and cells in a target specific manner. currently, many researchers are working on this issue by chemically modifying the oligonucleotides using various nanocarriers. commonly used drug delivery systems for respiratory diseases are polymer-based, lipid-based and peptide-based, and among these three, the lipid-based carriers are the most commonly used vectors for delivering rnai. they include solid lipid nanoparticles, cationic liposomes, lipidoids, solid nanostructured lipid carriers and ph-responsive lipids [ ] . liposomes are colloidal drug delivery systems consisting of a lipid layer encircling an aqueous centre. the drug is distributed as per its solubility in the lipid layer or hydrophilic core and these carriers enhance pharmacokinetics of the drugs [ ] . additionally, channel proteins might be developed into liposomes as well, that allow passageway of tiny-sized particles, for instance; ions, antibiotics, and nutrients. this leads to reduced degradation through proteolytic enzymes. owing to the concentration gradient difference, the drugs might diffuse via these channels as well. mccaskill and co-workers studied the systemic delivery of cationic liposomes formulated by hydration of freeze-dried matrix (hfdm) with sirna, to the lung epithelium. their study showed that sirna was delivered to ± % of murine lung cells when administered intravenously. it was observed that sirna was able to cause targeted gene and protein knockdown in most parts of the lungs. thus, these have the potential of being used for the treatment of lung epithelium diseases [ ] . another study reported the formulation of cationic liposomes from dialkyl cationic lipids such as, , -dioleoyl- -trimethylammoniumpropane (dotap) and studied their ability of delivering sirna to the lungs upon iv administration. the cationic liposomes made from n- propyl} carbamate hydroiodide (hapc-chol) showed increased buildup of sirna in lungs and decreased the expression of tie mrna [ ] . ozpolat and co-workers studied the utilization of neutral , -dioleoyl-sn-glycero- -phophatidylcholine based nanoliposomes in cancer therapy. they found it to be relatively safer and and times more efficacious than cationic liposomes and naked sirna in distributing sirna to cancerous tissues [ ] . another study reported the formulation of sterically stabilized cationic liposomes containing cpg-oligonucleotides, which studied their potential as anti-allergen and immunoprotectant. these liposomes enhance the duration of immune defense by cpg-oligonucleotides and offers protection by increasing their uptake by b-cells, dendritic cells and macrophages [ ] . li and co-workers formulated liposomes with anti-egfr aptamerconjugated-chitosan to deliver erlotinib and oxygen to reverse drug resistance caused by hypoxia in case of lung cancer. these liposomes are shown to have improved stability and additionally provided controlled discharge of drugs [ ] . garbuzenko and team formulated neutral and cationic liposomes to carry doxorubicin (dox), aso and sirna and compared the intratracheal delivery of both these liposomes with that of systemic administration. elevated peak concentration and extended retention time of both these liposomes were observed in case of intratracheal delivery. this study revealed the potential of both these liposomes in the treatment of lung cancer [ ] . mizuta et al., studied the potential of antisense phosphorothioate oligonucleotides in the treatment of influenza a virus. these oligonucleotides were found to be complementary to the translation codons of pb or pa genes (pb -as or pa-as) of influenza a virus rna polymerase. therefore, they formulated liposomes incorporated with pb as, which were able to reduce viral growth in lungs [ ] . in another study, otsuka and team prepared vitamin a-coupled liposomes incorporated with sirna for the treatment of pulmonary fibrosis by targeting myofibroblasts. these liposomes inhibit collagen-specific chaperone heat specific protein (hsp ) [ ] . niosomes are defined as vesicles made from non-ionic surfactants that are used for targeted delivery of drugs, whereby they also prevent the loss of drug, as these causes localized delivery of drugs. these molecules make a bi-layered structure in which the lipophilic regions project away from the aqueous solvent, while the hydrophilic regions remain in touch with the aqueous solvent [ ] . these can be used as carriers for effective gene transport because of better stability and small size. a study reported the formulation of cationic lipid , -di (teradecyloxy)propan- -amine, aqualene and polysorbate niosomes by solvent emulsification-evaporation method. these niosomes were able to prevent dna degradation and helped in its entry into cells. another study used cationic niosomes, which were composed of span , dota and pegylated lipid for the intracellular transport of sirna/mirna. niosomes are also incorporated with rna that lead to efficient gene silencing in human mesenchymal stem cells [ ] . particles with - nm size range are termed as nanoparticles which have been newly employed in targeted drug delivery [ ] . the small size range allows the nanoparticles to behave as drug carriers, which in turn allows them to reach any part of the human body [ ] . these can be broadly categorized as polymeric, inorganic or lipid based nanoparticles [ ] . polymeric nanoparticles have potential applications in the diagnosis of diseases and drug delivery due to their controlled drug release, theranostics, target specificity and better therapeutic index [ ] . this method of drug delivery depends on the biocompatibility and biodegradability of the polymer. kumar et al., demonstrated chitosan ifnγ gene nanoparticles as therapeutic substances against allergic asthma as well as prophylaxis. on intranasal administration, the nanoparticles were taken up by macrophages and bronchoepithelium cells, reducing airway hyper-responsiveness and th cytokine levels via stat signaling pathway [ ] . for over years, non-methylated cpg-oligonucleotides (cpg-odn) have been used as potential substances in the management of allergic asthma. however, these molecules cannot be delivered in high doses due to their undesirable side-effects such as, septic shock due to elevated cytokine level in the body [ , ] . to overcome the side effects, nm poly (lactic-co-glycolic) acid encapsulated with cpg-odn was found to improve th response and reduced eosinophilic count in pulmonary system of derp allergen immunocompromised mice [ ] . furthermore, polyethylenimine based sirna delivery for targeting t cells has been reported by xie et al. this has been demonstrated in a murine model for allergic asthma [ ] . chitosan nanoparticle based imiquimod cream with natriuretic peptide receptor sirna (sinpr) was applied to the ova-sensitized mice which showed decrease in ahr, eosinophil count and levels of pro-inflammatory cytokines including il- and il- [ ] . cystic fibrosis (cf) remains one of the lung disease targets for gene therapy. since , clinical trials with viral and non-viral vectors have been carried out [ ] . osman et al., demonstrated a novel pe-gylated cell penetrating peptide (cpp) nanoparticles with glycosaminoglycan (gag) that were efficiently delivered in vivo with superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid, compared to non-pegylated complexes. therefore, peg-gat technology is a successful approach against mucoobstructive lung diseases [ ] . in vivo lewis lung carcinoma was studied for aerosol delivery of polyethyleneimine (pea) conjugated akt sirna. the study reported that the lung tumor progression was inhibited weeks post the aerosol delivery [ ] . sung et al., demonstrated non-covalently bound pegylated connective transforming growth factor (ctgf) complex for lung fibrosis. on intratracheal administration of the copolymer and sirna targeting ctgf, a significant decrease in ctgf expression, inflammatory cytokines and collagen deposition were observed in a mice model for lung fibrosis [ ] . nafee et al., developed antisense oligonucleotide, -o-methyl-rna (omr), a telomerase inhibitor loaded onto chitosan nanoparticles against lung cancer. it was observed that omr reduces % of the telomerase activity in a lung cancer cell lines [ ] . nascimento et al., reported on a type of epidermal growth factor receptor (egfr)-chitosan nanoparticles for the co-delivery of mad sirna and cisplatin. this co-delivery had higher therapeutic effect crossing chemico-drug resistance barrier in lung cancer leading to apoptosis and mitotic failure [ ] . these are phospholipid based matrix useful to entrap hydrophobic and hydrophilic drugs with improved pharmacological action of drug and a better pharmacokinetics profile [ ] . bae et al., demonstrated quantum dots (qds) incorporated solid lipid nanoparticles (sln) for synergistic therapeutic activity of sirna-paclitaxel against human lung carcinoma. the synergistic activity promotes caspase mediated apoptosis and downregulates the expression of bcl- . the fluorescence of qds helps to observe the in situ nanoparticle translocation [ ] . taratula et al., reported multifunctional lipid nanoparticles delivered via inhalation, consisting of an anticancer drug (paclitaxel or doxobrubicin), multidrug resistant associated peptide (mrp) mrna targeting sirna, along with sirna for bcl mrna, which supresses nonpump cellular resistance. the effective delivery of the drug and sirna induced cell death of lung tumor cells by targeted gene silencing [ ] . inorganic nanoparticles are synthesized from gold, silver or platinum and are cost effective over polymeric nanoparticles due to less viability against microbial degradation. tarantula et al., reported mesoporous silica nanoparticles (msn) that can deliver drug molecules to incise cancer cells. it is a conjugated delivery system containing doxorubicin and cisplatin as anticancer drugs along with sirna to target mrp and bcl mrna. this delivery system has been reported to enhance the cytotoxic effects of the anti-cancer drugs [ ] . conde and coworkers studied gold nanoparticles modified with sirna targeting c-myc to mouse lungs using rgd peptide (arg-gly-asp) via intratracheal instillation. the peptide adheres to the cells and proliferates by binding to integrin avb which is an angiogenesis marker. this delivery system suppressed c-myc which further inhibited tumor proliferation [ ] . a list of sirna-based therapeutics that are under clinical trials is shown in table . from the first report on antisense oligonucleotides to the recently reported rna interference (rnai), numerous scientific communications have been published reporting promising results of oligonucleotide therapeutics in respiratory disorders [ ] . oligonucleotides, short dna or rna molecules, are emerging therapeutic modalities for various common respiratory diseases [ ] . major obstacles in successful delivery of rnai are sirna translocation across the plasma membrane and its subsequent release from the endosomal compartment. these biomacromolecules are susceptible to degradation by ubiquitous nuclease [ ] and hold a negative surface charge. to overcome these obstacles, extensive efforts are required to focus on the development of effective formulation that maintains the local drug concentration for longer duration and prevents fast clearance of sirna [ ] . in comparison to the other delivery systems, the use of bioadhesive polymeric materials gained less attention in the development of gene delivery formulations. few natural and synthetic polycations, especially mucoadhesive chitosan, have been explored for pulmonary sirna delivery [ ] . chitosan is a biodegradable and non-toxic polymeric material [ ] investigated to transfer plasmid dna to the pulmonary epithelial cells to express in desired proteins [ ] [ ] [ ] . mucoadhesive chitosan-based nanoparticles have been widely investigated for pulmonary delivery of sirna and gene silencing [ ] [ ] [ ] . it has cholesterol-lowering property and also used in wound healing. it is established as an ideal polymeric material for the delivery of dna to mucosal tissues due to its good mucoadhesive characteristics and the ability to increase paracellular transport by modulating tight junctions [ ] . intranasal administration of sins -sirna expressing plasmids (shrna) nanochitosan formulations resulted in decreased viral titers, airway reactivity, and inflammation in respiratory syncytial virus (rsv) infected animal models [ , ] . bivas-benita et al., delivered a dna plasmid encoding eight hla-a* -restricted t-cell epitopes from mycobacterium tuberculosis formulated in chitosan nanoparticles to hla-a transgenic mouse model via the pulmonary route. dna containing chitosan nanoformulations induced maturation of dendritic cells. pulmonary administration of dna plasmid containing chitosan nanoparticles has increased ifn-γ secretion [ ] . glud et al., investigated pulmonary gene silencing effect of small interfering locked nucleic acid (silnas), targeting enhanced-greenfluorescent-protein (egfp) in lung bronchoepithelium upon intravenous delivery of naked silnas and intranasal delivery of naked silna or chitosan based silna mucoadhesive nanoparticles. a significant reduction in egfp protein expression was observed after intravenous administration of naked silna in egfp-transgenic mice. intranasal administration of silna-chitosan nanoparticles also yielded similar effects. however, intranasal administration of naked silna did not cause a knockdown [ ] . to establish a relationship between structure and properties of chitosan-pdna polyplexes, koping-hoggard et al., compared polyplexes of ultrapure chitosan of preferred molecular structure with those of optimized polyethylenimine polyplexes. dnachitosan-complex delivery to the pulmonary epithelium detected gene expression of the reporter vectors luciferase and lac-z in the lung h after intratracheal administration in mice models. polyplexes of ultrapure chitosan was found to be nontoxic at higher doses [ ] . a patent (us b ) has been granted to rolland and mumper for their invention entitled 'chitosan related compositions and methods for delivery of nucleic acids and oligonucleotides into a cell'. this invention reported a non-viral method for the delivery of nucleic acids and oligonucleotides to cells [ ] . chitosan-sirna nanoparticles to deliver rnai based on the formation of inter-polyelectrolyte complexes between sirna duplexes and chitosan are reported to have rapid uptake of cy -labeled nanoparticles into nih t cells followed by accumulation over a h. nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (egfp) was demonstrated in both h human lung carcinoma cells and murine peritoneal macrophages. nasal administration of this formulation showed effective rna interference in bronchiole epithelial cells of mice models [ ] . intranasal vaccination with chitosan based plasmid dna induced significant peptide and virus specific cytotoxic t lymphocytes responses in balb/c mice [ ] . a substantial decrease in rsv titer and antigen load has been recorded in balb/c mice vaccinated with a cocktail of rsv cdnas in chitosan nanoparticulates [ ] . mao et al., developed chitosan-dna nanoparticles using a complex coacervation process, for pulmonary delivery of genes that carried a polyepitope plasmid encoding eight known t-cell epitopes derived from mycobacterium tuberculosis antigens. a significant increase in transfection efficiency ( -fold) was observed from conjugated nanoparticles as compared to the transferrin conjugation ( -fold) in hek cells and hela cells [ ] . surface functionalized dendritic cell targeted chitosan nanoparticles have been examined for the nasal dna immunization against severe acute respiratory syndrome cov (sars-cov). dendritic cell c-type lectin receptor (dec- ) is a c-type lectin receptor found in dendritic cells for recognition and uptake of pathogens. raghwanshi et al., developed a bifunctional fusion protein vector of truncated corestreptavidin fused with anti-dec- single chain antibody. the fusion protein were bound to the biotinylated chitosan nanoparticles. plasmid dna encoding nucleocapsid protein was loaded in chitosan nanoparticles. intranasal dendritic cell targeted nanoparticles enhanced mucosal iga and systemic igg levels against nucleocapsid proteins [ ] . poly (lactide-co-glycolide) (plga) is another class of biocompatible and biodegradable polymer explored to achieve efficient pulmonary gene expression [ ] . the addition of polyethyleneimine, a cationic polymer to the plga nanoparticles exhibited high positive charge density when protonated in aqueous solutions [ ] . it is regarded to be a promising polymer candidate as a non-viral vector for delivery of dna and oligonucleotides [ , ] . dna-loaded plga nanoparticles holding polyethyleneimine on their surface have been reported as nonviral gene vector to the human airway submucosal epithelial cell line, calu- . the study reported the presence of dna in endolysosomal compartment for a period of h. the results established the potential of nanoparticles in gene delivery to the lung epithelium [ ] . dendrimers (synthetic polymers) are of utmost importance in pharmaceutical drug discovery and development. these are polymers with moderately low toxicity in comparison to lipid-based vectors and have the advantage of versatility for chemical modification. the in vitro knockdown efficiency through sirna or antisense oligonucleotide (aon) delivered via dendriplexes to a lung alveolar epithelial cells was reported previously [ ] . dendrimers are drug delivery systems having a three-dimensional, star-shaped, branched macromolecular network. these nanocarriers possess low polydispersity index, are biocompatible and have good water solubility. the dendrimers consist of an exterior and an interior layer. the interior layer is responsible for the controlled release mechanisms, reduced drug toxicity and improved drug encapsulation efficiency. while the exterior layer contains functional groups responsible for conjugation of targeting moieties and drugs. due to such unique properties, dendrimers are becoming an useful drug delivery system [ ] . in a study, poly (amidoamine) (pamam) dendrimer nanocarriers (dncs) were synthesized and the effect of pegylation on the interaction of these nanoparticles was evaluated on both in vitro and in vivo models of the pulmonary epithelium. the transport of dncs was found to be increased from the apical to the basolateral sections across polarized calu- monolayers as the surface density of peg increased. this behavior was attributed to a significant reduction in charge density upon pegylation. the results showed that pegylation can potentially modulate the pulmonary epithelial transport and internalization of dncs which will further serve as an effective platform for targeting the lung tissue to treat the respiratory diseases [ ] . hatano and co-workers synthesized a series of carbosilane dendrimers with hemagglutinin binding peptide and evaluated their activity against influenza virus. the prepared dendrimers were found to exhibit strong anti-viral activity against human viruses [ ] . dendrimeric nanomaterials were prepared by chemical modifications and then optimized for targeted delivery of small interfering rna (sirna) to pulmonary vasculature. the poly (propylenimine) and poly (amido amine) dendrimers were substituted with different lengths of alkyl chains using combinatorial approach. the dendrimers were observed to have the potential to act as an efficient targeting agent for the pulmonary delivery of rna [ ] . polymeric micelles are capable of encapsulating water insoluble dna, proteins and drugs, and therefore help in targeted delivery. the structural as well as functional features of polymeric micelles are similar with natural transport system like lipoproteins and virus. the most important focus of development of micelles is to address the major problem associated with drugs i.e., drug resistance. for this purpose, chemical manipulations were done and their effects were assessed on the cellular interaction, bio-distribution, encapsulation and release of the polymeric nanocarriers. hydrophobic drugs can be transported to the target site at concentrations far more than their inherent water solubility by trapping them in the core of a micelle [ ] . in a study, pluronic ® p /f mixed micelles (pmm) were prepared and evaluated for their potential in the delivery of poorly watersoluble drugs to lungs. the pmm were loaded with budesonide (bud) and were then assessed for their delivery, transport and stability in pulmonary-relevant media. after in vitro evaluation, formulations were further evaluated for pulmonary bio-distribution and efficacy in vivo via intra-tracheal administration in rats. results showed excellent stability of pmm in vitro, which may be due to the smaller size of pmm as a result of which, the pmm did not interact with mucin and thus diffused effectively through artificial mucus. overall, the results of the study demonstrated pmm as inhalable formulation that can be an important platform for targeted delivery of water insoluble drugs in respiratory diseases [ ] . another approach for sustained delivery of the drugs to the lungs utilized chitosan-based micelles which has been found to be safe and can effectively deliver the protein-based drugs. these drugs are required to be delivered to the special cells which can be done with the help of nanocarriers like micelles [ , ] . for improving the cell-specific delivery and efficiency, a modified self-assembled micelle interfering rna nanoparticles (samirna) were synthesized. the nanoparticles were designed to contain hydrophobic lipid and hydrophilic polymer on each ends of sirna. these are capable of forming micelle in the solution spontaneously after administration [ ] . this study demonstrated that samirna nanoparticle is a stable sirna silencing platform with less toxicity for effective in vivo targeting of genes involved in the pathogenesis of respiratory diseases [ ] . gaber et al., synthesized beclomethasone dipropionate (bdp)loaded micelles using poly-(ethylene oxide)-block-distearoyl phosphatidyl-ethanolamine (mpeg-dspe) polymer and evaluated them for sustained release. the study observed that entrapment efficiency up to % can be achieved with bdp-loaded polymeric micelles. along with high encapsulation efficiency, sustained release behavior, comparable inhalation properties, and increased biocompatibility of these synthesized polymeric micelles can be useful in utilizing them as a versatile delivery system in the treatment of chronic obstructive pulmonary disease and asthma [ ] . this technique merges sensitivity provided by nucleic acid amplification with the specificity provided by dna-dna hybridization for identifying viruses like adenoviruses which are responsible for causing acute respiratory diseases [ ] . furthermore, the microarray technique targeting gyrb/pare genes can be used to identify bacterial species from cultural isolates and can be employed for the diagnosis of acute upper respiratory infections. this technique proves to be advantageous over the conventional pcr method as it can identify numerous pathogens from the clinical samples [ ] . these are small, double-stranded synthetic odn molecules consisting of transcription factor binding sites that are involved in the regulation of transcription. after entering into the cells, these combine with nuclear transcription factors and inhibit their attachment to consensus sequence in target genes. also, decoy odn targeting transcription factor stat- reduces airway inflammation due to allergens and airway hyper-reactivity in asthma [ ] . these are a new category of chemically engineered oligonucleotides that are the synthetic analogues of mirna. after iv administration, these target mir- , mir- and mir- that led to the decrease in mirna levels in lungs, liver, heart, kidney and intestines where they cause prolonged silencing of endogenous mirna [ ] . adam (a disintegrin) and neuropeptide s receptor (npsr ) may represent novel targets for aso. adam deletion by aso reduces the expression of proteins like alpha-actin and promotes their apoptosis. therefore, by targeting both these genes, these may be used as potential therapeutics for asthma [ ] . oligonucleotides therapy has several potential clinical applications. therapeutic importance of oligonucleotides can be well understood by their potential uses in various disorders like neurodegenerative disorders, respiratory disorders, diabetic retinopathy, and cancer. oligonucleotides do not enter into the cell through diffusion as they are large entities. various delivery methods can be utilized for the effective delivery for oligonucleotides therapy. although, sufficient information is available on biodistribution and overall pharmacokinetics of oligonucleotides, substantial studies are required to understand the cellular and intracellular behavior of oligonucleotides [ ] . despite the approval of monoclonal antibodies like mepolizumab, omalizumab, and reslizumab, oligonucleotides therapy might be preferable over these, as the monoclonal antibodies are invasive due to their parenteral administration [ ] . alternatively, oligonucleotides can be locally administered into the airway through inhalation by aerosols which are a lesser invasive method as compared to the monoclonal antibodies. additionally, threatening hypersensitivity reactions are also a major drawback of the monoclonal antibodies [ , ] . at present, over s generation antisense oligonucleotides are in the clinical development process for an assortment of oncological, neurological, metabolic and cardiovascular conditions [ ] . antisense oligonucleotides are conditional on nuclease susceptibility in systemic circulation, which leads to a short half-life, rapid renal excretion, and passive diffusion via cell membranes and also are limited for negatively charged antisense oligonucleotides [ ] . further detailed studies are required to overcome such problems and thus novel drug delivery approaches like nanoformulations are the need of the hour to combat such snags. although, clinical trials against different disease conditions are ongoing for various oligonucleotide therapies, there are very limited number of clinical studies against chronic inflammatory respiratory diseases. in the past few years, many oligonucleotide therapies have been approved by fda [ ] . some of these were for complex neurological diseases like spinal muscular atrophy and for duchenne muscular dystrophy [ ] . such successful examples may lead the way for the therapeutic utilization of oligonucleotide therapies like antisense and aptamers against chronic inflammatory respiratory diseases. moreover, strategies for chemical modifications of sugars, nucleotides, or phosphate backbone are obligatory for the stability enhancement and reduction of toxicity [ ] . synergistic approaches of the combination of two or more antisense oligonucleotides can enhance the efficacy against respiratory diseases at a lower dose as reported in the studies on rodent models [ ] . the delivery of oligonucleotides as therapeutic regimen against chronic inflammatory respiratory diseases can be done by utilizing a nanocarrier, where oligonucleotides could be incorporated. these can further determine the cellular interaction and tissue distribution of used oligonucleotide. furthermore, chemical modification of the oligonucleotide with a targeting ligand can also be studied [ ] . in a nutshell, several drug delivery systems can be employed for the delivery of oligonucleotides like polymer/lipid-based nanoparticles, target specific ligand-oligonucleotide conjugates and antibody conjugates. advancement in the novel approaches like oligonucleotidebased microarray techniques, decoy oligodeoxynucleotides, antagomirs, and adam and npsr targeting are some strategies that can be useful as the potential treatment strategies for respiratory diseases. trend in approval of oligonucleotide-based therapy by fda also showed the importance of oligonucleotides. moreover, the problems associated with drug delivery can also be resolved by use of various novel drug delivery methods as described in this review for instance via development of liposomes, niosomes, nanoparticles, mucoadhesive targeting and dendrimers etc. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. interactions with the macrophages: an emerging targeted approach using novel drug delivery systems in respiratory diseases increasing complexity and interactions of oxidative stress in chronic respiratory diseases: an emerging need for novel drug delivery systems multi-drug resistant mycobacterium tuberculosis & oxidative stress complexity: emerging need for novel drug delivery approaches delivery of rnai therapeutics to the airways-from bench to bedside emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease oligonucleotides: a multi-targeted approach for the treatment of respiratory diseases antisense oligonucleotides: the next frontier for treatment of neurological disorders rna therapeutics: beyond rna interference and antisense oligonucleotides sirna versus mirna as therapeutics for gene silencing micrornas as therapeutics for future drug delivery systems in treatment of lung diseases nanotechnology: advancing the translational respiratory research micrornas: synthesis, mechanism, function, and recent clinical trials aptamer chemistry aptamers and their biological applications cpg oligonucleotides as cancer vaccine adjuvants oligonucleotide therapeutic approaches for huntington disease oligonucleotide therapeutics in neurodegenerative diseases respirable antisense oligonucleotides: a new drug class for respiratory disease therapeutic antisense oligonucleotides against cancer: hurdling to the clinic antisense oligonucleotide therapy in diabetic retinopathy inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide oligonucleotide compositions and methods for treating disease including inflammatory conditions antisense oligonucleotides against nonstructural proteins ns and ns of respiratory syncytial virus and uses thereof method of treating inflammatory lung disease with suppressors of cpg oligonucleotides methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines lipid-based vectors for sirna delivery an imperative note on novel drug delivery systems efficient biodistribution and gene silencing in the lung epithelium via intravenous liposomal delivery of sirna effect of cationic lipid in cationic liposomes on sirna delivery into the lung by intravenous injection of cationic lipoplex nanomedicine based approaches for the delivery of sirna in cancer sterically stabilized cationic liposomes improve the uptake and immunostimulatory activity of cpg oligonucleotides co-delivery of oxygen and erlotinib by aptamer-modified liposomal complexes to reverse hypoxia-induced drug resistance in lung cancer intratracheal versus intravenous liposomal delivery of sirna, antisense oligonucleotides and anticancer drug antisense oligonucleotides directed against the viral rna polymerase gene enhance survival of mice infected with influenza a treatment of pulmonary fibrosis with sirna against a collagen-specific chaperone hsp in vitamin a-coupled liposomes niosome: a future of targeted drug delivery systems advances of non-ionic surfactant vesicles (niosomes) and their application in drug delivery nanotechnology in respiratory medicine directed self-assembly of nanoparticles drug delivery systems: an updated review polymeric nanoparticles: a study on the preparation variables and characterization methods chitosan ifn-γ-pdna nanoparticle (cin) therapy for allergic asthma cutting edge: modulation of airway inflammation by cpg oligodeoxynucleotides in a murine model of asthma improving the therapeutic index of cpg oligodeoxynucleotides by intralymphatic administration development of a poly (lactic-co-glycolic acid) particle vaccine to protect against house dust mite induced allergy targeted delivery of sirna to activated t cells via transferrin-polyethylenimine (tf-pei) as a potential therapy of asthma prevention of airway inflammation with topical cream containing imiquimod and small interfering rna for natriuretic peptide receptor antisense oligonucleotide eluforsen improves cftr function in f del cystic m pegylated enhanced cell penetrating peptide nanoparticles for lung gene therapy poly(ester amine)-mediated, aerosol-delivered akt small interfering rna suppresses lung tumorigenesis noncovalenly pegylated ctgf sirna/pdmaema complex for pulmonary treatment of bleomycin-induced lung fibrosis treatment of lung cancer via telomerase inhibition: self-assembled nanoplexes versus polymeric nanoparticles as vectors for '-o-methyl-rna overcoming cisplatin resistance in non-small cell lung cancer with mad silencing sirna delivered systemically using egfr-targeted chitosan nanoparticles nanoparticle-mediated pulmonary drug delivery: a review optically traceable solid lipid nanoparticles loaded with sirna and paclitaxel for synergistic chemotherapy with in situ imaging nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and sirna innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and sirna in vivo tumor targeting via nanoparticlemediated therapeutic sirna coupled to inflammatory response in lung cancer mouse models novobrantseva, sirna and the lung: research tool or therapeutic drug? oligonucleotide therapy for obstructive and restrictive respiratory diseases biodistribution of phosphodiester and phosphorothioate sirna running interference: prospects and obstacles to using small interfering rnas as small molecule drugs nonviral pulmonary delivery of sirna application of chitosan and its derivatives in nanocarrier based pulmonary drug delivery systems dnapolycation nanospheres as non-viral gene delivery vehicles carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens chitosan as a nonviral gene delivery system. structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo polycation-based nanoparticle delivery for improved rna interference therapeutics rna interference in vitro and in vivo using a novel chitosan/sirna nanoparticle system the influence of polymeric properties on chitosan/sirna nanoparticle formulation and gene silencing the potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. iii: effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro respiratory syncytial virus infection in fischer rats is attenuated by short interfering rna against the rsv-ns gene inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns gene pulmonary delivery of chitosan-dna nanoparticles enhances the immunogenicity of a dna vaccine encoding hla-a* -restricted t-cell epitopes of mycobacterium tuberculosis naked silna-mediated gene silencing of lung bronchoepithelium egfp expression after intravenous administration chitosan related compositions and methods for delivery of nucleic acids and oligonucleotides into a cell nasal delivery of chitosan-dna plasmid expressing epitopes of respiratory syncytial virus (rsv) induces protective ctl responses in balb/c mice intranasal ifngamma gene transfer protects balb/c mice against respiratory syncytial virus infection chitosan-dna nanoparticles as gene carriers: synthesis, characterization and transfection efficiency dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein biodegradable polymeric nanocarriers for pulmonary drug delivery poly(lactic acid)-poly(ethylene glycol) nanoparticles as new carriers for the delivery of plasmid dna design and gene delivery activity of modified polyethylenimines characterization of commercially available and synthesized polyethylenimines for gene delivery plga-pei nanoparticles for gene delivery to pulmonary epithelium poly(amidoamine) dendrimer nanocarriers and their aerosol formulations for sirna delivery to the lung epithelium application of dendrimers for the treatment of infectious diseases dendrimer nanocarriers for transport modulation across models of the pulmonary epithelium synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally functionalized with hemagglutinin-binding peptide dendrimer-inspired nanomaterials for the in vivo delivery of sirna to lung vasculature nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases in vitro/in vivo investigation on the potential of pluronic(r) mixed micelles for pulmonary drug delivery microencapsulated chitosan nanoparticles for lung protein delivery sustained delivery by leucinemodified chitosan spray-dried respirable powders attenuation of fibrosis in vitro and in vivo with sparc sirna self-assembled micelle interfering rna for effective and safe targeting of dysregulated genes in pulmonary fibrosis characterization of polymeric micelles for pulmonary delivery of beclomethasone dipropionate use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections a review of antisense therapeutic interventions for molecular biological targets in asthma silencing of micrornas in vivo with "antagomirs cellular uptake and intracellular trafficking of oligonucleotides mepolizumab for severe eosinophilic asthma (dream): a multicentre, double-blind, placebo-controlled trial infusion-related and hypersensitivity reactions of monoclonal antibodies used to treat colorectal cancer-identification, prevention, and management a randomised, open-label, parallel group phase study of antisense oligonucleotide therapy in acromegaly an overview of the clinical application of antisense oligonucleotides for rna-targeting therapies fda-approved oligonucleotide therapies in antisense technologies. improvement through novel chemical modifications multitargeted approach using antisense oligonucleotides for the treatment of asthma the delivery of therapeutic oligonucleotides supplementary data to this article can be found online at https:// doi.org/ . /j.cbi. . . . key: cord- - jweuq authors: mongia, aanchal; jain, stuti; chouzenoux, emilie; majumda, angshul title: deepvir -- graphical deep matrix factorization for"in silico"antiviral repositioning: application to covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: jweuq this work formulates antiviral repositioning as a matrix completion problem where the antiviral drugs are along the rows and the viruses along the columns. the input matrix is partially filled, with ones in positions where the antiviral has been known to be effective against a virus. the curated metadata for antivirals (chemical structure and pathways) and viruses (genomic structure and symptoms) is encoded into our matrix completion framework as graph laplacian regularization. we then frame the resulting multiple graph regularized matrix completion problem as deep matrix factorization. this is solved by using a novel optimization method called hypalm (hybrid proximal alternating linearized minimization). results on our curated rna drug virus association (dva) dataset shows that the proposed approach excels over state-of-the-art graph regularized matrix completion techniques. when applied to"in silico"prediction of antivirals for covid- , our approach returns antivirals that are either used for treating patients or are under for trials for the same. the problem of matrix completion has been deployed in numerous applications of computer science and bioinformatics. one can mention, for instance, recommender systems [ ] , wireless sensor networks [ ] and mimo channel estimation [ ] . matrix completion also forms the foundation of many biological interaction problems including gene expression imputation [ ] , drug-target interaction prediction [ , , ] , mirna disease association [ , ] and gene disease association [ ] . as seen in the aforementioned studies, matrix completion is turning out to one of the most promising approaches for modelling biological interactions. matrix completion for in silico drug re-positioning is an established topic [ , ] . in such context, the matrix rows relates to drugs while the columns correspond to the diseases. the information about a drug being effective for a disease is encoded with a one into the matrix and zero, otherwise. the matrix is only partially filled since information about all drug disease associations are not known. matrix completion amounts to filling the missing entries by relying and interpolating over the available knowledge. our recent work [ ] introduced a dataset of antiviral drug virus association (dva). it shows how the matrix completion framework can be used to computationally predict the drugs that could be effective against sars-cov- (severe acute respiratory syndrome coronavirus ), the virus responsible for the ongoing pandemic, covid- (corona virus disease- ). in the aforesaid dataset, we manually curated drug-virus associations from drugbank.ca. we collected all the anti-viral drugs proved to be effective against viruses infecting humans along with the similarity information between the viruses (genomic structure-based similarity) and drugs (chemical structurebased similarity). the dataset contains both dna and rna viruses known to infect human beings. state-of-the-art off-the-shelf matrix completion techniques were then employed to solve the association prediction problem. it was found that graph regularised techniques, including similarities based on genomic structure of viruses and chemical structure of antiviral drugs, performed better than the non-regularised competitors. the most successful techniques were graph regularized versions of matrix completion [ ] , binary matrix completion [ ] and (shallow) matrix factorization [ ] . in this present work, we propose to solve the problem of drug-virus association prediction via graph regularized deep matrix factorization. we propose a novel theoretically sounded algorithm for this task. moreover, since our final goal is to repurpose drugs for covid- , an rna virus, we have pruned our dva dataset to consists only rna viruses. dna viruses are not meaningful in this context [ ] , hence we will not be considering them in this work. furthermore, in addition to the graph based on chemical structure similarity of drugs [ ] , one more similarity graph has been built on the mode of action of the drugs. similarly, an additional similarity graph on viruses is built, based on the human symptoms caused by the virus' infection. the current work uses the symptom based similarities along with the genomic similarities used in the prior work [ ] . a prior study on graph regularised deep matrix factorization [ ] was based on the alternating direction method of multipliers (admm) approach [ ] . a major issue with admm is that the convergence guarantees are rather mild in such challenging non convex scenario. furthermore, admm method requires the resolution of costly inner steps as well as the introduction of several intermediary variables that may be detrimental to the practical efficiency of the method. owing to the aforesaid reasons, we propose a novel resolution scheme called hypalm (hybrid proximal alternating linearized minimization), relying on the recent techniques developed in [ , , ] . to summarize the main contributions of our proposed work are as follows: • an original graph structure introducing (i) symptoms based viral similarity in the virus-virus graph, (ii) mechanism-of-action based drug similarity in the drug-drug graph, leading to the so-called multi graph regularized deep matrix factorization (grdmf) model; we call the approch grdmf throughout this paper. • a novel optimization method, named hypalm, for addressing the resulting matrix factorization problem with sounded convergence guarantees on its iterates. in this subsection, we study the choice of technique to solve the grdmf problem ( ) . in the past work [ , ] , the proposed formulation has been solved using admm (alternating direction method of multipliers) [ , ] . we introduce here a novel resolution strategy named hypalm. in contrast with admm, the iterates of hypalm are guaranteed to converge to a stationnary point of the non-convex problem. the hypalm algorithm steps are depicted in the methods section of the paper. we randomly drop % of association values in the drug-virus association matrix and report the auc (area under the roc curve) and aupr (area under the precision recall curve) on the test entries (averaged over runs of cross validation) after solving -layer and -layer grdmf problem with admm (first two columns) and hypalm (last two columns). as can be seen, hypalm solution gives much better prediction results, for both and layers, and both evaluation metrics . hence, owing to the better performance and the assessed convergence guarantees, we propose to retain hypalm in the remaining of the paper for our simulations with the grdmf model. we compare the performance of our method with other recent graph regularized matrix completion techniques such has graph regularized matrix completion (grmc) [ ] , graph regularized binary matrix completion (grbmc) [ ] and graph regularized techniques like graph regularized matrix factorization (grmf) [ ] . the authors of [ ] show that grmf and grbmc perform well in predicting drug-virus association and hence estimating drugs for sars-cov- . we evaluate the performance of -layers and -layers graph regularized deep matrix factorization model (grdmf) with the similarities provided by the method from [ ] and by the newly proposed models grdmf- l and grdmf- l. to quantitatively measure and compare the performance of the proposed and other benchmark algorithms, we carry out -fold cross validation on the drug-virus association dataset in settings and observe the standard metrics such as mean auc and aupr. the first setting (cv ) corresponds to randomly hiding % association entries as the test set in the association matrix, while the other two settings cv and cv correspond to randomly hiding % viruses and drugs in the data as test set respectively. such a validation technique is a standard to evaluate the accuracy in association/interaction prediction problems [ , ] . as can be observed from table , both version of our algorithm (grdmf- l and grdmf- l) perform much better than the state-of-the-art in terms of aupr and gives comparable results in terms of auc. this metric is more important than auc because it penalizes more highly ranked false positives so that only highly ranked drug-virus pairs in prediction would be recommended for biological or chemical tests. it can be seen that the prediction results do not improve much in the -layers version. hence, we use the -layers grdmf for further experiments, as it presents lower complexity. let us analyse deeper the results in table . the dataset contains more drugs ( ) than viruses ( ) . in cv settings, when entries are randomly omitted, without skipping drugs and viruses our method performs the best. there is enough data and hence our method does not overfit. the results improve from one (grmf) to two and three layers. in the cv setting, when viruses are omitted, the size of the dataset reduces. the proposed deeper method overfits and hence performs poorly compared to shallower techniques. cv also reduces the size of the dataset by omitting drugs, but since the number of drugs are significantly larger than the number of viruses, the effect is not pronounced on our proposed algorithm and we see an improved prediction. we then measure the precision and recall at top k (pre@k and rec@k, k = , k = and ) drug recommendations for viruses while implementing leave-one-out-cross validation (loocv) by hiding (i.e., leaving out) the association profile of every virus. performing loocv tells us how well is the algorithm doing in terms for predicting drugs for current known viruses. this is the most realistic setting, where all the drugs and all the viruses (barring the test virus) are considered. one can see that our algorithm gives comparable precision values and exhibits the best recall at all values of k. this appears as the most realistic case since, on average, there are to drugs for each virus. table : precision@k and recall@k for association prediction for k= and with multiple similarities used for drugs and viruses. the values for the grdmf model parameters µ, k , k , k and for the hypalm parameters α, ϑ have been set by performing cross-validation on the training set (see supplementary material for the list of retained values). moreover, we set the number of iterations k = in hypalm, which appears sufficient to reach stability of the iterates. the time taken by the proposed approach is comparable to the state-of-the art techniques and has been reported in table . all experiments have been performed on a system with intel(r) core(tm) i x -based processor and gb ram. we also illustrate in figure the practical convergence profile of our algorithm hypalm, on an example. table : execution times (in seconds) for association prediction for all techniques. in order to quantitatively assess the necessity/importance of each of the similarity models at use, we conducted an ablation study where we used various combinations of similarity (graph regularizations) matrices within the model to predict % of the test association matrix, which were randomly hided as test set (cross validation setting cv ). the results have been reported in table . here, s d corresponds to chemical structure based similarity for drugs and s d corresponds to the similarity based on mechanism of action of drugs. similarly, s v corresponds to the genomic structure based similarity and s v corresponds to symptomatic similarity between the viruses. let us make a few observations from this study. firstly, taking into account the new similarities gives the best prediction results. secondly, when we use the chemical structure based drug similarity (s d ) and vary the viral similarities from s v to s v (columns and ), we observe that symptomatic similarity (s v ) yields better results than genomic similarity (s v ) and hence contributes more towards the prediction task. similar behavior appears when using the genomic similarity (s v ) for viruses and varying the kind of drug similarity used (columns and of table ). in other words, our results assess that the new similarities contribute more towards the drug recommendation and greatly improve the prediction results when taken into account in combination with the previous ones. lastly, we notice that simply replacing the older similarities with the new ones (column of table ) improves the results. hence, this ablation study establishes the benefits of the new similarity models that we introduced. in this subsection, we predict top- drugs using the best performing solution grdmf- l and the baseline algorithms for the novel coronavirus (sars-cov- ). we display the results in table laninamivir taribavirin simeprevir taribavirin dolutegravir chloroquine taribavirin paritaprevir as c table : top- ranked recommendations/drugs predicted for sars-cov- by the dva computational methods. as can be observed, table reports the drug recommendations for the novel corona virus (ncov/sars-cov- ) using the proposed algorithm and other baselines. the results from our proposed algorithm are sensible. ribavarin is an fda approved antiviral being considered as a treatment for covid either by itself [ ] or as a part of combination [ ] . remdesivir is a promising candidate for treating covid having shown efficacy against sars-cov- . the antiviral has been approved for emergency use by the us fda [ , ] ; it has been approved by the drug controller general of india (dgci) to treat mild to moderate covid- patients . the science behind the mechanism of remdesivir's action for inhibiting covid has been thoroughly studied [ ] . [ ] showed that umifenovir (brand name arbidol) is effective as a prophyalactic against covid infection. it has also found to be effective in the treatment of covid affected subjects [ , , , ] . dgci has approved it for phase trials in india . favipiravir, a russian drug for influenza, has also been undergoing trials in japan; it has been sent to + countries for covid trials . [ ] explains the scientific reason behind the prospective success of this antiviral. baloxavir marboxil has undergone trial in china for treating sars-cov . interferon alfa- a in combination with umifenovir have been found to be effective against covid infected pneumonia [ ] . in silico docking studies shows the potential of baloxavir marboxil [ ] ; [ ] suggests that this antiviral can be effective if administered within hours of the onset of infection. several in silico docking studies have shown dolutegravir may be effective in treating covid infection [ , ] . trials for chloroquine and alternately hydroxychloroquine was once suspended by who based on a controversial article, but they were resumed within a few days of suspension . this antimalarial drug has been found to be effective against corona in multiple studies [ ] ; the scientific reason behind the efficacy of this drug against covid has been explained in [ ] . geldanamycin and laninamivir, predicted by our algorithm has not been considered for treating covid patients. the benchmark techniques grmc, grbmc and grmf have three drugs common to ours -ribavarin, remdesivir and umifenovir. these three techniques also predict an anti-hepatitis c drug -sofosbuvir; this drug was known to be effective against the coronavirus family [ ] ; it's mechanism of action against covid has also been studied [ ] . evidence from clinical studies have corroborated the same [ ] . grmc and grbmc also predicts chloroquine (as do we); but grmf is unable to predict it. note that the results shown in table are ranked. the ranking is important, since it gives us a faith in the efficacy of the drugs. our method predicts drugs under trial among the top . the benchmarks only recommend drugs in correct order. in fact, had we looked at the top recommendations, only our algorithm would have recommended all of them correctly. the benchmark algorithms predict three common drugs -telaprevir [ ] , boceprevir [ ] and simeprevir [ , ] . these drugs have the potential to be considered for covid clinical trials. all the three benchmarks predict the antibody palivizumab; there is no evidence in medical literature to support its usage for treating covid patients or using it as a prophylactic. they also predict taribavirin, which is similar to ribavirin, and is used for treating hepatitis-c; it has not been considered for clinical trials for sars-cov . grmf predicts a unique drug -paritaprevir. an in silico docking study [ ] showed that it has the potential for treating covid much like telaprevir, boceprevir and simeprevir. the summary of the findings are in the following only our proposed algorithm predicts drugs that are under trial (remdesivir, ribavarin, umifenovir, chloroquine, favipiravir and interferon alfa- a). two of the benchmarks predict drugs that are under trial (remdesivir, ribavarin, umifenovir, sofosbuvir and chloroquine); one of them (grmf) misses out on chloroquine. it is encouraging to observe that all the techniques (proposed and benchmarks) are predicting common drugs -remdesivir, ribavarin, umifenovir. ai researchers all across the world are contributing towards fighting covid . this is our humble contribution towards the same goal [ , ] . in this paper, we have proposed a novel optimization approach to solve the drug-virus association prediction problem. the contribution of this work is two-fold. biologically, we have extended the phenomena of solving drug-virus association prediction for drug re-positioning using multiple similarities (graphs) and introduced new similarity measures for both drugs (based on mechanism of action) and viruses (based on symptoms). technically, the proposed novel technique brings together the benefits of deep learning (using deep matrix factorization) and multiple graph learning (by regularization with multiple graphs). the algorithmic solution to the problem, relying on the novel hypalm approach, does not require the setting of any extra hyper-parameter and has sounded convergence guarantees. we have made the software publicly available at https://github.com/aanchalmongia/deepvir. a significant amount of research has been done in formulating drug re-positioning as a machine learning task. various approaches such as network diffusion, supervised classification, neighborhood based prediction, clustering and matrix completion have been used in the past to address the said problem. of these matrix completion techniques have turned out to be the most promising approach [ ] . various biological applications which model the problem of drug-re-positioning using matrix completion include: • drug target interaction prediction: the problem is to predict interaction score between a drug and target protein in a matrix assumed to have partial interaction data with drugs on rows and proteins on columns [ , , , , , ]. • drug disease association prediction: one predicts the probability that a certain drug will interact with a disease or not in a partially filled drug-disease association matrix. [ , , , ] . • drug-drug interaction prediction: the drug-drug relationship is detected through a symmetrical drugdrug matrix/network [ ] . this helps predict drugs similar to the ones known to be effective against a pathogen/disease. attempts have also been made to propose a machine learning approach for predicting drug-likeness of a molecule to distinguish potential drugs from small molecules that lack drug-like features [ ] . in addition to drug re-positioning, other biological interaction problems have also be modeled as matrix completion with the common goal of discovering or improving treatment of human diseases, for example we can cite: • gene disease association prediction: disease and gene features are used to learn genes for diseases as a gene-disease association prediction [ , , ] . this assists deciphering genetic basis of human diseases and better understanding of gene functions, interactions, and pathways. • protein-protein interaction prediction: interactions are estimated in a protein-protein network [ , ] . kshirsagar et al. [ ] deploy a matrix completion variant to model interactions between host (human here) proteins and pathogen (viruses causing infectious diseases here) proteins. this helps identifying the interaction between viral proteins and the human proteins, enabling deeper understanding of infectious diseases (which may involve biologically similar pathogens). motivated by such works, the problem of drug virus association prediction was considered in [ ] . although solvable by any computational approach, the authors propose to tackle it using matrix completion motivated by the success of matrix completion in the above-mentioned applications. among all the methodologies compared in [ ] , graph regularized matrix factorization based technique (grmf) provided the best results for the validation setting where drugs are predicted for novel viruses. this impels us to come up with an improved algorithm built upon grmf and leverage the benefit of deep learning and various kinds of metadata associated with drugs/viruses into the current method to aid drug re-positioning for viruses (including sars-cov- ). the goal of matrix completion is to recover all the entries of a matrix given a subset of known entries. assume that y is a masked / undersampled version of the complete matrix x. then, the problem can be expressed as: here, m denotes the mask which is element-wise multiplied to x and has ones at positions where the values are known and zeros elsewhere. in general, the problem is under-determined with infinitely many solutions. however, when the sought matrix x is of low-rank, several efficient resolution methods exist. the simplest of them is the matrix factorization approach [ , ] . although known to work empirically, a theoretical understanding of factorization based completion is relatively recent [ ] . in this approach to matrix completion, x is factored into a product of two low-rank matrices u and v . formally, this is expressed as: the factor matrices (u and v ) are obtained by minimizing the following objective function: with · f the frobenius norm. the setting of the size of the latent factors u and v allow to impose rank constraints on their product. motivated by the success of deep learning in numerous fields [ , , , ] , the shallow (two factor) models have been extended to deeper versions. the general factorization problem when the matrix is completely observed have been proposed in [ , ] . the solution to matrix completion via deep factorization has been proposed very recently, in [ ] . this can be formally expressed as follows, in the -layers case: hereagain, the size of the latent factors u , u and v control the rank of the resulting product. one can extent to deeper layer formulations ( -layers and -layers) by factorizing x into and matrices respectively. the first factor matrix u (of size m × k ) contains the representation of the row elements while the last factor matrix v (of size k × n) is associated to its column representations in the latent space. the values for k and k here represent the number of latent factors, and are typically set using cross-validation. matrix factorization yields non-convex optimization problems which may be difficult to solve. a more direct approach to low rank matrix completion is to directly estimate matrix x under low-rank constraints. since the minimization of the rank leads to np hard problems, one usually replaces it by its convex proxy, the nuclear norm [ , ] , defined as the sum of singular values. the problem formulation is then given as follows: in regular matrix completion, the entries in the unknown matrix are assumed to be real-valued. in communications and signal processing, this is actually never the case as those values are almost always quantized. this led to the quantized matrix completion problem [ , ] . one extreme case is binary matrix completion [ ] where the matrix entries are represented by only one bit. binary matrix completion is particularly appropriate for modeling biological interactions, so unsurprisingly studies in the past have used it for microbe disease association [ ] . in all the aforesaid approaches, there was no scope for incorporating associated metadata, to constrain the completion problem and reduce the indeterminacy. in problems of biological imputations, there are often several sources of associated metadata. for example, in drug target interaction, one can compute the simcomp score between the drugs based on their chemical structure as a drug-drug similarity measure [ ] . similarly, similarities between target protein can be found a priori by computing the smith-waterman score between the amino acid sequences of the target proteins [ ] . studies have shown that the incorporation of associated metadata can improve the prediction results considerably [ ] . there can be several approaches to model the associated metadata, in the framework of matrix completion. one of the most promising approaches is to model the associations as graphs. this led to weighted graph regularised matrix factorization [ ] . the formulation was expressed as: hereabove, µ ≥ is the parameter penalizing the graph regularization terms, and tr(·) denotes the trace of a matrix. furthermore, the cost function in ( ) incorporates laplacian weight matrices l r and l c imposing prior correlation between row entities and column entities, respectively. those matrices are obtained from known similarity factors s r and s c , according to d r and d c are diagonal degree matrices for the corresponding similarity matrices s r or s c , that is, their i-th diagonal entry equals d ii r = j s ij r and d ii c = j s ij c . we recently proposed a deep formulation for ( ) in [ ] which amounts to solving: graph regularised version of nuclear norm minimization was also proposed in [ ] and was employed for drug target interactions [ ] . the formulation is given by: all the aforementioned graph regularised techniques have been compared in our prior work [ ] in the context of drug-virus association prediction. in this work, we assume the unknown matrix x to be a complete drug-virus association matrix, with drugs along the rows (m drugs) and viruses along the columns (n viruses). it is a binary matrix with each entry x ij either equal to zero, representing no proven association between the corresponding drug and virus or to one, denoting that the i th drug is known to be effective against the j th virus. we set y as the observed matrix, corresponding to a sampled version from x. the information of the known association positions is encoded in the masking binary operator m . in our proposed technique, multi-graph regularization is incorporated in the deep matrix factorization formulation with the aim to incorporate the metadata associated with the drugs and viruses in the form of similarity information as shown below: hereabove, with n d and n v the number of available similarity matrices (s ℓ d ) ℓ and (s ℓ v ) ℓ , for row entities (i.e. drugs) and column entities (i.e., viruses). the sizes of matrices u , u and v are m × k , k × k and k × n respectively, where k ≥ and k ≥ are the numbers of latent factors assumed to be involved. note that our formulation relies on the observation that computing the laplacians associated to the sums n d ℓ= s ℓ d and nv ℓ= s ℓ v is actually equals to the sum of all the individual laplacians involved [ ] , given by ( ) . in our previous work on drug-virus association prediction [ ] , we only consider one kind of similarity for drugs (based on the chemical structure of the drugs) and viruses (based on the genomic sequence of the viruses), so that n d and n v were equal to one. in this work, we propose to integrate a second type of similarities for drugs and for viruses by taking into account the mechanism of action of drugs and symptoms of viruses, so that n d = n v = . the sought drug-virus association matrix x to be recovered is assumed to be a positive entry matrix with a low-rank structure as similar drugs are known to affect biological systems (target pathways) in a similar fashion by having a similar mechanism of action [ ] , which motivates its formulation as the product of rectangular matrices, x = u u v and the addition of the positivity constraint on it. we deploy the sparsification method to process the similarity matrices (as done in [ , ] ) using p-nearest neighbor graph which is obtained by taking into account only the similarity values which correspond to the nearest neighbors for each drug/virus. p here is set by performing cross-validation on the training set. the minimization problem ( ) is non-convex. the multi-linear structure of the involved operators motivates us for using the alternating minimization algorithm from [ ] . this algorithm has been shown to be very powerful, in several applications such as seismic signal recovery [ ] , phase retrieval [ ] and hyperspectral imaging [ ] . it can be seen as an alternating majorization-minimization (mm) technique [ ] , including the powerful proximity operator [ ] which allows to stabilize and speed up the convergence. in order to apply this method, one needs to introduce explicitly the variable x in the problem formulation, as follows: with the advantage is to remove the complicated nonlinear constraint u u v ≥ while not deteriorating the solution (for ϑ > sufficiently large). function f is then the sum of convex terms involving only a single parameter, and a quadratic, thus differentiable, non convex coupling term weighted by ϑ. we are thus in the framework depicted in [ ] (see also [ ] for a particular case with two variables), so that we can apply the block coordinate variable metric forward-backward to solve problem ( ) . this amounts to updating the four variables x, u , u and v sequentially, combining gradient (possibly with preconditioning) and proximal updates (see [ ] for more details about the proximity operator). as emphasized in [ ] , the two first quadratic terms can actually be processed either through proximal or gradient descent updates, without altering the convergence of the method. this nice flexibility allows us to propose the following alternating hybrid scheme, that we called hypalm for the resolution of ( ): • (update of x) here, we perform a gradient step over the first quadratic term of ( ), followed by a proximal step over the remaining terms: hereabove, ς > is a parameter of the algorithm, and α is a stepsize that must be chosen within the interval ] , [ to secure the convergence of the method. the maximum operation in ( ) is taken entrywise, so it is equivalent to a simple cropping of the negative values. • (update of u ) we solve the following minimization problem, equivalent to evaluate the proximity operator of f restricted to the variable u , at the previous version of u denoted u : by taking the gradient of the above expression with respect to u equals to zero, we obtain: with i the identity matrix. system ( ) takes the form of a sylvester equation for which efficient solvers are available. • (update of u ) similarly to the latter update, we compute: with u the previous version of u . this leads to the resolution of the sylvester equation: • (update of v ) hereagain, we solve the minimization problem: with v the previous version of v . this leads to solving the sylvester equation: iterating over the above four steps, with the simplified setting ς = , leads to algorithm , where we denote sylv{a, b, c} the solution of the sylvester equation ax + xb = c. the convergence of the iterates to a stationary point of problem ( ) is guaranteed as a consequence of the theoretical analysis in [ , ] . it should be noted that the solution for deeper layers ( -layers, -layers, etc) of hypalm method for the grdmf problem can easily be obtained in a similar fashion to the -layers framework shown above. v (k− ) (u (k− ) v (k− ) ) ⊤ , ϑx (k) (u (k− ) v (k− ) ) ⊤ + u (k− ) } : : u (k) = sylv{((u (k) ) ⊤ u (k) ) − , ϑv (k− ) (v (k− ) ) ⊤ , ((u (k) ) ⊤ u (k) ) − u (k− ) + ϑ((u (k) ) ⊤ u (k) ) − (u (k) ) ⊤ x (k) (v (k− ) ) ⊤ } in this subsection, we describe in detail how each of the new similarities between drugs and viruses is obtained. for drugs, we relied on the chemical structure-based simcomp scores [ ] to represent drug-drug similarity as in [ ] . we also integrate an additional type of similarity by finding cosine similarities between one hot encoded representation of drug class based on mechanism of action of the drug. similarly, we used the genomic structurebased d * distance based on onf (oligonucleotide frequency) measure [ ] as similarities between the viruses. to obtain the second similarity measure, we calculate the cosine similarity between one hot encoded representations of symptomatic profiles of viruses i.e. symptoms caused by the virus. both the drug class information and the file encoding which symptoms are caused by the virus (metadata used to generate new similarity measures) are available as supplementary. hence, the drug similarity matrix (of size × ) and virus similarity matrix (of size × ) are fixed and encode the metadata available. a hybrid alternating proximal method for blind video restoration alignment-free oligonucleotide frequency dissimilarity measure improves prediction of hosts from metagenomically-derived viral sequences convex analysis and monotone operator theory in hilbert spaces anti-hiv and anti-hcv drugs are the putative inhibitors of rna-dependent-rna polymerase activity of nsp of the sars cov- (covid- ) minimal-uncertainty prediction of general drug-likeness based on bayesian neural networks probabilistic low-rank matrix completion from quantized measurements proximal alternating linearized minimization for nonconvex and nonsmooth problems distributed optimization and statistical learning via the alternating direction method of multipliers. foundations and trends in machine learning exact matrix completion via convex optimization the power of convex relaxation: near-optimal matrix completion predicting mirna-disease association based on inductive matrix completion stcdg: an efficient data gathering algorithm based on matrix completion for wireless sensor networks lorslim: low rank sparse linear methods for top-n recommendations a block coordinate variable metric forwardbackward algorithm predicting drug-target interactions using probabilistic matrix factorization a survey on the computational approaches to identify drug targets in the postgenomic era ewout van den berg, and mary wootters. -bit matrix completion. information and inference: a similarity-based machine learning methods for predicting drug-target interactions: a brief review the trial of chloroquine in the treatment of corona virus disease (covid- ) and its research progress in forensic toxicology remdesivir: a review of its discovery and development leading to emergency use authorization for treatment of covid- ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study quantitative structure-activity relationship and molecular docking revealed a potency of anti-hepatitis c virus drugs against human corona viruses a novel approach to quantized matrix completion using huber loss measure computational prediction of drug-target interactions using chemogenomic approaches: an empirical survey drug-target interaction prediction with graph regularized matrix factorization both boceprevir and gc efficaciously inhibit sars-cov- by targeting its main protease simcomp/subcomp: chemical structure search servers for network analyses insights from nanomedicine into chloroquine efficacy against covid- the challenges of deploying artificial intelligence models in a rapidly evolving pandemic triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial sofosbuvir as a potential alternative to treat the sars-cov- epidemic a systematic framework for drug repositioning from integrated omics and drug phenotype profiles using pathway-drug network gene expression prediction using low-rank matrix completion novel coronavirus treatment with ribavirin: groundwork for an evaluation concerning covid- walking the interactome for prioritization of candidate disease genes playing with duality: an overview of recent primal-dual approaches for solving large-scale optimization problems multitask matrix completion for learning protein interactions across diseases computational screening and identifying binding interaction of anti-viral and anti-malarial drugs: toward the potential cure for sars-cov- mcmda: matrix completion for mirna-disease association prediction weakly supervised deep matrix factorization for social image understanding testing covid- therapies to prevent progression of mild disease. the lancet infectious diseases neighborhood regularized logistic matrix factorization for drug-target interaction prediction simeprevir suppresses sars-cov- replication and synergizes with remdesivir artificial intelligence cooperation to support the global response to covid- computational drug repositioning using low-rank matrix approximation and randomized algorithms predicting drug-target interaction using deep matrix factorization computational prediction of drug-disease association based on graph-regularized one bit matrix completion. biorxiv drug-target interaction prediction using multi-graph regularized deep matrix factorization. biorxiv deep matrix completion on graphs: application in drug target interaction prediction drug-target interaction prediction using multi graph regularized nuclear norm minimization a computational approach to aid clinicians in selecting anti-viral drugs for covid- trials deepmc: deep matrix completion for imputation of single-cell rna-seq data inductive matrix completion for predicting gene-disease associations viral nucleic acids a nonconvex regularized approach for phase retrieval a preconditioned forward-backward approach with application to large-scale nonconvex spectral unmixing problems euclid in a taxicab: sparse blind deconvolution with smoothed l /l regularization computational evidence on repurposing the anti-influenza drugs baloxavir acid and baloxavir marboxil against covid- missed opportunities on emergency remdesivir use making deep neural networks right for the right scientific reasons by interacting with their explanations silico studies on therapeutic agents for covid- : drug repurposing approach. life sciences favipiravir strikes the sars-cov- at its achilles heel, the rna polymerase. biorxiv deep learning in medical image analysis bmcmda: a novel model for predicting human microbe-disease associations via binary matrix completion identification of common molecular subsequences deep learning for nlp (without magic) guaranteed matrix completion via non-convex factorization majorization-minimization algorithms in signal processing, communications, and machine learning an integrated drug repurposing strategy for the rapid identification of potential sars-cov- viral inhibitors a deep matrix factorization method for learning attribute representations arbidol: a potential antiviral drug for the treatment of sars-cov- by blocking the trimerization of viral spike glycoprotein? massive mimo channel estimation for millimeter wave systems via matrix completion drug-target interaction prediction via dual laplacian graph regularized matrix completion the anti-influenza virus drug, arbidol is an efficient inhibitor of sars-cov- in vitro global convergence of admm in nonconvex nonsmooth optimization low-rank matrix completion for inference of protein-protein interaction networks network-based global inference of human disease genes arbidol/ifn-α b therapy for patients with corona virus disease : a retrospective multicenter cohort study. microbes and infection effectiveness of arbidol for covid- prevention in health professionals drug repositioning based on bounded nuclear norm regularization structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir manifold regularized matrix factorization for drug-drug interaction prediction predicting drugdisease associations by using similarity constrained matrix factorization predicting drug-protein interaction using quasi-visual question answering system key: cord- -w mij c authors: mamidala, estari; davella, rakesh; gurrapu, swapna; shivakrishna, pujala title: in silico identification of clinically approved medicines against the main protease of sars-cov- , causative agent of covid- date: - - journal: nan doi: nan sha: doc_id: cord_uid: w mij c the covid- pandemic triggered by sars-cov- is a worldwide health disaster. main protease is an attractive drug target among coronaviruses, due to its vital role in processing the polyproteins that are translated from the viral rna. there is presently no exact drug or treatment for this diseases caused by sars-cov- . in the present study, we report the potential inhibitory activity of some fda approved drugs against sars-cov- main protease by molecular docking study to investigate their binding affinity in protease active site. docking studies revealed that drug oseltamivir (anti-h n drug), rifampin (anti-tb drug), maraviroc, etravirine, indinavir, rilpivirine (anti-hiv drugs) and atovaquone, quinidine, halofantrine, amodiaquine, tetracylcine, azithromycin, hydroxycholoroquine (anti-malarial drugs) among others binds in the active site of the protease with similar or higher affinity. however, the in-silico abilities of the drug molecules tested in this study, further needs to be validated by carrying out in vitro and in vivo studies. moreover, this study spreads the potential use of current drugs to be considered and used to comprise the fast expanding sars-cov- infection. a new occurred human coronavirus is informed in december in wuhan, china. , afterward, the covid- underway spreading across the world, putting the whole world on high attentive. the world health organization (who) surveillance draft in january stated that, any traveller to wuhan, hubei province in china, two weeks earlier the onset of the signs, is supposed to be a covid- case. , on april st , , a total of , , confirmed infections were reported worldwide, with , , deaths with a increasing mortality rate of > . %. the world health organization (who) strategy to comprise the distribution includes the decrease of human-to-human dispersal by preventing the interaction between individuals, accordingly avoiding transmission extension events and interactive critical risk evidence to all populations. in india, the first case of the covid- was reported in kerala on january . as of april, , there are , cases and deaths as reported by the ministry of health and family welfare, government of india. covid- is a member of betacoronaviruses similar to the severe acute respiratory syndrome human coronavirus (sars hcov) and the also the middle-east respiratory syndrome human coronavirus (mers hcov). main protease (mpro) is the one of the greatest-characterized drug targets among coronaviruses. beside with the papain-like protease(s), this main protease enzyme is vital for processing the polyproteins that are translated from the viral rna. this crucial function of main protease in virus duplication makes this enzyme a capable target for the expansion of inhibitors and possible treatment remedy for infection of novel coronavirus. to date, no precise therapeutic medicine or vaccine has been accepted for the management of human coronavirus. some clinical trials, it has been described that anti-hiv drugs and chloroquine phosphate, an anti-malarial drug, has a assured therapeutic effect on the covid- . in specific, chloroquine phosphate is suggested to treat covid- related pneumonia in larger inhabitants in the future. subsequently, other clinical trials suggested that hydroxychloroquine supplementary with azithromycin is very operative in the management of the covid- . this encouraged us to accomplish a systematic study on some clinically approved medicines using molecular docking and reinvestigate their biological efficacies and pharmacological properties. however, there is no organized study on the inhibition of the coronavirus by clinically approved drugs to the best of our knowledge. hence, the present study was aimed to molecular docking studies of clinically approved drugs against the main protease of sars-cov- . for molecular docking, auto-dock . software was used. the free energy (dg) binding of sars-cov- viral protease with the selected fda approved drugs was created by means of this molecular docking package. docking is a computational simulation method of a ligand binding to a receptor or enzyme and expects the favored orientation of binding of one molecule to the second to form a steady complex. to predict the attraction and activity of binding of the minor molecule to their enzyme targets by using scoring functions docking is used. therefore, docking shows significant role in the rational design of medicines. the sensitivity of docking calculations concerning the geometry of the involvement ligand displays that even minor changes in the ligand structure can lead to big changes in the geometries and scores of the subsequent docked poses. anti-viral and anti-malarial medications were recognized as potential coronavirus inhibitors from diverse literature evaluations. total fda approved drugs were selected for molecular docking with main covid- protease. among the approved drugs, drugs are anti-h n drugs, are anti-tb drugs, are anti-hiv- drugs and are anti-malarial drugs are selected from pubchem database. the three-dimensional structure files of the selected fda approved drugs were downloaded in sdf format from the pubchem were used for molecular docking. molecular d structures of selected fda approved drugs are shown in table . zanamivir (anti-h n ) isoniazid (anti-tb) meanwhile this protease has its crystal structure in a state that signifies the pharmacological target for the progress of new medicines to treatment diverse infectious diseases. the preparation of the target enzyme lu with the auto-dock tools software intricate addition of all h atoms to the enzyme, which is a step essential for accurate calculation of fractional atomic charges. the ligand and all water molecules were detached to make the structure for docking. gasteiger charges are considered for each atom of the protein in autodock . instead of kollman charges, which were used in the earlier versions of this package. for ligand conformational incisive, we take the 'lamarckian-genetic algorithm (lga)', which is a mixture of a genetic algorithm and a native search algorithm. this algorithm initially builds a population of entities, being a diverse casual conformation of the docked enzyme. each distinct protein is then mutated to attain a slightly diverse translation and alternation and the local search algorithm then achieves energy minimizations on a userspecified amount of the population of individuals. the entities with the low subsequent energy are moved to the succeeding generation and the procedure is then repetitive. this algorithm is called lamarckian while every novel group of entities is allowable to receive the local search variations of their parents. to get many docked structures, auto-dock was run numerous times, and used to examine the expected docking energy. rapid energy assessment was attained by pre-calculating nuclear affinity capacities for every atom in the compound molecule. the binding sites of the target enzyme for these molecules in the autogrid process were designated on the patterns of founded ligand-binding pockets. auto-dock tools deliver various approaches to examine the outcomes of docking-simulations such as, structural resemblance, and other limitations like inter-molecular energy, visualizing the binding site and its energy and inhibition constant. the energy of interaction of every atom in the ligand was met. for each ligand, best postures were made and scored using auto-dock . scoring purposes. computational approaches for drug discovery and development are proven to be effective and time efficient, as they are not based on difficult laborious works. the protein-ligand docking elucidates the mechanism of inhibition along with the specificity and efficiency of that ligand as an inhibitor. the association of drug candidate (ligand) to its target receptor is a fundamental binding reaction and the aim of the computer-aided drug discovery is to find small molecules having strong inhibitory or activating action against the biological targets. the strength of inhibition or activation is elucidated through binding affinity. oseltamivir and zanamivir, two fda approved drugs docked with sars-cov- main protease and obtained binding energy is − . kcal/mol and - . kcal/mol respectively (table- figure ). the results identified that oseltamivir is potential inhibitor of the sars-cov- main protease. earlier one study has reported that oseltamivir is a prodrug of oseltamivir carboxylate, a potent and selective inhibitor of the neuraminidase glycoprotein essential for replication of influenza a and b viruses. binding energy (kcal/mol) interaction of residues forming h bonds isoniazid, rifampin, ethambutol and pyrazinamide are clinically approved drugs were docked with binding energy - . , - . , - . and - . kcal/mol respectively against sars-cov- protease ( twenty-four fda approved anti-hiv drugs were docked with sars-cov- protease. among the twenty four drugs, four drugs maraviroc, etravirine, indinavir and rilpivirine were showed more potential inhibitors of sars-cov- main protease with binding affinity - . , - . , - . and - . kcal/mol respectively ( (figure ) . a joint research team of the shanghai institute of materia medica and shanghai tech university performed drug screening in silicon and an enzyme activity test, and they reported anti-hiv agents with potential antiviral activity against sars-cov- on january , . covid- protease ( lu ) with anti-hiv drugs, maraviroc (a), etravirine (b) , indinavir (c) and rilpivirine (d). (c) indinavir seventeen clinically approved anti-malarial drugs were docked with sars-cov- protease. out of seventeen, seven drugs were showed more potential inhibitors of sars-cov- main protease. figure ). as these residues play an important role during protein-ligand interaction, they may serve as a biomarker during the drug discovery process. this study focused on identification of potential inhibitors against sars-cov- from corona virus to control the viral replication. outcomes from the in silico molecular docking study maintained the great inhibitory efficacy of the one anti-h n drug (oseltamivir), one anti-tb drug (rifampin), four anti-hiv drugs (maraviroc, etravirine, indinavir, rilpivirine) and seven anti-malarial drugs (atovaquone, quinidine, halofantrine, amodiaquine, tetracylcine, azithromycin, hydroxycholoroquine) since they could launch h bonds with different amino acid residues that caused in an inhibition of sars-cov- protease activity with higher binding affinity ranging from (- . to - . kcal/mol). thus, the projected binding interactions of the dynamic molecules with the protease by the docking study evidently established their inhibitory strength towards catalytic response of the protease. this study provides the support of the repurposed drugs, which may be helpful for the treatment of novel coronavirus disease and can serve as potential drug candidates to curb the ongoing and ever enlarging covid- pandemic. since all the drugs used in this study are of known pharmacokinetics standards and approved by fda for human use they do not need to undergo specific long term clinical trials and therefore can fasten up the process of the therapeutics development. coronavirus fits to a set of viruses which can contaminate vertebrate animals and human. it has slaughtered thousands of individuals around the world with growth in mortality rate each single day. digestive, central nervous system, liver, respiratory systems of humans and animlas hampered by this virus infection. our study was focused on the fda approved drugs against the main protease in coronavirus, as a possible beneficial target for the management of coronavirus. lu (pdb id) is the major protease in covid- that has been relocated and structured in pdb newly and is available to everybody in the world (figure ). for the proteolytic maturation of virus, the protease is precise significant. protease has been studied as a possible target to avoid the extent of contamination by inhibiting viral polyprotein cleavage via blocking active sites of the protein. with this new finding of protease assembly in covid- , has providing an enormous chance to recognize possible drug candidates for the management of coronavirus. in this study, we have applied a computational approach of fda approved drugs in order to find a specific therapeutic possible agent against covid- . we have selected fda approved antiviral, anti-h n , anti-tb and anti-viral drugs and retrived directly from the pubchem (national library of medicine). molecular docking was accomplished with the drugs against covid- structure. molecular docking is a computational technique which aims to find non-covalent binding among protein (receptor) and a ligand/inhibitor (small molecule). for recognized binding site, the docking expects the method of interaction among a target protein and a ligand. binding energy proposes the attraction of an exact ligand and asset by which a ligand interacts with and binds to the pocket of a target protein. a drug with a lesser binding energy (Δg) is chosen as a probable drug candidate. in order to recognize the effect of active antiviral drugs on covid- , fda approved antiviral compounds were selected and performed molecular docking against covid- . docking results of sars-cov- protease with selected drugs out of the selected showed best docking score and were found to be best molecules at the target site of the protein. the present study concludes that, thirteen clinically approved drugs are identified as potent inhibitors against sars-cov- protease activity. these outcome afford a strong foundation for the use of these drugs are for corona management. moreover, the dynamic ligands inhibited the catalytic response of protease by blocking the residues of amino acids intricate in the processing and strand transmission reactions. the interactions by the structural model at the protease active site can afford a valuable guide for additional strategies for structurebased medicines and development of new operative inhibitors of sars-cov- protease. therefore, the effect of these inhibitors can be further revealed through in vitro and in vivo analysis in the termination of intracellular replication of corona virus, prior to the use as drugs in humans. clinical features of patients infected with novel coronavirus in wuhan, china. the lancet genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan. emerging microbes & infections a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster. the lancet epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet covid- ) situation report - government of india. covid- cases from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design. the febs journal targeting zoonotic viruses: structure-based inhibition of the c-like protease from bat coronavirus hku -the likely reservoir host to the human coronavirus that causes middle east respiratory syndrome (mers). bioorganic & medicinal chemistry hydroxychloroquine and chloroquine for treatment of sars-cov- (covid- ). inopen forum infectious diseases chloroquine and hydroxychloroquine as available weapons to fight covid- docking assisted design of novel -adamantanyl- -thiazolylimino- -arylidene- -thiazolidinones as potent nsaids virtual screening for hiv protease inhibitors: a comparison of autodock and vina critical assessment of the automated autodock as a new docking tool for virtual screening design of new cognition enhancers: from computer prediction to synthesis and biological evaluation influenza virus neuraminidase inhibitors. the lancet a joint research team of the shanghai institute of materia medica and shanghai tech university discover a group of old and traditional chinese medicines that may be efficacious in treating the novel form of pneumonia computational characterization of structural and functional roles of dreb a, dreb b and dreb c in enhancing cold tolerance in rice plant potential inhibitor of covid- main protease (mpro) from several medicinal plant compounds by molecular docking study in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel uk- , ), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr with broad-spectrum anti-human immunodeficiency virus type activity maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with ccr -tropic hiv- infection. the journal of infectious diseases maraviroc for previously treated patients with r hiv- infection all the authors are gratefully acknowledge to department of zoology, kakatiya university, warangal, telangana, india for providing bioinformatics lab to carry out this study. no funding to declare. conflict of interest declared none. key: cord- - q un rl authors: giri, allan; das, ankita; sarkar, ajoy k.; giri, ashok k. title: mutagenic, genotoxic and immunomodulatory effects of hydroxychloroquine and chloroquine: a review to evaluate its potential to use as a prophylactic drug against covid- date: - - journal: genes environ doi: . /s - - - sha: doc_id: cord_uid: q un rl hydroxychloroquine (hcq) and chloroquine (cq) are two anti-malarial drugs that are now being extensively used by front-line healthcare workers and other common people as a prophylactic drug against the corona virus disease − (covid- ) in india and as well as in many parts of the world. while only a few in vitro studies have pointed to some efficacy of these drugs as a prophylactic against covid- , to date, there are no clinical studies that have established any clinical efficacy of these drugs as a prophylactic. these drugs are commonly used for the treatment of rheumatoid arthritis (ra) and systemic lupus erythematosus (sle) because of its immunomodulatory effects. previously, we have evaluated the genetic toxicology of different drugs and chemicals including antimalarial drug cq both in vitro and in vivo. thus, we recognize the need to critically review the mutagenic, genotoxic, and immunomodulatory effects of these drugs, to find out whether it is safe to use as a prophylactic drug against covid- . existing literature suggests that cq can induce mutagenic and genotoxic effects in multiple test systems and both the drugs have immunomodulatory effects. there was no data available to evaluate the mutagenicity and genotoxicity for hcq. however, during metabolism about % of both the drugs remain unchanged and about % of the drugs are metabolized into two metabolites, desethylchloroquine and bisdesethylchloroquine by the action of the cytochrome p (cyp) enzymes in the liver. both hcq and cq are immunomodulatory drugs and have the potential to suppress normal immune system activation. in this review, we have elucidated the mechanism of immunomodulation by both hcq and cq and highlighted the mutagenic and genotoxic effects from the available literature. this article is written with the sole objective that the reader will be able to recognize the adverse effects of these drugs when consumed by healthy individuals as a prophylactic. current literature indicates that healthy individuals should refrain from the use of these drugs until further investigation. the novel severe acute respiratory syndrome corona virus (covid- ) pandemic has now become a nightmare throughout the world. this pandemic has caused serious health crisis not only among the poor nations but also across the world's advanced countries. researches all across the globe are trying to find an effective drug that would show promising results to prevent or to treat and control the covid- . recently, scientists have pointed out that the novel covid- was transmissible in aerosol [ ] . thus, it puts the healthcare workers at risk who works in close proximity with covid- patients. this demanded the need for a prophylactic drug against covid- amongst healthcare workers. hydroxychloroquine (hcq) and chloroquine (cq) are two antimalarial drugs that remain the universally accepted drugs for the treatment of rheumatoid arthritis and systemic lupus erythematosus [ ] [ ] [ ] [ ] . these two drugs have shown some results in inhibiting the novel covid- in vitro [ , ] . a recent study demonstrated that certain cell types when treated with hcq or cq and then exposed to the novel covid- strain, presented antiviral activity and that hcq was more potent than cq [ ] . on the other hand, another in vitro study found out that cq was potent than hcq at all four different multiplicities of infection to act as antiviral when exposed to covid- post-incubation with these drugs [ ] . additionally, cq was able to act as anti-viral both pre and post-infection against the covid- in vitro [ ] . these findings may have led to the proposal and optimistic use of hcq and cq as prophylactics. yet historically, we have seen that in vitro studies don't always translate in vivo or human subjects. for instance, despite the strong evidence of cq as a prophylactic against influenza a and b in vitro, cq was not effective to prevent either influenza a or b in the human subjects [ ] . rather, dizziness, nausea, and diarrhea were more common in the cq group compared to the control (placebo) group. another study showed that ebola virus replication was successfully inhibited in vitro by cq, however, it failed in guinea pig models in vivo [ ] . yet another study showed cq enhanced chikungunya virus replication in vivo when in fact cq had been shown to have an effective inhibitory effect in vitro [ ] . thus, to date, with the lack of any controlled clinical trials, the clinical effectiveness of these drugs as prophylactics against covid- in vivo remains unanswered. like any other drugs, these drugs also comes with certain risks. so, we mustn't overlook the toxicological risks while making a rational decision of using these drugs as prophylactics. previously, we have extensively reviewed and evaluated the genetic toxicology of antimalarial, analgesics, antipyretic drugs including cq [ ] [ ] [ ] . cq and hcq are both water-soluble drugs that are absorbed rapidly in the gut and have a long elimination half-life in the plasma of and h respectively [ ] . multiple authors, including us, have reported in vitro and in vivo evidence of cq-induced genotoxicity in the mammalian system. these drugs also possess immunomodulatory roles that have the potential to suppress the activation of the immune system in healthy individuals [ ] [ ] [ ] . considering the current situation, there is an urgent need for clinical studies to evaluate the clinical efficacy of hcq and cq as a prophylactic drug against covid- . we have not included the long-term side effects of these drugs since it is unlikely that prophylactic use of these drugs would be for a long time. here in this review, we mainly aim to critically review and discuss the mutagenic, genotoxic, and immunomodulatory aspects of hcq and cq using the available literature. mutagenic and genotoxic effect table summarizes the mutagenic, genotoxic and carcinogenic effects of cq in multiple test systems. it is interesting to note that there is almost no report on the mutagenic and genotoxic effects of hcq both in vitro and in vivo. however, both hcq and cq have a very similar, flat aromatic core structure with a basic side chain. the only difference is the presence of an additional hydroxyl (−oh) group in hcq. during metabolism, about % of both the drugs remain unchanged and about % of both the drugs are metabolized into two common metabolites desethylchloroquine and bisdesethylchloroquine by the action of the cytochrome p (cyp) enzymes in the liver [ ] . despite cq being recognized as more toxic than hcq, the tissue and plasma distribution of these two drugs were reported to be nearly identical when administered in equivalent dosage in humans [ ] . figure shows the comparison of structures and metabolism of hcq and cq as described by schrezenmeier and dorner [ ] . hcq produces two first-stage metabolites instead of one. one being desthylhyoxychloroquine and the other desthylchloroquine. desthylchloroquine is also the firststage metabolite product of chloroquine. both the firststage metabolites are further metabolized to a common product, bisdesthylchloroquine [ ] . while many authors have reported cq to induce mutagenic effects in bacterial systems [ - , , - , ] , few authors had found weak or no mutagenic associations in certain bacterial strains [ , , , , [ ] [ ] [ ] . positive mutagenic effects (either weak positive or positive) reported by several authors showed that cq is capable of inducing mutation in the salmonella strains ta , ta a, ta and ta , which are used to detects the frameshift mutations. during the mutagenicity assay, most studies did not find any significant differences in the revertant numbers either with or without metabolic activation [ , [ ] [ ] [ ] . cq also can interact with dna and produce an intercalated complex that may induced frameshift mutation by shifting the reading frame [ , ] . this also indicates it's dna damage and inhibition of dna repair potentials reported by several authors [ , , , , ] . cq is further reported to induce sex-linked recessive lethal mutation in drosophila melanogaster [ ] . these results indicate the mutagenic potentials of cq in bacterial systems. in a broad genotoxic review on several antimalarial drugs, cumulative pieces of evidence pointed out that cq is also a genotoxic drug [ ] . our study on cq has demonstrated genotoxic effect as measured by chromosomal aberrations (ca), sister chromatid exchange (sce), and micronuclei (mn) formations in vivo in mice [ ] . these results are in agreement with several other authors who have reported cq to be a genotoxic drug in both in vitro and in vivo systems [ - , , ] . cq has also been reported to induce oxidative stress in animal models [ ] . for instance, when cq administered intraperitoneally in rats, it induced dna breaks in the kidney within to weeks and in the liver within to weeks [ ] . chromosomal aberrations (ca) have also been long considered to be a predictor for cancer. rossner et al., [ ] reported a strong association between increased frequencies of ca in cells and an increased risk for cancer using a cohort of , subjects. ca along with other genotoxic effects like sce and mn as reported here, suggests that long-term use of cq can induce significant chromosomal damages which may lead to an increased risk of cancer in humans. cq is not considered carcinogenic due to inadequate evidence pointing to carcinogenicity in humans. yet, a well-controlled study by el-mofty et al., [ ] in egyptian toad showed that separately cq and primaquine can induced tumor formation in and % of the toads respectively. they further showed that cq and primaquine when used together the incidence of tumor rose to . % [ ] . this type of co-tumorigenic effect of cq was also observed in another study by reyes et al., [ ] where cq promoted the carcinogenic effect of a drug called ethynitrosourea on ependymal cells of rodents in vivo. the only report of cq induced aplasis and leukemia was observed in a patient treated with long term cq therapy [ ] . brambilla and martelli, [ ] showed that n-nitroso compounds, which are capable of inducing genotoxic effects and tumor formation in animal models, can be formed in the gastric environment when cq is used with nitrite drugs. so, the genotoxiccarcinogenic effect may be induced when nitrite drugs are taken along with amine drugs like cq and hcq. results presented in the table indicate that cq is mutagenic and genotoxic drug in both bacterial systems, and in vitro and in vivo on mammalian systems. table summarizes the available reports on the immunomodulatory aspects of hcq and cq in multiple test systems. both hcq and cq has been reported to inhibit the activation of the immune system in many ways. lysosomotropic drugs (like hcq and cq) can accumulate inside lysosomes and being basic they can increase the ph inside the lysosomes and prevent its normal functions [ , ] . these drugs can also cause lysosomal membrane destabilization and thus the release of lysosomal contents and enzymes inside the cells [ ] . lysosomes have an essential role in the exogenous (lysosomal) pathways of antigen presentation and therefore proper lysosome functions are essential for mhc class ii antigen processing and presentation. the intervention of hcq and cq in the exogenous pathway of antigen presentation has been presented in fig. [ ] . autophagy is an implied concept in immunity development. besides the degradation and recycling of endogenous substrates, the process of autophagy is a key mechanism used by cells to tackle intracellular pathogens [ ] . hcq and cq both can potentially inhibit the normal autophagy processes. autophagosomes require fusion with the lysosomes to start the process of degradation. the increased ph of lysosomes, due to hcq and cq intervention, inhibits the maturation of the autolysosome. the inhibition of autophagy has been further linked to the induction of apoptosis of memory t-cells, which is the basis of the mechanism of immunomodulation by these drugs in several autoimmune diseases [ , ] . mechanism of the inhibition of autophagy by hcq and cq which triggers apoptosis has been presented in fig. [ ] . hcq can further block endosomal activation of nadph oxidase (nox ) that normally generates the reactive oxygen species and involved in the proinflammatory response of the immune system [ ] . with a decreased activity of nadph oxidase, cells can phagocyte pathogens but can't degrade them inside the phagocytic vesicle. hcq can also function as an immunosuppressant by blocking steps in the t-cell activation pathway. hcq has been shown to inhibit transcription factor nfat (nuclear factor of activated t-cells) upon t-cell activation in vivo and block expression of co-stimulatory ligand cd i.e. cd l, which initiates t-cell dependent bcell proliferation and antibody formation [ ] . the probable mechanism of the interference in the t-cell activation by hcq has been presented in fig. [ , ] . hcq intervention can further down-regulate the cd marker in healthy controls by inhibiting calcium mobilization and dephosphorylation of nfat [ ] . cd functions as a costimulatory molecule for t-cell activation and proliferation. the same study showed that b-cell antigen receptor calcium signaling was also reduced by hcq intervention [ ] . patients on antimalarial drugs like hcq and cq have lower levels of il- , soluble cd , and il- receptors which is beneficial for those suffering from autoimmune diseases like sle and ra [ ] . studies have shown that cq, at doses that are expected in the serum of treated patients, was able to interfere with mitogenic-response of monocytes and this diminished mitogenic response was determined to be irreversible [ ] . furthermore, cq inhibited the generation of immunoglobulin-secreting cells by preventing the capacity of monocytes to secrete factors like il- [ ] . normal secretion of tnf-α, il- b, and il- by monocytes or macrophages was also inhibited by cq [ ] . hcq and cq can potentially inhibit toll-like receptor (tlr) signaling of tlr and tlr in antigen presenting cells (apcs) including dendritic cells, macrophages and b-cells. normally, upon activation of by nucleic acids, endolysosomal tlr and tlr are cleaved which in turn activates myd and triggers an innate immune response in the downstream cascade. this proteolytic cleavage is inhibited by the changes in the endosomal ph as a result of hcq and cq interference [ ] . the mechanism of the inhibition of endosomal tlr and tlr by hcq and cq has been presented in fig. [ ] . hcq and cq have further been shown to directly bind to nucleic acid, inhibiting tlr-nucleic acid interaction and preventing tlr activation [ ] , and cq has been shown to inhibit rna-mediated tlr activation [ ] . hcq treatment in vivo caused a significant reduction of the production of inf-α and tnf-α by the plasmacytoid dendritic cells by suppressing the activation of tlr and tlr [ ] . innate tlr signaling leads to the production of cytokines such as il- , tnf-α, and il- that eventually triggers the adaptive immune response. at clinical concentration, hcq can efficiently block tlr ligation and have an inhibitory effect on classswitched memory b-cells [ ] . fig. intervention of hcq and cq in the exogenous pathway of antigen presentation: exogenous antigens that are taken up either by endocytosis or phagocytosis requires fusion with lysosomes for the process of degradation. finally, the degraded antigen fragments are loaded onto the mhc class ii molecules for antigen presentation. lysosomotropic drugs like hcq and cq accumulate inside the lysosome and these drugs potentially increase the ph inside and disrupt lysosomal functions the cgas-sting is another pathway that is involved in the type i interferon response of innate immunity. cyclic gmp-amp synthase (cgas) is a nucleotidyltransferase that is activated to cgamp ( ′, ′ -cyclic gmp-amp dinucleotide) when dsdna, usually from a viral or bacterial origin, binds to it. cgmap then activates an endoplasmic reticulum membrane-associated protein known as the sting (stimulator of interferon genes). activation of sting leads to the activation of the transcription factor irf and nf-kb, which then can migrate to the nucleus to activate the type i ifns and cytokines [ ] . evidence suggests one-way hcq and cq can achieve the immunomodulatory effect is because of its ability to suppress the activation of this pathway by inhibiting ligand binding [ ] . the mechanism of the inhibition of cgas-sting pathway by hcq and cq has been presented in fig. [ ] . given all the above mechanisms, it is now clear that these drugs can effectively modulate cellular signaling. sometimes, this can be helpful but only when used by patients infected with covid- . for instance, covid- patients have usually higher levels of inflammatory cytokines which ultimately result in collateral damage to the host tissues. the use of hcq and cq has been reported to reduce the overproduction of il- b, il- , and granulocyte-colony stimulating factor (gm-csf). the reduction in endosomal acidification as a result of hcq and cq accumulation is believed to halt or reduce the disruption of viral particles and thus the release of infectious nucleic acid. the ace receptors of the lungs are required for sars-cov entry into the host cells. glycosylation of ace receptors is required for the translocation of ace receptors to the cell membrane. hcq has fig. inhibition of autophagy by hcq and cq triggers apoptosis: autophagy is often utilized by cells as a survival mechanism to prevent apoptosis. normal autophagy process includes the fusion of autophagosomes with the lysosome, followed by the degradation and recycling of the internal components like damaged organelles. accumulation of hcq and cq increases the ph inside the lysosomes and prevents the normal autophagy process. this prevention of autophagy triggers apoptosis in memory t-cells and this is one of the fundamental mechanisms of immunomodulation by hcq and cq. also, not shown in the diagram, inhibition of autophagy can also reduce antigen presentation via mhc class ii molecules fig. hcq interference in the t-cell activation pathway and transcription of cd : when t-cell receptor (tcr) is stimulated by antigen via mhc, a series of events leads to the activation of the phospholipase c, which then generates inositol triphosphate (ip ). ip induces the release of calcium from the endoplasmic reticulum (er). calcium acts as a secondary messenger to activate calcium-release-activated calcium channel (crac) for a steady influx of extracellular calcium. intracellular calcium binds to calmodulin and activates the phosphatase calcineurin (not shown in the diagram). calcineurin dephosphorylates and activates transcription factor nfatc . nfatc migrates the nucleus and triggers the transcription of nfatc . nfatc mrna is exported outside the nucleus where de novo synthesis of transcription factor nfatc occurs. nfatc then migrates back into the nucleus to triggers the transcription of cd . hcq can potentially interfere with intracellular calcium signaling and prevent dephosphorylation and activation of the transcription factor nfat. this is one of the mechanisms by which hcq interfere in t-cell activation and cd transcription also been shown to reduce this glycosylation step [ ] . these studies have together motivated healthcare workers to use these drugs as a prophylactic, postexposure prophylactic, and as a curative drug. unfortunately, these results were only successful in vitro. as we will discuss later, large clinical trials have found no evidence of the benefit of using these drugs. it is important to remember the metabolism of these drugs in vivo. both hcq and cq are metabolized in the liver by an enzyme known as cytochrome p (cyp) and the gene expression of this enzyme varies between different individuals as a result of the difference in nucleotide polymorphisms [ ] . furthermore, these polymorphisms have been associated with the formation of unstable enzymes and thus a decreased in its activity [ ] . some ethnicities have a complete absence of certain functional cyp enzymes like the cyp d , which is one of the important cyp enzymes that is actively involved in the metabolism of these drugs [ ] . an individual's p polymorphisms should be taken into account when considering to prescribe hcq since there is a clear association between different polymorphisms in cyp d and the blood concentration of hcq in sle patients [ ] . these polymorphisms have also been linked to the toxic accumulation of these drugs in the blood of the patients treated with hcq and cq [ ] . to date, there is still no evidence that points to any benefit of using these drugs against the covid- as a prophylactic. moreover, the potentials of these drugs as fig. endosomal tlr and inhibition by hcq and cq: mammalian toll-like receptors (tlr) and initiate immune response when it encounters microbial nucleic acids (only shown here is a viral particle). the ectodomain of tlr and are cleaved in the endolysosome (not shown) which then recruits myd , followed by the activation of irak, traf , and nf-kb. nf-kb then migrates to the nucleus and triggers the transcription of inflammatory cytokines. both hcq and cq can increase the ph of the endolysosome and interfere with the tlr and tlr cleavage and processing. furthermore, hcq and cq can directly bind to the microbial nucleic acids and prevent tlr-ligand interaction a post-exposure prophylactic or curative drug has also been called into question. recently, the food and drug administration (fda) of the united states (us) has revoked their emergency use authorization (eua) for emergency use of these drugs [ ] , and the world health organization (who) [ ] , along with national institute of health (nih) [ ] in the us had also stopped conducting further clinical studies because of sufficient evidence that these drugs provided no benefit. initially, a few in vitro evidence and small studies suggested that these medications might be helpful, however, large scale studies have found no such significance. the first clinical evidence of a positive result stems from a small study in china where subjects treated with cq were found to have a superior benefit over the control group suffering from covid- pneumonia [ ] . another small study in france with only subjects, who received hcq, showed a significant reduction in the viral load compared to the control group [ ] . the time to clinical recovery was also shown to be reduced in those under hcq treatment in a small randomized trial of just covid- patients [ ] . clearly, the small sample size was a major drawback in these studies. however, larger clinical randomized controlled trial has now been conducted and results have provided us with a different outcome. for instance, a recent study in brazil fig. cgas-sting pathway inhibition by hcq and cq: dna sensor cgas (cyclic gmp-amp synthase) can recognize dna of viral/bacterial origin and synthesize a dinucleotide molecule, cgamp ( ′, ′-cyclic gmp-amp dinucleotide). newly synthesized cgamp can activate an endoplasmic reticulum (er)-associated protein sting (stimulator of interferon genes). sting potentially activates the transcription factors irf and nf-kb, which can then migrate to the nucleus and lead to the transcription of type i ifns and cytokines. although hcq/cq doesn't directly bind to the active site of cgas, these molecules can occupy the minor grooves of dna molecule and prevent association with cgas with confirmed covid- patients, hcq treatment had no significant benefit over no treatment group [ ] . furthermore, chloroquine at high doses ( mg, twice daily) for consecutive days was associated with higher lethality and is now not recommended for critically ill patients [ ] . a large randomized clinical study investigating the post-exposure prophylactic role of hcq found that a higher dose of hcq did not prevent infection when treatment was initiated within days after exposure [ ] . another study showed us that in covid- patients those taking hcq only or hcq plus azithromycin had a death rate of . and . % respectively when compared to . % in the no-treatment group [ ] . most pathogenic microorganisms that infect humans are tackled satisfactorily by the innate and adaptive immune system of our body. healthy humans have an extraordinary capability to fight off infections caused by pathogenic microorganisms. the activation of innate immunity initiates the first line of defense until the more specific adaptive immunity develops. existing literature suggests that hcq and cq can potentially interfere with both innate and adaptive immune responses in multiple ways. here in this review, we have highlighted the known pathways where hcq and cq can intervene to achieve its immunomodulatory effects and also provided systematic diagrams for a better understanding of the affected pathways. furthermore, cq has been recognized to induce genomic instability by inducing mutagenic, genotoxic, and dna damages both in vivo and in vitro systems. most authors, including us, found weak to moderately strong mutagenic effects in different salmonella strains routinely used for mutagenicity screening of drugs and chemicals. moreover, almost all authors who worked on genotoxicity assays have reported positive genotoxic effects of cq in multiple test systems. this indicates that cq is a mutagenic and genotoxic drug. however, with a lack of sufficient studies on the genetic toxicology of hcq both in vitro and in vivo, the mutagenic and genotoxic effects of hcq remains inconclusive. current evidence cumulatively demonstrates that both hcq and cq are not effective against the covid- infection either as post-exposure prophylaxis or as a curative drug. no studies on the prophylactic role of these drugs have been evaluated to date. the world is going through tremendous turmoil because of the covid- pandemic. thus, we do recognize the importance of optimism and the implementation of any advancement in science during this emergency. thus, the use of these drugs post-infection might be useful, but this discussion is beyond the scope of this review. here, we are mainly concerned with the use of these drugs by healthy individuals as a prophylactic without any evidence. so, without any clinical or in vivo evidence, current literature suggests that healthy individuals should refrain from the use of these drugs as prophylactics until further investigation. aerosol and surface stability of sars-cov- as compared with sars-cov- hydroxychloroquine-induced toxic myopathy causing respiratory failure hydroxychloroquine cardiotoxicity presenting as a rapidly evolving biventricular cardiomyopathy: key diagnostic features and literature review mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model paradoxical effect of chloroquine treatment in enhancing chikungunya virus infection genetic toxicology of paracetamol and aspirin -a review comparative genotoxicity of six salicylic acid derivatives in bone marrow cells of mice comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine challenges and cares to promote rational use of chloroquine and hydroxychloroquie in the management of corona virus disease (covid- ) pandemic: a timely review modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory nonsmall-cell lung cancer cells hydroxychloroquine inhibits cd expression in cd + t lymphocytes of systemic lupus erythematosus through nfat, but not stat , signaling hydroxychloroquine inhibits calcium signals in t cells: a new mechanism to explain its immunomodulatory properties inhibition by chloroquine of uv repair in e. coli b mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test feasibility of testing dna repair inhibitors for mutagenicity by a simple method mutagenicity of antiamebic and anthelmintic drugs in the salmonella typhimurium microsomal test system mutagenicity examination of several non-steroidal anti-inflammatory drugs in bacterial systems the role of chloroquine supplementation in liquid holding recovery and ultraviolet lethality of escherichia coli strains the comparative responses of salmonella typhimurium ta and ta a to a range of reference mutagens and novel compounds testing of chloroquine and quinacnne for mutagenicity in drosophila melanogaster comparison of the ames assay and the induction of sister chromatid exchanges: results with ten pharmaceuticals and five selected agents aspects of chloroquine mutagenicity frameshift mutagenesis by chloroquine in escherichia coli and salmonella typhimurium cortinas de nava c. influence of the uvr repair system on the mutagenicity of antiparasitic drugs genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, aq- study of the evaluation of mutagenic effects of antimalarial drug chloroquine in ames salmonella assay choloroquine inhibition of repair of dna damage induced in mammalian cells by methyl methanesulfonate cytogenetic effects of chloroquine in human lymphocyte cultures simultaneous detection of chromosomal aberrations and sister-chromatid exchanges: experience with dna intercalating agents putative identification of functional interactions between dna intercalating agents and topoisomerase ii using the v in vitro micronucleus assay anti-malarial chloroquine stimulate p -apoptotic pathway in rat hepatocytes genotoxicity of chloroquine in rat liver cells: protective role of free radical scavengers action of chloroquine phosphate in rheumatoid arthritis. ii. chromosome damaging effect aplasia and leukaemia following chloroquine therapy the antimalarials quinacrine and chloroquine potentiate the transplacental carcinogenic effect of ethylnitrosourea on ependymal cells effects of low dose radiation and vitamin c treatment on chloroquine-induced genotoxicity in mice the carcinogenicity of some antimalarial drugs using the egyptian toad bufo regularisas a biological test animal effects of primaquine and chloroquine on oxidative stress parameters in rats animal toxicity and pharmacokinetics of hydroxychloroquine sulfate mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology spectrophotometer studies of the interaction of chloroquine with deoxyribonucleic acid variation of the supercoils in closed circular dna by binding of antibiotics and drugs evidence for molecular models involving interaction carcinogenicity of antibacterial, antiviral, antimalarial and antifungal drugs effects of chloroquine treatment on antioxidant enzymes in rat liver and kidney chromosomal aberrations in lymphocytes of healthy subjects and risk of cancer genotoxic and carcinogenic risk to human of drugnitrite interaction product immunosuppressive potential of antimalarials the relevance of antimalarial therapy with regard to thrombosis, hypercholesterolemia and cytokines in sle immune stimulation mediated by autoantigen binding sites within small nuclear rnas involves toll-like receptors and chloroquine inhibits production of tnf-alpha, il-beta and il- from lipopolysaccharide-stimulated human monocytes/macrophages by different modes the ectodomain of toll-like receptor is cleaved to generate a functional receptor hydroxychloroquine preferentially induces apoptosis of cd ro+ effector t cells by inhibiting autophagy: a possible mechanism for therapeutic modulation of t cells hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase hydroxychloroquine efficiently suppresses inflammatory responses of human class-switched memory b cells via toll-like receptor inhibition cutting edge: antimalarial drugs inhibit ifn-b production through blockade of cyclic gmp-amp synthase-dna interaction modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory nonsmall-cell lung cancer cells mechanism of action of hydroxychloroquine as an antirheumatic drug autophagy in immunity and inflammation inhibition of macroautophagy triggers apoptosis dna sensing by the cgas-sting pathway in health and disease smooth or risky revisit of an old malaria drug for covid- ? genetic polymorphism in cytochrome p d (cyp d ): population distribution of cyp d activity association of polymorphisms of cytochrome p d with blood hydroxychloroquine levels in patients with systemic lupus erythematosus guimarães l sp. and hutz m h. the effect of snps in cyp in chloroquine/ primaquine plasmodium vivax malaria treatment food & drug administration. letter revoking eua for chloroquine phosphate and hydroxychloroquine sulfate q&a: hydroxychloroquine and covid- national institutes of health. nih halts clinical trial of hydroxychloroquine. study shows treatment does no harm, but provides no benefit breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid- effect of high vs lowdoses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection a randomized clinical trial a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are grateful to the indian national science academy to provide the insa-senior scientist position to akg. authors are also grateful to the u.s. federal government for providing financial support to ag for graduate studies in kansas city university of medicine and biosciences and to the council of scientific and industrial research, new delhi for providing csir-net (jrf) to ad. our deep gratitude also to dr. pritha bhattacharjee, department of environmental sciences, university of calcutta for allowing her student ms. ankita das to work in this review and also for her valuable suggestions. all the figures in this review have been created with biorender.com there is no funding for this work.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicabe. the authors declares that he have no competing interests. key: cord- - n gylj authors: villoutreix, bruno o.; beaune, philippe h.; tamouza, ryad; krishnamoorthy, rajogapal; leboyer, marion title: prevention of covid- by drug repurposing: rationale from drugs prescribed for mental disorders date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: n gylj nan at present, no treatments or vaccines are available to treat or prevent the coronavirus sars-cov- infection. as drug development is time-consuming and costly, there is an urgent need to find a rationale for repurposing clinically approved compounds that could rapidly enter clinical trials. to guide the selection of molecules for the prevention of sars-cov- infection, we believe that there is great potential in exploring drugs prescribed for mental disorders. indeed, these past few weeks, an intriguing relationship appeared between sars-cov- infection and psychiatric disorders. patients with mental disorders were intuitively thought to be at increased risk of becoming infected: non-compliance to protective measures, delayed access to health services due to social discrimination, confined conditions in psychiatric units favoring dissemination of infections, and a high prevalence of high risk co-morbidities (diabetes, cardiovascular disorders, obesity). alarmed by these high risk situations, psychiatric departments in france created specialized covid- units dedicated to psychiatric patients. very much to our surprise, these units remained nearly empty during the lock-down period since only a small proportion of psychiatric patients were found to have covid- , suggesting that these patients, under treatment surveillance, may be at reduced risk of sars-cov- infection. this raised a compelling question: do the pharmacological treatment of these patients play a role in the observed protective effect? in order to rationalize our observations, we first identified the most commonly used drugs for in and out-patients (i.e., consumption of all the drugs in the psychiatric department of henri mondor hospital, creteil, france from to april ). we ended up with a list of drugs (table ) . we then mined the literature to find information about possible, at least in vitro, antiviral activity of these compounds. we also compared these drugs with published molecules known to have in vitro antiviral activities [ , ] using various chemoinformatics strategies (e.g., computation of molecular descriptors and compounds clustering carried out on j o u r n a l p r e -p r o o f about molecules with in vitro antiviral activities on various viruses including sars-cov- ). the reasoning here is that if a molecule x belonging to our list of most commonly prescribed drugs is not documented to have in vitro antiviral activity but can be chemically grouped (e.g., presence of identical substructures) with a molecule with known in vitro antiviral activity, it is highly likely that molecule x will also have antiviral activity (i.e., the so-called similarity principle in medicinal chemistry). among the most commonly prescribed psychotropic drugs, ten have documented in vitro antiviral activities, while four of the eight remaining compounds were found structurally very similar to compounds with known in vitro antiviral activity (table ) using various clustering approaches. for example, no publications about antiviral activity were found for alimemazine and cyamemazine but these molecules are very similar to promethazine which acts on ebola and mers [ ]) and chlorpromazine which acts on mers and sars [ ] . we also observed that of the most prescribed psychotropic drugs are cationic amphiphilic drugs (cads) which are known to perturb intracellular trafficking (table ). cads are characterized by hydrophobic-aromatic ring systems and a side chain that carries one (or more) ionizable amine functional group. to define the cad nature of our compounds, we computed pka and log p values as reported earlier [ ] with the chemaxon chemistry toolkit (https://chemaxon.com/). further, cads often induce phospholipidosis (drug-induced phospholipidosis or pld) in vitro [ ] and hundreds of drugs have already been tested in vitro or the property can be predicted in silico [ ] . the four molecules that are not cads or plp inducers are lithium (not an organic molecule), diazepam, lorazepam and valproate. in addition, nicotine which is largely prescribed to psychiatric patients to help them quit smoking, could be considered as a partial cad and is predicted to be a pld. this compound may not modulate intracellular trafficking but could act via binding to specific receptors such as the acetylcholine receptor (nachr) and possibly regulate the angiotensin converting enzyme (ace ) receptor [ ] . drugs against sars-cov- could operate at different stages of the virus lifecycle. yet, to protect the population, acting on the virus entry phases through drug repurposing represent an attractive solution. different strategies can be envisioned, from specific inhibition of some proteases and receptors, to mechanisms such as endocytosis. overall, our analysis suggests that the most commonly prescribed psychotropic drugs, including some antihistamine agents used as anxiolytics, possess in vitro antiviral activity ( table ). the vast majority of these drugs are cad and/or pld compounds. if we take the example of the highly debated anti-malarial chloroquine and hydroxychloroquine that have in vitro antiviral activity, these compounds are also cad and pld compounds (they display two ionizable amine groups while most often, psychotropic drugs contain only one ionizable amine group). these two anti-malarial drugs are lysosomotropic agent that accumulates in acidic organelles such as endosomes and lysosomes and neutralizes their ph thereby inhibiting the activity of some proteases such as cathepsins required for effective viral infection (nb: they also have other functions as most medicinal drugs). another example is chlorpromazine, related to the commonly prescribed alimemazine or cyamemazine (table ) . chlorpromazine is a cad and pld agent and is known to inhibit cell-cell fusion and disrupt clathrin-mediated endocytosis [ ] . in addition, several of the above mentioned commonly used psychotropic drugs may also act by interacting with some specific receptors. for instance, a number of psychoactive drugs (antipsychotics, antidepressants, anticonvulsants) and psychostimulants, all belonging to a variety of structural, pharmacological and therapeutic classes, can bind to sigma- receptors, and induce numerous molecular events [ ] . in summary, we propose that some of the drugs commonly prescribed to psychiatric patients could protect them from sars-cov- infection via the modulation of the endo-lysosomal pathway, membrane fusion and yet to be characterized interactions with specific receptors (e.g., nachr, ace and sigma receptors, fig. ). based upon the above analysis, we suggest that one of these cad molecules or a combination could be used as preventive treatment against sars-cov- infection, especially drugs with reduced adverse effects (e.g., low dosage nicotine patch associated with an antihistamine agent). further studies are currently ongoing in our groups to identify and test the above mentioned molecules in vitro and in clinical settings. this very simplified cartoon highlights sars-cov- cell entry. angiotensin-converting enzyme receptor (ace ), acetylcholine receptor (nachr), transmembrane surface serine protease (tmprss ). some graphical elements were taken from the free medical art collection distributed by the servier laboratory. j o u r n a l p r e -p r o o f antiviral activity of cationic amphiphilic drugs deja vu: stimulating open drug discovery for sars-cov- a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection identification of drugs inducing phospholipidosis by novel in vitro data emerging mechanisms of drug-induced phospholipidosis how does the quality of phospholipidosis data influence the predictivity of structural alerts? ) a nicotinic hypothesis for covid- with preventive and therapeutic implications clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace with the cytoplasmic tail deleted the molecular function of sigma receptors: past, present, and future we thank inserm and the foundation fondamental for supports. key: cord- -arnwk authors: gallimore, w. title: chapter marine metabolites oceans of opportunity date: - - journal: pharmacognosy doi: . /b - - - - . - sha: doc_id: cord_uid: arnwk abstract the marine environment provides an array of compounds often with unique molecular architectures boasting an equally wide array of bioactivities including anticancer, antiinflammatory, and antimicrobial activity. typically without the benefit of folklore therapeutic knowledge, marine organisms are collected, extracted, and fractionated to afford compounds that undergo evaluation with in vivo and in vitro assays en route to clinical applications. the pharmaceutical industry has benefited from research into marine metabolites with the development of marine-derived drugs including cytarabine, vidarabine, and ziconotide along with the more recently developed formulation carragelose, an antiviral spray. cosmetic applications incorporating marine extracts include abyssine and refirmar. research with macroinvertebrates, macroalgae, and microorganisms continue in the hope that drugs of the future will be culled from the oceans of the world. while obtaining a consistent and adequate supply of the bioactive compounds remains a challenge to be overcome, synthetic methods are being employed along with the application of biotechnological techniques to ensure that the drugs, when developed, will be in sufficient quantities for distribution to those who are in need. to gain an understanding of the importance of marine natural products chemistry in drug development g to be able to map the process involved in drug development from marine natural products g to gain an appreciation of the range of biological activities associated with compounds isolated from micro-and macroorganisms g to identify the marine-derived drugs which are undergoing clinical evaluation over % of the earth's surface is covered by vast expanses of ocean. its inhabitants are diverse with of the phyla occurring exclusively in the oceans with only one phylum (onychophora) being reported as present on land only [ ] . the marine environment provides an array of structurally unique and diverse constituents produced by an equally diverse consortium of marine organisms living on our coral reefs and in benthic communities. the marine organisms are highly variable in species, color, and morphology and belong to several phyla including porifera (sponges), ascidiacea (sea squirts), and octacorallia (soft corals). the metabolites of marine origin emanate from a variety of parts of the plants and animals and are thought to be produced as a form of chemical communication, defense, or to ward off potential predators [ À ] (figs. . À . ). the potential for a range of applications including anticancer, antibacterial, antiviral, antiinflammatory, antimalarial, antituberculosis activity, as well as pharmacological and industrial applications. the classes of compounds manufactured by marine organisms include alkaloids, terpenoids, shikimates, peptides, and polyketides [ À ] . temperature, salinity, ph, and dissolved oxygen concentrations in the water, thereby providing useful information to facilitate environmental studies [ , ] . caution should always be exercised in the collection of marine species. gloves should be worn in the collection and subsequent handling of specimens. scuba divers should be clad in wet suits to protect against the possible deleterious effects of chemicals being exuded into the water by the organisms being collected. the personal unfortunate experience (author's) of hours of severe discomfort and rashes as a result of collecting the sponge neofibularia nolitangere from a reef in discovery bay, jamaica, provides clear evidence regarding the level of respect which should be accorded to figure . student snorkeling to collect marine specimens. marine organisms whose chemistry is yet to be investigated. records are made of the depth, habitat, global positioning system coordinates (latitude and longitude), color, morphology, and associated organisms. an appropriate coding system should be employed to distinguish specimens. where possible, the specimens are photographed in situ as well as by the dockside (figs. . and . ) . a voucher specimen of each organism is usually preserved in % aqueous ethanol for the purpose of taxonomic identification. ascidians are usually preserved in seawater containing menthol crystals with more long-term storage in % formalin solution [ ] . it should be noted that the recollection of organisms has proved to be a challenge in some instances. an ascidian species, e.g., found to be thriving on the mangrove in the summer of one year could all but disappear from the ecological landscape or months later, while a healthy bed of algae may be short-lived if there are dynamic factors involved in their growth. for example, the occasional nutrient runoff or groundwater seepage event could provide the ideal environment for the growth of selected algal species. environmental factors are key in the marine landscape and often provide a source of frustration to the specimen collector. prior to extraction of the collected organism, the specimens may be frozen, air-dried, freeze-dried, or could be retained in the fresh state. the majority of the marine organisms are extracted fresh or frozen while the remaining specimens are lyophilized or dried in air before extraction [ ] . in some instances, dried algal species are ground to a powder prior to extraction as described by sansom and coworkers who isolated an antiproliferative bis-prenylated quinone from the alga perithalia capillaris [ ] . the extraction of marine organisms may be carried out using a range of organic solvents including hexanes, dichloromethane, acetone, ethyl acetate, as well as more polar solvents such as ethanol and methanol. in many instances, a mixture of polar and medium polarity or nonpolar solvents is utilized in the extraction protocol. for example, the extraction of the madagascar sponge monanchora dianchora was achieved in ch cl:meoh ( : ) to yield two polycyclic guanidine alkaloids [ ] . extractions are usually exhaustively performed over several days with at least three aliquots of the solvent being used. the solvent is then removed in vacuo by rotary evaporation. solvent partitioning is another strategy employed in the extraction of the organisms. this involves single one-step or two-step partitioning systems usually involving an aqueous phase portioned with a solvent immiscible with that phase. the kupchan and modified kupchan procedures are often employed in natural products as was described in the isolation of a diterpene from an axinella species [ ] . in this procedure, the concentration of the aqueous layer is progressively adjusted to afford three or four different fractions. complex partitioning procedures are also employed, albeit rarely so. simple partitioning has been most commonly employed with kupchan schemes being utilized with less frequency [ ] . chromatographic methods of separation include gravity column chromatography, flash column chromatography, and vacuum liquid column chromatography utilizing silica gel as the packing material. with silica gel, the components of the marine extract are separated on the basis of polarity of the compounds. as the polarity of the eluting solvent increases compounds of increasing polarity are eluted from the column with hydrocarbons, e.g., eluting before alcohols. the elution of the components of a column is monitored by using thin layer chromatography (tlc) plates which are spotted to show the sequence of elution of the compounds (figs. . and . ). bonded reverse phase silica is employed in instances where the constituents of the marine extract include polar metabolites. bonded phases include ods (c ), c , cyano, and diol columns. separation of constituents may also be effected using gel permeation chromatography which effects separation of constituents on the basis of the size of the compounds. in this regard, sephadex lh- is commonly utilized in marine natural products isolation work [ ] . resins such as biobeads, amberlite, xad- , and xad- are also utilized in separating components of relatively high polarity. the use of xad- in the separation of antiviral trisulfated triterpene glycosides from the sea cucumber staurocucumis liouvillei is one such example in marine natural products isolation work [ ] . the use of hplc employing a reversed phase stationary phase system is commonplace in marine natural products isolation work with c and c semipreparative and preparative columns being used. mplc and recycling hplc techniques are related techniques for purification of a range of metabolites including alkaloids, peptides, and terpenoids. tandem systems such as liquid chromatography-mass spectrometry systems are also employed to assist with dereplication efforts. unusual ms peaks in the profile suggest that novel components are present in the fraction or extract being evaluated. those fractions with unusual constituents may then become the focus of the research efforts. solidphase extraction methods are also employed in separating compounds. the structural identification of compounds isolated from the range of marine sources is facilitated by the use of spectroscopic techniques such as d and d nuclear magnetic resonance (nmr) spectroscopy and infrared (ir) spectroscopy. x-ray crystallographic techniques are also important in aiding in the determination of the stereochemistry of the compound. the identification of nanogram quantities of a novel compound is becoming increasingly more facile with the use of the cryoprobe, capillary probe, and mans probe [ ] . in vitro activities of marine metabolites have been investigated for a diverse range of cell systems including antiinflammatory, antimicrobial, and anticancer activities. crude extracts, fractions from crude extracts, as well as pure compounds are typically evaluated for biological activity. the in vitro biological evaluation of the isolated compound may be performed using cell lines from human subjects or animals. brine shrimp, fish, and sea urchin are among the organisms employed in the evaluation of compounds or extracts for ecological and therapeutic importance (figs. . and . ) . a summary of the biological activity of some of the organisms discussed in this section is presented in preclinical trials are an essential component of the process of evaluation of the therapeutic potential of a compound. these trials often include animal models such as rats, dogs and monkeys. the major sources of biologically relevant compounds have been found to be from sponges, coelenterates, algae, echinoderms, ascidians, molluscs and microorganisms [ ] . macroinvertebrates include sponges, ascidians, and soft coral. it has been found that the vast majority ( %) of novel compounds obtained from the marine environment have been sourced from the porifera and coelenterata (cnidaria) phyla [ ] . scheme . shows representative structures of compounds isolated from macroinvertebrates. macroinvertebrates include: . sponges . ascidians . soft coral sponges (porifera) are sedentary, filter feeding metazoans which utilize a single layer of flagellated cells (choanocytes) to pump water current through their bodies in a unidirectional manner. there are over species of sponges accounting for much of the epifaunal biomass. extracted fresh or freeze-dried, sponge extracts are an important source of biologically active compounds. these isolates exhibit an impressive array of biological activities, some of which are described here. one sponge which has gained a place in history due to the promising biological activity being displayed is halichondria okadai, the producer of halichondrin b, which underwent evaluation as an anticancer agent. okadaic acid, also from h. okadai, exhibited inhibitory activity against phosphatase- and phosphatase- a [ ] (fig. . ). agelaspin, an antitumor glycosphingolipid obtained from the marine sponge agelas mauritianus, demonstrated antitumor activity in vivo against murine b melanoma. this compound was also found to stimulate the immune system. a derivative of agelaspin, krn- , underwent clinical investigations for cancer immunotherapy [ ] . more recently, the extracts of another agelas sp., a. nakamurai, contained the compound agelasine d which exhibited high antibacterial activity [ ] . the deep water sponge discodermia dissoluta produced discodermolide, a polyhydroxylated lactone which exhibited anticancer activity, as well as immunosuppressive activity. it was found to stabilize microtubules in a manner similar to the drug taxol and underwent evaluation for use in tumors resistant to taxol [ , ] . dysidea arenaria was found to contain arenastatin a which showed potent activity against kb cell lines (ic pg/ml) [ ] . girolline is a substituted imidazole isolated from the sponge pseudaxinyssa cantharella which functions by inhibiting the termination step in eukaryotic protein synthesis. having entered phase clinical trials, it was withdrawn due to its adverse hypertensive effects seen in treated patients [ ] . mycalamides a and b are protein synthesis inhibitors isolated from the new zealand sponge mycale sp. in vivo activity against a coronavirus was observed in mice when treated with a % mycalamide mixture at a dosage of . μg/kg daily with % survival over a two-week period. pure mycalamide a inhibited the herpes simplex virus and polio virus type at a concentration of . μg/disk. mycalamide b was found to exhibit more potent antiviral activity and cytotoxicity than mycalamide a [ ] . the baculiferins i, j, l, and m from the marine sponge iotrochota baculifera have been found to inhibit human immunodeficiency virus- (hiv- ) with ic values between . and . μm [ ] . jasplakinolide, the first example of a cyclodepsipeptide isolated from a sponge, is a -membered macrocyclic depsipeptide from the jaspis sp. exhibiting in vitro antimicrobial activity at a minimum inhibitory concentration of melinacidins and gencidin antibacterial marinospora sp. lynamicins a-e antibacterial μg/ml against candida albicans. with a topical administration of % jasplakinolide solution, an effect similar to that of miconazole nitrate was achieved in vivo [ ] . discorhabdin r is a novel pyrroloiminoquinone isolated from the southern australian sponge negombata sp. and antarctic latruncula sp. which was found to display antibacterial activity against both gram-positive (staphylococcus aureus and micrococcus luteus) and gram-negative bacteria (serratia marcescens and escherichia coli), respectively [ ] . antibacterial activity against a strain of the bacterial parasite plasmodium falciparum was reportedly identified in monanchora arbuscula with the active agents being the batzellidine alkaloids (ic . À . μm) [ ] . an important isolate from a spongia sp. is the polyhydroxylated steroid, agosterol a, which functions by reversing multidrug resistance caused by the overexpression of two kinds of membrane glycoprotein in cancer cells [ ] . from the phylum cnidaria the genera sinularia and briareum have proven to be prolific sources of novel compounds. cembranoids, , -epidoxysteroids, sinulaflexiolides, and africanenes have been isolated from sinularia species [ ] (fig. . ) . examples of other species of soft corals include the taiwanese soft coral cespitularia taeniata which was extracted with ethanol to yield a group of verticillene diterpenoids including cespitulactam k. the compounds were evaluated against human epidermal carcinoma and murine l leukemia cell lines. cespitulactam k exhibited activity against the cancer cell lines ( . À . μg/ml) and also showed marked antimicrobial activity against m. luteus and cryptococcus neoformans [ ] . the methanol extract of the octocoral muricea austera showed in vitro activity against chloroquine-resistant p. falciparum and was found to contain a range of different classes of compounds including tyramine derivatives, steroidal pregnane glycosides, and sesquiterpenoids [ ] . cytotoxic dolabellane diterpenes were isolated from the formosan soft coral clavularia inflata var luzoniana and bioactivity against p cell lines with ed values between . and . μg/ml was observed [ ] . tunicates, sea squirts, or ascidians belong to the subphylum of tunicata (urochordata). they are so named because of their cellulose-containing protective tunic surrounding the organism. tunicates attach to a substratum, usually a marine solid surface such as a mangrove root, rocks, jetties, or even algal species (fig. . ). much like sponges and soft corals, ascidians have also been found to be a good source of bioactive agents. didemnin b, isolated from the tunicate trididemnum solidum, is one such bioactive compound, showing remarkable antiviral and cytotoxic activity. didemnin b demonstrated activity against p and l murine leukemia cell lines. it was advanced into preclinical and clinical trials and , but had to be withdrawn due to its harsh toxicity [ ] . aplidine, formally known as dehydrodidemnin, an isolate from the mediterranean tunicate aplidium albicans, is one such bioactive compound. being structurally related to didemnin b, aplidine was found to be up to more active and less toxic than didemnin b. it entered into phase clinical trials in under investigation for the treatment of solid tumors and non-hodgkin's lymphoma. broad spectrum activity was displayed in vitro and in vivo against leukemia, melanoma, breast, ovarian, colon, and lung (nonsmall cell) cancer. having advanced to phase clinical trials, aplidine affects protein synthesis through gtp-dependent inhibition of elongation factor -α [ ] . the extract of the palauan ascidian didemnum guttatum afforded the sulfonated serinolipid cyclodidemniserinol trisulfate which exhibits an antiviral effect by inhibiting hiv- integrase, an attractive target for antiretroviral chemotherapy [ ] . macroalgae belong to three main phyla: rhodophyta (red algae), chlorophyta (green algae), and phaeophyta (brown algae). biological activities identified in extracts and metabolites of algal origin include anticancer, antiobesity, neuroprotective, and antioxidant activity and scheme . shows chemical structures of representative bioactive compounds isolated from the macroalgae. a wide range of algal species are utilized in fresh or dried forms as food particularly in asian countries where folklore traditions govern their industrial and medicinal usage [ ] . macroalgae are the source of agar, carrageenan, and alginate, which are all of importance in the food industry. the range of compounds isolated from algal sources has been variable. representative examples of bioactive constituents from macroalgae are mentioned below. cytotoxic activity has been identified in α, -dihydroxypachydictyol a, a diterpenoid compound from a dictyota sp. collected on bangsaen beach in thailand. antimalarial activity was also found in the diterpene isolated from this extract when the compound was tested with malarial parasites [ ] . stypolactone, an isolate from the brown alga stypopodium zonale, was found to exhibit weak cytotoxic activity in vitro when evaluated with a- and h- cell lines [ ] . zonaquinone acetate, obtained from jamaican populations of s. zonale, displayed in vitro activity against breast and colon cancer cell lines [ ] . specimens of taonia atomaria produced atomarianones a and b which were reportedly found to be cytotoxic against nsclc-n and a- cell lines [ ] (fig. . ) . crude extracts of algal species have been found to exhibit a range of biological activities. for example, aqueous extracts of gracilaria corticata and sargassum oligocystum exhibit bioactivity against cancerous human leukemia cells [ , ] while a methanol extract of plocamium telfairiae was observed to display bioactivity against ht- colon cancer cells [ ] . antiinflammatory activity was found in the green alga from which -( , -dibromophenoxy)- , -dibromoanisol was isolated. this activity was identified using a snake toxin-induced mouse limb model [ ] . also exhibiting antiinflammatory activity is a mixture of phytosterols obtained from dunaliella tertiolecta. when administered in a sheep model of inflammation-induced cytokine production, an inhibitory effect was observed [ ] . polyphenolic extracts from the red alga laurencia undulatea displayed antiinflammatory activity in vivo. these extracts served to inhibit asthmatic reactions in mice sensitized and challenged with ovalbumin which was used to induce murine allergic reactions in test subjects [ ] . antiinflamatory agents floridoside and d-isofloridoside from the south korean alga l. undulatea were found to inhibit free radical oxidative stress at ic values between and μm [ ] . biologically active compounds have been isolated from the brown seaweed dictyota cervicornis from which was obtained sulfated polysaccharides with powerful anticoagulant activity [ ] . antioxidant activity, evaluated using the dpph method, was reported in phenolic isolates of halimeda monile when liver injury was induced in a rat model. the phenolic fraction was administered over a -day period and led to protective effects against chemicals harmful to the liver [ ] . with ic values between . and . μm, potent antimalarial activity against the human malarial parasite p. falciparum was identified in new macrolides bromophycolides j, m, n, o, p, and q from the red algae callophycus serratus [ ] collected in fiji. the marine alga halimeda tuna was studied by koehn and coworkers, leading to the isolation of halitunal, a diterpene displaying in vitro antiviral activity against murine coronavirus a [ ] . ecologically important roles are played by some compounds from alga sources. for example, halimedatrial, a diterpene isolated from halimeda lamouroux, exhibited toxicity toward reef fishes and appeared to be a feeding deterrent. antimicrobial activity was also reported from this compound [ ] . almost % of all bioactive marine compounds currently being studied are obtained from marine microorganisms [ ] . these microbes are found in swabs from the surfaces of marine plants and animals, suspended in the water from geothermal vents and deep water environments, or on sediment surfaces. they thrive in a variety of environments including locales characterized by high pressures of up to atmospheres, high temperatures, and high salinities. efforts at culturing some of the microorganisms have met with varying degrees of success. the ability to propagate these microorganisms in an economically feasible way will be of great significance as potent bioactive metabolites are discovered [ ] (figs. . À . ). marine microorganisms are found . on the surface of marine plants and animals . suspended in water . on sediment surfaces historically, terrestrial microbes have been a potent source of pharmaceutical agents with the seminal discovery of penicillin. the discovery of new antibacterial agents is a serious priority because of the development of potent resistance to current antibiotics on the market. marine bacteria produce a wide variety of secondary metabolites for the purpose of defending themselves against other microbes. scheme . shows structures of representative compounds from microorganisms associated with marine specimens. marine bacteria which produce compounds of biological significance include pseudoalteromonas species which was found to produce , , , -tetrabromo- , -diphenyl diol, an inhibitor of methicillin-resistant s. aureus. the class of -methoxypyrrole-containing compounds, the tambjamines, isolated from p. tunicata, was found to be active antifungal, immunosuppressive, and antimicrobial agents. biologically active compounds from marine bacteria also include streptomyces species from sediment and fish gut from which anticancer (e.g., halichomycin and δ-indomycinone) and antibacterial agents (e.g., phenazines) have been obtained [ À ]. vibrio species obtained from sponge specimens have produced phenolic and trisindole compounds with antibacterial activity [ , ] . a micromonospora sp. obtained from a soft coral produced thiocoraline, a compound exhibiting anticancer activity [ ] . marine fungi have also been known to produce compounds with a range of bioactivities including antiviral, antifungal, enzyme inhibition, and anticancer and antibacterial activities. the isolation and cultivation of fungi from the marine environment is of critical importance for propagation of the microbes from which biologically relevant compounds may be obtained. protocols have been established for this work [ ] . fungal species which have produced antibacterial compounds include corallospora pulchella isolated from sand. this species produced melinacidins and gencidin [ ] . anticancer activity has been reported from metabolites of aspergillus sp. (including the aspergillamides and fumiquinazolines) and penicillium sp. sourced from a marine alga which was found to contain pentostatins and communesins among other compounds [ , ] . antiviral activity, attributable to the presence of halovirs, was identified in a scytalidium sp. collected from a seagrass species. potent antiviral activity against h. simplex virus (type ) was observed and may be acting by binding directly to the virus [ ] . actinomycetes have been the source of a wide range of antimicrobial agents, the most common of which include tetracycline and streptomycin. other bioactive compounds originating from actinomycetes include antitumor and antimicrobial agents. a marinospora sp. produced a group of bisindole pyrroles, lynamicins aÀe, which exhibited biological activity against gram-positive and gram-negative species. importantly, activity was also shown against drug-resistant pathogens including methicillin-resistant s. aureus [ ] . anticancer activity against lung, colon, and breast cancer cell lines was exhibited by isolates from the fermentation of a streptomyces sp. (mbg- - - ). tartrolon d was found to be the bioactive agent [ ] . microalgae are found in seven phyla. these include chlorophyta, phaeophyta, rhodophyta, crystophyta, cryptophyta, eugelophyta, and pyrrhophyta. the blue-green algae, cyanophyta, are cyanobacteria which have been found to share characteristics with eukaryotic algae. these microalgae produce compounds with a high degree of structural diversity and species, such as lyngbya majuscula, have produced a vast array of biologically active compounds [ ] . curacin a, e.g., isolated by gerwick and coworkers in [ ] , was found to function by disturbing microtubule assembly, thereby functioning as a lead compound in chemotherapy. microcystis aeruginosa is the source of potent protein phosphatase- and phosphatase- a inhibitors identified in microcystins [ ] . other microalgal species under examination include dinoflagellates which produce an array of bioactive toxins including saxitoxin and maitotoxin which function by blocking or activating sodium/calcium channels. challenges exist with respect to the culturing of these organisms due to relatively low proliferation rates and the large quantities of culture required to obtain small amounts of bioactive compounds. diatoms, microscopic unicellular colonial algae, grow at a faster rate and are amenable to culturing but few bioactive metabolites have been identified from these microalgae [ ] . some marine compounds sourced from microbes are of clinical significance, undergoing evaluation as potential pharmaceutical agents. the marine-derived drug pipeline, almost nonexistent in decades gone by, now has a range of candidates at various stages of development as shown in table drugs in phase three clinical trials include tetrodotoxin, a guanidinium alkaloid under the trademark name tectin obtained from the pufferfish [ ] . affecting the sodium channels, this drug is being investigated for the treatment of chronic pains (scheme . ). a depsipeptide from a tunicate, plitidepsin, is being tested by pharmamar in the treatment of a variety of cancers, namely leukemia, multiple myeloma, and lymphoma. another drug under evaluation by pharmamar for cytotoxic activity is zalypsis (pm ) sourced from a mollusc which targets the dna-binding capacity of diseased uterine, lymphoma, cervical, and endometrial cancer cells. the alkaloid-derived compound pm is another drug candidate from pharmamar being evaluated for its efficacy against a range of cancers including ovarian, breast, lung, acute leukemia, and endometrial cancer [ , ] . bryostatin i, from the bryozoan bugula neritina has been involved in a battery of clinical trials being investigated for its potency against cancer. it is currently under phase i evaluation as a treatment for alzheimer's [ ] . in the early years, the challenge associated with the supply of the drug was underscored by the fact that, in order to obtain g of a cgmp quality bryostatin i, tonnes of b. neritina had to be collected in californian waters [ , ] . the gene cluster of the uncultivated microbial symbiont of b. neritina, candidatus endobugula sertula has been successfully identified, thereby opening the potential for the supply of the compounds [ ] . kahalalide f, a cyclic depsipeptide, was found in the mollusc elysia rufescens as well as the green algae bryopsis sp. on which it feeds. this compound is currently in phase i/ii trials as a treatment against prostate cancer [ ] (fig. . ) . dmxba [( -( , -dimethylxybenzylidene)]-anabaseine is a derivative of anabeseine, an alkaloid found in marine worms. found to improve cognition in animal models, dmxba and other related compounds have demonstrated neuroprotective activity in both in vitro and in vivo screens. thought to have an effect on macrophage receptors, antiinflamatory activity was also observed in animal models. phase i evaluation of healthy males and schizophrenics have shown that dmxba has led to marked improvements in cognitive function [ ] . there are several marine compounds sourced from microbes which are of clinical significance. clinical trials are being conducted on plinabulin (npi- ), a vascular disrupting agent obtained from a marine fungal extract with potential for activity against multidrug resistant tumor cells. marizomib (salinosporamide a, npi- ), an isolate from a marine bacterium salinospora tropica, is a novel proteasome inhibitor which is currently under investigation for its efficacy against solid tumor models. the compound exhibits low cytotoxicity to normal cells and has significant potential for oral and intravenous administration [ ] . the ultimate goal of many marine natural products and synthetic chemists is that the isolated or synthesized molecule possesses therapeutic applications. there are several food and drug association (fda)-approved drugs of marine origin obtained from sponges, a fish, a cone snail, a mollusc, and cyanobacterium species, while yondelis (trabectidin) obtained from the ascidian ecteinascidia turbinata, has been approved in the european union. the antitumor effects of aqueous ethanol extracts of e. turbinata were observed from . in vitro trials had been carried out on a human cancer cell panel by the company developing the drug, pharmamar, and the national cancer institute. aquaculture of the ascidian proved to be the initial strategy used to obtain sufficient quantities for evaluation of the efficacy of the compound. semisynthetic procedures involving the fermentation of pseudomonas florescens are now currently employed in the pharmaceutical preparation of the drug which is sold in over countries, including south korea and russia, under the trade name yondelis. yondelis is also used in patients with relapsed platinum-sensitive ovarian cancer. this drug is currently under evaluation in phase ii for breast, prostate, lung, and pediatric cancers. the sponge tethya crypta (cryptotethia crypta) was the original source from which the drug cytarabine was developed. cytarabine is a synthetic analogue of the nucleoside which was originally isolated from the sponge. sold under the trade name cytosar-u, this cytotoxic agent inhibits deoxyribonucleic acid (dna) polymerase and dna synthesis. acute lymphocytic leukemia, non-hodgkin's lymphoma, and acute myelocytic leukemia are among the conditions being treated by this drug approved by the fda in [ ] . produced by fermentation of streptomyces griseus, cytarabine has limited bioavailability but improvements in the delivery system have been made [ ] . a slow-release liposomal form of cytarabine (depo cyle) has been approved in the united states and europe for the prolonged administration/exposure in cerebrospinal fluid. a related drug, vidarabine (vira-a), was developed from spongouridine and found use as an antiviral treatment for epithelial and superficial keratitis caused by the h. simplex virus types and . viral dna polymerase and dna synthesis of herpes are inhibited by this drug which was discontinued over years ago. this drug is still in use in europe for ophthalmological challenges. prialt (ziconotide) was obtained from a peptide ω-conotoxin mviia isolated from the cone snail conus magus. with a unique mode of action, this drug acts by reversibly blocking n-type calcium channels in some specific nerves in superficial layers of the spinal cord. this drug is used for the management of severe and chronic pains in patients suffering from cancer and acquired immunodeficiency syndrome who are unable to use or are unresponsive to other drugs such as morphine. ziconotide had to be synthesized using solid-phase peptide synthesis due to the insufficient quantities supplied by the cone snail, c. magus [ ] . the blockage of the spinal cord induced by this drug prevents the release of neurotransmitters responsible for pain from specific neurons. related conus peptides are undergoing evaluation in human clinical trials [ ] . brentuximab vedotin (sgn- ) is being marketed under the trade name adcetris by seattle genetics and has gained repute for the treatment of hodgkin and systemic anaplastic large cell lymphoma [ ] . this drug is an analogue of dolastatin , a compound isolated from the sea hare dolobella auricularia, which was later found to be produced by diet-associated cyanobacteria symploca hydnoides and l. majuscula. preliminary phase i and ii clinical trials of dolastatin and a related analogue were largely unsuccessful. antibody-drug conjugates function by selectively delivering the drug to the cancer cell by linking the dolastatin , e.g., to an antibody that targets a cell membrane protein on the surface of hodgkin's lymphoma cells. this technology has proven to be a seminal development. omega- fatty acids from fish oils are being marketed under the trade name lovaza by glaxosmithkline. used in the treatment of hypotriglyceridemia, the drug controls ethyl esters of eicosapentaenoic acid and docosahexaenoic acid and functions by lowering triglyceride levels. [ ] . eribulin mesylate (e ), with the trade name halaven was formulated from the macrolide halichondrin b sourced from the sponge h. okadai. studies related to the anticancer activity of simpler analogues of halichondrin b showed that the efficiency is retained leading to the development of eribulin mesylate which is more water soluble than the parent macrolide. now approved for use, potent and irreversible inhibition in cancer cells medicated by this drug resulted in the death of the cells by apoptosis. in the absence of tubulin, cell growth grinds to a halt. related compounds are currently being evaluated in phase ii trials [ ] . one of the more recent formulations on the market is carragelose, an antiviral nasal spray which functions by creating a physical antiviral barrier in the nasal cavity. the company marinomed biotechnologie gmbh, utilized iotacarrageenan, sulfated polysaccharides found in the rhodophyceae seaweed as well as other seaweeds. the product is effective against the early symptoms of the common cold [ ] . it should be noted that, in addition to the pharmaceutical applications of marine-sourced therapies, a range of cosmetic applications also exist and are thriving industries. the foray into cosmetic applications was led by estee lauder with the antiaging skin care remedy resilience which contains an extract from the caribbean sea whip pseudopterogorgia elisabethae. the active antiinflammatory and analgesic agents are the pseudopterosins, tricyclic diterpene glycosides, which have been found to inhibit pla and -lipoxygenase. derivatives of the pseudopterosins underwent phase i and ii trials to examine wound healing efficiency but the lipophilic and insoluble nature of the compounds have served to limit its potential as an effective drug. compounds from this group of tricyclic diterpene glycosides also underwent preclinical evaluation as antiinflammatory drugs [ ] . abyssine is marketed as a product used to soothe and reduce irritation in skin sensitive to ultraviolet b light as well as chemical and mechanical attack. it consists of an extract from an alteromonas species and contains a high molecular weight polymer with two different oligosaccharides (exopolysaccharide), while seacode represents another exopolysaccharide which occurs as a mixture of extracellular glycoproteins and other glucidic exopolymers produced by fermentation of a pseudoalteromonas sp. this product has been found to improve skin roughness after up to four weeks of administration. refirmar, a recent product to be introduced, was obtained from an intracellular extract from a fermentation of a new pseudoalteromonas sp. isolated from a deep ( m) hydrothermal vent in portugal's exclusive economic zone, extraction of the cultured biomass afforded a mixture of macromolecules which inhibit muscle contraction. the hydrating and antiaging potential of the product has been evaluated in vivo and in topically applied formulations [ ] . the area of marine natural products chemistry has clearly developed leaps and bounds as evidenced by the relatively large number of marine-derived drugs undergoing evaluation as potential therapeutic agents. buoyed by the potential for the development of natural products from the sea, research work continues to advance with the discovery of new bioactive compounds and new applications for previously isolated molecules [ À ]. the supply issue, however, remains one of critical importance as it relates to the development of drugs from a marine organism. for example, ( ) spongistatin has been reported to be highly cytotoxic. it has been deemed to be the most active of all natural and synthetic compounds investigated by the national institute of cancer (usa). three tonnes of the sponge yielded . mg of the compound. another collection and processing of kg of the sponge afforded mg of the compound. this isolation work facilitated structure elucidation work. the ic value for this compound was evaluated at m in colon cancer cells and m for breast cancer cell lines [ ] . synthetic approaches to the compound have been presented by research groups including petit and coworkers [ , ] . total synthesis of biologically active marine compounds is often fraught with its attendant challenges due to the length of multistep synthetic procedures and the general complexity of the structural motifs which must take into account stereochemical considerations. propagation through mariculture and aquaculture are also being studied to determine the viability of using these approaches to deal with the challenges associated with procuring sufficient quantities for clinical trials and subsequent formulation into drugs [ ] . the timeline from discovering the drug, leading to the entry into the market typically spans a -to -year period during which time the capital injection is considerable, often necessitating support from the large pharmaceutical entities which are sometimes hesitant about making investments which may not yield significant financial rewards [ ] . the caribbean region, being an important source of marine species with which much research work has been carried out, is not likely to become the recipient of the potential benefits to be derived from the development unless more research work in this area is undertaken in the region with support from the appropriate collaborators. in the future, it is expected that new strategies will be employed to ensure the supply of large quantities of the target compounds. these include optimization or fermentation techniques for propagation of microbes, including mixed fermentation methods. biotechnological approaches are likely to include whole genome sequencing, genome mining, genetic engineering, chemoenzymatic synthesis, and in vitro enzymatic synthesis in the hope that new therapeutic drugs will come from our seas [ ] . . if you were required to evaluate an extract for its potential as a drug, what approach would you adopt? . silica gel chromatography is essential for the purification of organic compounds. identify three methods of chromatography. . design a form which could be used to document information when collecting a specimen. trends in the discovery of new marine natural products from invertebrates over the last two decades À where and what are we bioprospecting? marine natural products: metabolites of marine algae and herbivorous marine molluscs marine natural products: metabolites of marine invertebrates marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products drugs and cosmetics from the sea marine natural products and their potential applications as anti-infective agents biogeography of sponge chemical ecology: comparisons of tropical and temperate defenses temperature and spatiotemporal variability of salicylihalamide a in the sponge haliclona sp sources of secondary metabolite variation in dysidea avara (porifera: demospongiae): the importance of having good neighbors chemical mediation of interactions among marine organisms marine advanced technology education-marinetech.org. date accessed a survey of deep-water coral and sponge habitats along the west coast of the us using a remotely operated vehicle. noaa technical memorandum nos nccos isolation of marine natural products an antiproliferative bis-prenylated quinone from the new zealand brown alga perithalia capillaris ptilomycalin d, a polycyclic guanidine alkaloid from the marine sponge monanchora dianchora a new cycloamphilectene metabolite from the vanuatu sponge axinella sp bastadin and bastadin o-sulfate esters from ianthella basta: novel modulators of the ry r fkbp receptor complex two new cytotoxic and virucidal trisulfated triterpene glycosides from the antarctic sea cucumber staurocucumis liouvillei drugs from the sea from anti-fouling to biofilm inhibition: new cytotoxic secondary metabolites from two indonesian agelas sponges marine natural products and related compounds in clinical and advanced pre-clinical trials discodermolide, a new bioactive polyhydroxylated lactone from discodermia dissolute arenastatin a, a potent cytotoxic depsipeptide from the okinawan marine sponge dysidea arenaria baculiferins aÀo, o-sulfated pyrrole alkaloids with anti-hiv- activity, from the chinese marine sponge iotrochota baculifera marine pharmocolgy in : marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antitubercolosis, and antiviral activities; affecting the cardiovascular, immune, and nervous systems and other miscellaneous mechanisms of action bioactive guanidine alkaloids from two caribbean marine sponges agosterol a, a novel polyhydroxylated sterol acetate reversing multidrug resistance from a marine sponge of spongia sp nitrogen-containing verticillene diterpenoids from the taiwanese soft coral cespitularia taeniata antiplasmodial metabolites isolated from the marine octocoral muricea austera cytotoxic constituents from the formosan soft coral clavularia inflata var. luzoniana a guide to the common edible and medicinal sea plants of the pacific islands novel diterpenes with cytotoxic, anti-malarial and anti-tuberculosis activities from a brown alga dictyota sp an interesting diterpenoid from the brown alga stypopodium zonale antiproliferative activity and absolute configuration of zonaquinone acetate from the jamaican alga stypopodium zonale atomarianones a and b: two cytotoxic meroditerpenes from the brown alga taonia atomaria in vitro antitumor activity of gracilaria corticata (a red alga) against jurkat and molt- human cancer cell lines anticancer activity of sargassum oligocystum water extract against human cancer cell lines methanolic extracts of plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in ht- human colon carcinoma cells biological importance of marine algae a mixture of phytosterols from dunaliella tertiolecta affects proliferation of peripheral blood mononuclear cells and cytokine production in sheep anti-asthmatic effect of marine red alga (laurencia undulata) polyphenolic extracts in a murine model of asthma inhibitors of oxidation and matrix metalloproteinases, floridoside, and d-isofloridoside from marine red alga laurencia undulata biological activities of sulfated polysaccharides from tropical seaweeds free phenolic acids from the seaweed halimeda monile with antioxidant effect protecting against liver injury antimalarial bromophycolides jÀq from the fijian red alga callophycus serratus halitunal, an unusual diterpene aldehyde from the marine alga halimeda tuna isolation of halimedatrial: chemical defense adaptation in the calcareous reef-building alga halimeda screening for new metabolites from marine microorganisms δ-indomycinone: a new member of pluramycin class of antibiotics isolated from marine streptomyces sp rare phenazine l-quinovose esters from a marine actinomycete a novel antimicrobial substance from a strain of the bacterium vibrio sp marine natural products. . trisindoline, a new antibiotic indole trimer, produced by a bacterium of vibrio sp. separated from the marine sponge hyrtios altum thiocoraline, a new dipsipeptide with antitumor activity produced by a marine micromonospora. . taxonomy, fermentation, isolation, and biological activities methods for isolation of marine-derived endophytic fungi and their bioactive secondary products corollospora pulchella, a marine fungus producing antibiotics, melinachidins iii, iv and gancidin w new cytotoxic sequiterpenoid nitrobenzoyl esters from a marine isolate of the fungus aspergillus halovirs a-e, new antiviral agents from a marine-derived fungus of the genus scytalidium lynamicins a-e, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete tartrolon d, a cytotoxic macrodiolide from the marine-derived actinomycete streptomyces sp. mdg- - - continuing studies on the cyanobacterium lyngbya sp.: isolation and structure determination of -norlyngbyapeptin a and lyngbyabellin d structure of curacin a, a novel antimitotic, antiproliferative, and brine shrimp toxic natural products from the marine cyanobacterium lyngbya majuscula structure and biosynthesis of toxins from blue-green algae (cyanobacteria) the odyssey of marine pharmaceuticals: a current pipeline perspective the bryostatins identification of the putative bryostatin polyketide synthase gene cluster from "candidatus endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan bugula neritina development of cytarabine prodrugs and delivery systems for leukemia treatment industrial natural product chemistry for drug discovery and development conus peptides: biodiversity-based discovery and exogenomics marketed marine natural products in the pharmaceutical and cosmeceutical industries: tips for success marine natural products: a way to new drugs towards a more step-economical and scalable synthesis of spongistatin to facilitate cancer drug development efforts antineoplastic agents. isolation and structure of spongistatin aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process development and economics mariculture trials with mediterranean sponge species. the exploitation of an old natural resource with sustainable and novel methods marine natural products: a new wave of drugs? key: cord- - gkrsk authors: zhang, rui; hristovski, dimitar; schutte, dalton; kastrin, andrej; fiszman, marcelo; kilicoglu, halil title: drug repurposing for covid- via knowledge graph completion date: - - journal: nan doi: nan sha: doc_id: cord_uid: gkrsk objective: to discover candidate drugs to repurpose for covid- using literature-derived knowledge and knowledge graph completion methods. methods: we propose a novel, integrative, and neural network-based literature-based discovery (lbd) approach to identify drug candidates from both pubmed and covid- -focused research literature. our approach relies on semantic triples extracted using semrep (via semmeddb). we identified an informative subset of semantic triples using filtering rules and an accuracy classifier developed on a bert variant, and used this subset to construct a knowledge graph. five sota, neural knowledge graph completion algorithms were used to predict drug repurposing candidates. the models were trained and assessed using a time slicing approach and the predicted drugs were compared with a list of drugs reported in the literature and evaluated in clinical trials. these models were complemented by a discovery pattern-based approach. results: accuracy classifier based on pubmedbert achieved the best performance (f = . ) in classifying semantic predications. among five knowledge graph completion models, transe outperformed others (mr = . , hits@ = . ). some known drugs linked to covid- in the literature were identified, as well as some candidate drugs that have not yet been studied. discovery patterns enabled generation of plausible hypotheses regarding the relationships between the candidate drugs and covid- . among them, five highly ranked and novel drugs (paclitaxel, sb , alpha -antiplasmin, pyrrolidine dithiocarbamate, and butylated hydroxytoluene) with their mechanistic explanations were further discussed. conclusion: we show that an lbd approach can be feasible for discovering drug candidates for covid- , and for generating mechanistic explanations. our approach can be generalized to other diseases as well as to other clinical questions. anteed. on the other hand, de novo development and approval of an effective antiviral therapy can take more than a decade. in the absence of an effective vaccine or other therapies, there have been significant efforts in repurposing drugs approved for other diseases for covid- treatment, some of which have been tested in clinical trials (e.g., dexamethasone [ ] , hydroxychloroquine and lopinavir/ritonavir [ ] ). computational approaches to drug repurposing have also garnered much attention to accelerate discovery of therapies for covid- [ , ] . common computational drug repurposing methods include drug signature matching, molecular docking, genome-wide association studies, and network analysis [ ] . these data-driven approaches involve systematic analysis of various types of biological and clinical data (e.g., gene expression, chemical structure, genome and protein sequences, and electronic health records) to generate hypotheses regarding repurposed use of approved or investigational drugs [ ] . the potential of recent advances in artificial intelligence (ai) and machine learning for covid- drug repurposing has also been highlighted [ ] and several studies using these techniques have reported promising results [ ] [ ] [ ] [ ] . in particular, approaches leveraging network medicine [ ] principles and biological knowledge graphs have been emphasized [ ] . most of these computational approaches have focused on biological data, such as gene expression, protein-protein and drug-target interactions, and used sars-cov- -related data. however, covid- -specific data is meaningful in the context of the larger body of diverse knowledge underpinning medicine and life sciences, a primary source of which is the biomedical literature. while some covid- drug repurposing studies incorporated some literature-based knowledge [ , ] , their focus has remained largely covid- -specific. we argue that efficiently and safely repurposing drugs to treat covid- requires more effective integration of literature-based knowledge with biological data collected via high-throughput methods. in this paper, we propose a novel literature-based discovery [ , ] ap-proach for covid- drug repurposing. similar to related work [ ] , we cast drug repurposing as a task of knowledge graph completion (or link prediction). we use a large, literature-derived biomedical knowledge graph constructed from semmeddb [ ] as well as covid- research literature [ ] , as our data source. we use several state-of-the-art, neural network-based algorithms [ ] [ ] [ ] for the task, and also complement these approaches with an approach based on discovery patterns [ ] . furthermore, we highlight the role of discovery patterns in search of mechanistic explanations for the proposed drugs. unlike most approaches that focus on covid- -specific knowledge [ , ] , we consider a larger body of biomedical knowledge, as captured in the pubmed bibliographic database as well as in the covid- research literature. our results show that our approach can identify known drugs that have been used for covid- and discover other novel drugs that can potentially be repurposed for covid- . significant computational work has already been done to prioritize fdaapproved drugs for repurposing to treat covid- [ , ] . for the most part, these studies can be categorized as molecular docking-based drug screening studies and network-based studies, the majority of them belonging to the former category. in molecular docking studies, small molecules in compound libraries are screened for effectiveness against the host proteins in the sars-cov- host interactome. many studies of this kind have been reported, and some of the proposed drugs such as ritonavir, ribavirin, remdesivir, oseltamivir, have been used in practice and many are being evaluated in clinical trials [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while not as common as docking studies, network-based approaches to drug repurposing have also been explored. in one early study, a virus-related knowledge graph which consists of drug-target and protein-protein interactions and similarity networks from publicly available databases (e.g., drugbank [ ] , chembl [ ] , biogrid [ ] ) was constructed and network-based machine learning and statistical analysis were used to predict an initial list of covid- drug candidates. this list was narrowed down based on text mining from the literature and gene expression profiles from covid- patients, and a poly-adpribose polymerase (parp ) inhibitor cvl , was proposed for therapeutic use against covid- [ ] . cava et al. [ ] used gene expression profiles from public datasets to construct a protein-protein interaction network in conjunction with pathway enrichment analysis to identify potential drugs, including nimesulide, thiabendazole, and fluticasone propionate. in another study, network proximity analyses of drug targets and hcov-host interactions in the human interactome were used to prioritize potential repurposed drugs, including melatonin, mercaptopurine, and sirolimus, which were validated by enrichment analyses of drug-gene signatures and transcriptome data in human cell lines. potentially useful drug combinations (e.g., melatonin plus mercaptopurine) were also suggested [ ] . a follow-up study combined network medicine approaches based on human interactome with clinical patient data from a covid- registry to show that melatonin was associated with reduced likelihood of a positive sars-cov- laboratory test [ ] . the approach was further extended to explore deep learning [ ] . a comprehensive knowledge graph of drugs, diseases, and proteins/genes (named cov-kge) was constructed by combining molecular interaction information from the literature with knowledge from drugbank. a knowledge graph embedding model, named rotate [ ] was used to represent the entities and the relationships in the knowledge-based in low-dimensional vector space. using the ongoing covid- trial data as a validation set, high-confidence repurposed drug candidates (including dexamethasone, indomethacin, niclosamine, and toremifene) were identified, and further validated via an enrichment analysis of gene expression and proteomics data in sars-cov- -infected human cells. another study used node vec graph embeddings and variational graph autoencoders for the same purpose [ ] . [ ] evaluated three algorithms (graph neural network, network proximity, and network diffusion) on a network of drug protein targets and disease-associated proteins for covid- drug repurposing. while they obtained low correlations across the three algorithms, an ensembling approach that combined the predictions of all algorithms was shown to outperform the individual methods, ranking ritonavir, chloroquine, and dexamethasone among the most promising candidates. some limited literature knowledge relevant to covid- has been incorporated to network-based approaches; however, their focus remains largely on structured molecular interaction information encoded in databases. literature-based discovery (lbd) [ , ] is a method of automatic hypothesis generation pioneered by swanson [ ] . based on the concept of "undiscovered public knowledge", lbd seeks to uncover valuable hidden connections between disparate research literatures, and has been proposed as a potential solution for the problem of "research silos" (the view that scientific research areas are largely isolated from one another). the primary lbd paradigm is the so-called abc model. in the open discovery form of this model, a relationship between two concepts a and b is known in one research area and another relationship between concepts b and c is known in another, and a potential relationship between concepts a and c is proposed. conversely, in closed discovery, relationship ac is known, and a concept b is proposed as an explanation for the relationship ac. extensions to abc model have also been proposed, such as discovery browsing model that aims to elucidate more complex relationship paths between biomedical concepts [ , ] . most applications of lbd have been in the biomedical domain, beginning with swanson's discovery of fish oil as a treatment for raynaud disease [ ] , a hypothesis supported subsequently by clinical studies. while early lbd systems focused primarily on term cooccurrence [ , ] , semantic relations have been widely used in later years for representing scientific content of biomedical publications [ , [ ] [ ] [ ] . more recently, distributed vector representations based on term or semantic relation co-occurrence have been gaining popularity [ ] [ ] [ ] . drug repurposing has been one of the prominent applications of lbd [ , [ ] [ ] [ ] [ ] [ ] [ ] . for example, hristovski et al. [ ] used semantic discovery patterns following the abc model to identify potential therapeutic uses for drugs. zhang et al. [ ] used discovery patterns and semmeddb relations to identify potential prostate cancer drugs. cohen et al. [ ] used a vector representation approach based on semantic relations to predict a small number of active agents within a large library screened for activity against prostate cancer cells. knowledge graphs are represented as a collection of head entity-relation-tail entity triples (h,r,t), where entities correspond to nodes and relations to edges between them. knowledge graph completion (or link prediction) is the task of predicting unseen relations between two existing entities or to predict the tail entity given the head entity and the relation (or head entity given the tail entity and the relation). recent approaches to knowledge graph completion rely on knowledge graph embedding methods [ ] , which learn a mapping from nodes and edges to continuous vector space that preserve the proximity structure of the knowledge graph and are amenable to application of machine learning methods. such methods include translational models, which use distance-based scoring functions (e.g., transe [ ] , transh [ ] , rotate [ ] ), and semantic matching models, which use similarity-based scoring functions (e.g., rescal [ ] , distmult [ ] , complex [ ] , and holographic embeddings (hole) [ ] ). graph convolutional networks [ , ] as well as methods that use context-based encoding approach (kg-bert [ ] , stelp [ ] ) have also been recently proposed. knowledge graph embedding techniques based on a network of drug, disease, and gene/protein entities, have been used to support drug repurposing for rare diseases [ ] . graph convolutional networks were used to model drug side effects resulting from drug-drug interactions [ ] . a multimodal graph of proteinprotein interactions, drug-protein target interactions and drug-drug interactions was constructed from publicly available datasets. sang et al. ( ) [ ] constructed low-dimensional knowledge graph embeddings from semmeddb relations and trained a long short-term memory (lstm) model using known drug therapies from therapeutic target database [ ] , proposing potential drugs us-ing the trained model. in this section, we first describe our data sources and the preprocessing steps that were taken to construct a literature knowledge graph from these data sources. next, we discuss the knowledge graph completion methods that we used to predict candidate drugs for covid- as well as the discovery patterns used for providing mechanistic explanations. lastly, we detail various evaluation schemes that we used to automatically validate our predictions. a workflow diagram illustrating our approach is provided in fig. . we constructed our biomedical knowledge graph primarily from semmeddb [ ] , a repository of semantic relations automatically extracted from biomedi-cal literature using semrep natural language processing (nlp) tool [ , ] . semrep-extracted relations are in the form of subject-predicate-object triples (also called semantic predications) and are derived from unstructured text in concepts are enriched with semantic type information (disease or syndrome, pharmacologic substance, etc.) and the relations are linked to the supporting article and sentence. semmeddb has supported a wide range of computational applications, ranging from gene regulatory network inference [ ] to in silico screening for drug repurposing [ ] and medical reasoning [ ] , and has also found widespread use for literature-based knowledge discovery and hypothesis generation [ , , [ ] [ ] [ ] . in its most recent release (version , dated / / ) , semmeddb contains more than m relations from more than in this work, we focused on a subset of semantic relations derived from the combination of pubmed and cord- datasets, predicted to be accurate and informative for drug repurposing. first, we eliminated relations involving generic biomedical concepts (i.e., relations in which both subject and object were present in a generic concept table of semmeddb such as pharmaceutical preparations) and relations with identical subject and object arguments. next, we excluded a subset of predicate types that were not expected to be useful for drug repurposing, such as part of and process of. the predicate types used are affects, associated with, augments, causes, coexists with, complicates, disrupts, inhibits, interacts with, manifestation of, predisposes, prevents, produces, stimulates, and treats. lastly, we also excluded the relations in which the sub- in the second step, we eliminated uninformative semantic relations using loglikelihood ratio (g ) and network degree centrality for the concepts (in-degree and out-degree). we assigned each semantic relation a g score indicating how strongly the terms within a triple are associated with each other [ ] . a high g score means that the observed and expected frequencies are significantly different, indicating that the triple is less likely to occur by chance. for computational purposes, we created two three-dimensional contingency tables with indices i, j, and k. the first table (ot) holds observed frequencies of a triple from the knowledge graph and the second table (et) contains the expected values assuming independence of terms in each triple. g was then calculated using the equation where n ijk is the cell i, j, k in ot, m ijk is the cell i, j, k in et, and t = n ijk . next, we normalized all three measures (g , in-degree, and out-degree) to the range [ , ] and summed them up into a final score. the lower the score, the more specific and informative the relation is. for example, the relation operative surgical procedures-treats-woman with high score is more general than relation interleukin- -affects-autoimmune diseases. we kept all relations for which the score value was less than a threshold value α. we manually tuned the α value to achieve a balance between specificity of relations and their variability. we kept % of all relations with the lowest score in the data set. we also kept all biomedical concepts that refer to covid- terms (cuis: c , c , c , c , c , c , c ). at the end of the preprocessing stage, the knowledge graph consists of nodes and relations. the precision of semantic predications generated by semrep vary by domain (e.g., molecular interactions are less precise than clinical relationships). to improve the precision of the relations used for drug repurposing, we extended the semrep accuracy classifiers previously proposed [ , ] . we fine-tuned a collection of transformer-based pretrained language models to classify semantic predications as correct vs. incorrect. we used the following models: vanilla bert (base size, cased and uncased) [ ] , biobert [ ] , bioclinicalbert [ ] , bluebert [ ] , and pubmedbert [ ] . to extend the coverage of our existing classifiers, we used predications annotated as correct vs. incorrect with respect to their source sentences. we leveraged annotations from a previous study [ ] (cohen's κ of . ) and annotated new predications. annotation guidelines generated in the previous study was used. two of the authors (hk and mf) and two health informat-ics graduate students annotated predications containing predicates of interest absent in the prior study (fleiss' κ = . , indicating moderate agreement). the resulting annotated set was split into / / training/validation/test sets. hyperparameters were determined empirically and the learning rate was set to × − , the batch size was , the maximum number of epochs was set to but early stopping was employed. optimization was done using the adam optimizer [ ] with decoupled weight decay regularization using betas ( . , . ) and decay . . the pooled output from the bert model was fed through a linear layer to produce logits that then underwent a softmax transformation to return class probabilities. a single tesla v gpu was used to train the models. we compared the performance of various above-mentioned transformers. the best classifier was then used to filter incorrect semantic predications. consider a knowledge graph g = (e, r, e), where e refers to a set of entities, r denotes a set of possible relations, and t stands for a set of triples in the form (h)ead-(r)elation-(t)ail, formally denoted as {(h, r, t)} ⊂ e × r × e. the aim of knowledge graph completion is to infer new triples (h , r , t ) such that h , t ∈ e and r ∈ r. in this setting, the knowledge graph completion problem could be represented as a ranking task in which we learn a prediction function ψ(h, r, t) : e × r × e → r which generates higher scores for true triples and lower scores for false triples. we explored three classes of knowledge graph completion methods: transe [ ] and rotate [ ] for translational models, distmult [ ] and complex [ ] for semantic matching models, and stelp [ ] for context-based encoding. these methods differ in the way that they encode entities and relations in a knowledge graph into a low-dimensional vector space (i.e., kg embedding). such distributed vector representation can be used for downstream reasoning and machine learning tasks. transe [ ] describes a triplet (h, r, t) as a translation between head entity h and tail entity t through relation r in a continuous vector space, i.e., h + r ≈ t, where h, r, t ∈ r d is the embedding of h, r, and t, respectively. to measure plausibility of relations transe employs a distance-based score function we choose transe because of its simplicity and good prediction performance. however, transe is able to model only one-to-one relations and fails to embed one-to-many, many-to-one, and many-to-many relations. to solve this problem, many other solutions have been proposed including rotate [ ] . rotate treats each relation in a complex vector space as a rotation from the head entity to the tail entity, i.e., s(h, r, t) = |h • r − t| l , where • is a hadamard product. we selected rotate as a counterpart to transe, as transe reportedly does not perform well on some data sets (e.g., b k family of data sets), which require symmetric pattern modeling. distmult [ ] is the simplest approach among semantic matching models. semantic triple encoder for link prediction (stelp) [ ] , is a contextbased encoding approach to knowledge graph completion. at its core is a siamese bert model that leverages sharing one set of weights across two models to produce encoded, contextual representations of the predications that are then where d is the set of correct triples, n (tp) is the set of corrupted triples for where γ is a scaling factor for the contribution of the contrastive loss. at inference, stelp considers every entity-context combination for a given partial predication, (h, r) to find (t) or (r, t) to find (h), and ranks every pair using the sum of the positive class probability and the scaled negative euclidean distance. we replaced the vanilla base bert model proposed in the stelp paper with biobert, trained on biomedical literature corpora. the unique predications remaining after preprocessing were each corrupted to produce five negative predications for a total of negative predications and a grand total of predications. the hyperparameters were set to the same values as in the original stelp paper and the learning rate was set to e- , the batch size was , the contrastive loss scaling factor was . . optimization was done using adam with decoupled weight decay with betas ( . , . ) and decay . . training was run for training iterations. ranking was done by adding the scaled contrast score to the positive class probability and entities ordered in descending rank order. all preprocessing was done using custom bash and python scripts. transe, rotate, distmult, and complex link prediction models were implemented in pytorch using the dgl-ke package [ ] for learning large-scale kg embeddings. the bert models were based on huggingface bert implementations using pytorch. pre-trained weights for biobert (biobert-base v . (+ pubmed m)) , bioclinicalbert , pubmedbert and bluebert (bluebert-base, uncased, pubmed+mimic-iii) came from various sources associated with each paper. stelp was also implemented using a combination of a hug-gingface bert model and pytorch. our source codes are also publicly available . discovery patterns are defined as a set of constraints that need to be satisfied for the discovery of new relations between concepts [ ] . herein, we used discovery patterns for two purposes. first, we explored open discovery patterns to identify drugs that can be repurposed for covid- . second, we used closed discovery patterns to propose plausible mechanisms for drugs identified via knowledge graph completion methods described above. discovery patterns are expressed in terms of predication pairs (or predication chains). in particular, we focused on the following discovery pattern: druga -inhibits|interacts with -conceptb and / / ). we focus on the latter category in our qualitative evaluation below. we semi-automatically generated a ground truth drug list, similar to the approach in other computational drug repurposing studies for covid- [ ] . we downloaded the interventions used in covid- drug trials from clinicaltrials.gov using the following search: https://clinicaltrials.gov/ct /res this yielded a set of clinical trials. we extracted the interventions from these studies and mapped the intervention terms to umls cuis using metamap (v ) [ ] and filtered the resulting concepts by their semantic groups [ ] , keeping only those concepts with the semantic group chemicals & drugs. we also considered the additional semantic types therapeutic procedure and gene or genome, which also appeared for some concepts in intervention lists. we removed the duplicates and some general concepts (e.g., therapeutic procedure, placebo) as well as incorrect mappings, which resulted in a final list of concepts. the automatic evaluation (below) was performed against this set. time slicing is an evaluation technique often used in literature-based discovery and link prediction tasks [ ] . the idea is to train models on data prior to a specific date and test them on data after that date and evaluate whether links that formed only after the cutoff date can be predicted from the trained model. in this study, we trained our models on semantic relations extracted from publications dated / / or earlier and tested whether they can predict the drugs that have been proposed for covid- since then or have been evaluated in clinical trials. this date was selected as cutoff, as it is the date on which who declared covid- a pandemic. it is also a date by which enough biological knowledge about sars-cov- had accumulated, although covid- therapies were still in their infancy, making it a suitable cutoff for time slicing experiments. all five link prediction models were automatically assessed using a link prediction evaluation protocol proposed by bordes et al. [ ] . suppose that x is a set of triples, Θ e be the embeddings of entities e, and Θ r be the embeddings of relations r. in the first, corruption step, we go through a set of triples and for each triple x = (h, r, t) ∈ x replace its head and tail with all other entities in e. each triple is corrupted exactly |e| − times. formally, the corrupted triple is defined as: where h = h and t = t. we employ the filtered setting protocol not taking into account any corrupted triple that already appears in the kg. in the second, scoring phase, original and corrupted triples are tested using the constructed classifier ψ. intuition behind this is that the model will assign a higher score to the original triple and a lower score to the corrupted triple. in the third, evaluation phase, the proposed link prediction models are assessed using three measures: mean rank (mr), mean reciprocal rank (mrr), and hits@k measure. mr is an average rank assigned to the true predication, over all predications in a test set: where rank h i and rank t i denote the rank position: where the indicator function i[p ] is iff p is true, and otherwise. mrr is the average inverse rank for all test triples and is formally computed as: hits@k measures the percentage of predications in which the true triple appears in the top k ranked triples, where k ∈ { , , }; formally: our aim was to achieve low mr and high mrr and hits@k. in addition, we also performed a qualitative evaluation. one of the authors (mf) used neo j browser to assess the plausibility of some of the drugs highly ranked by the knowledge completion models, guided by literature search and review, and following the closed and open discovery paradigms. we report the performance of the semantic relation accuracy classifier as well as the knowledge graph completion methods in this section. the full table of results for the comparison of various bert models for the accuracy classifier is included below ( table ) the link prediction results for all employed models are presented in table . for mr a lower score is considered better, for all others a higher score is considered better. the score for each method is the mean value over all triplets in the testing set. sion, l norm, learning rate η = . and regularization coefficient λ = × − . model training was limited to epochs. relatively small number of relations ( ) ensure that all entities and relations can be smoothly embedded into the same vector space. next, we use t-sne (t-distributed stochastic neighbor embedding) [ ] algorithm to graphically represent embeddings of computed concepts in a twodimensional space (figure ). t-sne algorithm enables reduction of highdimensional data into a low-dimensional space such that similar concepts are presented by nearby points. the plot demonstrates relatively good co-localization of selected concepts, especially for suspected covid- and paclitaxel. our results indicate that more complex knowledge graph completion models might be less efficient in drug repurposing tasks. theoretical considerations suppose that transe is outperformed by its successors [ , , ] . however, differences in performances among distmult, complex, and rotate are relatively small. all three models achieved low performance on mrr, hits@ , inherently model only one-to-one relations and fails to represent one-to-many, many-to-one or even many-to-many relations, it shows its efficiency in embedding a large-scale complex biomedical knowledge graph, such as the extended semmeddb used here. empirical evidence shows that distmult and complex usually perform well for high-degree entities, but fails with low-degree entities [ ] . because we eliminated highly frequent concepts due to their lack of informativeness, it is possible that this is reflected in lower performance scores of both models. the context-encoding model, stelp, showed rather poor performance in evaluation. one possibility is that the model was only able to learn high-level groupings for the predicates. this is likely the case as it was observed the model versus affects covid- etc., it did not learn more granular features that allow it to differentiate between subjects within the context of treats covid- . however, analysis of the t-sne embedding and the qualitative evaluation show that the model mostly clustered the ground truth drugs into a couple of large clusters. to further compare the drug rankings between transe and stelp, we performed the wilcoxon signed-rank test (p = . ), which indicates that no correlation was found between how the two models were ranking novel predications. spearman's rank correlation between the novel predication rankings for both models was found to be − . and this further supports the results of the wilcoxon test. table and table show that there is very little agreement between transe and stelp, particularly in the top rankings for each model. it is worth noting that there were items in common in the top rankings for both models. ranked triples for the specified model, calculating the absolute difference between the rankings from the two models for each of those triples, and calculating the statistics. for example, the triples that transe ranked as the top triples we gathered, the absolute differences of rankings between transe and stelp for those triples were calculated, and the statistics were calculated from those differences. the can be possible to explore larger graphs than that explored in this work. on the other hand, with adequately large computational resources, it may be possible to optimize stelp hyperparameters and train over multiple random seeds to generate a model that obtains better results than transe or rotate, which are limited by their smaller representational capacity. discovery patterns based on semantic relations provide an intuitive way of exploring potential mechanistic links between biological phenomena. neo j and cypher, its query language, are powerful tools that complement semantic relations nicely in quickly pinpointing promising research directions, although massive graphs present some challenges for effective query and retrieval. in addition, a domain expert is needed to sort out some of the noise in semantic relations (some of it obvious) due to text mining errors. however, given that predictions made by the knowledge completion models above are largely opaque, a human-in-the-loop discovery browsing approach based patterns [ , ] remains an effective alternative to these more complex approaches, and also complements them by providing potential explanations. the following classes of drugs have been used for the management of covid- so far: antivirals (e.g., remdesivir), antibodies (e.g., convalescent plasma), anti-inflammatory agents (e.g., dexamethasone), immunomodulators (e.g., interleukin inhibitors), anticoagulants (e.g., heparin), antifibrotics (e.g., tyrosine kinase inhibitors), and adjuvants (e.g., vitamin d) [ , ] . in addition, several trials have studied antimalarials (e.g., hydroxychloroquine) and antiparasites (e.g., ivermectin), but evidence from trials do not support their use. the knowledge graph completion models did not predict antivirals, antimalarials, and antiparasites, except for antivirals from the class neuraminidase inhibitors and the antimalarial artemisone. all the other drug classes and most of their members were predicted by the models. dexamethasone, currently considered the most effective drug for reducing mortality in patients receiving oxygen, was the highest ranking drug from the rotate model. it is possible that the models missed specific antivirals and antiparasites due to their mechanism of action, which usually involves binding to specific receptors, a relation type on which semrep does relatively poorly. despite this issue, qualitative assessment of the drugs predicted by the models was overall positive. using the open discovery pattern approach, we identified five promising drugs that were ranked highly and were not, to our knowledge, discussed in the literature, which we discuss below (paclitaxel, sb , alpha -antiplasmin, pyrrolidine dithiocarbamate, and butylated hydroxytoluene). the same approach also yielded other highly ranked substances, which are currently evaluaetd in clinical trials, such as quercetin, melatoninm losartan, estradiol, and simvastatin. note that the knowledge graph completion models predicted of these drugs (excluding sb , alpha -antiplasmin, and pyrrolidine dithiocarbamate). figure shows the resulting network from this discovery pattern generated by neo j browser. paclitaxel is used to treat several cancer types, including ovarian cancer, breast cancer, lung cancer, cervical cancer, and pancreatic cancer. it stabilizes the microtubule polymer and protects it from disassembly. chromosomes are thus unable to achieve a metaphase spindle configuration. this blocks the progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the g -phase of the cell cycle without cell division [ ] . the following patterns support the paclitaxel discovery: . paclitaxel-inhibits-interleukin- -causes-covid- . paclitaxel-inhibits-nf-kappa b-associated with-covid- . paclitaxel-inhibits-interleukin- , beta-associated with-covid- . paclitaxel-inhibits-granulocyte colony-stimulating factorassociated with-covid- . paclitaxel-inhibits-interleukin- -predisposes-covid- . paclitaxel-inhibits-interleukin- -predisposes-covid- . paclitaxel-inhibits-thromboplastin-associated with-covid- the first six patterns support a role for paclitaxel in alleviating the cytokine storm of covid- , triggered by dysfunctional immune response and mediating widespread lung inflammation. paclitaxel may plausibly help as an immunosuppressive therapy to immunomediated damage in covid- [ ] . thromboplastin (pattern ) is a complex enzyme found in brain, lung, and other tissues and especially in blood platelets and functions in the conversion of prothrombin to thrombin in the clotting of blood and may be elevated in patients with covid- . as pulmonary microvascular thrombosis plays an important role in progressive lung failure in covid- patients, paclitaxel may reduce the state of hypercoagulability by acting as an inhibitor of thromboplastin [ ] . the final pattern involves the interaction of paclitaxel with tlr . paclitaxel is known to have high affinity for tlr receptors. sars-cov- spike protein binds with human innate immune receptors, mainly tlr , increasing secretion of il- and tnf-α and neuroimmune response. this suggests that paclitaxel may dislocate sars-cov- spike proteins [ , ] . sb is a specific inhibitor of p α, which suppresses downstream activation of mapkap kinase- , involved in many cellular processes including stress and inflammatory responses and cell proliferation. the following patterns support the sb discovery: ity, especially in patients with comorbidities such as hypertension, diabetes, and coronary heart disease [ ] . the following patterns support the alpha -antiplasmin discovery: . alpha -antiplasmin-inhibits-plasmin-predisposes-covid- . alpha -antiplasmin-inhibits-fibrinogen-associated with-covid- . alpha -antiplasmin-interacts with-igy-associated with-covid- more specifically, plasmin may cleave a newly inserted furin site in the s protein of sars-cov- , which increases its infectivity and virulence in covid- . in addition, fibrinogen levels are higher in covid- patients and may contribute to hypercoagulability [ ] . by inhibiting plasmin and fibrinogen (first two patterns), alpha -antiplasmin may confer protection to covid- . in addition, pattern suggests a mechanism of protection via immunoglobulin y (igy). in the immunology field, igy against acute respiratory tract infection has been developed for more than years. several igy applications have been effectively confirmed in both human and animal health. igy antibodies extracted from chicken eggs have been used in bacterial and viral infection therapy. igy production has been proposed as immunization as an adjuvant therapy in viral respiratory infection caused by covid- infection [ ] . chicken immunized with alpha -antiplasmin and the peptide-specific antibody (igy) was isolated from the egg yolks of hens that could be used as potential protections for covid- patients [ ] . pyrrolidine dithiocarbamate is a family of drugs used for metal chelation, induction of g phase and cell cycle arrest. it binds to zinc and the resulting complex can enter the cell and inhibit viral rna-dependent rna polymerase [ ] . it is supported by the following patterns: . pyrrolidine dithiocarbamate-inhibits-nf-kappa b-associated with- . pyrrolidine dithiocarbamate-inhibits-interleukin- associated with-covid- . pyrrolidine dithiocarbamate-inhibits-tnf protein, humanassociated with-covid- the mechanisms suggested here are similar to those observed for the previous drugs. pyrrolidine dithiocarbamate contains antioxidants and prevents inflammatory changes. it inhibits the expression of il- and tnf, and nf-κb in the virus-infected chorion cells through its antiviral activity. it has been proposed for the treatment of influenza [ ] and it may have potential as a therapeutic option for covid- . butylated hydroxytoluene is a lipophilic compound useful for its antioxidant properties. it is widely used to prevent free radical-mediated oxidation in fluids and other materials and is generally recognized as safe as a food additive. it has been postulated in the past as an antiviral drug. open discovery identified the following relevant patterns: . butylated hydroxytoluene-inhibits-cd protein, human- . butylated hydroxytoluene-inhibits-free radicals-associated with- . butylated hydroxytoluene-inhibits-tnf protein, humanassociated with-covid- . butylated hydroxytoluene-inhibits-hydrogen peroxideassociated with-covid- the first pattern indicates butylated hydroxytoluene as an inhibitor of cd . studies have shown that the cd + cells were detected in the lung of patients with asthmatic and eosinophilic pneumonia, suggesting a crucial role for cd in the pathogenesis of such inflammatory diseases. cd is, potentially, a new therapeutic target for patients with intractable inflammatory disorders and tumors [ ] . therefore, by inhibiting cd , butylated hydroxytoluene may halt potential inflammatory responses in covid- . however, cd does not appear to be a major player in the physiopathology of covid- (the query "cd and covid- " did not return any results in pubmed). nonetheless this is noteworthy, because this pathway is suggested as a novel and important pathway for all immune responses [ ] . the crucial role of free radicals in covid- has been acknowledged and an antioxidative therapeutic strategy for covid- has been suggested [ ] . along these lines, patterns - point to antioxidant function of butylated hydroxytoluene by scavenging free radicals and inhibiting reactive oxygen species [ ] . our approach relies on accuracy of the predications extracted by semrep. semrep precision is about . and its recall around . [ ] . while the accuracy classifier helped us improve the accuracy of the predications used, the remaining errors were still significant, impacting the knowledge graph completion task. in addition, despite aggressive filtering, the graph formed by the relations in extended semmeddb is very large, making it difficult to apply computationally intensive models like stelp. in this study, we examined a sub-graph which, inevitably, results in a loss of information available to knowledge graph completion techniques. while we were still able to apply modeling techniques to a fairly large sub-graph focusing on drug repurposing, there exists a larger, complementary sub-graph that may provide further drug candidates. as noted above, the transe model benefited from hyperparameter tuning using a grid search method to find an optimal configuration. similarly, stelp would likely benefit from a similar tuning to find an optimal configuration. for example, a single linear layer was used on the pooled output from the biobert model to produce the logits when increasing the representational capacity of the linear layer, by depth or width, might allow for stelp to develop a richer model of the underlying space formed by the biobert contextualized embeddings. our methods were limited to knowledge from the literature. other types of biological data (e.g., protein-protein interactions, drug-target interactions, gene/protein sequences, pharmacogenomic and pharmacokinetic data) are likely to benefit identification of drug candidates, as shown to some extent by other studies [ ] , as well as our prior work [ ] . however, the computational resources needed for training models based on such massive data can be prohibitive. transe and similar methods seem more promising in that respect. lastly, with our in silico approach, we can of course only propose drug candidates for repurposing. to evaluate whether these drugs could indeed act as therapeutic agents for covid- , clinical studies are needed. however, the fact that we were able to identify some drugs known to have some benefit for covid- (e.g., dexamethasone) via purely computational methods that rely only on automatically extracted literature knowledge is encouraging. in this study, we proposed an approach that combines literature-based discovery and knowledge graph completion for covid- drug repurposing. unlike similar efforts that largely focused on covid- -specific knowledge, we incorporated knowledge from a wider range of biomedical literature. we used state-of-the-art knowledge graph completion models as well as simple but effective discovery patterns to identify candidate drugs. we also demonstrated the use of these patterns for generating plausible mechanistic explanations, showing the complementary nature of both methods. the approach proposed here is not specific to covid- and can be used to repurpose drugs for other diseases. it can also be generalized to answer other clinical questions, such as discovering drug-drug interactions or identifying drug adverse effects. as covid- pandemic continues its spread and disruption around the globe, we are reminded how the spread of infectious diseases is increasingly common and future pandemics ever more likely. innovative computational methods leveraging existing biomedical knowledge and infrastructure could help us plan for, respond to and mitigate the effects of such global health crises. drug repurposing is a key piece of this response, and our approach provides an efficient computational method to facilitate this goal. sb -inhibits-interleukin- -causes sb -inhibits-tnf protein sb -inhibits-interleukin- , beta-associated with sb -inhibits-nf-kappa b-associated with sb -inhibits-interleukin- -causes sb -inhibits-granulocyte-macrophage colony-stimulating factor -associated with sb -inhibits-macrophage colony-stimulating factorassociated with similarly to paclixatel, all patterns involving sb point to a potential inhibition of the hyperinflammatory response in covid- of the protein kinases p α in inflammation and innate immunity was found when the compound sb suppressed tumor necrosis factor (tnf) production in monocytes, and this resulted in inhibition of septic (infammatory) shock alpha -antiplasmin is a serine protease inhibitor responsible for inactivating plasmin draft landscape of covid- candidate vaccines placebo-controlled study of azd for the prevention of covid- in adults statement on astrazeneca oxford sars-cov- vaccine, azd , covid- vaccine trials temporary pause safety and immunogenicity of an rad and rad vector-based heterologous prime-boost covid- vaccine in two formulations: two open, non-randomised phase / studies from russia researchers highlight 'questionable' data in russian coronavirus vaccine trial results dexamethasone in hospitalized patients with covid- -preliminary report effect of hydroxychloroquine in hospitalized patients with covid- : preliminary results from a multi-centre, randomized, controlled trial current status of covid- therapies and drug repositioning applications covid- drug repurposing: a review of computational screening methods, clinical trials, and protein interaction assays drug repurposing: progress, challenges and recommendations artificial intelligence in covid- drug repurposing, the lancet digital health a data-driven drug repositioning framework discovered a potential therapeutic agent targeting covid- networkbased drug repurposing for novel coronavirus -ncov/sars-cov- a network medicine approach to investigation and population-based validation of disease manifestations and drug repurposing for covid- repurpose open data to discover therapeutics for covid- using deep learning network medicine: a networkbased approach to human disease literature based discovery: models, methods, and trends emerging approaches in literature-based discovery: techniques and performance review semmeddb: a pubmed-scale repository of biomedical semantic predications cord- : the covid- open research dataset translating embeddings for modeling multi-relational data rotate: knowledge graph embedding by relational rotation in complex sspace semantic triple encoder for fast open-set link prediction exploiting semantic relations for literature-based discovery a sars-cov- protein interaction map reveals targets for drug repurposing discovery of sars-cov- antiviral drugs through large-scale compound repurposing analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods anti-hcv, nucleotide inhibitors, repurposing against covid- virtual screening and repurposing of fda approved drugs against covid- main protease using integrated computational approaches to identify safe and rapid treatment for sars-cov- fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study drugbank: a knowledgebase for drugs, drug actions and drug targets chembl: a large-scale bioactivity database for drug discovery biogrid: a general repository for interaction datasets in silico discovery of candidate drugs against covid- predicting potential drug targets and repurposable drugs for covid- via a deep generative model for graphs network medicine framework for identifying drug repurposing opportunities for covid- fish oil, raynaud's syndrome, and undiscovered public knowledge., perspectives in biology and medicine graph-based methods for discovery browsing with semantic predications semantic medline for discovery browsing: using semantic predications and the literature-based discovery paradigm to elucidate a mechanism for the obesity paradox an interactive system for finding complementary literatures: a stimulus to scientific discovery using concepts in literature-based discovery: simulating swanson's raynaud-fish oil and migraine-magnesium discoveries using the literature-based discovery paradigm to investigate drug mechanisms exploring relation types for literature-based discovery context-driven automatic subgraph creation for literature-based discovery reflective random indexing and indirect inference: a scalable method for discovery of implicit connections finding schizophrenia's prozac emergent relational similarity in predication space embedding of semantic predications combining semantic relations and dna microarray data for novel hypotheses generation using literature-based discovery to identify novel therapeutic approaches predicting high-throughput screening results with scalable literature-based discovery methods exploiting literature-derived knowledge and semantics to identify potential prostate cancer drugs a new method for prioritizing drug repositioning candidates extracted by literature-based discovery literature-based discovery of new candidates for drug repurposing knowledge graph embedding: a survey of approaches and applications knowledge graph embedding by translating on hyperplanes a three-way model for collective learning on multi-relational data., in: icml embedding entities and relations for learning and inference in knowledge bases complex embeddings for simple link prediction holographic embeddings of knowledge graphs convolutional d knowledge graph embeddings modeling relational data with graph convolutional networks kg-bert: bert for knowledge graph completion a literaturebased knowledge graph embedding method for identifying drug repurposing opportunities in rare diseases modeling polypharmacy side effects with graph convolutional networks gredel: a knowledge graph embedding based method for drug discovery from biomedical literatures ttd: therapeutic target database the interaction of domain knowledge and linguistic structure in natural language processing: interpreting hypernymic propositions in biomedical text broad-coverage biomedical relation extraction with semrep the unified medical language system the unified medical language system (umls): integrating biomedical terminology augmenting microarray data with literature-based knowledge to enhance gene regulatory network inference a reasoning and hypothesis-generation framework based on scalable graph analytics enabling discoveries link prediction on the semantic medline network are abstracts enough for hypothesis generation? investigating the role of interleukin- beta and glutamate in inflammatory bowel disease and epilepsy using discovery browsing keep up with the latest coronavirus research extending the log-likelihood measure to improve collocation identification, master's thesis mining biomedical literature to explore interactions between cancer drugs and dietary supplements evaluating active learning methods for annotating semantic predications bert: pre-training of deep bidirectional transformers for language understanding biobert: a pre-trained biomedical language representation model for biomedical text mining proceedings of the nd clinical natural language processing workshop transfer learning in biomedical natural language processing: an evaluation of bert and elmo on ten benchmarking datasets domain-specific language model pretraining for biomedical natural language processing adam: a method for stochastic optimization training knowledge graph embeddings at scale an overview of metamap: historical perspective and recent advances aggregating umls semantic types for reducing conceptual complexity visualizing data using t-sne a capsule network-based embedding model for knowledge graph completion and search personalization pharmacologic treatments for coronavirus disease (covid- ): a review pathophysiology, transmission, diagnosis, and treatment of coronavirus disease (covid- ): a review how taxol/paclitaxel kills cancer cells the trinity of covid- : immunity, inflammation and intervention covid- : coagulopathy, risk of thrombosis, and the rationale for anticoagulation the role of tlr in chemotherapy-driven metastasis is tolllike receptor involved in the severity of covid- pathology in patients with cardiometabolic comorbidities? what goes up must come down: molecular basis of map-kap kinase / -dependent regulation of the inflammatory response and its inhibition elevated plasmin (ogen) as a common risk factor for covid- susceptibility igy-turning the page toward passive immunization in covid- infection purification of human α -antiplasmin with chicken igy specific to its carboxy-terminal peptide antiviral function of pyrrolidine dithiocarbamate against influenza virus: the inhibition of viral gene replication and transcription a new therapeutic target: the cd -myl system in immune responses tackle the free radicals damage in covid- understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (bht): a review we thank françois-michel lang, leif neve, and jim mork for their assistance with processing the cord- dataset with semrep and providing updates to semmeddb. we acknowledge tom rindflesch for his encouragement with the project. key: cord- -bvtchcbt authors: domingo-espín, joan; unzueta, ugutz; saccardo, paolo; rodríguez-carmona, escarlata; corchero, josé luís; vázquez, esther; ferrer-miralles, neus title: engineered biological entities for drug delivery and gene therapy: protein nanoparticles date: - - journal: prog mol biol transl sci doi: . /b - - - - . - sha: doc_id: cord_uid: bvtchcbt the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the design of new chemical entities (nce) for diagnosis and treatment of human diseases has relied on the discovery of active chemical drugs from a diverse library of compounds or from naturally occurring molecules. , further chemical modifications improve pharmacokinetic properties to obtain a final product with a known mechanism of action and decreased toxicity. nonetheless, using such approaches, the final products present low specificity for their target molecules, interacting with many other molecules and accumulating in some tissues, disturbing the correct homeostasis of the system. in some cases, the adverse effects of drug administration exceed pharmacological effect and despite the concise mechanism of action of the drug over the target molecule representing an improvement in the patient's state, the treatment has to be prevented or discontinued. in fact, although a maintained steady increase in the number of launched nce has been observed in the last years, the question arises whether this classical approach has already exhausted the discovery of innovative molecules. on the other hand, macromolecular new biological entities (nbe) have been used to supplement cellular deficiencies or to inhibit cellular pathways exploiting their relatively specific mode of action. proteins and peptides have been obtained first from their natural source or produced as recombinant versions after the development of genetic engineering techniques in the late s. however, the delivery of biological entities is sometimes hampered by its low half-life in the bloodstream by unspecific degradation, resulting in an expensive and ineffective process. nevertheless, some solutions have already been explored for biopharmaceuticals to increase solubility and stability and to reduce immunogenicity including postranslational modifications such as glycosylation and covalent conjugation of polyethylene glycol. thus, one of the main objectives in the use of drugs (for either nce or nbe) is the need to optimize the delivery system to reduce the pharmacological dose which would consequently represent a concomitant reduction in toxicity and cost. in that scenario, new delivery approaches have been implemented using biological interactions such as antigen-antibody binding (immunoliposomes) or more sophisticated interactions including the binding between nutrient concentrator sparc (secreted protein acidic and rich in cysteine) and albumin in the treatment of some types of cancer (abraxane ). , proteins can be then used for their targeting qualities as molecular delivery vehicles both for the specific delivery of drugs or nucleic acids in gene therapy approaches and by themselves as therapeutic molecules. one of the interesting characteristics of proteins is their ability to form intermolecular driven complexes as sophisticated and structurally perfect as in the case of viral capsids. in addition, through the use of genetic engineering, recombinant proteins can be tuned to include additional properties to optimize drug delivery and nucleic acid delivery in gene therapy. in this chapter, the main available strategies to develop protein-based nanovehicles or biopharmaceuticals will be described. in this context, several parameters will be defined such as proper formulation, stability, immunogenicity, and delivery to the correct cell type and cell compartment. modular protein engineering, virus-like particles (vlps), and other self-assembling entities are envisioned as modulatable novel protein nanoparticles able to include many desirable properties in the correct delivery of drugs and nucleic acids. finally, some successful examples of protein nanoparticles on the market will be described in addition to protein products currently in clinical trials and under preclinical research in order to envision which type of protein nanoparticles will be available soon on the market. with the therapeutic molecule to generate a vehicle capable of being transported in the blood if a systemic administration is needed and retaining a significant stability before reaching the target cell. , in addition, the biological system poses specific barriers that have to be overcome such as membranes (cytoplasmic, endocytic, and nuclear), degradation (protease degradation induced by acid denaturalization in lysosomes, cytosolic proteosomes, and nucleases), cytosolic transport, and nuclear entry if necessary. , for central nervous system therapies, the blood-brain barrier (bbb) represents the main bottleneck, and for that, a specific strategy has to be designed. furthermore, the therapeutic complex has to be flexible enough in order to release the therapeutic molecule in the specific cell compartment. thus, several protein motifs have been described to overcome each and every process described earlier so that a modular multifunctional protein can be generated including those modules that are necessary to achieve its goal. in order to get a rational construction of the multifunctional vector, each step has to be carefully taken into account so as to overcome every step which is needed to achieve its final goal (table i) . the dna/rna condensation or drug interaction with the protein vector is a critical step in the formulation of protein nanoparticles for gene therapy. they have to remain attached to the vector during the whole transport process through the body and the cell until it can be released in the desired localization within the target cell. highly positively charged peptides containing a large number of arginines or polylysines have been used to promote electrostatic interactions since nucleic acids are highly negatively charged molecules. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] natural dna-condensing proteins as nuclear histidines or protamines can also be used to bind nucleic acids. [ ] [ ] [ ] [ ] protamine, which is the protein that replaces histidines during the spermatogenesis process, is a sperm chromatin component and just as the histidines do, it has very high dna condensation ability to protect nucleic acids form cytosolic endonucleases. , in addition, as soon as the complex reaches the cellular nucleus, protamine is degraded by chromatinremodeling proteins, releasing the transported dna allowing its expression. , in contrast, polycationic dna condensation modules such as polylysines and polyarginines-even they can present higher dna condensation ability depending on the polycationic chain length-usually present lower dna-releasing ability, interfering negatively with the accessibility of cellular transcription factors and dna expression capacity. all these dna condensation modules described above interact with any dna that is incubated in an unspecific way. however, there are proteins such as gal that are able to recognize specific dna sequences [ ] [ ] [ ] and that permit to bind and condensate specific dna sequences in the final vector. in many cases, the multifunctional protein vector is in vivo administrated by the systemic route in order to travel in the blood and reach the target cells. that exposes the vector to all blood components, making it susceptible to be degraded. thus, it is completely necessary that the vector remain in the blood long enough to be able to reach the target cells. it has also been described that naked dna has an estimated half-life in blood of minutes ; so protein nanovehicles in gene therapy, among other properties, are intended to protect nucleic acids from degradation. one important factor when the vector is exposed to the blood is that it can be recognized by the immune system components and produces an immune response against the vector. thus, it is also very important to try to make the vector as less antigenic as possible in order to avoid being degraded or even being toxic to the organism. peptide uptake or internalization involves a step before the protein binding to the cell surface. this attachment can be either specific or unspecific but in all cases the promotion of its internalization is required. positively charged peptides usually bind the cellular surface by unspecific electrostatic interactions with the negatively charged cell surface proteoglicans. this kind of peptides can be used in the multifunctional protein if specific targeting is not required. cell-penetrating peptides (cpps) have been widely described as unspecific cell-binding and internalization peptides [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (see also the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume). however, specific interactions can be obtained by incorporating cell receptor ligands if cell or tissue targeting is required for the therapeutic action. moreover, some of those ligand-receptor interactions promote the ligand-receptor complex internalization. many peptides have been described in the literature as receptor-specific ligands so any of them can be added to the multifunctional proteins in order to confer them cell specificity. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the most natural specific ligands that can also be used for cell targeting are monoclonal antibodies. , [ ] [ ] [ ] in addition, if no specific peptides are available for an intended target, new specific binding peptides can be found by using phage display or combinatorial chemistry. . endosomal escape several internalization pathways are possible depending on the vector properties, , including endocytosis (clathrin/caveolae-mediated, clathrin/ caveolae-independent), macropinocytosis, and non-endocytic pathways. it is known that more than one internalization pathway can be performed at the same time but usually the peptide-based vector uses endocytic pathways. moreover, it seems that proteins that interact with a specific cellular receptor are internalized by the clathrin-mediated endocytic pathway. most of the generated endosomal vesicles will converge to late endosomes that eventually will fuse with cellular lysosomes. , remaining in the cellular endosomes, the multifunctional protein will be degraded, so it is strictly necessary that the internalized multifunctional proteins be released into the cellular cytoplasm escaping from degradation. several peptides have been described that are able to promote endosomal escape and can be classified into two types depending on their escape mechanism: fusiogenic peptides and histidine-rich peptides. the fusiogenic peptides are small peptides that have hydrophobic amino acids (aa-s) interspersed at constant intervals with negatively charged aa-s. , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] thus, when early endosomes become late endosomes, their low ph induces a conformational change in the peptide, which adopts a alpha-helix structure, in an amphipathic structure able to fuse with the endosomal membrane, leading to pore formation and releasing all the endosomal content into the cell cytoplasm. the histidine-rich peptides are small peptides with a high histidine content whose endosmolytic activity is mediated by a mechanism called ''proton sponge''. , , [ ] [ ] [ ] [ ] when the endosomal ph becomes low in late stages, the imidazole groups of the histidines are protonated and attract endosomal cl À ions, buffering against the proton pump. thus, the endosomes collapse by an osmolytic swelling process and the endosomal content is released to the cell cytoplasm. further details are given in the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume. once the protein has achieved the cellular cytosol, it can be degraded by cellular proteases or by the cellular proteosome system. it is important to avoid this process, especially if the protein has to reach the cellular nucleus. if the final target of the nanoparticle is the cellular cytoplasm, it is necessary that it remain there at least long enough to perform its therapeutical action. several peptide proteosome inhibitors have been described that are able to avoid this type of protein degradation. by adding these peptides to the final protein vector it is possible to protect it and enhance cytoplasmatic stability. epstein-barr virus nuclear antigen (ebna ) contains a proteosome inhibitor consisting of glycine-alanine repeats able to prevent proteosomal proteolysis. it has been shown that a minimum of aa-s gly-ala repeats are necessary to achieve such protective activity. [ ] [ ] [ ] if the protein vector is carrying nucleic acids (dna or rna), degradation by the cytosolic endonucleases has to be taken into account, so it is also very important to protect this nucleic acid in order to maintain its integrity. some dna/rna condensing peptides as protamines also protect the dna against cytoplasmic endonucleases and enhance its stability as has been described above. the cellular cytoplasm is a very crowded and compartmentalized environment where cellular organelles and cytoskeleton make the free diffusion of macromolecules such as protein vectors difficult. however, cytoskeleton elements such as microtubules are used by endosomes and other cytosolic macromolecules for intracytosolic mobility. dyneins have been described as being capable of carrying those macromolecules and endosomes along the microtubules in a retrograde transport toward the nucleus. some small peptides that are able to bind dyneins have been identified. they can be added to the multifunctional protein vector in order to mediate an intracytosolic mobility toward the cellular nucleus. several dynein-binding proteins have been identified in viruses that are able to use this transport system. comparing those protein sequences, a consensus peptide sequence (kstqt) that is able to bind to the dynein lc light chain has been identified. molecules lower than kda/ - nm are able to enter in the cellular nucleus by passive diffusion. however, macromolecules higher than kda/ - nm generally require an active transport system through the nuclear pore system. this transport mechanism generally requires a specific targeting signal peptide named nuclear localization signal (nls). these signaling peptides are usually rich in basic aa-s, which are recognized by the cellular importines and actively transported through the nuclear pore. , monopartite or bipartite nls sequences which are nls peptides that have one or two nls recognized sequences respectively have been described. thus, these peptidic sequences can be added into the final multifunctional protein if nuclear localization is required in order to express a carried dna. it has been reported that a single nls sequence is sufficient to transport the vector to the nucleus and that a large number of nls sequences can result in inhibition of its activity. one of the most used nls signal peptides are fragments derived from the - aa-s of the simian virus sv large tumor antigen (t-ag). other nls sequences can be found in gal , protamines, or tat. , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it is important that when the transported dna reaches the cellular nucleus, it has to be released in order to be accessible to the nuclear transcription factors and achieve the desired expression level. thus, while designing the multifunctional protein vector, this aspect has to be taken into account. once the dna has been released in the cell nucleus, it will be necessary to control its expression level depending on which therapeutic action is being promoted. when the goal is to kill a cell as in cancer therapies, the uncontrolled dna expression levels would not be a problem. however, when a specific protein expression level is required, achieving good control is very important. some expression systems have been developed that can be pharmacologically regulated by oral drug formulation. cell-specific promoters and enhancers can be also used in order to confer high cell specificity to the therapy. , d. ways to get over the bbb the bbb is a hermetic barrier that only allows nonlipophilic molecules smaller than da to cross it. however, some human proteins such as insulin, transferrin, insulin-like growth factor, or leptins are able to go across it by receptor-mediated transporters. thus, the most important factor limiting central nervous system-targeting therapeutics is the presence of the bbb. finding the way to cross it will be the main challenge. some peptides have been described that are able to reach the brain crossing the bbb. moreover, it has been seen that they can be associated with another molecule and transported through the barrier. thus, they could be interesting candidates to be included in the multifunctional vectors if central nervous system targeting is required. , , , antibodies have also been described that bind transferrin and insulin receptors and that are able to cross the bbb efficiently. they can be conjugated with large molecules, allowing its translocation to the central nervous system. , , [ ] [ ] [ ] synthesis, and rational design the development of genetic engineering techniques has increased the natural repertoire of proteins for the design of useful and/or valuable proteins with the aim to obtain new proteins with desired functions. there are three main strategies leading to the construction of engineered proteins: (a) direct evolution, (b) de novo protein design, and (c) rational design. directed evolution has developed quickly to become a method of choice for protein engineers in order to create enzymes having desired properties for all kind of processes. over the past decade, this technique has become a daily part of the molecular toolbox of every biochemist. this is emphasized by the increasing number of publications about the subject. in nature, evolution and creation of new functionalities is achieved by mutagenesis, recombination, and survival of the fittest. directed evolution mimics this and is a process of iterative cycles of producing mutants and finding the mutant with the desired properties. mutations can be introduced at specific places using site-directed mutagenesis or throughout the gene by random mutagenesis. several mutagenesis techniques have been developed in order to avoid codon bias. , the first technique used to mimic evolution was dna shuffling. this method is based on the mixing and subsequent joining of different related small dna fragments in order to form a complete new gene. in the process of shuffling, the recombination frequency is dependent on the degree of homology. a high level of recombination is important to get all possible combinations of mutations. since recombination can be biased, several methods to overcome problems arising from the use of shuffling in the early years were tackled by novel strategies, all having their own advantages and disadvantages. the products obtained by these methods have to be screened for desired qualities and not all of them can be easily screened. de novo protein design offers the broadest possibility for new structures. it is based on searches for amino acid sequences that are compatible with a three-dimensional protein backbone template using in silico techniques. several research groups in the field have applied in silico methods to design the hydrophobic cores of proteins, with the novel sequences being validated with experimental data. in silico protein design has allowed novel functions on templates originally lacking those properties, modifying existing functions, and increasing protein stability or specificity. beyond any doubt, intense research activities are ongoing in the field, the potential of which is simply enormous. so far there have been numerous examples of full sequences designed ''from scratch'' that were confirmed to fold into the target three-dimensional structures by experimental data. the zinc-finger protein designed by dahiyat and mayo was the first one to appear by this method. rational design of proteins is based on the modification or insertion of selected amino acids or domains in a polypeptide chain backbone to obtain proteins with new or altered biological functions. when using that strategy, a detailed knowledge of the structure and function of the backbone protein is needed to make desired changes. this generally has the advantage of being inexpensive and technically feasible. however, a major drawback of this approach is that detailed structural knowledge of a protein is often unavailable or it can be extremely difficult to predict the effects of various mutations. modular engineering enables, by using simple dna recombinant techniques, the construction of chimerical polypeptides in which selected domains, potentially from different origins, provide the required activities. an equilibrate combination and spatial distribution of such partner elements has generated promising prototypes, able to deliver expressible dna or molecules to tissue culture but also to specific cell types in whole organisms. modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of dna or drugs present an attractive approach for the development of self-assembling vectors for targeted gene or drug delivery. one of the first examples was described by the group of uherek et al. they combined a cell-specific target module (antibody fragment specific for the tumor-associated erbb antigen), a dna-binding domain (gal ), and a translocation domain for endosomal escape. in this context, many strategies for the construction of safer vehicles are being explored and the number of nonviral prototype vectors for gene and drug delivery is noticeably increasing. here, the common steps that an approach like this might explore are presented ( fig. ) . when designing a new protein for drug or gene delivery there are many critical aspects, namely (a) design of the vehicle itself, required functions, stability, etc.; (b) production of the protein, suitable expression system, purification procedure, scaling up process, etc.; (c) characterization of the vehicle by physicochemical and functional tests; and finally (d) the administration route and regulatory guidance for biological products. although all these aspects belong to different disciplines, they have to be overviewed together. here, the major needs of a modular protein for gene and drug delivery are presented. to enhance the physicochemical stability of the cargo molecules and their resistance to nuclease/protease-mediated degradation, protein vehicles should ideally exhibit, like their natural counterparts (viruses), nucleic-acid binding and condensing properties. such abilities are, in general, conferred by cationic segments of the main scaffold molecules that interact with nucleic acids, mainly through electrostatic interactions. in addition, such complexes need to efficiently release the nucleic acid in the nucleus (if the cargo is a therapeutic gene), for which endosomal escape is required. such functions have been found in some peptides in many natural molecules and they are suitable for functionalizing protein vehicles. the ability to bind a particular cell type with high specificity is especially significant in a systemic delivery in which appropriate biodistribution and tissue targeting are essential. for nuclear targeting, only naked short nucleic acids can freely enter the nucleus of nondividing cells via free diffusion through the nuclear pore. large molecules require active transport mediated by nlss that are often found in viral proteins. because the molecular mass of plasmidic dna varies from to to mda, dna that is to be expressed, and essentially any macromolecular complex for nucleic acid delivery, requires nlss. the role and types of functional modules peptides used for all these purposes will be discussed in depth in the following sections. in vivo experiments finally, which protein or peptide is better for a given cargo is to be determined empirically and only few rules can be taken literally. , c. production of protein nanoparticles some steps in the production of a protein-based vehicle after molecular cloning such as protein production and protein purification might be experimentally labor intense with a variable success rate. for that reason, when small proteins are needed, solid-phase peptide synthesis guarantees the process. however, the classical procedure of biological production allows scaling up the process in most of the cases and the production of larger polypeptides and fulllength proteins. generally, in protein nanoparticle approaches, the protein is composed by different modules of natural sources such as the cell-penetrating peptide transactivator of transcription (tat) derived from the tat of the human immunodeficiency virus (hiv) or artificial sequences not present in any organism such as the polylysine dna-condensing sequence. once it has been defined which modules will be part of the protein, it is important to define the order they will have in the final construct. it has been demonstrated by boekle and coworkers using melittin conjugated to polyethylenimine (pei) that depending on the side of the linkage (c-or n-terminus), the lytic activity could be changed. some other modules have the need to be in a determined position for its correct function. when producing a protein for gene or drug delivery, it is important to know the origin of its domains to choose the most suitable expression system for its production. for instance, if any module naturally carries a posttranslational modification that is essential for its biological function, the expression system chosen will have to be able to reproduce the same crucial modification. the main biological production systems for protein drugs are described below. escherichia coli is the most widely used prokaryotic organism for the expression of recombinant proteins. the use of this host is relatively simple and inexpensive. added advantages include its short duplication time, growth to high cell densities, ease of cultivation, and high yields of the recombinant product. however, since it lacks fundamental prerequisites for efficient secretion, recombinant proteins manufactured by e. coli systems are mainly produced as inclusion bodies. , moreover, posttranscriptional modifications are not achieved with this system. there are many examples of proteins for gene delivery produced in e. coli with probed efficiency. , like e. coli, yeasts can be grown cheaply and rapidly and are amenable to high-cell-density fermentations. besides possessing complex posttranslational modification pathways, they offer the advantage of being neither pyrogenic nor pathogenic and are able to secrete more efficiently. species established in industrial production procedures are saccharomyces cerevisiae, kluyveromyces lactis, pichia pastoris, and hansenulapolymorpha. s. cerevisiae is the best genetically characterized eukaryotic organism among them all and is still the prevalent yeast species in pharmaceutical production processes. in spite of their physiological advantageous properties and natively high expression and secretion capacity, the employability of yeasts in some cases, however, might reach a limit, particularly when the pharmacological activity of the product is impaired by the glycosylation pattern. in such cases, either a postsynthetic chemical modification has to be considered or the employment of more highly developed organisms. most examples of nanoparticles produced in yeast are for vlps. animal cell expression systems show the highest similarity to human cells regarding the pattern and capacity of posttranslational modifications and the codon bias. however, their culture is more complicated and costlier and usually yields lower product titers. among the known systems, insect cells infected by baculovirus vectors have reached popularity since they are considered to be more stress-resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. for commercial application, scale-up related questions have to be solved. [ ] [ ] [ ] preferably applied in pharmaceutical production processes are mammalian systems like chinese hamster ovary (cho) cells and baby hamster kidney (bhk) cells. these systems are genetically more stable and easier to transform and handle in scale-up processes, to grow faster in adherent and submerged cultures, and to be more similar to human cells and more consistent in their complete spectrum of modification. in some cases, mammalian cell systems can be the only choice for the preparation of correctly modified proteins. peptides, being complex and unique complex molecules with regard to its chemical and physical properties, can be produced synthetically by the solidphase method. , this technology can be used to avoid problems related to biological production. general advantages of synthetic peptides are that they are very stable compounds, solid-phase chemistry produces highly standardized peptides, and the crucial polycation component is provided by a ''natural'' polycation, thus minimizing toxicity. however, some disadvantages related to synthetic peptides have been reported such as the difficulty to synthesize long and well-folded oligopeptides, peptides with multiple cysteine, methionine, arginine, and tryptophan residues due to technical limitations or production cost. when working with protein nanoparticles, it is very important to characterize them physically and functionally in order to understand their behavior. the size and charge of protein/cargo particles are crucial properties which influence rates of diffusion, binding to polyanionic components of connective tissues, transversal of anatomical barriers, binding of serum proteins, attachment to cells, and mechanisms of endocytosis, among other factors. stability in physiological salt solutions is a key issue for in vivo delivery, as salt is found everywhere in the body. mixing a multivalent polycation and dna results in electrostatic binding of both molecules, with charge neutralization of dna and a particle formation named conjugate. charge neutralization can be easily seen by retardation gel assays and particle formation by dynamic light scattering (dls). dls is a good method to see particle formation but not to quantify relative number of particles of different sizes. to visualize particles, many groups have used transmission electron microscopy (tem) , with good results while others have used fluid particle image analyzer (fpia) to photograph individual particles in physiological solutions. the net charge of protein/cargo particles is an important variable. generally, optimal gene delivery for cell lines requires a net positive charge but, as stated previously, it has to be determined empirically. one of the best techniques to determine the net charge is by calculating the zeta potential that measures the electrophoretic mobility of particles. despite the fact that physical characterization is a key element, understanding and testing the functionality and pharmacokinetics of a gene or drug is the most important part of its development process. most of the initial tests are done using cell lines in in vitro experiments using reporter genes, rna, or drugs. , quantifying the percentage of transfected cells or drug-induced changes is a very valuable tool to evaluate nanoparticle performance in both nuclear and cytoplasmic delivery, respectively. in addition, in vitro experiments may be designed to select a candidate for the in vivo experiments from a group of possible therapy vectors. the quantitative kinetics of particle binding, the molecular basis of particle interactions with target cell membranes, the efficiency of particle internalization, and endosomal escape are all poorly understood. interaction of particles with plasma membranes prior to protein internalization can be either unspecific or specific. untargeted delivery normally is the consequence of electrostatic interactions between anionic ligands in the cell surface and cationic components of the vehicle. on the other hand, targeted delivery to specific membrane molecules is a more sophisticated approach. it aims to improve cell specificity and efficiency, by directing to molecules, only expressed or overexpressed in a particular cell type, that initiate internalization by endocytosis. targeting moieties include many types of molecules and is discussed afterwards. internalization of particles, its mechanisms, and kinetics are not well known and most studies about nanoparticle delivery do not focus on this aspect. there are several endocytic pathways each initiated by different ligands. enhancing the delivery by addition of chloroquine, a synthetic molecule used primarily for the prophylaxis and treatment of malaria that disrupts endosomes, is an accepted parameter to demonstrate endosomal localization of particles. endosomal escape is the area most intensively investigated but is poorly understood. an important practical point to note is that some reagents that are used can be toxic. to enhance this step, anionic fusogenic peptides can be used. these peptides fuse to membranes in an acidic-dependent manner causing its disruption. in gene delivery approaches, translocation of dna expression plasmids into the cell nucleus involves an active, energy-dependent process through the nuclear pore complex. directly injected dna into the cytosol is usually, but not always, poorly transferred to the nucleus , and because of that, the use of proteins carrying cationic nuclear-localizing sequences (such as that of sv large t antigen) has been widely used to overcome this step. iv. natural self-assembling protein nanoparticles: vlps ideal drug delivery and gene therapy vehicles must accomplish some desired features such as appropriate packaging size for its cargo, target cellspecificity, safe and efficient cargo delivery, and protection against immune recognition, or capability to escape immune recognition. moreover, these vehicles must avoid inflammatory toxicity and rapid clearance. in this context, viral vectors have been exploited as one of the vehicles of choice. viruses are nano-sized ( - nm) supramolecular nucleoproteinbased entities, covered or not with a lipid bilayer (enveloped/nonenveloped viruses) that satisfy, into relatively simple structures, outstanding properties and functions that are relevant to drug and gene delivery. viruses are able to recognize and interact specifically with cells by receptor-mediated binding, internalize, escape from endosomes, and uncoat and release nucleic acids in different cellular compartments. they are also capable of transcribing and translating their viral proteins to self-assemble into new infectious virus particles and exit the host cell. , [ ] [ ] [ ] despite all these relevant properties of viral vectors or some other rising vehicles in drug and gene delivery such as cationic liposomes, their therapeutic use presents some limitations and risks because of the complexity of production, limited packaging capacity, insertional mutagenesis and gene inactivation, low probability of integration, reduced efficacy of repeat administration or reduced expression overtime, unfavorable immunological recognition or strong immune response against vehicle and transgene, inflammatory toxicity, and rapid clearance. , in this context, virus capsids or vlps, produced by recombinant capsid proteins but lacking the viral genome, have noticeably emerged as a safer alternative to viral vectors. a. structure of protein self-assembled nanovehicles vlps are classically described as self-assembling, nonreplicative and nonpathogenic, highly organized supramolecular multiprotein nanoparticles (coats) (ranging from to nm) that can be formed from the minimal spontaneous self-assembling of one or more viral structural capsid proteins. it has been described that the self-assembling process of the structural viral proteins for vlp formation involves both spontaneous assembly, under favorable experimental conditions, and the requirement of scaffold proteins as catalysts. , therefore, vlps are considered protein ''coats'', ''shells'', or ''boxes'' that lack the viral genome, still conserve the structure, morphology, and some properties of viruses. some of these properties such as cellular tropism and uptake, intracellular trafficking, membrane translocation, and transfer of nucleic acids or molecules across the cytoplasmic, endosomal, and nuclear membranes are important for drug delivery and gene therapy. , , , [ ] [ ] [ ] usually, the degree of similarity of vlps and their viruses depends on the number of proteins incorporated into the constructs. , since the first description in of the viral dna packaging into mouse polyomavirus (mpyv) vlps and its transduction in vitro, vlps of different viruses such as papillomaviruses, [ ] [ ] [ ] hepatitis b, c, and e viruses, [ ] [ ] [ ] polyomaviruses, vlps offer some structure, dynamics, characteristic features, and functions that make them appealing bionanomaterials to be exploited in the biomedicine arena as drug and gene delivery vehicles and are discussed in detail afterward. on the one hand, viral coat proteins have the ability to spontaneously selfassemble, which ensures the formation of highly organized, regular, repetitive structurally stable, and very low morphological polydisperse particles that provide useful properties to be used as scaffolds for bioimaging, synthesis of bionanomaterials, and as nanocarriers in drug and gene therapy. in addition, homogeneity of particle size and composition is a desired production factor when developing therapeutic molecules. the overexpression of structural viral proteins in a convenient expression system renders recombinant proteins capable of being folded and assembled in discrete organized nanoparticles with a defined size corresponding to the natural capsid geometry. [ ] [ ] [ ] moreover, even though vlps are structurally stable particles, some biochemical and structural studies have observed that viral capsids and bacteriophages may show some structurally dynamic properties varying in shape, size, or rearrangements of the coat proteins, in response to different factors such as ph. [ ] [ ] [ ] [ ] on the other hand, vlps are considered biologically safe nanostructures since they are not infectious (lack of viral genome) and do not replicate, representing a safer alternative to viral vectors. , [ ] [ ] [ ] [ ] however, they can elicit immune and inflammatory responses, especially when repeated administration is needed. it has to be also noted that when used in vaccination, vlps could show excellent adjuvant properties and the majority of vlps stimulate strong cellular and humoral immune responses as direct immunogens. it has been suggested that recombinant vlps derived from infection of insect cells with baculovirus or even those derived from prokaryotic systems could be contaminated with different residual components of these host cells, contributing those impurities to the adjuvant properties. one interesting property of vlps is that coat viral proteins present an enormous elasticity and adaptability to be modified chemically and/or by protein genetic engineering , , to incorporate multiple directed functionalities, in order to be addressed in biomedical applications such as drug delivery or gene therapy. it has been recently reviewed that chemically and/or genetically modified vlps, including cpmv, ccmv, ms , m bacteriophages, and other virus-based nanoparticles, , could maintain their structural integrity and improve their physical stability and, moreover, these modifications could also confer desired cell-targeting properties to the nanovehicle. [ ] [ ] [ ] , , vlps can be successfully engineered with spatial precision to incorporate (attached or genetically displayed on the surface) targeting tissue-specific ligands such as epidermal growth factor (egfr) and antibodies, or other molecules such as oligonucleotides, peptides, gold, and other metals, target proteins, carbohydrates, polymers, fluorophores, quantum dots, drugs, or small molecules. , , moreover, one of the potential benefits of such modifications is that the specific geometric rearrangement confers precise recognition patterns. , furthermore, accessibility of the materials carried within the particle and the ability of inclusion and separation of nucleic acids, small molecules, and unusual cargoes with appropriate charge is another outstanding feature and key advantage of vlps that has also made them excellent vessels for gene and drug delivery. , as described above, vlps can be used as empty nanocarriers to transport molecules chemically attached on their surface or can be loaded ex vivo with therapeutic small molecules such as drugs, dnas, mrnas, sirnas, oligonucleotides, quantum dots, magnetic nanoparticles, or proteins. , , vlps of different papillomavirus and polyomavirus have been widely characterized and used for directed delivery in biomedical applications. , , , , , osmotic shock and in vitro self-assembling of vlp subunits in the presence of the cargo have been the two main strategies used to packaged nucleic acid or other small molecules. it has to be taken into account that some attachment of the cargo on the vlp surface can occur. besides, diversity of natural tropism including liver for hepatitis b vlps, spleen for some papillomavirus and polyomavirus vlps, antigen-presenting cells for certain papillomavirus vlps, and glial cells for human polyomavirus jc (jcv) vlps, among others is one of the key advantages offered by vlps providing a wide spectrum of specific targeting and distribution profiles depending on the directed application. although each vlp has its own characteristic receptors, entry pathway, and intracellular trafficking, it has been demonstrated that tropism of vlps could be customized, modifying the residues identified as ligands of the cellular receptor on vlps' surface or even varying the delivery routes. , , another key advantage of vlps is that they can be easily produced by using a wide range of hosts and expression systems, each of them with its own conditionings. in the past years, there has been an increasing need to improve and optimize efficient large-scale production systems, process control and monitoring, and up-and down-streaming processes. , , , production of vlps usually involves transfection of the cell host expression system of choice with a plasmid encoding one or more viral structural proteins, further and rigorous purification for the removal of immunogenic cellular contaminants, and quality control of the produced vlp and encapsulation of the cargo ex vivo before administration. , the most frequent and convenient expression systems, adaptable to large-scale processes are ( ) yeast cells , , ( ) bacteria , , ( ) green plants infected with modified viruses , , and ( ) cell-free systems. , the preparative and large-scale manufacture of vlps in some of these hosts has been reviewed by pattenden et al. and can be classified into two main methods of bioprocessing: in vivo and in vitro systems. in addition, the capability of in vitro dissociation and reassociation of vlps contribute to the application of easy and more accurate purification methods than those of viral vectors. , furthermore, depending on the expression system, the resulting vlp might be significantly different even though expressing the same viral proteins. thus, a broad spectrum of vlps could be customized depending on the vlp type, the number of proteins needed for vlp assembling, and the targeted final application. , as described above, vlps have great potential as nanocarriers in drug and gene delivery. at the same time, although there is an increasing flow of developments in this area, these vehicles also present some limitations that should be addressed and taken into account, such as residual cellular components, variable yield of functional vlps after disassembly/reassembly process, immunostimulation and unsuitability for repeated administration, tolerance to the transgene, ineffective therapeutic molecule loading, and low transfection rates. protein nanoparticles engineered for drug delivery and gene therapy due to their versatile nanoparticulate structure and morphology, and nonreplicative and noninfecting nature combined with their natural immunogenic properties and ease production, vlps have principally emerged as an excellent alternative tool to attenuate viruses for vaccination. and ebola virus. , although vlp-based vaccines have been primarily developed for their use against the corresponding virus, in the last decades genetic engineering or chemical modifications have been applied in order to generate chimeric vlps. thus, on the one hand, commonly short heterologous peptide epitopes or full proteins that are unable to form vlps or that are unsafe for vaccination have been presented on surface-exposed loops or fused to n-or c-exposed termini of structural viral capsid proteins on vlps. , , different hpv, - hbv, , parvovirus, , and chimeric polyoma vlps have been engineered , and tested for different applications including vaccination against viral or bacterial diseases, against virus-induced tumors, and more recently, for immunotherapy of nonviral cancer. , on the other hand, chemical bioconjugation for covalent coupling of protein epitopes and small molecules to lysines, cysteines, or tyrosine residues of vlp surfaces has been applied in viral or cancer vaccines. chackerian et al. have demonstrated the efficient induction of protective autoantibodies using self-antigen conjugation to hpv vlps. it is important to point out that vlps can also be engineered to incorporate heterologous cell-specific ligands to cell receptors, thus altering their cellular tropism. , , , this great convertibility and flexibility of vlps to be modified (chemically and/or genetically), their high stability, natural and diverse tropism, their nanocontainer properties, and their ability to enter in the cell and incorporate, bind, and deliver nucleic acids and small molecules have positioned vlps as appealing entities not only for vaccination applications but also for a broad spectrum of other diverse and emerging applications in nanomedicine and nanotechnology such as immunotherapy against cancer, , gene therapy delivery of therapeutic genes into specific cells, , , , , , and targeted delivery of drugs and small molecules using vlps as nanocarriers. , domingo-espÍn et al. although there is no commercial vlp as vector in gene therapy, since the initial work in of uncoating polyoma pseudovirus in mouse embryo cells as gene delivery vector and the establishment in of the viral dna packaging into mpyv vlps and its transduction in vitro, different vlps such as hbv and hepatitis e virus, hpv and polyomavirus nanoparticles , , have been modified toward the specific delivery of therapeutic genes and proteins in different target cells, organs, and tissues in vitro and in vivo by systemic injection or oral administration. for example, recombinant vp -based polyomavirus vlps can encapsulate in vitro exogenous dna, and deliver it by cell surface sialic acid residues to human brain cells and fetal kidney epithelial cells. furthermore, vlps have recently emerged as novel nanocarriers or nanocontainers to store unnatural cargos, deliver modified oligonucleotides, synthetic small interfering rnas, and plasmids expressing short hairpin rnas as therapy to downregulate gene expression. , in this context, chou et al. have recently described the use of jcv vlps as an efficient vector for delivering rnai in vitro using murine macrophage raw . cells and in vivo using balb/c mice in silencing the cytokine gene of il- without significant cytotoxicity for systemic lupus erythematosus gene therapy. one of the key aspects in targeted gene and drug delivery is cell-specific delivery. it is important to point out that vlps are tunable nanoparticles that can also be chemically or genetically engineered to modify their natural cellular tropism in order to diversify the range of therapeutic applications in targeted gene or drug delivery. , some effective approaches to modify the natural cellular tropism include: ( ) genetic engineering of vlp chimeras incorporating heterologous cellspecific short peptides that contain recognition sites of target cell receptors. in this context, polyoma and papillomavirus, with solved atomic structures of their major structural capsid proteins, have been extensively used to obtain chimeric vlps as delivery vector systems. , however, this approach has some bioprocessing limitations such as low production levels as a consequence of vlp modification, alterations of size and properties of the vlps that could affect the structural interactions and conformations for vlp assembly, disassembly and packaging, and low transduction efficiencies. ( ) chemical bioconjugation of purified vlps with epitope-containing peptides , or a wide range of small molecules conferring cell-specific targeting such as transferrins, folic acid, or other targeting molecules. as an example, cmpv vlps have been successfully conjugated with tfn using ''click'' chemistry and with nhs-ester-derivatized folic acid, demonstrating both as internalized into hela cells and kb cells, respectively. , ( ) high-throughput library and directed evolution method is a rational approach that has been recently used to engineer viral vectors with the desired tropism properties. ( ) pseudotyping, which consists of replacing the envelope protein of one virus species by the envelope protein of another virus species. ( ) modification of the delivery route of the vlps. it has been shown that the levels of expression of b-galactosidase in heart, lung, kidney, spleen, liver, and brain are different depending on the delivery route of polyomavirus vp vlps. the great accessibility and reactivity showed by vlps, as well as their ability to serve as nanocarriers, which made them suitable to be exploited in gene therapy, have also been applied to targeted drug delivery. genetic modification and/or chemical functionalization of exposed amino acid residues on the capsid surface in order to attach small molecules, such as markers or bioactives molecules, is one of the most common approaches applied to target drug delivery. , as an example, canine parvovirus (cpv) vlps produced in a baculovirus expression system and exhibiting natural tropism to transferrin receptors (tfrs) were chemically modified on accessible lysines of the capsid surface with fluorescent dye molecules and delivered to tumor cells. derivatization of cpv-vlps did not interfere with the binding and internalization into tumor cells. , one limitation of vlps in gene therapy is the low efficiency of gene transduction due to inefficient dna packaging. however, a recent study presented a novel in vivo dna packaging of jcv vlps in e. coli that effectively reduced human colon carcinoma volume in a nude mouse model. in this study, the exogenous plasmid dna was transformed into the jcv vp expressing e. coli. the packaging of the second plasmid occurs simultaneously as the in vivo assembly of the jcv vlp. even though it is still not clear how the plasmid dna molecules are encapsidated in the vlp, the authors showed that gene transduction efficiency by their in vivo package system was about % in contrast to the - % of gene transduction efficiency achieved by the in vitro osmotic shock system. in addition, the administration of exogenous proteins may induce the immune system response, reducing therapy effectiveness or causing undesirable secondary effects, albeit immunological response of protein nanoparticles can be modulated. spontaneous protein self-assembly to form ordered oligomers is a common event in biology. it can prove advantageous in terms of genome-size minimization, formation of large structures, stabilization of complexes, and inclusion of functional features. it has been widely documented that cellular oligomer proteins as well as viral capsids are stabilized by several weak noncovalent interactions as hydrophobic interaction, electrostatic energy, and van der waals forces. [ ] [ ] [ ] these interactions result in a complex quaternary structure described by three symmetry point groups named cyclic (cn), dihedral (dm), and cubic (t, o, i). , the development of computational techniques to predict protein-protein interactions using solved d protein structures makes it possible to predict and/or strengthen experimental data performing in in silico approaches. furthermore, its use opens up the possibility to design proteins not only displaying specific biological functions but also interesting intermolecular interactions to obtain increased multivalency in the resulting complexes. moreover, it should be considered that not only whole proteins can self-assemble in smart nanoparticles; oligopeptides are also capable of forming organized structures. many applications are possible due to the enormous quantity of different combinations and features that can be exploited with peptides. , furthermore, protein-protein interactions are not the unique parameters involved in particle formation, nucleic acid-peptide interactions, salt concentration, order of mix, and ratio between nucleic acid and protein can also strongly influence the condensation process. , due to their natural tendency to self-assemble forming highly ordered structures, viruses provide a wide variety of scaffold proteins which are used as gene/drug carriers. among them, vlps have been reviewed in the previous section. however, simple bacterial proteins can be also utilized as carriers for gene delivery. for example, heat shock proteins (hsp) from hyperthermophilic archeaon methanococcus jannaschii can assemble in a small structure of subunits having an octahedral symmetry. these nm structures are stable at high temperature, up to c, and wide range of ph. residue modifications are allowed to elicit specific attachment of small molecules. , in bacteria, bacterial microcompartments (bmc) which are intracellular organelles consisting of enzymes encapsulated within polyhedral, protein-only shells, somewhat similar to viral capsids, have been described. bmcs are composed of a few thousand copies of a few repeated protein species (including one or more enzymes involved in specific metabolic pathways), and with sizes of around - nm in cross section. the general role of bmcs is to confine toxic or volatile metabolic intermediates, while allowing enzyme substrates, products, and cofactors to pass. the first described bmc, the carboxysome, was isolated in the early s , and has been found to contain both co -fixing ribulose bisphosphate carboxylase/oxygenase (rubisco) , and carbonic anhydrase [ ] [ ] [ ] enzymes. carboxysomes' function is to enhance autotrophic co fixation at low co levels. other bmcs were later identified in cyanobacteria and some chemoautotroph bacteria. among them, bmc proteins have been later found to be encoded in the propanediol utilization operon (pdu) of the heterotroph salmonella and by an operon for metabolizing ethanolamine (eut) in enteric bacterial species, including salmonella and escherichia. salmonella enterica forms a polyhedral organelle during growth on , -propanediol ( , -pd) as a sole carbon and energy source, but not during growth on other carbon sources. , the pdu organelles' function is to minimize the harmful effects of a toxic intermediate of , -pd degradation (propionaldehyde). [ ] [ ] [ ] other studies have shown that a polyhedral organelle is involved in ethanolamine utilization (eut) by s. enterica. the function of the eut microcompartment is to metabolize ethanolamine without allowing the release of acetaldehyde into the cytosol, therefore minimizing the potentially toxic effects of excess aldehyde in the bacterial cytosol [ ] [ ] [ ] and also preventing volatile acetaldehyde from diffusing across cell membrane. so far, about proteins containing bmc domains have been identified, covering at least different bacterial phyla. the typical bmc protein consists of approximately amino acids, with an alpha/beta fold pattern. , some individual bmc proteins self-assemble to form hexamers, which further assemble side by side to form the flat facets of the shell. , , the formation of icosahedral, closed shells from such flat layers was elucidated in part by structural studies in carboxysomes: some bmc proteins assemble to form pentamers, which are located at and form the vertices of the icosahedral shell. mechanisms directing enzyme encapsulation within protein-based bmcs have been studied during the last years. it has been described that, in some carboxysomes, protein ccmm is used as a scaffold to form interactions between both shell proteins and enzymes, , through a ccmm c-terminal region with homology to the small subunit of rubisco. other studies revealed that pdu shells can self-assemble without needing interior enzymes and that carboxysomes can self-assemble in vivo when rubisco has been deleted. regarding properties of the encapsulated enzymes, in the pdu bmc some of the internal enzymes are encapsulated by specific n-terminal targeting sequences. , in this line, sutter and colleagues described a conserved c-terminal amino acid sequence that mediates the physical interaction of an iron-dependent peroxidase (dyp) or a protein closely related to ferritin (flp) with a specific type of bmc (encapsulins). in another example, an icosahedral enzyme complex, lumazine synthase (aals) from bacillus subtilis and aquifex aeolicus, was engineered to encapsulate target molecules by means of charge complementarity and can also be modified to give different characteristics to the assembled structure. moreover, enzymatic subunits, like e of pyruvate dehydrogenase from bacillus stearotermophilus, can be modified to be used in gene delivery. e peptides naturally form a dodecahedron of subunits of nm in diameter allowing modification for drug-like accommodation. the assembling/disassembling of these structures can be modulated by changing the operative ph in the experimental environment. these nanoparticles can also be functionalized with antigens for vaccine development. , according to these results, specific targeting sequences could be of use in biotechnological applications to package proteins inside the stable selfassembled icosahedral shell of bmcs, offering appealing opportunities to manipulate in the laboratory such nanocages to fill them with therapeutic molecules. the simplicity of this system makes it very attractive for engineering studies to design, mimicking nature, new applications in biotechnology, providing a new, intriguing platform of microbial origin for drug delivery. bovine serum albumin (bsa) is able to form microspheres after sonochemical treatment in aqueous medium. chemical effects of ultrasound radiation and coupling with an anticancer drug such as taxol (paclitaxel) led to the assembling of a spherical carrier with an average diameter of nm. bsa particles resulting from s-s bonds, due to ho radical formation, are able to release the encapsulated taxol in cancer tissue with best results if compared with mere taxol treatment. this drug for breast cancer treatment is commercially available. , also little cationic peptides can lead to self-assembling particles. among others, arginine-rich cationic peptides are widely known as good tools for gene delivery. for example, purified r -tailored gfp in solution is described to form nanodisk particles nm in diameter. this structure is proved to be induced by the arg tails and is able to bind and condense dna. these nanodisks are also able to deliver dna toward the nucleus where the reporter gene is expressed. on the other hand, the expression of recombinant proteins over physiological rates can cause a bad functioning of cellular quality control system, leading to self-organizing, pseudo-spherical, protein aggregates known as inclusion bodies. these mechanically stable nanoparticles, ranging from to nm in diameter, were considered for a long time as undesired bio-products. recently, it became clearer that they are suitable for medical approaches when utilized as scaffold surface to promote cellular proliferation. [ ] [ ] [ ] one of the most difficult goals for a foreign gene delivery is to reach the nucleus. an approach to overpass this obstacle is by fusing an nls in a nonessential position of a dna-binding protein. such type of modification has been described for a tetracycline repressor protein (tetr) fused with an sv nls. the tetr-nls affinity and specificity to teto dna sequence is exploited to form spontaneous protein-dna complexes which allow an enhancing of dna transportation into the nucleus and subsequent expression of foreign genes, combining the two peculiar characteristics of each fusion component. there is still a tremendous gap between progresses made in protein-based nanoparticle research for drug delivery and clinical reality. hundreds of publications in basic research describe the combination of two or more functional elements in a single protein nanoparticle, by which the delivery of a carried drug is enhanced. these agents act by improving critical steps in the drug delivery process, such as increasing the systemic stability or tissue specificity, favoring internalization, endosomal escape, and entry into the nucleus, or transporting therapeutic material through the bbb, in in vitro and in vivo studies. besides the human recombinant therapeutic proteins currently on the market (or functional segments of them), there are also some fusion proteins approved for clinical use (most by incorporating an antibody fragment or a ligand to enhance cell specificity). sadly no gene therapy trials have so far used full protein carriers in vivo, but rather peptide-functionalized vehicles. bottlenecking the gap between research and clinical application, the us fda/european medicines agency (emea) only approves human proteins, to avoid the risk of an immune response that could affect not only the effectiveness of the nanoparticle but also challenge patients' health. another critical factor is the administration route, where the protein is degraded before arriving at the target; this problem could be solved or minimized by the use of protein d-isomers, pegylation, or the design of protecting groups for labile sites. despite the current situation mentioned above, there are many good examples of multifunctional modular proteins that, when carrying therapeutic material, can improve the prognosis in vivo in animal models for different diseases. these examples are reviewed below, along with those few protein nanoparticles that are currently on the market or in clinical trials. albumin is a natural protein transporter of hydrophobic molecules throughout plasma that has been approved by the fda to reversibly bind water-insoluble anticancer agents, as is the case of albumin-bound (nab) paclitaxel, abraxane . this albumin-nab technology-based drug is in use in patients with metastatic breast cancer who have failed combination therapy, and it is the first protein nanoparticle approved by the fda. albumin potentiates paclitaxel concentration within the tumor by increasing paclytaxel endothelial transcytosis through caveolae formation. it also contributes to the fact that tumors secrete an albumin-binding protein sparc (also called bm- ) to attract and keep albumin-bound nutrients inside the tumor cell. the albuminpaclitaxel complex was not formally considered a nanoparticle in the united states (due to an average size of nm) but only so in europe. apart from whole recombinant therapeutic proteins being currently commercialized, there are also some examples of vehicles formed by chimerical proteins with target ligands already in the market. dab il- (denileukin diftitox or ontak) is a fusion of diphtheria toxin catalytic and translocation domains for lethal effect and interleukin- (il- ) to gain cell specificity in the treatment of persistent or recurrent t-cell lymphoma. belatacept (bms- ) is a ctla -ig fusion protein formed by the cytotoxic t-lymphocyteassociated antigen joined to an immunoglobulin g fc fragment fusion protein, developed by bristol-miers-squibb. etanercept (enbrel) fusion tumor necrosis factor receptor (tnfr), which binds and inhibits specifically tnf activity, to an immune globulin g fc, to prevent inflammation mediated by tnf in autoimmune diseases like arthritis and psoriasis. on the other hand, fusion proteins which include an antihuman epidermal growth factor receptor (her ) monoclonal antibody that binds tumor cell surfaces, among them the so-called ''trastuzumab'' (commercialized as herceptin by roche), associated to dm- , an antimitotic drug, aimed at improving the treatment of breast cancer. finally, vlps, that is, empty viral entities formed by the self-assembly of a viral capsid protein, are the only truly protein nanoparticles (architectonically speaking) which are currently used in clinical practice. hbsag recombinant protein of hbv expressed in yeast and the capsid l recombinant protein of hpv (types , , , and ) administered currently as vaccines tend to form spontaneously vlps that elicit t and b immune response. recently, there have been preclinical and clinical trials to test the security and efficacy of vlp vaccines against chikungunya and seasonal influenza virus (http://www. medpagetoday.com/meetingcoverage//icaac/ ), respectively. influenza vlp vaccines have proven to provide complete protection against h n flu pandemics, within a record preparation time when compared to months for traditional vaccines. the use of vlps as a delivery system for drugs or nucleic acids in gene therapy is still under investigation. drugs and proteins may be transformed through pegylation, a process that can assist them in overcoming some of the potential problems that delay the adoption of protein nanoparticles for clinical use. the covalent attachment of peg can reduce immunogenicity and antigenicity by hiding the particle from the immune system, can increase the circulating time by reducing renal clearance, and can also improve the water solubility of a hydrophobic particle. the use of pegylation has been approved for commercial use by the fda and emea, and some examples of pegylated protein products are adagen (peg-bovine adenosine deaminase), the first pegylated protein approved by the fda in , pegasys (peg-interferon alpha), and oncaspar (peg-l-asparaginase). the majority of protein nanoparticles studied in clinical trials (http://clinicaltrias.gov) are fusion proteins composed of a therapeutic protein/peptide and a target cell-specific ligand. an example is alt- , a biologic compound composed of il- genetically fused to a humanized soluble t-cell receptor directed against the p -derived antigen. the clinical trials evaluated whether directing il- activity using alt- to the patient's tumor sites that overexpress p results in clinical benefits (nct , nct ). another ligand joined to il- is l , a tumor-targeted immunocytokine constituted of a single chain fragment variable (scfv) directed against the ed-b domain of fibronectin, one of the most important markers for neoangiogenesis. l -il- is in a phase i/ii study for patients with solid tumors and renal cell carcinoma (rcc) (nct ). l has also been fused to tnfa with the intention to target tnfa directly to tumor tissues resulting in high and sustained intralesional bioactive tnfa concentrations. the l tnfa is under clinical trial using isolated inferior limb perfusion (ilp) with the standard treatment with melphalan mg/l limb volume in subjects affected by stage iii/iv limb melanoma (nct ). ngr-htnf is another bifunctional protein which combines a tumor-homing peptide (ngr) that selectively binds to amino peptidase n/cd highly expressed on tumor blood vessels, thus affecting tumor vascular permeability, and htnf, with direct anticancer activity. ngr-htnf is undergoing clinical trials as a single agent to treat different cancers, as well as in combination with chemotherapy agents. another strategy to direct a therapeutic protein to the target cell is through fusion to a growth factor receptor ligand. an example is tp- , a recombinant chimerical protein composed of the egfr binding ligand (tgf-a) and a genetically engineered form of the pseudomonas exotoxin, pe- , to treat recurrent grade iv malignant brain tumors (nct ). many clinical trials are based on a therapeutic protein fused to a targeting antibody, as is the case of apc . this drug stimulates the immune system and stops cancer cells from growing by the combination of biological therapies with bevacizumab , an already approved monoclonal antibody that locates tumor cells and kills them in a specific way (nct ). there are also many putative protein drugs against cancer which include antibodies antiintegrins (e.g., cilengitide and imgn ), sometimes in combination with classical therapies. a recently developed tool, the nanobodies or single domain antibodies, have several advantages: small size (only - kda), which lowers the possibility of triggering immune response, safety in clinical trials (nct ), and is easy to be joined to different kinds of compounds. all these features make nanobodies competent drugs against different diseases, and have been tested in vivo as bifunctional proteins associated to a prodrug, very efficient in mice cancer xenografts. even though cpps are very useful tools to deliver drugs and in gene therapy (see the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume), their toxicity and endosomal entrapment slows their inclusion for systemic delivery in clinical trials. nevertheless, there are a few examples of use to prevent undesirable cell proliferation in coronary artery bypass grafts, as is the case of a cpp (r-ahx-r) ahxb-pmo conjugate targeted to human c-myc to be applied ex vivo. the trial, in phase ii, has been completed in (nct ). another case is psorban , a product patented for the treatment of psoriasis based on a cyclosporine-polyarginine conjugate of local application, which circumvents the specificity problem of intravenous (i.v.) application. it is in clinical trial phase iii, but not yet in the market. finally, kai- , a pkcd inhibitor peptide conjugated to tat to function as an intravenous drug for the treatment of acute myocardial infarction, is currently in phase b clinical trial (nct , kai pharmaceuticals). there are many proteins, often organized as nanoparticles, that when associated to a drug, therapeutic protein, peptide, or nucleic acid increase the therapeutic efficacy of a cargo alone in the treatment of various diseases. some of them proved effective in animal models, which are discussed in more detail in this section, with relevant examples listed in table ii . these nanoparticles may simply be (a) a cpp to promote nonspecific internalization, - (b) a peptide to confer cargo specificity by joining a receptor distinctive of a cell type, including scfvs or peptides obtained by phage display, and (c) a mixture of both, since as observed in several studies the cpp does not reduce ligand specificity and increases nanoparticle potency. [ ] [ ] [ ] complex and multifunctional vehicles including endosomal escape peptides enhance the therapeutic potency of the complex, or other domains that allow their selective activation in certain contexts. , apart from the cases listed in table ii , the spectrum of additional examples of multidomain protein nanoparticles tested in vivo is wide, and a considerable proportion of them include cpps, mainly tat and polyarginines. a classical tat fusion protein is the transducible d-isomer ri-tatp c ' fusion protein that activates p protein in cancer cells, but not in normal cells. ri-tatp c treatment in terminal peritoneal carcinomatosis and peritoneal lymphoma preclinical models results in significant increases in life span (higher than sixfold) and full recovery from the disease. there are also several studies in vivo using tat-fused therapeutic proteins which have proven effective in treating tumors [ ] [ ] [ ] and cerebral ischemia , when applied intraperitoneally (i.p.). regarding polyarginines, kumar and colleagues have presented two different models in which a bifunctional peptide formed by nine arginines ( r) and a specific ligand constitute an effective sirna vehicle. in the first model, a chimerical peptide derived from rabies virus glycoprotein (to confer neuronal specificity) fused to d-arginines (rvg- r), was able to transport si-rna across the bbb and silence specific gene expression in the brain when applied intravenously. in the second model, a cd -specific single-chain antibody was conjugated to oligo- -arginine peptide (scfvcd - r) for t cell-specific antiviral sirna delivery in humanized mice reconstituted with human lymphocytes. in hiv-infected humanized mice, this treatment controlled viral replication and prevented the disease-associated cd t cell loss. moreover, it effectively suppressed viremia in infected mice. some other examples of polyarginines in tumor models are -d-arginines fused to a tumor-suppressor peptide, which stopped tumor growth in hepatocellular carcinoma-bearing mice when applied intraperitoneally, and also colesteryl oligoarginines carrying vegf sirna, which inhibited tumor growth in colon adenocarcinoma after local application. another bbb-crossing peptide is g , which is able to transport nanoparticles loaded with loperamide. in general, the partner fusion peptide can confer specificity instead of penetrability, as is the case of egfr fab fragment associated to liposomes that contain anticancer drug, which increases efficiency of anticancer effect in egf overexpressing xenograft tumors ; in addition, rgd- c-doxorubicin in human breast xenografts increases efficacy and diminishes toxicity. in many conjugates, the therapeutic peptide of the chimerical proteins is a toxin. anthrax lethal toxin has been modified to be activated by methaloproteases, and it has probed to be effective for human xenografted tumors such as melanoma, lung, and colorectal cancer. anthrax toxin has also been associated to antibodies or growth factors for lethal effects specifically on cancer cells. the specific cytotoxicity desired to treat a tumor might derive from a tissue factor, which promotes clotting to restrict blood supply in tumor vessels, fused to peptides that provide specificity, like v-cam antibodies, fibronectin, and integrin ligands. eventually, drug activity may decrease when conjugated to a carrier protein, although if the entry of the drug is favored, the overall balance of activity can be much more efficient. on the other hand, the use of noncovalent bond drug carrier could avoid interfering with the activity of the drug. an important issue in a preclinical study to be considered for a clinical trial is the administration route. in in vivo experiments, most of the protein nanoparticles are administered by local or intraperitoneal injection, avoiding systemic spreading and clearance in the vascular system, in a way very similar to in vitro experiments. the fda and emea, on the other hand, will preferentially approve i.v. and oral administrations rather than intraperitoneal or local injections except for very accessible tissues. another relevant issue is the number of active domains to be included in a therapeutic protein carrier, an issue that seems to be relevant for the functionality of the construct. for example, the cpp neutralization of a ligand may depend on the cpp/ligand ratio that is in the vehicle. it has also been observed that the integrin binding power of rgd-containing motives increases with the number of rgd domains over the monomer until a maxim of four moieties. another example is tat activity empowerment when attached to molecules that form tetramers, such as beta-galactosidase and p- . some multidomain protein carriers allow the drug entrance only in selected target cells by tailored smart selective mechanisms. for instance, cpps neutralized by polyanions are activated and enter the cells when they are released by metalloproteases or by lowering the ph, both situations being very common in tumors. cpp-morpholino oligomer (pmo) nanoparticles have also shown their effectiveness in treating viral infections by inhibiting viral replication, as demonstrated with the carrier (r-ahx-r) ahxb-pmo administered i.v. in animal models infected with picornaviruses, i.p. in mice infected with coronaviruses and flaviviruses, and the carrier r f c-pmo administered also i.p. in mice infected with ebola virus. furthermore, it has also been shown in some of these studies that the efficacy of the treatment is dependent on the incorporation of arginine-rich peptides in the nanoparticle. a good example of how a cpp can improve the internalization of a therapeutic protein is the case of insulin. the instability and low absorption in the digestive tract of insulin prevents its oral administration, even though it would be very convenient for a daily administrated drug. in recent studies, noncovalent conjugation of insulin to different cpps enhances its absorption without toxic intestinal effect, l-penetratin being the most efficient as insulin carrier. among the protein nanoparticles tested in vivo, it is worth making special mention of trojan horses generated in pardridge's laboratory to cross the bbb, through a strategy of fusing within a chimerical peptide the therapeutic protein which has to reach the cns to a monoclonal antibody against the human insulin receptor (hirmab). this trojan horse is very potent for humans and primates, and has proven effective to transport b-glucuronidase, a-l-iduronidase, gdnf, abeta amyloid peptides, paroxonase, etc., with potential benefits in diseases like mucopolysaccharidosis type vii, hurler syndrome, parkinson, alzheimer, and organophosphates toxicity, respectively. there are also promising results when protein nanoparticles have been tested as carriers for gene therapy in vivo, some examples being listed in table ii . in this regard, the use of modular proteins generated by insertional mutagenesis of b-galactosidase condensing the sod gene are able to protect neurons against ischemic injury ; a bifunctional galactosylated polylysine is able to conjugate plasmid dna and to differentially promote expression in hepatocytes that display asialoglycoprotein receptor ; a suicide multidomain protein particle formed by herpes simplex virus thymidine kinase (hsv-tk) conjugated to transferrin (tf) by a biotin-streptavidin bridging, which, administered i.v. in k massively metastasized nude mice, was able to reduce tumor size and to increase mouse survival. in this chapter, proteins and peptides have been envisioned as potent biotechnological tools for the development of new biocompatible biological entities that can be used as therapeutic agents by themselves or as nanovehicles for the delivery of associated drugs. proteins are nanostructures that can form complex high-order entities such as vlps, resulting in appropriate cages for the internalization of therapeutic molecules. in addition, the design of modular proteins displaying selected functions has been possible by using in silico approximations to the feasibility of recombinant protein production. this approach has demonstrated the versatility of such molecules in the generation of novel delivery nanovehicles opening up the possibility of new functional combinations to enhance the specific interaction with the target tissue. such tunable specificity in the delivery of drugs, nucleic acids, or other proteins is one of the main properties that make multifunctional proteins appealing as more rational delivery vehicles. the presence on the market of such complex entities, which started with the approval of insulin for the treatment of diabetes, has been increasing over the past years, and this tendency is expected to continue. in fact, there are some products in clinical trials that will probably end up being approved and some more are being explored in preclinical experiments which might enter in clinical trials. identifying actives from hts data sets: practical approaches for the selection of an appropriate hts data-processing method and quality control review natural products in the process of finding new drug candidates when analoging is not enough: scaffold discovery in medicinal chemistry dose-toxicity models in oncology is declining innovation in the pharmaceutical industry a myth? the impact of pegylation on biological therapies anticancer activity of celastrol in combination with erbb -targeted therapeutics for treatment of erbb -overexpressing breast cancers soon-shiong p. sparc expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbate-based docetaxel in multiple xenografts as a function of her and sparc status pharmacokinetics of plasmid dna in the rat instability, stabilization, and formulation of liquid protein pharmaceuticals peptide-guided gene delivery artificial viruses: a nanotechnological approach to gene delivery approaches to transport therapeutic drugs across the blood-brain barrier to treat brain diseases multifunctional protein nanocarriers for targeted nuclear gene delivery in nondividing cells synthetic and natural polycations for gene therapy: state of the art and new perspectives structure-activity relationships of poly(l-lysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery systemic circulation of poly(l-lysine)/dna vectors is influenced by polycation molecular weight and type of dna: differential circulation in mice and rats and the implications for human gene therapy a novel dnapeptide complex for efficient gene transfer and expression in mammalian cells branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of dna polyplexes comparative gene transfer efficiency of low molecular weight polylysine dna-condensing peptides low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers protamine sulfate enhances lipid-mediated gene transfer protamine-induced condensation and decondensation of the same dna molecule evaluation of nuclear transfer and transcription of plasmid dna condensed with protamine by microinjection: the use of a nuclear transfer score the protamine family of sperm nuclear proteins membrane-active peptides for non-viral gene therapy: making the safest easier enhancement of msh receptor-and gal -mediated gene transfer by switching the nuclear import pathway target cell-specific dna transfer mediated by a chimeric multidomain protein: novel non-viral gene delivery system a multi-domain protein system based on the hc fragment of tetanus toxin for targeting dna to neuronal cells refined solution structure of the dna-binding domain of gal and use of j( cd, h) in structure determination immune responses to gene therapy vectors: influence on vector function and effector mechanisms peptide-assisted traffic engineering for nonviral gene therapy cell-penetrating peptides: a reevaluation of the mechanism of cellular uptake cell penetrating peptides: overview and applications to the delivery of oligonucleotides modular protein engineering in emerging cancer therapies oligomers of the arginine-rich motif of the hiv- tat protein are capable of transferring plasmid dna into cells tat-mediated delivery of heterologous proteins into cells tat peptide-mediated cellular delivery: back to basics a truncated hiv- tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus cellular uptake [correction of utake] of the tat peptide: an endocytosis mechanism following ionic interactions the design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters delivery of short interfering rna using endosomolytic cell-penetrating peptides conjugate for efficient delivery of short interfering rna (sirna) into mammalian cells cell penetration by transportan cellular translocation of proteins by transportan integrin-mediated vectors for gene transfer and therapy inhibition of tumor growth by rgd peptide-directed delivery of truncated tissue factor to the tumor vasculature hiv coreceptor downregulation as antiviral principle: sdf- alpha-dependent internalization of the chemokine receptor cxcr contributes to inhibition of hiv replication cxcr , inhibitors and mechanisms of action the transferrin receptor part ii: targeted delivery of therapeutic agents into cancer cells improved gene delivery into neuroglial cells using a fiber-modified adenovirus vector systemic genetic transfer of p waf- and gm-csf utilizing of a novel oligopeptide-based egf receptor targeting polyplex specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in scid mice a new n-acetylgalactosamine containing peptide as a targeting vehicle for mammalian hepatocytes via asialoglycoprotein receptor endocytosis transvascular delivery of small interfering rna to the central nervous system a novel peptide, plaeidgielty, for the targeting of alpha beta -integrins a synthetic peptide vector system for optimal gene delivery to corneal endothelium secretin-mediated gene delivery, a specific targeting mechanism with potential for treatment of biliary and pancreatic disease in cystic fibrosis a synthetic peptide containing loop of nerve growth factor for targeted gene delivery neurotensin-spdp-poly-l-lysine conjugate: a nonviral vector for targeted gene delivery to neural cells identification of peptides that target the endothelial cell-specific lox- receptor selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier anti-gad antibody targeted non-viral gene delivery to islet beta cells novel challenges in exploring peptide ligands and corresponding tissue-specific endothelial receptors from combinatorial chemistry to cancer-targeting peptides cell surface adherence and endocytosis of protein transduction domains influenza virus hemagglutinin ha- n-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-dna complexes: toward a synthetic virus-like gene-transfer vehicle the influence of endosomedisruptive peptides on gene transfer using synthetic virus-like gene transfer systems ph-dependent bilayer destabilization by an amphipathic peptide mechanism of leakage of phospholipid vesicle contents induced by the peptide gala association of a ph-sensitive peptide with membrane vesicles: role of amino acid sequence gala: a designed synthetic ph-responsive amphipathic peptide with applications in drug and gene delivery design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers new basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors the third helix of the antennapedia homeodomain translocates through biological membranes trojan peptides: the penetratin system for intracellular delivery histidine-rich peptides and polymers for nucleic acids delivery membrane permeabilization and efficient gene transfer by a peptide containing several histidines histidine containing peptides and polypeptides as nucleic acid vectors characterization of the gene transfer process mediated by histidine-rich peptides the proteasome metabolizes peptide-mediated nonviral gene delivery systems inhibition of ubiquitin-dependent proteolysis by a synthetic glycine-alanine repeat peptide that mimics an inhibitory viral sequence a minimal glycine-alanine repeat prevents the interaction of ubiquitinated i kappab alpha with the proteasome: a new mechanism for selective inhibition of proteolysis cis-inhibition of proteasomal degradation by viral repeats: impact of length and amino acid composition recognition of novel viral sequences that associate with the dynein light chain lc identified through a pepscan technique mechanisms of nuclear protein import gene delivery: a single nuclear localization signal peptide is sufficient to carry dna to the cell nucleus epstein-barr virus nuclear antigen forms a complex with the nuclear transporter karyopherin alpha identification of the human c-myc protein nuclear translocation signal a chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the egf receptor the requirement of h histones for a heterodimeric nuclear import receptor core histones and linker histones are imported into the nucleus by different pathways nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells the carboxyl amino acids of sv vp are essential for its nuclear accumulation pentapeptide nuclear localization signal in adenovirus e a identification of domains involved in nuclear uptake and histone binding of protein n of xenopus laevis competition between nuclear localization and secretory signals determines the subcellular fate of a single cug-initiated form of fgf the human poly(adpribose) polymerase nuclear localization signal is a bipartite element functionally separate from dna binding and catalytic activity two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence long-term pharmacologically regulated expression of erythropoietin in primates following aav-mediated gene transfer rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-dsred-express liverrestricted expression of the canine factor viii gene facilitates prevention of inhibitor formation in factor viii-deficient mice blood-brain barrier delivery targeting the central nervous system: in vivo experiments with peptide-derivatized nanoparticles loaded with loperamide and rhodamine- in vivo protein transduction: delivery of a biologically active protein into the mouse genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular trojan horse for delivery across the human blood-brain barrier genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier gdnf fusion protein for targeted-drug delivery across the human blood-brain barrier directed evolution: selecting today's biocatalysts novel methods for directed evolution of enzymes: quality, not quantity chemical and biochemical strategies for the randomization of protein encoding dna sequences: library construction methods for directed evolution rapid evolution of a protein in vitro by dna shuffling protein design automation advances in protein structure prediction and de novo protein design: a review solution structure and dynamics of a de novo designed three-helix bundle protein de novo protein design: fully automated sequence selection modular protein engineering for non-viral gene therapy a modular dna carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery gene therapy progress and prospects: non-viral gene therapy by systemic delivery using nuclear targeting signals to enhance non-viral gene transfer delivery of bioactive molecules into the cell: the trojan horse approach synthesis of cell-penetrating peptides and their application in neurobiology complexes of plasmid dna with basic domain - of the hiv- tat protein are transferred to mammalian cells by endocytosis-mediated pathways molecular organization of protein-dna complexes for cell-targeted dna delivery role of molecular chaperones in inclusion body formation recombinant protein expression in escherichia coli advanced genetic strategies for recombinant protein expression in escherichia coli recombinant expression systems in the pharmaceutical industry the major capsid protein, vp , of human jc virus expressed in escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous dna into human kidney cells neuroprotection from nmda excitotoxic lesion by cu/zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector yeast cells allow high-level expression and formation of polyomavirus-like particles mammalian cell culture systems for recombinant protein production insect cell culture for industrial production of recombinant proteins large-scale mammalian cell culture glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency efficient gene delivery to primary neuron cultures using a synthetic peptide vector system cell-penetrating dna-binding protein as a safe and efficient naked dna delivery carrier in vitro and in vivo synthetic peptides as non-viral dna vectors exploration of peptide motifs for potent non-viral gene delivery highly selective for dividing cells engineering nuclear localization signals in modular protein vehicles for gene therapy the role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells rotavirus-like particles: a novel nanocarrier for the gut efficient accommodation of recombinant, foot-andmouth disease virus rgd peptides to cell-surface integrins virus entry: open sesame high efficiency polyoma dna transfection of chloroquine treated cells plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers a nuclear localization signal can enhance both the nuclear transport and expression of kb dna efficacy of a peptidebased gene delivery system depends on mitotic activity biological gene delivery vehicles: beyond viral vectors virus-like particles-universal molecular toolboxes virus engineering: functionalization and stabilization viruses and their uses in nanotechnology scaffolding proteins and their role in viral assembly towards the preparative and large-scale precision manufacture of virus-like particles virus-sized vaccine delivery systems virus-like particles in vaccine development advances in the development of virus-like particles as tools in medicine and nanoscience vaccination, immune and gene therapy based on viruslike particles against viral infections and cancer virus-like particles as a vaccine delivery system: myths and facts gene transfer by polyoma-like particles assembled in a cell-free system papillomavirus-like particles induce acute activation of dendritic cells papillomavirus virus-like particles as vehicles for the delivery of epitopes or genes virus-like particle vaccines and adjuvants: the hpv paradigm a novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis b virus-like particle essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis e virus recombinant hepatitis c virus-like particles expressed by baculovirus: utility in cell-binding and antibody detection assays murine pneumotropic virus chimeric her /neu virus-like particles as prophylactic and therapeutic vaccines against her /neu expressing tumors in vitro and in vivo targeted delivery of il- interfering rna by jc virus-like particles cell-type specific targeting and gene expression using a variant of polyoma vp virus-like particles molecular cloning and expression of major structural protein vp of the human polyomavirus jc virus: formation of virus-like particles useful for immunological and therapeutic studies packaging of small molecules into vp -virus-like particles of the human polyomavirus jc virus chimeric polyomavirus-derived virus-like particles: the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast saccharomyces cerevisiae hamster polyomavirusderived virus-like particles are able to transfer in vitro encapsidated plasmid dna to mammalian cells virus-like gene transfer into cells mediated by polyoma virus pseudocapsids immunity against both polyomavirus vp and a transgene product induced following intranasal delivery of vp pseudocapsid-dna complexes virus-like particles: designing an effective aids vaccine lentivirus-based virus-like particles as a new protein delivery tool parenteral administration of rf - / / rotavirus-like particles in a one-dose regimen induce protective immunity in mice canine parvovirus-like particles, a novel nanomaterial for tumor targeting tumor targeting using canine parvovirus nanoparticles norwalk virus-like particles as vaccines virus-based nanoparticles (vnps): platform technologies for diagnostic imaging quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy high-resolution structure of a polyomavirus vp -oligosaccharide complex: implications for assembly and receptor binding the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry structures of virus and virus-like particles fabrication of novel biomaterials through molecular self-assembly bacteriophage capsids: tough nanoshells with complex elastic properties maturation of a tetravirus capsid alters the dynamic properties and creates a metastable complex the use of virus-like particles for gene transfer virus-like particles as vaccines and vessels for the delivery of small molecules recombinant virus like particles as drug delivery system virus-like particles as immunogens virus-like particles: passport to immune recognition manipulation of the mechanical properties of a virus by protein engineering viruses as building blocks for materials and devices adaptations of nanoscale viruses and other protein cages for medical applications blocking oncogenic ras signaling for cancer therapy assessment of cell type specific gene transfer of polyoma virus like particles presenting a tumor specific antibody fv fragment microbial production of virus-like particle vaccine protein at gram-per-litre levels recombinant baculoviruses as mammalian cell gene-delivery vectors production of core and virus-like particles with baculovirus infected insect cells production of fmdv virus-like particles by a sumo fusion protein approach in escherichia coli virus-like particles production in green plants an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles immunotherapeutic polyoma and human papilloma virus-like particles human hepatitis b vaccine from recombinant yeast evaluation of hbs, hbc, and frcp virus-like particles for expression of human papillomavirus e oncoprotein epitopes advances in methods for the production, purification, and characterization of hiv- gag-env pseudovirion vaccines nanotechnology in vaccine delivery protection against lethal challenge by ebola virus-like particles produced in insect cells ebola virus-like particles protect from lethal ebola virus infection activation of dendritic cells and induction of t cell responses by hpv l /e chimeric virus-like particles are enhanced by cpg odn or sorbitol vaccination trial with hpv l e chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (cin / ) hpv l e chimeric virus-like particles induce specific hla-restricted t cells in humans after in vitro vaccination chimeric virus-like particles for the delivery of an inserted conserved influenza a-specific ctl epitope chimeric hepatitis b virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against bacillus anthracis parvovirus b empty capsids as antigen carriers for presentation of antigenic determinants of dengue virus recombinant virallike particles of parvovirus b as antigen carriers of anthrax protective antigen conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies a vaccine against nicotine for smoking cessation: a randomized controlled trial efficient gene transfer using the human jc virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model a top-down approach for construction of hybrid polymer-virus gene delivery vectors dna and gene therapy: uncoating of polyoma pseudovirus in mouse embryo cells nanoparticles for the delivery of genes and drugs to human hepatocytes dna vaccineencapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration efficient delivery of rna interference effectors via in vitro-packaged sv pseudovirions reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle an investigation into the use of human papillomavirus type virus-like particles as a delivery vector system for foreign proteins: n-and c-terminal fusion of gfp to the l and l capsid proteins coupling of antibodies via protein z on modified polyoma virus-like particles conjugation of an antibody fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold molecular engineering of viral gene delivery vehicles filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo nuclear entry mechanism of the human polyomavirus jc virus-like particle: role of importins and the nuclear pore complex hybrid virus-polymer materials. . synthesis and properties of peg-decorated cowpea mosaic virus the power of two: protein dimerization in biology bottom-up design of biomimetic assemblies measuring the forces that control protein interactions recent progress in understanding hydrophobic interactions binding mechanisms in supramolecular complexes structural symmetry and protein function modeling experimental design for proteomics smart and genetically engineered biomaterials and drug delivery systems molecular designer self-assembling peptides nanoparticulate architecture of protein-based artificial viruses is supported by protein-dna interactions giant dna molecules exhibit on/off switching of transcriptional activity through conformational transition the small heat shock protein cage from methanococcus jannaschii is a versatile nanoscale platform for genetic and chemical modification comparative ultrastructure of the thiobacilli functional organelles in prokaryotes: polyhedral inclusions (carboxysomes) of thiobacillus neapolitanus association of carbonic anhydrase activity with carboxysomes isolated from the cyanobacterium synechococcus pcc a novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell isolation of a putative carboxysomal carbonic anhydrase gene from the cyanobacterium synechococcus pcc the control region of the pdu/cob regulon in salmonella typhimurium the -gene ethanolamine (eut) operon of salmonella typhimurium encodes five homologues of carboxysome shell proteins the propanediol utilization (pdu) operon of salmonella enterica serovar typhimurium lt includes genes necessary for formation of polyhedral organelles involved in coenzyme b( )-dependent , -propanediol degradation pdua is a shell protein of polyhedral organelles involved in coenzyme b( )-dependent degradation of , -propanediol in salmonella enterica serovar typhimurium lt protein content of polyhedral organelles involved in coenzyme b -dependent degradation of , -propanediol in salmonella enterica serovar typhimurium lt pdup is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in b -dependent , -propanediol degradation by salmonella enterica serovar typhimurium lt dna polymerase i function is required for the utilization of ethanolamine, , -propanediol, and propionate by salmonella typhimurium lt glutathione is required for maximal transcription of the cobalamin biosynthetic and , -propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, , -propanediol, and propionate in salmonella typhimurium lt microcompartments for b -dependent , -propanediol degradation provide protection from dna and cellular damage by a reactive metabolic intermediate conserving a volatile metabolite: a role for carboxysome-like organelles in salmonella enterica protein structures forming the shell of primitive bacterial organelles bacterial microcompartment organelles: protein shell structure and evolution atomic-level models of the bacterial carboxysome shell structure and mechanisms of a protein-based organelle in escherichia coli a multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria analysis of carboxysomes from synechococcus pcc reveals multiple rubisco complexes with carboxysomal proteins ccmm and ccaa analysis of a genomic dna region from the cyanobacterium synechococcus sp. strain pcc involved in carboxysome assembly and function synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric rubisco species short n-terminal sequences package proteins into bacterial microcompartments structural basis of enzyme encapsulation into a bacterial nanocompartment a simple tagging system for protein encapsulation multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly thermostability and molecular encapsulation within an engineered caged protein scaffold ph-triggered disassembly in a caged protein complex characterization and activity of sonochemically-prepared bsa microspheres containing taxol-an anticancer drug paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (r ) peptide the nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation surface cell growth engineering assisted by a novel bacterial nanomaterial nanostructured bacterial materials for innovative medicines development of a selfassembling nuclear targeting vector system based on the tetracycline repressor protein unraveling mysteries of the multifunctional protein sparc a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection recombinant h n viruslike particle vaccine elicits protective immunity in ferrets against the pandemic h n influenza virus properties, production, and applications of camelid singledomain antibody fragments efficient cancer therapy with a nanobody-based conjugate effect of cell-based intercellular delivery of transcription factor gata on ischemic cardiomyopathy morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters in vivo delivery of the caveolin- scaffolding domain inhibits nitric oxide synthesis and reduces inflammation a non-covalent peptide-based carrier for in vivo delivery of dna mimics targeting cyclin b through peptide-based delivery of sirna prevents tumour growth antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors selective inhibition of erbb -overexpressing breast cancer in vivo by a novel tat-based erbb -targeting signal transducers and activators of transcription -blocking peptide design of a tumor-homing cell-penetrating peptide a tumor-homing peptide with a targeting specificity related to lymphatic vessels penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors killing hiv-infected cells by transduction with an hiv protease-activated caspase- protein development of elastin-like polypeptide for thermally targeted delivery of doxorubicin treatment of terminal peritoneal carcinomatosis by a transducible p -activating peptide antitumor effect of tat-oxygen-dependent degradation-caspase- fusion protein specifically stabilized and activated in hypoxic tumor cells the - amino acid sequence of the beta-domain of von hippel-lindau gene product is sufficient to inhibit renal tumor growth and invasion dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity protein kinase c delta mediates cerebral reperfusion injury in vivo in vivo delivery of a bcl-xl fusion protein containing the tat protein transduction domain protects against ischemic brain injury and neuronal apoptosis t cell-specific sirna delivery suppresses hiv- infection in humanized mice cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo cancer treatment by targeted drug delivery to tumor vasculature in a mouse model matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature anthrax fusion protein therapy of cancer comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cellpenetrating peptide tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide improved targeting of the alpha(v)beta ( ) integrin by multimerisation of rgd peptides probing the impact of valency on the routing of arginine-rich peptides into eukaryotic cells cell-penetrating and cell-targeting peptides in drug delivery tumor imaging by means of proteolytic activation of cell-penetrating peptides tat peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors cell penetrating peptide conjugates of steric block oligonucleotides usefulness of cell-penetrating peptides to improve intestinal insulin absorption biopharmaceutical drug targeting to the brain gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake in vivo gene delivery to tumor cells by transferrin-streptavidin-dna conjugate the authors appreciate the financial support received through grants bfu - from micinn, ps from fiss, and sgr- from agaur. the authors also acknowledge the support of the ciber de bioingeniería, biomateriales y nanomedicina (ciber-bbn), an initiative funded by the vi national r&d&i plan - , iniciativa ingenio , consolider program, ciber actions and financed by the instituto de salud carlos iii with assistance from the european regional development fund. protein nanoparticles engineered for drug delivery and gene therapy key: cord- -gxhhwqdd authors: abadie, r.; dombrowski, k. title: “caballo”: risk environments, drug sharing and the emergence of a hepatitis c virus epidemic among people who inject drugs in puerto rico date: - - journal: harm reduct j doi: . /s - - -z sha: doc_id: cord_uid: gxhhwqdd background: sharing drug injection equipment has been associated with the transmission of hcv among pwid through blood contained in the cooker and cotton used to prepare and divide up the drug solution. while epidemiologists often subsume this practice under the sharing of “ancillary equipment,” more attention should be paid to the fact that indirect sharing takes place within the process of joint drug acquisition and preparation. methods: we employed an ethnographic approach observing active pwid (n = ) in four rural towns in puerto rico in order to document drug sharing arrangements involved in “caballo”, as this practice is locally known. we explored partners’ motivation to engage in drug sharing, as well as its social organization, social roles and existing norms. findings: findings suggest that drug sharing, is one of the main drivers of the hcv epidemic in this population. lack of financial resources, drug packaging, drug of choice and the desire to avoid the painful effects of heroin withdrawal motivates participants’ decision to partner with somebody else, sharing injection equipment—and risk—in the process. roles are not fixed, changing not only according to caballo partners, but also, power dynamics. conclusion: in order to curb the hcv epidemic, harm reduction policies should recognize the particular sociocultural contexts in which people inject drugs and make decisions about risk. avoiding sharing of injection equipment within an arrangement between pwid to acquire and use drugs is more complex than assumed by harm reduction interventions. moving beyond individual risk behaviors, a risk environment approach suggest that poverty, and a strict drug policy that encourage users to carry small amounts of illicit substances, and a lack of hcv treatment among other factors, contribute to hcv transmission. while hcv-related deaths in the united states seem to have declined recently [ ] , research suggests the existence of an emerging hepatitis c virus (hcv) epidemic among people who inject drugs (pwid) in non-urban areas [ ] , which is likely associated with the transition from oral prescription opioid use to injection and often with the transition from prescription opioids to heroin [ ] . rates of hiv and hcv among pwid are on a divergent trajectory. hiv prevalence has been declining for almost a decade, while hcv has increased over the same period [ ] . nowhere in the united states are these trends more prevalent than in puerto rico, a us territory, where recent epidemiological data suggest epidemic levels of hcv among pwid, with % prevalence in metropolitan san juan and . % in rural areas [ , ] . in the us, hcv prevalence among pwid ranks highest in puerto rico, at a similar level as the worst cases in the global south [ , ] . epidemiological studies of hcv transmission routes among pwid show that the virus can be transmitted not only though blood contained in shared syringes but also by sharing the cooker and cotton used to prepare the drug solution [ ] [ ] [ ] . yet, epidemiologists often misunderstand the dynamics of hcv transmission among pwid, subsuming the common use of a cooker, or a filtering cotton ball, as the shared use of "ancillary equipment" [ , ] . as social scientists have noted, equipment sharing is often a proxy for preparing and dividing drugs [ , ] . based on a study of pwid in colorado, koester ( ) suggests that indirect sharing, which happens when powder drugs are diluted in water before been divided up in a cooker with the help of a syringe, is an efficient way of distributing drugs among injection partners. using the calibration on the syringe barrel allows participants to compare the syringe contents' , effectively ensuring an equitable distribution. this type of indirect sharing which happens more often than direct syringe sharing, when one syringe is shared from one user to another [ ] (friedman et al. ) has been found to be a common feature among pwid in a variety of social contexts [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in a social network study of pwid in brooklyn, new york curtis and colleagues ( ) found that indirect sharing is extensively practiced within a network particular social networks, contributing to define a user position in it. those that engage in drug sharing often, are the "regulars" or insiders who are placed at the center of the network, while those that do engage occasionally sometimes coming from other neighborhoods or sporadically over the weekends, tend to occupy a peripheric position [ ] . a similar study by zule with pwid in san antonio shows that participants drug sharing arrangements are asymetric relationships, with the person that provides the drug in a position to direct drug preparation and the person receiving the drug solution unable to avoid the indirect sharing of injection equipment [ ] . a qualitative study of pwid in tajikistan shows that while few users report direct syringe sharing, joint drug acquisition and preparation is common, particularly in outdoor settings or if participants experience withdrawal symptoms [ ] . another study in viet nam shows that drug sharing practices are driven by heroin price and accessibility as well as a punitive approach to drug use that penalizes drug possession [ ] . in a multi-year study of puerto rican pwid in new york city and bayamon, in san juan found that, recent migrants from the island exhibit a much higher frequency of indirect drug sharing than native users [ ] . an ethnographic study conducted among pwid in san juan extends these observations by exploring the motivations and social roles involved in drug sharing arrangements [ ] . finlinson ( ) shows that the price and packaging of drugs and access to drug preparation materials along with power differences among partners shape the process by which drugs are prepared and injected. other ethnographic studies demonstrated that drug sharing is facilitated by the type of heroin available. for example, bourgois documents how homeless pwid in san francisco use "black tar, " a sticky variety of heroin, originating in mexico, that is extremely hard to divide up without preparing it, which, in turn, encourages the sharing of injection equipment [ , ] . departing from epidemiological views that focus on individual behaviors, these authors make a valuable contribution to the understanding of hcv risk among pwid by showing how particular social contexts, from the ways in which drugs are acquired, to social roles, cultural norms, drug policies and power dynamics among those involved in "caballo" shape hcv risk. these findings complicate traditional epidemiological views shaping harm reduction initiatives, suggesting that forms of indirect sharing within the process of jointly acquiring and using drugs, are not easily modified by knowledge about hcv transmission, or the access to new injection equipment alone. as critics have suggested, the focus on individual behaviors at the expense of local social contexts in which pwid live and make decisions about risk, might obscure how social, structural and environmental contexts shape drug use and related harms [ ] [ ] [ ] [ ] [ ] . nested within a study of social networks and hiv/ hcv risk among pwid in rural puerto rico, we propose an ethnographically informed approach to "caballo", the joint acquisition and sharing of drugs, as a window into the social production of an hcv epidemic among pwid. while drug sharing arrangements among pwid have been amply documented, an ethnographic study of caballo in puerto rico will illuminate the social context behind the joint acquisition and use of drugs and its related epidemiological risk. this paper utilizes ethnographic data from pwid recruited into a multi-phase study of social networks and hiv/hcv risk in four rural towns in the mountainous area of central puerto rico. in the first phase of this study we collected demographic and sociometric data on pwid in rural puerto rico, as well as information on injection behaviors, particularly on the sharing of syringes and injection equipment. in addition, rapid blood testing for hiv and hcv were conducted using insti rapid hiv antibody tests (biolytical laboratories) and oraquick hcv rapid antibody tests (orasure technologies). it was during the administration of a national health behavioral survey (nhbs) questionnaire [ ] that we learned for the first time about "caballo". consistently, we heard from participants that while they carried their own syringe and avoided sharing it with others, they could not avoid using the same cooker within the process of drug sharing. participants described the ways in which they divided up the drug solution in the cooker, using a backloading method to distribute it among partners. all participants insisted that they "always used their own cooker" during the preparation. of course, since there are two or more caballo partners and only one cooker available, this was not possible. after this phase of data collection, we were left with many questions about the social organization and the meaning of drug sharing among this population. to explore these issues, we conducted an ethnographic study with (n = ) participants randomly recruited from the first phase. during the participant observation, each participant was followed -with their consent-for up to two weeks, documenting practices related to joint drug acquisition and use. yet, our ethnographic observations were broadly constructed, focusing not only on drug sharing practices, but in describing their everyday lives and the strategies they used in order to afford their drugs of choice. for example, we had ample opportunity to observe participants' hustling, in car washes, or guarding a parking lot, or begging at the entrance of banks or dollar stores or other venues with heavy foot traffic. after partnering with el punto en la montana, the only syringe exchange provider in the area, we earned participants' trust and were allowed to join them at the "chutin" a spanglish deformation of shooting gallery. in so doing, we were able to collect data on their drug sharing practices. we paid particular attention to the ways in which caballo partners talked about acquiring and sharing drugs in an attempt to convey the norms or hidden scripts regulating caballo arrangements. in addition, we conducted in depth interviews to understand how pwid in our study described the social practice of "caballo", the joint acquisition and later use of drugs. some of the questions we posed were what is caballo, when do participants do it and with whom. we used our initial observations to iteratively refine our research questions. since we had observed during the course of the ethnographic data collection that sometimes arguments emerge during caballo, we asked participants about potential problems or conflicts associated with this practice and we also inquired about participants' views on drug sharing related hcv risk and their perceived ability to enact changes to prevent its transmission. one limitation of our data collection is that for security reasons, ethnographic observations were conducted during the day and until dusk and only during working days. abandoned and dilapidated houses without electricity, shooting galleries are seldom used at night but are more used early in the day -as soon as drug selling spots are open-and on weekends when "regulars' are joined by more occasional users. despite the limits imposed by fieldwork dynamics we believe that our observations of caballo and the social context in which it occurs were not affected by these constraints. fieldnotes were transcribed while in-depth interviews were transcribed and translated. all personal identifiers were removed. maxqda software was employed to manage coding. codes were developed to convey the wide arrange of themes in the data set: caballo, drug acquisition, motivations, social roles, norms and expectations and injection setting, among others. these codes were then iteratively revised and re-organized until they represented higher-level axial codes describing participants' caballo experiences [ , ] . following strauss' grounded theory approach [ ] (strauss and corbin ) the interpretation of the data emerged intuitively without imposing a pre-existing theoretical framework. the study received irb approval through the (omitted due to blind review) and the (omitted due to blind review). participants provided written consent at the study office prior to enrollment in the study and were compensated for their time and travel expenses. participants' sociodemographic background (table ) . participants had a mean age of . years and the sample had a standard deviation of . years. the sample is overwhelmingly male ( . %) and heterosexual ( . %). three-quarters were unemployed at the time, and almost one-half lived in poverty and had completed high school or a higher educational level. one in five were married or living together and the same number had been homeless during the past year. almost all participants ( . %) were currently covered by health insurance, with a large majority having "la reforma, " the local version of medicare/medicaid. around one in six ( . %) had participated in a drug treatment program but only one participant had enrolled during the past year. participants had been injecting for a mean of . years with a standard deviation of . . age at first injection is . years with a standard deviation of . . almost four out of ten reported injecting two or three times a day while one in three injected four times or more a day. while hiv prevalence in this population is extremely low ( . %), hcv prevalence reaches epidemic levels, with almost nine out of ten study participants testing positive reactive for the virus ( . %). less than one in ten ( . %) declared having used a syringe after somebody else had used it the last time they injected with somebody, and a large majority ( . %) admitted using a sterile syringe. almost three-quarters ( . %) used a cooker, cotton, or water that somebody had previously used, while one in three ( . %) divided drugs with a syringe that had been previously used by somebody else. in rural puerto rico, two or more pwid often pool funds necessary to acquire and later share drugs. most participants in our study have as a drug of choice heroin "droga" and cocaine "perico", "speedball". speedballs have more heroin than cocaine, a usual way in which participants talk about their drug mix is by identifying it by the ratio of heroin to cocaine. for example, they would say " - " meaning one bag of cocaine and two of heroin. other users might prefer three bags of heroin and one of cocaine " - ". in turn, this preference is also reflected in drug sharing arrangements. the drugs are mixed together in a cooker dissolved in water, and the resulting drug solution is shared usually through backloading, removing the plunger in a syringe and squirting the content using the tip of the needle of a loaded syringe, before placing it back. this practice is locally known as "caballo" (literally, horse). participants do not recall the origin of the name, "caballo" but suggest that the same expression is used on the island in situations where people pool resources to acquire and later consume goods together, usually food but also transportation. an ethnographic fieldnote taken at the shooting gallery a few blocks away from where our office was located, describes two study participants, pablito and cesar cayey engaging in a "caballo": pablito and cesar had bought their drugs of choice at the one of the local "puntos", the drug selling spot, only a few blocks away from the dilapidated house that served as shooting gallery. they briefly discussed how the drugs would be divided up agreeing that since they had contributed the same amount of money, each would receive equal parts of the drug solution. however, since pablito, was helping cesar inject because his partner was unable to find his own veins, pablito would be in charge of the preparation. the cooker, a small sized tin cup provided by the local syringe exchange provider, el punto en la montana and a plastic water bottle were laid out on the cement floor by pablito, who was in charge of the preparation. the plastic clear blue bag containing cocaine was opened and placed carefully on the cooker along with the contents of three colored metallic paper envelopes with heroin. water from a plastic bottle was loaded into a syringe and then discharged into the cooker. the content was then mixed with the back of a syringe making sure it was completely diluted. although the cocaine is a white powder and the heroin a light brown, grey or creamy color, when mixed with water, the solution turned into a brown color. without heating-it is believed to "eat up" the drug because when heated some of the content evaporates leaving a more powerful concentration, but less quantity-pablito skillfully "recogio" or loaded the preparation with the help of a syringe with its needle inserted into a small cotton ball no larger than the head of a match to filter any impurities. with the syringe "full", half of its content was then "echado" distributed into cesar's syringe by removing the plunger on the back. after verifying that both syringes had exactly the same amount by holding them side by side, using the lines on the side of the syringes as a guide, the plunger in cesar's syringe was placed back. both syringes were now, half full, having divided a loaded syringe used to divide drugs up, in two equal parts. far from a ritual practice to strengthen a bond between injection partners, caballo seems to be a consciously made strategic decision to maximize drug access among pwid. drugs are jointly acquired and then divided up among pooling partners. the most common arrangement is to distribute the drug solution according to the monetary contributions of each member proportionally. partners can share drugs at "brazo partido" a local expression translated as half and half, or %- %, if they contributed equally, although it is possible that the partner that contributes the most agrees to divide drugs equally, particularly if they have a long-standing relationship with the caballo partner. josephine, a -year-old woman who started injecting in her teens, provides a description of the advantages of this practice: "look, let's suppose that i want to use two and one [two bags of heroin and one bag of cocaine] and that you have and i put [in for] the heroin. ' we put everything together in the cooker, and then we divide it in the syringe, half and half, and we get cured. that's it. " while most pwid in the study would prefer to avoid caballo if they could, particularly, for high frequency users, the economic demands make it extremely hard to go during the day without partnering with another user to acquire and use drugs. with a large proportion in unemployment, receiving meager social security checks, working at the lower levels of the local drug trade, or engaging in side hustles, it becomes extremely difficult for pwid in our area to secure the whole amount every time they need to inject. opioid users dread the painful effects of heroin withdrawal, or what they call "being sick, " characterized by bodily pain and discomfort, nausea, coldness, shivers, and diarrhea that leave them "unable to function. " only "la cura", the cure, another dose of heroin, would stop or prevent these symptoms form occurring. faced with limited resources to "get cured" the user has to make a choice between partnering with somebody in a caballo or going it alone and hustling until they can afford the whole dose they need. entering into a caballo arrangement, enables them to feel normal again, while they can keep hustling to get their next dose. while the rewards of going alone might be higher because participants get their full dose, so are the associated costs because users have to battle their withdrawal symptoms while they come up with the money. and the longer it takes for participants to secure the resources to afford their dose, the worst their withdrawal symptoms become, offering a powerful incentive to enter into a drug sharing agreement with another user. to avoid heroin withdrawal, if possible, caballo partners prefer to acquire their drug of choice in a punto close to where they can use it, without delay. but in relatively small rural locations, drug choices are restricted, particularly in relation to package size. locally, heroin can be bought in bags of five or ten dollars and cocaine comes in "cinquillos" fives, or $ , but if caballo partners can get a hold on a car, they usually pool up $ or $ each for gas and head to nearby caguas o san juan where bags cost the same but are two or up to three times larger. this drug packaging makes the trip worthwhile, even considering the gasoline costs and that time spent on the hour round trip could have been used "revuleando", hustling, at home. the frequency of caballo changes from participant to participant; some engage in caballo almost every time they use, while others do it less frequently. since caballo partners tend to inject smaller drug amounts, they also need to engage in this practice more often. speedball also provides more opportunities to share drugs because one partner might have cocaine but not heroin, while other might be in the opposite situation, in this situation, one solution is pooling resources and then dividing up the drug solution. pwid in our study declared that they prefer not to do caballo for their first dose of the day, as it would significantly decrease the amount of heroin received and thus stave off withdrawal symptoms for a shorter amount of time. walter, a -year-old user who injects two or three times a day, explains this choice with the use of a metaphor: "it's like pizza: if you eat two pieces you are going to feel full but if you eat only one, or just a bite, you will feel hungrier sooner. " however, here economic considerations play a role. if access to a sufficient dose to prevent the onset of withdrawal symptoms is not available, users might resort to caballo early in the day. those who have been successful in securing their own first dose might decide to engage in caballo later in the day in order to maximize resources, and can afford to engage in the practice more selectively. caballo is also affected by a particularly punitive version of the war on drugs adopted by the island since the ′s. most of our study participants have been jailed at least once in their lifetime, often for non-violent drug offences [ ] . having no more than a few bags can lead to heavy prison sentences under the "possession with intent" to distribute charges. to avoid problems, pwid tend to carry small drug amounts with them, which, in turn, encourages drug sharing. caballo can be structured along defined social roles, with important epidemiological repercussions. a primary partner directs the preparation and distribution of the drug solution, usually keeping the cooker and cotton used to share drugs. the soaked filter and the drug residue left in the cooker can be later re-used adding a little bit of water for another shot. usually, this role is occupied by the user that contributed the most to the caballo. these roles are not static, it is possible for one user to be a primary partner in one caballo but engage in another drug sharing arrangement later on, either with the same partner or a different one, without being in charge of the process. in addition, partners might follow different strategies while seeking to jointly acquire and use drugs. "fixers" do caballo with a limited number of trusted injection partners in their network, usually kin, or others with whom they have close relationships, from school age friends, to neighbors or those with whom they have shared drugs extensively in the past. by minimizing the number of partners and routinizing sharing expectations, this strategy ensures access to resources while limiting the potential problems associated with doing caballo with strangers. other pwid take on the role of "maximizers, " entering into caballo with as many partners as possible, increasing their opportunities to access drugs by multiplying potential partners. sometimes maximizers only know their caballo partners because they have seen them around, in puntos, or shooting galleries, or because they have done a caballo in the past. the downside is that this choice also increases the potential problems associated with the transaction-robbery, cheating, hoarding. yet, neither of these are fixed strategies. a pwid might have been a maximizer but, over time, begun doing caballo with a limited number of partners, and the opposite also happens. jail, drug treatment, quitting drug use, and migration can all affect a person's social networks and their ability to engage in caballo. of course, this is only an approximate typology, and some users are neither "fixers" nor "maximizers" but operate in between these extremes. whether users might be primary partner or not, or rely on a "fixer" or a "maximizer" strategy, they all seek in their interactions some kind of fair play, adhering to the norms that regulate drug sharing in the community. bebe, in his late s, washes cars at a local gas station in addition to taking turns as a drug dealer in the only drug selling spot in town. he explains the need for reciprocity: "i tend to avoid caballo, if i have all the money i need for my dose i get the drug and i get your money and even if you don't have enough, i share with you because i know what it is to be 'sick. ' how much do you have, i would ask? four dollars [an insufficient amount for a / caballo]. fine! let's go! and we share. but then i remember that in other occasions it was me that had only four bucks and i were really sick and he had his full dose and he decided not to help me out, leaving me sick to fend for myself. i still decided to help him but i tell him right away: 'see, before you told me you couldn't help me because you had enough for yourself and now you are desperate, see how the world is round? yesterday it was me [that needed] and today it's you. come here, i'll fix it!'". caballo partners who consistently demand more than their fair share are labeled "problematic" and tend to be excluded. "tricksters" are also avoided. according to participants, tricks are very common among pwid, such as pocketing the money that participants have pooled to do caballo. this is viewed very negatively, not only because it causes a lack of trust among partners but also because it deprives users of the "cure" they need, forcing them to hustle for money again before they can have their dose. tampering with "bags, " for example by taking a cut, is also negatively viewed but is judged less severely than the first situation. participants also worry about others pulling a "water shoot, " a trick in which an injector uses deception to substitute drugs with water. a similar and more frequent trick involves placing not one but two cotton balls in the cooker, hiding the one used to filter the drug under the finger holding the cooker, and leaving the other with only a trace of drug for the other injector to use. personality issues and past disagreements also play a role in selecting potential partners. those users with more social connections will be able to find more suitable partners for a caballo while being able to reject those partners deemed less desirable. the reverse is also true: those users with fewer social connections might not have as many options. caballo partnerships are often shaped by asymmetric dynamics involving gender and other forms of power disparities among prospective members. these in turn affect risk taking and drug related harms. caballo partners try to manage hiv/hcv by serosorting, but most assume that there is no point in asking about their partners' hiv status, because "they will lie to you. " perhaps because they are in a more vulnerable position, women tend to ask their injection partners about their hcv status more often than men do. women are also more likely to avoid entering in a caballo with somebody if they know the prospective partner has a positive status. this study outlines how the social practice of caballo has contributed to the production of an hcv epidemic among pwid in rural puerto rico. results show that . % tested positive reactive to hcv, a result that has been confirmed by other epidemiological surveys in the area [ ] . pwid tend to avoid direct sharing of syringes; only . % reported having used a needle after somebody else had employed it, and . % used a sterile needle the last time they used drugs with somebody. on the other hand, participants often engaged in indirect sharing: . % divided drugs with a cooker or cotton that had been used by somebody else, and . % divided drugs with a syringe that had been used by somebody else. these forms of indirect sharing are linked to the practice of caballo and the need to divide up jointly acquired drugs, which in turn increases the risk of hcv among this population. while pwid in our study tend to avoid direct sharing, indirect sharing is driven by the mode of drug acquisition, drug packaging and pricing, a reliance on speedball, the need to avoid heroin withdrawal, and a strict drug policy that encourage users to carry small amounts of illicit substances, among other factors. the finding that participants attempt to manage hiv/ hcv transmission risk during caballo by serosorting has been replicated in other studies of pwid [ ] [ ] [ ] . however, the epidemic levels of hcv in this population suggest that this strategy has serious limitations and that individual behaviors alone are insufficient to curb hcv transmission. findings illustrate the need to understand social epidemics such as hcv among pwid, leaving behind individual-centered models prevalent in public health. decades of ethnographic studies with this population have shown that what appears to be the product of individual behaviors is better comprehended as a result of the particular social contexts in which pwid live and make decisions about risk [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moving beyond public health's emphasis on individual behaviors, the notions of "risk environment" [ ] and "syndemics" [ ] have recently been used to analyze the interplay of micro-level dynamics and larger structural forces in accounting for risk outcomes. while both approaches share some features, rhodes ( ) suggests that a risk-environment approach-combining insights from political economy, social epidemiology, and the sociology of health-can be productive for understanding how the relationship between individuals and environment can produce drug harms, thus contributing to a social science of "harm reduction. " [ ] . our findings show that poverty and economic dispossession have been found to be an important driver of indirect sharing among pwid. high frequency users might spend $ or even more a day, not an insignificant sum considering that puerto rico has a per capita income more than half of the poorest us states like missouri or mississippi [ ] . the same relationship between poverty and drug sharing has been replicated in numerous studies [ , , ] . in a study of homeless pwid in san francisco bourgeois ( ) finds that if users cannot afford their dose in full, indirect sharing allows users to regulate their dose intake in order to minimize the risk of experience heroin withdrawal, with some participants using half or even a third of a bag each time. in turn, using small doses encourages higher frequency of drug injection, making drug sharing more likely. our findings have shown that roles within drug sharing arrangements are not fixed. this aspect has been documented also by finlinson [ ] suggesting that roles change within the process of acquiring and dividing up drugs. according to this author, a primary partner in charge of dividing up the drug solution using their own equipment, might become a secondary partner receiving a drug solution that has been divided using up somebody else's equipment in another caballo arrangement. these practices facilitate the transmission of hcv within a population, complicating harm reduction efforts. one of the most distinctive features of indirect sharing in our study is that the drug preparation is usually loaded from the cooker using a single syringe, and then distributed to the injecting partners through backloading. while indirect sharing offers a fair method for equitable distribution [ ] , participants in our study do not seem to favor drawing the drug solution directly from the cooker, because of the perception that it makes it harder to ensure an equal share of the drug. it is possible that this preference might be driven by a lack of trust among injection partners and that in different social contexts, other ways of dividing up the drug solution might be preferable. a study of puerto rican pwid in new york and puerto rico show that pwid in new york city take turns to draw the drug solution from the cooker while those on the island distributed it through backloading as well as taking turns [ ] . the fact that our findings correspond not to metropolitan san juan, but rural puerto rico might account for extensive use of backloading. as other authors have shown, the joint acquisition and use of drugs appear to be shaped by particular cultural norms, which in turn are an adaptive response to a particular risk environment [ ] . examining the social context in which pwid use drugs and make decisions about risk is critical for the successful adoption of harm reduction strategies to reduce hcv [ ] [ ] [ ] [ ] . while harm reduction policies that emphasize the distribution of injection equipment are a valuable component in syringe exchange programs, findings suggest that preventing the sharing drug preparation equipment while sharing drugs might be more complex that behavioral health models suggests [ ] . indirect sharing occurs not because pwid lack information about hcv risk or do not possess knowledge about safe injection practices. this perspective has been criticized for blaming the victim [ ] while obscuring the role of social and risk environments in shaping drug sharing arrangements [ , ] . in addition, local implementation of effective harm reduction recommendations regarding hcv transmission among pwid is hampered by larger political and socio economic processes such as the colonial status of puerto rico that deprives the island of the resources it needs to tackle an hcv epidemic, while enacting an aggressive version of the war on drugs and subsequent incarceration that furthers the impact of hcv within its population [ ] . furthermore, the subjection to us patent laws and drug pricing ensures that despite the fact that a new hcv treatment is available (in the form of a three-month-long direct-acting antiviral regimen), the cost of that treatment is approximately $ , . in the continental us, the cost of this treatment is covered by most private insurers or by medicare/medicaid; in puerto rico, few pwid have access to private insurance, and hcv treatment is not covered by la reforma, a local version of medicare/medicaid for hiv-negative individuals who constitute the majority of the pwid population [ ] . the extremely low hcv coverage among pwid [ ] continues to fuel the epidemic as hcv-negative users are more likely to enter caballo arrangements with somebody infected by the virus. expanded hcv treatment coverage would not only reduce the incidence of hcv among this population but also would contribute to provide certain measure of "herd immunity" protecting those that are hcv negative [ ] . similarly, access to harm-reduction-based interventions such as medically assisted treatment and syringe exchange provision have been shown to be protective against hcv transmission [ ] [ ] [ ] [ ] [ ] [ ] [ ] . unfortunately, these resources are severely lacking in puerto rico, particularly in rural areas [ ] . a recent study showed that rural pwid have less access to syringe exchange in rural areas than those in urban settings, although both have had to resort to acquiring syringes from drug stores, peers, and drug dealers [ ] . understanding the social factors behind the hcv epidemic in puerto rico has a renewed urgency. as the island was starting to recover from the aftermath of hurricane maria in september it was hit by the arrival of covid- , a pandemic with an epicenter in wuhan china that has currently stricken most of the world. while the lasting effects of covid- are not yet well known, it is likely the pandemic will reinforce the devastating effects of hurricane maria which furthered a protracted economic crisis on the island, while exposing deep political corruption and mismanagement practices, [ , ] severely affecting the provision of health services pwid rely on, like medically assisted treatment and syringe exchange providers, leading the way to the worsening of an already existing hcv epidemic. increasingly, harm reduction programs will need to consider the impact of large-scale natural disasters, or pandemic events, and its effects on the risk environment of pwid, already afflicted by severe forms of poverty, social suffering, and structural violence [ ] . one limitation of this study is that findings are based on a sample that is overwhelmingly male. while this distribution mirrors not only the composition of the parent study but also of other studies of pwid in puerto rico that seem to be gendered, the relative lack of female participants raises questions about how gender dynamics and power imbalances might shape drug sharing arrangements among pwid in rural puerto rico. more research is needed to explore this issue. we believe that despite this limitation, the study of the social organization of drug sharing arrangements makes an important contribution to the understanding of hcv risk among this population. this study shows that drug sharing plays an important role in the hcv transmission among pwid in rural puerto rico. while participants avoided direct sharing of syringes, they were forced to share the cooker and cotton used to divide and inject the drug solution. drug sharing occurs not only within the joint acquisition and use of injection drugs, but in a particular risk environment that contributes to hcv risk. poverty, drug packaging and pricing, a reliance on speedball, the need to avoid heroin withdrawal, and a strict drug policy that encourage users to carry small amounts of illicit substances, among other factors fuels the hcv transmission. this finding complicates harm reduction interventions based on the distribution of injection equipment or information about safe injection practices alone, suggesting that preventing indirect sharing practices should consider the social context in which pwid acquire and use drugs. deaths associated with hepatitis c virus infection among residents in states and the district of columbia estimating the number of persons who inject drugs in the united states by meta-analysis to calculate national rates of hiv and hepatitis c virus infections risk factors for hcv infection among young adults in rural new york who inject prescription opioid analgesics hiv infection among persons who inject drugs: ending old epidemics and addressing new outbreaks understanding differences in hiv/hcv prevalence according to differentiated risk behaviors in a sample of pwid in rural puerto rico prevalence and correlates of hepatitis c virus infection among street-recruited injection drug users in global estimates of prevalence of hcv infection among injecting drug users county-level vulnerability assessment for rapid dissemination of hiv or hcv infections among persons who inject drugs, united states sharing of drug preparation equipment as a risk factor for hepatitis c des jarlais dc. meta-analysis of hepatitis c seroconversion in relation to shared syringes and drug preparation equipment prevalence and correlates of indirect sharing practices among young adult injection drug users in five u risk of hepatitis c virus transmission through drug preparation equipment: a systematic and methodological review risk of transmission associated with sharing drug injecting paraphernalia: analysis of recent hepatitis c virus (hcv) infection using cross-sectional survey data sharing cookers and cottons are surrogates for drug sharing drug sharing among heroin networks: implications for hiv and hepatitis b and c prevention social capital or networks, negotiations, and norms? a neighborhood case study ethnography, epidemiology, and public policy: needle-use practices and hiv- risk reduction among injecting drug users in the midwest migration and hiv risk behaviors: puerto rican drug injectors in new york city and puerto rico syringemediated drug sharing among injecting drug users: patterns, social context and implications for transmission of blood-borne pathogens injecting equipment sharing in russian drug injecting dyads it's not what you know but who you know": role of social capital in predicting risk injection drug behavior in a sample of people who inject drugs in baltimore city hiv risk behaviors associated with the injection process: multiperson use of drug injection equipment and paraphernalia in idu networks prescription opioid injection among young people who inject drugs in new york city: a mixedmethods description and associations with hepatitis c virus infection and overdose street-level drug markets: network structure and hiv risk. soc netw risk and reciprocity: hiv and the injection drug user drug preparation, injection, and sharing practices in tajikistan: a qualitative study in kulob and khorog drug injecting and hiv risk among injecting drug users in hai phong, vietnam: a qualitative analysis trends in hiv seroprevalence and needle sharing among puerto rican drug injectors in puerto rico and new york: - injecting shared drugs: an observational study of the process of drug acquisition, preparation, and injection by puerto rican drug users social misery and the sanctions of substance abuse: confronting hiv risk among homeless heroin addicts in san francisco the textures of heroin: user perspectives on "black tar" and powder heroin in two us cities addressing the "risk environment" of injection drug users: the mysterious case of the missing cop syndemics and public health: reconceptualizing disease in a bio-social context risk environment": a framework for understanding and reducing drug related harm the social production of hepatitis c risk among injecting drug users: a qualitative synthesis back then" and "nowadays": social transition narratives in accounts of injecting drug use in an east european setting needle acquisition patterns, network risk and social capital among rural pwid in puerto rico etnographer's toolkit: designing and conducting ethnographic research treatment scaleup to achieve global hcv incidence and mortality elimination targets: a cost-effectiveness model grounded theory research: procedures, canons, and evaluative criteria it ruined my life": the effects of the war on drugs on people who inject drugs (pwid) in rural puerto rico hepatitis c serosorting among people who inject drugs in rural puerto rico injection-related hepatitis c serosorting behaviors among people who inject drugs: an urban/rural comparison hepatitis c virus serosorting in people who inject drugs: sorting out the details stories of pain and the problem of aids prevention: injection drug withdrawal and its effects on risk behavior beyond prevention? injecting drug user narratives about hepatitis c do practice approaches go far enough in shifting focus from the individual? addiction the pastoral clinic: addiction and dispassion along the rio grande addicted, pregnant, poor needle exchange and the geography of survival in the south bronx enabling environments and the reduction of drug related harm: re-framing australian policy and practice intravenous drug use and hiv infection in miami social and structural violence and power relations in mitigating hiv risk of drugusing women in survival sex work perception of risk and safety within injection settings: injection users' reasons for attending a supervised injecting facility in vancouver structural violence and structural vulnerability within the risk environment: theoretical and methodological perspectives for social epidemiology of hiv risk among injection drug users and sex workers critical medical anthropology risk environments and drug harms: a social science for harm reduction approach small area income and poverty estimates drug sharing among intravenous drug users attitudes toward needle "sharing" among injection drug users: combining qualitative and quantitative research methods joint drug purchases and drug preparation risk behaviors among puerto rican injection drug users injection norms and practices among migrant puerto rican people who inject drugs in new york city: the limits of acculturation theory social structural factors that shape assisted injecting practices among injection drug users in vancouver, canada: a qualitative study navigating environmental constraints to injection preparation: the use of saliva and other alternatives to sterile water among unstably housed pwid in london safer environment interventions": a qualitative synthesis of the experiences and perceptions of people who inject drugs a systematic review of rural-specific barriers to medication treatment for opioid use disorder in the united states the theory of planned behaviour: reactions and reflections the political economy of aids among drug users in the united states: beyond blaming the victim or powerful others the process of drug injection: applying ethnography to the study of hiv risk among idus commentary on harris & rhodes: discouraging syringe re-use by addressing drug injectors' everyday suffering puerto rican syndemics: opiates, overdoses, hiv, and the hepatitis c virus in a context of ongoing crisis to enroll or not to enroll?: a researcher struggles with the decision to involve study participants in a clinical trial that could save their lives treatment access is only the first step to hepatitis c elimination: experiences of universal anti-viral treatment access in australia convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year submit your research ? effectiveness of structural-level needle/syringe programs to reduce hcv and hiv infection among people who inject drugs: a systematic review state hcv incidence and policies related to hcv preventive and treatment services for persons who inject drugs-united states des jarlais dc. a systematic review and meta-analysis of interventions to prevent hepatitis c virus infection in people who inject drugs the promise of treatment as prevention for hepatitis c: meeting the needs of people who inject drugs? recommendations for the management of hepatitis c virus infection among people who inject drugs combination interventions to prevent hcv transmission among people who inject drugs: modeling the impact of antiviral treatment, needle and syringe programs, and opiate substitution therapy retention in buprenorphine treatment is associated with improved hcv care outcomes tackling the health challenge posed by hepatitis c in puerto rico: a call for immediate public health actions differential access to syringe exchange and other prevention activities among people who inject drugs in rural and urban areas of puerto rico the battle for paradise: puerto rico takes on disaster capitalists aftershocks of disaster: puerto rico before and after the storm. chicago: haymarket books the everyday violence of hepatitis c among young woman who inject drugs in san francisco aids and accusation: haiti and the geography of blame the social value of drug addicts: uses of the useless springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - unadw authors: ogidigo, joyce oloaigbe; iwuchukwu, emmanuel a.; ibeji, collins u.; okpalefe, okiemute; soliman, mahmoud e. s. title: natural phyto, compounds as possible noncovalent inhibitors against sars-cov protease: computational approach date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: unadw at present, there is no cure or vaccine for the devastating new highly infectious severe acute respiratory syndrome coronavirus (sars-cov- ) that has affected people globally. herein, we identified potent phytocompounds from two antiviral plants momordica charantia l. and azadirachta indica used locally for the treatment of viral and parasitic infections. structure-based virtual screening and molecular dynamics (md) simulation have been employed to study their inhibitory potential against the main protease (m(pro)) sars-cov- . a total of compounds from m. charantia l. and a. indica were identified. the top six phytocompounds; momordicine, deacetylnimninene, margolonone, momordiciode f , nimbandiol, -hydroxyazadiradione were examined and when compared with three fda reference drugs (remdesivir, hydroxychloroquine and ribavirin). the top six ranked compounds and fda drugs were then subjected to md simulation and pharmacokinetic studies. these phytocompounds showed strong and stable interactions with the active site amino acid residues of sars-cov- mpro similar to the reference compound. results obtained from this study showed that momordicine and momordiciode f exhibited good inhibition potential (best mmgba-binding energies; − . and − . kcal/mol) against the m(pro) of sars-cov- when compared with fda reference anti-viral drugs (ribavirin, remdesivir and hydroxychloroquine). per-residue analysis, root mean square deviation and solvent-accessible surface area revealed that compounds interacted with key amino acid residues at the active site of the enzyme and showed good system stability. the results obtained in this study show that these phytocompounds could emerge as promising therapeutic inhibitors for the m(pro) of sars-cov- . however, urgent trials should be conducted to validate this outcome. communicated by ramaswamy h. sarma severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel strain of the coronavirus which is considered a global public health emergency (who, ; read et al., ) . it was first discovered in december in the city of wuhan, hubei province, china, and has since spread across the globe affecting over million people with over . million deaths as of october . in humans, sars-cov- is believed to cause mild respiratory infections, such as those observed in the common cold to severe pulmonary disease with an extreme case of death (khan et al., ; lu et al., ) . studies suggest that the sars-cov- can be transmitted through infected droplet (respiratory secretions) and close person-to-person contact (khan et al., ) where it binds to the primary target cells such as enterocytes and pneumocytes, coronaviruses, respectively (wu et al., ) . coronaviruses main protease (m pro ) also known as -chymotrypsin-like protease ( cl pro ) is an important protein that is required for proteolytic maturation of the coronavirus (wu, et al.) . the main protease is considered the "achilles hill" of coronaviruses (fehr & perlman, ) inhibiting the activity of this enzyme could block viral replication. moreover, the m pro is highly conserved across coronaviruses thus, the main proteases are considered an attractive target for the development of effective antiviral drugs in structure-based drug design for the treatment of sars-cov- and other coronaviruses (garc ıa-fern andez et al., ; krishnan & anirudhan, ; zhang et al., ) . currently, there is no known cure or vaccine for the covid- . a rapid search is on-going for the development of vaccines and new antiviral drugs for the effective treatment of the highly transmissible sars-cov- . many researchers are attempting to find new inhibitors for the sars-cov- main protease m pro both from synthetic and naturally active compounds (elfiky, ; khan, et al., ) . several drugs have been repurposed and used as a frontline therapy for sars-cov- and relief of symptoms for patients infected with covid , and these drugs include hydroxychloroquine antimalaria and anti-hiv drugs. on the may , the fda reference drug an emergency use authorization of remdesivir for the treatment of hospitalized patients with covid- (fda, ). yet the clinical safety and efficacy against covid- remain to be fully established. several crystal structures of sars-cov- c-like proteinase have been solved and made are available on protein databank. however, only one complex structure of sars-cov- c-like proteinase (pdb: w ) was bound to a broad spectrum noncovalent covalent inhibitor (x ) in data bank (https://www. rcsb.org). it has been observed that most of the current research effort focus on finding potential sars-cov- mpro covalent inhibitors while the possibilities to identify non-covalent inhibitors remain less investigated. for that reason, we decided to screen new natural phytocompounds against the w crystal structure to find potential non-covalent pharmacophores against sars-cov- c-like protease. previous studies have shown that covalent and non-covalent inhibitors have been designed and discovered to inhibit c proteinases and therefore to treat related diseases. these inhibitors can be used as guidance to design drugs for sars-cov- (zhavoronkov et al. ). active compounds have been obtained by covalent bonding with the cysteine at the catalytic site. however, side effects and toxicity can often be caused by covalent bonding ( zhavoronkov et al. ) . thus, prevents their use for clinic therapy. hence, for sars-cov- treatment, it is more attractive to design and discover noncovalent inhibitors. virtual screen by molecular docking of chemical databases is one of the most powerful approaches to discover non-covalent inhibitors. we report herein the screening of potential phyto non-covalent inhibitors against sars-cov- c-like proteinase. medicinal plants provide an excellent source for antiviral drugs due to fewer side-effects, low cost, bioavailability and easy availability along with less potential to cause resistance (mukherjee et al., ) . hence, the utilization of herbal drugs in viral infections is an important future therapeutic strategy. medicinal plants contain numerous phytochemicals responsible to exhibit the antiviral effect. two commonly used anti-viral herbs: momordica charantia l. (cucurbitaceae) and azadirachta indica a. juss. (meliaceae) are potent medicinal plants used in traditional medicine in nigeria and tropical countries for the treatment of many ailments including inflammatory, anti-diabetic, parasitic and viral infections. traditionally, these plants have been reported to exhibit potent antiviral activities by inhibiting several viruses including herpes simplex virus, hepatitis b virus, smallpox virus and human immunodeficiency virus (liu et al., ; mohammad et al., ; pongthanapisith et al., ) . considering the wide potent antiviral activity of these plants, it is, however, possible that potent non-inhibitors could emerge from these plants against sars-cov- main protease m pro . in this study, virtual screening of potent medicinal bioactive compounds from plants of m. charantia l. and a. indica as potential inhibitors against sars-cov- main protease m pro was carried out. based on the docking scores, six compounds we ranked and preceded for molecular dynamics (md) simulations and pharmacokinetic studies. three-dimensional x-ray crystallographic structure of sars-cov- m pro was downloaded from the rcsb protein data bank (pdb). the sars-cov m pro bound to a noncovalent inhibitor with pdb id: w (resolution . Å) (zhavoronkov et al., ) was used for this study. before the virtual screening, the protein structures were prepared using the 'protein preparation wizard' workflow in the schrodinger suite. this involved the addition of hydrogen atoms to the protein, assignment of bond orders, and deletion of unnecessary water molecules. the key water molecules interacting with the active site residues of the enzyme were retained. sidechains were added, disulphide bonds were formed, missing atoms were added, and the partial charges were assigned. energy minimization was done using opls_ (optimized potentials for liquid simulations) force field. as the downloaded protein was co-crystallized with x (n-( - a non-covalent inhibitor, the ligand-binding site was used to define the active site of the protein. receptor grid generation workflow was used to define a grid (box) around the ligand, to keep all the functional residues in the grid (sastry et al., ) . the residue used for preparations is cys . structural preparation -dimensional structures of compounds from m. charantia and a indica were identified and retrieved from the pubmed literature as test ligand molecules, two antiviral drugs and anti-malarial drug were retrieved from pubchem database as a reference. ligprep, pre-processing of the ligands was done, which includes the formation of tautomers and ionization states at ph . ± . using epik, hydrogen atoms were added, charged groups were neutralized and geometry of the ligands was optimized. virtual screening of compounds was performed in three stages involving a high throughput virtual screening (htvs), standard precision (sp) and extra precision (xp) using the using glide tool (grid-based ligand docking with energetics) algorithm implemented in schrodinger (friesner et al., ) . stage one and two, htvs and sp employ the self-scoring function, whereas xp reduces the intermediate conformations and thoroughness of the torsional refinement and sampling. a total of compounds and three reference fda drugs was docked against each target protein with the co-ligands x and a-ketoamide was used as a positive control. next, top scorers were set forth for sp docking, and the output of sp docking was put forward in xp docking with positive control. all the results were analysed in xp visualizer. compounds with the highest binding energies against sars-cov- m pro was selected for further md and in silico pharmacokinetic studies. the six top screened phyto-ligands (of the compounds) with high binding energy, three fda reference drugs and the apo-enzyme (apo) of the target was subjected to md simulation. the graphical processing unit (gpu) version of amber software package was used to perform the molecular dynamic (md) simulations of all the systems. optimization of the systems was carried out using antechamber (wang et al., ) and leap module of amber to ensure all the appropriate parameters were available for md simulations. the protein parameters were assigned using ff sb (perez et al., ) version of the amber force field. the leap module of amber was applied for adding the missing hydrogen atoms and counter ion to neutralize the systems. the systems were suspended with an orthorhombic water box of tip p (jorgensen et al., ) to restrain the protein within Å of the box edge. long-range electrostatics were treated with the particle-mesh ewald method (kholmurodov et al., ) implemented in amber with direct space and a a van der waals cut-off while periodic boundary conditions were adopted. before the commencement of md, initial and final minimization, gradual heating and the equilibration steps were performed. the final md production was performed as previously reported (emmanuel et al., b; ibeji, ) . the cpptraj and ptraj modules (roe & cheatham, ) of the amber suite was used to carry out post-analysis such as radius of gyration, root mean square deviation (rmsd), root mean square fluctuation (rmsf) and solventaccessible surface area (sasa). structural visualization and plots were done using ucsf chimera software package molecular modelling tool and origin data analysis software version (seifert, ) (http://www.originlab.com). mm/gbsa a key tool used in predicting macromolecular stability and protein-ligand binding affinity (genheden & ryde, ; zhou & madura, ; zhou et al., ) were applied. it gives an idea of the binding mechanism which equally includes the contribution of enthalpy, entropy to the molecular recognition and ligand-protein association (genheden & ryde, ) . the binding free energy was averaged over snapshots which were extracted from the ns trajectory as described by the set of equation. where e gas is the gas-phase energy; e int is the internal energy; e ele stands for the coulomb and e vdw are the van der waals energies. e gas is obtained from the ff sb force field terms. the solvation free energy, g sol , is split into polar and non-polar solvation state of contribution. here the polar solvation, g gb, contribution is calculated by solving the gb equation while the non-polar solvation contribution, g sa is estimated from the sasa calculated with the water probe radius of . Å. t and s are the temperatures and the solute entropy, respectively. to examine the individual amino acid contribution to the total binding free energy between the natural products and main protease, the per-residue free energy decomposition analysis was computed at the atomic level using the mm/gbsa method in amber . a comparative evaluation of the pharmacological properties of the natural phytocompounds and fda reference drugs was performed. this was done by predicting their individual pharmacokinetic (admet) properties with online and offline tools. the bioactivity of the various compounds was estimated using selected and offline tools. the prediction tools employed include molsoft program (http://molsoft.com/ mprop/), molinspiration cheminformatics (husain et al., ) , protox webserver (banerjee et al., ) and the osiris datawarrior property explorer (l opez-l opez et al., sander et al., ) . more than one prediction tools were used for precise validation and reproducibility of comparative analysis. the pharmacokinetic (admet) properties of the studied compounds were evaluated using molsoft online program, this is to investigate if they adhered to lipinski's rule of five (ro ). an additional step was equally taken to validate the obtained pharmacokinetic properties by employing data warrior property explorer (sander et al., ) , this program helps to predict ligand efficiency (le) (abad-zapatero, ; hopkins et al., ) , lipophilic ligand efficiency (lle) and lipophilicity-correlated ligand efficiency (lelp) (hopkins et al., ; johnson et al., ) . toxicity of compounds reveals their safety state for consumption. thus, in silico prediction of oral toxicity gives a faster route of obtaining information about doses that could be toxic in animals. in the present study, we screened compounds from m. charantia l. and a. indica as potential inhibitors against sars-cov- m pro (pdb id: w ) using structure-based drug design. this approach is based on computationally fitting compounds into the active site of the target protein, followed by a ranking of these compounds based on their low binding energies and interaction with the residues of the binding pocket. the docking scores of the compounds were when compared with a standard m pro inhibitor, a-ketoamide b and x . the a-ketoamide b is a broad-spectrum inhibitor with an ic value of . ± . lm against purified recombinant sars-cov- m pro . a comprehensive list of the docked phytocompounds can be found in the supplementary file (supplementary material table s ). thus, among the phytocompounds and antiviral drugs screened (supplementary material table s ), phyto ligands exhibited appreciable binding energies against the sars-cov- m pro and strong interactions within the binding pocket. the top six hit phyto-compounds were momordicine, deacetylnimninene, margolonone, momordiciode f , nimbandiol, -hydroxyazadiradione with binding energies of . , . ,À . , À . , À . and À . kcal/mol, respectively. also, the fda reference anti-viral drug remdesivir and ribavirin showed binding energies of À . and À . kcal/mol, respectively (table ). in comparison, standard m pro inhibitor; a-ketoamide b and x displayed binding energies of À . and À . kcal/mol, respectively. the results show that all the lead compounds were tightly bound into the substrate-binding pocket, through a good number of hydrogen bonds as well as hydrophobic interactions ( figure s ). the glide d interaction suit was used to reveal the interacting residue information. in particular, the best phyto ligand "momordicine" (m. charantia) was seen to interact with the target enzyme with a binding energy of À . kcal/mol. momordicine formed five hydrophobic interactions with leu , met , cys , met , leu , pro and established two hydrogen bonds with glu and asn ( figure s (a)). the second lead phytocompound i.e. deacetylnimninene (a. indica) showed two hydrogen bonds with residues cys and glu and eight hydrophobic interactions with cys , met , pro , tyr , cys , met , leu and pro ( figure s (b)). furthermore, it was observed that the fda antiviral drug; remdesivir exhibited the highest binding affinity among all tested compounds with a binding affinity of À . kcal/mol (table ) . this displayed three hydrogen bond interactions with cys , gln and thr residues and eight hydrophobic interaction with key residues (cys , met , tyr , pro , phe , leu , cys , met , leu , pro , val , ala ) ( figure s (g)). notably, the top lead phyto ligand and redemsivir were able to establish hydrogen bond and hydrophobic interactions with residues crucial for the inhibition of sars-cov- replication similar to phyto ligands and the standard m pro inhibitor, alpha-ketoamide and x ( figure s (h,j)). the sars-cov- m pro substrate-binding site mainly consists of a cysteine-histidine dyad (his and cys ) which controls the catalytic activity of sars cov- m pro . also, our share similar binding interaction patterns with a previous study on virtual screening of novel non-covalent inhibitors against sars-cov m pro (liu et al., ) . inhibition by these compounds reflects the possible inhibitory tendencies against the covid- . sars-cov- m pro with other papain-like proteases is important for processing the polyproteins into various non-structural proteins by cleaving at specific sites that are translated from the viral rna. several key amino acids in the active site of m pro enzyme have been documented to be leu, gln, ser, ala, gly along with the cys-his dyad which marks the cleavage site. based on the results obtained we further subjected the phyto ligands and the antiviral drugs remdesivir and ribavirin to md simulation to establish the mode of interaction with mpro protease. previous reports have suggested main protease (m pro , clpro) as a very attractive target for coronaviruses due to the crucial role it plays in processing the translated polyproteins of the viral rna . to verify the dynamics of the protein-ligand complex a ns md simulation was run, to reveal the conformational trends which occurred in the protein's backbone to the initial conformations. from figure , all the studied systems showed a favourable stability state during the ns of the md simulation after converging at around ns. from the average rmsd of all the systems in table , the apo-enzyme exhibited almost the lowest average rmsd when compared with the ligand-bound systems. however, exceptions exist in the case of hydroxychloroquine with almost a close mean rmsd ( . Å) value to that of the apo ( . Å) and deacetylnimbinene with lower rmsd of . Å. the average rmsd observed in all the systems revealed a tractable structural trend in the natural products and the reported fda reference drugs. the slightly lower average rmsd of the apo corroborate with the reported structural behaviour of proteases (munsamy et al., ) . the observed collective marginal structural deviation could be an indication of the characteristic mechanistic inhibitory mode of mpro. previous reports suggested that proteases exhibit consistent open/close and twisting dynamic motion mechanisms at the flap, flexible domain and hinge regions which promotes ligand binding (munsamy et al., ) . it is therefore suspected that this reported structural motion is transmitted to the entire protein structure which resulted in a higher conformational deviation of the complexed systems relative to the apo. hence the observed higher structural deviation of the ligandbound systems relative to the apo is an indication of inactivity of the viral mpro due to ligand binding. although high rmsd is an indication of atomic deviation, which suggest structural instability, based on the reported structural/conformational behaviour of proteases, the high rmsd of the bound systems relative to the apo does not suggest structural instability but rather a functional display in line with the reported structural behaviour of proteases. this indicates viral mechanism of viral protein inactivity. from the plots, momordicine stabilized immediately after attending convergence and maintained this stabilized trajectory till the end of the simulation whereas margolonone fluctuated from - ns within an rmsd between . and . Å and eventually reassumed stability till the end of the simulation whereas the rmsd of the other systems remained below Å. however, the lower average rmsd value of deacetylnimbinene and hydroxychloroquine when compared with the apo and observed little conformational changes in momordicine and margolonone were not of much different and does not indicate that ligand binding causes serious conformational changes. the structural dynamics of the ligands to the mpro at the active site was further investigated by tracking the ligand confirmation and/or orientations during the md simulation. the ligand rmsd indicate the stability of the ligands concerning the protein and its active site residues. as it is anticipated that binding pocket architecture could influence the mobility, orientation and interaction of drug molecule with protein residues. from figure (a -c ) remdesivir exhibited serious orientational/positional deviation to the other ligands examined, however, all the natural products exhibited lower conformational deviation relative to the reference drugs with ribavirin maintaining a very similar conformational trajectory with all the natural products. the plot revealed that momordicine fluctuated from to ns before stabilizing again. on the other hand, momordicoide f elicited higher structural deviation to the starting conformation while exhibiting higher structural deviation relative to the other natural products and the reference drugs except for remdesivir. a large conformational change was exhibited by remdesivir at the beginning of the simulation before attaining and maintaining stability; this initial wide conformational change of remdesivir could be a result of the orientational adjustment to properly fit into the binding pocket. to further gain insight into the conformational trends which resulted to the high conformation deviation of remdesivir in the ligand rmsd plot, a visual analysis/representation of the average conformational orientation of all the ligand at the active site of mpro was done. as shown in figure , remdesivir (blue) drifted towards the edge of the pocket and does not have contact with virtually all the ligands in the pocket except ribavirin. however, a look at the pocket, revealed that the rest of the ligands have a common contact point(s). the nature of motion exhibited by these ligands could have a relationship with the most favourable optimal orientation needed for most suitable interaction with active site residues. at the long run remdesivir has the highest average rmsd followed by momordicoide f and finally hydroxychloroquine this is an indication of close conformational/orientational behaviour of the natural product with that of the reference drugs as both ligand types did not have much difference in conformational trend. this minor difference in the mean rmsd values of all the simulated systems indicates that the binding of the ligand does not severely perturb the global structure of the protease. to gain insight into the nature and feature of the fluctuations displayed by the backbone atoms of the proteins, the c-a rmsf was investigated. rmsf plots reveal the precise conformational transition within the residues that make up the proteins secondary structure during md simulation. it further shows the nature of residual fluctuation and motion to ligand binding and unbinding. a higher value of rmsf indicates an increase in flexibility while a lower value correlates with a decline in flexibility. figure (a-c) shows that the residues in all the systems maintained a relatively similar pattern but varied levels of fluctuations. as indicated in table , all the systems had a near approximate value of average rmsf with deacetylnimbinene, momordicine and nimbandiol exhibiting the lowest residual fluctuation. however, regions of interest can be noticed in the plots, these are areas where the residues exhibited peak fluctuations. the nature/degree of residual drift from their mean positions in these interesting regions has been indicated with d images in the plots in figure for clarity. these are residues in the active site the play crucial role in system stabilization. they are residues that are actively involved in protein-ligand interactions(ibeji, ; olotu et al., ). these residues with residue numbers - , - , - , - , - exhibited wider and unusual fluctuations relative to others. to gain further insight into the degree and distinctiveness of these fluctuations in these regions, we performed an active site analysis of the rmsf of these residues and the results are presented in figure (a -c ). a close look at the average active site rmsf, table indicates that the phytocompounds have similar average active site rmsf values to those of the reference drugs. it was also observed that higher active site rmsf of the entire ligand systems when compared with apo corroborates with initially mentioned open/close and twisting mechanism of ligand inactivation of the viral protease. however, nimbandiol and momordicine had a very low average active site rmsf, this could be due to nonoptimal orientation for maximum interaction with the active site residues. remdesivir equally exhibited lower mean active site rmsf, though not too far from that of the apo. this lower mobility in the active site rmsf of these compounds could be suggestive of the need for ligand optimization. this further broadened the understanding of the mobility of the crucial residues in the active site which is the location of the flap and hinge regions. rog measures the degree of compactness of the backbone carbon atoms of a protein and accounts for the level of mobility of a protein structure which is an indication of stability in the protein backbone structure. the conformational shift/alteration initially suggested to be the mechanism of activity of proteases in the rmsd plots is validated by the results obtained in the rog plot. rog gives an idea of type/ nature structural deviation exhibited by the rmsd plot. the average rog values from the plot in figure revealed that the apo has an rog value that is slightly the lowest ( . Å) relative to the other systems. this low rog value which suggests the reduction in mobility validates the mechanism of ligand binding at the active site of protease and the entire proteases. as the previous report has suggested the ligand-binding mechanism to be via open/close and twisting motion which induces a high level of conformational deviation in protein d structure. elevated average rog indicates a decrease in compact structural packing which suggests increased mobility (emmanuel et al., a) and a stable state favourable for ligand binding to proteases. the nature of the trajectories exhibited by the rog plot of the ligand-bound relative to the apo is suggestive of the mechanism of ligand inactivation of proteases. hence, inhibiting the activity of this enzyme will inhibit viral replication and transcription. figure (d) shows a superimposed d images of the mpro starting structure, that of -hydroxyazadiradione complex with the highest mean rog value ( . Å) and that of the apo at the end of the simulation. the image reveals a comparative minor level of the structural deviation which could be suggestive of the level of compact packing of the protein. residual mobility which induces side-chain re-orientation was further studied by examining the intermittent burial and exposure of the hydrophobic and hydrophilic residues during the time duration of the md simulation. protein folding and unfolding could correlate with the re-orientation of the side chains from the hydrophilic phase to the hydrophobic phase. hence structural transformation of the surface residues towards the hydrophobic core could be indicative of protein unfolding whereas the inward transition of these surface residues suggests protein folding. this parameter was employed to examine protein activity and inactivity as indicated in figure . previous reports have revealed that high sasa values connote decrease in the exposure of the buried hydrophobic residues which indicates the reduction in systems stability while expulsion of the buried hydrophobic residues from the hydrophobic core indicates an increase in system stability figure (b ) and (c) figure (c ). (emmanuel et al., a) . from average sasa values in table which were obtained in figure , it can be observed that all the natural products have slightly lower average sasa values to the apo except for -hydroxyazadiranone with slightly higher average sasa values than the apo. two of the reference drugs; remdesivir and hydroxychloroquine individually produced the highest respective average sasa values relative to the natural products except for -hydroxyazadiranone thus indicating that the natural products were more stable than the drugs. therefore, this lower sasa values in the natural products systems relative to the apo and the two reference drugs systems are indicative of an increase in hydrophobicity induced by ligand binding. the results obtained here corroborate with the observations made from the rmsd, rmsf and rog plots. this structural unfolding due to the exposure of the hydrophobic residues as observed from the lower average sasa values correlates with the initially mentioned open/close and twisting mechanism of the protein activity and inactivity. conformation transition from the folded state to the unfolded state could indicate the inhibitory mechanism of ligand-bound protease. figure (d) is a d representation of an mdc-bound mpro (with almost lowest average sasa value); showing the solvent assessable surface (sas). it reveals that the sas (hydrophilic) occupy the smaller area around the ligand at the active site than the non-sas (hydrophobic). this trend at the active site is believed to be pass on to the entire protein residue. previous studies have suggested that inhibition of the activity of proteases equally inhibit the viral replication and transcription and henceforth viral survival and disease progression (ibeji, ; olotu et al., ) . this gives an idea of the interaction that is likely to be harnessed to design novel and more potent anti-viral inhibitors that will block further viral protein replication. we went further to investigate the energetic inhibitory potency of the selected natural product in comparison with reference drugs. mm/ gbsa has recently grown as a very popular computational tool for estimation of the binding propensity of chemical compounds to protein. the results obtained in table revealed thermodynamic interaction of ligands to protein and hence suggests the capacity of the ligand to block the replication and transcription of the viral protein which indirectly indicate the inhibitory prowess of the compound. our finding as indicated in table shows that all the selected reference drugs and the natural product have favourable binding free energy, efficient enough for good binding interaction. as outlined in the table, the three fda reference drugs gave binding free energy (dg bind ) that are close to the values obtained for most of the natural products except for momordicine and momordiciode f with very high binding À . and À . kcal/mol, respectively. this higher binding energy exhibited by these two compounds could be due to the similarity in their structural features. and this large binding energy of these two compounds is suggestive of the need for further studies to identify the unique moieties which are responsible for high energetic interaction that produced this high-affinity binding. some literature has highlighted crucial residues that play a vital role in the binding interaction of compounds with mpro with inhibitors, some examples are the catalytic residues his and cys . the decomposition of the total energy into electrostatic and van der waals (figures - ) has equally enabled us to obtain vital information about more residues that contributed to the energetic interaction and stabilization of the studied compounds to the main protease. the estimation was essential to identify crucial active site residues and their respective energy contributions towards favourable inhibitory activities of various compounds and stabilization of the protein. from the plot of the per-residue energy contribution prominent residues with most outstanding total energy (with a threshold from . kcal/mol) drawn from van der waals, electrostatic, polar solvation and non-polar solvation are indicated in table with their corresponding energy values. the consistent reoccurrence of some residues in addition to the earlier mentioned catalytic residue his and cys is suggestive of the crucial role they play in the inhibitory activity of different compounds to mpro proteases; this will equally provide insight into the design of compounds that distinctively target these important residues. the dominance of electrostatic and van der waals in the total energy outcome cannot equally be overemphasized as the plot also depict the level of the contribution of the individual residues to these energy types. to further unveil the interaction dynamics in protein-ligand complexes, we proceeded to visually analyse the nature and/or type of interactions that produced the strong high-affinity binding in the various systems. different bond types indicated in the right of figures - were noticed between the active site residues of mpro and various ligands due to diverse constituent functional groups in the ligands that interacted concurrently. this enabled us to identify crucial residues that played an active role in the binding interaction between the ligands and the protein. from the visual analysis, the dominant interactions include conventional hydrogen bonds, carbon bonds, charge-charge, pi-alky, pi-sulphur, pi-pi t-shaped and pi-pi stacked bonds. from the residue interactions, it can be observed that a "consistent" conventional hydrogen bond, carbon bond and pi-alkyl interactions were common in all the systems apart from -hydroxyazadiradione which did not interact with mpro via conventional hydrogen bond. visual inspection in figure (a) revealed that a conventional (nh-o) hydrogen bond occurred between ala of mpro and oxygen atom of deacetylnimbinene. in another case (figure (b) ), a conventional (nh-o) hydrogen bond was again observed between gln of mpro and oxygen atom of momordicine whereas an oxygen and sulphur atoms in met of mpro equally established another conventional (oh-o) hydrogen bond with an oh group in momordicine. in figure (c), nimbandiol equally had two conventional (nh-o, oh-o) hydrogen bond; nh group in met interacted with an oxygen atom in nimbandiol, while the oh group in nimbandiol interacted with an oxygen atom in cys . in figure (a), only one conventional (nh-o) existed between oxygen atom of margolonone and nh group of main proteases. in figure (b) , two different oh groups in momordicoide f maintained conventional (oh-o) hydrogen bonds with one oxygen atom in glu and cys . among the reference drugs (figure ), hydroxychloroquine has two conventional (nh-o) hydrogen bonds; one from the nh group in gln interacting with the oxygen atom on hydroxychloroquine while another nh group from hydroxychloroquine interacted with an oxygen atom in met (figure (a) ). ribavirin and remdesivir have three conventional hydrogen bonds each; this is suggestive of a stronger high-affinity binding. in ribavirin (figure (b) ), three conventional (nh-o) hydrogen bonds occurred between this compound and main protease; nh from asn and glu interacted with different oxygen atoms in ribavirin, while nh in ribavirin interacted with an oxygen atom in the main protease. finally, in figure (c), nh-o and nh-n conventional interactions were observed between remdesivir and main protease. here nh from gly and ser interacted with different oxygen atoms in remdesivir while another nh group in remdesivir interacted with an oxygen atom in glu . visual observation indicates that conventional hydrogen bond did not occur in -hydroxyazadiradione, but trends from the occurrence of this strong high affinity short distant bonding interactions suggest that it is required for the stabilization of the protein-ligand complexes. hence further structural optimization is needed to improve the binding dynamics of -hydroxyazadiradione to mpro active site. the types of reoccurring bonding interaction are indicative of variations in ligand positioning and proximity to active site residues of the proteins. table gives a brief description of the residues that played an active role in the binding interaction of this compounds to mpro and will pave way for the design of novel inhibitors that will specifically target this enzyme. from the table, met consistently interacted with all the inhibitors and could be very vital residue for the design of novel inhibitors the pharmacological profiles of the natural products were when compared with the minimum and maximum acceptable range and those of reference drugs by predicting their pharmacokinetic (admet) properties as indicated in tables and . it is worth mentioning that the ld as a parameter is very vital in dictating variable toxicities of compounds that are taken via oral routes (lipinski, ) . hence elevated ld depicts an increase in toxicity whereas a decline in ld correlated with a decrease in toxicity(b). we performed the toxicity class and labelling study with protox webserver; it uses the globally harmonized system for characterizing the toxicity class and labelling of chemicals (banerjee et al., ) . as estimated and indicated in table . ribavirin an fda reference drug has the highest ld which could be suggestive of high oral toxicity. however, ribavirin is reported with good bioactivity, bioavailability and no reported case of toxicity. the observation made from ribavirin is indicative of the promising attributes of the studied compounds as possessing the good quality to pass toxicity test. the estimation of cytotoxicity of the compounds with protox revealed a hepatotoxicity and cytotoxicity values similar and close to those of the reference drugs thus revealing their respective safety state at consumption. this hepatotoxicity and cytotoxicity values corroborates with the findings in ld . a good drug candidate is expected to have an mw threshold ˂ da (g/mol) based on lipinski's ro (lipinski, ; lipinski et al., ; omran & rauch, ; veber et al., ) . several studies have revealed a link between the molecular weight (mw) of drugs and its toxicity, wherein the note: the residues were colour-coded such that the once that were not absent in more than five systems were coloured. the colour coding is such that residues that were absent in five systems have five of the id uncoloured, while the once that were absent in four had four of its id uncoloured, the same happened for three and so on. the once that were absent in more than five systems were not coloured. the catalytic residue (cys ) was equally double coloured to reveal where it occurred. the second catalytic residue (his ) occurred consistently. higher the mw of a drug, the higher the toxicity whereas compounds with lower mw produces reduced toxicity (chapman et al., ) . therefore lower molecular weight is better (omran & rauch, ) because high mw in drugs decreases the concentration of the compounds at the intestinal epithelium surface and thus diminish. from table , all the compounds obeyed lipinski's ro of mw threshold ˂ da (g/mol), but, surprisingly, momordicoside f had a mw more than da and remdesivir which is an already approved drug that has no reported case of toxicity equally had an mw more than da. this could be suggesting a need for optimization the log p gives an idea of the hydrophobicity of chemical entities, it is universally defined as the negative of the logarithm of the partition coefficient between n-octanol and water (c octanol /c water ) (chapman et al., ) . hence an increase in log p is an indication of a decline in aqueous solubility, which results in a reduction in absorption. previous reports have revealed the chemical entities with log p range of À . to . tend to exhibit a high level of absorption while values greater than . and quite lower than À . possess low hydrophilicity, poor permeability and absorption (llorach-pares et al., ; remko, ; remko et al., ) . as presented in table , all the natural products and the approved drugs exhibited an acceptable level of solubility which will enhance absorption and distribution. although momordicine has a value that is slightly higher than the acceptable threshold (sander et al., ) , the value is within the range that is suggested to tend towards being well absorbed. this indicates that these studied compounds did not violate lipinski's rule of (log p < ). we took another step to use log s parameter to further evaluate the aqueous solubility of the studied compounds. the estimation is very crucial because it determines the oral bioavailability of drugs in line with membrane permeability (bennion et al., ) . studies available on about % of existing drugs has revealed the acceptable threshold for log s to be between to À . a look at table , section on log s indicates that all the studied compounds were within the suggested acceptable range for log s, this finding goes further corroborate the deductions made from the evaluation of log p. an important observation to be noted is the value of log p and log s of momordicine. though it is said to be within the range of chemical entities that are with the range described to tend to exhibit a high level of absorption. however, it is slightly above the acceptable range for log p and very close to the limit for the acceptable range for log s. this is there suggestive of the need for structural optimization to be done on this compound. the topological polar surface area (tpsa) parameter totals the polar atoms at the surface which are primarily oxygen and nitrogen in addition to the hydrogen atoms that are attached to them. this parameter predicts cell permeation ability of chemical compounds; it is therefore put that the lower the tpsa value the better (ertl et al., ; prasanna & doerksen, ). the metrics describes the size and volume of compounds which is an indication of physiological transport across the tight junction of the lipid bilayer membrane. these lipid bilayer membrane transport routes include the git and the blood-brain barrier (shityakov et al., ) . therefore, the increase in tpsa is suggested to diminish the transportation capability of drugs which in turn affect their biological activities (daga et al., ) . the table revealed that all the selected natural products adhered to the acceptable threshold tpsa value of Å . one is left to wonder why two of the approved drugs ribavirin and remdesivir with tpsa values of . and . , respectively, had values quite above that of the threshold and are still able to permeate cells and give reasonable efficacy. we suspect that there could have been other structural modification which was implemented in this compound; however, this observation is outside the scope of this present study. the results that we have obtained however suggests that the studied compounds possessed better physiological transport qualities than the approved drugs. hydrogen bonding is connected with constituent oxygen and nitrogen moieties and largely related with tpsa which is an indirect indication of polarity and the capacity of hydrogen bonding (di & kerns, ; prasanna & doerksen, ). the descriptors for hydrogen bonding are several hydrogen bond donors (hbd) and acceptors (hba) present in a molecule. these parameters have been extensively employed in the evaluation of druglikeness of compounds. lipinski's ro states that for a drug to be orally active, it must have an hbd count of and hba of (doak et al., ) . as predicted from the table, all the natural products and the approved drugs pass the test for oral bioactivity, however, remdesivir with hbd of once again left an impression for structural optimization. the drug-likeness of the compounds under investigation was further assessed using le, lle and lelp. these metrics have previously been employed to optimize ligands and equally identify ligands with improved binding efficiency for physiological targets (abad-zapatero, ; hopkins et al., ; johnson et al., ) . the suggested acceptable range for potential drug candidate for each of the parameters are le > $ . kcal/mol/heavy atom, lle > $ while optimal drug lelp value ranges between À and (abad- zapatero, ; hopkins et al., ; johnson et al., ) . most of the compounds were within the indicated range for some of the investigated parameters, however, a few exceptions exist which is suggestive of further structural optimization. as indicated, deacetylnimbinene fell short of all the studied parameters, this a clear indication of a need for critical structural optimization. on an additional note, nimbandiol has an le value that is close to the acceptable >$ . , but the lle and lelp values are somehow out of the required range thus suggesting the need for structural improvement. momordicoside and remdesivir exhibited le value that is slightly lower than the acceptable range. in the case of lle and lelp, only ribavirin had lle and lelp values within the suggested range. but of least importance is lack of metrics for momordicine coupled with the shortcomings on some of the required qualities, this, therefore, raises a need for additional structural optimization to be carried to improve the drug-likeness properties of these compounds. coronaviruses main protease (m pro ) is an essential protein critically required for proteolytic maturation of the coronavirus. in this study, compounds and fda drugs were screened from m. charantia l. and azadirachta indica for possible therapy against sars-cov- mpro using structure-based drug design and md approach. analysis from the root mean square fluctuations revealed that the higher active site rmsf of the phytocompounds when compared with apo m pro corroborates with the reported open/close and twisting mechanism of ligand inactivation of the viral protease. furthermore, two of the phyto ligands momordicine and momordiciode f , gave binding free energy that is higher when compared with fda drugs (ribavirin, remdesivir and hydroxychloroquine). analysis of the per-residue additional showed that studied compounds interact with these key amino acid residues in the active site of the main protease, suggesting that these phytocompounds could emerge as ideal candidate's inhibitors against sars-cov- m pro and another virus protease. pharmacokinetics suggests that the phytocompounds possessed better physiological transport qualities when compared with the reference drugs. our results present urgent attention since there are no drugs obtainable against sars-cov- . these findings may provide insight for developing new treatments for sars-cov- . ligand efficiency indices for effective drug discovery protox-ii: a webserver for the prediction of toxicity of chemicals predicting a drug's membrane permeability: a computational model validated with in vitro permeability assay data pharmaceutical toxicology: designing studies to reduce animal use, while maximizing human translation physiologically based pharmacokinetic modeling in lead optimization. . rational bioavailability design by global sensitivity analysis to identify properties affecting bioavailability biological assay challenges from compound solubility: strategies for bioassay optimization oral druggable space beyond the rule of : insights from drugs and clinical candidates ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study broadening the horizon: integrative pharmacophore-based and cheminformatics screening of novel chemical modulators of mitochondria atp synthase towards interventive alzheimer's disease therapy deciphering the 'elixir of life': dynamic perspectives into the allosteric modulation of mitochondrial atp synthase by j , a novel drug in the treatment of alzheimer's disease fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties coronaviruses: an overview of their replication and pathogenesis coronaviruses extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes two variants of the major serine protease inhibitor from the sea anemone stichodactyla helianthus, expressed in pichia pastoris the mm/pbsa and mm/gbsa methods to estimate ligand-binding affinities the role of ligand efficiency metrics in drug discovery synthesis, molecular properties, toxicity and biological evaluation of some new substituted imidazolidine derivatives in search of potent anti-inflammatory agents molecular dynamics and dft study on the structure and dynamics of n-terminal domain hiv- capsid inhibitors lipophilic efficiency as an important metric in drug design comparison of simple potential functions for simulating liquid water identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach a smooth-particle mesh ewald method for dl_poly molecular dynamics simulation package on the fujitsu vpp uptake of heavy metals in batch systems by sulfurized steam activated carbon prepared from sugarcane bagasse pith lead-and drug-like compounds: the rule-of-five revolution experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings new cucurbitane triterpenoids and steroidal glycoside from momordica charantia computer-aided drug design applied to marine drug discovery: meridianins as alzheimer's disease therapeutic agents datawarrior: an evaluation of the open-source drug discovery tool outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle proceedings of the ninth international conference on mobile computing and ubiquitous networking screening of indian medicinal plants for acetylcholinesterase inhibitory activity egress and invasion machinery of malaria: an in-depth look into the structural and functional features of the flap dynamics of plasmepsin ix and x probing gallate-mediated selectivity and high-affinity binding of epigallocatechin gallate: a way-forward in the design of selective inhibitors for anti-apoptotic bcl- proteins acid-mediated lipinski's second rule: application to drug design and targeting in cancer grid-based backbone correction to the ff sb protein force field for implicit-solvent simulations antiviral protein of momordica charantia l. inhibits different subtypes of influenza a. evidence-based complementary and alternative medicine topological polar surface area: a useful descriptor in d-qsar novel coronavirus -ncov: early estimation of epidemiological parameters and epidemic predictions molecular structure, lipophilicity, solubility, absorption, and polar surface area of novel anticoagulant agents molecular structure, pka, lipophilicity, solubility, absorption, polar surface area, and blood brain barrier penetration of some antiangiogenic agents datawarrior: an open-source program for chemistry aware data visualization and analysis protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments originpro . : scientific data analysis and graphing software: software review analysing molecular polar surface descriptors to predict blood-brain barrier permeation molecular properties that influence the oral bioavailability of drug candidates structure, function, and antigenicity of the sars-cov- spike glycoprotein antechamber: an accessory software package for molecular mechanical calculations a new coronavirus associated with human respiratory disease in china crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors potential non-covalent sars-cov- c-like protease inhibitors designed using generative deep learning approaches and reviewed by human medicinal chemist in virtual reality a pneumonia outbreak associated with a new coronavirus of probable bat origin relative free energy of binding and binding mode calculations of hiv- rt inhibitors based on dock-mm-pb/gs computational analysis of the cathepsin b inhibitors activities through lr-mmpbsa binding affinity calculation based on docked complex acknowledgements c.u.i. and e.a.i. are thankful to chpc (http://www.chpc.ac.za) for operational and infrastructural support. no potential conflict of interest was reported by the authors. key: cord- -k gnrdw authors: yıldız-peköz, ayca; ehrhardt, carsten title: advances in pulmonary drug delivery date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: k gnrdw pulmonary drug delivery represents an attractive, non-invasive administration option. in addition to locally acting drugs, molecules that are intended to produce systemic effects can be delivered via the pulmonary route. several factors need to be considered in the context of delivering drugs to or via the lungs—in addition to the drug itself, its formulation into an appropriate inhalable dosage form of sufficient stability is critical. it is also essential that this formulation is paired with a suitable inhaler device, which generates an aerosol of a particle/droplet size that ensures deposition in the desired region of the respiratory tract. lastly, the patient’s (patho-) physiology and inhalation manoeuvre are of importance. this special issue brings together recent advances in the areas of inhalation device testing, aerosol formulation development, use of in vitro and in silico models in pulmonary drug deposition and drug disposition studies, and pulmonary delivery of complex drugs, such as vaccines, antibiotics and peptides, to or via the lungs. drugs have been inhaled for therapeutic and recreational purposes since ancient times. the development of modern-day inhalers, e.g., pressurised metered-dose inhalers (pmdis) and more recently, dry powder inhalers (dpis), jet and vibrating mesh nebulisers (vmns), and soft mist inhalers (smis), has given pulmonary drug delivery a momentum boost that transformed a therapeutic niche into a market predicted to hit us$ . billion by [ ] . when delivering medicines to or via the lungs, not only the drug but also the formulation, the device and the patient need to be considered. we attempted to cover all these topics, while at the same time putting a spotlight on the up and coming areas of pulmonary drug delivery research. for this special issue, we selected publications written by authors hailing from countries-many of whom are regular contributors to our pulmonary drug delivery workshop series (www.pulmonarydrugdelivery. org). due to the variety of subjects covered, this has become the most comprehensive special issue published by pharmaceutics to date. adorni and colleagues studied different nebulisers taking into account their aerosol output, aerosol output rate, mass median aerodynamic diameter (mmad) and fine particle fraction according to the european standard en - , using sodium fluoride as a reference formulation [ ] . a correlation between the aerosol quality and the nebulization rate was identified. the respirable delivered dose and respirable dose delivery rate were also determined. this study might be helpful when choosing a nebuliser depending on the drug, therapy regime and patient. pmdis account for two-thirds of sold inhalers, however, due to technological advancements and environmental concerns, dpis emerged as the preferred medical device for the treatment of a diverse range of respiratory disorders. many dpis contain powder mixtures of coarse carrier particles and micronised drug particles with aerodynamic particle diameters of - µm. it is estimated that only - % of the drug reaches the deep lung while % of the drug is lost in the oropharyngeal sphere and % is not released from the carrier due to interparticulate adhesive forces. lechanteur and evrard have reviewed carrier-free particles, which are characterized by a sugar-based core encompassed by a corrugated shell layer produced by spray drying [ ] . special attention is given to the relation between the morphology (characterized by a corrugated surface) and lung deposition performance. a different approach to overcoming the limitations of conventional carrier-based dry powders was followed by benke et al. [ ] . they report the development of an interactive physical blend of a surface-modified carrier and spray-dried meloxicam potassium with suitable shape and size for pulmonary delivery. the nonsteroidal anti-inflammatory drug was used with the intention to provide local anti-inflammatory effects to decrease the progression of cystic fibrosis (cf) and chronic obstructive pulmonary disease (copd). in vitro and in silico studies resulted in high lung deposition, confirming that the interparticle interactions were indeed reduced in the novel formulation. rashid and co-workers, on the other hand, followed the traditional approach and formulated a lactose carrier-based dry powder formulation of glucagon for pulmonary delivery [ ] . they investigated l-leucine and magnesium stearate as dispersibility enhancers and found the highest fine particle fraction (fpf) for the formulation to contain mg stearate ( %) and large carrier lactose, whereas leucine was not a suitable excipient for the pulmonary delivery of glucagon. liquid formulation of fluticasone instead of dry powders were studied by dogbe et al., in order to improve the biopharmaceutical performance of the drug [ ] . the study compares liposomes and cyclodextrin (cyd) complexes in vitro and in vivo in mice. the in vitro tests showed no cytotoxic effects of either formulation. fluticasone liposomes resulted in up to -times higher lung concentration in comparison with free drug after intranasal administration. fluticasone hydroxypropyl-cyclodextrin complexes also showed higher lung accumulation than the free form after inhalation, however, this effect was not as pronounced as those observed with the liposomes. seven publications in this special issue cover various aspects of the inhalation of anti-infectives to treat lung infections. inhaling antibiotics allows for high target site concentrations, whilst minimising systemic exposure and toxicity. nonetheless, only a handful of antibiotics are currently marketed as nebulisable solutions or dry powders, and almost exclusively for the use in cf. future inhaled antibiotic trials should therefore focus on disease areas outside of cf, e.g., non-cf bronchiectasis, drug-resistant non-tuberculous mycobacterial infections, ventilator-associated pneumonia, post-transplant airway infections and tuberculosis (tb). therefore, an increased number of drugs as well as novel drugs must be studied as well as other formulations. banaschewski and hofmann [ ] have reviewed research into completed inhaled development programs, as well as ongoing research into inhaled therapies for both non-tb mycobacterial lung disease and tb. they conclude that preclinical and clinical studies have shown that inhalation therapy, complementary to current guidance-based therapy strategies, are clinically beneficial for all types of mycobacterial infections. however, an open-minded approach should be followed to continue investigating potential additions to the antimycobacterial therapeutic arsenal. in two papers, sibum and colleagues report the formulation, characterisation and stability testing of high-dose dry powders of isoniazid with little or no excipient for the treatment of tb [ , ] . initially, isoniazid was jet milled and spray dried with and without the excipient l-leucine. however, milling isoniazid did not yield a suitable formulation and spray drying the pure drug resulted in particles too large for pulmonary administration. when % l-leucine was added, respirable particles could be produced by spray drying but their storage stability was poor at higher relative humidity [ ] . the stability was later improved by using trileucine instead of l-leucine. the optimal formulation contained % trileucine w/w and had a maximum fine particle dose of mg when a nominal dose of mg was dispersed from the cyclops ® dry powder inhaler [ ] . in a case also using isoniazid, wyszogrodzka-gaweł et al. developed a theranostic approach to tb treatment and diagnosis that allows for imaging of the lungs by mri [ ] . metal-organic framework (mof) fe-mil- -nh nanoparticles were loaded with isoniazid using factorial design of spray-drying with poly(lactide-co-glycolide) and leucine. the formulation thus obtained had mri contrast capabilities, aerodynamic properties suitable for lung delivery, modified drug release and was taken up by macrophages. rossi and co-workers, in an attempt to treat mycobacterial lung infections, studied inhalable antibiotic powders targeting alveolar macrophages [ ] . their sodium hyaluronate-based formulation contained two antibiotics (i.e., rifampicin and isoniazid) and the efflux pump inhibitor, verapamil and was produced by spray drying. the sub-micron-sized particles had a high fine particle fraction, showed a sustained release profile, were not toxic towards macrophages and achieved more than % reduction in bacterial viability in susceptible and resistant m. tuberculosis strains in vitro. isoniazid was introduced in . bedaquiline, on the other hand is a relatively novel oral anti-tb drug that was approved in the us in by fast-track accelerated approval and is on the world health organization's list of essential medicines. bedaquiline, however, has a black-box warning of increased risk of death and arrhythmias. hence, momin et al. developed inhalable bedaquiline dry powder particles with the intention of reducing the systemic side-effects [ ] . bedaquiline was processed by spray drying and the resulting microparticles were stable during one-month of storage. spray-dried bedaquiline was non-toxic in respiratory epithelial cell cultures and effectively inhibited the growth of m. tuberculosis in vitro. antimicrobial peptides (amps) are being considered as alternatives to conventional antibiotics. amps do not only have direct antimicrobial activity, but also modulate the immune system and wound repair, making them of interest in cf therapy. forde and colleagues studied whether prodrugs of amps (pro-amps) can be delivered by vmn and whether modifications of pro-amp had an effect on the delivery [ ] . nebulisation did not alter amps' physical characteristics and antimicrobial activity. approximately % of the nominal dose was delivered in a spontaneous breathing setting, with higher delivery rates observed in a mechanically-ventilated model. these results demonstrated the feasibility of amp delivery using a vmn and also that the prodrug modification is not detrimental. vaccines against bacterial diseases may directly reduce antibiotic use through reduction of disease incidence. thus, immunisation has the potential to reduce antibiotic use. vaccine delivery via mucosal surfaces is an interesting alternative to parenteral vaccination and in many cases resembles the route taken by the microorganism when entering the body. hellfritsch and scherließ in their review provide an introduction to respiratory vaccination, formulation approaches and application strategies [ ] . another disease that could benefit from the advantages that inhalation therapy offers in terms of reduced systemic drug burden is lung cancer. parvathaneni et al. in their study investigated the anti-tumour effects of liposomally-encapsulated pirfenidone in vitro [ ] . pirfenidone, a repurposed anti-fibrotic drug, was encapsulated in cationic liposomes. the formulation was successfully aerosolised by a jet nebuliser and showed promising anti-tumour effects in various human lung cell lines compared to free pirfenidone. particle deposition in the lungs is associated with the breathing patterns of the patient and also pathophysiological changes due to lung diseases. in their study, farkas and colleagues measured realistic inhalation profiles of mild, moderate, and severe copd patients and simulated the deposition patterns of the symbicort ® turbuhaler ® in comparison to data generated from healthy control subjects [ ] . they found an association between the amount of drug deposited within the lungs and disease severity. the results from this study suggest that to receive a similar lung concentration, severe copd patients would require much higher doses than healthy individuals. tailoring the shape and size of fibre-like aerosols to achieve targeted pulmonary drug delivery with increased deposition efficiency is an interesting concept. shachar-berman et al. calculated the transport and deposition characteristics of fibres under physiological inhalation conditions in silico using computational fluid dynamics (cfd) simulations [ ] . aerosol deposition was quantified as a function of the equivalent diameter (dp) and geometrical aspect ratio (ar). they found that high ar fibres in the narrow range of dp = - µm mainly deposited in the upper airways, whereas fibres in the range of dp = - µm penetrated all the way to distal lung regions. to prolong the duration of the effect in the lungs, increasing the drug's affinity to lung tissue is an important strategy for drug development. however, differences in lung structure and blood flow affect local pulmonary drug disposition. himstedt and co-workers studied regional lung distribution of four drugs (i.e., salmeterol, fluticasone propionate, linezolid and indomethacin) after intravenous administration in rats [ ] . in addition, a semi-mechanistic model was employed to describe the observed tissue drug concentrations. the in silico model was able to explain the pulmonary pharmacokinetics of the two neutral and one basic model drug based on their tissue specific affinities (kp) and organ blood flow. the pulmonary pk of indomethacin, however, could not be modelled, suggesting that acidic drugs have different pulmonary pk characteristics. in their paper, salomon et al. studied the activity of carnitine transporter octn , which is associated with asthma and other inflammatory lung diseases. they studied freshly isolated human alveolar type i (ati)-like epithelial cells in primary culture and several respiratory epithelial cell models [ ] . [ h]-acetyl-l-carnitine uptake and pharmacological inhibition was determined in ati-like, ncl-h , a and calu- cells. it was concluded that octn is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium, however none of the tested cell lines are optimal surrogates for primary cells in carnitine transport studies. pulmonary drug delivery research is usually mainly concerned with administering aerosols to the lungs. the non-deposited, exhaled dose, however, can be a significant health hazard in both clinical and homecare settings. in two publications, mcgrath and colleagues used nebulised albuterol sulphate solution when they investigated fugitive aerosol emissions from two commercially available nebulisers in combination with an open or valved facemask or using a mouthpiece with and without a filter [ ] and during high flow nasal cannula (hfnc, see below for more on hfnc) therapy [ ] , respectively. it was shown that the mmad of the fugitively-emitted aerosols was less than µm, while the initially generated aerosols were between and µm. a facemask combination resulted in the highest time-averaged fugitively-emitted aerosol concentrations, whereas a filter on the exhalation port of the mouthpiece yielded the lowest concentrations. in the hfnc study, fugitive aerosol emissions were influenced by the interface type, patient and supplemental gas-flow rate, with fugitive aerosol mmad decreasing with an increasing flow rate. these findings are important in developing policy and best practice for risk mitigation from fugitive emissions. 'foamy' alveolar macrophages (fam) may be indicators of drug-induced phospholipidosis. currently, orally administered amiodarone is used to induce pulmonary phospholipidosis. patel et al. in their study investigated if pulmonary delivery of amiodarone in rats could be established as a novel phospholipidosis-induced fam model in comparative inhalation toxicology [ ] . a high dose of aerosolised amiodarone caused transient pulmonary inflammation, however, only oral delivery resulted in fam. high flow nasal cannula (hfnc) is widely utilized to support critically ill adults, paediatrics and neonates. through the continuous delivery of oxygen at high flow rates that meet or exceed patients' inspiratory flow, hfnc improves oxygenation, respiratory rates, patient comfort, and tolerance during therapy. as hfnc becomes more widely employed, the technique is also being considered for aerosol drug delivery. ji et al. have identified the ratio of nasal cannula gas flow to patient inspiratory flow as a primary independent predictor of inhaled dose. when the ratio was < , the inhaled dose was higher than those with ratio > . the inhaled dose was also more consistent with quiet and distressed breathing with ratio < [ ] . in a separate study, alcoforado and co-workers observed that both flow and active heated humidity inversely impacted aerosol delivery through hfnc. nonetheless, aerosol administration across the range of commonly used flows can provide measurable levels of lung drug deposition in healthy adult subjects [ ] . ji and colleagues retrospectively analysed study data on hfnc-delivery epoprostenol (iepo), utilised to improve oxygenation in mechanically ventilated patients with severe hypoxemia comorbid with pulmonary hypertension or right heart dysfunction [ ] . their data suggest that iepo via hfnc can improve oxygenation in adult patients and supports the need for a larger prospective randomised control trial to further evaluate the efficacy of iepo via hfnc. in this special issue, a cross-section of current research in the field of pulmonary drug delivery is published. there is still a lot of work to be done in the areas of inhaler devices and formulation development, particularly, with regards to dry powder and colloidal systems. a severe limitation in this field of research is the small number of excipients fda/ema-approved for inhalation. topical delivery of antibiotics appears to be an area that has attracted a lot of interest in recent years and is likely to make an even bigger impact in the treatment of pulmonary infections in the future. moreover, viral lung diseases such as covid- are a challenge and delivering antivirals by inhalation might be an approach worth considering. in addition to infectious diseases, conditions such as lung cancer are being actively researched in the context of inhalation drug delivery. the respiratory route can also be utilised to achieve mucosal vaccination against bacterial or viral infections. furthermore, there is a tangible shift from lab-based experiments towards in silico studies, e.g., in the areas of deposition modelling and physiology-based pharmacokinetic modelling. in the foreseeable future, however, the computer-based approached will need to be based on real-life data generated in actual experiments in the lab or the clinical setting. in the context of data generation, scientists should focus on novel techniques to study the fate of inhaled drugs, in order to allow in vivo/in vitro correlations and predictions. the authors declare no conflict of interest. inhalation delivery of complex drugs-the next steps aerosolization performance of jet nebulizers and biopharmaceutical aspects influence of composition and spray-drying process parameters on carrier-free dpi properties and behaviors in the lung: a review development of an innovative, carrier-based dry powder inhalation formulation containing spray-dried meloxicam potassium to improve the in vitro and in silico aerodynamic properties excipient interactions in glucagon dry powder inhaler formulation for pulmonary delivery pharmaceutical benefits of fluticasone propionate association to delivery systems: in vitro and in vivo evaluation inhaled antibiotics for mycobacterial lung disease characterization and formulation of isoniazid for high-dose dry powder inhalation dispersibility and storage stability optimization of high dose isoniazid dry powder inhalation formulations with l-leucine or trileucine an inhalable theranostic system for local tuberculosis treatment containing an isoniazid loaded metal organic framework fe-mil- -nh -from raw mof to drug delivery system sodium hyaluronate nanocomposite respirable microparticles to tackle antibiotic resistance with potential application in treatment of mycobacterial pulmonary infections inhalable dry powder of bedaquiline for pulmonary tuberculosis: in vitro physicochemical characterization, antimicrobial activity and safety studies vibrating mesh nebulisation of pro-antimicrobial peptides for use in cystic fibrosis mucosal vaccination via the respiratory tract systematic development and optimization of inhalable pirfenidone liposomes for non-small cell lung cancer treatment simulation of airway deposition of an aerosol drug in copd patients in silico optimization of fiber-shaped aerosols in inhalation therapy for augmented targeting and deposition across the respiratory tract towards a quantitative mechanistic understanding of localized pulmonary tissue retention-a combined in vivo/in silico approach based on four model drugs octn -mediated acetyl-l-carnitine transport in human pulmonary epithelial cells in vitro investigation of the quantity of exhaled aerosols released into the environment during nebulisation investigation of fugitive aerosols released into the environment during high-flow therapy comparison of oral, intranasal and aerosol administration of amiodarone in rats as a model of pulmonary phospholipidosis the ratio of nasal cannula gas flow to patient inspiratory flow on trans-nasal pulmonary aerosol delivery for adults: an in vitro study impact of gas flow and humidity on trans-nasal aerosol deposition via nasal cannula in adults: a randomized cross-over study epoprostenol delivered via high flow nasal cannula for icu subjects with severe hypoxemia comorbid with pulmonary hypertension or right heart dysfunction key: cord- - at gelt authors: han, namshik; hwang, woochang; tzelepis, konstantinos; schmerer, patrick; yankova, eliza; macmahon, méabh; lei, winnie; katritsis, nicholas m; liu, anika; schuldt, alison; harris, rebecca; chapman, kathryn; mccaughan, frank; weber, friedemann; kouzarides, tony title: identification of sars-cov- induced pathways reveal drug repurposing strategies date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: at gelt the global outbreak of sars-cov- necessitates the rapid development of new therapies against covid- infection. here, we present the identification of approved drugs, appropriate for repurposing against covid- . we constructed a sars-cov- -induced protein (sip) network, based on disease signatures defined by covid- multi-omic datasets(bojkova et al., ; gordon et al., ), and cross-examined these pathways against approved drugs. this analysis identified drugs predicted to target sars-cov- -induced pathways, of which are already in covid- clinical trials(clinicaltrials.gov, ) testifying to the validity of the approach. using artificial neural network analysis we classified these drugs into distinct pathways, within two overarching mechanisms of action (moas): viral replication ( ) and immune response ( ). a subset of drugs implicated in viral replication were tested in cellular assays and two (proguanil and sulfasalazine) were shown to inhibit replication. this unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials. to date, the majority of small molecule and antibody approaches for treating sars-cov- related pathology are rightly rooted in repurposing and are focused on several key virus or host targets, or on pathways as points for therapeutic intervention and treatment. this has been underpinned by the unprecedented pace of scientific research to uncover the molecular bases of virus structure, and the mechanisms by which it gains access to cells before replication and release of new virus particles. the emergence of global proteomics datasets is now propelling our understanding of these mechanisms through which the virus interacts with host cell proteins, determining the directly interacting proteins (dip) (gordon et al., ) and differentially expressed proteins (dep) . such interactome outputs, to understand the disease mechanism of covid- , we investigated which biological processes sip sub-networks are implicated in, for sars-cov- proteins ( structural proteins, non-structural proteins and accessory factors of the virus genome). we analysed several parameters: ( ) the subcellular localization of the proteins; ( ) the differences between the h and h timepoints (figures b and c) ; and ( ) the biological processes that the proteins act in. we found significantly stronger relevance of rna metabolism at h. we observe that the viral proteins n (nucleocapsid), nsp (non-structural protein ), orf (open reading frame ) and orf of sars-cov- interact with ribosomal proteins in the hidden layer of our sip network, indicating that they may have a possible influence on rna metabolism. the n and nsp proteins are known to drive viral replication (gordon et al., ) . more interestingly, orf and are the only two proteins of sars-cov- that are distinct from other coronaviruses (tang et al., ) . we observed that orf was enriched in the endoplasmic reticulum (er) ( figure c ), which may be significant as the er is the intracellular niche for viral replication and assembly (romero-brey and bartenschlager, ) . there were key proteins in the hidden layer that did not have strong enrichment in known biological pathways ('other') and that actively interacted with 'virus replication' proteins at h. further study on the unknown proteins found individual links to rna binding (atp a , mrto and nhp l ), host-virus interaction (ace , cxcr , derl , gnb l , hspd , kdr, krt , sirt and tmprss ), histones (h afz, hist h ps and wdtc ), viral mrna translation (mrps ) and er-associated responses (atf , cftr, derl and ins). we next confirmed statistically that virus-related pathways are enriched in the top enriched go-terms as well as rna-and er-related processes ( figure d ). the differences between two time points were also confirmed. in summary, our pattern analysis in the sip sub-networks revealed biological pathway changes during the course of infection, with prominent increases in proteins involved in virus replication by h. an in silico drug simulation on the key pathway of sip network identifies drug candidates to identify drugs targeting the key pathways, we conducted a network-based in-silico drug efficacy simulation (guney et al., ) on the key pathways of sip network at h and h after infection. we collected , approved drugs from publicly available databases (chembl (mendez et al., ) and drugbank (wishart et al., ) ). this virtual screening identified drugs (table s ) that are predicted to target the key pathways of sip network, of which ( . %) were specific to the h timepoint, ( %) were specific to the h timepoint and ( . %) were common to both timepoints. we then checked the anatomical therapeutic chemical (atc) code (available for drugs only) to determine the therapeutic areas for which specific drugs have been developed. the top clinical areas against which these approved drugs are used for were cancer, sex hormone signalling, diabetes, immune system, bacterial disease and inflammatory/rheumatic disease ( figure s ). interestingly, % of the drugs have been tested in phase or clinical trials for infectious diseases, and half of these were hiv trials; further drugs have been tested in trials for inflammatory ( %) and respiratory ( %) disease. among the identified drugs, ( %) are now in covid- clinical trials (clinicaltrials.gov, ) (table s ) . to determine the significance of this finding, we asked what the likelihood would be of this number of drugs being identified as hits by chance and found that, by comparison, only of , drugs were in the covid- clinical trials (clinicaltrials.gov, ) . a hypergeometric test returned a p-value of . e- , demonstrating the reliability and validity of our computational approaches. further drugs that we identified have also been reported as being potential candidates against covid- (courtney j. mycroft-west, dunhao su , yong li , scott e. guimond, timothy r. rudd and elli, gavin miller, quentin m. nunes, patricia procter, antonella bisio, nicholas r. forsyth, jeremy e. turnbull, marco guerrini, david g. fernig, edwin a. yates, ; criado et al., ; kuleshov et al., ; kumar, arun; c.s, sharanya; j, abhithaj; miribrahim sajid, javeria tariq, sheharbano awais, zehranaseem, samirashabbir balouch, ; shin et al., ) . thus, drug efficacy simulation has revealed drugs in total that are either in covid- clinical trials or being considered as potential drug candidates in pre-clinical studies, supporting the strength of our approach. in addition, we have discovered additional drugs that could provide novel opportunities for repurposing as covid- therapeutics. the full list of approved drugs along with their safety profile and moa are shown in table s . to investigate the mechanism of action (moa) for the drugs in the context of covid- , we used self-organizing map (som), a type of artificial neural network, to analyse the relationship between the drugs and the key pathways (termed drug-pathway association). after the unsupervised training of som, the distance between the adjacent neurons (pathways) was calculated and presented in different coloured hexagons, which illustrates the probability density distribution of data vectors (drug-pathway association score) (vesanto and alhoniemi, ) ( figures a and s ). based on the distance, we applied the davies-bouldin (db) index to separate the key pathways into clusters ( figure b ). these clusters of pathways and drugs identified two moa categories: ( ) virus replication (vr) and ( ) immune response (ir) ( figure c ). the som also mapped drugs into each neuron (the number of drugs per neuron is shown in figure d and drug names are shown in figure e ). notably, out of the drugs that are in covid- clinical trials (clinicaltrials.gov, ) were in the vr moa category while only drugs were in the ir ( figure d ). finally, we identified mechanistic roles and connections for the drugs and their target proteins, and mapping the drugs into pathway clusters ( figure e ). a more extensive analysis of information about each drug is given in table s . we next sought to identify the precise proteins, within the sip network, targeted by each of the drugs. we found that of the , proteins targeted by the drugs, most ( %) are targeted by a single drug ( figure s a ). however, there are proteins ( . %) that are targeted by or more drugs ( figure s a ). to establish whether there is a pathway relationship between these proteins, we interrogated their molecular function. figure s b shows that the most enriched categories of function for these proteins were heme, microsome, oxidoreductase and monooxygenase, all of which are related to nicotinamide adenine dinucleotide phosphate (nadp) and nitric oxide (no) synthesis. as no is important for viral synthesis (and because nadp affects no production), this could provide a potential mechanism by which these drugs might alter viral infection (kwiecien et al., ; lind et al., ; wang et al., ) . based on these findings we decided to validate in cellular assays, five drugs (ademetionine, alogliptin, flucytosine, proguanil and sulfasalazine) with good safety profiles which are functioning within this pathway. to assess whether these five drugs are able to reduce sars-cov- infection, we performed an initial screening using the vero e cell line, where we observed that of the drugs, proguanil and sulfasalazine, showed significant antiviral effects without any noticeable cellular toxicity at the indicated doses ( figures a and s a ). we then focused on these two drugs, expanding our validation using different cellular models (vero e and calu- ). treatment of vero e and calu- cells with proguanil and sulfasalazine illustrated strong anti-sars-cov- effects (represented by reductions of the envelope and nucleocapsid gene rnas) in a dose dependent manner, mirroring the results of the initial screen (figures b-e, s b-e). importantly, no significant effect on cellular viability was observed at any tested dose ( figures s f-h) . the effective concentration of sulfasalazine is comparable to maximal plasma concentrations achieved routinely in patients with rheumatoid arthritis or inflammatory bowel disease(iarc working group on the evaluation of carcinogenic risk to humans, ). to further investigate the anti-sars-cov- impact of these two drugs, we examined the status of recently discovered intracellular pathways directly associated with sars-cov- infection and cytokine production . indeed, treatment with either proguanil or sulfasalazine significantly reduced the phosphorylation of mapkapk (p-mk , t ) ( figure f ), an important component of the p /mitogen-activated protein kinase (mapk) signalling pathway, which has been shown to be activated via sars-cov- infection and stimulate cytokine response . importantly, treatment of calu- and vero e cell lines with proguanil and sulfasalazine led to a significant downregulation of the mrna of key cytokines (figures g-j and s ), which are dictated by the p /mapk signalling pathway and shown to become elevated during sars-cov- infection and replication (cxcl , ifnb and tnf-a). hence, the above results solidify the promising anti-sars-cov- effects of the two drugs, both at the viral as well as the molecular level. to understand why sulfasalazine and proguanil are effective against sars-cov- infection, but others functioning in the same pathway were not ( figure a ), we looked more closely at the targets of each drug. figure shows that sars-cov- orf binds to gammaglutamyl hydrolase (ggh) and regulates the synthesis of no, which is necessary for viral synthesis. an additional auxiliary pathway, mediating the synthesis of nadp, can also affect no production, although indirectly. sulfasalazine and proguanil impinge on both of these pathways: sulfasalazine targets the nfkb inhibitors nfkbia and ikbkb as well as cyp enzymes, whereas proguanil targets dhfr and cyp enzymes plus interacting partners. in this way these two drugs might more effectively target no production and thus disrupt viral replication. by contrast, the three drugs that were not effective against sars-cov- infection (flucytosine, alogliptin and ademetionine) only affect one of the two pathways. this analysis thereby highlights the possibility that targeting no production through multiple pathways may be the reason for the efficacy of sulfasalazine and proguanil in reducing viral replication. here we have used a series of computational approaches, including bespoke methods for data integration, network analysis, computer simulation and machine learning, to identify novel sars-cov- induced pathways that could be targeted therapeutically by repurposing existing and approved drugs ( figure s ). although network analysis is increasingly being used for analysis of genetic datasets to uncover disease signatures (barabási et al., ) , a few key aspects of our approach were essential in uncovering these new targets, including agnostic construction of the sip network and application of novel algorithms (previously used in other industries including social media). in addition, the use of artificial neural networks to understand systematically the mechanism of action for the drugs was vital to this investigation. our analysis identifies approved drugs, along with their moa, that may effective against covid- (table s ). we are confident that these drugs have a potential for repurposing for covid- , since out of the drugs have already entered clinical trials, testifying to the predictive power of our approach. an important part of our analysis is the use of already approved drugs. this allows for the rapid advancement of the most promising of the drugs which are not already in clinical trials. we identify two drugs, sulfasalazine and proguanil, that can reduce sars-cov- viral replication in cellular assays, raising the exciting possibility of their potential use in prophylaxis or treatment against covid- . both of these drugs function through the no pathway and have the potential to target more than one pathway necessary for no production. safety is a particularly important consideration, since such drugs will be prescribed to any covid- positive-case individuals who may have a broader range of underlying medical conditions and may not be hospitalised at the time of taking the drug. sulfasalazine and proguanil have the potential to be used prophylactically or therapeutically. both drugs are well established and well tolerated drugs (nakato et al., ; nikfar et al., ) . sulfasalazine is already in use as an anti-inflammatory drug against autoimmune disorders. given this drug has anti-viral activity (figure ) , this raises the possibility that sulfasalazine may act as an anti-viral and also an anti-inflammatory, if used against covid- . proguanil is in used against malaria in combination with atovaquone. it has an excellent safety profile and is well tolerated when used as a prophylactic and in treatment. a complementary study to ours, that uses large-scale compound screening in cultured cells, has recently uncovered molecules which have a partial effect on viral infectivity, of which show a dose dependent reduction of viral replication (riva et al., ) . this list of drugs does not overlap with ours, with only two of our approved drugs being present in this list. neither sulfasalazine nor proguanil are amongst them. the main reason for this apparent disparity is that only % of the compounds tested by riva et al. are approved, whereas % of our drugs are approved. this highlights the major difference in the two studies: our in-silico studies identify potential anti-viral drugs that are already approved and therefore at an advanced stage of repurposing, whereas riva et al. have identified compounds validated in african green monkey cells, most of which are either in pre-clinical or phase - clinical trials. our study has shed unanticipated new light on covid- disease mechanisms and has generated promising drug repurposing opportunities for prophylaxis and treatment. our data-driven unsupervised approach and biological validation has uncovered approved drugs not currently in clinical trials, which can be investigated immediately for repurposing and two drugs that show promise as anti-viral drugs. we expect this resource of potential drugs will facilitate and accelerate the development of therapeutics against covid- . (a) the schematic depicts our strategy of constructing a sars-cov- -induced protein (sip) hidden network through data integration and network construction of directly-interacting proteins (dips) and differentially expressed proteins (deps), followed by identification of drugs that target key pathways in this network (b) the sars-cov- orf sub-network shows the extent of the hidden layer that is revealed through the network analysis (c) percentage of shortest paths between the dip and dep that are via - proteins at h versus h. computer programming scripts that were used in this study are available from https://github.com/wchwang/covid . high-confidence sars-cov- -human ppis (gordon et al., ) were used as dip. lc-ms/ms data at hours, hours after sars-cov- infection and no infection as a control were analysed to identify differentially expressed proteins (dep) (|log fc| > . , fdr-bh padj-value < . ). sip network was constructed of all shortest paths between dip and dep in a human proteinprotein interaction network from string database (szklarczyk et al., ) . only interactions with a confidence score of greater more than medium ( . ) were used. all shortest paths between dip and dep were found using the python package networkx (hagberg et al., ) . networks were visualized using gephi . . (bastian et al., ) (figure s ) . multiple network centrality algorithms were deployed to identify key proteins in sip networks. eigenvector centrality was used to identify the most influential proteins in the network. degree centrality was used to identify the hub proteins in the network. betweenness centrality was used to identify the bottleneck proteins in the network. random walk with restart was used to identify proteins which are influenced by sars-cov- . the algorithms were implemented in the python package networkx (hagberg et al., ) . permutation tests were performed , times to identify significant proteins for each of the network centrality algorithms. for each permutation test, a random network that has the same degree distribution as the sip network was generated. if a protein has less than permutation p-value . for each of the network centrality algorithms, we considered it a key protein. key proteins of sip network were tested for enrichment of jensen disease (pletscher-frankild et al., ) and gene ontology (go biological process) terms. enrichment analyses were performed using enrichr (kuleshov et al., ) . key networks were built using interactions between the key proteins of the sip network at hours and hours after infection. when visualising the key networks, subcellular localization of key proteins and enriched pathways of hidden layer proteins was added (figures b and c) . subcellular localization information for key proteins was found using compartment database (binder et al., ) . among the available datasets in the compartment database, 'knowledge channel' data with a confidence score of greater than four was used. to identify enriched functions of the hidden layer proteins, the hidden layer proteins were tested for enrichment of reactome pathway terms. most hidden layer proteins belonged to the pathways "metabolism of rna", "cell cycle" and "immune system" so we retained only these pathways for key network visualisation. the key networks visualization was carried out using circos (krzywinski et al., ) . approved drugs were collected from chembl (mendez et al., ) and drugbank (wishart et al., ) . drug-target interaction information was collected from drugbank(v . ) (wishart et al., ) , stitch(confidence score > . ) (szklarczyk et al., ) and cheng, et al(cheng et al., ) . network-based in-silico drug efficacy simulation was conducted for key proteins from the sip network at -hour and -hour. given k, the set of key proteins from sip networks, and t, the set of drug targets, the network proximity(equation ( )) of k with the target set of t of each approved drug where d(k, t) the shortest path length between nodes k ∈ k and t ∈ t in the human ppis (cheng et al., ) was executed. to assess the significance of the distance between a key protein of sip network and a drug ! ( , ), the distance was converted to z-score based on permutation tests by using the permutation tests were repeated times, each time with two randomly selected gene lists with similar degree distributions to those of k and t. the corresponding p-value was calculated based on the permutation test results. drug to sars-cov- associations with a zscore of less than − were considered significantly proximal (cheng et al., ) . biological pathways were collated for the drugs we identified by in-silico drug efficacy simulation. to do this, proteins targeted by the drugs in sip network were tested for enrichment of reactome pathway terms using g:profiler (raudvere et al., ) . since reacome pathway hierarchy contains main overarching parent pathways and more specific child pathways nested within these, in cases where child pathways were among enriched pathways, the parent pathway term was removed from the enriched pathways list. finally, reactome pathways for drugs were identified. based on these identifications, a matrix containing drugs and reactome pathways was generated for drug-pathway association. this matrix was constructed using the f score (f = (precision x recall)/(precision + recall) from the pathway enrichment analysis. self-organizing map (som) (kohonen, ) was used to analyse moa of the drugs. the data used in training was the f score matrix for drug-pathway associations ( pathways by drugs). after the som training, davies-bouldin index (dbi) (davies and bouldin, ) was calculated based on the u-matrix to determine the best patterning among partitions ( figure a ). k-means algorithm were then used in order to find the pathway clusters ( figure b ). the som component maps of pathways were analysed based on the clustering result and mapped into two moa categories based on the biological functions ( figure s ). the som model also labelled each neurons with the drugs ( figures d and e ). the som toolbox package (vatanen et al., ) for matlab was used for this analysis. the frequency of drug-protein targeting was counted. permutation tests were then performed times to identify the significance threshold for the frequency of drug-protein targeting ( figure s a ). for each permutation test, the drugs among all the drugs which we used for the in silico drug efficacy simulation were randomly selected. then, the number of drugs targeting the same protein was calculated for all of the randomly selected drugs. the proteins frequently targeted in the sip network than randomised network were then tested for enrichment of uniprot keywords ( figure s b ). infection experiments were performed under biosafety level conditions. sars-cov- (strain münchen- . / / ) isolate was propagated in vero e cells in dmem supplemented with % fbs. for infection experiments in vero e and calu- cells, sars-cov- (strain münchen- . / / ) at moi= . pfu/cell for hours. all work involving live sars-cov- was performed in the bsl- facility of the institute for virology, university of giessen (germany), and was approved according to the german act of genetic engineering by the local authority. vero e and calu- cells were seeded using x cells in -well plates. the following day cells were treated for hour prior to infection with the indicated doses of ademethionine ( μm, selleckchem), alogliptin ( μm, selleckchem), flucytosine ( μm, selleckchem), proguanil ( nm- μm, selleckchem), sulfasalazine ( nm- μm, selleckchem), ifn-a ( u/ml), dmso (sigma) or mock and infected with sars-cov- at moi of . in serumfree dmem at °c for hours before rna or protein lysis. infection experiments were performed under biosafety level conditions. quantitative rt-pcr analysis rna was isolated using the rneasy mini (qiagen). sars-cov- replication (e-gene and ngene rna) and gene expression of the cytokines cxcl , ifnb and tnf-a was quantified by rt-qpcr. for cdna synthesis, rna was reverse-transcribed with the superscript vilo cdna synthesis kit (invitrogen, - ) . the levels of specific rnas were measured using the abi real-time pcr machine and the powerup™ sybr™ green master mix (applied biosystems, ) according to the manufacturer's instructions. Δct values were determined relative to the gapdh and ΔΔct values were normalized to infected dmso treated samples. error bars indicate the standard deviation of the mean from three independent biological replicates. all primer sequences are listed in table below. cytotoxicity was performed in vero e and calu- cells using neutral red (abcam, ab ) and mtt assay (roche) respectively, according to the manufacturer's instructions. cytotoxicity was performed in vero e and calu- cells with the indicated compound dilutions and concurrent with viral replication assays. all assays were performed in biologically independent triplicates. western blot analysis x vero e cells either mock-infected or infected and treated with dmso or proguanil ( μm) or sulfasalazine ( μm) for hours, were resuspended and lysed in whole cell xsds sample buffer ( xsds sample buffer: mm tris-hcl, ph = . , . % glycerol, . % sds, . mm bromophenol blue), supplemented with ml -mercaptoethanol, protease inhibitors (sigma), and phosphatase inhibitors (sigma) and boiled for min at °c. - μg of protein was separated on sds-page gels, and blotted onto polyvinylidene difluoride membranes (millipore). western blot experiments were performed using the following antibodies: gapdh (abcam, ab ), phospho-mapkapk- (thr , cell signalling, ), goat anti-rabbit (abcam, ab ) and anti-mouse-hrp (cell signalling, s). statistical analyses performed were specified in figure legends. differences were considered significant for p-values < . . network medicine: a network-based approach to human disease gephi: an open source software for exploring and manipulating networks compartments: unification and visualization of protein subcellular localization evidence proteomics of sars-cov- -infected host cells reveals therapy targets the global phosphorylation landscape of sars-cov- infection network-based prediction of drug combinations anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: critical role of ppar-γ signaling pathway sars-cov- clinical trials nadph-generating dehydrogenases: their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditions. front glycosaminoglycans induce conformational change in the sars-. biorxiv lessons from dermatology about inflammatory responses in covid- a cluster separation measure a sars-cov- protein interaction map reveals targets for drug repurposing clinical characteristics of coronavirus disease in china network-based in silico drug efficacy screening extrapulmonary manifestations of covid- exploring network structure, dynamics, and function using networkx hostile takeovers: viral appropriation of the nf-κb pathway some drugs and herbal products the self-organizing map circos: an information aesthetic for comparative genomics the covid- gene and drug set library. ssrn electron enrichr: a comprehensive gene set enrichment analysis web server update drug repurposing to identify therapeutics against covid with sars-cov- spike glycoprotein and main protease as targets: an in silico study lipid peroxidation, reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress -induced gastric injury inducible nitric oxide synthase: good or bad? chembl: towards direct deposition of bioassay data sars-cov- : cytokine storm and therapy a systematic review and meta-analysis of the effectiveness and safety of atovaquone -proguanil (malarone) for chemoprophylaxis against malaria a meta-analysis of the efficacy of sulfasalazine in comparison with -aminosalicylates in the induction of improvement and maintenance of remission in patients with ulcerative colitis diseases: text mining and data integration of disease-gene associations g:profiler: a web server for functional enrichment analysis and conversions of gene lists ( update) discovery of sars-cov- antiviral drugs through large-scale compound repurposing endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly papain-like protease regulates sars-cov- viral spread and innate immunity stitch : augmenting proteinchemical interaction networks with tissue and affinity data string v : protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets on the origin and continuing evolution of sars-cov- selforganization and missing values in som and gtm clustering of the self−organizing map nitric oxide regulates endocytosis by s-nitrosylation of dynamin nitric oxide and redox mechanisms in the immune response drugbank . : a major update to the drugbank database sulfasalazine or proguanil-treated vero e cells at indicated concentrations for three hours prior to infection with sars-cov- for hours. (g-j) rt-qpcr analysis of the indicated mrnas from calu- cells pre-treated with proguanil or sulfasalazine at indicated concentrations for three hours prior to infection with sars-cov- for hours. statistical test: student's t test key: cord- - odu lus authors: chen, daohong; qi, eric yining title: innovative highlights of clinical drug trial design date: - - journal: transl res doi: . /j.trsl. . . sha: doc_id: cord_uid: odu lus clinical trials serve as the gold standard to evaluate the efficacy and safety of tested drugs prior to marketing authorization. nevertheless, there have been a few challenging issues well noted in traditional clinical trials such as tedious processing duration and escalating high costs among others. to improve the efficiency of clinical studies, a spectrum of expedited clinical trial modes has been designed, and selectively implemented in contemporary drug developing landscape. herein this article presents an update on the innovated human trial designs that are corroborated through coming up with approval of notable therapeutic compounds for clinical utilization including delivery of several blockbuster products. it is intended to inspire clinical investigators and pharmaceutical development not only timely communicating with the regulatory agencies, but also insightful translating from cutting-edge scientific discoveries. the randomized clinical trials (rcts) of sequential three phases have been traditionally regarded as an official paradigm during drug development for decades. while phase i study is to define the tolerability, pharmacokinetics and adverse effects, subsequently phase ii and then iii studies are to examine the therapeutic efficacy in exploratory and confirmatory manners respectively [ ， ] . although rcts historically played and are still playing a decisive role in evaluating efficacy and safety of a therapeutic agent prior to the marketing authorization, the implementing practice also came up with several challenges including tedious processing duration, ever-escalating costs and lack of subgroup differentiation for maximizing clinical benefits [ , ] . to circumvent these problems, the significant advancement in disease biology and clinical pharmacology has inspired emergence of a spectrum of innovated clinical trial designs in contemporary drug developing landscape [ , , ] . overlapping with the essential principles of classic rcts, these recently emerged clinical studying models are characterized by an impressive list of additional strengths such as improved time and cost effectiveness among others [ ] . based upon the progresses in bio-assays of clinical pharmacology, the usa food and drug administration (fda) made a policy change known as hatch-waxman act in , which officially determined the human pharmacokinetic bio-equivalence (be) to replace traditional rcts for developing generic medications [ ] . as a result, the be policy was also implemented for chemical generics by european union in [ ] . consistently in , chinese state council initiated a campaign requiring the quality of generic drugs be reevaluated through running clinical be studies, compared to that of original reference products [ ] . in the field of innovative medicine, dramatic breakthroughs from life science have revolutionized our understanding in a number of aspects of disease biology, including therapeutic targets and diagnostic bio-markers [ ， ], thus inspiring some flexible modification of traditional rcts in order to deliver novel medicine to the patients in need more efficiently [ ] . accordingly in late , the st century cure act was passed into law by usa congress, and instructed fda to update the adaptive design guidance for investigational drugs and biological therapies [ ] . to date, certain modes of these updated trial protocols have exceptionally contributed to timely translating contemporary scientific discoveries into innovative drugs that addresses unmet clinical needs [ ， ] . in this light, the article herein highlights an array of outstanding developments in the perspective of drug trial design, being corroborated by notable successes in the clinical settings. it has been well recognized that pharmacological effects of drugs are dependent on their targeting tissue concentration which is usually proportional to their distribution in circulation system, known as bio-availability. of note, the latter can be affected by various pharmacokinetic-associated factors including active compound, formulation, manufacture, drug-drug interaction, among others [ , ] . in this sense, major pharmacokinetic parameters, such as area under curve (auc) and maximal concentration (cmax), are utilized as the surrogate parameters to compared efficacy and safety of a generic agent with those of the reference product in clinical be studies [ ] . most drugs are thereby accepted to be therapeutically equivalent when their auc and cmax fall in the range between of %- % regarding limits of % confidence interval [ ] . while having been significantly contributing to development of most chemical generics, be is recently noted as an efficient approach that can not be completely replaced by in vitro methodology even in some high solubility and high permeability products [ ] . in parallel, be study is frequently applied for comparing certain innovative agents in terms of formulation changes or fixed dose combinations; for example just being approved in , consensi consists of a combination of amlodipine and celecoxib to treat concomitant hypertension and osteoarthritis [ ] . realistically for evaluating a generic version of the polysaccharide drug heparin, the activities of anti-factors xa and iia instead of pharmacokinetic parameters are used to be the official standard for human be trials [ ] . in consistent with the policy of fda, european medicines agency (ema) has recently accepted the be investigation in healthy human subjects for generic development of low molecular weight heparin (lmwh) without requiring rcts in thromboembolism prone patients anymore [ ] . besides, utility of be study is being expanded into the field of evaluating biosimilar products such as antibodies and fusion proteins which represent much larger and more complex molecules [ ] . interestingly based upon a human be investigation in healthy volunteers and non-inferiority rcts in the patients, the biosimilar version of etanercept, a medication of fusion protein neutralizing inflammatory cytokine tumor necrosis factor- (tnf), has just be approved for managing rheumatoid arthritis (ra), psoriatic arthritis, and ankylosing spondylitis [ ] . contemporary pharmaceutic innovation is facing a serious realty of declined successful rates from research and development, in contrast to much more new compounds/new targets for examination in clinical investigation. in this regard to reduce a wast of the resources, fda issued a guideline of an alternative approach for first-in-human trials in , which was termed exploratory investigational new drug application (eind, or phase ) allowing a flexible amount of data needed for ind application based upon the specific circumstances of each proposed human trial [ ] . tending to bridge the gap between preclinical studies and traditional clinical development, phase investigation differs from phase i trial in fewer human subjects, short processing time and no tolerability test. as a result, phase study can accelerate the "goor-no go" decision making prior to a formal rct, namely improving the efficiency of identifying drug-like candidates and terminating non-promising compounds [ , ] . the strengths of phase study have been validated in various aspects of drug development including intra-target micro-dosing, clinical pharmacology, vulnerable populations, among others [ ] . interestingly, micro-dosing of insulin through limb artery injection was capable of achieving targeted control of local glucose level with minimal systemic exposure of the drug [ ] ; it offered insights into local application of medications, in particular for those with narrow therapeutic windows, to deliver clinical efficacy precisely while remarkably diminishing the systemic toxic effects [ ] . impressively, an unique role of phase trial has been recently highlighted in the drug development against brain tumor. as a cell cycle-associated kinase inhibitor, azd was revealed to have a strong anti-glioblastoma efficacy and poor blood-brain barrier (bbb) penetration in preclinical studies. it was through a phase investigation demonstrating that the compound substantially crossed bbb and induced the expected biomarker alterations clinically, which thus made a moving-forward decision for this project [ ] . besides, microdosing study appears particularly beneficial for vulnerable populations in which new drug trials are restricted, including children, liver/kidney function impaired, mutipharmacy, among others [ ] . in contrast to a classical clinical trial of sequential three-phases with interval times in between for analysis to determine the next design, seamless development integrates the three phase-processing into a comprehensive clinical study without gaps [ , ] . while reserving the core strength of traditional clinical trials in terms of defining efficacy and safety regarding tested compounds, seamless clinical study significantly improves time and cost efficiency to translate the scientific breakthroughs into innovative medicine [ ] . a seamless phase i/ii design is to examine the toxicity and efficacy in one trial, during which a reasonable dosing set need to be screened out at the first stage, and then forwarded to evaluate the efficacy at the second stage [ , ] . in a similar sense, a seamless phase ii/iii trial is performed with multiple dosing levels of the investigated drug at the exploratory stage to define the most efficacious dose based upon some surrogate end-points such as objective response rate (orr), and this dose is then continued into the confirmatory stage for further testing without a timing gap, to obtain more definitive end-points such as overall survival (os) [ , ] . given the flexibility for modification upon interim analysis, seamless development allows to study accumulating data from the ongoing clinical trial for early assessment of toxicity, efficacy or futility, and thus to decide the terminating or continuing/expanding treatment arms accordingly for further investigation [ ] . it was through a well-designed seamless clinical trial that the programmed death (pd- )-blocking antibody keytruda achieved accelerated approval by fda [ , ] . in this scenario, a dosing-escalation study was carried out at the first stage of the trial, to evaluate safety, tolerability and possible efficacy (orr) in a spectrum of patient cohorts with advanced cancer of various types [ , ] . based upon the interim analysis, the cohorts of melanoma and non-small cell lung cancer (nsclc) were determined to be expanded for the continuing clinical trial on treatment with keytruda at the selected dosages of mg/kg or mg/kg [ , ] . as a result, keytruda was demonstrated to confer a therapeutic benefit for the patients with melanoma (orr: %~ %) and nsclc (orr: . %); moreover, with the bio-marker stratification of pd-l + above %, an even better orr ( . %) was achieved in the subset of patients with nsclc [ ] . additionally, beyond successful application for oncological drug development, seamless clinical study has substantially contributed to therapeutic innovation in other medical fields [ ] . of note, the efficiency of clinical developing inhaled indacaterol, a long-acting  -agonist for treatment of chronic obstructive pulmonary disease (copd), was clearly improved through a seamless clinical trial [ , ] . historically human disease is categorized and managed according to anatomic location of body organs and histological types of pathology， which forms a solid basis for therapeutic indications designed in conventional clinical drug development [ ] . however, since the rise of targeted medicine in recent two decades, the landscape of pharmaceutical innovation has been transformed to modulate an aberrant biological pathway that can contribute to pathogenesis across a number of diseases [ ] , such as a gene mutation occurred in various neoplastic disorders [ ] . intriguingly, a mutational target is typically present only in a portion of the patients with each tumor type due to inter-personal heterogeneity, thus inspiring emergence of basket trials in the clinic [ , ] . in this light, a targeted compound is simultaneously examined across numerous disease baskets, for example different cancer types, in order to determine not only whether the drug is efficacious but also what tumor types and more precisely which patient subsets are sensitive to this therapy [ , ] . the first stage of a basket trial is to select the therapeutically sensitive disease types and patient sub-populations, with a bio-marker-based companion diagnosis if possible [ , ] . following interim analysis, the futile tumor type baskets are terminated, and the efficacious subjects are enriched from the responsive baskets, to be thus forwarded to further clinical investigation at the second stage [ , ] . as a selective tropomyosin receptor kinases (trk) inhibitor, larotrectinib represents the newly approved medicine targeting a wide spectrum of tumor types, reflecting a dramatic success of basket clinical study [ , ] . aberrant trk activation drives oncogenic pathogenesis, and is expressed in more than distinct tumor types. nevertheless, except a few types of rare neoplasms such as congenital fibrosarcomas, trk alterations occur in very low frequencies in common cancers of various tissue/cell lineage origins, for instance below % in lung adenocarcinoma [ ] . based upon a basket trial design to investigate the clinical efficacy of larotrectinib, the subjects were selectively enrolled from patients with the aberrant trk activity in tumors across histological types. impressively, larotrectinib was demonstrated to be well tolerated, conferring an orr of % and a median progression-free survival (pfs) of . months [ , ] . as such, this novel targeted agent stands out to address the unmet clinical need of combating the rare neoplasms and numerous common tumor types with the rare genetic mutations [ , ] . besides the validation in field of anti-cancer drug development, basket clinical has been proposed to go beyond oncology into cardiology, such as therapeutic innovation for preserved ejection fraction (hfpef) across diverse etiologies, pathobiologies, and clinical presentations [ ] . to identify the best therapeutic benefit(s) of a testing drug, the flexibility of adaptive clinical trials can be implicated in indication changes based upon accumulating data or/and cutting-edge scientific discoveries [ ] . depending on bio-medical contexts, such dynamic updating of clinical applications may be pre-planned or inspired by certain serendipitous observations, through shifting from a therapeutic hypothesis to an alternative one or multiple ones [ , ] . in this sense, a study design allows to examine beyond the intend-to-treat disease, thus being re-purposed to an alternative indication according to the initial assignment upon lack of efficacy or safety issues [ ] ; otherwise, a clinical trial can also be optimized to cover more therapeutic directions with the emerged evidence of certain additional efficacy [ , ] . in the era of precision medicine, there has been a consensus that therapeutic outcomes can vary significantly among subgroups of patients with differential genetic profiles [ , ] . accordingly taking the advantage of interim analysis based on novel biomarker approach for detecting the pathogenesis-specific molecular alteration(s), an adaptive clinical study can select the drug-sensitive sub-population from patients with initially targeted disease or an alternative indication, to continue the investigation for an optimized therapeutic efficacy [ ] . as a selective -phosphodiesterase inhibitor, sildenafil was designed to manage angina pectoris in the initial clinical trial. disappointingly this compound appeared non-efficacious in relieving anginal pain, with a side-effect of inducing penile erection [ ] . inspired by this serendipity, the clinical development was successfully re-directed to come up with a major innovative product of anti-erectile dysfunction [ , ] . in the field of oncology, crizotinib was initially identified as a potent inhibitor of mesenchymal-epithelial transition factor (met), thus tending to treat the relevant neoplasms such as nsclc and gastrointestinal tumors [ ] . whereas this compound was then revealed to suppress anaplastic lymphoma kinase (alk) as well, and impressively to exert a clear efficacy of tumor shrinkage in the nsclc with alk rearrangement during a phase i clinical study. in this regard, the therapeutic indication of crizotinib was re-focused and successfully developed through following-up clinical trials, to be a precisely targeted medication for managing a subset of lung cancer patients with alk aberrations [ , ] . interestingly in recent years, besides serving as an exceptional type of medicine for controlling tape diabetes, the clinical trials of sodium glucose cotransporter (sglt ) inhibitors are being expanded or re-positioned to manage heart failure and protect renal function through blood glucose loweringdependent or/and -independent mechanisms [ , ] . orphan drugs are developed to treat a particular spectrum of medical conditions with low morbidity, termed rare disease, resulting from genetically linked pathogenesis in most cases. of note, definition of rare disease varies among different countries depending on epidemic incidence of each illness geographically [ , ] . to address this largely unmet clinical need and meanwhile to deal with the challenging issue of limited market size for profits, fda announced an incentive policy known as the orphan drug act (oda) in , offering research & development funding, market exclusivity, among other attractive benefits [ ] . since then there were similar policies coming forth from other countries, encouraging an increased interest in this regard for pharmaceuticals; orphan drugs have been representing more than % of innovative medications marketed through last three decades [ ] . interestingly in recent years, certain orphan drugs are revealed to be overlapped with blockbuster products, which appear more notable in the field of oncology [ , ] . in this scenario, a novel medicine can be initially approved for a rare disease, and subsequently go beyond to treat additional types of disease; vice versa a conventional drug may occasionally obtain orphan status upon expanding its therapeutic indications toward a rare medical condition [ , ] . clinical development of orphan drugs often involves expedited trial designs described above, and may even be further simplified or alternatively designed under particular circumstances [ , ] . for instance, the approval of orphan drug xuriden was based upon a minimal seamless trial for weeks with only four patients, to treat hereditary orotic aciduria representing a rare disease of only cases worldwide [ ] . in addition, a viral inhibitor tecovirimat has recently been authorized to treat smallpox upon the waive of clinical efficacy study due to lack of patients with the naturally occurred disease. setting the first record in regulatory history, this innovative medication was exceptionally approved based on positive results from the preclinical efficacy studies in animal models of rabbits and monkeys, along with a phase i trial of clinical pharmacology and safety in healthy human subjects [ ] . impressively, certain orphan drug-based clinical trials can also come up with a broad spectrum of efficacy covering multiple indications. as a hallmark success of targeted medicine with orphan status, to combat philadelphia chromosomepositive chronic myelogenous leukemia (cml) imatinib was launched onto the market through the accelerated approval following a phase ii trial consisting of three open-label, single-arm clinical studies [ , ] . moreover, imatinib was subsequently evaluated in a basket clinical trial and demonstrated to be efficacious for an array of extra-therapeutic indications beyond cml, dramatically transforming this orphan drug toward being a blockbuster medication [ ] . besides, an interesting basket clinical study has recently been designed to investigate a target-specific monoclonal antibody for controlling an array of complement-mediated rare disorders, including bullous pemphigoid, antibody-mediated rejection of organ transplants and and warm autoimmune hemolytic anemia [ ] . clinical trial plays an indispensable role in evaluating efficacy and safety of therapeutic agents prior to marketing for human use, and has been constantly co-evolving along with the dynamic interactions between cutting-edge scientific discoveries and regulatory policy updating [ , ] . in this light, a number of agile clinical developing modes were designed through past decades, and impressively some of them have been further corroborated upon the contemporary achievements evidenced by successfully delivering innovative medicine to the patients in a more efficient manner [ ] . while human bioequivalence study is increasingly contributing to evaluation of emerging formulation and bio-similar agents besides chemical generics [ ] , several adaptive trial designs have been capable of translating the scientific breakthroughs into novel therapeutic benefits with shorter processing time and lower financial costs, to address the unmet clinical needs [ , ] . moreover with the assistance of bio-marker-based companion diagnosis, certain innovative trial designs have been streamlined to precisely confer selective therapeutic efficacy to the responding subgroups of patients with an array of serious diseases such as cancer and beyond [ , ] . of note, to preserve the strength of clear defining efficacy and safety of tested drugs, the innovative designs of clinical study are substantially overlapped with classic trial protocols of three phases which still serve as the mainstream approach of clinical investigation [ , ] . realistically, the medications through expedited processing appeared having a higher rate of post-approval black-box warnings than that of regularly approved drugs in terms of safety issues [ ] . it has accordingly been proposed that single-armed phased ii studies to be accepted only for accelerated approval (aa) applications regarding refractory diseases. otherwise, interim analysis of ongoing phase iii trials may be able to support aa processes, with the follow-up studies required to provide further evidence for the drug's effects on human bodies [ ] . whereas precision medicine-inspired trials represent an unique highlight in contemporary clinical studies to optimize the therapeutic efficacy for preferable subsets of patients with certain diseases upon enrichment strategies [ , ] , the validated biomarkers are limited and even much less than established drug-target molecules [ , ] . moreover, there still is a lack of the bio-markers to predict drugtriggered adverse events such as heparin-induced thrombocytopenia or immune checkpoint inhibitor-resulted hyper-progressive disease [ , ] . hence, it takes a dialectic perspective to appreciate the high efficiency of these innovated designs, with mindful efforts on circumventing their imperfectness. replaces rcts biosimilar enoxaparin [ ] biosimilar etanercept [ ] phase trial exploratory trial azd [ ] with microdosing midazolam ddi [ ] seamless trial integrated trial keytruda [ , ] without phase gaps indacaterol [ ] basket trial one trial for numerous larotrectinib [ ] types of disease tafamadis [ ] therapeutic shifting therapeutic sildenafil [ ] re-purpose indication during a trial crizotinib [ , ] empagliflozin [ ] orphan drug various expedited xuriden [ ] trial designs tecovirimat [ ] imatinib [ , ] assessing the gold standard--lessons from the history of rcts cancer clinical trials: the rear-view mirror and the crystal ball adaptive designs for clinical trials chemisimilars and biosimilars: is clinical testing fit for purpose? make up a missed lesson-new policy to ensure the interchangeability of generic drugs in china targeted therapy : resistance and re-sensitization adaptive design clinical trials: a review of the literature and clinical trials.gov adaptive clinical trials: advantages and disadvantages of various adaptive design elements the new european medicines agency guideline on the investigation of bioequivalence investigating the discriminatory power of bcs-biowaiver in vitro methodology to detect bioavailability differences between immediate release products containing a class i drug consensi) for hypertension and osteoarthritis bioequivalence of a biosimilar enoxaparin sodium to clexane ® after single mg subcutaneous dose: results of a randomized, double-blind, crossover study in healthy volunteers the introduction of biosimilars of low molecular weight heparins in europe: a critical review and reappraisal endorsed by the italian society for haemostasis and thrombosis (siset) and the italian society for angiology and vascular medicine (siapav) focus on biosimilar etanercept -bioequivalence and interchangeability microdosing and other phase clinical trials: facilitating translation in drug development phase trail of azd in first-recurrence glioblastoma patients intra-target microdosing-a novel drug development approach: proof of concept, safety, and feasibility study in humans cellular immunity augmentation in mainstream oncologic therapy adaptive clinical trial designs in oncology using model-based "learn and confirm" to reveal the pharmacokinetics-pharmacodynamics relationship of pembrolizumab in the keynote- trial pembrolizumab keynote- : an adaptive study leading to accelerated approval for two indications and a companion diagnostic intergrating indacaterol dose selection in a clinical study in copd using an adaptive seamless design an efficient basket trial design dual targeting autoimmunity and cancer: from biology to medicine efficacy of larotrectinib in trk fusion-positive cancers in adults and children basket trial of trk inhibitors demonstrates efficacy in trk fusion-positive cancers a next-generation trk kinase inhibitor overcomes acquired resistance to prior trk kinase inhibition in patients with trk fusion-positive solid tumors innovative clinical trial designs for precision medicine in heart failure with preserved ejection fraction the role of serendipity in drug discovery translating recent results from the cardiovascular outcomes trials into clinical practice: recommendations from the central and eastern european diabetes expert group (ceedeg) the serendipitous story of sildenafil: an unexpected oral therapy for erectile dysfunction development of crizotinib, a rationally designed tyrosine kinase inhibitor for non-small cell lung cancer sodium glucose cotransporter- inhibition in heart failure: potential mechanisms, clinical applications, and summary of clinical trials orphan drugs and their impact on pharmaceutical development challenges in orphan drug development and regulatory policy in china clinical research for rare disease: opportunities, challenges, and solutions fda approves ultra-orphan drug on a -patient trial oral tecovirimat for the treatment of smallpox gleevec for the treatment of chronic myelogenous leukemia: us. food and drug administration regulatory mechanisms, accelerated approval, and orphan drug status phase ii, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases combined integrated protocol/basket trial design for a first-in-human trial safety related label changes for new drugs after approval in the us through expedited regulatory pathways: retrospective cohort study accelerated approval of cancer drugs: improved access to therapeutic breakthroughs or early release of unsafe and ineffective drugs? antxr , a stem cellenriched functional biomarker, connects collagen signaling to cancer stem-like cells and metastasis in breast cancer heparin-induced thrombocytopenia hyperprogressive disease in patients with advanced non-small cell lung cancer treated with pd- /pd-l inhibitors or with single-agent chemotherapy we thank our colleagues for encouragement. the authors declare no conflict of interest. key: cord- - ftg bfx authors: guo, kai; wang, zhihan; gao, pan; pu, qinqin; wu, min; huang, canhua; hur, junguk title: identification of repurposal drugs and adverse drug reactions for various courses of coronavirus disease (covid- ) based on single-cell rna sequencing data date: - - journal: nan doi: nan sha: doc_id: cord_uid: ftg bfx with more than . million people infected coronavirus disease (covid- ), caused by novel severe acute respiratory syndrome coronavirus (sars-cov- ), poses a critical threat to human health. there is no proven vaccine or specific drug to date, which highlights the urgent need for rapid development of therapeutics for covid- . to identify potentially repurposable drugs, we employed a systematic approach to mine candidates from u.s. fda approved drugs and pre-clinical small-molecule compounds by integrating the gene expression perturbation data by chemicals from the library of integrated network-based cellular signatures (lincs) project with publically available single-cell rna sequencing dataset from mild and severe covid- patients. we identified fda approved drugs that have the potential to be effective against sars-cov- infection, of which are currently undergoing clinical trials to evaluate their efficacy against covid- . in conclusion, we have identified a list of repurposable anti-sars- cov- drugs using a systems biology approach. coronavirus disease (covid- ) is a highly contagious respiratory disease resulting from a life-threatening novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ). it has spread rapidly throughout the globe, causing . million infections and thousand of deaths by early may [ , ] . sars-cov- is an enveloped rna virus that belongs to the genus betacoronavirus of the family coronaviridae, which includes well-known severe acute respiratory syndrome coronavirus (sars-cov) as well as middle east respiratory syndrome coronavirus (mers-cov) [ ] . the advancement in the management of these coronaviruses and other viruses like influenza virus h n and ebola infections have provided insight into treating covid- . more than active clinical trials for covid- are being performed [ , ] . chloroquine [ , ] and its hydroxyl analogue hydroxychloroquine [ ] , lopinavir/ritonavir [ ] [ ] [ ] , and remdesivir [ , ] , developed for treating malaria, human immunodeficiency virus (hiv) and ebola virus, respectively, have provided some benefits to treat covid- and are being tested in ongoing trials. although covid- is less fatal than the sars and mers, older patients with comorbidities tend to experience more severe symptoms, making them more vulnerable. the majority of sars-cov- infected patients displayed mild symptoms and generally have a good prognosis, classified as mild covid- [ , ] . however, a large proportion of patients, especially among older men with underlying chronic diseases, have rapidly progressed to severe covid- and suffered from respiratory distress requiring emergent medical interventions [ ] . unfortunately, there is no evidence from randomized clinical trials supporting vaccines or efficient treatment for covid- [ , ] . additionally, recent studies have shown the important roles of host immune responses in protection and the pathogenesis of respiratory viral infections, for instance, sars-cov, mers-cov, and influenza a viruses [ , ] . liao et al. [ ] reported that increased immune cell recruitment in covid- patients suggested a crucial role of cd + t cells in successful viral control, and proposed support therapeutic strategies that target the myeloid cell compartment to treat covid- -associated inflammation. however, little is known about drug screens of disease-relevant cell types. drug repurposing is an essential and universal strategy in the development of new drugs [ ] . it may facilitate the discovery of new mechanisms of action for existing drugs, which is less time-consuming and cost-effective let alone the existing pharmaceutical supply chains for formulation and distribution [ , ] . considering that an rna virus exhibits a considerable degree of sequence variation, drugs targeting host factors may cause less mutational resistance with more effective and broad anti-virus spectrum potential. hence, there is an urgent need to identify potential therapeutics with new strategies for emerging infectious diseases, and repurposing clinically assessed drugs represents one of the most practicable strategies for the rapid identification of treatments to combat covid- . in this study, we analyzed a publicly available single-cell rna sequencing (scrna-seq) dataset of bronchoalveolar lavage fluid (balf) collected from mild and severe cases as well as bulk rna-seq of balf in covid- patients from different experiments ( figure ). data mining was performed by using the library of integrated network-based cellular signatures (lincs) [ ] , a drug perturbation database, to identify potential therapies for covid- disease. a total of candidates of different courses of covid- independent of cell subtypes were identified, of which were in clinical trials of covid- , including lopinavir/ritonavir [ ] , dexamethasone, niclosamide, lenalidomide, hydrocortisone, metformin, atorvastatin, sildenafil, and verapamil. subsequently, we utilized the side effect prediction based on l (sep-l ) project to predict adverse drug reactions (adrs) and constructed drug-adr association [ , ] . our findings may aid in the rapid preclinical and clinical evaluation of these therapeutics and can provide an important drug discovery pipeline to accelerate and facilitate the development of potential treatments for covid- . the gene-barcode matrix files of all donors containing mild cases and severe cases (lung balf) and healthy control (lung tissues) were downloaded from the ncbi gene expression omnibus database (accession id: gse ) [ ] . all expression matrices were loaded in r statistical analysis platform using seurat v [ ] and keeping cells with gene number between and , unique molecular identifier (umi) count above and mitochondrial gene percentage below . . a total of , cells collected from three healthy subjects, three mild covid- patients, and three severe covid- patients, were used for the analyses. we also collected a list of differentially expressed genes (degs) in sars-cov- -infected lung balf using a bulk rna-seq analysis to compare against the single-cell-based data. this deg list was obtained from the chinese national genomics data center (https://bigd.big.ac.cn/; accession id: cra ) [ ] . the lognormalize method in seurat was used for normalizing filtered gene-barcode matrix. principal component analysis (pca) was done by using the top , most variable genes. then uniform manifold approximation and projection (umap) was performed on the top principal components for visualizing the cells, and graph-based clustering was performed on the pcareduced data with seurat v [ ] . mast in seurat v was used to perform differential analysis. degs were identified by comparing each cluster between all of the three groups. genes with average log fc > . and adjusted p-value < . were deemed as degs. degs were first sorted by the log fc values and then the upregulated and downregulated genes were chosen to identify drugs and compounds against the lincs database using the connectivity map linked user environment (clue) platform [ ] . the drug connectivity score (cs) with a negative value smaller than - was used to determine candidate drugs and compounds. covid- database from the international clinical trials registry platform (ictrp) (https://www.who.int/ictrp/en/, updated on may th , ) was searched for clinical trials information associated with these drugs. both on-label and off-label adverse drug reactions (adrs) of the candidate drugs were collected from the sep-l database (https://maayanlab.net/sep-l /). the sep-l data include on-label adrs of fda-approved drugs collected from sider [ ] and off-label adrs from the pharmgkb database [ ] based on the post-marketing adr reports in the fda adverse event report system (faers). our study highlighted the identification of different therapeutic effects in the varied disease course. with the high variability of the cellular compartments underlying disease progression, our drug repurposing profiles from major cell subtypes included t, b, and nk cells, macrophages, and epithelial cells. a total of scrna-seq balf samples, including healthy cases, mild cases, and severe cases, were collected from publicly available scrna-seq data (supplemental table s ). after quality filtering, approximately , gene expression values from , cells were collected. the clustering analysis identified six major clusters of macrophage, nk figure s ), which was determined based on the unique signature genes cd (macrophage cell), il r and cd (cd + t cell), cd a (cd + t cell), ms a (b cells), tppp (epithelial cells), respectively (supplemental figure s ). we then compared these six major clusters across the healthy, mild, and severe covid- cases and identified differentially expressed genes between any of the two courses (supplemental tables s -s ). connecting to the lincs database of small-molecule perturbations on gene expression, we identified candidate drugs and compounds that can reverse these upregulated and downregulated genes via the clue platform. the closer the cs is to - , a score indicating a complete reversal, the higher chance identification of drug-adverse effect associations with upregulated or downregulated degs, in other words, drugs may show a better response to reverse expression of degs upregulated or downregulated in major cell subtypes in the balf. there were a total of candidates selected out by clue with cs lower than - based on degs among all three comparisons between two courses (supplementary table s ). to enable prioritization of known drugs for preclinical and clinical evaluation for the therapy efficiency of sars-cov- , a summary of the most among major cell subtypes during the patients' disease course and the publicly disclosed clinical trial phases are annotated in tables - . supplementary table s provides the complete list of potential anti-coronavirus agents from the current analysis, focusing on the fda approved drugs and experimental agents that have been already tested in clinical trials. to select candidates for mild cases, drugs and compounds were ranked according to their css (supplementary table s ), candidate drugs were identified compared to controls (mild vs healthy group), and of them involved in more than one cell subtype ( (table ) , including hiv protease inhibitors lopinavir/ritonavir [ ] combination (phase ), glucocorticoid receptor agonist dexamethasone (phase / ) [ ] , dna replication inhibitor niclosamide (phase / ) and antineoplastic lenalidomide (phase ). the tubulin inhibitor flubendazole, widely used in treating intestinal parasites, is a potent inducer of autophagy initiation and can decrease infection of dendritic cells with the hiv [ ] . azacytidine could partially reverse the aberrant dna methylation, a phase i clinical trial in combination with chemotherapy has been conducted to assess its therapeutic effects in children with leukemia, and in combination with apr- for myelodysplastic syndrome is in phase clinical trial [ ] . the bcl inhibitor abt- exhibits potential pro-apoptotic and antineoplastic activities [ , ] . lopinavir is widely used for the treatment of hiv, formulated in combination with ritonavir that can increase the half-life of lopinavir [ ] [ ] [ ] . repurposing analysis in severe covid- patients potent drugs were also selected in severe cases compared to controls (severe vs healthy group) according to their average cs between the replicates, and of them involved in more than one cell subtype ( figure b , supplementary tables s & s ) . as listed in table protein synthesis inhibitor brefeldin-a has used inhibit entry of some viruses, like human papillomavirus and polyomavirus [ ] , and egress of others, such as herpesviruses and paramyxoviruses [ ] . indirubin, an active ingredient of traditional chinese medicine (tcm) "danggui longui wan", has potent activity against myelocytic leukemia [ ] and therapeutic potential on iav-infection [ ] . a total of candidate drugs were identified in severe cases compared to mild ones (severe vs mild group), of them involved in more than one cell subtype ( figure c , supplementary tables s & s ). as listed in table , nine drugs (those for which drugs selected out in three separate cell types or more), including fostamatinib (syk inhibitor), ver- (hsp inhibitor), ku- (mtor inhibitor), pik- (pi k inhibitor), linsitinib (igf- inhibitor), tak- (p mapk inhibitor), y- (rho-associated kinase inhibitor), az- (raf inhibitor) and lestaurtinib (flt inhibitor). in this group, except lopinavir, we also following listed drugs in clinical trials for the treatment of covid- in table syk inhibitor fostamatinib produced clinically-meaningful responses for adult persistent and chronic immune thrombocytopenia in two parallel, phase randomized trials [ ] . hsp inhibitor ver- regulates kaposi's sarcoma-associated herpesvirus lytic replication and highlights the potential to be a novel antiviral agent [ ] . flt inhibitor lestaurtinib obtained orphan drug approval from the fda for acute myeloid leukemia [ ] and in a phase ii trial of advanced multiple myeloma and phase i trials of prostate cancer. as shown in figures a & b and supplemental table s , lopinavir was the only one identified in all three comparisons, and interestingly, ritonavir was common in two analyses. there were additional common drugs, such as sb- , abt- , jte- , brefeldin-a, pkcbetainhibitor, indirubin, gw- , flubendazole, tyrphostin-ag- , memantine, calyculin, kinetin-riboside, ascorbyl-palmitate, on- , mirin, verrucarin-a, emetine, tpca- , rhokinase-inhibitor-iii[rockout], pd- and nvp-auy . for example, the glycogen synthase kinase inhibitor sb- acts as neuroprotectant [ ] and prevents cardiac ischemia [ ] . jte- is a cannabinoid receptor inverse agonist producing anti-inflammatory effects [ ] . to further demonstrate the usefulness of this strategy, we have accomplished the identification of therapeutic drugs by transcriptional changes in balf of covid- patients with a bulk rna-seq data [ ] . ten efficient candidates were identified using the same analysis pipeline, two of which, including glycogen synthase kinase inhibitor sb- and ppar receptor antagonist gw- , were also included in the single-cell-based candidate lists (supplemental table s ). further investigations are necessary to characterize the adrs which are a central consideration during drug development [ ] . therefore, we conducted a computational approach using the sep-l database to predictive relationships between drugs and the emergence of adrs (supplemental tables s s ). figure shows a heatmap of the top drug-adr association for on-label ( figure a ) and off-label ( figure b ) adrs. these findings highlighted drug-adr associations and may lead to inform clinical decisions regarding treatments for covid- . covid- has spread rapidly, and no proven vaccine or drug has yet been identified to treat it. generally speaking, there are several ways to control or prevent emerging coronavirus disease, including antivirals, small-molecule drugs, biologics, and vaccines [ , ] . due to the lack of effective therapeutic agents and long development cycles of vaccines, it is, therefore, reasonable to consider repurposing existing drugs and compounds for covid- . drug repurposing is a potentially important strategy for the discovery of existing medicines to tackle covid- [ ] . gordon et al. [ ] identified high-confidence sars-cov- human protein-protein interactions for drug repurposing. an additional study [ ] tested the antiviral activity of fda approved drugs against sars-cov- that previously shown to inhibit sars-cov and mers-cov. another research team [ ] conducted a high-throughput analysis of the reframe library to identify candidates existing drugs that prevent the covid- virus from replicating in mammalian cells. in a study [ ] based on public data of patients with pulmonary fibrosis and the database lincs, several drugs were identified on covd- targets ace . host immune responses are particularly important in the protection and pathogenesis of the respiratory viral infections like sars-cov, mers-cov, and influenza a viruses [ , ] . liao et al. [ ] observed that t and nk cells accumulated, epithelial cells decreased in covid- patients compared to controls, meanwhile, macrophages dysregulated and the cell compartments differed in mild and severe disease courses, more t and nk cells decreased in severe cases but cd + t cells increased in mild cases. however, drug screening of disease-relevant cell types is still unclear. here, we analyzed publicly available data of covid- patients and performed data mining by using the lincs l database to identify potential therapies for covid- disease and sep-l database to predict side effects. our approach is different from previous methods for drug repurposing for coronavirus, since it does not merely rapidly identify likely effective therapeutic agents in preventing or treating covid- , but tries to filter out specific medications during the patients' disease courses. furthermore, the data and transcriptome are derived from human samples from real patients, two independent sets of experiments. lastly, we explore the underlying risk factors associated with some side effects of the candidates. overall, our data will guide the future development of therapies for the different durations of covid- and other viral respiratory infections. this study has several limitations to note. first, the public scrna-seq data had a small number of clinical samples (n= ) without available patient information, which makes comparisons between studies difficult. with the fast pace and expected large number of published literature using other patient samples, our candidate lists may need to be revised. second, our findings may not apply to children because all the sequencing data were from adults. future work on large-scale data mining would help us in better identification of antiviral drugs. the pandemic of covid- represents the greatest global public health crisis in this generation. so far, no proven vaccines and therapies have been identified. based on the study, we thoroughly investigated potential candidates for the treatment in covid- progression and predicted some possible adverse effects. the findings can guide additional repurposing studies, tailored for different stages of disease progression. the authors declare that they have no competing interests. jh, ch, mw and kg designed the project, collected data, performed analysis, and prepared figures. kg and zw collected data, prepared figures, and wrote the manuscript. pg performed analysis and prepared tables. qp revised the manuscript. the work was partially supported by the national institutes of health (p gm ) pilot grant to j.h. all the codes and data are available at https://github.com/guokai /covid / table . a list of potential drugs for treating covid- based on lincs database and degs between mild and healthy samples in b, cd + t, cd + t, epithelial, nk cells, and macrophage. connectivity scores were calculated from the clue platform. asterisk (*) represents the clinical trial for its efficacy in covid- disease. (+) indicates drugs meeting the sc < - criteria, while (-) indicates drugs not meeting the criterion. table . a list of potential drugs for treating covid- based on lincs database and degs between severe and mild samples in b, cd + t, cd + t, epithelial, nk cells, and macrophage. connectivity scores were calculated from the clue platform. asterisk (*) represents the clinical trial for its efficacy in covid- disease. (+) indicates drugs meeting the sc < - criteria, while (-) indicates drugs not meeting the criterion. input publicly available scrna-seq data and transcriptomic data of balf in covid- patients against the lincs database by using the clue platform. candidates are selected which can world health organization. coronavirus disease (covid- ) outbreak a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding pharmacologic treatments for coronavirus disease (covid- ): a review therapeutic options for the novel coronavirus ( -ncov) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid- characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention immune responses in influenza a virus and human coronavirus infections: an ongoing battle between the virus and host immunopathogenesis of coronavirus infections: implications for sars the landscape of lung bronchoalveolar immune cells in covid- revealed by single-cell rna sequencing renovation as innovation: is repurposing the future of drug discovery research? drug repurposing for viral infectious diseases: how far are we? drug repurposing: progress, challenges and recommendations a next generation connectivity map: l platform and the first , , profiles an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid- (elacoi) drug-induced adverse events prediction with the lincs l data network-based assessment of adverse drug reaction risk in polypharmacy using high-throughput screening data comprehensive integration of single-cell data transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients normalization and variance stabilization of single-cell rna-seq data using regularized negative binomial regression a side effect resource to capture phenotypic effects of drugs data-driven prediction of drug effects and interactions dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential a phase study of azacitidine combined with chemotherapy in childhood leukemia: a report from the tacl consortium an inhibitor of bcl- family proteins induces regression of solid tumours the bh mimetic abt- targets selective bcl- proteins and efficiently induces apoptosis via bak/bax if mcl- is neutralized human papillomavirus type infection of human keratinocytes requires clathrin and caveolin- and is brefeldin a sensitive antiviral activity of brefeldin a and verrucarin a molecular mechanisms of indirubin and its derivatives: novel anticancer molecules with their origin in traditional chinese phytomedicine anti-inflammatory effects of indirubin derivatives on influenza a virus-infected human pulmonary microvascular endothelial cells fostamatinib for the treatment of adult persistent and chronic immune thrombocytopenia: results of two phase , randomized, placebo-controlled trials hsp isoforms are essential for the formation of kaposi's sarcoma-associated herpesvirus replication and transcription compartments results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with flt mutant aml in first relapse selective smallmolecule inhibitors of glycogen synthase kinase- activity protect primary neurones from death delayed cardioprotection afforded by the glycogen synthase kinase inhibitor sb- occurs via a katp-and mptp-dependent mechanism at reperfusion the cannabinoid cb receptor inverse agonist jte- suppresses spontaneous itch-associated responses of nc mice, a model of atopic dermatitis clinical and economic burden of adverse drug reactions broad anticoronaviral activity of fda approved drugs against sars-cov- <em>in vitro</em> and sars-cov <em>in vivo</em> repurposing didanosine as a potential treatment for covid- using single-cell rna sequencing data key: cord- - r s authors: farhoudian, ali; baldacchino, alexander; clark, nicolas; gerra, gilberto; ekhtiari, hamed; dom, geert; mokri, azarakhsh; sadeghi, mandana; nematollahi, pardis; demasi, maryanne; schütz, christian g.; hash-emian, seyed mohammadreza; tabarsi, payam; galea-singer, susanna; carrà, giuseppe; clausen, thomas; kouimtsidis, christos; tolomeo, serenella; radfar, seyed ramin; razaghi, emran mohammad title: covid- and substance use disorders: recommendations to a comprehensive healthcare response. an international society of addiction medicine practice and policy interest group position paper date: - - journal: basic clin neurosci doi: . /bcn. .covid . sha: doc_id: cord_uid: r s coronavirus disease (covid- ) is escalating all over the world and has higher morbidities and mortalities in certain vulnerable populations. people who use drugs (pwud) are a marginalized and stigmatized group with weaker immunity responses, vulnerability to stress, poor health conditions, high-risk behaviors, and lower access to health care services. these conditions put them at a higher risk of covid- infection and its complications. in this paper, an international group of experts on addiction medicine, infectious diseases, and disaster psychiatry explore the possible raised concerns in this issue and provide recommendations to manage the comorbidity of covid- and substance use disorder (sud). oronavirus disease (covid- ) is a new member of the coronavirus family that infect humans . it first emerged in the wuhan region of china in november (lai shih, ko, tang, & hsueh, ) . by march , the world health organization (who) assessed the global situation of covid- as a pandemic. patients with cardiovascular diseases, chronic respiratory diseases, people aged or older, and males have a higher risk of mortality than the rest of the population huang et al., ; wang et al., ) . frequently reported clinical symptoms at the onset of the disease include pyrexia ( %- %), cough ( %- %), myalgia or fatigue ( %- %), and shortness of breath ( %) huang et al., ; wang et al., ) . sore throat and, less commonly, sputum production, headache, hemoptysis, and diarrhea have also been reported (chan et al., ) . in more severe cases, covid- can cause pneumonia, severe and acute respiratory syndrome, and sometimes ( %- % of all infected cases) death (world health organization, b) . currently, the medications used for severe cases of covid- include chloroquine phosphate (gao, tian, & yang, ) , hydroxychloroquine sulfate (javadi et al., ) , lopinavir/ritonavir (li & de clercq, ; lim et al., ) , oseltamivir (li & de clercq, ; vetter, eckerle, & kaiser, ) , and ribavirin (li & de clercq, ) . but none have been approved by regulatory authorities for use against covid- . the most common strategies, as advised by who, include preventative measures such as quarantine and limitations of movement in infected areas (hellewell et al., ; wu, leung, & leung, ) , interruption of human-to-human transmission, early identification and isolation, providing appropriate care for patients, identifying and reducing transmission from the animal source, and minimizing the social and economic impact through the new coronavirus created a complex situation for all sections of the communities around the world. health care providers are in the frontline of intervening to stop the spread of covid- . meantime people who use drugs (pwud) are at increased risk during this pandemic since they are a stigmatized and marginalized populations. health service providers who are providing different needs for pwud in treatment and/or harm reduction settings should always keep themselves safe with using standard ppe based on the who recommendations. additionally pwuds live in crowded locations and so screening and early identification of covid- patients are important to break the cycle of transmission. it is recommended that protocols for opioid substitution therapy modify with complete adherence to patients' safety regarding both opioid drug risks and covid- infections. it is important to have in mind that different stages of ost needs different approaches. pwuds are more vulnerable to stress and other mental health problems. this makes psychological interventions such as cognitive-behavioral therapy and other modalities very important to have for pwuds during these difficult and challenging times to assist and sustain treatment. medical conditions such as respiratory illness, renal insufficiency, chronic pain and cardiovascular disorders are also important medical conditions that should be addressed appropriately among pwuds with covid- . health service providers in both fields of addiction treatment and covid- treatment and prevention systems should be aware regarding special situations arising in the overlap of drug use and covid- illness. march, april , vol , num [covid- ] farhoudian, a., et al. ( ) . covid- and substance use disorders. bcn, ( ), - . multispectral partnerships (world health organization, a). bai and colleagues mentioned some covid- transmission from asymptomatic patients as a challenge for preventive activities (bai et al., ) . in most countries, people who use drugs (pwud) are stigmatized and marginalized population with lower access to healthcare. they suffer from poorer health, weaker immune function, chronic infections, various issues with respiratory, cardiovascular, and metabolic systems, as well as a range of psychiatric comorbidities (ahern, stuber, & galea, ; stuber, galea, ahern, blaney, & fuller, ) . pwud are a marginalized group who experience high rates of morbidities, three to five times higher compared to the general population (o'connell, ) . cheung et al. estimated that the risk of death among young pwud homeless women in toronto is to times higher than their housed counterparts (bohnert & ilgen, ; fernandez-quintana et al., ) . substance use imposes different health problems, which may complicate superimposed infection with co-vid- . for instance, chronic high alcohol consumption significantly increases the risk of acute respiratory distress syndrome (mccarthy et al., ) . during the h n epidemic, a history of opium inhalation had been identified as a risk factor for admission to an intensive care unit (icu) with confirmed h n (tabarsi et al., ) . additionally, it is essential to understand how pwud differently perceive danger and risk-taking behaviors during an epidemic, making them more risk averse (manfredi & d'onofrio, ; rhodes, ) . pwud have a higher rate of smoking and different studies estimated the current smoking rate of more than % among them (duan et al., ; sutherland et al., ; weinberger, gbedemah, & goodwin, ) . several studies found smoking a significant risk factor for middle east respiratory syndrome (mers) transdmission (alraddadi et al., ; nam, park, ki, yeon, kim, & kim, ; sherbini iskandrani, kharaba, khalid, abduljawad, & hamdan, ) . a literature review did not reveal even one article focusing on substance use disorder (sud) and covid- . therefore, a group of international experts on addiction medicine, infectious diseases, and disaster management teamed up to explore the comorbidity of covid- infection with substance use disorder and identify the necessary recommendations for health service providers and policymakers in this situation. although the majority of covid- infections are mild, the number of severe cases in a pandemic has the potential to overwhelm any healthcare system. consequently, health authorities may be required to repurpose health services and facilities away from pwud. when such an incident occurs, a business continuity protocol will cover several contingency measures so that organizations supporting pwud will continue to provide their essential services. a response to both covid- and drug use involves government, different sectors of the community and health authorities (who director-general's opening remarks at the media briefing on covid- on march , ) to implement evidence-based prevention programs as well as engaging different stakeholders for policy coordination (volkow, poznyak, saxena, gerra, & unodc-who informal international scientific network, ) . generally, drug use prohibition and criminalization approaches result in higher stigmatization and discrimination against pwud (boyd & macpherson, vancouver area network of drug users, ; santos da silveira, andrade de tostes, wan, ronzani, & corrigan, ) . this approach puts pwud at a higher risk of viral transmission. governments, health authorities, and other relevant stakeholders should identify the provision of services for pwud as essential services to support a comprehensive and proactive response to the challenges that covid places on this population, especially when they are under treatment (ekhtiari et al., ). pwud have poor access to health services due to stigma and discrimination (ahern, stuber, & galea, ; salamat, hegarty, & patton, ) . they are among the pervasive hard-to-reach populations. for example, studies show that drug use is one of the significant barriers to taking the influenza vaccine (bryant et al., ; kong, chu, & giles, ) . many homeless pwud communities live in crowded groups in shelters and or shooting galleries with no or minimal air conditioning facilities. additionally, poor hygiene, risky behaviors such as sharing drug-using paraphernalia and intoxication put pwud at higher risk of covid- infection. one of the other risk factors for pwud and people who inject drugs (pwid) is their weaker immune system due to a range of factors. these factors include long-term/high-dose administration of opioid drugs (liang, liu, chen, ji, & li, ; sacerdote, franchi, gerra, leccese, panerai, & somaini, ) , malnutrition (haber, demirkol, lange, & murnion, ; vila et al., ) , homelessness (haber et al., ) , and longterm alcohol and methamphetamine use (nelson, zhang, bagby, happel, & raasch, ; roy et al., ) . despite lacking evidence for introducing hiv as a risk factor for covid- (british hiv association, ), there are some concerns regarding the access to treatment services for people living with hiv/aids (plwha) and their adherence to antiretroviral therapy (dadkha, mohammadi, & mozafari, ) . this situation could finally increase the rate of mortality among plwha. on the other hand, respiratory infections among pwud are common and, in many cases, do not present with recognized symptoms of these diseases (dimassi & rushton, ; bradley drummond et al., ; gordon & lowy, ) . tuberculosis is another respiratory infection that is more common among pwud (perlman, salomon, perkins, yancovitz, paone, & jarlais, ) even in high-income countries heuvelings et al., ) . care providers are at the front line of any outbreak response. they are not only at the risk of infection but are also prone to burnout and psychological distress. in a study conducted on frontline staff involved in the severe acute respiratory syndrome (sars) epidemic, it was found that they had high levels of burnout, psychological distress, and posttraumatic stress (maunder et al., ) . this situation is compounded with evidence that counselors and therapists for pwud are well-known as having a higher rate of burnout (vilardaga et al., ) during usual practice. staff working in harm reduction settings, where most of the health service providers are peer groups, should be adequately supported. this support should prevent cross-viral exposure, psychological distress (hashemian et al., ; lancee, maunder, & goldbloom, ) , psychiatric disorders (tang, pan, yuan, & zha, ; , discrimination (gilchrist et al., ) , and physical and psychological violence (world health organization, b). concerns regarding infection and the above mentioned stressful events may affect their effectiveness in an outbreak (abolfotouh, alqarni, al-ghamdi, salam, al-assiri, & balkh, ) . all staff should have access to personal protection equipment (ppe). they should perform hand hygiene frequently, use alcohol-based hand rub/gels if their hands are not visibly soiled or with soap and water when they are visibly soiled. they must keep at least one meter distance from affected individuals, wear a medical mask when in the same room with an affected individual, and dispose of the material immediately after use. they should clean their hands immediately after contact with respiratory secretions, cover the nose and mouth with a flexed elbow or disposable tissue when coughing and sneezing, and refrain from touching eyes, nose, or mouth with potentially contaminated hands (world health organization, b). also, they must avoid close contact with anyone that has fever or cough (world health organization, b) and finally improve airflow in living space by opening windows as much as possible (world health organization, b). self-isolation of individual staff is paramount if there are signs of an infection (heymann & shindo, ; world health organization, a) . pwud staff still need to retain their crucial role at a distance either through digital technology or phone and continue their pwud management and treatment plan, such as the provision of daily ost medication (tschakovsky, ). there is no convincing evidence that the paraphernalia and devices for drug use are the primary sources of virus transmissions in the latest epidemics of coronaviruses (alagaili, briese, amor, mohammed, & lipkin, ) . however, as the main source of viral transmission has been defined to occur through the droplets, it makes sense to advise pwud populations to avoid sharing cigarettes, pipes , water pipes and hookahs, and so on (knishkowy & amitai, ; munckhof, konstantinos, wamsley, mortlock, & gilpi, ) . one should continue providing clean needle and syringes and 'take-home' naloxone (thn) when appropriate. infected patients are most virulent during the prodromal period. in the case of being mobile and carrying on usual activities, they play an essential part in spreading the infection to the other parts of the community (heymann & shindo, ). in such conditions, it is imperative to have an effective mechanism for the active and rapid detection of signs and symptoms and patient's isolation (hellewell et al., ; shamaei et al., ) . during the h n pandemic in , one of the risk factors for death or admission at intensive care units was a delay in diagnosis (tabarsi et al., ) . early detection in pwud can be difficult as covid- symptoms could be confused with a withdrawal syndrome (dimassi & rushton, ; bradley drummond et al., ; gordon & lowy, ) . it is highly recommended that a mechanism be implemented for the frequent screen-ing of covid- in pwud within harm reduction and treatment settings (salamat, hegarty, & patton, ; van olphen, eliason, freudenberg, & barnes, ). any pandemic affects illicit drug distribution networks (rahimi movaghar, farhoudian, rad goodarzi, sharifi, yunesian, & mohammadi, ) . sometimes this situation persuades pwud to seek treatment services for help, but usually, they switch to a more hazardous consumption. the iranian covid pandemic generated the highest incidence of mortality secondary to methanol toxicity (at least dead from drinking toxic alcohol in iran after coronavirus cure rumor, ; tainted alcohol claims more lives than coronavirus in khuzestan province, iran, ). however, opioid substitution therapy (ost) provision of controlled medication has become the main focus of the continuity plans around pwud to make sure that such provision is not interrupted during the covid- lockdown strategies (being imposed by several governments). any close personal contact may be harmful and risky for covid- transmission. methadone syrup and buprenorphine tablets or film are often provided to pwud after bringing out their blister packs. despite no evidence, this action might increase the chance of viral transmission by exposing both staff and pwud. it is recommended that dispensing clinics be trained to handle the process of tablet delivery with minimum hand contact. take-home doses of medications can be provided for more extended periods in situations of quarantine, selfisolation, or lockdown and health service disruptions. the maximum time for take-home doses of drugs is recommended when the dose and social situation are stable. treatment seeking individuals should be adequately informed about the changes in the practice, and they should receive appropriate support in case of uncertainty and concerns. however, decisions should be taken on a case by case basis. in summary, individuals under buprenorphine maintenance treatment (bmt) can receive accelerated take-home doses after two weeks of initiation. in particular, the people can receive this protocol who are at least on mg methadone or mg daily buprenorphine and have no signs of withdrawal symptoms, do not experience craving (mokri, ekhtiari, edalati, & ganjgahi, ) , are abstinent based on self-reporting, and provide negative toxicological tests. this condition should be reviewed every days if the individuals provided with take-home doses are not showing the stability mentioned above. buprenorphine take-home doses are probably safer than methadone take-home doses. if the person is in isolation and unable to pick up their medication personally, it can be delivered to their homes, or they can authorize someone else to collect the medication. opioid substitution therapy (ost) is among a category of treatment modalities that is normally considered to need regular and frequent supervision of patients, especially early in treatment. it is recommended that a more flexible ost program needs to be taken into account during the covid pandemic . given the safety profile of buprenorphine, it would seem to be the preferred substitution treatment for individuals who want to initiate treatments. it is faster and safer (maremmani & gerra, ) to reach an effective maintenance dose of buprenorphine compared to methadone, in fact it can be done on the first day of treatment. some of the medications under consideration for the treatment of covid- can significantly inhibit and/or stimulate methadone metabolism, puting patients at the risk of withdrawal or toxicity (lüthi, huttner, speck, & muelle, ; winton & twilla, ) . methadone specifically in high doses may prolong qt interval and cause fatal arrhythmias (krantz, lewkowiez, hays, woodroffe, robertson, & mehle, ) . possible cardiomyopathy in infected patients may increase the chance of torsade's de pointes arrhythmia (lüthi et al., ) and, particularly if combined with chloroquine which also prolongs the qt interval. withdrawal symptoms from buprenorphine are milder than that of methadone in case of interruption to the supply of medication, at least in the short term. where available, the long acting (monthly) subcutaneous injections are an alternative to providing take home doses. even transdermal buprenorphine should be considered where no other alternatives exist. multiple patches can be given simultaneously if necessary to achieve a therapeutic dose for opioid dependence treatment. additionally, benzodiazepine prescription for myalgia or stressful circumstances due to covid- may also increase the risk of toxicity during methadone mainte-nance treatment (mmt). during the pandemic period, it is more likely that individuals with drug use disorders or those who are in treatment seek out benzodiazepines or other tranquillizers (dorn, yzermans, & van der zee, ; fassaert et al., ) . benzodiazepines misuse may mask signs and symptoms of covid- infection and could escalate respiratory distress. patients are at a higher risk of methadone overdose in the initial stabilization period of methadone prescription (cornish, macleod, strang, vickerman, & hickman, ; degenhardt, randall, hall, law, butler, & burns, ) . for mmt patients, the authors do not recommend relaxing the methadone dose protocol at this phase of treatment however they do suggest avoiding unnecessary visits and rigor, on a case by case basis. if accelerated induction is necessary, an additional dose of - mg can be followed by a further dose if someone has been observed hours after their initial methadone dose. if they are still experiencing withdrawal at this time, they can safely be given a further dose. for buprenorphine, individuals can be rapidly inducted to optimal maintenance doses ( - mg daily). clinicians should consider increasing the dose if the individuals are still experiencing daily cravings, ongoing opioid use, or opioid withdrawal. however, clinicians should be sensitized in the differentiation between withdrawal syndromes, including myalgia, insomnia, sweating, fatigue, and nausea with signs and symptoms of viral covid- infection. pupil size is the best guide to distinguish opioid withdrawal from the symptoms of covid- as this infection does not affect pupil size. it should be possible to see pupil size even with video consultations. although the prescription period of anti-viral treatment is usually less than two weeks and the induction of hepatic metabolic enzymes takes more than the regular time for antiretroviral therapy (art) prescription, the clinicians should be careful about the changes of methadone level in these patients during and more specifically after termination or discharge of the treatment for co-vid- . change from split doses to multiple daily doses is a strategy in patients who receive antiviral therapy. as a result of the induction of methadone metabolism, some patients may need a mild increase in their previous methadone dosage after a few days of initiating antiviral treatment. for buprenorphine, double doses can be given every other day for people who are not considered safe to receive take-home doses. in exceptional situations, some patients on mmt or bmt fulfill the criteria for completion of their ost. termination is a stressful process (berger & smith, ) and needs close supervision and constant consultation. besides, the emotional distress associated with opioid withdrawal may increase the risk of suicidal ideation. termination of mmt and bmt increases the stress, so more attendance at treatment centers are needed, and it is not recommended during the covid- epidemic. some people who use opioids may wish to cease their opioid use during the outbreak, either due to reduced availability of opioids or the difficulty accessing treatment services. the most straightforward approach to detoxification, if available, would be single high dose buprenorphine. doses ranging from to mg have been used for this purpose (ahmadi, jahromi, ghahremani, & london, ) . alternative approaches include clonidine or a combination of symptomatic medications (world health organization, ). individuals with moderate to severe signs of cov-id- infection need medications consisting of a cocktail of art, antimicrobials, and analgesics. these medications may interfere with urine or saliva test results. for instance, quinolones (e.g. moxifloxacin, lomefloxacin, norfloxacin, ofloxacin, ciprofloxacin), rifampin, tolmetin (a non-steroidal anti-inflammatory drug) may yield a false-positive result in opiates urine drug screening (reisfield, goldberger, & bertholf, ). chloroquine demic, the clinicians should assess the benefits of the urinary or saliva testing at this critical circumstance, especially as this will potentially increase unnecessary risks due to close contacts. in this pandemic, it seems that information is spreading more extensively and rapidly in comparison to the sars outbreak in . however, this condition may result in a worsening of public fear, panic, and distress. social isolation may also make individuals susceptible to more psychological distress. consequent economic depression after a pandemic also causes uncertainty and threats to future welfare (strong, ) . the unpredictable future is exacerbated by myths and misinformation that are often driven by fake news and public misunderstanding (bao, sun, meng, shi, & lu, ) . some patients will experience grief over the loss of loved ones. the relationship between adverse life events and brain stress systems have a prominent role in addiction disease (koob, (koob, , (koob, , . pwud are much more vulnerable to stress and crisis followed by lapse and relapse to ex-drug users (goeders, ; koob et al., ; milidvojevic & sinha, ; somaini et al., ) . as a result of stressful events and disasters, mental health problems emerge or exacerbate (farhoudian, hajebi, bahramnejad, & katz, ; farhoudian, rahimi movaghar, rad goodarzi, younesian, & mohammad, ) . in such circumstances, healthy individuals may start drug use (farhoudian et al., ; somaini et al., ) , and several patients may relapse into their previous drug use and start their high-risk behaviors (brandon, vidrine, & litvin, ; farhoudian et al., ; rahimi movaghar, et al., ) . anxiety, worry, depression, irritability, and anger in pwud should be considered as a prodromal sign of lapse or relapse into a new episode of drug use. psychosocial interventions are a vital element in the treatment of pwud, especially in people using stimulants and having mental problems (de crescenzo et al., ) . in this period, internet-based psychotherapy is highly recommended as a replacement. internet consultation, including phone calls, video chat, and short messages, have great potential to make psychological assessment and treatment more cost-effective. computer-assisted therapy appears to be as effective as a face-toface treatment for treating anxiety disorders and depression (taylor & luce, ) . although it requires some equipment and knowledge, it offers a good alternative for more isolated locations, which is relevant in this pandemic. negative emotional states, including fear, anxiety, and boredom, as well as social withdrawal and or isolation, are the main emotions that patients will experience during the covid- pandemic. cbt has been recognized as one of the most beneficial interventions for pwud (lee & rawson, ) . stress reduction as a technique of cbt, either alone or in combination with pharmacotherapies, may prove beneficial in increasing quality of life and reducing cravings and promoting abstinence in clients seeking treatment for sud (goeders, ) . clinicians should help their patients to identify, manage, and reduce their negative emotional states associated with relapse and apply techniques of behavioral activation compatible with specific circumstances of each patient. coping skills training and crisis intervention are the most common types of cbt interventions to be recommended. matrix model is a multi-element package of therapeutic strategies to produce an integrated outpatient treatment experience (rawson & mccann, ; rawson et al., ) . treatment is delivered in an intensive outpatient program primarily in structured group sessions targeting the necessary skills. it is recommended that the meetings could be held individually instead of group format, hoping to lessen the risk of covid- infection. the recommended parts based on the manual (services among iranian people living with hiv and aids: a qualitative study, ) for the period of covid pandemic include: . rp : taking care of yourself; . rp : emotional triggers; . rp : recognizing stress; . rp : reducing stress; . rp : acceptance; . rp : coping with feelings and depression; . rp elective c: recreational activities. incentive-based treatment approaches (i.e. contingency management [cm] ) are effective interventions in reducing addictive behaviors in pwud (ainscough, mcneill, strang, calder, & brose, ; benishek et al., ; lee & rawson, ; messina, farabee, & rawson, ; rawson et al., ) . evidence also supports the cm beneficial effect on the treatment of these individuals targeting infectious disease control (herrmann , matusiewicz, stitzer, higgins, sigmon, & heil, ) . to take advantage of cm in the prevention of cov-id- , the desired behaviors (e.g. washing hands every hour, cleaning hands, etc.) and their scores or prizes (e.g. take-home doses) should be clearly defined and inserted into the list, just like other desired behaviors (e.g. negative urine test). perceived social support from relatives and friends is a major predictor for retention in treatment for pwud shirinbayan, rafiey, vejdani roshan, narenjiha, & farhoudian, ) and the main factor of psychological resilience to disaster (radfar, nematollahi, & arasteh, ; rodriguez-llanes, vos, & guha-sapir, ) . considering the importance of family support, clinicians are advised to engage family and care providers more than ever during the pandemic. attracting other sources of social support such as guaranteed wages and an increase in social security payments will help the individual to pass this period with a better outcome. opioids such as methadone are respiratory depressants, and tolerance develops very slowly and incompletely. when patients under mmt acquire covid- , they should be more closely monitored for both worsening respiratory functions and methadone toxicity. abrupt cessation of methadone must be avoided because anxiety and agitation due to withdrawal syndrome may induce or worsen cardiorespiratory complications (friedman, kamel, perez, & hamada, ; kienbaum, thurauf, michel, scherbaum, gastpar, & peters,, ) . the prevalence of kidney impairment in hospitalized covid- patients is high, and renal insufficiency increases the risk for in-hospital deaths (cheng et al., ) . studies indicate that heroin users, especially pwid, suffer from nephropathy (cunningham, brentjens, zielezny, andres, & venuto, ; do sameiro faria, sampaio, faria, & carvalho, ; may, helderman, eigenbrodt, & silva, ) . other studies confirm that individuals using amphetamine (ginsberg, ertzman, & schmidt-nowara, ; rifkin, ) , cocaine (merigian & roberts, ; norris et al., ; sharff, ) , alcohol (de marchi, cecchin, basile, bertotti, nardini, & bartoli, ; perneger, whelton, puddey, & klag, ) , and potent cannabis (abodunde, nakda, nweke, & veera, ; gudsoorkar & perez jr, ) are more likely to suffer from renal failure. it might be logically concluded that people with a history of drug consumption are more prone to contract renal insufficiency when they are infected to covid- ; however, there is not any revealing evidence so far. evidence suggests that renal insufficiency does not affect the metabolism of methadone in mmt patients (murtagh, chai, donohoe, edmonds, & higginson, ) . despite this issue, patients in acute renal failure due to covid- should be monitored for signs of methadone toxicity because of other reasons for renal insufficiency. heart diseases increase the risk factors of death due to covid- to % in affected individuals with hypertension, . % in diabetics, and . % in patients with other cardiovascular diseases (murtagh et al., ) . individuals with a history of alcohol or drug use are more likely to have cardiac pathology. excessive alcohol consumption (fabrizio & regan, ; mirijello et al., ) , amphetamine (giv, ; o'neill et al., ) , heroin (routsi et al., ) , and cocaine (barton duell, ) are all associated with the increased risk of cardiac pathology. contracting covid- sometimes can result in moderate to severe pain including myalgia, sore throat, and headache that requires pain management. it is recommended that acute pain in pwid with covid- is managed in consultation with pain or addiction specialists. people who use opioids regularly will require additional opioids for the management of pain (athanasos, smith, white, somogyi, bochner, & ling, ; doverty et al., ) . buprenorphine as a high-affinity partial agonist of mu-receptors has an analgesic effect in divided doses, but stops effecting other opioid analgesics and hinders acute pain management in case of necessity (harrington & zaydfudim, ) . in this case, buprenorphine can be ceased and opioid analgesics used or buprenorphine can be continued and non-opioid medications such as clonidine, pregabalin/gabapentin and ketamine can be used (goel et al., ) . health services will need to rapidly adapt to the cov-id- situation. they will need to establish a mechanism of making decisions quickly and under stress, to identifying the essential services to be continued, to develop new mechanisms of patient flow (including screening, batching and referral), to redistribute staff from non-essential roles, and maintaining the continuity of essential supplies (communications, ) . to reduce the risk of transmission, it is generally recommended that nonessential services close, or make their services available by telephone or on-line. when face-to-face services are required, some modifications may need to be made to the service system, for the identification of cases, the protection of staff, the reduction of transmission, and to ensure the continuity of essential services (interim guidline for healthcare facilities, ). when health services remain open in a pandemic, they should first invite all visitors to wash their hands before they touch anything. then they should screen all new visitors with whatever sars-cov- screening mechanism is appropriate for the local conditions. this may include a combination of temperature (where possible measured with a non-touch thermometer), clinical symptoms (cough, shortness of breath, sore throat), and epidemiological criteria (recent travel, contact with cases, health care worker). where, possible, patients meeting the testing criteria should be tested on-site and then directed to isolate themselves awaiting the results. for testing and any subsequent clinical interaction, staff should wear personal protective equipment (ppe) to protect themselves from transmission. if the client is coughing, it is preferable they should also wear a surgical mask (world health organization, c). transmission is through to be mostly via droplet spread when people who are infected sneeze, cough or talk. staff and patients should wash their hands frequently and be careful what they touch. surfaces should be cleaned after they have potentially contaminated. depending on the availability of ppe and the risk in the local community, it may be appropriate for staff to wear masks and gloves, or even gowns and eye protection. patients with symptoms should wear a mask to prevent transmission through cough and sneezing. patients can be divided into three risk groups, those with confirmed sars-cov- virus, patients who meet criteria for testing awaiting test results, and other patients with differing levels of ppe depending on the availability of ppe. preferably, patients with different risk levels should be treated in different parts of the health service. staff and patients should keep a distance from each other (world health organization, c). in addition to providing ost, services should take the opportunity to encourage cessation of smoking by prescription of nrt, and by the distribution of naloxone and overdose resuscitation. in preparation of staff members being sick or isolated awaiting test results, each staff member involved in ost treatment should have at least one other staff member who can continue their role if they are sick. where possible, staff may separate into different teams who have even less contact, so that if one person is sick then the risk of all needing to isolate themselves is reduced (guide on business continuity planning for covid- , february, ). pwud are a marginalized hard-to-reach population living in crowded groups with lower access to healthcare. they usually suffer from poorer health, weaker immune function, chronic infections, as well as various issues with physical and psychiatric comorbidities. consequently, they have a higher risk of contracting co-vid- and its transmission and casualties. we believe that substance use and covid- have a complicated relationship with each other. in summary, we suggest the following items: health authorities should develop and apply specific strategies for pwud for early covid- identification and patient isolation, interrupting transmission, providing appropriate care, attending medical issues, and minimizing negative social impact. health authorities are responsible for providing adequate healthcare for pwuds. they may be required to repurpose and reorient health services through a business continuity team. this team implements evidence-based programs and makes decisions on how the organizations will continue to provide their services. also, they make sure that all ost patients have adequate access to their opioid drugs. treatment sectors should provide essential requirements, as well as software and programs tailored to their own clients' needs. staff may also teach the patients the hygiene rules, self-monitoring for signs of illness, and rapid reporting of the disease in case of occurrence. a mechanism for frequently screening for signs and symptoms of infection should be established. internet and mobile-based social media communications should be considered as the first-line approaches for education and appropriate interventions. opioid users face increased challenges; some concerns are about their take-home doses and repetitive visits that make it impossible for them to stay at home. this pandemic could be considered as an extraordinary circumstance; the clinicians should facilitate ost protocol for clinically stable patients and cancel all group-based interventions or therapies. healthcare workers in substance use treatment facilities are also facing a higher risk of infection, burnout, distress, psychiatric disorders, discrimination, and violence. the essential right for each service provider, no matter a peer group or professional service provider, is to be safe and secure, in both physical and mental health aspects. misinformation, social isolation, ensuing economic depression, and possible grief reactions may result in exacerbation of public fear, panic, and distress that can be followed by lapse and relapse in ex-drug users. stress reduction, crisis interventions, coping skills training, motivational interviewing, and tailored and modified relapse prevention interventions, modification in contingency-based management for rewarding virus transmission preventive behaviours, attracting family support, managing patients' vocational problems are the main helpful psychosocial interventions. in this period, internet-based psychotherapy and phone counseling are highly recommended. there are many medical considerations regarding pwud that other physicians in charge of the management of co-vid- treatment should keep in their minds. clinicians should be careful in the differentiation between withdrawal signs and symptoms and those of covid- infection. pwud may have different clinical manifestations due to various etiologies. healthcare providers should consider different possible manifestations and, more importantly, avoid any type of medical stigma or discrimination against pwud. pwud regularly self-medicate their physical and mental problems with drugs, which may mask critical covid- symptoms. a number of drug-drug interactions between substance of use, addiction treatment medications, and covid- medications must be considered in terms of toxicity, withdrawal, and exacerbation of fatal side effects. there is also a possible overlap of pathological laboratory results of the cbc and liver enzymes in pwud and people with covid- infection. histories of renal failure, cardiovascular and metabolic diseases are more likely to emerge in pwud that put them at higher risk of morbidity and mortality after contracting covid- pain management in pwud, specifically opioid users and patients under ost, has some complexity, which calls for the involvement of joint expertise. the study has been conducted with no funds from external sources. original idea, wrote initial topics and headlines, and the first draft: ali farhoudian and seyed ramin radfar; participated in the literature review, writing, editing, and revision of the report and reached consensus on the conclusion: all authors. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. we are immensely grateful to dr. richard rawson, professor at integrated substance abuse programs, university of california, los angeles, and dr. richard schottenfeld, senior research scientist at yale school of medicine, for their comments during the process of writing the manuscript. the views expressed are those of the author (s) and not necessarily dr. richard rawson and or dr. richard schottenfeld. authors also respectfully dedicate the article to the souls of all healthcare providers who lost their precious lives in the fight against covid- . cannabinoid hyperemesis syndrome presenting with recurrent acute renal failure an assessment of the level of concern among hospital-based health-care workers regarding mers outbreaks in saudi arabia stigma, discrimination and the health of illicit drug users. drug and alcohol dependence single high-dose buprenorphine for opioid craving during withdrawal contingency management interventions for nonprescribed drug use during treatment for opiate addiction: a systematic review and meta-analysis. drug and alcohol dependence waterpipe smoking as a public health risk: potential risk for transmission of mers-cov risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia methadone maintenance patients are cross-tolerant to the antinociceptive effects of very high plasma morphine concentrations -ncov epidemic: address mental health care to empower society. the lancet chronic cocaine abuse and dilated cardiomyopathy prize-based contingency management for the treatment of substance abusers: a meta-analysis voluntary versus prescribed termination of methadone maintenance opioid substitution treatment planning in a disaster context: perspectives from emergency management and health professionals in aotearoa/new zealand forensic medicine and toxicology: drug addiction management of the opiate abstinence syndrome opioid substitution treatment planning in a disaster context: perspectives from emergency management and health professionals in aotearoa/new zealand efavirenz treatment and falsepositive results in benzodiazepine screening tests understanding links among opioid use, overdose, and suicide at least dead from drinking toxic alcohol in iran after coronavirus cure rumor community engagement-the harms of drug prohibition: ongoing resistance in vancouver's downtown eastside prevalence and risk factors for unrecognized obstructive lung disease among urban drug users relapse and relapse prevention determinants of influenza vaccination in hard-to-reach urban populations a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-march epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study kidney impairment is associated with in-hospital death of covid- patients. medrxiv comment on covid- from the british hiv association (bhiva). ( ). british hiv association. retrieved february from covid- : operational guidance for maintaining essential health services during an outbreak. world health organization pain responses in methadone-maintained opioid abusers risk of death during and after opiate substitution treatment in primary care: prospective observational study in uk general practice research database heroin nephropathy: a clinicopathologic and epidemiologic study knowledge and attitude towards hiv/aids among college students in ardabil comparative efficacy and acceptability of psychosocial interventions for individuals with cocaine and amphetamine addiction: a systematic review and network meta-analysis renal tubular dysfunction in chronic alcohol abuse--effects of abstinence barriers and facilitators to the uptake of tuberculosis diagnostic and treatment services by hard-to-reach populations in countries of low and medium tuberculosis incidence: a systematic review of qualitative mortality among clients of a state-wide opioid pharmacotherapy program over years: risk factors and lives saved prescription of benzodiazepines in general practice in the context of a manmade disaster: a longitudinal study suicidal intentionality among deaths due to acute reaction after drug consumption: a five-year follow-up study methadone-metabolism, pharmacokinetics and interactions severe intraoperative hypertension and opioid-resistant postoperative pain in a methadone-treated patient breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of cov-id- associated pneumonia in clinical studies staff regard towards working with substance users: a european multi-centre study amphetamine intoxication with coagulopathy, hyperthermia, and reversible renal failure: a syndrome resembling heatstroke exposure to amphetamines leads to development of amphetamine type stimulants associated cardiomyopathy (atsac) the impact of stress on addiction bacterial infections in drug users the perioperative patient on buprenorphine: a systematic review of perioperative management strategies and patient outcomes coronavirus (covid- ) and people living with hiv in scotland a new differential diagnosis: synthetic cannabinoids-associated acute renal failure enterprise singapore management of injecting drug users admitted to hospital. the lancet buprenorphine maintenance therapy hinders acute pain management in trauma stress among iranian nurses in critical wards feasibility of controlling covid- outbreaks by isolation of cases and contacts contingency management interventions for hiv, tuberculosis, and hepatitis control among individuals with substance use disorders: a systematized review effectiveness of interventions for diagnosis and treatment of tuberculosis in hard-to-reach populations in countries of low and medium tuberculosis incidence: a systematic review covid- : what is next for public health? the lancet clinical features of patients infected with novel coronavirus in wuhan interim guidline for healthcare facilities cocaine administration increases cd / cd lymphocyte ratio in peripheral blood despite lymphopenia and elevated corticosterone prevalence of hepatitis b and c infections and their associated risk factors in addict prisoners of central provinces of iran num [covid- ] [micro sign]-opioid receptor blockade in opioid-addicted patients during barbiturate-induced anesthesia for acute detoxification water-pipe (narghile) smoking: an emerging health risk behavior factors influencing the uptake of influenza vaccine vary among different groups in the hard-to-reach population. australian and new zealand a role for brain stress systems in addiction brain stress systems in the amygdala and addiction addiction is a reward deficit and stress surfeit disorder addiction as a stress surfeit disorder torsade de pointes associated with very-high-dose methadone severe acute respiratory syndrome coronavirus (sars-cov- ) and corona virus disease- (covid- ): the epidemic and the challenges prevalence of psychiatric disorders among toronto hospital workers one to two years after the sars outbreak a comprehensive review of opioid-induced hyperalgesia a systematic review of cognitive and behavioural therapies for methamphetamine dependence therapeutic options for the novel coronavirus ( -ncov) a simple laboratory parameter facilitates early identification of covid- patients. medrxiv opioid system modulates the immune function: a review case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr high concentration of chloroquine in urine gives positive result with amphetamine cedia reagent methadoneinduced torsade de pointes after stopping lopinavir-ritonavir covid- clinical guidance for the cardiovascular care team modeling the interplay between human behavior and the spread of infectious diseases opioid-induced abnormal pain sensitivity epidemiological and clinical characteristics of sars-cov- and sars-cov: a system review buprenorphine-based regimens and methadone for the medical management of opioid dependence: selecting the appropriate drug for treatment long-term psychological and occupational effects of providing hospital healthcare during sars outbreak chronic sclerosing glomerulopathy (heroin-associated nephropathy) in intravenous t's and blues abusers the use of the methadone/metabolite ratio (mmr) to identify an individual metabolic phenotype and assess risks of poor response and adverse effects: towards scientific methadone dosing cocaine intoxication: hyperpyrexia, rhabdomyolysis and acute renal failure treatment responsivity of cocaine-dependent patients with antisocial personality disorder to cognitive-behavioral and contingency management interventions central and peripheral biomarkers of stress response for addiction risk and relapse vulnerability alcoholic cardiomyopathy: what is known and what is not known relationship between degree of craving and different dimensions of addiction severity a cluster of tuberculosis associated with use of a marijuana water pipe the use of opioid analgesia in endstage renal disease patients managed without dialysis: recommendations for practice high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea alcohol abuse, immunosuppression, and pulmonary infection cocaine use, hypertension, and end-stage renal disease dying in the shadows: the challenge of providing health care for homeless people acute amphetamine cardiomyopathy in a drug addict effect of smoking on oxygen saturation in healthy sedentary men and women qt prolongation and torsades de pointes among methadone users: reports to the fda spontaneous reporting system tuberculosis in drug users risk of end-stage renal disease associated with alcohol consumption current research on methamphetamine: epidemiology, medical and psychiatric effects, treatment, and harm reduction efforts factors influencing access and use of care and treatment serČvices among iranian people living with hiv and aids: a qualiČtative study behaviors influencing human immunodeficiency virus transmission in the context of positive prevention among people living with hiv/acquired immunodeficiency syndrome in iran: a qualitative study tainted alcohol claims more lives than coronavirus in iran's khuzestan the matrix model of intensive outpatient treatment. behavioral health recovery management a comparison of contingency management and cognitive-behavioral approaches for stimulant-dependent individuals an intensive outpatient approach for cocaine abuse treatment: the matrix model false-positive' and 'false-negative' test results in clinical urine drug testing risk theory in epidemic times: sex, drugs and the social organisation of 'risk behaviour amphetamine-induced angiitis leading to renal failure measuring psychological resilience to disasters: are evidencebased indicators an achievable goal? environmental health characterization of interference with commercial Δ -tetrahydrocannabinol immunoassays by efavirenz (glucuronide) in urine acute cardiomyopathy and cardiogenic pulmonary edema after inhaled heroin use opioid drug abuse and modulation of immune function: consequences in the susceptibility to opportunistic infections buprenorphine and methadone maintenance treatment of heroin addicts preserves immune function false-positive interferences of common urine drug screen immunoassays: a review same clinic, different conceptions: drug users' and healthcare professionals' perceptions of how stigma may affect clinical care the stigmatization of drug use as mechanism of legitimation of exclusion services among iranian people living with hiv and aids: a qualitative study drug abuse profile-patient delay, diagnosis delay and drug resistance pattern-among addict patients with tuberculosis renal infarction associated with intravenous cocaine use middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data predictors of retention in methadone maintenance therapy: a prospective multi-center study psychobiological responses to unpleasant emotions in cannabis users epidemic psychology: a model the association between multiple domains of discrimination and self-assessed health: a multilevel analysis of latinos and blacks in four low-income new york city neighborhoods tobacco and ecigarette use amongst illicit drug users in australia. drug and alcohol dependence factors associated with death or intensive care unit admission due to pandemic influenza a (h n ) infection prevalence and related factors of post-traumatic stress disorder among medical staff members exposed to h n patients computer-and internet-based psychotherapy interventions methadone maintenance treatment: best practices in case management. toronto: centre for addiction and mental health department of health and human services, counselor's family education manual -matrix intensive outpatient treatment for people with stimulant use disorders covid- : a puzzle with many missing pieces protein malnutrition impairs the immune control of trichinella spiralis infection burnout among the addiction counseling workforce: the differential roles of mindfulness and values-based processes and work-site factors drug use disorders: impact of a public health rather than a criminal justice approach pain among high-risk patients on methadone maintenance treatment clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan cigarette smoking quit rates among adults with and without alcohol use disorders and heavy alcohol use sudden cardiac arrest in a patient on chronic methadone after the addition of azithromycin department of mental health, substance abuse, world health organization, international narcotics control board guidelines for the psychosocially assisted pharmacological treatment of opioid dependence. geneva: world health organization who director-general's opening remarks at the media briefing on covid- - coronavirus disease (covid- ) situation report - coronavirus disease (covid- ) outbreak: rights, roles and responsibilities of health workers, including key considerations for occupational safety and health infection prevention and control during health care when novel coronavirus ( ncov) infection is suspected: interim guidance infection prevention and control during health care when covid- is suspected: interim guidance nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention pathological findings of covid- associated with acute respiratory distress syndrome. the lancet respiratory medicine cocaine use disorder is associated with changes in th /th /th cytokines and lymphocytes subsets a novel coronavirus from patients with pneumonia in china covid- in wuhan: immediate psychological impact on health workers. medrxiv coronavirus disease (covid- ): a perspective from china key: cord- -r p helx authors: aggarwal, sadhna; verma, sumit singh; aggarwal, sumit; gupta, subash chandra title: drug repurposing for breast cancer therapy: old weapon for new battle date: - - journal: semin cancer biol doi: . /j.semcancer. . . sha: doc_id: cord_uid: r p helx abstract despite tremendous resources being invested in prevention and treatment, breast cancer remains a leading cause of cancer deaths in women globally. the available treatment modalities are very costly and produces severe side effects. drug repurposing that relate to new uses for old drugs has emerged as a novel approach for drug development. repositioning of old, clinically approved, off patent non-cancer drugs with known targets, into newer indication is like using old weapons for new battle. the advances in genomics, proteomics and information computational biology has facilitated the process of drug repurposing. repositioning approach not only fastens the process of drug development but also offers more effective, cheaper, safer drugs with lesser/known side effects. during the last decade, drugs such as alkylating agents, anthracyclins, antimetabolite, cdk / inhibitor, aromatase inhibitor, mtor inhibitor and mitotic inhibitors has been repositioned for breast cancer treatment. the repositioned drugs have been successfully used for the treatment of most aggressive triple negative breast cancer. the literature review suggest that serendipity plays a major role in the drug development. this article describes the comprehensive overview of the current scenario of drug repurposing for the breast cancer treatment. the strategies as well as several examples of repurposed drugs are provided. the challenges associated with drug repurposing are discussed. breast cancer, one of the most common cancer in women, is globally diagnosed by physical examination, breast scan and tissue biopsies. a total of . million new cases of breast cancer were identified in , of which ∼ , cases died. this accounted for approximately % of all cancer deaths among women globally [ ] . in , the highest numbers of breast cancer cases were reported from asia ( , , ) followed by europe ( , , ) , north america ( , , ) , latin america and caribbean ( , ), africa ( , , ) and oceania ( , ) [ ] . although very common in women, breast cancer rarely occurs in men [ ] . a vast heterogeneity in breast cancer molecular profiling has been reported [ ] . based on the er/pr and her expression, breast cancer can be of types: (i) er/pr+, her +; (ii) er/ pr−, her +; (iii) er/pr+, her −; and (iv) er/pr−, her − [ ] . the most aggressive breast cancer is er/pr−, her − (also called triple negative breast cancer, tnbc) because no receptor is present on these tumors. it is estimated that % of the breast cancers are hormone receptor positive ( %) and can be treated with corresponding therapies [ , ] . in addition to hormone receptors, other biomarkers used for clinical, pathological and molecular characterization of breast cancer include ki , p , ca , cea, brca /brca etc. [ ] . these molecular biomarkers in breast cancer are used for diagnosis, staging and grading, therapeutic intervention, prognosis as well as clinical management of recurrent and metastatic cases [ ] . are given as neoadjuvant therapy and only trastuzumab is continued post-surgery [ , ] . surgery is mostly followed by chemotherapy or radiotherapy to further destroy the remnant micrometastatic cancer cells that have escaped the breast or lymph nodes, to decrease the chances of recurrence and to increase the overall patient survival [ , ] . the adjuvant (additional) therapies i.e. hormone or targeted therapy are provided to breast cancer patients depending on the expression of hormone receptor or target protein, respectively [ ] . the er/pr + breast cancer patients are given tamoxifen and aromatase inhibitor together or alternatively after the surgical resection [ , ] . likewise, trastuzumab (herceptin) is mostly recommended to the her + breast cancer patients [ ] . tnbc patients are difficult to treat, with unfavorable prognosis and are generally administered with the standard chemotherapy along with the parp inhibitors or the dnatargeting platinum drug (carboplatin) [ , ] . another strategy is based on the holistic approach of patient recovery i.e. complementary and alternative medicine. in the holistic approach, the patients are advised for several life style changes like exercise, yoga, meditation, acupuncture and/or ayurvedic treatments in addition to the standard therapies [ ] . the united states fda has approved a number of drugs for the breast cancer therapy (table ) . however, these drugs are very costly and produce numerous side effects [ , ] . the common reported side effects in patients include fatigue, headaches, pain, numbness, dental issues, lymphedema, musculoskeletal symptoms, heart problems, blood clots, menstrual and menopausal symptoms, infertility, bone loss, and memory loss. these side effects are hard to be tolerated by the patients who are already weakened by the disease. eventually these also affect the decision for further choice of treatment and impair the quality of life. this necessitates the development of novel drugs for breast cancer therapy. we have previously reviewed the overall process involved in cancer drug development [ , ] . the drug development for breast cancer like other cancer types is a multi-step process involving the designing, synthesis, characterization, testing for efficacy and toxicities, and approval. overall, the process is lengthy, involving huge investment of money. it is estimated that the development of a new drug usually requires - years of time and approximately - billion dollars. on the top of all this, nearly % of the drugs fail during the clinical trial stage due to unexpected lack of appropriate efficacy, unacceptable side effects or regulatory norms [ , ] . therefore, the approaches to expedite the drug development process are required. one approach might be finding new uses for old clinically approved drugs (drug repositioning). the drug repositioning (aka drug repurposing, drug reprofiling, drug re-tasking, drug redesigning, drug resorting, drug reindication, indication switching, therapeutic switching) can be defined as exploring the new uses for an old clinically approved drug, with reduced risk, time and cost. drug repositioning circumvents the usual route of substantially higher rates, slow pace and side effects etc. [ ] [ ] [ ] . it is becoming an attractive concept for pharmaceutical industries. it is estimated that the repositioning for drug development requires much lesser time ( - years) and money ($ . billion) with at least known and approved side effects; and higher probability of success [ , [ ] [ ] [ ] [ ] . repositioning of old drugs offer rapid transition from bench to bedside as already approved by fda and other regulatory norms, passed through the clinical trials and are approved for human use. these drugs have well defined pharmacodynamics, pharmacokinetics, dose, side effects, metabolic profiles and targeted molecular pathway etc [ , ] . here, the clinical development can directly start from the phase ii trial to assess the efficacy for new indication/disease target. the successful example of drug repositioning includes sildenafil (viagra) and thalidomide. viagra was originally launched for hypertension and angina pectoris patients by pfizer ( ) . however, the patients developed penile erections as side effects in phase i clinical trial. the drug was later developed for erectile dysfunctions. in , the total sale of viagra was . billion dollars [ ] . similarly, the sedative thalidomide was serendipitously repurposed for erythema nodosum leprosum (enl) in and for multiple myeloma and other cancers in [ ] [ ] [ ] [ ] . recent advances in genomics, proteomics, transcriptomics and metabolomics have provided vast and deep knowledge about the molecular and metabolic alterations that occurs in cancers. the fundamentals of drug repurposing through the integration of system biology and bioinformatics depends on basic concepts i.e. activity based and in silico drug discovery [ ] . activity based drug repurposing is the experimental approach where the drug candidate is evaluated for the anticancer activity directly. the drug that is structurally similar would tend to have similar target, shared biological activity and indications [ ] . in the two diseases where same metabolic pathway or signaling is affected, then the drugs that target the specific pathway can be used in both of the diseases irrespective of their structural dissimilarity [ , ] . the drug that shows strong efficiency for any off-target (side effect) in one disease can be explored further i.e. the off-target binding and effect can be used as the novel indication of that drug in some other disease [ ] [ ] [ ] . in silico drug repurposing approach is based on the association and/or overlaps in the disease-gene-drug paradigm. the large data about drug-disease, gene expression (microarrays) or protein-protein interactions or gene-protein interactions or signaling pathway mapping, signature matching, genome-wide association studies (gwas) can be used for providing drug-disease gene association networking by the systematic integration and coordination of computation and bioinformatics, modeling (such as docking for structural modeling), experimentation, statistics, machine learning etc. [ ] [ ] [ ] [ ] . the various data resources are available that can be used for bio-informatics based exploration followed by experimental validation for any drug repurposing possibilities in any disease. the similar gene expression profile for drug phenotype in patients with different diseases as mapped by connectivity map (cmap), library integrated network based cellular signatures (lincs), expression signatures may have similar therapeutic applications [ ] [ ] [ ] . other important public data resources that are used for network-based drug repositioning includes gene set enrichment analysis (gsea) for drug-drug similarity network [ ] ; drugbank [ ] , online mendelian inheritance in men (omim) [ ] and geo [ ] for predicting drug-disease network; kegg [ ] , string [ ] , biogrid [ ] , happi [ ] and reactome [ ] for pathway and/or proteinprotein interactions; stitch drug-gene/protein database [ , ] , ttd therapeutic target database [ ] , sfinx for drug-drug interactions [ ] ; and sider for drug side effects [ ] . also, the recently reported 'drug repurposing from control system theory (decost)' is a comprehensive platform for drug repurposing that encompasses various limitations in the previous databases like variation in number of copies of gene of interest, mutations, lack of reference for normal range of gene expression etc. in different diseases [ ] . vigorously exploited 'network-based approach' by integrating the mentioned resources for identifying potential target, pathway and drug has been reported as the most potential way for drug repositioning [ ] . in the following section, we discuss the clinically approved drugs that were originally used for diseases other than breast cancer. however, these drugs are now being used or explored for breast cancer therapy. the clinically approved repositioned drugs for breast cancer are grouped based on their mode of action (table ). these drugs are diverse in terms of their chemical structures (fig. ) . abbreviations: cdk: cyclin-dependent kinase; erd: er down-regulator; lhrh: luteinizing hormone-releasing hormone; serd: selective estrogen receptor degrader; serm: selective estrogen receptor modulator; er: estrogen receptor; pr: progesterone receptor, her: human epidermal growth factor receptor. these are dna damaging agent that alkylate the guanine base thereby rendering them unable to bind to complementary strand. this leads to inhibition of dna replication and cancer cell growth. the alkylating agents affect all phases of cell cycle. cyclophosphamide is a known immuno-modulator that inhibits the suppressive regulatory t cells and enhances the effector t cells in tumor microenvironment. it shows the biphasic effect i.e. at low doses it imparts the immuno-suppressive function and at higher doses it functions as an alkylating agent leading to the death of tumour as well as the lymphoid cells [ , ] . usually mg/m² cyclophosphamide is intravenously (iv) administered to breast cancer patients in combination with other chemotherapeutics (docetaxel, paclitaxel, doxorubicin). the recommended regimens for robustly healthy breast cancer patients include cycles of cyclophosphamide, methotrexate and -fluorouracil (cmf) or cycles of adriamycin and cyclophosphamide (ac) [ , ] . thiotepa is a derivative of n,n',n"-triethylenephosphoramide (tepa) that was launched ( ) as immunosuppressive drug for transplantation in hematological diseases [ ] . subsequently, the drug was recommended for solid tumors in [ ] and for breast cancer ( . to . mg/kg iv repeated every - weeks) in [ ] . thiotepa in combination with vinblastine, adriamycin, and halotestin (vath) was effective in patients with relapse after adjuvant therapy and in metastatic breast cancer patients [ ] . anthracyclins are antibiotics that intercalates between the adjacent base pairs of dna in such a manner that it forms anthracyclin-dna-topoisomeraseii ternary complex. the ternary complex disrupts the resealing activity of enzyme leading to inhibition of dna and rna synthesis in highly replicating cancer cells [ , ] . some of the common anthracyclins are doxorubicin, daunorubicin, epirubicin and idarubicin. these are generally extracted from streptomyces bacterium. approved for medical use in , doxorubicin ( -hydroxylated version of daunorubicin) [ ] was originally extracted from streptomyces peucetius [ ] . there was a long lapse of years between first clinical usages of anthracyclins to their proven relevance in breast cancer treatment. however, once established the chemotherapeutic regimens that contained doxorubicin [usual dose: - mg/m² intravenously (iv) every days] were used regularly with dose-intense and dosedense schedules. some of the regimens containing doxorubicin are ac (adriamycin, cyclophosphamide), tac (taxotere, ac) and fac ( fluorouracil, ac) [ ] . unlike doxil (the pegylated form of doxorubucin), myocet is the non-pegylated liposomal version of doxorubicin that is approved for treatment of metastatic breast cancer in combination with cyclophosphamide in europe and canada [ ] . antimetabolites are the chemo-drugs that interfere with the metabolic pathways in the cancer cells primarily by acting as structural analogues to important cellular metabolites. -flourouracil ( -fu) is a pyrimidine antimetabolite, an uracil analogue that inhibits the activity of thymidylate synthetase by converting -fu to fdump and futp, instead of uracil to thymidine conversion. therefore, -fu not only inhibits the dna synthesis in the cell but also inhibits rna synthesis due to fdutp incorporation in rna; leading to highly toxic effects on the growth of rapidly multiplying cells like cancer cells. klein and colleagues introduced -fu for medical use in for the treatment of self-healing, squamous cell tumors of skin i.e. keratoacanthomas (kas) [ ] . -fu was subsequently used for other cancer types including breast cancer [ ] . general drug dosage of -fu is mg/m or mg/m iv on day and day of every -day cycle (six cycles in total). lokich et al ( ) first introduced the infusion of -fu in combination with methotrexate (m) for breast cancer [ ] . since then various regimes of combination therapy containing -fu (f) with other drugs like doxorubicin (a), cisplatin (c), cyclophosphamide (cx) and epirubicin (e) (ecf, cmf, caf) have been continuously used for treating metastatic breast cancer [ ] . the other combinations such as -fu with paclitaxel in [ ] and high dose -fu/paclitaxel with doxorubicin [ ] were found successful for breast cancer treatment. methotrexate is a folate/folic acid antagonist that binds to the dihydrofolate reductase (dhfr) enzyme and inhibits the synthesis of building blocks of dna and rna itself. folic acid is converted to dihydrofolate by dhfr, which is then reduced to tetrahydrofolate (thdf) for further action by thymidine synthetase. methotrexate binding to dhfr decreases the synthesis of purines and pyrimidines in the cells that inhibits the cell cycle progression in s-phase. methotrexate was discovered in s as a substitute to folic acid, because the cases with decreased leukemic cell count (acute lymphoblastic leukemia) due to dietary deficiency of folic acid were being presented in clinics [ , ] . methotrexate was first shown to remit breast cancer in by jane c wright [ ] . in s and s, methotrexate was being used as single cytotoxic agent for advanced breast cancer treatment [ ] . however, gianni and bonadonna group ( ) showed the first treatment with cycles of cmf combination in mastectomised women with advanced cancer and positive lymph nodes and reported only . % treatment failure with acceptable toxicity [ , ] . in fact, cmf regimen was the first ever schedule for breast cancer treatment. further, cmf was found to be equally effective even at cycles. immunosuppressive activity of methotrexate has been explored for its clinical use in autoimmune diseases like rheumatoid arthritis [ ] . general dosage of cmf regimen is defined as - cycles of cyclophosphamide ( mg/m ); methotrexate ( mg/m ), and fluorouracil ( mg/m ) at -day intervals. capecitabine, a pro-drug for -fu, is another pyrimidine antimetabolite. the conversion of capecitabine to -fu requires the three systematic enzymatic reaction cascades through intestine, liver and tumour cells. the enzyme that catalyzes the last step of conversion is highly expressed in tumour cells as compared to the normal cells. therefore, the tumour selective conversion of capecitabine to -fu prevents the systemic exposure of body to -fu [ ] . also, capecitabine is easier to administer and safer than -fu with better efficacy. the capecitabine was first used against colon cancer in [ ] . capecitabine ( mg/m given orally twice daily for days followed by a -day rest period in a -day cycle for cycles) is generally used to treat paclitaxel or docetaxel resistant advanced and metastatic breast cancers [ ] . it is used either alone or in combination with cabazitaxel [ ] , vinorelbine [ ] or ixabepilone [ ] . gemcitabine is a pro-drug that gets tri-phosphorylated inside the cell (dfdctp) by sequential enzyme catalyzed reactions. this dfdctp masquerades as an analogue of cytidine and gets incorporated in the newly synthesized dna generating irreparable error that inhibits dna replication leading to cell death [ , ] . gemcitabine was primarily manufactured in by larry hertel's group at eli lilly and company to be used as an anti-viral drug against enteroviruses [coxsackievirus b (cvb )]. the drug was also used against cvb , ev , human rhinoviruses (hrvs), human immunodeficiency virus (hiv), hepatitis c virus (hcv), poliovirus, influenza virus, zikv and mers-cov [ , ] . it was then pre-clinically tested for its anti-tumour attribute. the drug was approved by fda for pancreatic cancer in [ ] and for non-small lung cancer in [ ] . finally, gemcitabine was approved for metastatic breast cancer in in combination with paclitaxel [ , ] . after anthracycline-containing adjuvant chemotherapy failure, gemcitabine ( mg/m² iv infusion over min on days and of each -day cycle) and paclitaxel ( mg/m² on day as a h infusion before gemcitabine) combination is used as the first-line of treatment against metastatic cancer. other combinations such as gemcitabine/vinorelbine (gemvin), gemcitabine/cisplatin (gemcis), gemcitabine/capecitabine (gemcap) have also shown increased response rate and overall survival in pretreated metastatic breast cancer patients [ ] . palbociclib is a cdk / inhibitor that halts the progression of cells from g -to s-phase. palbociclib ( mg, -day cycle with aromatase inhibitor) is used as the targeted therapy against er + /her − advanced and metastatic breast cancer in conjunction with hormone therapy. the open label paloma (palbociclib: ongoing trials in the management of breast cancer) clinical trials were designed recently with either aromatase inhibitor (letrozole) (paloma- or paloma- trials) [ , ] or fulvestrant hormone therapies (paloma- trails) [ ] . an improvement in the overall survival of metastatic breast cancer patients was reported in paloma- phase iii clinical trials. inhibiting cdk / activity delays the resistant to hormone therapy and significantly improves the progression free survival (pfs) of patients [ , ] . the hormonal therapy or endocrine therapy is usually given for - years. this therapy either directly targets the hormone (estrogen and/or progesterone) production or negatively regulates the functional effects in hormone sensitive breast cancer patients (er + and/or pr + ). selective estrogen receptor modules (serms) serve as anti-estrogens by binding to the hormone receptors as antagonists. the widely used serms that have been repositioned as breast cancer drugs are tamoxifen ( ), toremifene ( ) and raloxifene ( ) . tamoxifen is the oldest serm that has been in use for more than years for early stage breast cancer treatments in pre-and post-menopausal women. toremifene and raloxifene are equally effective but safer alternatives of tamoxifen that are used in only post-menopausal women with advanced breast cancer [ , ] . multiple outcomes raloxifene evaluation (more) clinical trial was an osteoporosis treatment trial in postmenopausal women, with secondary objective of evaluating the effects on breast cancer risk reduction. more lead to the designing of further clinical trials such as continuing outcomes relevant to evista® (core), raloxifene use for the heart (ruth), and study of tamoxifen and raloxifene (star) [ ] . aromatase inhibitor hormone therapy is administered only in postmenopausal women to treat er + early and/or late stage breast cancer [ ] . it acts on the aromatase enzyme that still produces estrogen hormone in fat tissue of post-menopausal women or women without active ovaries. thus, the aromatase inhibitor reduces the amount of estrogen in post-menopausal women with breast cancer that would otherwise feed the breast cancer cells for further growth. the aromatase inhibitors (anastrazole, exemestane and letrozole) were initially used for ovary stimulation and induction of ovulation in infertile females or polycystic ovary syndrome. aromatase inhibitors can be used as neoadjuvant or adjuvant therapy, mostly alone or in combination, which were introduced as an alternate to tamoxifen in postmenopausal patients [ ] . selective estrogen receptor degrader (serd) such as fulvestrant, are pure anti-estrogens that blocks estrogen receptor and degrades the receptor without any agonist effect [ ] . fulvestrant was first used in as a 'serd hormone therapy' against hr + her − advanced and metastatic breast cancer in post-menopausal women that were resistant to other hormone therapy [ ] . it is used in combination with cdk / inhibitors like palbociclib (paloma- ) and ribociclib (monaleesa- ) and anti-pi k/akt/mtor pathway drugs such as pictilisib (fergi) and buparlisib (belle- and belle- ) [ ] . luteinizing hormone releasing hormone (lhrh) analogs interfere with the signaling mechanism that activates the estrogen synthesis in ovaries causing temporary menopause. in , goserelin was used for the assisted reproduction and prostate cancer treatment. goserelin was then approved for the treatment of pre-menopausal women with hormone sensitive breast cancers in . it is used alone or in combination with other hormone therapies [ ] . goserelin is currently under phase ii clinical trial as an additional drug into the standard neoadjuvant therapy for tnbc patients. the goserelin phase ii trial is expected to complete by [ ] . everolimus is an mtor kinase inhibitor that inhibits the pi k/akt/ mtor signaling pathway. everolimus was originally approved for renal cancer in , as immunosuppressant during renal transplants in and for pancreatic cancer in . a phase iii clinical trial 'breast cancer trial of oral everolimus- (bolero- )' that included everolimus in combination with exemestane was successfully completed in leading to the approval of everolimus by us fda for the treatment of hr + , her − advanced metastatic cancers that are resistant to letrozole or anastrazole [ , ] . mitotic inhibitors terminate the cell division or mitosis by disrupting the microtubule dynamics. this leads to g /m phase cell cycle arrest or inhibition of spindle formation. some of the common examples of mitotic inhibitor include docetaxel, paclitaxel and vinblastine. while docetaxel and paclitaxel induces g /m cell cycle arrest, vinblastine is known to inhibit spindle formation. docetaxel and paclitaxel are used as neoadjuvant or adjuvant therapy as single agent or in combination with other chemotherapeutic agents for the treatment of early, advanced and metastatic breast cancer in pre-and postmenopausal women. paclitaxel was isolated from pacific yew in . it was used as drug for arterial restenosis. docetaxel and paclitaxel were initially used as therapeutics in prostate and ovarian cancer ( ) respectively. thereafter, docetaxel ( mg/m² iv h after doxorubicin and cyclophosphamide weeks x cycles) and paclitaxel ( mg/m² iv over h weeks times with doxorubicincontaining regimen) were repositioned as chemotherapy adjunct in breast cancer treatment regimen [ ] [ ] [ ] . taxanes are now used as the part of standard chemotherapy in metastatic breast cancer. numerous combinatorial chemotherapy containing taxanes are used in routine practice for treating breast cancer by clinicians around the globe [ ] . vinblastine is a naturally occurring vinca alkaloid found in white flowered periwinkle, vinca rosea. this was discovered by robert noble and charles t beer in . the discovery of vinblastine is a beautiful example of serendipity in drug development. the group rather aimed to evaluate the anti-diabetic effect of extract in rats and observed pseudomonas mediated septicemia that was accompanied with the rapid wbc fall and granulocytopenia. further study in this direction showed peripheral granulocytopenia and leukopenia in vinca rosea extract treated rats. finally, they reported carcinostatic activity of vinca rosea extract/vinblastine in rats with transplantable mammary adenocarcinoma and sarcoma [ , ] . vinblastine was approved for lymphoma in . also, since s, vinblastine ( - mg/mm iv once weekly or every other week) in combination with mitomycin or mvp (mitomycin c, vinblastine and cisplatin) has been used as chemotherapy against advanced and metastatic breast cancer [ , ] . with a drastic increase in the number of new cases, the cost of the cancer treatment is rising even at a higher speed. this is because the disease demands molecular dissection at gene and protein level to find newer strategies, appropriate targets and corresponding drugs. this requires a lot of time and the use of high-end techniques by pharmaceutical companies thereby increasing the overall financial investments and eventually the cost of the drug [ ] . the antibody-based immunotherapies or cell-based cart therapy, dc vaccine, act types of drug treatment might prove promising. however, these therapies are unaffordable by most patients in developed as well as developing countries. this necessitates the development of drugs with higher efficacy, lower side effects and practically lower cost. one of the smart ways is to repurpose an old, existing and approved drug for a newer indication. the advantage of this approach is that the approved drugs have existing information about molecular targets, off-targets, modes of action, safety level and side effects. this would not only save a lot of time that is otherwise required for discovery, designing, clinical trials and approvals of a new drug but also reduce the overall cost of anticancer drug. this will cut the cost involved in preclinical development and phase i trial. further, the old drugs would be off-patent and hence cheaper than the initial costs. in this article, we discussed in detail the advantages of drug repurposing for breast cancer treatment. we provided several drugs that have been successfully repurposed for breast cancer treatment. the triple negative breast cancer (tnbc) is highly heterogeneous, aggressive and complex form of breast cancer without expression of er, pr, or her receptors. tnbc is untreatable with regular hormone therapy. however, the combination of chemotherapies and the drug repositioning approach has offered promising outcomes by preclinical studies. flunarizine, a n-ras inhibitor, has been approved for migraine or vertigo. flunarizine has also shown promise in tnbc mouse models by inducing autophagy [ ] . a recent publication showed that the combination of metformin and hemin, used for type diabetes or porphyria respectively, could inhibit the growth of breast cancer cells. bioinformatics tools revealed that the bach expression is significantly elevated in tnbc and the hemin sensitizes the tnbc to metforminmediated degradation of bach [ ] . the completion of repurposing drug in oncology (redo) project has provided evidence for the new uses of drugs for breast cancer. originally these were discovered for indications other than breast cancer [ ] . these drugs include mebendazole (anti-helminthic), cimitedine (anti-acid), nitroglycerins (heart attack preventing), itraconazole (anti-fungal), and diclofenac (anti-inflammatory). other drugs such as l-nmma (tilarginine acetate, a nitric oxide synthase inhibitor) [ ] , pro-viral integration moloney virus kinase (pim)- inhibitors (olaparib) [ ] , l-asparginase [ ] , and fenofibrate [ ] are being repurposed for breast cancer. these drugs were originally used in patients with cardiogenic shock, against viral infection, leukemia, helminthic infection, and in patients with high serum cholesterol and triglycerides, respectively. implementation of network pharmacology in examining the potential of natural herbs i.e. ayurvedic formulations and traditional chinese medicines (tcm) are also clinching the attention of researchers for anticancer drug repositioning. these are based on multi-targeted synergistic drug approach instead of one target-one drug approach and thus appear to be promising for breast cancer therapy. the integration of super-computation, simulations, network pharmacology and bioinformatics can detect the target and efficacy of herbs. triphala is the mixture of at least ayurvedic formulations meant for the treatment of many diseases. the bioactive-target-pathwaycancer type networking revealed the link of triphala with cancer types including breast cancer through bioactive and targets. this information can be explored further for either bioactive-or target-based drug repositioning and/or new drug development. however, some challenges and concerns associated with drug repurposing for breast cancer therapy require thorough consideration. the breast tumor heterogeneity, poorly defined molecular signatures, and poorly identified drug dosage provides a roadblock to the drug repurposing. moreover, the current strategies often ignore tumor grade. it is crucial to search the new therapeutic strategies for certain very stringent and hard to treat molecular subgroups of breast cancers such as basal subtype, tnbc, mbc and tumors resistant to standard treatment therapy. the molecular alterations in tnbc, mbc, and resistant tumor types should also be thoroughly investigated. aforementioned bach inhibition by targeting the energy generating mechanism of rapidly growing cells is indeed the novel approach against otherwise resistant-breast cancer. understanding and targeting the tumour microenvironment instead of tumor itself appears more promising in detailing the heterogeneity in breast cancer patients. the overlapping area that consists of tumour and normal cells in tumor microenvironment should be thoroughly characterized at the molecular level [ , ] . identifying the molecular basis of super-responders and non-responders also holds valuable insight for deeper exploration [ ] . another area that needs attention while strategizing drug repositioning for breast cancer is personalized medicine i.e. to extend the repositioning strategy to treat upto single patient design, as the patients under same molecular subgroup often presents further variations leading to unexpected response. structurally similar drugs may, at times, target functionally dissimilar protein hence pathway driven repurposing strategy is better for multi-targeted diseases like cancer. the orphan drugs generally have short or no patent and thus repurposing them is associated with lower cost. repurposing the off-patent drugs eventually blocks the patenting rights on repurposed drug and hence future investors [ ] . inclusion and exclusion criteria for the selection of trial group for repurposed drug is very crucial as different physiological responses are anticipated in comparison to the original group. for example, the pregnant women (first trimester) with cancer are excluded for thalidomide treatment due to risk of amelia and phocomelia [ ] . drug companies that expand the repositioning in similar therapeutic areas, such as reusing ovarian cancer drug as breast cancer drug, had success rate of % in comparison to % success rate when explored in different area [ ] . a comprehensive, large-scale data mining and research is required before jumping on to actual repurposing procedure in order to save the time and finances because smallest of errors in computation or simulations can mislead the entire study. faultless selection of most relevant pharmacological target using most appropriate database (tcga and metabric) for data mining and accurate repurposing strategy is indispensable for the successful drug repositioning for breast cancer. repurposed drugs mainly include the phase iii clinical trial, which still is the most challenging phase due to the longest duration, huge investments and inclusion of largest number of patients as compared to the phase i and phase ii clinical trials. the repurposed drug for breast cancers usually does not work in monotherapy but as poly-pharmacology/combinations. the parp inhibitors are synthetic lethal with brac mutation [ ] . hence, initially proving the repurposed drug efficacy as single drug agent becomes difficult. the toxicity of repurposed drug in pretreated patients or in combination therapy is unknown. for example, valproate-doxorubicin treatment caused toxicityinduced death of two patients in a group of cancer patients [ ] . the overall success rate of development of new drugs ( %) and repositioning of drugs does not vary much as the ultimate efficacy remains same [ ] . also, the repositioned drug anyhow requires clinical re-assessment for optimizing its efficacy and cytotoxicity if the route of administration or drug dosage is different from the older indication; thereby increasing the overall cost of repositioning scheme. nonetheless, the animal model used for drug testing does not represent the exact patient phenotype and hence is less predictive of efficacy in real [ ] . despite many benefits of drug repositioning, much attention is required to lower the drug dosage and toxicity without mitigating efficacy and resolve above discussed limitations for cost effective and more efficient drug development. in this article, we have comprehensively explained the current scenario of repurposed drugs for breast cancer. we have discussed in detail the need and strategies of drug repositioning in breast cancer and several classical examples of drugs that have been repositioned as breast cancer chemotherapy. the futuristic potential of non-cancer drugs that are under investigations for breast cancer as well as the challenges and bottlenecks of drug repositioning were also discussed. we thus conclude that comprehensive approach of selecting the most appropriate gene-protein-pathway-target-drug modeling via integration of system biology and bioinformatics holds the high potential of providing more efficient, safer and cost-effective chemotherapeutics for treatment of even the most stringent forms of breast cancer (metastatic and triple negative). the scientists, researchers and clinicians must continue to work together for extension of the present pharmacological databases, multi-omics and bioinformatics tools. we hope that this will lead to the development of novel drugs and eventually fighting the deadly breast cancer. none. global cancer statistics : globocan estimates of incidence and mortality worldwide for cancers in countries male breast cancer proteomics of breast cancer for marker discovery and signal pathway profiling breast cancer subtypes based on er/pr and her expression: comparison of clinicopathologic features and survival molecular profiling currently offers no more than tumour morphology and basic immunohistochemistry breast cancer treatment: a review molecular markers for breast cancer: prediction on tumor behavior novel molecular markers for breast cancer treatment of breast cancer recent advances in the treatment of breast cancer breast cancer: conventional diagnosis and treatment modalities and recent patents and technologies the prevention, detection, and management of breast cancer longer against shorter collaborative, long-term effects of continuing adjuvant tamoxifen to years versus stopping at years after diagnosis of oestrogen receptor-positive breast cancer: atlas, a randomised trial advances in novel drug delivery strategies for breast cancer therapy breast cancer: a review for the general surgeon beyond trastuzumab: novel therapeutic strategies in her -positive metastatic breast cancer seminars in cancer biology xxx (xxxx) xxx-xxx safety profile of pertuzumab with trastuzumab and docetaxel in patients from asia with human epidermal growth factor receptor -positive metastatic breast cancer: results from the phase iii trial cleopatra toxicity of antiestrogens adjuvant endocrine therapy for premenopausal women with breast cancer senn, m. panel, personalizing the treatment of women with early breast cancer: highlights of the st gallen international expert consensus on the primary therapy of early breast cancer olaparib for metastatic breast cancer in patients with a germline brca mutation homologous recombination deficiency (hrd) status predicts response to standard neoadjuvant chemotherapy in patients with triple-negative or brca / mutation-associated breast cancer use of complementary and alternative medicine and breast cancer survival in the health, eating, activity, and lifestyle study breast cancer management: past, present and evolving serendipity in cancer drug discovery: rational or coincidence? cancer drug discovery by repurposing: teaching new tricks to old dogs how to improve r&d productivity: the pharmaceutical industry's grand challenge drug repositioning: identifying and developing new uses for existing drugs drug repurposing: progress, challenges and recommendations a systematic framework for drug repositioning from integrated omics and drug phenotype profiles using pathway-drug network can you teach old drugs new tricks? new drug development in the united states from drug repurposing from an academic perspective repurposing metformin for the prevention of cancer and cancer recurrence sildenafil: an orally active type cyclic gmp-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction thalidomide embryopathy thalidomide is an inhibitor of angiogenesis antitumor activity of thalidomide in refractory multiple myeloma phase ii trial of bevacizumab, thalidomide, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer recent advances in drug repositioning for the discovery of new anticancer drugs drug repurposing in malignant pleural mesothelioma: a breath of fresh air? drug repurposing: far beyond new targets for old drugs drug repositioning: a machine-learning approach through data integration a survey of current trends in computational drug repositioning network-based inference methods for drug repositioning precision oncology: an overview biological databases for human research cancer systems biology: a network modeling perspective the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease the connectivity map: a new tool for biomedical research identifying network of drug mode of action by gene expression profiling gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles drugbank . : shedding new light on drug metabolism online mendelian inheritance in man (omim), a knowledgebase of human genes and genetic disorders ncbi geo: archive for functional genomics data sets-update kegg: new perspectives on genomes, pathways, diseases and drugs string v : protein-protein interaction networks, integrated over the tree of life the biogrid interaction database: update happi- : a comprehensive and high-quality map of human annotated and predicted protein interactions reactome: a database of reactions, pathways and biological processes stitch: interaction networks of chemicals and proteins stitch : zooming in on protein-chemical interactions therapeutic target database update : a resource for facilitating target-oriented drug discovery evaluation of usage patterns and user perception of the drug-drug interaction database sfinx the sider database of drugs and side effects decost: a new approach in drug repurposing from control system theory unveiling the role of network and systems biology in drug discovery clinical pharmacokinetics of cyclophosphamide old-school chemotherapy in immunotherapeutic combination in cancer, a low-cost drug repurposed pathologic complete response with neoadjuvant doxorubicin and cyclophosphamide followed by paclitaxel with trastuzumab and pertuzumab in patients with her -positive early stage breast cancer: a single center experience docetaxel and cyclophosphamide as neoadjuvant chemotherapy in her -negative primary breast cancer clinical studies on triethylenephosphoramide and diethylenephosphoramide, compounds with nitrogen-mustard-like activity thiotepa in treatment of advanced breast cancer vinblastine, adriamycin, thiotepa, and halotestin (vath): therapy for advanced breast cancer refractory to prior chemotherapy a critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin anthracyclines: selected new developments, current medicinal chemistry the evolution of drug discovery: from traditional medicines to modern drugs the complete drug reference liposomal doxorubicin (caelyx, myocet) clinical applications for topical -fluorouracil in the treatment of dermatological disorders five years clinical experience with -fluorouracil curt, -fluorouracil and methotrexate administered simultaneously as a continuous infusion. a phase i study continuous -fluorouracil in the treatment of breast cancer fluorouracil-based combinations in the treatment of metastatic breast cancer addition of -fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines the pharmacology and clinical use of methotrexate an evaluation of folic acid antagonists in adults with neoplastic diseases: a study of patients with incurable neoplasms systemic chemotherapy for advanced breast cancer combination chemotherapy as an adjuvant treatment in operable breast cancer years' follow up of randomised studies of adjuvant cmf in operable breast cancer: cohort study the management of rheumatoid arthritis in adults identification of the cytosolic carboxylesterase catalyzing the ′-deoxy- -fluorocytidine formation from capecitabine in human liver design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates -fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue a unified definition of clinical anthracycline resistance breast cancer a multicentre dose-escalating study of cabazitaxel (xrp ) in combination with capecitabine in patients with metastatic breast cancer progressing after anthracycline and taxane treatment: a phase i/ii study phase ii trial of weekly intravenous vinorelbine in first-line advanced breast cancer chemotherapy randomized phase iii trial of ixabepilone plus capecitabine versus capecitabine in patients with metastatic breast cancer previously treated with an anthracycline and a taxane pharmgkb summary: gemcitabine pathway cellular pharmacology of gemcitabine preclinical characteristics of gemcitabine gemcitabine, a broadspectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion new first-line therapy for pancreatic cancer activity of gemcitabine in non-small-cell lung cancer: results of the japan gemcitabine group (a) phase ii study single-agent gemcitabine is active in previously treated metastatic breast cancer computer-assisted drug design randomised phase ii trial of gemcitabine plus vinorelbine vs gemcitabine plus cisplatin vs gemcitabine plus capecitabine in patients with pretreated metastatic breast cancer palbociclib and letrozole in advanced breast cancer palbociclib plus letrozole as first-line therapy in estrogen receptorpositive/human epidermal growth factor receptor -negative advanced breast cancer with extended follow-up palbociclib in combination with fulvestrant in patients with hormone receptorpositive, human epidermal growth factor receptor -negative advanced breast cancer: paloma- subgroup analysis of japanese patients overall survival with palbociclib and fulvestrant in advanced breast cancer fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, her -negative metastatic breast cancer that progressed on previous endocrine therapy (paloma- ): final analysis of the multicentre, double-blind, phase randomised controlled trial toremifene for breast cancer: a review of years of data raloxifene hydrochloride for breast cancer risk reduction in postmenopausal women continuing outcomes relevant to evista: breast cancer incidence in postmenopausal osteoporotic women in a randomized trial of raloxifene aromatase inhibitors in premenopausal women with breast cancer: the state of the art and future prospects the history and mechanism of action of fulvestrant overall survival with fulvestrant plus anastrozole in metastatic breast cancer a review of fulvestrant in breast cancer goserelin: a review of its use in the treatment of early breast cancer in premenopausal and perimenopausal women neoadjuvant strategies for triple s. aggarwal, et al. seminars in cancer biology xxx (xxxx) xxx-xxx negative breast cancer:' state-of-the-art' and future perspectives everolimus in the treatment of metastatic breast cancer everolimus in postmenopausal hormone-receptor-positive advanced breast cancer in vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles taxanes for breast cancer: an evidence-based review of randomized phase ii and phase iii trials the role of taxanes in the treatment of breast cancer docetaxel and paclitaxel in the treatment of breast cancer: a review of clinical experience role of chance observations in chemotherapy: vinca rosea mitomycin c and vinblastine in the treatment of advanced breast cancer first-line and salvage therapy of metastatic breast cancer with mitomycin/vinblastine mitomycin c, vinblastine and cisplatin (mvp): an active and well-tolerated salvage regimen for advanced breast cancer induction of n-ras degradation by flunarizine-mediated autophagy effective breast cancer combination therapy targeting bach and mitochondrial metabolism redo_db: the repurposing drugs in oncology database nitric oxide inhibitors hit target for triple-negative breast cancer pim is responsible for il- -induced breast cancer cell emt and stemness via c-myc activation asparagine bioavailability governs metastasis in a model of breast cancer anticancer properties of fenofibrate: a repurposing use pathway-based drug repositioning for breast cancer molecular subtypes rethinking cancer: current challenges and opportunities in cancer research characteristics of exceptional or super responders to cancer drugs more cuts loom for us science thalidomide: a review of approved and investigational uses renovation as innovation: is repurposing the future of drug discovery research? parp inhibitors: synthetic lethality in the clinic european lung cancer working, valproate-doxorubicin: promising therapy for progressing mesothelioma. a phase ii study can the pharmaceutical industry reduce attrition rates? drug repurposing in oncology: compounds, pathways, phenotypes and computational approaches for colorectal cancer this work was supported in part by funds from the science and engineering research board (ecr/ / ), and university grants commission [ - / (bsr)] to scg. ssv was supported by a fellowship from dbt new delhi (dbt/ /bhu/ ). key: cord- -gsnjlhjd authors: dhanani, jayesh; fraser, john f.; chan, hak-kim; rello, jordi; cohen, jeremy; roberts, jason a. title: fundamentals of aerosol therapy in critical care date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: gsnjlhjd drug dosing in critically ill patients is challenging due to the altered drug pharmacokinetics–pharmacodynamics associated with systemic therapies. for many drug therapies, there is potential to use the respiratory system as an alternative route for drug delivery. aerosol drug delivery can provide many advantages over conventional therapy. given that respiratory diseases are the commonest causes of critical illness, use of aerosol therapy to provide high local drug concentrations with minimal systemic side effects makes this route an attractive option. to date, limited evidence has restricted its wider application. the efficacy of aerosol drug therapy depends on drug-related factors (particle size, molecular weight), device factors, patient-related factors (airway anatomy, inhalation patterns) and mechanical ventilation-related factors (humidification, airway). this review identifies the relevant factors which require attention for optimization of aerosol drug delivery that can achieve better drug concentrations at the target sites and potentially improve clinical outcomes. the main goal of aerosolization is to achieve high drug concentrations in lung tissue. aerosol therapy has been used as part of the treatment for a variety of respiratory diseases [ ] . indeed, there is also significant interest in the utilization of the respiratory system as a portal for systemic therapy [ ] of conditions that are not purely respiratory in nature. factors such as a large surface area, thin air-blood barrier and vascular epithelium coupled with low first-pass metabolism and enzymatic activity could achieve high bioavailability for aerosolized drug therapy [ ] . the possibility of achieving very high local drug concentrations at the therapeutic site for respiratory pathology, rapid onset of action and lower systemic side effects [ ] has thus led to a renewed interest in the field of aerosolized drug therapy in intensive care. datura administration in india, tobacco in ancient south america and smoking pipes from north american indians are some of the early uses of airways as a route for systemic drug delivery [ , ] . vaporized opium was used as a treatment for cough. anticholinergic properties of inhaled herbal preparations were used to treat asthma and inhaled epinephrine was first used around [ ] . aerosolized therapy is used for many therapies now including bronchodilators and corticosteroids, with a particular interest in antibiotic administration reemerging recently. although there are references to the use of inhaled penicillin as early as [ ] , the first randomized controlled trial of inhaled antibiotics was first reported in cystic fibrosis (cf) patients in . in critical care, endotracheal antibiotic administration was first reported in the s [ ] , when klastersky et al. reported that endotracheal polymyxins were effective for prevention of ventilator-associated pneumonia in tracheostomized patients [ ] [ ] [ ] . following these and other studies it was noted that there were adverse effects such as bronchospasm and poor tolerance [ ] as well as concerns regarding emergence of drug resistance associated with prolonged (> weeks) endotracheal administration and pharyngeal aerosolization [ ] . this led to a reduction in the use of inhaled antibiotics. even so, some investigators continued to prescribe intratracheal antibiotics in the critically ill patient, often successfully, especially in drug-resistant pneumonias [ , ] . antibiotic instillation practices were used in some early studies, but this practice was largely abandoned in the s. subsequent use of bench models enabled an improved understanding of the aerosolization factors such as optimal ventilator parameters, device position in the circuit and effects of humidity to enable optimal therapy [ ] [ ] [ ] . this work was then supplemented with antibiotic studies in experimental pneumonia that demonstrated higher lung tissue concentrations of antibiotics [ ] . the later development of 'new generation' devices such as the ultrasonic nebulizer and the vibrating mesh nebulizer (vmn) encouraged further study and application of aerosol therapy in critical care because of the ability of these devices to consistently generate desired aerosol particle sizes which are considered optimal for deep lung penetration [ , , ] . previously, the formulation of drugs used for aerosolization was the reconstituted form of compounds developed for parenteral administration. these were poorly tolerated by patients due to hyperosmolarity and added preservatives (i.e. phenols), which induced bronchial irritation and bronchospasm, leading to abandonment of this route of therapy. these formulation issues were particularly problematic for antibiotics until the s, when aerosolized tobramycin was evaluated in patients with cf chronically infected with increasingly resistant pseudomonas aeruginosa [ , ] . a number of highquality studies using preservative-free and iso-osmolar formulations of tobramycin showed improvements in lung function, a decreased exacerbation rate and reductions in sputum bacterial load [ ] [ ] [ ] . these results have encouraged further developments in the application of aerosolized antibiotics in non-cf patient populations such as critical care. in the critically ill patient, certain anatomicophysiological changes can significantly affect the pharmacokinetics (pk)-pharmacodynamics (pd) characteristics, thus causing dosing difficulties [ ] . mechanically ventilated patients pose a challenge for the effective delivery of aerosolized drugs [ ] . these various factors need to be considered and optimized to achieve desired therapeutic outcomes with aerosolized drug therapy [ ] . the research interest in aerosol drug therapy in critically ill patients is not yet reflected in the bench-to-bedside transfer of knowledge. one report mentions that up to % of intensivists are routinely prescribing aerosol medications [ ] . this report also highlighted the lack of application of scientific principles during therapy and indicated the need for education and research in the bench-tobedside transfer of knowledge [ ] . in another study, every fourth critically ill patient and every fifth ventilated patient received aerosol therapy [ ] . a recent international survey performed in europe, asia, australasia and north america showed that although % of icus practice antibiotic nebulization, very few actually follow the recommendations [ ] . given the commonness of use of aerosolization in critical care, yet the uncertainty over the optimal approach for administration, this article aims to discuss the essential concepts related to aerosolized drug therapy in critical care. an aerosol is defined as a suspension of liquid or solid in a gaseous medium [ ] . for successful aerosolization, consideration of the aerosol system is required. the aerosol system includes the drug, the aerosol device, the disease (i.e. the target site) and the patient's respiratory system, with the ventilator being an additional factor in mechanically ventilated patients. the aim of the aerosol system is to produce aerosols with characteristics suitable for drug delivery to the lungs. drug deposition, absorption, metabolism and elimination are essential determinants of the pharmacokinetic profile resulting from the aerosol system. key expressions used to evaluate the aerosol system performance include [ ] the following: emitted dose (ed)-the amount of drug exiting the delivery device. fine particle fraction (fpf)-the mass of particles below a cut-off diameter [ ] . the overall efficiency of the aerosol system is a composite of the ed, the dose delivered to the lung (fpf as a surrogate marker) and lung bioavailability. the ed and fpf are normally determined in vitro and are governed by particulate properties and device design. the bioavailability of the drug is influenced by patient factors such as airway and lung anatomy, drug permeability across membranes, metabolism of the drug and phagocytic clearance in the lung [ ] as well as fpf. the efficacy of the aerosolized drug depends on the dose deposited at the target site of action as well as its distribution in the lungs [ ] . deposition in the airways can occur by inertial impaction, gravitational sedimentation or diffusion (brownian motion) (fig. ) . because of the turbulence and high air velocity associated with aerosolization, an inertial impaction method is predominant in the first branchings of the airway [ ] . this proximal region is the target for aerosol therapy for diseases such as copd, asthma and ventilatorassociated tracheobronchitis. in the distal five to six airway generations, however, sedimentation predominates due to lower air velocity [ ] . at the alveolar level, minimal air velocity means no effect of impaction will occur and a combination of sedimentation and diffusion will influence drug deposition [ ] . most aerosolized particles for therapeutic purposes are in the range of - μm and diffusion is the predominant mechanism for lung deposition. the optimal technique for aerosolization is important to achieve distal airway and alveolar deposition. factors affecting aerosolized drug delivery in the critically ill patient drug concentrations in lung tissue are affected by the aerosolized dose administered, patient factors, device factors and the formulation of the drug. mechanical ventilation (mv) introduces additional elements such as the circuit and the ventilator and associated factors. for the purposes of describing the factors affecting aerosol therapy, critically ill patients could be classified into two groups: ventilated patients and non-ventilated patients [ ] [ ] [ ] [ ] . figure shows the factors conducive for effective aerosol drug delivery in the critically ill mechanically ventilated and non-mechanically ventilated patient groups. airflow and tidal volume influence the effect of airway anatomy on aerosol deposition. patients suffering from airway obstruction such as asthma or copd have impaired mucociliary clearances and mucous retention [ ] . for drugs with poor trans-mucous permeability (e.g. aerosolized aminoglycosides) this could mean reduced drug delivery and hence impaired efficacy, although this is yet to be confirmed in clinical studies [ ] . chronic inflammation may result in airway remodelling, which changes the dynamics of airflow [ , ] , and impaired mucociliary clearance, thus reducing the pulmonary drug deposition [ , ] . these changes lead to a proximal shift in the airway deposition pattern of the aerosols [ ] . significance-abnormal airways and impaired mucociliary clearance serve as a barrier to effective aerosolized drug therapy when the target site is the lung parenchyma. the airflow is not homogeneous throughout the lungs even in health. the result in an upright patient is that the apical portions of the lungs receive lung deposition of the order of a : higher ratio compared with the basal regions [ ] . this difference is significantly reduced in the supine position [ ] . moreover, most lung diseases are regional which adds to the heterogeneity to regional airflow, an important determinant of aerosol deposition [ ] . for example, it has been shown that there is lower deposition in areas of poor air flow (i.e. atelectatic lungs) [ ] . in the area of antibiotics, there is a large body of work with experimental pneumonia models which have demonstrated that lung tissue concentrations of nebulized amikacin, using a ultrasonic nebulizer, was significantly higher than the concentrations resulting from administration via the intravenous route [ , ] . indeed, even though deposition of nebulized drug decreased with more severe pneumonia, it still resulted in higher lung tissue concentration than that achieved from intravenous administration. figure illustrates this phenomenon. the same group also demonstrated that nebulized amikacin resulted in greater bactericidal activity leading to greater sterility rates compared with the intravenous route [ ] . when compared with continuous intravenous [ , , , , , , , , , , , , ] . niv non-invasive ventilation, hme heat and moisture exchanger, pmdi pressurized metered dose inhaler, aad adaptive aerosol device, vmn vibrating mesh nebulizer, dpi dry powder inhaler, peep positive end-expiratory pressure fig. effects of regional lung aeration and pneumonia on drug concentration in lungs. a relationship of lung aeration (%) to pulmonary concentration of amikacin (μg/g) for different routes of administration. b relationship of route of drug administration to pulmonary concentration of amikacin (μg/g) for different severities of pneumonia. pulmonary concentrations derived from homogenized lung tissue specimens measured by an immunoenzymatic method. figure derived from elman et al. [ ] infusion of ceftazidime, frequent nebulization achieved higher lung tissue concentrations with better bactericidal effects in an experimental model of pseudomonas pneumonia [ ] . significance-differences in regional lung aeration may explain some of the variability in therapeutic outcomes amongst different lung diseases. in a critically ill, spontaneously breathing patient, air flow is likely to be turbulent leading to impaction in the proximal airway. for drugs dependent on lung deposition for their effect, this results in a decreased pharmacological effect. in contrast, laminar flow patterns are considered to enable optimal lung deposition [ ] . in the critically ill patient, certain mv settings (e.g. square wave airflow pattern) enable generation of laminar airflow to improve drug deposition in the lungs. on the other hand, lower flows may reduce the ed when dry powder inhalers (dpis) are used [ ] . using pressurized metered dose inhalers (pmdis) with valved holding chambers (vhcs) or spacers could mitigate this effect. significance-whilst a laminar flow pattern would be beneficial for aerosolized drug delivery, mechanistic data need to be confirmed using clinical trials. diseases such as pneumonia and other inflammatory lung diseases result in deficiencies of lung surfactant both in content and/or effect [ , ] . drugs with high solubility will probably have a uniform dispersion compared with insoluble drugs. inferentially the soluble drugs are likely to have longer and more effective lung residence times, thus improving drug potency [ ] . surfactant deficiency is associated with atelectasis, which in turn impairs drug deposition [ ] . studies on surfactant replacement therapy in acute lung injury and ards, however, have failed to demonstrate benefit and may even be deemed harmful [ , ] . aerosolized surfactant therapy has been studied as a mucokinetic agent in specific conditions [ ] . its application for this purpose in critically ill patients needs further study. significance-uncertain benefits requiring further studies to demonstrate effects of surfactant. a major fraction of the aerosolized drug is entrapped in the mucous in the conducting airways. factors such as particle size, solubility, lipophilicity and charge govern the ability of the drug to penetrate this mucous barrier. for example, steroids and antimicrobial agents are seen to have reduced trans-mucous transport [ , ] . atelectasis is a common occurrence in the majority of critically ill patients. this may have adverse effects on drug deposition and may result in heterogeneous distribution in the lung [ ] . significance-both, mucous and atelectasis serve as a barrier to effective aerosolized drug therapy. a detailed discussion on the effect of device-related factors has been reviewed elsewhere [ , ] . appropriate particle sizes are important to enable adequate concentrations at the target site. particle size also determines the mechanism of deposition in the respiratory system [ ] . particles that distribute deep in the smaller airways (< μm) are reported to have up to % deposition efficiency [ , ] . smaller particles ( - μm) are considered to have the optimal droplet size for efficient deposition in the alveolar airspaces, for systemic delivery [ ] . in this regard, the efficiency of the aerosol device can be defined to be the ability to generate the aerosol in the desired particle size range. pmdis with spacers or vhcs have demonstrated superior deposition efficacy over nebulizers in various studies [ ] [ ] [ ] , although the vhcs cannot be used for mechanical ventilators due to their inability to trigger/ activate the device. dpis have no propellant, are inherently breath-synchronized/activated and produce little variation in particle size. these features may make dpis the preferred delivery device. in critically ill patients, however, poor respiratory reserve and diminished patient efforts are barriers to achieving the desired respiratory pattern for effective use of dpis. the dpis also vary widely in their efficacy [ ] and their use in mechanically ventilated patients is not typically possible with a standard set up [ ] . thus, dpis are presently used in stable and unventilated patient groups. both pmdis and dpis are limited by the formulations available to be delivered by these devices. nebulizers are different devices that are used to transform liquid formulations and suspensions into an aerosol form. these devices can be used to deliver larger volumes of a drug as an aerosol either intermittently or continuously, for prophylaxis or treatment purposes. depending on their mechanism of operation, there are three types of nebulizers: jet, ultrasonic and smns. jet nebulizers are the cheapest and simplest, albeit being inefficient in drug delivery [ ] . their drawbacks are noise, poor dosing control and the requirement for changes in the ventilator settings such as airflow and tidal volume; although improvements have been made in the form of reservoirs and new baffles that ensure more optimal particle sizes. breath-enhanced versions of the jet nebulizers could increase fpf, improve drug delivery and reduce drug loss. there are limited studies evaluating the efficiency of these newer jet nebulizers and data are certainly lacking in critical care settings [ ] . newer ventilators have in-built nebulization systems which improve the efficiency by synchronizing nebulization with the respiratory cycle. ultrasonic nebulizers are infrequently used and also have limitations [ , ] . they are expensive, large in size, increase concentration of the drug during nebulization and can cause thermal inactivation of the nebulized drug. mesh nebulizers are the result of improvement in nebulizer technologies. although more efficient and with significant advantages, there is a dearth of human studies using mesh nebulizers. despite major improvements in the technology there is a need to reduce the cost of these devices. table compares and contrasts the principles, advantages and disadvantages of different nebulizers. significance-where possible, pmdis with spacers should be used. dpi use is likely to be limited in critical care. for nebulizers, the device should be selected according to the formulation used and the desired site of deposition and effect. the rate and extent of absorption of the aerosolized substances is dependent on the molecular weight, ph, electrical charge, solubility and stability. macromolecules < kda are observed to be better absorbed (in minutes) in the bloodstream following inhalation in the airways (e.g. insulin, molecular weight (mw) . kda) [ ] . however, macromolecules > kda are absorbed slowly over hours (e.g. albumin, mw kda) [ ] . molecules with mw > kda may need an absorption enhancer for absorption in the alveoli [ ] . significance-depending on the desired site of action, appropriate drug formulations should be used alongside delivery devices that would generate a suitable particle size. a combination of helium and oxygen (heliox) reduces gas density and increases aerosol deposition, particularly in the peripheral lung [ ] . with pmdis, heliox has been reported to increase aerosolized drug delivery during mv [ ] . however, with jet nebulizers heliox also increases the nebulization time, requiring higher gas flows to compensate for the low-density gas [ ] . in an experimental study, there was no increase in lung deposition of nebulized ceftazidime in bronchopneumonic lungs compared with healthy lungs [ ] . significance-further investigations and large-scale trials are needed to evaluate the effect of heliox in critical illness. because of a variety of proposed mechanisms, patient sputum is thought to cause aminoglycoside inactivation resulting in 'sputum antagonism' [ ] . significance-uncertain, therefore the effect of sputum antagonism requires further in-vivo investigation. current data from cf patients support use of inhaled aminoglycosides [ , ] . aerosol therapy is routinely used in mechanically ventilated patients, both invasive and non-invasive, and is inherently challenging due to the interplay of a variety of factors [ ] . however, not all nebulization techniques are comparable. the patient position, formulation, temperature, endotracheal tube size, presence of airway obstruction or ventilatory asynchrony, flow pattern, respiratory rate, dose and frequency applied or position of the nebulizer in the circuit are important factors that influence delivery to the lung. the higher the turbulence, the lower the drug deposition in the distal airways. optimal settings of nebulization are not tolerated by many patients (such as those with severe hypoxemia, associated with ards or pneumonia) and require the addition of deep sedation and relaxation, which prolongs the duration of mv. disposition in unilateral pneumonia might be imbalanced. currently, nebulizers and pmdis, with and without spacers, are two types of devices available for use in mechanically ventilated patients. depending on the site of action, devices producing an appropriate particle size should be used [ ] . nebulizers take a considerably longer time to deliver a standard dose as compared with other devices. there is also a variation in efficiency between nebulizer types and between nebulizers in different batches [ ] . this effect is accentuated when coupled with the effects of different ventilator modes and lung mechanics [ ] . inadequate cleaning and disinfection of the nebulizer increases the risk of nosocomial pneumonia [ ] . compared with jet nebulizers, vmns could increase the drug delivery by - -fold [ ] , although as discussed previously the nebulizer choice is dependent on the formulation and the desired delivery site. pmdis are easy to administer, require less staff time, provide reliable dosing and have minimal risk of bacterial contamination when compared with nebulizers. when used with a collapsible spacer in the circuit, the circuit does not need to be disconnected [ ] . pmdis are also more cost-effective than nebulizers [ ] . although only bronchodilators and anti-inflammatory agents are available for this device, it is seen that using pmdis significantly reduces overall costs of care and could be equally effective in the treatment of inflammatory airways disease such as asthma and copd [ , [ ] [ ] [ ] [ ] [ ] . in-vitro studies have shown improved aerosol delivery with large spacers compared with that with small spacers for pmdis and vmns [ ] . published recommendations for the correct methods of their use are available [ ] . others have shown modest improvement in the aerosol delivery [ ] . significance-pmdis are possibly more effective than nebulizers. vmns appear superior to other nebulizer types although the choice should be dependent on the drug formulation properties and the desired deposition site. at this time, there is insufficient evidence to support the use of either delivery method over the other [ ] . the use of spacers with pmdis needs further clinical trial to test the efficacy. in-vitro studies [ ] using adult ventilators have shown that, when using vibrating mesh and ultrasonic nebulizers as well as the pmdi, a position cm from the y-piece in the inspiratory limb of the circuit yields the highest drug delivery. in a constant flow pattern of ventilation, the vmn connected to the endotracheal tube could be as effective [ ] . however, jet nebulizers seem to perform better when positioned closer to the ventilator, possibly due to the effect of the continuous gas flow 'charging' the circuit, which functions as an aerosol reservoir [ ] . for non-invasive ventilation (niv), using the vmns position after the exhalation port is more efficient for drug delivery compared with that before the exhalation port [ ] . significance-the best position for the aerosol generator may be cm from the y-piece in the inspiratory limb. invivo studies are required to make definitive conclusions. although the endotracheal tubes and tracheostomy tubes present certain similarities, the tracheostomy tube is shorter and more curved than an endotracheal tube. in patients who are not mechanically ventilated, a tpiece interface between the tracheostomy tube and the nebulizer has been demonstrated to be more effective than a tracheostomy mask [ , ] .preferably, the inner cannula should be removed before nebulization particularly for the smaller sized tubes [ ] because smaller diameter airways lead to an increase in the resistance to airflow, resulting in increased drug deposition in the artificial airways and tracheobronchial region [ , ] . significance-for aerosolized drug delivery, larger size artificial airways are better. in mechanically ventilated patients, a temperature of - °c (average °c) and relative humidity of - % are required to prevent heat loss [ ] . humidification also prevents drying of secretions, mucous plugging and consequently atelectasis. there are two major methods of humidification-active and passive. active methods include a heated humidifier (hh) and passive methods include a heat and moisture exchanger (hme). humidification is thought to have a significant effect on aerosol drug delivery. because of the hygroscopic effects of humidification, there may be a - -fold growth in particle size as they pass through airways. this increase in size may reduce peripheral lung drug deposition and hence pharmacological efficacy [ ] . compared with humidified conditions, drug delivery can be doubled in non-humidified conditions [ ] . it is recommended that hh should be ceased for the duration of therapy. of interest, in an in-vitro non-mechanically ventilated model, using the excipient enhanced growth (eeg) of sub-micrometre particles, one group has demonstrated increased aerosol deposition in the airways and lungs [ , ] . further investigations of this method are required to harness its effect in mv. the hme is a physical barrier and should not be placed between the delivery device and the patient. the particulate air filter in the expiratory limb, used to protect the ventilator and the flow meter, could get saturated resulting in airflow obstruction. it is recommended that the filter should be changed after every nebulization treatment [ , , ] . significance-using hme or a particulate air filter with nebulization could result in air flow obstruction. awareness and routine changing of air filters after each nebulization should be performed. ventilator breath characteristics have an important effect on the efficacy of aerosol delivery. slower inspiratory flows, long inspiratory times [ ] and tidal volumes > ml (using a pmdi) [ ] correlate well with improved aerosol delivery. higher bias flow is seen to reduce the delivery efficacy of nebulizers [ ] . decelerating flow pattern is considered inferior to constant flow pattern for drug delivery [ ] . the effect of ventilation mode is negligible for pmdis [ ] . the delivery efficiency in patients on niv is seen to be comparatively less [ ] . however, it must be remembered that specific techniques of ventilation may in themselves produce a greater benefit than the relative detriment of drug delivery (e.g. in niv and asthma). hence, in acute asthma, niv plus nebulization is more effective than nebulization alone [ ] . a prescribed ventilatory pattern may not be practical in the critically ill patient. the most effective combination of tidal volume, flow and other ventilator parameters for aerosol delivery can be calibrated to the drug and delivery device using in-vitro models [ ] . significance-tidal volumes > ml may enhance aerosolized drug delivery. niv results in effective therapy despite reduced drug delivery in conditions like asthma. ventilator settings optimal for nebulization, however, could lead to patient-ventilator dyssynchrony in severely hypoxemic patients (e.g. due to severe pneumonia)-thus requiring deep sedation, which may increase the duration of mv. positive end-expiratory pressure (peep) is a commonly used ventilator setting as part of the lung protective ventilatory strategy in severe lung diseases [ ] . peep has significant effects on regional ventilation and perfusion [ ] and hence could influence the pk of an aerosolized drug. in an animal model using radiotracers, peep was found to enhance aerosol clearance. this could be due to the stretching of the alveolar epithelium and enhancing the distribution of aerosol into the bloodstream [ ] . significance-peep is potentially beneficial, although further data are needed to quantify the effect on aerosolized drug delivery. the choice of one antimicrobial against another should consider efficacy data, costs, local antimicrobial resistance patterns and drug availability. aminoglycosides require tissue concentrations > -fold higher than the mic to be maximally effective. because airway inflammation could increase systemic absorption and the molecular weight is low, serum aminoglycoside concentrations should be monitored to avoid systemic toxicity. beta-lactams are rapidly cleared from airways, requiring frequent administration. colistin is administered in its anionic (methanesulfonated) form-colistimethate. despite high doses (up to million units of colistimethate every hours ( mg of colistimethate, equivalent to mg of colistin base)) as administered in colonized patients with bronchiectasis, lung epithelial lining fluid concentrations are not above mg/l after hours (upper threshold of eucast mic breakpoint for pseudomonas) or even above mg/l after hours in many patients (eucast mic breakpoint for klebsiella sp. and acinetobacter baumannii). therefore, high doses ( million units every hours) should be considered in pneumonia. despite delivery of drugs via the inhaled route, significant extrapulmonary drug losses may mean that the actual amount of drug delivered might be less than % of the ed into the trachea and even less will reach the alveolar space [ ] . this factor should be taken into account when calculating dosing regimens. a number of animal studies have been useful to better understand the mechanistic principles of aerosol therapy. guillon et al. [ ] showed effective teicoplanin nebulization during mv with good pk properties compared with the intravenous route. others successfully nebulized ceftazidime to achieve high local concentrations [ , ] . further studies are required to quantify the exact dosing amount and schedule using pk studies. doses should be different in patients with colonization, tracheobronchitis or pneumonia. increasing doses (e.g. million units of colistin) require longer periods of nebulization (~ hour) which is not well tolerated by patients suffering from ards. significance-the inhaled drug dose is likely to be significantly higher than expected due to concerns about drug losses. further pk-pd studies are required to guide inhaled drug dosing. most of the drug losses occur in the exhalation phase of ventilation. to minimize this loss, the actuation of the inhaler or nebulizer could be matched with inspiration [ ] . however, the use of the spacer-pmdi combination negates the effect of lack of breath synchronization [ ] . the effect of breath synchronization on aerosol deposition is unproven. using radiolabelled aerosols, dubus et al. [ ] showed that there is no significant increase in aerosol deposition in neonatal ventilation with breath synchronization. further investigations are thus needed to evaluate the effects of breath synchronization on aerosol deposition. in any event, devices which introduce synchronization of drug delivery facilitate tolerance. significance-breath actuation of the drug delivery devices has the potential to improve drug delivery. however, trial-based data are required to establish efficacy in aerosolized drug deposition. high-flow nasal oxygen therapy is becoming a widely prevalent therapy in intensive care [ ] . a number of factors influence the nebulization therapy in patients using high flow, which was studied recently in an invitro model [ ] : . position of the nebulizer-a position distant from the humidifier (closer to the patient) improved delivery of the drug upstream. . nebulizer type-vmns demonstrated improved delivery as compared with jet nebulizers, although the nebulizer choice is dependent on the formulation and desired site of action. . airflow-the delivery of respirable mass is lower with higher airflow and improves at a lower airflow. . patient efforts-converse to the effect of airflow with a high-flow oxygen system, in situations mimicking respiratory distress (i.e. increased patient inspiratory airflow) the delivery was in fact better. an open mouth, on the contrary, had no significant difference to closed mouth with respect to drug delivery. significance-limited data suggest better drug delivery using vmns at a lower airflow even in patients with respiratory distress. further in-vivo studies need to be performed using high-flow oxygen therapy devices. contemporary applications of aerosol therapy in critical care: focus on antibiotics table summarizes the common applications of aerosol therapy in critical care. aerosolized bronchodilators and corticosteroids have been effectively utilized in critical care. aerosolized antibiotics are quickly gaining more data to support their position in the critical care armamentarium. with improvements in drug formulation and delivery devices, more is now known about the optimal conditions required for effective aerosolized therapy as summarized in fig. . despite these developments there are concerns that best evidence for administration is not being applied, particularly for aerosolized antibiotic therapy [ , ] . clinical and experimental study data for aminoglycosides and colistin are perhaps most numerous for antibiotics in critical care [ ] . aminoglycosides are concentrationdependent antibiotics whereby the bactericidal effect is best described by the c max /mic ratio. studies have shown that intravenous aminoglycosides penetrate poorly into the epithelial lining fluid [ , ] . in an escherichia coli inoculation pneumonia model, aerosolized amikacin was seen to achieve significant lung concentrations [ ] . figure is an illustration of this phenomenon. on the other hand, with repeated administration, there was no accumulation effect and hence no toxicity concerns with aerosolized amikacin [ ] . in experimental studies, the serum concentration of amikacin was higher when aerosolized amikacin was used in a pneumonia model [ ] compared with that of healthy lungs [ ] . moreover, a combination of intravenous and aerosolized aminoglycosides has not been shown to increase cure rates compared with that of aerosolized antibiotic alone. thus, for the treatment of ventilator-associated pneumonia, aerosol therapy alone may be adequate without the need for intravenous therapy, decreasing the risk of systemic toxicity [ ] . colistin, also a concentration-dependent antibiotic, is another antibiotic used widely in aerosolized form. colistin aerosolization is not approved by the fda and is not licensed for human use in china. like aminoglycosides, colistin has poor lung penetration when given intravenously. experimental studies have shown that a rapid and high bactericidal effect can be achieved with aerosolized colistin [ ] . figure illustrates this phenomenon. as demonstrated by lu et al. [ ] , with low serum concentrations resulting from aerosolized colistin in an inoculation pneumonia model, the risk of toxicity is minimal. in a prospective observational study, lu et al. [ ] demonstrated similar clinical cure for patients with vap where susceptible p. aeruginosa or a. baumannii were treated with only intravenous colistin and mdr strains were treated with nebulized colistin. combined intravenous aminoglycoside and aerosolized colistin has not been shown to be superior to aerosolized colistin alone although implemented worldwide. the benefit from the use of aerosolized colistin instead of systemic colistin is to avoid nephrotoxicity, and this was further confirmed in one randomized clinical trial [ ] . observational cohort studies report less adverse events than randomized clinical trials. indeed, there is potential to cause systemic toxicity (e.g. nephrotoxicity by aminoglycosides) or local toxicity in the form of airway irritation, cough and often bronchospasm [ ] , worsening hypoxemia (and secondary arrhythmias) as well as pulmonary injury when using aerosol therapies [ ] . ventilator malfunction and obstruction of expiratory filters have been reported and contraindicate the use of drugs with lipid components or lactose sugar in the formulation (such as zanamivir or lipid-based amphotericin formulations), and careful monitoring of the potential increase of airway pressure and oxygen saturation is required to anticipate severe adverse events [ ] . modification of ventilator parameters for appropriate jet nebulizer use (table ) is not tolerated by some patients, increasing the work of breathing and ventilator dyssynchrony (requiring additional sedation). poor tolerance may preclude the use of aerosolized antibiotics in patients with pao /fio < mmhg or high peep requirements. initial concerns regarding drug resistance as a result of intratracheal or nebulized use of antibiotics (polymyxin b) have been investigated and do not appear to be supported, with aerosolized antibiotics using newer devices no more likely than intravenous therapy to confer bacterial resistance [ ] . this was probably linked to previousgeneration nebulizers and the technique of administration (instillation, pharyngeal aerosolization, etc.). however, this finding must be interpreted reservedly because no longterm follow-up has been performed. tolerance of aerosolization is different when drugs are nebulized for different durations of time. as a consequence, this might limit use of aerosolization in patients with ards or severe hypoxemia, such as severe pneumonia (in contrast to ventilator-associated tracheobronchitis), who often have poor tolerance. when high doses of colistin are nebulized, the infusion volume may represent an hour of nebulization and many patients require added sedatives or relaxation (with potential increased risk of myopathy or hypotension). this requirement would be associated with a prolonged mv period and extra length of stay [ ] . further clinical trials should therefore use pre-defined outcome parameters (rather than surrogates), control by hypoxemia and careful recording of adverse events. environmental contaminations resulting from aerosolization of drugs in an open circuit system pose a small but significant risk to the caregivers. using expiratory filters with valves in the aerosol delivery devices could minimize this. this occupational risk exposure should be assessed and interventions to mitigate the risks should be implemented [ ] . when using aerosolized antibiotics, it is recommended to change the filter after every therapy. optimizing the aforementioned factors could lead to effective drug delivery. however, it is important to realize that aerosolization of medications does not automatically lead to beneficial drug effects and may in fact be harmful, as shown in some studies [ , ] . aerosol therapy provides effective drug delivery in the critically ill patient. careful consideration of the various elements that affect pharmacological effect of aerosolized therapies is essential to derive optimal therapeutic benefit. effective drug delivery alone does not ensure successful aerosol drug therapy. it is crucial that the drug in its aerosolized form should have efficacy in the specific disease condition to derive clinical benefit. good quality data and clinical experience support use of bronchodilators such as salbutamol, anti-infectives such as tobramycin, aztreonam and colistin, and antiinflammatory agents such as budesonide. although with application of principles it is possible to provide aerosol drug delivery, the effectiveness of the therapy in disease conditions is yet to be proven. this is because there is a scarcity of high-quality trial-based data in this area to quantify how effective these agents are in the critically ill patient. given the challenges of effective treatment of the critically ill patient, it is necessary to optimize as many factors as possible for effective drug delivery. hence, it is important that guidelines for aerosol therapy are developed. it is envisaged that as the technologies become mature through rigorous evaluation, a diverse range of aerosol therapies with unique advantages (i.e. controlled release/sustained release or direct targeting) and or for specific indications may be possible. table optimization of ventilator parameters required for aerosolization of antibiotics modified from lu et al. [ ] • nebulizer placement-in the inspiratory limb cm proximal to y-piece • expired aerosolized particles collected in a filter hme heat and moisture exchanger, rr respiratory rate, i:e inspiratory: expiratory ratio, vt tidal volume fig. comparison of lung concentration (measured by hplc) and bacterial burden of colistin between aerosolized and intravenous administration. samples taken hour after the third aerosol in the aerosol group and the fourth infusion in the intravenous group and hours after the bacterial inoculation. diagram derived from data of lu et al. [ ] respiratory medicine efficacy of inhaled human insulin in type diabetes mellitus: a randomised proof-of-concept study respiratory pharmacology and toxicology pharmacokinetics of inhaled drugs a history of nebulization history of aerosol therapy: liquid nebulization to mdis to dpis treatment of asthmatic attacks by inhalation of adrenaline endotracheal gentamicin in bronchial infections in patients with tracheostomy endotracheal antibiotics for the prevention of tracheobronchial infections in tracheotomized unconscious patients. a comparative study of gentamicin and aminosidin-polymyxin b combination aerosol polymyxin and pneumonia in seriously ill patients prevention of gram-negative bacillary pneumonia using polymyxin aerosol as prophylaxis. ii. effect on the incidence of pneumonia in seriously ill patients a review of the therapeutic efficacy of aerosolized and endotracheally instilled antibiotics endotracheal aminoglycosides in gram negative pneumonia. a preliminary report endotracheal tobramycin in gram-negative pneumonitis aerosol therapy in mechanically ventilated patients: recent advances and new techniques in vitro evaluation of aerosol bronchodilator delivery during mechanical ventilation: pressure-control vs. volume control ventilation aerosol delivery and modern mechanical ventilation: in vitro/in vivo evaluation aerosolized antibiotics for ventilator-associated pneumonia: lessons from experimental studies influence of nebulizer type, position, and bias flow on aerosol drug delivery in simulated pediatric and adult lung models during mechanical ventilation american college of chest p, american college of asthma a, et al. device selection and outcomes of aerosol therapy: evidence-based guidelines efficacy of aerosolized tobramycin in patients with cystic fibrosis intermittent administration of inhaled tobramycin in patients with cystic fibrosis. cystic fibrosis inhaled tobramycin study group efficacy and safety of aerosolized tobramycin in cystic fibrosis augmented renal clearance in the icu: results of a multicenter observational study of renal function in critically ill patients with normal plasma creatinine concentrations inhalation therapy in invasive and noninvasive mechanical ventilation aerosol therapy during mechanical ventilation: an international survey aerosol therapy in intensive and intermediate care units: prospective observation of critically ill patients intratracheal administration of antimicrobial agents in mechanically ventilated adults: an international survey on delivery practices and safety introduction in aerosol science: technology and applications what the pulmonary specialist should know about the new inhalation therapies particle size analysis in pharmaceutics: principles, methods and applications particle and device engineering for inhalation drug delivery deposition of inhaled particles in the lungs clinical aerosols. i. characterization of aerosols and their diagnostic uses pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications the science of nebulised drug delivery inhaled antibiotics to treat lung infection bronchodilator delivery with metered-dose inhalers in mechanically-ventilated patients the role of tracheobronchial mucus in drug administration to the airways effect of mucus on transepithelial drug delivery deposition, retention, and clearance of inhaled particles therapeutic aerosols and airway secretions theoretic analysis of sites of aerosol deposition in the human lung regional deposition of aerosolized pentamidine. effects of body position and breathing pattern inhaled antibiotics: dry or wet? lack of lung tissue and systemic accumulation after consecutive daily aerosols of amikacin in ventilated piglets with healthy lungs influence of lung aeration on pulmonary concentrations of nebulized and intravenous amikacin in ventilated piglets with severe bronchopneumonia lung deposition and efficiency of nebulized amikacin during escherichia coli pneumonia in ventilated piglets mechanical ventilation-induced air-space enlargement during experimental pneumonia in piglets nebulized ceftazidime in experimental pneumonia caused by partially resistant pseudomonas aeruginosa mode of breathing-tidal or slow and deep-through the i-neb adaptive aerosol delivery (aad) system affects lung deposition of ( m)tc-dtpa is inhalation rate important for a dry powder inhaler? using the in-check dial to identify these rates pulmonary inflammation disrupts surfactant function during pneumocystis carinii pneumonia changes in pulmonary surfactant during bacterial pneumonia pulmonary drug metabolism, clearance, and absorption exogenous pulmonary surfactant for acute respiratory distress syndrome in adults: a systematic review and meta-analysis exogenous natural surfactant for treatment of acute lung injury and the acute respiratory distress syndrome effects of aerosolized surfactant in patients with stable chronic bronchitis: a prospective randomized controlled trial aerosol therapy for children air and soul: the science and application of aerosol therapy deposition of inhaled particles in the human respiratory tract and consequences for regional targeting in respiratory drug delivery the lungs as a portal of entry for systemic drug delivery nebulizers versus pressurized metered-dose inhalers in preschool children with wheezing metered-dose inhaler with spacer instead of nebulizer during the outbreak of severe acute respiratory syndrome in singapore combining treatment with pressurized metered dose inhalervalved holding chamber (pmdi+vhc) with dosimetric therapy via a breath actuated nebulizer (ban) in patient titration for obstructive lung diseases (thoracic) are outcomes the same with all dry powder inhalers? use of dry powder inhalers in acute exacerbations of asthma and copd medication nebulizer performance. effects of diluent volume, nebulizer flow, and nebulizer brand randomized controlled trial of a breath-activated nebulizer in patients with exacerbation of copd guidelines for aerosol devices in infants, children and adults: which to choose, why and how to achieve effective aerosol therapy time to peak insulin level, relative bioavailability, and effect of site of deposition of nebulized insulin in patients with noninsulin-dependent diabetes mellitus drug delivery via the respiratory tract inhalable drugs for systemic therapy deposition in asthmatics of particles inhaled in air or in helium-oxygen improvement in aerosol delivery with helium-oxygen mixtures during mechanical ventilation the effect of heliox on nebulizer function using a beta-agonist bronchodilator intravenous versus nebulized ceftazidime in ventilated piglets with and without experimental bronchopneumonia: comparative effects of helium and nitrogen aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum inhaled tobramycin effectively reduces fev decline in cystic fibrosis. an instrumental variables analysis long-term safety and efficacy of tobramycin in the management of cystic fibrosis aerosol deposition in mechanically ventilated patients. optimizing nebulizer delivery aerosol delivery during mechanical ventilation: from basic techniques to new devices contaminated medication nebulizers in mechanical ventilator circuits. source of bacterial aerosols the distribution of costs of care in mechanically ventilated patients with chronic obstructive pulmonary disease inhaled bronchodilator administration during mechanical ventilation: how to optimize it, and for which clinical benefit? effect of albuterol on expiratory resistance in mechanically ventilated patients inhaled bronchodilator administration during mechanical ventilation inhalation therapy with metered-dose inhalers and dry powder inhalers in mechanically ventilated patients in vitro performance of spacers for aerosol delivery during adult mechanical ventilation effect of a spacer on pulmonary aerosol deposition from a jet nebuliser during mechanical ventilation metered dose inhalers versus nebulizers for aerosol bronchodilator delivery for adult patients receiving mechanical ventilation in critical care units evaluation of aerosol generator devices at locations in humidified and non-humidified circuits during adult mechanical ventilation influence of inspiratory flow pattern and nebulizer position on aerosol delivery with a vibrating-mesh nebulizer during invasive mechanical ventilation: an in vitro analysis in vitro comparison of five nebulizers during noninvasive ventilation: analysis of inhaled and lost doses an in vitro evaluation of aerosol delivery through tracheostomy and endotracheal tubes using different interfaces albuterol delivery via tracheostomy tube aerosol delivery through tracheostomy tubes: an in vitro study aerosol delivery to ventilated newborn infants: historical challenges and new directions airway resistance and deposition of particles in the lung humidification during invasive and noninvasive mechanical ventilation: model of the deposition of aerosol particles in the respiratory tract of the rat. ii. hygroscopic particle deposition the use of condensational growth methods for efficient drug delivery to the lungs during noninvasive ventilation high flow therapy targeting aerosol deposition to and within the lung airways using excipient enhanced growth influence of inspiratory flow rate, particle size, and airway caliber on aerosolized drug delivery to the lung aerosol delivery from a metered-dose inhaler during mechanical ventilation. an in vitro model the mask for noninvasive ventilation: principles of design and effects on aerosol delivery reversal of bronchial obstruction with bi-level positive airway pressure and nebulization in patients with acute asthma the role of aerosolized antimicrobials in the treatment of ventilator-associated pneumonia national heart l, blood institute actn. higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome effect of positive end-expiratory pressure on regional ventilation distribution during mechanical ventilation after surfactant depletion pulmonary clearance of radiotracers after positive end-expiratory pressure or acute lung injury nebulized and intravenous colistin in experimental pneumonia caused by pseudomonas aeruginosa aerosol route to administer teicoplanin in mechanical ventilation: in vitro study, lung deposition and pharmacokinetic analyses in pigs comparison of lung tissue concentrations of nebulized ceftazidime in ventilated piglets: ultrasonic versus vibrating plate nebulizers aerosol deposition in neonatal ventilation high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure aerosol therapy in adults receiving high flow nasal cannula oxygen therapy global survey on nebulization of antimicrobial agents in mechanically ventilated patients: a call for international guidelines aerosol therapy during mechanical ventilation: an international survey penetration of gentamicin into the alveolar lining fluid of critically ill patients with ventilator-associated pneumonia nebulized antibiotics study g. efficacy of high-dose nebulized colistin in ventilatorassociated pneumonia caused by multidrug-resistant pseudomonas aeruginosa and acinetobacter baumannii efficacy and toxicity of aerosolised colistin in ventilator-associated pneumonia: a prospective, randomised trial high incidence of bronchospasm with regular administration of aerosolized pentamidine pulmonary toxicity of inhaled aerosolized prostacyclin therapy-an observational study nebulized antibiotics study group. nebulized ceftazidime and amikacin in ventilator-associated pneumonia caused by pseudomonas aeruginosa reduction of bacterial resistance with inhaled antibiotics in the intensive care unit occupational risk prevention in aerosol therapy inhaled dornase alfa (pulmozyme) as a noninvasive treatment of atelectasis in mechanically ventilated patients randomized, placebo-controlled clinical trial of an aerosolized beta( )-agonist for treatment of acute lung injury albuterol delivery by different nebulizers placed in different positions in a pediatric ventilator in vitro model williams rd ro. current therapies and technological advances in aqueous aerosol drug delivery combined inhaled salbutamol and mannitol therapy for mucus hyper-secretion in pulmonary diseases administration of aerosolized antibiotics in cystic fibrosis patients update on new pulmonary therapies tolerability and pharmacokinetics of orally inhaled zanamivir: a randomized study comparing rotacap/rotahaler and rotadisk/diskhaler in healthy adults aerosolized liposomal amphotericin b: prediction of lung deposition, in vitro uptake and cytotoxicity pharmacokinetic considerations in critically ill patients guidelines for the diagnosis and treatment of pulmonary hypertension international right heart failure foundation scientific working group. right heart failure: toward a common language pulmonary arterial hypertension: new insights into the optimal role of current and emerging prostacyclin therapies predictors of outcome in patients with ards treated with inhaled epoprostenol hypotension in response to iloprost, a prostacyclin analogue clinical review: airway hygiene in the intensive care unit inhalational therapies for the icu adverse effects of acetylcysteine on human and guinea pig bronchial asthma in vivo and on human fibroblasts and leukocytes in vitro safety of administering dry powder mannitol to stimulate sputum clearance in intubated intensive care patients with sputum retention: a pilot study (thoracic) ventilator-induced lung injury: historical perspectives and clinical implications effects of surfactant depletion on regional pulmonary metabolic activity during mechanical ventilation consensus on surfactant and inhaled nitric oxide for ards effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial resolution of severe ischemia-reperfusion injury post-lung transplantation after administration of endobronchial surfactant authors' contributions jd conceptualized the article, collected data, drafted, revised and submitted the manuscript. jff participated in the initial design and edited the manuscript. h-kc helped in the final editing of the manuscript. jr helped in the final editing of the manuscript. jc helped with design of figures and tables and in the final editing of the manuscript. jar participated in the design, drafting, editing and submission of the manuscript. all authors read and approved the final manuscript.competing interests jr has received funding for consulting from bayer. none of the other authors have any competing interests, of a financial or non-financial nature.author details key: cord- -kl uvrbw authors: bordet, régis title: is the drug a scientific, social or political object? date: - - journal: therapie doi: . /j.therap. . . sha: doc_id: cord_uid: kl uvrbw nan randomized controlled trials are based on two principles: comparison and comparability. these two principles allow dispensing first with the natural evolution of the disease, particularly in infectious diseases, and second the placebo effect, which can now be corroborated by brain imaging studies. without respect for these two principles, it is impossible to establish the role of the drug in the clinical effects observed when testing a new pharmacological approach. paradoxically, the methodological rigour inherent to controlled trials and the role of the pharmaceutical industry in promoting these trials have ended up bringing discredit to this approach, which is, however, the best method for establishing the proven effect of a drug, in part of the population. the craze that has arisen for the distribution of hydroxychloroquine, based on a very preliminary study and the analysis of a cohort without a comparator group, was, from this point of view, very emblematic of the fact that belief can quickly replace scientific demonstration. this is not new and already exists for alternative therapeutic methods that refuse to fall under the caudine forks (so named in reference to the battle at the end of which the romans had to submit to the samnites) of clinical trial methodology. this attitude has, however, taken an emotional and claiming side in the context of the anxiety and fear generated by the covid- pandemic and its lethal risk. empiricism seems to be trying to take again a top position over the rational approach, which some consider too dogmatic, especially in critical situations. is it necessary, because it is urgent, to abandon all the scientific principles of clinical trials that have previously led to undeniable therapeutic progress? undoubtedly not, which does not mean that the experimental design of the trials should not be adapted, in an approach reminiscent of charles peirce and william james theorising pragmatism as a third way transcending the empiricism/rationalism opposition. for more than thirty years, french medical pharmacologists and therapists have been contributing to an international movement of methodological diversification through a think tank (called "ateliers de giens") bringing together the academic, industrial and institutional worlds. pragmatic trials, adaptive trials using the bayesian approach, studies with external comparators, trials on small samples, taking into account secondary assessment criteria and the use of biomarkers are all methodological innovations that aim to make the framework of controlled trials more flexible in order to speed up or improve the evaluation of drugs, without abandoning the major and basic principle of comparison [ , ] . the media outburst against methodology rightly clashes with the convictions of the vast majority of health professionals, who have perhaps not sufficiently integrated the fact that drugs, which they consider above all as a scientific object that is their prerogative, have also become an issue that the social body has taken up. it is above all through the drug risk and its often-mediatized affairs that society has integrated drugs as a social fact [ ] . the so-called "mediator" affair has had a major impact on populations, in this case the french one, with the realization that drugs can be also dangerous, even in the long term. the front pages of newspapers and magazines devoted to medicines and their risks have been booming, like the questions addressed to the regional pharmacovigilance centres. the development of social networks has had an amplifying effect on the emergence of medicines as a social issue, by bringing it out of the circle of health professionals, previously considered as the only "knowers". some years ago, several episodes of reporting adverse drug reactions linked to changes in thyroid hormone formulation have emerged as a viral relay on social networks. social demand may lead regulatory agencies to re-evaluate positions that were initially scientifically based, or to speed up market entry. recently, the film " beats per minute" recalled the interactions, sometimes violent, between patient associations, pharmaceutical industry and regulatory agencies in order to bypass the usual marketing rules, in view of the mortality caused by hiv. this is reminiscent of what we are experiencing today with molecules reputed to be potentially effective against sars-cov- , recalling what we have previously seen with other products, such as baclofen in alcohol use disorders. while society may request answers from public authorities and healthcare professionals regarding the risks/benefits balance of drugs, social facts may also influence the medical use or diversion of medicines, or even investment in their development. the cult of thinness, advocated year-round in all magazines, was undoubtedly the main factor that triggered the misuse of the mediator, replacing dexfenfluramine, which, a few years earlier, had been withdrawn from the market because of its valvular risk. our modern societies, based on the over-emphasis on performance, generate a diversion of drugs, particularly psychotropic ones, for the purpose of cognitive doping. however, anxiety induced by loss of social bearings or by suffering at work linked to the search for profitability at all costs explains the over-consumption of psychotropic tranquillizers or antidepressants. the social context may therefore explain the emergence of a pattern of use or an increase in the prescribing or consumption of a particular drug, which then escapes rational, evidence-based use, necessitating regulatory measures to control prescribing or dispensing. the current health crisis has highlighted the immediate link between the announcement of preliminary or hypothetical results on the efficacy of a particular drug against coronavirus and its dispensing, and often consumption, outside of any regulatory framework. taking into account the social dimension of the drug reveals, however, a contradiction in the demand of the social body, which has been amplified during the health crisis. on the one hand, there is an expectation for information that has been scientifically validated by health professionals. in the space of one month, the pharmacovid site [ ] set up on march was consulted nearly , times, with many questions asked by the current health crisis may be leading to a turning point, with drugs becoming a political football. it is first of all an object in the international political games, because it is clear that states have lost their sovereignty over the production of medicines, which, like other manufactured products, has become one of the goods of globalized capitalism. the current crisis, however, has made us aware that drugs are above all a common good, since they affect one of the essential elements for humanity, namely health. if tomorrow a vaccine or treatment against coronavirus is found, will we witness a war between states to control supply, as happened with masks? will the world balance between states be altered? will the ability to control drug policy in all its aspects (innovation, rapid assessment, production) become a diplomatic weapon or even a propaganda tool for external or internal propaganda? because drugs have also become a domestic political issue, as we have seen with chloroquine and hydroxychloroquine, with partisan confrontations or positions taken without scientific basis by a head of state announcing his belief in the interest of chloroquine, in a country that was the first to create a regulatory agency. the balance between political power and regulatory agencies may be shifting, as shown by the announcement by the president of the united states of america, in person, of the authorisation of an antiviral by the fda, developed by an american company, even though the results of clinical trials are still contradictory. it is the same with a nationalist attitude for the priority delivery of vaccine. will drug policy tomorrow become a subject of electoral debate, on a par with fiscal or educational policy, and an argument for economic nationalism? how can we reconcile this irruption in the public or political debate and the maintenance of a sufficient level of expertise to avoid falling into the populism we know in other fields, with its share of false or truncated information? the participants of giens xxxv round table clinical research. from single-arm studies to externally controlled studies. methodological considerations and guidelines quelle utilisation et quelle hiérarchisation pour les critères de jugement secondaires social pharmacology: a new topic in clinical pharmacology pharmacovid plateform: questions and answers for the public about drugs in the context of the covid- key: cord- - t ykc authors: altay, ozlem; mohammadi, elyas; lam, simon; turkez, hasan; boren, jan; nielsen, jens; uhlen, mathias; mardinoglu, adil title: current status of covid- therapies and drug repositioning applications date: - - journal: iscience doi: . /j.isci. . sha: doc_id: cord_uid: t ykc summary the rapid and global spread of a new human coronavirus (sars-cov- ) has produced an immediate urgency to discover promising targets for treatment of covid- . drug repositioning is an attractive approach that can facilitate the drug discovery process by repurposing existing pharmaceuticals to treat illnesses other than their primary indications. here, we review current information concerning the global health issue of covid- including promising approved drugs and ongoing clinical trials for prospective treatment options. in addition, we describe computational approaches to be used in drug repurposing and highlight examples of in-silico studies of drug development efforts against sars-cov- . over the past few centuries, outbreaks caused by bacteria like yersinia pestis and vibrio cholerae (the causative agents of plague and cholera, respectively(cohn and kutalek, ; pechous et al., ) and also by viral infectious agents such as influenza viruses, ebolaviruses, sars-cov- , mers-cov, the zika virus, and lately, sars-cov- , have undermined public trust in the ability for modern science to predict and prevent global pandemic threats. accordingly, studying historical epidemics can help us identify patterns of viral outbreaks and design a plan to prepare for next pandemic. provide a better picture of statistics related to asymptomatic infections for epidemiologic analysis. the phylogeny, virology, and epidemiology of sars-cov- is being studied extensively. at the genome level, sars-cov- has · % homology to sars cov- cov- and other coronaviruses, and its relative ease of sample acquisition and study, it has been widely accepted that drug repositioning is a promising approach to make available an effective, safety-assured treatment in a timely manner. in this review, we summarize diagnosis approaches, risk groups, available treatment options, and drug repositioning studies related to covid- . prior to efficient treatment, precise diagnosis and classification of patients based on disease severity and their probable vulnerability to covid- is crucial. diagnosis has several steps that can be prioritized based on the provision of facilities during an outbreak. generally, evaluation of symptoms and particular laboratory features that are associated with worse cases come first. considering antihypertensive medications, there are advocates for both use and cessation of consumption. it is still unclear whether the high mortality rate of patients with hypertension comorbidity is due to the pathology of disorder or the treatment used to cure it such as angiotensin-converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs). ace has been shown to be a co-receptor for viral entry and pathogenesis of sars-cov. (li et al., ) there is considerable evidence which shows the escalated expression level of ace in the heart, brain, and even in urine after treatment with arbs; however, there is limited evidence showing changes in serum or pulmonary ace levels. (patel and verma, ; zhang et al., c) in addition, serological, radiological, and histo-morphological similarities of covid- -associated acute respiratory distress syndrome (ards) and connective tissue disease (ctd-ild) propose the postulation of triggering or simulating a form of organ-specific autoimmunity in predisposed patients. also, some patients showed high titer antiphospholipid antibodies, including anticardiolipin antibodies and anti-β glycoprotein antibodies. (zhang et al., d) the immunosuppressive therapy in identified patients with autoantibodies may prevent the development of respiratory failure. with regard to treatment, immunological and pharmaceutical investigations are still ongoing. no specific therapies for covid- is approved by u.s. food and drug administration (fda) so far but many previously approved drugs, as an efficient approach to drug discovery named drug repurposing, is being tested on covid- . at the time of writing, interventional studies have been registered in clinicaltrials.gov (https://clinicaltrials.gov/) related to covid- and this number is increasing progressively (table s ). in particular, the ongoing clinical trials sponsored by who named solidarity will compare different treatment options, namely remdesivir; lopinavir/ritonavir dual treatment; lopinavir/ritonavir dual treatment with interferon beta -alpha; and chloroquine or hydroxychloroquine (paused temporarily due to the concerns raised about the safety of the drug) against standard of care. as of may th, , more than countries have been confirmed to contribute in this investigation. the interventional drugs in clinical trials can be classified based on their nature and complementary effect. in this regard, antivirals, antiparasitic drugs, immunosuppressors, immunomodulators, some well- known drugs, and nutritional drugs, in addition to combination therapies are considered on ongoing studies for treatment, supportive care or prevention. one can barely see the same mechanism of action inside and outside each group (table ) but many drugs are discovered for a specific disease and repurposed later for another disorder. figure illustrates a general path which is being traversed by clinicians these days although, the results may not be desirable in some cases due to the emergent nature of sars-cov- . in-silico drug repurposing methodologies have accelerated the studies in drug discovery through the use of data mining approaches, bioinformatics techniques, and predictive models for determining the efficacy and safety of the drugs. however, there is still a long way to reach the high success rates with repurposed drugs (e.g. repurposed candidates succeeded - % -depending on the similarity between indicated and repurposed therapeutic areas -and the novel molecular entity success rate is ~ % and reduce the possibility of hypothesis risk will be essential to increase the success rate of repurposed drugs in the product development process. a computation-based methodology, namely viral-track, applied to bronchoalveolar lavage samples from severe and mild covid- patients unveils a remarkable impact of the virus on the immune system of severe patients compared to mild cases, including replacement of the tissue resident alveolar macrophages with recruited inflammatory monocytes, neutrophils, and macrophages and an altered cd + t cell cytotoxic response.(bost et al., ) further efforts on large-scale datasets will lead to reveal many mechanistic aspects of viral infections yet to be resolved and will help to develop more efficient therapy strategies. due to urgent need, we posit that drug repurposing is the leading method for drug discovery against covid- , whether through wet lab techniques and/or system biology approaches. available clinical trials at both clinicaltrials.gov (https://clinicaltrials.gov/) and who solidarity includes the investigation of previously approved drugs for different indications. taking into account that in the past two decades three coronaviruses emerged from animal reservoirs (the virus family which used to cause mild to moderate upper-respiratory tract illnesses) and each one of them was cause of global concern, a common treatment may prevent coronavirus to become another lethal annual re-occurrent threat on top of seasonal influenza. data for this review were identified by searches of pubmed, google scholar, and references from relevant articles using the search terms "sars-cov- ", "covid- ", "drug repurposing", "antiviral therapy", "clinical trials", "covid- therapy", and "pandemi". only articles published in english were included. this work was supported by knut and alice wallenberg foundation. the funders had no role in study design, data collection, data analysis, interpretation or writing of the report. mortalities. in some outbreaks, the exact duration is not precisely determined (nd) because the outbreak died out and recurred multiple times in the same region and era. aggravating factors, laboratory-based tests, and candidate treatments. reinfection could not occur in sars-cov- infected rhesus macaques. biorxiv remdesivir for the treatment of covid- -preliminary report broad-spectrum agents for flaviviral infections: dengue, zika and beyond clinical features of patients infected with novel coronavirus in wuhan a second, non-canonical rna-dependent rna polymerase in sars coronavirus structure of mpro from covid- virus and discovery of its inhibitors potent human neutralizing antibodies elicited by sars-cov- infection. biorxiv host-directed therapies for bacterial and viral infections predicting new molecular targets for known drugs assessing activity and middle east respiratory syndrome coronavirus papain-like and c-like proteases using luciferase- based biosensors docking and scoring in virtual screening for drug discovery: methods and applications tocilizumab for the treatment of chimeric antigen receptor t cell-induced cytokine release syndrome a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury drug repurposing: a better approach for infectious disease drug discovery? angiotensin-converting enzyme is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses cancer patients in sars-cov- infection: a nationwide analysis in china the reproductive number of covid- is higher compared to sars coronavirus genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding ongoing clinical trials for the management of the covid- pandemic inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines renovation as innovation: is repurposing the future of drug discovery research? case-fatality rate and characteristics of patients dying in relation to covid- in italy drug repurposing from an academic perspective. drug discov today ther strateg covid- and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? jama pneumonic plague: the darker side of yersinia pestis drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy protocol for traumadornase: a prospective, randomized, multicentre, double-blinded, placebo-controlled clinical trial of aerosolized dornase alfa to reduce the incidence of moderate-to-severe hypoxaemia in ventilated trauma patients drug repurposing: progress, challenges and recommendations a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication transmission of -ncov infection from an asymptomatic contact in germany pharmacologic treatments for covid- ): a review use of genome-wide association studies for drug repositioning critically ill patients with covid- with convalescent plasma a review of the herald pandemic wave: importance for contemporary pandemic response strategies covid- drug therapy-potential options covid- : combining antiviral and anti-inflammatory treatments. the lancet infectious diseases detection of novel coronavirus by rt-pcr in stool specimen from asymptomatic child, china. emerging infectious diseases on the origin and continuing evolution of sars-cov- mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir systems biology based drug repositioning for development of cancer therapy discovery of therapeutic agents for prostate cancer using genome-scale metabolic modeling and drug repositioning remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( - ncov) in vitro unique epidemiological and clinical features of the emerging novel coronavirus pneumonia (covid ) implicate special control measures remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial bacille calmette-guérin (bcg) vaccination and covid- : scientific brief evolving status of the novel coronavirus infection: proposal of conventional serologic assays for disease diagnosis and infection monitoring identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen single-cell metagenomics: challenges and applications structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design a familial cluster of infection associated with the novel coronavirus indicating potential person-to-person transmission during the incubation period multiscale modelling of drug mechanism and safety crystal structure of sars-cov- main protease provides a basis for design of improved α- ketoamide inhibitors the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease (covid- ): the experience of clinical immunologists from china coagulopathy and antiphospholipid antibodies in patients with covid- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china sars-cov- viral load in upper respiratory specimens of infected patients jakafi ® , jakavi ® jak inhibition rheumatoid arthritis plague outbreaks era key: cord- -ua udlc authors: koch, oliver; sheehy, susanne; sargent, catherine; democratis, jane; abbas, sarah; schiefermueller, jurgen; angus, brian j. title: antiviral drugs date: - - journal: side effects of drugs annual doi: . /s - ( ) - sha: doc_id: cord_uid: ua udlc publisher summary this chapter discusses the adverse effects of antiviral drugs used against cytomegalovirus, herpesviruses, hepatitis viruses, against hiv, and against influenza viruses. the cidofovir, drug active against cytomegalovirus, has been associated with bronchiolitis obliterans. aciclovir and valaciclovir has been reported with renal insufficiency. adefovir , a drug active against hepatitis viruses, is associated with the fall in creatinine clearance in patients with lamivudine-resistant hbe antigen (hbeag)negative disease. drugs active against hiv are comprehensively reviewed as in combination, nucleoside analogue reverse transcriptase inhibitors, nucleoside analogue reverse transcriptase inhibitors, and protease inhibitors. in a randomized controlled trial of indinavir, saquinavir and lopinavir in combination with low-dose ritonavir in patients, median total cholesterol increased by . mmol/l in the patients with the highest minimum drug plasma concentrations. in patients with aids-associated aids dementia complex taking optimal stable background antiretroviral therapy including either abacavir or placebo, there was significantly more nausea in those who took abacavir. respiratory cidofovir has been used to treat adenovirus pneumonia in four pediatric lung transplant recipients ( c ). two developed bronchiolitis obliterans, one of whom underwent retransplantation with a good outcome. the other developed tracheomalacia and required continuous low-flow oxygen and positive pressure ventilation through a tracheostomy. urinary tract of six hemopoietic stem cell transplant recipients, who were given cidofovir mg/kg/week for weeks, then on alternate weeks for a minimum of four (range - ) doses, three developed adenovirus hepatitis, two had colitis and one had nephritis ( c ). all had cd þ selected grafts and/or graft-versus-host disease and all had a cd count under  /l. the patients were pre-hydrated and brian j. angus antiviral drugs and given probenecid and intravenous immunoglobulin - g/kg/week followed by . mg/kg on alternate weeks for a minimum of four doses. two patients died of infection and four responded. none required withdrawal of treatment because of adverse effects. baseline creatinine was slightly raised ( µmol/l) in the patient with hepatitis and improved with cidofovir. most of the patients had transient increases in creatinine as the number of doses increased. cidofovir mg/kg/week or mg/kg on alternate days adjusted to creatinine clear ance was used to treat hemopoietic stem cell transplant recipients with invasive ade noviral infection, including three with pneu monia, one with hepatitis, four with hemorrhagic colitis and three with hemor rhagic cystitis ( c ). all had concomitant intravenous immunoglobulin, and those with colitis had in addition one or more of oral immunoglobulin, ribavirin, or donor lymphocyte infusion. they varied in their degree of immunocompromise and the pre sence of graft-versus-host disease and some had leukocyte-depleted allografts. six died. there was nephrotoxicity in two cases. in a retrospective study of recipients of hemopoietic stem cell transplants, mean age years, who were given a mean of . (range - ) weekly doses of cidofovir mg/ kg without probenecid for bk virus-asso ciated hemorrhagic cystitis, % had graft versus-host disease and were taking corti costeroids at the time of diagnosis ( c ). there was no significant rise in creatinine in ; had renal insufficiency, but in all cases there were other potential causes. in only cases could cidofovir have played a role. in those with normal renal function, - % of the cidofovir was recovered from the urine, raising the possibility of therapeutic intravesicular administration. aciclovir psychiatric hemodialysis has been successfully used to treat an aciclovir induced psychosis ( a ). • a -year-old man with rectal carcinoma and end-stage renal failure on hemodialysis was given intravenous aciclovir. he developed delirium, visual and auditory hallucinations, disorientation in place and time, and impaired recent memory. he recovered fully after consecutive days of hemodialysis. urinary tract reports of renal insuffi ciency associated with the use of intravenous aciclovir continue to appear ( a ). in a retrospective review of children there was no effect of dose per kilogram, age, or sex on nephrotoxicity ( c ). the predictors of aciclovir nephrotoxicity were the concomitant use of nephrotoxic drugs and impaired glomerular filtration rate (gfr) at baseline. fetotoxicity it has been suggested that maternal use of aciclovir may be associated with necrotizing enterocolitis in the neonate ( a ). immunologic an acute allergic reaction to valaciclovir has been described ( a ). • a -year-old woman developed general malaise, diffuse pruritus, angioedema of the hands and feet, and reduced consciousness and faintness within minutes of taking valaciclovir mg for genital herpes. skin prick tests for aciclovir were positive. urinary tract as with aciclovir, renal insufficiency has been reported with valaciclovir ( a ) • a -year-old japanese man with cardio myopathy and chronic renal disease was admitted with anuria, systemic edema, and renal dysfunction after taking valaciclovir g tds days for herpes zoster infection with a non-steroidal anti-inflammatory drug. he was briefly dialysis-dependent but made a full recovery. adefovir (sed- , ; seda- , ; observational studies in patients with lamivudine-resistant hbe antigen (hbeag) negative disease, adefovir mg alone (n = ) or in combination with lamivudine (n = ) caused a fall in creatinine clearance requiring a modification in adefovir dose in two patients on combined therapy; both had underlying liver cirrhosis ( c ). one developed reduced liver function and biopsy showed steatohepatitis. one on adefovir monotherapy developed gastric cancer. five on combined therapy with underlying cirrhosis developed hepatocellular carcinoma, but this was not statistically significant. adefovir mg/day in combination with lamivudine mg/day has been used in patients with recurrent hepatitis b virus infec tion after liver transplant ( c ). all patients with chronic hepatitis b pre-transplant underwent a period of preoperative treat ment and peri-operative prophylaxis with lamivudine and hepatitis b immunoglobulin with or without adefovir if lamivudine-resis tant. no patient had adverse effects necessi tating drug withdrawal, but one required a dosage adjustment because of a rise in creati nine from to µmol/l. three had a recurrence of hepatocellular carcinoma, two of whom died and months after starting adefovir therapy and one of whom continued to be positive for hepatitis b virus dna. drug-drug interactions entacavir the interaction of entacavir mg/day with adefovir mg/day has been studied in a fixed-sequence crossover study in heal thy adults ( c ). the results suggested that combination therapy can be safely adminis tered without the need for dosage adjust ment of either drug. there was headache in nine subjects and dysmenorrhea in two when they took entecavir alone. see 'drugs active against influenza viruses: ion channel inhibitors', below. drug-drug interactions adefovir the interaction of entacavir mg/day with ade fovir mg/day has been studied in a fixedsequence crossover study in healthy adults ( c ). the results suggested that com bination therapy can be safely administered without the need for dosage adjustment of either drug. there was headache in nine subjects and dysmenorrhea in two when they took entecavir alone. lamivudine (sed- , , seda- , ; seda- , ) observational studies in treatmentnaive hbeag-positive children who took lamivudine and high-dose interferon alpha a combination therapy, flu-like symptoms and anorexia were the commonest adverse effects ( and %); weight loss, nausea, vomiting, arthralgia, and loss of hair were also noted ( c ) . in an open study of the pharmacokinetics of lamivudine in patients receiving peri toneal dialysis, eye redness (n = ) and diar rhea (n = ) were the commonest adverse events ( c ). comparative studies in a double-blind, phase iii, randomized, controlled trial of lamivudine mg/day (n = ) versus tel bivudine mg/day (n = ), creatine kinase activity was raised in patients receiv ing lamivudine ( . %) and telbivudine ( . %) and fell spontaneously during drug treatment to grade or lower in % of those taking lamivudine and % of those taking telbivudine ( c ). grade or rises in transaminases during treatment were more frequent with lamivudine than with telbivudine. observational studies high-dose ribavirin during an outbreak of severe acute respira tory syndrome in toronto was associated with a high rate of adverse events: anemia (odds ratio [or] = . ; % confidence interval [ci] = . , . ), hypomagnesemia (or = ; % ci = . , ), and bradycar dia (or = . ; % ci = . , . ) ( c ). the risks of anemia, hypomagnesemia, and bra dycardia attributable to ribavirin were %, %, and % respectively. the authors concluded that the use of high-dose ribavirin is appropriate only for the treatment of infectious diseases for which ribavirin has proven clinical efficacy, or in the context of a clinical trial. they further stated that ribavirin should not be used empirically for the treatment of viral syndromes of unknown origin. skin occasional rashes in areas of drug contact and conjunctival irritation occurred when aerosolized ribavirin was used for months in an infant with immunodeficiency ( a ). musculoskeletal in a double-blind, phase iii, randomized, controlled trial of lami vudine mg/day (n = ) versus telbivu dine mg/day (n = ), raised creatine kinase activity was more common in patients who took telbivudine and it fell sponta neously during drug treatment to grade or lower in % of those who took telbivu dine and % of those who took lamivu dine. myopathy (characterized by muscle pain, weakness, and moderately raised crea tine kinase activity before and during treat ment) was reported in one patient after telbivudine therapy for months. when telbivudine was withdrawn, the creatine kinase activity normalized within month and the symptoms resolved over - months ( c ). cardiovascular syncope occurred in a young man after he took tenofovir, emtricitabine and nevirapine for primary human immunodeficiency virus- (hiv- ) infection for weeks and resolved after withdrawal of the antiretroviral drugs ( a ). metabolism the hemochromatosis gene polymorphism hfe c> g and possibly mitochondrial haplogroup j gave relative protection against lipoatrophy during antiretroviral drug therapy in a trial in which patients were randomized to didanosine þ stavudine or zidovudine þ lamivudine, combined with efavirenz and/ or nelfinavir in aids clinical trials group (actg) sub-study a s ( c ). stavudine had a less favorable effect on lipid profile and caused more lipoatrophy than abacavir ( % versus . %) in a ran domized, open trial, stratified by viral load and cd cell count, in which adults with hiv infection were assigned to stavudine (n = ) or abacavir (n = ), both com bined with lamivudine and efavirenz ( c ). there were dose-related increases in total cholesterol, ldl cholesterol, and triglycerides in an open study in seronegative volun teers, with persistent increases weeks after withdrawal ( c ). in a randomized controlled trial of indina vir, saquinavir and lopinavir in combination with low-dose ritonavir in patients, med ian total cholesterol increased by . mmol/l in the patients with the highest minimum drug plasma concentrations ( a ). gastrointestinal in a retrospective obser vational study of highly active antiretroviral therapy (haart), of patients who took indinavir in combination with zidovudine and lamivudine developed nausea and were significantly more likely to stop taking the treatment than those who were taking zidovudine þ lamivudine þ tenofovir ( c ). in a long-term follow-up study of asymptomatic hiv-positive participants for weeks taking a combination of indinavir, lamivudine, and zidovudine, stopped treat ment because of adverse effects, of which nausea ( %), diarrhea ( %), and abdom inal pain ( %) were the most common ( c ). the most common adverse events in of patients in a prospective study of once-daily saquinavir þ ritonavir and two nucleoside reverse transcriptase inhibitors (nrtis) were abdominal discomfort, diar rhea, and vomiting ( c ). similar findings were seen by other investigators ( a ). liver in hiv/hepatitis c co-infected patients, failure to achieve a sustained viral response, nrti therapy, didanosine, and stavudine were significantly associated with worsening of hepatic fibrosis in ( %) ( c ). after multivariate analysis, didanosine (or = . ; % ci = . , . ) and failure to have a sustained viral response (or = . ; % ci = . , ) remained significantly associated with worsening of fibrosis. in a retrospective study of hiv-posi tive subjects ( % men), first-line therapy was efavirenz, lamivudine, and zidovudine; women of child-bearing potential were given nevirapine instead of efavirenz. an efavirenz-based regimen was used in and received a nevirapine-based regimen ( c ) . during the first year subjects took isoniazid prophylaxis and received antituberculosis therapy ( months of rifam picin, isoniazid, pyrazinamide, and etham butol followed by months of rifampicin and isoniazid). of a random sample of tested, % were hepatitis b surface anti gen (hbsag)-positive. there was grade or worse hepatotoxicity in subjects ( %) and had a first episode of grade or hepatotoxicity. antituberculosis therapy (adjusted hazard ratio [hr] = . ; % ci = . , ) and hbsag (adjusted hr = . ; % ci = . , . ) were strongly associated with hepatotoxicity. however, hepatotoxicity had little impact on symp toms, the need for hospitalization and the need for a change in antiretroviral drug regimen. the use of isoniazid preven tive therapy during antiretroviral drug therapy did not increase the risk of hepatotoxicity. three adults developed nodular regen eration of the liver while taking haart regimens containing atazanavir, fosampre navir, and indinavir ( a ). pancreas in the large eurosida study there was no association between an increased incidence of pancreatitis and cumulative exposure to antiretroviral drugs generally, or to didanosine and stavudine in particular ( c ). there were ( presumptive) pancreatic events in individuals during person-years ( . per person-years). the incidences among those with no, or less, and over years of exposure to antiretroviral drugs, including stavudine and didanosine, were . , . , and . per person-years, respectively. in multivariate analysis, higher baseline cd cell counts were associated with a reduced risk of pancreatitis. urinary tract of hiv-positive patients who started to take tenofovir, ( %) had reduced renal function ( c ). multivariate analysis showed a significant association between reduced renal function and concurrent use of amprenavir and di danosine, age over years and lower baseline weight. there was an association between concomitant use of tenofovir and amprenavir and reduced kidney function in patients (or = . ). acute interstitial nephritis was seen on renal biopsy in three hiv-positive patients taking amprenavir þ tenofovir; it resolved after drug withdrawal ( a ). in a randomized controlled trial of in dinavir, saquinavir, and lopinavir in combi nation with low-dose ritonavir in patients, there was no apparent dose-related association with renal adverse events ( c ). immunologic an allergic reaction with possible cross-reactivity to didanosine and tenofovir has been reported ( a ). • a -year-old woman tested positive for hiv- and was given a once-a-day regimen including tenofovir plus emtricitabine and efavirenz. after days she developed a diffuse rash with fever and glossitis. efavirenz was withdrawn but her conditions worsened over the next days, so both tenofovir and emtrici tabine were withdrawn, with dramatic resolu tion of symptoms within hours. after days she was given zidovudine plus didanosine and ritonavir-boosted fosamprenavir. after week she again developed a diffuse rash and the treatment was withdrawn. she restarted zidovudine and didanosine days later and within a few days the rash appeared again. under observation she was given zidovudine and atazanavir and after days lamivudine and low-dose ritonavir. she remained well, except for mild hyperbilirubinemia, during the next months. fetotoxicity there is equivocal evidence of a relation between in utero nrti exposure and mitochondrial dysfunction in hivnegative children born to hiv-infected women. in hiv-negative children, possible cases with unexplained signs of mitochondrial dysfunction according to the enquête perinatale française criteria were identified in a retrospective review ( c ). associations between possible mitochondrial dysfunction and both overall in utero nrti exposure and the trimester of first in utero nrti exposure were estimated by exact logistic regression. cases (n = ) were significantly more likely to be boys and to be born in earlier years than non-cases (n = ). there was no association between overall in utero nrti exposure and mitochondrial dysfunction. in unadjusted models there were higher odds of first in utero exposure in the third trimester to lamivudine (or = . versus unexposed; % ci = . , ) and to zidovudine þ lamivudine (or = . versus unexposed; % ci = . , ) among cases than noncases. when adjusted for year of birth, the odds of first exposure in the third trimester to lamivudine (or = ; % ci = . , ) and zidovudine þ lamivudine (or = . ; % ci = . , ) were significantly higher among cases than non-cases. incomplete data precluded control of possible confounding by maternal viral load and psychoactive drug use. reports of renal toxicity with the combination of tenofovir and didanosine in children suggest that this combination should be avoided ( a ). combination of saquinavir with darunavir þ ritonavir is currently not recommended as plasma concentrations of darunavir are increased ( c ). buprenorphine atazanavir and atazanavir þ ritonavir both resulted in increased metabolite concentrations of buprenorphine, and dosage reduction of buprenorphine is recommended; there was no change in the concentrations of the protease inhibitors ( c ). warfarin in two patients who were taking a non-nucleoside reverse transcriptase inhibitor (nnrti), nevirapine, or a protease inhibitor, nelfinavir or lopinavir þ ritonavir, and two nucleoside analogues, high doses of warfarin were required to maintain therapeutic inrs ( a ). warfarin has two enantiomers, r-warfarin and s-warfarin, which are substrates of cyp a (r-warfarin), cyp a (r-warfarin), and cyp c (s-warfarin gastrointestinal in patients with aidsassociated dementia adc taking optimal stable background antiretroviral therapy including either abacavir or placebo there was significantly more nausea in those who took abacavir ( c ). immunologic three patients developed painful lymphadenopathy shortly after starting to take abacavir, mimicking immune reconstitution syndrome ( a ). they also had a fever and a rash and all were hla-b* -positive. they recovered after withdrawal of abacavir. abacavir hypersensitivity occurred in a -year-old man after he switched from a twice-daily to a once-daily regimen ( a ). in a -year-old woman, abacavir hyper sensitivity presented as acute fibrinous and organizing pneumonia, with dyspnea, hypoxia, and bilateral infiltrates ( a ). susceptibility factors genetic a sequence variation in the hiv reverse transcriptase codon has been associated with host hla-b* in hiv-infected, antiretroviral drug-naive adults, and the relation between the codon variation and premature abacavir withdrawal was investigated in treated individuals ( c ). only one of subjects with b* harbored virus with the clade b 'wild-type' amino acid v, compared with of who did not have b* . the sensitivity and specificity of codon substitutions for predicting hla-b* were % and % respectively, and the positive and negative predictive values were % and . % respectively. the authors argued that the reverse transcriptase codon could be adopted as a simple, low-cost screening method to identify individuals who could be safely treated with abacavir when detection of hla-b* is not rapidly and easily available. drug-drug interactions alcohol three patients had possible reactions to alcohol while taking abacavir ( a ). one had a disulfiram-like reaction (nausea, facial flushing, tachycardia) repeatedly on rechallenge with alcohol. another described a feeling of being drunk after small amounts of alcohol. a third had malaise after increasing his alcohol intake. the authors suggested that abacavir might inhibit alcohol dehydrogenase. urinary tract fanconi syndrome and nephrogenic diabetes insipidus associated with didanosine have been reported ( a ). • a -year-old man developed polydipsia, poly uria, fatigue, and weight loss after taking dida nosine, lamivudine and boosted atazanavir for years. he had hypophosphatemia, hypouricemia, hyperchloremic metabolic acidosis with a normal anion gap, normoglycemic glycosuria, and low-molecular-weight protei nuria. the plasma antidiuretic hormone (adh) concentration was high at . pg/ml (reference range . - . pmol/l). the didano sine was replaced with another protease inhi bitor and the other medications remained unchanged. he improved slowly. drug-drug interactions ganciclovir it has been suggested that ganciclovir and its prodrug valganciclovir inhibit purine nucleoside phosphorylase (pnp) in a similar manner to tenofovir and increase didanosine concentrations, reducing its efficacy ( a ). • a -year-old woman with hiv and cyto megalovirus enteritis was given valganciclovir, lamivudine, didanosine, and lopinavir þ ritona vir. after months, her viral load fell to less than copies/ml and the cd þ cell count was  /l. over the next months her viral load remained suppressed, but the cd þ cell count fell to  /l and she had symptoms of didanosine toxicity. didanosine was replaced with abacavir, leading to complete recovery of the cd þ cell count and resolu tion of symptoms. reduction of the dosage of didanosine or substitution with an alternative antiretro viral drug may be necessary when ganciclo vir is used. metabolism lipodystrophy tended to occur more often in hiv/hepatitis c co-infected patients who had severe weight loss than in other patients ( % versus %), and patients who had persistent weight loss over % for weeks after the completion of anti-hepatitis c virus (hcv) therapy were more likely to be taking a stavudine-based antiretroviral therapy ( c ). in another study patients with lipoatrophy had higher drug exposure to stavudine than controls ( c ). this was reflected in the higher geometric concentration ratios ( . and . respectively) and a higher percentage of ratios over . , representing a drug concen tration above the normal population curve ( % versus %). in addition, the duration of stavudine therapy was independently asso ciated with lipoatrophy. in a multivariate ana lysis, both duration of stavudine therapy and a concentration ratio over . independently correlated with lipoatrophy. changes in body habitus occur when sta vudine is withdrawn. in hiv-positive women who stopped taking stavudine for over . years, there were significantly smaller reductions in hip and thigh circum ferences compared with the reductions that occurred at - . years after stavudine withdrawal ( c ). stavudine reduces insulin sensitivity and causes mitochondrial toxicity in healthy sub jects. in participants without a personal or family history of diabetes who were randomized to stavudine - mg bd or placebo for month, insulin sensitivity was significantly reduced by stavudine ( c ). in addition, muscle biopsies in those who took stavudine showed significant reductions in mtdna/nuclear dna, but there were no changes in placebo-treated subjects. p mag netic resonance spectroscopy studies of mito chondrial function correlated with measures of insulin sensitivity. in patients in a retrospective study, symptomatic hyperlactatemia in ( %) was associated with stavudine median dura tion months ( c ). nine patients ( . %) died; those who died had a higher mean lactate concentration ( . versus . mmol/l) and mean alt activity ( versus u/l) at the time of diagnosis than those who sur vived. those who died had a lower mean weight than those who survived ( versus kg). by logistic regression, mortality was associated with patients whose body weight was under kg (or = . ; % ci = . , ) and whose serum lactate was over mmol/l (or = ; % ci = . , ). dosage regimens a meta-analysis of clinical trials conducted before and after regulatory approval of stavudine has shown that a dosage of mg bd has equivalent antiviral efficacy, with some evidence of lower rates of peripheral neuropathy and lipoatrophy, to the standard dosage of mg bd ( m ). it has been suggested that this is the most appropriate dose in resourcelimited settings ( m ). reducing the dosage of stavudine by one-half increased fat mtdna and bone density and decreased lactate concentrations in a study of patients already taking a standard dose ( c hematologic hemoglobin a can rise in hiv-infected patients, possibly because of therapy ( c ). in cross-sectional and cohort studies, hemoglobin a was often raised in untreated patients, but a further rise during treatment was specifically attributable to zidovudine. the concentration of hemoglobin a may be high enough to lead to a misdiagnosis of beta-thalassemia. genotoxicity micronucleated reticulocyte frequencies have been measured as a mar ker of chromosomal damage in hivinfected mother-infant pairs, of whom women had taken prenatal zidovudine and antiretroviral drugs without zidovudine ( c ). all the infants received zidovudine for weeks. venous blood was obtained from women at delivery and from infants at - days, - weeks, and - months of life; cord blood was collected immediately after delivery. ten cord blood samples (controls) were obtained from infants of hiv-negative women who did not receive antiretroviral therapy. there were -fold increases in micronucleated reticulocytes in women and infants who received zidovudine-containing antiretroviral therapy prenatally and no increases in the other women and infants. micronucleated reticulocytes in the zidovudine-exposed neonates fell over the first months of life to values comparable to cord blood controls. the authors concluded that transplacental zidovudine exposure is genotoxic in humans and they recommended long-term monitoring of zidovudine-exposed infants. teratogenicity a possible association between first trimester exposure to zidovudine and an increased risk of hypospadias based on one cohort study has been reported ( c ). among live births to women, defects were identified in babies ( . defects per live births). the rate of defects was . per live births with first-trimester antiretroviral drug exposure, . per live births with exposure later in pregnancy, and . of live births with no antiretroviral drug use. only genital abnormalities, specifically hypospadias, were significantly increased among babies born to women with firsttrimester exposure to antiretroviral drugs ( of male live births) compared with the two other groups ( of male live births). logistic regression suggested that use of zidovudine in the first trimester was associated with hypospadias (adjusted or = ; % ci = . , ). pregnancy in a pharmacokinetic study of three doses of zidovudine mg -hourly in pregnancy in six subjects, plasma zidovudine concentrations were substantially lower than previously reported during continuous intravenous therapy ( a ). in another study in four women who took an initial mg dose followed by two mg doses -hourly, the zidovudine auc and concentrations increased approximately in proportion to the increase in dose but varied - times ( a ). in both cohorts, the pharmacokinetic results suggested erratic absorption. combined exposure to zidovudine plus co-trimoxazole caused a clinically signifi cant suppression of humoral immune responses to influenza immunization in hiv-positive patients with cd counts above  /l ( c ). ribavirin ribavirin did not inhibit the formation of zidovudine triphosphate in peripheral blood monocytes in patients over weeks ( c urinary tract in a pilot, open, noncomparative add-on study, in which patients who had failed treatment with at least two thymidine-associated resistance mutations were given tenofovir mg/day for weeks in addition to their current failing antiretroviral regimen (n = ), one developed de fanconi syndrome at week and two developed grade one hypopho sphatemia ( c ). in a multicenter, observational, retro spective study of hiv-positive patients taking tenofovir, patients ( . %) stopped taking tenofovir because of adverse events, including nausea, vomiting, diarrhea, and headache. there was no association between any abnormal basal analytical parameter and a greater probability of stop ping treatment ( c ). in a single-center, cross-sectional evalua tion of b microglobinuria as a marker of proximal renal tubule damage in hivinfected children, tenofovir was significantly associated with very high abnormal values ( c in an evaluation of the effectiveness and adverse effects of a simplification regimen with tenofovir, lamivudine, and efavirenz in haart-experienced hiv- -infected subjects with sustained viral suppression, had psychiatric adverse effects related to efavirenz, leading to drug withdrawal in most cases; the symptoms included nightmares, insomnia, nervousness, and anxiety ( c ). metabolism actg study was a ran domized, placebo-controlled, double-blind study designed to compare three protease inhibitor-sparing antiretroviral drug regi mens (zidovudine þ lamivudine þ abacavir; zidovudine þ lamivudine þ efavirenz; zidovudine þ lamivudine þ abacavir þ efa virenz) in the initial treatment of hiv- infection in subjects ( c ). there were modest rises in serum triglycerides, ldl cholesterol, and hdl cholesterol in the two efavirenz-containing arms com pared with the triple-nucleoside arm. urinary tract a renal calculus that occurred in a -year-old man taking efavirenz contained efavirenz metabolites ( %) and about % of unspecified proteins ( a ). this was a between-the eyes adverse effect of type a, definitively implicating efavirenz ( h ). breasts in cohort study from cambodia, of patients developed gynecomastia while taking a regimen containing efavirenz ( c ). susceptibility factors genetic efavirenz is metabolized by cyp b . the pharmacokinetics of efavirenz have been studied in children with a g-to-t polymorphism at position of the cyp b gene, which affected its oral clearance ( c ). children with the t/t genotype had a slower oral clearance rate than those with the g/t genotype and the g/g genotype. the fastest clearance was found in children under years of age with the g/g genotype. the association between efavirenz-induced psychosis and a genetic polymorphism in cyp b has been reported in a child ( a ). • a -year-old hiv-positive girl taking lopinavir mg bd, ritonavir mg bd, stavudine mg bd, didanosine mg/day, and efavirenz mg/day developed an overt psychosis. her serum efavirenz concentration was - times higher than expected and she had a heterozygous gene polymorphism encoding for the cyp b isoenzyme, which has previously been associated with reduced clearance of efavirenz. the psychotic symptoms resolved gradually after withdrawal of efavirenz. antimalarial drugs two healthy volunteers who took amodiaquine plus artesunate and efavirenz had significant asymptomatic rises in liver transaminases, which did not occur in the absence of efavirenz ( c ). voriconazole in a randomized, placebocontrolled, two-period, multiple-dose within-group, fixed-dose sequence study of the interaction of voriconazole mg bd with efavirenz mg/day in healthy men, repeated doses of efavirenz substantially reduced the steady-state mean auc and c max of voriconazole by % and % respectively ( c ). repeated therapeutic doses of voriconazole moderately increased the steady-state mean auc and c max of efavirenz by % and % respectively. when voriconazole was co-administered with efavirenz, the incidence of adverse events was similar to that with efavirenz alone. liver hepatotoxicity is a major problem with nevirapine ( r ). the frequency of large increases in liver enzymes in patients taking efavirenz is - %, whereas in patients taking nevirapine it is - %. a warning about the increased risk of hepatotoxicity in antiretroviral-naive patients who start to take nevirapine-containing combination antiretroviral therapy has been issued based on cd cut-off values and sex. however, it is unclear whether this higher risk also applies to stable virolo gically suppressed patients. a meta-analysis of four published randomized studies in patients, including virologically suppressed patients who switched to nevirapine-con taining regimens with a follow-up of at least months, has shown that the risks of hepatotoxicity within the first months were % and % in those with low and high cd counts, respectively, with a com bined or of . ( % ci = . , ) ( m ). the risk of hepatotoxicity at any time dur ing the study was similar in the groups, with a combined hr of . ( % ci = . , . ). the authors concluded that virologically suppressed patients who switch to nevira pine do not have a significantly higher risk of hepatotoxicity or rash when stratified by sex and cd cell count. the aim of a retrospective study was to determine whether these recommendations are of use in preventing adverse effects ( c ). drug-naive patients (n = ) who started treatment with nevirapine were divided into two groups: those with high or low cd counts (n = and respec tively). there were rashes in patients in the high-cd group and in of those in the low-cd group and hepatotoxicity in and patients respectively. the authors concluded that the advice not to use nevir apine in drug-naive patients at increased risk of toxicity on the basis of sex and cd cell count does not seem to be useful in preventing adverse effects. in a retrospective study, patients ( % men) received nevirapine-based haart regimens ( c ). during % of treatments, there were grade or greater increases in transaminase activities, an overall incidence rate of . cases per person-years. this led to treatment with drawal in . % of cases. in a retrospective study of over pregnant women taking nevirapine-contain ing regimens, % started or continued nevirapine during the first and second trimesters ( ca ). concurrent chronic hepatobiliary disorders slightly increased the likelihood of hepatotoxicity. of seven patients who had liver dysfunction, six pre viously had had hepatitis c and gall blad der disease. skin widespread vitiligo after erythroderma has been attributed to nevirapine ( a ). • a -year-old man developed erythroderma, a high fever and hepatitis after taking nevirapine for month. there was jaundice and a confluent macular rash on the arms, legs, trunk, and face. histology showed a parakeratotic epidermis with focal spongiosis, necrotic keratinocytes and vacuolar degeneration of the basal layer, together with moderate edema and a perivascular mononuclear cell infiltrate, with some eosinophils in the upper dermis. the lesions faded on withdrawal of nevirapine and administration of oral glucocorticoids, but took around months to finally subside. • a -year-old girl took a nevirapine containing regimen for months and had recurrent episodes of fever, cough, sore throat, nausea, and vomiting - weeks apart. a chest x-ray showed mild bronchial wall thickening with left lower lobe atelectasis or a mild infiltrate. her temperature was °c and she had a tachycardia of /minute. she had a generalized maculopapular confluent blanching rash, but no target lesions or blisters, conjunctival injection, nasal congestion, a hyperemic posterior pharynx, and several bilateral non-tender cervical lymph nodes. the liver was enlarged by cm but the spleen was not palpable. the white blood cell count was .  /l, with a marked eosinophilia ( %). she was successfully treated with intravenous immunoglobulin and antiretroviral drug withdrawal. nevirapine-induced stevens-johnson syn drome has been reported as having been misdiagnosed as viral keratitis ( a ). immunologic in a retrospective study of trough plasma concentrations of nevirapine and five oxidative metabolites in patients with rashes or liver function abnormalities during the first weeks of treatment and controls matched for glucocorticoid use, cd cell count, sex, race, and hepatitis b/c status, casecontrol pairs were studied ( c ). women had significantly greater exposure than men to nevirapine and four of the five metabolites at week , but the plasma concentrations were comparable by week . there were no strong relationships between plasma concentrations of nevirapine or any of its five metabolites and case-defining events. the authors commented that systemic exposure to -hydroxynevirapine and -carboxynevirapine, hypothesized to be reactive intermediates for immunemediated adverse reactions, were comparable between the cases and controls and were comparable in proportion to nevirapine exposure. pregnancy in a retrospective, five-center comparison in hiv- -infected women who took nevirapine as part of combination antiretroviral therapy during pregnancy, of eligible women ( . %) developed a rash and ( . %) developed hepatotoxicity, including with a co-existent rash, giving a combined incidence of potential cases of nevirapine toxicity during pregnancy ( . %) ( c ). alternative causes of rash or hepatotoxicity were suspected in cases, and only mothers ( . %) stopped taking nevirapine. of the women who started taking nevirapine during pregnancy, ( . %) developed a rash and ( . %) hepatotoxicity. only of women ( . %) with nevirapine exposure before pregnancy had had a rash. susceptibility factors genetic hla typing and demographic and immunological susceptibility factors for reactions to nevirapine and efavirenz have been studied in hiv-positive patients with rashes ( c ). isolated rashes were significantly associated with hla-drb . there were no cases of liver toxicity nor any association with the percentage of cd cells. in hiv- -positive individuals, of whom were japanese, % of those who had hypersensitivity reactions to nevirapine had hla-cw , compared with only % of the others and - % of the general japa nese population ( c ). in the former group, four patients, including one who developed hepatotoxicity, had hla-cw* and one had hla-cw* . among the others, three patients had hla-cw* . there were no significant differences in the fre quencies of other hla alleles between the two groups. drug dosage regimens nevirapine has a long half-life and could be given once a day, but the risk of rashes and concerns over liver toxicity preclude the routine use of once-daily dosing. however, tolerance to high concentrations of nevirapine can develop when doses are increased slowly during the start of therapy. it is therefore theoretically possible that the benefits of once-daily dosing could be achieved without excess toxicity by switching to once-daily nevirapine after several months of twice-daily administration ( r ). however, in the daufin study, a twicedaily regimen of zidovudine mg þ lami vudine mg þ nevirapine mg was compared with a once-daily regimen of lamivudine mg þ tenofovir mg þ nevirapine mg ( c ). the study was stopped after early virological failure was observed in of once-daily patients. resistance mutations accumulated during treatment, with high rates of k r muta tions and severe nnrti resistance profiles indicative of continuing viral replication caused by suboptimal nevirapine plasma trough concentrations, possibly due to non adherence. non-b-subtype infection (sub types a and c not stated) was observed in of patients with virological failure. the authors suggested that once-daily dosing can be introduced after induction of viral suppression has been achieved with a twice-daily regimen. co-administration of nevirapine mg/day with fluconazole mg/day results in markedly increased trough plasma nevirapine concentrations compared with nevirapine alone ( c ). in a retrospective study in patients who were given nevirapine-based therapy with or without fluconazole or mg/day, mean nevirapine concentrations were . mg/l without fluconazole and . with fluconazole. one patient taking fluco nazole developed hepatitis. six of those who did not take fluconazole developed nevira pine-related rashes. there were no differ ences in -week antiviral efficacy between the two groups. liver rises in serum unconjugated bilirubin concentrations were reported in the pivotal phase iii trial. the mechanism is thought to be direct inhibition of bilirubin conjugation by competitive inhibition of udp glucuronosyltransferase. patients with polymorphisms in the ugt a gene are more likely to develop hyperbilirubinemia ( r ). urinary tract indinavir causes nephro lithiasis and renal impairment as a result of crystallization in the urinary tract and resultant inflammation ( a ). continuation of the drug with some improvement in renal function is possible with drug concentration monitoring. co-factors such as concomitant co-trimoxazole therapy and environmental temperature increase the risk ( r ). using indinavir þ ritonavir at the lower doses of and mg bd seems to reduce these adverse effects. hematuria and flank pain each occurred in patients in a study of asymptomatic hiv-infected individuals who took indina vir in a long-term follow-up study over weeks ( c ). hair and nails paronychia, alopecia, curling of straight hair, and ingrowing toenails have all been attributed to indinavir ( r ). pregnancy in a study of pregnant women taking indinavir, two women and eight infants developed hepatotoxicity and had increased concentrations of indinavir, suggesting increased intestinal/hepatic cyp a activity during pregnancy ( a ). metabolism of pregnant women taking nelfinavir developed gestational diabetes compared with none taking zidovudine monotherapy and two of taking nrtis and nnrtis; the risk of gestational diabetes was increased in those with hepatitis c or who had begun haart before pregnancy ( c ). nails paronychia has been reported in a case report occurring during nelfinavir treatment ( a ). drug-drug interactions fluticasone cush ing's syndrome and adrenal suppression can be caused if protease inhibitors increase systemic glucocorticoid concentrations. iatrogenic cushing's syndrome has been attributed to ritonavir by an interaction with fluticasone ( a ). • a -year-old girl who had taken various antiretroviral drugs eventually took stavudine, lamivudine, and ritonavir and then used inhaled fluticasone þ salmeterol for bronchiectasis. after months she had excessive weight gain, increased appetite, fatigue, facial edema, marked acne, stretch marks on her limbs and abdomen, hypercholesterolemia, hypertriglyceridemia and amenorrhea. cushing's syndrome was attributed to an interaction of fluticasone with ritonavir, which was changed to efavirenz. there was a gradual improvement within - days, with reduced edema and stretch marks and return of menstruation. a similar case involved a -year-old girl ( a ). observational studies in the aspire study, of healthy volunteers who took saquinavir þ ritonavir for months developed grade gastrointestinal adverse effects and seven had hyperbilirubinemia ( c ). in the aspire study there was hyperbilirubinemia in of healthy volunteers. tipranavir is a non-peptide protease inhibitor approved for use in patients with resistant strains of hiv ( r , m , r ). its pharmacokinetics, efficacy, and adverse effects in children and adolescents have been reviewed ( r ) . it can be used in combination with ritonavir. observational studies in a -week multi-center, double-blind, randomized, dose-finding trial of ritonavir-boosted tipra navir in patients, the most common adverse events were diarrhea, nausea, vomiting, fatigue, and headache ( c ). hematologic tipranavir boosted with ritonavir caused an increased risk of bleed ing in of hiv-infected patients with hemophilia ( c ). intracranial hemorrhage has been reported in a patient taking tipranavir ( a ). liver tipranavir is associated with an excess of grade / rises in liver enzyme compared with other ritonavir-boosted protease inhibitors ( r ). pancreas acute pancreatitis associated with hypertriglyceridemia has been reported in a patient taking tipranavir þ ritonavir ( a ). • a -year-old man taking tenofovir mg/day, trizivir (zidovudine, lamivudine, and abacavir) one tablet bd, and tipranavir mg bd þ ritonavir mg bd drank six standard alcoholic drinks days before admission and developed marked tenderness in the epigastric region and a raised serum lipase at iu/l (reference range below iu/l). an abdominal computed tomography (ct) scan showed pancreatic edema with peripancreatic fluid consistent with acute pancreatitis. ultrasonography showed a mildly dilated common bile duct with no evidence of cholelithiasis and ct cholangiography showed no evidence of gall-stone pancreatitis. the presumed diagnosis was alcohol-induced pancreatitis. he was managed conservatively by withdrawal of medications, intravenous fluids, and slow resumption of oral intake. however, within days he again developed severe epigastric pain. he denied further alcohol use. the serum concentration of triglycerides was mmol/l, and retrospective testing of blood samples taken during the earlier illness also showed marked hypertriglyceridemia. tipranavir þ ritonavir was withdrawn, efavirenz given, and tenofovir and trizivir continued. his triglyceride concentrations fell to . mmol/l weeks later and . mmol/l months later without specific intervention. skin porphyria cutanea tarda has been reported after the introduction of tipranavir þ ritonavir to a backbone of tenofovir and lamivudine ( a ). • a -year-old heterosexual woman switched to tipranavir-containing therapy after virological failure. after days she developed a rash accompanied by nausea, vomiting, malaise, and hyperamylasemia. all antiretroviral drugs were withdrawn, she improved, and treatment was restarted. although the lesions were resolving, several blisters appeared on her hands, mainly on the fingers, along with itching and skin fragility on her arms. aciclovir was ineffective and month later the itch and blisters worsened. urine concentrations of protoporphyrin and coproporphyrin were about and times higher than normal. drug resistance virological response rates in tipranavir-treated individuals were reduced when isolates with substitutions at amino acid positions i , v , m , i , q , d , v , or i were present at baseline ( c ). in addition, virological response rates to tipranavir were reduced when the number of baseline protease inhibitor mutations was five or more. individuals who took tipranavir without concomitant enfuvirtide and had five or more baseline protease inhibitor mutations began to lose antiviral response at weeks - . however, individuals taking enfuvirtide with tipranavir achieved more than . log reductions in viral load from baseline at weeks, even if they had five or more baseline protease inhibitor mutations. virological response rates to tipranavir were reduced when the baseline phenotype for tipranavir had a greater than threefold shift in the % effective concentration (ec ) from reference. the most common protease inhibitor mutations that were associated with virological failure were l i/v/s, i v, l v/i/f, m v/i/l v t, v l, and i v. drug-drug interactions interactions with tipranavir þ ritonavir have been reviewed ( r ). tipranavir is metabolized by cyp a and tipranavir þ ritonavir in vitro inhibits cyp a , cyp c , cyp c , cyp d , and cyp a and induces glucuronidase and p glycoprotein. inhibitors ritonavir-boosted tipranavir, alone and in combination with comparator protease inhibitors, has been studied in hiv-infected patients ( c psychiatric oseltamivir-induced worsening of delirium has been reported in an -year old man, whose symptoms resolved days after withdrawal ( a ). japanese authorities advised against using oseltamivir in adolescents aged - years after two suicides during and more than reports of neuropsychiatric events identified during post-marketing sur veillance, including delirium, convulsions, and encephalitis ( r ). however, it is not clear whether these events were due to the influenza or the drug; in phase iii trials, there were similar incidences of neurologi cal and psychiatric events in both treated and untreated patients ( r ). of patients enrolled in a japanese neuramini dase inhibitor treatment study, had neuropsychiatric symptoms, in cases before the start of treatment ( c ). the us food and drug administration (fda) and european medicines agency (ema) have advised doctors to monitor patients for abnormal behavior throughout treat ment ( r ). gastrointestinal acute hemorrhagic colitis has been associated with oral oseltamivir in a -year-old man, who developed abdominal pain, diarrhea and hematochezia after taking two doses of oseltamivir ( a ). in a systematic review of three trials of neuraminidase inhibitors for preventing and treating influenza in children, of whom had laboratory-confirmed influenza, those who took oseltamivir had vomiting more often than untreated children but withdrawal was rarely required ( m ). drug resistance resistance occurs in under % of healthy adults but occurs more often in children, from . % up to % in one study, although a lower dosage regimen was used in that study. resistance is often seen among immunocompromised patients. it was reported in two of eight patients infected with h n and was associated with a fatal outcome. resistance is associated with loss of fitness ( r ). in widespread resistance emerged in h n influenza, but it remains to be seen whether its circulation is sustained after the emergence of oseltamivir-sensitive swine vh n . susceptibility factors genetic there was no evidence of a difference in auc ! of oseltamivir or its active metabolite oseltamivir carboxylate between japanese subjects and caucasian subjects, or between children aged - years old and adults ( c ). systematic reviews a meta-analysis of the adverse effects of zanamivir in children showed that it was no worse than placebo ( m comparative studies in a randomized controlled trial in patients with chronic hepatitis c treated with interferon-alfa a alone (n = ), with amantadine mg bd (n = ), with ribavirin (n = ) or with amantadine þ ribavirin (n = ), there was a sustained virological response in %, %, %, and % respectively ( c ). this was statistically different between interferon þ amantadine and triple therapy but not between interferon þ ribavirin and triple therapy. the spectra and frequencies of adverse effects were similar in all four treatment arms. however, six patients withdrew because of adverse effects, three of them in an arm containing amantadine. in two groups of non-responders with hcv genotype chronic infection taking interferon and ribavirin, with or without amantadine mg/day, viral load fell more markedly in the group taking triple therapy including amantadine, but the response rates at the end of treatment were not signifi cantly different ( c ). although analysis of the viral ion channel p showed selective pressure during therapy, no specific resi dues appeared to be linked to the effect of amantadine on the virus. the authors sug gested that this implied that the antiviral effect of amantadine is non-specific and related to reduced endosomal acidification and therefore reduced transport of hepatitis c by a ph-dependent pathway. in a multicenter study of non-respon ders with chronic hepatitis c randomized to interferon monotherapy (n = ), dual ther apy with ribavirin (n = ) and triple therapy with additional amantadine mg/day (n = ), amantadine did not increase the frequency or severity of adverse effects ( c ). in patients with chronic hepatitis c genotype b, with high viral loads treated with interferon-beta for weeks followed by interferon-alfa b, ribavirin, and amantadine mg/day for weeks, there was a sustained virological response in . only one patient had to stop taking amantadine, because of the adverse effect of light-headedness ( c ). in renal transplant recipients with chronic hepatitis c, doses of ribavirin monotherapy (n = ) or ribavirin þ amantadine (n = ) were adjusted according to creatinine clearance ( c ). there was no difference between treatment groups with respect to liver enzymes, hepatitis c viremia, liver his tology, or renal function. anemia, the main adverse effect, was most notable in those with a creatinine clearance below ml/ minute. other adverse effects included leukopenia, mood disorders and profuse sweating. only one of those who received combination therapy were still taking aman tadine after months; two completed treatment but with ribavirin alone. four of those taking monotherapy completed treat ment. neither regimen was clearly superior to no treatment, but the study was small and probably underpowered. sensory systems in a post-marketing surveillance study of patients with a new diagnosis of corneal disease and new prescriptions for amantadine over years, ( . %) of patients developed fuchs dystrophy (corneal edema) ( c ). the relative risk of corneal edema was . ( % ci = . , . ); in patients ( . %) the diagnosis was made in the first month. drug resistance amantadine is unreliable in the management of influenza because of the emergence of widespread resistance. this is notable in the h n subtype and most h n subtypes, but some seasonal h n remains sensitive to amantadine ( r treatment of adenovirus pneumonia with cidofovir in pediatric lung transplant recipients infections in t-cell-depleted allogeneic hematopoietic stem cell transplantation: high mortality in the era of cidofovir low-dose cidofo vir treatment of bk virus-associated hemor rhagic cystitis in recipients of hematopoietic stem cell transplant acyclovir-induced neuropsychosis success fully recovered after immediate hemodialy sis in an end-stage renal disease patient acute renal failure caused by intravenous acyclovir for disseminated var icella zoster virus infection determi nants of aciclovir-induced nephrotoxicity in children entérocolite ulcéronécrosante chez un nouveau-né à terme. rôle de l'acy clovir? [necrotizing enterocolitis in a full term infant. is acyclovir involved? immediate allergy from valacyclovir oliguric acute renal failure following oral valacyclovir therapy adding-on versus switching-to adefovir therapy in lamivudine-resistant hbeag negative chronic hepatitis b adefovir dipivoxil therapy in liver transplant recipients for recurrence of hepatitis b virus infection despite lamivudine plus hepatitis b immu noglobulin prophylaxis absence of a pharmacokinetic interaction between entecavir and adefovir lamivudine and high-dose interferon alpha a combination treatment in naive hbeag-positive immunoactive chronic hepatitis b in children: an east mediterra nean center's experience pharmacokinetics of lamivu dine in subjects receiving peritoneal dialysis in end-stage renal failure telbivu dine versus lamivudine in patients with chronic hepatitis b adverse events associated with high-dose ribavirin: evidence from the toronto out break of severe acute respiratory syndrome long-term therapy with aerosolized riba virin for parainfluenza virus respiratory tract infection in an infant with severe com bined immunodeficiency syncope as a prob able side effect to combination antiretro viral therapy initiated during primary hiv infection kallianpur araids clinical trials group and a s study teams. hemochromatosis gene poly morphisms, mitochondrial haplogroups, and peripheral lipoatrophy during anti retroviral therapy del río labcde (aba cavir vs. d t (stavudine) plus efavirenz) study team. less lipoatrophy and better lipid profile with abacavir as compared to stavudine: -week results of a randomized study antiretroviral drug levels and interactions affect lipid, lipoprotein, and glucose metabolism in hiv- seronegative subjects: a pharmacoki netic-pharmacodynamic analysis maxcmin and trial groups. phar macokinetics of two randomized trials evaluating the safety and efficacy of indina vir, saquinavir and lopinavir in combination with low-dose ritonavir: the maxcmin and trials assess ment of adverse events associated with anti retroviral regimens for postexposure prophylaxis for occupational and nonoccu pational exposures to prevent transmission of human immunodeficiency virus for the protocol study groupefficacy, safety, and tolerability of long-term combination antiretroviral therapy in asymptomatic treatment-naive adults with early hiv infection clinical and pharmacokinetic data support once-daily low-dose boosted saquinavir ( , milli grams saquinavir with milligrams ritonavir) in treatment-naive or limited protease inhibitor-experienced human immunodeficiency virus-infected patients dose reduction effective in alleviating symptoms of saquinavir toxicity french national agency for research on aids; viral hepa titis-hc -ribavic study team. progres sion of fibrosis in hiv and hepatitis c virus-coinfected patients treated with inter feron plus ribavirin-based therapy: analysis of risk factors hepato toxicity in an african antiretroviral therapy cohort: the effect of tuberculosis and hepa titis b nodular regenerative hyperplasia of liver as a consequence of art the role of antire troviral therapy in the incidence of pancrea titis in hiv-positive individuals in the eurosida study amprenavir and di danosine are associated with declining kid ney function among patients receiving tenofovir acute interstitial nephritis of hiv-positive patients under atazanavir and tenofovir therapy in a retrospective analysis of kidney biopsies possible allergic cross-reaction to didanosine and tenofovir in an hiv- -infected woman seage rd gr. in utero nucleoside reverse transcriptase inhibitor exposure and signs of possible mitochondrial dysfunc tion in hiv-uninfected children adverse events experi enced by three children taking tenofovir and didanosine in combination phar macokinetic interaction between darunavir and saquinavir in hiv-negative volunteers interaction between buprenor phine and atazanavir or atazanavir/ritona vir pos sible antiretroviral therapy-warfarin drug interaction factors in aids dementia com plex trial design: results and lessons from the abacavir trial b* -associated abacavir hypersensitivity on the basis of sequence variation in hiv reverse transcriptase are disulfiram-like reactions associated with abacavir-containing antiretroviral regi mens in clinical practice? fanconi syndrome and nephrogenic diabetes insipidus associated with didanosine ther apy in hiv infection: a case report and literature review cd þ cell count decline despite hiv suppression: a prob able didanosine-valganciclovir interaction severe weight loss in hiv/hcv-coinfected patients trea ted with interferon plus ribavirin: incidence and risk factors stavudine plasma concentrations and lipoatrophy hernández-mora dine discontinuation to anthropometric mg, novoa sr, quintana fb, lahoz jg, changes among hiv-infected women complicating the abacavir hypersensitivity reaction abacavir hypersensitiv ity reaction after switching from the twicedaily to the once-daily formulation acute fibrinous and organizing pneumonia as a rare presen tation of abacavir hypersensitivity reaction a simple screening approach to reduce effects of a nucleoside reverse transcriptase inhibitor, stavudine, on glucose disposal and mitochondrial func tion in muscle of healthy adults risk factors for mortality in symptomatic hyperlactatemia among hiv-infected patients receiving antiretroviral therapy in a resource-limited setting systematic review of clinical trials evaluating low doses of stavudine as part of antiretroviral treat ment safety of stavudine in the treatment of hiv infection with a special focus on resource-limited settings blunted humoral response to influenza vaccination in patients exposed to zidovu dine plus trimethoprim-sulfamethoxazole aids clinical trials group s study team. pharmacokinetic evaluation of the effects of ribavirin on zidovudine triphosphate formation: actg s study team efficacy and safety of tenofovir double-dose in treatment-experi enced hiv-infected patients: the teno plus study castillo muñoz a, toxicity in hiv-infected subjects: a rando-baños roldán u, artacho criado s. analisis mized, controlled study increased haemoglobin a percentage in hiv infection: disease or treatment? elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to pre vent mother-to-child transmission of hiv assessment of birth defects according to maternal therapy among infants in the women and infants de las causas y factores predictivos de discon tinuacion del tratamiento con tenofovir en pacientes vih pretratados increased beta- microglobulinuria in human immunodeficiency virus- -infected children and adolescents treated with tenofovir neuropsychiatric side effects of efavirenz therapy pharmacokinetics of oral zidovudine administered during labour: a preliminary study teratogenicity risk of antiretro viral therapy in pregnancy efficacy and safety of a once daily regimen with efavirenz, lami vudine, and didanosine, with and without food, as initial therapy for hiv infection: the eladi study effectiveness and safety of simplification therapy with once-daily tenofovir, lamivu dine, and efavirenz in hiv- -infected patients with undetectable plasma viral load on haart metabolic effects of protease inhibitor-spar ing antiretroviral regimens given as initial treatment of hiv- infection (aids clin ical trials group study a ) efavir enz urolithiasis anecdotes that provide definitive evidence positive outcomes of haart at months in hiv-infected patients in cambodia efavirenz pharmacokinetics in hiv- -infected chil dren are associated with cyp b -g t polymorphism psychosis in a -year-old hiv-positive girl with an increased serum concentration of efavirenz hepatotoxicity due to a drug interaction between amodia quine plus artesunate and efavirenz pharmacokinetic interaction between voriconazole and efavirenz at steady state in healthy male subjects liver toxicity induced by non-nucleoside reverse tran scrptase inhibitors hepatotoxicity of nevirapine in virologically suppressed patients according to gender and cd cell counts risk of side effects associated with the use of nevirapine in treatment-naive patients, with respect to gender and cd cell count reasons for disconti nuation of nevirapine-containing haart: results from an unselected population of a large clinical cohort safety issues about nevirapine administration in hiv-infected pregnant women widespread viti ligo after erythroderma caused by nevira pine in a patient with aids nevira pine-associated rash with eosinophilia and systemic symptoms in a child with human immunodeficiency virus infection nevirapine induced stevens-johnson syndrome in an hiv patient case-control exploration of relationships between early rash or liver toxicity and plasma concentra tions of nevirapine and primary metabo lites safety of nevirapine in pregnancy hla-drb * associated with cutaneous hypersensitivity induced by nevirapine and efavirenz hla-cw primarily associated with hypersensitivity to nevirapine once-daily nevirapine dosing: a pharmacokinetics, efficacy and safety review once-daily dosing of nevirapine in haart plasma nevirapine levels, adverse events and efficacy of antiretroviral therapy among hiv-infected patients concurrently receiving nevirapine-based antiretroviral therapy and fluconazole phar macokinetics of darunavir (tmc ) and atazanavir during coadministration in hivnegative, healthy volunteers alo pecia associated with ritonavir-boosted ata zanavir therapy pharmacokinetic interaction between rifampicin and ritonavir-boosted atazanavir in hiv-infected patients increased incidence of cutaneous mycoses after haart initiation: a benign form of immune reconstitution disease? fosamprenavir calcium plus ritonavir for hiv infection experience of indinavir/ritonavir / mg twice-daily highly active antiretro viral therapy-containing regimen in hiv- infected patients in bamako, mali: the nogoma study lack of indinavir-associated nephrological complications in hivinfected adults (predominantly women) with high indinavir plasma concentration in abidjan indinavir: the forgotten hivprotease inhibitor. does it still have a role? néphropathie tubulo-interstitielle associée a l'indinavir dermatologic adverse effects of antiretroviral therapy: recognition and management pharmacokinetics and safety of indinavir in human immunodefi ciency virus-infected pregnant women obste tric and perinatal complications in hivinfected women. analysis of a cohort of pregnancies between paronychia in an hiv-infected patient under nelfinavir ther apy iatrogenic cushing's syndrome in a adolescent with aidss on ritonavir and inhaled fluticasone. case report and literature review cushing syndrome and severe adrenal suppression caused by fluti casone and protease inhibitor combination in an hiv-infected adolescent pharmacokinetics of saquinavir with atazanavir or low-dose ritonavir administered once daily (aspire i) or twice daily (aspire ii) in seronegative volunteers tipranavir: a new option for the treatment of drug-resistant hiv infection tipranavir: the first nonpeptidic protease inhibitor for the treatment of protease resistance tipranavir: a review of its use in the management of hiv infec tion tipra navir: a new protease inhibitor for the pediatric population efficacy and safety of three doses of tipranavir boosted with ritonavir in treatment-experienced hiv type- infected patients increased risk of bleeding with the use of tipranavir boosted with ritonavir in haemophilic patients intracranial haemorrhage possibly related to tipranavir in an hiv- patient with cryptococcal meningitis tipranavir: a new protease inhibitor for the treatment of antiretroviral-experienced hiv-infected patients acute pancreatitis caused by tipranavir/ritonavir-induced hypertriglycer idaemia porphyria cutanea tarda in an hiv- -infected patient after the initiation of tipranavir/ritonavir: case report food and drug administration analysis of tipranavir clini cal resistance in hiv- -infected treatmentexperienced patients mechanisms of pharmacokinetic and pharmacodynamic drug interactions associated with ritonavir enhanced tipranavir pharmacokinetics, safety, and efficacy of tipranavir boosted with ritonavir alone or in combination with other boosted protease inhibitors as part of optimized combination antiretroviral therapy in highly treatmentexperienced patients oseltamivir-induced delirium in a geriatric patient tamiflu and neuropsychiatric disturbance in adolescents the role of oseltamivir in the treatment and prevention of influenza in children a comparison of the effectiveness of zanamivir and oseltami vir for the treatment of influenza a and b acute hemorrhagic colitis associated with oral administration of osel tamivir for the treatment of influenza a symmonds-abrahams m. neur aminidase inhibitors for preventing and treating influenza in children antivirals for influenza similarity in pharmacokinetics of oseltami vir and oseltamivir carboxylate in japanese and caucasian subjects induction doses of interferon alpha- a in combination with ribavirin and/ or amantadine for the treatment of chronic hepatitis c in non-responders to interferon monotherapy: a randomized trial hepatitis c virus p membrane protein quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine a randomized trial of induction doses of interferon alone or in combination with ribavirin or ribavirin plus amantadine for treatment of nonresponder patients with chronic hepatitis c triple therapy of initial high-dose interferon with ribavirin and amantadine for patients with chronic hepatitis c combination therapy with ribavirin and amantadine in renal trans plant patients with chronic hepatitis c virus infection is not superior to ribavirin alone postmarketing sur veillance of corneal edema, fuchs dystrophy, and amantadine use in the veterans health administration key: cord- -kjgpriow authors: scalia, santo; haghi, mehra; losi, vanessa; trotta, valentina; young, paul m.; traini, daniela title: quercetin solid lipid microparticles: a flavonoid for inhalation lung delivery date: - - journal: eur j pharm sci doi: . /j.ejps. . . sha: doc_id: cord_uid: kjgpriow purpose: the aim of the present work was to develop solid lipid microparticles (slms), as dry powders containing quercetin for direct administration to the lung. methods: quercetin microparticles were prepared by o/w emulsification via a phase inversion technique, using tristearin as the lipid component and phosphatidylcholine as an emulsifier. the quercetin slms were characterised for morphology, drug loading ( . % ± . , which corresponded to an encapsulation efficiency of . %), particle size distribution, response to humidity, crystallinity, thermal behaviour and in vitro respirable fraction. furthermore, the toxicity and the in vitro transport of the slms on an air liquid interface model of the calu- cell line were also investigated using a modified twin-stage impinger apparatus. results: results showed that quercetin slms could be formulated as dry powder suitable for inhalation drug delivery ( . ± . % fine particle fraction ⩽ . μm) that was absorbed, via a linear kinetic model across the calu- monolayer ( . ± . % over h). in addition, quercetin slms were shown to be non-toxic at the concentrations investigated. interestingly, no apical to basolateral transport of the micronised quercetin was observed over the period of study. conclusions: these observations suggest quercetin diffusion was enhanced by the presence of the lipid/emulsifying excipients in the slms; however further studies are necessary to elucidate the exact mechanisms. quercetin is a flavonoid, a natural substance with a phenolic structure. flavonoids can be divided into various classes on the basis of their molecula r structure with quercetin belonging to the flavone class, which has a planar structure due to the double bond in the central ring of the -phenylchr omen- -one ( -phenyl- -benzopyran- -one) backbone. this flavonoid group can be found in abundance in onions, apples, broccoli, and berries (nijveldt et al., ; formica and regelson , ) . quercetin pharmacokine tics and bioavailability in humans has previousl y been studied graefe et al., ). an important effect of quercetin is its ability to scavenge for oxygen-der ived free radicals (heijnen et al., ) , given its antioxidant properties at low concentrations (robaszkiew icz et al., ) . furthermor e, in vitro and in vivo experiments have also shown that flavonoids possess potential anti-inflammatory, anti-allergic , antiviral, anti-carcinogeni c and anti-asthma tic propertie s (pettinari et al., ; wu et al., ; li et al., ; davis et al., ; gang et al., ; . specifically, for the lungs, cell culture studies have shown that quercetin can reduce infectivity of target cells and replication against a wide variety of respiratory viruses, including herpes simplex virus and adenovirus (chiang et al., ) , coronavirus (debiaggi et al., ) , parainfluenza and respiratory syncytial virus (kaul et al., ) , rhinovirus (dimova et al., ) , and severe acute respirato ry syndrome (chen et al., ) . furthermor e, there is evidence that quercetin plays a critical role in the amelioration of the pathogenic process of asthma in a murine model (mice) (park et al., ) , due to the alteration of specific cytokine production (th /th ) and transcriptor factors (t-bet and gata- ) gene expression in ova-induced asthma model mice, suggesting quercetin could be used as a new therapeutic approach to allergic airway diseases. more recently, when given orally to guinea pigs, quercetin has been found to reduce hyper-reacti vity of airways, one of the main attributes of allergic asthma (joskova et al., ) ; causing significant broncodilation . furthermore, a report suggests that flavonoids such as quercetin and luteolin could stimulate cl À secretion by activating an entry step of cl À across the basolater al membrane through na + /k + / cl À co-transpor ter; contributing to maintenanc e and/or production of airway surface liquid by regulatin g cl À secretion in airway epithelial cells (asano et al., ) , very important in chronic obstructi ve pulmonary disease. quercetin is sparingly soluble in water and has poor bioavailability. as a result, the clinical applicati on of the drug is greatly restricted. previous studies have tried to resolve this issue by producing nanocrystals with enhanced dissolution (kakran et al., ; li et al., ). an alternative approach would be to incorporate the drug into a lipid matrix (li et al., ) . the production of solid lipid micropartic les (slms) has been previously studied as a respiratory drug delivery vehicle for both poorly water-soluble drugs, such as budesonide ) and for more water-solub le drugs, such as salbutamol (scalia et al., ) . there are many advantages in using slms (jaspart et al., ) , with the most significant being the ability to control release after deposition. furthermore, slms should be well tolerated in vivo since they are made of physiolog ical compounds . of course, the toxicity of the surfactants and other excipients, used for their manufacture, needs to be considered. while solid lipid nanoparticl es are currently attracting a lot of attention in the research communi ty, solid lipid micropatrtic les have been rather unexploited, especially for inhalation drug delivery scalia et al., ; jaspart et al., ) , where particles should have an optimum aerodynamic diameter between and lm (patton, ) . mezzena et al. ( ) and scalia et al. ( ) used this concept to deliver budesonide and salbutamol, two anti-asthma drugs, respectively, using glycerol behenate as the solid lipid component. in other studies, dellamary et al. ( ) , used dipalmitoyl phosphatidylch oline, a biocompatible lipid and component of normal lung surfactant, to modulate immunoglo bulin release while sanna et al. ( ) applied the same concept for the formulat ion of slms to be used as carrier for lung delivery, using compritol as lipophilic component and poloxamer as emulsifying agent. the aim of this study was to investigate the potential use of solid lipid micropartic les containing quercetin delivered as a dry powder for inhalation. the physicochemi cal characteristics of this formulation was investigated together with aerosol performanc e, in vitro cell toxicity and transport studies, using a calu- adenocarcinoma cell line grown using an air-interface model. micronised quercetin hydrate (referred to as quercetin hereafter) and tristearin were supplied by sigma aldrich (steinheim, germany). phosphatidylchol ine was supplied by cargill (hamburg, germany). ammonium acetate was supplied by ajax finechem pty ltd. (sydney, australia). acetic acid was purchased from analar vwr international (poole, england) and edta di-sodium salt was supplied by aps finechem (sydney, australia). the calu- cell line (htb- ) was purchased from the american type cell culture collection (attc, rockville, usa). dulbecco's modified eagle's medium, fetal bovine serum, hanks balanced salt solution, phosphate buffer saline, hepes, trypsin-edta solution ( . g/l trypsin, . g/l edta), l-glutamine solution ( mm), nonessential amino acids solution and cellytic m cell lysis ( mm tris-hcl, ph , mm nacl, % np- , . % sodium deoxycholat e, . % sds) were supplied by invitrogen (sydney, australia). transwel l cell culture supports ( . cm polyester, . lm pore size) were obtained from corning costar (lowell, ma, usa). all solvents were analytica l grade and were obtained from sigma (sydney, australia). water was purified by milli-q reverse osmosis (molsheim, france). for the development of slms loaded with quercetin, melt o/w emulsification techniqu e was employed, since it circumvents the use of organic solvents jaspart et al., ) . this approach is environmentall y friendly, but also eliminates the potential for residual solvent in the final dosage form. quercetin ( g) was dissolved into a melted ( - °c) lipid phase ( . g tristearin) and the phase inversion was obtained adding hot ( - °c) milliq water ( ml), containing . g of phosphat idylcholine as surfactant. the mixture was maintained at °c and was subjected to high-shear mixing ( , rpm for min, using a t- ultra-turrax; ika-wer k, staufen, germany). addition ally, the o/w emulsion was sonicated at a constant duty cycle ( khz) for min, using a probe (microson xl ultrasonic cell disruptor, misonix, farmingd ale, ny) at a power input of w. the emulsion was quickly cooled to a room temperature , using an ice bath and the formed micropartic les recovered by freeze-drying at À °c (hetosicc, heto lab equipment, saint-julie, canada). the amount of quercetin entrapped in the slms was determined by heating ( °c for min) and sonication ( min) of the micropar ticles ( - mg) in ethanol in sealed glass vials. the obtained sample was diluted to volume ( ml) with methano l, filtered and assayed by high performanc e liquid chromatograp hy (hplc). physical and chemical characterisati on . . . . particle size analysis. the particle size distribution of the slms was analysed using laser diffraction (malvern mastersizer , malvern instruments ltd., uk). samples of powder (ca. mg) were dispersed using the scirocco dry dispersion unit (malvern, uk) with a feed pressure of bar and feed rate of %. samples were analysed in triplicate, with an obscurati on value between . % and % and a reference refractive index of . . . . . . scanning electron microscopy. the morphology of slms particles was studied using a field emission scanning electron microscope (zeiss ultra plus, carl zeiss pty ltd., sydney, australia ). the samples were sputter coated with gold to a thickness of nm, under an argon atmosph ere prior to analysis. samples were dispersed onto carbon sticky tabs and images were taken at random locations . . . . . differential scanning calorimetry. the thermal response of quercetin raw material and slm powders were studied using differential scanning calorimetry (dsc e; mettler-tol edo, schwerzenbach, switzerland ). samples ( - mg) were crimp-sealed in dsc sample pans and exposed to a °c min À temperature ramp between and °c. exothermal and endothermi c peak temperature s, onset temperat ure and heat of enthalpy (dh) for each peak were determined using stare software v. . x (mettler toledo, greifensee, switzerla nd). . . . . x-ray powder diffraction. the x-ray powder diffraction (xrpd) pattern for the slms was analysed using a d x-ray powder diffractome ter (siemens, munich, germany). measurements were conducte d at °c, using cu ka radiation at ma and kv, with angular increment of . °s À . . . . . dynamic vapour sorption. dynamic vapour sorption (dvs) was used to investiga te the relative moisture sorption and stability of the quercetin raw material and slms, with respect to humidity. samples (ca. mg) were added to glass sample pans, which were placed in the sample chamber of a dvs (dvs- , surface measurement systems ltd., london, uk). each sample was dried at % rh before being exposed to % rh increments for two - % rh cycles ( °c). equilibrium moisture content at each humidity step was determined by a dm/dt of . % min À . chemical analysis of quercetin was performed by high-performance liquid chromatograp hy (hplc) using a shimadzu prominence uflc system equipped with an spd- a uv-vis detector, lc- at solvent delivery unit, sil- a ht autosampler (shimadzu corporation , japan) and novapak c column ( lm,  . mm, waters, ireland). the hplc settings were as follow: detection wavelength nm, flow rate . ml min À , injection volume ll, and retention time of . min. the mobile phase was a mixture of acetate buffer and acetonitrile ( : , v/v) and mm edta. standard linearity was obtained between . and lg ml À . the aerosolisation of quercetin slms were analysed using an aerolizer Ò dry powder inhalation (dpi) device (novartis, surrey, uk) and a next generation impactor (ngi) (copley scientific, nottingham, uk) according to the method specified for dry powder inhalers in the pharmacopoei a (british pharmacop oeia, ). briefly, prior to testing, all eight ngi collection cups were coated with silicon oil to eliminate particle bounce. mg ± . mg of quercetin slms were weighed and filled in size gelatine capsules (capsugel, sydney, australia ) and placed in the aerolizer Ò . a mouthpiece adaptor fitted into a united states pharmacopei a (usp) throat was used to connect the aerolizer Ò to the ngi. the particles were aerolised at l min À for s. the device containing the capsule, throat and the stages were then washed separately with methano l and the samples were analysed using hplc. the mass balance was calculated as the recovery from the stage deposition, capsule and device. in addition, the fine particle dose (fpd) (drug recovered from stages to filter) ( . lm) and the fine particle fraction (fpf) (fpd/total dose  ) were calculated . the toxicity of quercetin slms was assessed by measuring the viability of calu- cells in a liquid covered culture, following days drug exposure to increasing drug concentratio ns, as previousl y described (haghi et al., ) . briefly,  cells were seeded per well in a volume of ll into a well plate. cells were incubated overnight at °c in % co atmosphere . after h, ll of increasing in addition, measureme nt of the transepithe lial electrical resistance (teer) was performed using evom voltohmm eter (world precision instrument, usa) connected to stx- chopstick electrodes. measure ments were taken per well after the experiment to confirm the maintenance of barrier integrity over h of experiment. subtracting the resistance of a cell-free insert and correcting the value for the surface area of the transwell calculated the resistance. . . . in vitro drug diffusion/transp ort studies using air interface calu- cell culture the transport of quercetin from the slms was studied using a calu- lung epithelial model. the culture condition s for the calu- cell air interface model were as previously described (haghi et al., ) . briefly, calu- cells were grown and subcultur ed in dulbecco's modified eagle's medium (f- containing % (v/v) fetal calf serum, % (v/v) non essential amino acid solution and % (v/v) l-glutamine solution). cells were then seeded on transwel l polyester inserts at a density of  cells cm À . after h, medium was removed from the apical chamber and the basolater al medium replaced every alternate day with fresh medium to establish an air-interf ace model over - days. prior to transport studies the viability and integrity of the cell monolayer with respect to quercetin slms was evaluated. a modified in vitro aerosol testing apparatus (twin stage impinger tsi, radleys, essex, uk) that allows the attachment of a transwell containing calu- epithelial cells was used to study the mechanis m of drug deposition, dissolution and diffusion/transp ort (haghi et al., (haghi et al., , . before the drug transport studies, the tsi flow rate was set to l min À using a rotary vein pump and a calibrated flow meter. the quercetin slm sample was weighed ( mg ± . mg) into a size gelatine capsules, which was then insert into an aerolizer Ò device and the pump activated for s to disperse the powder. the tsi was then disassembled and the transwel l removed. after deposition of the powder, the transwell was placed into a well plate containing ll modified buffer (hbss supplem ented with hepes ( % v/v), and ethanol % ( % v/ v) at ph . ) to measure the quercetin transport. at min time points the transwells were moved to new well containing fresh modified buffer. after h the surface of the monolayer was washed with transport buffer to account for the drug remaining on cell surface and the cells were lysed with cell lytic solution to allow the measure ment of the drug in the cells. the lysates were centrifuged at , rpm for min at °c. the supernatant was collected and diluted to an appropriate volume. all the samples were analysed by hplc. the initial amount deposited on the cell layer was calculated from the sum of the drug passed through the cell monolay er, the mass retained on the monolay er at the end of the experime nt and the mass of drug inside the cells. experime nts were randomised for dose and repeated three times. data was subjected to statistical analysis (spss . ), using an-ova one-way analysis to examine for significant differenc e. significant differences between formulations were analysed using multiple comparisons (tukeys post hoc test) and p values of < . were considered to be significant. for the preparation of the slms, tristearin and hydrogenated phosphatidy lcholine were selected respectively as lipidic material and surfactant, based on a previous study on the developmen t of quercetin-lo aded lipid micropartic les for dermal application ). in addition, phosphatidy lcholine is a physiological compatible emulsifier (phosphatidylcholine is the major component of lung endogenous surfactant). the quercetin content of the slms was . % w/v ± . , which corresponded to an encapsulation efficiency of . % (encapsulation efficiency is based on the recovered amount of quercetin in the microparticles as a function of the theoretical amount present in the powder when taking into account the excipients content) . the particle size distribut ion of the as supplied micronised quercetin and quercetin slms are shown in fig. with analysis of the data indicating median volume diameters (d . ) of . ± . lm and . ± . lm, respectively. further analysis suggested % of the slms had a diameter . ± . lm. scanning electron microscop y images of the slms and as-supplied quercetin are shown in fig. a and b, respectivel y. in general the slms particles had a spherical morphology and a size in line with particle size analysis, while raw quercetin was columnar in shape and larger than lm. it is important to note that particle size data is represented as volume distribution and makes a series of assumptions (i.e. uniform spheres), while sem is a 'morphological' techniqu e. this is reflected by the differences observed in particle size when compared with sem images, where particles appear to have an elongated or plate-like morphology. particle size distribution for the slms was of an appropriate size for respiratory delivery (patton, ) and similar to that measured using laser diffraction. the thermal response of the slm components; tristearin, phosphatidylchol ine and quercetin, as well as the prepared slms are displayed in fig. . tristearin is a commercial monoacid triglyceride. generally, triglycerides exhibit three different polymorphic forms characterised by particular chain packing and thermal stability: alpha (a), beta-prime (b ), and beta (b) (hernqvist, ) . in this specific experiment, tristearin presented an endotherm corresponding to the melting of the b phase at . °c, similar to previous literature (lutton, ) . phosphati dylcholine is a surface-active molecule. its transition temperat ure from crystalline to mesomo rphic (liquid crystalline) was visible at around . °c, in agreement with previously reported values (rutven and mcelhaney, ) . the dsc thermogram of quercetin raw material exhibited an endothermi c peak at °c, typical of melting, in line with literature data (borghetti et al., ) . another smaller endothermic peak is visible at around °c related to a decompo sition process with water loss (da costa et al., ) . the dsc thermogram of the prepared slms showed a noticeable reduction in the melting point in comparis on with quercetin raw material, followed by an endothermi c response. these results are supported by various previous studies conducted on the phospholipid complexes of some phytoconsti tuents such as silybin, puerarin curcumin, naringenin and gallic acid (maiti et al., ; semalty et al., ) . it is hypothes ised that the main exothermic event is due to the melting of the primary excipient, tristearin and the simultaneou s dissolution of the drug and phospholipid in the melt. the significance of the dsc data is that the quercetin appears incorporate d into the matrix; an essential requiremen t with respect to stability and release profile modification. the diffractogra m of raw quercetin and quercetin slms measured using xrpd is shown in fig. . the xrpd pattern of the starting material indicated a highly crystalline structure, as evident from the sharp peaks observed at , , and of h values. this was in good agreement with a previous study of quercetin (sri et al., ) . in comparison, the slms had no characteristic peaks of a crystalline structure suggesting the particles to be amorphous. the incorporati on of the drug into the lipid matrix is likely to enhance stability of the drug and control release after deposition. this is reflected in the dvs and in vitro experiments presented in sections . . and . , respectievely . dry powder inhalers are inherentl y susceptible to moisture sorption and thus elevated relative humidity (rh). to further study the slms, the powders were exposed to two - % rh cycles using a dvs. time vs. mass plots as a function of humidity are shown in fig. a while the moisture sorption isotherm for the first humidity ramp (cycle ) is shown in fig. b . in general, all of the formulat ion components were reversible in terms of moisture sorption/des orption suggesting no irreversible polymorphic change or crystalisation phenomena . the moisture sorption was highest for phosphat idylcholine ( . % at % rh) compare d with . % and . % for quercetin and tristearin, respectively. the moisture sorption of the slms was . % at % rh. the reduction in moisture sorption for the slms in comparison to the phosphatidy lcholine (which equates for $ % of the slm particle mass) is most likely due to the contributing effects of the drug and tristearin (which make up the majority of the particle) and the presence of phosphatidylchol ine on the surface of the particles (contributing to the high humidity sorption of slms). the aerosolisation performanc e of quercetin slms was studied using the ngi and the data is presented in fig. . analysis of the stage deposition data, capsule and device recovery indicated that . % ± . % of the loaded dose was recovered across all stages (indicating good mass balance). in addition, the fine particle dose (fpd) and fine particle fraction (fpf) were calculated to be . ± . lg and . ± . %. the fpd and fpf represented the dose and percentage drug concentratio ns with an aerodynamic diameter less than . lm, suitable for inhalatio n delivery. the dose response cytotoxicity profile of quercetin slms on calu- cells was established and is shown in fig. . calu- cell cultures were exposed to a range of slms concentratio ns (from a minimum of . nm to a maximum of , nm) over a h treatment period. cells viability was calculated with reference to the untreated cells, where average absorbance was normalised to % viability. the viability assay demonst rated that calu- cells could tolerate a wide range of quercetin concentrations . the ic value could not be determined across the concentratio n range and thus, the slms should be considered non-toxic for calu- cells. teer of the calu- monolayer was measured after min of exposure to quercetin, quercetin slms, tristearin and phosphat idylcholine. this was performed to determine if any of the components in the formulation could change the integrity of the epithelial cells. it was found that there was no significant difference (p > . ) between the control cells and cells treated with quercetin ( ± x cm ), quercetin slms ( ± x cm ), tristearin ( ± x cm ), phosphatidy lcholine ( ± x cm ) or co-depositio n of tristearin and phosphatidylch oline ( ± x cm ). although to the authors' knowledge, no previous viability studies have used calu- epithelial cells, flavonoids are ubiquitous in the human diet, are highly bioavailable, and have been extensive ly studied with regard to their pharmacology and toxicology (rossand and kasum, ) . furthermor e, multiple laboratory clinical trials point to an excellent safety profile of quercetin, including studies using systemic, oral, and intravenous administrat ion (hard et al., ) . consequentl y in this study it could safely be assumed that quercetin slms are not toxic for the concentr ations studied. . . in vitro drug diffusion/tra nsport studies using an air interface calu- cell culture the percentage of drug transported through the calu- cell grown on the transwel l in the aic configuration and attached to the modified tsi apparatu s is shown in fig. . drug concentr ations were measured and expresse d in terms of percent total recovery througho ut the experiments and data was plotted as mean cumulative percentage (±standard deviation ) of drug transport over h. data analysis demonstrat es a linear kinetic profile, with a maximum of . ± . % drug transported after h from slm formulation. importan tly, quercetin was not detectable in the basal chamber h after deposition of the micronised quercetin micro particles . such observations suggest that the rate-limiting step in uptake of quercetin slms was linked to the solubility, presumably due to the amorphous nature of quercetin trapped within the solid lipid matrix, since the flux is independen t of quercetin concentration remaining on the cell monolayer. after deposition of slms on the calu- cells grown in the aic model, the concentratio n of quercetin transported into the basal fig. . quercetin slms aerosol deposition by next generation impaction. s -s represents ngi stages cut off diameters: s > . ; s > . ; s > . ; s > . ; s > . ; s > . ; s > . ; s > . lm, respectively. compartme nt at set time points was measured and the cumulati ve mass and percentage transport calculated. fig. depicts the percentage of drug transported, remaining on the cells and trapped within the cells, from the single quercetin and quercetin slms. after min . ± . % of quercetin slms and > . ± . % of quercetin alone was still found to be on the cells surface, while . ± . % and . ± . % (from slm formulation and quercetin alone, respectively ) was found 'trapped' inside the cells. only . ± . % of drug from the slm formulation was transported across the calu- and was found in the basal compartment. no quercetin raw material was transported to the basal compartme nt. phosphatidylchol ine has previously been shown to have skin penetration enhancing characterist ics in other studies (saija et al., ) . specifically, a previous study has also shown that didecanoyl-l-alpha-p hosphatidylchol ine enhances the nasal absorption of insulin, in an in vitro rabbit nasal mucosa model, by facilitating a paracellul ar passage through a reversible opening of tight junctions (carstens et al., ) . here we studied the deposition and transport of both quercetin slm and micronis ed quercetin, alone. the quantities deposited were different between the slm ( . lg) and micronised material ( . lg) since we attempted to achieve equivalent deposited masses of powder at the epithelia (i.e. including the lipid components ). in addition, the higher mass of micronised sample was additionally tested to study if micronised material would be transported at all. only quercetin from slm produced any epithelial transport across the cell line (although some internalizat ion of quercetin was observed for the micronized samples). the enhancement effect achieved by the lipid micropartic les could be traced to a more effective delivery of quercetin to the cells due to increased contact surface between the encapsul ated substance and the cells. in addition, since the lipid microparticles enable slow release of quercetin at the cell surface over time, the extent of decompo sition/oxidativ e degradation is reduced compared to the free drug, which is immediately available. lipid carrier with incorporated drugs has been demonstrat ed to increase the absorption and circulation in the body versus standalone compound s (maeda, ) . for nontoxic enhancer s that have proven successful in in vivo studies, additional mechanis ms to paracellular enhancement have been suggested (gizurarson and bechgaard, ) . for instance, increased cell membrane permeability caused by enhancer incorporation into the luminal cell membrane has been suggested (muranishi, ) , but how this could induce drug transcellular absorption is unclear. although this study indicates that the absorption of quercetin is enhanced by the presence of phosphat idylcholine or tristearin, the real mechanism is still unclear. it is possible that the phospholipid may be incorporated into the luminal cell membrane, and this may constitute the primary step in the mechanism by which phosphatidylcholine affects tight junction permeability. furthermore, changes in the lipid environm ent of the cell membrane may affect the basic transport properties of the calu- epithelium. additional studies are needed to evaluate this possibility. this study has investigated the formulation and aerosol delivery to the lung of solid lipid quercetin microparticles as a potential active pharmaceuti cal ingredient for asthma therapy. we have demonstrat ed slm particle technology can be used to deliver this naturally derived flavonoid for the treatment of range of respira-tory diseases. the effect of the absorption enhancing non-toxic excipients in increasing the permeabilit y of quercetin has also been highlight ed; making this approach significant, since the raw micronis ed quercetin was not transported across epithelia. to the authors knowled ge no previous studies have investigated quercetin transport mechanis m using calu- cell epithelial monolayer. this is an important aspect of the study since the lung epithelia will be the first barrier after deposition in the airways. further studies are necessary to elucidate the exact mechanism. due to the natural physiologica l composition of the quercetin and its components in the final slms, this system has the potential to be used as a 'bio-comp atible' delivery system to modulate the release of inhaled dry powder formulat ions. quercetin stimulates na+/k+/ cl À cotransport via ptk-dependent mechanisms in human airway epithelium quercetin/betacyclodextrin solid complexes prepared in aqueous solution followed by spraydrying or by physical mixture transport of insulin across rabbit nasal mucosa in vitro induced by didecanoyl-l-alphaphosphatidylcholine binding interaction of quercetin- -beta-galactoside and its synthetic derivatives with sars-cov cl(pro): structure-activity relationship studies reveal salient pharmacophore features in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids thermal characterization of the quercetin and rutin flavonoids quercetin reduces susceptibility to influenza infection following stressful exercise effects of propolis flavonoids on virus infectivity and replication rational design of solid aerosols for immunoglobulin delivery by modulation of aerodynamic and release characteristics safety-assessment of -methoxyquercetin as an antirhinoviral compound for nasal application: effect on ciliary beat frequency review of the biology of quercetin and related bioflavonoids liposomal quercetin: evaluating drug delivery in vitro and biodistribution in vivo intranasal administration of insulin to humans pharmacokinetics and bioavailability of quercetin glycosides in humans time-and passage-dependent characteristics of a calu- respiratory epithelial cell model deposition, diffusion and transport mechanism of dry powder microparticulate salbutamol, at the respiratory epithelia re-evaluation of the kidney tumors and renal histopathology occurring in a -year rat carcinogenicity bioassay of quercetin flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups polymorphism of triglycerides -a crystallographic review solid lipid microparticles: formulation, preparation, characterisation, drug release and applications acute bronchodilator effect of quercetin in experimental allergic asthma fabrication of quercetin nanocrystals: comparison of different methods antiviral effect of flavonoids on human viruses anti-inflammatory effect and mechanism of proanthocyanidins from grape seeds enhancement of gastrointestinal absorption of quercetin by solid lipid nanoparticles the polymorphism of tristearin and some of its homologs the enhanced permeability and retention (epr) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting curcuminphospholipid complex: preparation, therapeutic evaluation and pharmacokinetic study in rats solid lipid budesonide microparticles for controlled release inhalation therapy quercetin inhalation inhibits the asthmatic responses by exposure to aerosolizedovalbumin in conscious guinea-pigs quercetin pharmacokinetics in humans absorption enhancers flavonoids: a review of probable mechanisms of action and potential applications quercetin regulates th /th balance in a murine model of asthma inhalation delivery of therapeutic peptides and proteins effect of polyphenolic compounds on the proteolytic activities of constitutive and immuno-proteasomes online: vol. iii, tso, information & publishing solutions antioxidative and prooxidative effects of quercetin on a cells dietary flavonoids: bioavailability, metabolic effects, and safety the mesomorphic phase behaviour of lipid bilayers influence of different penetration enhancers on in vitro skin permeation and in vivo photoprotective effect of flavonoids preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration preparation and in vitro evaluation of salbutamol-loaded lipid microparticles for sustained release pulmonary therapy incorporation of quercetin in lipid microparticles: effect on photo-and chemical-stability preparation and characterization of phospholipid complexes of naringenin for effective drug delivery preparation and characterization of quercetin and rutin cyclodextrin inclusion complexes glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating lps-induced nf-kappa b key: cord- - ol i authors: singh, bhupinder; lohan, shikha; sandhu, premjeet s.; jain, atul; mehta, surinder kumar title: functionalized carbon nanotubes and their promising applications in therapeutics and diagnostics date: - - journal: nanobiomaterials in medical imaging doi: . /b - - - - . - sha: doc_id: cord_uid: ol i carbon nanotubes (cnts) have attracted much attention from researchers worldwide in recent years due to their high aspect ratio, high surface area, and excellent material properties, such as electrical and thermal conductivities and mechanical strength. these rolled-up seamless cylinders of graphene sheets possess nanosized hollow-tube-shaped structures. the cnts can be single-walled, double-walled or multi-walled, depending upon the number of graphene layers from which a single nanotube is composed. the cnts, favoring encapsulation of drug molecules or by possible attachment of theranostic agents on the nanotube walls, have enabled their use in controlled drug delivery, and in targeting of drug molecules to specific sites such as the lymphatic system, brain, ocular system, and cancerous tissue. this chapter provides an overview of various types of cnts, methods utilized for their commercial production, and the functionalization approaches employed in drug-delivery applications. in addition, the chapter also endeavors to provide a thoughtful insight into the toxicity and regulatory concerns that need to be addressed before the cnts can be launched in the market. cnts can be broadly classified into two types based on their wall structure, that is, swcnts and mwcnts (foldvari and bagonluri, ) . in swcnts, a single graphene sheet is folded to form a cylindrical closed structure, whereas in case of mwcnts, around À sheets of single-walled nanotubes are rolled upon each other to form a multilayered structure. in addition, the swcnts may further be categorized based on the differences in the arrangements of their carbon atoms. the armchair arrangement is typically characterized by the chairs perpendicular to the tube axis, whereas a v-shape perpendicular to the tube axis is characteristic of the zigzag arrangement. the unique electrical, conductive properties and metallic characteristics are primarily determined by their degree of chirality. this forms the basis of designing a variety of nanoelectronic and diagnostic instruments (tessonnier and su, ) . beside structural differences, the cnts also differ from each other dimensionally. a coaxial structure, containing two concentric graphene cylinders with higher thermal and chemical stability than single-walled nanotubes has been explored recently. these are termed double-walled cnts. apart from this, several variants in cnt shapes have been seen including carbon nanohorns (cnhs), nanobuds, and nanotorus, which have enormous applications in drug delivery due to the suitability of their characteristic modified structure. figure . diagrammatically depicts the various types of cnts employed in drug delivery. further, cnts are also classified depending upon their methods of preparation, structural modifications, and solubility properties, such as functionalized cnts, cnts dispersed in solvent, surfactant-coated cnts, and conjugated cnts, which may include drugs, monoclonal antibodies, ligands, etc. heating carbon black and graphites in a controlled flame environment has been successfully utilized for the preparation of cnts for the last two decades (iijima, ) . however, nanotubes synthesized by this method are irregular in size, shape, mechanical strength, quality, and purity owing to their uncontrollable natural environment. of late, a number of artificially developed methods have been extensively utilized for their synthesis, including catalytic chemical vapor deposition (cvd), electric arc discharge, and laser ablation (awasthi et al., ) as depicted in figures . À . respectively, and table . . additionally, several newer techniques, plasma-enhanced cvd, thermal cvd, laser-assisted cvd, high-pressure cvd, cobalt-molybdenum catalytic (comocat) process, and high-pressure carbon monoxide (hipco) disproportionation process have been developed for high-quality cnt production (beg et al., ) . different methods of preparation produce cnts with different physical and mechanical properties. the types of methods usually differ from one another in terms of the type of cnts produced, solubility, mechanical properties, quality, purity, and yield. modification of cnts by introduction of a drug molecule or ligand onto the walls and/or sides of cnts is referred to as cnt functionalization (lay et al., ) . this functionalization may be utilized for their enhanced biocompatibility, enhanced encapsulation tendency, and multimodal drug delivery and imaging electric arc discharge method for the production of cnts. laser ablation technique for the preparation of cnts. (liu et al., ) . several studies on the fate of nanotubes in the body have suggested that the functionalized cnts loaded with drug molecules could easily pass into the cells and further into the cell nucleus, thus attaining targeted drug delivery both at cellular and nuclear levels (mehra et al., ) . functionalization can be of two types: noncovalent (or adsorption) or covalent. many small, as well as large, drug molecules can be adsorbed noncovalently onto the surfaces of cnts. in this manner, cnts act as nanoreservoirs to adsorb the drug molecules by hostÀguest interaction. forces that govern such adsorption are the hydrophobic and πÀπ stacking interactions between the chains of the adsorbed molecules and the surfaces of cnts. in the case of lipophilic drug moieties, the hydrophobic forces are the main driving forces for the loading of such drugs into or onto cnts. the presence of charge on the nanotube surface due to chemical treatment can enable the adsorption of the charged molecules through ionic interactions (liu et al., ; chen et al., ) . noncovalent functionalization of cnts is particularly attractive as it offers the possibility of attaching chemical modifications without affecting the electronic network of the tubes. such functionalization can be achieved simply by exposing the cnts to vapors containing functionalization species that noncovalently bond to the nanotube surface. using this functionalization process, surfactants, polymers, and biomolecules like dna, sirna, proteins, and peptides can be successfully loaded onto the surfaces of cnts (figure . ). covalent functionalization gives the more secure conjunction of drugs or functional groups (balasubramanian and burghard, ) . in order to achieve such functionalization, cnts can be oxidized using strong acids, resulting in the reduction of their length while generating carboxylic groups, thus increasing their dispersibility in aqueous solutions. alternatively, addition reactions of hydrophilic groups to the cnts external walls and tips can also make them soluble in water. to achieve such a type of functionalization with therapeutic molecules like methotrexate, chemical reactions like , -cycloaddition can be employed. complete control over such chemo-or region-selective additions, however, is somewhat tricky to achieve, as it involves particular groups such as cyclic compounds, halogens, arynes, or carbenes. moreover, such reactions often require extreme conditions for covalent bonding. further, characterization of such functionalized nanotubes to determine the precise functionalization location and mode of addition are also very difficult. it may broadly be classified as end-defect functionalization or side-wall functionalization. end-defect functionalization involves oxidation at the "end" tips, whereas covalent binding of surfactants, proteins, and peptides on the surface of cnts is referred to as side-wall functionalization. figure . schematically represents the types of functionalization possible on the cnt surface. cnts used in pharmaceutical applications need to be characterized extensively to determine their fundamental properties. the characteristic properties include diametric size, shape, purity, solubility, electromechanical properties, and thermal conductivity (foldvari and bagonluri, ) . quite a few techniques are being employed for characterizing these nanotubes, each with its pros and cons. the preliminary investigation of the morphology and impurities present in the cnt structure are presented by the use of scanning electron microscopy. transmission electron microscopy gives elucidation of the structural arrangement of cnt drug composites and provides qualitative information on size, shape, and structure of cnts. atomic force microscopy provides a three-dimensional ( d) surface profile desirable for determining the surface topology of cnts. other commonly employed techniques include thermogravimetric analysis, infrared spectroscopy, nuclear magnetic resonance (nmr), raman spectroscopy, h -nmr, and dynamic light scattering. lately, functionalized cnts have been exploited for the delivery of biomolecules and drugs to desired sites. their ability to readily cross the plasma membrane for the transport of cargo molecules renders them as interesting nanovehicles. to achieve a desired response cellular internalization needs to be extensively studied. scientists across the globe have proposed different pathways for its cellular internalization. this internalization by the cell also depends on the physical properties, surface charge, and chemical functionalization of these cnts. the other parameters influencing its interactions include degree of dispersion and formation of supramolecular complexes (lacerda et al., ) . two most commonly understood mechanisms are described below. referred to as insertion, passive diffusion, or nanoneedle mechanism, this process is quite instantaneous and cnts diffuse across the lipid bilayer in a noninvasive manner. the uniqueness of cnts in terms of high aspect ratio, cylindrical shape, and elongated form favors translocation across the barriers. the two-step process involves accommodation of the cnts on the lipidic bilayer followed by transmembrane configuration (lopez et al., ) . well-individualized mwcnts have been shown to cross cell membranes by means of an energy-dependent process employing the use of atp. some scientists have also reported that cnts of approximately nm are able to fit into caveolae and clathrin vesicles, while larger-sized cnts are taken up by means of the macro-pinocytosis pathway (mu et al., ) . figure . clearly demonstrates the receptor-mediated endocytosis mechanism for surface-engineered cnts. the receptor-mediated endocytosis mechanism for surface-engineered cnts. cnts have recently emerged as efficient carriers in the arena of drug delivery. table . provides a bird's eye view on the applications of cnts in the delivery of various therapeutic agents against diseases like cancer, and genetic and infectious disorders. an explicit account of diverse drug-delivery applications of cnts is enumerated below. the unique capability of cnts and their functional counterparts to penetrate into cells makes them interesting vehicles for the delivery of small-molecule drugs. in addition to their ability to carry one or more therapeutic agents, their recognition capacity, optical imaging signals, and targeted delivery have also been exploited for the successful development of these delivery systems. both hydrophilic and hydrophobic molecules can be bound to cnt by means of amide/ester linkages. in addition, various polymers are also grafted to their surface. researchers have also reported that pegylated mwcnts may be exploited as an efficient drug carrier to overcome multidrug resistance (mdr). these mwcnts can target and specifically accumulate in mdr tumor cells. besides, these mdr cells cannot remove intracellular mwcnts thereby effecting more efficient drug delivery. functionalized nanotubes have been employed on a large scale for targeted delivery of nucleic acids, proteins, antibodies, drugs, and other therapeutic agents to their respective sites of action. the use of cnts for targeted delivery is primarily accepted in treating various malignant disorders, which include choriocarcinoma, carcinoma of the cervix, breast cancer, prostate cancer, brain gliomas, and testicular tumors (thakare et al., ) . as drug-loaded functionalized cnts encounter problems in the release of their drug contents, these have been encapsulated in novel membrane microcapsules made up of an alginateÀpoly-l-lysine-alginate membrane (degim et al., ) . excellent drug-release profile and enhanced safety and effectiveness, owing to protection from the external harsh environment, have been exploited for their use in targeted delivery. in addition, functionalization of chitosan on their surface enhances cell attachment to the sidewalls of the nanotubes, resulting in the desired targeted release to the cells, and improved drug absorption. such systems have significant potential for the delivery of drugs, peptides, and nucleic acids. antibody-mediated drug delivery, despite being a popular drug-delivery technique, suffers from a major hiccup of antibody specificity on binding with drug molecules. nanotubes, in this regard, are at a specific advantage as these do not alter this antibody specificity and can deliver the drug at the targeted site (prakash and kulamarva, ) . monoclonal antibody-functionalized swcnts have been employed for tumor targeting and for diagnostic imaging. an important prerequisite is the compatibility of the cnts with the targeting antibodies as well as nanotubes. also, the antigenicity of antibodies must remain unaltered after their attachment to the nanotubes and subsequently to their desired site of action. in addition, this approach can be employed for the delivery of both hydrophilic and lipophilic drugs. one of the functionalization techniques, that is, pegylation, has recently gained immense importance owing to its enhanced biocompatibility, solubility, and reduced toxicity (bottini et al., ) . in addition, monoclonal antibodies on swcnts functionalized with peg have the distinct ability to target the cd cell surface receptor on b-cells selectively. this antibody-mediated approach has thus been proved to be an ideal alternative in targeting drug molecules to cancer cells. lymphatic targeting is essential to protect the body against a range of infectious diseases and malignant disorders. in addition, it helps in targeting drug molecules to the reticuloendothelial system. over the past several years, various approaches have been attempted for lymphatic targeting of the drug molecules, for example, controlled-release and magnetic microspheres. these systems, however, cause capillary blockade and chemoembolism owing to their large size. researchers have now shown that lymphatic targeting of drugs with the use of magnetic nanotubes is better accepted and employed for lymphatic targeting. because of their chemical and mechanical stability, incorporation, and controlled drug release, these cnts can be chemically modified to achieve the desired targeting effect. in addition, yang et al. ( ) indicated that the size specificity allows for effective uptake into the lymphatics. cnts, functionalized with folic acid in conjugation with magnetic nanoparticles, have shown better targeting ability to cancer cells in the lymph nodes. in addition, these mwcnts can be retained at the target site by means of an externally placed magnetic field. brain targeting of drug molecules is hampered by the impermeable bloodÀbrain barrier, which restricts the entry of substances so as to maintain the internal milieu of the brain. in addition, the presence of enzymes in the brain degrades the few neuropharmaceutical agents that manage to cross the impermeable barrier. conventional drug-delivery systems for that matter, fail to deliver drugs effectively into the brain. of late, cnts have been exploited for the brain targeting of drug molecules, primarily because of their inherent ability to cross the impermeable barrier (yang et al., ) . in this regard, mwcnts are quite effective in delivering these agents to the inner environment of brain microglial cells. recently, au/cnt hybrid nanomaterials have been exploited for detection of dopamine and ascorbic acid owing to the biocompatibility of aunps and unique electronic properties and ease of surface modification of cnts (vinoth et al., ) . additionally, magnetic properties bestowed by these nanotubes have been exploited for treating neurodegenerative disorders. in combination with nerve growth factors, these cnts also enable cells to differentiate into neurons and, thus, provide effective management for neurodegenerative disorders. the potential of cnts has also been used for ocular drug delivery. these help in specific and local targeting of drug molecules to the retinal site. examples of drugs which have been delivered for ocular delivery of drugs include antibiotics, anticholinergics, mydriatics, etc. (sinha and yeow, ) . however, limited research work has been carried out in this area so far, thus calling for further exploration of cnt applications in ocular delivery. the rampant spread of cancer in society has posed a great challenge for scientists across the globe. the biggest challenge, therefore, is to treat malignant cells without their "spillover" to neighboring cells. conventional chemotherapy has the disadvantage of nonspecificity and in more than % of cases normal cells are destroyed along with the cancerous tissues. cnts, in this regard, have been investigated for in vivo distribution and highly effective tumor targeting for cancer therapy in mice. investigations are being done on the biodistribution of radio-labeled swcnts in mice by in vivo positron emission tomography, ex vivo biodistribution, and raman spectroscopy. it has been found that swcnts that are functionalized with phospholipids bearing polyethylene glycol (peg) are surprisingly stable in vivo. cnts loaded with chemotherapeutic agents have been shown to achieve relatively higher uptake by cancerous tissue without affecting collateral tissues. consequently, nanotubes may also be beneficial in dosage reduction by localizing dose distribution at the tumor site. this can further be fortified by functionalizing the drug-containing cnts with an antibody molecule and targeting it to the antigen of cancer cells. recently, cnts have been reported to exhibit their own cancer-curing properties as well when exposed to an infrared light source (zhou et al., ) . such heated tubes, especially mwcnts, when placed at a tumor site, specifically destroy malignant cells and seem to act as a tumoricidal agent. attachment of cnts to folic acid, a tumor marker, has also been used for programmed cell death. this process of cancer treatment is generally referred to as photothermal therapy. similarly, mwcnts doped with nitrogen gas have been found to induce thermal ablation causing apoptosis (i.e., programmed death) of cancer cells upon irradiation with an infrared beam (torti et al., ) . it can thus be concluded that the antitumor activity of cnts might be due to heat transduction, which leads to less cellular cytotoxicity (figure . ) . literature reports also suggest increased tumoricidal activity of mwcnts when functionalized with dna and sirna. swcntÀsirna complexes have been demonstrated to have effective and prolonged suppression of tumor growth in comparison to earlier available tools for sirna delivery. it has been observed that functionalized swcnts are an effective solution in reducing the progression of human myelogenous leukemia. apart from the delivery of pharmaceuticals and/or nucleic acids to malignant cells, recent investigations have proved their importance in radiotherapy for cancer treatment by increasing the rate of oxygen uptake to malignant cells, thereby making the therapy more efficacious. apart from the cnts, cnhs are under investigation for exploring their application in chemotherapy. watersoluble cnhs have also been screened for the delivery of anticancer agents with some promising results (murakami et al., ) . the application of cnts as reinforcing agents has been mainly attributed to their excellent and unique electrical and mechanical properties (erik and tsu-wei, ) . cnts may be the best tissue engineering candidate among numerous other materials of natural or synthetic origin for tissue scaffolds. in addition, they are less dense, highly flexible, and have a very high young's modulus representing good stiffness. these properties are utilized to make lighter scaffolds with very high strength (iijima et al., ) . it has also been shown by various researchers that the material surfacefree energies also play an important role in influencing cell adhesion, leading to greater tissue regeneration. surface functionalization of these cnts further imparts to them biocompatibility and biodegradability, ideal for their use in the body. controlled delivery of drugs and genetic material such as dna, genes, and/or antibodies has challenged pharmaceutical scientists for years. of late, cnts have attracted much attention due to their ability to deliver drug molecules to a specific site in a controlled manner (luo et al., ) . studies carried out by yang et al. ( ) on amine-functionalized mesoporous silica nanotubes (nh -msnts), with additional functionalization by cds quantum dots (qds), have been found shown to deliver anti-inflammatory drugs in a controlled manner. furthermore, carboxyl functionalized mwcnts (mwcnt-cooh) along with nanohybrid hydrogels have also been employed as controlled drug-delivery vehicles. such systems show low micropore densities and large mesh sizes with an increase in mwcnt-cooh content. the ph responsiveness conferred by the unique combination upon contact with water produces controlled drug-release profiles. recently, "smart bio-nanotubes," a new generation of nanomaterials, have been developed. these trilayered structures are able to regulate the rate of drug release by tailoring the thicknesses of protein lipid and protein coats. highly efficient electrophoretic and iontophoretic pumping has been accomplished through functionalized cnt membrane due to the high charge density, dramatically quick flow of drugs through the cnt cores, and small pore dimensions (degim et al., ) . cnt membranes are usually integrated with the drug molecule to obtain a switchable transdermal delivery device. these highly energy-efficient programmable devices offer minimal skin irritation without any skin barrier disruption. utilizing this concept, mwcnts for transdermal delivery of nicotine have been fabricated through the cvd approach. improper absorption, antigen-induced hypersensitivity, anaphylactic reactions, and hypersensitivity due to vaccine adjuvant are some of the serious drawbacks associated with vaccine delivery. among the new approaches suggested for delivery of vaccines, cnts have also been tried for vaccine delivery, where these help in improving vaccine action due to their adjuvant action (bianco et al., ) . in addition, cnts, when conjugated with antigenic peptides, can act as a new system for safe and effective delivery of synthetic vaccines. gene delivery or gene therapy is an approach in which a defective gene, which is the cause of some chronic hereditary disease, is corrected by introducing a dna molecule into the cell nucleus. cnts have been quite widely exploited for improving gene delivery owing to their capability of replacement of damaged/ missing genes, and transportation of dna into cells (bianco, ) . attempts have been made for treating gene defects by transporting grafted genes with the help of nanotubes (pan et al., ). ammonium-functionalized cnts have been demonstrated to enhance gene therapy in comparison with dna alone . research has also indicated that sirna and swcnt complex can be easily taken up by splenic immune-recognizing cells such as cd c cells, cd b cells, and gr- cd b cells to induce the immune response for the particular gene. it has also been observed that when single-stranded dna is bound to swcnts, dna probes are protected from enzymatic cleavage and interference from nucleic-acid-binding proteins. studies have shown that an swcnt-modified dna probe can target a specific mrna inside living cells, thus causing increased self-delivery capability and intracellular biostability, compared with free dna probes. in contrast to the traditional methods available for gene delivery, cnts provide distinct advantages like enhanced capability to penetrate into the cells owing to their needle-like shape, hydrophobic surface, and their electrical properties. in addition, their capacity to achieve controlled release, influence on transitions of dna/sirna, and ability to monitor the therapeutic effects provides this conjugate with great potential for applications in the field of genetic engineering (cheung et al., ) . infectious diseases, such as tuberculosis, leishmaniasis, severe acute respiratory syndrome, and flu (swine, bird, and avian), have always been a critical public health issue with global concerns. these infectious agents show a high level of resistance against numerous antivirus and antibacterial drugs. recently, functionalized cnts have shown promising outcomes in the treatment of these diseases owing to their ability to easily conjugate drugs like amphotericin b (vinoth et al., ) , dapsone, etc. (mehra et al., ) . conjugation of amb to these f-cnts has been shown to have reduced toxicity and enhanced antimycotic efficiency. in addition, their targeted deliveries to macrophage cells have also been indicated by researchers. furthermore, cnts themselves might possess antimicrobial activity as bacteria, such as escherichia coli may be absorbed onto the surface of the cnts. the cnt might induce oxidation of the intercellular oxidant glutathione, resulting in increased oxidative stress on the bacterial cells and thus eventual cell death. the potential role of cnts as antioxidants is still an emerging area of research. cnts, and in particular carboxylated swcnts, have been reported as antioxidants in nature and may possess useful biomedical applications in preventing chronic ailments, aging, and in food preservation as well. such potential calls for more investigations of different forms of cnts to develop their precious effect as free radical scavengers. antitumor immunotherapy consists of stimulating the patient's immune system to act against malignant tumor cells. studies have shown that cnts can be effectively used as carriers for a cancer vaccine or a therapeutic antibody as a drug. biosensors in the field of diagnostics have made an enormous impact on basic scientific research and healthcare. these detect chemical, physical or biological quantity and transduce it in the form of a signal. fast electron transfer rate, wide potential window, flexible surface chemistry, and good biocompatibility of cnts give them excellent property for their use in biosensors. for example, cnts have been effectively coupled with glucose-oxidase biosensors for blood sugar control in diabetic patients. various mechanisms and properties of cnts have been utilized in construction of these molecular machineries. figure . elucidates different sensing mechanisms of cnt-based biosensors. lately, research has revealed that cnts can also generate qds, or may behave like qds. basically, these are semiconductor nanocrystals with nanosize range. they are diode substances capable of emitting light of various colors. due to their light-emitting property, they have potential applications in imaging various body parts (lim et al., ) . cnts are capable of mimicking these lightemitting nanoparticles. cisplatin and epidermal growth factors (egfs) have been attached to swcnts, specifically to target squamous cell cancer (bhirde et al., ) . also, swcnt-qdÀegf bioconjugates have been shown to possess better cell internalization properties compared to plain cnts. sensing mechanism of cnt-based biosensors. in comparison to bulk materials, nanotubes have unique properties including an exponentially high aspect ratio that leads to toxicity. reduction of size consequently makes the nanomaterial surface more reactive on itself (i.e., aggregation) and its surrounding environment (i.e., biological components). accumulation of nanosized material may also cause increased uptake into tissues, thus influencing the critical biological function of cells. besides, metal traces available in nanotubes are also another causative agent for toxicity (beg et al., ) . exposure to cnts causes multifunctional defects in various body organs and organ systems. early systemic exposure of mwcnts causes temporary organ injury, particularly to lungs and heart, attributable largely to their delayed clearance from the body due to strong agglomeration. persistent accumulation of agglomerated mwcnts in the lungs initiates inflammatory events (takagi et al., ) . similarly, swcnts have been found to affect the cns due to a high degree of accumulation in the spinal cord or dorsal root ganglia. furthermore, swcnts significantly decrease the dna content when the cells were exposed to disperse swcnt bundles due to their accumulation in the nuclear material. also, exposure to swcnts has resulted in ultrastructural and morphological changes in cultured skin cells (yacobi et al., ) . comparative evaluation of toxicity studies has revealed that mwcnts are more toxic as compared to swcnts, and induce massive loss of cell viability through programmed cell death. this toxicity of mwcnts is primarily due to oxidative stress, cellular toxicity due to formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability as depicted in figure . . cnts also exhibit primary genotoxicity due to the direct interaction of particles with cells, or secondary genotoxicity due to the generation of an excess of ros. mwcnts have genotoxicity effects due to dna damage through ros, leading to increased mutation frequencies (sargent et al., ) . recently, researchers have warranted the elucidation of an alternative, nonoxidative stress-mediated pathway of cellular damage. physical interference of cnt with cellular and extracellular constituents is also one of the potentially relevant mechanisms of damage. this physical interference may cause alterations of vital cellular processes, leading to various degrees of cellular injury, and in some cases even to cell death. despite the aforementioned literature reports, the present knowledge of cnt toxicity is inadequate and contradictory, thus still requiring more extensive toxicity, safety, and efficacy studies on animal models including humans. also, effects of cnt aggregation, size, length, functionalization, metal impurities, and polymers on safety require more extensive research. functionalization of swcnts and mwcnts and its effects on aggregation and consequently genotoxicity also need to be evaluated (uo et al., ) . the biodistribution of cnts is not significantly influenced by the route of administration. researchers have shown that these cnts distribute quickly throughout the whole body, with preferential organs being the stomach, kidneys, and bone. approximately % of cnts are shown to excrete into the urine and % in the feces. recently, analyses of urine samples by workers have revealed that both swcnt and mwcnt are excreted as intact nanotubes. the preferred sites of accumulation were found to be the stomach, kidneys, or bones, but no tissue damage or distress was reported. functionalizations of these swcnts have also been shown to prolong the swcnt blood circulation, reduced uptake in the reticuloendothelial system, and complete clearance from major organs (shvedova et al., ). awareness of nanotechnology has dramatically risen in recent years among lawmakers, regulators, and environmental activists. accordingly, the question of whether, and how well, to regulate nanotechnology is not new. to date, increasing interest in the field of development and nanotechnology applications of nanomaterials has had an impact on development of strict regulatory norms in their production and use in animals as well as in humans. as per the reports of the us environmental protection agency (arepalli, ) , cnts require major regulatory concern over toxicity as well as environmental safety (lewinski, ) . france is in the process of publishing a series of technical guidance documents related to nanotechnologies including cnts as fr , fr , and fr . similarly, the uk government has committed toward ehs research, primarily with new studies on safety issues of specific nanomaterials, in particular nanosilver and cnts with the unites states as uk , uk , uk , and oecd . of late, the national environmental policy act (nepa) of united states has issued certain regulations for cnts and other nanomaterials. the primary considerations for regulating these nanomaterials include product quality assessment (i.e., quality control and manufacturing) and product safety assessment (i.e., biodistribution, clearance, metabolism, and toxicity). cnts have been proposed and explored as multipurpose innovative carriers for drug delivery and diagnostic applications. as a consequence, in a very short time span, cnts have drawn the attention of nanotechnologists, from industry as well as academia. in the last two decades, remarkable work has been carried out on the use of cnts for biomedical applications. functionalization of cnts has further opened new perspectives in the application of cnts in drug delivery. attachment of organic moiety to nanosized tubes has facilitated their use for diagnostic as well as targeting purposes, especially in cancer therapy and infectious disease treatment. however, despite their promising role in nanomedicine, cnts still require extensive research investigations to guarantee their safety profile in drug delivery. toxicity studies, therefore, are critical to establish the full in vivo potential of cnts for drug delivery before their real application and marketing. physiological, physicochemical, and molecular processes need to be considered for understanding of clinical and preclinical toxicity of cnts. regardless of knowledge gained in recent years on nanotoxicology; scientists have not yet been able to precisely forecast the behavior and biokinetics of cnts. furthermore, before the pharmaceutical commercialization of cnts, strict regulations are mandatory taking into their ambit the environmental, health and safety issues. however, as alarmingly high numbers of reports are piling up, it can be rationally anticipated that cnts have a golden future in drug delivery. multiwalled carbon nanotube-doxorubicin supramolecular complexes for cancer therapeutics laser ablation process for single-walled carbon nanotube production synthesis of carbon nanotubes growth, structure, and properties of graphite whiskers advancement in carbon nanotubes: basics, biomedical applications and toxicity cobalt-catalysed growth of carbon nanotubes with single-atomic-layer walls polymer functionalized single walled carbon nanotubes mediated drug delivery of gliotoxin in cancer cells targeted killing of cancer cells in vivo and in vitro with egf-directed carbon nanotube-based drug delivery distribution and clearance of peg-single-walled carbon nanotube cancer drug delivery vehicles in mice carbon nanotubes for the delivery of therapeutic molecules applications of carbon nanotubes in drug delivery peg-modified carbon nanotubes in biomedicine: current status and challenges ahead noncovalent engineering of carbon nanotube surfaces by rigid, functional conjugated polymers dna and carbon nanotubes as medicine hyaluronate tethered, "smart" multiwalled carbon nanotubes for tumor-targeted delivery of doxorubicin carbon nanotubes for transdermal drug delivery aligned multi-walled carbon nanotube-reinforced composites: processing and mechanical characterization soluble single-walled carbon nanotubes as longboat delivery systems for platinum(iv) anticancer drug design carbon nanotubes as functional excipients for nanomedicines: i. pharmaceutical properties structural flexibility of carbon nanotubes single-shell carbon nanotubes of -nm diameter production of cnt-taxol-embedded pcl microspheres using an ammonium-based room temperature ionic liquid: as a sustained drug delivery system chemical vapor deposition of carbon nanotubes: a review on growth mechanism and mass production carbon nanotubes as nanomedicines: from toxicology to pharmacology functionalized carbon nanotubes for anticancer drug delivery nanotechnology policy and environmental regulatory issues selection of quantum dot wavelengths for biomedical assays and imaging supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery drug delivery with carbon nanotubes for in vivo cancer treatment carbon nanotubes in biology and medicine: in vitro and in vivo detection, imaging and drug delivery solubilizing carbon nanotubes through noncovalent functionalization. insight from the reversible wrapping of alginic acid around a single-walled carbon nanotube development and characterization of dexamethasone mesylate anchored on multi walled carbon nanotubes. drug target understanding nature's design for a nanosyringe advances in bioapplications of carbon nanotubes dual targeted delivery of doxorubicin to cancer cells using folate-conjugated magnetic multi-walled carbon nanotubes carbon nanotube nanoreservior for controlled release of anti-inflammatory dexamethasone pharmaceutical and biomedical applications of surface engineered carbon nanotubes endosomal leakage and nuclear translocation of multiwalled carbon nanotubes: developing a model for cell uptake effect of functionalisation of cnt in the preparation of hapÀcnt biocomposites solubilization of singlewall carbon nanohorns using a peg-doxorubicin conjugate filamentous growth of carbon through benzene decomposition synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems hydroxyapatite-magnetite-mwcnt nanocomposite as a biocompatible multifunctional drug delivery system for bone tissue engineering recent advances in drug delivery: potential and limitations of carbon nanotubes. recent pat functionalized carbon nanotubes in drug design and discovery macrophages targeting of amphotericin b through mannosylated multiwalled carbon nanotubes the targeted delivery of anticancer drugs to brain glioma by pegylated oxidized multi-walled carbon nanotubes modified with angiopep- enhanced anticancer activity of multi-walled carbon nanotube-methotrexate conjugates using cleavable linkers potential pulmonary effects of engineered carbon nanotubes: in vitro genotoxic effects enhanced neuronal cell differentiation combining biomimetic peptides and a carbon nanotube-polymer scaffold antibacterial activity and cytotoxicity of multi-walled carbon nanotubes decorated with silver nanoparticles mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: two faces of janus? carbon nanotubes for biomedical applications reversible targeting and controlled release delivery of daunorubicin to cancer cells by aptamer-wrapped carbon nanotubes induction of mesothelioma in p / mouse by intraperitoneal application of multiwall carbon nanotube carbon fibrils, method for producing same and composition containing same recent progress on the growth mechanism of carbon nanotubes: a review carbon nanotubes in cancer theragnosis thermal ablation therapeutics based on cn(x) multi-walled nanotubes toxicity evaluations of various carbon nanomaterials sirna delivery with functionalized carbon nanotubes simultaneous detection of dopamine and ascorbic acid using silicate network interlinked gold nanoparticles and multiwalled carbon nanotubes targeted delivery of amphotericin b to cells by using functionalized carbon nanotubes covalently combining carbon nanotubes with anticancer agent: preparation and antitumor activity nanoparticle effects on rat alveolar epithelial cell monolayer barrier properties magnetic lymphatic targeting drug delivery system using carbon nanotubes fluorescent mesoporous silica nanotubes incorporating cds quantum dots for controlled release of ibuprofen pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating alzheimer disease targeted delivery and controlled release of doxorubicin to cancer cells using modified single wall carbon nanotubes cancer photothermal therapy in the near-infrared region by using single-walled carbon nanotubes key: cord- -eja fkwv authors: iftikhar, hafsa; ali, hafiza nayyer; farooq, sadia; naveed, hammad; shahzad-ul-hussan, syed title: identification of potential inhibitors of three key enzymes of sars-cov using computational approach date: - - journal: comput biol med doi: . /j.compbiomed. . sha: doc_id: cord_uid: eja fkwv the recent outbreak of coronavirus disease- (covid- ) continues to drastically affect healthcare throughout the world. to date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection. therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against sars-cov- for lead identification and repurposing prospects. this study addresses the identification of small molecules that specifically bind to any of the three essential proteins (rdrp, cl-protease and helicase) of sars-cov- . by applying computational approaches we screened a library of compounds also containing fda-approved drugs against these viral proteins. shortlisted hits from initial screening were subjected to iterative docking with the respective proteins. ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds. finally, to minimize chances of false positives, we performed docking of the identified molecules with irrelevant proteins of diverse classes thereby ruling out the non-specific binding. three fda-approved drugs showed binding to cl-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation. a drug-like molecule showed binding to rdrp in its catalytic pocket blocking the key catalytic residues. two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis. this study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects. covid- after its emergence in china, continues to spread across the globe leading to global health emergencies and accounting for devastating economic and social impacts [ , ] . as of april , , over . million people have been confirmed with covid- leading to over , deaths since the emergence of the infection [ ] . to date, no specific treatment regimen is available against covid- . however, previously approved antiviral drug remdesivir and an antimalarial drug chloroquine have shown promising effects, at least in reducing the duration of symptoms of covid- in limited clinical studies [ , ] . sars-cov- that causes this disease belongs to the family coronaviridae and genus coronavirus [ ] . angiotensin-converting enzyme (ace ) that is expressed on the cellular surfaces of different organs including lungs has been identified as the primary receptor of the virus in addition to the role of another host protease enzyme, transmembrane serine protease (tmprss ) in viral entry [ ] [ ] [ ] . the viral genome consists of , base pairs that encode different structural and non-structural proteins. the structural proteins include the membrane protein, spike, envelope and nucleocapsid. non-structural proteins include nsp -nsp . nsp (papain-like protease), nsp ( -chymotrypsin-like protease " -cl-protease"), nsp (rna-dependent rna polymerase), nsp (helicase), nsp (n -methyltransferase) and nsp ( -o-methyl transferase) are important viral enzymes while nsp -nsp are regulatory proteins. the sars-cov- orf a and orf ab genes encode polyprotein a (pp a) and polyprotein ab (pp ab), respectively. these polyproteins are further processed by protease enzymes to produce different functional proteins. in this regard, cl-protease cleaves the polyprotein at different specific positions thereby producing individual functional proteins [ ] . sars-cov- is a positive sense rna virus that requires rnatemplated rna synthesis. rna-dependent rna polymerase (rdrp) catalyzes the synthesis of new viral rna and plays a crucial role in the sars-cov- replication cycle [ ] . similarly, sars-cov- helicase (nsp ) is also crucial for the viral replication and proliferation as it catalyzes the unwinding of duplex rna and dna into single strand nucleic acid chain during replication [ ] . considering an urgent need of containing the pandemic, repurposing of previously approved drugs to use against sars-cov- is the first choice in the fight against this virus, as such drugs are not required to pass through most of the steps of an extensive drug testing process. in this regard, several recent studies have been conducted using computational methods to screen libraries of approved drugs or drug-like molecules to identify potential inhibitors of different viral proteins, particularly, rdrp and cl-protease [ ] [ ] [ ] [ ] [ ] . moreover, high throughput in vitro screening of fda approved drug libraries followed by in vivo validation has also been applied on limited hits [ , ] . many of the reported virtual screening based studies have targeted the fda approved drugs and/or dug-like compound libraries. since a vast range of compound libraries exist therefore, multiple efforts to screen diverse compound libraries are important to explore a large chemical landscape to identify potential inhibitors or the lead structures to design inhibitors of sars-cov- . here, we applied a computer aided drug discovery approach by targeting three important enzymes (rdrp, cl-protease and helicase) of sars-cov- and identified three fda-approved drugs and three other drug-like molecules as potential therapeutics. structures of sars-cov- rdrp and helicase were modeled on the basis of amino acid sequence homology using swismodel as crystal structure of these proteins are yet to be solved [ ] . the genome sequence of sars-cov (ncbi reference sequence: nc_ . ) was used to model the structures of rdrp and helicase [ ] . the model accuracy and its stereochemical quality was assessed using the procheck analysis tools, which predicts the quality of modeled structure on the basis of distribution of backbone phi/psi angles with reference to the ramachandran plot [ ] . recently published crystal structure of sars-cov- cl-protease (pdb id, lu ) was used for screening and docking simulations [ ] . first, a library of compounds also containing several fda approved drugs was screened through mtiopen screen automated virtual screening platform using the integrated vina autodock program against all three proteins [ ] . hits from virtual screening were shortlisted on the basis of binding energy based vina empirical scoring function using À kcal mol À as the threshold [ ] . in this regard, , [ ] and compounds were shortlisted against cl-protease, rdrp and helicase, respectively and were downloaded from the zinc database [ ] . in parallel, fda approved antiviral drugs with random targets were selected and their structure files were obtained from drugbank in the sdf file format [ ] . format of these compounds were converted from smiles into pdb using an online tool (online smiles translator, nci) [ ] . the autodock tools . . program was used to prepare ligand and protein structures for further docking [ ] . in ligand preparation, gasteiger charges were computed, nonpolar hydrogen atoms were merged, torsion angles for all rotatable bonds were set as flexible and the file was saved in the pdbq format. similarly, protein files were prepared using automated functions of the autodock tools that added all hydrogen atoms, computed gasteiger charges, merged non-polar hydrogen atoms and created pdbqt files. each of the shortlisted compounds was subjected to iterative docking with the respective protein using the autodock . . program [ ] . docking was performed by lamarckian genetic algorithm (lga) method using , generations and by applying at least iterations. pre-calculation of grid parameters for each protein was performed by using the autogrid program. during docking, the size of grids was kept at maximum covering the whole surface of the protein to allow the ligand to bind in an unbiased binding pocket. the docking results were analyzed on the basis of a combination of binding energy, clustering score, shape complementarity, functional significance of the binding pocket and favorable interactions including h bonds and hydrophobic interactions. we used integrated clustering tool of the autodock . . program to cluster different docked structures of a ligand on the basis of root mean square deviation (rmsd) of Å. the binding energy of every docked structure was predicted by the autodock program. thirty percent population of the docked structures in a cluster with À . kcal mol À binding energy was used as a cutoff for the first ranking of iterative docking using the autodock tools program. a cluster containing the highest number of docked structures was then selected. to further illuminate h bonds and hydrophobic interactions the ligplot þ program was used [ ] . the binding pockets with known functional significance on the basis of previously reported experimental data were considered. shape complementarity was superficially accessed by visual analysis of the docked structures. to further rule out non-specificity, protein families were selected randomly from pfam to check for the specificity of the drug with a particular target. the details of the representative proteins from each family are given in table s . eight drugs or organic molecules (table ) , which showed binding to any of the three target proteins of sars-cov- , were subjected to docking simulation with all the proteins by using the autodock . . program. pre-calculation of grid parameters for each protein was performed by using the autogrid program. to rule out non-specific binding, docking was performed by lamarckian genetic algorithm (lga) method. the significance measure (probability of obtaining a better binding score by chance) for a drug was calculated by comparing the binding scores obtained for specific target (three sars-cov proteins) with the binding scores for structures belonging to random protein families. recently published x-ray structure of cl-protease (pdb id: lu ) was used for virtual screening [ ] . a library consisting of compounds also including fda approved drugs was screened and after table predicted binding scores of grazoprevir with different hcv ns / a x-ray structures. predicted binding score with grazoprevir À . À . À . À . À . applying first ranking with predicted binding energy cutoff of À . kcal mol À , compounds were shortlisted. detailed iterative docking and subsequent analysis resulted in the identification of three sars-cov- cl-protease binding compounds as potential inhibitors. rimantadine is an anti-flu fda-approved drug with generic name as flumadine. the known target of rimantadine is the m-protein of influenza-a virus [ ] . we observed that rimantadine bound to sars-cov- cl-protease at its substrate-binding site blocking about half of the binding pocket. two highly conserved amino acid residues his- and cys- are the main catalytic residues of cl-protease of corona viruses, however, many residues around these catalytic residues also play key role in the binding of peptide substrate and its subsequent catalysis. for example, his- and phe- stabilize the position of glutamine present at the p position (gln-p ) of the peptide substrate by developing two h bonds between the nΣ atom of his- and main carbonyl oxygen of phe- with the oΣ and nΣ atoms of gln-p , respectively [ ] . in our docked structure of rimantadine, the adamantyl ring of the drug perfectly fits in the sub-pocket of the substrate binding site occupying the space between his- and ph- of the protease and stabilized by hydrophobic and van der waals interactions (fig. a) . in the previously reported structure of his- mutant of sars-cov cl-protease in complex with its peptide substrate, carbonyl oxygen of gln-p of the substrate occupies position in the oxyanion hole formed by the amide groups of gly- and cys- [ ] . in the docked structure the nitrogen atom of amino group of rimantadine occupies the position close to the oxyanion hole and is stabilized by four potential h bonds with backbone nitrogen of cys- , carbonyl oxygen of leu- and backbone nitrogen and side chain oxygen of ser- (fig. b) , thereby blocking the critical residues of the protease required for catalysis. another drug, bagrosin was observed to bind at a potential allosteric site of the protease. bagrosin is an anti-epileptic drug that belongs to a class of hydantoin derivatives (anticonvulsant compounds). it works by blocking the voltage-gated sodium channels of neurons and causes inhibition of calcium flux across neuronal membranes thereby stabilizing neurons. compared to other hydantoin derivatives, bagrosin has been reported to be safer in clinical trials [ , ] . sars-cov cl-protease has been reported to form a homodimer resulting in better catalytic efficiency as compared to its monomeric form [ , ] . the recently reported x-ray structure of sars-cov cl-protease is very similar to that of sars-cov forming a dimer. the dimer formation is primarily mediated by several hydrophobic interactions between domain- of each monomer ( fig. a ) [ ] . we observed that bagrosin bound in a hydrophobic pocket in domain- of the protease and can potentially interfere with the dimer formation. in the docked structure, the phenanthryl ring of the compound perfectly fits in a hydrophobic pocket while the polar hydantoin ring is stabilized with two h bonds with backbone nitrogen and oxygen of lys- ( fig. b and c) . another fda approved drug, grazoprevir was also found to bind at the allosteric site of sars-cov- protease and can potentially interfere with the dimer formation of the protease ( fig. d and c) . grazoprevir is known as hepatitis c protease (ns / a) inhibitor and is used to treat hepatitis c in combination therapy [ ] . several x-ray structures of the complexes of grazoprevir and hepatitis c virus (hcv) ns / a have been reported [ , ] . in addition to performing docking of grazoprevir with sars-cov cl-protease, we also performed its docking with (hcv) ns / a and reproduced the binding interactions reported in different x-ray structures. predicted binding score obtained through docking simulations of grazoprevir with different ns / a variants is given in table . the binding score of grazoprevir with sars-cov cl-protease was predicted to be À . , suggesting the potential stronger binding as compared to binding with hcv ns / a. rdrp has been considered as an important target for the discovery of direct acting antiviral therapeutics against positive sense rna viruses. in this study, we used homology modeling to model the structure of sars- table identifying the specificity of the drug-target interactions using a significance score based on binding potential to random targets. all of its side chain parameters were within the accepted limit of structure quality (fig. s and tables s and s ). preliminary virtual screening and subsequent analysis resulted in the identification of compounds potentially binding to the protein. these compounds were subjected to iteratively docking by selecting complete surface of the protein to map the binding. final analysis resulted in the identification of one compound, casopitant, tentative generic name rezonic. casopitant is a neurokinin- receptor antagonist and is under development to manage chemotherapy induced nausea [ ] . two aspartate residues, asp- , and asp- are the main catalytic residues of rdrp. these two residues are highly conserved in all corona viruses [ ] . casopitant bound at the catalytic site of the rdrp enzyme (fig. a) . the binding is stabilized by several favorable interactions including three h bonds between carbonyl oxygen of the acetyl piperazine ring and backbone nitrogen of ala- , and between the fluorine atoms of trifluoromethyl-phenyl moiety and the side chain nitrogen atoms of arg- and arg- . the ligand perfectly fits in the catalytic cavity completely covering the two catalytic aspartate residues (fig. a and b ). sars-cov- helicase is considered an important target for therapeutic intervention due its sequence conservation among all corona viruses [ ] . a typical cov helicase consists of a zinc-binding domain, which is separated by stalk from the main catalytic domains a and a. it incorporates distinct dna and atp binding sites. in addition to these two important binding sites, the β -β loop encompassing residues - plays a crucial role in nucleic acid unwinding [ ] . we homology modeled the structure of sars-cov- helicase using the swiss-model online tools for virtual screening. the sars-cov- helicase sequence showed . % sequence identity with helicase of sars-cov (pdb id, jyt) that was used as a template in homology modeling. according to the ramachandran plot analysis, % of the residues were in the allowed region with over % in the most favorable region, validating the quality of the modeled structure ( fig. s and tables s and s ). we identified that meclonazepam, a drug-like molecule with reported sedative, anxiolytic and anti-parasitic effects [ ] , bound to the viral helicase. according to the docking results, meclonazepam binds to helicase at a pocket primarily formed by the β -β loop and the binding is stabilized by several h bonds and hydrophobic interactions (fig. a and b) . another drug-like molecule, oxiphenisatin was found to bind sars-cov helicase also in a binding pocket primarily formed by the β -β loop. the binding was stabilized by several favorable interactions including five h bonds and hydrophobic interactions involving primarily the residues from the β -β loop (fig. a and b) . in order to minimize chances of false positive results in the docking simulations, we calculated a significance measure for the binding scores of prominent drug-protein pairs. the significance measure is calculated by comparing the binding scores obtained for potential targets of a drug with the binding scores of structures belonging to random protein families with the respective drug and counting the number of times out of that we see better docking value when docking the respective drug with the random protein families [ ] . the significance measure enabled us to identify more promiscuous compounds where even the targets with most favorable docking score have an unfavorable significance measure (meaning that the drug is sticky and is interacting with many proteins with high probability). we set the cutoff value of the significance measure at . also to include slightly less specific compounds taking into account the identification of leads to develop more specific potential drugs. drug-protein pairs with significance measure less than . were considered as specific (those with measure less than . are highly specific) as compared to the other pairs tested ( table ). the two docked structures with binding score higher than À . showed the significance measure higher than . indicating non-specific binding of these ligands. over the last decade, computer aided drug discovery has emerged as a fundamental tool in drug discovery and design process [ ] [ ] [ ] [ ] . identification of compounds that bind and inhibit normal function of proteins involved in viral replication has been the most successful strategy in direct acting antiviral drug discovery [ ] [ ] [ ] [ ] . in this study, we used a virtual screening based strategy to identify already approved drugs or drug-like molecules that can bind to any of the three key viral enzymes, cl-protease, rdrp and helicase, and potentially inhibit the function of these enzymes. virtual screening of over drugs and subsequent docking simulation resulted in the identification of three cl-protease binding drugs, one rdrp binding molecule and two helicase binding molecules. one of the identified cl-protease binding drugs, rimantadine, showed binding at the catalytic site blocking the critical catalytic residues and half of the catalytic pocket. the two other compounds bound in a pocket present in domain iii of the protease and can potentially interfere with the formation of dimerit is well established that cov cl-proteases are catalytically active primarily in dimeric form [ ] . one rdrp binding compound, casopitant was identified which perfectly fits in the catalytic cavity of the enzyme and completely blocks the two critical catalytic residues, asp- and asp- . casopitant is not an approved drug but it is in the process of development towards its approval. we identified two helicase-binding molecules, meclonazepam and oxiphenisatin both of these molecules bind at a pocket primarily formed by the β -β loop of the enzyme. this loop has been reported to play a crucial role in the catalytic activity, nucleic acid unwinding, of the helicase enzyme [ ] . three of the identified compounds are already approved drugs that potentiate for their repurposing towards sars-cov- after in vitro and in vivo validation. moreover, all of these compounds represent leads for chemical modifications to develop new potential therapeutics. for instance, rimantadine binds to cl-protease and fits in half of the substratebinding pocket of the enzyme. this drug also shows non-specific binding to other proteins as determined from its specificity score ( . ) after performing its docking simulation with irrelevant proteins. its nonspecific binding could be attributed to its smaller size as several side effects of this drug have also been reported [ ] . nevertheless, this docked structure provides necessary information for potential chemical modifications to incorporate chemical groups to fill the space of the binding pocket potentially leading to enhance the binding affinity and specificity. in specificity score measurement, we set the cutoff value of the significance measure at . considering two aspects. firstly, to identify leads for chemical modification to synthesize more specific inhibitors and secondly, not to reject a ligand with good docking score even with moderate potential off target effects as in many cases a drug with several adverse effects is also acceptable when the question of life saving arises. for example, rimantadine is an fda approved drug to use against flu despite its several side effects. recently, several studies have been conducted using computational methods to identify already approved drugs as potential inhibitors of sars-cov- . elfiky et al. reported the binding of four approved antiviral drugs to rdrp of sars-cov- , including currently under clinical trial drug remdesivir [ ] . rdrp is a highly significant drug target due to its conservation across positive sense rna viruses. however, this study was highly focused on nucleotide inhibitors and only eight already known drugs were screened against sars-cov- rdrp using a less vigorous docking program autodockvina. wang et al. recently conducted a study to screen a library consisting of diverse approved drugs against the sars-cov- cl-protease and identified binding of several antiviral drugs, an anticancer drug, carfilzomib and an antibiotic, streptomycin [ ] . this study is computationally vigorous as it also includes molecular dynamics simulation to approximate binding free energy. however, docking simulations using the autodock program provides quite a reliable approximation for further in vitro and in vivo testing as elfiky et al. utilized only the less vigorous program of autodock (autodock vina) to identify potential inhibitors and one of their identified drugs is currently in clinical trials, suggesting the reliability of docking. in our studies we performed computational screening by targeting three important enzymes of sars-cov- including rdrp, cl-protease and helicase, to identify not only the already approved drugs for repurposing but also the drug candidates or lead structures that can be chemically modified to develop potential drugs. moreover, we incorporated an additional aspect of specificity score obtained by performing docking simulation of every identified compound with irrelevant proteins. a similar study was recently reported by mirza et al. incorporating screening of drug candidates against three key enzymes of sars-cov- and reported several potential inhibitors of these enzymes [ ] . however, they performed docking by selecting a very specific area around the predicted catalytic sites of the protease and rdrp, and the atp binding site of helicase ignoring allosteric sites. these enzymes also incorporate allosteric sites that can be targeted for potential drug discovery. for example, cl-protease is known to be catalytically active in its dimeric form and any inhibitor of dimerization could be of high significance. similarly, helicase incorporates several distinct sites including dna and atp binding sites, the zinc-binding domain and the β -β loop involved in nucleic acid unwinding process. in our studies, during docking, the whole surface of the protein was selected to allow the ligand to bind in an unbiased binding pocket and results were analyzed considering the known functional significance of the binding pocket that resulted in the identification of potential allosteric inhibitors of these enzymes as well. moreover, several other studies have also been reported contributing to the efforts in identifying approved drugs for repurposing or drug candidates or leads to develop drugs against sars-cov- . overall, this computer-aided study represents a contribution to the efforts against sars-cov- and provides information about potentially repurposable drugs and drug-like molecules or lead structures that can be chemically modified to develop into potential drug candidates for further in vitro and clinical investigation. the authors declare that they have no conflict of interest. declining public health protections within autocratic regimes: impact on global public health security, infectious disease outbreaks, epidemics, and pandemics impact of school closures for covid- on the us healthcare workforce and net mortality: a modelling study covid- ) situation report number remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor cryo-em structure of the -ncov spike in the prefusion conformation covid- infection: origin, transmission, and characteristics of human coronaviruses the sars-coronavirus nsp þ nsp complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension the nonstructural proteins directing coronavirus rna synthesis and processing structural elucidation of sars-cov- vital proteins: computational methods reveal potential drug candidates against main protease, nsp rna-dependent rna polymerase and nsp helicase anti-hcv, nucleotide inhibitors, repurposing against covid- computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- identification of novel compounds against three targets of sars cov- coronavirus by combined virtual screening and supervised machine learning fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study broad anti-coronaviral activity of fda approved drugs against sars-cov- in vitro and sars-cov in vivo, biorxiv the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro swiss-model: homology modelling of protein structures and complexes a novel coronavirus associated with a respiratory disease in wuhan of hubei province, china procheck: a program to check the stereochemical quality of protein structures structure of mpro from covid- virus and discovery of its inhibitors mtiopenscreen: a web server for structurebased virtual screening autodock vina, improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading zinc -ligand discovery for everyone drugbank: a comprehensive resource for in silico drug discovery and exploration online smiles translator and structure file generator, national cancer institute, (last time visited on may th autodock and autodocktools : automated docking with selective receptor flexibility ligplotþ: multiple ligand-protein interaction diagrams for drug discovery current status of amantadine and rimantadine as anti-influenza-a agents: memorandum from a who meeting structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design chemistry and anticonvulsive effect of new hydantoin derivatives treatment of childlike epilepsy with bagrosin, or bagrosin sodium an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy evaluating the c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery crystal structure of sars-cov- main protease provides a basis for design of improved alpha-ketoamide inhibitors grazoprevirþ elbasvir for the treatment of hepatitis c virus infection the molecular basis of drug resistance against hepatitis c virus ns / a protease inhibitors structural and thermodynamic effects of macrocyclization in hcv ns / a inhibitor mk- casopitant: a novel nk -receptor antagonist in the prevention of chemotherapy-induced nausea and vomiting delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp upon atp hydrolysis characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites an integrated structure-and system-based framework to identify new targets of metabolites and known drugs computational methods in drug discovery computational screening and design for compounds that disrupt protein-protein interactions the pharmacophore concept and its applications in computer-aided drug design applications of computer-aided approaches in the development of hepatitis c antiviral agents evolution of efficacious pangenotypic hepatitis c virus therapies the efficacy and safety of direct-acting antiviral regimens for hcv/hiv co-infection: a systematic review and network meta-analysis potential targets for therapeutic intervention and structure based vaccine design against zika virus an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors amantadine and rimantadine for influenza a in children and the elderly this study was supported by an internal funding from lahore university of management sciences. one of the authors, hafsa iftikhar was supported by the higher education commission of pakistan in the form of phd stipend. supplementary data to this article can be found online at https://doi. org/ . /j.compbiomed. . . key: cord- -ezsy mx authors: rahman, hamidur; hossain, md. rakib; ferdous, tahmina title: the recent advancement of low-dimensional nanostructured materials for drug delivery and drug sensing application: a brief review date: - - journal: j mol liq doi: . /j.molliq. . sha: doc_id: cord_uid: ezsy mx in this review article, we have presented a detailed analysis of the recent advancement of quantum mechanical calculations in the applications of the low-dimensional nanomaterials (ldns) into biomedical fields like biosensors and drug delivery systems development. biosensors play an essential role for many communities, e.g. law enforcing agencies to sense illicit drugs, medical communities to remove overdosed medications from the human and animal body etc. besides, drug delivery systems are theoretically being proposed for many years and experimentally found to deliver the drug to the targeted sites by reducing the harmful side effects significantly. in current covid- pandemic, biosensors can play significant roles, e.g. to remove experimental drugs during the human trials if they show any unwanted adverse effect etc. where the drug delivery systems can be potentially applied to reduce the side effects. but before proceeding to these noble and expensive translational research works, advanced theoretical calculations can provide the possible outcomes with considerable accuracy. hence in this review article, we have analyzed how theoretical calculations can be used to investigate ldns as potential biosensor devices or drug delivery systems. we have also made a very brief discussion on the properties of biosensors or drug delivery systems which should be investigated for the biomedical applications and how to calculate them theoretically. finally, we have made a detailed analysis of a large number of recently published research works where theoretical calculations were used to propose different ldns for bio-sensing and drug delivery applications. by the world health organization [ ] . these three research works from the last three years are just some of many more recent research works done with ldns in the search for a biosensor. besides the application of ldns as drug and biomolecule sensors, their biocompatibility leads them to another crucial biomedical use, drug delivery systems. the drug delivery systems play an essential role in the pharmaceutical field by reducing the severe adverse effects of the various drug, especially chemotherapeutic anticancer drugs, by delivering the drugs to the targeted cell area. earlier in , xibo pei and his co-workers used polyethylene glycol attached graphene oxide (pgo) nanosheet as a dual-drug delivery system for antitumor drugs cisplatin (pt) and doxorubicin (dox) [ ] . in vivo data from their work confirmed that compared to the toxicity of free pt and dox drugs itself, pgo-pt/dox dual-drug delivery system displayed a significantly reduced toxicity. also, in vitro data from their study confirmed most prominent cell apoptosis and necrosis for pgo-pt/dox system compared to the free drugs and showed a higher growth inhibition property. the graphene and go nanosheets were further used as a drug delivery system for many other anticancer drugs including βlapachone [ ] , fluorouracil [ ] , camptothecin [ ] etc. and many other biological drugs including plasmid dna [ ] [ ] [ ] , sirna [ ] [ ] [ ] , proteins [ ] [ ] [ ] etc. other than the graphene nanosheets (gns), e. czarniewska and his team found that the oh functionalized bn (bn-oh-n) nanosheets showed very low cytotoxicity in both in vivo and in vitro hence bnnss can be applied in the platform like bio-imaging and drug delivery [ ] . furthermore, hollow bn spheres with anticancer drug paclitaxel were observed to suppress the tumour growth significantly with an increased prostate cancer cell apoptosis and decreasing cell viability [ ] . another allotrope of bn, bnnt functionalized with mesoporous silica was also found to perform as a potential drug delivery system for another anticancer drug dox to treat prostate cancer by increasing cancer cell killing ability [ ] . other than these gns and bn allotropes, other nanomaterials, e.g. metal-organic frameworks [ ] , phosphorene [ ] , gold nanoparticle [ ] etc. displayed their ability to carry many drugs by reducing adverse effects with lower cytotoxicity. figure : comparison between the normalized experimental and dft simulated absorption spectra of ssz in water. reproduced with permission from ref. [ ] . copyright springer nature, . choose the functionals and basis sets selectively, in case of the first-principles calculations through a computer simulation package. besides spectroscopic resemblance, dft theory also predicted the reactivity and reaction mechanism successfully with accordance to the experimental observations [ ] . the dft theory was also applied by m. darvish ganji et al. to observe the drug release mechanism using cnt [ ] . furthermore, they have also observed the interaction energy of - . kcal/mol in case of the interaction of benzene molecules with the graphene surface where it was observed as - . kcal/mol experimentally. due to these conveniences with the experimental results, first-principles calculations holds a potential application to explore various biosensor devices and drug delivery systems at the first place in a short time with a reduced cost. finally, considering a significant increase in the drug adsorption related work in the last years from figure , herein we have made a comprehensive review on the recent advancement of first-principles calculation on the applications of ldns in the biomedical fields including drug sensors and drug delivery. we have analyzed the properties which can be investigated through the first-principles calculations and application of those properties in search for promising drug sensors and drug delivery systems for different kind of drugs, especially the anticancer drugs. furthermore, we have explored the adverse effects of the drugs and several specific properties of the nanostructures, which provides it with an advantage to perform as a drug sensor or delivery system. finally, we have analyzed a wide range of research works that were reported in the last few years searching for promising drug sensors or delivery systems using first-principles calculations. biomedical fields like drug sensor and drug delivery. in this section, we will make a brief discussion on the properties of the drug sensors and drug delivery systems which can be calculated theoretically using computer simulation packages. the first thing first-principles calculations allows researchers is to optimize and relax their structures into the global minimum energy using many existing quantum mechanical theories like dft, md etc. with their respective exchange correlational functional basis sets. from the optimized geometry, it is possible to calculate the bond length and bond angle between the constituent atoms in the structures. these are essential parameters to investigate from which the interaction between the drug molecules and respective adsorbents can be confirmed, and their type of interactions can be identified. the different vibrational mode of the constituting atoms of a material can be observed through the ir spectroscopy. as it denotes every individual vibrational mode of a material, it is often used to identify the material hence works as a fingerprint of the material [ ] . by investigating the imaginary frequencies in the ir spectrum, researchers also achieve the information about the stability of the structures and the possibility of finding them into nature or synthesizing them into the laboratory. if any imaginary frequency for a vibrational mode in the structure is observed from the theoretical calculations, there will be a minimal possibility to find that material into nature as the naturally existing materials can not possess any imaginary frequencies. to investigate the structural stability of a nanostructure system, the cohesive energy ( ) of nanostructures can be calculated as [ ] : where n is the total number of constituent's atoms of the nanostructure, a and b represents the different constituent atoms of the nanostructure and and respectively represents their energies with x and y be the total number of them in the nanostructure. uv-vis spectrum usually provides researchers with information about the range of wavelength the material will absorb. the spectrum is also associated with the electronic bandgap of the material where a broader spectrum represents a wider bandgap of the material. besides, it is also useful to investigate the interaction of a biomolecule or drugs with the adsorbent material as an introduction of the new energy states usually creates a noticeable change in the spectrum [ ] . furthermore, the right shift of the spectrum or simply redshift due to the drug adsorption indicates with a decrease in the bandgap with an increase in the conductivity of the sensor material and an enhanced sensitivity towards the drug molecule [ ] . the nuclear magnetic resonance (nmr) usually allows researchers to investigate the magnetic field around an atomic nucleus. however, it is further applied extensively in structural biology to study the structure and dynamics of a biological sample. in drug sensing and drug delivery applications, nmr can also be used to investigate the chemical shift due to the interaction of an adsorbate molecule with an adsorbent [ ] . to investigate the adsorption process between the adsorbent and an adsorbate molecule, one of the most reliable and often used method is to examine the adsorption energy ( ) due to the adsorption which can be calculated as [ ] , where, represents the energy of adsorbate adsorbed adsorbent nanostructures and both and represents the isolated energy of the adsorbent and adsorbate, respectively. here a negative value for the adsorption energy represents a stable adsorption process, whereas the positive value for the adsorption energy represents no adsorption process. besides, if the adsorption energy value is less than ev, then the adsorption process is considered as weak physisorption, and no chemical interaction can be found between the adsorbate and adsorbent [ ] . but if the adsorption energy exceeds ev, then a strong chemical interaction is considered to have occurred between the adsorbate and adsorbent, and the adsorption process is then levelled as chemisorption. hence, more negative the value of adsorption energy gets, more strongly the system is considered to be bounded, representing a higher stable system. so, for the drug delivery and drug sensor devices, the adsorption process between the drug molecules and adsorbents are expected to have a negative value for the adsorption energy where in case of drug delivery systems the adsorbent is required to make a strong interaction with the drug molecule. but in the drug sensor devices, weak physisorption is desired as the strong chemical interactions often lead to a higher recovery time which is not suitable for the drug sensor device. several important information about the adsorption can also be found from the minimum distance between the adsorbate and adsorbent. a smaller distance between the adsorbate and absorbent often lead to a higher charge transfer between the adsorbent and the adsorbate molecules. hence the sensitivity of the adsorbent materials towards the adsorbate molecules can be understood which plays an important role in a drug sensor device development [ ] . several studies also claimed that a smaller adsorbent-adsorbate distance leads to a stronger interaction between and provides them with higher stability [ ] . to investigate the thermodynamical nature of the interaction, first-principles calculations allows calculating the change in the gibbs free energy ( ) using [ ] , where g represents the sum of electronic and thermal free energies. from the definition of the gibbs free energy, a negative change in the gibbs free energy represents a spontaneous interaction between the adsorbate molecule and adsorbent hence a negative change in desired in case of both drug delivery and drug sensor devices. the change in enthalpy ( ) can be calculated from [ ] , where h represents the sum of electronic and thermal enthalpies. a positive change in the enthalpy labels the reaction between the adsorbent and adsorbate as an endothermic reaction, whereas the negative changes in the change of enthalpy lead to an exothermic reaction. finally, the change in entropy ( ) can be calculated using, where t is the considered temperature depending on the systems. in most of the cases, the changes in entropy become negative due to the adsorption of a drug molecule by an adsorbent material which is because of the constrained movement of the structures due to their newly formed intermolecular bondings. j o u r n a l p r e -p r o o f . . homo and lumo energy the homo energy ( ) represents the energy state of the highest occupied molecular orbital (homo) whereas the lumo energy ( ) stands for the energy state of the lowest unoccupied molecular orbital (lumo). these are significant properties for a drug sensor or drug delivery systems as the difference between the homo and lumo energy, which is often expressed as h-l energy gap ( ), represents the conductive nature of the system where the conduction electron population (n) can be defined as [ ] , where, hence, by definition, a smaller h-l energy gap represents a higher conductivity which is desired for a drug sensor device. therefore, to inspect the sensitivity of the adsorbent towards the adsorbate, change in the h-l energy gap due to the adsorption plays an important role. for a drug sensor, if the h-l energy gap decreases significantly due to the adsorption of the drug on the nanostructure, then the nanostructure is considered to be sensitive towards the drug. this increase in the conductivity produces a detectable electrochemical signal from which the adsorption process can be confirmed, and the drug can be detected. besides just the homo and lumo orbital, first-principles calculations allow investigating all possible states for a given energy range by allowing to calculate the dos spectrum. hence the h-l energy gap can also be investigated from the dos spectrum. another vital application of dos spectrum for the drug sensor devices is that the interaction between the drug and the adsorbent can also be confirmed from the alteration of the dos spectrum due to the adsorption of the drug molecule. the resistance of a structure towards the deformation can be investigated from the global chemical hardness (η), which can be calculated from [ ] : hence, a higher value for the global hardness indicates higher chemical stability for the structure with reduced chemical reactivity. the global softness (s) has an inversely proportional relationship with the global hardness which can be expressed as [ ] , hence, opposite to the global hardness, an increase in the global softness indicates an increase in the chemical reactivity with the reduced chemical stability of the structure. using the koopmans' theorem, the chemical potential (µ) can be calculated as [ ] : the chemical potential also can be used to determine the stability of the investigated structures besides its another potential application to determine the direction of electron transfer from the adsorbate to adsorbent [ ] . the electrophilicity index ( ) from parr et al. also can be calculated using the formula [ ] : the electrophilicity can also be used to investigate the structural stability, reactivity and toxicity of a nanostructure where, similar to the global softness, an increasing value the electrophilicity index also indicates an increase in the chemical reactivity [ ] . the negative value of the chemical potential is known as the electronegativity ( ) of the structure and can be calculated as, to investigate the charge transfer between the adsorbate molecules and the adsorbent, the natural bond orbital (nbo) based charge transfer and the original basis function-based mulliken charge transfer can be calculated from theoretical calculations using computer simulations. these investigations of the charge transfer play an essential role to the development of a drug sensor device by observing if the adsorbent nanostructures are capable of making a detectable electrochemical signal on the presence of a drug molecule. to understand the charge transfer between the adsorbate and adsorbent more extensively, the quantum mechanical atoms in molecule (aim) analysis can be studied where the charge transfer between the adsorbate and adsorbent atoms can be observed from the bonded paths and the bond critical points (bcp), an appearance of which can confirm the charge transfer between the adsorbate and adsorbent atoms. the type of interaction can also be understood from aim analysis using several other parameters including the electron density at bcp ( ) and its laplacian ( ), kinetic electron energy density ( ), potential electron energy density ( ), and the total electron energy density ( ) which is the sum of kinetic and potential electron energy density [ ] . a closed-shell interaction can be confirmed from the positive value of the where the negative value represents a shared interactions. besides, if the negative ratio of the kinetic electron energy density to the potential electron energy density ( ) is greater than then the interaction is considered to be non-covalent and electrostatic in nature where < represent a covalent nature of the interaction. furthermore, at the bcp, if both and becomes negative, the interaction is considered as a strong covalent interaction following by weak electrostatic interaction for the positive value of both and [ ] . but if the becomes positive and remains negative, then the interaction can be labeled as a partial covalent and partial electrostatic interaction in nature. the molecular electrostatic potential (mep) map represents the regions where a positive test charge can be attracted or repelled. the positive electrostatic potential region, which is often coloured as blue in the mep map represents the regions with low electron density where a positive charge gets repelled by the atomic nuclei. furthermore, the negative electrostatic region is coloured as red in the mep map, which represents the region with higher electron density where a positive test charge can be attracted by the atomic nuclei. the interaction of a drug molecule with the adsorbent usually alters the electron density in the system hence an interaction can be detected from this alteration in the mep map which can further be used in the drug sensor device development. it is also possible to determine the direction of the charge flowing through the adsorbate and adsorbent from the calculation of the change in fractional charge ( ) using pearson's method [ ] : where µ and η represent the global chemical potential and chemical hardness, respectively. in this case, if the value for appears to be negative, then it can be concluded that the charge will flow towards the adsorbent from the adsorbate where a positive value will indicate the charge transfer to the adsorbate from the adsorbent. j o u r n a l p r e -p r o o f the dissolvability of the nanostructures into a polar medium like water etc. can be investigated using dipole moment (dm) which plays an integral part for drug delivery devices. an increasing dm represents a higher charge asymmetry for the structures which is desired in case of the drug delivery systems so that they can attract more random electrons and dissolve into the polar mediums. besides vacuum, theoretical calculations allow calculating the properties of nanostructures in many other mediums, e.g. water, acid, methanol etc. which plays a crucial role on investigating the behaviour of the drug sensors and drug delivery systems in many different targeted environments. for example, it is crucial to examine the performance of an anticancer drug delivery systems in a low ph cancerous cell region for a better understanding to the drug release performance of the nanostructures in the targeted region. the solubility and stability of the nanostructures in any solvent medium can be further investigated in terms of the solvation energy. for example, negative solvation energy of a nanostructure in a solvent medium represents spontaneous solvation and higher stability of that nanostructure in that solvent [ ] . hence for the drug delivery application, the solvation energy is essential to be negative in the polar solvent mediums like water etc. the solvation energy can be calculated from, where, represents the total energy of the structure in the solvent medium and represents the energy of the structure in the gaseous medium. in both drug delivery and drug sensor devices, the drug release mechanism plays a crucial to study the efficiency of the device. this can be observed from the interaction of the drug molecule with the adsorbent in the targeted environment. for example, if the adsorption energy of an anticancer drug on a nanostructure decreases or become positive in the low ph cancerous cell environment, the nanostructure is considered to desorb and release the drug ideally in a low ph environment. graphene is a d single-layer hexagonal network of carbon atoms bonded with sp hybridization. this honeycomb shaped allotrope of carbon along with its zero bandgap, exceptional hardness and other superior structural, electronic, and optical properties lead scientists to apply this material into many diverse fields like solar cells, gas sensor, biosensors, drug delivery system etc. [ ] [ ] [ ] [ ] [ ] . after the first synthesis of graphene by novoselov et al., from the university of manchester using the scotch tape method [ ] , several synthesis methods like exfoliation of natural graphite [ ] , chemical vapour deposition (cvd) [ ] , reduction of graphene oxide (go) [ ] etc. showed promising future in the graphene fabrication industry. though the method relating to the reduction of go into gns provides excellent cost-effectiveness, cvd can offer a large area on the gnss. the experimental c-c bond length of the graphene was observed as . - . Å [ ] with several other superior properties like very high strength with the value of young's modulus of tpa [ ] , the high thermal conductivity of w/mk [ ] , the electrical conductivity of s/m [ ] , and high carrier mobility at the room temperature as , cm /vs for carrier densities below × cm [ ] . besides these unique properties, the biocompatibility and reduced cytotoxicity of gnss made itself a promising candidate for the drug sensor and delivery applications [ , ] . but pristine gns shows weak interaction towards many drugs and amino acids which can be significantly enhanced by altering its properties with impurity atom doping. besides doping, the hexagonal ring of the graphene was also modified into a pentagonal and heptagonal ring to create a new type of nanomaterial named as phagraphene which shows promising applications in the drug delivery applications [ ] . besides, the theoretical calculations with gns showed significant similarity with experimental results, e.g. in figure we have optimized graphene quantum dot (gqd) using dft with b lyp/ - g (d,p) level of theory which showed comparable bond length, bond angles etc. with the experimental observations. thus the research on the theoretical implementation of pristine, doped or functionalized graphene in the field of drug delivery and drug sensor become very popular in recent years. β-lapachone (β-lap), a potential chemotherapeutic anti-cancer agent, possess an excellent value as it selectively kills or stops the growth of various tumour cells responsible for pancreas, prostate head and neck cancers [ ] [ ] [ ] . β-lap, containing naphthoquinone fused to a methylated hydropyran moiety, was originally j o u r n a l p r e -p r o o f isolated from a south american tree named lapacho. to carry this anti-cancer agent into the cancerous cell, the feasibility of using pristine and metal-doped (pt, au) graphene was investigated by zahra khodadadi et al. by a first-principles investigation [ ] . they observed the adsorption properties of β-lap into pure gns first, and then it's dipole moment to investigate the solubility into a polar medium like water. pristine graphene had a c-c bond length of . Å with a bond angle of ° and sp hybridization. but these properties were further modified by decorating metal atoms (pt, au) into the gns. the pt-c and au-c bond length for pt and au decorated gnss were increased to . Å and . Å respectively with a variation in the sp hybridization as the bond angles decrease to ° and ° respectively. but these modifications into the structural properties enhanced the adsorption properties of β-lap into the pt and au decorated graphene and their solvation properties into polar mediums. the adsorption energies of pt and au decorated graphene was increased as - . kcal/mol and - . kcal/mol which was - . kcal/mol for pristine graphene. hence it can be understood that the stability of the graphene was enhanced due to the metal doping. further, the dipole moment of the β-lap adsorbed graphene was increased by . and . debye by pt and au doping respectively while it was only . debye for the pristine gns. hence, β-lap was expected to dissolve into the polar medium more efficiently with an increased reactivity. so, it can be concluded that the metal-doped graphene performs better as a β-lap drug carrier rather than pristine graphene itself. doxorubicin, another chemotherapeutic drug for several cancer treatments, comes with high toxicity which causes many physical difficulties like hypertension and even a heart failure [ ] [ ] [ ] . m. z. tonel et al., made an investigation on the adsorption of doxorubicin on gns using dft theory with gga-pbe functional applied in the siesta code to decrease the side effects of the drug [ ] . they have found the binding energy of the doxorubicin with the pristine graphene can be as high as . kcal/mol with an adsorbate-adsorbent distance of . Å. however, this small adsorbate-adsorbent distance allowed a charge transfer of - . e between them. but depending on the orientation of the doxorubicin, the binding energy can drop down to . kcal/mol with an adsorbate-adsorbent distance of . Å. so, depending on the most stable orientation, they have reported stable adsorption of doxorubicin drug on gnss which were previously inconspicuous for carbon-based nanomaterial in many experimental and theoretical studies [ ] [ ] [ ] . aluminium (al) doped nanostructures were reported to adsorb -fluorouracil (fu), an anti-cancer drug for gastrointestinal, stomach etc. cancer treatment, strongly adsorption with an enhanced drug loading capacity [ , ] . to bring these research further, m. vatanparast et al., theoretically studied aln and bn doped gqd, another low toxic structure with high biocompatibility, as an efficient nanomaterial for the fu drug delivery [ ] . supporting the literature, the aln doped gqds showed some strong adsorption to the fu with a noticeable adsorption energy of - . kcal/mol for aln-fu gqd structure. in their work, they have worked with c h gqd whose central ring was further replaced by b-n, b-p, al-n and al-p bonds. besides high adsorption energy for the aln-fu gqd structure, negative values for the gibbs free energies of all other doped structures confirmed the spontaneous adsorption of fu with adsorption energies higher than that of the pristine gqd. further restriction to the translation degree of freedom lead to a decrease in the entropy with predicted exothermic adsorption with negative values for the change in enthalpy. these confirmed the ability of the pristine and doped gqds to load the fu drug. finally, an increase in the dipole moment up to . debye for aln-fu gqd, highest among the investigated structures, represents aln doped gqd's better solubility to the polar medium and enhanced reactivity towards fu drug made itself a potential candidate to carry fu to the target sites. carrier [ ] . the functionalization of the gns modified the adsorption process of the tegafur towards the structures significantly with a maximum value of the adsorption energy as - . kcal/mol for g-co/tg along with a minimum of - . kcal/mol for g-cooh/tg structure. the solvation energy of the g-cooh/tg structure was also found as - . kcal/mol with a - . kcal/mol of interaction energy. besides g-cooh, g-oh also showed very strong adsorption towards the tegafur with a remarkable adsorption energy of - . kcal/mol and solvation energy, and interaction energy of - . , - . kcal/mol respectively. though the dipole moment of the pristine gns is zero but increases significantly with the functionalization and hits as high as . debye for the tegafur adsorbed g-co structure. on the other side, the energy gap was reduced to as low as . ev for the tegafur adsorbed co functionalized gns while it was . ev for the pristine gns nanosheet. these indicated the batter reactivity of the tg adsorbed functionalized gns sheet with a higher polarity towards a polar medium like water which was further supported by the global parameters like chemical potential, global hardness, electrophilicity investigations. as it can be observed from figure that many anti-cancer drugs have structures akin to the nucleobases as the vital role of these drugs is to slow the growth rate of cancer cells by interfering their dna synthesis [ ] . this led c. rungnim et al., to investigate the adsorption properties, along with edge effect of the gnss, of three nucleobases with similar structures like anti-cancer drugs, mercaptopurine, thioguanine, and fluorouracil, on a graphene flake considering the weak dispersion forces caused by van der waals interaction [ ] . the lack of accuracy of many gga functional with dft due to their underestimation of π-π interaction lead rungnim et al., to use several functional like m - x, mp , ccsd(t) etc. [ ] . to validate their theoretical model, they have first investigated the adsorption properties of nucleobase uracil and then the anti-cancer drug akin to the uracil, fluorouracil, on the benzene ring. it was observed that the o on uracil and fluorouracil pointing to the benzene ring, and uracil/fluorouracil parallel to the benzene ring has better stability than any other investigated orientations. for o pointing to the benzene ring, benzene/uracil and benzene/fluorouracil configurations have adsorption energies as high as - . kcal/mol and - . kcal/mol respectively which becomes - . kcal/mol, and - . kcal/mol respective for uracil/fluorouracil parallel to the benzene ring. a favourable result from the meta gga based m - x functional led them to study the adsorption behaviour of the nucleobases towards the c , c , and c gnss. they have observed an edge effect which gradually decreased with the increased size of the graphene nanoclusters. less edge effect was observed in case of the adsorption of the nucleobases in the middle of the carbon clusters and depending on the alignment and position this effect can reduce the adsorption energies by < kcal/mol. further, the edge effect of c is almost as much as c lead them to adsorb mercaptopurine, thioguanine, and fluorouracil anti-cancer drugs in a parallel position with different alignments in the c nanosheet. finally, the adsorption energies of fluorouracil, mercaptopurine, and thioguanine were found around - . , - , and - kcal/mol respectively made c a promising candidate for the delivery of these drugs. adenine, another nucleobase with a structure akin to many drugs and biomolecule, showed enhanced adsorption properties on the ni decorated gnss compared to the pristine gns which was observed on an investigation made by s. gholami et al., [ ] . for a plane alignment of adenine to the pristine graphene surface, the adsorption energy was found as - . kcal/mol with a graphene-adenine distance of . Å and a charge transfer of - . e (mulliken) only. the adsorption energy was dramatically increased to a maximum of - . kcal/mol with an adsorbate-adsorbent distance of . Å, and a charge transfer of + . e (mulliken) in the ni decorated gns, where ni was adsorbed above the hexagonal ring of graphene with a strong chemical interaction of - . kcal/mol. adsorption of the adenine on the ni decorated graphene also decreased the bandgap and fermi level of the structure to . ev and - . ev respectively from a value of . ev and - . ev for ni-g itself. though the pristine graphene showed a weak interaction towards the pristine gns, decorating it with a ni atom increased its interaction significantly. hence the ni decorated graphene can be considered as a potential candidate for delivery of the drugs with structures similar to the adenine. furthermore, the adsorption of guanine nucleobase was investigated by a. s. rad et al., on pristine and al-doped graphene (alg) using dft theory with b lyp hybrid functional and - g basis set [ ] . it was observed that the doping of al to the pristine gns significantly alters the adsorption properties towards guanine. for pristine gns, the adsorption energy of guanine was observed only as - . kcal/mol with an adsorbateadsorbent distance of . Å. this allowed them to transfer a little amount of charge of - . e (based on mulliken charge transfer analysis), or - . e (based on natural population analysis) between adsorbate and the adsorbent. this was significantly enhanced by the doping of al atom to the pristine gns. for a specific orientation of guanine, o atom from guanine above al atom from alg, the adsorption energy increased as much as - . kcal/mol with an adsorbate-adsorbent distance of . Å only which allows them to transfer + . e (mulliken) amount of charge. for other orientations the adsorption energies were observed as - . , - . , - . , - . kcal/mol with an adsorbate-adsorbate distance of . Å, . Å, . Å, and . Å respectively. furthermore, along with the adsorption energy and charge transfer, the dipole moment of the alg structures was also enhanced significantly with a maximum of . debye. this excellent modification in the adsorption energy, charge transfer and dipole moment made alg nanostructure a suitable adsorbent or sensor for the guanine. other than the anti-cancer drugs, graphene was also reported to work as a drug delivery vehicle for -aminopyridine or dalfampridine, often marketed as ampyra with a chemical name of c h n-nh , helps to overcome walking difficulties for the adults by increasing nerve signal conduction through blocking the potassium channels on it [ , ] . but ampyra has several side effects like headaches, dizziness, nausea, insomnia etc. and an increased dose of it can also cause seizures [ ] . to reduce the possibility of a seizure, najme dastani et al. proposed pristine and functionalized gns (f-gns) for the transportation of ampyra using quantum mechanical calculations [ ] . they choose the dft theory with m - x functional and - g (d, p) basis set to optimize the structures due to its excellent agreement with the experiment. for gns, they observed c-c bond length as . Å while it was found as . Å experimentally [ ] . this encouraged them to study several f-gns (g-cooh, g-co, g-oh, and g-o) with the same functional and basis set. they have studied several essential properties like adsorption energies, homo-lumo energy gaps, chemical potential, dipole moment and several global parameters for both gaseous and aqueous phase. it was found that the dipole moment in the aqueous phase rose to . debye from a maximum of . debye in the gaseous phase. for the functionalized gnss this dramatic change on the dipole moment was continued and was found as high as . debye for g-cooh/ampyra complex. finally, the highest negative adsorption energies of - . kcal/mol for g-cooh/ampyra complex among the pristine gns and f-gns in the aqueous phase made g-cooh the best candidate for the transportation of ampyra drug. this was further supported by the lowest value for the energy gap as . ev, global hardness as . ev and the highest value for the change in global electrophilicity as . ev. besides the application of graphene as a potential sensor for the drugs and nucleobases, to understand the interaction of graphene with other biomolecules, the adsorption properties of amino acids on the gnss were also reported using first-principles investigations. b. saha et al. investigated the interaction of four amino acids (alanine, serine, phenylalanine and tyrosine) into pristine and b and/or n doped functionalized (-oh, -nh , -cooh) gnss [ ] . it was observed that the functionalized gnss interacted with the amino acids with interaction energy ranging from . - . kcal/mol depending on the adsorbent structures and dopants. furthermore, though the doping of b and/or n atoms changed the adsorption properties of amino acids to the -oh and nh functionalized graphene, it hardly affected the -cooh functionalized gns. on another dispersion corrected dft study, h. t. larijani et al., investigated the adsorption of several amino acids (glycine, histidine and phenylalanine) on pristine, defected and oxidized gnss [ ] . they have found that every interaction of the amino acids with the monolayer gnss was physisorption and the gnss is a potential candidate for the bio-related applications. as pyridinic n, pyrrolic n, and graphitic n [ ] . in their investigation with dft theory along with pbe functional, they have used nitrogen-doped d gnss along with pristine gns with hexagonal vacancy. the dft theory with pbe functional gave the c-c bond length as precise as . Å for the pristine gns. among three types of interaction between the amino acids and the substrates, the side chains perpendicular to the substrates was used as it was most energetically favourable. due to the adsorption of the several amino acids arginine, histidine, aspartic acid, asparagine, and tyrosine, to the pristine gns, the adsorption energy was found as low as - . kcal/mol for the aspartic acid with a maximum as - . kcal/mol for the arginine adsorption. but these values for the adsorption energies increased dramatically in case of the nitrogen atoms doping. for four nitrogendoped pyridinic n, this was found as high as - . kcal/mol for histidine with a minimum of - . kcal/mol for the arginine adsorption. this was again reduced in case of the hydrogenated nitrogen-doped structure disproportionally and finally for hydrogenated pyrrolic n it ranges between - . kcal/mol to - . kcal/mol. despite this low adsorption values for the hydrogenated structures, it increased dramatically again in case of a hydrogenated n-edge. for hydrogenated pyrrolic n-edge, it went as high as - . kcal/mol for arginine with a minimum value of - . kcal/mol for tyrosine adsorption. hence it was concluded that a pyridinic n nanosheet with a hydrogenated n-edge works best for the adsorption of amino acids. the modification of the hexagonal ring of the gns into the pentagonal and heptagonal ring made a noble kind of nanosheet known as phagraphene is also studied as a potential drug delivery nanomaterial for adrucil by r. bagheri et al., [ ] . the small adsorption energy of - . kcal/mol for phagraphene made it incompatible for the adrucil drug delivery. however, the adsorption energies increased dramatically to the - . , - . , and - . kcal/mol due to the doping of b, al and si atoms respectively. however, noticeable change in the electronic properties of b doped phagraphene made it unsuitable for adrucil drug delivery and despite having enormous adsorption energy, al-doped phagraphene showed a considerable response time of . × s. hence the sidoped graphene with a moderate adsorption energy of - . kcal/mol with a short response time of . s, was predicted to be a potential drug delivery system for adrucil drug. cnt is an allotrope of carbon made by a hexagonal network of carbon atoms which is cylindrically rolled up into a tubular shape. ever since its discovery, numerous research works were done on its various unique properties including exceptional mechanical strength, electrical, optical, and thermal properties etc. [ ] [ ] [ ] [ ] [ ] . similar to the gns, the sp hybridized carbon-carbon bond of cnt lead it to one of the strongest material with young's modulus of gpa and a tensile strength ranging from gpa to gpa [ ] . the density of the cnt was observed experimentally as large as ∼ . g/cm with a low room temperature resistivity of ∼ mΩ-cm [ ] . the thermal conductivity of cnt was predicted as high as ∼ w/mk at room temperature along with an electrical conductivity ranging from to s-cm - [ , ] . cnt can be classified in many subclasses but most simply into two, single-walled cnt (swcnt) and multi-walled cnt (mwcnt). where the swcnt is a single layer of graphene, mwcnt is a multi-layer of graphene rolled up cylindrically into a tubular shape as it is shown in figure [ ] . now, based on the chiral vector defined as (where n and m are the integer number of steps along the unit vectors and ) and chiral angle, defined as the rolling angle between the graphene and axis of the tube, cnt can be further classified into three chiralities as zigzag , armchair , and chiral ( ) [ ] . after the first synthesis of cnt through arc-discharge method [ ] , over the years cnt was reported to be synthesized using many techniques including chemical vapour deposition (cvd) [ ] , laser ablation [ ] , j o u r n a l p r e -p r o o f plasma jet [ ] and many more [ ] [ ] [ ] [ ] . though the arc-discharge method reports growth of swcnt with % figure : schematic diagram of various kind of cnt made by rolling up d graphene sheet. reproduced with permission from ref. [ ] . copyright elsevier science ltd., . purity and laser ablation method with %, cvd with - % purity still widely used due to its cost-effective solution for the mass production [ , ] . this wide range of methods to synthesize cnt along with a wide range of structural variation and unique properties led cnt to be applied in many fields like aerospace industry [ ] , electronics [ ] , energy harvesters [ ] , gas sensors [ ] , biosensors [ ] , drug delivery [ ] etc. among these widespread applications of cnt, our particular interest is in its adsorption ability of biomolecules (both for sensing and delivery purpose) as it offers biocompatibility in this field and has the ability to penetrate cells along with excellent stability, high reliability, faster response time, high adsorption strength towards the bio-molecules etc. [ ] . starting from small bio-molecules like glucose sensing [ ] , cnt can be used to sense and deliver several therapeutics, anaesthetic, anti-depression etc. drugs to the human body [ ] . to acquire the desired sensing and drug delivery performance, sometimes cnt is required to be doped or fictionalized by impurity atoms, and several studies have successfully reported enhanced performance towards biosensors and drug delivery due to the functionalization and doping to cnt [ ] [ ] [ ] . finally, due to the recent advancement on the accuracy of computer simulation methods with approximation theory like dft, e.g. a theoretically optimized swcnt is shown in figure , numerous research works were put forward on investigating cnt as an efficient system for the bio-molecule sensing and drug delivery. in this section, we aim to review recent research work exploring the bio-molecule sensing and drug delivery efficiency of the cnt. a first-principles study conducted by h. shaki et al. reported the adsorption of the penicillamine (pca), an anti-cancer drug with several vital side effects, on the pristine and functionalized swcnt [ ] . considering the dispersion terms, for pristine swcnt the pca drug make weak physisorption with a maximum adsorption energy of - . kcal/mol and an adsorbent-adsorbate distance of . Å. but this interaction was further enhanced by adding an extra -cooh functional group to the pristine swcnt. with -cooh functionalized swcnt (f-swcnt), the maximum adsorption energy of pca towards f-swcnt was observed as high as - . kcal/mol for a particular orientation of pca with a maximum dipole moment of . debye and an energy gap of . ev. thus, this study reveals that the -cooh f-swcnt performs better the pca drug delivery system, whereas all interactions were exothermic and stable with negative adsorption energies. these results were further compared with the md simulations, which successfully supported the result obtained from the dft investigations. isonicotinic acid hydrazide (inh), simply known as isoniazid, is a prodrug for the treatment of the infectious disease tuberculosis (tb) caused by mycobacterium tuberculosis (mtb) bacteria [ ] . as inh drug often comes with side effects like peripheral neuropathy, nausea, aplastic anaemia etc. n. saikia et al. proposed swcnt as a drug carrier to the target area [ ] . as the si doping in the cnt is less reported in the literature than the b/n doping, better electronic properties of si lead them to use si-doped swcnt to carry the inh drug using dft theory and molecular docking [ , ] . in a van der waals interaction corrected investigation it was found that inh parallel to the ( , ) swcnt have better stability with more adsorption energy of . kcal/mol compared to the perpendicular alignment of the inh with ( , ) swcnt with an adsorption energy of . kcal/mol. though the adsorbate-adsorbent distance of . Å was unchanged for both parallel and perpendicular alignment, electronic band gap decreased in the perpendicular alignment slightly to . ev compared to . ev for parallel alignment. for covalent functionalization of inh to the ( , ) swcnt, the adsorption energy was drastically increased to . kcal/mol with a change in the inh-( , ) swcnt distance of . Å and a change in bandgap as . ev. the quantum descriptors also reported an increase in the reactivity due to the covalent functionalization of inh. hence, ( , ) swcnt have a potential future as a drug delivery system for isoniazid drug. ', '-difluoro-deoxycytidine (dfdc) or simply know as gemcitabine is a popular therapeutic option to treat numerous cancers including breast cancer, pancreatic cancer and non-small cell lung cancer [ ] [ ] [ ] [ ] . it comes with relatively fewer side effects but as severe as fatal pulmonary toxicity and acute lung injury [ , ] . this lead h. moradnia et al., to investigate the pyrrolidine functionalized ( , ) swcnt (f-swcnt) as an efficient drug delivery system for gemcitabine drug using dft theory with m x functional and - g (d,p) basis set [ ] . in water solution, for a particular orientation ‗complex e', the gemcitabine was adsorbed towards f-swcnt with an adsorption energy of - . kcal/mol and a dipole moment of . debye with a minimum adsorbate-adsorbent distance of . Å. for another orientation complex a, the adsorption energy was slightly decreased to - . kcal/mol but with an increase in the dipole moment to . debye. the energy gap of the complex a and e was observed as . ev and . ev respectively. the global hardness of the structures ranges between . ev to . ev where the electrophilicity had a range between . ev to . ev. these findings lead them to declare the f-swcnt as a potential drug delivery system for the gemcitabine which was further confirmed through the aim, nbo, and dos analysis. flutamide, an anticancer drug with chemical name '-nitro- '-trifluoromethyl-isobutyranilide is a widely used drug to treat prostate cancer often comes with side effects like severe gynecomastia, liver toxicity etc. [ ] [ ] [ ] . to transfer this drug directly to the cancerous cell, to minimize the side effects, m. kamel et al., analyzed the potential of ( , ) swcnt for efficient delivery of flutamide drug, and how co-solvents like ethanol affects the interaction between the flutamide drug and ( , ) swcnt [ ] . in the water solution for a particular orientation labelled as configuration , the most negative adsorption energy value was observed with a minimum adsorbate-adsorbent distance and highest value for dipole moment. the values were - . kcal/mol, . Å, and . debye, respectively. so the flutamide drug made physisorption towards the swcnt, and the nature of the interaction was exothermic. the values of solvation energy indicated a spontaneous solvation with a range of - . to - . kcal/mol and the least value was for the configuration . a spontaneous flow of electron was observed from the adsorbent to the adsorbate with positive values of . ev and . ev for the charge transfers (Δn) and the energy change of the acceptor (Δe b (a) ). finally, from the md simulations, it was found that the addition of ethanol with . molar concentration increases the stability of the simulation system. however, another study lead by this group, m. kamel et al., investigated the cooh functionalized swcnt to use as a drug carrier for flutamide drug [ ] . in that study, they have found that the flutamide drug can be adsorbed in the swcnt with a maximum adsorption energy of - . kcal/mol in the water solvent where the dipole moment of the structure was found as . debye with a bandgap of . ev. the same cooh functionalized swcnt was used to investigate the carmustine drug delivery ability by the same group but this time lead by r. khorram [ ] . the performance was almost similar as for flutamide drug with a maximum adsorption energy of - . kcal/mol along with a dipole moment of . debye, the energy gap of . ev, and interaction energy of - . kcal/mol. an oral drug, metformin (mf), is used as a first-line medication to treat type diabetes especially patient with breast cancer, prostate cancer, and pancreatic cancer [ ] [ ] [ ] . to minimize the side effects, m. s. hoseininezhad-namin et al., investigated pristine, si and al-doped ( , ) swcnts as a promising mf drug carrier [ ] . weak physisorption of mf towards pristine ( , ) swcnt was observed with an adsorbateadsorbent distance of . Å, charge transfer of . e (nbo analysis), and adsorption energies of - . kcal/mol (b lyp) and - . kcal/mol (ωb xd) where an increment in the adsorption energy through ωb xd functional clearly indicated the role of dispersion in the adsorption of mf drug toward the pristine swcnt. however, doping the swcnt with si and al significantly increased the adsorption energy of mf to - . kcal/mol and - . kcal/mol, respectively in the solvent phase. the reactivity of the mf-doped swcnt configurations also increased in the solvent phase with an increment to the dipole moment to . debye and . debye for the mf adsorbed si and al doped swcnts respectively when it was only . debye for the adsorption of mf towards pristine swcnt. this increment in the reactivity was further supported by the global reactivity descriptors like chemical potential, global hardness and softness, and electrophilicity. hence si doped swcnt with a moderately high adsorption energy but with lowest band gap as . ev and highest reactivity in the solvation phase made itself most promising candidate for the mf drug delivery between pristine, si and al doped ( , ) swcnt. a theoretical investigation using dft theory with b lyp hybrid functional and - g (d, p) basis set, was made on the ca doped ( , ) swcnt to inspect its ability to perform as a drug carrier for atropine drug delivery [ ] . atropine is a drug taken by an injection to the muscle to treat several types of nerve agent and also performs as an antipode to the several poisonings [ ] . atropine drug was adsorbed by ca-swcnt with adsorption energy of - . kcal/mol for d-atropine, and - . kcal/mol for l-atropine with a dipole moment of . and . debye respectively. the homo-lumo energy gap was observed around . ev for both structures with a charge transfer of around + . e from the atropine to the ca-swcnt, which was confirmed by the nbo analysis. the solubility of the atrophine+ca-swcnt in the water medium was established through the negative solvent energy with a value of - . kcal/mol for d-atropine and - . kcal/mol for l-atropine. hence the ca-swcnt was predicted as a potential drug carrier for the atropine drug. ketamine, an n-methyl-daspartate (nmda) receptor antagonist, is clinically often used as an anaesthetic with limited use as an analgesic, anti-depression and anti-inflammatory drug, comes with numerous side effects like psychoactive effects, peripheral effects and neurotoxicity [ ] . so, the urgency of rapid determination of ketamine lead r. zhiani et al., to investigate swcnt and cooh, conhch functionalized swcnt as a sensor to detect ketamine in the gaseous medium along with several other polar mediums [ ] . it was found that the adsorption of ketamine in functionalized swcnt causes a change in the bond length of c-o, n-h and c=o groups noticeably. further, binding energy in the gaseous medium was observed as - . kcal/mol and - . kcal/mol for cooh and conhch functionalized swcnt. this value for binding energy was further increased as - . , - . , and - . kcal/mol in chloroform, methanol, and water medium respectively for cooh functionalized swcnt and became - . , - . , and - . kcal/mol respectively for conhch functionalized swcnt. furthermore, negative values for the change in enthalpy and gibbs free energies indicated spontaneous exothermic adsorption of ketamine in the functionalized swcnt. the response of swcnt to the ketamine was finally confirmed through the noticeable change in fermi energy to the positive values. finally, though the cooh functionalized swcnt responses better to the ketamine in the gaseous phase, conhch shows slightly better response towards ketamine in other medium like chloroform, methanol and water. another anti-inflammatory and analgesic, ibuprofen (ibp), comes with several side effects like indigestion, nausea, vomiting etc. [ , ] . hence c. parlak et al., made a first-principles investigation using dft theory along with b lyp/ m x functional - g(d) basis set and on the adsorption properties of ibp into si-doped swcnt and fullerene [ ] . the literature on the accuracy of m x functional to calculate the adsorption energies but the overestimation of the bandgap lead them to use b lyp hybrid functional for bandgap calculations [ , ] . after the correction of basis set superposition error (bsse), for a specific alignment of ibp where the o atom of (c-o) group of ibp was placed above si atom to the cnt, the adsorption energy was observed as - . kcal/mol with an adsorbate-adsorbent distance of . Å and bandgap . ev. furthermore, due to the adsorption of ibp, the work-function of si-doped swcnt increased by . % with a change in the fermi energy level from - . to - . ev. thus strong chemisorption with a change in the fermi energy constitutes swcnt a potential sensor for ibp drug detection. besides the swcnt as a drug sensor and delivery system, multi-walled cnt (mwcnt) was also investigated as the potential drug sensor for several drugs. d. shahabi et al. investigated several pristine and sulfur-doped swcnt along with several mwcnt (double-walled and triple-walled cnt) as potential drug sensor for the fluoxetine (fx), a widely used antidepressant drug [ ] . it was observed that sulfur atom slightly affected the adsorption processes of fx towards the swcnt where for most stable structure, ( , ) sulfur-doped swcnt, the adsorption energy in the gas (liquid) phase was observed as - . (- . ) kcal/mol while the fx drug was adsorbed inside the sulfur-doped swcnt, and only - . kcal/mol while the fx was adsorbed in the outside of the sulfur-doped swcnt. further to reduce time consumption, molecular docking study was initiated instead of dft to investigate the adsorption behaviour of fx towards systems with a large number of electrons like from ( , ) swcnt to ( , ) swcnt, double-walled cnt, triple-walled cnt. it was found that the stability was decreased from ( , ) swcnt with an increase in the chirality and the most stable structure, ( , ) swcnt, had adsorption energy of - . kcal/mol while the fx drug was adsorbed inside the ( , ) swcnt. the stability was increased slightly for the double and triple-walled cnt and the adsorption energies was found as - . kcal/mol and - . kcal/mol for ( , ) double-walled and ( , ) triple-walled cnt while the fx drug was adsorbed inside the cnt. however, another study lead by c. p. sousa et al., reported that the acetaminophen drug was also adsorbed inside an mwcnt more favourably than external surface [ ] . also, the functionalization of the mwcnt with polyethyleneimine polymer enhanced the stability of the acetaminophen adsorbed f-mwcnt complex with an adsorption energy of - kcal/mol while it was - kcal/mol for the adsorption of acetaminophen towards the pristine mwcnt. so, it was observed that both acetaminophen and fluoxetine could be sensed strongly with the mwcnt. other than directly the drug molecule itself, cnt was theoretically investigated to perform as an efficient sensor for several other molecules like bio-molecules, amino acids and lactic acid. the adsorption of five small bio-molecules co, h o no, so and po towards the swcnt was reported by y. xu et al., through the vibrational spectrum analysis using dft with gga-pbe level of theory [ ] . in another study, m. yoosefian et al. investigated ability of the pd doped swcnt to adsorb an amino acid, histidine, using dft and hf theory with b lyp hybrid functional [ ] . they reported strong chemisorption of the histidine towards the pd-swcnt with a maximum adsorption energy of - . kcal with an adsorbent-adsorbate distance of . Å. finally, the interaction of -hydroxypropanoic acid or simply know as lactic acid (la) with swcnt with different chirality was observed with a dft-d study from a. n. chermahini et al. [ ] . in their research, they have found that la interacts more with ( , ) swcnt than other swcnt with higher chirality with an adsorbate-adsorbent distance of . Å and maximum adsorption energy of - . kcal/mol in the gas phase and - . kcal/mol in the solvent phase. the solvation energy for la adsorbed ( , ) swcnt was observed as - . kcal/mol with an h-l energy gap of . ev. hence the ( , ) swcnt can work as a potential adsorbent for the la. so, it can be concluded that the swcnt can perform as a potential adsorbent for amino acids, lactic acid and biomolecules. akin to graphene and cnt another sp hybridized allotrope of carbon atoms with closed d structure, fullerene, got eminent attention on the field of biosensor and drug delivery application recently. fullerene was observed in the literature as early as in from h. p. schultz where he predicted a new possible topological structure of c h with truncated icosahedron shape with vertices until the first prediction of c fullerene came from e. osawa in [ , ] . finally, in , h. kroto et al. unintentionally observed the c fullerene while they were performing an experiment by vaporizing the carbon atom from a solid graphite surface into a high-density helium flow to understand the mechanism of long carbon chain formations in star and space [ ] . as shown in figure , the c fullerene is consists of pentagons along with hexagons like a soccer ball where pentagons cannot be adjacent as each of them is surrounded by five hexagons. with a fixed number of pentagons, fullerenes can be found with variable hexagons, and the closest stable one to c fullerene is c fullerene with pentagons and hexagons. though c is claimed to be the smallest stable fullerene structures, c unstable fullerene structure was reported to be stabilized in several ways, including metal doping [ ] [ ] [ ] . due to the mass commercial production of c fullerene, there is a wide range of methods available to synthesize it. after the discovery of c fullerene from h. kroto et al., the first macroscopic production of c was reported by w. krätschmer et al. in from electric arc heating of pure graphitic carbon soot in a helium atmosphere of around torr [ ] . following by the method of w. krätschmer et al., c was further produced by h. ajie et al. in from the evaporation of carbon rods but this time by heating it resistively [ ] . later in , growth of fullerenes was reported from r. f. bunshah et al. by electron beam evaporation and sputtering of graphite [ ] , further in , extraction of c ion beam was reported from s. chuanchen et al. from a hollow cathode ion source [ ] , and many more methods were used including the usage of low-pressure diffusion flame from p. hebgen et al. [ ] , arc discharge method from m. caraman et al. [ ] . besides this wide range of j o u r n a l p r e -p r o o f synthesis, due to their unique physical [ ] , electrical [ ] , optical [ ] and chemical properties [ ] of fullerenes lead them to many fields with potential applications in ion storage [ ] , photovoltaics [ ] , sensors [ ] , supercapacitors [ ] etc. despite fullerene's insolubility to the water which was further settled by functionalizing or doping with impurity atoms, it is soluble in a wide range of common solvents in room temperature, and of them were reported r. s. ruoff et al. for c fullerene back in [ ] . hence, moderated solubility in the polar mediums along with several other enchanted properties like hydrophobicity, biocompatibility [ ] etc. made fullerenes one of the front liner in the biological applications like antibacterial activity [ ] , antiviral activity [ ] , drug delivery [ ] , drug sensing [ ] etc. in this section, we aim to review the recent scientific (a) (b) figure : (a) c and (b) c fullerene nanostructures optimized using dft theory with b lyp hybrid exchange correlational functional and - g, - g (d,p) basis sets respectively. reports on the drug delivery and sensing application of carbon fullerenes which were made by the first-principles investigation. as several drugs require interaction with dna and rna, these drugs sometimes have a structure like dna and rna nucleobases which made the investigations on the adsorption properties of the nucleobases towards an adsorbent so crucial in drug delivery application. so, following the trend, a. s. rad made the firstprinciples investigation using dft theory on the adsorption properties of a purine derivative nucleobase, adenine, on the cr and ni-doped c fullerene [ ] . considering the dispersion forces and exchange-correlation energies, all structures were optimized using meta-hybrid functional ωb xd and a gga functional perdew-burke-ernzerhof (pbe) respectively along with - g (d, p) basis set which is widely used to investigate the drug delivery systems. chemisorption of the adenine nucleobase towards the both cr and ni-doped c fullerenes were reported with negative adsorption energies as high as - . and - . kcal/mol respectively with a charge transfer of . e and . e between the adenine and respective doped structure observed from nbo analysis. however, the adsorption process of adenine was spontaneous and exothermic in both cr and ni-doped structures with negative values for change in enthalpy (Δh) and gibbs free energy (Δg) ranging - . of fu with c fullerene using dft theory with dispersion corrected b lyp-d functional and - g* basis set [ ] . a very weak interaction of fu was observed towards the pristine c fullerene with a maximum adsorption energy of - . kcal/mol along with a h-l energy gap of . ev and an inferior charge transfer of . e (from nbo analysis). this interaction was further enhanced by doping a b atom to the fullerene structure. for b doped c fullerene the adsorption energy of fu was observed as high as - . kcal/mol with a decreased bandgap of . ev along with a significantly increased charge transfer of . e ( from nbo analysis). finally, they have reported a successful release of the fu drug to the low ph (< ) targeted area with a cancerous cell. similar to the adsorption behaviour of fu towards c fullerene, y. gökpek et al., observed a weak physisorption interaction between -phenylpyridine ( -phpy) and pristine c fullerene while the adsorption energy was increased significantly due to the doping of c fullerene with b or si [ ] . the dispersion corrected (with m x functional) dft study found that -phpy was adsorbed on a pristine c with an adsorption energy of - . kcal/mol in the water phase where this value was significantly increased to - . and - . kcal/mol for b and si-doped c fullerenes (bc and sic ) respectively. the solvation energy of -phpy was also increased to - . and - . ev for bc and sic structures respectively while it was only - . ev for pristine c fullerene. the h-l energy gap was decreased from . ev for -phpy-c to . and . ev for the phpy-bc and -phpy-sic respectively with an nbo charge transfer of - . e and - . e respectively. these adsorption properties of -phpy with pristine and b or si-doped c fullerenes was slightly diminished in the gaseous phase. observing these enhanced properties for the -phpy-bc and -phpy-sic systems led them to conclude bc and sic as potential structures to carry -phpy into drug delivery applications. the sic system was again subjected to study by m. moradi et al., but this time accompanied by alc to investigate their interactions with phenylpropanolamine (ppa) drug [ ] . ppa is used as a most used decongestant and also marked as a potentially hazardous drug due to its side effects like hypertension, cardiovascular side effects including a potential risk of hemorrhagic stroke [ ] [ ] [ ] . similar to -phpy, ppa had weak physisorption with pristine c fullerene structure with an adsorption energy of - . kcal/mol with a . % decrease in the h-l energy gap to . ev. this interaction was significantly enhanced due to the doping of c with al atom, where the adsorption energy of ppa was increased to - . kcal/mol with an . % decrease to the bandgap to . ev. but the most stable structure to adsorb ppa was found as si-doped c structure with the highest adsorption energy of - . kcal/mol with a significant . % drop in the energy gap to . ev. this fall in the bandgap increased the electrical conductivity of ppa-sic complex, which made sic structure most sensitive towards the ppa detection. but in another study lead by a. s. ghasemi et al., the al-doped c fullerene showed a promising behavior to detect and carry penicillamine drug which is commonly used to treat wilson's disease and rheumatoid arthritis [ ] [ ] [ ] . al-c adsorbed penicillamine with an adsorption energy of - . kcal/mol but increasing the dipole moment to as high as . debye and decreasing the work function as low as . ev. this increase in the reactivity was further confirmed by the global reactivity descriptors where the ionization potential along with the electrophilicity and global hardness decreased with an increase in the global softness for the al-c complex. the rising importance on the detection of the illicit drug to the medical community and law enforcing agency lead s. bashiri et al., to investigate pristine and several doped c fullerenes for its sensing ability to a central nervous system (cns) stimulant, amphetamine (aa) which is medically used to treat attention deficit hyperactivity disorder but is a habit-forming and highly potential drug for abuse [ , , ] . the pristine c fullerene showed a good sensitivity towards the aa drug with a low bandgap of . ev and a work function of . ev but with weak physisorption with an adsorption energy of - . kcal/mol. doping the fullerene with b, al, ga, si, and ge significantly increased the adsorption energy whereas the most stable structure was al-c with adsorption energy as high as - . kcal/mol while it was - . and - . for ga and b doped c fullerenes respectively. but the most promising performance was found from si and ge doped c fullerenes with a j o u r n a l p r e -p r o o f moderate adsorption energy of - . and - . kcal/mol but with a . % and . % reduction to the work function down to . ev for both case. hence si and ge doped c fullerenes were concluded as a potential sensor for the aa drug detection. c. parlak et al. further studied these structures for a drug sensor and carrier for antiviral drug amantadine, which is used to treat or prevent influenza-a, parkinson's disease but comes with side effects like corneal edema [ ] [ ] [ ] . all doped structures showed a promising performance as a sensor and carrier for the amantadine drug with adsorption energies ranging around - to - kcal/mol which was further increased in the water solution and energy gap around ev which was further reduced in the water solution. besides doping an adsorbent with impurity atoms, a noticeable change in the adsorption properties can be found due to the solvents, different isomers of the adsorbate drugs etc. which was further analyzed by o. ergürhan et al., in their investigation on the interaction of hydroquinone (hq) with pristine and doped c fullerenes [ ] . [ ] . for interaction between the -nh head of inh drug and a b atom of c b n heterofullerene, the adsorption energy was observed as - . kcal/mol with a significant decrease of . % to the bandgap which became . ev. for a drug delivery system of ibp drug e. alipour et al. modified c fullerene by substituting its five carbon atoms by n atoms and one tm atoms between fe, co, and ni which formed tmn c system [ ] . while nin c was reported to have a higher sensitivity towards ibp drug, nin c , con c , and fen c adsorbed ibp with acceptable adsorption energies of - . , - . , and - . kcal/mol, respectively including - . kcal/mol for the pristine c fullerene hence was considered potential systems for the ibp drug delivery. besides drug delivery and drug sensor, c fullerenes were also studied by m. f. kaya et al. to perform as a sensor for the phenylalanine (phe) amino acid, a high concentration of which in the blood can lead to mental retardation [ , , ] . using m x/ - g (d) level of theory, the maximum binding energy of the phe towards pristine c fullerene, alc and, sic was calculated as - . , - . , and - . kcal/mol respectively in the gas phase, except pristine c which was further increased to - . , - . , and - . kcal/mol respectively in the water solvent phase. the doping of al and si atoms reduces the bandgap of the structures j o u r n a l p r e -p r o o f significantly. for phe-c the bandgap in the gas(water) phase was observed as . ( . ) ev which dropped down as much as . ( . ) and . ( . ) ev for the al and si-doped c fullerenes with an nbo charge transfer of - . e and - . from n atom of phe to the al and si atoms of the adsorbent respectively. due to the alternation in the adsorption properties of phe towards alc and, sic made them a potential sensor for the phe amino acid while pristine c suffered from weak adsorption. currently, a popular method to synthesis the graphene is to reduce the go into gns [ ] . but the synthesis of the go was dated back to when b. c. brodie reported the first synthesis of go during his investigation on the various properties of graphite [ ] . this brodie synthesis method along with a slightly improved model from l. staudenmaier in was mostly used to synthesis until another improved model was proposed by w. s. hummers and r. e. offeman in by oxidizing the graphite with a water-free mixture of potassium permanganate (kmno ), sodium nitrate (nano ), and concentrated sulfuric acid (h so ) which is popularly known as the hummers method and is one of the most popular methods to synthesis go till now [ , ] . based on these methods till now there is an unambiguity on the exact structure of go, but this nonconductive hydrophilic allotrope of carbon lead it to be applied extensively in biomedical and therapeutic applications including the drug delivery and sensing [ , ] . pristine go was found to be a potential drug delivery system for sumatriptan (stt), a medicine used to treat migraine headaches [ ] . the dispersion corrected dft study lead by z. jafari et al. found that go adsorbed stt drug with acceptable adsorption energy to be used as a drug delivery system while the drug was reported to induce many chest symptoms [ ] . in the water phase, the stt drug was adsorbed with an adsorption energy of - . kcal/mol to go with a dipole moment of . debye and solvation energy of - . kcal/mol. the observed h-l energy gap of stt as . ev was further reduced to . ev for stt-go while it was . ev for go. a - . e charge transfer was observed from go to stt with a fermi energy level of - . ev. these interactions were further supported by the global reactivity descriptors where global hardness was increased to . ev with a decrease to the global softness and the electrophilicity to . ev - and . ev respectively. this result from the interaction of stt drug with go leads them to conclude go as a potential carrier for the stt drug. like other allotropes of carbon, go was further reported as a potential drug system for several anticancer drugs. z. hasanzade et al. made a dft investigation on the interaction of ellipticine (ept) anticancer drug with pristine go nanosheet (gons) and observed the effects of ph and solvents on the interaction using various solvents, e.g. water, ethanol and dimethyl sulfoxide (dmso) [ ] . in the gaseous environment, the ept drug was adsorbed with a maximum adsorption energy of - . kcal/mol where the maximum rise in the dipole moment was observed as . debye. in the water phase, a significant decrease in the adsorption energy was observed with a maximum of - . kcal/mol along with a considerable increase in the dipole moment to . debye where ept was dissolved with solvation energy ranging from - . to - . kcal/mol for different orientation of ept. the effects of dmso and ethanol was almost comparable with water as the adsorption energy and dipole moment were ranged between - . to - . kcal/mol and . to . debye for dmso. in contrast, for ethanol, it was - . to - . kcal/mol and . to . debye, respectively. as ept failed to alter the e g of gons, it may not work as a sensor for ept. still, it can be a potential drug delivery system which was further supported by the interaction of ept with gons in low ph regions as cancerous cells were reported to be slightly acidic. for another anticancer drug doxorubicin (dox) delivery, p. gong et al. decorated go with fluorine (fgo) to enhance the drug release mechanism in low ph regions [ ] . however, a high dox loading j o u r n a l p r e -p r o o f capacity was observed for the water-soluble fgo where non-covalent bonds were dominated in the loading and release mechanism of dox hence made fgo a promising candidate for the dox delivery. in their other work, they reported the synthesis of fgo with amino-polyethylene glycol and reported its potential use in the dox and another anticancer drug camptothecin delivery [ ] . besides the purely sp hybridized gns, another sp and sp hybridized allotrope of carbon, graphyne was also studied for medical applications using first-principles calculations. the graphyne nanosheet (gpns) was reported as a potential drug carrier for anticancer drug -fluorouracil ( -fu) and quercetin [ ] . in the surface of gpns, the most stable interaction of -fu was observed with an adsorption energy of - . kcal/mol where quercetin was adsorbed with - . kcal/mol. a charge transfer of . e and . e was observed for between the gpns and, -fu and quercetin respectively with a maximum drop of . % and . % in the energy gap lead to . ev and . ev respectively. with the increasing reactivity observed from the quantum reactivity descriptors, an increase in the adsorbent-adsorbate distance due to the protonation ensured the ability of gpns to deliver -fu and quercetin to the target cancerous cell. gpns was also investigated as a sensor to sense the cytosine and guanine nucleobases [ ] . gpns adsorbed the cytosine and guanine with an adsorption energy of - . and - . kcal/mol with a significant amount of . % and . % reduction in the h-l energy gap, respectively. this reduction in the bandgap indicated the increase in the electrical conductivity due to the adsorption of the nucleobases to the gpns ensured the cytosine and guanine sensing ability of the gpns. another graphyne like structure with carbon atoms is graphdiyne was reported to be synthesized by guoxing li et al. in by a cross-coupling reaction using hexaethynylbenzene on the copper surface [ ] . graphdiyne was also subjected to study as a potential drug delivery system for anticancer drugs. the interaction of two anticancer drugs sorafenib (stb) and regorafenib (rtb) with graphdiyne nanosheet (gdns) was investigated by u. srimathi et al. using dft theory with gga based pbe functional [ ] . stb that is used to treat thyroid [ ] and kidney cancer [ ] often come with several side-effects like alopecia, arthralgia, hypertension etc. [ ] [ ] [ ] whereas rtb is used to treat liver and colorectal cancer [ , ] but sometimes comes with deadly side effects with severe liver damage [ ] . stb was adsorbed by the gdns with a maximum adsorption energy of - . kcal/mol with solvation energy of - . kcal/mol and eg of . ev when for rtb these values were observed as - . kcal/mol, - . kcal/mol and . ev. a further alteration in the band structure was observed due to the adsorption of stb and rtb. finally, in the low ph regions similar to the cancer cells regions, the bond length between the gdns and stb, rtb increased noticeably, which ensured the drug release in the low ph regions. another anticancer drug, flutamide (flu), which was reported to make weak physisorption on cnt, a study by v. nagarajan et al. revealed strong chemisorption of flu drug with the graphdiyne nanotube (gdnt) [ ] . in the most stable interaction, flu was adsorbed by the gdnt with an adsorption energy of - . kcal/mol and solvation energy of - . kcal/mol along with a . % reduction to the energy gap to . ev which lead to a charge transfer of . e between the flu and gdnt. due to the interaction of the flu with gdnt, the chemical potential and global hardness decreased with an increase in the electrophilicity, which indicated the enhancement in the reactivity of the structures. while the minimum adsorbent-adsorbate distance was observed as . Å, this was raised up to . Å for lowering the ph by protonation, which ensured the drug release capability of gdns in slightly acidic cancerous cell environment. hence, gdns was concluded as a potential flu drug delivery system. the interaction of several other drugs like azathioprine, mesalamine, and hyoscyamine to be delivered to the targeted area using gdnt was also reported by the same group [ ] . in a first-principles investigation using dispersion corrected dft theory showed that the gdnt strongly adsorbed the azathioprine drug with an j o u r n a l p r e -p r o o f journal pre-proof adsorption energy of - . kcal/mol in its most stable position whereas the mesalamine and hyoscyamine were adsorbed with an adsorption energy of - . and - . kcal/mol respectively with solvation energy ranging from - . to - . kcal/mol. though a . % decrease in the energy gap to . ev was observed for the azathioprine drug adsorption on gdnt, it further dropped to . and . ev respectively for the mesalamine, and hyoscyamine with . % and . % decrease respectively. the reactivity of the structures was also studied using quantum reactivity descriptors. finally, a significant increase in the adsorbent-adsorbate bond length in the lower ph region ensured the drug release at the targeted site. the interaction of bcl with c h benzene at a very high temperature of ο c created a new type of graphite-like nanosheet but less stable than graphite known as bc , a theoretically relaxed structure of which is displayed on figure [ , ] . this bc nanosheet (ns) and nanotube (nt) was further studied by a. r. moosavi-zare et al. to sense an illicit cns stimulant amphetamine (aa) which was also reported to be adsorbed by pristine and si, ge doped c fullerenes [ , ] . it was observed that the bc nt adsorbed aa more strongly than the bc nt with an adsorption energy of - . kcal/mol where it was - . kcal/mol for the bc ns. despite stable adsorption, bc ns and nt were not considered as a sensible sensor for aa drug as a very poor decrease of . % and . % in the energy gap was observed. this was surmounted by doping bc with a si atom which reduced the energy gap by . % and . % for bc ns and nt respectively in case of aa adsorption. besides the si-doped bc ns and nt was found to adsorb aa drug more strongly with an adsorption energy of - . and - . kcal/mol respectively. despite this most stable and sensible for aa drug, si-doped bc nt suffers from a considerable recovery time of . s where it was only . s for si-doped bc ns. hence sidoped bc ns was considered as a potential sensor to sense the amphetamine drug. bc nt was further modified into bc n nt to work as a sensor for β-keto-amphetamine or simply cathinone (ct) [ ] . akin to the pristine bc ns and nt, bc n nt also suffers from a reduction in the energy gap by only % while it adsorbed ct with adsorption energy as high as - . kcal/mol. though al-doped bc n nt solved the problem with a . % reduction in the energy gap with a very strong adsorption of ct drug with an adsorption energy of - . kcal/mol, it suffers from a considerable recovery time. besides, si-doped bc n nt had a recovery time of . s and was considered as most sensible to the ct with a . % decrease in the energy gap due to the adsorption of ct with an adsorption energy of - . kcal/mol. finally, the interaction of an amino acid glycine with bc n nt was also reported by a. soltani et al. [ ] . glycine made strong chemisorption with the sidewall of bc n nt with a dipole moment ranging from . to . debye for different orientations resulting an increased reactivity due to the adsorption of glycine which was further verified from the global reactivity parameter analysis. another graphene alike d nanosheet, bnns made of boron and nitrogen atoms, is alternatively known as -white graphene‖ due white colour and graphene-like sp bonded hexagonal honeycomb structure with d h symmetry as shown in figure . the bnns is highly popular for its higher thermal and chemical stability compared to graphene while it has a very high melting point e.g. the hexagonal bnns (bnns) platelets showed thermal stability up to ~ o c in an investigation made by n. kostoglou et al. [ ] . the thermal conductivity of the bnns is also comparable to the graphene as the thermal conductivity for the bn nanoribbon is observed as ∼ - w m − k − at the room temperature [ ] . on the other hand, while graphene works as a zero bandgap semiconductor material, with a large bandgap of ~ . ev bnns works as an insulator or wide bandgap semiconductor material which help bnns to exhibit several unique optical properties [ ] . besides, the bnns j o u r n a l p r e -p r o o f can be synthesized with the large surface using numerous methods including the popular cvd methods for a large scale production [ ] [ ] [ ] . these alluring properties and sufficiently available synthesis methods made bnns to be applied as a catalyst [ , ] , dielectric material [ , ] , high temperature application [ ] , uv photodetector [ ] , energy storage device [ ] , sensor [ ] [ ] [ ] [ ] etc. finally, the biocompatibility of bnns [ ] leads them to the biomedical applications like drug sensors and drug delivery which will be reviewed in this section of our study. it was previously observed that many anticancer drugs have a structure like nucleobases as the operation of these drugs includes interactions with dna and rnas. several of these anticancer drugs -mercaptopurine (mp), -thioguanine (tg) and -fluorouracil (fu) which have structures akin to nucleobases adenine, guanine and uracil were subjected to be studied by m. vatanparast et al. for an efficient drug delivery system as they have a significant amount of side effects and they came up with bnns on this purpose [ ] . all three anticancer drugs made spontaneous adsorption to the bn, which was confirmed from the negative values for the adsorption energy and [ ] . it was observed that the among × , × , and × bn, thymine was adsorbed more strongly towards × bn with an adsorption energy of - . kcal/mol which was further increased to - . kcal/mol in case of an al atom doping. hence the size of the nanosheet along with doping it with impurity atoms plays a significant role in the drug adsorption. furthermore, using van der waals corrected dft theory (dft-d ), s. d dabhi et al. proposed a sensor to sense all dna and rna nucleobases using one-dimensional armchair and zigzag boron nanoribbons (bnnr) which can also be further applied for dna sequencing purpose [ ] . the adsorption profile of the nucleobases adsorbed in parallel with the armchair bnnr (a-bnnr) was observed as g > t > a > u > c with adsorption energies ranging from - . to - . kcal/mol with an adsorbent-adsorbate distance ranging as . Å to . Å. the a-bnnr responded towards all the nucleobases significantly by decreasing the h-l energy gaps ranging as . ev to . ev while it was . ev for the a-bnnr alone. the least h-l energy gap was observed for c + a-bnnr complex as . ev indicated that a-bnnr was most sensitive towards cytosine (c) and least towards adenine (a). on the contrary to the h-l energy gap, the work function of the a-bnnr increased noticeably with figure : hexagonal bnns optimized using dft theory with b lyp hybrid exchange correlational functional and - g basis set. adsorption of the nucleobases. the zigzag bnnr (z-bnnr) also followed the same trend upon the adsorption of nucleobases. but the adsorption energies were this time observed as g > c > a > t > u with a range of - . to - . kcal/mol. z-bnnr was also most sensitive towards the c with a least h-l energy gap . ev following by g, u, t, and a. here, the work function again increased upon the adsorption of the nucleobases towards the z-bnnr. hence the bnnr may not function as a work function based sensor but holds the potential of an electronic sensor to detect nucleobases. in several other theoretical studies, the performance of bnns was compared with other bn nanostructures for several other drug sensing. k. nejati et al. made a first-principles investigation using dft theory with b lyp hybrid functional on the interaction of cathinone drug with bnns, zigzag ( , ) bnnt, and b n nanocage (bnnc) [ ] . though the bnns showed a moderate sensitivity towards cathinone, it failed to adsorb the drug sufficiently. for different orientations of cathinone, bnns adsorbed with adsorption energies of - . and - . kcal/mol with a decrease in the h-l energy gap . % and . % respectively. the same trend was j o u r n a l p r e -p r o o f observed with bnnt while it failed to adsorb cathinone if shows sensitivity towards the drug. though bnnt still showed more enhanced performance than bnns by adsorbing the drug with adsorption energies as - . and - . kcal/mol for different orientations, with a decrease in the h-l energy gap by . % and . %. the nbo charge transfer was also greater in the bnnt from cathinone as . e and . e while it was only . e and . e respectively for bnns. the most alluring sensing properties towards cathinone was observed from bnnc while it adsorbed the drug with adsorption energies as - . and - . kcal/mol with a significant decrease in the h-l energy gap as . % and . % respectively. the nbo charge transfer from cathinone to bnnc was observed as . e and . e made it a potential candidate for the development of an electronic sensor to detect cathinone drug. besides, all three nanostructures benefit from a short recovery time of . s for bnnc following by . s for bnnt, and . ns for bnns at the room temperature of k. the bnns, bnnt, and bnnc also showed the same sensing properties towards another anticancer chemotherapeutic drug, mercaptopurine (mp), which was revealed by s. a. aslanzadeh from dft calculations using b lyp/ - g (d) level of theory [ ] . alike from cathinone, bnns also showed inferior adsorption properties towards mp with an adsorption energy of - . kcal/mol with a decrease in the h-l energy gap only by . %. this time also the most sensitivity and the most stable adsorption of mp was observed with bnnc. bnnc adsorbed the mp drug with maximum adsorption energy - . kcal/mol while the range of decrease in h-l energy gap was observed as . % to . %. but due to this strong adsorption, this time the bnnc suffered from an unrealistic high recovery time of . seconds. besides this time, the most promising adsorption behaviour of mp drug was observed from bnnt. bnnt adsorbed the drug with a decent maximum adsorption energy of - . kcal/mol while the decrease in h-l energy gap was observed from . % to . % for different orientations of the drug. besides, the bnnt also benefits from a shorter recovery time of . seconds at the room temperature of k. hence, bnnt holds the most potentiality in the development of an electrochemical sensor to detect mp drug. after the discovery of cnt in [ ] , the novel properties of bnns lead to the successful discovery of another polymorph of graphitic bn, bnnt in by n. g. chopra et al. [ ] shortly after its theoretical prediction in [ ] . by that time, the availability of many different methods to grow cnt and bns lead bnnts to be synthesized using many popular methods like arc discharge [ ] , ball mailing method [ ] , laser ablation method [ ] , and chemical vapour deposition (cvd) [ ] . being made of bns the bnnt also shares the similar properties as bnns like high thermal and chemical stability [ , ] , high thermal conductivity [ ] , high oxidation resistance [ ] and a wide insulating bandgap of ~ . ev [ ] . besides, having a tubular structure provided it excellent mechanical strength with the elastic modulus in tpa level as cnt [ ] . these properties made its applications as nanocomposites [ ] , nano light source [ ] , oil-polluted water treatment [ ] , radiation shielding [ ] , sensor [ , , ] etc. finally, bnnts were found to be non-toxic to the human body and biocompatible, which lead it to many biomedical applications [ ] [ ] [ ] [ ] . an efficient sensor for the broadly used sulfonamide (sa) antibacterial drugs is crucial to the medical community as it causes an adverse reaction to the % of its general consumers which rises % to the patients with human immunodeficiency virus (hiv) [ ] . hence z. rahmani et al. made a first-principles investigation on the bnnt and several other doped (al, si) bnnt using dft theory along with m - x functional and - g (d) basis set to study their sa drugs sensing ability [ ] . unlike the majority of the drug sensors, in this case, pristine bnnt performed as a better sa drugs sensor than si and al-doped bnnt. in the most stable position, bnnt adsorbed sa drugs with an adsorption energy of - . kcal/mol with an % reduction to the h-l energy gap. though the value for adsorption energy increased dramatically for al-doped bnnt to - . j o u r n a l p r e -p r o o f kcal/mol, it failed to decrease the energy gap significantly, which was only . %. hence with this poor sensing ability but strong adsorption of sa drug made al-bnnt a decomposition agent for sa drugs. for, si-bnnt the sa drug was adsorbed with an adsorption energy of - . kcal/mol at the most stable orientation but increased the h-l energy gap by . % which was enhanced in another orientation where sa decreased h-l energy gap by . % but was adsorbed with an adsorption energy of - . kcal/mol only. so, si-bnnt is not ideal to be used either as a sensor or to decompose sa drug. the popular anticancer drug -fluorouracil (fu) which was reported to be carried out by several carbon and boron-based nanoclusters, is also reported to make strong chemisorption with zigzag and armchair bnnt. from a first-principles investigation lead by a. soltani et al., it was observed that the ( , ) zigzag bnnt adsorbed fu more strongly with a reduction in the h-l energy gap significantly with an increase in the dipole moment (dm) compared to the ( , ) zigzag bnnt [ ] . the ( , ) zigzag bnnt adsorbed fu with an adsorption energy of - . kcal/mol with a dm of only . debye and an increase in the h-l energy gap by . % whereas the ( , ) zigzag bnnt adsorbed fu with an adsorption energy of - . kcal/mol with an increased dm of . debye and decrease in the h-l energy gap by . %. so they further doped ( , ) zigzag bnnt with al, ga and ge to enhance the adsorption of fu to bnnt. the most promising result was observed for al-doped ( , ) zigzag bnnt (al-bnnt) where it adsorbed fu strongly with chemisorption of - . kcal/mol with a significant decrease in the energy gap by . % where the dm was observed as high as . debye. after that, a promising result was also observed from the ga-bnnt where it adsorbed fu with an adsorption energy of - . kcal/mol and decreased the h-l energy gap by . % with a dm of . debye. among doped bnnt the least promising performance was observed from ge-bnnt but better than the pristine ( , ) zigzag bnnt. ge-bnnt adsorbed fu with an adsorption energy of - . kcal/mol with a cut to the h-l energy gap by . % and an increase in the dm to . debye. these increase in the reactivity was further verified from a decrease in the global hardness and changes in the global reactivity parameters accordingly. though the adsorption process of fu drug to the ( , ) zigzag bnnt wasn't spontaneous as it was seen from the change in gibbs free energy (Δg), for al and ga doped bnnt it was spontaneous with a negative value for Δg. and the adsorption process of fu to all adsorbent was exothermic with a decrease in the entropy. so analyzing the results, they came to a conclusion that ga-bnnt can be used as a potential drug delivery system for fu for its enhanced nbo charge transfer properties where al-bnnt can be a potentially stable sensor fu with high adsorption energy and reduction in the h-l energy gap. besides zigzag bnnt, k. shayan et al. investigated the interaction of fu with a wide number of armchair bnnt [ ] . though the armchair bnnts adsorbed fu with less adsorption energies compared to the zigzag bnnts, from a detailed analysis of thermodynamical parameters, charge transfer, and global reactivity descriptors it was revealed that armchair bnnts also could be a potential drug delivery systems to deliver fu drug. the dft theory with m - x and b lyp exchange correlational functional with - g* basis set was also employed to investigate the -oh functionalized ( , ) zigzag bnnt (f-bnnt) as a drug delivery system for another anticancer drug carmustine (bcnu) [ ] . bcmu is a potential anticancer drug that is used to treat many kinds of brain cancers for over few decades, but sometimes it induces unwanted side effects like acute interstitial pneumonitis, myocardial ischemia etc. [ ] [ ] [ ] . using the b lyp hybrid exchange correlational functional, it was observed that the bncu interacted with f-bnnt with a maximum adsorption energy of - . kcal/mol in the gas phase which decreased to - . kcal/mol in the water phase. this interaction energy was increased as high as - . (- . ) kcal/mol in the gas (water) phase, while the dispersion corrected m - x functional was used. this significant difference in the adsorption energies using two different functional indicated that the weak van der waals (vdw) interactions played an important role in the bncu adsorption on f-bnnt. this was then confirmed from the md simulations where vdw energy increased to - . kcal/mol from - . kcal/mol for a j o u r n a l p r e -p r o o f single drug molecule with an increase in the temperature to k from k. the adsorption process was spontaneous and thermodynamically possible with an exothermic one in both gaseous and water phase. but the adsorption of bncu barely affected the charge transfer and electronic properties with an increase in the h-l energy gap. besides the reactivity of the structures was also decreased with an increase in the global hardness of the bncu adsorbed f-bnnt. the biocompatibility and non-toxicity of bnnts encouraged s. ghahremani et al. to encapsulate an anticancer drug floxuridine (fudr) into a ( , - ) armchair bnnt [ ] . fudr is an anticancer drug to treat colorectal cancer and often causes side effects like toxicity, diarrhoea etc. which requires immediate medical attention and hospitalization [ ] [ ] [ ] . fudr was encapsulated inside the ( , - ) bnnt with interaction energy of - . kcal/mol only. but the encapsulation of fudr decreased the energy gap to . ev from . ev, which was occurred due to a significant increase in the lumo energy to - . ev from - . ev. more importantly, the dm of the fudr encapsulated ( , bnnt increased significantly to the . debye while it was . for the ( , bnnt. this increase in the dm indicated an enhanced reactivity of the structure in a polar medium. this enhancement in the reactivity was also reflected in the quantum reactivity descriptors where the global hardness of the structure was increased to . ev where it was . ev for the ( , - ) bnnt alone and the global softness increased to . ev - from . ev - along with an increase in the electrophilicity to . ev from . ev. from nbo analysis, it was observed that the electron is most like to flow towards bnnt from fudr which made fudr an electron donor and bnnt an electron acceptor. the weak physisorption was further confirmed from the qtaim analysis where it was observed that the interaction between the fudr and bnnt was weak van der waals interactions with a small value for the electron density and a negative value for where five hydrogen bond was also observed. these promising results lead them to propose a drug carrier for the fudr based on ( , bnnt to reduce the unwanted effects of the fudr drug in our body. besides the anticancer drug delivery, the sensing properties of bnnt on anti-fever and pain-reducing drugs like paracetamol (pa) and phenacetin (ph) was also observed from several studies as the overdose of these drugs may induce some liver and kidney damage [ ] . the interaction of both pa and ph with several armchair bnnt with different chirality was investigated by h. ghasempour et al. using dispersion corrected dft-d theory with gga functional [ ] . interestingly, instead of adsorbing pa, it was desorbed in the inside of ( , ) bnnt with a positive adsorption energy of . kcal/mol while it was adsorbed strongly with an adsorption energy of - . kcal/mol in the outside of the ( , ) bnnt. but the reverse effect was observed in case of the ( , ) and ( , ) bnnt where pa was adsorbed more stably inside the bnnt with an adsorption energy of - . and - . kcal/mol respectively while it was - . and - . kcal/mol respectively for the adsorption of pa on the outside of ( , ) and ( , ) bnnts. again more enhanced properties on sensing pa like a high drop in the h-l energy gap and an increase in the dm was observed while the drug was in the outside of ( , ) and ( , ) bnnts. the exact same adsorption properties like desorption inside ( , ) bnnt but more stable adsorption inside ( , ) and ( , ) bnnts, more decrease in the energy gap and more increase in the dm while the drug was adsorbed on the outer surface of ( , ) and ( , ) bnnts was also observed for the ph adsorption on the bnnts. the more effect on the pa adsorption towards bnnt was further investigated by z. iranmanesh-zarandy et al. using md simulations in terms of change in nanotube's size, length, chirality and temperature [ ] . the binding free energy of pa was increased with an increase in the diameter of the bnnt. the binding free energy of pa towards the ( , ) bnnt with diameter . Å was observed as - . kcal/mol while it was gradually increased to - . kcal/mol for the ( , ) bnnt with a diameter of . Å. but a significant decrease in the stability was observed in cases like an increase in the length of bnnt and chirality along with a minor decrease in the stability for increasing temperature. recently several computational works also had been done on investigating bnnt as a sensor for several harmful chemical compounds, a long exposure of which may induce harmful effect to the human body. b. makiabadi et al. investigated the sensing properties of ( , ) zigzag bnnt and three al-doped b al nnt with a diameter ranging . - . Å using b lyp/ - ++g(d) level of theory, to sense carcinogenic compounds nitrosamine (na) and thionitrosamine (tna) which can make an entry to the human body through cigarette smoke, water, foods etc. and induce gastric and oesophageal cancer [ ] [ ] [ ] [ ] . both bnnt and b al nnt adsorbed na and tna with stable negative adsorption energies along with a decrease in the h-l energy gap hence increase in the conductivity, and increase in the reactivity by a decreasing chemical hardness with increasing chemical softness and electrophilicity. b al nnt showed more stable adsorption of na and tna with adsorption energy (bsse corrected) ranging - . to - . kcal/mol while it was - . to - . kcal/mol for the bnnt. besides more charge transfer from na and tna to bnnt was observed than b al nnt and all the bonds between the adsorbent and adsorbate were covalent in nature. g. fan et al. proposed another sensor for acetone based on zigzag bnnts from a dispersion corrected dft-d study which can have a potential application on the detection of diabetes as a patient with diabetes produces more acetone in their body than a normal person [ ] . it was observed that the pristine bnnts adsorbed acetone with decent stability but hardly able to sense it which was further improved by doping impurity atoms to the bnnt hence an acetone sensor can be developed with flexible sensing properties based on the necessity. besides, the interaction of the oxazole and isoxazole with bnnt was investigated by j. kaur et al. as many drugs have a structure similar to these heterocyclic aromatic organic compounds hence plays an essential role in the development of many drugs [ ] . though the ( , ) zigzag bnnt adsorbed oxazole in both gaseous and solvent phase with an adsorption energy of - . and - . kcal/mol respectively, it adsorbed isoxazole only in the solvent phase with an adsorption energy of - . kcal/mol. but the ( , ) armchair bnnt desorbed both oxazole and isoxazole in both gaseous and solvent phase with a positive value for the adsorption energy. a higher reactivity with a significantly increased dm was also observed for the ( , ) bnnt than ( , ) bnnt. hence the solvent and the chirality of the bnnt affects the adsorption properties of oxazole and isoxazole significantly. prior to the advancement of the cnt and c fullerenes, the investigation on the bnnt and boron nitride nanocages (bnnc) also started with almost the same timeline. research on the bnncs got immense attention after a report from f. jensen and h. toftlund stating that b n nanocage offers higher stability compared to the c fullerenes [ ] . shortly after that, in the first accidental observation of the nested bnnc was reported by . stephan et al. during doping the cnt with b and n atoms using the electric arc-discharge method [ ] . furthermore, many groups reported the growth of the bnnc using various methods including the formation of octahedral b n bnnc ( figure ) using electron beam irradiation by d. golberg et al. [ ] , the formation of b n using an arc-melting method by t. oku et al. [ ] etc. being made of boron nitrides, bnncs also benefit from the unique properties shown by other bn polymorphs like wide bandgap, high resistance to the oxidation, high thermal and chemical stability etc. which lead its application as good lubrication [ ] , wastewater treatment [ ] , hydrogen storage [ ] , gas sensors [ ] etc. bnncs are also reported to have a potential application in the anticancer drug sensing and delivery system. thioguanine or -thioguanine (tg), a drug that has clinical applications to treat several types of acute leukaemia, have many unwanted side effects like bone marrow suppression, liver toxicity etc. [ ] [ ] [ ] . to reduce these adverse effects on the human body, m. noormohammadbeigi et al. investigated pristine b n nanocage to work as a potential nano-drug delivery vehicle for tg delivery [ ] . considering non-covalent calculations, they chose m - x functional along with widely used - g (d,p) basis set and dft theory as the m - x functional have a higher percentage of hartree-fock exchange ( %). the fascinating property upon tg j o u r n a l p r e -p r o o f adsorption on b n was the decrease in the h-l energy gap by a noticeable change ranging . - . % which was confirmed from the dos spectrum of structures hence made a significant rise to the electrical conductivity of tg adsorbed structures. besides this sensitivity towards tg, b n was also spontaneously adsorbed tg by strong chemisorption with an exothermic process. the adsorption energy of the tg on b n was in a range of - . to - . kcal/mol for different orientations and isomers of the drug in the gaseous phase. however, this range increased into - . to - . kcal/mol in the water solvent phase with a significant increase in the dm to as high as . debye. higher charge polarization transfer from tg to b n was also observed in the water phase. these properties made them conclude b n nanocage as a nano drug delivery system for tg drug. ifosfamide (ifo) is another anticancer drug which has a potential application to treat several kinds of cancer and tumours including testicular cancer, small cell lung cancer, ovarian cancer etc. and have relatively fewer side effects [ ] . but sometimes, it is capable of inducing life-threatening toxicities like cardiac toxicity, hemorrhagic cystitis, nephrotoxicity, encephalopathy and neuropathy [ ] . a. soltani et al. investigated the interaction of ifo with pristine b n and carbon atom doped b n c and b n c fullerenes which revealed that those unwanted side effects of ifo might be reduced by using bn fullerenes as a nano-drug delivery system for the ifo [ ] . besides the dft theory, they chose one gga and another hybrid functional with a high percentage of hf (hartree-fock) exchange as gga pbe- was observed to have results closer to the experimental outcomes for b n , and the hybrid m - x was used widely for b n c , graphene and other nanosystems. unlike many other nanostructures, pristine b n fullerene adsorbed ifo drug with strong chemisorption of - . kcal/mol in its most stable orientations which was further enhanced in the water solution where it became - . kcal/mol. this figure : b n nanocage relaxed using dft theory with b lyp hybrid exchange correlational functional and - g (d,p) basis set. interaction between the ifo and bnnc became stronger due to the substitution of carbon atoms, and the interaction energies were observed as - . and - . kcal/mol while the adsorbents were b n c and b n c fullerenes, respectively. the dm for the ifo adsorbed structures increased in the solvent medium and was observed as high as . debye for b n c and . debye for b n c fullerene. these properties ensured the ifo drug loading ability of b n c and b n c and the biocompatibility of the structures were increased with a decrease in the toxicity which was confirmed from an increasing reactivity observed from the increasing chemical potential and electrophilicity with a decrease in the global hardness. besides delivering the tg and ifo anticancer drugs, bnnc was also reported to be worked as a potential sensor candidate for another anticancer drug cisplatin (cp) which was revealed from a first-principles investigation made by s. onsori et al. [ ] . it was observed that at the most stable interaction, the b n nanocage adsorbed cp drug with an adsorption energy of - . kcal/mol. but one of the key features for sensor development, decrease in the h-l energy gap, was observed with the highest decrease of . %, which was analyzed from the dos spectrum. for the different orientation of cp drug the reduction in the h-l energy gap ranges from . % to . % which ensured that b n nanocage could detect cp drug in various orientations efficiently with an increase in the conductivity to produce a detectable signal. besides the recovery time of the sensor was also found as low as ~ ms ensured that pristine b n nanocage could be a potential candidate for cp drug sensor without any modification, doping or decoration. furthermore, bnnc was considered to be a potential sensor to detect another anticancer drug α-cyano- -hydroxycinnamic acid (chc) [ ] . frontier molecular orbital analysis with dos revealed that due to the adsorption of chc the h-l energy gap of b n nanocage decreased by a minimum of . % to a maximum of . % for different orientations which ensured its sensitivity towards chc drug. almost the same sensitivity was also observed in the water solvent where the decrease in the h-l energy gap was observed as . % to . % for different orientations of chc on b n nanocage. besides, it adsorbed chc with a maximum adsorption energy of - . kcal/mol, which leads the sensor to be suffered by a moderately large recovery time of . sec at the room temperature. hence the b n nanocage can be considered as a potential sensor candidate to detect cp and chc drug. in addition, bnnc was studied to design a nano-vehicle for an anti-inflammatory drug like celecoxib (cxb) and a sensor for metformin (mf) that is used to treat type diabetes. first, n. abdolahi et al. investigated the adsorption properties of cxb drug adsorption on b n fullerene (bnnc) and found that bnnc can be a potential candidate for the nano-vehicle of cxb to transfer it into the targeted cell [ ] . the rising importance on the drug delivery system is associated with adverse effects of cxb like hypertension, diarrhoea, gastrointestinal effects besides its use as a cox- selective nonsteroidal anti-inflammatory drug (nsaid) [ ] [ ] [ ] . the bnnc firmly bound the cxb drug by strong chemisorption with an adsorption energy of - . kcal/mol in a vacuum and with - . kcal/mol in water medium at its most stable state. besides the bnnc was also sensitive towards the cxb drug as a decrease of . % in the h-l energy gap was observed from the dos spectrum analysis. due to the adsorption of cxb, the reactivity of the structure also increased with a decreasing global hardness, chemical potential, and electrophilicity along with an increase in the global softness while the dm of the structure was also increased to as high as . debye from zero for the pristine symmetric b n fullerene which raised the potentiality of bnnc as a nano-vehicle for cxb in drug delivery application. in another study, a.s. ghasemi et al. initiated an investigation lead by dispersion corrected dft-d theory to find the drug delivery capability of pristine and doped b n and b n fullerenes to deliver mf but ended up with finding sensor application of bnnc to sense mf [ ] . without any doping or functionalization of b n and b n fullerenes, both interacted with mf strongly with an adsorption energy of - . and - . kcal/mol respectively with a decrease in the h-l energy gap by . % and . %. besides the reactivity of the structures increase with an increase in the dm up to . debye along with an increase in global softness and decrease in the global hardness, chemical potential, and electrophilicity. hence the pristine b n and b n fullerenes can be potential candidate in the sensor development of mf drug. due to doping b n fullerenes with al, si, ge and ga, while the most stable interaction of mf was observed with alb n with a large adsorption energy of - . kcal/mol, geb n was most sensitive towards mf drug with a decrease in the h-l energy gap by . % with an acceptable adsorption energy of - . kcal/mol. hence the study concludes geb n fullerene as the most potential nanostructure to work as a biosensor for mf drug. the popular pyridine nucleobase uracil like anticancer drug fluorouracil (fu) was further adsorbed with the quasi-planar b boron nanosheet ( figure ) in order to investigate if the b can be used as a novel drug carrier system for the fu drug [ ] . all the quantum mechanical calculations were carried out by timedependent dft (td-dft) theory, and for the exchange correlational functional they depended on the hybrid tpssh functional with - +g (d) basis set. first, the fu drug failed to generate any detectable signal where no noticeable change in the h-l energy gap was observed while the drug interacted with the concave and convex surface of b nanosheet with weak interaction energy of - . to - . kcal/mol. but these interactions were greatly enhanced while the drug interacted with its o atom on the edge of the b nanosheet. while a single molecule was adsorbed on the edge of b nanosheet, in the gaseous medium the adsorption energy was observed as high as - . kcal/mol with maximum deformation energy of . kcal/mol. the b also detected fu with a noticeable decrease in the h-l energy gap of % with an nbo charge transfer of . a.u. the dm of these structures was also observed as high as . debye, which indicated an increase in the reactivity in the polar medium. while instead of one fu, two fu molecule was adsorbed on the b nanosheet, the adsorption energy per molecule was slightly decreased with a maximum value of - . kcal/mol but increased the deformation energy as high as . kcal/mol. despite a less interaction the sensitivity and reactivity increased significantly where the decrease in the h-l energy gap was as high as % with nbo charge transfer ranging . to . a.u. while the dm was observed as high as . debye. besides, a more strong interaction of fu with b was observed in the aqueous solution where the adsorption energy was as high as - . kcal/mol with an increased reactivity where the highest dm was observed as . debye, but no significant change in the h-l energy gap was observed. hence these enhanced adsorption properties in the polar medium made b nanosheet a potential candidate for the nano-drug carrier of fu. besides, drug delivery application, b nanosheet was further investigated by c. xiao et al. in a search for an electrochemical sensor development to sense metronidazole (ml) drug [ ] . this antibiotic drug is used to treat a number of diseases including bacterial vaginosis, perineal crohn's disease, trichomoniasis etc. but an extensive usage of it can lead to unwanted carcinogenic effects on the human body [ ] [ ] [ ] . the dft theory based b lyp-gcp-d / - g* level of theory was applied to optimize the structures which further found that like fu, ml also interacted more strongly with the b nanosheet by its edge with adsorption energy as high as - . kcal/mol while the adsorption process was spontaneous with a negative value for the change in gibbs free energy as - . kcal/mol. but the maximum adsorption energy of the complexes was slightly decreased to - . kcal/mol with an increase in the solvation energy to - . kcal/mol. despite a very slight change in the work function of the b nanosheet due to the adsorption of ml, the change in the h-l energy gap was dramatic with the highest drop by . % to . ev from . ev ensuring a significant increase in the conductivity of the structure. this significant decrease in the h-l energy gap also caused an nbo charge transfer of . e between the ml and b nanosheet which will be able to induce a detectable electrochemical signal and can be used to develop a potential sensor to sense ml drug. the recovery time of the sensor was further found to be as low as . s for the most stable adsorption in the room temperature. using the different sensing properties towards the four dna nucleobases adenine, guanine, cytosine, thymine, the quasi-planar b nanosheet was further proposed for an application as a dna sequencing device by a dft investigation from a. rastgou et al. [ ] . it was observed that the b nanosheet was most sensitive towards the cytosine with its edge than other nucleobases with a massive decrease in the h-l energy gap by . %. for the thymine, this decrease in the h-l energy gap was observed within the range . % to . % and was the second most sensitive structure towards b nanosheet. the sensitivity of adenine and guanine was almost similar towards the b nanosheet, but the sensitivity was slightly higher for adenine with a decrease in the energy gap by . % to . % following by . % to . % for guanine although two orientations can decrease the j o u r n a l p r e -p r o o f energy gap by . % and . %. hence the highest peak in the conductivity will be observed for cytosine following by the thymine, adenine and guanine from which these dna nucleobases can be identified. though the electronic sensitivity was observed as cytosine>thymine> adenine ~ guanine, the energetic sensitivity of b nanosheet was observed as adenine>guanine> cytosine>thymine. another quasi-planar boron nanocluster, bowl-shaped b nanostructure was proposed to function as a drug delivery system for amantadine drug as it often comes with unwanted adverse effects like insomnia, depression, hallucinations etc. besides its application to treat parkinson disease and influenza a [ ] [ ] [ ] [ ] . like b nanosheet the strongest interaction of amantadine was observed on the edge of b nanostructure with an adsorption energy of - . kcal/mol while for other orientations it ranged between - . to - . kcal/mol ensuring strong chemisorption for every orientation. the amantadine adsorption by b nanostructure further got stronger on the water solvent while the range of the adsorption energy became - . to as high as - . kcal/mol. besides b nanostructure was more sensitive to amantadine by its edge but still not sufficient to develop a sensor as the maximum drop in the h-l energy gap was observed as only . % while for the other orientations this was observed with a range . % to . %. finally, the polarity and reactivity of the b nanostructure increased significantly with a major increase in the dm as high as . debye from . debye for the pristine b nanostructure. for other orientations, the dipole moment was also almost similar within the range of . to . debye. this increase in the polarity and reactivity with decent adsorption energy which also increased significantly in the aqueous solution made b nanostructure a potential candidate as a nano-drug delivery system for amantadine drug. as usual, metal oxide (mo) nanostructures were also studied as a drug delivery system for several therapeutic anticancer drugs. r. khorram et al. investigated the drug delivery properties maghemite (γ-fe o ) nanostructure to deliver carmustine (bcnu) anticancer therapeutic drug which was previously discussed in this study to be delivered using -oh functionalized ( , ) zigzag bnnt (f-bnnt) [ ] . like f-bnnt, bcnu was j o u r n a l p r e -p r o o f also made a weak interaction with maghemite with a maximum adsorption energy - . kcal/mol while other orientation of the drug it was also observed as low as - . kcal/mol. from thermodynamical parameter analysis, it was observed that the drug adsorption process was spontaneous and exothermic in nature as the change in the gibbs free energy and enthalpy was negative. the dm of the structures increased significantly to as high as . debye due to the adsorption of bcnu from . debye for the maghemite which increased the polarity and reactivity of the structures noticeably, and this played a crucial role into the delivery of bcnu using maghemite. the aim analysis was also done to investigate the type of interaction between the drug and maghemite which revealed the presence of partial covalent interaction between them. besides, nbo analysis identified the bcnu as electron donor where maghemite was a receiver, and this was further verified from a more negative value of chemical potential for maghemite as - . ev compared to - . ev for bcnu. this work with maghemite besides f-bnnt will surely advance the study to find a nano-drug carrier for bcnu drug. besides drug delivery, another study from s. onsori and e. alipour proposed another mo, zn o nanocluster, to develop an electrochemical sensor for another anticancer drug cisplatin (cp) with brand name platinol for which another sensor was discussed previously on this study using b n nanocage [ ] . the zn o nanocluster made a stronger interaction with cp compared to the b n nanocage but with a less sensitivity where the maximum adsorption energy and decrease in the h-l energy gap were observed as - . kcal/mol and . % respectively. the adsorption process was spontaneous with a negative value for the change in gibbs free energy as high as a - . kcal/mol with a maximum change transfer of . e between the cp and zn o nanocluster. the sensitivity of the zn o nanostructure towards cp was enhanced in the aqueous solution with a maximum drop in the h-l energy gap by . % with a slight reduction in the maximum adsorption energy to - . kcal/mol. the sensor also had a short recovery time of . sec in the combination of which made zn o nanostructure a promising sensor candidate for cp drug. the isonicotinic acid hydrazide (inh) with brand name isoniazid, a prodrug to treat the deadly infectious disease tuberculosis (tb), which was previously discussed to be delivered using swcnt by n. saikia et al., was also proposed to be delivered using another mo framework mg o nanocage by i. ravaei et al. [ ] . the pristine mg o nanocage made a strong interaction with inh drug with a maximum adsorption energy of - . kcal/mol but failed to alter the reactivity and electronic properties with no noticeable change in the global reactivity parameters and h-l energy gap of the structures. this problem was overcome by replacing an mg atom by al atom to the mg o nanocage, which decreased the h-l energy gap to . ev from . ev. the h-l energy gap was further reduced to . ev by . % due to the adsorption of inh drug on al-mg o nanocage with an increase in the reactivity by lowering the global hardness and chemical potential. due to the doping of al atom, the interaction of the inh drug also got stronger with an increase in the adsorption energy to as high as - . kcal/mol. besides from aim and nbo analysis, it was revealed that the interaction between the drug and carrier was partially covalent in nature and the electrons were transferred from inh drug to the al-mg o nanocage. in another first-principles investigation, m. rezaei-sameti et al. investigated the interaction of another mo, be o nanocluster with mercaptopyridine (mcp) drug [ ] . interestingly, pristine be o nanocluster showed more encouraging adsorption properties towards mcp than sc and ti-doped be o nanocluster in order to develop a nano-drug carrier system for mcp drug with maximum adsorption energy and dm of - . kcal/mol and . debye respectively. doping the nanocluster with sc and ti increased the adsorption energy to as high as - . kcal/mol but decreased the maximum dm to . debye. but the doping of sc and ti increased the sensitivity of the be o nanocluster towards the mcp drug by reducing the h-l energy gap significantly, which was not observed noticeably for pristine be o nanocluster. hence, those nanoclusters can hold future potential for the sensor development of mcp drug. furthermore, the adsorption process was spontaneous, j o u r n a l p r e -p r o o f exothermic, ordered for all investigated structures except the mcp adsorption with s atom towards the pristine be o nanocluster with a positive value for the change in gibbs free energy. besides mcp, another mo nanocluster, zno single-walled nanotube (swnt) was also proposed to be a potential sensor candidate for protein tyrosine nitration (ptn), -nitrotyrosine, which can be produced during the post-translational modifications in proteins and cause several human disorders like rheumatoid arthritis, parkinson's disease, alzheimer etc. [ ] . the interaction of the ptn increased significantly with the increasing chirality of the zno swnts. the maximum binding energy of the ( , ) zno swnt was observed as . kcal/mol while it was increased to . kcal/mol for zno swnt. finally, a noticeable alteration in the electronic band structure of the zno swnt indicated its sensitivity towards ptn. the interaction of isoniazid (inh) was also studied with the pristine and ni-doped ( , ) armchair gannts by m. rezaei-sameti et al. using the dft cam-b lyp/ - g(d) level of theory [ ] . it was observed that the pristine and ni-doped gannts adsorbed the inh drug with an adsorbate-adsorbent distance of . - . Å. the pristine gannt adsorbed the inh strongly with adsorption energies ranging - . to - . kcal/mol for different orientations for the drug which hadn't increased due to the doping with ni. the adsorption processes were exothermic in nature and thermodynamically favourable. all interactions were spontaneous and ordered with negative values for the change in gibbs free energy and entropy. the h-l energy for all of the investigated structures were ranging between . ev and . ev and a decrease in the energy gap was observed in the nidoped structures with an increase in the conductivity. besides, the work function of the investigated structures was observed as - . to - . ev. from the nbo analysis, it was also revealed that the inh worked as the electron donor in the inh-gannt complexes with a positive value for which agreed with the positive values for maximum electronic charge transfer ( ). these alluring adsorption properties may lead ( , ) armchair gannts to a potential sensor candidate for the inh drug. the sensor for another drug, benzoylethanamine (bea) or β-keto-amphetamine, was reported by a. hosseinian et al. using aln nanoclusters [ ] . another sensor for bea was previously discussed in this review besides, the aln nanosheet also has a very short recovery time of . s in the room temperature. though the aln nanoclusters hadn't shown better properties than the si-doped bc n nanotube, as pristine structures they showed better sensing properties than pristine bc nanotube, and these nanoclusters can be considered as potential candidates to develop a sensor for bea drug. besides the sensor for bea, aln nanocage was also reported to have a potential application in the development of a work function based sensor for antiparkinsonian drug amantadine (am) drug whose delivery was previously discussed by z. noroozi et al. [ , ] . at its most stable state, the aln nanocage adsorbed the am drug with an adsorption energy of - . kcal/mol with an adsorbent-adsorbate distance of . Å. though a charge transfer of . e from am to the nanocage which was confirmed by nbo analysis, am hardly able to decrease the h-l energy gap of the nanocage upon its adsorption. the decrease in the energy gap was observed by only . % leading no significant change in the conductivity of the nanocage. but the adsorption of am significantly decreased the work function by . % to . ev from . ev for pristine aln nanocage. hence the aln was considered to have potential on the development of a work function based biosensor for am drug. on the other hand, the bnnc was observed to decrease its h-l energy gap by . % to . ev on the adsorption of am drug. besides, bnnc also adsorbed the am drug with a decent adsorption energy of - . kcal/mol. hence the bnnc was considered as a potential electrochemical sensor to detect am drug. both aln and bn sensors were benefited from a shorter recovery time of s and . s at a high temperature of k, and their interactions with the am drug was electrostatic and covalent in nature, respectively. the similar kind of j o u r n a l p r e -p r o o f response was also observed on the adsorption of acetylsalicylic acid (asa) drug on the aln and bnnc [ ] . here, the asa was also adsorbed by aln nanocage strongly with an adsorption energy of - . kcal/mol but failed to alter the electronic properties of aln nanocage. besides, this time aln nanocage also suffered from an unrealistic large recovery time of . sec. on the other side, asa was adsorbed on the bnnc with a relatively weak adsorption energy of - . kcal/mol but decreased the h-l energy gap by . %. besides the bnnc also benefitted from a short recovery time of ~ seconds. again, the bnnc could not work as a work function based sensor as it increased the work function by % to . ev from . ev. hence bnnc can only be considered as a potential electrochemical sensor to detect asa. the result from adsorption of asa and am on aln and bn was also extended by r. padash et al. for -aminopyridine ( -ap) drug and exactly the same trends were followed [ ] . the al n nanocage was again adsorbed the -ap drug with the highest adsorption energy of - . kcal/mol followed by - . kcal/mol for b n nanocage, and - . kcal/mol for both al p and b p nanocages. following the same trend, while adsorption of -ap on bnnc decreased the h-l energy gap only to . ev from . ev, it hadn't decreased at all from . ev for alp nanocage. on the other side, the h-l energy gap was reduced to . ev from . ev on the adsorption of -ap towards bnnc following by . ev from . ev for bp nanocage. hence it was concluded that the sensitivity of the bn and bp was higher than aln and alp towards -ap made bn and bp structures a potential sensor candidate to detect -ap. finally, the aln nanoclusters were also proposed to be applied as a sensor for anticancer drug purinethol (pe) or mercaptopurine, by s. a. javarsineh et al. using dft theory with b lyp hybrid functional and the - g (d) basis set [ ] . in early s pe drug was first evaluated to treat leukaemia, but this hepatotoxic drug comes with several serious adverse effects like bone-marrow depression, abnormal liver function etc. which sometimes requires withdrawal of the drug from the human body which indicates rising importance for the pe sensor [ ] . the aln nanocage made spontaneous chemisorption with the pe drug with a maximum adsorption energy of - . kcal/mol. also, it showed relatively good sensitivity towards the pe drug with a maximum decrease in the h-l energy gap by . % to . ev with a maximum charge transfer of . e from drug to the nanocage. these made aln nanocage a promising candidate for the pe drug, but unfortunately, it suffers from a considerable recovery time of . seconds. on the other hand, the aln nanocone made a relatively weak interaction with pe with a maximum adsorption energy of - . kcal/mol but showed relatively higher sensitivity towards the pe drug with a maximum decrease in the h-l energy gap by . % to . ev. besides, the aln nanocone also benefits from a comparatively shorter recovery time of . seconds on the adsorption of pe in room temperature. hence, the aln nanocone holds a potential in the development of an electrochemical sensor to detect pe drug. b. khodashenas et al. used the gelatin-coated gold nanoparticles (gnps) to investigate the nanostructure's size effects on the curcumin drug encapsulations with the drug release mechanism using dft theory with b lyp hybrid functional and lanl dz basis set along with an experimental mechanism [ ] . in order to do that, they have first synthesized gnps with three different sizes ( , , and nm) and then coated them with gelatin. the characterization of the structures was further ensured by the ft-ir spectroscopy, uv-vis spectroscopy, dynamic light scattering, and scanning electron microscopy. from the dft investigation, a further strong interaction between the gnp and gelatin was revealed with a binding energy of - . kcal/mol and the gelatin works as an electron donor in the complex. finally, it was observed that the efficiency of the curcumin drug encapsulations was increased slightly with an increase in the nanocluster size. for au-gelatin ( nm) structure the efficiency was . % while the least was observed for the au-gelatin ( . nm) as . % following by . % for the au-gelatin ( . nm). besides the drug release rate was observed higher at a low ph ( . ) environment compared to the relatively neutral environment with . ph. j o u r n a l p r e -p r o o f investigate the drug delivery properties of silver nanoparticles for the aromatic organic compounds, anthraquinone derivatives, which induced a potential interest in the biological applications in recent years [ ] . they have prepared the anthraquinone derivative , -dimethoxy -nitro- -methylanthracene- , -dione (dmnmad) using an available method in the literature [ ] and the silver nanoparticles were synthesized solution combustion method. for the quantum mechanical calculations, they have used the b pw functional and lanl dz basis set with dft theory. from the surface-enhanced raman spectroscopy spectrum it was observed that the theoretical values from the dft calculations for different vibrational modes of the structures were very close to the experimental values which ensured the validity of first-principles calculations for this system. further from the dft calculations the h-l energy gap of the dmnmad was found as . ev which decreased to the . ev in the ag-dmnmad complex. this alteration in the electronic properties of dmnmad holds a promise in the future drug delivery of anthraquinone derivatives which was further investigated using uv-vis spectroscopy, molecular electrostatic potential etc. another micromolecule, poly (amido-amine) (pamam) dendrimers, recently had attention for working as a drug delivery system, electrochemical sensor etc. and the interaction of pamam with gnps was investigated by m. b. camarada using dft theory with b lyp hybrid exchange correlational [ ] . it was observed that the pamam interacted with the gnps with maximum interaction energy of . kcal/mol (bsse corrected) with an adsorbent-adsorbate distance ranging as . to . Å. this interaction increased the dm of the complex significantly to a maximum of . debye from . debye with a maximum decrease in the h-l energy gap to . ev from . ev. these alterations in the electronic properties indicate a promising application of au-pamam in the field of drug delivery and sensor. d. cortés-arriagada et al. used another graphene-like nanosheet, phosphorene, to investigate the interaction of nucleobases which plays an important role in designing drug molecules [ ] . for the dft calculations, they have used dft-d methods with pbe functional and def -svp basis sets. the nucleobases were observed to be adsorbed on the surface of phosphorene nanosheet more strongly than the gns where the most stable interaction was observed with guanine (g) nucleobase. the range of adsorption energies for the nucleobases on gns was observed on a range . to . kcal/mol which increased as . to . kcal/mol on the phosphorene nanosheet with weak long-range electrostatic closed-shell interactions. the stability was further increased while the nucleobases worked as a base pair and the most stable pair was c-g pair on the adsorption towards phosphorene with an adsorption energy of . kcal/mol following by . and . kcal/mol for a-t and a-u bases. during the interactions of nucleobases with phosphorene, a significant contribution to the van der waals dispersion energy was observed, and it was in the range of . to . kcal/mol for single nucleobases while was . to . kcal/mol for base pairs adsorption on phosphorene. but the adsorption of nucleobases almost failed to alter the electronic properties of phosphorene but increased the dm to as high as . debye in the base pairs and . debye for the single nucleobases while it was zero for the phosphorene nanosheet alone. these properties hold potential for the phosphorene nanosheet in the development of biosensors, nano-drug carrier vehicles or to be applied in dna/rna sequencing. the phosphorene nanosheet was further investigated by u. saikia et al. to find an efficient electrochemical sensor or nano-drug delivery vehicle for acetaminophen drug or more commonly known as paracetamol [ ] . it was observed that phosphorene adsorbed acetaminophen drug slightly more strongly compared to graphene with an adsorption energy of - . kcal/mol and an adsorbent-adsorbate distance of . Å following by - . kcal/mol and . Å for graphene. but the phosphorene clearly outnumbered the graphene and bnns in terms of charge transfer and dm which was . e and . debye respectively for phosphorene following by . e and . debye for graphene, and . e and . debye for bnns though bnns adsorbed j o u r n a l p r e -p r o o f acetaminophen more strongly than phosphorene with an adsorption energy of - . kcal/mol with a lower adsorbent-adsorbate distance of . Å. however, the most alluring interaction properties were observed for acetaminophen adsorption on the silicene, a two-dimensional nanosheet made of silicon atoms. when the drug was adsorbed in parallel with silicene, it was adsorbed with an adsorption energy of - . kcal/mol but with a small adsorbent-adsorbate distance of . Å. this lead to a higher amount charge transfer between the drug and nanosheet of . e along with a significantly increased dm of . debye. for the perpendicular orientation of the drug with silicene, a more stable interaction was observed with an adsorption energy of - . kcal/mol but with a slightly less charge transfer and dm of . e and . debye respectively. hence, the silicene surely holds a potential to be applied as a nano-drug carrier for acetaminophen drug, whereas other structures may work as a sensor to detect acetaminophen drug which can be explored through experimental research. after analyzing these vast number of research works, ldns can be concluded as promising candidate for the bio-sensing and drug delivery related applications. the biocompatibility and a great variety of the ldns with significant noble properties made these materials to work as sensors and delivery systems for many different types of drugs and biomolecules. besides, the quantum mechanical calculations have shown a considerable accuracy on predicting the different properties of the nanomaterials in the nanoscale range with a close resemblance to the experimental outcomes. furthermore, these first-principles calculation methods hold a promising future with the increasing computational abilities, which will significantly assist experimental researchers in developing any biomedical devices in a considerably short period of time during an emergency-response-situation. we have observed that the first-principles calculations allow researchers to calculate a wide range of essential properties from an atomic perspective to firmly understand the interaction of drug molecules with the nanomaterials. for the drug delivery systems, it also allows investigating the drug release mechanism in the targeted cell region. the excellent mechanical and electronic properties along with the biocompatibility of carbon-based ldns, e.g. graphene, cnt, fullerenes etc. made themselves evenhanded to be investigated more extensively than other nanomaterials to be applied in the stable drug sensor and drug delivery systems for various kind of drugs which show significant adverse effects in the human and animal bodies. among other nanomaterials, bnns and its allotropes, e.g. bnnt, bnnc showed a comparable mechanical strength, enhanced thermal stability, and other properties similar to the carbon-based nanomaterials which are being applied in the development of highly durable biosensors and drug delivery systems. other nanostructures like quasi-planar b , phosphorene, gold nanoparticles, metal nitrides etc. were investigated less extensively but showed promising sensing behaviour towards many drugs and biomolecules. especially, phosphorene, semimetal and post-transition metal nanostructures were studied to detect many toxic nerve agent, kidney biomarkers etc. hence, these nanomaterials can further be investigated to develop biosensors to detect many other drugs and biomolecules. in conclusion, the wide variety of ldns with many alluring novel properties holds a great potential in biomedical applications where first-principles investigations with rapidly increasing computational abilities will assist researchers in understanding these systems from the atomic perspective at a low cost with considerable accuracy. [ ] nanofood for thought, nat. nanotechnol. ( ) there's plenty of room at the bottom c : buckminsterfullerene a nontransferring dry adhesive with hierarchical polymer nanohairs role of dimensionality and quantum confinement in p-type semiconductor indium phosphide quantum dots synthesis, assembly and applications of semiconductor nanomembranes graphene-enabled and directed nanomaterial placement from solution for large-scale device integration non-lithographic direct patterning of carbon nanomaterial electrodes via electrohydrodynamic-printed wettability patterns by polymer brush for fabrication of organic field-effect transistor high-pressure-assisted design of porous topological semimetal carbon for li-ion battery anode with high-rate performance carbon nanomaterials for non-volatile memories combinatorial search for optimal hydrogen-storage nanomaterials based on polymers nanomaterials and nanostructures for efficient light absorption and photovoltaics in situ characterization of electrochemical processes in one dimensional nanomaterials for energy storages devices nanomaterials at work in biomedical research are carbon nanotubes safe? advanced hybrid nanomaterials for biomedical applications spontaneous insertion, helix formation, and hydration of polyethylene oxide in carbon nanotubes nano-bio interactions between carbon nanomaterials and blood plasma proteins: why oxygen functionality matters how to design preclinical studies in nanomedicine and cell therapy to maximize the prospects of clinical translation graphene in the sky and beyond synthesis, characterization of graphene oxide wrapped silicon carbide for excellent mechanical and damping performance for aerospace application grafting carbon nanotubes directly onto carbon fibers for superior mechanical stability: towards next generation aerospace composites and energy storage applications application of nanomaterials in civil engineering bt -nanomaterials and their applications nanotechnology: advantages and drawbacks in the field of construction and building materials nanotechnology: the future of fire safety nanotechnology innovations for the construction industry application of nanoparticles in percutaneous delivery of active ingredients in cosmetic preparations nanomaterials and regulation of cosmetics measurement of the elastic properties and intrinsic strength of monolayer graphene mechanics of carbon nanotubes optical properties of hexagonal boron nitride epitaxial growth of a silicene sheet phosphorene: an unexplored d semiconductor with a high hole mobility germanene: a novel two-dimensional germanium allotrope akin to graphene and silicene large-gap quantum spin hall insulators in tin films the potential environmental impact of engineered nanomaterials nanoscience and nanotechnologies: opportunities and uncertainties safe handling of nanotechnology science policy considerations for responsible nanotechnology decisions fabrication, mechanical properties, and biocompatibility of graphene-reinforced chitosan composites preparation of carbon nanotube bioconjugates for biomedical applications the biocompatibility of carbon nanotubes nanomaterials and biocompatibility: carbon nanotubes and fullerenes bt -nanotechnology in drug delivery borophene is a promising d allotropic material for biomedical devices biocompatibility of boron nitride nanotubes: an up-date of in vivo toxicological investigation chemical sensors: definitions and classification state of the art in alcohol sensing with d materials ultrasensitive detection of nucleic acids using deformed graphene channel field effect biosensors enhanced surface plasmon resonance (spr) signals based on immobilization of core-shell nanoparticles incorporated boron nitride nanosheets: development of molecularly imprinted spr nanosensor for anticancer drug, etoposide fabrication of graphene film composite electrochemical biosensor as a pre-screening algal toxin detection tool in the event of water contamination pegylated nano-graphene oxide as a nanocarrier for delivering mixed anticancer drugs to improve anticancer activity anticancer efficacy and subcellular site of action investigated by real-time monitoring of cellular responses to localized drug delivery in single cells synthesis and drug-delivery behavior of chitosan-functionalized graphene oxide hybrid nanosheets water-soluble poly(n-isopropylacrylamide)-graphene sheets synthesized via click chemistry for drug delivery graphene based gene transfection graphene oxide-polyethylenimine nanoconstruct as a gene delivery vector and bioimaging tool engineered polyethylenimine/graphene oxide nanocomposite for nuclear localized gene delivery enhanced chemotherapy efficacy by sequential delivery of sirna and anticancer drugs using pei-grafted graphene oxide functionalized graphene oxide mediated adriamycin delivery and mir- gene silencing to overcome tumor multidrug resistance in vitro fabrication of mpegylated graphene oxide/poly( -dimethyl aminoethyl methacrylate) nanohybrids and their primary application for small interfering rna delivery folic acid-conjugated graphene oxide loaded with photosensitizers for targeting photodynamic therapy graphene multilayers as gates for multi-week sequential release of proteins from surfaces multilayered graphene nano-film for controlled protein delivery by desired electro-stimuli non-cytotoxic hydroxylfunctionalized exfoliated boron nitride nanoflakes impair the immunological function of insect haemocytes in vivo hollow boron nitride nanospheres as boron reservoir for prostate cancer treatment boron nitride nanotubes functionalized with mesoporous silica for intracellular delivery of chemotherapy drugs chiral nanoporous metal-organic frameworks with high porosity as materials for drug delivery black phosphorus nanosheet-based drug delivery system for synergistic photodynamic/photothermal/chemotherapy of cancer a novel nanoparticle vector for tumor directed drug delivery probing the solute-solvent interaction of an azo-bonded prodrug in neat and binary media: combined experimental and computational study experimental and theoretical studies of novel azo benzene functionalized conjugated polymers heteroleptic copper(i) complexes of -scorpionate‖ bis-pyrazolyl carboxylate ligand with auxiliary phosphine as potential anticancer agents: an insight into cytotoxic mode nmr and dft investigations of structure of colchicine in various solvents including density functional theory calculations transition-metal-free formal crosscoupling of aryl methyl sulfoxides and alcohols via nucleophilic activation of c-s bond molecular origin of drug release by water boiling inside carbon nanotubes from reactive molecular dynamics simulation and dft perspectives first principles study on the boron-nitrogen domains segregated within ( , ) and ( , ) single-wall carbon nanotubes: formation energy, electronic structure and reactivity high selectivity of cyclic tetrapyrrole over tetrafuran and tetrathiophene toward toxic chemicals; a first-principles study theoretical insight of polypyrrole ammonia gas sensor an ab initio study of the b boron nanocluster for application as atmospheric gas (no,no ,n o,nh ) sensor boron trifluoride interaction studies on graphdiyne nanotubes -a firstprinciples insight electronic properties of acetaminophen adsorbed on d clusters: a first principles density functional study a study on quercetin and -fluorouracil drug interaction on graphyne nanosheets and solvent effects -a first-principles study dft and md investigations on the functionalized boron nitride nanotube as an effective drug delivery carrier for carmustine anticancer drug amantadine antiparkinsonian drug adsorption on the aln and bn nanoclusters: a computational study a dft study on the geometrical structures, electronic, and spectroscopic properties of inverse sandwich monocyclic boron nanoclusters conbm (n = . ; m = - ) chemical hardness in density functional theory ordering of wave functions and eigenenergies to the individual electrons of an atom boron nitride nanotube (bnnt) as a sensor of hydroperoxyl radical (ho ): a dft j o u r n a l p r e -p r o o f study theoretical study of solvent and co-solvent effects on the interaction of flutamide anticancer drug with carbon nanotube as a drug delivery system hexagonal boron nitride nanosheet as novel drug delivery system for anticancer drugs: insights from dft calculations and molecular dynamics simulations behavior of ylides containing n, o, and c atoms as hydrogen bond acceptors absolute hardness: companion parameter to absolute electronegativity adsorption of oxazole and isoxazole on bnnt surface: a dft study -structural health monitoring of processes related to composite manufacturing quantum dots sensitized graphene: in situ growth and application in photoelectrochemical cells graphene and graphene oxide as new nanocarriers for drug delivery applications investigation of the solvent effect, molecular structure, electronic properties and adsorption mechanism of tegafur anticancer drug on graphene nanosheet surface as drug delivery system by molecular dynamics simulation and density functional approach recent advances in graphene based gas sensors recent advances in graphene-based biosensors electric field in atomically thin carbon films, science ( -. ) highly conducting graphene sheets and langmuir-blodgett films large area, few-layer graphene films on arbitrary substrates by chemical vapor deposition synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide chemical nature of boron and nitrogen dopant atoms in graphene strongly influences its electronic properties superior thermal conductivity of single-layer graphene thermal properties of graphene and nanostructured carbon materials approaching ballistic transport in suspended graphene cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts direct electrochemistry of glucose oxidase assembled on graphene and application to glucose detection phagraphene: a low-energy graphene allotrope composed of - - carbon rings with distorted dirac cones induction of apoptosis by β-lapachone in human prostate cancer cells efficacy of betalapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target nqo selective killing of cancer cells by β-lapachone: direct checkpoint activation as a strategy against cancer studying metal-doped graphene nanosheet as a drug carrier for anticancer drug β-lapachone using density functional theory (dft) doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review risk factors for doxorubicin-lnduced congestive heart failure doxorubicin pathways: pharmacodynamics and adverse effects a first-principles study of the interaction of doxorubicin with graphene fabrication and characterization of a triple functionalization of graphene oxide with fe o , folic acid and doxorubicin as dual-targeted drug nanocarrier controlled release of doxorubicin from graphene oxide based charge-reversal nanocarrier adsorption of ampyra anticancer drug on the graphene and functionalized graphene as template materials with high efficient carrier b n fullerene as a carrier for -fluorouracil anti-cancer drug delivery: dft studies a dft study of -fluorouracil adsorption on the pure and doped bn nanotubes aln and alp doped graphene quantum dots as novel drug delivery systems for -fluorouracil drug: theoretical studies process research and development of a dihydropyrimidine dehydrogenase inactivator: large-scale preparation of eniluracil using a sonogashira coupling -fluorouracil: mechanisms of resistance and reversal strategies screening of the structural, topological, and electronic properties of the functionalized graphene nanosheets as potential tegafur anticancer drug carriers using dft method recent and new targets for small molecule anti-cancer agents how strong is the edge effect in the adsorption of anticancer drugs on a graphene cluster? benchmark theoretical study of the π-π binding energy in the benzene dimer adsorption of adenine on the surface of nickel-decorated graphene study on the structure and electronic property of adsorbed guanine on aluminum doped graphene: first principles calculations sustained-release fampridine for symptomatic treatment of multiple sclerosis theoretical study of functionalized single-walled carbon nanotube ( , ) with mitoxantrone drug dalfampridine: review on its recent development for symptomatic improvement in patients with multiple sclerosis phonon dispersion curves of a bc honeycomb j o u r n a l p r e -p r o o f epitaxial sheet adsorption of amino acids on boron and/or nitrogen doped functionalized graphene: a density functional study trends of amino acid adsorption onto graphene and graphene oxide surfaces: a dispersion corrected dft study computational explanation for interaction between amino acid and nitrogen-containing graphene si-doped phagraphene as a drug carrier for adrucil anticancer drug: dft studies helical microtubules of graphitic carbon strength and breaking mechanism of multiwalled carbon nanotubes under tensile load, science ( -. ) quantum transport in carbon nanotubes nanotube optoelectronic memory devices thermal conductance of an individual single-wall carbon nanotube above room temperature highly dense and perfectly aligned single-walled carbon nanotubes fabricated by diamond wire drawing dies unusually high thermal conductivity of carbon nanotubes carbon nanotube (cnt)-based composites as electrode material for rechargeable li-ion batteries: a review carbon nanotubes: a review on structure and their interaction with proteins advances in the science and technology of carbon nanotubes and their composites: a review catalytic growth of carbon microtubules with fullerene structure large-scale purification of single-wall carbon nanotubes: process, product, and characterization gas-phase synthesis of swnt by an atmospheric pressure plasma jet one-pot synthesis of carbon nanofibers from co synthesis of large arrays of well-aligned carbon nanotubes on glass large-scale production of single-walled carbon nanotubes by the electric-arc technique gas-phase catalytic growth of single-walled carbon nanotubes from carbon monoxide carbon nanotube growth by pecvd: a review a review featuring the fundamentals and advancements of polymer/cnt nanocomposite application in aerospace industry a review on carbon nanotube field effect transistors (cntfets) for ultra-low power applications a review on the use of carbon nanotubes nanofluid for energy harvesting system recent development in nanocarbon materials for gas sensor applications recent uses of carbon nanotubes & gold nanoparticles in electrochemistry with application in biosensing: a review applications of carbon nanotubes in drug delivery: a comprehensive review how do carbon nanotubes serve as carriers for gemcitabine transport in a drug delivery system? quaternized polymer-single-walled carbon nanotube scaffolds for a chemiresistive glucose sensor carbon nanotubes for the delivery of therapeutic molecules biomolecule-functionalized carbon nanotubes: applications in nanobioelectronics platinum(iv) prodrugs entrapped within multiwalled carbon nanotubes: selective release by chemical reduction and hydrophobicity reversal the application of carbon nanotubes in target drug delivery systems for cancer therapies modeling the interaction between anti-cancer drug penicillamine and pristine and functionalized carbon nanotubes for medical applications: density functional theory investigation and a molecular dynamics simulation tuberculosis and the tubercle bacillus si-doped single-walled carbon nanotubes interacting with isoniazid-a density functional and molecular docking study effect of b/n co-doping on the stability and electronic structure of single-walled carbon nanotubes by firstprinciples theory a randomized trial in patients with gemcitabine refractory pancreatic cancer. final results of the conko study efficacy and safety profile of gemcitabine in nonsmall-cell lung cancer: a phase ii study activity of gemcitabine in patients with non-small cell lung cancer: a multicentre, extended phase ii study gemcitabine plus paclitaxel versus paclitaxel monotherapy in patients with metastatic breast cancer and prior anthracycline treatment fatal pulmonary toxicity resulting from treatment with gemcitabine severe acute lung injury induced by gemcitabine a density functional theory-based analysis of the structural, topological and electronic properties of gemcitabine drug adsorption on the pyrrolidine functionalized single-walled carbon nanotube flutamide-induced hepatotoxicity: report of a case series severe gynecomastia due to anti androgens intake: a case report and literature review mechanism of action and pure antiandrogenic properties of flutamide assessment of the adsorption mechanism of flutamide anticancer drug on the functionalized single-walled carbon nanotube surface as a drug delivery vehicle: an alternative theoretical approach based on dft and md assessment of solvent effects on the interaction of carmustine drug with the pristine and cooh-functionalized single-walled carbon nanotubes: a dft perspective metformin use and prostate cancer in caucasian men: results from a population-based case-control study metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer antidiabetic therapies affect risk of pancreatic cancer quantum chemical study on the adsorption of metformin drug on the surface of pristine, si-and al-doped ( , ) swcnts, phys. e low-dimensional syst calcium-doped single-wall nanotubes (ca/swcnts) as a superior carrier for atropine drug delivery: a quantum-chemical study in gas and solvent phases chapter -drugs involved in drug-facilitated crime-pharmacological aspects ketamine and ketamine metabolite pharmacology: insights into therapeutic mechanisms functionalized single-walled carbon nanotube for ketamine sensing: dft and md studies an overview of clinical pharmacology of ibuprofen intermolecular and low-frequency intramolecular raman scattering study of racemic ibuprofen adsorption of ibuprofen on silicon decorated fullerenes and single walled carbon nanotubes: a comparative dft study density functional investigation of co and no adsorption on tm-decorated c fullerene a dft study on the functionalization of c fullerene with , -benzoquinone dft, nbo and molecular docking studies of the adsorption of fluoxetine into and on the surface of simple and sulfur-doped carbon nanotubes computational modeling of functionalized multi-walled carbon nanotubes dispersed in polyethylenimine for electrochemical sensing of acetaminophen absorption behavior of small biomolecules on carbon nanotube by density functional theory pd-doped single-walled carbon nanotube as a nanobiosensor for histidine amino acid, a dft study a dft-d study on the interaction between lactic acid and single-wall carbon nanotubes topological organic chemistry. polyhedranes and prismanes geometrical and electronic properties of the clusters of c cage doped with alkali metal atoms first-principles study of no adsorption on c fullerene stabilization of the c cage by encapsulation of h + and he + ions solid c : a new form of carbon characterization of the soluble all-carbon molecules c and c fullerene formation in sputtering and electron beam evaporation processes extraction of c cluster ion beam synthesis of fullerenes and fullerenic nanostructures in a lowpressure benzene/oxygen diffusion flame arc discharge instalation for fullerene production covalent fullerene chemistry, science ( -. ) electron-phonon coupling and the electrical conductivity of fullerene nanotubules fullerene-styrene random copolymers. novel optical properties fullerene chemistry for materials science applications fullerene materials for lithium-ion battery applications bt -perspectives of fullerene nanotechnology endohedral fullerenes for organic photovoltaic devices electrocatalytic properties and sensor applications of fullerenes and carbon nanotubes synthesis of c /graphene composite as electrode in supercapacitors, fullerenes, nanotub. carbon nanostructures solubility of fullerene (c ) in a variety of solvents antibacterial activity of fullerene water suspensions: effects of preparation method and particle size synthesis of a fullerene derivative for the inhibition of hiv enzymes a tumoral acidic ph-responsive drug delivery system based on a novel photosensitizer (fullerene) for in vitro and in vivo chemo-photodynamic therapy electrochemical behavior of an antiviral drug acyclovir at fullerene-c -modified glassy carbon electrode study on the electronic structure of cr-and ni-doped fullerenes upon adsorption of adenine: a comprehensive dft calculation a density functional theory study on the interaction between -fluorouracil drug and c fullerene adsorption mechanism, structural and electronic properties: -phenylpyridine & undoped or doped (b or si) c theoretical study on the phenylpropanolamine drug interaction with the pristine, si and al doped [ ] fullerenes, phys. e low-dimensional syst phenylpropanolamine: a potentially hazardous drug adverse drug effects attributed to phenylpropanolamine: a review of case reports phenylpropanolamine-induced hypertensive seizures a dft study of penicillamine adsorption over pure and al-doped c fullerene penicillamine, a new oral therapy for wilson's disease penicillamine in severe rheumatoid arthritis utility of extrinsic [ ] fullerenes as work function type sensors for amphetamine drug detection: dft studies fluorescence polarization immunoassay detection of amphetamine, methamphetamine, and illicit amphetamine analogues ge doped c fullerenes amantadine: the journey from fighting flu to treating parkinson disease amantadine caused corneal edema conformational and electronic properties of hydroquinone adsorption on c fullerenes: doping atom, solvent and basis set effects fullerene c containing porphyrin-like metal center as drug delivery system for ibuprofen drug application of c b n heterofullerene in the isoniazid drug delivery: dft studies, phys. e low-dimensional syst theoretical insight of alpha amino acid phenylalanine adsorption on pristine and decorated fullerenes adsorption behavior of amphetamine on the inorganic bc nanotube and nanosheet: dft studies the electronic response of pristine, al and si doped bc n nanotubes to a cathinone molecule: computational study a dft study of adsorption of glycine onto the surface of bc n nanotube potential of graphene oxide as a drug delivery system for sumatriptan: a detailed density functional theory study multifunctional fluorescent pegylated fluorinated graphene for targeted drug delivery: an experiment and dft study density functional theory calculations and molecular dynamics simulations of the adsorption of ellipticine anticancer drug on graphene oxide surface in aqueous medium as well as under controlled ph conditions investigation on graphdiyne nanosheet in adsorption of sorafenib and regorafenib drugs: a dft approach flutamide drug interaction studies on graphdiyne nanotube -a first-principles study adsorption behavior of cytosine and guanine nucleobases on graphyne nanosheets: a dft study a dft study on the detection of isoniazid drug by pristine, si and al doped c fullerenes brain dysfunction in phenylketonuria: is phenylalanine toxicity the only possible cause? pathogenesis of cognitive dysfunction in phenylketonuria: review of hypotheses on the atomic weight of graphite verfahren zur darstellung der graphitsäure preparation of graphitic oxide biomedical applications of graphene and graphene oxide new horizons for diagnostics and therapeutic applications of graphene and graphene oxide chest symptoms after sumatriptan: a two-year clinical practice review in consecutive migraine patients fluorinated graphene as an anticancer nanocarrier: an experimental and dft study architecture of graphdiyne nanoscale films phase ii trial of sorafenib in advanced thyroid cancer sorafenib for the treatment of advanced renal cell carcinoma dermatologic symptoms associated with the multikinase inhibitor sorafenib meta-analysis of dermatological toxicities associated with sorafenib sorafenib in radioactive iodine-refractory well-differentiated metastatic thyroid cancer regorafenib (bay - ) in advanced colorectal cancer: a phase i study regorafenib as second-line therapy in hepatocellular carcinoma acute liver failure due to regorafenib may be caused by impaired liver blood flow: a case report interaction of imuran, pentasa and hyoscyamine drugs and solvent effects on graphdiyne nanotube as a drug delivery system -a dft study a novel graphite-like material of composition bc , and nitrogen-carbon graphites calculation of electronic and structural properties of bc , phys. rev utility of extrinsic [ ] fullerenes as work function type sensors for amphetamine drug detection: dft studies thermal and chemical stability of hexagonal boron nitride (h-bn) nanoplatelets, vacuum thermal transport in hexagonal boron nitride nanoribbons synthesis of monolayer hexagonal boron nitride on cu foil using chemical vapor deposition synthesis of large-area multilayer hexagonal boron nitride for high material performance large scale growth and characterization of atomic hexagonal boron nitride layers nanorice-like structure of carbon-doped hexagonal boron nitride as an efficient metal-free catalyst for oxygen electroreduction doped h-bn monolayer as efficient noble metal-free catalysts for co oxidation: the role of dopant and water in activity and catalytic de-poisoning synthesis and characterization of hexagonal boron nitride as a gate dielectric on the use of two dimensional hexagonal boron nitride as dielectric boron nitride (bn) and bn composites for high-temperature applications high operating temperature and low power consumption boron nitride nanosheets based broadband uv photodetector sandwich-structured pvdf-based composite incorporated with hybrid fe o @bn nanosheets for excellent dielectric properties and energy storage performance d hexagonal boron nitride ( d-hbn) explored for the electrochemical sensing of dopamine ultra-sensitive hall sensors based on graphene encapsulated in hexagonal boron nitride study the gas sensing properties of boron nitride nanosheets boron nitride nanosheets as a platform for fluorescence sensing biocompatibility of boron nitride nanosheets thymine adsorption on two-dimensional boron nitride structures: first-principles studies nucleobases-decorated boron nitride nanoribbons for electrochemical biosensing: a dispersioncorrected dft study a comparative dft study on the interaction of cathinone drug with bn nanotubes, nanocages, and nanosheets adsorption of mercaptopurine drug on the bn nanotube, nanosheet and nanocluster: a density functional theory study theory of graphitic boron nitride nanotubes mass-production of boron nitride double-wall nanotubes and nanococoons synthesis of boron nitride nanotubes at low temperatures using reactive ball milling nanotubes in boron nitride laser heated at high pressure cvd growth of boron nitride nanotubes rate theory of yield in boron nitride nanotubes synthesis and characterization of ropes made of bn multiwalled nanotubes isotope effect on the thermal conductivity of boron nitride nanotubes boron nitride nanotubes: pronounced resistance to oxidation stability and band gap constancy of boron nitride nanotubes young modulus, mechanical and electrical properties of isolated individual and bundled single-walled boron nitride nanotubes electrically insulating polymeric composites with boron nitride nanotubes as fillers eu-doped boron nitride nanotubes as a nanometer-sized visible-light source superhydrophobic and superoleophilic boron nitride nanotube-coated stainless steel meshes for oil and water separation polyethylene/boron nitride composites for space radiation shielding boron nitride nanotube based nanosensor for acetone adsorption: a dft simulation detection of phosgene by sc-doped bn nanotubes: a dft study boron nitride nanotubes are noncytotoxic and can be functionalized for interaction with proteins and cells boron nitride nanotubes: biocompatibility and potential spill-over in nanomedicine boron nitride nanotube-based biosensing of various bacterium/viruses: continuum modelling-based simulation approach boron nitride nanotube as a delivery system for platinum drugs: drug encapsulation and diffusion coefficient prediction practical issues in the management of hypersensitivity reactions a density functional theory outlook on the possible sensing ability of boron nitride nanotubes and their al-and si-doped derivatives for sulfonamide drugs boron nitride nanotubes for delivery of -fluorouracil as anticancer drug: a theoretical study the chemotherapy of brain tumors: clinical experience with severe diffuse interstitial pneumonitis induced by carmustine (bcnu), chest. myocardial ischemia associated with high-dose carmustine infusion theoretical study of encapsulation of floxuridine anticancer drug into bn quality of life and survival with continuous hepatic-artery floxuridine infusion for colorectal liver metastases toxicities and complications of implanted pump hepatic arterial and intravenous floxuridine infusion adjuvant perioperative hepatic arterial mitomycin c and floxuridine combined with surgical resection of metastatic colorectal cancer in the liver electrochemical behavior of acetaminophen and its detection on the pani-mwcnts composite modified electrode theoretical studies of the paracetamol and phenacetin adsorption on single-wall boron-nitride nanotubes: a dft and md investigation molecular dynamics simulation of paracetamol drug adsorption on boron nitride nanotube: effects of temperature adsorption of the nitrosamine and thionitrosamine molecules as carcinogen compounds on the bn and b al n nanotubes: a dft study nitrosamine and related food intake and gastric and oesophageal cancer risk: a systematic review of the epidemiological evidence a study on carcinogenesis of endogenous nitrite and nitrosamine, and prevention of cancer ab initio investigation of pristine and doped single-walled boron nitride nanotubes as acetone sensor structure and stability of c and b n isomers doping graphitic and carbon nanotube structures with boron and nitrogen, science ( -. ) octahedral boron nitride fullerenes formed by electron beam irradiation formation and structure of b n clusters boron nitride nanocages synthesized by a moderate thermochemical approach controlled fabrication of ultrathin-shell bn hollow spheres with excellent performance in hydrogen storage and wastewater treatment fullerene nanocage capacity for hydrogen storage b n nano-cage as potential sensor for no detection treatment of adult acute leukemia with arabinosylcytosine and thioguanine hepatic veno-occlusive disease caused j o u r n a l p r e -p r o o f by -thioguanine -thioguanine treatment of psoriasis: experience in patients theoretical investigation of thioguanine isomers anticancer drug adsorption treatment on b n nanocage ifosfamide-pharmacology, safety and therapeutic potential side effects of ifosfamide a comparative theoretical study on the interaction of pure and carbon atom substituted boron nitride fullerenes with ifosfamide drug a computational study on the cisplatin drug interaction with boron nitride nanocluster interaction of α-cyano- -hydroxycinnamic acid drug with inorganic bn nanocluster: a density functional study adsorption of celecoxib on b n fullerene: spectroscopic and dft/td-dft study celecoxib associated esophagitis: review of gastrointestinal side effects from cox- inhibitors cyclooxygenase- inhibition with celecoxib enhances antitumor efficacy and reduces diarrhea side effect of cpt- adsorption behavior of metformin drug on boron nitride fullerenes: thermodynamics and dft studies quantum chemical assessment of the adsorption behavior of fluorouracil as an anticancer drug on the b nanosheet borophene as an electronic sensor for metronidazole drug: a computational study healing of perineal crohn's disease with metronidazole is metronidazole carcinogenic? dna sequencing by borophene nanosheet via an electronic response: a theoretical study a computational study for the b bowl-like nanostructure as a possible candidate for drug delivery system for amantadine amantadine in the treatment of parkinson's disease amantadine as treatment for dyskinesias and motor fluctuations in parkinson's disease specific structural alteration of the influenza haemagglutinin by amantadine the computational study of the γ-fe o nanoparticle as carmustine drug delivery system: dft approach a theoretical investigation on the adsorption of platinol drug on a zno nanocluster: solvent and density j o u r n a l p r e -p r o o f functional effect aim and nbo study of isoniazid drug delivery by mgo nanocage the capability of the pristine and (sc, ti) doped be o nanocluster to detect and adsorb of mercaptopyridine molecule: a first principle study exploring the sensitivity of zno nanotubes to tyrosine nitration: adft approach interaction of isoniazid drug with the pristine and ni-doped of ( , ) armchair gannts: a first principle study a computational study on the purinethol drug adsorption on the aln nanocone and nanocluster benzoylethanamine drug interaction with the aln nanosheet, nanotube and nanocage: density functional theory studies electronic and work function-based sensors for acetylsalicylic acid based on the aln and bn nanoclusters: dft studies p) nanocages for drug-delivery systems? a dft study on the adsorption property of -aminopyridine drug gelatin-gold nanoparticles as an ideal candidate for curcumin drug delivery: experimental and dft studies the clinical pharmacology of -mercaptopurine adsorption of , -dimethoxy- -nitro- -methylanthracene- , -dione onto silver nanoparticles first tdae approach in quinonic series: synthesis of new -substituted , -dimethoxy- , -anthraquinones dft investigation of the interaction of gold nanoclusters with poly(amidoamine) pamam g dendrimer phosphorene as a template material for physisorption of dna/rna nucleobases and resembling of base pairs: a cluster dft study and comparisons with graphene we thankfully acknowledge the higher education quality enhancement program (heqep) subproject cp- , university grant commission (ugc) of bangladesh, and the world bank for the financial assistance to set up the computational physics (cp) research lab in the department of physics at jahangirnagar university. also, we would like to give our special gratitude to ahmad azuad yaseer and shawon mahmud for their valuable assistance with the graphical content creation. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: j o u r n a l p r e -p r o o f graphical abstract:in this review article, we have analyzed the application of low-dimensional nanostructured materials in the biomedical applications like drug delivery systems and drug sensors development.j o u r n a l p r e -p r o o f key: cord- - am l ms authors: nan title: spr date: - - journal: pediatr radiol doi: . /s - - - sha: doc_id: cord_uid: am l ms nan dear colleagues, i confess i haven't read many "welcome letters" at the beginning of the spr program book over the years. perhaps the only defensible benefit of this is that there is no preconception about the content of this message…or the length. i will be brief. this meeting is about building bridges…bridges from our past to the future and bridges between all of us who believe fundamentally in maintaining or improving the health of our children. the content, which is detailed on subsequent pages, speaks for itself. this material will be presented during the sessions with an appreciative look back at past accomplishments-the legacy of our subspecialty-with a vision to the future of pediatric imaging. we can only measure how broad and deep our successes have been by connecting with these beginnings. looking beyond the titles (and the speakers), i think you will see that the material is not only about techniques and tactics but about ideas, insights, energy, all conspiring in the creative process … an aggregate for excellence in pediatric imaging. the content is also punctuated by a strong presence of our clinical colleagues. again, this builds bridges. how can we maintain and expand these relationships? moreover, the connections between science and clinical practice are evident in the structured blending of scientific papers and topical presentations by both imaging and clinical experts. this blending is also "fraternal" in that there will sometimes be disagreement and critical commentary, but this is essential in the advancement of medicine. support and criticism make a stronger mortar. in the end, this gathering is about fostering a connected community, including technologists, nurses, physicists and other allied health experts including industry experts. finally, the emblem of pediatric radiology has always been embossed by cooperation, passion, commitment, and humanistic care. i believe the program content, the presenters and you, the participants, all embrace this. i hope that you will feel the spirit and the passion of the meeting and all of us will in many ways be better able to care for children because of this-even if you never read this message! donald p. frush the gold medal of the society for pediatric radiology is our most distinguished honor. the spr medal is awarded to pediatric radiologists who have contributed greatly to the spr and our subspecialty of pediatric radiology as a scientist, teacher, personal mentor and leader. marilyn goske has always wanted to make a differenceand what a difference she has made! her role as an educator, and her lifelong commitment to improving training for residents, fellows, faculty, medical staff and radiologic technologists has resulted in many wonderful initiatives that have benefited all in pediatric radiology. the work she is most proud of-the cleveland clinic web based curriculum, working with the leadership of spr's philanthropic campaign for children, launching the image gently campaign and the pediatric research component within the american college of radiology's dose index registry share a common theme: educating others in providing the best care possible for children. born in berea, ohio, marilyn's father, george, was a chemical engineer. her mother, cornelia aka "corky", loved writing as one of the first women journalists for the associated press and later teaching, passions she passed on to her daughter. while marilyn was blessed with a strong female role model in her mother, it was her brother, james, who was her cheerleader, always pushing her to dream big. he encouraged her to follow in his footsteps first at ohio university, then on to the ohio state college of medicine to pursue an md degree during an era when nursing would have been a more conventional goal. marilyn met her husband rick on a double date in college-unfortunately, they were with different dates! luckily, they were able to get together for an actual date with each other months later. they quickly became engaged and married within a year of that first true date. when rick started his residency in internal medicine, marilyn transferred to the university of connecticut school of medicine in farmington. it was here that she met her first pediatric radiologist-and what a giant-mike ozonoff! when rick moved on to a neurology residency in rochester, new york, marilyn followed and met another pediatric radiology giant: beverly wood, at strong memorial hospital. beverly proved to be a wonderful teacher, mentor, co-researcher and lifelong friend. marilyn describes beverly as inspirational and "fearless" in trying new technologies. it was during her time in rochester that marilyn went to her first spr meeting and, not surprisingly, won the caffey award for her work on "experimental neonatal intraventricular hemorrhage: clinical, radiographic and pathologic features." by then marilyn had two young children and moved on to the private sector, practicing part-time for several years first in rochester, then in cleveland, ohio. her years in private practice were particularly helpful in learning the importance of patient oriented service-and paved the way for her intuitive public relations strategies when designing the image gently campaign in later years. dr. goske was asked to join the cleveland clinic in , as the first full-time section head of pediatric radiology. it was here that she built a new section and spearheaded the web based education program for pediatric radiology residents with co-founder janet reid. this important free web site with modules is used widely by over radiology residencies nationally and internationally. her passion for education continued, inspiring her to complete a medical education fellowship focused on professionalism within the cleveland clinic lerner college of medicine. her work towards this fellowship has led to many creative educational initiatives including yearly educational summits at the spr. she was named chair of the professionalism committee of the rsna where she along with her committee have sponsored interactive workshops on this topic dear to her heart. dr. goske's energy and effective leadership skills brought her to become involved in the society for pediatric radiology, first as the coordinator for spr's first video-taped course in . mentors diane babcock and carol rumack proposed her for the nominating committee. this was followed by chair of the membership committee, where she organized the first formal survey of the society, then as a board member, then as secretary, and finally as president and chair of the board of directors, completing years on the spr board. working together with stuart royal, she successfully energized the campaign for children raising funds for the research and education foundation of the spr and expanded the work of prior presidents in further organizing the corporate support committee. marilyn's years as president and chairman of the board of the spr were highly successful with many unique strategic goals. she was instrumental in the founding of the junior spr. she led the wonderful spr national meeting in miami which included the first educational summit to enhance knowledge in adult learning and resident competencies. most people would rest after completing their arduous year as president but as chairman of the board, marilyn was just beginning! she moved to cincinnati children's hospital, joining the radiology department and was named the dr. corning benton endowed chair for radiology education where she got to work with dr. janet strife, another influential mentor and friend. acknowledging spr's long focus on reducing radiation doses in the imaging of children but concerned about the lack of change in practice by a majority of radiologists despite increasing reports of possible side effects, marilyn developed a public relations and awareness campaign. her goal was to inspire all to work towards decreasing radiation exposure to children when possible. with the help of many, she founded the alliance for radiation safety in pediatric imaging and the image gently campaign, initially focusing on ct. her ability to encourage numerous experts and societies to work together and get involved in "child sizing the amount of radiation used" has resulted in a groundswell of research and activity in this area. currently organizations with over , members have joined the alliance including international societies. the web site, www.imagegently.org, has been immensely successful filled with free information pamphlets in over languages, pqi projects, and modules for parents, physicians, and technologists. the image gently campaign has received several awards including the associations advance america honor roll, rt image magazine group with the "most influence in radiology" and the most effective philanthropy program from aunt minnie. image gently has spawned the creation of the adult-focused image wisely campaign. the alliance has been named by the joint commission, u.s. food and drug administration, and the american medical association in their influential statements on radiation dose as providing much needed guidance and information. marilyn's exceptional talent is inspiring and coordinating experts in multiple fields to work together towards common goals. she continues to work hard on the image gently campaign with more safety and quality messages planned for the coming years. she is also proud of her work with the acr dose index registry and quality improvement registry in ct scans in children in working toward developing diagnostic reference levels with a talented consortium of pediatric radiologists, medical physicists and technologists. she has acted as a national and international expert in her work with the international atomic energy agency, the world health organization and the national council on radiation protection in medicine and the fda. dr. goske's multiple committee appointments are taken seriously, and her work is always meticulous, well thought out, and brought to successful completion. she has been an active member of numerous national and international societies including the john caffey honorary society, acr, rsna, espr, aawr, and scorch. it is important to remember that dr. goske is also a successful researcher with numerous grants obtained through the spr ref including the thorne griscom education award and the rsna scholar grant. she has published over peer-reviewed articles, electronic publications, chapters, and presented scientific exhibits as well as given numerous scientific presentations. an articulate and engaging speaker, she has been invited to give over lectures locally, nationally and internationally. while marilyn has been very focused on her work with the spr, she believes that it is her amazing family and their love that really fuels her life. her husband rick is an internationally known neurologist and researcher in multiple sclerosis. her adult children jamie and brian, both in manhattan, remain close, and spending quality time together as a family remains the joy of her life. whether it is relaxing together in florida cooking or fishing, or taking an exotic vacation to india, being with rick, jamie and brian makes her the happiest. marilyn's genius is partly refusing to take "no" as an answer. along the way, at every turn there were those who believed that what she wanted to do couldn't be done. her approach was to draft the nay-sayers to the team and charge ahead with their willing and enthusiastic help. daniel burnham might have been talking about marilyn and not about his plan for the city of chicago when he said: make no little plans; they have no magic to stir men's blood and probably themselves will not be realized. make big plans; aim high in hope and work, remembering that a noble, logical diagram once recorded will not die, but long after we are gone be a living thing, asserting itself with evergrowing insistence. as an amazing change agent, inspirational leader, and wonderful role model, the spr is proud to honor marilyn goske with the gold medal. she made big plans! dorothy i. bulas, md pioneer honorees were first acknowledged in as a means to honor certain physicians who made special contributions to the early development of our specialty. it is time to reevaluate the meaning of the pioneer honoree. the subspecialty of pediatric radiology has been in existence now for more than years. we are beyond "the early development"; we must recognize other pioneering paths and should consider contributions to the subspecialty beyond the bounds of a modality, a technique, an observation or a change in practice. whatever this advancement is, it must be forged with vision, innovative ideas, and the ability to enable and sustain science and application. george s. bisset, iii, m.d. why george bisset? has he been part of the pediatric radiology landscape these last ten years? been part of the dialogue that has been increasingly influential across all of radiology, a conversation steeped in a deep tradition of excellence in diagnosis and treatment, and the safety and welfare of our children? been a leader in science and application? part of the landscape? no. but he has been beyond that and has worked tirelessly within the horizon, surveying…a step before, but guiding us on towards our destiny. a conversant? part of the dialogue? maybe. but he has been defining thought and concept upon which such conversation is born and nurtured. part of the science and application? yes, as much as anyone who promotes, who facilitates and sustains discovery, then here we are. horizons, innovation, and the gift of en-abling…what else is needed to define a true pioneer? how was this done? simply stated, george bisset has devoted at least the last decade to the advancement of our specialty in truly novel ways through his leadership, especially in rsna and the abr. in the rsna, as the scientific program committee chair several years ago he was instrumental in the conception, development and implementation of the integration of scientific papers and refresher course topics. this has been a resounding success, is currently used in other categories during rsna and is a model for other meetings, including the annual meeting for the society for pediatric radiology over the past few years. pediatric radiology was first in this effort. george continued to endorse topics that were marquees for pediatric radiology over the year in his education role on the rsna board of directors. he endorsed and implemented the pediatric campus concept at the rsna. early returns are that this was an extremely successful model to consolidate experts in pediatric radiology (and those interested in this subspecialty), pertinent science, education and administration. george is now the president of rsna, perhaps the most widely respected scientific and educational organization for our profession across the globe…and i would argue, with more promise for our future success in pediatric radiology than has ever existed. and george bisset, who through two terms as the pediatric trustee for the board of trustees for the abr, again, was on the horizon of a critical, sometimes perilous, and complete transformation of our certification examination process, always mindful of his constituency and colleagues, his duty as a physician, and the public and patients. this required delicate diplomacy, forward thinking, professionalism, and enlistment of a cadre of experts from within our subspecialty to assure excellence in pediatric radiology through abr certification. he was also a leader in the development, validation and implementation of the imagerich computer based examination model (the pediatric caq) now the standard for the new abr examinations. with these successes in mind, who better to embody the concept of bridging horizons that is the theme for this entire meeting? if you were looking for more numbers and accolades, i apologize. here are some: more than contributions to medical and scientific literature, advancing care through pediatric body ct and mr imaging research, a litany of presentations and invited lectures, vice chairs, chairs, chiefs, boards of directors, committee member and committee leader, clinical excellence including as a pediatric cardiologist and interventional radiologist, a superb speaker and author.… all are on his cv but i believe serve really as signposts for his gifts, some of those mentioned above, that a cv simply cannot convey. he could have played it safe with all of these successes on his cv. but pioneers don't play it safe. they are on the horizon, too busy defining thought and enabling (our) advancement-building bridges. i believe it is time to reevaluate the meaning of the pioneer honoree and i have the greatest honor and pleasure of introducing george s. bisset, iii for the pioneer award for . linked with past awardees, he continues an exceptional legacy and i don't believe his explorations and discoveries are finished… donald p. frush, md the society bestows presidential recognition awards on members or other individuals whose energy and creativity have made a significant impact on the work of the society and its service to its members. in , david kushner was recognized by the spr with its first presidential recognition award for his vision and foresight in working with both the american college of radiology (acr) and the society for pediatric radiology (spr) in developing an important new relationship and for his service to the spr. in summarizing his considerable efforts for that award, i noted that he "contributed substantively to the increased visibility of the spr within the acr. his tenure as our treasurer placed our organization on a firm financial foundation." with the current award, the society recognizes his indefatigable continuing efforts on our behalf including: his work with the acr: . establishing a pediatric radiology caucus at the annual acr meeting, . convincing the acr of the value of managing specialty societies by making the spr its first successful new model for imaging society management, . advocating tirelessly for pediatrics and children's health within the acr by serving on the council steering committee and then as acr council vice speaker and speaker, . helping establish the first pediatric commission, assuring that pediatric issues will receive support of the college and its resources while serving on the board of chancellors of the acr for the past five years. the spr's "image gently" campaign was a beneficiary of this pediatric commission of the board of chancellors, . continuing to shepherd and contribute to the pediatric component of the acr practice guideline process. his work with the spr: since his earlier award, david has served as: foundation from to , including the launch of the formal fundraising effort, "the campaign for children," . spr president - , organizing and running a very successful meeting in savannah, . chair of the board of directors of the spr from to , including leading a strategic planning process that resulted in a new, more focused division of labor amongst board members and defined board responsibilities. david was born in fargo, north dakota, received a ba from the university of minnesota, and received his medical education at the university of pennsylvania. this was followed by two years of training in pediatrics at children's hospital, boston. he then did a two-year fellowship at the national institute of health in bethesda, performing research in embryology and teratology. he returned to massachusetts general hospital for training in diagnostic radiology. this was followed by a year of residency in pediatric radiology at children's hospital boston, followed by a one-year fellowship. he then became director of the pediatric radiology section at massachusetts general hospital, a position he held from to . from to , david was chief of the division of diagnostic imaging and radiology at children's national medical center in washington, dc attracting a strong faculty, training many fellows and promoting research. during that time, he served as a volunteer radiologist and pediatrician to inner city healthcare systems aiding the indigent and homeless, and developing telemedicine capabilities linking free clinics with radiology experts. in , our man inside the beltway moved a bit outside by accepting the medical directorship of radiology at the children's hospital of the king's daughters in norfolk, virginia, and professor of radiology and pediatrics at the eastern virginia medical school. he assures me that life there is good, being a bit more "laid back" with fishing and sailing just outside the door. he also finds time for italian cooking and practicing jazz on his several guitars. fortunately for all of us in the spr, david is close enough to our central office and the acr that he will be able to continue work on our behalf for many years to come. the society bestows presidential recognition awards on members or other individuals whose energy and creativity have made a significant impact on the work of the society and its service to its members. the spr presidential award is given in recognition of stuart's numerous significant and outstanding contributions to the spr over many years of service. the awardee is selected by the honors committee, a committee comprised of the three most recent past presidents of the society. dr. royal is a proud native of birmingham, alabama. he is a second generation physician who came naturally to his desire to care for children as the son of a pediatrician, arnold royal, who took care of children in the birmingham community until he was years old. dr. royal attended rice university in houston, texas followed by md and ms degrees from the university of alabama at birmingham. he subsequently moved to san francisco, where he completed a pediatric internship followed by a diagnostic radiology residency at the university of california, san francisco. dr. royal credits dr. charles gooding at ucsf for influencing his decision to pursue a career in pediatric radiology. during his internship stuart observed dr. gooding make a plain film diagnosis of tapvr, type on a severely ill and perplexing newborn, and he was immediately hooked into radiology. while at ucsf dr. royal was also appointed as a national institute of health research fellow in the department of radiology. following residency, stuart completed a fellowship in pediatric radiology at the children's hospital medical center in boston. from boston, stuart returned to his roots in birmingham, alabama in , where he was appointed as a pediatric radiologist at the university of alabama and subsequently the children's hospital in birmingham. in recognition of his outstanding leadership skills and accomplishments at the children's hospital, dr. royal was appointed as the radiologist-in-chief in , and subsequently the harry m. burns endowed chair of pediatric radiology. he also holds appointments as clinical professor of radiology and pediatrics at the university of alabama at birmingham and serves on the children's hospital board of trustees. at alabama dr. royal has earned the high esteem of his colleagues, referring physicians, and staff for his outstanding clinical acumen as a diagnostic radiologist and for his undaunting commitment to excellent care of children. colleagues describe stuart as one who fosters a strong work ethic, high commitment to teaching, and sincere compassion for children. in , stuart was the recipient of the children's advocate award by childcare resources for improving the quality of care and access to radiological services for underserved children in birmingham. stuart has been married to the love of his life, barbara royal, for the past years. stuart and barbara are the proud parents of two very accomplished children, jeremy a budding radiologist in training at the university of alabama, and rachael, who has an mba and works as a vice president for moody's in new york. stuart and barbara are also the proud grandparents of three grandchildren. in conversation, stuart is quick to pull out his iphone and share the latest pictures of family members while recounting their latest activities and milestones. throughout his professional career, dr. royal has worked tirelessly to advance the mission of the society for pediatric radiology. he is past president and chairman of the board of the spr and has served on numerous spr committees. he ran a highly successful spr meeting in new orleans in . those in attendance will recall the jubilant parade stuart led through the streets of new orleans to culminate the meeting. as president and then chair of the spr board, stuart played a critical and instrumental role in bringing the spr management contract under the umbrella of the acr. the synergy achieved by the spr-acr relationship has yielded results well beyond a simple management contract. pediatric radiology and spr now have a voice at the "radiology table." stuart has also been a strong advocate for supporting translational research to advance the care of children via imaging. to help achieve this goal, he has worked aggressively to secure increased funding for the society of pediatric radiology research and education foundation. following the launch of the ref's campaign for children in , stuart made it his personal mission to work with the leadership of the society, both past and present, to discuss major gifts to the foundation. through stuart's personal effort, the foundation received pledges for many significant leadership gifts, including from spr pioneers drs. hooshang taybi and ed singleton and from himself and barbara. the spr is highly fortunate to have benefited from stuart's numerous contributions and dedication to the care of children. the society is very proud to bestow the president's award on dr. stuart a. royal. the society extends honorary membership to individuals outside of pediatric radiology who have made outstanding contributions to the care of children. this evening, dr. harvey l. neiman, whose leadership of the american college of radiology is resoundingly praised, is the recipient of the honorary member award. for , as in when his contributions were similarly recognized, dr. neiman's selection by the society for pediatric radiology honors committee was made in appreciation for the strength of his efforts to further the spr's philosophy, goals, and programs for responsible diagnosis and treatment of the young patient as embodied in the acr and spr's "image gently" campaign. image gently has succeeded not only in raising awareness of the great diagnostic benefits we can offer to pediatric patients but also directs us to acknowledge the downside of overzealous diagnostic efforts where excessive radiation becomes a risk. importantly, the "image gently" campaign, an upbeat, positive program rather than a punitive one, a smile rather than a frown, makes pediatric and all radiologists aware that their best practice reflects balanced, educated, up-to-date utilization of state-of-the art technology with exercise of responsible leadership in protecting the pediatric patient. for adults, awareness of the need for patient protection is communicated in image wisely. dr. neiman's vision and successful achievements are evident on every page of his curriculum vitae. a consummate strategist in assembling teams to make forward-looking goals a reality, harvey now stands at the top of our specialty as the first physician executive director of the american college of radiology. at this time in big-business medicine, as we see the physician, leader of the patient care team, being diminished to one of many "providers," it is so important for our patients' well-being for us to recognize the obligations commanded by our training, clinical experience and commitment. dr. neiman's recognition of the need for physicians' leadership in improving the quality of patient services and his development of programs in all areas of the college's activities have been just short of miraculous-image wisely for adults, quality and safety including the performance guidelines and accreditations, education, government relations, economics, imaging metrix, acrin, and the new radiology leadership institute-to name only a few. all have contributed significantly to the care of our patients and the stature of our specialty. dr. neiman was born in detroit and attended mumford high school. from wayne state university, he received his b.s. in and his md in . harvey's postgraduate training was at the university of michigan, where he was a resident in radiology ( - ), chief resident ( , and a - fellow in angiography (cardiovascular radiology), receiving abr certification in and a caq in vascular and interventional radiology in february . dr. neiman often expresses his gratitude to and profound respect for his mentor and beloved chief at the university of michigan, dr. william martel. dr. neiman was chief of cardiovascular radiology at walter reed army hospital and a lecturer in cardiovascular radiology at the afip from - . in , he joined the northwestern radiology faculty, rising to professor in , and for ten years he headed up the section of angiography and sectional imaging, advancing its technology and honing the skills of northwestern's radiology residents. harvey also offered a highly sought-after fellowship in interventional radiology, us, and ct. in , dr. neiman left northwestern to assume the chair in medical imaging at the western pennsylvania in pittsburgh. i was the first woman to have completed his fellowship in us, ct, and interventional radiology at northwestern and accompanied him to pittsburgh. his tenure at west penn attests to his talent in making his visions a reality: the department became a highly respected, successful academic private practice notable in many areas including ultrasound, breast and women's imaging, and interventional radiology. harvey instituted an excellent radiology residency program in as well as fellowship programs in in the areas of excellence noted above. during the years since harvey received his md, he has been awarded honors from many national, international, and specialty societies, has been an invited lecturer over times on ultrasound, interventional radiologic, radiologic educational, management, turf issues, disruptive and new technology topics to name just a few. dr. neiman, who was a founder of the sru (society of radiologists in ultrasound), has to his credit peer-reviewed articles, scientific presentations and exhibits, a text co-authored with dr. james yao, angiography of vascular disease ( ) , and book chapters. he has received many honors including fellowship in the american college of radiology, american institute of ultrasound in medicine, society of radiologists in ultrasound and the society of cardiovascular and interventional radiology (now sir). as part of his strong commitment to the future leaders of radiology, for diagnostic radiology he has served as a member of the residency review committee of the accreditation council for graduate medical education. he has been a member of the american college of radiology and its committees and commissions for many years including the commissions on education, ultrasound, and economics. he also served as chair of the commissions on ultrasound and economics. from to , he was a member on the acr board of chancellors, serving as its chairman - . he was president of the radiology advocacy alliance from to . in , nine years ago, dr. neiman became the acr's executive director. he currently serves in this position, where his excellent business skills, knowledge of health policy and economic issues, and strong administrative background have furthered our specialty. his goal, to ensure that the acr's resources benefit all radiologists and patients across all economic strata, is evident in his actions at the college. harvey has a devoted, wonderful family that often included me and my youngest daughter on many pittsburgh occasions. his beautiful, elegant wife of many years, ellie neiman, is here tonight to celebrate with him the spr's recognition of his many achievements. dr. neiman has two accomplished, lovely daughters, jennifer, extremely successful in her marketing career, and hilary, an attorney. jennie's husband, dr. seth kligerman, one of many young radiologists whom harvey has mentored, is on the radiology staff at the university of maryland. how harvey has had time between, through, and among all of these achievements to have become mentor, colleague, and friend to me and to so many others who have been inspired by his ability to see into the future and to shape it in a positive way is remarkable. now that dr. neiman has taken all of radiology under his wing, not just its component parts, the future of our specialty, one of the best, can be assured but also recognized for its centrality to all of medicine. it is my honor and privilege to introduce to you harvey l. neiman md, facr as this year's society for pediatric radiology honorary member. the singleton-taybi award is given in honor of edward singleton and hooshang taybi, in recognition of their personal commitment to the educational goals of the spr. initiated in , the award is presented annually to a senior member of the spr whose professional lifetime dedication to the education of medical students, residents, fellows, and colleagues has brought honor to him/her and to the discipline of pediatric radiology. it comes as no surprise to those who know him that dr. daneman, "dr. d" as some of us call him, has been named the recipient of the singleton-taybi award in recognition of his many years of dedication to the education of residents, fellows, and colleagues. born in south africa in , he received his medical degree at the university of the witwatersrand, johannesburg, receiving the harwood-nash award for the most successful student in surgery. initially, dr. d thought he would become a pediatric surgeon; but after passing the part i examination offered by the royal australasian college of surgeons, he changed his mind and began his training in diagnostic radiology. he chose a radiology residency at the royal prince alfred hospital, in sydney, australia. this included a year in pediatric radiology at the royal alexandra hospital for children in sydney where his interest and love of pediatric radiology began. dr. d then had the foresight to pursue pediatric radiology fellowship training at the hospital for sick children in toronto, canada. after completing the fellowship, he was immediately offered a position as staff radiologist at "sick kids." he became director of body imaging in and radiologist-in-chief in serving in that capacity for years. his management style was simple but effective. he chose staff that were young, but smart and innovative. he nurtured them and provided them with all the tools they needed to become successful professionals, like him. but contributing to his own department was not enough for him. he also found the time and strength to contribute, teach, train, and help pediatric radiologists in the most remote portions of the globe in every continent, which resulted in recognition from prestigious organizations in places such as south america, israel, europe, taiwan and australia: he is an honorary member of the european society for pediatric radiology and the sociedad latinoamericana de radiologia pediatrica as well as other national societies. dr. d is an "institution" inside the great institution that is sick kids. his teaching is unique and praiseworthy in being enthusiastic, provocative, and fun at the same time. his lectures have been regarded as both instructive and practical by his students and trainees due to his special gift of making the most complicated things look as simple as possible. in sharing his diagnostic knowledge and know-how, he passes his own, innate teaching spirit on to his apprentices. he has earned several awards for this, including the outstanding teacher award granted by the university of toronto fellows at sick kids for the past consecutive years. dr. d receives numerous invitations to present at national and international meetings and symposia and has been invited as a visiting professor to more than a hundred institutions across the globe. he does not only teach us the ins and outs of pediatric radiology, but he makes sure that we learn to love it and understand the importance not only of good practice but also the imperative to pass knowledge on by teaching and publishing. dr. d is someone who inspires us to reach beyond our limits, someone we want to emulate. he shares his knowledge, his wisdom, and his advice freely. he shares with us the most incredible secrets of his own career, so we understand from his personal experience. dr. d never tells you what to do, he suggests to you, in an incredible articulate fashion, what you want to do yourself. dr. d has been and is for many of us, more than an educator, more than a mentor, he is our "coach." well before this concept was introduced into medicine by a. gawande , dr. d intuitively had the vision to "coach" his trainees, trying to get the best out of them, without pressure, but with love and passion, and especially emphasizing the importance of achieving a worklife balance in order to prevent the now so common "stress and burnout" affecting the radiology community . he warned us that many high achievers reach their goals only at the expense of their personal lives, but dr. d has been as successful personally as he is professionally. his wonderful wife of years, louise, his two daughters and his recently newborn granddaughter serve as sources of strength and pride. he is a truthful and generous friend to many, both in and out of radiology. it is not uncommon for many of us, who came through sick kids, to come back and visit and be invited to his house to share a wonderful dinner with other invitees, who may be radiologists from north america or from other parts of the globe visiting sick kids to learn from him. dr. daneman's research has widely influenced the field of pediatric radiology. examples include the work of dr. daneman and his colleagues on intussusception, which has promulgated the use of ultrasound for diagnosis, and the use of air enema for reduction. this approach has been adopted as standard practice at many institutions in north america and across the globe. to share his research with others in the field, dr. daneman has authored or co-authored more than publications, including peer reviewed articles and book chapters on a wide range of topics related to the imaging of children. dr. d is one of those rare people who are irreplaceable. he is a superb teacher, a gifted academician, a capable administrator, and a person called "friend" by so many of us. we are thrilled and proud to present our society's singleton-taybi award to dr alan daneman in recognition of his lifelong accomplishments and personal commitment to the educational goals of the spr. we cannot imagine anyone more deserving of this award than dr. d. thank you "coach"! monica epelman, md and oscar navarro, md john caffey, md - dr. caffey was regarded throughout the world as the father of pediatric radiology. his classic textbook, pediatric x-ray diagnosis, which was first published in , has become the recognized bible and authority in its field. the seventh edition of this book was completed several months before his death in . it has been among the most successful books of its kind in the medical field. dr. caffey was born in castle gate, utah on march , . it is interesting that he was born in the same year that roentgen discovered the x-ray. dr. caffey was graduated from university of michigan medical school in , following which he served an internship in internal medicine at barnes hospital in st. louis. he spent three years in eastern europe with the american red cross and the american relief administration, and returned to the united states for additional training in medicine and in pediatrics at the universities of michigan and columbia, respectively. while in the private practice of pediatrics in new york city at the old babies hospital of columbia university college of physicians and surgeons, he become interested in radiology and was charged with developing a department of pediatric radiology in . he frequently expressed appreciation and admiration for the late ross golden, chairman of radiology at columbia presbyterian hospital, who allowed him to develop a separate department of diagnostic radiology without undue interference, and who was always available to help and advise him. dr. caffey's keen intelligence and inquiring mind quickly established him as the leader in the fields of pediatric x-ray diagnosis, which recognition became worldwide almost instantaneously with the publication of his book in . dr. caffey received many awards in recognition of his achievements. outstanding among these were the mackenzie davidson medical of the british institute of radiology in , the distinguished service award of the columbia presbyterian medical center in , the outstanding achievement award of the university of michigan in , the howland award of the american pediatric society in , the jacobi award of the american medical association in , and the gold medal award of the american college of radiology in . he had been a member of the american journal of roentgenology. he was a counselor of the society for pediatric radiology and was an honorary member of the european society of pediatric radiology. dr. caffey's contributions to the pediatric radiologic literature were many. he was instrumental in directing attention to the fact that a prominent thymic shadow was a sign of good health and not of disease, an observation that literally spelled the end to the practice of thymic irradiation in infancy. infantile cortical hyperostosis was described by him and is called "caffey's disease." dr. caffey in first recognized the telltale radiographic changes that characterize the battered child, and his students helped disseminate his teachings about these findings. it was dr. caffey who first recognized and descried the characteristic bony changes in vitamin a poisoning. he recognized and described the findings associated with prenatal bowing of the skeleton. in , three years after his retirement from babies hospital, he joined the staff of the children's hospital of pittsburgh as associate radiologist and as visiting professor of radiology and pediatrics at the university of pittsburgh school of medicine. although dr. caffey came to children's hospital and the university of pittsburgh in an emeritus position, he worked daily and on weekends throughout the years he was there. in pittsburgh, he made four major new contributions to the medical literature. he described the entity, "idiopathic familial hyperphosphatasemia." he recognized and described the earliest radiological changes in perthes' disease. he called attention to the potentially serious effects of shaking children, and used this as a subject of his jacobi award lecture. he described, with the late dr. kenny, a hitherto unrecognized form of dwarfism that is now known as the caffey-kenny dwarf. the john caffey society, which includes as its members pediatric radiologists who have been intimately associated with dr. caffey, or who have been trained by his students, was established in . this society is now among the most prestigious in the field of radiology. his book and the society named in his honor will live on as important memorials to this great man. his greatness was obvious to all who worked with him. he was warm, kind, stimulating, argumentative, and above all, honest in his approach to medicine and to x-ray diagnoses. his dedication to the truth was expressed in his abiding interest in the limitations of x-ray signs in pediatric diagnosis and in his interest in normal variation in the growing skeleton. he was concerned with the written and spoken word and was a skilled semanticist. his book and his articles are masterpieces of language and construction. he stimulated and was stimulated and loved by all who had the privilege of working with him. radiology and pediatrics have lost a great man, but they shall ever have been enriched by his presence. interstitial lung disease, which is more common in infants than older children, is defined as a rare heterogeneous group of parenchymal lung conditions primarily due to underlying developmental or genetic disorders. affected infants typically present with clinical syndromes characterized by dyspnea, tachypnea, crackles, and hypoxemia. mainly due to a lack of evidence based information regarding underlying pathogenesis, natural history, imaging findings, and histopathologic features of interstitial lung disease, the understanding of interstitial lung disease in infants has been limited in the past. however, in recent years, the understanding of interstitial lung disease in infants has been substantially improved primarily due to: ) advances in imaging technology for better detection; ) improvement of thoracoscopic techniques for lung biopsy; ) established pathologic criteria for consistent diagnosis; and ) development of new classification system based on underlying etiology of the interstitial lung disease. in fact, several forms of interstitial lung disease in infants that exhibit distinct clinical, radiological, and pathological patterns are currently emerging. the overarching goal of this article is to review a new classification system, imaging findings, and pathological correlation of interstitial lung disease in infants. improved understanding of this often challenging disorder can aid in early and accurate diagnosis, which in turn, will result in improved patient care. large airway disease in pediatric patients: impact of advanced post-processing techniques catherine m. owens, bsc mbbs mrcp frcr the introduction of multidetector row computed tomography (mdct) scanners has altered the approach to imaging the pediatric thorax. in an environment where the rapid acquisition of ct data allows general hospitals to image children instead of referring them to specialist pediatric centers, it is vital that general radiologists have access to protocols appropriate for pediatric applications. this lecture will focus on the main principles of volumetric ct imaging that apply generically to all mdct scanners and in particular we describe the reconstruction techniques for imaging the pediatric thorax and the low-dose protocols used in our institution on a -slice dual source ct scanner. examples of important clinical applications with the impact and added value of post processing are also given. neoplasms, by definition, comprise an abnormal uncoordinated proliferation of cells that persists even after the inciting stimulus as ceased. the resulting mass may be benign or malignant and arise from any tissue that is normally found in the location where the mass develops. thus, tumors of the chest may arise from bone, lung, pleura, lymphatics, muscle, etc. whether benign or malignant, chest masses may be incidental findings on imaging obtained for other reasons. this presentation will focus on malignant tumors of the chest, address the imaging characteristics and staging of the most common chest malignancies and discuss characteristics that may aid in distinguishing these lesions from their corresponding benign or infectious counterparts. included in this presentation will be the most common chest wall malignancies (ewing family of tumors and rhabdomyosarcoma), mediastinal malignancies (lymphoma, germ cell tumors, and neurogenic malignancies) and pulmonary primary malignancies (pleuropulmonary blastoma and carcinoid). the changing appearance of selected tumors in patients treated with new targeted therapies will be introduced. lung disease is the most common chronic disease of childhood, but young children cannot perform the breathing maneuvers required for the most commonly used method for assessing lung function, spirometry. ct provides exquisite structure information about the lung but concerns regarding the long-term consequences of the relatively high radiation dose limit its use particularly in the pediatric population. magnetic resonance imaging (mri) has the potential to provide regional information about the lung without the use of ionizing radiation. while conventional proton mri has found widespread clinical application in most organs of the body, mri of the lung lags behind because the lung is intrinsically difficult to image with mri. the strength of the mr signal depends on the physical density of protons in the tissue being imaged and the local environment of the protons. the lung has a low physical density and thus a low proton density so little mr signal is generated by the lung. furthermore, the magnetic susceptibility effects from its many air-tissue interfaces cause what little signal is generated to rapidly decay so that the lung typically appears dark on conventional proton mr images. a variety of strategies have been developed to overcome the inherent difficulties of mri of the lung, resulting in recent substantial improvements in image quality. additionally by administering an inhaled gaseous contrast agent, such as the hyperpolarized noble gases helium- or xenon- , direct visualization of lung airspaces in an mr image is possible. a number of unique strategies for evaluating the structure and function of the human lung using hyperpolarized gas mri have been developed. although the level of structure detail possible with lung mri may never equal that of ct, mri may nonetheless has the potential to provide clinically useful information and be a sensitive, effort independent test of pediatric lung disease. for a matter of time, we will focus in this presentation only on the following: intestinal malrotation a normal visceral situs can be inferred sonographically in relation to the right-sidedness of the superior mesenteric vein, to the retromesenteric location of d and to the right iliac position of the ileocecal valve. conversely, intestinal malrotation is likely when the aforementioned features are reversed. in addition, cdu can display the whirlpool pattern in case of midgut volvulus or internal hernia, alleviating the need for preoperative opacification. the reliability of us in diagnosing intussusception is well documented since the early s. the value of us in predicting the success or failure of pneumatic reduction and/or bowel necrosis is more debatable, based upon a coexisting bowel occlusion, the presence of interloop fluid, bowel wall changes (intramural air, dilated mural vascular channels), absent blood flow at cdu. the continuous down-grading of us in comparison to ct, and the opposite conclusions of various series regarding imaging of pediatric appendicitis are based upon different prerequisites and definitions. historically and in most usa institutions, sonography reports are either negative (entire normal appendix), positive (abnormal inflamed appendix), or equivocal (non-visualization or partial visualization of appendix). the equivocal group is then logically investigated by a subsequent abdominal ct. in europe, some usa centers, and in our practice, us reporting include groups and take into account ancillary findings: . normal appendix (blind-ended, lamellated, compressible, < mm in diameter, without peristalsis; . appendix not depicted, no secondary signs; . appendix not depicted, with one of the following: hyperchoic mesenteric fat, fluid collection, local dilated small bowel loop; . appendix inflamed. group represents most cases of perforated appendicitis, groups and the negative sonogram. ct is then indicated only in obese patients and to assess the feasibility of percutaneous interventions. inflammatory bowel disease in the recent literature, mr enterography is often preferred to ct enterography. small bowel series look prehistoric and us is rarely mentioned. sonography however is very valuable both for screening children presenting with abdominal pain, diarrhea, weight loss, or gi bleeding and for following the course of the disease and searching for complication. hypervascularization has been proved to parallel the disease activity. initially mentioned by dr. rita teele, the interest of us for differentiating high-intermediate/low varieties of imperforated anus has been re-emphasized more recently. a perineal rectal cul de sac distance of mm is quoted as the significant cut-off value. us can also display rectourinary fistulae outlined by air. update on mdct and mri of hepatobiliary disease in children: what's new lisa h. lowe, md a variety of disorders may affect the pediatric liver. recent advances in histopathological knowledge and imaging techniques have led to important changes that radiologists must be aware of in order to allow for an accurate limited differential, and in some cases, specific, diagnosis. this presentation will focus on recent developments that have lead to a better understanding of the embryopathogenesis for fibropolycystic liver diseases (including choledochal cysts and caroli disease), histopathological findings that have led to new classification systems for of pediatric vascular anomalies, technological advances and contrast agents in magnetic resonance imaging that are useful to characterize and limit the differential diagnosis of hepatic masses. diagnostic errors in pediatric abdominal imaging: diagnostic pearls and pitfalls george a. taylor, md this presentation reviews the types of diagnostic errors in abdominal imaging occurring over a -year period in an academic pediatric radiology practice. radiologists engage in two interrelated processes when interpreting imaging studies: perception and analysis. failures in perception (failure to identify an important finding) are a common source of diagnostic error in pediatric imaging, while failures in the analytic portion of the process (over-or faulty interpretation of a finding) are not as common. under-interpretation of findings can be related to a number of perceptual and visual phenomena including visual isolation where attention is selectively focused on a main area of the image while less or no attention is given to secondary areas, and satisfaction of search which occurs when additional lesions remain undetected after detection of an initial lesion. many analytic errors are the result of commonly used heuristics or shortcuts in reasoning. these include the availability heuristic in which likelihoods are based on memory of a similar case, the framing effect in which a different diagnosis is reached based on how the information is presented, and the anchoring heuristic in which the initial impression is difficult to change, despite conflicting new information. another recognized pitfall is blind obedience, in which a diagnostician stops thinking when confronted by authority. this authority can be human or technical (reliance on a laboratory value). finally, diagnostic errors can result from an attitude of overconfidence. examples of these heuristics and strategies to minimize cognitive errors will be discussed. marta hernanz-schulman, md, faap, facr this session will consider abdominal masses that present in the neonatal period, spanning developmental, inflammatory and neoplastic conditions. time constraints do not allow an exhaustive list or description, but the more important or frequent lesions are discussed. the presentation is subdivided by systems. the renal section discusses various conditions presenting with hydronephrosis, such as ureteropelvic junction obstruction and duplication anomalies, followed by autosomal recessive polycystic kidney disease and multicystic dysplastic kidney, cystic entities commonly presenting in the perinatal period. neoplastic renal entities include lesions with benign behavior, such as ossifying renal tumor of infancy, with the discussion extending to entities with very poor prognosis such as clear cell sarcoma and rhabdoid tumor, while discussing the congenital mesoblastic nephroma, its histologic subtypes and the differences in their presentation, imaging findings and clinical behavior. suprarenal lesions include the adrenal hemorrhage, congenital neuroblastoma and subdiaphragmatic sequestration. hepatic lesions include developmental anomalies that present as mass lesions, such as choledochal cysts, vascular lesions such as congenital and infantile hemangiomas, and neoplastic lesions such as the mesenchymal hamartoma and hepatoblastoma. differences in clinical presentation, imaging characteristics and behavior of the lesions are discussed. the section on pancreatic lesions discusses pancreatic cysts and pancreaticoblastoma. gi tract and mesenteric lesions include duplication cysts, lymphangioma, and meconium pseudocyst, and their relationship to bowel obstruction and persistent perforation. ovarian cysts can present as large masses in neonatal girls, and should be high in the differential diagnosis of large masses encountered in female infants; the imaging characteristics of simple and complicated cysts are described, as well as their course and potential complications. pediatric procedures: from imaging to intervention the spectrum of vascular anomalies in pediatric patients: multimodality imaging evaluation and current treatment patricia e. burrows, md vascular anomalies are categorized into two main groups, vascular tumors and vascular malformations. genetic and molecular regulation of vascular genesis of angiogenesis, and mutations responsible for some of the vascular malformations, have been delineated. in order to implement future targeted treatment of vascular lesions, accurate diagnosis is important. imaging modalities that are effective in distinguishing the various types of vascular anomalies and demonstrating the extent include ultrasonography with doppler interrogation, mri and various forms of mr vascular flow imaging, conventional angiography and venography. techniques used to image lymphatic channel anomalies, conventional lymphangiography, lymphoscintigraphy and infrared fluorescent lymphangiography. in this presentation, common forms of vascular anomalies will be described and rare or recently recognized anomalies will be mentioned. current treatment of the different forms of vascular anomalies will also be discussed, including pharmacotherapy using beta blockers, angiogenesis inhibitors and mtor inhibitors. endovascular techniques used in treating vascular malformations, including embolization and sclerotherapy will be presented. pediatric vascular disease is extremely varied, with a wide range of conditions requiring diagnostic or therapeutic intervention. technological improvements in non-invasive imaging modalities such as mri and ct have reduced the need for diagnostic angiography; however, with advances in interventional techniques, arteriography in the pediatric patient is now often performed for therapeutic reasons. pediatric arteriography presents unique issues and challenges. tremendous variability in patient size and physical maturity limits the ability to standardize technical aspects of performing arteriography. in addition, radiation protection, sedation/anesthetic support, monitoring of fluid balance, and maintaining patient warmth must be considered. a regimented protocol for assessment of the pediatric patient must be followed, with review of the indications for the study requested, and review of patient-specific issues such as coagulation profile, concurrent medical disease, patient weight, and anesthetic concerns. appropriate patient monitoring is imperative to ensure patient safety. vascular access can be quite challenging. ultrasound and micropuncture access techniques have tremendously improved successful access while reducing associated complications. the smallest catheter that can accomplish procedure objectives should be used. for most diagnostic cases, french systems can be used for children> kilograms, while french catheters are preferred in those < kg. intraprocedural heparinization ( - iu/kg) is also more often used, especially in children weighing less than - kg. rates and volumes of contrast injected for pediatric arteriography are not standardized, as in adult patients. in general, contrast dose should be limited to - ml/kg, and - ml/kg in premature infants and neonates. all these new technique are less invasive, improve patients' outcomes and reduce morbidity. they are also cost-effective as patients are discharged home earlier and recover faster from the intervention. the future holds promising new technologies such as high-intensity focused ultrasound (non-invasive method of thermal ablation) and nanoparticles for drug delivery. pediatric interventional radiology will continue to be an essential part of these minimally invasive therapies. musculoskeletal imaging: from planning to performance kirsten ecklund, md the purpose of this talk is to review advanced mr imaging techniques currently being used in the evaluation of pediatric musculoskeletal tumors. the goals of these techniques include improved image resolution and quality, lesion tissue characterization, and increased acquisition speed. diffusionweighted (dw) and perfusion imaging will be emphasized; however, whole body, metallic artifact mitigation, and volumetric sequences will also be discussed. dw mri is based upon the brownian motion of water within extra and intra-cellular spaces which depends upon tissue cellularity. dwi can aid in the differentiation of benign from malignant lesions, which generally have restricted diffusion. there is even greater potential for dwi in the assessment of tumor response to therapy. the apparent diffusion coefficient (adc) maps are critical to accurate interpretation of diffusion sequences. adc maps distinguish between restricted diffusion and t effect, both of which appear bright on dwi. both qualitative and quantitative tissue assessments can be made with dwi. challenges for dwi in the pediatric musculoskeleton include susceptibility artifacts from bone, motion vulnerability, and geometric distortion at larger fields of view. our current protocols and parameters for dwi will be presented. contrast-enhanced (dce) mr using one of a variety of vendor specific sequences. qualitative and quantitative assessments of inflow and distribution of contrast have been shown to help differentiate between benign and malignant lesions and to evaluate drug efficacy during therapy. this technique is especially promising in those patients undergoing antivascular and antiangiogenic therapy. tal laor, md congenital abnormalities of the musculoskeletal system can result in alterations of limb size, configuration, and/or segmentation. these disorders often affect both the osteocartilaginous skeleton as well as the surrounding soft tissues and can be localized or diffuse. in this session, we will focus on the imaging features of several congenital abnormalities that result in a small or short limb, in altered configuration of a limb, or in abnormal segmentation. deformities of both upper and lower limbs will be examined. like congenital abnormalities, developmental disorders of the pediatric musculoskeletal system can be limited to a single area or can affect numerous sites within the body. for example, neonatal brachial plexopathy is a localized disorder that produces characteristic musculoskeletal alterations about the shoulder girdle and elbow of affected children. the alterations of morphology and function of the shoulder develop over time with growth of the child and change in response to a variety of therapies. we will review the features of developmental anomalies of the pediatric musculoskeletal system and evaluate the role that imaging plays in the initial evaluation and in the subsequent assessment of these children during treatment. multimodality imaging of skeletal trauma in children: using all of the tools peter j. strouse, md skeletal trauma is a common indication for imaging throughout the pediatric age range. newborns may suffer birth trauma. infants and toddlers may be subject to abusive injury. children of all ages may suffer accidental injury. older children and adolescents are increasing hurt in sporting activity and vehicular accidents. fracture patterns vary with maturation of the child. interference with normal growth is a potential complication. imaging of skeletal trauma begins with radiography. proper anatomic and age specific radiographic technique assures optimal diagnostic yield. radiography suffices in most cases to diagnose fracture or confirm normalcy. "clinical correlation" aids in diagnosis. ultrasound, ct, mri and nuclear medicine may play a role in specific instances where plain radiographs are non-diagnostic or to better delineate certain fractures. arthrography and conventional tomography have occasionally been used in the past and tomosynthesis may prove useful. follow-up radiographs may be useful for diagnosis or confirmation of some fractures. this presentation will focus on the imaging of acute skeletal injury. technique and approach for plain radiography will be emphasized. specific indications and roles for ancillary imaging techniques will be defined and illustrated with representative cases. although classically thought of as a disease of adulthood, stroke is much more common in the pediatric population than was once appreciated. this may be due to many factors, not the least of which is increased awareness due to the presence of subspecialty stroke teams now fairly commonplace in many children's hospitals, and the fairly recent advent of more advanced imaging technique such as diffusion-weighted imaging (dwi) and its routine use in imaging the central nervous system (cns) in the child and adolescent. causes of stroke in children can be protean, and range from idiopathic on one end of the spectrum, to traumatic on the other, with many causes in between, many of which may not be intuitive to the clinician without further research. moyamoya disease and its many causes, such as sickle cell disease (scd), trisomy and neurofibromatosis type i (nf i) can all lead to stroke in children, as can congenital clotting deficiencies such as factor v leiden deficiency and congenital cardiac lesions with their resultant shunting of blood between the left and right cardiac circulations. although usually arterial in nature, strokes may arise from the venous system in clinical scenario of venous thrombosis with resultant venous infarctions. factors contributing to venous thrombosis in children and adolescents can be due to dehydration (especially in the very young), severe iron deficiency anemia, inflammatory bowel disease and exogenous hormone ingestion such as is seen with oral contraceptives (ocp) in young women. advanced imaging techniques for neuroimaging in pediatric patients: where are we now? blaise v. jones, md the past decade has seen a large number of advanced imaging techniques introduced to the clinical armamentarium of the pediatric radiologist. from the development of multidetector ct scanners that can obtain whole head diagnostic studies in less than s to the routine use of t mr imaging, technical advances have dramatically changed our ability to diagnose and manage neurological disorders in children. however, all of these advances are not of equal clinical utility, and it is imperative that the pediatric radiologist be well versed in their judicious and appropriate application. this presentation will discuss the effective use of volume ct scanning, cta, swi, asl, fmr, pmr, and other advanced imaging techniques in the diagnosis of neurological disorders presenting in childhood. at the conclusion of the presentation the attendee will have a better understanding of how to ideally apply these technologies in practice. a spectrum of abnormality in pediatric neck: practical imaging choices and interpretation caroline d. robson, mbchb learning objectives: . become familiar with an optimized imaging approach for head and neck infections . recognize the complications of head and neck infections . recognize the utility and interpretation of imaging for neck masses this talk will cover the imaging approach and interpretation of findings in head and neck infection and neck masses. infection includes acute complicated sinusitis, coalescent mastoiditis, neck infection and local and intracranial complications. optimized imaging protocols and image interpretation for neck masses will also be discussed and illustrated. acute complicated sinusitis is diagnosed when acute sinusitis is accompanied by orbital symptoms (e.g. proptosis) and/or mental status changes, seizures or other neurological findings. coalescent mastoiditis is diagnosed when otomastoiditis is accompanied by tenderness and/or swelling over the mastoid process. ct and mr provide complementary information. ct is obtained with contrast. mr sequences include fatsuppressed t , t , diffusion, and fat-suppressed contrastenhanced t weighted images with mr venography. intracranial complications include epidural abscess, subdural empyema, meningitis, cerebritis, brain abscess, venous thrombosis and venous infarction. the limitations and usefulness of ct in the diagnosis of neck abscess will be illustrated. the imaging approach to masses depends on patient age, and the size and location of the mass. us, ct, mr, and nuclear medicine studies provide complementary information. as for infection, optimized imaging approaches and key imaging features for various masses will be discussed. embryology and diagnostic approach in spinal dysraphism l. santiago medina, md, mph and esperanza pacheco-jacome, md congenital anomalies of the spine are malformations that can be confusing due to the complexity of their embryology, and to the sometimes unclear classifications and terminology. the purpose of this review is to give a clear and basic understanding of the different stages of the embryological development of the spinal cord, starting with the bilaminar disc in the first week of gestation. during the second week, the formation of a trilaminar disc (gastrulation), the notochord, and the formation of the neural tube or neurulation. also, a review of the development of the distal cord: conus medullaris, filum terminale, ventriculus terminalis, by a different mechanism, canalization and retrogressive differentiation. beside the embryological review, a case correlation will be presented using mr imaging to demonstrate these malformations. open spina bifida entities include meningocele and myelomeningicele. closed or occult spinal dysraphism (osd) is characterized by a spinal anomaly covered with skin and hence with no exposed neural tissue. osd spectrum includes dorsal dermal sinus, thickened filum terminale, diastematomyelia, caudal regression syndrome, intradural lipoma, lipomyelocele, lipomyelomeningocele, anterior spinal meningocele and other forms of myelodysplasia. several studies have shown that mri and ultrasound have better overall diagnostic performances (i.e., sensitivity and specificity) than plain radiographs for detection of occult spinal dysraphism. for h n , most patients had mild illness but a small percentage required mechanical ventilation and icu admission. the high risk groups include children < years old and those with chronic medical conditions in particular neurodevelopmental impairment. pediatric mortality was . % of all deaths associated with the pandemic reported in the u.s. in both conditions, the most prominent radiographic and ct features were airspace disease including ground glass opacities (ggo) and consolidation, commonly with multi-focal and bilateral involvement. pleural effusion, adenopathy and cavities were absent. in some patients with viral infection, respiratory symptoms may be mild but are complicated by neurological manifestations. a brief review of mri features in h n related encephalopathy including acute necrotizing encephalopathy (ane) will be given. bernard f. laya, do tuberculosis (tb) is a worldwide major public health problem with one-third of the world's population being infected. it is a leading cause of death and disability from infection worldwide. children are amongst the most vulnerable group because of their immature immune status. a child usually gets tb infection after being exposed to a sputum-positive adult. depending on many factors, the infection can lead to latency or tb disease. it can affect virtually any organ in the body and can be devastating if left untreated. tb in children remains a diagnostic challenge. in addition to history of tb exposure, signs and symptoms, laboratory and microbiologic tests, medical imaging remains a valuable tool in its diagnosis. although findings are nonspecific, the radiograph is the most commonly ordered initial imaging tool for screening and diagnosis of pulmonary and musculoskeletal involvement. computed tomography and magnetic resonance imaging offer more detailed assessment especially in cranial and abdominal involvement. medical imaging is also utilized to follow up patients during or after anti-tb treatment. knowledge of the common imaging patterns, pitfalls and dilemma are very important in establishing the diagnosis of tb in children. the pathophysiology of pediatric tb will be discussed as it correlates with imaging findings. the wide spectrum of imaging manifestations in various modalities will be presented. imaging updates along with pitfalls and dilemma in the interpretation will also be discussed. tb can affect almost every organ system but the author will present cases that are more commonly encountered. and concurrent ct/ pet-ct (k . ) panels. there were more indeterminate nodule predictions by pet-ct (n of ; %) and concurrent ct/pet-ct (n ; %) than by ct alone (n ; %). the overall accuracy of ct alone was %, pet-ct alone % and concurrent review %. worst case sensitivity and specificity were % and % for ct alone, % and % for pet-ct alone, and % and % for concurrent ct/pet-ct. conclusions: pet-ct assessment of pulmonary nodules in children is feasible but limited by non-diagnostic quality ct images and atelectasis caused by sedation. subjective assessment of nodules by pet-ct does not appear to improve the ability to distinguish benign from malignant histology in children with solid malignancies. semi-quantitative nodule assessment using the standardized uptake value may improve the performance of pet-ct in this setting and will be investigated in the future. purpose or case report: nec is the most common lifethreatening medical/surgical emergency of the gastrointestinal (gi) system in neonates, with an incidence up to % in infants weighing < g. with advances in treatment of nec, increased survival rates result in rise in post-nec gi complications such as feeding intolerance. development of post-nec bowel strictures results from healing of involved bowel and can result in bowel obstruction. it has been routine to study the bowel of infants after medical treatment for nec by contrast enema and small bowel follow-through prior to initiating feeding. however, in order to "image gently" we are attempting to decrease the radiation exposure to these patients. we postulate that in patients with no abnormal bowel dilation prior to initiation of feeds, the incidence of colonic stricture would be so low that routine enemas would be unnecessary and could be eliminated from the workup. recorded as present or absent in anatomic abdominal regions defined as: and -from the dome of the diaphragm to top of l to the right and left of midline, respectively; and -from top of l to the iliac crest to the right and left of midline, respectively; -from the iliac crest to the top of the sacrosciatic notch; -below the top of the sacrosciatic notch. we assessed the frequency of findings in each region and how often findings in regions and were associated with findings in regions - . % confidence intervals were calculated. results: the fewest pertinent findings were present in region in . % ( / ) ( % ci: . - . %) of radiographs. findings included: abnormal bowel % (n ), bowel gas paucity . % (n ), pneumatosis . % (n ), inguinal hernia . % (n ) and osseous abnormalities . % (n ). pertinent findings were present in region in . % ( / ) ( % ci: . - . %). findings included: abnormal bowel . % (n ), bowel gas paucity gas . % (n ), pneumatosis . % (n ), free air . % (n ), and abnormal bowel with pneumatosis . % (n ). among patients with an abnormality in region , ( . %) also had an abnormality within at least one of regions through . among the patients with an abnormality in region or , ( . %) also had an abnormality within at least one of regions through . catheter/tube tips were located in region in . % (n ) and region in . % (n ) of radiographs, respectively. pneumatosis was present most frequently in regions ( . %), ( . %), and ( . %). free air was present most frequently in regions ( . %), regions and ( . % each). conclusions: our preliminary data suggest that pertinent findings on neonatal portable abdominal radiographs are rarely isolated to the pelvis, implying that gonadal shielding of regions and should not compromise diagnostic accuracy. purpose or case report: the purpose of this study was to determine the sensitivity, specificity, and positive and negative predictive value of ultrasound in diagnosing appendicitis when the appendix is visualized, using three diagnostic categories: positive, negative, and equivocal. the -category diagnostic accuracies for appendiceal diameter and radiologist impression were compared. methods & materials: a retrospective study was performed evaluating all right lower quadrant ultrasound reports dictated over a -month period. included studies were interpreted as positive, negative, or equivocal for appendicitis. report impressions that did not specify one of these categories and studies where the appendix was not seen were excluded. the pathologic diagnosis of appendicitis was considered the gold standard for a positive diagnosis. because virtually all pediatric surgical cases in the region are referred to our hospital, it was assumed that the patient did not have appendicitis if surgery was not performed. logistic modeling using appendiceal diameter as the independent variable established cutoff diameters of ≤ mm negative, > mm positive, and <x≤ mm equivocal. sensitivity, specificity, and positive and negative predictive values were calculated for both methods. results: of the patients imaged during the study period, ( %) met the inclusion criteria; ( %) reports did not categorize the impression and in ( %) studies the appendix was not seen. surgery was performed on ( %) subjects, of which ( %) had a pathologic diagnosis of acute appendicitis. using the radiologist impression, there were true positives (tp), false positives (fp), false negatives (fn), and true negatives (tn), for a sensitivity of % ( % ci - %), specificity of % (ci - %), ppv of % (ci - %), and npv of % (ci - %). there were ( %) cases reported as equivocal; of these, ( %) had a pathologic diagnosis of appendicitis. using appendiceal diameter, there were tp, fp, fn, and tn, for a sensitivity of % (ci - %), specificity of % (ci - %), ppv of % (ci - %), and npv of % (ci - %). there were ( %) cases categorized as equivocal; of these, ( %) had a pathologic diagnosis of appendicitis. conclusions: the use of diagnostic categories enables high ppv and npv with a relatively low number of equivocal cases. using appendiceal diameter alone results in higher ppv than that of the radiologist impression, but also yields a higher number of equivocal cases. purpose or case report: intussusception is an obstructive condition that a segment of the bowel invaginates into adjacent bowel and is an urgency that needs to be reduced as soon as possible to prevent bowel ischemia or necrosis. when it occurs in the infants and young child, it usually develops idiopathically and reduced by the technique such as air enema under the fluoroscopic guidance or saline enema under the ultrasound guidance. the success rate of the non-surgical reduction has reported over %. the pneumatic reduction is to introduce air into the patients' rectum via a ballooned foley catheter by hand pump. a mercury manometer connecting with the foley catheter calibrates the pressure of insufflation, and the recommended maximum pressure is mmhg. the hydrostatic reduction is to drip saline from a bag at the height of ft that may be different according to the institute's guidelines. until recently, in both techniques, the precise measurement of the intraluminal pressure was not possible. with the purpose of intraluminal pressure monitoring, we introduced a specific digital pressure gauge that can measure both pneumatic and hydrostatic pressure in various units and can give the collected data to our workstation. with the data, we purpose to make the curve of intraluminal pressure during the procedure and to compare the pattern and difference in both techniques of reduction, which permits suggesting the better technique for reducing the intussusception. from february , we performed the intraluminal pressure monitoring in children; cases of pneumatic reduction and cases hydrostatic reduction. the results indicated that the curves were similar in both techniques. however, the hydrostatic reduction tended to need longer time and higher pressure difference than the pneumatic reduction. we assumed that a higher flow rate and more fluctuating introduction of pressure in the pneumatic reduction made the difference between both methods. purpose or case report: anecdotal evidence suggests that some pediatric brain tumors, particularly pilocytic astrocytomas, demonstrate greater enhancement on delayed post-contrast t weighted mr imaging compared to early post-contrast imaging. our goal was to compare immediate post-contrast imaging with delayed post-contrast imaging in pediatric brain tumors. methods & materials: the research protocol was granted exempt status by the institutional review board (comirb# - ). between november and march , all patients for whom an mri of the brain was ordered to follow-up known brain tumor or to evaluate new brain tumor underwent a modified protocol that included both immediate and min delay post-gadolinium images. the modified protocol sequence order was dwi, t mprage, contrast injection, immediate post-contrast t mprage, t , flair, min delayed post-contrast t mprage. this sequence allowed both immediate and delayed post-contrast imaging to be performed without scanner downtime. a total of unique patients met inclusion criteria of an enhancing primary brain tumor with the delayed imaging performed within a window of - min. these studies were evaluated independently by pediatric neuroradiologists (ns and lf) and a radiology resident (jm) to compare the degree of lesion conspicuity and edge discrimination, and to determine an overall preference. in cases of discrepancy between reviewers, final grading was determined by group consensus. results: delayed post-contrast images were preferred in / tumors ( %), immediate post-contrast images were not preferred in any case, and / cases were graded as equivalent ( %). notably, delayed imaging was preferred for / pilocytic astrocytomas ( %). in / cases ( %), the improvement in conspicuity and edge discrimination on delayed imaging was felt to have a potential impact on clinical management, e.g. by extending resectable or treatable tumor margin. conclusions: delayed post-contrast imaging can be performed easily by reordering existing mri protocols, and no scanner downtime is required for this protocol change. our results indicate that pediatric brain tumors (particularly pilocytic astrocytomas) often enhance more conspicuously on delayed imaging, that no diagnostic confidence is lost on delayed imaging, and that clinical management may be more accurately guided. -directions) were retrospectively reviewed, after irb approval. both sporadic (n ) and nf- associated optic gliomas (n ) were included. tumors confined to intraconal optic nerve were excluded due to technical limitation of epi dwi within the bony orbit. apparent diffusion coefficient (adc) of the solid portions of the tumor was measured using the region of interest method both at baseline and on follow-up mri. results: for the patients, required treatment (surgery and/or chemotherapy) for symptomatic tumor progression ( sporadic opg and cases of nf- ). median follow-up interval was . years (range: months to . years, sd . years). there was no difference in adc values between sporadic and nf- associated opg. opg that required therapy had significantly higher baseline mean adc ( x - mm /s, sd . ), whereas opg with low mean adc ( x - mm /s, sd . ) remained clinically asymptomatic or stable (stratified mann-whitney test, p . ). an increase in mean adc of x - mm /s doubled the odds the patient will require treatment (or . , % ci: . - . , p< . ). enhancement patterns did not predict tumor behavior or nf- status (fisher's exact test, p . ). conclusions: significantly higher mean adc was seen in opg that required therapy for tumor progression. mri may be a useful prognostic tool in further defining a subset clinically significant sporadic or nf- associated opg. purpose or case report: fast brain mri imaging has recently been introduced for assessing brain ventricles in patients with ventriculo-peritoneal shunts (vps). however, there is minimal literature on how this method could be beneficial for other indications. compared to ct, this method reduces lifetime cumulative radiation dose and has multiplanar capabilities. it is much faster than full brain mri, reducing exam costs and potentially reducing the need for sedation. however, fast brain mri has lower resolution than a standard brain mri and cannot detect hemorrhage. the goal of our study is to retrospectively assess additional clinical applications for fast brain mri. methods & materials: patients who had undergone fast brain mri were found in xenobase, a database which references deidentified electronic medical records at our institution. xenobase was then used to identify subgroups by diagnosis. one of the most common diagnoses was arachnoid cyst. after irb approval, patients with fast brain mri and arachnoid cyst were identified so that their fast brain mri could be compared to any full brain mri and to clinical outcomes. fast brain mri findings were excluded if there was not a full brain mri within year. agreement with full brain mri, and documented effect on clinical management were coded for statistical review. results: between and , there were arachnoid cyst patients with fast brain mri exams. in members of this group, fast brain mri was used alone or in comparison with a full brain mri in the decision to follow or discharge the patient. exams failed to report a pituitary cyst and mass effect which were reported on prior exams. patients had no complications in the year following their fast brain mri. the most common complication was headache ( cases). conclusions: fast brain mri reduces radiation exposure, time, cost, and the need for sedation in vps patients. these benefits may cautiously be extended to other patient groups also requiring routine imaging, such as those with arachnoid cysts. disclosure: the authors disclose that they will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: susceptibility-weighted imaging (swi) is a new technique that exploits susceptibility differences in different tissues, to provide a different type of tissue contrast. swi is a gradient echo sequence utilizing both phase and magnitude data to achieve exquisite sensitivity to tissue magnetic susceptibility effects. it is extremely sensitive to even minute amounts of paramagnetic substances and therefore for detection of blood products (hemosiderin, ferritin), deoxygenated blood, calcium, iron, and small vein depiction. it is particularly suited for vascular imaging, especially in cerebral ischemia. swi has been shown to be more sensitive in detecting cerebral microbleeds than is conventional t *-weighted gradient-recalled echo imaging (gre) partly because of its inherent sensitivity, increased spatial resolution and the thinner slices acquired. recent studies have shown that swi detected more microbleeds in more patients, irrespective of their location and that swi may be more sensitive in detecting traumatic lesions than ct or mri. methods & materials: between january and october we have performed swi on pediatric patients (from day to yrs old) affected with different pathologies. mri was performed using either a -or . -t magnet (achieva ; philips healthcare, best, the netherlands). among these patients also received a classic gre sequence. results: our experience confirms a greater sensibility of swi than gre in detecting microbleedings and larger bleedings. swi is much more helpful in patients younger than year since it can distinguish hemorrhagic and non hemorrhagic punctuate white matter lesions detected as hyperintensities on t -w images. it is more accurate in distinguishing petechial hemorrhages and ischemic foci in patients with germinal matrix hemorrhages. swi can provide important diagnostic information not only in vascular pathologies but also in tumors showing the altered tumor microvascularity, the degree of intratumoral necrosis and the presence of subtle defects of the bbb within the surrounding parenchymal tissue. the technique also allows the detection of possible bleedings in infectious lesions. conclusions: swi is a valuable technique in the evaluation of a wide variety of intracranial pathologies in the pediatric population and its use is advisable on a routine basis. purpose or case report: a common problem with pediatric mr imaging is motion. diffusion-weighted imaging (dwi) is highly sensitive to the effects of both rigid-body motion and brain pulsation. the resulting motion artifacts can manifest in several ways, and include blurring (motion between volumes), slice 'drop-outs' (motion during the diffusion encoding), and aliasing (motion during the parallel imaging/ghost calibration scan). in order to improve image quality in pediatric dwi and avoid the potential for repeat scans, we have implemented our grappa-accelerated diffusion tensor (dt)-epi sequence on over , pediatric patients at our institution, and have developed tailored reconstruction software to correct for large motion and reduce the need for general anesthesia (ga). methods & materials: grappa-accelerated epi dti data was collected on patients at . t and , patients at t between the ages of month and years old. with these data, we investigate the use of a method to select the best grappa and ghost calibration weights; d rigid-body realignment with importance weighting; a new volume rejection criterion for large motion; together with other procedures to lower image noise and reduce phase artifacts. all image reconstruction was performed using compiled threaded matlab code on the vendor's (ge) multiprocessor reconstruction hardware. results: using the maximum motion detected from the d realignment procedure on the , dti exams, % of patients did not move (either through co-operation or ga, denoted here by < mm of motion); % of patients moved between - mm; % between - mm; % between - mm; and % between - mm. except for patients in total, the motion correction steps employed here were extremely robust in correcting for motion. in addition, the motion correction steps employed here do not blur or negatively affect the data on non-moving patients. conclusions: our proposed integrated reconstruction scheme suggests that correcting for different types of motion at different stages of the dti acquisition is critical to provide scans of diagnostic quality. apart from the very rare instance ( . %) where the patient moved continuously an extensively throughout the scan, our motion correction procedures employed here are extremely robust. using this scheme, we show that highly diagnostic motion-corrected dti data can routinely be read on our patient database within min. . participating sites followed mutually agreed upon vendor-specific dti protocols used for neonates in clinical practice: ss-epi; b ;~ mm voxel; - directions; te~ msec; acquisition; parallel imaging; multi-channel ( - ) head coil. tensor data was analyzed at one site using internally developed software in idl. b and eddy current distortion were assessed by analyzing grid line distortion on acr slice ; deviations from linearity were measured in the phase encoding direction. acr slice was used to assess snr of the center and periphery of the b image, and fractional anisotropy (fa) of the phantom, which should approach . deviations from specified sequence parameters were recorded. results: both siemens scanners had some image degradation due to vibration susequently corrected by the vendor. nonlinear gridline distortion was highest on the ge followed by philips but was corrected with the use of nd order shimming. eddy current distortion was consistently smaller than acquisition voxel size after image registration. te varied from - msec; acquisition bandwidth range was - hz/pixel. the mm slice thickness was problematic on clinical ge scanners resulting in non-isotropic voxels. across vendors, snr was consistently higher in the image periphery than center by a factor of . - . ). the greatest difference was showed in patients with fhc. no association was found between the value of ldl-c and imt. the . % received drug treatment at the time of evaluation. in dbt- a no association was found between the imt and the levels of hba c and lipids. and in the ob group found no association between imt and bmi z score. fhc %, % of dbt- and % of ob showed atherosclerotic plaques and intima irregularities. conclusions: the imt ultrasonography was able to demonstrate that patients with chronic diseases, with increased cardiovascular risk in adulthood, showed early alterations of carotid intima media in children. this allows clinical applications of preventive and therapeutic guidance. a benchmark for precision of flow assessment was determined from differences between aortic and pulmonary arterial flows, which should agree. the precision of flow when utilizing a slow flow structure (vein) and a fast flow structure (artery) was then determined, serving as an indicator of the precision of slow flow measurements. this was done in pairs for the upper body (svc flow vs difference between ascending and descending aortic flow), lower body (ivc flow vs descending aortic flow) & total body (svc flow+ivc flow vs ascending aortic flow). paired t-tests were used to identify significant differences in the respective arterial and venous flow rates. with an f-test, the relative precision of the arterial flow (determined from aortic and pulmonary arterial flow) was compared against the precision of agreement between upper, lower, and total body flows, each of which has a slow flow venous component, thus assessing precision of venous flow measurement. results: arterial & venous flow measurements were strongly correlated for the upper body (r . ), lower body (r . ) and total body flow (r . ); net aortic and net pulmonary trunk flow rates were also tightly correlated (r . ). moreover, there was no significant difference in the precision of arterial & venous flow measurements (t-test, p . ). lower venc ( cm/s or lower) improves the accuracy and precision associated with low velocity venous flow, a finding that tended to but it did not reach statistical significance. conclusions: with -d pc mri, the accuracy and precision of venous flow quantification are comparable to arterial flow quantification, though venc settings may improve venous flow assessment. disclosure: dr. tariq has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the spatial resolution requirements for imaging tiny target vessels in neonates have so far precluded its use of cemra in this patient population. the purpose of our study, therefore, was to explore the potential of high resolution cemra at . t in neonates with suspected congenital vascular anomalies methods & materials: neonates (mean age . days, range - days; mean weight . kg, range . kg- . kg) underwent cemra at . t. cemra was performed under controlled apnea on a -channel . t system using either a -channel head-neck coil ( pts) or a -channel adult knee coil ( pts). gd contrast delivery was calculated based on a total dose of . mmol /kg. parallel imaging factors of - were used, generating . x . x . mm voxels in an acquisition time of - s. images were scored for quality and the presence of artifacts independently by two observers. the cemra series were evaluated to identify pulmonary arterial and venous anatomy. extra-pulmonary vascular anatomy was divided into sections for image quality assessment on mpr. results: the mra data sets had high mean image quality and low mean artifact grading scores. no significant difference was evident between the two observers for scoring image quality and artifact (p . ), and there was good interobserver agreement (k . , % ci: . - . ). the pulmonary arteries were confidently visualized to at least the nd order branches in all patients, at least the rd order branches in ( %). out of evaluated segments, ( %) were graded as the vessel "very well visualized with excellent definition of vessel" pa abnormalities were diagnosed in patients and pv anomalies in patients. correlation was made with open surgical findings in cases, confirming the cemra diagnoses with no discrepancy between the operative and imaging conclusions. the results of our study suggest that high resolution cemra at . t is extremely promising in neonatal cchd and, in combination with echocardiography, may often provide comprehensive information for treatment planning. with appropriate use of physiological motion compensation, parallel imaging and coil configuration, voxel volumes less than . mm are routinely achievable. purpose or case report: it is not well understood whether transfusion-dependent children (mainly those who have hereditary anemias subject to excessive iron levels from regular blood transfusions) with no or mild cardiac iron overload and without signs of heart failure exhibit early changes in cardiac dysfunction. this study aims to evaluate intramyocardial deformation and subsequent cardiac dysfunction in such patients using the cardiovascular magnetic resonance technique of myocardial tagging as well as assess iron overload by another cardiovascular magnetic resonance technique of t star (t *). we hypothesize that transfusion dependent children undergoing chelation therapy will be at high-risk for cardiomyopathy despite normal or slightly elevated iron levels and no signs of heart failure due to progressive myocardial deformation as assessed by myocardial tagging; that is, we expect abnormal mechanical parameters to exist despite t * values which correspond to normal or slightly elevated myocardial iron levels. methods & materials: patients (mean age yrs) requiring regular blood transfusions for various reasons (e.g. thalassemia, sickle cell disease, other anemias) were investigated using harmonic phase magnetic resonance tagging and t * imaging. various myocardial mechanical parameters were quantified and correlated to cardiac iron levels as measured by t *. results: children with no overt signs of heart failure (as measured by ejection fraction and volume indices) exhibited normal t * iron levels in combination with global circumferential abnormal myocardial strain and children exhibited regional strain. conclusions: this preliminary data suggests that transfusiondependent children without overt signs of heart failure exhibit early signs of cardiac dysfunction (an effect of strain as assessed by myocardial tagging) in direct relation to normal or slightly elevated myocardial iron levels (as measured by t *). a larger sample size of children could support the idea of abnormal myocardial mechanics being a diagnostic imaging marker for the development of myocardial iron overload and eventual iron-mediated cardiomyopathy. the choice of coil and field of view varied according to the age and size of the patient. imaging was performed on a . t magnet (philips mri system). after the cine gradient echo time of flight sequences were acquired, the dynamic keyhole mr angiographic sequence was applied in the coronal orientation. this sequence commenced - s after initiation of the first bolus administration of - ml of undiluted gadopentetate dimeglumine ( . mmol/ml, magnevist) at a rate of - ml/sec followed by - ml of saline at a rate of ml/sec. image acquisition was repeated multiple times without delay for s during shallow breathing. contrast material was administered through a peripheral or central venous line, and sometimes via simultaneous bilateral extremity injections. rarely manual hand injection was performed. the parameters were as follows: centra k-space filling, acquired voxel size - . x - . x - , reconstructed voxel size . - x . - x - . , number of dynamics - , keyhole percentage - %, sense (parallel imaging) factor x - x , dynamic times - . s, and injection rate - cc/second. image analysis was based on original contrast-enhanced data sets, mip and volume rendering. the quality of the mrv was graded by a subjective scale from - ( excellent, no limitations; nondiagnostic). comparison with unenhanced mr venographic sequences and doppler ultrasound were available in all cases. results: d mrv provided diagnostic information on patency of venous segments in all cases, including the presence of venous occlusion or stenosis, the segments involved, and collateral venous pathways. conclusions: d mr venography using the method outlined is a reliable means of estimating central venous thrombosis in children, using short dynamic scan times with high spatial and temporal resolution, creating an effect similar to conventional venography. paper #: pa- analysis of optimal phase interval for prospective ekg-triggered cardiac ct in children prakash masand, md, texas children's hospital, savivek bogale, lorna browne, rajesh krishnamurthy purpose or case report: evaluate high temporal resolution cine mri of the heart in children to determine relationship of heart rate to the optimal motion free interval during the cardiac cycle, and to derive a chart depicting optimal reconstruction intervals across a range of heart rates which could be applied for prospective ekg triggered ct imaging of the heart. methods & materials: pediatric patients who had high temporal resolution cine steady state free precession sequence ( frames acquired across a single cardiac cycle) performed as part of a mri/mra of the heart from - were included. a single reader analyzed the high temporal resolution cine data to determine the location and duration of the motion-free phase interval in systole and diastole for heart rates ranging from - bpm. the following information was collected: age, heart rate, duration of systole and diastole, ekg phase and phase percentage of motion-free interval, and duration of motion-free interval. heart rates were grouped in ranges of ( - , - , - etc.), with at least patients in each group. wilcoxon sum rank test was performed to compare the static durations of systolic and diastolic phases for each heart rate group. two-tailed p-values< . were considered to indicate statistical significance. a chart matching heart rate range to the optimal phase of image acquisition and reconstruction was created. results: optimal reconstruction intervals for motion-free imaging of the heart in systole and diastole, and single best phase for a given heart rate are as follows: (please see table ) mean duration of the motionless phase in systole and diastole for a given heart rate is as follows: (please see table ) conclusions: this is the first time that variability in location of the motion-free interval within the cardiac cycle with respect to heart rate in children has been studied in rigorous fashion. this chart will have immediate application in the so-called 'target scan', which uses prospective ekg triggered acquisition of data in combination with half-scan reconstruction to minimize radiation dose with respect to retrospective ekg gated imaging. results: a total of patients were found to have aberrant pda by ccta. nine patients had a pda emanating from the brachiocephalic artery to the pulmonary arteries and one patient had pda emanating from the carotid or the subclavian artery according to the operative report. all ten patients had echocardiography in addition to ccta. nine of the ten patients underwent surgery. one patient was lost to follow up. comparison between ccta and echocardiography to operative report showed the following: out of the echocardiograms agreed completely with ccta and operative report. out of the echocardiograms did not report aberrant pdas which were seen on ccta and confirmed during surgery. in one patient, the echocardiogram reported a double aortic arch which was found to be an aberrant pda from the left brachiocephalic artery; a finding that changed the surgical management and pre-surgical planning. the results were statistically significant with a p value of < . . conclusions: diagnostic accuracy of ductus arteriosus anatomy is important in pre-surgical planning and management of patients with ductal dependent pulmonary circulation. cardiac ct angiography is more accurate as compared to echocardiography in diagnosis of aberrant patent ductus arteriosus. ) in patients younger than year of age. the prospective ecg-gated protocol scanned % of the cardiac cycle with msec padding in patients with heart rates greater than bpm. two experienced radiologists evaluated the image quality and course of the coronary arteries. image quality was assessed based on the degree of homogeneity of vascular enhancement, presence of stair-step artifacts, image signal and noise as well as the overall subjective image quality based on a point scale. the course of the left and right coronary arteries was also evaluated based on a point scale, focusing on how well the evaluator was able to visually follow the coronary artery from its origin to its termination. radiation dose was recorded and compared. results: the mean score of the homogeneity of vascular enhancement, stair step artifacts, image noise and signal, overall image quality, and score given to visualization of the course of the coronary arteries showed no significant difference (p> . ) in the retrospective and prospective ecg gated groups. the mean estimated effective dose was significantly lower for the prospective ecg gated group compared to the retrospective group, ( . msv vs . msv) respectively (p< . ). conclusions: prospective ecg-gated cardiac mdct provides comparable assessment of coronary anatomy, image quality with significantly less radiation dose when compared to the retrospective ecg-gated mdct. prospective ecg gated cardiac mdct is a powerful adjunct to the treatment and surgical planning of pediatric patients with congenital heart disease less than yr of age with lower radiation dose. methods & materials: pediatric neuroradiology lectures were recorded and made available to radiology residents at a university program through on-line streaming video viewed through an internet link. topics included brain tumors, phakomatosis, and congenital brain malformations. one lecture per week was recommended prior to case conferences that reviewed the same topic. pre-and post-tests and a feedback survey were administered. nonparametric paired sign test and analysis of covariance were used to evaluate changes in test scores overall and according to feedback responses. spearman's partial rank correlation coefficient was used to evaluate the relationship between the number of viewed videos and test scores. results: twenty-nine residents completed the pre-test and the post-test. the means (sd) scores were . % ( . %) and . % ( . %) respectively. there was a significant improvement in test scores (p . ). residents that agreed/strongly agreed that the streaming technology lectures were convenient had greater improvement than those who did not ( . vs. - . %, p . ). similarly, those who agreed/strongly agreed that being able to replay a lecture was helpful had greater improvement than those who did not ( . vs. - . %, p . ). finally, those who agreed/ strongly agreed that the streaming technology lecture format was a better teaching tool had greater improvement than those who did not ( . vs. - . %, p . ). significant positive correlation between number of videos watched postconference and improvement was present (spearman's rho . , p . ). conclusions: on-line streaming video with live case conferences enhances radiology resident learning of pediatric neuroradiology. step (mpps) software provided an accurate measurement of scan time. visual charting made gaps in utilization apparent. technologists and nursing notes correlated to gaps identified barriers and opportunities for improvement. nursing and anesthesiology reduced redundancies. standardized protocols lead to more consistent scan times. appointment access for sedated mri was measured by the third available appointment. results: manually entered data points were time consuming, inconsistent and unreliable. the process improvement was most effective when fewer more reliable data points were used to evaluate the effect of change. the program resulted in a reduction of appointment access for sedated mri from days to days with no change in the hours of operation. magnet utilization was increased from % to %. induction outside the scan room provided the most efficient process tested. we ranked first in utilization in a children's healthcare cooperation of america (chca) survey as measured by exams per scanner. patient preference for a.m. scheduling was shown by survey and corroborated by scheduling data. consistent scan times were achieved by protocol standardization augmented by indication driven decision support. conclusions: a consise definition of mr utilization established a metric that was used in the process cycle of analyze-optimize-measure. anesthesia induction outside the magnet was the most efficient practice but required collaboration between nursing, mr technologists and anesthesiology. protocol standardization were valuable aspects of process improvement essential to optimizing parallel sedation. these adjustments reduced appointment access from days to days, increased utilization from % to % and produced a number one rank in utilization by chca survey. we exploited the synonym option in epic order entry to translate indications into procedures mapped to specific protocols for mri neuroimaging. the order screen allows the provider to enter an indication. that indication is linked through a synonym option to a specific exam and protocol. the recommendation is based upon institutionally created clinical care guidelines, and can be accepted with a single click to complete the order. the requesting provider retains the option to override the recommendation. an step in process development utilized an order queue established within the epic inbasket. a pediatric radiologist monitored the queue and communicated with referring providers to obtain additional history and educate toward best imaging practice. these interactions facilitated development of a robust index of clinical indications used to create the synonym pool. results: mri neuroimaging indications were expanded into a robust data set linked to specific mri exams aligned with specific protocols. the synonym option within epic created the opportunity for the requesting provider to simply enter an indication which drives the procedure and recommended protocol. provider satisfaction has been high and concurrence with recommendations nearly universal. conclusions: indication driven order entry was achieved through the synonym option in order entry within the epic emr. imaging recommendations are based upon institutional clinical care guidelines developed through consensus. a robust compilation of pediatric mri neuroimaging indications has been created and linked to specific exams and protocols. compliance with the indication driven recommendation has been high. modifications of the current system are currently under development for all cross sectional modalities and organ systems. paper #: pa- cost-effectiveness of routine neonatal renal ultrasound in non-syndromic complex congenital heart disease elfrides traipe, tch, extraipe@texaschildrens.org; jill v. hunter, marthe munden purpose or case report: to assess the prevalence of abnormal renal ultrasound in non-syndromic complex congenital heart disease (cchd) and assess the cost-effectiveness of routine renal ultrasound in this population. we restrospectively reviewed the initial neonatal renal ultrasound and any subsequent renal imaging in patients with non-syndromic cchd. etiologies included hypoplastic left heart syndrome (hlhs), transposition of the great vessels (tga), coarctation of the aorta (coa), truncus arteriosos (ta), double outlet right ventricle (dorv) with or without patent ductus arteriosus. patients were recruited consecutively as part of a prospective trial for pre-and post-operative magnetic resonance imaging of the brain. results: the neonatal pre-operative renal ultrasounds were analyzed in female and male patients. only of the patients showed any congenital renal anomaly. this patient was born with hypospadias, that would have routinely stimulated neonatal follow up. conclusions: knowledge of embryology would not lead us to anticipate a high co-incidence of congenital renal and cardiac pathology. based on this statement and our findings, our recommendation to improve cost-effectiveness is not to perform routine neonatal renal ultrasound in non-syndromic cchd, but only if otherwise clinically indicated. purpose or case report: to conduct a meta-analysis of the diagnostic performance of contrast enhanced voiding urosonography (cevus) in comparison to voiding cystourethrography (vcug) or direct radionuclide cystography (drnc). a literature search was conducted for studies published on cevus in the pediatric age group. studies were included if the ultrasound contrast agents (usca) levovist® (bayer-schering pharma, germany) or sonovue® (bracco, italy) were used and enough data was available to extract x tables. if the cevus was compared to both vcug and drnc in the same patients the results for each were analyzed separately. a bivariate hierarchical model that takes into account the heterogeneity in cevus sensitivity and specificity in different studies was used for the assessment of the summary diagnostic metrics. the summary roc curve was derived and presented graphically from the parameters of the model. additionally, the % confidence intervals (ci) and the positive (lr+) and negative (lr-) likelihood ratios were calculated. results: out of publications only comparative studies fulfilled the inclusion criteria. these encompassed cevus studies in comparison with vcug and with drnc with regards to detection of vesicoureteric reflux. in studies the usca levovist® and in sonovue® were used. a total of children with pelvi-ureteral-units (puus) were included in the meta-analysis. cevus compared to vcug and drnc had a sensitivity of % (ci: - ) and a specificity of % (ci: - ) with lr+and lr-of . and . , respectively. the performance of cevus was better when compared to drnc than to vcug (sensitivity %, specificity % versus % and %, respectively. the meta-analysis of the diagnostic performance of cevus regarding the urethra included patients ( boys). excellent imaging of urethral anatomy was reported in over % of the patients. however, currently there is only one comparative study with patients available. in this study % sensitivity and % specificity were reported. conclusions: sufficient evidence is available clearly demonstrating the high diagnostic performance of cevus compared to vcug or drnc regarding the detection or exclusion of vur in children. these findings combined with the absence of radiation should be convincing reasons for promoting the widespread use of cevus in children. disclosure: dr. darge has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: prenatal ultrasound (us) has increased identification of infants with asymptomatic renal pelvic dilatation. society for fetal urology (sfu) grading is used in the sonographic evaluation of pediatric hydronephrosis. based on us findings, a nuclear medicine diuretic renogram may assess renal function, which could result in operative intervention. standardized protocols for diuresis renography, the "welltempered renogram," already exist; however, no current study has assessed effect of intravenous hydration (iv) status with us in the evaluation of childhood hydronephrosis. our study assesses the effect of hydration on sfu grading. in this prospective irb approved study, pediatric patients diagnosed with pelvicaliectasis requiring a diuretic renogram were recruited to undergo pre and post hydration renal us. a urinary catheter was placed followed by renal us pre and post iv hydration ( ml/kg normal saline bolus). imaging was performed by the same sonographer on the same us machine. a well-tempered renogram was then performed. all images were reviewed by two blinded radiologists, one pediatric radiologist who assigned sfu grades to each kidney. results: data were collected from studies, with ages ranging from weeks- years, with an average age of months. there were unique patients. of these, underwent a single renogram, underwent two renograms, and underwent renograms. one patient had a solitary kidney due to mcdk. thus, there were usable paired sonograms ( kidneys) for analysis. sfu grades were compared in the pre-and post-hydration us for each kidney. two-sided statistical tests were done to assess whether sfu grades changed significantly after hydration (sign test). of ( %) kidneys remained the same grade post hydration. when there was a difference, most demonstrated an increase ( of kidneys and p< . ). no change in sfu grade pre-and post-hydration differed by more than . only kidney went from grade to grade . sfu above grade is considered clinically significant. no kidney that was grade - pre-hydration became grade - post-hydration. when sfu is dichotomized grade - vs. - , there was no significant change in grade from pre to post hydration (p ). conclusions: hydration does not appear to have a clinically significant effect on sfu grade. therefore, performance of a "well-tempered" us is unnecessary. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. study type was significantly associated with both total and weighted score (both p < . ). rus was better and mag was worse than vcug, rnc, and dmsa, which did not differ from each other. other factors associated with worse total scores included patient age - years (p< . ) and non-white race (p . ). gender, prior testing history, wait time, and parent education were not associated with total scores. in the multivariate model, rus remained the best, mag the worst, and dmsa, vcug, and rnc in the middle (p< . ). compared directly, dmsa and vcug total score did not differ (p . ). conclusions: this study documents significant differences among gui studies with respect to the patient and family experience, but there was no overall difference between dmsa and vcug. these findings may be useful to aid decision-making when considering gui for pediatric patients. we retrospectively reviewed the imaging findings of consecutive patients with histologic diagnosis of cd ( males, females; mean age . years; age range - years) who underwent mre between / / and / / . the mre was performed in a siemens avanto . tesla scanner. standard departmental volume of polyethylene glycol and fluid were administered for bowel distention. the imaging protocol included dwi with eight b values ranging between sec/mm and sec/ mm and gadolinium enhanced dynamic d vibe (volume interpolated breath hold exam) in the coronal plane. the studies were qualitatively evaluated in a blinded fashion by two board certified radiologists. disease activity was defined as bowel wall thickening and enhancement in the gadolinium enhanced images. dwi abnormality was defined as bowel thickening, increase signal on dwi images and decrease signal on adc maps of the ileum. intra voxel incoherent motion (ivim) dwi parameters were used as quantitative biomarkers for the analysis of slow diffusion (d) and fast diffusion fraction (f). results: gadolinium enhanced vibe images identified abnormal thickening and enhancement of the ileum in / ( %) patients. dwi identified abnormal signal in / ( %) patients. the sensitivity and specificity of the qualitative dwi for identifying ileitis, as shown by gadolinium enhanced imaging, were % and %, respectively. quantitative analysis showed statistically significant difference in ivim maximal values for f (fast diffusion fraction) between abnormal (mean . , std . ) and normal (mean . , std . ) ileum segments (p . ). there was statistically significant difference in ivim maximal values for d (slow diffusion) between abnormal (mean . μm /ms, std . μm /ms) and normal (mean . μm /ms, std . μm /ms) ileum segments (p . ). abnormal loops of bowel had decreased slow and fast diffusion parameters. conclusions: diffusion weighted imaging has excellent sensitivity and specificity for the detection of active ileitis in pediatric cd. furthermore, quantitative ivim model parameters provide effective biomarkers for this condition. ivim dwi has the potential to assess bowel inflammation without intravenous contrast enhancement and further increase our understanding of cd. methods & materials: forty pediatric patients (median age . years, range . - . ) with suspected (n ) or confirmed ibd (n ) were included and underwent gastroileocolonoscopy with biopsies followed by mre (median interval days, range - ). the mre results were compared with macroscopic and microscopic assessment of the ileum. the clinical importance of the mre results was registered. results: crohn disease (cd) was diagnosed in cases, ulcerative colitis (uc) in , and ibd unclassified (ibdu) in three. macroscopic ileitis was detected in / ( %) of cd cases and in / ( %) of uc (backwash ileitis). microscopic inflammation was found in another four cd cases and one ibdu patient. in total, discrepancy between macroscopic and microscopic inflammation was found in cd, uc and one ibdu patients. the sensitivity of mri was % (against macroscopy and/or microscopy) to % (against macroscopy alone), while the specificity was % and %, respectivley. mre findings was decisive for diagnosis in / ( %) and led to treatment adjustments in / ( %) in the following six months. conclusions: mre is a reliable method for imaging of intestinal inflammation in pediatric ibd, and can be supportive or essential for clinical treatment decisions. results: of children with ec, had ct imaging of the abdomen and pelvis. these children ranged in age from months to years (mean . years +/− . ) with a male predominance (n , %). the most common presenting symptoms were abdominal pain (n ), bloody diarrhea (n ), and rectal bleeding (n ). ec was characterized as a dense and predominant eosinophilic inflammatory infiltrate in the lamina propria and/or epithelium without granulomas. ct scans were abnormal in ( %). no colonic luminal contrast was present in patients, and in one of these, the colon appeared normal. abnormal ct findings included cecal wall thickening (n , %), mucosal enhancement without colonic wall thickening, (n , %), mesenteric lymph node enlargement (n , %), terminal ileal thickening (n , %), jejunal and ileal thickening (n , %), and pneumatosis (n , %). of the patients with cecal involvement, primarily involved the cecum with less severe or no ileal or downstream colonic involvement. pneumatosis extended along the length of the colon with rectal predominance. conclusions: the predominant ct finding in our ec series was wall thickening, most severe in the cecum with variable extent downstream with mild or no involvement of the terminal ileum. although there is overlap, these findings are different from the most common patterns encountered with ulcerative colitis or crohn disease and should raise the possibility of ec in children presenting with abdominal pain and bloody diarrhea. purpose or case report: timely identification of childhood arterial ischemic stroke (ais) is critical to development of acute treatment strategies. we present our experience prior to and following development of a pediatric stroke alert system (sas). through multi-disciplinary collaboration in a tertiary care setting, a pediatric sas was established in . we describe the system, imaging protocol evolution, and impact upon the time between admission and mri initiation (time-to-mri) in patients with childhood ais. of patients in our stroke database (comirb # - ), % met inclusion criteria for stroke alert initiation (acute focal neurological deficit within h). eleven pre- and nine post- patients met criteria. we compared the time-to-mri between these two groups, utilizing a two-tailed t-test. results: the pediatric sas has two phases: i-neurological evaluation and ii-imaging and treatment consideration. phase i stroke alert is initiated when a child presents with an acute focal neurologic deficit. if neurology confirms stroke symptoms and ct head is negative for an alternative etiology, a stroke alert is called prompting an emergent brain mri. if mri confirms an acute stroke, hyperacute therapies are considered. initial mri protocol included dwi, t , flair, d tof cow mra, d tof neck mra and fat saturated t neck imaging. after internal quality review, t mprage brain and contrast enhanced d neck mra were added. the sequence order was also altered so diagnostic sequences were scanned first (dwi and cow mra). there was a trend towards decreased time-to-mri in the post- group (mean min, sd +/− ) as compared to the pre- group (mean min, sd +/− ; p . ). conclusions: institution of a pediatric sas improved urgent neurologic evaluation and demonstrated a trend towards shorter time-to-mri. ongoing quality review has enhanced imaging quality and decreased time-to-mri. continued refinement of pediatric sas's will be critical to the success of recently funded phase i clinical trials in the evaluation of hyperacute therapies. results: there were female and male infants. the mean post-gestational age at presentation was days (range - days), while the corrected age was less than days for all patients. patients presented with seizures and signs of infection; presented with lethargy and later proved to have protein c deficiency. mri was performed - days from presentation in these patients. another patient with known protein c deficiency underwent mri at d for followup of screening us abnormalities. there were a total of deep cerebral white matter lesions: frontal, parietal, temporal lobe. lesions were fluid signal cavities with restricted diffusion. larger lesions had dependent debris. all lesions had associated hemorrhage and most lesions had evidence of adjacent small vessel venous thrombosis. lesions imaged after gad showed peripheral enhancement. three lesions were seen to increase in size on follow-up imaging. three patients, with meningitis confirmed via microbiology and with presumed meningitis by csf counts, underwent surgical aspiration of a total of lesions. all specimens were sent for pathology and culture and were negative for microorganisms. conclusions: recognizing the mr appearance of necrosis and liquefaction after deep white matter cerebral venous infarction in neonates can distinguish this entity from cerebral abscess and potentially avoid an unnecessary neurosurgical aspiration procedure. all four children were initially treated with aspirin but experienced recurrent events on therapy. all four were subsequently anticoagulated. two children have remained on warfarin for - years without recurrent events, while the other two had recurrent events despite adequate anticoagulation. these two children underwent uncomplicated coil embolization of the affected vertebral artery segment, and they have remained symptom-free for five and months since then. conclusions: dava was diagnosed by ca in / patients. all four children with dava in our series suffered recurrent strokes despite aspirin therapy. two of the four experienced further strokes on anticoagulation, necessitating endovascular therapy. these findings suggest that dava in children may require ca to diagnose, and that it may be refractory to standard adult therapies. ongoing multicenter efforts in childhood ais should further evaluate the diagnostic approach and recurrence risk of childhood dava. ) and cerebral gray matter abnormalities were present in ( %). posterior fossa lesions were seen on us in . % , but mastoid views were included in only % of the centrally read us. conclusions: in the largest extreme preterm cohort to date with near-term mri and serial us, % had mod-severe wma on brain mri, similar to previous reports. cerebellar abnormalities were detected more frequently by mri than by us. neurodevelopmental outcomes at - months and school age will assess the relative and combined values of mri and us as outcome predictors. , an analysis technique based on task-free resting state fmri recording, can be useful in assessing disruption of connectivity in certain disease states, including epilepsy. in healthy control subjects, functional connectivity reveals strong bilateral interhemispheric connectivity in such system as sensory-motor, visual, auditory as well as dorsal attention and default mode networks. in patients with epilepsy associated with unilateral diffuse hemispheric disease such data is limited. differences in the pattern of activation would suggest alteration in connectivity in these entities. this finding would impact the typical interpretation of this data that is becoming routinely collected for epilepsy pre-surgical evaluation. methods & materials: siemens (erlangen, germany) system, -tesla (trio) scanner was used for imaging (epibold sequence, te ms, flip angle °). resting state fmri scan were performed in both awake and anesthetized patient. awake patients were instructed to relax and rest while keeping their eyes open. analysis was performed using functional connectomes project scripts based on afni and fsl software packages. resting state data were analyzed for connectivity with the following seeds: somatomotor, visual, auditory, and default mode (posterior cingulated cortex (pcc)). results: we applied this technique to evaluate patients with hemispheric seizure disorders, including rasmussen's, neonatal infarct and migration disorders. all the subjects demonstrated some deviation from typical interhemispheric connectivity with a spectrum of findings. the figure below shows connectivity patterns in a patient with cortical dysplasia. while some interhemispheric connectivity remained in somatomotor (sm) and auditory (a ) systems, it was disrupted in visual (v ) and default mode (pcc) networks. variable patterns were found across the cases that corresponded to lesion side, supportive of disruption in interhemispheric connectivity as measured by fmri. conclusions: resting state functional connectivity patterns are well documented in healthy subjects. these results suggest that interhemispheric connectivity disruption is a typical feature of unilateral diffuse hemispheric disease though variable in presentation, either being limited to select systems or demonstrating broad disconnect between the two hemispheres. these results should be carefully considered when evaluating data for pre-surgical epilepsy evaluation. purpose or case report: premature birth is associated with white matter injury leading to a wide ventricular system. however,normative standards for ventricular size are lacking for this particular group.aims: we aimed to, in a controlled, population based norwegian cohort of ex-prematures without major handicaps, and for men and women separately,to ) create standards for radiological indices of ventricular dilatation, )investigate associations of these measurements with subjectively assessed ventricular size, ) examine differences in ventricular size between ex-prematures and healthy controls methods & materials: the initial birth cohort included neonates, birth weight below g (low birth weight)born within hordaland county, norway, between april st and august th . of eligible survivors (without major handicaps)underwent mr examination during the period january to may . of these were expremature (born before gestational age weeks) and were included in this sub-study. based on t weighted images, the ventricular size was subjectively judged as being normal, mildly, moderately or severely dilated by an experienced paediatric neuroradiologist, while objective measurements were performed in a blinded fashion, by a second observer (sma) using an imaging software program (nordic ice®). results: the normative standards for the ventricular system in ex-premature young adults showed wide variations, in particular for the occipital horns. the agreement between subjective and objective assessment of ventricular size was good. ex-prematures had smaller heads than those born term (control group). there was no difference in ventricular size between the two groups, even after adjusting for head size. ex-premature males had larger ventricles than females; however, the difference disappeared after adjusting for head size. conclusions: young adults born prematurely with a birth weight below g do not have larger lateral ventricles than healthy controls born term, even after correcting for a smaller head size. paper #: pa- best practice for reproducibility when measuring t *: implications for liver and cardiac iron assessment mark ferguson, md, radiology, seattle children's hospital, markferg@uw.edu; randolph otto, seth d. friedman purpose or case report: patients with red blood cell transfusion-dependent conditions receive high amounts of iron that can lead to abnormal iron accumulation in tissues resulting in organ damage. while the liver is the dominant excess iron storage organ, iron related cardiotoxicity is a leading cause of morbidity and mortality in patients with transfusion-dependent thalassemia. therefore, accurate determination and tracking of tissue iron levels in both the liver and the myocardium is important for patient prognostication as well as monitoring treatment changes. while multi-echo gradient echo mri (t *) is widely used and validated method employed for iron assessment, less attention has been given to derived metrics. specifically, the literature almost exclusively reports and uses the mean value for t * from a pixel-wise (pw) map. infrequently used is the median. the median is a potentially superior metric than the mean because it is insensitive to outliers. outliers will always occur in data because of either noise or imperfect vessel exclusion. to compare mean versus median on reproducibility of t * measurement, subjects who had paired heart/liver measurements were examined. the entire liver (excluding vessels) and the interventricular septum myocardium were traced on representative images from each series. mean and median t * values were generated from the pixel maps. r * ( / t *) and coefficient of variation (cv) were computed on a patient-by-patient basis. these measures were then summarized for the group. results: markedly higher r * values were observed in both heart and liver using median summary measures (liver: t − . , p . , heart: t − . , p . ). these findings were accompanied by lower cv's (better reproducibility) for the median approach (liver: t . , p . ; heart: t . , p . ). conclusions: the consistent difference in derived t * values between the methods (median>mean) should be considered when comparing derived r * values to established normal ranges. cv data support that using the median as the final summary metric will always outperform mean metrics for measuring change in r *. this finding has immediate implications for the scientific literature and for guiding therapeutic management over time. results: twelve children (age mo- yo; m:f : ) with gsds ( abca mutations, sp-c mutations, undefined mutation) and children (age wk- yo; m:f : ) with other dlds (including pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia of infancy, lymphocytic interstitial pneumonia, lipoid pneumonia, diffuse alveolar damage, granulomatous infection, capillaritis and other pulmonary hemorrhage syndromes) were identified. ct findings with highest sensitivity for gsds were ground glass attenuation ( %), parenchymal cysts ( %), and interstitial thickening ( %). parenchymal cysts, honeycombing, and pectus excavatum were more specific for gsds compared to other dlds (p< . ). the combination of either parenchymal cysts and honeycombing or ground glass attenuation and pectus excavatum provided the highest specificity ( %) but low sensitivity ( %). the combination of parenchymal cysts and ground glass attenuation provided good specificity ( %) and modest sensitivity ( %). no combination of findings provided both high sensitivity and specificity. conclusions: ground glass attenuation is the most sensitive finding for gsds, while parenchymal cysts, honeycombing, and pectus excavatum are more specific findings for gsds than other chronic dlds of childhood. however, no single finding or combination of findings on chest ct is both highly sensitive and specific for gsds, and chest ct cannot substitute for genetic testing or lung biopsy for the differentiation of gsds from other dlds. pes were observed in scans of patients without a history of congenital heart disease (chd), and pes in scans of patients with a history of chd. z-axis scan lengths for the chest ct exams ranged from . - . cm. a z-axis scan length of cm centered . cm below the carina captured all pes in all patients, and a length of cm centered . - cm below the carina in patients with chd. a z-axis scan length of cm centered cm below the carina was sufficient to capture at least one pe in all patients, and a length of cm centered - cm below the carina in patients with chd. the radiation effective dose of the chest ctpa exams ranged from - msv. limiting the z-axis scan length on ctpa exams to cm or cm would have resulted in a % or % decrease in z-axis coverage, respectively, and estimated radiation effective dose reduction of - % due to less radiation exposure to the intrathoracic structures, thyroid gland and upper abdominal viscera. conclusions: limiting the z-axis scan length coverage for ctpa exams based on a model of the typical anatomic distribution of pes relative to the reference level of the carina permits a substantial reduction of radiation dose in children without reducing the sensitivity for detection of pulmonary emboli. purpose or case report: to determine whether the addition of multiplanar reformation mdct images affects reader performance parameters and provides added diagnostic value compared to the use of axial ct mdct images alone for diagnosing pe in children. this was an institutional review board-approved retrospective study of consecutive pediatric patients who underwent ctpa for clinically suspected pe. two faculty pediatric radiologists and two radiology residents independently reviewed each study initially using only axial mdct images and later using mpr mdct images in any x-, y-, or z-axis for detecting pe. diagnostic accuracy, confidence level, and interpretation time of mpr mdct images were compared to axial mdct images using mcnemar's test and paired t-tests. the kappa coefficient was calculated to assess interobserver agreement. diagnostic accuracy was compared between faculty pediatric radiologists and radiology residents by logistic regression whereas confidence level, interpretation time, and added diagnostic value were evaluated with analysis of variance (anova). results: the final study cohort consisted of ctpa studies from children ( m/ f; mean age . years). nine ( %) of ctpa studies were found to have pe. diagnostic accuracy in correctly detecting pe ranged from . to % (mean . %), with no significant differences between the use of axial and mpr mdct images. logistic regression indicated no significant difference in diagnostic accuracy of detecting pe between faculty pediatric radiologists and radiology residents for axial mdct images (p . ) or mpr mdct images (p . ). confidence level and interobserver agreement were significantly higher and average interpretation time was longer in evaluating pe with mpr mdct images compared to axial mdct images for all reviewers (p<. ). compared to faculty pediatric radiologists, significantly greater increases in confidence level, interobserver agreement, interpretation time, and added diagnostic value using mpr mdct images compared to axial mdct images to diagnose pe were found for radiology residents (p< . ). conclusions: use of mpr mdct images in diagnosing pe on ctpa in children significantly increases confidence, interobserver agreement, and interpretation time among faculty pediatric radiologists and radiology residents. because mpr mdct images provide significantly greater improvements in reading parameters for residents than for faculty members, their routine use should be encouraged for trainees. paper #: pa- chest ct in children, anesthesia and atelectasis beverley newman, md, radiology, stanford university, bev.newman@stanford.edu; elliot krane, terry e. robinson purpose or case report: in spite of advances in ct equipment and speed, sedation/ anesthesia is required in many young children for optimal quality ct for detailed parenchymal evaluation; resultant atelectasis is a common and important quality issue. our purpose was to evaluate the safety and effectiveness of a standardized lung recruitment technique. methods & materials: with irb approval and parental informed consent, controlled ventilation, low dose, chest ct's (cooperative effort between anesthesia, pulmonology and radiology) were performed in children ( had - cts) ( f, m; ages . - . yrs, mean . yrs). indications included cystic fibrosis ; ciliary dyskinesia ; chronic or interstitial lung disease ; evaluate pulmonary metastases . ct parameters were - kvp, - mas, iv contrast . various prior methods employed by the pediatric anesthesiologists to maintain lung inflation had unpredictable results (a brief survey showed / nonintubated anesthetized cases had problematic atelectasis). a standardized intubation technique was therefore adopted: .use of a tight fitting face mask during induction and iv placement, inspiratory pressures of - and peep of . . introduction as early as possible using an appropriately sized cuffed endotracheal tube. . alveolar recruitment maneuvers- - s breaths to cm h o/ ( - in st cases). . three breaths at / , inspiratory breathold followed by - cm on th breath for scout and inspiratory scan, and complete ventilator disconnection for expiratory scan. recruitment breaths repeated before each scan. two experienced readers reviewed and scored the images on a point scale for overall quality and atelectasis. results: all studies were completed safely with no procedural complications. one child had propofol-related postoperative emergence delirium. all ct scans were diagnostically good to excellent with small subsegmental atelectasis in ( / were the initial cases with lower recruitment pressures) and segmental atelectasis in . cases had prior cts, without this technique, that were suboptimal due to moderate procedural atelectasis, in spite of tracheal intubation in the majority of cases. conclusions: an intubation lung recruitment technique can be performed safely and consistently by different individuals using a standardized protocol. procedural atelectasis that affects quality is reliably absent and repeat sequences are not needed. obtaining a high-quality dynamic airway imaging study is critical for accurate interpretation and subsequent medical decision-making. the ideal mri sleep study is one that allows successful completion while maintaining spontaneous breathing without artificial airway, which can be an anesthesia challenge. dexmedetomidine has been shown to have sedative properties paralleling natural sleep with minimal respiratory depression. we hypothesized that dexmedetomidine compared to propofol would have less effect on upper airway tone and airway collapsibility and provide favorable conditions with less airway interventions required during dynamic mri airway imaging in children with osa. in this prospective study, we examined the requirement for airway intervention for propofol ( - mcg/kg/m) and dexmedetomidine( - mcg/kg/h) in children and adolescents with osa. severity of osa was analyzed by overnight polysomnography. for children with history of mild osa there was no intervention unless oxygen saturation decreases below %; while for children with history of moderate/severe osa, an artificial airway was placed when oxygen saturation decreased below %. results: demographics and osa severity by polysomnography were comparable. requirement for artificial airway by severity of osa as documented by polysomnography will be shown. mri sleep studies required airway intervention in / ( %) children in the dexmedetomidine group versus / ( %) children in the propofol group. mri sleep studies were successfully completed without the use of artificial airways in children ( %) in the dexmedetomidine group versus children ( %) in the propofol group. conclusions: safe and effective anesthetic management is a key factor in obtaining good quality mr images of the airway. although there was no statistical significant difference in the need for airway intervention between drugs, dexmedetomidine provided acceptable sedation for mri sleep studies with less airway intervention in children with osa. dexmedetomidine may be the preferred agent for sedation during mri sleep studies in children, and may offer benefits to children with sleep disordered breathing requiring anesthesia or sedation for other diagnostic imaging studies. an open mouth and administration of cpap resulted in smaller ap diameter of the retroglossal airway compared to images without cpap due to cpap pressure pushing the tongue posteriorly. in patient volume of oral cavity anterior to the tongue increased from . ml to . ml. meanwhile, the ap diameter of the retroglossal airway decreased from . to . mm ( % decrease). in patient the mouth was initially closed but parted when the pressure of cpap was added with the oral volume increasing from . ml to . ml. the ap measure of the retroglossal airway decreased from . mm to . mm ( % decrease). in patient the mouth was then closed and cpap reapplied resulting in an ap measurement of . mm ( % increase). the ap diameter difference between cpap and no cpap were tested with paired t-test, but were not statistically significant (p . ). conclusions: positive airway pressure on a patient by full facemask and an open mouth can have an adverse effect on the retroglossal airway. this adverse effect is an important consideration in the use of positive airway pressure to support airways for osa, or during emergency resuscitation when a full facemask is used. paper #: pa- the purpose or case report: a nanoparticle blood pool iodinated contrast agent (nctx) has been designed and tested in preclinical animal models. we report data in animal models exemplifying its advantages over conventional contrast in the setting of ct pulmonary angiography methods & materials: nctx blood pool nanoparticles of nm diameter with an encapsulated total iodine concentration of~ mgi/ml were administered by intravenous injection to mice, rabbits, dogs, pigs and sheep. (these studies were actually conducted for other purposes and a review of the data revealed the similarities that motivated this paper.) total injected volumes were~ ml/kg in large animals, and as high as ml/kg in small animals to provide satisfactory vessel enhancement. iohexol or iopamidol was administered for comparative studies with conventional contrast. in a subset of pigs, iatrogenic pulmonary arterial emboli were introduced prior to contrast administration. toxicity studies were conducted in mice and monkeys. results: the visualization of pulmonary vessels using nctx blood pool nanoparticles was generally at least equivalent to using conventional contrast, and superior in several cases, particularly in small veins and when bolus timing of the conventional contrast was suboptimal. in all cases, satisfactory vessel enhancement was achieved for a duration of several hours following a single infusion of nctx blood pool nanoparticles. there was no evidence of renal toxicity, and only transient elevation of hepatic enzymes at relevant dose levels. conclusions: nctx nanoparticle blood pool agents demonstrate several advantages over conventional glomerularfiltered iodinated contrast agents for ct pulmonary angiography in animal models, including no nephrotoxicity, no dependence on bolus injection technique, superior depiction of small veins, and capability of re-imaging for follow-up studies without needing contrast re-injection. potential applications in human pediatric subjects include the diagnostic and post-therapeutic evaluation of cardiopulmonary anomalies and pulmonary embolism, especially in patients with renal insufficiency or tenuous vascular access. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- cardiovascular image quality using a nanoparticle ct contrast agent: preliminary studies in a pig model rajesh krishnamurthy, radiology, texas children's hospital, rxkrishn@texaschildrens.org; ketan ghaghada, prakash masand, abhay divekar, eric hoffman, ananth annapragada purpose or case report: image quality in a separate study using a long circulating, liposomal-based nanoscale blood pool iodinated contrast agent (nctx) suggests clinical utility in pediatrics, potentially reducing difficulties in contrast-ct of children with congenital heart disease (chd) including the size of intravenous cannula, need for accurate timing, inability to simultaneously opacify multiple targets of interest (requiring repeated contrast administration and/or repeated imaging). methods & materials: six pigs (average weight kg) were imaged after slow intravenous infusion of nctx ( mg i/ml) at an iodine dose of approximately mg i/ kg ( . ml/kg). retrospective ekg gated ct imaging was performed h later using a -slice dual-source ct scanner at and kvp. two radiologists analyzed and graded (on a -point scale with : unreadable, : excellent) images aimed at anatomic structures relevant to chd. quality of images obtained at and kvp were compared. uniformity of contrast opacification was measured using a roi-based ctnumber method at various intracardiac and extracardiac sites and mean non-uniformity was calculated. results: there was excellent agreement between the two readers on all counts at kvp. kvp images received lower scores for coronary morphology ( / ), and aortic valve visualization ( . / ), but were comparable in other aspects. pulmonary artery and pulmonary vein branch visualization extended up to the th generation in all cases. visualization of coronary artery branches was possible up to the second generation, with good arteriovenous separation. subtle morphologic features including crista terminalis, thebesian valve, foramen ovale, membranous septum, and chordae of the mitral valve were demonstrated in all cases. automated functional analysis and myocardial mass quantitation was feasible in all cases. there was no significant difference in blood pool attenuation between the atria, ventricles, and extracardiac vasculature on quantitative assessment. no image artifacts were visible on the reconstructed images. conclusions: these findings suggest that nctx promises to be superior to conventional contrast agents for ct imaging of complex congenital heart disease, due to the absence of nephrotoxicity, avoidance of repeated contrast administration, and reduced number of scans performed. avoiding the need for accurately timed scans precludes the need for large bore intravenous access. these attributes make it a promising agent that warrants further studies. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- theoretical cost and x-ray dose reduction in pediatric congenital heart disease imaging by the use of a nanoparticle contrast agent robert bell, the university of texas-houston; rajesh krishnamurthy, gabriela espinosa, christopher petit, ananth annapragada purpose or case report: the purpose of this study is to determine the effective, population averaged reduction in costs and radiation dose that can be achieved in the diagnosis of congenital heart disease by use of a nanoparticle long circulating blood pool contrast agent. methods & materials: a markov model of the decision tree followed at the texas children's hospital in the image based diagnosis of congenital heart disease was constructed in treeage software. the model included ct angiography, mr angiography, cardiac catheterization, and echocardiography diagnostic modalities. patient records, accumulated between and were examined to inform the model. the radiation dose and cost for each step were encoded as penalty functions. markov simulations were run for two decision trees: ( ) utilizing ct angiography and ( ) replacing conventional ct angiography with blood-pool agent based ct angiography. the overall population x-ray dose and accrued cost was calculated for each pass through the model. results: x-ray dose distributions for the example populations showed substantial reductions per ct study, as much as %. averaged over the population, since a sizeable fraction of patients are diagnosed without ever being exposed to any x-ray based modality, reductions were more modest, but still substantial. costs per ct study were slightly higher when the blood pool contrast agent was used. when the diagnostic probability using the blood pool agent increased, it led to an automatic overall cost reduction. conversely when the diagnostic probability remained unchanged, costs rose, commensurate with the increased cost of the contrast agent. conclusions: the use of a blood pool contrast agent for ct angiography leads to substantial reduction in radiation dose in the setting of congenital heart disease. cost reductions are more modest, and are driven almost completely by the reduction in the number of mr and invasive angiography procedures resulting from increased diagnostic success using blood pool based ct angiography. the model as constructed does not account for potential workflow changes that might result from the use of a new contrast agent. actual reductions realized may therefore be higher. disclosure: dr. annapragada has indicated that he is a stock holder and consultant for marval biosciences inc. paper #: pa- frequencies and patterns of situs discordance in chest and abdomen justin boe, stanford, justinj.boe@gmail.com; beverley newman, shreyas vasanawala, frandics chan purpose or case report: incidence of situs anomalies, including heterotaxy and situs inversus, is estimated at . % of population. as the first step in the segmental analysis of structural heart disease, the determination situs position is of fundamental importance. abdominal situs, as defined by splenic position and morphology, and cardiac situs, as defined by atrial morphology, are usually but not always in agreement. echocardiographers also employ the relative position of the great arteries and vein at the hiatus to determine cardiac situs. we evaluate the frequencies of discordances among abdominal, hiatal and cardiac situses. methods & materials: with retrospective irb approval, imaging records from to were reviewed for the diagnosis of cardiac situs inversus and heterotaxy. patients who had cardiac ct or mri were included. images were evaluated on a d-processing station by a cardiac radiologist. cardiac situs was determined by the morphology of the atrial appendages. when an atrial appendage was not adequately visualized, cardiac situs was assessed by the relative position of the main pulmonary artery and bronchi. hiatal situs was determined by the relative position of the aorta and the systemic venous return, and abdominal situs by the position and morphology of the spleen. results: thirty-five cases were identified, with cardiac ct and mri. patients' age ranged from day to years old. in the abdomen, the numbers of situs inversus, asplenia, and polysplenia were ( %), ( %), and ( %). for the heart, the numbers of situs solitus, inversus, rightisomerism, and left-isomerism were ( %), ( %), ( %) and ( %). the abdominal and cardiac situses were discordant in ( %) cases. polysplenia had the highest number of discordance with the heart. hiatal situs was discordant with the abdomen in cases ( %) and with the heart in ( %) cases. conclusions: situs disagreement between the abdomen and the heart is not uncommon and they should be documented separately in radiology reports. hiatal situs, as used by echocardiographer, disagrees with the cardiac situs in a quarter of the cases. it should be used with caution in the segmental analysis. paper #: pa- diminished asl intracranial perfusion in children with neurofibromatosis type kristen yeom, md, stanford university, kyeom@stanford. edu; cynthia campen, patrick barnes purpose or case report: neurofibromatosis type (nf ), a neuro-cutaneous syndrome affecting / children is associated with moyamoya syndrome (mms). however, no comparisons of cerebral perfusion in patients with nf and nf -associated mms to healthy controls exist. we hypothesize cerebral blood flow (cbf), as measured by magnetic resonance imaging (mri) arterial-spin-labeled (asl), is diminished in children with nf compared to healthy controls, with the lowest levels seen in patients with nf -associated mms. methods & materials: twenty children aged - years with nf , four with mms, and age-matched controls underwent asl cbf on a t magnet. pseudocontinuousspin-echo-asl technique was used. measurements were taken bilaterally in cerebral cortical-subcortical regions, and the deep gray nuclei. trends in measurements as a function of disease severity were tested with the jonckheere-terpstra test for ordered alternatives. a bonferroni-adjusted p-value less than . was considered significant. results: we identified / areas with significantly diminished asl cbf (ml/ g/min) in patients with nf (midrange), and nf -associated mms (lowest) compared to healthy controls (highest). these included the: thalami (left: p . , right: p . ); superior/middle temporal lobes (left: p . , right: p . ); temporooccipital lobes (left: p . , right: p . ); occipital poles (left: p . , right: p . ); centrum semiovale (left: p . , right: p . ); and left parietal lobe (p . ). conclusions: cerebral perfusion diminishes in a graded fashion in children with nf and nf -associated mms, particularly in the posterior circulation and the mca-pca posterior watershed zones. future studies may demonstrate an important role for asl in the presymptomatic diagnosis of cerebral vasculopathy, and the definition of nf -related vasculopathy patterns. paper #: pa- cingulate gyrus mri sign in pediatric nf patients: a novel imaging marker nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; nabila hai, rhea udyavar , amir noor, gilbert l. vezina, maria t. acosta purpose or case report: we observed a magnetic resonance imaging (mri) signal abnormality in the anterior cingulate gyrus of pediatric patients with neurofibromatosis type (nf ). the cingulate gyrus could play a role in cognitive deficits of nf patients. the first objective here is to document inter-rater reliability scores for visual detection of this sign. the second objective is comparing adc values of the cingulate gyrus in areas of visually abnormal mri signal in nf patients to matched normal mris to confirm a pathophysiological basis of the visual mri sign. methods & materials: retrospective analysis, irb approved, nf patients and matched controls. in the visual assessment part, two blinded neuroradiologists rated presence or absence of mri signal abnormality in the cingulate gyrus in three different age groups of nf patients mixed with normal controls. cohen's kappa inter-rater reliability coefficients were calculated. the same blinded neuroradiologists evaluated the cohort one year later, this time by agreement at the workstation. in the adc measurements part, two researchers, one blinded, manually placed roi's in the anterior and posterior cingulate regions of nf patients and their matched controls, and student t-test was used to assess for significance of differences in measured values. results: cohen's kappa for the three age groups showed very good agreement (kappa coefficients were either . or . ). rater agreement at the workstation was %. all subjects with a positive finding also had nf and the sign was not seen in any of the normal controls. the prevalence of the sign was %. adc measurements showed significantly higher adc values in the anterior cingulate gyrus of nf patients when compared to normal controls and also when compared to the posterior cingulate gyrus in nf patients. conclusions: our results show that visual t /flair mri abnormalities in the anterior cingulate gyrus are present in % of patients with nf from ages to years. adc measurements confirm a pathophysiological basis for this finding. future correlation with clinical manifestations, such as learning and behavioral manifestations in patients with nf , are under way to further evaluate the clinical importance of this finding. tract-based spatial statistical analysis of diffusion tensor imaging in pediatric patients with mitochondrial disease seth friedman, phd, seattle children's, seth.friedman@ seattlechildrens.org; andrew v. poliakov, sandra l. poliachik, dennis w. shaw purpose or case report: often diagnosed at birth or in early childhood, mitochondrial disease presents with a variety of clinical symptoms, particularly in organs and tissues that require high energetic demand such as brain, heart, liver, and skeletal muscles. in a group of pediatric patients identified to have complex i or i/iii deficits, but with white matter tissue appearing qualitatively normal for age, we hypothesized that quantitative dti analyses might unmask deficits in microstructural integrity. methods & materials: dti and structural mr brain imaging data were collected in pediatric patients with confirmed mitochondrial disease and clinical control subjects matched for age, gender, scanning parameters, and date of exam. paired tract-based spatial statistics (tbss) were performed to evaluate differences in fractional anisotropy (fa) and mean diffusivity (md). results: in patients with mitochondrial disease, significant widespread reductions in fa values were shown in white matter tracts. md values were significantly increased in patients, having a sparser distribution of affected regions compared to fa. results of tbss statistical analysis will be shown. to be shown in green is the mean fa skeleton which represents the centers of main white matter tracts. all results p<. . red and yellow represent a significant increase, blue and light blue represent a significant decrease. conclusions: despite qualitatively normal appearing white matter tissues, patients with confirmed mitochondrial disease have widespread microstructural changes measurable with quantitative dti. this supports the evaluation of such metrics in other populations where gross imaging features may be normal. to extend our studies to patients with other plp mutations, we analyzed the brains of male pmd patients (ranging in age from to ) and female carriers for whom the plp genotype had been determined and analyzed by mri. for each patient we measured, white matter volume (wmv) and the intercaudate distance (icd). the mri data were correlated with functional disability scores (fds) using a system we developed for clinical evaluation of pmd patients and which was validated by assessments of pmd patients. brain volume and segmentation were measured using nih image . . the average number of coronal slices analyzed from each patients mri was slices. when graywhite contrast was not adequate, then the intercaudate distance (icd) and intercaudate ratio were measured as described in caon et. al., ( ) . results: comparison of the mr measurements and the fds demonstrated that white matter volume inversely correlates with functional disability, suggesting that the initial disability does correlate with the extent of myelination. the intercaudate distance also correlated with the fds, and may usefully substitute when gray-white matter segmentation is not possible. conclusions: pmd is a clinically and genetically heterogeneous disease caused by mutations in the gene encoding the major cns myelin protein, proteolipid protein (plp). myelin is a major target of disease pathogenesis in most cases of pmd, but how the various mutations cause clinical disability is not fully understood. our data demonstrate that the extent of brain white matter atrophy, measured directly by volumetric fractionation, or indirectly by analyzing the intercaudate ratio, is significantly correlated with the patient's functional disability. white matter atrophy is thus the main cause of clinical disability in patients with pmd of all ages and mutation type. paper #: pa- maturational effects on language localization in children demonstrated by fmri susan palasis, md, children's healthcare of atlanta at scottish rite, spalasis@yahoo.com; binjian sun, laura l. hayes, richard a. jones purpose or case report: language localization is of paramount importance when contemplating surgery in children with intractable epilepsy or brain tumors. the potential risk of injury to language centers in the developing pediatric brain needs to be weighed against the potential benefits of surgery. in the past, language localization was crudely and invasively determined using the wada test. most institutions are now transitioning to non invasive localization using functional mri (fmri). the purpose of our study was to analyze language localization relative to age in children using age appropriate language paradigms and fmri. methods & materials: forty three healthy, english speaking, right handed children underwent fmri evaluation for language localization. the studies were performed on a t system. three novel age appropriate language block paradigms were utilized, targeted both to expressive and receptive language processing. these paradigms were the auditory category decision task (audcat), the auditory description decision task (addt), and the listening task. the spatial statistical maps generated by the fmri data were fused to the d anatomical mri dataset. language areas were localized and statistical analysis was performed with age as the variable in a general linear model. results: our results demonstrate a distinct trend in language localization and lateralization with brain maturation. in the young age groups (less than years) the localization tended to be less focused and bilateral in the frontal and temporal regions of the brain. in the older age groups (greater than years), language became more localized and lateralized to the expected left sided pattern. the findings were more robustly demonstrated with the addt task and were statistically significant (p< . ). conclusions: our study clearly demonstrates the plasticity of language centers in the maturing pediatric brain. this observation is significant for neurosurgical planning and rehabilitation in the pediatric population. ( ) no slc a mutations were found in , and subjects, respectively. significantly higher association with slc a mutations was found in bilateral eva+v/c dysplasia ( / ). double mutations of slc a is more often associated with combined eva+ v/c dysplasia, while a single mutation with eva only. cochlear aplasia without eva ( / ) and snhl with normal imaging ( / ) are less likely associated with slc a mutation. conclusions: slc a mutation is highly associated with eva and v/c dysplasia. once eva with or without v/c dysplasia are found at imaging, genetic investigation is recommended for slc a mutation because of possible thyroid involvement. moderate-severe hie who were randomized to cooling ( . °c for h). there were in the hypothermia group and in the control group. all mris were reviewed by a cental reader masked to the clinical findings, groupings, and outcomes. the mri findings were scored according to pattern and extent of injury, including involvement of the cerebral hemispheres, basal ganglia, thalami, internal capsules, and other structures. brain injury scores were correlated with death or disability at months postnatal age. results: no mri abnormalities were observed in of infants ( %) in the hypothermia group and in of infants ( %) in the control group (p . ). infants in the hypothermia group had fewer areas of injury ( %) as compared with the control group ( %, p . ). there were of the infants with death or disability at months. the brain injury score correlated with outcome of death or disability (p . ) and disability among survivors (p . ). conclusions: fewer areas of brain injury on mri were observed following whole-body hypothermia. the mri brain injury score is a marker of death or disability at months following hypothermia for term hie. . presence or absence of the "red dot" on fa color maps was correlated to clinical (ataxia, oculomotor abnormalities etc.) and morphological data, and to fa and md measurements. results: the "red dot" was absent in js and hgpps (genetic cross wiring impairment diseases) and present in coma and wvs (no reported gene abnormalities so far) as in normal controls. js and coma presented on mri molar tooth appearance. hgpps presented "split pons" appearance. js and coma patients presented oculomotor apraxia, wvs and hgpps palsy of the horizontal gaze. mirror movements were found in js and in wvs. ipsilateral responses are present in hgpps. wvs presented multiple cranial nerves impairment. in js, fa and md values of scp, pt and pc were significantly lower than in normal controls (p > . ). in hgpps high fa and low md were found in pc and pt (p > . ) and normal in scp. conclusions: the "red dot" absence is unrelated to morphological or clinical abnormalities. absence of the "red dot" is associated to abnormal measurements of fa and md in pc and pt( low in js and high in hgpps). these findings indicate a pivotal role for the pc in the physiopathology of these diseases. the "red dot" absence seems to be a marker of genetic cross wiring diseases. in this view, coma and wvs should not be considered as part of these diseases. sonographic predictors of intermittant testicular torsion in the pediatric patient jennifer williams, md, pediatric radiology, texas children's hospital, jlwilli @texaschildrens.org; marthe munden purpose or case report: intermittent testicular torsion (itt), defined as sudden onset unilateral scrotal pain with spontaneous resolution, is difficult to confirm both clinically and sonographically. the purpose of this study was to determine if sonographic predictors exist for diagnosing itt in the pediatric patient. methods & materials: a search of the pacs data system for patients presenting with suspected intermittent testicular torsion was performed. fifteen patients with a total of episodes presenting over a year period were found. a retrospective review of the medical records for clinical presentation, surgical outcome, and comorbidities was performed. scrotal ultrasound images and reports were reviewed for testicular size and echotexture, testicular flow, epididymal appearance, vascular bundle appearance, and presence of hydrocele. results: an abnormal appearance of the vascular bundle was found in % of episodes ( / ). initial absence of testicular flow followed by reperfusion during the scan was seen in % of episodes ( / ); % had increased flow ( / ), % had decreased flow ( / ), and % had normal flow ( / ) . nine of the patients had surgery; of these were found to have evidence of itt and was found to have acute testicular torsion. of patients with itt, % ( / ) had an abnormal vascular bundle. testicular flow was not initially visualized but returned during the exam in % of patients ( / ), was increased in % of patients ( / ) and was decreased in % patients ( / ). conclusions: itt is a difficult diagnosis. the most reliable sonographic indicator is an abnormal spermatic cord, found in % of episodes and % of surgically proven itt. dedicated views of the spermatic cord must be obtained in order to differentiate an abnormal epididymis from an engorged vascular bundle (the so-called pseudomass). attention to testicular flow is of particular importance. while visualization of a transition from no or decreased testicular flow to normal flow during the sonogram is certainly diagnostic of itt, increased testicular flow should not lead to false reassurance. purpose or case report: testicular torsion is a common acute condition in boys requiring prompt and accurate diagnosis. the objective was to evaluate ultrasound accuracy and findings, and clinical predictors in testicular torsion in boys presenting to the stollery pediatric emergency department (ed) with acute scrotal pain. methods & materials: retrospective review of us, surgical and ed records for boys aged month to years, presenting with acute scrotum from to , was performed. age, demographics, clinical symptoms, physical findings, us and surgical techniques, findings and diagnoses were recorded. surgical results and follow-up were used as the gold standard as all pediatric urology in our region is performed at our centre. results: patients presented to ed with acute scrotum with the following diagnoses: testicular torsion, possible torsion-detorsion, torsion of appendix testes, epididymo-orchitis, and other. of us performed, boys had torsion confirmed by surgery. there were inconclusive us reports, none of which had torsion at surgery or follow-up. the false positive rate of us was . % ( patients), and there were no false negatives. six torsion patients had no us. median time from ed to us and surgery for torsion patients was and min. six patients had non-salvageable testes. diagnostic accuracy of us compared to surgery was % for torsion and % for other. sonographic heterogeneity was seen in % of patients with testes that the surgeon felt were non-viable at surgery and % of patients with viable testes (p . ). sudden-onset scrotal pain ( %), abnormal position ( %) and absent cremasteric reflex ( %,) were most prevalent in torsion patients. conclusions: color doppler us is accurate and sensitive for diagnosis of torsion in the setting of acute scrotum. despite heterogeneity on pre-operative us, many testes were felt to be salvageable at surgery. rate of salvage of torsion was high. common symptoms and findings of torsion were sudden onset of pain, abnormal testicular position and absent cremasteric reflex. paper #: pa- diagnostic twists of tubal torsion srikala narayanan, md, children's national medical center, snarayan@childrensnational.org; anjum n. bandarkar, dorothy bulas purpose or case report: fallopian tube torsion is a rare cause of acute pelvic pain in a young female and requires prompt diagnosis for immediate surgical intervention. our purpose is to review varied imaging findings of surgically proven cases of tubal torsion. methods & materials: retrospective review of our data base from to revealed cases of surgically proven fallopian tube torsion. ages ranged from to years of age. all had pelvic ultrasound performed, cases had additional ct performed for acute pelvic pain. results: us findings included thickened dilated tubular hypoechoic structure ( ), cystic mass ( ); adnexal ( ), midline ( ). five cases had normal ovaries bilaterally ( with paratubal cysts). ct imaging findings include dilated, fluid filled, thickwalled tube with internal hyperdensity ( hu) likely debris/ hemorrhage in case. additional findings included cystic adnexal mass ( cases), beak sign ( case) and increased vascularity ( case). secondary signs included free fluid ( ), peritubular fat stranding ( ), vascular congestion and thickening of the broad ligament ( ) and enlarged draining vein ( ). laparoscopic salphingectomy was performed in cases (including cases with isolated tubal torsion). laparoscopic detorsion was performed in a total of cases. in addition, laparoscopic cyst drainage was performed in out of these cases. detorsion with paratubal cystectomy and hemorrhagic ovarian cystectomy was performed in of the cases. conclusions: imaging diagnosis of tubal torsion can be difficult. it can occur in isolation with a dilated thickened tubular structure adjacent to a normal ovary or potentially mimic appendicitis, pyosalpinx, complex adnexal cyst or cystic adnexal neoplasm. presence of normal ovaries, beaked tapered tubular structure with intratubal fluid level and hemorrhage may help in making the diagnosis. it is important to recognize this entity in a patient with acute pelvic pain to facilitate prompt tubal sparing surgery. paper #: pa- adjusted renal length in pediatric bone marrow transplant recipients nicholas bodmer, md, university of washington, nbodmer@gmail.com; teresa chapman, sangeeta hingorani, marguerite parisi purpose or case report: bilateral nephromegaly has been observed in the bone marrow transplant (bmt) patients at our institution. this study aims to quantify this observation, thereby providing radiologists with an adjusted baseline agedetermined renal growth curve for bmt patients. methods & materials: a retrospective clinical chart and imaging review was performed on patients who underwent bmt between and and who had abdominal imaging including the kidneys. ultrasound, ct, and mri exams were used for renal length measurement. renal lengths were assessed for each age group, first as an average length of all the patients within that age group overall, and subsequently as an average renal length by age group divided into the following time frames after transplantation: - days, - days, - days, and + days. clinic chart information collected included bun, creatinine, weight, and medication use. results: renal length was measured using imaging cases, distributed across each age group as follows: - months, n ; months- . years, n ; . - . years, n ; . - . years, n ; . - . years, n ; . years and higher, n . renal lengths were greater, on average, within every age group, compared with previously established normative age-related renal lengths (rosenbaum et al.) . the augmented renal lengths universally were observed in the - day post-transplantation timeframe. return to normal renal lengths typically occurred by months post transplant. clinic chart review revealed that the majority ( %) of patients received nephrotoxic medication within two weeks of imaging. conclusions: pediatric bmt patients have larger kidneys in the absence of known renal disease than age-matched peers. a revised, age-based renal length chart for post-bmt patients has been generated which should help prevent the misdiagnosis of nephromegaly in this population, eliminating unnecessary diagnostic evaluations. multiple etiologies to explain renal enlargement in these patients are possible, including fluid overload, nephrotoxic medication, or direct effect of the transplant. purpose or case report: mr urography can be a comprehensive exam for anatomical and functional pediatric renal evaluation. quantification of renal function may benefit when dynamic contrast enhanced images can be obtained at high spatiotemporal resolution and with minimal respiratory motion artifacts. though respiratory triggering may decrease motion artifacts, it results in loss of temporal resolution by a factor of about three. a two-echo gradient echo sequence with segmented outer k-space sampling and view-sharing/dixon image reconstruction (disco, differential subsampling with cartesian ordering) was chosen as a starting point due to its high temporal resolution. it was then modified to enable respiratory triggering while maintaining temporal resolution of one temporal frame every one to two respirations, with segments of k-space only acquired in the expiratory phase of respiration. imaging parameters were: °flip angle, ± khz bandwidth, tr~ . , matrix x , fov - cm, slice thickness mm, and x spatial acceleration. with irb approval and informed patient consent consecutive patients referred for mri renal function evaluation were recruited (age range; . to . years, mean±sd: . ± . years; males % females %), and scanned on a ge t mr using a -channel torso array with the respiratory-triggered high spatiotemporal resolution technique to extract regional gfr maps. two readers by consensus assessed image qualitative snr, motion artifacts and volumetric fat-water suppression performance. results: data acquisition was obtained to completion in all subjects without triggering failure. temporal resolution was approximately s for two respiratory cycles. no case had major fat suppression failure, whereas minor fat suppression failure was seen in % ( % c.i. to %). all cases had diagnostically acceptable snr. no motion artifacts were noted in / cases, while some artifacts with ghosting in / cases. regional gfr maps could be successfully extracted for each patient without the need for image registration. attached figure shows image quality. conclusions: view-sharing offsets loss of temporal resolution from respiratory triggering. thus, high spatiotemporal resolution renal dynamic contrast enhanced respiratory triggered images can be obtained with minimal motion artifacts in a pediatric clinical setting to evaluate renal function. disclosure: dr. chowdhury has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. methods & materials: the study cohort was selected from the irb approved children's oncology group aren b study. cases are evaluated for pre-operative wt rupture based on central review of surgical/pathology findings. wt cases with rupture were matched to wt controls by age and tumor weight (within months and g). ct scans were independently reviewed by blinded radiologists, for presence/absence of rupture and the following ct signs: poorly circumscribed mass, perinephric fat stranding, peritumoral fat planes obscured, retroperitoneal fluid, ascites beyond cul-de-sac, peritoneal implants, ipsilateral pleural effusion, intratumor hemorrhage. sensitivity, specificity of ct for assessing pre-operative wt rupture was determined. the relationship between ct signs and rupture was assessed by mcnemar's test, and the most predictive ct signs determined by backward selection multivariate logistic regression. results: sensitivity, specificity for detecting wt rupture were: reviewer - . %, . %, reviewer - . %, . %. kappa coefficient for interobserver agreement was substantial: . (p< . ). all ct signs tested, except peritoneal implants and intratumoral hemorrhage, had significant association with tumor rupture (p< . ). for reviewer , ascites and fat stranding around tumor were most predictive (odds ratio . and . , p< . ). for reviewer , ascites and retroperitoneal fluid were most predictive (or . and . , p< . ). conclusions: ct has high specificity but relatively low sensitivity for detecting preoperative wt rupture. the presence of ascites beyond cul-de-sac is the best indicator of preoperative rupture, followed by fat stranding and retroperitoneal fluid. paper #: pa- the failed pyeloplasty: evaluation with mr urography damien grattan-smith, children's healthcare of atlanta, damien.grattansmith@mac.com; ricahrd jones, stephen little, wolfgang cerwinka, hal scherz, andrew kirsch purpose or case report: to identify imaging characteristics associated with failed pyeloplasty seen with mr urography. we have performed mr urography in children following pyeloplasty. from this group, children had follow-up surgical intervention with repeat pyeloplasty or balloon dilatation of the upj. imaging features reviewed included degree of hydronephrosis, calyceal transit times, renal transit times, signal intensity versus time curves, as well as functional analysis based on volumetric and patlak differential function and change in the asymmetry index. results: all children who underwent a second surgical procedure had delayed calyceal transit times. the degree of hydronephrosis and renal transit times were either stable or worse when compared to pre-operative evaluation. functional derangement could show stability, slight improvement or deterioration. the asymmetry index estimated the severity of the obstruction. conclusions: mr urography is valuable in the evaluation of children who have undergone pyeloplasty. the calyceal transit time appears to be the most reliable discriminator when comparing successful and failed pyeloplasty. calyceal transit times may be prolonged before the hydronephrosis becomes progressive. disclosure: dr. grattan-smith has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: initial attempts at interpreting functional mr urography (fmru) can be challenging. a time intensive navigation through a multitude of both subjective and objective functional results is necessary to render a useful interpretation. this is a guided review of fmru, noting the important functional findings in high-grade unilateral pelvicalyceal dilatation (pcd), in the absence of ureterectasis, with a contralateral normal kidney allowing for an optimal functional comparison. methods & materials: a retrospective functional evaluation of cases with unilateral pelvicalyceal dilatation (pcd), without prior pyeloplasty, was conducted. the fmru studies were carried out according to a standard protocol and post-processing using the chop-fmru software. this included iv hydration, bladder catheterization and iv furosemide administration. fifteen minutes after diuretic administration, a dynamic coronal d fat saturated t sequence was performed in a supine position over min. a sagittal d t and delayed single coronal t , both fat saturated, followed in a supine and/or prone position. the following functional features were evaluated: visualization of the ureter, the presence of a contrast-urine level and swirling of contrast in the dilated renal pelvis. the functional results included in the analysis were calyceal transit time (ctt), renal transit time (rtt), time-to-peak (ttp), parenchymal volume (pv), differential renal functions (volumetric-vdrf, patlak-pdrf and volumetric patlak-vpdrf) and the difference between vdrf and pdrf. results: patients were comprised of males and females with an age range of . - . years (median . yrs). of the kidneys with pcd, the ureter was visualized in , during the dynamic sequence, / during supine delay and / only in prone position. a contrast-urine level was present in of the dilated systems, and swirling in . the ureter was visualized during dynamic sequence in all contralateral normal kidneys and at no time was swirling or a contrast-urine level identified. the average functional parameters are seen in table . a statistically significant (p< . ) difference between the normal and dilated pelvicalyceal systems was achieved in ttp, pdrf and vpdrf for this small sample size. conclusions: awareness of multiple functional features and the range of calculated results may aid in subsequent combined interpretation of the fmru with the morphologic analysis. disclosure: dr. lecompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. and . demographics, clinical presentation , diagnostic studies and treatment outcomes were evaluated. post-procedure imaging was evaluated for clot burden reduction (patency) and residual venous stenosis by two-reader consensus. results: ten patients ( male; female, mean age years, range - ) presenting with acute upper extremity swelling and pre-procedure imaging revealing % occlusion of the axillary and subclavian veins received successful endovascular therapy. all patients underwent infusion catheter placement for thrombolysis with tissue plasminogen activator or urokinase. patients received additional pharmacomechanical treatment. angioplasty was also performed in all patients. the mean treatment duration was h (range - ). post-procedural imaging revealed that of patients achieved - % patency (clot burden reduction) and patient achieved - % patency. the residual venous stenosis was graded: patients had - % stenosis, patients had - % stenosis and patients had - % stenosis. all patients were discharged on full anticoagulation therapy with low molecular weight heparin. patients had surgical rib resection postthrombolysis with an average length of time from thrombolytic therapy to surgery being days (range - ). patients had re-thrombosis events during the follow-up period (mean months; range - ), with one re-thrombosis event occurring within one week of thrombolytic therapy, prior to surgery and the other two occurring - weeks post-rib resection. there were no procedure related complications. one patient was lost to follow-up after initial successful catheter directed therapy. conclusions: percutaneous endovascular techniques such as pharmacomechanical thrombolysis and angioplasty appear to be feasible and safe options for paget schroetter syndrome in otherwise healthy adolescent patients. in attempt to prevent rethrombosis and chronic symptoms, we refer all patients for adjunctive surgical decompression. future larger studies are needed to address optimal strategies for these patients. combined d fluoroscopy image guided percutaneous intervention with real-time optical sensing at the tip of a needle for tissue characterization rami nachabe, philips, rami.nachabe@philips.com; john m. racadio, drazenko babic, ross schierling, jasmine hales, benno hendriks purpose or case report: to investigate the feasibility and potential of real-time tissue characterization at the tip of a needle with diffuse optical spectroscopy (dos) sensing capabilities during d fluoroscopy guidance using cone beam ct and dedicated needle path planning software. methods & materials: a c-arm x-ray system that combines fluoroscopy and d imaging from a cone beam ct was used to image a woodchuck with hepatocellular carcinoma (hcc). the imaging system enabled needle path planning, which was used to perform insertion and navigation of a needle toward the liver tumor. the needle was integrated with optical fibers for real-time tissue spectral sensing at its tip. optical spectra measurements were obtained continuously as the needle passed through healthy liver tissue and then into the tumor. from the diffuse optical spectra measurements, the following clinical parameters were extracted for tissue characterization: blood volume fraction, blood oxygenation, lipid volume fraction and tissue light scattering (related to tissue density). the tissue parameters were compared for healthy liver and tumor using the kruskal-wallis test. results: the tissue density of the healthy liver was lower than that of the tumor. higher blood and lipid volume fractions as well as oxygenation levels were observed in the healthy liver as compared to the tumor. all differences were statistically significant (p< . ). additionally, a much wider heterogeneity in tissue density was observed in the tumor as opposed to the healthy liver. conclusions: differences in tissue properties between tumor and healthy liver enable discrimination between these two types of tissues. adding real-time optical sensing at the tip of a needle to d fluoroscopy image guidance is a feasible technique that complements the imaging information with relevant physiological parameters; it facilitates more precise definition of tumor boundaries despite any target motion during needle insertion. disclosure: dr. racadio has disclosed that he is a consultant for philips healthcare and receives travel reimbursement. rami nachabe, drazenko babic and benno hendriks are employees of philips healthcare. methods & materials: two children aged months and months were treated at this institution for liver failure resulting from urea cycle disorders, with a hepatocyte transplant procedure. the recipient liver was irradiated prior to transplant to facilitate engraftment. the procedure involves the injection of prepared hepatocytes from a suitably screened, compatible donor, via a main portal vein branch into the recipient liver. in both procedures access to the umbilical vein was achieved by the surgery service and a french arterial sheath was placed. a french angled catheter was used for diagnostic runs and to access the right and left main portal vein. a french fogarty catheter (edwards lifesciences, irvine, ca) was placed to isolate each portal vein branch in turn and hepatocytes injected using hand injections. pressures in the main, right and left portal veins were measured and hand injections of contrast made at regular intervals. careful attention must be paid for evidence of pruning of portal branches, indicating occlusion of small portal branches, or portal to hepatic vein shunting. if shunting is seen, infusion must be stopped as embolism of hepatocytes into pulmonary arteries may result with serious clinical sequelae results: in both patients, the desired number of hepatocytes were successfully delivered into the recipient liver. in both cases, mild pruning of the portal vein branches was evident at the end of the procedure. portal vein presssures remained steady. there was no venographic or clinical evidence of pulmonary arterial embolization. conclusions: the interventional radiologist plays a central role in the hepatocyte transplant procedure. familiarity with catheterizing portal branches from an umbilical vein approach, measuring venous pressures, using small occlusion catheters and recognizing venographic end points such as portal vein pruning and portal to hepatic vein shunting are necessary to the safe and successful completion of this new technique. purpose or case report: the aim of the study was to evaluate the trends in term of type of tube placed, number of procedures per year, number and age of the patients as well as the number of procedures per patient and the interval of time between two placements, and finally the irradiation burden borne by the patients. methods & materials: after reb approval the radiologic files of the patients who underwent naso-duodenal-jejunal (ndj) or gastro-jejunal (gj) or jejunal (j) tube placement under fluoroscopy over the past five years ( to ) were extracted from the ris and reviewed. the results were tabulated as a single batch and stratified by year. results: eighty-nine patients representing procedures ( ndj, , gj, j) were included. only patients underwent a single procedure. the average number of procedures per patient was . with a maximum of during the study period. the average patient's age was . months (sd . , median . ). the average fluoro time per procedure was . min (sd . , median . ). the average interval between two procedures was days (sd , , median ). the average fluoroscopy time per patient combining those having a single procedure and those having multiple ones, was . min (range . to . , sd . , median . ). conclusions: fluoroscopic placement of enteric tubes delivers a significant amount of irradiation. our data led to two interventions with respect to insertion and management of the tubes. on one hand, when the attempt pursued by a radiologist is not successful after min of fluoroscopy other strategies should be considered including another operator or an alternative technique for tube positioning. on the other hand, information will be distributed toward the clinicians and nurses in order to improve the management of these tubes and avoid fortuitous displacement which was responsible of a significant amount of repeated procedures leading to undue irradiation. purpose or case report: to evaluate drug elution pharmacodynamics of doxycycline in an albumin-based solution, as used in percutaneous imaging-directed therapy of aneurysmal bone cyst (abc) and microcystic lymphatic malformation (lm) methods & materials: doxycycline mixed with % human serum albumin (hsa), and doxycycline mixed with saline solution (both mg/ml) were evaluated using a fluid diffusion chamber system over h, recording ph and doxycycline concentration. static ph and doxycycline concentrations were recorded every min for the first min, then every min for a total of h, averaged over trials in each of the hsa and saline systems. statistical analysis evaluated standard deviation and rate of change for the trials in each system. drug elution dynamics data were correlated with clinical experience in the doxycycline/albumin treatment of patients ( treatments) with aneurysmal bone cyst (abc) and patients with lymphatic malformation microcysts. results: drug elution was linear in both the hsa and saline systems, with statistically significant (p<. ) slower elution drug release from the albumin system as compared with the doxycycline and saline solution, both over and h. purpose or case report: to describe a successful interventional radiologic approach to the management of paget schroetter syndrome presenting as acute arm swelling in adolecent athletes. methods & materials: institutional review board approval was obtained for this retrospective study. five patients aged to years (mean . years) were treated at this institution over a year period all presenting with acute arm swelling (july -july ). ultrasound confirmed subclavian vein thrombosis in all cases. all were treated with placement of an infusion catheter (ev , plymouth, mn), infusion of tissue plasminogen activator (tpa) at a rate of mg/hour overnight and aspiration of remaining clot with a "trellis" (bacchus vascular, santa clara, ca, usa) thrombectomy device. results: clot was successfully removed in all five patients. complete clearance of clot was confirmed by contrarst venography in all cases. in four patients balloon angioplasty of a narrrowing at the junction of the subclavian and brachiocephalic veins was carried out. in one, the thrombus recurred within h. the patient was retreated the next day with aspiration of clot using the "trellis" device and an infusion catheter placed with low dose ( . mgs/hour) tpa commenced until surgical review; this patient was operated on within h of final thrombolysis. all patients were seen by a vascular surgeon with an interest in this condition. all underwent surgical decompression; at end of the study period all patients were asymptomatic. conclusions: interventional radiologic management of acute axillo-subclavian thrombosis due to paget schroetter syndrome is safe and highly successful in the adolescent population. early recurrence of thrombus is not uncommon and prompt surgical consultation with a view to early surgical decompression is recommended. purpose or case report: diagnostic reference levels (drl) or target radiation dose ranges for pediatric ct scans are needed in the u.s. the first u.s. pediatric ct dose index registry (quircc) within the american college of radiology recorded estimates of patient radiation dose using a new method (ssde) based on body width(bw) for the purpose of developing diagnostic reference levels (drl). in addition to developing drl at the th percentile, the purpose of this study was to determine the ssdes associated with the lower range of acceptable image quality through subjective image quality evaluation. methods & materials: six children's hospitals participated in a retrospective review of abdominal ct with iv contrast on patients < yrs of age. from exams, each site submitted de-identified images for selected cases based on ssde and patient width. a total of cases were selected from the lowest, first quartile and median ssde. six investigators reviewed images from each case under identical viewing conditions and rated them for subjective quality according to a score sheet and reference scale of images with known quantum mottle. cases were considered non-diagnostic if at least of reviewers ranked them as such. results: first, second, and third quartile ssde and ctdi-vol values from sites for each bw will be shown. / cases were ranked non-diagnostic by the reviewers. / non-diagnostic cases were below the th percentile based on ssde. / of "non-diagnostic" cases had ssde less than the th percentile. the unacceptable case with ssde above the th percentile ( cm, ssde . mgy) was due to subcutaneous metal implant with artifact. the quircc th percentile using ctdivol for a yr old is . mgy which is % lower than the acr ct accreditation data's published th percentile. conclusions: this consortium developed target dose ranges (drls) for ct of the abdomen with iv contrast for routine exam indications based on evaluation of image quality that establish lower and upper ranges ( - percentile) of patient dose(using ssde) associated with clinically acceptable images. this study demonstrates that pediatric radiologists in this consortium are comfortable interpreting images at or above the percentile ssde and judged all but one image within this target range as diagnostically acceptable. table ). with the exception of neonate chest, most used age-based techniques; only two centers reported using thickness. no survey used grids for wrist images, while / of the surveys used grids for chest and abdomen exams in -year-olds. at the most common sid there was up to a kvp variation ( year-old chest ap) and up to -fold variation in mas ( year old scoli lat). only two surveys used equipment that displayed the new iec exposure index. conclusions: participants report variability in the techniques and methods used to acquire common radiographic studies, reflecting differences between detector types and users. radiologists, technologists, medical physicists, manufacturers, and the fda have an opportunity to work together to standardize the techniques based on detector type to optimize radiation exposure for pediatric radiographic exams. disclosure: dr. don has indicated that he performs contract research for carestream and that he is on the speaker's bureau for siemens and receives an honoraria. purpose or case report: this study assesses community adoption of ct radiation dose guidelines after a -year international initiative to reduce medical radiation exposure in children. size-specific dose estimates (ssde) from community pediatric body ct scans are compared to ssde from matched scans obtained at a children's hospital that adheres to image gently campaign principles. we reviewed pediatric ct scans ( chest (c), abdomen/pelvis (ap), chest/abdomen/ pelvis (cap)) transferred from community imaging centers to our university children's hospital between july and february . community scans were acquired with variable parameters and reconstructed with traditional filtered back projection (fbp). comparison was made to children's hospital ct scans, performed in accordance with principles of the image gently campaign. because iterative reconstruction (ir) software was added to our scanner during the study, enabling us to reduce ctdivol by %, children's hospital scans were divided into two groups: a) scans obtained with standard weightbased pediatric protocols and fbp (october -october ; c, ap, cap) and b) scans obtained with reduced-dose weight-based pediatric protocols and blended ir/ fbp (october -april ; c, ap, cap). ctdivol and greatest lateral dimension were recorded from each scan and were used to calculate ssde. mean ssde from community scans was compared to mean ssde from children's hospital groups a and b. statistical analysis was performed with student's t-test. results: patient age range was - years in both community and children's hospital groups. mean ssde for community c, ap, and cap scans was . , . , and . times higher than mean ssde for matched scans in control group a (p< . ) and . , . , and . times higher than mean ssde for matched scans in control group b (p< . ). conclusions: ssde was significantly higher for community pediatric body ct scans than for matched scans performed at a children's hospital that adheres to image gently campaign principles. results suggest that more community outreach and education are required in implementation of low-dose ct protocols outside of children's hospitals. concurrent use of ir provides a means of achieving even greater ssde reduction than is possible with fbp alone and should be encouraged. paper #: pa- optimization of tube voltage and current in size-based pediatric ct imaging: a phantom study boaz karmazyn, md, radiology, riley hospital for children, bkarmazy@iupui.edu; yun liang, keith kaser, peter johnson, mervyn cohen purpose or case report: determine the change in ct dose index (ctdivol) required to maintain the same quantum mottle noise when using lower tube voltages ( and kvp) relative to kvp in different sized cylinder water phantoms (cwp) representing a wide range of pediatric body sizes. we performed mdct scans of , , , and cm cwp. thirty scans were performed for each phantom. the tube currents ranged from to mas with increments of mas, and the tube voltage levels were , , and kvp. the noise (standard deviation in hu) was measured using center region of interest (roi) that was % of phantom's area. two other rois (each % of the area) were placed at the center and periphery of the phantom images to measure noise gradient. results: in the smallest ( cm) cwp, approximately the same noise level was maintained with all three tube voltages without a significant change in ctdivol. for the , , and cm phantoms, the average ctdivol needed to be increased by %, %, and %, respectively, to maintain same noise level when the voltage was decreased from to kvp. the average ctdivol needed to be increased by %, % and % to maintain the same noise level in the , , and cm cwp when the tube voltage was decreased from to kvp. the difference between central and peripheral noise increased on average by . %, . %, . %, and . % in the cwp of , , , and cm, respectively. in each cwp, the central to peripheral noise difference was more pronounced (up to . % more) with decrease in kvp from to or . conclusions: noise measurements in the water phantom model indicate that tube voltage could be decreased from to in cwp of cm without significant change in ctdivol. it is also possible to decrease the voltage from to kvp with a minimal (< average %) increase in dose in cwps of , , and cm. the noise gradient increases with larger cwp and smaller kvp. paper #: pa- comparison of radiation dose estimates, image noise, and scan duration in pediatric body imaging using -row and -row ct jennifer johnston, md, radiology, cincinnati children's hospital medical center, jhtai@yahoo.com; daniel j. podberesky, erin angels, terry t. yoshizumi, greta toncheva, donald p. frush purpose or case report: to compare effective dose (ed) estimates, image noise, and scan duration for pediatric chest, abdomen and pelvis protocols using -row and -row ct scanners in various acquisition modes. methods & materials: organ doses were measured using mosfet dosimeters. dose, scan duration, and noise measurements were made in a -year-old anthropomorphic phantom for conventional helical, -detector helical and volume acquisition modes for chest, abdomen and pelvis protocols on a -row ct, and for helical mode on a row ct (aquilion one and aquilion , toshiba medical systems, otawara, japan) using similar scan parameters representing currently used clinical protocols. mean organ doses from three runs for each protocol, in combination with icrp tissue weighting factors, were used to obtain ed for each protocol. noise was measured as the standard deviation of hounsfield units in equivalent locations at levels for each protocol with an roi tool. ed and noise were compared with a paired t-test or sign test. results: compared to helical acquisitions on the -row ct, ed of all tested acquisition modes on the -row volume ct were significantly lower for chest, abdomen/pelvis (ap) and chest/abdomen/pelvis (cap) protocols (table) . scan durations were lower across the board on the -row volume ct. compared to acquisitions on the -row ct, noise was in general similar to those on -row ct protocols, but some acquisition protocols on the -row ct produced greater noise (table) , specifically volume acquisition for chest ct and -detector helical and volume modes for ap and cap protocols. conclusions: dose savings can be achieved for chest, ap and cap ct examinations on a -row ct scanner compared to helical acquisition on a -row ct, with shorter scan durations. image noise was in general comparable between protocols. although noise differences between some modes did reach statistical significance, the impact on overall image quality will need to be studied further. paper #: pa- the observed to expected total fetal lung volume as a predictor of short-and long-term morbidity in surviving infants with congenital diaphragmatic hernia emily stenhouse, the royal hospital for sick children, emilysten@doctors.org.uk; neil patel, judith simpson, watt andrew, gregor walker, carl davis purpose or case report: observed-to-expected total fetal lung volume (o:e tflv) is a validated mr measure which we have previously demonstrated to be significantly reduced in non-surviving infants with congenital diaphragmatic hernia (cdh). our aim was to investigate the relationship between o:e tflv and short-and long-term morbidity outcomes in surviving infants with cdh. methods & materials: a retrospective analysis of cases of isolated left-side cdh referred to our institution for fetal mr evaluation between - weeks. mr imaging studies were performed on a . t philips system using a phased array body coil. the observed tflv was calculated by multiplying the summed area of the region of interest by the section thickness. the expected tflv was calculated with a formula previously described in the literature using the gestational age of the fetus. the observed tflv was expressed as a percentage of the expected tflv at a given gestation. morbidity outcome data was obtained from the case records of all surviving infants. specific measures of illness severity relating to short-term intensive care management and long-term outpatient management were recorded. differences in o:e tflv between outcome groups were assessed by t-test. results: liveborn infants with isolated left-side cdh and antenatal mr scans were identified. scans were performed at - weeks gestation. infants survived to discharge; gestation . ( . - ) weeks, birth weight . ( . - . ) kg. median length of admission was ( - ) days, median duration of follow-up was . ( . - . ) years. o:e tflv was significantly lower in non-surviving infants; vs. %, p . . o:e tflv was significantly lower in infants who received high frequency oscillation ventilation (hfov) versus those who were conventionally ventilated ( % vs %, p . ). o:e tflv was also significantly lower in those infants who had a length of admission greater than the median of days ( % vs. %, p . ). o:e tflv trended lower with other measures of increased morbidity; inhaled nitric oxide use, patch repair of diaphragm, rehospitalisation within year, supplemental feeding at discharge, gastro-oesophageal reflux, and developmental delay. conclusions: as well as predicting survival, lung volume measurement by o:e tflv is a promising predictor of outcome and morbidity in surviving infants with cdh. further studies in larger populations are required to provide quantitative predictive risk data. characterization of the inherent acoustic noise of a dedicated nicu mri system jean tkach, phd, cincinnati children's hospital medical center, jean.tkach@cchmc.org; yu li, ron g. pratt, christopher villa, beth m. kline-fath, charles dumoulin purpose or case report: we have developed a small foot print . t mri scanner specifically for neonatal imaging that can be easily installed in a neonatal intensive care unit (nicu). the scanner has a maximum patient bore diameter of . cm (without rf coil), and roughly twice the gradient performance of the best conventional adult whole-body . t mr systems. it is known that sensory stimulation such as acoustic noise can elicit autonomic instability in both term and preterm neonates. the inherent noise properties of the nicu mri system were measured as part of the initial safety evaluation of the system and compared against that of a conventional . t mri system. to evaluate the inherent acoustic noise characteristics of the nicu mri scanner, sound pressure level (spl) measurements were performed on it and on a conventional adult sized whole body . t hdx ge mri system (ge healthcare, waukesha, wi). a brüel & kjaer model sound level meter (brüel & kjaer sound & vibration measurement a/s, denmark) was used to perform the spl measurements for several different mr acquisitions (spin echo, gradient echo, fast rf spoiled gradient echo, fully balanced steady state free precession, gradient echo echo planar, and diffusion weighted) using acquisition parameters consistent with clinical protocols. the mr sequences, acquisition parameters, noise measurement equipment and methodology were identical for the two mr systems. the maximum spl in units of a weighted decibels (dba) was recorded for each of the mr acquisition/mr system combinations evaluated. results: the maximum spl values measured during each of the mr acquisitions were lower for all sequences (average . dba (range - dba)) for the nicu mri unit as compared to the conventional mri scanner ( table ). the average measured maximum spl value, reported in dba, across all acquisitions was . ± . for the nicu scanner, and . ± . for the conventional mri scanner. the highest spl values were measured for the diffusion-weighted sequence: and dba, for the nicu and conventional mri scanner respectively. conclusions: because of the smaller dimensions of the gradient coils in the nicu mri system, acoustic noise is less than that of conventional mri scanners despite the superior gradient performance of the smaller coils. the lower inherent acoustic noise level of the nicu system provides improved safety for the neonate, and facilitates siting of the unit in the nicu. disclosure: dr. tkach has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. paper #: pa- late neurologic events in extremely premature infants carlos guevara, md, radiology, duke university, cjg @duke.edu; brett bartz, caroline l. hollingsworth, caroline w. carrico, michael c. cotten, charles m. maxfield purpose or case report: germinal matrix hemorrhage (gmh) is a major complication of prematurity. persistence of germinal matrix and immature neurovascular autonomic regulation in the premature neonate is thought to predispose to gmh. most gmh in premature population occurs during the first days of life, and yet the persistence of the germinal matrix to weeks gestation may allow for post-natal gmh outside of the immediate perinatal period. to our knowledge, this is the first systematic review of late gmh (after the first week of life) in a large population of extremely preterm neonates (less than weeks of gestation). this irb approved retrospective review included patients weighing less than g or born at less than weeks of gestation from through . the study population included infants who had a head ultrasound (hus) within the first week of life and at least one follow hus after the first week of life. all hus were reviewed by three experienced pediatric radiologists for the presence and grade of ich or late developing hemorrhagelike lesions (hll). infants with and without hll were evaluated for several clinical variables, including neurodevelopmental outcomes (bayley scales). results: average gestational age of study population was . weeks. the incidence of gmh in the first week of life was % grade , . % grade , . % grade / , and . % posterior fossa. new echogenic foci (hll) at the caudothalamic groove were seen in . % after the first week of life. % of these lesions were bilateral. a four-fold increase in incidence of hll was seen in infants < g compared to those> g. higher grade hemorrhages were not seen in this patient population, although % of infants had late posterior fossa hemorrhages. the clinical course of infants with hll trended towards a higher incidence of stressors, but this was not statistically significant. the psychomotor development index scores were lower than those infants without hemorrhage. conclusions: small hll at the caudothalamic groove are common in extremely preterm infants after the first week of life. higher grade ( - ) hemorrhages were not seen. there were no cases of intraventricular extension and no direct complications. if isolated, this finding necessitates no follow-up imaging, but may be associated with poor neurodevelopmental outcome. disclosure: dr. guevara has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: tof/apv is a rare congenital heart lesion in which pulmonary arteries may become aneurysmally dilated and compress adjacent airways. pulmonary arterioplasty is often required to relieve tracheobronchial compression in addition to intracardiac repair. the purpose of this study was to review pre and postnatal imaging findings and their impact on patient management and clinical course. methods & materials: a retrospective database search identified infants with tof/apv between - ( fetal diagnosed cases and diagnosed postnatally). for fdc, prenatal ultrasound (us) and fetal mri were correlated with postnatal ct for the size of the central pulmonary arteries, airway compression, and presence / distribution of air trapping/atelectasis. for all cases postnatal ct findings (between - days of age) were correlated with clinical management and outcome. results: prenatal diagnosis of tof/apv was suggested sonographically, based on dilated central pas, between - weeks gestational age (ga). fetal mri, performed between - weeks ga confirmed the diagnosis and aneurysmal central pas and demonstrated air trapping &/or atelectasis in / with normal appearing lungs in fetus. size of the pas ( / ) and presence and distribution of lung abnormality ( / ) correlated closely between fetal mri and postnatal ct, although detailed visualization of the central airway/ vascular relationships were better defined on ct. fetal mri identified an unexpected diaphragmatic hernia (dh) not seen on us. for the pnd cases, ct showed aneurysmal pas and airway compression with air trapping &/or atelectasis in / infants. seven infants with airway obstruction on ct required pulmonary arterioplasty; infant with no air trapping did not have arterioplasty. / operative patients survived, one with concomitant dh died at age days due to hemorrhagic shock. one fdc was inoperable due to poor cardiac function and died at age days. conclusions: prenatal mri correlates well with postnatal ct for assessing pulmonary artery size and location and severity of lung abnormality in patients with tof/apv, this allows for appropriate management planning and may negate the need for an immediate postnatal ct. ct accurately depicts the location and extent of airway compression and resultant air trapping or atelectasis, serving to guide the need for and extent of the arterioplasty procedure. paper #: pa- craniosynostosis syndromes: prenatal findings by us and mri eva rubio, md, cnmc, rubioeva@yahoo.com; anna blask, alexia egloff, dorothy bulas purpose or case report: craniosynostosis with associated malformations is a feature of several related syndromes resulting from a fgfr or twist genetic mutation. syndromes include apert, crouzon, pfeiffer, and carpenter syndromes. our purpose was to review imaging findings which aid in suggesting the diagnosis prenatally. we retrospectively reviewed prenatal us and mri findings in cases with prenatal ( with postnatal/molecular) diagnosis of a craniosynostosis syndrome: cases of apert, case of carpenter, and cases of pfeiffer syndrome. results: / cases were correctly diagnosed prenatally. in the second trimester findings may be subtle, with mild calvarial changes; digit abnormalities, in particular, may elude the imager in unsuspected cases. although the diagnosis could be made with either modality, the full spectrum of abnormalities was best appreciated using a combined imaging approach of mri and us. by us many salient features were depicted: turribrachycephaly/trigonocephaly/cloverleaf ( / ); syndactyly ( / ); polydactyly ( / ). agenesis of the corpus callosum was identified by us in ( / ) cases. conversely, mri, performed in all cases, contributed additional observations not well seen by us: the fetal airway was well delineated in all cases ( / ); a low lying spinal cord was noted ( / ), midface hypoplasia ( / ) and migrational/sulcation abnormality ( / ). additional findings of absent ductus venosus with biliary atresia ( / ), abdominal wall defect ( / ) and renal anomalies ( / ) were seen with both modalities. reimaging in later pregnancy depicted important changes ( / ), including worsening hydrocephalus and resolution of suspected airway occlusion. conclusions: us and mri are complementary modalities in evaluating fetuses with craniosynostosis. airway patency, midface hypoplasia, spinal cord abnormalities and intracranial abnormalities are often better seen with mri. fetal activity, digits, bone detail, and cardiac anomalies are better appreciated by us. findings may be subtle in the second trimester. repeat imaging in later pregnancy may reveal specific information affecting delivery planning. paper #: pa- pcpra best paper hyperpolarized carbon- mrsi for pediatric disease john mackenzie, md, department of radiology and biomedical imaging, ucsf, john.mackenzie@ucsf.edu; yi-fen yen, linda nguyen, jeffrey gu, john kurhanewicz purpose or case report: to study the potential of carbon- mr spectroscopic imaging ( c-mrsi)-a radiation free molecular imaging strategy-for the detection and treatment monitoring of pediatric disease. methods & materials: the potential of c-mrsi to detect pediatric disease was tested in rodent models of pediatric arthritis. animals were induced with arthritis and subsequently given intravenous hyperpolarized c-pyruvate, and imaged. the amount of c-lactate produced from pyruvate in normal and arthritic joints was measured both at single points in time and dynamically at either or tesla. the c-mrsi data were compared with clinical measures of arthritis, cell stimulation studies, and joint changes on conventional anatomic mri and histology. results: alterations in lactate production as measured by c-mrsi appear to depict sites of arthritis and correlate with other more established but potentially less reliable or more invasive measures of disease status. imaging robust mouse models of pediatric disease may be feasible at telsa. this method may also be translated from high-field to clinical equipment with reasonable hardware and software modifications that allow detection of hyperpolarized c compounds. c-mrsi depicts increased lactate production at specific regions of inflammation within arthritic joints and is confirmed by histological inspection and anatomic mri. on average, lactate production is increased by % in areas affected by inflammation. conclusions: the intravenous injection of hyperpolarized carbon- compounds and subsequent imaging with c-mrsi provides a unique molecular imaging strategy to noninvasively monitor pediatric disease. this non-invasive imaging strategy may eventually provide clinical utility for several pediatric diseases involving inflammation, infection and tumor. disclosure: dr. mackenzie has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. methods & materials: using the hemangioma-vascular malformation clinic registry at cincinnati children's hospital, we searched for patients diagnosed with khe whose evaluation included mri. twenty such patients were found, although three of the patients had no pre-therapy mris. the imaging studies were reviewed by the authors with assessment of the following characteristics: location, margin definition, soft tissue involvement, and pre and post contrast signal intensity. results: location: lesion location was as follows: trunk ( ), extremity ( ), extremity plus trunk ( ), and head/neck ( ). signal: all lesions were dark on t weighted sequences with diffuse enhancement after contrast administration. the majority of the lesions were bright on t weighted sequences, but there were cases that had heterogenous to low t signal (with all involving the retroperitoneum). of the cases, only one had both high arterial and venous flow by mri. margin definition: four of the lesions had well defined borders (greater than % well circumscribed) with minimal to no adjacent infiltration/edema. two of those four cases were exophytic masses. the remaining cases were poorly defined lesions with adjacent infiltrative fluid signal intensity and enhancement. tissue/organ involvement: tissue/organ involvement was counted if abnormal fluid-signal intensity or enhancement was identified at that site. review of these cases showed fifteen patients with muscular involvement. dermal and subcutaneous involvement was observed in all but cases, with the uninvolved lesions being isolated and deep. additional sites of suspected involvement included bone ( ), pleura ( ), penis ( ), and pancreas ( ). conclusions: khe is a rare neoplasm of infancy with a spectrum of features by mri. poorly defined lesions are much more frequent than well-circumscribed masses. however, pathologic correlation of such infiltrative margins is usually not available as treatments after biopsy are primarily medical rather than surgical. common additional mri features include predominant involvement of muscle, subcutaneous fat, and skin over viscera and bone with lesions generally showing increased t signal and enhancement. is dedicated chest ct needed in addition to pet ct for evaluation of pediatric oncology patients? ibrahim tuna, montefiore medical center, dristuna@yahoo. com; jeffrey levsky, jeremy rosenblum, rosanna ricafort, benjamin taragin purpose or case report: to evaluate the diagnostic accuracy of low dose ct performed during pet-ct as compared to dedicated chest ct in the assessment of pulmonary findings in children with malignancy. the institutional review board approved this hipaa compliant research. pediatric oncology patients, ages between - , with known solid malignant tumors who were referred to pet-ct and standard chest ct within days for staging or assessment of treatment response between - and - were eligible for this retrospective study. radiology reports were reviewed for potential discrepancies. two radiologists re-evaluated the standard chest ct and low dose chest ct portion of the pet ct of the discordant cases, while comparing with the most recent prior studies. studies were scored for pulmonary nodules, bony metastasis, adenopathy, and pleural effusions. true discrepancies were assessed by a panel of pediatric oncologists to judge whether the differences in reports might lead to a significant change in management. results: ( female, male) patients were identified. radiologic reports of different patients ( female, male) had potential discrepancies based on review of the reports. the primary tumors were rhabdomyosarcoma (n ), hodgkin's lymphoma (n ) and others (n ). reevaluation of the original images showed true discrepancies in . % ( / total ). in studies, the discrepancy had no clinical significance. in studies, a pulmonary nodule was identified on standard chest ct which was not described on the pet-ct. both of these patients had rhabdomyosarcoma. one of these patients had findings that pediatric oncologists considered significant enough to alter patient management. conclusions: we found a low false negative rate for clinically significant findings on the low dose portion of pet-ct as compared to standard chest ct. in the future, improvements in acquisition technique and post processing of the ct portion of the pet-ct may further improve its diagnostic utility, obviating the need for a routine separate diagnostic ct, thereby minimizing radiation exposure in these young patients. methods & materials: low-dose cta examinations were performed in pediatric patients over a three year period to evaluate suspected vascular traumatic injury with some patients receiving scans of more than one area of the body. areas scanned in this include the head and/or neck (n ), chest (n ), abdomen and/or pelvis (n ), upper extremity (n ) and lower extremity (n ). in of these patients, suspected vascular injury was due to a history of either blunt (n ) or penetrating (n ) trauma. patients were referred directly from the emergency department, while were inpatients and the remaining were referred from an outpatient setting. patients ( f: m) ranged in age from to years old (mean age ). studies were performed on a -channel mdct scanner with or kv, to mas, . to . mm section thickness, reconstructed with % overlap, and . to . pitch. contrast medium was power-injected using weight-based protocols to optimize iodine delivery. exams were interpreted on a workstation using advanced imaging techniques. patient radiation dose was calculated in all cases. clinical outcome was assessed through a month follow-up when possible. results: all studies were technically adequate. . % (n ) of studies revealed no vascular injury, while . % (n ) revealed acute vascular pathology. vascular injuries included vascular occlusion (n ), vasospasm (n ), narrowing/dissection (n ), pseudoaneurysm (n ), and transection (n ). extravascular traumatic findings were demonstrated in . % (n ), including fractures, lung injury, soft tissue hematomas, and a ruptured baker's cyst. of the patients with acute vascular findings, . % (n ) underwent surgical management (including for vascular injury), while . % (n ) were managed conservatively. one patient with active extravasation was managed with angiographically-guided embolization. in no case was catheter angiography required to confirm cta findings. conclusions: low dose cta is a reliable means to screen pediatric patients emergently for acute vascular injury. vascular and non-vascular pathology can be diagnosed noninvasively for efficient patient management. paper #: pa- elasticity measurement by acoustic radiation force impulse (arfi) technique of normal liver, kidney and spleen in healthy children mi-jung lee, radiology, severance children's hospital, mjl @yumc.yonsei.ac.kr; myung-joon kim purpose or case report: there are many previous studies about using acoustic radiation foce impulse (arfi) value to measure the elasticity of tissue, mainly the liver in adult patients. however, there was limited study about arfi measurement in the children. the purpose of this study is to evaluate the arfi value in the normal liver, kidney and spleen in healthy children and to evaluate the effect of sex, age, and body mass index (bmi). the study prospectively enrolled healthy pediatric volunteers who are under years old, and underwent abdominal ultrasonography and arfi between july and august . arfi velocity measuring was performed by - mhz linear probe for children under years old and - mhz convex probe for older children. arfi velocity was measured three times at each organ. however this measurement was stopped if the child cannot tolerate. results: two hundred two children (m:f : ; mean age, ± . years) were enrolled. and arfi measurement was performed only two time for some organs in three children. the mean arfi value was . ± . m/s in liver, . ± . m/s in right kidney, . ± . m/s in left kidney, and . ± . m/s in spleen. arfi velocity was not different between boys and girls. however, arfi velocity was different between right and left kidneys (p . ). the arfi value of right kidney, left kidney and spleen was correlated with age, height, weight and bmi (p< . ). however, the arfi value of liver was not correlated with these parameters. conclusions: arfi measurement is feasible in children with only three times acquisition for each abdominal organ. the mean arfi velocity was increased according to the age, height, weight and bmi in kidney and spleen, but it was constant in liver. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: diagnostic image quality can be achieved over a wide range of radiation exposure in digital radiography. "exposure factor creep" or "dose creep'" in which technologists tend to increase dose to avoid the appearance of noise has been well described. using the alara principle, acceptable images can be achieved while minimizing dose. at our institution "dose creep" has been observed in bedside pediatric chest radiography. to address this we coupled a data mining tool with a continuous quality improvement (cqi) initiative which educates individual technologists on appropriate technique. methods & materials: radiation dose in digital radiography is estimated from an exposure index, a proprietary format that varies among manufacturers. our institution uses a fuji computed radiography system which calculates an s, or sensitivity value, that provides an approximation of the radiation dose to the imaging plate, using an inverse scale. overexposed bedside chest radiographs were defined by a s value less than . a data-mining program was developed to extract from the dicom header the s value and other relevant information, on a monthly basis. these data were used to provide training and feedback on a one-on-one-basis. results: with ad hoc feedback and group training initiatives prior to implementation of this new system, approximately . % ( / ) of bedside chest radiographs were overexposed over a four month period. after one-on-one intervention with the technologists, preliminary findings reveal a trend towards fewer overexposed radiographs with approximately . % ( / ) with s< . conclusions: our tool provides a simple method for systematically identifying overexposed radiographs and the corresponding responsible technologists. we anticipate that this personalized educational program will continue to reduce the proportion of overexposed radiographs and thus the radiation dose to our pediatric patients. purpose or case report: ensuring radiation protection for children undergoing ct scans is challenging due to rapidly changing technology, differences in ct equipment and potential lack of understanding of unique aspects of scanning children. the joint commission has named technologists' training as an "action" item. we developed online training modules to fill potential gaps in ct technologists' education. methods & materials: four modules were created by pediatric radiologists, radiologic technologists and medical physicists; were developed by education/training experts from major ct vendors (ge, philips, toshiba, siemens) through the medical imaging technology alliance. modules were created as microsoft word documents containing de-identified images and edited by education specialists at the american society of radiologic technologists and the alliance for radiation safety in pediatric imaging. they were converted to audio/video format using question/answer narration. vendor modules were created in microsoft powerpoint format and edited. all modules were converted into adobe captivate learning program to achieve uniformity of appearance. modules are hosted on the asrt server and linked to the image gently website. a certificate may be printed as documentation of completion. results: all modules are available at www.imagegently.org. two introductory modules discuss basics of ct equipment and medical physics related to radiation dose in children. the third and fourth modules discuss dose-saving strategies for neu-roct and body ct. four vendor-produced modules address unique aspects of equipment design such as automatic exposure control and dose saving strategies for children. conclusions: through collaborative efforts with medical imaging professionals and vendors, we have developed free online modules addressing radiation protection for children. ct technologist training in specific dose saving strategies for children is variable and limited. these modules have the potential to improve ct technologists' understanding of equipment. end confusion which focused attention on improving communication with patients and families. there is little research regarding health literacy (hl) in radiology. the purpose of our study was to determine if an educational intervention (brochure) improves hl for parents whose child will undergo a fluoroscopic study. methods & materials: an education exemption was obtained from the irb. a multidisciplinary team developed brochures for fluoroscopic procedures. participants were randomly selected and asked to complete a survey to assess their knowledge of the procedure and use of radiation both before and after reading a brochure. a final survey to rate and gain feedback about the brochure was completed. results: median age of children whose parents participated (n ) was years. vcug was most commonly performed ( %). prior to the brochure, % of participants knew the name of the test their child was having. after the brochure, % knew the name (p < . ). prior to the brochure, % felt informed about the test, whereas % felt informed after (p<. ). test scores showed an improvement in parent knowledge about the procedure with a median increase of points after the brochure (scale of - ; p<. ). even after reading the brochure, % of parents wanted more information. prior to the brochure, % of parents knew the test involved radiation compared to % afterwards (p<. ). parents improved their understanding of the relative amount of radiation compared to background from % before to % after the brochure (p<. ). overall, % rated the brochure > on a -point scale with % rating the brochure (p<. ). written feedback was uniformly excellent. conclusions: improving hl for parents is part of the mission of radiology medical professionals. our study demonstrates that there is room for improvement in communicating with parents about fluoroscopy. straightforward information for parents provided as a brochure improves their understanding of radiologic fluoroscopic procedures. paper #: pa- compendium of resources for radiation safety in medical imaging anum minhas, duke university, anum.minhas@duke.edu; donald frush purpose or case report: diagnostic imaging, including ionizing radiation modalities, maintains a foremost role in evaluation of medical disorders. there is increasing awareness and need for information across varied sectors about low level radiation and potential risks. many medical/scientific organizations have resources discussing radiation risk and management. however, there is no one resource compiling the same available information. methods & materials: websites, including those of national and international medical organizations (e.g., acr, "image gently" alliance, iaea) were reviewed for information on radiation dose, risk, justification, optimization, guidelines (which included general information about improvement in quality and dose reduction without specific mention of optimization techniques), appropriateness criteria, and general principles of radiation safety for radiography, fluoroscopy/angiography, and ct. this information was divided by modalities and separated into adult and pediatric populations. information from organizations that were not arbitrarily considered to be national (e.g., subspecialty society, regional organization, individual institution/practice) was not reviewed. the resources were then organized into tables, organized by modality. websites with training modules were noted as well. results: websites were explored. overall, less information is available about medical radiation safety in children compared to adults. across both, most information is available on ct, then fluoroscopy, and finally radiography. across all groups and modalities, there is no information available for patients/parents on optimization, appropriateness, or guidelines, with the exception of adult radiography where there were some guidelines. conclusions: this compendium on medical imaging radiation serves as a collective resource for communities including the public and regulatory organizations. additionally, the compendium can be used to determine redundant or deficient areas, providing opportunities for more comprehensive and efficient efforts in medical radiation protection for patients. inappropriate and cloned histories in children: how big a problem is it? leann linam, md, radiology, uams/ach, llinam@uams. edu; chetan c. shah, s bruce greenberg purpose or case report: acr standards require appropriate clinical history for obtaining imaging examinations. cloning clinical histories is a federal violation. our purpose is to determine the frequency of inappropriate histories (ih) and/or cloning histories (ch) at a tertiary children's hospital. methods & materials: three pediatric moc radiologists reviewed clinical histories for radiographs obtained at a tertiary children's hospital on randomly selected dates ( weekdays and weekend day) for appropriateness and cloning. appropriate histories have associated icd- codes. cloning is defined by identical clinical histories occurring on consecutive days and could be clinically appropriate or inappropriate. only the first patient radiograph on a day was included. χ testing was performed to determine significant differences. results: % ( / ) of exams had ih. ih were significantly more common in inpatients than outpatients (p< . ). nicu examinations accounted for % of all ih and were significantly more frequent than other inpatient locations (p . ). the cvicu examinations accounted for % of all ih and was the second most common patient location for ih, but not significantly different from other inpatient locations (p . ). the increased frequency in ih on the weekend reflects a change in patient mix with fewer outpatient examinations performed than on weekdays and was not significant (p . ). the most common ih included: evaluate ett or evaluate lungs ( each). cloning only occurs in inpatients and was combined with ih in % of patients with ch. the nicu accounted % of ch which was significantly greater than other inpatient locations (p . ). conclusions: in radiographs had ih which can lead to misdiagnoses or nonpayment by insurance companies. inpatients, especially the nicu were the most common patient locations. cloning was also a common problem and was frequently combined with ih. identifying the extent of ih allows for corrective educational measures to be instituted which should improve compliance with existing medical and legal standards for ordering radiographs. paper #: in vivo validation of size-specific dose estimates (ssde) through breast entrance skin dosimetry (esd) during pediatric chest ct angiography sjirk westra, md, radiology, massachusetts general hospital, swestra@partners.org; xinhua li, mannudeep kalra, bob liu, suhny abbara purpose or case report: ssde is a new ct dose measure that corrects scanner console ct dose index (ctdi) for cross-sectional body diameter, being a better estimate of absorbed dose in individual patients of varying body size. ssde has been developed through phantom studies and computer simulations of ct dose, but has not yet been validated in vivo. the purpose of our study was to determine correlation between ssde and measured breast entrance skin dose (esd) for pediatric chest cta across a variety of scanning techniques, scanner models and patient sizes. methods & materials: our study was irb-approved, with waiver of written informed consent. during consecutive chest cta exams done on different scanners over a period of years, we measured mid-sternal esd as an approximation of breast dose with skin dosimeters, which was also expressed as mammogram equivalents. for each scan, we recorded patient age, weight, effective ma, kvp, console ctdivol- cm and dlp- cm (from which we calculated age-adjusted effective dose (ed)). we measured effective chest diameter Ø to convert ctdi to ssde, and we correlated ssde with measured breast esd, using linear regression. we evaluated image quality with regard to answering the clinical question. (table) , due to systematic introduction of automatic exposure control, low kv and high pitch scanning techniques. all studies were of diagnostic image quality to address the clinical question. conclusions: ssde is a valid measure of ct dose in pediatric patients undergoing chest cta over a wide range of scanner platforms, techniques, and patient sizes, and may be used to model breast and other organ dose, and to document results of dose reduction strategies over time. purpose or case report: the purpose of this project was to create an automated system capable of quantifying slice-byslice ct image quality and radiation dose data based on patient size. the information generated from this system should enable size-specific optimization of ct scan parameters in order to obtain images of diagnostic quality at the lowest possible radiation doses. methods & materials: a mathematical model was developed to predict ct image noise based on kvp, effective mas, and water-equivalent diameter of the patient. a conical water phantom was used to calibrate the model on multiple scanners and accounting for different operational modes and scan parameters, including tube voltage (kvp), tube current (effective mas), bowtie filter, and focal spot size. a software application was created to process image data from the scout topogram and incorporate dicom metadata from the axial images. a database and data viewing application were developed to display individual and aggregate study data. all of these systems were integrated and automated to enable real-time monitoring of image quality and radiation dose as a function of patient size. results: since the completion of the automated system, ct exams have been processed. a search application allows the user to find an individual study or a collection of studies based on parameters such as body part imaged or study protocol. the viewing application displays slice-by-slice patient diameter, radiation dose, and image quality for each study. radiation dose estimates are adjusted for patient size, yielding size-specific dose estimates. the application also graphs individual study data compared to those of comparative studies that are included in the search. conclusions: we have successfully developed an automated system that monitors ct image quality and radiation dose data based on patient size. the system enables simultaneous real-time monitoring of all studies performed on all ct scanners at our institution. specifically, the system enables size-specific radiation dose estimates at every scan level. this system will be used to guide protocol adjustments in order to optimize ct image quality and thus optimize radiation dose. disclosure: dr. larson has disclosed that he has a patent application in process through cchmc for ct radiation dose reduction. purpose or case report: at many institutions, ct scan parameters for children are determined by patient age or weight. aapm task group recommends cross sectional body dimension, such as patient width to determine size specific dose estimates. the purpose of our study was to develop prediction models of body width based on patient age and weight and compare these models with actual measured body widths for children undergoing body ct. methods & materials: children's hospitals participated in a -month retrospective review of abdominal ct scans on patients < years of age after local irb approval. recorded values included patient width(cm) from an axial image at the level of the splenic vein, patient age (yrs) and patient weight (lbs). a regression model for predicting patient width as a function of age and weight was determined. results: exams, had all measurements. both age and weight were significant predictors of patient width (p<. ). there was also a significant interaction between weight and age (p<. ), indicating that the relationship between patient width and weight depended on the age of the patient. the r for the regression model for predicting patient width from age and weight individually were . and . respectively. the r for the model including both age and weight and their interaction was . leaving % of the variation unexplained. the regression equation for this model is: patient width . + . x age(yrs)+ . x weight(lbs)- . x age x weight. despite the r of . for the model using both age and weight, the average error (rmse) for predicting patient width compared to a direct measurement of width was . cm. the plot of observed minus predicted values (residuals) versus predicted values indicates that the best model (combination of weight and age) results in measurable errors of predicted patient width relative to direct measurement. conclusions: a combination of both patient age and weight results in a more accurate patient width prediction than using age or weight alone. while age and weight can be used to predict body width, this is not sufficiently accurate for generating ct protocols. therefore, direct measurement of body width form either physical measurement on the patient or from the scout view or an axial image is preferred to select appropriate scan parameters for pediatric abdominal ct. paper #: pa- automated size-adjusted dose monitoring for pediatric ct dosimetry olav christianson, clinical imaging physics group, olav. christianson@duke.edu; ehsan samei, donald frush purpose or case report: the potential health risks associated with low levels of ionizing radiation have created a movement in the radiology community to minimize radiation dose during ct imaging; this is especially important for pediatric patients due to their increased sensitivity to radiation. it is thus essential to accurately assess the risks to pediatric patients undergoing ct imaging. current efforts to monitor radiation dose, however, are limited because they do not account for differences in risk from ionizing radiation due to variability in patient size, age, and gender. in this context, we developed an automated size-adjusted dose monitoring program capable of performing patient-specific risk estimation to facilitate protocol optimization. methods & materials: dicom routing software was used to send dose reports and scout images to an image repository on a dosimetry server. optical character recognition was used to extract dose-relevant data from dose reports; patient size was determined from corresponding scout images. based on anatomical location, risk estimation conversion coefficients (qfactors) were determined for each series in the dose reports. the q-factors were adjusted according to patient size, age, and gender and then multiplied by the dlp to estimate the risk to each patient. this process was applied to the cohort of pediatric patients undergoing ct examination at our institution. to evaluate the impact of including patient size, age, and gender, risk estimates were obtained excluding and including the dependencies on size, age, and gender. the results were computed in units of cancer incidence per cases exposed (cpt). results: the average patient-generic risk estimate for a pilot group of patients undergoing body ct was . ± . cpt. by including patient size, the risk estimate was increased to . cpt± . cpt. by including patient age and gender, the average risk estimate was further increased to . cpt± . cpt. conclusions: we developed a new size-adjusted dose monitoring program for pediatric ct dosimetry. comparisons between patient-generic and our new patient-specific risk estimates show that failure to consider patient size, age, and gender resulted in risk estimates that were too low by a factor of seven. additionally, the increase in standard deviation we observed demonstrates that our method of including patient size, age, and gender is sensitive to the inherent variability in the patient population. disclosure: dr. christianson has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: treatment of prenatally diagnosed lung masses is controversial, with many specialists recommending elective surgical removal in the first year of life because of a reported or perceived increased risk of infection and malignancy, while other centers recommend a conservative approach to management. the natural history of unresected lung masses is not clear. in our center, our standard recommendation is prophylactic resection of asymptomatic lesions, although not all families choose this option. we asked whether respiratory morbidity increased during the time prior to elective resection of prenatally diagnosed lung masses. methods & materials: ninety-eight pregnant women carrying fetuses with chest masses were imaged by ultrasound (us) and magnetic resonance imaging (mri). medical records of the liveborn infants were retrospectively reviewed. results: fetal diagnosis of a lung mass was made at a mean of weeks gestation (range - wks). intrauterine fetal demise was documented in pregnancies. there was one elective termination of pregnancy. three infants were lost to follow up. thus, outcomes were available for children ( % m, % f) with prenatally diagnosed lung masses. significant respiratory morbidity (rm) was defined as the occurrence of pneumonia, asthma, chronic coughing or wheezing, or respiratory symptoms severe enough to require an emergency room visit or hospitalization. of the children who had surgical removal of their lung mass, ( %) had rm prior to surgery. fifteen out of children did not have surgery but have been followed expectantly, and of ( %) developed some form of rm. fifteen of ( %) infants had immediate and significant rm (tachypnea, grunting, increased work of breathing, increased oxygen requirements or need for intubation) in the newborn period leading to urgent surgery (range of age at surgery: - d; mean . d). of the initially asymptomatic infants, ( %) developed rm prior to elective removal of the mass (range - weeks, mean weeks). of the lesions removed, histology revealed: cystic adenomatoid malformation (ccam) %, ccam + sequestration %, sequestration %, congenital lobar emphysema (cle) %, ccam+cle %, other %. conclusions: the risk of respiratory morbidity appears to be increased during the time prior to elective resection of prenatally diagnosed lung masses, which may be important for parents and pediatric specialists to consider when deciding whether to remove an initially asymptomatic lung mass. purpose or case report: it is now accepted that fetal mri with its superior tissue resolution can be very helpful in clarifying anomalies detected during obstetrical ultrasound. this is particularly the case with intracranial abnormalities, although indications are expanding. the current english medical literature, though, appears to be focused on evolving mri techniques and how mri compares to ultrasound with regards to image quality and detection of additional findings which may alter the diagnosis. however, we found no study specifically evaluating the clinical relevance and impact of the information obtained by fetal mri to the specialists who counsel and treat these patients. a "satisfaction and clinical impact" survey was created and sent to all the members of our fetal diagnosis and treatment group, asking specifically how the clinicians rated their satisfaction with this type of imaging, its influence on their counseling and on various clinical decisions, both prenatal and postnatal. results: we received responses from specialists in different clinical disciplines. the greatest number of respondents came from our obstetricians ( %), many of whom perform their own ultrasounds, and from members of our medical geneticists/genetic counselors ( %), although % of respondents were from various other clinical disciplines, both medical and surgical. there was a surprisingly high degree of satisfaction overall with the quality of the images and with the type and amount of information provided. most respondents indicated they felt fetal mri was "moderately" or "extremely" useful for their particular clinical decisions, and most respondents agreed that fetal mri impacted "moderately" or "significantly" on counseling and management of these pregnancies. impact appeared greatest on the counseling of the parents and their decision to terminate/pursue the pregnancy, and the least impact was on issues around delivery. conclusions: fetal mri, in addition to providing images of better quality, particularly in certain conditions, has clinical value in that it directly impacts on the counseling of parents and on clinical decisions. - . ultrasound reports were reviewed to determine sonographic diagnoses. selected patients from this cohort underwent mri using ge . tesla magnet without contrast (sequences included ssfse, fiesta, fgre or dual echo in planes). the images were reviewed and multiple characteristics were assessed for specifiying the area of obstruction. the features included: presence of normal fluid-filled bowel, small rectum for gestational age, signal of meconium in the rectum, and meconium filled dilated bowel. results: cases of sonographically suspected bowel obstruction were identified during the study period; of these underwent fetal mri. of these cases, had normal mri and postnatal outcomes, cases did not have postnatal findings available, and had postnatal meconium peritonitis but no obstruction. one case of congenital chloride diarrhea was diagnosed by fetal mri. a variety of bowel abnormalities were observed amongst the remaining cases. proximal obstruction was diagnosed in cases: jejunal atresia (n ) and multiple atresia (n ). distal obstruction was diagnosed in cases: ileal atresia (n ), meconium plugging (n ), closed gastroschisis (n ), enteral duplication cyst (n ), and imperforate anus (n ). characteristic patterns of features were identified amongst these cases that specified the location of obstruction. these patterns of findings allowed accurate localization of the level of obstruction in all cases when compared to postnatal findings. distal obstruction was characterized by normal fluid-filled small bowel and high t signal in distended loops. jejunal atresia was characterized by multiple loops of dilated bowel with high t signal primarily in the left upper quadrant. small rectum for gestational age was not consistently associated with proximal or distal atresia. conclusions: evaluation of fetal mri with attention to specific features allows localization of bowel obstruction. this may aid in counseling and postnatal management, including the need and type of postnatal imaging study. early diagnosis and treatment of ph may prevent clinical deterioration. pvt may produce a spectrum of imaging appearances, which has not been fully recorded in the literature. the goal of this paper is to review the spectrum of imaging appearances of neonates and survivors of neonatal pvt with special emphasis on the role of us and to correlate these findings with the clinical findings including outcome. methods & materials: a retrospective review of consecutive neonates admitted between - and diagnosed with pvt was conducted. diagnosis was established by us at a mean age of days (range: - ). health records, initial and follow-up (f/u) imaging were reviewed. findings were classified as non occlusive, single branch, pvt (grade ); occlusive pvt (grade ) and pvt with extensive parenchymal ischemia (grade ). results: pvt was diagnosed in patients, of whom were followed up to for years or longer. twelve patients were excluded due to liver disease, expired and were lost to f/u. of the in whom f/u was available, at the time of initial diagnosis, grade pvt was present in , all were on the left. grade pvt was diagnosed in and grade pvt in . on f/u physical exam, findings were unremarkable in / patients. liver function tests (lft) and thrombophilia assessment were available in children, mild lft abnormalities were noted in and children had evidence of thrombophilia. us exams were available in / children. among the survivors of neonatal pvt, us was regarded as normal in only children; showed left lobar atrophy (lla), had slowly progressive splenomegaly without other signs of ph, and developed clinically significant ph requiring shunting. conclusions: pvt has a wide spectrum of imaging appearances, it is possibly underdiagnosed and clinically unsuspected. varying degrees of lla are likely a sequela of clinically silent left pvt. us is a sensitive method for the detection of disease and assessment of progression. paper #: pa- fetal mri in arthrogryposis hedieh eslamy, md, radiology, lucile packard children's hospital, hkeslamy@gmail.com; erika rubesova, louanne hudgins, britton rink, richard a. barth purpose or case report: to present the fetal mri findings in fetuses with a prenatal diagnosis of arthrogryposis and correlate with postnatal outcome or autopsy results. arthrogryposis refers to contractures involving more than one joint which often represent deformational changes secondary to decreased or absent fetal movement. prognosis varies widely dependent on diagnosis, ranging from isolated contractures in amyoplasia to lethality in some cases. we hypothesized that fetal mri may demonstrate central nervous system (cns) pathology and muscle abnormalities which are important for predicting postnatal outcome. methods & materials: we identified fetuses with a diagnosis of arthrogryposis between january and october . all had fetal mri which was performed on a ge . tesla magnet, with ssfse, fiesta and fgre sequences in planes. the fetal mri's were evaluated for cns and muscle abnormalities. the extremities were evaluated for: muscle mass, increase in subcutaneous fat (indicative of muscle atrophy), and extremity joint positioning. these findings were subsequently correlated with the clinical exam of the neonates, pathology in the abortus and karyotype when available. results: results of fetal us, amniocentesis, fetal mri and post-natal or post-termination outcomes will be summarized. five fetuses had ≥ limb joint contractures. a sixth case had neck hyperextension and lateral flexion associated with akinesia and hydrops. on mri, no structural brain or spine abnormalities were identified. the abnormalities detected in the extremities were: severe decrease in muscle mass associated with increased subcutaneous fat ( cases); normal muscle mass ( cases); moderate decreased muscle mass associated with increased subcutaneous fat ( case). in the cases that delivered, the diagnoses were amyoplasia ( ) and distal arthrogryposis ( ). in a fourth case that underwent elective termination, autopsy was consistent with amyoplasia. two cases are pending delivery. conclusions: while fetal mri can be useful to rule out cns anomalies, it may also provide important information on decreased muscle mass as an important prognostic sign in a fetus with arthrogryposis. in our series, severely decreased muscle mass was predictive of amyoplasia, and joint contractures limited to hands and feet with preserved proximal muscle mass was predictive of distal arthrogryposis. both diagnoses are associated with relatively good prognosis and usually normal intelligence. purpose or case report: the purpose of this study is to assess the effects of iterative reconstruction technique (irt) on image quality metrics measured in child-sized anthropomorphic phantoms as kvp is changed. methods & materials: ct scans were performed on anthropomorphic phantoms with sizes of , & years (atom phantoms, cirs, norfolk virginia) using low dose pediatric chest protocols ( . , & msv) to determine baseline noise and dose levels. subsequently three voltage levels ( , & kvp) were used while adjusting mas to maintain baseline ctdivol and without mas adjustment which allowed varied ctdivol. images were reconstructed using % filtered back projection (fbp) and blends fbp: ir ( : , : , & : ) . parameters including ctdivol, dose length product, scan length, kvp, and mas, were recorded for each scan. image noise, contrast:noise (cnr), and signal:noise (snr) data were recorded from rois in phantoms and dilute iodine contrast filled syringes ( , , . %). results: as kv is lowered from to , image noise is doubled if mas is not increased to maintain ctdivol, and cnr is increased but snr is decreased due to the increased image noise. as kvp is lowered from to , image noise is increased nominally ( - %) if mas is increased to maintain ctdivol; therefore the increase in cnr and decrease in snr is negligible. ctdivol is reduced > % in all phantom scans as kv is reduced from to . irt reduces image noise by up to % [range - %] in all phantom sizes and in clinical images. as ctdivol is maintained in patient scans, image noise, cnr, and snr are reduced in patients (p< . ), resulting in improved image quality. conclusions: when lowering kvp, compensation with increases in mas is necessary to maintain ctdivol. however, lower target ctdivol can be achieved when adding irt as image noise can be decreased. for these phantoms, cnr and snr improved using all [selected] levels of ir, even when kv was reduced, resulting in lower ctdivol in phantoms. at all kvp settings when irt is applied, image noise is reduced, resulting in improved cnr and snr for all phantoms. disclosure: dr. bardo has indicated she is in the speaker's bureau and receives an honorarium from koninklijke philips. paper #: pa- adaptive iterative dose reduction in evaluation of the pediatric abdomen with ultra-helical -channel mdct jeffrey hellinger, md, stony brook university, jeffrey. hellinger@yahoo.com; bernice hoppel, richard mather, monica epelman purpose or case report: radiation reduction is paramount for pediatric patients. ultra-helical -channel mdct allows for rapid acquisitions at low dose. we evaluated the ability of a new adaptive iterative dose reduction algorithm (aidr) to reduce noise in low-dose ultra-helical pediatric abdominal ct scans. aidr is an iterative algorithm that adaptively reduces noise in the raw and image domains while preserving image structure. the raw data from consecutive low-dose pediatric abdomen exams was gathered. a dose simulation tool which adds noise to raw projection data was employed to simulate tube current at / of baseline ma. data were reconstructed with both standard filtered back projection and with aidr. regions of interest were drawn in the liver and lumbar musculature to determine the signalto-noise (snr), contrast-to-noise (cnr) and overall diagnostic quality of each data set. statistical significance was determined using a student's t-test. subjective image quality was evaluated by two reader blind review using a five point scale ( excellent, unacceptable). results: the snr and cnr were significantly lower for the % dose reduction datasets compared to the original filtered back projection reconstructions (snr: . vs . , p< . ; cnr: . vs . , p . ). when aidr was applied to the % dose reduction data, the snr and cnr improved to be superior to the native case (snr: . vs . , p< . ; cnr: . vs . , p . ). the average image quality score for the low dose datasets with aidr was . compared to . with standard filtered back projection at the baseline ma conclusions: aidr significantly improves the image quality of pediatric abdominal ct images. with a simulated % reduction in dose, aidr produces images with significantly greater snr and cnr. the subjective image quality scores for aidr showed dramatic improvement over standard filtered back projection. aidr processing algorithms with ultra-helical mdct will allow % reduction in radiation exposure while achieving the same diagnostic quality as compared to routine pediatric abdomen mdct radiation protocols with filtered back projection processing algorithms. purpose or case report: to explore incorporating asir into pediatric head ct protocols, to reduce patient radiation dose while maintaining image quality. methods & materials: an alderson rando head phantom was estimated to approximate the size of a -year-old child's head, and was scanned at decreasing % ma intervals ( to %, to ma) relative to this institution's age-based head ct protocols. each of these studies was then was reconstructed at % asir intervals ( % to %), and a mm roi was obtained in a consistent location behind the frontal bone to estimate noise (sd). using this phantom data, our ventriculoperitoneal (vp) shunt follow-up ct protocol was modified, and patients were scanned at % asir with approximately % ma reductions relative to our normal age-based mas. these asir studies were then anonymously compared to older non-asir head ct studies from the same patients (with identical kvp/slice thickness) by two blinded attending pediatric neuroradiologists. all studies were evaluated subjectively for diagnostic utility ( - ), sharpness ( - ), noise ( - ), and artifacts ( - ). - mm rois were drawn in consistent locations to estimate noise in air, bone, csf, and white matter (wm). results: the phantom study suggested similar same noise levels at % ma/ % asir ( . ) and % ma/ % asir ( . ). patients (average , range to years) were then scanned at approximately % ma reductions, with an average of days (range to days) between the asir study and prior non-asir study. the average ctdivol and dlp values of the % asir studies were . mgy and . mgy-cm, and for the non-asir studies were . mgy and . mgy-cm, representing statistically significant decreases in the ctdivol ( . %, p . ) and dlp ( . %, p . ) values. there were no significant differences between the asir studies and non-asir studies in respect to diagnostic acceptability (p . ), sharpness (p . ), or noise (p . ). there was a non-significant trend that the asir studies had a lower artifact score ( . vs . , p . ). there was good to perfect (kappa . to . ) agreement. the asir studies had statistically significant decreased csf noise ( . vs . , p . ), but no noise differences were seen in air (p . ), bone (p . ), or wm (p . ). conclusions: our findings suggest that asir can provide dose reductions in pediatric head ct without affecting image quality. purpose or case report: to validate the t map as a noninvasive quantitative biomarker of fatty infiltration of muscles and to determine whether the t map can differentiate between boys with dmd and healthy boys. methods & materials: two groups of boys with similar ages (range - years) were evaluated: boys with dmd (mean age . years) and healthy boys (mean age . years). mr images were performed at t. fatty infiltration of the pelvic and thigh muscles on t -weighted images (wi) was graded from to . on t maps with and without fat suppression, the muscle with the greatest fatty infiltration on t -wi was selected, and a region of interest was placed to obtain t values. t values from t maps with fat suppression were subtracted from values of t maps without fat suppression and designated as the "t fat value." t fat values were obtained from the same muscles in all boys. comparison was made between the t fat values of the two groups. the upper reference limit of the reference interval (ri) of t fat values was obtained from the control group to establish the normal range and applied to both groups to determine the accuracy of the t map. results: the gluteus maximus muscle had the greatest fatty infiltration on t -wi. median t fat value was . msec for dmd ( % ri . , range . - . ) and . msec for the control group ( % ri . , range . - . ). when applied to the two groups, the upper reference limit of the ri for control patients yielded % sensitivity, % specificity, % positive predictive value, and % negative predictive value. conclusions: utilization of t maps for the quantitative measurement of fatty infiltration of muscles can clearly differentiate between dmd and normal control boys with a high degree of accuracy and precision. this advanced noninvasive technique may potentially replace invasive muscle biopsies currently used for diagnosis. purpose or case report: prior work has shown that the gluteus maximus muscle has the greatest t relaxation time on mr imaging using t mapping in boys with duchenne muscular dystrophy (dmd). however, an increased t value on t relaxation time mapping may reflect both fatty infiltration and inflammation of the muscle. fatty infiltration characteristically follows inflammation in this disease process. therefore, the purpose of this study was to determine the contribution of each component (fat and inflammation) within gluteus maximus muscles and to correlate each component to clinical assessments. methods & materials: forty-six boys with dmd (ages: - years) were recruited. mr imaging of the pelvis using t maps with and without fat suppression were performed. the t map "fat values" (t value calculated from the t map without fat suppression [fs] minus t map with fs) and the t map "inflammation value" (t value from the t map with fs) were obtained. clinical assessments typically used to evaluate dmd patients (including clinical functional score, ft run, gower score, and step-up time) were also performed. spearman correlation coefficients between fat and inflammation values and the clinical assessments were calculated. results: there was a statistically significant correlation between the fat value of the gluteus maximus muscle and each clinical assessment test (p< . ). however, the inflammation value of the gluteus maximus muscle did not correlate with any clinical assessment. conclusions: in dmd, the amount of fatty infiltration of the gluteus maximus muscle has excellent correlation with clinical assessment. the amount of inflammation of the gluteus maximus muscle, however, does not correlate with clinical function. therefore, further study is needed to determine whether components (fatty infiltration or muscle inflammation) of the single most involved muscle reflect the components of all the muscles of the pelvis and thighs and whether the cumulative muscle involvement of each component represents clinical disease severity. utility of contrast-enhanced mr imaging in children with osteonecrosis: does gadolinium help? lamya atweh, md, radiology, texas children's hospital, laatweh@texaschildrens.org; robert c. orth, wei zhang, r. paul guillerman, herman kan purpose or case report: at our institution, gadolinium contrast-enhanced mr sequences are often obtained to assess epiphyseal and non-epiphyseal osteonecrosis in children. several studies have shown that dynamic contrast-enhanced sequences may provide prognostic information about long-term complications and healing of osteonecrosis. to our knowledge, no studies have determined the added value of routine post-contrast mr imaging in assessing acute complications related to chronic osteonecrosis. the purpose of this study was to evaluate the utility of intravenous gadolinium contrast in the mri identification of complications in children with an established diagnosis of osteonecrosis. methods & materials: patients were restrospectively identified (age range: . years to . year; m:f : ) with an imaging diagnosis of chronic osteonecrosis who underwent contrast-enhanced mr studies between / and / . the pre-and post-contrast mr images were consensus reviewed by two caq pediatric radiologists. pre-and post-contrast images were reviewed at separate times. the pre-contrast images were available during the review of post contrast images. studies were assessed for: osteonecrosis location (epiphyseal, non-epiphyseal osteonecrosis, or both), joint effusion, marrow edema, and epiphyseal collapse. % confidence interval (ci) and cohen's kappa coefficient(κ) was calculated to assess observed agreement. results: the diagnosis of osteonecrosis without complicating features was made in . % (ci: . - . %) ( / ) of pre-contrast studies and . % (ci: . %- . %) ( / ) of post-contrast studies. when chronic osteonecrosis with complicating features was identified,pre-and postcontrast images idenfied joint effusion in . % ( / ) and . % ( / ) (κ . , p< . ); marrow edema in . % ( / ) and . % ( / ) (κ . , p< . ); and epiphyseal collapse in . % ( / ) and . % ( / ) (κ . , p< . ), respectively. myositis or muscle strain was incidentally diagnosed in . % ( / ) pre-contrast and . % ( / ) post-contrast (κ . , p < . ) studies. conclusions: the high observed agreement between the pre-and post-contrast mr images shows that the addition of intravenous gadolinium may not be necessary in the majority of children with chronic osteonecrosis. paper #: pa- systematic protocol for assessment of the validity of bold mri in a rabbit model of inflammatory arthritis at . tesla michael chan, bhsc, university of toronto, mw.chan@ utoronto.ca; afsaneh amirabadi, anguo zhong, antonella kis, rahim moineddin, andrea s. doria purpose or case report: blood oxygen level-dependent (bold) mri has the potential to identify regions of early hypoxic and vascular joint changes in inflammatory arthritis. at this point, there is no standard protocol for data analysis of bold mri measurements in musculoskeletal disorders. standardization of the technique is paramount to compare results between studies and assess the validity of this technique in tissues outside the blood-brain barrier. our objective is to optimize bold mri reading parameters in a rabbit model of inflammatory arthritis by determining the diagnostic accuracy of ( ) statistical threshold values (r> . vs r> . ), ( ) summary measures of bold mri contrast [ the mean of the % bold signal differences within the region of interest (roi) (diff_on_off) and the percentage of suprathreshold voxels within the roi (pt%)], and ( ) voxel activation algorithm (positive, negative, and positive_negative). methods & materials: using bold mri protocols with a carbogen stimulus on a . t magnet, we imaged injected and contralateral knee joints of juvenile rabbits at baseline, and days , and after a unilateral intra-articular injection of carrageenin. nine non-injected rabbits served as controls. receiver operating characteristic (roc) curves were plotted to determine the diagnostic accuracy of the reading parameters. the bold measures from [(injected knee-control knees)/control knees] were counted as positive cases, while the bold measures from [(contralateral knees-control knees)/control knees] were regarded as negative cases. areas under the curve (aucs) were calculated to determine the most accurate parameters. results: using diff_on_off and positive_negative activations as constants, r> . was found to be more accurate than r> . (p . at day ). comparison of diff_on_off and pt% yielded no statistically significant difference (p> . ). finally, positive_negative activations for diff_on_off and negative activations for pt% using r> . were the most diagnostically accurate (auc . , p< . at day , and auc . , p< . ). conclusions: from the results of this study, the most diagnostically accurate and clinically relevant reading parameters included the use of a more lenient threshold of r> . , a diff_on_off measure of bold contrast, and a positive_negative voxel activation algorithm. pt% may used as an ancillary measure of bold contrast. quantitative versus semi-quantitative mr imaging of cartilage in blood-induced arthritic ankles andrea doria, md, phd, diagnostic imaging, the hospital for sick children, andrea.doria@sickkids.ca; ningning zhang, carina man, pamela hilliard, ann marie stain, victor blanchette purpose or case report: to cross-sectionally compare the ability of a scoring system (semi-quantitative method) with a manual segmentation technique (quantitative method) to evaluate the status of the articular cartilage of growing ankles of children with blood-induced arthritis. methods & materials: boys, with hemophilia (a, n ; b, n ) and with von willebrand disease, median age (range, - ) underwent a high resolution mri protocol at . tesla, x-rays, and physical examination using the hemophilia joint health score (hjhs) system. two blinded radiologists scored the mri examinations for cartilage items (horizontal component: surface erosions, scores - and vertical component: cartilage degradation, scores - ) according to the semiquantitative method (international prophylaxis study group mri scale). an experienced operator applied a validated quantitative d-mri method (horizontal components: ac, vc, vctab, thctab; vertical component: thccab) to corresponding high resolution mr images of ankles. results: internal correlation of the semi-quantitative method components was substantial (r . , p< . , tibia) to high (r . , p< . , talus) in any site of investigation, but it was site-specific with the quantitative method, being significant only in the talar trochlea (r . , p< . ). external correlation of corresponding components of the semiquantitative and quantitative methods was moderate (r . , p . ) to poor (r . , p . ) for horizontal components, and non-existent for vertical components. components of the semi-quantitative method highly correlated with lifetime number of previous ankle bleeds (r . - . , p< . ), pettersson x-ray (r . - . , p< . ), and hjhs scores (r . , p< . ). this correlation was poor (r . , p . ) to moderate (r − . , p . ) for horizontal components of the quantitative method. conclusions: the biologic concepts of the semi-quantitative and quantitative mri methods are distinct for assessment of ankles. the semi-quantitative method is valid for assessing cartilage changes in cross-sectional studies of blood-induced arthropathy, however the quantitative method is suboptimal or less powerful for this purpose. paper #: pa- shoulder mr arthrography in skeletally immature patients nancy chauvin, md, department of radiology, the children's hospital of philadelphia, chauvinn@email. chop.edu; camilo jaimes, victor ho-fung, diego jaramillo purpose or case report: there has been a well documented increase in sports participation in children which has lead to an increase in sports-related injuries. to date, there are no studies describing the value of shoulder mr arthrography compared with the gold standard, arthroscopy. we retrospectively reviewed mr shoulder arthrograms obtained in pediatric patients between and who underwent subsequent shoulder arthroscopy. interpretation of the images was performed by three pediatric radiologists who were blinded to the arthroscopy findings. images were evaluated in consensus and independently. assessment included evaluation of the osseous structures, labral-ligamentous complex, joint space and the rotator cuff interval. the mr results were compared with reported surgical findings. sensitivity and specificity were calculated. results: nine patients were excluded due to technical reasons. of the remaining patients, were boys ( . - . years, mean . years) and were girls ( . - . years, mean . years). at arthroscopy, patients ( %) had injury to the anterior inferior glenoid labrum. mr sensitivity was % for depiction of bankart-type injuries with a specificity of %. patients ( %) had hill sach lesions and mr had sensitivity of % with specificity of %. superior labrum anterior posterior (slap) tears ( %) were identified at arthroscopy with mr sensitivity of % and specificity of %. overall, mr arthrography had a positive predictive value of % for identification of a surgical lesion. agreement between the observers was high. interobserver reliability was calculated with an intraclass correlation coefficient (icc)of . with a cronbach's alpha of . . conclusions: mr shoulder arthrography can accurately depict labral and osseous injury and provides pertinent preoperative information. a novel multi-channel mr coil for improved pediatric elbow coil imaging suraj serai, phd, cchmc, suraj.serai@cchmc.org; randy giaquinto, kathleen emery, charles dumoulin purpose or case report: single flex coils or adult size coils are currently used for imaging the pediatric elbow. this frequently results in uncomfortable patient positioning, motion, poor fat suppression, low snr and there is currently lack of a dedicated pediatric elbow coil in the commercial market. our goal was to explore the usefulness of a new coil array dedicated for pediatric elbow imaging and to compare quantitative & qualitative imaging findings to commercially available coils. methods & materials: an eight channel elbow coil was designed. the coil frame was designed to be rigid and lightweight. seven identical loop coils were built into a polycarbonate frame and an eighth coil built into a paddle that fits into the top frame. the coil elements were constructed with heavy copper to provide a high q-factor and increased snr. the complete coil including electronics & covering, weighs only . kg. mr imaging under irb approval was performed on a ge . t scanner using a routine clinical elbow protocol including t w, pdw, t w, fat-sat, non-fat-sat, d & d sequences. subjects were positioned feet-first with the elbow on the side & were subjectively assessed for comfort level. images obtained from the new coil & from the current commercial coils were compared for snr. results: scan positioning was reported to be comfortable. snr was between - % higher as compared to the routine coils. fat saturation was uniform, indicating that the magnetic susceptibility of the coil is well-matched to human anatomy. anatomical detail depiction was subjectively better for anatomic features such as trochlea. detection & diagnostic confidence of elbow disorders were improved with the new coil & greatly decreased motion artifacts were observed. conclusions: the new pediatric elbow coil provided excellent image quality, patient acceptance and clinical performance improvements over existing coils. the open coil design also allows for imaging of the elbow in a partially flexed position or in a cast. the advantages provided by the new coil are expected to include shortened image acquisition times (via parallel imaging) & increased snr. disclosure: dr. serai has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. incremental value of knee radiography in the interpretation of pediatric knee mri yen-ying wu, texas children's hospital, yxwu@ texaschildrens.org; robert c. orth, wei zhang, r. p. guillerman, herman kan purpose or case report: the acr appropriateness criteria recommendation for the imaging work-up of knee pain is radiography followed by mri. in many cases, mri is performed prior to review of radiographs or the referring subspecialist does not feel radiographs add value, particularly when ligamentous injury is suspected. the purpose of this study is to determine if radiography adds incremental value in the interpretation of knee mr studies electively referred by pediatric sports medicine and orthopedic subspecialists. knee mri studies referred from pediatric sports medicine physicians or pediatric orthopedic surgeons between / and / (n , ages - years, m:f : ) with accompanying radiographs were identified. patients were separated into groups based on mri findings: normal, ligamentous injury, or osteochondral injury (osteochondral lesions, bone contusions/fracture, and avulsion injury). knee radiographs were consensus reviewed by two caq pediatric radiologists blinded to mri findings and categorized into the same groups. radiograph and mri findings were compared and categorized into groups: neutral if radiograph and mri findings were the same, misleading if findings were discordant, or helpful if radiographs improved mr interpretation. the latter group was analyzed for impact on mr diagnosis. results: for knee radiographs, were normal, showed ligamentous injuries, and showed osteochondral injuries. when radiographs were interpreted as normal (n ), by mr % were normal, % had ligamentous injury, % had osteochondral injury, and % had both ligamentous and osteochondral injury. when radiographs were interpreted as ligamentous injury (n ), by mr % were normal and % had ligamentous injury. when radiographs were interpreted as osteochondral injuries, by mr % had ligamentous injury, % had osteochondral injury, and % had both ligamentous and osteochondral injury. subset analysis of true positive radiographs (n ) found % to be helpful and % to be neutral in mr diagnosis. for radiographs considered helpful, % resulted in a change in mr diagnosis. in regards to the influence of radiographs on mr interpretation, % ( / ) were misleading, % ( / ) were neutral, and % ( / ) were helpful. conclusions: a minority of pediatric knee radiographs aided mr diagnosis, and none resulted in a change in diagnosis. pediatric knee mri and interpretation should not be predicated on radiologist review of knee radiographs in this subset of patients. paper #: pa- sonographic evaluation of pediatric skeletal lesions: is it worthwhile? henrietta rosenberg, md, radiology, the mt. sinai medical school, henrietta.rosenberg@mountsinai.org; amish patel, neil lester purpose or case report: the purpose of this paper is to demonstrate how ultrasound(us) may serve as a readily available, cost-effective, non-invasive, non-ionizing, practical tool for the evaluation of a variety of skeletal abnormalities in the pediatric age range. we reviewed the clinical and imaging findings in patients seen during the past years in whom us demonstrated abnormalities related to the skeletal system, excluding patients with hip joint effusions or ddh. results: us proved useful in the following situations: evaluation hard superficial immobile mass (osteoma shin) ( ), absent medial end clavicle on x-ray in region of neck mass (us showed abc medial end clavicle)( ), to determine if soft tissue mass involves adjacent bone nodular fasciitis surrounding clavicular head ( ), for diagnosis and followup fracture (displaced/non-displaced) in infants ( ), diagnosis osteomyelitis in patients with cellulitis ( ), question of fracture underlying cephalohematoma or subgaleal hematoma ( ), rib mass (osteochondroma) ( ) or mass costochondral junctions (contour deformities costochondral cartilage) ( ), firm posterior knee mass (baker's cyst) ( ), firm anterior knee mass (septated cystic mass suprapatella region due to rheumatoid disease) ( ), immobile hard scalp mass due to epidermoid cranial vault ( ), painful mass occipital bone with soft tissue components extending through the skull externally and internally due to langerhan's histiocytosis ( ), indeterminate mass clavicle clinically thought to be post-traumatic sequellae, resolved on follow-up ( ), assessment craniosynostosis ( ), for differentiation of pathological entity from normal anatomic structure (lump on back of slender baby proved to be normal posterior spinous process) ( ). conclusions: us is worthwhile for evaluation of wide range of pediatric skeletal abnormalities and helps to determine if the a lesion is one that is "touch" or "don't touch". to maximize diagnostic accuracy, the imager should have thorough knowledge of the clinical history, physical findings, laboratory and other imaging findings. in equivocal cases or in those patients in whom the field of view (fov) is insufficient for complete visualization of an obvious lesion or if malignancy is suspected, us serves to triage those patients in whom further imaging is necessary. high incidence of vertebral fractures in children with acute lymphoblastic leukemia months after the initiation of therapy mary ann matzinger, md frcp(c), university of ottawa, matzinger@cheo.on.ca; nazih shenouda , brian lentle, josée dubois, helen r. nadel purpose or case report: vertebral fractures due to osteoporosis are a potential complication of childhood acute lymphoblastic leukemia (all). to date, the incidence of vertebral fractures during all treatment has not been reported methods & materials: we prospectively evaluated children with all during the first months of leukemia therapy. lateral thoracolumbar spine radiographs were obtained at diagnosis and months. vertebral bodies were assessed for incident vertebral fractures using the genant semi-quantitative method, and relevant clinical indices such as spine bone mineral density (bmd), back pain and the presence of vertebral fractures at diagnosis were analyzed for association with incident vertebral fractures. results: of the children, ( %, % confidence interval [ci] % to %) had a total of incident vertebral fractures, of which ( %) were moderate or severe. thirteen of the children with incident vertebral fractures ( %) also had fractures at the time of diagnosis. vertebral fractures at diagnosis increased the odds of an incident fracture at months by an odds ratio of . ( % ci . to . , p . ). in addition, for every . standard deviation reduction in spine bmd z-score at diagnosis, there was . -fold increased odds for incident vertebral fracture at months ( % ci . to . %, p . ). conclusions: children with all have a high incidence of vertebral fractures months after diagnosis, and the presence of vertebral fractures and reductions in spine bmd zscores at diagnosis are highly associated clinical features. purpose or case report: to provide objective measures of acetabular morphology utilizing volume-rendered ct and to better characterize normal acetabular development in adolescents. implications for the diagnosis of femoroacetabular impingement (fai) will be discussed. methods & materials: hips in consecutive patients ( female, male; ages - years) who underwent abdominal and pelvic ct for non-hip related complaints were retrospectively examined. examinations were performed for a variety of complaints, including abdominal pain, nephrolithiasis, vomiting etc. patients with obvious hip pathology were excluded. pelvic rotation was eliminated, and pelvic inclination was measured and corrected to °u tilizing a volume rendered ct model. measurements of femoral head diameter (fhd), anterior femoral head coverage (fhca), and posterior femoral head coverage (fhcp) were obtained. femoral head area (fha) was defined as π(fhd/ ) . percent anterior femoral head coverage (%fhca) was defined as (fhca/fha)* . percent posterior femoral head coverage (%fhcp) was defined as (fhcp/fha)* . acetabular version by volume-rendered ct (avvr) was defined as (fhcp/fhca). results: average pelvic inclination angle (sd) was . ( . ) for females and . ( . ) for males. average (sd) %fhca was . ( . ) for males and . ( . ) for females. average (sd) avvr was . ( . ) for males and . ( . ) for females. among males, average avvr decreased with subject age. on the other hand, there was little change in average avvr with age among females. conclusions: average avvr is greater for females than males, and this difference becomes more striking with increasing subject age. this represents an unexpected finding given the reported increased incidence of "pincer" type fai among females. characterization of acetabular morphology among adolescents with clinical fai should consider subject age and gender. in this regard, volumerendered ct is capable of providing an objective measure of acetabular morphology. mistakes in musculoskeletal plain film interpretation james crowe, pediatric radiology, texas children's hospital, jecrowe@texaschildrens.org; george s. bisset purpose or case report: to evaluate the mistakes made by trained pediatric radiologists when interpreting radiographs of the extremities obtained for the evaluation of outpatient acute pain (mostly post-traumatic). we retrospectively evaluated all radiographs and associated interpretations obtained during a month period from april , , to october , , of the elbows, wrists, knees and ankles in pediatric outpatients who presented with acute pain in the affected area. all radiographs were previously interpreted by a caq-certified pediatric radiologist varying in experience from year to years. abnormals were identified, including elbows, wrists, knees and ankles. all radiographs were determined to be "as dictated", missed significant finding, or overcall. attention was focused on the missed findings and overcalls. results: findings were as follows: elbow radiographs- missed findings and overcalls, wrist radiographs- missed findings and overcalls, knee radiographs- missed findings and overcalls, ankle radiographs- missed findings and overcalls. this resulted in a total of missed findings ( . % of abnormals) and overcalls ( . % of abnormals). of the misses, % were fractures. the highest mistake percentage occured in the ankles where the combined misses and overcalls approached %. this was also the location where we found the highest percentage of missed fractures ( . %) conclusions: when just abnormal cases were considered, fully trained pediatric radiologists have a mistake rate of approximately . %, if misses and overcalls are included. from a quality improvement perspective, we will review all of the types of misses and overcalls to expose common themes. longitudinal assessment of osteoporosis in a blood-induced hemophilia rabbit model using quantitative ultrasound kuan-chieh wang, university of toronto, kc.wang@ utoronto.ca; afsaneh amirabadi, anguo zhong, christopher tomlinson, andrea s. doria purpose or case report: the reduction of physical activities in hemophilic patients may lead to bone demineralization and consequent osteoporosis. quantitative ultrasound (qus) is free of ionizing-radiation, relatively inexpensive, and easy to use that making this technique suitable for follow-up of hemophilic children with clinical suspicion of osteoporosis. to our knowledge, no previous study has investigated the value of qus for longitudinal assessment of growing bones in an animal model which is paramount for clinical translation of the technique once change in measurements could relate to either the baseline pathology or physiologic bone growth variability. the objective of this study is to investigate the intra-and inter-operator reliability of qus over time, and its ability to discriminate bone loss in pathologic vs control knees of a rabbit model of blood induced arthritis. methods & materials: sixteen juvenile white new zealand rabbits distributed into two groups: received intraarticular blood injections over weeks (n pathologic and contralateral knees), and noninjected rabbits were used as controls (n knees). midshaft tibia speed-of-sound (sos) was measured at baseline, and weeks and of the experiment. two operators scanned each site twice at each time point. qus measurements were compared to microct (reference standard) on week to validate the study results. results: the sos measured in the control group increased significantly (p< . ) over the week period. there was not such an increase in the arthritis sos value (p> . ). in both groups the overall intra-operator coefficient of variation of sos measurements was % at baseline and decreased to % at week likely due to increased tibia size. the inter-operator reliability was % at baseline and % at week . with regard to the effect of bone growth on qus measurements for the control group (n ), sos values increased by . m/s, whereas for the pathologic group (n ), they only increased by m/s. statistically significant differences in ratios of sos between final/baseline results were noted (p . ) between the pathologic and control groups. conclusions: the longitudinal use of qus has an acceptable intra-and inter-operator reliability. even accounting for the significant impact that bone growth has on qus measurements over time, qus can differentiate pathologic from control knees in the proposed animal model and holds potential for clinical use in the assessment of osteoporosis in hemophilic children. methods & materials: the study was approved by the institutional review board. pediatric patients with abdominal tumors ( malignant and benign lesions) underwent diffusion-weighted mr imaging (dwi) on clinical . t (n ) and t (n ) mri scanners. adc maps were generated from b dwi and adc values were retrospectively and independently measured by two radiologists. adc values of benign and malignant tumors were compared with the welch two sample t-test. a p value of . was considered to indicate statistical significant differences. in addition, a receiver operating curve analysis (roc) was performed to determine the optimal cut-off adc value for differentiating benign and malignant tumors. results: the mean adc value (mm /sec) of benign tumors was . x - for the first reader and . x - for the second reader. the mean adc value (mm /sec) of malignant abdominal tumors was . x - for the first reader and . x - for the second reader. the differences between benign and malignant tumors were statistically significant (p< . for both readers). roc analysis revealed an optimal cut-off adc value for differentiating malignant and solid tumors as . x - mm /sec. conclusions: diffusion-weighted imaging with adc maps can be used to differentiate between benign and malignant pediatric abdominal tumors. creation of a database to evaluate imaging findings in long-term survivors of pediatric malignancy alexander towbin, md, radiology, cincinnati children's hospital medical center, alexander.towbin@cchmc.org; seth hall purpose or case report: over the past years, there have been significant improvements in the treatment of pediatric malignancies. improved therapy has led to an increase in the number of long-term survivors. many of these survivors are now experiencing late effects as a result of the original disease process or its treatment. these late effects are frequently identified on imaging. the purpose of this study is to create a database of the imaging findings of long-term survivors of pediatric malignancy in an attempt to begin to classify the findings and identify associations. methods & materials: after irb approval, the institutional cancer registry was searched to identify all patients younger than years of age who were diagnosed with a solid tumor between and . patients were included in the database if they survived for more than years from the date of their initial diagnosis. the electronic medical record system was then used to obtain demographic and treatment information for each included patient. the dictated reports from all cross-sectional imaging studies evaluating the chest, abdomen, or pelvis performed more than two years from the date of diagnosis were then reviewed. each positive imaging finding was classified by the involved organ. results: after querying the institutional cancer registry, patients were identified who met the inclusion criteria for this database. the most common neoplasms were neuroblastoma, wilms tumor, and astrocytoma. of the included subjects, had imaging of the chest, abdomen, or pelvis. overall, reports were evaluated and classified. findings were most commonly identified in the lungs, musculoskeletal system, kidneys, liver, and lymph nodes. conclusions: a database examining the late effects in longterm survivors of pediatric malignancies was created. this database has the potential to help identify the radiologic manifestations of the complications of cancer therapy and thus help guide rationally determined long-term risk-benefit ratios in the treatment of pediatric malignancies. imaging followup of lymphoma in pediatric patients: is pelvic ct necessary? javier lopez bueno, md, children's hospital of eastern ontario, jlopezbueno@cheo.on.ca; nishard abdeen purpose or case report: pelvic ct is often included in the imaging followup of patient with lymphoma before, during and after treatment to assess response to treatment and monitoring for relapses. while such followup is expected to improve detection of relapse, there is little objective evidence of its effectiveness in lymphoma. anecdotally, there are few pelvic relapses in pediatric patients with lymphoma regardless of primary site. we hypothesize that pelvic ct could be avoided as part of the followup without adverse impact on survival or in the detection rate of relapses, and with subsequent significant reduction in the radiation dose, particularly to the gonads. methods & materials: research ethics board approval was obtained. patients diagnosed with lymphoma and with at least one year of followup at our tertiary care pediatric hospital were included. sex, age, type of lymphoma, stage, primary site, site of relapse if any as well as the number of ct scans of the head, neck, chest, abdomen and pelvis were recorded. results: a total of patients met study criteria. there were males and females, with an average age of . years (range - years). eighteen patients had hodgkin disease ( %) and eleven had non-hodgkin lymphoma ( %). mean length of followup was . years (range - years). an average of . pelvic scans per patient were performed for surveillance (range - ). three relapses were detected. of these only one was in the pelvis, in a patient whose initial t cell non-hodgkin lymphoma was extensive and involved the neck, chest, abdomen and pelvis. conclusions: this study suggests a low incidence of pelvic relapse in pediatric patients with lymphoma. the routine use of pelvic ct in surveillance protocols may therefore be of little benefit while imposing a significant radiation burden. our study is limited by small sample size and short length of followup. further large scale studies are required. (esft) is performed by measuring the size of the tumors before and after chemotherapy. the proposed method of measuring tumor size, however, differs amongst recist . (response evaluation criteria in solid tumors), who (world health organization) and cog (children's oncology group) response criteria. in our project, we assessed whether response classification differs between the three different methods. methods & materials: after irb approval, we retrospectively analyzed mri studies of patients with ewing sarcoma who were treated at stanford and ucsf medical centers. tumor size was assessed before and after therapy. tumor measurements were obtained using recist . (longest single diameter), who (longest diameter and perpendicular diameter), and cog criteria (three measurements to calculate tumor volume). tumor response was assessed by the differences in sizes of the tumors before and after treatment using four response categories: progressive disease (pd), stable disease (sd), partial response (pr), and complete response (cr). concordance between the three response classification systems was assessed using cohen's kappa (k) coefficient and percentage of disagreement per response category. results: the k statistic for concordance in cog/who, cog/ recist and recist/who were . , . and . respectively. disagreement rates for recist/who, cog/ who, and cog/recist were . , . , and . % respectively. using tumor volume, twenty-six patients were reclassified: twenty-four cases of stable disease coded by recist were reclassified as progressive disease by cog and two cases of partial response coded by recist were reclassified as complete response by cog. conclusions: this study demonstrates poor agreement between the recist . and cog response criteria in esft. given the degree of discordance between response criteria in esft, evaluation of the prognostic impact of each of these classification systems may guide selection of the optimal system for future use in this disease. imaging recognition of chylous ascites following surgery for abdominal neuroblastoma zeyad metwalli, md, baylor college of medicine, metwalli@bcm.edu; r. p. guillerman, heidi v. russell, eugene s. kim purpose or case report: surgical resection is a standard part of multimodality treatment of neuroblastoma, the most common abdominal malignancy of infancy and early childhood. chylous ascites is a rarely reported complication of surgery for abdominal neuroblastoma, and is likely underrecognized, posing the risk of nutritional deterioration and sepsis. to facilitate early diagnosis and institution of appropriate therapy, we present the salient imaging findings of the largest known series of chylous ascites following surgery for abdominal neuroblastoma. methods & materials: all patients with abdominal neuroblastoma complicated by post-operative chylous ascites over a five-year period at a large children's hospital were identified by a database search. a retrospective review of the imaging studies and clinical charts was conducted. results: chylous ascites developed following surgical resection of abdominal neuroblastoma in of patients, with the diagnosis made between postoperative days and . four cases were high-risk neuroblastoma and one was intermediaterisk neuroblastoma. all cases involved resection of an adrenal mass and dissection around the abdominal great vessels. all cases manifested with abdominal distention on physical exam, and ascites was suspected clinically in cases. computed tomography (ct) in all cases revealed a large volume of ascites of near-water attenuation (range of − to . hounsfield units). the cases imaged with ultrasound (us) showed hypoechoic or anechoic ascites without septations. the chylous ascites resolved after - months of treatment with dietary fat restriction, medium chain triglycerides, intravenous octreotide, or peritoneal catheter drainage. conclusions: chylous ascites is an under-recognized complication of surgical resection for abdominal neuroblastoma, occurring in % of patients in this series. the diagnosis is supported by the demonstration on ct or us of a large volume of ascites causing abdominal distention - weeks post-operatively. the ascites is typically near-water in attenuation rather than fatty in attenuation and should not be misattributed to peritonitis, hemorrhage, bowel leak, or early tumor recurrence. cervical spine injuries in patients with suspected physical abuse nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; zarir p. khademian, tanya hinds, katherine deye, allison m. jackson, eglal shalaby-rana purpose or case report: to evaluate the incidence and nature of cervical spine injuries and relationship to posterior fossa abnormalities in children who underwent brain and cervical spine mri as part of the clinical workup for suspected physical abuse. methods & materials: authors retrospectively analyzed records of eighty-five children less than three years of age who were documented by the child protective services at a level one pediatric trauma center over a period of four years ( ) ( ) ( ) ( ) ( ) . only patients who underwent both mri imaging of the cervical spines (c-spine) in addition to brain imaging as part of the clinical workup were included. cspine and posterior fossa of brain mris were independently reviewed by two pediatric neuroradiologists, both blinded to clinical details. c-spine abnormalities (bone marrow edema, cord edema, intrathecal blood, disc pathology, soft tissue/ ligamentous injury, vascular injury) were documented and correlated with abnormalities seen in the posterior fossa (blood, brainstem edema, cerebellar edema). results: at this time, / patients have been reviewed. twenty patients ( %) had both cervical spine injuries and posterior fossa abnormalities. there were no patients with isolated cervical spine injuries without posterior fossa abnormalities, but there were five patients ( . %) that had posterior fossa abnormalities in the absence of c-spine injuries. fifteen patients ( . %) did not have any spinal or posterior fossa imaging abnormality. none of the patients had bone marrow edema, disc pathology, or intrathecal blood. one patient had vascular neck injury and cord edema. conclusions: our results show that the incidence of cervical spine injury in children under investigation for abusive head trauma is as high as %. our data show further that cervical spine injury predicted posterior fossa injury in all patients, while presence of posterior fossa injury predicted concomitant c-spine injury in only %. the incidence of c-spine trauma we found in these patients is higher than reported elsewhere in the literature and may impact whether or not routine c-spine mri will be included in national imaging guidelines for children under investigation of abusive head trauma. pediatric skull fracture andre loyd, phd, biomedical engineering, duke university, aml @duke.edu purpose or case report: skull fractures are often seen in the setting of non accidental trauma (abuse) abuse, and are usually attributed to falls from heights above m. part of the difficulty in assessing height is due to uncertainties in actual distance. objective: to determine what types of skull fractures can occur in pediatric and adult post-mortem human specimens during controlled impacts on hard surfaces from various heights. methods & materials: skull fracture patterns in postmortem human specimens from a unique bank of pediatric specimens ( -week gestation to -years-old, n ) were subjected to controlled drops from both arbitrarily low heights ( and cm) and high heights ( m) onto an aluminum platen. the specimens were dissected from the neck at the occipital condyles and intracranial were sealed inside the head using pmma. the heads were dropped on to five different impact locations. fractures were identified using palpation and high resolution mdct. results: no specimens between -weeks-gestation and days-old sustained fractures from the - cm drops. three out of four ( %) specimens ages between -and -months old fractured due to the or cm drops. the -and -year-old specimens and all adult specimens survived the - cm drops. all specimens subjected to the m drop fractured. the specimen between -months and -months sustained either a linear fractures or diastatic fractures from the cm and cm drops. the results indicate that some aged infants and young children can sustain skull fractures by being dropped or falling from relatively low heights. drops, as low as cm, can cause linear and diastatic fractures in pediatric skulls. the presences of compliant sutures and fontanelles in neonatal heads allow the head to deform during impact. these data add very important information to mechanisms of skull fractures across ages, including ages in which child abuse is a consideration. evaluation of a new classification system for temporal bone fractures in children aimed at increasing prognostic value badriya al-qassabi, md, mcgill university, albahlania @ yahoo.com; lucia carpineta, rania ywakim, bahar torabi, andrew m. zakhari, lily h p. nguyen purpose or case report: to compare a new classification of temporal bone fractures which specifically evaluates involvement of the otic capsule against the traditional classification system (transverse versus oblique versus longitudinal), to evaluate whether this new classification is able to better identify patients at risk of adverse otologic outcome and neurologic complications in the pediatric population. methods & materials: a retrospective hospital chart review was performed by ent colleagues searching for all patients with temporal bone fractures seen at our center over the past years. this was followed by a blinded review of the ct heads by a resident and a trained pediatric radiologist with neuro expertise. these cts were evaluated for petrous involvement, otic capsule involvement and any associated intracranial lesions. this information was then correlated with clinical outcome measures including post-traumatic hearing deficit, facial nerve palsy, persisting csf leak and global neurologic sequelae. the new classification was compared to the traditional one, and specifically analysed for the ability to better predict the clinical outcomes. results: expectedly, pediatric temporal bone fractures were infrequent and otic involvement even more rare. fractures with involvement of the otic capsule (versus otic sparing) were found more frequently in boys. they were also more likely to be associated with immediate otologic signs and neurologic findings on presentation. these fractures also had the highest association with conductive hearing deficit (> %) and were twice as likely as otic sparing fractures to be associated with immediate facial nerve palsy and with more important concomitant intracranial injuries such as midline shift. conclusions: while our numbers are small, our results suggest a trend that when temporal bone fractures show involvement of the otic capsule, there is higher risk of adverse otologic outcome and neurologic complications even in the pediatric population. absence of a causal relationship between mr detected subdural hematomas (sdh) in neonates with hypoxic-ischemic encephalopathy (hie) deniz altinok, children's hospital of michigan; jay shah, harut haroyan, gulcin altinok, nitin chouthai purpose or case report: the existing controversy regarding subdural hemorrhages noted in patients with hie is an important discussion in the medical, legal and child-welfare realms. it is our goal to provide additional information to this critical debate through mr findings on patients with clinically diagnosed hie. methods & materials: all patients born with clinically diagnosed hie, and treated at children's hospital of michigan in the past years were examined; those with head mri taken within days of life were selected. in total, patients fit the criteria, this included: males and females, and an age range of - days at scan (average age of days at scan). all traumatic births, coagulopathies, and other pertinent clinical findings were noted. mr imaging was reviewed and reported by a blinded pediatric neuroradiologist, these reports were then compared to the "original read". results: all patients were confirmed radiologically to have hie. the causes of hie in all cases examined were either intrauterine/delivery asphyxia, aspiration, or congenital disease. of these cases, the findings were: sdh, parenchymal hemorrhages, intraventricular hemorrhages, cephalohematomas, subarachnoids, large subcutaneous hemorrhage and instance of mca stroke. all patients with mr detectable sdh had or more confounding factors ( meningitis, coagulopathies, chest compressions, cardiac malformation, pph, severe pulmonary hemorrhage requiring transfusion of plasma and prbc). conclusions: it has been hypothesized that sdh is often found incidentally in children diagnosed with hie, this is however a dubious conclusion considering our results. in fact, the presence of sdh and hie concomitantly is low even when including a population with traumatic births such as ours. sports-related concussion in children: an mri and mrs study kim cecil, phd, cincinnati children's hospital medical center, kim.cecil@cchmc.org; todd a. maugans, james l. leach, mekibib altaye purpose or case report: the pathophysiology of sportsrelated concussion (src) is poorly understood, especially for children. following src and mild traumatic brain injury in adults, a few mri and proton mrs studies have identified axonal injury with declines in the neurometabolite n-acetyl aspartate (naa). we wanted to examine a src adolescent population with proton mrs, diffusion tensor imaging (dti) and other mri methods within h of concussion and with short term followup to determine if there were differences in imaging metrics with age and sex matched healthy control participants. methods & materials: twelve children, ages - years, who experienced src were evaluated with impact neurocognitive testing, t -weighted mri, susceptibility weighted imaging (swi), dti, proton mrs, and phase contrast angiography (pca) at less than h, days and days or greater post-concussion. healthy, age and sex matched controls for each src participant were recruited and evaluated at a single time point. quantitative imaging metrics included fractional anisotropy, metabolite concentrations, and global cerebral blood flow (cbf). group comparisons were examined by paired t-test or wilcoxon signed rank test. correlational data employed spearman rank correlation. results: impact results revealed significant differences in initial total symptom score (tss), and reaction time (rt) for the src group compared with the control group, with tss resolving by a mean of days and rt at days. no evidence of structural injury was observed qualitatively for either group. analyses between groups or over time within the src group found no decreases in naa or elevation of lactic acid upon mrs, and no changes in fractional anisotropy upon dti. within the src group, significant changes in the global cbf were observed. improvement towards control values occurred by days for % and by days for % of src group participants. conclusions: pediatric src affects global cbf without evidence of structural or metabolic injury. predictive value of high resolution mr imaging of brain and sella in children with clinical optic nerve hypoplasia for hypopituitarism charles glasier, radiology, arkansas childrens hospital, glasiercharlesm@uams.edu; raghu h. ramakrishnaiah, julie shelton, chetan c. shah, paul h. philips purpose or case report: to review the spectrum of cns abnormalities and their incidence in children with optic nerve hypoplasia and to calculate the sensitivity and specificity of magnetic resonance imaging in predicting endocrine abnormalities. methods & materials: this is an irb approved retrospective study of children with clinical optic nerve hypoplasia who underwent mri of the brain and orbits as part of the clinical workup in a tertiary care pediatric hospital. high resolution mri studies were performed on . tesla scanners. mri studies were reviewed for optic nerve hypoplasia, absent or ectopic posterior pituitary, absent pituitary infundibulum, absent septum pellucidum, migration anomalies and hemispheric injury.radiologists were blinded to patients endocrinologic status.all patients had clinical evaluation by a pediatric neuro-ophtalmologist and endocrinologist. a standardized panel of serologic testing that included serum cortisol, acth, tsh, and free t levels were performed on all patients. statistical analysis was performed to determine the sensitivity and specificity of mr findings in predicting endocrinologic deficiency. results: study included children( males and females) who had clinical optic nerve hypoplasia. the mean age of the study population was yr (sd: . yr). children had unilateral and children had bilateral optic nerve hypoplasia by mri. children had absent posterior pituitary bright spot and had ectopic posterior pituitary, had absent infundibulum, had complete callosal agenesis, partial callosal agenesis and had callosal thinning. had absent septum pellucidum. had hypopituitarism. of the patients with hypopituitarism had abnormal abnormal pituitary on mri, had absent septum pellucidum, and child had migration abnormality. none had corpus callosal abnormality. the sensitivity and specificity of mri in predicting hypopituitarism by demonstration of abnormal pituitary is % and % respectively. the positive predictive value and the negative predictive value is % and % respectively. among the patients with normal endocrinologic function, none had pituitary abnormalities on mri. conclusions: pituitary abnormalities are the most common intracranial abnormality in patients with optic nerve hypoplasia followed by absent septum pellucidum. detection of pituitary abnormalities by the mri has high specificity and high negative predictive value for endocrine abnormality. paper #: pa- ct imaging pearls for shunted pediatric brains srikala narayanan, md, children's national medical center, snarayan@childrensnational.org; nadja kadom purpose or case report: shunted pediatric patients frequently present emergently with symptoms that could indicate shunt malfunction, such as headache and vomiting. here, we present imaging pearls on non-contrast head ct in shunted children. methods & materials: illustration of each of the following: . shunt tip and volume averaging-consider location of side holes and use of multiplanar reformatted images. . shunt at burr hole-consider radiolucent shunt parts. . shunt rupture in the neck-remember to investigate the lower extracranial shunt parts. . shunt in cyst/subdural shunt (vs dislocation)-consider primary shunt location in a cyst rather than shunt dislocation. . enlarged temporal horns-look for it. in infants occipital horns may dilate first. . enlarged rd ventricle-look for bulging of lateral walls. . sulcal effacement-use the "three shades of gray" rule. . small cisterns-detecting shape distortion can help. . periventricular edema-easily overlooked because of similar low density compared to ventricular fluid. . slit-ventricle -requires cautious reporting. conclusions: careful evaluation of ct images in shunted pediatric patients can reveal important clues for making an accurate diagnosis, even when prior images are not available. successful treatment of mice with creatine transporter deficiency kim cecil, phd, cincinnati children's hospital medical center, kim.cecil@cchmc.org; diana m. lindquist, matthew r. skelton, gail j. pyne-geithman, joseph f. clark purpose or case report: creatine transporter deficiency (ctd) is an untreatable x-linked mental retardation syndrome with severe cognitive and speech impairment. patients are identified by an absence of creatine in the brain on mr spectroscopy (mrs) and distinguished from two creatine synthesis deficiency syndromes with genetic testing. for ctd, the absence of the transporter (slc a ) prevents creatine from crossing the blood brain barrier and entering brain cells. a brain specific ctd knockout mouse was developed replicating key features of the human disease and establishing an animal model for treatment of ctd. we report the successful treatment of the ctd knockout mouse and present confirmation by mrs. methods & materials: brain specific knockout and littermate control mice were randomly assigned and treated with on one of three supplements: agentx (confidential), creatine or maltodextrine as placebo. h and p mrs data were collected on a t mr system (bruker). mice (n ) were studied with mrs after weeks of supplementation. single voxel h data were acquired on a ul voxel covering the cerebrum using a double spin echo sequence. p data were acquired with an isis sequence from the same voxel. metabolite quantification was performed with jmrui and compared between groups and over time with statistical tests for significance (t-tests, anova). results: creatine and phosphocreatine levels in the brain were all significantly higher after weeks supplementation of agentx in knockout mice, compared to creatine and placebo fed knockout mice (phosphorus mrs [ ul brain voxel] with phosphocreatine (pcr) ( ppm) observed only in agentx treated knockout mice. adenosine triphospate (atp) gamma (− . ppm), alpha (− . ppm) and beta (− ppm) peaks are noted in all three knockouts. conclusions: successful treatment was achieved in a slc a brain specific knockout mouse for the second largest known cause of x-linked mental retardation in humans, ctd. disclosure: dr. cecil has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. prevalence of abusive injuries in siblings and contacts of abused children kenneth feldman, md, general pediatrics/children's protection program, university of washington/seattle children's, kfeldman@u.washington.edu purpose or case report: siblings and children who share a home with a physically abused child are thought to be at high risk for abuse. however, rates of injury in these children are unknown. disagreements between medical and cps professionals are common and screening is highly variable. our objective was to measure the rates of occult abusive injuries detected in contacts of abused children using a common screening protocol. this was a multi-center, observational cohort study of child abuse teams who shared a common screening protocol. data were collected for all children < years undergoing evaluation for physical abuse and their contacts. for contacts of abused children, the protocol recommended physical examination for all children < years, skeletal survey and physical exam for children < months, and physical exam, skeletal survey and neuroimaging for children < months old. results: among , children evaluated for abuse, met criteria as "physically abused" and these had contacts. for each screening modality, screening was completed as recommended by the protocol in approximately % of cases. of contacts who met criteria for skeletal survey, new injuries were identified in ( . %). none of these fractures had associated findings on physical examination. physical examination identified new injuries in . % of eligible, examined contacts. neuroimaging failed to identify new injuries among imaged, eligible contacts less than months old. twins were at significantly increased risk of fracture relative to other non-twin contacts ( . % vs . %, or . ). conclusions: these results support physical examinations and skeletal survey, regardless of physical examination results, for contacts of abused children < months of age. too few children had cranial imaging to change recommendations to image contact children less than months old. even for children where no injuries are identified, these results demonstrate that abuse is common among children who share a home with an abused child. they support including contacts in evaluations and interventions (foster care, safety planning, social support) designed to protect physically abused children. the project was supported by the health resources and services administration/maternal shown that pediatric rib fractures may be a marker for significant intrathoracic injury. this information has been used to suggest that children with rib fractures and no underlying intrathoracic injury may have sustained them due to insufficient bony mineralization and minor trauma rather than inflicted injury. methods & materials: irb approval was obtained for a retrospective review of all children under years of age with imaging diagnosis of rib fracture over a -year period at two university hospitals. children with prior thoracotomy, previously recognized metabolic bone disease, and prematurity < weeks were excluded. medical records were reviewed and children with documented abuse or accidental trauma were evaluated. children with indeterminate injury mechanisms were excluded. sixty-six patients with rib fractures were included in analysis, due to abusive injury and due to accidental trauma. children were analyzed for associated intrathoracic, abdominal or intracranial injury, additional fractures and retinal hemorrhage. results: abused children were younger ( . +/− . months) than accidentally injured children ( . +/− . months, p< . ). children with rib fractures due to accidental trauma had a higher incidence of intrathoracic injury compared to those due to abusive injury ( % vs %,p< . ). there was no difference in the incidence of abdominal or intracranial injury between groups. mortality and icu admission rates were similar. abused children had a higher total number of rib fractures (mean . vs . , p< . ) and were more likely to sustain additional fractures outside of the thoracic cavity ( % vs %, p< . ). conclusions: abuse is a more common cause of rib fractures in young children than accidents. children with rib fractures due to abusive trauma are less likely to have intrathoracic injury compared to those sustaining rib fractures due to accidental trauma. this suggests differences in mechanism of injury between groups. pediatric elbow fractures: a different angle on an old topic shannon zingula, md, pediatric radiology, cincinnati children's hospital medical center; kathleen emery, christopher g. anton purpose or case report: the most common elbow fractures classically reported in pediatric orthopedic texts are supracondylar (sc) ( - %), lateral condylar (lc) ( %), and medial epicondylar (me) fractures ( %) with fractures of the proximal radius (including but not limited to fractures of the radial neck) being relatively uncommon ( - %). our experience at a large children's hospital suggests a different distribution. purpose: ) to describe the frequency of different elbow fracture types in a large pediatric population, and ) to determine the fracture types that were occult on initial radiographs but detected on follow-up. methods & materials: review of medical records identified children, median age years and interquartile range for age of - years (range, . - years) diagnosed with elbow fractures at our institution from october through july . initial and follow-up radiographs were reviewed in blinded fashion independently by two experienced pediatric musculoskeletal radiologists to identify fracture type(s) on initial and follow up radiographs. note was made of fractures identified on follow up only. results: the most common fractures included sc (n , %), radial neck (rn) (n , %), and lc fractures (n , %). as compared to classically referenced incidences, rn fractures were seen significantly more (p< . ) and me fractures (n , %) significantly less (p . ) than would be predicted. in patients without fracture seen on initial films, occult fractures were seen on follow up; sc (n , %) and rn fractures (n , %) were most common. the frequency of rn fractures compared to the overall group ( % vs. %) approached but did not reach statistical significance (p . ). patients with one fracture had additional fractures seen on follow-up not seen initially with olecranon fractures most frequent (n , %.) this was significantly more common than the number identified on initial radiographs (n , %) (p< . ). conclusions: sc fractures are the most frequent elbow fracture seen initially and in follow up followed by rn and lc fractures in a distribution different than classically described. the relatively high frequency of rn and olecranon fractures detected on follow up speaks to their potentially occult nature. careful attention to these areas is warranted in patients with initially normal radiographs. purpose or case report: previous studies have found that fractures involving the spine, hands and feet are rare on skeletal surveys for suspected child abuse, leading some authors to suggest eliminating views of these regions from the initial skeletal survey protocol. the purpose of this study was to assess this recommendation by performing a historical review of these injuries in a population undergoing screen-film based skeletal surveys for suspected abuse. this cross-sectional, retrospective irb approved study reviewed the reports of the initial skeletal surveys of all children < years of age with suspected abuse imaged between april, and december, . infants underwent skeletal surveys according to acr standards acquired on a mammographic type screen-film imaging system with at least line pairs per millimeter resolution. studies in toddlers were performed using a par speed screenfilm system. results: % ( / ) of all skeletal surveys demonstrated positive findings, and % ( / ) had > fracture. . % ( / ) of all studies had fractures involving the spine, hands or feet. of all positive skeletal surveys, . % ( / ) had fractures involving the spine, hands or feet, and . % ( / ) of all patients with > fracture on skeletal survey had fractures involving these regions. conclusions: these data, acquired in the screen-film era, suggest that fractures of the spine, hands and feet may not be rare in infants and toddlers in cases of suspected child abuse. the benefits of eliminating views of these regions from the initial skeletal survey should be carefully weighed against the cost of missing these potentially important injuries in at-risk pediatric populations. purpose or case report: dating fractures is critical in cases of suspected infant abuse, but there are little scientific data to guide radiologists, and dating is generally based on personal experience and conventional wisdom. we previously reported a scientific scheme for dating fractures in infants based on an analysis of subperiosteal new bone and callus formation in birth-related clavicular fractures. we hypothesize that when used as a guide this system can significantly improve the ability of radiologists to accurately date fractures in young infants. methods & materials: radiographs of presumed birthrelated clavicular fractures in infants - months were reviewed by pediatric radiologists with (reader a) and (reader b) years experience in two reading sessions separated by one year. for the first read, no guidelines were provided. training was carried out prior to the second session, and readers were given the dating scheme as a guide during fracture analysis. readers were asked to provide an estimate of the minimum and maximum fracture age in both sessions. the primary outcome was whether or not the reader's estimated range for fracture age included the actual fracture age. a secondary outcome was the width of the estimate of fracture age. these outcomes were compared across the two reading sessions. results: the rate of correct response significantly increased after training for each reader (reader a: % to %, p<. ; reader b: % to %, p . ). the width of estimated fracture age after training was significantly smaller for each reader (reader a: mean width days to days, p<. ; reader b: days to days, p . ). conclusions: our results suggest that the ability of a radiologist to accurately date fractures can improve significantly when provided with a scientifically based system outlining patterns of fracture healing. this scheme can be applied in radiologic practice and may prove particularly useful in cases of suspected abuse, where fracture dating often has forensic implications. purpose or case report: to demonstrate the acute and subacute features of proximal femoral physeal fractures in the abused child. also to demonstrate how to recognize this injury in patients with unossified femoral heads. the database of patients with suspected non-accidental trauma, accumulated over years, was reviewed. out of a total of patients ( %) were proven to be cases of non-accidental trauma, as determined by the child abuse pediatrician. from these patients, the cases of proximal femur growth plate fractures were identified. results: patients with proximal femur growth plate fractures were identified for a prevalence of . %. one patient had bilateral proximal femoral fractures, for a total of fractures in patients. were boys, were girls with ages ranging from . mos to yrs mos. in patients, the fracture was revealed on imaging performed because of refusal to bear weight; in the other patients, the fracture was found during imaging for the skeletal survey. the fracture was on the left side in cases and on the right side in (the patient with bilateral fractures). in all of the fractures, there was lateral displacement of the femoral shaft. in fractures, the femoral head was not yet ossified simulating the appearance of a dislocation. location of the femoral head in the hip joint was verified by ultrasound or ct (ct abdomen had already been done in patient) thus delineating the presence of a physeal fracture. / fractures were salter-harris i and the other were salter-harris ii fractures. the fracture was acute in cases and subacute in cases. in these subacute cases, periosteal reaction and/or calcifying subperiosteal hemorrhage was present in , and irregularity and scalloping of the metaphysis was present in the other . conclusions: proximal femoral growth plate fractures are quite uncommon in non-accidental trauma. the injuries are typically salter-harris i or ii fractures, seen more often in the healing phase. in the presence of an unossified femoral head, the laterally displaced femoral shaft can simulate hip dislocation; this can be clarified with hip sonogram. purpose or case report: in recent years, metal stents have been used to overcome airway obstruction in children for whom no better surgical option is available. these devices are not designed for use in the airway, however, and may cause significant complications. bioabsorbable airway stents may avoid some of the problems associated with metal stents. methods & materials: this is a retrospective review of all endoluminal insertions of bioabsorbable airway stents at a single institution from april to september . custom-made polydioxanone stents of various sizes (ella dv, ella, czech republic) were used. results: twelve stents were inserted in the airways of seven children. indications were: recurrent obstruction after slide tracheoplasty ( ), persistent airway compression after correction of a congenital cardiac lesion ( ), collapse of stem cell supported tracheal homograft, tracheomegaly following fetal balloon insertion, and syndromic tracheobronchomalacia (tbm). eleven stents (diameters to mm) were placed in the trachea and one in the left main bronchus. two stents had to be removed and replaced for technical reasons (one was too long and the other too narrow). the child with syndromic tbm died when treatment was withdrawn because she could not be weaned from the ventilator. the remaining children are alive at a median follow-up of nine months (range to months). the granulation tissue response was similar to that seen after placement of metal stents. the stents were observed to absorb gradually over a period of approximately three months, requiring serial stenting in two children. conclusions: bioabsorbable airway stents are more difficult to insert than metal stents. they cause similar early complications, especially granulation tissue formation, but appear to avoid potential long-term complications of metal stents, including vascular erosion and growth limitation. disclosure: dr. mcclaren has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: renal angiomyolipomas (amls) in tuberous sclerosis complex (tsc) grow at a faster rate, exhibit a wider and more problematic range of symptoms, and hemorrhage more frequently than sporadic amls. we examined the efficacy of prophylactic embolization of renal amls in tsc in decreasing tumor size, alleviating symptoms, and preventing hemorrhage while preserving renal function. we retrospectively reviewed the charts and imaging studies of consecutive patients who underwent transarterial, transcatheter embolization of amls. tumor volume was measured from available ct or mri imaging before and after embolization. pre-and postembolization symptoms and creatinine levels were documented. results: patients had available follow-up imaging at a mean of months post-embolization. the mean preembolization tumor volume was ml and postembolization was ml; median decrease in volume was %. using the schwartz method, the mean glomerular filtration rate before embolization was calculated to be . ml/min/ . m . after embolization the mean value was statistically unchanged at . ml/min/ . m . none of the patients experienced renal hemorrhage or symptom recurrence during the follow-up period. conclusions: selective embolization of renal amls in patients with tsc decreases tumor volume, relieves symptoms and reduces the risk of future hemorrhage while preserving renal function. etoh was injected percutaneously with g needle; transductal ablation performed through a f micropuncture sheath. drug volumes, technical difficulties, percentage reduction in saliva production, family reported clinical significance, and complications were recorded. results: salivary gland ablation (sga) included bilateral smg and slg ablation without parotid gland ablation in cases, and with unilateral parotid gland ablation in cases. one case of bilateral parotid gland ablation following surgical resection of bilateral smgs. mean etoh dose for smg . ml, and . ml for slg. one case of focal skin necrosis was noted; no other complications. patient families reported response to sga in / cases ( %) with mean saliva production of %. greatest health and family impact was reported with elimination of hospitalizations for recurring aspiration pneumonia ( cases), elimination of choking in bed ( cases) , and improved patient sense of self-hygiene in cases. one complication occurred with temporary marginal mandibular nerve paralysis (resolution in months). conclusions: percutaneous and transductal sga is feasible, safe, and effective in this small patient series, offering an alternative to surgical salivary gland resection, or treatment option following failed surgical intervention. paper #: pa- mr-guided procedures in children: initial experience joao amaral, md, diagnostic imaging, the hospital for sick children, joao.amaral@sickkids.ca; michael temple, dimitri parra, philip john, bairbre connolly purpose or case report: the primary purpose of this study was to review our initial experience with mr-guided procedures in children. our secondary objective was to share some aspects on how to start an mr-guided program in a tertiary pediatric center. patients with lesions identified only on magnetic resonance (mr) imaging were selected to undergo an mr-guided procedure. patients' demographic data, primary diagnosis, referring team's clinical suspicion, lesion's anatomical location, tissue adequacy for pathology, final diagnosis and clinical follow up were reviewed. aspects of starting a program of mr-guided procedures, safety concerns, imaging and technical challenges, and mr compatible materials were also addressed. results: to date, procedures ( bone biopsies, soft tissue biopsy and pre-surgical needle localization) were performed in patients during months. there were girls and boys with a mean age of . years ( y mo- yrs). one patient had a nasopharyngeal carcinoma, cardiofacial syndrome, wilm's tumor and had no previous medical issues. the clinical suspicion for procedures in patients was metastatic disease and for procedures in patients was primary malignancy or infection. lesions were located in the tibia ( -metaphysis and diaphysis), femur ( -metaphysis and epiphysis), thigh ( -soft tissues), sacrum ( ) and retroperitoneum ( ). all biopsies provided adequate tissue for diagnosis. needle localization and hook deployment was also accurate. malignancy was excluded in all patients. final diagnosis included chronic recurrent multifocal osteomyelitis (crmo), osteomyelytis, fibrous tissue, osteoid osteoma, and scar tissue. mean follow up was . months. no patient required a second procedure to confirm the diagnosis. conclusions: mr with its unique soft tissue resolution and lack of ionizing radiation is an excellent method to guide interventions in children. one of the greatest advantages of this method is the precise target localization especially in lesions located in the bone marrow or lesions better identified on mr. special safety measures, specific mr compatible material (needles, surgical instruments), dedicated imaging techniques to reduce or increase material/needle artifact and careful technique are paramount. - . m, f; . - months, mean . months, median months, mode months. sonographic approach expanded as our experience grew over months. studies performed by a single pediatric radiologist. bilateral sonography included: interscalene and supraclavicular neck, nerve roots at neural foramina, cervical spinal canal, diaphragm during spontaneous respiration, rhomboid muscle, serratus anterior muscle, posterior shoulder, all performed and interpreted blind to other imaging. results: interscalene and supraclavicular neck evaluated in all patients. all exhibited echogenic interscalene portion of brachial plexus.size and extent of traction neuroma varied. nerve roots at foramina noted in axial and coronal planes. in cases enlarged root(s)noted. cervical spinal canal studied in patients: cord oscillated normally, no syrinx, cord concentric in canal. intracanalicular traction pseudomeningoceles on concurrent ct myelography or mri were not apparent on us. in cases a "clumped" retracted nerve root on the cervical cord was later found to correspond to a pseudo-meningocele on ct myelogram. otherwise, cervical spinal canal us was unremarkable in cases. diaphragm motion was evaluated in patients during spontaneous respiration; no phrenic nerve palsy. rhomboid muscle was evaluated for atrophy in patients; had atrophy. the rhomboids are innervated by the dorsal scapular nerve which arises solely from c , prior to c joining the brachial plexus. intact rhomboid indicates that the central c root is intact. serratus anterior muscle, innervated by the long thoracic nerve (c ,c ,c ), was evaluated for atrophy in patients; had atrophy. dynamic evaluation of the posterior shoulder looking for posterior laxity was evaluated in patients; had laxity. posterior shoulder dislocation or subluxation is a known sequela of brachioplexopathy which sometimes requires muscle transfer when the child is older. conclusions: comprehensive us evaluation of perinatal brachioplexopathy detects: extent of traction neuromafibroma from the interscalene region peripherally toward clavicles (important for neurosurgeon), thick nerve roots, phrenic nerve diaphragm palsy, muscle atrophy from denervation, and posterior shoulder subluxation. us misses: intracanalicular traction pseudomeningoceles. paper #: alt- impact of the image gently campaigns in adult-focused hospitals: a survey of practice leaders brett bartz, duke university medical center; donald frush, kimberly applegate, michael callahan, laura coombs, marilyn goske purpose or case report: the alliance for radiation safety in pediatric imaging is an organization that uses social marketing to promote radiation protection for children and effect change across radiology practices. the impact of the alliance's image gently campaigns on practice patterns in radiology practices has yet to be assessed, especially outside of freestanding children's hospitals. the purpose of this investigation was to assess the impact of the image gently campaigns on academic and private practices/institutions that treat children but primarily serve adults. a web-based survey was emailed to leaders in radiology practices (n ) who do not practice at freestanding children's hospitals utilizing the acr's pred database. the survey consisted of questions designed to measure the recognition and impact of the image gently campaigns, including the impact on practice patterns. results: a total of practice leaders in u.s. states and territories responded for a response rate of . %. the majority ( %) of sites image pediatric patients in their practices. respondents consisted of department chairs ( %), group presidents/ceos ( %), and division chiefs ( %). the majority ( %) of respondents described their practice as a hospital-based private practice without a dedicated pediatric radiology division. the vast majority ( %) of respondents was familiar with the image gently campaigns; % of respondents reported that image gently had effected a change on how they imaged children. specifically, respondents (%) reported that the campaign caused a modification to lower dose protocols for head ct ( %), chest ct ( %), and abdominal/pelvic ct ( %). slightly more than half of respondents ( %), however, estimated that the image gently campaign resulted in no modification of pediatric fluoroscopy exposure. conclusions: to our knowledge, this is the first survey evaluating the impact of the image gently campaigns. there is near universal recognition of the campaigns, which have impacted practice patterns beyond the freestanding children's hospital in ct, but not in fluoroscopy. reliability of shear-wave velocity using different frequencies in acoustic radiation force impulse (arfi) elastography mi-jung lee, radiology, severance children's hospital, mjl @yumc.yonsei.ac.kr; suyon chang, myung-joon kim purpose or case report: although there are many studies about acoustic radiation force impulse (arfi) measurement, standard protocol has not been established. and a new probe with high frequency has been developed which can be applied for pediatric patients. the purpose of this study was to assess the reliability of shear-wave velocity (swv) at various depths using different frequencies to suggest standard measurement in arfi elastography. methods & materials: arfi elastography of both the elasticity phantom and normal liver was performed at different depths ( - cm) with convex ( - mhz) and linear ( - mhz) probes. ten valid swv measurements at each depth were performed. it was repeated ten times with the phantom and it was done in healthy volunteers (m:f : , age - years; mean . ). the mean value and standard deviation of swv were calculated. results: in both the elasticity phantom and the liver, variability of swv was different between the depths in both probes. the depth with lower variability in the phantom was and cm with the convex probe and cm with the linear probe. in the liver, the depth with lower variability was cm with the convex probe and and cm with the linear probe. in comparison of two probes, the linear probe showed lower variability at and cm depth in the phantom and at cm depth in the liver whereas the convex probe showed it at cm depth in both the phantom and the liver. conclusions: in arfi elastography, measurement of depth shows different variability in both low and high frequency probes. to obtain the most reliable measurement of swv, using high frequency probe is recommended for - cm depth and using low frequency probe is recommended for - cm depth. disclosure: dr. lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. imaging -year-old fetuses sabah servaes, children's hospital of philadelphia; teresa victoria, ann johnson, sandra kramer, richard markowitz, diego jaramillo purpose or case report: to demonstrate normal anatomy and pathology of medical museum specimens without disturbing the specimens. methods & materials: nine fetal specimens from a medical museum were imaged with ct and mri ( . t and . t) when possible with the specimens in their preserving fluid and containers. results: the fetal specimens are estimated to be approximately years old. one specimen is from the first trimester, seven are from the second trimester, and one is from the third trimester. normal anatomical structures at various stages of development including the brain (and varied sulcation pattern), lungs (lobar anatomy), and skeletal structures (several developmental features such as the ossification centers, perichondrial structures, and marrow cavitation) can be evaluated using imaging without causing harm to the specimens. pathologic entities including anencephaly and sirenomelia are also evaluated demonstrating features of these entities. conclusions: imaging historical fetal specimens provide an opportunity to evaluate normal developmental changes and pathological entities and also to gain a better understanding of the museum pieces without damaging the museum specimens. pediatric ct interpretations: does a tertiary care radiologist make a difference? wendy d. ellis, monroe carell jr. children's hospital at vanderbilt university; sumit pruthi, david johnson, christopher eakins, chang yu, marta hernanz-schulman purpose or case report: to determine whether a substantive difference exists between the pediatric imaging reports of community radiologists and reinterpretations by tertiary care radiologists at a free-standing children's hospital; and how those interpretations were related to the final diagnosis. methods & materials: this retrospective review examined the computed tomography (ct) reports of all pediatric patients referred to our tertiary care children's hospital over a month period ( / / - / / ). the outside reports and the requested second interpretation reports were compared and their content categorized as "agreement" vs. "disagreement: major or minor". a representative sample of major disagreements in which there was reliable followup information was correlated with the final diagnosis to determine if there was added value provided by the reinterpretation. results: ct scans from patients were submitted for reinterpretation. disagreements were found in / cases ( . %); with . % ( / ) classified as major disagreements. among the neurologic cases, major disagreements occurred in patients ( . %) and minor disagreements in patients ( . %). among the body scans, major disagreements occurred in cases ( . %) and minor disagreements in cases ( . %). in the cohort of cases reviewed for final diagnosis, the second read interpretation was more accurate in . % of cases with a p-value of < . (neurologic . %, p < . ; body . %, p < . ). conclusions: in our review, discrepancy rates between community and tertiary care radiologists in interpretation of pediatric ct scans were substantial, with discrepancies occurring in more than % of cases. further review of the cases for final diagnosis, showed that a significant number of the tertiary care interpretations were more accurate. possibilities that may account for this discrepancy include subspecialty training and elapsed time since performance of the study, which might provide additional clinical data in some cases. diagnostic ct scans performed at outside institutions should not be repeated considering added radiation burden to the child and additional expense. our data indicates there is added value to the reinterpretation which impacts the accuracy of the report (as assessed by the final diagnosis), and should be recognized by payors as integral to optimal patient care. ionizing radiation exposure from radiography in the neonatal intensive care unit-per-patient cumulative effective doses amaya basta, radiology and biomedical imaging, ucsf; jesse courtier, john mackenzie purpose or case report: to better understand the levels of exposure to ionizing radiation for infants in the neonatal intensive care unit (nicu). we retrospectively collected the number and types of radiographs performed per infant in our nicu by searching our radiology information system database over a five-year period. we focused on the most common examinations ( % of all radiographs) and assigned each an estimated equivalent dose based on published literature: chest and abdomen . micro sieverts (μsv), one-view chest . μsv, abdomen . μsv, twoview chest . μsv, two-view abdomen μsv. we then calculated a cumulative equivalent dose (ced) for each infant based on the number of each type of examination they received. descriptive statistics were generated to depict the distribution of number of examinations and ced. results: over five years, , infants cared for in our nicu received at least one radiograph of the chest and/or abdomen. the number of examinations obtained on these infants was . , , , (mean, median, minimum, maximum). the st quartile was and the rd quartile was examinations. the cumulative equivalent dose these infants received was . , . , . , , . μsv (mean, median, minimum, maximum) . the st quartile was . and the rd quartile was . μsv. two hundred infants ( . % of the study population) received a ced of over μsv. conclusions: descriptive statistics provide a valuable assessment for the broad range of radiation that infants receive in the nicu. although the distribution is skewed towards a low level of exposure, a subset of patients ( . %) received a ced of over μsv. identification of factors that cause infants to enter this group will be important for future dose reduction strategies. poster #: cr- congenital cardiac fibroma: a case report earic bonner, meharry medical college, ebonner @email. mmc.edu; seth crapp, david parra purpose or case report: a -week-old male presented to his pediatrician with a ii/vi systolic ejection murmur along the left sternal border. he had mild tachypnea without cyanosis. his oral intake was adequate with no evidence of failure to thrive. he was referred to a pediatric cardiologist who performed an ecg and a transthoracic echocardiogram. the ecg showed normal sinus rhythm at beats per minute with no abnormalities. the transthoracic echocardiogram showed a x x mm homogeneous mass originating from the anterior free wall of the right ventricle, and mild dilation of the right ventricle. mild dynamic subpulmonary stenosis and a secundum atrial septal defect were also noted. although the murmur was significantly louder at one month follow-up, a repeat echocardiogram did not reveal any increase in the size of the mass. at months of age, a cardiovascular magnetic resonance imaging (cmri) study under general anesthesia was performed. cmri revealed a x x mm cardiac tumor that was causing narrowing of the right ventricular outflow tract. the tumor was hypointense on t -weighted imaging and hyperintense on t weighted imaging, with positive delayed enhancement. these findings, along with the size and location of the mass, are consistent with a diagnosis of a cardiac fibroma. chest mra, that was also performed, showed normal extracardiac vascular anatomy with no evidence of peripheral branch pulmonary stenosis. cardiac fibromas do not usually increase in size; however, the concern is the child's risk of arrhythmias. frequent holter monitoring was recommended for this patient. considerations were also made for an electrophysiology study in the next - years to determine the risk of ventricular ectopy. at that point, the patient can be assessed for the possibility of resection of the fibroma. purpose or case report: treatment of pulmonary atresia is complex and demands intricate solutions. one solution is the creation of a conduit between the right ventricle and the main pulmonary artery. the lifespan of these conduits is limited by progressive occlusion over time, which can be treated with endovascular stent placement in lieu of surgical re-intervention. however, these stents are at high ( %) risk for fracture, typically at the stent waist. the radiologist should be aware of this complication, as they may be the first to identify it on chest radiograph. the purpose of this electronic poster is to familiarize radiologists with this entity by presenting cases of stent fracture and migration. methods & materials: over a month period, we identified three children with rv-pa stent fractures and associated stent migrations on chest radiography. imaging analysis was focused on the appearances of these fractured stents. patient management and outcomes were reviewed. results: three children, males, female (ages , , and years) were found to have asymptomatic rv-pa conduit stent fractures with fragment migration. one chest xray was performed in the er for fever and cough; one was pre-op for gi surgery; one was done to confirm abnormal findings seen on a routine cardiac echo. the time between stent placement and fracture detection ranged from to months. two patients had stent fractures and embolizations to the right ventricle that required open surgery to remove stent fragments. the third patient had embolization to both pulmonary arteries, but did not require treatment. all patients did well. conclusions: stent fractures and migrations are a relatively common complication of rv-pa conduit stent placement. pediatric radiologists need to be aware of this complication in order to provide value-added interpretations. purpose or case report: we describe the case of a week stillborn fetus with a . cm diameter craniopharyngioma detected by ultrasonography. a g p woman in her third decade had ultrasonographic examination showing hydrocephalus, polyhydramnios and an intracerebral mass. the nature of the mass was uncertain and intracerebral hemorrhage was considered. the pregnancy was terminated at weeks gestation. at postmortem examination the decedent was a g male fetus with a head circumference of . cm and a crown-rump length of . cm. anterior and posterior fontanelles appeared large. no other external abnormality was found. the placenta was unremarkable and cytogenetics on placental tissue showed a normal male karyotype. examination of fetal viscera was remarkable for mildly underweight adrenal glands ( . g, expected . g) and hepatomegaly ( . g, expected . g). intracranial csf was increased in volume. there was a suprasellar . cm diameter somewhat gritty, but smooth-surfaced tumor. the brain and tumor together weighed g. the floor of the cranium and sella turcica were grossly normal. histologic examination of the tumor showed an adamantinomatous type craniopharyngioma with characteristic epithelium, stellate reticulum, focal keratinizing squamous epithelium and calcification. pre-and postnatal mri of caudal regression syndrome claire b. beaumont, md, university of arkansas for medical sciences, cbbeaumont@uams.edu; nafisa k. dajani, leann e. linam purpose or case report: caudal regression syndrome is a rare form of caudal dysplasia characterized by a spectrum of findings including agenesis of the lumbosacral vertebra, multiple orthopedic deformities in the lower limbs, as well as anomalies of the gastrointestinal and genitourinary tracts. the mechansim of caudal regression syndrome is not completely understood but is believed to be secondary to a defect in the induction of caudal elements. mri is a valuable tool for identifying the specific anomalies involved with caudal regression syndrome on a case-by-case basis. the following is a case from our institution which includes both pre-and postnatal mri. unsuspecting tuberous sclerosis diagnosed on neonatal cranial ultrasound vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; puneet gupta, richard thomas, vaseem iqbal, jan najdzionek. purpose or case report: tuberous sclerosis (ts) is a rare autosomal dominant genetic disorder causing hamartomatous proliferation in number of organ systems. because the classical triad of epilepsy, mental retardation and adenoma sebaceum is not commonly seen on clinical examination, imaging plays a central role in the diagnosis and treatment of tuberous sclerosis. central nervous system features of ts include subependymal nodules, cortical tubers, subependymal giant cell astrocytoma, white matter bands and cysts. in patients with ts, cerebral involvement in the form of subependymal nodules is seen in % to % and white matter abnormalities are noted in % to % of cases. knowledge of expected radiological features is thus important in making the correct diagnosis. recent studies have indicated that earlier appearance of brain lesions indicate a greater risk of mental retardation and a more severe clinical course. we present a case of a -day-old neonate who was referred to us with concerns for hydrocephalus. the cranial ultrasound demonstrated multiple echogenic subependymal nodules of varying sizes and mild asymmetry of the ventricles. the differential diagnosis included ts, torch infections, and x-linked subependymal heterotropia. areas of increased echogenicity were noted within the white matter of the left frontal lobe, which favored ts. subsequently, an mri was performed to validate these findings and assess for additional white matter lesions. the mri showed classic manifestations of ts that included periventricular lesions and streaky, linear, wedge-shaped hyperintensities on flair imaging. a noncontrast ct scan was also performed which revealed classic calcified subependymal nodules. cardiac rhabdomyoma and renal angiomyolipoma are the other recognized manifestations of ts and were respectively excluded by subsequent echocardiogram and renal ultrasound. pyloric atresia with epidermolysis bullosa: fetal mri diagnosis with postnatal correlation arnold c. merrow, md, radiology, cincinnati children's hospital medical center, carl.merrow@cchmc.org; jason s. frischer, anne w. lucky purpose or case report: pyloric atresia (pa) is an uncommon disorder, accounting for % of congenital gastrointestinal atresias. up to % of cases have associated anomalies, the most common of which is epidermolysis bullosa (eb). prenatal findings have been reported sonographically for each of these anomalies, both in isolation and in the rare case of association. a case of isolated pa has been reported by fetal mri. we present the first reported case of pa with eb diagnosed by fetal mri with corroborative postnatal imaging and surgical findings. the mother of this child was initially referred to the fetal care center of cincinnati at weeks gestation for a possible myelomeningocele diagnosed by prenatal ultrasound at an outside facility. these ultrasound images were not available for review at the time of our workup. a fetal mri was the first study to be obtained at our institution. the mri showed no myelomeningocele or brain anomalies. the stomach was moderately enlarged throughout the exam and did not empty. subjective polyhydramnios was also noted. no duodenal dilation was seen, and there was minimal fluid in the distal bowel loops. this constellation of findings raised concern for pyloric atresia, resulting in a careful search for any sign of epidermolysis bullosa due to a known association of these disorders. prominent debris was seen layering dependently in the amniotic fluid and in the dilated fetal stomach, and the external ears were abnormally small and misshapen. the pa-eb association was proposed as the underlying diagnosis based on our mri findings. it was also postulated that skin blistering over the lumbosacral spine at the time of the prior outside ultrasound could have mimicked a myelomeningocele, thus prompting the referral to our center. at delivery, the baby had numerous skin defects, and the ears were malformed. an abdominal radiograph obtained after nasogastric tube placement and air injection showed no gas beyond the stomach. a pyloric ultrasound showed a distended stomach without a patent pyloric channel to the duodenal bulb, consistent with pyloric atresia. a skin biopsy confirmed epidermolysis bullosa, and the patient underwent a resection of the pa with gastroduodenostomy. the baby subsequently expired less than two weeks later, most likely due to sepsis based on wound cultures and autopsy results. our case demonstrates the ability of fetal mri to diagnose this rare condition and highlights the key imaging manifestations of the pa-eb association. disclosure: dr. merrow has indicated that he is an author for amirsys and receives a royalty accordingly. purpose or case report: we demonstrate a case where the changing position of the contrast filled appendix lead to the diagnosis of malrotation, with review of the embriology of intestinal rotation. a newborn preterm female presented with a golf ball sized umbilical mass, that reduced by itself, thought to represent an umbilical hernia vs omphalocele. she was unstable to undergo an upper gi exam under fluoroscopy, therefore a limited contrast study was performed at bedside and was inconclusive for malrotation. subsequent nicu radiographs showed changing position of the appendix filled with residual contrast, visiting all quadrants of the abdomen in a random pattern over a few days period. this confirmed our suspicion for malrotation. it is well know that in malrotation the position of the cecum can be variable, most commonly located in the right upper quadrant or left lower quadrant. to our knowledge it has not been described yet that the changing position of the appendix can lead to the diagnosis of malrotation. through this case we display the embriology of the intestinal rotation and the radiologic signs of malrotation. poster #: cr- mr imaging patters of liver transplant complications in the pediatric population edward richer, md, emory university, richerej@gmail. com; adina alazraki, jonathan loewen purpose or case report: pediatric liver transplantation is a relatively common surgery, with more than transplants in the united status annually. the spectrum of post transplant complications has been previously described, primarily utilizing ultrasound. as mri has become a more widely used technique in pediatric imaging, and ultrasound findings may be non-specific, knowledge of mr imaging patterns is an important adjunct in the post-transplant evaluation. we present a spectrum of complications, including vascular, biliary, hepatic parenchymal, and systemic complications. methods & materials: using an electronic record system, we identified pediatric patients with prior liver transplantation who subsequently underwent abdominal mri at our institution and were found to have a post transplant complication. patient management and outcomes were reviewed. results: our review of a subset of the available patients shows vascular complications to be the most commonly encountered abnormality at our institution, including hepatic artery stenosis/thrombosis, and portal vein stenosis/ thrombosis, cavernous transformation of the portal vein. biliary complications were relatively common, including bilary stenoses and bilomas. hepatic parenchymal and systemic complications, such as ptld, were less common. we demonstrate the mr imaging patterns of these complications. conclusions: pediatric liver transplantation is a relatively common surgery, and the mri appeance of post transplant complications warrants illustration as abdominal mri becomes more widely used in pediatric imaging. we present a pictorial review of common patterns of complication. imaging of progressive familial intrahepatic cholestasis (pfic) matthew d. dobbs, md , radiology, vanderbilt university medical center, matthew.dobbs@vanderbilt.edu; sumit pruthi, stephanie e. spottswood purpose or case report: progressive familial intrahepatic cholestasis (pfic) is a relatively rare pediatric liver disease due to a genetic mutation (abcb gene on chromosome q - ) in a bile salt export protein causing cholestasis leading to chronic inflammation within the biliary system. the diagnosis is made clinically with detection of a low ggt in the face of an elevated bilirubin and alkaline phosphatase. genetic testing confirms the diagnosis. one of the subtypes, type , was shown in to be highly related to the development of hepatocellular carcinoma. the vast majority in children in this study developed hcc at less than years of age. radiological contribution to the management of these chronic liver disease patients is to perform surveillance imaging to detect hcc. due to the rarity of this condition, almost no reports exist in the radiological literature describing the imaging features or management of this condition. our presentation will review the imaging findings in our small population of pfic type patients on us, ct, and mri. we will also review suggested surveillance imaging techniques and imaging algorithms. renal rhabdoid mimics wilms tumor vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; paul montgomery, jan najdzionek, vaseem iqbal purpose or case report: in the past most pediatric renal tumors have been classified together under the umbrella of wilms tumor. however, over the last decade with advancement in imaging, several distinctive imaging features specific to renal tumors have been recognized which aid in their classification as being distinct pathologically. we present a case of rhabdoid tumor where in the primary tumor arose from the kidney. it had classical imaging features of wilms tumor. we want to highlight that even with the most sophisticated imaging techniques, specific renal tumors cannot always be diagnosed with preoperative imaging and how this alters the management and prognosis for child with a renal mass. in our case, the postoperative findings, pathology and immunohistochemical techniques confirmed a rhabdoid tumor. differentiation of these two tumors is essential since in patients with rhabdoid tumor survival is poor with -year overall survival rates of % for stages i and ii and % for stages iii, iv, and v. on imaging, there are several features that suggest the diagnosis of rhaboid tumor. these include subcapsular fluid collections, linear calcifications outlining tumor lobules, and vascular invasion. also, a pertinent feature of rhabdoid tumor due to its aggressive nature is the presence of lung metastasis ( %) and synchronous malignant brain lesions ( %). these findings were not present on our case, which led us in formulating a diagnosis of wilms. our patient is unusual in the fact that the local renal findings and absence of metastasis, synchronous malignant lesions, and vascular invasion led us to an incorrect diagnosis of wilms tumor. in conclusion, we would like to stress that diagnosis of rhabdoid tumor of the kidney on imaging presents a challenge because of its imaging similarity to wilms tumor. ectopic ureters in young infants: mru findings shin-lin shih, md, department of radiology, mackay memorial hospital; yi-fang chen, chun-chao huang, fei-shih yang purpose or case report: to localize the terminations of ectopic ureters by mri methods & materials: mr urography (mru) was conducted in four female patients with hydroureter and a suspected ectopic orifice. mr imaging was performed with a t mr scanner (achieva; philips). the imaging protocol mainly consisted of a single-shot t -weighted turbo spin echo sequence with a slice thickness of mm and multiplanar reformations. the ages of the four patients were day, days and months (for two). the latter two patients presented with urinary tract infection. the newborn patients presented with abnormal prenatal examination. the pertinent findings and descriptions of a variety of renal anomalies were described. results: the locations of the ectopic ureters were two in the vagina, one in the uterus and one in the bladder neck. the associated renal anomalies were a right duplex kidney in four, a left duplex kidney in one, a left ectopic dysplastic kidney in one and vesicoureteral reflux in one (confirmed by vcug). conclusions: mru may demonstrate the exact point of termination of an ectopic ureter and also the associated renal anomalies. poster #: cr- acquired polycystic kidneys in neuroblastoma survivors richard bellah, , radiology, the children's hospital of philadelphia, bellah@email.chop.edu; bernard kaplan, camilo jaimes, yael p. mosse, jill p. ginsberg, kevin e. meyers purpose or case report: neuroblastoma (nbl) is the most common extracranial solid malignancy of childhood. with current therapy, the prognosis and long term survival of patients affected by this condition has dramatically improved. nevertheless, the treatment for nbl may account for some complications further in life. in patients with neuroblastoma, acute renal failure can occur usually as a result of a thrombotic microangiopathy associated with bone marrow transplantation. in addition, end-stage renal disease has been reported in long-term survivors of nbl. this exhibit describes and illustrates the first case series of five patients with treated nbl in whom the imaging features of polycystic kidney disease (pkd) developed over time, and in some cases, as progressive renal failure ensued. methods & materials: medical and imaging records were reviewed (irb approved) of patients with treated nbl in whom pkd became apparent during the course of followup imaging. results: five patients displayed findings of pkd on us and/or ct. three of the five patients (where images were available) had normal renal imaging at time of nbl diagnosis. the mean age at nbl diagnosis was . years (range . - . yr). the mean age at time pkd was detected was . years (range - yrs). none of the patients had a family history of pkd, or had previously undergone dialysis. all patients received chemotherapy and total body irradiation prior to bone marrow transplantation. four patients survived nbl therapy but eventually developed end-stage renal disease. conclusions: an association between acquired pkd and nbl has not been previously reported. the etiology of this observation is still unclear, but a toxic insult is likely to account for the renal changes. further research is needed to establish the epidemiology, prognosis, and etiology of this association. abnormal migration of the retention anchor suture in a case following gastrostomy tube insertion surendra narayanam, mbbs, dmrd, dnb, division of image guided therapy, department of diagnostic imaging, the hospital for sick children, nrssbabu@gmail.com; joao amaral, luke toh, bairbre connolly, vicente deoliveira, dimitri parra purpose or case report: during percutaneous gastrostomy tube placement, retention anchor suture(s) are deployed into the stomach to tack the anterior gastric wall to the abdominal wall. in our practice the thread of the retention anchor suture is cut at days and the metallic portion passes pre rectum. we report an interesting and very rare migration of the metallic portion of the retention anchor suture in post-primary gastrostomy tube insertion. an -month-old girl, with a mitochondrial disease and severe hypotonia underwent percutaneous gastrostomy placement. during the procedure the retention anchor suture thread snapped and the metallic portion of the suture remained within the stomach. day post procedure, the child became uncomfortable, so a gastrostomy tube check was performed. the suture was not visible in the abdomen on abdominal x-ray or fluoroscopically. on close review of the images, the suture was found projected over the distal esophagus. initial impression was the anchor suture had refluxed into the esopahgeal lumen. careful attempts were made to remove it along with the nasogastric tube, from above under fluoroscopic control. however on withdrawal of the nastogastric tube, the retention anchor suture moved enbloc with the nasogastric tube. once removed the retention anchor suture was confirmed to be within the nasogastric tube. this case illustrates the importance of examining the chest x-ray carefully before assuming a retention anchor suture has passed. to understand the appropriate post procedural radiographic workup and its technique for timely diagnosis. . to learn the potential complications of delayed diagnosis. pediatric retroperitoneal synovial sarcoma ahmad aouthmany, university of toledo medical center, ahmad.aouthmany@utoledo.edu; asif abdullah purpose or case report: pediatric synovial sarcoma most commonly affects the extremities, especially the lower thigh and knee region; other primary sites such as the retroperitoneum have been only infrequently reported. we report an extremely rare case of a retroperitoneal synovial sarcoma masquerading as retroperitoneal hematoma in a -year-old white female with non-traumatic back pain and non-contrast enhanced ct findings of right quadratus lumborum and psoas region presumed hematoma. coagulation studies revealed factor xi deficiency also known as hemophilia c. however, on follow-up imaging, the presumed retroperitoneal bleed persisted and a subsequent mr examination revealed a solid enhancing mass. ct, mr, and fdg-pet findings as well as a brief histopathology are discussed. our case is rare in the regards that the tumor occurred in an uncommon retroperitoneal location in a pediatric patient and was mimicking a retroperitoneal hematoma which posed a significant diagnostic challenge. despite a rare entity, synovial sarcoma among other sarcomatous lesions maybe considered in the differential consideration of a spontaneous retroperitoneal hematoma even in hemophiliac patients. longitudinal bracket epiphysis michael jubang, geisinger, mjjubang@geisinger.edu; farzad sedaghat, william j. malone, george wu, william mirenda purpose or case report: longitudinal bracket epiphysis is a rare anomaly with multiple synonyms such as delta bone, triangular bone, and congenital angular deformity. the purpose of this case report poster is to discuss an -month-old male born with an adducted right great toe with a broad nail and a notch in the center of the distal phalanx. the review will discuss radiographic findings, the natural progression of the disease, the treatment options, the mri findings used for pre-surgical planning, and associated pathology. whole body mri in pediatric non oncologic diseases: pictorial review ramy el jalbout, md, radiology, chu sainte justine, ramy.jalbout@yahoo.com; vijay moorjani purpose or case report: with the advances in scanning techniques and the scanning sequences, the role of wbmri is expanding. mri has a great role in the pediatric population owing to its inherent advantages namely lack of radiation, high tissue specificity, and high diagnostic yield at the level of the entire body under a single sedation. unlike the application of wbmri in the assessment of metastasis and bone marrow involvement in leukemia, its role in systemic diseases is yet to be further investigated. certain diseases such as crmo are very often multifocal. the extent of osteonecrosis in patients on steroids, dermatomyositis and the lesions related to child abuse are very often wide spread in the skeleton. we intend to present some of the findings of these pediatric systemic and multifocal diseases on wbmri. chronic relapsing multifocal osteomyelitis (crmo): crmo can be acute or chronic and is multifocal. the abnormality manifests as high signal intensity. wbmri can guide for the best site for biopsy and provides monitoring for response to treatment. osteonecrosis: only few small studies evaluated the usefulness of wbmri in the diagnosis of both the symptomatic and asymptomatic sites of osteonecrosis in all patients on steroid therapy. wbmri is more sensitive than conventional radiographs. the abnormalities are typically geographical areas of high stir signal intensity. myopathies: wbmri has also the role of detecting the extent of idiopathic inflammatory myopathies such as dermatomyositis in the entire skeleton. child abuse: wbmri has a low sensitivity for the highly specific fractures that are pathognomonic for child abuse. conclusions: wbmri is a useful examination in the pediatric patient that is radiation free, quick and allows imaging of the entire body. it is an adjunct to dedicated mris to look for multifocality and extent of systemic diseases such as crmo, osteonecrosis in patients on steroids and dermatomyositis. it has a great potential as a screening examination but at the same time can detect both the symptomatic and the asymptomatic lesions in the bone marrow and muscles that are otherwise not seen on conventional radiography. it also allows guidance for biopsy and monitors response to treatment. mobile "cerebroliths" in hemihydranencephaly: a case report usha d. nagaraj, md, the ohio state university medial center, usha.nagaraj@osumc.edu; brent adler purpose or case report: hydranencephaly is a congenital central nervous system disorder manifested by the replacement of the cerebral hemispheres with a thin membranous sac filled with cerebrospinal fluid and necrotic debris. hemihydranencephaly is an extremely rare brain condition in which the vascular anomaly is unilateral, with fewer than cases previously reported in the literature. this is a case of a -month-old male who presented to the ophthalmologist for evaluation of possible leukocoria of the right eye. the patient had a history of a difficult vaginal delivery that required forceps delivery with possible associated trauma to the right eye. dilated fundoscopic exam revealed retinal calcifications. this caused a clinical concern for retinoblastoma and ct and mri of the orbits were obtained. ct demonstrated profound dilatation of the left lateral ventricle with only a thin rim of cortex surrounding it. there was some midline shift to the right with mild dilatation of the right lateral ventricle. the thalami and brainstem were spared. there were multiple soft tissue bodies that layered in the dependent portion of the left lateral ventricle, which were isodense to grey matter. mri revealed similar findings consistent with hemihydranencephaly involving the left cerebral hemisphere. there were multiple round soft tissue masses that measured up to cm in size that layered posteriorly in the left lateral ventricle. these masses were isointense to grey matter on t and hyperintense on t . when the patient was placed with his head turned to the left, these masses moved to the dependent portion of the left lateral ventricle. the orbits were normal on both ct and mr. these soft tissue collections are presumed to be mobile collections of infarcted brain tissue. this unusual appearance has not been described in the radiology literature. we review the ct and mr findings and review the relevant literature. purpose or case report: citrullinemia type i is a rare inborn error of urea cycle metabolism resulting in hyperammonemia. in the classic form, the newborn presents with poor feeding, vomiting, progressive lethargy and signs of increasing intracranial pressure - days after birth, rapidly progressing to apnea, coma and death if left untreated. we present a case of a term infant who presented to the hospital on the th day of life with a typical history of poor feeding and profound hypotonia. upon admission he had multiple episodes of apnea and hemodynamic instability prompting intubation and intensive support. laboratory evaluation revealed multiple abnormalities, most notably, hyperammonemia ( umol/l) and elevated citrulline (> umol/l). mri of the brain performed on the th day of life showed findings consistent with term hypoxic ischemic encephalopathy with restricted diffusion in bilateral rolandic cortex and subcortical white matter, bilateral caudate heads and lenticular nuclei, bilateral insular cortex, and bilateral cerebral peduncles. the genu of the corpus callosum, bilateral deep frontal white matter, and the left parietal white matter also demonstrated restricted diffusion suggesting infarction secondary to thrombosis of deep intramedullary veins. an area of restricted diffusion in the right parietal cortex was suspicious for superficial venous infarct. review of the literature reveals that this case of neonatal citrillunemia has unique mri findings. while our patient had diffusion changes with some shared similarities to the previous two cases in the literature, there are also findings consistent with deep intramedullary venous thrombosis and infarction. poster #: cr- duplicated internal auditory canal: a rare anomaly of the temporal bone ahmad aouthmany, university of toledo medical center, ahmad.aouthmany@utoledo.edu; asif abdullah purpose or case report: duplicated internal auditory canal (iac) is a rare anomaly of the temporal bone, which is usually associated with sensorineural hearing loss. only a few cases have been previously described in literature. we describe an extremely rare case of duplicated right internal auditory canal in a six month-old patient with a history of down syndrome. a six month-old male with trisomy presented with profound bilateral sensorineural hearing loss. the patient failed the newborn hearing screening tests. past medical history was unremarkable for recurrent ear infections. on focused physical examination, the auricles were normal appearing. external auditory canals were patent bilaterally revealing clear and translucent tympanic membranes. patient did not reveal a facial palsy. subsequently, a high resolution computed tomography (hrct) of the temporal bone was performed. duplicated appearance of the right internal auditory canal with separation of facial and vestibulocochlear segments was noted. the facial nerve canal demonstrated normal caliber while there was significant narrowing of the cochlear canal near the fundus. significant stenosis of the vestibulocochlear segment of the duplicated iac was identified at the porus acousticus. dehiscent right posterior semicircular canal was also seen. an enlarged right vestibule was also noted. a single iac was identified on the contralateral side with significant stenosis at the porus acousticus. high-resolution magnetic resonance imaging of iac was recommended which revealed normal appearance of the bilateral cochlear and vestibular nerves. duplication of the iac is an extremely rare anomaly involving a redundant osseous canal extending from the cerebellopontine angle through the otic capsule bone toward the labyrinth or cochlea. a duplicated iac may or may not be associated with congenital sensorineural hearing loss secondary to aplasia or hypoplasia of the vestibulocochlear nerve. to evaluate for structural abnormalities that may preclude cochlear implantation, it is important to evaluate pediatric patients with sensorineural hearing loss radiologically. although hrct is the best imaging modality for evaluation of osseous iac, the iac contents are best viewed on mri in oblique sagittal planes of the iac using a -d volumetric steady state sequence. neuroimaging in hemiplegic migraine: cases and review of the literature nicholas v. stence, md, children's hospital colorado-radiology, nicholas.stence@childrenscolorado.org; sita kedia, john a. maloney, jennifer armstrong-wells, timothy bernard purpose or case report: hemiplegic migraine (hm) is a rare variant of migraine with aura. it is characterized by a motor deficit lasting up to h that is fully reversible. little neuroimaging data for hm exists in the literature. we report our experience with two pediatric cases of hemiplegic migraine. we also review published cases of pediatric hm with abnormal findings on neuroimaging. methods & materials: cases and presented to our institution with severe headache (ha), acute right-side weakness, aphasia, and altered mental status (ams), which did not resolve after h. magnetic resonance imaging (mri) and genetic testing are reviewed for these cases. the literature was reviewed for pediatric cases with neuroimaging changes during hm attacks. results: initial mri, including diffusion-weighted imaging (dwi), was negative in both patients within h of onset. repeat mris at h (case ) and h (case ) were both positive for mild hyperintensity on trace diffusion images, and corresponding reduced diffusion on adc maps, involving regions of the cortex and juxtacortical white matter in left middle cerebral artery distributions. these findings completely resolved at months in both cases. mr angiograms (mra) were negative in both cases. case had a family history of migraines and was found to have an unreported mutation in atp a gene at a highly conserved location in vertebrates. case had a family history of hm and was found to have an indeterminant mutation in the cacna a gene. infectious, metabolic and hypercoagubility work up was negative. case required inpatient rehabilitation and at year follow up was requiring speech therapy. case resolved completely. in the literature, cases of hemiplegic migraine with neuroimaging changes were reported. all cases had prolonged hemiplegic migraines (symptoms> h) and showed cerebral edema with or without restricted diffusion. conclusions: all eight hm cases in the literature with abnormal findings on neuroimaging had prolonged attacks. mris for our two cases and two cases reported in the literature were initially normal at admission. mild swelling and restricted diffusion developed in our two cases after h, and resolved on follow up mris. subtle findings on diffusion and t imaging may lag behind the clinical picture in hm, therefore serial neuroimaging may be useful in individuals with prolonged symptoms. most cases eventually show resolution clinically and on mri. correlation of neurosonographic anatomy with matching mr scan planes denise castro, hospital for sick children, denisecastro @ gmail.com; pam rasalingham, omar islam, don soboleski purpose or case report: new high-resolution mr sequences have allowed for exquisive anatomic detail and enables reconstruction of images in any scan plane desired. this ability allows for precise matching of mr image planes with the standard oblique coronal, sagittal and axial images obtained during routine neurosonography. the purpose of this poster is to correlate the morphology demonstrated on neurosonography with the mr image, utilizing this ability in order to enhance our understanding of the neuroanatomy distinguishable on sonographic imaging. we believe this will allow a better appreciation of the subtle differences in echotexture of neuroanatomic structures which are often ignored or overlooked on neurosonography and help improve our detection of subtle sonographic abnormalies. ectopic cerebellum in the posterior cranial fossa: report of a case and review of the literature usha d. nagaraj, md, the ohio state university medical center, usha.nagaraj@osumc.edu; daniel boue, lisa martin purpose or case report: cerebellar heterotopia is a common congenital anomaly frequently encountered in the form of cell rests around the fourth ventricle. however, isolated well-differentiated cerebellar ectopia is extremely rare. of the previously reported cases in the literature, only have presented as a discrete, extraaxial mass and none have been described in the posterior cranial fossa. we present a case of a -year-old male who initially presented with persistent daily headaches. physical exam including a detailed neurologic exam was within normal limits. non-contrast computed tomography (ct) of the brain was initially performed, demonstrating no abnormalities. further work-up with magnetic resonance imaging (mri) was performed, which revealed a well-defined, extra-axial mass superior to the cerebellum and inferior to the tentorium, immediately beneath the vein of galen. the mass was isointense to grey matter on t and t sequences and there was no significant enhancement on post-contrast images. there was mass effect on the vermis and the cerebellar tonsils were displaced mm below the foramen magnum. neurosurgery was consulted and the mass was removed for diagnosis and treatment of the patient's symptoms. the mass was easily identified intra-operatively and gross total resection was accomplished successfully. pathologic analysis of the mass revealed well-formed cerebellar tissue without evidence of neoplasia. to the best of our knowledge this is the only case of ectopic cerebellum presenting as a discrete extra-axial mass in the posterior cranial fossa. our case shows that an extra-axial mass that parallels grey matter on all sequences can be a presentation of ectopic cerebellum. we describe the ct and mri findings, surgical and histopatholgical results and review the relevant literature. pediatric isodense acute subdural hemorrhage jeffrey s. kao, md, msee, university of kansas-wichita, run boston@gmail.com; debbie desilet-dobbs purpose or case report: the density (attenuation coefficient) of subdural hemorrhage (sdh) in computed tomography (ct) is important in assessing the acuity of sdhs. an acute sdh is traditionally described as hyperdense and then becoming isodense in approximately weeks when entering the subacute phase. in this report, we document the case of a pediatric patient with the new appearance of an acute sdh within h of the prior ct that was isodense. greater than % of the collection was isodense, with a small focus of hyperdensity. acute sdhs are known to be isodense to gray matter in patients with anemia (wp smith, am j neurorad ). however, the hemoglobin and hematocrit was within normal limits. in addition, acute sdhs that are only a few hours old can have a mixed hyperdense and hypodense appearance because of uncoagulated blood before clotting takes place (j provenzale, ajr ) . thus, an acute sdh can have an isodense appearance in a non-anemic patient. radiologists should consider the possibility of an acute sdh with an isodense appearance, especially in case of possible non-accidental trauma where timing of an injury is important. undifferentiated sarcoma of the esophagus in an year-old male: case report and radiologic/pathologic correlation michael e. daniel, md, ut southwestern / children's medical center dallas, michael.daniel@utsouthwestern. edu; lisa sutton, sandy cope-yokoyama, neil j. fernandes purpose or case report: mesenchymal neoplasms of the gastrointestinal (gi) tract occur infrequently in the adult and are extremely rare in the pediatric population. the occurrence of these lesions in the esophagus is limited to a collection of case reports in the available literature. most esophageal mesenchymal tumors in the pediatric gi tract are benign leiomyomas. the vast majority of malignant mesenchymal tumors in children are categorized as either sarcomas or gastrointestinal stromal tumors (gist). we report a case of a high grade undifferentiated sarcoma of the distal esophagus in an year-old male. while this tumor most closely resembles a gist, the immunohistochemical profile of the lesion is not typical of any distinct mesenchymal neoplasm. a review of the literature demonstrates a single case report of a likely benign undifferentiated mesenchymal neoplasm of the distal esophagus in an adolescent. to our knowledge, this is the first reported case of an undifferentiated esophageal sarcoma in a pediatric patient. we provide radiologic and pathologic features of the above lesion, and review the typical imaging and pathologic characteristics of mesenchymal gi neoplasms. potential airway management issues in sedated children kimberly fagen, md, ms, children's national medical center, kfagen@childrensnational.org; nadja kadom, ira cohen purpose or case report: many pediatric imaging studies require sedation. it has been shown that a variety of health care professionals other than anethesiologists may provide sedation, including advanced practice registered nurses, nurse practitioners, physician assistants, fellow level trainees, emergency medicine physicians, intensivists, pediatricians and, last but not least, radiologists. moderate sedation, also called "conscious sedation", does generally not require an anesthesiologist as there is usually adequate spontaneous ventilation and no airway intervention required. however, in case of a complication during the imaging study intubation may become necessary. for patients with certain congenital or acquired conditions emergent intubation may be very difficult and should be brought to the attention of an anesthesiologist prior to inducing moderate sedation. the four "d's" is a quick way to assess potentially difficult airways that necessitate consultation with anesthesia prior to moderate sedation: dentition (incisor/tooth size, dental alignment, and macroglossia), distortion (swelling from infection, tumor, or trauma), disproportion (hyoid-chin ratio, such as with micrognathia), and dysmobility (jaw or cervical spine movement issues, i.e. trauma or atlanto-occipital instability). presence of some of these features may be an indication to consider general anesthesia for sedation; at the very least, anesthesiologist's awareness of a potentially difficult intubation adds to patient safety during moderate sedation. purpose or case report: lymphangiomatosis describes the presence of multiple lymphangiomas often with multiorgan involvement; typically bones, spleen, mediastinum and lungs. although lymphangiomatosis has been described in patients ranging from birth up to years, it most frequently presents in childhood. the lesions can occur in any tissue in which lymphatics are normally found, with a predilection for neck and chest involvement. the clinical presentation is variable including pleural or pericardial effusion, hemoptysis, protein wasting enteropathy, peripheral edema, hemihypertrophy and disseminated intravascular coagulopathy. the coexistence of lytic bone lesions and chylothorax serves as an important diagnostic clue. we describe typical radiographic, ct and mri findings in the appropritate clinical setting that narrow the differential diagnosis and raise concern for this rare entity as the etiology for the patient's symptoms. we report a -year-old girl and year-old boy with pulmonary lymphangiomatosis with typical presentation and imaging findings. results: bilateral interstitial infiltrates, pericardial and pleural effusions are evident on chest radiograph. sampling of the pleural fluid demonstrates chylous effusion. ct scans of the thorax reveal diffuse smooth thickening of interlobular septa and bronchovascular bundles with extensive infiltrative involvement of mediastinal fat. osseous and splenic lesions are demonstrated both on ct and mr. differential diagnosis includes interstitial edema, lymphoma and sarcoidosis. conclusions: the natural history of pulmonary lymphangiomatosis is characterized by progressive growth and compression of adjacent structures. therapy should aim to decrease the compressive effects, to control chylous effusions, and to maintain cosmesis. the success of surgical resection is limited by inability to separate lymph collections from normal structures. characteristic clinical and radiographic presentation, chylothorax, and extrathoracic lymphatic dysfunction should prompt a consideration of lymphangiomatosis and prevent delay in diagnosis. aortic arch congenital anomalies: what the radiologist needs to know luana stanescu, radiology, seattle children's hospital, stanescu@u.washington.edu; stephen done purpose or case report: . review classic imaging findings in congenital aortic arch anomalies which can improve detection on radiographs and barium esophagogram . describe pertinent embryologic basis of the radiologic findings . describe correlative imaging findings on ct and/or mri in dedicated cases . describe common diagnostic pitfalls methods & materials: after obtaining institutional irb approval we reviewed various patients presentations with this condition and analyzed images to characterize this particular entity and it's manifestations for better definition of diagnostic criteria. results: radiographs and barium esophagogram: algorithmic approach in reviewing chest radiographs in order to improve detection of aortic arch anomalies; classic findings and common pitfalls. cross-sectional imaging (ct and mri): what the surgeons need to know before surgical repair; detection of associated cardiac anomalies. sample cases: double aortic arch, double aortic arch with complete or partial atresia of one of the arches. conclusions: major teaching points of this exhibit are: . review of classic features of congenital aortic anomalies on radiographs, esophagogram, ct and mri with pertinent embryologic basis . describe the utility of various imaging modalities in congenital aortic anomalies, emphasizing common pitfalls. cardiovascular and mediastinal imaging in children with unexpected clinical presentation shunsuke nosaka, md, radiology, national center for child health and development, nosaka-s@ncchd.go.jp purpose or case report: children with cardiovascular and mediastinal diseases can be congenital or acquired in etiology. they usually present with straightforward clinical course. in certain situation, however, some of the children show unexpected clinical presentation predominantly with those of neighboring organs such as respiratory tract, hepatobiliary system, and gastrointestinal tract. these unexpected presentations can be the cause of delay in proper diagnosis and treatment. the purpose of this exhibit is demonstrate a variety of imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation. this exhibit is case based presentation of cardiovascular and mediastinal imaging in children including tips, pitfalls and lessons learned among patients presented with unexpected clinical presentation. diagnostic imaging modalities for cardiovascular disease usually consist of various combinations of plain radiography, ultrasound, ct, mr imaging, fluoroscopy, nuclear medicine, and angiography. the general concept of alara-as low as reasonably achievable-should always be utilized when radiation-producing modalities are indicated in children. the diseases included will be double aortic arch found during workup for the cause of aspiration pneumonia, unilateral pulmonary vein atresia presented with recurrent episodes of pneumonia, severe mitral regurgitation secondary to chordal rupture mimicking fluminant hepatic failure, myocarditis initially present as acute abdomen, cardiomyopathy as unusual initial presentation of neuroblastoma, and thymolipoma mimicking gradual development of cardiomegaly. conclusions: it is important for radiologist to be familiar with imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation. cardiac embryology made easy: a novel teaching approach using claymation andrew phelps, children's hospital boston, aphelpsmd@ gmail.com; purpose or case report: congenital heart disease can be an intimidating subject for radiology residents, and cardiac embryology is key to its understanding. however, this can be an equally intimidating topic to teach! various diagrams and animations are available in textbooks and online, but much like advanced origami, many of these resources suffer from being visually too complex for the first-time learner. to overcome this teaching obstacle, i created my own cardiac embryology animations using modeling clay and incorporated them into a comprehensive didactic lecture on congenital heart disease. methods & materials: cardiac embryology animations were created using modeling clay, a digital camera, and microsoft powerpoint. surface and cross-sectional views were generated, depicting the key events in cardiac embryology: heart tube formation, cardiac looping, chamber division, truncus arteriosus division, and pulmonary venous connection. example models are shown in figure . results: in this lecture, the animations are presented alongside actual embryonic heart photographs. the lecture then uses the embryology knowledge as a basis to explain the common congenital heart diseases and their mri appearances. examples of septal defects, ventricular hypoplasia, and transposition of the great arteries are presented, among others. conclusions: understanding cardiac embryology is required in order to approach congenital heart disease in a logical fashion. modeling clay animations are a cheap and easy way to simplify this complex topic. arterial tortuosity syndrome: an introduction to the clinical and radiologic manifestations in the pediatric population neal desai, umkc som, neal @gmail.com; suchit patel, ayushi gupta, marius hubbel, doug rivard purpose or case report: . to describe the clinical findings of arterial tortuosity syndrome and give a brief discussion of the disease process. . to describe the radiologic manifestations of arterial tortuosity syndrome. . to give a brief discussion of loeys-dietz syndrome-a disease with similar arterial findings, but with unique molecular characteristics from arterial tortuosity syndrome. . to use this knowledge to help establish the diagnosis and reduce mortality. methods & materials: arterial tortuosity syndrome overview • epidemiology • molecular basis • pathophysiology • review of signs, symptoms and presentation • brief discussion of treatment differential diagnosis of arterial tortuosity syndrome • loeys-dietz syndrome-similarities and differences radiologic findings and discussion • chest radiograph • computed tomographic angiography • magnetic resonance angiography-neck • magnetic resonance angiography-head • conventional angiography making a diagnosis • sample case report • review questions conclusions: arterial tortuosity syndrome is a rare disease whose chief manifestation is severe cardiovascular connective tissue defects. due to the nature of these defects and the significance of rapid intervention, it is important to be aware of and recognize the radiologic manifestations associated with arterial tortuosity syndrome in the presence of appropriate clinical history to help offer a better prognosis to the patient. dynamic pulmonary computed tomography for evaluation of cardiopulmonary disease shilpa v. hegde, md, arkansas childrens hospital, university of arkansas, shilpavhegde@gmail.com; s. bruce greenberg purpose or case report: dynamic pulmonary computed tomography (dpct) is a wide-detector ct technique that allows for continuous chest imaging during respiration. when combined with intravenous contrast, the technique is a unique tool for evaluation of cardiopulmonary abnormalities in children with cardiopulmonary abnormalities. the purpose of this poster is to illustrate the technique of dpct for evaluation of cardiopulmonary disease in children with congenital heart disease and persistent respiratory distress. methods & materials: methods and materials: dpct exams with intravenous contrast were performed on infants with a history of congenital heart disease and palliative surgery. four continuous msec gantry rotations were obtained with respiratory rates set at /minute. the imaging was accomplished during the time of a single respiratory cycle. kvp and low ma resulted in effective dose of≈ . msv. eight respiratory phases were reconstructed to create d and mpr cine loops for evaluation of cardiopulmonary abnormalities. results: cardiopulmonary abnormalities were detected in all patients. patency of sano shunt, blalock tausig shunt or patent ductus arteriosus stent was established. intimal thickening was identified in one sano shunt. hypoplastic branch pulmonary arteries were present in infants and pulmonary vein thrombosis in infant. left bronchomalacia was identified in four of five infants and best or only identified on the expiratory phase of respiration. left lung air trapping was present in two patients. conclusions: dpct with intravenous contrast is the ideal study for evaluation of the post-operative infant with congenital heart disease and persistent respiratory distress. the role of low-dose ct angiography in the evaluation of renovascular hypertension in children jessica kurian, md, chop, kurianj@email.chop.edu; monica epelman, kassa darge, els nijs, jeffrey hellinger purpose or case report: historically, the evaluation of renovascular hypertension has been accomplished via us and conventional angiography. based on the reported adult experience we introduced renal ct angiography (cta) for the evaluation of renovascular hypertension in mid- . our institution has a robust, well-established protocol, which results in reproducible, high quality images. we aim to present our imaging strategies for the evaluation of these patients and to discuss and illustrate the role of low-dose cta with -d imaging as a noninvasive alternative in the evaluation of pediatric renovascular hypertension. methods & materials: we used our department information system to identify pediatric patients (< years of age) who had documented renovascular hypertension confirmed either by conventional angiography and/or surgery during a -year period. we present our protocol and discuss the indications, limitations and benefits of renal cta. ct thin slice data, obtained employing dose reduction strategies, was reviewed and reconstructed in d and d renderings. pertinent us and mr studies as well as demographic and clinical data were reviewed and recorded. several causes for renovascular hypertension were documented and relevant ct angiographic findings were selected for presentation. results: radiation dose ranged . - msv. fibromuscular dysplasia was the most common diagnosis followed by neurofibromatosis type . vascular pathology included stenoses, beading, occlusions, and aneurysms. disease was noted in the extraparenchymal renal arteries in approximately % of the cases. the choice of the imaging modality for the investigation of renovascular hypertension in pediatric patients remains controversial. in the authors' experience, cta with -d imaging is a valuable, non-invasive diagnostic tool for the evaluation of pediatric renovascular hypertension. low dose protocols can reduce the radiation exposure associated with ct. this method can spare patients the complications associated with conventional angiography. fetal mri: brain, head and neck malformations-a pictorial essay sumit singh, md, children's hospital of wisconsin, sumitsingh @yahoo.com; mohit maheshwari, teresa c. gross kelly, tushar chandra, ibrahim s. tuna, craig johnson purpose or case report: the purpose of the exhibit is to illustrate various brain, head and neck massses/vascular anomalies on fetal mri. we will also briefly discuss the normal fetal brain anatomy as seen on fetal mri. methods & materials: major indications for fetal mri include evaluation of inconclusive sonographic findings in cases of cns malformations. in our institute patients are scanned on . t mr scanner. a body surface six channel phased array coil is used to maximize signal to noise. all the scans are checked by a neuroradiologist to make sure adequate plane imaging of the brain or other lesion in question were performed. plane scanning of the fetal body is also performed for the laterality determination of the lesion and also screen for other congenital anomalies. results: prenatal usg is frequently inconclusive for evaluation of complex fetal brain and head and neck anomalies. most studies suggest that mri after first trimester is safe. in addition, advent of rapid mri sequences like single shot fast spin echo (ssfse) have helped in reducing scan time and motion artifacts leading to availability of diagnostic quality images. these have led to increasing use of mri as supplemental tool to further investigate inconclusive fetal sonographic findings. mri provides better anatomical delineation of these complex abnormalities. it helps in making appropriate diagnosis with high confidence and aids in appropriate obstetric and prenatal/neonatal surgical planning or intervention. this educational exhibit will illustrate few common fetal anomalies. these will include agenesis of corpus callosum, malformation of cortical development, posterior fossa malformations, ventriculomegaly, in-utero stroke, orbital abnormalities and some fetal neck masses/ vascular malformation. correlation and confirmation with the postnatal mri will also be provided for some cases. conclusions: technical and therapeutic advances have driven the development of fetal mri. it is an important adjunctive tool for prenatal imaging in those instances in which a complex anomaly is suspected by sonography, when fetal surgery is contemplated, or when a definitive diagnosis cannot be determined. it has prognostic implications and may help in optimal and timely obstetric and neonatal management. purpose or case report: this educational report will provide a review of the imaging appearance of intradiaphragmatic and subdiaphragmatic pulmonary sequestrations on fetal mri. the proposed pathophysiology, review of sequestration subtypes, and surgical management options will also be described. case examples will be provided to illustrate the fetal mr imaging findings of these variants of pulmonary sequestration that help support the diagnosis. specifically a "triangle sign" of t hyperintense tissue directed toward the diaphragm will be demonstrated. illustrative case examples will be placed in the context of a differential diagnosis for subdiaphragmatic masses seen on prenatal imaging. imaging signs that help make a diagnosis of these pulmonary sequestration variants and separate this entity from other lesions will be emphasized. poster #: edu- mri of the fetal head and neck masses alok jaju, md, mallinckrodt institute of radiology, alokjaju@gmail.com; joshua shimony, per amundson purpose or case report: fetal magnetic resonance imaging (mri) is a useful problem solving tool for abnormalities detected by prenatal ultrasound (us). masses of the head and neck region can vary from benign incidental lesions to devastating neurological lesions and life threatening tumors. we share our experience in characterizing these lesions by prenatal mri, that can have a bearing on follow up imaging, perinatal management and overall prognosis. we did a retrospective review of all fetal mri studies performed at our tertiary care children's hospital between / and / , to identify fetuses with head and neck masses. we reviewed the maternal demographic and clinical data, prenatal ultrasound, fetal outcomes and post natal imaging (when available). results: out of the fetal mri studies, had dominant head and neck masses. majority were encephaloceles ( occipital, parietal). the remaining included variety of masses such as nasal glioma, teratoma ( ), epidermoid cyst, hemangioma and lymphatic malformation ( ) . mri played a useful role in distinguishing encephaloceles from other masses based on underlying bone defect and intracranial extension. it also helped in characterizing other masses based on location and signal characteristics. the presence and degree of airway compromise was determined. intracranial anomalies associated with encephaloceles including callosal dysgenesis, cerebral and cerebellar hypoplasias, migrational disorders and spinal anomalies were also correctly identified. conclusions: we present the prenatal mr imaging findings of a spectrum of head and neck lesions, correlating with prenatal ultrasound, postnatal imaging and clinical or pathological outcomes. purpose or case report: the immaturity of the cns in neonatal infants makes neurologic assessment difficult. neuroimaging plays an essential role in the assessment of brain injury by helping to indentify the injury and expected neurologic outcome. cranial ultrasound (us) is usually the first neuroimaging modality used since the technique is portable, does not involve radiation and can be used sequentially. magnetic resonance imaging (mri), however, is the most sensitive imaging modality for the detection of hypoxic brain injury. the goal of this presentation is to compare the us and mri performed within a -hour interval, and evaluate these findings to improve the interpretation of the us which is usually the first methodology used to evaluated these patients. we performed a retrospective review of the neonatal imaging studies with us and mri performed within -hour interval on preterm and term newborns with clinical history of hypoxia-ischemia. the imaging findings of the two modalities, mri and us, were correlated with the pattern and severity of the injury and brain maturity. results: diffuse white matter abnormalities were observed in % of the patients by us or mri. the ultrasound identified diffuse increased echogenicity which did not show correlation with mri in % of patients. focal white matter abnormalities were better identified by mri on non-cavitary leukomalacia which is the most common pvl observed in premature neonates with low birth weight and the most difficult to identified using us. cavitary leukomalacia showed strong agreement in both methodologies. the mri identified % more cases of intraventricular hemorrhage, however, the corresponding increase in hemorrhage was of minimal clinical significance. in most cases extra axial hemorrhage was better identified by mri. conclusions: after viewing this exhibit, the viewer will gain a better appreciation and understanding of the neuroimaging characteristics of hypoxia-ischemia in us and mri, and thus improving the interpretation of the us which is usually the first imaging modality used to evaluate this patient population. purpose or case report: the most common thoracic lesions found on prenatal imaging, congenital pulmonary airway malformation (cpam), bronchopulmonary sequestration (bps), and congenital diaphragmatic hernia (cdh), usually have characteristic imaging findings previously described in detail. however, common entities presenting with atypical findings and rarer thoracic entities do occur and can be characterized by fetal magnetic resonance (mr) imaging. the purpose of this educational exhibit is to show examples of atypical presentations of common thoracic lesions and more unusual thoracic entities on fetal mr. when applicable, prenatal mr is compared with prenatal ultrasound, postnatal imaging, operative findings, or pathology. methods & materials: using a radiology information system database, the reports of all fetal mr exams at our institution from january through january were reviewed. when unusual thoracic findings were described in the report, all prenatal and postnatal images (when available) were evaluated. in the cases selected, medical charts were reviewed for operative findings and pathologic reports. results: the cases to be described, both pulmonary and extrapulmonary in location, include: hybrid lesion in a horseshoe lung, cpam extending across the midline, bilateral bps, bps located within the mediastinum, bps located within the leaves of the diaphragm, ectopia cordis and cdh as components in pentalogy of cantrell, cdh with herniation of liver into the pericardium, elongated esophageal duplication cyst, chest wall lymphatic malformation, and tight double aortic arch causing congenital high airway obstruction syndrome (chaos). conclusions: after studying this educational exhibit, the reader will be acquainted with a variety of unusual fetal pulmonary and extrapulmonary lesions, with emphasis on fetal mr. prenatal and postnatal imaging findings in megacystis-microcolon-intestinal hypoperistalsis syndrome (mmihs) mary kitazono, chop, mkitazono@gmail.com; richard bellah purpose or case report: to review the classic constellation of findings seen in prenatal and postnatal imaging of megacystis-microcolon-intestinal-hypoperistalsis syndrome (mmihs), as well as to illustrate additional imaging features that are variably seen in this syndrome. the imaging database at our children's hospital was searched for all cases of mmihs diagnosed since . all available prenatal and postnatal imaging studies were reviewed in patients with a diagnosis of mmihs, and representative images are provided with a description of the findings. results: since , patients ( girls, boy) have been diagnosed with mmihs at our institution, including on prenatal mri and us. the characteristic prenatal imaging findings include marked urinary bladder distension, bilateral pelvicaliectasis, and dilated, tortuous ureters, as well as a diminutive colon containing no or minimal t w-hyperintense meconium on mri. postnatal imaging studies also characteristically demonstrate a massively distended urinary bladder (with no apparent mechanical cause of obstruction) as well as a small, unused colon with dilated, hypoperistaltic small bowel seen proximal to the microcolon. additional findings which are variably seen include intestinal malrotation, stomach and esophageal hypoperistalsis or aperistalsis, gastroesophageal reflux, and biliary stasis. conclusions: although a rare syndrome, the constellation of imaging findings in mmihs is pathognomonic, and recognition of the classic pattern of findings can allow the radiologist to make a diagnosis of mmihs in both the in-utero and postnatal setting. early diagnosis is essential for allowing prenatal counseling regarding this generally fatal disorder, as well as to optimize early management options. purpose or case report: gastric mass lesion are uncommon. this presentation is an educational review of pediatric gastric mass lesions including gastro-intestinal stromal tumor (gist), inflammatory myofibroblastic tumor (pseudotumor). burkitt's lymphoma, squamous cell carcinoma, gastric teratoma, gastric varices, gastric hamartoma, gastric polyp and hypertrophic pyloric stenosis (hps). clinical presentation is varied with upper gi bleeding, feeding intolerance, pain, weight loss and fatigue manifesting. the imaging work-up might initially have been endoscopy or ultrasound. cross section imaging (ct mr) can be invaluable. the role and impact of fdg pet on the management, staging and follow up of the oncologic pathology will be emphasized. imaging findings in megacystic microcolon intestinal hypoperistalsis syndrome, a rare disease kiery braithwaite, pediatric radiology, emory-egleston, kieryb@yahoo.com; kiery braithwaite, paula dickson, marianne m. ballisty purpose or case report: megacystis microcolon intestinal hypoperistalsis (mmih) syndrome is a rare congenital form of severe functional intestinal obstruction which is more commonly found in females. the presenting clinical and imaging features of this disease can often mimic other causes of proximal bowel obstruction in the neonate. in combination with its common association with intestinal malrotation, the clinical picture of mmih syndrome may be confusing at times. awareness of additional imaging features characteristic of mmih syndrome may help the radiologist suggest this diagnosis. the purpose of this study is to enhance the ability of the pediatric radiologist to suggest this rare diagnosis by recognizing this unusual constellation of imaging features. we retrospectively reviewed the clinical data and imaging studies of four patients with mmih syndrome at our institution. imaging studies included plain radiography, ultrasonography, fluoroscopy, and cross sectional imaging. the initial presentation and clinical outcome was also reviewed. results: the clinical presentations of our patients, who were all female, were somewhat varied but typically included symptoms of intestinal obstruction. the diagnosis of mmih syndrome was made in our patients from the first few weeks of life through early childhood. the four patients demonstrated imaging features characteristic of this disease including a very large dilated bladder, severe bilateral hydroureteronephrosis, gaseous distention of the stomach and proximal small bowel, intestinal hypoperistalsis, and a very small colon. the clinical course of these patients that we observed was also quite variable, with some patients dying in neonatal period while another patient continues to do reasonably well at years old after a multi-organ transplant. conclusions: mmih syndrome is a rare and frequently lethal disease. the ability of the pediatric radiologist to recognize this constellation of imaging findings can help the clinical team arrive at a diagnosis of mmih syndrome. more prompt diagnosis can aid in the development of a long term management plan for the patient and in counseling the family regarding the prognostic implications of this disorder. pathologies of omphalomesenteric duct remnant: radiologic-surgical correlation swapnil bagade, md, pediatric radiology, mallinckrodt institute of radiology, bagades@mir.wustl.edu; geetika khanna, rebecca hulett purpose or case report: . to facilitate understanding of embryology of the omphalomesenteric(vitelline) duct and normal anatomy of the umbilicus. . review the spectrum of omphalomesenteric duct malformations and diversity of clinical presentations of these remnants. . illustrate the imaging findings of omphalomesenteric remnants, from the common such as meckel's diverticulum to the uncommon such as the omphalomesenteric duct cyst, with surgical correlation. methods & materials: cases with complications of persistent omphalomesenteric duct were collected from the joint surgery/radiology conferences at a tertiary level children's hospital. imaging features were correlated with intraoperative findings. conclusions: preoperative diagnosis of complications related to the omphalomesenteric duct remnants can be challenging because clinical and imaging features overlap with other etiologies of acute abdomen. knowledge of the embryologic, clinical, radiologic, and surgical characteristics of omphalomesenteric duct remnants will aid in early and accurate diagnosis. neonatal bowel obstruction-a pictorial essay tanmay patel, university of kentucky; harigovinda challa purpose or case report: bowel obstruction is the most common abdominal emergency in the newborn period and in most cases is secondary to a congenital anomaly requiring early surgical intervention. however not every case of abdominal distension or dilated bowel is secondary to mechanical bowel obstruction or underlying surgical condition. radiologic imaging forms a central role in the work up of newborns with suspected intestinal obstruction. the role of the radiologist is to identify whether or not mechanical obstruction is present; if obstruction is identified on initial radiographs, to determine the level of obstruction, and finally to identify the etiology of obstruction. initial plain radiographic evaluation also helps to determine the subsequent diagnostic or therapeutic approach. methods & materials: a retrospective review of multiple radiographic and fluroscopic examinations in patients with diagnosis of neonatal bowel obstruction was performed at kentucky children's hospital. multiple examples of classical imaging findings were compiled and placed into a pictorial review. results: neonatal intestinal obstruction generally presents with nonspecific symptoms such as abdominal distention, vomiting, or failure to pass meconium depending on the level of obstruction and time of occurrence of underlying congenital lesion/atresia in the intrauterine life. initial plain radiographs of the abdomen reveal dilated bowel loops when obstruction is present. high intestinal obstruction is suspected when only few dilated loops are identified, while multiple dilated bowel loops are seen in low obstruction. most cases of high obstruction may not need another diagnostic imaging test. all cases of distal intestinal obstruction require water soluble enema to identify the etiology of obstruction. in conditions like functional immaturity of the colon, and meconium ileus water soluble enema is therapeutic and thus surgery can be avoided in most cases. the objective of this presentation is to present an educational exhibit of classical imaging findings of various types of neonatal bowel obstructions, and how to differentiate between them. conclusions: bowel obstruction is the most common abdominal emergency in the new born period. most cases are secondary to a congenital surgical condition and early diagnosis and treatment significantly reduces mortality and morbidity. radiographic evaluation plays a central role in the diagnosis and treatment of these conditions. poster #: edu- d t -weighted mrcp in the pediatric population-a pictorial review nathan egbert, mbbs mph, university of michigan, nathaneg@med.umich.edu; jonathan r. dillman, peter j. strouse purpose or case report: to demonstrate the utility of d t -weighted magnetic resonance cholangiopancreatography (mrcp) in the pediatric population, and to illustrate the mrcp findings of various conditions affecting in the pediatric pancreaticobiliary system. we identified all mrcp exams performed on pediatric patients (< years of age) from january , through august , by searching institutional electronic medical records. we then identified representative d t -weighted mrcp images of various conditions affecting the pediatric pancreaticobiliary system. results: representative d t -weighted mrcp images (including source, maximum intensity projection, and volume rendered images) from the following conditions will be presented: abnormal biliary narrowing/stricture (including sclerosing cholangitis, anastomotic strictures following kasai procedure & liver transplantation, and "pseudostricture"), biliary atresia, choledochal cyst (including various subtypes, based on todani classification), choledocholithiasis & cholelithiasis, congenital anomalies of the pancreaticobiliary system (including pancreas divisum and anomalous pancreaticobiliary junction), pancreatobiliary system trauma (including main pancreatic duct transection), and other rare conditions affecting the pancreaticobiliary system (including rhabdomyosarcoma of the biliary tree). conclusions: d t -weighted mrcp has become an extremely useful tool in the evaluation of children with suspected disorders of the pancreaticobiliary system. since mrcp has distinct advantages over alternative diagnostic techniques, such as endoscopic retrograde cholangiopancreatography (ercp) or percutaneous cholangiography, including lack of ionizing radiation and noninvasiveness, mrcp is a much preferred initial study for pediatric pancreaticobiliary imaging. this pictorial review is intended to highlight the d t weighted mrcp appearances of various pancreaticobiliary conditions occurring in the pediatric population. purpose or case report: magnetic resonance enterography (mre) is rapidly emerging as an important imaging tool for the diagnosis and follow-up of inflammatory bowel disease (ibd). its lack of ionizing radiation makes this imaging modality especially vital to the pediatric population. using a casebased approach, we will demonstrate the usefulness of diffusion-weighted imaging (dwi) as part of a comprehensive mre protocol for the assessment of ibd in children. the basics of dwi will be discussed with particular attention to abdominopelvic techniques. the role of mre dwi for the evaluation of pediatric crohn disease (cd) and ulcerative colitis (uc) will be reviewed using a case-based approach. key images from pertinent imaging studies will be identified by searching institutional electronic medical records and presented with relevant clinical data. results: a review of pediatric mre examinations suggests dwi can be used to detect the following: ) small and large bowel segments affected by ibd (both cd and uc) ) abdominopelvic abscesses (including within the mesentery, body wall, iliopsoas muscle, and liver) ) abnormal lymph nodes ) sacroiliitis ) perianal disease (including abscesses and other penetrating complications). conclusions: dwi has the potential to play a very important role in the diagnosis and follow-up of pediatric ibd. this mre technique is particularly useful for detecting a variety of disease-related complications. as the exact meaning of bowel wall restricted diffusion is poorly understood to date, continued investigation will be necessary to determine the clinical and histologic significance of this finding. cases of cf involving the gi tract were collected from clinical workflow encounters of the authors and from the main hospital medical records database. relevant imaging studies were reviewed for known gi manifestations of cf. these imaging studies were correlated with clinical histories and available intraoperative and pathologic findings. results: cf involvement of the gi tract presents over a wide range of ages, organs involved, and associated symptoms. these manifestations can generally be divided anatomically into those involving the alimentary tract, hepatobiliary system, and pancreas. alimentary tract manifestations consist of meconium ileus in uncomplicated and complicated forms (with the latter including secondary intestinal atresia, volvulus, and perforation with meconium peritonitisdistal intestinal obstruction syndrome, constipation, rectal prolapse, duodenal fold thickening, and appendiceal dilation. hepatobiliary disorders secondary to cf include microgallbladder, cholelithiasis, biliary ductal abnormalities, neonatal hepatitis, and cirrhosis (including complications such as portal vein thrombosis and ascites). pancreatic expressions of cf include fatty infiltration, calcifications, and cysts/ cystosis, frequently in the setting of malnutrition and/or stooling abnormalities. this exhibit will demonstrate the spectrum of clinical and radiologic gi findings in this disease from the fetal and neonatal period through adolescence across a range of imaging modalities. conclusions: gastrointestinal manifestations of cystic fibrosis occur frequently in the pediatric population and may be the earliest clinical expression of the disease. familiarity with the variety of gastrointestinal imaging findings of cystic fibrosis can expedite appropriate diagnosis and therapy, particularly in those children in whom the primary disease is not clinically suspected. beyond acute appendicitis: imaging of additional pathologies of the pediatric appendix kelly dietz, md, cincinnati children's hospital; arnold c. merrow, daniel j. podberesky, alexander j. towbin purpose or case report: primary acute appendicitis (or appendiceal inflammation caused by a superimposed bacterial infection in the setting of appendiceal obstruction) is by far the most common pathology of the appendix, and imaging evaluations to exclude this diagnosis occur daily in the pediatric radiology setting. the clinical and imaging differential diagnosis in a patient with right lower quadrant pain and suspected appendicitis is a broad but well-recognized list that predominantly involves structures adjacent to the appendix including the ovaries, small and large bowel, and ureters. there are, however, less common pathologies primarily involving the appendix which can create an imaging diagnostic dilemma in the setting of right lower quadrant symptoms. our goal is to review the imaging and clinical manifestations of these less commonly encountered appendiceal abnormalities. methods & materials: cases of appendices that were abnormal by imaging but ultimately determined not to be due to primary acute appendicitis were collected from clinical encounters by the authors as well as through a search of the radiology and pathology report databases. clinical course, surgical findings, and pathology reports (if available) were subsequently reviewed through the main hospital medical records system. results: the collected cases demonstrate a wide range of additional pathologies of the appendix outside of primary acute appendicitis. a variety of imaging modalities were employed in the workup of these cases. examples reviewed in this exhibit include crohn's disease, ulcerative colitis, cystic fibrosis, carcinoid tumor, inguinal hernia with incarceration, retained foreign body, pinworm infestation, and ileocolic intussusception. conclusions: despite the frequency of primary acute appendicitis, there is a differential diagnosis when an abnormal appendix is found by imaging. familiarity with these alternative diagnoses may be particularly helpful in guiding management of the patient whose clinical presentation is not typical for primary acute appendicitis. methods & materials: a hospital pacs database search from the past years for patients with bws. selected cases, with multimodality imaging, were cross-referenced with pathology reports from patient records database. results: intricate abdominal pathologies are depicted utilizing multimodality imaging, such as plain films, us, ct, mri and pet/ct, and with pathologic correlation. cases with highlight the following: liver: hepatoblastoma, nonspecific hepatobiliary cysts, multiple hemangiomas mimicking metastatic disease; adrenal: dysplastic organomegaly mimicking neoplasm; pancreas: diffuse and focal hyperplasia in the setting of hyperinsulinism, organomegaly; renal: neprocalcinosis, including medullary sponge kidney, nephroblastomatosis, organomegaly; adnexal: ectopic paraovarian adrenal tissue mimicking metastatic lymph node; urinary bladder: benign fibro-uroepithelial polyp. conclusions: diagnosis of bws can be difficult when the classic clinical and radiological findings are not present. these few cases highlight the unusual abdominal pathologies, so when detected, a radiologist can aid in the appropriate diagnosis and help guide therapy for these young patients. this poster will discuss pharmaceuticals the fda considers investigational for their intended use. disclosure: dr. lecompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. radiologic-pathologic review of pancreatic masses encountered at a tertiary pediatric hospital over a -year period no kwak, md, radiology, long island jewish medical center, kwak_nb@yahoo.com; karen naar, jeanne choi-rosen, lee collins, sukhjinder singh, anna thomas purpose or case report: review of pathologically proven pancreatic masses in pediatric patients encountered at a tertiary pediatric hospital over a -year period. describe the key morphologic features and other pertinent findings using various imaging modalities. correlate pathologic and radiologic findings. methods & materials: illustrate the various imaging characteristics of pathologically proven pancreatic masses including pseudocyst, pancreatoblastoma, solid pseudopapillary tumor, acinar cell carcinoma, ductal adenocarcinoma, lymphoma, pancreatic neuroblastoma, and inflammatory myofibroblastic tumor. correlate pathologic and radiologic findings. identify the key imaging features that allow narrower differential diagnosis. results: pancreatoblastoma and solid pseudopapillary tumor are the more commonly encountered pediatric primary pancreatic tumors. both are bulky and heterogeneously enhancing tumors with solid and cystic elements. pancreatoblastoma occurs more commonly in young children. internal hemorrhage and fibrous capsule favor solid pseudopapillary tumor which more commonly occurs in adolescent girls. ductal adenocarcinoma, acinar cell carcinoma and an inflammatory myofibroblastic tumor, which were pathologically proven in our pediatric patients, are exceedingly rare entities. the imaging findings of these cases and their pathology when available will be presented, as well as a quick literature review of these rare tumors. illustration and correlation of the pathologic and radiologic findings. conclusions: pancreatic masses in children are rare but in general have a better prognosis than in adults. salient imaging findings for the various tumors encountered at a tertiary care center with pathologic and radiologic correlation. evaluation of hepatoblastoma with gadoxetate disodium-typical, atypical, pre and post treatment evaluation arthur b. meyers, radiology, cincinnati children's hospital, arthurbmeyers@yahoo.com; alexander j. towbin, daniel j. podberesky purpose or case report: gadoxetate disodium (gd-eob-dtpa) is a hepatobilliary mri contrast agent that is widely used in adults for characterization of liver tumors and is being increasingly used in pediatric patients. hepatoblastoma is the most common primary hepatic malignancy of childhood. the purpose of this presentation is to describe our experience with the use of this agent in the mri evaluation both before and after initiating therapy in patients with hepatoblastoma. methods & materials: the radiology report system at our institution was queried for all patients with pathology proven hepatoblastomas who underwent a liver mr with administration of gadoxetate disodium between / / and / / . the mr imaging characteristics of the patient's primary hepatoblastoma pre-and post-therapy (when available) and post treatment findings (when available) were reviewed. results: mri studies in different patients were reviewed. the patients ranged in age from months to years. patients had pre and post treatment evaluation with gd-eob-dtpa enhanced mri, patient had only pretreatment evaluation and patients had only post treatment evaluation. of the hepatoblastomas did not take up gd-eob-dtpa during the hepatocyte phase and were therefore low signal intensity during the hepatocyte phase of imaging. this was useful in the pretreatment evaluation of hepatoblastoma, particularly in defining the relationship of the tumor to hepatic and portal veins. post treatment gd-eob-dtpa imaging allowed characterization of the biliary anatomy and demonstrated the communication of a postoperative fluid collection with the biliary tree, consistent with biloma. atypical hepatoblastoma showed uptake of gd-eob-dtpa on hepatocyte phase imaging, similar to what has been described in adults with atypical hepatocellular carcinoma. conclusions: gadoxetate disodium enhanced mri is useful in the imaging evaluation of hepatoblastoma, particularly in defining the relationship of tumor to vascular and biliary anatomy and in characterizing post-treatment complications. disclosure: dr. meyers has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. imaging of the gallbladder and biliary tree in pediatric age group ihsan mamoun, md, cleveland clinic, ihsanmamoun@ yahoo.com; s. pinar karakas, unni udayasankar, neil vachhani, ellen park purpose or case report: interactive educational exhibit to illustrate the embryology, anatomical variants as well as congenital and acquired diseases of the bile ducts and gallbladder in pediatric patients. methods & materials: a)the embryology of the gallbladder and biliary tree will be demonstrated with diagrams. b) imaging techniques for gallbladder and biliary tree including us, ct, mri, ercp and intraoperative cholangiogram will be discussed. c)imaging findings of various lesions with special emphasis on key findings that can lead to accurate diagnosis will be discussed. d)an appropriate list of differential diagnosis will be provided. e)an algorithm for the assessment of suspected biliary pathology will be presented. f)the exhibit will be interactive and the reader will answer questions about the discussed entity, related imaging algorithm and management. results: a)discuss congenital anomalies including duplicated and septated gallbladder, choledochal cyst, caroli disease, situs abnormalities and biliary atresia. b)discuss infectious and inflammatory conditions including cholecystitis, kawasaki's disease, sclerosing cholangitis and hepatitis. c)discuss iatrogenic complications including post transplant biliary stricture and leak. d)discuss benign and malignant neoplasms invoving the gallbladder including polyps, ptld and rhabdomyosarcoma. conclusions: this exhibit will demonstrate a logical approach to imaging of the congenital and acquired diseases of the gallbladder and biliary tree based on the embryology and underlying pathology. postnatal work up of congenital uronephropathies-a pictorial essay harigovinda r. challa, radiology, university of kentucky, hch @uky.edu purpose or case report: the use of obstetric ultrasound routinely in the prenatal care has lead to the discovery of many fetal anomalies. uronephropathies in the newborn represent one of the largest groups of anomalies amenable to neonatal management. since these uropathies are detected mostly in asymptomatic patients the treatment is mainly preventive. the pediatric radiologist has a key role in the post natal work up and management of these patients with prenatally diagnosed neprhouropathies and familiarity with the congenital urinary tract abnormalities is necessary. methods & materials: a retrospective review of multiple radiographic, sonographic and fluroscopic examinations performed in the newborn babies and infants with prenatal diagnosis of urinary tract abnormalities was performed at kentucky children's hospital. multiple examples of classical imaging findings were compiled and placed into a pictorial review. results: numerous anomalies can be detected in utero, including anomalies of renal number, position, morphology, collecting system dilation and bladder, urethral abnormalities. of these postnatal work of congenital hydronephrosis is the most common routinely encountered clinical entity. renal ultrasound is the initial examination in the evaluation in all cases of prenatal hydronephrosis, which is best performed around postnatal day . if collecting system dilatation persists on postnatal ultrasound, further imaging work up with vcug, radionuclide imaging may be required depending on degree of dilatation. conclusions: uroneprhopathies are increasingly detected in the prenatal life with increasing use of obstetric ultrasound. the objective of this presentation is to demonstrate in a pictorial essay of different neprhouropathies and their workup in newborns. isolated fallopian tubal torsion: causes, imaging findings, and how to suggest the diagnosis jesse courtier, md, ucsf dept of radiology, jesse. courtier@ucsf.edu; amaya m. basta, rebecca maine, pierre-alain cohen, shinjiro hirose, john d. mackenzie purpose or case report: the purpose of this educational report is to describe the rare entity of isolated fallopian tubal torsion in the pediatric population and depict the cross sectional imaging findings that help make a diagnosis and guide management. the proposed pathophysiology, predisposing factors, and surgical management will be described. an illustrative case example of -year-old female patient will be provided with surgical correlation. the exhibit will review imaging findings on us, ct and mri that help support the diagnosis including, dilated tubular structure in the pelvis, normal ovaries, and corkscrewing and beaking of the proximal fallopian tube. isolated fallopian tubal torsion will be placed in the context of a differential diagnosis for girls presenting with pelvic pain and the imaging signs that help make a diagnosis of isolated tubal torsion and separate this entity from other causes of pediatric pelvic pain will be emphasized. multimodality imaging characteristics of genitourinary rhabdomyosarcoma rhea udyavar, md, george washington university medical center, rudyavar@gwmail.gwu.edu; amir noor, pranav k. vyas purpose or case report: in this pictorial essay, we will demonstrate salient imaging features of mr, us, and ct modalities for the diagnosis of genitourinary rhabdomyosarcoma in male (n ) and female (n ) children ages - years, evaluated at our institution over the past years. background information, including tumor biology, staging, and treatment will also be discussed. the swollen scrotum: ultrasound technique and differential diagnosis kelli r. schmitz, md, oregon health & science university, schmitzk@ohsu.edu; roya sohaey purpose or case report: to review the ultrasound protocol for the performance of scrotal ultrasound and illustrate the ultrasound appearance of conditions resulting in scrotal swelling in pediatric patients. a retrospective review of the imaging database at a tertiary pediatric referral center was performed to identify pediatric patients who presented with scrotal swelling and underwent diagnostic ultrasound. when available, surgical/pathologic correlation was obtained. results: a variety of pathologic processes result in scrotal swelling. causes illustrated include: testicular torsion, epididymitis/orchitis, hydrocele, varicocele, inguinal hernia, trauma, adrenal rest, and testicular or paratesticular neoplasm. conclusions: the causes of scrotal swelling are myriad, including infectious/inflammatory, developmental, traumatic, and neoplastic etiologies. in children, the clinical presentation of a swollen scrotum is nonspecific, and ultrasound plays a key role in making the correct diagnosis. experiences of starting a functional mr urography program at a university hospital: trials and tribulations steven l. blumer, bsc, montefiore medical center/albert einstein college of medicine, sblumer@montefiore.org; ibrahim tuna, amanda north, benjamin taragin, netta blitman, terry l. levin purpose or case report: starting a functional mru program can be challenging as there are numerous potential hurdles to overcome. this presentation describes the process of starting a functional mr urography (fmru) program at a university hospital and discusses the difficulties encountered starting such a program. selecting a sufficient patient referral base, resolving common and uncommon technological issues, and education of clinicians, patients and technical staff are some of the challenges that will be discussed. conclusions: awareness of the common pitfalls in fmru imaging and close partnering with referring physicians can make establishing a functional mru program easier. despite many potential obstacles, the benefit of exquisite anatomical and functional information provided by fmru in children, without exposure to ionizing radiation, greatly outweighs any challenges. disclosure: dr. blumer has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. pictorial review of ultrasound findings in boys presenting to emergency department/urology with acute scrotum teresa liang, faculty of medicine, university of british columbia, teresaliang @gmail.com; peter metcalfe, william sevcik, michelle noga purpose or case report: testicular torsion is a common acute condition in adolescent boys. rapid and accurate diagnosis is critical. diagnosis is currently based on history, physical findings, and ultrasound (u/s) with doppler. the objective of this poster is to demonstrate ultrasound findings from a retrospective review of acute scrotum over years, and to demonstrate some pitfalls of the technique with regard to testicular torsion diagnosis. we reviewed the u/s, surgical and ed records at the stollery children's hospital for boys aged month to years, presenting with acute scrotum from july , to . age, demographics, clinical symptoms, and physical findings, u/s and surgical techniques, findings, diagnoses and follow-up were also recorded. results: patients presented to uah stollery ed with acute scrotum: were diagnosed with testicular torsion ( inguinal torsion), were suspected of a torsion-detorsion, torsion of appendix testes, epididymitis/orchitis, and other diagnoses including hydroceles, varicoceles, epididymal cysts, abscesses, cellulitis and hematomas. for the patients who had ultrasound, % sensitivity and % specificity for testicular torsion. the ultrasound findings including size, vascularity and echogenicity associated with both salvageable and necrotic testicles including use of color and pulse doppler will be reviewed. the sonographic findings and pictorial examples associated with the more common acute scrotum etiologies will be presented. sonographic findings from problematic cases (those with inconclusive ultrasound reports or false positive reports) will also be addressed. conclusions: ultrasound imaging problem case examples and characteristic findings of common acute scrotum presentations at stollery hospital at the university of alberta are reviewed in this poster. primary and secondary amenorrhea in pediatric patients: from the beginning to the end cesar cortes, md, miami children's hospital, n c @ hotmail.com; yanerys ramos, ricardo restrepo, alejandro diaz, lorena sequeira, edward lee purpose or case report: to describe the role of imaging in evaluating patients with primary and secondary amenorrhea and to illustrate the normal imaging findings of the reproductive organs in the pediatric population as well as the imaging findings of the different etiologies causing amenorrhea. a search of the literature is done to determine the different etiologies of amenorrhea and the role of imaging in their evaluation. first, we will focus on the normal physiologic hormonal influence and changes of the girl's reproductive organs since birth until adolescence on ultrasound and mri. images of the normal appearance of the female reproductive organs as well as imaging findings of the different common and uncommon etiologies of amenorrhea will be shown. then, specific reference will be made to crucial related concepts such as minipuberty of infancy, latest criteria for polycystic ovarian disease and ovarian failure syndrome among others. finally, the treatment, either medical or surgical will be briefly discussed. results: causes of amenorrhea in children range from disorders affecting the hypothalamus, pituitary gland, adrenal glands, and ovaries, as well as uterine and vaginal structural abnormalities. even though history and clinical exam are essential in evaluating a patient with amenorrhea, the pediatric radiologist plays a pivotal role helping guide the area to be imaged and thus the modality that should be used. mri and ultrasound are the main modalities in the evaluation of amenorrhea. conclusions: ultrasound and mri are the main imaging modalities used in the evaluation of amenorrhea in children and are usually part of the work up. amenorrhea in children can have implications in girl's fertility allowing pediatric radiologists to play an important role in helping not only the patient but also their offspring. imaging of mullerian duct anomalies in children kelly k. horst, md, radiology, university of michigan, khorst@med.umich.edu; maryam ghadimi mahani, deepa pai, jonathan r. dillman, peter j. strouse purpose or case report: the purpose of this educational exhibit is to provide an up-to-date appraisal of mullerian duct anomalies presenting in the pediatric population. the appearances of anatomic variants on ultrasound and mri will be used to illustrate the strengths and potential pitfalls of these imaging modalities. methods & materials: patients who have previously undergone ultrasound and/or mri in the course of their clinical workup within the university of michigan health system (umhs) were identified using electronic medical records. imaging reports were reviewed by a single author in order to identify relevant imaging findings (interesting anatomic variations, associated anomalies, etc.). pertinent images from these imaging examinations were de-identified and saved to a secure hard drive. the medical record was accessed by a single researcher to obtain relevant information regarding the patients' clinical presentations. in cases of corrective surgery, pathology reports were reviewed, if available, for correlation with the imaging findings. results: cases of mullerian duct anomalies were reviewed within the pediatric population. clinical manifestations were correlated with imaging appearances. conclusions: mullerian duct anomalies represent a range of developmental variants. although functioning ovaries and age-appropriate external genitalia are characteristic, there may be anomalies ranging from uterine and vaginal agenesis, to duplication of the uterus and vagina, to minor uterine cavity abnormalities. müllerian malformations are frequently associated with abnormalities of the renal and axial skeletal systems, and pediatric patients in particular may present with these associated anomalies. menstrual abnormalities may represent a more typical presentation in the adolescent age group. this is in contrast to the adult population, which may be more likely to present with infertility. the variation in clinical presentations make mullerian duct anomalies difficult to diagnose and, because surgical techniques for correction and treatment depend on the underlying anatomy, understanding these variants in the context of imaging studies is important to their diagnosis and management. patient had radiographs which showed an irregular left humeral metaphysis with an associated fracture. patient had a phase bone scan that showed slightly increased uptake on the angiographic and blood pool phases and increased activity on the delayed phase in the right femur. radiographs showed a moth eaten appearance of the right femur with soft tissue swelling. patient had radiographs that showed periosteal reaction in the right tibia with an associated fracture. patient , in addition to radiographs, had an mri that showed osteomyelitis of the left humerus and scapula with an associated subperiosteal abscess. patient had multi focal osteomyelitis that was demonstrated on radiographs by irregular cortices and periosteal reaction involving the upper and lower extremities. conclusions: neonatal osteomyelitis is an uncommon entity that can have severe complications if not diagnosis and treated promptly. it is important to review cases and to review the appearance of neonatal osteomyelitis on multiple modalities. radiographs will usually demonstrate periosteal reaction and possibly soft tissue swelling. additional studies may be obtained to evaluate for complications, such as abscesses or involvement of the joint space. purpose or case report: review the epidemiology of ddh. describe the critical diagnostic imaging findings of ddh. understand the role of imaging accompanying treatment. methods & materials: images including radiographs, ultrasound, ct and mri will be used to demonstrate the current and historical role of imaging in caring for patients with ddh. discussion of the importance of reducing radiation exposure when choosing imaging studies will be included. results: radiographs and ultrasound are used primarily in making the diagnosis of ddh. ultrasound and mri are most often used during the course of treatment to assess its effectiveness. mri is increasingly utilized without sedation for patients in spica cast. conclusions: imaging is critical in the care of patients with ddh. pediatric musculoskeletal ultrasound of the proximal lower extremity (pelvis to thigh) julia rissmiller, md, dept of radiology, children's hospital boston, julia.rissmiller@childrens.harvard.edu; howard christianson, michael j. callahan purpose or case report: to review indications for ultrasound of the proximal lower extremity (pelvis, hip and thigh), and to illustrate the practical use of ultrasound in evaluation of the proximal lower extremity, emphasizing the sonographic appearance of various musculoskeletal disorders. ultrasound is a well-established modality for the evaluation of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children. in general, ultrasound has a more limited role for the primary evaluation of other pediatric musculoskeletal disorders including trauma, articular and periarticular diseases and tumors or tumor-like processes. advantages of ultrasound, a relatively non-invasive technique, include excellent spatial resolution, low cost, lack of ionizing radiation, lack of need for sedation, and the ability to image the patient in real-time. the major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications. we present ultrasound examples of pathology involving the proximal lower extremity (pelvis, hip and thigh). cases include developmental hip dysplasia, hip effusion, osseous metastasis to the iliac bone, osteomyelitis of the hip, femoral acetabular impingement, rectus femoris hernia, vascular malformation, ewing's sarcoma and myositis ossificans. results: a range of images from pediatric diagnostic ultrasounds performed of the proximal lower extremity (pelvis to thigh) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders. conclusions: ultrasound is an excellent modality for evaluating the proximal lower extremity in children, beyond the current indications of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children. a multi-modality pictorial review of lesions of the epiphysis in infants and children ernesto i. blanco, md, st. christopher's hospital for children, eiblanco @gmail.com; jacqueline urbine, evan geller, peter pizzutillo purpose or case report: to review the imaging spectrum of epiphyseal lesions in infants and children. a retrospective review of our imaging database was performed to identify studies with either primary lesions of the epiphysis or processes that affect the epiphysis. results: multiple epiphyseal lesions were elucidated primarily by radiography, with cross-sectional imaging included where clinically necessary. congenital lesions include the epiphyseal dysplasias represented here by chondrodysplasia punctata. epiphyseal infarction may due to multiple etiologies including slipped capitol femoral epiphysis, developmental dysplasia of the hip, sickle cell disease, or idiopathic reasons. neoplasms may occur in the epiphysis, including chondroblastoma and histiocytosis. traumatic lesions include fracture and avulsion. osteomyelitis can occur in the epiphysis as well. pseudolesions that mimic pathology will also be reviewed. other pathologies that can affect the epiphysis include juvenile idiopathic arthritis and hemophilia. conclusions: a wide spectrum of congenital and acquired pathologies may affect the epiphysis in the infant and child. plain radiography, computed tomography, and magnetic resonance imaging all contribute to the diagnosis of these varied lesions. purpose or case report: we aim to present the spectrum of common and uncommon hip disorders in pediatric population. we will formulate a systematic approach and present a flowchart to workup and characterize hip diseases. methods & materials: relevant imaging appearances of normal as well as pathological hip will be presented. normal hip anatomy will be discussed through anatomic drawings and radiological images (plain radiographs, ct, usg, and mri). we will illustrate the various anatomic landmarks, measurements and lines on plain radiographs and ultrasound of hip. results: evaluation of limp and hip pain in the pediatric population has undergone rapid evolution. surgical treatment for these disorders continues to be refined, and our ability to identify patients along the spectrum of disease continues to improve. yet, despite our advances, obtaining an accurate diagnosis can remain challenging, especially in the setting of mild structural abnormalities. many imaging studies can be used to evaluate the bones and soft tissues, but conventional radiography is the primary imaging modality for most clinical conditions. plain radiographs usually are obtained first because they are sensitive and specific for a wide range of bone pathology. more sophisticated imaging modalities including radionuclide scintigraphy (bone scan), ultrasonography (usg), computed tomography (ct) and magnetic resonance imaging (mri) are reserved for specific clinical situations. each of these imaging modalities has specific advantages and disadvantages. it is the aim of this review to guide in selecting and interpreting the appropriate imaging modality for a variety of common disorders. this exhibit will illustrate imaging features of developmental dysplasia of hip, perthes disease, slipped capital femoral epiphyses, hip malformations in syndromes, femoral acetabural impingement, labral disorders, septic arthrits and other disorders. the role of various imaging modalities in evaluation of these disorders will be discussed, along with common imaging pearls and pitfalls. conclusions: a systematic approach is necessary for evaluation of pediatric hip disorders. familiarity with normal appearances, pitfalls and specific imaging of these entities is essential for proper diagnosis and management. osteoid osteomas: a pain in the "night" diagnosis nancy k. laurence, md, the children's hospital of philadelphia, nkang @gmail.com; monica epelman, richard markowitz, camilo jaimes, diego jaramillo, nancy chauvin purpose or case report: a common benign bone-forming lesion, osteoid osteoma comprises approximately % of all benign bone tumors. the tumor is composed of a nidus of vascular osteoid tissue and woven bone lined by osteoblasts, frequently with considerable surrounding inflammation. the radiolucent nidus surrounded by variable degrees of reactive sclerosis usually leads to a straightforward diagnosis; however, sometimes the diagnosis of osteoid osteoma can be challenging, as it may have a non-specific and misleading appearance on different imaging modalities, particularly on mri. the purpose of this exhibit is to review the typical and atypical features of osteoid osteomas on different imaging modalities. we present diagnostic dilemmas of osteoid osteomas from our institution and how imaging characteristics can aid in diagnosis. we performed a retrospective review of our imaging database to identify cases of typical and atypical osteoid osteomas, with special emphasis on cases which posed a diagnostic dilemma on imaging. results: when osteoid osteomas occur in atypical locations the diagnosis can be elusive. when located in the intraarticular space there is often minimal or absent cortical thickening and there may be a joint effusion with synovial hypertrophy. phalangeal lesions may cause extensive bone marrow edema and surrounding soft tissue swelling. both of these types of osteoid osteomas can be mistaken for infection. the recently described "ct vessel" or "vascular groove" sign, a low density vascular groove adjacent to the nidus, is highly specific for osteoid osteoma. in the authors' experience, a rim of sclerosis surrounding the nidus may aid in diagnosis on mri and can be identified as an outer hypointense halo on all sequences. we illustrate the findings in cases of atypical osteoid osteomas which may be difficult to diagnose including intraarticular, phalangeal, and vertebral osteoid osteomas. we also show examples of the newly described sign which has high specificity for osteoid osteoma. conclusions: imaging findings in osteoid osteomas can be misleading and cause misdiagnosis, especially in atypical cases. knowledge of their appearance in atypical locations and specific findings can aid in the correct diagnosis. ultrasound of normal entheses in the growing skeleton nancy chauvin, md, department of radiology, the children's hospital of philadelphia, chauvinn@email.chop. edu; pamela f. weiss, monica epelman, diego jaramillo purpose or case report: ultrasound is an underutilized modality in the evaluation of the pediatric musculoskeletal system. evaluation of tendon insertions about the elbow, knee and foot can be easily performed with ultrasonography. a good knowledge of the age dependent normal ultrasound appearance of the entheses is crucial in order to evaluate for pathology, such as trauma or ethesitis-related arthritis. this exhibit will serve to provide the reader with a practical approach to imaging when assessing tendon insertions. optimal patient positioning and transducer selection will be discussed. in addition, important anatomic landmarks will be described to allow for reproducibility and avoiding pitfalls. methods & materials: transverse and longitudinal ultrasound images of entheseal insertion sites were performed on healthy girls and boys between the ages of and years. ultrasound of the elbow was performed while in full extension and the insertions of the common flexor and common extensor tendons were evaluated. the quadriceps and patellar insertions were imaged with patients in the supine position, with the knees flexed at degrees. the achilles tendon and plantar fascia insertion were evaluated with the patient prone, with the feet hanging off the edge of the table. results: tendons demonstrated the expected fibrillar pattern with parallel echogenic lines. the appearance of the entheses changed as the insertion matured from sonolucent cartilage to echogenic bone. conclusions: using a systematic approach and knowledge of the normal anatomy, sonography of the tendons of the elbow, knee and foot can easily be performed in children. pediatric musculoskeletal ultrasound of the distal lower extremity (knee to ankle) howard christianson, md, radiology, children's hospital boston, howard.christianson@childrens.harvard.edu; julia rissmiller, michael j. callahan purpose or case report: ultrasound is a well-established technique in children for evaluation of the painful hip, developmental dysplasia of the hip, soft tissue infection, palpable masses and foreign bodies. in general, ultrasound has a somewhat more limited role for the primary evaluation of several other pediatric musculoskeletal disorders in the setting of trauma, articular and periarticular diseases and tumors and tumor-like conditions. inherent advantages of ultrasound include excellent spatial resolution, a lack of ionizing radiation, a relatively non-invasive technique and lack of a need for sedation. real-time imaging allows problem solving not available with other modalities which is well suited for musculoskeletal applications, particularly in the setting of trauma. the major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications. the purpose of this study is to illustrate the practical use of ultrasound in the evaluation of the distal lower extremity (knee to ankle) emphasizing the sonographic appearance of various musculoskeletal disorders. examples include: ) cystic lesions around the joints: baker's cyst, synovial cyst, ganglion cyst and suprapatellar bursitis; ) infectious processes: pretibial, subperiosteal and intramuscular abscess; ) tumor and tumor like lesions: nerve sheath tumor, tumoral calcinosis; ) trauma related injuries: sinding larsen johansson, tibialis anterior muscle herniation, hematoma. methods & materials: cases selected for presentation from a series of diagnostic musculoskeletal ultrasounds performed at our institution. results: a range of images from diagnostic ultrasounds performed of the distal lower extremity (knee to ankle) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders. conclusions: selected musculoskeletal ultrasounds of the distal lower extremity are presented to familiarize the audience with the sonographic appearance of various musculoskeletal disorders and to highlight the tremendous potential of ultrasound in evaluating musculoskeletal disease in children and adolescents. role of conventional and dynamic contrast enhanced magnetic resonance imaging in diagnosis of hemihypertrophy syndromes in children shrey k. thawait, md, phd , radiology, yale university-bridgeport hospital, sthawai @jhmi.edu; gaurav k. thawait, sally e. mitchell, laura m. fayad, john a. carrino, kate puttgen purpose or case report: hemihypertrophy syndromes in children are complex and there is some overlap among these conditions. hence, establishing a diagnosis can be challenging. identification of the correct vascular anomaly associated with these overgrowth disorders helps to correctly classify the disease into one of the several syndromes, which in turn guides management. in this educational poster, we will review the definition, clinical presentation, conventional magnetic resonance imaging (mri) and contrast enhanced magnetic resonance angiography and venography (mra / mrv) features of hemihypertrophy syndromes in children. methods & materials: . learn the diagnostic criteria for overgrowth syndromes in children such as klippel-trenaunay syndrome (kts) and parkes weber syndrome (pws) with special emphasis on associated vascular anomalies. . gain knowledge of high resolution mri technique for evaluation of vascular anomalies associated with the hemihypertrophy syndromes. . understand the additional value of dynamic contrast enhanced mra / mrv in the differentiation of the hemihypertrophy syndromes in the pediatric age group. results: . mri technique for a dedicated "vascular anomaly protocol" consisting of fat saturated t weighted, pre contrast axial t weighted, and post contrast triplanar t weighted fat saturated imaging will be described. . special emphasis will be provided on dynamic contrast enhanced mra/mrv. . conventional and dynamic mri features of clinically proven cases of hemihypertrophy syndromes will be demonstrated. conclusions: systematic mri interpretation utilizing a dedicated vascular anomaly protocol enables the radiologist to correctly identify the hemihypertrophy syndrome, and provide detailed extent of disease. correlative ultrasound, mri imaging and physical examination of elbows in hemophilic children andrea s. doria, md, the hospital for sick children-diagnostic imaging, andrea.doria@sickkids.ca; frederico xavier, arun mohanta, carina man, ningning zhang, pamela hilliard purpose or case report: .to report a systematic ultrasound (us) protocol for assessment of hemophilic elbows. . to discuss advantages and disadvantages of us and mri for evaluating hemophilic elbows in comparison with physical examination. .to illustrate us and mri findings and associated pitfalls in hemophilic joints. background: the value of physical examination for assessment of early arthropathic changes in hemophilic joints is unknown. us does not require sedation in young children, but involves operator training and standardized technique. mri is the reference standard imaging modality for assessment of pathology in hemophilic joints. standardization of a systematic protocol for data acquisition and interpretation of us findings and understanding of the correlation of findings between physical examination, us and mri in hemophilic elbows is essential for the use us as an outcome measure both in clinical practice and research. so far such information is not available for growing elbow joints. methods & materials: eight hemophilic boys (age range/ median, - / years) with a history of prior elbow bleeds underwent us and mr imaging, and physical examination on the same day. corresponding images on us and mri were highlighted to illustrate abnormalities and pitfalls. soft tissues (effusion/hemarthrosis,synovial hypertrophy,hemosiderin deposition) changes were characterized as small, moderate, or large. erosions, cartilage and subchondral abnormalities were graded based on depth or extent of articular changes. results: . us is helpful for discriminating synovial hypertrophy, joint effusion/hemarthrosis, and large hemosiderin deposition which otherwise generates susceptibility artifacts on gradient-echo mri obscuring adjacent tissues. . us can visualize erosions, cartilage and subchondral abnormalities at the joint periphery. however,differentiation between subchondral cysts and erosions is usually unfeasible by us. . prior knowledge of the degree of joint maturation is essential for an accurate evaluation of cartilage loss by us. . physical examination has limitations for assessment of early joint changes in contrast to us. conclusions: us can be useful for assessing hemophilic elbows, with advantages over mri in the evaluation of soft tissues. further development of an us-mri atlas on normal cartilage in growing joints is needed for definition of the value of us in the assessment of minimal osteochondral abnormalities. digital atlas of skeletal surveys of common skeletal dysplasias shawn parnell, mbbs, md, dnb, radiology, seattle children's hospital, shawn.parnell@seattlechildrens.org; corey wall, edward weinberger purpose or case report: skeletal dysplasias are conditions of abnormal bone and cartilage growth which result in short stature. developing expertise in the radiographic evaluation of skeletal dysplasias can be difficult, as more than dysplasias exist. exhaustive description of individual dysplasias can be found in hard copy textbooks, without the ability to compare individual dysplasias side by side. by providing radiographic images and descriptive text of thirteen common skeletal dysplasias and two comparative normal skeletal surveys, we aim to facilitate understanding of the terminology and highlight the differences in imaging appearances one may commonly encounter in interpreting skeletal dysplasias. methods & materials: initial skeletal surveys and/or follow up radiographs obtained for evaluation of skeletal dysplasias at our institution from to were compiled and reviewed for best quality images. selected images for each case were labeled according to body part and view, to include ap and lateral views of the spine and skull and ap views of the extremities and pelvis. for neonates, ap and lateral babygram images were used. the software program used for viewing the atlas, written in c#, may be freely downloaded. it permits linked scrolling and resizing of the images, and simultaneous comparison of different cases is available. cases may be viewed as unknowns or in a selfteaching mode. results: radiographic images for thirteen common skeletal dysplasias and two comparative normal skeletons (neonate and child) are provided within an interactive digital atlas. cases include achondroplasia, pseudoachondroplasia, cleidocranial dysplasia, thanatophoric dysplasia, diaphyseal dysplasia, multiple epiphyseal dysplasia, osteopetrosis, osteogenesis imperfecta, multiple hereditary exostoses, dysostosis multiplex, fibrous dysplasia, asphyxiating thoracic dysplasia (jeune syndrome), and spondyloepiphyseal dysplasia. conclusions: by displaying radiographic images of several common skeletal dysplasias in an interactive and comparative format with descriptive text, understanding of basic radiographic terminology and appearances will be facilitated. purpose or case report: . to classify various pediatric msk soft tissue masses . to describe pathogenesis, imaging appearances and differential diagnosis of these lesions methods & materials: radiology and clinical medical records were reviewed and pediatric patients with musculoskeletal soft tissue masses were identified. representative images were collected as examples of each lesion. the lesions were then classified into different groups based on the similar pathology and etiology. brief discussion is done for each of these masses with their multimodality imaging appearances. results: the search yielded pediatric soft tissue masses of multiple different etiologies, including post-traumatic (hematoma, fat necrosis, fibromatosis coli, myositis ossificans), inflammatory or infectious (cellulitis, abscess, granuloma annulare, retained foreign bodies), pseudotumors (synovial cysts, ganglion cysts, vascular malformations) and neoplastic lesions (fatty, vascular, neural, fibrous, muscular). multiple different imaging modalities were used to evaluate these masses, including ultrasound, ct and mri. representative examples of different lesions and their appearances on different imaging modalities will be presented and an organized approach to the diagnosis of these lesions will be discussed. conclusions: musculoskeletal soft tissue masses are relatively common in children. majority of these are benign; however, up to % of these lesions can be malignant "sarcomas". multiple different imaging modalities often provide complimentary information in the work-up of these lesions. despite multimodality imaging approach, tissue diagnosis or short interval follow-up is still often required when the mass does not show typical features of a benign etiology. pediatric radiologists should be familiar with various pediatric msk soft tissue masses and their imaging appearances, and should be able to guide appropriate management. results: elbow mri examinations were reviewed on children aged months to years with ( %) of these investigating clinical instability in children. mechanism of injuries included congenital dislocation ( %), traumatic dislocation ( %), fracture or avulsion ( %) and other injuries ( %). the patient's with congenital elbow dislocations most commonly presented with radial head dislocation and associated dysplasia or flattening, effusion and less frequently dysplasia of the olecranon or capitellum. patient's with traumatic dislocations were frequently associated with ligamentous or capsular disruption, bone oedema and epicondylar avulsion with effusion, loose osseous bodies and fractures less often. the epicondylar avulsions and ligamentous or tendon injuries occurred equally often in those few patients with unspecified injury mechanism. conclusions: a number of the bony, ligamentous, articular and developmental anomalies evident on elbow mri have been illustrated highlighting the importance of careful and systematic review of all elbow structures when presented with a child with elbow instability. accurate identification of these abnormalities is vital to facilitate their appropriate management. methods & materials: from our computerized radiology information system, we retrieved all patients that have foot ultrasound for evaluation of vertical or oblique talus deformities in the last years ( / - / ). the us was performed by a pediatric radiologist using a high resolution linear and tight convex curve probes with foot in neutral, plantar flexion and dorsiflexion. all medical charts, ultrasound scans and foot radiographs were reviewed by a pediatric radiologist. results: we identified nine patients' with foot deformities who were suspected of vertical or oblique talus and were evaluated by ultrasound. seven patients are male; two of them had initial foot radiographs that were not diagnostic. two female patients had unilateral oblique talus deformity. there were patients with vertical talus deformity; three of them had bilateral deformities. conclusions: us can directly visualize the unossified navicular, the talar cartilage and their alignment. dynamic us can. ultrasound evaluation of costal chrondral pathologies in children presented as anterior chest wall mass or pain nucharin supakul, md, radiology, riley hospital for children, tanyasupakul@yahoo.com; boaz karmazyn purpose or case report: to summarize our experience with the use of ultrasonography (us) for evaluation of costal cartilage pathology presented as anterior chest wall mass. methods & materials: from our computerized radiology information system, we retrieved all patients that have chest wall ultrasound for evaluation of a mass in the last . years ( / - / ) . the us was performed by a pediatric radiologist using a localized scan with high resolution linear probe. all medical charts, pathology results, ultrasound scans and other imaging studies were reviewed by a pediatric radiologist. results: ten patients were found with costal chrondral pathologies. nine patients presented with anterior chest wall mass and one with chest wall pain. eight patients had angular deformity of a single costal cartilage and one patient had biopsy proven osteochrondroma, presented with anterior chest wall mass. one patient had a non-union fracture after motor vehicle accident, presented with anterior chest wall pain. in patients with rib deformity, the mass was non-tender. nine patients had prior imaging study including chest x-rays (n ), ct scan (n ), breast mr (n ). all these studies were negative. conclusions: us optimally demonstrated costal cartilage abnormities. chest radiographs and cross sectional studies were negative. we therefore recommend using high resolution chest wall us in children with negative chest radiograph and anterior hard chest wall mass. challenges in pediatric marrow imaging-boning up on current mr techniques srikala narayanan, md, division of radiology, children's national medical center, snarayan@childrensnational.org; neha kwatra, nabile safdar purpose or case report: a wide range of pathologies demonstrate similar findings when imaged using conventional mr sequences. however, pediatric musculoskeletal imagers are increasingly leveraging newer techniques to add specificity to their diagnoses when abnormal marrow signal is detected. the purpose of this educational exhibit is to review the application of current mr techniques to pediatric marrow imaging across the spectrum of normal, variant, and pathologic processes. methods & materials: cases with potentially overlapping imaging appearances on conventional mr sequences, including hematopoetic marrow, sickle cell disease, osteomyelitis, chronic recurrent multifocal osteomyelitis, and infiltrative neoplasms, will be presented. the basis of various mr techniques including chemical shift imaging, "whole body" marrow imaging, diffusion weighted imaging, and fat-water separation techniques such as dixon or ideal (ge) will be reviewed. the strengths and weaknesses of such techniques in differentiating between infection, neoplasm, and normal variation will be emphasized through the case examples. challenges and pitfalls in the imaging of these pathologies using such techniques will be discussed. results: current mr imaging techniques add specificity to diagnoses of marrow pathology which are otherwise difficult to differentiate using traditional sequences alone. the use of opposed phase imaging can be helpful in differentiate hematopoietic marrow or infection from infiltrative and neoplastic conditions. "whole body" marrow imaging may serve as an alternative to other modalities which involve significant radiation exposure. the use of diffusion weighted imaging is a promising, but developing, technique being applied to marrow pathology. conclusions: pediatric bone marrow mr imaging is a challenging area for a vast majority of the radiologists. understanding normal developmental bone marrow changes and being aware of the pitfalls is crucial to render accurate diagnosis. current techniques such as ideal, chemical shift imaging, and "whole body" mri have a potentially important role in further characterization of marrow abnormalities. radiologists beware: unusual imaging manifestations in child abuse eglal shalaby-rana, mbbs (hons), children's national medical center, erana@childrensnational.org; allison m. jackson, tanya hinds, katherine deye purpose or case report: to present less common imaging manifestations of injuries in child abuse that may not be readily recognized as possibly abusive injury. methods & materials: through bi-monthly review of cases with the child protection team over a period of years, the imaging studies of patients with suspected non-accidental trauma were recorded. of the pts with suspected nonaccidental trauma, outcomes were available in patients. the child protection team concluded ( %) were cases of non-accidental trauma with reasonable medical certainty. this data base was reviewed for less common injuries that were found in these medically confirmed cases of child abuse. results: less common manifestations of abuse identified by radiographs included salter-harris injuries in the proximal humerus, and proximal femur. pelvic fractures were rare and when present were associated with sexual abuse. severe chest wall injury, with associated rib fractures, causing complete or near-complete white-out of the chest was occasionally encountered. soft tissue injures, such as hematomas were found in various locations in the body including the buttocks and anterior abdominal wall, were imaged on ultrasound and ct. paraor prevertebral injuries, with or without associated bone injury were identified; one infant presented with retropharyngeal soft tissue swelling. mri identified cervical spine injuries which included ligamentous injury and intrathecal hematomas. conclusions: while classic metaphyseal lesions and rib fractures are the most common, specific injuries documented by radiologic work up of suspected non-accidental trauma, less common injuries to the soft tissue and skeletal system may occur as a result of child abuse. the ability of the radiologist to recognize these uncommon manifestations of demonstrated in axial, coronal, and sagittal planes. the ligament of interest will be denoted by arrows. at the conclusion of the anatomy section, there will be a self assessment exam. the participants then will be asked to identify the ligament. if answered correctly, a summary slide will be displayed and, if common, images of the relevant pathology will be demonstrated. if an incorrect answer is indicated, a slide will appear denoting the incorrect answer with explanation. conclusions: hopefully, with review of this educational exhibit, the participant will have a better understanding of the relevant ligamentous anatomy of the ankle and hindfoot. purpose or case report: the purpose of this educational exhibit is to demonstrate the pathologic sonographic findings, one might encounter in the pediatric ankle. a systematic methodological approach including patient positioning, transducer orientation and sonographic technique are vital for ideal sonographic assessment of the pediatric ankle. using a data search program from a large academic institution, pediatric ankle ultrasounds performed in the last years were reviewed. pathologies include trauma, inflammation/infection, masses and congenital abnormalities. examples of normal anatomy will be included particularly when demonstrating ligament and tendon pathology. the normal side was often assessed for comparison purposes. results: ankle sonography is a useful modality to evaluate commonly encountered pathologies in the pediatric ankle. radiographically occult fractures may be discovered. ligament and tendon pathology, such as tears of the anterior talofibular ligament, high ankle sprain and peroneus longus tendon tears, can be easily detected. signs of infection that can be radiographically occult such as subtle periosteal reaction or fluid collections can be identified. finally, "lumps and bumps" can be characterized. for example, one of the most commonly encountered masses in the pediatric ankle is a ganglion cyst which can be well characterized sonographically. awareness of imaging pitfalls is also critical to avoid misdiagnosis and to guide appropriate management. conclusions: with basic ultrasound skills and knowledge of normal anatomy, sonography of the pediatric ankle is a useful modality to evaluate soft tissue structures and other pathologies. it is comparable to mri and allows for dynamic evaluation without need for anesthesia. resonance angiography (mra) using time resolved imaging is a relatively new technique that has become increasingly utilized in the diagnosis of vascular anomalies. we will describe the technique used at our institution, time resolved imaging of contrast kinetics (tricks, ge healthcare, milwaukee, wi), and the parameters that can be adjusted to optimize the exam. we will review key imaging features of hemangiomas and vascular malformations in various modalities, with a special emphasis on the tricks appearance. we performed a retrospective review of all the tricks studies performed at our institution for suspected vascular anomalies. in addition to the mr imaging features, we specifically analyzed the t weighted with fat saturation post tricks enhancement and the temporal tricks enhancement pattern. we reviewed all additional imaging including plain film, ultrasound, and ct and correlated the radiographic imaging with the available clinical and histopathologic features. results: we present illustrative cases of hemangiomas, kaposiform hemangioendothelioma, venous malformations, arteriovenous malformations, lymphatic malformations, and other pitfall lesions. we propose a diagnostic algorithm that relies heavily on the post contrast t weighted with fat saturation post tricks enhancement pattern and the temporal tricks enhancement pattern. conclusions: time resolved contrast-enhanced mra has become an increasingly important adjunct in the diagnosis of vascular anomalies. optimization of the exam technique and familiarity of the tricks imaging appearance is essential and can often assist in accurate lesion characterization. purpose or case report: vertical expandable prosthetic titanium rib (veptr) is increasingly used in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children. in contrast to spinal fusion surgery, veptr allows for growth while stabilizing the deformity. we review the indications, pre-operative imaging, normal radiographic appearance, and complications of this device. methods & materials: on review of the literature, the indications for veptr have expanded in the past several years to include thoracic insufficiency, idiopathic and neuromuscular scoliosis, and chest wall defects. we illustrate the normal radiographic appearance of the three common configurations of veptr (cradle-to-cradle assembly, cradle with lumbar extension assembly; cradle-to-ala hook assembly). we discuss the potential complications of veptr, including infection, rib fracture, dislodged hardware, and neurological injury, with an emphasis on imaging diagnosis. results: there is a relatively high rate of reported complications with veptr in the literature. therefore, awareness of the growing number of indications, as well as the expected and unexpected appearance of this device, aids in radiographic diagnosis of complications. conclusions: vertical expandable prosthetic titanium rib (veptr) is gaining acceptance in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children. recognition of the indications, normal radiographic appearance, and complications of this device will facilitate timely and accurate diagnosis. disclosure: dr. philips has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. spectrum of pediatric spinal neoplasms: an interactive tutorial benjamin t. haverkamp, md, radiology, university of missouri-kansas city, haverkampbt@umkc.edu; salvador f. iloreta, maha jarmakani, lisa lowe, seth gibson purpose or case report: the objective of this educational electronic exhibit is to provide the radiologist with an approach to pediatric spinal neoplasms. emphasis will be placed on narrowing the differential diagnosis using a combination of lesion location, characteristic imaging findings, relevant history, and associations. the exhibit format will include a casebased review of various pediatric spinal neoplasms, radiologicpathologic correlation, and a brief discussion of imaging findings useful in guiding surgical management. an interactive selfassessment exam will be presented at the end of the exhibit. results: a review of the specific etiologies will be presented with classification as intra-and extra-medullary and extradural lesions. radiologic images will be related to gross and microscopic pathology. conclusions: after viewing this exhibit, the learner will be able to: . recognize the clinical, imaging, and pathologic characteristics of pediatric spinal neoplasms. . understand relevant imaging findings useful in surgical management. . test their understanding of the presented material through an interactive exam. poster #: edu- craniosynostosis jason tsai, mbchb, children's hospital boston, jason. tsai@childrens.harvard.edu; diana p. rodriguez purpose or case report: to review the normal developmental appearance of the cranial sutures with computed tomography (ct) and to describe ct findings of the various forms of craniosynostoses. in this irb-approved retrospective study we reviewed ct images of subjects diagnosed with craniosynostosis between and september . we included patients with single-suture synostosis, isolated bilateral coronal synostosis, pansynostosis, and combined craniosynotoses. additionally, we identified individuals with normal appearing sutures from to years of age imaged with head ct to describe the pattern of normal development of the cranial sutures. results: a description of the normal developmental ct appearance of the cranial sutures using computed tomography has been provided. of the group of patients with craniosynostosis the following variables were recorded: age at presentation, the pattern of sutural fusion, skull shape, presence of hydrocephalus, genetic testing, and types of surgical correction. conclusions: we have demonstrated the normal developmental ct appearance of the cranial sutures and the ct patterns of the various forms of craniosynostoses, with clinical, genetic and surgical correlation. posterior fossa tumours: a pictorial review sam byott, md, manchester children's hospital, sambyott@ hotmail.com; neville wright, vivian tang, abdu shabani, stavros stivaros purpose or case report: posterior fossa tumours account for - % of childhood brain tumours. the most common differentials include pilocytic astrocytoma, medulloblastoma and ependymoma. mr imaging is crucial to diagnosis, staging and identification of complications such as hydrocephalus and haemorrhage. soft tissue characteristics alongside tumour location, invasion and clinical history facilitate radiological discrimination prior to surgery. however, there is significant clinical equipoise with regards to the imaging appearances in a significant proportion of cases making definite diagnosis difficult. the aim of this study is to evaluate the radiological findings and correlate with histological data. this will allow identification of the key morphological features that discriminate different tumours. these can then be presented to educate fellow radiologists. methods & materials: radiology pacs and patient notes were used to collate radiological, histological and clinical data. results: there were patients presenting at our institution with posterior fossa tumours. had pilocytic astrocytomas, had medulloblastomas and had ependymomas. one patient had an atypical teratoid rhabdoid tumour (atrt). traditional features alongside more advanced mr characteristics were correlated with histology, and the features allowing for discrimination of tumour types are presented in this pictorial review. conclusions: posterior fossa tumours have a highly variable radiological appearance. we present a range of appearances and describe the important morphological features that allow radiological discrimination of tumour type. poster #: edu- dt imaging of the pediatric spine teresa c. gross kelly, children's hospital of wisconsin, tkelly@chw.org; ibrahim s. tuna, mia s. kelly, tushar chandra, sumit singh, mohit maheshwari, hervey d. segall purpose or case report: some abnormalities of the pediatric spine can be challenging. we have discovered that in many such cases, diagnosis of spinal lesions can be faciliated by using the dt weighted sequence. the purpose of this educational poster is to demonstrate the remarkable usefulness of dt weighted images for delineating pathology of the pediatric spine. methods & materials: lesions of the spine that will be reivewed in this educational exhibit will be categorized as: ( ) vascular ( ) due to infection/inflammation ( ) neoplastic/ neurogenic ( ) congenital ( ) traumatic/iatrogenic ( ) endocrine/metabolic. the imaging characteristics of lesions found in the pediatric spine will be described and the utility of d t -weighted mr sequences for the evaluation of these lesions will be discussed. finally the role of imaging in the treatment planning of abnormalities of the pediatric spine will be addressed. results: this educational exhibit will provide numerous examples of how d t -weighted imaging can elucidate diagnosis of lesions involving the spine. examples include enhancement of the cauda equina in guillain barre syndrome, lipomatous malformations, spondylolysis in children with low back pain, thecal cysts, filar cysts, metastasis, hydromyelia and ventriculus terminalis. conclusions: d t -weighted images of the spine performed in the sagittal plane with coronal and axial reformations, as well sagittal oblique reformations (scotty dog reformations) for evaluation of spondylolysis, can facilitate the evaluation of lesions involving the pediatric spine. the normal pediatric spine: a pictorial review of mr anatomy and development in the infant, child and adolescent ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; teresa c. gross kelly, tushar chandra, mohit maheshwari, sumit singh, hervey d. segall purpose or case report: radiological evaluation of the pediatric spine can be more challenging in child than in the adult patient due to the wide range of normal anatomic variants and synchondroses, combined with the unique effects of trauma in children. mri is an excellent imaging modality for the evaluation of the pediatric spine. however, in order to provide an accurate interpretation of acute posttraumatic changes in the pediatric spine, particularly in the setting of abusive head trauma, a fundamental knowledge of normal anatomy, variants and pathology of the pediatric spine is required. the aim of this educational exhibit is to illustrate normal mri anatomy of the spine in the infant, child and adolescent. methods & materials: this exhibit will first describe basic spinal embryology and development of the vertebra and spinal cord, followed by mri depiction of the developmental anatomy of the spine from infancy through adolescence. the changing appearance of the spinal canal, spinal cord and vertebral bodies with age will be illustrated using normal cases from the radiology database. sagittal and transverse diameter of vertebral bodies, thickness of the dural thecal sac, dimensions of the spinal canal, normal bone marrow signal changes, vertebral body heights, level of conus medullaris, prevertebral and paraspinous soft tissues and epidural fat thickness will be described and changes according to age will be pointed out. results: in early life, the spinal cord extends to the inferior aspect of the bony spinal column. because the vertebral bodies grow longitudinally faster than the spinal cord does, the conus medullaris may change. ossification of the vertebral bodies and posterior elements is nearly complete by age , with a resultant decrease in the spinal canal diameter. the nucleus pulposus becomes smaller after years and spans approximately half the disk space in the sagittal plane. the spinal cord is elliptical in cross section in the cervical spine and demonstrates a difference in signal between the normal gray and white matter of the spinal cord which should not be mistaken for intramedullary pathology. conclusions: a solid understanding of normal spine anatomy and embryological development is essential in evaluation of pediatric spine, mainly in the setting of trauma. familiarity with normal anatomic variants is essential to provide an accurate interpretation of pathology in the pediatric spine. spectrum of intracranial cystic lesions in infants and children ernesto i. blanco, md, st. christopher's hospital for children, eiblanco @gmail.com; eric faerber purpose or case report: to review the imaging spectrum of intracranial cystic lesions in the pediatric population. methods & materials: a retrospective review of our imaging database was performed to identify studies obtained in which the findings included intracranial cystic lesions. results: multiple cystic lesions were elucidated primarily by computed tomography or magnetic resonance imaging. these lesions can be divided into nonneoplastic and nonneoplastic tumor-associated cysts. the nonneoplastic cysts, which is the largest group, include: cavum septi pellucidi and cavum veli interpositi, choroid plexus cyst, enlarged peri-vascular spaces, pineal cyst, the large spectrum of arachnoid cysts, colloid cyst, epidermoid cyst, rathke cleft cyst, and porencephalic cyst. nonneoplastic tumor-associated cysts include: craniopharyngioma, optic glioma, pilocytic astrocytoma, hemangioblastoma, and ganglioglioma. conclusions: intracranial cystic lesions are relatively common entities in the pediatric population. a wide spectrum of nonneoplastic and nonneoplastic tumor associated pathologies are presented using both computed tomography and magnetic resonance imaging. kelly, sumit singh, ibrahim s. tuna, hervey d. segall purpose or case report: the aim of this educational exhibit is to provide a comprehensive review of imaging features, classification and management of pediatric spinal cord tumors. we also aim to elicit the differences between pediatric spinal cord tumors and their adult counterparts. we will summarize the differences between the individual tumors based on histological cell types and the pertinent implications on management and outcome methods & materials: this exhibit will provide an overview of the common as well as uncommon tumors of the pediatric spinal cord. various classification systems for these tumors-anatomical as well as histological will be discussed. we will illustrate the relevant imaging findings that can help in differentiating these tumors. results: pediatric spinal cord tumors account for % to % of all pediatric central nervous system tumors. mri is the mainstay for the initial diagnosis as well as the post surgical evaluation and surveillance of these tumors. pediatric and adult spinal cord tumours differ both in terms of anatomical location as well as histology. the disease and treatment related morbidities are also different in children as compared to adults. astrocytomas, ependymomas, glioneural tumors and csf metastasis represent the vast majority of cord neoplasms in the pediatric age group. some of cord tumors may also be associated with inherited syndromes (like neurofibromatosis type ) or may have genetic predisposition. these would also be discussed. we will also illustrate and discuss common non neoplastic spinal masses that may mimic tumors. conclusions: pediatric spinal cord tumors have varied clinical presentations, imaging appearance and outcome. this review would improve the understanding of these tumors thereby helping in diagnosis, management and follow up of these uncommon neoplasms. multi-modality imaging of pediatric head and neck lesions jason au, md, oklahoma university health sciences center, jasonmau@gmail.com; anthony alleman, mahmoud elkaissi, roy jacob purpose or case report: the purpose of this study is to present a side by side comparison of the multi-modality imaging features of pediatric masses. using cases that have been imaged with multiple modalities, the exhibit will delineate the sonographic, mr, and ct appearance of congenital, infectious, and neoplastic head and neck lesions in the pediatric population. methods & materials: a restrospective search of pacs was performed on studies completed at the oklahoma university medical center on the oklahoma university health science center campus from january to the present. ultrasound, ct, and mr examinations were selected that depicted relevant pediatric head and neck pathology. all studies were de-identified prior to image export. results: over twenty representative cases of pediatric infections, fibrous tumors, cystic neoplasms, vascular malformation, bony tumors, developmental anomalies, and other neoplasms were selected for inclusion. results: pictorial review of cases including the following representative cases: myelonmeningocele associated with arnold chiari malformation, lipomyelomeningocele, tethered cord with spinal lipoma/fibrofatty filum, tethered cord and dermal sinus tract, and chiari i with syringohydromyelia. several unique cases including the following will be presented as well: thoracic meningocele with arnold chiari malformation, terminal myelocystocele, diastematomyelia, and myelomeningocele without arnold chiari malformation. while mri demonstrates the cranio-cervical junction and the cervicothoracic spinal cord better than ultrasound, ultrasound often allows for improved resolution of the distal spinal cord, lumbosacral spinal canal, and spinal dysraphism structures near the skin surface in the neonate. conclusions: congenital spinal malformations are complex and variable in imaging appearance. it is important to understand the classification in order to determine the appropriate management and prognosis. in the neonatal period imaging should be performed with ultrasound and mri studies, as they may provide different and complementary information. conclusions: hypoxic ischemic injury is a common condition resulting in a wide spectrum of severe neurological defects. while in the past treatment only consisted of supportive care for hii, recent advances have yielded promising treatment options if initiated within a limited time window. thus due to the severity of the disease and the need for rapid intervention, it is important to recognize radiological manifestations of hii along with its clinical signs and symptoms to offer a better prognosis to the patient. craniosynostosis: looking beyond the sutures tushar chandra, md, children's hospital of wisconsin, drtusharchandra@gmail.com; teresa c. gross kelly, mohit maheshwari, sumit singh, ibrahim s. tuna, hervey d. segall purpose or case report: the aim of this educational exhibit is to provide a framework upon which the diagnosis of the various types of craniosynostosis can be facilitated. our goal is to provide an efficient way to evaluate craniosynostosis for the radiologist in clinical practice. we plan to accomplish this goal by providing a succinct review of the sutures, an overview of the various classification schemes for craniosynostosis and potential complications associated with premature sutural closure. the role of imaging in the evaluation of craniosynostosis will be described and the features of craniosynostosis that are most important to the craniofacial surgeon will be elucidated. finally, surgical strategies for the repair of craniosynostosis and postoperative findings will be described. results: some of the forms of craniosynostosis may have a genetic basis, but many are spontaneous in nature. untreated progressive craniosynostosis can lead to inhibition of brain growth, and an increase in intracranial pressure. mdct with mip and d surface reformations is the preferred modality for diagnostic evaluation of craniosynostosis. it is also a robust modality for post operative assessment and long-term follow up. mri is a useful adjunct for assessment of associated intracranial anomalies and complications. timely and appropriate imaging is essential to assess for potential complications of craniosynostosis which may include intracranial hypertension, anomalies of external and middle ear, hydrocephalus, chronic tonsillar herniation, cranial base deformity, impaired venous drainage, enlarged emissary foramina and veins and optic atrophy. on the other hand, positional plagiocephaly should not be misinterpreted as craniosynostosis. surgical management is typical for nonsyndromic craniosynostosis, which involves correction of craniosynostosis between three to six months of age. conservative management is the mainstay for syndromic craniosynostosis. postoperative follow up imaging for surveillance for ventricular size and signs of raised intracranial pressure are necessary. conclusions: craniosynostosis is a challenging area of pediatric neuroimaging. knowledge of the sutural anatomy, an understanding of the potential intracranial complications caused by premature sutural closure, as well as the role that imaging plays in presurgical planning, can provide a practical way for the radiologist to evaluate craniosynostosis in a fast-paced clinical setting. poster #: edu- the perinatal brain and spinal cord-imaging across a life border: a case-based approach anand dorai raju, md, radiology university of tennessee, araju@uthsc.edu; harris l. cohen, matthew whitehead, asim choudhri purpose or case report: to review normal and abnormal perinatal ultrasound (us) and magnetic resonance (mr) imaging findings and note their significance for the analysis of the fetal and neonatal brain as well as spinal cord and vertebral column using a case based approach. to highlight us and mr capabilities in allowing correct perinatal diagnosis of congenital and acquired central nervous system abnormalities. methods & materials: cases will be shown of normal and abnormal anatomic findings in fetal and neonatal brain and spinal cord imaging. key teaching points necessary for the diagnosis of such brain abnormalities as ventriculomegaly, chiari malformations, holoprosencephaly, and agenesis of the corpus callosum as well as dandy walker malformations and avms will be discussed. intraventricular hemorrhage, periventricular leukomalacia, anoxic injuries and infectious abnormalities will be reviewed. abnormal anatomic findings in fetal and neonatal spine evaluations for congenital and acquired abnormalities and key teaching points necessary for the accurate diagnosis of tethered cord, myelomeninocele, caudal regression syndrome, hydromyelia, diastomatomyelia and sacrococcygeal teratoma will be reviewed. some diagnostic difficulties and controversies will be addressed. conclusions: ultrasound aided by mri can provide ready diagnosis to many central nervous system abnormalities involving fetuses and neonates. ever improving perinatal imaging experience and technique allow for better prenatal as well as postnatal diagnosis. cases showing such imaging and key points helping such imaging diagnoses will be reviewed. overview of imaging of pediatric extraocular orbital tumors srikala narayanan, md, division of radiology, children's national medical center, snarayan@childrensnational.org; nadja kadom, gilbert l. vezina purpose or case report: to show the spectrum of benign and malignant extraocular orbital tumors in children. methods & materials: we reviewed the cross-sectional imaging of orbit (ct and mr) done in the last years. specific imaging signs of extraocular tumors including benign and malignant tumors such as hemangiomas, lymphangiomas, optic nerve glioma, optic nerve sheath meningioma, pseudotumors, rhabdomyosarcoma, orbital myofibroma, eosinophilic granuloma and neuroblastoma metastases will be shown. important imaging features that should be considered when formulating a differential diagnosis will be described. conclusions: the spectrum of diseases affecting pediatric orbit is substantially different from what we see in the adults. it is not easy always to differentiate between different tumors. important imaging characteristics will help us towards better differential diagnosis. in this exhibit, we will illustrate ultrasound anatomy of the neonatal spinal cord. discussion of the normal anatomic variants and pathological conditions of the spinal cord will be provided. representative images of a variety of common and uncommon pathological conditions of the spine will be presented to illustrate teaching points. in abnormal cases, follow up mri images will also be illustrated for comparison. results: ultrasound is a robust screening modality for evaluation of the lumbosacral spine in neonates. it is cheaper, readily available, safer first line imaging modality in neonates suspected to have spinal malformations. under able and well trained operator, diagnostic accuracy of spinal ultrasound approaches mri. however, mri remains the gold standard for imaging evaluation of spine. normal variants that simulate disease processes like ventriculus terminalis, prominent filum terminale and central echo complex will be presented. congenital malformations of the cord such as tethered cord, hydromyelia, lipoma, diastematomyelia, myelomeningocele, lateral meningocele and presacral masses will also be discussed. conclusions: ultrasound is a very useful screening technique for evaluation of pathological conditions of lumbosacral spine in neonates. this review would improve the understanding of utility and limitations of ultrasound in evaluation of neonatal spinal malformations. purpose or case report: although mri is the standard for detecting epilepsy and brain tumor abnormalities, pet-ct is performed to ascertain metabolism related to epileptogenic regions or characterize tumor metabolic activity. asymmetric metabolism often correlates to structural abnormalities like cortical dysplasia. metabolic activity often correlates with tumor aggressiveness or grade. fdg pet is commonly used to assess seizure and tumor metabolism. the lesser utilized amino acid pet tracers (c methionine, fdopa) show increasing value with lower grade tumors due to high tumor to normal tissue contrast. literature is accumulating regarding c methionine (cmet) in the detection of lesions like cortical dysplasia and its ability to delineate low grade seizure related tumor lesions. despite the established fdg and accumulating cmet literature, little information exists about the imaging seen with both in pediatrics. as these studies are increasingly viewed as part of fusion mri images, there is more scrutiny of focal metabolism correlating with mri findings and less interpretative reliance on abnormality based solely on asymmetry. methods & materials: review of patients who underwent cmet and fdg brain pet-ct was performed. each was imaged on a philips scanner and had prior mri. studies demonstrating a variety of tumors, postoperative findings of residual or recurrent tumor, and pseudoprogression were selected. epilepsy cases with structural cortical abnormalities or seizure-associated tumors were also selected. cmet and fdg studies were analyzed by pediatric neuroradiologists and the imaging findings correlated with prior mri and any pathology or follow-up imaging. pictorial galleries of the cmet and fdg imaging patterns were created. results: pathologically proven low-grade glial tumors showed increased cmet uptake and no hypermetabolism on fdg. high-grade tumors showed increased uptake on cmet and hypermetabolism on fdg. patients with residual or recurrent tumors showed uptake similar to their original tumor. granulation tissue and pseudoprogression changes showed increased uptake on cmet and no hypermetabolism on fdg. epilepsy surgery patients with cortical dysplasia or low grade glial tumors showed increased uptake on cmet and fdg hypometabolism. conclusions: this study illustrates the variety of findings on cmet and fdg pet-ct in pediatric patients clinically evaluated for brain tumor and epilepsy. this atlas provides readers with a guide to the appearance of these findings on an emerging imaging technique. pediatric head and neck neoplasms: a multimodality pictorial review alok jaju, md, mallinckrodt institute of radiology, alokjaju@gmail.com; marilyn j. siegel purpose or case report: neck masses are common in children and most occur in the suprahyoid region. knowledge of the fascial spaces involved in conjunction with imaging features can help in diagnosis. in this pictorial review, we present a multimodality imaging approach based on anatomy of the suprahyoid fascial spaces for evaluation of pediatric neck tumors. methods & materials: radiology information system (ris) at our tertiary care children's hospital was queried to identify patients with suprahyoid neck masses who had imaging performed between july and present. a variety of conditions having congenital, inflammatory, neoplastic, or vascular origin were identified and the anatomic location in the neck as well as imaging and clinical findings were retrospectively reviewed. results: the imaging evaluation included ultrasound, ct and mri. lesions arose within the following fascial spaces of the suprahyoid neck: superficial, carotid, masticator, submandibular, sublingual, parotid, parapharyngeal, visceral, retropharyngeal and prevertebral. key imaging features important in diagnosis included lesion vascularity, calcification, necrosis and bone invasion. we discuss and illustrate these imaging findings and relate them to specific suprahyoid fascial spaces. specific lesions include vascular and lymphatic malformations, teratoma, nerve sheath tumors, thyroglossal duct and branchial cleft cysts, pleomorphic adenoma, dermoid cyst, ranula, lymphadenopathy, abscess, lymphoma, rhabdomyosarcoma, neuroblastoma and nasopharyngeal carcinoma. conclusions: knowledge of fascial spaces of the suprahyoid compartment and key imaging features on multiple modalities can aid in the diagnosis of pediatric neck masses. pediatric sinusitis: spectrum of imaging findings with clinicopathologic correlation roy jacob, md, university of oklahoma, drjacobr@gmail. com; paul digoy, robert s. glade, anthony alleman purpose or case report: the clinical spectrum of sinusitis in children can range from uncomplicated bacterial sinusitis to invasive fungal sinusitis. most cases respond favorably to medical management. however, complications occasionally occur due to the spread to adjacent structures. imaging plays an important role in characterizing the disease and guiding the clinical and surgical planning and treatment. this electronic presentation outlines the following- . review radiologic anatomy and unique characteristics of pediatric sinuses. . review the clinical features, pathophysiology, and microbiology of sinusitis. . review of ct and mri imaging characteristics of sinusitis with representative cases such as complicated sinusitis and invasive fungal sinusitis. . review the treatment approaches of sinusitis. methods & materials: a retrospective search of pacs was performed on studies completed at the ou children's hospital in oklahoma city for the last three years. ct and mr examinations were selected that depicted relevant disease processes. corresponding nasal endoscopic pictures were obtained from cases which required surgical management. all studies were de-identified prior to image export. results: over fifteen representative cases of the clinical spectrum of sinusitis and its complications were selected for inclusion. conclusions: this educational exhibit provides a concise review of imaging, clinical features, and treatment of pediatric sinusitis. findings will be richly illustrated with radiological and clinical images. microcephaly or hydrocephalus. knowing the embryology of the cerebellum and th ventricle is important to perceive the development of posterior fossa malformations and to further understand the imaging findings. several classifications schemes have been proposed from a pure embryologic to an imaging-based approach using some essential findings such as the size of the posterior fossa, the presence of csf collection or expansion of csf space, and the size and morphology of the cerebellum. mr is the gold-standard for adequately access and characterize the posterior fossa structures. this pictorial essay will review the mr findings of some of the most common posterior fossa malformations including dandy-walker malformation, persistent blakes pouch, mega cisterna magna, arachnoid cyst, paleocerebellar hypoplasia, cerebellar agenesis, cerebellar and pontocerebellar hypoplasia, cerebelar cortical malformations, isolated brainstem hypoplasia/dysplasia and chiari malformations. we will provide a practical approach to the mr findings of posterior fossa malformations in children. conclusions: mr plays a crucial role in identifying and characterizing malformations of the posterior fossa structures. it should give a logical approach to these complex malformations thus guiding the refereeing physician into the clinical approach and in determining further investigations. results: neuroimaging features of abnormal thalami as encountered in the pediatric population were detailed, and wherever applicable, the relevance of additional mr imaging sequences and techniques to determine etiology was described. while there was considerable overlap in imaging appearances, making a precise diagnosis was found to be challenging in difficult cases, and by and large, a stepwise approach was successfully formulated and used to: . diagnose the more emergent conditions and to . devise a management algorithm for the less acute abnormalities. conclusions: bilateral thalamic lesions are occasionally encountered in pediatric neuroimaging and have a limited differential; a good knowledge base and adequate technique are imperative to tease out the precise diagnosis and institute appropriate management. cortical developmental abnormalities in pediatric seizure patients ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; mohit maheshwari, teresa c. gross kelly, sumit singh, tushar chandra, hervey d. segall purpose or case report: to describe various cortical malformations with illustrative examples. we will also briefly discuss the embryology, genetic basis, classification schemes and characteristic imaging findings . methods & materials: this exhibit will illustrate three main categories of cortical malformations: neuronal proliferation, migration and organization. understanding of this complex topic would be facilitated by brief discussion on the embryological basis and proposed genetic causes of some of these cortical malformations. classification schemes on embryology and imaging will be discussed. characteristic imaging findings of these malformations will be discussed and examples from the authors database will be shown. results: neuroimaging in pediatric seizures is challenging. mri is considered the imaging modality of choice because of superior soft tissue contrast and better ability to characterize the pathologic process. we will also discuss the dedicated seizure protocol which is used in our institute. pet-ct imaging can also provides additional information in cases where mri is negative, inconclusive or does not correlate with eeg/clinical findings. brief discussion on advanced imaging techniques will also be presented. malformations are frequently detected in infancy. however, if the initial mri scan performed in infancy is negative, a repeat scan after years of age may be helpful. conclusions: evaluation of cortical malformation in seizure patients still remains a challenging area of pediatric neuroimaging. reviewing of the embryological basis, classification schemes and characteristic imaging findings would improve the understanding the cortical malformations and interpreting the images. poster #: edu- sprs best poster cystic neonatal lesions associated with the spinal cord: discussion and differential diagnosis for these uncommon lesions jacob pirkle, md, jpirkle@mc.utmck.edu, james boyd, brian dupree purpose or case report: to review intradural cystic neonatal spine lesions and discuss the various causes and appearance of these lesions. this poster presentation provides a brief review of neonatal cystic spine lesions, including their etiologies, and presents the targeted audience (radiology resdients, fellows, and practicing radiologists) a helpful differential diagnosis of these lesions based upon their imaging appearance. methods & materials: a brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material. images from several cases of cystic neonatal spine lesions are presented. results: a brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material. images from several cases of cystic neonatal spine lesions are presented. conclusions: neonatal spine ultrasound is often performed to evaluate for abnormalities related to the presence of sacral dimples, cutaneous stigmata, skin tags, hairy tufts, during the evaluation of other congenital anomalies, or when prenatal ultrasound/mri demonstrates an abnormality warranting postnatal follow-up. the identification of cystic spinal cord lesions is relatively rare in the neonate. however, the etiology of these lesions can often be deduced or surmised based upon the location and the imaging appearance of the lesion. the most common cause of a cystic intramedullary spinal lesion is ventriculus terminalis, with a reported incidence of . %. additional lesions include transient dilatation of the central canal, filar cyst, syringohydromyelia, intramedullary arachnoid cyst, and myelomalacia related to in utero/birth trauma. extremely rare etiologies in the neonate include epidermoid/dermoid, cavernous malformation, intranatal cystic infections etiologies, neuroepithelial cysts, and cystic neoplasms. mimics include diastematamyelia, spinal lipomas, and intramedullary hematomas. numerous imaging examples of these lesions are provided in the accompanying poster. brain mri in peroxisomal disorders: a pictorial essay bruno p. soares, md, radiology and biomedical imaging, university of california at san francisco, bruno.soares@ucsf. edu; leonardo vedolin, guido gonzalez purpose or case report: our presentation aims to illustrate the brain mri patterns in peroxisomal disorders. peroxisomes are intracellular organelles involved in important cellular processes including beta-oxidation of very-longchain fatty acids and plasmalogen production. peroxisomal disorders can be categorized into disorders of peroxisomal biogenesis, in which the peroxisomes are abnormally formed and several peroxisomal functions are deficient, and in defects involving a single peroxisomal function, in which the structure of the peroxisome is intact. disorders of peroxisomal biogenesis include zellweger syndrome, neonatal adrenoleukodystrophy, infantile refsum disease and rhizomelic chondrodysplasia punctata. numerous disorders are caused by loss of a single peroxisomal function including x-linked adrenoleukodystrophy and acyl-coa oxidase deficiency. clinical findings in peroxisomal disorders include dysmorphic features, hepatic dysfunction, neurodevelopmental delay, retinopathy and hearing impairment. methods & materials: pictorial essay illustrating brain mri patterns in peroxisomal disorders, including disorders of peroxisomal biogenesis and disorders with loss of a single peroxisomal function. results: brain abnormalities in peroxisomal disorders have a wide spectrum of patterns. neuronal migration disorders with abnormal myelination are typically seen in zellweger disease and neonatal adrenoleukodystrophy. specifically, the association of abnormal myelination with germinolytic cysts is suggestive of zellweger syndrome. classic x-linked adrenoleukodystrophy typically shows posterior central white matter involvement and symmetric demyelination also involving the corticospinal tracts and corpus callosum. a similar pattern of white matter involvement is seen in acyl-coa oxidase deficiency and infantile refsum disease. conclusions: brain mri helps narrow the differential diagnosis and guides subsequent evaluation in infants presenting with clinical features concerning for peroxisomal disorders. therefore, knowledge of the brain mri patterns in peroxisomal disorders is important for the radiologist interpreting neuroimaging studies. clots in tots: role of imaging in diagnosis of acute stroke and its causes in children asif abdullah, c.s. mott children's hospital of the university of michigan, asifa@med.umich.edu; ellen hoeffner, augusto elias purpose or case report: stroke is a major cause of morbidity and mortality in children. long-term neurologic deficits occur in % to % of infants and children after arterial ischemic stroke. limited awareness regarding pediatric stroke among physicians and in general community is a major concern. imaging plays crucial role in the diagnosis of pediatric stroke. the goal of this presentation is to provide awareness to the reader about the role of imaging in childhood stroke and its myriad causes in children. we will provide a case based approach to imaging diagnosis of acute pediatric stroke based on three categories: ( ) arterial ischemic stroke, ( ) cerebral venous thrombosis, and ( ) hemorrhagic. arterial ischemic stroke (ais) is classified according to the pediatric stroke classification (psc). psc includes eight subtypes of ais: ( ) sickle cell disease, ( ) cardioembolic disease, ( ) moyamoya syndrome, ( ) cervical arterial dissection, ( ) stenoocclusive cerebral arteriopathy, ( ) other determined etiology, ( ) multiple probable etiologies, and ( ) undetermined etiology. we will describe the role of computed tomography (ct) and magnetic resonance imaging including angiography (mri/mra) in identifying these causes in relation to available clinical data. the etiologies of cerebral venous thrombosis related infarction would be discussed from an imaging perspective with a case-based approach with emphasis on mrv and swi techniques. finally, we will focus on hemorrhagic causes of childhood stroke such as vascular malformation, aneurysm, neurocutaneous disorders, coagulopathy, and a variety of other causes from an imaging standpoint. perfusion imaging in pediatric stroke demonstrates flow within the brain and can detect areas that are at risk of ischemia; however, further studies in the pediatric population need to be validated for the role of this technique in pediatric stroke. results: the most important factors in the diagnosis of childhood stroke are causal investigation, appropriate laboratory tests, and imaging studies. imaging is frequently the first step in the evaluation of an acutely ill child. conclusions: pediatric stroke is a debilitating disease that requires urgent multidisciplinary approach for diagnosis and treatment. in cases of both ischemic and hemorrhagic origin, the radiological approach to be obtained in emergency setting leads to the initial screening and the first therapeutic possibility. methods & materials: this exhibit will illustrate the characteristic imaging findings of vascular anomalies in the head and neck region. vascular anomalies are divided into vascular tumors and vascular malformations which include slow flow malformations (capillary malformations, venous malformations, lymphatic malformations and their combinations) and high flow malformations (arteriovenous fistula and arteriovenous malformations). complex malformations are also seen in several syndromes including klippel-trenaunay syndrome, phace syndrome, etc. cases from author's database will be used for illustration. results: a review of clinical manifestations, characteristic imaging findings and interventional treatment strategies in cases of head and neck vascular anomalies will be presented with pre and post treatment imaging features. ultrasonography and mri are the mainstay in diagnosis of these malformations. ct scan and catheter angiography may occasionally be needed for diagnosis and treatment planning. various imaging findings and main treatment options will be listed. conclusions: head and neck vascular malformations are common in pediatric population. understanding the characteristic imaging findings and clinical presentation is essential in evaluating the vascular malformations. interventional procedures are generally the preferred treatment modality, either alone or in association with surgery in majority of these cases. isolated cortical diffusion restriction in pediatric brain mri ihsan mamoun, md, cleveland clinic, ihsanmamoun@ yahoo.com; sarah stock, s. pinar karakas, unni udayasankar, janet r. reid purpose or case report: diffusion-weighted imaging continues to emerge as a powerful neuroimaging tool. isolated cortical restricted diffusion is a particularly striking pattern with specific differential in the pediatric population. we aim to review this specific imaging pattern supplemented by case examples and key physiologic and imaging concepts. methods & materials: review the concept of diffusion restriction a) pathophysiology b) specific imaging appearances pictorial review of pediatric conditions that lead to cortical restricted diffusion: a) post ictal change b) infection-i. meningoencephaliitis ii. herpes c) hypoxic ischemic injury d) infarct: venous and arterial e) posterior reversible leukoencephalopathy f) mitrochondrial cytopathy g) metabolic: hypoglycemia. discuss certain artifacts. summary table and differential clues conclusions: the pattern of isolated cortical restricted diffusion has specific differential diagnosis in the pediatric population. the radiologist should be aware of this as use of dwi continues to grow. this exhibit with familiarize the reader with common conditions that specifically affect the cortex and produces true restricted diffusion. methods & materials: high resolution ct scan and mri are mainstay of diagnosis and assessment in patients with sensorineural hearing loss. in this exhibit we will present a pictorial review of ct scan and mri images of various causes of sensorineural hearing loss (snhl) that are seen on imaging. reviewing the embryologic basis of these anomalies would enable better understanding of this complex subject. results: the new system classifies these malformations according to descending order of severity into complete labyrinthine aplasia, cochlear aplasia, common cavity, cystic cochleovestibular malformation or incomplete partition-i (ip-i), cochleovestibular hypoplasia, and incomplete partition-ii (ip-ii). there is a lot of confusion in literature pertaining to mondini deformity. the new classification divides incomplete partition into ip-i representing cystic cochleovestibular malformation and ip-ii representing the classic mondini deformity with three components (cystic cochlear apex, dilated vestibule, and large vestibular aqeduct). recently a subclassification of ip-i and ip-ii has been proposed (subdividing into typical and atypical subtypes)[ ]. this will be discussed briefly. isolated large vestibular aqueduct without associated cochlear abnormalities will also be discussed. we will discuss the relevant embryology with correlations of malformations to the timing of embryologic insult. conclusions: the new classification system provides precision in description of inner ear malformation. this also helps in providing a uniform scale for comparison of effectiveness of cochlear implant for different malformations. purpose or case report: congenital cranial nerve anomalies often present as sensory and/or motor deficits of unknown etiology in the pediatric age group. the early recognition of a definitive cranial nerve abnormality using high-resolution imaging can focus further clinical investigation and shorten the time to diagnosis. methods & materials: to promote appropriate recognition of cranial nerve anomalies, we present the imaging findings of the most commonly affected cranial nerves and provide correlation with clinical presentation. all studies were performed on a . t magnet with dedicated high resolution imaging of cranial nerve exit zones. results: ours is a tertiary care pediatric hospital with an extensive neuroimaging database. we intend to review all known cases of cranial nerve anomalies from the prior years and present interesting and representative images including optic nerve hypoplasia as part of septo-optic dysplasia, kallman syndrome, duane retraction syndrome, and mobius syndrome. conclusions: congenital cranial nerve anomalies present with varied symptomatology including anosmia, impaired vision, occulomotor deficits, and hearing loss. additionally, clinical manifestations of cranial nerve anomalies can be difficult to recognize in the pediatric age group. effective imaging and prompt diagnosis is crucial to initiate appropriate clinical management. purpose or case report: mr is the standard for evaluation of tumors or epilepsy. pet-ct imaging is often performed to ascertain metabolic asymmetries related to epileptogenic regions or to better characterize the metabolic activity of tumors. a baseline for normals with pet-ct fdg- and c methionine does not exist. methods & materials: retrospective review was performed of the pediatric patients who underwent pet-ct with c methionine and fdg. representative studies were selected for patients imaged during infancy (< yr), early childhood ( - ), childhood ( - ), late childhood ( - ), teenage ( - ). c methionine and fdg studies were analyzed for normal patterns of uptake and any trends identified across the stratified age groups. representative pictorial image galleries of the c methionine and fdg imaging patterns through development were created. results: the pattern of radiotracer uptake on c methionine differed from that of fdg. the c uptake remained low level throughout development compared to fdg uptake, which was robust in much of the cortex. the cortical fdg uptake within the frontal lobes progressively increased with age. the c uptake within the brainstem and thalamus was equal to cortex throughout development. the fdg uptake within the basal ganglia was equal to cortex while the brainstem and thalamic uptake was generally less than cortex. several anatomic structures showed robust c uptake not seen on fdg. these included the lacrimal, submandibular and parotid glands. incidentally, the pituitary gland and hippocampus consistently showed c uptake equal to cortex contrary to their appearance on fdg. our institutional protocol regarding the performance of combination c methionine and fdg brain pet-ct studies is presented. conclusions: this study illustrated the normal appearance of brain pet-ct imaging performed with c methionine and fdg in a representative cohort of the pediatric patients through development. normal variance imaging patterns and developmental trends seen with each radiotracer was demonstrated. the pediatric cerebellum: a pictorial review of normal anatomy using mri and diffusion tensor imaging ibrahim s. tuna, md, radiology, children's hospital of wisconsin, dristuna@yahoo.com; sumit singh, teresa c. gross kelly, mohit maheshwari, tushar chandra, hervey d. segall purpose or case report: the aim of this educational exhibit is to illustrate normal anatomical and functional anatomy of the cerebellum in the pediatric patient. the cerebellum receives sensory input from the brain and spinal cord and integrates this information to coordinate motor control. in addition, the cerebellum also plays a role in some cognitive functions such as attention and language. the first step toward understanding how cerebellar abnormalities can lead to neurological dysfunction, is to provide a solid understanding of the neuroanatomy and functional pathways of the cerebellum. we will describe basic cerebellar embryology, the various cell types and gross anatomy using mr images as well as dti fiber tractography. methods & materials: this exhibit will describe the microstructure, gross anatomy and functional pathways of the cerebellum through illustrations, mr images, diffusion tensor imaging (dti) and pathological correlation. first embryology of the cerebellum will be described, followed by mri depiction of the developmental anatomy of the cerebellum from infancy through adolescence. finally dti tractography images will be used to delineate functional pathways to and from, as well as within, the cerebellum. pathological specimens will be photographed to further illustrate gross anatomy. results: afferent white matter pathways travel mainly via the inferior and middle cerebellar peduncles. the main efferent cerebellar white matter pathway is through the superior cerebellar peduncle. transverse fiber tracts are present in the vermis. there are mainly two main systems of cerebellar white matter fibers which are easily visualized with dti color mapping; however more anterior components of dti tracts are intermixed with afferent white matter projections following the middle cerebellar peduncle. conclusions: knowledge of the precise neuroanatomy and white matter tracts of cerebellum may elucidate our ability to comprehend the clinical manifestations of cerebellar diseases in children. a solid understanding of normal cerebellar anatomy, development and functional fiber tracts in the pediatric patient can provide a baseline that may help predict the clinical outcome of various diseases or interventional procedures. gastroesophageal reflux scintigraphy: a low radiation alternative to gerd evaluation in children vikas menghani, md, pediatric radiology, women's and children's hospital, drvikasmenghani@gmail.com; feraas jabi, jan najdzionek, vaseem iqbal purpose or case report: gastroesophageal reflux disease (gerd) is among the common causes for failure to thrive, recurrent cough and aspiration in children. early diagnosis of gerd is essential in avoiding long-term sequelae such as growth delay, chronic lung disease, esophageal stricture, and esophagitis. gastroesophageal reflux scintigraphy, a noninvasive imaging modality, has been applied for detection of gerd and gastric emptying in children over the past few decades. the radiation burden is considerably small given that a very low dose of radioactivity via a short half-life radioisotope like technetium- m tagged to oral sulfur colloid is administered to a patient. this feature makes reflux scintigraphy especially attractive as the patient can be scanned for prolonged and delayed periods without increasing radiation dose permitting not only identification but also assessment of severity of gerd. characterizing gerd severity is essential in determining how aggressive the pediatrician should be with therapy. gastroesophageal reflux scintigraphy also allows a child to be fed their regular meal tagged to radiopharmaceutical without altering food taste. qualitative and quantitative parameters like gastrointestinal transit and gastric emptying time can be measured, respectively. scintigraphy is highly sensitive to low grade reflux making it very desirable for monitoring response to therapy. while scintigraphy like all other imaging modalities,has limitations, it continues to be an excellent technique for gerd identification and characterization as well as in monitoring response to gerd therapy. the pediatric kidney-a review of common and uncommon renal anomalies ruby lukse, staten island university hospital, drjosemorey@gmail.com; josé morey, jeremy neuman, arnold brenner, oren herman, adam bernheim purpose or case report: renal parenchymal imaging in nuclear medicine has long been performed with mtcdimercaptosuccinic acid (dmsa) due to its sufficient binding to the renal tubules to permit renal cortical imaging. dmsa is of particular value when high-resolution images of the renal cortex are needed. this poster will be a pictorial review of common and uncommon congenital anomalies evaluated on dmsa imaging, such as horseshoe kidney, pelvic kidney, sshaped kidney and crossed-fused ectopia. the poster will also correlate planar imaging findingss with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). purpose or case report: in this poster we will review the differential diagnoses of congenital anomalies that give the appearance of hydronephrosis on renal imaging of the pediatric patient. we will show a pictorial review of both common and uncommon congenital anomalies such as congenital megaureter, ureterocele, uretero-pelvic junction (upj) obstruction, uretero-vesicular junction (uvj) obstruction and posterior urethral valves (puv). we will also review common mimickers of hydronephrosis such as multicystic dysplastic kidney (mcdk) and pseudo-obstruction secondary to bladder overdistention. the poster will also correlate planar imaging finds with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). the pediatric bone scan-a review of neoplastic pathology shrita smith, staten island university hospital, drjosemorey @gmail.com; josé morey, jeremy neuman, arnold brenner, daniel klein, purpose or case report: bone imaging continues to be the second greatest-volume of nuclear imaging procedure performed today, offering the advantage of total body examination, low cost, and high sensitivity. the diagnostic utility, sensitivity, specificity and predictive value of m-tc bone imaging of malignant conditions have long been established. in fact, more than , , bone scans were performed in the united states in . in this poster we will review the current indications for planar bone imaging for the evaluation of malignant and benign neoplasms in the pediatric population, such as osteoid osteoma, langerhan cell histiocytosis (lch), osteoblastoma, ewing's sarcoma, lymphoma, osteosarcoma and osseous/hepatic metastatic disease from neuroblastoma. the poster will also correlate planar and single-photon emission computed tomography (spect) imaging findings with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). the many faces of duplex kidneys on dmsa scans-a pictorial essay neha kwatra, children's national medical center, nskwatra@childrensnational.org; massoud majd purpose or case report: renal duplication is the most common malformation of the urinary tract and is often seen in children with urinary tract infections (uti). the purpose of this study is to learn to recognize duplex kidneys on dimercaptosuccinic acid (dmsa) scintigraphy, review their entire spectrum of findings and correlate with other imaging modalities. methods & materials: dmsa scintigraphy is routinely performed in the nuclear medicine department with a single-head gamma camera (siemens e.cam, schaumberg, illinois). about . h after injection of dmsa, posterior and posterior oblique images are obtained using parallel and pin hole collimators. differential renal function is also calculated. dmsa scan reports containing the words "duplex" or "duplicated" from - were populated using a radiology search engine (montage health care solutions inc.). the images were then reviewed in pacs and representative examples were selected for the poster. the scans were evaluated for renal position, size, contour, any evidence of duplication and parenchymal damage. results: patterns of duplication included non complicated duplex kidney recognized by asymmetric renal size and a prominent cortical bar separating the two moieties, complicated duplex systems with hydronephrosis, scarring or pyelonephritis of one or both moieties. a small nonfunctioning upper moiety was sometimes evidenced by just an indentation along the superomedial aspect of the larger lower moiety. cases with bilateral duplex kidneys were also seen. illustrative examples of each will be provided. correlating findings on other imaging modalities will also be included. conclusions: establishing the diagnosis of duplex kidney on a dmsa scan requires a careful systematic review of the images. the findings can be subtle and it is important for the radiologist to recognize them. correlation with other modalities such as ultrasound or voiding cystogram can be complementary. the assessment of parenchymal function of the upper and lower moieties separately on dmsa scintigraphy can be of immense value in patient management and in choosing surgical options. poster #: edu- f-fdg pet/ct imaging of pediatric brain tumors, neurofibromatosis (nf ) and non-lymphomatous head and neck tumors. lisa states, md, radiology, chop, states@email.chop.edu; purpose or case report: this educational poster will review the current literature and summarize the value of f-fdg pet/ct in standard clinical practice in the evaluation of pediatric brain tumors, nf plexiform neurofibromas and malignant peripheral nerve sheath tumors, and nonlymphomatous head and neck tumors. normal variants and pitfalls will be reviewed. comparison with other pet tracers will be briefly discussed. case examples will be used to illustrate the value of f-fdg pet/ct in grading, staging, assessment of therapeutic response and detection of residual or recurrent disease in various pathologic entities. results: examples of cases will include: benign brain tumor, residual brain tumor in the post-operative bed, brain metastasis, malignant peripheral nerve sheath tumor in nf , head and neck rhabdomyosarcoma, mandibular osteosarcoma, and infection. conclusions: an understanding of the value of pet molecular imaging is essential to the success of the next phase of hybrid imaging with pet/mri which has the potential to play an important role in the development of new diagnostic and therapeutic approaches for the treatment of pediatric brain tumors, nf , and pediatric head and neck tumors. disclosure: dr. states has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the pediatric bone scan-a review of non-malignant pathology josé morey, staten island university hospital, drjosemorey@ gmail.com; jeremy neuman, arnold brenner, vinh phan, cheryl lin purpose or case report: bone imaging continues to be the second most performed nuclear imaging procedure, offering the advantage of total body examination, low cost, and high sensitivity. the diagnostic utility, sensitivity, specificity and predictive value of m-tc bone imaging of benign conditions have long been established. in fact, more than , , bone scans were performed in the united states in . in this poster we will review the current indications for planar bone imaging for the evaluation of non-malignant diseases in the pediatric population, such as acute osteomyelitis secondary to salmonella enterobacteriaceae and tubercle bacillus (tb), chronic osteomyelitis, reflex sympathetic dystrophy, spondylolysis, bone infarcts in the setting of sickle cell disease, fractures (occult/stress), ankylosing spondylitis, dermatomyositis and non-accidental trauma. the poster will also correlate planar and single-photon emission computed tomography (spect) imaging findings with appropriate additional imaging including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (alara) principle set forth by the american college of radiology (acr). purpose or case report: we review the radiologic features of pathologic conditions linked to diesel exposure. the hydraulic fracturing ("fracking") technique is increasingly used in many areas of the country to extract natural gas from rock formations. diesel fuel, or fluids containing diesel, are one component of fracking fluid and create a potential for ground water contamination and risk to air quality. the toxic effects of diesel exhaust are described in the literature, and include asthma, hydrocarbon pneumonitis, and leukemia. there are no scientific data currently available on the effects of chronic diesel ingestion. methods & materials: multi-modality examples of pathology were obtained from a radiology database at a tertiary care pediatric hospital. the specific cases displayed are not known to have diesel exposure, but are intended to serve as representative examples of the type of pathology that may be encountered in the setting of chronic diesel exposure. results: imaging findings of asthma include hyperexpansion, atelectasis, peribronchial thickening, and air-trapping. hydrocarbon pneumonitis may demonstrate low attenuation consolidation and subsequent pneumoatocoeles with ct. leukemia may present on plain radiographs with lucent metaphyseal bands and with marrow infiltration on mri. conclusions: in conjunction with other symptoms not necessarily evaluated in the radiology department, including rhinitis, laryngitis, acute coronary syndrome, and dementia, the radiologist may suggest the diagnosis of diesel toxicity, particularly in populations that may be at high risk of exposure. pediatric radiology in the philadelphia region: a historical review* richard markowitz, md, children's hospital of philadelphia, markowitz@email.chop.edu purpose or case report: the specialty of pediatric radiology in the philadelphia region has grown and evolved over the past eight decades originating from early "visiting" radiologists to drs. hope and kirkpatrick, the "giants" of the s and ' s, to over fifty practicing pediatric radiologists today. clinical excellence, commitment to teaching, and advancement of knowledge through research remain the goals and ideals, much as they were many years ago. philadelphia has been a fertile home and environment for this evolution, mostly because of outstanding leaders and role models who have trained and influenced generations of pediatric radiologists. developments and leadership at the children's hospital of philadelphia, st. christopher's hospital for children, and a.i. dupont institute are highlighted. the purpose of this poster is to tell the story of the growth and development of pediatric radiology in this area and to explore the intellectual origins, professional "genealogy," and legacies left by those who created and those who have carried on this tradition. *note: this material is based on a previously published article: pediatric radiology ( ) : - and "addendum" (pediatric radiology : - ), but never presented at spr. superficial lumps and bumps henrietta k. rosenberg, md, radiology, the mt. sinai medical school, henrietta.rosenberg@mountsinai.org; diane belvin, neil lester purpose or case report: superficial soft tissue masses in the pediatric age range can be quite challenging to the pediatrician and the imager. the purpose of this presentation is to demonstrate the efficacy of duplex/color doppler ultrasound for the diagnosis and follow up of a large gamut of superficial lumps and bumps. methods & materials: we reviewed our experience during the past years using ultrasound to evaluate superficial soft tissue masses that had been encountered in many parts of the body, from the skull to the soles of the feet, in a large group of patients ranging in age from newborn to years. all sonograms were performed after obtaining pertinent clinical information as well information regarding the clinical characteristics of each of the masses, e.g. location, consistency (firm [solid], compressible [cystic]), fixed or easily movable, smooth or irregular surface, tenderness. the masses were palpated by the imaging team and duplex/color doppler ultrasound was performed. comparison sonographic views of the opposite side were obtained as needed. clinical followup and surgical/pathological correlation was obtained in most of the patients. results: most of the masses were benign and included a wide variety of etiologies. most often, us was sufficient for assessment of soft tissue masses if the entire mass was included in the field of view. if the lesion was too large for the field of view or malignancy was suspected, ct/mri were required preoperatively. nuclear medicine studies are reserved for midline masses likely due to ectopic thyroid and pet was used for more complete evaluation of a lesion that was likely malignant. conclusions: duplex/color doppler ultrasound (us) is the modality of choice for evaluation of superficial lumps and bumps! this modality allows for rapid acquisition of information without the use of ionizing radiation, intravenous contrast material, or sedation/anesthesia. reliable information can be rapidly acquired regarding the size, shape, borders, location, internal consistency, vascularity, vascular encasement/displacement. correlation of the ultrasound and clinical findings helps narrow differential diagnosis. sonography helps to determine what is the next best step: watchful waiting (clinical observation, follow-up us), surgical resection, or us guided interventional procedure. present day imaging of down syndrome rupa radhakrishnan, md, radiology, university of cincinnati college of medicine, radhakrp@ucmail.uc.edu; alexander j. towbin purpose or case report: down syndrome is a common genetic condition characterized by unique physical traits and multisystem anomalies. the purpose of this exhibit is to portray the imaging findings of down syndrome and discuss with illustrative examples, the use of imaging in multidisciplinary management. methods & materials: published literature was reviewed to identify the multisystem imaging findings in down syndrome. the electronic medical record system was then searched to find illustrative case examples from our institution. results: in patients with down syndrome, abnormalities can be found in the musculoskeletal, cardiovascular, respiratory, gastrointestinal, and central nervous systems. abnormalities can range from emergent, life threatening conditions such as malrotation with midgut volvulus to chronic conditions such as scoliosis. examples of abnormalities from each organ system and the modalities used for diagnosis and management are described. cardiovascular system: echocardiogram and cardiac mri and ct are useful in evaluating congenital heart disease associated with down syndrome. respiratory system: micrognathia with macroglossia and hypotonia predisposes patients to sleep apnea which can be evaluated with dynamic mri. chest ct demonstrates subpleural cysts which are characteristic of this syndrome. gastrointestinal system: fluoroscopy and/or radiographs are the mainstay in diagnosing many gastrointestinal disorders including duodenal atresia, malrotation, annular pancreas, imperforate anus, and hirschsprung disease. central nervous system: choroid plexus cysts may be identified on prenatal ultrasound in a fetus with down syndrome. imaging is used in the evaluation of epilepsy, hearing loss and alzheimer disease that is more common in these individuals. musculoskeletal system: multiple skeletal anomalies can be present in patients with down syndrome. radiographs are often used as the method of identifying and, if needed, following the anomalies. prenatal imaging: increased nuchal translucency is the earliest imaging finding. other features of down syndrome can be identified on prenatal ultrasound or mri. prenatal imaging is helpful in determining the prognosis of the fetus and in guiding management. conclusions: modern day multidisciplinary management has improved quality of life and survival in individuals with down syndrome. imaging plays a critical role in guiding management in these individuals. imaging the spectrum of lymphatic malformations in the pediatric patient andrew schapiro, md, radiology, university of wisconsin, aschapiro@uwhealth.org; kara gill, bradley maxfield purpose or case report: lymphatic malformations (lm) occur as a result of abnormal development of the lymphatic system during embryogenesis. as % of lm present by years of age, these lesions represent an important pediatric entity. lm can often be suspected clinically in an infant with the classic presentation of an asymptomatic, soft mass in the head, neck, or axilla. however, myriad presentations are possible as lm occur in numerous other anatomic locations, can be multiple, and can be a component of mixed vascular malformations. in addition, the true extent of lm is often not apparent clinically. given these considerations and the implications for proper management, imaging plays an important role in the assessment of lm. the purpose of this exhibit is to review the spectrum of radiographic, ct, sonographic, and mr imaging findings of a variety of lm presentations. methods & materials: cases of lymphatic malformation in pediatric patients identified at a single institution over the past ten years with available imaging were reviewed utilizing pacs. results: images of lm involving the head and neck, chest, abdomen, retroperitoneum, extremities, and skeletal system were identified. in addition, cases of lymphangiomatosis and mixed venolymphatic malformation were identified. various imaging modalities including radiography, ct, sonography, and mr were represented. conclusions: adequate knowledge of the imaging characteristics of lm across multiple modalities enables proper diagnosis, assessment of disease extent, and guidance of appropriate therapy in pediatric patients. results: ct and mr imaging findings in nine cases will be presented. they include ) congenital absence of the inferior vena cava with thrombosis of the external iliac vein secondary to venous stasis ) pyelophlebitis complicating ruptured appendicitis ) left iliac vein thrombosis in a patient with may-thurner syndrome ) splenic vein thrombosis complicating pancreatitis ) splenic vein thrombosis following splenectomy ) renal vein thrombosis in an infant of a diabetic mother ) adrenal vein thrombosis as the presenting sign of antiphospholipid syndrome ) budd-chiari syndrome associated with underlying myeloproliferative disease ) iliac vein thrombosis as a manifestation of behcet's syndrome (hughes-stovin syndrome, a variant of behcet's syndrome, which presents with systemic venous thrombosis and pulmonary artery aneurysms will also be discussed). conclusions: thrombosis of large abdominal and pelvic veins in children and adolescents is uncommon. certain conditions, both congenital and acquired, predispose to the development of venous thrombosis. ct/mr imaging defines the extent of thrombosis, and demonstrates additional findings that may elucidate the nature of the underlying condition leading to clot formation. purpose or case report: because abnormal gait in a young child has a wide range of causes, imaging plays a critical role in establishing the definitive diagnosis. the purpose of this exhibit is to review the clinical clues (age, duration, laboratory markers) and imaging findings of the causes of abnormal gait in a toddler and to assess the strengths and limitations of radiographs, ultrasound, magnetic resonance imaging (mri), and computed tomography (ct). methods & materials: cases, from a single institution experience with various causes for abnormal gait in a toddler, are reviewed and categorized into congenital, traumatic, inflammatory, neoplastic, or neuromuscular etiologies. results: there are various causes of abnormal gait in a toddler. the congenital causes include spinal dysraphism, proximal and distal skeletal deformities and dysplasias. the traumatic causes include non-accidental trauma, toddler's fracture, foreign body, and soft tissue injuries. the inflammatory causes include juvenile idiopathic arthritis, transient synovitis, and infection, including osteomyelitis, septic arthritis, discitis, cellulitis, and abscess. the neoplastic causes include various neurogenic, bone, and soft tissue tumors. the neuromuscular causes include cerebral palsy and spinal bifida. the combination of clinical presentation, supporting laboratory findings, and classic imaging findings help to distinguish the possibilities and often allows confident diagnosis. conclusions: knowledge of imaging findings and clinical factors can demystify the diagnosis of abnormal gait in a toddler. familiarity with the clinical presentation can ensure the performance of the appropriate diagnostic studies, timely diagnosis, and effective treatment. nonaccidental causes should never be overlooked. ultrasonography has become an important tool in the radiologist's armamentarium, augmenting radiography, mri, and ct. approximately different contrast agents for mri and ct are now commercially available for use. although most of them are fda approved in adults, information on usage and safety in children is not readily available. the most important reason is lack of controlled studies in children, especially for the age of - years. however, the lack of fda approval has not limited the use of these promising agents in children. in fact, there is widespread off-label use of these agents in most major pediatric hospitals in the country. based on a review of relevant literature in children, and based on a survey of radiology faculty at major pediatric hospitals, this poster will address the gap between approved use and reality in the setting of pediatrics. results: using a tabular format, this poster will provide a list of mr and ct contrast agents that are available for clinical use, their relevant clinical properties (ionic or nonionic, viscosity, linear or macrocyclic, degree of relaxivity for mri, iodine concentration for ct, cost, dosage, halftime, incidence of allergic reactions, nephrogenic systemic fibrosis and other adverse reactions), fda approval status (for ages - days, days- years, and - years), common pediatric applications, and contrast injection protocols for common applications. conclusions: to enlighten imaging personnel about usage and safety of contrast agents in children. disclosure: dr. krishnamurthy has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. a pictorial essay and literature review of the spleen in sickle cell disease david hindson, md, boston medical center, david. hindson@bmc.org; heather imsande, philippa sprinz, ilse castro-aragon purpose or case report: the morbidity and mortality of sickle cell disease (scd) results from acute and chronic infarction events that affect almost every organ. repeated infarction has some of its greatest visual and physiologic impact within the spleen. continuous hemolysis, sequestration and vaso-occlusion within the spleen result in loss of splenic function early in life and frequently autosplenectomy thereafter. by years of age, approximately % of children with hemoglobin ss disease will have diminished splenic function, putting them at increased risk for infections. treatments for scd have evolved over the last years, and among others include penicillin prophylaxis and immunizations, hydroxyurea and transfusion therapy (or hypertransfusion program). imaging findings are a reflection of the different treatments and their efficacy. methods & materials: our institution cares for a large group of patients with sickle cell disease, from birth to adulthood. this offers an unprecedented opportunity to document the imaging findings of the spleen with different treatment regimens, and over many years. the splenic size and morphology can be followed, by ultrasound, in a very straightforward way. we have compiled a pictorial essay of the various imaging characteristics of spleens from infants to adults. we also performed a literature review to compare and supplement the findings of our images. results: there is a spectrum of imaging findings in the spleen of patients with scd that changes from birth to childhood. the findings range from the normal appearance of a spleen to a calcified spleen, and include regenerative nodules, fibrosis, altered parenchymal echotexture, increased echogenicity, and changes in size, including enlargement secondary to sequestration. the ultrasound characteristics not only change with advancing age, but also appear to depend on whether or not the patient has received specific treatments, and at what age treatment was initiated. conclusions: the ultrasound appearance of the spleen in patients with scd is variable. treatments such as blood transfusions and hydroxyurea, patient compliance with therapy and type and severity of the disease are some of the factors that affect imaging characteristics. cystic fibrosis: not just for children cindy miller, md, radiology, yale-new haven hospital, cindy.miller@yale.edu purpose or case report: cystic fibrosis has been recognized for hundreds of years with the first descriptions of it including such anecdotes as mothers licking the foreheads of their children and knowing that if it tasted salty, an early death could be predicted. it was not until that the disease was first named by dr. dorothy andersen, and for the following years, treatment was largely supportive, and imaging was essentially done with plain films alone. in with the elucidation of the cftr gene, there was an explosion of knowledge which included the range of increased awareness and understanding of the suspected etiology, imaging findings and significance of the ductus bump. the contribution of d imaging for evaluation of the pediatric central airways jessica kurian, md, the children's hospital of philadelphia, kurianj@email.chop.edu; monica epelman, david a. mong purpose or case report: evaluation of the central airways in children has historically been accomplished by flexible bronchoscopy, an invasive technique associated with inherent risks and complications. multidetector ct (mdct) with volume rendering offers a noninvasive alternative for airway evaluation. in this educational exhibit, we will review imaging techniques and clinical applications of mdct for the assessment of large airway maladies in children. methods & materials: mdct imaging in children with a variety of tracheobronchial disorders is reviewed. for each entity, the characteristic clinical features are described, and key imaging features are illustrated. emphasis is placed on the contribution of d techniques for characterizing complex airway anomalies. dose reduction strategies are also highlighted. results: the entities reviewed in this exhibit include, but are not limited to, congenital anomalies of tracheobronchial branching, airway malformations associated with situs, and congenital or acquired airway compression and/or obstruction. conclusions: mdct with volume visualization is a useful adjunct for evaluation of the pediatric central airways in a variety of pathologies. as a noninvasive technique, it avoids sedation risks and spare patients from complications associated with conventional flexible bronchoscopy. low dose protocols should be used to minimize radiation exposure. bronchopulmonary foregut malformation that result from abnormal budding of the primitive foregut. currently, many such anomalies are initially detected by prenatal ultrasound and are further delineated by fetal magnetic resonance imaging (mri), while others may be incidentally detected on postnatal radiologic examinations or later in life in the setting recurrent pulmonary infection. imaging plays a very important role in the diagnosis and characterization of these lesions and assists surgical planning. the purpose of our educational exhibit is to illustrate the common and uncommon radiologic appearances of cpams using various imaging modalities, including radiography, computed tomography, prenatal and postnatal ultrasound, and prenatal and postnatal mri. methods & materials: all pediatric and adult cpam (including both sequestration and ccam) patients were identified using electronic medical records. pertinent imaging reports (including radiography, prenatal and postnatal ultrasound, ct, and prenatal and postnatal mri) were reviewed by a single author in order to identify relevant imaging findings. relevant images from these imaging examinations were de-identified and saved to a secure hard drive. medical records were accessed by a single researcher to obtain relevant demographic information as well as data regarding the patients' clinical presentations. in cases of corrective surgery, operative and pathology reports were reviewed, if available, for correlation with the imaging findings. results: cases of pediatric and adult cpam were identified and presented in a variety of clinical contexts. their appearances were reviewed through multiple imaging modalities. conclusions: congenital pulmonary airway malformations are varied in their clinical presentation and imaging appearance. the purpose of this pictorial essay is to enhance understanding of their diagnosis and to use a multidisciplinary approach in order to highlight imaging aspects that may alter clinical management. disclosure: dr. horst has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. the imaging evaluation of cystic lung disease in children: an evidence-based approach jordan caplan, md, pediatric radiology, lucile packard children's hospital, stanford university, caplan@stanford. edu; beverley newman purpose or case report: the goal of the poster is to provide a framework for use when confronted with cystic lung disease in a child. methods & materials: the differential diagnosis for the types and causes of cystic lung disease in children will be presented using an evidence-based, age appropriate approach. categories of disease discussed and illustrated with case examples will include: a. congenital cystic bronchopulmonary malformations b. infectious cysts c. autoimmune/inflammatory/vasculitic disease with cavitating lesions d. neoplastic conditions e. collagen/soft tissue abnormalities f. mimics of cystic lung disease results: the pathophysiology, imaging appearance, and demographics of the above entities will be reviewed with attention to relevant recent literature. important educational points include the differentiation of bronchopulmonary malformations from neoplasm, notably pleuropulmonary blastoma (ppb), the relationship between lung cysts and ppb, and the management and surveillance of lung cysts in children. conclusions: an evidence-based approach to the broad spectrum of causes of cystic lung disease in children is a useful starting point in forming a concise and pertinent differential diagnosis. an understanding of the pathophysiology, imaging appearance, and demographics of these entities is essential in guiding patient management. pediatric interstitial lung disease (ild): a pictorial review with radiologic and pathologic correlation hollie west, md, diagnostic radiology, vanderbilt university, hollie.c.west@vanderbilt.edu; melissa a. hilmes, sudha p. singh, jennifer soares, lisa young purpose or case report: while adult interstitial lung disease is a well-described and fairly well understood group of disease processes, pediatric interstitial lung disease (ild) remains a subject of uncertainty and misunderstanding for many clinicians and radiologists. confusion surrounding the phenomenon of pediatric ild stems not only from the rarity of the disease, but also from the extensive list of disease entities that can produce ild, the existence of certain patterns that are restricted to infants and children and the fact that patterns of ild manifest differently in a child's developing lung than in an already developed adult lung. imaging plays an important role in diagnostic work-up of this disease and can guide lung biopsy in specific patient populations. methods & materials: the irb approved retrospective study will show patients at our institution over a year period diagnosed with various types of ild, including pulmonary insterstitial glycogenolysis (pig), diffuse neuroendocrine cell hyperplasia (nehi), surfactant deficiency diseases, and lung diseases associated with other systemic processes such as downs syndrome and inflammatory bowel disease. we will include patients with biopsy proven ild and will provide examples of the major ilds, including clinical, radiologic and pathologic correlation. our pictorial review will describe the radiologic patterns associated with the different forms of ild, emphasizing what the radiologist needs to know and how to be helpful to a multidisciplinary team in the diagnosis and treatment of these diseases. results: the study will report the frequency of ild at our institution, including a breakdown of the various subtypes of ild. we will show examples of the subtypes with correlative chest radiography, computed tomography, and pathology. we plan to highlight specific differentiating factors between the different diseases and demonstrate how a radiologist can be helpful in collborating with clinicians in diagnosing and treating these diseases. conclusions: pediatric ild can be a confusing topic for radiologists. increasing knowledge and awareness of these diseases, their clinincal presentation, work up, and treatment is important for pediatric radiologists who work as part of of a multidiciplinary team. poster #: sci- ct radiation dose delivered by community hospitals and imaging centers stephen little, children's healthcare of atlanta, stephen. little@choa.org; damien grattan-smith, bonnie johnson purpose or case report: to evaluate and compare ct radiation dose for pediatric abdominal and cranial ct examinations performed by community hospitals and imaging centers. methods & materials: consecutive ct examinations ( cranial, abdominal) from community hospitals and imaging centers were reviewed following transfer of care. the examinations were performed between january and july . consecutive ct examinations ( cranial, abdominal) performed at our own institution were also reviewed. ctdivol and dlp were obtained from the dose report for each examination ( cm-phantom for abdominal exams, cm-phantom for cranial exams). patient age and weight were obtained from the medical record. results: average ctdivol for abdominal ct performed by local community hospitals and imaging centers was . mgy, while average ctdivol was . mgy for abdominal ct performed at choa. there was a wide variation in ct radiation dose delivered. while some sites delivered a ct radiation dose comparable to our own, others delivered a substantially greater dose. in fact, % of pediatric abdominal ct exams performed by local community hospitals and imaging centers exceeded the notification value recommended by the aapm ( mgy using the cm phantom). low kvp technique for imaging small children was infrequent. multi-phase examinations were more often performed, resulting in additional elevation in ct radiation dose when dlp is considered. average ctdivol delivered by local community hospitals and imaging centers for cranial ct was mgy compared to a ctdivol of mgy for cranial ct performed at choa. % of pediatric cranial ct exams performed by local community hospitals and imaging centers exceeded the notification value recommended by the aapm ( mgy for - years, mgy for > years). conclusions: despite ongoing efforts at education, there is wide variation in ct radiation dose delivered for pediatric abdominal and cranial ct examinations performed by local community hospitals and imaging centers. appropriate use of dose check software on newer scanners may help reduce the number of children subjected to excessive ct radiation dose. ultimately, each site performing pediatric ct must take responsibility for minimizing radiation dose while producing diagnostic quality exams. the impact of adaptive statistical iterative reconstruction on ct image quality parameters -a phantom study karen thomas, md, radiology, hospital for sick children, karen.thomas@sickkids.ca; nancy ford, angjelina protik, paul babyn purpose or case report: to quantify the effect of adaptive statistical iterative reconstruction (asir) on ct image quality parameters. methods & materials: phantom (catphan ) studies were performed on a ge hd -slice scanner to investigate the impact of a) % asir compared to routine filtered back projection using variable kvp ( - ) and mas ( - ), and b) incremental asir % ( , , , , %), scanning at mas and variable kvp ( - ). pitch, acquisition fov and detector width were kept constant. image noise, spatial and contrast resolution, contrast noise ratio (cnr) and wiener spectrum analysis were performed on . mm ax, mm ax mpr and mm cor mpr series. results: % asir resulted in a mean decrease in noise of % ( . mm ax), % (ax mpr) and % (cor mpr) and improvement in cnr of - %. incremental advantage was seen with stepwise increase in asir %. however, application of asir was associated with a small reduction in spatial resolution ( - % at % asir). low contrast detectability (lcd) improved except at the smallest target lesion size. image quality effects at very low mas and at high asir % will be presented. conclusions: image noise reduction and improvements in cnr and lcd with asir hold considerable potential for dose reduction in pediatric ct. this study provides quantitative data that may be used to design asir-enhanced protocols with consideration of diagnostic task, balancing image quality benefits and potential pitfalls. pictorial essay on cardiac mr for congenital heart disease on t mr scanner with rf multi-transmit technology (tx) taylor chung, md, diagnostic imaging, children's hospital & research center oakland, taylorchung @gmail.com purpose or case report: this is a pictorial essay (e-poster) to show artifacts on cine ssfp images pre-tx and post-tx upgrade on congenital heart disease cardiac mr; to illustrate methods prior to tx-upgrade to minimize artifacts. disclosure: dr. chung has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. revisiting the relationship between anthropometric parameters and left ventricular mass abdullahi adamu, md, phd, ahmadu bello university, scorpion kd@yahoo.com purpose or case report: the purpose of this study was to find the correlation between anthropometric parameters and left ventricular mass in normal adolescents and young adults. methods & materials: healthy individuals in the age range to years ( males and females) were included in this study. anthropometry was performed with standard anthropometry kit and measurements of height, weight, body surface area (bsa), upper arm circumference and upper hip circumference were taken. echocardiography was performed and the american society of echocardiography (ase)-recommended method was employed for calculation of left ventricular mass (lvm). statistical analysis was performed using statistica . (stat soft, usa). results: the mean value of lvm for all our subjects was found to be . ± . g. there was significant correlation between lvm and height (r . , p< . ), weight (r . , p< . ) and bsa (r . , p< . ). correlation with upper arm circumference was moderate (r . , p< . ), while it was found to be weak with upper hip circumference (r . , p< . ). diagnostic. both field strengths can be used successfully for cardiac and vascular imaging. the decision as to which to use is weighted by local availability and the relative requirement for detailed vascular vs intra-cardiac imaging. disclosure: dr. nguyen has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. poster #: sci- color coded d cardiac cta of congenital heart disease: a five year experience nhi huynh, md, radiology, st. joseph hospital and medical center, e.nhihuynh@gmail.com; randy richardson purpose or case report: post-processing of cardiac computed tomography angiograms can be performed on a commercially available workstation to create color coded d volume rendered images of the segmented heart and great vessel anatomy in patients with congenital heart disease. these studies optimally demonstrate complex anatomy, streamlining communication between members of the healthcare team and providing a tool for communicating complex anatomy and treatment options with families. these studies have been ordered with more frequency over the past five years. we retrospectively reviewed the types of congenital heart disease demonstrated by cardiac d cta over the past five years at a congenital heart center. methods & materials: color coded cardiac cta postprocessing was performed from ecg gated prospective and retrospective cta data on a commercially available workstation for / patients over the past three years. the anatomy was initially segmented and colored into individual parts of the anatomy of the heart and great vessels as follows rv purple, lv light red, aorta red, pulmonary arteries blue, systemic veins and right atrium aqua, pulmonary veins and left atrium pink, pda or collaterals green, airway yellow, coronary arteries neutral. the anatomy was then reassembled and images obtained every °in a °rotation for display. results: d color coded cta images were used in the treatment and care of congenital heart patients for the following types of congenital heart diseases: cases of complex anatomy (tga, truncus arteriosus, hlhs, tricuspid atresia, tof…), coronary artery anomalies, cases of pulmonary atresia or stenosis, cases of systemic and venous anomalies, cases of coarctation or interruption of the aortic arch, and tracheobronchial tree anomalies. conclusions: color coded cardiac cta post-processing is an effective and viable method for demonstrating anatomy in complex congenital heart patients. it is an excellent tool for demonstrating anatomy which is difficult to see by echocardiography such as: coronary artery anomalies, pulmonic atresia, aortic arch coarctation or interruption, and tracheobronchial anomalies and/or stenosis. neuroimaging in the evaluation of hie in term neonates post hypothermia therapy julio m. araque, md, radiology, medical college of georgia, jaraque@georgiahealth.edu; jatinder bhatia, leann vanlandingham purpose or case report: to illustrate and review the potential utility of brain mri, ct and ultrasound in hypoxic ischemic encephalopathy in newborns treated with hypothermia. neuroimaging studies including brain ultrasound, ct and mri of fifteen term newborns treated in our institution with therapeutic hypothermia, since april were evaluated retrospectively. more relevant lesions are depicted and the diagnostic and prognostic value of the findings is discussed and compared with a review of the literature. results: recent studies showed that patients treated with cooling had a more favorable prognosis than was suggested by the clinical grade of encephalopathy compared with infants treated with standard care. our institutional protocol includes the performance of mri, and ultrasound. ct is performed when is a clinical impossibility of perform mri. brain ultrasound was performed in all the patients. mri scans were obtained in neonates. ct was obtained in patients. all mri studies included dwi. the utility of dwi and adc maps as an aid in diagnosis of non-ischemic lesions is becoming increasingly established. mri evidence of brain injury was visible on basal ganglia in cases with negative ultrasound. abnormal signal intensity in the posterior limb of the internal capsule coexists with lesions in the basal ganglia and thalami have been associated with abnormal motor outcome. the remaining newborns did not develop significant mri evidence of brain injury. it has been suggested that the ability of mri to predict subsequent neurological impairment is unaltered by therapeutic hypothermia. further research is needed for defining the relation between mri findings and cooling. it is possible that imaging findings might be delayed in cooled infants. conclusions: mri offers the highest sensitivity in detecting anoxic injury of the neonatal brain. mr biomarkers in combination with clinical markers may identify patients with adverse outcome with therapeutic implications. disclosure: dr. araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. purpose or case report: to correlate bowel wall diffusionweighted imaging (dwi) apparent diffusion coefficient (adc) values with multiple mr enterography (mre) and clinical findings in pediatric small bowel crohn disease. methods & materials: pediatric crohn disease patients with mre exams containing diffusion-weighted imaging and demonstrating terminal ileitis were identified. minimum bowel wall adc values were tested for correlation/association with other mri findings and clinical parameters (including laboratory values). results: there is negative correlation between adc value and degree of bowel wall thickening (r (−) . ; p . ). lower adc values were significantly associated with striated pattern of arterial phase postcontrast enhancement (p . ), greater degree of arterial phase postcontrast enhancement (p . ), and presence of stricture (p . ). adc values were not associated with diseased bowel length, degree/pattern of delayed postcontrast enhancement, degree of mesenteric inflammation or fibrofatty proliferation, or clinical markers of inflammation. conclusions: restricted diffusion in pediatric small bowel crohn disease is associated with other mri findings of that are suggestive of active disease, including degree of bowel wall thickening and degree and pattern of arterial phase postcontrast enhancement. our data also suggests that dwi may be useful when attempting to characterize small bowel strictures as either predominantly inflammatory or fibrotic, although further investigation is needed. quantification of blood flow into and out of the liver with d phase contrast mri in the pediatric patient binh huynh, md, radiology, stanford, bhuynh@stanford. edu; shreyas vasanawala, albert hsiao purpose or case report: the ability to probe blood flow dynamics in the liver may aid management of children with liver disease, including shunt fractions in portal hypertension and arterial flow fraction in diffuse liver disease. the purpose of this study is to evaluate the ability to measure blood flow into and out of the liver with time resolved volumetric ( d) phase contrast mri in the pediatric patient. methods & materials: nineteen consecutive patients were retrospectively identified who underwent d flow imaging through the level of the hepatic vessels on . t and t magnets. a software enabling d flow program was utilized to first assess for the feasibility of measurement of flow in the hepatic artery (ha), portal vein (pv), splenic vein (spv), superior mesenteric vein (smv), supra (sivc) and infrahepatic (iivc) inferior vena cava. if measurable, calculations were performed to evaluate for internal consistency by comparing the sum of smv and spv flow to pv flow. calculations were then performed to compare hepatic inflow (pv+ha) to hepatic outflow (sivc-iivc) and for the percentage of pv and ha contribution to hepatic inflow. results: of the nineteen patients, all of the above mentioned six vessels were visualized and measurable in two patients, both of which were imaged on the . t magnet. in the remaining patients, flow measurements were limited by respiratory motion artifacts obscuring the smaller vessels, and severe eddy currents, particularly in patients imaged with the t magnet. the evaluation for internal consistency demonstrated an average of . % ( . % & - . %) difference between smv+spv and pv flow. hepatic inflow was found to closely match the measured hepatic outflow with an average difference of . % ( . % & . %). the portal vein was found to contribute . % and . % to hepatic inflow, while the hepatic artery contributed . % and . %. conclusions: measurement of hepatic flow with phase contrast mri is more challenging than assessment of thoracic flow. when respiratory artifacts are minimal, vessels can be identified and measurements have internal consistency and good agreement between hepatic inflow and outflow at . t. conversely, flow measurements were limited at t by eddy currents. thus, ongoing efforts are aimed at mitigating respiratory motion artifacts at . t. poster #: sci- mri findings in post-fontan hepatopathy adina alazraki, md, radiology, emory university/children's healthcare of atlanta, adina.alazraki@choa.org; pinar bulut, kiery braithwaite, miriam vos, rene romero, nitika a. gupta purpose or case report: as advances in congenital heart disease continue to improve both mortality and quality of life, associated complications are becoming more prevalent. amongst patients who have had fontan repair for hypoplastic left heart syndrome, tricuspic atresia, or other right heart dysfunction, it is well known that liver disease is a complication. we describe the mri findings in post-fontan patients and propose mri as a useful tool to the hepatologist's evaluation of these patients. methods & materials: irb approval was obtained for a retrospective review of patients who underwent fontan repair and were subsequently referred for hepatology evaluation between - . all but one patient was scanned on a siemenstriotrim t magnet; one patient was scanned on a ge twinspeed . t magnet with an equivalent protocol due to orthodontics. a standardized departmental protocol was utilized. mri findings were correlated with age at surgery and years since surgery. mr images were reviewed independently by pediatric radiologists and compared with the dictated report in the patients record. results: patients underwent mri of the abdomen. patients had mri incompatible hardware and patients were not scanned secondary to insurance denial. patients were divided into groups based on elapsed time since surgery: less than years, - years, - years, and greater than years.(table ) mr images were evaluated for the presence of fibrosis, congestion and any other hepatic abnormalities. fibrosis was determined based on a specific pattern of delayed reticular enhancement in combination with liver morphology. congestion was deemed present if there was increased t signal in the liver parenchyma or periportal regions in combination with cloud-like enhancement on dynamic post-contrast images. all patients demonstrated morphologic changes in the liver with varying degrees of hepatic fibrosis and hepatic congestion. fibrotic changes were often non-uniform, and thus could be underdiagnosed by biopsy. interestingly, patients, %, had focal arterially enhancing lesions speculated to represent vascular proliferative lesions, however, none warranted biopsy. conclusions: it is established that patients who undergo fontan develop hepatic abnormalities. mri is a reliable, non invasive technique that accurately demonstrates these findings. mri may be a more sensitive method to evaluate the etiology and full extent of hepatic disease. poster #: sci- complications within the interventional radiology division of a tertiary care children's hospital: initiatives for ongoing quality and practice improvement brian dillon, children's hospital boston, brian. dillon@childrens.harvard.edu; pamela sanborn, yolanda milliman-richard, darren orbach, stephan voss purpose or case report: between and , procedure-related complications occurring within the division of interventional radiology at our institution were recorded and classified according to level of severity. the goals of this study were to determine rates of procedurebased complications based on severity, to establish thresholds for complications, and to determine whether measurable trends in complications over time were evident. methods & materials: between and , , interventional procedures were performed within the division of interventional radiology at our institution. adverse events were characterized both according to level of severity (using an institutional point severity scale), and with brief descriptions of individual events. adverse events were reviewed monthly at the division's morbidity and mortality conference, with respect to procedure type and operator. based on review of our interventional radiology data and benchmarks rates used for diagnostic errors, threshold complication rates were established by consensus between the department quality improvement committee and the division of interventional radiology. for severe events (level and ) there is no allowable threshold; all such events were subjected to both internal and institutional review. results: the overall complication rate was less than % for all procedures performed. the complication rates for the respective severity levels were: level ( . ), level ( . ), level ( . ), level ( . ), and level ( . ). the severity of a given complication was not associated with procedural complexity. no operator-specific trends were identified. conclusions: since , the society of interventional radiology has offered guidelines and strategies for improving safety and quality in interventional radiology. however, no specific benchmark data or procedural recommendations are available for pediatric interventional procedures. our results demonstrate rates of complications well below published overall complication rates for interventional radiologic procedures. this database of procedure-based complications serves as a foundation for a quality improvement program that allows review of complications with respect to specific procedure types, individual operators, and procedural complexity, in an effort to institute an ongoing and continuous process of quality improvement within interventional radiology. purpose or case report: dysosteosclerosis (dss), an extremely rare dense bone disease, features short stature and fractures and sometimes optic atrophy, cranial nerve palsy, developmental delay, and failure of tooth eruption in infancy or early childhood consistent with osteopetrosis (opt). bone histology during childhood shows unresorbed primary spongiosa from deficient osteoclast action. additionally, there is remarkable progressive flattening of all vertebrae mimic ppi blocking mineralization. during ehdp treatment for gaci, in our patient prolonged high dose ehdp resulted in severe skeletal deformity resembling hypophosphatasia which was reversable with drug stoppage. methods & materials: a -year-old boy with gaci referred for profound, acquired, progressive skeletal deformity. he was receiving mg/day of ehdp and was wheelchair bound. we studied him and his response to stopping ehdp. results: skeletal radiographic findings resembled pediatric hypophosphatasia with pancranial synostosis, widened physes with metaphyseal osteosclerosis, "tongues" of radiolucency, along with cupping and fraying, and long-bone bowing. in addition there were large intra and extraarticular calcifications. radiographic features of bp-induced opt included femoral erlenmeyer flask deformity and osteosclerosis (lumbar sine dxa z-score + . ). biochemical parameters of mineral homeostasis were essentially normal although serum osteocalcin was low and he had markedly elevated serum levels of creatine kinase and trap- b consistent with osteopetrosis (opt). after stopping ehdp, he improved quickly with remarkable healing of his rachitic appearing skeleton and decreased joint calcifications. conclusions: our patient with gaci had profound skeletal deformities from high-dose ehdp therapy that significatly improved with drug stoppage. magnetic resonance imaging in the evaluation of infants with hypoxic ischemic encephalopathy julio m. araque, md, radiology, medical college of georgia, jaraque@georgiahealth.edu purpose or case report: to illustrate and review a spectrum of brain abnormalities of infants with hie. defining the most useful approaches and mri sequences, to facilitate identification and early diagnosis of lesions with the potential to predict outcome and abnormal neurodevelopment. methods & materials: reviewed available evidence on mri strategies for evaluating infants with hypoxic ischemic encephalopathy. different cases illustrating lesions are presented and discussed for proper diagnosis correlating physiopathology and imaging appearance. more relevant findings are depicted with didactic illustrations. identifying studies where new techniques such as dwi, adc, dti, swi, or mrs adds significant diagnostic value to the overall interpretation. results: mri is routinely performed as a very sensitive method for detection of hie lesions. advanced mr techniques, such as dti, dwi, adc, mrs, swi offer the possibility of detecting injuries at a time when intervention is theoretically possible. the understanding of the physiopathology allows for prediction of the location and extent of lesions, facilitating identification and appropriate classification. the identification of infants with potentially abnormal neurodevelopment, offers the opportunity to provide therapeutic neurodevelopmental interventions in early childhood. mrs is the best mr biomarker to predict neurodevelopmental outcome in asphyxiated full-term neonates. brain metabolite ratios and regional adc values may vary between mr systems and coils. development of normal values for each institution is required, and support of physicists is mandatory. conclusions: mri continues to evolve as a valuable adjunctive tool routinely obtained in nearly all cases of hie. advanced mri techniques increase sensitivity of conventional t and t -w images and outperform computer tomography and ultrasound for confirming the diagnosis of hypoxic-ischemic brain injury or providing prognostic information for the care of patient with hie. disclosure: dr. araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the food and drug administration (fda) as investigational for intended use. posterior fossa abnormalities in children amit gupta, mbbs, radiodiagnosis, r.n.t. medical college, udaipur, rajasthan, india, amitsensation@yahoo.co.in purpose or case report: the aim of this exhibit is to demonstrate various conditions involving the posterior fossa in children with emphasis on importance of embryologic development of cerebellum in reaching a correct diagnosis. methods & materials: this pictographic presentation displays the imaging features of cases encountered in our clinical practice on . tesla magnetic resonance (mr) imaging. results: with the advent of mr imaging, there has been a revolution in identification and characterization of malformations of the brain this is especially true in posterior fossa, where the sensitivity and specificity of mr imaging with its multidimensional imaging capability are far superior to those of computed tomography (ct) in the detection of subtle morphologic abnormalities. however, there is still a great deal of confusion regarding their classification, terminology, and spectrum of expression and this is where neuroembryology is of great help. this exhibit demonstrates : ) review of embryology and normal anatomy of cerebellum. ) mr appearance of spectrum of conditions involving posterior fossa in children which includes developmental abnormalities (dandy-walker complex, arnold chiari malformations, cerebellar dysplasia/ hypoplasia, joubert's syndrome, etc.), cysts (arachnoid cyst, giant cisterna magna etc.), tumours (medulloblastoma, ependymoma, hemangioblastoma etc.) and miscellaneous conditions. significantly reduces dose ( / of other gadolinium based contrast agents), and doesn't require trigger imaging. conventional mri provides important information regarding the anatomical extent, size, and relation to critical anatomical structures thus when combined with twist, mri provides the best information without use of radiation in children. functional connectivity mri in pediatric brain tumor patients with and without epilepsy andrew v. poliakov, phd, radiology, seattle children's hospital; david bauer, edward novotny, seth d. friedman, dennis shaw, jeff ojemann purpose or case report: functional connectivity mri (fcmri) is a way to evaluate cortical networks across different modalities such as motor, sensory, vision, and the default mode network using functional magnetic resonance imaging. fcmri relies on correlation in fmri image intensity that occurs between functionally connected regions. this effect can be seen in awake as well as anesthetized patients. we evaluated these pathways in pediatric patients with brain tumors. methods & materials: patients were randomly selected from our tumor database. inclusion criteria included age less than , history of brain tumor resection, and complete fcmri data. imaging was performed on a t siemens trio system. functional mri data were acquired as part of a clinical imaging protocol over . - min using a gradient echo, echo-planar sequence. preprocessing of fmri data followed by independent component analysis (ica) was performed using fsl software. functional connectivity analysis was performed using software provided by functional connectomes project, based on afni and fsl software packages. correlation maps were produced by extracting the bold time course from a seed region, computing the correlation coefficient between that time course and the time course from all other brain voxels, correcting for multiple sampling and degrees of freedom and thresholded at a z value of . . results: fourteen patients were included in the study, eight female and six male. tumor types include ganglioglioma ( ), pleomorphic xanthoastrocytoma ( ), juvenile pilocytic astrocytoma ( ), ependymoma ( ), anaplastic astrocytoma ( ), glioblastoma multiforme ( ), and primitive neuroectodermal tumor ( ). seven patients had tumor-associated epilepsy, and seven patients did not. the figure shows connectivity patterns in the motor network in patients without (a) and with (b) epilepsy. in the patients without epilepsy, functional connectivity was often displaced but not decreased or absent. in the patients with epilepsy, we observed decreased or absent functional connectivity. similar results were found for default mode network: connectivity was diminished or absent in the patients affected by epilepsy. conclusions: fcmri is a novel technique that may prove useful for evaluation and presurgical planning by giving us insight into how tumors disrupt function. functional connectivity was often displaced but relatively preserved in the patients without epilepsy. it was disrupted or absent in the patients with epilepsy. poster #: sci- corpus callosum dti measurements in neurofibromatosis type and normal controls nadja kadom, md, radiology, children's national medical center, nkadom@childrensnational.org; amir noor, rhea udyavar, marine bouyssi-kobar, iordanis evangelou, maria t. acosta purpose or case report: many patients with neurofibromatosis type (nf ) have corpus callosum enlargement; pathogenesis and underlying pathophysiology are unclear. the goal of our study is to investigate the pathophysiological basis of corpus callosum enlargement in nf patients through mri diffusion tensor (dti) measurements. methods & materials: retrospective study, irb approved. patients consecutively selected from institutional data base; inclusion criteria: established diagnosis of nf , brain imaging with dti sequence, abnormally high corpus callosum to skull ratio; excluded were patients with complications of nf that could affect size of the corpus callosum. age and gender matched normal controls were randomly selected from the radiology data base. roi were placed manually over the corpus callosum for dti measurements using dti-studio by two independent researchers, one blinded to diagnosis. results: fifteen nf patients and matched controls were analyzed. the corpus callosum to skull ratio was found to be significantly different between the experimental and control group (p . ). for nf patients we found: a trend to lower apparent diffusion coefficient (adc, p . ), significantly higher radial diffusivity (p . ), significantly lower axial diffusivity (p . ), and significantly lower fractional anisotropy (fa, p . ). conclusions: the significantly lower axial diffusivity in nf can indicate that there are more crossing fibers in the corpus callosum of nf patients than in normal controls. further studies using comparative dti tractography may be helpful in further investigating this stipulation. the significant increase in radial diffusivity can be explained by a variety of factors, including thinner myelin sheaths, increased interstitial fluid, smaller axons, or a combination thereof. the trend of lower adc may indicate low axonal diameter, as adc has been shown to more strongly correlate with axonal diameter without the myelin sheath. in future studies we will correlate abnormal corpus callosum dti markers with cognitive functions in nf patients to see if relationships exist that can be used as predictors of cognitive deficits in nf patients. screening for vitamin d deficiency in children with suspected non-accidental fracture conor kain, md, tripler army medical center; veronica rooks, laura keller, jordan pinsker, allyson cordoni, sarah frioux purpose or case report: determine if routine screening of vitamin d levels after suspected non-accidental fracture detects vitamin d deficiency and changes clinical outcomes. methods & materials: after irb approval we reviewed all skeletal surveys performed at tripler army medical center (tamc) in the last years and selected the children who were evaluated for suspected non-accidental fracture. we determined if -hydroxyvitamin d [ (oh)d] level was requested for these patients and characterized the provider's clinical suspicion of vitamin d deficiency as high or low. per the institute of medicine report and endocrine society guidelines we defined vitamin d deficiency as a (oh)d level of less than ng/ml. we calculated the prevalence of children with low (oh)d levels whose providers had low clinical suspicion for vitamin d deficiency. results: skeletal surveys were done at tamc from november to july . were performed after identifying a suspected non-accidental fracture. of these patients children from ages to months had (oh)d levels requested. for children whose providers had a low pre-test suspicion for vitamin d deficiency, the prevalence of vitamin d deficiency was . % ( % binomial ci . - . , of cases. these results indicate that at least one out of every three hundred children evaluated for nonaccidental fracture could have vitamin d deficiency despite a low clinical suspicion by their provider, although the actual rate is likely much higher given that we found one in eight cases. the child we identified with a low vitamin d level whose provider had no suspicion for rickets was treated with ergocalciferol and continued to be evaluated for abuse. conclusions: routine vitamin d level screening after nonaccidental fracture may detect vitamin d deficiency in children for whom there is low clinical suspicion. as our population resides at a low latitude and receives greater than average sun exposure, the rate of deficiency in children with suspected non-accidental fracture may be much greater in other areas. comet tails and dirty shadows: the secrets behind artifacts in pediatric ultrasound adam edelstein, pediatric radiology, massachusetts general hospital; anuradha shenoy-bhangle, katherine nimkin purpose or case report: to review common ultrasonographic artifacts, explain what causes them, and show how they can be used to aid in diagnosis in a variety of pediatric conditions, including less common entities. methods & materials: ultrasonographic images in patients less than years of age were reviewed. cases were selected that showed classic artifacts which helped with the diagnosis of a variety of entities. results: ultrasound artifacts include comet tail, reverberation, ring down and "dirty" shadowing. these can be used to help characterize a variety of pediatric conditions including gossypiboma, bezoar, subcutaneous foreign body, complications of nec, and staghorn calculus. artifacts can also be used to confirm the presence of stool or bowel gas. conclusions: familiarity with ultrasonographic artifacts is critical for tissue characterization and can help narrow the differential diagnosis in difficult pediatric cases. cardiac cta: non-vascular ring tracheobronchial compression secondary to enlarged patent ductus arteriosus in infants with congenital heart disease. nhi huynh, md, radiology, st. joseph hospital and medical center, e.nhihuynh@gmail.com; todd chapman, randy richardson purpose or case report: tracheobronchial compression or narrowing secondary to a vascular ring has been well documented. the purpose of this study is to describe the frequency of airway compression secondary to an enlarged patent ductus arteriosus detected by ccta without the presence of a vascular ring. methods & materials: a retrospective study of ccta exams in infants was performed over the period between / / and / / . ccta was performed with a -slice mdct, with ekg gating, followed by three-dimensional reformations. results: of the congenital heart disease infant patients, there are patients with tracheobronchial compression or narrowing. of these patients, patients reported to have patent ductus arteriosus as the primary cause of tracheobronchial compression or narrowing. approximately % of patients with airway compression in patients with congenital heart disease are secondary to an enlarged and/or tortuous patent ductus arteriosus. none of these cases were due to a vascular ring. of these patients, , , and patients demonstrated to have mild, moderate, and severe airway compression respectively. conclusions: tracheobronchial compression or narrowing secondary to vascular ring with a patent ductus arteriosus has been well documented. in this study, we demonstrate that a significant percentage of airway compression in patients with congenital heart disease without a vascular ring is due to a tortuous enlarged patent ductus arteriosus. cardiac cta is uniquely equipped to evaluate airway compression due to an enlarged patent ductus arteriosus and can help improve patient care in congenital heart disease patients with respiratory symptomatology. pediatric liver mr elastography: a primer suraj serai, phd, cchmc, suraj.serai@cchmc.org; daniel j. podberesky, alexander j. towbin purpose or case report: a wide variety of pediatric liver disorders may be complicated by the development of liver fibrosis and ultimately cirrhosis. with early interventions, the progression to hepatic fibrosis can be slowed, halted, and in some cases reversed. liver biopsy has long been considered the gold standard for assessing the presence and degree of liver fibrosis. however, liver biopsy has disadvantages, due to its potential sampling error, risk of complications, relatively high cost, intra-and inter-observer variability, and, in general, poor acceptance by pediatric patients and their parents. mr elastography (mre) is a relatively new, non-invasive technique that provides a safe, rapid and cost-effective method for objectively evaluating of a wide variety of hepatic diseases by quantitative stiffness evaluation of the liver-parenchyma. the purpose of this exhibit is to review our clinical experience with this technique and illustrate the application of liver mre in the pediatric population at our medical center. methods & materials: a review of pathogenesis and staging of liver fibrosis in children and current methods available for assessing liver fibrosis will be provided. a review of mre physics and technique, including the specific liver mre protocol used at our institution will be illustrated. we will review widely-used and emerging clinical indications for liver mre, as well as benefits and limitations to the technique, supported by brief literature review. results: in addition to sharing our liver mre technique, we will illustrate clinical case examples from our institution of a variety of liver disorders including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, storage disorders, cardiac disease, and idiopathic elevated liver enzymes. conclusions: this educational exhibit will review our experience with liver mre, a safe, newly available technique which will play an increasingly important role in the noninvasive evaluation of pediatric liver disease. poster #: sci- spectrum of tuberculosis in children amit gupta, mbbs, radiodiagnosis, r.n.t. medical college, udaipur, rajasthan, india, amitsensation@yahoo.co.in purpose or case report: the aim of this exhibit is to present a spectrum of tuberculosis (tb) in the human body which commonly involves pulmonary, nervous, musculoskeletal, gastrointestinal and genitourinary systems. this pictographic presentation displays the imaging features of tb cases encountered in our clinical practice with reference to plain x-rays, ct and mri as appropriate. results: with the advent of the newer modalities, the utility of the plain skaigram has been largely limited a initial screening tool only. whereas ct scores over mri in pulmonary tb (parenchymal disease, lymphadenopathy, pleural effusion, empyema, miliary disease) and abdominal tb (spectrum from mesenteric lymphadenitis to visceral involvement), the magnetic resonance (mr) imaging is much better in diagnosing cns tb (tuberculoma, abscess, meningitis, subdural empyema and myelitis). in musculoskeletal and genitourinary tb, ct and mr imaging may be preferred based on the stage of disease and the character of the lesion. cardiac involvement (pericarditis) is among the less common affections of tb. conclusions: tuberculosis is a multisystem disease that can affect virtually any part of the body from head to toe. tb demonstrates a variety of clinical and radiologic findings and has a known propensity for dissemination from its primary site and therefore can mimic numerous other disease entities. hence it is imperative for radiologists to understand the typical disease distribution, patterns and imaging manifestations of tb. janet l. strife, md carol m. rumack md ole a. eklof, md clement c. faure, md andres giedion, md denis lallemand, md arnold lassrich md donald r. kirks, md beverly p. wood, md hooshang taybi md marta hernanz-schulman, md, facr m. ines boechat, md, facr neil d. johnson, mbbs dorothy i. bulas, md *deceased singleton-taybi award investigator award this award is given to the author of the best paper presented by a resident or fellow at the spr meeting md ricardo faingold, md andrea doria, md nina m. menezes, phd anthropometric parameters are a strong determinant of lvm in healthy individuals kiyarash mohajer, pierangelo renella, paul j. finn purpose or case report: despite theoretical advantages of higher field strength ssfp cine imaging, time-resolved magnetic resonance angiography (tr-mra), and high resolution contrast-enhanced mra (ce-mra) were performed. two readers independently evaluated the data for image quality, vessel and cardiac chamber definition, and presence of artifacts. snr and cnr were calculated. results: % of ssfp cine images at t were rated as good or excellent quality with % having mild and % having moderate artifacts (k . ) % of arterial and venous phase ce-mra images were considered good or excellent cardiac chamber definition was considered good or excellent in % of arterial and venous phase ce-mra images (k . ). % ce-mra images showed good or excellent definition of the thoraco-abdominal vessels on average, both readers scored cine ssfp images higher at . t and cemra images higher at . t. overall diagnostic performance was high at both field strengths. conclusions: mri of pediatric patients with chd and vascular abnormalities at . t is feasible. relative to . t, snr and cnr are both improved at higher field strength and higher resolution cemra is achievable t are more prevalent, they rarely render cine imaging non-poster # exclusion criteria were lack of correlating us or follow up information. two pediatric radiologists blinded to us findings reviewed the mr images and analyzed the contents of abdominal wall defect, organ location and attachment; spine anomalies; umbilical cord and limb anomalies. results: our search yielded patients. all fetuses had ventral wall defects, small thorax and eviscerated liver and bowel. in two cases kidneys were in extracorporeal location. in / there was no membrane covering extruded organs. in five mr showed organs attached to the placenta or uterine wall (mainly bowel and liver) mahmoud al-hawary and, by adolescence, paradoxical metaphyseal osteopenia with thin cortical bone. reports of consanguinity indicate autosomal recessive inheritance our studies, spanning ages - mo, showed weight %, but length diminishing from~ % to − . sd. head circumference was + sd. she had frontal bossing, blue sclera, normal teeth, genu valgum, and unremarkable joints. radiographs showed orbital and facial sclerosis, basilar thickening, "bone-in-bone" appearance in the pelvis, sclerotic long bone ends, and fractures of ribs and extremities. progressive metaphyseal widening occurred as vertebrae changed from ovoid to flattened and became beaked anteriorly. consistent with opt, serum pth concentrations reflected dietary calcium levels. serum bone alkaline phosphatase, osteocalcin, and trap b were sub-normal. iliac crest contained excessive primary spongiosa and no osteoclasts. splice sites and exons were intact for the genes encoding cholride channel , t-cell immune regulator , opt-associated transmembrane protein the hallmarks include stippled epiphyses, nasal hypoplasia, and hypoplastic distal phalanges and developmental delay. punctate calcifications are seen not only in the epiphyses but also in the paravertebral regions. paravertebral puncta are commonly associated with defective ossifications in the cervical spine. the malformation of the cervical spine causes spinal canal stenosis and instability, which occasionally necessitate surgical intervention none of the cases had brain infarction. conclusions: tortuousity and luminal narrowing of the cervical arteries is a common finding in cdp-bt. this previously unknown malformation is an important factor to discriminate patients at increased risk of cerebral ischemia, particularly in patients undergoing surgical intervention. disclosure: dr. okabe has indicated that she will discuss or describe severe skeletal toxicity from protracted etidronate therapy for generalized arterial calcification of infancy william h. mcalister, md, mallinckrodt institute of radiology campbell sheen purpose or case report: generalized arterial calcification of infancy (gaci) is an autosomal recessive disorder caused by deactivating mutations within the gene for ectonucleotide pyrophosphatase phosphodiesterase- (enpp ). enpp on osteoblasts, chondrocytes, and vascular smooth muscle cells hydrolyzes nucleotide triphosphates to nucleotide monophosphates and inorganic pyrophosphate (ppi) can time-resolved contrast-enhanced mra (twist) classify soft tissue vascular anomalies in the head and neck in children accurately? aylin tekes, md children from - years of age were enrolled. twist and conventional mri was performed (triplanar t -weighted [t -w] imaging with fat saturation, pre-contrast axial t -weighted [t -w] imaging, and post contrast triplanar fat-suppressed t -w imaging). twist was performed in coronal plane using blood-pool mr contrast agent (ablavar-lantheus) to enhance image quality and spatial resolution of mra. two pediatric neuroradiologists evaluated all patients in two different sessions, days apart: one session conventional mri with contrast was evaluated, in the second session twist was evaluated. clinical evaluation and/or percutaneous venogram/lymphogram data were the gold standard. results: our patients had diagnosis of infantile hemangioma (n ), venous malformation (n ), and lymphatic malformation (n ). twist alone could accurately classify / , conventional mri with contrast could accurately classify / . conventional mri with contrast combined with twist could accurately classify all cases. conclusions: twist offers high temporal resolution in the order of seconds, and provides functional data about the dynamics of contrast enhancement comprising the arterial, venous and delayed venous phases kiery cr- , sci- edu- , edu- edu- , edu- , pa- edu- , pa- a- , pa- , pa- , pa- , pa- edu- , edu- , edu- , edu- , edu- , edu- , edu- edu- , edu- , edu- , edu- , edu- , edu- suraj sci- , pa- , pa- edu- pa- , edu- , pa- the society for pediatric radiology gratefully acknowledges the support of the following companies in presenting the th annual meeting and postgraduate course: cme committee reviewers for this activity have disclosed any relevant financial relationships. no conflicts of interest exist.abuse and offer potential mechanisms of injury may help make the diagnosis of child abuse. the pediatric elbow-mri findings with multimodality correlation michael guandalini, md, royal children's hospital; murray bartlett purpose or case report: to describe and illustrate elbow abnormalities identified by mri performed in a cohort of pediatric patients with multimodality correlation. methods & materials: retrospective review of mri elbow studies performed at the royal children's hospital, melbourne between and . the studies were reviewed by a pediatric musculoskeletal radiologist and pediatric radiology fellow with patient demographics, clinical indication, findings and selected images recorded. results: elbow mri examinations were reviewed on children aged months to years ( boys, girls) with equal numbers of left and right sides examined. clinical indications included previous trauma in cases ( %) and nontraumatic conditions in ( %). the most common traumatic indication was suspected or confirmed fractures or avulsions ( %) followed by osteochondral or cartilage injuries ( %), growth arrest ( %), loose bodies ( %) and ligament injuries ( %). hemophilia ( %) was the most frequent nontraumatic indication followed by neoplasm ( %). mild to severe arthropathy, fractures, physeal growth arrest, subluxations, osteochondral lesions and loose bodies were the most frequently demonstrated abnormalities. ligament strains and tears, bone oedema, neuromuscular abnormalities, infections and several neoplasms including lipomas, vascular/lymphatic malformations and bone tumors also featured. conclusions: this pictorial review illustrates the broad range of abnormalities one might expect to encounter on pediatric elbow mri studies, highlighting the major features and corresponding appearances on ct and plain x-ray. spectrum of patellar tendon avulsive injury on mri in children: differentiation between acute and chronic avulsive injuries of the inferior patellar pole and tibial tuberosity zeyad metwalli, md, baylor college of medicine, metwalli@ bcm.edu; herman kan, scott rosenfeld, r. p. guillerman purpose or case report: the extensor mechanism of the knee is an intricate component of the joint and is frequently injured in pediatric athletes. due to the strength of the patella tendon, trauma to the anterior knee is often manifested by avulsive injuries, which may occur on an acute or chronic repetitive basis. purpose: this pictorial review will illustrate differentiating radiographic and mri features of acute and chronic avulsive injuries of the pediatric knee. outline: . anatomy and physiology a. discuss the anatomic differences of the pediatric and adult knee extensor mechanism b. pathophysiology and biomechanical basis for chondro-osseous avulsion injuries versus tendon tears in the skeletally immature. purpose or case report: the purpose of this educational exhibit is to demonstrate the magnetic resonance imaging (mri) appearance of the ankle and hindfoot ligaments using an interactive approach. methods & materials: a tesla siemens mri scanner with a multichannel ankle coil was utilized in the acquisition of images of ankle and hindfoot. three dimensional volume acquisition proton density images will be used to demonstrate the ligamentous anatomy of the ankle and hindfoot in axial, axial oblique, coronal, and sagittal planes. results: the exhibit will begin with an interactive review of the ankle and hindfoot ligamentous anatomy with each ligament poster #: edu- cns imaging findings in hemophagocytic lymphohistiocytic syndrome rupa radhakrishnan, mbbs, md, dnb, radiology, university of cincinnati college of medicine, radhakrp@ucmail. uc.edu; marcia k. kukreja, alexandra filipovich, alexander j. towbin purpose or case report: hemophagocytic lymphohistiocytosis (hlh) is a rare, life threatening condition caused by an uncontrolled proliferation of activated lymphocytes and histiocytes with high levels of inflammatory cytokines. the organs most commonly involved in this disorder include the liver, spleen, lymph nodes, bone marrow and central nervous system (cns). the purpose of this exhibit is to review the cns imaging findings associated with hlh, its complications, and its management. the published literature was reviewed to identify the potential imaging findings hlh. the electronic medical record system was then searched to find illustrative case examples from our institution. cases demonstrating the primary imaging findings as well cases highlighting complications of the disease or its therapy were selected. results: cns involvement is common in hlh with approximately % of patients demonstrating neurological symptoms. ct findings of cns involvement include diffuse parenchymal atrophy, low attenuation lesions in the white matter and calcifications. mr findings include diffuse leptomeningeal and perivascular enhancement, t hyperintense lesions with nodular or rim enhancement as well as confluent white matter lesions, and diffuse parenchymal volume loss of the cerebrum and cerebellum. restricted diffusion has been demonstrated in some lesions. ring enhancing parenchymal lesions have been described representing active demyelination. intracranial hemorrhage may occur as a result of thrombocytopenia and coagulation abnormalities. sepsis with opportunistic organisms can involve the cns and produce intracranial findings such as parenchymal abscesses. cns changes, such as posterior reversible encephalopathy syndrome, are also seen with the commonly used immunomodulatory regimen used in the treatment of hlh. conclusions: this exhibit will aid the viewer in identifying the cns imaging findings of hlh as well as the complications of the disease and its therapy. while the cns imaging findings are not specific, they may help the radiologist formulate a diagnosis in association with the other clinical and imaging findings; furthermore, imaging can help the clinical team in managing the disease and its complications. methods & materials: medical records of our pediatric patients with palpable head masses over the last years, were reviewed and images were collected. correlation of us of these lesions with other imaging modalities and/or pathologic diagnosis was done. results: us appearances of various head masses including congenital/developmental (encephalocele, meningocele, dermoid, occipital protuberance), traumatic (cephalhematoma, subgaleal hematoma, calvarial fracture), inflammatory/infectious (sebaceous cyst, histiocytosis, dermatitis), vascular (malformations, pseudoaneurysm) and neoplastic (benign and malignant lesions including metastases) etiologies, will be illustrated with case based approach. mri and/or ct or tissue diagnosis can be problem solving. role of ultrasound guidance for percutaneous procedures (biopsy, sclerotherapy) will also be described. conclusions: ultrasound can play an important role in the delineation, diagnosis and guiding further management of pediatric palpable head masses. us can differentiate various scalp lesions and suggest the underlying calvarial defect or involvement to some extent, helping to narrow the differential diagnosis for such lesions. color doppler us can be useful to detect vascularity within the lesion or vascular lesions. given that us is often requested for the evaluation of palpable head masses, pediatric radiologists should be familiar with their sonographic features. posterior fossa malformations-a pictorial review rui santos, md, bc children's hospital, ruiradiologia@gmail. com; khalid khashoggi, angela t. byrne purpose or case report: posterior fossa malformations are a group of central nervous system anomalies that may be detected during pregnancy or present early infancy with features that include hypotonia, developmental delay, mutations responsible for the disease and proposals as to mechanism of action of the mutation with respect to disease manifestations. this preceded the development of hypotheses regarding the relationship between genotype and phenotype and the attempt to utilize imaging modalities that could better assess disease activity as it related to functional status. the purpose of this exhibit is to briefly review the history pre- and to focus on the numerous ways in which the understanding has improved since that time. conclusions: . there are over different mutations of the cftr gene responsible for cystic fibrosis with varying prevalence throughout the world. . the class of mutation often dictates its particular mechanism of action. . there is some relationship between genotype and phenotype-particularly with respect to pancreatic involvement. . newer imaging modalities including ct and mri with or without hyperpolarized helium are better predictors of disease severity than is plain film. imaging pulmonary tuberculosis in infants: what are the most useful diagnostic radiological findings? handan cakmakci, pediatric radiology, dokuz eylul university hospital, handancakmakci@gmail.com; nevin uzuner, filiz tetik purpose or case report: early diagnosis and treatment are very important for infants with tuberculosis. infantile pulmonary tuberculosis is more symptomatic, and the risk of severe and life-threatening complications such as tuberculous meningitis or miliary tuberculosis is higher. bacteriologic confirmation of the disease in children is difficult and in younger infants (< months), the tuberculin skin test is frequently negative. therefore, radiological findings play important role in diagnosing tuberculosis in infants. the purposes of this study are to identify chest x-ray and lung ct findings in pulmonary tuberculosis of infants and consider the most useful diagnostic findings of these age group patients.methods & materials: chest radiographs and chest ct images of infants who were diagnosed in our hospital from to were retrospectively reviewed. the study group included boys and girls ranging in age from to months (mean age, months). chest x-ray and computed tomography images were analyzed considering air space consolidation, nodular lesions, cavitating lesions, mediastinal enlargement, hyperinflation, bronchial narrowing, atelectasis pleural effusion on plain radiography and additional mediastinal calcific or caseating lymph nodes on ct images.results: air space consolidation was seen on out of chest x-ray and computed tomography images. nodular lesions were seen out of chest x-ray and computed tomography images. cavitating lesion was seen on out of chest x-ray and computed tomography images. mediastinal enlargement suggesting lymph node was seen out of chest x-ray and computed tomography images. hyperinflation, bronchial narrowing was seen out of chest x-ray and computed tomography images. atelectasis, pleural effusion was seen out of chest x-ray and out of computed tomography images. mediastinal caseating lymph nodes, mediastinal calcific lymph nodes were seen out of computed tomography images. conclusions: frequent and the most useful diagnostic radiological findings of pulmonary tuberculosis in infants are mediastinal or hilar lymphadenopathy with central necrosis and air space consolidations. disseminated nodules including miliary lesions and airway complications are also detected in this age group. ct can show detailed parenchymal lesions and tuberculous lymphnodes especially calcified ones. the ductus bump: radiographic findings of this normal variant and differential diagnoses anusuya mokashi, staten island university hospital, anusuya.mokashi@gmail.com; jeremy neuman, cheryl lin purpose or case report: the ductus bump: review of radiographic findings, differential diagnoses and current controversies. the ductus bump was first described in by berdon et al as a transient physiologic mass in the chest in newborn infants. some controversy remains as to the exact etiology and clinical significance. although initially thought to represent a dilated ductus arteriosus, recently it has been suggested that it actually represents a ductus arteriosus aneurysm that spontaneously resolves. others contend it represents dilation of the infundibulum of the closing ductus. regardless of etiology, the time of discovery, location, and rapid resolution are characteristic of this entity. in this presentation we will review the radiographic and echocardiogram findings of the ductus bump, as well as discuss the differential diagnosis. the frontal radiographic findings are a round mass to the left of the vertebral spine projecting from the mediastinum near the aortic arch. this mass does not indent the esophagus and it cannot be seen on the lateral view. it is classically said to resolve within the first few days of life. the controversy regarding the etiology has also led to some disagreement involving the clinical significance and appropriate follow up, which will also be discussed. after reviewing this educational poster, the reader will have conclusions: abnormalities of the posterior fossa are often difficult to differentiate solely on the basis of their radiologic appearances alone. however, an accurate diagnosis is essential for proper treatment planning and genetic counselling. therefore it is imperative for radiologists to be well versed with the normal anatomy and development of cerebellum so as to correctly diagnose the various posterior fossa abnormalities.poster #: sci- imaging of oculoauriculofrontonasal syndrome with low-dose -dimensional computed tomography paritosh c. khanna, md, radiology, seattle children's hospital, pkhanna@uw.edu; kelly evans, gisele ishak, joseph gruss, michael cunningham, anne hing purpose or case report: oculoauriculofrontonasal syndrome (oafns) combines elements of abnormal morphology of the frontonasal and maxillary processes of the face. the aim of our exhibit is to demonstrate the low-dose computed tomography (ct) features of this syndrome, in seven patients who have been followed at seattle children's hospital (sch) over years. we underscore the imaging features of this condition, and describe additional features including bony nasal abnormalities not previously described in the literature, to improve imaging recognition of this spectrum. we present d ct imaging features of a series of eight patients with oafns. in keeping with the alara (as low as reasonably achievable) concept and the image gently recommendations (www.imagegently. org), ct head and face studies were obtained on six of eight patients at sch, while two had prior exams at outside institutions. using a -slice multidetector ct scanner (ge lightspeed vct, waukesha wi), low-dose ct ( kv, mas or lower depending on age) of the head and face was obtained. planar bone window and d surface rendered images were analyzed. results: our series of patients demonstrated bifid nasal bones, uni-or bilateral mandibular hypoplasia, temporomandibular and zygomatic dysplasia and bony external auditory canal abnormalities. one patient had an interfrontal bone with a frontal bony defect that was contiguous with the metopic suture. we describe additional previously unidentified ct anomalies of the nasal bones, anterior nasal spine and nasal septum. these structures are involved in all patients who had ct imaging available, although unique features are present in each case. conclusions: ct is the mainstay of imaging of craniofacial anomalies in the post-natal period, both pre-and postoperatively. in addition to our low-dose ct imaging findings of oafns, novel nasal bone anomalies identified by our group serve to identify a new subset of patients with this syndrome and may help refine the phenotype of the oafns spectrum. key: cord- -xo zyi authors: burry, lisa d.; barletta, jeffrey f.; b.pharm, david williamson; kanji, salmaan; maves, ryan c.; dichter, jeffrey; christian, michael d.; geiling, james; erstad, brian l. title: it takes a village… contending with drug shortages during disasters date: - - journal: chest doi: . /j.chest. . . sha: doc_id: cord_uid: xo zyi nan the world health organization (who) designates access to essential drugs as a critical concern due to persistent shortages and escalating costs. , drug shortages are a function of demand and supply mismatches that can be affected by manufacturing, distribution, as well as regulatory, economic, or political considerations. prior to the coronavirus disease (covid- ) pandemic, numerous countries frequently documented such shortages, mostly with injectable drugs, to include antimicrobials, anaesthetics, cardiovascular and neurologic drugs, nutrition, electrolytes, and cancer chemotherapy. , the covid- pandemic dramatically demonstrates how large and sudden surge in demand can lead to shortages when local, national, and international supply chains cannot keep pace, in particular medications with limited therapeutic alternatives for critical care. we highlight the available literature on possible causes and mitigation strategies to manage shortages of critical care drugs from the local, institutional level to the global stage in a worldwide crisis such as a pandemic. the covid- pandemic resulted in international shortages of multiple critical drugs. - sedatives, analgesics and paralytics needed to support critical care were particularly affected by the surge in volumes of patients requiring mechanical ventilation, combined with high dose requirements often used to manage severe hypoxemia. the result was a quick depletion of limited "just-in-time" inventories at many hospitals. this was exacerbated by the inability to access supplies to match these new demands in either local, national or international networks. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are also disparity concerns at the international level, such as preferential access and hoarding by higher-income countries, and, conversely, ineffective stock management, notification systems and demand forecasting in low-and middle-income countries. , - further complicating the issue are the absence of international surveillance and notification systems for medication demands and a lack of globally-accepted best practices for dealing with shortages. supply-chain disruptions leading to medication shortages are more likely to occur when there is limited diversification in the development and distribution of required active pharmaceutical ingredients (apis). the problem is further exacerbated when the manufacturing process is arduous or lengthy, or the supply of raw materials is disrupted. for example, approximately % of active ingredients required for pharmaceutical compounding hail from india and china, one of which was the first country impacted by covid- . additionally, over % of new molecular entities (nmes) are patented with varying degrees of market exclusivity and availability by country. of nmes launched between to in countries, the percent available by country varied from a high of % in the united states (us) to less than % in other countries. there are also additional concerns with the supply of generic medications, which comprise at least % of the market in canada, germany, netherlands, the us and the united kingdom (uk). in a study of non-prescription, generic oral forms approved by the food and drug administration (fda) since , only % had a manufacturer approved by four or more of seven non-us j o u r n a l p r e -p r o o f regulators evaluated. of the % of medications with no fda-approved generic, only % were available from at least one manufacturer approved by one of the seven non-us regulators. the international pharmaceutical federation (ipf) held a summit involving multiple stakeholders in to address the persistent issue of drug shortages. the ipf recommended the compilation of publicly-available lists of known national drug shortages and the creation of national surveillance bodies to gather and share information about demand. the goal was to develop mitigation strategies for times of shortage, remove unnecessary variability in regulatory practices, and develop a global process for determining vulnerable products. a scoping review evaluated publicly available listings of drugs at risk of shortage on an international level, providing a listing by country of publicly available databases, along with frequency of updating and whether reporting was mandatory or voluntary. while many countries have mandatory reporting, only seven countries required daily updating: australia, belgium, canada, czechia, latvia, portugal, sweden, and the us. two multi-country collaborations were identified that analyzed the causes of shortages and provided frameworks to ensure patient safety: the south american institute of government in health and the cost research collaboration in europe. , , based on these networks' example, a larger international task force could reasonably facilitate preparation, mitigation and coordination and minimize the impact of shortages at the front lines of care. the root causes of drug shortages involve every aspect of the supply chain, from manufacturing to delivery to an individual institution's case mix and preferences. , in the us, several measures to improve reporting of production interruptions have been implemented, but regulatory bodies cannot mandate a pharmaceutical company into drug production. the fda drug shortages task force report described three major root causes. the first was the lack of incentive for manufacturers to produce less-profitable drugs. many of these shortages involve older or generic drugs whose manufacturers contend with intense price competition and uncertain revenues. in an analysis of data from - , injectables identified in shortage were in the rd percentile by cost of all marketed drugs. the decision to increase manufacturing of a less-profitable but needed drug could force production tradeoffs with more-profitable drugs. the second factor described was the expense of maintaining mature quality management systems. many drugs in shortage have been available for more than years, and quality maintenance requires continual technical improvement, requiring investments that markets may not reward in the short term. a failure to comply with required production standards or good manufacturing practice (gmp) regulations, however, can result in supply interruption and consequently drug shortages. finally, logistical and regulatory challenges may make it difficult for markets to recover after supply disruptions. as drug supply chains become longer and more fragmented (with increased overseas production), the ability to increase production rapidly during a shortage is diminished. additional causes of drug shortages also require consideration. health systems typically obtain their drug products through wholesale distributors. restrictive distribution methods may limit product availability to suppliers j o u r n a l p r e -p r o o f or systems that comply with manufacturer agreements based on market approval requirements. shortages can also occur when a disproportionate number of hospitals in a region use the same wholesale distributor. most institutions utilize "just-in-time" inventory practices for budgetary reasons; this inventory strategy is highly reliant on a functional supply chain, making them vulnerable to unexpected shortages during a natural disaster or pandemic. furthermore, rumors of a disruption in the supply chain can lead to hoarding, potentially preventing drugs from reaching regions where affected patients are concentrated. the impact of rumors and fears on supply chains was dramatically illustrated to the world with the "great toilet paper panic of " where sales ballooned to > % nearly overnight. while individual institutions may have good intentions when ordering increased stock in anticipation of future events, the cumulative impact of such practices across an entire region or country can be enormous. next, allocations for some drugs are based on historical usage, meaning institutions are restricted to ordering only their usual allotment even if patient volumes escalate. local shortages can therefore occur with drastic increases in hospital census (e.g., a large number of ventilated patients requiring deep sedation) or when use of a medication increases for a new indication (e.g., azithromycin, hydroxychloroquine, or tocilizumab). last, in the us, distribution of remdesivir, a novel antiviral with activity against covid- , fell under federal control for use consistent with an emergency use authorization. uncertainty about access has led to calls for transparency from multiple organizations. careful assessment and forecasting of essential drugs is a vital component of an icu surge plan. , the american college of chest physician's task force for mass critical care (tfmcc) recommends that institutions should stock necessary drugs to support icu care and be prepared to provide emergency mass critical care for up to days. however, the stock quantities suggested by the tfmcc now require reexamination, given the greaterthan-expected demand to manage mechanically-ventilated covid- patients as described in italy, the uk and the us where institutional supplies were quickly "burned" (personal communication). national stockpiles are available in some countries, but drugs are typically not available for immediate use and represent only a fraction of the essential drugs required to provide critical care (e.g., common antimicrobials) (personal communication). there are prediction models available to quantify the amount of drug required to support an icu surge, but these are largely based on assumptions pertaining to expected census (i.e., size of the surge), estimated use (i.e., percentage of patients receiving the drug), and estimates of daily dose and duration of therapy. , several factors specific to the covid- pandemic illustrate the challenges with estimating drug needs and the need for real time data for predicting drug supplies. first is the variance reported in key outcomes that dictate drug usage. for example, one large case series from new york city noted a median (iqr) duration of mechanical ventilation (for survivors) of ( - ) days, while data from the seattle region reported a median of ( - ) days. , these differences would greatly influence the amount of sedatives, analgesics and paralytics required to facilitate mechanical ventilation, especially given a very high reported daily drug consumption per patient that far exceeds the average needs for ventilated patients, leading to rapid supply depletion. next is the highly volatile state of prescribing based on less-rigorous sources of data and non-comparative trials. the explosion of information, ranging from traditional journalism to social media and hearsay, has led to drastic shifts in drug demand. for example, lopinavir-ritonavir, an older antiretroviral agent, was described as a potential therapy given its in vitro activity against sars-cov and was used widely early in the pandemic. demand for lopinavir-ritonavir promptly decreased following the publication of a single randomized trial that demonstrated no benefit to its use. similarly, the perceived benefit of hydroxychloroquine has changed throughout the pandemic, with initial use largely influenced by a single small, non-randomized study. as subsequent studies have failed to demonstrate benefit, the demand for hydroxychloroquine decreased. another challenge with estimating drug usage during the pandemic is that doses and treatment durations for newer or off-label medications are not readily available, making it challenging to estimate needed quantities. dosing strategies for tocilizumab, an il- receptor antagonist, have been described including a weight-based dose of - mg/kg or a fixed non-weight-based dose of mg. no guidance is provided regarding the most appropriate weight metric to use (e.g., total, ideal, or adjusted body weight) or if there is benefit with larger doses. as such, real-time data are needed to optimize the process of estimating supply requirements. this type of drug supply planning must occur not just at the institutional level but at regional and national levels, permitting coordination of the critical care response with input from scientific advisory committees and experts. similarly, incident command systems must include pharmacy experts in operations, logistics and planning. regional and national multidisciplinary teams that proactively plan ahead for alternatives and prepare recommendations for substitution or conservation strategies before supplies are exhausted are more robust than any single-center effort. , , , , regional and national dashboards that include drug quantities available can provide valuable information when drug shortages do arise and can allow drugs to be moved to where the patients are concentrated. when supplies within an institution are exhausted, borrowing is common between hospitals within the same health system, but very few countries or regions have organized methods for borrowing (personal communication). this process becomes more complex when hospitals are from different systems, especially during a pandemic when hospitals are unlikely to "return" that medication (i.e., a vial for a vial) and have to compensate monetarily. additional concerns arise when new products are obtained (e.g., the acquisition of % propofol in a country where % propofol is routinely used); there are significant patient safety efforts required to ensure compatibility with technology such as bar coding, pump programming and electronic documentation. vendor support is needed to ease the burden of making safe product changes within their systems. pharmaceutical manufacturing involves multiple contributors involved in processes that include the synthesis of apis and inactive excipients, the production of an administrable form, packaging and labelling. the manufacturing of biologic drugs involves the use of microorganisms, plants or animals, resulting in a longer and more complicated process. as noted, pharmaceutical and api manufacturers are at the heart of the supply chain and play a major role both in the creation and the mitigation of drug shortages. manufacturing difficulties related to j o u r n a l p r e -p r o o f regulatory or quality issues, including product sterility, impurities, or degradation, are among the most common causes of drug shortages ( figure ). , , , , in addition, pharmaceutical manufacturers often depend on a limited number of api suppliers; any disruption in production of ingredients, such as due to a regulatory concern or a natural disaster, will consequently affect manufacturing. , production interruptions and capacity concerns, such as line breakdowns, are also potential causes of shortages, especially for sterile parenteral drugs. , , [ ] [ ] [ ] [ ] these drugs demand highly specialized production lines, are expensive to produce, and are typically among the drugs needed to manage mass critical care. this results in low profit margins, and very few manufacturers are consequently interested in producing them. , given the lack of incentives to produce these drugs and limited competition among generic drug manufacturers, the number of producers is generally low. countries such as canada and the us often depend on only one or two suppliers of essential drugs, including many critical care drugs, and production interruptions rapidly cause shortages. this was illustrated in with propofol, where two of three manufacturers in the us simultaneously shut down production for regulatory reasons. , in the context of shortages, pharmaceutical manufacturers may also have to deal with internal competition, as many essential products will generally share the same production lines. in the case of biological drugs such as vaccines, limited manufacturing options due to the complexity of production may also escalate shortages. as the pharmaceutical and especially api production are highly concentrated in a limited number of countries, pandemics, unplanned large-scale political events, and natural disasters have the potential to cause global shortages. capacity reduction and lockdown of pharmaceutical or api production sites because of governmentimposed restrictions or staffing shortages during a quarantine may halt production or natural disasters may disrupt the manufacturing plant itself. fear of local shortages or a surge in consumption, as seen with covid- , can push governments to impose export restrictions on exports, further preventing drugs from reaching patients. , unforeseen increases in demand, such as for the off-label use of hydroxychloroquine, may also inflict shortages and impact the management of patients previously stabilized on indicated therapy. pharmaceutical manufacturers, regulatory bodies, and professional organizations need to collaborate to assess acute and long-term needs and determine products to be prioritized in production. , as regulatory and quality problems are often at the heart of drug shortages, continuous improvement of pharmaceutical processes is fundamental to avoid these difficulties and ensure the continuous supply of products. manufacturers have shortage prevention and management plans in place, as well as internal procedures and forecasting to track and avoid shortages in normal circumstances. in the case of unforeseen shortages, pharmaceutical manufacturers should plan to increase manufacturing capacity to favor those agents needed to manage critical patient populations. manufacturers can further decrease the risk of shortages using methods to increase system resiliency, such as decentralising production to multiple sites rather than one single site, investing in redundancy of critical production steps, and ensuring availability of active and inactive pharmaceutical ingredients through optimal inventory management and the development of relations with alternative producers. regulatory agencies in north america and europe have legislation in place to ensure that drug shortages are reported in a timely manner, but manufacturers j o u r n a l p r e -p r o o f must also maintain transparent real-time inventories and actively participate in the rapid notification of shortages on national registries. at the hospital pharmacy level, strategies for mitigating drug shortages involve identifying drugs at risk and anticipating their demand and supply, balancing inventory with allocations from distributors while implementing conservation and therapeutic alternative strategies. shortages are sometimes predictable and anticipated (e.g., pre-planned production gaps for maintenance), allowing for increased stockpiling of medication and planning for larger allocations of alternatives. for example, the manufacturing of premixed unfractionated heparin infusion bags was deemed by the fda in to be non-compliant with gmp regulations, resulting in a drug shortage in multiple countries. hospitals had time to consider other suppliers, alternative products and the possibility of local preparation. unfortunately, most drug shortages usually have little advanced warning, as seen during the covid- pandemic. sedatives, opioid analgesics, and paralytics have been among the most vulnerable classes of drugs in short supply in this pandemic. , professional societies must be engaged in the process of developing and disseminating guidelines to list class alternatives and aid broad distribution, rather than individual institutions working in isolation. all potential supply chain options should be explored, including different manufacturers and distributors, requiring the formation of partnerships with regulatory bodies. interchangeable products, such as therapeutic alternatives within the same class (e.g., morphine instead of fentanyl), may be readily available and can be considered at the bedside. conservation strategies may involve different routes of administration (e.g., enteral opioids instead of intravenous) or alternative drugs from other classes (e.g., midazolam instead of propofol). we illustrate an example of applying potential alternatives and conservation options for the use of analgesics, sedatives and paralytics in the setting of drug shortages in table . specific recommendations for therapeutic alternatives and conservation strategies for many essential drug classes are detailed in other publications. , opportunities to minimize drug wastage must also be considered (e.g., dose rounding, central batch drug pharmacy preparation, selection of appropriate vial sizes and concentrations). in the icu, therapeutic escalation strategies can be employed to conserve sedatives and opioid analgesics, using intermittent enteral dosing as the preferred option, followed by intermittent intravenous dosing and finally continuous infusions only when required. , additional strategies include ensuring that the lowest effective dose is being used (e.g., by employing sedation and analgesic targets), critically evaluating how essential a medication is for each patient (e.g., agents for stress ulcer prophylaxis), drug rotation (i.e., not depleting the supply of one drug but instead rotating between drug alternatives while awaiting new shipments), and prioritization (e.g., reserving selected drugs for specific situations). while the impact of drug shortages for covid- may focus on critical care areas, conservation strategies may apply to other areas that require similar drugs (e.g., operating rooms, palliative care). for example, smaller vial sizes of propofol may be less vulnerable to shortages and could be reserved for procedural sedation or within the operating room, while larger vial sizes are conserved for icu patients requiring prolonged sedation. we must recognize that conservation strategies and therapeutic alternatives may not always be possible, may not represent best practices, or could involve the use of locally unfamiliar medications that may compromise patient safety. each institution should engage administrators, educators and bedside clinicians to evaluate the risks and benefits of proposed strategies in their institution. in addition to efficacy and safety, the feasibility, suitability and logistics must also be considered within the institution. any change in clinical practice will require time and resources to disseminate information and educate staff. hospitals using electronic health records with prescriber order entry may need to develop new ordering pathways and program intravenous pumps for new drugs, concentrations or bag sizes (e.g., changing from % to % propofol). during a global health crisis like the covid- pandemic, there are always competing interests that must be considered in addition to drug shortages. in these times, it can be expected that drug shortages will occur at the same time as disruption of other essential resources, such as staff and space, as well as the availability of personal protective equipment (ppe), intravenous pumps and supplies for medication preparation and administration. some strategies that mitigate drug shortages may negatively impact other essential resources. for example, twice or thrice daily intermittent dosing of subcutaneous unfractionated heparin for thromboembolic prophylaxis requires more frequent ppe usage to support the room entries for nurses, compared to a single daily dose of enoxaparin. consolidating medication administration times to coincide with other activities (e.g., routine assessments, obtaining laboratory samples) can further reduce room entries. when considering conservation strategies for drugs or other resources, consultation with end users is essential. we sought to highlight the issues associated with anticipating and managing shortages of essential critical care drugs from global, national, regional and institutional perspectives during a disaster. important issues associated with drug shortages have been reported in the literature in the last five years, providing us with an understanding of the root causes of these shortages and potential mitigation strategies. figure summarizes take-home messages that stakeholders must consider for future steps during a global disaster. dealing with shortages of essential drugs during a global disaster teaches us "that coordination, communication and transparency should be fundamental principles in all actions between stakeholders at the regional, national and global level" to mobilized from essential therapies to the front lines of patient care. • may cause tachycardia, hypertension and hypersalivation; has minimal respiratory depression effects • ketamine is a dissociative agent thus patients may not appear "sedated" • bis values will not change and may even increase with ketamine administration • may cause hallucinations on emergence (that requires coadministration of benzodiazepines) • potential for accumulation exists with prolonged administration secondary to active metabolite (with / potency of parent compound), lipophilicity, end-organ dysfunction and drug interactions • ideal or adjusted body weight is suggested for weightbased dosing remifentanil • short half-life ( - minutes) requires administration via continuous infusion • does not accumulate in hepatic or renal failure • ideal body weight is suggested for weight-based dosing in obesity sufentanil • approximately times as potent as fentanyl • limited data with use in the intensive care unit • drug interactions present related to cytochrome p system • ideal body weight is suggested for weight-based dosing in obesity abbreviations: bis =bispectral index; cpot = critical-care pain observation tool; nsaids = non-steroidal antiinflammatory agents j o u r n a l p r e -p r o o f • may cause tachycardia, hypertension and hypersalivation; has minimal respiratory depression effects • ketamine is a dissociative agent thus patients may not appear "sedated" • bis values will not change and may even increase with ketamine administration • may cause hallucinations on emergence • potential for accumulation exists with prolonged administration secondary to active metabolite (with / potency of parent compound), lipophilicity, end-organ dysfunction and drug interactions • ideal or adjusted body weight is suggested for weightbased dosing phenobarbital • potential for drug interactions related to cytochrome p system • may consider conversion to oral therapy ( : conversion) once stabilized abbreviations: rass = richmond agitation-sedation scale j o u r n a l p r e -p r o o f while cisatracurium is the agent of choice for many institutions, in the setting of drug shortages, consider reserving cisatracurium (and atracurium) for patients with endorgan dysfunction and use vecuronium or rocuronium when both kidney and liver function are preserved addressing the global shortage of, and access to, medicines and vaccines report by the director-general background medicines shortages: global approaches to addressing shortages of essential medicines in health systems medicine shortages: gaps between countries and global perspectives essential icu drug shortages for covid- : what can frontline clinicians do? potential of chloroquine and hydroxychloroquine to treat covid- causes fears of shortages among people with systemic lupus erythematosus the coronavirus outbreak could disrupt the u.s. drug supply. council on foreign relations us pharmaceutical innovation in an international context new analysis shows that more medicines worldwide are available to u.s. patients. the catalyst paying for prescription drugs around the world: why is the u.s. an outlier? the commonwealth fund affordability and availability of off-patent drugs in the united states-the case for importing from abroad: observational study fip addressing global medicines shortages situation of essential medicines at risk of supply shortage with emphasis on south american countries european medicines shortages research network-addressing supply problems to patients (medicines shortages drug shortages: root causes and potential solutions ashp guidelines on managing drug product shortages emergency use authorization of remdesevir: the need for a transparent distribution process preparing your icu for disaster response. society of critical care medicine managing icu surge during the covid- crisis: rapid practice guidelines definitive care for the critically ill during a disaster: a framework for optimizing critical care surge capacity. from a task force for mass critical care summit meeting epidemiology, clinical course and outcomes of critically ill adults with covid- in new york city: a prospective cohort study lancet covid- in critically ill patients in the seattle region-case series a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- surge capacity logistics. care of the critically ill and injured during pandemics and disasters: chest consensus statement neilipovitz d; ontario covid- icu drug task force. therapeutic alternatives and strategies for drug conservation in the intensive care unit during times of drug shortages: a report of the ontario covid- icu drug task force a toolkit for improved understanding and transparency of drug shortage response in canada the drug shortage crisis in the united states: causes, impact, and management strategies drug shortages in developed countries -reasons, therapeutic consequences, and handling medication shortages during the covid- crisis: what we must do economic and technological drivers of generic sterile injectable drug shortages us propofol drug shortages: a review of the problem and stakeholder analysis why are there so many drug shortages? drug shortages in the united states: are some prices too low? the reality of drug shortages--the case of the injectable agent propofol indian pharma threatened by covid- shutdowns in china new regulatory strategies to manage medicines shortages in europe sedation of mechanically ventilated covid- patients: challenges and special considerations baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region, italy recommendations for alternative analgesic and sedative agents in the setting of drug shortages key: cord- -q egnzwf authors: khan, md. arif; mahmud, shafi; alam, a. s. m. rubayet ul; rahman, md. ekhtiar; ahmed, firoz; rahmatullah, mohammed title: comparative molecular investigation of the potential inhibitors against sars-cov- main protease: a molecular docking study date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: q egnzwf recent outbreak of novel coronavirus and its rapid pandemic escalation in all over the world has drawn the attention to urgent need for effective drug development. however, due to prolonged vaccine and drug development procedure against a newly emerged devastating sars-cov- virus pathogen, repurposing of existing potential pertinent drug molecules would be preferable strategy to reduce mortality immediately and further development of new drugs to combat overall global covid- crisis in all over the world. herein, we have filtered prospective drug candidates through literature review. assessing evidences from molecular docking studies, it was clearly seen that, epirubicin, vapreotida, and saquinavir exhibited better binding affinity against sars-cov- main protease than other drug molecules among the potential inhibitors. however, ns molecular dynamics simulation indicated the less mobile nature of the docked complex maintaining structural integrity. our overall prediction findings indicate that epirubicin, vapreotida, and saquinavir may inhibit covid- by synergistic interactions in the active cavity and those results can pave the way in drug discovery although it has to be further validated by in-vitro and in-vivo investigations. communicated by ramaswamy h. sarma following detection of sars-cov- in late december in wuhan, capital of central china's hubei province, it has quickly spread across the globe (tang et al., ) and this highly spreading and devastating infection, covid- has killed , , people worldwide (https://www.worldometers. info/coronavirus/#countries, n.d.). named after the related structured severe acute respiratory syndrome-related coronaviruses, sars-cov- has a genome sized$ kb with the specific pattern, orf ab encoding the large replicase polyprotein pp ab and forming the non-structural proteins (nsp - ) as well as the structural proteins (s, e, m, and n) (khailany et al., ) . moreover, the sars-cov- produced accessory proteins, encoded by orf a, orf , orf a, orf b, orf , and orf genes (khailany et al., ) . two cysteine proteases, namely the main protease (m pro ) or the c-like protease ( cl pro ) and the secondary papain-like protease (pl pro ) mainly process the replicase complex (rna-dependent rna polymerase and the helicase) that ultimately facilitate the viral replication, where m pro and pl pro cleaves at and three sites of the polyprotein, respectively. wu et al. ( ) reported that among the non-structural proteins of sars-cov- , cl pro plays a key role in the replication and maturation step. based on these facts, the m pro is a more promising drug target against sars-cov- than the pl pro , as well as for potential drug targets; scientists generally choose proteins and enzymes of covs vital for the replication process. in addition, previous studies have also shown that the main proteases of different coronaviruses are highly conserved in terms of both sequences and d structures . these features, together with its functional importance, have made cl pro an attractive target for the design of anti-corona viral drugs than other corona viral proteins. until now, no clinically approved drug is available for coronavirus (sars-cov- ) treatment (battegay et al., ) . scientists are trying to solve the puzzle through drug repurposing techniques, which is a promising option considering the global pandemic caused by the virus (pushpakom et al., ) . drug repurposing, an alternative option for approved or investigational drugs outside their defined indication, has a lower risk of failure and drastically reduces the time frame for development, facilitating prompt clinical decisions at lower costs development (cherian et al., ) . in the recent reports, we have come across some of the scientific endeavors, most of which are related to drug repurposing, to find effective drugs, nucleotide analogs (chien et al., ; kirchdoerfer, ) , and peptide binder . for example, ritonavir and lopinavir, which were under phase ii/iii clinical trials for mers-cov (chan et al., ; kim, won, et al., ) , have activity against hcov- e, hcov-nl and animal covs (kim, won, et al., ) , and can be an excellent repurposed drug for sars-cov- because the drug binding sites and % protein sequences were similar in sars-cov and sars-cov- main protease . also, researchers have currently reported using the drug repurposing approach based on the molecular docking and dynamics study where the key target proteins are cl protease, rna dependent rna polymerase (rdrp), and spike proteins (elfiky, b; muralidharan et al., ; smith & smith, ; tahir ul qamar et al., ; yu et al., ) . in this study, we explore potential drug candidate(s) against sars-cov- by performing the virtual screening based on drug repurposing concept, using available commercial medicine ( potential drugs against other viruses) that can eventually, be predicted to act against the main protease (m pro ), or -c like protease ( cl pro ). due to the lack of confidence in giving the accurate binding energy, binding mode, solvation effect, constant motion effect, and entropy energy during molecular docking (sethi et al., ) , a molecular dynamics study was preferred. in this study, the crystal structure of the main protease of sars-cov- was extracted (pdb id: lu ) which has similar binding sites of sars-cov (rose et al., ) . the protein structure was cleaned and prepared by discovery studio (san diego: accelrys software inc, ) and pymol (delano, ) software package. the cleaned protein structure was prepared and minimized with gromos b force field with the aid of swiss pdb viewer (kaplan & littlejohn, ) . the ligand molecules were retrieved from pubchem database (kim, thiessen, et al., ) after a deep literature review process. the ligand structure was optimized and cleaned with the help of mmff force field (halgren, ) with the steepest gradient algorithm. molecular docking was performed to understand the binding affinity with the ligand and drug compounds. the ligand were converted in pdbqt format to make them acceptable format for employing docking in autodock vina (trott & olson, ) . the center of the grid box was x:- . , y: . , z: . and dimensions were (x: . , y: . , z: . ) Å. finally the docked pose were analyzed with the aid of discovery studio and pymol. the molecular dynamics simulation was run in yasara application (krieger et al., ) . the amber force field (case et al., ) was employed for the minimization of the drugprotein complex and transferable intermolecular interaction potential points (tip p) were used for the addition of na and cl ions. the box size of the cell was set as ( . Â . Â . ) Å with a periodic boundary condition. a cut off the radius of angstroms was used for the calculation of short-range van der waals and coulomb interaction. the particle mesh ewalds was used to calculate long-range electrostatic interaction with a physiological condition at k, ph . , . % nacl (krieger et al., ; krieger & vriend, ) . the simulation was run for ns and root mean square deviation (rmsd), root mean square fluctuation (rmsf), the radius of gyration (rg), solvent-accessible surface area (sasa). moreover, all the trajectory further used to calculate mm-posson-boltzman surface area or mm-pbsa binding energy for binding free energy calculation where positive energy denotes favorable binding (dash et al., ; krieger & vriend, ) . among drug molecules only three showed better binding energy while interacting with main protease (table ) . epirubicin, formed five hydrogen bond at phe , glu , gln , asn , pro and one hydrophobic bond at pro position. on the other hand, vapreotida and main protease complex stabilized by several hydrogen bond at gln , asp , arg , asp , ile and one pi-pistacked bond phe , one pi-pi-t shaped at his , one alkyl pro and two pi-alkyl bond at val , ile . besides, main protease and saquinavir complex stabilized by six hydrogen bond at ile , phe , gln , thr , pro , phe and six hydrophobic bond at asn , phe , ile , val , pro , ile (table and figure ) . the rmsd value of docked complex analyzed from simulated trajectory to find out whether the complex remain stable or complex rigidity. from the figure , it can be observed that, all three chemical drugs had lower pic than . angstrom during whole simulation time however, epirubicin showed less flexibility till ns and thereafter, fluctuated for the next ns. the epirubicin and main protease protein from sars-cov- were stabilized and then reached in a stabilized state. on the other hand, vapreotida and saquinavir had similar profile till ns and after then, it exhibited lower rmsd value. it can be denoted from figure , that all the complex stabilized despite have some minor fluctuation but they were way below . angstrom. on the other hand, root means square fluctuation of the drug-protein complex can help to understand the flexibility in the protein region. from figure it can be understood that all three drug molecules exhibit a similar level of flexibility over the protein region. most of the residue did not have a higher peak in rmsf therefore responsible for the complex overall stability. however, figure indicates that the amino acid; ser (helix), gly (helix), arg (helix), glu (beta turn), leu (beta turn), glu (helix), arg (helix), his (helix), asn (beta hairpin), arg (helix), lys (beta turn), tyr (beta hairpin), pro (beta hairpin), thr (beta hairpin), arg (beta turn), phe (beta turn), thr (beta turn), met (helix), lys (helix), gln (helix), asn (beta turn), gly (beta turn), arg (beta turn), phe (helix), arg (helix), gln (helix), ser (beta turn), gly (beta turn), val (beta turn), thr (beta turn), phe (beta turn), gln (beta turn) from all the three complex were most flexible than other residue in the system. the complex compactness and rigidity can be assessed through a radius of gyration. figure denoted that, vapreotida and main protease has lower rg value than saquinavir and epirubicin which indicates the degree of compactness of the vapreotida. moreover, epirubicin and protein complex is less compact than the other two docked complex as they had higher rg value among three complex. on the other hand, higher sasa value indicates the high degree of expansion in the protein surface area. the saquinavir had a slightly higher peak than the other two drug molecules till ns. subsequently, saquinavir, vapreotida, epirubicin had a similar trend to ns which indicates protein surface area remain same during this simulation time. however, epirubicin had a lower sasa peak than two complexes after ns which may be the result of complex area expansion. both saquinavir and vapreotida had similar sasa profiles for the rest of the simulation time. furthermore, we have calculated mm-pbsa binding energy by taking each trajectory from the simulated protein complex to understand the relation between ligand binding and structural changes (figure ) . the main protease complex and epirubicin exhibited lower binding energy than other two complexes. the rest two complex had higher binding energy which indicates better binding with main protease of sars-cov- . molecular modelling especially docking and dynamic simulation study has become essential part in modern drug discovery pipeline (bappy et al., ; elfiky, a; islam et al., ) . moreover, time and lab cost can significantly reduce by narrowing the drug molecule choices in in silico based drug discovery process (talele et al., ) . besides, this combinatorial computational approaches allow the researcher to get the binding insights of the drug molecules to the target protein and rationally design the therapeutic targets (mullard, ) . among the selected drugs from this study, epirubicin can have a negative impact on chronic hbv patient through reactivation of the viral genome (xu et al., ) . the epirubicin drug molecules showed À . kcal/mol energy while binding with the main protease of sars-cov- which is the highest binding energy among the drug molecules. this might be the result of several hydrogen bonding that was found such as phe , glu , gln , asn , and pro at the active cavity of the main protease enzyme. researcher suggests that non-covalent binding at the active site of the target protein may lead to possible inhibition and blockage of the protein (hasan et al., ) . on the other hand, the somatostatin analog, vapreotide (vap) was used to control tumor growth, progression, and metastasis. saquinavir is a protease inhibitor of hiv has shown in-vitro effect against sars and mers coronaviruses (feng et al., ) . multiple hydrogens and hydrophobic bonds for the vapreotida and saquinavir may be responsible for the tight binding and better affinity among the drug molecules hossain, oany, et al., ) . molecular dynamics study of screened drug molecules were checked to evaluate their stability at atomic scale. the average root mean square deviation (rmsd) of epirubicin, vapreotida, saquinavir are . , . , . Å which indicates complex stability throughout the entire simulation (islam et al., ) . additionally, the average radius of gyration for these three complexes were found as . , . , . Å and did not deviate much. besides, the mean of the surface area of the three complexes was . , . , . Å . it was cleared from figure that simulation analysis of top three therapeutic drug molecules consistent with overall stable nature and complex rigidity . in this ground, it is clearly seen that epirubicin, vapreotida, and saquinavir may inhibit covid- by synergistic interactions among the potential inhibitors against sars-cov- main protease and those results pave the way in drug discovery although it has to be further validated by in vitro and in vivo investigations. scientists have chosen drug repurposing techniques as alternative options for approved or investigational drugs outside their defined indication for ease of utilization in an emergency with minor associated risk and clue for prompt target drug development and application with minimum cost involvement. bioinformatics approaches, such as molecular docking and dynamics study with probable drug candidates binding to key target proteins of pathogens for neutralization and inhibition are widely acceptable effective techniques to validate the drug repurposing approach. we have evaluated potential drug candidates against the main protease of the sars-cov- to understand their binding sites and structural stability. the nature of the binding mode of drug compound found as stable and molecular dynamics simulation study supports their conformational rigidity. as these outcomes solely came from combining computational algorithms, it requires further validation of these findings conducting further in-vitro wet-lab experimentation followed by in-vivo rapid trials for further use of the drugs individually or in combination to reduce mortality during covid- pandemics globally. moreover, based on these data, further, the experimental study can aid in getting suitable new drug molecules for combating covid- . discovery studio modeling environment. release . extensive immunoinformatics study for the prediction of novel peptide-based epitope vaccine with docking confirmation against envelope protein of chikungunya virus: a computational biology approach -novel coronavirus ( -ncov): estimating the case fatality rate-a word of caution the amber biomolecular simulation programs treatment with lopinavir/ritonavir or interferon-b b improves outcome of merscov infection in a nonhuman primate model of common marmoset perspectives for repurposing drugs for the coronavirus disease nucleotide analogues as inhibitors of sars-cov- polymerase structure-based identification of potent vegfr- inhibitors from in vivo metabolites of a herbal ingredient the pymol molecular graphics system, version . . schr€ odinger llc natural products may interfere with sars-cov- attachment to the host cell sars-cov- rna dependent rna polymerase (rdrp) targeting: an in silico perspective synergistic inhibition of breast cancer by co-delivery of vegf sirna merck molecular force field. i. basis, form, scope, parameterization, and performance of mmff molecular-docking study of malaria drug target enzyme transketolase in plasmodium falciparum d portends the novel approach to its treatment treating diabetes mellitus: pharmacophore based designing of potential drugs from gymnema sylvestre against insulin receptor protein identification of potential inhibitor and enzyme-inhibitor complex on trypanothione reductase to control chagas disease assessment of structurally and functionally high-risk nssnps impacts on human bone morphogenetic protein receptor type ia (bmpr a) by computational approach swiss-pdb viewer (deep view) genomic characterization of a novel sars-cov- pubchem substance and compound databases combination therapy with lopinavir/ritonavir, ribavirin and interferona for middle east respiratory syndrome halting coronavirus polymerase fast empirical pka prediction by ewald summation making optimal use of empirical energy functions: force-field parameterization in crystal space new ways to boost molecular dynamics simulations potential inhibitors against -ncov coronavirus m protease from clinically approved medicines exploring the potent inhibitors and binding modes of phospholipase a through in silico investigation supersized virtual screening offers potent leads computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- drug repurposing: progress, challenges and recommendations the rcsb protein data bank: integrative view of protein, gene and d structural information molecular docking in modern drug discovery: principles and recent applications repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface structural basis of sars-cov- clpro and anti-covid- drug discovery from medicinal plants successful applications of computer aided drug discovery: moving drugs from concept to the clinic on the origin and continuing evolution of sars-cov- autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods epirubicin directly promotes hepatitis b virus (hbv) replication in stable hbv-expressing cell lines: a novel mechanism of hbv reactivation following anticancer chemotherapy computational screening of antagonists against the sars-cov- (covid- ) coronavirus by molecular docking the first-in-class peptide binder to the sars-cov- spike protein the authors declares that they have no conflict of interest. the authors did not receive any external funding. md. http://orcid.org/ - - - key: cord- - s e i authors: neumann, natalie r.; chai, peter r.; wood, david m.; greller, howard a.; mycyk, mark b. title: medical toxicology and covid- : our role in a pandemic date: - - journal: j med toxicol doi: . /s - - - sha: doc_id: cord_uid: s e i nan medical toxicology and infectious disease are not specialties traditionally associated with one another. pandemics, however, have a way of disrupting convention, and in this era of modern medicine, our specialty has much to offer. when a major medical crisis occurs for which there is no known cure, several phenomena may ensue. the public, fearful and increasingly connected to and influenced by social media, the internet, and television, may experiment with self-medication. institutional bodies, desperate to advance care, may abandon the conventional mechanisms that ensure medication safety in order to facilitate the rapid approval and dissemination of novel pharmacotherapy. when these developments are considered within the context of our specialty, our role becomes clear. as medical toxicologists we serve as a fund of knowledge for our healthcare colleagues and the public: we provide physicians with information regarding antidotal therapy, drug-drug interactions, and novel therapeutics. we advise the public on an individual and community level through poison control centers and public outreach. as a specialty, we have the knowledge base and the position with respect to our peers and our society to monitor, prevent, and manage the toxicities born of a pandemic. accepting that medical toxicology has a role to play in an outbreak, we must also acknowledge that pandemics are not new; the covid- epidemic is unlikely to be the last humankind will ever face. therefore, the intent of this piece is to frame the role of our field in responding to pandemics not just today, but in the years to come. the first and most obvious function of a medical toxicologist in a pandemic is to recognize and manage the acute and chronic toxicities associated with therapy. in the case of the sars-cov- coronavirus outbreak, extensive attention has been paid, appropriately, to chloroquine and hydroxychloroquine toxicity [ , ] . poisoning from agents with antiviral activity is a high priority, and as such, jmt features a well-timed review by chary et al. on the adverse effects of nucleotide analogues, protease inhibitors, and monoclonal antibodies as prescribed for covid- [ ] . given that our understanding of the epidemic evolves on a daily, even hourly basis, some of the information presented may require recurrent reassessment. nevertheless, it serves as a helpful preliminary guide for clinicians. treatment-associated toxicity, it should also be noted, encompasses not only medically sanctioned therapies, but also those interventions the general population unearths and applies from less reputable sources. examples of potentially fatal home-brewed regimens used to treat coronavirus have included drinking bleach (sodium hypochlorite), insufflating cocaine, and consuming bootlegged alcohol adulterated with methanol [ ] [ ] [ ] . providing expertise with respect to the supervising editor: trevonne thompson, md * natalie r. neumann natalie.neumann@cuanschutz.edu treatment of toxicities born of the use and misuse of approved and alternative therapies is a service our field can readily provide to an otherwise overburdened medical system. determining which of these poisoned patients may remain at home will not only reduce ed visits and hospital admissions but also limit microbial transmission. mastery in medical toxicology is predicated on an understanding of pharmacokinetics and pharmacodynamics. here again, our specialty has a service to offer other medical fields. anticipating and mitigating the adverse interactions which occur between those medications a patient takes routinely, and those he or she is prescribed to treat an infection, may spare hospital resources, physician confusion, and patient lives [ ] . hydroxychloroquine, for example, is a known cytochrome p inhibitor that has been demonstrated to increase the serum concentration of medications which may be fatal in overdose [ ] . remdesivir belongs to the nucleotide analog class of medications typically associated with mitochondrial inhibition. the idea of a mitochondrial poison being prescribed to thousands of patients within days could reasonably make a practitioner nervous. concern is diminished, however, once one understands the range and timeframe of toxicity, and the fact that mitochondrial rna polymerase inhibitors do not act synergistically with other mitochondrial toxins [ ] [ ] [ ] . understanding when and when not to be concerned about drug-drug interactions may help mitigate diagnostic uncertainty and prevent hazardous polypharmacy. relatedly, there is a role for toxicologists as a voice of caution with respect to novel therapeutic administration when information on toxicokinetics and drug safety is lacking. the non-randomized, non-blinded application of anti-malarial medications to thousands of severely ill patients with multiple comorbidities may be reasonably questioned. similarly, the decision to administer on a large scale an antiviral agent that is undergoing fda approval at an unprecedented pace is worthy of dialog [ ] . by discussing with colleagues what level and kind of information is necessary to conclude that a drug is safe in any given patient population, we may reduce reflexive and potentially dangerous prescribing patterns. participating in the design and implementation of clinical trials involving these agents may also provide opportunities to minimize risks to individuals under study and to obtain the safety data we need to protect the broader patient population. medical toxicologists are also capable of addressing the question of what infection itself does to drugs. the impact of medications on patient outcomes and viral infectivity is debated frequently and is yet another a discussion to which medical toxicology can contribute. a classic example is the ongoing debate surrounding ace receptor density and the use of non-steroidal anti-inflammatory drugs and angiotensinconverting enzyme inhibitors in the setting of coronavirus infection [ ] [ ] [ ] . less commonly explored, however, is the impact a virus or bacteria may have on drug metabolism. covid- , for example, is associated with hepatic derangements including transaminitis and microvesicular steatosis [ ] [ ] [ ] [ ] . these findings have raised concerns that dosing regimens should be altered in the setting of infection [ ] . but hepatic injury does not necessarily correlate to abnormal pharmacokinetics, and in this case, as with others, the mechanism of injury may not interfere at all with drug metabolism [ ] . interpreting the nature of end-organ dysfunction as it pertains to therapeutic drug administration and overdose is well within the wheelhouse of medical toxicology and represents both a service our field may provide and a potential research frontier. it should also be recognized that by optimizing our standard practices we may improve outcomes and reduce strain on the healthcare system. antidote stocking, a challenge at the best of times, may become more difficult as a pandemic escalates [ ] [ ] [ ] . ensuring adequate access to antidotal therapy, ideally prior to an epidemic's peak, may save lives and spare hospital resources. recommendations for antidote stocking are available in an expert consensus document by dart et al. from [ ] . similar guidance is available in the united kingdom through the national poisons information service and royal college of emergency medicine [ ] . consideration may also be given to unconventional treatment regimens that allow appropriate patients to be treated at home and avoid hospital admission (e.g., oral fomepizole for toxic alcohol exposure, oral n-acetylcysteine after acetaminophen ingestion, etc.). lastly, poison centers, historically considered an underutilized source for reporting adverse drug reactions, may be more mindful of tracking and reporting toxicity from novel therapies [ ] . indeed, individual toxicologists can do the same and encourage their colleagues to use medwatch, the fda's medication safety reporting system, or the uk's mhpra yellow card reporting system [ ] [ ] [ ] . a final contribution our field can make is to monitor and potentially reduce the number of overdoses that might occur as a pandemic progresses. colleagues in europe have noted a precipitous drop in the number of ingestions reported since the institution of national and regional lockdowns. they are concerned that even though suicide rates may have decreased temporarily, if social isolation persists for months, citizens' mental health may deteriorate, leading to a rapid uptick in overdoses just as beds become scarce. patients with substance use disorders, already at higher risk of suicide and overdose, may be disproportionately affected, especially if they are unable to access opioid agonist therapy during quarantine [ ] [ ] [ ] . transitioning to innovative models of continued care, be they telehealth visits or extended prescriptions for buprenorphine, may improve patient outcomes and reduce hospital strain. identifying other at-risk populations and encouraging all providers to intervene early may save lives, generate constructive research, and inspire novel paradigms of care. surmounting a crisis requires the engagement of all parties. while medical toxicology may not be traditionally associated with viral pandemics, our field nevertheless has a part to play. chary et al's up-to-date review is a must-read for front-line caregivers, policy makers, and even patients [ ] . it reminds us of what is known and what is speculated, and it represents but one way in which we can help. optimizing the treatment of poisoned patients, minimizing potentially dangerous polypharmacy, clarifying situations of diagnostic uncertainty, and limiting strains on hospital resources are all skills that we bring to the table. these acts may not inspire celebrity, but they are positive actions that can quietly improve outcomes in a highly pressurized, exceptional setting. funding information peter r chai is funded by nih k da ; investigator-initiated research grants from gilead sciences, philips biosensing and e-ink corporation; and the hans and mavis lopater psychosocial foundation. medical experts explain the use and potential adverse effects of chloroquine and hydroxychloroquine for covid- man fatally poisons himself while self-medicating for coronavirus, doctor says. the new york times covid- : therapeutics and their toxicities blue ridge poison center warning people not to drink bleach: cbs news iran reports leap in death toll bbc news adverse drug reactions as cause of admission to hospital: prospective analysis of patients influence of hydroxychloroquine on the bioavailability of oral metoprolol gs- and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses mitochondrial toxicity of antiviral drugs mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir gilead sciences statement on request to rescind remdesivir orphan drug designation are patients with hypertension and diabetes mellitus at increased risk for covid- infection? the lancet respiratory medicine fda advises patients on use of nonsteroidal anti-inflammatory drugs (nsaids) for covid- a crucial role of angiotensin converting enzyme (ace ) in sars coronavirusinduced lung injury liver injury in covid- : management and challenges clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china pathological findings of covid- associated with acute respiratory distress syndrome epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study covid- and the liver: little cause for concern insufficient stocking of poisoning antidotes in hospital pharmacies availability of antidotes at acute care hospitals in ontario national audit of antidote stocking in acute hospitals in the uk expert consensus guidelines for stocking of antidotes in hospitals that provide emergency care royal college of emergency medicine and national poisons information service reporting of adverse drug reactions by poison control centres in the us medwatch: the fda safety information and adverse event reporting program the importance of physicians identifying and reporting adverse drug events yellow card risk factors for adolescent suicide and suicidal behavior: mental and substance abuse disorders, family environmental factors, and life stress suicide and substance abuse understanding links among opioid use, overdose, and suicide publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest none. key: cord- -x qbqy authors: liu, gengqi; lovell, jonathan f.; zhang, lei; zhang, yumiao title: stimulus-responsive nanomedicines for disease diagnosis and treatment date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: x qbqy stimulus-responsive drug delivery systems generally aim to release the active pharmaceutical ingredient (api) in response to specific conditions and have recently been explored for disease treatments. these approaches can also be extended to molecular imaging to report on disease diagnosis and management. the stimuli used for activation are based on differences between the environment of the diseased or targeted sites, and normal tissues. endogenous stimuli include ph, redox reactions, enzymatic activity, temperature and others. exogenous site-specific stimuli include the use of magnetic fields, light, ultrasound and others. these endogenous or exogenous stimuli lead to structural changes or cleavage of the cargo carrier, leading to release of the api. a wide variety of stimulus-responsive systems have been developed—responsive to both a single stimulus or multiple stimuli—and represent a theranostic tool for disease treatment. in this review, stimuli commonly used in the development of theranostic nanoplatforms are enumerated. an emphasis on chemical structure and property relationships is provided, aiming to focus on insights for the design of stimulus-responsive delivery systems. several examples of theranostic applications of these stimulus-responsive nanomedicines are discussed. one of the challenges of traditional drug delivery systems is the lack of specific targeting capability, which leads to dose-limiting side effects [ , ] . some drugs can be rapidly metabolized, and low bioavailability necessitates large injection doses but unsatisfactory therapeutic effects. for example, it is generally necessary to increase the dosage of anticancer drugs for the desired therapeutic effect, to an extent that also causes undesirable side effects [ , ] . in addition, orally administered medicines need to evade the risk of degradation in the acidic/enzymatic environment in the gastrointestinal tract [ , ] . all of these problems can potentially be addressed with stimulus-responsive nanotechnology. utilizing specific microenvironmental conditions at diseased sites or alternatively by using external stimuli, nanotherapeutics can release active therapeutic ingredients specifically at desired spots in a controlled and targeted manner. as early as , tanaka described the phase transition of polyacrylamide polymers [ ] , and in the same year blumenthal reported thermosensitive liposomes [ ] . findings such as these laid the groundwork for stimulus-responsive drug delivery systems, which have been developed for more than years. stimulus-responsive nanomaterials have been designed for a differences between the microenvironment of diseased tissues (e.g., arthritic tissues, tumor cells, and other diseases) and normal tissues can be used as endogenous stimuli to trigger drug release. endogenous stimuli include lower ph values in tumor cells, high concentrations of glutathione (gsh) and reactive oxygen species (ros) in inflammatory tissues and cancer tissues, and specific enzymatic activity at the target sites [ , ] . in addition to these endogenous stimuli above, exogenous stimuli such as magnet, light, and ultrasound can realize real-time tracking of drug distribution, disease diagnosis and disease management. stimulus-responsive systems responsive to one single stimulus or multiple stimuli have been used to treat or diagnose a variety of diseases, such as cancers (colon cancer [ , ] , breast cancer [ , ] , lymphoma [ , ] ), inflammatory diseases (inflammatory bowel disease [ , ] , neurodegenerative diseases [ ] ), diabetes [ , ] and others. some nanomedicines that could be considered stimulus-responsive have successfully gone into clinical trials. thermodox (thermosensitive doxorubicin liposomes) used heat to release the cargo from liposome [ ] ; other successful examples include opaxio [ ] (involving on enzyme-degradable polymer), nanotherm [ ] (based on magnetic iron oxide nanoparticles), and auroshell [ ] (based on thermosensitive gold nanoshells). stimulus-responsive systems also have potential for bioimaging. they can enhance the contrast in specific diseased area in response to internal or external stimuli for real-time monitoring. imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] hydrazone int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ , ] ester int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] acetal imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ , ] cis-aconityl imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] orthoester imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amide-bonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on not-carcinogenic tissues were also reduced because of such rational design [ ] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] diselenium int. j. mol. sci. , , x for peer review of besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ , ] thioether selenium tellurium besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ , ] vinyldithioether int. j. mol. sci. , , x for peer review of besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] pba/pbe besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) containing s-s bonds. at similar gsh concentrations ( mm), % of encapsulated dox was released by paur-se-se micelles within h, while % of the drug was released by paur-s-s micelles. in addition, the antitumor effect of dox-loaded paur-se-se was six fold higher than that of s-s analogues due to faster cleavage of the diselenium bond and enhanced drug release [ ] . in a recent study, a new type of redox-responsive drug delivery system was developed based on the nano-metal-organic framework. its core structure was formed by zirconium ions (zr + , metal nodes), , -disulfanylterephthalic acid (bdc-(sh) , organic ligand) and benzoic acid (ba, modulator), the sulfhydryl group on the anticancer drug, -mercaptopurine, formed a disulfide bond with bdc-(sh) . this disulfide-based nanomaterial exhibited excellent intracellular drug release ability, and its cytotoxicity to cancer cells was shown to be three times higher than normal cells [ ] . ros-responsive systems are also an effective strategy to control drug release by utilizing the ros accumulated in inflammation sites or other diseased tissues [ ] . it has been shown that the concentration of ros in inflammatory tissue is - times higher than that in normal tissue [ ] . there are two chemical mechanisms commonly used in ros-responsive systems. one is ros-induced non-breaking hydrophobic-hydrophilic transition and the other is ros-induced structural dissociation ( table ). ros can oxidize chalcogens (e.g., s, se, te) [ , ] , causing oxygen atoms to form covalent bonds with chalcogens, and polarizing groups to form hydrogen bonds with environmental water molecules. thus, hydrophobic-hydrophilic transition of the carrier backbone was induced without damaging the chemical structure of the drug carrier. among the chalcogen elements, tellurium-containing compounds have more potential than other chalcogen elements due to their lower electronegativity and lower toxicity [ ] . additionally, it has been shown that tellurium-containing compounds have higher oxidation responsiveness than selenium-containing compounds and sulfur-containing compounds [ , ] . xu et al. reported a synergistic therapeutic nanoplatform using near-infrared light-responsive cisplatin for cancer therapy. cisplatin (cddp) and indocyanine green (icg) were simultaneously loaded in nanocarriers made of amphiphilic tellurium-containing block copolymer peg-pute-peg. tellurium atoms in nanocarriers can be easily oxidized by ros (stimulated by near-infrared laser) produced by indocyanine green. tellurium oxidation weakens the coordination with cisplatin, thereby releasing cisplatin and achieving better anti-tumor effect [ ] . in addition, ros can react with chemical structures such as thioketal (tk), phenylboronic acid/ester (pba/pbe), oxalate ester [ , ] , vinyldithioether [ ] and proline oligomers, leading to the cleavage of these structures. in general, tk groups are easily oxidized to generate acetone and two other thiol-containing fragments in the presence of ros. a novel amphiphilic peptide-drug conjugate for cancer targeted therapy has recently been reported. hydrophilic cyclic peptides and hydrophobic cytotoxin epothilone b (epo b) were linked by ros responsive tk groups. when nanotherapeutics entered tumor cells, tk groups could be cleaved due to high levels of intracellular ros, thereby releasing epo b, which effectively inhibited cell proliferation [ ] . h o can specifically oxidize pbe/pba and subsequently induce the formation of borate ester and hydroxybenzyl alcohol [ ] . in addition, amino acids such as proline, histidine and arginine are also found to be susceptible to ros-mediated and metal-catalyzed oxidation [ , ] . for example, murthy et al. developed thioketal-based nanoparticles that could degrade selectively in the presence of ros in inflamed intestinal tissue. in responsive to abnormally high levels of ros in the sites of intestinal inflammation, thioketal nanoparticles could effectively deliver and release sirna to diseased sites, protecting mice from dextran sodium sulphate (dss)-induced colitis [ ] . in addition to treating inflammation, ros-responsive systems can also be used for cancer treatment. ge et al. designed ros-responsive nanoparticles made up by a thioketal linker (tl), poly (ε-caprolactone) and poly (n,n-dimethylacrylamide) (pcl-tl-pdma). amphiphilic di-block copolymers with pcl-b-poly ( -guanidinoethyl methacrylate) (pcl-pgema) were used to encapsulate the anticancer drug paclitaxel and a photosensitizer, achieving efficient cell uptake and enhanced anticancer efficacy [ ] (figure ). complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo ) and glucose oxidase (gox) have also been studied [ ] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [ ] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. cell uptake under nm light irradiation was improved. a synergistic effect of photodynamic therapy (pdt) and chemotherapy was achieved through efficient cell uptake and drug release. reproduced with permission from the publisher of corresponding reference [ ] . redox stimulus-responsive systems need high sensitivity and accuracy for fast triggered responses in diseased tissues, but stability under normal conditions. however, considering the complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo ) and glucose oxidase (gox) have also been studied [ ] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [ ] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. for example, a new type of polymer poly (ethylene glycol) diacrylate (pegda) have been designed that could form spherical polymeric nanoparticles when mixed with peptide. the nanoparticles can be hydrolyzed by the overexpressed matrix metalloproteinase in lung lesions and then release the drugs [ ] . in addition, it has been shown that the use of protease triggered drug release can enhance the therapeutic effect of drugs, and also reduce the toxicity and other side effects of drugs [ ] . cathepsin-b is normally expressed in the lysosomes of human cells, but is also overexpressed in many invasive and metastatic cancers. therefore, cathepsin-b has been used as one of the targets of the enzyme-responsive systems. the amino acid sequence of gly-phe-leu-gly (gflg) can be degraded in the presence of cathepsin-b, so it can be applied for the development of nanodrug carriers. for example, peg, anticancer drug cisplatin and gflg peptide were used to be assembled for cisplatin prodrug units. icg was used to co-assemble with cathepsin-responsive cisplatin multidrug nanoplatforms. after cell uptake, the gflg peptide structure was degraded by cathepsin b in lysosomes, which in turn released icg and cisplatin prodrugs for cancer treatment [ ] . among many kinds of matrix metalloproteinases, mmp has been one of the most widely explored for stimulus-responsive systems because it is overexpressed in cancer tissues. mmp -acting substrates have been used for drug delivery and molecular imaging. for example, torchilin et al. synthesized an octapeptide (gly-pro-leu-gly-ile-ala-gly-gln) as a mmp -sensitive linker for the conjugation of long-chain peg to liposomes and encapsulation of the cell-penetrating peptide tatp. when octapeptide (gplgiagq) was cleaved by mmp , tatp was exposed, giving rise to the increased uptake of liposome particles by tumor cells [ ] . in , similar structures were also used in drug-loaded liposomes for pancreatic cancer treatment [ ] . to improve permeability, antitumor efficacy and biodegradability, a size-shrinkable drug delivery system has been designed based on polysaccharide-modified dendrimers, which contained poly (amidoamine), hyaluronic acid and mmp -sensitive peptide linker (plglag). the system retained good stability in blood circulation, and underwent degradation upon the action of mmp , leading to enhanced uptake in tumor cells [ ] . in addition, polypeptide gplgvrg [ , ] , polypeptide gplgvrgdg [ ] and triglyceride monostearate (tgms) [ ] have also been synthesized for mmp sensitive drug delivery systems. phospholipase is another therapeutic target that can be overexpressed in infectious diseases, inflammatory diseases and tumors. in several inflammatory diseases or cancer cell types, the expression of phospholipase a (spla ) has been shown to be higher [ ] [ ] [ ] [ ] . pla is often used as a trigger condition in liposomal drug delivery systems, which specifically hydrolyzes sn- ester bonds in phospholipids, resulting in direct release of active drugs or hydrolysis of liposomes [ ] . for example, colchicine-containing phosphatidylcholinase-responsive liposomes have been developed that are stable in human blood circulation. at the same time, liposomes could release colchicine-containing fatty acids upon the action of high levels of phospholipase a . the latter further hydrolyzed and released colchicine analogues by non-specific enzymes [ ] . besides, in a recent report, an enzyme reactive liposome containing , -dipalmitoyl-sn-glycerin- -phosphocholine (dppc) and -o-stearyl- -retinoic acid receptor (rar)-c -sn-glycerin- -phosphoglycerol with c -rar as a prodrug was designed. in the absence of spla , the ic of c -rar prodrug in mt- breast cancer cell line was µm, while the ic of c -rar prodrug decreased to µm after adding spla , indicating that the prodrug was hydrolyzed into c -rar and lyso-o-spg by spla , in response to phospholipase [ ] . oxidoreductase has also been used as a potential target of stimulus-responsive systems due to its important role in intracellular oxidative environment. gox is a kind of oxidoreductase tested for stimulus-response systems. it catalyzes the oxidation of β-d-glucose to d-glucose-δ-lactone and hydrogen peroxide with oxygen as electron acceptor [ , ] . glucose responsive systems have been assessed for the treatment of diabetes [ , ] . for example, willner et al. recently used zeolite imidazolium frameworks (zif- nmofs) as a glucose-responsive carrier to release insulin and vascular endothelial growth factor (vegf) aptamers, which inhibited angiogenesis by binding to vegf proteins. zif- was stable under neutral physiological conditions but degraded under acidic conditions. insulin or vegf aptamers and gox were integrated into zif- nmofs as biocatalysts. gox catalyzed the aerobic oxidation of glucose to produce gluconic acid. local acidification of nmofs degraded zif- , thereby releasing insulin and vegf aptamers [ ] . besides, nqo has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [ , ] . in a recent report, a nqo -responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo , both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo triggered the self-immolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [ ] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by -fold in the tumor environment [ ] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated , -naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated -amino- , -naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [ ] (figure ) . nmofs degraded zif- , thereby releasing insulin and vegf aptamers [ ] . besides, nqo has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [ , ] . in a recent report, a nqo -responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo , both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo triggered the selfimmolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [ ] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by fold in the tumor environment [ ] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated , -naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated -amino- , -naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [ ] (figure ). in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [ ] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [ , ] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e and oligo (p-phenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [ ] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with p-glycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [ ] (figure ). solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [ ] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [ , ] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e and oligo (pphenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [ ] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with pglycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [ ] ( figure ). the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [ ] . the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [ ] . in addition to photothermal therapy and pdt, light-responsive strategies have also been applied in the design of prodrug systems and drug delivery carriers. light-responsive structure is generally a robust strategy because it is easy to control with good accuracy and minimal invasiveness. light-responsive systems for drug release are often based on the cleavage of prodrugs with light-sensitive structures or the changes of photosensitive molecules upon light stimulation, so as to release the conjugated or encapsulated drugs [ , ] . lovell et al. developed near-infrared light-responsive liposomes doped with hexyloxyethy-pyropheophorbide (hpph) entrapped into the bilayers. the anticancer drug doxorubicin (dox) was encapsulated inside the light-sensitive liposomes. when nir light was irradiated at the target site, hpph-liposomes opened the bilayer structure and then released dox, but when nir light was off, the stable bilayer structure of hpph-liposomes was reformed [ ] . common photolytic functional groups include o-nitrobenzyl derivatives (onb), anthracene [ ] , coumarin esters [ ] , arylmethyl [ ] and pyrenylmethyl ester [ ] (table ) . among these, o-nitrobenzyl derivatives have been widely used because of their fast photolysis rate and simple synthesis process. for example, willner et al. designed a light-responsive and ph-responsive dna microcapsule. light-responsive capsules are assembled by o-nitrobenzyl phosphate groups with dna layers used to stabilize the microcapsule shell (table ) . when stimulated by light, o-nitrobenzyl groups were cleaved, causing the degradation of microcapsules and the release of the cargo [ ] . another report showed that the o-nitrobenzyl linker containing carbamate bond had a better photolysis rate and a slower hydrolysis rate than other o-nitrobenzyl derivatives (containing an ester bond or amide bond), so that it could maintain higher stability in vivo and can degrade faster after light stimulation [ ] . besides, baker et al. chose onb group as the core of photosensitive protective group and conjugated with poly (amidoamine) (pamam) dendrimer to develop a drug delivery platform for methotrexate (mtx) loading. it was demonstrated that this photochemical approach can be used for in vivo delivery of anticancer drugs [ ] . ultraviolet-visible (uv-vis) light could be used for the trigger of o-nitrobenzyl derivatives and aryl methyl photolysis functional groups; however, it is difficult to achieve tissue penetration for better therapeutic performance. compared with uv and visible light, near-infrared light with wavelengths in the - nm range can penetrate into deeper tissues. nir could be used to cleave, isomerize or rearrange molecules through the two-photon absorption (tpa) process and nir-uv upconversion (uc) process. the theoretical analysis on the principle of tpa process was first proposed in s [ ] . however, the generation of tpa requires high excitation energy, and there are still some problems in practical applications. in the uc process, the luminescence center can absorb the energy of two or more photons, and then generate one emitted photon whose energy is higher than that of a single excited photon. additionally, unlike the tpa process, each process of photon absorption is like a second-order element reaction due to the existence of a real intermediate state in the uc process, which has a higher probability than the simultaneous absorption of two molecules [ , ] . upconversion nanoparticles (ucnps) based on rare earth ions provide a new method for the nir light-responsive system. for example, zou and his colleagues designed a near-infrared light-triggered nanocomposite peg-nmab-pla-ucnps (pnp-uc) to achieve photo-controllable release of dox for cancer treatment. under the irradiation of nm nir light, -nitrobenzyl group was triggered for its cleavage to release dox. compared with other ucnps-based nir responsive systems, this new nanocomposite has many advantages, such as high drug encapsulation efficiency, fast light responsiveness at low power density and less heating effect, showing potential in pharmaceutical and biomedical applications [ ] . common photoisomerization materials include azobenzene-based materials, spiropyran (sp)-based materials, and diarylethene-based materials (table ) . azobenzene is an azo group containing an aryl group, with cis and trans isomers. it shows a significant π-π transition in the uv region and a faint π-π transition in the visible region. therefore, upon irradiation of uv light, the trans structure of azobenzene changes into cis structure. additionally with heat or visible light, azobenzene can change from cis structure to trans structure [ ] . liu and colleagues assembled azobenzene-functionalized dna strands using ucnp to construct nano-pumps and load dox into nano-pumps. with nm nir irradiation, the continuous rotational inversion motion of azo molecules caused dna hybridization and de-hybridization, thereby leading to the release of dox. this novel light-responsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ring-closed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] coumarinyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] arylmethyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] pyrenylmethyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] o-nitrobenzyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] diarylethene [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [ ] ( table ). the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [ ] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [ ] (table ) . the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [ ] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings such as alkene bridges (such as cyclopentene [ ] , thiazole [ ] , imidazole [ ] , furan [ ] ) or using six-membered rings (such as benzoquinone [ ] , coumarin fluorophores [ ] , benzothiazole [ ] ). in addition, some light-sensitive molecules-vitamin b derivatives [ , ] , ruthenium complexes [ ] , etc.-have also been applied to light-sensitive systems. still, further development of light-stimulated systems is hindered by some factors. for example, for the treatment of solid tumors, penetration depth, effective area, power intensity and irradiation time are some important factors to be considered for the light-responsive therapy. in addition, many photosensitizers and photothermal agents have inherent toxicity or photo-toxicity. ongoing research is still making efforts for the application of light stimulation systems for clinical advances. temperature is a frequently studied stimuli for responsive delivery systems. inflammatory pathological sites and the hyperthermic nature of tumors can be used as internal stimuli [ , ] . another strategy is to increase the temperature by applying an external heat source. ideally, the thermal response nanocarrier should maintain its stability at body temperature and release the drug rapidly when the diseased site is heated. thermal stimulus-responsive polymers usually have lower critical solubility temperature (lcst) or upper critical solubility temperature (ucst), and the former is more widely used. for example, poly (n-isopropylacrylamide) (pnipam) and its derivatives have been used in the design of thermal stimulation response systems because their corresponding lcst is about • c, which is close to the physiological temperature of human body [ ] [ ] [ ] . a core-shell drug delivery system was designed to improve the solubility of drug by encapsulating hydrophobic drugs in nanoparticles with thermo-responsive materials as the shell. luo et al. developed a core-shell micelle that used hydrophilic thermo-responsive material pnipam as a shell for the encapsulation of the drug of paclitaxel (ptx). both drug and shell were conjugated by diselenide bond (pnipam-sese-ptx). the nanoparticles exhibited dual characteristics of temperature-responsiveness and redox-responsiveness, with high loading capacity ( . %) and encapsulation efficiency ( . %) [ ] . compared with pnipam that cannot be degraded in vivo, temperature-sensitive oligomers containing oligo (ethylene glycol) (oeg) potentially has better biocompatibility. jayakannan et al. developed a dual stimulus-responsive amphiphilic copolymer formed by copolymerization of hydrophobic polymerizable monomers with methacrylamide based on hydrophobic -pentadecylphenol (pdp) and oligopolyethylene glycol (peg) methacrylate, which thus was temperature sensitive and enzyme sensitive. the nanoparticles formed from the amphiphilic copolymer, which remained stable in normal tissues in vivo with less than % drug release, while above lcst, the nanoparticles had an up to % drug release rate in two hours due to the thermal response property of acrylamide copolymers. in the presence of esterase, the drug release rate could exceed % within h. such type of dox-polymer showed better anticancer efficacy [ ] . in addition to thermosensitive hydrogels and thermosensitive polymers, temperature-sensitive liposomes have also been investigated. since yatvin first proposed the concept of temperature-sensitive liposomes (tsl) in the late s [ ] , tsl has been attracting much attention from molecular design to clinical applications. thermodox is an example of temperature-sensitive liposomes, which was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is - fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [ ] (figure ). was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is - fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [ ] (figure ). for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperatureresponsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in [ ] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperature-responsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in [ ] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [ , ] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [ ] (figure ) . nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [ , ] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [ ] ( figure ). magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in [ ] . in , mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus (aav ) gene vector through heparin for c s cells and mice in vivo [ ] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [ ] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [ ] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, highintensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasoundmediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [ ] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [ ] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in [ ] . in , mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus (aav ) gene vector through heparin for c s cells and mice in vivo [ ] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [ ] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [ ] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, high-intensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasound-mediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [ ] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [ ] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of selfimmolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of self-immolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of selfimmolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. the self-immolation mechanism can be categorized into electron rearrangement and intramolecular cyclization. electron rearrangement is mainly based on the structure of quinone or its derivatives (such as thioquinone methide or azaquinone methide). the mechanism of electron rearrangement includes , -elimination, , -elimination, , -elimination and β-elimination. the electron rearrangement reaction usually contain aromatic ring structure with hydroxyl [ , ] , amino [ , ] or mercaptan groups [ , ] . when they are masked by protective groups, the electron rearrangement process of these functional groups is inhibited. when the external stimulus triggers the cleavage of the protective groups, these functional groups undergo irreversible self-immolation process driven by positive entropy or the generation of stable products. common protection groups that can be triggered by various stimuli are summarized in table . self-immolative spacers based on electron rearrangement are mostly based on , -elimination or , -elimination [ , ] . additionally, , -cleavage may occur in para amino (or hydroxy) cinnamyl alcohol or coumarin alcohol. in contrast, self-immolative spacer groups based on continuous combinations (such as , -elimination of naphthalene or , -elimination of biphenyls) usually do not result in the release of drug groups because the high energy barrier destroys the aromaticity, and the repulsion of the hydrogen atom in adjacent biphenyl prevents the formation of the planar structure needed for electron rearrangement [ ] . the cyclization reaction is based on alkyl chain or aromatic spacer groups [ ] [ ] [ ] . once the cleavage is triggered, the nucleophilic attack of carbonyl or electrophilic aliphatic carbon atoms could result in the cyclization of spacer groups. as of the electron rearrangement, the self-immolation cyclization is driven by the formation of positive reaction entropy and thermodynamic stable products (such as -membered and -membered rings). additionally, the triggers of self-immolation include chemical reagents, enzymes, light and others. self-immolative linkers that can be triggered by chemical reagents; boc(tert-butoxycarbonyl), fmoc( -fluorenylmethyl) and -oxobutyl carbamate are typically ph-responsive for organic synthesis [ ] . boc and fmoc are protecting groups for protecting amino groups, and the fmoc is the only widely used amino acid protecting group of carbamates which can be dissociated in weak base condition. these functional groups undergo self-immolation under some specific triggering conditions (trifluoroacetic acid, piperidine), which in turn releases the attached cargo [ , ] . redox-triggered self-immolative linkers could be broadly divided into three categories by triggers: transition metal (e.g., zn, pd) based reagents [ ] [ ] [ ] , reducing reagents (dtt, gsh or tcep) [ ] , and oxidant (e.g., h o ) [ ] [ ] [ ] [ ] . disulfide bonds are the most widely used reductant-responsive linkers. wu et al. employed α, α-dimethyl groups with disulfide bonds, so that p-dithiobenzyl (dtb) intermediates could maintain faster self-immolation rate and improve the stability. this novel self-immolative linker is expected to promote the design of targeted drug delivery systems and achieve traceless drug release [ ] . similarly, self-immolative linkers with h o as trigger condition have also been widely used in stimulus-responsive systems in various fields. recently, clausen et al. reported the synthesis of arylboronic acid-based hydrogen peroxide-responsive methotrexate and aminopterin prodrugs. this new prodrug can deliver drugs to the lesion of chronic rheumatoid arthritis, so that the side effects of the drug on normal cells were significantly reduced [ ] . enzyme sensitive self-immolative linkers are also commonly used in enzyme responsive systems. plasmin can induce the hydrolysis of tripeptides, which in turn causes the cleavage of self-immolative linkers and release of drugs [ , ] . penicillin g amidase (pga) and bovine serum albumin (bsa) can be used to trigger the cleavage of phenylacetamide or carbamate bonds to release the linked cargo [ , ] . β-galactoside and β-glucuronide are functional groups that can be cleaved by β-galactosidase and β-glucuronidase, respectively, and these two functional groups can also be combined with other self-immolative linkers [ ] [ ] [ ] [ ] . in addition, dibenzyl phosphate derivatives are also used for enzymatic (alkaline phosphatase) self-immolative design for the delivery and release of fluorescent probes and prodrugs [ , ] . enzymatically activated self-immolation is usually slow and requires synergy with other self-immolative linkers. light-activated self-immolation does not require an additional step for synergy. a variety of similar structures based on nitrobenzyl groups can undergo self-immolation under uv light [ , ] . examples of self-immolative structure triggered by nir light are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] h + ph [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] piperidine/ morpholine ph [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] zn/acoh redox [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] dithiothreitol redox [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] thiols redox [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] plasmin enzyme [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] penicillin-g-amidase (pga) enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] bsa enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] β-glucuronidase enzyme [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] β-galactosidase enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] alkaline phosphatase enzyme [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] nir light light [ , ] conventional drug delivery systems lack controlled drug release schemes whereas stimulus-responsive drug delivery systems or drug conjugates with rational design can release loaded drugs at specific sites triggered by various endogenous or exogenous stimuli. the human body is a complex collection of various microenvironments, so occasionally it may not suffice if only one stimulation condition is used for the design of stimulus-responsive systems. in this section, we mainly describe the combination of dual or multiple stimuli-responsive conditions for the design of nanoplatforms to improve the specificity and accuracy of nanotherapeutic systems. although single stimulus-responsive systems can solve the problems of specificity and side effects to some extent, the microenvironment within tumor tissues is complex. by contrast, dual or multiple stimuli-responsive nanocarriers can better detect subtle changes in diseased tissues. the traditional combination of ph and temperature has certain defects, such as possible premature drug release in blood or normal tissue species [ , ] , and the ph responsive system might be affected by various factors. to solve the problem of premature leakage, cui et al. made n-isopropylacrylamide-methacrylic acid-octadecyl acrylate (nipam-maa-oda) copolymer liposomes as nanodrug carriers that can be responsive to dual stimulates of ph and temperature [ ] . in recent years, the combination of light and ph has also become a popular choice for dual response systems. for example, researchers have conjugated ph-sensitive i-motif dna (converted from single-stranded structure to c-quadruplex structure in acidic ph environment) to gold nanostars (gns) with as used as a targeting structure. it has been discovered that a-gns/dna/dox nanocomposites have strong photothermal conversion ability, and the combination of ph and nir irradiation can effectively trigger drug release. this nanocomposite showed good stability in not-carcinogenic tissues, and therapeutic effect and biocompatibility were achieved using the construct for combined chemoand photo-therapy [ ] (figure ). in addition to the combination with photothermal therapy, ph responsive systems can also be combined with the uv light-responsive systems. host-guest interaction of β-cyclodextrin with azobenzene has also been studied, but most supramolecular polymer drug carriers are single stimulus-responsive with low delivery accuracy [ ] [ ] [ ] . chen et al. recently constructed supramolecular polymers harnessing host-guest interaction between β-cyclodextrin and azobenzene. β-cyclodextrin was combined with ph-sensitive hydrophilic poly( -(dimethylamino) ethyl methacrylate, and azobenzene was modified with hydrophobic poly(ε-caprolactone), enabling nanodrug micelles to be responsive to both ph and uv light. additionally, higher anticancer activity and stronger cancer cell inhibition than free dox could be achieved using this method [ ] . dual stimuli-responsive system using ph and redox is also a common combination. with low ph and high gsh levels in tumor cells, a strong controlled release effect can be achieved. recently, ph/redox-responsive mixed polymeric micelles formed by self-assembly of two amphiphilic diblock copolymers (poly(ethylene glycol) methyl ether-b-poly(β-amino ester)) (mpeg-b-pae) and poly(ethylene glycol) methyl ether-grafted disulfide bond-poly(β-amino ester) (pae-ss-mpeg) have been developed, which released drugs at low ph and high gsh concentration in tumor cells [ ] . in addition, there are also many combinations, such as ph/ros responsive system [ , ] , ph/enzyme responsive system [ , ] and others. as a significant characteristic stimulus condition in vivo, the combination of ph with other stimulus has shown promise for cancer treatments. redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [ ] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [ ] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [ , ] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir and dox to obtain stable nanoparticles (ir /dox@ptk). ir was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [ , ] . upon the irradiation of nm nir light, ir could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( o ) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [ ] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( o )-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [ ] (figure ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [ , ] and other ph-based multiple stimuli-responsive systems [ ] [ ] [ ] . redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [ ] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [ ] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [ , ] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir and dox to obtain stable nanoparticles (ir /dox@ptk). ir was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [ , ] . upon the irradiation of nm nir light, ir could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( o ) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [ ] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( o )-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [ ] (figure ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [ , ] and other ph-based multiple stimuli-responsive systems [ ] [ ] [ ] . inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [ , ] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [ , ] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. the characteristic conditions within the microenvironment of inflammatory tissues include lower ph value and higher ros concentration [ , ] . therefore, ph/ros dual stimulus-responsive system is the most widely used treatment strategy for inflammation therapy [ , ] . for example, almutairi et al. designed a ros-reactive dextran-drug conjugate (nap-dex) and blended nap-dex with an acid-sensitive acetal-dextran polymer (ac-dex) to obtain ph/ros dual stimulus-responsive nanoparticles. the ros-responsive pba-modified anti-inflammatory drug naproxen was used. when the nanodrug was stimulated by h o and acidic environment, the ac-dex and pba structure of the nanodrug micelle was cleaved, thereby releasing naproxen (nap) for the treatment of inflammatory tissues. dual stimuli-responsive nanodrug micelles are more effective in scavenging ros. compared with free naproxen, dual stimuli-responsive nanoparticles reduced the levels of proinflammatory cytokines il- and tnfα by times and times respectively [ ] . in addition to lower ph and higher ros concentrations, inflammation of tissues tends to have slightly increased local temperature ( - • c above ambient temperature) [ ] . therefore, the temperature/ph dual stimulus-responsive system has also been applied in the treatment of inflammation [ , ] . a microbead system was designed with a porous poly (d, l-lactic-co-glycolic acid) (plga) shell coupled with a gelatin plug (thermal-responsive switch). additionally, n-palmitoyl chitosan (npcs) (ph-responsive switch) was employed as a core. vancomycin was loaded in the nanoparticles as the inflammatory therapeutic drug. this dual stimuli-responsive drug mbs could release the drug only in the presence of both stimuli (temperature and ph), preventing unexpected drug release due to accidental stimulus [ ] . inflammatory bowel disease (ibd) is a chronic condition of idiopathic inflammation. ibd can affect the entire gastrointestinal tract (gi) and increases the risk of colorectal cancer [ ] . the incidence of ibd worldwide has increased and effective treatment approaches are needed. therefore, drug delivery systems utilizing ibd microenvironment characteristic conditions (low ph, colonic enzymes, and high ros concentration) as responsive factors have attracted extensive attention [ , ] . li et al. reported a nano-platform of oxidatively sensitive dextran (oxidex) with its exterior modified by chitosan (cs) and then further encapsulated by ph-sensitive hydroxypropyl methylcellulose acetate succinate (hpmcas). the intestinal-specific antibiotic rifampicin was used as a model drug to form ph/ros dual stimulus-responsive nanodrug composites. this novel nanodrug composite remained stable in the upper gastrointestinal tract, but hpmcas were cleaved in the intestine, thereby releasing nanodrug particles. triggered by higher ros levels, rifampicin is released into inflamed tissues. compared with traditional enteric drug formulations, this nanodrug composite effectively reduced the permeability of drugs at the intestinal epithelium, preventing non-specific absorption and side effects [ ] (figure ). in addition, some oral drug formulations responsive to ph have been used for clinical treatments. examples include -aminosalicylic acid encapsulated in capsules made from copolymers of acrylic acid derivatives and methyl methacrylate (eudragit ® ), such as salofalk ® , calitoflak ® , claversal ® , pentasa ® and other brands [ ] [ ] [ ] . other inflammation examples include neuroinflammation, which represents an abnormal condition of central nervous system in many neurodegenerative diseases [ ] . some serious neurodegenerative diseases include alzheimer's disease, epilepsy, huntington's disease and parkinson's disease [ ] [ ] [ ] . strategies for the treatment of these diseases are desired and some ongoing efforts have been made on stimulus-responsive systems that can target excessive ros concentrations in neuroinflammatory tissues [ , ] . oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include ( ) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, ( ) the permeability in the intestine, and ( ) drug molecules can be absorbed into the systemic circulation through the intestine [ , ] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [ ] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include ( ) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, ( ) the permeability in the intestine, and ( ) drug molecules can be absorbed into the systemic circulation through the intestine [ , ] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [ ] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [ ] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [ ] , various cancers [ ] [ ] [ ] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir can be used for photothermal treatment on cancer cells [ ] (figure ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl -aminocaproicacid) (pa aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l - ), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa aca and l - formed a loosely cross-linked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [ ] . stimulus-responsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [ ] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [ ] , various cancers [ ] [ ] [ ] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir can be used for photothermal treatment on cancer cells [ ] (figure ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl -aminocaproicacid) (pa aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l - ), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa aca and l - formed a loosely crosslinked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [ ] . stimulusresponsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [ , ] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [ , ] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [ ] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albumin-poly( -(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by - fold compared to non-stimulus-responsive nanoprobe and icg [ ] (figure a ). photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [ ] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albuminpoly( -(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by - fold compared to non-stimulus-responsive nanoprobe and icg [ ] (figure a ). [ ] . (b) ros significantly enhanced the ultrasound signal [ ] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [ ] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [ ] . (e) pet imaging enabled realtime monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [ ] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [ ] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [ , , ] (figure b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of [ ] . (b) ros significantly enhanced the ultrasound signal [ ] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [ ] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [ ] . (e) pet imaging enabled real-time monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [ ] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [ ] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [ , , ] (figure b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of ph value owing to the ph-sensitive coating. when the concentration of hydrogen ions decreased, the ultrasound imaging was significantly enhanced [ ] . magnetic resonance imaging (mri) is a powerful technology for diagnosis and bioimaging of diseases because of its non-invasiveness, reasonable imaging time, deep tissue penetration and high resolution [ , ] . zhang et al. recently developed a triple stimulus (gsh/ph/nir)-responsive nanocarrier based on magnetic hollow porous carbon nanoparticles (mhpcn). the outer shell of mhpcn consists of two layers: the inner layer with iron oxide (fe o ) as contrast agent for mri and the outer layer with fluorescent carbon nanodots as the outer layer. dox was selected as a model drug and folic acid (fa) was crosslinked for targeting purpose. this new triple stimulus-responsive nanodrug carrier can achieve synergistic photothermal/chemical therapy of tumor guided by mri under the synergistic stimulation of low ph, high concentration of gsh and external nir light in tumor cells [ ] . in addition, a strategy using magnetic resonance/near-infrared fluorescence (mr/nirfl) was developed to determine the location of tumors. using ph and nir as stimuli, chemo/photothermal therapy could be achieved for efficient cancer nanotheranostics [ ] . yang et al. used polyphenols as phase transfer agents to increase the solubility of hydrophobic magnetic nanoparticles. meanwhile, polyphenols also promoted the self-assembly of nanoparticles. the core of this assembly is atp/ph responsive for the dual imaging of mr/fl and photothermal therapy. when the nano-assembly entered the tumor microenvironment with lower ph value and higher concentration of atp, they showed enhanced mr contrast (due to the strong binding affinity of atp to fe o ) and significantly altered fluorescence signal (the protonation of hydroxyl groups in tannic acid (ta). thus, functional activities in tumors could be tracked using this mr responsive nanoplatform [ ] (figure c ). photoacoustic (pa) imaging is an emerging biomedical imaging modality with deep penetration depth, high spatial resolution, and high selectivity that combines the advantages of optical imaging and ultrasound imaging [ ] . when laser irradiation is applied to biological tissues, pa signals are generated in the tissues, and the detailed information of human internal tissues could be collected and reconstructed in computer. tan et al. designed a nitric oxide (no)/ph activatable theranostic nanoprobe (datn), taking advantage of high concentration of nitric oxide and low ph in tumor microenvironment. benzo [c] [ , , ] thiadiazole- , -diamine was used for the stimulus responsive π-receptor-π-donor (d-π-a-π-d) molecules. in the tumor microenvironment, datn could turn on pa signals for tumor specific pa imaging in vivo. using these dual stimuli, the pa signal intensity of datn was . times higher than that no responsive system and times higher that of acid responsive system [ ] (figure d ). pu recently reported the synthesis of a fluoro-photoacoustic polymeric renal reporter (fprr) for noninvasive near-infrared fluorescence (nirf) and pa dual imaging of drug-induced acute kidney injury (aki) in mice. fprr consists of dextran (used to promote renal clarity), hemicyanine cyoh (used for imaging), and γ-glutamate (enzyme-responsive moiety). fprr exhibited high renal clearance efficiency in living mice, it also enhanced nirf and pa signals for real-time molecular imaging of aki after the enzyme responsive moiety is cleaved. this nanoplatform showed potential for early detection of aki in clinics [ ] . positron emission tomography (pet) can provide precise biological information on metabolic processes with whole-body penetration and excellent sensitivity at the molecular level using radiopharmaceuticals. it has been widely used in clinical disease treatment and diagnosis [ ] . when used in combination with computed tomography (ct), the clinical effect of pet can be improved for disease diagnosis such as tumors and heart diseases [ ] . for example, hyaluronic acid (ha)-functionalized mos was designed with the surface modified by pei and cu (positron emitter with a half-life of . h). the resulting mos -pei-ha multifunctional nanoplatform was endowed with the property of triple stimuli (ph/haase/nir) responsiveness. this new nanocarrier can be used as contrast agent for pet imaging, providing an effective way to monitor the treatment process and optimize disease management plan [ ] (figure e ). chen et al. developed a multimodal imaging synergistic therapy based on amphiphilic iron oxide-gold janus nanoparticles (fe o -au-jnps) in response to ph stimulus. it was shown that under the guidance of pet, mr and pa multimodal imaging, fe o -au-jnps could effectively carry out ros-mediated cancer treatment. after intravenous injection of [ cu] radiolabeled jnp vesicles, pet imaging showed that the drug vesicles remained stable in the blood, but gradually dissociated in the liver, showing reduced hepatotoxicity. pet imaging modality provided effective real-time monitoring of the whole treatment process [ ] . computed tomography (ct) collects information of anatomical structures via x-ray scans and cross-sectional images are reconstructed by computer operation. due to its penetration capability and fast scanning speed, ct is one of the most widely used clinical imaging methods. it is also usually used in combination with mri, pa and other imaging modalities to achieve better imaging results. for example, as coronavirus disease (covid- ) is spreading worldwide, ct has served as an important imaging tool for the assessment of covid- . ct scans can spot abnormalities of lungs such as multiple opacities (denser, more profuse and confluent) [ ] . in other applications such as cancer diagnosis and treatment, ct also plays a significant role and is mostly used in combination with other imaging approaches. for example, yu et al. recently developed bismuth nano-raspberries (bi-bsa nrs) modified by bsa. bi-bsa nrs have high drug loading efficiency (~ %) and can release the cargo in response to ph and nir stimuli. the efficiency of ct contrast agent is improved by infrared thermal and pa. % tumor treatment rate was achieved by chemo-photothermal therapy. in conclusion, this bi-bsa nrs have potential for the application of multimodality imaging and combination therapy [ ] (figure f ). in summary, nanotechnology-based therapeutics have developed rapidly in recent years, and a wide variety of novel strategies have been explored. particularly, stimulus-responsive nano-systems warrant attention as they show promise in targeting and controlled release in response to endogenous or exogenous stimuli. owing to their unique advantages, stimulus-responsive systems have been widely investigated as a strategy well-suited for nanotherapeutics. furthermore, dual/multiple stimulus-responsive systems have been investigated for more accurate disease diagnosis and management. self-immolative linkers with different cleavage and self-immolation rates hold potential as a versatile technology to address controlled drug release. stimulus-responsive systems have shown promise for the treatment of various cancers, ibd, neuroinflammation, gastrointestinal diseases, bio-imaging and many others. however, the translational potential, especially for the highly complex chemically modified systems, is a concern. drug-device combinations for external stimuli are more complicated than drug-alone approaches from regulatory perspectives. for example, for cancer treatment, stimulus-responsive systems treating only local tumors would require one to find advantages over the well-established modalities of surgery, radiotherapy and other ablation approaches, which do not have the complications of drugs. in addition, toxicity of not only the api, but also the carrier system, is another factor for consideration. nevertheless, with smart and rational design, stimulus-responsive nanotechnology adds a new weapon to drug delivery and molecular imaging arsenal, and warrants further research and development. funding: this work is supported by the start-up grant at tianjin university (y.z.) and one-thousand young talent program (y.z.). the authors declare no conflict of interest. stimuli-responsive nanomaterials for biomedical applications remotely triggerable drug delivery systems external triggering and triggered targeting strategies for drug delivery overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors bacteria-mediated tumor therapy utilizing photothermally-controlled tnf-α expression via oral administration emerging frontiers in drug delivery collapse of gels and critical endpoint design of liposomes for enhanced local release of drugs by hyperthermia ph-responsive nanoparticles for drug delivery integrated nanoparticles to synergistically elevate tumor oxidative stress and suppress antioxidative capability for amplified oxidation therapy targeted mmp- responsive chimeric polymersomes for therapy against colorectal cancer development of a theranostic prodrug for colon cancer therapy by combining ligand-targeted delivery and enzyme-stimulated activation stimuli-responsive polymeric prodrug-based nanomedicine delivering nifuroxazide and doxorubicin against primary breast cancer and pulmonary metastasis stimuli-responsive mesoporous silica nps as non-viral dual sirna/chemotherapy carriers for triple negative breast cancer telomerase responsive delivery of doxorubicin from mesoporous silica nanoparticles in multiple malignancies: therapeutic efficacies against experimental aggressive murine lymphoma doxorubicin loaded ph responsive biodegradable aba-type amphiphilic peg-b-aliphatic polyketal-b-peg block copolymer for therapy against aggressive murine lymphoma bifunctional succinylated ε-polylysine-coated mesoporous silica nanoparticles for ph-responsive and intracellular drug delivery targeting the colon rational design of polyphenol-poloxamer nanovesicles for targeting inflammatory bowel disease therapy rational design of a stimuli-responsive polymer electrode interface coupled with in vivo microdialysis for measurement of sialic acid in live mouse brain in alzheimer's disease glucose-and h o -responsive polymeric vesicles integrated with microneedle patches for glucose-sensitive transcutaneous delivery of insulin in diabetic rats functionalized gold nanoparticle-polypyrrole nanobiocomposite with high effective surface area for electrochemical/ph dual stimuli-responsive smart release of insulin lyso-thermosensitive liposomal doxorubicin: an adjuvant to increase the cure rate of radiofrequency ablation in liver cancer in vitro and in vivo metabolism of paclitaxel poliglumex: identification of metabolites and active proteases efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme feasibility study of particle-assisted laser ablation of brain tumors in orthotopic canine model ph-sensitive nano-systems for drug delivery in cancer therapy the relevance of tumour ph to the treatment of malignant disease extracellular ph distribution in human tumours causes and consequences of tumour acidity and implications for treatment biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment targeted delivery of small and macromolecular drugs stimuli-responsive nanomedicines for overcoming cancer multidrug resistance a. ph-dependent equilibrium swelling properties of hydrophobic polyelectrolyte copolymer gels ph-sensitive dissociable nanoscale coordination polymers with drug loading for synergistically enhanced chemoradiotherapy synthesis of a triple-responsive double hydrophilic block copolymer prodrug using a reducible raft-atrp double-head agent probe-inspired nano-prodrug with dual-color fluorogenic property reveals spatiotemporal drug release in living cells hollow chitosan-silica nanospheres as ph-sensitive targeted delivery carriers in breast cancer therapy micelles based on acid degradable poly(acetal urethane): preparation, ph-sensitivity, and triggered intracellular drug release acetal-functionalized pillar ( )arene: a ph-responsive and versatile nanomaterial for the delivery of chemotherapeutic agents doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages synthesis of highly ph-responsive glucose poly(orthoester) facile fabrication of double-walled polymeric hollow spheres with independent temperature and ph dual-responsiveness for synergetic drug delivery incorporation and controlled release of silyl ether prodrugs from print nanoparticles acetal-linked pegylated paclitaxel prodrugs forming free-paclitaxel-loaded ph-responsive micelles with high drug loading capacity and improved drug delivery micromotors spontaneously neutralize gastric acid for ph-responsive payload release a transistor-like ph nanoprobe for tumour detection and image-guided surgery multistimuli-responsive pegylated polymeric bioconjugate-based nano-aggregate for cancer therapy intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both ph and reduction conditions smart drug delivery systems: from fundamentals to the clinic noninvasive imaging of tumor redox status and its modification by tissue glutathione levels intracellular microenvironment responsive pegylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release glutathione metabolism and its implications for health gradient redox-responsive and two-stage rocket-mimetic drug delivery system for improved tumor accumulation and safe chemotherapy bond energy data summarized enhanced bioreduction-responsive biodegradable diselenide-containing poly(ester urethane) nanocarriers thioether phosphatidylcholine liposomes: a novel ros-responsive platform for drug delivery self-assembled redox dual-responsive prodrug-nanosystem formed by single thioether-bridged paclitaxel-fatty acid conjugate for cancer chemotherapy synthetic applications of organotellurium chemistry γ-ray-responsive supramolecular hydrogel based on a diselenide-containing polymer and a peptide ultra-sensitive ros-responsive tellurium-containing polymers near-infrared light stimuli-responsive synergistic therapy nanoplatforms based on the coordination of tellurium-containing block polymer and cisplatin for cancer treatment hydrogen peroxide-responsive micelles self-assembled from a peroxalate ester-containing triblock copolymer in vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles visible light-induced singlet oxygen-mediated intracellular disassembly of polymeric micelles co-loaded with a photosensitizer and an anticancer drug for enhanced photodynamic therapy nanoparticles formed from rgd-epothilone b conjugate for targeted cancer therapy synthesis of a phenylboronic ester-linked peg-lipid conjugate for ros-responsive drug delivery glutathione-responsive nanoscale mofs for effective intracellular delivery of the anticancer drug -mercaptopurine stimuli-responsive nanocarriers for drug delivery the smart drug delivery system and its clinical potential proline isomerization-regulated tumor microenvironment-adaptable self-assembly of peptides for enhanced therapeutic efficacy ros-cleavable proline oligomer crosslinking of polycaprolactone for pro-angiogenic host response orally delivered thioketal nanoparticles loaded with tnf-α-sirna target inflammation and inhibit gene expression in the intestines ros-responsive polymeric nanocarriers with photoinduced exposure of cell-penetrating moieties for specific intracellular drug delivery enzyme-responsive nanoparticle systems proteases: multifunctional enzymes in life and disease enzyme-responsive hydrogel microparticles for pulmonary drug delivery polymer-directed enzyme prodrug therapy. i. hpma copolymer-cathepsin b and pk as a model combination cascade-promoted photo-chemotherapy against resistant cancers by enzyme-responsive polyprodrug nanoplatforms matrix metalloprotease -responsive multifunctional liposomal nanocarrier for enhanced tumor targeting an mmp- responsive liposome integrating antifibrosis and chemotherapeutic drugs for enhanced drug perfusion and efficacy in pancreatic cancer mmp- -sensitive ha end-conjugated poly(amidoamine) dendrimers via click reaction to enhance drug penetration into solid tumor smart asymmetric vesicles with triggered availability of inner cell-penetrating shells for specific intracellular drug delivery peg-sheddable polyplex micelles as smart gene carriers based on mmp-cleavable peptide-linked block copolymers modular design and facile synthesis of enzyme-responsive peptide-linked block copolymers for efficient delivery of doxorubicin matrix metalloproteinase-responsive pegylated lipid nanoparticles for controlled drug delivery in the treatment of rheumatoid arthritis phospholipase a enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention expression of group iia secretory phospholipase a increases with prostate tumor grade overexpression of group ii phospholipase a in human breast cancer tissues is closely associated with their malignant potency presence of secretory group iia and v phospholipase a and cytosolic group ivα phospholipase a in chondrocytes from patients with rheumatoid arthritis phospholipase a s in cell injury and death phospholipidic colchicinoids as promising prodrugs incorporated into enzyme-responsive liposomes: chemical, biophysical, and enzymological aspects phospholipase a -susceptible liposomes of anticancer double lipid-prodrugs a new glucose oxidase fromaspergillus niger: characterization and regulation studies of enzyme and gene kinetics of glucose oxidase excretion by recombinant aspergillus niger glucose-responsive nanoparticles for rapid and extended self-regulated insulin delivery erythrocyte-membrane-camouflaged nanoplatform for intravenous glucose-responsive insulin delivery glucose-responsive metal-organic-framework nanoparticles act as "smart" sense-and-treat carriers nad(p)h:quinone oxidoreductase (dt-diaphorase) expression in normal and tumor tissues efficacy of beta-lapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target nqo cytosolic nqo enzyme-activated near-infrared fluorescence imaging and photodynamic therapy with polymeric vesicles a unique case of breast carcinoma producing pancreatic-type isoamylase glycosidase activated release of fluorescent , -naphthalimide probes for tumor cell imaging from glycosylated 'pro-probes' factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles functional nanomaterials for phototherapies of cancer light-activated ros-responsive nanoplatform codelivering apatinib and doxorubicin for enhanced chemo-photodynamic therapy of multidrug-resistant tumors applications of light-responsive systems for cancer theranostics biologically active molecules with a "light switch porphyrin-phospholipid liposomes permeabilized by near-infrared light tunable luminescent lanthanide supramolecular assembly based on photoreaction of anthracene a self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery photocontrolled nuclear-targeted drug delivery by single component photoresponsive fluorescent organic nanoparticles of acridin- -methanol photoinduced reversible worm-to-vesicle transformation of azo-containing block copolymer assemblies prepared by polymerization-induced self-assembly light-responsive and ph-responsive dna microcapsules for controlled release of loads photolabile linkers: exploiting labile bond chemistry to control mode and rate of hydrogel degradation and protein release a photochemical approach for controlled drug release in targeted drug delivery anti-stokes shift luminescent materials for bio-applications upconversion and anti-stokes processes with f and d ions in solids copolymer upconversion nanocomposites for triggered drug release in vitro and in vivo light-controlled tools a dna-azobenzene nanopump fueled by upconversion luminescence for controllable intracellular drug release photo, ph and redox multi-responsive nanogels for drug delivery and fluorescence cell imaging diarylethenes for memories and switches reversible photoswitching of triplet-triplet annihilation upconversion using dithienylethene photochromic switches synthesis and comparative photoswitching studies of unsymmetrical , -diarylcyclopent- -en- -ones photochromic properties of terarylene derivatives having a π-conjugation unit on central aromatic ring photoswitchable organocatalysis: using light to modulate the catalytic activities of n-heterocyclic carbenes reporting the release of caged species by a combination of two sequential photoreactions, a molecular switch, and one color of light a contribution to the design of molecular switches: novel acid-mediated ring-closing−photochemical ring-opening of , -bis(heteroaryl)quinones (heteroaryl = thienyl, furanyl, pyrrolyl) dual-mode fluorescence switching of photochromic bisthiazolylcoumarin synthesis and photochromism of a novel diarylethene compound containing terminal benzothiazole unit light-responsive platform for launching therapeutic agents on command drug delivery via cell-conveyed phototherapeutics photoactivation of anticancer ru complexes in deep tissue: how deep can we go? chem. a eur temperature-responsive smart nanocarriers for delivery of therapeutic agents: applications and recent advances multifunctional up-converting nanocomposites with smart polymer brushes gated mesopores for cell imaging and thermo/ph dual-responsive drug controlled release release kinetics of benzoic acid and its sodium salt from a series of poly(n-isopropylacrylamide) matrices with various percentage crosslinking the targeted behavior of thermally responsive nanohydrogel evaluated by nir system in mouse model thermoresponsive poly(n-vinyl caprolactam)-coated gold nanoparticles: sharp reversible response and easy tunability dual-stimuli-responsive paclitaxel delivery nanosystems from chemically conjugate self-assemblies for carcinoma treatment enzyme and thermal dual responsive amphiphilic polymer core-shell nanoparticle for doxorubicin delivery to cancer cells thermosensitive liposomes modified with poly(n-isopropylacrylamide-co-propylacrylic acid) copolymers for triggered release of doxorubicin magnetism in medicine thermoresponsive polymer brush-functionalized magnetic manganite nanoparticles for remotely triggered drug release controlled release of doxorubicin loaded within magnetic thermo-responsive nanocarriers under magnetic and thermal actuation in a microfluidic channel magnetically stimulated drug release using nanoparticles capped by self-assembling peptides magnetic microspheres: a model system for site specific drug delivery in vivo improved method of recombinant aav delivery for systemic targeted gene therapy magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo acoustic cavitation: a possible consequence of biomedical uses of ultrasound microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications lipid/plga hybrid microbubbles as a versatile platform for noninvasive image-guided targeted drug delivery in situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging ultrasound-responsive conversion of microbubbles to nanoparticles to enable background-free in vivo photoacoustic imaging short and long term efficacy of high intensity focused ultrasound therapy for advanced hepatocellular carcinoma extracorporeal high intensity focused ultrasound ablation in the treatment of patients with solid carcinomas in china: an overview a novel connector linkage applicable in prodrug design polymeric persulfide prodrugs: mitigating oxidative stress through controlled delivery of reactive sulfur species alkylamine-substituted perthiocarbamates: dual precursors to hydropersulfide and carbonyl sulfide with cardioprotective actions development of a cysteine-conjugatable disulfide fret probe: influence of charge on linker cleavage and payload trafficking for an anti-her antibody conjugate small molecule as fluorescent probes for monitoring intracellular enzymatic transformations engineered ph-responsive mesoporous carbon nanoparticles for drug delivery reactive oxygen species (ros)-responsive polymersomes with site-specific chemotherapeutic delivery into tumors via spacer design chemistry rational design of latent fluorophores from water-soluble hydroxyphenyltriazine dyes suitable for lipase sensing disassembly kinetics of quinone-methide-based self-immolative spacers that contain aromatic nitrogen heterocycles supramolecular hydrogels for enzymatically triggered self-immolative drug delivery quinone-methide species, a gateway to functional molecular systems: from self-immolative dendrimers to long-wavelength fluorescent dyes development of a drug-release strategy based on the reductive fragmentation of benzyl carbamate disulfides synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery real-time monitoring of drug release single-triggered ab self-immolative dendritic amplifiers elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release prodrug strategies based on intramolecular cyclization reactions oxidation degradable aliphatic polycarbonates with pendent phenylboronic ester syntheses and kinetic studies of cyclisation-based self-immolative spacers programmable microcapsules from self-immolative polymers self-immolative linkers as caps for the design of gated silica mesoporous supports intramolecular cyclization for stimuli-controlled depolymerization of polycaprolactone particles leading to disassembly and payload release use of dithiasuccinoyl-caged amines enables cos/h s release lacking electrophilic byproducts -bis(hydroxymethyl)aniline as a building block for oligomers with self-eliminating and multiple release properties convergent synthesis of geometrically disassembling dendrimers using cu(i)-catalyzed c−o bond formation disulfide-based self-immolative linkers and functional bioconjugates for biological applications dual-responsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species exponential molecular amplification by h o -mediated autocatalytic deprotection of boronic ester probes to redox cyclers effects of electronics, aromaticity, and solvent polarity on the rate of azaquinone-methide-mediated depolymerization of aromatic carbamate oligomers synthesis and evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis stabilizing p-dithiobenzyl urethane linkers without rate-limiting self-immolation for traceless drug release design and synthesis of new protease-triggered co-releasing peptide-metal-complex conjugates self-immolative polymers activity-based optical sensing enabled by self-immolative scaffolds: monitoring of release events by fluorescence or chemiluminescence output a gadolinium chelate for detection of β-glucuronidase: a self-immolative approach synthesis and antitumor properties of bqc-glucuronide, a camptothecin prodrug for selective tumor activation benzyl ether-linked glucuronide derivative of -hydroxycamptothecin designed for selective camptothecin-based anticancer therapy aryl sulfate is a useful motif for conjugating and releasing phenolic molecules: sulfur fluorine exchange click chemistry enables discovery of ortho-hydroxy-protected aryl sulfate linker an improved fluorogenic substrate for the detection of alkaline phosphatase activity synthesis, and pharmacokinetic evaluation of phosphate and amino acid ester prodrugs for improving the oral bioavailability of the hiv- protease inhibitor atazanavir light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers sterically-controlled self-immolation in phosphoramidate linkers triggered by light single uv or near ir triggering event leads to polymer degradation into small molecules multi-stimuli-responsive self-immolative polymer assemblies red and near-infrared light-cleavable polymers increasing materials' response to two-photon nir light via self-immolative dendritic scaffolds ph-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor ph and thermo dual-stimuli-responsive drug carrier based on mesoporous silica nanoparticles encapsulated in a copolymer-lipid bilayer facile construction of i-motif dna-conjugated gold nanostars as near-infrared and ph dual-responsive targeted drug delivery systems for combined cancer therapy light-triggered reversible assemblies of azobenzene-containing amphiphilic copolymer with β-cyclodextrin-modified hollow mesoporous silica nanoparticles for controlled drug release light-triggered specific cancer cell release from cyclodextrin/azobenzene and aptamer-modified substrate controlled drug release from cyclodextrin-gated mesoporous silica nanoparticles based on switchable host-guest interactions dual stimuli-responsive supramolecular self-assemblies based on the host-guest interaction between β-cyclodextrin and azobenzene for cellular drug release dual ph/redox-responsive mixed polymeric micelles for anticancer drug delivery and controlled release reactive oxygen species synergistic ph/h o -responsive poly(l-lactic acid)-block-poly(sodium -styrenesulfonate)/citrate-fe(iii)@zif- hybrid nanocomposites for controlled drug release intracellular enzyme-triggered assembly of amino acid-modified gold nanoparticles for accurate cancer therapy with multimode a smart drug delivery system responsive to ph/enzyme stimuli based on hydrophobic modified sodium alginate enzyme/ph dual-responsive polymer prodrug nanoparticles based on -hydroxycamptothecin-carboxymethylchitosan for enhanced drug stability and anticancer efficacy glutathione and reactive oxygen species dual-responsive block copolymer prodrugs for boosting tumor site-specific drug release and enhanced antitumor efficacy smart unimolecular micelle-based polyprodrug with dual-redox stimuli response for tumor microenvironment: enhanced in vivo delivery efficiency and tumor penetration gsh and light dual stimuli-responsive supramolecular polymer drug carriers for cancer therapy dual-stimuli-responsive microparticles ir- dye loaded tumor targeting theranostic nanoparticles for nir imaging and photothermal therapy sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye nir-triggered multifunctional and degradable nanoplatform based on an ros-sensitive block copolymer for imaging-guided chemo-phototherapy photoactivatable organic semiconducting pro-nanoenzymes dual stimuli-guided lipid-based delivery system of cancer combination therapy non-magnetic injectable implant for magnetic field-driven thermochemotherapy and dual stimuli-responsive drug delivery: transformable liquid metal hybrid platform for cancer theranostics multi-stimuli responsive mesoporous carbon nano-platform gated by human serum albumin for cancer thermo-chemotherapy reduction/temperature/ph multi-stimuli responsive core cross-linked polypeptide hybrid micelles for triggered and intracellular drug release synthesis of temperature, ph, light and dual-redox quintuple-stimuli-responsive shell-crosslinked polymeric nanoparticles for controlled release reactive oxygen species in inflammation and tissue injury inflammation responsive logic gate nanoparticles for the delivery of proteins a dual ph/redox responsive copper-ligand nanoliposome bioactive complex for the treatment of chronic inflammation a ph/ros dual-responsive and targeting nanotherapy for vascular inflammatory diseases inflammation-responsive drug-conjugated dextran nanoparticles enhance anti-inflammatory drug efficacy responsive polyelectrolyte complexes for triggered release of nucleic acid therapeutics dual thermo-and ph-sensitive network-grafted hydrogels formed by macrocrosslinker as drug delivery system thermo and ph dual actuating smart porous anodic aluminum for controllable drug release injectable microbeads with a thermo-responsive shell and a ph-responsive core as a dual-switch-controlled release system inflammatory bowel disease bioresponsive drug delivery systems in intestinal inflammation: state-of-the-art and future perspectives reactive oxygen species responsive nanoplatforms as smart drug delivery systems for gastrointestinal tract targeting ph and reactive oxygen species-sequential responsive nano-in-micro composite for targeted therapy of inflammatory bowel disease nanoparticulate drug delivery systems targeting inflammation for treatment of inflammatory bowel disease drug delivery strategies in the therapy of inflammatory bowel disease short-term adverse effects of -aminosalicylic acid agents in the treatment of ulcerative colitis inflammation in neurological disorders: a help or a hindrance? neuroscience van der valk, p. inflammation in neurodegenerative diseases role of immunity and inflammation in the pathophysiology of neurodegenerative diseases dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic dna damage in huntington disease positively charged polyprodrug amphiphiles with enhanced drug loading and reactive oxygen species-responsive release ability for traceable synergistic therapy core-cross-linked nanoparticles reduce neuroinflammation and improve outcome in a mouse model of traumatic brain injury a review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules insulin in hospital and home dual stimuli-responsive nanoparticle-incorporated hydrogels as an oral insulin carrier for intestine-targeted delivery and enhanced paracellular permeation development of ph-responsive chitosan/heparin nanoparticles for stomach-specific anti-helicobacter pylori therapy oral delivery of ph-responsive alginate microbeads incorporating folic acid-grafted solid lipid nanoparticles exhibits enhanced targeting effect against colorectal cancer: a dual-targeted approach in vitro evaluation of ph-responsive nanoscale hydrogels for the oral delivery of hydrophobic therapeutics ph-responsive lignin-based nanomicelles for oral drug delivery oral nanostructured lipid carriers loaded with near-infrared dye for image-guided photothermal therapy a ph-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices amphiphilic semiconducting polymer as multifunctional nanocarrier for fluorescence/photoacoustic imaging guided chemo-photothermal therapy dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy nir-ii dye-based multifunctional telechelic glycopolymers for nir-iia fluorescence imaging-guided stimuli-responsive chemo-photothermal combination therapy site-selective in situ growth-induced self-assembly of protein-polymer conjugates into ph-responsive micelles for tumor microenvironment triggered fluorescence imaging dual-enzyme-loaded multifunctional hybrid nanogel system for pathological responsive ultrasound imaging and t -weighted magnetic resonance imaging polyphenol-inspired facile construction of smart assemblies for atp-and ph-responsive tumor mr/optical imaging and photothermal therapy nitric oxide-activated "dual-key-one-lock" nanoprobe for in vivo molecular imaging and high-specificity cancer therapy intelligent mos nanotheranostic for targeted and enzyme-/ph-/nir-responsive drug delivery to overcome cancer chemotherapy resistance guided by pet imaging dual-stimuli responsive bismuth nanoraspberries for multimodal imaging and combined cancer therapy microbubbles in medical imaging: current applications and future directions injectable microbubbles as contrast agents for diagnostic ultrasound imaging: the key role of perfluorochemicals dynamic solid-state ultrasound contrast agent for monitoring ph fluctuations in vivo molecular imaging by mri advances in high-field magnetic resonance imaging triple stimuli-responsive magnetic hollow porous carbon-based nanodrug delivery system for magnetic resonance imaging-guided synergistic photothermal/chemotherapy of cancer hp-β-cd functionalized fe o /cnps-based theranostic nanoplatform for ph/nir responsive drug release and mr/nirfl imaging-guided synergetic chemo/photothermal therapy of tumor noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain fluoro-photoacoustic polymeric renal reporter for real-time dual imaging of acute kidney injury the merging of biology and imaging into molecular imaging positron emission tomography basic science of pet and pet/ct self-assembled responsive bilayered vesicles with adjustable oxidative stress for enhanced cancer imaging and therapy correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -x f hn f authors: tzelepis, ilias; kapsetaki, stefania-elisavet; panayidou, stavria; apidianakis, yiorgos title: drosophila melanogaster: a first step and a stepping-stone to anti-infectives date: - - journal: curr opin pharmacol doi: . /j.coph. . . sha: doc_id: cord_uid: x f hn f following an expansion in the antibiotic drug discovery in the previous century, we now face a bottleneck in the production of new anti-infective drugs. traditionally, chemical libraries are screened either using in vitro culture systems or in silico to identify and chemically modify small molecules with antimicrobial properties. nevertheless, almost all compounds passing through in vitro screening fail to pass preclinical trials. drug screening in drosophila offers to fill the gap between in vitro and mammalian model host testing by eliminating compounds that are toxic or have reduced bioavailability and by identifying others that may boost innate host defence or selectively reduce microbial virulence in a whole-organism setting. such alternative screening methods in drosophila, while low-throughput, may reduce the cost and increase the success rate of preclinical trials. drosophila melanogaster: a first step and a stepping-stone to anti-infectives ilias tzelepis , stefania-elisavet kapsetaki , stavria panayidou and yiorgos apidianakis following an expansion in the antibiotic drug discovery in the previous century, we now face a bottleneck in the production of new anti-infective drugs. traditionally, chemical libraries are screened either using in vitro culture systems or in silico to identify and chemically modify small molecules with antimicrobial properties. nevertheless, almost all compounds passing through in vitro screening fail to pass preclinical trials. drug screening in drosophila offers to fill the gap between in vitro and mammalian model host testing by eliminating compounds that are toxic or have reduced bioavailability and by identifying others that may boost innate host defence or selectively reduce microbial virulence in a whole-organism setting. such alternative screening methods in drosophila, while low-throughput, may reduce the cost and increase the success rate of preclinical trials. a common countermeasure to the ever-growing antibiotic drug resistance is the production of new effective drugs. nevertheless, the rate of production of new antibiotics is steadily declining [ ] . one reason for this might be the chemical screening methods that rely solely on in vitro culture systems. traditionally, drug research is moving from in vitro small molecule screens to preclinical assessment in mammalian hosts. there are two problems with this approach: first, tests in mammals are costly and can usually be restricted to a few compounds at a time, and second, in vitro assays are inappropriate to capture the complexity of an infected host [ ] . live hosts are preferable because they enable drug toxicity and bioavailability assessment at the organismal level [ ] and [ ] . in addition, drugs that might interfere with the host microenvironment or microbial virulence per se can only be assessed upon the interaction of microbes with a host. thus, quality antiinfective drug assessment in simple model hosts might be a more effective way to identify new drug leads. in this review, we aim to examine the suitability of drosophila melanogaster as a model organism for anti-infective drug assessment due to its high degree of molecular, cellular and physiological conservation with humans, which allows the modelling of infections that recapitulate aspects of human disease [ , ] . in this respect, drosophila might fill the gap between in vitro screens and preclinical trials or be used directly, instead of in vitro screens. drosophila has a short life cycle of days from egg to sexually mature adult as compared to the . months of mice (table ) . large numbers of flies can be propagated quickly, since tens of females can produce hundreds of offspring within two weeks. the offspring become sexually mature very early in their adult life, enabling the life cycle to continue [ ] . due to its small size of mm in length thousands of flies can be contained in a space that would normally fit less than mice. in addition, fly food is usually made of grocery store ingredients such as cornmeal, yeast and sucrose, thus the cost of maintenance is quite low. moreover, there are no ethical concerns or regulated protocols for its use in biomedical research. as an advantage over caenorhabditis elegans, a popular invertebrate model host, drugs can not only be mixed in the fly food but also administered by injection (table ) . precise doses of - nl of drug solutions can customarily be injected in each fly [ ] and less than ml on a paper disc suffice to feed flies for hours [ ] . hence, only small quantities of drugs are required during experiments; yet another reason why drug tests in flies are not expensive. in addition, drosophila can be used for toxicological studies because the relative toxicity of chemicals in flies correlates well with that in mammals [ ] . finally, drosophila infection and inflammation can easily be studied in relation to aging overcoming the barrier of long experimental time [ ] . this is because drosophila maximum life span ranges between and days, with day of the fly roughly corresponding to year of humans. that is, flies exhibit aging effects as early as days post the onset of adulthood. drosophila has a long history as a model organism for genetics and a significant similarity with humans in terms of gene homologs. it has functional homologs for % of human disease related genes [ ] , more than any other invertebrate model host studied today (table and [ ] ). its genome is fully sequenced and is one of the bestannotated among eukaryotes. thus, many technologies have been developed and techniques are easily and commonly used, such as transgenesis, rna interference (rnai) technology and gene microarrays. doublestranded rnas have been synthesized for almost all genes and the tools are commercially available for the conditional inactivation of essentially any gene of interest in vivo or in cell culture [ ] . for example, drosophila cells have been used in genome-wide rnai screens to rapidly identify genes required for replication of influenza and dengue viruses [ , ] . furthermore, there are large collections of mutants and transgenic drosophila stocks maintained at bloomington and other stock centers around the world (http://flybase.org). moreover, the drosophila genome contains fewer genes than humans, and consequently, presents less overall genetic redundancy. this allows for an easier target identification, although multiple or modified drugs might be needed in mammals to affect the multiple gene variants. finally, a variety of genetic tools and markers are available today in order to study the role of microbial pathogenicity tissue-specifically using the gal /uas system [ ] . this is an advantage over other model hosts, because expression of any drosophila gene can be controlled time and tissuespecifically (table ) . for example, tissue-specific and temporal rnai allowed the identification of the jak/ stat signalling pathway as a regulator of the intestinal immune response and regeneration in the fruit fly [ ] . in addition, intestinal damage and regeneration can be studied by flip-out clones of cells emanating from intestinal stem cells [ , ] , as well as mitotic clones using either the b-galactosidase marker or the ''mosaic analysis with a repressible cell marker'' method [ ] . drosophila physiology and the immune system -conservation and significance for mammalian research several organs and specific cells fundamental to the immune response are highly conserved between flies and mammals. this is the most significant advantage over all other invertebrate model hosts studied today (table ) . flies have a defined brain that interacts with other organs, for example, the fat body and the intestine via cytokines and insulin peptides, respectively [ , ] . the fat body is the equivalent of the mammalian liver, an innate immunity and a metabolic organ [ , ] . the fly intestine bears many similarities with that of mammals in terms of cellular and molecular biology and epithelial architecture [ ] . plasmatocytes are the macrophage-like cells of drosophila that detect and phagocytose microbes and secrete cytokines and antimicrobial peptides [ ] . the muscle cells of the drosophila flight muscle, heart and intestine are stratified or smooth similarly to those of humans and share a role in host response to infection [ , ] . the drosophila trachea is an air-transporting organ with similarities to the human vasculature [ ] . finally, the nephrocytes and the malpighian tubules are kidney-like cells with a role in host defence [ , ] . the drosophila epithelia that are attached to the cuticle, as well as those of the intestine and the trachea are physical barriers to pathogen invasion and the first to respond to external microbes. should microbes invade anti-infectives these epithelia other local tissues, such as the drosophila flight muscle, respond to wound infection eliciting a localized host defence response orchestrated by the highly conserved jnk pathway [ ] . importantly, muscle responses to wound infection appear to be conserved in mice and in humans [ , ] . on the other hand, when bacteria enter and damage the intestine, they induce enterocyte regeneration, which serves as a defence response to protect the host [ ] . numerous conserved signalling pathways are involved in intestinal regeneration upon infection, including the wnt/wg, notch, hippo, jnk, insr/inr, k-ras/ras , jak-stat and the nf-kb pathways [ ] . in case microbes pass through intestinal or other barrier epithelia, additional mechanisms of protection take place. these include phagocytosis by the plasmatocytes, which are analogous to the mammalian macrophages, and the production of antimicrobial peptides by the fat body [ ] . many bacteria and fungi induce the toll and/or the immune deficiency (imd) pathways, which are the two highly conserved nf-kb pathways of the systemic drosophila immune response [ ] . viral infections elicit systemic immune responses via the universally conserved rnai mechanism. the drosophila small interfering rna pathway is activated by doublestranded viral rna or dna [ ] . moreover, dexd/h box helicases, cell autophagy as well as the conserved jak/stat, imd/tnf and/or the toll/tlr innate immune cascades play a crucial role in responding to viral rna in flies and mammals [ ] . many human bacterial pathogens have been studied in drosophila including the gram-positive bacteria enterococcus faecalis, staphylococcus aureus, steptococcus pneumoniae, bacillus cereus and listeria monocytogenes, and the gram-negative bacteria vibrio cholerae, serratia marcescens, pseudomonas aeruginosa, salmonella typhimurium, chlamydia spp., burkholderia cepacia, yersinia pseudotuberculosis, francisella tularensis, legionella pneumophila and mycobacterium marinum [ ] . of those, p. aeruginosa and m. marinum may suppress the innate immune response as part of their virulence repertoire [ , ] . interestingly, the antibiotics rifampicin, dinitrobenzamide, amikacin and isoniazid show good bioavailability, because when fed to the flies they alleviate systemic m. marinum infection. of special note, the success of the antituberculosis drugs isoniazid and pyrazinamide against the tuberculosis model microbe m. marinum is facilitated by a boost in host cell autophagy in flies and mammals [ ] . these data suggest that not only direct antibacterial efficacy but also innate immune induction share similarities between flies and mammals and can be exploited for pharmacological assessments in flies. intestinal p. aeruginosa induces damage and apoptosis of midgut enterocytes in drosophila, which in turn induces intestinal stem cell proliferation, a process that is however reversible upon bacteria clearance by the common food preservatives methyl paraben and propionic acid [ ] . strikingly, k-ras/ras oncogene expressing drosophila hindgut cells induce tumors and delaminate through the basal side of the epithelium upon p. aeruginosa infection, which is an additional process that can be inhibited by eliminating infection using food preservatives [ , ] . furthermore, -aminoacetophenone, a small chemical produced by p. aeruginosa, has been shown to reduce p. aeruginosa virulence in drosophila and mice [ ] . finally, researchers have exploited phages as anti-infectives against p. aeruginosa using drosophila. fruit flies infected with p. aeruginosa can be treated with bacteriophages mpk , mpk by feeding [ , ] . such findings encourage future assessment of food preservatives and natural or biological products, including bacterial metabolites and bacteriophages, as anti-infectives. apart from bacteria, human fungal pathogens can also inflict disease in flies. candida albicans, aspergillus fumigatus, aspergillus hyphae, cryptococcus neoformans, cunninghamella berthollethiae, scedosporium spp. and fusarium spp. have been studied in flies [ , ] . of those, the zygomycete c. berthollethiae has been meticulously studied in combination with chemical modifiers of iron in drosophila. enhancers of zygomycetes virulence traditionally used in humans, such as corticosteroids, increase iron supply, and iron availability through treatment with deferoxamine dramatically increases pathogenicity by zygomycetes. accordingly, iron starvation induced by treatment with the iron chelator deferasirox significantly protects infected flies [ ] . another common antifungal, voriconazole is potent against f. moniliforme and s. apiopermum infection in flies [ ] . moreover, combinatorial drug assessment assays in drosophila reveal a synergism between voriconazole and terbinafine against aspergillus fumigatus, similar to that seen in mammals [ ] . recently, another synergy was shown between tarcolimus and posaconazone in flies and mice against ryzopus oryzae [ ] . because all of the aforementioned treatments were administered by feeding in flies, while infections were either superficial or systemic, many antifungal drugs appropriate for humans may have good bioavailability and efficacy in flies. [ ] . the last three of those have also been studied in adult flies, thus allowing the assessment of treatments against them in a whole organism setting. pertinent to the identification of gene target against these viruses, drosophila nramp and its human homologue nramp have been identified as necessary for the entry of sindbis virus into the host cells [ ] . in addition, drosophila toll- has been identified similar to its mammalian ortholog tlr- as important for host defence to infection against vesicular stomatitis virus via the induction of cell autophagy [ ] . finally, west nile virus 'untranslated region-derived rna molecule, known as subgenomic flavivirus rna, suppresses the sirnainduced and mirna-induced rnai pathways in both mammalian and insect cells [ ] , indicating that rnaibased therapies might be a goal for the near future against insect-borne flaviviruses. despite the numerous advantages of d. melanogaster as a model organism for the study of anti-infectives, there are also several shortcomings. that flies are infected and maintained at a temperature of - c can be a problem for the study of pathogens and virulence factors that require the mammalian body temperature, that is, c [ ] . also, its inability to simulate human intestinal anaerobic microflora can be a disadvantage. while microaerophilic and aerotolerant bacteria might be used to infect flies, the presence of oxygen in the fly intestine prohibits fly infections with strict anaerobes, which are plentiful in the human gut [ ] . nevertheless, as with any microbe that is difficult to establish an infection with, specific virulence factors can be expressed or administered to flies to study their virulence. moreover, pharmacokinetic analyses are still problematic in insects as there is not a precise method to measure the levels of administered drug tissue-specifically and insect xenobiotic metabolism might be very different from that of mammals. furthermore, as opposed to mammals, drosophila lacks an adaptive immune system and specialized immune response cells, such as dendritic cells (dc), b and t lymphocytes, which are responsible for immunological specificity and memory [ ] . in addition, despite the significant conservation of the core of drosophila signalling pathways, some of them might be activated differently between flies and mammals. for example, the mammalian toll/tlr pathway that is directly activated by microbially associated molecular patterns, while the drosophila toll is activated indirectly through a cascade of proteases [ ] and the mammalian tlr- that is localized in intracellular membranes versus the plasma membrane-localized drosophila toll- [ ]. finally, high-throughput screening for anti-infectives has not been developed in drosophila and this is its major drawback as compared to other invertebrate hosts (table ) . in recent years, the conventional methods used in most pharmacological studies for the discovery of new therapeutic drugs are based either on screening of small molecule libraries for the capacity to induce a specific phenotype in vitro or in silico [ , ] . however, the efficacy of these methods is very low, because they lack the complex and dynamic host-pathogen interactions, which occur in vivo. consequently, the use of mammalian hosts in such studies is needed and seems to be very widespread and prevalent nowadays. even so, using a conventional animal model for this purpose can be time-consuming, laborious and expensive, not to mention the ethical concerns. exploiting alternative strategies, d. melanogaster is a very promising and useful host, which may cover this gap between the computational or cellular testing studies and the tests in mammals (figure ). while low-throughput drug assessment in drosophila has been proven meaningful, large-scale assessments might also be possible on the basis of protocols used for the identification of molecules that modify disease progression in fragile x syndrome though a screen of compounds in fmr -mutant flies [ ] and a screen of small molecules that identified reserpine as a sleep regulator [ ] . in addition, the fly can be used to assess drugs already approved for human use ( figure ). indeed, the efficacy of a number of licensed anti-infective agents has been evaluated in drosophila, demonstrating a significant correlation in drug efficacy between flies and mammals. therefore, the use of drosophila for anti-infective drug discovery may be a promising auxiliary tool for preclinical research. drosophila can be used either to validate candidate drugs or in combinatorial drug assessment assays to identify synergistic drug combinations. flies have significant similarities with humans enabling a facile and cost effective assessment of anti-infective drugs during the interaction of microbes with a host. hits selected from in vitro or in silico chemical screens can be further screened in drosophila survival or microbial colonization assays to select drug candidates that will have a higher success rate in preclinical trials. in addition, natural products, for example, microbial secondary metabolites and drugs approved in humans can be tested for the fist time combinatorially in flies to identify synergistic effects between two or more chemicals. antibiotics in the clinical pipeline in drosophila melanogaster as a model for human intestinal infection and pathology recombinagenic and mutagenic activities of fluoroquinolones in drosophila melanogaster chemotherapyinduced toxicity is highly heritable in drosophila melanogaster human disease models in drosophila melanogaster and the role of the fly in therapeutic drug discovery drosophila as a genetic model for studying pathogenic human viruses drosophila melanogaster as a model host for studying pseudomonas aeruginosa infection a quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes feeding or injecting a p. aeruginosa metabolite to flies and mice reduces virulence of its own species. an example of how bacterial metabolites from pathogens might help us fight infection with the same pathogens drosophila melanogaster as a model to characterize fungal volatile organic compounds intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in drosophila a systematic analysis of human disease-associated gene sequences in drosophila melanogaster glö ckner g, vilcinskas a: a comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host galleria mellonella new tools for the study of fungal virulence, pharmacology and immunology drosophila rnai screen identifies host genes important for influenza virus replication discovery of insect and human dengue virus host factors genome-wide rnai screen identifies genes involved in intestinal pathogenic bacterial infection synergy between bacterial infection and genetic predisposition in intestinal dysplasia cytokine/jak/stat signaling mediates regeneration and homeostasis in the drosophila midgut drosophila cytokine unpaired regulates physiological homeostasis by remotely controlling insulin secretion infection-induced intestinal oxidative stress triggers organ-to-organ immunological communication in drosophila rä met m: drosophila phagocytosis -still many unknowns under the surface involvement of skeletal muscle gene regulatory network in susceptibility to wound infection following trauma molecular architecture of the fruit fly's airway epithelial immune system bringing together components of the fly renal system immune response and anti-microbial peptides expression in malpighian tubules of drosophila melanogaster is under developmental regulation down-regulation of glutatione s-transferase a (hgsta ) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection regenerative inflammation: lessons from drosophila intestinal epithelium in health and disease the dna virus invertebrate iridescent virus is a target of the drosophila rnai machinery mycobacterium marinum infection in drosophila melanogaster for antimycobacterial activity assessment host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action an elegant demonstration of how two antimycobacterial drugs, apart from their direct antibiotic role, induce a conserved in flies and mammals autophagy-dependent host defence response immune response to bacteria induces dissemination of ras-activated drosophila hindgut cells bacterial clearance or pharmacological inhibition of jnk eliminates innate immune-induced enterocyte invasion ras-oncogenic drosophila hindgut but not midgut cells use an inflammation-like program to disseminate to distant sites antibacterial efficacy of phages against pseudomonas aeruginosa infections in mice and drosophila melanogaster drosophila models of anti-infective drug discovery tzelepis et al. www.sciencedirect.com current opinion in pharmacology antibacterial efficacy of temperate phage-mediated inhibition of bacterial group motilities bacteriophage treatments are simple to perform in flies. their specificity and effectiveness in both flies and mice is a promising way to develop biological anti-infective therapies drosophila melanogaster as a model organism for invasive aspergillosis drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis virulence studies of scedosporium and fusarium species in drosophila melanogaster toll-deficient drosophila flies as a fast, high-throughput model for the study of antifungal drug efficacy against invasive aspergillosis and aspergillus virulence kontoyiannis dp: tacrolimus enhances the potency of posaconazole against rhizopus oryzae in vitro and in an experimental model of mucormycosis in vivo combinatorial drug assays reveal that antifungals may work synergistically and in a similar fashion between flies and mice natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts this put drosophila in an excellent position for screening for pharmacological inhibitors of sindbis virus entry into the host cells noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells a human gut microbial gene catalogue established by metagenomic sequencing drosophila immune priming against pseudomonas aeruginosa is short-lasting and depends on cellular and humoral immunity evolutionary relationships, but functional differences, between the drosophila and human toll-like receptor families pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor screening for small molecule inhibitors of toxoplasma gondii identification of small molecules rescuing fragile x syndrome phenotypes in drosophila small-molecule screen in adult drosophila identifies vmat as a regulator of sleep funding from marie curie (gig-infectioncancer) and fontation sante (yasante ) to ya. key: cord- -y vg frb authors: montané, xavier; kowalczyk, oliwia; reig-vano, belen; bajek, anna; roszkowski, krzysztof; tomczyk, remigiusz; pawliszak, wojciech; giamberini, marta; mocek-płóciniak, agnieszka; tylkowski, bartosz title: current perspectives of the applications of polyphenols and flavonoids in cancer therapy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: y vg frb the development of anticancer therapies that involve natural drugs has undergone exponential growth in recent years. among the natural compounds that produce beneficial effects on human health, polyphenols have shown potential therapeutic applications in cancer due to their protective functions in plants, their use as food additives, and their excellent antioxidant properties. the possibility of combining conventional drugs—which are usually more aggressive than natural compounds—with polyphenols offers very valuable advantages such as the building of more efficient anticancer therapies with less side effects on human health. this review shows a wide range of trials in which polyphenolic compounds play a crucial role as anticancer medicines alone or in combination with other drugs at different stages of cancer: cancer initiation, promotion, and growth or progression. moreover, the future directions in applications of various polyphenols in cancer therapy are emphasized. the appearance of the severe acute respiratory syndrome coronavirus (sars-cov- ) in december last year and its very rapid spread around the world in early , known to cause covid- disease, has evidenced, among other things, the importance of investing in research to improve the people's quality of life or eradicate diseases that still do not have an effective treatment. as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , ′-trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cellsevents that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , -trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cells-events that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . curcuminoids are natural polyphenols that contain two phenol units joined through a linear diarylheptanoid. the presence of curcuminoids gives a yellow color to plants that contain these kinds of natural structures. the phenolic rings of curcuminoids are chemically modified with other chemical groups with the aim of overcoming some drawbacks of natural curcuminoids in clinical applications such as their poor solubility, low absorption, and bioavailability [ ] . among the curcuminoids, curcumin is one of the most known and studied structures with a high potential as medicine to treat different cancers, apart from also being useful in treating other types of diseases. nonetheless, the poor solubility of curcumin in water of acidic and physiological ph requires the use of diverse alternatives to avoid losing the effectiveness of curcumin as a medicine, such as the synthesis of other curcumin derivatives or the combination of curcuminoids with surfactants or co-surfactants. curcumin is a natural compound and the principal curcuminoid of turmeric plants, which is responsible for turmeric's yellow color [ ] . in addition to its applications in medicine, the use of curcumin has reached other fields. in the food industry, it has been used as a dietary supplement (it is sold as herbal supplement) or a food additive. additionally, it is used in cosmetics and other products. curcumin is commonly used in cancer therapies of different types of cancer: lung, cervix, prostate, breast, bone, and liver [ ] . nevertheless, the administration of free curcumin presents some drawbacks: poor solubility in water, instability in aqueous conditions, low bioavailability, and poor cellular uptake. to overcome these problems, two different solutions were attempted: - the synthesis of curcumin derivatives [ ] , and - the encapsulation of curcumin in different nanostructures ranging from liposomes to natural biopolymeric nanoparticles [ , ] . one of the curcumin derivatives used in breast and renal cancer therapies is dimethoxy curcumin. chen et al. recently proved that this curcumin derivative can be effective in the therapy of colon cancer cells due to causing the reduction of survivin expression and the enhancement of e-cadherin, a cell adhesion molecule, whose loss contributes to the formation of epithelial types of cancers such as carcinomas [ ] . recently, various research groups have reported that the combination of both curcumin and resveratrol can reduce the incidence of lung and prostate cancer [ , ] . lignans are diphenolic compounds found in a wide variety of plants including broccoli, beans, soybeans, rye, sesame seeds, pumpkin seeds, flax seeds, and some berries in very small amounts (µg of lignans per g of dry product) [ ] . their structure consists of two c -c units linked by β,β' bonds. lignans are one of the two main groups of phytoestrogens, which are well known for their good antioxidant properties. in fact, some antioxidant phytochemical compounds could be used as anticancer drugs as they are mimicking the functions of human hormones. some studies on rats showed that lignans prevent the growth of breast and prostate tumors [ , ] . numerous lignans could be considered as possible anticancer medicines due to their large pharmacologically valuable properties. among all of them, arctigenin, magnolol, and honokiol are the main lignans investigated in medicine. nonetheless, etoposide is a commercial lignin belonging to the podophilotoxin subfamily that is used in the treatment of different types of cancer such as lung cancer and breast cancer [ , ] . however, etoposide chemotherapy presents several side effects: low blood cell counts, vomiting, diarrhea, fever, loss of appetite, and alopecia. certain plants belonging to the family known as compositae produce arctigenin, especially the seeds of greater burdock (arctium lappa). some studies revealed that arctigenin inhibits the growth of various cancer cells: stomach, lung, liver, and colon, as well as leukocytes [ ] . at the same time, the addition of arctigenin intensifies the activity of caspase- , which is a protein that plays a crucial role in the death of carcinogenic cells. as a matter of fact, huang et al. demonstrated that the treatment of ovcar and skov ovarian cancers with arctigenin causes the apoptosis of cancer cells in vitro [ ] . one of the most used conventional anticancer drugs is doxorubicin, which is a medicine that belongs to the anthracycline family applied in the treatment of, among other cancers, bladder, stomach, ovaries, lung and thyroid cancers. however, doxorubicin exhibits side effects among which the most frequent are severe nauseas, vomiting, and alopecia [ ] . studies were conducted by lee et al. on adding natural products such as arctigenin to doxorubicin and determining the efficiency of both drugs in improving breast cancer treatment and reducing the side effects provoked by doxorubicin [ ] . the work concludes that the combination of arctigenin and doxorubicin induced the apoptosis of mda-mb- human breast cancer cells in vitro. the addition of arctigenin ameliorates the cellular uptake of doxorubicin, which causes the death of carcinogenic cells. another lignan that was tested in some studies on cancer therapy is magnolol. as its name indicates, magnolol is an isomer of honokiol found in magnolia bark [ ] . since ancient times, extracts from the bark of magnolia have been used in traditional chinese, korean, and japanese medicine. in the last decades, the research on the use of natural products in various cancer treatments has been focused on attempts of understanding mechanisms that induce the antitumor agents' response in the tumor cells [ ] . this year, su and co-workers elucidated the mechanism that reduces the endogen activity of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), which is a protein complex that controls dna transcription and cell survival. therefore, the cells that do not have regulated nf-κb can contribute to the onset and growth of various types of cancers. moreover, magnolol used in the treatment of colorectal cancer reduces the phosphorylation of protein kinase c delta type (pkcδ) and nf-κb, which are two proteins that are involved in tumour progression in vitro and in vivo [ ] . following the methodology used with other drugs, magnolol was co-encapsulated with trastuzumab, an anticancer drug commonly used in stomach or throat cancer therapies, and gold nanoparticles, building a nanocarrier cluster. the synthesized nanocarriers induced a specific photothermal near-ir response combined with targeted anticancer activity resulting in an improvement of magnolol cytotoxicity to breast cancer cells [ ] . as mentioned before, honokiol (also known as houpa or hnk) is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus of magnolias, which includes around species. honokiol, which has been used in traditional eastern herbal medicines as an analgesic and together with magnolol, obovatol, and -o-methylhonokiol in the treatment of anxiety and mood disorders, has a spicy odor [ ] . honokiol is most frequently taken orally. in nature, honokiol and magnolol isomers are found together. usually, the separation and purification of both compounds had always been complexed, and it is commonly limited to hplc. in , amblard and co-workers developed a method in which the authors protect the near hydroxyl groups in magnolol to produce a magnolol acetonide that can be simply separated from honokiol via flash chromatography over silica [ ] . recent studies suggest that honokiol could be an effective agent in cancer treatment due to its physical properties-honokiol's ability to easily cross the blood-brain barrier and the bloodcerebrospinal fluid barrier-and its high bioavailability. many research studies have shown that honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that p-coumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that pcoumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids have a wide spectrum of functions in plants: -flavonoids attract pollinating insects through the color or smell that they give to the plant or its flowers, -filtration of uv light, -protection against herbivorous predators, -protection against fungi, -they are involved in the hormone auxin transport, -regulation of the cell cycle, -pigmented blue colors given by anthocyanins are responsible for the resistance of plants to the photooxidation of uv light from the sun, and - in carnivorous plants, they attract prey. usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c ′ in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : -in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. -in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. - in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, -by stopping the progression and proliferation of cancer cells, and -by inducing cancer cells apoptosis. one of the latest investigations to clarify the mechanism of kaempferol as an anticancer drug against breast tumors was carried out by zhu et al. the authors mentioned that kaempferol induced apoptosis and dna damage in mda-mb- cancer cells by the upregulation of the phosphorylated form of the h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm) [ ] . da and co-workers tested kaempferol in prostate cancer cells [ ] . the authors concluded that the use of kaempferol against lncap prostate cancer cell lines led to cancer cells death and impeded cancer cell proliferation and invasion in a dose-dependent manner. quercetin is the most common flavonoid in human diet with an average daily consumption of - milligrams [ ] . quercetin is mainly found in red onions, kale, apples, grapes, broccoli, and tea. in red onions, quercetin represents around % of its dry weight. various in vitro and in vivo studies showed that quercetin is one of the most potent antioxidants of the flavonoid family [ ] , which makes it an ideal candidate for an anticancer drug. indeed, quercetin is the active ingredient of yang-yin-qing-fei-tang, which is a traditional chinese medicine. furthermore, quercetin exhibited cytotoxicity in various tumor cells, in breast, cervical, colon, liver, lung, gastric, prostate cancers, and in leukemia [ , ] . making use of the anticancer effects of quercetin, the most recent studies combined quercetin with other anticancer drugs with the aim of increasing the efficiency of cancer therapies. some examples are summarized below. one of the natural compounds that lately has been combined with quercetin in cancer therapy studies is curcumin. srivastavaa et al. showed that the mixture of quercetin and curcumin improved the inhibition of cancer cell proliferation by regulating the wnt/β-catenin signaling and promoting the carcinogenic cells death by distinct pathways [ ] . furthermore, sunoqrot and co-workers combined both curcumin and quercetin by preparing nanoparticles with encapsulated curcumin and a shell of quercetin covalently bonded with polyethylene glycol (peg) prepared in a one-pot procedure [ ] . once tested in vivo, these nanocarriers exhibited a controlled drug delivery of curcumin in physiological conditions, which makes it a potentially powerful tool in cancer therapy. it has also been observed that the addition of quercetin to docetaxel therapy in prostate cancer reduces the docetaxel resistance of carcinogenic cells. that increases the efficacy of cancer therapy resulting from an intensification of the apoptosis of cancer cells and the reduction of tumor proliferation and migration [ ] . flavones are a class of flavonoids with a chemical structure very similar to flavonols, from which they only differ in the non-hydroxyl substitution at the carbon -position of flavones ( figure ). flavones are basically found in herbs (parsley, thyme, chamomile, mint, chrysanthemum flowers) and red or purple plants and vegetables (apple skins, broccoli, cabbages, celery, onion leaves, carrots, and red peppers) [ ] . in plants, flavones usually act as defense mechanisms against diseases originated by pathogens. some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . to enhance the anticancer effects of luteolin, the flavone is usually used together with other anticancer drugs. ren and co-workers demonstrated that the application of luteolin in combination with oxalipatlin, a conventional anticancer drug used to inhibit the development of cancer cells, stopped the proliferation of gastric cancer cells in vitro by the upregulation of the activity of caspase- and bax proteins [ ] . the construction of nanocarriers containing anticancer drugs allows obtaining controlled drug delivery systems. by the encapsulation of luteolin in polymeric micelles, hu et al. developed a thermosensitive nanocarrier that demonstrated an improved apoptosis of colorectal cancer cells compared to the administration of free luteolin [ ] . flavanones are colorless ketones derived from flavone. flavanones are found in a wide variety of foods included in our daily diet and in herbs [ , ] in citrus fruits, flavanones are usually glycosylated by a disaccharide in position ( figure ). they present different functions in plants: taste-modifying properties (eriodictyol, homoeriodictyol and sterubin), and -they are responsible for the bitter taste in citrus fruits (naringin). in the last decades, flavanones have gained a lot of importance in medicine for their antioxidant activity, radical scavenging, cardiovascular, anti-inflammatory, antiviral, and anticancer effects [ ] . naringenin and hesperetin are the most often investigated for being anticancer drugs. nevertheless, some tests were carried out using other flavanones such as didymin and alpinetin [ , ] . naringenin is a flavanone predominating in oranges and grapefruits. it is also found in bergamot, sour orange, tomatoes, cocoa, water mint, beans, etc. [ , ] . in some of these fruits, narigenin is present in its glycosidic form: naringin (which has attached a disaccharide neohesperidose via a glycosidic linkage at carbon ). as it has been proven in several studies, naringenin induces cytotoxicity in various carcinogenic cells of breast, stomach, liver, cervix, pancreas, colon cancers, and in leukemia [ ] . nevertheless, its poor solubility and instability in physiological medium limits the medical applications of naringenin. to solve these drawbacks, akhter et al. reported the encapsulation of naringenin in plga (poly(lactide-co-glycolid acid)) nanoparticles. moreover, they suggested that the encapsulated naringenin showed higher cytotoxicity when compared with free naringenin due to a more controlled drug release [ ] . another option that could enhance the anticancer properties of naringenin involves the synthesis of naringenin derivatives [ ] . an alternative recent study demonstrated naringenin's effectivity as an anticancer drug in breast cancer treatment is due to the activation of the caspase- protein and caspase- enzymes [ ] , while kumar and co-workers showed in vivo that naringenin showed antitumor effects on skin cancer [ ] . hesperetin and hesperetin's -o-glycoside (also known as hesperidin) are the main flavonoids found in lemons and sweet oranges [ ] . hesperetin's anticancer properties against specific tumors are well documented in numerous research publications: -it inhibits glucose uptake in various cancer cell lines [ , ] , -reduces the nf-κb activity, which leads to a decrease in tumor progression [ ] , and -upgrades the apoptosis via the induction of intracellular ros formation [ ] . in a more recent study, the addition of hesperetin improves the activity of cisplatin, which is an anticancer drug that is commonly used to treat lung cancer [ ] . it was observed that hesperetin inhibits mdr protein (multidrug resistance protein ), which is associated with the resistance to cisplatin developed in a great number of patients subjected to cancer therapy. curiously, the administration of both naringenin and hesperetin were tested in vitro and in vivo trials to analyze the anticancer effects in human pancreatic cancer [ ] . for the first time, the authors reported that the combination of both naringenin and hesperetin could be used as a potential non-toxic cancer therapy system that stops pancreatic cancer development. flavanols or flavan- -ols are another group of monomeric flavonoids. catechin and its derivatives are included in this group. natural sources of flavan- -ols are mainly the "tea plant" (camellia sinensis), and some cocoas. therefore, they are highly present in the human diet in both beverages (tea) and solid foods (chocolates) [ , ] . since studies of flavanols have started in the course of the last century, it has been found that these compounds provide resistance against dangerous trespassers, including microbes, fungi, insects, and herbivorous animals [ ] . thereby, the flavanols' health benefits have been broadly studied in humans. some investigations suggest that the intake of cocoa flavanols could help in the prevention of cardiovascular and metabolic diseases. indeed, the european food safety authority approved cocoa products containing mg of flavanols because they "help to maintain the elasticity of blood vessels, which contributes to normal blood flow" [ ] . epigallocatechin gallate (epigallocatechin- -gallate or egcg) is a catechin that is mostly found in tea and one of the polyphenolic compounds most commonly found in nature; it is also the ester of epigallocatechin and gallic acid [ ] . the objective of finding a correlation between green tea intake and the risk of cancer onset has been a well-studied topic [ ] . as an obvious example, the study presented by guo et al. [ ] validated that the consumption of green tea-and therefore catechins-up to seven cups a day provided a small reduction in the prostate cancer risk. moreover, egcg has been tested against certain cancer cell lines. in ht- colorectal cell lines, egcg upregulated the activity of tfr (transferrin receptor), which is a carrier protein for transferrin, and inhibited the activity of the ferritin-h protein via the iron chelation activity in ht- colorectal cancer cells [ ] . in another example, the synergistic effect of egcg and trail (tumor necrosis factor (tnf)-related apoptosis-inducing ligand), a protein that causes cell death, intensifies the activity of both caspase and the death receptor , causing the death of sw and hct colon cancer cells [ ] . despite the fact that egcg is commonly found in nature, this flavanol shows some drawbacks that limit its applications in cancer therapy (poor stability, low absorption, and hepatotoxicity) [ ] . so, the encapsulation of egcg can be a promising solution to minimize the limitations of the egcg use [ ] . the (−)-epicatechin molecule is a flavonoid of which large quantities are found in cocoa [ ] . the use of epicatechin in cancer therapy has been emerging over the last decade in the attempt to overcome some of the drawbacks of egcg [ , ] . pereyra-vergara and co-workers studied the effects and mechanism of (−)-epicatechin in breast cancer cells [ ] . it was shown that the addition of (−)-epicatechin to carcinogenic cells results in the apoptosis of the two tested breast cancer cell lines (mda-mb- and mcf- ). moreover, the authors proved that (−)-epicatechin increased the intracellular ros production and intensified the activity of bcl associated agonist of cell death (bad) and bcl- -like protein (bax), proteins that are associated with cell apoptosis. isoflavones are another type of biological active flavonoids. isoflavones are mostly found in plants of the leguminosea family. this family includes many species that are of great importance in the human diet (peas, lentils, licorice, beans, chickpeas, and carob), in animal fodder (alfalfa, clover, and carob) and as ornamental plants (mimosa and false acacia) [ , ] . since isoflavones present estrogenic properties, plants use these kinds of compounds as part of their natural defense system against the overpopulation of herbivores by controlling their male fertility [ ] . moreover, these properties make isoflavones good complementary therapeutic options in treating menopause and its symptoms such as osteoporosis, anxiety, emotional instability, and headaches. genistein and daidzein are the most studied compounds of this subgroup in terms of medical applications. nevertheless, other isoflavones such as glabridin and alpinumisoflavone have raised interest as potential cancer medicines in various types of cancer such as breast, liver, or thyroid cancers [ , ] . the isoflavone most reported in medicine is genistein, which is a phytoestrogen compound produced in soybeans. genistein was for the first time isolated in . however, it was not until the end of the last century that researchers started to explore its potential beneficial effects on human health and its possible applications as a medical compound in a wide range of diseases, including cardiovascular diseases, osteoporosis prevention, diabetes, and some types of cancers [ ] . it has been proven that genistein is involved in the regulation of different genes that are associated with the onset of cancers by various mechanisms [ ] . in a recent research, hsiao et al. studied the effects and mechanisms of genistein against leukemia cell lines. in fact, the application of genistein to hl- leukemia cells revealed that this natural medicine kills the carcinogenic cells via two different pathways (endoplasmatic reticulum stress and mitochondria-dependent pathway) in vitro and in mouse xenograft models in vivo [ ] . furthermore, different authors studied the effects of genistein when it is combined with other anticancer drugs [ ] . in a recent investigation, liu et al. tested mixtures of genistein and cisplatin in varied concentrations as a plausible anticancer agent in the treatment of cervical cancer cells [ ] . the authors proved that the addition of genistein improved the chemotherapeutic activity of cisplatin, requiring a lower dose of the drug in cancer treatment, which led to a reduction in the therapy side effects. the second isoflavone most commonly found in nature, which similar to genistein is also isolated from soybeans, is daidzein [ ] . the chemical structure of daidzein is very similar to genistein, without the hydroxyl group at position (table ) . rigalli et al. studied in vitro the effects of daidzein use in breast cancer therapy [ ] . in one of those studies, they proved that daidzein downregulated the expression of multidrug resistance-associated protein (mrp ) in both michigan cancer foundation- (mcf- ) and mda-mb- breast cancer cell lines. the reduction of this protein's activity is very important because mrp is involved in transporting many of the chemotherapeutic drugs out of the cells (for example, doxorubicin or mitoxantrone). in another study in vivo, mice were inoculated with t breast cancer cells and then treated with daidzein administered orally for days. in this case, the highest dose of daidzein ( mg/kg) was required to observe a considerable decrease in tumor size. at the same time, the authors reported that the combination of daidzein with regular exercise promotes the breast cancer cells apoptosis via the fas/fasl-mediated mechanism [ ] . chalcones are a class of polyphenolic compounds that are characterized by the presence of an aromatic ketone and an enone in their central core. many fruits such as citrus and apples, vegetables such as tomatoes, potatoes, shallots, and bean sprouts, and some edible plants such as licorice contain chalcones [ ] . besides, chalcones can be synthesized in the form of base-catalyzed aldol condensation of benzaldehydes with acetophenones (for example, sodium hydroxide) [ ] . the most studied chalcone in the field of medicine is ellagic acid, which has been investigated as a potential antitumor agent [ , ] . ellagic acid is an antioxidant that is found in various natural resources: in oak species such as white oak (quercus alba) and european red oak (quercus robur) or in medicinal fungi (phellinus linteus). peaches, pomegranates, grapes, strawberries, raspberries, pecans, walnuts, and raw chestnuts also contain a considerable amount of ellagic acid [ ] . the anti-proliferative and antioxidative properties of ellagic acid have encouraged researchers to study the health benefits of this natural compound. for years, the effects of treating tumors with ellagic acid have been studied by the evaluation of various alternatives (chemical modifications of ellagic acid or its encapsulation among other options) [ ] . one of the last studies that examined the breast cancer treatment with ellagic acid was published by yousuf et al. [ ] . this work evaluated the capacity of numerous phytochemicals in addition to ellagic acid (capsaicin, tocopherol, rosmarinic acid, ursolic acid, limonene, caffeic acid, and ferulic acid) to inhibit the activity of cyclin-dependent kinase (cdk ), which is an important gene associated with cancer progression. among all the tested natural compounds, ellagic acid showed the highest binding affinity for cdk , decreasing the tumor proliferation. however, the encapsulation of ellagic acid to enhance its poor solubility combined with an improvement of its controlled delivery was attempted by some research groups [ , ] . in a recent work, pirzadeh-naeeni et al. reported the nanoencapsulation of ellagic acid in two different biopolymers (schizophyllan and chitin), which were then tested against mcf- breast cancer cells [ ] . in this case, the controlled release of ellagic acid improved the cytotoxicity when compared with non-encapsulated ellagic acid. it also reduced the progression of tumor cells. anthocyanidins are water-soluble pigments found in plants. they are responsible for leaves, flowers, and fruit colors. some fruits included in the human diet are rich in anthocyanins: blueberries, raspberries, black rice, and black soybeans (normally known as dark fruit). the term anthocyanin was coined in by ludwig clamor marquart, a german pharmacist, to denote the blue pigment of red cabbage (brassica oleracea) [ ] . table . summary of various polyphenols, their chemical structures, and their anticancer effects. resveratrol dna protection against reactive oxygen species (ros), trap the hydroxyl and superoxide groups and the free radicals produced into the cells. inhibition of a lung cancer cells with the activation of caspase- . u. s. department of health and human services public health service food and drug administration status: bulk ingredient for human prescription compounding. [ , ] other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). [ [ ] [ ] [ ] , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). improves the cellular uptake of doxorubicin and reduces its side effects. apoptosis of mda-mb- breast cancer cells. [ , ] of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). kappa-light-chain-enhancer of activated b cells (nf-κb) signaling through protein kinase c delta type (pkcδ) inactivation. upgrades the cytotoxicity of trastuzumab and increases the specificity to breast cancer cells. synergistic effects of honokiol and doxorubicin in breast cancer by suppressing the metastasis of carcinogenic cells and apoptosis induction. apoptosis of multiple myeloma cancer cells. [ , ] p-coumaric acid apoptosis of hct- colon cancer cells through ros mitochondrial pathway. inhibits the growth of snu- gastric cancer cells. [ [ ] [ ] [ ] kaempferol induces the apoptosis and dna damage in mda-mb- breast cancer cells by the upregulation of h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm). induces the apoptosis of lncap prostate cancer cells. impedes the proliferation of cancer cells. luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] u. s. department of health and human services public health service food and drug administration status: drug for further processing. promotes apoptosis of pancreatic cancer cells by increasing intracellular ros. damages dna of hela cervical cancer cells. inhibits the growth of cancer cells and induces its apoptosis. [ , ] luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] naringenin [ , ] naringenin apoptosis of breast cancer cells by the increase of the activity of caspase- and caspase- . suppression of skin cancer cells. [ , ] molecules , in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used anthocyanidins have varied functions in plants: attracting pollinating insects, preventing the freezing of fruits such as grapes, and protecting plants against harmful uv radiation [ ] . moreover, these kinds of compounds are widely used in the food industry (preparation of food coloring, a parameter for determining wine quality) and in medical industry (decreased risk of contracting various diseases such as obesity, improved memory and age-related deficiencies, or improvement of the immunological system) due to their chemical and physical properties [ ] . some anticancer properties of anthocyanidins extracted from the plant cyanomorium coccineum have been recently described by rescigno et al., which demonstrated the antiproliferative effect of anthocyanidins against different leukemia cell lines [ ] . delphinidin, which can be found in red cabbage, grapes, berries, and sweet potatoes among other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used as chemotherapy adjuvant agents in cancer therapies. however, the process of discovering the polyphenols' mechanisms of action as anticancer drugs and their interactions with tumors requires further studies in order for those natural compounds to improve the actual therapeutic strategies. the authors declare no conflict of interest. cancer nanotherapy: concept for design of new drug cancer statistics molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal advances in cancer therapy with plant based natural products new aspects of natural-killer-cell surveillance and therapy of cancer natural polymers: a source of inspiration discovery, development, and regulation of natural products. in using old solutions to new problems-natural drug discovery in the st century discovery, development, and regulation of natural products cancer chemoprevention with dietary phytochemicals dietary phytochemicals and human health phytochemicals modulate cancer aggressiveness: a review depicting the anticancer efficacy of dietary polyphenols and their combinations polyphenols as antimicrobial agents plant polyphenols: chemical properties, biological activities, and synthesis the pharmacology of avenanthramides: polyphenols ultrasound-assisted extraction of biologically active compounds and their successive concentration by using membrane processes stability and anti-proliferative properties of biologically active compounds extracted from cistus l. after sterilization treatments minireview-biological effects of resveratrol resveratrol and cancer: challenges for clinical translation resveratrol in cancer patients: from bench to bedside resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses in vitro and in vivo evaluation of the antitumor efficiency of resveratrol against lung cancer. asian pac phase i randomized, double-blind pilot study of micronized resveratrol (srt ) in patients with hepatic metastases-safety, pharmacokinetics, and pharmacodynamics a phase study of srt (resveratrol) with bortezomib for patients with relapsed and or refractory multiple myeloma resveratrol reduces the levels of circulating androgen precursors but has no effect on, testosterone, dihydrotestosterone, psa levels or prostate volume. a -month randomised trial in middle-aged men biological activities of curcuminoids, other biomolecules from turmeric and their derivatives-a review compound from natural sources, a true scientific challenge-a review synthesis of curcumin derivatives and analysis of their antitumor effects in triple negative breast cancer (tnbc) cell lines photo-triggered capsules based on lanthanide-doped upconverting nanoparticles for medical applications encapsulation for cancer therapy dimethoxy curcumin induces apoptosis by suppressing survivin and inhibits invasion by enhancing e-cadherin in colon cancer cells potential of curcumin and resveratrol as biochemical and biophysical modulators during lung cancer in rats evaluation of biophysical as well as biochemical potential of curcumin and resveratrol during prostate cancer lignans in food and nutrition flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis anticancer effects of a plant lignan -hydroxymatairesinol on a prostate cancer model in vivo irinotecan compared with etoposide in combination with platinum in previously untreated extensive stage small cell lung cancer: an updated systemic review a systematic review and pooled analysis of studies of oral etoposide in metastatic breast cancer molecular mechanisms of the action of arctigenin in cancer arctigenin promotes apoptosis in ovarian cancer cells via the inos/no/stat /survivin signalling nanoparticle delivery strategies to target doxorubicin to tumor cells and reduce side effects arctigenin enhances the cytotoxic effect of doxorubicin in mda-mb- breast cancer cells therapeutic applications of compounds in the magnolia family anticancer activity of natural compounds from plant and marine environment suppression of pkcdelta/nf-kappab signaling and apoptosis induction through extrinsic/intrinsic pathways are associated magnolol-inhibited tumor progression in colorectal cancer in vitro and in vivo near ir responsive targeted integrated lipid polymer nanoconstruct for enhanced magnolol cytotoxicity in breast cancer facile purification of honokiol and its antiviral and cytotoxic properties honokiol for cancer therapeutics: a traditional medicine that can modulate multiple oncogenic targets honokiol: a review of its anticancer potential and mechanisms synergistically enhanced antimetastasis effects by honokiol-loaded ph-sensitive polymer−doxorubicin conjugate micelles honokiol induces apoptosis in human lymphoid leukemia molt b cells the natural product honokiol induces caspase-dependent apoptosis in b-cell chronic lymphocytic leukemia (b-cll) cells honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis bioactivity of phenolic acids: metabolites versus parent compounds: a review dietary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden in situ antioxidant and antimicrobial activities of naturally occurring caffeic acid, p-coumaric acid and rutin, using food systems events associated with apoptotic effect of p-coumaric acid in hct- colon cancer cells effects of p-coumaric acid on microrna expression profiles in snu- human gastric cancer cells anti-proliferative properties of p-coumaric acid in snu- gastric cancer cells anthocyanins and other flavonoids research trends in flavonoids and health phenol content in sprouted grains tautomerism of flavonol glucosides: relevance to plant uv protection and flower colour enhancement of oxidative and drought tolerance in arabidopsis by overaccumulation of antioxidant flavonoids dietary flavonoids: bioavailability, metabolic effects, and safety role of fisetin in chemosensitization health-promoting components of fruits and vegetables in the diet the mechanism of anticancer action and potential clinical use of kaempferol in the treatment of breast cancer kaempferol suppresses proliferation and induces cell cycle arrest, apoptosis, and dna damage in breast cancer cells kaempferol promotes apoptosis while inhibiting cell proliferation via androgen-dependent pathway and suppressing vasculogenic mimicry and invasion in prostate cancer review of the biology of quercetin and related bioflavonoids fruits and vegetables in the prevention of cellular oxidative damage quercetin is the active component of yang-yin-qing-fei-tang to induce apoptosis in non-small cell lung cancer pharmacological basis and new insights of quercetin action in respect to its anti-cancer effects curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate wnt/β-catenin signaling and apoptotic pathways in a cells bioinspired polymerization of quercetin to produce a curcumin-loaded nanomedicine with potent cytotoxicity and cancer-targeting potential in vivo quercetin reverses docetaxel resistance in prostate cancer via androgen receptor and pi k/akt signaling pathways the herbal medicine sho-saiko-to inhibits proliferation of cancer cell-lines by inducing apoptosis and arrest at the g( )-g( )-phase medicinal importance, pharmacological activities, and analytical aspects of hispidulin: a concise report curcumin and apigenin-novel and promising therapeutics against chronic neuroinflammation in alzheimer's disease in vitro anti-inflammatory effect of apigenin in the helicobacter pylori-infected gastric adenocarcinoma cells antal, i. study on the pulmonary delivery system of apigenin-loaded albumin nanocarriers with antioxidant activity apigenin and cancer chemoprevention a plant flavone playing noble roles in cancer prevention via modulation of key cell signaling networks. recent pat. anti-cancer drug discov apigenin in cancer therapy: anti-cancer effects and mechanisms of action mutant p , stabilized by its interplay with hsp , activates a positive feed-back loop between nrf and p that induces chemo-resistance to apigenin in pancreatic cancer cells the stability and activity changes of apigenin and luteolin in human cervical cancer hela cells in response to heat treatment and fe + /cu + addition intestinal absorption of luteolin and luteolin -o-beta-glucoside in rats and humans molecular targets of luteolin in cancer apoptosis induced by luteolin in breast cancer: mechanistic and therapeutic perspectives luteolin suppresses the proliferation of gastric cancer cells and acts in synergy with oxaliplatin thermosensitive in situ gel containing luteolin micelles is a promising efficient agent for colorectal cancer peritoneal metastasis treatment polyphenols: a concise overview on the chemistry, occurrence, and human health a comprehensive review on flavanones, the major citrus polyphenols didymin, a dietary flavonoid glycoside from citrus fruits, induces fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo antiproliferative effect of alpinetin in bxpc- pancreatic cancer cells antiatherogenic properties of naringenin, a citrus flavonoid review of the flavonoids quercetin, hesperetin naringenin. dietary sources, bioactivities, and epidemiology inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma s- -implanted mice sonication tailored enhance cytotoxicity of naringenin nanoparticle in pancreatic cancer: design, optimization, and in vitro studies optimizing the flavanone core toward new selective nitrogen-containing modulators of abc transporters naringenin has a chemoprotective effect in mda-mb- breast cancer cells via inhibition of caspase- and - activities naringenin suppresses chemically induced skin cancer in two-stage skin carcinogenesis mouse model therapeutic potential of hesperidin and its aglycone hesperetin: cell cycle regulation and apoptosis induction in cancer models hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells natural compounds regulate glycolysis in hypoxic tumor microenvironment molecular mechanisms behind the biological effects of hesperidin and hesperetin for the prevention of cancer and cardiovascular diseases hesperidin: a promising anticancer agent from nature hesperetin reverses p-glycoprotein-mediated cisplatin resistance in ddp-resistant human lung cancer cells via modulation of the nuclear factor-κb signaling pathway combined administration of naringenin and hesperetin with optimal ratio maximizes the anti-cancer effect in human pancreatic cancer via down regulation of fak and p signaling pathway cocoa flavanols: natural agents with attenuating effects on metabolic syndrome risk factors scientific opinion on the modification of the authorisation of a health claim related to cocoa flavanols and maintenance of normal endotheliumdependent vasodilation pursuant to article ( ) of regulation (ec) no / following a request in accordance with article of regulation (ec) no epigallocatechin- -gallate (egcg): chemical and biomedical perspectives cancer prevention by tea: animal studies, molecular mechanisms and human relevance green tea and the risk of prostate cancer a systematic review and meta-analysis iron chelation properties of green tea epigallocatechin- -gallate (egcg) in colorectal cancer cells: analysis on tfr/fth regulations and molecular docking. evid-based complementary altern epigallocatechin- -gallate induces apoptosis as a trail sensitizer via activation of caspase and death receptor in human colon cancer cells the safety of green tea and green tea extract consumption in adults-results of a systematic review cancer therapeutics with epigallocatechin- -gallate encapsulated in biopolymeric nanoparticles flavor chemistry of cocoa and cocoa products-an overview antileukemic action of (−)-epicatechin in the spleen of rats with acute myeloid leukemia induction of apoptosis of sw human colon cancer cells by (−)-epicatechin isolated from bulnesia sarmienti apoptosis induced by (−)-epicatechin in human breast cancer cells is mediated by reactive oxygen species phenolic compounds as functional ingredients in beverages phytochemical mimicry of reproductive hormones and modulation of herbivore fertility by phytoestrogens. environ. health persp glabridin inhibits migration and invasion by transcriptional inhibition of matrix metalloproteinase through modulation of nf-kappa b and ap- activity in human liver cancer cells alpinumisoflavone inhibits tumor growth and metastasis in papillary thyroid cancer via upregulating mir- - p recent progress on biocompatible nanocarrierbased genistein delivery systems in cancer therapy the role of genistein and synthetic derivatives of isoflavone in cancer prevention and therapy genistein induces apoptosis in vitro and has antitumor activity against human leukemia hl- cancer cell xenograft growth in vivo anti-inflammatory and anticarcinogenic effect of genistein alone or in combination with capsaicin in tpa-treated rat mammary glands or mammary cancer cell line effects of genistein on anti-tumor activity of cisplatin in human cervical cancer cell lines nutritional epigenetic regulators in the field of cancer the phytoestrogens daidzein and equol inhibit the drug transporter bcrp/abcg in breast cancer cells: potential chemosensitizing effect synergetic inhibition of daidzein and regular exercise on breast cancer in bearing- t mice by regulating nk cells and apoptosis pathway a review on natural chalcones an update synthesis and anti-inflammatory activity of three nitro chalcones novel -amino chalcones: design, synthesis and biological evaluation antiproliferative and pro-apoptotic activities of -and -aminochalcones against tumor canine cells ellagic acid induces cell cycle arrest and apoptosis through tgf-β/smad signaling pathway in human breast cancer mcf- cells ellagic acid, sulforaphane, and ursolic acid in the prevention and therapy of breast cancer: current evidence and future perspectives ellagic acid controls cell proliferation and induces apoptosis in breast cancer cells via inhibition of cyclin-dependent kinase development of biodegradable nanoparticles for oral delivery of ellagic acid and evaluation of their antioxidant efficacy against cyclosporine a-induced nephrotoxicity in rats preparation of β-cd-ellagic acid microspheres and their effects on hepg cell proliferation a comparative study on schizophyllan and chitin nanoparticles for ellagic acid delivery in treating breast cancer anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits anthocyanin biosynthesis and degradation mechanisms in solanaceous vegetables: a review food applications and physiological effects of anthocyanins as functional food ingredients antiproliferative and antiviral extracts from sardinian maltese mushroom (cynomorium coccineum l.) updating the research on prodelphinidins from dietary sources influence of fruit juice processing on anthocyanin stability delphinidin induces apoptosis via cleaved hdac -mediated p acetylation and oligomerization in prostate cancer cells delphinidin inhibits bdnf-induced migration and invasion in skov ovarian cancer cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -gu nnv d authors: chandran, uma; mehendale, neelay; patil, saniya; chaguturu, rathnam; patwardhan, bhushan title: chapter network pharmacology date: - - journal: innovative approaches in drug discovery doi: . /b - - - - . - sha: doc_id: cord_uid: gu nnv d abstract the one-drug/one-target/one-disease approach to drug discovery is presently facing many challenges of safety, efficacy, and sustainability. network biology and polypharmacology approaches gained appreciation recently as methods for omics data integration and multitarget drug development, respectively. the combination of these two approaches created a novel paradigm called network pharmacology (np) that looks at the effect of drugs on both the interactome and the diseasome level. ayurveda, the traditional system of indian medicine, uses intelligent formulations containing multiple ingredients and multiple bioactive compounds; however, the scientific rationale and mechanisms remain largely unexplored. np approaches can serve as a valuable tool for evidence-based ayurveda to understand the medicines’ putative actions, indications, and mechanisms. this chapter discusses np and its potential to explore traditional medicine systems to overcome the drug discovery impasse. drug discovery, the process by which new candidate medications are discovered, initially began with random searching of therapeutic agents from plants, animals, and naturally occurring minerals (burger, ) . for this, they depended on the materia medica that was established by medicine men and priests from that era. this was followed by the origin of classical pharmacology in which the desirable therapeutic effects of small molecules were tested on intact cells or whole organisms. later, the advent of human genome sequencing revolutionized the drug discovery process that developed into targetÀbased drug discovery, also known as reverse pharmacology. this relies on the hypothesis that the modulation of the activity of a specific protein will have therapeutic effects. the protein that the drug binds to or interacts with is also referred to as a "target." in this reductionist approach, small molecules from a chemical library are screened for their effect on the target's known or predicted function (hacker et al., ) . once the small molecule is selected for a particular target, further modifications are carried out at the atomic level to ameliorate the lock-and-key interactions. this one-drug/onetarget/one-therapeutic approach was followed for the last several decades. the information technology revolution at the end of th century metamorphosed the drug discovery process as well (clark and pickett, ) . advancements in omics technologies during this time were used to develop strategies for different phases of drug research (buriani et al., ) . computational power was implemented in the discovery process for predicting a drug-likeness of newly designed or discovered compounds and ligandprotein docking for predicting the binding affinity of a small molecule with a protein three-dimensional structure. in silico tools were developed to predict other pharmacological properties of the drug molecules such as absorption, distribution, metabolism, excretion, and toxicity-abbreviated together as admet (van de waterbeemd and gifford, ; clark and grootenhuis, ) . the technological advancements triggered discovery efforts in a direction to discover more specific magic bullets that were completely against the holistic approach of traditional medicine. this magic bullet approach is currently in decline phase. the major limitations of this drug discovery approach are side effects and the inability to tackle multifactorial diseases. this is mainly due to the linearity of this approach. during the peak, historical time of drug discovery and development of natural productsÀbased drugs had played a significant role due to their superior chemical diversity and safety over synthetic compound libraries (zimmermann et al., ) . currently, it is estimated that more than one hundred new, natural productÀbased leads are in clinical development (harvey, ) . many active compounds (bioactives) from traditional medicine sources could serve as good starting compounds and scaffolds for rational drug design. natural products normally act through modulation of multiple targets rather than a single, highly specific target. but in drug discovery and development, technology was used to synthesize highly specific mono-targeted molecules that mimic the bioactives from natural compounds rather than understanding the rationale behind their synergistic action and developing methods to isolate the bioactives from natural resources. researchers understand that most diseases are due to dysfunction of multiple proteins. thus, it is important to address multiple targets emanating from a syndrome-related, metabolic cascade, so that holistic management can be effectively achieved. therefore, it is necessary to shift the strategy from one that focuses on a single-target, new chemical entity to one of a multiple-target, synergistic, formulation-discovery approach . this tempted the research world to go back and extensively explore natural sources, where modern pharmacology had begun. this renewed research focus indicates the need to rediscover the drug discovery process by integrating traditional knowledge with state-of-the-art technologies (patwardhan, a) . a new discipline called network pharmacology (np) has emerged which attempts to understand drug actions and interactions with multiple targets (hopkins, ) . it uses computational power to systematically catalogue the molecular interactions of a drug molecule in a living cell. np appeared as an important tool in understanding the underlying complex relationships between botanical formula and the whole body berger and iyengar, ) . it also attempts to discover new drug leads and targets and to repurpose existing drug molecules for different therapeutic conditions by allowing an unbiased investigation of potential target spaces (kibble et al., ) . however, these efforts require some guidance for selecting the right type of targets and new scaffolds of drug molecules. traditional knowledge can play a vital role in this process of formulation discovery and repurposing existing drugs. by combining advances in systems biology and np, it might be possible to rationally design the next generation of promiscuous drugs (cho et al., ; hopkins, ; ellingson et al., ) . np analysis not only opens up new therapeutic options, but it also aims to improve the safety and efficacy of existing medications. the postgenomic era witnessed a rapid development of computational biology techniques to analyze and explore existing biological data. the key aim of the postgenomic biomedical research was to systematically catalogue all molecules and their interactions within a living cell. it is essential to understand how these molecules and the interactions among them determine the function of this immensely complex machinery, both in isolation and when surrounded by other cells. this led to the emergence and advancement of network biology, which indicates that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biology and disease pathologies in the st century (barabási and oltvai, ) . during the first decade of the st century, several approaches for biological network construction were put forward that used computational methods, and literature mining especially, to understand the relation between disease phenotypes and genotypes. as a consequence, lmma (literature mining and microarray analysis), a novel approach to reconstructing gene networks by combining literature mining and microarray analysis, was proposed (li et al., ; huang and li, ) . with this, a global network was first derived using the literatureÀbased, cooccurrence method and then refined using microarray data. the lmma biological network approach enables researchers to keep themselves up to date with relevant literature on specialized biological topics and to make sense of the relevant large-scale microarray dataset. also, lmma serves as a useful tool for constructing specific biological network and experimental design. lmmaÀlike representations enable a systemic recognition for the specific diseases in the context of complex gene interactions and are helpful for studying the regulation of various complex biological, physiological, and pathological systems. the significance of accumulated-data integration was appreciated by pharmacologists, and they began to look beyond the classic lock-and-key concept as a far more intricate picture of drug action became clear in the postgenomic era. the global mapping of pharmacological space uncovered promiscuity, the specific binding of a chemical to more than one target (paolini et al., ) . as there can be multiple keys for a single lock, in the same way, a single key can fit into multiple locks. similarly, a ligand might interact with many targets and a target may accommodate different types of ligands. this is referred to as "polypharmacology." the concept of network biology was used to integrate data from drugbank (re and valentini, ) and omim (hamosh et al., ) , an online catalog of human genes and genetic disorders to understand the industry trends, the properties of drug targets, and to study how drug targets are related to disease-gene products. in this way, when the first drug-target network was constructed, isolated and bipartite nodes were expected based on the existed one-drug/one-target/onedisease approach. rather, the authors observed a rich network of polypharmacology interactions between drugs and their targets (yildirim et al., ). an overabundance of "follow-on" drugs that are drugs that target already targeted proteins was observed. this suggested a need to upgrade the singletarget single-drug paradigm, as single-protein single-function relations are limited to accurately describing the reality of cellular processes. advances in systems biology led to the realization that complex diseases cannot be effectively treated by intervention at single proteins. this made the drug researchers accept the concept of polypharmacology which they previously thought as an undesirable property that needs to be removed or reduced to produce clean drugs acting on single-targets. according to network biology, simultaneous modulation of multiple targets is required for modifying phenotypes. developing methods to aid polypharmacology can help to improve efficacy and predict unwanted off-target effects. hopkins (hopkins, (hopkins, , observed that network biology and polypharmacology can illuminate the understanding of drug action. he introduced the term "network pharmacology." this distinctive new approach to drug discovery can enable the paradigm shift from highly specific magic bulletÀbased drug discovery to multitargeted drug discovery. np has the potential to provide new treatments to multigenic complex diseases and can lead to the development of e-therapeutics where the ligand formulation can be customized for each complex indication under every disease type. this can be expanded in the future and lead to customized and personalized therapeutics. integration of network biology and polypharmacology can tackle two major sources of attrition in drug development such as efficacy and toxicity. also, this integration holds the promise of expanding the current opportunity space for druggable targets. hopkins proposed np as the next paradigm in drug discovery. polypharmacology expands the space in drug discovery approach. hopkins had suggested three strategies to the designers of multitarget therapies: the first was to prescribe multiple individual medications as a multidrug combination cocktail. patient compliance and the danger of drugÀdrug interactions would be the expected drawbacks of this method. the second proposition was the development of multicomponent drug formulations. the change in metabolism, bioavailability, and pharmacokinetics of formulation as well as safety would be the major concerns of this approach. the third strategy was to design a single compound with selective polypharmacology. according to hopkins, the third method is advantageous, as it would ease the dosing studies. also, the regulatory barriers for the single compound are fewer compared to a formulation. an excellent example of this is metformin, the first-line drug for type ii diabetes that has been found to have cancerinhibiting properties (leung et al., ) . the following years witnessed the application research of np by integrating network biology and polypharmacology. a computational framework, based on a regression model that integrates human proteinÀprotein interactions, disease phenotype similarities, and known geneÀphenotype associations to capture the complex relationships between phenotypes and genotypes, has been proposed. this was based on the assumption that phenotypically similar diseases are caused by functionally related genes. a tool named cipher (correlating protein interaction network and phenotype network to predict disease genes) has been developed that predicts and prioritizes disease-causing genes (wu et al., ) . cipher helps to uncover known disease genes and predict novel susceptibility candidates. another application of this study is to predict a human disease landscape that can be exploited to study the related genes for related phenotypes that will be clustered together in a molecular interaction network. this will facilitate the discovery of disease genes and help to analyze the cooperativity among genes. later, cipher-hit, a hitting-time-based method to measure global closeness between two nodes of a heterogeneous network, was developed (yao et al., ) . a phenotypeÀgenotype network can be explored using this method for detecting the genes related to a particular phenotype. a net-workÀbased gene clustering and extension were used to identify responsive gene modules in a conditionÀspecific gene network aimed to provide useful resources to understand physiological responses (gu et al., ) . np was also used to develop mirnaÀbased biomarkers (lu et al., ) . for this, a network of mirna and their targets was constructed and further refined to study the data for specific diseases. this process integrated with literature mining was useful to develop potent mirna markers for diseases. np was also used to develop a drug geneÀdisease comodule (zhao and li, ) . initially, a drug-disease network was constructed by information gathered from databases followed by the integration of gene data. the gene closeness was studied by developing a mathematical model. this network inferred the association of multiple genes for most of the diseases and target sharing of drugs and diseases. these kinds of networks give insight into new drug-disease associations and their molecular connections. during the progression period of network biology, natural products were gaining importance in the chemical space of drug discovery, as these have been economically designed and synthesized by nature for the benefit of evolution (wetzel et al., ) . researchers began analyzing the logic behind traditional medicine systems and devised computational ways to ease the analysis. a comprehensive herbal medicine information system that was developed integrates information of more than anticancer herbal recipes that have been used for the treatment of different types of cancer in the clinic, individual ingredients, and small organic molecules isolated from herbal medicines (fang et al., ) . this system, which was developed using an oracle database and internet technology, facilitates and promotes scientific research in herbal medicine. this was followed by the development of many databases that serve as a source of botanical information and a powerful tool that provides a bridge between traditional medicines and modern molecular biology. these kinds of databases and tools made the researchers conceive the idea of np of botanicals and their formulations to understand the underlying mechanisms of traditional medicines. we refer to such networks as "ethnopharmacological networks" and the technique as "network ethnopharmacology (nep)" (patwardhan and chandran, ) . shao li pioneered this endeavor and proposed this network as a tool to explain the zheng (syndrome of traditional chinese medicine (tcm)) and the multiple-targets' mechanism of tcm (li, ) . li et al. tried to provide a molecular basis for -year-old concept of zheng using a neuro-endocrine-immune (nei) network . zheng is the basic unit and key concept in tcm theory. it is also used as a guideline in disease classification in tcm. the hot (hans zheng in mandarin) and cold (re zheng) are the two statuses of zheng which therapeutically directs the use of herbs in tcm. chinese herbs are classified as hotÀcooling and are used to remedy hot zheng and coldÀwarming herbs that are used to remedy cold zheng. according to the authors, hormones may be related to hot zheng, immune factors may be related to cold zheng, and they may be interconnected by neurotransmitters. this study provides a methodical approach to understand tcm within the framework of modern science. later they reconstructed the nei network by adding multilayer information including data available on the kegg database related to signal transduction, metabolic pathways, proteinÀprotein interactions, transcription factor, and micro rna regulations. they also connected drugs and diseases through multilayered interactions. the study of cold zheng emphasized its relation to energy metabolism, which is tightly correlated with the genes of neurotransmitters, hormones, and cytokines in the nei interaction network ma et al., ) . another database, tcmgenedit, provides information about tcms, genes, diseases, tcm effects, and tcm ingredients mined from a vast amount of biomedical literature. this would facilitate clinical research and elucidate the possible therapeutic mechanisms of tcms and gene regulations (fang et al., ) . to study the combination rule of tcm formulae, an herb network was created using collaterals-related formulae . they developed a distance-based, mutual-information model (dmim) to uncover the combination rule. dmim uses mutual-information entropy and "between herb distance" to measure the tendency of two herbs to form an herb pair. they experimentally evaluated the combination of a few herbs for angiogenesis. understanding the combination rule of herbs in formulae will help the modernization of traditional medicine and also help to develop a new formulae based on the current requirement. a network targetÀbased paradigm was proposed for the first time to understand the synergistic combinations , and an algorithm termed "nims" (network tar-getÀbased identification of a multicomponent synergy) was also developed. this was a step that facilitated the development of multicomponent therapeutics using traditional wisdom. an innovative way to study the molecular mechanism of tcm was proposed during this time by integrating the tcm experimental data with microarray gene expression data (wen et al., ) . as a demonstrative example, si-wu-tang's formula was studied. rather than uncovering the molecular mechanism of action, this method would help to identify new health benefits of tcms. the initial years of the second decade of the st century witnessed the network ethnopharmacological exploration of tcm formulations. the scope of this new area attracted scientists, and they hoped nep could provide insight into multicompound drug discoveries that could help overcome the current impasse in drug discovery (patwardhan, b; . nep was used to study the antiinflammatory mechanism of qingfei xiaoyan, a tcm . the predicted results were used to design experiments and analyze the data. experimental confirmation of the predicted results provides an effective strategy for the study of traditional medicines. the potential of tcm formulations as multiple compound drug candidates has been studied using tcm formulations based np. tcm formulations studied in this way are listed in table . . construction of a database containing , natural product structures, followed by their docking to target proteins of fda-approved drugs, shows the amount of space shared in the chemical space between natural products and fda drugs (gu et al., a) . molecular-docking technique plays a major role in np. the interaction of bioactives with molecular targets can be analyzed by this technique. molecular dockingÀbased nep can be a useful tool to computationally elucidate the combinatorial effects of traditional medicine to intervene disease networks (gu et al., c ). an approach that combines np and pharmacokinetics has been proposed to study the material basis of tcm formulations (pei et al., ) . this can be extrapolated to study other traditional medicine formulations as well. in cancer research, numerous natural products have been demonstrated to have anticancer potential. natural products are gaining attraction in anticancer research, as they show a favorable profile in terms of absorption and metabolism in the body with low toxicity. in a study all of the known bioactives were docked for their property to interact with cancer targets (luo et al., ) . it was inferred that many bioactives are targeting multiple ejiao slurry regulates cancer cell differentiation, growth, proliferation, and apoptosis, and shows an adjuvant therapeutic effect that enriches the blood and increases immunity xu et al. ( b) xiao-chaihu decoction and da chaihu-decoction xchd treats diseases accompanying symptoms of alternating fever and chills, no desire for food or drink, and dry pharynx, while dchd treats those with symptoms of fullness, pain in abdomen, and constipation. dragon's blood used in colitis and acts through interaction with putative targets xu et al. ( a) protein targets and thus are linked to many types of cancers. np coupled to sophisticated spectroscopical analysis such as ultra-performance liquid chromatographyÀelectrospray, ionizationÀtandem mass spectroscopy (uplc-esi-ms/ms) is a useful approach to study the absolute molecular mechanism of action of botanical formulations based on their constituent bioactives (xu et al., a) . bioactiveÀtarget analysis has shown that some of the botanical formulations are more effective than their corresponding marketed drugÀtarget interactions . this indicates the potential of np to better understand the power of botanical formulations and to develop efficient and economical treatment options. the holistic approach of botanical formulations can be better explained by np. a study has reported this property by exemplifying a tcm formulation against viral infectious disease . not only does the formulation target the proteins in the viral infection cycle, but it also regulates the proteins of the host defense system; thus, it acts in a very distinctive manner. this unique property of formulations is highly efficient for strengthening the broad and nonspecific antipathogenic actions. thus, network-based, multitarget drugs can be developed by testing the efficacy of the formulation, identifying, and isolating the major bioactives and redeveloping a multicomponent therapeutic using the major bioactives based on synergism (leung et al., ) . np also serves to document and analyze the clinical prescriptions of traditional medicine practitioners . a traditional medicine network that links bioactives to clinical symptoms through targets and diseases is a novel way to explore the basic principles of traditional medicines (luo et al., ) . the network-based approaches provide a systematic platform for the study of multicomponent traditional medicine and has applications for its beneficial modernization. this platform not only recovers traditional knowledge, but it also provide new findings that can be used for resolving current problems in the drug industry . this section explains a handful of ethnopharmacological networks that were developed to understand the scientific rationale of traditional medicine. dragon's blood (db) tablets, which are made of resins from dracaena spp., daemonorops spp., croton spp., and pterocarpus spp., is an effective tcm for the treatment of colitis. in a study, an np-based approach was adopted to provide new insights relating to the active constituents and molecular mechanisms underlying the effects of db (xu et al., a) . the constituent chemicals of the formulation were identified using an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry method. the known targets of those identified compounds were mined from literature and putative targets that were predicted with the help of computational tools. the compounds were further screened for bioavailability followed by the systematic analysis of the known and putative targets for colitis. the network evaluation revealed the mechanism of action of db bioactives for colitis through the modulation of the proteins of the nod-like receptor signaling pathway (fig. . ) . the antioxidant mechanism of zhi-zi-da-huang decoction as an approach to treat alcohol liver disease was elucidated using np an and feng, ). an endothelial cell proliferation assay was performed for an antiangiogenic alkaloid, sinomenine, to validate the network targetÀbased identification of multicomponent synergy (nims) predictions. the study was aimed at evaluating the synergistic relationship between different pairs of therapeutics, and sinomenine was found to have a maximum inhibition rate with matrine, both through the network and in vitro studies. the discovery of bioactives and elucidation of the mechanism of action of the herbal formulae, qing-luo-yin and the liu-wei-di-huang pill, using np, has given insight to the design validation experiments that accelerated the process of drug discovery . validation experiments based on the network findings regarding cold zheng and hot zheng on a rat model of collagenÀinduced arthritis showed that the cold zhengÀoriented herbs tend to affect the hub nodes in the cold zheng network, and the hot zheng-oriented herbs tend to affect the hub nodes in the hot zheng network . np was used to explain the addition and subtraction theory of tcm. two decoctions: xiao chaihu and da chaihu were studied using np approach to investigate this theory. according to the addition and subtraction theory, the addition or removal of one or more ingredients from a traditional formulation resulted in a modified formula that plays a vital role in individualized medicine. compounds from additive herbs were observed to be more efficient on diseaseÀassociated targets (fig. . ) . these additive compounds were found to act on diseases through drug targets (li et al., a) . experimental verification of the antithrombotic network of fufang xueshuantong (fxst) capsule was done through in vivo studies on lipopoly-saccharideÀinduced disseminated intravascular coagulation (dic) rat model. it was successfully shown that fxst significantly improves the activation of the coagulation system through targets from four herbs (sheng et al., ) . np analysis of the bushenhuoxue formula showed that six components-rhein, tanshinone iia, curcumin, quercetin and calycosin-acted through targets for the treatment of chronic kidney disease. these predictions were validated using unilateral ureteral obstruction models, and it was observed that even though the individual botanicals showed a significant decrease in creatinine levels, the combination showed lower blood creatinine and urea nitrogen levels (shi et al., ) . the antidiabetic effects of ge-gen-qin-lian decoction were investigated using an insulin secretion assay, and an insulinÀresistance model using of the ingredients showed antidiabetic activity using np studies (li et al., b) . to confirm the predictions of the network of liu-wei-di-huang pill, four proteins-pparg, rara, ccr , and esr -that denote different functions and are targeted by different groups of ingredients were chosen. the interactions between various bioactives and their effect on the expression of the proteins showed that the np approach can accurately predict these interactions, giving hints regarding the mechanism of action of the compounds (liang et al., ) . experimental results confirmed that the core ingredients in modified simiaowan, obtained through network analysis, significantly increased huvec viability and attenuated the expression of icam- and proved to be effective in gout treatment (zhao et al., ) . the role of anthraquinone and flavanols (catechin and epicatechin) in the therapeutic potential of rhubarb in renal interstitial fibrosis was examined using network analysis and by conventional assessment involving serum biochemistry, histopathological, and immunohistochemical assays (xiang et al., ) . in silico analysis and experimental validation demonstrated that compound / of fructus schisandrae chinensis targets gba /shbg . np is a valuable method to study the synergistic effects of bioactives of traditional medicine formulation. this was experimentally shown on the sendeng- formulation for rheumatoid arthritis (fig. . ). data and network analysis have shown that the formulation acts synergistically through nine categories of targets (zi and yu, ) . another network that studied three botanicals, salviae miltiorrhizae, ligusticum chuanxiong, and panax notoginseng for coronary artery disease (cad), displayed their mode of action through targets, out of which are common among the botanicals (fig. . ) . these common targets are associated with thrombosis, dyslipidemia, vasoconstriction, and inflammation . this gives insight to how these botanicals are managing cad. another approach using np is the construction of networks based on experimental data followed by literature mining. this method is very effective for large space data analysis, which will help to derive the mechanism of action of the formulation. a network of qishenyiqi formulation having cardioprotective effects, constructed based on the microarray data and the published literature, showed that main compounds were found to act through pathways, out of which are immune and inflammation-related (li et al., c) . the mechanism of action for the bushen zhuanggu formulation was proposed based on lc-ms/ms standardization, pharmacokinetic analysis, and np (pei et al., ) . the efficacy of shenmai injection was evaluated using a rat model of myocardial infarction, genome-wide transcriptomic experiment, and then followed by a np analysis. the overall trends in the ejection fraction and fractional shortening were consistent with the networkÀrecovery index (nri) from the network . in order to develop an ethnopharmacological network, exploring the existing databases to gather information regarding bioactives and targets is the first step. further information such as target-related diseases, tissue distribution and pathways are also to be collected depending on the type of study that is going to be undertaken. the universal natural products database (unpd) (gu et al., a ) is one of the major databases that provides bioactives information. other databases that provide information regarding bioactives include cvdhd (gu et al., b) , tcmsp (ru et al., ) , tcm@taiwan (sanderson, ) , supernatural (banerjee et al., ) , and dr. dukes's phytochemical and ethnobotanical database (duke and beckstrom-sternberg, ) . the molecular structures of bioactives are usually stored as "sd" files and chemical information as smiles and inchkeys in these databases. any of these file formats can be used as inputs to identify the targets in protein information databases. binding database or "binding db" (liu et al., ) and chembl (bento et al., ) are databases for predicting target proteins. binding db searches the exact or similar compounds in the database and retrieves the target information of those compounds. the similarity search gives the structurally similar compounds with respect to the degree of similarity as scores to the queried structure. the information regarding both annotated and predicted targets can be collected in this way. this database is connected to numerous databases, and these connections can be used to extract further information regarding the targets. the important databases linked to binding db are uniprot (bairoch et al., ) , which gives information related to proteins and genes; reactome, a curated pathway database (croft et al., ) ; and the kyoto encyclopedia of genes and genomes (kegg), a knowledge base for systematic analysis of gene functions and pathways (ogata et al., ) . therapeutic targets database (ttd) (zhu et al., ) gives fully referenced information of targeted diseases of proteins, their pathway information, and the corresponding drug directed to each target. disease and gene annotation (dga), a database that provides a comprehensive and integrative annotation of human genes in disease networks, is useful in identifying the disease type that each indication belongs to (peng et al., ) . the human protein atlas (hpa) database (pontén et al., ) is an open database showing the spatial distribution of proteins in different normal human tissues. the information of the distribution of proteins in tissues can be gathered from hpa. the database also gives information regarding subcellular localization and protein class. an overall review of the methods to implement np for herbs and herbal formulations is also available, including a systematic review of the databases that one could use for the same (kibble et al., ; lagunin et al., ) . integration of knowledge bases helps data gathering for network pharmacological studies, and its knowledge base shows the inter-relationships among these databases (fig. . ) . the counts of entities, such as bioactives, targets, and diseases, can vary based on the knowledge bases that are relied on for data collection. an integration of knowledge bases can overcome this limitation. another factor that affects the counts of these entities is the time frame for data collection. this change occurs due to the ongoing, periodic updates of the databases. a network is the schematic representation of the interaction among various entities called nodes. in pharmacological networks, the nodes include bioactives, targets, tissue, tissue types, disease, disease types, and pathways. these nodes are connected by lines termed edges, which represent the relationship between them (morris et al., ) . building a network involves two opposite approaches: a bottom-up approach on the basis of established biological knowledge and a top-down approach starting with the statistical analysis of available data. at a more detailed level, there are several ways to build and illustrate a biological network. perhaps the most versatile and general way is the de novo assembly of a network from direct experimental or computational interactions, e.g., chemical/gene/protein screens. networks encompassing biologically relevant nodes (genes, proteins, metabolites), their connections (biochemical and regulatory), and modules (pathways and functional units) give an authentic idea of the real biological phenomena (xu and qu, ) . cytoscape, a java-based open source software platform (shannon et al., ) , is a useful tool for visualizing molecular interaction networks and integrating them with any type of attribute data. in addition to the basic set of features for data integration, analysis, and visualization, additional features are available in the form of apps, including network and molecular profiling analysis and links with other databases. in addition to cytoscape, a number of visualization tools are available. visual network pharmacology (vnp) , which is specially designed to visualize the complex relationships among diseases, targets, and drugs, mainly contains three functional modules: drug-centric, target-centric, and disease-centric vnp. this disease-target-drug database documents known connections among diseases, targets, and the usfda-approved drugs. users can search the database using disease, target, or drug name strings; chemical structures and substructures; or protein sequence similarity, and then obtain an online interactive network view of the retrieved records. in the obtained network view, each node is a disease, target, or drug, and each edge is a known connection between two of them. the connectivity map, or the cmap tool, allows the user to compare gene-expression profiles. the similarities or differences in the signature transcriptional expression profile and the small molecule transcriptional response profile may lead to the discovery of the mode of action of the small molecule. the response profile is also compared to response profiles of drugs in the cmap database with respect to the similarity of transcriptional responses. a network is constructed and the drugs that appear closest to the small molecule are selected to have better insight into the mode of action. other software, such as gephi, an exploration platform for networks and complex systems, and cell illustrator, a java-based tool specialized in biological processes and systems, can also be used for building networks . ayurveda, the indian traditional medicine, offers many sophisticated formulations that have been used for hundreds of years. the traditional knowledge digital library (tkdl, http://www.tkdl.res.in) contains more than , classical ayurveda formulations. approximately of these are popularly used at the community level and also as over-the-counter products. some of these drugs continue to be used as home remedies for preventive and primary health care in india. until recently, no research was carried out to explore ayurvedic wisdom using np despite ayurveda holding a rich knowledge of traditional medicine equal to or greater than tcm. our group examined the use of np to study ayurvedic formulations with the wellknown ayurvedic formulation triphala as a demonstrable example (chandran et al., a, b) . in this chapter, we demonstrate the application of np in understanding and exploring the traditional wisdom with triphala as a model. triphala is one of the most popular and widely used ayurvedic formulations. triphala contains fruits of three myrobalans: emblica officinalis (eo; amalaki) also known as phyllanthus emblica; terminalia bellerica (tb; vibhitaka); and terminalia chebula (tc; haritaki). triphala is the drug of choice for the treatment of several diseases, especially those of metabolism, dental, and skin conditions, and treatment of cancer (baliga, ) . it has a very good effect on the health of heart, skin, eyes, and helps to delay degenerative changes, such as cataracts (gupta et al., ) . triphala can be used as an inexpensive and nontoxic natural product for the prevention and treatment of diseases where vascular endothelial growth factor aÀinduced angiogenesis is involved . the presence of numerous polyphenolic compounds empowers it with a broad antimicrobial spectrum (sharma, ) . triphala is a constituent of about ayurveda formulations and it can be used for several diseases. triphala combats degenerative and metabolic disorders possibly through lipid peroxide inhibition and free radical scavenging (sabu and kuttan, ) . in a phase i clinical trial on healthy volunteers, immunostimulatory effects of triphala on cytotoxic t cells and natural killer cells have been reported (phetkate et al., ) . triphala is shown to induce apoptosis in tumor cells of the human pancreas, in both in vitro and in vivo models (shi et al., ) . although the anticancer properties of triphala have been studied, the exact mechanism of action is still not known. the beneficial role of triphala in disease management of proliferative vitreoretinopathy has also been reported (sivasankar et al., ) . one of the key ingredients of triphala is amalaki. some studies have already shown the beneficial effect of amalaki rasayana to suppress neurodegeneration in fly models of huntington's and alzheimer's diseases (dwivedi et al., (dwivedi et al., , . triphala is an effective medicine to balance all three dosha. it is considered as a good rejuvenator rasayana, which facilitates nourishment to all tissues, or dhatu. here we demonstrate the multidimensional properties of triphala using human proteome, diseasome, and microbial proteome targeting networks. the botanicals of triphala-eo, tb, and tc-contain , , and bioactives, respectively, according to unpd data collected during june . of these, a few bioactives are common among the three botanicals. thus, triphala formulation as a whole contains bioactives. out of these, bioactives were score- , based on binding db search carried out during june . eo, tb, and tc contain , , and score- bioactives, respectively ( fig. . ). the score- bioactives that are common among three plants are chebulanin, ellagic acid, gallussaeure, , -digalloyl-beta-d-glucopiranoside, methyl gallate, and tannic acid. this bioactive information is the basic step toward constructing human proteome and microbial proteome targeting networks. thirty-six score- bioactives of triphala are shown to interact with human protein targets in combinations (fig. . ) . quercetin, ellagic acid, , , , , -pentagalloylglucose and , , , -tetrakis-(o-galloyl)-beta-d-glucose are the four bioactives that interact with the maximum number of targets: , , and , respectively. the other major bioactives that have multitargeting property include catechin; epicatechin; gallocatechin; kaempferol; and trans- , ', ', , -pentahydroxylflavane. the major protein targets of triphala include alkaline phosphatase (alpl); carbonic anhydrase (ca ); coagulation factor x (f ), dna repair protein rad homolog (rad ); gstm protein (gstm ); beta-secretase (bace ); plasminogen activator inhibitor (serpine ), prothrombin (f ); regulator of g-protein signaling (rgs) , , and , tissue-type plasminogen activator (plat); and tyrosineprotein phosphatase nonreceptor type (ptpn ). the targets of triphala are associated with disease types, which include disease indications (fig. . ) . the major disease types in which triphala targets are associated include cancers, cardiovascular diseases, nervous system diseases, and metabolic diseases. analysis of existing data indicates that targets of triphala bioactives are involved in the different types of cancers making it the largest group of diseases, involving triphala targets. this linkage is through the interaction of bioactives and target proteins in different bioactiveÀtarget combinations. the types of cancers which are networked by triphala include pancreatic, prostate, breast, lung, colorectal and gastric cancers, tumors, and more. quercetin, ellagic acid, prodelphinidin a , and , , -benzenetriol are the important bioactives; and rad , bace , f , mmp , igf r, and egfr are the important targets that play a role in cancer. triphala shows links to indications of cardiovascular diseases through bioactives and targets. the cardiovascular diseases that are covered in the triphala network include atherosclerosis, myocardial ischemia, infarction, cerebral vasospasm, thrombosis, and hypertension. the bioactives playing a major role in cardiovascular diseases are quercetin, , , , , -pentagalloyoglucose, , , , -tetrakis-(o-galloyl)-beta-d-glucose, bellericagenin a , and prodelphinidin a , whereas the targets playing an important role are serpine , f , f , and fabp . triphala's network to nervous system disorders contains diseases in which the significant ones are alzheimer's disease, parkinson's disease, diabetic neuropathy, and retinopathy. in this subnetwork, bioactives interact with targets through different interactions. quercetin, , , , , -pentagalloyoglucose, , , , -tetrakis-(o-galloyl)-beta-d-glucose, and epigallocatechin- -gallate are the most networked bioactives whereas the most networked targets are bace , serpine , plat, aldr, ca . the association of triphala with metabolic disorders is determined by six bioactives that interact with seven targets. the major metabolic diseases come in this link are obesity, diabetic complications, noninsulin-dependent diabetes, hypercholesterolemia, hyperlipidemia, and more. the bioactives having more interactions with targets are ellagic acid, quercetin, and bellericagenin a , whereas the highly networked targets are igf r, fabp , aldr, and akr b . triphala bioactives are also linked to targets of other diseases comprising autoimmune diseases, ulcerative colitis, mccuneÀalbright syndrome, psoriasis, gout, osteoarthritis, endometriosis, lung fibrosis, glomerulonephritis, and more. the proteome-targeting network of triphala, thus, shows its ability to synergistically modulate targets that are associated with disease indications. this data is generated with the available information that included only one-fifth of the total number of bioactives. further logical analysis and experimental studies based on the network result are needed to explore the in-depth mechanism of action of triphala. for researchers in this area, these kind of networks can give an immense amount of information that can be developed further to reveal the real mystery behind the actions of traditional medicine. triphala is also referred to as a "tridoshic rasayana," as it balances the three constitutional elements of life. it tonifies the gastrointestinal tract, improves digestion, and is known to exhibit antiviral, antibacterial, antifungal, and antiallergic properties (sharma, ; amala and jeyaraj, ; sumathi and parvathi, ) . triphala mashi (mashi: black ash) was found to have nonspecific antimicrobial activity, as it showed a dose-dependent inhibition of gram-positive and gram-negative bacteria (biradar et al., ) . hydroalcoholic, aqueous, and ether extracts of the three fruits of triphala were reported to show antibacterial activity against uropathogens with a maximum drug efficacy recorded by the alcoholic extract (bag et al., ; prasad et al., ) . the methanolic extract of triphala showed the presence of active compounds using gc-ms and also showed potent antibacterial and antifungal activity (amala and jeyaraj, ) . triphala has been well studied for its antimicrobial activity against gram-positive bacteria, gram-negative bacteria, fungal species, and different strains of salmonella typhi (amala and jeyaraj, ; sumathi and parvathi, ; gautam et al., ; srikumar et al., ) . triphala showed significant antimicrobial activity against enterococcus faecalis and streptococcus mutans grown on tooth substrate thereby making it a suitable agent for prevention of dental plaque (prabhakar et al., (prabhakar et al., , . the application of triphala in commercial antimicrobial agents has been explored. a significant reduction in the colony forming units of oral streptococci was observed after % triphala was incorporated in a mouthwash formulation (srinagesh et al., ). an ointment prepared from triphala ( % (w/w)) showed significant antibacterial and wound healing activity in rats infected with staphylococcus aureus, pseudomonas aeruginosa, and streptococcus pyogenes (kumar et al., ) . the antiinfective network of triphala sheds light on the efficacy of the formulation in the simultaneous targeting of multiple microorganisms. also, this network provides information regarding some novel bioactiveÀtarget combinations that can be explored to combat the problem of multidrug resistance. among the bioactives of triphala, score- bioactives target microbial proteins of microorganisms. the botanicals of triphala-eo, tb, and tccontain , , and score bioactives respectively which showed interactions with microbial proteins. they act through modulation of targets which are associated with diseases such as leishmaniasis, malaria, tuberculosis, hepatitis c, acquired immunodeficiency syndrome (aids), cervical cancer, candidiasis, luminous vibriosis, yersiniosis, skin and respiratory infections, severe acute respiratory syndrome (sars), avian viral infection, bacteremia, sleeping sickness, and anthrax ( fig. . ). the microorganisms captured in the triphala antiinfective network includes candida albicans, hepatitis c virus, human immunodeficiency virus , human papillomavirus type , human sars coronavirus leishmania amazonensis, mycobacterium tuberculosis, staphylococcus aureus, plasmodium falciparum, and yersinia enterocolitica. in mycobacterium tuberculosis, dtdp- -dehydrorhamnose , -epimerase rmlc is one of the four enzymes involved in the synthesis of dtdp-l-rhamnose, a precursor of l-rhamnose (giraud et al., ) . the network shows that triphala has the potential to modulate the protein through four bioactives such as punicalins, terflavin b, -o-(s)-flavogallonyl- -o-galloylbeta-d-glucopyranose, and , -o-(s,s)-gallagyl-alpha/beta-d-glucopyranose. research on new therapeutics that target the mycobacterial cell wall is in progress. rhamnosyl residues play a structural role in the mycobacterial cell wall by acting as a linker connecting arabinogalactin polymer to peptidoglycan and are not found in humans, which gives them a degree of therapeutic potential (ma et al., ) . triphala can be considered in this line to develop novel antimycobacterial drugs. the network shows the potential of gallussaeure and -galloylgallic acid to modulate human immunodeficiency virus type reverse transcriptase. inhibition of human immunodeficiency virus at the initial stage itself is crucial and thus, targeting human immunodeficiency virus type reverse transcriptase, at the preinitiation stage is considered to be an effective therapy. protein e of human papillomavirus (hpv ) prevents apoptosis of figure . the microbial proteomeÀtargeting network of triphala. dark green verus are the botanicals of triphala and oval green nodes are the score bioactives. targets, diseases, and microorganisms are represented by blue diamond nodes, red triangle nodes, and pink octagon nodes, respectively. infected cells by binding to fadd and caspase and hence being targeted for development of antiviral drugs (yuan et al., ) . kaempferol of triphala is found to target protein e of hpv , which is a potential mechanism to control the replication of the virus. the network also shows triphala's potential to act on plasmodium falciparum. enoyl-acyl carrier protein reductase (enr) has been investigated as an attractive target due to its important role in membrane construction and energy production in plasmodium falciparum (nicola et al., ) while the parasite interacts with human erythrocyte spectrin and other membrane proteins through protein m aspartyl aminopeptidase (lauterbach and coetzer, ) . trans- , ', ', , -pentahydroxylflavane, epigallocatechin, and epicatechin can modulate both while epigallocatechin -gallate can regulate enoyl-acyl carrier protein reductase and, quercetin and vanillic acid can act on m aspartyl aminopeptidase. epigallocatechin -gallate can also target -oxoacyl-acyl-carrier protein reductase which is a potent therapeutic target because of its role in type ii fatty acid synthase pathway of plasmodium falciparum (karmodiya and surolia, ) . epigallocatechin -gallate and quercetin are the bioactives that have shown maximum antimicrobial targets interaction. while epigallocatechin -gallate shows interaction with -oxoacyl-(acyl-carrier protein) reductase, cpg dna methylase, enoyl-acyl-carrier protein reductase, glucose- phosphate -dehydrogenase, hepatitis c virus serine protease, ns /ns a and yoph of plasmodium falciparum, saccharomyces cerevisiae, and spiroplasma monobiae; quercetin acts on c-like proteinase ( cl-pro), arginase, beta-lactamase ampc, glutathione reductase, m aspartyl aminopeptidase, malate dehydrogenase and tyrosine-protein kinase transforming protein fps of escherichia coli, fujinami sarcoma virus, human sars coronavirus (sars-cov), leishmania amazonensis, plasmodium falciparum, saccharomyces cerevisiae, and thermus thermophiles. np has gained impetus as a novel paradigm for drug discovery. this approach using in silico data is fast becoming popular due to its cost efficiency and comparably good predictability. thus, network analysis has various applications and promising future prospects with regard to the process of drug discovery and development. np has proven to be a boon for drug research, and that helps in the revival of traditional knowledge. albeit there are a few limitations of using np for nep studies. this is because the bioactives form the foundation of any traditional medicine network. . absorption, distribution, metabolism, excretion, and toxic effects (admet) parameters associated with the bioactives/formulation when they are administered in the form of the medicine need to be considered in order to extrapolate in silico and cheminformatics data to in vitro and in vivo models. in silico tools that offer the prediction of these parameters can be depended on for this. but traditional medicines are generally accompanied by a vehicle for delivery of the medicine. these vehicles, normally various solvents-water, milk, lemon juice, butter, ghee (clarified butter), honey-that alter the solubility of the bioactives, play a role in regulating admet parameters. experimental validation studies are required to evaluate this principle of traditional medicine. . target identification usually relies on a single or a few databases due to the limited availability of databases with free access. this can occasionally give incomplete results. also, there may be novel targets waiting to be discovered that could be a part of the mechanism of action of the bioactives. to deal with this discrepancy in the network, multiple databases should be considered for target identification. integration of databases serving similar functions can also be a solution for this problem. in addition to this, experimental validation of the target molecules using proteinÀprotein interaction studies or gene expression studies will provide concrete testimony to the network predictions. . a number of traditional medicines act through multiple bioactives and targets. synergy in botanical drugs helps to balance out the extreme pharmacological effects that individual bioactives may have. the interactions of bioactives with various target proteins, their absorption into the body after possible enzyme degradation, their transport, and finally their physiological effect are a crucial part of traditional medicine (gilbert and alves, ) . however, in vitro assays or in silico tools are unable to give a clear idea as to the complete and exact interactions in a living organism. np is only the cardinal step toward understanding the mechanism of bioactives/formulations. but this gives an overview of the action of traditional medicine which can be used to design in vivo experiments and clinical trials. this saves time and cost of research and inventions. . it is observed that formulations are working by simultaneous modulation of multiple targets. this modulation includes activation of some targets and inhibition of other. in order to understand this complex synergistic activity of formulation, investigative studies regarding the interactions of ligands with targets are to be carried out. this can be achieved by implementing high-throughput omics studies based on the network data. network pharmacological analysis presents an immense scope for exploring traditional knowledge to find solutions for the current problems challenging the drug discovery industry. nep can also play a key role in new drug discovery, drug repurposing, and rational formulation discovery. many of the bioactiveÀtarget combinations have been experimentally studied. the data synthesis using np provides information regarding the mode of action of traditional medicine formulations, based on their constituent bioactives. this is a kind of reverse approach to deduce the molecular mechanism of action of formulations using modern, integrated technologies. the current network analysis is based on the studies that have been conducted and the literature that is available. hence, the data is inconclusive as a number of studies are still underway and novel data is being generated continuously. despite its limitations, this still is a favorable approach, as it gives insight into the hidden knowledge of our ancient traditional medicine wisdom. np aids the logical analysis of this wisdom that can be utilized to understand the knowledge as well as to invent novel solutions for current pharmacological problems. determination of antibacterial, antifungal, bioactive constituents of triphala by ft-ir and gc-ms analysis antibacterial potential of hydroalcoholic extracts of triphala components against multidrug-resistant uropathogenic bacteria--a preliminary report triphala, ayurvedic formulation for treating and preventing cancer: a review super natural ii--a database of natural products network biology: understanding the cell's functional organization the chembl bioactivity database: an update network analyses in systems pharmacology exploring of antimicrobial activity of triphala mashi-an ayurvedic formulation. evidence-based complement approaches to drug discovery omic techniques in systems biology approaches to traditional chinese medicine research: present and future network pharmacology: an emerging technique for natural product drug discovery and scientific research on ayurveda network pharmacology of ayurveda formulation triphala with special reference to anti-cancer property molecular mechanism research on simultaneous therapy of brain and heart based on data mining and network analysis anti-inflammatory mechanism of qingfei xiaoyan wan studied with network pharmacology chapter : network biology approach to complex diseases progress in computational methods for the prediction of admet properties reactome: a database of reactions, pathways and biological processes mechanism study on preventive and curative effects of buyang huanwu decoction in qi deficiency and blood stasis diseases based on network analysis an analysis of chemical ingredients network of chinese herbal formulae for the treatment of coronary heart disease in vivo effects of traditional ayurvedic formulations in drosophila melanogaster model relate with therapeutic applications ayurvedic amalaki rasayana and rasa-sindoor suppress neurodegeneration in fly models of huntington's and alzheimer's diseases tc;mgenedit: a database for associated traditional chinese medicine, gene and disease information using text mining antifungal potential of triphala churna ingredients against aspergillus species associated with them during storage rmlc, the third enzyme of dtdp-l-rhamnose pathway, is a new class of epimerase identification of responsive gene modules by networkbased gene clustering and extending: application to inflammation and angiogenesis use of natural products as chemical library for drug discovery and network pharmacology cvdhd: a cardiovascular disease herbal database for drug discovery and network pharmacology understanding traditional chinese medicine antiinflammatory herbal formulae by simulating their regulatory functions in the human arachidonic acid metabolic network evaluation of anticataract potential of triphala in selenite-induced cataract: in vitro and in vivo studies pharmacology: principles and practice online mendelian inheritance in man (omim), a knowledgebase of human genes and genetic disorders network pharmacology: the next paradigm in drug discovery vnp: interactive visual network pharmacology of diseases, targets, and drugs detection of characteristic sub pathway network for angiogenesis based on the comprehensive pathway network analyses of co-operative transitions in plasmodium falciparum beta-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding network pharmacology applications to map the unexplored target space and therapeutic potential of natural products triphala promotes healing of infected full-thickness dermal wound chemo-and bioinformatics resources for in silico drug discovery from medicinal plants beyond their traditional use: a critical review network-based drug discovery by integrating systems biology and computational technologies systems pharmacologybased approach for dissecting the addition and subtraction theory of traditional chinese medicine: an example using xiao-chaihu-decoction and da-chaihu-decoction a network pharmacology approach to determine active compounds and action mechanisms of ge-gen-qin-lian decoction for treatment of type diabetes traditional chinese medicinebased network pharmacology could lead to new multicompound drug discovery framework and practice of network-based studies for chinese herbal formula traditional chinese medicine network pharmacology: theory, methodology and application. chin constructing biological networks through combined literature mining and microarray analysis: a lmma approach understanding zheng in traditional chinese medicine in the context of neuro-endocrine-immune network herb network construction and comodule analysis for uncovering the combination rule of traditional chinese herbal formulae network target for screening synergistic drug combinations with application to traditional chinese medicine analysis on correlation between general efficacy and chemical constituents of danggui-chuanxiong herb pair based on artificial neural network network pharmacology study on major active compounds of fufang danshen formula a network pharmacology study of chinese medicine qishenyiqi to reveal its underlying multi-compound, multitarget, multi-pathway mode of action herb network analysis for a famous tcm doctor' s prescriptions on treatment of rheumatoid arthritis. evidence-based complement a novel network pharmacology approach to analyse traditional herbal formulae: the liu-wei-di-huang pill as a case study bindingdb: a web-accessible database of experimentally determined protein-ligand binding affinities network pharmacology study on major active compounds of siwu decoction analogous formulae for treating primary dysmenorrhea of gynecology blood stasis syndrome computational pharmacological comparison of salvia miltiorrhiza and panax notoginseng used in the therapy of cardiovascular diseases triphala and its active constituent chebulinic acid are natural inhibitors of vascular endothelial growth factor-a mediated angiogenesis computational identification of potential microrna network biomarkers for the progression stages of gastric cancer systems pharmacology strategies for anticancer drug discovery based on natural products multiscale modeling of druginduced effects of reduning injection on human disease: from drug molecules to clinical symptoms of disease drug targeting mycobacterium tuberculosis cell wall synthesis: genetics of dtdp-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dtdp-glucose to dtdp-rhamnose bridging the gap between traditional chinese medicine and systems biology: the connection of cold syndrome and nei network analysis and visualization of biological networks with cytoscape discovery of novel inhibitors targeting enoyl-acyl carrier protein reductase in plasmodium falciparum by structure-based virtual screening global mapping of pharmacological space rediscovering drug discovery network ethnopharmacology approaches for formulation discovery integrative approaches for health: biomedical research, ayurveda and yoga material basis of chinese herbal formulas explored by combining pharmacokinetics with network pharmacology the disease and gene annotations (dga): an annotation resource for human disease significant increase in cytotoxic t lymphocytes and natural killer cells by triphala: a clinical phase i study. evid. based complement alternat the human protein atlas as a proteomic resource for biomarker discovery evaluation of antimicrobial efficacy of herbal alternatives (triphala and green tea polyphenols), mtad, and % sodium hypochlorite against enterococcus faecalis biofilm formed on tooth substrate: an in vitro study evaluation of antimicrobial efficacy of triphala (an indian ayurvedic herbal formulation) and . % chlorhexidine against streptococcus mutans biofilm formed on tooth substrate: an in vitro study potent growth suppressive activity of curcumin in human breast cancer cells: modulation of wnt/beta-catenin signaling tcmsp: a database of systems pharmacology for drug discovery from herbal medicines anti-diabetic activity of medicinal plants and its relationship with their antioxidant property databases aim to bridge the east-west divide of drug discovery cytoscape: a software environment for integrated models of biomolecular interaction networks network pharmacology analyses of the antithrombotic pharmacological mechanism of fufang xueshuantong capsule with experimental support using disseminated intravascular coagulation rats a network pharmacology approach to understanding the mechanisms of action of traditional medicine: bushenhuoxue formula for treatment of chronic kidney disease triphala inhibits both in vitro and in vivo xenograft growth of pancreatic tumor cells by inducing apoptosis aqueous and alcoholic extracts of triphala and their active compounds chebulagic acid and chebulinic acid prevented epithelial to mesenchymal transition in retinal pigment epithelial cells, by inhibiting smad- phosphorylation evaluation of the growth inhibitory activities of triphala against common bacterial isolates from hiv infected patients antibacterial efficacy of triphala against oral streptococci: an in vivo study antibacterial potential of the three medicinal fruits used in triphala: an ayurvedic formulation dissection of mechanisms of chinese medicinal formula realgar-indigo naturalis as an effective treatment for promyelocytic leukemia a network study of chinese medicine xuesaitong injection to elucidate a complex mode of action with multicompound, multitarget, and multipathway phytochemical and pharmacological review of da chuanxiong formula: a famous herb pair composed of chuanxiong rhizoma and gastrodiae rhizoma for headache in silico analysis and experimental validation of active compounds from fructus schisandrae chinensis in protection from hepatic injury discovery of molecular mechanisms of traditional chinese medicinal formula si-wu-tang using gene expression microarray and connectivity map biology-oriented synthesis a network pharmacology approach to evaluating the efficacy of chinese medicine using genome-wide transcriptional expression data identifying roles of "jun-chen-zuo-shi" component herbs of qishenyiqi formula in treating acute myocardial ischemia by network pharmacology network-based global inference of human disease genes the study on the material basis and the mechanism for anti-renal interstitial fibrosis efficacy of rhubarb through integration of metabonomics and network pharmacology a systems biology-based approach to uncovering the molecular mechanisms underlying the effects of dragon's blood tablet in colitis, involving the integration of chemical analysis, adme prediction, and network pharmacology study on action mechanism of adjuvant therapeutic effect compound ejiao slurry in treating cancers based on network pharmacology alternative medicine. intech network pharmacological research of volatile oil from zhike chuanbei pipa dropping pills in treatment of airway inflammation navigating traditional chinese medicine network pharmacology and computational tools modularity-based credible prediction of disease genes and detection of disease subtypes on the phenotype-gene heterogeneous network small molecule inhibitors of the hpv -e interaction with caspase an integrative platform of tcm network pharmacology and its application on a herbal formula network understanding of herb medicine via rapid identification of ingredient-target interactions dbnei . : building multilayer network for drug-neidisease systems pharmacology dissection of the anti-inflammatory mechanism for the medicinal herb folium eriobotryae network pharmacology study on the mechanism of traditional chinese medicine for upper respiratory tract infection a network pharmacology approach to determine active ingredients and rationality of herb combinations of modifiedsimiaowan for treatment of gout a co-module approach for elucidating drug-disease associations and revealing their molecular basis deciphering the underlying mechanisms of diesun miaofang in traumatic injury from a systems pharmacology perspective network pharmacology-based prediction of the multi-target capabilities of the compounds in taohong siwu decoction, and their application in osteoarthritis a network-based analysis of the types of coronary artery disease from traditional chinese medicine perspective: potential for therapeutics and drug discovery therapeutic target database update : a resource for facilitating target-oriented drug discovery multi-target therapeutics: when the whole is greater than the sum of the parts studying traditional medicine that would hopefully get resolved in the future. the major limitations and possible solutions are listed: . nep currently relies on various databases for literature and bioactive mining. databases, though curated, may show discrepancies due to numerous sources of information, theoretical, and experimental data. moreover, the botanicals that undergo certain preparatory procedures during the formulation of the medicine may have its constituents that have chemically changed due to the procedures; like boiling, acid/ alkali reactions, interactions between the bioactives, etc. a way to navigate around this problem is to make use of modern, high-throughput chemical identification techniques like ultra-performance liquid chromatogra-phyÀelectrospray ionizationÀtandem mass spectroscopy (uplc-esi-ms/ms). this technique will help to identify the exact bioactives or the chemical constituents of the formulation, and will enrich the subsequent key: cord- -qxiynbl authors: nabi, bushra; rehman, saleha; aggarwal, sumit; baboota, sanjula; ali, javed title: nano-based anti-tubercular drug delivery: an emerging paradigm for improved therapeutic intervention date: - - journal: drug deliv transl res doi: . /s - - - sha: doc_id: cord_uid: qxiynbl tuberculosis (tb) classified as one of the most fatal contagious diseases is of prime concern globally. mycobacterium tuberculosis is the causative agent that ingresses within the host cells. the approved conventional regimen, though the only viable option available, is unfavorably impacting the quality of life of the affected individual. despite newer antibiotics gaining light, there is an unending demand for more therapeutic alternatives. therefore, substantial continuous endeavors are been undertaken to come up with novel strategies to curb the disease, the stepping stone being nanotechnology. this approach is instrumental in overcoming the anomalies associated with conventional therapy owing to their intriguing attributes and leads to optimization of the therapeutic effect to a certain extent. this review focusses on the different types of nanocarrier systems that are being currently explored by the researchers for the delivery of anti-tubercular drugs, the outcomes achieved by them, and their prospects. [figure: see text] tb, being one of the most prevalent infectious and contagious disease caused by mycobacterium tuberculosis (mtb), has a huge global burden accounting to about million new cases in . according to the who report , around . million deaths take place due to tb. additionally, also onethird of the world population is latently affected by it. the worst affected geography is the asian subcontinent with india reporting the highest tally [ ] [ ] [ ] [ ] [ ] [ ] . owing to the limited number of approved anti-tb drugs, the emergence of multi and extensively drug-resistant strains of tb has been a problematic issue. in the case of drug-susceptible cases, the initial phase consists of months of combinatorial treatment commonly called "drug cocktail" which consists of isoniazid (inh), rifampicin (rif), ethambutol (emb), and pyrazinamide (pza). this is further followed by a continuation phase of months comprising of inh and rif. however, bushra nabi and saleha rehman contributed equally to this work. there are drug-resistant strains that follow a different treatment regimen. the drug resistance occurs principally due to mutations occurring in drug target genes. the other causes might be attributable to the impermeability of the mtb cell wall or the activity of efflux pumps. multidrug resistance occurs when patients become resistant at least to inh or rif. on the other hand, extensive drug resistance occurs on developing resistance to inh and rif along with fluoroquinolones. in the case of inh resistance, the treatment regimen incorporates months of rif, emb, pza, and levofloxacin [ ] [ ] [ ] . an extensive literature survey was carried out on databases such as scopus, pubmed, sciencedirect, and google scholar. primarily research conducted on anti-tubercular nano-formulations within the duration of - was considered for incorporation within the manuscript. few breakthrough review articles, however, limited in number, have also been incorporated to give the readers a clear concept. the lungs appear as an attractive target for the therapeutic delivery of anti-tb drugs. the inhalational route of drug administration serves as the primary option for delivering dry powder inhalers or aerosols, since they provide a preferential accumulation of drugs at the target site in addition to being non-invasive and overcoming gastrointestinal drug degradation. they can thus also restrict drug exposure to healthy cells. however, they were also associated with certain loopholes such as bolus drug release culminating into severe lung toxicity, short residence time, and poor aqueous solubility [ ] . the other contemporary tb treatment includes oral and parenteral dosage forms. however, they lead to sub-therapeutic levels attributable to poor drug distribution to the lungs, ultimately causing drug-resistant strains [ ] . the commercially available conventional anti-tb therapy is associated with innumerable glitches such as metabolic instability, low solubility, low permeability, a large number of allied side effects especially hepatotoxicity, and high drug load [ , ] . moreover, the combination therapy also leads to complex dosing schedules frequently causing inadequate therapeutic effect [ ] . therefore, these drawbacks with conventional therapy led to the paradigm shift towards nanotechnology-engineered delivery systems. the nanocarrier systems have established their supremacy over conventional therapy since they have the ability to incorporate both lipophilic and hydrophilic drugs. they offer advantages such as the drastic reduction in size hence providing high surface volume ratio, the protection of drug moiety from degradation, the targeted and site-specific delivery, follow a controlled release mechanism, and could be easily functionalized to modify drug therapeutic profile. additionally, one of the prime outlooks might be the biocompatible nature of these systems [ , ] . furthermore, the lipid-based systems hold special significance, since the pathogen, i.e., mtb, relies on the lipidic membrane to ingress with the host body and egress from the same [ ] . furthermore, nanoparticulate systems possess intrinsic antimycobacterial activity along with serving as a vehicle for various anti-tubercular drugs, hence being instrumental in reducing the dosing frequency along with associated side effects. additionally, nanoformulations incorporating inorganic metals have antibacterial activity. silver and gold nanoparticles (nps) demonstrated promising in vitro antimycobacterial activity [ ] . moreover, targeted site-specific delivery could be obtained via inhalational nps [ ] . nanotechnology primarily focusses on "technology transfer," whereby novel techniques are implemented on a conventional treatment regimen to boost the therapeutic process. however, establishing the toxicity and safety profile of these nano-formulations is a daunting task and needs to be investigated further [ ] . therefore, the nano-based anti-tubercular therapy system overcomes the obsolete techniques of conventional regimen and revamps the drug delivery systems, hereafter promoting the healthcare. impetus must be laid on implementing novel prospects for restricting the boundaries for the propagation of tb. no doubt, plethora of anti-bacterial agents is existing, which bears activity against mtb; however, these synthetic antimicrobials are associated with unavoidable glitches, most prominent among them being drug resistance. consequently, researchers have been exploiting this domain to come up with novel techniques and anti-tb leads. hence, plant-derived alternatives suit fashion and serve as probable leads, which warrants tremendous exploration [ ] . researches have come to light of late, which explicitly establishes the prospects of herbal origin drugs when incorporated in nano-formulations. the different categories of nano-based systems that have been researched for the treatment of tb have been undermentioned. nps have gained impetus owing to their small dimension of around nm. however, they comprise primarily of three layers, i.e., the outer layer, which is functionalized by either polymers or metal ions which give specific characteristics to these nps, thus dividing them into different categories. the middle layer forms the shell of the nps, while the inner layer is the core [ ] . thus, this section on nps deals with their different types such as polymeric nps or metallic nps. polymeric nps have garnered speculations attributable to their remarkable credentials such as passive deposition in the target site, biocompatibility, physicochemical stability, and modified release. furthermore, the hydrophilic outer core and hydrophobic inner core allow for varied drugs to be entrapped within it [ ] . all these abilities explicitly justify the use of nps in tb therapy. therefore, nps have been the favorite among the researchers, and abundant of them are available in the public domain. praphakar et al. tailored rif silver nps on chitosan (cs)-grafted cetyl alcohol-maleic anhydride-pza polymer for ameliorated drug efficacy. it was observed that % of the drug was released within a span of days and the anti-bacterial activity was ameliorated by the formulation. the vero cell line was used for the cytotoxicity analysis, while thp- cells were employed for cell apoptosis assay. the results established the supremacy of nps [ ] . tenland et al. studied the effect of a novel antimycobacterial peptide, nzx, on the colonization of mtb. a slow release of . % was observed from the nps in the duration of h. primary macrophages and thp- cells used for uptake studies unraveled the fact that primary macrophage cell lines had greater cellular uptake. there was decline in cfu values, i.e., % reduction in the nzx group; % in the np group; and % in the rif-treated group, which was taken as standard, thus establishing the effectiveness of the prepared formulation [ ] . in the research carried out by bachhav et al., rif-loaded nps were fabricated by conjugation with gantrezan- and using ethyl cellulose as polymer. lymph-mediated lung targeting was elucidated from the fluorescence microscopy. a -fold increment in the auc/ minimum inhibitory concentration (mic) ratio was observed for the prepared np, whereas there is a . ± . % increase in relative bioavailability (ba). additionally, hepatotoxicity was augmented by the formulation [ ] . bhusal and colleagues developed glycan-coated magnetic nps for the detection of acid-fast bacilli in sputum by making use of np-based colorimetric biosensing assay. this biosensing technique might bear fruitful results through adequate implementation and facilitating the health care process. the outcome clarified that the designed system was in conformation with the established benchmark xpert mtb/rif in terms of specificity and selectivity [ ] . in the research carried out by saifullah et al., emb was loaded in graphene oxide-iron oxide magnetite nps. a sustained in vitro release was observed along with potent anti-tubercular activity [ ] . rawal et al. developed rif cs np to be delivered as an inhalational system. the safety analysis was carried out using j macrophage cells, whereby the % cell viability was calculated to be - % at a dose of . mg/ml. the formulation also did not show any trace of toxicity [ ] . in a different study envisaged by thomas et al., they developed alginate cellulose nanocrystal hybrid nps of rif. a slow initial drug release of only - % was observed in h, which gradually increased up to % in h on increasing the ph to . . the l cell lines were used to perform the mtt assay, which demonstrated the % cell viability to be nearing % [ ] . in a different study, viswanathan and his colleagues developed mannosylated gelatin np of licorice. the in vitro studies revealed enhanced cellular uptake in raw . from the formulation. furthermore, the pharmacokinetic (pk) study of the formulation showed the existence of the optimal drug level in the body even after h of drug administration. the in vivo antitubercular activity was confirmed since there was a significant decline in bacterial load in the lungs and spleen when mtb h rv-infected mice were treated with mannosylated np on the contrary to the untreated mice [ ] . malik and colleagues formulated poly (lactic-co glycolic) acid (plga) nps by loading bivalent h antigen, which is a fusion of mtb ag b and esat proteins. the developed nps were internalized by thp- human macrophages and later used for immunizing c bl/ j mice. there was a significant increase in the total serum igg level when compared with h alone. a . -fold increase in cytokine level and . -fold increment in il level were also observed demonstrating the efficacy of the nps [ ] . apart from the aforementioned studies, there were uncountable researches undertaken pertaining to np. however, few of them with outstanding outcomes have been elaborated in table . solid lipid nanoparticles (slns) are lipidic carrier systems encompassing solid lipids and surfactants. they have garnered recognition lately by virtue of their attributes, namely, easy to scale up, inhibit drug degradation, ameliorating the pharmacological profile of the drug, desirable physical stability, and controlled or modified drug release [ ] . in order to make the formulation accepted by the masses, numerous researches were undertaken. costa and associates fashioned a conventional sln formulation of inh and also by functionalization with mannose for contrasting evaluation. the outcome revealed that both the slns prepared curtailed the possibility of any toxicity on the human lung epithelial cell line (nci-h ) and differentiated thp- . nevertheless, the functionalized sln has enhanced uptake caliber in macrophagic cells and demonstrated receptor-dependent internalization [ ] . gaspar et al. developed rfb sln using two different lipid constituents, namely, glyceryl dibehenate and glyceryl tristearate. in vitro uptake studies from thp cells in the macrophagic system showed ± % for glyceryl dibehenate and ± % for glyceryl tristearate, directing preferable proportion towards glyceryl dibehenate. the cell viability study conducted on a and calu- cells gave evidence of low cytotoxicity nearing % [ ] . nemati et al., taking into consideration the astounding characteristics of sln, developed emb sln as an inhalational delivery system. a cells were employed for the evaluation of toxicity, which depicted no significant cytotoxicity. a sustained release of % was reported from sln, while the free drug has a release of % rif magnetic iron oxide np the np prepared was cross-linked with polyethylene glycol hybrid cs to form the gel beads, which established its potential applicability as an anti-tb system [ ] rif octanoyl cs np a sustained release of . ± . % was quantified from the np. in vitro biocompatibility and no significant cytotoxicity was observed. [ ] rif [ ] . shipli et al. fabricated rif sln, which was conjugated with lactoferrin. preliminary characterization was carried out. in vitro release studies exhibited enhanced output from the unconjugated system while the in vivo biodistribution study revealed a . -fold increment in ba and amplification in drug uptake from the conjugated system [ ] . in the study conducted by banerjee et al., dual drugloaded (rif and inh) lipid nanoparticulate system was tailored, which demonstrated sustained in vitro release pattern. the developed carrier system effectively localized within the different compartments of thp- . the in vivo studies revealed that the relative ba from the sln was . fold higher than the drug suspension, proving the effectiveness of the developed formulation [ ] . there are certain other pertinent researches that were carried out and governed by optimistic results as shown in table . nanostructured lipid carrier (nlc) is a newer generation of lipidic carrier systems that were devised to overcome the lacunas of sln, which restricts its extensive adoption. they are the amalgamation of both solid and liquid lipids, hence possessing diminution in crystallinity and a loosely packed matrix system. this leads to the overall increase in drug entrapment capability and superior stability necessitating its further investigation [ ] . innumerable studies have been steered globally, which established its futuristic commercial applicability, nevertheless demanding exhaustive research. carneiro et al. developed rif nlc functionalized with a tuftsin-modified peptide. the drug release profile showed an initial burst release followed by a controlled release. the fluorescent labeled nlc depicted improved cellular uptake, enhanced cell internalization, and a -fold increase in activity against mtb in contract to the free drug [ ] . nlc of different copper complexes was fabricated by sato and colleagues. the in vitro activity of the formulation against mtb h r v showed a value of . , . , and . folds along with a reduction in toxicity profile [ ] . table represents certain other researches which need to be brought forward. nanoemulsion (ne), being a robust carrier, incorporates a wide range of therapeutic agents along with offering stupendous advantages such as increased drug loading and stability, improved bioavailability, and controlled release. when administered via the oral route, nes not only enhance the solubility of the hydrophobic drugs but also prolong the git residence time and improve lymphatic uptake, thereby avoiding the first-pass metabolism [ ] . shah et al. took note of the qualities of ne and fabricated st-generation rif ne and its subsequent functionalization with cs and cs-folate. the inhalational efficiency was greater than % for the decorated ne coupled with high lung content, an increase in cell internalization, and improvement in cytotoxic profile [ ] . halicki et al. designed rif ne and conducted resazurin microtiter assay to determine its antimycobacterial potential. the mic value for the developed ne was . μg/ml, while the free drug was μg/ml. the incorporation of the drug within the lipidic framework of ne led to a decline in drug degradation, whereby improving the pharmacological drug profile [ ] . shobo and associates developed ne of pretomanid, which was to be administered intranasally. the peak concentration of the drug in the brain was estimated to be , . ng/g, which was significantly greater than the optimal concentration required, henceforth proving the effectiveness of the prepared formulation [ ] . [ ] rif the optimized formulation manifested anti-lipolytic potential and was stable in git media. a biphasic drug release pattern was observed. [ ] rfb reduction in mycobacterial load was evident. decline in growth index value for the treated animals when compared with control for liver was . ± . to . ± . , for spleen . ± . to . ± . , and for lung . ± . to . ± . . [ ] rif the inhalational delivery system was developed and optimized using the design of experiments. a high respirable fraction (> %) was confirmed, which established the efficiency of the formulation. a drug payload of - % was released within h of the inhalation. [ ] rfb the sln prepared was not susceptible to degradation by the gastric media, thereby protecting the drug entrapped within its core. a -fold increase in relative ba was observed from the sln in comparison with free drug. [ ] rif cs-coated sln revealed an increase in the mucoadhesive property of the formulation coupled with enhancement in permeability in alveolar epithelial cells a when compared with uncoated sln. [ ] drug deliv. and transl. res. liposome (lp) delivery systems, a lipid-based vesicular system, display tremendous intrinsic potentialities viz. biocompatibility, biodegradability, could easily be tailored and engineered, and immunogenic in addition to being formulated using phospholipids. lps are known to be engulfed by the macrophagic cells to meet their fate, hence being a probable candidate for anti-tb drugs since mtb invests the macrophages. these attributes account for their versatile nature and urge for further exploration [ , ] . liu et al. designed thermoresponsive lp in the hydrogel system for delivering inh at the target site for bone tb. local drug delivery systems offer stupendous advantage by maximal drug delivery at the target site and negligible systemic exposure. the in vivo studies manifested rapid drug availability in synovial fluid post-injection. furthermore, the cytotoxicity study conducted using the mtt assay affirmed no toxicity, thus substantiating the use of the prepared formulation in bone tb [ ] . hamed and his co-workers fabricated spray-dried nano-lps incorporated in microparticles of mxf for improved lung deposition of the drug. the surface was modified using -aminophenyl-alphad-manno-pyranoside, which facilitated improved drug uptake by alveolar macrophages. the biphasic drug release mechanism was observed from the formulation coupled with improved mic values [ ] . in the study carried out by viswanathan et al., inhalational lps were prepared from glycyrrhiza glabra, which contains licorice in it. the in vivo lung deposition studies in mice revealed that % of the drug directed reaches the lungs, while % still persists there post -h duration. the study also established the decline in bacterial count and growth in the lungs and spleen of tb infected balb/c mice. no toxicity was evident even on administering times the normal dose, thus paving the way for its use as an effective anti-tb drug [ ] . table puts forth a few additional commendable researches and their remarkable outcomes. micelles have principally been explored for hydrophobic drugs attributable to their amphiphilic nature [ ] . tripodo et al. tailored micelles of rif based on inulin modified with vitamin e. the mic values obtained post formulation administration showed superior activity against bacterial strains as compared with free drug. cytocompatibility on human alveolar macrophages demonstrated a higher value of more than % [ ] . prabhakar and his colleagues constructed polymeric micelles loaded with rif and inh. after a duration of days, the % drug release of inh and rif was found to be . % and . % respectively at ph . . lrp assay was performed to determine the anti-tb activity which depicted the inhibition of mtb h rv by the formulation. they also assessed the cell viability against l and u cells by [ ] soybean lecithin lp inh the research remarkably represents the worth of naturally occurring polysaccharides in promoting pulmonary and macrophage-targeted delivery of anti-tb drugs. [ ] dda/tdb lp fusion peptide of hspx, ppe , and esxv the effect of bcg vaccine was elevated by the use of the formulation, and it provided a synergistic effect. [ ] [ ] a comparison was drawn between the sln and nlc of the drugs. sln and nlc revealed . -and -fold increases in inh protection from degradation. effective intracellular trafficking of the drugs was observed from the lipid formulations nlc being better engulfed by the macrophage and having significantly higher distribution in the cytosol. [ ] pza pegylation was commenced. the drug release from conventional nlc was %, while pegylated nlc caused a decrease and was observed to be . %. nonetheless, the biphasic release was observed in both cases. [ ] rif mannosylated nlc demonstrated a decline in intracellular mycobacterium growth and an upsurge in uptake by bone marrow-derived macrophages. [ ] mtt assay. fluorescence microscopy corroborated cell deformation. furthermore, the in vivo localization study in zebrafish and hemolysis assay conformed to the standards and revealed its potential to enhance the therapeutic and pharmacological aspects of the drug [ ] . the rif-loaded pulmonary nano-based micellar system was developed by grotz et al. the in vitro aerodynamic study revealed the suitability in alveolar delivery. the in vitro bactericidal activity was increased up to . folds in mtb-infected thp- macrophages when compared with rif solution [ ] . other promising researches have been mentioned in table . in addition to the nano-formulations described above, the area has been laden with surplus other nanocarriers, which are yet to be investigated to their fullest or are underexploited. however, few potential researches carried out globally have been undermentioned in table . based on the results obtained from the researches carried out, it could rightly be said that the nano-based tubercular systems could revitalize the drug delivery process and improve the therapeutic profiling of the drugs. since tb is a condition affecting people globally with a soaring morbidity and mortality index, there is an upsurge in the researches to deduce novel approaches for combating the disease. focus ought to be laid on the development of newer antimicrobial peptides for therapeutic intervention and to overcome the lacunas of the conventional commercially available therapies [ , ] . photodynamic therapies could also be employed in drug-resistant cases, since they yield reactive oxygen species [ ] . gastric resistant systems and drug depot formulations [ ] core-shell micelles of pluronics rif and inh the micelles boosted the anti-bacterial ability in addition to enhanced drug permeability when equated with free drug. [ ] the optimized formulation showed a mic of . ± . mg/ml, and fluorescence intensity was . %, while % from the dye solution. [ ] lipopolysaccharide polyelectrolyte complex the result of ex vivo permeability studies demonstrated that the formulation significantly enhanced the permeability by folds when compared with free drug [ ] halloysite nanotubes inh the drug-loaded formulation exhibited superior in vitro biocompatibility towards caco- cells in comparison with free drug. [ ] nanocage zinc oxide the study measured the mic as . mg/ml, thereby establishing the potential of developed nanocage in overcoming tb. [ ] nanocomposites rif and pza the prepared formulations displayed sustained release of % and % for rif and pza, respectively, along with depicting the improved antimycobacterial activity. [ ] nano-lipomer rif the lipomer developed demonstrated rapid dissolution profile owing to its initial burst release. [ ] lipomer rif the study elucidated higher peyer's patch uptake by the formulation followed by significantly greater lung:plasma concentration ratio. [ ] nanosphere rif the aerosol formulation developed exhibited a significant delay in drug release by adapting to the sustained release mechanism. [ ] niosomes eth and d-cycloserine the formulation was optimized using bbd, which depicted superior bacterial inhibition property when compared with free drug. [ ] vesicles artemisone, clofazimine, and decoquinate among the varied formulations prepared by zyl et al., niosomes exhibited maximum percentage inhibition of % against mtb h rv laboratory strain. [ ] would give a radical solution to some of the relevant issues pertaining to the therapy [ ] . newer functionalization and surface modification techniques to suit the purpose should be repeatedly tried to pave the way for the development of newer products. it could also be rightly stated that herbal origin derivatives could be instrumental in delivering a holistic formulation with minimal side effects, though necessitating in-depth research. additionally, it could be formulated in conjugation with synthetic drugs proposing a synergistic relation, thus positioning them as a strong competitor in future researches. a major breakthrough in anti-tb dosage regimen will be nanocarrierbased depot formulation. this will be instrumental in improving patient compliance by reducing the dosing frequency and minimizing the side effects [ ] . furthermore, tb-associated diseases also cause a concern, e.g., in tb-associated hiv. hence, integrating nanotechnology to develop combination preparation to treat both the conditions would bear fruitful results. moreover, till date, not much has been explored in the arena of vaccine development. therefore, nanotechnologybased tb vaccine, if developed in the future, will prove to be a breakthrough in the field of drug discovery [ ] . additionally, ligand targeting is one arena that needs to be exploited completely such as mycolic acid like targeting ligands [ ] . repurposing of established drugs can be employed for establishing a newer treatment regimen. for example, fluoroquinolones and clofazimine all were repurposed drugs. there are multiple drugs in clinical trials for mdr-tb. however, to overcome this epidemic, improvement in the management of susceptible tb needs to be taken care of [ ] . the review anticipates the use of nanotechnology as a boon for the delivery of anti-tubercular drugs. a plethora of drugs, which is associated with issues hindering their pharmacological profiles, could be incorporated within these nanocarrier systems, henceforth leveraging an increase in therapeutic effectiveness. although the state of the art for these systems warrants to be exploited to its fullest to make these systems come to reality and hit the markets for commercial applicability. recent insights into mycobacterium tuberculosis through proteomics and implications for the clinic potential of dry powder inhalers for tuberculosis therapy: facts, fidelity and future a novel approach in treatment of tuberculosis by targeting drugs to infected macrophages using biodegradable nanoparticles nanomedicines towards targeting intracellular mtb for the treatment of tuberculosis combination drug therapy via nanocarriers against infectious diseases scrutinizing the drug resistance mechanism of multi-and extensively-drug resistant mycobacterium tuberculosis: mutations versus efflux pumps the world health organization standards for tuberculosis care and management inhalable nanotherapeutics to improve treatment efficacy for common lung diseases exploring the use of lipid based nano-formulations for the management of tuberculosis solid nanoparticles for oral antimicrobial drug delivery: a review recent therapeutic approaches for the management of tuberculosis: challenges and opportunities how can nanoparticles contribute to antituberculosis therapy? lipids in infectious diseases-the case of aids and tuberculosis how can nanoparticles contribute to antituberculosis therapy? barriers for the development, translation, and implementation of nanomedicine: an african perspective new agents for the treatment of drug resistant mycobacterium tuberculosis nanoparticles: properties, applications and toxicities development of extended-voyaging anti-oxidant linked amphiphilic polymeric nanomicelles for anti-tuberculosis drug delivery silver nanoparticle functionalized csg-(ca-ma-pza) carrier for drug deliv. and transl. res. sustainable anti-tuberculosis drug delivery effective delivery of the anti-mycobacterial peptide nzx in mesoporous silica nanoparticles exploring peyer's patch uptake as a strategy for targeted lung delivery of polymeric rifampicin nanoparticles nanoparticle-based biosensing of tuberculosis, an affordable and practical alternative to current methods nano-formulation of ethambutol with multifunctional graphene oxide and magnetic nanoparticles retains its antitubercular activity with prospects of improving chemotherapeutic efficacy rifampicin loaded chitosan nanoparticle dry powder presents: an improved therapeutic approach for alveolar tuberculosis synthesis and in vitro evaluation of alginate-cellulose nanocrystal hybrid nanoparticles for the controlled oral delivery of rifampicin mannosylated gelatin nanoparticles of licorice for use in tuberculosis: formulation, in vitro evaluation, in vitro cell uptake, in vivo pharmacokinetics and in vivo anti-tubercular efficacy single-dose ag b-esat -loaded poly(lacticco-glycolic acid) nanoparticles confer protective immunity against tuberculosis in vitro pharmacokinetic cell culture system that simulates physiologic drug and nanoparticle exposure to macrophages promising chitosan-coated alginate-tween nanoparticles as rifampicin coadministered ascorbic acid delivery carrier against mycobacterium tuberculosis surface layer modification of poly(d,l-lactic-co-glycolic acid) nanoparticles with targeting peptide: a convenient synthetic route for pluronic f −tuftsin conjugate system with embedded drug release and nanoparticle degradation sensor showing efficient rifampicin delivery into macrophages hpma-plga based nanoparticles for effective in vitro delivery of rifampicin development and in vitro release of isoniazid and rifampicin-loaded bovine serum albumin nanoparticles antitubercular activity of zno nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel surface-modified gatifloxacin nanoparticles with potential for treating central nervous system tuberculosis enhancing antimycobacterial activity of isoniazid and rifampicin incorporated norbornene nanoparticles magnetic iron oxide nanoparticles (mions) cross-linked natural polymer-based hybrid gel beads: controlled nano anti-tb drug delivery application development of novel octanoyl chitosan nanoparticles for improved rifampicin pulmonary delivery: optimization by factorial design antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against mycobacterium tuberculosis h rv strain enhanced delivery of -thioureidoiminomethyl pyridinium perchlorate in tuberculosis models with igg functionalized poly(lactic acid)-based particles microencapsulated solid lipid nanoparticles as a hybrid platform for pulmonary antibiotic delivery mannose-functionalized solid lipid nanoparticles are effective in targeting alveolar macrophages rifabutin-loaded solid lipid nanoparticles for inhaled antitubercular therapy: physicochemical and in vitro studies ethambutol-loaded solid lipid nanoparticles as dry powder inhalable formulation for tuberculosis therapy solid lipid nanoparticles for ocular delivery of isoniazid: evaluation, proof of concept and in vivo safety & kinetics assessment of lactoferrin-conjugated solid lipid nanoparticles for efficient targeting to the lung mechanisms of the effectiveness of lipid nanoparticle formulations loaded with antitubercular drugs combinations toward overcoming drug bioavailability in tuberculosis improved antibacterial function of rifampicin-loaded solid lipid nanoparticles on brucella abortus formulation, optimization and characterization of rifampicin loaded solid lipid nanoparticles for the treatment of tuberculosis solid lipid nanoparticle assemblies (slnas) for an anti-tb inhalation treatment-a design of experiments approach to investigate the influence of pre-freezing conditions on the powder respirability lipid nanocarriermediated drug delivery system to enhance the oral bioavailability of rifabutin mucoadhesive chitosan-coated solid lipid nanoparticles for better management of tuberculosis design of a nanostructured lipid carrier intended to improve the treatment of tuberculosis functionalized rifampicin-loaded nanostructured lipid carriers enhance macrophages uptake and antimycobacterial activity nanostructured lipid carriers for incorporation of copper(ii) complexes to be used against mycobacterium tuberculosis. drug des dev ther new active formulations against m. tuberculosis: bedaquiline encapsulation in lipid nanoparticles and chitosan nanocapsules comparative study of oral lipid nanoparticle formulations (lnfs) for chemical stabilization of antitubercular drugs: physicochemical and cellular evaluation role of peg in the surface modification and physicochemical characteristics of pyrazinamide loaded nanostructured lipid carriers targeted macrophages delivery of rifampicinloaded lipid nanoparticles to improve tuberculosis treatment nanoemulsions in drug delivery: formulation to medical application critical physicochemical and biological attributes of nanoemulsions for pulmonary delivery of rifampicin by nebulization technique in tuberculosis treatment alternative pharmaceutical formulation for oral administration of rifampicin enhanced brain penetration of pretomanid by intranasal administration of an oil-in-water nanoemulsion macrophages targeted drug delivery as a key therapy in infectious disease development and evaluation of a dry powder formulation of liposomeencapsulated oseltamivir phosphate for inhalation a thermo-responsive and self-healing liposome-in-hydrogel system as an antitubercular drug carrier for localized bone tuberculosis therapy enhanced antitubercular activity, alveolar deposition and macrophages uptake of mannosylated stable nanoliposomes inhalable liposomes of glycyrrhiza glabra extract for use in tuberculosis: formulation, in vitro characterization, in vivo lung deposition and in vivo pharmacodynamic studies targeted theranostic liposomes: rifampicin and ofloxacin loaded pegylated liposomes for theranostic application in mycobacterial infections conjugation of isoniazid to a zinc phthalocyanine via hydrazine linkage for ph-dependent liposomal controlled release preparation and characterization of isoniazid loaded crude soybean lecithin liposomes enhancement of the effect of bcg vaccine against tuberculosis using dda/tdb liposomes containing a fusion protein of hspx, ppe , and esxv development of extended-voyaging anti-oxidant linked amphiphilic polymeric nanomicelles for antituberculosis drug delivery drug delivery of rifampicin by natural micelles based on inulin: physicochemical properties, antibacterial activity and human macrophages uptake a versatile ph-responsive chitosan-g-polycaprolactone/maleic anhydride-isoniazid polymeric micelle to improve the bioavailability of tuberculosis multi-drugs pulmonary delivery of rifampicin-loaded soluplus micelles against mycobacterium tuberculosis preparation and characterization of antitubercular drugs encapsulated in polymer micelles experimental design-based optimization of lipid nanocarrier as delivery system against mycobacterium species: in vitro and in vivo evaluation lipopolysaccharide polyelectrolyte complex for oral delivery of an anti-tubercular drug halloysite nanotubes as tools to improve the actual challenge of fixed doses combinations in tuberculosis treatment zno nano-cages derived from zif- with enhanced anti mycobacteriumtuberculosis activities sericin-chitosan doped maleate gellan gum nanocomposites for effective cell damage in mycobacterium tuberculosis development of respirable rifampicin-loaded nanolipomer composites by microemulsion spray drying for pulmonary delivery rifampicin lipidpolymer hybrid nanoparticles (lipomer) for enhanced peyer's patch uptake self-assembled nanospheres for encapsulation and aerosolization of rifampicin formulation and optimization of long acting dual niosomes using box-behnken experimental design method for combinative delivery of ethionamide and d-cycloserine in tuberculosis treatment antimicrobial peptides as an alternative to anti-tuberculosis drugs host antimicrobial peptides: the promise of new treatment strategies against tuberculosis the potential application of photodynamic therapy in drug-resistant tuberculosis making the case: developing innovative adherence solutions for the treatment of tuberculosis mycolic acids, a promising mycobacterial ligand for targeting of nanoencapsulated drugs in tuberculosis new drugs and perspectives for new anti-tuberculosis regimens publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord- -vv pyft authors: khosravi-darani, kianoush; pardakhty, abbas; honarpisheh, hamid; rao, v.s.n. malleswara; mozafari, m. reza title: the role of high-resolution imaging in the evaluation of nanosystems for bioactive encapsulation and targeted nanotherapy date: - - journal: micron doi: . /j.micron. . . sha: doc_id: cord_uid: vv pyft nanotechnology has already started to significantly impact many industries and scientific fields including biotechnology, pharmaceutics, food technology and semiconductors. nanotechnology-based tools and devices, including high-resolution imaging techniques, enable characterization and manipulation of materials at the nanolevel and further elucidate nanoscale phenomena and equip us with the ability to fabricate novel materials and structures. one of the most promising impacts of nanotechnology is in the area of nanotherapy. employing nanosystems such as dendrimers, nanoliposomes, niosomes, nanotubes, emulsions and quantum dots, nanotherapy leads toward the concept of personalized medicine and the potential for early diagnoses coupled with efficient targeted therapy. the development of smart targeted nanocarriers that can deliver bioactives at a controlled rate directly to the designated cells and tissues will provide better efficacy and reduced side effects. nanocarriers improve the solubility of bioactives and allow for the delivery of not only small-molecule drugs but also the delivery of nucleic acids and proteins. this review will focus on nanoscale bioactive delivery and targeting mechanisms and the role of high-resolution imaging techniques in the evaluation and development of nanocarriers. nanotechnology is a multidisciplinary approach that employs a vast and diverse array of tools and techniques derived from engineering, physics, chemistry, and biology (sahoo et al., ) . within the past decade, there has been a flurry of new research, development and patent applications around nanoscaled technologies in the health area . one of the principal areas of nanotechnology is ''nanomedicine,'' which, according to the national institute of health (nih) nanomedicine roadmap initiative, refers to highly specific medical intervention at the molecular scale for diagnosis, prevention and treatment of diseases (nih roadmap initiatives, http://nihroadmap.nih.gov/initiatives.asp.). nanomedicine has significant potential for revolutionizing the diagnostics and therapeutics under the premise of developing smart nanodevices. the overall objective of nanomedicine is the same as it has been in medicine: to diagnose as accurately and early as possible, to treat as effectively as possible without side effects, and to evaluate the efficacy of treatment noninvasively (caruthers et al., ) . bioactive delivery nanosystems (nanocarriers) in general, and drug delivery in particular, constitute a significant domain of nanomedicine. most drugs have been formulated for the oral or injection delivery routes, which are not always the most efficient routes for a particular therapy. new bioactive materials, such as nucleic acids and proteins, require novel delivery technologies that will minimize side effects and lead to better patient compliance (hughes, ) . on the other hand, reformulating www.elsevier.com/locate/micron micron ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] old drugs can reduce side effects and increase patient compliance, thus saving money on health care system. furthermore, drug candidates that did not pass through the trials phases can be reformulated to be used with new carrier systems. advancements in nanoscience and technology have made it possible to manufacture and characterize sub-micrometric bioactive carriers on routine basis. the delivery of bioactives to various sites within the body and their release behavior is directly affected by particle size. compared to micrometersized carriers, nanocarriers provide more surface area and have the potential to increase solubility, enhance bioavailability, improve time-controlled release and enable precision targeting of the entrapped compounds to a greater extent (mozafari, ) . as a consequence of improved stability and targeting, the amount of material required for a specific effect when encapsulated in, or incorporated to, a nanocarrier is much less than the amount required when unencapsulated. this is particularly useful when dealing with expensive/rare bioactive materials. a timely and targeted release improves the effectiveness of bioactive compounds, broadens their application range and ensures optimal dosage, thereby improving costeffectiveness of the product. in general, reactive or sensitive material, such as polynucleotides and polypeptides, can be turned into stable ingredients through encapsulation or entrapment by nanocarrier systems (mozafari, ) . innovative nanocarriers can make it possible to use certain chemicals or biologicals that were previously impractical because of toxicities or because they were impossible to administer. for example, bioactive targeting is enabling the delivery of chemotherapy agents directly to tumors, reducing systemic side effects (hughes, ) . scientists are investigating new ways to deliver macromolecules that will facilitate the development of new biologic products such as bioblood proteins and biovaccines. similarly, the success of dna and rna therapies will depend on innovative bioactive delivery techniques (el-aneed, ) . in many occasions, the success of a bioactive compound is dependent on the delivery method. this importance is exemplified by the presence of more than companies based in the united states alone, which are involved with developing bioactive delivery platforms (d'aquino, ) . it should be noted that effective bioactive carriers range from nanosystems (e.g., drug-polymer conjugates and polymeric micelles) to microparticles in the range of mm. both nanoand microscale systems have been extremely important in developing various clinically useful bioactive delivery systems. for instance, while microcarriers can be useful for bioactive targeting to certain parts of the pulmonary tract, for systemic targeting the tumors nanocarriers are more effectual. for practical reasons, in this perspective, ''nanotechnology'' includes ''microtechnology'' and ''nanofabrication'' or ''nanomanufacturing' ' and its micro counterparts (park, ) . to describe what nanotechnology can do to manufacture nano-/ microdrug delivery systems, the manufacturing of nano/micro particles (or capsules) can be taken as an example. current methods of preparing nano-/microparticles are mainly based on double emulsion methods or solvent exchange technique (freitas et al., ) . the main problems with these methods are the low drug loading capacity, low loading efficiency, and poor ability to control the size distribution. utilizing nanotechnologies, such as nanopatterning, could allow manufacturing of nano-/microparticles with high loading efficiency and monodisperse size distribution (park, ) . this review focuses on the potential of nanotechnology in nanotherapy, including the recent status of nanocarriers for bioactive delivery and diagnostics and the role of highresolution microscopies in this regard. these technologies will extend the limits of current molecular diagnostics and permit development of personalized medicine. nanotechnology is a relatively new discipline, and although the full scope of its contributions to the field of human health care remains unexplored, recent advances suggest that nanotechnology will have a profound impact on disease prevention, diagnosis, and treatment (cheng et al., ; emerich, ; sahoo and labhasetwar, ; williams, ) . applications of nanotechnology in medicine are particularly promising and areas such as molecular imaging, disease diagnosis, bioactive encapsulation and targeted delivery at specific sites in the body are being intensively investigated and some products undergoing clinical trials (moghimi et al., ; shaffer, ; wilkinson, ) . encapsulation and targeting the bioactive agentsincluding drugs, vaccines, nutrients and cosmetics -and their protection from degradation and inactivation have been investigated extensively using microencapsulation systems. however, to provide targeted controlled release is a key functionality that can be provided much more efficiently by employing nanocarrier technologies (mozafari, ) . many of the current nanocarrier systems are in fact conventional drug delivery systems that happen to be in the nanometer range, such as nanoliposomes, polymeric micelles, nanoparticles, dendrimers, niosomes and nanocrystals (park, ) . in addition to reducing the frequency of drug administration and thus improving patient comfort, novel delivery systems would offer protection and improve the pharmacokinetics of easily degradable compounds, such as peptides and polynucleotides, which often have short half-lives in vivo (orive et al., ) . for the pharmaceutical industry the field of bioactive delivery represents a strategic tool for expanding drug markets, because new delivery technologies could reformulate/repackage classical drugs, offering a competitive edge after the upcoming patent expirations and avoiding competition from generics. it is noteworthy that % of the current global pharmaceutical market is related to the sale of products that include a bioactive delivery system (mazzola, ) . the efficiency of bioactive delivery to various parts of the body is directly affected by particle size. nanostructuremediated bioactive delivery, a key technology for the realization of nanotherapy, has the potential to enhance bioavailability, improve the timed release of bioactive molecules, and enable precision targeting (dass and su, ; dubin, ) . nanoscale delivery systems can be applied for pulmonary therapies (courrier et al., ) , as gene delivery vectors (senior, ; mozafari et al., ) , and for stabilization of bioactives that would otherwise degrade rapidly (lavan et al., (lavan et al., , . additional benefits of using targeted nanocarriers are reduced toxicity and more efficient distribution of bioactive material (ravi kumar, ) . anatomic features such as the blood brain barrier, the branching pathways of the pulmonary tract, and the tight epithelial junctions of the skin make it difficult for bioactives to reach many desired physiologic targets. nanocarriers will help to penetrate or overcome these barriers to bioactive delivery (hughes, ) . many bioactive materials are poorly water soluble, which results in a low bioavailability. micelles are currently under investigation as carrier vehicles of poorly soluble, hydrophobic bioactives (torchilin, ) . micelles solubilize these drugs by incorporating them into their hydrophobic core and thus increase the bioavailability. microemulsions have also been investigated for their potential to serve as a drug carrier vehicle, since their oil phase can contain a high payload of hydrophobic drugs (bagwe et al., ; lawrence and rees, ) . another possibility for the encapsulation and controlled release of lipidsoluble agents is the use of lipidic carriers with high lipid-phase to water-phase ratio, such as onion-shaped liposomes in the form of multilamellar vesicles (mlv). delivery of polynucleotides, as an important and new class of bioactives, is described below in a separate section. the anatomical changes and pathophysiological conditions of diseased or inflamed tissues offer opportunities for the delivery of various nanotechnological products. bioactive targeting can be achieved by taking advantage of these specific characteristics of abnormal tissues . an ideal targeting system should have long circulation time, should be present at sufficient concentrations at the target site, and should not lose its activity or therapeutic efficacy while in circulation (sahoo et al., ) . the increased vascular permeability coupled with an impaired lymphatic drainage in tumors allows an enhanced permeability and retention effect of the nanosystems in the tumors or inflamed tissues (hashizume et al., ; mcdonald and baluk, ; maeda et al., ; matsumura and maeda, ) . therefore, this pathophysiological opportunity allows extravasation of the nanosystems and their selective localization in the inflamed tissues (allen and cullis, ; hobbs et al., ) (fig. ) . the tendency of nanosystems to specifically localize in the reticuloendothelial system also presents an excellent opportunity for passive targeting of therapeutic agents to the macrophages present in the liver and spleen. this natural system, therefore, can be used for bioactive targeting for intracellular infections such as candiasis, leishmaniasis and listeria. the macrophages of the infected individual play a role in these diseases; consequently, if the macrophages are destroyed then will be the disease as well (daemen et al., ; davis, a ). an important body organ considered for site-specific nanotherapy is the brain. however, the therapeutic value of many promising bioactives for the treatment of various neurological disorders is diminished by the presence of the blood-brain barrier (calvo et al., ) . the blood-brain barrier is a unique membrane that tightly segregates the brain from the circulating blood (pardridge, ) . as a result of this very efficient protection, bioactive delivery to the brain is a challenge. nanotechnology offers a solution for using the numerous chemical entities for treating brain disorders that are otherwise not clinically useful because of the presence of the blood-brain barrier. nanoparticles can be effectively used to deliver relevant therapeutics to the brain (alyautdin et al., ; garcia-garcia et al., ) . encapsulation of bioactives into nanoparticles modifies cell and tissue distribution and leads to a more selective delivery of biologically active compounds to improve therapeutic efficacy and reduces toxicity (de kozak et al., ; feng et al., ; kattan et al., ) . consequently, various nanosystems can be successfully used as new bioactive carriers for delivery to the brain. in a recent study, visser et al. ( ) studied targeting of pegylated liposomes loaded with horseradish peroxidase and tagged with transferrin to the blood-brain barrier in vitro. they obtained effective targeting of liposomes loaded with protein or peptide drugs to the brain capillary endothelial cells and suggested that the system is an attractive approach for drug delivery to the brain. enhanced uptake efficiency has also been shown for gastrointestinal absorption (desai, ; hussain et al., ) and transcutaneous permeation (kohli and alpar, ) , with particles around nm and nm in size, respectively. however, such small particles traveling in the lungs may also have a greater chance of being exhaled. larger, compartmental or multilayered bioactive carriers can help with delivery to the pulmonary extremities. for instance, the outer layers of the carrier architecture may be formulated to biodegrade as the carrier travels through the pulmonary tract. as the carrier penetrates further into the lung, additional shedding will allow the encapsulated material to be released. biodegradable nanoparticles of gelatin and human serum albumin show promise for bioactive delivery to the lungs (brzoska et al., ) . another major site for bioactive transport and targeting is the skin. skin acts as a key target as well as a principle barrier for topical/transdermal (tt) bioactive delivery. the topical/ transdermal delivery route for drug administration has advantages over other pathways including avoiding the hepatic first pass effect, continuous drug delivery, fewer side effects and improved patient compliance (barry, ) . the interest of both the pharmaceutical and cosmetic industry for skin delivery has prompted the development and investigation of a wide variety of carrier systems with different physico-chemical characteristics. a major obstacle to tt drug delivery is low percutaneous penetration. the stratum corneum provides a principle barrier to tt delivery of applied bioactive and consists of corneocytes embedded in an inter-cellular lipid matrix composed of ceramides, free fatty acids, and cholesterol (schurer and elias, ) . several approaches have been used to weaken this skin barrier and to improve tt bioactive delivery (choi and maibach, ; elias et al., ; williams and barry, ) . among the most efficient tt bioactive delivery have been the nanotechnological approaches employing elastic vesicles and ethosomes of nanometric size ranges (elsayed et al., ; lopez-pinto et al., ) . in a study on pig skin, lopez et al. ( ) employed high-resolution, low-temperature scanning electron microscopy in order to detect the effect of nanoliposomes (ca. nm average size) in the protection of stratum corneum (sc) against a nonionic surfactant. the imaging technique enabled visualization of native and treated sc (incubated with nanoliposomes and octyl glucoside) without causing damage to the sc during sample preparation for the microscopic investigations (lopez et al., ) . inadequacy of conventional drugs in the treatment of many of the existing health problems and emergence of new challenges, including acquired immunodeficiency syndrome (aids) and severe acute respiratory syndrome (sars), make the requirement for potent therapeutic formulations a matter of urgency. a new class of bioactive therapeutic agents are based on the polynucleotide molecules and our increasing knowledge of genomics. these molecules, also known as nucleic acid drugs, have the potential to offer healing of human (and animal) diseases at their cause rather than only treating their symptoms. this is very important particularly in the case of hereditary diseases to make sure they are treated at the source and will not be passed to the next generations (mozafari et al., a) . polynucleotide-based therapeutics such as plasmids containing transgenes used in gene therapy, antisense and antigene oligonucleotides, ribozymes, dnazymes, dna and rna aptamers and small interfering rna (sirna), have been developed over the past years (crooke, ; mortazavi et al., ; stull and szoka, ; ulrich et al., ) . although most polynucleotide-based bioactives are in the early stages of clinical trials, they have emerged during recent years as promising therapeutic candidates able to act in a large range of diseases such as hereditary disorders, cancer, neurological and cardiovascular disorders, aids and other viral infections (mozafari et al., a; stull and szoka, ; ulrich et al., ) . during the past two decades, more than clinical studies in gene therapy have been reported. gene therapy is identified with the procedures used to insert the exogenous polynucleotides (dna, mrna, oligonucleotides) into cells or tissues to cure a disease or to improve the associated symptoms (ruozi et al., ) . gene therapy starts with the choice of therapeutic gene, although the most critical objective is the success in the gene transfer to the target tissue for which nanotechnology can play a crucial role. due to the limited ability of naked dna to enter cells and its susceptibility to enzymatic degradation, gene transfer (transfection) has mainly been achieved using a delivery vector. three main types of gene delivery systems have been described: viral vectors, nonviral vectors (in the form of nanoparticles, liposomes or dendrimers), and the direct injection of genetic materials into tissues using the so-called gene guns (goverdhana et al., ; labhasetwar, ; mortazavi et al., ; mozafari et al., a) . viral vectors are attractive in terms of the scientific strategy exploiting the natural targeting mechanisms that viruses acquired during the course of evolution. as a result, viral vectors based on retroviruses, adenoviruses and other viruses are currently the most efficient method for dna transfer into cells. however, these vectors could suffer from the serious difficulties of effective pharmaceutical processing and scale-up, and the possibility of the reversion of an engineered virus to the wild type (sahoo et al., ) . furthermore, viral vectors have other drawbacks such as the risk of recombination, immunogenicity and carcinogenity (crystal, ; tripathy et al., ) . therefore, synthetic vectors have potential advantages for gene transfer even if they show a lower efficiency than viral systems. liposomes and nanoliposomes, in particular the cationic ones, have become one of the most studied synthetic nonviral vectors frequently used in human gene therapy (audouy et al., ; eastman and scheule, ; igarashi et al., ) . the ability of cationic liposomes to mediate transfection was attributed to the intrinsic properties of these systems, namely spontaneous electrostatic interaction between the positively charged vesicles and the negatively charged dna molecules that ensures an efficient condensation of the polynucleotides. by modifying the lipid composition the liposome-polynucleotide complex can exhibit an appropriate charge that enhances the possibility of cellular uptake. in the case of cationic liposomes both fusion and endocytosis have been proposed as mechanisms for the dna or oligonucleotide uptake (de lima et al., ) . to efficaciously use these systems for in vivo gene transfer, the biological and the physicochemical properties of the liposomes/dna complex must be elucidated. microscopic techniques have proven to be useful in imaging and clarifying how the factors such as composition, carrier/dna ratio, configuration, size and polydispersity can affect the assembly and stability of the vector and its gene transfer ability. recently anionic nanoliposomes are becoming more popular as polynucleotide delivery vehicles due to the toxicity and some other complications associated with the cationic agents (mozafari et al., a mortazavi et al., ) . a method of incorporating polynucleotides to the similarly charged anionic liposomes, by the mediation of divalent cations, has been reported and is under development by mozafari and coworkers since (kahveci et al., ; zhdanov et al., ; mozafari, ; zareie et al., ; mozafari et al., , a mozafari et al., , a mozafari et al., , mortazavi et al., ) . this group studied the structure of the ternary complexes of liposome-ca + -dna morphologically using scanning probe and other microscopes (zareie et al., ; . in addition, the mechanism of calcium-induced dna interaction with liposomes containing zwitterionic lipids, as well as those containing anionic lipids, has been studied using light scattering and different microscopic techniques (zareie et al., ; . the problems of toxicity and scale-up to industrial levels have been addressed by a new technique, called the heating method, developed by mozafari et al. ( a mozafari et al. ( , and mortazavi et al. ( ) , in which no potentially toxic solvent or deleterious procedure is involved. applications of nanotechnology in human gene therapy have been reviewed extensively by davis ( b) , who described nonviral vectors based on nanoparticles (usually - nm in size) employed to transport plasmid dna. he emphasized that nanotechnology in gene therapy would be applied to replace the currently used viral vectors by potentially less immunogenic nanosize polynucleotide carriers. usefulness of high-resolution scanning probe imaging in the study of lipidic gene transfer vectors and the interaction between liposomes and dna molecules have recently been reviewed by mozafari et al. ( b) . liposomes and other carrier systems used in bioactive transport and targeted nanotherapy are explained in the following sections. lipid-based carrier systems, including liposomes and their nanoversions (nanoliposomes), are among the most promising encapsulation technologies employed in the rapidly developing field of nanotechnology. compared with other encapsulation strategies, such as chitosan-and alginate-based carriers, lipidbased nanoencapsulation systems have unparalleled advantages, including the ability to entrap material with different solubilities, the possibility of being produced using natural ingredients on industrial scales, and targetability (bummer, ; mozafari et al., ; yurdugul and mozafari, ) . lipid-based carriers can shield an ingredient from free radicals, metal ions, ph and enzymes that might otherwise result in degradation of the bioactive compounds. they impart stability to water-soluble material, particularly in high water-activity applications (gouin, ) . they can accommodate not only water-soluble material but also lipid-soluble agents, if required, simultaneously, providing a synergistic effect (suntres and shek, ) . another unique property of lipid-based nanocarriers is the targeted delivery of their content to specific areas within the body as well as in nonliving systems. in addition, lipid based nanocarriers may be targeted to the required site inside the body via active (e.g., by incorporation of antibodies) and passive (e.g., targeting based on particle size) mechanisms (mozafari and mortazavi, ) . the main lipid-based nanoencapsulation systems that can be used for the protection and delivery of various bioactive materials are explained below. the word liposome derives from two greek words, lipos (fat) and soma (body or structure), meaning a structure in which a fatty envelope encapsulates aqueous core(s) or compartment(s). a recent definition, proposed at a conference in the field of liposomology, describes liposomes as 'closed, continuous bilayered structures made mainly of lipid and/or phospholipid molecules' (mozafari et al., b; mozafari and mortazavi, ) . they are under intensive investigation and development by the pharmaceutical, cosmetic and food industries as micro-and nanocarrier systems for the protection and delivery of bioactive agents. recent studies suggest that liposomes are even naturally present in the very first food we take, breast milk (keller et al., ; keller, ) . liposomes are composed of one or more lipid and/or phospholipid bilayers and can contain other molecules such as proteins or polymers in their structure. a liposome composed of a number of concentric bilayers is known as a multilamellar vesicle (mlv), while one composed of many small nonconcentric vesicles encapsulated within a single lipid bilayer is known as a multivesicular vesicle (mvv). another type of liposome is known as a unilamellar vesicle (ulv), which contains a single lipidic bilayer (fig. ) . owing to the possession of both lipid and aqueous phases, liposomes can be utilized in the entrapment, delivery and release of both watersoluble and lipid-soluble material. the term nanoliposome has recently been introduced to exclusively refer to nanoscale lipid vesicles (mozafari and mortazavi, ) , since liposome is a general word covering many classes of lipid vesicles whose diameter range from around nm to several micrometers. nanoliposomes possess the same physical, structural and thermodynamic properties as the liposomes described above. manufacture of both liposomes and nanoliposomes requires input of energy to a dispersion of lipid/phospholipid molecules in an aqueous medium (mozafari and mortazavi, ) . the underlying mechanism for the formation of liposomes and nanoliposomes is basically the hydrophilic-hydrophobic interaction between phospholipids and water molecules (mozafari, ) . since liposomes are dynamic entities that tend to aggregate and/or fuse and as a result increase in size, vesicles prepared in nanometric size ranges may end up becoming micrometric particles upon storage. however, nanoliposomes should have sufficient stability to maintain their sizes and could be defined as 'bilayer lipid vesicles possessing and maintaining nanometric size ranges during storage and application' (mozafari and mortazavi, ) . the unique properties of liposomes have triggered numerous applications in different fields of science and technology, from basic studies of membrane structure/function to bioactive agent delivery. liposomes and nanoliposomes are particularly useful as efficient bioactive delivery devices because of their ability to pass through lipid bilayers and cell membranes. targeted therapy can also be achieved efficiently via liposomes and nanoliposomes employing passive or active targeting mechanisms (mozafari and mortazavi, ; mozafari, ) . active targeting is achieved by engineering carriers sensitive to different stimuli (e.g., ph, temperature, light, etc.) or conjugating the bioactive/carrier system to one or more targeting ligands such as tissue or cell-specific molecules. in a recent study, zhang et al. ( ) showed that pegylated (treated with polyethylene glycol) liposomes, linked to a monoclonal antibody for the human insulin receptor, led to widespread reporter expression in the brains of rhesus monkeys. passive targeting, on the other hand, uses the natural course followed by the bioactive-carrier complex upon being introduced to the body as the method of site-specific delivery and release of the bioactive agent. it is therefore based on the physicochemical properties of bioactive-carrier complex and physio-anatomical conditions of the body. the clearance kinetics and in vivo biodistribution of carrier systems depend on the physicochemical factors like size, charge and hydrophobicity and can be manipulated to enable passive targeting. archaeosomes are liposomes made from one or more of the polar ether lipids extracted from the domain archaea (archaeobacteria). many archaea live in environments including high salt concentrations, low ph values or high temperatures. hence their membrane structure is unique and enables them to survive in such hostile conditions. the core lipids (polar head groups removed) of archaea consist of archaeols (diethers) and caldarchaeols (tetraethers), wherein the regularly branched, -carbon repeating units forming the isoprenoid chains (usually carbons per chain in archaeols, and carbons per chain in caldarchaeols) are attached via ether bonds at the sn- , position of the glycerol carbons. in contrast to this, the core lipids found in bacteria and eucarya consist of unbranched (mostly) fatty acyl chains, often unsaturated, attached via ester bonds to the sn- , glycerol carbons. the polar moieties (archaeols are monopolar and caldarchaeols are bipolar) are similar to those (phospho, glyco, polyol, amino, hydroxyl groups) encountered in ester lipids, but phosphatidylcholine is rarely present in archaeal lipids patel et al., ) . although archaeosomes are a recent technology, they have already proven to be a safe delivery system for bioactive agents including drugs and vaccines (patel and chen, ) . compared with liposomes (which are made from ester phospholipids), archaeosomes are relatively more thermostable, and more resistant to oxidation and chemical and enzymatic hydrolysis. they are also more resistant to low ph and bile salts that would be encountered in the gastrointestinal tract . archaeosomes prepared from the total polar lipid extract or from individual purified polar lipids show promise as adjuvants that promote strong humoral and cytotoxic t-cell responses to encapsulated soluble antigens. therefore, there is a great potential for using archaeosomes in drug, vaccine and other bioactive material delivery applications. as is the case with liposomes, it is possible to incorporate ligands such as polymers to archaeosomes. it has been shown that incorporation of polyethyleneglycol and coenzyme q into archaeosomes can alter the tissue distribution profiles of intravenously administered vesicles (omri et al., ) . it has recently been reported that intravenous and oral delivery of nanometric-sized archaeosomes to an animal model was well tolerated with no apparent toxicity (omri et al., ) . the results of these studies are very promising for the utilization of archaeosomes in the encapsulation and delivery of different bioactive compounds. cochleates are small-sized and stable lipid-based carriers comprised mainly of a negatively charged lipid (e.g., phosphatidylserine) and a divalent cation such as calcium zarif, ) . they have a cigar-shaped multilayered structure consisting of a continuous, solid, lipid bilayer sheet rolled up in a spiral shape with little or no internal aqueous space. hydrophobic, amphiphilic, negatively or positively charged molecules can be delivered by cochleates. cochleates and their sub-micron versions (i.e., nanocochleates) have been used to deliver proteins, peptides and dna for vaccine and gene therapy applications and are able to cover unpleasant taste and smell of bioactive material intended for oral delivery (mannino and gould-fogerite, ; mozafari et al., ; . due to their nanometric size, stability and resistance to degradation in the gastrointestinal tract, nanocochleates have revealed great potential to deliver bioactive agents both orally and parenterally (mannino and gould-fogerite, ; zarif, ) . cochleates containing amphotericin b (amb) are now in development to enter phase i clinical trials, for both the oral and parenteral treatment of fungal infections (zarif, ) . the unique structure and properties of cochleates make them an ideal candidate for oral and systemic delivery of sensitive material including peptide and nucleic acid drugs. polymer materials potentially possess several desirable properties to be used as nanocarriers including biocompatibility, biodegradability, and functionalization capability. through functionalization and structural manipulation of polymer materials, bioactive molecules can be incorporated within the polymer. entrapping or encapsulating the drug within a polymer allows for greater control of the pharmacokinetic behavior of the bioactive molecule (hughes, ) . the bioactive can be released with a more ideal, near zero-order kinetic profile, which establishes a more constant flow of the encapsulated substance out of the carrier. this pharmacokinetic behavior maintains more appropriate steady levels of the bioactive material at the site of delivery. in contrast, conventional oral delivery typically follows first-order release kinetics where the release rate is proportional to the amount of material remaining in the carrier. landgraf et al. ( ) have compared the release kinetics of an anti-inflammatory agent taken orally by use of a macroporous copolymer carrier and a microporous copolymer carrier containing nanochannels. the macroporous bioactive carrier releases the encapsulant with an initial burst and follows first-order release kinetics. the microporous carrier structured with nanochannels steadily releases the biomaterial in near zero-order fashion. techniques that are used to couple the bioactive with the polymer include sequestering, conjugation, and micelle formation (duncan, ) . ulrich et al. ( ) have reviewed the biodegradable polymeric materials that show promise for bioactive delivery applications. biodegradable polymer nanoparticles, typically consisting of polylactic acid (pla), polyglycolic acid (pga), or a copolymer of pla and pga, are being investigated for the delivery of polynucleotides and polypeptides, vaccines, anticancer therapeutics, ocular drugs, and cytokines (hughes, ) . other polymers being investigated as nanoscale bioactive carriers include polyalkylcyanoacrylate, poly( -hydroxybutanoic acid) (phb), poly(organophosphazene), poly(ethylene glycol) (peg), poly(caprolactone) (pcl), poly(ethylene oxide) (peo), and copolymers such as pla-peg. synthetic polymers, such as peg, can be used to encapsulate biologic materials to create a more stable nanocarrier (hughes, ) . one example of a hybrid drug carrier is a liposome coated with peg, called a stealth liposome. conventional liposomes and nanoliposomes are typically cleared rapidly from the blood circulation. stealth liposomes, with peg coatings, can have prolonged circulation times (moghimi and szebeni, ) . the mechanisms behind this prolonged circulation are still being investigated. furthermore, polymers are being used to enhance the release characteristics of other carrier systems; as is the case with the coating of tablets with hydroxypropyl methylcellulose phthalate (hpmcp) nanoparticles (kim et al., ) . the nanoparticle-coated tablets show a decrease in release rate and a migration towards zero-order release kinetics as the particle size is decreased. dendrimers are a unique class of polymers, which provide another avenue for nanodelivery of different bioactive compounds. the word dendrimer is derived from the greek words dendri-(tree branch-like) and meros (part of) (gardikis et al., ) . dendrimers are considered as highly branched macromolecules; they are small in size, while their low polydispersity can contribute to the reproducibility of their pharmacokinetic behavior (cloninger, ) . dendrimers are fabricated from monomers using either convergent or divergent step-growth polymerization. the well-defined structure, monodispersity of size, surface functionalization capability and stability are properties of dendrimers that make them attractive nanocarrier candidates (hughes, ). an ideal dendrimer as bioactive delivery system, however, must also be nontoxic, nonimmunogenic and biodegradable (for a review, see aulenta et al., ) . the first complete dendrimer family which has been synthesized, characterized and commercialized is the poly(amidoamine) (pamam) dendrimers. they are characterized as safe and nonimmunogenic nanocarriers and have been used for the delivery of drugs and antisense nucleotides and in gene therapy, both in vitro and in vivo (frechet and tomalia, ) . bioactive molecules can be associated to dendrimers via either complexation or encapsulation as schematically shown in fig. . a conjugate carrier system composed of dendrimers and liposomes has recently been manufactured and characterized by gardikis et al. ( ) . niosomes are vesicles made of nonionic surfactant molecules and have been developed as controlled delivery systems in order to overcome the problems associated with some other nanocarriers such as sterilization, large-scale production and stability. they are liposome-like vesicles formed from the hydrated mixtures of cholesterol, charge inducing molecules and nonionic surfactants such as monoalkyl or dialkyl polyoxyethylene ether. the assembly into closed bilayers, both in the case of liposomes/nanoliposomes (mozafari and mortazavi, ) and niosomes, is not spontaneous. thermodynamically stable vesicles form only in the presence of proper mixtures of surfactants and charge inducing agents. the mechanism of vesicle formation upon use of nonionic surfactants is not completely clear. the most common theory is that nonionic surfactants form a closed bilayer in aqueous media based on their amphiphilic nature. formation of this structure involves some input of energy, for instance by means of physical agitation (e.g., by using the handshaking method; see baillie et al., ) or heat (e.g., by using the heating method; see mozafari, ; mozafari et al., a mozafari et al., , . in this closed bilayer structure, hydrophobic parts of the molecule are oriented away from the aqueous solvent whereas the hydrophilic head comes in contact with the aqueous solvent. niosomes resemble phospholipid vesicles (liposomes and nanoliposomes) and hence, enable entrapment of both hydrophilic and hydrophobic material. the low cost, stability and resultant ease of storage of nonionic surfactants have led to the exploitation of these compounds as alternatives to phospholipids (uchegbu and vyas, ) . niosomes can entrap hydrophilic drugs and other bioactives upon encapsulation or hydrophobic material by partitioning of these molecules into their hydrophobic domains. these vesicles can be formulated either unilamellar or multilamellar in structure. moreover, niosomes possess great stability, cost-effectiveness, and simple methodology for the routine and large-scale production without the use of hazardous solvents. uchegbu et al. ( a uchegbu et al. ( , b have studied the different phases and morphologies of niosomes (e.g., discomes and polyhedral niosomes) by employing confocal laser scanning microscopy. microscopic examinations enabled observation and identification of the spherical, helical, tubular and polyhedral niosomes (for a review, see uchegbu and vyas, ) . along with the more conventional polymer-and liposomebased formulations, silicon-based nanocarriers are emerging in the field of bioactive encapsulation and targeting (ferrari, ) . the most commonly investigated silicon-based materials for bioactive delivery are porous silicon and silica, or silicon dioxide. architectures include calcified nanopores, platinumcontaining nanopores, porous nanoparticles, and nanoneedles (hughes, ) . porous silicon was discovered more than years ago and since then has attracted much interest after the demonstration of its photoluminescence at room temperature (cullis et al., ; vaccari et al., ) . two aspects of porous silicon are of particular relevance for in vivo applications; namely: (i) it can be used as a sensitive biosensor for proteins, antigens, and dna, and (ii) it can be modified with a wide range of biological or organic molecules. these features should allow porous silicon to serve as a versatile biomaterial. although efforts in this area are still in early developmental stages, combining the biocompatibility of the material with its highly bio-sensitive capabilities leads to new applications in tissue-based bioassays, bioactive delivery, and health-monitoring applications. the demonstration in of porous silicon biodegradability in physiological environment (canham, ) with a dissolution rate dependent on the medium acidity, porous silicon morphology, porosity, and on the chemical surface derivatization, opened the way for its applications in biomedicine (e.g., see vaccari et al., ) . in the most basic sense, porous silicon is a network of air holes within an interconnected crystalline silicon matrix. the free volume inside pores can be loaded with a bioactive that will be released in the body following the dissolution of the matrix. this idea has been the guideline followed for the design of an implantable microsystem prototype as an anticancer device for the release of doxorubicin in the treatment of tumors (minotti et al., ; vaccari et al., ) . ordered mesoporous silica material with very high surface area and large pore volume appeared as a new member in the family of silica-based materials in (beck et al., ) . the pore size distribution of mesoporous silica is very narrow and can be modulated in the mesoporous region from to several nanometers, which has expanded the available pore sizes of zeolites. such properties would make these materials potentially very interesting as novel carriers for large drug molecules (! nm). recently, considerable investigations have been carried out for the applications of mesoporous silicas as nanocarrier systems for high bioactive loading capacity and sustained or controlled release (xue and shi, ; tang et al., ) . some examples of therapies being investigated for use with silicon-based delivery systems include porous silicon embedded with platinum as an antitumor agent, calcified porous silicon designed as an artificial growth factor, silicon nanopores for antibody delivery, and porous silica nanoparticles containing antibiotics, enzymes and dna (for a review, see hughes, ) . the carbon nanostructures, which have received much attention in recent years are hollow, cage-like architectures known as nanotubes and fullerenes, also called buckyballs because of their spherical structure resembling the geodesic domes of buckminster fuller (hughes, ) . single-wall nanotubes, multiwall nanotubes, and c fullerenes are common configurations. the size, geometry, and surface characteristics of these structures make them attractive for application as nanocarrier systems. carbon nanotubes are on the light spot of nanoscience and nanotechnology owing to their exceptional physical properties (zanella et al., ) . the electronic properties of single-wall carbon nanotubes are remarkable insofar as they can be either metallic or semiconducting, depending on their (n, m) indices or chirality (saito et al., ) . nanotubes are very stable molecules and their chemical inertness is due to the strong covalent sp bonds of the carbon atoms on their surface. surface-functionalized carbon nanotubes can be internalized within mammalian cells (shi kam et al., ) . much work with carbon nanotubes has involved composite materials. for example, temperature-stabilized hydrogels for bioactive delivery applications incorporate carbon nanotubes (li et al., ) . on the other hand, fullerenes have also shown bioactive targeting capability. tissue-selective targeting as well as intracellular targeting of mitochondria have been shown with the use of fullerene structures. furthermore, experiments with fullerenes have also shown that they exhibit antioxidant (lin et al., ) and antimicrobial activities (tsao et al., ) . carbon nanostructures containing ferromagnetic material are another means of bioactive targeting. these nanocarriers can be injected intravenously and then directed using an external magnetic field upon which they will travel through the blood vessels to the region of interest for treatment (berry and curtis, ) . the process of bioactive targeting using a magnetic delivery system is based on the competition between forces exerted on the nanocarrier by the blood flow and the magnetic forces generated by the externally applied magnetic field. when the magnetic forces exceed the linear blood flow rates in arteries ( cm/s) or capillaries ( . cm/s), the nanocarriers are retained at the target site and internalized by the cells of the target tissue (tartaj et al., ) . the particles should be small enough to remain in the circulation after injection and to pass via the capillary systems of organs and tissues avoiding vessel embolism. the nanocarrier surface can be grafted by cooh groups after the acid treatment. bioactive molecules and targeting ligands can be covalently attached to the nanocarriers via carboxyl groups (fig. ) . the ligands attached to the nanocarriers recognize individual components characteristic for cell-surface antigens (berry and curtis, ; wozniak et al., ) . the morphology and cytotoxicity of magnetic carbon nanoparticles have recently been evaluated by wozniak et al. ( ) using inverted microscope and atomic force microscopy (afm). modern nanocarrier systems such as nanoliposomes, niosomes, solid lipid nanoparticles (saupe and rades, ) , as well as silicon-, carbon-and polymer-based nanocarriers play an important role in controlled delivery of the bioactive agents to the desired site of action, limiting the side effects at nontarget sites (ruozi et al., ) . development of these nanosystems requires a rational characterization approach. certain parameters pertained to each of the newly developed nanocarrier systems must be thoroughly assessed before being approved for clinical applications. in the case of liposomes, for instance, parameters having critical importance on their in vivo performance such as morphology, size, polydispersity index, number of lamellae, zeta potential, bilayer fluidity, lipid composition, encapsulation efficiency, carrier-bioactive interaction and chemical stability must be studied. for these, various analytical techniques are being applied. dynamic light scattering (dls, also known as photon correlation spectroscopy, pcs) is used in the determination of particle size distribution, while the nuclear magnetic resonance (nmr) and the electron paramagnetic resonance (epr) are being applied to investigate the lamellarity, the permeability of the bilayer and the influence of particle size on the bioactive transport (for a recent review, see ruozi et al., ) . the microscopical approach is commonly used to characterize the structure/morphology/geometry of the nanocarriers. electron microscopy techniques have been widely used to measure the size and the size distribution of particles. several electron microscopy techniques can be employed for nanocarrier research. these include: (i) scanning electron microscopy (sem); (ii) transmission electron microscope (tem); (iii) negative stain electron microscopy (nsem); (iv) freeze fracture transmission electron microscopy (fftem). in particular, tem provides information on the size distribution and shape of nanocarrier systems. in addition to the configuration of nanocarriers, electron microscopy can also provide information on the interaction between particles (e.g., in the form of aggregation or fusion), different types of each nanocarrier (e.g., in the case of liposomes, mlv, ulv and mvv types; see fig. ), different phases of each carrier (e.g., discomes and polyhedral niosomes), location of the bioactive with respect to the carrier (e.g., internalized or attached to the surface) and the stability of the carrier systems in time. compared with other electron microscopes, sem is a less frequently used imaging technique, particularly in liposome research. however, several sem micrographs showing cells with absorbed liposomes have been published, which are very useful in determining mechanisms of cell-liposome interactions (e.g., see vinay et al., ) . unfortunately, nanocarriers may suffer structural perturbations as a result of the high vacuum conditions and the staining process required by some of the electron microscopes (see fig. ). in the previous years, besides the progress in sample preparation, other microscopical techniques have been developed. atomic force microscopy (afm), one of the techniques belonging to the family of scanning probe microscopes (spm) with dimensional resolution approaching Å , has revolutionized imaging of the nanosamples (binning et al., ; santos and castanho, ) . the most attractive characteristics of spm are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. the other spm technique is scanning tunneling microscope (stm) that was invented in by binnig and coworkers (binnig et al., a (binnig et al., , b and has since become established as a powerful tool for the study of micro-and nanoscale structures. compared with the other types of microscopy, stm has unique characteristics that include: (i) ultra-high resolution down to atomic dimensions; (ii) three-dimensional images with very high resolution especially in the vertical direction; (iii) a variety of operating conditions, such as vacuum, air, and liquids; (iv) observation range from micrometer to angstrom; (v) the ability to do tunneling spectroscopy (feng et al., ) . fig. depicts three-dimensional stm images of double-stranded dna molecules. major and minor grooves of dna are visible in fig. b . representative stm images of two-months old nanoliposomes are presented in fig. . these nanoscale images enabled characterization of the nanocarriers including evaluation of their size and stability; as indicated by their size and morphology variation in time (e.g., see mozafari et al., a) . afm, on the other hand, was developed in and has since been applied for imaging surfaces of different material including ceramics, metals, in addition to biological and pharmaceutical samples (binning et al., ; mozafari et al., b; ruozi et al., ; santos and castanho, ) . the afm ability to explore samples under variety of environmental conditions, including biological specimens in an aqueous/ physiological environment, or in air at different temperatures, or under controlled humidity, make it a very versatile characterization technique. in addition, it is possible to study samples at low temperatures using cryo-afm (prater et al., ; shao et al., ) . unlike the electron microscopical methods, which often require sophisticated sample preparation procedures, the sample preparation for afm is easy and fast and it allows the material to be preserved in its native state (mozafari et al., b) . afm can be operated in a number of different imaging modes depending on the nature of the interaction between its tip and sample surface. when the scanning is carried out keeping an approximately constant distance between probe and sample, afm operation is in the ''contact mode.'' new generations of afm also use another scanning mode called ''tapping mode'' or ''macmode'' (santos and castanho, ) . tapping mode afm provides high-resolution topography images with minimal damage to the sample surface. application of the additional measurement modes such as lateral force mode (lfm) and force modulation mode (fmm) are possible with afm (ruozi et al., ) . in addition to topographic imaging, and especially considering the versatility of the operation modes, afm can also provide information regarding micromechanical properties such as surface and adhesive forces (ruozi et al., ) . spm studies of phospholipid bilayers, for example, have been providing valuable insights into the micromechanical properties of biomembranes (liang et al., ) as well as biological processes such as formation of pili and bacterial conjugation (maeda et al., ) . as is the case with the electron microscopes, spm techniques also enable investigation of particle-particle interactions and the aggregation and fusion processes characteristics of certain nanocarriers (uvarov et al., ) . furthermore, stm in particular is very useful in determining the bilayer thickness of liposomes, nanoliposomes, niosomes and archaeosomes by its analytical ability in the vertical axis (zareie et al., ) . another useful application of the microscopical techniques in general, and spm in particular (due to their high resolution), is that they can identify both the density and the spatial distribution of ligands such as polymers, peptides, and antibody molecules anchored to the surface of the nanocarriers. as explained above, these ligands equip the nanocarriers with specific targeting mechanisms. for a rational development of nanocarriers as bioactive delivery systems, it is essential to characterize these systems as particles. the evaluation of nanocarrier morphology includes the characterization of shape, structure, surface morphology and size measurement of these particles. evaluation of size distribution of nanocarriers is important not only to study the physico-chemical properties and the stability of the preparations but also to identify the in vivo kinetics of these systems and in particular their ability to cross vessel walls and to be accumulated in target tissues (e.g., tumors or infected sites) in order to exert the desired effect. determination of nanocarrier size distribution is an obligatory quality control assay due to the following reasons: (i) the size distribution of bioactive delivery formulations is an important parameter with respect to the physical properties and stability (goren et al., ) ; (ii) size distribution, along with composition, defines plasma pharmacokinetics, biodistribution, and stability of nanocarriers and their associated/entrapped substances in plasma and other organs (barenholz and amselem, ) ; (iii) nanocarrier size is a major factor in their permeation through tumor microvessels and their local residence in tumor tissue (nagayasu et al., ) ; (iv) in pulmonary applications the deposition region of bioactive carriers depends mainly on density, shape, and size of the particles (mozafari et al., b) . each of the currently used particle size determination techniques has its own advantages and limitations. light scattering, for example, provides cumulative average information of the size of a large number of particles simultaneously. however, it does not provide information on the shape of the nanosystem and it assumes any aggregation of more than one particle as one single particle. spm and other microscopic fig. . two and three-dimensional scanning tunneling micrographs of nanoliposomes deposited on hopg (highly oriented pyrolytic graphite) and dried at room temperature under atmospheric pressure. nanoliposomes (indicated by arrows) were prepared by the heating method two months prior to imaging. techniques, on the other hand, make direct observation possible, and hence provide information on the shape of the nanocarriers as well as presence/absence of any aggregation/ fusion (mozafari et al., b) . the drawback of the microscopic investigations is that the number of particles that can be studied at any certain time is limited. with respect to a statistically meaningful analysis of size distribution of the nanocarriers, methods such as light scattering, which measure the average size of large number of particles, are more appropriate than microscopic techniques (mozafari and mortazavi, ) . it should be noted that spm techniques can assess samples in liquid or as adsorbed on a solid surface (partially or fully dehydrated) while the light scattering method evaluates particles in suspension. the general approach for the determination of size distribution of nanocarrier formulations should hence be to use as many different techniques as possible, or at least combine high-resolution imaging and particle sizing techniques together. as we gain more knowledge with respect to disease pathophysiology and cellular mechanisms, more specific bioactive materials are being developed. to use the specificity and potency of these bioactives, new carrier systems must be exploited. nanostructured delivery systems are promising candidates that will enable efficient and targeted delivery of novel bioactive compounds. systematic characterization of the nanocarriers is one of the main steps in the evaluation of their present and coming applications. in this respect, the microscopical approach enables direct visualization and provides valuable information about the geometry and morphology, size distribution and the superficial properties of the nanocarriers affecting their interaction with the bioactive material and target cells. the microscopical techniques in general, and spm in particular, enable us to identify both the density and the spatial distribution of targeting ligands such as polymers, peptides, and antibody molecules anchored to the surface of the nanocarriers. the localization and the way by which nanocarriers interact with the bioactive materials are very important to obtain a good in vivo applicability. moreover, force measurement is another interesting property of high-resolution microscopes such as spm, which provides significant information about the elastic, chemical and adhesion properties of nanocarriers. the information obtained through high-resolution imaging techniques are crucial in the rational design and formulation of optimal nanocarrier systems to be used in the modern field of nanotherapy. drug delivery systems: entering the mainstream significant entry of tubocurarine into the brain of rats by adsorption to polysorbate -coated polybutylcyanoacrylate nanoparticles: an in situ brain perfusion study in vivo characteristics of cationic liposomes as delivery vectors for gene therapy dendrimers a new class of nanoscopic containers and delivery devices improved drug delivery using microemulsions: rationale, recent progress and new horizons the preparation and properties of niosomes non-ionic surfactant vesicles quality control assays in the development and clinical use of liposome-based formulations novel mechanisms and devices to enable successful transdermal drug delivery a new family of mesoporous molecular sieves prepared with liquid crystal templates functionalisation of magnetic nanoparticles for applications in biomedicine surface studies by scanning tunneling microscopy tunneling through a controllable vacuum gap atomic force microscope incorporation of biodegradable nanoparticles into human airway epithelial cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases physical chemical considerations of lipid-based oral drug delivery-solid lipid nanoparticles long-circulating pegylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery bioactive silicon structure fabrication through nanoetching techniques nanotechnological applications in medicine nanotechnologies for biomolecular detection and medical diagnostics elastic vesicles as topical/transdermal drug delivery systems biological application of dendrimers pulmonary drug delivery systems: recent developments and prospects an overview of progress in antisense therapeutics. antisense nucl. acid drug dev transfer of genes to humans-early lessons and obstacles to success correlation of the structural and optical-properties of luminescent, highly oxidized porous silicon liposomal doxorubicin-induced toxicity: depletion and impairment of phagocytic activity of liver macrophages good drug therapy: it's not just the molecule-it's the delivery particle-mediated intravascular delivery of oligonucleotides to tumors: associated biology and lessons from genotherapy plasmid dna expression systems for the purpose of immunization biomedical applications of nanotechnology-implications for drug targeting and gene therapy intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis cationic lipid-dna complexes in gene delivery: from biophysics to biological applications gastrointestinal uptake of biodegradable microparticles: effect of particle size special delivery: pharmaceutical companies aim to target their drugs with nano precision the dawning era of polymer therapeutics cationic lipid: pdna complexes for the treatment of cystic fibrosis an overview of current delivery systems in cancer gene therapy the potential of metabolic interventions to enhance transdermal drug delivery deformable liposomes and ethosomes: mechanism of enhanced skin delivery nanomedicine-prospective therapeutic and diagnostic applications scanning tunneling microscopy of proteins on graphite surfaces nanoparticles of biodegradable polymers for clinical administration of paclitaxel cancer nanotechnology: opportunities and challenges dendrimers and other dendritic polymers microencapsulation by solvent extraction/evaporation: reviewing the state of the art of microsphere preparation process technology a relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles interactions of dendrimers with model lipid membranes assessed by dsc and raman spectroscopy correlation between physical characteristics and pharmacological behavior of doxorubicin-containing liposomes micro-encapsulation: industrial appraisal of existing technologies and trends regulatable gene expression systems for gene therapy applications: progress and future challenges openings between defective endothelial cells explain tumor vessel leakiness regulation of transport pathways in tumor vessels: role of tumor type and microenvironment nanostructure-mediated drug delivery. nanomed. nanotech recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics biosurfactant mel-a enhances cellular association and gene transfection by cationic liposome proceedings of the th nat. cong. biochem. istanbul, importance of nucleic acid-lipid interactions in functioning of the initial cell c- phase i clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles liposomes in nutrition liposomes in breastmilk swelling and drug release behavior of tablets coated with aqueous hydroxypropyl methylcellulose phthalate (hpmcp) nanoparticles potential use of nanoparticles for transcutaneous vaccine delivery: effect of particle size and charge nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery polymer microcarrier exhibiting zero-order release moving smaller in drug discovery and delivery small-scale systems for in vivo drug delivery microemulsion-based media as novel drug delivery systems synthesis of a novel gelatincarbon nanotubes hybrid hydrogel probing small unilamellar egg pc vesicles on mica surface by atomic force microscopy carboxyfullerene prevents iron-induced oxidative stress in rat brain liposomes as protective agents of stratum corneum against octyl glucoside: a study based on high-resolution, low-temperature scanning electron microscopy effect of cholesterol and ethanol on dermal delivery from dppc liposomes tumor vascular permeability and the epr effect in macromolecular therapeutics: a review micromanipulation of phospholipid bilayers by atomic force microscopy antigen cochleate formulations for oral and systemic vaccination in new generation vaccines a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs commercializing nanotechnology significance of blood vessel leakiness in cancer anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity stealth liposomes and long circulation nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties nanomedicine: current status and future prospects preparation of liposomal gene therapy vectors by a scalable method without using volatile solvents or detergents liposomes: an overview of manufacturing techniques liposome-derived model dna delivery system bioactive entrapment and targeting using nanocarrier technologies: an introduction mechanism of calcium ion induced multilamellar vesicle-dna interaction nanoliposomes: from fundamentals to recent developments formation of supramolecular structures by negatively charged liposomes in the presence of nucleic acids and divalent cations construction of stable anionic liposome-plasmid particles using the heating method: a preliminary investigation formation and characterisation of non-toxic anionic liposomes for delivery of therapeutic agents to the pulmonary airways construction of a gene delivery vector using anionic liposomes a review of scanning probe microscopy investigations of liposome-dna complexes recent trends in the lipid-based nanoencapsulation of antioxidants and their role in foods cytotoxicity evaluation of anionic nanoliposomes and nanolipoplexes prepared by the heating method without employing volatile solvents and detergents the size of liposomes: a factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs influence of coenzyme q on tissue distribution of archaeosomes and pegylated archaeosomes administered to mice by oral and intravenous routes short-term repeated-dose toxicity profile of archaeosomes administered to mice via intravenous and oral routes drug delivery in biotechnology: present and future vector-mediated drug delivery to the brain nanotechnology: what it can do for drug delivery in vitro assessment of archaeosome stability for developing oral delivery systems archaeosomes as drug and vaccine nanodelivery systems atomic force microscopy of biological samples at low temperature nano and microparticles as controlled drug delivery devices application of atomic force microscopy to characterize liposomes as drug and gene carriers nanotech. approaches to drug delivery and imaging the present and future of nanotechnology in human health care physical properties of carbon nanotubes an overview of the biophysical applications of atomic force microscopy solid lipid nanoparticles the biochemistry and function of stratum corneum lipids nano-dumpling with drug delivery potential nanomedicine transforms drug delivery cryoatomic force microscopy of filamentous actin nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into mammalian cells antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects alleviation of paraquat-induced lung injury by pretreatment with bifunctional liposomes containing a-tocopherol and glutathione studies on a new carrier of trimethylsilyl-modified mesoporous material for controlled drug delivery the preparation of magnetic nanoparticles for applications in biomedicine targeted polymeric micelles for delivery of poorly soluble drugs immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors in vitro action of carboxyfullerene non-ionic surfactant based vesicles (niosomes) in drug delivery the activity of doxorubicin niosomes against an ovarian-cancer cell-line and in vivo mouse-tumor models phasetransitions in aqueous dispersions of the hexadecyl diglycerol ether (c( )g( )) non-ionic surfactant, cholesterol and cholesteryl poly- -oxyethylene ether-vesicles, tubules, discomes and micelles polymeric systems for controlled drug release dna and rna aptamers: from tools for basic research towards therapeutic applications scanning tunneling microscopy study of cytochrome p b incorporated in proteoliposomes porous silicon as drug carrier for controlled delivery of doxorubicin anticancer agent targeted drug delivery in cancer therapy nanosystems in drug targeting: opportunities and challenges characterization of a novel costimulatory molecule: a - kd b cell surface protein provides accessory help to cd + t cells to proliferate and differentiate targeting liposomes with protein drugs to the blood-brain barrier in vitro the emerging nanomedicine landscape nanotechnology applications in medicine nanotechnology: a new look penetration enhancers magnetic nanoparticles of fe and nd-fe-b alloy encapsulated in carbon shells for drug delivery systems: study of the structure and interaction with the living cells plga/mesoporous silica hybrid structure for controlled drug release recent advances in micro-and nanoencapsulation of food ingredients ab initio study of pristine and si-doped capped carbon nanotubes interacting with nimesulide molecules scanning tunnelling microscopy investigation of liposome-dna-ca + complexes cochleates: new lipidbased drug delivery system cochleates: lipid-based vehicles for gene delivery -concept, achievements and future development nanocochleate cylinders for oral and parenteral delivery of drugs organspecific gene expression in the rhesus monkey eye following intravenous nonviral gene transfer lipid spin labeling and n.m.r. study of the interaction between polyadenylic acid:-polyuridilic acid duplex and egg phosphatidylcholine vesicles. evidence for involvement of surface groups of bilayer, phosphoryl groups and bivalent metal cations valuable assistance of professor e. piskin, dr. c. kocum, and dr. h. zareie in the stm studies is highly acknowledged. key: cord- - socw hp authors: ortega, miguel Ángel; guzmán merino, alberto; fraile-martínez, oscar; recio-ruiz, judith; pekarek, leonel; g. guijarro, luis; garcía-honduvilla, natalio; Álvarez-mon, melchor; buján, julia; garcía-gallego, sandra title: dendrimers and dendritic materials: from laboratory to medical practice in infectious diseases date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: socw hp infectious diseases are one of the main global public health risks, predominantly caused by viruses, bacteria, fungi, and parasites. the control of infections is founded on three main pillars: prevention, treatment, and diagnosis. however, the appearance of microbial resistance has challenged traditional strategies and demands new approaches. dendrimers are a type of polymeric nanoparticles whose nanometric size, multivalency, biocompatibility, and structural perfection offer boundless possibilities in multiple biomedical applications. this review provides the reader a general overview about the uses of dendrimers and dendritic materials in the treatment, prevention, and diagnosis of highly prevalent infectious diseases, and their advantages compared to traditional approaches. examples of dendrimers as antimicrobial agents per se, as nanocarriers of antimicrobial drugs, as well as their uses in gene transfection, in vaccines or as contrast agents in imaging assays are presented. despite the need to address some challenges in order to be used in the clinic, dendritic materials appear as an innovative tool with a brilliant future ahead in the clinical management of infectious diseases and many other health issues. infectious diseases are produced by pathogenic microorganisms, mainly bacteria, viruses, parasites, and fungi. since the dawn of civilization, these diseases have persisted as sources of human morbidity and mortality, representing the second cause of death worldwide and the main reason of b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [ , ] . pharmaceutics , , x for peer review of routes are prevalent ( figure ): the divergent strategy and the convergent strategy [ ] . in the divergent growth approach, first developed by tomalia et al. [ ] , the dendrimer synthesis proceeds inside-out from the core. the core reacts with abn monomeric units through the a functional group, while the b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [ , ] . a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [ ] [ ] [ ] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [ ] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [ ] , carbosilane [ ] , poly(phosphorhydrazone) (pph) [ ] , and polyester dendrimers [ ] (figure ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pharmaceutics , , x for peer review of a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [ ] [ ] [ ] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [ ] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [ ] , carbosilane [ ] , poly(phosphorhydrazone) (pph) [ ] , and polyester dendrimers [ ] (figure ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh or oh), figure a . pamam dendrimers exhibit appealing properties for biomedical studies [ ] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [ ] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [ ] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [ ] . however, they share the presence of multiple nh peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [ ] , figure d . they are often decorated with polar groups in order to achieve water solubility. they are pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh or oh), figure a . pamam dendrimers exhibit appealing properties for biomedical studies [ ] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [ ] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [ ] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [ ] . however, they share the presence of multiple nh peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [ ] , pharmaceutics , , of figure d . they are often decorated with polar groups in order to achieve water solubility. they are classified as "inorganic dendrimers" and exhibit relevant differences compared to traditional "organic dendrimers" such as pamam. furthermore, they have a great variability by altering the core and the amount and length of the branches. pph dendrimers, which can be quantitatively prepared up to generation [ ] , present phosphorus atoms throughout the entire dendritic scaffold and have been widely studied for biomedical applications [ ] , figure e . like carbosilane dendrimers, pph are also "inorganic dendrimers" with a huge variability in cores, branches, and peripheral groups, and require the attachment of polar groups in the periphery to become water-soluble. polyester dendrimers attract widespread attention in the biomedical field due to their biocompatibility and biodegradability. in particular, dendrimers based on , -bis(hydroxymethyl) propanoic acid (bis-mpa, figure f ) are commercially available. since the first reports in the early s, bis-mpa dendrimers have undergone an extraordinary increase in their structural complexity and control, which capitalized on a constant evolution of the synthetic strategies, from the traditional divergent and convergent routes to accelerated approaches based on chemoselective reactions [ , ] . since their first reports, dendrimers have been tested in a large number of in vitro and in vivo studies for multiple biomedical applications. the most explored use of dendrimers is their ability to carry drugs to the desired site of action, being an important resource in precision medicine [ ] . dendrimers protect the encapsulated or bound drug and allow the delivery to the desired site of action. as they can be customized, dendrimers improve the drug pharmacokinetics and solubility, control the drug release, enable more comfortable administration routes, and reach target sites with difficult accessibility such as the ocular system [ ] . another application that has raised great interest is the use of dendrimers in gene therapy. several dendrimers have been explored as non-viral vectors for dna and rna, enabling gene transfection to specific cells. this has been especially useful in in vitro cancer studies, where rna transfected to tumor cells can alter their mechanisms, making them more susceptible to treatment or hindering their uncontrolled division [ ] . on the other hand, dendrimers can act as immunomodulators, by either reducing or enhancing the immune response [ ] . the first approach can be very useful towards autoimmune diseases and allergies [ ] , while the second has been employed, for example, in cancer immunotherapy [ ] . the attachment of multiple antigen copies to the dendritic scaffold produces an increase in the immune response related to the multivalency and the decrease in the conformational freedom of the antigen. in infectious diseases, dendrimers can support the development of vaccines by acting as antigens carrier, providing stability, safety, and a sustained release. in addition, they can serve as adjuvants or can promote the uptake of the antigen by the antigen-presenting cells, thus enhancing its recognition and improving the effectiveness of the vaccine [ ] . another outstanding application is the use of dendritic materials in diagnosis [ ] , such as iron oxide magnetic nanoparticles decorated with dendrimers, which can be monitored through magnetic resonance, or the oxygen sensors, very useful in pathologies such as diabetes [ ] . dendrimers also enable a combined therapeutic and diagnostic action in a single platform, the so-called "theranosis" [ ] . the diagnostic capacity is provided by a specific molecule (e.g., a radionuclide) which, bound to the surface or encapsulated inside the nanoparticle, serves to detect its position in vivo by means of diagnostic imaging such as single photon emission computed tomography (spect). in order to fully benefit from the use of dendrimers as nanocarriers, it is essential to understand the mechanisms of interaction between the dendrimer and the different cargo ( figure ) [ ] [ ] [ ] : • encapsulation. the drug is physically trapped within the dendritic scaffold due to the spheroidal or ellipsoidal hollow cavities found between the different branches. these cavities are frequently hydrophobic, so they exhibit affinity towards drugs with poor water-solubility, and can also lead to h-bonding due to the presence of oxygen and nitrogen atoms. the main drawback of this approach is the tendency of the drug to rapidly leak in biological fluids, compared to a covalent conjugation approach [ ] . • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [ ] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [ ] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [ ] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. pharmaceutics , , x for peer review of • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [ ] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [ ] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [ ] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [ ] [ ] [ ] [ ] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section . the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia ( ) [ ] , more than , articles/patents have been published on dendritic materials, with an approximate increase of publications and patents per year since . key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [ ] . these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [ ] [ ] [ ] [ ] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section . the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia ( ) [ ] , more than , articles/patents have been published on dendritic materials, with an approximate increase of publications and patents per year since . key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [ ] . from the low rate of issued patents turning into commercial products, it becomes apparent that dendritic materials must face several challenges for the bench-to-bedside translation in the biomedical field. mignani et al. ( ) summarized the requirements to become a clinical candidate [ ] . to secure a successful development, the authors highlight the importance of complying with the good laboratory practice (glp) requirements to ensure the quality, reproducibility and reliability of in vitro and in vivo data. furthermore, the good manufacturing practice (gmp) is desirable, but emerges as one of the main challenges in dendrimer translation. indeed, dendrimer defects have been related to the failure of relevant preclinical trials [ ] . an accurate dendrimer synthesis and a thorough purification process are deemed necessary to ensure monodispersity and batch-to-batch reproducibility. this is a highly demanding challenge especially for high-generation dendrimers, multipurpose platforms, and large-scale production. many different strategies are currently explored to overcome these challenges in dendrimer translation, including engineering through critical nanoscale design parameters (cndps) [ ] ; accelerated synthetic approaches [ ] ; the improvement of analytical techniques, such as mass spectrometry; the accurate and simplified design of multipurpose platforms [ ] ; or the dendronization of materials to expand their uses [ ] . viruses are simple acellular organisms, which have coevolved with living beings to replicate and reproduce inside their cells, after the binding to specific receptors [ ] . nearly species of viruses have been reported; many of them can cause human diseases [ ] . some viral infections, such as respiratory ones, represent an important economic burden and a serious public health concern [ ] . antiviral drugs are the most common clinical tool to address these pathologies. to date, different drugs have been approved for the treatment of viral infections [ ] . however, some drugs have serious side effects, including nausea, insomnia, vomiting, allergic reactions, behavior disorders, cardiovascular complications, and dependency [ ] . opening new therapeutic windows, decreasing the side effects while maintaining their efficacy, is a key action. dendrimers contribute to the fight against viral infections [ ] , acting as microbicides per se or as drug nanocarriers, with relevant properties such as low systemic absorption, biocompatibility, water solubility, or simple formulation [ ] . the main uses of dendrimers in viral infections are herein addressed, the human immunodeficiency virus (hiv) being one of their most important targets. stis are highly prevalent worldwide, despite the efficient preventive tools (e.g., preservatives) [ ] . one of the most illustrative examples is the human immunodeficiency virus (hiv). hiv is responsible for the deterioration of immune cells, especially the target cd + t cells [ ] , thus aiding the entry of opportunistic pathogens that cause the acquired immunodeficiency syndrome (aids) [ ] . hiv transmission mainly occurs through body fluids exchange, mostly by sexual contact, but blood, breastfeeding or vertical transmission have also been described. according to the who, . million people were infected by hiv in [ ] . fortunately, around % decrease in infections was registered from to , but the treatment, diagnosis, and prevention remain as a global challenge, especially for developing countries with lack of resources. to date, antiretroviral therapy has shown an excellent outcome in the clinical management of aids. however, these drugs produce important side effects, including hiv resistance to the treatment [ ] . dendrimers represent an interesting alternative to minimize these side-effects and prevent the transmission of hiv and other viral or bacterial stis, figure [ ] . a promising approach relies on the use of dendrimers bearing anionic, sugar, or peptide moieties to prevent the entry of the virus in the target cell. these dendrimers block either the host cell or the viral receptors ( figure , top), such as the glycoproteins gp and gp located at the hiv envelope, two key proteins for the interaction and fusion of hiv with cd t cells. the most representative example is the anionic pll dendrimer spl [ ] . spl is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct ). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov- [ ] , enabling a fast-track development of tools to fight covid- . polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [ ] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [ ] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [ ] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [ ] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th response compared to th [ ] . pharmaceutics , , x for peer review of fusion of hiv with cd t cells. the most representative example is the anionic pll dendrimer spl [ ] . spl is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct ). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov- [ ] , enabling a fast-track development of tools to fight covid- . polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [ ] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [ ] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [ ] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [ ] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th response compared to th [ ] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a (ev ). ev belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [ ] . currently, neither vaccines nor therapies have been approved to prevent or treat ev infection, representing an important global problem but specially in the asian southeast [ ] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev in some clinical isolates in the low nm-high pm range [ ] . as ev is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev and hiv [ ] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov- [ ] , figure a ; ebola virus [ ] , figure b ; zika and dengue viruses [ ] , figure c ; hsv [ ] ; cytomegalovirus [ ] ; some flavivirus, such as the responsible of the japanese encephalitis [ ] ; and different human or aviary flu viruses [ ] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a (ev ). ev belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [ ] . currently, neither vaccines nor therapies have been approved to prevent or treat ev infection, representing an important global problem but specially in the asian southeast [ ] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev in some clinical isolates in the low nm-high pm range [ ] . as ev is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev and hiv [ ] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov- [ ] , figure a ; ebola virus [ ] , figure b ; zika and dengue viruses [ ] , figure c ; hsv [ ] ; cytomegalovirus [ ] ; some flavivirus, such as the responsible of the japanese encephalitis [ ] ; and different human or aviary flu viruses [ ] . bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [ ] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [ ] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of , multiresistant bacterial infections were estimated, being responsible for up to , deaths in [ ] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [ ] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [ ] or cystic fibrosis [ ] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [ ] . lectines are proteins which show a high specificity for sugars and bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [ ] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [ ] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of , multiresistant bacterial infections were estimated, being responsible for up to , deaths in [ ] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [ ] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [ ] or cystic fibrosis [ ] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [ ] . lectines are proteins which show a high specificity for sugars and their derivatives, being able to successfully recognize and agglutinate cells with glycosylated proteins or lipids. lecb binds to fucose, a mucin located in the epithelial mucosa, playing a key role in p. aeruginosa biofilm formation, in conjunction with leca, specific to galactose, although this binding is weaker and less important [ ] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd (ic = . µm) depicted in figure a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic ~ µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [ , ] . pharmaceutics , , x for peer review of less important [ ] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd (ic = . µm) depicted in figure a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic ~ µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [ , ] . one of the most representative examples of resistant bacteria is gram-negative bacteria. these microorganisms present a peptidoglycan wall located between the inner and the outer membranes, which is responsible for the higher resistance of gram-negative bacteria to immune system, and in case of lysis, promotes the release of proinflammatory substances known as lipopolysaccharides (lps), exacerbating the infection [ ] . this structure also confers gram-negative bacteria resistance to some external agents through multiple mechanisms, which are heterogeneous between species [ ] . examples of common mechanisms of bacterial resistance are the expelling of toxic residues that eliminate antibacterial agents; a decrease in bacterial permeability, through the alteration of the membrane channels; or the production of antibiotic inactivating enzymes [ ] . cationic dendrimers may be a solution to fight against resistant bacteria, as they can bind efficiently to the negatively-charged walls, destabilize it by displacing sodium and calcium ions and increase the membrane permeability [ , ] , figure bottom. however, despite their promising biocide activity, cationic dendrimers exhibit high toxicity towards mammal cells and require structural modifications to reduce their cytotoxicity, without affecting the efficacy [ ] . cationic antimicrobial peptides (amp) arranged as multiple antigen peptide (map) dendritic structures can also exert a potent antibacterial activity, decreasing the minimum inhibitory concentration (mic) and minimum bactericide concentration (mbc) of the peptide alone, while dramatically increasing the peptide stability to proteolysis [ ] . for example, a dendritic map structure based on the peptide qkkirvrlsa effectively inhibited diverse gram-negative bacteria (mic = - g/ml for e. coli atcc , p. aeruginosa atcc , and a clinical isolate of k. pneumoniae) [ ] . the higher local concentration of amp allows a multivalent binding and enhances the destabilizing effect of the bacterial membrane. dendrimers may also be used in the rapid diagnosis to discern between gram-negative or gram-positive bacteria, through a ph-dependent bacteria-selective aggregation occurring within min of adding the dendrimer to a bacterial suspension [ ] ; and as carriers of different drugs such as vancomycin or agents like led , both specific to gram-negative microorganisms [ , ] . chorioamnionitis is an infection in the amniotic liquid, which may cause neurological problems in the fetus due to the production of proinflammatory cytokines, escherichia coli being one of the most important etiological agents [ ] . this disease often occurs due to the ascent of microorganisms from vagina to uterus, although other pathways have also been reported like transplacental infection, retrograde seeding from the peritoneal cavity through the fallopian tubes or accidental invasive procedures [ ] . the use of antibiotics such as penicillins, cephalosporins, macrolides, and corticosteroids reduce the risk of developing chorioamnionitis [ ] . in this sense, dendrimers can increase their efficacy. for example, a study conducted by wang et al. ( ) in a guinean pig model demonstrated that hydroxyl-and amino-functional pamam dendrimers successfully encapsulate ampicillin [ ] . both dendrimers significantly decreased the uterus cytokines, compared to the usual therapy, but the amino-dendrimer exhibited a higher toxicity. dendrimers have been studied against other bacterial infections like chlamydia trachomatis, increasing the efficacy of vaccines by conjugating a peptide mimic of a chlamydial glycolipid antigen to a g -pamam-oh dendrimer [ ] ; some opportunistic agents, such as s. aureus, using different generation gn-pamam-nh dendrimers [ ] ; or genital infections, through a sustained and localized delivery of amoxicillin in the cervicovaginal region by pamam-peg dendritic hydrogels [ ] . overall, these studies show the potential role of dendrimers in bacterial infections, although a long road is still to cover, especially to decrease their cytotoxicity and increase their specificity. fungi are eukaryotic organisms responsible of a wide range of human infections. the prevalence of these diseases has increased in some countries, particularly in hospital areas and immunocompromised patients, candida, cryptococcus, pneumocystis, and aspergillus being the most representative families [ ] . data collected by the national nosocomial infections surveillance system (nnis) from january to april showed that up to % of nosocomial infections in eeuu were due to fungi, mainly candida species [ ] . however, current trends indicate the prevalence of aspergillus in different european states [ ] . the treatment of fungal infections is performed through antifungal (antimycotic) drugs, which produce some alterations in their cellular structures, thus inhibiting their development, viability and survival in a direct or indirect way. the most representative antifungal drugs include [ ] : polyenes (e.g., amphotericin b); azoles (e.g., imidazole, triazole); allylamines; lipopeptides; and miscellaneous agents, as griseofulvin, which inhibits microtubules and mitotic fuse, affecting cell division. unfortunately, the resistance developed by some fungi may hinder the clinical management of these infections [ ] . in addition, the great similarity between mammal and fungal cells can lead to cytotoxicity problems, being necessary to find molecules which selectively target fungal cells in a particular tissue [ ] . in this context, the use of nanoparticles like dendrimers may be an effective method to carry all these substances, maintaining their benefits and reducing their side effects. candida albicans, which is responsible of more than half of total fungal infections around the world [ ] , is often treated with ketoconazole, a dual-action drug capable of inhibiting both ergosterol synthesis and the transformation of spores to micellar infectious forms [ ] . however, ketoconazole is poorly water-soluble and can greatly benefit from the use of nanocarriers, which increase its bioavailability in the bloodstream. gn-pamam-nh dendrimers improve the administration of ketoconazole (up to -fold increase of antifungal activity using g dendrimer, compared to the drug alone), being even more efficient when used as hydrogel formulation [ ] ; as well as clotrimazole (up to -fold increase with g dendrimer) due to its hydrophobic and electrostatic interactions [ ] . similarly, these dendrimers have significantly improved the antifungal activity of amphotericin b, overcoming the low water solubility and nephrotoxicity issues [ ] . on the other hand, peptide dendrons have shown efficient anti-candida activity per se [ ] . the representative example shown in figure b , which displays four tryptophan residues in the periphery and a dodecyl chain in the focal point, produced % growth inhibition at µg/ml, as well as affected the biofilm viability and the hyphal and cell wall morphology. on the other hand, dendrimer-assisted gene therapy can prevent fungal infections. cationic pamam dendrimers, bearing -nh , -nme , and -nme + peripheral groups, were used with a ribozyme extracted from an intronic region of c. albicans, an rna molecule capable to cut other rna chain or even itself [ ] . these dendrimers inhibited the catalytic activity of candida ribozymes, with a generation-dependent activity (g > g > g ). however, the nature of the peripheral group did not produce a significantly different inhibition. consequently, the construction of the dendrimer depended on the size of the rna to inhibit and the charge ratio between dendrimer and rna [ ] . the use of rna:dendrimer complexes may have multiple applications, such as inhibiting protein synthesis, splicing or even rna delivery in an era where non coding rna are beginning to be used, thus showing the potential of dendrimers in targeted therapy [ ] . unfortunately, the diagnosis of fungal diseases is currently a challenge. the current golden standard for the detection of these infections relies on poorly sensitive and invasive methods such as cell culture and histopathological study of the infected tissue [ ] . new diagnostic tools are demanded, such as polymerase chain reaction (pcr), immunoassays, or tests capable of detecting specific fungal antigens such as beta-glycans [ ] . in this context, the use of nanostructures can play a key role in the development of new techniques that are more sensitive and effective in the early diagnosis of fungal infections. at present, very few studies report the use of dendrimers in the diagnosis of fungal diseases. takano et al. ( ) performed cdna microarray analysis using highly sensitive dendrimer-based technology in the detection of the rice pathogen magnaporthe grisea and the stage of infection in this pathogen [ ] . another potential approach is the dendrichips ® technology (dendris), relying on a phosphorous dendrimers coating which dramatically increases their sensitivity. this tool is capable of discerning up to respiratory bacterial pathogens from a single sample [ ] , and could be refined and targeted to other types of infectious agents such as fungi. parasites are organisms characterized by the need of other living organism or "host" in order to survive. parasites comprise an important variety of species of diverse complexity, from the simplest organisms such as protozoans to the more complex ones, such as plants [ ] . helminths and protozoans entail the main threat of human parasitosis; the clinical expression and its severity depend on the condition of the immune system of the host, as part of a tight interrelation [ , ] . prevention could be the most efficient mechanism of controlling parasitic infections but, despite the considerable efforts, there are no effective vaccines against any of the main parasites. accordingly, antiparasitic drugs are the pillar in protozoan control, when the simple prevention measures fail. however, the drug resistance of protozoans is becoming an alarming public health problem [ ] . dendrimers could be an effective tool in the early diagnosis or prevention of parasitosis, as well as a new treatment for some of these infections. protozoans comprise a diverse group of eukaryotic unicellular microorganisms that belong to protista kingdom. most common human infections caused by protozoans are related to plasmodium spp. and toxoplasma gondii, as well as trypanosoma and leishmania spp. protozoan parasites are responsible for a considerable mortality and morbidity all over the world, that affect more than million people [ ] . malaria is par excellence the main parasitic infection and it is caused by intracellular plasmodium parasites transmitted by mosquitoes of genus anopheles. approximately, % of world population lives in areas where malaria is transmitted, causing - million infections and . million deaths per year. in specific regions, such as sub-saharan africa, children below years-old conform % of the total deaths from malaria [ ] . an important epidemiological study of the prevalence of malaria in salomon islands revealed the high rate of asymptomatic patients, highlighting the need for a diagnostic tool with high sensibility and specificity to detect plasmodium [ ] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [ ] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp ) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [ ] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure . the role of dendrimers against helminthic schistosoma parasites has also been tested. the disease, known as schistosomiasis [ ] , begins when the larvae form penetrates in the organism through the skin and settle in mesenteric and pelvic veins of the host, turning into the adult form. here, the female parasites lay eggs, which could be eliminated through feces or urine or lead to complications like granulomas or intestinal, hepatosplenic and urogenital damage. this highlights the need for an early diagnosis. wright et al. ( ) confirmed the promising activity of magnetic particles coated with g -pamam-nh to concentrate the schistosoma circulating anodic antigen (caa), resulting in a -fold improvement in caa limits of detection for lateral flow assays [ ] . preventive measures like vaccines are key to stop the impact of schistosoma parasites in specific regions where they cause endemic infections. for example, infections by s. haematobium, s. japonicum, or s. mansoni affect over million people worldwide [ ] . lysine-decorated pamam dendrimers showed excellent behavior as vaccine vector, enhancing the immunoreactivity and efficacy of dna vaccine against s. japonicum infection [ ] . along the same lines, anderson et al. ( ) developed a dendrimer-based vaccine platform which encapsulate antigen-expressing replicon mrnas and generate a protective response towards others parasites such as toxoplasma gondii, and relevant viruses like ebola and h n influenza, with a single dose [ ] . the vaccine nanoparticle comprised an ionizable g -pamam dendrimer, a lipid-anchored peg and rna. these studies show the role of dendrimers in the development of new generation vaccines against different infections. specificity to detect plasmodium [ ] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [ ] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp ) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [ ] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure . leishmaniasis is a parasitic disease produced by leishmania protozoans that infects and multiplies in macrophage-rich organs and tissues of the reticuloendothelial system, mainly the liver and spleen. estimations indicate an increase of . to million cases per year [ ] . for decades, leishmaniasis has been treated with pentostam and glucantime, leading to drug resistance and serious side effects like pancreatitis. alternative broad-spectrum drugs with less toxicity, such as amphotericin b, have been used but they present disadvantages such as the high cost, low solubility, the side effects, and its less efficacy as antiparasitic agent. mannose-decorated g -ppi dendrimers improved the activity of amphotericin b for the treatment of leishmaniasis, reducing the toxicity by increasing the targeting in macrophage-rich organs [ ] . other nanocarrier, a dendritic-linear-dendritic hybrid based on peg and citric acid, figure c , also improved the solubility of amphotericin b ( times) and reduced the in vitro/in vivo toxicity. it resulted as potent as free amphotericin and glucantime in reducing the parasite burden and number [ ] . toxoplasmosis is a zoonosis caused by the ingestion of the parasite toxoplasma gondii. this disease has a chronic and silent course in the majority of population without immune system disorders, mainly causing symptoms such as low fever and muscular pain. this infection is usually treated with sulfadiazine [ ] , which has two disadvantages: it requires a high dose of the drug, which can produce severe side effects like high fever or allergic reactions; and it cannot reach target tissues where the parasite is typically localized. cationic g pamam dendrimers and anionic g . dendrimers efficiently solubilize sulfadiazine (up to molecules per dendrimer) and improve the penetration into the parasite, thus greatly reducing the required sulfadiazine dose and localizing the drug in muscle and brain, where t. gondii is usually present [ ] . the main dendrimer-drug interactions found were electrostatic, for cationic dendrimers, and hydrogen bonding, for anionic counterparts. furthermore, the anionic dendrimer showed intrinsic antiparasitic effect. other successful examples of antiparasitic dendrimers include pegylated pll dendrimers coated with chondroitin sulfate a, as targeted unimolecular micelles for the delivery of the antimalarial drug chloroquine phosphate [ ] ; and pamam dendrimers decorated with ethynil estradiol against trypanosoma cruzi, the parasite responsible for chagas disease, where the g dendrimer is times more effective than benznidazole at h and h (ic = . µm) [ ] . amoebae are eukaryotic protozoa extensively distributed in nature and human habitats, often acting as a host and reservoir of other microorganisms like giant viruses and some class of bacteria [ , ] . amoebae infecting humans are classified as parasitic, such as entamoeba organisms, or opportunistic free-living amoebae, such as acanthamoeba spp., balamuthia mandrillaris, sappinia diploidea, and naegleria fowleri (known as the brain-eating amoebae) [ ] . free-living amoebae do not require human infection for their life cycle, which presents two stages: trophozoite (active form) and cyst (inactive form). acanthamoeba and other free-living amoebae are responsible of serious diseases such as keratitis, encephalitis, and infections in immunocompromised patients in the central nervous system, skin, and lungs. fortunately, amoebae infections are relatively rare, although the high mortality associated with meningoencephalitis are of concern, mainly due to the late diagnosis and the lack of effective antimicrobial treatments [ ] . low generation cationic carbosilane dendrimers bearing ammonium or biguanide moieties have shown strong amoebicidal activity against trophozoites and cysts of acanthamoeba spp., figure d (ic = . mg/l; minimum cysticidal concentration mcc = mg/l) [ , ] . furthermore, they exhibit a synergistic effect with traditional drugs such as chlorhexidine, decreasing the required drug concentration - times using g dendrimers [ ] . the dendrimers target the amoeba membrane and produce inhibition and cell death. in this context, dendrimers could be a promising therapeutic alternative to properly manage amoebae infections. prions (prp) are infectious pathogens that cause neurodegenerative transmissible diseases such as spongiform encephalopathies, after a long period of incubation from to years [ , ] . prp sc are glycoproteins with an abnormal folding that are originated from a conformational change of normal prion proteins (prp c ), acquiring pathological properties [ ] . prion diseases can occur for two reasons: the infectious prion agent can transmit the pathological folding to the prp c ; or the prnp gen mutates, leading to a genetic variation of the prion disease [ ] . infectious prion diseases have gained importance since the epidemic bovine spongiform encephalopathy in united kingdom in that was transmitted to humans as a variation of the creutzfeldt-jakob prion disease [ , ] . the spongiform encephalopathies are named after the shape the brain acquires at final stages of the disease, caused by an increase of vacuoles and the holes that appear in the tissue. it is a rare but severe pathology, which leads to neuronal loss and, eventually, dementia. nowadays, there are some pharmacological interventions that delay the progression of prion diseases; however, there is no effective treatment to stop or avoid it in a significant way [ ] . in this field, nanomaterials such as dendrimers can exert a relevant activity, preventing the conversion of prp c to prp sc [ ] . due to the high affinity of amine groups to prions, phosphorus dendrimers decorated with tertiary amines in their surface are promising agents in the therapy against prion diseases, figure e [ ]. these dendrimers showed an inhibitory effect in the generation of prions (ic = (g ), . (g ) and (g ) µg/ml) and they had anti-infectious action in vitro and in vivo for some spongiform encephalopathies, some of them causing creutzfeldt-jakob disease [ ] . other dendritic families with even higher anti-prion activity include g -pamam-nh and g -ppi-nh (both ic = ng/ml) [ , ] . for example, ottaviani et al. ( ) showed that ppi glycodendrimers, comprising maltose or maltotriose units, prevented the aggregation of prp and aβ( - ) proteins due to the interference of dendrimers with the latency phase (nucleation) of the prion protein [ ] . dendrimers can also be useful for the diagnosis of prion infections. usually, elisa is used to determine whether the causing agent of the disease is a prion, but this method has important limitations as it cannot distinguish between prion chains [ ] . the nature of the prion agent will determine the etiology and course of the disease, changing the pathology parameters like the incubation period or the type of lesion [ ] . importantly, different generation pamam and ppi dendrimers were capable of distinguishing between the different types of prions [ ] . this study demonstrated that the susceptibility of a prion towards a particular dendrimer could be used to diagnose and predict the course of a disease. on the other hand, korri-youssoufi et al. developed an electrochemical biosensor comprising multiwalled carbon nanotubes modified with dendrimers and aptamers to detect prion proteins with a high sensibility (min. . pm) [ ] . in conclusion, dendrimers constitute a promising research line for fighting against prion diseases as they could slow down their progression and allow a reliable diagnosis of the responsible etiological agent. dendritic nanosystems could have a principal role as excipients in the pharmaceutical industry, providing that they are compatible, safe and effective nanocarriers in several administration routes [ ] . for example, ocular administration is still a challenge due to the unique physiology of the eye and the presence of numerous barriers. in this context, recent studies showed that dendrimers improved the time that a drug remains in the cornea after topical administration and they supply directed and sustained neuroprotection for retina [ ] . moreover, dendrimers could act as dna vectors, providing safety and reducing cytotoxicity compared to the viral vectors and intraocular injections used at present [ ] . dendritic polymers can easily improve the properties of different materials, such as cotton, to generate antimicrobial activity. for example, the addition of amine-functional dendrimers confers antibacterial activity against gram-positive bacteria (e. coli, p. aeruginosa) and gram-negative bacteria (s. aureus) and fungi (c. albicans) [ , ] , even after several washing cycles. sepsis is a life-threatening response to an infection, which happens when the immune system overreacts and starts to damage the patient's own tissues and organs. peptide-decorated pamam dendrimers inhibited the acetylation of transforming growth factor β-induced protein, thus improving the mortality and organ damage in the septic mouse model [ ] . additionally, gadolinium-containing g dendrimers can be used as mri diagnostic and prognostic biomarker of sepsis-induced acute renal failure [ ] . these studies open up a field in the treatment and diagnosis of sepsis with a not very high cost and that could be protective against infectious pathogens in certain circumstances like during hospital stays, surgeries or risky exposures. the design of metallodendrimers is a promising field to explore, which can open new avenues in the fight against infectious diseases [ ] . dendrimers are excellent platforms to control the attachment of a great variety of metal complexes with antimicrobial activity, such as silver(i), copper(ii), and zinc(ii), among others. the resultant metallodendrimers often exhibit a synergistic activity and improve the therapeutic response of the dendrimer or the metal complex alone. for example, demonstrated the impact of a single metal ion in the prevention of hiv infection [ ] . a carboxylate-decorated g ppi dendrimer, which inhibited hiv- infection of hec- a cells around %, reached % inhibition by attaching a single cu(ii) ion through its ethylenediamine core. the remarkable control on the dendritic structure enables an optimized antimicrobial activity, difficult to accomplish with other nanomaterials. the reader is referred to a recent review on this topic [ ] . dendrimers are an innovative tool in the treatment, prevention and diagnosis of serious infectious pathologies, as summarized in table . they emerge as a unique opportunity to overcome problems of traditional approaches, such as microbial resistance. nevertheless, the transition from the laboratory to the clinical practice still requires facing several challenges, such as the big scale production, the batch-to-batch consistency, the purity for clinical tests, or the regulatory obstacles. it should be kept in mind that most investigations have been conducted in experimental models in vitro or in animal models, so it is not possible to extrapolate these results to humans yet. however, the outstanding results obtained by vivagel ® in the prevention of viral and bacterial infections predict a brilliant future of dendritic materials in the fight against infectious diseases. [ ] prevention of prp aggregation [ ] diagnosis detect and distinguish between prion diseases [ ] author the importance of infection control and prevention in anesthesiology emerging infectious disease prevention: where should we invest our resources and efforts? las enfermedades infecciosas emergentes y reemergentes, un tema de interés para todos climate change and infectious diseases: from evidence to a predictive framework climate change: challenges and opportunities for global health contact structure, mobility, environmental impact and behaviour: the importance of social forces to infectious disease dynamics and disease ecology infectious disease-related laws: prevention and control measures emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes a guide to aid the selection of diagnostic tests drivers of irrational use of antibiotics in europe antibiotic resistance antimicrobial resistance: a global emerging threat to public health systems quantum dots in biological and biomedical research: recent progress and present challenges the consolidation of nanomedicine multifunctional nanoparticles for multimodal imaging and theragnosis combatting infections with nanomedicine advances in integrative nanomedicine for improving infectious disease treatment in public health designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications nanogel: a versatile nano-delivery system for biomedical applications recent progress in dendrimer-based nanomedicine development discovery of dendrimers and dendritic polymers: a brief historical perspective introduction to dendrimers and other dendritic polymers dendrimers: a novel approach for drug targeting characterization of dendrimers a new class of polymers: starburst-dendritic macromolecules simplifying the synthesis of dendrimers: accelerated approaches dendrimer chemistry: synthetic approaches towards complex architectures boas, u. dendrimers, dendrons, and dendritic polymers: discovery, applications, and the future advances in the chemistry of dendrimers poly(amidoamine) (pamam) dendrimers: synthesis and biological applications poly(lysine) dendrimers and other dendritic molecules from naturally occurring monomers poly(carbosilane) dendrimers and other silicon-containing dendrimers poly(phosphorhydrazone) dendrimers and other phosphorus-containing dendrimers bis-mpa dendrimers and other dendritic polyesters a review on comparative study of ppi and pamam dendrimers the role of branch cell symmetry and other critical nanoscale design parameters in the determination of dendrimer encapsulation properties large dipole moments of phosphorus-containing dendrimers chemistry of multifunctional polymers based on bis-mpa and their cutting-edge applications dendrimers: a novel carrier system for drug delivery pamam dendrimers as efficient drug and gene delivery nanosystems for cancer therapy functionalized branched polymers: promising immunomodulatory tools for the treatment of cancer and immune disorders peptide glycodendrimers as potential vaccines for olive pollen allergy targeted nanosystems: advances in targeted dendrimers for cancer therapy nanovaccines formulation and applications-a review dendrimers for diagnostic applications dendrimers with tetrabenzoporphyrin cores: near infrared phosphors for in vivo oxygen imaging dendrimers for theranostic applications dendrimer-protein interactions versus dendrimer-based nanomedicine dendrimers as drug carriers: applications in different routes of drug administration dendrimers as drug delivery vehicles: non-covalent interactions of bioactive compounds with dendrimers association of chemotherapeutic drugs with dendrimer nanocarriers: an assessment of the merits of covalent conjugation compared to noncovalent encapsulation dendrimers for gene delivery-a potential approach for ocular therapy? pegylated pamam dendrimers as a potential drug delivery carrier: in vitro and in vivo comparative evaluation of covalently conjugated drug and noncovalent drug inclusion complex function oriented molecular design: dendrimers as novel antimicrobials dendrimers and dendritic polymers as anti-infective agents: new antimicrobial strategies for therapeutic drugs dendrimers-revolutionary drugs for infectious diseases application of dendrimers for the treatment of infectious diseases in dendrimer chemistry: synthetic approaches towards complex architectures bench-to-bedside translation of dendrimers: reality or utopia? a concise analysis dendrimer drugs for infection and inflammation synthesis of heterofunctional polyester dendrimers with internal and external functionalities as versatile multipurpose platforms dendronization: a useful synthetic strategy to prepare multifunctional materials virus entry paradigms virus classification-where do you draw the line? update in viral infections novel activities of safe-in-human broad-spectrum antiviral agents a systems approach to study immuno-and neuro-modulatory properties of antiviral agents dendrimers and antivirals: a review evaluations of unformulated and formulated dendrimer-based microbicide candidates in mouse and guinea pig models of genital herpes diversification of the prevention of sexually transmitted infections réservoirs cellulaires et tissulaires du vih- : dynamique au cours de l'infection. virologie frequent detection and isolation of cytopathic retroviruses (htlv-iii) from patients with aids and at risk for aids accountability for the global health sector strategies the pharmacology of hiv drug resistance the potential of hiv- nanotherapeutics: from in vitro studies to clinical trials vivagel (spl gel): a candidate dendrimer-microbicide for the prevention of hiv and hsv infection astodrimer sodium, dendrimer antiviral, inhibits replication of sars-cov- in vitro development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent hiv- sexual transmission triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent hiv- sexual transmission carbosilane dendrons with fatty acids at the core as a new potential microbicide against hsv- /hiv- co-infection dendronized magnetic nanoparticles for hiv- capture and rapid diagnostic conjugated anionic peg-citrate g dendrimer with multi-epitopic hiv- vaccine candidate enhance the cellular immune responses in mice immunohistology of infectious diseases antiviral drug discovery for the treatment of enterovirus infections optimization of a class of tryptophan dendrimers that inhibit hiv replication leads to a selective, specific, and low-nanomolar inhibitor of clinical isolates of enterovirus a structure-activity relationship studies on a trp dendrimer with dual activities against hiv and enterovirus a . modifications on the amino acid nanocarbon-based glycoconjugates as multivalent inhibitors of ebola virus infection synthesis of highly efficient multivalent disaccharide/[ ]fullerene nanoballs for emergent viruses dendrimers functionalized with membrane-interacting peptides for viral inhibition peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in japanese encephalitis antiviral potential of -sialyllactose-and -sialyllactose-conjugated dendritic polymers against human and avian influenza viruses the healthy human microbiome bacterial-host interactions: physiology and pathophysiology of respiratory infection attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in : a population-level modelling analysis the significance of infection related to orthopedic devices and issues of antibiotic resistance review of the incidence and prognosis of pseudomonas aeruginosa infections in cancer patients in the s cystic fibrosis and pseudomonas aeruginosa: the host-microbe interface inhibition and dispersion of pseudomonas aeruginosa biofilms by glycopeptide dendrimers targeting the fucose-specific lectin lecb adaptive and mutational responses to peptide dendrimer antimicrobials in pseudomonas aeruginosa urbańczyk-lipkowska, z. design and studies of multiple mechanism of anti-candida activity of a new potent trp-rich peptide dendrimers reduction toxicity of amphotericin b through loading into a novel nanoformulation of anionic linear globular dendrimer for improve treatment of leishmania major ultrastructural study of acanthamoeba polyphaga trophozoites and cysts treated in vitro with cationic carbosilane dendrimers cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie diagnostic microbiology of the immunocompromised host resistance to antimicrobial peptides in gram-negative bacteria an overview of the antimicrobial resistance mechanisms of bacteria mesoporous silica nanoparticles decorated with polycationic dendrimers for infection treatment synthesis and in vitro evaluation of monodisperse amino-functional polyester dendrimers with rapid degradability and antibacterial properties the influence of surface modification on the cytotoxicity of pamam dendrimers antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification rapid and selective discrimination of gram-positive and gram-negative bacteria by boronic acid-modified poly(amidoamine) dendrimer a potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with led targeting qsec receptor to inhibit the virulence genes of gram negative bacteria pamam-dendrimer enhanced antibacterial effect of vancomycin hydrochloride against gram-negative bacteria development of a guinea pig model of chorioamnionitis and fetal brain injury micronutrients and intrauterine infection, preterm birth and the fetal inflammatory response syndrome mid-trimester preterm premature rupture of membranes (pprom): etiology, diagnosis, classification, international recommendations of treatment options and outcome inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using pamam dendrimers dendrimer-conjugated peptide vaccine enhances clearance of chlamydia trachomatis genital infection antimicrobial efficacy and mechanism of action of poly(amidoamine) (pamam) dendrimers against opportunistic pathogens injectable pamam dendrimer-peg hydrogels for the treatment of genital infections: formulation and in vitro and in vivo evaluation general epidemiology of invasive fungal disease international surveillance of bloodstream infections due to candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in in the united states, canada, and south america for the sentry program. the sentry participant group the changing face of epidemiology of invasive fungal disease in europe tissue penetration of antifungal agents alastruey-izquierdo, a. the global problem of antifungal resistance: prevalence, mechanisms, and management antifungal therapy in european hospitals: data from the esac point-prevalence surveys hydrogel of ketoconazole and pamam dendrimers: formulation and antifungal activity poly (amidoamine) dendrimers increase antifungal activity of clotrimazole prolonged drug delivery system of an antifungal drug by association with polyamidoamine dendrimers polycationic dendrimers interact with rna molecules: polyamine dendrimers inhibit the catalytic activity of candida ribozymes importance of size-to-charge ratio in construction of stable and uniform nanoscale rna/dendrimer complexes the cellular delivery of antisense oligonucleotides and ribozymes diagnosis of invasive aspergillosis: recent developments and ongoing challenges glycobiology of human fungal pathogens: new avenues for drug development large scale parallel analysis of gene expression during infection-related morphogenesis of magnaporthe grisea innovative dendrischips®technology for a syndromic approach of in vitro diagnosis: application to the respiratory infectious diseases one health: parasites and beyond human intestinal microbiota: interaction between parasites and the host immune response biochemical and molecular mechanisms of drug resistance in parasites a review of recent patents on the protozoan parasite hsp as a drug target genome sequence of the human malaria parasite plasmodium falciparum a large proportion of asymptomatic plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of temotu province, solomon islands: challenges for malaria diagnostics in an elimination setting performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) to detect malaria antigen development of a novel fluorophore for real-time biomonitoring system poly(amidoamine)-coated magnetic particles for enhanced detection of schistosoma circulating anodic antigen in endemic urine samples schistosomiasis: drugs used and treatment strategies pamam-lys, a novel vaccine delivery vector, enhances the protective effects of the sjc dna vaccine against schistosoma japonicum infection dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose leishmaniasis impact and treatment access surface-engineered dendrimeric nanoconjugates for macrophage-targeted delivery of amphotericin b: formulation development and in vitro and in vivo evaluation antibiotics for human toxoplasmosis: a systematic review of randomized trials nanomolar cationic dendrimeric sulfadiazine as potential antitoxoplasmic agent pegylated peptide dendrimeric carriers for the delivery of antimalarial drug chloroquine phosphate pérez-campos, e. in vitro activity of steroidal dendrimers on trypanosoma cruzi epimastigote form with pamam dendrons modified by "click"chemistry genetic and physiological interactions in the amoeba-bacteria symbiosis giant virus vs amoeba: fight for supremacy human infections caused by free-living amoebae pathogenic and opportunistic free-living amoebae: acanthamoeba spp., balamuthia mandrillaris, naegleria fowleri, and sappinia diploidea in vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against acanthamoeba castellanii uah-t c trophozoites in vitro anti-acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms transmissible spongiform encephalopathies and prion protein interconversions the prion concept and synthetic prions prion disease a new variant of creutzfeldt-jakob disease in the uk prion disease genetics toward therapy of human prion diseases nanomedicine for prion disease treatment: new insights into the role of dendrimers elimination of prions by branched polyamines and implications for therapeutics influence of surface functionality of poly(propylene imine) dendrimers on protease resistance and propagation of the scrapie prion protein kinetics of amyloid and prion fibril formation in the absence and presence of dense shell sugar-decorated dendrimers rapid typing of transmissible spongiform encephalopathy strains with differential elisa adaptation of the bovine spongiform encephalopathy agent to primates and comparison with creutzfeldt-jakob disease: implications for human health differentiating prion strains using dendrimers electrochemical aptasensor of human cellular prion based on multiwalled carbon nanotubes modified with dendrimers: a platform for connecting redox markers and aptamers dendrimers as pharmaceutical excipients: synthesis, properties, toxicity and biomedical applications dendrimeric systems and their applications in ocular drug delivery the antimicrobial activity of the cotton fabric grafted with an amino-terminated hyperbranched polymer preparation, characterization, and antimicrobial property of cotton cellulose fabric grafted with poly (propylene imine) dendrimer. cellulose dual peptide-dendrimer conjugate inhibits acetylation of transforming growth factor β-induced protein and improves survival in sepsis dendrimer-enhanced mri as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice metallodendrimers as a promising tool in the biomedical field: an overview hiv- antiviral behavior of anionic ppi metallo-dendrimers with eda core the authors declare no conflict of interest. key: cord- -u k i authors: vaja, rakesh; rana, meenal title: drugs and the liver date: - - journal: nan doi: . /j.mpaic. . . sha: doc_id: cord_uid: u k i the liver is a major organ with multiple functions. a number of drugs are metabolized by the liver during phase and reactions which include complex processes involving cytochrome p enzymes. genetic and acquired variability in cytochrome p activity may have profound effects on pharmacokinetics. additionally, drugs can also modify how the liver functions and cause dysfunction or even failure of the organ both by a direct effect on the liver or by alteration in liver blood flow. it is important to recognize the signs and symptoms of liver failure in patients and identify possible causes including drug interactions. furthermore, once a patient has been recognized to be suffering with liver dysfunction or failure drug choice and dosing regime will need to be rationalized. paracetamol overdose can have severe and life threatening consequences for patients due to its effect on liver function. it is the leading cause of acute liver failure in the uk, correct and early management is crucial and will be discussed within this article. the liver receives approximately % of cardiac output. uniquely it receives both arterial blood from the hepatic artery and venous blood from the portal veins. the portal vein supplies e % of hepatic blood flow but only % of oxygen supply, the remaining blood flow and oxygen supply being from the hepatic artery. anatomically the liver is divided into two lobes and further into functional lobules based around a central vein, which contains blood from the hepatic arterial and portal venous circulations. blood arriving to the liver flows into the sinusoids which are spaces lined by hepactocytes. blood then drains towards the centre of the lobule and the central vein then hepatic vein to return blood back to the heart via the inferior vena cava. it is the portal veins taking blood directly from the gut to the liver which allows for first pass metabolism, making the liver susceptible to ingested drugs as they are absorbed from the gastrointestinal tract and transported to the liver. the liver has a broad range of functions categorized in table . the liver metabolises a wide range of drugs the end result being to produce water soluble compounds which can be excreted in the bile. this results from phase reactions mediated by cytochrome p including oxidation, reduction and hydrolysis reactions. this is followed by phase reactions which are conjugative. the cytochrome p family are a group of enzymes found mainly in the liver, which perform oxidation and reduction reactions (phase ) using iron to enhance the water solubility of drugs to aid excretion. cyp enzymes are so named as they are bound to membranes within the cell and contain a haem pigment that absorbs light at a wavelength of nm when exposed to carbon monoxide. after reading this article you should: c understand the mechanisms of drug metabolism by the liver c have an appreciation of alterations to drug choice and dosing regimens in patients with liver disease due to their altered pharmacokinetics c know the management of a patient with paracetamol overdose there are many different isoforms of cyp , classified according to their amino acid sequencing into families, subfamilies and individual genes. their importance can be seen in certain subgroups that lack particular genes. an example pertinent to anaesthesia is deficiency in cyp d which metabolises codeine to morphine, these patients therefore find codeine ineffective. conversely there is a small subgroup of people of saudi arabian and ethiopian decent with very high expression of d who metabolize codeine into vast amounts of morphine. refer to table for more details. an individual more detailed breakdown of cyp genes is beyond the scope of this article. some drugs can induce or inhibit cyp enzymes which have the sequential effect on the metabolism of other drugs, either increasing or reducing it respectively. possibly the most important example is cyp a which metabolises many substrates and is induced by rifampicin, carbamazepine, phenytonin and dexamethasone. of interest to anaesthesia this will increase metabolism of opioids, benzodiazepines and local anaesthetics. another well cited example is the increased metabolism of the oral contraceptive pill and its reduction in efficacy. for a more exhaustive list of substrates, inducers and inhibitors see table (see table ). a number of non-cytochrome p dependent reactions occur in the liver eg oxidation of dopamine and alcohol and hydrolysis of amides and esters (eg lignocaine and pethidine respectively). a predominant rise in aspartate aminotransferase (ast) and alanine aminotransferase (alt) signals hepatocellular injury or death. this can be caused by drug reactions or toxicity (e.g. paracetamol), viral hepatitis, autoimmune hepatitis, alcoholic hepatitis, ischaemic hepatitis secondary to profound hypotension, and rare causes such as wilson's disease. an obstructive pattern has a rise predominantly in alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt), these are canalicular enzymes and suggest cholestasis. this is caused by obstruction, either calculi or tumour (primary biliary, pancreatic or metastases), and liver disease such as primary biliary cirrhosis. pharmacological causes include antibiotics, anabolic steroids and oral contraceptives. a mixed pattern can be seen in sepsis, some drug reactions, cholangitis, congestive cardiac failure and alcoholic liver disease. halothane hepatitis can cause raised liver enzyme assays, raised bilirubin and jaundice. an isolated rise in unconjugated bilirubin may be attributed to gilbert's syndrome or haemolysis. the sars-cov- virus uses angiotensin-converting enzyme (ace- ) receptors to gain entry into cells. liver particularly in the ductal region has abundance of these receptors, hence may be susceptible to sars-cov- elevated lft have been reported widely in hospitalized patients with covid- , the range of elevation is highly variable from to %. surprisingly, the pattern of elevation mimics hepatocyte damage (ast/alt higher than bilirubin or alp) rather than cholangiocytic damage, as would have been expected given the density of ace- receptors is higher in the ductal system. additionally low albumin has been seen and is a marker of severe disease. one study reported longer hospital stay in patients with elevated lft. an international registry has suggested that has many as % of patients with covid- may present with hepatic decompensation in absence of respiratory symptoms. the above findings hold important implications for anaesthetists, especially in preop assessments. most drugs given in anaesthesia and intensive care are given intravenously, thus having a bioavailability of . however some maybe given orally or nasogastrically and absorbed enterally. the absorption will be affected by delayed gastric emptying or reduced by diarrhoea and increased gastric transit time seen in liver failure. additionally if vasopressors are used there maybe splanchnic vasoconstriction with associated reduced absorption. volume of distribution is a theoretical calculated volume within which a dose of a drug is dissolved. hepatic dysfunction can cause fluid retention and will increase the volume within which drugs are present, particularly those which usually remain in the plasma, thus increasing their volume of distribution and reducing their plasma concentration. in liver disease, protein synthesis may be reduced. these proteins are important as binding sites for drugs and as such alter the amount of free drug available, volume of distribution, half life and duration of action. an important example is albumin. hypo-albuminaemia will increase the proportion of free drug which is active, therefore doses of highly protein bound drugs may need to be reduced, for example phenytoin and benzodiazepines, aspirin and warfarin. another protein produced by the liver, a acid glycoprotein binds basic drugs such as carbamazepine, propanolol, alprenolol and imipramine as well as steroids. bilirubin can also compete for protein binding sites, so raised levels can increase amount of free drugs, the effect however is less in vivo than in vitro. problems with absorption of enterally delivered drugs have been described. once absorbed these drugs undergo the "first pass effect" by the liver before reaching the systemic circulation. in liver failure the degree of metabolism will be reduced, therefore the extraction ratio will also be reduced and more drug will reach the systemic circulation, thus increasing bioavailability. prevalence of ultrarapid metabolisers metabolism of drugs in liver disease depends on liver blood flow. this can be reduced in a cirrhotic liver as portovenous shunting in the form of varices which are created and blood is diverted directly into the systemic circulation by-passing the liver. thus first pass metabolism is reduced. drug metabolism by the liver may also be reduced by the use of vasopressors on intensive care which reduce liver blood flow due to varying degrees of splanchnic vasoconstriction. the phase and reactions performed by the liver are affected and metabolism and thus extraction ratios are reduced. drugs can be divided into those with high extraction ratios > . , for example fentanyl and morphine and low extraction ratios < . such as lorazepam, diazepam and methadone. most drugs have low extraction ratios < . that is they have poor permeability and are metabolized by the liver but poorly extracted, therefore clearance is limited by reduced metabolism not by blood flow. those with high extraction ratios > . are highly permeable and clearance is dependent on blood flow. hepatic dysfunction is not uncommon within the intensive care setting affecting e % of critically ill patients depending on definitions used. there is currently no tool akin to renal clearance to indicate degree of liver dysfunction. therefore clinicians use liver function blood tests, international normalized ratio (inr), serum albumin and clinical scores such as the child pugh score act as a surrogate for function. more recently the model for end stage liver disease (meld score) and the meld-na have been used to more accurately predict the severity of liver dysfunction. , although their correlation with pharmacokinetic function not well understood. due to the alterations discussed in pharmacokinetics in liver dysfunction drug choices, dosages and frequency may need to be rationalized and altered accordingly. for example the induction dose and maintenance dose, for either anaesthesia or sedation, needs to be reduced. historically inhalational agents, particularly halothane have been implicated in causing hepatitis. the risk is related to the generation of trifluroacteyl chloride (tfa) by metabolism of agents, which is implicated in toxicity around % of administered dose of halothane is metabolized by the liver, more specifically by cytochrome p . this is a relatively high percentage when compared to more modern inhalational agents, for example, . % isoflurane, . % desflurane and % sevoflurane. even though sevoflurane undergoes % metabolism it does not generate tfa and hence is not linked to immune mediated injury sevoflurane metabolism produces fluoride which is not linked to hepatotoxicity. inhalational agents themselves cause a dose dependent reduction in hepatic blood flow (hbf). isoflurane and sevoflurane result in relatively lower reduction in hbf at mac as compared to desflurane. as long as hypotension is avoided and above effects are kept in mind desflurane is probably the safest choice of inhalational agent due to its' low rate of metabolism and rapid and predictable emergence from anaesthesia. there are two types of halothane hepatitis. type which is mild, transient and has a relatively high incidence ( e %). type caused by oxidative metabolism of halothane in the liver leading to fever, jaundice, and dramatically elevated serum transaminases. the compounds synthesized by oxidation then bind to trifluoroacetylate proteins in the hepatic endoplasmic reticulum causing cellular dysfunction, it is thought to occur in genetically predisposed individuals. the committee on safety of medicines in recommended the avoidance of halothane in patients with a history of previous adverse reactions, those who had received halothane within months unless clinically necessary, and those with a history of unexplained jaundice or pyrexia following previous halothane anaesthesia. iv anaesthetics: the induction agents have a marked effect on haemodynamics and may cause sudden precipitious fall in blood pressure. in clinical practice a standard induction dose need not be altered. however they should be titrated slowly to effect. there are no current recommendations on the use of tiva (total intravenous anaesthesia) in patients with liver disease. research is sparse and conflicting. some earlier reports suggested that inhalational anaesthesia results in smaller elevation of liver enzymes than tiva with propofol-fentanyl. a more recent study however suggested slightly lower rate of elevation in lft after using tiva. opiates morphine is metabolized by the liver to active metabolite morphine- -glucoronide which has potent analgesic properties, and morphine- -glucuronide, which has no analgesic properties but has adverse neurotoxic side effects such as confusion and respiratory depression. as both metabolites are excreted renally, they accumulate in renal failure. in liver failure morphine itself may accumulate as extraction ratio is reduced thereby enhancing further the effect of morphine. therefore dose of morphine should be reduced. the same is true of fentanyl and alfentanil dose, as although there is no active metabolite they also rely on hepatic metabolism. remifentanil may be good choice as its metabolism is by plasma esterases and it has no active metabolites. a review of pain management in patients with liver disease by the american association for study of liver diseases (aalsd) in states in general most opioids have prolonged half life. their recommendations include increase the dosing intervals ( e hr) and using immediate release preparations over extended release in july the medicine and healthcare products regulatory agency (mhra) produced a drug safety update that restricted the use of codeine in children. this was prompted by case reports of four children suffered serious harm following the administration of codeine in the immediate post-operative period. codeine should only be used to relieve acute moderate pain in children older than years and only if it cannot be relieved by other painkillers such as paracetamol or ibuprofen alone. a significant risk of serious and life-threatening adverse reactions has been identified in children with obstructive sleep apnoea who received codeine after tonsillectomy or adenoidectomy (or both). codeine is now contraindicated in all children younger than years who undergo these procedures for obstructive sleep apnoea. codeine is converted to morphine in the liver by the cyp d enzyme. the extent of conversion of codeine to morphine depends on genetic variations of cyp d . people can be classified as: poor; intermediate; extensive; or ultra-rapid metabolisers. poor metabolisers convert very little codeine into morphine and therefore have little or no pain relief, ultra-rapid metabolisers or extensive metabolisers have an excessive amount of morphine in their blood following ingestion of codeine. ethnic origin is an important factor in genetic variability. up to % of caucasians are poor metabolisers whereas up to % of patients of african origin may be ultra-rapid metabolisers. (table ) . this genetic variability leads to different plasma morphine concentrations in patients leading to different analgesic effects as well as side effects including respiratory depression. codeine is contraindicated in all patients of any age known to be cyp d ultra-rapid metabolisers and should not be used by breastfeeding mothers because it can pass to the baby through breast milk and potentially cause harm. nsaids are contraindicated for systemic use in most liver disease patients, because of increased bioavalibilty, the high risk of precipitating gastrointestinal bleeding and renal failure. pregabalin and gabapentin are not metabolized in the liver and can be considered for use. these drugs are renally excreted therefore patients with hepatorenal syndrome warrant cautious adminstration. , gabapentin is considered as first line non opiod drug for analgesia. among the tricyclic antidepressants nortryptilline appear safer than amitriptyline and imipramine. an increased dose of non-depolarising neuromuscular blockers (nmb) may be required in liver disease possibly due to altered pharmacology anaesthesia and intensive care medicine xxx:xxx protein binding and increased volume of distribution. however, those which are metabolized by the liver have a prolonged duration of action, atracurium as metabolized in the plasma has a more predictable duration of action. however, it is worth noting that in prolonged usage concentrations laudanosine from hoffmann degradation may accumulate with the potential to provoke epileptiform activity on electro encephalography (eeg). this is of greater concern if the patient has concomitant renal failure or impaired blood brain barrier. the metabolism of succinylcholine may be prolonged due to reductions in pseudocholinesterase concentrations, but clinically this is of little significance. suggamadex is a unique reversal agent for amniosteriodal nmb which acts by chelating the nmb. it is not metabolized and is excreted almost exclusively unchanged by the kidneys within h. data regarding use of sugammadex, in patients with liver dysfunction is limited. however as sugammadex is almost entirely excreted renally, no dose reduction is required in patients with mild to moderate liver dysfunction. in patients undergoing liver transplant sugammadex is able to reverse neuromuscular block maintained by rocuronium continuous infusion. however it is important to note that the sugammadex recovery time in this population was found to be considerably longer than in other surgical settings, and should be considered in clinical practice. dexmedetomidine is a highly selective alpha- receptor agonist, with analgesic, anxiolytic and sedative. it is primarily metabolized in the liver and may have a prolonged half-life in patients with liver disease. dexmedetomidine has potential protective effects on the liver and intestine during hepatectomy and intraoperative use of dexmedetomidine during liver surgery is subject to ongoing research. additionally patients with elevated bilirubin and bile salts secondary to jaundice may show bradycardias limiting its use. paracetamol overdose paracetamol is the commonest drug taken in overdose in the uk to date, it can result in liver failure and in some cases fatal. hepatocelluar necrosis can occur if as little as . g of paracetamol is ingested. normal pathways of metabolism are saturated and hepatic glutathione stores are exhausted. patients are often initially asymptomatic for the first h before reporting nausea, vomiting, right upper quadrant pain with progressive derangement of liver function tests (lfts) after h. the mhra produced new guidelines for treatment of paracetamol overdose in ; the changes included an updated nomogram and a simplified treatment schedule. the administration of acetylcysteine has previously been based on the rumack-matthew nomogram, which divided treatment groups into high and low risk. the dose was then calculated on a weight-based table, which increased the risk of drug error. the updated nomogram has a single treatment line (figure ) ; thereby eliminating the need for assessing whether the patient falls into the high-risk category. it also advises that in cases of staggered overdoses there should be no delay in the administration of acetylcysteine. another cause for concern was the adverse events that had been reported following the bolus dose of acetylcysteine, this has been addressed by increasing the duration over which it is infused, from min to min. acetylcysteine should be administered when the plasma paracetamol level is on or above a single treatment line joining points of mg/l at h and mg/l at h after ingestion. a baseline full blood count (fbc), urea and electrolytes (u&es), lfts and coagulation screen along with an arterial blood gas sample should also be done at the earliest opportunity. intravenous preparations of paracetamol were licensed in the uk in and it is routinely used in anaesthetic practice. however, there have been some concerns regarding the dosage and administration; especially in adults and children under kg, patients with pre-existing hepatic dysfunction and the elderly. children and infants weighing less than kg should receive the reduced dose of . mg/kg not exceeding the daily dose of mg/ kg, whilst those > kg can be prescribed up to mg/kg not exceeding the daily dose of mg/kg. both the mhra and the npsa have issued alerts, regarding the correct dose prescription as there have been reported cases of accidental overdose due to ml to mg conversion and the administration of g in adults weighing less than kg. the key issue is that with intravenous paracetamol plasma levels will peak immediately after administration. the traditional nomograms used to predict plasma levels after overdose refer to oral ingestion where the levels peak some hours after. indicators of severe paracetamol poisoning which is likely to require referral to a specialist liver centre include: inr of > . at h, > at h or > at hours; renal impairment (creatinine > micromol/l); hypoglycaemia; metabolic acidosis despite rehydration; hypotension despite resuscitation or encephalopathy. the only other treatment in fulminant liver failure is transplantation. paracetamol as analgesic in chronic liver disease given the potential of paracetamol to cause liver injury, there is a common misconception that these patients should never take paracetamol. however, various studies have shown that if taken in appropriate doses, paracetamol is one of the safest analgesics for patients with cirrhosis. limiting the total daily dosage to e g/day with thrice daily dosing is generally recommended. patients should be educated about over-the-counter and prescription medications that may also contain acetaminophen to avoid overdose. remdesvir: elevated liver enzymes are commonly observed in clinical trials of patients with remedevir. the elevated values rarely warrant treatment discontinuation. current recommendations suggest that if the enzymes are elevated to times or more above baseline, to discontinue the drug. tocilizumab: alt elevations are frequent but fulminant hepatitis is rare. the risk of re activation of hbv should be kept in mind if patient had chronic liver disease secondary to viral etiology. steriods: low dose dexamethasone is probably safe in patients with chronic stable liver diease. however use of methylprednisolone in high doses may reactivate hbv and increase risk of spontaneous bacterial peritonitis (sbp) in severe cases. hydoxychloroquine: data is limited but generally has not been associated with elevations in alt levels and is an extremely rare cause of drug induced liver injury. e changing patterns of causation and the use of transplantation in the united kingdom drug dosing considerations for the critically ill patient with liver disease sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor liver injury in covid- : the current evidence hypoalbuminemia predicts the outcome of covid- independent of age and co-morbidity liver injury during highly pathogenic human coronavirus infections high mortality rates for sars-cov- infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry differentiated effects of liver cirrohosis on albumin binding sites for diazepam, salicylic acid and warfarin anaesthesia for the patient with liver disease a model to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts meld-na (the new meld) and peri-operative outcomes in emergency surgery buggy donal j. pharmacology of anaesthetic agents ii: inhalation anaesthetic agents anaesthesia for patients with liver disease the effects of prolonged lowflow sevoflurane anesthesia on renal and hepatic function blood supply to the liver in the human after mac desflurane in comparison with isoflurane and halothane the effect of isoflurane or propofol anaesthesia on liver injury after partial hepatectomy in cirrhotic patients comparison of the postoperative liver function between total intravenous anesthesia and inhalation anesthesia in patients with preoperatively elevated liver transaminase levels: a retrospective cohort study pain management in patients with cirrhosis analgesics in patients with hepatic impairment: pharmacology and clinical implications sugammadex versus neostigmine after rocuronium continuous infusion in patients undergoing liver transplantation dexmedetomidine reduces intestinal and hepatic injury after hepatectomy with inflow occlusion under general anaesthesia: a randomized controlled trial bile acids induce arrhythmias: old metabolite office for national statistics) paracetamol overdose: an evidence based flowchart to guide management the therapeutic use of analgesics in patients with liver cirrhosis: a literature review and evidence-based recommendations care of patients with liver disease during the covid- pandemic: easl-escmid position paper liver injury in covid- : management and challenges covid- : abnormal liver function tests pharmacokinetics in disease states modifying hepatic and metabolic function key: cord- -o wx add authors: li, allen; li, ming k.; crowther, mark; vazquez, sara r. title: drug-drug interactions affecting drug levels of direct oral anticoagulants in the real world: a systematic review() date: - - journal: thromb res doi: . /j.thromres. . . sha: doc_id: cord_uid: o wx add background: direct oral anticoagulants (doacs) have emerged as safe and effective alternatives to vitamin-k antagonists for treatment and prevention of arterial and venous thrombosis. due to their novelty, pharmacokinetic doac drug-drug interactions (ddis) that result in clinical adverse events have not been well-documented. objective: this study aims to systematically review reported pharmacokinetic ddis resulting in clinical adverse events through documented observational evidence to better inform clinicians in clinical practice. methods: a comprehensive literature review of embase, medline, and ovid healthstar was conducted through march th, . two independent reviewers screened and extracted data from eligible articles according to pre-established inclusion and exclusion criteria. articles reporting bleeding or thrombotic outcomes in non-controlled (observational) settings resulting from suggested pharmacokinetic doac ddis were included. results: a total of citations were reviewed, of which were included following data extraction. the majority were case reports (n = ) documenting a single adverse event resulting from a suspected doac ddi, while the remaining papers were a case series (n = ) and cohort studies (n = ). the most commonly reported interacting drugs were amiodarone and ritonavir (bleeding), and phenobarbital, phenytoin, and carbamazepine (thrombosis). bleeding events more often resulted from a combined mechanism (p-glycoprotein and cyp a inhibition), whereas thrombotic events resulted from either combined or single p-glycoprotein/cyp a induction. conclusion: current literature evaluating the real-world risk of doac ddis is limited to few case reports and retrospective observational analyses. clinicians are encouraged to continue to report suspected drug interactions resulting in adverse events. warfarin has historically been a cornerstone for treating or preventing thrombosis in various settings. warfarin, due to its variable interpatient response, requires systematic monitoring of the international normalized ratio (inr) to ensure safety and efficacy. warfarin is subject to numerous food and drug interactions, and there is reasonable quality evidence that some of these interactions are associated with bleeding and/or thrombotic complications. , however, the universal availability of international normalized ratio (inr) testing can mitigate the impact of these interactions by allowing real-time dose adjustment. in the last decade, direct oral anticoagulants (doacs) have emerged as an effective and safe alternative to warfarin. [ ] [ ] [ ] doacs are administered in fixed doses and have fewer drug-drug interactions (ddis) compared to warfarin but are still subject to ddi-induced alterations in plasma concentrations that may result in bleeding or thrombotic events. all doacs (including dabigatran, rivaroxaban, apixaban, and edoxaban) are substrates of the efflux transporter permeability-glycoprotein (p-gp, also referred to as p-gp transportor, abcb , and p-gp multidrug transporter), which regulates the absorption of doacs from the gastrointestinal lumen and is also involved in their hepatic and renal excretion. additionally, rivaroxaban and apixaban have cyp -mediated metabolism (rivaroxaban primarily by cyp a with minor contribution by cyp j and apixaban primarily by cyp a with minor contributions by cyp c , cyp a , cyp c , and cyp c ). , drugs that induce p-gp or cyp a may reduce the plasma concentration of the doac; conversely, drugs that inhibit p-gp or cyp a may increase the plasma concentration of the doac. although fewer in number than warfarin, , , the clinical importance of pharmacokinetic doac ddis is increased as the lack of an "inr-equivalent" results in less testing and monitoringthus such interactions are likely to remain unnoticed until a complication occurs since doac levels are not routinely monitored. currently, pharmacokinetic studies, case reports, and guidance documents based on these reports in the literature propose several pharmacokinetic ddis for doacs demonstrated by significant alterations in plasma doac concentrations - but some occur in the absence of adverse clinical events. [ ] [ ] [ ] [ ] while the ability of p-gp and/or cyp a modifiers to alter doac drug levels is well-established, given the wider therapeutic index of the doacs relative to a narrow therapeutic index drug like warfarin, it is unknown if these pharmacokinetic alterations are sufficient to contribute to clinical adverse events. therefore, to aid clinicians in deciphering the clinical significance of doac ddis, this study seeks to systematically evaluate the existing body of literature of pharmacokinetic doac ddis that result in clinical adverse events (bleeding and thrombosis) in uncontrolled clinical settings. as the pharmacodynamic interactions between anticoagulants and antiplatelet medications, non-steroidal anti-inflammatory drugs (nsaids), and selective serotonin reuptake inhibitors (ssris) and selective norepinephrine reuptake inhibitors (snris) are known to enhance bleeding risk [ ] [ ] [ ] [ ] [ ] [ ] [ ] studies to be included in the review had to report either a thrombotic or bleeding event due to potential pharmacokinetic drug-drug interaction between a doac an another drug in a non-controlled clinical setting. studies that did not control for concurrent administration of ssris, nsaids, antiplatelets, thrombolytics, and other anticoagulants were excluded as these drugs can independently cause bleeding or as the result of pharmacodynamic interactions. for each study that was included, investigators evaluated the study quality (if applicable) using the newcastle-ottawa scale, and extracted data using precomposed data extraction templates on microsoft excel. extracted data included outcome observed (bleeding or thrombosis), doac involved, interacting drug, study population, coagulation or doac plasma concentration measurements (if available), and a drug-interaction probability score (dips) (if available). using a -item questionnaire, the dips tool considers a variety of factors including alternative causes, and previous documentation of the ddi. , it is a widely used reference standard for determining the probability of drug interactions and has been shown through a systematic review to overcome limitations present in other assessment instruments. , data synthesis involved utilizing tables and descriptive statistics to summarize data. the mechanism of the interaction was objectively identified using lexi-drugs wolters kluwer clinical drug information database. after our initial search among the databases, , articles were identified from our search. of these studies, a total of , duplicates were removed with , proceeding to title and abstract screening. of these, , articles were deemed ineligible, with articles moved to full-text review. a total of citations were excluded based on exclusion criteria, leaving for data extraction (figure , online supplement). of the included publications, were case reports, one was a case series, and the remaining two were large cohort studies. of the case reports, just over half (n= ) reported ddis resulting in thrombotic outcomes while the other half reported bleeding outcomes. the most frequently reported ddi for bleeding events in the case reports involved the combinations of amiodarone and dabigatran (via inhibition of p-gp) and ritonavir and rivaroxaban (via inhibition of both p-gp and cyp a ). the most frequently reported ddi for thrombosis involved the antiepileptic drugs phenobarbital, phenytoin, and carbamazepine (via induction of p-gp and/or cyp a ). five case reports included a statement involving a dips (drug interaction probability scale) score (table , online supplement). in cases that also reported laboratory values ( of reports), seven cases reported only coagulation laboratory tests, three reported only doac plasma concentrations,and three reported a combination of coagulation and doac-specific laboratory tests. in the bleeding cases reporting coagulation laboratory results, five reported the prothrombin time (pt), four reported the aptt, and two reported the inr; all results were above the reference range. for the thrombotic cases, one case reported an elevated pt but all j o u r n a l p r e -p r o o f other coagulation labs were within normal limits. six of the case reports reported doac-specific drug levels. all three bleeding cases reported above on-therapy range doac levels. one thrombotic case reported an on-therapy trough for apixaban in the presence of phenobarbital, yet a random plasma apixaban level quadrupled with the removal of phenobarbital. we found one case series by bortz et al, which evaluated a series of patients treated with rivaroxaban and carbamazepine.four of seven patients in this study developed some form of recurrent or extension of a thrombus during concurrent treatment ( table , online supplement). both cohort studies scored a perfect on the newcastle-ottawa scale for nonrandomized studies and assessed bleeding outcomes. chang, et al identified , taiwanese atrial fibrillation patients taking doacs during a -year period and analyzed them for bleeding events (defined as a hospitalization or an emergency department visit with a primary diagnosis of intracranial hemorrhage or gastrointestinal, urogenital, or other bleeding event) during doac therapy with and without the presence of cyp a and p-gp modifying drugs. results demonstrated that after inverse probability of treatment weighting using the propensity score including adjustment for several confounders, the use of dabigatran or rivaroxaban with amiodarone, fluconazole, or phenytoin was associated with a higher rate of major bleeding; apixaban combined with fluconazole or cyclosporine was associated with a higher rate of bleeding. (table , online supplement)pham et al, also evaluated bleeding outcomes. using the ibm watson marketscan databases, j o u r n a l p r e -p r o o f this study identified , patients that were prescribed a doac with concomitant verapamil, diltiazem, amlodipine, or metoprolol from janurary to july . bleeding rates were compared between patients that were prescribed known cyp a and p-gp transporter inhibitors (verapamil or diltiazem) with a doac to patients prescribed with non-interacting drugs (amlodipine or metoprolol) with a doac. dabigatran had statistically significant increases in bleeding risk when paired with diltiazem than with amlodipine and when paired with verapamil than with metoprolol. the rest of the comparisons did not reach statistical significance (table , online supplement).among both case reports and and the cohort study, unique drugs were implicated as a contributor to bleeding events. the most commonly identified mechanism of interaction was a drug that was a combined inhibitor of cyp a and pgp. for the case reports of thrombotic adverse events, eight unique drugs were implicated, with only three having a combined cyp a /p-gp induction mechanism. three reports of ddis causing bleeding events had an unclear mechanism (topical miconazole, loperamide, and phenytoin) ( only five of the cases reported doac-specific drug levels in the setting of adverse events, which reflects the limited availability of these laboratory tests. of note, doac plasma concentrations were above the published on-therapy reference ranges in all bleeding cases, as were the more widely available coagulation laboratory tests. recently, one italian center noted a six-fold increase in the trough doac concentration of patients hospitalized for covid- pneumonia who were also receiving antiviral therapy with ritonavir. no bleeding complications were reported in these patients; however, over half had the doac discontinued as a result of the concerning elevation in trough concentration. in our study, a doac-ritonavir ddi was responsible for two reported bleeding events, and given this risk, doac-specific labeling recommendations include avoiding this drug combination or employing a doac dose reduction. , , ritonavir is likely a contributor to the elevated doac concentrations in these covid- patients, but the scenario is likely multifactorial. in addition to antiviral therapy with ritonavir, some patients in the italian study were also receiving darunavir (a strong cyp a inhibitor) and/or azithromycin (a p-gp inhibitor) which could represent an additive ddi effect on doac concentrations. additionally, renal impairment is a common sequela of covid- infection and could impair doac clearance. in our study, all but one coagulation test was within normal limits for patients with thrombotic events. this reinforces the concept that standard coagulation tests may be helpful to qualitatively assess excess doac effect in the absence of doac-specific assays but may have limited utility in suspected doac failures or thrombotic events. the included cohort studies also warrants discussion. the chang, et al study's findings with amiodarone are consistent with the case reports in this analysis and with other literature finding no difference in adverse outcomes with the combination of apixaban and amiodarone. however, a systematic review showed no difference in clinical outcomes in patients taking any doac and amiodarone. the finding that phenytoin contributed to increased bleeding risk is paradoxical given the proposed however, this may be due to pham et al, using an active comparator group instead of a non-user group. overall, an interesting mechanistic finding in this study is that more ddi-related bleeding adverse events were likely resulting from a combination cyp a /p-gp inhibition mechanism, whereas thrombotic adverse events could have been either from a single cyp a inducer or a combined cyp a /p-gp inducer. a sub-analysis of the apixaban atrial fibrillation clinical trial found no significant differences in bleeding outcomes in patients taking apixaban and amiodarone, and as mentioned above, a systematic review showed no difference in clinical outcomes in patients taking any doac and amiodarone. the chang,et al study showed an increase in bleeding related to combinations of dabigatran-amiodarone and rivaroxaban-amiodarone, but not apixaban-amiodarone. as previously noted, amiodarone-dabigatran-related bleeding was cited in two case reports , . interestingly, the manufacturer prescribing information for both rivaroxaban and apixaban have been recently updated to allow the use of clarithromycin with these doacs, despite its being a strong inhibitor of both p-gp and cyp a . , pharmacokinetic data indicate that j o u r n a l p r e -p r o o f changes in doac exposure due to clarithromycin are not likely to be clinically relevant. , this is consistent with the findings of the chang, et al study. as most of the body of literature in this area consists of case reports, reporting bias potentially limits the scope and findings of our review. specifically, underreporting of clinical events has almost certainly occurred, making it difficult or impossible to identify all clinically relevant interactions resulting in adverse events. finally, other reports may have been published and not fallen into the search criteria for this systematic review. the safety and efficacy profile of a drug is often well tested and documented in clinical trials prior to reaching the market, however, its profile will continue to evolve and build after reaching the market. the topic of drug-drug interactions may be best addressed by a collaboration between already established governmental databases such as the united states food and drug administration adverse event reporting system, the pharmaceutical industry, and academia. one collaborative product could be the creation of a mandatory de-identified prospective registry. this could eliminate reporting bias by identifying clinically relevant ddis associated with adverse events in addition to the absence of clinically relevant ddis. efforts to make doac-specific laboratory monitoring tests more widely available would allow for more quantitative data in the setting of ddis. publicizing existing internal data and/or funding of prospective studies to investigate how doac-specific laboratory tests correlate to clinical adverse events would provide useful clinical guidance. as such collaboration between these agencies and academia is called upon and can promote efficient and expansive research towards drug-drug interactions. , j o u r n a l p r e -p r o o f the strengths of this study lie in the reporting of ddis that resulted in adverse events. many prior ddi studies have been conducted in highly controlled environments with healthy subjects, or drew conclusions from in vitro data. [ ] [ ] [ ] [ ] [ ] although our study had to utilize lower quality of evidence case reports and observational data, it offers insight toward the ddis that would most likely be encountered in a real-world clinical scenario. furthermore, we choose to look at studies comparing specific doacs with specific cyp a /p-gp modifiers. studies where doacs and modifiers are grouped together and assessed for risks may over estimate the risk for bleeding or thrombotic outcomes. current literature evaluating the real-world risk of doac ddis is limited to case reports and retrospective observational analyses. the most commonly reported interacting drugs to cause bleeding events were amiodarone and ritonavir via inhibition of p-gp and cyp a ; culprit drugs for thrombotic events were phenytoin and carbamazepine (via combined strong induction of p-gp and cyp a ), and phenobarbital (via strong induction of cyp a ). clinicians are encouraged to recognize multiple pathways of interaction and other clinical factors, assess the ddi literature appropriately, and identify and report potential interactions they encounter and to determine the likelihood of the validity of the interaction using validated tools such as the dips score (table ). j o u r n a l p r e -p r o o f reporting, resulting in very poor quality data in this clinically important area."  assess the relative contributions of drug absorption, metabolic and elimination pathways and the clinical significance of each. recognize drug-drug interactions may involve multiple pathways and multiple individual patient characteristics and consider the net effect on the patient.  when assessing drug-drug interaction literature, prioritize reports of human in vivo data in actual patients, particularly those reporting on adverse clinical outcomes as a result of a proposed drug-drug interaction.  utilize labeled recommendations for avoiding drug combinations that have been demonstrated to cause bleeding or thrombotic events and utilize recommended dose reductions according to product labeling.  apply the drug interaction probability scale (dips) to retrospectively assess the likelihood of a drug-drug interaction.  report drug-drug interactions that result in adverse events as per institutional protocol and to the food and drug administration adverse event reporting system (faers) as appropriate, and write up a report of the proposed ddi and adverse event for publication and contribution to the literature as a case report. j o u r n a l p r e -p r o o f systematic overview of warfarin and its drug and food interactions drug and dietary interactions of warfarin and novel oral anticoagulants: an update assessment of outcomes of treatment with oral anticoagulants in patients with atrial fibrillation and multiple chronic conditions: a comparative effectiveness analysis risks and benefits of direct oral anticoagulants versus warfarin in a real world setting: cohort study in primary care comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials guidance for the practical management of the direct oral anticoagulants (doacs) in vte treatment drug-drug interactions in an era of multiple anticoagulants: a focus on clinically relevant drug interactions new oral anticoagulants: their advantages and disadvantages compared with vitamin k antagonists in the prevention and treatment of patients with thromboembolic events drug-drug interaction studies of cardiovascular drugs involving p-glycoprotein, an efflux transporter, on the pharmacokinetics of edoxaban, an oral factor xa inhibitor decrease in the oral bioavailability of dabigatran etexilate after co-medication with rifampicin oral bioavailability of dabigatran etexilate (pradaxa((r)) ) after co-medication with verapamil in healthy subjects effect of rifampin on the pharmacokinetics of apixaban, an oral direct inhibitor of factor xa the effect of rifampin on the pharmacokinetics of edoxaban in healthy adults co-administration of rivaroxaban with drugs that share its elimination pathways: pharmacokinetic effects in healthy subjects effect of ketoconazole and diltiazem on the pharmacokinetics of apixaban, an oral direct factor xa inhibitor edoxaban drugdrug interactions with ketoconazole, erythromycin, and cyclosporine an openlabel study to estimate the effect of steady-state erythromycin on the pharmacokinetics, pharmacodynamics, and safety of a single dose of rivaroxaban in subjects with renal impairment and normal renal function european heart rhythm association practical guide on the use of non-vitamin k antagonist oral anticoagulants in patients with atrial fibrillation: executive summary measurement of dabigatran drug levels to manage patients taking interacting drugs: a case report reduced anticoagulant effect of dabigatran in a patient receiving concomitant phenytoin correlation between trough plasma dabigatran concentrations and estimates of glomerular filtration rate based on creatinine and cystatin c drug interaction as a predictor of direct oral anticoagulant drug levels in atrial fibrillation patients characteristics and outcomes in patients with venous thromboembolism taking concomitant anti-platelet agents and anticoagulants in the amplify trial prediction of major and clinically relevant bleeding in patients with vte treated with edoxaban or vitamin k antagonists rivaroxaban in patients stabilized after a stsegment elevation myocardial infarction: results from the atlas acs- -timi- trial (anti-xa therapy to lower cardiovascular events in addition to standard therapy in subjects with acute coronary syndrome-thrombolysis in myocardial infarction- ) apixaban with antiplatelet therapy after acute coronary syndrome predictors of gastrointestinal bleeding in older persons taking nonsteroidal anti-inflammatory drugs: results from the fda adverse events reporting system bleeding risk of patients with acute venous thromboembolism taking nonsteroidal anti-inflammatory drugs or aspirin selective serotonin reuptake inhibitor use and risk of gastrointestinal and intracranial bleeding the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration the newcastle-ottawa scale (nos) for assessing the quality of nonrandomised studies in meta-analyses proposal for a new tool to evaluate drug interaction cases consensus recommendations for systematic evaluation of drug-drug interaction evidence for clinical decision support increasing understanding regarding the risk of concomitant use of carbamazepine and direct oral anticoagulants association between use of non-vitamin k oral anticoagulants with and without concurrent medications and risk of major bleeding in nonvalvular atrial fibrillation association of oral anticoagulants and verapamil or diltiazem with adverse bleeding events in patients with nonvalvular atrial fibrillation and normal kidney function postoperative bleeding after administration of a single dose of rivaroxaban to a patient receiving antiretroviral therapy. drug safety -case reports gastrointestinal bleeding associated with rivaroxaban administration in a treated patient infected with human immunodeficiency virus dabigatran toxicity in a year old male who failed other anticoagulation methods dabigatran and amiodarone interaction: a serious hemorrhage case report. fundamental and clinical pharmacology drug interactions of dabigatran: report of one case. basic and clinical pharmacology and toxicology bleeding associated with coadministration of rivaroxaban and clarithromycin increased bleeding tendency from interaction between rivaroxaban and topical miconazole: case report spontaneous gross hematuria during dabigatran therapy for secondary stroke prevention probable drug-drug interaction between dabigatran and quinidine resulting in thrombin time rebound despite multiple idarucizumab doses development of left atrial thrombus after coadministration of dabigatran etexilate and phenytoin clinical challenges related to novel oral anticoagulants: drug-drug interactions and monitoring venous thromboembolism due to suspected interaction between rivaroxaban and nevirapine tocilizumab and mesenteric arterial thrombosis: effect per se and/or interaction with anticoagulants. fundamental and clinical pharmacology management of phenobarbital and apixaban interaction in recurrent cardioembolic stroke pulmonary embolism due to interaction between rivaroxaban and carbamazepine when not to choose apixaban: a case of apixaban failure xarelto(r) [package insert eliquis(r) [package insert bristol-myers squibb company induction of cyp a by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital induction of drug-metabolizing enzymes by phenobarbital in layered co-culture of a human liver cell line and endothelial cells in vitro predictability of drug-drug interaction likelihood of p-glycoprotein-mediated efflux of j o u r n a l p r e -p r o o f dabigatran etexilate based on [i] /ic threshold direct oral anticoagulant plasma levels' striking increase in severe covid- respiratory syndrome patients treated with antiviral agents: the cremona experience renal involvement and early prognosis in patients with covid- pneumonia laboratory assessment of the anticoagulant activity of direct oral anticoagulants: a systematic review amiodarone, anticoagulation, and clinical events in patients with atrial fibrillation: insights from the aristotle trial efficacy and safety outcomes of direct oral anticoagulants and amiodarone in patients with atrial fibrillation drug interactions with non-vitamin k oral anticoagulants-reply drug interactions with non-vitamin k oral anticoagulants drug interactions with non-vitamin k oral anticoagulants drug interactions with non-vitamin k oral anticoagulants efficacy and safety of rivaroxaban versus warfarin in patients taking nondihydropyridine calcium channel blockers for atrial fibrillation (from the rocket af trial) clinical safety outcomes in patients with nonvalvular atrial fibrillation on rivaroxaban and diltiazem application of physiologically based pharmacokinetic modeling to the prediction of drug-drug and drug-disease interactions for rivaroxaban the effects of clarithromycin on the pharmacokinetics of apixaban in healthy volunteers: a single-sequence crossover study postmarketing safety surveillance : where does signal detection using electronic healthcare records fit into the big picture? detection of drug-drug interactions through data mining studies using clinical sources, scientific literature and social media unifying drug safety and clinical databases playing well with others! initiating and sustaining successful collaborations between industry, academia and government . patients on direct oral anticoagulants (doacs) can encounter drug interactions . drug interactions can alter the amount of doac in a patient's system. health providers should continue to be cautious with potential doac interactions we would like to thank the librarians at mcmaster university, health sciences library for helping us conduct our literature search. no funding was provided for conduction of this study. al has no conflicts to disclose. ml has no conflicts to disclose. srv serves as an editorial consultant for uptodate®, which is a product of wolters-kluwer who also produces lexi-comp® drug interaction database. she is also a member of the board of directors for the anticoagulation forum. doctor crowther discloses data safety monitoring board work for bayer, advisory board work for servier canada , asahi kasei, and precision biologicals and preparation of educational materials and/or presentations for pfizer, csl behring and diagnostica stago. doctor crowther holds individual stocks in alnylam. doctor crowther has ongoing relationships with a number of for profit, and not for profit agencies and has worked on medicolegal cases involving anticoagulant therapy.j o u r n a l p r e -p r o o f key: cord- -vcmkz lh authors: mandsberg, nikolaj kofoed; christfort, juliane fjelrad; kamguyan, khorshid; boisen, anja; srivastava, sarvesh kumar title: orally ingestible medical devices for gut engineering date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: vcmkz lh orally ingestible medical devices provide significant advancement for diagnosis and treatment of gastrointestinal (gi) tract-related conditions. from micro- to macroscale devices, with designs ranging from very simple to complex, these medical devices can be used for site-directed drug delivery in the gi tract, real-time imaging and sensing of gut biomarkers. equipped with uni-direction release, or self-propulsion, or origami design, these microdevices are breaking the barriers associated with drug delivery, including biologics, across the gi tract. further, on-board microelectronics allow imaging and sensing of gut tissue and biomarkers, providing a more comprehensive understanding of underlying pathophysiological conditions. we provide an overview of recent advances in orally ingestible medical devices towards drug delivery, imaging and sensing. challenges associated with gut microenvironment, together with various activation/actuation modalities of medical devices for micromanipulation of the gut are discussed. we have critically examined the relationship between materials–device design–pharmacological responses with respect to existing regulatory guidelines and provided a clear roadmap for the future. introduction history of medicine has taught us that complex biological problems need not necessarily have to have a complex biological solutiona simple engineering device can do. medical devices, both with and without electronic interfaces, have already revolutionized the diagnosis and treatment of many diseases. from hip and cochlear implants [ , ] , to cardiac stents and pacemakers [ , ] , to vestibular prosthesis and insulin pumps [ , ] , all highlight tremendous engineering efforts for translating basic science into relevant patient-care technologies [ ] . also, miniaturization of technology has triggered development of non-invasive microdevices, including microrobots for cellular surgery and regeneration [ , ] , orally ingestible devices for insulin delivery, and microsensors for in vivo imaging and biopsy [ ] [ ] [ ] . this has ushered a new era of medical devices to 'explore and manipulate' complex biological microenvironment, which was otherwise restricted owing to bulky device size and poor biocompatibility. for instance, the first insulin pump invented in , the biostar, was the size of a microwave oven, thereby, limiting its usage to treatment of diabetic ketoacidosis [ , ] . nonetheless, the concept of ingestible microdevices for monitoring did exist as early as the s and was termed endoradiosonde. mackay and jacobsen "produced a small capsule that a person can swallow, and which contains the sensing transducer and the radio transmitter" and the "device successfully operated in the gastro-intestinal tract" [ ] . this review focuses on the design of such engineered ingestible microdevices for applications in sensing, drug delivery and gut microsampling (fig. a) . we chose 'device size' as an important distinction criteria as we noticed a strong correlation between size scale and associated biological application as shown in fig. b . despite advances in polymer engineering, leading to 'sustained release formulations', several challenges remain with contemporary oral capsules [ ] . for instance, a) many therapeutics are not absorbed in some or all parts of the gastrointestinal (gi) tract; b) drug absorption is limited by gi transit time; and c) any biological response is dependent on chemical stability of the formulation in the harsh gi environment. these challenges get amplified many folds in case of oral delivery of biologics, including proteins, peptides, hormones and nucleic acids [ , ] . biologics are relatively sensitive to external factors, including ph and temperature, ionic concentrations and denaturing conditions, like high acidity and proteasesconditions that are present in abundance in the gi tract [ ] . if biologics would somehow survive all of the above, the presence of a - μm thick mucus layer acts as a negatively-charged barrier, thereby, preventing the entry of biologics into the underlying gi epithelium [ ] . here, orally-ingestible microdevices (oims) are designed to protect the drug molecule and minimize the distance between the site of release and the epithelium. this prevents drug release in the intestinal lumen and limit exposure to above mentioned rate limiting factors (ph, microbes, continuous mucus secretion etc.). note that the term 'microdevice' is used irrespective of the size scale i.e. normally referring to both microscale (μm) and macroscale (mm), unless otherwise specified. at this point, it is important to understand how an orally administered drug interacts with the body. once ingested, the drug is dissolved in the intestinal fluids and can then be absorbed by: i) the transcellular pathway i.e. drug transport across the cells mainly via passive diffusion and carrier-mediated transport; or ii) the paracellular pathway i.e. drug transport between the cells through passive diffusion [ , ] . most oral drugs are absorbed by passive transport via the transcellular pathway [ ] . fig. presents an overview of challenges faced by a microdevice after oral ingestion. apart from the shared conventional oral drug delivery challenges (like ph, transit time, and enzymes), microdevices are strongly impacted by their size. therefore, minimizing accidental retention is of paramount importance. in a way, device size and shape determine its safe passage from the esophagus all the way to the colonthe gi section with the highest residence time of - days [ ] . but the journey in-between is also perilousincluding low ph in the stomach, risking an unwanted leaching of material, and various enzymes in the different sections of the gi tract (fig. ) . additional challenges include tissue wall localization due to constant motility and peristalsis in the gi tract. evidently, both device size and associated design play a key fig. . orally ingestible microdevices: a) timeline depicting the first appearance of multi-compartment sensing, drug delivery, and sampling devices to illustrate technological progression and the focus of our review; b) size scale as a selection criteria for oral device application in sensing, drug delivery and gut microsampling. role in governing safety profile and therapeutic efficacy of any orally ingestible device. the current generation of oims are of considerable interest with direct application in: ( ) understanding of metabolic and pathophysiological conditions linked to the gi tract; ( ) re-designing gut microbiota in modulating autoimmune diseases, like coeliac and crohn's disease; and ( ) oral delivery of biologics, like vaccines and hormones, which are otherwise administered via injections. this becomes important since escaping the gut metabolism does not guarantee high bioavailability as the absorbed drug is subjected to first-pass effect (with exception to those absorbed in terminal rectum) [ ] . clearly, to overcome above challenges, we need multidisciplinary fundamentals rooted in materials/polymer engineering, device design and integrated microelectronics, and robust assessment of pharmacological response in pre-clinical studies. in this regard, oral delivery of microdevices has emerged as a superior method for gut engineering, with a potential for high patient compliance, and the ability to combine multi-modalities onto a single-ingestible deviceall pointing towards a successful clinical translation. this article encompasses all the above multidisciplinary elements and provides an up-to-date assessment of orally-ingestible gi microdevices in vivo. we have taken this opportunity to not only discuss key-developments but also provide inputs on potential research areas more suitable for certain device types. both scientific publications and industrial patents are discussed to highlight key-elements necessary for a successful clinical translation. we have made a conscious effort in selecting state-of-the-art oim technologies, assessed their design-material-bioactivity relationship, and provided an in-depth discussion on the regulatory framework of u.s. food and drug administration (fda) and communautés européennes (ce) associated with the regulation of such oral microdevices. the idea of uni-directional drug release, similar to a transdermal patch, has influenced the development of oims. the concept of patches and microcontainers filled with drugs i.e. "reservoir containing a plurality of tiny pills" dates back to the sthanks to alejandro zaffaroni, the founder of alza corp, who was one of the major early pioneers in the field of controlled drug delivery systems [ , ] . microfabricated drug devices incorporate planar geometries with low aspect ratio, together with uni-directional release, as most are sealed from one end (i.e reservoir-like geometry). therefore, they offer drug protection and minimal shear stress induced by constant peristalsis and mucosal fluid gradients, resulting in higher drug bioavailability due to increased intestinal retention [ ] . chirra et al. developed planar microdevices (fig. a inset) , where a poorly permeable drug (acyclovir) was loaded in a peg-pmma hydrogel to improve the oral bioavailability in wild-type c bl/ mice [ ] . the area under the curve (auc), which represents the total drug exposure across time, was found to be approx. . times larger for the microdevices than for an oral solution with the same dose (fig. a) . to this end, we have also actively investigated oral delivery of drugs with another type of a passive microdevice, namely microcontainers [ ] [ ] [ ] . additional challenges faced by orally ingested microdevices traversing through the gi tract have been highlighted. images adapted from servier medical art by servier and licensed under a creative commons attribution . unported license. [ ] [ ] [ ] . recently, microcontainers were developed in poly-Ɛcaprolactone (pcl) in order to have a biodegradable oral drug delivery device in microscale [ ] . in a pharmacokinetic study in rats, the pcl microcontainers revealed trends towards a higher auc when compared to paracetamol in coated gelatin capsules ( ± and ± μg min ml − , respectively). in another study, nemeth et al. demonstrated drug loading by inkjet printing inside a planner pmma reservoir-type microdevices (fig. b ) [ ] . silanization of the microdevice was carried out to increase its hydrophobicity, which proved to allow drug loading using droplets of nine times the reservoir volume due to increased droplet confinement. this reduced the number of loading iterations required significantly. the effect of silanization on cell toxicity was further concluded by an mtt assay in a caco- cell model, and confirmed the devices to be 'safe to use'. later, drug loading and capping process were all conducted in a single platform using a picoliter dispenser by inkjet printing polymer/drug solutions on a silanized silicon substrate (fig. b ) [ ] . to do so, the authors took advantage of the coffee-ring effect by deposition of water droplets with % (w/v) eudragit® fs d on a hydrophobized substrate. although eudragit® polymers are non-biodegradable, they are fda approved for ph sensitive release of drugs in the gi tract [ ] . so far, these devices have not been tested in animals. while passive microdevices offer extensive flexibility in terms of fabrication, the very idea of a unidirectional release may take a hit when they are inside the gi tract. tissue wall localization requires constant motility and navigation through the~ m gi tract. the challenge is further amplified by periodic smooth muscle contractions from peristalsis, which pushes the luminal content through the upper gi tract, causing turbulent convective flow regimes of up to cm/s [ , ] . to counter this challenge, devices have been designed to penetrate or adhere to the mucus layer, such as by nanostraw patterning, to take advantage of these random flow patterns and stick to the intestinal wall. once adhered to the gi mucosa, nanopatterned devices create zones of cellular diffusion via topography-mediated adhesion together with chemical permeation enhancers, like cell-penetrating peptides, for enhanced drug absorption. however, over % of nanostraw devices, on average, attach facing the wrong way (by landing on the wrong side without surface indentation), or never attach at all [ ] . academically, researchers have overcome the situation by applying several, sometimes thousands, of these microdevices [ ] as expected, dose variability is inevitable leading to limited clinical translation potential [ ] . nonetheless, passive microdevices offer fast prototyping in terms of fabrication, which can potentially pave way for delivery of toxic drugs with a narrow therapeutic index. to make the efforts more relevant, suitable in vitro models need to be developed to provide realistic simulation of the gi microenvironment. an example of such in vitro model is a human gut-on-achip microfluidic device that enables human intestinal epithelial cells (caco- ) to be cultured in the presence of physiologically relevant luminal flow and peristalsis-like mechanical twists ( fig. c i-ii) [ ] . these conditions promoted formation of basal crypts and villi ( fig. c iii-iv) lined by all four epithelial cell linages of the small intestine (absorptive, goblet, enteroendocrine and paneth), thereby also promoting secretion of mucus, necessary for realistic models. furthermore, the intestinal epithelium inside the gut-on-a-chip device has been shown to support the presence of the microbiota that normally colonizes the human small intestine [ ] . such a microfluidic device has potential applications in modeling of human intestinal inflammation in vitro. furthermore, kim et al. implemented key characteristics of intestinal inflammation diseases, including destruction of intestinal villi and associated compromise of the permeability barrier [ ] . these damages are believed to origin from interplays between the intestinal epithelium, gut microbes and immune cells and changes in luminal flow due to altered peristalsis [ , ] the developed in vitro human gut-on-a-chip microfluidic device could potentially be further developed to work in a patient specific manner to advance personalized medicine in the futurepassive oims can play a key-role in developing personalized medicine and can be tested in these gut-on-a-chip systems as well [ , , ] for instance, workman et al. demonstrated induced pluripotent stem cells (ipscs) derived intestinal organoids to express intestine markers (post- days differentiation), and their association with ibd was studied upon exposure to the ifn-γ cytokine [ ] . a two-channel pdms mold was created where cell-monolayer was maintained in the top-channel ( μm high), followed by a thin porous membrane ( μm~pore size), and growth media being circulated in the bottom-channel ( μm high). a similar setup can be designed with passive oims for high-throughput screening of drugs as well as food allergensmuch like a miniaturized version of a -well plate. also, till date, it is not possible to co-culture living epithelial cells (like caco- ), together with gut microbes using conventional in vitro models, or even more sophisticated intestinal organoid cultures [ ] . passive oims can bridge this gap by acting as a 'gut-microbe carrier' for greater understanding of intestinal diseases and their pathophysiology. therefore, it is necessary to develop passive microdevices that can aid our current understanding of host microbiome together with relevant gut in vitro models. . . active microdevices for oral drug delivery: mucopenetration for therapeutic efficacy conventionally, all oral drugs must dissolve in the aqueous environment of the gi tract before they can be absorbed [ ] . such a seemingly simple process is a considerable challenge for compounds that are poorly water-soluble, or degradable in the lumen, such as biologics [ ] in such a situation, self-propelling or self-actuating microscale devices can play a key role by delivering therapeutics directly across the gi mucosa. several self-propelling microscale devices (i.e. micromotors) have been reported, which utilize a gas-evolution reaction towards selfpropulsion, resulting in mucopenetration/mucoadhesion. such a reaction can be initiated via acid hydrolysis of a metal (like zn) [ ] or hydrolysis of metal oxides (like mgo) in the gi environment [ ] . in fact, the ability to penetrate mucus without damaging the underlying gi epithelial layer has been a key-driver for micromotor technology in the gut. zhou et al. reported cylindrical micromotors with a length of~ . μm and~ . μm diameter, designed with multiple concentric layers for oral delivery of chemotherapeutics [ ] . the outermost layer is comprised of polyaspartic acid (paa), which is robust in acidic ph, but dissolves in the neutral ph of the intestine. in neutral solution, paa has a negative zeta potential (− . mv) by virtue of its carboxyl groups, which facilitates electrostatic binding with the amino groups of doxorubicin (dox). a thin fe layer underneath, allowed magnetic control and promoted galvanic corrosion of the zn particles core (fig. d i, ii). in an acidic environment, these produce hydrogen gas, which propels the dox incorporated micromotor at an average speeds of . ± . μm/s for a time period of ± s. the microrockets with dox-fe (loaded with . mg dox per mg microrocket) were dosed to mice in water and control groups were dosed with non-propelling microrockets (with dox-pt) in water or the same volume of ultrapure water. after min, the mice were euthanized and the gastric tissue was cleansed and evaluated with fluorescence imaging (fig. d iii-v). owing to the innate fluorescence of dox and propulsion effects of microrockets with dox-fe, these microrockets showed strongest fluorescent signal all over the gastric tissue, whereas only a very small and local signal was detected from the controls (ultrapure water and microrockets with dox-pt). this study showed that the propulsion effects were sufficient to impart enhanced penetration of the mucus layer, with no adverse effect to the underlying gi epithelium as confirmed via hematoxylin & eosin staining (fig. d vi-vii) . in another study, wei et al. showed active delivery of antigens for oral vaccination [ ] . mg-microparticles were coated with red blood cell membranes that displayed the staphylococcal α-toxin, and subsequently with a layer of chitosan to facilitate mucoadhesion (fig. e i) . finally, an enteric layer of eudragit® l - was provided to protect the drug from the acidic conditions in the stomach. when administered orally to mice, micromotors safely passed through the stomach, after which the enteric coating was dissolved and the motors were activated. images of intestines from mice dosed with the vaccine showed that the micromotors accumulated in the intestinal wall much better than nonmotorized particles (fig. e ii-iii). furthermore, the micromotors stimulated the production of iga antibodies against the staphylococcal αtoxin about ten times more than the static particles (fig. e iv) . micromotors have also been applied to treat local infections in the stomach. de Ávila et al. demonstrated micromotors loaded with clarithromycin (clr) for treatment of h. pylori infection in a mouse model [ ] . the micromotors were fabricated around a μm mgcore with a thin layer of tio around it. this was followed by a clrloaded plga layer and then a thin (~ nm) layer of chitosan. see fabrication scheme in fig. f i. the coating process intentionally left an opening (~ μm), which serves two purposes: firstly, it allowed the galvanic corrosion reaction between mg and gastric acid protons, which increased ph and produced hydrogen gas that propelled the micromotor. secondly, it imparted surface asymmetry, which causes uni-directional motion with speed: μm/s for up to min (fig. f ii) . finally, the authors concluded mucopenetrative efficacy based on intense fluorescent signals (via incorporation of did & fitc dyes), meaning that the micromotors were efficiently distributed and retained throughout the mouse stomach h after oral gavage (fig. f iii) . therapeutic efficacy was further confirmed by quantification of bacterial burden in the stomach. clearly, micromotors can also become highly relevant for phsensitive drugs where the bioavailability could benefit from delivery in the stomach rather than the small intestine. such a system could create a suitable physiological micro-environment, i.e. near-neutral ph, for drug release in the stomach. however, from a clinical translation perspective, there is an immediate need for long term study to demonstrate its safety, toxicity and foreign body response in the gut. further, the amount of drug loaded in such microdevices may be of relevance in murine studies. however, in case of larger animals, like porcine models, a significantly larger amount of devices and drug will be required to achieve similar therapeutic efficacy. while price parity is a different discussion all together, active microdevices need to address some of the above challenges before they can emerge as key-challenger for some of the well-established oral drug delivery technologies [ ] [ ] [ ] . key success criteria for oims in gi tract include: a) chemical stability in low ph and enzyme-rich environment; b) mechanical properties with a design which is conducive for soft tissue navigation (i.e. no sharp edges or exposed metal surface to cause an allergic reaction); and c) minimal toxicity or foreign body response. in case of electronic microdevices, radio-frequency (rf) radiated power and electromagnetic compatibility (i.e. the device does not affect itself or other devices in its vicinity) are of high importance [ , ] on-board energy storage systems can amount more than % of the mass of an oim, and in a way, govern overall toxicity [ ] . this is especially the case with unintended device retention, which is also one of the key-risks identified by the fdaa meta-analysis of wireless endoscopy suggests a pooled retention rate between . and . % [ ] . since, gastric transit time and risk of retention increases dramatically with the device size, it is vital to utilize flexible, biodegradable materials [ ] . ideally, a device must dissolve completely within h to prevent clinical obstructive symptoms. to this end, fda has established a list of materials generally regarded as safe (gras) [ ] , which includes gelatin, cellulose, pla, pva, various derivatives of plga and several other natural polymers and metals like fe and zn [ ] . readers interested to know more about biodegradable polymers are suggested to look elsewhere [ , ] fda regulation allows device size measuring mm × mm (like video capsule endoscope like pillcam®) [ ] as an infrequently dosed diagnostic devicethough such a size is not very practical for regular oral drug administration [ ] . li et al. presented a d printed macrodevice comprised of two concentric compartments [ ] . the authors explored fused deposition modeling (fdm) to d print a tablet (duotablet, . mm × . mm) with a controllable drug release profile (fig. a i) , achieved through its multilayer design; in this case, layers of polyvinyl alcohol containing glipizide for diabetes treatment. the use of d printing allowed for easy manufacturing which could be exploited in the field of personalized medicine with customized release profiles (fig. a ii inset) . for compatible drug and filament combinations (i.e. no chemical reaction between the drug and filament) duotablet's coating could protect the drug from degradation in the low ph environment of the stomach, or act as an initial high release rate followed by a more gradual release rate. furthermore, flexibility in in the d printing process makes it easy to dimensionalize the tablet as per requirement. another strategy for controlled drug release via an ingestible device includes ph sensitive poly(n-isopropylacrylamide-co-methacrylic acid) (pnipam-maa) nanoparticles loaded in polydimethylsiloxane containers (size mm) [ ] . upon ph decrease, these nanoparticles shrink, which opens up channels for drug release as shown in fig. b i-ii. however, subcutaneous implantation of an empty control device triggered an inflammatory reaction, suggesting a need for better material properties. to this end, liu et al. demonstrated an ingestible hydrogel device that can be ingested as a standard-sized pill, which swells rapidly into a large soft sphere, and maintains robustness under repeated mechanical loads in the stomach for up to one month (as demonstrated by large animal tests) with no signs of toxicity [ ] . macroscale devices routinely incorporate mucoadhesive coatings to aid correct device orientation and increase retention time. generally, the remainder of the device surface is made of a hydrophobic polymer such as cellulose acetate or polydimethylsiloxan (pdms) to impart greater protection against acidic/enzymatic degradation. overall, such a dual-feature design has shown to improve oral bioavailability [ , ] , including oral delivery of insulin [ ] . as an example, gastric patches comprising a mucoadhesive surface of chitosan, alginates or cellulose derivatives have been loaded with a drug [ ] . miyashita et al. utilized a self-unfolding stomach patch, which expanded five times when deployed in the gut towards patching of stomach wounds [ ] . in fact, it will be interesting to document adhesive forces between such polymeric origami structures to attain greater information on possible medical applications. to this end, terry et al. investigated adhesive forces of different capsule robot materials (polycarbonate, stainless steel and micro patterned pdms) against intestinal mucosa [ ] . mucoadhesion was evaluated using both tack and peel adhesion tests ex vivo. they found the peel adhesion to porcine mucosa ( . ± . mj cm − ) to be several orders of magnitude less than that of the commercial adhesive on a polypropylene surface ( ± mj cm − ). further, tack separation required higher force ( . ± . mj cm − and ± mj cm − ) than the peel mode; something that could potentially be utilized in the device design, especially towards delivery of hemostatic agents in the gi tract. while mucoadhesion will bring the device closer to the gi epithelium, a key-challenge remains in getting the drug across the thick mucus layer and tight junctions. this problem has been addressed by incorporation of microneedles over a mucoadhesive surface, activated by physio-chemical stimuli (i.e. unfold, puncture, expand, contract, float etc.), towards therapeutic payload delivery across the underlying gi epithelium. much like a transdermal microneedle technology, microneedle patches can be coated with a ph sensitive layer, which dissolves in the appropriate site of the gi tract to release drug-incorporated microneedles. in fact, rani therapeutics has developed several technologies to deliver macromolecules like proteins and antibodies. ranipill® is an ingestible capsule device containing a) a guide tube for device orientation for tissue penetration; b) delivery actuation mechanism; and c) a biodegradable release element with drug (i.e. microneedles) [ ] . abramson et al. also took advantage of the microneedle concept to design a luminal unfolding microneedle injection (lumi) device on top of a compressed spring [ ] . when the device enters the phneutral small intestine, a polymer that was immobilizing the spring gets dissolved, which leads to ejection of the device (fig. c iii) . packed inside a polymeric capsule, lumi comprises of a spring actuated -polymeric arm contraption made of polyethylene oxide and soluplus®, which causes the device to unfold and expand (fig. c iii) . at the end of each arm, multiple drug-loaded microneedles are forced into contact with the intestinal tissue. the arm length was designed to be long enough to ensure contact independent of the deployment orientation (fig. c iv) . furthermore, the optimized device caused penetration (force threshold of mn), but not tissue perforation (threshold of n . n). the microneedles can be loaded with . mg drug/ . cm , which is sufficient to illicit a clinical response, as demonstrated towards in situ delivery of insulin (fig. c v) . finally, upon successful delivery, the arms slowly degrade and the capsule separates into multiple parts to reduce the risk of mechanical obstruction. a different release mechanism was applied in an ingestible selforienting millimeter-scale applicator (soma), where re-coiling action of a stainless steel spring penetrated insulin needles across the gastric epithelium (fig. d iii) [ ] . compared to lumi, soma has the advantage that it delivers insulin across the gastric epithelium (fig. d iii) , and thus soma does not rely on gastric emptying to reach its target. this is an important advantage since gastric emptying typically occurs within - h, but can vary up to h in diabetic patients [ , ] . further, oral delivery of lumi was not possible yet and the results described in the published work relies on device deployment in swine jejunum in vivo. thus, it is still to be shown how lumi deals with ingestion and gastric emptying rate. irrespective of their bioavailability, both lumi and soma represent the growing area of self-actuating medical devices for gut engineering. this in itself is an important step, and gives rise to optimism regarding oral delivery of insulin to patients in the near future. despite improved bioavailability, these devices do not allow timecontrolled or sustained drug release, which can be an important parameter for clinical translation. however, for some applications it is of outmost importance to maintain a plasma concentration above the minimum effective concentration (mec) of the drug for - h, which often requires colonic absorption. intellicap® offers a new technology to obtain information about the colonic absorption profile of a given drug early in the development process [ ] . it is a swallowable device ( × mm) and can be filled with up to . ml of liquid formulation and programmed to deliver it over a period of h. intellicap® extrudes the drug via a piston moved by a spindle connected to a motor. the motor is controlled by an onboard microprocessor, which can be programmed before ingestion or via the wireless data exchange unit (fig. e i) . it has a built-in ph sensor that enables real-time localization in the gut by monitoring the ph profile, thus allowing for drug release in [ ] ]. reprinted with permission from aaas. e) intellicap® is a device for controlled release drug delivery, which can be preprogrammed, real-time controlled, or triggered at specific temperature/ph changes to deliver with different rates. f) balloon based delivery device. both the needle injection and liquid drug pumping are actuated by a balloon being inflated upon mixing of reactant a and b. a specific region of the gi tract. in a clinical study with healthy volunteers (n = ), an intellicap® device with diltiazem hcl was compared to the traditional diltiazem capsule. intellicap® reported a faster time (t max ) for maximum plasma concentration (c max ) and a higher c max . while, overall plasma profiles were found to be similar in many other aspects, intellicap® device can be a beneficial tool to assess colonic absorption. another interesting technology in terms of self-actuation is a balloon-actuated microneedle device [ ] . fig. f shows a schematic representation of this technology where a gas-evolution reaction proceeds when reactant a and reactant b get in contact. the resulting gas expands the polymer balloon, which pushes a needle sitting on top of it. compressible reservoirs with one or multiple liquid drugs can be connected to the needle, and upon balloon inflation, the drug is squeezed out. interestingly, upon completion of drug delivery, the balloon continues to expand, which leads to fracturing of the capsule fabricated from separate portions joined by seams. the seams can also be made from biodegradable material, causing them to work also in a springactuated version. this fracturing then facilitates safe passage through the gi tract. capsule endoscopy obtained clearance by the fda and ce certification in paving its way into clinical gastroenterology [ ] . although efforts worth two decades preceded this event, it was in that paul swain swallowed the first wireless capsule endoscope [ ] and the rest is history [ ] . the initial version of such a wireless endoscope had three major challenges: ever-changing capsule orientation in the wide colon, battery life, and visualization of the mucosa. these challenges were overcome by addition of a second camera monitor (forward and backward), additional battery, and vigorous bowel preparation prior to the ingestion. this resulted in development of a pillcam wireless capsule endoscope to do real-time gi tract monitoring [ ] . the second version of pillcam (colon ) had two major changes: the angle of view was widened from °to °and the recorder was updated to take advantage of artificial intelligence (ai) (fig. a i-ii) . further, incorporation of an adjustable frame rate extended the battery life to allow visualization all the way through the gi tract. lastly, ai detection of the location made it possible to notify the patient regarding possible interventions, such as ingestion of a prokinetic agent or a booster laxative. clearly, the pillcam technology is a promising tool to diagnose the presence and degree of inflammation in the small and large intestine, but it falls short for early diagnosis before occurrence of any visible symptoms. in this regard, mimee and nadeau reported an early stage detection system in the form of an ingestible micro-bio-electronic device (imbed) [ ] . the design of imbed comprises of a semi-permeable membrane that separates the luminal space from an inner chamber holding genetically modified probiotic e. coli nissle. this bacterial strain generates light upon sensing of certain blood markers, which is detected by photodetectors embedded in the electronics (fig. b i-iv) . this was utilized for detection of gi bleeding (induced with indomethacin) in murine fecal sample. further, a study in pigs demonstrated in situ blood detection in the stomach with a sensitivity and specificity of % after min increasing to % after min (fig. b v) . imbed offers a minimally invasive alternative to diagnose acute bleeding in the upper gi tract, which would otherwise require an endoscopic observation. also, it supports in situ biochemistry studies, which could lead to the discovery of labile biomarkers that would otherwise degrade before excretion in stool. despite clear advantages, the capsule is still too large for unmonitored ingestion ( mm in diameter and about cm long) and requires insertion via an orogastric tube. an alternative to imbed for in situ measurements would be sampling of the gi material for post-sampling analysis. to this end, a purely mechanical device (optimally by mm) that samples gastrointestinal material via a suction mechanism has been patented by biome oxford limited [ ] . one end of the device has sample collection openings, while the other holds a dissolvable filling material (e.g. natural polymers like cellulose and starch derivatives) (fig. c) . initially, this material is protected by a ph-sensitive coating, but upon entering the small intestine the coating is dissolved and the material is exposed. this causes the relaxation of a spring, and upon expansion it creates a partial vacuum in the sealed microchamber. the resulting pressure gradient forces the sample collection valves to open and gi fluid rushes into the sample chamber until the pressure difference has dropped below the threshold for valve opening. similarly, nejah et al. also developed a suction based sampling device, where a salt-holding chamber made with a semi-permeable membrane forms an osmotic pump, which drives the luminal fluids into the device (fig. d i) . also, the device can be controlled magnetically (fig. d ii) [ ] . the authors claimed that such a design promotes continuous sampling throughout the entire intestine as shown in fig. d iii-v ( . μl/h for almost h). however, the use of membrane filtering makes it complicated to retrieve absolute information, e.g. number of bacteria per volume, and so only allows for proportions between the present species. such a suction mechanism was also demonstrated towards intentional sensor retention towards long-term data collection in the gi system. nutech ventures has disclosed a patent describing a suction based device to achieve long term data collection by mechanical fastening. the device aims to overcome the challenges of attachment to the gi wall, such as irregularity, slippiness, chemically corrosiveness, and physiological activity from peristalsis [ ] . the opportunities to modify the device for sensing or manipulation are manifold. for sensing, trigger mechanisms such as heat, ph, pressure, analytes or flow rate could be implemented. the measurements can be wirelessly transmitted to the outside and/or it can be used to trigger the suction mechanism to induce anchoring in the desired region of the gi tract. alternatively, the suction can be preprogrammed to activate after a certain time. the sensors and communication system can be powered by a battery or with a power generator driven by peristalsis force. however, intestinal wall proximity prior to suction activation is still a challenge. since only one attempt at anchoring is possible, this is a critical part of the design. for this reason, two peristaltic pressure sensors are present on the device, which informs about the tissue proximity, and aids the decision about when to actuate. one of the major challenges for researchers in medical devices and robotic intervention, both in academia and in industry, is the process of converging a medical device prototype from the lab to the clinic. in order to ensure safety and effectiveness of new medical devices, the prototypes must obtain regulatory approvalsomething that needs to be conceptualized since the very ideation of the research project. typically, fda does not regulate specific materials for an implantable device, but evaluate the device as a whole. to do so, fda assigns three main regulatory classes: class i, ii and iii with increasing level of risk. as an example, wireless endoscopy falls under class ii risk profile. the european commission follows classes (i, iia, iib and iii). table provides an overview of the technologies discussed in this review with regard to their material composition, device design and therapeutic outcome. it is evident that the devices discussed in this article rely on complex fabrication proceduresoften involving both organic and inorganic materials serving multiple purposes. it can be observed from the detailed descriptions presented in table that the materials, or more specifically the polymers used in the production of these devices, are often chosen based on the properties needed for the fabrication schemes [ , ] . as depicted in table , several drug delivery technologies are biocompatible but not biodegradable. likewise, biodegradability will become a challenge if a device is being used for sensing or sampling. the tug-ofwar between toxicity and therapeutic efficacy can invariably be won by the body, thanks to our immune system, but is it the 'best in class' solution for a particular medical problem? characteristics such as t g , tanδ, young's modulus and solubility in specific solvents lead to the use of specific polymeric materials for very specific fabrication processes. in this manner, engineering polymers with high processability e.g. pmma, pc and peo or castable elastomers such as pdms are more compatible with the existing scalable microfabrication techniques [ , ] . on the other hand, production of microdevices by biodegradable and biocompatible polymers, such as polysaccharides (e.g. alginates, dextran, chitosan etc.), and protein-based polymers such as gelatin can hardly be scalable due to less favorable processability and more importantly, the costs of extraction and purification of such polymer from natural resources. clearly, device development seems to have been fabrication-driven, which leaves gaping holes in terms of material properties governing in vivo safety profile and even clinical studies further down the road. future oims need to be simple-despite their intrinsic complexityas well as easy to use, non-toxic, and with superior performance. the common notion of a disruptive innovation, if not being made for a resource-limited setting [ ] , needs to be checkeda medical devices, at the very least, should have a comparable traditional performance and higher ancillary performance to meet regulatory requirements [ ] . another major challenge is the testing of new devices in patients. absence of such a transparent framework often results in critical setbacks, as was revealed by the implant files, a global investigation into medical devices that were tested inadequately or not at all [ ] . the european union (eu) made an advancement to tackle this issue by revamping its medical device regulation (mdr) policy. previously in the eu, class i or low risk devices needed merely a "self-declaration" by the manufacturer. however, improvised eu's medical device regulation act, which will apply from may (postponed until may due to covid- crisis), puts stringent controls on active medical devices [ ] . invasive control systems, such as active therapeutic devices (with integrated or embedded diagnostic function), which were previously being assigned to class iib, will now adhere to the stringent requirements of class iii. the same is applicable for invasive devices (like the ones in gi tract) or devices involving therapeutics delivery (like the insulin pump on skin). clearly, this will drastically change the medical device development scenario in the eu, as compared to the us, where (k) filings facilitate regulatory clearance based upon substantial similarity with the previously approved medical devices (i.e. predicate device) [ ] . here in the eu, the implications of new mdr policy is yet to be seen, but it is safe to say that more often than before, a clinical trial will be required [ , ] in the near future, three main focus areas of development will include: i) safer power sources; ii) non-invasive microsampling d printed high temperature resin + cellulose acetate size- enteric coated capsule containing helical channels sampling of the gi tract contents and microbiome using osmotic pressure was tested in vitro and in vivo in pigs and primates biocompatible [ ] technologies; and iii) imaging modalities in vivo. self-activated microdevices, like micromotors, and stimuli-responsive polymeric origami structures will provide a low-power actuation scheme for specific gi applications. at the same time, controlled motion of a microdevice by wireless power transfer will gain traction [ ] though for gi applications, peristalsis is still a force to reckon with. biocompatible microfabrication technologies, like d printing, and large-scale production via additive manufacturing, will dominate microdevice design for exploring new applications in microsampling techniques. another major focus area will be development of in vivo imaging modalities in the gutboth ultrasound and magnetic resonance will have their fair share. in fact, ultrasound may have an additional advantage by providing physical actuation owing to the phenomenon of acoustic cavitation [ , ] . the next generation of such oral microdevices will demonstrate 'intelligence' by implementation of the 'perception-decision-action' loop architecture, which will further blur the lines between medical devices and microrobotics. this is not a distant futureby , the world will have three times as many smart connected devices as peopleand more and more medical devices and processes contain integrated sensors [ ] . this will give rise to smarter oims, which will be more patient-specific; thanks to ongoing progress in the ai and huge amount of patient specific data (which these devices will be able to generate/harness). finally, greater cooperation is needed between researchers and relevant stakeholders i.e. pharma companies, clinicians and drug/healthcare regulatory authorities. the evolution from endoradiosonde into a 'robotic pill' has already begun and will require us to rethink the 'patient-clinician-technology' relationship in the near future. the operation of the century: total hip replacement better speech recognition with cochlear implants pathology of second-generation everolimus-eluting stents versus first-generation sirolimus-and paclitaxel-eluting stents in humans a brief history of cardiac pacing prosthetic implantation of the human vestibular system past, present, and future of insulin pump therapy: better shot at diabetes control a review of in-body biotelemetry devices: implantables, ingestibles, and injectables medibots: dual-action biogenic microdaggers for single-cell surgery and drug release thread-like radical-polymerization via autonomously propelled (trap) bots (adv. mater. / ) biopsy using a magnetic capsule endoscope carrying, releasing, and retrieving untethered microgrippers clinical experience with the intraparenchymal intracranial pressure monitoring codman microsensor system image-guided interventions: technology review and clinical applications when oral agents fail: practical barriers to starting insulin robotics in the gut challenges and opportunities in the encapsulation of liquid and semi-solid formulations into capsules for oral administration non-invasive delivery strategies for biologics challenges for the oral delivery of macromolecules toward oral delivery of biopharmaceuticals: an assessment of the gastrointestinal stability of peptide drugs mucus as a barrier to drug delivery -understanding and mimicking the barrier properties integrative physiology of transcellular and paracellular intestinal absorption multiple facets of intestinal permeability and epithelial handling of dietary antigens oral absorption, intestinal metabolism and human oral bioavailability effects of gender, age, and body mass index on gastrointestinal transit times oral delivery of biologics using drug-device combinations some biological issues in oral, controlled drug delivery factors affecting drug absorption and distribution, anaesthesia intensive care med drug delivery system comprising a reservoir containing a plurality of tiny planar microdevices for enhanced in vivo retention and oral bioavailability of poorly permeable drugs from concept to in vivo testing: microcontainers for oral drug delivery microcontainers for oral insulin delivery -in vitro studies of permeation enhancement polymeric microcontainers improve oral bioavailability of furosemide biodegradable microcontainers -towards real life applications of microfabricated systems for oral drug delivery picoliter-volume inkjet printing into planar microdevice reservoirs for low-waste, high-capacity drug loading bottom-up fabrication of multilayer enteric devices for the oral delivery of peptides modeling the fluid dynamics in a human stomach to gain insight of food digestion simulation of peristaltic flow of chyme in small intestine for couple stress fluid fabrication of sealed nanostraw microdevices for oral drug delivery predicting oral drug absorption: mini review on physiologicallybased pharmacokinetic models gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human guton-a-chip natural history of recurrent crohns disease at the ileocolonic anastomosis after curative surgery inflammatory bowel disease organs-on-chips at the frontiers of drug discovery intestine-on-chip device increases ecm remodeling inducing faster epithelial cell differentiation models of the gut for analyzing the impact of food and drugs modeling radiation injuryinduced cell death and countermeasure drug responses in a human gut-on-a-chip article microfluidic organ-on-a-chip models of human intestine micromotor-enabled active drug delivery for in vivo treatment of stomach infection ph-induced precipitation behavior of weakly basic compounds: determination of extent and duration of supersaturation using potentiometric titration and correlation to solid state properties oral delivery of macromolecular drugs: where we are after almost years of attempts artificial micromotors in the mouse's stomach: a step toward in vivo use of synthetic motors micromotors for drug delivery in vivo: the road ahead self-propelled and targeted drug delivery of poly(aspartic acid)/iron-zinc microrocket in the stomach biomimetic micromotor enables active delivery of antigens for oral vaccination microencapsulation: methods and industrial applications oral drug delivery current status of selected oral peptide technologies in advanced preclinical development and in clinical trials radiation characteristics of ingestible wireless devices in human intestine following radio frequency exposure at , , , and mhz emerging batteryingestion hazard: clinical implications indications and detection, completion, and retention rates of small-bowel capsule endoscopy: a systematic review advances in materials and structures for ingestible electromechanical medical devices generally recognized as safe (gras), encyclopedia of toxicology: third edition is the product a medical device biomedical applications of biodegradable polymers biomaterials in clinical practice: advances in clinical research and medical devices video capsule endoscopy of the small bowel size, shape and other physical attributes of generic tablets and capsules preparation and investigation of controlled-release glipizide novel oral device with three-dimensional printing an ingestible self-orienting system for oral delivery of macromolecules a monolithic polymeric microdevice for ph-responsive drug delivery ingestible hydrogel device mucoadhesive intestinal devices for oral delivery of salmon calcitonin delivery of exenatide and insulin using mucoadhesive intestinal devices intestinal mucoadhesive devices for oral delivery of insulin intestinal patch systems for oral drug delivery controllable, and degradable origami robot for patching stomach wounds small intestine mucosal adhesivity to in vivo capsule robot materials rani therapeutics a luminal unfolding microneedle injector for oral delivery of macromolecules gastric emptying time of fluids and solids in healthy subjects determined by c breath tests: influence of age, sex and body mass index gastroparesis-related hospitalizations in the united states: trends, characteristics, and outcomes novel orally swallowable intellicap®device to quantify regional drug absorption in human gi tract using diltiazem as model drug pillcam colon capsule endoscopy: recent advances and new insights the history of time for capsule endoscopy an ingestible bacterial-electronic system to monitor gastrointestinal health a device for sampling gastro -intestinal material ingestible osmotic pill for in vivo sampling of gut microbiomes gastrointestinal sensor implantation system microfabricated drug delivery systems: from particles to pores microfabrication methods for biodegradable polymeric carriers for drug delivery system applications: a review polymer nanoengineering for biomedical applications advances in biomaterials, drug delivery, and bionanotechnology business models for frugal innovation in emerging markets: the case of the medical device and laboratory equipment industry how disruptive innovation can finally revolutionalize healthcare, innosight ind new medical device regulations: the regulator's view medical device recalls and the fda approval process the need for transparency of clinical evidence for medical devices in a flexible microsystem capable of controlled motion and actuation by wireless power transfer intelligent magnetic manipulation for gastrointestinal ultrasound the characterization of acoustic cavitation bubbles -an overview scanning the future of medical imaging key: cord- -r ygqmy authors: lapeyre-mestre, maryse; boucher, alexandra; daveluy, amelie; gibaja, valerie; jouanjus, emilie; mallaret, michel; peyrière, helene; micallef, joëlle title: addictovigilance contribution during covid- epidemic and lockdown in france date: - - journal: therapie doi: . /j.therap. . . sha: doc_id: cord_uid: r ygqmy abstract addictovigilance is a safety monitoring targeted at substances with potential for abuse and dependence. this vigilance was involved during the period of covid- epidemic due to the significant changes in access to drugs and psychological disruption caused by the pandemic and lockdown. this article aims to present the different steps implemented by the french addictovigilance network in collaboration with the french health authorities from march to may , including monitoring of potential harmful events, and scientific communication. the first events were identified through the continuity of the networking between the french addictovigilance centres and their partners: community pharmacies, general practitioners, specialized structures and emergency wards. as soon as the lockdown began, first cases of overdoses (lethal or not) were reported with opioids, mainly with methadone, and other opioids (heroin, oxycodone, tramadol or antitussive codeine). lockdown-related noteworthy events consisted in clinical cases or other relevant information for which lockdown clearly played an important role : among the many substances identified at least once, pregabalin, benzodiazepines, cannabis, cocaine and nitrous oxide were the most significant in terms of prevalence, seriousness or particularly specific to the lockdown context. despite significant decrease in the activity and travel limited to vital needs, community pharmacies continued to identify falsified prescriptions in this period, highlighting an increase in suspicious requests for pregabalin, codeine and tramadol. in parallel, the french addictovigilance network continued its communications efforts in the period, issuing a newsletter on tramadol, a press release on methadone and naloxone, and participating in the covid- frequently asked questions (faqs) of the french society of pharmacology and therapeutic website (https://sfpt-fr.org/covid ). covid- epidemic has been an important challenge for addictovigilance, and has proved that this monitoring is highly essential for alerting health professionals and health authorities to points of vigilance in the field of psychoactive substances. any safety monitoring system is part of a global approach aimed at identifying emergence or spread of a health risk. this health security approach involves the early detection of signals and their most rapid integration into an action system allowing an adapted, effective and early intervention to preserve the health of populations. in the context of pharmacovigilance and drug safety, new or unexpected adverse drug reactions should be detected as early as possible in order to further inform and secure the use of the drug, giving the general population and health professionals the opportunity of evidence-based information about these risks. in the context of covid- epidemic, the french regional pharmacovigilance centres network ensured this mission [ ] , with an assessment maintained in its continuity, based on a pharmacological and medical characterization of cases, shared with a population-based approach integrating pharmacoepidemiological methods when possible, contributing to optimizing the level of evidence. sharing and collaboration, both within and beyond the french pharmacology and therapeutics scientific community, was integral within these special weeks and beyond (see frequently asked questions [faq] at https://sfpt-fr.org/covid ) [ ] [ ] [ ] . in the addictovigilance context, the field is even wider and more heterogeneous [ ] [ ] [ ] . in the first weeks of epidemic spread, most of the interrogations were related to the disease itself and to drugs with supposed antiviral properties or interactions with the immune system. concerns about substances of abuse appeared as soon as lockdown occurred in france on march , . this article the french addictovigilance network was set up in the s, in order to benefit from a proactive vigilance system targeted at substances with potential for abuse and dependence (except tobacco and alcohol), and to participate in a proactive and coordinated manner in the activities of the world health organization (who) expert committee on drug dependence [ , ] . this vigilance is based on spontaneous notification by healthcare professionals of any serious case of misuse, abuse and drug dependence involving psychoactive substances, regardless of their nature or status [ , ] . in addition to this passive monitoring subject to under-reporting, other sources of information have been developed to improve vigilance: systematic data collection on falsified prescriptions from pharmacies ("ordonnances suspectes indicateur d'abus possible", osiap survey) [ , ] and on secure prescription forms for narcotic drug prescriptions ( "antalgiques stupéfiants et ordonnances sécurisées", asos survey) [ ] , systematic data collection from patients seen in addiction specialized structures ("observatoire des produits psychotropes illicites ou détournés de leur usage médicamenteux", oppidum survey) [ ] , analysis of toxicological data on chemical submission [ ] or on deaths in a medico-legal framework ("décès en relation avec l'abus de médicaments et de substances", drames survey) [ ] . addictovigilance can broaden the assessment of the potential for abuse and dangerousness of substances by specific analyses on large databases from the national health data warehouse [ , ] , or on ad hoc field studies [ ] [ ] [ ] [ ] [ ] [ ] . the identification of a potential signal from one or more of the sources described above makes it possible to anticipate an emerging problem and to assess its magnitude using a multi-source approach (fig. ) [ , ] . on march , in his first address on the extend of the epidemic in france, the french president announced, in a message broadcast to the nation, the implementation of travel restrictions, lockdown, and a state of emergency involving the redeployment of the entire healthcare sector to prioritize covid- care from the following day. among the different measures launched by the government, several ones were intended to ensure continuous access for care, while limiting outing to what was strictly necessary (urgent medical care). in these conditions, both public and private medical sectors (general and specialized practitioners, nurses, most of other health professionals) decreased their activities, together with addiction specialized structures, in order to insure social distancing and prophylactic barrier measures to reduce the risks of viral contamination. for example, in many areas in france, several first line harm reduction structures [ ] and addiction specialized centres modified their way of functioning, with limited access hours, redeployment of nurses and doctors for covid care, remote consultations, etc., all these changes leading to a degraded operating mode. some other structures may have also closed their doors, in particular those offering conviviality space with coffee and food for homeless and vulnerable isolated people, because of the impossibility to ensure social distancing. the rules for renewing prescriptions have been modified by several decrees (the first being published in the official journal on march , [ ] ), in order to prevent the health risks related to the abrupt interruption of chronic exposure to drugs, in a context of a reduced availability of prescribers during covid- epidemic. pharmacists were invited to issue even if the period of validity of a renewable prescription has expired, within the framework of the initially planned dosage, a number of boxes per prescription line guaranteeing the continuation of treatment, for a period not exceeding one month. these measures include specific provisions concerning medicinal products liable to be abused or misused, such as anxiolytic or hypnotic drugs, opioid maintenance drugs and other narcotic drugs or drugs falling under the regulations of narcotics. along the successive decisions of the president and government over time, these decrees were intended to be prolonged during the period of the national state of health emergency. table summarizes the different situations concerning psychotropic and narcotic drugs (at the date of may , ). from march , , some important problems rapidly appeared: because of the strict lockdown and repeated controls for any outing or trip, border shutdown for all extra european countries, but also with our immediate neighbours, drug trafficking has been drastically impacted, raising fears of an increase of episodes of withdrawal syndromes in the population of drug users. opioid maintenance treatment (omt) should be considered as an essential treatment during the covid- pandemic, as significant risks to the community exist with an interruption of the stable provision of opioid treatment. difficulties for omt drug provision have been expected with permanent changes of the prescribing and dispensing rules for narcotic drugs in the first days of lockdown, leading some patients to stock large amount of methadone at home. there may be an increased risk of opioid j o u r n a l p r e -p r o o f overdose arising from i) erratic access to omt, ii) erratic access to illicit opioid supplies and iii) increased access to takeaway doses of methadone, which would have required the systematic prescription for take-home naloxone supplies [ ] . there was also a growing concern about the risk of overdose with methadone (or of accidental exposure because of lockdown and provision of takeaway methadone at home), as methadone was already the first substance involved in drug abuse-related deaths before the disease outbreak, with an increasing trend in the last years [ ] . unfortunately, despite drug approval for forms of naloxone directly available without medical prescription in , the level of use of takeaway naloxone from specialized structures or community pharmacies remains very low [ ] . psychological disturbances may occur due to the lockdown, with an increasing risk of misuse and abuse of psychoactive drugs in the population of drug users (including patients on omt particularly vulnerable to these disruptions), but also in the general population [ ] . distress may result in some people increasing their substance use and subsequently require treatment (for example, alcohol use may increase). changes in illicit drug supply may occur due to a range of complex interacting factors, with an increased demand for services. alternately, some people who use drugs may be less likely to request services during the pandemic, with an escalation of substance use during a time of distress. some not evidence-based and potentially deleterious "guidelines" were launched in order to anticipate withdrawals, with several dangerous recipes for substitution or techniques to make provisions of narcotic drugs. such practices may bring new patterns of problematic use, including access to new psychoactive substances sold on the internet, with free home delivery services for using up stocks of illicit drugs. finally, in relation with the covid- itself, concerns arose about risk of drug-drug interactions and qt prolongation with methadone potentially combined with chloroquine and hydroxychloroquine or azithromycin. actually, when infected by sars-cov- , older people, men and those with medical comorbidities (chronic pulmonary disease, cardiovascular disease, cerebrovascular disease, diabetes and a compromised immune system) present a much higher likelihood of acute respiratory distress, renal failure and death. due to the respiratory and pulmonary tropism of sars-cov- , people who smoke or vape tobacco or cannabis products were expected to be more at risk of pulmonary complications. immune-suppressed people, for example, due to hiv infection or other chronic medication conditions, are also at increased risk for sars-cov- infection. consequently, drug users with these conditions may be a subgroup more at risk. we described the different events and facts collected and observed from mid-march to may , . in the first days of lockdown, several concerns emerged in the field. the first events were identified through the continuity of the networking between the french addictovigilance centres and their partners (i.e. community pharmacies, general practitioners, specialized structures and emergency wards). by the second week of lockdown, several cases of methadone overdoses for people at home were reported, and falsified prescription forms to obtain hydroxychloroquine and azithromycin were also identified as osiap by different pharmacies on the french territory. these early signals have been transmitted to national health authorities, leading to the implementation of a weekly specific monitoring of noteworthy cases or events related to the covid- , related to the lockdown, and of all falsified or abnormal prescription forms reported through the osiap survey during the period. this weekly monitoring was closely done between the french addictovigilance network and the ansm [ ] . the lessons of this weekly monitoring by conference calls and shared minutes of the meeting are presented in the following paragraphs. the fig. summarizes the highlights of this monitoring. detecting and identifying signals are a cornerstone for addictovigilance actors: they need to be able to label a piece of information received as a signal [ , , , , [ ] [ ] [ ] . signals suggesting a public health risk are collected and analysed in continuous manner in a surveillance process implemented by watchdog or public health structures, in a perspective of alert, anticipation and early action. in this framework, a signal is defined as a piece of information concerning a health phenomenon or exposure to a risk or hazard, which requires investigation in order to validate it and decide whether or not it should be considered as an alert. the signals observed in addictovigilance may be related to human cases (unusual deaths, symptoms or syndromes grouped in clusters); to psychoactive substances or associations thereof likely to have serious health consequences (presence of adjuvants, degree of purity, novelty of the substance or its usage) and to new ways of administration or new settings of use. monitoring such noteworthy events is an important issue in addictovigilance. simad-covid was the national periodical assessment with the aim to proactively monitor and share occurrence of fatal and non-fatal overdoses due to opioid medications (methadone, opioid analgesics) or opioid substances (heroin) or other illicit drugs (cocaine). as soon as the lockdown began, first cases of overdoses were reported with opioids, mainly with methadone, and to a less extent, with heroine and other opioid analgesics (oxycodone, tramadol) or cough syrups containing codeine. until may methadone was the most reported drug among overdoses. interestingly, several characteristics of methadone overdose have emerged: i) accessibility of methadone by storage from family/friends at home was often reported ii) occurrence of overdose among opioid naïve subjects (never previously exposed to opioids or return to use after cessation) iii) occurrence among vulnerable subjects (homelessness, migrants, patients with psychiatric comorbidities) iv) methadone used outside its labelling in france, for anxiolytic or analgesic purposes iv) take-home naloxone was exceptionally used in the period. it is important to note that during this period the price for street methadone remained relatively low, suggesting continued accessibility during the lockdown period compared to illicit drugs. heroin overdoses were also observed in several areas, often among previous heroin users (around - years old) leading to severe opioid toxidromes (acute renal failure, rhabdomyolysis, haemodialysis). the same trend was observed with cocaine leading to cardiogenic complications including a patient with covid- myocarditis. overdoses were reported among young adults after tramadol use alone or associated with other drugs (cannabis) or after concomitant codeine and promethazine use (purple drank). lockdown related noteworthy events "simad confinement"" consisted in clinical cases or other relevant information for which lockdown clearly played an important role, and concerned all other substances, whatever their nature (medications, illicit drugs, diverted drugs). during the lockdown period and until may , , reports were collected by the french addictovigilance centres all over the country, including oversea territories. among the many substances identified at least once in these reports, pregabalin, benzodiazepines (including z drugs), cannabis, cocaine and nitrous oxide (n o) were the most significant in terms of prevalence, seriousness or particularly specific to the lockdown context. -first signals of abuse of pregabalin (a gabapentinoid close to gabapentin, approved for the treatment of neuropathic pain, epilepsy and generalized anxiety disorder) were reported in france from with falsified prescriptions, medical nomadism and diverted use for psychoactive effect [ ] [ ] [ ] . the french addictovigilance monitoring of pregabalin has shown, at the end of , a dramatic increase in the number of cases of abuse, with the emergence of a population of young abusers. during the whole lockdown period and then afterwards, reports came from medical doctors who were urgently requested for prescription of pregabalin by young people, often minors, including migrants. this pregabalin addiction was not clearly identified before by these health professionals, since in the recent past reports came only from community pharmacists reporting abnormal prescription of lyrica ® . during the period, several cases of overdose were reported with pregabalin, including one requiring hospitalization with dyspnoea and hallucinations in a -year-old male. -benzodiazepines and z-drugs were expected to be highly consumed during the beginning of the lockdown in france, because of social isolation or psychological troubles due to the lockdown with the potential increase of marital conflicts and domestic violence. no withdrawal syndrome was reported (renewal of prescriptions was possible along the period), but abuse or misuse (with alcohol or other psychoactive substances) were reported. clonazepam alprazolam, oxazepam and zolpidem were the most frequently reported. -several reports concerned n o indicating persistent diverted use during the lockdown due to i) a shortage of other substances in some areas and ii) a need to consume due to inactivity. on the other hand, difficulties to easily obtain large quantities of n o cartridges led a -year-old male to abuse cocaine because of his craving. during the lockdown, it would appear that home deliveries have been made easier with internet orders. neurological complications with sensory-motor axonal polyneuropathies were also observed in the period, highlighting the spread of this new phenomenon of n o addiction that has appeared in recent months [ ] . -unexpectedly, reports concerning cocaine were numerous (more over than with heroine or cannabis), while supply constraints could be considered as the same as for other illicit substances. actually, this accessibility varied according to the regions, with cocaine easily available in some ones and with a wide disparity in cocaine concentration. the above described case of switching n o to cocaine illustrates this greater availability of cocaine, with modified supply chains (home delivery instead of buying on the street from dealers). -cannabis supply was expected to be more difficult during lockdown. some patients reported withdrawal symptoms due to supply difficulties or an increase in prices, while others abused cannabis in a context of anxiety related to the outbreak. cases of accidental poisoning in children under years of age who have accidentally ingested cannabis have been also reported. in addition to these most frequent substances, other reports confirm that after a short period of waiting, the drug trade has adapted to lockdown, and cases of abuse, misuse or deleterious consequences of use were reported with synthetic cathinone -mmc (n = ), amphetamines (n = ), lsd, ketamine and ghb (n = each). finally, even if the number of reports seems quite low, it should be borne in mind that there is often a delay in reporting (cases that have occurred since lockdown break have not been reported by may , ) and that under-reporting in this area is very significant [ ] . the two first reports collected through the osiap survey concerned out of date and falsified hydroxychloroquine prescription forms (presented during the first week of lockdown), in the context of media coverage about its hypothetic efficacy on sars-cov- [ , ] . this first signal has been forwarded to the ansm at the end of march. from this date, all suspected falsified prescription forms identified by community pharmacies and reported to the addictovigilance centres were centrally analysed weekly and compared to the information collected at the same period in . as a reminder, osiap is one of the national program implemented by the french addictovigilance network in the s to record all falsified prescriptions presented to a network of community pharmacies located all over the country [ , ] . this monitoring program has been useful to identify addictovigilance signals or characterize the abuse potential of prescription drugs [ , [ ] [ ] [ ] . usually, osiap are periodically collected each year (in may and november) on a voluntary basis by sentinel pharmacies [ ] . outside these proactive collection periods, osiap are continuously reported by community pharmacies, regional health authorities or medical/pharmacy councils. the osiap intensive data collection planned for may was cancelled due to the lockdown. between march and may , , falsified prescription forms were reported by community pharmacies to the french addictovigilance network, in a context of a significant decrease in the activity and travel limited to vital needs. this frequency must be considered with caution, as falsified prescriptions are often reported with a significant delay each year. in comparison, prescription forms were collected in the same period in , including the intensive data collection in may [ , ] . fig. presents the main frequently reported drugs during the covid- monitoring by weeks, compared to the same period in (estimated through the information available on may , ). during this period, the most frequently reported drugs were pregabalin, antitussive codeine syrup and analgesic codeine and tramadol. pregabalin and codeine syrups were mainly requested by a population of young males. this profile was similar to that observed in the covid and the lockdown noteworthy events, highlighting the emergence of a little-known population to health professionals [ ] [ ] [ ] . the french addictovigilance network has published a national newsletter on addictovigilance news for several years ("bulletin d'addictovigilance"), which was issued four times in (january, april, september and october) and once in (january). table summarizes the different topics discussed in these newsletters, which highlight the emergence or confirmation of addictovigilance signals in the recent months. in retrospect, the majority of bulletins have addressed substances that had been a problem during lockdown. throughout the lockdown and then, communication by the french addictovigilance network remained active with release of new national communications. the last issue of the national addictovigilance bulletin was entitled: "limitation of the prescription period of tramadol: how did we get there". this bulletin presented a summary of the data collected in france on tramadol between and and summarized the key elements which have led in particular to limit the duration of prescription of this drug. from april , , the maximum prescription period for analgesics containing tramadol has been reduced from to months. continuation of treatment beyond months will require a new prescription. following the results of the national addictovigilance monitoring of methadone, the french addictovigilance network has published a press release on the need to maintain access to methadone during the lockdown period, while ensuring the safety of its use. methadone is a mu opioid receptor agonist indicated for the substitution of opioid dependence. in france, for at least the past ten years, it has been the most frequently retrieved substance during the toxicological analyses of those involved in deaths linked to the excessive use of psychoactive substances (drames survey). the lockdown period may increase the risks linked to exposure to this drug in naïve-opioid subjects including children and those around them not treated with methadone. it should be remembered that the potentially lethal dose of methadone ingestion in a person who has never used opioids is estimated at mg/kg body weight. the press release focused on the risk of overdose, due to the larger dispensed quantities, methadone "storage", consumption of larger quantities of methadone or other respiratory depressants (alcohol, benzodiazepines, other opioids, etc.), resort to illegal obtaining, risk of overdose in the event of resumption of methadone after a few days off, risk of serious poisoning in children or naïve subjects. the press release also highlighted the risk of qt prolongation increased because high doses of methadone itself and because of combination with drugs or substances which also modify qt: domperidone, macrolides (erythromycin, clarithromycin, etc.), antidepressants (citalopram, escitalopram), antihistamines (hydroxyzine), antipsychotics (haloperidol, quetiapine), as well as drugs currently tested against covid- in hospitals (hydroxychloroquine, azithromycin, lopinavir/ritonavir) or other psychoactive substances such as cocaine. in order to minimize these risks, the press release insisted on warning about purchase of these drugs outside the pharmaceutical circuit, and on the need to report treatment with methadone in case of hospitalization for sars-cov- suspicion. the press release also insisted on the urgent need to increase the distribution of naloxone to methadone consumers (see brochure about where and how find naloxone; fig. ). on march , , the french society of pharmacology and therapeutics has launched a national faqs website at https://sfpt-fr.org/covid , focused on the proper use of drugs during the covid- pandemic [ ] . the french addictovigilance network has joined the scientific council and has participated to document the responses to each question related to addictovigilance. one topic of the faqs was about opioid maintenance treatment, because drugs approved in this indication (methadone and buprenorphine) should be considered as essential medications during the covid- pandemic, and significant risks to the community exist with an interruption of the stable provision of opioid treatment. another topic was related to the accessibility of naloxone take home in france. another topic gave information on the risk to switch to other substances (cannabidiol or gabapentin) to manage cannabis withdrawal or to switch to opioid analgesics outside medical management for non-cancer pain [ ] conclusion covid- epidemic has been an important challenge for addictovigilance. only part of the events that took place during this period have been reported to the french addictovigilance network, and it is likely that in the coming weeks or months the number of overdoses or deaths related to substance abuse will be higher than described in this article. this is of particular concern for methadone, heroin and pregabalin, but also for cocaine and nitrous oxide which seem to be more accessible than expected in this period. this addictovigilance monitoring has proved to be indispensable for warning health professionals at the local and regional level in order to limit the risk for users, and for alerting health authorities at the national level to points of vigilance in the field of psychoactive substances. adverse drug reactions of hydroxychloroquine: analysis of french prepandemic sars-cov pharmacovigilance data off-label" use of hydroxychloroquine, azithromycin, lopinavir-ritonavir and chloroquine in covid- : a survey of cardiac adverse drug reactions by the french network of pharmacovigilance centers french society of pharmacology t. non-steroidal anti-inflammatory drugs, pharmacology, and covid- infection genesis of an emergency public drug information website by the french society of pharmacology and therapeutics during the covid- pandemic signal identification in addictovigilance: the functioning of the french system social media mining for toxicovigilance: automatic monitoring of prescription medication abuse from twitter comment on: an insight into z-drug abuse and dependence: an examination of reports to the european medicines agency database of suspected adverse drug reactions from psychoactive medicines to addictovigilance in french public health code the french system of evaluation of dependence: establishment in a legal system safety signal detection by the french addictovigilance network: innovative methods of investigation, examples and usefulness for public health medical prescriptions falsified by the patients: a -year national monitoring to assess prescription drug diversion network of centers for e, information p. survey of forged prescriptions to investigate risk of psychoactive medications abuse in france: results of osiap survey tamperresistant prescription forms for narcotics in france: should we generalize them? surveillance system on drug abuse: interest of the french national oppidum program of french addictovigilance network french network of centers for e, information on p. chemical submission: results of -year french inquiry décès directement liés aux drogues interest of large electronic health care databases in addictovigilance: lessons from years of pharmacoepidemiological contribution ten-year trend of opioid and non-opioid analgesic use in the french adult population a capture-recapture method for estimating the incidence of off-label prescriptions: the example of baclofen for alcohol use disorder in france identification and tracking of addictovigilance signals in general practice: which interactions between the general practitioners and the french addictovigilance network? parachuting psychoactive substances: pharmacokinetic clues for harm reduction medical complications of psychoactive substances with abuse risks: detection and assessment by the network of french addictovigilance centres use of new psychoactive substances to mimic prescription drugs: the trend in france identifying life-threatening admissions for drug dependence or abuse (iliadda): derivation and validation of a model les caarud, lieux privilégiés d'émergence de signaux pour l'addictovigilance arrêté du mars complétant l'arrêté du mars portant diverses mesures relatives à la lutte contre la propagation du virus covid- intérêt de la mise à disposition de la naloxone auprès des usagers de drogues pour le traitement d'urgence de surdosage d'opioïdes améliorer la balance bénéfices/risques de la méthadone en respectant ses spécificités pharmacologiques psychopathological consequences of confinement pharmacovigilance et addictovigilance dans le contexte du covid- : une surveillance renforcée detection of signals of abuse and dependence applying disproportionality analysis early signal of diverted use of tropicamide eye drops in france pregabalin use disorder and secondary nicotine dependence in a woman with no substance abuse history patterns of gabapentin and pregabalin use and misuse: results of a population-based cohort study in france drug abuse monitoring: which pharmacoepidemiological resources at the european level? warning on increased serious health complications related to non-medical use of nitrous oxide use of multiple sources and capture-recapture method to estimate the frequency of hospitalizations related to drug abuse evidence of clonazepam abuse liability: results of the tools developed by the french centers for evaluation and information on pharmacodependence (ceip) network slow-release oral morphine sulfate abuse: results of the postmarketing surveillance systems for psychoactive prescription drug abuse in france example of an investigation of an "emergent" phenomenon in addiction vigilance: the case of methylphenidate medical prescriptions falsified by the patients: a -year national monitoring to assess prescription drug diversion pharmaciens d'officine, étudiants en pharmacie et demandes de médicaments à base de codéine : étude observationnelle disproportionality analysis for the assessment of abuse and dependence potential of pregabalin in the french pharmacovigilance database detecting the diverted use of psychoactive drugs by adolescents and young adults: a pilot study site de l'association française des centres d'addictovigilance the french addictovigilance network would like to acknowledge all persons in the addictovigilance centres who participated in the active monitoring during this period (all health professionals who reported cases during the period, and persons in charge of psychoactive drugs at the ansm (aldine fabreguettes, emilie monzon, charlotte pion, nathalie richard). authors have no competing interest to declare key: cord- - ffa djf authors: syatila ab ghani, nur; emrizal, reeki; makmur, haslina; firdaus-raih, mohd title: side chain similarity comparisons for integrated drug repositioning and potential toxicity assessments in epidemic response scenarios: the case for covid- date: - - journal: comput struct biotechnol j doi: . /j.csbj. . . sha: doc_id: cord_uid: ffa djf structures of protein-drug-complexes provide an atomic level profile of drug-target interactions. in this work, the three-dimensional arrangements of amino acid side chains in known drug binding sites (substructures) were used to search for similarly arranged sites in sars-cov- protein structures in the protein data bank for the potential repositioning of approved compounds. we were able to identify target sites for the repositioning of approved drug compounds as potential therapeutics for covid- . using the same approach, we were also able to investigate the potentially promiscuous binding of the compounds to off-target sites that could be implicated in toxicity and side effects that had not been provided by any previous studies. the investigations of binding properties in disease-related proteins derived from the comparison of amino acid substructure arrangements allows for effective mechanism driven decision making to rank and select only the compounds with the highest potential for success and safety to be prioritized for clinical trials or treatments. the intention of this work is not to explicitly identify candidate compounds but to present how an integrated drug repositioning and potential toxicity pipeline using side chain similarity searching algorithms are of great utility in epidemic scenarios involving novel pathogens. in the case of the covid- pandemic caused by the sars-cov- virus, we demonstrate that the pipeline can identify candidate compounds quickly and sustainably in combination with associated risk factors derived from the analysis of potential off-target site binding by the compounds to be repurposed. epidemics caused by novel infectious agents result in situations where no known treatment regimens are in practice. case management would therefore first rely on treating and alleviating the symptoms. the focus of the treatment would then move on to eradication of the infectious agent from the host and more in-depth therapeutic management. such an epidemic scenario presented itself in the city of wuhan, hubei province, china in late [ ] . the causative pathogen for the observed acute respiratory distress was later identified to be a novel human coronavirus (ncov ) named as severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] . although, many coronaviruses are found in bat reservoirs, it is probable that sars-cov- also has intermediate hosts such as pangolins and snakes [ ] . three months after it was first reported, the disease, named coronavirus disease (covid- ) , had progressed into a global pandemic. the fast spread of the disease was however paralleled by the speed that data regarding the disease and its causative agent were generated. in mid-january , the first genome sequence was deposited into genbank (https://www.ncbi.nlm.nih.gov/genbank/); by mid-july , more than , complete genomes with high coverage from samples throughout the world had been deposited in the gisaid database (http://www.gisaid.org/; http://epicov.org). while the rate of genome sequencing and data sharing is unprecedented, the rapid availability of structure data has also been equally impressive. in late september , more than structures of sars-cov- proteins had been deposited in the protein data bank (pdb) [ ] . despite the number of confirmed cases passing . million with more than million fatalities worldwide in early october , treatment options are still lacking for covid- although several vaccines have recently started their trials in july . this dire but data-rich scenario has led investigators to resort to drug repurposing strategies. although such efforts to reposition approved drugs to a new target can be explored in a clinical setting, we focus specifically on how computational approaches can feature prominently in the identification of the candidate compounds. various approaches have been deployed to explore the repertoire of known and approved compounds for covid- . zhou et al. utilized network-based analyses of drug targets and the virus-host interactions in the human interactome to list potential drugs to prioritize for repurposing [ ] . an even larger effort that generated a sars-cov- -human protein-protein interaction map was able to identify druggable human proteins that could be targeted by currently available fda approved compounds to be used as covid- treatments [ ] . side chain similarity comparisons [ , ] have been reported to be a potential starting point in drug repurposing efforts [ ] . for such an approach, the d arrangements of known drug binding sites are collected as a search database to identify similar sites in non-homologous structures thus implying the capacity to bind similar ligands. drug-target interaction prediction using structural data has remained a largely unexplored niche [ ] . the identification of possible alternative binding sites for an approved drug can also provide insights into their possible off-target effects. there is a clear urgency to discover and deploy suitable candidates that can be repositioned against targets associated with covid- . nevertheless, it is prudent to steer clear of adverse effects resulting from the poly-pharmacological actions of promiscuous drugs with the ability to bind to other targets [ ] . in this work, amino acid side chain similarity searching was utilized to propose alternative target sites in sars-cov- protein structures for drug repositioning. these searches were based on the premise that if a known drug binding site could be found in a sars-cov- protein, then that protein could also serve as an alternative target for the same drug. this same principle was then used to identify off-target sites that could present as side effects or result in some form of toxicity. the list of potential drugs derived from the side chain arrangements similarity searches was then used to propose structurally similar compounds that could also target the sites already identified for repositioning. our approach differs significantly from that reported by zhou et al. [ ] and gordon et al. [ ] which can serve as additional confirmatory analysis and complement the gaps in existing work. the details of these differences will be discussed in a later section. the downloaded sequences were clustered at % sequence similarity cut-off using the cd-hit program [ ]. members of individual clusters were sorted according to the x-ray crystallography resolution; the sars-cov- protein sequence with the higher resolution structure was selected as the cluster's representative. the pdb structures containing representative sequences were compiled together for further similarity searches against the dataset of known drug binding sites derived from protein-drug complexes in the pdb. for the selection of drug compounds, we further selected drug compounds that are: (i) currently undergoing clinical trials for covid - , and (ii) protein was used as the receptor structure for docking. the python script contains all the necessary commands that will be executed in the ucsf chimera command line to automatically pre-process structures and perform blind molecular docking. the pre-processing steps of the ligand and receptor structures include the removal of water molecules and ligands, assigning the partial charges for both standard and non-standard residues, as well as an additional energy-minimization step. the atomic partial charges for standard residues including standard amino acids, water and know ligands, as well as non-standard residues were assigned based on the amber ff sb force field (default), while the partial charges for non-standard residues were calculated using the antechamber module based on the am -bbc method. in the case of residues with missing side chains, the amino acid side chains were replaced based on information from a rotamer library. energy minimization was performed with steps of steepest descent minimization set to . molecular docking was carried out using a local installation of autodock vina and linked for use in ucsf chimera. blind docking was carried out instead of using the binding site as a reference point. therefore, a whole protein structure target was exhaustively searched for potential binding poses using the default settings for parameters such as exhaustiveness value (set to ) and maximum number of binding modes (set to ). the default box size was used to sample the ligand orientation where it automatically covers the entire protein receptor thus allowing for matches of binding poses to not only known binding sites, but also to other putative sites that have not been reported elsewhere. upon completion of the docking run using the python script, ucsf chimera loads a selection of docking poses for visualization where the docking poses are ranked according to the docking scores reported in kcal/mol with more negative values indicating better binding. the sites found from the sub-structural similarity search is also visualized. the ucsf chimera session for individual script runs were saved for further curation and analysis. the sites from the sub-structural similarity search were compared against the sites in pre-computed binding poses from molecular docking, where an overlap of at least three matched residues with poses of docking scores more negative than - . kcal/mol selected for further analyses. . . searching for potential off-targets from human for selected drugs proposed for covid- potential off-targets for selected drugs proposed for covid- were identified using three different methods. first, known human proteins bound to the selected drug compounds were obtained from the pdb through the 'advanced search' interface in the rcsb using the ligand pdb id as a query. the list of pdb structures retrieved were filtered to only contain pdb with organism denoted as 'homo sapiens'. second, human proteins with similarly arranged sites to drug binding sites for the selected drugs were retrieved from pre-compiled results for sub-structural similarity searches in drug reposer web server. third, human proteins with more than % sequence similarity to individual sars-cov- protein structures were retrieved from blastp searches against the pdb. the list of proteins retrieved was filtered to only contain proteins with sequences more than % sequence identity to the query sars-cov- protein. these human structures were then used for molecular docking against the selected compounds. molecular docking runs were conducted based on the above-mentioned protocol using python scripts executed in ucsf chimera. a compound's involvement in specific biological mechanisms and potential adverse effects upon interaction with the selected compounds were manually assessed and extracted from information queried and similar ligands were structurally aligned in the ucsf chimera interface [ ] . the queried and the similar ligands were individually searched against the drug reposer application database to retrieve results for sub-sructural similarity searches. both sets of results were compared and shared sars-cov- protein targets from the list of proteins (proteins containing sites similar to binding sites for both queried and similar ligands) were obtained for molecular docking against the corresponding ligand molecules with autodock vina using the above-mentioned protocol [ ] . . results and discussion in this study, sub-structural similarity searches and docking analyses were carried out to: (i) identify potential targets and drug binding sites in sars-cov- proteins; (ii) identify off-targets for proposed drug compounds for covid- ; (iii) identify other approved drugs with similar structure to proposed drugs that are potentially useful for covid- treatment. a total of sars-cov- proteins were obtained from the pdb that included the following proteins; adp ribose phosphatase (pdbid: w ), spike protein (pdbid: vsb), main protease (pdbid: lu ), nucleocapsid (pdbid: m m), nsp -nsp complex (pdbid: yhu), nsp replicase (pdbid: w b), nsp -nsp complex (pdbid: w h), nsp (pdbid: w ), orf a encoded accessory protein (pdbid: w ) and rna-dependent rna polymerase or nsp (pdbid: m ). the substructure similarity searching used in this work utilized the assam computer program which solves a maximal common subgraph problem to match similar d arrangements of amino acids in a dataset of protein structures [ ] . the arrangements of amino acids in d space are represented as graphs, where the graph nodes are the pseudo-atoms representing side chain groups and the graph edges are distances between the side chain groups. using this scheme, it is possible to match similar d arrangements, such as catalytic sites and ligand binding sites, in non-homologous structures. drug reposer is an extended application of the assam program that focuses on sub-structures that constitute the binding sites for approved drug molecules [ ]. at the time of writing, approximately a third of the proteins encoded in the sars-cov- genome have corresponding pdb structures. in anticipation that more structures will be deposited, we have enabled the analysis pipeline to be deployed to process new structures as and when they become available, based on the clustering of protein sequences and comparison to readily available structures. the results from the analyses reported in this work and those that will be carried out by the pipeline for new structures will be made accessible via a dedicated module of the drug reposer web application - http://mfrlab.org/drugreposer/covid /. the list of pdb ids with pre-computed results from sub-structural similarity searches and the sequence clusters are also available at the same resource. the search for covid- treatments has resulted in the registration of more than clinical trials in the clinicaltrials.gov database to explore the repurposing of more than twenty readily available drugs ( searching for sites in the sars-cov- protein structures (hit sites) that are geometrically similar to sites for approved drug compounds (query sites) using the drug reposer application [ ] had identified matches that included sites from protein-drug complexes with sequence identities lesser than % to the corresponding sars-cov- proteins (table ). these results show that the computational approach adopted in this study is able to find similarly arranged sites in unrelated proteins which could be an advantage when there are limited numbers of homologous structural models to be used for comparison of binding sites. in addition, the selection of matches to proteins with lesser than % sequence similarity could be indicative of function differences, thus potentially distinct pathways where the bound drugs could be repurposed to. the sites identified in the sars-cov- proteins were then docked with their corresponding drug compounds derived from the protein-drug complex data. molecular docking runs resulted in the identification of several poses with docking scores ranging from - . kcal/mol up to - . kcal/mol, which are congruent with the results of the drug reposer searches (table , figure ). of these potential interactions, six have been reported in other studies [ ] [ ] [ ] . the sub-structural similarity searches carried out revealed that six of the nine analysed sars-cov- contain multiple potential alternative binding sites for different compounds. for example, the nucleocapsid protein contains potential sites for losartan, ritonavir, darunavir and aspirin ( the hiv protease inhibitors -darunavir ( ), ritonavir (rit) and lopinavir (ab ) -inhibit the hiv aspartyl protease and prevents the cleavage of gag and pol proteins into their subsequent protein components [ ] . the potential antiviral activity of such inhibitors against coronaviruses had been previously studied; nelfinavir for example, had been reported to inhibit the replication of sars-cov and prevent cytopathic effects [ ]. lopinavir and ritonavir had been shown to improve clinical outcomes from sars-cov infections and are hypothesized to bind to the -chymotrypsin-like protein ( clpro) or main protease [ ] . our analysis also demonstrated the potential ability of lopinavir (pdbid: qhc) to bind to adp ribose phosphatase (pdbid: w ) with a docking score of - . kcal/mol at a position close to the known substrate binding site ( figure a ). we found that the nsp - complex (pdbid: w ) may potentially bind to ritonavir (rit) in a manner similar to that observed in the hiv protease (pdbid: sh ) while adp ribose phosphatase (pdbid: w ) could potentially bind to lopinavir (pdbid: qhc) with a high docking score (- . kcal/mol) ( figure a). a potential site for folic acid (fol) binding that is similar to the arrangement found in dihydrofolate reductase (pdbid: i ) was also found at the interaction site between domain iii (residue - ) of two monomers, where dimerization is crucial for protease function took place (pdbid: y g). we also found that the nucleocapsid might bind to losartan, darunavir and aspirin at the dimerization site between two monomers in a similar manner to the sars-cov- main protease. the drug reposer searches also identified similarly arranged sites between the indomethacin-bound prostaglandin e synthase (pdbid: z h) and the nsp protein (pdbid: w h) ( figure b ). an arrangement of amino acid residues that make up the indomethacin binding site in cyclooxygenase- (cox- ), also known as prostaglandin synthase (pdbid: cox), was also found to be similar to residues at the vicinity of docked indomethacin in the nsp -nsp complex (pdbid: yhu) ( response. in this context, the binding of indomethacin to these protein structures (nsp /nsp or nsp ) may also prevent potential inflammatory events. the same mechanism could be adopted by other nsaids like naproxen (nps), that might recognize similar sites from cox- (pdbid: nt ) in the adp ribose phosphatase (pdbid: w y), as indicated from the sub-structural similarity we have uncovered. these sub- structural similarities to a known indomethacin binding site may explain the mechanism for studies that have reported the ability of nsaids to bind to sars-cov- proteins [ ] although the atomic level details of such interactions have not yet been reported. . . adp ribose phosphatase of nsp as potential target in sars-cov- our analysis revealed that the adp ribose phosphatase of nsp from sars-cov- has the most number of d residue arrangements that are similar to the binding sites in known drug targets compared to other sars-cov- proteins (table , figure ). all the identified sites are within the substrate binding sites with the docking scores for the different poses ranging from - . to - . . in this case, the known adp ribose phosphatase -apr complex was used as a control to obtain reasonable docking scores that could be considered acceptable based on predicted binding poses between the adp ribose phosphatase and the substrate, apr. the molecular docking with energy minimization steps resulted in several binding poses with docking scores ranging from - . to - . , with all sites located within the actual binding site for apr. the adp ribose phosphatase of non-structural protein (nsp ) is likely to be targeted by anti- retrovirals and several other drugs more than any other sars-cov- structures, particularly at the active site of the structure (figure a ). this finding is in agreement with recent computational screening for the drug binding ability of sars-cov- proteins which highlighted the promiscuity of nsp in binding to other molecules at the adp ribose binding site [ , ]. the de-adp ribosylation activity of nsp suppresses the expression of host innate immunity genes such as interferon and interleukin related genes [ ] . disruption of nsp function will allow for the host immune system to respond normally to the infection. sub-structural similarity searches and molecular docking runs have revealed the potential binding sites for darunavir ( ) that originally targeted hiv protease (pdbid: dh ), as well as chloroquine (clq) that originally targeted quinone reductase (pdbid: fgl) and indicated for malaria and rheumatoid arthritis, onto the adp ribosylation site of nsp (pdbid: w ) (table , figure ). despite the similarity of these sites in terms of their d arrangements, the similarity of their molecular functions is unlikely to be related. the docking results indicate that hiv protease inhibitors and nsaids are among the existing drugs that could potentially be repositioned against adp ribose phosphatase and several non-structural proteins for treatment of covid- . the similarly arranged residue patterns observed between the binding poses in sars-cov- proteins from docking simulations and those from available drug-bound protein complexes allow us to infer the similarities of the binding mechanisms shared by these proteins despite the lack of sequence similarities. . . potential off-targets of approved drugs proposed for covid- the binding of drug compounds to off-target sites in proteins other than their intended targets can lead to unexpected pharmacological outcomes including the activation or disruption of molecular functions that cause adverse effects or other unexpected conditions [ , ]. however, off-target effects are not necessarily negative and it is this same concept that is in use to repurpose approved compounds for alternative indications based on the availability of similar of binding sites shared among proteins involved in distinct disease pathways [ , ] . we deployed the same substructure searching methodology to identify off-target sites for the drugs being explored as covid- treatments. an assam search of the human protein structures in the pdb using the drug binding sites we have identified was used as a means to investigate whether the use of these drugs could alter other pathways. the searches led us to a compilation of potential off-target sites and/or effects for eleven approved compounds (table ). the substructural similarity searches for potential off-target sites in human proteins using the drug reposer application was able to identify several proteins that have similar geometry to the binding site of a drug proposed for repositioning against sars-cov- targets (section . . ). the same data also allowed us to compile potential repurposing opportunities of these drugs for other indications including covid- ( table and table ). non-homologous proteins that share similarly arranged sites for a particular drug molecule are more likely to be considered as off-targets because they may have different molecular function and are involved in distinct pathways that may not be associated with the original target disease. recent computational studies have proposed several hiv protease inhibitors [ , , ] , nsaids [ ] , and losartan [ ] as potential therapeutic agents for covid- . although we can confirm the presence of potential binding sites to these drugs on sars-cov- proteins, we were also able to identify potential off-target sites where these drugs may alternatively bind in the structures of human proteins (table ) . (table ). peripheral neuropathy due to the neurotoxicity of hiv protease inhibitors have been reported as complications resulting from anti-hiv treatment [ ] . one potential off-target protein that might cause such symptoms is the herc protein (pdbid: kci). disruption of this protein causes reduction of e ap activity that has been implicated in neurodevelopment disorders such as angelman syndrome and autism [ ] . losartan targets the angiotensin type ii receptor, however, it may also bind to the drug metabolizing cytochrome p (pdbid: x ) that has a similarly arranged site in ceruloplasmin (pdbid: kcw) ( table , figure b ). ceruloplasmin has been implicated with parkinson's disease where disruption of the oxidative activity by ceruloplasmin causes increased iron levels in the brain that is correlated to parkinson's [ , ] . on the other hand, it was also reported that losartan could be useful for parkinson's where it might be able to reduce oxidative stress and neurodegeneration [ ] thus warranting further investigations regarding the neuroprotective benefits of losartan. the function inhibition of certain off-target proteins may provide coincidental antiviral effects (table ) . other than potentially targeting the sars-cov- proteins, nsaids such as naproxen (nps), indomethacin (imn) and aspirin (ain) may also interact with host proteins involved in mounting the defense against viral infections. for example, we found that naproxen might be able to bind polypyrimidine tract-binding protein (ptbp ) (pdbid: qm ) based on the similarity of the binding site for naproxen in serum albumin (pdbid: po ) (table , figure c ). the ptbp protein had been shown to activate the replication of picornaviruses and coronaviruses through binding to its rna binding domain [ , ] , thus binding of naproxen to its binding site could potentially block viral replication. other nsaids like the indomethacin and aspirin might also induce antiviral properties by binding to myeloperoxidase, which is a part of host defense system (table ) . the protein acts as tissue damage factor that induces secondary bacterial lung infections causing the acute respiratory distress syndrome seen in influenza [ ] . decreased function of myeloperoxidase had been shown to potentially decrease inflammatory damage and lung viral load [ ]. sars-cov- proteins that may share a similar fold to human proteins were also considered as potential off-targets. in this case, sars-cov- proteins that retrieved a human protein by blastp alignment with more than % sequence identity is a possible indication of fold similarity. these protein structures were then analyzed to ascertain whether they contained a similar sub-structure arrangement as the sars-cov- protein that is being targeted for drug repositioning (table ) . table human proteins with more than % sequence identity to sars-cov- proteins retrieved by a blastp search of the pdb database. the sars-cov- adp-ribose phosphatase from nsp has a similar sequence to human adp-ribose binding protein and both share a similar mechanism of adp ribose binding (figure ) . the sars-cov- spike protein is found have sequence similarities to the irap protein with both being associated to the renin- angiotensin pathway. no human sequences with possible fold similarities were detected for the main protease and non-structural proteins that include the nsp , nsp and nsp which are conserved in viruses. the binding of approved drugs or inhibitors to interleukin a and mineralocorticoid receptors that have similar sequences to the nucleocapsid and nsp respectively, could prevent inflammation by the immune response [ , ], a known complication of covid- (table ). this would mean that such drugs could target both the virus and the host in parallel with potentially therapeutic results. . . other compounds with potential as covid- therapeutics based on ligand structure similarity it is known that similar drugs may require a similar binding environmentand can have similar inhibitory effects, thus making it possible that a target protein can interact with a set of drug molecules with similar structures [ ] . with this premise, the structures of the drugs proposed for repositioning against sars-cov- targets (proposed drugs) were used as a reference point to find other drug molecules with similar structures (matched drugs). this was carried out using the ligand search interface in the pdb that is based on the comparison of pharmacophores. the search identified matches with similar structures to the input queries -quinacrine, vardenafil, lenalidomide, pomalidomide, amprenavir and methotrexate ( table ). with the exceptions of methotrexate, which has structural similarities to folic acid (clinicaltrials.gov ids: nct and nct ), and lenalidomide, which is related to thalidomide (clinicaltrials.gov id: nct ), none of these compounds are involved in any known clinical trials for covid- at the time of writing. molecular docking targeting the sars-cov- proteins using both the proposed and matched drugs (shared sars-cov- protein targets) resulted in several binding poses that is indicative that the matched drugs can potentially bind to sars-cov- proteins in a similar manner as the proposed drugs (table , figure ). our analyses found that both darunavir and amprenavir can potentially bind to the same sars-cov- site (p , g , i , v , and d ) in nsp ( figure e , table ). darunavir, when docked on nsp , has a molecular binding affinity of - . kcal/mol. amprenavir, when docked at the similar site ( figure e ), also has a molecular binding affinity of - . kcal/mol (table ) . some structurally similar drug molecules are intended for similar indications. both darunavir and amprenavir ( figure e ) are protease inhibitors that have been used for the treatment of hiv. however, amprenavir is useful against infections that exhibit resistance to other protease inhibitors used in hiv treatment [ ] . thus, amprenavir might confer an advantage in a scenario where the protein target from sars-cov- develops resistance towards darunavir. both chloroquine and quinacrine ( figure a ) have been indicated for the treatment of systemic lupus erythematosus as well as other diseases [ ] . a study comparing the oculotoxicity of chloroquine and quinacrine in the management of lupus erythematosus found that quinacrine exhibits less oculotoxicity compared to chloroquine if taken at low doses [ ]. thus, quinacrine might be a less toxic alternative compared to chloroquine with regard to any ophthalmologic side effects. sildenafil and vardenafil ( figure d) vardenafil, amprenavir and methotrexate had been reported to potentially bind to sars-cov- proteins through structural analyses [ , ] . to our knowledge, the potential use of quinacrine, lenalidomide, and pomalidomide for covid- have not been reported elsewhere in the context of binding ability through structural analyses. furthermore, the finding that quinacrine is a readily available compound that has yet to be explored or proposed for covid- is novel to this work. should the current candidate drug molecules proposed for covid- clinical trials fail at any stage of the process, these structurally similar drug molecules can be investigated as potential alternatives. it is not unexpected that the use of these structurally similar compounds could be used in concert as a cocktail for more effective therapy [ ]. indicate regions containing residues within less than . Å to docked drug molecules. the orange shaded areas indicate regions containing residues that form the binding sites identified by drug reposer. . distinction from other covid- drug repurposing efforts and future directions in this work, all drugs that have been proposed for clinical trials were analyzed using the drug reposer pipeline to find their potential binding sites in any sars-cov- protein by virtue of having similar d arrangements of amino acid residues to the known target sites. it is not unexpected that our results will overlap or have parallels with the outcomes of other studies that have been recently published or are ongoing. however, the results presented here and in the covid- drug reposer resource, will also provide the relevant supporting insights regarding why or how a particular drug may be effective while at the same time, have the added advantage of presenting the potential capacity for off-target interactions that may cause or explain any side effects upon administration. the human proteins based on the analysis of association networks between human and sars-cov- proteins [ ] . in comparison to our analyses, there are two drugs that overlap with our results, chloroquine (targeting sigma -receptor:nsp ) and indomethacin (targeting ptges :nsp ). this study was intended to develop a pipeline to identify drug compounds that could be repositioned against sars-cov- targets using the available structural information in the pdb. this pipeline was also able to identify potential side effects or toxicity associated with those compounds that arose from off-target binding. integrating the data to pharmacophore matching tools allowed other similarly structured drug compounds to be identified that also had the potential to be repositioned against sars-cov- targets. the information derived from such analyses could be used as a means of decision making to prioritize down- stream experimental validation and assays. this study does not provide any experimental evidence validating the binding of the proposed repositioned drugs to sars-cov- proteins. the results of this study should not be regarded as an explicit treatment recommendation or protocol for covid- . a limited set of existing drugs extracted from lists of those currently undergoing or planned for covid- trials was used in this work. the analyses reported only utilized data of compounds that were structurally present as a standalone ligand in the pdb. both these factors restricted the number of potential candidates that could be proposed for repurposing. despite these limitations, our analyses yielded target sites for repurposing of which only had been mentioned in other studies. it is clear that the work reported here could be extended to include all known drug binding sites in the pdb. although the current targets for repositioning in this study considers only sars-cov- proteins, the pipeline can be integrated to network analyses methods to identify human proteins that could also yield therapeutic effects for covid- . furthermore, this study can also be extended to include other sars-cov- structures as and when they become available. such data will be updated via the specific drug reposer resource for covid- . the fastest and safest route to providing drug treatments for covid- would be to reposition approved compounds against targets from this newly described disease. at the time of writing, the search for effective covid- treatments is still ongoing. despite being subject to the availability of associated protein coordinate structure data in the pdb, the use of amino acid d side chain based sub-structure comparisons have proven to be a feasible means of identifying candidate compounds to be repositioned for covid- . our analyses yielded potential sites in sars-cov- proteins and drug compounds that could be repurposed for covid- . it is clear that the use of structural data from the pdb is able to provide high quality mechanistic level details for strategizing the selection of candidate compounds to be repurposed. the capacity to not only identify new target sites, but also identify potential off-target sites, provide a deeper level of context for the decision making process to safely proceed with exploring specific compounds to be repurposed for the new disease. acknowledgments we thank the malaysia genome institute for the use of computational resources. this the authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript clinical features of patients infected with novel coronavirus in wuhan, china the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars recent progress and challenges in drug development against covid- coronavirus (sars-cov- ) -an update on the status protein data bank: the single global archive for d macromolecular structure data network-based drug repurposing for novel coronavirus -ncov/sars-cov- a sars-cov- protein interaction map reveals targets for drug repurposing sprite and assam: web servers for side chain d-motif searching in protein structures imaaagine: a webserver for searching hypothetical d amino acid side chain arrangements in the protein data bank drug reposer: a web server for predicting similar amino acid arrangements to known drug binding interfaces for potential drug repositioning drug repositioning or target repositioning: a structural perspective of drug-target-indication relationship for available repurposed drugs drug promiscuity in pdb: protein binding site similarity is key cd-hit suite: a web server for clustering and comparing biological sequences firdaus-raih m. sprite and assam : web servers for side chain d-motif searching in protein structures autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading ucsf chimera- a visualization system for exploratory research and analysis uniprotkb/swiss-prot trial reporting in clinicaltrials.gov -the final rule structure based drug discovery by virtual screening of compounds against the crystal structures of six key sars-cov- proteins computational view toward the inhibition of sars-cov- spike glycoprotein and the cl protease potential drugs targeting nsp protein may corroborates a promising approach to combat sarscov- virus nature to nurture-identifying phytochemicals from indian medicinal plants as prophylactic medicine by rational screening to be potent against multiple drug targets of sars-cov- therapeutic options for the novel coronavirus ( -ncov) hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus coronaviruses-drug discovery and therapeutic options coronavirus nsp , a critical co-factor for activation of multiple replicative enzymes structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors indomethacin and resveratrol as potential treatment adjuncts for sars-cov- /covid- inhibition of cyclooxygenase blocks human cytomegalovirus replication repurposing naproxen as a potential antiviral agent against sars-cov- the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection large-scale detection of drug off-targets: hypotheses for drug repurposing and understanding side-effects oxidative stress in malaria parasite-infected erythrocytes: host-parasite interactions -acyl pyrroles: mimicking acetylated lysines in histone code reading an engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications pde inhibitors -pharmacology and clinical applications years after sildenafil discovery folate and antifolate pharmacology inhibitors and structure-activity relationships of human cytochrome p c and implications in drug development structural basis for catalytic and inhibitory mechanisms of human prostaglandin reductase ptgr mechanisms of peroxisome proliferator activated receptor γ regulation by non-steroidal anti-inflammatory drugs inhibition of viral macrodomain of covid- and human trpm by losartan tay-sachs disease mutations in hexa target the α chain of hexosaminidase a to endoplasmic reticulum-associated degradation perspectives on progressive strategies and recent trends in the production of recombinant human factor viii down-regulation of the tumor suppressor gene c-terminal src kinase: an early event during premalignant colonic epithelial hyperproliferation different origin of adipogenic stem cells influences the response to antiretroviral drugs deficiency in serine protease inhibitor neuroserpin exacerbates ischemic brain injury by increased ceruloplasmin and iron in alzheimer's disease and parkinson's disease: a synopsis of recent studies selenocysteine lyase-mediated selenium recycling pathway leads to metabolic syndrome in mice activation of picornaviral iress by ptb shows differential dependence on each ptb rna-binding domain contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection influenza virus infection causes neutrophil dysfunction through reduced g-csf production and an increased risk of secondary bacteria infection in the lung perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections neurologic complications of hiv- infection and its treatment in the era of antiretroviral therapy a comparison of neurodegeneration linked with neuroinflammation in different brain areas of rats after intracerebroventricular colchicine injection risk of stroke associated with nonsteroidal anti-inflammatory drugs. vasc health a case of parkinsonism worsened by losartan: a probable new adverse effect proteomic investigations of human herc mutants: insights into the pathobiology of a neurodevelopmental disorder importance of the brain angiotensin system in parkinson's disease binding protein affects coronavirus rna accumulation levels and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites parp and parp cross-regulate macrophage activation via stat adp-ribosylation at ) receptor is the enzyme insulin-regulated aminopeptidase ephrinb are critical for its use as an alternative receptor for nipah virus altered inhibitory synapses in de novo gabra and gabra mutations associated with early onset epileptic encephalopathies brain penetrant p αmapk inhibitor candidate for neurologic and neuropsychiatric disorders that attenuates neuroinflammation and cognitive dysfunction the role of the mineralocorticoid receptor in inflammation: focus on kidney and vasculature interleukin- a (il- a), a key molecule of innate and adaptive immunity, and its potential involvement in covid- -related thrombotic and vascular mechanisms advances in the development of shape similarity methods and their application in drug discovery pharmacology and clinical experience with amprenavir current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review antimalarial therapy for lupus erythematosus: an apparent advantage of quinacrine vardenafil for treatment of men with erectile dysfunction: efficacy and safety in a randomized, double-blind, placebo-controlled trial comparison of clinical trials with sildenafil, vardenafil and tadalafil in erectile dysfunction pomalidomide and its clinical potential for relapsed or refractory multiple myeloma: an update for the hematologist lenalidomide and thalidomide: mechanisms of action -similarities and differences molecular basis for pharmacokinetics and pharmacodynamics of methotrexate in rheumatoid arthritis therapy new approaches to cancer chemotherapy with methotrexate adverse drug reaction to methotrexate: pharmacogenetic origin potential covalent drugs targeting the main protease of the sars-cov- coronavirus nanoparticles for combination drug therapy key: cord- -pmf stua authors: jourdan, jean‐pierre; bureau, ronan; rochais, christophe; dallemagne, patrick title: drug repositioning: a brief overview date: - - journal: j pharm pharmacol doi: . /jphp. sha: doc_id: cord_uid: pmf stua objectives: drug repositioning, that is, the use of a drug in an indication other than the one for which it was initially marketed, is a growing trend. its origins lie mainly in the attrition experienced in recent years in the field of new drug discovery. key findings: despite some regulatory and economic challenges, drug repositioning offers many advantages, and a number of recent successes have confirmed both its public health benefits and its commercial value. the first examples of successful drug repositioning mainly came about through serendipity like acetylsalicylic acid, thalidomide, sildenafil or dimethylfumarate. conclusion: the history of great‐repositioned drugs has given some solutions to various pathologies. serendipity is not yet useful to find repositioning drugs. drug repositioning is of growing interest. nowadays, a more rational approach to the identification of drug candidates for repositioning is possible, especially using data mining. drug repositioning lies in repurposing an active pharmaceutical ingredient that is already on the market for a new indication. although this strategy has a number of drawbacks and offers certain challenges, it also possesses many advantages, including helping to overcome the attrition currently experienced in the field of new drug discovery. [ ] the history of medicine has been marked by several examples of repositioned drugs, including some very old drugs. most of them came out purely through serendipity. new methods have now been developed, based in particular on data mining, to identify new candidates for drug repositioning. many startups are entirely focused on developing this concept, a journal has been devoted to it (drug repurposing, rescue, and repositioning; ma liebert inc., boston, ma, usa), as well as an annual international conference (the th edition was held in washington dc in ). this article provides a brief overview of repurposing and particularly evokes its most recent scientific basis as well as the new tools, and especially the computational ones, used to render it more efficient. although some historical examples exist, drug repositioning (also referred to as drug repurposing, reprofiling, redirecting, switching, etc.) is a relatively recent concept that appears to have emerged in with an article by ashburn and thor, who provided an initial definition. [ ] they defined drug repositioning as the process of finding new uses for existing drugs, sometimes but not necessarily when they fall into the public domain and become generic drugs. the number of articles devoted to this concept grew exponentially from onwards, [ ] to reach in (based on a search of pubmed on november, th for titles or abstracts containing the terms 'drug repositioning' or 'drug repurposing' and the publication year ). this original definition of drug repositioning has since been extended to include active substances that failed the clinical phase of their development on account of their toxicity or insufficient efficacy, as well as drugs withdrawn from the market because of safety concerns. it should not, however, include substances that have not yet been subjected to clinical investigation. in particular, this rules out substances held in chemical libraries by academic and industry research groups to be screened to identify those with new biological properties, different from the properties for which they were originally designed and synthesised. such compounds usually require hit-to-lead chemistry, in order to optimise the new therapeutic effect sought. similarly, selective optimisation of side activities (sosa), as proposed by professor wermuth, falls outside the scope of repositioning. [ ] in the sosa approach, the biological properties deemed responsible for a drug's adverse effects in a particular indication are isolated and amplified through chemical modification so that the modified drug can be proposed for a new indication. the concept of drug repositioning thus excludes any structural modification of the drug. instead, repositioning makes use in a new indication of either the biological properties for which the drug has already been approved (possibly according to a different formulation, at a new dose or via a new route of administration), or the side properties of a drug that are responsible for its adverse effects. drug repositioning relies on two main scientific bases: ( ) the discovery, through the human genome elucidation, that some diseases share sometimes common biological targets and ( ) the concept of pleiotropic drugs. the description of the elements associated to the complex interplay between diseases, drugs and targets with in silico approaches (data mining, machine learning, ligandbased and structure-based approaches) is one of the key for drug repurposing. [ ] it is now possible to describe diseases by their molecular profile (e.g. the genes, biomarkers, signalling pathways, environmental factors etc. . . involved in their pathogenicity) and to use computational methods, especially data mining, to determine the degree of similarity between diseases that share a number of these molecular features. [ ] for example, parkinson's disease and alzheimer's disease share genes and four signalling pathways [ ] . the existence of protein targets common to several diseases suggests that a given drug might have efficacy against both conditions. most drugs are now phenotypically well characterised in terms of their primary therapeutic effects and their (usually unwanted) side effects. this array of effects results from pleiotropic interactions between the drug and several (primary and secondary) biological targets. a drug can therefore have efficacy against a disease other than the one for which it was initially designed if one of its secondary targets has a role in the new disease. note also that these pleiotropic interactions make it is possible to develop drugs with multiple effects, all intended, that act in synergy to provide greater clinical efficacy, for example, pan-kinase inhibitors used in oncology. like diseases, drugs can be analysed for phenotypic similarity, regardless of their therapeutic indication. if two drugs indicated in different diseases have a high similarity score, they may be effective in both indications. [ ] advantages of drug repositioning drug repositioning has a number of interrelated advantages. they essentially consist of the simplification in the regulatory procedures for introducing a previously approved drug on the market, especially in certain countries such as the united states. this procedure takes into account data previously acquired, in particular on the drug's safety and toxicity, which can make the initial phases of development for a repositioned drug considerably faster, [ ] and therefore cheaper (by over % according to naylor), [ ] and increases the chances of introducing it on the market (by % compared with a novel drug, according to thayer). [ ] one important consideration, however, is that, due to the level of safety required for a drug is highly dependent on its indication, the adverse effects of a drug will be proportionately less acceptable when repositioned for a disease that is less serious or severe than its original indication. [ ] any change in formulation, dosage, or route of administration will require re-examination of the drug's safety profile under these new conditions, in what will be a new medicinal product. the main challenges faced by repositioners lie in the relatively weak intellectual property protection afforded to such medicinal products, which can reduce their return on investment and discourage companies from developing them. [ ] as the drug concerned has already been patented as a new chemical entity, subsequent medicines containing the same entity can only be protected by a new application patent, possibly backed up by a new formulation process. patents on applications are necessarily narrower than those for a new chemical entity in terms of the therapeutic uses they cover. for example, they cannot always prevent generic products containing the same drug being prescribed off-label for the patented application. they are also weaker, in particular in the face of a potential legal challenge on the basis that the new indication was predictable from data in the scientific literature. [ ] the relative weakness of the protection provided by these patents may nevertheless be offset by certain advantages granted to companies repositioning drugs for the treatment of orphan diseases (defined in europe as those with a prevalence no higher than in ), such as fee reductions and a guaranteed period of market exclusivity. [ ] some companies (such as apteeus in france) specialised in 'personalised' drug repositioning, by screening candidate drugs on cells derived from patients with orphan diseases. as mentioned above, the scientific literature abounds with studies aimed at demonstrating the value of an existing drug in another indication, yet the website http://drugrepur posing.info lists only cases in which a repositioned drug made it to the market (accessed on january ). it was estimated in that repositioned drugs generated $ billion in sales worldwide; that is, approximately, one-quarter of the pharmaceutical industry's annual revenue, with five such drugs each generating over $ billion in their new indication. [ ] aspirin the oldest example of drug repositioning is without doubt acetylsalicylic acid (figure ) . initially marketed by bayer in as an analgesic, aspirin was first repositioned in the s, at low doses only, as an antiplatelet aggregation drug. [ ] it is still widely used today in this second indication to prevent cardiovascular events, based on the work of vane, for which he was awarded the nobel prize in medicine in . [ ] previously, dr lawrence craven, a general practitioner at glendale memorial hospital, california, had observed that aspirin, administered for analgesia for his tonsillectomy patients, had the adverse effect of increasing bleeding. aspirin inhibits platelet aggregation by irreversibly inhibiting platelet cyclooxygenase (cox- ), an enzyme responsible for the formation of the prostaglandin precursor of thromboxane a , a potent stimulator of platelet aggregation. aspirin's analgesic and anti-inflammatory effects stem from its inhibition of cyclooxygenase (cox- ), in particular vascular cox- , an enzyme involved in the synthesis of prostaglandins that generate pain and inhibit platelet aggregation. at low doses (< mg/day), aspirin has partial selectivity for cox- and exerts its antiplatelet aggregation effect, which at higher doses is circumvented by concomitant cox- inhibition. aspirin's inhibition of cox- , regardless of the dose administered, is also responsible however for its harmful effects on the gastrointestinal tract, due to this enzyme's role in the synthesis of prostaglandins involved in its cytoprotection. aspirin may soon be repositioned again, this time in oncology. it has been shown that daily administration of aspirin for at least years can prevent the development of many cancers, and in particular colorectal cancer [ ] aspirin's protective effect against cancer is thought to result from cox- inhibition, thus blocking the antiapoptotic effect of cox- in malignant cells and promoting their apoptotic death. [ ] although this second potential repositioning of aspirin offers great promise from a public health point of view, there is little encouragement for it from the pharmaceutical industry, probably due to the intellectual property issues outlined above. it is well known that the world health organization (who) banned thalidomide (figure ) in due to its teratogenicity, which affected thousands of victims worldwide, and continues to affect a second generation, mainly through its use as an antiemetic for pregnant women. despite this, dr jacob sheskin from hadassah university, jerusalem, demonstrated in its dramatic efficacy against erythema nodosum leprosum, an autoimmune complication of leprosy. thalidomide, which achieves this effect by inhibiting the synthesis of the proinflammatory cytokine tumour necrosis factor-alpha (tnf-a), was consequently repositioned by celgene in as an orphan drug for complications of leprosy. [ ] its use must obviously be accompanied by drastic measures to prevent exposure to the drug during pregnancy, including stringent contraceptive measures. this example illustrates how even drugs with an exceptionally poor toxicity profile can be repositioned if the new indication is a rare disease (the estimated incidence of leprosy is cases per year according to http://www.orpha.net, accessed november, th ). like aspirin, thalidomide was repositioned for a second time in the field of oncology. investigation of the mechanism underlying thalidomide's teratogenicity demonstrated its antiangiogenic activity, responsible for the arrested limb development (phocomelia) that occurs after in utero exposure. this activity led to research into its potential use to block or destroy blood vessels supplying malignant tumours, culminating in in its second repositioning as a first-line treatment for multiple myeloma. the same precautions apply, and in europe, the prescribing and dispensing of thalidomide is subject to specific monitoring. sildenafil (figure ) is an example of a pharmaceutical substance that was repurposed before it reached the market. sildenafil was initially investigated by pfizer in as a potential antihypertensive drug. it was shown to produce vasodilation and to inhibit platelet aggregation by inhibiting phosphodiesterase type- (pde ), the enzyme that degrades cgmp. in light of these properties, the focus was shifted onto its potential in the treatment for angina. an unexpected side effect emerged during clinical trials conducted in the united kingdom, in the form of penile erections. this effect is the consequence of vasodilation, by inhibiting pde and consequently blocking cgmp degradation. sildenafil only produces an erection however in the presence of sexual stimulation, leading to the release of nitric oxide (no), which in turn leads to cgmp production. it differs in this way from amyl nitrite, contained for example in 'poppers', which provokes no release and therefore, unlike sildenafil, can be termed an aphrodisiac. this physiological effect led pfizer to market sildenafil in for erectile dysfunction, under the brand name viagra â , generating peak annual sales in this indication in excess of $ billion. [ ] sildenafil was subsequently repositioned for a second time. pfizer continued to study the potentially therapeutic effects of inhibiting pde and proposed exploiting the vasodilation it produces to treat pulmonary arterial hypertension, at one-fifth of the dose used in erectile dysfunction. the idiopathic form of pulmonary hypertension is considered a rare disease ( - million cases per year) and is potentially fatal, which explains why it was relatively easy for pfizer to obtain approval for a second sildenafil product (revatio â ) in in this new indication. [ ] unlike aspirin and thalidomide, which act on different biological targets in the various indications for which they have been approved, and can therefore be described as pleiotropic, sildenafil acts on the same target (pde ) to treat both erectile dysfunction and pulmonary arterial hypertension. dimethyl fumarate (figure ) was first synthesised in . for years, it was only known as a mould inhibitor to protect leather and a cause of allergies, which led to a ban on its use in europe in . nevertheless, dimethyl fumarate has also been marketed as a drug since . it is commonly used in germany to treat psoriasis, under the brand name fumaderm â . having discovered that dimethyl fumarate's anti-inflammatory activity was mediated by increased expression of nrf -dependent antioxidative genes, biogen proposed its use in another autoimmune disease, multiple sclerosis (ms), at higher doses. it was marketed for this indication in , under the brand name tecfidera â . it constituted a major advance in the treatment of ms, because it could be taken orally and is less cardiotoxic and hepatotoxic than the other drugs used in this condition, although it does expose patients to the risk of developing progressive multifocal leukoencephalopathy. in , this product generated almost $ billion in annual revenue. [ ] finally, it should be noted that unlike the previous examples, dimethyl fumarate was not repositioned to make use of a side effect considered undesirable in the treatment of psoriasis, but on the basis of the similarity between the molecular profiles of psoriasis and ms. if there is one field in which attrition in the introduction of new drugs is particularly evident, it is alzheimer's disease, for which no new drugs have been marketed since galantamine in . it is undoubtedly for this reason that so many drug repositioning trials have been and continue to be conducted in this field, albeit unsuccessfully to date. in , over drugs were undergoing clinical trials for this disease. [ ] one-quarter ( ) of these drugs have already been marketed and are therefore being investigated for potential repositioning (table ) . methylthioninium chloride (methylene blue) can also be added to this list, as it has been used to treat malaria and methaemoglobinaemia and is always used as an antiseptic to treat urinary tract infections and non-infectious conjunctivitis. many mechanisms of action that have already been exploited in other therapeutic fields have been reinvestigated in alzheimer's disease for their potential to offer symptomatic treatments (e.g. levetiracetam, cannabinoids, lithium and neuroleptics) or disease-modifying therapies (e.g. insulins, dihydropyridines, nicotine and sartans). the high proportion of existing drugs in the alzheimer's disease pipeline is a good illustration of the fields that rely heavily on the concept of drug repositioning (table ) . the repositioning of drugs for a therapeutic indication other than the one for which they were initially marketed is a growing trend. an illustrative and very recent example of useful repositioning is the study led in marseille which showed that chloroquine and hydroxychloroquine could constitute useful weapons against covid- . [ ] the first examples of repositioned drugs largely came out through serendipity. information technology, and cheminformatics in particular, have now made it possible to facilitate and accelerate the process. data mining is one current method for identifying candidate drugs for repositioning. [ , , ] by defining descriptors from databases for both drugs and diseases, this technology can be used to establish drug-disease pairs and construct models to predict new pairs, which will undoubtedly yield new candidates for repositioning through a more rational process. the main aim of drug repositioning is to combat the attrition and rising costs which, according to 'eroom's law', [ ] are having a dramatic effect on the number of new drugs entering the pharmaceuticals market. however, this approach must be considered as an add-on rather than an alternative to the search for novel drugs. none. all authors participate to this work. all authors revised, added figures, tables and polished it in english. drug repurposing: progress, challenges and recommendations drug repositioning: identifying and developing new uses for existing drugs drug repositioning and repurposing: terminology and definitions in literature selective optimization of side activities: the sosa approach on the integration of in silico drug design methods for drug repurposing systematic drug repositioning for a wide range of diseases with integrative analyses of phenotypic and molecular data laying in silico pipelines for drug repositioning: a paradigm in ensemble analysis for neurodegenerative diseases can you teach old drugs new tricks? therapeutic drug repurposing: repositioning and rescue drug repurposing|october scientific commercial value of drug repurposing opportunities for drug repositioning from phenome-wide association studies database identifies fda-approved drugs with potential to be repurposed for treatment of orphan diseases fabuleux hasards: histoire de la d ecouverte de m edicaments inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection thalidomide -a revival story alzheimer's disease drug development pipeline chloroquine and hydroxychloroquine as available weapons to fight covid- drug repositioning for alzheimer's disease based on systematic "omics" data mining a standard database for drug repositioning diagnosing the decline in pharmaceutical r&d efficiency key: cord- -z ucnwk authors: achilonu, ikechukwu; iwuchukwu, emmanuel amarachi; achilonu, okechinyere juliet; fernandes, manuel antonio; sayed, yasien title: targeting the sars-cov- main protease using fda-approved isavuconazonium, a p -p α-ketoamide derivative and pentagastrin: an in-silico drug discovery approach date: - - journal: j mol graph model doi: . /j.jmgm. . sha: doc_id: cord_uid: z ucnwk the sars-cov- main protease (m(pro)) is an attractive target towards discovery of drugs to treat covid- because of its key role in virus replication. the atomic structure of m(pro) in complex with an α-ketoamide inhibitor (lig b) is available (pdb id: y g). using y g and the prior knowledge that protease inhibitors could eradicate covid- , we designed a computational study aimed at identifying fda-approved drugs that could interact with m(pro). we searched the drugbank and pubchem for analogs and built a virtual library containing ∼ conformers. using high-throughput virtual screening and ligand docking, we identified isavuconazonium, a ketoamide inhibitor (α-ki) and pentagastrin as the top three molecules (lig b as the benchmark) based on docking energy. the Δg(bind) of lig b, isavuconazonium, α-ki, pentagastrin was - . , - . , - . , - . kcal/mol, respectively. molecular dynamics simulation revealed that these ligands are stable within the m(pro) active site. binding of these ligands is driven by a variety of non-bonded interaction, including polar bonds, h-bonds, van der waals and salt bridges. the overall conformational dynamics of the complexed-m(pro) was slightly altered relative to apo-m(pro). this study demonstrates that three distinct classes molecules, isavuconazonium (triazole), α-ki (ketoamide) and pentagastrin (peptide) could serve as potential drugs to treat patients with covid- . with over . million infections and deaths today ( th may ), the world is witnessing a calamitous viral pandemic caused by a new strain of a coronavirus, scientifically referred to as sars-cov , the causative agent of corona virus disease . retroviral sars-cov- shares ~ % genome similarity to the sars coronavirus [ , ] . the viral infection is believed to have originated in china with the initial epicentre in wuhan, a city in the hubei province of china [ , ] . the actual date of origin of the infection and first patient is still unknown; however, it is believed that this virus may have crossed from wild animals to humans, a possible zoonotic virus that is originally from bats, akin to the african ebola and the lassa fever viruses [ ] . over the course of time the epicentre, the epicentre has moved from china to italy and currently the epicentre in the usa. at the time of writing this article there is no known vaccine or treatment option available. even though the death rate is lower than the historical coronavirus-associated sars, the recovery rate seems to be relatively protracted. this has resulted in straining health care systems globally and sub-saharan africa may become the next epicentre if the infection is not controlled effectively. in most countries around the world, the only means of control is by instating a nationwide lockdown. countries, such as south africa, that took this initiative, however disastrous it was on the economy, have witnessed a slower infection rate, from % to less than % (between th march and th of april ). as viral disease outbreaks are not often foreseen, covid- is a global emergency and there is a race against time to produce either a vaccine and/or effective drugs to curb the global plague of covid- [ ] . several drug treatments have been proposed worldwide. chloroquine/hydroxychloroquine, an fda-approved drug that was used to treat malaria has been proposed, in combination with zinc, to be effective in eradicating sars-cov- in patients [ ] [ ] [ ] [ ] [ ] [ ] . this is because chloroquine is an endocytosis blocker and acts as an ionophore that facilitates the entry of zn + into cells such as t-lymphocytes and zn + is known to inhibit coronavirus and arterivirus rna polymerase [ ] . other antiviral agents including remdesivir, lopinavir, ritonavir and interferon α, have also been proposed as possible treatment alternatives against covid- [ , ] . currently, several countries have begun chloroquine and antiretroviral treatment trials; however, some of the clinical information emanating from these trials is still anecdotal and cannot be justified as the treatment of choice [ ] . as the world continues to grapple with the outbreak of the sars-cov- virus, the most logical approach to treating this infection is by accelerated rational j o u r n a l p r e -p r o o f drug discovery [ ] using a combination of computational modelling and empirical studies [ , ] , such as in autoimmune disease drug discovery [ ] . however, this is only possible if there are empirically determined crystal structures of key druggable targets in the virus. in this instance, m pro , a viral protease, represents a prime target because it is critical for processing viral polyproteins and viral maturation inside infected host cells [ ] . of concern are unconfirmed reports in asia of people who have recently recovered from covid- infection and have tested positive for a second time. this may point to the fact that vaccines alone may not be the best strategy for dealing with this pandemic. this, therefore, underscores the need for a rational approach to the discovery of drugs to treat the current scourge of covid- . proteases are attractive targets in a rational approach to covid- drug discovery. this is because most retroviruses depend on key enzymes, such as proteases, for processing of their polyprotein precursors [ ] [ ] [ ] . zhang et al. [ ] recently published a paper on the structure of the sars-cov- main protease (sars-cov- m pro ) with accession code y g and deposited it in the protein data bank in march [ ] . a notable feature of this atomic structure is that a derivative of an α-ketoamide inhibitor is bound to the enzyme and this must, therefore, serve as the seeding point that drives the rational drug design approach towards anti-sars-cov- drug discovery. this atomic structure, in addition to the other ~ pdb-deposited structures related to covid- , can serve as a template to discover other potential inhibitors using computational/machine learning (artificial intelligence) studies. currently, several databases are curating several million compounds, some of which are fda-approved drugs as well as drugs that are at the final stages of clinical trials with published outcomes. databases such as drugbank [ , ] and pubchem [ , ] offer structural biologists the opportunity to x-ray millions of compounds that can be validated theoretically as having potential bioactivity towards sars-cov viral enzymes. drug repurposing, which describes the identification and development of an existing drug for a new indication [ ] [ ] [ ] , is rationalised by computer-aided drug design. computer-aided drug design and discovery has become progressive in recent times, primarily due to state-ofthe-art advances in algorithms that simulate, to near-reality, the structure and function (behaviour) of biomolecules, especially regarding biomolecular interactions. this also has been aided with the development of supercomputing technologies with incredible capacities to perform these calculations at an astonishing speed. several studies have described the success of computer-aided drug repurposing towards the discovery of a new generation of j o u r n a l p r e -p r o o f anti-cancer therapeutics [ ] . in the event of an outbreak of disease at epidemic or pandemic scale, drug-repurposing is an answer to accelerated ("warp-speed") discovery of drugs to mitigate the devastating effect of highly communicable disease, such as the ebola virus outbreak and sars-cov- pandemic. studies have also suggested that drug-repurposing will greatly enhance and enable the discovery of existing drugs that can treat the clinical stage of tsars-cov- infections [ ] [ ] [ ] [ ] [ ] . a recent study by wan et al. [ ] using analysis based on decade-long structural studies of the coronavirus suggested that remdesivir could be repurposed for the treatment of the virus, targeting the main-protease. hoffmann et al. [ ] also recently suggested that a known protease inhibitor can be repurposed to block the entry the sars-cov- virus, an event that depends on ace (angiotensin-converting enzyme ) and tmprss (transmembrane protease, serine ) receptors. most of these studies have suggested repurposing fda-approved drugs; hence, empirical studies backed by computational studies may be ongoing to prove this concept. this study typifies the contribution of computational biology and chemistry in the race towards successful rational drug design discovery. our work showcases the value of biocomputational studies and that it may be used effectively and reliably as a tool to validate the results obtained using other biochemical and biophysical techniques. in light of this, our study validated the study by zhang et al. [ ] and other studies that proposed to repurposing [ ] of currently available drugs such as such as remdesivir, a drug that was initially designed, although ineffective, against the ebola virus [ , ] . we could have screened the entire drugbank and other publicly available small molecule databases using high-throughput virtual screening (htvs), but we chose to begin with two classes of protease inhibitors (i) the ketoamide inhibitors and (ii) known antiretrovirals including remdesivir, saquinavir, atazanavir using computational modelling approaches. we used htvs, induced-fit ligand docking and molecular dynamics simulation studies to identify additional classes of plausible fda-approved drugs as possible drug candidate to treat covid- . the conceptual framework of our study is illustrated in fig. . the α-ketoamide derivative (lig b) by zhang et al. [ ] and eleven fds-approved antiretrovirals (table s ) were submitted to the pubchem and drugbank databases for analog search. similar compounds were extracted in a structured data file (sdf) format and submitted to the ligprep module implemented in maestro v for ligand preparation, which involves energy minimisation using opls force field. the algorithm was set to generate possible states of the molecules at ph . ± , while accurately predicting the pka of these states at the set ph using the epik module of the algorithm. the ligands were also desalted and possible tautomeric states (~ tautomers/ligand) were further generated at ph . ± . additionally, specific chiral centres were retained (for molecules with multiple chiral centres), while other chiral centres were varied during the ligand preparation to return chemically sensible structures. these generated molecules were saved as a compressed maestro file. the atomic coordinate for the sars-cov- m pro (pdb id y g) was extracted from the rcsb-pdb database and submitted to the protein preparation wizard module implemented in maestro. the entire structure was energy-minimised by assignment of accurate protonation state at physiological ph and hydrogen atoms were added to the crystal structure using the default parameters. the stereochemistry of the side chains was checked to ensure that no major perturbations were induced while preparing the structure. a grid file of the receptor was prepared using maestro for the htvs. more than molecules were prepared using the ligprep algorithm and were submitted to the highthroughput virtual screening (htvs) module implemented in maestro. three steps of the virtual screening workflow were used, beginning with the htvs, the standard protocol (sp) j o u r n a l p r e -p r o o f and finally the extend protocol. the option for mm/gbsa was not applied at this step. the lipinski adme filtering was not applied, but the qikprop filtering was applied during the htvs. the ligand docking step in the htvs performed initial docking of the entire set of more than molecules and % of the htvs-docked ligands were further subjected to sp-docking protocol. this rigorous and systematic process generated docked potential hits that were scored using glide docking scores. top scoring ligands in each class of drug were extracted and re-submitted to the induced-fit docking (ifd) module implemented in the maestro v algorithm, which employs a mixed molecular docking and dynamic protocol. briefly, the standard ifd protocol was applied to the selected (centroid) amino acid side chains ( - , - , , - , , - , - , , - ) in an implicit solvent model using the opls_ force field. h-bond and metal ion constraints were applied to both the initial and re-docking stages. ring conformational sampling with a . kcal/mol energy barrier, as well as a non-planar conformation penalty on amide bonds was applied to the ifd protocol. the scaling for both receptor and ligand was set at . with a maximum of allowable poses per ligand. residues within Å of the docked ligand were further refined using prime refinement algorithm implemented in maestro v . prime energy was used to rank the refined proteinligand complexes. the receptor structures within kcal/mol of the minimum energy structure were submitted for a final round of glide docking and scoring. each ligand was redocked into every single refined low-energy receptor structure in the subsequent second docking step using the default glide xp settings. molecular dynamics simulation was carried out using gpu-enabled desmond [ ] [ ] [ ] engine implemented in maestro v . the complex corresponding to the top-scoring pose for each ligand or the un-complexed (apo) protein was saved as a pdb file and submitted to the linux (ubuntu) computer for the desmond high-performance molecular dynamics simulations studies. this study has two main phases; namely, system building (solvation and ionisation) and production. the system builder module implemented in the desmond algorithm was used to solvate the system using the tip p explicit solvent model with the opls_ force field. the model was placed in an orthorhombic water box (distance from the box face to the outermost protein/ligand atom = Å, box angle α = β = γ = °). the box j o u r n a l p r e -p r o o f volume was minimised, and counter ions added to neutralise the system, making sure the ions are placed at least Å from each ligand. the system was physiologically conditioned by adding . m nacl into the solvent box. after the solvation and ionisation phase in the explicit solvent model was completed, the system was submitted to the molecular dynamics production phase. this phase of md simulation is divided into eight distinct stages with specified parameters. the first seven stages involve the equilibration phase and is composed of short simulation steps. step is a final, long simulation stage. a total of ns production stage was carried out. in the first stage, the type and parameters of the solvated system were detected. in stage , a ps simulation was carried out using brownian dynamics under nvt conditions at k, while placing restraints on solute heavy atoms. stage involved a ps simulation under nvt conditions at k with restraints on heavy atoms. stages , and (the pocket solvation at stage was omitted) employed short simulation steps ( , and ps, respectively) under npt conditions (at k and restraints on heavy atoms for stages and ). no restraints were placed on heavy atoms at stage . the final production stage at constant temperature ( k) was carried out at stage , for ns. binding free energy (Δg bind ) calculations were carried out using the molecular mechanics/generalised born solvent area (mm/gbsa) method [ ] [ ] [ ] implemented in amber in order to gain more insight into the binding of the ligands to m pro . briefly, the free energy of binding of lig b, α-ki, pentagastrin and isavuconazonium to m pro were calculated by averaging snapshots of the simulated complexes (from ns molecular dynamics simulation). Δg bind of ligands at the m pro active site was calculated using ( ) where Δg rl , Δg r , and Δg l represent the free energies of complex, receptor, and the ligand, respectively. the free energy (g) of each state was calculated using the following equations: the ff sb force field terms were used to estimate the gas phase energy (e gas ), which is the sum of the internal energy (e int ); coulomb energy (e ele ) and the van der waals energies (e vdw ). the energy contribution from the polar states (g gb ) and non-polar states (g sa ) were employed to evaluate the solvation free energy (Δg sol ). the solvent accessible surface area (sasa in Å ) was also used to derive the non-polar solvation energy (g sa ) using a water probe radius of . Å, while the contribution from polar solvation (g gb ) was determined by solving the generalised born equation, where the total entropy of the solute and temperature is represented by s and t, respectively. to obtain the contribution of each residue to the total binding free energy profile between the peptidomimetics and m pro , per-residue free energy decomposition was carried out at the atomic level for imperative residues using the mm/gbsa method in amber . in order to further study the effect of ligand binding on dynamics of the cα atoms, we the rationale behind performing molecular docking is to make a systematic prediction of the ideal pose or conformation of a ligand in a protein's binding site, which could be taken table s and fig. s ). the table . we will focus mainly on the four peptidomimetics (indicated in table ) that were subsequently submitted for md simulation studies. fig. s shows the perresidue energy decomposition plots of the four ligands. isavuconazonium appears to have more amino acid residues contributing towards its stability within the active site when compared to the other three ligands. each of the complexes between m pro and the four peptidomimetic ligands, as well as the non- (table s ) showed that the overall total energy, potential energy, temperature and volume were stable throughout the ns simulation period (average slope ± . -unit/ps). all the m pro -ligand complexes showed comparable cα rmsd in comparison with the non-liganded system (fig. a) . overall, the system reached equilibrium around ns with an average cα rmsd of . Å (± . Å) after an initial rapid increase from to . ns (fig. s ) . the system converged and remained stable beyond ns. this is further affirmed by the representative d structures of the complexes superimposed on the apo-m pro (fig. b) . the shown the inhibitor (an α-ketoamide inhibitor derivative) to be effective against the virus. using y g as a template, we aimed to generate theoretical extrapolations on plausible inhibitors of sars-cov- m pro capable of being introduced as experimental drugs for the treatment of covid- . we have utilised accessible databases, including pubchem and drugbank, to search for analogs of antiretrovirals and ketoamide inhibitors that can serve as (i) a template for rational drug design and (ii) fda-approved drugs that can be introduced immediately to treat the disease and (iii) a proof of a concept of using any known drug to treat this viral pandemic. molecular modelling has, in the past, proved to be a viable option in accelerating drug discovery and complements experimental studies in drug discovery [ ] [ ] [ ] . our hypothesis is linked to the fact that a crystal structure of a protein provides a wealth of opportunity for a computational approach to drug design [ , ] . in addition, a druggable target must also be critical for the biochemistry of the target pathogen-in this case, the sars-cov- . we did not initially intend to specifically select protease and non-nucleoside reverse transcriptase inhibitors. we used prior conception on the fact that studies [ ] seem have suggested that antiretroviral drugs (arvs) may be capable of treating sars-cov- virus, itself being a retrovirus. hence, we used classes of pis and nnrti as search templates to identify candidate analogs in the molecules databases including drug bank and pubchem. to cast our net as wide as possible in searching for this best-fit molecule, we extracted over molecules, generated over conformers (isomers) and critically assed more that initial hits using induced-fit ligand docking. we tested the stability of the top-scoring (using lig b as the benchmark) molecules with a ns molecular dynamics simulation. we used the glide emodel parameter implemented in maestro v to score and rank our ligands. our htvs protocol in the maestro modelling algorithm is designed to rationalise and filter the j o u r n a l p r e -p r o o f number of ligands that were carried over to the second step, which is ifd followed by free binding energy calculation using mm/gbsa and md simulation. we did not apply lipinski's rule of five [ ] penalties because most of these molecules are peptidomimetics, which are often larger than g/mol and may not pass the absorption parameter of this rule due to their molecular weights [ ] . however, we excluded molecules with reactive functional groups and ensured that the molecules conformed with the physiological conditions in terms of the pka at ph ~ . . this is because ligands with reactive functional groups may form covalent bonds with m pro , therefore resulting in a false positive interaction [ ] [ ] [ ] . the although the coordinates of y g indicate that m pro is a homodimer with two active sites per subunit; for this study, we used a single subunit because we observed that it is only ser in subunit a that is within possible non-bonding interaction distance ( . - . Å) with the ligand in subunit b (and vice versa). there was no initial indication that ser makes any interaction with the active site, which largely depends on the nature of the ligand. from the architecture of the m pro active site, one is tempted to predict that several water molecules will be involved in binding of any ligand at this site. the structure of y g illustrates that two water molecules form either direct or indirect interactions with the lig b. our ifd studies showed that some of the known antiretrovirals and their analogs may interact with m pro based on docking scores and emodel energies. however, remdesivir, which is proposed as a potential covid- drug, had binding energy values more than - . kcal/mol, which is higher when compared with pentagastrin, p -p ketoamide derivative (α-ki), and isavuconazonium. these drugs are fda-approved and may be optional protease [ ] . the presence of catalytic histidine residues in proteases is well-documented [ ] [ ] [ ] [ ] . one remarkable feature of the ifd algorithm is the ability to predict a realistic interaction that is comparable with experimental models [ ] [ ] [ ] . a comparison of the modelled interaction between m pro and lig b with the experimental interaction with the experimental interaction ( y g) shows that the rmsd between the experimental pose and the theoretical pose is . Å. therefore, we have strong reasons to accept the results generated in this study as "close to reality" as possible and allow rational inferences to be made from this study. all the top-scoring ligands bind within Å of the catalytic cys , which could be a target for covalent inhibition [ , ] of the sars-cov- m pro . a large amount of useable and inferable information can be obtained from a md simulation study. md simulation gives us first-hand information on the stability of the ligand within the ligand-binding site and how the binding of these ligands impact on the conformational dynamics of the receptor, especially when compared with apo-m pro (unliganded m pro ). in order to validate our study, we used the desmond molecular dynamics engine to generate trajectories of m pro in a complex with lig b, pentagastrin, α-ki, and isavuconazonium. although it is advisable to test all the ligands listed in table contact with the catalytic his and cys residues. therefore, our top candidate drugs may be a competitive inhibitor of m pro . our results further affirm that the binding of the ligand affects the dynamics between these two residues and each ligand exerted a specific landscape of dynamics between these two residues. cys and his may both be involved in the binding and stabilisation of the ligands within the m pro catalytic site [ ] because these two catalytic residues are within Å of the ligands in the binding pocket. this substantial piece of information may be a molecular insight into the mode of inhibition of this enzyme. in conclusion, we have used a computational approach which includes htvs, ifd, mm/gbsa free binding energy calculations and md simulation to study potential drug candidates for covid- . we also used pca to deconvolute the dynamics between the cα atoms as a function of ligand binding. zhang et al. [ ] laid the foundation of this study by depositing the coordinates of the atomic structure of sars-cov- m pro ( y g) in complex with a derivative of an α-ketoamide inhibitor (lig b table s . six analogs of lig b [ ] are isavuconazonium, p -p ketoamide derivative (α-ki), pentagastrin, bromocriptine, ceftolozane and cobicistat (see table ). j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f green bars represent a fraction of time for h-bond (categorised into backbone acceptor; backbone donor; side-chain acceptor; side-chain donor). hydrophobic interactions are categorised into π-cation; π-π*; and other, non-specific interactions. the stacked bar charts are normalised throughout the ns trajectory. therefore, the fraction of interaction can be construed to be percentage time, which could be more than %. severe acute respiratory syndrome-related coronavirus-the species and its viruses, a statement of the coronavirus study group a pneumonia outbreak associated with a new coronavirus of probable bat origin updates on wuhan novel coronavirus epidemic world health organization declares global emergency: a review of the novel coronavirus (covid- ) a clinical guide to viral haemorrhagic fevers: ebola, marburg and lassa. tropical doctor attempt to understand public health relevant social dimensions of covid- outbreak in poland. available at ssrn chloroquine and hydroxychloroquine as available weapons to fight covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro zn + inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture remdesivir as a possible therapeutic option for the covid- the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection a structure-based drug discovery paradigm. international journal of molecular sciences potential therapeutic agents for covid- based on the analysis of protease and rna polymerase docking in silico identification and docking-based drug repurposing against the main protease of sars-cov- , causative agent of covid- celgene and exscientia enter -year ai drug discovery collaboration focused on accelerating drug discovery in oncology and autoimmunity crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors retroviral proteases and their roles in virion maturation viral proteinases. annual review of biochemistry drugbank: a knowledgebase for drugs, drug actions and drug targets drugbank: a comprehensive resource for in silico drug discovery and exploration pubchem substance and compound databases pubchem as a public resource for drug discovery. drug discovery today drug repositioning and repurposing: terminology and definitions in literature. drug discovery today computer-aided drug repurposing for cancer therapy: approaches and opportunities to challenge anticancer targets inventing new therapies without reinventing the wheel: the power of drug repurposing receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus in silico studies on therapeutic agents for covid- : drug repurposing approach virtual screening and repurposing of fda approved drugs against covid- main protease. life sciences fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study covid- treatment by repurposing drugs until the vaccine is in sight. drug development research sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor repurposing antiviral protease inhibitors using extracellular vesicles for potential therapy of covid- gpu performance as of desmond performance on a cluster of multicore processors : desmond molecular dynamics system calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models combined molecular mechanical and continuum solvent approach (mm-pbsa/gbsa) to predict ligand binding. perspectives in drug discovery and design theory and applications of the generalized born solvation model in macromolecular simulations bio d: an r package for the comparative analysis of protein structures vmd: visual molecular dynamics rational drug discovery revisited: interfacing experimental programs with bio-and chemo-informatics. drug discovery today computational methods to rationalize experimental strategies in biocatalysis adme-tox in drug discovery: integration of experimental and computational technologies. drug discovery today structure-based drug design high-throughput crystallography for lead discovery in drug design systematic review of the efficacy and safety of antiretroviral drugs against sars, mers or covid- : initial assessment lead-and drug-like compounds: the rule-of-five revolution. drug discovery today: technologies absorption of peptide and peptidomimetic drugs assay interference by chemical reactivity apparent activity in high-throughput screening: origins of compound-dependent assay interference. current opinion in chemical biology reactive compounds and in vitro false positives in hts. drug discovery today high-molecular-weight protein (pul ) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable evidence for histidine in the active site of papain the importance of the active site histidine for the activity of epoxide-or aziridine-based inhibitors of cysteine proteases. chemmedchem: chemistry enabling drug discovery four plasmepsins are active in the plasmodium falciparum food vacuole, including a protease with an active-site histidine use of an induced fit receptor structure in virtual screening. chemical biology & drug design fully automated molecular mechanics based induced fit protein− ligand docking method novel procedure for modeling ligand/receptor induced fit effects structural basis of inhibition of cysteine proteases by e- and its derivatives vinyl sulfonate esters and vinyl sulfonamides: potent, irreversible inhibitors of cysteine proteases the drug-target residence time model: a -year retrospective conformational adaptation in drug-target interactions and residence time discovery of potential multi-target-directed ligands by targeting host-specific sars-cov- structurally conserved main protease targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and ′-o-ribose methyltransferase prediction of the sars-cov- ( -ncov) c-like protease ( cl pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates targeting the sars-cov- main protease using fda-approved isavuconazonium, a p -p α-ketoamide derivative and pentagastrin: an in-silico drug discovery approach ikechukwu achilonu * *corresponding author email: ikechukwu.achilonu@wits.ac.za i.a conceived the study, performed the parts computational studies and wrote the manuscript. a.e.i performed mm/gbsa and per-residue energy decomposition analysis, o.j.a performed and interpreted the pca, dccm and other statistical analysis using the r statistical environment. y.s. co-conceived the study. m.f assisted in setting up the desmond molecular dynamics simulation engine and analysed the results. all authors reviewed the manuscript. the authors declare no competing interests. supplementary information accompanying this article can be found at https://... • this article describes the application of computational modelling towards the identification of potential sars-cov- main protease fda-approved inhibitors that may potentially be employed as an experimental drug for the treatment of covid- .• we hereby state that we have had no prior discussions or consultations with any board member of your journal regarding this publication.• the authors wish to declare that we have no competing financial and/or non-financial interests in relation to the work described in this manuscript as stated by journal of molecular graphics and modelling policy on competing for interest. key: cord- - fsqgkw authors: zolla, lello title: proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: fsqgkw the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailor-made molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency. in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins lello zolla department of environmental science, university of tuscia, piazzale università, viterbo, italy the most abundant proteins in serum, such as albumin and igg, act as molecular sponges that bind and transport low molecular weight proteins/peptides and drugs. in the near future, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development, and interactomics, the branch of proteomics which is concerned with identifying interactions between proteins, will allow researchers to (i) know the specific protein changes that occur in biological compartments in response to drug administration; (ii) design small novel therapeutic molecules that can have extended half-lives if carried by plasma protein in the blood stream. advances in these fields will open new avenues of tailormade molecular therapy, reducing present limitations on treatment arising from toxicity and inefficiency.in this short review we report and discuss the most recent developments arising from the use of proteomic tools in blood plasma protein research, looking at the identification of proteins found in plasma as well as their interactions with small molecules such as drugs, peptides, organic chemicals and metals. we believe this research demonstrates that proteomic technologies, and in particular pharmacoproteomics, interactomics and post-translational modification analysis, could be instrumental in the design of new tailor-made drugs leading to substantial improvements in molecular therapy. blood plasma is the most complex human-derived proteome. because of this complexity, and the enormous range of concentration encountered across the population of protein components, spanning in excess of ten orders of magnitude, whole blood plasma is the most difficult specimen to analyze, and this creates serious challenges for proteomics. much progress has already been made in this field and new directions have been put forward and discussed as part of the hupo plasma proteome project (ppp), to focus efforts on the remaining challenges [ ] . the ppp initiative has three stated long-term goals (i) to make a comprehensive analysis of the protein constituents of plasma; (ii) to determine the extent and source of variation in an individual's plasma over time; and (iii) to determine the extent of variation in plasma between individuals within and across populations [ ] . blood plasma is known to contain proteins derived from blood cells and other body tissues that may have ended up there through cell death or damage (causing proteins to be released from normal cells), or they may come from aberrant protein secretions from tumor cells. in a recent investigation [ ] , the examination of the plasma protein component categories revealed that many of the proteins detected in plasma are normally associated with cells (i.e. they are not known plasma proteins). these 'cellular leakage proteins' were categorized according to their original location and function. intracellular proteins accounted for up to % of the proteins identified, while membrane-associated proteins, including those proteins that are membrane-based but not known to be released in plasma (i.e. receptors, coreceptors and adhesion molecules) [ ] accounted for another %. another % of the proteins were found to be of cellular origin, and are either secreted or occupy an extracellular location, and % were identified as specific cytokines or cytokine-related proteins. all these proteins are generally considered passenger proteins (some more transient than others) that utilize plasma for transportation, localization and mediation of cellular responses. overall, this group is the least characterized but possibly the most interesting one in terms of potential to yield biomarkers, with protein concentrations believed to range from low mg/ml to pg/ml levels, and possibly extending to levels below the detection limits of traditional elisa assays ( pg/ ml). the 'classic' plasma proteins, those whose activity is specifically localized in the plasma, such as human serum albumin (hsa), complement components and apolipoproteins, make up only % of the total protein found in plasma. approximately % of the proteins identified, however, had no known function [ ] . hence, with such a diverse population of proteins derived from a range of sources we might expect that the analysis of the extracellular proteome of proteins circulating in the plasma and the cell-based proteome are necessary and complementary for an exhaustive plasma investigation. one strategy applied in several recent examples involves using a secondary tissue or fluid of interest to first identify potential candidates for biomarkers and then screening the complementary plasma sample for their presence. such an investigation is highly desirable because disease markers present in plasma may include proteins with significant potential for early disease diagnosis, containing information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases [ ] . thus, a broad inventory of plasma proteins (both qualitative and quantitative) could be used for the identification of putative protein markers for any diagnosable disease as well as for the development of new therapeutic products [ ] . quantitatively speaking, the core plasma protein is albumin, representing about % of the total plasma protein content (in the order of - g/l). immunoglobulins (igs) represent - % of the total protein mass [ ] . low-abundance plasma proteins from tissue leakage and cytokines are present in the range of picograms to nanograms per milliliter. the most abundant proteins in serum, such as albumin, igg and transferrin, are known to act as the carriers for hormones, lipoproteins and many other proteins, lipids and metals. they may, in fact, be described as molecular sponges, which bind and transport low molecular weight (lmw) protein/peptide species preventing their rapid clearance by the renal system, thereby extending their half-life in the blood stream. although it has been estimated that % human plasma proteins do not display associations with the major proteins, about % have been found in association with igg and % of proteins have also been found with hsa codepleted groups, suggesting the possibility of weak overlapping interactions between some proteins and both hsa and igg (see fig. ). moreover, because proteins in the circulatory system are exposed to a variety of proteases or chemical oxidations [ , ] , plasma is rich in peptides, the so-called 'peptidome' (see box ) from which about peptides have been revealed [ ] , prevalently associated with the carrier proteins. if igs are retained in the sample during peptide-based proteomic studies, they can act as a generous source of random peptide sequences contributing to a large library of 'background' species that, because of their abundance ( mg/ml sample concentration) and variety, greatly complicate the process of peptide detection and identification. plasma protein interactions appear to exhibit little dependence on protein size. in fact, small plasma proteins such as the basic proline-rich peptide p-e (ib- , mw = da, igg codepleted protein) show more interaction specificity than large proteins such as cardiac titin isoforms (mw = , , da) that are typically found in more than one protein interaction group [ ] . this kind of information, concerning the interactions between human plasma proteins, should be useful for further studies in human blood systems/network biology, such as evaluating possible protein contaminants in therapeutic protein products prepared from human plasma, and for the design of analytical approaches to deplete high abundance plasma proteins effectively. additionally, it could be used to help design proteins with a reduced rate of clearance from the circulatory system, particularly important when small proteins are used as therapeutic agents [ ] . normally, small uncomplexed proteins and peptides (i.e. less than kda) are rapidly cleared from the circulation through enzymatic degradation, uptake by the reticuloendothelial system or by glomerular filtration, which discriminates on the basis of molecular size and charge [ ] . it is believed that the circulation half-life of this lmw fraction is directly related to its binding affinity to large high abundant carrier proteins. drug discovery today volume , numbers / december box the array of peptides normally present within the circulatory proteome is termed the 'peptidome' , and could be a rich source of cancer-specific diagnostic information because it provides a record of the cellular and extracellular enzymatic events that take place at the level of the cancer-tissue microenvironment. this new information archive seems to show that most peptides in vivo are bound to high-abundance proteins such as albumin. measuring panels of peptidome markers might be more sensitive and specific than conventional biomarker approaches. a biomarker is a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or a disease. a biomarker may also be used to see how well the body responds to a treatment for a disease or condition, to measure the progress of disease or the effects of treatment. the use of proteomic technologies in the field of drug discovery and development. toxicoproteomics is the use of global protein expression technologies to better understand environmental and genetic factors, both in the episodes of acute exposure to toxicants and in the long-term development of disease. primary aims in toxicoproteomics are the discovery of key-modified proteins, the determination of affected pathways, and the development of biomarkers for eventual prediction of toxicity. the use of proteomic technologies and tools, such as phosphorylation-site-specific antibodies, to assess and monitor the phosphorylation state(s) of various proteins involved in signaling pathways in cells. a method that allows for the quantitative measurement of proteins in two separate populations by mass spectrometry via the differential tagging of proteins in each population at cysteine residues with heavy or light isotope reagents. protein microarrays are composed of a series of immobilized spots, containing a homogeneous or heterogeneous 'bait' molecule. a spot on the array can represent an antibody, a cell or phage lysate, a recombinant protein or peptide or a nucleic acid. the array is queried with either a probe (a labeled antibody or ligand), or an unknown biological sample (e.g. a cell lysate or serum sample) containing analytes of interest. by directly or indirectly tagging the query molecules with a signal-generating moiety, a pattern of positive and negative spots is generated. a combinatorial ligand library is composed of millions of diverse hexapeptide baits, able to capture aspecifically all peptides/ proteins in any given proteome. in this way they concentrate the 'low-abundance' proteome, while drastically cutting the concentration of the most abundant compounds. it is based on the concept of a 'one-bead, one-peptide' approach. free-flow electrophoresis (ffe) is electrophoresis carried out in an aqueous medium without using any solid matrix, such as acrylamide. it is useful for the separation of a wide variety of charged analytes like low-molecular weight organic compounds, peptides, proteins, protein complexes, membranes, organelles and whole cells in aqueous media under native and denaturing conditions. the analyte is injected into a thin, laminar separation buffer film (which defines the electrophoretic mode such as zone electrophoresis, ief or isotachophoresis) and is deflected by an electric field perpendicular to the flow direction. differential scanning calorimetry differential scanning calorimetry measures the difference in the amount of heat required to increase the temperature of a changed biological sample with respect to an unchanged one (reference) as a function of temperature. the difference in thermograms of blood plasma between normal and diseased individuals is not related to the concentrations of the most abundant plasma proteins but, rather, seems to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such a behavior is consistent with the 'interactome' hypothesis. interactomics is a fusion science of biology, informatics and engineering which provides a global view of protein family interaction networks. it involves the study of both the interactions and the consequences of those interactions between and among proteins, and other molecules within a cell and can be used to compare networks of interaction between and within species to see how the traits of such networks are varied and conserved. the interactome network includes certain calculated parameters that weigh the reliability of a given interaction (i.e. the 'edges' of the interactome network) between two proteins, and also qualify the functional environment around any given protein (i.e. the 'nodes' of the interactome network). some of the strategies devised to retard the clearance of therapeutic proteins include the covalent attachment of polyethylene glycol or dextran chains or by protein-protein crosslinking. genetic modification has also been used to create chimeras of the therapeutic protein of interest with long-lived plasma proteins like albumin or igg [ ] . over , different proteins have been estimated to be commonly present in the plasma, most of which are at very low relative abundances [ ] . the dynamic range of currently available proteomic techniques such as mass spectrometry (ms) or d gel electrophoresis ( -de) spans three to four orders of magnitude and as such does not approach the ten orders of magnitude represented by the plasma proteins. thus, a significant challenge for proteomic analysis of plasma is how to reveal the low-abundance proteins. the strategies that have been most frequently used to overcome the dynamic range problem are to fractionate the plasma proteome into smaller subsets, and/or to deplete one or more of the major proteins. immunoaffinity is the most efficient way to deplete proteins and so these methods are most widely used [ ] [ ] [ ] . immobilized antibodies packed in columns or cartridges capture several the most abundant proteins lowering the protein content down to % of the initial amount. although nontarget proteins are removed in this process, it has been shown to be reproducible [ ] . multiple orthogonal separation steps have also been used, such as strong cation exchange chromatography followed by reverse phase-liquid chromatography (rp-lc) [ ] or insolution isoelectric focusing (ief) [ ] . recently, a novel separation approach has been proposed, using d free-flow electrophoresis (ffe), separating proteins and peptides in solution according to their pi before lc-ms/ms [ ] . a commercial combinatorial ligand library (proteominer) is now available, enabling researchers to pick out the low-abundance proteins [ ] . another innovation is the microflow mf , a device to prefractionate complex low volume, low-abundance samples that can also enrich for very specific species of proteins based on charge and/or size either in native or in denaturing format [ ] . all these strategies concomitantly remove proteins/peptides associated with the highly abundant proteins targeted for depletion. a recent study [ ] focused on the binding properties of six of the most abundant serum proteins -to investigate the small peptides/proteins that may be bound to them. each of the proteins targeted was found to be capable of binding several different peptide/proteins ( proteins from a total of unique peptides), many of which are clinically useful biomarkers. these results suggest that, on the one hand, albumin or igg depletion before protein identification may actually eliminate many valuable biomarkers but, on the other hand, the identification of biomarkers, through the selective isolation of protein bound to the more abundant proteins in serum, could be a novel proteomic strategy. because the general and specific sample losses increase with each separation stage, the best solution to this dilemma is to maximize the efficiency of each separation stage and minimize several dimensions required for the characterization of complex mixtures. as stated above, proteomic approaches may be utilized for disease classification as well as for the development of novel biomarkers related to prognosis, diagnosis and choice of therapeutic regimen. it is generally accepted that these biomarkers will not originate from classic plasma secretions but, most probably, from leakage, secretion or shedding of proteins from the specific affected tissue, cell type or cellular pathway. nevertheless, blood is still the logical choice of biospecimen and has become the most frequently used biomarker discovery matrix to date, because the driving force of biomarker discovery is the development of blood-based assays for early detection and prediction of therapeutic response [ ] . it follows that if protein biomarkers have been identified in biopsied tissues, researchers will then be able to examine the blood to determine whether these biomarkers are in circulation. the great advantage being that blood tests are much less invasive than biopsies. the downside is that once such proteins are released into around liters of plasma, their concentration is extremely low. hence, the need to extend the dynamic range of protein detection and this is where proteomics comes into its own. although blood is a very convenient and noninvasive fluid to monitor for biomarkers, it poses many challenges from the perspective of protein detection. probably the greatest potential application for proteomics lies in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. among the challenges facing clinical proteomics is the ability to link protein expression profiles to specific disease phenotypes and the identification of relevant biomarkers to develop diagnostic tools [ ] . in this sense, proteomics is an expansion of reductionist biology, where single proteins are analyzed in a high throughput fashion to arrive at an understanding of the entire system. the intention is that, once the human blood proteome has been fully described, several biomarkers will emerge for each particular disease state that can be used to provide a 'fingerprint' of that disease and here, there are obvious implications for blood donor testing [ ] . there are already systematic searches underway to look for plasma proteins that are biological indicators or biomarkers for cancer (see refs. [ , ] ). if a suite of biomarkers were to be available for early detection, stratification into distinct subtypes and monitoring of progression or response to therapy we could expect significant improvements in clinical outcomes for cancer patients. the intention is to develop panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy, possibly detectable in a single proteomics experiment. despite the recent progress in proteomic technologies based on ms, however, the discovery of novel clinical assessment tools has been slow. this is partly because of the inherent difficulties in working with blood. it is hoped that a better understanding of the limitations of blood for comparative protein profiling and an appreciation of the advantages of cancer tissue or cancer cell secretomes will greatly enhance the progress [ ] . a comparative proteome study of the body fluids of schizophrenia patients has been carried out to look for biomarkers or associated proteins in an effort to understand the etiology of schizophrenia. it was found that protein expression of the ttr tetramer and apolipoprotein e (apoe) was downregulated by up to . and . times, respectively, in schizophrenia patients compared to normal controls [ ] . a study that revealed potential diagnostic cancer biomarkers used blood from sjl (selected by james lambert) mice, in which the growth of rcsx lymphoma cells induces an inflammatory response by stimulating v b +t cells [ ] . after the depletion of albumin and immunoglobulin, mouse nr proteins were identified in sjl mouse plasma in a single experiment. most were found to be upregulated (e.g. acute phase reactants) but some proteins were found to be unique to the tumor-bearing mouse plasma (i.e. haptoglobin, proteosome subunits, fetuin-b, - - zeta and mage-b antigen). while it remains to be seen whether a similar unique profile occurs in human lymphomas, recent studies have demonstrated that in sepsis and cancer, the interalpha inhibitor proteins (iaips), a family of structurally related serine protease inhibitors, are present in relatively high concentrations in human plasma, suggesting their suitability as potential biomarkers [ ] . it would seem logical to assume that a better understanding of the biological mechanisms of drug toxicity and the development of therapeutic resistance would lead to the development of improved therapeutics with significant utility in clinical research. in this context, pharmacoproteomics, the use of proteomic technologies in the field of drug discovery and development (see fig. ), is an emerging science. protein drug-activity biomarkers can be defined as specific protein changes in total protein content in biological compartments that occur in response to drug administration [ ] . in contrast to genomic approaches, the application of pharmacoproteomics, by virtue of being stimulus-induced, may be more amenable to systematic experimental manipulation that could ultimately result not only in validation of a biomarker, but also in characterizing its behavior under specific conditions [ ] . in association with chemical compound library screening strategies, pharmacoproteomics is expected to accelerate the discovery of new lead compounds for future drugs. it should also play an important part in preclinical studies by providing information on the mode of action and the eventual deleterious side effects of drugs. hopefully, ongoing and future proteomic studies will open up new avenues of tailor-made molecular therapy, reducing present limitations associated with treatment toxicity and efficiency. ms-based proteomics is ideal in this respect, because it offers a targeted way to identify hundreds of protein or peptide biomarkers simultaneously, with the obvious potential for discovering drugactivity markers because there is often no a priori knowledge of the particular proteins that are likely to change [ ] in response to drug administration [ , ] . while sample enrichment narrows the dynamic range over which molecules of interest must be detected, the improvements in separation technology and ms have extended the ability to find and identify proteins over an ever wider range of relative protein concentrations. ion trap instrumentation has been superseded by the greatly improved linear ion trap mass spectrometers. at the same time, there has been a steady decrease in the diameters of lc-capillary columns that has resulted in increased sensitivity and ability to quantify proteins. this has improved analysis by increasing, by orders of magnitude, the detectable dynamic range of a protein that would not have been picked up previously in samples of similar size. as such, at this time, lc-fticr-ms (fourier transform ion cyclotron drug discovery today volume , numbers / december theoretical example of differential screening of plasma proteins from healthy, ill and treated (responding and resistant) patients by d gel electrophoresis. the appearance or disappearance of protein spots as well as quantitative variations of protein expression can be observed. resonance ms) technology provides the most sensitive and powerful measurement platform and though some significant potential exists for further increasing its dynamic range, this applies to those cases limited by sample complexity, rather than detection limits. recently, however, differential scanning calorimetry has provided a new window into the plasma proteome. thermograms of plasma from diseased individuals not only were found to differ dramatically from those of normal (control)_individuals, but also differed between sufferers of different illnesses, at least for the three diseases examined (arthritis, lyme disease and lupus). each disease appears to have a distinctive and characteristic thermogram. these radically different thermograms seen for different diseased states seem to arise from binding interactions involving as yet unknown biomarkers with the more abundant plasma proteins, particularly albumin. such behavior is consistent with the 'interactome' hypothesis, suggesting a novel use for calorimetry as a diagnostic tool [ ] . for a careful approach to clinical design and data analysis it is important to maximize the chance of discovering meaningful drug-activity markers. the first step is to establish a link between drug administration and the resulting biologically quantified change [ ] . this strong foundation should set the stage for any data-driven hypothesis generation and testing, and further establish the utility of such a multidisciplinary tool in expanding the frontiers of clinical research. too often there is no information available on the tissue under investigation, in these circumstances protein-profile changes in response to drug treatment can serve as evidence of cell pharmacodynamic activity, and such data can be used to establish a minimum dose for early clinical studies. more broadly, because targeted proteomics can also be hypothesis generating, detecting a drug-induced change can also lead to new lines of research and inquiry in the field of drug development or pathophysiology. a clear understanding of proteomic variability under controlled conditions is crucial to enable future clinical studies to be appropriately designed and to improve the contextual interpretation of data obtained from pharmacoproteomic studies. it is also important to establish a suitable time period between drug administration and analysis primarily to allow sufficient time for a tissue-targeted drug to reach a pharmacokinetic steady state before exploring protein profile changes [ ] . this is to ensure minimal interference from extraneous factors on endogenous variability, thus maximizing the probability of identifying even subtle drug-induced changes. the variability between individual subjects could potentially affect observations in unpaired comparison studies, such as diseased versus healthy volunteers, and should be taken into account [ ] . in other words, we cannot assume that pharmacodynamic responses to identical chemical or biochemical stimuli will be similar among healthy individuals and that these responses will differ significantly and consistently in patients with pathological disorders, because a variety of factors will influence the profile of each individual [ ] . this aspect requires further investigation to ascertain how useful biomarkers will ultimately prove to be and what consequence this will have for drug development. pharmacoproteomics is not limited to the extracellular proteome analysis of blood plasma, because cell-based proteome analysis also has an important role in this field. in this context, post-translational modifications (ptm) of proteins have a significant impact. some potential modifications are known, and they include phosphorylation, glycosylation, acetylation, myristoylation, palmitoylation, methylation, sulfation, prenylation and ubiquitylation (http://www.abrf.org/index.cfm/ dm.home). during the past two decades, sensitive ms methods have been refined to determine the type and site of protein modifications that can seldom be predicted from a genomic sequence [ ] . some of the protein modifications are regulatory and reversible, most notably phosphorylation which controls protein functions such as localization, complex formation, stability and activity through different mechanisms [ ] . as a result of extensive research, we now know that about % of all human proteins can be modified by phosphorylation, while only . % of the phosphorylated residues are tyrosine [ ] . protein phosphorylation is one hallmark of the protein-protein interactions (ppis) underlying signaling networks and, in many cases, it is aberrant protein kinase activity that drives diseaseassociated derangements in signaling pathways. the aim would be to design drugs that effectively disrupt this aberrant proteinphosphorylation-based enzymatic activity and epigenetic phenomena. pharmacoproteomics, or the tailoring of therapy based on proteomic knowledge, is expected to take a central role in this process [ ] . a recent study describes a cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, with which researchers were able to screen for inhibitors of multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level [ ] . protein microarrays that examine protein-protein recognition events (i.e. phosphorylation) in a global, high-throughput manner have been used to profile cellular signal pathways in a way not possible with gene arrays [ , ] . the activity levels of the proteins in a particular pathway can thereby be assessed in 'real time', to tailor treatment to each patient's cellular 'circuitry' [ ] [ ] [ ] . the advantage of protein microarrays lies in their ability to provide a 'map' of known cellular signaling proteins that generally reflect the state of information flow through protein networks in individual specimens. the identification of crucial nodes or interactions within the network (see later) is a potential starting point for drug development and the design of individual therapeutic regimens [ ] [ ] [ ] . accurate annotation of peptide modifications through unrestrictive database searches can reveal post-translational modifications, as well as sequence polymorphisms [ ] . monitoring different phosphoprotein levels will also help to identify treatment-acquired resistance to chemotherapy. the effect of imatinib on the tyrosine phosphoproteome in bcrabl positive leukemia cell lines has been examined by proteomic approaches. the investigation revealed sites of tyrosine phosphorylation corresponding to different proteins [ ] . affinity column chromatography using an immobilized pyrido( , -d)pyrimidine derivative has been successfully used to select protein kinases that bound to the matrix and were then identified using ms, demonstrating the fact that pyrido( , -d)pyrimidine is a kinase inhibitor with low specificity [ ] . a similar methodological approach was applied to study the p kinase inhibitor sb , demonstrating that cyclin g-associated kinase (gak) and ck were almost as potently inhibited as p a [ , ] . protein expression subsequent to treatment with the hdaci trichostatin-a has been studied in human pancreas ductal carcinoma cell lines using -de and maldi-tof ms [ , ] . trichostatin-a appears to upregulate proteins which promote cell death and downregulate proteins that favor cell growth. similarly rc modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis and gene expression in colon carcinoma cells [ ] . the pharmacoproteomic approach was found to be particu-larly useful for the identification of molecular alterations implicated in type diabetes, and for further characterization of existing or new drugs [ ] . research has shown that several physiological factors lead to changes in both the plasma proteome and the peptidome, including stress, sleep, sport training, eating meals and pregnancy. exposure to toxic agents, including drugs, can also be detected using proteomics leading to a distinct field of study known as toxicoproteomics [ ] . recently, the proteins in the plasma of workers exposed to benzene were analyzed [ ] . two drug discovery today volume , numbers / december human erythrocyte protein-protein interaction network (interactome). nodes of the network are the known rbc proteins and links connecting the nodes correspond to the known interactions [ ] . significantly upregulated protein profiles were found in workers exposed to the toxic chemical: the t cell receptor b chain and matrix metalloproteinase- . thus, the plasmatic t cell receptor b chain may be a useful indicator for the early detection of exposure to benzene. comparative proteome analysis of human plasma allowed qian et al. [ ] to identify proteins that were significantly increased after lipopolysaccharide administration. a study of the plasma of workers occupationally exposed to polycyclic hydrocarbons revealed several proteins as markers of such an exposure [ ] . using the same technique, truncated forms of a -antitrypsin were discovered in serum samples of patients presenting with severe acute respiratory syndrome [ ] . in another study involving patients with the same syndrome, samples revealed a total of differential spots, most of them corresponding to acute phase proteins [ ] . the authors also identified proteins such as peroxiredoxin ii, not previously detected in plasma -de. the investigation of plasma protein interactions with metals remains a relatively new and challenging arena for toxicoproteomics. current studies include the identification of the protein carrier and how to determine toxic metal concentrations, which requires sensitive analytical techniques and powerful equipment. thankfully, the search for suitable quantitative techniques in the field of drug design and proteomics may be almost over with the development of inductively coupled plasma ms (icp-ms) [ ] . another relevant field for pharmacoproteomic applications is that of elucidating the biological mechanisms of drug resistance. in drug-resistant cells alternative protein forms appear that prevent the drug binding to active sites and/or executing signaling effects independent of the regulated native protein forms. the methodology of proteomics seems highly applicable to the search for drug-resistant protein forms (drug-resistance proteomics). in this context the plasma proteome in aspirin (acetylsalicylic acid [asa])-sensitive and asa-resistant coronary ischemic patients has been analyzed. the expression of one isotype of the fibrinogen g chain and three isotypes of haptoglobin was increased in asa-resistant patients as well as that of three vitamin d binding protein (dbp) isotypes. it has been suggested that dbp regulates the inhibitory effect of asa on platelets, by reducing the inhibitory effect of asa on thromboxane a production [ ] . in the field of oncology, proteomics is becoming widely used for the identification of tumor-specific protein markers, and pharmacoproteomics has found a place in the evaluation of chemotherapy, particularly for the characterization of drugresistance mechanisms [ ] . identifying important interactions between blood proteins is emerging as another focus of blood-based proteomic research. many proteins circulate in blood, not as single entities but as multicomponent complexes, and as such comprise the blood 'interactome' [ ] . protein-protein interactions (ppi) information can be extracted from the currently available databases of interactions (in silico approaches). little is currently known about the interactions of any given protein in the blood or whether these interactions are biologically relevant. examining protein complexes will, however, often yield information about protein function. the identification of such interactions may bring to light important information for designing new small molecule therapeutics. this conceptual framework contrasts with the single protein (or single pathway) approach that has previously dominated biology. the combination of proteomic and in silico approaches allows one to not only identify disease and/or drug-related changes in the proteome but also predict the changes in the protein interactome network that are associated with disease-related change of function. it can also predict modifications associated with ptm changes occurring as a result of a specific disease or stage of disease, as well as drug or gene therapy treatment. these linked approaches represent the future of clinical identification of a specific disease, its current stage or severity, and the effects and side effects of various treatments. clearly, an interactome of plasma proteins is far from reality at present, because of its complexity, while the simplicity of the human erythrocyte cell structure has instead made it an optimal cell for proteomic study. in fact, while nucleated cells contain , - , proteins [ , , ] , red blood cells (rbcs), which lack nuclei and other organelles, contain far fewer. a comprehensive list of rbc proteins known to date contains entries obtained using proteomics technology [ ] . the resulting ppi network is depicted in fig. [ ] . the network presented here was derived from protein interaction data obtained from the unified human interactome (unihi) [ ] . the correlation reported by unihi is derived from gene expression experiments and represents a measure of confidence for the interaction [ ] . as the knowledge of ppis continues to increase owing to advances in proteomics research we expect that, in addition to a deeper understanding of the erythrocyte protein complement and how it relates to function, it will also be possible to understand how changes in the rbc proteome and interactome affect the development of erythrocyte disorders [ ] . the promise of proteomic-based profiling, as opposed to gene transcript profiling alone, is that the resulting prognostic signatures are derived from drug targets (proteins) not genes, so the pathway analysis provides a direction for therapeutic mitigation. the only dark cloud on the horizon may be that extending the use of pharmacoproteomics (beyond molecular network analysis for patient-tailored therapy to include risk stratification or 'predispostional testing') is likely to raise a larger societal issue, particularly in a climate of cost containment. the powerful combination of pharmacoproteomics and interactomics may win through despite costs, furnishing new targeted therapies for disease prevention as well as for posttherapy monitoring. in short, these techniques not only provide a window on the presence or severity of a specific disease, and drug and gene therapy treatment, but can also be used to identify disease-and/ or drug-related changes in the proteome. they can also predict those changes in the protein interactome network that are associated with disease-related changes of function [ ] . without doubt, these linked approaches represent the future for the cost effective and noninvasive identification of specific clinical diseases, their prognosis and effective treatment. hupo plasma proteome project: challenges and future directions the human proteome organization plasma proteome project pilot phase: reference specimens, technology platform comparisons, and standardized data submissions and analyses characterization of the human blood plasma proteome utilizing human blood plasma for proteomic biomarker discovery oxidation of proteins: basic principles and perspectives for blood proteomics proteomic analysis of rbc membrane protein degradation during blood storage composition of the peptide fraction in human blood plasma: database of circulating human peptides peptide and protein drug delivery to and into tumors: challenges and solutions effects of genetic engineering on the pharmacokinetics of antibodies toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome differences among techniques for high-abundant protein depletion immunoaffinity separation of plasma proteins by igy microbeads: meeting the needs of proteomic sample preparation and analysis evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry a novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis the proteominer in the proteomic arena: a non-depleting tool for discovering low-abundance species prefractionation, enrichment, desalting and depleting of low volume and low abundance proteins and peptides using the mf an investigation into the human serum interactome protein biomarker discovery and validation: the long and uncertain path to clinical utility proteomics and disease -the challenges for technology and discovery clinical proteomics: revolutionizing disease detection and patient tailoring therapy mining the plasma proteome for cancer biomarkers strategies for plasma proteomic profiling of cancers proteomics-driven cancer biomarker discovery: looking to the future dysregulation of retinoid transporters expression in body fluids of schizophrenia patients comparative plasma proteome analysis of lymphoma-bearing sjl mice proteomic characterization of inter-alpha inhibitor proteins from human plasma proteomic strategies for individualizing therapy of acute myeloid leukemia (aml) pharmacogenomics and pharmacoproteomics in the evaluation and management of short stature from genomics to proteomics mass spectrometry-based proteomics mass spectrometry-based clinical proteomics calorimetry outside the box: a new window into the plasma proteome the impact of systems approaches on biological problems in drug discovery identifying pharmacodynamic protein markers of centrally active drugs in humans: a pilot study in a novel clinical model alpha cell function in health and disease: influence of glucagon-like peptide- technologies and methods for sample pretreatment in efficient proteome and peptidome analysis quantitative phosphoproteomic analysis of signaling network dynamics signaling - and beyond technology insight: pharmacoproteomics for cancerpromises of patient-tailored medicine using protein microarrays high-content single-cell drug screening with phosphospecific flow cytometry antibody arrays in cancer research protein chip technology reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front progress in protein and antibody microarray technology protein microarrays as tools for functional proteomics experimental design and analysis of antibody microarrays: applying methods from cdna arrays clinical proteomics: translating bench side promise into bedside reality protein biochips: a new and versatile platform technology for molecular medicine accurate annotation of peptide modifications through unrestrictive database search profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry chemical proteomic analysis reveals alternative modes of action for pyrido -dpyrimidine kinase inhibitors an efficient proteomics method to identify the cellular targets of protein kinase inhibitors proteomic analysis of pancreatic ductal carcinoma cells treated with -aza- -deoxycytidine proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin a a proteomic approach for evaluating the cell response to a novel histone deacetylase inhibitor in colon cancer cells pharmacoproteomic approach to the study of drug mode of action, toxicity, and resistance: applications in diabetes and cancer toxicoproteomics: serum proteomic pattern diagnostics for early detection of drug induced cardiac toxicities and cardioprotection proteomic analysis of plasma proteins of workers exposed to benzene comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons the use of proteomics in the discovery of serum biomarkers from patients with severe acute respiratory syndrome plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry the application of inductively coupled plasma mass spectrometry in pharmaceutical and biomedical analysis relationship between vitamin d binding protein and aspirin resistance in coronary ischemic patients: a proteomic study the abc's (and xyz's) of peptide sequencing proteomics of organelles and large cellular structures the human red blood cell proteome and interactome centrality measures for the human red blood cell interactome unihi: an entry gate to the human protein interactome a graph theoretic approach to testing associations between disparate sources of functional genomics data transfusion medicine in the era of proteomics this work was supported in part by the italian national blood centre, istituto superiore di sanità, rome, italy (directed by dott. giuliano grazzini). the author would like to thank dr g.m. d'amici for his help and dr j. scarpa for english revision. key: cord- -r y sgb authors: tiwari, nidhi; upadhyay, jyoti; nazam ansari, mohd; joshi, rohit title: novel β-coronavirus (sars-cov- ): current and future aspects of pharmacological treatments date: - - journal: saudi pharm j doi: . /j.jsps. . . sha: doc_id: cord_uid: r y sgb the novel coronavirus outbreak has reported to be rapidly spreading across the countries and becomes a foremost community health alarm. at present, no vaccine or specific drug is on hand for the treatment of this infectious disease. this review investigates the drugs, which are being evaluated and found to be effective against ncovid- infection. a thorough literature search was performedon the recently published research papers in between january to may , through various databases like “science direct”, “google scholar”, “pubmed”,“medline”, “web of science”, and “world health organization (who)”. we reviewed and documented the information related with the current and future aspects for the management and cure of covid- . as of st july, a total of , , confirmed cases of coronavirus and , deaths have been reported world-wide. the main clinical feature of covid- ranges from asymptomatic disease to mild lower respiratory tract illness to severe pneumonia, acute lung injury, acute respiratory distress syndrome (ards), multiple organ dysfunction, and death. the drugs at present used in covid- patients and ongoing clinical trials focusing on drug repurposing of various therapeutic classes of drug e.g. antiviral, anti-inflammatory and/or immunomodulatory drugs along with adjuvant/supportive care. many drugs on clinical trials shows effective results on preliminary scale and now used currently in patients. adjuvant/ supportive care therapy are used in patients to get the best results in order to minimize the short and long-term complications. however, further studies and clinical trials are needed on large scale of population to reach any firm conclusion in terms of its efficacy and safety. novel-coronavirus (ncovs) is spherical in shape, enveloped with single-stranded rna, and considered as the largest viral . coronaviruses (covs) belongs to group of zoonotic viruses having enormous diversity of animal hosts i.e. birds & humans. the infection caused by cov varies variably and greatly from asymptomatic-symptomatic to severe disease, depending on the species of virus and the host. this virus subdivided into four families: α, β, γ, δ-coronavirus. the γ, δ-covs are mainly infected birds, whereas α, and β-covs largely infected the mammals (yin and wunderink, ) . earlier, global spread of two β-coronavirus such as severe acute respiratory syndrome coronavirus (sars-cov), origin from foshan (china) in november (zhong et al., ) and middle east respiratory syndrome coronavirus (mers-cov) occurred in saudi arabia in june (zaki et al., ) . at present, a novel coronavirus (sars-cov- ) reported first case in china (wuhan) on december , later on february , world health organization (who) given named as covid- (huids et al., ; who, ) . it has spread rapidly, affected many countries all over the world and declared by who, as covid- emergency/outbreak, a pandemic (dong et al., ) . sars-cov- is highly infectious and transmittable diseases, highly vulnerable to entire population worldwide via a directly contact with infected person through respiratory droplets (zheng, ) . according to recent evidences, this epidemic also known as zoonotic disease, a disease transmitted from animals to humans. several genealogical analyses documented that novel cov considerably identical to the sequence of bat sars-like coronavirus (benvenuto et al., ) .the replication and transcription phase of sars-cov- starts when virus (host) enters into the cell and interacting with spike proteins (s-protein) present at the surface of target receptor. mainly two target receptors are present within the cell to which virus binds either with angiotensin converting enzyme- (ace- ) receptor or with the serine protease tmprss (hoffmann et al., ) . however, exact mechanism is still unknown and need to investigate whether novel coronavirus employs ace- and tmprss for the entry into the cytoplasm. novel-coronavirus infection is a community transmission disease, primarily affected person to person, via respiratory droplets (li et al., ) . sometimes these droplets can land on objects and surfaces such as tables, doorknobs and handrail. in most of the cases, person can become infect by touching these objects and then touching their eyes, nose and mouth. the most common symptoms are fever, dry cough, tiredness, nasal congestion, sore throat and develop breathlessness. in severe cases patient symptoms can be characterized to organ failure i.e. kidney & respiratory.however, geriatrics population and person with preexisting medical condition (diabetes, hypertension,heart and lung problems) chances are higher for developing symptoms (rawat et al., ) . apart from these symptoms this infection can be associated with lymphopenia, interstitial pneumonia and elevated levels of pro-inflammatory cytokines like as interleukins (ils), interferons (ifns), granulocyte-colony stimulating factor (g-csf), tumor necrosis factor (tnf-α), etc. this condition sometimes also called "cytokine storm" can induce patient's death (guo et al., ) . currently, only symptomatic treatments are available to minimize the symptoms like self-isolation/quarantine, supplemental oxygen, mechanical ventilation, paracetamol for fever and antibiotics only for hospitalized patients for control bacterial co-infection. till date, there are no vaccines or drug therapy approved by the u.s. food and drug administration (fda) for the treatment of this pandemic virus covid- (βcoronavirus). due to varying nature of this virus, development of novel pharmacological treatment takes several years. many scientist or researchers work day and night and trying to focus on drug repurposing, which already are in use in some other conditions for competently control and spread of pandemic situation all over the world (kruse, ) . from this review, our aim is to provide a basic overview about currently available drugsand drugs under clinical trial for covid- patients. based on many evidences from laboratory, animal and clinical studies,all are focusing on anti-viral, anti-inflammatory and immune-modulators drugs for the treatment of the novel-coronavirus. here, we discussed all currently used treatment, under clinical development and outlook clinical trial for the treatment of covid- . patients with covid- characterized by abnormal in respiration rate (hypoxia), hear rate, fever,irregular neutrophil counts (neutropenia), and higher level of plasma cytokines i.e. interleukins (il- , il- , etc.), and tnf-α. all these symptoms are the reason to promote disease severity. sars-cov- virus detected in both types i and ii pneumocytes present in the ciliated epithelial cells of nasal, bronchial and bronchiolar mucosae of respiratory system. type i pnemocytes is responsible for gaseous exchange whereas type ii has main role in the production and secretion of surfactants. destruction of both types of pnemocytesis, resulting in hypoxia and alveolar collapse. ace- act as a target receptor for the treatment of covid- infection, whereas tmprss protein, enhance the transmissibility of the virus. increase in the production of leukocytes (neutrophil) leads to the formation of highly reactive oxygen species (ros), and oxidative stress inside alveoli, resulting in the formation of mucus (cough) and alveolar damage. figure describes the potential targets with pathogenesis pathway for broad-spectrum antiviral agents, anti-inflammatory and immuno-modulatory agents in treatment of covid- . antiviral drugs are beneficial for several infectious diseases such as herpes, hepatitis, hiv, influenza and flu like symptoms (razonable, ) . researchers and scientists are endeavoring to find drugs, which are effective in the treatment of covid- . drug repurposing is widely acceptance at present, because of immediate impact of ncovs outbreak into the society and public health emergency concern (muralidharan et al., ) . many drugs that approved by usfda for some other disease conditions are under preclinical and clinical development for the management of covid- . here, we review and examine all the antiviral drug therapies and its relevance in the treatment of coronavirus as per available pharmacokinetic and pharmacodynamics data of clinical studies. several studies has been documented that antiviral drugs delivered abruptly after beginning of symptoms and continue last up to days (sputum peak around - days). these drugs minimizing the chances of infection to others by reducing release of virus from the hostand replication into the respiratory secretions of infected person, and diminishrisk of infection by prophylactic targeted treatment (mitja andclotet, ) . classification, mechanism of action, adverse effects and therapeutic uses of all antiviral agents (currently used and under clinical development) against covid- are shown in table . lopinavir (lpv) and ritonavir (rtv) are effective agents that inhibit the protease activity of coronavirus. lpv and rtv act as an antiretroviral used for the treatment of aids/hiv virus (hiv- & hiv- ). rtv enhances the plasma concentration of lpv through inhibition of cytochrome p (cyp a)-mediated metabolism of lpv (soliman et al., ) . previous studies reported that lpv blocks virus replication in the later stages steps of mers-cov cycle (de wilde et al., ) and abate the activity of sars-cov by inhibition of proteinase activity (hurst and faulds, ) . in a randomized, controlled and open-label trial involving adults sars-cov positive patients, patients were divided into two groups (lpv-rtv treated & standard-care group). there is no remarkable changes were observed in both the groups in terms of improvement and mortality. et al., ) . another study reported that, patients were assigned randomly in phase -trial and administered days combination of lpv ( mg), rtv ( mg) and ribavirin ( mg) in every h with ifn beta- b to improve the antiviral response. this combination seems to be better, superior and provides best output to lpv-rtv alone and also diminished the virus desquamation, reduces symptoms of patients, and often promoting discharge of patients in the mild-moderate covid- (hung et al., ) . another invitro study reported that ribavirin, analogue of guanosine nucleotide having wide spectrum of antiviral activity, used along with lpv/rtv to treat sars-cov- viral infection in china (chictr ) . earlier this combination was wellestablished for the treatment of sars-cov infection along with minimal risk of acute respiratory distress syndrome (ards) and mortality rate (chu et al., ) . moreover, future clinical studies are needed to check this combination with other antiviral-combination against sars-cov- . the drug was developed in , for the treatment of ebola and marburg virus infections. rdv is a prodrug of nucleotide (adenosine) analogue and having a broad-spectrum antiviral activity. in body, it is metabolized into active metabolite gs- . the antiviral mechanism of rdv is to inhibit rna-dependent rna polymerase (rdrp) enzyme, necessary for virus replication (al-tawfiq et al., ) . earlier invitro studies reported that rdv inhibits single stranded rna virus replication such as sars-cov and mers-cov (agostini et al., ) .another mouse model study reported that combination of rdv and ifn-beta is better than lpv& rtv for the treatment of mers-cov (sheahan et al., ) . currently preclinical study reported that association of rdv & chloroquine is greatly efficient for preventing sars-cov- infection . in addition, phase clinical trials conducted in participants to check efficacy and safety of the rdv (loading dose, mg on day only followed by mg maintenance dose, od for days) in covid- patients (cao, ) . rdv shows promising output in some of the us patients with covid- (holshue et al., ) . furthermore, beigel et al. reported remdesivir shows possible efficacy better as compared to placebo group in hospitalized patients for the treatment of sars-cov- virus. a randomized, doubleblind and placebo-controlled clinical trial has been conducted and the patients received remdesivir i.v. {day loading dose ( mg) + daily maintenance dose ( mg) up to additional days}. patients are assigned randomly and total of, infected patients are enrolled and divided into two groups, remdesivir group: patients and placebo group: patients. the overall time to recovery rate is better in remdesivir and also lowers infection in respiratory system as compared to placebo. in addition, various evidences suggested serious adverse event i.e acute respiratory failure and sudden death in few patients and strict monitoring is required to manage dose at the time of treatment. from this report, we can conclude antiviral treatment always need supportive care or supplemental oxygen to prevent other negative outcomes (brigel et al., ) . recently, hetero pharma company launched covifor vials, brand name of remdesivir, a game-changer drug for treatment of covid- . this drug is restricted used in patients who already have kidney problems, pregnant and lactating women (https://www.pharmaceutical-technology.com/news/cipla-hetero-remdesivir-covid- /). hence, further research is requisite to evaluate its pharmacological and pharmacodynamics characteristics against sars-cov- virus. favipiravir (avigan) is an antiviral drug, firstly developed by japan in the year , for treatment of influenza virus infections (furuta et al., ) . it is a nucleotide (guanine) analogue, & converted into active metabolite by phosphorylation, then inhibits the synthesis of rna (rdrp) and virus mutagenicity (vanderlinden et al., ) . it has been documented by invitro study that fpv inhibits sars-cov- in vero e cell (ec = . μm) (du and chen, ). an open & controlled study designed to determine the efficacy of fpv ( mg bd, on day followed by mg bd on - days) vs lpv/rtv ( mg/ mg bd) along with ifn-α b, bd) for the treatment of covid- . preliminary results showed that there is significant difference between viral clearance & chest improvement rate . in india, glenmark is the first company to initiates the phase trials on fpv by enrolled participants, duration of treatment maximum of days and total study duration of an individual maximum upto days, with mild to moderate covid- under restricted emergency used. recently, on june , glenmark permitted fpv, under brand name of fabiflu tablets ( mg bd on first day followed by mg bd from day to day ) into the market for treatment of mild to moderate cases of covid- patients (baravkar et al., ) . umifenovir (arbidol) is a dual-acting direct antiviral/host-targeting agent used as a prophylaxis in the influenza and respiratory infections. a cohort study enrolled adult patients characterized by lymphopenia showed no significant difference in the results obtained between the survival and nonsurvival groups by receiving treatment with antiviral, antibiotic and glucocorticoid therapy. however, there is a significant data and less mortality rate for those patients who receive arbidol . this combination of drug used as an antiretroviral in the treatment of hiv/aids. darunavir is a hiv protease inhibitor and cobicistat enhances the plasma concentrations of darunavir by blocking its metabolism by cyp enzyme (deeks, ) . preclinical studies documented that this combination showed beneficial effects in the sars-cov- virus infections (lin et al., ; omotuyi et al., ) . the effectiveness and safety concern of darunavir/cobicistat combination is being evaluated under development of clinical trials phase by enrolling covid- patients and estimated completion of study on december , . lastly, the primary outcomes measures shows the clearance rate of virological salivation, less throat infections and decreases respiratory secretion at day (time frame: day after first randomization) (lu, ) . initially, camostatmesilate and nafamostatare used in postoperative reflux esophagitis and in chronic pancreatitis (uno, ) . camostatmesilate andnafamostat, both drugs act on attractive target tmprss , a serine protease inhibitor and block the entry of the virus into the lung alveolar cells (hoffmann et al., ) . recently, camostat mesilate enters into the phase trials by taking participants (administered dose approx. mg for days, tid). the outcomes obtained like changes in covid- symptoms (frequency and severity), body temperature and changes entry in sars-cov- viral load. ivermectin is a fda approved drug, used as an anti-parasitic and in the treatment of many types of virus infections such as hiv, dengue, influenza and zika (caly et al., ) . recent study showed that ivermectin, inhibits the growing of virus in cell culture approximately for hrs. till date, test is only carried out in-vitro, for confirmation, strong clinical trials are needed to evaluate its safety and efficacy in the treatment of covid- patients (scavone et al., ) .clinical studies of ivermectin in covid- patients are underway by enrolling confirmed cases of covid- , as per hospital protocol. this study is in progress and might be getting its primary outcomes about its safety and clinical efficacy by theend of july (budhiraj and mishra, ) . earlier data about coronavirus (sars & mers) showed, cell infiltration and cytokine storm (cs) leads to acute cell lung injury, adrs and death of many patients (channappanavar and perlman, ; chousterman et al., ) . recently, covid- infection, which is due to sars-cov- virus, accompanied by rapid replication of virus and a massive release of cytokine profile as same in other two reported coronavirus sars and mers (teijaro, ) . currently, study documented that cytokines like interleukins (il- b, il- , il- , il- & il- ); g-csf; ifns; interferon-γ-inducible protein (ip ); monocyte chemoattractant protein (mcp ),tumor necrosis factor (tnfα), vascular endothelial growth factor (vegf), platelet derived growth factors (pdgf)and other pro-inflammatory mediators are increase in covid- patients (huang et al., ) . another studies reported extensively elevation in the level of il- in critically severe poorly in covid- infected patients (ruan et al., ; zhou et al., ) . classification, mechanism of action, adverse effects and therapeutic uses of all anti-inflammatory and immunomodulatory drugs(currently used and under clinical development) against covid- are listed in table . it is an anti-malarial and over the counter (otc) drug, activity similar to chloroquine. recently, it was reported that hcqs shortened the time to clinical recovery on the sars-cov- patients. due to its interferon blocking property, it has diminished the immune response to a viral infection. this immunomodulatory property makes its useful in autoimmune disorder such as rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). the safety profile of hcq is superior as compared to chloroquine during long term used in covid- patients and little concern about drug-drug interactions (yao et al., ) . several invitro studies reported cqs/hcqs are used as an effective treatment in sars-cov- virus. as they are weak bases and enhance ph of endosomal in host cell organelles, inhibit fusion of autophagosome-lysosome and inactivates enzyme require for replication of virus. they also have a potential to deactivate ace- glycosylation, for which coronavirus may enters into the cell (ferner and aronson, ) . currently studies showed that hcq administered for - days, effective in clearing viral nasopharyngeal carriage of sars-cov- in most of the covid-macrolide antibacterial drug was shown to be successful in the treatment of zika and ebola viruses by in-vitro studies (madrid et al., ; bosseboeuf et al., ) . previous studies reported that combination of hcq and azithromycin showed synergistic effect and prevent respiratory tract infections when administered by viral infection patients (bacharier et al., ) . recently, world health organisation (who) updated information on june , about safety and efficacy of chloroquine (cqs)/hydroxychloroquine (hcqs) and compared it with standard care treatment for covid- patients. the overall outcomes showed that there is no significant difference in case of mortality when compared with standard care treatment. in addition, some reports evaluated hcqs cause's serious adverse effects like prolongation of qt interval, ventricular fibrillation, arrhythmia and sometimes leads to abrupt death. the safety and efficacy assurance for cqs/hcqs, there is necessitate that other clinical studies are needed to be performed and exemplify with safety concern (cochrane, ). further, us fda summarized details on july with safety concern, and permitted used of cqs/hcqs is now accessible to take care of patients with covid- but some strict monitoring are required while treatment as it shows various heart related problems, blood and lymph disorders, renal injury and hepatic failure (us fda, ). interferons are types of cytokines effective in the management of infections & infectious diseases (arnaud, ) . it is a broad-spectrum antiviral, antiproliferative & immunomodulatory drug interacting with the toll-like receptors and inhibits viral replication and protein synthesis (uematsu and akira, ) . there are three types of ifn (α, β, and γ). ifn-β shows high potent specificity as compared to ifn-α in reducing the replication of mers-cov (hart et al., ; shalhoub, ) . in human lung tissue (shalhoub, ) . further, several future studies are being conducted to determine its efficacy & safety along with other antiviral drug in treatment of covid- . interleukin plays a major role in the pathogenesis of all three coronavirus sars-cov- , mers & sars-cov- infections (lau et al., ) . recent study reported several pro-inflammatory mediators plays a major role in the lung damage such as interleukins (il- , il- etc) and inflammatory markers (d-dimer, ferritin, and c-reactive protein) (conti et al., ; kuppalli and rasmussen, ) . several ils antagonist drugs are under ongoing trials to check its safety and efficacy against sars-cov- viral infections. anakinra is a recombinant il- receptor antagonist and may be helpful in covid- patients. it prevents the binding of il- α and il- β to interleukin- receptors, which is used in the treatment of autoimmune disorders (cavalli et al., ) . recently, retrospective cohort study showed high dose of anakinra ( mg/kg, bd,iv) produces beneficial and efficacious effects in % covid- infected patients associated with ards (cavalli et al., ) . in addition, anakinra alleviated hyperinflammation and respiratory distress in sars-cov- infected patients (tufan et al., ) . another studies reported anakinra ( mg repeated after h, sc for days) administered along with remdesivir ( mg ld, followed by mg after h, iv for days) used in the treatment of respiratory dysfunction in covid- patients (franzetti et al., ) . however, several clinical trials in large population are required for confirmation of its efficacy. a human monoclonal antibody particularly targets and neutralizes il- β thus blocking its binding with il- receptors (dhimolea, these classes of drugs blocks interleukin (il- ) mediated signaling pathways by binding competitively with soluble and membrane bound il- receptors (sil- r and mil- r). il- involved in various physiological process like activation of t-cells, b-cell, hematopoietic cell proliferation and differentiation, hepatic acute-phase protein synthesis initiation and also immunoglobulin secretion (tanaka et al., ) . it is a monoclonal antibody, firstly initiates and tested by china to decrease the lung related complications in patients with covid- . a randomized and retrospective study showed that tocilizumab plays a beneficial role as an adjunctive therapy in covid- patients. preliminary data proved that percentage of lymphocyte, concentration of crp, and computed-tomography clarity changes and reduces the hospitalization duration of these patients (xu et al., ) . at present, three clinical trials are under advancement to assess its safety and efficacy alone or in combination with some other drugs by taking participants with sars-cov- infection (barrett, ; de paris, ; regeneron pharmaceuticals, infected patients (henriksen, ) . it is a humanized recombinant igg anti-cd monoclonal antibody, used for the treatment of moderate to severe plaque psoriasis, rheumatoid arthriris and autoimmune disorders (saavedra et al., ) . intially, it was developed by biocon in bengaluru and approved in in psoriatic patients. recently, biocon repositioning the drug into patients with moderate to severe covid- associated with cytokine release syndrome (crs) and acute respiratory distress syndrome (ards). in addition, drug controller general of india (dcgi) allowed use of alzumab ( mg/ ml injection) in sars-cov- patients as an emergency treatment and capable to save numerous seriously sick patients with covid- (https://www.pharmaceutical-technology.com/news/biocon-itolizumab-approval/). itolizumab is also acts as a immunomodulator and binds with cd receptor along with suppress many proinflammatory mediators (cytokines) and downregulates the t-cell activation at site of inflammation (saavedra et al., ). an open-label randomized and controlled study performed by biocon, in participants associated with moderate to severe symptoms of sars-cov- virus. the study being conducted in two groups, first group taken itolizumab+ supportive care while second group only with supportive care. the overall outcomes showed there is no mortality rate and all patients are recovered well as compared to second group. further, clinic-pathological observations i.e release of pro-inflammatory cytokines (il- & tnf-α) significantly decrease in first group when compared to second group (jolla, ; uttamani et al., ) . moreover, several clinical studies are required to confirm its safety and efficacy in covid- patients. it is a vasoactive intestinal polypeptide (vip) analogue used in the treatment of erectile dysfunction, acute lung injury and besnier-boeck-schaumann disease. the significance of aviptadil in ards, because it has been documented by animal studies that vip is extensive concerted in the lungs and inhibits nmda-induced caspase- activation, il and tnf-α production and also protects against acidic (hcl)-induced lung edema. in the various animal models, aviptadil protects lungs and other organs from damage or failure by restoring barrier function at the alveolar interface (petkov et al., ; said, ) . in a phase clinical trials,conducted under european regulatory authority (era), patients were successfully treated with intravenous infusion of aviptadil on escalating doses and restores all the functioning of pulmonary hypertension, acute lung injury and ards. few adverse effects related to cardiovascular system such as change in blood pressure, heart rate or ecg was noticed. eculizumab is a monoclonal protein based monoclonal antibody (mab) used to treat paroxysmal nocturnal hemoglobinuria (pnh), atypical hemolytic uremic syndrome (ahus), and neuromyelitis optica (wong and kavanagh, ) . the main target of eculizumab is complement protein c , preventing cleavage to c a and c b, and the formation of the terminal complement complex c b- , which is involved in many autoimmune diseases (gralinski et al., ) . based on case study of patients with sars-cov infection and also confirmed severe pneumonia and ards treated with i.v. infusion of eculizumab along with anticoagulant therapy (enoxaparin iu/day s.c), antiviral therapy (lpv mg/day + rtv mg/day), hydroxychloroquine mg/day, ceftriaxone g/day iv, vitamin c g/day for days. all patients showing decrease in the inflammatory markers including c-reactive protein (crp) (diurno et al., ) . more clinical study is needed for confirmation its safety and efficacy as ananti-inflammatory in the treatment of covid- . jak are the intracellular enzymes located on cellular membrane, transmits signal by binding with various cytokines and growth receptors. after binding with receptor, enzymes phosphorylation occurs, which modulating the changes in the gene expression and thus, activates the signal transducers and activators of transcription proteins (stats) (seif et al., ) . jak pathway is key enzyme and plays a crucial role in the activation of immune system, hematopoiesis and inflammation. inhibitors of jak, prevents the enzymes phosphorylation and activation of stats proteins (o'shea et al., ) . it is reversible and competitive inhibitor of jak & jak , and firstlyaccepted by us fda in the treatment of autoimmune disorder i.e. rheumatoid arthritis. it also have broad immunosuppressive effect, and used in many autoimmune disorders (winthrop, ) . it blocks the enzymes protein no significant adverse effect reported in baricitinib-treated patients (cantini et al., ) . further studies are needed to confirm its clinical safety and efficacy in the treatment of covid- infection. others jak inhibitors are also under clinical development and data are being evaluated in the treatment of covid- are ruxolitinib and tofacitinib (smith and prosser, ) . isotretinoin,a retinoid derivative of vitamin a, is used in the treatment of severe acne. it is the strongest down-regulator of ace- receptors. it has been documented that isotretinoin is a protease inhibitors and can be a taken as a target therapy in covid- (wu et al., a) . based on the above mechanism, isotretinoin currently tested under phase clinical trials in the adult patients with positive sars-cov- viral infections.current outcomes showing there is large difference at the onset of clinical signs and symptoms treated patients with isotretinoin, standard therapy or combined therapy and checked by on a daily basis. corticosteroid therapy is only used as supportive care in patients of covid- with refractory shock or ards (wu et al., b) . according to who, corticosteroids are recommended only for some conditions like asthma, chronic obstructive pulmonary disease (copd), and septic shock (who, b) . earlier studies showed corticosteroids i.e. methyl-prednisolone directly not supported the patients having symptoms of viral-pneumonia in covid- (jin et al., ) , only act as an antiinflammatory and reduces inflammatory mediators i.e. interleukin (il- &il- ), mcp , th chemokine,ip etc. (sung et al., ; wong et al., ) . dexamethasone, another cheap and widely steroid drug used in the treatment of many inflammatory disease conditions and disorders like asthma, severe skin allergies, chronic obstructive lung disease and certain cancers. recently, it has been documented that dexamethasone could be lifesaving drug in critically ill patients with covid- (who ). a retrospective cohort study showed daily administered of dexamethasone ( mg upto days) reduces the mortality rate among patients with covid- who needed supplemental oxygen and mechanical ventilation. there is no advantage of dexamethasone observed in patients who did not necessitate mechanical support or/ ventilation (horby et al., ) . it is an antibody-rich plasma, collected from the patients who already have recovered from diseases caused by virus e.g. polio, measles, ebola, h n influenza, hepatitis b, and now currently in sars-cov- responsible for covid- . on may , , us fda permitted issued guidelines to healthcare providers, make ensure the safety and efficacy of investigational product by collecting plasma from recovered patients under demand of public health emergency (stephen and hahn, ) . a newly reports showed that use of plasma therapy in five ventilated coronavirus patients, all patients improved after one week of transfusion. moreover, this report was not a clinical trial, further research is essential to confirm its potential and managing patients in sars-cov- infected patients (https://clinicaltrials.gov/ct /show/study/nct ). bronchodilators are preferred only as a supportive care for covid- patients associated with copd, asthma and other respiratory disorders (lippi and henry, ) . due to concern of safety precautions, both standard nebulizers and metered dose inhalers (mdi) are risky to covid- patients, as it requires opening of ventilator circuit. regular administration of bronchodilator may be needed, to minimize the virus transmission to others multiple health care patient. in order to alleviate these hazards, aeroneb solo (aerogen©, galway, ireland) has been successfully made for covid- patients which continuously delivers medication via inhalation without circuit opening and requires minimal health-care workers for handling (miller and epstein, ) . these are the agents that breakdown plasminogen to plasmin on the surface of thrombi, thus produced fibrinolysis (chapin and hajjar, ) . a study was done in seriously unwell patients, already suffered with ards and respiratory collapse/failure. patients administered with alteplase along heparin improves pao /fio (p/f) ratio momentary approximately by - % (moore, ). further, detailed studies are required to check its clinical efficacy among large population. others supportive fibrinolytic agents are also being evaluated for covid- patients e.g. defibrotide (fabio, ) . coronavirus patients have a higher risk of venous thromboembolism (vte), deep vein thrombosis (dvt) and pulmonary embolism (porfidia andpola, ) . coagulopathy is one of the main reasons of mortality in the majority of covid- patients. a recent report suggested anticoagulant therapy such as low molecular weight heparin (lmwh) administered to patients showed low mortality risk (kollias et al., ) . aspirin is also used as a prophylaxis treatment in case of post-discharge vte covid- patients(ash, ). further, additional data needed regarding its efficacy and safety in covid- patients. due to pandemic of covid- disease, no vaccine or drug is approved to eliminate the virus. drug development and discovery is broad and extensive process, due to utmost necessity of the treatment, drug repurposing is suitable for treatment of covid- infection. at present, numerous drugs are ongoing clinical trial and tested directly in patients with covid- , strict monitoring is mandatory at the time of treatment of patients to assure its safety and efficacy of unknown profile of drugs. based on the exposed and revealed structure and transmission mechanisms of novel coronavirus, the available treatment option is antiviral, anti-inflammatory & immunomodulatory drugs. this review provides detailed description of all available in-vitro & in-vivo studies, mechanism, clinical spectrum of disease, symptoms and safety concern profile of currently available treatment and concerning drugs, which are progress under clinical development. in conclusion, while waiting for the most effectual pharmacological treatment with less adverse effects, many trials are currently ongoing and will available around at the end of , according to clinical trials government website. hope, their consequences will definitely provide us good outcome and help worldwide in significant the greatest approach to cure or treat covid- along with symptoms and complications. the authors declare that they have no conflict of interests. this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. table classification, mechanism of action, adverse effects and therapeutic uses of anti-inflammatory &immunomodulatory agents against covid- name of drug(s) coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease remdesivir as a possible therapeutic option for the covid- the interferons: pharmacology, mechanism of action, tolerance and side effects covid- and vte/anticoagulation: frequently asked questions version . early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial corona virus and covid- treatment of moderate to severe coronavirus disease (covid- ) in hospitalized patients remdesivir for the treatment of covid- -preliminary report the -new coronavirus epidemic: evidence for virus evolution biocon's drug itolizumab gets approval to treat covid- . updated on azithromycin inhibits the replication of zika virus to study the effectiveness of ivermectin with standard of care treatment versus standard of care treatment for covid cases experimental treatment with favipiravir for covid- : an open-label control study. engineering (beijing) the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro baricitinib therapy in covid- : a pilot study on safety and clinical impact a phase randomized, double-blind, placebo-controlled, multicenter study to evaluate the efficacy and safety of remdesivir in hospitalized adult patients with severe a trial of lopinavir-ritonavir in adults hospitalized with severe covid- clinical features and short-term outcomes of patients with corona virus disease interleukin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology fibrinolysis and the control of blood coagulation cytokine storm and sepsis disease pathogenesis role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings cipla and hetero launch remdesivir to treat covid- in india targeted update: safety and efficacy of hydroxychloroquine or chloroquine for treatment of covid- ross r, frydasi. induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): antiinflammatory strategies convalescent plasma trial in covid- patients. nih u.s library of medicine the national institutes of health (nih) covid- treatment guidelines panel provides recommendations for dexamethasone in patients with covid- cohort multiple randomized controlled trials open-label of immune modulatory drugs and other treatments in covid- patients -sarilumab screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture darunavir/cobicistat/emtricitabine/tenofoviralafenamide: a review in hiv- infection eculizumab treatment in patients with covid- : preliminary results from real life asl napoli nord experience an interactive web-based dashboard to track covid- in real time response to" dose rationale for favipiravir use in patients infected with sars-cov- use of defibrotide to reduce progression of acute respiratory failure rate in patients with covid- pneumonia chloroquine and hydroxychloroquine in covid- interleukin- receptor antagonist anakinra in association with remdesivir in severe coronavirus disease : a case report favipiravir (t- ), a broad spectrum inhibitor of viral rna polymerase hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak-an update on the status interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays effectiveness of interleukin- receptor inhibitors in the management of patients with severe sars-cov- pneumonia: an open-label sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor first case of novel coronavirus in the united states clinical features of patients infected with novel coronavirus in wuhan the continuing -ncov epidemic threat of novel coronaviruses to global health: the latest novel coronavirus outbreak in wuhan, china triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) clinical trial shows itolizumab reduces mortality in patients hospitalized with covid- . equillium thromboembolic risk and anticoagulant therapy in covid- patients: emerging evidence and call for action therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china a glimpse into the eye of the covid- cytokine storm delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia molecular modeling evaluation of the binding effect of ritonavir, lopinavir and darunavir to severe acute respiratory syndrome coronavirus proteases chronic obstructive pulmonary disease is associated with severe coronavirus disease (covid- ) potential therapeutic agents against covid- : what we know so far efficacy and safety of darunavir and cobicistat for treatment of covid- evaluation of ebola virus inhibitors for drug repurposing baricitinib: a novel fda-approved small molecule inhibiting janus kinases. pharmaceuticals safe bronchodilator treatment in mechanically ventilated covid- patients: a single center experience use of antiviral drugs to reduce covid- transmission fibrinolytic therapy to treat ards in the setting of covid- infection: a phase a clinical trial computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- phase multicenter, randomized, double-blind, placebo-controlled study to assess the efficacy and safety of canakinumab on cytokine release syndrome in patients with covid- -induced pneumonia darunavir disrupts critical nodes in metastable -ncov-rbd/ace- complex the jak-stat pathway: impact on human disease and therapeutic intervention vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension venous thromboembolism and heparin use in covid- patients: juggling between pragmatic choices, suggestions of medical societies case study: -year-old female presenting with shortness of breath antiviral drugs for viruses other than human immunodeficiency virus an adaptive phase / , randomized, double-blind, placebo-controlled study assessing efficacy and safety of sarilumab for hospitalized patients with covid- clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china an anti-cd monoclonal antibody (itolizumab) reduces circulating il- vasoactive intestinal peptide in pulmonary arterial hypertension current pharmacological treatments for covid- : what's next? the role of jak-stat signaling pathway and its regulators in the fate of t helper cells interferon beta- b for covid- comparative therapeutic efficacy of remdesivirand combination lopinavir, ritonavir, and interferon beta against mers-cov covid- drug therapy-potential options boosted protease inhibitors and the electrocardiographic measures of qt and pr durations coronavirus (covid- ) update: fda encourages recovered patients to donate plasma for development of blood-related therapies severe acute respiratory syndrome: report of treatment and outcome after a major outbreak il- in inflammation, immunity, and disease cytokine storms in infectious diseases covid- , immune system response, hyperinflammation and repurposing antirheumatic drugs toll-like receptors and type i interferons camostatmesilate therapy for covid- fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems therapeutic modalities in the management of covid- : a worldwide landscape distinct effects of t- (favipiravir) and ribavirin on influenza virus replication and viral rna synthesis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro who welcomes preliminary results about dexamethasone use in treatingcritically ill covid- patients world health organization, a. draft landscape of covid- candidate vaccines clinical management of severe acute respiratory infection (sari) when covid- disease is suspected the emerging safety profile of jak inhibitors in rheumatic disease plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome anticomplement c therapy with eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease pneumonia in wuhan, china analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods effective treatment of severe covid- patients with tocilizumab in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) mers, sars and other coronaviruses as causes of pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia sars-cov- : an emerging coronavirus that causes a global threat epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study key: cord- -pjyvg w authors: hrkach, jeff; langer, robert title: from micro to nano: evolution and impact of drug delivery in treating disease date: - - journal: drug deliv transl res doi: . /s - - - sha: doc_id: cord_uid: pjyvg w over the past years, drug delivery breakthroughs have enabled the approval of several important medicines. often, this path starts with innovation from academic collaborations amongst biologists, chemists, and engineers, followed by the formation of a start-up company driving clinical translation and approval. an early wave featured injectable (i.e., intramuscular, subcutaneous) biodegradable polymeric microspheres to control drug release profiles for peptides and small molecules (e.g., lupron depot®, risperdal consta®). with these early successes for microspheres, research shifted to exploring systemic delivery by intravenous injection, which required smaller particle sizes and modified surface properties (e.g., pegylation) to enable long circulation times. these new innovations resulted in the nanoparticle medicines doxil® and abraxane®, designed to improve the therapeutic index of cytotoxic cancer agents by decreasing systemic exposure and delivering more drug to tumors. very recently, the first sirna lipid nanoparticle medicine, patisiran (onpattro®), was approved for treating hereditary transthyretin-mediated amyloidosis. in this inspirational note, we will highlight the technological evolution of drug delivery from micro- to nano-, citing some of the approved medicines demonstrating the significant impact of the drug delivery field in treating many diseases. the complexity of human disease biology presents many challenges for the development and approval of new medicines. it is very difficult to design drug molecules with activity specific for a single biological pathway unique to diseased cells; with a pharmacokinetic profile with optimal onset and/or duration of action; or without detrimental side effects due to systemic exposure. in addition, patient compliance in taking medicines can offer significant challenges. drug delivery systems offer the possibility to design and engineer levels of control beyond the drug molecule's inherent properties to overcome the aforementioned challenges. until the mid- s, many thought that controlled release drug delivery was limited to small molecules. pioneering research by folkman and langer led the way for sustained release of proteins and other macromolecules [ ] . biodegradable polymers such as the poly(lactide-coglycolide) (plg) family degrade by hydrolysis in the body and can be engineered to encapsulate drug molecules in microspheres suitable for subcutaneous or intramuscular injection. modification of polymer molecular weight or composition, drug loading levels, and particle size can be utilized to tune the rate of polymer and microsphere degradation and corresponding drug release profiles. leuprorelin, or leuprolide, is a gonadotropin-releasing hormone that was initially approved to treat prostate cancer in in an unencapsulated form and required daily injections due to its inherent pharmacokinetic profile. in , lupron depot®, a plg microsphere with encapsulated leuprolide acetate, was initially approved for the treatment of prostate cancer as a once-monthly intramuscular injection. this dramatic reduction in the frequency of injections led the way for many additional plg microsphere-based medicines. although not a long-term commercial success, in , nutropin depot® was approved by the fda for the treatment of growth failure due to a lack of adequate endogenous growth hormone secretion. this product comprised -kd recombinant human growth hormone encapsulated in a plg microsphere which permitted dosing once or twice a month, as opposed to the typical daily subcutaneous injection dosing regimen [ ] . a much larger success for a plg microsphere product is risperdal consta®. risperidone is an anti-psychotic medicine used to treat schizophrenia and bipolar disorder. administered as a daily oral medication, patient compliance is critically important as missed doses can lead to relapse and increased psychotic symptoms. in , the fda approved risperdal consta®, a controlled release form of risperidone encapsulated in plg microspheres that enabled dosing once every weeks by intramuscular injection. compared with the parent oral risperidone, less frequent dosing and improved compliance with administration by a health care professional rather than patient self-administration has made risperdal consta® a major success for treating these brain disorders [ ] . the many successes of controlled release microsphere-based medicines led innovators to explore the potential of drug delivery for systemic administration to reach specific sites of disease, with particular interest in targeting tumors for treating cancer. this cannot be achieved with microspheres, because they would be rapidly cleared from plasma. as a result, research efforts shifted toward reducing particle sizes (diameters < nm) and surface modification using poly(ethylene glycol) to avoid immune recognition and clearance. seminal papers in the field of nanomedicine were published in the early s on pegylated liposomes [ ] and polymeric nanoparticles [ ] . these efforts built upon the pioneering research by maeda and matsumura in the mid- s which demonstrated enhanced permeability and retention of macromolecules based upon their size owing to tumor leaky vasculature and poor lymphatic drainage [ ] . the first fda-approved cancer nanomedicine was doxil®, a pegylated liposome containing doxorubicin. it was initially approved in for the treatment of aidsrelated kaposi's sarcoma, and subsequently for treating ovarian cancer and multiple myeloma (in combination with bortezomib). doxil increased doxorubicin circulation halflife by approximately -fold and tumor drug levels up to fold in preclinical models [ ] . by containing doxorubicin within the liposome and targeting more drug to tumor, doxil also showed an improved toxicity profile in patients, by greatly reducing the cardiovascular side effects of free doxorubicin [ ] . ten years after the initial approval of doxil, a new nanoparticle-based cancer drug received its first approval. in , abraxane®, a nanoparticle albumin-bound paclitaxel, was initially approved for treating breast cancer. it has also been approved for treating non-small cell lung and pancreatic cancers. due to its poor solubility, paclitaxel (taxol®) was originally developed by being dissolved in cremophor el, a polyoxyethylated castor oil, which can lead to severe anaphylactic or hypersensitivity reactions. by solubilizing paclitaxel in albumin nanoparticles, abraxane eliminated the need for cremophor and improved the tolerability profile of paclitaxel, with the highest label recommendation for taxol at mg/ m , whereas abraxane can be dosed as high as mg/m [ ] . the first two waves of drug delivery enabled products with enhanced utility, compliance, and therapeutic window relative to the existing therapeutic agent. current drug delivery innovations feature ambitious efforts to deliver large macromolecules to specific disease locations on timelines generally thought impossible. the developments of many of today's most innovative therapeutics are utilizing nanoparticle drug delivery systems, and for our scope, we will focus on rna nanomedicines. in , fire and mello started a new revolution for gene therapy and gene editing, beginning with the statement, "experimental introduction of rna into cells can be used in certain biological systems to interfere with the function of an endogenous gene" [ ] . significant research has gone into developing short interfering rna (sirna) for rna interference to turn off destructive biological pathways. unmodified rnas require drug delivery systems as they are not stable in biological fluids and do not readily penetrate into target cells where their action is required to reprogram the production of proteins. building off the success of doxil and other liposomal or lipid nanoparticles, major efforts focused on the optimization of lipid nanoparticles for the delivery of rna [ ] . twenty years later in , the first sirna product was approved in . onpattro® (patisiran) is a lipid nanoparticle containing sirna to stop the toxic production of transthyretin in patients with polyneuropathies induced by hereditary transthyretin amyloidosis [ ] . a second rna wave has focused on developing messenger rna (mrna) therapies to promote healthy protein expression to treat diseases. a major potential advantage of mrna therapies is the possibility to treat patients regardless of any particular genetic mutation. for cystic fibrosis, mutations in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene can impact cftr protein folding and capability to regulate salt transport. the most debilitating impact of cf and primary cause of mortality is the inability to clear mucus from the lungs, causing build-up and decreased lung function. multiple programs, one of which is in early clinical development [ ] , are focused on inhaled mrna to encode normal cftr protein to restore healthy lung function. these approaches entail encapsulation of mrna in lipid nanoparticles which are then inhaled as nebulized solutions to allow delivery to the upper airways where cftr protein is produced. in addition to therapeutic treatments, mrna vaccines offer a promising opportunity due to their high potency, capacity for rapid development, and safe administration [ ] . these potential advantages for mrna vaccines are currently being played out in real time, as the covid- pandemic is driving innovation and implementation in an effort to rapidly develop an mrna vaccine. several academic and industry groups around the world are putting a huge effort into developing therapeutics and vaccines for covid- . one company whose efforts merit highlighting at this point is moderna. on january , , the chinese authorities shared the genetic sequence of the novel coronavirus. on january , , the nih vaccine research center and moderna's infectious disease research team finalized the sequence for the sars-cov- vaccine and moderna mobilized toward clinical manufacture. the first clinical batch was completed on february , , and underwent analytical testing; it was shipped on february , , from moderna and delivered to nih from the company's manufacturing facility in days from sequence selection. the first participant in an nih-led phase clinical study was dosed on march , [ ] . such a timeline from virus sequencing to human dosing has never been achieved and was likely never thought possible. a critical enabling element to this impressive achievement was the development of a robust lipid nanoparticle delivery platform with the capability to be applied across mrna sequences for developing vaccines as well as therapeutic mrna modalities [ , ] . from rather modest beginnings, drug delivery technologies are now enabling a vast landscape of new and innovative therapies. from academic labs to large biopharmaceutical companies, the collaboration between scientists, engineers, and clinicians will continue to advance therapies with drug delivery systems to address ongoing unmet patient needs. over the past years, we have witnessed an incredible evolution in drug delivery. earlier generations featured improved versions of existing drugs utilizing controlled release microspheres and long-circulating nanoparticles for cancer treatments. today, innovative nanotechnologies are core to the development of novel medicines to treat a range of serious diseases. endosomal escape and sustained pharmacology and safety in nonhuman primates. mol ther. ; ( ): - . publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. robert s. langer is one of institute professors at mit; being an institute professor is the highest honor that can be awarded to a faculty member. dr. langer has written approximately articles. he also has nearly issued and pending patents worldwide. dr. langer's patents have been licensed or sublicensed to over pharmaceutical, chemical, biotechnology and medical device companies. he is the most cited engineer in history (h-index with over , citations according to google scholar). polymers for the sustained release of proteins and other macromolecules fda: nutropin depot prescribing information systematic review of long-acting injectables versus oral atypical antipsychotics on hospitalization in schizophrenia prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes biodegradable long-circulating polymeric nanospheres a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs reduced cardiotoxicity and comparable efficacy in a phase iii trial of pegylated liposomal doxorubicin hcl (caelyxtm/doxil) versus conventional doxorubicin for firstline treatment of metastatic breast cancer nab-paclitaxel dose and schedule in breast cancer potent and specific genetic interference by double-stranded rna in caenorhabditis elegans liposomal drug delivery systems: from concept to clinical applications the onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs translate bio: safety and tolerability of a single dose of mrt , a nebulized cftr mrna therapeutic mrna vaccines-a new era in vaccinology moderna: moderna announces first participant dosed in nih-led phase study of mrna vaccine (mrna- ) against novel corona virus optimization of lipid nanoparticles for intramuscular administration of mrna vaccines a novel amino lipid series for mrna delivery key: cord- -zgrtux i authors: amin, sk. abdul; jha, tarun title: fight against novel coronavirus: a perspective of medicinal chemists date: - - journal: eur j med chem doi: . /j.ejmech. . sha: doc_id: cord_uid: zgrtux i the ongoing novel coronavirus disease (covid- ) pandemic makes us painfully perceive that our bullet shells are blank so far for fighting against severe human coronavirus (hcov). in spite of vast research work, it is crystal clear that the evident does not warrant the commercial blossoming of anti-hcov drugs. in this circumstance, drug repurposing and/or screening of databases are the only fastest option. this study is an initiative to recapitulate the medicinal chemistry of severe acute respiratory syndrome (sars)-cov- (sars-cov- ). the aim is to present an exquisite delineation of the current research from the perspective of a medicinal chemist to allow the rapid development of anti-sars-cov- agents. coronaviruses (covs) typically cause mild to severe respiratory and intestinal infections in mammals, including humans [ ] [ ] . it belongs to the family coronaviridae which comes under the order nidovirales [ ] [ ] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. the first two genera (i.e., alpha-and betacoronavirus mainly infect mammals, whereas, gammacoronaviruses and deltacoronaviruses foul avian species. more than years have passed since the identification of first human cov (hcov) was documented as a respiratory tract modulator [ ] [ ] . in december , several clusters (epidemiologically associated with a seafood and animal market in wuhan, china) of patients suffering fever, illness, severe respiratory tract infections and pneumonia of unknown origin were reported [ ] [ ] [ ] [ ] [ ] [ ] . this finally leaded to the isolation of a novel coronavirus ( -ncov) and the disease recently named as covid- . world health organization (who) already characterized covid- as world pandemic [ ] . this infection has spread over to countries and territories [ ] . before covid- outbreak, there were six species of hcovs that were reported for their association with respiratory tract infections ( table ) . [ table [ ] . the seventh strain of hcov is novel coronavirus ( -ncov aka sars-cov- ) which is taxonomically belongs to the betacoronavirus genre and possesses high nucleotide sequence similarity with sars-cov and mers-cov [ ] [ ] [ ] [ ] [ ] . sars-cov- is a positive-sense single-stranded rna viruses surrounded by an envelope (figure ). sars-cov- , about , bp single-stranded rna virus, utilizes host cellular components to accomplish its physiological affairs such as viral entry, the assembly and budding of virions, genomic replication, and protein synthesis, subsequently executes pathological damage to the host [ ] [ ] [ ] . thus, punctuating any juncture of viral life cycle by small molecules, peptides, vaccines or physical elements may potentially gain therapeutic benefit to host. depending on several viral targets (figure ) related to the stages of viral life cycle, novel anti-viral agents may be designed and discovered. nonetheless, different structure-based modeling techniques and numerous ligandbased computational techniques may be fruitful strategy to design newer inhibitors against sars-cov- [ ] [ ] [ ] . meanwhile, the hefty menace posed by current outbreak of covid- , it is obvious that the scientific community is looking for effective drugs within plausible time. the coherent development and well organised strategies remains the only hope to triumph the battle against partially known sars-cov- . now, repurposing of existing anti-viral drugs based on previous ground work of closely related coronavirus and rapid screening of drug databases is one of the strategic and economic ways to eradicate covid- pandemic [ ] [ ] [ ] . the traditional bioinformatics and chemo-informatics approaches readily generated new data into sars-cov- research at an explosive pace. considering the severity of the spread of covid- , this study is in-line with the concept of finding the chemo-types to expedite the process of anti-hcov drug discovery. here, an exquisite picture of the recent research including target-based and biological screening is provided. we includes virtual (in silico) as well as experimental (in vitro) screening approaches in response to sars-cov- reported until april . the main aim is to provide the scientific community with an overview of the medicinal chemistry of sars-cov- to allow the rapid development of antiviral agents. this work, a part of our rational drug design and discovery [ ] [ ] [ ] [ ] [ ] [ ] [ ] , is an initiative to pave the way of anti-sars-cov- drug discovery paradigm that could help to facilitate the global efforts to fight against covid- . the details structural biology of the sars-cov- virus is yet to be discovered. it contains a , bp, single-stranded positive sense rna genome encapsulated within a membrane envelope ( figure ) [ ] [ ] [ ] . it recruits multi-subunit replication machinery [ ] . the genome of sars-cov- comprises about , nucleotides with ten open reading frames (orfs). the ′ terminal regions encodes for several structural proteins including spike (s), membrane envelope (e) and nucleocapsid (n) proteins (figure ) whereas, the ′ terminal orf ab responsible for two viral replicase polyproteins pp a and pp b. upon proteolytic cleavage of these two viral replicase polyproteins fabricate sixteen non-structural proteins (nsp) (figure ) [ ] . [ table may be placed here] the spike glycoprotein of coronavirus is the main conciliator of entry into the host cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this spike glycoprotein contains (i) a large ecto-domain, (ii) a single-pass transmembrane anchor, and (iii) a short c-terminal intracellular tail [ ] . the ecto-domain consists of a receptor-binding unit s and a membrane-fusion s stalk. basically, the receptor-binding unit s binds to a specific cell surface receptor via its receptor binding domain (rbd), whereas the trimeric s fuses the viral membranes and host cell to enable the entry of viral genomes into host cells (figure ). researchers already identified angiotensin converting enzyme (ace ) as a functional receptor for sars-cov [ ] [ ] [ ] [ ] . the crowned shaped spike glycoprotein of covs binds directly to ace on the host cells surface and plays critically in virus infection. ace is expressed widely with conserved primary structures throughout the animal kingdom. ace from fish, amphibians, reptiles, birds, to mammals can potentially interact with rbd of sars-cov- [ ]. therefore, blocking of the rbd and ace interaction is an obvious therapeutic intervention to treat diseases caused by covs. specific antibodies or small molecular inhibitors can disrupt the interaction of rbd with ace . among the set of sixteen non-structural proteins, nsp is identified to play a pivotal role in the life cycle of sars-cov- replication as well as maturation (figure ) . being a key component, the nsp is termed as main protease (mpro). like other rna viruses, the functional significance of this mpro or chymotrypsin-like protease ( clpro) of sars-cov- emerges as an attractive drug target for the development of anti-viral agents. recently, the d structure of clpro of sars-cov- was reported [ ] . like other coronaviruses mpro, it also consists of three domains. the domain i (comprising of - amino acids) and ii rdrp active site is appointing two successive aspartate residues projected from a beta-turn structure making them surface accessible through the nucleotide channel. as configured to sars-cov, the robson [ ] performed a preliminary bioinformatics studies to propose a synthetic vaccine and peptidomimetic antagonist against the spike glycoprotein of sars-cov- . the author employed q-uel language to perform the bioinformatics approach. krsfiedllfnkv was identified as a well conserved sequence motif that corresponds to the known cleavage sites of the sars virus. this sequence motif formed the basis for design of specific synthetic vaccine epitope and peptidomimetic agent [ ] . a group of scientists from the cairo university, egypt predicted covid- spike binding site to a cell-surface receptor namely glucose regulated protein (grp ) by employing structural bioinformatics in combination with protein-protein docking [ ] . notably, the region iv was supposed to be a pivotal driving force for grp binding (predicted binding affinity: - . kcal/mol). prediction of this binding site sheds light on the mode of envelope protein recognition by the grp substrate-binding domain for future endeavours [ ] . in a molecular modeling study to explore potential inhibitors of rna binding to n terminal domain (ntd) of nucleocapsid protein (n protein), sarma et al [ ] pointed out two potential hits namely zinc and zinc (figure ). the authors employed two ntd structures of n proteins namely ofz and ssk. firstly, a set of diverse compounds from asinex and maybridge library were docked. then compounds for each of the targets were prioritized with significant docking scores. further mm-gbsa binding free energy, pharmacokinetic properties (qikprop), drug-likeness (swissadme) and molecular dynamics (md) studies were performed to screen the compounds. out of these two potential hits, one compound was a theophylline derivative. since theophylline derivative is commonly used as a bronchodilator, hence, the author further screened approved bronchodilators against the n protein rna binding site of covid- [ ] . the approved bronchodilators showed mm-gbsa binding affinity in the following order: formeterol> terbutaline > ipratropium bromide >tiotropium bromide > theophylline > salbutamol. recently, native or non-native protein-protein interactions (ppis) are emerged as a target for structure-based screening of small molecule. it may be an alternative drug design paradigm which could accelerate anti-viral drug discovery against various pathogens [ ] [ ] [ ] . since the orthostatic/allosteric stabilization of non-native ppis of sars-cov- nucleocapsid protein results abnormal protein oligomerizaion and finally leading to loss of viral activity. scientists from national chung hsing university, taiwan acclaimed non-native ppis of n-terminal domain of the mers-cov nucleocapsid protein (mers-cov n-ntd) [ ] . they reported a crystal structure of mers-cov n-ntd in a non-native dimeric configuration which turned a target for virtual screening of orthosteric stabilizers from acros and zinc drug databases. this finding provides valuable insight and further motivations into the design of new anti-virals based on stabilizing a non-native protein interaction interface of n protein [ ] . gupta and co-workers [ ] employed computational techniques to explore the best possible structure of the sars-cov e protein present in the pdb database. the author reported that e protein of sars-cov- is a pentameric protein comprised of α-helices and loops. near about , compounds were docked whereas compounds were prioritized with significant affinities or docking scores. the docking study with e protein of sars-cov- and phytochemicals like belachinal, macaflavanone e &vibsanol divulged that amino acids such as v and f play crucially in the binding interactions. the functional behaviour of e protein of sars-cov- after ns molecular dynamics studies revealed that α-helix and loops of e protein escapades random movement and modulates normal ion channel activity to succour the pathogenesis in human and other vertebrates. unlikely the random manoeuvre of the e protein of sars-cov- gets reduced after binding with inhibitors [ ] . khan et al [ ] pinpointed three fda-approved drugs such as remdesivir, saquinavir and darunavir) and two small molecules (flavone and coumarin derivatives) as possible inhibitors of clpro by target-based virtual screening. a number of , compounds were docked and top primary hits were distinguished by significant affinities or docking scores. then the binding interaction between the active site residue of clpro and the selected compounds were extensively scrutinized using the plif module in moe. further, md simulation and binding free energy calculations were recorded to evaluate the dynamic behaviour, stability of protein-ligand contact, and binding affinity of the hit compounds [ ] . kandeel and al-nazawi [ ] reported statistics of pairwise sequence comparison matrix among the main protease (mpro) of covs. a high pairwise sequence alignment identity ( . %) was found between sars-cov- and sars-cov- mpro, whereas only . % identity was exposed for sars-cov- and mers-cov mpro. in consequence, the number of amino acid differences between sars-cov- and sars-cov- mpro was only , while it was between sars-cov- and mers-cov mpro. an early virtual screening (vs) study of fda approved drugs (retrieved from selleckchem inc.) against the first resolved sars-cov- mpro crystal structure (pdb: lu ) was performed. that helps the repurposing of already approved drugs to eradicate covid- [ ] . detailed scanning of the binding mode of these drugs with sars-cov- mpro conferred that hydrophobic and hydrogen bonding interactions were the main imperators for binding. interestingly, telbivudine (figure ) , an anti-hepatitis b virus agent, bind with the sars-cov- mpro through hydrogen bond interactions with amino acid residues s and q . moreover, a broad spectrum antiviral agent, ribavirin (figure ) , interacted with the sars-cov- mpro by forming hydrogen bonds with side chain amino acid residue q and the backbone amino acid residue t . ribavirin is officially approved against respiratory syncytial virus (rsv) infection. it is also used in combination with interferon α b against hepatitis c virus. moreover, it also exhibited potency against sars-cov infection [ ] . in a study to distinguish potential active herbs against -ncov, ma et al. leading to viral eradication [ ] . in another study, elfiky [ ] reported sars-cov- rdrp targeted molecular docking study of some anti-polymerase drugs which have been approved for use against various viruses. not surprisingly, ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovirexhibited promising binding affinity against sars-cov- rdrp. these results were also consistence with previous one [ ] . guanosine derivative (idx- ), setrobuvir, yak has potential to block the sarscov- strain. since these drugs have already passed the adme and toxicity measurements these may be used as a new therapeutic drug candidate against sars-cov- . lung and co-workers [ ] identified theaflavin, a polyphenolic constituent present in black tea, hydrophobic interactions were found to be the driving force in binding. theaflavin formed hydrogen bonds with d , r and r of sars-cov- rdrp. additionally, a π-cation interaction was found with r [ ] . calligari et al [ ] autodockvina scoring − . kcal/mol) was recognised as the best autodock vina scoring drug. in spite of hcv main protease measure very low identity with the sars-cov- homolog (only . %), thus this finding was thunderbolt [ ] . therefore, it may be inferred that the similar topology of active site of both proteases was the main driving force in binding of simeprevir. it fitted well into the two hydrophobic pockets fringed the catalytic dyad h -c of sars-cov- mpro. it was also fitted into the hydrophobic loop f -f . moreover, the binding of simeprevir to sars-cov- protease was anchored by three hydrogen bonding interactions with e , g and n [ ] . in the same article, the homology modelling of sars-cov- s protein by the aid of itasser server was done by using sars-cov spike (pdb: wrg, × , xlr) as a template. global pairwise sequence alignment measured that sars-cov- s protein shares about % of its primary sequence with its homolog in addition an overall similarity of %. autodock vina molecular docking of the homology modelled structure of s protein predicted umifenovir (db ), enfuvirtide (db ), and pleconaril (db ) as potential inhibitors [ ] . in a study to repurposing existing drugs against current pandemic covid- , wu et al [ ] predicted some potential drugs acting on a certain target or multiple targets of sars-cov- . bioinformatics based homology modelling was utilised to build possible targets such as viral mpro, plpro, rdrp, helicase, spike, etc. next, these modelled proteins and human relative proteins including human ace and type-ii transmembrane serine protease enzymes were forwarded to systematically analyse and screen zinc drug database (zdd) along with database of traditional chinese medicine and natural products and the database of commonly used anti-viral drugs [ ] . this study seems to be very interesting due to its deep discussions and vast target predictability. the lead candidates emerged from this study required in vitro and in vivo studies for further conformations. the new drug discovery and development takes more than five to ten years of investigations as well as cost billions of dollars. thus, drug repositioning is the only cheap strategy to respond immediately. fda-approved drugs justify safe alternatives if at least modest anti-sars-cov- activity can be achieved. currently, academia and industry personnel are involved in the testing of -(i) approved drugs and/or (ii) drug candidates in clinical trials. the in vitro screenings of fda approved drugs as well as the compounds which are currently under clinical trials phases were well documented [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the need for further in vivo testing to facilitate drug discovery efforts against sars-cov- . nafamostat ( figure ) is a potent membrane fusion inhibitor of mers-cov [ ] . now, it has been found to inhibit the -ncov infection (ec besides, this is agent marketed as an anti-protozoal drug [ ] and also reported to possess antiviral against a broad range of viruses [ ] . in the same article, wang et al [ ] remdesivir have been illustrated few years ago [ ] . now, it has been found to effectively block sars-cov- infection at low-micromolar concentration [ ] . it exhibited half-maximal effective concentration (ec ) of . μm against -ncov in vero e cells with half cytotoxic concentration (cc ) > μm; selectivity index (si) > . ). remdesivir also inhibited virus infection in sars-cov- sensitive human liver cancer huh- cells [ ] . a widely-used anti-malarial drug chloroquine (cq, figure ) has been used for more than years [ ] , now, found to shows clinical potency against covid- . [ figure may be placed here] the molecular mechanism of chloroquine against sars-cov is known [ ] [ ] . very recently, wang et al [ ] reported time-of-addition assay that explained the function of cq (ec = . μm; cc > μm; si > . ) at both entry as well as at post-entry stages of the novel corona virus infection in vero e cells. the same group of researches further evaluated the in vitro anti-sars-cov- effect of hydroxychloroquine (hcq, figure ) sulphate, a derivative of cq, in comparison to cq [ ] . hydroxychloroquine sulphate [ ] was introduced long before, first synthesized in [ ] . upon introduction of a hydroxyl group into cq resulted in about % less toxic agent than cq in animals. both cq and hcq are weak bases and restrict the virus infection by-(i) triggering endosomal ph which is essential for virus/cell fusion and (ii) interfering with the glycosylation of ace receptor and spike protein of coronavirus [ ] . additionally, both cq and hcq obstructed the sars-cov- transport from early endosomes (ees) to endolysosomes (els). interestingly, it exhibited very distinct binding mode than the other covalent or peptidomimetic clpro inhibitors [ ] . in vitro screening of drugs was screened against hcovs infection [ ] . immunofluorescence analysis with an antibody specific for the viral n protein of sars-cov- was scored for each drug treated cells. the dose-response curve (drc) was developed after analysing the confocal microscope images of both viral n protein and cell nuclei. remdesivir (sars-cov- ic = . µm), lopinavir (sars-cov- ic = . µm) and chloroquine (sars-cov- ic = . µm) were used as reference drugs. consequently, drugs showed good activities with ic ranges of . to μm. an anti-helminthic drug, niclosamide, and a corticosteroid used to treat asthma and allergic rhinitis, ciclesonide, emerged as sars-cov- inhibitors with ic of . μm and . μm, respectively. notably, niclosamide reduces mers-cov replication by inhibiting skp activity leading to enhancement in autophagy [ ] . thus, a similar mechanism may be introduced by niclosamide to hamper sars-cov- infection [ ] . zhang et al [ ] reported structure-based design, synthesis and activity assessment of α-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication. these showed good inhibitory properties against the isolated proteases, viral replicons, virus-infected huh cells. nearequipotency against the enteroviruses, alphacoronaviruses, and betacoronaviruses was observed upon optimization of the p substituent of the α-ketoamides. the cyclopentylmethyl and cyclohexylmethyl at the p substituent disposed low-micromolar ec values against the three virus genera in cell cultures. compound r was found promising against mers-cov in virus-infected huh cells [ ] . by dint of high similarity among the clproteases of coronavirus, it is awaited that compound r is expected to display good anti-viral activity against covid- in near future. meanwhile, the clinical trial studies of some molecules have been started against covid- mainly through drug repurposing [ ] ; those are depicted in table . the medicinal chemistry of sars-cov- infection is still in its infancy, with target specific lead molecules are yet to identify. this study deals with the information currently available on potential targets for therapeutic invention and screening of new compounds or drug repurposing against sars-cov- (figure ). as the d structure of the sars-cov- c-like protease bears % identity with its ortholog from (sars-cov). interestingly, the residues involved in the catalysis, substrate binding and dimerization of clpro are % conserved. in addition, the polyprotein pp ab sequences are highly similar ( % identity). depending upon the alike substrate specificities and high identities, we are of the opinion that the previous progress of specific sars-cov inhibitors development can undertaking a course of action on the design and discovery of inhibitors against sars-cov- . our group have already explored the structural properties important for sars-cov viral clike protease inhibitors [ ] . recently in a collaborative work, our research team suggested the implications of naphthyl derivatives against sars-cov- plpro enzyme though in-depth ligandreceptor interaction analysis [ ] . we have already predicted some in-house glutamine-based molecules to use as a seed for drug design and optimization against plpro of sars-cov- [ ]. in fact, some other anti-viral drugs can also be taken into consideration. in this regards, target-based vs is one of the most important approaches used for drug repurposing. the computational analyses are not subordinate but are a right choice to enrich the basal knowledge during the long process leading to drug development (figure ) . until any clear-cut treatment approach is prescribed for covid- , the use of already approved drugs is only alternative strategy. howbeit, relatively limited computational resource or biased in silico screening may scattered the linearity of drug discovery of novel coronavirus. in the near future, the virtual hits may serve as a promising drug like molecule against sars-cov- after details in vitro and in vivo laboratory investigations. moreover, the availability of x-ray crystal structures of the important viral proteins will trigger more exhaustive docking calculation of diverse chemotypes. it is crystal clear that the prevention of covid- requires strong and sustainable global collaborative work [ ] . data sharing is exigent to fill the knowledge gaps on this global pandemic. further progress of the scientific understanding regarding the structural and molecular biology of sars-cov- will legitimate the shape of lead compounds to achieve therapeutic goals. the development of medicinal chemistry through bioinformatics and chemo-informatics studies remains indispensable with a bit of savoir faire. the authors have no conflict of interests. sk. abdul amin is a senior research fellow at department of pharmaceutical technology, jadavpur university, kolkata, india. he is working under the guidance of tarun jha. his research area includes design and synthesis of small molecules with anti-cancer and anti-viral properties, computational chemical biology, and large-scale structure-activity relationship analysis. he has published sixty two research/review articles in different reputed peer-reviewed journals and three book chapters. he enjoys a good conversation on science, regional history, contemporary art and books. tarun jha, a professor at department of pharmaceutical technology, jadavpur university, kolkata, india, has supervised ph.d. students and guided nine research projects funded by different organizations. he has published more than research articles in different reputed peer-reviewed journals. his research area includes design and synthesis of anti-cancer small molecules. prof. jha is a member of the academic advisory committee of national board of accreditation (nba), new delhi, india. structure of mpro from covid- virus and discovery of its inhibitors coronaviruses -drug discovery and therapeutic options structure, function, and evolution of coronavirus spike proteins coronaviruses post-sars: update on replication and pathogenesis a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease clinical features of patients infected with novel coronavirus in wuhan cryo-em structure of the -ncov spike in the prefusion conformation novel coronavirus of pneumonia in wuhan, china: emerging attack and management strategies the global spread of -ncov: a molecular evolutionary analysis coronavirus -ncov: a brief perspective from the front line recent discovery and development of inhibitors targeting coronaviruses the novel coronavirus: a bird's eye view network-based drug repurposing for novel coronavirus -ncov/sars-cov- advance of promising targets and agents against covid- in china déjà vu: stimulating open drug discovery for sars-cov- . drug discovery today the outbreak of sars-cov- pneumonia calls for viral vaccines sars-cov orf b-encoded nonstructural proteins - : replicative enzymes as antiviral targets genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases potential interventions for novel coronavirus in china: a systematic review fighting the coronavirus outbreak therapeutic options for the novel coronavirus fast identification of possible drug treatment of coronavirus disease - (covid- ) through computational drug repurposing study molecular docking and dynamics simulation of fda approved drugs with the main protease from novel coronavirus structural insight into the viral clike protease inhibitors: comparative sar/qsar approaches design of aminopeptidase n inhibitors as anti-cancer agents exploration of structural and physicochemical requirements andsearch of virtual hits for aminopeptidase n inhibitors synthesis, anticancer activity, sar and binding mode of interaction studies of substituted pentanoic acids exploring the structural aspects of ureido-amino acid-based apn inhibitors: a validated comparative multi-qsar modelling study histone deacetylase (hdac ) inhibitors as anticancer agents: a review matrix metalloproteinase- (mmp- ) and its inhibitors in cancer: a minireview covid- : the role of the nsp and nsp in its pathogenesis structural basis of severe acute respiratory syndrome coronavirus adp-ribose- ''-phosphate dephosphorylation by a conserved domain of nsp genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding structure of the rna-dependent rna polymerase from covid- virus discovery of baicalin and baicalein as novel, natural product inhibitors of sars-cov- cl protease in vitro structure analysis of the receptor binding of -ncov genomic characterization of a novel sars-cov- evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission angiotensin-converting enzyme is a functional receptor for the sars coronavirus a highly conserved cryptic epitope in the receptor-binding domains of sars-cov- and sars-cov structural basis of receptor recognition by sars-cov- structure of the sars-cov- spike receptor-binding domain bound to the ace receptor functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses anti-hcv, nucleotide inhibitors, repurposing against covid- preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov- ( -ncov, covid- ) coronavirus covid- spike-host cell receptor grp binding site prediction medhib ( ) in-silico homology assisted identification of inhibitor of rna binding against -ncov n-protein (n terminal domain) emerging roles of allosteric modulators in the regulation of protein-protein interactions (ppis): a new paradigm for ppi drug discovery ppi inhibitor and stabilizer development in human diseases site-directed fragment-based screening for the discovery of protein−protein interaction stabilizers structure-based stabilization of non-native protein−protein interactions of coronavirus nucleocapsid proteins in antiviral drug design in-silicoapproaches to detect inhibitors of thehuman severe acute respiratory syndrome coronavirus envelope protein ion channel identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach virtual screening and repurposing of fda approved drugs against covid- main protease study on screening potential traditional chinese medicines against -ncov based on mpro and plp rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds insilico studies on therapeutic agents for covid- : drug repurposing approach ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study the potential chemical structure of anti-sars-cov- rna-dependent rna polymerase molecular investigation of sars-cov- proteins and their interactions with antiviral drugs analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods identification of antiviral drug candidates against sars-cov- from fda-approved drugs remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro fda approved drugs with broad anti-coronaviral activity inhibit sars-cov- in vitro the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro teicoplanin potently blocks the cell entry of -ncov in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay nitazoxanide: a new thiazolideantiparasitic agent nitazoxanide: a first-in-class broad-spectrum antiviral agent current knowledge about the antivirals remdesivir (gs- ) and gs- as therapeutic options for coronaviruses. one health chloroquine: mechanism of drug action and resistance in plasmodium falciparum new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine is a potent inhibitor of sars coronavirus infection and spread current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review rein t ( ) skp attenuates autophagy through beclin -ubiquitination and its inhibition reduces mers-coronavirus infection endoribonuclease from sars cov- . vww deposited diffraction resolution: . Å the crystal structure of papainlike protease of sars cov- w c deposited it is a challenging task to identify effective anti-covid- drugs urgently. an exquisite picture of the recent research including target-based and biological screening is provided. experimental screening approaches in response to -ncov is highlighted. it is an initiative from the perspective of a medicinal chemist to provide information about potential targets and drug repurposing against covid- . the authors have no conflict of interests. key: cord- - fhhuzg authors: hoffman, paul s. title: antibacterial discovery: st century challenges date: - - journal: antibiotics (basel) doi: . /antibiotics sha: doc_id: cord_uid: fhhuzg it has been nearly years since the golden age of antibiotic discovery ( – ) ended; yet, we still struggle to identify novel drug targets and to deliver new chemical classes of antibiotics to replace those rendered obsolete by drug resistance. despite herculean efforts utilizing a wide range of antibiotic discovery platform strategies, including genomics, bioinformatics, systems biology and postgenomic approaches, success has been at best incremental. obviously, finding new classes of antibiotics is really hard, so repeating the old strategies, while expecting different outcomes, seems to boarder on insanity. the key questions dealt with in this review include: ( ) if mutation based drug resistance is the major challenge to any new antibiotic, is it possible to find drug targets and new chemical entities that can escape this outcome; ( ) is the number of novel chemical classes of antibacterials limited by the number of broad spectrum drug targets; and ( ) if true, then should we focus efforts on subgroups of pathogens like gram negative or positive bacteria only, anaerobic bacteria or other group where the range of common essential genes is likely greater?. this review also provides some examples of existing drug targets that appear to escape the specter of mutation based drug resistance, and provides examples of some intermediate spectrum strategies as well as modern molecular and genomic approaches likely to improve the odds of delivering st century medicines to combat multidrug resistant pathogens. infectious diseases are a leading cause of death worldwide, yet the miracle antibiotics developed to combat them are being lost to the rapid emergence of drug resistance. modern medicine is heavily dependent on antibiotics that enable many procedures including abdominal surgeries, bone marrow and organ transplants, managing immune compromised patients and cancer patients and for routine hip and knee replacements [ , ] . while one might presume that new antibiotics would be discovered at a sufficient pace to replace those rendered obsolete by resistance, this has not happened. worse, the few antibiotics that recently entered the clinic are derivatives of older drugs and are susceptible to the same existing drug resistance mechanisms. it is hard to imagine that the last new chemical class of antibiotic for treating infections caused by gram negative (superbug) bacteria (gnb) was the quinolones, discovered over years ago. and, it is not much better for the gram positives (gp) with linezolid and daptomycin discovered nearly years ago. while fleming and waksman are well known discoverers of antibiotics (penicillin and streptomycin, respectively), it is unlikely that anyone today knows someone that delivered a new class of antibiotic from bench to clinic. thus, unlike most disciplines of science and technology, there are no experts to advise us. worse, there seems to be no cumulative knowledge gained from the many failures upon which to build a winning strategy [ ] . while it has been argued that the discovery of antibiotics is one of the greatest achievements in medicine in the th century; our failure to build a sustainable antibiotic discovery platform has left us with one of the greatest liabilities, and now in the st century, one of the greatest challenges. some blame the lack of funding or of effort, but in retrospect the effort was substantial, particularly by the pharmaceutical industry in the 's, and most agree that massive investments would have made little difference. while much has been written recently to explain these failures [ ] [ ] [ ] [ ] ; few have provided tactical approaches that might fare any better [ ] . emergence of drug resistance remains the greatest concern and biggest challenge to future discovery efforts. many believe it is impossible to create an antibiotic to which bacteria cannot evolve resistance and based on some recent experience, the therapeutic window for any new one is projected to be less than five years-a major disincentive. if indeed antibiotic resistance (mutation-based drug resistance) is the major challenge; then, why have we not focused on identifying drug targets less prone to mutation-based resistance? with scarce resources and many suggestions for how to find next generation antibiotics, where do you place your bets [ ] ? this review examines current discovery approaches and their intrinsic liabilities; challenges some myths associated with antibiotic resistance; and concludes by exploring promising st century discovery strategies along with some examples of antibacterials that might escape the specter of drug resistance. there are many headwinds and challenges to antibacterial discovery and delivery of new medicines to the clinic that are not discussed herein (societal, regulatory and marketing) that readers are directed elsewhere for more comprehensive coverage [ ] [ ] [ ] [ ] . most of the antibiotics used in the clinic today were derived from natural products (np) identified in screens of environmental material using variations of the waksman platform strategy [ ] . synthetic antibacterials trace back to paul ehrlich who identified chemical compounds like salvarsan that was active in the treatment of syphilis. later, domagk discovered the dye prontosil and its active metabolite sulfanilamide that inhibited folic acid biosynthesis. this work led to development of the sulfa drugs [ ] . medicinal chemistry and the advent of high throughput screens of compound libraries (target based) replaced np searches in the mid 's and reintroduced the idea of synthetic chemistry as a strategy for creating next generation antibacterials. advances in computational biology (bioinformatics, systems biology, and metagenomics) and structural biological technologies (crystal structures of protein targets, ligand screens via docking software, the so called designer drug strategy) have led to a third discovery strategy-synthetic biology. while all these technologies engender some optimism, the main reason for the antibiotic discovery void in the first place is that creating new chemical classes is really hard. is repeating history a viable strategy?-probably not. one definition of insanity is repeating the same task over and over again while expecting a different outcome. this is especially applicable to antibiotic discovery research where much emphasis has been placed on finding new natural product (np) antibiotics [ ] . after all, the vast majority of antibiotics used in the clinic are derivatives of nps. the insanity with continuing on this path is that for all of these np antibiotics, resistance mechanisms predated their therapeutic use and this is unlikely to change [ , , ] . moreover, we know from past discovery efforts that nps tend to be redundant-with the same targets and mechanisms of action appearing over and over again [ ] . casting a wider net will not change this reality, because the number of broad spectrum drug targets is quite small [ ] . hypothesis. the number of antibiotic classes is limited by the number of broad spectrum drug targets. it follows that the number of resistance mechanisms will generally parallel the number of antibiotic classes. hence, modifying the genes associated with secondary metabolism, while potentially improving an existing scaffold, is unlikely to produce new chemical classes of antibacterials. another issue with nps is that since they have evolved in natural environments and not in the human milieu, most nps are structurally complex and as such are by default poor pharmacophores (pk/pd), requiring much medicinal chemistry to improve bioavailability, to reduce drug metabolism or to reduce intrinsic toxicities. the pharmaceutical industry abandoned np divisions long ago in favor of high throughput screens of compound libraries against novel drug targets (another failure, see below); or, more productively to create next generation derivatives of existing antibiotics, a strategy that has worked well and continues today as judged by the number of analogues in the current pipeline. despite the continuum of second, third and fourth generation β-lactams (penicillin, cephalosporin and carbapenem derivatives) [ ] , by far the largest group of antibiotics used in the clinic today, has not solved the resistance problem originally pointed out by alexander fleming in the late 's. while logic dictates that we should abandon further development of this class, advances in pairing them with β-lactamase inhibitors (clavulinate, avibactam, and vaborbactam to mention a few) has restored their effectiveness and extended their clinical lifetimes [ ] . since β-lactamases and β-lactam targets (penicillin binding proteins, pbp) share conserved structural attributes that can be altered via mutation, resistance will eventually defeat them, but for now this strategy is both viable and profitable. the current antibacterial pipeline is populated with new derivatives based on the scaffolds for quinolones, tetracyclines, macrolides and aminoglycosides [ ] . new chemistries around existing scaffolds has the potential to overcome current resistance mechanisms by improving binding complexity with the drug target, and or improve pharmacological properties. in this regard, the ketolides were supposed to overcome resistance to macrolides, but the tradeoff is increased toxicities. however, given our experiences with np antibiotics in general, it is unlikely that continued searches for new ones, regardless of where one looks, will be any more successful than past efforts. in the mid 's, the pharmaceutical industry turned to molecular biology and genomics to sort through microbial genomes for novel drug targets that could be cloned and their products configured into assays amenable to robotic high throughput screens of massive compound libraries (target-based platform). after all, new medicines were found in library screens in other therapeutic areas, including antivirals [ ] . the fatal assumption in antibacterial screens was the belief that antibiotic-like material was present in these chemical libraries [ ] [ ] [ ] [ ] . in reality, they contained little that resembled antibiotics, because antibiotics are often of high molecular weight and far more complex than can be synthesized by chemists. worse, most of these compounds were poor pharmacophores (hydrophobic with poor pk/pd properties) and failed both lipinski's rule of five and hergenrother's rules for penetration of gn bacteria [ ] [ ] [ ] . unfortunately, efforts to assemble libraries composed of more "antibiotic like material" have yet to deliver an antibacterial to the clinic. what does "antibiotic like" really mean anyway? the experiences of glaxosmithkline, astrazeneca and others are well documented and today there seems to be little enthusiasm for revisiting this approach [ , , ] . however, the lists of potential therapeutic drug targets identified in these bioinformatics initiatives and others remain to be exploited. one of the targets in the gsk screen, fabi, an enoyl-acp reductase and rate-limiting step in fatty acid biosynthesis is the target of a novel therapeutic (afabicin, formerly debio- ) that is active against staphylococcus [ ] . importantly, the failure of hts to deliver new therapeutics to the clinic should not be confused with whether or not synthetic antibacterials can be developed de novo as part of a rational or designer drug strategy (see later section). every healthcare worker can tell you why antibiotic resistance is bad for patients and why antimicrobial stewardship (judicious use) is so important, but few have any deep understanding of the diversity of underlying molecular and genetic mechanisms. this has created a "chicken little" effect; where strong opinions based on shallow thinking, perpetuation of myths and intolerance of new ideas have unnecessarily complicated the discovery process. much of antibiotic theory has evolved over nearly years of history and mostly from experiences with nps. antibiotics and their respective resistance mechanisms have co-evolved over millions or even billions of years of internecine microbial warfare that is ongoing in the environment. these resistance mechanisms are encoded in dna and can be disseminated throughout the microbial world via mobile genetic elements and horizontal transfer [ ] . it is not surprising that soil and aquatic microbes transfer these resistance determinants to human and animal pathogens, often in clinical settings where antibiotics are in heavy use. there is no good outcome to the continued use of nps and as discussed previously it is unlikely that new ones will fare any better. this is best exemplified by the emergence of resistance (mcr- family) to colistin, a polymyxin class antibiotic that has been around for decades, but little used, until the emergence of carbapenem resistance in klebsiella (kpc) and other carbapenem resistant enteric superbugs (cre) made it the drug of last resort [ ] . the rapid and global emergence of mcr- just underscores the point. the resistance problem is further exacerbated by globalization, increasing population density and international travel that has brought us mcr- as well as the ndm- β-lactamase resistance determinant [ ] . the covid- pandemic, unfortunately, is another example of the accelerated global spread of biological agents. it follows from the previous paragraph on nps, that synthetic antibacterials that inhibit targets "mother nature" has yet to find would require millions of years of evolution for resistance determinants to emerge. note that this hypothesis also holds true for new chemistries against established drug targets, provided that they are also not susceptible to existing resistance mechanisms. a good example is linezolid, a synthetic antibacterial discovered nearly years ago and more recent derivatives (tedizolid), that inhibit protein synthesis in gp bacteria [ ] . one might think that synthetic antibacterials would enjoy a long clinical life. unfortunately, synthetic classes of antibacterials, including linezolid, are often defeated by mutation-based drug resistance. mutations occur naturally (probability of~ in ) in dna and since microbial infections involve hundreds of billions of bacteria, microbes tend to win the probability game. survival is the prime directive for any lifeform and in particular, the stress of antibiotics on bacteria, especially at sub-inhibitory levels, provides strong selection for these resistant variants to emerge and flourish. it should be pointed out that mutation-based drug resistance also underlies resistance for nearly all nps. this reality led eric lander and john p. holdren to conclude in a summary of their report by the committee on antimicrobial resistance to the president's council on science and technology that: "in the fight against microbes, no permanent victory is possible: as new treatments are developed, organisms will evolve new ways to become resistant" [ ] . this grim prediction is certainly supported by a recent clinical trial of promising synthetic boron containing heterocycle leucyl trna synthetase inhibitor which was halted due to rapid emergence of drug resistance [ ] . this novel drug inhibited a non-essential proof-reading region of the essential enzyme in which mutations could accumulate and thereby defeat the inhibitor without total loss of function [ ] . the lessons learned here include: ( ) focus on the essential catalytic center of a prospective drug target, ( ) ensure amino acid conservation within this region (resists mutation), and ( ) confirm by co-crystallization that leads indeed are binding within the catalytic pocket of the target. it is also helpful early on to explore possible off target activities against human drug targets related to a particular enzyme class. attention to these details might lead to medicines that slow the inevitable pace to drug resistance. it raises another critical question "can we find drug targets that by catalytic mechanism are capable of escaping mutation-based drug resistance? targets that might defeat mutation based drug resistance do exist. one example is the lipid ii and lipid iii targets associated with cell wall biosynthesis [ ] . these targets are composed of lipid and not amino acids and are the target of a new np antibiotic teixobactin that is in early clinical development for treatment of infections caused by gp bacteria [ ] . another type of target that had not been previously considered is pyruvate: ferredoxin oxidoreductase (pfor) whose catalytic center is highly conserved through evolution and for which there are no np inhibitors [ ] . the pfor catalytic mechanism involves a uniquely positioned and contorted vitamin cofactor (vitamin b or thiamine pyrophosphate, tpp) [ , ] . redox cycling via iron/sulfur centers activates the vitamin to enable binding of substrate pyruvate. the nitrothiazolide class of synthetic antibiotics (fda approved nitazoxanide and analogue amixicile in clinical development) deactivates the vitamin via a proton abstraction mechanism that is dependent on a functional enzyme [ , ] . two key points here are that mutations to the enzyme (loss of function) or that alter the vitamin, a small molecule, are lethal ( ). tpp is also a cofactor in many enzymes (pyruvate dehydrogenase [pdh] and pyruvate carboxylase), but these are not inhibited by nitazoxanide or amixicile [ , ] . importantly for nitazoxanide, there are no toxicities in humans or mitochondria which lack the drug target. importantly, in over years of clinical use, there are no reports of drug resistance with nitazoxanide. a hint at possible resistance to nitazoxanide comes from studies of lab generated resistant mutants of human parasite giardia, where whole genome sequencing found no single correlating mutation among resistant strains [ ] . from this study and others, it is possible to suggest that second site compensatory mutations may lead to metabolic shifts that produce a "tolerance like phenotype", much like over expression of efflux systems incrementally raise mic for quinolones. is this evolution at work? it could be, as efflux buys time for mutations in gyrase and topoisomerase genes to manifest. alternatively, as noted with nitazoxanide tolerance in helicobacter pylori, it may be an example of the microbe "faking it", i.e., tinkering with limited metabolic regulatory choices to enable survival [ ] . in this case, tolerance can be overcome by increasing the therapeutic dose, i.e., one that the microbe cannot overcome by tinkering. the jury is still out though, as only time will tell if such tolerance mechanisms can be inherited. there are several caveats from the pfor example that might be useful in identifying new targets: ( ) pfor is an ancient enzyme highly conserved through evolution; ( ) the vitamin cofactor is uniquely positioned and tightly bound within the enzyme; ( ) the critical amino acids required for cofactor function are absolutely conserved based on analysis of available sequences of pfor enzymes (> ) despite whether pfor is a dimer of high molecular weight monomers or like in helicobacter pylori, a dimer comprised of multiple subunits [ ] ; and ( ) all of these traits are found in the entire broad family of the alpha-keto-acid oxidoreductases (multiple essential targets). perhaps most important in this example is that nitazoxanide and amixicile work by a "theft" mechanism that requires a functional enzyme and cofactor. the phenotype to the affected pathogen is one of slow starvation (pyruvate) with no definable selective mechanism or means to overcome inevitable death by starvation. in retrospect, few searches for new drug targets considered mutation frequency as a criterion for prioritization. finding catalytic centers that are highly conserved and potentially druggable can be relatively straightforward, but like pfor, would likely yield targets of limited spectrum. "highly conserved" generally means that mutations in these regions lead to loss of function or lethality. these potential targets can be catalogued by comparative genomics (blastp approach) by comparing genomes (gene products) from a target group of pathogens. by setting the probably of the match (identity) bar to be more stringent (e − or e − ) creates a shorter list of protein matches whose amino acid sequences are highly conserved [ , ] . further analysis would include amino acid conservation within the catalytic center along with other evaluative criteria that includes chemical space, catalytic mechanism, participation of cofactors and uniqueness from orthologues found in humans or mitochondria. crystal structure of the enzyme is crucial in this process and in silico screening of chemical libraries becomes an early first step in finding developable leads. a number of recent examples can be used to guide this process [ , , ] . the power of computational biology and chem-informatics has yet to be fully exploited, so this area has room to grow. an advantage of this approach is that objective criteria replace age old empiric rationale (guesswork) in choosing drug targets. however, the reality of finding new broad spectrum drug targets is close to nil. this is because microbial genomes are small and the major targets for broad spectrum antibiotics are already known (protein synthesis, dna and rna synthesis, cell wall biosynthesis and a few central metabolic pathways) [ ] [ ] [ ] [ ] [ ] . as discussed below, we need to consider targets of more limited spectrum. the number of broad spectrum drug targets among microbes that meet the criteria of essential and non-redundant is predicted to be quite small, perhaps fewer than several hundred [ , ] . most targets are already known and come with the baggage of preexisting antibiotic resistance mechanisms and the reality that they will cause collateral damage to normal flora when deployed. the problem with narrow spectrum antibiotics is that clinicians do not use them when the pathogen responsible for an infection is not known or is potentially polymicrobial. broad spectrum antibiotics are a hedge and provide liability coverage in the event of an adverse outcome. this reluctance also plagues new antibacterials that enter the clinic, another headwind to drug development. ironically, getting a new broad spectrum antimicrobial through the food and drug administration (fda) today is nearly impossible because for each indication a separate clinical trial is generally required and the additive costs are prohibitive. antibacterials currently in clinical development (gepotidacin) and those that have recently entered the clinic like oritavancin, dalbavancin and tedizolid are examples where the potential use is purposefully limited [ ] . in the distant past, the spectrum for an antimicrobial was often established by the process of "off label" use (trial and error), but today, hospital practice/malpractice and liabilities have essentially ended this strategy. we can expect in the future that the number of indications for antibacterials will continue to be limited by development costs [ ] . perhaps a more productive antibacterial strategy is to focus on groups of microbes sharing common essential drug targets as exampled by pfor. in this example, nitazoxanide exhibits the same spectrum as metronidazole, providing broad anaerobic coverage (gp and gn), treatment of infections caused by anaerobic human parasites and potentially treatment of infections caused by members of the epsilon proteobacteria (helicobacter and campylobacter). along these lines, developing antimicrobials that target either gp or gn bacteria would exploit new drug targets unique to each, such as teixobactin for gpb. similarly, drugs that are selective against mycobacterium tuberculosis are highly desirable since resistance is a major problem [ ] . recent new chemical classes of anti-tubercular therapeutics include the nitroimadazole delamanid that targets the f deazaflavin nitroreductase [ ] . similarly, pathogen selective therapeutics such as ridinilazole, a novel synthetic drug in clinical development for treatment of colitis caused by clostridioides difficile, are developed in response to an urgent clinical need [ ] . in this case, ridinilazole targets cell division by an unknown mechanism that is likely to uncover a novel drug target and mechanism. microbiome studies support limited collateral damage with ridinilazole and the frequency of drug resistance appears to be low [ ] . since there is an urgent need to develop gn-selective therapeutics, focusing on targets unique to them and vetted by objective criteria (including resistance to mutation) should be encouraged. indeed, ongoing drug discovery efforts are targeting lps biosynthesis (lpxc, lepb, msba) [ ] , essential enzymes associated with beta-barrel assembly machine (bam), lipoprotein assembly (lol), and even the periplasmic disulfide bond isomerase (dsba, dsbc, dsbd) enzymes [ ] [ ] [ ] [ ] [ ] . unfortunately, lead compounds targeting lps biosynthetic systems have proven toxic and reminds us that finding new chemistries of low toxicity remains a significant challenge. focus on anti-virulence strategies, particularly for gn superbugs, is now gaining momentum as inhibitors are emerging against secretion systems, pili, motility, quorum sensing and toxins [ , ] . conceptually, if virulence systems contribute to antibiotic resistance or tolerance, inhibition of these systems might result in regulatory changes that restore or enhance susceptibility to mainline antibiotics. as better rapid diagnostics come onboard, narrow or limited spectrum antibacterials will see increased usage, and when treating polymicrobic infections, therapeutic strategies would likely mix and match the appropriate therapeutics. even better, we might consider creating bi-functional or hybrid antibacterials that provide dual function that likely will extend both coverage and timelines to resistance. repurposing of drugs, especially fda approved medicines, for use as antibacterials has gained in interest since they skip the early preclinical and clinical development stages [ ] . it is also believed that such drugs would also come with novel modes of action and perhaps escape preexisting drug resistance mechanisms. perhaps the most studied of these compounds is auranofin, a drug developed to treat rheumatoid arthritis and cancer, that is a potent inhibitor of thioredoxin reductase found in many human pathogens including clostridioides difficile [ ] . for repurposed drugs in general the primary indication is still a secondary target of the new application. in order to gain therapeutic efficacy (serum levels greater than mic), repurposed drugs would likely be administered at concentrations exceeding levels required for efficacy of the original use. in reality, drugs that actively target human enzymes, often come with undesirable side effects or toxicities. still, for treatment of infections such as tuberculosis, gonorrhea or by c. difficile and h. pylori, repurposed drugs in combination with other therapeutics might aid overcoming drug resistance. the goal of this review was to point out basic science challenges to the discovery of new classes of antibacterials and to provide some suggestions on how to move forward in a more productive manner. we know that the "waksman platform" for discovery of new nps and the "hts-target based platform" for screening chemical libraries have both failed to deliver new antibacterials. does repeating these strategies boarder on insanity? from a business perspective, investment in antibacterial discovery is mitigated by concerns over rapid emergence of drug resistance and loss of investment. if indeed mutation-based drug resistance is the greatest impediment to the discovery effort, then why not work backwards from the problem as a discovery strategy? this review provides a few examples of such drug targets (pfor and lipid ii) and respective inhibitors that shows that this approach has merit. structure based drug design has yielded therapeutics in other areas including hiv/aids (protease inhibitors), antivirals (zanamivir for influenza), and even inhibitors of cyclooxygenase, so why not for antibacterials? bioinformatics can be used to collate essential targets and provide a probability score for evaluating risk for mutation based drug resistance. structure based drug design tools have been steadily evolving and their use in evaluating three dimensional structure and chemical space for ligand binding can be integrated with in silico screens of synthetic chemical libraries to produce early leads. lead optimization and structure activity relationships (sar) can be modeled and synthetic chemistry and chem-informatics can be used to optimize candidates in areas of pk/pd, drug metabolism and toxicology, essential to building safe medicines. it should be obvious that antibacterial development in the future will require a wide range of biological, chemical, computational and pharmacological disciplines working towards a common goal to progress st century medicines. while the pharmaceutical industry used to contain this expertise, we need to find a way to fund such initiatives that also removes the shareholders and corporate return on investment metrics from the equation. while carb-x and other related initiatives are critical in the clinical development of antibacterials via cost sharing with startup companies, the real bottleneck occurs much earlier in the early discovery process. if years of effort has not yielded a novel class of gn therapeutic, one might imagine the failure rate for any new initiative might be close to %, not a viable strategy for "bean" counters. we have to ask "how much are we willing to invest in order to get one new antibiotic to the clinic?" we already know many ways not to make an antibiotic, perhaps now we can find more creative and imaginative ways to produce them. perhaps if we present the antibiotic crisis as on the scale of the covid- pandemic, maybe it would engender greater attention and resource allocation. funding: this research received on external funding. the author declares on conflict of interest. who will develop new antibacterial agents? attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in : a population-level modelling analysis antibacterial drug discovery: some assembly required challenges of antibacterial discovery revisited drugs for bad bugs: confronting the challenges of antibacterial discovery eskapeing the labyrinth of antibacterial discovery challenges of antibacterial discovery time for a change: addressing r&d and commercialization challenges for antibacterials towards a new business models for r&d for novel antibiotics. drug resist a comprehensive regulatory framework to address the unmet need for new antibacterial treatments impediments to discovery of new antimicrobials with new modes of action hidden antibiotics: where to uncover? antibiotics: past, present and future reducing the bottleneck in discovery of novel antibiotics a. β-lactams and β-lactamase inhibitors: an overview. cold spring harb critical analysis of antibacterial agents in clinical development structure-based design of hepatitis c virus inhibitors experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings predictive compound accumulation rules yield a broad-spectrum antibiotic the challenge of converting gram-positive-only compounds into broad-spectrum antibiotics innovative therapies for acute bacterial skin and skin-structure infections (absssi) caused by methicillin-resistant staphylococcus aureus: advances in phase i and ii trials mechanisms of antibiotic resistance collateral damage of using colistin in hospitalized patients on emergence of colistin-resistant escherichia coli and klebsiella pneumoniae colonization and infection coexistence of plasmid-mediated mcr- and bla ndm- genes in a klebsiella pneumoniae clinical strain in vietnam a review of its properties, function, and use in critical care report to the president on combating antibiotic resistance bacterial resistance to leucyl-trna synthetase inhibitor gsk develops during treatment of complicated urinary tract infections a new antibiotic kills pathogens without detectable resistance new approaches to antimicrobial discovery antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of helicobacter pylori and selected anaerobic bacteria and parasites, and campylobacter jejuni synthesis and antimicrobial evaluation of amixicile-based inhibitors of the pyruvate-ferredoxin oxidoreductases of anaerobic bacteria and epsilonproteobacteria amixicile: a novel inhibitor of pyruvate: ferredoxin oxi-doreductase shows efficacy against clostridium difficile in a mouse infection model resistance formation to nitro drugs in giardia lamblia: no common markers identified by comparative proteomics helicobacter pylori porcdab and oordabc genes encode distinct pyruvate:flavodoxin and -oxoglutarate:acceptor oxidoreductases which mediate electron transport to nadp systematic identification of selective essential genes in helicobacter pylori by genome prioritization and allelic replacement mutagenesis antibactr: dynamic antibacterial-drug-target ranking integrating comparative genomics, structural analysis and experimental annotation approaches to the structure-based design of antivirulence drugs: therapeutics for the post-antibiotic era design, synthesis biological and evaluation of novel aryldiketo acids with enhanced antibacterial activity against multidrug resistant bacterial strains rational design of drug-like compounds targeting mycobacterium marinum melf protein postgenomic strategies in antibacterial drug discovery antibacterial drug discovery in the resistance era antibacterial discovery and development-the failure of success? antibiotic discovery: where have we come from, where do we go? antibiotics an overview of new antitubercular drugs, drug candidates, and their targets from discovery to its use for pulmonary multidrug-resistant tuberculosis (mdr-tb) ridinilazole: a novel antimicrobial for clostridium difficile infection enhanced preservation of the human intestinal microbiota by ridinilazole, a novel clostridium difficile-targeting antibacterial, compared to vancomycin countering gram-negative antibiotic resistance: recent progress in disrupting the outer membrane with novel therapeutics a novel inhibitor of the lolcde abc transporter essential for lipoprotein trafficking in gram-negative bacteria monoclonal antibody targeting the β-barrel assembly machine of escherichia coli is bactericidal disrupting gram-negative bacterial outer membrane biosynthesis through inhibition of the lipopolysaccharide transporter msba the small molecule nitazoxanide selectively disrupts bam-mediated folding of the outer membrane usher protein thinking outside the bug: molecular targets and strategies to overcome antibiotic resistance defeating antibiotic-resistant bacteria: exploring alternative therapies for a post-antibiotic era drug repurposing for the treatment of bacterial and fungal infections repurposing auranofin as a clostridioides difficile therapeutic key: cord- -u fdicgz authors: majumder, joydeb; minko, tamara title: targeted nanotherapeutics for respiratory diseases: cancer, fibrosis, and coronavirus date: - - journal: adv ther (weinh) doi: . /adtp. sha: doc_id: cord_uid: u fdicgz systemic delivery of therapeutics for treatment of lung diseases has several limitations including poor organ distribution of delivered payload with relatively low accumulation of active substances in the lungs and severe adverse side effects. in contrast, nanocarrier based therapeutics provide a broad range of opportunities due to their ability to encapsulate substances with different aqueous solubility, transport distinct types of cargo, target therapeutics specifically to the deceased organ, cell, or cellular organelle limiting adverse side effects and increasing the efficacy of therapy. moreover, many nanotherapeutics can be delivered by inhalation locally to the lungs avoiding systemic circulation. in addition, nanoscale based delivery systems can be multifunctional, simultaneously carrying out several tasks including diagnostics, treatment and suppression of cellular resistance to the treatment. nanoscale delivery systems improve the clinical efficacy of conventional therapeutics allowing new approaches for the treatment of respiratory diseases which are difficult to treat or possess intrinsic or acquired resistance to treatment. the present review summarizes recent advances in the development of nanocarrier based therapeutics for local and targeted delivery of drugs, nucleic acids and imaging agents for diagnostics and treatment of various diseases such as cancer, cystic fibrosis, and coronavirus. lung diseases are one of the main causes of death among both men and women worldwide. the mortality rates for lung diseases have been increasing by each year. [ , ] therefore, methods of developing new therapeutic solutions as well as improving the current therapies for the common lung diseases such as asthma, cystic fibrosis, chronic obstructive pulmonary disease, lung cancer, and coronavirus infections remain the main focus in the fields of targeted drug delivery. the widely utilized conventional drug delivery methods usually induce adverse side effects. [ , ] recent development of nanoscale-based systems opens a door for a doi: . /adtp. better delivery of therapeutics by addressing the limitations of conventional therapy. nanocarrier-based drug delivery systems can increase bioavailability of poorly water-soluble therapeutics and address other barriers and shortcomings of traditional drugs. [ ] while majority of the marketed drugs are poorly water soluble, which limits their administration at high doses, [ , ] nanoscale-based drug delivery systems were found to improve solubility and increase therapeutic efficacy of free non-bound drugs. [ , ] similarly, biomacromolecular therapeutics such as nucleic acids (dna, small interfering rna, antisense oligonucleotides, etc.) are usually degraded in the biological fluids and difficult to deliver at their target site. [ , ] however, delivery of nucleic acids via nanocarrier based systems increased their stability and concentration at the target site. [ ] [ ] [ ] [ ] [ ] moreover, nanoscale drug delivery systems can be administered via different routes, such as intravenous, [ , ] oral, [ , ] and inhalation [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] routes. furthermore, nanoscale delivery systems are less toxic and immunogenic than the traditional viral vector-based gene delivery systems. [ ] [ ] [ ] because of such handful advantages, researchers around the world have been applied significant efforts in recent years to develop various nanosized carriers for targeted delivery of therapeutics. over the decade, several nanocarrierbased therapeutics were also approved by fda for clinical application. [ , ] thus, the recent development of wide spectrum nanoscale systems introduced a new way in diagnosis, treatment and prevention of diseases. previously, we formulated main requirements and basic concept of effective drug and nucleic acid delivery systems for effective treatment of diseases including cancer. [ ] to enhance treatment effectiveness, the advanced system should provide for a ) protected delivery of active components in order to prevent their degradation during its journey to targeted cells; ) targeted transport specifically to the site of action with the aim of limiting adverse side effects of treatment upon healthy organs, tissues, and cells; ) modulation of pump drug resistance with the purpose to prevent drug efflux from the diseased cells; ) suppression of nonpump resistance in order to overcome other resistance mechanisms non related to drug efflux pumps; and ) controlled release of active components in a predefined desired manner. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] these main requirements concretized for cancer treatment and general composition of advanced proapoptotic drug delivery system are shown in figure . in this review, we summarize recent reports on the development of various nanotherapeutics including nanocarrierbased drugs and nucleic acids for the treatment of lung diseases with emphasis on lung cancer, idiopathic pulmonary, and cystic fibrosis and recent coronavirus infections. [ , , , , [ ] [ ] [ ] [ ] [ ] targeted drug delivery of therapeutics is aimed at transporting of the administered active component predominately at a desired site of action limiting its accumulation in healthy organs and tissues. this goal can be achieved by both passive and active targeting of drugs. [ , [ ] [ ] [ ] in passive targeting, high molecular weight substances are accumulated in targeted cells because of specific pathophysiological characteristics of the diseased cells and surrounding microenvironment. for instance, passive targeting to solid tumors is dependent on the enhanced permeability of vessels that supply blood, oxygen, and nutrients to the tumor and limited lymphatic drainage from the tumor environment. this phenomenon was termed as the enhanced permeability and retention (epr) effect. [ ] a schematic representation of the epr effect is displayed in figure . however, the efficiency of passive targeting is limited and the conditions responsible for the epr effect is not attributed for all diseased tissues which often develop specific mechanisms for resisting traditional treatment approaches. [ ] on the other hand, active targeting is achieved mainly by decorating the surface of the nanocarriers with targeting moieties such as antibodies, [ ] proteins, [ ] peptides, [ ] aptamers, [ ] [ ] [ ] lectins, carbohydrates, and glycoproteins, [ ] small molecules, [ ] [ ] [ ] [ ] [ ] etc. which have strong affinity to their cellular binding partners such as tumor figure . a schematic representation of the enhanced permeability and retention (epr) effect. the leaky vasculature and dysfunctional lymphatics of tumors allow the preferential accumulation and retention of high molecular weight nanoparticles in solid tumors. reproduced with permission. [ ] copyright , hindawi. antigens, cell surface receptors, tumor vasculature. [ ] such active targeting nanocarriers are designed to increase the accumulation of therapeutics at their site of actions with limiting exposure to other healthy organs thereby reducing the risk of adverse side effects. [ ] [ ] [ ] [ ] [ ] [ ] nanocarrier-based passive and active targeting of cancerous cells and their advantages are presented in figure . several receptor proteins (such as receptors specific for folate, lhrh, transferrin, etc.) are often overexpressed in tumor cells. a schematic demonstration of various overexpressed cell surface receptors in lung under pathological conditions is . passive and active targeting cancer cells. passive targeting depends on the enhance permeability and retention (epr) effect. active targeting may be achieved by enabling the uptake of nanotherapeutics by receptor mediated endocytosis. for instance, nanoparticles may be decorated with a ligand targeting receptors (or other molecules) overexpressed on the plasma membrane of cancer cells. outlined in figure . western blot analysis revealed the presence of the targeting receptor proteins such as folate receptor alpha (fra), epidermal growth factor receptor (egfr), integrin etc. in different lung cancer cells as shown in the right panel of figure . folic acid, transferrin etc. were widely explored as affinity ligands for targeting many tumors cells. [ ] [ ] [ ] for example, recently, researcher developed a gold nanocarrier loaded with drug aurimmune cyt- and functionalized with tumor necrosis factor alpha (tnf-) using polyethylene glycol (peg) linker for treating lung cancer. tnf-was used as both targeting and therapeutic agent. [ ] bind- is another nanocarrier based therapeutics which was investigated in phase ii clinical trial for patients with non-small cell lung cancer. [ ] bind- is a polylactic acid (pla) based nanocarrier system wherein the anticancer drug docetaxel was entrapped. the surface of this system was coated with peg and targeting ligands against prostate-specific membrane antigen (psma) which is usually abundant in prostate cancer cells as well as in the non-prostate cancers, such as nsclc. [ ] figure a represented a schematic illustration of bind- composed of a hydrophobic pla polymeric core and a hydrophilic peg corona decorated with small-molecule targeting ligands, and an encapsulated anticancer drug docetaxel. [ ] the ct scans acquired from a patient suffering from a primary cholangiocarcinoma revealed regression of the lung metastases after two cycles of bind- treatment ( figure b ). [ ] these results indicated that bind- was clinically active and nontoxic for nsclc. lung cancer is the prime cause of cancer death worldwide. chemotherapy, radiation therapy and their combination with surgery are the current therapeutic options for all type of lung cancers. [ ] in most cases of lung cancer chemotherapy, drugs are often administered intravenously, and they can circulate throughout the body affecting both normal and cancer cells. over the last two decades, various nanoparticles (such as metal-based nanoparticles, lipid-based nanoparticles, polymeric nanoparticles, etc.) were explored for targeted therapeutic delivery and diagnostic applications (or combination of both in one theranostic system). [ , , ] however, the translational application of naked metallic nanoparticles (as imaging contrast agents) is limited due to their toxicity. [ , ] therefore, lipid and polymer-based nanoparticles have received more attention of the researchers for drug delivery and theranostics applications. in this review, we will summarize recent reports on the development of lipid and polymer based nanocarriers for targeted delivery of drugs and nucleic acids for the treatment of lung cancer. drugs can be either encapsulated physically or bonded chemically through linker with the nanocarriers and can be delivered to almost all organs because of their small size and ease of penetration of many biological barriers. over the years, a broad range of nanocarriers were evaluated for targeted delivery of several anticancer drugs for lung cancers. few examples of lipid-based nanoparticles as well as polymeric nanoparticles for targeted drug delivery applications have been summarized below. lipid-based nanoparticles: lipid-based nanoparticles possess unique benefits necessary for drug delivery application. lipidbased nanoparticles have an advantage of being the least toxic among other nanocarriers with a substantial progress in the fields of drug and nucleic acid delivery using lipid-based nanoassemblies. [ ] here, we have summarized recent reports of several lipid-based nanosystems including liposome, nanostructured lipid carriers (nlcs), and micelles and their application as targeted drug delivery systems. liposomes: liposome is a type of lipid-based nanoparticle with a bilayer structure comprised of phospholipids, phosphatidylcholine, cholesterol, etc. (figure ). liposome can incorporate lipid-soluble drugs in its lipid bilayer structure as well as encapsulate water-soluble drugs in its inner aqueous core. [ ] liposomes were widely investigated for drug delivery applications because of their hydrophobic and hydrophilic drug loading capability as well as their biocompatibility properties. [ , , , , , [ ] [ ] [ ] [ ] [ ] doxil is the first liposomal drug which got fda approval as an anticancer nanotherapeutics in . [ ] over the last two decades, researchers have explored various liposomal formulations (such as temperature-sensitive liposomes, [ ] cationic liposomes, [ ] and archaeosomes, [ ] etc.) for drug and gene delivery applications ( figure ). for example, song et al. developed a multifunctional liposome based complex system for in vivo treatment of nsclc. the authors loaded an anticancer drug epirubicin inside the aqueous core of the liposome and an anti-metastatic drug honokiol into the lipid bilayer of the formulation. the surface of the liposome was further conjugated with a somatostatin targeting peptide octreotide which can binds to somatostatin receptors overexpressed in cancer microenvironment and facilitate targeted drug delivery. this complex liposomal system showed improved in vivo anticancer activity. [ ] cisplatin is one of the widely used drugs for the treatment of lung cancer. however, it showed nephrotoxicity in patient with high doses. [ ] devarajan et al. reported a liposomal formulation of cisplatin (namely the impact of the nanotherapeutic bind- , a polymeric nanodrug carrier, encapsulating docetaxel, and targeting prostate specific membrane antigen on human drug metastases from a primary cholangiocarcinoma. a) a schematic representation of bind- composed of a biodegradable and hydrophobic pla polymeric core and a hydrophilic peg corona decorated with small-molecule targeting ligands, and an encapsulated anticancer drug (docetaxel). adapted with permission. [ ] copyright , dovepress. b) representative axial images from contrast-enhanced ct scans obtained from a patient with lung metastases at baseline and at day after two treatment cycles of bind- . red circles indicate locations of metastatic lesions observed in the baseline ct scan. adapted with permission. [ ] copyright , american association for the advancement of science. lipoplatin) which showed less nephrotoxicity when compared with free non-bound cisplatin. [ ] paclitaxel is another anticancer drug which was widely used for the treatment of various cancers including lung cancer. in a phase i clinical trial in nsclc patients, a liposomal formulation of paclitaxel showed enhanced therapeutic efficacy. [ ] in a recent study, researchers prepared a liposomal formulation containing both cisplatin and paclitaxel for the treatment of lung cancer. in a phase iii trial, this liposomal formulation displayed improved therapeutic activity and reduced nephrotoxicity in nsclc patients. [ ] in the last decade, researchers have developed a special type of liposomesarchaeosomes, which are made with ether lipids unique to the domain of archaeobacteria. [ ] achaean-type lipids consist of archaeol (diether) and/or caldarchaeol (tetraether) core structures. the membrane of this type of liposomes is made with both conventional phospholipids and bipolar lipids ( figure ). archaeosomes can be made using standard procedures used for liposome preparation including a sonication of hydrated film, extrusion, and detergent dialysis. in contrast to conventional liposomes, archaeosomes remain stable at acidic ph, high temperature and pressure, resist oxidative degradation and can be sterilized by autoclaving. a stability of archaeosomes at a wide physiological (or lower) temperature range opens a possibility of encapsulating thermally stable compounds. however, the uptake of archaeosomes by phagocytic cells can be up to -fold greater when compared with conventional liposomes, which may be considered as their disadvantage. nanostructured lipid carriers: nlcs are another widely used lipid-based nanocarrier for targeted drug and gene delivery applications. nlcs are composed of biodegradable and biocompatible lipids and usually prepared by mixing a liquid lipid mixture containing unsaturated lipid or oils to a solid lipid ( figure ). among the lipid-based nanocarrier, nlcs have the advantages of ease of manufacturing processes, drug protection during the storage, low toxicity, biodegradation, etc. these and other benefits of nlcs made them a promising therapeutic delivery system in recent years. [ , , , ] here, we have summarized few examples of nlc-based therapeutic nanomedicines for drug and gene delivery. for instance, guo et al. [ ] prepared a dual drug paclitaxel (ptx) and -demethylnobiletin (dmn) loaded and cetuximab (cet) functionalized cet-ptx-dmn-nlcs for the combination therapy of lung cancer. the treatment of the cet-ptx-dmn-nlc inhibited the growth of the lung cancer cells as compared to the single ptc-nlc and dmn-nlc treatments. the authors also observed remarkable inhibition of in vivo lung tumor for the treatment of this dual drug containing cet-ptx-dmn-nlc. [ ] wang et al. [ ] developed a dual drug loaded nlc system for the treatment of lung cancer. this dual drug loaded ptx/dox nlc displayed × higher activity as compared to that of single drug ptc-nlc and dox-nlc treatment as revealed in cytotoxicity assay in ncl-h cells. also, in vivo study of this dual drug nlc on a non-small cell lung cancer mice model showed improved the anticancer activity. [ ] micelles: micelles consist of lipid molecules arranged in a spherical form in polar solvents (e.g., water). polar groups of lipids form an outer shell of the nanoparticles in polar solvent system while lipid hydrophobic tails create an inner core of micelles. in contrast to liposomes, micelles usually have a single hydrocarbon chain ( figure ). in , kim et al. prepared nanosized micelles (genexol-pm) loaded with the anticancer drug paclitaxel for the treatment of nsclc. [ ] this nanotherapeutic was found to deliver higher paclitaxel dose with reduced drug toxicity as well as exhibited significant antitumor activity in the treatment of advanced nsclc. a series of nanocarrier systems which included liposomes, micelles, quantum dots, mesoporous silica nanoparticles, dendrimers, and peg polymers were prepared and examined in our laboratory in order to find out the suitable nanocarrier for local and inhalation delivery of anticancer drugs to the lungs. we investigated organ distribution and retention of all these nanocarriers in the lung and observed higher accumulation of liposomes and micelles based nanocarriers in lungs as compared to that of mesoporous silica nanoparticles, quantum dots, and dendrimers. [ ] we found a significant enhancement of anticancer activity of doxorubicin when it was delivered to mice bearing lung tumor by inhalation by liposome-based system. this study revealed that lipid-based nanocarriers such as liposomes with higher accumulation of drug in lungs and longer retention time were more suitable and effective than non-lipidbased nanocarriers in treating lung cancer by inhalation. [ ] polymeric nanoparticles: polymeric nanoparticles are usually prepared by self-assembly of various block-copolymers with alternate hydrophobic unit between blocks. various biodegradable polymers such as poly (lactic-co-glycolic) acid (plga), polycaprolactone, poly (lactic acid) (pla), chitosan etc. were widely used for the preparation of polymeric nanoparticles mainly because of their biocompatibility and controlled release properties. while core-shell of such polymeric nanoparticles can encapsulate hydrophobic drugs, the surface of the polymeric nanoparticles can be modified for receptor targeted drug delivery. [ , ] in the past two decades, many polymeric nanoparticles have been investigated for treatment of different diseases including lung cancers and enhancing efficacy of anticancer drugs. [ ] in a recent report, hu et al. [ ] developed paclitaxel encapsulated polycaprolactone/poly (ethylene glycol)/polycaprolactone nanoparticles for the combined treatment of lung cancer with chronomodulated chemotherapy. this combination treatment showed better inhibition of tumor growth in vivo. wang et al. [ ] developed a new strategy for delivering drug loaded polymeric nanoparticles at the disease site using mesenchymal stem cells (msc) as carrier. docetaxel was encapsulated in the nanoparticles and tested in vivo. inhibition of the tumor growth was observed in the animal's experiment, which also revealed the translocation of nanoparticles from msc to cancer cells. researchers developed peg modified and taxane encapsulated polylactic acid figure . glutathione stimuli responsive organic plga-ss-peg nanocarriers for targeted delivery of anticancer agent homoharringtonine (hht) to lung cancer cells. a) core-shell structured nanoparticles were synthesized using the solvent evaporation approach of oil-in-water. the plga nanomedicine release was triggered by reduced glutathione (gsh) overexpressed in tumor cells after receptor mediated endocytosis. b) in vivo antitumor efficacy of plga nanomedicine. treatment with plga-ss-peg nanoparticles reduced the tumor volume significantly when compared with non-treated and treated with free non-bound hht groups. adapted with permission. [ ] copyright , springer. nanoparticles for lung cancer treatment. the authors observed significant improvement of the efficacy of chemoradiation therapy in an a lung tumor xenograft model. [ ] in another report, investigators developed a polymeric nanoparticle system comprised of block copolymers of peg and polylactic acid and encapsulated paclitaxel and cisplatin for treatment of lung cancer. [ ] tseng et al. prepared gelatin based polymeric nanoparticles conjugated with biotinylated egf (begf) motif for egfrtargeted drug delivery. [ ] the authors observed enhanced cellular uptake of this polymeric nanoparticle in egfr overexpressing cancer cell lines such as lung cancer cells. zhang et al. [ ] studied glutathione stimuli responsive organic plga-ss-peg nanocarriers for targeted delivery of anticancer agent homoharringtonine (hht) to lung cancer cells. a relatively high level of glutathione reduced (gsh) in cancer cells caused disulfide-bond (ss) breakage in a reductive manner and release of hht inside cancer cells. this nanocarrier showed a reasonable biocompatibility and was further decorated with an epidermal growth factor receptor (egfr) aptamer as a targeting moiety. after endocytosis of this nanosystem by lung cancer cells, the high level of glutathione in tumor cells stimulated the release of the loaded drug. finally, this multifunctional and stimuli responsive nanocomplex inhibited the growth of human lung cancer cells and displayed better therapeutic efficacy when compared with the free non-bound anticancer agent (figure ) . combined lipid-polymer nanoparticles: in addition of nanocarriers prepared mainly with lipids or polymers, complex anisotropic nanoparticles containing lipid and polymeric structures (as well as polymers with different properties) were prepared. [ ] [ ] [ ] [ ] [ ] [ ] as pierre-gilles de gennes pointed in his nobel prize lecture, [ ] similarly to the ancient roman god of gates janus who was portrayed with two faces-one facing the past, and one facing the future, janus particles also have two distinct parts with antagonistic properties (figure ). one such janus structure with two faces (lipid and polymeric) was tested in our laboratory for inhalation lung delivery of a mixture of lipoand hydrophilic drugs namely curcumin and doxorubicin. [ ] these janus particles were synthesized from binary mixture of biodegradable and biocompatible materials and evaluated for cytotoxicity and genotoxicity. the inhibition of lung tumor growth by the combination treatment was significantly higher when compared with either free drugs or nanoparticles containing only one drug. this study showed that such janus particle could be explored for the simultaneous co-delivery of hydrophilic and hydrophobic drugs. carbon nanomaterials: carbon nanomaterials are a new class of nanosized materials comprised of sp hybridized carbon atoms with hexagonal structure. common carbon nanomaterials include d fullerenes, d carbon nanotubes (cnts), and d graphene such as graphene oxide. [ ] among the carbon figure . suppression of lung tumor growth in mice treated by inhalation with janus nanoparticles containing anticancer drug(s). a) ancient roman god of gates janus who was portrayed with two faces (photo by a. kokorin on behance). b) representative optical, c) scanning electron and d) fluorescence microscope images of anisotropic biodegradable biphasic polymer/lipid janus nanoparticles. polymeric phase of nanoparticles was labeled with fitc (green fluorescence); lipid phase was labelled with dir (red fluorescence). e) representative optical and f) magnetic resonance images of untreated (control) and treated mice four weeks after tumor instillation. g) changes in lung tumor volume after beginning of the treatment with nanoparticles containing doxorubicin (dox), curcumin (cur), and both drugs. mice were treated twice per week. means ± sd are shown. reproduced with permission. [ ] copyright , acs publications. nanomaterials, cnts have received significant attention in the past two decades for their high surface area, biocompatibility and drug-loading capacity which made them suitable for wide range of applications such as drug delivery, tissue engineering, biosensors, cosmetic products etc. [ , ] . there are mainly three types cnts namely single wall-, double wall-and multi wall-carbon nanotubes with diameter up to nm and lengths up to microns size. [ , ] surface of these cnts can be functionalized with hydrophobic and hydrophilic drugs for their targeted delivery applications. carbon nanomaterials such as cnts were explored as an attractive systems in targeted drug delivery application. in , kim et al. developed peg-coated carbon nanotube system loaded with small molecule bcl- inhibitor abt- for its targeted delivery to lung cancer cells. [ ] the authors investigated cellular uptake, apoptosis, and cytotoxicity this peg-cnt-abt nanotube system in lung cancer a cells and observed bcl- -mediated apoptosis of lung cancer cells. the peg-cnt-abt system also excreted improved cytotoxic activity in a cells when compared with treatment by free non-bound abt . the drug loaded nanotubes represented an effective system for inducing bcl- -mediated apoptosis in lung cancer cells. in , cirillo et al. reported a ph-responsive nanohybrid system comprised of multi-walled carbon nanotubes and chitosan for delivery of methotrexate to lung cancer cells. [ ] this chitosan coated cs-mwcnt nanohybrid system displayed its ph-responsive behavior and showed faster and higher release of the drug methotrexate in acidic (ph . ) versus neutral (ph . ) environments. such a nanohybrid system showed reduced drug toxicity in normal lung mrc- cells while it exerted anticancer activity in lung cancer h cells. mesoporous silica nanoparticles: nanosized silica particles also known as mesoporous silica nanoparticles (msns) have been investigated in the past two decades for various drug and gene delivery application. because of large pore size, high surface area, good chemical stability, biocompatibility, and ease of surface modification with targeting ligands, msns based nanomaterials were extensively studied for various therapeutic delivery applications. [ , , , , ] for example, wang et al. designed a nanosized drug delivery system containing anticancer drug paclitaxel into the core-shell of mesoporous silica nanoparticle (pac-csmsn) for the treatment of lung cancer. [ ] this csmsns formulation improved the adsorption of the poorly water-soluble drug paclitaxel. the authors found pac-csmsn system was more effective in promoting cell apoptosis in a lung cancer cells than the free drug. this pac-csmsn system was administered for three consecutive days in animals and no indication of inflammation was observed in the lung biopsy. all these results indicated that such pac-csmsn system has the potential for inhalation delivery of paclitaxel for the treatment of lung cancer. , jing-hua sun et al. prepared another msns system for codelivery of a photosensitizer chlorin e (ce ) and a drug doxorubicin (dox) for both photodynamic therapy and chemotherapy of lung cancer. [ ] the anticancer drug doxorubicin was encapsulated into the pores of msns system while ce was conjugated with the msns through covalent bonding. treatment with these dox@msns-ce hybrid nanoparticles increased the level of cellular reactive oxygen species and exerted synergistic therapeutic effect in lung cancer a cells when compared with treatment by each individual component. gold nanoparticles: gold nanoparticles (aunps) are one of the extensively used inorganic nanocarriers for various biomedical applications including drug and gene delivery. because of high atomic number, stable nature and surface plasmon resonance properties, aunps can serve as stable contrast agents for photothermal therapy and medical imaging. [ , ] moreover, aunps possess unique physiochemical characteristics such as high biocompatibility and low-toxicity as well as aunps are non-immunogenic which made gold nanoparticles as an attractive nanocarriers for various biomedical applications. [ ] [ ] [ ] in , qian et al. conjugated cetuximab (c ), a targeting agent for epidermal growth factor receptor (egfr) with aunps for the treatment of egfr positive non-small cell lung cancer (nsclc). [ ] this c -aunps inhibited proliferation and migration of a cells and accelerated apoptosis in a cells as compared to treatment with free c alone. the activity of c -aunps was higher in a cells with higher egfr expression than in h cells with low egfr expression. treatment of nude mice bearing tumor xenografts with c -aunps showed significant suppression of tumor size. such egfrtargeted aunps system can be a promising strategy for targeted delivery of therapeutics in egfr positive nsclc cells. in , ramalingam et al. conjugated doxorubicin on the surface of gold nanoparticles through polyvinylpyrrolidone linker for the treatment of human lung cancer cells. [ ] these dox-pvp-au nanoparticles inhibited the growth of human lung cancer cells more effectively than both the pvp-aunps and free drug. treatment with these nanoparticles induced early and late apoptosis as well as upregulated expression of tumor suppressor genes in the human lung cancer cells. this dox-pvp-au nanoparticle system represents a promising drug delivery approach for lung cancer therapy. cell based drug carriers: biocompatibility, biodegradability, and cytotoxicity of synthetic nanocarriers represent a substantial problem. moreover, exogenous carriers potentially may induce immune responses. consequently, drug carriers prepared from human live cells or their derivatives attract a considerable attention in recent years. such carriers demonstrate native targeting mechanisms and controlled release of the encapsulated drug molecules. several types of human cells have been considered for a targeted drug delivery for treatment of cancers and variety of other pathological conditions, such as cardiovascular and inflammatory diseases. [ , ] major characteristics of different cell types used for drug delivery are presenting in the figure . erythrocytes are usually used for a systemic drug delivery and do not possess intrinsic tropism. in contrast, platelets, neutrophils, adipose cells, macrophages, and stem cells can be used for targeted delivery of therapeutics because of their native tropism to tumors, circulating tumor cells, sites with inflammation, and hypoxic conditions as well as microorganisms. [ ] a strategy of drug loading into human cells used for therapeutic delivery is selected based on the nature and properties of drugs, loading capacity and release mechanisms. therapeutic agents can be loaded into cellular cytoplasm of carrier cells, attached to their surface by a membrane insertion, nonspecific noncovalent or targeted interaction as well as covalent coupling methods. the release of payload may be continuous (e.g., after slow hydrolysis of a prodrug and exocytosis) or triggered by various internal in vivo signals (glucose, hormones, cytokines, and other biomolecules, ph, and changes in cell shape) or external stimuli (light, ultrasound, magnetic field, temperature, etc.). [ ] a comprehensive list of cell based carriers designed for intraperitoneal, intratracheal, intravenous, subcutaneous and left anterior descending delivery of therapeutics developed during the last decade is presented in the open access review by lutz et al. [ ] gene therapy has become a promising therapeutic option in recent time for lung cancer. several different nanocarriers (such as dendrimer, micelles, gold nanoparticles, liposomes, lipid nanoparticles, auroliposome etc.) were explored as carriers of nucleic acids with effective results. [ , , , , , , , , , , , [ ] [ ] [ ] [ ] examples of carriers used for the delivery of nucleic acids are presented in figure . nucleic acids used as gene therapeutics are negatively charged because they are composed of few, several or many nucleotides with phosphate backbones carried one negative charge per residue. [ ] consequently, they often delivered as conjugates formed with positively charged (cationic) carriers. such conjugation not only protects gene material from degradation in the blood stream and improves pharmacokinetics of the resulting complex, but also neutralizes positive charge of highly toxic anionic carriers limiting their cyto-and genotoxicity. [ ] however, an encapsulation inside nanocarriers or direct conjugation of native or modified nucleic acids via different (preferably cleaved inside targeted cells, e.g. s─s) bonds are also used to form a stable system for an effective gene delivery. small chunks of nucleic acids can be modified to decrease their negative charge and encapsulated inside nanocarriers. in our laboratory, the dna backbone of all bases in antisense oligonucleotides (aso) was pethoxy modified in order to make the entire aso neutral and increase their incorporation efficacy into liposomes. [ ] such modification also enhanced nuclease resistance of aso. liposomal aso were successfully used to suppress pump and nonpump resistance of cancer cells. [ , , , ] it should be stressed, that treatment of cancer with nucleic acids alone (e.g., sirna, antisense oligonucleotides) in most cases demonstrate a pretty limited anticancer effect. however, a combination of anticancer drug(s) with nucleic acids targeted to the drug efflux pumps, antiapoptotic, and other cancer cell defensive proteins/mrnas is expected to substantially enhance anticancer efficacy of both anticancer drugs and nucleic acids. such a concept of advanced proapoptotic anticancer delivery system was first developed and tested in the laboratory of professor minko at rutgers university almost years ago. [ , ] possible structures of such multifunctional nanoparticles are presented in figure . in several such systems, figure . various cell types employed for drug delivery. redrawn from. [ , ] where nucleic acids (sirna and antisense oligonucleotides) were used as suppressors of drug efflux pumps (pump drug resistance) and antiapoptotic cellular defense (non-pump resistance). [ , ] lipid-based nanoparticles: lipid-based nanoparticles such as liposomes, nlcs, and various polymeric nanoparticles are widely used to protect and deliver nucleic acids ( figure ) . few examples of both liposome and nlc based nucleic acid delivery systems are discussed below. liposomes: gopalan et al. prepared dotap/cholesterol based nanocarrier system for direct delivery of tumor suppressive gene at the tumor site. this system was effective and nonimmunogenic. [ ] in an early study, our group prepared a multicomponent liposomal delivery system for improving anticancer activity of doxorubicin against multidrug-resistant human non-small-cell lung cancer cells. this multi-component liposomal system was included doxorubicin as an anticancer drug, antisense oligonucleotide (aso) as a suppressor of pump resistance for mrp mrna and another aso as a suppressor of nonpump resistance for bcl mrna. [ , , , , ] antisense oligonucleotides were p-ethoxy modified to decrease their charge, enhance nuclease resistance, and increase incorporation efficacy into liposomes. we reported successful intracellular delivery of both doxorubicin and asos to lung cancer cells. also, this liposomal treatment increased anticancer efficacy of doxorubicin and inhibited synthesis of both mrp and bcl proteins. this multicomponent liposomal system displayed -fold higher cytotoxicity as compared to both free and liposomal doxorubicin treatment against the resistant lung cancer cells and could be used for the enhancement of anticancer activity of doxorubicin against multidrug-resistant lung cancer cells. in a similar study, we used a complex liposomal drug delivery system containing anticancer drug doxorubicin and both mrp and bcl targeting antisense oligonucleotides for inhalation treatment in lung cancer cells. [ ] while empty liposome, free antisense oligonucleotides and their combination treatment showed almost no influence on viability of lung cancer cells; liposome targeted to both mrp mrna and bcl mrna significantly inhibited the growth of the lung cancer cells. we evaluated this complex liposomal system on an orthotopic murine model of human lung cancer and the results revealed its higher chemotherapeutic efficacy with lower side effects as compared to that observed for individual treatment of each component. [ ] nanostructured lipid arriers: garbuzenko et al. reported a multi-functional nanostructured lipid carrier (lhrh-nlc-sirnas-tax) composed of an anticancer drug paclitaxel (tax), a peptide analog targeted to luteinizing hormone-releasing hormone (lhrh) receptor and a pool of sirnas as inhibitors of different types of egfr-tks. this lhrh-nlc-sirnas-tax nanoparticle system was investigated in various human lung cancer cell lines and in vivo on an orthotopic nsclc mouse model and displayed good organ distribution, stability, solubility, and improved anticancer activity when compared with free individual drugs and non-targeted therapy. [ , , ] han et al. developed a multifunctional blc system for delivery of anticancer drug doxorubicin (dox) and green fluorescent protein plasmid (pegfp) dna as a prototypical nucleic acid in lung cancer cells. the authors prepared dox and pegfp encapsulated nlc and modified its surface with transferrin-targeting motif. transferrin-modified and dox and pegfp co-encapsulated nlc system showed higher in vitro and in vivo transfection of plasmid dna than the other control treatment. these results also indicated that such multifunctional nlc system could be an effective method for both drug and gene delivery for the treatment of lung cancer. [ ] in another work, han et al. prepared an nlc system for delivery of plasmid-containing enhanced green fluorescence protein (pegfp) in lung cancer cells. the authors prepared the pegfp-loaded nlc and decorated its surface with transferrin (tf) targeting ligands. this tf-nlc/pegfp showed higher transfection efficiency as observed in in vitro and in vivo studies than the non-targeted nlc/pegfp-suggesting this nlc system could be a promising vehicle for gene therapy in lung cancer. [ ] polymeric and hybrid nanoparticles: chitosan-based polymeric nanoparticles were widely used for the delivery of nucleic acids. because of ionizable sidechain amino groups in chitosan, it has cationic nature which made chitosan polymer a good vehicle for the delivery of anionic agents such as sirna, dna etc. okamoto et al. developed a chitosan-based nanoparticle for delivery of pcmv-luc gene into the lung cancer cells. [ ] nafee et al. prepared a chitosan based nanocarrier system loaded with an antisense oligonucleotide such as -o-methyl-rna for the treatment of lung cancer. the authors evaluated the inhibitory function of telomerase after treating the nanocarrier in lung cancer cells and observed % reduction of the telomerase activity in a lung cancer cells. these chitosan nanoparticles were safe and effective for the treatment of lung cancer. [ ] dhananjay et al. developed a polymeric nanoparticle system comprised of pei-peg copolymer for the delivery of akt shrna in lung cancer cells. [ ] both service and internally anionic polypropylenimine tetrahexacontaamine (ppi) and polyamidoamine (pamam) dendrimers were successfully used for the delivery of nucleic acids into cancer cells. [ ] [ ] [ ] it was found that dendrimers and additional caging of resulting nanoplexes protected nucleic acids from degradation and effectively delivered genetic material inside cancer cells. delivered sirna demonstrated high intracellular activity and effectively knocked down gene expression and synthesis of targeted proteins. carbon nanomaterials: carbon nanomaterials such as cnts decorated with positively charged polymers were investigated as gene carrier in recent years for specific delivery of nucleic acids. [ , ] podesta et al. prepared amino-functionalized mwcnt system for delivery of sirna and tested it using an animal xenograft and orthotopic breast tumor models. [ ] treatment with this mwnct-sirna complex significantly delayed the growth of tumor and increased the survival of the tumor bearing animals. varkouhi et al. developed mwcnt systems decorated with cationic pei (cnt-pei) and cnt-pyridinium for the delivery of sirna to lung cancer cells. [ ] both these cnts displayed cytotoxicity effect and gene silencing activity in h human lung cancer cells, while non functionalized cnts did not show any such effects. mesoporous silica nanoparticles: mesoporous silica nanoparticles have been investigated as a delivery vehicle of various cargo molecules including nucleic acid therapeutics. for example, dilnawaz et al. developed msn based system for co-delivery of anticancer drug doxorubicin and sirna in lung cancer cells. [ ] this combinational treatment enhanced in vitro cellular uptake, cytotoxic effect in a lung cancer cells. in , song yinxue et al. developed a complex msn system comprised of a polyphenolic drug myricetin (myr), sirna specific to multidrug resistance protein (mrp- ) and a targeting ligand folic acid (fa) in order to improve delivery efficiency of myr in nsclc cells. [ ] this targeted myr-mrp- /msn-fa nanoparticles showed significant cellular uptake and reduced viability of a , nci-h lung cancer cells when compared with free drug and other controls. in vivo results revealed that this system was more effective in suppressing the tumor growth and it might be an attractive therapeutic strategy for the treatment of nsclc. gold nanoparticles: gold nanoparticles possess good biodistribution, physiological stability and low cytotoxicity which made them an attractive vehicle for delivery of various payloads including large biomolecules such as nucleic acids. [ ] over the years, researchers have explored both non-covalent and covalent conjugation of the nucleic acids such as sirna, oligonucleotides etc. on the surface of aunps for their effective transportation to the target cells. for instances, conde et al. developed a peg modified gold nanoparticle system by conjugating of rgd peptide and cmyc sirna on the surface of gold nanoparticles and tested this system on mice bearing cmt/ lung carcinoma tumors. [ ] the authors observed downregulation of the c-myc oncogene and significant inhibition of lung tumor growth after the treatment with si-rna/rgd aunps. recently, an innovative hybrid formulation (so-called auroliposomes) consisting of liposomes loaded with -nm gold nanoparticles (aunps) was developed and used for the sirna delivery ( figure ). it was found that auroliposomes modulated the intracellular uptake and silencing efficacy leading to the enhanced suppression of tumor growth in vivo when compared with conventional liposomes. [ ] nanocarriers have the potential to enhance the diagnosis of diseases. recently, nanobased materials and methods have emerged as novel diagnostic tools for several diseases. over the years, various nanocarriers were explored for the delivery of imaging (or both imaging and therapeutic) agents for diagnosis of many diseases including lung cancer. [ ] [ ] [ ] for example, researchers designed folic acid functionalized dendrimers containing gold nanocarrier as cancer-targeted imaging probes for computed tomography (ct) imaging of lung cancer cells. [ ] ct imaging after nanoparticle uptake revealed the presence of these gold nanocarriers in the lysosomes of lung adenocarcinoma cells. in another study, researchers developed ultrasmall ( . ± . nm) gadolinium containing nanoparticles (so called ultra-small rigid platforms or usrps) for enhancing ultrashort echo time ( ms) proton mri of the lung. [ ] these nanoparticles were prepared using , , , -tetraazacyclododecane- , , , -tetraacetic acid (dota) as a chelator and was delivered by the intratracheal instillation. the authors observed the substantial (> %) enhancement of mri signal in the lungs for almost . h after instilling the solution of the nanoparticles. erten et al. prepared a dextran core-based stealth pegylated liposomes containing anticancer drug doxorubicin, iron oxide as an mri contrast agent and boron dipyrromethene (bodipy) fluorescence stain for imaging theranostics applications. [ ] the authors observed strong ability of these liposomal nanoparticles of enhancing both types of imaging in the in vivo murine model of lewis lung cancer. in another report, lowery et al. labeled a tumor targeted doxorubicin loaded liposomes with alexa fluor for imaging of lung tumor. [ ] an hvggssv peptide with a selective binding to irradiated tumors was used as a targeting moiety in order to deliver the anticancer drug and imaging agent specifically to irradiated tumors limiting their accumulation in the normal tissues. these liposomes ( nm) contained maleimide and amine functionalized peg chains for the conjugation of the cysteine containing peptide and the n-(succinyl)-fluorophore, respectively. doxorubicin in theranostic liposomes was loaded by the ph gradient. the authors studied these fluorophore labeled irradiated tumor targeted liposomes in murine model of lewis lung cancer. they found that such a radiationguided tumor-targeted delivery of liposomes enhanced the delivery of the fluorophore and anticancer drug specifically to irradiated tumors in the lungs, effectively induced cell death and limited cell proliferation within lung tumors finally inducing a delay in tumor growth and destruction of tumor blood vessels and increase of apoptosis in lung tumor cells. hvggssv targeting peptide also increase the accumulation of an entire system in irradiated tumors enhancing imaging quality. cystic fibrosis is an inherited disorder, caused by mutations of the cystic fibrosis transmembrane conductance regulator (cftr) gene. over-production of mucous in the lungs causes airway obstruction resulting in infectious diseases such as cystic fibrosis (cf). [ ] typically, heterogeneous and large molecular weight oligomeric gel-forming mucin glycoprotein are produced in cf. gene therapy is the mainstay therapy to inhibit the mutation of cftr protein. gene therapies involve delivery of sirna, dna etc. into cells to rescue the function of the defective cftr gene. usually, viral and non-viral vectors are employed to transfer correct copies of cftr gene in the effected cells in lungs. [ ] because of small size, nanocarriers have emerged as an effective vehicle for delivery of gene through the mucus barriers. nanoscale carriers can be used as vectors for gene therapy due to their less immunogenic and good gene transport capacity. [ ] nanocarrier based non-viral vectors are easy to prepare as compared to that of viral vectors. [ ] in recent years, researchers attempted to develop various nucleic acid based nanocarrier to affect mutation of cftr gene to change the composition of mucin as well as to minimize the mucin production. [ , ] in an early research, konstan et al. developed a dna nanoparticle for the treatment of cystic fibrosis and observed effective transfer of vector gene. [ ] in order to overcome the mucus barrier, recently, suk et al. prepared a densely peg-coated dna nanoparticle system, which can penetrate extracorporeal human cystic fibrosis to deliver its payload. [ ] this nanocarrier displayed better gene transfer after intranasal administration to mice as compared to other carriers. minko et al. prepared a liposomal--tocopherol (lat) formulation for the treatment of hypoxic lung injury in rats. the authors evaluated antioxidant and antiapoptotic activity of this lat in rats with severe hypoxia and observed significant antihypoxic effects. [ ] it was found that, treatment with lat of rats under severe hypoxic conditions (breathing of % of oxygen within two hours) normalized lung phospholipid composition, inhibited lipid peroxidation, suppressed genes responsible for the development of lung damage and improved breathing pattern. finally, such a treatment two-times decreased the mortality of the animals under severe hypoxic conditions. lately in our lab, a similar liposomal system for inhalation delivery of prostaglandin e (pge ) was developed for treatment of pulmonary fibrosis. [ ] this liposomal system was evaluated for local delivery of pge using a standard bleomycin-induced murine model of idiopathic pulmonary fibrosis. the results revealed that liposomes were accumulated in higher amount in lungs after inhalation delivery when compared with intravenous administration. besides, this inhalation treatment reduced fibrotic injury in the lung tissues. these data probed that the inhalation administration of liposomal form of pge can be an effective therapy for cystic fibrosis in the lungs. to further improve inhalation treatment of idiopathic pulmonary fibrosis (ipf) by liposomal pge , sirnas targeted to major proteins responsible for the lung damage under ipf (mmp , ccl , and hif a) were added to the nlc based nanoparticles containing pge and tested on the similar experimental model of lung fibrosis using inhalation delivery. [ ] this enhanced advance system was more effective in the treatment of ipf when compared with sirna and pge delivered separately. another combination of drugs in one nlc-based nanoparticle system was recently tested for the treatment of lung manifestation of cystic fibrosis (cf). [ ] the system included lumacaftor for the correction of correct p.phe del mutation (the loss of phenylalanine at position ) and cftr potentiator ivacaftor for increasing the open probability of cftr channels. this system was tested in vitro using cf cells and in vivo on homozygote/homozygote bi-transgenic mice with spontaneously developed cf. the system was delivered in vivo by inhalation. the results showed a high efficacy of the proposed treatment of the lung manifestation of cf. wang et al. prepared rapamycin and azithromycin loaded polymeric nanocarrier via nanoprecipitation method. [ ] nanocomposite microparticles (ncmp) were formulated from this nanoparticle for the inhalation delivery of antibiotics in the form of dry powder aerosols. these, nanocomposite microparticles displayed aerosol dispersion characteristics indicating their deposit in the lungs. viral infections in respiratory systems such as in lungs have become a worldwide public health threat in recent years. several emerging positive-stranded rna coronaviruses [ , ] such as severe acute respiratory syndrome coronavirus (sars-cov), [ ] [ ] [ ] middle east respiratory syndrome coronavirus (mers-cov) [ ] etc. not only threatened public health, but also caused international epidemics in the past two decades. recent outbreak of coronavirus infection caused by the severe acute respiratory syndrome-coronavirus- pathogen has seriously threatened public health all over the world. the taxonomic name "severe acute respiratory syndrome coronavirus " (sars-cov- ) given by the international committee on taxonomy of viruses (ictv) became official to refer to this virus strain. on february , the world health organization (who) officially named the "coronavirus disease " as "covid- ". [ ] the genome of sars-cov- is a bp with single-stranded rna (ss-rna). the complete genome sequence of sars-cov- is available in the national center for biotechnology (ncbi) database, with id nc_ . [ , ] covid- is characterized by severe respiratory disease along with mild to high fever, cough, and shortness of breath. covid- has been considered as an emerging disease and on march , the outbreak of this disease has been declared as global pandemic by the who. [ , ] this virus has been found to spread from person to person mainly through respiratory droplets, cough, sneeze, etc. [ ] causing severe acute respiratory distress syndrome (ards). as of september , , this virus has already infected more than thirty million people and caused deaths with billions of people are at risk around the world. [ ] despite repeated outbreaks of sars-cov in and mers-cov in , no potent vaccines and anti-viral drugs are commercially available against these viral infections-mainly due to the fact that the outbreaks of these viruses were rapidly contained and did not reappear. [ ] therefore, there are no effective treatment for the ongoing pandemic of covid- , a close subtype of sars-cov. [ ] because of constant emergence of new viruses including current sars-cov- infection, there is an urgent need for the development of potent and broad-spectrum vaccines and antiviral drugs for effective control of viral diseases. since the first report of sars-cov- infection in late december in , both researchers and clinicians have been attempted clinical trials of several known antiviral drugs, their combination as well as development of vaccine in patients with confirmed covid- disease. this review is mainly focused for summarizing recent developments of nanotherapeutics for respiratory diseases including sars-cov, mers-cov, and covid- . therefore, other types of therapeutic and diagnostic methods such as small molecule antiviral therapeutics, anti-sars-cov- antibody treatments, convalescent plasma therapy etc. [ ] [ ] [ ] [ ] which have been discussed elsewhere are out of the scope of this review. briefly, we will summarize recent innovation of nanobased diagnostics such as nanoparticle-based pcr and anti-body test as well as nanoparticle-based therapeutic approaches for covid- . diagnostic tests are essential not only for monitoring every stage of a disease, but also to identify new patients with that illnessespecially for an outbreak of viral disease. typical diagnosis methods for viral diseases include nucleic acid detection of the viral genome in clinical samples. currently, covid- has been diagnosed by real-time polymerase chain reaction (rt-pcr) test for the detection of viral genome, serological, and immunological assays for the detection of anti-sars-cov- antibody in patient samples as well as chest computed tomography (ct) imaging for screening abnormal observations in chest scans. [ , ] however, most of these methods are laborious and time-consuming processes. therefore, there is an urgent need for developing timeeconomic, easily performed and point-of-contact test for the detection of this virus. because of similar size and shape of sars-cov- virus with the synthetic nanoparticles, researchers have attempted to develop nanoparticle based diagnostic methods for covid- . for examples, huang et al. developed a rapid, easily operated and cost-effective detection of the igm antibody produced in serum sample of patient with covid- . [ ] the authors prepared a colloidal gold nanoparticle-based lateral-flow (aunp-lf) system composed of various low-cost inorganic nanomaterials. the aunp-lf strip was developed for the sample test by coating an analytical membrane with the sars-cov- nucleoprotein followed by conjugating anti-human igm antibody. this method can detect the sars-cov- virus in min using only µl of serum sample of the patient. the authors have figure . schematic representation for the selective naked-eye detection of sars-cov- rna mediated by the suitably designed aso-capped gold nanoparticles. this naked eye detection method involves the isolation of the viral rna from the clinical swab sample from covid- patient and then incubation of the viral rna samples with aso capped gold nanoparticles for min. at the end, rnase h is added in the viral composite of aso-capped gold nanoparticles and the resulting mixture is incubated for min at c to get the visual precipitate. reproduced with permission. [ ] copyright , acs publications. evaluated the specificity of this detection method against the results of widely used tr-pcr's test. this aunp-lf assay has a great potential for large-scale and fast detection of covid- disease specially during this pandemic period. [ ] in another report, moitra et al. developed gold nanoparticle based colorimetric assay for naked-eye detection of sars-cov- virus present in patient samples. [ ] the authors decorated the gold nanoparticles (aunps) with thiol-modified antisense oligonucleotides (asos) which are specific for n-gene (nucleocapsid phosphoprotein) of sars-cov- virus. the use of rnaseh in this detection helps to cleave the rna-dna hybrid resulting in the visually detectable precipitation from the experimental solution. this aunp system can detect the presence of sars-cov- stain in the isolated rna samples within min. thus, this method can be a very promising for visual detection of covid- positive patient without the use of typical instrumental procedures as outlined in figure . [ ] there are no clinically approved therapeutics by the u.s. food and drug administration (fda) to prevent or treat the covid- disease. however, clinical trials of many known antiviral drugs are ongoing in patient with confirmed covid- disease. [ , ] for instance, a newly developed antiviral drug remdesivir previously effective against ebola virus diseases which interferes with viral rna polymerase (rdrp) and arrests viral replication [ ] was repurposed for the treatment of covid- infections. [ ] human trials showed promising clinical improvements in ≈ % of patients. [ ] recent studies showed that the sars-cov- virus has similar size range ( - nm) and spherical shape like the synthetic nanoparticles. [ ] therefore, nanosized spherical therapeutics can be promising to detect and neutralize coronaviruses as previously evaluated for sars-cov, mers-cov etc. various nanoparticle based therapeutic approaches have been discussed here for mers-cov, sars-cov, and covid- diseases. for instance, huang et al. prepared gold nanorod complex of heptad repeat (hr ) peptide inhibitors for middle east respiratory syndrome coronavirus (mers-cov) disease. [ ] this gold nanorod complex was biocompatible and metabolically stable and displayed -fold higher inhibition of membrane fusion between host cells and mers-cov via hr /hr -mechanism as compared to that of the free inhibitor treatment. this the gold nanorod based anti-viral system showed a great promise in treating mers-cov infection. lin et al. prepared a virus-like hollow nanoparticle comprised of biodegradable polymer and a viral antigen along with an adjuvant. [ ] this plasmid like nanoparticle was capable of delivering of both antigens and stimulator of interferon genes agonist adjuvant to induce potentiation to the immune cells. the authors observed that this nanoparticle-based mers-cov vaccine was effective against a lethal dose of mers-cov infection as compared to control treatment in a mers-cov-permissive transgenic mouse model. the potency of this nanoparticle-based vaccine for mers-cov was demonstrated, and this study provides a new outline for developing nanocarrier based vaccine for viral pathogen. loczechin et al. prepared seven different carbon quantum dots (cqds) and investigated their anti-viral activity against the human coronavirus hcov- e infections. [ ] these cqds displayed a concentration-dependent virus inactivation and cqd produced from -aminophenylboronic acid showed better activity with ec . ± . µg·ml − . the mechanistic studies revealed that interaction of the surface functional groups of the cqds with entry receptors of the hcov- e virus resulted in inhibition of the infection. these results suggested such cqds systems might be explored for developing anti-viral therapeutics for other coronavirus infections (figure ) . [ ] polymeric nanoparticles consisting of poly (ethylene glycol)block-poly(lactide-coglycolide) (peg-plga) were developed for delivery of diphyllin, a novel vacuolar atpase blocker for its antiviral activity against the feline coronavirus infection. [ ] treatment with these nanoparticles significantly reduced toxicity and enhanced antiviral effect of diphyllin. these nanoparticles were well tolerated as revealed in animal study in mice following high-dose intravenous administration. the results of the study indicated that such diphyllin nanoparticles could be explored as effective host-targeted antiviral therapeutics for other coronavirus infections. coleman et al. developed a novel strategy for preparing spike nanoparticles which in combination with adjuvants produced high titer anti-bodies in mice against both the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) infections. [ ] the results showed that these spike nanoparticles were able to neutralize the antibody responses in mice-suggesting a step towards nanovaccine development. [ ] recently, fabric material-based face mask containing hydrophilic absorbent layers and hydrophobic barrier layers was constructed. [ ] this fabric masks were found to show equivalent or better filtration and adsorption of nanoparticle like aerosols than the commercial n respirators. the aerosols were composed of fluorescent labeled virus like nanoparticles for tracking their transmission through the fabric masks. the authors evaluated different combinations of common fabric materials using forced convection air flux with pulsed aerosols. this fabric masks can be used to protect from the inhalation of such viruses. in an early study, li et. al reported that treat-ment based on rna interference (rnai) exhibited antiviral immunity in mammals. [ ] in , sohrab s. et al. designed and developed a series of lipid and polymeric nanoparticles for delivery of therapeutic sirna for the treatment of mers-cov infection. [ ] since sars-cov- is a single strand rna virus like other coronaviruses, therefore inhibiting the life cycle of sars-cov- viruses via silencing their viral mrna in the host cells by rna interference might be an effective therapy for covid- . [ ] each group of people received second vaccination after days of the first vaccination. systematic side effects such as headache, fatigue, pain at the injection site etc. were observed in half the participants particularly after second vaccination with higher dose treatment. initial results revealed that treatment of mrna- induced immune responses in all participants and the antibody responses were higher for the higher dose treatment participants. recently, corbett et al. studied this mrna- vaccine in nonhuman primates and observed that treatment with this vaccine candidate increased neutralizing antibody levels that were higher than in human convalescent-phase serum sample. [ ] the mrna- vaccine is now under further evaluation for covid- . bnt b is another nanoparticle encapsulated mrna vaccine candidate that encodes receptor binding domain (rbd) of spike glycoprotein of sars-cov- . recently, mulligan et al. conducted a placebo-controlled phase / clinical trial of nucleosidemodified mrna vaccine which was formulated in a lipid-based nanoparticle system for targeting rbd of spike glycoprotein of sars-cov- virus (clinical trial identifier: nct ). [ ] the authors performed a randomized and placebo-controlled trial of bnt b vaccine in healthy adults. there were participants for each of the dose level , , and µg of the vaccination and nine participants in placebo with bnt b increased the sars-cov- neutralizing titers with dose level in the serum sample. thus, nanosized therapeutics such as nanoformulation of anti-viral drugs, nanovaccines, etc. can be promising options for the diagnosis and treatment of such viral infection. as summarized in this review, the past two decades have witnessed substantial amount of work in the development and applications of nanocarrier based systems for targeted delivery of drugs, gene, imaging agents etc. as well as nanoparticle-based diagnostics for various respiratory diseases. several preclinical and clinical investigations revealed that nanocarrier-based systems address many limitations of conventional therapy not only by site-specific delivery of therapeutics at the lung tissue, but also reducing the drug availability into other organs thereby reducing adverse side effects. besides, nanocarrier based systems demonstrated sustain and control release of the therapeutics than the burst release observed in systematic delivery of therapeutics in lungs. moreover, nanosized carriers have a potential to overcome the mucus barrier and poor lung penetration associated with various respiratory diseases. similarly, recent development of various nanoparticle-based detection methods for coronavirus infection showed a great promise in the development of time-economic diagnosis of covid- disease. while such nanoscale systems promise new therapeutic options for respiratory diseases, still the most challenging task is their safety assessment. preparation of an appropriate size of nanoparticles in each batch of synthesis is also challenging. many such challenges need to be overcome in order to translate the nanotherapeutics into clinical practice. though nanotechnology can find a way for further application of nanotherapeutics against the covid- diseases, still more research needs to be conducted for evaluation of nanosized therapeutics for covid- . it has been considered that the effective vaccine against covid- will be available in - months. therefore, early diagnosis, effective treatment etc. are essentials to mitigate the spread of this infection before any clinically approved vaccine comes in the market. finally, the following should be mentioned. because the major result of covid- infections is acute respiratory distress syndrome, the discussed nanomedicines for treatment lung hypoxia and fibrosis potentially can be used for the treatment of covid- in combination with other antiviral actions. such pilot investigations have been recently initiated in our laboratory. european lung white book theranostics sub-cell drug discovery today molekulare virologie theranostics this work was supported in part by grants (r ca and r ca ) from the national institutes of health (nih). some figures were created with biorender.com and using servier medical art images, which are licensed under a creative commons attribution . unported license (https://smart.servier.com). key: cord- -aofva ab authors: li, qizhang; wang, zhiying; zheng, qiang; liu, sen title: potential clinical drugs as covalent inhibitors of the priming proteases of the spike protein of sars-cov- date: - - journal: comput struct biotechnol j doi: . /j.csbj. . . sha: doc_id: cord_uid: aofva ab in less than eight months, the covid- (coronavirus disease ) caused by the sars-cov- (severe acute respiratory syndrome coronavirus ) virus has resulted in over , , confirmed cases and over , deaths around the world. with the increasing worldwide spreading of this disease, the lack of effective drugs against sars-cov- infection makes the situation even more dangerous and unpredictable. although many forces are speeding up to develop prevention and treatment therapeutics, it is unlikely that any de novo drugs will be available in months. drug repurposing holds the promise to significantly save the time for drug development, since it could use existing clinic drugs to treat new diseases. based on the “steric-clashes alleviating receptor (scar)” strategy developed in our lab recently, we screened the library of clinic and investigational drugs, and identified nine drugs that might be repurposed as covalent inhibitors of the priming proteases (cathepsin b, cathepsin l, and tmprss ) of the spike protein of sars-cov- . among these hits, five are known covalent inhibitors, and one is an anti-virus drug. therefore, we hope our work would provide rational and timely help for developing anti-sars-cov- drugs. since its outbreak in december , the covid- (coronavirus disease ) disease from the infection of the sars-cov- (severe acute respiratory syndrome coronavirus ) virus has caused over , , confirmed cases and over , deaths in over countries/regions as of august , (https://coronavirus.jhu.edu). although most covid- patients can recover from the disease, some of the severe patients might suffer from long-term health issues including irreversible lung damages [ ] and fertility compromise [ ] . in addition to the devastating health crisis, the ongoing covid- pandemic is also inflicting heavy losses on the global economy due to city lockdowns and glitches in supply chains (https://www.imf.org/en/topics/imf-and-covid ). facing the escalating risk from covid- , the whole world is intensively working on the discovery of prevention and treatment options for sars-cov- infection [ ] . according to the statistics of the according to a recent study [ ], both the endosomal cysteine proteases cathepsin b/l (catb/l) and the transmembrane protease serine type (tmprss ) can prime the s protein of sars-cov- . meanwhile therefore, similar with sars-cov [ ] and mers-cov historically, the drug discovery practice mainly focuses on non-covalent drugs due to potential off- target effects and toxicity issues of irreversible covalent drugs however, recent years have witnessed the resurgence of covalent drugs because many people have realized that compared to non- covalent drugs, covalent drugs might have extra advantages including: (i) better biochemical efficiency since they are more competitive than non-covalent endogenous substrates and co-factors [ ]; (ii) lower patient burden and less drug resistance due to lower and less frequent dosing to help the discovery of covalent drugs, we previously established a "steric-clashes alleviating receptor (scar)" strategy [ ] for the in silico docking and screening of covalent drugs enlightened by in silico protein design org) containing approved and in-trial drugs with known warhead groups targeting cysteine (catb/catl) or serine (tmprss ). then, scardock was used to computationally screen potential covalent inhibitors of human catb, catl, and tmprss . after careful filtering and evaluation, we identified five (trapoxin b, neratinib, hki- , domatinostat and (z)-dacomitinib) potential covalent inhibitors for catb, three (neratinib, hki- and (z)-dacomitinib) for catl the d structures of the indicated proteins were downloaded from the rcsb database the small molecule inhibitors were extracted from the complex structures mentioned above. the structures were visually checked, and incorrect bonds/atoms were manually corrected in iqmol (version . . ). mgltools (version . . ) was used to generate the corresponding pdbqt files for the small molecules were docked into the corresponding pockets of the proteins with autodock vina (version . . ) [ ]. the docking process did not consider the flexibility of the protein. the space coordinates of the s atom (for cys) or the o atom (for ser) in the wild-type protein were used for calculating the atom distances of the bonding atoms in the warhead groups. the distance cutoff between the bonding atoms and the s/o atom in the protein was set to . Å, indicating that the conformation with a distance above . Å is not accepted. since the results (table ) had distances between . - . Å, no score punishment for steric conflict was applied for the cases in this study. for each ligand, top poses were used for evaluation following the scardock protocol [ ], these reactive residues were computationally mutated to glycine to generate the scar proteins for docking from the rcsb database, we obtained four x-ray structures of human catb and seventeen x-ray structures of human catl (supplementary table ). to identify the most suitable structures for supplementary table ), the protein structure from csb (pdb id) was chosen for catb, and the protein structure from mae (pdb id) was chosen for catl. the human tmprss structure was obtained from the swiss-model repository since there were no x-ray structures available. these structures were then used for scardock screening, and after distance and score filtering, we identified five potential covalent inhibitors for catb overall, three cysteine covalent warheads and two serine covalent warheads were observed in the identified drugs (figure f) trapoxin b contains an epoxide warhead. for this warhead, the nucleophilic attack might occur on the ring carbon next to the carbonyl carbon, and then a covalent bond can form between the sulfur atom of cys and the bonding carbon of the oxirane moiety, accompanied by the ring opening and the formation of a hydroxyl group (figure f & figure a). neratinib (hki- ) and hki- contain a nitrile warhead. for this warhead, a covalent thioimidate bond might form at the electrophilic nitrile carbon after the attack of the cysteine sulfur atom (figure b & c). domatinostat ( sc- ) and (z)- dacomitinib are amide-based ligands. a covalent bond might form between the β-carbon and the cysteine sulfur atom (figure d & e) three drugs, i.e. neratinib (hki- ), hki- and (z)-dacomitinib, were identified as potential this result is not unexpected though, since catb and catl are homologous and have very similar d structures (figure b & c). thus, neratinib (hki- ), hki- and (z)- dacomitinib are potential covalent inhibitors for both catb and catl. although the docked poses were slightly different on these two proteins, the warheads of these drugs were also at the positions suitable for covalent bonding (figure f-h) )-boceprevir and (r)-boceprevir, were identified as potential tmprss covalent inhibitors (figure a). lodoxamide, aceneuramic acid and aleplasinin possess an α-ketoacid group, whereas (s)-and (r)-boceprevir have an α-ketoamide group both of these groups could be used as covalent warheads targeting serine (figure f) figure b, the warheads in these inhibitors are positioned well for the covalent bonding between the bonding atoms and the hydroxyl group of tmprss-ser the ongoing covid- pandemic caused by the sars-cov- virus is undoubtedly reminding us his warning was not a hoax. the high infection rate and mortality ratio of covid- are unexpected [ ], and the sars-cov- virus has made a difficult year for a lot of people in the world. although this virus would unlikely kill over million people, it is still posing an unprecedent threat to both the health and the economy of the whole world due to the shortage of effective prevention and treatment therapeutics so far during the infection of coronaviruses, the spike (s) protein mediates host recognition and binding however, the s protein needs to be cleaved and primed by the host cell before the virus can enter and hijack the host cell catl, and tmprss of the human cell can prime the s protein of sars-cov- targeting these priming proteases might be an effective choice to disrupt the infection of this virus. based on our recent work [ , , ], we adopted the scardock protocol to repurpose clinic drugs as potential inhibitors of these priming proteases in this study. we identified several clinic drugs that might be useful as the covalent inhibitors of catb more interestingly, all these drugs are covalent pan-her (human epidermal growth factor receptor) kinase inhibitors targeting the nucleophilic cysteine in the atp binding site of egfr and/or her [ ]. this fact indicated that these three molecules are electrophilic, and it is highly possible that they can form covalent bonds with the indicated cysteine residues of catb and catl if the non-covalent binding affinity is high enough. nontheless, we want to note that in our docking results, the nitrile groups however, there were docked poses of these two drugs with suitably positioned acrylamide groups in less optimal poses (supplementary figure ). in addition, domatinostat ( sc- ) and trapoxin b were identified as potential catb covalent inhibitors. interestingly, both domatinostat ( sc- ) and trapoxins b are the inhibitors of histone deacetylases (hdacs), and they have been used for the treatment of hki- ), hki- and dacomitinib might be the most as of the identified hits of tmprss , the most attractive drug might be boceprevir, which is a first- generation inhibitor of hepatitis c virus non-structural protease (hcv ns ) [ ]. based on the docking score and the scar enriching score (table ), (s)-boceprevir might be better than (r)- boceprevir. since boceprevir has anti-virus activity aceneuramic acid is used for the treatment of hereditary inclusion body myopathy [ ], and aleplasinin was developed to treat alzheimer disease [ ]. a note is that the screening of tmprss inhibitors was based on a homology model of human tmprss , which might make the result not as reliable as the catb/l cases. however a theoretical method to evaluate if a ligand could bind to two different proteins is to compare the similarity of the binding pockets of the target proteins presumably, the protein-ligand interactions between a same ligand and different proteins should be similar, although the extent to which should vary on a case-by-case basis therefore, to validate the binding potential of our computational hits with the target proteins, the ligand-protein interactions were generated this analysis also showed that (s)-boceprevir formed more interactions with tmprss than (r)-boceprevir did, which, in agreement with their docking scores and scar enriching scores taken together, using our scardock protocol, we identified nine drugs that might be repurposed as the covalent inhibitors of the priming proteases of the s protein of sars-cov- . among these nine drugs, neratinib (hki- ), hki- , dacomitinib, and boceprevir might be highly potential with moderate side effects did the computational work this work was supported by the grants from national natural science foundation of china ( , ) covid- ): lessons from severe acute respiratory syndrome and middle east respiratory syndrome rising concern on damaged testis of covid- patients evaluation and treatment coronavirus deconstructing the drug development process: the new face of innovation a bibliometric review of drug repurposing drug repurposing: progress, challenges and recommendations more than clinical trials launch to test coronavirus treatments remdesivir for the treatment of covid- -preliminary report cov- cell entry depends on ace and tmprss and is blocked by a clinically proven tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss the resurgence of covalent drugs drug discovery considerations in the development of covalent inhibitors ligand conformational bias drives enantioselective modification of a surface-exposed lysine on hsp identification of the clinical development candidate mrtx , a covalent krasg c inhibitor for the treatment of cancer discovery of covalent ligands via noncovalent docking by dissecting covalent docking based on a "steric-clashes alleviating receptor (scar discovery of novel inhibitors of human s- adenosylmethionine decarboxylase based on in silico high-throughput screening and a non- radioactive enzymatic assay potential covalent drugs targeting the main protease of the sars- cov- coronavirus repurposing clinical drugs as adometdc inhibitors using the scar strategy trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase optimization of , -disubstituted- -(arylamino)quinoline- -carbonitriles as orally active irreversible inhibitors of human epidermal growth factor receptor- kinase activity irreversible protein kinase inhibitors a novel molecular mechanism to explain mutations of the hcv protease associated with resistance against covalently bound inhibitors zinc -ligand discovery for everyone rosetta : an object-oriented software suite for the simulation and design of macromolecules autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading specialized roles for cysteine cathepsins in health and disease a review of small molecule inhibitors and functional probes of human cathepsin l hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers real estimates of mortality following covid- infection rapid repurposing of drugs for covid- the p /p ′ sites affect the substrate cleavage of tnf-α converting enzyme (tace) small-molecule egfr tyrosine kinase inhibitors for the treatment of cancer retracted: design and synthesis of chap , trapoxin b and hc-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors phase i study of domatinostat ( sc- ), a class i histone deacetylase inhibitor in patients with advanced hematological malignancies the effect of the first- generation hcv-protease inhibitors boceprevir and telaprevir and the relation to baseline ns resistance mutations in genotype : experience from a small swedish cohort efficacy of lodoxamide eye drops on mast cells and eosinophils after allergen challenge in allergic conjunctivitis clinical trial data available for ux , aceneuramic acid extended-release the apparent upa/pai- paradox in cancer: more than meets the eye identify drug repurposing candidates by mining the protein data bank. briefings in proteins are shown in gray ribbons. the nucleophilic cysteines are shown in sticks and colored in green. (f) reaction mechanisms of the amide figure . structures of the scardock hits for catb. the bonding atoms in the warheads are pointed out by arrows docked poses of the identified hits of catb (a-e) and catl (f-h). the proteins are shown in surface or ribbons, and the drugs are shown in sticks. the cysteine residues for covalent binding are colored in yellow. the displayed items for catb are trapoxin b (a) the displayed items for catl are neratinib (hki- ) (f), hki- (g) and (z)-dacomitinib (h). the putative covalent bonding atoms in the drugs are the binding details between the scardock hits and catb/catl. the docked poses are as same as in figure . for catb: trapoxin b (a), neratinib (hki- ) (b), hki- (c), domatinostat ( sc- ) (d) and (z)-dacomitinib (e); for catl: neratinib (hki- ) (f) the proteins are shown in ribbons with key residues shown in sticks, and the drugs are shown in sticks boceprevir and (r)-boceprevir. (b) the docked poses of the hits in tmprss . the proteins are shown in surface or ribbons, and the drugs are shown in sticks. the serine residues for covalent binding are colored in red. the putative covalent bonding atoms in the drugs are indicated by arrows. (c) the binding details between the scardock hits and tmprss . the proteins are shown in ribbons with key residues shown in sticks, and the drugs are shown in sticks the plots were prepared with ligplot+. ligands and protein side chains are shown in ball-and-stick representation, with the ligand bonds colored in purple. hydrogen bonds are shown as green dotted lines, while the spoked arcs represent protein residues making nonbonded contacts with the ligands. the red circles and ellipses indicate protein residues that are in equivalent d positions when the structural models are superposed. the pdb codes of the experimental complex structures were shown in the parentheses competing financial interests: the authors declare no competing financial interests. key: cord- -pwi i authors: khan, abbas; ali, syed shujait; khan, muhammad tahir; saleem, shoaib; ali, arif; suleman, muhammad; babar, zainib; shafiq, athar; khan, mazhar; wei, dong-qing title: combined drug repurposing and virtual screening strategies with molecular dynamics simulation identified potent inhibitors for sars-cov- main protease ( clpro) date: - - journal: j biomol struct dyn doi: . / . . sha: doc_id: cord_uid: pwi i the current coronavirus (sars-cov- ) pandemic and phenomenal spread to every nook and cranny of the world has raised major apprehensions about the modern public health care system. so far as a result of this epidemic, , , confirmed cases and , deaths are reported. the growing infection rate and death toll demand the use of all possible approaches to design novel drugs and vaccines to curb this disease. in this study, we combined drugs repurposing and virtual drug screening strategies to target clpro, which has an essential role in viral maturation and replication. a total of fda approved anti-hiv drugs, and traditional chinese medicines (tcm) database were screened to find potential inhibitors. as a result, saquinavir, and five drugs (tcm , tcm , tcm , tcm , and tcm ) from the tcm database were found as promising hits. furthermore, results from molecular dynamics simulation and total binding free energy revealed that saquinavir and tcm target the catalytic dyad (his and cys ) and possess stable dynamics behavior. thus, we suggest that these compounds should be tested experimentally against the sars-cov- as saquinavir has been reported to inhibit hiv protease experimentally. considering the intensity of coronavirus dissemination, the present research is in line with the idea of discovering the latest inhibitors against the coronavirus essential pathways to accelerate the drug development cycle. communicated by ramaswamy h. sarma. the coronaviruses (covs) are placed within four genera, alphacoronavirus, beta-coronavirus, gamma-coronavirus and deltacoronavirus (a, b, c, and d) of subfamily orthocoronavirinae. however, the members of beta-coronavirus such as severe acute respiratory syndrome coronavirus (sars-cov and sars-cov ) and the middle east respiratory syndrome coronavirus (mers-cov) have been involved in pneumonia epidemics in the st century. sars-cov , mers-cov and sars-cov outbreaks caused by zoonotic viruses (lau et al., ; reusken et al., ); with the case fatality ratio of % (cheng et al., ; lee et al., ; zaki et al., ) , % (de groot et al., ; zaki et al., ) ; and %, respectively. the recent outbreak and phenomenal spread of sars-cov- to every nook and cranny of the world ( . million infections and deaths) compelled world health organization (who) to declare it pandemic on march , . the virus causing covid- was named -ncov ( novel coronavirus) on january , by who zhu et al., ) , however the international virus classification commission (ictv) on february , , named this virus as severe acute respiratory syndrome coronavirus (sars-cov- ). this threat of global concern to humanity, mainly due to the unavailability of proper treatment pushed the investigators to discover and design therapeutic drugs and vaccines to combat the coronavirus. infection symptoms include fever, dyspnea, shortness of breath, and cough, whereas severe cases lead to kidney failure and death (rothan & byrareddy, ) . the sars-cov- is composed of spike (s) protein, membrane (m) protein, envelope (e) protein, and nucleocapsid (n) protein. in anti covs therapies, the main focus is to boost the human immune system or to block the binding of spike protein with the receptor proteins. therefore, the treatments based on targeting coronavirus rely on blocking of virus binding to receptors, inhibition of virus replication, prevention of the synthesis of viral rna and inhibition of the virus self-assembly process (bosch et al., ; khan et al., ; omrani et al., ) . to combat the latest covs threats, the researchers are following multiple approaches for novel drug development (zumla et al., ) and testing the efficacy of existing drugs. in one approach, experiments were performed to test the existing broad-spectrum antiviral drugs (ribavirin, interferons, and cyclophilin inhibitors) against sars-cov- (chan et al., ; zumla et al., ) . the use of these antiviral drugs is approved against viral infections, and their dosages, efficacy, metabolic characteristic, and side effects are well known. however, there is a possibility that this "broad-spectrum" treatment against covs would not be effective. in the second approach, molecular databases were subjected to highthroughput screening for potential drug molecules to combat infection caused by a coronavirus (de wilde et al., ; dyall et al., ) . this strategy was proved successful in the discovery of lopinavir/ritonavir for the treatment of hiv. some researchers are using the genomic and pathological information of covs for the development of a new specific drug from scratch. theoretically, this is an effective approach; however, it is very time consuming and might cost several years or even more than ten years (khan et al., ; omrani et al., ) . structural bioinformatics based approaches are the fastest way for finding the potential molecules from the marketed drugs or bioactive compounds for effective treatment of sars-cov- . main-protease or clpro inhibition is a promising target to control the recent sars-cov- infection due to its essential role in viral maturation and replication (ul qamar et al., ) . clpro has three important domains i-iii, which correspond to positions - , - , and - , respectively. there is a connecting loop that corresponds to position - , which connects domains ii and iii. the structure of clpro has an important catalytic dyad consist of his and cys . in this study, the protein of sars-cov- ( clpro, also named -chymotrypsin-like protease) was subjected to drug repurposing and virtual screening for potent drug identification followed by molecular dynamics simulation and binding free energy calculation. our findings revealed that saquinavir, which is an hiv protease inhibitor reported experimentally (kim et al., ) and tcm , are promising hits, which need in vitro validation for antiviral effects. we hope this study will provide useful information for the clinical treatment of novel coronavirus associated pneumonia. (b) the electrostatic potential of clpro and displays the important active site residues in the sticks, including the two essential residues his and cys (catalytic dyad). protein databank (http://www.rcsb.org/) (rose et al., ) was used for retrieval of clpro ( lu ) crystal structure. using the protein preparation implemented in schr€ odinger software (schr€ odinger, llc, new york, ny), the structure was prepared and optimized. the opls_ force field was used for protein-energy minimization. for ligands preparation such as assigning appropriate ionization, stereochemistry, ring conformations, and tautomer (release, ; schrodinger, ) , a ligprep module was used. apbs tool (lerner & carlson, ) implemented in pymol was used for electrostatic potential calculation. drug repositioning or repurposing approach is used to speed up the drug development cycle by finding a new therapeutic application for a marketed drug that has been licensed for a particular use (sleigh & barton, ) . this approach was fruitful in the case of sildenafil for leprosy, erectile dysfunction, and pulmonary hypertension, and multiple myeloma thalidomide (hernandez et al., ) . literature mining was carried out to collect anti-hiv drugs for screening against clpro (sars-cov- ). multiple drugs were retrieved from drugbank database. a total of drugs were shortlisted for screening against the clpro (sars-cov- ). schr€ odinger binding site was used for finding the binding site of proteins using the default parameters, and the generated maps show the binding cavity. the identified binding sites have the descriptions regarding hydrogen bonding, a degree of exposure and enclosure, size, linking site points, tightness, hydrophobic and hydrophilic nature. the grid with dimensions Å Â Å Â Å was generated. the final active site grid identified was based on the experimentally reported residues by a recent crystallographic study and the maps generated by schr€ odinger maestro. three steps of virtual screening (htvs, sp, and xp) were used to screen the anti-hiv and tcm compounds databases. furthermore, the bioactivity of these compounds was predicted by using molinspiration cheminformatics tool. molinspiration is an efficient tool that has been used by several studies ($ ) to predict bioactivity results. top hits from anti-hiv drugs and tcm database were subjected to molecular dynamics simulation using the amber package (case et al., ) . the antechamber was used to generate the drugs topologies.tip p water model was to solvate the system, and na þ counter ions were used to neutralizing the system. two steps energy minimization of the system followed by heating and equilibration was performed. particle mesh ewald (pme) algorithm was applied to calculate the long-range electrostatic interactions (price & brooks iii, ) . for van der waals interactions, a . nm cutoff values were set and also for short-range columbic, respectively. a total of ns md simulation was performed with a time step of fs. the behavior of the ligand-protein complex and stability were analyzed. post-simulation analysis such as rmsd, rmsf, rog and hydrogen bonds occupancy were performed using cpptraj and ptraj (roe & cheatham iii, ) . the script mmpbsa.py was used to calculate the free binding energy for all the protein-ligand complexes (chen et al., ; hou et al., ; miller iii et al., ; sun et al., ) ; considering snapshots from md trajectories using the following equation: in this equation, dg bind represents total free binding energy, while others show the free energy of complex, the protein, and the ligand. specific energy term contributes to the whole free energy was calculated by the equation: g bond , g ele and g vdw specify interactions among bonded, electrostatic, and van der waals states. in contrast, g pol and g npol represent the polar and non-polar interaction to the table . a list of anti-hiv drugs used for docking against the proteinase enzyme of coronavirus (sars-cov- ). targeted virus(es) free energy presumed through precise gb (generalized born). this free energy calculation method is widely used by different studies to understand the binding energy of different ligands wang et al., ) . clpro structure retrieval and preparation the crystallographic structure ( amino acids long) of the clpro (sars-cov- ) was retrieved from the rcsb database using accession id: lu . the structure of clpro was analyzed for missing residues and refinement. the structure was subjected to energy minimization and structure preparation using maestro. the structure of clpro has three domains, as given in figure (a). these domains i-iii correspond to position - , - , and - , respectively. while there is a connecting loop corresponds to position - , which connects domains ii and iii. all these domains are coloured differently. the ligand in the active site (yellow colour (sphere)) has also been shown in figure (a). the electrostatic potential of the clpro shows that the active site region possesses a strong electronegative potential, while weak electronegative potential around the active site can also be observed. however, the weak electropositive and neutral potential is distributed too. the active site grid (x¼ À . , y ¼ . , z ¼ . ) was generated, and important residues were identified. important active site residues his , met , tyr , phe , leu , asn , cys , his , met , glu , leu , phe , asn , and gln were identified and shown in figure (b). the binding cleft is at the same place as in the previously reported sars with the two critical residues his and cys forms a catalytic dyad. in the current study, the repurposing of anti-hiv drugs against the sars-cov- main protease was carried out using structure-based screening methods. anti-hiv drugs such as lopinavir-ritonavir are reported to be active against the sars-cov- . clinical trials of these drugs on different groups of patients showed significant improvement. thus, anti-hiv drugs possess the potential to work against srs-cov- targets. therefore, immediate testing of anti-hiv drugs may lead to a discovery of potent inhibitor against the sars-cov- . a list of anti-hiv drugs ( these drugs were prepared for molecular docking against the proteinase enzymes of coronavirus (sars-cov- ). the docking scores were ranged from À . kcal/mol to À . kcal/mol. all the conformations were analyzed for the best interactions with the key residues. initial criteria were set to shortlist the drugs based on binding affinity and interactions with the dyad residues (his and cys ). the docking score À . kcal/mol was reported for the hiv protease inhibitor drug saquinavir (figure ). this drug has been reported to inhibit the hiv proteinase enzymes too. in this case, (sars-cov- ), the saquinavir formed six hydrogen bonds, including two with the central dyad residues (his and cya ). previous reports on different sars, such as the octa-peptide, also stressed the blocking of these two residues. other interactions include four hydrogen bonds with glu , gln , met , and gly , while some pi-alkyl interactions, including one with cys and other residues, are also formed. thus, we speculate that the clinical testing of figure . rmsds of all the systems including clpro-squanavir (red), clpro- (green), clpro- (blue), clpro- (cyan), clpro- (magenta) and clpro- (navy). the x-axis shows the time in nanoseconds while the y-axis shows the rmsd in Å. saquinavir should be done at the earliest. it is also reported by different scientists that hiv drugs could be potentially used against the recent coronavirus. virtual screening of tcm database against the clpro (sars-cov- ) furthermore, we also performed virtual screening of the traditional chinese medicines database (tcm). a total of , compounds were screened after excluding the drugs violating the lipinski rules of five. important residues in the active site (his and cys ) were targeted. the top five compounds based on the docking scores were selected. the best docking score, À . kcal/mol, was reported for the compound ( ). more than ten bonds, including hydrogen bonds, van der waals, electrostatics, and pi-alkyl interactions, were formed. among the seven hydrogen bonds, two hydrogen bonds with the residue his were also formed, which is a key residue in the active site dyad. among the other phe , leu , asn , his , glu , and thr also formed hydrogen bonds with the ligand ( ). on the other hand, compound , with the docking score À . kcal/mol formed a strong hydrogen bond with the key residue his . the compound also formed five hydrogen bonds with the other active site residues (glu , arg , gln , and thr ). the other three compounds ( , , and ) were also analyzed for the interaction with the key residues. it can be seen from figure that his is mainly involved in the interaction with the ligands. table is showing the d structures of the top five compounds with their docking scores. cross validation of our predicted compounds by comparing with the experimentally tested compounds showed that the saquinavir and tcm possess a similar scaffold with myricetrin and scutellarin which are flavonoids and reported to have strong inhibitory effects by targeting clpro. on the other hand, the four compounds (tcm , tcm , tcm and tcm ) from tcm database shares structural similarity with scutellarein, dihydromyricetin, quercetagetin, myricetin, , -dihydroxyflavone, , -dihydroxyflavone, chrysin, herbacetin and baicalein which are flavonoids and testes to have strong inhibitory effects in an experimental condition by targeting clpro (chen & du, ; dai et al., ; yang et al., ) . their structural analyses showed that these compounds mainly form bonds with phe and glu while our compounds specifically target the catalytic dyad with supplementary interactions with the other active site residues. thus, we speculate that our compounds could also show the same potential activity against the clpro in the experimental assay because our compounds are also plants derived extracts and belongs to a group of flavonoids. furthermore, results obtained from the server shows that all these shortlisted compounds are active against the protease target. from the scores it can be seen that saquinavir with score . possesses strong inhibitory effects against the proteases while the others the reported score for tcm (À . ), tcm (À . ), tcm (À . ), tcm (À . ) while tcm possess (À . ) bioactivity score against the protease target. thus, these results strongly suggest that the shortlisted compounds could efficiently inhibit the clpro in the experimental setup and could be tested for clinical trials. figure . rmsfs of all the systems including clpro-squanavir (red), clpro- (green), clpro- (blue), clpro- (cyan), clpro- (magenta) and clpro- (navy). the x-axis shows the time in nanoseconds while the y-axis shows the rmsf in Å. the right panel shows the secondary structure of clpro. to understand the conformational and dynamics features of the selected hits against the clpro of sars-cov- molecular dynamics (md) simulation is an imperative method to explore the behavior of each system in real-time. dynamics features of the six selected systems (saquinavir, , , , , and were calculated after ns simulation. for stability rmsd, while to evaluate the flexibility at residue level rmsf of the complexes were calculated to comprehend the overall stability and flexibility of the system. the stability of all the six systems was calculated by using root mean square deviation (rmsd). we tested the stability of the docked drugs in the active pocket and its effects on the stability of the whole system. the results suggest that all the six systems were stable with acceptable deviation (as shown in figure ). the average rmsd for all the systems was found to be between and Å. saquinavir- clpro exhibited an average rmsd of . Å soon after reaching ns; the system attained stability remains uniform for the rest of the time. it can be seen that the complex clpro- remained stable, but after ns, the system showed an acceptable deviation for a little time. after that, the system gained stability and entered the production stage. for a complex clpro- , the system showed a slight deviation between and ns but remained stable during the course of the simulation. on the other hand, clpro- and clpro- reported divergence between and ns, but the system soon gained stability. similarly, in the case of clpro- , the examination of rmsd of the ca backbone has an average rmsd of . Å. the system shows a deviation in figure . rogs (radius of gyration) of all the systems including clpro-squanavir (red), clpro- (green), clpro- (blue), clpro- (cyan), clpro- (magenta) and clpro- (navy). the rmsd for the first ns while remained uniformed for the rest of the simulation time. thus, these results revealed the stable internal motions and negligible fluctuations during the course of the simulation. the differences in the rmsd of each system is due to the binding and unbinding of the ligand at different time intervals. also, the systems with more stable behaviour show that the ligand remained intact, and the system entered the production phase soon when compared to the others. aside, the binding of small molecules affects the system differently as the ligand-binding orientation changes over the simulation time. the rmsf depicts the flexibility at residues level. as shown in figure , the pattern of flexibility is almost similar and was recorded around - Å in all secondary components except loop regions with slightly fluctuated residues. however, active site residues seem stable in the course of the simulation, which is due to the ligand recognition by the ligandbinding regions. it can be seen that clpro-squanavir and clpro- showed an average rmsf of . Å with significant fluctuations in the regions - , - , - , and - . a similar pattern of fluctuation was also observed for systems including clpro- , clpro- , and clpro- . however, the average rmsf remained below the Å. only the system clpro- wed higher fluctuation than the rest, but the residual flexibility pattern was similar. the average rmsf for all the five systems was around - Å, as clearly seen in figure (left panel). these findings here show that the target protein is stabilized by binding of all five chosen drugs docked against it. thus the binding of ligand has significantly affected the residue fluctuation, which is due to the internal residues disturbed by the binding of different ligands, and this both correlated and non-correlated motions are affected. the secondary structure components are also given in figure (right panel). the radius of gyration (rog) conveys the information of stable and unstable folding protein while interacting with ligands. less compactness (more unfolded) shows higher rog values, whereas low rog values indicate strong compactness and higher structural stiffness (more folded). thus, rog was determined to assess the system's compactness over time. as shown in figure , the simulated complexes show gyration scores between and Å. in the case of clpro-squanavir, the gyration score for the initial ( ) frames was reported to be . Å. however, a little fluctuation between ( - ) frames was observed, but soon after , the values become lower and uniform. in the case of clpro- , clpro- , clpro- , and clpro- showed an average gyration of - . Å. similarly, clpro- also showed similar fluctuation, but in the last few frames, the values fluctuated a little higher, which is assumed to be in an acceptable range. the binding and unbinding of ligands greatly affected the overall compactness of the complexes. as given in figure , in clpro-squanavir, clpro- , and clpro- complexes, the hydrogen bond with his was reported in %, % and % of the trajectories. hydrogen bond with cys residue was reported in % trajectories only in the clpro-squanavircomplex. furthermore, leu and glu were found in almost all complexes. however, another important residue gln was found in % of the clpro-squanavir trajectories, % in clpro- , while % in clpro-tcm trajectories. other active site residues were not detected in a significant population of the md trajectories. binding free energy mm/gbsa, a popular approach, was used to estimate the binding free energy of all the six systems. the binding free energy determines the binding affinity between ligands and clpro. each energy term, including van der waals energy, electrostatic energy, polar solvation energy, solvent accessible surface area energy, and total binding free energy of all the systems are given in table . it can be seen that the clpro-saquinavir system possesses the highest total binding energy of À . kcal/mol. thus it confirms that saquinavir, an anti-hiv drug that has been reported to inhibit hiv protease experimentally, possesses strong inhibitory effects against the clpro of sars-cov- . recently published results also shortlisted saquinavir as a potential candidate, but they only performed ns simulation (khan et al., ) , which could not reflect better results than what we revealed by adding -folds more simulation time. furthermore, the top five hits from tcm virtual screening results were also subjected to the free binding energy as reported by the virtual screening results tcm was reported with the best docking score and interaction. herein, tcm also possesses the best total binding energy, . kcal/mol, against the clpro. also, the other hits from including tcm , tcm , tcm , and tcm possess total binding energy of À . kcal/ mol, À . kcal/mol, À . kcal/mol and À . kcal/ mol. while the other energy terms such as van der waals energy, electrostatic energy, polar solvation energy, solventaccessible surface area are given in table , thus these results strongly suggest that saquinavir and tcm should be tested experimentally against the sars-cov- at earliest. to date, a total of , , confirmed cases and , deaths are reported from the sars-cov- epidemic, which indicates that the current viral treatment is not satisfactory, and more diverse studies are needed for the incorporation of various natural and fda approved compounds for the effective treatment of sars-cov- . further, our existing capacity for treating zoonotic coronavirus infections is still in the trial, and the available resources seem very limited to fight this life-threatening hazard. although extensive research efforts have been initiated during and outbreaks of sars and mers-cov, respectively, however, no effective drugs so far have been synthesized, nor the matter may be taken seriously to prevent the future pandemics of zoonotic coronavirus. drug repositioning is a 'universal strategy' for neglected diseases due to reduced number of clinical trial steps required could reduce the time and cost of the medicine to reach the market, existing pharmaceutical supply chains could facilitate the 'formulation and distribution' of the drug, the known possibility of combining with other drugs could enable more efficient treatment, repositioning could encourage the development of 'new modes of action for old drugs and new types of medicines, eliminating 'activation obstacles' from early stages of study could allow the project to progress rapidly towards disease-oriented development (pushpakom et al., ) . one of the primary reasons why no prototype coronavirus inhibitor has progressed to the (early) preclinical stage so far is due to the transient nature of this epidemic. like the sars virus years ago, the new sars-cov- , and the new evolving coronaviruses that may continue to pose a threat to global public health in the future. hence, discovering broad-spectrum inhibitors that can reduce the symptoms of human coronavirus infection remains a formidable subject of study. considering the time-consuming process of designing and documenting antiviral medications, proven therapies for certain diseases may be the only fastest therapeutic choice for emerging infectious diseases. the prescription has ample experience and dosage for most of those medications that have been formulated, and their health and adme properties are well known. in this study, based on the results of bioinformatics analysis, we targeted clpro from sars-cov- using drugs repurposing (anti-hiv drugs) virtual drugs screening (tcm) approaches to shortlist the most potent compounds for the possible treatment. the results of the entire article stressed the potential inhibitory role of saquinavir and tcm , which are based on computer-based virtual drug screening. we have not conducted further in vivo and in vitro antiviral experiments yet, because we want to share our results with scientists in anti-sars-cov- research as soon as possible. this study will help to repurpose drug design, perform in vivo and in vitro evaluations for candidate drugs obtained in this study, and prepare for clinical trial applications. c-like protease ( clpro), also called the main protease, is an attractive target for the treatment of sars-cov- because of its role in the viral replication cycle. the current study focused on structure based approaches repurposed anti-hiv drugs and screened traditional chinese medicines databases against the active site residues of clpro. initial analysis such as molecular docking scores and interactions with important residues shortlisted one drug (saquinavir) from anti-hiv drugs while five compounds from tcm database.these compounds were then subjected to molecular dynamics simulation and post-simulation analysis. among the six complexes subjected to molecular dynamics simulation saquinavir, tcm showed the highest hydrogen bond occupancy. the rest were also found to have good activity against the clpro. in addition, the bioactivity of these compounds were predicted which reported saquinavir as strong potential inhibitor followed by the others.binding free energy calculations of all the complexes suggested that these compounds significantly interacting and binds with the receptor. thus, here from all the analyses, we suggest that saquinavir and tcm should be tested experimentally for possible treatment of sars-cov- . ak, zb, mk, and ssa conceptualized the study and did the analysis. aa, ms, ss wrote the manuscript. ak, ss and ssa revised the manuscript and improved the write-up. dqw is an academic supervisor. he supervised the study. the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the amber biomolecular simulation programs broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus assessing the performance of the mm/pbsa and mm/gbsa methods. . capability to predict protein-protein binding free energies and rerank binding poses generated by protein-protein docking potential natural compounds for preventing sars-cov- ( -ncov) infection. preprints emerging coronaviruses: genome structure, replication, and pathogenesis severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection structure-based design, synthesis and biological evaluation of peptidomimetic aldehydes as a novel series of antiviral drug candidates targeting the sars-cov- main protease commentary: middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection giving drugs a second chance: overcoming regulatory and financial hurdles in repurposing approved drugs as cancer therapeutics characterization of domain-peptide interaction interface: prediction of sh domain-mediated proteinprotein interaction network in yeast by generic structure-based models clinical features of patients infected with novel coronavirus in wuhan. the lancet structure of mpro from covid- virus and discovery of its inhibitors. biorxiv deeplearning-based target screening and similarity search for the predicted inhibitors of the pathways in parkinson's disease allosteric ligands for the pharmacologically important flavivirus target (ns ) from zinc database based on pharmacophoric points, free energy calculations and dynamics correlation identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach saquinavir, an hiv protease inhibitor, is transported by p-glycoprotein severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a major outbreak of severe acute respiratory syndrome in hong kong apbs plugin for pymol mmpbsa.py: an efficient program for end-state free energy calculations ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study a modified tip p water potential for simulation with ewald summation drug repurposing: progress, challenges and recommendations maestro. schr€ odinger, llc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. the lancet infectious diseases ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data the rcsb protein data bank: integrative view of protein, gene and d structural information the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak schrodinger software suite (p. ). schr€ odinger assessing the performance of mm/pbsa and mm/gbsa methods. . accuracies of mm/pbsa and mm/gbsa methodologies evaluated by various simulation protocols using pdbbind data set structural basis of sars-cov- clpro and anti-covid- drug discovery from medicinal plants the systematic modeling studies and free energy calculations of the phenazine compounds as anti-tuberculosis agents drugbank . : a major update to the drugbank database traditional chinese medicine in the treatment of patients infected with -new coronavirus (sars-cov- ): a review and perspective isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china coronaviruses -drug discovery and therapeutic options the authors declare no conflict of interest. key: cord- - cjr rol authors: chan, marion m.; chen, rensa; fong, dunne title: targeting cancer stem cells with dietary phytochemical - repositioned drug combinations date: - - journal: cancer lett doi: . /j.canlet. . . sha: doc_id: cord_uid: cjr rol the tumor microenvironment is complex with the cancer stem cell (csc) as a member within its community. this population possesses the capacity to self-renew and to cause cellular heterogeneity of the tumor. cscs are resistant to conventional anti-proliferative drugs. in order to be curative, it is imperative that cscs must be eliminated by cancer therapy. a variety of dietary phytochemicals and repositioned drugs can act synergistically with conventional anti-cancer agents. in this review, we advocate the development of a novel approach, namely combination therapy by incorporating both phytochemicals and repositioned drugs to target cscs. we cover select dietary phytochemicals (curcumin, resveratrol, egcg, genistein) and repurposed drugs (metformin, niclosamide, thioridazine, chloroquine). five of the eight (curcumin, resveratrol, egcg, genistein, metformin) are listed in “the halifax project”, that explores “the concept of a low-toxicity ‘broad-spectrum’ therapeutic approach that could simultaneously target many key pathways and mechanisms” [ ]. for these compounds, we discuss their mechanisms of action, in which models their anti-csc activities were identified, as well as advantages, challenges and potentials of combination therapy. in the course of its lifespan, a somatic cell undergoes changes in genome and epigenome due to intrinsic (cell-developmental) and extrinsic (environmental) factors. when accumulated changes disrupt its regulation of cell growth and death, the resulting uncharted growth leads to cancer. carcinogenesis is a multi-step process: initiation, promotion, progression and metastasis. the "hallmarks of cancer" denote alterations in cancer cell physiology [ ] . indeed, only a very limited number of genes ( or so), covering a dozen signaling pathways on the processes of cell fate, cell survival and genome maintenance, are relevant and designated as "driver" mutations [ ] . histologically, a tumor is heterogeneous, consisting of cancer cells at various stages of differentiation, as well as other cell types (fibroblasts, immune cells, endothelial cells). these cells behave as members of a community; the tumor microenvironment (niche) is a complex interactive network [ ] . cancer stem cell (csc) is the cell within a tumor that possesses the dual capacity to self-renew and cause the heterogeneous lineages of cancer cells that comprise the tumor [ ] . conventional chemotherapy targets the bulk of proliferating cancer cells. examples are paclitaxel for several cancer types and imatinib (tyrosine kinase inhibitor) for chronic myelogenous leukemia (cml). however, cscs are resistant to these anti-cancer drugs [ ] . a curative cancer therapy must acknowledge csc plasticity and their complete elimination. many csc-targeting dietary phytochemicals can act synergistically with conventional anti-cancer agents. recently, investigators discovered csc-targeting repurposed drugs [ ] . we advocate here a novel approach: combination therapy with the pairing of csc-targeting phytochemicals and repositioned drugs. our interest in ovarian cancer led us to phytochemical-drug combinations [ , ] . anti-cancer studies have demonstrated benefits of specific phytochemical combinations over individual compounds [ ] . dietary phytochemicals and repositioned drugs should provide easier access towards clinical use, because of the former's "generally regarded as safe" (gras) status and the latter's prior food and drug administration (fda) approval. this innovative approach is advantageous: both compounds target cscs, an activity insurmountable by current anti-cancer agents. for csc targeting, we recommend four dietary phytochemicals (curcumin, resveratrol, egcg, genistein) and four repurposed drugs (metformin, niclosamide, thioridazine, chloroquine). (see fig. applications of csc-targeting phytochemical-drug combinations. furthermore, five (curcumin, resveratrol, egcg, genistein, metformin) are listed by an international task force of clinicians and scientists of "the halifax project", with dedicated objective to "explore the concept of a low-toxicity 'broad-spectrum' therapeutic approach that could simultaneously target many key pathways and mechanisms" for cancer treatment [ ] . block et al. ( ) stated: "among approaches, curcumin, genistein, resveratrol and egcg boast a wealth of fundamental research" [ ] . ( we are aware investigators may disagree with our selection of phytochemicals; others will also be cited.) all eight have efficacy, are low cost and free of intellectual property constraints. these attributes make sure the combinations can be widely affordable, and are economical compared to current chemo-and targeted therapies. different from conventional anti-cancer drugs with unique molecular targets, dietary phytochemicals and repurposed drugs are pleiotropic. each has a multitude of cellular targets (as shown later in compound description). multilateral targeting of molecular and cellular pathways will inhibit csc growth or induce csc differentiation, and hinder the csc's ability to develop resistance. in this case, "promiscuous drugs" is a virtue; "dirty" might be better [ , ] . a phytochemicaldrug combination is advantageous for synergistic effects. such pairing allows lower effective doses than using a single compound for activity. lower doses mean less potential toxicity and fewer side effects. for a particular cancer, various combinations can be explored to look for the ideal pair at optimal doses. for clinical use, the combination can be used alone (if both cscs and bulk cancer cells are eliminated), or as adjuvant to conventional cancer therapy (the combination targets cscs and conventional therapy eliminates the bulk cancer cells). if the approach of csc-targeting dietary phytochemical-repositioned drug combination is important, what are the reasons for its being to present a more comprehensive picture, we start with considerations to csc methodologies on which various observations were made, in the "evolving concept" of csc [ , ] . we follow with compound description, and end with challenges and the need of applying csc-targeting phytochemical-drug combinations in the current climate of cancer therapy. human cscs were first discovered in leukemia and then characterized in solid tumors. utilizing an array of mechanisms, cscs are insensitive to conventional cancer treatments, but these mechanisms can be targets of phytochemicals and drugs (as described later) [ ] [ ] [ ] [ ] [ ] . (see fig. for key mechanisms of csc resistance to therapy.) cscs may be quiescent and exhibit a slow proliferating nature. they may possess an efficient dna repair system, activated by genomic instability and regulated by epigenetic modifications via histone deacetylases. pro-survival signaling pathways may be activated (hedgehog, wnt, notch, pi k, nfκb). cscs may have high levels of anti-apoptotic molecules. for drug resistance, both the activation of drug transporters, with high level expression of atp-binding cassette (abc) membrane transporters for drug efflux, and the enzyme aldehyde dehydrogenase (aldh), for drug metabolic activities, have been detected. in addition, their niche has less reactive oxygen species (ros), thus less susceptible to radiation therapy. one notable csc-associated feature is epithelial-mesenchymal transition (emt), a process when epithelial cells lose polarity and become less adhesive [ ] . with these changes the cells become invasive, migratory and mesenchymal-like with "stemness" properties. the process involves the activation of transcription factors (snail, twist) and change in marker proteins (e-to n-cadherin). first seen in embryogenesis, emt has been viewed as reappearance of embryonic features in cancer cells. the process is reversible and has been linked to cscs for tumor heterogeneity and metastasis. emt involvement with cscs is under active research [ ] . cscs can be identified by in vitro and in vivo methods. they are isolated via fluorescence-activated cell sorting (facs) by their surface antigens or detoxification capacity. cscs express identifying cell surface antigens, for example, ovarian cscs express cd (prominin- ); these molecules can serve as biomarkers allowing their isolation by facs. aldh is a detoxification enzyme that oxidizes aldehydes to carboxylic acids, for further metabolism or excretion via the liver. its activity has been utilized for csc selection by facs, using a fluorescent substrate (aldefluor) [ ] . the "side population" (sp) assay identifies cscs apart from the main population of cancer cells in flow cytometry, by their fast expulsion of a fluorescent dye (hoechst ). however, cell surface markers for csc selection are also present in normal stem cells and other cell types, they are not unique to cscs [ ] . both sp and aldh selections were originally developed for hematopoietic stem cells (hscs) and then adapted to cscs. spheroid culture is another method for identifying cscs. a cancer cell (transformed due to mutations) exhibits anchorage independence, a characteristic allowing it to form a cell colony in the absence of substratum, as shown by soft agar colony formation assay (normal cells die in the absence of attachment via the process of anoikis). for serum-free spheroid cultures (spheres), cancer cells grow as spheroids in suspension (in untreated plastic ware) when provided with specific growth factors (insulin, epidermal growth factor, basic fibroblast growth factor), unlike ordinary tissue culture where cells usually grow as a monolayer (with fetal bovine serum for growth factors and other components). spheroids mimic the three-dimensional nature of tumors with limited oxygen available to the internal cells. this hypoxic condition favors expression of pluripotency maintaining transcription factors, thus promoting the differentiation to cscs and facilitating the state of "stemness" [ ] . spheroids can form secondary and tertiary spheres in subsequent cultures. cscs known as "neoplastic sphereforming cells" are measured by enumerating secondary spheres generated when re-plating the spheres, based on the assumption that the secondary spheres are clones [ ] . the technique of spheroid culture was originally developed for neurobiology and then adapted to cscs [ ] . apart from in vitro methods, the "gold standard" for csc is "neoplasia-initiating cells" that regenerate detectable neoplastic populations as xenografts in immunodeficient mice in vivo [ ] . cscs, isolated from either a fresh tumor or an established cell line, are transplanted to initiate tumor growth in immunocompromised recipient animals. different models are available: athymic nude mice, non-obese diabetic/ severe combined immunodeficiency (nod/scid) mice, and nod/scid interleukin- receptor gamma chain null (il rg −/− ) (nsg) mice. the abundance of cscs in a tumor sample can be estimated as the xenotransplantation yield, which confirms the presence of cscs by limiting dilution analysis [ ] . many compounds (natural, natural-derived, synthetic) have been reported as csc-targeting [ , , ] . two strategies are available for compound identification: a pathway-specific approach and a general high throughput, mass screening approach. the first yielded cyclopamine from the plant corn lily veratrum californicum; it targets glioblastoma cscs via the hedgehog pathway but can act as a teratogen that causes cyclopia. the second yielded salinomycin from the bacterium streptomyces albus; it is toxic to humans so its use is confined to the poultry industry, as a coccidiostat for treating a parasitic protozoan disease [ ] . for clinical uses, medicinal chemists must modify to remove teratogenicity or toxicity. identified compounds must target only cscs and not normal stem cells. even though cscs differ from the bulk cancer cells, they may share gene expression and signaling pathways with normal stem cells. csc-targeting phytochemicals and drugs are discussed in the following sections. dietary phytochemicals have advantages over other compounds [ ] : ( ) they usually have very low or no toxicity, in contrast to most chemotherapeutic drugs; ( ) they are present in commonly consumed food that is readily available to most people in daily life; ( ) they have shown potential as adjuvants to chemotherapy. originally, they were studied for cancer chemo-prevention [ ] . as initiation (chemoprevention) and growth (cancer chemotherapy) may share common molecular mechanisms, they were applied to cancer therapy. furthermore, phytochemicals can overcome drug resistance in ovarian and other cancers [ ] . they act on genes and non-coding gene regulatory pathways (microrna) [ ] . most importantly, phytochemicals target cscs [ ] [ ] [ ] [ ] [ ] [ ] . commenting on cscs and dietary phytochemicals, kim et al. ( ) wrote: "a diet-induced shift from deregulation to regulation in cancer stem cells could have profound influence on cancer relapses and therefore is of immense societal importance" [ ] . instead of diet, here we propose the use of csc-targeting phytochemical-drug combinations for cancer therapy. our laboratories have been studying the phytochemicals curcumin, quercetin and egcg. these polyphenols act synergistically with cisplatin (conventional anti-cancer drug) in growth inhibition of drugresistant ovarian cancer cell lines [ , ] . these compounds are active against cscs in vitro, as well as against cancer and other diseases in animal models. some are in clinical trials, though to date none has been approved for cancer treatment. here, we discuss four csc-targeting phytochemicals: curcumin, resveratrol, egcg and genistein. (see table for select cancer clinical trials of the four phytochemicals, from https://clinicaltrials.gov/.) curcumin, a diferuloylmethane, is the biological principle from the indian spice turmeric that gives curry powder the yellow color. turmeric is produced from rhizome of the plant curcuma longa; it has multiple utilities and health benefits in traditional indian medicine (from insect bites to wound healing). a widely studied property of curcumin is anti-inflammation. curcumin inhibits many pro-inflammatory gene products, including enzymes (inducible nitric oxide synthase, inos), transcription factors (nuclear factor kappa b, nfκb), cytokines (tumor necrosis factor, tnfα) and chemokines (interleukin- , il- ) [ ] . using the lipopolysaccharide (lps)-induced murine sepsis model, we reported curcumin ingestion by gavage down-regulated inos gene expression in the murine liver [ ] . curcumin may suppresses carcinogenesis by down-regulating inflammation, which supports cancer cell survival, proliferation and invasion [ ] . another widely studied anti-cancer property of curcumin is csctargeting [ ] [ ] [ ] [ ] [ ] . (see table for select cellular targets of phytochemicals and drugs in cscs.) cscs that resist conventional anti-cancer drugs are susceptible to curcumin. curcumin acts on stem cell signaling pathways implicated in the process of carcinogenesis, including wnt, notch, hedgehog, and signal transduction and activator (stat). for example, in hepatocellular carcinoma cscs, tsai et al. ( ) reported that curcumin inhibited sp, invasion, emt and reduced tumor size and lung metastasis in a nude mice xenograft model. immunoblots revealed that the sphingosine -phosphate receptor (sipr ) signaling pathway was inhibited [ ] . in cell culture, subramaniam et al. ( ) reported that curcumin-induced apoptosis of esophageal cancer cells and decreased esophageal csc spheroid size and number. the molecular mechanism was inhibition of the notch pathway, seen as a decrease in rna and protein expression of γ secretase, notch- protein and its ligand jaggard- [ ] . the investigators also found down-regulated oncomir mirna (mir- , mir- a) and up-regulated tumor suppressor mirna (let- a mirna) in the curcumin-treated esophageal csc spheroids. similar to the esophageal example, for colorectal cscs, ramasamy et al. ( ) reported curcumin-induced epigenetic modifications, including the methylation of epidermal growth factor receptor (egfr) promoter, expression of microrna oncomirs relevant to metastasis (for emt), as well as suppression of csc markers (cd , aldh + ) [ ] . thus, curcumin modifies epigenetically by inducing specific methylation changes and regulating microrna expression. huminiecki et al. ( ) have reviewed the functional genomic studies of curcumin from microarray, methylation array, microrna array to rna-seq and concluded that curcumin has "powerful effects" on gene expression, including "genes involved in cell signaling, apoptosis, and the control of cell cycle" [ ] . curcumin can induce csc differentiation [ ] . zhuang et al. ( ) reported that curcumin promoted differentiation of glioma cscs and induced autophagy, using immunofluorescence to show decrease in stemness markers (cd ) and increase in differentiation markers (βiii tubulin) [ ] . curcumin can alter chemo-sensitivity. it synergizes with conventional drugs and has specificity for cscs. in vitro, curcumin enhanced cisplatin effects on non-small cell lung csc [ ] . we found that curcumin ( μm daily for days via medium change) reduced sp of the c rat glioma cells, possibly via inhibition of the drug transporter [ ] . in vivo, curcumin enhanced paclitaxel effects on brain tumor cscs; it sensitized breast cscs to mitomycin c in a nude mice xenograft model [ ] . several reasons have been proposed to explain the csc-specific action and low adversity towards normal stem cells [ ] . curcumin uptake may be greater in cscs, and curcumin may target the csc microenvironment which obviously differs from those of normal stem cells. for example, buhrmann et al. ( ) reported that curcumin sensitized colorectal cscs towards fluorouracil ( -fu) and suppressed the crosstalk between cscs and stromal fibroblasts in the tumor niche, using co-culture and assaying emt markers by immunoblotting [ ] . resveratrol is a stilbene ( , ′, -tri-hydroxy-trans-stilbene) from grapes, peanuts and pine nuts, and is present in red, but not white, wine because it is associated with the skin of grapes. it is a pleiotropic agent with multiple cellular targets. for example, it inhibits the mammalian target of rapamycin (mtor) pathways and cyclooxygenase (cox) enzymes to play roles in inflammatory diseases, diabetes, obesity, cardiovascular diseases, neurological disorders and cancer [ ] [ ] [ ] . resveratrol also activates amp-activated protein kinase (ampk, a key nutrient sensor) and sirtuin deacetylases, leading to lifespan extension in yeasts, nematodes, fruit flies, fish and obese mice (but not normal mice) [ ] . it retards the ageing process in mammals, similar to caloric restriction. furthermore, resveratrol is a phytoalexin (compound produced by plants against pathogens, such as fungal infections to the grape plant) and we found this property can be harnessed for inhibiting the growth of human cutaneous fungal species, thus potentially useful for tinea (ringworm, athlete's foot) [ ] . in cancer, resveratrol can target cscs. park and pezzuto ( ) have reviewed the molecular alterations resulting from resveratrol intervention in different breast, prostate, lung and colorectal cancer models [ ] . the general approach in these studies is to isolate the cscs, assay the effects of resveratrol in vitro and in vivo (comparing cscs with the bulk cancer cells) and determine which molecular mechanisms are modulated. for example, fu et al. ( ) reported resveratrol cytotoxicity to breast cscs in vitro [ ] . mechanistically, immunofluorescence and immunoblot of microtubule-associated protein light chain (lc ) and β catenin proteins showed that it induced autophagy and suppressed the wnt pathway. resveratrol also inhibited spheroid formation, decreased aldh + cells and decreased tumor size in murine xenografts. shankar et al. ( ) reported resveratrol inhibited pancreatic csc spheroid formation via the induction of apoptosis, inhibition of mrna expression of the pluripotency maintenance transcription factors (oct , nanog), as well as interference with emt [ ] . similar inhibition was also observed in a transgenic mouse model of pancreatic ductal adenocarcinoma. with glioblastoma multiforme, found that cscs pre-treated with resveratrol in vitro were less tumorigenic, as the xenografted scid mice survive better than the untreated counterpart [ ] . the investigators also performed microarray analyses and shrna confirmation to compare the effects of resveratrol on cscs (cd + ) and non-cscs (cd -). resveratrol inhibited "stemness" gene expression and induced csc differentiation via suppression of the stat pathway. resveratrol, like curcumin, can be csc-specific and spare the normal stem cells. pandey et al. ( ) reported that resveratrol blocked gene expression of fatty acid synthase and inhibited breast csc growth, but the enzyme in normal human mammary epithelial cells was not susceptible [ ] . fu et al. ( ) reported resveratrol was cytotoxic to breast cscs but not normal breast epithelial cells [ ] . similarly, sayd et al. ( ) reported that resveratrol blocked enzymatic induction of sirtuin activity and inhibited the growth of glioma cscs, but had no effect on normal neural stem cells (from fetal brains) [ ] . epigallocatechin gallate (egcg), a polyphenol (flavone- -ol) from the plant camellia sinensis, is the major component in green tea. its many health benefits include cancer chemoprevention and treatment, reduction of atherosclerosis, hypercholesterolemia, alzheimer's and other ageing-related diseases. before cscs, egcg was first studied for its effect on normal stem cells. chen components, including egcg, inhibited normal rat neurosphere adhesion and migration [ ] . egcg has pleiotropic activity on multiple cellular targets, both genetic and epigenetic (including dna methyltransferase, nfκb, ampk signaling pathway) [ ] [ ] [ ] . it can act on its target with very impressive sensitivity, kd of . nm for the kd laminin receptor [ ] . in cancer, egcg can target cscs. it inhibited aldh activity, spheroid formation, and expression of "stemness" genes (oct and nanog) in human neuroblastoma cscs [ ] . similar results were observed in breast cscs. egcg treatment significantly decreased the expression of nanog, the number of aldh + cells, and the tumor volume in mouse xenografts [ ] . furthermore, chen et al. ( ) reported egcg inhibited the spheroid formation of colorectal cscs, the mechanism being inhibition of the wnt pathway, as shown by a decrease in β catenin in immunoblot [ ] . egcg works in combination with other phytochemicals and conventional drugs to enhance anti-cancer effects, targeting bulk cancer cells and cscs [ ] . it synergized with quercetin to decrease spheroids, inhibited activation of emt and increased apoptosis in prostate cscs [ ] . it synergized with temozolomide to decrease spheroids and pglycoprotein synthesis in glioma cscs [ ] . we found that egcg acted synergistically with cisplatin to inhibit the cisplatin-resistant ovarian cancer cell line c in cell culture [ ] . in vivo, the egcg and cisplatin combination decreased tumor formation in xenografts of head and neck, and nasopharyngeal cscs [ , ] . it induced chemo-sensitivity to cisplatin by enhancing apoptosis and inhibiting the phosphorylation of stat in nasopharyngeal cscs [ ] . these findings suggest its use as an adjuvant in cancer therapy [ ] . genistein, an isoflavone ( , , -trihydroxyisoflavone) from the legume plant glycine max, is found in soybeans. it has been classified as a phytoestrogen because it binds estrogen receptor. genistein is the active ingredient in soy-rich food that contributes to the lower rates of prostate and breast cancers in china and japan, as compared to western countries. besides as anti-cancer agent, genistein has other health benefits, including osteoporosis, heart diseases and cognition [ ] [ ] [ ] [ ] . genistein has multiple cellular targets and acts on a spectrum of protein tyrosine kinases and dna topoisomerase ii. it targets cscs in solid tumors. huang et al. ( ) reported genistein inhibited spheroids, "stemness" (oct and nanog gene expression) and reduced xenograft tumor volume of gastric cscs [ ] . genistein also inhibited drug transporter and extracellular signal-regulated kinase (erk) pathway in these cscs. in both breast and prostate cscs, genistein down-regulated the hedgehog pathway (reducing gli gene expression) and decreased spheroid formation in cell culture and tumor volume in xenografts [ , ] . in chronic myelogenous leukemia (cml), genistein reduced the leukemic progenitor cells by inhibiting expression of the tyrosine kinase coded from the breakpoint cluster region/abelson murine leukemia viral oncogene homolog (bcr/abl) fusion gene [ ] . however, as a protein tyrosine kinase inhibitor, genistein targets both leukemic stem cells and normal stem cells. genistein acts synergistically with conventional anti-cancer agents; it also targets the csc microenvironment. it overcame docetaxel resistance, and decreased the tumor size of docetaxel-resistant prostate cscs (than either compound administered separately) [ ] . breast adipose tissue contributes to breast cancer development. montales et al. ( ) found that genistein acted on the csc niche and inhibited the differentiation of mammary stromal fibroblast-like cells to adipocytes [ ] . the mechanism was inhibition of pparγ and fatty acid synthase gene expression. interestingly, the investigators discovered genistein's biphasic effect, only seen in low dose ( nm) but not high dose ( μm). montales et al. ( ) also found the effect of genistein to be transferrable [ ] . when fed to mice at concentrations present in soy protein isolate ( mg/kg food), their sera could inhibit the spheroid formation of human breast cscs when added at - % in cell culture. this suggests the presence of "csc inhibiting factors" in the circulation after genistein consumption. furthermore, genistein has transgenerational effect. maternal dietary supplementation with genistein leads to dna hyper-methylation in the embryo, and this methylation state is maintained until adulthood. the color change in the fur of genistein-fed agouti mice indicates an epigenetic biosensor for nutritional and environmental alterations on the fetal genome. phytochemicals such as genistein modify the epigenome and the effect starts early in embryogenesis [ ] . fda-approved small-molecule drugs for some diseases/disorders have been shown to target cscs by the process of drug repurposing. the nih national center for advancing translational sciences (ncats) defines: "repurposing generally refers to studying drugs that are already approved to treat one disease or condition to see if they are safe and effective for treating other diseases". the rationale is to expedite drug discovery. ncat estimates years for a new drug development. therefore, the strategy is "to reduce this time frame, decrease costs and improve success rates …. many agents approved for other uses already have been tested in humans, so detailed information is available on their pharmacology, formulation and potential toxicity. because repurposing builds upon previous research and development efforts, new candidate therapies could be ready for clinical trials quickly …" (https://ncats.nih.gov/preclinical/repurpose#learn-more). drug repurposing, also known as drug repositioning, drug re-tasking, drug reprofiling or therapeutic switching, is a way to obtain fast and cost-efficient drug discovery, since this strategy effectively enables the preclinical studies to be bypassed. resources (drugsurv, drug repurposing hub) are available for this "new tricks for old drugs" [ , [ ] [ ] [ ] . investigators can perform experimentation in silico, in vitro or in vivo in search for new targets of existing drugs. for cscs, drug libraries are screened in assays of csc inhibition, as shown below for niclosamide and thioridazine. here, we discuss four csc-targeting drugs: metformin, niclosamide, thioridazine and chloroquine. probably the most promising discovery is metformin, a synthetic biguanide derived from the herb french lily (galega officianalis) [ ] . it is an inexpensive and long-approved drug for type diabetes, prescribed to an estimated million individuals worldwide. it modulates energy metabolism by targeting ampk and other ampk-independent effects. besides diabetes, metformin has been shown to be applicable to cardiovascular and neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . it is in a clinical trial for longevity and ageing [ ] . metformin was explored for csc-targeting because it targets multiple signaling pathways (wnt, ampk, nfκb) [ , ] . metformin has been used in combination therapy. for example, it synergized with the conventional combination of -fluorouracil ( -fu), epirubicin and cyclophosphamide (fec chemotherapy) in inhibiting breast cscs derived from spheroids [ ] . it enhanced the effect of denosumab, an antibody against rankl (receptor activator of nfκb ligand, a cytokine), in inhibiting breast csc spheroids [ ] . it enhanced the sensitivity of pancreatic cscs to gemcitabine in vivo, significantly reducing the tumor volume in murine xenografts [ ] . when given in combination, metformin and doxorubicin showed the advantage of targeting both breast cscs and non-stem cancer cells, by reducing the overall tumor growth and tumor remission in nude mice [ ] . synergy was found with paclitaxel and carboplatin in prostate and lung cscs murine xenografts [ ] . for phytochemicals, montales et al. ( ) reported that metformin and genistein targeted colon csc spheroid formation and proliferation, and the combination increased the effectiveness of -fu [ ] . ning et al. ( ) reported that both curcumin and metformin individually inhibited pancreatic csc spheroids [ ] , the two compounds should be tested as a combination. these examples suggest a combinational strategy for metformin to target cscs and cancers [ ] . the next most promising existing drug is niclosamide, an anthelminthic against tapeworms [ ] . it is also active against viruses (severe acute respiratory syndrome, sars virus), possibly because it blocks proton carriers. niclosamide was explored for csc-targeting based on the general association that a developmental stage of tapeworm, the metacestode, resembles cancer in having uncontrolled proliferation, invasion and metastasis, and can be difficult to kill without collateral damage to the surrounding host tissues [ ] . niclosamide can block multiple csc signaling pathways, including wnt, notch, stat and nfκb [ ] . wieland et al. ( ) selected niclosamide as the lead for glioblastoma targeting after screening the member killer plate compound library [ ] . simultaneous inhibition of multiple pathways (wnt, notch, mtor, nfκb) was indicated when niclosamide pretreatment inhibited glioblastoma xenograft development in nude mice. from drugs in the lopac chemical library, [ ] and yo et al. ( ) [ ] selected niclosamide for its inhibition of breast and ovarian cscs, respectively. niclosamide inhibited spheroid formation and reduced tumor volume of xenografts in nod/scid mice. it acted on cisplatin-resistant ovarian cscs. spheroids from patient-derived cd + and aldh + ovarian cancer were susceptible to niclosamide, with inhibition of the wnt pathway (shown by β catenin immunoblot) [ ] . furthermore, it complements platinum drugs. niclosamide in combination with carboplatin inhibited primary ovarian cscs [ ] and in combination with cisplatin inhibited breast csc spheroid formation and reduced tumor size of xenografts in nude mice [ ] . thioridazine, a phenothiazine, is dopamine receptor antagonist originally prescribed as an anti-psychotic drug for schizophrenia in the s. withdrawn by norvatis in due to cardiac side effects, the generic version is still available. interest in thioridazine has recently been revived as anti-microbial against methicillin-resistant staphylococcus aureus (mrsa) and multidrug-resistant mycobacterium tuberculosis [ ] . thioridazine targets cscs. it can inhibit cscs from a spectrum of origins: myeloid leukemia, glioblastoma, lung, liver, ovarian and breast cancers [ ] [ ] [ ] [ ] [ ] . it inhibits csc spheroid formation and induces apoptosis in vitro, and the treated cells show reduced xenograft tumor volume in mice. sachlos et al. ( ) selected thioridazine in their screening for acute myeloid leukemia (aml) csc-specific but not normal pluripotent stem cell-reactive molecules from compounds of the nih clinical collection and canadian collection [ ] . thioridazine was found to induce aml differentiation; it also synergized with cytosine arabinoside (cytarabine, arac) in aml csc killing. in glioblastoma cscs, it induced autophagy (seen as expression of biomarker lc ) [ ] . in lung, hepatoma and breast cscs, it induced apoptosis in association with activation of caspases, inhibition of "stemness" gene expression (oct , cd ), and inhibition of the mtor pathway, respectively [ ] [ ] [ ] . synergy with conventional drug has been shown: thioridazine was co-delivered with doxorubicin in mixed polymeric micelles to target both breast cscs and bulk cancer cells [ ] . we found that thioridazine and curcumin may act synergistically in inhibiting spheroids from ovarian cancer cells, with the combination more effective than either compound alone. (see fig. for inhibition of spheroids by thioridazine and curcumin). chloroquine, a -aminoquinoline, is an anti-malarial [ ] . first discovered in b y hans andersag at bayer, it was ignored until rediscovery by us army during world war ii. highly effective and well tolerated, chloroquine remains the drug of choice for malaria. its accumulation within lysosomes raises organelles' ph and inhibits lysosomal function in the malaria parasite. chloroquine is also used for immunosuppression in rheumatoid arthritis. chloroquine, either independently or in combination with conventional drugs, has efficacy against several cancers. it inhibits cscs from a spectrum of origins: glioma, liver, pancreatic, urothelial, ovarian and breast cancer [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it inhibits autophagy and spheroid formation; it induces csc apoptosis. these effects have been demonstrated in primary glioma cscs, cd + liver cscs enriched by deprivation of oxygen and nutrients, pancreatic cscs, urothelial carcinoma cscs, and breast cscs. the csc-targeting chloroquine shows synergistic effects with conventional therapy [ ] . it sensitizes radiation and chemotherapeutic agents in cancer [ , ] . for example, chloroquine increased the cytotoxicity of gemcitabine-mitomycin combination against urothelial carcinoma cscs [ ] . it synergized with gemcitabine to decrease tumor volume in pancreatic csc xenografts [ ] , and with carboplatin in breast csc xenografts [ ] . we discuss four phytochemicals and four drugs that target cscs, and view them as the most promising for a novel approach of phytochemical-drug combination. there are others. exploring csc-targeting phytochemicals is a growing field. other dietary phytochemicals include quercetin, a flavonol found in common fruits and vegetables such as apples, cranberries and onions; sulforaphane, an isothiocyanate found in cruciferous such as broccoli, cauliflower and kale; indole- -carbinol, another compound from cruciferous vegetables; and cucurbitacin i, from cucumber. non-dietary phytochemicals include parthenolide from herbal medicine feverfew plant (tanacetum parthenium), celastrol from traditional chinese medicine thunder god vine (tripterygium wilfordii), berberine from herbal medical plant chinese goldthread (coptis chinensis), oxymatrine from chinese herbal medical plant sophora flavescens, silybin (silibinin) from milk thistle (silybum marianum), and gossypol from cotton plant (genus gossypium) [ ] . similarly, the list of csc-targeting existing drugs is expanding. other repurposed drugs include anti-allergy drug tranilast, cholesterollowering statins (simvastatin and lovastatin), and anti-alcoholism drug disulfiram [ , , , , ] . we anticipate more to come. for challenges we start with the safety issue. with gras status dietary phytochemicals are apparently safe. for example, when curcumin was given to cancer patients at mg/day for months, only minor adverse effects were observed. daily oral doses of egcg, for weeks at mg/day in volunteers, caused only minor adverse effects. a single oral dose of resveratrol at g in volunteers caused only minor adverse effects [ ] . with prior fda approval, repurposed drugs are also presumably safe. although individual compounds are safe, for the selected synergistic csc-targeting phytochemical-drug combination, pharmacokinetics and pharmacodynamics features, toxicity profiles, are still needed because this information is lacking. current clinical trials may yield useful data. (see table for phytochemical clinical trials.) molecular targets of phytochemicals are under scrutiny. recent findings suggest that promiscuity (multiple targets) results from perturbation of cellular membranes, leading to alteration of multiple membrane protein functions [ ] . this multi-target nature of polyphenolic phytochemicals hamper therapeutic development because, in high throughput drug screening assays, phytochemicals (including curcumin, resveratrol, egcg and genistein) are known as "pan-assay interference compounds" (pains) and "invalid metabolic panaceas" (imps) for their non-specific inhibitions [ ] [ ] [ ] . whereas there may be truth to the designation, the fact that phytochemicals (such as curcumin) will cause global changes in gene expression shows there are more than mere non-specific protein interactions [ ] . nonetheless, multi-target interactions, specific or non-specific, at the protein or nucleic acid level, will be a plus in csc-targeting as long as normal stem cells are spared. additional concerns are phytochemical bioavailability in vivo and product quality. for some polyphenolic compounds, effects in vivo, although significant, are less prominent than ones observed in vitro [ ] . the bioavailability issue has been addressed with enhancing compounds, such as piperine from the spice black pepper (piper nigrum), which amplifies the effect of phytochemicals (including curcumin). bioavailability can be aided by formulations and special delivery systems, such as nanoparticles for curcumin and micelles for niclosamide [ , [ ] [ ] [ ] . curcumin is an example which we managed to devise a measure to enhance bioavailability. by oral gavage to mice on empty stomach, we demonstrated in vivo effect: curcumin inhibition of the inflammatory inos in murine liver (beneficial outcome) and exacerbation of murine visceral leishmaniasis, a protozoan parasitic disease (detrimental outcome) [ , ] . another concern is reliability of the source of phytochemicals. consistent and unadulterated phytochemicals are essential, for example, curcumin as curcumin c complex from sabinsa and egcg as polyphenon e from mitsui norin (as used in some clinical trials, see table ) [ ] . (repurposed drugs, as synthetic compounds, do not have this problem.) yet another approach is the synthesis of better analogs and derivatives (such as difluorinated-curcumin and various chloroquine analogs), but they are beyond the scope of this review [ , , , ] . discrepancies in phytochemical research results are another challenge. whereas we found curcumin inhibited sp in rat glioma [ ] , found curcumin induced human glioma cscs [ ] . kakarala et al. ( ) reported curcumin inhibited both breast cscs and normal human breast stem cells, thus affecting both cscs and normal stem cells [ ] . these differences can be attributed to variations in dosage, length of treatment periods, grade and stability of compounds used, or other experimental conditions [ ] . polyphenolic phytochemicals have antioxidant capacities against reactive oxygen species (ros), but under special conditions they exhibit pro-oxidant capacity [ ] . whereas ros are harmful in general, the removal of too much ros interferes with bodily functions, as seen in the warning presented by scientists to the food industry [ ] . related to dosage is the concept of hormesis [ ] , which suggests the fundamental nature of the dose-response curve is neither linear nor threshold, but u-or j-shaped. a low dose stimulatory response is the hormetic effect, representing overcompensation in response to disruptions in homeostasis. a biphasic dose response is observed. at high concentrations, phytochemicals can be toxic, whereas sub-toxic doses may induce adaptive stress responses. hormetic mechanisms of action have been proposed to underlie many of the health benefits of phytochemicals [ ] . protective effect of resveratrol is only observed in low dose but not high dose in a mouse colon cancer model [ ] . similar dose effects are seen in genistein [ ] . for repurposed drugs, most research results indicate csc-targeting, there are discordance. sancho et al. ( ) reported metformin-resistant pancreatic cscs [ ] and xin et al. ( ) reported metforminresistant liver cscs [ ] . in these studies, the effective dose of metformin is much higher than other drugs or phytochemicals. moreover, asiedu et al. ( ) reported heterogeneity in metformin response in both patient-derived and cancer cell lines [ ] . chloroquine is "a double-edged sword of autophagy", causing severe kidney damage [ ] . whether it is the compound's pleiotropic nature that leads to unexpected adverse effects is still unclear. for repurposed drugs, studies are needed to determine that they can reach the csc niche [ ] . combinational treatment has a long history in cancer chemotherapy. the first successful example is the treatment of childhood leukemia with "vamp", four drug combination (vincristine, amethopterin, -mercaptopurine, prednisone) in [ ] . our approach continues this tradition. we recommend four phytochemicals (curcumin, resverestrol, egcg, genistein) and four drugs (metformin, niclosamide, thioridazine, chloroquine). this choice is in sync with selections by the halifax project, aiming at multi-targeted compounds for cancer prophylaxis and treatment [ ] . we advocate a novel approach of csc-targeting dietary phytochemical-repurposed drug combinations and look forward towards clinical applications. in the united states, cancer drug prices are getting out of hand. the march report to president donald trump stated: "the president's cancer panel concluded that addressing the dramatic rise of cancer drug prices must be made a national priority. … innovative drugs offer new hope for patients to achieve long-term remissions-even cures-but virtually all new cancer drugs enter the market with a price tag that exceeds $ , per year and, increasingly, much higher" (https://prescancerpanel.cancer.gov/report/drugvalue). instead of new cancer drugs, the older repurposed drugs in combination with dietary phytochemicals are inexpensive and may work just as well. cancer is a worldwide problem affecting all nations rich and poor. on this topic, sullivan et al. ( ) warned: "treating cancer with the latest drugs and techniques is costly and will not improve survival globally" [ ] . they asserted: "cancer is on the rise" and concluded the focus should be "on building infrastructure, and delivering affordable, equitable and effective care". our idea of csc-targeting phytochemical-drug combination fits seamlessly in this scenario as effective and affordable cancer treatment for the global population. for world health organization (who), cancer is non-communicable chronic disease, and the increase in cancer is due to an aging population and lifestyle changes. there is an urgent need for affordable cancer treatment. whether independently administered or in combination (as adjuvant with current therapy), development of a csc-targeting dietary phytochemical-repurposed drug combination will be affordable, effective, and low toxicity. our goal is to increase the scientific community's awareness and build a momentum towards conducting well-controlled clinical trials, done under uniform experimental conditions and in a double-blinded manner, to fully validate this novel combinatorial approach. since we propose to combine dietary phytochemicals and repurposed drugs, i.e. non-patentable natural products and patent-expired older drugs, we envision financial supports for this endeavor have to come from governmental and philanthropic resources. the novel approach is ready for action. none declared. designing a broad-spectrum integrative approach for cancer prevention and treatment hallmarks of cancer: the next generation tumorigenesis: it takes a village cancer stem cells-perspectives on current status and future directions: aacr workshop on cancer stem cells dietary phytochemicals target cancer stem cells for cancer chemoprevention existing drugs and their application in drug discovery targeting cancer stem cells inhibition of growth and sensitization to cisplatin-mediated killing of ovarian cancer cells by polyphenolic chemopreventive agents epigallocatechin- -gallate delivers hydrogen peroxide to induce death of ovarian cancer cells and enhances their cisplatin susceptibility mechanisms of combined action of different chemopreventive dietary compounds: a review promiscuous drugs compared to selective drugs (promiscuity can be a virtue) commentary: novel therapies for cancer: why dirty might be better cancer stem cells: an evolving concept cancer stem cells: basic concepts and therapeutic implications multiple drug resistance in cancer revisited: the cancer stem cell hypothesis tumourinitiating cells: challenges and opportunities for anticancer drug discovery targeting cancer stem cells to suppress acquired chemotherapy resistance cancer stem cells (cscs) in drug resistance and their therapeutic implications in cancer treatment chemical approaches to targeting drug resistance in cancer stem cells concise review: stem cells and epithelialmesenchymal transition in cancer: biological implications and therapeutic targets aldehyde dehydrogenases and cancer stem cells cancer stem cell definitions and terminology: the devil is in the details eyes wide open: a critical review of sphere-formation as an assay for stem cells targeting cancer stem cells with defined compounds and drugs the use of natural products to target cancer stem cells implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds cancer chemoprevention with dietary phytochemicals overcoming drug resistance by phytochemicals dietary intervention by phytochemicals and their role in modulating coding and noncoding genes in cancer targeting cancer stem cells with phytochemicals novel strategies targeting cancer stem cells through phytochemicals and their analogs phytochemicals as innovative therapeutic tools against cancer stem cells therapeutic effectiveness of anticancer phytochemicals on cancer stem cells emerging importance of dietary phytochemicals in fight against cancer: role in targeting cancer stem cells cancer stem cells: potential target for bioactive food components in vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties new insights into therapeutic activity and anticancer properties of curcumin the role of cancer stem cells in the anti-carcinogenicity of curcumin targeting cancer stem cells by curcumin and clinical applications curcumin and cancer stem cells: curcumin has asymmetrical effects on cancer and normal stem cells targeting colorectal cancer stem cells using curcumin and curcumin analogues: insights into the mechanism of the therapeutic efficacy curcumin suppresses phthalate-induced metastasis and the proportion of cancer stem cell (csc)-like cells via the inhibition of ahr/erk/sk signaling in hepatocellular carcinoma curcumin induces cell death in esophageal cancer cells through modulating notch signaling the functional genomic studies of curcumin curcumin promotes differentiation of glioma-initiating cells by inducing autophagy curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p and cyclin d -mediated tumour cell inhibition in non-small cell lung cancer cell lines curcumin inhibits the side population (sp) phenotype of the rat c glioma cell line: towards targeting of cancer stem cells with phytochemicals curcumin improves the tumoricidal effect of mitomycin c by suppressing abcg expression in stem celllike breast cancer cells curcumin suppresses crosstalk between colon cancer stem cells and stromal fibroblasts in the tumor microenvironment: potential role of emt the pharmacology of resveratrol in animals and humans the molecular targets of resveratrol effects of resveratrol, curcumin, berberine and other nutraceuticals on aging, cancer development, cancer stem cells and micrornas lifespan and healthspan extension by resveratrol antimicrobial effect of resveratrol on dermatophytes and bacterial pathogens of the skin resveratrol inhibits breast cancer stem-like cells and induces autophagy via suppressing wnt/beta-catenin signaling pathway resveratrol inhibits pancreatic cancer stem cell characteristics in human and krasg d transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition resveratrol suppresses tumorigenicity and enhances radiosensitivity in primary glioblastoma tumor initiating cells by inhibiting the stat axis resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase sirtuin- activity is required for glioma stem cell proliferation arrest but not necrosis induced by resveratrol capillary electrophoretic determination of the anine, caffeine, and catechins in fresh tea leaves and oolong tea and their effects on rat neurosphere adhesion and migration multitargeted therapy of cancer by green tea polyphenols new insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin -gallate absorption, metabolism, anti-cancer effect and molecular targets of epigallocatechin gallate (egcg): an updated review anti-cancer effect of egcg and its mechanisms epigallocatechin gallate inhibits sphere formation of neuroblastoma be( )-c cells epigallocatechin- -gallate inhibits stem-like inflammatory breast cancer cells epigallocatechin- -gallate inhibits colorectal cancer stem cells by suppressing wnt/beta-catenin pathway synergistic enhancement of anticancer effects on numerous human cancer cell lines treated with the combination of egcg, other green tea catechins, and anticancer compounds inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics egcg inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of p-glycoprotein inhibition epigallocatechin- -gallate attenuates head and neck cancer stem cell traits through suppression of notch pathway epigallocatechin- -gallate inhibits nasopharyngeal cancer stem cell self-renewal and migration and reverses the epithelial-mesenchymal transition via nf-kappab p inactivation egcg inhibits the growth and tumorigenicity of nasopharyngeal tumor-initiating cells through attenuation of stat activation green tea polyphenol epigallocatechin- -gallate (egcg) as adjuvant in cancer therapy multi-targeted therapy of cancer by genistein the effect of genistein aglycone on cancer and cancer risk: a review of in vitro, preclinical, and clinical studies understanding genistein in cancer: the "good" and the "bad" effects: a review soy isoflavone: the multipurpose phytochemical (review) genistein-inhibited cancer stem celllike properties and reduced chemoresistance of gastric cancer genistein decreases the breast cancer stem-like cell population through hedgehog pathway genistein inhibits the stemness properties of prostate cancer cells through targeting hedgehog-gli pathway selection of myeloid progenitors lacking bcr/abl mrna in chronic myelogenous leukemia patients after in vitro treatment with the tyrosine kinase inhibitor genistein repression of mammary adipogenesis by genistein limits mammosphere formation of human mcf- cells repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells maternal genistein alters coat color and protects avy mouse offspring from obesity by modifying the fetal epigenome can you teach old drugs new tricks? drugsurv: a resource for repositioning of approved and experimental drugs in oncology based on patient survival information the drug repurposing hub: a next-generation drug library and information resource repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions repurposing metformin: an old drug with new tricks in its binding pockets metformin against cancer stem cells through the modulation of energy metabolism: special considerations on ovarian cancer metformin targets multiple signaling pathways in cancer metformin ameliorates core deficits in a mouse model of fragile x syndrome metformin as an anti-cancer agent: actions and mechanisms targeting cancer stem cells a trial for the ages metformin synergizes -fluorouracil, epirubicin, and cyclophosphamide (fec) combination therapy through impairing intracellular atp production and dna repair in breast cancer stem cells metformin inhibits rankl and sensitizes cancer stem cells to denosumab metformin increases sensitivity of pancreatic cancer cells to gemcitabine by reducing cd + cell populations and suppressing erk/p s k signaling metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells bulk pancreatic cancer cells can convert into cancer stem cells(cscs) in vitro and compounds can target these cscs combinational strategies of metformin and chemotherapy in cancers multi-targeted therapy of cancer by niclosamide: a new application for an old drug the genomes of four tapeworm species reveal adaptations to parasitism niclosamide, an old antihelminthic agent, demonstrates antitumor activity by blocking multiple signaling pathways of cancer stem cells anticancer effects of niclosamide in human glioblastoma drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells growth inhibition of ovarian tumor-initiating cells by niclosamide inhibition of wnt/beta-catenin pathway by niclosamide: a therapeutic target for ovarian cancer combined niclosamide with cisplatin inhibits epithelial-mesenchymal transition and tumor growth in cisplatin-resistant triple-negative breast cancer thioridazine: resurrection as an antimicrobial agent? br identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data thioridazine has potent antitumor effects on lung cancer stem-like cells roles of dopamine receptors and their antagonist thioridazine in hepatoma metastasis thioridazine inhibits angiogenesis and tumor growth by targeting the vegfr- /pi k/mtor pathway in ovarian cancer xenografts co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies chloroquine or chloroquine-pi k/akt pathway inhibitor combinations strongly promote gammairradiation-induced cell death in primary stem-like glioma cells autophagy contributes to the survival of cd + liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine gemcitabine and mitomycin induced autophagy regulates cancer stem cell pool in urothelial carcinoma cells autophagy inhibition reduces chemoresistance and tumorigenic potential of human ovarian cancer stem cells the autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing dna break repair time to use a dose of chloroquine as an adjuvant to anti-cancer chemotherapies concise review: emerging drugs targeting epithelial cancer stem-like cells repurposing established compounds to target pancreatic cancer stem cells (cscs) phytochemicals perturb membranes and promiscuously alter protein function chemistry: chemical con artists foil drug discovery can invalid bioactives undermine natural product-based drug discovery? the essential medicinal chemistry of curcumin bioavailability and bioefficacy of polyphenols in humans. ii. review of intervention studies targeting cancer with nano-bullets: curcumin, egcg, resveratrol and quercetin on flying carpets, asian pac enriched inhibition of cancer and stem-like cancer cells via stat- modulating niclocelles a synergistic combination of curcumin, epicatechin gallate and resveratrol, repolarizes tumor-associated microglia/macrophages, and eliminates glioblastoma (gbm) and gbm stem cells long-term use of an antiinflammatory, curcumin, suppressed type immunity and exacerbated visceral leishmaniasis in a chronic experimental model curcumin induces glioma stem-like cell formation targeting breast stem cells with the cancer preventive compounds curcumin and piperine antioxidant polyphenols in cancer treatment: friend, foe or foil? antioxidants in foods: state of the science important to the food industry hormesis: from marginalization to mainstream: a case for hormesis as the default dose-response model in risk assessment cancer chemoprevention: evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice myc/pgc- alpha balance determines the metabolic phenotype and plasticity of pancreatic cancer stem cells liver label retaining cancer cells are relatively resistant to the reported anti-cancer stem cell drug metformin patient-and cell type-specific heterogeneity of metformin response chloroquine in cancer therapy: a double-edged sword of autophagy a history of cancer chemotherapy cancer patients need better care, not just more technology we thank drs. david axelrod and dongming sun for helpful comments. we are grateful to claire chen, chase christensen, nana haruna, avery lee and tanjida tasmin for assistance in manuscript preparation. mmc received grant support from american institute for cancer research. rc was the recipient of undergraduate research fund from aresty research center at rutgers university. df received grant support from new jersey health foundation (pc - ). key: cord- - zgljet authors: garcía-serradilla, moisés; risco, cristina; pacheco, beatriz title: drug repurposing for new, efficient, broad spectrum antivirals date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: zgljet emerging viruses are a major threat to human health. recent outbreaks have emphasized the urgent need for new antiviral treatments. for several pathogenic viruses, considerable efforts have focused on vaccine development. however, during epidemics infected individuals need to be treated urgently. high-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics. repurposed drugs can bypass part of the early cost and time needed for validation and authorization. in this review we describe recent efforts to find broad spectrum antivirals through drug repurposing. we have chosen several candidates and propose strategies to understand their mechanism of action and to determine how resistance to antivirals develops in infected cells. viruses are a constant threat to humans, animals and plants. there are hundreds of viruses that can cause disease in humans but there is no treatment for most of them. emerging or reemerging viruses are a public health concern (howard and fletcher, ) . some of these pathogens are the rift valley fever virus (rvfv), dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), ebola virus (ebov), sars-and mers-cov, zika virus (zikv), crimean congo hemorrhagic fever virus (cchfv), severe fever with thrombocytopenia syndrome virus (sftsv), chikungunya virus (chikv) and influenza a virus (iav). recent outbreaks of these viruses are summarized in table . international travel and migrations, globalization of commerce, technology and industry, agriculture development or climate changes are favoring the emergence and reemergence of viruses that could easily spread and potentially become a pandemic (jones et al., ) . a clear example of the potential of an emerging virus to become a pandemic is the emergence of human immunodeficiency virus (hiv) in the s (http:// aidsinfo.unaids.org/) (sharp and hahn, ) . there is no cure for hiv, although antiretroviral treatment can control the virus ( barre-sinoussi et al., ) . in the case of influenza virus there are both vaccines and antiviral drugs. however, these viruses constantly change and antiviral resistance emerges . in , human infections with a new avian influenza virus in china caused considerable concern due to the pandemic potential of this virus (su et al., ) . the experts consider that the virus could gain the ability to spread among people, triggering a global epidemic (su et al., ; tanner et al., ) . due to all these reasons, we urgently need new, efficient, broadspectrum antivirals (howard and fletcher, ; schor and einav, ) . the traditional strategy in drug development of one virus one drug, based in the development of molecules that specifically target a viral protein, has been very successful for certain viruses like hiv or hepatitis c virus (hcv) (barre-sinoussi et al., ; li and de clercq, ) . in fact, most approved antiviral treatments target a viral protein, and thus are usually quite virus specific. however, direct acting antivirals (daa) have some drawbacks. the development of new therapeutic compounds is time and resources consuming (dimasi et al., ) . the whole process from the discovery of a new drug that can potentially treat a specific disease or condition to its approval to be used in humans can take several years (on average to years) (nosengo, ) . the first stage, drug discovery, can take between to years. then preclinical studies, usually in animal models, are carried out. these studies that are aimed to test the efficiency and safety of a molecule in a suitable model before it is tested in humans can go on for about years. if a molecule has shown promising results up to here, then clinical trials in humans are started. these trials, from phase i to phase iii, usually last about years. finally, if a molecule successfully passes all the process it needs to get the approval from the appropriate agency. at the end it is estimated that only about % of the candidate molecules successfully pass the whole process. adding to this the cost of drug development, about - billion dollars or more in some cases (dimasi et al., ) , the one virus one drug strategy is not the most cost efficient approach. in addition, the use of daas can easily lead to the appearance of drug resistant viruses due to the elevated mutation rate of viruses (iyidogan and anderson, ; menendez-arias et al., ; perales et al., ) . to circumvent these limitations other strategies aim to develop molecules that target host cellular factors that are needed for viral replication. although viruses can also find ways to use alternative routes to replicate in a host, emergence of resistant viruses is less likely (lin and gallay, ; zeisel et al., ) . in addition these approaches focused in host cellular pathways needed for viral replication can favor the development of broad spectrum antivirals. there are families of viruses that share certain cellular pathways. thus, drugs that inhibit a specific cellular target could work for several viruses. as a drawback drugs that inhibit a host cell target have higher chances of producing undesirable side effects in the patient compared to drugs that target a viral protein. drug repurposing or repositioning is an alternative approach that can lead to less costly and faster approval of new treatments for viral infections (mercorelli et al., ) . a clear advantage of drug repositioning over traditional drug development is that since the repositioned drug has already passed a significant number of tests, its safety is known and the risk of failure is reduced. although repositioned drugs can most likely skip phase i clinical trials, they still need to go through phase ii and iii trials to test their efficacy for the new condition (nosengo, ) . in some instances these drugs might have lost their patents, as is the case of generic medicines. in these cases, when a new formulation or a new medical use exists for an "old" drug, second medical use patents can be obtained for them. on the other hand, increasing implication of practicing physicians and researchers at academic centers in clinical trials can also help in the repurposing of these drugs (nosengo, ) . additionally, there are thousands of drugs that have already passed clinical trials that confirm their safe use in humans. however, for some reasons, they lack or have poor efficiency to treat the condition under scrutiny and have not reached the market. these collections of drugs are another source of potential antivirals. several drugs in the market have been tested for their potential use as broad spectrum antiviral treatments ( table ). many of these compounds have shown promising results in preclinical studies. below we summarize the main results and antiviral mechanisms of selected candidates for repositioning as broad spectrum antiviral drugs. digoxin is a cardiac glycoside or cardiotonic steroid that has been used for treating certain heart conditions including heart failure, atrial fibrillation, atrial flutter, and paroxysmal atrial tachycardia (gheorghiade et al., ) . cardiac glycosides comprise a large family of natural compounds that share a common steroid structure. digoxin was first isolated from foxglove plant, digitalis lanata, in , although foxglove plant extracts have been used from ancient times to treat heart conditions (gheorghiade et al., ) . digoxin has been shown to block the na + /k + atpase, raising intracellular na + levels. the increase in intracellular na + leads to an increment in intracellular ca + due to inhibition of na + /ca + exchanger. elevated intracellular ca + ultimately leads to an increased contractibility of the cardiac muscle (smith, ) . inhibition of the na + /k + atpase has been shown to have an effect in multiple signaling cascades through modulation of neighboring tyrosine kinases, including src, ultimately affecting gene expression (nesher et al., ) . thus, cardiac glycosides are in the spotlight for drug repurposing. some studies have shown the potential use of digoxin and other cardiac glycosides to treat cancer (prassas and diamandis, ) . cardiac glycosides can induce apoptosis in diverse cancer cells by multiple mechanisms including inhibition of na + /k + atpase, suppression of nuclear factor-kappab and inhibition of topoisomerase ii (ishida et al., ; vaklavas et al., ) . by performing a chemical screen with an insect cell-based reporter system, the cardiac glycoside digoxin was identified as a specific inhibitor of the retinoic acid receptor-related orphan nuclear receptor (rorγt) transcriptional activity (huh et al., ) , which is required for induction of il- transcription and for the manifestation of t h -dependent autoimmune disease in mice. this study indicated that digoxin and its derivatives could be used as therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease. recent studies have shown that digoxin and other cardiac glycosides can have a potential use as antivirals. wong and colleagues reported the suppression of hiv- replication by digoxin (wong et al., ) . digoxin was shown to accomplish its antiretroviral effects by two mechanisms: ) inducing oversplicing of hiv- rna which reduces the accumulation of viral rnas encoding for structural proteins needed for new virion assembly, and ) selectively inhibiting the expression of hiv- regulatory protein rev. these studies showed that digoxin exerted its anti-hiv effect by altering the function of a subset of sr proteins, a conserved family of serine and arginine-rich proteins involved in rna splicing. a screening of a drug library for compounds inhibiting late steps of hiv- replication cycle also identified several members of the cardiac glycoside family (laird et al., ) . the cardiac glycosides digoxin and ouabain have also been shown to impair replication of human cytomegalovirus (hcmv) and herpes simplex virus (hsv) that belong to the family of dsdna viruses herpesviridae (dodson et al., ; hartley et al., ; kapoor et al., ) . inhibition happens at an early post-entry step and produces a (grosso et al., ) . although the precise mechanism of action is not fully clear, these drugs altered viral mrna processing, blocking replication before viral dna synthesis. some cardiac glycosides like procillaridin a, bufallin, covallatoxin and digitoxin are able to inhibit hepatitis b virus (hbv) in cell cultures (okuyama-dobashi et al., ) . in this study, however, digoxin did not show an anti-hbv effect. digoxin also inhibits alphaviruses (singlestrand positive-sense rna viruses) like chikungunya, ross river virus and sindbis virus, as well as the unrelated mammalian orthoreovirus (polysegmented double-strand rna virus) and vesicular stomatitis virus from the rhabdoviridae family (negative-sense rna virus) (ashbrook et al., ) . in this study digoxin was shown to impair chikungunya infection at an early post-entry step. some coronaviruses like feline infectious peritonitis virus, murine hepatitis virus, and mers-cov are inhibited by ouabain and bufalin (burkard et al., ) . the antiviral effect of these cardiac glycosides was observed only when the drug was added prior to infection. infection was not affected when the drugs were added h post-infection, suggesting that for these viruses the drugs were acting during the entry step. sunitinib is a small molecule that inhibits multiple tyrosine kinase receptors like vascular endothelial growth factor receptor vegfr, platelet-derived growth factor receptors pdgfrα and pdgfrβ, fibroblast growth factor receptor , and epidermal growth factor receptor (mendel et al., ; sun et al., ) . sunitinib has been approved by the fda for the treatment of some cancers (atkins et al., ) . recent studies have shown that sunitinib may also have broad spectrum antiviral activity. intracellular trafficking of viruses relies on the endocytic and exocytic cellular pathways. these processes usually require signal transduction, suggesting that kinase inhibitors may have antiviral activity by blocking the endocytic or exocytic pathways. binding of the hcv core protein to adaptor protein (ap- ) μ subunit (ap m ) is essential for hcv assembly. phosphorylation of ap by adaptor associated kinase (akk ) and cycling g-associated kinase (gak) regulate this interaction (neveu et al., . it has also been shown that ap and ap co-traffic with hcv viral particles within the cell (bekerman et al., ) . these studies showed that sunitinib prevents hcv entry and assembly, apparently through an inhibitory effect on aak and gak, with no effect on hcv rna replication. erlotinib, another anticancer drug, also inhibits hcv entry and assembly. similarly, sunitinib and/or erlotinib can restrict infection by denv and ebov in vitro and in a murine animal model and have potent in vitro antiviral activity against zikv, wnv, chikv, junin virus (junv) and respiratory syncytial virus (rsv) (bekerman et al., ; pu et al., ) . sunitinib has also been shown to inhibit hcmv infection in cell culture (cai et al., ) and hiv- infection of resting cd t cells (guo et al., ) . although these studies showed the potential antiviral activity of sunitinib, this drug can also inhibit protein kinase r (pkr) and ′ ′oligoadenylate synthetase (oas)/rnase l system that act as antiviral effectors in response to type i interferons (jha et al., ) . the capacity of sunitinib to block these two innate immunity pathways could hamper its potential use as antiviral. the development of more selective gak inhibitors could bypass this hurdle. in this sense, a screening of a library based on a bicyclic, heteroaromatic flat scaffold designed to discover novel ligands of gak, led to the identification of a hit compound based on a isothiazolo[ , -b]pyridine scaffold (kovackova et al., ) . some isothiazolo[ , -b]pyridine derivatives with low nanomolar affinity for gak exhibited inhibitory activity against hcv in cell culture, acting at the level of viral entry and assembly (kovackova et al., ) . chloroquine (cq) is a weak base -aminoquinolone derivative that can easily enter the cells and accumulate in acidic compartments like endosomes, lysosomes or trans-golgi network vesicles raising their ph (thome et al., ) . in the clinic cq has been used for the treatment of non-resistant malaria (slater, ; white, ) . additionally cq has been proven to have anti-inflammatory properties, and has been approved for the clinical management of some autoimmune diseases such as rheumatoid arthritis and lupus erythematosus (rainsford et al., ) . many viruses use acidic organelles at some point of their replication cycle. this has prompted the study of cq and its hydroxyl analogue hydroxychloroquine (hcq) as potential antiviral drugs. cq and hcq have been tested and proved to have in vitro, and in some instances in vivo, antiviral activity against several viruses, as described below. two main mechanisms of action have been described for the antiviral activity of cq (al-bari, ). on the one hand, many viruses use acidic endocytic vesicles, like endosomes or lysosomes, to enter the host cell. the acidification of these vesicles triggers conformational changes in the envelope proteins of the virus that induce the fusion of the viral and cellular membranes and the delivery of the virion content into the cell. cq raises the ph of these vesicles impeding the conformational changes needed for viral entry or uncoating and blocking the infection. cq has also been shown to impair maturation of some viral proteins in the golgi network by blocking the action of ph-dependent enzymes, like proteases or glycosyltransferases, which are needed for the maturation process. the antiretroviral effect of cq in cell cultures has been shown to occur through the inhibition of the glycosylation of hiv viral particles (savarino et al., ) , while the antiviral activity of cq against sars-cov seems to be related to the poor glycosylation of the sars-cov receptor ace (vincent et al., ) . cq has been reported to inhibit denv replication by blocking the cleavage of the prm protein that occurs in acidic compartments (boonyasuppayakorn et al., ; randolph et al., ) . cq and some of its derivatives have been reported to have strong antiviral activity against sars-cov and mers-cov in different cell lines with ic ranging from . to . μm (de wilde et al., ; dyall et al., ; keyaerts et al., ; vincent et al., ) . however, in a sars-cov mouse model cq treatment did not reduce virus titer in the lungs (barnard et al., ) some studies have reported inhibitory effect of cq against influenza a viruses (iav) h n and h n in vitro at concentrations similar to doses used for malaria treatment (ooi et al., ) . however, the susceptibility of iav to cq seems to depend on the ph requirements of the viruses and the electrostatic potential of hemagglutinin (di trani et al., ) . in a mouse model of infection with iav, cq treatment dramatically increased the survival of infected mice when administered therapeutically but not when administered as a prophylactic treatment (yan et al., ) . furthermore, a randomized, double-blinded placebo controlled clinical trial did not protect against influenza infection (paton et al., ) . dengue virus and wnv replication has also been reported to be blocked by cq in cell culture (boonyasuppayakorn et al., ; farias et al., ) . a recent study has demonstrated the efficacy of cq in the prophylactic and therapeutic treatment of aotus monkeys inoculated with denv (farias et al., ) . however, a double-blinded, placebocontrolled clinical trial in vietnam failed to show a beneficial impact of cq treatment in the outcome of the infection except for a modest reduction in the percentage of patients that developed dengue hemorrhagic fever in the cq-treated arm (tricou et al., ) . chloroquine also exhibits antiviral activity against zikv, another member of the flaviviridae family, in different cell lines with an ic around . - . μm (delvecchio et al., ) . additionally, some studies have demonstrated that chloroquine can protect mice against zikv infection shiryaev et al., ) . cq has also been reported to inhibit chikv in cell cultures in a dose dependent manner when added at an early point during infection (sourisseau et al., ) . although in this study the therapeutic index of the drug in cell cultures was low, the results pointed at the potential use of cq for the treatment of chikv infection. however, a clinical trial in the french reunion island during an outbreak of chikv to test the efficacy and safety of cq treatment did not show evidence of a significant antiviral effect in cq treated patients compared to placebo treated patients (de lamballerie et al., ) . this lack of antiviral effect could be due to the high viral load in the serum of patients during the acute phase of the infection. the rather narrow therapeutic index of cq leaves little room for a dose adjustment that could allow the use of cq as a therapeutic treatment in acute chikungunya infection. nonetheless, in cell cultures, where the viral titer is lower, cq strongly inhibits chikv infection at concentrations achieved in plasma of individuals on cq treatment for malaria, which suggest that cq could be used as a prophylactic treatment during chikungunya outbreaks to prevent transmission (khan et al., ) . however, a recent study in non-human primates showed that chloroquine treatment exacerbated the disease and/or suppressed the antiviral immunity in the chloroquine treated macaques compared to the placebo group (roques et al., ) . cq and hcq were both shown to inhibit hiv replication in monocytic and t cell lines as well as in monocytes and primary t cells at a post-transcriptional step (sperber et al., ; tsai et al., ) . cq can also inhibit other viruses like cchfv (ferraris et al., ) , hepatitis a virus (hav) (bishop, ) and ebov (madrid et al., ) in cell lines. additionally, cq has been shown to protect mice against a challenge of a deadly dose of ebov (madrid et al., ) . in cell culture cq has shown potent antiviral activity against a broad range of viruses and in some cases efficacy in animal model of infection, as discussed above. however, clinical trials aimed to test the efficacy of cq as antiviral has shown very limited antiviral effect. the rather narrow therapeutic index of chloroquine might be behind the limited antiviral effect of chloroquine in clinical trials. the development of chloroquine derivatives with lower toxicity could improve their effectiveness for the treatment of viral infections. the immunomodulatory activity of cq can also contribute to the control of viral diseases reducing the excessive release of cytokines and other proinflammatory mediators. cyclophilin a (cypa) is a peptidylprolyl isomerase that is expressed in the cytosol (schmid, ) . cyclosporin a (csa) is a cyclic undecapeptide with immune suppressive activity that mainly targets cellular cyclophilins (cyps) (handschumacher et al., ; schreiber, ) . the cyp-csa complex inhibits the phosphatase activity of calcineurin needed for the nuclear translocation of the nuclear factor of activated t cells (nfat), which eventually leads to the block of transcription of cytokines and inhibition of t cell activation (matsuda and koyasu, ) . csa is one of the drugs approved by the fda for immunosuppressive therapy to avoid rejection in organ transplants (hartono et al., ) . csa has been reported to have antiviral activity against a wide range of viruses including human papilloma virus (hpv) (bienkowska-haba et al., ), vesicular stomatitis virus (vsv) (bose et al., ) , vaccinia virus (vv) (damaso and moussatche, ) , hiv- (franke et al., ; thali et al., ; wainberg et al., ) , and hcv (kaul et al., ; nakagawa et al., ; yang et al., ) . although the immunosuppressive activity of csa does not make it a good candidate for antiviral treatment, some csa derivatives, like alisporivir, nim and scy- , with reduced immunosuppressive properties that retain the ability to bind cyclophilin have been developed and proved to conserve the antiviral activity (flisiak et al., ; ma et al., ; paeshuyse et al., ; watashi et al., ) . the replication of certain viruses like hiv, hcv, hpv, vsv, vv or influenza virus relies on their interaction with cyclophilins at certain steps of their replication cycle. thus, the antiviral activity of cyclosporine a (csa) and some of its nonimmunosuppressive analogs against these viruses has been shown to be related to its ability to bind cellular cyclophilins and inhibiting the interaction with the viral proteins (bienkowska-haba et al., ; bose et al., ; damaso and moussatche, ; franke et al., ; kaul et al., ; nakagawa et al., ; thali et al., ; wainberg et al., ; yang et al., ) . csa also blocks the replication of diverse coronaviruses (de wilde et al., (de wilde et al., , pfefferle et al., ) . the nucleocapsid protein of sars-cov specifically binds cypa (luo et al., ) , and cypa has been found to be incorporated into the sars-cov particle (neuman et al., ) . however, sirna knock-down of cellular cypa and cypb, the main targets of csa, did not have an effect in the infectivity of this virus, suggesting that either these cyclophilins are not required for viral replication or that the remaining levels of cellular cyclophilins are enough to support normal replication (de wilde et al., ) . additionally csa and some csa analogs have been shown to inhibit hbv entry into cells (watashi et al., ) . however, this inhibitory activity was independent on the binding to cypa or calcineurin, and correlated to the ability of csa to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (ntcp), a membrane protein that has been proposed to be the hbv receptor. cypa has been reported to interact with influenza a virus matrix protein m suppressing viral replication in cell cultures and animal models liu et al., ) . the role of cypa in the viral cycle is unclear. although it seems to act at several steps of the viral life cycle, the isomerase activity of cypa does not seem to be implicated (liu et al., ) . overexpression of cypa inhibits m translocation into the nucleus (liu et al., ) while depletion of cypa accelerated the replication of the virus (liu et al., b) . additionally cypa has been shown to increase degradation of m though the ubiquitin proteasome system (liu et al., b) . the effect of csa on influenza a virus infection has also been investigated. csa-treated mice were administered a dose of influenza a virus that would be lethal for untreated mice, but they survived (schiltknecht and ada, ) . csa has also been reported to inhibit the propagation of several strains of influenza a virus in cell cultures blocking a late step of the replication cycle by mechanisms that might implicate cypa-dependent and -independent pathways (hamamoto et al., ; liu et al., a; ma et al., ) . globally these results point at non-immunosuppressive csa analogs as promising broad-spectrum antivirals. the most advanced csa analogue is alisporivir or debio that has been evaluated in phase ii and phase iii clinical trials with hcv infected patients (clinicaltrials.gov identifier: nct and nct ) with promising results on their safety and efficacy. further studies to evaluate the efficacy of csa analogs with reduced immunosuppressive activity as antivirals in clinical trials are assured. the antibacterial effect of silver metal has been known from ancient times (barillo and marx, ) . recent advances in nanotechnology have allowed the introduction of silver nanoparticles (agnps) for biomedical applications (burdusel et al., ) . agnps can be prepared by several techniques, including physical, chemical and biological methods. depending on the size and the technique used to prepare the nanoparticles their properties and toxicity can vary (thorley and tetley, ; ullah khan et al., ) . agnps have been proven to have broad antimicrobial activity against gram positive and gram negative bacteria (kim et al., ; morones et al., ) . in addition, some studies have shown that agnps also have antiviral activity against a broad range of viruses including herpes simplex virus (baram-pinto et al., ) , influenza virus (papp et al., ; xiang et al., ), hepatitis b virus (lu et al., , hiv- lara et al., a, b; sun et al., ) , rift valley fever virus (borrego et al., ) , dengue virus (murugan et al., ; sujitha et al., ) and tacaribe virus (speshock et al., ) . the antiviral mechanism of action of agnps is not well defined. agnps are thought to interfere with virus-cell attachment and entry into the target cell or by directly inducing structural changes in the virion turning it non-infective xiang et al., ) . additionally, agnps can enter the cell and exert their antiviral activity by interfering with cellular proteins needed for viral replication or by direct interaction with viral proteins. silver ions (ag + ) can react with thiols and phosphate groups present on proteins and nucleic acids inhibiting different steps of the replication cycle (lara et al., a) . agnps formulated as topical vaginal gels could be used to prevent the transmission of sexually transmitted infections like hiv and hsv. studies in an in vitro system using human cervical tissue cultures that simulate in vivo conditions have shown encouraging results inhibiting hiv transmission (lara et al., b) . although the use of agnps for biomedical applications seems promising, further studies are needed to better understand their potential fig. . experimental strategy to study the mechanism of action of antiviral drugs in single cells and to investigate the origin of antiviral drug resistance. toxicity and long term effects on human health and in the environment. the toxicity of agnps is highly related to the release of ag + , which directly depends on the size of the nanoparticles. the smaller the size the higher the amount of ag + released. ag + has been reported to alter mitochondrial function (chappell and greville, ; kone et al., ) . moreover, exposure of cells to agnps induce the production of high levels of reactive oxygen species (ros) and jnk and p activation leading to mitochondria-dependent cell apoptosis (hsin et al., ) . therefore, further studies are needed to better understand the mode of action of agnps, their cell specificity and toxicological issues in order to generate new and more effective compounds as well as the use in combination with other drugs in the treatment of different viral diseases. high content imaging (hci) is a popular and basic tool of early drug discovery in multiple disease research areas (carpenter, ) . hci was developed with the contribution of major advances in robotics, imaging and automated image processing (moffat et al., ) . hci is already a fundamental technology for antiviral screening and validation (bernatchez et al., ; hoenen, ; lowen et al., ; mudhasani et al., ; panchal et al., ; tan et al., ) . moreover, recent developments in light and electron microscopy are having a significant impact in virology. in particular, live-cell microscopy, super-resolution microscopy, correlative light and electron microscopy (clem) and d imaging are now fundamental tools for studying virus-cell interactions (bykov et al., ; fernandez de castro et al., ; francis and melikyan, ; risco et al., ; witte et al., ) . microscopy is also a powerful tool for studying antivirals. with clem, scientists can study events at the level of single cells in culture (fernandez de castro et al., ; hellstrom et al., ; tenorio et al., ) and infected animals (lowen et al., ) . clem is starting to show its potential for testing antivirals because changes in viral structures assembled in cells where the treatment was effective and in those where the antiviral drug failed can be now studied in detail (berger et al., ; lowen et al., ; martinez et al., ) . cell sorting and clem in combination with proteomics, transcriptomics and lipidomics, will be exceptionally informative in identifying new targets for antiviral drugs, to characterize the mechanism of action of old and new antiviral compounds and to understand how resistance to antivirals is developed in cells (fig. ) . the strategy for these studies can be as follows: duplicated cell cultures infected with a fluorescent virus and treated in the presence or absence of an antiviral drug are processed either by clem or cell sorting. fluorescence microscopy will reveal interesting features in single cells that are then processed for ultrastructural analysis. electron microscopy will show how viral structures are affected by the antiviral drug. in parallel, key cell factors involved in cell resistance to infection or in viral escape to the antiviral drug will be identified by comparing fluorescent and nonfluorescent cells separated by cell sorting and studied by proteomics, transcriptomics and lipidomics. for these studies, cells that differentiate into tissues and d organoids (liu et al., ; takebe et al., ; walters et al., ) will provide more physiologically relevant cellular systems to study virus infection and to test antivirals. emerging viruses are a major threat to human health and the rate at which new pathogenic viruses are emerging has accelerated in the past fifty years. the frequent outbreaks have highlighted the urgent need for new antiviral treatments. generally, antiviral drug development has focused on targeting viral components to block virus entry, replication, morphogenesis and propagation, or on the modulation of the host immune response (zhu et al., ) . drug repurposing is an important alternative. in this review we have chosen several repurposed drugs with proven antiviral activity. with different mechanisms of action, digoxin, sunitinib, chloroquine, cyclosporine a and silver nanoparticles are promising candidates for broad spectrum antivirals that could be used in a combined antiviral therapy. drug repurposing is now facilitated by a number of resources (pollastri and campbell, ) , such as pathogen target bioinformatics resources, public data repositories of screening data, structural biology resources and compound collections, such as the library of small molecules from the national institutes of health (http://www.nihclinicalcollection.com) (ashbrook et al., ; cao et al., ) or the canadian drugbank (https://www.drugbank. ca/), a large drug database commonly used by computational drug repositioning methods. drugbank contains fda approved drugs and those in clinical trials and is frequently updated with new information. with new and powerful screening assays and prediction tools, in silico structure-based screening of these large chemical libraries is identifying potential inhibitors of viral infections including repurposed compounds that target a variety of viral proteins and host factors (abu bakar and ng, ; barrows et al., ; dowall et al., ; martinez et al., ; yuan et al., ) . another strategy that will surely reveal more promising candidates is based on comparing data from databases of pathogens (brown and patel, ; sharma et al., ) with databases of drugs, e.g., drug information from the national library of medicine (https://www.nlm.nih.gov/learn-about-drugs.html). imaging studies combined with functional assays will show the localization and intracellular effects of these new inhibitors (panchal et al., ) . combination therapies with more than one drug against more than one target will be necessary to minimize problems of resistance to repurposed antiviral drugs. the authors declare no conflict of interest. nonstructural proteins of alphavirus-potential targets for drug development targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases antagonism of the sodium-potassium atpase impairs chikungunya virus infection sunitinib maleate inhibition of herpes simplex virus type infection by silver nanoparticles capped with mercaptoethane sulfonate silver in medicine: a brief history bc to present evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice past, present and future: years of hiv research a screen of fda-approved drugs for inhibitors of zika virus infection anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects daclatasvir-like inhibitors of ns a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting zika virus target cell cyclophilins facilitate human papillomavirus type infection examination of potential inhibitors of hepatitis a virus uncoating amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity potential application of silver nanoparticles to control the infectivity of rift valley fever virus in vitro and in vivo requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype a standard database for drug repositioning biomedical applications of silver nanoparticles: an up-to-date overview atp a -mediated src signaling inhibits coronavirus entry into host cells correlative light and electron microscopy methods for the study of virus-cell interactions in vitro combination of anti-cytomegalovirus compounds acting through different targets: role of the slope parameter and insights into mechanisms of action a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs image-based chemical screening effect of silver ions on mitochondrial adenosine triphosphatase effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affinity for cellular cyclophilins on chikungunya acute infection and chloroquine treatment. vector borne zoonotic dis cyclosporin a inhibits the replication of diverse coronaviruses screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cyclophilins and cyclophilin inhibitors in nidovirus replication chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models different ph requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza a viruses mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna innovation in the pharmaceutical industry: new estimates of r&d costs inhibitors of the sodium potassium atpase that impair herpes simplex virus replication identified via a chemical screening approach repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection interaction of silver nanoparticles with hiv- chloroquine interferes with dengue- virus replication in u cells antiviral activity of chloroquine against dengue virus type replication in aotus monkeys metallothioneins for correlative light and electron microscopy threedimensional imaging of the intracellular assembly of a functional viral rna replicase complex evaluation of crimean-congo hemorrhagic fever virus in vitro inhibition by chloroquine and chlorpromazine, two fda approved molecules the cyclophilin inhibitor debio- shows potent anti-hepatitis c effect in patients coinfected with hepatitis c and human immunodeficiency virus live-cell imaging of early steps of single hiv- . infection specific incorporation of cyclophilin a into hiv- virions contemporary use of digoxin in the management of cardiovascular disorders suppression of adenovirus replication by cardiotonic steroids genistein interferes with sdf- -and hiv-mediated actin dynamics and inhibits hiv infection of resting cd t cells cyclosporin a inhibits the propagation of influenza virus by interfering with a late event in the virus life cycle cyclophilin: a specific cytosolic binding protein for cyclosporin a ionic contra-viral therapy (icvt); a new approach to the treatment of dna virus infections immunosuppressive drug therapy. cold spring harb correlative light and electron microscopy enables viral replication studies at the ultrastructural level mycophenolic acid inhibits hepatitis c virus replication and acts in synergy with cyclosporin a and interferon-alpha luciferase-expressing ebolaviruses as tools for screening of antivirals emerging virus diseases: can we ever expect the unexpected? the apoptotic effect of nanosilver is mediated by a ros-and jnk-dependent mechanism involving the mitochondrial pathway in nih t cells digoxin and its derivatives suppress th cell differentiation by antagonizing rorgammat activity repurposing of approved cardiovascular drugs current perspectives on hiv- antiretroviral drug resistance suppression of antiviral innate immunity by sunitinib enhances oncolytic virotherapy global trends in emerging infectious diseases human cytomegalovirus inhibition by cardiac glycosides: evidence for involvement of the herg gene essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics ex vivo modeling of the effects of mycophenolic acid on hiv infection: considerations for antiviral therapy severe acute respiratory syndrome coronavirus by chloroquine assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells antimicrobial effects of silver nanoparticles silver ion (ag+)-induced increases in cell membrane k+ and na+ permeability in the renal proximal tubule: reversal by thiol reagents selective inhibitors of cyclin g associated kinase (gak) as anti-hepatitis c agents minocycline suppresses dengue virus replication by down-regulation of macrophage migration inhibitory factor-induced autophagy a novel cell-based high-throughput screen for inhibitors of hiv- gene expression and budding identifies the cardiac glycosides mode of antiviral action of silver nanoparticles against hiv- pvpcoated silver nanoparticles block the transmission of cell-free and cell-associated hiv- in human cervical culture drug repurposing of minocycline against dengue virus infection protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in cem cells current therapy for chronic hepatitis c: the role of directacting antivirals cyclophilin a protects mice against infection by influenza a virus chloroquine, a fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice curing a viral infection by targeting the host: the example of cyclophilin inhibitors cyclophilin a interacts with influenza a virus m protein and impairs the early stage of the viral replication cyclosporin a inhibits the influenza virus replication through cyclophilin a-dependent and -independent pathways cyclophilin a restricts influenza a virus replication through degradation of the m protein generation of functional organs from stem cells countering zika virus: the usamriid response silver nanoparticles inhibit hepatitis b virus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance a systematic screen of fda-approved drugs for inhibitors of biological threat agents abacavir and mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase, have profound and synergistic anti-hiv activity imaging the alphavirus exit pathway antiviral drug discovery: broad-spectrum drugs from nature mechanisms of action of cyclosporine in vivo antitumor activity of su , a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship nucleoside/nucleotide analog inhibitors of hepatitis b virus polymerase: mechanism of action and resistance drug repurposing for viral infectious diseases: how far are we? minocycline inhibits west nile virus replication and apoptosis in human neuronal cells minocycline neuroprotects, reduces microglial activation, inhibits caspase induction, and viral replication following japanese encephalitis opportunities and challenges in phenotypic drug discovery: an industry perspective the bactericidal effect of silver nanoparticles high content image-based screening of a protease inhibitor library reveals compounds broadly active against rift valley fever virus and other highly pathogenic rna viruses fighting arboviral diseases: low toxicity on mammalian cells, dengue growth inhibition (in vitro), and mosquitocidal activity of centroceras clavulatum-synthesized silver nanoparticles specific inhibition of hepatitis c virus replication by cyclosporin a the digitalis-like steroid hormones: new mechanisms of action and biological significance proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap m essential for viral assembly ap- -associated protein kinase and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets can you teach old drugs new tricks? hepatitis b virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide in vitro inhibition of human influenza a virus replication by chloroquine the non-immunosuppressive cyclosporin debio- is a potent inhibitor of hepatitis c virus replication in vitro mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis c virus infection in vitro and in vivo development of high-content imaging assays for lethal viral pathogens inhibition of influenza virus infection by multivalent sialic-acidfunctionalized gold nanoparticles chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial resistance of hepatitis c virus to inhibitors: complexity and clinical implications the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors target repurposing for neglected diseases novel therapeutic applications of cardiac glycosides feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases acidotropic amines inhibit proteolytic processing of flavivirus prm protein three-dimensional imaging of viral infections paradoxical effect of chloroquine treatment in enhancing chikungunya virus infection anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors in vivo effects of cyclosporine on influenza a virusinfected mice protein folding. prolyl isomerases join the fold repurposing of kinase inhibitors as broad-spectrum antiviral drugs chemistry and biology of the immunophilins and their immunosuppressive ligands unraveling the web of viroinformatics: computational tools and databases in virus research origins of hiv and the aids pandemic. cold spring harb repurposing of the antimalaria drug chloroquine for zika virus treatment and prophylaxis chloroquine: mechanism of drug action and resistance in plasmodium falciparum digitalis. mechanisms of action and clinical use characterization of reemerging chikungunya virus inhibition of human immunodeficiency virus type replication by hydroxychloroquine in t cells and monocytes interaction of silver nanoparticles with tacaribe virus epidemiology, evolution, and recent outbreaks of avian influenza virus in china green-synthesized silver nanoparticles as a novel control tool against dengue virus (den- ) and its primary vector aedes aegypti discovery of -[ -fluoro- -oxo- , -dihydroindol-( z)-ylidenemethyl]- , -dimethyl- h-pyrrole- -carboxylic acid ( -diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase silver nanoparticles fabricated in hepes buffer exhibit cytoprotective activities toward hiv- infected cells minocycline attenuates hiv infection and reactivation by suppressing cellular activation in human cd + t cells vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation inhibition of dengue virus replication by mycophenolic acid and ribavirin cell-based flavivirus infection (cfi) assay for the evaluation of dengue antiviral candidates using high-content imaging the pandemic potential of avian influenza a(h n ) virus: a review reovirus sigmans and muns proteins remodel the endoplasmic reticulum to build replication neo-organelles functional association of cyclophilin a with hiv- virions chloroquine: modes of action of an undervalued drug new perspectives in nanomedicine host genes and influenza pathogenesis in humans: an emerging paradigm a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults inhibition of human immunodeficiency virus infectivity by chloroquine nanosilver: new ageless and versatile biomedical therapeutic scaffold common cardiovascular medications in cancer therapeutics chloroquine is a potent inhibitor of sars coronavirus infection and spread the effect of cyclosporine a on infection of susceptible cells by human immunodeficiency virus type generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter the treatment of malaria concepts in light microscopy of viruses digoxin suppresses hiv- replication by altering viral rna processing inhibitory effects of silver nanoparticles on h n influenza a virus in vitro inhibition of a/human/hubei/ / (h n ) influenza virus infection by silver nanoparticles in vitro and in vivo anti-malaria drug chloroquine is highly effective in treating avian influenza a h n virus infection in an animal model cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro structure-based discovery of clinically approved drugs as zika virus ns b-ns protease inhibitors that potently inhibit zika virus infection in vitro and in vivo host-targeting agents for prevention and treatment of chronic hepatitis c -perspectives and challenges broad-spectrum antiviral agents neuroprotective and anti-human immunodeficiency virus activity of minocycline this work was supported by a research grant from the spanish ministry of economy, industry and competitiveness (bio - -r, mineco-feder to cr). bp was recipient of a post-doctoral contract from the severo ochoa center of excellence program. key: cord- -mirh gn authors: hickey, anthony j. title: emerging trends in inhaled drug delivery date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: mirh gn ideally, inhaled therapy is driven by the needs of specific disease management. lung biology interfaces with inhaler performance to allow optimal delivery of therapeutic agent for disease treatment. inhalation aerosol products consist of the therapeutic agent, formulation, and device. the manufacturing specifications on each of the components, and their combination, allow accurate and reproducible control of measures of quality and in-vitro performance. these product variables in combination with patient variables, including co-ordination skill during inhaler use, intrinsic lung biology, disease and consequent pulmonary function, contribute to drug safety and efficacy outcomes. due to the complexity of pulmonary drug delivery, predicting biological outcomes from first principles has been challenging. ongoing research appears to offer new insights that may allow accurate prediction of drug behavior in the lungs. disruptive innovations were characteristic of research and development in inhaled drug delivery at the end of the last century. although there were relatively few new inhaled products launched in the first decade of the new millennium it was evident that the earlier years of exploration resulted in maturation of commercially successful technologies. a significant increase in new and generic products has occurred in the last decade and technical, regulatory and disease management trends are emerging. some of these developments can trace their origins to earlier periods of creativity in the field while others are a reflection of advances in other areas of basic and computer sciences and engineering. select biological and technical advances are highlighted with reflections on the potential to impact future clinical and regulatory considerations. high quality, pharmacologically mechanistic targeted inhaled therapy has become the primary strategy for pulmonary disease management over the last seventy years. these modern therapies can, in many cases, trace their origins to the earliest records of anecdotal use of inhaled natural products in india and china [ ] . the last decade has been characterized by the innovative technologies of the previous quarter century maturing into commercial products or being fine-tuned to meet development, quality, safety or efficacy needs. as this evolution of inhaled medicines has occurred the opportunity space has been filled leaving little to explore in the context of conventional pulmonary disease targets. a disruptive technological approach will likely come from the demands of as yet unmet medical need. in this regard, rare and infectious lung diseases are receiving considerable attention and the field of systemic drug targeting through pulmonary drug delivery remains in its infancy. figure summarizes the requirements of matching the therapeutic agent to the desired treatment outcome while considering the overall host system barriers (deposition and clearance mechanisms) and target (receptor or pathogen) biology guiding selection or development of a suitable drug delivery technology. the therapy, biology and technology each introduce complex variables that must be taken into account to optimize efficacy and safety. the nature of the therapeutic agent (small molecule, nucleic acid or other biological) imposes specific limits on formulation and device based on stability and performance considerations. specialized formulation technologies may be required to modulate the physico-chemical behavior and biological disposition of the therapeutic agent. device technology is generally protecting lung integrity during an overwhelming immune response might play in reducing mortality [ , ] . understanding response to infection and the role of co-morbidities may give greater insight into the role of the pulmonary immune system in both protection from, and exacerbation of lung damage. this will have significant implication for future efforts in vaccine and immunomodulator delivery [ ] . the intent of this review is not to describe comprehensively inhaled dosage forms. thorough reviews of this nature, some of which are referenced below, are available in the literature. indeed, the author has recently edited two volumes on this topic [ , ] . the perspective taken below is to highlight key developments and speculate on directions that may drive future advances in inhaled drug delivery. this approach is intended to complement the existing literature. inhaler design has seen little change in the last decade but some features have resulted in improvements that offer new opportunities for disease treatment. the most significant improvement in nebulizer technology was the development of vibrating mesh systems [ ] . the most prominent of these are the eflow (pari) and ineb (philips) [ , ] . while the technologies themselves were developed over several decades the applications seem to be increasing the range from very small (individual micrograms, treprostinil, iloprost) to large (hundreds of milligrams, tobramycin) doses of drug delivered [ , }. in addition, a unique opportunity has been explored of evaporating vibrating mesh nebulizer output to create an aerosol that can be introduced through a nasal cannula. this j o u r n a l p r e -p r o o f aerosol increases peripheral lung deposition though the use of excipients that are subject to hygroscopic growth [ ] . dry powder inhaler technology has three major components, the formulation, the metering system and the aerosol dispersion features [ ] . all commercially available inhalers are actuated passively in response to the patients' inspiratory flow. regulatory approval and commercialization of advair  /seretide  (glaxosmithkline) was one of the most significant developments at the beginning of the millennium [ , ] . the combination salmeterol xinafoate/fluticasone dipropionate product was the first dry powder product for maintenance therapy of asthma, notably containing a long-acting  -agonist, unlike earlier combination products. subsequently, this product was also approved for the treatment of chronic obstructive pulmonary disease. the complexity of the drug-lactose blend and diskus  inhaler was challenging to manufacture and in the interval since approval has proven difficult to reproduce rapidly as a generic product [ ] . despite these challenges mylan received us fda approval for its generic salmeterol xinafoate/fluticasone dipropionate in . the combination of two drugs in a single powder was both an exciting new therapeutic development and a formidable test of quality and performance controls. in this context, one of the most significant advances of the last decade was the approval of ellipta  devices that are capable of delivering two drugs independently from two separate blister strips, thereby avoiding the arduous task of ensuring combination drug stability and optimizing performance of two drugs from a single formulation [ , ] . as will be discussed in the next section the difficulty of dispersing micronized dry powders has been recognized since modern dpis were developed in the s and s [ , ] . often tortuous paths and baffles have been inserted in dpis to help de-agglomerate drug but historically this was not possible without the presence of lactose in the formulation [ ] . the challenge has been to generate sufficient energy on the inspiratory flow of the patient to disperse micronized particles alone. the use of a cyclone classifier as part of a passive inhaler design has allowed a device driven approach to disperse micronized particles [ ] . this has opened the possibility for relatively high dose delivery of antimicrobials, as required for the treatment of infectious diseases. figure b show the cyclops device [ ] . the powder is drawn from a metering blister built into the device through a cyclone classifier such that only respirable particle can pass into the airstream to the patient. this is a simple disposable inhaler, approximately the size of a credit card, resulting in ease of storage and transport of bulk product and offering a discreet form factor for patient convenience. [ ] . these are important to the products they serve but they do not change the overall operating principles of the device. perhaps the most substantial new developments in pmdi technology is the addition of counters and exploration of electronic patient data recording systems that may ultimately lead to major steps forward in personalized medicine [ ] . it should be noted that as the vigorous debate on climate change and global warming agents begins to focus on limiting their use another transition may be required from existing hfa propellants to alternatives with lower global warming potential. if history repeats itself this will support another period of innovation. the preparation of formulations for inhalation is constrained by the device employed for drug delivery. nebulizers and soft mist sprays employ solutions or suspensions in an aqueous medium. dry powders inhalers contain formulations of solid drug with excipients that enhance performance. metered dose inhalers employ solutions or suspensions in a non-aqueous medium. nebulizer solutions and suspensions use limited formulation additives. however, recently the use of liposomes to aid in drug preparation and delivery and to aid in targeting of macrophages has advanced formulation options [ , ] .the use of ethanol in the solutions delivered from the respimat™ soft mist inhaler, e.g. tiotropium (spiriva®, boehringer ingelheim) enhances the solubilization properties of the medium and facilitates a hand held aqueous based system with accurate and reproducible performance in delivering very low doses (< µg) on a single breath analogous to a metered dose inhaler [ , ] . dry powder inhaler products began their modern development with a characteristic respirable drug particle blend in lactose that allowed delivery of small drug doses (< mg) from j o u r n a l p r e -p r o o f large total powder masses (< mg) [ ] . these lactose blends were employed due to the large forces of interaction that typified micronized powders and prevented their accurate metering and reproducible dispersion [ ] . many highly successful products have been manufactured that conform to this general description. a breakthrough occurred in the late s when it was observed that low density particles prepared by spray drying exhibited low forces of interaction and were easily dispersible [ ] . since these particles could be manufactured with the presence of small quantities of excipient it was possible to deliver large drug doses. figure a illustrates the low density, collapsed hollow sphere appearance of the early low-density particles [ , ] . figure b shows the whiffle ball appearance of pulmosphere™ tobramycin in the tobi  podhaler  product (novartis) [ ] . this low-density morphology allows the delivery of mg of drug ( capsules, mg/capsule) increasing the previous limit on dry powder drug delivery by orders of magnitude. however, a formulation conundrum was exposed with this major advance in dry powder drug delivery. in order to deliver a large mass of low-density powder, large volumes are required. hence the need for capsules in the tobramycin product. this may be acceptable for certain severe disorders but increasing the number of actuations/breaths required to deliver the dose increases the possibility of lack of patient adherence to the therapy. recently, it was observed that micronized particles may be prepared in a manner that forms aggregates that mimic low density particles and may, therefore, exhibit reduced interparticulate forces sufficient to allow their dispersion [ ] .this represents a compromise between low volume and low density that allows for accurate and reproducible dispersion in a minimum number of actuations/breaths that would pose less of a challenge to patient adherence. figure c shows a stable aggregate of micronized particles of clofazimine with j o u r n a l p r e -p r o o f sufficient interstitial space to provide the agglomerate low-density performance and ease of dispersion characteristics. metered dose inhalers have had a standard composition for decades, namely canister, propellant formulation, valve and actuator [ ] . there have been advances in each of these most significantly the shift from chlorofluorocarbon propellants to hydrofluoroalkanes [ ] . the latter required considerable reformulation efforts but the transition is now complete. the new hfa formulations have lower velocities and smaller droplets that maximize lung deposition while minimizing oropharyngeal deposition [ ] . as the reformulation effort was occurring it j o u r n a l p r e -p r o o f became evident that the excipient options were even more limited than had been the case with cfcs. in a recent development, knowledge gained from dry powder formulation has been transferred to metered dose inhalers. the addition of low-density lipid particles to hfa as a dispersant for micronized drug has allowed for a range of new metered dose inhaler products to come to market [ ] . the environmental concern over ozone depletion that was the cause of the cfc to hfa transition has been superseded by concern over global warming potential [ , ] . unfortunately, hfas have significant global warming potential. as a consequence, the search for new propellants is underway that will extend the future of metered dose inhalers in the event of new environmental regulation on global warming [ ] . figure depicts two important characteristics of disease management using inhalers. on the xaxis the dose that has been delivered from different inhalers, depicted on the left, and the number of breaths required to inhale the dose of drug, shown on the right. while there are differences in the performance of these inhalers with respect to aerodynamic particle size distribution, the parameter governing lung delivery, for the purposes of this discussion it is sufficient to assume that all deliver respirable aerosols (< m) [ ] . nebulizers have long been known to be capable delivering large doses, > mg. the introduction of vibrating mesh nebulizers allowed for much shorter delivery times on large doses ( - mins) and much lower therapeutic doses than historically possible on individual breaths as is the case in the treatment of pulmonary arterial hypertension with treprostinil and iloprost. dry powder inhalers were limited initially by the need for lactose excipient to facilitate the dispersion of micronized drug. however, the advent of low-density particles extended the dose to > mg while increasing the number of breaths/actuations required to deliver the dose. the range of pmdi dosing has remained fairly constant over several decades. broadening the range of accurate and reproducible dosing is a significant achievement of the last decade. figure a depicts schematically the dimensional representation of an alveolus and its blood supply. figure b shows a transverse section through this alveolus depicting the proximity of the capillary blood supply to the alveolus and the presence of lumenal alveolar macrophages. these images are frequently depicted as textbook representations. however, figure b also points out that the milieu in which these anatomical features exist also contains interstitium and lymphatics, shown as the shaded region. as a consequence, the usual j o u r n a l p r e -p r o o f discussion of disposition of drug in and from the lungs would benefit from an additional level of scrutiny that is rarely considered, disposition within lung tissue not simply from the airways to the vasculature. a number of disease states that might be treated with aerosols are not associated with airways. potent drugs that are given in low doses and penetrate readily though the lung mucosa to the blood supply may be suitable for treating diseases that require systemic delivery. the most recent development in inhaled insulin to treat diabetes is afrezza  (mannkind). this product employs a dry powder formulation including a unique carrier particle composed of the excipient fumaryl diketopiperazine [ ] . additionally, loxapine, an antipsychotic for the treatment of schizophrenia, has been delivered from an evaporation condensation device, staccato  , in the adasuve product (alexa) [ , ] . the delivery of systemically acting agents continues to be a promising area of opportunity for future aerosol products. recent interest in pulmonary arterial hypertension and idiopathic pulmonary fibrosis focuses attention on the transport of drug at the cellular level in the regions of the lungs between airway and blood vessel lumens [ , ] . residence time of drug at therapeutic concentrations is crucial to the effectiveness of drugs to target the local manifestations of these diseases. when placed in the context of the illustration in figure [ ] [ ] [ ] . the disease manifests as extracellular biofilms associated with bronchiectasis and intracellular disease in lung macrophages [ ] . the recent approval of arykace  (liposomal amikacin) for the treatment of non-tuberculous mycobacteria represents a thoughtful approach to matching device, formulation to the needs of the disease. the liposomes are taken up by infected macrophages and penetrate biofilms sufficiently to treat the infection and improve lung function [ ] . clofazimine offers a unique potential to intervene in this disease as the drug spontaneously targets macrophages and local delivery as an aerosol obviates systemic side effects arising from other routes of administration [ ] . development of new therapies for ntm j o u r n a l p r e -p r o o f treatment capitalizes on the decades of research into development of inhaled therapy for tuberculosis [ ] . this brief review of example disease targets and their therapy illustrates the range of dosing that is required and can now be achieved from very low, single microgram, to high, several hundred mg, doses that support efficacious concentrations of drugs at different sites in the lungs not simply the airways. as changes to aerosol products have occurred new guidance from regulatory agencies was necessary to accommodate the needs of industry. in the last ten years many new guidances to industry on generic products have been issued [ ] . the initial challenge was substitutable hfa products for the existing cfc products with the first albuterol product being approved in [ ] . subsequently, complex combination products such as the salmeterol xinafoate/fluticasone dipropionate (advair) took longer to consider with the guidance appearing in [ ] . perhaps the most significant event of the last decade was the first revision to the draft guidance ( ) on chemistry manufacturing and controls relating to testing of dry powder and metered dose inhaler products in [ , ] . also, the weight of evidence approach advocated by the food and drug administration gives applicants insight into expectations that can be pursued in formal discussions at various stages of development. the demand for an approach to bioequivalence has been a driver of industry activities over the last ten years [ ] . two consensus meetings can be noted in this context. the first occurred in and framed the issues surrounding pharmaceutical and bio-equivalence [ ] . more recently, a workshop considered the foundations of an inhaled biopharmaceutical classification system (ibcs) that might be used, as was the original oral bcs is, to guide product development and illuminate regulatory review [ ] . these events facilitated discussion of standard methods to assure quality and safety in the context of product performance but they also encompassed efforts to develop predictive approaches that would conform to the philosophy of quality by design. this will continue to be a focus for inhaled drug product development and regulation. new observations in biology may emerge from the evolution of thought over decades of scientific endeavor or from sudden disruptive events that expose new challenges and illuminate underlying mechanisms. two examples can be given, recognizing that many more may be in the literature to illustrate this point. it has recently been observed that the fibroblast cytoskeleton may play a role in asthma. this has implications for the function of muscle function in conducting airways. protein tyrosine phosphorylation has been reported to regulate actin polymerization in smooth muscle contractile stimulation. consequently, the role of protein/threonine phosphorylation in modulating actin dynamics has been investigated with a specific focus of ste -like kinase (slk), a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation and migration [ ] . the major finding of this study was that slk mediates actin cytoskeleton j o u r n a l p r e -p r o o f reorganization and may facilitate force transmission between contractile units and the extracellular matrix. since bronchoconstriction as a response to airway hyper-reactivity is mediated by muscle tone in asthma this may offer a new target for bronchodilatation. the recent observation that caspase, an important protein in defending from bacterial infection, may become overactive and provoke a damaging inflammatory reaction has implications for allergic asthma therapy [ ] . caspase- causes cell death which results in the release of cell contents and is a key driver of inflammation in asthma. caspase- , the human equivalent of caspase- has never been implicated in asthma so it is a promising possible target for new drugs to treat this disease. the recent covid- pandemic caused by the sars-cov virus has one significant and alarming clinical outcome, the rapid progression in susceptible individuals to bilateral interstitial pneumonia [ , ] . major effort has been expended in preventing deterioration of lung function including, in the extreme, the use of ventilators to support gaseous exchange [ ] . however, since understanding of the pathogenesis of disease is increasing, it is evident that being able to ensure the patency of the lungs is a key to survival. the historical precedents of zanamivir (relenza, gsk) and ribavirin to treat influenza and respiratory syncytial virus indicate that lung delivery of therapeutics has merit for respiratory viral infections [ ] [ ] [ ] . however, to prevent the manifestations of disease seen in covid- early intervention with agents that prevent viral uptake and/or replication and can target affected areas of the lung in therapeutic concentrations is desirable. examples would include aerosol delivery of antibodies, antisense oligonucleotides or antivirals, such as remdesivir [ , ] . in addition, the observation that in countries where bacille calmette guerin (bcg, attenuated mycobacterium bovis) immunization j o u r n a l p r e -p r o o f for tuberculosis is conducted the population has a lower incidence of covid- mortality raises the potential for a directed vaccine strategy [ ] . further research is required to address some of the inconsistencies in this initial observation. however, if evidence based on rigorous interrogation of the data supports any protective effect conferred by bcg, it should be noted that mycobacterial antigens delivered as aerosols induce a systemic and lung mucosal immune response. [ ] [ ] [ ] . moreover, work continues on the pharmaceutical properties of inhaled bcg vaccine [ ] and a recombinant viral vector approach is also under development for antigen a, a mycobacterial secreted subunit protein [ ] . raising these speculative opportunities for potential clinical intervention simply highlights new biological challenges posed by our evolving understanding of the pathophysiology of disease. the disease biology will drive both new therapeutic agent development and the technological needs to address them. as the potential disease applications for aerosol therapy expand innovation will necessarily follow. there has been continuous development of novel inhaled medicines for decades. the last ten years has seen a maturation of technologies and some novel excursions to expand the application of inhalation technology to more effectively treat pulmonary diseases. there have been developments in each of the major categories of inhaler that have enabled a wider range of doses and improved the accuracy and precision of dosing from which it can be inferred that quality has improved. the safe and efficacious new products improve management of pulmonary diseases for which aerosols have long been used, such as asthma and more recently illustrations, not to scale, of (a) ellipta  dual blister strip mechanism to allow independent drug delivery in combination products and (b) cyclops micronized drug particle high dose delivery device* showing the cyclone classifier dispersion mechanism. *courtesy of dr. anne de boer. pulmonary drug delivery: an historical overview in controlled pulmonary drug delivery targeting inhaled therapy beyond the lungs updated clinical classification of pulmonary hypertension idiopathic pulmonary fibrosis acute lung injury: epidemiology, pathogenesis and treatment severe acute respiratory syndrome corona virus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenge radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study mucosal immunization via the respiratory tract inhalation aerosols, physical and biological basis for therapy mesh nebulizers have become the first choice for new nebulized pharmaceutical drug developments lung deposition of inhaled tobramycin with eflow/lc plus nebulizer in healthy and cystic fibrosis patients pharmacokinetics and safety of tobramycin administered by the pari eflow rapid nebulizer in cystic fibrosis the potential for inhaled treprostinil in the treatement of pulmonary arterial hypertension inhaled iloprost for the treatment of pulmonary hypertenstion high-efficiency generation and delivery of aerosols through a nasal cannula during non-invasive ventilation dry powder inhalation: past present and future the diskus tm : a review of its positioning among dry powder inhaler devices advair combination treatment with fluticasone propionate/salmeterol in the treatment of asthma between batch pharmacokinetic variability inflates type i error rate in conventional bioequivalence trials: a raondomized advair diskus clinical trial the ellipta dry powder inhaler: design, functionaility, in vitro dosing performance and critical task compliance by patients and caregivers assessment of the bioequivalence of two dpis containing fluticasone and salmeterol, in patients with controlled or partly controlled asthma the history of therapeutic aerosols: a chronological review dry powder inhaler formulation the cyclops for pulmonary delivery of aminoglycosides; a new member of the twincer family advances in metered dose inhaler technology: formulation development advances in metered dose inhaler technology: hardware development amikacin liposome inhalation suspension (alis) penetrates non-tuberculous mycobacterial biofilms and enhances amikacin uptake into macrophages development of liposomal ciprofloxacin to treat lung infections the respimat softmist inhaler: implications of drug delivery characteristics for patients soft mist inhalers in pharmaceutical inhalation aerosol technology, third edition fundamentals of dry powder inhaler technology, in particles and nanoparticles in pharmaceutical products, hg merkus large porous particles for pulmonary drug delivery fundamental effects of particle morphology on lung delivery: predictions of stokes' law and the particular relevance to dry powder inhaler formulation and development density and shape factor terms in stokes' equation for aerodynamic behavior of aerosols development of an inhaled dry powder formulation of tobramycin using pulmonsphere tm technology excipient-free pulmonary delivery and macrophage targeting of clofazimine via air jet micronization transition to cfc free metered dose inhalers -into the new millennium the evolution of pressurized metered dose inhalers co-suspension delivery technology in pressurized metered dose inhalers for multi-drug dosing in the treatment of respiratory diseases environmental impacts of healthcare and pharmaceutical products: influence of produc design and consumer behavior environmental impact of inhalers for respiratory diseases: decreasing the carbon footprint while preserving patient-tailored treatment preclinical and clinical considerations in "inhaled pharmaceutical product development perspectives technosphere insulin: defining the role of technosphere particles at the cellular level loxapine inhalation powder (adasuve), pharmacy and therapeutics in vitro aerosol characterization of staccato loxapine lady windermere syndrome inhaled antibiotics for mycobacterial lung disease non-tuberculous mycobacteria an underappreciated cause of geriatric lung disease general overview on non-tuberculous mycobacteria, biofilms and human infection targeting liposomal drug delivery to monocytes and macrophages clorazimine inhalation suspension for the aerosol treatment of pulmonary non-tuberculous mycobacterial infections drug delivery systems for tuberculosis prevention and treatment regulatory strategy in "inhaled pharmaceutical product development perspectives us food and drug administration ( ) guidance, fluticasone dipropionate and salmeterol xinafoate guidance metered dose inhaler and dry powder inhaler guidance metered dose inhaler and dry powder inhaler guidance. revision bioequivalence of inhaled drugs: fundamentals, challenges and perspectives demonstrating bioequivalence of locally acting orally inhaled drug products (oips): workshop summary report scope and relevance of a pulmonary biopharmaceutical classification system aaps/fda/usp workshop march - th ste -like kinase-mediated control of actin polymerization is a new mechanism for thin filament associated regulation of airway smooth muscle contraction use of non-invasive ventilation for patients with covid- : a cause for concern? direct measurement of the anti-influenza agent zanamivir in the respiratory tract following inhalation oral versus inhaled ribavirin therapy for respiratory syncytial virus infection after lung transplantation a novel inhaled dry powder formulation of ribavirin allows for efficient lung delivery in healthy participants and those with chronic obstructive pulmonary disease in phase i study emerging drugs for respiratory syncytial virus infection compassionate use of remdesivir for patients with severe covid- is global bcg vaccination coverage relevant to the progression of sars-cov pandemic? med hypothesis immunization by a bacterial aerosol pulmonary immunization using antigen -b polymeric microparticles to boost tuberculosis immunity pulmonary vaccine delivery design and optimization of a temperature stable dry powder bcg vaccine alternate aerosol and systemic immunization with a recombinant viral vector for tuberculosis mva a: a phase i randomized controlled trial key: cord- -ahl ql authors: van buuren, nicholas; tellinghuisen, timothy l; richardson, christopher d; kirkegaard, karla title: transmission genetics of drug-resistant hepatitis c virus date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: ahl ql antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. for hiv and hepatitis c virus (hcv), combination therapy has proved life-saving. the targets of direct-acting antivirals for hcv infection are ns / a protease, ns a phosphoprotein and ns b polymerase. differential visualization of drug-resistant and -susceptible rna genomes within cells revealed that resistant variants of ns / a protease and ns a phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. confocal microscopy revealed that rna replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. no hcv antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. however, effective inhibitors of hcv polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs. in a recent triumph of modern science and medicine, patients chronically infected with hepatitis c virus (hcv) now receive multidrug regimens that are often curative and have low toxicity (lawitz et al., ; afdhal et al., ; dhaliwal and nampoothiri, ) . over the past two decades, researchers have developed and tested thousands of antiviral compounds with varying efficacies and toxicity profiles that have ultimately lead to the fda approval of powerful combination therapies (lawitz et al., ; scheel and rice, ) . several different direct-acting antivirals (daas) that target the ns / a protease, ns a phosphoprotein, or ns b rna-dependent rna polymerase of hcv have been approved for use in the clinic (afdhal et al., ; dhaliwal and nampoothiri, ; manns and von hahn, ; younossi et al., ) . ideally, the knowledge gained in developing hcv antivirals that are effective and not prone to the outgrowth of drug resistance will be applied to other viruses as well. the emergence of drug-resistant variants follows basic evolutionary principles, requiring spontaneous mutations as well as selective pressure, so that beneficial mutations increase the progeny size of genomes that bear them. the genetic diversity in rna viral genomes results from the high error frequencies incurred by rna-dependent rna polymerases, which occur at approximately  À errors for each nucleotide synthesized (sanjuán et al., ) . given the iterative copying of positive and negative strands, much higher cumulative error frequencies are observed, even during a single cycle of infection (sanjuán et al., ; acevedo et al., ) . when more than one mutation is required to confer drug resistance, the outgrowth of drug resistance can be delayed (bloom et al., ) . as a result, treatment with combinations of drugs can be extremely effective at suppressing siblings. if the drug target is, for example, a subunit of an oligomeric complex and subunits from different genomes have the opportunity to mix, chimeric oligomers often form. at the time of its creation, the drug-resistant genome will be a minority species, and such chimeras would be predominantly composed of the drug-bound, susceptible components thus incapacitating the entire oligomeric structure. such 'phenotypic masking' was originally invoked to explain the very low frequency of foot-and-mouth-disease escape variants following selection with neutralizing antibodies when passaged at high multiplicities of infection (mois) (holland et al., ) . our goal was to screen the hcv-encoded viral proteins that are current targets of antiviral compounds to determine the intracellular dominance relationships between drug-resistant and drug-susceptible genomes. the high cost to viral fitness of sofosbuvir-resistant variants is sufficient to explain its high barrier to resistance. there are currently no antivirals directed against hcv core protein; however, it is likely to be a dominant drug target. we used differential hybridization of rna probes to detect two different genomic rnas in a single cell by confocal microscopy and by flow cytometry. this analysis showed the cis-dominance of hcv viruses that are resistant to inhibitors of either ns / a protease or ns a phosphoprotein, consistent with the rapid outgrowth of drug-resistance in patients of these two inhibitor classes. newly mutated drug-resistant genomes first arise within cells that are pre-populated by drug-susceptible genomes. to mimic such mixed infections, we have previously employed co-infection of cultured cells with drug-susceptible and drug-resistant viruses at high mois to ensure mixed infection (tanner et al., ; mateo et al., ) . for hcv, it is not practical to use high mois to achieve co-infection due to the difficulty of obtaining sufficiently high-titer viral stocks. thus, we needed to develop an approach to distinguish between uninfected, singly infected and co-infected cells in relatively sparsely infected cell populations ( figure a) . to detect individual genomes in infected cells, a single-molecule fluorescence in situ hybridization (fish) approach was used. a recently developed branched dna probe technology allows the generation of sufficiently sensitive rna probes to identify single molecules within cells, but requires approximately nucleotides of differential probe hybridization to achieve specificity (affymetrix inc, ) . to create a viral strain with this extreme dissimilarity from wild-type virus, we tested the viability of three different codon-altered versions of the jfh variant of hcv ( figure b) . each mutated version contained - nucleotide changes that did not alter the protein sequence (figure -figure supplements - ). of these codon-altered (ca) variants, ca- was inviable, ca- showed reduced viral protein accumulation, and ca- showed accumulation of both viral protein and rna to abundances equivalent to those of the wild-type virus ( figure c) . recently, detailed analysis of covarying nucleotides within the hcv coding region has identified the location of several previously unknown functional rna secondary structures (pirakitikulr et al., ) . interestingly, ca- contains two such regions and ca- contains one, which correlates with decreasing viability, while ca- contains no such regions (figure -figure supplements - ) (pirakitikulr et al., ) . thus, subsequent experiments were performed only with ca- . this variant, now termed 'ca' virus, contains synonymous mutations over a -nucleotide region that spans the coding sequences for most of ns and the n-terminus of ns (figure to test the sensitivity of rna fish probes generated against the positive-and negative-strands of wild-type (wt) and codon-altered (ca) viruses, both confocal microscopy and flow cytometry analyses were employed. branched dna technology allowed the labeling of each target rna with as many as fluorophores ( figure a ) (affymetrix inc, ) . huh- . . cells were infected with either wt or ca viruses, subjected to fish and visualized by confocal microscopy. wt and ca probe sets specifically targeted either the positive-sense ( figure b ) or the negative-sense vrna ( figure c ) of their corresponding virus. additionally, we tested whether flow cytometry efficiently resolved cells transfected with different vrnas; transfection was used to maximize the yield of each population. we resolved cells transfected with wt vrna (figure di) , transfected with ca vrna (ii), a mixture of these two cell types (iii) and cells co-transfected with both wt and ca vrnas (iv). thus, figure . construction of codon altered jfh . (a) cell cultures coinfected with two strains of hcv result in four populations: uninfected, two types of singly infected, and coinfected cells. (b) three segments of the jfh genome, that were roughly nucleotides in length and had altered codon usage, were designed using geneart algorithms and synthesized. these genome fragments were then cloned into the jfh strain of hcv. specific rna probes could be used to monitor the fate of drug-susceptible and drug-resistant viruses in co-infected cells. to test the genetic properties of viruses that are resistant to ns / a inhibitors, we employed the original ns / a inhibitor, biln- ( figure a ) (lamarre et al., ) . like other ns / a inhibitors, biln- treatment rapidly allows the selection of drug resistant variants both in tissue culture and in patients (lamarre et al., ; lin et al., ) . given the ease of outgrowth of drug-resistant variants, we hypothesized that ns / a was not a dominant drug target and that drug resistance would be genetically dominant. ns -d a is the prototypic mutation associated with resistance to ns / a inhibitors. asp is in close proximity to the protease active site ( figure b ). the ability of the ns -d a mutation to confer resistance to biln- was confirmed in both the wt and ca backgrounds ( figure -figure supplement ). as shown schematically in figure d , the ability to track cells that are uninfected (u), singly infected with drug-susceptible virus (s), infected with both susceptible and resistant virus (s + r) and singly infected with drug-resistant virus (r), can reveal dominance relationships during co-infection. in the absence of a drug, all viral populations should be present. however, in the presence of a drug, three outcomes are possible depending on the genetic outcome within the r + s population. if drug resistance were trans-dominant ( figure e ), the drug-resistant virus would rescue the drugsusceptible genomes and all viruses in r + s cells would survive in the presence of the drug. s cells would drop into the u population, and r cells would survive. if drug resistance were cis-dominant ( figure f ), only the r viruses in the r + s cells would survive, because the drug-resistant proteins would be unable to rescue the s viruses in the same cell. consequently, the r + s cells would drop into the r population. if drug susceptibility were dominant ( figure g ), all viruses in the r + s cells would be cleared, and the r + s cells, like the s cells, would drop into the u population, and only the r cells would continue to replicate. to determine the dominance relationship between biln- -susceptible and the biln- resistant virus, huh- . . cells were infected with ca and wt-d a viruses ( figure c ). cells were infected for hr at mois such that all four populations were represented, followed by hr of continued incubation in the absence or presence of mm biln- . cells were then harvested, fixed, co-stained with wild-type and codon-altered rna probe sets and analyzed by flow cytometry. all four cell types appeared in the absence of biln- ( figure h ,i). in the biln- -treated samples ( figure j ,k), the susceptible s population shifted to the u cells as expected. the s + r cells, on the other hand, shifted to the r population upon drug treatment. thus, the drug-resistant viral genomes in the co-infected cells could replicate, but could not rescue the drug-susceptible ones. data from this and replicate experiments ( figure i ,k) confirmed the quantitative shift of s + r cells into the r population upon drug treatment. we conclude that, for the ns / a target, drug-resistant genomes are cis-dominant for the : ratio of s and r viruses tested here. we also tested whether over-expressed drug-resistant ns / a precursors could rescue biln- -susceptible virus ( proteins are drug targets, drug-resistant products will enhance the propagation of only those genomes that encode them, allowing powerful selection for drug resistance. for ns b polymerase inhibitor sofosbuvir, the few resistant viral variants that arise in patients are highly attenuated. to investigate whether a related compound, r (klumpp et al., ) , exacted a similar cost to viral fitness to drug-resistant variants, we attempted to recover r -resistant viruses for dominance testing. jfh was passaged for multiple rounds of infection in the . each mutation was introduced independently into the jfh genome and rna transfections were performed. the t a genome was the only variant to show any viral rna production by or days post-transfection. we noticed that f l and t a were always isolated together. to test whether these mutations could together increase viral fitness, jfh viruses were generated that contained both mutations. viruses with the mutations separate or together were passaged extensively in the presence of r . occasional resistant outgrowths were observed, but none conferred sustained growth ( figure -figure supplement c). thus, like sofosbuvir, the poor viability of mutant viruses resistant to r precludes the ability to perform further genetic analysis but provides an excellent paradigm for antiviral development. transmission genetics and phenotypic dominance of drug-resistant ns a variant y n ns a is highly oligomeric (sun et al., ; tellinghuisen et al., ) and we were curious as to whether drug resistance or drug susceptibility would be dominant during viral infections. this idea seemed promising because exogenously expressed ns a has a dominant-negative effect on the growth of hcv replicons (graziani and paonessa, ) . additionally, the ns a inhibitors, as a class, display ec 's in the low picomolar range (gao, ) , making them among the most potent antiviral compounds ever identified. assuming uniform inhibitor concentrations in cells and in medium, it has been estimated that only a small fraction of ns a molecules should be bound to drugs under inhibitory conditions (sun et al., ; gao et al., ) . thus, it seemed mechanistically likely that drug-bound ns a proteins from drug-susceptible viruses could be dominant inhibitors of ns a encoded by newly arising drug-resistant ones. however, ns a inhibitors have generally demonstrated low barriers to resistance in patients. our goal was gain mechanistic insight into this dichotomy. the structures of two such potent ns a inhibitors, sr (also known as bms- ) (lemm et al., ) and daclatasvir (gao et al., ) are shown in figure a . mutations of tyr to asp or his confer resistance to a broad array of ns a inhibitors (gao et al., ) . tyr is located near an ns a dimer interface shown in the crystal structure ( figure b ) . thus, this interface is postulated to be part of the binding site for the ns a inhibitor class. the y n and y h mutations were introduced into both the wild-type and codon-altered viruses. as shown in figure c , the y h mutation conferred resistance to both sr and daclatasvir while the y n mutation conferred resistance only to sr . to test whether susceptibility to ns a inhibitors was dominant in the context of viral infections, we analyzed u, s, s + r and r cell populations by flow cytometry as previously performed for the ns / a inhibitor in figure . huh- . . cells were coinfected with ca and wt-y n viruses for hr ( figure d ). cells were then treated with dmso or nm sr for hr, harvested, fixed, costained for wt and ca vrnas and analyzed by flow cytometry. in the absence of the ns a inhibitor, all four populations, u, s, r + s and r were observed ( figure e ,f). in the presence of sr , the s population of cells dropped into the u population as expected. as was the case in figure , the co-infected r + s population of cells dropped into the r population. thus, resistance to ns a inhibitor sr in the context of viral infection was genetically dominant and the lack of rescue of the s virus with which it was mixedly infected shows that drug resistance is also cis-dominant. hcv infected cells become resistant to superinfection upon expression of non-structural proteins (schaller et al., ; tscherne et al., ) . due to this superinfection exclusion, it is likely that all coinfected cells arise through nearly synchronous infection throughout the course of the experiment. to control for any effects on selection that may occur due to the differential timing of coinfections that occurs over the initial hr incubation period, we performed the same experiment with higher titer virus and a single cycle of infection in the absence of drug. huh . . cells were infected at an moi of focus-forming unit (ffu)/cell with ca and wt-y n viruses and incubated for only hr before drug treatment. cells were then incubated in the absence and presence of nm sr for an additional hr. in this case, we also observe cis-dominance of drug resistant wt-y n genomes, indicating that asynchronous coinfection has no effect on selection ( figure g) . finally, the cis-dominance of daclatasvir-resistant wt-y h was observed when coinfected with drug susceptible virus (s) in the absence and presence of daclatasvir ( figure h ). we conclude that ns a, despite being an oligomeric species is not a dominant drug target. instead, genomes resistant to ns a remain drug resistant in co-infected cells but do not rescue drug-susceptible viruses present in the same cell. this is consistent with the observed outgrowth of viruses that are resistant to ns a both in cultured cells and in patients, and with an earlier report that at least some functions of ns a act exclusively in cis (fridell et al., ) . one hypothesis that could mechanistically account for cis-dominant drug resistance is that ns a molecules expressed from different alleles may not freely associate in mixed oligomers. as previously demonstrated, two different ns as expressed from the same rna can associate, while ns a molecules expressed from different constructs could not (berger et al., ) . we were curious whether the dominance phenotypes were altered if we forced ns a alleles to mix. to test whether exogenously expressed drug-susceptible ns a proteins could co-assemble with drug-resistant ns a, we utilized the previously described hcv plasmid that expresses ha-tagged and gfp-tagged ns a within the same polyprotein but does not support genome replication ( figure a) . constructs that contained all combinations of drug-susceptible ns a (s) and the drug-resistant y n variant (r) were created. upon transfection, all tagged proteins were expressed and can be observed in figure (input, panels b,c). immunoprecipitation with anti-ha antibodies revealed that the gfptagged and ha-tagged ns a proteins were present in the same complexes in the presence or absence of sr . therefore, as has been shown previously, mixed oligomers can form upon coexpression within the same polyprotein (berger et al., ) . furthermore, these interactions are not disrupted by drug treatment or by drug-resistant mutations ( figure b ,c). to determine whether there were any functional consequences of mixed oligomer formation, we visualized cells that expressed mixed oligomers using confocal microscopy. all s and r combinations of ns a co-localized at discrete membrane-associated complexes characteristic of hcv infection in the absence of drug ( figure d , top panel). however, in the presence of sr , membrane-associated complex formation was inhibited in r:s and s:s expressing cells and observed only in r:r expressing cells ( figure d, bottom panel) . the dispersal of ns a signal upon drug treatment in the presence of s protein makes ns a protein appear less abundant ( figure d ). however, the immunoblots demonstrate that no such decrease in expression occurs as we observe equal levels of ns a protein independent of allele or the presence of drug ( figure b ,c). one hallmark of hcv infection is the accumulation of cytoplasmic lipid droplets (miyanari et al., ; romero-brey et al., ) . electron microscopy performed by high-pressure freezing and freeze-substitution, to preserve membrane structure, revealed many lipid droplets in the cytoplasm of cells expressing s:s, r:r and s:r combinations of ns as in the absence of inhibitor ( figure e,f) . however, in the presence of sr , only the r:r cells displayed the accumulation of lipid droplets ( figure e , g). therefore, using both assays, the presence of drug-susceptible ns a prevented drug-resistant phenotypes from being displayed, and thus drug-susceptibility was genetically dominant. this confirmed our original hypothesis that ns a had the potential to be a dominant drug target. one of the most likely mechanisms for cis-dominance, when the benefit of a particular gene product accrues only to the genome that encodes it, is physical isolation. we hypothesized that hcv genomes co-infecting the same cell might be physically isolated from each other. to test this possibility, confocal microscopy was used to identify and localize negative vrna strands in genome-specific rna replication complexes ( figure a ). the majority of negative strands of the two different viruses were found to be discrete. identification and quantification the vrna puncta in coinfected cells was determined computationally using volocity software. this program determined the number of negative strand puncta per cell per strain and quantified how many puncta overlapped ( figure b ). this value was low even for the positive stranded vrnas, which are present in the cytoplasm at much higher frequencies ( figure a,b) . as a positive control for colocalization, we performed a similar experiment but additionally stained for ns a or core in addition to minus strand vrnas. we would expect minus strand vrna and ns a to colocalize strongly, as ns a is present inside replication complexes. alternatively, core is not localized directly within replication complexes, but is present within packaging complexes and on lipid droplets, which are nearby. volocity was used to count negative strand vrnas, and then asked, how many of those puncta were touching ns a or core. representative images demonstrating each of the pairwise comparisons demonstrate that nearly % of all minus strand vrnas were touching ns a while fewer than half of the minus strand vrnas were touching core ( figure c,d) . these data support the hypothesis that, upon co-infection, drug-resistant and drug-susceptible rna genomes create independent membranous web structures, limiting the mixing of ns / a and ns a proteins and their precursors. this scenario is modeled schematically in figure e . failure of ns a proteins to mix during infection is a likely explanation for the cis-dominance of drug resistance observed in cultured cells (figure ) . these circumstances account for the ready outgrowth of drug resistance in patients (gao et al., ) , even though ns a is highly oligomeric. (berger et al., ) were created to co-express ha-and gfp-tagged ns a alleles. huh -lunet-t cells were transfected with ptm-dual-ns a constructs that contained two drug-resistant (r), two drug susceptible (s), or mixed alleles of ns a. proteins from transfected cell extracts that were incubated in the absence (b) or presence (c) of sr were subjected to sds-page without further fractionation (input) or after immunoprecipitation with anti-ha antibodies (ip aha). the gel was subjected to immunoblotting with gfp or ha antibodies as indicated. (d) cells were transfected with dual-ns a constructs in the absence or presence of sr for hr. cells were then fixed, stained with anti-ha antibodies and visualized by confocal microscopy. representative images from over cells are presented. (e) cells transfected with dual-ns a constructs that expressed drug-susceptible (s) and drug-resistant (r) alleles as shown were prepared for electron microscopy by highpressure freezing and freeze-substitution and visualized by transmission electron microscopy. even when drug-resistant ns a was overexpressed in a precursor form, no rescue of drug-susceptible virus was detected (figure -figure supplement c ). it has previously been shown that when ha-and gfp-tagged ns a molecules were expressed on different constructs such as those depicted in figure a , no mixed complexes were formed (berger et al., ) . thus, even though high-order ns a oligomers are formed in infected cells, it is unlikely that these are mixed-allele oligomers, preventing dominant inhibition of drug-resistant hcv. due to the highly mutagenic nature of rna viruses and the large number of genomes and antigenomes generated during infection, a high barrier to drug resistance is extremely difficult to achieve. this has led to abandoned usage and development of many otherwise promising antivirals. to decrease the frequency with which drug-resistant variants arise, combinations of antivirals that, individually, exhibit low barriers to resistance are often used. when drug-resistant variants are first formed intracellularly, through error-prone rna replication, they arise in a population that includes parental and sibling drug-susceptible viruses. several genetic relationships between drug-resistant and drug-susceptible genomes are possible. first, the drug resistance of the new variants has the potential to be genetically dominant, and rescue both resistant and susceptible viral genomes. alternatively, drug resistance can be cis-dominant, with the drug-resistant products rescuing only the genomes that encode them. finally, the drug-resistant genome can fail to benefit any genomes in the cell because the drug-susceptible products present in the same cell are dominant inhibitors. the daas targeting ns / a protease of hcv were the first to be discovered (lamarre et al., ) and the first to reach the clinic jacobson et al., ) . it was soon realized that, both during the growth of hcv replicons in cultured cells and in phase ii clinical trials, drug-resistant viruses were generated rapidly (lin et al., ) . nonetheless, in , further advances led to fda approval of telaprevir and boceprevir poordad et al., ) . the anticipation, which proved to be correct, was that inhibitors of ns / a would prove useful in combination therapies (feld et al., ; lawitz et al., ) . we have used flow cytometry to identify cell populations that are co-infected with hcv that is susceptible or resistant at a : ratio, to protease inhibitors. within these cells, the drug-resistant genomes replicated, but the drugsusceptible genomes did not. we therefore conclude that ns / a inhibitor resistance is cis-dominant (figure ) , which should allow the rapid and specific selection for outgrowth from its cell of origin. cis-dominance of drug resistance was also not originally anticipated while targeting ns / a. the original characterizations of the ns / a protease suggested that cleavage of the ns / a junction occurred in cis, but that cleavages at the a/ b, b/ a and a/ b junctions could all occur in trans (bartenschlager et al., ) . we felt that it was therefore, more likely, that drug-resistant ns / a could rescue drug-susceptible virus within the same cell. ns / a is not known to assemble into high-order oligomers in the same manner as ns a, and we therefore did not anticipate drug-susceptible ns / a would be trans-dominant. furthermore, a trans cleavage assay demonstrated that a ns b- b polyprotein could be cleaved by ns / a supplied in trans (romero-brey et al., ) . however, the trans-cleavage system does not result in membranous web formation that would accompany genome sequestration. other groups have reported a different result, that defective ns mutants cannot be rescued in trans by replicons with functional ns / a (kazakov et al., ; appel et al., ) . our interpretation of these studies is that ns / a is likely physically able to figure continued (c) huh . . cells were coinfected with ca and wt-y n at a moi of ffu/cell for hr. cells were costained to visualize core or ns a together with ca and wt vrnas. representative cells are displayed demonstrating all pairwise comparisons analyzed for colocalization. (d) volocity was used to quantify the number of vrnas per cell, the number of colocalized vrnas, as well as the number of vrna puncta touching ns a or core. (e) depiction of the clonal nature of individual rna replication sites (adapted from figure in zayas et al., ] ). membrane invaginations house either drugresistant (red) or drug-susceptible (blue) genomes. in the model, the rna replication sites are segregated and therefore only the rna from drugresistant virus is amplified in the presence of inhibitors of rna replication. it is visually suggested that ns a molecules that bring core protein to lipid droplets for viral assembly mix on this surface and that this could lead to genetic dominance of drug susceptibility at a packaging step. cleave in trans in cells, but requires access to the alternate precursor proteins in order for this to occur. therefore, cis-dominance of drug-resistance is likely the result of a lack of free-mixing of ns / a encoded by different vrnas within the same cell. ns a emerged as an hcv drug target through a chemical genetics screen for compounds that inhibited hcv growth but did not target the ns / a protease or the ns b polymerase (gao et al., ) . the ease with which resistant viruses were selected suggested that drug resistance was either dominant or cis-dominant. this was somewhat surprising, given that ns a is oligomeric and the ns a inhibitors are extremely potent, and have been postulated to function at sub-stoichiometric ratios to ns a protein (gao, ) . indeed, when drug-susceptible and drug-resistant ns a protein were co-expressed in the present study, hetero-oligomers formed and the biological phenotypes of the drug-susceptible protein were dominant ( figure ) . nonetheless, single-cell analysis of cells coinfected at a : ratio with ns a inhibitor-susceptible and -resistant viruses showed, as with the ns / a inhibitor, that resistance to both sr and daclatasvir was cis-dominant ( figure ) . what does cis-dominant resistance mean mechanistically? one potential mechanism is physical sequestration of the rna replication complexes of the two co-infecting genomes. genome-specific rna probing of co-infected cells revealed that both the negative strands and positive strands from the two viruses were present at physically distinct locations ( figure ). it is therefore highly probable that membrane-associated proteins such as hcv ns / a and ns a do not mix within individual rna replication complexes. however, not all mutations in a particular viral product should lead to the same defect with the same genetic properties. for example, we show here that viruses that are defective in a function of ns a in rna replication complexes are not rescued and have no effect on the outgrowth of drug-resistant variants. however, ns a also plays an important role in packaging and assembly of mature hcv particles on lipid droplets (miyanari et al., ; boson et al., ) . lipid droplets are large and form adjacent to rna replication complexes. in figure e , we have depicted the possibility that ns a molecules encoded by distinct rna replication complexes might mix on the surface of these lipid droplets. however, if replication of the drug-susceptible genomes is inhibited, contribution of their encoded proteins to any oligomers on the surface of lipid droplets should be minimal. in this vain, a hypothetical ns a inhibitor that allowed rna replication but inhibited the function of ns a in particle assembly might have different genetic properties than the ns a inhibitors currently in use. viral capsids have especially interesting genetic properties, often intermixing within co-infected cells. defective capsid proteins of poliovirus, hbv and hiv have been shown to be dominant inhibitors of wild-type viruses (crowder and kirkegaard, ; tanner et al., ; tan et al., ; tan et al., ; trono et al., ; pettit et al., ; lee et al., ; müller et al., ; checkley et al., ) . thus, when antiviral targets are capsid proteins, drug susceptibility can be genetically dominant by suppressing the outgrowth of drug-resistant virus within the cell in which it is first generated (crowder and kirkegaard, ; tanner et al., ; kirkegaard et al., ) . for hcv, very few inhibitors of capsid function have been identified, and their inhibition of viral growth is not sufficiently robust to make genetic experiments possible (kota et al., ) . it is therefore not yet possible to test if, as we hypothesize, drug-susceptible virus will prove to be a dominant inhibitor of drug resistance. consistent with this hypothesis, however, epitope-tagged hcv core protein can form mixed disulfide-bonded core oligomers (kushima et al., ) . the success of combination therapy for hcv and the efficacy of the individual constituents illustrate some of the weapons in the arsenal of antiviral strategies. future directions are likely to include, as well, the rational design of antivirals with high barriers to resistance such as those that hyper-stabilize oligomers and the prediction of daa targets that impart a high fitness cost to drug resistance. huh . . cells were a gift from dr. michael gale jr (university of washington) and were cultured in dmem (sigma, st. louis, mo) supplemented with % fetal bovine serum (omega, tarzana, ca), penicillin/streptomycin (invitrogen, grand island ny), non-essential amino acids (invitrogen), and glutamax (invitrogen). huh -lunet-t cells were a gift from dr. ralf bartenschlager (university of heidelberg) and were cultured in dmem supplemented with % fetal bovine serum, penicillin/ streptomycin, non-essential amino acids, glutamax and mg/ml zeocin (invitrogen). cell line identification was performed using str profiling services available through the stanford functional genomics facility. alignments were generated using huh as a reference. cell lines were screened for mycoplasma contamination using the mycoalert mycoplasma detection kit (lonza). the plasmid pjfh was a gift from dr. michael gale jr (kato et al., ) . this plasmid contains a synthesized genome length copy of the jfh strain of hcv (genotype a). to produce cell-culturederived hcv particles (hcvcc), pjfh was digested with xbai (new england biolabs). the linearized plasmid was then used as a template for in vitro transcription with the megascript high yield transcription kit (ambion). vrna was purified using trizol (invitrogen) and electroporated into huh . . cells as previously described to generate hcvcc cultures (wakita et al., ) . following a period of amplification, hcvcc cultures were converted to human serum media as described previously (steenbergen et al., ) . human serum media comprised dmem supplemented with % heat inactivated human serum (omega), penicillin/streptomycin, non-essential amino acids and glutamax. antibodies recognizing hcv core (abcam), gapdh (santa cruz biotechnologies), gfp (life technologies) and ha (genscript) were purchased from the individual suppliers. antibodies recognizing ns a were described previously (lindenbach et al., ) . to construct codon-altered strains of hcv, we subjected three approximately -nucleotide fragments of the jfh genome through the geneart codon optimization algorithm offered by life technologies. the genome fragments were composed of nucleotides - (ca- ), - (ca- ), and - (ca- ). all three codon-altered genome fragments were synthesized by life technologies and cloned into the pjfh plasmid by restriction digestion and ligation with t dna ligase (invitrogen). the resulting plasmids: pjfh -ca- , pjfh -ca- and pjfh -ca- , were used to produce hcvcc cultures as described above. to create drug-resistant hcvcc cultures, two subcloning plasmids were created by pcr by amplifying nucleotides - or - of the pjfh plasmid with taq polymerase (new england biolabs) and ligating the pcr products into pcr . (invitrogen). the resulting plasmids, pcr . - - and pcr . - - were used as templates for site-directed mutagenesis using the quikchange site-directed mutagenesis kit (agilent technologies). pcr . - - -y n was generated using the forward primer '-cctatcaattgcaatacggagggccag tgcgcgcc- ' and the reverse primer '-ggcgcgcactggccctccgtattgcaattgatagg- '. pcr . - - -y h was generated using the forward primer '-cctatcaattgccatacg-gagggccagtgcgcgcc- ' and the reverse primer '-ggcgcgcactggccctccgtatgg-caattgatagg- '. pcr . - - -d a was generated using the forward primer '-aaatccatcgccttcatcccc- ' and the reverse primer '-ggggatgaaggcgatggatttggc- '. these mutated hcv genome fragments were cloned into pjfh or pjfh -ca using restriction digestion and ligation with t dna ligase (invitrogen). hcvcc cultures were generated as described above. the plasmids ptm_ns - b_ns a-ha_ a_ns a-gfp_jfh (referred to as ptm-dual-ns a) and ptm_ns - b_ns a-gfp (referred to as ptm-ns - b) were the generous gifts of dr. ralf bartenschlager (university of heidelberg). the d a and y n mutations were cloned into the ptm-ns - b plasmid using the quikchange lightening mutagenesis kit using the primers described above. the ns a alleles of the ptm-dual- a plasmid were first separated by removing an rsrii fragment containing most of the ns a-gfp allele to create ptm-dual- a-drsrii and pcdna -ns a-gfp-rsrii. site-directed mutagenesis was performed using the quikchange lightening kit on ptm-dual- a-drsrii or on pcdna -ns a-gfp-rsrii independently. the rsrii fragments containing wildtype ns a or ns a-y n were then cloned back into the ptm-dual- a-drsrii vectors to create all combinations of wild-type ns a and ns a-y n ptm-dual- a. vrna was harvested from cells using trizol (invitrogen) or collected from hcvcc culture supernatants using the qiaamp vrna mini kit (qiagen). a standard curve was generated using in vitro transcribed hcv vrna. qrt-pcr was performed using the quantitect sybr-green rt-pcr kit (qiagen) and the qrt-pcr forward '-ctggcgactggatgcgtttc- ' and reverse '-cgcattcctccatc tcatca- ' primers. alternatively, the following ca-specific primers were used: forward '-gtggtg tccatgaccggca- ' and reverse '-ggtcacggggcctctcagt- ', or the following wt-specific primers were used: forward '-gtggtgagtatgacggggc- ' and reverse '-cgtgaccg-gaccccgtaag- '. samples were analyzed on a real-time pcr machine (applied biosystems). wt vrna target probes recognizing the ns region of either the positive or negative strand were designed and synthesized by affymetrix. these probes were specifically designed to avoid detection of codon-altered jfh viral rna. additionally, probes were designed to recognize the corresponding region of the negative or positive strand jfh -ca vrna. these ca target probes were specifically designed not to recognize the wt vrna. huh . . cells were infected with wt or ca hcvcc particles for hr. infected cells were fixed with % formaldehyde solution (sigma) and subjected to rna in situ hybridization (ish) using the viewrna cell assay kit (affymetrix) according to the manufacturer's protocol. cells were co-stained with both ca and wt vrna target probe sets in all experiments. cells were visualized on a leica sp confocal microscope. protein and vrna colocalization was performed on cells coinfected with jfh -ca and jfh -y n for hr. following infection, cells were fixed and stained using the view-rna cell plus assay reagents. core and ns a were visualized using the antibodies described above at a to dilution followed by the anti-mouse-alexaflour- secondary antibody at to dilution. quantification of colocalization was performed using volocity software (perkin elmer). briefly, we defined vrna puncta as objects larger than . mm . objects larger than . mm were broken into subunits based on total volume. objects sharing . mm of mutual space were quantified as mutual. due to the localization patterns of core and ns a, spot counting algorithms were not appropriate. total vrna objects and as well as the total number of vrna objects touching ns a or core were quantified. huh -lunet-t cells were transfected with ptm-dual-ns a constructs using branched polyethylenimine (sigma-aldrich) at a ratio of : . at hr post-transfection, cells were treated with nm sr or a dmso control. at hr post-transfection, cells were fixed with % paraformaldehyde, stained with anti-ha antibodies and dapi and visualized on a leica sp confocal microscope. a more detailed description of the rna fish microscopy methods are provided in bio-protocol (van buuren and kirkegaard, ) . huh -lunet-t cells were transfected with ptm-dual-ns a constructs using the polyethylenimine transfection reagent. at hr post-transfection, cells were treated with dmso or nm sr . at hr post-transfection, cells were harvested using an enzyme-free cell dissociation buffer (life technologies) and facs sorted for gfp-positivity on a facs aria cell sorter. gfp-positive cells were resuspended in % bsa in pbs then placed into a mm deep hat and high-pressure frozen using a leica empact . frozen samples were then freeze substituted in % osmium tetroxide and . % uranyl acetate in acetone using a leica emafs at À ˚c for hr, warmed to À ˚c in . hr at ˚c/ hr and held for hr then warmed to ˚c in hr at ˚c/hr and held for hr. the samples were then washed two times in acetone, then in propylene oxide for min each. samples are infiltrated with embed- resin (ems cat# ) mixed : , : , and : with propylene oxide for hr each, leaving samples in : resin to propylene oxide overnight rotating at room temperature. the samples are then placed into embed- for three hours then placed into taab capsules with fresh resin and placed into a ˚c oven overnight. sections were taken between and nm, picked up on formvar/carbon coated mesh copper grids, then contrast stained for s in . % uranyl acetate in % acetone followed by staining in . % lead citrate for min. cells were visualized using the jeol jem- kv microscope and photos were taken using a gatan orius k  k digital camera. huh . . cells were either transfected with wt and/or ca vrna as previously described or infected with wt and/or ca hcvcc particles. coinfections were performed by infecting with each virus for or hr followed by treatment with either mm biln- for hr or nm sr for hr. cells were harvested with trypsin and fixed with the flowrna fixation and permeablization kit. cells were then costained with ca and wt vrna target probe sets using the flowrna kit (affymetrix) and analyzed on the scanford facscan flow cytometer. data was analyzed and processed using flowjo software. mutational and fitness landscapes of an rna virus revealed through population sequencing ledipasvir and sofosbuvir for untreated hcv genotype infection quantigene flowrna assay. a novel rna ish multicolor application for flow cytometry efficient rescue of hepatitis c virus rna replication by transcomplementation with nonstructural protein a boceprevir for previously treated chronic hcv genotype infection kinetic and structural analyses of hepatitis c virus polyprotein processing daclatasvir-like inhibitors of ns a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication permissive secondary mutations enable the evolution of influenza oseltamivir resistance daclatasvir prevents hepatitis c virus infectivity by blocking transfer of the viral genome to assembly sites the capsid-spacer peptide gag processing intermediate is a dominant-negative inhibitor of hiv- maturation complete three-dimensional structures of picornaviral rna-dependent rna polymerases trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses coronaviruses resistant to a c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance daclatasvir plus sofosbuvir for hcv infection general catalytic deficiency of hepatitis c virus rna polymerase with an s t mutation and mutually exclusive resistance towards '-modified nucleotide van oral direct-acting agent therapy for hepatitis c virus infection: a systematic review treatment of hcv with abt- /r-ombitasvir and dasabuvir with ribavirin combining oncolytic virotherapy and cytotoxic therapies to fight cancer distinct functions of ns a in hepatitis c virus rna replication uncovered by studies with the ns a inhibitor bms- chemical genetics strategy identifies an hcv ns a inhibitor with a potent clinical effect antiviral activity and resistance of hcv ns a replication complex inhibitors hcv ns a replication complex inhibitors dominant negative effect of wild-type ns a on ns a-adapted subgenomic hepatitis c virus rna replicon virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions telaprevir for previously untreated chronic hepatitis c virus infection cell culture and infection system for hepatitis c virus hepatitis c virus rna replication depends on specific cis-and trans-acting activities of viral nonstructural proteins origins of combination therapy for tuberculosis: lessons for future antimicrobial development and application my cousin, my enemy: quasispecies suppression of drug resistance ná jera i. . the novel nucleoside analog r ( '-azidocytidine) is a potent inhibitor of ns b-dependent rna synthesis and hepatitis c virus replication in cell culture ml , a small molecule inhibitor of dimerization of the core protein of hepatitis c virus (hcv) a disulfide-bonded dimer of the core protein of hepatitis c virus is important for virus-like particle production an ns protease inhibitor with antiviral effects in humans infected with hepatitis c virus sofosbuvir for previously untreated chronic hepatitis c infection ribavirin, to treat chronic infection with hepatitis c virus genotype in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the cosmos randomised study a strongly transdominant mutation in the human immunodeficiency virus type gag gene defines an achilles heel in the virus life cycle discovery of potent hepatitis c virus ns a inhibitors with dimeric structures in vitro resistance studies of hepatitis c virus serine protease inhibitors, vx- and biln : structural analysis indicates different resistance mechanisms complete replication of hepatitis c virus in cell culture novel therapies for hepatitis c -one pill fits all? suppression of drug resistance in dengue virus the r k substitution in hiv- subtype c is more deleterious for integrase enzymatic function and viral replication than in subtype b the lipid droplet is an important organelle for hepatitis c virus production hiv- gag processing intermediates trans-dominantly interfere with hiv- infectivity processing sites in the human immunodeficiency virus type (hiv- ) gag-pro-pol precursor are cleaved by the viral protease at different rates the coding region of the hcv genome contains a network of regulatory rna structures boceprevir for untreated chronic hcv genotype infection three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication ns a domain and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication viral mutation rates analysis of hepatitis c virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes understanding the hepatitis c virus life cycle paves the way for highly effective therapies human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a -fold increase in hcv japanese fulminant hepatitis type titers resensitizing daclatasvir-resistant hepatitis c variants by allosteric modulation of ns a l f and v a sofosbuvir-associated hepatitis c virus ns b substitutions genetically altering the thermodynamics and kinetics of hepatitis b virus capsid assembly has profound effects on virus replication in cell culture the interface between hepatitis b virus capsid proteins affects self-assembly, pregenomic rna packaging, and reverse transcription dominant drug targets suppress the emergence of antiviral resistance exploiting genetic interference for antiviral therapy structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase hiv- gag mutants can dominantly interfere with the replication of the wild-type virus detection and differentiation of multiple viral rnas using branched dna fish coupled to production of infectious hepatitis c virus in tissue culture from a cloned viral genome treatment with ledipasvir and sofosbuvir improves patient-reported outcomes: results from the ion- , - , and - clinical trials coordination of hepatitis c virus assembly by distinct regulatory regions in nonstructural protein a we thank drs. yury goltsev and garry nolan for advice on fluorescent cell sorting-based visualization of rna, drs. michael gale jr. and ralf bartenschlager for the generous donation of reagents and dr. peter sarnow for critical reading of the manuscript. we would like to acknowledge the work of drs. jeannie spagnolo and ernesto mendez for initiating hcv research in our laboratory. this work was supported by funding to kk from nih u ai (jeffrey glenn, pi), an nih director's pioneer award and the alison and steve krausz innovation fund. nvb was supported by the canadian institutes for health research ncrtp-hepc training program and the american liver foundation. electron microscopy was performed in a facility supported in part by arra award number s rr - from the national center for research resources (ncrr). the cell sciences imaging facility used for confocal microscopy was supported by arra award number s od from the ncrr. the contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the ncrr or the national institutes of health. key: cord- - kz iws authors: qutayba almerie, muhammad; daniel kerrigan, david title: the association between obesity and poor outcome after covid- indicates a potential therapeutic role for montelukast date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: kz iws it is widely believed that infection with the sars-cov virus triggers a disproportionate immune response which causes a devastating systemic injury, particularly in individuals with obesity, itself a chronic, multi-organ inflammatory disease. immune cells accumulate in visceral adipose tissue and together with paracrine adipocytes release a wide range of biologically active cytokines (including il- β, il , il and il ) that can result in both local, pulmonary and systemic inflammation. a more intense ‘cytokine storm’ is postulated as the mechanism behind the extreme immune response seen in severe covid- . it is striking how dangerous the combination of obesity and covid- is, resulting in a greater risk of icu admission and a higher mortality. furthermore, patients from a bame background appear to have increased mortality after sars-cov infection; they also have a higher prevalence of central obesity and its metabolic complications. in the absence of an effective vaccine, the therapeutic potential of immune-modulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid- . montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il- expression in inflammatory cells. it has also been shown to decrease elevated levels of il- β and il in humans with viral upper respiratory tract infections compared with placebo-treated patients. in addition, in silico studies have demonstrated a high binding affinity of the montelukast molecule to the terminal site of the virus’s main protease enzyme which is needed for virus rna synthesis and replication. montelukast, which is cheap, safe and widely available would appear to have the potential to be an ideal candidate drug for clinical trials, particularly in early stage disease before irreparable tissue damage has already occurred. hypothesis: through a direct anti-viral effect, or by suppression of heightened cytokine release in response to sars-cov , montelukast will reduce the severity of immune-mediated multiorgan damage resulting from covid- , particularly in patients with central obesity and metabolic syndrome. in the absence of an effective vaccine, the therapeutic potential of immune-modulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid- . montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il- expression in inflammatory cells. it has also been shown to decrease elevated levels of il-  and il in humans with viral upper respiratory tract infections compared with placebo-treated patients. in addition, in silico studies have demonstrated a high binding affinity of the montelukast molecule to the terminal site of the virus's main protease enzyme which is needed for virus rna synthesis and replication. montelukast, which is cheap, safe and widely available would appear to have the potential to be an ideal candidate drug for clinical trials, particularly in early stage disease before irreparable tissue damage has already occurred. through a direct anti-viral effect, or by suppression of heightened cytokine release in response to sars-cov , montelukast will reduce the severity of immune-mediated multiorgan damage resulting from covid- , particularly in patients with central obesity and metabolic syndrome. it is widely believed that infection with the sars-cov virus triggers a disproportionate immune response which causes a devastating systemic injury, particularly in individuals with obesity, itself a chronic, multi-organ inflammatory disease. immune cells accumulate in visceral adipose tissue and together with paracrine adipocytes release a wide range of biologically active cytokines (including il- , il , il and il ) that can result in both local, pulmonary and systemic inflammation - . a more intense 'cytokine storm' is postulated as the mechanism behind the extreme immune response seen in severe covid- . it is striking how dangerous the combination of obesity and covid- is, resulting in a greater risk of icu admission and a higher mortality , suggesting that the already heightened background inflammatory process resulting from obesity might prime the immune system for a more catastrophic response to sar-cov infection. furthermore, patients from a bame background appear to have increased mortality after sars-cov infection , an observation that has at least partly ben explained by the higher prevalence of central obesity and its metabolic complications in this group , . in the absence of an effective vaccine, exploring the therapeutic potential of immunemodulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid- . montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il- expression in inflammatory cells . it has also been shown to decrease elevated levels of il-  and il in humans with viral upper respiratory tract infections compared with placebo-treated patients . in addition, in silico computer modelling studies have demonstrated a high binding affinity of the montelukast molecule to the sars-cov virus's main protease enzyme substrate-binding pocket that is involved in virus rna synthesis and replication , . evidence supporting montelukast as a candidate covid- therapy in individuals with obesity: patients affected by severe obesity share a common physiological response with patients with covid- as both have raised concentrations of pro-inflammatory cytokines (e.g., tnf-a, il and il ) and t-helper- cytokines (il , il ) [ ] [ ] [ ] . whilst it is impossible to exclude increased viral burden as the driver for this inflammatory response in covid- , there is evidence that some patients develop 'cytokine storm (release) syndrome', described as an uncontrolled and overwhelming release of proinflammatory mediators by an overly-activated immune system , , . an overwhelming production of proinflammatory mediators in the lungs causes localised pulmonary injury characterised by diffuse alveolar damage with epithelial and endothelial apoptosis, and thrombosis due to dysregulated coagulation and fibrinolysis. these mediators may also leak into systemic circulation leading to cardiac, renal and hepatic inflammation and multiorgan failure . the background chronic inflammatory state associated with obesity could act as a catalyst for the a massive inflammatory response to sars-cov by priming the immune system, triggered by the 'second hit' of covid- , and explaining why those with obesity are more likely to become severely ill, require icu care and die. severe lymphopenia with hyperactivated proinflammatory t cells and decreased regulatory t cells are commonly seen in critically ill covid- patients supporting the likelihood of a dysregulated immune response to the viral infection . leukotrienes (ltc , ltd , and lte ) are peptide-conjugated lipids that are prominent products of activated eosinophils, basophils, mast cells and macrophages , . they are generated de novo from cell membrane phospholipid-associated arachidonic acid via the -lipoxygenase pathway. known to cause contraction of bronchial smooth muscle, leukotrienes are now also recognised as potent inflammatory mediators that initiate and propagate a diverse array of biologic responses including macrophage activation, mast cell cytokine secretion and dendritic cell maturation and migration , . animal studies indicate a significant role for leukotrienes in adaptive immunity, particularly the induction of th response in the lung via their effect on dendritic cells and cytokine generation (mainly il ), the recruitment and/or activation of effector cells (especially eosinophils and mast cells), pulmonary inflammation, microvascular permeability and pulmonary fibrosis , . montelukast , (montelukast sodium: c h clno s) is a leukotriene receptor antagonist which demonstrates a marked affinity and selectivity to the cysteinyl leukotriene receptor type- , thus it elicits substantial blockage of any ltd leukotriene-mediated effect, resulting in reduced inflammation and relaxation of smooth muscle. it has also been shown to diminish the pulmonary response to antigen, tissue eosinophilia and the number of inflammatory cells expressing il- . montelukast decreases elevated levels of il-  and il in nasal washouts in humans with viral upper respiratory tract infections compared with placebo-treated patients . montelukast was first approved for clinical use by the us fda in . it is currently licensed to treat asthma, exercise-induced bronchospasm and allergic rhinitis. montelukast mg is given orally once daily (oral bioavailability %). it is highly metabolised in the liver by cytochrome p . the drug and its metabolites are almost exclusively excreted in the bile and into the faeces. with its prominent effect in reducing leukotriene-mediated cytokine release montelukast would have the potential to moderate the background inflammation associated with obesity and the body's inflammatory response to sars-cov . coronaviruses are enveloped viruses with a positive rna genome. the virus contains at least four structural proteins: spike (s) protein, envelop (e) protein, membrane (m) protein, and nucleocapsid (n) protein , . sars-cov- entry into the host cells is mediated by an interaction between the spike protein and angiotensin-converting enzyme (ace ) receptors , , cell surface proteins highly expressed in the lung, heart and kidney. wu et al performed in silico, target-based virtual ligand screening for sars-cov- main proteins including the main protease, spike protein, rna-dependent rna polymerase and papain-like protease. the authors screened approved drugs from a subset of the zinc database which contains compounds . they also screened a natural product database of , chemicals separated from traditional chinese herbals and known anti-viral drugs. their computer modelling study found that montelukast should demonstrate binding energy to the virus's main protease enzyme. it fitted well into the active pocket of the main protease, in which hydrophobic amino acids would create a relatively hydrophobic environment to contain the drug and stabilise its conformation . similarly, a report by farag et al predicted a high affinity of montelukast to the same binding domain in the main protease enzyme with hydrophobic-hydrophobic and hydrogen bond-interactions. farag et al focussed on targeting the sars-cov- main protease enzyme at its two potential binding sites in the main substrate-binding pocket: the central site and terminal site. the authors screened > , fda-approved drugs to identify potential hits based on drug molecules' binding energies, binding modes and the predicted interaction of amino acids with the enzyme's substrate binding pockets. whilst antiviral drugs unsurprisingly had the best binding to the central site of the main protease enzyme substrate-binding pocket, montelukast demonstrated a very high affinity for the terminal site, better than any other drug tested at this site whist drug binding in in silico modelling studies such as these may not translate into clinical effect, the protease enzyme is essential for the sars-cov- virus rna synthesis and replication and if montelukast was shown to confer benefit in clinical studies it may be, at least in part, due drug-induced disruption of the virus replication cycle. one of the main treatment strategies for covid- is to identify new targeted antiviral drugs based on the genomic information and pathological characteristics of sars-cov- . these drugs are likely to be the most effective against the virus. however, anti-viral drug development and registration is time-consuming, the drug might not be available for the current outbreak and it is likely to be very expensive, an important factor particularly in poorer countries afflicted by covid- . an alternative, faster strategy is 'drug repurposing'. this relies on identification of potential therapeutic characteristics of drugs currently licensed for other purposes. there are many clinical trials enrolling patients with covid- worldwide investigating the potential benefits of established antimicrobial, antiviral and anti-inflammatory drugs . the strong association between the pro-inflammatory state found in metabolic syndrome and obesity and a more aggressive clinical course in covid- suggests a potential treatment role for drugs that inhibit cytokine release and macrophage activation. there is, however, so far no convincing clinical evidence to support the use of corticosteroids, one of the most widely utilised anti-inflammatory agents. if anything, there is concern about patients being harmed with such a treatment , . anti-cytokines (interleukin inhibitors, e.g., tocilizumab) have also attracted much interest recently with a few ongoing clinical trials assessing their role . however, these monoclonal antibodies are expensive and like corticosteroids have the potential for promoting secondary infections. cost and complications are likely to limit their application on a global scale, other than selective use in severely ill patients , . we believe there is a good theoretical basis to consider montelukast as a therapeutic candidate with a hypothetical dual mode of action through suppression of cytokine activity and interference with viral replication. to date, the cochrane covid- study register does not identify any studies exploring the clinical benefits of montelukast. whilst it is unlikely that montelukast would be effective at curing covid- or reversing severe immune-modulated multiorgan damage, it could have a powerful role in preventing disease progression when used at an early stage in the disease, reducing the need for hospital admission, or the requirement for the care of hospitalised patients to be escalated up to level critical care. it may shorten the course of mild to moderate covid- with the attendant social, economic and healthcare system benefits that would bring. furthermore, it has a good safety profile. but perhaps most importantly, if montelukast was shown to be of clinical benefit in well-conducted studies it is a widely applicable treatment, accessible to all patients, and at just over £ per month , not just available to those who live in nations with firstworld healthcare resources that can afford highly expensive new drugs. neither of the authors have any conflicting interests and neither receive funding from any third party or external source. professor kerrigan was the original creator of the hypothesis linking potential therapeutic benefits of montelukast in covid- , with the inflammatory state associated with obesity. both authors were involved in background research and the writing of the manuscript. this paper did not require ethics approval. regulation of adipose tissue inflammation by interleukin circulating levels of mcp- and il- are elevated in human obese subjects and associated with obesity-related parameters inflammatory cytokines in general and central obesity and modulating effects of physical activity features of , hospitalised uk patients with covid- using the isaric who clinical characterisation protocol are more black, asian and minority ethnic people dying with covid- than might be expected? southall and brent revisited: cohort profile of sabre, a uk population-based comparison of cardiovascular disease and diabetes in people of european, indian asian and african caribbean origins is ethnicity linked to incidence or outcomes of covid- ? montelukast, a leukotriene receptor antagonist, inhibits the late airway response to antigen, airway eosinophilia, and il- -expressing cells in brown norway rats effect of montelukast on pro-inflammatory cytokine production during naturally acquired viral upper respiratory infections (vuris) in adults analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods identification of fda approved drugs targeting covid- virus by structure-based drug repositioning (pre-print) dysregulation of immune response in patients with covid- in wuhan, china. clin infect dis clinical features of patients infected with novel coronavirus in wuhan clinical features of cases with coronavirus disease covid- : consider cytokine storm syndromes and immunosuppression pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses cysteinyl leukotrienes: multifunctional mediators in allergic rhinitis leukotriene e and granulocytic infiltration into asthmatic airways singulair (montelukast sodium) us fda label coronaviruses -drug discovery and therapeutic options structural insights into coronavirus entry a pneumonia outbreak associated with a new coronavirus of probable bat origin structure, function, and antigenicity of the sars-cov- spike glycoprotein zinc: a free tool to discover chemistry for biology a real-time dashboard of clinical trials for covid- clinical evidence does not support corticosteroid treatment for -ncov lung injury immunosuppression for hyperinflammation in covid- : a double-edged sword? can we use interleukin- (il- ) blockade for coronavirus disease (covid- )-induced cytokine release syndrome (crs)? why tocilizumab could be an effective treatment for severe covid- ? key: cord- -bnl i oy authors: wright, gerard d title: q&a: antibiotic resistance: where does it come from and what can we do about it? date: - - journal: bmc biol doi: . / - - - sha: doc_id: cord_uid: bnl i oy nan antibiotics target essential bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. there are five major antibiotic targets: the bacterial cell wall, the cell membrane, protein synthesis, dna and rna synthesis, and folic acid (vitamin b ) metabolism ( figure ). these bacterial targets are different or nonexistent in eukaryotic cells (including those of humans), which means that antibiotics are relatively nontoxic drugs. for example, the β-lactam antibiotics such as penicillins, cephalosporins and carbapenems block the synthesis of the bacterial cell wall. this structure is absent in higher organisms but is essential for bacterial survival. the bacterial ribosome is the target of the tetracycline, aminoglycoside, macrolide and other antibiotics, and is sufficiently different from the eukaryotic ribosome that cross-inhibition does not occur. resistance to antibiotics occurs through four general mechanisms: target modification; efflux; immunity and bypass; and enzyme-catalyzed destruction ( figure ). target modification can occur through mutation of the targets themselves -for example, the topoisomerases that are the target of the fluoroquinolone antibiotics -or by the production of enzymes that modify antibiotic targets, as, for example, in ribosomal methylation. vancomycin resistance is a version of target modification where new biosynthetic machinery is engaged to alter cell-wall structure. efflux occurs through a large family of protein pumps that eject antibiotics from inside the cell. in immunity, antibiotics or their targets are bound by proteins that prevent the antibiotic binding to its target. arguably, the most specific and evolved mechanism of antibiotic resistance are enzymes that recognize antibiotics and modify them in such a way as to eliminate the functional characteristics that enable them to interact with their targets. for example, β-lactamases hydrolytically cleave the core β-lactam ring that is characteristic of the class and essential to antibiotic action. resistance to antibiotics was recorded even before the first clinical use of penicillin in the early s. in the intervening years, resistance to all classes of antibiotics has emerged, and there are no antibiotics for which resistance does not exist. there are two general strategies for resistance. one comprises mechanisms that transfer resistance vertically from a bacterium to its progeny. examples are mutations in chromosomal genes that give rise to drug-insensitive products, such as the point mutations in the genes encoding dna gyrase or topoisomerase iv that result in resistance to fluoroquinolone antibiotics such as ciprofloxacin. the second strategy includes the actions of genes that can be transmitted both vertically to progeny and horizontally to other bacteria, even those of different genera. these genes are located on mobile genetic elements such as plasmids, which can carry one or more resistance genes. many of the β-lactamase genes that confer resistance to the penicillin, cephalosporin, penem and monobactam antibiotics are located on such elements, as is the glycopeptide-resistance gene cluster vanhax, which provides resistance to vancomycin. the prevalence and mobility of resistance genes in previously sensitive pathogenic bacteria has now reached crisis levels in many cases because new antibiotics are no longer being developed at a rate that can keep pace with microbial evolution. in the past two decades we have witnessed: • the rise of so-called extended spectrum β-lactamases (esbls), which are mutants of enzymes that previously could only inactivate penicillins but now have gained activity against many cephalosporins; • carbapenemases such as kpc and ndm- that inactivate all β-lactam antibiotics; • plasmid-mediated (and thus horizontally disseminated) resistance to fluoroquinolone antibiotics; • the spread of virulent mrsa (methicillin-resistant staphylococcus aureus) in the community; • the rise of multi-drug resistant neisseria gonorrhoea; • the emergence and global dissemination of multi-drug resistant acinetobacter baumannii, pseudomonas aerugi nosa, klebsiella pneumoniae and enterobacteriaceae; • the spread of extensively drug resistant mycobacterium tuberculosis; • the development of resistance to the two newest antibiotics to be approved for clinical use -daptomycin and linezolid. resistance is relentless and unavoidable as long as we use antibiotics. antibiotic resistance is the evolutionary response to the strong selective pressure that results from exposure to these compounds. the horizontal dissemination of resistance genes into bacterial species and genera that are not themselves intrinsically resistant, as well as the maintenance of resistance mutations vertically through populations is likely to be the result of contemporary use of these drugs in the clinic and on the farm. support for this hypothesis is the infrequency of antibiotic resistance in collections of pathogenic bacteria that pre-date the antibiotic era. nevertheless, antibiotic resistance is a natural phenomenon. it has been recognized for decades that the resistance mechanisms that have emerged in the clinic parallel those that are intrinsic to the bacteria that produce antibiotics. recent studies of non-pathogenic soil bacteria have revealed that the majority of environmental bacteria tested are multi-drug resistant. this reflects the fact that these microbes live and have evolved in an environment where small bioactive molecules, some toxic, some benign, are plentiful and diverse. bacteria have simply evolved to interact with them and control their biological effects. pathogens, on the other hand, are often more virulent forms of our commensal bacteria and simply have not been exposed to the diversity and types of small molecules found in the environment; as a result, they have not required the gamut of resistance genes found in some environmental bacteria. furthermore, the genes and proteins responsible for resistance in environmental bacteria are homologous to those found circulating in pathogens, strongly suggesting contemporary horizontal gene transfer. opportunistic pathogens with environmental reservoirs -for example, p. aeruginosa and a. baumannii -are highly drug resistant and have a remarkable capacity to acquire new resistance genes. the environment is therefore a large reservoir of potential resistance genes: the environmental 'resistome' . given the vast numbers of bacteria on the planet and the massive selection pressure provided by antibiotics, the movement of antibiotic-resistance elements from benign, but resistant, microbes into previously susceptible pathogens is simply a matter of time and opportunity. antibiotics themselves are the source of the evolutionary pressure that eventually renders them obsolete. limiting exposure of microbes to antibiotics therefore makes good sense to reduce the opportunity for the selection and dissemination of resistance. the inappropriate use of antibiotics by clinicians and the agricultural community needs to be curtailed. over the past several years, the medical community in particular has made concrete efforts to curb the improper use of antibiotics. the european union has taken the lead in limiting the nontherapeutic use of antibiotics in food animals. robust surveillance networks that span the clinic and the farm need to be supported in order to monitor the impact of resistance and the emergence of new threats in real time. in north america, efforts such as the strategies to address antimicrobial resistance act seek to diminish antibiotic use in agriculture and improve surveillance. furthermore, there have been several successful campaigns to educate the public on the importance of antibiotics and the proper use of these drugs. while none of these efforts is perfect, there is much to be celebrated and encouraged. these measures all serve to reduce antibiotic use and, as a result, delay the emergence of resistance. furthermore, by decreasing selection pressure, the opportunity for the rise of particularly clinically challenging or virulent organisms should be lessened. all strategies that reduce the incorrect use of antibiotics are welcome, but in the end new drugs will always be needed because of the inevitability of resistance. unfortunately, in the developing world, access to antibiotics is frequently not regulated and their use in agriculture is often rampant. these facts make antibiotic stewardship especially challenging. in an era of rapid intercontinental travel, pathogens are no longer geographically contained and can move from country to country with ease. the recent examples of transcontinental spread of the severe acute respiratory syndrome (sars) virus from guangdong province in china to hong kong and then canada in , and the ndm- carbapenemase, which inactivates all β-lactam antibiotics and appears to have originated in the indian subcontinent but is now found in north america, the uk and europe, make the point. the growing problem of resistance fuels a continuous need for new antibiotic drugs. the enterobacteria that produce carbapenemase are just one example of antibiotic-resistant enterobacteria. other gram-negative pathogens resistant to virtually all antibiotics include multi-drug resistant a. baumannii and p. aeruginosa. the expanding problem of mrsa, and the global challenge of extensively drug-resistant m. tuberculosis (also called extreme drug-resistant m. tuberculosis), require new therapies. there are some promising new candidates on the horizon, especially for the treatment of infections caused by gram-positive pathogens such as mrsa and enterococci. as already mentioned, two new drugs active against this microbial spectrum -daptomycin and linezolid -have been introduced in the past decade. tigecycline, a third-generation semi-synthetic tetracycline antibiotic approved in , also has activity against mrsa. the semi-synthetic glycopeptide antibiotic telavancin recently received approval in the united states and the fifth-generation cephalosporin ceftobiprole is available in some european countries and canada. however, there are few candidates in late-stage clinical trials suitable for the problem of gram-negative pathogens. here, often the choice of last resort is colistin, an antibiotic discovered more than years ago and seldom used in the past because of adverse affects, including kidney toxicity; however, it is now increasingly used. there are a number of reasons, some economic, for the paucity of new antibiotics. they include challenging and shifting processes of government regulatory approval that add to the risk for the pharmaceutical industry. furthermore, considerations of return on investment favor drugs for chronic diseases, which are taken by patients over long periods of time, often decades. in contrast, antibiotics cure disease and are taken for short periods of time. other reasons for the decline in antibiotic discovery and development are scientific. the first wave of antibiotics discovered five decades ago have been termed the 'low hanging fruit' . despite the discovery of numerous compounds with antibiotic properties in the years since, few have had the requisite properties to become drugs. most antibiotics are natural products or their derivatives that have been isolated from soil bacteria. some researchers have suggested that this source might now be exhausted. furthermore, the promise of the genomic era and the reality of hundreds of available bacterial genomes have so far failed to deliver the hoped-for new molecular targets for antibiotics. other new technologies, such as highthroughput screening of libraries of synthetic molecules, have not resulted in new drugs, although this may reflect a poor choice of chemical classes in the screens, emphasizing molecules more active in human biology than as antibiotics. test compounds were often skewed in favor of small lipophilic molecules with physical properties meeting the criteria of lipinski's rule of . however, though helpful in assessing the prospect of a compound to be an orally active drug for human disease, this strategy has been shown to fail when searching for antibiotics. well, natural chemicals have significant advantages. although the first antibiotics introduced into the clinic were the synthetic sulfonamides, the majority of antibiotics in current clinical use are bacterially produced natural products or their derivatives; only a few are completely synthetic in origin. the reasons for this in part reflect the history of antibiotic discovery postpenicillin, and the relative ease of discovery of suitable molecules through screening the products of soil microbes compared with libraries of synthetic compounds. many of these 'natural' antibiotics have desirable drug-like qualities, such as good bioavailability, the ability to cross the cell membrane (and outer membrane in the case of compounds with gram-negative activity) and the ability to evade efflux systems, and chemical structures that favor binding to vital cellular targets, supporting the idea that natural products encompass privileged structures in antibiotic drug discovery. however, the increasing difficulty of identi fying new chemical compounds with equally suitable drug-like characteristics from natural sources has caused natural-product-based screening programs to fall out of favor in many pharmaceutical firms over the past few decades. instead, the ability of parallel synthesis methods to generate hundreds of thousands of synthetic molecules suitable for modern high-throughput screening has shifted the focus in favor of synthetic molecules in commercial antibacterial drug discovery. the advantages of synthetic compounds are not insignificant: pure lead molecules can easily be produced in quantity and quality suitable for clinical trials, and are relatively easily modified to improve target affinity. however, after two to three decades of emphasis on such molecules and millions of dollars spent on high-throughput in vitro and cell-based screens, no new synthetic antibiotics have emerged. linezolid, the one synthetic antibiotic to be brought to market in the past decade, was discovered using traditional medicinal chemistry in a research program with a plant-disease focus in the early s. they do have great advantages, although a direct comparison of the success and failure of synthetic as against natural product libraries is unfair. microbial natural products have evolved over millennia to interact with biological molecules, whereas the synthetic chemical libraries used in antibiotic drug-discovery screens were generally developed with a focus on eukaryotic drug-discovery campaigns, as noted earlier. efforts to develop physical-property rules for antibiotics and to incorporate natural-product-like chemical complexity in libraries of synthetic chemicals will no doubt improve success in identifying new synthetic antibiotic leads. ironically, at the same time that the pharmaceutical industry was abandoning natural-product libraries, university researchers were making remarkable advances in understanding the molecular details of natural-product biosynthesis by bacteria. many bacteria, especially the actinomycete group of common environmental bacteria, are prodigious producers of natural products. these are termed secondary metabolites to contrast with molecules of primary metabolism, such as carbohydrates, amino acids and so on. secondary metabolites range in molecular weight from around daltons (da) to up to , da and they have diverse biological activities, including induction of cell death (antibiotics such as tetracycline, vancomycin and daptomycin, and anticancer agents such as adriamycin), iron sequestration (for example, enterobactin), facilitation of cell-cell communication (γ-butyrolactones), protection from oxidizing agents (phenazines), and a host of others. the bacterial natural products that are most important as antibiotics include polyketides, such as the macrolides and tetracyclines, and non-ribosomal peptides -that is, peptides that are not synthesized on ribosomes -which include β-lactams and glycopeptides such as vancomycin. these are produced in the cell in assembly-line fashion on large dedicated enzyme platforms called, respectively, polyketide synthases and non-ribosomal peptide synthetases. following assembly the compounds are then 'decorated' by a series of modifying enzymes, such as glycosyltransferases. the end result is a molecule of often complex structure, with multiple chiral centers and functional groups such as sugars, halogens, sulfates, acyl groups and others. in general, bacterial genes that encode the production of natural products are clustered together in the genome, greatly facilitating analysis and prediction of biosynthetic pathways and structures. indeed, several software packages (for example, np.searcher) have been developed based on rules-based understanding of natural-product biosynthesis. the availability of cheap, rapid genome sequen cing means that time-consuming construction and screening of gene libraries for natural-product clusters can now be bypassed. genome sequencing has also revealed a hitherto unrealized richness in the quantity and variability of natural-product biosynthetic clusters. sequenced genomes of bacteria of the actinomycetes class reveal to biosynthetic clusters in each organism. furthermore, natural-product producing bacteria from non-soil environments are being investigated and these have already resulted in new chemical matter, suggesting that there is a fantastic wealth of untapped chemical diversity waiting to be discovered. perhaps some of this diversity will include new antibiotic chemical scaffolds. we are in a remarkably productive time for naturalproduct research that is serving to reinvigorate interest in this sector. at the same time, the application of synthetic biology approaches to this field could serve to improve issues of yield and expand chemical diversity. yes. first, existing discarded antibiotics can be reexamined. the development of daptomycin is instructive. daptomycin was discovered by the antibiotic group at eli lilly in the s, but was not fully developed because of toxicity concerns. the antibiotic was obtained by researchers at cubist in and by altering the dosing, this group was able to bring the antibiotic to market in , since when it has proved highly successful in treating infections caused by gram-positive pathogens. certainly, there are other 'old' antibiotics discovered by the pharmaceutical industry but not developed at the time that could be resurrected as leads for new drugs. a second option is the combination of antibiotics with each other and with other drugs to improve efficacy. infectious-disease physicians often combine antibiotics in an effort to achieve synergy, and this well-established practice has resulted in formulated drug combinations, such as co-trimoxazole (trimethoprim and sulfa methoxazole). combination of antibiotics with non-antibiotics deserves investigation as well. several natural products have been discovered by satoshi omura's group that potentiate the activities of antibiotics such as imipenem in s. aureus by unknown mechanisms. other antibiotic adjuvants are inhibitors of resistance mechanisms. the tremendous commercial and clinical success of augmentin (ampicillin together with the βlactamase inhibitor clavulanic acid) and other similar combinations speaks to the power of such combinations. our growing understanding of the mechanisms of resistance should fuel such approaches. inhibitors of efflux pumps, for example, have been discovered, and though challenging to implement in organisms with multiple redundant systems, are worthy of continued investigation. finally, other strategies orthogonal to antibiotics must be on the table. we should never forget vaccines as proven and outstanding protective agents against infectious diseases. bacterial viruses (bacteriophages) were used extensively to treat bacterial infections in the former soviet union and could find new application in the face of outbreaks of multi-drug resistant bacteria, especially in settings such as hospital infections. the use of enhancers of innate immunity, such as cationic antimicrobial peptides, is also an approach worth investigating. we need antibiotics to maintain our current standard of health care. as already stated, resistance is a natural evolutionary phenomenon that cannot be stopped. through judicious use of current drugs and the development of new ones, the pace of resistance development can be controlled without impairing our ability to control disease. the need for new drugs is, however, acute. antimicrobial stewardship alone cannot fulfill our requirement for new antibiotics. we are in a remarkably exciting time for fundamental research in antibiotics. the rapidity of genome sequencing, the maturing of our knowledge of natural product biosynthesis, a growing understanding of the physical properties of ideal antibiotics, the development of new strategies to develop synthetic compounds with improved antibiotic properties, and the possibilities of synthetic biology combine to suggest that we are entering a highly productive period of antibiotic discovery. the challenges of moving these advances into the clinic fast enough to keep pace with resistance are significant, but with concerted effort between scientists, funders, industry, regulators and clinicians, i believe they can be overcome. where can i go for more information? antibiotic-resistant bugs in the st century -a clinical super-challenge ecology and evolution of antibiotic resistance bad bugs, no drugs: no eskape! an update from the infectious diseases society of america developing a new resource for drug discovery: marine actinomycete bacteria antibiotics for emerging pathogens emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study has the era of untreatable infections arived? beta-lactams and beta-lactamase-inhibitors in current-or potential-clinical practice: a comprehensive update the antibiotic resistome: the nexus of chemical and genetic diversity xanthoradones, new potentiators of imipenem activity against methicillin-resistant staphylococcus aureus, produced by penicillium radicum fki- - : i. taxonomy, fermentation, isolation and biological properties research in the author's lab on antibiotic resistance is supported by a canada research chair, the canadian institutes of health research and the natural sciences and engineering research council. key: cord- -exbdg x authors: guarnieri, michael; brayton, cory; tyler, betty m. title: a long-term study of a lipid-buprenorphine implant in rats date: - - journal: j vet med doi: . / / sha: doc_id: cord_uid: exbdg x animal models to study opiates are of growing interest. we have examined the short-term safety of buprenorphine implants in fischer f /ntac rats treated with excess doses of a cholesterol-triglyceride suspension of buprenorphine. a single injection of . mg/kg afforded clinically significant blood levels of analgesia for days. chemistry, hematology, coagulation, and urinalysis values with - to -fold excess doses of the drug-lipid suspension were within normal limits. histopathology findings were unremarkable. the skin and underlying tissue surrounding the drug injection were unremarkable. here we report the results of a long-term follow-up study of female rats injected with . and . mg/kg. the -month evaluation showed no abnormal findings that could be attributed to the drug or lipid suspension. these results confirm the safety of cholesterol-triglyceride carrier systems for subcutaneous drug delivery in laboratory animals and suggest that this model may be used to study long-term effects of opiate therapy. models of long-acting drug therapy in laboratory animals have been managed by adding drugs to the feed or water supplies. [ , ] the utility of this strategy decreases when the food or water mixture may be released inadvertently to the environment or the drug is highly regulated, such as controlled substance. alternative approaches have focused on longacting drug implants made by combining a drug with biodegradable implants composed of lipids or polymers [ ] . polymers have been studied as dug carriers for neurooncology [ ] . side effects generally have been modest and localized when the polymer is implanted into neural tissue [ ] . less is known about biodegradable polymers for subcutaneous (sc) delivery of chemotherapy. moderate to severe inflammatory reactions have been reported for sc implants of polymer-opiate constructs [ ] [ ] [ ] [ ] [ ] [ ] . fewer adverse events have been reported with lipid drug carriers [ ] . kent described an implantable cholesterol matrix that delivered large molecules such as insulin and growth hormone [ ] . grant and coworkers demonstrated that a phospholipid-morphine liposome had prolonged activity and greater safety in mice than the free drug [ ] . lipid-based carrier strategies have been refined for the delivery of several opiates [ ] . pontani and misra described a cholesterol-triglyceride matrix for the long-term delivery of drugs to treat chronic pain and opiate addiction [ ] . cholesterol-triglyceride vehicles appeared to provide a promising carrier to examine the delivery of antibiotics, antiinflammatory drugs, and analgesics for pain management in animals. yet, solid cholesterol-based implants can be sequestered by interstitial tissues and create drug-containing depots with uncertain release kinetics [ ] . subsequent studies have demonstrated that cholesterol suspensions provide consistent release kinetics with little evidence of tissue sequestration effects [ ] . we examined the safety of cholesterol-triglyceride suspensions of buprenorphine in mice and rats using us food and drug administration (fda) target animal safety (tas) drug-development protocols. histopathology examinations were performed on mice and rats treated with up to tenfold excess of the intended dose of drug and control animals following -and -day drug trials [ ] [ ] [ ] . the examinations, including studies of the sc area of drug implantation, were unremarkable. there were no significant differences between the drug-treated and control animals. to examine the longterm safety of the lipid suspension, we dosed groups of female rats with the intended dose ( . mg/kg), . mg/kg of drug, and control, drug-free matrix. the rats were maintained for months and euthanized to provide long-term clinical pathology data. as shown in the following report, there were no significant differences between the weights, laboratory chemistry, hematology, and necroscopy reports in drug-treated and control rats. studies were approved by a johns hopkins university institutional animal care and use committee (iacuc). the iacuc protocol complies with the national research council's guide for the care and use of laboratory animals and fulfills the requirements of the association for the assessment and accreditation of laboratory animal care, international program. fischer f (f /ntac) rats, - -week-old, female, - g, were obtained from taconic farms (hudson ny) and housed in an environmentally controlled room which maintained the temperatures of to ∘ f. monthly health surveillance was conducted by a soiled-bedding sentinel system. sentinel rats were negative for pneumonia virus of mice, reovirus, sendai virus, lymphocytic choriomeningitis virus, rat coronavirus, sialodacryoadenitis virus, rat parvovirus, kilham rat virus, toolan h parvovirus, rat theilovirus, cilia-associated respiratory bacillus, pneumocystis carinii, mycoplasma pulmonis, and pinworms throughout the study. the facility maintained a relative humidity of to % with a -hour light/ -hour dark cycle from am to pm. animals were group housed (up to per cage) during the quarantine and acclimation period based on group/sex designation. the animals were quarantined and acclimated for six days prior to dosing. no disease-related signs were noted during the quarantine/acclimation period. prior to being placed on test, a clinical veterinarian approved the animals for study use. all animals appeared normal prior to dosing. after the drug injection, rats were housed per cage for days to prevent redosing by coprophagy. on day , rats were housed per cage. housing consisted of soft fiber contact bedding (carefresh natural bedding; ferndale wa) ad libitum access to harlan teklad certified global rodent diet c (harlan teklad, indianapolis in) and ad libitum access to drinking water (baltimore city water system, baltimore md) in disposable water bottles. the animals were provided ad libitum access to harlan teklad certified global rodent diet c (harlan teklad, indianapolis in). rats were provided enrichment devices of polycarbonate red tubes (bio services, uden, nl). their static microisolator cages were changed weekly. design. the label dose of . mg/kg of buprenorphine, which provides - days of clinically significant blood levels of drug, was established in bioequivalence trials and efficacy studies using male and female rats [ ] . the female rats in this study were divided into groups of rats provided . , . , and . mg/kg buprenorphine. cage side evaluations were conducted twice daily in the morning and afternoons for two weeks by a veterinarian who was blind to the dose group. rats were evaluated at weekly intervals by staff veterinary services thereafter. rats were scheduled for harvesting and clinical pathology studies at year and actual months. anomalies or abnormalities discovered during necroscopy were further examined by histopathology. the hypothesis was that rats treated with the highest dose would have tissue changes near the injection site not found in the control group. to limit stress associated with constraining conscious animals for sc injections, rats were anesthetized with an intraperitoneal (ip) solution of ketamine, mg/kg, and xylazine, mg/kg, in a saline solution containing . % ethyl alcohol. each rat was injected with the designated dose of test article or buprenorphine-free control suspension before they recovered from anesthesia. the dose was administered sc on the mid-dorsal area about cm rostral to the surgical incision using a gauge needle (bd, franklin nj) attached to a ml bd tuberculin syringe. following dose administration, animals were transferred to a clean cage on a heating pad until recovered. once the animal regained consciousness and demonstrated normal movement, and with the absence of signs of distress, it was returned to its home cage. posttreatment distress was not observed. pathology. at the months' time point, euthanasia was administered by co inhalation and blood collection by and exsanguination cardiocentesis, followed by a thoracotomy. hematology examination (cbc) included red blood cell (rbc) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (wbc) count, differential blood cell count, and blood smear. serum chemistry tests included glucose, urea nitrogen, creatinine, total protein, albumin, globulin (as calculation), total cholesterol, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, calcium, sodium, potassium, chloride, and phosphate. necropsy was then performed for each animal. tissues for histopathology were immersion fixed in individually labeled containers containing % neutral-buffered formalin. the transfer was performed and documented by the histology lab. containers were labeled with study number, date, group number, and animal number. organ weights included adrenals, brain, heart, kidneys, liver, ovaries, spleen, and uterus with cervix. histopathology was assessed on any gross lesions, dorsal skin surrounding the injection site as previously described, and esophagus, heart, kidneys, liver, lung, lymph nodes, and spleen. statistical analyses (mean, standard deviations, n) were conducted for organ weight and clinical pathology data comparing treated groups to the control group. because of the lack of variation in the data (see the results) further analyses including one-way analysis of variance (anova) and dunnett's t-test ( , ) were not used. all rats survived to the scheduled termination date. signs of excessive grooming and self-gnawing were noted in one rat in the . mg/kg dose group at days - posttreatment. the observer noted the findings as a comment on the animal's chart. there was no evidence of an open wound. since day , there were no remarkable changes in animal behavior. the outcome weight of the three groups was similar: ± g, ± g, and ± g for the rats in the . , . , and . mg/kg dose groups, respectively. there was no significant difference in organ weights of liver, spleen, heart, kidneys (both), brain, adrenal glands (both), uterus + cervix, and ovaries (both). there was no significant difference in the hematology values between the three groups. average group differences were noted between the two clinical chemistry values. blood urea nitrogen (bun) values were . ± . , . ± . , and . ± . mg/dl for the control, . and . mg/kg groups, respectively. alkaline phosphatase (alp) values were . ± . , . ± . , and . ± . (u/l), respectively. on gross examination, rats in the vehicle control group and in the . mg/kg had mild dorsal neck/interscapular skin excoriations or mild crusting. injection sites could not be distinguished from surrounding skin in other rats. on microscopic examination one rat in the control group and one rat in the . mg/kg dose group had mild ulceration and chronic dermatitis involving the dorsal neck/interscapular skin, but mild-moderate hemorrhage and serosuppurative crust that were consistent with more recent injury, possibly related to conspecific trauma such as aggressive neck grooming by cage mates. other findings included one rat in the control group: cystic uterine (endometrial) polyp less than mm diameter, and one rat in the . mg/kg dose group: ovarian cyst ∼ cm diameter. mild cardiac changes (inflammation, degeneration, and fibrosis), mild nephropathy with pigment in tubule epithelium, mild lung inflammatory changes, and micromineralization of lung vasculature were observed in all groups. pigmented macrophages in lymph nodes and spleen were observed in all rats. one rat in the . mg/kg group had an ovarian cyst and one in the vehicle control group had a uterine polyp. pigment at various sites was consistent with hemosiderin, hematoidin, and or ceroid/lipofuscin. no unique microscopic lesions were associated with the test article. the objective of this study was to evaluate the long-term safety of a lipid suspension of buprenorphine for delivery of an opiate analgesic in female f rats. female rats were chosen for a long-term trial because female rats may have increased susceptibility to opiates [ ] , although sex differences have not been fully determined. the results confirmed that lipid-based delivery system caused no significant adverse effects in a f rat model. one rat illustrated signs of nausea at days - , signs that have been observed previously [ ] . the incidence of nausea in this model appears similar to the observed incidence of nausea in human patients treated with opioid analgesia [ ] . there were no remarkable differences in hematology parameters between control and drug-treated rats. clinical chemistry values for serum bun and alk in the rats given . mg/kg dose were decreased compared to control values and in rats given the intended dose of . mg/kg. the clinical significance of these differences remains uncertain. weight loss has been cited as a deterrent to the use of postsurgical buprenorphine analgesia, and it has been linked to significant morbidity secondary to gastrointestinal blockage associated with hardwood bedding [ , ] . a number of reports between and described weight loss in rats treated with buprenorphine without reference to the bedding used in the experiment [ , ] , or they report using hardwood bedding without reference to previous reports associating hardwood bedding with pica [ ] . previous studies have demonstrated that the risk of pica-related gastric distress can be controlled by the appropriate choice of bedding [ ] . there were no significant differences between the organ weights or appearances of the drug-treated rats. body weights also were similar. the studies reported here confirm this observation. the results of this study are compromised by the failure to include male rats and use of higher doses of drug. it did not include a saline-control group to establish the safety of the lipid suspension. there do not appear to be clinically significant treatmentrelated effects following a subcutaneous injection of an extended release lipid suspension of buprenorphine at . and . mg/kg dose. although several clinical pathology findings exceeded normal limits, there were no correlated changes or findings in body weights, clinical observations, organ weights, or microscopic evaluation of tissues. the data used to support the findings of this study are available from the corresponding author upon request. michael guarnieri owns a significant financial interest in animalgesic labs. voluntary ingestion of nut paste for administration of buprenorphine in rats and mice oral self-administration of buprenorphine in the diet for analgesia in mice new developments in long-acting injectable nanoformulations local delivery of rapamycin: a toxicity and efficacy study in an experimental malignant glioma model in rats brain biocompatibility of a biodegradable, controlledrelease polymer in rats pharmacokinetic comparison of sustained-release and standard buprenorphine in mice duration of action of sustained-release buprenorphine in strains of mice evaluation of a sustained-release formulation of buprenorphine for analgesia in rats safety and clinical effectiveness of a compounded sustained-release formulation of buprenorphine for postoperative analgesia in new zealand white rabbits clinical efficacy of sustained-release buprenorphine with meloxicam for postoperative analgesia in beagle dogs undergoing ovariohysterectomy pharmacokinetics of formulations of buprenorphine in macaques (macaca mulatta and macaca fascicularis) engineering solid lipid nanoparticles for improved drug delivery: promises and challenges of translational research cholesterol matrix delivery system for sustained release of macromolecules prolonged analgesia and decreased toxicity with liposomal morphine in a mouse model pharmacokinetics of ammonium sulfate gradient loaded liposome-encapsulated oxymorphone and hydromorphone in healthy dogs a long-acting buprenorphine delivery system subcutaneous implants for long-acting drug therapy in laboratory animals may generate unintended drug reservoirs thermal latency studies in opiate-treated mice subcutaneous implants of buprenorphine-cholesterol-triglyceride powder in mice safety studies of post-surgical buprenorphine therapy for mice subcutaneous implants of a cholesterol-triglyceride-buprenorphine suspension in rats sex differences in opioid antinociception unanticipated adverse events associated with an extended-release buprenorphine toxicity study in fischer rats opioid-induced emesis among hospitalized nonsurgical patients: effects on pain and quality of life pica behavior associated with buprenorphine administration in the rat pica behavior associated with buprenorphine administration in the rat adverse effects on growth rates in rats caused by buprenorphine administration correlation between body weight changes and postoperative pain in rats treated with meloxicam or buprenorphine influence of buprenorphine analgesia on post-operative recovery in two strains of rats funding for this research was supplied by the maryland biotechnology center biotechnology development awards, maryland industrial partnerships (mips), and by animalgesic labs. key: cord- -vcp o xn authors: destefano, vincent; khan, salaar; tabada, alonzo title: applications of pla in modern medicine date: - - journal: nan doi: . /j.engreg. . . sha: doc_id: cord_uid: vcp o xn polylactic acid (pla) is a versatile biopolymer. pla is synthesized with ease from abundant renewable resources and is biodegradable. pla has shown promise as a biomaterial in a plethora of healthcare applications such as tissue engineering or regenerative medicine, cardiovascular implants, dental niches, drug carriers, orthopedic interventions, cancer therapy, skin and tendon healing, and lastly medical tools / equipment. pla has demonstrated instrumental importance as a three-dimensionally ( d) printable biopolymer, which has further been bolstered by its role during the coronavirus disease of (covid- ) global pandemic. as an abundant filament, pla has created desperately needed personal protective equipment (ppe) and ventilator modifications. as polymer chemistry continues to advance, so too will the applications and continued efficacy of pla-based modalities. mechanical properties of pla may vary given the molecular weight of the polymer and degree of crystallinity. mechanical properties are also governed by the stereochemical configuration of the molecular structures of the pla backbone. given that lactide monomers are chiral, mechanical properties can be manipulated through the polymerization of d-lactide, llactide, d, l-lactide or meso-lactide. the molecular weight may also be changed through the addition of functional groups to the backbone. these functional groups may include the addition of hydroxyl species, lactic acid, and water. as a result, the user may modify the configurations of the pla polymer backbone so as to achieve the desired properties. improved mechanical properties of pla can be achieved with semi-crystalline pla over an amorphous assembly of the polymer. semi-crystalline pla exhibits the following properties: approximate tensile modulus of gpa, approximate tensile strength - mpa, flexural modulus of gpa, and flexural strength of mpa. pla is an advantageous biopolymer with relatively high strength and high modulus. further properties are presented in table . pla exhibits a proportional increase in tensile modulus and molecular weight. with an average molar mass increase from to kda, the tensile modulus increases by a factor of . however, pla in engineering applications may have mechanical limitations. pla has poor toughness resulting in a quite brittle material displaying less than % elongation prior to breakage. given these toughness metrics, pla toughness may result in failure at high-stress levels inducing plastic deformation. this may limit the orthopedic use of pla in fixation plates and screws. pla embodies the physical characteristics and processability of a thermoplastic. thermoplastic polymers may be heated or reheated and cooled to alter for the desired morphology. in contrast, a thermoset polymer has increased heat resistance and is irreversibly set upon hardening. pla is a semi-crystalline polymer that exhibits a glass transition temperature (t g ) of ℃ and has a melting point (t m ) at ℃. both t g and t m are vital in describing pla's physical parameters such as density, rheology, and heat capacity. molecular weight, primary structure, optical characterization, as well as crystallinity are critical to pla's physical properties. pla's physical properties are summarized in table . the widespread usage of pla can be attributed to its versatility. mechanical properties of pla could potentially be tuned in several methods. polymer crystallinity defines physical properties such as hardness, modulus, tensile strength, stiffness, and the melting point. semicrystalline pla, specifically, may be modified through plastic additives, which can reduce t g , t m , and overall crystallinity. semi-crystallinity of the polymer is usually characterized by the percent weight per volume of poly(l-lactic acid) (plla) in the sample: a % w/v of plla is deemed semicrystalline. lower values result in an amorphous polymer. pla may also be blended with other polymers, including, but not limited to, polyethylene, polypropylene, chitosan, polystyrene, polyethylene terephthalate, and polycarbonates. blending with pla-based stereocomplexed polymers, such as plla and plda, has demonstrated improved thermal stability and decelerated degradation rate. as a polymer, pla can also be altered structurally through branches, leading to the formation of, for examples, grafted and cross-linked polymers. copolymerization with glycolic acid is a frequently used method known to yield favorable material properties. the hydrophilicity of pla can be particularly enhanced through copolymerization with polyethylene glycol (peg). pla maleation has proven to be an effective method to modify the properties of pla/ poly(ε-caprolactone) (pcl) immiscible blends. peroxide compounds may be added to pla/pcl to induce chemical interfacial cross-links, which in turn improve blend component compatibilities. shin et al. utilized the compatibilizing agent, glycidyl methacrylate (gma), such that gma acted as a monomeric compatibilizer and a reactive agent interfacing pla and pcl phases. interfacial adhesion was also improved by the addition of pla-g-ma to blends of pla and starch improving the compatibility of blend constituents. these additions are conducted through the addition of carbon nanotubes, ceramic nanoparticles, natural fibers, and cellulose. manufacturing techniques may take advantage of the strong interactions between pdla and plla blocks, which result from the formation of stereocomplex crystallization as well as improved mechanical properties and thermal stability. moreover, slower degradation rates, which can be essential in drug delivery applications, may be achieved. modulation in the synthesis of plla and pdla blocks may aid in the production of hydrogels, nanoparticles, and micelles. the following physical properties must be considered for tuning pla biodegradability. polymer degradation rate will increase with an increasingly hydrophilic material, and thus, plla has a slower degradation rate than poly(d,l-lactic acid) (pdlla) due to crystalline regions. the geometry of an implantable or pla derived device will affect the biodegradation rate as it will directly relate to the surface area present to the solution of the bulk material. degradation rate will further decrease with an increase in molecular weight, which in turn implies a lower concentration of carboxyl end groups. semicrystalline pla is less susceptible to degradation than amorphous configurations as crystalline pla is less subject to hydrolysis. the hydrolysis rate can be increased with the addition of acidic compounds. basic molecules may neutralize carboxyl end groups and enhance degradation through base catalysis. plasticizers increase water diffusion increasing the degradation rate. sterilization is of particular importance for pla manufactured for implantation and can be accomplished through beta or gamma irradiation techniques. these irradiation techniques result in reactions such as chain scission or cyclization, lowering the molecular weight and thereby increasing the degradation rate. it must be considered that bulk pla fabricated through extrusion, injection molding, or other processing techniques may decrease the molecular weight and thus enhance degradation. degradation rate is a major property of pla as the kinetics of its breakdown dictate its performance in a variety of applications. first and foremost, it is important to mention that pla possesses the ability to degrade naturally in situ, which benefits medical operations for several reasons, such as the reduction of surgical interventions. prolonged degradation could also be achieved for the purpose of controlled drug release. degradation rate is dependent upon a number of factors: polymer composition, ph, device geometry, molecular weight, crystallinity, addition of drugs and/or additives, sterilization, mechanical stress, and fabrication processing. pla solubility affects manufacturing and processing characteristics. pla is noted to be soluble in dioxane, acetonitrile, chloroform, methylene chloride, , , -trichloroethan, and dichloroacetic acid. pla exhibits partial solubility when heated to boiling temperatures in ethylbenzene, toluene, acetone, and tetrahydrofuran. pla is not soluble in water, alcohols, ethyl acetate, or linear hydrocarbons. oksman et al. reported utilizing cellulose whiskers to modulate pla properties. microcrystalline cellulose (mcc) and pla were used to develop novel nanostructured composites through compound extrusion. mcc treated with n,n-dimethylacetamide in combination with lithium chloride aided in the swelling of mcc and separation of cellulose whiskers. these suspended cellulose whiskers were injected into the pla melt during the extrusion process. peg was utilized as a processing aid. this material combination demonstrated an % increase in elongation to break. oksman et al. noted future studies will investigate process optimization to avoid thermal degradation of the composite. pla processing may result in a group of polymers, which include homopolymers plla and poly(d-lactic acid) (pdla), which are synthesized from mixtures of pure l-or d-lactic acid. the copolymer pdlla will result in a racemic mixture. predictably this varying stereochemistry impacts material properties where plla and pdla are generally semicrystalline, while the copolymer pdlla is generally amorphous. polymer crystallinity modulates mechanical properties such as hardness, tensile strength, stiffness, and melting points. the constituent monomer of pla is the optically active molecule, lactic acid (ch -chohcooh). pla can be found in two chiral configurations, l-lactic acid and d-lactic acid. stereoisomers provide variation in pla types. pla is a degradable substance, whereby breakdown occurs through hydrolysis of the polymer chain. the rate of hydrolysis is dependent upon molecular weight, crystallinity, morphology, and rate of water diffusion. the mechanism of pla autocatalysis is propagated by terminal carboxylic acid groups. due to its relatively slow degradation rate, pla can remain in vivo for typically - years. degradation rate may be accelerated with increased temperature as well as with heightened acidity. molecular weight is also reported to influence pla degradation. proteins and cells exhibit limited surface interaction with pla given its hydrophobicity. this may reduce the potential of pla scaffolds to promote cell ingress. pla's hydrophobic nature may also instigate an inflammatory response of local tissue. moreover, given its relatively inert chemical nature, side-chain additions, bulk modifications, and surface manipulations are difficult to achieve. the synthesis of pla occurs through the polymerization of lactide monomers. the lactide stock is obtained through bacterial fermentation of a renewable source such as corn starches, sugarcane, or beet sugar. synthesis procedures can entail direct oligomer polycondensation, solid-state polycondensation, or simply by addition polymerization of the primary chain, which yields low molecular weight intermediates that can later be processed to obtain pla. direct polymerization may be carried out through azeotropic dehydration polycondensation or enzymatic polymerization. these processes facilitate the direct production of pla from lactide monomers. ring-opening polymerization techniques are commonly used to create pla from low molecular weight pre-polymer, followed by depolymerization resulting in a lactide molecule. the site of ring-opening results in the formation of pla. the polymerization of pla facilitated through ring-opening yields higher molecular weight products. this synthesis procedure requires high purity lactide and heavy metal catalysts which increase the cost of production. the company mitsui toatsu chemicals creates pla in industrial quantities utilizing azeotropic dehydration, reacting % l-lactic acid and tin catalysts. these reactants are added to an organic solvent in a distilling reactor. high heat reactions require the use of an organic solvent with a high boiling point. this process is carried out for hrs at °c, vaporizing water. a Å molecular sieve allows for the reuse of the distilled solvent. water levels diminish to ppm as polymerization occurs for - hours at °c. the final product has a molecular weight of ⨉ kda. the pla end product can be used for injection molding, film extrusion and forming, blow molding, thermoforming, and fiber spinning. the resulting pla is superior for thermal processing over its counterparts, poly-hydroxyl-alkanoate (pha), peg, and pcl. pla crystallinity may vary upon manufacturing processes. the polymer is generally an amorphous thermoplastic. amorphous molecular geometry is a result of pla chains with variable cross-linkages. consequently, it is optimal to synthesize pla as a copolymer of both the l and d stereoisomer configurations. the l configuration of plla accounts for the majority of crystalline properties. pla is entirely amorphic when the polymer has greater than % of the d stereoisomer configuration (pdla) by weight. despite pla's relatively low crystallinity, homopolymer pdla and plla structures constitute a variety of crystal types. pla can be configured in an ɑ-crystalline structure (the most common configuration) which is composed of an ortho, pseudo-orthorhombic arrangement, existing in ( ) helical form of pla chains. while a variety of forms can be generated above ℃ (ɑ-crystalline structure included), only the ɑform can be synthesized at temperatures below ℃. an alternative molecular framework exists in ɑ'-crystallinity which is fabricated at - ℃. the ɑ'-crystalline structure has comparable molecular geometry to the ɑ-crystalline form, however, the defining contrast being ɑ'-crystallinity exists in looser packing of crystal fibers. as a result, ɑ'-crystallinity displays decreased modulus, as well as barrier properties. differential scanning calorimetry (dsc) conducted on pla samples revealed that the ɑ'-crystallinity structure displays a brief exotherm just prior to the melting peak. this exothermic behavior preceding melting indicates a conformational change from ɑ'-crystallinity structure to ɑ-form. additionally, ß-crystalline molecular structures have been fabricated in a comparable methodology to hot-drawing, which elongates the material while exposed to high temperatures. ß -form structures are unique in that they have a ( ) helical configuration which permits trigonal molecular arrangements. ßstructures have diminished molecular stability in comparison to their ɑ-form counterparts, indicated by a ℃ depression in melting point temperature. the ß -form unit cell composition preserves density, however, there is a substantial reduction in unit cell volume from . g/cm to . g/cm . given the unit cell volume changes between ɑ-form and ß -molecular structures, new steric relationships may be introduced that yield less thermodynamically stable products. pla plays a pivotal role in fulfilling various tissue engineering and regenerative medicine treatment strategies. one example is its capability to foster hard tissue regrowth in bone grafting procedures. the approach of current technologies focuses on the integration of tissue-engineered bone with native bone. the synthesized material is reported to encourage osteogenesis and angiogenesis with the neighboring tissues. however, several publications agree that the material utilized in these modalities suffer from a lack of physiological function. complications arise largely from a broad array of biological factors that must be considered before implantation of the biomaterial. for instance, scaffolds are intended to induce cell processes, including, but not limited to, migration, delivery, proliferation, and differentiation. concurrently, the material has to demonstrate an ability to integrate with the surrounding environment. moreover, optimal performance is achieved when it can withstand immune responses and circumvent degradation into deleterious by-products. pla combined with hydroxyapatite (ha) has shown promise in this field. ha can independently stimulate osteogenesis through activation of osteoblasts and pre-osteoblastic cells. , ha is normally blended with macro-materials as micro-or nano-sized crystals. a composite of pla and ha affects the unique properties of each substance. pla is already known to influence the physical and mechanical characteristics of ha, and, concomitantly, ha can enhance the flexural strength of pla. previous studies have explored the applications of pla/ha blends. hatano et al., for example, evaluated the mechanical nature of a pla/ha composite. the material was synthesized in accordance with an affordable, user-friendly hotpressing technique. results suggested a pla/ha blend that was % ha by weight possessed an elastic modulus comparable to the value seen in human cortical bone -roughly gpa. nonetheless, unpredicted shortcomings were experienced and documented. increasing the concentration of ha above %, however, undermined the inherent structural parameters. one potential explanation may be due to the dearth of pla polymers that ha crystals may aggregate or bind to. additionally, the blend portrayed very poor structural integrity and mechanical function under in vitro stresses. pla/ha blends were subjected to solutions that emulated physiological ph and isotonic salt content, which consequently reduced measurements. hatano et al. conjectured ph and salt triggered the blends to dissolve, providing more mobility, thus, less fixation on the polymer, for ha crystals. it is worth mentioning that this study did not perform in vivo experimentation, which leaves the impact of osteoblasts, osteoclasts, and other cells on the material unaddressed. pla offers excellent bioresorption capabilities which allows the polymer to integrate with host cells and tissues. this feature is particularly useful for bone grafting. as a result of the defects associated with pla-based bone substitutes, efforts have shifted to tailor pla properties for regenerative therapies. indeed, the current paradigm focuses on the design of tissueengineered bone in lieu of a complete artificial replacement. findings in this regard have given way to optimism. pla loaded with high molecular weight ha particles accelerates osteoblast development from precursor cells. several other effects of ha on the interaction of pla with cells have been reported. for example, russias et al. investigated changes in the physical properties of pla generated by ha insertion. pla was combined with ha through a highvelocity stream containing micro-ha-particles. this methodology led to a homogenous distribution of ha across the pla surface. data indicated a -fold increase in surface roughness of pla/ha composites compared to pla. in addition, a % reduction in the water contact angle was observed. these modifications are interpreted as of high importance as they enhance protein adsorption and interaction with the extracellular environment. improved wettability leads to greater hydrophilicity, giving way to the aforementioned advantages. moreover, the rough topography demonstrated greater pre-osteoblast proliferation and differentiation. the authors mentioned this phenomenon most likely occurred from heightened integrin bindings between native cells and the biomaterial. most of all, russias et al. showed a viable method of fabrication that increases osteoblastic activity, while decreasing osteoclastic-related breakdown -two qualities favorable for bone growth. the capabilities of pla can be further explained through their utility in forming new bone. specifically, scaffolds of pla may reach this outcome from the assistance of glass such as calcium phosphate glass. calcium phosphate glass has a chemical composition capable of alteration, which enables the rate of degradation to be tailored to that of bone. further features include being highly biocompatible due to low cytotoxicity. pla/calcium phosphate glass scaffolds have been manifested through the following procedure. the foam formation process may be used to create these scaffolds. this process entails the insertion of the glass into a pla solution to enable the union of these two components. as a result, calcium phosphate glass can be developed in a homogeneous fashion. additionally, the pla/calcium phosphate glass provides the desired mechanical strength as well as the appropriate interaction for the cells and the material ( figure ). scaffolds may be improved through the modulation of the following characteristics: porosity, mechanical strength, and bioactivity. the pla/calcium phosphate composite was biocompatible in that it effectively integrated with host tissues. pla controls demonstrated poor cell adherence. the depth and diameter of porous interconnected veins, in the pla/calcium phosphate composites, encouraged the ingress and proliferation of osteoblasts. these composites demonstrated superior angiogenesis and nutrient sequestration when compared to unmodified pla. upon addition of bioglass to the pla composite, there was a % increase in porosity from % to %. compressive yield testing revealed that pla/calcium phosphate composite demonstrated superior properties compared to the pla control. the test resulted in mpa for glass infused composite compared to . mpa for the pla foam. this % increase represented a successful transfer of the desired calcium phosphate glass traits. the composite also exhibited a higher level of resistance to failure, as well as an increase in compressive modulus, which may be attributed to the glass's ample adhesion. the pla/calcium phosphate composite was able to achieve a value of . mpa, whereas the pla control presented . mpa. pla tissue engineering scaffolds may also be used to regenerate epithelial cells. polyglycolic acid (pga)/pla blends have drawn interest in the treatment of short bowel syndrome. in this disease state, the jejunal-ileal length is truncated by an average of % and may result in nutrient deficiencies. the efficacy of tissue grown in this manner may be tested through electrophysiology and histology. pla coating of pga sheets can be treated to experimental stents. these stents were placed into cardiovascular disease rat models, whereby success was observed through type i collagen integration of of the total scaffolds. the organoid units themselves were made of a core of mesenchymal stromal cells surrounded by an epithelium. results indicated out of scaffolds contained notable cyst dimensions, of which were coated with collagen. authors proposed that these outcomes may be related to the immunogenicity and high permeability of collagen type i. electrophysiological testing indicated active ion-transport of scaffold grown mucosal cells, and comparable epithelium barrier strength. histological evaluation is demonstrated in figure . crypt presence and adequate epithelial morphology suggest the potential future utility of pga/pla scaffolds for epithelial regrowth. novel forms of pla-based scaffolds, intended for vasculature, have also been researched due to their tunable bioresorption. plga has been successful in experimental cardiac patches. these patches are capable of anisotropic electrophysiological functions of native tissue and may provide relief in conditions such as myocardial infarctions. electrospun pla/chitosan copolymers have demonstrated promise in fostering the growth of cardiomyocytes. efficacy was demonstrated through phenotypically appropriate cell elongation, as well as the presence of troponin i and sarcomeric ⍺-actin. patients suffering from coronary artery disease may require bypass surgery which requires an autograft of saphenous arteries or veins. moreover, autografts impose risks of patient infection and donor site pain. cardiovascular implant failure may result in restenosis years post-operation. as a result, these obstacles provide for an opportunity to create a novel treatment, or improved implant to mitigate current shortcomings. synthetic pla grafts can provide appropriate mechanical support and offer versatile biodegradable qualities favorable for healing periods. the degradation of pla endothelial grafts resulted in . % mass loss after days and . % loss after days of in vitro testing. pla grafts maintain mechanical support despite gradual resorption, thus enabling appropriate cell growth. endothelial cell viability was observed when exposed to pla extract, further confirming pla meets biocompatibility requirements for arterial grafting. additionally, pla facilitated the growth of human adipose stem cell monolayers. through mtt analysis, significant variability in pla cellular viability was not found. with the evolution of pathologies and antibiotic-resistant bacteria, the need for new pharmaceuticals is as dire as ever. drug discovery, however, has not progressed mainly due to the abundance of failed clinical trials. as a result, researchers are more inclined to explore delivery strategies that enhance drug efficiencies rather than create novel medications. the performance of a drug is principally dictated by the physiological responses of the body following administration. once in the body, the drug must overcome metabolic and immune processes. these challenges have led to the design of novel drug carrier systems that transport pharmaceuticals through biological barriers. drug delivery systems usually consist of a biodegradable polymer loaded with therapeutic agents. pla boasts several advantages that have led to the design of effective drug delivery strategies. one of the primary attributes that make pla favorable for drug carriers is its biodegradability. pla can dissolve readily in extracellular environments. in addition to this capability, the degradation rate may be modulated to yield a desired effect. particularly, for drug carrier systems, the kinetics of this breakdown may be prolonged to sustain a continuous release of medicinal agents. this provides sufficient time for the drug to induce its effect, which is pivotal as this therapeutic strategy could potentially be diminished by metabolic processes. drugs low in molecular weight are of primary concern given their increased likelihood to be filtered from the bloodstream through the hepatic and renal systems. to overcome these challenges, drugs have been loaded onto carriers, which target specific sites in the body. pla has been reported to be an excellent drug delivery material as it can withstand dissolving over an extensive period of time. this quality of pla has been demonstrated in anti-cancer drug compounds. maji et al. evaluated the performance of tamoxifen encapsulated in poly(lactic-coglycolic acid) (plga) nanoparticles. results indicated an enhanced bioavailability compared to non-loaded control samples. in addition, tamoxifen/plga composites were shown to improve the anti-tumor effects of the drug. similar findings were reported by hrkach et al. in a study that observed the delivery capability of docetaxel loaded on pla. data suggested a greater concentration of docetaxel within targeted tumors in comparison to free-docetaxel administration. consequently, the anticancer performance of the drug was improved, leading to reduced tumor growth. while pla shows promise in various cancer treatments, pla is also utilized in a variety of implant procedures. with an adaptable degradation rate, pla can deliver antibiotic agents to sites of implantation. this modality is particularly useful to limit risks of infection post-operation, decreasing the probability of implant failure. pla is a versatile biomaterial that possesses the capability to have its properties modified. as a polymer, pla assumes several structural isomers, each with unique characteristics. additionally, pla molecules can be altered chemically through interactions with adhesive proteins. adsorption of certain proteins determines the cells and tissues the polymer may come into contact with once placed in situ. this property has been beneficial in drug delivery applications. post-intake, physiological responses inevitably dictate the fate of the drug molecules. with pla, pharmaceuticals may target specific cells and avoid biological obstacles. lv et al. demonstrated advanced chemotherapy against drug-resistant leukemia cells using daunorubicin-loaded pla nanoparticles. some leukemia cells secrete agents that stymie anti-cancer drug performance. combined with pla, daunorubicin is able to bypass said inhibitors. moreover, pla aids the transport of medications across membranes and physiological barriers. as a result, intake of the drug in targeted cells is improved. additionally, pla has demonstrated potential in bypassing the blood brain barrier (bbb). diffusion of drug particles across the bbb has been reportedly increased by pla composites. pla is also well known for its positive impacts on drug specificity. although drug particles have an intended medicinal purpose, unforeseen side effects may transpire. this may occur as a consequence of interactions between the drug and non-targeted cells. pla can be coupled with drug particles to curtail the incidence of side effects and improve drug efficiency, which could potentially lead to lesser dosages. a recent study demonstrated increased drug specificity when the particles were loaded onto pla containing magnetite (fe o ). the magnetic pla composite was then subjected to a magnetic field, which improved drug accuracy. the publication conveyed promising findings that may aid in the development of highly specific medications. given the widespread cautions concerning a myriad of drugs, it is vital that this research area continues to be explored. even with the above advantages of pla-based drug delivery systems, the therapeutic efficiency of such strategies can still be improved through manipulation of associated microparticles. zhao et al. demonstrated the potential of this approach using pomegranatestructured composite microspheres composed of ibuprofen-loaded mesoporous silica nanoparticles (msn) inserted into plla microspheres. results suggested drug release rates may be regulated through adjustment of the msn and ibuprofen concentrations. consequently, prolonged drug release using this technique may be fruitful especially for treatments involving long-term therapeutic effects. in another study, zhao et al. showed favorable outcomes may be obtained through modifications of the loading strategy onto the polymer matrix. according to the results, programmable release of anti-inflammatory agents and anti-tumor agents on a short-term and long-term scale, respectively, can be simultaneously performed in the same drug delivery system. findings as such prove to be of high value due to the enhanced efficiency of the pharmaceutical effects. pla-based drug delivery systems have shown promise for therapeutic strategies in cancer treatments. due to its adaptable degradation rate, pla scaffolds offer a highly desirable feature for drug delivery systems: long-term drug release. given the understanding that inflammation may lead to tumor regrowth, it is critical to mitigate inflammatory responses post-tumor resection operations. , dual-drug delivery polymer scaffolds are an effective method to induce more favorable tumor microenvironments. early inhibition of inflammation can help optimize the performance of antitumor agents. yuan et al. demonstrated the advantageous usage of an electrospun composite plla fibrous scaffolds immobilized with bicarbonate, doxorubicin, and ibuprofen for hepatic cancer therapy. electrospun fiber-based scaffolds have previously been limited against hepatocellular carcinoma for two reasons: ( ) a dearth of control over the release of drugs at specific time periods; and ( ) minimal responsiveness to extracellular ph changes. yuan et al. addressed the latter issue through incorporation of sodium bicarbonate-loaded mesoporous silica particles into the plla scaffold. these modified particles provide a degree of sensitization within the polymer to acidic environments, which triggered drug release. strategic loading of ibuprofen on the scaffold surface offered control of inflammation at an early time frame. the reduced inflammation contributed to enhanced efficiency of the antitumor agent doxorubicin. insertion of doxorubicin deeper within the scaffold led to sustained release of the antitumor drug. results from yuan et al. indicated the potential of ph-sensitive mediated drug release plla scaffolds in tumor recurrence prevention. dual-drug delivery could potentially lead to improved anti-inflammatory and anti-tumor outcomes. moreover, temporal manipulation of drug release can create environments suitable for more profound therapeutic effects. , similar findings were reported by pan et al. and in a study that investigated the inhibition of inflammation capability of implanted electrospun pla fibers loaded with ibuprofen. sustained release of ibuprofen from the fibers led to increased efficiency in later-stage inflammation inhibition. thus, short-and long-term modulation of inflammation via drug-loaded delivery systems may be attained. cervical carcinoma is a major causation of cancer-related deaths amongst females on a global scale. current treatment options, including chemotherapy, have proven success in clinic, however, patient complications from side effects are well-documented. in addition, chemotherapeutic agents suffer from several shortcomings, such as low bioavailability and subpar delivery efficiency. the advent of nano drug delivery systems has garnered tremendous attention as a possible solution to address these concerns. this is likely due to the controlled drug release and enhanced anti-tumor effects seen in doxorubicin-loaded pla-based scaffolds, such as the one described by niu et al. pla stereocomplexes displayed drug delivery capabilities in cervical carcinoma mouse models and in vitro analyses. sustained doxorubicin release contributed to improved tumor cell uptake of the anti-tumor agents. as a result, tumor cell activity was decreased with the drug delivery system compared to non-loaded drug performance. increased tumor inhibition rates were also exhibited by doxorubicin-loaded stereocomplex micelles. figure . schematic of design of doxorubicin-loaded stereocomplex micelle and administration to mouse subject. pla can also serve a significant role in tissue regenerative medicine. similar to drug delivery systems targeting carcinomas, pla scaffolds may hold bioactive drugs that facilitate wound healing. drug-loaded pla scaffolds, however, could potentially be limited due to unintended changes in the properties of the scaffold and/or drugs during the synthesis process. moreover, surface modification of the scaffolds can be difficult to achieve given the relatively inert nature of pla. the latter issue is of large importance as the polyesters may compromise the therapeutic strategy. thus, the surface requires a stimulus to increase the polymer's overall cytocompatibility and function in vivo. various biofunctionalization methods have been developed to improve these drawbacks. based on results published by cheng et al., bioactive ginsenoside-rg was effectively combined with biomolecules, such as basic fibroblast growth factor (bfgf), in an electrospun plga scaffold through mussel-inspired poly(dopamine) (pda) coating. incorporation of bio-signaling compounds led to enhanced cell adhesion and cell proliferation on the scaffold. improved interactions between the scaffold and cells developed a favorable environment that mediated drug-therapy. the composite drug-biomolecule scaffold demonstrated a synergistic effect that encouraged early wound healing of rabbit ear wounds as well as long-term inhibition of hypertrophic scar formation. pla scaffolds loaded individually by either the drug or growth factor showed less efficient healing and inhibition capabilities compared to the composite scaffold. cheng et al. also mentioned the preservation of the original scaffold properties. though the polyesters' chemical stability is not conducive for optimal cellular interactions, their adjustable biodegradability, mechanical properties, and porosity remain favorable, and thus, should not be altered. the methodology presented by cheng et al. is a promising approach in tissue regenerative medicine as enhanced tissue repair may be achieved compared to single-drug-loaded scaffolds. proper tendon repair is a crucial aspect of post-tendon surgery healing. however, adhesions and scar formation are a natural part of the healing process, which may lead to several complications, including hindered joint mobility. due to its tunable degradation rate, plla provides advantages that may serve well in tendon repair. however, the hydrophobicity of the polymer reduces the drug efficiency of hydrophilic particles loaded into its structure. micro-sol electrospinning offers a solution that combines hydrophilic mitomycin-c (mmc) into hyaluronan (ha) hydrosols loaded onto plla fibers. this technique, outlined by zhao et al., created a drug delivery system that protects the function of mmc. most importantly, the novel approach led to the development of a scaffold that provided controlled release of mmc, stimulating intrinsic tendon healing, while concomitantly inhibiting scar tissue formation. zhao et al. exemplify the versatility of pla in terms of its applications. modifications to its structure and addition of various molecules refine its function for desirable outcomes. due to its structural adaptability and biocompatibility, pla has attracted much attention to dentistry-based applications. pla stimulates successful osseointegration of dental implants with native oral hard tissue. while pla is not the standard material for dental implantology, implants designed to hold drug loaded pla polymers have demonstrated beneficial outcomes. bone regeneration, for example, is possible using pla-containing composite materials. pla also plays a role in dental resins essential for several restoration procedures. resins are evaluated based on their mechanical function, stability, and appearance. improved mechanical properties may be achieved using pla composite scaffolds in resin. ranjbar et al. observed increased flexural strength, modulus, and compressive strength in pla/al o nano-scaffolds compared to traditional resins. the composite material was synthesized through crosslinking the polymer with al o nanoparticles. moreover, pla combined with other polymers has been utilized to address post-operative complications. plga membranes have reportedly provided enhanced oral bone regeneration in rats. similarly, plga membranes facilitated improved bone healing in calvarial defects in rabbits. plga scaffolds have also shown regeneration of damaged oral tissue. the drug delivery capabilities of pla have been applied to clinical purposes in dental medicine. plga particularly is able to serve an endodontic role through sustained delivery of antibiotics during root canal procedures. pla offers potential advantages for biodegradable orthopedic devices. pla suture devices were first patented in , and have since found uses in resorbable fracture fixation plates. polymer fixation enables higher quality imaging due to radiolucent properties thus reducing potential artifact formation. pla copolymers can be generated to modulate degradation properties, such as the introduction of chitosan-based materials. pla / chitosan-based copolymers offer increased material strength and prolonged degradation time. in vivo studies indicate sufficient pla strength is demonstrated for - weeks post-implantation. these findings suggest pla may be used effectively for internal bone fixation. pla has also proven to maintain robust sterility which helps to mitigate infection. orthopedic implantables composed of pla may be infused with osteogenic or anabolic bioactives to encourage cell proliferation. biodegradable fixation devices offer a remedy to potential osteopenia that may result from stress shielding that occurs with metallic implants. pla based scaffolds may be d printed and precisely tailored for porosity sizes / depth, as well as connectivity to optimize osteoinductive capabilities. pla copolymer degradation products are non-toxic, however, degradation products may increase resorption site acidity. chitosan copolymers are effective in neutralizing byproduct acidification. pla biodegradation is advantageous in that no further surgeries are required for implant removal. pla degradation rates can be modified to provide for the gradual transition of loading from implant to bone. orthopedic implants composed of pla have demonstrated tremendous success in animal models. pla copolymers integrated with proteolipid (mucopeptide n-acetyl muramoyl hydrolase phosphatidylinositol , -diphosphate) has promoted osteogenesis in rats and dogs. furthermore, sutures and fixation rods composed of pla have excelled in mending mandibular fractures in dogs. rabbit osteotomies have been successfully reduced via resorbable selfreinforced, fibrillated pla ( l/ d)/ (sr-pla ) fixation rods. with a tensile strength between . - . mpa and flexural strength between - mpa, l-pla is an excellent biomaterial candidate for orthopedic implants. furthermore, l-pla with a molecular weight of at least kda has routinely demonstrated adequate mechanical strength for implantation. l-pla rods have improved hydrophobicity which may serve to retard degradation rates. ultra-high-strength pla has demonstrated effectiveness in osteochondral reconstruction and healing in soft tissue cases. these high strength pla blends have proven efficacy in enhanced fixation and repair of tendons and ligaments. pins, suture anchors, and screws composed of pla and copolymers have facilitated anterior cruciate ligament (acl) reconstruction, where suture anchors serve to reattach tendons and ligaments to bone. pla has been manufactured into highly specific and modified porous scaffold or cell carriers tasked with rebuilding extracellular ligamentous matrix components. flexibility in manufacturing provides for permeable scaffolds that adequately sequester nutrients and promote cellular ingress while leaving exit pathways for cellular waste removal. figure . illustration of the components of the scaffold designed for ligament, cartilage, and bone regeneration. a challenge of pla orthopedic scaffolds is that pla surfaces lack a viable epitope for cell attachment. pla surfaces must undergo modification to promote specific cell or protein attachment. it is imperative to increase surface bioactivity within the pore channels. while pla embodies various advantageous characteristics, it may struggle with orthopedic applications due to low fatigue strength, creep, poor adhesion, and potentially poor biocompatibility in specific cases. pla's brittle nature is a result of its slow crystallizing nature. inadequate reactivity in the pores of pla scaffolds may fail to promote cell ingress and may also need an autograft to see scaffolds. autografts are not without complications as they may result in increased donor site pain, bleeding, necrosis, extended healing times, and an increased risk of infection. lastly, if scaffolds are seeded with poor donor site stock they may compromise the effectiveness of scaffold treatment. bacterial colonization and subsequent biofilm formation may result in detrimental infections which require surgical debridement. pla films reinforced with magnesium (mg) have demonstrated osteogenic properties and promote bone cell ingress. despite bone promoting properties, pla integrated with mg may allow for the inception of biofilms. gonzález-martin et al. reported an initial reduction in bacterial biofilm formation in pla films integrated with % mg. however, this ratio of pla and mg is merely deemed bacteriostatic and not bactericidal, as after hours of incubation biofilm formation of pla/mg scaffolds surpass that of pla control. karakurt et al. attempt to mitigate pla's poor antibacterial surface properties through the addition of immobilized saccharides. utilizing plasma post-irradiation grafting techniques, immobilized glucosamine (glcn) and chondroitin sulfate (chs) are added to modulate surface properties, chemical composition, surface topography, and hydrophilicity. following these modifications, pla films displayed bactericidal effects against escherichia coli and staphylococcus aureus strains. it was found that the integration of clcn and chs into pla films was bactericidal yet no synergistic effects were reported. optimal antibacterial effects for chs were seen against e. coli, while glcn was superior against s. aureus. this proposed surface modification may hasten the emergence of pla based medical devices to market. three-dimensional ( d) printing has emerged as a promising fabrication modality with a broad array of applications in the field of medicine. d printing may be characterized as an additive manufacturing process that gives way to rapid prototyping. the profound contributions of this state-of-the-art technology to medicine are tied to its ability to produce a tangible model from a preliminary virtual model. this capability is frequently utilized for pre-operative planning as well as educational purposes. d printed organ models aid surgeons develop an effective treatment strategy as the analogs are designed with a morphology and anatomy according to diagnostic images. device testing and medical training are also well-documented benefits provided through this technique. due to its widespread accessibility, ease of use, low cost, and speed, d printing offers the opportunity to design customized devices, addressing complications associated with patient variance. the growing popularity of this modality in addition to efforts to personalize medicine is likely to expand its usage across extreme scenarios. recent studies have already explored the significance of d printing within organ printing and personal protective equipment manufacturing. , equally important to the function of a d printer is the filament of choice. various materials possess unique properties that may be exploited to fulfill a specific outcome. pla is one of the most common filaments available for d printing. pla offers several advantages that bolster the effectiveness of additive manufacturing. encouraging results suggested increased infill density and a single layer of aluminum spray significantly increased the tensile strength of pla parts made by fdm. mechanistically, increased infill density provides for strong bonds between pla layers, while added aluminum improves thermal conductivity. future studies will investigate pla aluminum d printed composites for their wear resistance. pla has demonstrated sufficient strength and mechanical characteristics suitable for a variety of clinical applications. pla has demonstrated an ability to withstand external loads experienced by surgical retractors. rankin et al. produced a pla-based surgical retractor using d printing methods. the fabricated retractor tolerated n of tangential force. additional results suggested the pla instrument exhibited adequate durability to support forces presented during operation. pla has also been the desired filament for other tools, including needle drivers, hemostats, forceps, and scalpel handles. due to its low allergenicity and safety, pla can be used as a solution to a broad range of clinical circumstances with minimal adverse physiological reactions. moreover, pla may be repeatedly sterilized without significant compromise of its physical and chemical properties. as a result, the cost of production for several d printed instruments is substantially low compared to their pre-existing counterparts. rankin et al. estimated the price of their d printed retractor may be a tenth of the cost for the stainless steel equivalent. furthermore, when under duress, pla fractures in a predictable manner. rankin et al. loaded five d printed retractors till failure. data indicated each retractor failed at the same stress value as well as in the same position. this quality is particularly useful for d printed materials that may be inserted or implanted into the body. d printing combined with computer-aided design (cad) software can produce replicable components or whole tools intended for clinical performance. moreover, rapid prototyping permits the development of customizable instruments tailored to patient anatomical measurements. the surgical relevance of d printed models using pla has been illustrated by a great number of studies describing self-produced prototypes. for example, pla-based space holders designed for esophageal retraction demonstrated adequate functionality in porcine models. the space holders widened the aperture of the esophagus to provide enhanced exposure of lesions. the larger field of view aided suture placement, which could potentially lead to higher quality closure of wounds. given its prevalence, d printing may serve pivotal roles in times of disaster. historically, d printing has led to the development of models, prostheses, surgical aids, implants, and scaffolds. modern technological advancements have expanded its applications to critical care equipment. in response to the covid- pandemic, d printing has been utilized to create ventilators that could potentially accommodate more than one patient. clarke reported a d printed circuit splitter that enabled ventilation of two patients using a single ventilator. the findings demonstrated in this publication convey the value of care feasible from d printing during emergency situations. moreover, unprecedented demands on the healthcare system could lead to protective personal equipment (ppe) shortages. d printing offers a rapid means for mass production of protective gear. in addition, ppe may be customized to the sizes and shapes of wearers. cai et al. demonstrated a swift, effective protocol for the fabrication of personalized n masks. while supply restoration is of large importance, quality gear that may be tailored to an individual's unique features is a breakthrough development that could potentially maximize comfort and fit on top of personal safety. modular d printed ppe designs can be readily found on the nih d print exchange. as a commonly printed filament, pla has played a fundamental role in the rapid manifestation of these designs. as a result, pla should be considered for future manufacturing of personal equipment. reports have documented deficiencies associated with pla in biomedical applications. particularly, brittleness has been cited pervasively as a defect to the material's mechanical nature. as a result, studies have focused on methods to obtain adequate functional capability as well as robustness throughout implantation and industrial transportation, respectively. pla also exhibits low toughness and delayed crystallization. despite widely available raw materials to synthesize pla, none of the manufacturing techniques are simple with straightforward execution. pla synthesis requires controlled reaction conditions such as temperature, pressure, and ph to ensure proper catalyst function. pla requires long polymerization times which results in high energy consumption. as a result, a criticism put forth in the literature is pla manufacturing, in some instances, may result in high costs. while pla is biodegradable, the application of this biomaterial can be limited by slow degradation kinetics. the pla interface has poor hydrophilicity prior to degradation. while pla is versatile, once formed, pla exhibits poor mechanical ductility. pla has also displayed poor thermal stability. pla's degradation can also be a disadvantage if an implant is intended as a permanent fixture. pla has the potential to spread communicable pathologies. pla may also undergo denaturation when exposed to alcohol. despite these challenges, current research has shown that pla deficits can be addressed, and when modified for one of the aforementioned applications, pla may be a useful tool in biomedical science. , polylactic acid, pla, has an exemplary history as a biomaterial. as a thermoplastic, users have demonstrated an ability to manufacture highly specific products for key problems in healthcare. provided pla's uses in d printing, as well as its eco-friendly traits, it is likely pla will be a contributing biomaterial for the foreseeable future. the covid- pandemic has made clear the usefulness of bulk thermoplastics that can be printed into any desired product. perhaps these principles will take root in field hospitals in distressed countries, military hospitals, disaster relief, or medicine practiced in space. lactic acid: production, applications, nanocomposites, and release studies physical and mechanical properties of pla, and their functions in widespread applications -a comprehensive review effect of molecular weight and crystallinity on poly(lactic acid) mechanical properties high molecular weight polylactic acid polymers properties of lactic acid based polymers and their correlation with composition poly(lactic acid) modifications. progress in polymer science a perspective on polylactic acid-based polymers use for nanoparticles synthesis and applications pla maleation: an easy and effective method to modify the properties of pla/pcl immiscible blends. colloid and polymer science compatibilization of immiscible poly(lactic acid)/poly(εcaprolactone) blend through electron-beam irradiation with the addition of a compatibilizing agent manufacturing process of cellulose whiskers/polylactic acid nanocomposites parameters affecting cellular adhesion to polylactide films an overview of polylactides as packaging materials poly(lactic acid) crystallization. progress in polymer science nano-hydroxyapatite coating promotes porous calcium phosphate ceramic-induced osteogenesis via bmp/smad signaling pathway improvement of surface bioactivity of poly(lactic acid) biopolymer by sandblasting with hydroxyapatite bioceramic fabrication and mechanical properties of pla/ha composites: a study of in vitro degradation effect of surface roughness on proliferation and alkaline phosphatase expression of rat calvarial cells cultured on polystyrene development and cell response of a new biodegradable composite scaffold for guided bone regeneration studies of brush border enzymes, basement membrane components, and electrophysiology of tissue-engineered neointestine application of collagen scaffold in tissue engineering: recent advances and new perspectives poly(lactic acid) as biomaterial for cardiovascular devices and tissue engineering applications engineering a freestanding biomimetic cardiac patch using biodegradable poly(lactic-co-glycolic acid) (plga) and human embryonic stem cell-derived ventricular cardiomyocytes (hesc-vcms) composite poly(lactic acid)/chitosan nanofibrous scaffolds for cardiac tissue engineering an innovative approach towards d-printed scaffolds for the next generation of tissue-engineered vascular grafts drug development in the era of precision medicine -protein and peptide delivery through respiratory pathway colloidal drug carriers: achievements and perspectives pomegranate-structured electrosprayed microspheres for long-term controlled drug release. particle & particle systems characterization polylactic acid (pla) controlled delivery carriers for biomedical applications preparation and characterization of tamoxifen citrate loaded nanoparticles for breast cancer therapy preclinical development and clinical translation of a psma-targeted docetaxel nanoparticle with a differentiated pharmacological profile from lactic acid to poly(lactic acid) (pla): characterization and analysis of pla and its precursors poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering novel nanocomposite of nano fe( )o( ) and polylactide nanofibers for application in drug uptake and induction of cell death of leukemia cancer cells self-coated interfacial layer at organic/inorganic phase for temporally controlling dual-drug delivery from electrospun fibers synergistic mediation of tumor signaling pathways in hepatocellular carcinoma therapy via dual-drug-loaded ph-responsive electrospun fibrous scaffolds tumor-triggered controlled drug release from electrospun fibers using inorganic caps for inhibiting cancer relapse full-course inhibition of biodegradationinduced inflammation in fibrous scaffold by loading enzyme-sensitive prodrug controlled drug delivery by polylactide stereocomplex micelle for cervical cancer chemotherapy surface biofunctional drug-loaded electrospun fibrous scaffolds for comprehensive repairing hypertrophic scars optimization of intrinsic and extrinsic tendon healing through controllable water-soluble mitomycin-c release from electrospun fibers by mediating adhesion-related gene expression a systematic study and effect of pla/al o nanoscaffolds as dental resins: mechanochemical properties degradable polymers may improve dental practice poly(lactic acid) fiber: an overview. progress in polymer science orthopaedic applications for pla-pga biodegradable polymers impact of pla/mg films degradation on surface physical properties and biofilm survival. colloids and surfaces antibacterial activity and cytotoxicity of immobilized glucosamine/chondroitin sulfate on polylactic acid films threedimensional printing surgical instruments: are we there yet? clinical efficacy and effectiveness of d printing: a systematic review role of innovative d printing models in the management of hepatobiliary malignancies organ printing: the future of bone regeneration? three-dimensional printed circuit splitter and flow restriction devices for multiple patient lung ventilation using one anaesthesia workstation or ventilator emerging technology and applications of d printing in the medical field investigation of tensile properties of sprayed aluminium based pla composites fabricated by fdm technology d printed surgical instruments: the design and fabrication process an ad hoc three dimensionally printed tool facilitates intraesophageal suturing in experimental surgery special issue: d printing for customized design and d printing of face seal for an n filtering facepiece respirator key: cord- -flgwxc authors: kipshidze, nicholas; iversen, patrick; porter, thomas r.; kipshidze, nodar; siddiqui, fakiha; dangas, george; fareed, jawed title: targeted, site-specific, delivery vehicles of therapeutics for covid- patients. brief review date: - - journal: clin appl thromb hemost doi: . / sha: doc_id: cord_uid: flgwxc definitive pharmacological therapies for covid- have yet to be identified. several hundred trials are ongoing globally in the hope of a solution. however, nearly all treatments rely on systemic delivery but covid- damages the lungs preferentially. the use of a targeted delivery approach is reviewed where engineered products are able to reach damaged lung tissue directly, which includes catheter-based and aerosol-based approaches. in this review we have outlined various target directed approaches which include microbubbles, extracellular vesicles including exosomes, adenosine nanoparticles, novel bio-objects, direct aerosol targeted pulmonary delivery and catheter-based drug delivery with reference to their relative effectiveness for the specific lesions. currently several trials are ongoing to determine the effectiveness of such delivery systems alone and in conjunction with systemic therapies. such approaches may prove to be very effective in the controlled and localized covid- viral lesions in the lungs and potential sites. moreover, localized delivery offered a safer delivery mode for such drugs which may have systemic adverse effects. definitive covid- therapies have yet to be identified, despite the many ongoing trials globally. the results based on early, small, underpowered, non-controlled studies and a few open label randomized-controlled trials (rcts) have been discouraging. for example, one trial evaluating lopinavir-ritonavir for covid- therapy, noted that an even higher dose than what was administered may be required to achieve therapeutic efficacy. only pharmaceuticals have demonstrated signals toward efficacy. remdesivir has been shown to reduce the total amount of time in icu required per patient (remdesivir for the treatment of covid- -preliminary report;j.h. beigel, k.m. tomashek, l.e. dodd, a.k. this article was published on may , , at nejm.org. doi: . /nejmoa , and dexamethasone had reduced deaths by % in patients who needed treatment with invasive ventilation and by % in those needing supplemental oxygen (eu clinical trials register: eudract - - clinical trials.gov: nct ). however, almost all the proposed pharmaceutical therapies for covid- pose at least a few possible adverse effects while citing the need for increased dosing. targeted drug delivery of these promising pharmaceuticals might prove to be more effective, enhancing the local effect while attenuating their off-target side effects. sars-cov- has an affinity for host cell factors, which are primarily expressed in human lung tissue: ace- and tmprss . , considering that covid- primarily affects the lungs in patients with ards, we propose a targeted drug delivery strategy using different types of drug delivery vehicles including microbubbles, extracellular vesicles (ev), nanoparticle drug carriers, liposomes, viral vectors, perfluorocarbon droplets, catheter-based and aerosol-based approaches. it should be noted that while there has been substantial research on local drug delivery vehicles in cancer and cardiovascular medicine, broadly; there appear to be only a handful of reports that describes using different carriers for targeted delivery in patients with covid- . - in the following, some of the newer approaches for targeted drug delivery in covid- patients are discussed. it may also apply to other bacterial or viral pneumonias not associated with covid- . microbubbles specifically have shown to adhere to sites of damaged vascular endothelium and thus may be a method of systemically targeting delivery of therapeutics to damaged lungs with sars-cov- . for example, perfluoro butane gas microbubbles (pgmc) with a coating of dextrose and albumin efficiently bind to different pharmaceuticals. these . to mm particles bind to sites of vascular injury. further, the perfluoro butane gas is an effective cell membrane fluidizer. the potential advantages of microbubble carrier delivery include none to minimal (additional) vessel injury through delivery, no resident polymer to degrade leading to eventual inflammation, rapid bolus delivery, and repeated delivery. microbubble carriers were successfully used in different animal models and clinical trials to deliver antisense oligonucleotide and/or sirolimus to the injured vascular bed. [ ] [ ] [ ] the formulation of microbubbles and therapeutics is easy and could be performed in a hospital pharmacy, considering they are currently used widely to enhance diagnostic ultrasound imaging. we propose to use microbubbles with pro-inflammatory cytokine (tnf-a, il- , or il- ) inhibitors as an approach to mitigate the ards-induced cytokine storm. one possible drug is tocilizumab, which is an il- inhibitor, which shows some early promise as a possible covid- therapeutic. , tocilizumab may enhance the metabolism of drugs utilizing the cytochrome p system, which warrants further investigation into whether the same effects are attenuated or maintained when given locally versus systemically. , extracellular vesicles (ev) kumar and colleagues describe the use of extracellular vesicles (evs), which are a family of natural carriers in the human body. evs play a critical role in cell-to-cell communications and can be used as unique drug carriers to deliver protease inhibitors to treat covid- . though the authors of the reported investigations concluded that certain limitations need to be overcome as well as understanding the mechanism to control targeted delivery. specifically, the isolation and drug encapsulation techniques employed to engineer evs could result in the loss of functional properties of the evs, such as the destruction of surface proteins. these unintended changes could lead to nonspecific interactions with other cells, leading to off-target effects, toxicity, and suboptimal efficacy. recently, the efficacy in mitigating inflammation was demonstrated through the targeted delivery of adenosine and of multidrug formulations. bioconjugation of adenosine to squalene produces a prodrug-based nanocarrier, which, after nano formulation with vit e, yields stable multidrug nanoparticles. this nanoparticle improves the bioavailability of both drugs with significant pharmaceutical activity in models of acute inflammatory injury. there are several clinical studies planned to use this technology in patients with novel bio-objects a group of researchers has succeeded in engineering a new kind of microscopic bio-object that may one day be used for personalized diagnostics and targeted delivery of drugs. the object consists of a genetically modified e. coli bacterium and nano-erythrosomes (small vesicles made of red blood cells), and it demonstrates a substantial improvement in motility over previous designs. direct pulmonary delivery (e.g., aerosol, inhalers, etc.) is a more selective mode of drug delivery that typically requires a lower quantity of drug to achieve an effective dose. currently, studies are enrolling patients to study the feasibility and efficacy of this approach -the nosarscovid phase ii trial (nct ) and the pulsed inhaled nitric oxide for treatment of patients with mild or moderate covid- expanded access program (nct ). pulmonary drug delivery can provide the following advantages: quick onset of action coupled with ease and convenience of administration; the pulmonary dose is significantly lower than the oral dose; and degradation of the drug in the liver can be avoided. on the other hand, the following drawbacks are often associated with pulmonary drug delivery: improper dosing, stability problems, and difficulty in producing the optimum particle size. in addition, not all drugs can be delivered via a pulmonary route due to formulation difficulties. given that acoustically activatable perfluoro propane droplets permeate across endothelial barriers to reach the interstitial spaces and even intracellular locations. this may be an ideal method of enhancing nitric oxide delivery to regions of abnormal ventilation to perfusion mismatch in covid- pneumonia. however, it should be noted that inhaler-based delivery is an aerosol-generating procedure and therefore and it should be performed with extreme caution and the appropriate personal protective equipment (ppe). heparin and low-molecular weight heparin have been used in the anticoagulant management of covid- . heparin also exhibit antiviral properties and localized delivery in the lung may have therapeutic effects including both the antiviral and anticoagulant effects. the inhaled formulations of heparins have been reported before in animal models such as dogs. special devices such as jet and ultrasonic nebulizers have also been used to deliver heparin to intrapulmonary sites. a recent article has reported on the intrapulmonary delivery of heparin in covid- patients and call for randomized clinical trial to validate the clinical efficacy of this approach. localized delivery of heparin may also results in anti-inflammatory, cytoprotective and membrane stabilizing effects. other modalities may include heparin mist in different formulations to produce eco effects. catheter-based local drug delivery of antivirals (liquid remdesivir) and/or pro-inflammatory cytokine inhibitors (tocilizumab) can be performed trans-pulmonary, with a swan-ganz catheter. the drug can be administered through the pulmonary artery to target pulmonary vasculature, which is safe and feasible. this does not require imaging and could be performed bedside. another aspect recently revealed is the prothrombotic state in some covid- patients. systemic antithrombotic and thrombolytic therapy has been anecdotally used with scattered reports of acute improved oxygenation. therefore, catheter-directed thrombolysis could be another approach through localized thrombolysis. another approach is the recently, commercially available intravenous microbubbles, which has been utilized for ultrasound-targeted sonothrombolysis in acute myocardial infarction and in pulmonary thromboembolic disease. , the mechanism for sonothrombolysis appears to be cavitation induced thrombus dissolution in addition to vasodilation induced by endothelial shear-release of nitric oxide. given the growing evidence that the most detrimental sars-cov- reactions are primarily within the respiratory system, localized targeted delivery of therapeutics may prove advantageous over a systemic approach, provided that bioavailability to the target tissue can be proven/verified. several approaches have been discussed, in which microbubbles are the only carrier to have been used in clinical practice and therefore are the most promising. adenosine nanoparticles and novel bioobjects are other classes of drug carriers under either preclinical development or in early clinical investigation. beyond carriers, there are several delivery methods to also consider. for example, direct inhalation or pulmonary delivery are a highly selective modes requiring less dosage, and there are several ongoing trials and expanded access programs to evaluate this approach. finally, there are several catheter-based drug delivery approaches, including catheter-directed thrombolysis to treat viral coagulopathies (pulmonary embolism, micro-thrombosis) induced by sars-cov- infection. the nebulized drug delivery systems provide a practical and clinically feasible approach for the localized delivery of heparins and other drugs in the management of pulmonary lesions with high viral load. further investigations through well-designed, timely clinical studies for targeted site-specific therapy will demonstrate evidence toward the best carriers, delivery methods, and approach (i.e. combination systemic and local delivery versus stand-alone). all authors equally contributed to the work in this manuscript. the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: tp has served as a consultant for lantheus medical imaging, inc. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: tp has received speaker fees from bracco diagnostics inc. tp has received research equipment support from phillips healthcare. a trial of lopinavir-ritonavir in adults hospitalized with severe covid- liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor detection of retained microbubbles in carotid arteries with real-time low mechanical index imaging in the setting of endothelial dysfunction successful recanalization of thrombotic occlusion in pulmonary artery stent using sonothrombolysis. case (phila) therapeutic application of contrast ultrasound in st elevation myocardial infarction: role in coronary thrombosis and microvascular obstruction sonothrombolysis in st segment elevation myocardial infarction treated with primary percutaneous coronary intervention repurposing antiviral protease inhibitors using extracellular vesicles for potential therapy of covid- squalene-based multidrug nanoparticles for improved mitigation of uncontrolled inflammation detection of sars-cov- in different types of clinical specimens targeted vascular delivery of antisense molecules using intravenous microbubbles novel site-specific systemic delivery of rapamycin with perfluorobutane gas microbubble carrier reduced neointimal formation in a porcine coronary restenosis model clinical applications of ultrasonic enhancing agents in echocardiography american society of echocardiography guidelines update tocilizumab treatment incovid- : a single center experience effective treatment of severe covid- patients with tocilizumab interleukin- and cytochrome-p , reason for concern? a physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of cyp a by il- targeted transthoracic acoustic activation of systemically administered nanodroplets to detect myocardial perfusion abnormalities nanoerythrosome-functionalized biohybrid microswimmers anticoagulant effects of inhaled unfractionated heparin in the dog as determined by partial thromboplastin time and factor xa activity characterization of heparin aerosols generated in jet and ultrasonic nebulizers nebulised heparin as a treatment for covid- : scientific rationale and a call for randomised evidence key: cord- -flkjekyo authors: hijikata, atsushi; shionyu‐mitsuyama, clara; nakae, setsu; shionyu, masafumi; ota, motonori; kanaya, shigehiko; shirai, tsuyoshi title: knowledge‐based structural models of sars‐cov‐ proteins and their complexes with potential drugs date: - - journal: febs lett doi: . / - . sha: doc_id: cord_uid: flkjekyo the world health organization (who) has declared the coronavirus disease (covid‐ ) caused by the novel coronavirus sars‐cov‐ a pandemic. there is, however, no confirmed anti‐covid‐ therapeutic currently. in order to assist structure‐based discovery efforts for repurposing drugs against this disease, we constructed knowledge‐based models of sars‐cov‐ proteins and compared the ligand molecules in the template structures with approved/experimental drugs and components of natural medicines. our theoretical models suggest several drugs, such as carfilzomib, sinefungin, tecadenoson, and trabodenoson, that could be further investigated for their potential for treating covid‐ . the newly identified coronavirus (sars-cov- ) causes severe pneumonia (coronavirus disease -covid- ) and has rapidly spread across the world from the initial outbreak point in wuhan, china, in late [ ] . it has become a global health emergency, and on march , , the world health organization (who) declared a pandemic status of this novel coronavirus outbreak. since no approved drug that is specifically targeted to this virus exists at this point in time, drug repositioning/repurposing is thought to be the most effective and feasible approach toward this clear and present threat, and researchers have initiated studies by employing various means in order to find potential therapeutics [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the sars-cov- genome is very close to that of the severe acute respiratory syndrome coronavirus (sars-cov) [ ] . from the past efforts to cure rna virus infections, including the experiences from the sars and middle east respiratory syndrome (mers) epidemics, several potential target proteins and drugs have been proposed [ , ] . the c-like (main) proteinase, surface glycoprotein [ ] , and rna-dependent rna polymerase are thought to be the most promising targets for anti-covid- therapeutics. for example, the anti-hiv drug lopinavir/ritonavir, which has been proposed to treat sars [ , ] , is expected to be effective toward sars-cov- c-like proteinase [ , ] . additionally, the antiviral drug remdesivir is expected to target the rna-dependent rna polymerase [ ] . the recent studies on drug repositioning/repurposing involve a variety of computational methods, such as network analysis, text mining, machine learning, and structure-based drug repositioning (sbdr) [ ] [ ] [ ] [ ] [ ] [ ] . among these methods, sbdr is the most promising to find abbreviations ace , angiotensin i-converting enzyme ; mers, middle east respiratory syndrome; rbd, receptor-binding domain; sars-cov, severe acute respiratory syndrome coronavirus; sbdr, structure-based drug repositioning; who, world health organization. specific drugs toward a defined target protein, and it prompted the quick structure analyses of sars-cov- c-like proteinase and surface glycoprotein [ , ] . although structure analyses of many other sars-cov- proteins would soon follow, predictions of other protein structures with homology/knowledgebased (theoretical) methods would be required until structure analyses are completed, especially for the proteins currently out of focus as drug targets. in the presented study, therefore, the homology models of sars-cov- proteins and their ligand complexes were comprehensively constructed. also, the structural models of the complexes between sars-cov- proteins and potential drugs were proposed by comparing the ligand molecules of the proteins and approved, experimental, or natural drugs. the amino acid sequences of sars-cov- proteins (table ) were retrieved from the refseq database at ncbi [ ] , and structural modeling templates were sought with the sird system (http://sird.nagahama-i-bio.ac.jp/sird/), which accepted multiple query sequences and sought for similar sequences (more than % sequence identity to query) with known structures in the protein data bank (pdb) [ ] by using blast [ ] . this system also sought for the templates of protein complex structures, in which two or more proteins in the multiple query were associated or any ligand bound to query proteins. the coordinates of template structures were obtained from the pdb [ ] and were rendered into the biological quaternary structures. initial structural models were constructed by using mod-eller [ ] . in some cases, the resultant models contain residues with rare dihedral angles (ramachandran outliers), rare shape of side chains (rotamer outliers), and short atom-atom distances (atomic crashes). then, the models were further modified by iteratively applying molecular dynamics and geometry minimization procedures of phenix [ ] to eliminate aforementioned outliers, and finally, manual model modifications with visual inspection on coot for resolving rotamer outliers or atomic crashes [ ] . the model quality was evaluated with molprobity [ ] . the percentages of rotamer outlier, ramachandran outlier, and crash score were monitored for each model to achieve less than %, . %, and , respectively. modeling of sars-cov- protein complexes with potential drugs the molecular formula of , drugs in total was retrieved from the kegg database [ ] and the drugbank database [ ] . the molecular formula of , metabolites in total, which have been used for natural medicines (natural drugs), was obtained from the knapsack database [ ] . the structures of the ligand molecules in the known complex structures, as sought in the template search process, were exhaustively compared with that of the drugs by using complig [ ] . complig matches molecular graphs and evaluates the similarity score of two molecules a and b as min{m selected drug molecules were built into the protein models by superposing drug molecules to known (original) ligand molecules with complig. according to the graph matching results, the dihedral angles in the drug molecules were adjusted toward the corresponding angles in the original ligand molecules, and corresponding atoms were superposed between drug and known ligand by fixing the coordinates of the latter. the models of protein-drug complexes were further modified with phenix and coot. the constraints for drug molecules were generated by using the elbow application in phenix. hydrogen bonds were evaluated with canonical parameters (constraints were relaxed by . Å and degrees) [ ] by using chimera [ ] . the protein-ligand complexes were also assessed by using the dsx score function [ ] . the sars-cov- genome encodes genes (open reading frames), and the polyprotein from orf ab is processed into proteins (polypeptides) through cleavages by the papain-like proteinase and c-like proteinase activities [ , , ] . as a result of template search, the appropriate structural templates were found for sars-cov- proteins among a total of , and their homology models were constructed ( table ). the unmodeled proteins included those from very short orfs, namely nsp ( amino acid residues), orf b ( residues), and orf ( residues) and probable membrane proteins (nsp , orf a, orf , m, and orf ), which were annotated by the sosui server [ ] . since a considerable amount of structural studies have already been done for sars-cov and mers-cov proteins, most of the available templates were from these viruses, and they had high-sequence similarity (more than %) to sars-cov- proteins. two proteins, namely papain-like proteinase (nsp ) and nucleocapsid phosphoprotein, could not be modeled into a single structural model. as a result of homology search, template structures covering the entire protein were unavailable. template structures were separated into and fragment/domain models, respectively. consequently, the coverages of structural model were % and % of total residues for papain-like proteinase (nsp ) and nucleocapsid phosphoprotein, respectively ( table ) . the third region of papain-like proteinase (nsp ), nsp , c-like proteinase, nsp , endo-rnase, surface glycoprotein, envelope protein, and c-terminal region of nucleocapsid phosphoprotein was modeled into homo-multimer. as the hetero-multimeric models, nsp and nsp were modeled into hetero- mer; rnadependent rna polymerase, nsp , and nsp were modeled as hetero-tetramer ( : : stoichiometry); 'to- ' exonuclease and nsp formed hetero-dimer; '-o-ribose methyltransferase and nsp also formed hetero-dimer; and homo-trimer of surface glycoprotein was modeled in complex with human angiotensin i-converting enzyme (ace ) ( table ) . although the models of sars-cov- protein would be useful for structure-based virtual screening, potential drugs for these proteins were sought by rather simple knowledge-based method in the presented study. the ligand molecules that were complexed with the homologs of sars-cov- protein in the pdb were extracted, and structurally similar molecules to the ligands were sought among the approved/experimental drugs retrieved from the kegg database [ ] and the drugbank database [ ] . many of the approved drugs, such as morphine, aspirin, or penicillin, have been adapted from natural medicines [ , ] . the molecules in the natural medicines are expected to serve as argent therapeutics. therefore, the ligand structures were also compared with the components of natural medicines (natural drugs) registered in the knapsack database [ ] . the original ligand molecules and the detected drug molecules are summarized in table . a total of ligand molecules were matched to approved/experimental and natural drugs, and the complex models of the sars-cov- proteins with several promising drugs, those with high similarity score or placed in higher ranking, were constructed as follows. c-like proteinase is involved in the processing of viral polyprotein [ ] . this enzyme is one of the most extensively studied drug targets and thus analyzed in complex with various peptide mimetic inhibitors [ ] [ ] [ ] [ ] . unexpectedly, these ligands did not show very high similarity to known drug molecules ( table ) . as a peptide mimetic drug, carfilzomib showed highest score to the template ligand (ligand code azp) of clike proteinase homolog (fig. a) . however, the similarity score between the ligand and the drug was only . . carfilzomib is the irreversible proteasome inhibitor targeted to the subunits with chymotrypsin-like activity and has been approved for refractory multiple myeloma or waldenstr€ om's macroglobulinemia [ , ] . a complex model of carfilzomib-sars-cov- c-like proteinase was constructed. in the model, carfilzomib formed a parallel b-sheet with his -glu , and side chains of his , cys , met , leu , phe , and gln contributed major interactions (fig. b,c) . these residues were conserved between the template (sars-cov) and the model (sars-cov- ) proteins. carfilzomib covalently binds to active site threonine through epoxy moiety, and the epoxy moiety is also reactive with thiol group of cysteine. although a possible covalent linkage between carfilzomib and the catalytic cys of sars-cov- c-like proteinase was not explicitly modeled, the epoxy moiety was placed close to the catalytic residue in this model. the fitness of the ligand to sars-cov- c-like proteinase in the model was evaluated by the dsx function, and the score of carfilzomib, - . , was even better than that (À . ) of inhibitor n in the complex crystal structure of sars-cov- c-like proteinase (pdb id lu ). surface glycoprotein is used for a viral entrance into the host cell, and its cell surface receptor is human angiotensin i-converting enzyme (ace ) [ ] . ace, a homolog of ace sharing % amino acid sequence identity, is a major target of hypertension medicating drugs, and several ace-drug complexes have been reported [ ] [ ] [ ] . lisinopril, enalaprilat, and captopril, which show similar structures to each other ( fig. a) , have been targeted toward ace and approved for hypertension treatments [ ] [ ] [ ] [ ] . in the structural complex models, these drugs were bound to the protein through a zn + coordination with glu , his , and his (fig. b,c) . these residues were conserved between the template (ace) and the model (ace ) structures. the drug molecules also formed electrostatic interactions with arg and arg even though these residues were not conserved between ace and ace (gln and ser in ace). the sars-cov- surface glycoprotein interacted with ace through the receptor-binding domain (rbd), while the bound drugs had no direct interaction to the rbd (fig. c) , suggesting that those drugs would not directly interfere the host-pathogen interaction. the dsx scores for lisinopril, enalaprilat, and captopril in the models were À . , À . , and À . , respectively. these scores were considerably inferior to that (À . ) of the specific inhibitor mln- in the crystal structure of human ace complex (pdb id r l) [ ] . the complex of '-o-ribose methyltransferase (nsp ) and nsp is involved in the modification of the viral rna caps [ ] . the structure of '-o-ribose methyltransferase subunit was determined in complex with sadenosyl-l-methionine (ligand code sam), -methylguanosine- '-triphosphate- '-guanosine (gtg), and sinefungin (sfg) [ ] [ ] [ ] . among these ligands, sadenosyl-l-methionine is used for a therapeutic against depression, liver disorders, fibromyalgia, and osteoarthritis [ ] , but also is an authentic substrate for this enzyme. sinefungin is a natural drug produced by streptomyces griseolus and experimentally used as antibiotics [ ] [ ] [ ] (fig. a) . the residues of '-o-ribose methyltransferase, ser , asp , asn , asp , and met , were involved in the major interactions with sinefungin (fig. b,c) . these residues were conserved among the template proteins (sars-cov and betacoronavirus) and sars-cov- . as the drugs similar to these ligands, several investigational adenosine a receptor agonists, namely tecadenoson, selodenoson, and trabodenoson, were found (fig. a) . these molecules share adenosine moiety, and this moiety interacts with the aforementioned conserved residues in the complex models. the dsx scores of sinefungin, tecadenoson, selodenoson, and trabodenoson were À . , À . , À . , and À . , respectively. the scores of sinefungin and selodenoson were comparable to that (À . ) of the genuine substrate, s-adenosyl-l-methionine, in the complex crystal structure with mers-cov '-o-ribose methyltransferase (pdb id yni). in the presented study, knowledge-based models of sars-cov- proteins were constructed by homology modeling and comparison of the known ligands with drugs. since a considerable number of structure analyses have already reported for coronavirus proteins including those of sars-cov, % ( / ) of the sars-cov- proteins could be modeled based on highly similar ( % sequence identity and % coverage on average) templates (table ) . several drugs were suggested to bind to the sars-cov- targets ( table ). the procedure employed in the presented study should largely limit the extent of search (because it depends on the presence of ligands in known complex structures). however, it is noteworthy that the binding of suggested drugs to the homologous proteins of the sars-cov- targets would be probable because of the presence of structural evidences. the complex models were constructed for several high-scored and/or high-ranked drugs. unexpectedly, no drug was detected for one of the most promising drug targets, c-like proteinase, with a similarity score higher than . . in the previous study, the score more than . was suggested to be required for highly similar interactions between ligand and protein [ ] . it is implied that the inhibitors bound to the c-like proteinase in the known structures are considerably deviated from most of the approved protease-targeted drugs. for example, the anti-hiv drug lopinavir/ritonavir, which was expected to target sars-cov- clike proteinase [ , ] , showed only limited similarity (score . ) to the known ligand (ligand code axp) of sars-cov c-like proteinase ( fig. a) . one possible reason for the low similarity to drugs is that the protease inhibitors tend to have higher molecular weight and thus their molecular structures showed large variety. another reason would be that a majority of the protease inhibitory drugs are targeted toward serine or zinc proteases [ , ] . also, the expected drug lopinavir/ritonavir has been designed for hiv protease, which is aspartic protease. these proteases are structurally distinct from c-like proteinase known to be a cysteine protease. this observation implies that structure optimizations would likely be required for repurposed drugs for sars-cov- c-like proteinase. consequently, the presented study suggested carfilzomib, which has been targeted toward threonine protease and approved for multiple myeloma treatment [ , ] , as a marginally resembling drug. the model showed, however, carfilzomib fits well (even better than the specific inhibitor n , according to the dsx score) into the active site by forming considerable stabilizing interactions and no severe steric hindrance (fig. c) . another potential target is the complex of surface glycoprotein and ace to prevent virus entry into the cell [ , ] . many hypertension drugs are targeted to ace, and the presented study highlighted the approved drugs, namely lisinopril, enalaprilat, and captopril, as potential ligands for ace . an expectation in advance was to find a drug that bound to ace and also interfered the interactions between surface glycoprotein and ace . however, as the models revealed, the drug-binding site of ace existed inside a deep cleft in the center of ace molecule, and the ligands do not interact directly with the surface glycoprotein (fig. c) . the human ace has been demonstrated to change its conformation from open to close forms (pdb ids r l and r , respectively) upon inhibitor binding [ ] . thus, if this conformational change involves the interface to the rbd of surface glycoprotein, a drug bound to the drug-binding site might interfere with the binding between ace and surface glycoprotein indirectly. this allosteric inhibition mechanism, however, is not highly expected because no obvious conformational change was observed in the interface region when comparing the open and close conformations of ace . most of the predicted drugs are targeted toward ace (not ace ), and ace and ace diverge considerably in their amino acid sequences ( % identity). the fitness of the drugs was evaluated to be lower than the specific inhibitor to ace . therefore, effects of the predicted ace drugs on preventing surface glycoprotein-ace interactions would not be highly promising. another target presented results highlighted was '-o-ribose methyltransferase (nsp )-nsp complex, which is less focused as a target of drug repurposing. '-o-ribose methyltransferase is required to finalize the cap structure, me gpppa 'ome , of coronavirus rnas by transferring a methyl group to ' oh group of ribonucleotide from s-adenosyl-l-methionine [ , ] . the cap structure is essential for viral mrnas to be translated and escape from innate immune system in the host cell. thus, inhibition of this enzyme might prevent virus propagation. despite the overall sequence identities between templates (sars-cov or human betacoronavirus) and sars-cov- enzymes were relatively low (~ %), the residues interacting with the drugs were conserved. among the suggested drugs for this enzyme, sinefungin is a naturally occurring and verified inhibitor of '-o-ribose methyltransferase. since a toxicity was detected [ ] , however, appreciation of this natural drug should be carefully considered. although tecadenoson has been examined in a clinical trial for atrial fibrillation, and passed phase ii test, final results were not formally reported at this point of time [ ] . trabodenoson was designed for treating ocular hypertension and primary open-angle glaucoma [ ], but had failed in the phase iii clinical trial test due to lack of superiority over placebo. selodenoson was designed to control heart rate [ ] , and it seems still in a developmental stage. since tecadenoson and trabodenoson appeared to have cleared the phase i tests, these drugs would worth examining against covid- . the dsx score suggested the fitness of sinefungin or selodenoson is comparable to the genuine substrate of the '-o-ribose methyltransferase. several structure determinations of sars-cov- proteins, for example, endo-rnase (pdb ids vww and vw ), nucleocapsid phosphoprotein ( vyo), and nsp (rna-dependent rna polymerase)-nsp -nsp complex (pdb id bv ) have been reported after the modeling of the presented study was executed. although many of the other proteins should be under analyses undoubtedly, it would take considerable time before all structures of potential targets are experimentally elucidated. during the period until the structural determinations, theoretical models might be useful. the presented structural models are freely available from the binds webpage (https://www.bind s.jp/sars-cov- /) and also deposited in the bsm-arc repository (bsm ) [ ] . this work was partly supported by the platform project for supporting drug discovery and life science mo, sk, and ts conceived and designed the study. ah, cs, sn, ms, and ts constructed the models. ah, cs, and ts wrote the manuscript. all authors commented on the manuscript. a new coronavirus associated with human respiratory disease in china return of the coronavirus: -ncov drug treatment options for the -new coronavirus ( -ncov) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies repurposing of clinically approved drugs for treatment of coronavirus disease in a -novel coronavirus ( -ncov) related coronavirus model discovering drugs to treat coronavirus disease (covid- ) cryo-em structure of the -ncov spike in the prefusion conformation therapeutic options for the novel coronavirus ( -ncov) traditional chinese medicine is a resource for drug discovery against novel coronavirus (sars-cov- ) potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review sars and mers: recent insights into emerging coronaviruses from sars to mers, thrusting coronaviruses into the spotlight treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings potentially highly potent drugs for -ncov. biorxiv nelfinavir was predicted to be a potential inhibitor of -ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus drug repositioning: identifying and developing new uses for existing drugs drug-target network disease networks. uncovering disease-disease relationships through the incomplete interactome a survey of current trends in computational drug repositioning drug repurposing: progress, challenges and recommendations drug databases and their contributions to drug repurposing sars-cov- protein models crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation announcing the worldwide protein data bank basic local alignment search tool comparative protein structure modeling using modeller phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: more and better reference data for improved all-atom structure validation kegg for integration and interpretation of large-scale molecular data sets drugbank . : a major update to the drugbank database knapsack family databases: integrated metaboliteplant species databases for multifaceted plant research classification of ligand molecules in pdb with fast heuristic graph match algorithm complig three-dimensional hydrogen-bond geometry and probability information from a crystal survey ucsf chimera-a visualization system for exploratory research and analysis dsx: a knowledgebased scoring function for the assessment of proteinligand complexes coronavirus polyprotein processing sosui: classification and secondary structure prediction system for membrane proteins a historical overview of natural products in drug discovery natural products as reservoirs of novel therapeutic agents mechanisms and enzymes involved in sars coronavirus genome expression crystal structures reveal an induced-fit binding of a substrate-like azapeptide epoxide to sars coronavirus main peptidase structure-based design, synthesis, and evaluation of peptide-mimetic sars cl protease inhibitors peptide aldehyde inhibitors challenge the substrate specificity of the sars-coronavirus main protease design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus c-like proteases potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma sars-cov- protein models a angiotensin-converting enzyme is a functional receptor for the sars coronavirus crystal structure of the human angiotensinconverting enzyme-lisinopril complex structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-i-converting enzyme (ace) innappropriate renin secretion unmasked by captopril (sq ) in hypertension of chronic renal failure effect of n-[(s)- -carboxy- -phenylpropyl]-l-ala-l-pro and its ethyl ester (mk- ) on angiotensin converting enzyme in vitro and angiotensin i pressor responses in vivo effect of a new angiotensin converting enzyme inhibitor mk and its lysine analogue on the components of the renin system in healthy subjects from peptides to peptidases: a chronicle of drug discovery ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase biochemical and structural insights into the mechanisms of sars coronavirus rna ribose '-o-methylation by nsp /nsp protein complex crystal structure and functional analysis of the sars-coronavirus rna cap '-o-methyltransferase nsp /nsp complex structural basis and functional analysis of the sars coronavirus nsp -nsp complex mischoulon d & as work group of the american psychiatric association council on same) for neuropsychiatric disorders: a clinician-oriented review of research antileishmanial effect of sinefungin and its derivatives amino acid metabolism and transport mechanisms as potential antifungal targets antiviral effect of sinefungin on in vitro growth of feline herpesvirus type the druggable genome structure-based view of the druggable genome angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor in vitro reconstitution of sars-coronavirus mrna cap methylation molecular mechanisms of coronavirus rna capping and methylation a , a new adenine-containing antifungal antibiotic. i. discovery and isolation tecadenoson: a novel, selective a adenosine receptor agonist trabodenoson, an adenosine mimetic with a receptor selectivity lowers intraocular pressure by increasing conventional outflow facility in mice anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents, and innovative approaches the biological structure model archive (bsm-arc): an archive for in silico models and simulations key: cord- -f w m authors: ortega, joseph t.; serrano, maria luisa; jastrzebska, beata title: class a g protein-coupled receptor antagonist famotidine as a therapeutic alternative against sars-cov : an in silico analysis date: - - journal: biomolecules doi: . /biom sha: doc_id: cord_uid: f w m the pandemic associated with severe acute respiratory syndrome coronavirus type (sars-cov ) and its disease named covid- challenged the scientific community to discover effective therapeutic solutions in a short period. repurposing existing drugs is one viable approach that emphasizes speed during these urgent times. famotidine, a class a g protein-coupled receptor antagonist used for the treatment of gastroesophageal reflux was recently identified in an in silico screening. additionally, a recent retrospective clinical report showed that the treatment with famotidine provided a good outcome in patients infected with sars-cov . a clinical trial testing effectiveness of famotidine in combination with hydroxychloroquine is currently ongoing in the united states (us). in the s, famotidine was described as an antiviral agent against human immunodeficiency virus (hiv). interestingly, some hiv protease inhibitors are presently being used against sars-cov . however, it is not clear if famotidine could be effective against sars-cov . thus, by using a computational analysis, we aimed to examine if the antiviral effect of famotidine could be related to the inhibition of proteases involved in the virus replication. our results showed that famotidine could interact within the catalytic site of the three proteases associated with sars-cov replication. however, weak binding affinity of famotidine to these proteases suggests that a successful famotidine therapy could likely be achieved only in combination with other antiviral drugs. finally, analysis of famotidine’s pharmacokinetic parameters indicated that its effect against sars-cov infection could be reached only upon intravenous administration. this work will contribute to the pharmacological knowledge of famotidine as an antiviral agent against sars-cov . the severe acute respiratory syndrome coronavirus type (sars-cov ) pandemic known as covid- has affected more than million people worldwide as of june [ ] . with an original epicenter in the wuhan city in china, reported in december , the virus spread all around the world in less than four months [ ] . since the outbreak began, several advances were made in the therapeutic management of sars-cov infection [ ] . all these advances are associated with a better understanding of the viral pharmacological targets and the clinical outcomes of the applied treatments. we focused our interest on the proteases associated with the replication cycle of sars-cov . the replication cycle of sars-cov involves two viral proteases, the main protease ( clpro) and papain-like protease (plpro), and one human host protease, transmembrane serine protease type-ii (tmprss ) [ ] [ ] [ ] [ ] . the viral currently available viral protease inhibitors. in addition, we analyzed the pharmacokinetic profiles of these drugs to predict their successful use as anti-sars-cov therapeutics. the homology structural model of human tmprss (np_ . ) was built by using the tools of the swiss-model modeling server and the deepview/swiss-pdbviewer . software [ ] . the best model for tmprss was obtained using the crystal structure of serine protease hepsin with an inhibitor bound (protein data bank (pdb) identifier (id): ce ) as a template. hydrogen atoms were added and partial charges were assigned for the energy refinement. the obtained model was subjected to molecular dynamic (md) simulations using namd . [ ] , as described in ortega et al. [ ] using the charmm force field [ ] and gasteiger charges. the obtained structure represents the lowest energy frame of the md simulations. the quality of the model was validated via prosaweb [ ] and procheck programs [ ] . the coordinates for the sars-cov main protease, papain-like protease, and hiv protease were obtained from the protein data bank, pdb ids lu , wuc, and lzs, respectively. the pdb files were optimized by removing co-crystallized molecules and all crystallographic water molecules before being used for further computational analysis. hydrogens were added and partial charges were assigned to all atoms. the obtained pdb files for each protein were further submitted to restrain the molecular mechanics refinement. all molecular dynamic simulations described in this study were performed with namd . [ ] and vegazz . . . software [ , ] as described in ortega et al. [ , ] . for each protein, the substrate-binding site located in the catalytic site was identified by using the achilles blind docking server [ ] with a respective ligand. the three-dimensional ( d) structure of each inhibitor was obtained from pubchem. molecular docking was performed with vina/vegazz . . . with iterations for each compound. results were prioritized according to the predicted binding free energy in kcal/mol. the results collected from the docking simulation were visualized via the biovia discovery studio visualizer . . software. a comprehensive analysis of physicochemical descriptors, parameters related to administration, distribution, metabolism, and elimination (adme), drug-like nature, and medicinal chemistry for famotidine and other protease inhibitors was carried out using swissadme tools [ ] . these tools were accessed through the website at http://www.swissadme.ch. moreover, a two-dimensional ( d) representation for the chemical structures of all drugs examined in this report are shown in table . table . chemical structure of the compounds evaluated as protease inhibitors. chemical structure the viral and host proteases play a pivotal role in the sars-cov replication. the viral sars-cov genome codifies two proteases: the main protease ( clpro) and papain-like protease (plpro). both enzymes were studied as possible targets for antiviral drugs [ , ] . as reported, hiv inhibitors such as lopinavir and darunavir could block the hiv main protease [ ] , while antivirals such as ribavirin, naphthalene derivative, and phenylthioacetic acid derivatives block the plpro [ ] . however, it is not clear whether these compounds could be used in combating the sars-cov infection. thus, despite testing well-known antiviral drugs, the search for new anti-sars-cov therapeutics is urgently required. a drug-repurposing strategy provides an opportunity to discover new effects for already existing fda-approved drugs, and it promises the shortest time to begin a clinical trial [ ] . retrospective clinical studies showed that famotidine, a histamine type receptor antagonist, could improve the clinical outcome in patients diagnosed with covid- [ ] . in addition, recent computational analysis of therapeutic targets for sars-cov reported that famotidine could be a hit compound. [ ] . thus, we hypothesized that famotidine can inhibit the proteases involved in the viral replication. to gain a deeper understanding if famotidine indeed can interact with the catalytic site of these enzymes, we performed detailed in silico analysis. the main protease, also named chymotrypsin-like protease ( clpro), of sars-cov shares~ % homology with clpro of sars-cov. structurally, these proteases are also very similar, showing only subtle differences (root median squared of . , in the cα atoms) [ ] . moreover, both enzymes functionally belong to a cysteine protease group. the sars-cov main protease has a binding site with a catalytic dyad formed by his and cys , and several subsites designated as s (his , glu , cys , gly , his , and phe ), s (cys , his , and thr ), s (met , met , and his ), s (met and glu ), and s (gln , met , and glu ) ( figure a ). the main protease is responsible for the proteolysis of pre-proteins associated with the viral replication machinery such as rna-dependent rna polymerase, a helicase, and an exoribonuclease, among others. prior reports showed that anti-hiv drug lopinavir could block the activity of both sars-cov and sars-cov proteases [ , , ] . thus, in this work, we used a priori docking with lopinavir to define the substrate-binding site in the sars-cov main protease [ , ] . after determining the binding site coordinates, we analyzed the binding pattern and binding free energy of famotidine to the main sars-cov protease. the results obtained for famotidine were compared with two hiv protease inhibitors, lopinavir and darunavir (table ). both lopinavir and darunavir showed lower binding free energy (− . kcal/mol and − . kcal/mol, respectively) as compared to famotidine (− . kcal/mol), which is likely related to the chemical features of each inhibitor. nevertheless, each compound (famotidine, lopinavir, and darunavir) could interact with the key residues, his and cys , related to the catalytic activity of the main protease ( figure b -d). lopinavir and darunavir were able to produce hydrogen bonds, π-π, and π-sulfur interactions, with hys and cys residues of the catalytic center and with residues of the subsites s , s , and s (met , his , his , met , pro , arg , gln , and thr ) ( figure c ,d, respectively). famotidine adopted a distinct binding mode as compared to lopinavir and darunavir. it also produced hydrogen bonds, π-π, and π-sulfur interactions with residues of the catalytic center (hys and cys ), but it interacted with different residues of the subsites s , s , and s (thr , thr , hys , thr , ser , phe , leu , ser , cys , hys , and glu ) ( figure b ). the detailed interaction patterns of lopinavir, darunavir, and famotidine with the main protease are shown in the d diagrams in figure . the papain-like protease (plpro) is also a cysteine protease. however, in contrast to the main protease, plpro possesses an asp/his/cys catalytic triad within the substrate-binding site critical for its catalytic activity ( figure a ; the active site is shown in blue and the catalytic triad is shown in yellow) [ ] . these residues (asp , his , and cys ) are located within two main domains of plpro. cys is located within the central domain, which is mostly composed of α-helices, while his and asp are located in the c-terminal region, which is composed of a β-sheet [ ] . plpro is responsible for the cleavages of the n-terminus of the polyprotein to release nsp , nsp , and nsp , non-structural proteins associated with the viral replication [ ] . furthermore, plpro also has other functions such as de-ubiquitination and de-isgylation (isg: interferon-stimulated gene) related to antagonizing the host's innate immunity. compounds described as inhibitors of the sars-cov plpro could potentially inhibit the activity of the sars-cov plpro [ ] . thus, we performed the molecular docking analysis to determine the binding free energies and the binding pattern of such compounds to the sars-cov plpro, and we compared the results to those obtained for famotidine. firstly, using the crystal structure of plpro (pdb id: wuc) and castp . software, we performed topological analysis of the substrate-binding pocket of the sars-cov plpro [ ] . this pocket exhibits a solvent-accessible area of~ Å and it involves the following residues: trp , asn , cys , tyr , leu , gly , arg , met , ser , ala , tyr , gly , his , tyr , thr , and asp . next, we performed a priori docking using a common antiviral drug ribavirin as a ligand to confirm our analysis and to define the binding site coordinates. then, we performed molecular docking of famotidine, ribavirin, and two other compounds described as the sars-cov plpro inhibitors ( figure b -e, respectively; table ). our results indicated that all the evaluated compounds could interact in the substrate-binding site via several electrostatic interactions and hydrogen bonds. ribavirin mainly interacted with the following residues: asp , ser , ala , tyr , tyr , thr , and asp via van der waals interactions, hydrogen bonds, salt bridges, sulfur-x, and π-alkyl interactions. the binding free energy of ribavirin to the sars-cov plpro was − . kcal/mol, while the sars-cov inhibitors showed slightly lower binding free energy (− . kcal/mol). the main interactions of these inhibitors with the sars-cov plpro involved the following residues: leu , asp , pro tyr , tyr , and thr . among the analyzed compounds, famotidine exhibited the highest binding free energy (the lowest affinity) of − . kcal/mol. the main interactions between famotidine and the sars-cov plpro within the binding site involved the following residues: asp , arg , ser , ala , pro , pro , and tyr , and they occurred mostly via hydrogen bonds. all the interactions between the sars-cov plpro and the inhibitors evaluated in this work are shown in the d diagrams in figure . table . binding free energies calculated for famotidine and other protease inhibitors upon docking to the sars-cov papain-like protease. pubmed id binding free energy (kcal/mol) biomolecules , , of the viral replication cycle of sars-cov starts with an interaction between the viral spike protein and the angiotensin-converting enzyme (ace ) receptor expressed on the surface of the target host [ ] . then, proteolytic priming occurs in the viral spike protein, allowing the exposure of the fusion motive related to the endosome formation that allows the release of the viral rna into the host cytosol [ , ] . the human host protease tmprss is involved in this processing of sars-cov [ ] . as reported, protease inhibitors derived from benzoic acid such as nafamostat and camostat showed an antiviral effect against sars-cov in vitro by blocking tmprss protease activity [ , ] . this protein contains an intracellular domain (residues to ), transmembrane spanning domain (residues - ), low-density lipoprotein receptor domain (ldlra: residues - ), and two extracellular domains. to date, there is no crystal structure solved for tmprss . thus, we developed a homology model by using a crystal structure of a serine protease hepsin (pdb id: ce ) as a template. the homology model of tmprss showed two extracellular domains: the cysteine-rich domain (crd) (residues - ) and the serine protease domain (spd) (residues - ), with the presence of ser as a catalytic residue ( figure a ). the quality of our homology model was validated using the validation server, and it is depicted with the ramachandran and the prosa plots shown in figure b and c. the ramachandran plot denotes the phi and psi angles of each residue that are related to its structural configuration and indicates if these residues are in an energetically favorable conformation. the assessment of the ramachandran plot indicates a good overall geometry for the model, with~ % of the residues in the most favored regions. on the other hand, the prosa plot contains the z-scores of all experimentally determined protein chains in the current pdbs. in this plot, groups of structures from different sources (x-ray, nmr) are distinguished by different colors. our model has a z-score of − . for the overall quality (black dot denoted with yellow circle), which is within the range of scores typically found for native proteins of similar size. thus, this model is suitable for molecular docking calculations. in our molecular docking analysis, we used antivirals nafamostat and camostat as positive controls ( figure c ,d, respectively). both compounds interacted within the active site of tmprss with the following residues: his , leu , asp , ser , cys , trp , gly , gly , and cys via electrostatic interactions and hydrogens bonds. the binding free energy for nafamostat was − . kcal/mol and that for camostat was − . kcal/mol (table ). famotidine interacted with his , asp , gly , ser , ser , and gly within the catalytic site mostly via hydrogen bonds ( figure b ). the binding free energy for famotidine (− . kcal/mol) ( table ) was higher than for nafamostat and camostat, indicating its lower affinity to tmprss as compared to two other drugs. the detailed interactions between tmprss and the inhibitors evaluated in this work are shown in the d diagrams in figure . several reports published in the s showed that class a gpcr antagonists, including famotidine, could block hiv replication in vitro [ , ] . however, the exact mechanism related to famotidine inhibition was never determined, mainly due to the approval of more potent hiv inhibitors. interestingly, hiv has a protease that plays a pivotal role in virus replication by processing the viral polyprotein into mature and functional proteins [ ] . the hiv protease is an aspartyl protease and, together with the reverse transcriptase and the integrase, they are the main targets for hiv therapy [ ] . there are several drugs approved by the fda as hiv protease inhibitors. for example, lopinavir and darunavir are commonly used as anti-hiv therapeutics. as described earlier in this report, these two antiviral drugs could bind to the main protease of sars-cov [ ] . we hypothesized that the earlier observed antiviral effect of famotidine against hiv could be related to its interaction with the hiv protease. in order to evaluate this hypothesis, we performed molecular docking of famotidine to the hiv protease. for this analysis, we used the crystal structure of lopinavir-bound hiv protease (pdb id: q k) and of darunavir-bound hiv protease (pdb id: ll ) ( figure c ,d, respectively). our results indicated that both hiv inhibitors have lower binding free energy (higher affinity) (− . kcal/mol and − . kcal/mol, respectively) than famotidine (− . kcal/mol) ( table ). the hiv inhibitors produced more electrostatic interactions like π-π stacking and π-alkyl interactions with the active site of hiv protease than famotidine. nevertheless, famotidine via hydrogen bonds was able to interact with several residues within the active site, including gly , which plays a critical role in the structural geometry of the active site ( figure b ) [ ] . the binding poses and the main interactions with the hiv protease for all the examined inhibitors are shown in figure . all drugs examined in this work could interact with their targets to produce an inhibitory effect. thus, pharmacodynamically, these drugs should act as inhibitors of the respective proteases in sars-cov . however, often, the results obtained by in silico analyses and in vitro experiments cannot be reproduced in vivo, likely due to drug pharmacokinetic parameters that cannot be fully assessed in the early, experimental stage. in order to gain a deeper understanding if the pharmacokinetic parameters of the sars-cov protease inhibitors could be related to positive outcomes in the therapy, we analyzed the adme parameters of famotidine and compared with several known antiviral drugs such as ribavirin, lopinavir, and nafamostat, which were evaluated against sars-cov . the adme parameters were determined by using the tools available on the swissadme server. the assessment of "drug likeness" in pharmaceutical research is associate with lipinski's "rule of five" that established guidelines for the characteristic of permeable compounds through biological membranes. swissadme used six physicochemical parameter derivatives from lipinski's "rule of five" represented as radar plots of molecule properties. the red area represents "good" property space for oral bioavailability, and the red bold line represents values of calculated properties of the analyzed molecule. when a compound satisfies the "rule of five", it has molecular properties similar to those of typical bioavailable drugs. however, there are some exceptions from the "rule of five" for certain drug classes (i.e., antibiotics, antifungals, vitamins, and cardiac glycosides). all the analyzed drugs are fda-approved and their pharmacokinetics parameters are well known. in figure , we summarize the main adme parameters in the radar representations for each drug property, including lipophilicity, size, polarity, solubility, saturation, and flexibility. interestingly, only ribavirin and famotidine exhibited all the properties characteristic for orally available drugs. of note, famotidine is more polar than ribavirin. all examined compounds except lopinavir have low predicted absorption in the gastrointestinal tract. the water solubility of a drug is related to its absorption, formulation, and administration pathway. thus, we evaluated the water solubility of lopinavir, nafamostat, ribavirin, and famotidine by using three predictors. the swissadme server uses two topological methods to predict aqueous solubility, the esol (estimated solubility) model and the model adapted from ali et al. [ , ] . the third predictor for solubility was developed by silicos-it. all predicted values are shown as decimal logarithms of the molar solubility in water (log s). the values obtained for each inhibitor are shown in table . ribavirin and famotidine have higher water solubility with logs (esl) of − . and − . , respectively than lopinavir and nafamostat (logs (esl) of − . and − . , respectively). lopinavir showed the lowest water solubility among the four analyzed drugs. interestingly, lopinavir has a very flexible structure and larger size as compared to the other three inhibitors. on the other hand, nafamostat has a higher number of unsaturated bonds than the other analyzed drugs. both the size and the high content of unsaturated bonds are likely related to the lower solubility of lopinavir and nafamostat. all the analyzed drugs are fda-approved and their pharmacokinetics parameters are well known. in figure , we summarize the main adme parameters in the radar representations for each drug property, including lipophilicity, size, polarity, solubility, saturation, and flexibility. interestingly, only ribavirin and famotidine exhibited all the properties characteristic for orally available drugs. of note, famotidine is more polar than ribavirin. all examined compounds except lopinavir have low predicted absorption in the gastrointestinal tract. figure . the analysis of pharmacokinetic parameters of protease inhibitors. chemical structures and administration, distribution, metabolism, and elimination (adme) parameters for famotidine, ribavirin, lopinavir, and nafamostat, drugs that were evaluated as sars-cov inhibitors, are shown. figure . the analysis of pharmacokinetic parameters of protease inhibitors. chemical structures and administration, distribution, metabolism, and elimination (adme) parameters for famotidine, ribavirin, lopinavir, and nafamostat, drugs that were evaluated as sars-cov inhibitors, are shown. the colored zone is a suitable physicochemical space for oral bioavailability obtained using the swissadme software. the compounds were scored based on the structure and whether these parameters fit into the values established for each indicator related to oral bioavailability. lipo (lipophility), size (size), polar (polarity), insolu (insolubility), insatu (instauration), and flex (flexibility). the d structures of each drug are shown with the specific atoms represented as follows: carbon in gray, oxygen in red, nitrogen in blue, and sulfur in yellow. in the late stage of the disease, the sars-cov replication occurs mainly in the lungs. thus, in order to produce a positive effect, an antiviral compound needs to reach the pulmonary tissue. high gastrointestinal absorption of a drug would be advantageous for fast tissue distribution. in fact, fda-approved drugs currently used for treatment of sars-cov such as chloroquine, camostat, and lopinavir have high gastrointestinal absorption. however, in the case of lopinavir, reaching the pulmonary tissue could be delayed due to its high volume of distribution, which is associated with its tight binding to plasma proteins [ ] [ ] [ ] [ ] . on the other hand, famotidine has low gastrointestinal absorption; however, due to its high polarity, it could be given intravenously. the intravenous route of drug administration increases the probability of reaching the tissue target. thus, if administered intravenously, famotidine has a better chance to be a successful anti-sars-cov drug in the late stage of infection than lopinavir. thus, in order to develop efficient anti-sars-cov therapy, the pharmacokinetic parameters associated with drug absorption, metabolism, and distribution need to be carefully taken into account. initially, sars-cov antiviral treatment strategies were adopted based on the prior clinical approaches used for combating the viral respiratory infections, mainly the sars-cov infection. these strategies included treatments with interferon-alpha, lopinavir/ritonavir combination, ribavirin, and chloroquine [ ] [ ] [ ] . however, the outcomes of these treatments were not as good as expected, mainly due to low viral clearance and side effects. thus, multiple research groups currently focus on basic research approaches using in silico and in vitro screenings to quickly find new therapeutic alternatives [ , ] . in a pandemic situation, time is crucial. thus, adopting already existing drugs for identifying new functions of these drugs, known as drug repurposing, could decrease the time and cost of developing new therapy [ ] . the screening of large libraries of compounds already approved by the fda against the main targets in sars-cov can assure an accelerated start to clinical trials [ ] . the sars-cov replication cycle offers several steps that could be used as molecular targets for antiviral drugs. the most evaluated are viral entry, the main protease, the papain-like protease, and the rna-depended rna polymerase [ ] [ ] [ ] . the computational and in vitro analyses of these targets and specific antiviral drugs are ongoing and some were already reported [ ] . based on the results of these studies, some clinical trials of antiviral drugs against sars-cov are currently in progress. in this study, by using computational analyses, we examined the therapeutic potential of famotidine as an anti-sars-cov drug. structurally, famotidine is a member of , -thiazoles, a sulfonamide, and a member of guanidines. this histamine type receptor antagonist was never tested as an antiviral agent against coronavirus. however, as reported in the s, famotidine could inhibit hiv replication in vitro, but its mechanism of action was never clarified [ , ] . recently, in silico analysis of the sars-cov targets and their potential new inhibitors suggested that famotidine could possibly target the sars-cov main protease [ ] . thus, in this study, we analyzed if famotidine could also inhibit other proteases involved in the viral replication and, thus, similarly to ribavirin, act as a broad-antiviral agent. ribavirin, a purine nucleoside analogue demonstrated efficacy against variety of dna and rna viral infections [ , ] . the overwhelming success of ribavirin is largely related to its excellent performance in synergy with standard or pegylated interferon-alpha in chronic hepatitis c virus (hcv) infection [ , ] . our in silico analysis revealed that famotidine can interact with the sars-cov main protease with a binding free energy of − . . moreover, it could also interact with two other proteases involved in sars-cov replication, the viral plpro and human host tmprss . however, the binding free energies for these proteases (− . and − . , respectively) were higher (lower affinities) than for the main protease. nevertheless, these results indicate that famotidine could bind non-specifically to all proteases involved in virus replication with rather low binding affinity. thus, it seems that, similarly to ribavirin, the best therapeutic effect of famotidine could be achieved in combination therapy with other antiviral drugs [ , ] . the binding affinity of famotidine to the viral and host proteases could be related to its chemical structure that lacks multiple aromatic rings as compared with other inhibitors, thus showing higher binding free energy (tables - ) . these aromatic rings produce electrostatic interactions such as π-π interactions and π-alkyl interactions with the residues in the catalytic site of the enzyme that stabilize the compound and decrease the binding free energy. the molecular docking score functions are used to assign a binding free energy value that could be in relation to the binding efficacy of the molecule to the active site. in this work, we refer to this efficacy as an affinity for the binding site. however, further in vitro assays are necessary to validate this data and to establish a correlation between the affinity values and experimental drug binding constants for each enzyme. nevertheless, the high binding free energy of famotidine reflects its low target specificity. thus, to enhance the potential positive outcome of famotidine therapy, it is highly likely that it has to be administered together with other antiviral drugs. the combination of famotidine and hydroxychloroquine could be a valid approach to treat sars-cov infection. hydroxychloroquine elevates the ph of acidic intracellular organelles, such as endosomes and lysosomes, essential for membrane fusion. thus, this drug could inhibit endosome-mediated viral entry [ , ] . in addition, ph elevation impairs enzymes involved in protein posttranslational modifications. indeed, hydroxychloroquine could inhibit sars-cov cellular entry through changing the glycosylation pattern of ace receptor and spike protein [ ] . thus, hydroxychloroquine is a non-specific antiviral drug that could improve the effectiveness of other antiviral drugs when administered simultaneously. multiple studies evaluating a combination of hydroxychloroquine with other antiviral drugs are ongoing (nct , nct , nct ). for example, a small open-labeled, non-randomized clinical trial from france, evaluating hydroxychloroquine, demonstrated its positive effect in combination with azithromycin [ ] . however, larger randomized controlled clinical trials are needed to attain deeper knowledge about the positive effect of these drugs combination. another clinical trial evaluating hydroxychloroquine in combination with camostat recently began in germany (nct ). a combination of these two drugs should produce a synergistic effect by inhibiting two main mechanisms associated with the early stage of viral entry. a combination therapy of hydroxychloroquine with famotidine could possibly achieve synergy by blocking both the early infection stage with hydroxychloroquine and the late stage with famotidine. the analysis of pharmacokinetic parameters of famotidine revealed that the anti-sars-cov effect of this drug could likely be achieved only if famotidine is administered intravenously. famotidine exhibits low gastrointestinal absorption and it has a low volume of distribution; thus, if given orally, it would most likely not be able to reach the target respiratory pathway to produce an antiviral effect. therefore, drugs with low gastrointestinal absorption and low volume of distribution such as famotidine and nafamostat should be given via intravenous injection in the clinical setup. on the other hand, compounds such as hydroxychloroquine or ribavirin that have high gastrointestinal absorption and high volume of distribution [ ] should achieve therapeutic effect upon oral administration. unexpectedly, anti-hiv drug lopinavir, despite its high gastrointestinal absorption, proved to be ineffective against sars-cov infection. the poor outcome of lopinavir treatment could be related to other pharmacokinetic parameters of this drug such as high metabolism and low bioavailability [ , ] . although the volume of distribution of lopinavir upon its oral administration is reasonably small (~ . l) [ ] , this drug tightly binds to plasma proteins (over %) [ ] . thus, its plasma concentration of free drug is likely too low to reach the pulmonary tissue to target sars-cov . thus, in order to develop successful effective antiviral therapy targeting sars-cov , the pharmacokinetic parameters should be carefully taken into consideration. altogether, in this study, we showed that famotidine could be used as an antiviral agent against sars-cov , targeting proteases involved in the virus replication, mostly the main protease, as well as the viral plpro and human host tmprss . based on our analysis, it is unlikely that famotidine would produce a strong antiviral effect. it is rather expected that famotidine should be administered together with other antiviral drugs in order to produce synergistic effect to combat sars-cov infection. although our results obtained using in silico analysis need further experimental validation, the potential of famotidine as an inhibitor of proteases implicated in viral replication is already supported by the published data obtained from retrospective studies, which showed the beneficial effect of famotidine against sars-cov . in addition, one clinical trial aiming to evaluate the safety and effectiveness of famotidine against sars-cov is already ongoing. however, taking into account the pharmacokinetic parameters of famotidine, we predict that it could only be effective as an anti-sars-cov drug when given intravenously in the clinical health facility by healthcare providers. a review of coronavirus disease- (covid- ) a review of sars-cov- and the ongoing clinical trials covid- : review of epidemiology and potential treatments against expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle drug development and medicinal chemistry efforts toward sars-coronavirus and covid- therapeutics sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor a sars-cov- protein interaction map reveals targets for drug repurposing the viral, epidemiologic, clinical characteristics and potential therapy options for covid- : a review an update on the status of covid- : a comprehensive review prediction of the sars-cov- ( -ncov) c-like protease ( cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates molecular mechanism of evolution and human infection with sars-cov- . viruses designing of improved drugs for covid- : crystal structure of sars-cov- main protease m(pro) unrevealing sequence and structural features of novel coronavirus using in silico approaches: the main protease as molecular target drug repurposing: progress, challenges and recommendations expanding the frontiers of existing antiviral drugs: possible effects of hiv- protease inhibitors against sars and avian influenza a trial of lopinavir-ritonavir in adults hospitalized with severe covid- review of early and emerging options. open forum infect. dis. , , ofaa efficacy of chloroquine and hydroxychloroquine in the treatment of covid- new and future drug development for gastroesophageal reflux disease the swiss-model workspace: a web-based environment for protein structure homology modelling scalable molecular dynamics with namd antiviral activity of flavonoids present in aerial parts of marcetia taxifolia against hepatitis b virus, poliovirus, and herpes simplex virus in vitro charmm general force field: a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins procheck-a program to check the stereochemical quality of protein structures vega-an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming the retinoid and non-retinoid ligands of the rod visual g protein-coupled receptor high-throughput parallel blind virtual screening using bindsurf swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds famotidine use is associated with improved clinical outcomes in hospitalized covid- patients: a retrospective cohort study analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods structure-based design of antiviral drug candidates targeting the sars-cov- main protease treatment of severe acute respiratory syndrome case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr ul-haq, z. identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach activity profiling of sars-cov- -plpro protease provides structural framework for anti-covid- drug design potential inhibitors against papain-like protease of novel coronavirus (sars-cov- ) from fda approved drugs role of changes in sars-cov- spike protein in the interaction with the human ace receptor: an in silico analysis severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response candidate drugs against sars-cov- and covid- the effect of histamine type receptor antagonists on human immunodeficiency virus (hiv) replication: identification of a new class of antiviral agents effect of the oral anti-ulcer agent, cimetidine, on hiv type replication the structural biology of hiv- : mechanistic and therapeutic insights hiv- antiretroviral drug therapy. cold spring harb interactions of different inhibitors with active-site aspartyl residues of hiv- protease and possible relevance to pepsin estimating aqueous solubility directly from molecular structure revisiting the general solubility equation: in silico prediction of aqueous solubility incorporating the effect of topographical polar surface area plasma protein binding: from discovery to development plasma protein binding in drug discovery and development pharmacokinetic-pharmacodynamic analysis of lopinavir-ritonavir in combination with efavirenz and two nucleoside reverse transcriptase inhibitors in extensively pretreated human immunodeficiency virus-infected patients covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics the first days of novel coronavirus (sars-cov- ) outbreak: recent advances, prevention, and treatment coronavirus disease (covid- ): current status and future perspectives network-based drug repurposing for novel coronavirus -ncov/sars-cov- structural elucidation of sars-cov- vital proteins: computational methods reveal potential drug candidates against main protease, nsp polymerase and nsp helicase rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds drug targets for corona virus: a systematic review potential interventions for novel coronavirus in china: a systematic review research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases mechanism of action of ribavirin in the treatment of chronic hepatitis c treating viral hepatitis c: efficacy, side effects, and complications targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial key: cord- -fbxtj ij authors: boccaletti, valeria; cortelazzi, chiara; fantini, carolina; tognetti, elena; fabrizi, giuseppe; pagliarello, calogero; di nuzzo, sergio title: acute generalized exanthematous pustulosis following paracetamol ingestion in a child date: - - journal: pediatr allergy immunol doi: . /pai. sha: doc_id: cord_uid: fbxtj ij nan to the editor, acute generalized exanthematous pustolosis (agep) is a rare, sometimes life-threatening, cutaneous reaction more frequent in adults than in children ( ) caused by drugs in more than % of cases ( ) . a -yr-old philippino boy was admitted to primary children's medical center with malaise, temperature of °c and a -day history of pustular eruption. his mother reported that days prior he had been suffering from a sore throat and fever and was given two doses of an over-the-counter (otc) product (originating from the philippines) containing paracetamol (acetaminophen). clinical examination revealed oropharingeal erythema, red strawberry tongue, cheilitis, and a severe itchy erythema of the trunk that extended to the arms and legs, covered with hundreds of white micropustules. inguinal folds were spared, as the abdominal one. somewhere small islands of intact skin were seen intermingled with affected skin (fig. a) . the patient was hospitalized in the pediatric clinic, and paracetamol was discontinued. after h, his condition worsened and the micropustules coalesced with the pustulation spreading to involve previously unaffected skin (fig. b) . fever reached °c. the boy was given ampicillin-sulbactam mg/kg, and a bland emollient lotion was used on the body surface. on the third day, fever subsided and general conditions started to improve (fig. c) . the skin completely healed by desquamation in days. laboratory investigations showed leukocytosis ( /l) with striking neutrophilia (up to . /l) and elevated crp ( . mg/l). tests for influenza a and b virus, metapneumovirus, coronavirus, adenovirus, bocavirus, rsv, mycoplasma pneumonia, cmv, ebv, group a streptococcus pyogenes, and toxoplasma gondii were all negative. only group a streptococci on throat swab was found positive. due to the rapid healing, no skin biopsy was carried out. a diagnosis of acute generalized exanthematous pustolosis (agep) was carried out (score referring to the agep validation score of the european severe cutaneous adverse reactions study group, indicating high probable diagnosis of agep) ( ). we feel we can rule out group a streptococci as the origin of the rash because of the prompt resolution of the eruption after discontinuation of paracetamol that characterized every other drug-induced agep. three months later, the patient was submitted to patch tests containing paracetamol - % (with readings extended at and h) not using the commercialized form provided by the patient which was no more available at the moment of the test. they failed to reveal sensitization. no relapses at and months follow-up. paracetamol, one of the most used drugs in europe and in the united states, is widely used in both prescription and otc products to reduce pain and fever. the fda adverse events reporting system database, during the fourth quarter of , recorded several possible adverse events for this drug, so all products containing paracetamol appeared on the watch list (http://www.fda.gov/drugs/drugsafety/ucm .htm). the reports concerned cases of severe skin reactions (stevens-johnson syndrome, toxic epidermal necrolysis, agep). agep is caused by drugs in at least % of cases ( ); the drugs concerned more often are antibiotics (macrolides, quinolones, pristinamycin, aminopenicillins, antibacterial sulfonamides), antimycotics (terbinafine, ketoconazole), calcium channel blockers (diltiazem), analgesics, antipyretics (paracetamol), and antimalarials (hydroxychloroquine) ( ). contact sensitivity has been reported in some cases ( ) , and the role of infectious agents coxsackievirus b , cmv, parvovirus b , chlamydia, and escherichia coli has been suggested in some reports especially in children ( ) . other times, it can be attributed to factors such as food, essential oils, food supplement, and chemicals for professional use ( ) . the diagnosis is substantially clinical, supported by laboratory examination and skin biopsy. agep is supposed to represent a delayed hypersensitivity reaction in which patch tests with the suspected culprit drug may be of value. tests can be positive in up to % of the patients as reported in recent series ( ) . a broad range of cutaneous diseases causes pustular reactions such as bacterial folliculitis, subcorneal pustular dermatosis (sneddon-wilkinson disease), immunoglobulin a pemphigus, toxic epidermal necrolysis (ten), and drug reaction with eosinophilia and systemic symptoms (dress) ( ), but agep is a separate entity and must be distinguished especially from generalized pustular psoriasis (von zumbusch type): abrupt onset, rapid course, and spontaneous healing as the culprit drug is stopped together with non-recurrence and absence of personal and family history of psoriasis may help. kardaun et al. ( ) have also proposed a spectrum of differential histopathologic features. considering the usual benign and self-limited course, greater awareness of agep as a clinical entity in pediatric patients is needed to improve prompt clinical recognition and avoid useless invasive examinations such as skin biopsy and unnecessary systemic treatment: the association of pustules, hyperpyrexia, and leukocytosis can be misunderstood as acute infective disease, but a pustule not always means bacterial infection. awareness to rare but potentially serious cutaneous adverse reactions to paracetamol is needed. acute generalized exanthematous pustulosis in children acute generalised exanthematous pustulosis acute generalized exanthematous pustulosis (agep) -a clinical reaction pattern acute generalized exanthematous pustulosis mercury-induced acute generalized exanthematous pustulosis misdiagnosed as a drug-related case cutaneous adverse drug reactions" are not always drug-induced a multicenter study to determine the value and safety of drug patch tests for three main classes of severe cutaneous adverse drug reactions acute generalized exanthematous pustulosis induced by paroxetine in an adolescent girl the histopathological spectrum of acute generalized exanthematous pustulosis (agep) and its differentiation from generalized pustular psoriasis key: cord- -a kfw authors: orienti, isabella; gentilomi, giovanna angela; farruggia, giovanna title: pulmonary delivery of fenretinide: a possible adjuvant treatment in covid- date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: a kfw at present, there is no vaccine or effective standard treatment for severe acute respiratory syndrome coronavirus- (sars-cov- ) infection (or coronavirus disease- (covid- )), which frequently leads to lethal pulmonary inflammatory responses. covid- pathology is characterized by extreme inflammation and amplified immune response with activation of a cytokine storm. a subsequent progression to acute lung injury (ali) or acute respiratory distress syndrome (ards) can take place, which is often followed by death. the causes of these strong inflammatory responses in sars-cov- infection are still unknown. as uncontrolled pulmonary inflammation is likely the main cause of death in sars-cov- infection, anti-inflammatory therapeutic interventions are particularly important. fenretinide n-( -hydroxyphenyl) retinamide is a bioactive molecule characterized by poly-pharmacological properties and a low toxicity profile. fenretinide is endowed with antitumor, anti-inflammatory, antiviral, and immunomodulating properties other than efficacy in obesity/diabetic pathologies. its anti-inflammatory and antiviral activities, in particular, could likely have utility in multimodal therapies for the treatment of ali/ards in covid- patients. moreover, fenretinide administration by pulmonary delivery systems could further increase its therapeutic value by carrying high drug concentrations to the lungs and triggering a rapid onset of activity. this is particularly important in sars-cov- infection, where only a narrow time window exists for therapeutic intervention. coronaviruses (covs) are rna viruses. they may infect both humans and animals, leading to lethal and contagious diseases, such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). the recent severe acute respiratory syndrome coronavirus- (sars-cov- ) (or coronavirus disease- ) is characterized by high genetic homology with sars-cov and mers-cov [ ] , sharing with them . % and . % nucleotide identity, respectively. several studies have shown that sars-cov predominantly infects airway and alveolar epithelial cells, vascular endothelial cells, and macrophages. sars-cov- and sars-cov use the same receptor, angiotensin-converting enzyme (ace ), for infection, indicating that the same cell types are targeted and infected [ , ] . in sars-cov- infection, the early onset of a rapid viral replication may cause extensive apoptosis of epithelial and endothelial cells and vascular leakage. this frequently leads to acute lung injury and lethal inflammatory responses [ ] . although antiviral drugs, glucocorticoids, and mechanical ventilation have been used, there is no specific treatment for covid- at the moment [ ] . therefore, in the absence of a standard therapeutic intervention, the urgent treatment of pulmonary inflammation to prevent acute lung injury is needed. pulmonary formulations of anti-inflammatory drugs could represent a good option in combination with systemic antiviral drugs or glucocorticoids. in pulmonary administration, the drugs are directly carried to the airway and alveolar epithelia in high concentration, providing rapid onset of the therapeutic response with prompt relief of lung occlusion and respiratory distress symptoms. nevertheless, the drugs used by pulmonary administration in covid- should be characterized by low toxicity to avoid injury to the airway and alveolar epithelia already damaged by the viral infection. fenretinide-(n-( -hydroxyphenyl) retinamide)-is a semisynthetic derivative of all-trans-retinoic acid. it is endowed with many pharmacological features, including anti-inflammatory and antiviral activities, prevention of obesity and type- diabetes [ ] , and the well-known antitumor activity on a wide range of tumors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, its low toxicity profile has been proved in many clinical trials, also in long term treatments [ , , ] . therefore, due to its poly-pharmacology, fenretinide administration by pulmonary formulations may be expected to be protective against acute lung injury (ali)/ acute respiratory distress syndrome (ards) caused by sars-cov infection and could represent a useful tool in a multimodal therapy aimed at establishing a rapid anti-inflammatory and antiviral effect. pulmonary hyper inflammation is a common feature in covid- patients. when sars-cov is inhaled and infects epithelial cells in the lungs, an immune response is activated by the local dendritic cells that phagocytose the virus and circulate to the regional lymph nodes where they present antigens to t cells. activated t cells migrate to the lungs, where they mount the immune response. helper t cells secrete cytokines that regulate and assist the immune response; cytotoxic cd + t cells (ctls) recognize and kill the infected cells. they release pro-inflammatory cytokines, perforins, and granzymes to induce programmed cell death [ ] . however, the cytokines produced by ctls can damage uninfected as well as virus-infected cells, and their massive production may cause detrimental effects, leading to acute lung injury. sars-cov- infection is characterized by a rapid viral replication that triggers an amplified immune response with the activation of a cytokine storm. the consequent massive inflammation and increase in vascular permeability induce an abnormal accumulation of neutrophils, macrophages, inflammatory monocytes, and lymphocytes in the lung alveoli, further increasing cytokine production [ ] . in this condition, the regulation of the immune response is lost, and the cytokine storm is further activated. without therapeutic intervention, the development of ali/ards and permanent alterations in lung functions may result in dire consequences [ ] (figure ) . moreover, the circulation of cytokines to other organs can lead to multi-organ damage. figure . schematic representation of the progression of coronavirus disease- . after an incubation period, severe acute respiratory syndrome coronavirus- (sars-cov- ) starts a rapid replication in the lung airway and alveolar epithelial cells. this triggers an immune response with cytokine production, excessive inflammation, and further amplification of the immune response that triggers the cytokine storm. acute lung injury (ali)/ acute respiratory distress syndrome (ards) may arise with dire consequences. fenretinide is the most investigated retinoid due to a significant antitumor activity on a wide range of tumor types [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] combined with a favorable toxicological profile [ , , ] . besides its antitumor activity, multimodal interactions with several biological mechanisms endow fenretinide with other pharmacological properties, including anti-inflammatory activity, antiviral activity, and prevention of obesity and type- diabetes [ ] . the anti-inflammatory activity of fenretinide has been largely proved, and its mechanism of action has been characterized in different pathological conditions. in inflamed tissues, where the macrophages are characterized by a significantly higher ratio of arachidonic acid (aa) vs. docosahexaenoic acid (dha) than in normal tissues, fenretinide induced normalization of the aa/dha ratio, thus inhibiting phosphorylation of erk / and decreasing the expression of inflammatory cytokines [ ] . moreover, fenretinide's ability to downregulate the production of aa and increase the levels of dha provided attenuation of inflammation in cystic fibrosis [ ] , osteoporosis [ ] , and in a model of spinal cord injury [ ] . in a mouse model of allergic asthma, fenretinide inhibited lypopolysaccaride (lps)-induced expression of various th and th cytokines, including ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , interleukin (il)- , tnf-α, and inos [ ] . in lps-exposed mouse brain microvascular endothelial cells (bend. cells), fenretinide showed inhibitory effects on the release of pro-inflammatory cytokines (il- β, mcp , inos, and tnf-α). fenretinide may inhibit nf-κb signaling by reducing its nuclear translocation via downregulation of ikkβ and iκbα phosphorylation [ ] . this hampers the secretion of inflammatory modulators that is closely associated with the activation of nf-κb signaling. in mouse monocyte/macrophage cells infected with aggregatibacter actinomycetemcomitans, fenretinide suppressed jak-stat, pi k-akt, pkc, and downstream nf-κb signaling pathways, therefore attenuating il- β, il- , and pge proinflammatory cytokine expression [ ] . fenretinide has been demonstrated to induce reactive oxygen species (ros) generation and oxidative stress in tumor cells as a part of its cytotoxic mechanism [ , ] . in contrast, it has not increased ros in normal cells, such as fibroblasts, peripheral blood mononuclear cells [ ] , and normal and activated peripheral lymphocytes [ ] . therefore, its anti-inflammatory activity in non-tumor tissues is not counterbalanced by the ros-inducing activity, and no detrimental effect can be expected when fenretinide is applied to non-tumor tissues. the antiviral activity of fenretinide has been proved in different virus types. in a screening study using a library of pharmacologically active compounds (lopac) of bioactive compounds against the respiratory syncytial virus (rsv), fenretinide was identified as a specific blocker [ ] . in the dengue virus (denv), fenretinide has shown high activity both in vitro and in vivo [ ] . its mechanism of action includes inhibition of denv non-structural protein (ns ) recognition by host nuclear import proteins importin-α/β ; upregulation of transcripts representing the protein kinase r-like endoplasmic reticulum kinase (perk) arm of the unfolded protein response (upr); phosphorylation of eukaryotic translation initiation factor α (eif α) with consequent translation attenuation and induction of an antiviral state [ ] . fenretinide inhibited the steady-state accumulation of viral genomic rna and reduced viremia when orally administered in a murine model of denv infection. the molecular target responsible for this antiviral activity was distinct from other known inhibitors of denv and appeared to affect other members of the flaviviridae, including the west nile, modoc, and hepatitis c viruses [ ] . fenretinide inhibited the zika virus (zikv) in mammalian cell culture and reduced both viremia and brain viral burden in a murine model. this antiviral activity was correlated with a significant decrease in the abundance of zikv rna. it was found that fenretinide reduced rna synthesis without direct inhibition of the viral polymerase or chemical destabilization of membrane-associated replication complexes, rather, fenretinide acted via an indirect mechanism mediated by a host factor [ ] . in both zikv and denv, fenretinide inhibited the non-structural protein (ns ), which contributed to pathogenesis, by antagonism with the production of interferon-i (ifn-i) [ ] . as it is well-known, ifn-i promotes an early protective host mechanism against viral infection, by driving an antiviral state in non-immune cells and activating antiviral immune responses. therefore, the reestablishment of ifn-i production by fenretinide can block the early onset of viral infection [ ] . the delayed release of ifn-i is well known in sars-cov and mers-cov infections as mechanisms to hinder the body's antiviral response [ ] . the viral mechanisms of ifn-i evasion are multifaceted, including sequestering and shielding rna within double-membrane vesicles, modification of viral mrna -cap structures, and specific targeting of antiviral cellular pathways [ , ] . in sars-cov and mers-cov, inf-i production is protective only at the early stages after infection; at later time points, on the contrary, when exaggerating immune response takes place, ifn-i and inflammatory cytokines become pathogenic [ ] . no studies on the ability of fenretinide to block inf-i evasion in coronavirus have been reported so far, but, by analogy with flaviviridae, some fenretinide activity on the mechanisms regulating the inf-i evasion in coronavirus might be expected. fenretinide treatment has been found to inhibit hiv infection by perturbing cell membrane structure and inhibiting crucial early events in the hiv fusion process [ ] . fenretinide indeed is known to modify the biosynthesis and metabolism of ceramides, therefore, changing localized membrane domain organization. cell membrane modifications might alter the trafficking of virions through the endocytic pathway, thus leading to non-productive infection [ , ] . sars-cov- predominantly infects airway and alveolar epithelial cells, vascular endothelial cells, and macrophages by linking angiotensin-converting enzyme (ace ) that is abundantly expressed in the human lungs. after linkage to ace , the viral entry into the host cells is mainly mediated by endocytosis. consequently, inhibition of cell entry may be expected by treatment with fenretinide. it has been observed that the endocytic pathway of sars-cov- also involves autophagy with the formation of autophagosomes and autolysosomes [ ] . autophagy represents an essential cellular process, decreasing virus infections. beclin (becn ) is one of its key regulators. mers-cov replication results in reduced becn levels, with a consequent block of the fusion of autophagosomes and lysosomes [ ] . thus, autophagy-inducing, becn -increasing agents may have antiviral effects [ ] . fenretinide has demonstrated autophagy-inducing ability in different cell types. in human mammary carcinoma cell line mcf- , fenretinide triggered an autophagic cell death pathway mediated by an increase in beclin expression [ ] . in human pancreatic cancer cells, fenretinide induced autophagy by ros increase [ ] . in gastric carcinoma hcg , autophagy was stimulated by the accumulation of dihydroceramide induced by fenretinide [ ] . another target for antiviral activity is the akt /mtor pathway. indeed, a kinome analysis of mers-cov-infected cells has detected phosphorylation changes of several regulatory kinases, including akt and mtor. these changes have been correlated to autophagy [ , ] . the pharmacological use of akt -and mtor-pathway inhibitors has shown promise for antiviral therapy against several viruses, including mers-cov [ ] . the inhibitory effect of fenretinide in akt and mtor pathways has been reported in tumor cells [ ] and in monocytes/macrophages [ , ] . all these findings clearly show the involvement of fenretinide in multiple pathways, controlling viral replication. therefore, even in the absence of specific studies on sars-cov- , an indirect antiviral activity of fenretinide may be supposed through the induction of an "antiviral environment", hindering supportive steps of viral replication, both at a cellular level and in the infected tissues. fenretinide has been well-tolerated in many clinical trials aimed at evaluating its antitumor activity in pediatric and adult cancers. oral administrations of fenretinide in corn oil gelatin capsules ( mg/m for days) or in a lipid matrix (lym-x-sorb, mg/kg for days) have been well-tolerated at high doses and for protracted periods of time. no significant hematological alterations or dose-limiting side effects have been obtained [ , ] in these studies. many other clinical trials with fenretinide have confirmed its tolerability [ , , ] . a phase ii study reported thrombocytopenia, anemia, hepatosplenomegaly in patients with myelodysplastic syndromes orally treated with fenretinide at mg/day for weeks and escalated to mg/day for other weeks [ ] . thrombocytopenia was also observed in a phase i study in some patients with hematologic malignancies after administration of fenretinide ( mg/m ) as a continuous intravenous infusion for five consecutive days [ ] . in a very recent study in mice, oral fenretinide led to splenomegaly and increased the circulating leukocytes [ ] . a trial aimed at evaluating the effect of fenretinide as a chemopreventive agent included patients that received a -year fenretinide oral treatment. endpoints considered for safety assessment indicated that fenretinide was endowed with good tolerability. indeed, the most common adverse events were diminished dark adaptation ( . %) and dermatologic disorders ( . %). less common events were gastrointestinal symptoms ( . %) and disorders of the ocular surface ( . %). these symptoms occurred during fenretinide treatment and recovered over time [ , ] . in covid- patients, the rapid evolution of the disease requires prompt treatment because only a narrow time window exists for therapeutic intervention. in this regard, pulmonary drug delivery offers the advantage of carrying the bioactive molecule in direct contact with the pathological lung epithelia, thus ensuring a rapid onset of the therapeutic response. high local drug concentrations may be easily obtained by pulmonary administration with a concomitant increase of the pharmacological effect but without the side effects elicited by other administration routes. drugs administered by enteral or parenteral routes, indeed, need to reach the blood circulation to be distributed to tissues and organs and enter the pathological site. this general distribution may provide extensive side effects, particularly when high drug administration doses are required to achieve a therapeutically active concentration of the drug in the pathological site. the increased pharmacological effect provided by high drug local concentrations and the decreased toxicity due to lack of a systemic distribution may improve the overall therapeutic efficiency of the drugs administered by pulmonary route, as demonstrated in several respiratory diseases, such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease [ ] . the inhalation devices play a crucial role in the effectiveness of pulmonary drug administration. the most common devices are nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers), metered-dose inhalers, and dry powder inhalers [ ] . the selection of the inhalation device depends on the physicochemical characteristics of the drug and its formulation. liquid formulations are administered by nebulizers and metered-dose inhalers, and solid formulations by dry powder inhalers. in each case, the inhalation device provides aerosol particles whose size can control the extent of inhaled drug accumulation and the site of drug deposition within the airways. smaller particles achieve a greater total drug accumulation in the lungs and farther distal airway penetration compared with larger particles. particles smaller than µm in diameter may flow in the airstream beyond the retro-pharynx and reach the trachea. particles of - µm in diameter are deposited in the upper respiratory tract at the level of the trachea and tracheal bifurcation. particles smaller than µm in diameter deposit in the lower airway and alveolar epithelia [ , ] . then, the modulation of intrapulmonary deposition through the control of the aerosol particle size can appreciably improve the inhalation drug therapy (figure ). the pulmonary administration of drugs mainly provides a local therapy but may also provide a systemic therapy when the physicochemical characteristics of the drugs can support their absorption through the alveolar epithelium at extents suitable to elicit systemic effects. indeed, the large surface area, extensive vascularization, and single-cell barrier in the alveoli make the lungs an appropriate portal for the systemic absorption of molecules, such as insulin, human growth hormone, etc. [ ] . pulmonary delivery of fenretinide could be a valuable tool in covid- due to the possibility of obtaining a very high drug concentration in the airway and alveolar epithelia, thus triggering a rapid onset of local anti-inflammatory response. at the same time, the ability of fenretinide to induce an "antiviral environment" could further enhance its therapeutic efficacy (figure ). in order to be effective, pulmonary fenretinide formulations should provide aerosol particle size smaller than µm, for deposit in the lower airway and alveolar epithelia, where the infection process is amplified by the extensive vascularization. moreover, after deposition, they should trigger a rapid drug release to speed up the onset of the therapeutic activity. such formulations require fenretinide solubilization in an aqueous phase, and the adequate solubilization degree to provide high concentrations of the bioavailable drug, in the lungs, after inhalation. unfortunately, the hydrophobic character of fenretinide strongly hinders its aqueous solubilization. moreover, the possibility to use solubilizing agents, such as tensides or water-mixable co-solvents, is severely restricted, by tolerability issues, in the formulations destined to inflamed lungs. highly tolerated, aqueous fenretinide formulations have been obtained by complexation with cyclodextrins [ ] or encapsulation in nanomicelles [ ] . complexation with -hydroxypropyl beta-cyclodextrin has increased fenretinide aqueous solubility from . mg/ml (pure drug) to . mg/ml (complex). the aqueous formulation of the complexed drug, administered by the parenteral route, was well-tolerated and increased the drug bioavailability and antitumor activity in mouse models of different tumor types [ ] . nanoencapsulation in phosphatidylcholine-glyceryltributyrate nanomicelles has increased fenretinide's aqueous solubilization up to . mg/ml (nanoencapsulated drug). the intravenous administration of the nanomicelles in mice bearing tumor xenografts showed enhanced drug bioavailability and antitumor activity [ ] . moreover, high tolerability was demonstrated by the absence of adverse effects after repeated administrations and for protracted periods of time. therefore, the in vivo tolerability and the ability to provide high fenretinide solubilization levels suggest that complexation with cyclodextrins [ ] or encapsulation in nanomicelles [ ] can be valuable means for preparation of safe and efficient pulmonary fenretinide formulations. there is presently no vaccine or documented specific, standard treatment against covid- . most of the potential drugs are being investigated for safety and efficacy in sars-cov- infection, but, until now, no drugs are validated to have significant efficacy in the clinical treatment of covid- patients in large-scale studies. current treatments are using drugs that were originally designed for other pathologies and have been repurposed for covid- trials. these include antiviral and immunomodulating drugs designed to boost the innate immune response or to inhibit the inflammatory processes, causing lung injury. drugs for symptomatic control are also employed. antiviral drugs act on the coronavirus by direct mechanisms, inhibiting key viral enzymes responsible for genome replication or hindering viral entry to human cells. remdesivir is the most promising, among the antiviral drugs. it exhibits broad-spectrum antiviral activity against rna viruses. in previous studies, it showed antiviral activities, both in vitro and in vivo, against different coronaviruses, including sars-cov and mers-cov [ , ] . in a recent study, remdesivir inhibited sars-cov- in vivo [ ] . ivermectin is an anti-parasitic agent, which has proved to exert antiviral activities toward both hiv and dengue virus [ ] . recently, an in vivo study put in evidence its capability to reduce viral rna up to fold in sars-cov- infection [ ] . lopinavir/ritonavir are protease inhibitors. they are used in combination in the treatment of patients with hiv infection. several clinical, preclinical, and in vitro studies performed on sars-cov and mers-cov proved that the lopinavir/ritonavir combination was effective against these viruses [ ] [ ] [ ] . hydroxychloroquine is active against malaria as well as autoimmune diseases (such as rheumatoid arthritis, lupus erythematosus). it has been recently investigated as a potential broad-spectrum antiviral drug for its ability to increase the endosomal ph, which prevents virus-cell fusion [ ] . hydroxychloroquine has been shown to specifically inhibit the replication of sars-cov by interfering with the glycosylation of its cellular receptor ace [ ] . recent in vitro studies revealed its ability to effectively reduce the viral copy number of sars-cov- [ ] . immunomodulating drugs able to stimulate innate antiviral immune responses are now repurposed for the treatment of covid- . natural killer (nk) cells represent a highly specialized lymphoid population of the innate immune system with potent activity against virus-infected cells. migration of nk cells and macrophages to the lungs has been demonstrated to play a major role in the clearance of sars-cov [ ] . indeed, the innate response itself can control sars-cov infection, without the help of ctls and antibodies, particularly in the early stages of the infection process. type i interferons are secreted by virus-infected cells and represent a host protective mechanism. recombinant interferons, used alone or in combination with other drugs, have provided a broad-spectrum antiviral activity against hcv, respiratory syncytial virus, sars-cov [ ] , and mers-cov [ ] . recent trials are assessing their safety and efficacy in covid- . drugs interfering with the il- pathway are used to attenuate the inflammatory response in covid- [ ] . elevated il- levels are predictive of poor prognosis in patients with ards [ ] . the pathway of il- signaling occurs through il- receptors, which are expressed predominantly on neutrophils, monocytes, macrophages, and some lymphocytes [ ] . tocilizumab and siltuximab are monoclonal antibodies against il- . tzls- is an anti-il- receptor antibody, and sarilumab an il- receptor antagonist. they all are designed to inhibit the binding of interleukin- to its receptors, thus alleviating cytokine release syndrome. thalidomide has been repurposed for the treatment of covid- due to its anti-angiogenic, anti-inflammatory, and anti-fibrotic activity. its anti-inflammatory activity is mainly based on the inhibition of tnf-α production. thalidomide has shown promise in the treatment of multiple inflammatory diseases, such as lupus erythematosus and crohn disease [ ] . preclinical studies proved that thalidomide was effective in treating mice infected with influenza virus h n. it decreased the infiltration of inflammatory cells and inhibited the production of inflammatory cytokines [ ] . recent studies are evaluating its efficacy in sars-cov- [ ] . lenalidomide is a derivative of thalidomide. it is currently used in the maintenance therapy of multiple myeloma due to its immunostimulatory effect on nk cell number and function combined with an acceptable toxicity profile. lenalidomide has been proven to limit the amount of pro-inflammatory cytokines, such as tnf-α, il- , il- , il- , and increased il- and ifnγ [ , ] . many studies have demonstrated the lenalidomide's strong anti-angiogenic activity [ ] [ ] [ ] [ ] . with respect to thalidomide, lenalidomide is expected to provide a superior activity and decreased side effects in covid- treatment. glucocorticoids have been used in sars-cov and mers-cov infections to restrain lung inflammation and immune responses [ ] [ ] [ ] . their use has provided side effects, such as secondary bacterial infections, osteoporosis, and prolonged viral clearance. glucocorticoids, such as methylprednisolone, are now under evaluation in covid- for effectiveness and safety [ ] . pulmonary administration of fenretinide, in combination with the drugs currently used in covid- treatment, could represent a new, effective tool to improve the efficacy of the single drugs and produce a strengthened overall pharmacological response. fenretinide is particularly suitable to be used in combination because its poly-pharmacology may reinforce the activity of several drugs, and its high tolerability may enable large adjustments of the administered dose without toxicity concerns. the combination of fenretinide with the antiviral drugs used in covid- is expected to improve the treatment efficacy due to the ability of fenretinide to induce an "antiviral environment" that can behave as an adjuvant to the specific antiviral activity of the drugs. in combination with the immunomodulating drugs, fenretinide may contribute to enhance the innate immune response due to its stimulating effect on nk cells' proliferation and cytotoxicity [ , ] . moreover, its anti-inflammatory activity, largely demonstrated in several models, may boost the attenuation of the inflammatory response induced by the immunomodulating drugs. in particular, the ability of fenretinide to decrease il- [ , ] should improve the efficacy of the drugs blocking the il- pathway, such as tocilizumab, siltuximab, sarilumab, tzls- . the combinations of thalidomide or lenalidomide with fenretinide are expected to be very effective due to the multiplicity of mechanisms involved in the anti-inflammatory activity of both types of drugs. particularly, the decrease of tnf-α [ , ] and the antiangiogenic effect [ , ] provided by fenretinide, added to the same effects provided by thalidomide and lenalidomide, should boost the anti-inflammatory response elicited by the combination. fenretinide and lenalidomide were evaluated, by intravenous administration, in a mouse model of neuroblastoma. high tolerability and antitumor activity were obtained with this combination [ ] . in regard to the administration route, the drugs to combine with the pulmonary fenretinide are expected to be administered by enteral or parenteral routes, depending on the form of the formulations available on the market. however, the pulmonary administration route would be the best option because, as already reported for fenretinide, the local drug administration in the pathological site would provide a rapid onset of the therapeutic response. moreover, in addition to local therapy, systemic therapy may also be obtained if the drug absorption through the alveolar epithelium can take place at a suitable extent. the utility of fenretinide anti-inflammatory activity, associated with its high tolerability, has been repeatedly demonstrated in lung-related pathologies, such as cystic fibrosis, allergic asthma, and chronic lung infections. its antiviral activity in the zika virus, dengue virus, respiratory syncytial virus, hepatitis c virus, and hiv has been largely proved and attributed to several mechanisms of action. although there is no direct evidence of fenretinide application in covid- , the multiple inhibition mechanisms demonstrated in the studied viruses and the involvement of fenretinide in multiple pathways controlling viral replication strongly suggest that fenretinide could provide an indirect antiviral activity towards sars-cov- , as well as other virus types, by means of the induction of an "antiviral environment" hindering infection. therefore, both the anti-inflammatory activity and the ability to induce an "antiviral-environment" indicate that fenretinide may have supportive adjuvant utility in treating covid- . fenretinide administration by means of pulmonary formulations may further improve its therapeutic value by providing high drug concentrations in the pathological lung epithelia and a fast onset of the drug activity, which is particularly important in sars-cov- infection, where only a narrow time window exists for therapeutic intervention. moreover, the pulmonary administration of fenretinide, in combination with the drugs that are currently used in sars-cov- infection, could represent a new, effective tool in covid- treatment. the authors declare no conflict of interest. identification of a novel coronavirus causing severe pneumonia in human single-cell rna expression profiling of ace , the receptor of sars-cov- a pneumonia outbreak associated with a new coronavirus of probable bat origin understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools clinical features of patients infected with novel coronavirus in the mechanisms of fenretinide-mediated anti-cancer activity and prevention of obesity and type- diabetes clinical development of fenretinide as an antineoplastic drug: pharmacology perspectives phase i trial and pharmacokinetics of fenretinide in children with neuroblastoma phase i trial of fenretinide delivered orally in a novel organized lipid complex in patients with relapsed/refractory neuroblastoma: a report from the new approaches to neuroblastoma therapy (nant) consortium. pediatr. blood cancer a phase ii study of fenretinide in patients with hormone refractory prostate cancer: a trial of the cancer therapeutics research group phase ii trial of fenretinide (nsc ) in patients with recurrent small cell lung cancer fifteen-year results of a randomized phase iii trial of fenretinide to prevent second breast cancer phase ii study of oral capsular -hydroxyphenylretinamide ( -hpr/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: a report from the children's oncology group high plasma levels of fenretinide ( -hpr) were associated with improved outcome in a phase ii study of recurrent ovarian cancer: a study by the california cancer consortium phase ii study of fenretinide (nsc ) in adults with recurrent malignant gliomas: a north american brain tumor consortium study phase ii trial of fenretinide in advanced renal carcinoma. investig. new drugs phase i trial of oral fenretinide in children with high-risk solid tumors: a report from the children's oncology group (ccg ) phase i clinical trial of fenretinide (nsc ) in advanced solid tumors quis custodiet ipsos custodes? regulation of cell-mediated immune responses following viral lung infections new insights into the immune molecular regulation of the pathogenesis of acute respiratory distress syndrome fenretinide corrects the imbalance between omega- to omega- polyunsaturated fatty acids and inhibits macrophage inflammatory mediators via the erk pathway cystic fibrosis fatty acid imbalance is linked to ceramide deficiency and corrected by fenretinide fenretinide prevents the development of osteoporosis in cftr-ko mice fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of allergic asthma fenretinide attenuates lipopolysaccharide (lps)-induced blood-brain barrier (bbb) and depressive-like behavior in mice by targeting nrf- signaling fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by aggregatibacter actinomycetemcomitans preferential eradication of acute myelogenous leukemia stem cells by fenretinide reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of t-cell lymphoid malignancies implication of mitochondria-derived ros and cardiolipin peroxidation in n-( -hydroxyphenyl)retinamide-induced apoptosis replication-competent influenza virus and respiratory syncytial virus luciferase reporter strains engineered for co-infections identify anti-viral compounds in combination screens a nuclear transport inhibitor that modulates the unfolded protein response and provides in vivo protection against lethal dengue virus infection novel dengue virus inhibitor -hpr activates atf independent of protein kinase r-like endoplasmic reticulum kinase and elevates levels of eif α phosphorylation in virus infected cells the bioactive lipid -hydroxyphenyl retinamide inhibits flavivirus replication anti-viral activity of n-( -hydroxyphenyl) retinamide ( -hpr) against zika virus nuclear import inhibitor n -( -hydroxyphenyl) retinamide targets zika virus (zikv) nonstructural protein to inhibit zikv infection the many faces of the flavivirus ns protein in antagonism of type i interferon signaling ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes to sense or not to sense viral rna-essentials of coronavirus innate immune evasion antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology fenretinide inhibits hiv infection by promoting viral endocytosis proc. natl. acad. sci targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid- skp attenuates autophagy through beclin -ubiquitination and its inhibition reduces mers-coronavirus infection autophagy and viruses: adversaries or allies? fenretinide induces autophagic cell death in caspase-defective breast cancer cells cytotoxic responses to n-( -hydroxyphenyl)retinamide in human pancreatic cancer cells dihydroceramide delays cell cycle g /s transition via activation of er stress and induction of autophagy anti-viral potential of erk/mapk and pi k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis a pharmacologic target for autophagy regulation a new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors the chemopreventive retinoid hpr impairs prostate cancer cell migration and invasion by interfering with fak/akt/gsk beta pathway and beta-catenin stability pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite -oxo-fenretinide oral fenretinide in biochemically recurrent prostate cancer: a california cancer consortium phase ii trial results of a phase i-ii study of fenretinide and rituximab for patients with indolent b-cell lymphoma and mantle cell lymphoma phase ii trial of fenretinide [n-( -hydroxyphenyl) retinamide] in myelodysplasia: possible retinoid-induced disease acceleration phase i study of fenretinide delivered intravenously in patients with relapsed or refractory hematologic malignancies: a california cancer consortium trial fenretinide treatment accelerates atherosclerosis development in apoe-deficient mice in spite of beneficial metabolic effects safety of the synthetic retinoid fenretinide: long-term results from a controlled clinical trial for the prevention of contralateral breast cancer pulmonary administration: strengthening the value of therapeutic proximity to study the attitudes, beliefs and perceptions regarding the use of inhalers among patients of obstructive pulmonary diseases and in the general population in punjab systematic review of errors in inhaler use: has patient technique improved over time? chest drug delivery to the lungs: challenges and opportunities de fougerolles, a. nanobodies® † nanobody is a registered trademark of ablynx nv. as inhaled biotherapeutics for lung diseases a novel nanomicellar combination of fenretinide and lenalidomide shows marked antitumor activity in a broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro ivermectin is a specific inhibitor of importin alpha/beta-mediated nuclear import able to inhibit replication of hiv- and dengue virus the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro treatment with lopinavir / ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavir-ritonavir in adults hospitalized with severe covid- effects of chloroquine on viral infections: an old drug against today's diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread positive rt-pcr test results in patients recovered from covid- cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd + t cells are important in control of sars-cov infection treatment of sars with human interferons il- trans-signaling via the soluble il- receptor: importance for the pro-inflammatory activities of il- interleukin- displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury the il- /sil- r complex as a novel target for therapeutic approaches thalidomide-induced teratogenesis: history and mechanisms anti-inflammatory effect of thalidomide on h n influenza virus-induced pulmonary injury in mice a review of sars-cov- and the ongoing clinical trials lenalidomide acts as an adjuvant for hcv dna vaccine anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide mechanism of action of lenalidomide in hematological malignancies antiangiogenic in vivo effect of lenalidomide (cc- ) in myelodysplastic syndrome with del ( q) chromosome abnormality and its relation to the course of disease orally administered lenalidomide (cc- ) is anti-angiogenic in vivo and inhibits endothelial cell migration and akt phosphorylation in vitro the anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions systematic review of treatment effects corticosteroid therapy for critically ill patients with middle east respiratory syndrome adjunctive glucocorticoid therapy in patients with septic shock effects of n-( -hydroxyphenyl)-retinamide on the number and cytotoxicity of natural killer cells in vitamin-a-sufficient and -deficient rats retinoids, breast cancer and nk cells anti-angiogenic properties of chemopreventive drugs: fenretinide as a prototype fenretinide as an anti-angiogenic agent in neuroblastoma key: cord- - rlya w authors: castells, mariana c. title: drug allergy labeling and delabeling in the coronavirus disease era: what is important and what do we need to know date: - - journal: ann allergy asthma immunol doi: . /j.anai. . . sha: doc_id: cord_uid: rlya w nan drug allergy labeling and delabeling in the coronavirus disease era what is important and what do we need to know at the time of this writing, april , , more than . million people worldwide are infected and more than , have died because of coronavirus disease (covid- ) induced by the novel severe acute respiratory syndrome coronavirus infection, and this figure will surely multiply in the next few weeks according to dr anthony fauci, head of the national institute of allergy and infectious diseases. severe acute respiratory syndrome coronavirus uses angiotensin-converting enzyme- (ace- ) (a type i transmembrane metallocarboxypeptidase) as its preferred entry receptor. the expression of ace- is high in alveolar and epithelial tissues in the lungs and the gastrointestinal tract. drugs which affect the expression of ace- (such as ace inhibitors and angiotensin receptor blockers) are being investigated as possible risk factors for the severity of coronavirus disease . nonsteroidal antiinflammatory drugs are being similarly investigated. clinical trials with azithromycin and hydroxychloroquine, antiretroviral drugs, and antieinterleukin- are ongoing in an attempt to improve disease outcomes before a vaccine can be available. this provides a glimpse of the complexities of this disease and reveals the importance of identifying candidate drugs for clinical trials that may save lives. it follows in importance to identify patients with allergy who are at risk, if treated, and who may need desensitization. understanding the mechanisms of drug allergy is key, given that the classification of drug hypersensitivity continues to expand. cytokine stormelike reactions with elevated interleukin- can be seen in patients treated with chemotherapy and monoclonal antibodies and are now part of a broader definition of anaphylaxis, allowing for better management and treatment options. the timing of this issue of the annals is highly relevant given that it is dedicated to broadening our understanding of the scope of drug allergy in the general population. various tools can be used in personalized medicine to confirm or refute specific drug allergy status through delabeling. these standardized diagnostic interventions can allow both children and adults to safely take the drug for which they had been previously labeled as allergic, thereby resulting in the removal of this label. the topics covered in this issue provide the necessary and updated knowledge for all allergists involved in labeling and delabeling procedures, aiming to broaden drug choices and treatment options for patients in this unknown world of covid- pandemic and other disease states. our first question is: who is labeled as drug allergic in the general population and what can be done to uncover true drug allergy? a review by macy provides data on a large cohort of more than million members of kaiser health care, with % reported to have a drug allergy and more than % having antibiotic allergy. in this drug allergy cohort, twice as many patients are females. age of more than years and increased body mass index were found to be associated with drug allergy. the review also discusses whether drug allergy and hypersensitivity are due to increased use, given that countries with lower rates of antibiotic use have a lower prevalence of antibiotic allergy. inappropriate use of antibiotics is still high in the setting of dental procedures. target populations for receiving a drug allergy label include the following: ( ) children with approximately , visits to the emergency department reported annually for adverse drug events with penicillins, cephalosporins, and sulfamethoxazole-trimethoprim as the most frequent medications; and ( ) hospitalized patients with cancer, of whom % have a label of antibiotic allergy. what are the tools for the labeling or delabeling of a drug allergy? for individuals with penicillin-associated anaphylaxis, penicillin skin testing with penicilloyl-polylysine before oral amoxicillin mg oral challenge (if skin test negative) is the avenue proposed by the author; however, with the lack of minor determinants, sensitization is not addressed. for patients with a history of benign cutaneous reactions, single oral dose of amoxicillin is recommended. what are the benefits and dangers of a drug allergy label? in a review by solenki, the author reviewed self-reported penicillin allergy, which accounts for at least % of the population, and observed that, among these individuals claiming to be allergic to penicillin, more than % are not truly allergic and can tolerate penicillin. these discrepancies were reviewed, which included initial mislabeling at the time of the clinical event, such as associated symptoms of viral infections, including urticaria and gastrointestinal adverse effects of antibiotics. many drug allergies are not long-lived and the natural resolution of penicillin allergy was reviewed. the author validated current diagnostic tools for the diagnosis of penicillin, cephalosporins, and other antibiotics allergies. multicentered clinical trials are needed to validate skin testing predictive values and to assess the value of new tools, such as specific immunoglobulin e and basophil activation test. how to detect children with true penicillin allergy? vyles et al provide a review that describes that most allergies in pediatric patients are self-reported and often clinically inconsistent with true allergy. the rate of parent-reported adverse drug reactions ranges from % to %, and most of these so-called allergic reactions are attributed to beta-lactam antibiotic derivatives, anti-inflammatory drugs, and other antibiotics. nonimmediate rashes occurring after several days of treatment are the most frequently reported symptoms. although skin testing, followed by oral challenge, is the safest way to identify true immunoglobulin emediated allergy in children with high-risk allergy symptoms, risk stratification and direct oral challenge of low-risk patients is becoming a standard. of interest are studies, which reported that both parents and physicians were reluctant to utilize penicillin class antibiotics after the penicillin allergy label was removed because of fear of an allergic reaction. the authors concluded that current and future efforts should focus on preventing penicillin allergy labels that can carry over into adulthood, providing education and decision support in the electronic medical record, and testing low-risk drug administration strategies in low-risk patients. integrating penicillin allergy management into stewardship efforts with the government and third-party payer incentives should be the long-term goal for penicillin allergy delabeling at the population level. what is the current understanding of drug hypersensitivity and allergic reactions? jakubovic et al provide a broad and updated review of the current knowledge by reviewing the classical model of drug hypersensitivity reactions and comparing this with the current and more customized classification based on phenotypes, endotypes, and biomarker profiles. this approach allows for the classification of reactions to chemotherapy drugs, monoclonal antibodies, and new small molecules. complementing the gell and coombs classification drug allergy phenotypes allows for the description of classical and atypical clinical symptoms, such as cytokine stormelike manifestations in the context of drug exposure, timing, and severity. the endotypes look at the mechanisms, and also the molecular and cellular targets, whereas biomarkers are used as diagnostic tools. biomarkers such as skin testing, tryptase, and basophil activation test provide the signature for the different endotypes. as more mechanisms of drug allergy are uncovered and new biomarkers become available, they can be incorporated into this flexible classification, guiding clinicians toward an optimal approach for patient labeling or delabeling, treatment, and management. what is the evidence for, and how can recommendations be made for labeling and delabeling? are there models for these recommendations? shaker et al, on behalf of the joint task force for allergy practice parameters (jtfpp), provided a review of the recommendations for anaphylaxis treatment. the authors introduced grading of recommendations assessment, development, and evaluation (grade), a new method of evidence appraisal and translation, which has emerged as a leading approach to anaphylaxis guidelines development. grade creates explicit processes for evaluating the broad evidence based on a specific, structured, and answerable clinical question. randomized controlled trials begin the evaluation process as high certainty, whereas observational studies begin as low certainty. evidence may be downgraded depending on the following considerations: ( ) the risk of bias, ( ) imprecision, ( ) inconsistency, ( ) indirectness, and ( ) publication bias. through this methodology, evidence and certainty are clearly and simply described as "very low," "low," "moderate," or "high." the jtfpp has been producing grade guidelines since , and the jtfpp anaphylaxis grade is focused on the practice of anaphylaxis prevention through identification and mitigation of risk factors for biphasic anaphylaxis and evaluation of the use of supplemental glucocorticoid and/or antihistamine premedication for immunotherapy, radiocontrast media and chemotherapy. in contrast to grade, good practice statements include the administration of epinephrine as first-line treatment for uniphasic and/or biphasic anaphylaxis. a good practice statement may be used when there is a high certainty that a recommendation will be more beneficial than harmful, though there is little direct evidence. grade is prescriptive, explicit, and transparent and requires expert judgment and consensus of guideline groups as evidence is evaluated and translated into recommendations. what is the practical approach to drug allergy labeling and delabeling? louisias and wickner provided a review on the playground and available tools for drug allergy delabeling. the authors indicated that large-scale drug allergy delabeling is influenced by multiple factors, such as changing cultural moors, easily adapted tools to delabel, and electronic health record (ehr) crosstalk. current functionalities of ehrs' drug allergy sections are often at odds with providing reliable, updated, expert, safe, and affordable care. they reported that up to % of patients had at least drug allergy listed in their ehr, and many had up to ; nobody removed duplicates or delabel drugs with nonallergic symptoms. the authors indicated the need to uncover the integral components of drug allergy delabeling programs that can be tailored and disseminated, incentivized by insurance companies and hospitals, and standardized nationally. one study estimated penicillin allergy delabeling programs could have cost savings of $ , per year in tertiary care center pediatric emergency departments, thus underscoring the economic incentives of delabeling. allergists need to challenge every drug allergy label and to recognize drug allergy and hypersensitivity symptoms using the new framework of phenotypes and endotypes supported by biomarkers. providing risk stratification is key to safe delabeling procedures and to help provide management options including desensitization to patients who are truly allergic. minimizing inappropriate use, recording accurate intolerances, delabeling whenever possible, and adhering to essential elements of effective stewardship will solve the antibiotic allergy epidemic. renin-angiotensin-aldosterone system inhibitors in patients with covid- coronavirus disease (covid- ): epidemiology, virology, clinical features, diagnosis, and prevention hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial nih clinical trial of remdesivir to treat covid- begins: study enrolling hospitalized adults with covid- in nebraska tocilizumab in covid- pneumonia (tocivid- ) penicillin allergy diagnosis and management of anaphylaxis in precision medicine hypersensitivity reactions to therapeutic monoclonal antibodies: phenotypes and endotypes addressing the epidemic of antibiotic "allergy" over-diagnosis doctor, i am allergic to penicillin children with reported penicillin allergy: public health impact and safety of delabeling drug hypersensitivity in the fast lane: what clinicians should know about phenotypes, endotypes, and biomarker making the grade in anaphylaxis management: toward recommendations integrating values, preferences, context, and shared decision making drug allergy delabeling in the clinical setting: an all-handson-deck opportunity division of rheumatology, immunology, and allergybrigham and women's hospital boston, massachusetts mcastells@bwh.harvard.edu key: cord- - ifwtlp authors: smith, sherri a.; waters, nigel j. title: pharmacokinetic and pharmacodynamic considerations for drugs binding to alpha- -acid glycoprotein date: - - journal: pharm res doi: . /s - - -x sha: doc_id: cord_uid: ifwtlp according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, and as such the importance of plasma protein binding primarily resides in its impact on pharmacokinetics and pharmacodynamics. of the major plasma proteins, alpha- -acid glycoprotein (aag) represents an intriguing one primarily due to the high affinity, low capacity properties of this protein. in addition, there are marked species and age differences in protein expression, homology and drug binding affinity. as such, a thorough understanding of drug binding to aag can help aid and improve the translation of pharmacokinetic/pharmacodynamic (pk/pd) relationships from preclinical species to human as well as adults to neonates. this review provides a comprehensive overview of our current understanding of the biochemistry of aag; endogenous function, impact of disease, utility as a biomarker, and impact on pk/pd. experimental considerations are discussed as well as recommendations for understanding the potential impact of aag on pk through drug discovery and early development. according to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, whether it is the intended pharmacological target, or action at an undesired site with potential toxicological consequences. the importance of plasma protein binding primarily resides in its impact on pharmacokinetic properties such as clearance (cl) and volume of distribution (v ss ), with serum albumin, lipoproteins and alpha- acid glycoprotein (aag) being the major proteins involved in sequestering drugs in plasma ( ) . aag also known as orosomucoid (orm), is a member of the acute phase protein (app) family, first described in ( ) . it has a single polypeptide chain consisting of amino acids with five glycan chains, accounting for~ % of its total molecular weight (~ - kda) ( , ) . aag is formed primarily in the liver and circulates from . to . mg/ml in the plasma of healthy humans ( ) . levels in healthy animals are generally lower compared to healthy humans (table i) . in most disease states including inflammation, infection, and cancer, aag levels increase from to -fold in humans ( ) , and show a much broader fold of induction in animals from to -fold depending on animal species and disease (table i) . while the biological role of aag remains unclear, it has been demonstrated to regulate immunity and play a role in both pro-and anti-inflammatory response ( , ) . aag has long been used as a clinical biomarker, and the potential to expand its - c a n c e r . ± . ( . - . ) infection . ± . ( . - . ) ( ) application for disease diagnosis, prognosis, and characterization has grown given the recent advances in proteomics and high resolution mass spectrometry ( ) ( ) ( ) ( ) . while aag represents a relatively small portion (~ - %) of the total plasma proteins, compared to~ % composition of albumin, it can play a significant role in drug binding and pharmacokinetics (pk) ( ) . aag is considered a high affinity/low capacity plasma protein, whereas albumin is considered low affinity/high capacity. aag is a highly acidic protein with a very low isoelectric point (pi) ranging from . to . ( ) . this property enables aag to bind mainly basic drugs (i.e. lidocaine, propranolol, verapamil) but it may also bind to neutral lipophilic molecules (i.e. steroid hormones) and to acidic drugs (i.e. phenobarbital), whereas albumin is mostly implicated in binding to the latter charge type ( , ) . most drugs bind to both plasma proteins with varying degrees of affinity. the extensive and variable sialylation of aag is what drives the low and wide pi range, a property that can impact drug affinity, and ultimately pk ( ). since aag levels increase in most disease states ( ) , drugs with a high affinity may demonstrate higher binding (lower fraction unbound, f u ) and altered pk properties (e.g. lower total cl), lower v ss . given the known species differences in aag abundance and drug affinity there is a growing body of work where pk in preclinical species did not accurately predict pk in human, and several of these case studies are discussed herein. incorporating the ontogeny of aag may also enable more accurate predictions of pk in neonate and infant patients ( ) . we provide a rationale for testing the extent and affinity of drug binding to aag and albumin in the drug discovery process to aid in prospective human pk prediction efforts (fig. ) . research is still lagging in the characterization of higher species aag which could help better predict pk in human. furthermore, there are experimental factors that are emerging as critical to the accurate determination of aag-drug binding in vitro. these aspects are also discussed in this review. albumin, aag, and lipoproteins are considered the most relevant plasma proteins in terms of drug binding ( ). the range of albumin levels is slightly lower in animals compared to human. while levels of aag are also relatively lower in most animals compared to human in the healthy state, levels are much more variable in the diseased setting as described below and in table i . human albumin and aag levels in table ii provide units of mg/ml and μm to serve as a quick reference when considering stoichiometry with drug concentrations, assuming a single drug binding site model. generally, in rodents ( . - . mg/ml) and mini-pigs ( . - . mg/ml) aag values are lower compared to human (table i ). an exception to this generalization was reported in mice, however no explanation was suggested by the authors ( ) . in dogs, aag demonstrates highly variable levels ( . - mg/ml) ( ) , though generally lower mean values ( . - . mg/ml) have been reported ( ) ( ) ( ) . there is little characterization of monkey aag in the literature, though it has also been reported to be lower in cynomolgus monkey ( . mg/ml) compared to human ( ) . the conventional pig is the only species where aag levels have been reported to be higher ( . - . mg/ml) ( , ) compared to human. however, this finding has not been consistently observed, lower aag values of . mg/ml ( ) and . mg/ml ( ) have also been reported in the conventional pig. in most species, aag behaves as a positive acute phase protein (app), increasing in response to stimuli, including infection, inflammation, and cancer (table i) . the acute phase response (apr) is considered part of the innate (non-specific) defense system that offers protection prior to the adaptive (specific) immune response. the magnitude of response can differ across species and disease setting. minor, moderate, and major aaps are characterized by increases in protein expression by . - , - , and > -fold, respectively. in mice aag is considered a major positive app as levels increase up to -fold following stimuli ( , ) , whereas in human the response is relatively moderate with increases of~ - fold ( , ) . in the domestic cat, aag increases are moderate (~ - fold) in the oncology setting, whereas a more robust response has been reported with infections (up to -fold) ( ) ( ) ( ) . mixed responses have been reported in the conventional pig and the minipig. in the conventional pig, aag has been shown to behave as both a negative app with levels declining to~ . - . -fold normal values ( ) and as a positive app in response to infection and inflammation, with levels increasing - -fold ( ) . in both conventional pigs ( ) and minipigs ( , , ) there was no aag response following acute stimuli (lps or turpentine injection), despite increases in other apps in the conventional pig (tnfα and il- ) and in minipigs (c-reactive protein (crp), serum amyloid a (saa), haptoglobin (hp), pig major acute phase protein (pmap)). christoffersen et al. ( ) hypothesized the aag response in the minipig may have different sensitivity in the acute versus chronic setting, an observation previously reported for other apps (saa and hp) in cattle ( ) . in a chronic inflammatory setting, obese and mildly diabetic minipigs fed a high fat diet had elevated (~ -fold) aag levels ( ) . changing housing conditions also caused up to a -fold increase in aag in minipigs (as well as increases in crp, saa, hp and pmap), and it is unclear when levels return to baseline, a finding that should be considered in acclimation, experimental design and data interpretation ( ) . neither the negative app response nor lps insensitivity of porcine aag have been reported in any other species to date. however, it is not unprecedented for a given app to behave differently across species. for example, while crp and saa are considered major positive apps in human and pig, crp and saa are not affected in mice and rats, respectively. another example of differential species response is with transferrin, which acts as a negative app in human, and a positive app in mice ( , ) . there have been mixed reports regarding gender differences in aag levels. while there may be statistical differences, they are relatively modest compared to the differences observed in disease or developmental settings. booker et al. ( ) reported statistically significant differences in aag levels between newborn human males ( . mg/ml) and females ( . mg/ml) ( table iii) . the opposite trend was observed in adult humans; aag levels of . and . mg/ml in males and females, respectively ( ) . similarly, blain et al. ( ) reported slightly lower levels in human males ( . mg/ml) compared to female ( . mg/ml). female minipigs generally have slightly lower, though overlapping ranges, of aag levels compared to males ( ) . no gender difference was observed in dog aag levels ( . mg/ml) ( ) . ontogeny aag levels range from undetectable in the developing human fetus, to . - . mg/ml in cord blood ( , , ) , up to . mg/ml at birth ( , , ( ) ( ) ( ) ( ) , steadily increasing to . - . mg/ml at - months ( , , ) , and achieving adult levels ( . - . mg/ml) by - months of age ( , , ) (table iv) . similarly, aag is undetectable (< . mg/ml) in the cord blood of dogs, thus significantly lower compared to adult dogs ( . mg/ml) ( ) . the opposite trend was observed in conventional pigs, with . mg/ml reported in the fetal pig, . mg/ml in day newborns, . mg/ml in week olds, and . - . mg/ml in adults ( , ) . consistent with this pattern, heegaard et al. ( ) reported aag levels of . mg/ml in newborn pigs ( - days old) that declined to . mg/ml in adults. aag comprises about % of the total plasma proteins in the newborn pig, whereas only about . % in the adult pig. this property has not been reported in mini-pigs or any other laboratory animal to date. limited data have been published on fetal and neonatal plasma levels of aag in other species. however, liver aag levels in rat have also been shown to vary in development ( ) . pregnancy can also impact aag levels. in human aag levels are lower in the pregnant female and continue to decline throughout pregnancy until birth when they begin to climb back to pre-pregnancy values ( , , ) . wood and wood ( ) reported the same values in female non-pregnant healthy volunteers and pregnant women, however the study size was relatively small (n = ). the opposite has been reported in the pregnant dog ( ) and in rhesus monkey ( ) , with aag levels about -fold and -fold higher in the pregnant animal, respectively. given the striking differences between fetal, newborn, and adult aag levels, it may be important to understand placental transfer and the milk to plasma ratio (m/p) for drugs that bind to aag. fleishaker and mcnamara ( ) described a diffusional model to assess drug distribution in milk, showing that the in vitro drug binding to serum and milk protein reasonably predict m/p drug ratio in vivo. the same authors tested the model in lactating rabbits using propranolol, a compound known to bind with high affinity to aag. to mimic the disease setting, rabbits were dosed with bovine aag and propranolol pk parameters were evaluated. the diffusional model was able to accurately predict the decrease in propranolol m/p from . to . before and after aag administration. importantly, a roughly proportional reduction in total plasma cl ( %) counteracted the decrease in f u ( %), maintaining consistent cl u rate and total drug levels in milk. to improve the prediction of f u in human infants mcnamara and alcorn ( ) considered the ratio of aag and albumin in cord blood of newborns and adult blood. the corresponding ratios for aag and albumin employed were . ( . mg/ml cord divided by . mg/ml adult) and . ( mg/ml cord divided by mg/ml adult). prediction of f u in newborns was better for drugs that predominantly bind to albumin. the average predicted and observed ratios (newborn/adult), of f u were . and . , respectively, for drugs that predominantly bind to albumin (n = drugs in study set). the average predicted and observed ratios (newborn/adult), of f u were . and . , respectively, for drugs that predominantly bind to aag (n = drugs in study set). for the majority of drugs, the f u in newborns was under-predicted, / and / drugs that predominantly bind to aag and albumin, respectively. possible explanations for the disparity suggested by the authors include changes in drugligand affinity associated with age as well as increased free fatty acids and bilirubin in the newborn that can contribute to decreased drug binding. in addition, the under prediction may be due to inaccurate (falsely high) aag and albumin newborn levels employed in the model. while aag levels are generally more variable (higher dynamic range) compared to albumin, this alone cannot explain the trend in under prediction. three genes (aag-a, aag-b, and aag-b′), located on chromosome , encode human aag (haag) ( ) . aag-a encodes orm and is expressed in the liver at > -fold that of aag-b and aag-b′. aag-b and aag-b′ are identical in structure, differ from aag-a by amino acids, and encode orm ( ) . aag shares significant homology with human immunoglobulin g (igg) and the epidermal growth factor (egf)-binding domain of the egf receptor ( , ) . aag-a (orm ) is polymorphic with three closely related genetic variants: f , f , and s, differing by < amino acid residues, and generally referred to as f *s in table v ( , ) . aag-b and aag-b′ (orm ) encode the genetic variant a. most individuals possess a mixture of these variants ( ) . f + s + a is the most common phenotype ( %), followed by f + a ( %) and s + a ( %). the molar ratio of f *s to a is~ - : in healthy individuals ( ) . the ratio can increase up to : in the disease setting since the f *s variant is inducible ( ) . no gender related differences have been observed in expression of these variant forms ( ) . x-ray crystallography showed two common binding pocket lobes between the f *s and a variants, while the f *s variant possesses a unique third lobe making it more promiscuous for drug binding ( , ) . drug binding properties have been shown to differ amongst these variants ( ) . for example, the basic drug imipramine was shown to bind more strongly to a variant, whereas warfarin more strongly to the f and s variants. for most drugs, binding to genetic variants has not been well characterized since protein binding studies are routinely conducted on a pooled supply of healthy human plasma or in the whole plasma of individual subjects/patients. proteins routinely undergo post-translational modifications that can impact physiological function and half-life (t / ). glycosylation, the addition of oligosaccharide chains (glycans) is one of the most abundant post-translational modifications, with an incidence of~ % in eukaryotic proteins ( ) . aaps are particularly susceptible to glycosylation. glycosyltransferases and glycosidases are responsible for building the precursors to glycans, a process highly vulnerable to changes in disease state ( ) . oligosaccharyltransferases then transfer glycans to the polypeptide chain at asparagine (n-linked) or serine/threonine (o-linked) residues, the former of which exhibit a common pentasaccharide core. the glycan bonds occur in either α or β configuration allowing for more structural diversity. the antiinflammatory and immunomodulatory properties are directly impacted by glycan composition which, in turn, changes throughout the various stages of inflammation. the heavily sialylated glycans make aag one of the most acidic plasma proteins. there is a high level of heterogeneity resulting in a very low but wide pi ranging from . to . which in turn can impact drug binding and aag t / ( ) . desialylation can result in an increase in pi range from . to . ( , ) . human aag contains n-linked glycans of the polypeptide backbone, each of which can form a variety of bi, tri, or tetra-branches all potentially further expressing sialic acid moieties ( ) . despite thousands of potentially unique glycan combinations associated with aag, only about - glycan combinations are observed in the plasma of healthy humans ( , ) . however, in the disease state many more glycan modifications have been detected under the regulation of inflammatory cytokines, tumor necrosis factor (tnfα), interleukin- (il- ) and il- and its utility as a biomarker will be described later ( ) . aag offers two drug binding sites for basic drugs, one for acid drugs ( ) , and up to for steroids ( ) . drug binding to aag is reportedly mediated predominantly via hydrophobic interaction with some data suggesting potential for electrostatic interaction. evidence to support the latter includes the observation of stereoselective binding in propranolol isomers ( ) . desialylation and lower plasma ph have been shown to decrease drug binding to aag ( , ) . propranolol binding was reduced and progesterone binding unchanged with desialylated aag. homologous aag genes have been observed across mammals including rodents, cats, dogs, pigs, monkeys and humans (table vi) . mouse, rat, rabbit, and pig aag genes sharẽ %, %, % and % homology, respectively, with human aag ( , ) . while there are three human aag genes, there is only one gene reported in rat and two to three in mouse depending on strain or source. despite the presence of multiple ( ) . two disulfide bridges have been described in human and pig aag, with only one in rat. various binding sites have been characterized across species; in human acidic, basic, and steroid binding sites have been reported, whereas cows and dogs lack the acidic binding site ( ). aag belongs to a family of apps mainly generated in liver parenchymal cells at elevated levels within - h of injury (i.e. infection, inflammation, burns, cancer). apps by definition are proteins that change in response to injury by > % in plasma ( ) . examples of positive apps include ceruloplasmin, aag, and serum amyloid a (saa), all increasing levels in humans by about %, -fold, and > -fold, respectively, in the diseased state ( ) . negative apps include albumin, transferrin, and insulin-like growth factor i, which modestly decline in plasma in the diseased state. aag is a member of lipocalins, a superfamily of extra-cellular transporters that bind and transport small hydrophobic endogenous and exogenous chemicals. upregulation of apps enhances local inflammation by aiding in recognition of microbes, directing leukocytes, and increasing blood flow to the site of insult while minimizing inflammatory responses elsewhere ( ) . the rapid and high magnitude of response, as well as the short t / , are properties characteristic of apps and hypothesized to be key elements in innate immunity. homeostasis is kept in check with redundancy such that a given app may impact multiple pathways while multiple apps may provide overlapping biological function. the inflammatory cytokines, tnfα, il- and il- , have been shown to regulate aag, in addition to the other apps including c-reactive protein (crp), haptoglobin (hp), saa and hemopexin ( ) . the function of aag is still poorly understood, however, as part of a cytokine mediated feedback mechanism it has been implicated in both anti-and pro-inflammatory modulation ( , ) . monocyte activation and induction of t-cell proliferation ( ) as well as activation of tnfα, il- and il- secretion ( ) ( ) ( ) have been associated with the pro-inflammatory role of aag. su et al. ( ) proposed a positive feedback mechanism of apps whereby inflammation is amplified in response to tnfα-mediated synthesis of aag-stimulated monocytes and vice-versa. the anti-inflammatory role of aag has also been reported. aag inhibits neutrophil chemotactic response associated with stimulation of n-formylmethionyl-leucyl-phenylalanine (fmlp) and the inflammatory peptide complement component c a ( , ) . aag was shown to modulate release of free radicals regardless of treatment time, whether aag was introduced prior to or post neutrophil activation ( ) . multiple in vivo septic shock models in rodents have demonstrated the protective effect of aag when dosed prophylactically to animals challenged by tnfα or endotoxin ( ) . the role of aag in angiogenesis was studied using an ex vivo rat model ( ) . following aortic excision, macrophages respond by rapidly (within minutes) increasing tnfα levels, peaking at h, and remaining elevated throughout angiogenesis. tnfα guides overexpression of aag within h, with levels peaking after - days and sharply declining thereafter. as with inflammation, aag (and tnfα) has both pro-and anti-angiogenic effects depending on the context. early in the angiogenesis process, aag plays an inhibitory role via modulation of mitogen-activated protein kinases, whereas later in the process aag promotes angiogenesis via vascular endothelial growth factor regulation. as an app, levels of aag typically increase within h of injury and begin to decline several days post amelioration. increased aag levels have been reported in the serum of breast, lung, and ovarian cancer patients ( ) . in a study comparing aag levels in lung cancer patients versus individuals with no known cancers, results showed % sensitivity and % specificity and aag levels correlated with relapse-free survival ( ) . in the case of hepatocellular carcinoma (hcc), diagnosis can be challenging due to other liver conditions (i.e. cirrhosis) presenting similar abnormalities, however increased aag levels are more pronounced with hcc providing a potential basis to differentiate these diseases ( ) . the proportion of breast cancer patients with increased aag levels increases with disease progression, for example % and % of stage ii and iv patients respectively, had elevated levels compared to % in healthy donors ( , ) . in most disease states, aag is modified both quantitatively as described above, as well as qualitatively, a phenomenon described by alminquist and lausing ( ) after comparing serum glycoproteins of cancer patients versus healthy donors. relative to the associated polypeptide backbone, the heterogeneity in glycan composition and structure make it a good system for characterization and correlation with disease state ( ) . some commonly exploited glycoproteins used in clinical cancer biomarker tests include carcinoembryonic antigen (cea), cancer antigen (ca- ), ca- - , and prostate-specific antigen (psa), for the diagnosis of colorectal, ovarian, pancreatic and prostate cancers, respectively. technological advances in mass spectrometry and proteomics have led to an improved understanding of glycan structure and function. glycans are relatively more abundant compared with their associated proteins, often times multiple copies per glycoprotein, as is the case for aag known to have - associated glycans depending on species (table vi) . not only are they associated with the cancerous tissue but they can also be detected in serum, making its use as a biomarker more feasible in the clinical setting. in addition, the same modified glycan may be associated with more than one glycoprotein affording multiple opportunities/patterns for detection. modifications of the glycans associated with these biomarkers are relatively more specific and selective than the epitope itself, allowing for more accurate means for distinguishing healthy versus diseased tissue, and disease progression ( ) . for example, testing serum levels of modified glycans associated with psa enable the distinction between benign prostatic hypertrophy and cancerous prostate ( ) . in breast cancer, the serum glycosylation pattern not only distinguishes healthy from diseased tissue but also differentiates between malignant and non-malignant tumors and disease stage ( ) . in inflammatory diseases including rheumatoid arthritis and asthma, aag glycans are more branched compared to healthy subjects ( ) . patients suffering from acute inflammation, infection, burns, and tissue damage all showed an asialylated carbohydrate-deficient variant of aag ( ) . human aag t / is relatively short,~ - days ( , ), compared to albumin, - days ( ) . aag turnover is dependent on sialic acid residues and terminal galactose groups. mccurdy ( ) studied the impact of glycosylation on in vivo cl of human derived aag in rabbit following intravenous injection. the terminal t / of native human aag was h (consistent with the t½ value of h reported by regoeczi et al. ( ) . altered/reduced or absence of glycosylation lowered the t½ to h and h, respectively. cl of native aag was . ml/h/kg, whereas much higher values were observed for aag forms with altered ( ml/h/kg) or absent glycosylation ( ml/h/kg). the steady-state volume of distribution (v ss ) was ml/kg for native aag, whereas much higher values were observed with altered ( ml/kg) or absent glycosylation ( ml/kg). the absence, reduction or alteration of n-linked glycosylation resulted in a marked increase (> -fold) in renal elimination compared to native aag. these studies support that the dispositional properties of aag are dependent on disease state since the biochemistry of aag is altered with disease as described earlier. therefore, if a given drug binds to aag to a high extent, it is possible underlying differences in the pk of the drug between healthy and diseased populations may be attributable to aag. drug binding to plasma, tissue(s) and intended target are critical parameters to predict pk and pharmacodynamics (pd). however, optimizing protein binding to plasma in the drug discovery setting is scientifically unsound ( , ) . nearly % of the fda approved drugs prior to are classified as highly bound (> % or f u < . ), and this trend has increased in recent years with % of new drugs classified as highly bound, % of which have f u < . ( ) . while the free drug hypothesis describes that the free concentration drives activity as it can cross cellular membranes to reach its target, the focus should be to optimize cl u and permeability. as described above, albumin, aag, and lipoproteins are considered the most important plasma proteins involved with drug binding. while albumin has a higher drug capacity due to its relative abundance in plasma, aag levels are lower and with high affinity drugs saturation may occur. the saturation of aag may or may not be buffered by albumin depending on the drug binding affinity for albumin. plasma protein levels can change with disease state, a decrease in albumin and an increase in aag are generally observed, which may impact protein binding and pk. both albumin and aag levels are significantly lower in the newborn, with newborn:adult ratios of about . and . , respectively ( ), a factor that should be considered when predicting pk in the very young pediatric population. a proposed flowchart to ascertain the impact of aag as a potential covariate for pk variability early in drug development is shown in fig. . routine screening in human and laboratory animal species at a single low concentration ( - μm) is typically performed in early drug discovery as pk data in preclinical species is acquired. as a program advances towards development candidate selection with preliminary projections of human pk and dose, there is value in assessing concentration dependency and the identity of the major plasma proteins involved in drug binding. if the extent of binding is high (> % bound) under single concentration assay conditions, further characterization of the binding constants for aag and albumin is warranted particularly if human free fraction is lower relative to animals or if concentration dependency is observed. if there is high affinity to human aag it may be worthwhile to assess k d in other species to build additional confidence in human pk predictions. multiple routine in vitro analytical procedures exist to assess the extent and affinity of drug binding to proteins. a recently published industry white paper (di et al., ) provides a comprehensive review of commonly used protein-binding practices, challenges, and recommendations ( ) . the intention in this section of the manuscript is to suggest use of control compounds and to describe a source for erroneous fraction unbound values that has been overlooked. as with any assay it is good practice to include control compounds that are assessed along with test compounds to ensure a properly functioning assay. if data for control compounds are not available for a given assay/species one can still monitor the value for the control over time to ensure its consistency. when conducting definitive assays the use of multiple control compounds is advised since multiple factors can influence binding and some are compound specific. literature values for propranolol and warfarin are summarized across species in table vii . the intent here is to provide references for acceptable free fraction values for control compounds across species. propranolol was selected because it has moderately high binding to human plasma proteins, however the affinity is higher for aag relative to albumin by approximately two orders of magnitude ( ) , therefore, propranolol can serve as a control for compounds that preferentially bind to aag. the reported propranolol f u values range from . to . in human plasma, a -fold difference, and outside what is typically deemed normal assay variability. the acceptable assay f u value for propranolol should be within . to . in healthy human plasma. warfarin was selected because it is highly bound to both aag and albumin ( , ge life sciences application note aa). it is helpful to include a highly bound control compound since they generally require longer incubation time to achieve equilibrium. while the reported warfarin f u values range from . to . in human plasma, more than a -fold difference, the absolute difference is low. the majority of references indicate a narrower range, therefore the acceptable assay f u value for warfarin should be within . to . , a range similar to that reported in the recently published white paper ( ) . it is not advised to select a compound with moderate or low protein binding to serve as a control because the f u values are generally more variable. based on recent studies ( , ) it is possible the large range in human f u values is due to the blood collection and storage procedure. in the clinical setting, f u is typically measured in the plasma or serum of patients from blood collected in vacutainers. the collection procedure and storage of blood can have a significant impact on protein binding results. it has long been reported that plasticizers can disrupt the binding of drugs to aag ( ) ( ) ( ) . for example, the plasticizer tris ( -butoxyethyl) phosphate (tbep), used to soften rubber stoppers in vacutainers, was shown to disrupt aag binding to the basic drugs lidocaine and quinidine ( ) . polyvinyl chloride (pvc) bags containing the plasticizer diethylhexyl phthalate (dehp) are routinely used in blood collection. butler et al. ( ) reported an average of a two-fold increase in f u for drugs known to bind to aag when blood was collected in these pvc bags versus blood collected in vacutainers. more recently, experiments were conducted to show the correlation between dehp levels and change in f u with drugs that bind to aag ( ) . when blood was immediately transferred from terumo® bags to vacutainers the dehp levels were low ( - μm) but steadily increased with storage after days (up to μm) and days ( - μm). dehp can easily leach into the contents of bags since it is not chemically bound to the pvc. as expected, f u was higher ( - -fold) with drugs known to bind to aag when tested using plasma containing high levels of dehp. the shift in f u was significantly reduced, though not eliminated, when the blood was immediately transferred to vacutainers. results generated using blood products collected/stored in plasticizer containing bags should be interpreted with caution as the f u values may over-estimate true in vivo values. in most instances when protein binding is measured clinically, vacutainers (no/minimal dehp exposure) are used since small (< ml) blood volumes are collected. this is in contrast to the relatively larger volumes (up to ml) collected in bags for donation and/or non-clinical research purposes. it should be noted that the blood from animals is typically collected in smaller vessels not containing dehp, therefore the over-estimation of f u is less likely to occur in animal blood. studies may be warranted to assess effects on drug binding with other commercially available vacutainers as a precaution. despite what has been reported in the literature for decades, bags containing plasticizers known to disrupt aag-drug binding continue to be widely used for blood collection with the intended use in both research and in the clinical setting. dehp is essential in maintaining the shelf life of blood products up to days as it protects the membrane of the red blood cell ( ) . transfusion recipients routinely receive blood that has been collected and stored in these bags. clinical effects with regard to drug displacement have not been reported. other blood collection bags have been developed though it is unclear if they have an effect on aag-drug binding. multiple classes of drugs have been reported to bind to aag. examples of the relationship between aag binding and lipid solubility and/or electrostatic interactions have been reported for benzodiazepines, phenothiazine neuroleptics, beta blockers, anthracycline derivatives, antihistamines, and analgesics ( , ) . here we focus on several well-studied examples where the drug-aag binding affected pk and/or pd in the clinical oncology setting. vismodegib was approved for the treatment of metastatic basal cell carcinoma by the food and drug administration (fda) in . pk characteristics exhibited in the phase i clinical trial were unexpected based on preclinical allometric scaling. following a single oral dose, exposure was higher than predicted with a very low apparent cl and a long t / of about - days ( ) . cross species in vitro data showed high plasma protein binding (≥ % bound) and low metabolic turnover in hepatocytes of all species except monkey, which agreed well with in vivo cl values ( ) . mechanistic pk modeling was employed to explain the roles of plasma protein binding, solubility-limited absorption, and low metabolic cl in contributing to the unusual clinical pk properties ( ) . in vitro studies revealed far lower vismodegib solubility, . mg/ml at higher ph range . - . , compared to~ . mg/ml at ph . ( ) . the impact of solubility was manifested in saturation of oral absorption. there was no increase in mean steady-state concentration (c ss ), . , . and . μm, with increase in oral dose from to and mg, respectively ( ) . the free fraction of vismodegib remained constant at . ± . % across all dose groups ( ) . however, in a separate study, a . -fold increase in free fraction of vismodegib was reported between a single mg dose ( . ± . %) versus repeat daily mg doses ( . ± . %) ( ) . vismodegib plasma protein binding properties were further characterized by isothermal titration calorimetry (itc) and surface plasmon resonance (spr) with both procedures showing higher vismodegib-aag affinity to the human isoform relative to the rat isoform ( ) . by itc the k d values of vismodegib-aag were . and μm in human and rat, respectively. by spr the k d values of vismodegib-aag were μm and not detectable in human and rat, respectively, whereas the k d values for vismodegib-albumin were similar in human and rat ( and μm, respectively). in vitro experiments showed a negative correlation between aag concentration and target engagement, whereby supplementing physiologically relevant concentrations of aag resulted in a dampening of hh signaling via gli -luciferase reporter assay ( ) . there was a high correlation (r = . , slope . ) between aag and total vismodegib c ss in plasma samples from cancer patients, suggesting the role of plasma protein binding in vismodegib drug disposition ( ) . perhaps even more compelling was the intra-patient parallel changes in total vismodegib concentrations with changes in aag. no correlation was found with albumin levels and vismodegib concentrations ( ) . saturation of aag has been proposed to be a key determinant in the non-linear pk of vismodegib given that aag is a high affinity-low capacity protein and near stoichiometric levels of vismodegib and aag are reported ( ) . despite the high affinity and resultant low free fraction, there remains sufficient unbound vismodegib available to interact with target to demonstrate pharmacological effect. in order to understand the mechanism(s) of non-linear pk, healthy human subjects received either a single oral dose or daily oral doses of mg vismodegib, followed by an iv microtracer dose of μg [ c]-vismodegib h post first or last (day ) oral dose ( ) . aag levels were within close range in the two dose groups to eliminate need for correction. cl and v ss values after a single iv dose were . ml/h and . l, roughly - -fold lower (depending on number of species employed in model) and -fold lower, respectively, compared to preclinical allometric scaling predictions ( ) . relative to day , cl and v ss increased % and %, respectively on day , while t / remained unchanged at~ - days. mean free fraction increased . -fold after days oral dosing ( . ± . %) compared to a single dose ( . ± . %), a finding consistent with the observations previously described ( ) . correcting for protein binding of vismodegib, the unbound cl and v ss values were relatively similar on day and . absolute bioavailability was . % following a single oral dose and declined to . % after days of repeat dosing, a finding attributed to slow absorption and limited intestinal solubility ( ) . given the non-linear pk with long t / , a phase ib clinical study was conducted to determine if vismodegib steady-state concentrations could be maintained with less frequent dosing ( ) . three oral dosing schedules were evaluated: mg once daily (qd), once weekly (qw) or three times per week (tiw), all after having received a loading dose of vismodegib ( mg qd, days). after steady-state was achieved for the alternate dose schedules, vismodegib levels declined in the qw and tiw groups relative to qd, however the decline in unbound vismodegib ( % and %, respectively) was greater than the decline in total vismodegib ( % and %, respectively). only the qd dose schedule maintained unbound vismodegib concentrations sufficient to achieve target pathway inhibition (ic ) of glioma-associated oncogene (gli ) previously described ( ) , therefore the recommended dose and schedule was maintained at mg qd. one additional point of consideration is the discrepancy between in vitro and ex vivo free fraction values reported for vismodegib in human plasma,~ - % and < . - . %, respectively ( , , , ) . this discrepancy may be attributed to the collection and storage of blood products as described herein and in recent publications ( , ) . free fraction values increased sharply for aag-binding drugs when human blood was exposed to plasticizer dehp; for example, vismodegib free fraction increased from . % when collected in vacutainer (no/minimal plasticizer), to . % and . % when collected and stored in terumo® bags containing plasticizer for < and days, respectively. additionally, vismodegib free fraction increased from . to . % when human plasma was spiked with μm plasticizer, a concentration one could expect to measure in blood after several weeks storage in terumo® or similar bags. ucn- ( -hydroxystaurosporine) is a small molecule protein kinase inhibitor. single agent clinical trials were initiated for multiple oncological indications in the late s, followed by combination studies with other anti-cancer agents. the pk parameters in preclinical species (mouse, rat and dog) ranged as follows: moderate to high cl roughly to % hepatic blood flow, high v ss to l/kg, and moderate t / from to h ( ) . clinical pk were not predicted by allometry and exhibited low cl ( ml/h), low v ss ( l) and very long t / (> h) ( ) . while ucn- ( μg/ml) is considered highly bound to plasma proteins in preclinical species with free fraction ranging from . to . %, the free fraction was substantially lower in human plasma at < . % ( ) . ucn- free fraction was < . % or . % when incubated with physiologically relevant levels of haag ( mg/ml) or albumin ( mg/ml), respectively, showing the preferential binding to aag. in vitro studies showed marked increase in ucn- free fraction with increases in concentration approaching stoichiometric levels of aag ( ) . the association constant (k a ) was × l/mol in haag (roughly equivalent to k d of . nm), whereas in dog the k a was~ -fold lower at . × l/mol ( , ) . sparreboom et al., further characterized the role of aag in the pk of ucn- ( ) . with an increase in dose ranging from . to mg/m /day iv infusion over h, there was an increase in cl, . ml/h to . ml/h, respectively, a linear increase in v ss , . l to . l, respectively, and less than proportional ( . -fold) increase in auc ∞ , (area under the curve extrapolated to infinity) to , mg*h/l. cl trended (r = . ) with pre-dose aag levels, despite a relatively small data set (n = ). it is proposed the increase in cl in humans is due, at least in part, to the increase in free fraction once aag becomes saturated. cl in dogs had demonstrated no dependence on dose from . to . mg/kg ( , ) , a finding consistent with the notable difference in ucn- -aag k d in human versus dog. the k d of haag-ucn- is roughly orders of magnitude lower compared to k d haag-vismodegib ( ) . in addition, ucn- does not appear to bind significantly to albumin given the comparable k d values of ucn- to aag vs human plasma, vs × l/mol, respectively ( ) . saturation of aag and the differential affinity to aag and albumin are likely to contribute to the non-linear pk of ucn- . as with vismodegib the haag-unc- k d differed considerably from that of the preclinical species leading to poor predictive accuracy with allometric methods. ucn- has a slow dissociation rate which may reduce v ss , further hindering free drug from target interaction ( ) , in contrast to the preclinical observations of high v ss , high tumor:plasma ratios, and decline in tumor volume ( ) . ucn- has not advanced in the clinic due to unpredictable pk and off-target kinase inhibition ( ) . imatinib is a selective inhibitor of bcr-abl, platelet-derived growth factor receptors, and c-kit receptor tyrosine kinases ( ) . approval was granted for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors by the fda in and . imatinib-aag binding is concentration-dependent with a reported k a of . × l/ mol, roughly equivalent to k d of . μm, while imatinibalbumin binding is considerably weaker with a k a of . × l/mol, roughly equivalent to k d of μm ( , ) . adding to the complexity is the differential binding of imatinib to various human aag isoforms, the k a reported above is for the f -s variant, whereas binding was much weaker for the a variant (unpublished data) ( ) . separately incubating μm of the aag variants f -s and a with μm imatinib resulted in % and % free fraction, respectively. imatinib exhibits linear pk in patients ( ) with low oral cl ranging from to l/h ( , , ) , long t / of h and high oral bioavailability > %. a proportional increase in auc is observed with oral doses from to mg. pk parameters are similar between single and repeat doses, showing . -to . -fold accumulation at steady-state. correlations between imatinib pk and abcb genotype, body weight and aag levels was shown in patients ( , ) . despite linear pk in total imatinib, a non-linear relationship exists between free fraction and total imatinib concentrations in plasma as a result of high affinity to aag and~ -fold weaker affinity to albumin ( ) . elevated levels of aag in patients have been linked with delayed or lack of response to imatinib treatment as well as potential resistance mechanism ( , ) . in a clinical study with cml patients, approximately half exhibited elevated aag levels positively correlating with disease progression and white blood count. in the chronic, accelerated, and blast crisis phases of disease, , and % of these patients, respectively, were increasingly likely to have higher aag levels. the impact of the plasticizer dehp on imatinib free fraction in human plasma was also assessed ( ) . imatinib free fraction increased from . % when collected in vacutainer (no/minimal plasticizer), to . % and . % when collected and stored in terumo® bags containing plasticizer for < and days, respectively. additionally, imatinib free fraction increased from . to . % when human plasma was spiked with μm plasticizer. the plasticizer-free free fraction values reported by ingram et al. ( ) are similar (~ %) to those reported in the package insert (novartis pharmaceuticals corporation, reference t - ). pinometostat (epz- ) is a first-in-class, small molecule inhibitor of dot l and was the first member of the novel histone methyltransferase inhibitor class to enter phase clinical trials in both adult and pediatric mll-r leukemia patients. consensus preclinical predictions across multiple diverse methods suggested pinometostat would be a moderate-to-high cl compound in human with estimates ranging from to ml/min/kg, and species-invariant time approaches showed cross-species congruence in time-concentration profile. however, during early development, the observed cl in human was shown to be markedly lower than that determined in preclinical species. the majority of interspecies scaling and allometric methods over-predicted human cl of pinometostat with fold errors ranging from to ( ) , characterized by 'vertical allometry'. the - -fold difference in free fraction between rat and human provided the basis for the improved prediction using the free fraction corrected intercept (fcim) method. the unambiguous species difference in cl was not related to qualitative differences in metabolic pathways or routes of elimination, but instead to cross-species differences in plasma protein binding. concentration dependence in protein binding was observed in human plasma, over a relevant concentration range, which was less apparent in the preclinical species. this, along with in vitro kinetic determinations, suggested the saturable binding of pinometostat to aag. the equilibrium dissociation constant (k d ) for pinometostat binding to human aag was measured as . μm indicating a high affinity interaction. by comparison, prototypical aag ligands such as dipyridamol, disopyramide and thioridazine have k d values of . , . and μm respectively. furthermore, there is the disproportionately higher expression of aag in human plasma relative to preclinical species, which is likely a contributing factor alone, irrespective of potential speciesspecific differences in aag affinity. it has been suggested when the k d for a given drug-aag binding is low, and more than a log order lower relative to the k d for albumin, the pk may exhibit non-linearity ( ) . if the drug also has a high affinity to albumin, a high capacity protein, fluctuations in free fraction will be minimal. if the drug has a low affinity to albumin, the non-linear effect may be exacerbated when drug levels are near stoichiometric with aag, since aag is a low capacity protein and may become saturated. given the known differences in abundance and homology across species for aag, allometric scaling may not be suitable for human pk prediction when there are differences in k d , something that can easily be measured in vitro now that aag of preclinical species are commercially available, though still limited in supply. monitoring aag and/or free drug concentrations, as well as phenotyping the genetic variants of aag in patients may be warranted in special circumstances to better understand pk and pd. in fig. , we propose a flowchart for incorporation of protein binding assessment in drug research and development. investigations are ongoing to propose more reliable procedures for the collection and storage of blood for future use in drug free fraction measurements (personal communications with q solutions holdings, llc). special consideration should be given to pregnancy and pediatric populations since aag levels are substantially lower until about months post-natal. pbpk models have shown predictive utility with incorporation of aag and albumin levels and drug binding parameters. quantitative and/or qualitative analysis of aag may prove useful as a biomarker for disease diagnosis and prognosis, with the potential to serve as discerning criteria to improve likelihood of successful treatment. since aag behaves as a positive apr protein in most species (except pig), with levels increasing to varying extents in the disease setting, it may be helpful to consider when comparing pk in healthy versus diseased populations or in translating pk/pd relationships across species. role of protein binding in pharmacokinetics preparation and properties of an acid glycoprotein prepared from human plasma drug binding to human alpha- -acid glycoprotein in health and disease characterization of ligand binding sites on the alpha -acid glycoprotein in humans, bovines and dogs role of α -acid glycoprotein in therapeutic antifibrotic effects of imatinib with macrolides in mice role of α acid glycoprotein in the in vivo resistance of human bcr-abl + leukemic cells to the abl inhibitor sti nonspecific resistance to a lethal gram-negative infection the acute phase response in the rodent serum binding of tolterodine and its major metabolites in humans and several animal species alpha- -acid glycoprotein concentrations and protein binding of propranolol in sprague-dawley and dark agouti rat strains treated by phenobarbital stress-induced acute phase response rankl inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-tnfα or anti-il- therapies synthesis of acute phase proteins in rats with cirrhosis exposed to lipopolysaccharide pig α -acid glycoprotein: characterization and first description in any species as a negative acute phase protein the influence of age and health status on the serum alpha- -acid glycoprotein level of conventional and specific pathogen-free pigs the major serum protein of fetal and newborn pigs: biochemical properties and identification as a fetal form of α -acid glycoprotein the acute phase response of acid soluble glycoprotein, alpha ( )-acid glycoprotein, ceruloplasmin, haptoglobin and c-reactive protein, in the pig time course of increased plasma cytokines, cortisol, and urea nitrogen in pigs following intraperitoneal injection of lipopolysaccharide age-and sex-associated effects on acute-phase proteins in gottingen minipigs serum α- acid glycoprotein and serum amyloid a concentrations in cats receiving antineoplastic treatment for lymphoma value of α -acid glycoprotein in the diagnosis of feline infectious peritonitis critical assessment of the diagnostic value of feline α -acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach changes in concentrations of serum amyloid a protein, a- -acid glycoprotein, haptoglobin, and c-reactive protein in feline sera due to induced inflammation and surgery performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases alpha acid glycoprotein (aag) levels in healthy and pregnant beagle dogs binding of oxprenolol and propranolol to serum, albumin and alpha -acid glycoprotein in man and other species c-reactive protein and α- -acid glycoprotein levels in dogs infected with ehrlichia canis dog alpha- -acid glycoprotein: purification and biochemical characterization serum a- -acid glycoprotein concentrations in dogs with pyometra in vivo effects of recombinant human interleukin- in primates: stimulated production of platelets alpha -acid glycoprotein concentrations and propranolol binding in elderly patients with acute illness study of the expression of the genetic variants of human alpha -acid glycoprotein in healthy subjects using isoelectric focusing and immunoblotting effects of development, aging, and renal and hepatic insufficiency as well as hemodialysis on the plasma concentrations of albumin and alpha -acid glycoprotein: implications for binding of drugs laser nephelometry of orosomucoid in serum of newborns: reference intervals and relation to bacterial infections α acid glycoprotein binds to imatinib (sti ) and substantially alters its pharmacokinetics in chronic myeloid leukemia patients determinants of plasma alpha- -acid glycoprotein (aag) concentrations in health alpha- -acid glycoprotein immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses a study of serum glycoproteins in cancer sweetening the pot: adding glycosylation to the biomarker discovery equation carbohydrate structure and differential binding of prostate specific antigen to maackia amurensis lectin between prostate cancer and benign prostate hypertrophy the degree of branching of the glycans of alpha( )-acid glycoprotein in asthma. a correlation with lung function and inflammatory parameters the plasma proteins: structure, function and genetic control investigation of the binding of various tricyclic neuroletpics and antidepressants to alpha -acid glycoprotein steroid-protein xix. complex formation between a -acid steroid hormones measurement and analysis of unbound drug concentrations protein binding predictions in infants transferrin and a -macroglobulin-i are circulating acute phase reactants in the mouse acute phase proteins in cattle: discrimination between acute and chronic inflammation perioperative changes in α -acid glycoprotein concentrations in infants undergoing major surgery changes in plasma drug binding and alpha -acid glycoprotein in mother and newborn infant sex-related differences in the plasma protein binding of lignoncaine and diazepam plasma protein binding of disopyramide in pregnant and postpartum women, and in neoates and their mothers drug binding to plasma proteins during human pregnancy and in the perinatal period, studies on cloxacillin and alprenolol serum concentrations of acute-phase proteins in healthy term and preterm infants from birth to age months changes in serum albumin and alpha- acid glycoprotein concentrations during pregnancy: an analysis of fetal-maternal pairs changes in α -acid glycoprotein serum concentrations and glycoforms in the developing human fetus effects of age on the serum concentration of α -acid glycoprotein and the binding of lidocaine in pediatric changes in plasma alpha -acid glycoprotein and albumin concentrations during late pregnancy in rhesus monkeys the expression of α- -acid glycoprotein mrna during rat development. high levels of expression in the decidua effect of altered serum protein binding on propranolol distribution into milk in the lactating rabbit structure and expression of the genes coding for human alpha -acid glycoprotein the carbohydrate units of human plasma alpha -acid glycoprotein sequence similarity between egf receptor and a -acid glycoprotein human plasma alpha -acid glycoprotein-biochemical properties, the amino acid sequence and the structure of the carbohydrate moiety, variants and polymorphism molecular aspects of human alpha- acid glycoprotein -structure and function the genetic polymorphism of human alpha -acid glycoprotein investigation of genetic variants of alpha- acid glycoprotein by ultra-performance liquid chromatography-mass spectrometry structural insights into differences in drug-binding selectivity between two forms of human alpha -acid glycoprotein genetic variants, the a and f *s form the . -a crystral structure of alpha -acid glycoprotein (orosomucoid) solved by uv rip reveals the broad drug-binding activity of this human plasma lipocalin evidence for differences in the binding of drugs to the two main genetic variants of human α -acid glycoprotein on the frequency of protein glycosylation, as deduced from analysis of the swiss-prot database alpha- -acid glycoprotein (agp) as a potential biomarker for breast cancer orosomucoid (alpha -acid glycoprotein) phenotyping by use of immobilized ph gradients with m urea and immunoblotting. a new variant encountered in immunoblotting changes of alpha -acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively alpha aacid glycoprotein (orosomucoid): pathophysiological changes in glycosylation in relation to its function binding of antiarrhythmic drugs to purified human a -acid glycoprotein factors affecting the measurement of lidocaine protein binding by equilibrium dialysis in human serum in vitro binding of propranolol and progesterone to native and desialyated human orosomucoid evidence for the existence of multiple alpha -acid glycoprotein genes in the mouse molecular cloning of cdnas corresponding to two genes of alpha -acid glycoprotein and characterization of two alleles of agp- in the mouse cloning of double-stranded cdna and kinetics of induction of mrna levels following acute inflammation molecular cloning and nucleotide sequence of complimentary dna encoding rabbit a -acid glycoprotein cloning and developmental regulation of alpha acid glycoprotein in swine influence of inflammation of serum concentration, molecular heterogeneity and drug binding properties of canine alpha- -acid glycoprotein differential expression of the mouse alpha -acid glycoprotein genes (agp- and agp- ) during inflammation and aging expression of human alpha- -acid glycoprotein genes in cultured cells and in transgenic mice analysis of the five glycosylation sites of human α -acid glycoprotein serum c-reactive protein levels in disease acute-phase proteins and other systemic responses to inflammation acute phase proteins: structure and function relationship acute phase proteins molecular biology, biochemistry and clinical applications lymphocyte stimulation in vitro by orosomucoid glycoprotein du ran d g. al pha -a cid g ly copro tein pot entia tes lipopolysaccharide-induced secretion of interleukin- beta, interleukin- and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages unstimulated peripheral blood mononuclear cells from patients with the hyper-igd syndrome produce cytokines capable of potent induction of creactive protein and serum amyloid a in hep b cells effects of alpha -acid glycoprotein on tissue factor expression and tumor necrosis factor secretion in human monocytes alpha -acid glycoproteininduced tumor necrosis factor-alpha secretion of human monocytes is enhanced by serum binding proteins and depends on protein tyrosine kinase activation comparison of the effects of purified human alpha -acid antichymotrypsin and alpha -protease inhibitor on nk cytotoxicity: only alpha -protease inhibitor inhibits natural killing serum and urinary alpha- acid glycoprotein in chronic renal failure differential effects of a -acid glycoprotein on bovine neutrophil respiratory burst activity and il- production protection by alpha -acid glycoprotein against tumor necrosis factor-induced lethality the acute phase reactant orosomucoid- is a bimodal regulator of angiogenesis with time-and context-dependent inhibitory and stimulatory properties expression of the genetic variants of human alpha- -acid glycoprotein in cancer monitoring the therapy of lung cancer with alpha- -acid glycoprotein serum protein biomarkers screening in hcc patients with liver cirrhosis by icat-lc-ms/ms early effects of adjuvant tamoxifen therapy on serum hormones, proteins and lipids elevated serum acute phase protein levels as predictors of disseminated breast cancer pharmacokinetics of intravenously administered i-labelled human alpha -acid glycoprotein turnover of plasma orosomucoid im man levels of α acid glycoprotein and ceruloplasmin predict future albumin levels in hemodialysis patients in vivo clearance of alpha- acid glycoprotein is influenced by the extent of its n-linked glycosylation and by its interaction with the vessel wall carbohydrate mediated elimination of avian plasma glycoprotein in mammals the effect of plasma protein binding on in vivo efficacy: miconceptions in drug discovery do we need to optimize plasma and tissue binding in drug discovery? rational use of plasma protein and tissue binding data in drug design industry perspective on contemporary protein-binding methodologies: considerations for regulatory drug-drug interaction and related guidelines on highly bound drugs the importance of ph control for accurate assessment of in vitro protein binding α -acid glycoprotein and serum binding in healthy and diseased dogs species differences in distribution and prediction of human v (ss) from preclinical data stereoselective disposition of propranolol in rabbits. role of presystemic organs and dose a comparison of pharmacokinetics between humans and monkeys development of a high throughput equilibrium dialysis method age and propranolol stereoselective disposition in humans disposition of warfarin enantiomers and metabolites in patients during multiple dosing with rac-warfarin tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands human alpha- -glycoprotein and its interactions with drugs impact of blood collection method on human plasma protein binding for compounds preferentially binding to α -acid glycoprotein effect of the plasticizer dehp in blood collection bags on human plasma fraction unbound determination for alpha- -acid glycoprotein (aag) binding drugs interferences from blood collection tube components on clinical chemistry assays the effect of the plasticizers tbep (tris-( -butoxyethyl)-phosphate) and dehp (di-( -ethylhexyl)-phthalate) on beta-adrenergic ligand binding to alpha -acid glycoprotein and mononuclear leukocytes interference with measurements of certain drugs in plasma by a plasticizer in vacutainer tubes drug-protein binding interferences caused by the plasticizer tbep hexamoll dinch plasticized pvc containers for the storage of platelets determination of gdc- , a small molecule inhibitor of the hedgehog signaling pathway, in human plasma by solid phase extraction-liquied chromatographic-tandem mass spectrometry preclinical assessment of the absorption, distribution, metabolism and excretion of gdc- ( -chloro-n-( -chloro- -(pyridin- -yl)phenyl)- -(methylsulfonyl) benzamide), an orally bioavailable systemic hedgehog signaling pathway inhibitor pharmacokinetics of hedgehog pathway inhibitor vismodegib (gdc- ) in patients with locally advanced or metastatic solid tumors: the role of alpha- -acid glycoprotein binding phase trial of hedgehog pathway inhibitor vismodegib (gdc- ) in patients with refractory, locally advanced or metastatic solid tumors pharmacokinetic dose-scheduling of hedgehog pathway inhibitor vismodegib (gdc- ) in patients with locally advanced or metastatic solid tumors identification, characterization, and implications of species-dependent plasma protein binding of the oral hedgehog pathway inhibitor vismodegib (gdc- ) single and multiple dose intravenous and oral pharmacokinetics of the hedgehog pathway inhibitor vismodegib in healthy female subjects pharmaokinetic-phamacodyanic analysis of vismodegib in preclinical models of mutational and ligand-dependent hedgehog pathway activation pharmacokinetcs and pharmacodynamics of a novel protein kinase inhibitor, ucn- phase i trial of -hour continuous infusion ucn- in patients with refractory neoplasms unpredicted clinical pharmacology of ucn- caused by specific binding to human alpha -acid glycoprotein altered pharmacokinetics of a novel anticancer drug, ucn- , caused by specific high affinity to alpha -acid glycoprotein in human review of ucn- development: a lesson in the importance of clinical pharmacology effects of a -acid glycoprotein on the clinical pharmacokinetics of -hydroxystaurosporine pharmacokinetics, distribution, metabolism and excretion of [ h]ucn- in rats and dogs after intravenous administration inhibition of c-kit receptor tyrosine kinase activity by sti , a selective kinase inhibitor pharmacokinetic-pharmacodynamic relationships of imatinib and its main metabolite in patients with advanced gastrointestinal stromal tumors selective binding of imatinib to the genetic variants of human alpha -acid glycloprotein clinical pharmacokinetics of imatinib effect of st john's wort on imatinib mesylate pharmacokinetics population pharmacokinetics and pharmacogenetics of imatinib in children and adults imatinib disposition and abcba (mdr , pglycoprotein) genotype population pharmacokinetics of imatinib and the role of alpha-acid glycoprotein determination of alpha- acid glycoprotein in patients with ph+ chronic myeloid leukemia during the first weeks of therapy with st relationship between elevated levels of the alpha acid glycoprotein in chronic myelogenous leukemia in blast crisis and pharmacological resistance to imatinib (gleevec) in vitro and in vivo mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha- -acid glycoprotein and permeability-limited hepatic uptake conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -da ypiy authors: mônica vitalino de almeida, sinara; cleberson santos soares, josé; lima dos santos, keriolaine; emanuel ferreira alves, josival; galdino ribeiro, amélia; trindade tenório jacob, Íris; juliane da silva ferreira, cindy; celerino dos santos, jéssica; ferreira de oliveira, jamerson; bezerra de carvalho junior, luiz; do carmo alves de lima, maria title: covid- therapy: what weapons do we bring into battle? date: - - journal: bioorg med chem doi: . /j.bmc. . sha: doc_id: cord_uid: da ypiy urgent treatments, in any modality, to fight sars-cov- infections are desired by society in general, by health professionals, by estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding covid- : knowledge is the means to discover or to produce an effective treatment against this global disease. scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of with over , published articles related to the new coronavirus. three great lines of publications related to covid- were identified for building this article: the first refers to knowledge production concerning the virus and pathophysiology of covid- ; the second regards efforts to produce vaccines against sars-cov- at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat covid- by drug repurposing. in this review, the drugs that have been repurposed so far are grouped according to their chemical class. their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions. this can help identifying anti-sars-cov- promising therapeutic agents. the world is facing a huge challenge in the coronavirus disease (covid- ) pandemic: how to fight an enemy without weapons in terms of therapy? unfortunately, even before the severe acute respiratory syndrome coronavirus (sars-cov- ) worldwide spread, there were no clinical treatments nor prevention strategies available for any human coronavirus. it is understandable that both society and researchers urge the discovery of new compounds or even of a drug that is commercially available that can be employed by physicians mainly for patients with the extreme presentation of covid- . there is also urgency in the discovery of medicine with prophylactic action to prevent the entry of the virus in host cells after exposure. vaccine research experts already indicate that rescue from sars-cov- will come from a long but effective journey to produce a vaccine. while this is not a reality, the scientific community, including medicinal chemists and doctors who accompany patients, are trying to identify therapeutic alternatives. this is a meritorious attitude: the commitment with the protection of humanity. nevertheless, the rigorous feature of science in the discovery of a new drug cannot be disregarded, even during a pandemic and in the face of the urgent demand for a treatment, to avoid eventual mistakes and spurious hope. the increase in studies related to sars-cov- during the first semester in has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs. it is noteworthy the increase in outbreaks of sars-cov ( ) and mers-cov ( ), with accelerated production of knowledge on these hcovs, which has been very useful for ongoing investigations on sars-cov- . one example is the availability of technological devices that allowed the fast sequencing of sars-cov- genome and the elucidation of a promising antigen target, the s glycoprotein. nonetheless, the development of a human vaccine can take years, especially because employing emergent technologies requires extensive safety tests and expansion to large scale production in order to assist the world population, as demanded in the case of the covid- pandemic. the development of new medicine also demands many years of research that involve stages of reasonable planning, synthesis, structural characterization, formulation of prototypes, preclinical and clinical trials. therefore, the literature highlights, as alternative treatments for covid- , the repurposing of drugs, which is fast and useful in emergencies such as the one experienced today. the repurposing of drugs means the use of broad-spectrum medicine for a new disease, once its metabolic characteristics, doses, potential efficacy and adverse effects are pre-established due to drug studies extracellular liquid volume and arterial pressure of the human body. it is largely expressed in fifteen human tissues, including ciliated bronchial epithelial cells and type ii pneumocytes form pulmonary alveoli, the main location of lesions caused by sars-cov- . , after ace receptor-binding, a conformational alteration occurs in protein s allowing the fusion between the viral envelope and the host cell membrane via endosomal. then, sars-cov- releases the rna into the cytoplasm to be translated into viral replicase polyproteins pp a and pp ab, which are processed by cl pro and pl pro proteases, respectively. the cleavage products are nsps that form the transcription and replication complex. next, the positive rna strand is translated into a template of negative strand that allows the synthesis of new genomics and sub genomics mrnas. these mrnas are translated and transcribed producing structural and accessory proteins. viral proteins and rna genomic are put together in virions at endoplasmic reticulum and golgi complex, finally transported through vesicles and released from the cell host for infecting new cells. , the covid- symptomatology starts after the virus is installed in host cells. in general, the symptoms include lasting and high fever, dry cough, shortness of breath, muscles aches or tiredness, sputum production, headaches, and a small percentage of individuals presented gastrointestinal symptoms such as diarrhoea and vomit. the incubation period of sars-cov- , from exposure to first symptoms, lasts to days. the pre-symptomatic stage lasts from - days (possibly more) before the beginning of symptoms. the post-symptomatic stage lasts at least days after the beginning of symptoms and days after lowering of fever and improvement of respiratory symptoms. there are many unanswered questions such as the duration of potential immunity of both symptomatic and asymptomatic individuals when infected with sars-cov- . it is noteworthy that efficient strategies to fight the disease should not depend on the symptoms of patients, once asymptomatic or pre-symptomatic subjects can play an important role in the direct and indirect transmission to others, as demonstrated by arons et al. this investigation reports that half the residents in a nursing facility, who tested positive, were asymptomatic when tested and probably contributed to the transmission to other residents. thus, control strategies focused on symptomatic residents were not sufficient to prevent transmission once sars-cov- had been introduced in the facility. laboratory exams of infected patients showed alterations in haematology and biochemistry. it was verified the increase of leukocytes and the reduction of lymphocytes; increased d-dimer and erythrocyte sedimentation rate (esr), prolongation in prothrombin times (pt), followed by increase in bilirubin levels, aspartate transaminase (ast), alanine transaminase (alt), creatinine, lactate dehydrogenase (ldh), protein c reactive (pcr), hypoalbuminemia (low albumin), microcytosis and thrombocytopenia. in addition, inflammatory factors that indicate the immune condition of patients, such as interleukins (il) il- , il- , il- , and il- and the tumoral necrosis factor-α (tnf-α) become elevated. plasma levels of granulocyte-colony stimulating factor (gcsf), protein induced by interferon gamma, monocyte chemoattractant protein- (mcp- ), macrophages inflammatory protein α and tnf-α also display significant increase. potential risk factors or comorbidities that can lead to complications of covid- include elderly individuals (specially above years of age), cardiovascular issues, cerebrovascular, chronic pulmonary diseases, immunocompromising, renal problems, hepatic disease, hypertension, diabetes and obesity. [ ] [ ] [ ] [ ] [ ] [ ] there is a notorious concern regard the medicine administered to fight these comorbidities because some of them can lead to greater expression of ace , such as treatments for diabetes or hypertension. this may favour or even aggravate covid- infection. these facts justify the urgency of research that contemplate alternative therapeutic targets such as calcium channels blockers for hypertensive individuals as suggested by fang et al. however, there is little clinical evidence on the risk of treating covid- patients with therapies that induce greater expression of ace . further investigation is necessary to explore whether these medicines inhibit or trigger the viral entry into the cells of an infected host. a frequent report in epidemiological data regarding the mortality of covid- concerns the sex of individuals, as men are the predominant fatal victims of the disease. therefore, being of the male sex is considered a bad prognostic factor for infection. , a possible explanation lies in the relation between gonadal hormones and the expression of ace enzymes or even an alleged vitamin d deficiency, according to vignera et al. the latter suggest monitoring of serum levels of testosterone and vitamin d in infected patients for a better understanding of the different fatality rates between sexes, including the hypothesis that women's hygiene justify a lesser rate of infection. understanding the pathogenic effects of sars-cov- for the different organs affected by the disease has also been object of investigation, such as gut-lung crosstalk. data from research conducted thus far indicate that the infection caused by sars-cov- is not only capable of causing pneumonia, but it can also damage other organs such as the heart, the liver, the kidneys and organic systems such as the blood and the immune system. , , patients with the extreme form of the disease frequently manifest lymphopenia, , hepatic insufficiency and viral sepsis diagnostic, whose complications can be related to the severity of the cases and the mortality of patients. , there are reports that the eventual death of such patients is due to multiple organ insufficiency, acute respiratory distress syndrome (ards), cardiac insufficiency, arrhythmia and renal insufficiency. , therefore, great attention is necessary to the disease's potential damage to multiple organs and to therapeutic alternatives to fight covid- , given that some of these alternatives can have side effects on organs initially unrelated to the respiratory system, but that may be susceptible to a systemic compromise prompted by the virus once the treatment has begun. hence, it is possible to observe the existence of different forms of aggravating the disease. in this regard, wang et al. recommend the creation of a system to categorize patients with the severe form of covid- . several investigations report that all hcovs, sars-cov, mers-cov and sars-cov- induce exaggerated immune responses in the host, which are associated to the severity of pulmonary pathology and might lead to the development of acute respiratory distress syndrome (ards) or death. the incidence of the extreme form of the infection is associated with cytokine storm syndrome, characterized by high plasma concentration of several interleukin, inflammatory cytokine, inflammatory chemokines, among other factors that cause infiltrated inflammatory in the organs. , , survivors of this excessive response by the immune system can develop long-term fibrosis and pulmonary damages that might culminate in functional injuries to these organs, thus reducing the patient's quality of life. during the development of drugs to fight microorganisms, the adoption of strategies that allow the design of molecules to act against specific biological targets of bacteria, parasites or viruses is preferred. therapies for covs can be divided into several categories, based on specific paths: ( ) covs proteins or functional enzymes that are essential for viral replication; ( ) structural proteins of the virus that prevent its binding to the respective receptors in human cells or its assembly process; ( ) some viral factor that restores the host's inherent immunity and; ( ) host-specific enzymes or receptors, that prevent the entry of the virus in the host cells. , so far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-covs therapies (fig. ) . investigations by wu et al. through bioinformatics, analysed possible sars-cov- therapeutic targets. the proteins coded by this virus were verified and compared to proteins coded by other covs. the results enabled the detection of structural similarities to sars-cov, from which it was possible to conduct homology modelling to build proteins for sars-cov- . among the targets were spike (s) glycoprotein, nsp (rna-dependent rna polymerase -rdrp), enzyme helicase ( cl pro and pl pro ), tmprss, orf a factor and ace presents in the host cells. these targets have pivotal roles in the development of the virus and have great influence on its pathogenicity, hence, some details are provided next. molecular modelling showed that spike (s) glycoprotein is a transmembrane protein of approximately to kda type i whose n-terminal turns to the exterior of the virus and its cterminal segment turns to the interior of the virus. the typical structure of covs is given by the assemble of a bulbous projection of a corolla as trimers of protein s and it is cleaved into two important subunits from the pathogenic perspective: s and s . sars-cov and sars-cov- (s) glycoprotein share about % of amino acid identity and enable the entry of the virus in the host cells. therefore, s glycoprotein present in covs has been considered a promising biological target for antiviral mechanisms. , the moment when the virus approximates the target cell prompts the recognition by the receptor-binding domain (rbd) in the s glycoprotein of its receptor, which leads to the binding to subunit s . next, the subunit s allows fusion of viral and cellular membranes, which enables entry in the cell and the release of viral rna genome. , , some investigations suggest that the strong binding affinity between s protein and ace is essential for viral entry, hence, ace is also relevant for the development of drugs. , molecules that bind to the surface of the virus can destabilize the formation of s glycoproteins and interfere both with the trimerization of the protein and with the continuity of the life cycle of covs. several studies have been conducted on s protein to clarify its sars-cov- structure and its binding process as well as to evaluate its relevance as target for in-silico and in-vitro assays on molecules for anti-sars-cov- therapies. one study conducted by hoffman et al. investigated how the sars-cov- s protein facilitates viral entry in the target cells and how this process could be blocked. results showed that ace is used as receptor for the entry of sars-cov- in host cells and that the spread of this cov in the infected host depends on the activity of tmprss (a cellular serine protease responsible for initiating the binding process between s protein and ace ). this process can be blocked with clinically approved tmprss inhibitor. prior to this, the relevance of tmprss was highlighted in the dissemination of several types of viruses such as influenza a and other covs, which also makes it a relevant target for covid- therapeutic intervention. [ ] [ ] [ ] [ ] [ ] [ ] [ ] binding between s proteins and ace receptors was corroborated through x-ray crystallography conducted by lan et al. to elucidate the interaction between the sars-cov- rbd and ace at a higher resolution. in spite of different interactions with ace , the sars-cov- rbd /ace and sars-cov rbd /ace interfaces share a substantial similarity regarding the surface area, the number of interacting residues and the networks of hydrophilic interactions. such similarity strongly points to a convergent evolution of both sars-cov- and sars-cov rbd structure which improves the binding affinity for the same receptor, the ace . the non-conserved rbd regions in s protein, such as subunit s , could be potential targets for cross-reactive antibodies. considering rbd as a critical region for receptor binding, antibodies that target the conserved epitopes in the rbd are also good candidates for the development of highly potent cross-reactive therapeutic agents against several species of covs, including sars-cov- . investigations on ligands obtained from drugbank . used molecular docking to identify target regions in the pockets of the quaternary structure of sars-cov- s glycoprotein (from protein data bank-pdb). six pockets present in s glycoprotein deserve further investigation in medicinal chemistry due to suitable features for small molecule binding. among the six pockets, the eight best ligand candidates from drugbank were all binding pocket # , which contained residues of amino acids proline, leucine, lysine, asparagine, phenylalanine, glycine, threonine, glutamine, alanine, methionine and tyrosine. one of the best ligands was the drug saquinavir, an antiviral from the class of protease inhibitors, used in anti-hiv therapy. nsps are involved in the rna transcription, translation, protein synthesis, processing and modification, viral replication and infection of the host. significant functional proteins, cl pro , pl pro , helicases and rdrp are important targets for the development of small-molecule inhibitors, due to their biological function and vital enzyme active site. factors nsp , nsp c and orf a are related to assistance to the immune evasion of sars-cov- . interaction between nsp and the host ribosomal subunit induce the degradation of mrna, allowing the virus to develop resistance to the host innate immunity. binding between orf a and bone marrow matrix antigen (bst- ) inhibits activity and blocks bst- glycosylation. these results suggest that all three structures are potential targets for antiviral medicine. proteases pl pro and cl pro mediate the proteolytic cleavage of polypeptides produced by βcoronavirus sars after genome transcription, thus generating other proteins. the cl pro , known as nsp , cleavages several non-structural proteins of importance for viral replication and the maturation of nsps, which is essential in the life cycle of the virus. therefore, it is an attractive biological target for that has been inhibited in-silico by several antiviral, anti-inflammatory and anti-hypertensive drugs from the database zinc (fda). in addition, docking and molecular dynamic studies conducted by qamar et al. showed that non-toxic natural products formed strong bonds with sars-cov- catalytic dyad cis -his of cl pro . moreover, the proteinase pl pro is responsible for cleavages of n-terminus in the replicase polyprotein to release nsp , nsp and nsp , which are essential for correcting virus replication significant to antagonize the host's innate immunity. analysis of the docking model showed that ribavirin formed hydrogen bonds with residues gly , gln , tyr , asp as well as hydrophobic interactions between tyr and the pl pro residue. these results indicate ribavirin as a powerful pl pro enzyme inhibitor, which means it has promising features for anti-covid- therapy given the inhibition of a likely pl pro therapeutic target. helicase (nsp ) has been identified as a promising target for antiviral drug discovery, particularly against sars-cov- . it is a multifunctional protein necessary for a wide range of biological processes, such as genome replication, recombination and dislocation of proteins related to chromatin and nucleic acid remodelling. for covs, helicase is indispensable for viral replication. in studies on molecular modelling, several antibacterial, antifungal and antiviral drugs were analysed and presented elevated affinity to helicase, suggesting it as a good target for sars-cov- therapy. rna-dependent rna polymerase (rdrp -also nominated nsp ) catalyses the viral rna, which performs a key role in the replication/transcription complex of sars-cov- , possibly aided by nsp and nsp complex as cofactor. , nsp has been studied as potential target for several sars-cov and mers-cov inhibitors, due to its importance for viral control. satisfactory results of rdrp inhibition by several ligands were presented in the modelling studies by gao et al. yin et al. those ligands included antiviral analogous to nucleotides, such as remdesivir, which already shows great potential in the treatment of covid- infections. in addition, some nonstructural proteins, including nsp b, nsp e, nsp , nsp , nsp , nsp , nsp , nsp and nsp , also stood out as useful targets due to their significant role in the synthesis and replication of viral rna. cl pro is key enzyme for covs, also called main protease (m pro ), that plays a pivotal role in mediating viral replication and transcription, making it an attractive target for anti-sars-cov- drugs. such claim is reinforced by studies by jin et al. after the virtual screening of n inhibitor. results show that n ( ) is a time-dependent irreversible inhibitor of this enzyme and that a stable covalent bond is formed between n and cl pro . high-throughput screening (hts) was applied to , drugs and drug candidates, demonstrating that ebselen ( ), px- ( ) and carmofur ( ) are all able to covalently bind to cl pro do sars-cov- , with ic that varied from . to . µm (fig. ) . it is likely that a part of the hits identified by hts are bonded to the catalytic cysteine of cl pro through their sulfhydryl groups. in-vitro studies on antiviral activity were performed to corroborate the results. real time quantitative pcr (qrt-pcr) demonstrated that ebselen and n had the strongest antiviral effects at a concentration of μm treatment in sars-cov- infected vero cells. after plaque-reduction assay, the dose-response curves suggested that both could penetrate cellular membrane to access their targets. this result strongly supports the hypothesis that developing a single antiviral agent targeting cl pro or in combination with other therapies could provide an effective first line of defence against all covs related diseases. in relation to sars-cov- therapy, some of the aforementioned targets have been explored for both new drug proposition as well as for sars-cov- drug repurposing. our focus is on this last type, and for each medicine, the putative mechanism of action and viral target will be described trying to find an understandable rational therapy even for an immediate illness situation like covid- pandemic. carmofur ( ). as previously mentioned, sars-cov- is an enveloped virus, whose nucleocapsid consists of a positive rna genome surrounded by multiple copies of nucleocapsid protein. this virus, after entry in the host cell, replicates fast the viral genome with new virion production. the rna replication into the cell host depends on enzymes and substrates for rna synthesis, such as ribonucleotides (adenine, guanine, cytosine or uracil) that have nitrogenous bases in the purine or pyrimidine classes. compounds can mimic these chemical structures and interfere with the formation or use of one of these essential normal organism metabolites. the interference is generally prompted by enzyme inhibition in the biosynthetic pathway of the metabolite or by incorporation, as a false building block, into vital macromolecules such as proteins and polynucleotides. so, this class of therapeutic agents is called antimetabolites. , diverse antimetabolites have been indicated as promising anti-sars-cov- . they are described next. pyrimidine derivatives are aromatic organic compounds necessary for all life forms. examples of pyrimidine derivatives are nitrogenous bases cytosine ( ), uracil ( ) and thymine ( ) (fig. ) . they are found in dna and rna and participate in the metabolic process that involves carbohydrate and lipids. , these heterocyclic rings share two nitrogen atoms at and positions, but display variations between themselves, such as an amine group at -position in the cytosine and a methyl at -position in the thymine. from the pharmacologic perspective, nitrogenous bases are investigated as pharmacophores and are found in the structure of many drugs and experimental substances with various activities, such as antitumoral, antibacterial, antiparasitic, and antiviral. , regarding antiviral activity, there are several approved drugs that are classified as pyrimidine nucleotide biosynthesis inhibitors (pnbi) because, after phosphorylation, they are incorporated either into the dna or into the rna and inhibit hosts or pathogenic enzymes, such as polymerases. therefore, the likely mechanism of action of some pyrimidine derivative drugs has been considered for repurposing. some pyrimidine derivatives with antiviral activity are often formulated as prodrugs. this format solves issues of high polarity in its final structure prompted by the phosphonic acid, which interferes with pharmacological properties and causes low cellular permeability and low oral bioavailability. one compound appointed as potential anti-sars-cov- is the -fluorouracil ( ) ( -fu) (fig. ) , a heterocyclic aromatic amine similar to uracil (u) that presents a fluorine-carbon bond at -position. this compound is used in the treatment of oesophageal cancer, stomach cancer, breast and colon cancer. the similarity between -fu and uracil allows the direct action on nuclei acid as it is incorporated into the genetic material and inhibits replication. tests with -fu as monotherapy confirmed its failure against any coronaviruses. the reason proposed to such failure relied on the fact that coronaviruses rna proofreading activities involve a ' → ' exoribonuclease in the nsp , which removes -fu during replication and metabolism. hence, the combination between -fu and deoxyribonucleoside and deoxyribose was suggested so that, after its insertion in the rna, it escapes rna proofreading and prompt lethality and/or lethal mutagenesis in the virus. despite the proposition of using a widely marketed drug to treat several types of cancer, which means it has well-established efficiency and safety, no other type of test has been made to confirm its efficacy against sars-cov- . therefore, further experiments are necessary to explore -fu potentialities. another antitumoral drug considered for its anti-sars-cov- potential is gemcitabine (gct) ( ) (fig. ) , an analogue of deoxycytidine whose pharmacological action is triggered after the intracellular transformation into triphosphate gemcitabine. the latter competes with endogenous nucleoside triphosphates by incorporation into the genetic material, thus inhibiting dna synthesis. initially, gct was developed for antiviral activity, however, initial results caused it to be redirected for anticancer therapy. it became, then, widely used against non-small cell lung cancer, pancreas, bladder and breast cancers as well. [ ] [ ] [ ] in-vitro analyses of gemcitabine hydrochloride inhibited mers-cov and sars-cov, with a ce of . μm and . μm, respectively, in addition to low cell toxicity for vero e cells. , these data are indicative of a possible activity against sars-cov- , but complementary preclinical investigations are necessary before clinical trials. albeit considered a safe drug under predetermined doses, gct adverse effects are noteworthy and include myelosuppression and disruption of liver functions. in february , the european union (eu) approved baricitinib ( ) as second-line oral treatment for mild to severe active rheumatoid arthritis in adults. a differential feature of baricitinib structure is the azetidine ring bearing an ethylsulfonyl, beyond an acetonitrile group at -position. the same ring binds to the n atom at -position in the pyrazole, which, in its turn, binds to the pyrimidine conjugated to a pyrrole ring. this medicine can modulate human innate and adaptive immune system. based on this property, presumably, one of the important mechanisms of action of baricitinib in the treatment of rheumatoid arthritis is the inhibition of the il- / jak / jak pathway. the promising nature of baricitinib and other small molecule inhibitors against sars-cov- was pointed by richardson et al. through in-silico tests using benevolent ai. the authors evaluated compounds to show that sunitinib ( ) and erlotinib ( ) inhibit ap -associated protein kinase (aak ) interrupting the virus entry to the cells and the intracellular assembly of new viral particles (fig. ) . regarding these two antitumor drugs, it is known that sunitinib is an oral oxindole multitargeted kinase inhibitor that inhibits certain tyrosine kinases including vascular endothelial growth factor receptors (vegfr types and ), platelet-derived growth factor receptors (pdgfr-α and pdgfr-β), stem cell factor receptor (kit), fms-like tyrosine kinase- (flt ), glial cell-line derived neurotrophic factor receptor (ret) and the receptor of macrophage-colony stimulating factor (csf r). concerning erlotinib, it was developed as reversible and highly specific small-molecule tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of epidermal growth factor receptor (egfr), thereby inhibiting autophosphorylation and blocking downstream signalling. however, these oncological drugs have serious adverse effects such as diarrhoea, loss of appetite and skin rashes. in addition, high doses of these medications can aggravate those effects. in relation to baricitinib, its anti-sars-cov- potential was explained in three ways: aak inhibition like sunitinib and erlotinib; the kinase associated to cyclin g, which is another endocytosis regulator; and the janus kinase, that inhibits the action of cytosines that triggers the inflammatory process. because baricitinib can inhibit aak at the therapeutic dose ( or mg/day), the drug is indicated for clinical trials. it is highlighted that baricitinib is not indicated for patients with neutropenia or lymphopenia, once it lowers rates of neutrocytes and lymphocytes, which can lead the disease to progress and increase anaemia. furthermore, treatment with baricitinib can reactivate varicella-zoster, herpes simplex and epstein-barr viruses. this implicates in a conflict between the potential effect and the adverse effects of baricitinib against covid- to prevent aggravating the disease and the mortality of patients. an analogue of adenosine, galidesivir (gsv) ( ) is a broad-spectrum antiviral drug that blocks viral rna polymerase by replacing a natural nucleotide with galidesivir triphosphate. this alteration prompts changes in electrostatic interactions and prevents the formation of the rna elongated strand. , adenosine and gsv differ in that galidesivir has one carbon at -position in the pyrimidine ring and nitrogen in the ribose ring, whereas adenosine has one nitrogen in the former and oxygen in the latter (fig. ) . it is noteworthy that gsv has not been approved for clinical trial and is an experimental drug in advanced stages of development. gsv was first developed against hepatitis c (hcv) but first clinical trials were conducted to ensure its safety (in healthy individuals) and efficacy against yellow fever. furthermore, gsv displayed in-vitro and in-vivo antiviral activity against filoviridae, alphavirus, bunyavirus, arenavirus, paramyxovirus, flavivirus, orthomyxovirus, picornavirus and sars and mers coronaviruses. , , recent in-silico studies have shown the existence of a strong bond between gsv and sars-cov-rdrp to demonstrate the capacity of alterations in rna polymerase, which can eradicate the virus. although, preclinical and clinical trials are necessary to either confirm or deny this hypothesis. it is noteworthy that investigations have pointed the inactivity of gsv against sars-cov- at concentrations lower than mΜ. the existence of antiviral activity against other coronaviruses indicates that more investigations on gsv against sars-cov- are required to elucidate its potential activity in advanced testing. next, sofosbuvir (sbv) ( ) is an example of successful nucleotide prodrug, approved by the food drug administration (fda) since , against chronic hepatitis c infections. sbv is also combined with other antiviral drugs, such as ledipasvir, velpatasvir and voxilaprevir. , the structural similarity between sbv (fig. ) and uridine allows that drug to act on hcv rdrp, incorporate itself into the viral rna and terminate the synthesis of the nucleotide sequence. structural analysis of sbv revealed that its elevated potential is partly due to the presence of the 'phosphate, which terminates the primary enzyme transformation monophosphate inhibitor. the antiviral activity has been explored against other viruses through in-vitro and in-silico studies and shown potential for inhibiting the dengue virus, yellow fever, telbivudine (tbv) ( ) (fig. ) is a thymidine nucleoside analogue used with specific activity against the hepatitis b virus (hbv). it starts acting after phosphorylation by cellular kinases, which results in the active metabolite, telbivudine '-triphosphate, enabling dna polymerase and inhibiting viral replication. the hydroxyl at -position in the sugar β-l- '-desoxirribose provides specificity to hbv polymerase. suggesting repurposing tbv to fight covid- was prompted by virtual screening to find drugs that act on viral m pro . among other results were ribavirin, tbv and two vitamins, cyanocobalamin (b ) and nicotinamide (b ). researchers suggest that these four drugs can be combined and used against covid- , once they are safe, marketed and approved by the authorities. notwithstanding, the suggestion of repurposing these drugs requires more information, including on drug interaction parameters. in spite of well-tolerated and safe for monotherapy, associating tbv and ribavirin, another antiviral drug, can increase hepatotoxic activities of tbv. , it is also important to consider the elevated risk of resistance to tbv, which and pyrimidine derivatives drugs: -fluorouracil ( ); gemcitabine ( ); baricitinib ( ); sunitinib ( ); erlotinib ( ); galidesivir ( ); sofosbuvir ( ) ; telbivudine ( ). purine is a and -membered bicyclic ring. similar to pyrimidines, purine derivatives are essential to life. they are basic constituents of nitrogenous bases adenine (a) ( ) and guanine (g) ( ) (fig. ) . the safety of rdv for humans infected with ebov was evaluated in the democratic republic of congo. results confirmed its safety but did not point rdv as the best therapeutic option, once its mortality rate reached % of treated group. it has been proved that gs- inhibits epidemic and zoonotic hcov. of viral loads and weight loss in murine. therefore, rdv is a potential drug to treat mers-cov infections. regarding covid- , rdv was used to treat the first us case. the patient was years old, had slight cough, low fever and no evidence of pneumonia at day of the disease. when the clinical symptoms became worse, the patient was given vancomycin and cefepime. as the symptoms worsened, intravenous treatment with rdv was administered at day , and vancomycin and cefepime were no longer administered. at day , the patient displayed clinical improvement, unfortunately details on the doses and duration of treatment were not provided. after this first case, a clinical trial with a larger number of covid- patients was conducted. this study of efficacy involved patients infected with sars-cov- who displayed saturation equal or inferior to % while they were breathing ambient air or receiving oxygen support. the treatment lasted days, patients were given mg intravenously on day , followed by mg daily for the remaining days of treatment. follow up of patients treated for days indicated that, after the first dose of rdv, % improved oxygen support whereas % of patients got sicker, % were discharged and mortality rate was %. the most common adverse events ( % of patients) were increased hepatic enzymes, diarrhoea, rash, renal impairment, and hypotension. some limitations were noted in the study, such as the small size of the cohort, the short duration of follow-up and the lack of information on the patients. hence, the efficacy of rdv requires validation by the ongoing randomized, placebo-controlled trials. one advantage of repurposing rdv is the availability of data on safety and pharmacokinetics, which were obtained previously at phase clinical trial. in addition to the promising results shown by rdv, other purine analogues have been investigated for sars-cov . ganciclovir (gcv) ( ) also named, according to its chemical structure, -( , -dihydroxy- -propoxymethyl) guanine (fig. ) , is a guanine analogue, similar to acyclovir, except for the bond between the methyl group and one hydroxyl. gcv inhibits the human herpesvirus and is also indicated in the treatment of cytomegalovirus infections related to acquired immunodeficiency syndrome (aids). gcv is converted into ganciclovir triphosphate by cellular kinase, which inhibits dgtp and disrupts viral dna synthesis due to substitution of various adenosine bases in the dna chain. recently, gcv was used to treat covid- patients in china. the drug was administered with other antivirals, such as oseltamivir and kaletra. as a descriptive study, the relation between gcv as key factor in clinical outcome of the patients ( % of which were discharges) was not possible. therefore, gcv efficacy as monotherapy or part of combined therapy is yet necessary for more robust investigations. valganciclovir ( ) (fig. ) is the antiviral prodrug of gcv taken by mouth. it is indicated for the same treatments as gcv (cytomegalovirus in people who have acquired immunodeficiency syndrome, gastrointestinal disorders related to aids). the drug has great bioavailability and is converted by hydrolysis into ganciclovir. using valganciclovir in its oral form enables clinical treatment and makes patients more comfortable. [ ] [ ] [ ] the mechanism of action is the same of gcv. valganciclovir was computationally evaluated for covid- . the assay with the main proteins coded for sars-cov- allowed the determination of possible binding targets, of which were proteins and host targets. valganciclovir, one of the drugs used in the study, was presented as a possible anti-sars-cov- therapeutic drug due to its high binding affinity to two wellestablished viral targets. the first target was pl pro , indispensable enzyme in viral replication; the second, rdrp, conserved nsp in coronavirus, which is vital for its replication/transcription. therefore, valganciclovir could be a significant antiviral drug to treat sars-cov- . but there are no clinical reports on valganciclovir used to treat covid- in addition to what has been reported about gcv. hence, its efficacy is yet to be confirmed as anti-sars-cov- therapeutic. tenofovir (tfv) ( ) is another adenine analogue pointed as promising covid- therapeutic (fig. ) , it is also called tenofovir disoproxil fumarate or alafenamide tenofovir (taf). approved by the fda in , tfv is a prodrug used to treat hiv and cases of nucleoside resistance. tfv is an analogue reverse-transcriptase inhibitor (ntrti). inside cells, tfv is phosphorylated and competes with deoxyadenosine '-monophosphate (d-amp), thus preventing the formation of dna. once incorporated into a growing dna strand, it causes premature termination of dna transcription and prevents viral replication. , modelling and docking studies evaluated the antiviral effects of tfv and verified a strong bond to sars-cov-rdrp, which can disrupt this polymerase and terminate the viral infection. however, in-vitro tests showed that tfv lacks apparent antiviral effect at concentrations inferior to μm for sars-cov- . in spite of lukewarm in-vitro and in-silico outcomes, an ongoing clinical study on tfv (chictr ), expected to end in june , aims at assessing the effect of the combination tenofovir + emtricitabine (cytidine analogue) related to lpv/r in covid- patients. in addition to the efficacy of treatment, clinical trials can validate the prevalence of adverse effects related to the toxicity of tfv in patients. tfv is also a powerful nephrotoxic drug causing damage to proximal tubular cells. in spite of that, interrupting the treatment is sufficient to improve adverse effects, which makes monitoring of patients essential. heterocyclic compounds with different heteroatoms such as nitrogen, sulphur and oxygen can present different pharmacological properties. one such property is to serve as analogue of nitrogenous bases of nucleic acids, such as triazoles, which have a five-membered ring of two carbon atoms and three nitrogen atoms. this aromatic ring can assume two isometric forms, , , -triazole and , , -triazole. the former is stable under acid and basic conditions and becomes more reactive when binding to electronegative elements. , triazoles are important and stand out for their various biological activities, such as anticancer, antituberculosis, anti-inflammatory, antimicrobial, and antiviral. specifically for the latter action, triazole-based derivatives have shown promising invitro activity against coronavirus, probably by cl pro inhibition. ribavirin ( ) (fig. ) is a powerful triazole-based antiviral analogue to guanosine. it presents a wide range of pharmacological activities related to several viruses, for instance: herpes simplex virus, human immunodeficiency (hvi- ), influenza, respiratory syncytial (rsv) and hepatitis c. , the drug was initially used in to treat syncytial virus in children, generally combined with interferon (inf). however, ribavirin treatment presents undesirable adverse effects, like lowering of haemoglobin, which limit clinical use. its action mechanism relies on the inhibition of enzyme inosine monophosphate dehydrogenase, necessary in the synthesis of guanosine triphosphate, which prevents viral dna and, mainly, rna replication. the necessary concentration for in-vitro inhibition of rsv and influenza ranges from - μg/ml. site similar to other sars-pl pro inhibitors. the formation of hydrogen bonds and π-π stacking were also predicted. these findings suggest that ribavirin as a powerful pl pro inhibitor. nonetheless, investigation on triazole derivatives for anti-sars-cov- therapy are still preliminary. on the other hand, favipiravir (fpv) ( ) (fig. ) is a prodrug, approved in in japan, to treat cases of influenza a and b that displayed resistance to first line drugs. it has provided results that indicate a promising character and is currently undergoing clinical trials against covid- . its antiviral efficacy has also been investigated in different countries to fight ebola and lassa, for example. the molecular structure of the drug consists of a pyrazine heterocyclic ring with fluorine at -position, carboxamide at -position and a double bond between oxygen and carbon , which renders its analogue to guanine ( ). , the metabolization of the prodrug into its active form, favipiravir-ribofuranosyl- '-triphosphate, requires intracellular ribosylation and phosphorylation. fpv therapeutic targets are rdrp enzymes, necessary in viral transcription and replication, and its inhibition blocks synthesis of viral rna for a spectrum of viruses, including human coronavirus. a different investigation compared patients treated with fpv plus interferon inhalation to lpv/rtv. patients under fpv therapy responded better to the progression of the disease with accentuated viral depuration. also, the incidence of nausea and vomit was higher for lpv/rtv. in addition to these clinical trials, the antiviral activity of fpv against sars-cov- was also evaluated but no clear antiviral effect was noted for doses lower than μm. an in-vitro study using molecular docking focused on the binding properties of sars-cov- protein structures to antiviral agents, including oseltamivir. the study showed that molecules form bonds to sars-cov- crystal proteins. however, data did not show oseltamivir as the best structure because lopinavir, asunaprevir and remdesivir interacted with more than two protein structures in the virus. hence, they are likely more promising than oseltamivir. notwithstanding, we suggest further look into oseltamivir against other enzyme targets since different studies achieved positive results regarding its use as anti-sars-cov- . nelfinavir ( ) (fig. ) is a safe anti-retroviral drug largely used for hiv- protease inhibition with strong in-vivo activity. generally, nelfinavir is combined with other anti-retroviral medication as part of a highly active antiretroviral therapy (haart) that reduces significantly the viral load by increasing cell number to mm - cd (+) lymphocytes. the drug is prescribed for children, young individuals, adults and pregnant women. , nelfinavir and its active metabolite m strongly bind to serum proteins, displaying optimal tissue distribution. a frequent adverse effect is light to moderate diarrhoea, reported for to % of patients. the sars-cov outbreak in several countries triggered the search for antiviral drugs active against the disease. among the drugs likely to inhibit sars-cov, nelfinavir stands out in all assays. the mechanism of action suggested for nelfinavir involves preventing sars-cov replication after its entry in the host cell and disrupting virion production. based on results from previous studies as well, nelfinavir was considered a likely therapy for covid- after its indication for clinical trials as a promising anti-sars drug. recently, , drugs were evaluated for their binding affinity to sars-cov- m pro . among the compounds, drugs were selected based on the docking score and three-dimensional atazanavir ( ) (fig. ) is an antiretroviral drug protease inhibitor used to treat hiv infections with in-vitro inhibitory concentration of , - , nmol. compared to other protease inhibitors, atazanavir has the advantage of allowing a daily posology regimen with a favourable metabolic profile and low frequency of adverse effects. , several hiv- resistant to protease inhibitors are still sensitive to in-vitro atazanavir, which is considered safe and well tolerated. the atazanavir acts to inhibit hiv- protease, which is indispensable in the processing of polyproteins precursors of viral structures and prevents the formation of infectious and mature viral particles. the good activities reported for this drug as well as the search for safe and fast therapy for captopril ( ) (fig. ) is an angiotensin-converting enzyme inhibitor (acei). it is a zinc metallopeptidases inhibitor that converts angiotensin-i into angiotensin-ii, an essential function that regulates arterial pressure. it is predominantly indicated as vasodilator in patients with cardiac insufficiency. this drug was suggested as potential antibiotic capable of inhibiting zinc succinyls/dipeptidase by blocking its zinc catalytic center. , tolerance to captopril has been largely investigated; its single dose by mouth is well-established and confirms the pharmacological activity in the short term ( - minutes) at the cellular level. this capacity is related to captopril transport mainly through plasma proteins such as albumins with absorption rate between - %. reported adverse effects are neutropenia, proteinuria, dysgeusia and cough, but less frequent for low doses. , some investigations have suggested captopril as possible covid- treatment. serafin et al. indicated captopril as potential for inhibiting the bond between human sars-cov- and ace- and reduce severe pneumonia symptoms. in-silico studies using molecular docking were conducted with fda-approved drugs capable of binding to the main active site in proteinase cl pro . two drugs were identified as ligands for the enzyme active site: captopril and disulfiram. the former binds to the active site at the same position of n inhibitor (a standard inhibitor that reacts irreversibly in the same site with cl pro cys ). it is, thus, suggested that captopril binds to the same site of n , obstructing the function of cys -his catalytic dyad. captopril probably inhibits the enzyme in two stages. initially, it establishes non-covalent bonds to sites in the enzyme targets, then, a reaction takes place between the critic groups, which results in a more stable inhibitor complex. the hypothesis is that captopril can bind covalently to cl pro cys . although the potential of captopril on the enzyme has been demonstrated, therapeutic use against covid- is controversial, once the drug induces overexpression of ace- -the main receptor used by sars-cov- to entry the cells. therefore, combination with other drugs, such as angiotensin-ii receptor blockers, needs analysis to clarify the effects of captopril in covid- treatment. the cyclosporin a (csa) ( ) (fig. ) is isolated from the fungus beauveria nivea and was approved for use by the fda in . this drug has been used for decades to prevent organ rejection and to treat t cell-associated autoimmune diseases such as behcet's disease, psoriatic arthritis, lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus or interstitial lung disease. such drug exerts its immunosuppressive function and anti-inflammatory effects by inhibiting the transcription of genes required for t cell proliferation, notably interleukin- . [ ] [ ] [ ] due to the severity of covid- , csa can be potential to prevent hyperinflammation-induced lung injury. in this regard, it is known sars-cov nps induces the expression of interleukin- via nuclear factor of activated t cell (nf-at) activation, which can trigger the cytokine storm seen in patients with severe covid- status. another advantage presented by csa in relation to other antiinflammatory drugs is its already known anti-cov action against all genus, including sars-cov, [ ] [ ] [ ] at low and non-cytotoxic micromolar concentrations verified in cell culture assays. this antiviral property is thought to be mediated by the inhibition of cyclophilin-a-dependent viral assembly as well as inhibition of the nf-at pathway or even by genetic or pharmacological specific inhibition of cyclophilin-d, hindering the viral replication. , as already reported, sars-cov and sars-cov- are very similar ( . % sequence identity). teicoplanin ( ) (fig. ) is an antibiotic used against gram-positive bacteria with major compounds at different side chains. it prevents polymerization of peptidoglycans and inhibits the development of the cell-wall, thus prompting cell death. it is a big molecule that has displayed antiviral activity on an early stage of the viral life cycle by inhibiting the low-ph cleavage of the viral spike protein by cathepsin l in the late endosomes thereby preventing release of viral rna and replication. this compound has already shown inhibitory activity against ebola virus, mers-cov and sars-cov. recent investigations have suggested teicoplanin as alternative treatment for covid- after an in-vitro assay achieved ic value of . µm, thus proving its efficacy against sars-cov- . these results need to be confirmed through randomized clinical trials, which are still to be conducted. , , using antibiotics to fight viruses, albeit completely ignored, can become useful to treat covid- . , some studies report the possibility of repurposing drugs like terconazole, which displayed good in-vitro results against mers-cov and sars-cov, dasabuvir ( ) (fig. ) is a drug from the naphthalene class and phenyl-naphthalene subclass due to the bond between its naphthalene ring and a phenyl group. dasabuvir is a first line drug used as combined therapy for chronic hepatitis c. dasabuvir is a non-nucleoside inhibitor that binds to nps b (non-structural protein b -rdrp) and induces conformational change that makes rdrp incapable of elongating the viral rna. repurposing of this drug can be useful as sars-cov- therapy due to its antiviral activity. dasabuvir was subjected to docking studies against sars briefly, dasabuvir forms π-cation interactions with lys a (present in the ace ), π-π interactions with phe b (s protein residue) and hydrogen bonds to ace residues glu a and asp a and gly b and ser b (s protein residue). the authors highlight the importance of repurposing drugs as new therapeutic alternatives not only for the new coronavirus but for the next viral outbreaks. darunavir ( ) (fig. ) is a benzene derivative that has been evaluated for repurposing against covid- . this drug is an antiviral used in the treatment of hiv- infections. it provides a great genetic barrier to resistance and is highly active against resistant strains of hiv- that are not susceptible to other protease inhibitors. darunavir is administered orally as pills or suspension and is often used with low doses of ritonavir as part of a combined art protocol. its mechanism of actions works by protease inhibition. darunavir establishes high affinity bonds to hiv- protease forming a stable complex, thereby selectively inhibiting polyprotein gag-pol coded by the virus. this prevents the formation of mature viral particles. fda-approved drugs against cl pro , rdrp, helicase, exonuclease ′ a ′, endornase e ′-o-ribose methyltransferase. among the best drugs in the assay, darunavir was a surprise because, despite inhibiting viral proteinase, the study showed that it binds to the replication complex components of sars-cov- with inhibitory potency kd < nm. one example is rdrp, whose kd value was . nm and exonuclease ′ to ′ with k d value of . nm. a docking study was conducted by sang et al. as in-silico evaluation of anti-hiv drugs in their interaction capacity to proteinase cl pro . results suggest that all drugs have higher binding affinity to sars-cov- cl pro than to the homolog sars-cov proteinase. among the evaluated drugs, indinavir and darunavir displayed the highest docking scores, therefore, they were subjected to molecular dynamic (md) simulations, free binding energy calculations and molecular mechanics poisson-boltzmann surface area (mm-pbsa) to detail molecular interactions between inhibitors and proteinase. the data suggest darunavir had better binding affinity to sars-cov- cl pro with binding affinity of - , kj / mol. in addition, darunavir bind to sars-cov- cl pro via contact residues and to sars-cov cl pro via residues. this difference explains the lower binding energy values between darunavir and sars-cov cl pro . it was also noted hydrogen bonds between darunavir and sars-cov- cl pro but none for indinavir and this proteinase. because hydrogen bonds are important in the stability of the inhibitor-enzyme complex, darunavir is probably more promising against covid- . finally, a different in-silico assay was conducted by pant at al. to assess a large variety of compounds ( ) from several data banks and potential compounds from fda-approved drugs. the compounds were tested against sars-cov- cl pro . darunavir was among the best fda-approved drugs, with a score of - . . all data collected from in-silico studies still require experimental studies to validate the anti-sars-cov- activity of darunavir. a research conducted by dyall et al. performed a robust in-vitro assay and showed that the drug dasatinib ( ) (fig. ) , a kinase signalling inhibitor developed to treat human cancers, inhibited mers-cov and sars-cov, exhibiting ec values . and . , respectively. this study also revealed that kinase signalling may also be important for replication of this hcovs. nevertheless, the authors reported that dasatinib may be valuable against coronaviruses infections if a dosing regimen that minimizes immunotoxicity while still blocking viral replication can be defined. results indicated this drug as a likely therapeutic alternative against sars-cov- infection. an in-silico study carried out by qiao et al. showed that dasatinib, among others, is one of the most promising drugs for the inhibition of sars-cov- cl pro . more preclinical and clinical studies are required to prove whether dasatinib is really promising for covid- patient treatment. imatinib ( ) (fig. ) is an oral anticancer agent that inhibits the activity of some tyrosine kinases, most prominently the bcr-abl fusion oncoprotein (whose overactivation can lead to chronic myeloid leukaemia, cml), c-kit (involved in gastrointestinal stromal tumours development), platelet-derived growth factor receptor (pdgfr), and the native abl kinase, which has a ubiquitous expression and plays important roles in several biological processes. in addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during cov infection), by interfering with virus-cell fusion, and other rna viruses including coxsackie virus, hepatitis c virus, ebola, among others, mainly by blocking viral entry or egress from the host cell. besides, this drug showed activity against sars-cov and mers-cov, both phylogenetically related to sars-cov- . in this regard, it is reported that imatinib has anti-cov activity in two points of the virus life cycle. in the early phases of infection, it inhibits virion fusion with the endosome and subsequent release into the cytoplasm, thus preventing viral entry and viral replication via abl-mediated cytoskeletal rearrangement. in a later phase of the infection, abl protein expression, which is inhibited by this drug, enables sars-cov and mers-cov replication, which suggests that abl is a new host cell protein required for viral growth. furthermore, evidences suggest that imatinib can modulate the immune response by sundry mechanisms, [ ] [ ] [ ] for several diseases, such as rheumatoid arthritis, asthma, and crohn's disease. this information insinuates that such drug might perform its potentially beneficial immunomodulatory role as a treatment alternative for covid- pneumonia. in addition, the use of imatinib as treatment appears to be reasonable from an economic point of view and its high availability in hospitals, since this drug is well tolerated and the risk of severe adverse effects is relatively low, especially in short-term administration. it is also recognized that adverse effects, mostly mild to moderate in intensity, will be easily controlled by dose reduction or discontinuation. in light of this information, tatar and turhan used the docking methodology to better understand the mechanism of inhibition of the sars-cov- n protein with antiviral compounds. based on this study results, imatinib was one of the highly binding affinities performed against the aforementioned target, with the lowest micromolar ki values among the compounds evaluated. in line with this study, an in-vitro research carried out by weston et al. found fda approved drugs that inhibited sars-cov- at non-cytotoxic concentrations. the authors indicate imatinib as one of the hits, since it exhibited ic value of . μm. they subsequently determined the mechanism of action, demonstrating this drug inhibits fusion of covs with cellular membranes, precluding their entry. this result indicates imatinib use against sars-cov- . however, its efficacy and safety need to be better confirmed in further preclinical and clinical trials in order of elect him as candidate drug in the treatment of covid- . synthesized for the first time by jean francois rossignol in the beginning of the s, the acetyloxy-n-( -nitro- -thiazolyl) benzamide, sold under the name nitazoxanide (ntz) ( ) (fig. ) , is the result of a structural modification in the antiparasitic niclosamide when a benzene ring is replaced with a nitrothiazole. , developed and sold as antiparasitic, ntz is also a first line broad spectrum antiviral with good results against parainfluenza, coronavirus, rotavirus, hepatitis and other respiratory infections. [ ] [ ] [ ] [ ] following oral administration, ntz is absorbed in the intestine, where it is rapidly hydrolysed by plasma esterase into the active metabolite tizoxanide. the mechanism of action of ntz varies according to the pathogen. in relation to antiviral activity, ntz blocks the maturation of the viral hemagglutinin at the post-translation stage in treatments against influenza. in treatments against hcv (hepatitis c), it activates protein kinase r (pkr), which leads to phosphorylation of the eukaryotic initiation factor- α thereby preventing translation. cao et al. conducted an in-vitro evaluation of ntz against recombinant murine coronavirus expressing the firefly luciferase (mhv- afls). the strand was pivotal to triage the drugs with likely anti-cov activity. the first assay resulted in molecules among which was ntz. the antiviral effect of ntz verified for mouse astrocytoma (dbt) and fibroblasts ( cl- ). the dbt cells infected with mhv- afls were treated with ntz at µm for hours, after which the viral titer (tcid ) was determined and viral n protein was subjected to western blot. results show the strong inhibitory effect of ntz on the viral titer. in-vitro studies on ntz or its metabolite tizoxanide were also conducted to verify efficacy against different coronaviruses. the replication of canine coronavirus (strain k ) in a cells, for instance, was blocked by the tizoxanide with ic of µg/ml. on the other hand, ntz inhibited the viral n protein in bovine coronavirus l (βcov-l ) and human enteric coronavirus (hecov- ) with approximate values of . µg/ml. , ntz is also responsible for inhibiting pro-inflammatory cytokines, such as tnf-α, il- , il- nitazoxanide ( ) drugs. more recently, molecules with a quinoline group have been widely investigated as treatment for the new coronavirus (sars-cov- ), such as chloroquine (cq) ( ) and hydroxychloroquine (hcq) ( ) (fig. ) that belong to the quinoline class and aminoquinoline subclass. both are quickabsorption synthetic drugs approved to treat malaria (plasmodium falciparum) by several regulating agencies in the world. cq and hcq are water soluble; the latter is more soluble due to presence of hydroxyl group. they are currently used to treat autoimmune diseases such as lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis as they have immunomodulatory and antithrombosis properties. [ ] [ ] [ ] therefore, these drugs could be useful against covid- due to the elevated levels of cytokine caused by cov infections in humans. the mechanism of action of cq for anti-malarial treatment is not entirely clear, but the interference with the digestion of haemoglobin by the parasite has been suggested. hcq has a similar mechanism, however, in regard to sars-cov- , clinical trials showed it to be safer. [ ] [ ] [ ] therefore, hcq, rather than cq, is used against sars-cov- . recent studies report antiviral activity of cq and hcq as they impair viral entry and release in different in-vitro and in-vivo models. , a factor that can also justify viral mechanisms is the aminoquinoline bioaccumulation in the tissues, as defended by patil, singhal & masand. a factor that facilitates viral replication is the acidic ph of endosomes, lysosomes and golgi complex of the host. thus, cq is promising because it increases the ph of intracellular vacuoles, binds to the cellular receptors, changes the glycosylation and because of its selective and reversible immunomodulator effect on human cd + t cells. hcq exerts similar mechanism of action: a) increases the ph; b) modulation of activated immune cells; c) reduces the number of proinflammatory cytokine and other mediators to control inflammation. it has also been suggested by roldan et al. a likely involvement of hcq in iron homeostasis during sars-cov- infection, which is a similar mechanism to other viral infections in humans. [ ] [ ] [ ] the little difference between the therapeutic and the toxic dose of cq is also known, and poisoning is related to cardiovascular complications that can be fatal. using either cq or hcq, then, requires strict prescription and self-medication is not advised. based on the recovery data, it was concluded that hcq is not effective in patients admitted with covid- . this highlights the importance of randomized clinical trials and the collection of clear data on the efficacy and safety of medications. ivermectin ( ) (fig. ) is an fda-approved broad-spectrum antiparasitic agent used in the treatment of tropical diseases, such as onchocerciasis, lymphatic filariasis, strongyloidiasis and lice. there is also evidence of its effectiveness in the management of myiasis, trichinosis, malaria, leishmaniasis, trypanosomiasis, chagas disease and schistosomiasis as well as bed bugs, inflammatory skin lesions, epilepsy, neurological diseases, tuberculosis and some cancers. it is known that ivermectin is capable of inhibiting the bond between a virus and the nuclear transport mediated by the superfamily of importin proteins (imp α/β ) based on the fact that sars-cov- is a rna virus deeply related to sars-cov, studies on sars-cov proteins have revealed a potential role for imp α/β during infection in signal-dependent nucleocytoplasmic shutting of the sars-cov nucleocapsid protein. furthermore, the sars-cov accessory protein orf has been shown to antagonize the antiviral activity of the stat transcription factor by sequestering imp α/β on the rough er/golgi membrane. considering the ivermectin nuclear transport inhibitory activity, such drug is widely believed as a promising therapeutic approach against sars-cov- . recently, caly et al. a different mechanism of action that consolidates the use of ivermectin against covid- is its immunomodulatory property. the inflammatory response (proinflammatory cytokine) is exaggerated in patients with the extreme case of the disease, which is likely explained by the hypoxiainducible factor (hif- α) that is activated by the virus when no inhibitory medication is administered. this understanding can be explained by the study conducted by kosyna et al. , whose lab tests with and without ivermectin aimed to examine whether the properties of the bond between hif- α and imp α/β and between hif- and nuclear localization signals were affected by the hypoxia mechanism on the cellular level. the authors concluded that ivermectin inhibited both imp α/β and hif- α. regarding this hinders its indication and clinical decisions. thus far, data indicate ivermectin is useful at the early stages of the disease, even the epidemiologic profile of covid- shows significant differences regarding the age of patients for the affected countries and patients with comorbidity. therefore, large scale randomized clinical trials are necessary to standardize clinical, laboratory and image evaluations, as well as combined drug therapy with vitamins and zinc, for example. financially, ivermectin is inexpensive and its doses and protocols are well established for different purposes. in addition, this drug has little side effects. indole, also named benzo [b] pyrrole, is a planar bicyclic heteroaromatic, whose ten π electrons move across its structure making this chemical group behave as a weak base. , the indole ring is the most abundant heterocyclic in nature and is commonly found in biologically active natural products, such as vegetables and seafood. it is also present in the structure of the essential amino acid tryptophan, which interferes with protein synthesis and with the regulation of physiological mechanisms such as precursors for serotonin and vitamin b . and adding the indole ring to spirothiazolidinones conducted to better influenza a/h n inhibition. now, the antiviral activity of indole and its derivatives for covid- therapy is supposed. arbidol ( ) is also a promising drug to fight covid- (fig. ) . this drug is classified as antiviral and has been used for there is high expression of eca , identified as human receptor for virus entry. notwithstanding, a different clinic trial concluded that arbidol monotherapy is best for the patient than lpv/rtv. despite the results, both investigations recognize their own limitations due to small cohort size and lack of placebo-control group. according to the authors, these limitations are inherent to pandemic times when placebo-control groups are difficult to conduct due to life-threatening conditions. most studies with arbidol use mg/ *day , , following the chinese guidelines. previous investigations on the pharmacokinetics of arbidol in healthy chinese patients showed that a single mg oral dose is sufficient for cmax de ~ , μm. this value was obtained through invitro assays that pointed the drug as effective and promising against sars-cov- . hence, clinical trials are still necessary to confirm the efficacy of arbidol at elevated doses to treat covid- . rizatriptan (rzt) ( ) (fig. ) is used to treat migraine and is a selective receptor of serotonin ( -ht) type b and d, structurally and pharmacologically related to other selective antagonists at these receptors. its structure is based on an indole ring replaced with methyltriazole at -position and unsubstituted ethanamine replaced with methyl at -position, the substitution sites are the same of melatonin (mlt). after virtual triage through molecular dock at spike-ace interface, ligations π-cation, interactions π-π and hydrogen bonds were identified between rzt and the sars-cov- protein complex. as one of the outstanding compounds in the analysis, in-vitro tests are still necessary. it is noteworthy that overdosing of the drug can trigger dizziness, fainting, cardiac issues, hypertension, bradycardia and vomiting. despite the safety of the drug in regular doses, there are no reports on either in-vitro or in-vivo tests to support the theoretical data and the antiviral action thus far. one last indole derivative that could be repurposed to treat covid- is melatonin (mlt) ( ) (fig. ) . mlt is classified as a hormone and nutraceutical, as it is naturally produced by the pineal gland and released into the bloodstream. it regulates the sleep-wake cycle as well as our mood, learning and memory, fertility, reproduction and the immune system. from a chemical perspective, it is an indole derivative with a methoxy group at -position and one ethylacetamide at -position. regarding its antiviral potential, mlt acts indirectly through anti-inflammatory, antioxidant and immune modulating activities. an investigation with murine infected with semliki forest virus (sfv) and west nile virus (wnv) showed the efficiency of mlt in reducing mortality rates for these viruses as well as in reducing the levels of pro-inflammatory cytokines. the anti-inflammatory and antioxidant properties of mlt point to its antiviral effects in humans. based on computational data, zhou et al. suggested using combined medication to fight sars-cov- . one such combination involves mlt plus mercaptopurine in synergic action against the following targets: pl pro , ace , c-jun signal and anti-inflammatory vias. therefore, experimental studies on modifications of ace pathways caused by mlt are useful to understand this drug. on the other hand, it has been suggested that using this neuro-hormone can mitigate the extreme form of the disease, the acute respiratory syndrome that has caused most deaths by sars-cov- cases. despite its safety for humans, the lack of data on the relevance of its use for covid- patients emetine ( ) is an approved anti-protozoal drug used against amebae with reported inhibitory activity for enterovirus infections, zika virus and ebola by interfering with the process of viral replication and entry in host cells. emetine is an isoquinoline alkaloid that presents methoxy groups in its structure (fig. ) . studies also confirm emetine has in-vitro activity against coronaviruses, including sars-cov and mers-cov. assays to clarify the activity of these drugs and the mechanism involved in the antiviral action of emetine both in isolation and combined to other drugs. another alkaloid candidate to repurpose against covid- is homoharringtonine (hht) ( ) (fig. ) . hht is an fda-approved drug in semi-synthetic form known as omacetaxine. this drug displays antitumoral activity in the treatment of myeloid chronic leukaemia. the mechanism of action implicates the ribosomal bond to prevent protein translation. in addition to antitumor activity, there are data in the literature that describe antiviral activity of hht against several types of viruses including covs. , a recent in-vitro evaluation of anti-sars-cov- activity displayed ec of . µm. however, the mechanism of action is not yet clear, which demands further investigation on ideal doses of hht to achieve the clinical results expected of a covid- therapeutic drug. the first reports on tetraethylthiuram disulfide, disulfiram (dsf) ( ) (fig. ) , date back to . however, only in the s that dsf would become popular when it was discovered that it could form copper chelates which favoured the death of micro-organisms and enabled treatment of intestinal parasites. [ ] [ ] [ ] in , dsf alcohol sensitivity was discovered accidentally and it was soon used in the clinical treatment of alcohol dependence. , dsf is used to treat alcohol dependence because it irreversibly inhibits the acetaldehyde dehydrogenase enzyme and modifies cysteine residues in its active site. this change prompts the formation of a disulphide bond between two cysteine residues in the active site. dsf effectiveness is based on its similarly to several proteins yielding a range of biological activities, such as antitumoral, , antimicrobial, and anti-sars and mers-cov. adding to the list of drugs to be repurposed against sars-cov- , recent studies indicate that dsf is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues that suppress the natural cycle of the enzymes, suggesting broad-spectrum characteristics. , covs have two viral enzymes, m pro and pl pro , that are cysteine protease involved in the formation of structural and non-structural proteins that constitute the viruses and favour control of host cells. different assays, such as proteolytic and binding synergy assays, were also conducted and described. although outcomes indicate dsf for anti-mers-cov and anti-sars-cov therapy, to the present date ( th june ), no other article was published claiming the availability of the compound as promising anti-sars-cov- . recently, an announcement was published on the oxford university website on the results of one randomized evaluation of covid- therapy. more specifically, the study focused on dexamethasone ( ), a corticosteroid with fluorine at -position (fig. ) . the drug is mostly used as anti-inflammatory, which works by inhibiting vasodilation, reducing leukocyte migration to the inflammation site and increasing vascular permeability. immunotherapy is an effective intervention in viral infections. most attempts at immunotherapy were successful in fighting viruses similar to sars-cov- . the principal methods include vaccine, neutralizing antibodies (nabs) candidates and convalescent plasma. , , , , , , in addition, according to the evidences from viral infections (ebola, influenza, sars and mers), immunotherapeutic interventions can reduce viral load and mortality rate of patients. , development of either monoclonal (mabs) or polyclonal (pabs) neutralizing antibodies is a commonly adopted immunotherapeutic alternative due to its specificity, purity, low contamination by blood-transmitted pathogens and relative safety. however, there are limitations to the use of nabs once its development and large-scale production for clinical use are a complex, expensive and slow process. promising scientific investigations have suggested using mabs or pabs as prophylactic and therapeutic measures against influenza and hcovs, such as mers-cov and sars-cov. targets reported as promising for hcovs immunotherapy were cytokine, s -receptor-binding domain (s -rbd), s n-terminal domain (s -ntd) and some other region of subunit s in order to block the rbds bonds to their respective receptors and to interfere either with s -mediated membrane fusion or with the entry in the host cells, thus inhibiting infection. these researchers have encouraged the development of nabs with cross reactivity potential and/or cross neutralization effect on sars-cov- infections, as shown by tian et al. data suggest that mab cr can be developed as therapeutic candidate, either isolated or combined with neutralizing antibodies to prevent and treat covid- , given that it could potently form bonds with sars-cov- rbd (kd of . nm). a different study by wang et al. reported the discovery of a human mab ( d ) that promoted cross neutralization of sars-cov and sars-cov- in a culture of cells through an independent receptor-binding inhibition mechanism that targets a conserved epitope on the spike hcovs rbd mentioned above. it is also reported the on-going investigation of convalescent plasma or immunoglobulin as last resource to improve the survival rate of patients with several viral infections such as h n avian influenza, a possible explanation for the efficacy of convalescent plasma is that immunoglobulin antibodies in the plasma of recovered patients can suppress viremia. shen et al. reported that five patients with extreme symptoms of covid- received blood transfusion containing convalescent plasma with specific sars-cov- antibodies. after a series of blood transfusions, the improvement in clinical status of patients was observed. viral loads also decreased and became negative within days after the transfusion, and sars-cov- -specific elisa and nabs titers increased following the transfusion. in spite of the limited sample, the authors concluded that convalescent plasma transfusion benefited patients infected with sars-cov- . therefore, testing safety and efficacy of transfusing convalescent plasma in patients infected with sars-cov- can be of value. , among the modalities of immunotherapy, vaccines are expected to be more promising, hence the global engagement in their production. over the last decade, the scientific community and the vaccine industry had to answer urgently to the epidemics of h n , ebola, zika and, more recently, sars-cov- . vaccine development is an expensive and slow process with high risks of failure, which often motivate developers to follow a linear sequence of steps with several breaks for data analysis and fabrication processes. therefore, it is fundamental that vaccines be developed through faithful methods even if it takes longer to move them onto clinical trials or to make a large number of doses available, a challenge during a pandemic. developing efficient vaccines for sars-cov- will be essential to reduce the severity of the disease, viral shedding and transmission to control future outbreaks. prior to the covid- pandemic, multiple strategies were used to generate vaccines for the first hcovs (sars-cov and mers-cov). several studies related to sars-cov vaccine production targeting the protein s, due to its function in the receptor binding and fusion to the host membrane, were successful in animal tests against that coronavirus. - these vaccines employed live-attenuated virus vaccines, killed virus, dna vaccines and viral vector vaccines. theoretically, these techniques could be applied to develop sars-cov- vaccines given their similarities from both the genomic perspective and the mechanisms employed in the invasion and infection of host cells. gao et al. promoted the pilotscale production of a purified inactivated sars-cov- virus vaccine candidate (picovacc), which induced sars-cov- -specific nabs in mice, rats and non-human primates. in addition, three immunizations using two different doses ( μg or μg per dose) in macaques provided partial or complete protection against the sars-cov- challenge, respectively, without observable antibodydependent enhancement of infection. these data reinforce the use of picovacc in the next steps of clinical trials targeting sars-cov- still for the present year. given the magnitude of the covid- pandemic, it has become indispensable to work as fast as possible to develop vaccines for global distribution. however, protocols are necessary to safekeep the population's health. hence, before allowing human testing of covid- vaccines, regulatory organizations must evaluate their safety against a series of virus strains and more than one animal model. they also must demand preclinical evidences that experimental vaccines prevent infectioneven if it means waiting weeks or months for models to become available. this is time well-spent, once testing vaccines without investing the due amount of time to completely understand the risks can lead to setbacks for current and future pandemics. repurposing potential relies on the mechanism for other viruses, such as hepatitis c, by inducing conformational changes that compromise rdrp activity. it was also possible to identify benzene derivatives used to treat cancer (dasatinib and imatinib) and one antiviral (darunavir), but predominant in-silico and some in-vitro outcomes demand more conclusive studies. representative of benzoic derivatives, ntz displayed significant inhibitory activity against pro-inflammatory cytokines, which can benefit control of ards, in spite of an unclear mechanism against sars-cov- . quinoline derivatives, hcq and cq, were some of the first drugs investigated. after several in-silico, in-vitro and in-vivo assays, based on results presented by recovery to the present date, unfortunately, these drugs were proven ineffective in hospitalized covid- patients. ivermectin represents macrolide derivatives and is suggested as promising due to both immunostimulatory activity and inhibitory activity on nuclear transport when administered at the early stages of the disease. among indole derivatives, arbidol was pointed out as useful for reducing viral binding and releasing of intracellular vacuoles that contain the virus. nonetheless, clinical trials are still necessary after dose adjustment for better outcomes. both indole derivatives (emetine and hht), in spite of promising results, were only submitted to in-silico and in-vitro tests, thus demanding further investigation on their toxicity and mechanism of control. hence, the currently available data on the classes of drugs investigated here revealed that drugs were considered promising mainly after in-silico tests only. in fact, the inefficacy of some of these drugs became evident after in-vitro or in-vitro tests, as cq and hcq, whose clinical trials failed to confirm the so-expected anti-sars-cov- activity. it is necessary to highlight the importance of clinical trials with drugs considered promising in theoretical studies, with due calm and openness to question and refute hypotheses, in the absence of scientific evidence to support their use to treat covid- . similarly, the careful analysis of practices involving patients with the extreme form of the disease is also important to identify new alternatives, such as the results recently published by recovery on dexamethasone. it is also expected that detailed clinical trials are conducted with some of the drugs described in this article as potential anti-sars-cov- , for instance sofosbuvir, origin and evolution of pathogenic coronaviruses the covid- vaccine development landscape sars-cov- vaccines: status report recent discovery and development of inhibitors targeting coronaviruses analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods drug repurposing for viral infectious diseases: how far are we? tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study virtual screening and repurposing of fda approved drugs against covid- main protease baricitinib as potential treatment for -ncov acute respiratory disease remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites coronaviruses -drug discovery and therapeutic options development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation of human coronaviruses oc and e coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a review of coronavirus disease- (covid- ) emergence of novel coronavirus -ncov: need for rapid vaccine and biologics development a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization. coronavirus disease (covid- ) pandemic advances in virus research covid- : a promising cure for the global panic genotype and phenotype of covid- : their roles in pathogenesis molecular evolution of human coronavirus genomes recent progress and challenges in drug development against covid- coronavirus (sars-cov- ) -an update on the status genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding structure analysis of the receptor binding of -ncov mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence visualizing speech-generated oral fluid droplets with laser light scattering pitfalls of judgment during the covid- pandemic a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study origin of viruses: primordial replicators recruiting capsids from hosts the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor renin-angiotensin-aldosterone system inhibitors in patients with covid- renin-angiotensin-aldosterone system and covid- infection covid- infection: origin, transmission, and characteristics of human coronaviruses review of the clinical characteristics of coronavirus disease (covid- ) mild or moderate covid- the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreakan update on the status clinical features of patients infected with novel coronavirus in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical characteristics of coronavirus disease in china clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study are patients with hypertension and diabetes mellitus at increased risk for covid- infection? comorbidities and multi-organ injuries in the treatment of covid- the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases sars-cov- and viral sepsis: observations and hypotheses novel coronavirus infection (covid- ) in humans: a scoping review and meta-analysis sex-specific sars-cov- mortality: among hormone-modulated ace expression, risk of venous thromboembolism and hypovitaminosis d probiotics and covid- : one size does not fit all clinical characteristics of deceased patients with coronavirus disease : retrospective study pathological findings of covid- associated with acute respiratory distress syndrome liver injury in covid- : management and challenges endothelial cell infection and endotheliitis in covid- comorbidities and multi-organ injuries in the treatment of covid- coronavirus infections and immune responses longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients reducing mortality from -ncov: host-directed therapies should be an option ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study coronavirus membrane fusion mechanism offers a potential target for antiviral development pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid- structure of the sars-cov- spike receptor-binding domain bound to the ace receptor structural basis of receptor recognition by sars-cov- a possible strategy to fight covid- : interfering with spike glycoprotein trimerization the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies evidence that tmprss activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry protease inhibitors targeting coronavirus and filovirus entry structural basis of sars-cov- clpro and anti-covid- drug discovery from medicinal plants structure of the rna-dependent rna polymerase from covid- virus structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir structure of mpro from sars-cov- and discovery of its inhibitors efficiency of incorporation and chain termination determines the inhibition potency of ′-modified nucleotide analogs against hepatitis c virus polymerase protective effect and potential mechanisms of wei-chang-an pill on high-dose -fluorouracil-induced intestinal mucositis in mice higher order structures in purine and pyrimidine metabolism human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy pyrrolopyrimidines: an update on recent advancements in their medicinal attributes synthesis and evaluation of anti-tumor activity of novel triazolo[ , -a] pyrimidine on cancer cells by induction of cellular apoptosis and inhibition of epithelial-to-mesenchymal transition process a facile one pot synthesis of novel pyrimidine derivatives of , -benzodiazepines via domino reaction and their antibacterial evaluation high antiparasitic activity of silver complexes of , -dimethyl- , , -triazolo[ , a]pyrimidine new carbocyclic n -substituted adenine and pyrimidine nucleoside analogues with a bicyclo[ . . ]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and x-ray crystallography new prodrugs of two pyrimidine acyclic nucleoside phosphonates: synthesis and antiviral activity -fluorouracil upregulates cell surface b -h (pd-l ) expression in gastrointestinal cancers a new animal model of intestinal mucositis induced by the combination of irinotecan and -fluorouracil in mice -fluorouracil in combination with deoxyribonucleosides and deoxyribose as possible therapeutic options for the coronavirus, covid- infection the safety and efficacy of gemcitabine for the treatment of bladder cancer depletion of sirt sensitizes human non-small cell lung cancer cells to gemcitabine therapy by inhibiting autophagy gemcitabine for recurrent ovarian cancer -a systematic review and meta-analysis repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection drug repositioning is an alternative for the treatment of coronavirus covid- an efficient synthesis of baricitinib janus kinase inhibitor baricitinib modulates human innate and adaptive immune system in vivo antitumor activity of su , a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship the egf receptor family as targets for cancer therapy janus kinase inhibitor baricitinib is not an ideal option for management of covid- protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx galidesivir limits rift valley fever virus infection and disease in syrian golden hamsters therapeutic options for the novel coronavirus ( -ncov) new nucleoside analogues for the treatment of hemorrhagic fever virus infections remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro genotype and subtype profiling of psi- as a nucleotide inhibitor of hepatitis c virus selected nucleos(t)ide-based prescribed drugs and their multi-target activity mechanism of activation of psi- and its diastereoisomer psi- evaluation of sofosbuvir (β-d- ′-deoxy- ′-α-fluoro- ′-β-cmethyluridine) as an inhibitor of dengue virus replication yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo the fda-approved drug sofosbuvir inhibits zika virus infection beyond members of the flaviviridae family, sofosbuvir also inhibits chikungunya virus replication sofosbuvir as repurposed antiviral drug against covid- : why were we convinced to evaluate the drug in a registered/approved clinical trial antiviral β-l-nucleosides specific for hepatitis b virus infection. in: frontiers in viral hepatitis treatment of chronic hepatitis b: focus on telbivudine safety and efficacy of telbivudine for the treatment of chronic hepatitis b. ther clin risk manag -trisubstituted- h-purine)- -chalcone derivatives as potent anti-gastric cancer agents: design, synthesis and structural optimization novel phosphodiesterases inhibitors from the group of purine- , -dione derivatives as potent modulators of airway smooth muscle cell remodelling novel butanehydrazide derivatives of purine- , -dione as dual pde / inhibitors with potential anti-inflammatory activity: design, synthesis and biological evaluation the purines: potent and versatile small molecule inhibitors and modulators of key biological targets synthesis and biological evaluation of novel hiv- protease inhibitors with pentacyclic triterpenoids as p -ligands seley-radtke, design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity remdesivir in covid- : a critical review of pharmacology, pre-clinical and clinical studies the epidemiology, diagnosis and treatment of covid- therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys a randomized, controlled trial of ebola virus disease therapeutics broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov first case of novel coronavirus in the united states compassionate use of remdesivir for patients with severe covid- pharmacologic treatments for coronavirus disease (covid- ) oral ganciclovir for patients with cytomegalovirus retinitis treated with a ganciclovir implant drugbank . : a major update to the drugbank database epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study incidence, risk factors, and outcome of cytomegalovirus infection and disease in patients receiving prophylaxis with oral valganciclovir or intravenous ganciclovir after umbilical cord blood transplantation pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir in allogeneic stem cell transplant patients with graft-versus-host disease of the gastrointestinal tract the first days of novel coronavirus (sars-cov- ) outbreak: recent advances, prevention, and treatment tenofovir disoproxil fumarate -triazole-containing hybrids as leads in medicinal chemistry: a recent overview medicinal attributes of , , -triazoles: current developments synthesis and anticancer activity of long chain substituted , , -oxadiazol- -thione, , , -triazol- -thione and , , -triazolo[ , -b]- , , -thiadiazine derivatives chemical synthesis and biological evaluation of triazole derivatives as inhibitors of inha and antituberculosis agents synthesis, antiinflammatory and analgesic activity of -[ -(substituted benzylideneamino)- -(substituted phenoxymethyl)- h- , , -triazol- -yl thio] acetic acid derivatives synthesis and biological evaluation of some novel triazol- -ones as antimicrobial agents design, synthesis, antiviral and cytostatic activity of ω-( h- , , -triazol- -yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues synthesis, biological evaluation and molecular modeling of a novel series of fused , , -triazoles as potential anti-coronavirus agents treatment options for covid- : the reality and challenges compounds with therapeutic potential against novel respiratory ribavirin in the treatment of sars: a new trick for an old drug? inhibition of novel β coronavirus replication by a combination of interferon-α b and ribavirin remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro anti-hcv, nucleotide inhibitors, repurposing against covid- ebola virus dynamics in mice treated with favipiravir favipiravir (t- ), a broad-spectrum inhibitor of viral rna polymerase favipiravir versus arbidol for covid- : a randomized clinical trial. medrxiv [epub ahead of print a possible pharmaceutical treatment for covid- experimental treatment with favipiravir for covid- : an open-label control study. engineering an antiviral for covid- ? covid- -the search for effective therapy coronavirus disease (covid- ): current status and future perspectives role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report a trial of lopinavir-ritonavir in adults hospitalized with severe covid- triple combination of interferon beta- b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an openlabel, randomised, phase trial molecular modeling evaluation of the binding abilities of ritonavir and lopinavir to wuhan pneumonia coronavirus proteases oseltamivir treatment of influenza in children therapeutic management of covid- patients: a systematic review in silico studies on therapeutic agents for covid- : drug repurposing approach synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus nelfinavir was predicted to be a potential inhibitor of -ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation atazanavir for the treatment of human immunodeficiency virus infection influence of atazanavir on the pharmacodynamics and pharmacokinetics of gliclazide in animal models therapy with atazanavir plus saquinavir in patients failing highly active antiretroviral therapy: a randomized comparative pilot trial predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov- ) through a drug-target interaction deep learning model déjà vu: stimulating open drug discovery for sars-cov- state-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in sars-cov- fda-approved thiolreacting drugs that potentially bind into the sars-cov- main protease, essential for viral replication cyclosporin a and cardioprotection: from investigative tool to therapeutic agent modulation of innate immunity by cyclosporine a the use of cyclosporine a in rheumatology: a comprehensive review cyclosporine a: a valid candidate to treat covid- patients with acute respiratory failure? the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits the replication of diverse coronaviruses human coronavirus nl replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir cyclophilins and cyclophilin inhibitors in nidovirus replication identification of antiviral drug candidates against sars-cov- from fda-approved drugs cyclosporin a is a potential inhibitor of sars-cov- . researchgate (preprint) cyclosporine therapy in cytokine storm due to coronavirus disease (covid- ) clinically significant drug interactions with cyclosporin glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) discovery and development of safe-in-man broadspectrum antiviral agents teicoplanin: an alternative drug for the treatment of covid- ? fighting viruses with antibiotics: an overlooked path current epidemiological and clinical features of covid- ; a global perspective from china pharmacokinetics and safety of co-administered paritaprevir plus ritonavir, ombitasvir, and dasabuvir in hepatic impairment dasabuvir: a non-nucleoside inhibitor of ns b for the treatment of hepatitis c virus infection in silico studies on therapeutic agents for covid- : drug repurposing approach darunavir: a review in pediatric hiv- infection darunavir: a review of its use in the management of hiv- infection discovering drugs to treat coronavirus disease (covid- ) therapeutic options and critical care strategies in covid- patients; where do we stand in this battle? majallahi danishkadahi pizishkii isfahan insight derived from molecular docking and molecular dynamics simulations into the binding interactions between hiv- protease inhibitors and sars-cov- clpro peptide-like and small-molecule inhibitors against covid- computational view toward the inhibition of sars-cov imatinib: a breakthrough of targeted therapy in cancer coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions abl tyrosine kinase regulates hepatitis c virus entry productive replication of ebola virus is regulated by the c-abl tyrosine kinase abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion sars coronavirus spike protein-induced innate immune response occurs via activation of the nf-κb pathway in human monocyte macrophages in vitro imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury imatinib stimulates prostaglandin e and attenuates cytokine release via ep receptor activation imatinib mesylate induces clinical remission in rheumatoid arthritis kit inhibition by imatinib in patients with severe refractory asthma long-standing remission of crohnʼs disease under imatinib therapy in a patient with crohnʼs disease imatinib might constitute a treatment option for lung involvement in covid- european leukemianet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia principal long-term adverse effects of imatinib in patients with chronic myeloid leukemia in chronic phase investigation of n terminal domain of sars cov nucleocapsid protein with antiviral compounds based on broad anti-coronaviral activity of fda approved drugs against sars-cov- in vitro and sars-cov in vivo nitazoxanide: a first-in-class broad-spectrum antiviral agent nitazoxanide: a new thiazolide antiparasitic agent nitazoxanide/azithromycin combination for covid- : a suggested new protocol for early management therapeutic potential of nitazoxanide against newcastle disease virus: a possible modulation of host cytokines nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus the anti-hepatitis c agent nitazoxanide induces phosphorylation of eukaryotic initiation factor α via protein kinase activated by double-stranded rna activation a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs nitazoxanide suppresses il- production in lpsstimulated mouse macrophages and tg-injected mice a systematic review on use of aminoquinolines for the therapeutic management of covid- : efficacy, safety and clinical trials the possible mechanisms of action of -aminoquinolines (chloroquine/hydroxychloroquine) against sars-cov- infection (covid- ): a role for iron homeostasis? chloroquine: modes of action of an undervalued drug cardiovascular risks of hydroxychloroquine in treatment and prophylaxis of covid- patients: a scientific statement from the indian heart rhythm society chloroquine and hydroxychloroquine as available weapons to fight covid- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro of chloroquine and covid- viral infection and iron metabolism hepcidin and the iron-infection axis iron and infection comparison of the antiviral activity in vitro of some non-steroidal antiinflammatory drugs antihistaminics, local anesthetics, and other amines as antiviral agents effect of chloroquine on the growth of animal viruses in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine a systematic review on the efficacy and safety of chloroquine for the treatment of covid- chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial hydroxychloroquine in the management of critically ill patients with covid- : the need for an evidence base association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state observational study of hydroxychloroquine in hospitalized patients with covid- this national clinical trial aims to identify treatments that may be beneficial for people hospitalised with suspected or confirmed covid- ivermectin: enigmatic multifaceted 'wonder' drug continues to surprise and exceed expectations ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of hiv- and dengue virus ivermectin is a potent inhibitor of flavivirus replication specifically targeting ns helicase activity: new prospects for an old drug repurposing ivermectin to inhibit the activity of sars cov helicase: possible implications for covid therapeutics influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import nuclear localization of dengue virus (denv) - non-structural protein ; protection against all denv serotypes by the inhibitor ivermectin the broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β heterodimer nucleocytoplasmic transport of nucleocapsid proteins of enveloped rna viruses severe acute respiratory syndrome coronavirus orf antagonizes stat function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro the importin α/β-specific inhibitor ivermectin affects hif-dependent hypoxia response pathways antiviral treatment of covid- hydroxychloroquine and ivermectin: a synergistic combination for covid- chemoprophylaxis and treatment? therapeutic potential of ivermectin for covid enhancing immunity in viral infections, with special emphasis on covid- : a review safety of high-dose ivermectin: a systematic review and meta-analysis five-membered heterocycles: indole and related systems. modern heterocyclic chemistry a review on recent developments of indole-containing antiviral agents discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication medicinal applications of (benz)imidazole-and indole-based macrocycles medicinal chemistry of indole derivatives: current to future therapeutic prospectives synthesis and antiviral activity of some novel indole- -carboxylate derivatives superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones arbidol combined with lpv/r versus lpv/r alone against corona virus disease : a retrospective cohort study arbidol monotherapy is superior to lopinavir/ritonavir in treating covid- the anti-influenza virus drug, arbidol is an efficient inhibitor of sars-cov- in vitro pharmacokinetics of single and multiple oral doses of arbidol in healthy chinese volunteers rizatriptan vs sumatriptan in the acute treatment of migraine ação da melatonina no tecido cartilaginoso covid- : melatonin as a potential adjuvant treatment protective effects of melatonin in mice infected with encephalitis viruses melatonin: its possible role in the management of viral infections-a brief review network-based drug repurposing for novel coronavirus -ncov/sars-cov- lungs as target of covid- infection: protective common molecular mechanisms of vitamin d and melatonin as a new potential synergistic treatment emetine protects mice from enterovirus infection by inhibiting viral translation emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine tracing the journey of disulfiram: from an unintended discovery to a treatment option for alcoholism supervised disulfiram as adjunct to psychotherapy in alcoholism treatment the clinical use of disulfiram (antabuse®): a review disulfiram inhibits the in vitro growth of methicillin-resistant staphylococcus aureus disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of francisella tularensis structural basis for inactivation of giardia lamblia carbamate kinase by disulfiram disulfiram/copper markedly induced myeloma cell apoptosis through activation of jnk and intrinsic and extrinsic apoptosis pathways disulfiram is a direct and potent inhibitor of human o -methylguanine-dna methyltransferase (mgmt) in brain tumor cells and mouse brain and markedly increases the alkylating dna damage disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes coronaviruses -drug discovery and therapeutic options inhibition of urease by disulfiram, an fda-approved thiol reagent used in humans the microcirculation and inflammation: site of action for glucocorticoids the convalescent sera option for containing covid- the possible of immunotherapy for covid- : a systematic review perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease- (covid- ) potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review advances in respiratory virus therapeutics -a meeting report from the th isirv antiviral group conference a mouse model for mers coronavirus-induced acute respiratory distress syndrome neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential effective treatment of severe covid- patients with tocilizumab neutralizing antibodies against sars-cov- and other human coronaviruses potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody a human monoclonal antibody blocking sars-cov- infection treatment with convalescent plasma for influenza a (h n ) infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h n ) virus infection evaluation of convalescent plasma for ebola virus disease in guinea the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol treatment of critically ill patients with covid- with convalescent plasma convalescent plasma as a potential therapy for covid- developing covid- vaccines at pandemic speed a decade after sars: strategies for controlling emerging coronaviruses severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus sars coronavirus: a new challenge for prevention and therapy coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus development of an inactivated vaccine candidate for sars-cov- don't rush to deploy covid- vaccines and drugs without sufficient safety guarantees the authors would like to thank facepe (fundação de amparo à ciência e tecnologia none. all authors researched data for the article, contributed substantially to discussion of the content, wrote the article and reviewed and edited the manuscript before submission. key: cord- -yznlgzir authors: varanko, anastasia; saha, soumen; chilkoti, ashutosh title: recent trends in protein and peptide-based biomaterials for advanced drug delivery date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: yznlgzir nan they also increase the occurrence of dose-dependent side effects. the lack of target specificity made it impossible to deliver a therapeutic to the organ of interest without impacting other cells, which caused off-target toxicity. in the s, scientists began to focus on developing drug delivery systems (figure ) . in the next thirty years, they established the principles of drug diffusion, dissolution, and pharmacokinetics [ , ] . research then shifted toward prolonging drug release and increasing the drug's retention time in circulation [ ] . scientists also focused on how to specifically deliver drugs to a disease site by engineering systems for local drug release, passive drug accumulation in the diseased tissue, and active targeting [ ] . this research led to the first fda approval of a drug delivery system -liposomal amphotericin b-in for treatment of fungal infections [ ] . since then, controlled delivery has been leveraged to improve the bioavailability and efficacy of numerous therapeutics [ ] . biomaterials have been critical to the success of drug delivery systems. many drug formulations employ biomaterials to extend the therapeutic window by both sustaining its release from the formulation and slowing its elimination from the body. beginning in the late s, scientists used synthetic materials, especially polymers, as drug carriers [ , ] . these systems successfully extended the drug's circulation time by increasing its molecular weight to slow renal excretion. biodegradable systems were also engineered to prolong drug release, thus reducing the frequency of administration [ ] . despite these accomplishments, synthetic materials can have high immunogenicity or toxic degradation products [ ] . furthermore, there is limited control over the stereochemistry, structure, and molecular weight of synthetic polymers, which impacts the drug's biodistribution and pharmacokinetics [ , ] . the production of synthetic polymer drug carriers can be difficult and expensive to scale up [ ] . figure : conjugation of the cyclic pentapeptide crgdfk and the photodynamic agent chlorin e (ce ) to sf was achieved using a simple acid-amine coupling reaction and the resulting conjugate was doped with -fluorouracil ( -fu) using genipin peptide as a crosslinker. adapted with permission from [ ] . the adaptable mechanical properties and thermal stability of sf make it an ideal candidate material for forming hydrogels and scaffolds. its degradation can be tuned by controlling the self-assembly of sf which can stably encapsulate the therapeutics and release them on demand. kaplan and colleagues have extensively studied the abilities of sf and its hybrids to form hydrogels. they developed a method to synthesize a thixotropic silk nanofiber hydrogel from aqueous solution, which otherwise requires an organic co-solvent [ , ] . the injectable nanofiber hydrogel stably entraps dox, solidifies in situ, and demonstrates ph-triggered sustained release of dox [ ] . kaplan et al. also used an injectable sf-hydrogel system to sustain the delivery of anti-vascular endothelial growth factor (anti-vegf) therapeutics [ ] , and small molecule drugs [ ] . however, the prolong gelation time (weeks-months) of sf hydrogel acts as a major roadblock for their practical application. to decrease the gelation time gong et al. synthesized another class of thixotropic hydrogels by blending regenerated sf and hydroxyl propyl cellulose (hpc), a natural cellulose ether approved by fda [ ] . hpc has lower critical solution temperature (lcst) of about °c above which it precipitates in water. the j o u r n a l p r e -p r o o f hpc-sf blend gelled at °c within h. results from confocal laser scanning microscopy (clsm), raman spectroscopy, and c-nmr spectroscopy suggested that the conformational transition of sf from random coil to β-sheet during phase separation resulted in gel formation through β-sheet crosslinking and immobilization of the molecules of sf and hpc in the dispersed phase. the blended hydrogel encapsulated mice fibroblasts and protected them against high shear force during injection, which suggests that the hydrogel can be used for cell delivery [ ] . germershaus et al. developed heuristics to decipher protein interactions with sf using protamine and polylysine as model proteins [ ] . the author concluded that the interaction between protein and sf arises primarily from entropy-driven complex coacervation, which depends on the ionic strength of the solution and presence of kosmotropic and chaotropic salts. sf-hydrogels have also been fabricated with various nanoassemblies including sfnps, carbon nanotubes and hybrid nanoparticles of different materials. mao and colleagues encapsulated curcumin-loaded cationic nanoparticles of rrr-α-tocopheryl succinate-grafted-εpolylysine conjugate into a sf-hydrogel, which promoted the penetration of curcumin into the thickening corneum of psoriatic mice and thus inhibited skin inflammation. compared to % of curcumin released from the nanoparticle, only % was released from the mixed hydrogelnanoparticle system. the author concluded that this slow release of curcumin might be due to adherence of the nanoparticles into the sf hydrogel, making it difficult for the embedded curcumin to diffuse out of the gel. this delayed release profile resulted into significant accumulation of curcumin in the stratum corneum at the h [ ] . wu et al. incorporated salinomycin and paclitaxel-loaded sf-nanoparticles into a sf hydrogel to inhibit cancer stem cell and tumor growth. the dual drug-loaded hydrogel had homogeneous drug distribution and exhibited increased tumor inhibition compared to the single drug-loaded hydrogel, as evidenced by fewer cd +cd + tumor cells in vivo. because paclitaxel and salinomycin interacted differently with sf, their release profiles were different, with paclitaxel showing sustained release and salinomycin an initial burst release [ ] . gangrade et al. introduced carbon nanotubes into sf hydrogels to construct an on-demand, tumor-targeting system [ ] . they synthesized folic acid functionalized, dox-loaded, single-walled carbon nanotubes (swcnt) and incorporated them into an sf hydrogel matrix. only % of dox was released from the composite material over a period of five days under physiological conditions. however, intermittent exposure to near-infrared light stimulated on-demand dox release (~ %) due to the photothermal property of swcnt. he et al. developed an injectable silk fibroin nanofiber hydrogel system complexed with upconversion nanoparticles and nano-graphene oxide (sf/ucnp@ngo) for upconversion luminescence imaging and photothermal therapy [ ] . the naluf :er + ,yb + upconversion j o u r n a l p r e -p r o o f nanoparticles were complexed with the nano-graphene oxide and then doped into an aqueous sf solution to form a hybrid hydrogel system. sf/ucnp@ngo hydrogels efficiently ablated t breast cancer cells via the photothermal effect both in vitro and in vivo. recombinant techniques can also be utilized to modulate the properties of silk-based scaffolds and hydrogels. anderson et. al. recombinantly synthesized slp segments conjugated to a human fibronectin segment and cell attachment domain [ ] . crystal structure characterization of (gagags)-based slps revealed the hydrophobic domains of silk collapsed to form anti-parallel β-sheets that assembled into crystallite whiskers at the nanometer scale. this self-assembled material is mechanically tough, can undergo processing in the presence of bioactive molecules, and can be processed for into thin films, hydrogels, and three-dimensional scaffolds. schacht et al. fabricated highly porous foams made from recombinant spider silk protein eadf (c ) and a variant containing an rgd motif using a salt-leaching technique [ ] . in contrast to other salt-leached silk scaffolds, the swelling behaviors of these scaffolds as measured by discovery v stereomicroscope were low, and the mechanical properties were suitable for soft tissue engineering. the compressive moduli of the foam in a hydrated state was . ± . kp at a protein concentration of % (w/v). the pore size and porosity of the foams were optimized by altering the salt crystal size, thus rendering them suitable to adhere and culture fibroblasts. as silk fibroin can self-assemble into hydrogels from an aqueous solution, it has been widely used for ocular delivery of various drugs ranging from small molecules to antibodies and other therapeutic proteins. in one such example, silk hydrogel formulations of bevacizumab, a clinically used antibody as angiogenesis inhibitor showed sustained release of the antibody over a three months' period in an intravitreal injection model in dutch-belted rabbits [ ] . the bevacizumab concentration in the vitreous humor on day using hydrogel formulation at both standard ( . mg / µl injection) and high dose ( . mg / µl injection) was equivalent to the levels achieved with positive control on day ( . mg bevacizumab/ µl injection). [ ] . this concentration is estimated to be the therapeutic threshold based on the current dosage of injection/month. these gels also got degraded after months, indicating a repetitive dosing may be possible. additionally, the propensity of sf to bind positively charged molecules through electrostatic interaction can be exploited for topical drug delivery on the eye surface. dong et al. electrostatically coated an ibuprofen-encapsulated liposome of cationic lipids with sf for ocular drug delivery [ ] . the sf being a potential mucoadhesive biopolymer aided in retention of the drug on eye surface and facilitated its sustained release. recently a phase ii clinical trial within the protein. additionally, disulfide bonding, electrostatic interactions, and hydrogen bonding provide keratin with mucoadhesive properties, which make it a useful material for delivery of drugs to the gastrointestinal tract [ ] . furthermore, keratins have been employed as -smart‖ materials for stimulus responsive drug delivery. the large number of carboxyl groups within their sequence makes them phsensitive so that in response to an increase in ph, release their cargo in a controlled manner as these groups become deprotonated [ ] . additionally, their rich cysteine content renders them redox-responsive. tuning the number or level of crosslinking of disulfide bonds within the protein alters the duration of drug release from a keratin-based material. similarly, keratin is responsive to changes in glutathione (gsh) concentration. this is particularly useful for delivery of chemotherapeutics to metastatic cancer cells, which have significantly higher concentrations of gsh than healthy cells. moreover, the high lysine and arginine content in some keratins enables their cleavage by high concentrations of trypsin [ ] , which is frequently overexpressed in inflamed tissue [ ] . thus, keratin could be a useful material when targeting injured or tumorous tissues. due to its unique material properties, keratin has been used to create nanoparticles that encapsulate and sequester a therapeutic cargo before releasing it in response to biological stimuli. these nanoparticles can stably carry therapeutics via electrostatic interactions, hydrogen bonding, disulfide bond formation, or chemical conjugation. keratins are durable and remain stable in the bloodstream, which increases the half-life of its cargo [ ] . positively charged drugs electrostatically adsorb to the surface of negatively charged keratin nanoparticles; this interaction provides long-term, sustained release of the drug from its carrier. zhi et al. first explored this strategy by complexing the model drug chlorhexidine (chx) with keratin nanoparticles generated by ionic gelation [ ] . carboxylate groups on the nanoparticle surface stabilized the polyanion complex with chx. this interaction provided a remarkable chx encapsulation efficiency of . % and a loading content of . %. zhi et al. demonstrated that for hours, the drug was released in a ph-dependent manner with greater release observed at neutral and slightly acidic ph, which indicates the utility of this system to deliver chemotherapeutics to the acidic tumor microenvironment [ ] . can be loaded with dox via electrostatic interactions [ ] . these nanoparticles were designed to exploit keratin's responsiveness to ph and glutathione concentration to release the drug, which is a useful strategy to treat solid tumors, which have a ph of . - . and gsh concentration of . - mm, which is approximately -fold greater than the gsh concentration of healthy tissue [ ] . kdnps were created with a desolvation method that destabilizes keratin with ethanol and causes it to aggregate into nanoparticles that are crosslinked with glutaraldehyde. in an environment with a low ph or high concentrations of glutathione, kdnps experienced a stark increase in zeta potential and underwent a negative-to-positive charge conversion. the positive charge facilitated internalization by cells and electrostatically repelled the drug, thus accelerating its release (figure ) . error! reference source not found.moreover, the charge conversion destabilized the nanoparticles, causing them to aggregate and accumulate at the tumor due to the enhanced permeation and retention (epr) effect, which further enhanced their anti-cancer potency [ ] .the stimulus-responsive properties of kdnps were expanded by li et al., whoe engineered triple stimuli-responsive kdnps via drug-induced ionic gelation. in addition to releasing dox in response to changes in ph and glutathione concentration, these nanoparticles broke down in the presence of high concentrations of trypsin, which digested the peptide bonds within the keratin [ ] . similarly, keratin graft poly(ethylene glycol) nanoparticles have also been explored as glutathione-responsive vehicles; dox-hcl entrapped in the disulfide-crosslinked keratin core was released in response to increased intracellular glutathione concentrations [ ] . due to its exquisite and versatile loading capacity, keratin has also been harnessed to create bimodal nanoformulations that combine chemotherapeutics and photodynamic therapy. martella et al. functionalized high molecular weight keratin with the photosensitizer chlorin-e (ce ) and induced spontaneous nanoparticle formation by mixing the protein with paclitaxel, a chemotherapeutic that aggregates with the hydrophobic residues in keratin [ ] . this is an attractive bottom-up nanoparticle fabrication strategy, as it did not require any toxic crosslinkers or downstream purification steps for nanoparticle fabrication. when administered to an osteosarcoma cell line, the nanoparticles localized to the cell lysosome. although ce typically experiences a drop in fluorescence in acidic conditions, the keratin protected the photosensitizer and no decrease in fluorescence was observed. further, the nanoparticle formulation j o u r n a l p r e -p r o o f transported the paclitaxel in a three-dimensional tumor model system without reducing the drug's potency [ ] . keratin nanoparticles have also been engineered for mucoadhesive drug delivery. kerateine (ktn) and keratose (kos) are two forms of keratin that have been extracted and processed in its reduced and oxidized forms, respectively. cheng et al. demonstrated that, by altering the ktn:kos ratio, the mucoadhesive properties and thus drug release, gastric retention time, and bioavailability of keratin nanoparticles could be tuned [ ] . an evaluation of the hydrophobicity, surface charge, and terminal groups of the nanoparticles revealed that the mucoadhesive properties of ktn were dominated by electrostatic interactions, whereas those of kos were primarily due to hydrogen bonding with gastric mucin. moreover, cheng et al. discovered that gastric retention time decreased with an increase in kos, and release of the model drug, amoxicillin, increased with a larger proportion of ktn due to its ph responsiveness [ ] . keratin-based films have been widely explored for biomedical applications due to the presence of cell attachment sites, their biocompatibility, and their large surface area. these mechanically and chemically stable materials have been useful for delivering a variety of drugs and peptides. in an early study of keratin films, fuji et al. extracted keratin from human hair in the absence of surfactant, thus creating a water-soluble film comprised primarily of α-keratins [ ] . alkaline phosphatase, a model enzyme, was incorporated into the film by mixing it with the keratin prior to gelation. the biochemical properties and bioactivity of alkaline phosphatase were maintained for two weeks after loading [ ] . while keratin films have excellent biocompatibility, they lack mechanical strength. thus, many studies combine keratin with other materials or treat the protein with a crosslinking agent to improve its rigidity, stiffness, and stability. in one example, keratin was blended with sf to create a composite film for the delivery of bowman-birk inhibitors (bbi), synthetic peptides designed to inhibit elastase in wound healing applications [ ] . sf provided structural stability to the film while keratin governed the degradation and bbi release rates. using fitc -tagged bovine serum albumin (bsa) as a model protein, vasconcelos et al. demonstrated that, although the sf was compact and rigid, increasing the percentage of hydrolytic keratin could increase the rate of fitc-bsa release by film degradation and diffusion [ ] . another study used transglutaminase (tgase) to crosslink keratin films to improve the mechanical strength and chemical stability of the material [ ] . treatment with tgase resulted in more compact network correlated with the amount of iodoacetamide used, though it was not directly proportional due to the binding affinity of the model drug to the keratin [ ] . a disulfide shuffling strategy was used by cao et al. to explore how disulfide bond formation impacts drug release. they cleaved intramolecular disulfide bonds with a reductive agent (e.g., cysteine) to free thiol groups that could then form intermolecular disulfide bonds ( figure a ) error! reference source not found. [ ] . this tactic increased the mechanical strength of the hydrogel, reduced its gelation time, and required a lower amount of keratin for gelation. the release rates of ciprofloxacin and dox were inversely proportional to the level of cysteine in the hydrogels. hydrogels that entrapped ciprofloxacin also demonstrated zero-order release kinetics in pbs (figure b & c) . moreover, when these hydrogels were exposed to increasing concentrations of glutathione, the degradation rate increased in response to the more rapid degradation of disulfide bonds ( figure d & e) [ ] . these studies indicate that the degradation rate of keratin hydrogels can be altered for specific disease states and drugs. ph-responsive keratin hydrogels have also been designed to reversibly swell in response to their environment to modulate the release of their cargo. in many of these examples, keratin has been combined with itaconic acid, n-isopropyl acrylamide, or methacrylic acid to achieve ph responsiveness [ ] [ ] [ ] . more recently, peralta ramos et al. reported a stimulus-responsive hydrogel that did not depend on chemical grafting to achieve its mechanical properties and phresponsivity [ ] . this was credited to a novel synthesis strategy during which disulfide bridges were broken and the α-keratin reorganized. at an acidic ph, the carboxyl groups were protonated and formed hydrogen bonds, which created a higher fraction of β-sheet conformations and kept the gel in a collapsed state. at a basic ph, hydrogen bonds broke to create more sites for water adsorption, thus forcing chain reorganization and allowing the material to swell [ ] . villanueva et al. expanded upon this work by incorporating antimicrobial zno nanoplates into the ph-responsive hydrogel [ ] . upon administration to a chronic wound, the hydrogel swelled in response to the basic environment and locally released the biocidal agent. as the wound healed and the presence of microbes decreased, the gel collapsed and inhibited the release of zno [ ] . this study demonstrates how keratin hydrogels can be used to adjust the delivery of a therapeutic agent in response to alterations in ph. albumin is the most abundant protein in human plasma and has a set of properties that make it a unique molecular carrier for drugs: (i) it is a natural physiological carrier of native ligands and nutrients; (ii) it bypasses systemic clearance and degradation by the body's own innate mechanisms, so that it has an exceptionally long half-life of days in humans, and similarly long half-lives in most animal species [ ] [ ] [ ] [ ] ; (iii) it preferentially accumulates at sites of vascular leakiness; (iv) it is highly internalized and metabolized by rapidly growing, nutrient-starved cancer cells; and (v) it is biodegradable and has no known systemic toxicity. because of these properties especially its extraordinarily long plasma circulation, albumin has attracted a lot interest as a carrier for diverse drugs. three naturally derived albumin molecules have been used to address the delivery challenges associated with small molecule drugs. ovalbumin (ova), a highly functional food protein with a molecular weight of kda and isoelectric point (pi) of . , is a monomeric phospho-glycoprotein consisting of amino acid residues [ ] . each ova molecule has one internal disulfide bond and four free sulfhydryl groups. ova was originally chosen as a carrier for drug delivery because of its availability, low cost, ability to form gel networks and stabilize emulsions and foams, and ph-and temperature-sensitive properties [ ] . bovine serum albumin (bsa), which has a molecular weight of kda and a pi of . in water (at °c), has also been widely used for drug delivery because of its abundance, low cost, ease of purification, unusual ligand-binding properties, and wide acceptance in the pharmaceutical industry [ ] [ ] [ ] [ ] [ ] [ ] . however, due to possible human immunologic response to ova and bsa in vivo, human serum albumin (hsa) is now exclusively used for drug delivery [ ] . hsa, which has a mw of . kda, is the most abundant serum protein with a concentration of - g/l in human serum. it is long-circulating with an exceptionally long in vivo half-life of ~ days [ ] [ ] [ ] ] . about - g of albumin is synthesized by liver hepatocytes daily and released into circulation [ ] . when albumin extravasates into tissue, it is naturally recycled and returned to the vascular space via the lymphatic system. the same approximate mass of - g of albumin entering the intravascular space is also catabolized daily. hsa is a carrier of a wide variety of endogenous and exogenous compounds and facilitates the colloidal solubilization and transport of j o u r n a l p r e -p r o o f hydrophobic molecules, such as long chain fatty acids, and variety of other ligands, including bilirubin, hormones, amino acids, metal ions, and drugs [ , ] . hsa is a soluble, globular, monomeric protein consisting of amino acid residues. it contains cysteinyl residues that form one sulfhydryl group and disulfide bridges [ , , ] . figure a shows the crystal structure of albumin and the sites where ligands can bind [ ] . three distinct features of albumin make it ideal for use in drug delivery applications: binding, trafficking, and recycling. binding: each class of drugs has a distinct binding affinity to different binding sites of albumin. dicarboxylic acids and sterically demanding anionic heterocyclic molecules (e.g., warfarin) bind to sudlow's site i (figure a ), whereas aromatic carboxylic acids with a single negatively charged acid group separated by a hydrophobic center (e.g., diazepam, ibuprofen) bind to sudlow's site ii [ ] . hsa has seven long-chain fatty acid binding sites (fa - in figure a , asymmetrically distributed throughout its three domains) that promote binding of up to two moles of fatty acid per mole of hsa under normal physiological condition [ ] [ ] [ ] . compounds with p-isothiocyanate (p-scn) or nhs ester (n-hydroxysuccinimide) functional moieties covalently react with albumin's lysine, with lys residue being the most reactive [ , ] . however, due to multiple other (at least ten) surface accessible lysine residues in hsa, reaction with lysine residues leads to poorly defined conjugates with a range of stoichiometries [ , ] . alternatively, the solvent accessible cysteine in hsa can be selectively modified with a prodrug containing a maleimide functionality, as (i) % of circulating serum albumin possess one free cysteine; (ii) other major serum proteins lack a solventaccessible free cysteine; and (iii) the th cysteine residue of albumin (pk a ~ ) has the most reactive thiol group in human plasma [ , ] . trafficking: albumin naturally transcytoses across the vascular endothelium, which normally creates an impermeable barrier to most plasma proteins. this process is attributed to the kda vascular endothelium receptor sialoglycoprotein (gp ). albumin binds to gp and forms a cluster in association with cav- , the main protein critical to caveolae formation. clustered albumin-gp receptors and compounds bound to albumin are then internalized and transported to the basolateral membrane to complete transcytosis [ ] . interestingly, modified albumins show preferential binding to gp and gp [ ] . preferential internalization of albumin in cancer cells has also been correlated with albumin binding to sparc (secreted protein acidic and rich in cysteine). for example, immunohistochemical staining detected stromal j o u r n a l p r e -p r o o f sparc in ∼ % of non-small cell lung cancer (nsclc) cases, and increased chemotherapeutic efficacy of albumin-bound paclitaxel was correlated with high stromal sparc reactivity of nsclc cells [ ] . recycling: the long half-life of albumin is attributed to the neonatal fc receptor (fcrn), a widely distributed intracellular receptor responsible for salvaging albumin from cellular catabolism [ ] . fcrn binds to albumin in the acidic endosome, diverts it from the lysosomal degradation pathway, and the complex is exocytosed. albumin is released from fcrn at extracellular ph and enters circulation through the lymphatics, thus prolonging its half-life. albumin also avoids renal clearance by reabsorption through megalin and cubilin receptormediated endocytosis in the renal proximal tubule. in situ albumin-binding drugs have been designed to covalently react with or noncovalently bind to endogenous albumin, which capitalizes on the long-circulating property of the protein to enhance the pk and hence pd of the drug. indeed, exploitation of endogenous serum albumin has several advantages over the use of exogenous albumin: first, although commercial exogenous albumin can be isolated with high yield and purity, it is often contaminated with pathogens. second, the cost, effort, and time required for manufacturing exogenous albumin is altogether avoided. third, as no external macromolecular carrier is involved, the quality control associated with endogenous albumin is comparable to small molecule drug candidates. irreversible covalent bond formation between endogenous albumin molecules and a therapeutic payload can be utilized to enhance the pharmacokinetics of the latter. kratz et al. pioneered this strategy by synthesizing a prodrug that selectively reacts with the th cysteine residue of circulating serum albumin after systemic administration, which improves the drug's plasma half-life and protects it from premature degradation. the therapeutic cargo (e.g., dox) was anchored with a thiol reactive maleimide group via a ph-sensitive hydrazone bond and a carefully optimized alkyl spacer. upon intravenous injection, the highly reactive maleimide group formed an irreversible thioether bond with the thiol group of the albumin. the ph-sensitive hydrazone bond facilitated the liberation of the covalently attached dox from albumin after en endocytosis by cells. aldoxorubicin, also known as dox-emch (figure b & c) is currently j o u r n a l p r e -p r o o f under various phases of clinical trial for the treatment of soft tissue sarcomas (nct and [ ] ) and small cell lung cancer (nct ). inspired by dox-emch, many other albumin-binding prodrugs have been developed. these prodrugs often consist of an anticancer drug, a maleimide group as the thiol-binding moiety, and a cleavable linker [ ] . native albumin-binding ligands such as fatty acids can be directly conjugated to therapeutics, which results in docking of the drug to endogenous albumin upon systemic administration. a notable example of this class is semaglutide, an fda approved drug that is a glucagon-like peptide- (glp- ) analog with an octadecyl fatty diacid conjugated to the epsilon amine of a lysine residue in the peptide via a glutamyl ethylene glycol spacer [ ] . the plasma half-life (t / , seven days in humans) and glp- receptor (glp- r) binding affinity (k d ~ . nm) of semaglutide make it suitable for once-weekly administration for treatment of type diabetes [ , ] . this was an important advancement, as its predecessor liraglutide -which contains a hexadecyl monoacid conjugated to the same lysine residue of glp- via a γ-glutamic acid spacer-has a t / of only . days in humans, and is hence only approved for once-daily treatment of type diabetes, despite having a three-fold higher affinity for glp- r (k d ~ . nm) than semaglutide. this dramatic improvement was attributed to both the spacer and ligand chemistry of the glp- analog in semaglutide, which influenced in vivo albumin binding [ ] . other therapeutic payloads are bound to endogenous hsa by conjugation of the drug to fatty acids through various cleavable linkers [ , ] . synthetic small molecules can also bind endogenous albumin [ ] . most drugs and small molecules that interact with hsa are anionic, although a few cationic drugs have detectable affinity [ ] . evans blue (eb), an aromatic dye with four anionic charges, reversibly binds to serum albumin with a micromolar affinity. the affinity of eb for albumin allows % of the injected dose to be retained in the blood; thus, it can be used to quantify total plasma volume of a test subject [ ] . albumin-bound eb can also be used to assess blood brain barrier (bbb) permeability, as the bbb is impermeable to the complex in healthy, non-pathological conditions [ ] . eb derivatives with various affinities to albumin have been used to deliver therapeutic cargo [ , ] . yamamoto et al. developed an o-toludine eb analogue that chelates gadolinium (iii), as a t -weighted mri contrast agent for imaging of blood vessels [ ] . labeling with different pet isotopes (e.g., ga, cu) [ ] [ ] [ ] . chen et al. evaluated labeled neb in mice as a blood pool imaging agent under various pathological conditions, including myocardial infarction (mi) and serum leakage from permeable or abnormal blood vessels. sentinel lymph nodes (slns) were visualized in all pathologically diagnosed breast cancer patients within . − . ( . ± . ) min [ ] . by analyzing ga-neb pet/ct images. to identify molecular features required for albumin binding, the neri group screened a dnaencoded chemical library comprising > oligonucleotide-conjugates of potential albumin-binding molecules coded with unique six-base-pair sequences for identification [ ] . the hsa-binding tags were chosen through systematic evolution of ligands by exponential enrichment (selex). the selection, amplification, and microarray analysis of the pool suggested that following structural features are important for albumin binding: (i) the presence of a -phenylbutanoic acid moiety is essential; (ii) a butanoyl acid moiety increases albumin affinity as propanoyl and pentanoyl acid residue was not enriched in the pool; and (iii) substitution at the para position of the phenyl ring with a hydrophobic functionality increases the affinity towards albumin. albumin-binding domains (abds) are small protein domains that non-covalently associate with serum albumin. abds are structurally robust and stable, as evidenced by their ability to withstand denaturation at extremely low ph ( . ) and high temperatures. and have also been explored for drug delivery. abds are used for drug delivery by either fusion with a therapeutic protein at the gene level if the protein is recombinantly produced or are chemically synthesized to conjugate small molecule drugs for systemic administration. sjöbring and colleagues isolated a -kda albumin binding protein fragment from streptococcal protein g. this -amino acid native abd (abdn) is a -helix bundle and has nanomolar affinity for hsa [ , ] . using abdn as a template, jonsson et al. identified an engineered version of abdn from a phage library. specifically, they targeted residues in a combinatorial protein engineering strategy to identify an albumin -binding sequence with improved hsa affinity. the high -affinity abd (abdh) has superior thermal stability and binds hsa with femtomolar affinity. chilkoti and colleagues recombinantly fused abdn and abdh to the n-terminus of a hydrophilic, thermally responsive chimeric polypeptide (cp) that contained multiple c-terminal cysteine residues [ ] . the recombinant polypeptides formed highly monodisperse micellar nanoparticles upon covalent conjugation of multiple copies of hydrophobic dox molecules to the cysteine residues through the ph-sensitive hydrazone bond (using maleimide-thiol chemistry) ( figure a) . the dox-loaded abdn/abdh-decorated polypeptide micelles -termed abd-j o u r n a l p r e -p r o o f journal pre-proof cp-dox-bind both human and mouse serum albumin with high affinity, as seen by native page and isothermal titration calorimetry (figure b & d) . hence, upon systemic injection, they instantaneously bind endogenous albumin with high affinity and are coated with an albumin corona. the albumin decorated abd-cp-dox nanoparticles had significantly superior pk in both murine and canine models compared to a negative control, cp-dox nanoparticles that do not present abd domains on the surface of the nanoparticle (figure e & f) the long plasma circulation of the abd-cp-dox nanoparticles also resulted in higher accumulation in tumors than the cp-dox nanoparticles, and these nanoparticles also showed better tumor regression, and a wider therapeutic window than the cp-dox nanoparticles. these results clearly show the enhancement in pk and pd conferred by decorating a drug-loaded nanoparticle with endogenous albumin. furthermore, the same group took advantage of the native sortase reaction to directly install dox onto the abd (abd-dox) through a ph-sensitive linker, without forming nanoparticles [ ] . abd-dox bound to both human and mouse serum albumin with nanomolar affinity, had a terminal t / of . h in mice, and increased tumor accumulation by ~ fold compared to free dox ( figure a ). in multiple mouse xenograft models, abd-dox resulted in greater tumor regression than aldoxorubicin, dox-prodrug under clinical development that was designed to covalently bind endogenous serum albumin. other notable examples of albumin-based delivery systems involve the genetic fusion of abd to various therapeutic proteins including affibodies [ , ] , human soluble complement receptor type [ ] , single chain antibody-drug conjugates [ ] , insulin-like growth factor ii [ ] , immunotoxins [ ] , and respiratory syncytial virus subgroup a (rsv-a) g protein (g na) [ ] . j o u r n a l p r e -p r o o f albumin-bound therapeutics can also be formulated ex vivo with purified albumin molecules prior to administration. in contrast to other physiological proteins, albumin tolerates a wide range of ph (stable in the range of - ), temperatures (can be heated at °c for up to h), and organic solvents [ ] . these properties have motivated researchers to covalently and non-covalently append various payloads, including radioisotopes (e.g., f, ga, in) and anticancer drugs (e.g., dox, curcumin, methotrexate), to albumin for imaging and delivery purposes [ , ] . however, modification of exogenous albumin with drugs is often associated with suboptimal drug loading, as denaturation and albumin molecule crosslinking potentially compromise its physiological behavior. smith et al. addressed these challenges by synthesizing an albumin-polymer-drug conjugate with high drug loading efficiency [ ] . using a convergent, reversible addition−fragmentation chain transfer (raft) polymerization reaction, they synthesized a polymeric prodrug of panobinostat, an fda-approved anticancer agent, with hpma (n- -hydroxypropylacrylamide). multiple copies of panobinostat were attached to the polymeric prodrug, which was then conjugated to albumin using nhs chemistry under physiological conditions, thereby minimally affecting the albumin molecule ( figure b ). the first albumin-drug conjugate that entered phase i/ii trial was a methotrexate-albumin (mtx-hsa) conjugate synthesized using covalent coupling between carboxylate of the mtx and lysine residues of hsa. seventeen patients not amenable to standard care were treated with up to eight injections given in weekly intervals. tumor responses were seen in three with no sign of toxicity and drug accumulation. the mtd for weekly administration was found to be × mg/m and a injection of mg/m every weeks was recommended for a further study [ ] . however, in a subsequent phase ii study no objective responses were seen, although eight patients did not have disease progression (stable disease) for up to months (median days) [ ] . a phase ii study showed combination of mtx-hsa with cisplatin as an effective treatment modality against urothelial carcinomas with an acceptable toxicity profile [ ] . patients were treated with a loading dose of mg/m of mtx-hsa followed by a weekly dose of mg/m starting on day . tumor response was observed in patients. complete response (cr) and partial response (pr) was observed in patient each (overall response rate: %) but no follow up clinical investigation was undertaken. paclitaxel, an fda approved paclitaxel formulation for the treatment of multiple cancer types [ , ] . abraxane was developed by american bioscience using the so-called nab-technology in which paclitaxel and human serum albumin are passed through a jet under high pressure to form nanoparticles with a mean diameter of nm. several albumin nanoparticle of small molecule drugs has been developed using nab technology and are under various phases of clinical trials [ ] . nab-rapamycin is currently under phase i clinical trial to treat non-muscle invasive bladder cancer (nct ) and under phase ii clinical trial to treat progressive high-grade glioma (nct ). nab-docetaxel proved to be effective against hormone refractory prostate cancer (nct ) and metastatic breast tumors (nct ). nab- , a novel albumin formulation of thiocolchicine dimer exhibits dual inhibition of tubulin polymerization and topoisomerase i activities. it showed antiangiogenic and vascular targeting activities and was found to be effective against solid tumors and lymphomas (nct ). lin et al. reported a method to simultaneously encapsulate paclitaxel by denaturing albumin with sodium borohydride at high urea concentration, followed by addition of hydrophobic drugs to the denatured protein, and diluting the mixture to spontaneously refold albumin and form nanoparticles [ ] . this method avoids chemical crosslinkers and high-pressure emulsification techniques. by adding a cell-penetrating peptide to the dual drug-loaded albumin nanoparticles, they achieved bbb penetration through interaction with sparc, which resulted in greater survivability in a u orthotopic glioma model [ ] . albumin has also been derivatized into a diblock format in which a hydrophilic albumin block gets exposed at the surface of the hybrid nanoparticle and a synthetic hydrophobic polymer, such as polycaprolactone, forms the core. to achieve this, jiang et al. synthesized two different polymers based on ring-opening polymerization: poly(oligo-(ethylene glycol) methyl ether acrylate)-poly(ε-caprolactone) (poegma-pcl) and maleimide-functionalized polycaprolactone (mi-pcl). mi-pcl was conjugated to the free cysteine on bovine serum albumin (bsa-pcl) [ ] . the authors formulated hybrid nanoparticles by co-assembling poegma-pcl, bsa-pcl, and curcumin with increasing albumin content. cellular uptake of the nanoparticles in cancer cell lines increased with albumin content [ ] . additionally, albumin nanoparticles can be decorated with targeting ligands, such as mannose [ ] , folic acid [ ] , antibodies [ , ] , and aptamers [ ] , to provide greater receptor specificity. recombinant albumin is another alternative to animal-derived albumin and is increasingly being used in exogenous formulations. direct genetic fusion to recombinant albumin enables one-step fabrication of therapeutic proteins. for instance, rlx-fp, a genetically encoded fusion protein linking recombinant human albumin with human coagulation factor ix, has a -fold longer j o u r n a l p r e -p r o o f plasma half-life than unmodified coagulation factor ix (fix) products. an advanced phase iii clinical trial (prolong- fp) demonstrated the long-term safety, tolerability, and efficacy of rix-fp for prophylactic and on-demand treatment of bleeding episodes in children [ ] . li et al. synthesized an albumin-lidamycin conjugate by recombinant dna technology. lidamycin, a cyclic enediyne antibiotic is ~ fold more potent than dox against multiple cultured cancer cell line and it consists of an apoprotein (ldp) and an enediyne chromophore (ae) that can be separated and reassembled in vitro [ ] . a dna fragment encoding hsa-ldp was constructed and the fusion protein was purified from pichia pastoris using imac. the hsa-ldp conjugate was then reconstituted with the ae to form the albumin-lidamycin conjugate that had a sub nanomolar ic value in various cancer cell lines, significant tumor retention , and potent in vivo tumor regression efficacy against mouse hepatoma h model [ ] . other recombinant albumin proteins include genetic fusions with interleukin- [ ] and interferons [ , ] . albuferon® aka albinterferon-α- b was developed as a fusion protein of albumin and interferonα- b (infα- b) and is under phase iii for the treatment of hepatitis c infection (nct ). several other phase iii trials are under progress aimed at evaluating the efficacy and safety of albinterferon-α- b [ ] . however, high-yield synthesis of pure recombinant albumin and albumin fusions requires a complex procedure and specialized yeast expression system that are not readily available to most researchers. nguyen et al. made significant progress by purifying recombinant hsa from a bacterial expression system using maltose-binding protein and protein disulfide isomerase [ ] . but an optimized bacterial expression system to purify recombinant albumin is still needed. collagen is the most abundant protein in mammals, making up nearly one-third of all proteins in the body. a diverse family of structural proteins, collagen is a major component of the extracellular matrix (ecm) and connective tissue. it has a broad spectrum of functions, including cell adhesion, cell migration, tissue scaffolding, and repair [ ] . to date, thirty different classes of collagen have been identified and characterized [ ] , though not all are relevant for this discussion. the most abundant class of collagen -fibrillar collagen-represents more than % of human collagen and includes types i, ii, iii, v, and xi. this class is a major component of skin, hair, ligament, tendon, cartilage, bone, and placenta. other common types of collagens, like iv and viii, construct the network structure of basement membranes [ , ] . before discussing their molecular architecture and biomedical applications, it is important to define the term -collagens.‖ gelatin, a thermally degraded, collagen-derived product, is also termed j o u r n a l p r e -p r o o f ‗‗collagen'' in the literature despite losing the characteristics of real collagen, and will hence be discussed separately [ ] . although the molecular architectures and functions of collagens vary greatly, they all share the same tertiary structure -the collagen triple helix [ ] . under the electron microscope, native collagen shows a thread-like structure -fibrilsconsisting of collagen molecules organized in three interlocking polytripeptide chains that form a triple helix , with each chain having the general sequence giy-x-y, where x and y are occupied by proline (pro) and ( r)hydroxyproline with the highest statistical frequency. each collagen polypeptide chain is twisted around a threefold screw axis and exists in a secondary structure analogous to the left-handed polyproline ii-helix. the three polyproline ii-helices are held together by hydrogen bonding, which repeats periodically between the glycine amide in one helix and the carbonyl group of the amino acid residue of the neighboring helix. the three polyproline ii-helices form a right-handed triple helix stabilized by hydrogen bonds [ , , ] . with tunable self-assembly, gel-forming ability, biodegradability, and responsiveness to external stimuli, collagens have been increasingly used for drug delivery and tissue regeneration [ , [ ] [ ] [ ] . current research is focused on developing short, bioinspired model collagens, termed as collagen-mimetic peptide (cmps) or collagen-like peptides (clps) [ , ] . clps are short, synthetic peptides arranged in the same triple-helical conformation as native collagens. clps have been used: (i) to elucidate the triple helix structure and the molecular forces responsible for this architecture; (ii) to target pathological collagen in vivo through triple helix hybridization; and (iii) as a bioactive domain to construct smart biomaterials through hierarchical self-assembly. each of these applications will be discussed separately as they are relevant for drug delivery. to exploit clps to fabricate biomaterials, it is critical to elucidate the intrinsic parameters affecting the sequence-based thermal stability of the triple helix. in contrast to native collagen, clps exhibit reversible phase transition behavior. their slow folding rate upon denaturation and small size allow researchers to thermodynamically characterize the folding and melting processes of clps by x-ray crystallography, atomic force microscopy (afm), light scattering, and circular dichroism (cd) and nuclear magnetic resonance (nmr) spectroscopy. many researchers have synthesized and studied polypeptides with gly-x-y sequences that fold into a triple-helical structure. persikov et al. investigated the role of guest amino acid residues at the x and y positions on the thermal stability of the triple helix [ ] . they documented that the guest j o u r n a l p r e -p r o o f triplets gly-x-hyp and gly-pro-y can be used to quantitate the conformational propensities of all amino acids to form a triple helix at the x and y positions in a (gly-pro-hyp) persikov proposed that the triple-helical structure of clps is the direct result of the propensity of amino acids to adopt a polyproline ii-like conformation, which is driven by the high propensity of ionizable residues in the x position for interchain hydrogen bonding and a low propensity of bulky residues in the y position for effective solvation. building upon this concept, many studies have deciphered the structure-function relationship of the clp phase transition [ , , ] . to improve the stability of the triple-helical structure of clps, c-terminal covalent crosslinking was performed using an interchain cystine knot derived from collagen type iii [ ] [ ] [ ] . such covalent modification also includes the crosslinking of three α-chains at the cterminus using various templates such as cis,cis- , , -trimethylcyclohexane- , , -tricarboxylic acid (kta) and tris( -aminoethyl)amine-(succinate-oh) (tren) [ , ] . comparative analysis using cd and nmr spectroscopy revealed that the flexibility of the tren scaffold is superior to that of the kta scaffold for the induction of triple helicity [ ] . however, to avoid cumbersome covalent crosslinking, simple noncovalent crosslinking strategies have been developed based on: (i) interchain metal bridging by functionalization of the terminus of clps with a chelating ligand [ , ] and (ii) hydrophobic interactions using a single saturated hydrocarbon or lipid tail [ ] [ ] [ ] . the thermal stability of the triple helix of lipid-modified clps increased as the monoalkyl chain length increased from c to c [ ] . furthermore, introducing amino acids with guest residues that have a cγ substitution with a highly electronegative atom, such as fluorine and chlorine, in place of hydroxyproline also increased triple helix stability [ ] [ ] [ ] [ ] . it is important to note that the triple-helical structure restricts the sequence space available for synthetic clps, and until recently, was thought to be intolerant to substitution of glycine in the gly-x-y triplet. multiple recent reports have shown that modification of glycine with a thioamide, nitrogen atom, or azaglycine (azgly) yielded comparable or better stability of clps compared to their non-substituted counterparts. another recent approach incorporated metal-binding sites at the ends of clps to drive thermodynamically stable, nanomicro scale self-assemblies of various shapes [ , ] . zheng et al. elucidated the role of surface electrostatics and hydrogen bonding on the stability of the triple helix and provided computational tools for de novo design of clps beyond the gly-x-y triplet [ ] . taken together, these structure-function studies indicate that nature may not have optimized the stability of the collagen triple helix, and that there is plenty of room for further improvement through precise design of clps beyond tandem arrays of the gly-x-y triplet sequence. although the major impediment in clp research is deciphering the triple helix conformation and supramolecular assembly formation (discussed in the next section), biomedical applications of monomeric clps have gained attention in recent years using denatured collagen. degradation of the ecm is a crucial element governing the progression of tissue remodeling in many life-threatening diseases, including cancer, cardiovascular diseases, and organ fibrosis, as well as prevalent debilitating conditions, such as arthritis and intervertebral disc degeneration. during tissue remodeling, collagen molecules within the collagen fibers and networks are degraded by proteases, such as mmps or cathepsin, and denature at body temperature. in a seminal work, yu and coworkers demonstrated that unfolded, single-stranded clps with a sequence (gpo) n (where n= - ) have a high propensity for binding denatured collagen through a -strand hybridization process‖, which is similar to the binding of complementary dna strands [ , ] . due to the high serum stability of such collagen -hybridizing peptides [ ] and their affinity for native collagen [ ] , they have been used to selectively stain native collagen both in vitro and in vivo by fluorescently labeled clps [ ] [ ] [ ] [ ] [ ] . fluorescently labeled clps can also target solid tumors through the triple helix hybridization process, as high mmp- activity in tumor tissue exposes denatured collagen [ ] . because homotrimeric clps have little driving force for collagen hybridization, clps must be thermally dissociated by heating at °c to the monomeric state before binding to a collagen substrate. to avoid the preheating step, the yu group developed a sequence (gfo) by replacing the hydroxyproline with a fluoroproline (f) [ ] . this new sequence cannot self-trimerize at body temperature but maintains the ability to hybridize with natural collagen chains. by appending an octa-glutamic acid residue at the nterminus of the (gpo) , the authors attracted vascular endothelial growth factors (vegfs) to the collagen-binding site of the endothelial cells through charge-charge interactions, which resulted in tubulogenesis [ ] . triple helix hybridization has also been used to impart collagen-targeting properties to nanoparticles [ , ] and for sustained release of nucleic acids from collagen films and depots [ , ] . recently, the hubbell lab recombinantly fused a collagen-binding domain (cbd) to interleukin- (figure ) , a potent cytokine that stimulates the innate and adaptive immune system (cbd-il- ). intravenously administered cbd-il- preferably accumulated in the tumor stroma and provided a sustained intratumoral level of interferon-γ, thereby eliciting superior anti-tumor effects and fewer off-target toxicities when compared to naked il- [ ] . in the past few decades, the wealth of knowledge accumulated about the structure of collagen has driven a surge in research focused on the supramolecular assembly of collagenlike peptides. in the previous section, we described how various molecular interactions can stabilize the formation of a triple helix. in this section, we delve deeper into the molecular determinants of higher-order self-assembly of clp--based biomaterials -beyond the triple helix-and how they can be used in drug delivery. we have organized this section by the specific molecular stimuli that trigger self-assembly. cysteine knot in clp self-assembly: in an early example, raines and coworkers used two cysteine knots to covalently link three clps, one of which protruded out to produce a sticky end during triple helix assembly [ ] . the sticky ends forced the trimers to configure themselves in a head-to-tail fashion, which resulted in a long, single collagen triple helix. such collagen-like fibrils were much longer ( nm) than the native type i collagen ( nm). in a similar approach, the koide group developed a synthetic clp system in which three clps were preorganized in a staggered configuration that was locked in place by two cysteine knots [ ] . cd, ultrafiltration, and laser diffraction analysis indicated that the staggered trimers formed large supramolecular architectures through intermolecular triple helix formation. however, hydrogels made of these collagen fibers did not successfully form due to concentration-dependent aggregation. to address this limitation, yamazaki et al. appended a hydrophilic arginine residue at the cysteine knotted end of the synthetic clps [ ] . the disulfide-linked trimers of the gly-x-y triplet repeats formed hydrogels by spontaneous intermolecular triple helix formation upon cooling. the thermal sol-gel transition is reversible, and the design of the peptides can tune the transition temperature. koide's group also incorporated an integrin-binding sequence gfoger into one clp strand of the same knotted trimeric base unit. the supramolecular structure exhibited adhesion to human dermal fibroblasts that was comparable to natural collagens [ ] . to further tune the gel properties, ichise et al. appended multiple end-to-end disulfides through crosslinking of chemically synthesized triple-helical clp. rheology showed that gel stiffness is controlled by the number of cysteine residues up to three, at which point it plateaus. with the incorporation of an integrin α β -binding sequence, the peptide polymer showed receptorspecific cell binding in vitro. moreover, cell signaling activity and biodegradability of such a system can be tuned by altering the content of integrin binding ligand in the peptide polymer and by varying the weight percentage of clp [ ] . π-π interaction in clp self-assembly: the maryanoff group introduced an aromatic-aromatic recognition motif at either the n-or c-terminus of each peptide chain of the clp triple helix to drive the supramolecular assembly of clps [ , ] . the aromatic π-π interaction facilitated head-to-tail stacking of clps into a micrometer-sized fibrillar structure, as evidenced by cd, h nmr, dynamic light scattering (dls), tem, afm, and computational energy dynamics. the fibrillar clps induced the aggregation of human blood platelets, an ability lacking in less organized clps, with nearly the same potency as native type i collagen [ ] . kar et al. investigated how the presence of aromatic residues on neither end, one end, or both ends of a collagen model peptide affected the kinetics of self-association. clps with aromatic residues at both termini self-assembled and aggregated too quickly to be monitored by turbidimetry at a concentration of mg/ml [ ] . similar interactions, such as ch-π interaction between amino acids (proline and hydroxyproline) and aromatic residues (phenylalanine and tyrosine) and cation-π interaction between a positively charged n-terminal arginine and a c-terminal phenylalanine, were also introduced to fabricate collagen-like fibrillar structures in a head-to-tail manner [ , ] . hydrophobic interaction in clp self-assembly: hydrophobic interactions also contribute to clphybrid self-assembly and collagen-mimicking properties. the field and tirrell research groups independently synthesized clp-hybrids by conjugating collagen peptides with single or double hydrocarbon tails. lipidation of clps increased the triple helix stability, triggered self-assembly of the clp-hybrid in spherical micelles, induced the formation of lipid film, and promoted cell adhesion [ , ] . to induce even higher-order assembly, the tong research group designed a series of clps consisting of four segments: an n-terminal palmitoyl segment, a β-sheetforming domain, a polylysine spacer, and a bioactive clp domain [ ] . a clp with the sequence (gpo) gfoger(gpo) and a c-terminal c hydrocarbon tail self-assembled into micrometer-long fibers with a nm diameter in aqueous solution. self-assembled peptide nanofibers made of triple-helical clp constructs with bioactive gfoger sequences recognized and adhered to several integrin receptors and promoted cell spreading, thereby mimicking native collagen [ ] . electrostatic interaction in clp self-assembly: the conticello and chaikof research groups first presented evidence of a self-assembled, fibrous, collagen-like structure with a well-defined dperiodicity using lateral electrostatic interaction through a sticky-ended nucleation strategy, j o u r n a l p r e -p r o o f reminiscent of that commonly used in the assembly of dna [ ] . they used a synthetic peptide sequence (prg) (pog) (eog) consisting of three distinct segments: a central tetrameric pog sequence flanked by an arginine-containing positively charged domain at the n-terminus and a glutamic acid-containing negatively charged domain at the c-terminus (figure ). tem suggested that in the absence of thermal annealing, non-banded fibers are formed. in contrast, fiber growth was observed within several hours after thermal annealing. a nucleation-growth mechanism was followed by the self-association of multiple triple helices, which was driven by electrostatic interactions between oppositely charged terminal residues and hydrophobic interactions between central domains from neighboring triple helices. building upon this concept, o'leary et al. proposed a new sequence (pkg) (pog) (dog) by replacing arginine and glutamic acid with lysine and aspartic acid, respectively [ ] . they argued that arginine has a higher propensity to hydrogen bond with a carbonyl group of a neighboring strand compared to lysine, which inhibits its ability to form a salt bridge with the carboxylate group of the glutamic acid and thus forms homogenous collagen mimetic nanofibers. the modified peptide sequence formed both homogeneous fibers and hydrogels, and the hydrogel was degraded by collagenase at a rate similar to that for natural collagen. raines's group adapted this approach to generate extended fibers a few microns in length with a highly symmetrical and uniform -tesselated‖ geometry that was dictated by the precise placement of lysine-aspartate salt bridges in the staggered triple helix building blocks. the extended geometry of this construct maximizes the surface area available to support the growth and maturation of cells [ ] . notably, the banding periodicity of these peptide fibers ( nm) is significantly longer than the length of the peptide itself ( nm) [ ] . this elongation suggests that the units driving self-assembly are not the individual triple-helical peptides but oligomers of multiple triple helices that are preorganized such that their assembly into mature fibers leads to periodic packing and charge distribution, as visualized by tem. this contrasts with the native type i collagen fiber, in which staggered assembly of individual collagen molecules is responsible for the periodic banding pattern and suggests that true mimicry of the natural collagen assembly process has yet to be achieved. hydroxyproline (o-propionate hydroxyproline, p e ) that shows ph-sensitive folding [ ] . melting and folding experiments using cd indicated that introducing - units of the unnatural amino acid p e into the backbone had little effect on triple helix stability at acidic and neutral ph, although the rate of folding into the triple helical structures were different. they also observed that a peptide in which all seven hydroxyproline residues were substituted with p e underwent ph-triggered folding with an even lower transition temperature than the parent clps [ ] . research group led by chmielewski and hsu also placed metal-binding ligands at various points along the clp backbone for chelation-controlled higher-order assembly. they appended histidine or iminodiacetic acid to the termini of the (pog)n backbone and upon adding divalent metal ions, such as ni + , zn + , and cu + , the clps co-assembled into petal-like microstructures with a periodic banding at the nanometer scale [ ] . subsequent work from the chmielewski group demonstrated that a diverse range of distinct structures, including microflorettes, stacked sheet microsaddles, and fiber-like meshes, could be made by varying the length of the (pog)n core of the peptides and the metal ion concentration. [ ] . as demonstrated by hsu and coworkers, a similar metal-promoted supramolecular assembly can also be obtained by introducing histidine residues at both termini as well as into a clp backbone lacking hydroxyproline. a variety of higher-order structures were obtained, ranging from the nano-to micro-scale [ ] . metal-induced self-assembly is advantageous over lateral electrostatic interaction because diverse supramolecular structures can be obtained under mild physiological conditions. a repertoire of unsatisfied metal-ligand interactions are also available in these selfassembled structures that enable incorporation of functional peptide sequences [ ] [ ] [ ] [ ] for biomedical applications, including d culture of human endothelial cells, protein purification, and fluorescence tagging. temperature -triggered self-assembly of clps: kiick and co-workers recently reported the temperature -triggered self-assembly of clp-hybrid materials [ ] [ ] [ ] [ ] . they appended a synthetic polymer, poly(diethylene glycol methyl ether methacrylate) (pdegmema) or human tropoelastin -derived elastin-like polypeptide (elp, to be discussed in a later section) with clps. both pdegmema and elps are thermally responsive and exhibit lower lcst-phase behavior. above a transition temperature (t t ), they undergo reversible phase transition from a soluble phase to an insoluble coacervate, which drives the self-assembly of the clp-hybrid into vesicular-or platelet -like structures. conversely, triple helix formation of the clp chains drives the formation of a bilayer of the supramolecular assembly (figure ) [ , ] . it is important to note that the t t of the short elp sequence is drastically reduced in the clp-elp hybrid due to formation of a clp-triple helix that increases the local concentration of the elp. above the j o u r n a l p r e -p r o o f melting temperature of the clps, these nanostructures disassemble into unimers. moreover, the clp-elp vesicle can target type ii collagen, which is overexpressed in various diseases, including cancer, through triple helix hybridization. these vesicles exhibited sustained release of an encapsulated fluorophore over a period of three weeks, and complete release was triggered by destroying the clp -helical structure with heat [ ] . gelatin is a well-known, biodegradable, biocompatible, and nonimmunogenic biomaterial obtained either by partial acid or alkaline hydrolysis or by thermal or enzymatic degradation of type collagen [ ] . gelatin is composed primarily ( - %) of proteins, with minerals and water constituting the rest. gelatin is used in a wide range of food, cosmetic, and pharmaceutical applications [ , ] . the triple helix conformation of gelatin endows it with high thermal stability in solution [ ] . the use of gelatin has gained popularity for the following reasons: (i) gelatin is readily available and inexpensive; (ii) gelatin is highly soluble and easy to use; (iii) gelatin's structure is highly similar to that of collagen and contains peptide binding motifs for cell attachment; (iv) gelatin is biocompatible, biodegradable, and does not induce antigenicity or at the sequence level, gelatin molecules contain repeating (gly-x-pro)n triplets, where x represents the amino acid (usually lysine, arginine, methionine, or valine), that is responsible for the triple helical structure of gelatin. gelatin is a polyampholyte that is ~ % positively charged (lysine and arginine), ~ % negatively charged (glutamic and aspartic acid), and ~ % hydrophobic (leucine, isoleucine, methionine and valine) [ ] . another important trait shared by collagen and gelatin is the low frequency of aromatic amino acids -tryptophan, tyrosine, and phenylalanine-in their primary sequence. the low frequency of these amino acids is the primary reason for the low antigenicity and toxicity of both gelatin and native collagen [ ] . commercially, two different types of natural gelatin -cationic type a & anionic type b-are j o u r n a l p r e -p r o o f available and differ based on the details of the manufacturing process -collagen hydrolysisused to obtain them [ ] . gelatin a is obtained from porcine skin following acid pre-treatment, which minimally hydrolyses the amide groups of glutamine and asparagine and results in a macromolecule with a pi ranging from to . conversely, gelatin b is extracted from osseincut bovine hide-and is pre-treated at alkaline conditions, which leads to hydrolysis of asparagine and glutamine to aspartate and glutamate, respectively, thus resulting in a lower pi of - . recombinant gelatin, which can serve as a substitute for the natural protein, is also commercially available [ , ] . however, unlike collagen, gelatin suffers from mechanical instability and has high rate of degradation. it is also highly susceptible to several proteases. gelatin nanoparticles (gnps) prepared without crosslinking were unstable and formed aggregates [ , ] . for these reasons, crosslinking of gelatin-based materials is required to give it mechanical stability, shape, and enhanced circulation time in vivo [ , , ] . crosslinking of gelatin can improve its thermal and mechanical stability, as well as lower its degradation in vivo. crosslinking, which modifies cleavage sites of gelatin molecules, inhibits enzyme-substrate interactions and hence increases the resistance to degradation [ ] . various crosslinking agents, including aldehydes, genipin, carbodiimide/n-hydroxysuccinimide, and enzymes, have been explored to covalently crosslink gelatin [ , ] . the crosslinking density, defined as bloom number, of the gelatin biomaterials can be altered by the crosslinking reaction conditions and/or increasing the concentration of crosslinker, thus regulating its physicochemical and biomedical properties. in addition, the mechanical properties of gelatin can also be tuned by temperature and ph. at a sufficiently high concentration, gelatin forms a semisolid hydrogel at lower temperatures. as the gelatin solution cools to below °c, a random coil to triple helix transition, promoted by stable hydrogen bond formation, occurs [ ] . changes in ph affect the melting temperature of gelatin and hence the propensity for gel formation. moreover, changes in ph modify ionic interactions between gelatin monomers and consequently affect hydrogen bondmediated crosslinking [ ] . physical crosslinking is also possible and can reversibly modulate gel strength and has the advantage that it does not need enzymes or chemical reagents to effect crosslinking [ ] . various methods such as desolvation, coacervation, emulsification, nanoprecipitation, and layer-by-layer coating have also been used to fabricate gelatin-based systems for drug and gene delivery [ , ] . gelatin nanoparticles (gnps) range from to nm in size [ ] . colloidal stability, drug release, drug encapsulation, tissue distribution, and cellular uptake are strongly dictated by the charge and size of gnps. the effects of various parameters, such as temperature, ph, j o u r n a l p r e -p r o o f cosolvents, degree of crosslinking, and type of gelatin, on the physicochemical properties of gnps have been investigated by several groups [ ] [ ] [ ] . a temperature of °c was found to be optimal to attain a narrow size distribution of gnps because at q high temperature ( °c), gelatin forms a random coil, which destroys the self-assembly and the nanoparticle while at room temperature, gelatin forms a highly viscous semi-solid. in contrast, at °c, a controlled uncoiling of the triple-helical structure results in monodisperse gnps. the size and dispersity of gnps is highly dependent on solution ph, and ph and ph were found to be optimal for nanoparticle formation of cationic type a and anionic type b gelatins, respectively. increasing the degree of crosslinking results in greater polydispersity and a reduction in particle size. gnps and their hybrids have been explored to encapsulate both hydrophobic and hydrophilic bioactive molecules including antimalarial drugs [ ] , such as chloroquine phosphate; anti-cancer drugs [ , ] , such as paclitaxel, dox, cisplatin, curcumin, and methotrexate; growth factors [ ] , such as osteogenic bone morphogenetic protein- (bmp- ), and angiogenic basic fibroblast growth factor (bfgf); anti-inflammatory drugs [ ] , such as ibuprofen; photo-responsive dyes [ , ] ; anti-hiv drugs [ ] such as didanosine; and therapeutic nucleic acids [ , ] . in all cases, an on-demand release profile was achieved by controlling the degree of crosslinking, ph, temperature, and fabrication method and thus the physicochemical properties of the gnps. for example, amjadi et al. designed a ph-responsive nanocarrier by decorating the gnps with (methoxy poly (ethylene glycol)-poly (( dimethylamino) ethyl methacrylate-co-itaconic acid) (pgnps) (figure a ) [ ] . the nanoparticles ( nm diameter) were encapsulated with dox and betanin with high efficiency (loading capacities of . % and . %, respectively), and demonstrated ph-and temperature -responsive drug release, and synergistically inhibited the growth of mcf- breast cancer cell lines. in another example, rose bengal -conjugated gelatin nanoparticles (rb-gnps) were developed for antimicrobial photodynamic therapy ( figure b ) [ ] . rb-gnps generated singlet oxygen that damaged the microbial cell membrane, which demonstrated their potential for treating multi-drug-resistant microbial infections. gelatin microparticles (gmps) have been extensively investigated for many biomedical applications. solorio et al. engineered self-assembled, gelatin microsphere-incorporated human mesenchymal stem cell (hmsc) sheets [ ] . the microspheres were loaded with growth factor tgf-β , distributed within the hmsc sheets, and released chondrogenic growth factor to the j o u r n a l p r e -p r o o f interior of the sheets, thus enabling spatially homogenous differentiation of the hmscs. the differentiation rate could be tailored by adjusting the microsphere crosslinking levels, which controls the release kinetics of the growth factor. this system not only produces hmsc sheets with superior mechanical properties but also decreases the pre-implantation culture time. in addition, the hmscs are continuously exposed to growth factor by microparticles, which prevents loss of the chondrogenic phenotype in vivo. similar controlled release of growth factor and proteins from gmps to stimulate tissue regeneration have been achieved for vascular endothelial growth factor (vegf) [ ] , bone morphogenetic protein- (bmp- ) [ ] , basic fibroblast growth factor (bfgf) [ ] , and matrix metalloproteinases (mmps) [ ] . in all cases, the release kinetics were dependent on the degree of crosslinking of gmps. it is well known that the use of most hydrophobic drugs is limited by their poor aqueous solubility, short circulation time, low bioavailability, and narrow therapeutic window. gmps have been used to address some of these challenges. for instance, the antibacterial activity of curcumin -a hydrophobic polyphenol derived from turmeric with a wide range of biological properties-is negligible at concentrations up to mg/ml. however, curcumin encapsulated in gmps at mg/ml reduced the microbial population by . , . , . , and . log counts (cfu/ml) for l. monocytogenes, s. enterica, s. aureus, and e. coli, respectively [ ] . gmps encapsulated with noscapine, an anticancer drug that targets human nsclc, demonstrated first order release kinetics and increased the plasma half-life of the drug by ~ -fold with an accumulation of ~ % drug in the lungs [ ] . kim et al. used a microfluidic approach to fabricate monodisperse gmps with a microshell structure for embolization, a minimally invasive, nonsurgical procedure that deliberately blocks a blood vessel [ ] . uniform gelatin emulsion precursors were fabricated using the microfluidic technique and consecutively crosslinked by inbound diffusion of glutaraldehyde from the oil phase to the suspending droplets of gelatin precursor. the authors also performed a model micromechanics study in an artificial blood vessel, which revealed that the microshell structure results intorapid degradation of the gmps within a narrow time period leading to burst release of the drug under chemoembolic conditions. gelatin fibers are used for various biomedical applications such as tissue-engineering and wound-healing, due to their morphological resemblance to the structure of the native ecm. gelatin fibers can be fabricated by several methods, such as melt spinning, wet spinning, gel spinning, electrospinning, centrifugal spinning, and solution/melt blow spinning. multiple processing parameters, including solution properties, humidity, and temperature, can influence the morphology of nanofibers and release rate of the entrapped drug. for instance, nanofibers fabricated by electrospinning of poly (d, l-lactide-co-glycolide) (plga) and gelatin were loaded with fenbufen. the hydrophilicity of the fibers was enhanced by increasing the gelatin content, which in turn increased the release rate of fenbufen. in contrast, the addition of a crosslinker (glutaraldehyde vapor) reduced the drug release rate [ ] . vitamins a and e incorporated in gelatin nanofibers via electrospinning promoted the proliferation of fibroblasts, increased the expression of collagen-specific genes, and supported wound healing [ ] . another research group demonstrated that genipin-crosslinked gelatin fibers loaded with human vegf sustained viability, promoted endothelial differentiation, attracted human mesenchymal stem cells (hmscs) and stimulated early angiogenesis [ ] . chemically modified gelatin has been extensively used as hydrogel because of its tunable mechanical and biochemical properties. gelatin-methacryloyl (gelma), a synthetic, photosensitive crosslinkable derivative of gelatin, is widely used as a precursor to fabricate gelatin hydrogels and is commercially available as a bio-ink for d printing. various crosslinking methods, such as ultraviolet stereolithography, gamma irradiation, and two-photon polymerization, have been used to fabricate gelatin-based hydrogel and scaffolds [ ] . light-activated crosslinking of methacrylated gelatin in air or in an aqueous solution resulted in an injectable hydrogel that supported hmsc growth and tgf-b -induced chondrogenesis [ ] . mazaki et al. developed furfurylamine-conjugated gelatin (gelatin-fa) that was rapidly crosslinked by visible light with rose bengal [ ] . hybrid gelatin hydrogels can also be made from various synthetic polymers, including plga, poly (hydroxyethyl methacrylate), and polyvinyl alcohol, or natural polymers, such as chitosan and alginate. composite hydrogels consisting of gelatin, polyvinyl alcohol, and the anticancer drug cisplatin were synthesized as a controlledrelease drug delivery system. in vivo, inhibition of tumor growth was similar for hydrogels containing a low dose of cisplatin and conventional intraperitoneal administration of high doses of free cisplatin [ ] . kinsella and colleague fabricated various alginate-gelatin bio-printable composite hydrogels that resemble the microscopic architecture of native tumor stroma [ ] [ ] [ ] . d-printed hydrogels embedded with breast cancer cells, and fibroblasts promoted the self-assembly of breast cancer cells into multicellular tumor spheroids (mcts), which were viable for more than days [ ] . gelatin -based tissue bioadhesives, fabricated alone or with other synthetic and natural polymers, have also been used in drug delivery. composite tissue adhesives, developed by carbodiimide -mediated crosslinking of gelatin and alginate, were used to entrap the antiinflammatory drugs bupivacaine and ibuprofen, and the resulting adhesive was used for local pain management [ , ] . hydrophilicity of the adhesive and its swelling properties control the release of drugs from this matrix. the incorporation of a drug -bupivacaineimproved the bonding strength of the adhesive, whereas incorporation of ibuprofen adversely affected the bonding strength. this is due to the inert nature of bupivacaine and the reinforcing effect of its fibrous crystals. in contrast, ibuprofen reacts with the crosslinking reagent -carbodiimideand decreases the crosslinking density and hence bonding strength of the adhesive networks [ ] . to address this issue, n-hydroxysuccinimide can be added during crosslinking of gelatin and alginate to decrease the carbodiimide content without compromising the bonding strength. the resulting patch is less cytotoxic (due to low carbodiimide content) and can be used for controlled release of various antibiotics such as clindamycin, vancomycin, and ofloxacin [ ] . like collagen, elastin is a key protein found in the ecm and connective tissues. it is abundant in organs that require elasticity and resilience for their function, including skin, ligaments, lungs, and blood vessels [ ] . its inherently disordered structure provides elastin with unique properties that impact its recombinant production and purification, drug-loading capacity, and in vivo behavior. the soluble precursor to elastin, tropoelastin, is composed of alternating hydrophobic and hydrophilic domains that undergo enzymatic crosslinking via their lysine residues in the extracellular space. the resulting elastin fibrils are durable and insoluble. although these characteristics are critical for its function in biological tissue, they were a hindrance during early efforts to manipulate elastin as a biomaterial [ ] . consequently, soluble and recombinant forms of elastin were explored. the most widely studied elastin-based materials used for drug delivery are elastin-like polypeptides (elps) [ ] . elps are biopolymers with the val-pro-gly-x-gly pentapeptide motif, in which x represents any amino acid besides proline [ ] . perhaps the most interesting property of elps is their thermal responsiveness, as they demonstrate lcst phase behavior. in an aqueous solution, they are soluble below a characteristic transition temperature (t t ). above their t t , elps reversibly phase transition and forms an insoluble, polymer-rich phase. this behavior is thermodynamically driven by the j o u r n a l p r e -p r o o f favorable entropy of demixing above the t t (figure a & b) [ ] . spontaneous mixing occurs when the gibbs free energy change is negative. for an elp to be solubilized, water molecules must be ordered along the polypeptide chain. this interaction results in a negative entropy term. a small temperature term compensates for this hit in entropy, which keeps the gibbs free energy change negative and allows the elp to stay in solution. however, as the temperature term increases, elp-water mixing becomes more energetically unfavorable. the entropy term becomes dominant, causing the elp to aggregate into a -coacervate‖ phase. the temperature at which this phenomenon occurs is dependent on multiple features of the elp and the solution environment [ , ] the elp's molecular weight and concentration in the aqueous solution are inversely related to its t t , which is also impacted by the guest residue. high concentrations of hydrophobic residues, such as lysine or valine, depress the t t and high concentrations of hydrophilic residues, such as alanine or glycine, increase the t t [ ] . elps retain their lcst behavior when recombinantly fused to a drug, targeting moiety, or other molecule, but the t t can be similarly affected by the surface hydrophobicity and charge of its fusion partner [ ] . the t t of an elp solution can be easily quantified by optical turbidity measurements of an elp solution at nm (od ) as a function of solution temperature. as the temperature is increased, the elp will transition from an optically transparent solution to an opaque mixture within a narrow, - °c temperature window. the t t can be graphically determined at the inflection point of an od vs temperature plot, which is typically a sigmoidal curve (figure c ) [ ] . because the t t is concentration dependent, it should be analyzed over a range of application -relevant concentrations. the lcst behavior of elps can also be used in a chromatography-free purification process termed inverse transition cycling (itc). initially developed by meyer and chilkoti, this procedure cycles cell lysate through a series of hot and cold centrifugation steps [ ] . after insoluble cell debris is removed from the lysate via centrifugation, the elp-containing supernatant is heated above the t t of the elp. kosmotropic salts, which interact more favorably with the water along the elp chain, may be added to facilitate the phase transition and promote aggregation. the solution is centrifuged to pellet the elp while soluble contaminants remain in the supernatant. the elp pellet is then dissolved in cold buffer and centrifuged below the t t , which causes insoluble contaminants to precipitate while the elp remains in solution. this process is rapid and inexpensive, avoids the use of harsh chemicals, and can be scaled up [ ] . elps have hence been used as a purification tag for other proteins [ ] . after itc, the elp can be cleaved from the protein of interest using a proteolytic cleavage site or self-cleaving intein that is incorporated between the elp and protein [ , ] . j o u r n a l p r e -p r o o f though small molecule drugs and peptides demonstrate potency and -in the case of peptides-specificity, their efficacy is frequently hindered by their rapid clearance from circulation or poor solubility (kalepu & nekkanti, ) , necessitating frequent dosing [ , ] . [ ] scientists have addressed these limitations by conjugating macromolecular carriers to these therapeutics to improve their circulation time, target-site accumulation, and solubility [ , ] . soluble elp unimers have been recombinantly fused to therapeutic peptides and chemically conjugated to small molecule drugs for these purposes [ ] [ ] [ ] . elp fusions benefit from the large molecular weight of the elp, which extends the circulation time of the fusion partner, and enhance the solubility of the drug, and improve their biodistribution, and pharmacokinetic profiles. compared to synthetic drug carriers, elps benefit from monodispersity and a lack of toxicity [ ] . despite the advantages of employing elps as soluble macromolecular carriers, they lack the stealth properties provided by some hydrophilic polymers, such as poly(ethylene glycol) and zwitterionic polymers [ ] . these -stealth‖ polymers create a hydration sphere around the payload and polymers, which improves the pharmacokinetic profile of the conjugate by protecting it from opsonization and premature clearance from circulation [ ] . recently, banskota and coworkers modified the canonical elp sequence to impart stealth behavior to biopolymers [ ] . they designed zwitterionic polypeptides (zipps), which are composed of repeat units of the val-pro-x -x -gly motif, where x and x are positively and negatively charged amino acids, respectively ( figure a) . like elps, zipps can be recombinantly expressed in e. coli, and exhibit the favorable properties of elps including monodispersity, ability to recombinantly be fused to a therapeutic peptide, and sequence-level control of their properties. additionally, zipps can be purified without chromatography by inverse transition cycling, similar to elps. zipps provide greater solubility to their payload than elps as a result of their charged residues [ ] . most importantly, zipps had a -fold longer half-life and - -fold greater bioavailability compared to an uncharged elp control. consequently, when glp- was j o u r n a l p r e -p r o o f fused to either a zipp or a mw-matched uncharged elp control, the zipp achieved . -fold longer blood glucose control, which demonstrates that the stealth behavior of zipps can increase the efficacy of their therapeutic cargo (figure b ) [ ] . owing to their ease of production and thermal phase behavior, elps are useful materials for design of self-assembling nanostructures and nanoparticles to deliver a variety of therapeutic agents. elp nanoparticle self-assembly is driven by hydrophobic interactions. depending on the design, the hydrophobic component might be either an elp segment or its conjugated hydrophobic cargo. elp amphiphiles, which contain two connected elp blocks with different hydrophobicities and hence t t , form micelles at temperatures between the t t of each block. at phenylalanine and valine were the guest residues. due to the presence of ionizable residues in the sequence, the size and shape of the self-assembled nanoparticles were ph-sensitive. above the cmt, these elp fusions self-assembled into spherical and cylindrical morphologies [ , ] . conticello et al. later designed triblock elps, which were made of a hydrophilic block between hydrophobic end blocks [ ] . the hydrophobic blocks phase transitioned and aggregated to act as -crosslinks‖ in an elastomeric network. j o u r n a l p r e -p r o o f subsequently dreher and coworkers systematically explored the design parameters that controlled elp nanoparticle self-assembly by creating a library of ten diblock elps [ ] . they discovered that the temperature at which unimers assemble into micelles depends upon the length of the hydrophobic domain. in contrast, micelle diameter is controlled by the length of the copolymer and the ratio of hydrophobic to hydrophilic blocks. dreher et al. also incorporated a functional ligandthe tumor-homing ngr peptide sequence at the terminus of the hydrophilic segment of a diblock elp. these micelles were readily internalized by cells that recognized the displayed ligand, which demonstrates that the elp diblock design can be engineered for targeted drug delivery [ ] . several models have contributed to our understanding of diblock elp self-assembly. elps based on the t t values of each elp domain. they found that although the cmt was dependent on the hydrophobic domain, the bulk transition temperature was dependent on the hydrophilic block [ ] . a second, theoretical model developed by hassouneh et al. used polymer physics to study micelle assembly. upon examination of six diblock elps, they found that these copolymers form -weak‖ micelles with dense cores and unstretched coronas, which is unique for diblock polymer micelles [ ] . the initial studies on diblock elp micelle targeting were expanded by the development of dynamic affinity modulation, a strategy in which local hyperthermia induces micelle formation to increase the avidity of a low-affinity ligand that is fused to the n-terminal end of the hydrophilic segment of diblock elp for site-specific accumulation and targeting. simnick and coworkers first used this strategy in a proof -of -concept study in which a low-affinity ligand specific to α v β integrin was recombinantly fused to the n-terminus of an elp [ ] . cysteine residues were incorporated at the c-terminus for drug conjugation. at temperatures below the cmt of the elp, the fusion protein existed as a soluble unimer and the low-affinity ligand interacted weakly with the α v β integrin. in the presence of local hyperthermia, the elp underwent temperature -triggered self-assembly into spherical micelles, thus increasing the valency and avidity of the ligand such that it could efficiently bind to and be taken up by cells [ ] . this strategy was then modulated to achieve more specific cell internalization of cell-penetrating peptides (cpps) [ ] . cpps are arginine -rich sequences that promote uptake by electrostatically interacting with the cell membrane of multiple cell types. cpps have been conjugated to nanoparticle surfaces to improve their internalization but lack specificity. macewan and chilkoti addressed this issue by incorporating five arginine residues onto a diblock elp. they found that below the cmt, the number of arginine residues was below the threshold of six necessary to induce cell uptake [ ] . local hyperthermia drove the formation of micelles that displayed a high valency of arginine on their surfaces, resulting in -fold increase in cell internalization [ ] . the design of these nanoparticles and their thermal -switch‖ allow for control over the site of action of elp nanoparticles, which can reduce off-target drug delivery and mitigate systemic toxicity. furthermore, adding these targeting moieties did not disturb micelle formation. hassouneh and coworkers showed that when proteins ~ kda in size are incorporated at the end of the hydrophilic block, the diblocks still self-assembled [ ] . diblock elps have also been designed for applications beyond cancer, including specific targeting to liver cells [ ] and lacrimal gland cells [ ] . to improve tumor-specific drug delivery of diblock elp micelles, callahan and coworkers engineered a diblock elp that disassembled in the acidic tumor microenvironment [ ] . histidine, which has a pka near physiological ph, was incorporated into the hydrophobic block. these micelles assembled at °c, but upon exposure to the low ph conditions mimicking the tumor microenvironment (ph ~ ), the histidine became ionized. consequently, the t t of the hydrophobic block increased, and it was no longer energetically favorable for elps to selfassemble. the micelles broke down into elp unimers, resulting in better penetration and accumulation of the elp in the tumor compared to ph insensitive elp micelles. this study demonstrated that diblock elps can be engineered to overcome the diffusion barrier many nanocarriers face when delivering drugs to tumors [ ] . mackay and coworkers have adapted diblock elps nanoparticles for two-phase rapamycin release by nonspecifically entrapping the drug in the nanoparticle core as well as specifically displaying the drug on the nanoparticle corona [ , ] . this type of display was achieved by appending fk binding protein (fkbp ), the cognate protein target of rapamycin, to the hydrophilic block of the elp fusion. the entrapped rapamycin was rapidly released upon administration, while the release of fkbp -bound rapamycin was prolonged. the two-phase release increased the terminal t / of rapamycin to . h compared to . h for nanoparticles without fkbp -bound rapamycin [ ] . the nanoparticle formulation also reduced off-target toxicity compared to the free drug. in the aggressive breast cancer model mda-mb- , treatment with the nanoparticle resulted in more potent reduction in tumor volume and extended survival time [ ] . in a model for the chronic autoimmune disease sjögren's syndrome, the treatment downregulated inflammation in the endocrine gland and reduced lymphocytic infiltration into the lacrimal gland [ ] . other elp nanoparticle designs fuse an elp segment with a peptide or protein to drive self-assembly. in an early example of this method, mcdaniel and coworkers incorporated repeats of the sequence (xg y ) z onto the elp c-terminus, where x is a hydrophobic residue [ ] . relative to the elp, the (xg y ) z domain is only a few percent by mass in the diblock. despite the low mass fraction of the second segment, it was surprising and notable that these highly asymmetric amphiphiles self-assembled into cylindrical micelles with lengths that could be controlled by altering the identity of x or the value of y-the number of glycine spacers [ ] . mackay et al. further explored how small proteins and peptides impact nanoparticle assembly. they discovered that fusing an scfv -a single chain antibody-to a hydrophilic elp block drives the fusion to self-assembly into a worm-like micelle, with the elp forming the corona [ ] . despite being entrapped in the core, the scfv remained active due to the low packing density of the corona. these nanoworms outperformed their antibody equivalent in an in vivo xenograft model of lymphoma [ ] . another study by mckay and coworkers was driven by the amphipathic nature of l f, whose four phenylalanine residues orient face-to-face to create vesicles with nm thick lamellae and a nm radius [ ] . l f maintained its anti-inflammatory properties despite being in the vesicle lamellae. this study demonstrates how amphipathic peptides may be used to drive elp vesicle formation, which can be leveraged to encapsulate soluble therapeutics into the aqueous vesicle interior. hydrophobic drug conjugation can also trigger elp self-assembly into nanoparticles, masking the drug inside the core, thus increasing the aqueous solubility of the drug and protecting it from premature release and degradation. this results in improved drug half-life, biodistribution, tumor accumulation, and efficacy while reducing off-target toxicity. an early example of this strategy was developed by chilkoti and coworkers. in their bottom-up nanofabrication method, termed attachment directed assembly of micelles (adam), a hydrophobic (cgg) domain was conjugated to the c terminus of a hydrophobic elp [ ] to create a -chimeric polypeptide‖ (cp). the cysteine-rich domain of the cp enabled the attachment of up to eight maleimide -functionalized small molecules (figure ) . when the hydrophobicity of the small molecule exceeds a threshold (log ≥ ~ . ), it provides the conjugate sufficient amphiphilicity to drive self-assembly [ ] . this platform was adapted for drug delivery by chemical conjugation with dox to create cp-dox nanoparticles, which self-assemble at physiological ph at concentrations above µm. dox is rapidly released upon exposure to an acidic ph ( ) , which mimics the conditions of late endosomes and lysosomes. a single injection of cp-dox had -fold higher maximum tolerated dose and reduced tumor size nearly -fold compared to free drug, indicating that this nanoparticle formulation can be harnessed to j o u r n a l p r e -p r o o f enhance potency while reducing off-target effects such as cardiotoxicity. adam has also been leveraged to deliver the hydrophobic chemotherapeutics paclitaxel [ ] and niclosamide [ ] . while the original adam strategy was successful at enhancing the efficacy and safety of hydrophobic chemotherapeutics, many cancer drugs are hydrophilic and cannot be encapsulated by this strategy. to address this, the chilkoti and coworkers developed a new strategy by the design of an asymmetric triblock polypeptides [ ] . atbps consist of three segments-a hydrophilic elp segment that is fused to a hydrophobic elps segment that in turn is fused to a drug attachment (ggc) segment. the atbp is deigned to retain its temperature triggered amphiphilicity even upon conjugation of hydrophilic small molecules with a logd ≤ . that the drug-conjugated atbp self-assembles into rod-shaped micelles above its cmt, which is designed to be below body temperature. they showed that conjugation of hydrophilic drugs, such as gemcitabine, to the cysteine residues of the atbp did not abrogate the formation of micelles, and these drug-loaded atbp micelles had greater efficacy in an aggressive in vivo model of colon cancer compared to free drug [ ] . while cysteine-maleimide covalent conjugation can incorporate a structurally diverse set of drugs, molecules, and imaging agents into the nanoparticle's core, this strategy relies on functionalization of a naturally occurring amino acid. these residues can be promiscuously distributed across other peptides that may be appended to the elp such as targeting peptides or proteins, and, consequently, site-specific drug attachment for these constructs is challenging. to address this concern, costa and coworkers inserted p-acetylphenylalanine, an unnatural amino acid, onto an elp micelles that displayed a functional nanobody [ ] . the nanobody contains a disulfide bond that is essential for its stability and functionality, so that site specific covalent conjugation of a drug by introduction of cysteine residues in a drug attachment was not possible. the p-acetylphenylalanine residue provided a biorthogonal ketone moiety to sitespecifically conjugate dox via a ph-sensitive bond without affecting the disulfide bond of the nanobody. as a result, this platform enabled site-specific drug conjugation while enabling the display of a functional nanobody on an elp nanoparticle [ ] . [ ] . fatty acids have also been incorporated by a post-translation modification into elps to drive nanoparticle assembly. luginbuhl and coworkers incorporated a myristoyl group at the nterminus of an elp that presented a peptide substrate for n-myristoyltransferase. the enzyme was co-expressed with the elp from a bicostronic plasmid in e. coli that grown with exogenous myristic acid added to the culture medium [ ] . this led to close to % myristoylation of the elp in e. coli, and the myristoylated elp (m-elp) formed spherical or rod-like micelles depending on the elp chain length. chemotherapeutic drugs could be physically loaded into the micelles via hydrophobic interactions with the fatty acid, which resulted in a greater encapsulation efficiency than that possible with diblock elps [ ] . this strategy thus provides a useful mechanism for entrapping drugs without covalent conjugation, making it possible to deliver drugs that do not contain an active functional group for conjugation, or drugs that lose potency upon conjugation. elps have been similarly combined with silk to form silk-elastin like protein (selp) nanoparticles [ , ] . nanoparticle self-assembly is driven by both hydrophobic interactions and hydrogen bonding between the silk blocks. the radius of the nanoparticle is controlled by altering the elp guest residue as well as the ratio of silk blocks to elp blocks [ ] . xia and coworkers demonstrated that these nanoparticles can be used to deliver hydrophobic small molecule drugs by loading dox into the nanoparticles [ ] . dox was entrapped in the core by hydrophobic interactions with the silk blocks. the selp nanoparticles were endocytosed into the cell, thus allowing drug to accumulate in the cytoplasm. the cytotoxicity of the selp-dox nanoparticles was . -fold greater than that of free dox [ ] . elps provide a useful platform for sustained drug release due to their lcst behavior. their t t can be precisely tuned by modulating the molecular weight and guest residue of the sequence, making it possible to engineer the elp with a t t below physiological temperature. the elp thus remains soluble at room temperature, enabling injection through narrow-bore syringe, but rapidly coacervates upon exposure to body temperature, forming a subcutaneous depot. because the t t is concentration -dependent and the boundary layer of the coacervate is diluted by interstitial flow, the depot slowly undergoes surface-to-core dissolution. as it dissolves, the therapeutic cargo is released into circulation. this sustained -release platform, coupled with the ability of elps to impede renal filtration, extends the half-life of small molecules and peptides to which the elp is fused. prolonged -release systems are advantageous for diseases that require long-term treatment, such as cancer or chronic illnesses. liu et al. systematically altered elp parameters to adapt the molecule for in situ radiotherapy [ , ] . they recombinantly fused a hydrophobic elp, comprised of repeats of a val-pro-gly-val-gly pentapeptide, to a tyrosinerich domain that could be radiolabeled with i. an intratumoral injection of the i-elp was retained in the tumor for more than one week. the elp shielded the radionuclide from dehalogenase degradation and increased its tumor retention time [ ] . liu and coworkers found that doubling the molecular weight of the elp increased tumor retention time by -fold; however, the effect plateaued with the t t once the elp surpassed repeats. similarly, increasing the injection concentration of i-elp -fold resulted in -fold greater tumor retention [ ] . furthermore, i-elps with seven repeats of the hydrophobic, tyrosine-rich peptide domain self-assembled into rod-shaped micelles and formed long-lasting -seeds‖. the seed-like particles exhibited potent anti-tumor efficacy, remarkable intratumoral retention, and minimal toxicity [ ] . the coacervate was further stabilized by radiation-induced crosslinking of the tyrosine residues in the hydrogel, which resulted in retention of % and % of its radioactivity over days in a prostate and pancreatic tumor model, respectively [ ] . more recently, wang and coworkers engineered an elp depot for the sustained release of interferon-α, an anti-cancer protein that has been clinically hindered due to its short half-life and dose-dependent toxicity [ ] . fusion of the protein to repeats of a val-pro-gly-val-gly pentapeptide resulted in near zero-order release of ifn-α over the course of a month. compared to free ifn-α and ifn-α fused to a soluble elp, the depot-forming ifn-α-elp exhibited greater antitumor efficacy in a melanoma model. the fusion was well tolerated and demonstrated little renal toxicity, key advantages over free ifn-α [ ] . the enhanced pharmacokinetics of glp are attributed to the increased residence time of depot-forming glp -elp compared to the soluble control or glp alone. adapted with permission from [ ] . elp depots have also been extensively engineered for the treatment of diabetes. antidiabetic peptides such as insulin and glucagon-like peptide- (glp- ) have short half-lives that necessitate frequent and repeated injections to achieve long-term blood glucose control. there is hence a need for sustained -release systems that prolong the efficacy of these peptides, and which reduce the need for multiple injections and improve patient compliance. glp- to and elp that was designed to coacervate at body temperature. the depots were retained in the subcutaneous space and lowered blood glucose levels for up to days [ ] . luginbuhl and coworkers expanded upon this work and optimized the elp parameters for depot retention and glp- half-life [ ] . they discovered that elps with t t values of approximately °c and molecular weights greater than kda achieved near zero-order release kinetics for glp- . the optimized glp -elp fusion formed a depot in the subcutaneous space upon injection as a solution and provided glycemic control in three different diabetic mouse models for up to days after a single injection (figure ) . in a cynomolgus monkey model, circulating levels of the elp fusion were detected for - days post-injection. glp- delivery has also been achieved by introducing protease cleavage sites between oligomeric repeats of glp- on an elp. once these so-called -protease-operated depots‖ are injected subcutaneously, proteases under the skin free glp- from the elp in the depot. this system extended blood glucose control for days [ ] . the antidiabetic protein fibroblast growth factor (fgf ) has also been fused to an elp by gilroy et al. to improve its release, half-life, and production [ ] . fusion to the elp enhances fgf solubility, thus allowing it to be expressed in the bacterial cytosol rather than inclusion bodies and consequently avoiding costly, complex refolding steps during purification. the elp-fgf depot was retained in the subcutaneous space for up to days and achieved glycemic control in an ob/ob mouse model for up to days. in a long-term study, treatment with an elp-fgf fusion resulted in % reduction in mean serum insulin and triglyceride levels when mice received subcutaneous injections every days for up to days [ ] . other elp depots have been engineered for the treatment of dry eye disease [ ] , infectious illnesses [ ] , and inflammation [ ] . resilin is another elastomeric protein that has been extensively engineered for biomedical applications. natural resilin is a rubbery protein with remarkable elasticity that is derived from the exoskeletons of insects and arthropods, where it is a critical component of locomotive systems such as jumping and flight [ ] . it possesses high resilience and extensibility, low stiffness, and the ability to efficiently store energy [ ] . these unique mechanical properties, coupled with its biodegradability, make resilin an attractive protein for regenerative medicine and drug delivery [ ] . after ardell and anderson identified tentative resilin gene sequences in drosophila melanogaster [ ] , elvin and coworkers engineered the first example of a recombinantly expressed resilin-like polypeptide (rlp) in [ ] . the resulting polypeptide, termed rec resilin, is composed of the first exon of the d. melanogaster cg gene, which contains seventeen copies of the ggrpsdsygapgggn motif [ ] . rec -resilin possesses the favorable mechanical properties of native resilin with the added benefit of customizability provided by its recombinant synthesis. upon photochemical crosslinking of the tyrosine residues, rec -resilin demonstrated up to % resilience that is comparable to that of many synthetic rubbers [ ] . dros . an is composed of sixteen repeats of an eleven amino acid motif found in anopheles gambiae while dros is composed of sixteen copies of a fifteen amino acid motif in the first j o u r n a l p r e -p r o o f exon of the cg gene from which rec -resilin is derived [ ] . similar to rec -resilin, these rlps were highly resilient, elastic, and capable of photo-crosslinking [ ] . rlps are intrinsically disordered polypeptides that contain high concentrations of glycine, which provides them with high flexibility and makes secondary structure formation entropically unfavorable. the high proline content of rlps also prevents secondary structure formation, as the bulky residue reduces hydrogen bonding [ ] . further, unlike other elastin-mimetic proteins, rlps contain high frequencies of polar and charged residues and are thus hydrophilic. rlps are responsive to a variety of stimuli, including temperature [ , ] . rlps demonstrate dual phase behavior (dpb), an unusual property characterized by both a lcst and upper critical solution temperature (ucst), the temperature above which the rlp is fully miscible in solution. the dpb of rlps is fully reversible and largely dependent on the molecular composition and architecture of the rlp. their ucst behavior is also strongly impacted by ph [ ] . at ph values greater than the pi, the charged residues enhance rlp solubility and the ucst increases. at low ph values, carboxylic acid residues on the rlp become protonated, which reduces the molecule's solubility and drives up the ucst [ , ] . the tunable dpb of resilin is useful to fabricate modular drug delivery systems, described in the next section. the unique, temperature-driven phase behavior of rlps provides numerous opportunities for designing nanoparticles, whose size, shape, and aggregation number can be tuned to create drug delivery vehicles for a variety of applications. li et al. initially described the factors that impact rlp nanoparticle formation using a novel rlp based on twelve repeats of a fifteen amino acid consensus motif derived from d. melanogaster [ ] . they replaced the tyrosine residues with phenylalanine and methionine, which can be leveraged for subsequent chemical modification. to form nanoparticles, the polypeptide was heated to its lcst of °c, at which point it irreversibly aggregated [ ] . li and coworkers found that the lcst was concentration-and ph-dependent; transition temperature decreased with increasing concentration or decreasing ph (figure ) . interestingly, the presence of salt improved rlp solubility and increased the transition temperature, which is the opposite of elps (figure ) . this phenomenon is attributed to the ionizable residues in the rlp. the resulting nanoparticles can be tuned to have diameters ranging from nm below the transition temperature up to nearly nm when heated beyond their transition temperature [ ] . this study demonstrates how the temperature-triggered phase behavior of rlps can be exploited to design nanoparticles with precision. block copolypeptides that incorporate rlps have also been used to create nanoparticles. the self-assembly of diblock copolymers containing both ucst and lcst blocks is strongly impacted by temperature. at low temperatures, the ucst block is relatively hydrophobic and aggregates at the core while the lcst block remains soluble. as the temperature increases, the ucst block becomes hydrophilic and relocates to the nanoparticle corona as the lcst block aggregates [ ] [ ] [ ] . this phenomenon motivated weitzhandler et al. to combine rlps with elps to better characterize temperature-triggered phase behavior of ucst-lcst block co-polypeptides with the goal of nanoparticle formation [ ] . using an rlp composed of repeat units of the gln-tyr-pro-ser-asp-gly-arg-gly octapeptide and an elp, they identified two principles that affect nanoparticle formationthe rlp:elp mass ratio and elp hydrophilicity. with an increase in the rlp:elp ratio, they observed an increase in the nanoparticle size. in contrast, the hydrophobicity of the elp impacted morphology; a hydrophobic elp resulted in spherical micelles, while a hydrophilic elp promoted the formation of worm-like structures [ ] . [ ] . dzuricky and coworkers expanded upon this work to observe how nanoparticle morphology impacted cell uptake [ ] . a fibronectin type-iii (fn ) -binding domain that binds the α v β integrin was fused to the c-terminus of an rlp-elp diblock copolymer. they found that the fn domain did not perturb self-assembly of the rlp-elp diblock copolymer, so that it was displayed on the nanoparticle surface upon self-assembly (figure ) . the tunable morphology and high avidity of the rlp-elp-fn nanoparticles significantly improved the binding affinity to the α v β integrin via multivalency. they found that shape played an important role in promoting multivalency though the avidity effect. while a spherical rlp-elp micelle that presented a fn a) j o u r n a l p r e -p r o o f domain showed a respectable -fold increase in affinity for the α v β integrin compared to the monovalent rlpelp-fn unimer, the worm-like rlp-elp-fn nanoparticles showed a remarkable , -fold increase in affinity for the integrin (figure ) [ ] . the combination of morphology and high avidity yielded a picomolar k d , a remarkable achievement considering that a therapeutic antibody against the same integrin exhibits a k d of only nm. consequently, the worm-like rlp-elp-fn was more readily taken up by cells than the antibody [ ] . these studies demonstrate that rlp-elp nanoparticles can be harnessed for robust, specific cellular targeting and uptake of therapeutic agents. because of their exquisite stimulus responsiveness and mechanical properties, rlps have been engineered as hydrogels for many biomedical applications. although many of the hydrogels were designed for use in tissue engineering applications, the principles derived from these studies can are also useful for the design of rlp-based controlled drug delivery systems. charati and coworkers reported an early example of a rlp hydrogel [ ] . to test its utility for biomedical applications, they incorporated several biologically active domains into the sequence to control cell adhesion, hydrogel degradation, and biocompatibility. upon purification from e. coli, the protein was crosslinked with [tris(hydroxymethyl) phosphine] propionic acid (thpp), which linked the lysine residues in the rlp sequence. the resulting hydrogel was mechanically stable and capable of supporting hmscs and mouse nih- t fibroblasts [ ] . li and coworkers later evaluated the mechanical properties of rlp hydrogels more precisely and showed that the extent of thpp crosslinking impacts factors such as swelling ratio, elastic shear modulus, and resilience [ ] . su and coworkers adapted rlps for cell adhesion by combining cell binding domains from fibronectin with a consensus sequence derived from a. gambiae. after it was crosslinked with tris(hydroxylmethyl)phosphine, the novel rlp scaffold exhibited mechanical properties consistent with those of rec -resilin and a compression modulus comparable to that of cartilage [ ] . the material supported mscs at % viability for up to days, and the cells exhibited extensive spreading on the material surface [ ] . while this hydrogel was primarily engineered j o u r n a l p r e -p r o o f for tissue engineering applications, it provides an example of how peptides can be appended to the rlp sequence for other biomedical applications such as drug delivery. other moieties, such as heparin [ , ] , vegf-mimicking peptides [ ] , and chitin -binding domains [ ] , have similarly been appended onto rlps. in a reducing environment (b) , dextran release was not mw-dependent. in a non-reducing, pbs environment (c), dextran release was dependent on mw. adapted from [ ] . taking advantage of the recombinant nature of rlps, su et al. engineered their novel rz -rgd hydrogel to respond to changes in redox [ ] . they used , 'dithiobis(sulfosuccinimidyl propionate) (dtssp), a redox-responsive crosslinker, to create rlp hydrogels that degrade in a reducing environment ( figure a) . various molecular weights of fitc-labeled dextran were loaded into the crosslinked rz -rgd hydrogel. in reducing conditions, there was no observed difference in the release rates of dextrans with different molecular weights. in non-reducing conditions, release rate decreased with an increase in dextran mw (figure b ) [ ] . the redox-responsiveness of this novel hydrogel makes it a promising candidate for tumor-specific drug delivery. other -smart‖ rlp hydrogels have been designed to degrade in response to elevated mmp concentrations [ ] . when an mmp-sensitive domain was incorporated into a rlp sequence, the hydrogel underwent % degradation in the presence of physiological levels of mmps over the course of rlp hydrogels without the mmp-sensitive domain did not degrade [ ] . because the concentration of mmp-sensitive domains within the rlp hydrogel may be modulated to control degradation, this design can be adapted for drug delivery to tissues with elevated mmp concentrations, such as tumors or wound sites. resilin has also been combined with other proteins, including elastin, collagen, silk, and peptide amphiphiles (pas), to enhance the properties of rlps. bracalello and coworkers designed a chimeric polypeptide comprised of an rlp, elp, and clp in tandem, which they termed the rec polypeptide [ ] . the purified material formed highly aligned fibrils that, over time, self-assembled into large fibers and fiber bundles. the fibers and bundles possessed a young's modulus of . - mpa, which demonstrated that the material retained the elastic properties of resilin and elastin [ ] . more recently, okesola and coworkers developed a highly tunable, multicomponent hydrogel using a covalent co-assembly strategy to combine an rlp with a peptide amphiphile (pa). this approach enabled control over the hierarchical self-j o u r n a l p r e -p r o o f assembly of the rlp hydrogel and enhanced its mechanical properties. the hydrogels were synthetized using a thiol-ene photoclick reaction and, depending on the pa concentration, assembled into a variety of nanostructures. in the absence of pa, the rlp hydrogels formed nanospheres upon photo-crosslinking, a low concentration of pa resulted in co-assembled beaded strings, and a high concentration of pa created co-assembled nanofibers. the photoclicked hydrogels maintained the remarkable elastic and energy-storing properties of rlp hydrogels, thus demonstrating that the co-assembly strategy can improve the mechanical properties of more brittle proteins such as pas. these results further support that rlp materials can be precisely tuned to achieve desirable properties for a drug release system. silk-elastin-like polypeptides, or selps, are block co-polymers consisting of tandem repeats of silk-like and elastin-like blocks [ ] . these hybrid polypeptides combine the mechanical strength and chemical stability of silk with the unique thermal responsivity of elps, resulting in a material that can assemble into a variety of architectures ranging from nanomaterials to hydrogels [ ] . like many other biopolymers, these materials are recombinantly produced, offering precise control over their length, the ratio of slp to elp blocks, and the ability to introduce biologically active molecules into the sequence [ ] . an interesting characteristic of selps is their ability to undergo an irreversible sol-gel transition under physiological conditions, creating a matrix in situ that is useful for local delivery [ ] . selps have been engineered to self-assemble into micellar structures that can be used as nanocarriers for drugs. the core of the micelle is formed by the slp blocks which form intermolecular hydrogen bonds to stabilize the micelle while the relatively hydrophilic elp forms the corona [ ] . the hydrodynamic radius of the micelle can be tuned by altering the slp to elp block ratio or by altering the choice of guest residue in the elp block [ ] . the self-assembly of selp nanoparticles may also be triggered by encapsulation of a hydrophobic drug. xia et al first explored this concept by mixing dox with selps with a slp to elp block ratio of : , : or : [ ] . the dox hydrophobically interacted with the silk j o u r n a l p r e -p r o o f domains, driving the formation of nanoparticles with hydrodynamic radii between and nm ( figure a) . these nanoparticles are in the appropriate size range to be passively targeted to solid tumors via the epr effect [ ] . this approach is attractive as it avoids the need for harsh solvents and provides a respectable . % drug loading efficiency [ ] . dox-loaded selp nanoparticles enter hela cells via endocytosis rather than diffusion like free dox (figure b ). this allowed for potent accumulation of the drug in the cytoplasm and nearly -fold greater cytotoxicity than the free drug [ ] . more recently, parker et al developed mucoadhesive selp nanoparticles for transmucosal drug delivery [ ] . to identify potential mucoadhesive sequences, they generated a series of selps with a : slp to elp block ratio with four different guest residues -cys, arg, lys, and glu. these amino acids were chosen due to the potential of charged residues and thiol groups to interact with mucus. ans, a hydrophobic, solvatochromatic dye, was loaded into the nanoparticles via simple mixing to serve as both a model compound and reporter. in an in vitro biosimilar mucus model, the selp with cys guest residues demonstrated the strongest affinity for mucus, followed by selps containing lys, arg, or glu as the guest residue, respectively. interestingly, when incubated with mucus-secreting ht -mtx cells, the cys-containing selp demonstrated little cell retention, suggesting it may have mucolytic properties. meanwhile, selps with charged guest residues were well retained by the cells, indicating their potential for transmucosal delivery. perhaps the most interesting property of selps is their ability to undergo a sol-gel transition under physiological conditions. these materials can be engineered to be soluble at room temperature, at which point drugs and other therapeutic molecules can be loaded by simple mixing without the use of toxic or denaturing solvents. upon injection, the material coacervates to form a polymeric matrix in situ. this property, along with the biocompatibility and j o u r n a l p r e -p r o o f sequence control of selps, makes them an attractive material for engineering local, controlled drug delivery systems. this approach was first explored by cappello et al in . in this study, fluorescently labeled macromolecules of varying molecular weights were loaded into selp hydrogels and their release rates were monitored [ ] . for all compounds, release from the hydrogels was near first-order; however, there was no correlation between the release kinetics and the molecular weight of the molecule. this was attributed to the large size of the hydrogel pores, which are not small enough to impede diffusion [ ] . the release rate decreased with an increase in selp concentration. the hydrogels were biocompatible when implanted in vivo with no sign of immunological activity or inflammation [ ] . dinerman et al further evaluated the diffusion behavior of molecules out of selp hydrogels by using cytochrome c, theophylline, and vitamin b as model compounds [ ] . they observed fickian, diffusion-controlled release behavior wherein diffusion slowed with an increase in molecular weight and polymer volume fraction [ ] . due to the simplicity of drug loading and their gelation behavior, selps are also attractive materials for gene delivery. nucleic acids can be incorporated into the selp hydrogel by physical mixing, and upon injection, the selp hydrogel protects its cargo from rapid degradation while controlling the rate and location or release. this concept was first explored by megeed et al in using an selp containing positively charged residues. the negatively charged dna molecules electrostatically adsorbed onto the selp before injection [ ] . they discovered that the release of dna was inversely related to the selp concentration and gelation time and that the ionic strength of the buffer directly correlated with dna release rate; as ionic strength increases, the greater concentration of counter ions disrupt the electrostatic interaction between the selp and dna, facilitating the release of the dna [ ] . in a later study, megeed et al discovered that the release rate of plasmids from the selp hydrogel was inversely proportional to plasmid mw. furthermore, plasmid conformation impacted release rate, j o u r n a l p r e -p r o o f with linearized plasmids diffusing from the hydrogel fastest followed by supercoiled and circular plasmids, respectively [ ] . the hydrogel maintained the stability and bioactivity of plasmid dna for days and adenoviruses for days. in an in vivo model with athymic nude mice bearing subcutaneous mda-md- tumors, mice treated with selps loaded with a plasmid for luciferase exhibited greater gene expression than mice treated with naked dna [ ] . these studies aid the groundwork for selp design for dna delivery to treat cancer. hatefi et al loaded selp- k, an selp comprised of four slp and elp blocks with one elp block containing a lys substitution, with the adenoviruses ad.gfp or ad.cmv.lacz that encode green fluorescent protein (gfp) or β-galactosidase, respectively [ ] . in an in vitro study, the selp hydrogel maintained the release of the adenoviruses over a day period with only a - % loss in virus activity ( figure a ) [ ] . the ad.gfp-loaded selp was intratumorally showed significantly lower gfp expression than tumors receiving the selp formulation ( figure b ) [ ] . this study demonstrated the potential of selps for adenoviral delivery to solid tumors. the ability of selp- k to deliver adenoviruses was further tested in a head-to-head comparison with poloxamer , a commercially available synthetic copolymer used for gene delivery. both platforms were used to deliver a genetically encoded prodrug with an antiviral vector to a xenogenic model of hnscc. compared to poloxamer and free virus, selp- exhibited the highest initial gene expression in tumors followed by prolonged expression for up to three weeks [ ] . this high and sustained level of gene expression resulted in the greatest tumor size reduction and survivability across all groups. furthermore, selp- k showed superior safety than poloxamer , as adipocytic necrosis was observed in animals treated with the latter [ ] . a subsequent study demonstrated that, compared to free virus, virus-loaded selps also had reduced systemic toxicity [ ] . more recent studies have provided new insights into the stability of viral vectors in selp hydrogels. two different selps were loaded with the oncolytic adenovirus ad-Δb -kox and evaluated with afm imaging to visualize the interaction between the adenovirus and selp j o u r n a l p r e -p r o o f nanofibers. afm showed that as adenovirus concentration increased, the density of the selp nanofibers also increased despite their fixed concentration [ ] . this is likely attributed to the brownian motion of the adenovirus during assembly, as it acts as a stimulus for selp network formation. although most adenoviruses exhibited a round morphology for the first half-hour of being attached to selp, they began to deform after approximately h. this is likely due to multiple strong interactions forming between adenovirus and different selp fibers [ ] . it was also discovered that selp analogues with shorter elastin units exhibited lower temperature responsivity and more rapid elastase degradation [ ] . with this fundamental understanding of selp degradation and adenovirus complexation, better gene delivery systems can be engineered from this material. selp hydrogels have also been used for sustained release of polysaccharides. the ghandehari group synthesized positively charged selp hydrogels that could electrostatically interact with an anionic polysaccharide -semisynthetic glycosaminoglycan ether (sage)-that is an anti-inflammatory drug for mucosa restoration. unfortunately when sage is administered, it does not achieve a high enough therapeutic concentration due to its insufficiently long residence time [ , ] . to solve this problem for treatment of radiationinduced proctitis (rip), the ghandehari group designed an in situ gelling enema ( figure a ) [ ] . in a murine model of rip, sage was present in the rectal tissue of mice h after receiving a sage-loaded selp enema, while it was undetectable in mice receiving sage with saline ( figure b ) [ ] . the ghanedhari group further adapted the selp platform for the sustained release of intravesically delivered sage for treatment of interstitial cystitis (ic) model. they found that employing a softer selp with a slower gelation time could provide an analgesic effect for h without exacerbating the discomfort associated with ic, whereas stiffer gels could reduce the capacity of or obstruct the bladder [ ] . j o u r n a l p r e -p r o o f more recently, researchers have focused on the design of de novo artificial polypeptides as drug carriers. these genetically engineered polypeptides are conformationally disordered, hydrophilic, and inert. like other protein polymers, they are recombinantly expressed and purified in bacterial systems, providing exquisite control over their length and sequence. they can also be genetically fused to therapeutic peptides or proteins to increase the half-life, stability, and biodistribution of their therapeutic partner. in addition to their stability and nonimmunogenicity, these hydrophilic biopolymers have long plasma circulation, making them an intriguing alternative to pegylation. in the last decade, two polypeptide sequences in particular-xten and pashave gained traction as biopolymeric drug carriers. the first xten molecule was designed and synthesized by schellenberger et al in [ ] . to create a stable, soluble and unstructured stealth polymer, they eliminated hydrophobic amino acids known to contribute to aggregation or secondary structure formation, positively charged molecules that could bind cell membranes, and amide-containing residues that reduce stability. these criteria limited their pool of potential amino acids to sixalanine, glutamate, glycine, proline, serine and tyrosine. schellenberger et al then screened a library of nonrepetitive, randomized sequences to obtain an -residue biopolymer with optimal expression levels, yield, solubility, and stability [ ] . this original xten molecule was genetically fused to exenatide, an antidiabetic peptide that has an in vivo half-life of . h. fusion to xten increased the peptide's half-life to h in cynomolgous monkeys, with a predicted half-life of h in a kg human (figure ) [ ] . xten similarly enhanced the terminal half-life of gfp, human growth hormone, glucagon and factor vii. since this initial study, different mw variants of xten have been produced [ ] and different functional groups for drug conjugationincluding azide, maleimide, and alkyne groups -have been introduced into the polypeptide [ ] . xten has been similarly employed to counteract nocturnal hypoglycemia in diabetic patients. geething et al fused the biopolymer to gcg, a peptide hormone that converts glucagon stores into glucose but suffers from a minutes-long half-life and poor stability in liquid formulations [ ] . in addition to improving the stability of gcg -fold, fusion to xten prolonged the efficacy of gcg in a fasted beagle dog model. gcg-xten provided strong resistance to a glucose challenge h after prophylactic administration without affecting baseline blood glucose level [ ] . the effect tapered off at h post-administration, whereas free gcg was ineffective h post-administration. xten also remarkably enhanced the pharmacokinetic profile of teduglutide, a glucagon-like peptide analog used to treat an inflammatory malabsorption disorder in the small intestine with a half-life of ~ h in humans [ ] . the teduglutide-xten fusion achieved a h half-life in cynomolgous monkeys, with a predicted half-life of h in humans, further indicating how xten can be employed for once-monthly administration. the formulation also reduced the occurrence of ulcerations and presence of inflammatory cytokines j o u r n a l p r e -p r o o f in the small intestine [ ] . additionally, this study was the first instance where the payload was chemically conjugated to xten; in this case, teduglutide was chemically conjugated via a cterminal cysteine residue. since then, xten has been chemically conjugated to other therapeutics including antivirals [ ] and imaging agents such as annexin [ ] to improve their circulation time. xtenylation has been used to improve th p to human growth hormone (hgh), which typically suffers from an hours-long half-life and requires daily administration. somavaratan , an xtenylated formulation of hgh, achieved a half-life of hr, a - fold increase compared to recombinant hgh in a phase i clinical trial (nct ) [ ] . interestingly, fusion to xten reduced the potency of hgh -fold, yet the prolonged tissue exposure xten provided a to -fold increase in efficacy compared to recombinant hgh [ ] . in a phase ii clinical trial vista study (nct ), patients treated with somavaratan demonstrated increasing height velocity with increasing doses of the drug. and dose-dependent adverse events were minimal [ ] . unfortunately , somavaratan failed to achieve the primary endpoint of non-inferiority versus genotropin, a recombinant formulation of hgh, in a phase iii velocity clinical trial (nct ) for pediatric patients with growth hormone deficiency. a phase ii clinical trial (nct ) has also been completed for growth hormone deficiency in adults. these studies demonstrate the promise of xten in improving drug half-life, bioavailability, and efficacy. [ ] . more recently, a von-willebrand factor independent fviii-xten fusion, termed bivv , has demonstrated good safety and efficacy in early stage clinical trials (nct ) and is currently recruiting for phase (nct ) [ , ] . xtenylation of fviii increases its plasma half-life three to four-fold relative to recombinant fviii, achieving a half-life of up to h in cynomolgous monkeys [ ] . this resulted in four-fold longer hemostatic control, suggesting that this formulation could provide clotting protection for patients for up to one week [ ] . xten's large size has also been harnessed to take advantage of the epr effect to passively deliver anticancer therapeutics to tumors. to demonstrate its utility, haeckel et al designed a multicomponent fusion protein to improve the delivery of killin, a cytostatic and cytotoxic protein [ ] . despite the promising antitumor effects of killin, its expression in bacterial systems was impossible due to its toxicity. c-terminal fusion to xten deactivated the protein so it could be stably expressed and purified. upon administration, xten would further serve to enhance the circulation time of killin and exploit the epr effect for tumor-specific targeting. a matrix metalloproteinase cleavage site was incorporated between the xten and killin, allowing the killin to be released and activated upon tumor penetration. to improve cell membrane penetration and dna binding, a cell penetrating peptide (cpp) was also added onto j o u r n a l p r e -p r o o f the c-terminus of killin [ ] . the fusion protein was preferentially taken up into cancer cells with high mmp- or mmp- expression. this xten-killin-cpp fusion also arrested growth and induced concentration-dependent apoptosis in killin-sensitive cell lines, with the effect peaking at h after treatment [ ] . this study demonstrates the potential of xten as a carrier for anticancer therapeutics. this concept has recently been adapted by amunix to develop xtenylated protease-activated t-cell engagers, or xpat. in this technology, the bispecific t-cell engager blincyto, which engaged cd and and cd , is xtenylated to reduce its immunogenicity. tandem protease recognition sites are incorporated between the xten chain and tandem scfvs of the bispecific t-cell engager to promote its release upon protease exposure. this technology has been adapted to target her + (amx- ) and egfr+ cells [ ] . in recent pre-clinical studies, these fusions achieved picomolar affinity ec s against cell lines expressing their target proteins while reducing t-cell toxicity , -fold. they also induced significant proteasedependent tumor regression in xenograft models. her -and egfr-xpat were also well tolerated by cynomolgous monkeys, with a -fold greater c max for the her -xten fusion and a -fold greater c max for the egfr-xpat than their non-xtenylated counterparts [ ] . a second recombinant polypeptide, termed pas, was engineered by xl-protein gmbh company in and is conceptually similar to xten [ ] . both are intrinsically disordered, hydrophilic polypeptides that are designed to enhance the plasm half-life of fused proteins and peptides [ ] . pas are reported to be non-immunogenic, biodegradable, and are produced in bacterial systems [ ] . their recombinant synthesis enables precise control over their sequence and length enable gene-level fusion to a therapeutic peptide or protein of interest in order to enhance the pharmacokinetic profile of the fusion partner [ ] . whereas xten incorporates negatively charged residues and consists of pro, tyr, glu, gly, ala and ser, pas do not contain charged amino acids and their sequence is limited to pro, j o u r n a l p r e -p r o o f journal pre-proof ala and ser [ ] . in the initial study published by xl-protein gmbh company, pas was recombinantly fused to three proteins -a recombinant fab region of a humanized anti-her antibody, human interferon a b, and human growth hormone-to evaluate its utility as a drug delivery carrier. fusion to pas increased the hydrodynamic volume of the cargo up to -fold and extended the blood circulation of the fusions [ ] . the plasma half-life and auc of pasylated ifn was -fold and -fold greater than the non-pasylated ifn and the protein's binding affinity for its receptor was not affected by fusion to pas (figure a) . similarly, pasylated hgh exhibited a -fold greater plasma half-life than control hgh and exhibited a fold greater growth promoting effect than native hgh on an equimolar basis ( figure b ) [ ] . since this initial study, pasylation has been employed to enhance the half-life and pharmacokinetics of numerous therapeutics. like other biopolymers, many applications that harness pasylation require long-lasting therapeutics to reduce dosing frequency and side effects. as such, xl-protein gmbh company has developed several formulations to improve the therapeutic efficacy of several peptides for the treatment of many diseases, including exendin for type ii diabetes, hgh for pediatric growth hormone deficiency, and anti-inflammatory agents for rheumatoid arthritis [ ] . many of these fusions are currently in preclinical trials. other groups have also explored pasylation as a strategy to improve the circulation and efficacy of interferons for the treatment of multiple sclerosis. in a study conducted by harari et al, a superagonistic analog of ifnβ, ynsα , was fused to the c terminus of a amino acid pas polypeptide chain [ ] . when administered to a transgenic mouse model harboring humanized ifnar receptors (hybnar model), pas-ynsα exhibited a -fold increase in half-life compared to ynsα alone without impacting the biological activity of the superagonist. despite being administered at an overall -fold lower dosage than ifnβ alone, pas-ynsα outperformed the control group in an experimental autoimmune encephalomyelitis model, as indicated by a decrease in infiltrating macrophages in the central nervous system and an increase in regulatory t cells [ ] . in a separate study by zvonona et al, pas was fused to the j o u r n a l p r e -p r o o f journal pre-proof c-terminus of ifn-β b and resulted in a -fold increase in hydrodynamic radius compared to free ifn-β b [ ] . the fusion protein also notably increased ic -fold compared to free ifn-β b, an accomplishment considering pegylation of the same interferon decreases its activity. furthermore, this formulation was stable when stored for months at - °c, indicating how pasylation can contribute to the maintenance of biological stability [ ] . similarly, pasylation has contributed to the improved half-life and efficacy of numerous proteins including leptin [ , ] , erythropoietin [ ] , conversin [ ] and clotting factor viii [ ] . pasylation has also been harnessed to improve the delivery of nanocarriers, most notably for the treatment of cancer. in one study, pas or residues long were recombinantly fused to ferritin, an iron storage protein that self-assembles into a hollow sphere and binds internalizing transferrin receptor i, which is upregulated on many cancer cells [ ] . pasylation stabilized the ferritin during purification and drug loading, resulting a greater yield of nanocarriers and -fold higher dox loading. the half-life of the pasylated nanocarrier was fold greater than that of the control nanocarrier and -fold greater than that of free dox [ ] . the same research group further developed this platform by incorporating an mmp-cleavable linker between pas and ferritin. this design resulted in a - fold decrease in affinity between ferritin and its receptor, transferrin receptor i. thus, healthy cells with low expression of transferrin receptor i were not affected by the encapsulated dox [ ] . upon exposure to high mmp concentrations in the tumor microenvironment, the pas shield was cleaved from the ferritin, increasing the affinity of the nanocarrier for its target. in mice bearing xenogeneic paca- pancreatic tumors, treatment with the pasylated formulation led to -fold greater tumor regression than a non-pasylated nanocarriers and -fold greater regression than seen with free dox, respectively [ ] . these studies indicate how pasylated nanocarriers can be used to improve drug potency while reducing off-target effects. pasylation is also useful for nucleic acid delivery by providing stealth behavior to the delivery system. morys et al conjugated short pas sequences only four repeats long to polyethylenimine, a cationic polymer frequently employed in nucleic acid delivery systems [ ] . the pas provided enough hydrophilicity to prevent aggregation and dissociation of the pei/dna polycomplexes. furthermore, pasylation of the pei-dna complex prevented interaction with blood serum proteins, preventing polyplex opsonization and clearance in vivo [ ] . although this system needs further optimization for in vivo gene transfer, this study indicates that pasylation is potentially a useful strategy for improving the stability and delivery of nucleic acids. gliadin is a gluten protein extracted from wheat. its monomeric form, which has a mw of - kda, is up to % soluble in alcohol, and its polymeric form, which has a mw of kda, contains a high level of disulfide bonds that renders it largely insoluble [ ] . as such, gliadin materials show excellent stability without the need for toxic crosslinking agents. gliadin is rich in neutral residues that provide it with mucoadhesive properties and make it an ideal platform for oral drug delivery [ ] . gliadin also contains many lipophilic residues that allow it to protect its cargo for prolonged release and hydrophobic interaction with tissues, including skin. due to its apolarity, gliadin has been most useful as a carrier for hydrophobic or amphiphilic drugs [ ] . the solubility, hydrophobicity, and bioadhesive properties of gliadin contribute to its ability to sustain the release of therapeutics in drug delivery applications. polymeric gliadin has been widely explored as a material for nanoparticulate systems targeting the gastrointestinal mucosa. arangoa et al. demonstrated that upon oral administration of the lipophilic molecule carbazole in a gliadin nanoparticle to mice, the gliadin delivery vehicle improved drug adsorption through the stomach mucosa [ ] . this phenomenon resulted in a sustained carbazole concentration in circulation and improved bioavailability. further research evaluated gliadin as a nanoparticulate carrier for the anti-cancer drug cyclophosphamide [ ] . after loading cyclophosphamide onto the nanoparticles with a remarkable % loading efficiency, gulfam et al. demonstrated that the drug was released in a two-step manner. after a brief period of rapid release from the nanoparticles, cyclophosphamide was steadily released over the course of h and induced apoptosis in breast cancer cells, which highlighted the utility of this system utility as a carrier for anti-cancer therapeutics [ ] . because gliadin nanoparticles improve drug penetration into the gastric mucosa, it has been extensively engineered to protect and deliver antibiotics to the stomach to treat helicobacter pylori infections [ ] . ramteke et al. loaded gliadin nanoparticles with clarithromycin and omeprazole via a desolvation technique [ ] . the nanoparticles showed sustained release of the drugs at an acidic ph that mimicked conditions in the stomach and were more effective in eradicating h. pylori in vitro than free drug. in a similar study, umamaheshwari et al. demonstrated that gliadin nanoparticles increased the gastrointestinal residence time of amoxicillin, thus necessitating a lower dose to eradicate h. pylori infection than the free drug in a mongolian gerbil model [ ] . these studies indicate that the bioadhesive properties of gliadin can be leveraged to improve gastric absorption and drug availability. another abundant source of plant protein is the soybean, from which soy protein isolate (spi) is extracted. spi contains glycinin and β-conglycinin, which compose nearly % and % of the protein, respectively [ ] . these proteins exist as globular structures in an aqueous environment and aggregate upon the addition of a crosslinking agent or solvent [ ] . this can drive the formation of hydrogels, polymer blends, or microspheres. moreover, because spis are enriched in glutamine, aspartic acid, and leucine that offer a variety of secondary bonding interactions, many types of drugs can be incorporated onto the proteins for delivery purposes. [ ] . engineered soy nanoparticles to sequester and relase curcumin [ ] . they prepared the nanoparticles using a desolvation technique and achieved an encapsulation efficiency of %. curcumin release from the nanoparticles followed a biphasic trend. initial burst release was observed as the nanoparticles swelled, followed by sustained release [ ] . the pharmacokinetics, gastrointestinal uptake, and encapsulation efficiency of soy nanoparticles have also been improved by combining the protein with other materials such as folic acid [ ] , zein [ , ] , or alginate [ ] . spi films have also been engineered to release a variety of drugs to treat bacterial infections and promote wound healing. in one example, peles et al. incorporated the antibiotic gentamicin into the matrix of an spi film prepared by solvent-casting. after an initial period of burst release, gentamicin exhibited prolonged release over the course of four weeks [ ] . other studies have explored various crosslinking agents to improve the structural integrity and degradation kinetics of spi films. to circumvent the toxicity associated with chemical crosslinkers, song et al. employed genipin, a natural crosslinking agent, to control the swelling ratio and enhance the strength of the film [ ] . films crosslinked with genipin exhibited slowed release rates of bsa, the model protein drug, compared to spi films without genipin. furthermore, the film exhibited ph-sensitivity that could be employed for delivery to the gastrointestinal tract [ ] . chen et al. also designed spi films to release drug in the gastrointestinal tract by using formaldehyde as a crosslinking agent [ ] . formaldehyde crosslinking increased the tensile strength of the film. methylene blue -a model drug-and rifampicin-and antibiotic -interacted strongly with the spi such that they were not released until bulk erosion occurred, which was accelerated by gastric enzymes [ ] . another ph-sensitive spi film was engineered by vaz et al. using glyoxal as a crosslinking agent for spis produced via a melt extrusion technique [ ] . because the pi of soy protein isolate is between . - . , rapid release of the model drug theophylline was observed at a ph of , as there was little interaction between the drug and soy. at physiological ph, however, soy protein isolate was negatively charged and more strongly interacted with the drug than in acidic conditions , thereby slowing its release [ ] . spi fibers have also been investigated as a drug delivery scaffold. xu et al. studied the sorption, kinetics and thermodynamics of drug release from spi fibers for three different drugs [ ] . as the temperature was increased, the diffusion coefficient of drug sorption onto the fibers increased. for metformin and diclofenac, sorption and release relied on the ionic interactions j o u r n a l p r e -p r o o f between the drug and spi scaffold. conversely, these parameters were governed more strongly by van der waals forces for drugs such as -fluorouracil. for all model drugs, burst release was controlled by altering the drug-loading concentration. because release relies on the affinity of the drug for the spi, a lower loading concentration resulted in less burst release [ ] . collectively, these studies demonstrate that spi vehicles can be designed and tuned to deliver a variety of therapeutics. zein, the major storage protein in corn, is a low-molecular weight protein in maize endosperm [ ] . depending on its molecular weight and extraction process, zein is classified asα-zein, β-zein, γ-zein, or δ-zein. the most abundant form, α-zein, is a -to -kda protein that forms a triple super-helix [ ] . though the structure is not fully elucidated, small-angle xray scattering data suggests that nine or ten homologous, helical repeating units arrange into an anti-parallel helix that is stabilized by hydrogen bonding [ , ] . all classes of zein are rich in alanine, leucine, proline, and glutamine residues, but α-zein is the most frequently used class in drug delivery applications due to its abundance. though zein contains both hydrophilic and hydrophobic regions, its large fraction of non-polar residues makes it insoluble in water without the addition of alcohol, urea, or anionic or alkaline surfactants [ ] . the hydrophobicity of zein also renders it mucoadhesive [ ] . zein has been incorporated into -smart‖ hydrogels to control the release of drugs in response to a biological or environmental stimulus. liu et al. generated complex hydrogel beads composed of zein and pectin [ ] . the hydrophobic zein suppressed bead swelling in physiological conditions, while pectin protected the zein from protease degradation in the gastrointestinal tract, thus promoting colon-specific drug delivery [ ] . zein has also been engineered into an injectable, biodegradable gel that forms in situ. gao [ ] . when administered to venous malformations as a sclerosant, the zein-saib hydrogel provided four days of prolonged drug release [ ] . zein hydrogels have also been designed for sustained release of chemotherapeutics and reduce their off-target effects. cao et al. demonstrated that an intratumoral injection of an in situ zein hydrogel extended the release of entrapped dox, resulting in increased anti-tumor efficacy [ ] . they also showed that dox release could be modulated by adjusting the zein concentration [ ] . most zein-based materials for drug delivery have been microspheres or nanoparticles. zein is rich in nonpolar residues, which allows it to stably complex with drugs and entrap them in j o u r n a l p r e -p r o o f the nanoparticle core. the first reported zein microparticle was designed by matsuda et al. to improve the therapeutic index of polysaccharide-kureha (ps-k), a protein polysaccharide that stimulates anti-cancer immune activity [ ] . monodisperse microspheres were generated by sonication and produced -µm microspheres that could be phagocytosed by macrophages [ ] . subsequent studies by this group involved combinations of zein with three other anti-cancer drugs to improve their pharmacokinetics, which demonstrates the versatility of the platform [ ] . since then, zein microparticles have been engineered to deliver antibiotics such as ciproflaxin [ ] , anti-parasitic drugs such as ivermectin [ ] , and corticosteroids such as prednisolone [ ] . muthuselvi et al. investigated how the solvent used for microsphere fabrication impacts drug release [ ] . using the cardiotonic glycoside gitoxin as a model drug, they fabricated zein microspheres with ethanol, methanol, and isopropanol mixtures. while microspheres made with methanol and isopropanol rapidly released the drug over four days, microspheres prepared with ethanol exhibited biphasic release. following an initial phase of burst release, drug release was sustained for h before tapering off into a slower, final stage of release [ ] . their ability to harness the epr effect provides another reason for the use of zein nanoparticles in chemotherapeutics. in one example, lai et al. loaded zein nanoparticles with fluorouracil for delivery to the liver [ ] . when labeled with rhodamine-b intravenously administered, the zein nanoparticles increased liver-targeting efficiency % vs. free rhodamine b and -fluorouracil uptake in the liver was ~ -fold higher than that of free drug [ ] . dong et al. loaded zein nanoparticles with dox and achieved % loading efficiency [ ] . dox release was ph-dependent, with more rapid release occurring in acidic conditions. confocal laser scanning microscopy in hela cells showed that the zein nanoparticles were effectively endocytosed into the cytoplasm and that smaller nanoparticles could enter the nucleus [ ] . improve the anti-cancer potency of statins using lovastatin as the model drug [ ] . lovastatin-loaded zein nanoparticles promoted anti-proliferative activity in hepg cells by accumulating in the cytoplasm during the g /m and pre-g phases of mitosis [ ] . further, zein nanoparticles have improved the pharmacokinetics, solubility, and potency of anti-tuberculosis drugs-including rifampicin, pyrazinamide, and isoniazid [ ] -and vitamin d [ ] . caseins are milk proteins that evolved from a family of secreted calcium (phosphate)binding phosphoproteins [ ] . accounting for nearly % of all bovine milk proteins, caseins j o u r n a l p r e -p r o o f exist in four different forms -αs , αs , β-, and κ [ , ] . though they are all amphiphilic and proline-rich, the four caseins differ in their overall amino acid, phosphorous, and carbohydrate content [ ] . caseins are naturally unstructured, which endows them with the flexibility to exhibit intermolecular interactions. consequently, they assemble into micelles - nm in diameter with negatively charged surfaces [ ] . the size distribution of these micelles is readily impacted by environmental factors, including ph, ionic strength, and hydrostatic pressure, due to their amphiphilicity and lack of tertiary structure [ , ] . although they withstand high temperatures of up to ~ °c, they are quickly destabilized by acidic environments, which makes them a good option for oral delivery [ ] . because casein naturally takes on a micellar conformation, it has been readily adapted as a drug nanocarrier. an early example of this idea was demonstrated by semo et al. they encapsulated the fat-soluble vitamin d in the core of casein micelles and demonstrated that the casein barrier protected vitamin d from uv degradation [ ] . soon after this finding was published, casein micelles were used to entrap poorly soluble chemotherapeutics, such as curcumin. in this study, sahu and coworkers used steady state fluorescence spectroscopy to show that curcumin shows hydrophobic interactions with casein. the complexes self-assembled into micelles ~ nm in diameter, which were efficiently internalized by hela cells [ ] . the ph-sensitivity of casein nanoparticles also makes them useful for gastrointestinal delivery. the livney group loaded β-casein nanoparticles with the chemotherapeutic drug mitoxantrone to create stable nanoparticles - nm in diameter that clustered to trap the drug between micelles as well as within their cores [ ] . these micelles rapidly disintegrated in an acidic microenvironment, suggesting their potential for targeting gastric cancer [ ] . casein micelles have similarly been adapted to improve the bioavailability and targeting of other drugs, such as dox [ ] , folic acid [ ] and flutamide [ ] . casein films have also been applied to tablets to slow drug release. these films, which are typically prepared with transglutaminase as the crosslinker, exhibit remarkable tensile strength, and are gradually hydrolyzed by chymotrypsin [ ] . abu diak and coworkers coated diltiazem-hcl tablets with casein using four different plasticizing agents and ultimately derived a continuous tablet coat with oleic acid as the olasticizer [ ] . at ph . , casein-coated tablets slowed the release of diltiazem-hcl compared to uncoated tablets, which released % of drug within h. the rate at which the coated tablets released the drug decreased with increasing post-coating heat treatment temperature. similarly, at ph . , only coated tablets that underwent j o u r n a l p r e -p r o o f post-coating heat treatment at °c successfully showed sustained drug release, which was likely due to casein crosslinking under these conditions [ ] . the favorable hydrophilicity and biocompatibility of casein, coupled with the availability of reactive sites for chemical modification within its sequence, make it an ideal material for forming hydrogels. in an early example, song and coworkers created genipin-crosslinked casein hydrogels to encapsulate bsa [ ] . hydrogel swelling and subsequent bsa release was governed by the environmental ph. at ph . , there was minimal hydrogel swelling and the bsa remained entrapped in the gel. when the ph was increased to . , the hydrogel swelled and released greater amounts of bsa, making this hydrogel useful for intestinal delivery. the swelling behavior was similarly influenced by the degree of crosslinking by genipin [ ] . in a later study, the same group employed microbial transglutaminase to crosslink casein hydrogels into a fractal network structure compared to uncrosslinked casein hydrogels [ ] . vitamin b , was loaded into the hydrogel under mild conditions and was slowly released from the hydrogel at ph . . the rate of release slowed with increased crosslinking [ ] . casein-drug composites have also been designed to improve the solubility and dissolution of various drugs. millar and corrigan first demonstrated that freeze-dried compacts made from sodium caseinate and acidic casein enhanced the dissolution rates of chlorothiazide and hydrochlorothiazide -fold and . -fold, respectively. the improvement in dissolution was related to the complexation with casein and processing of the drug [ ] . millar et al. further characterized the dissolution rate of the compacts over a range of compositions and found that the dissolution rate of acidic casein was half that of sodium caseinate [ ] . using ibuprofen as the model drug, they showed that acidic casein compacts significantly slowed drug dissolution compared to sodium caseinate compacts. they attributed the difference to the changing form of casein that took place due to microenvironmental ph differences in the boundary layer. acidic casein was also more viscous and rigid than sodium caseinate, which decreased its solubility and reduced drug diffusivity [ ] . a subsequent research group demonstrated that acidic casein and sodium caseinate could be mixed with other materials, such as hydroxypropylmethocellulose matrices, to slow drug dissolution [ ] . iron binding proteinsmost notably transferrin and ferritinhave been widely explored as drug delivery vehicles due to the increased need for iron in many diseases, including cancer. in these applications, iron binding proteins serve to both improve the pharmacokinetics and stability of j o u r n a l p r e -p r o o f their cargo, but also to target the drug by taking advantage of the body's natural iron transport mechanisms. transferrin is an abundant glycoprotein that plays a critical role in the systemic transport of iron. as a natural iron-chelating agent, it reversibly binds one or two ferric ions to maintain their solubility and prevent toxic free-radical generation [ ] . it also promotes iron endocytosis by binding the transferrin receptor , which is upregulated on malignant cells to meet their increased iron needs [ ] . transferrin receptor is also expressed on brain endothelial cells, making transferrin an attractive option for delivering therapeutic cargo across the blood brain barrier [ ] . it should be noted that, due to its unique targeting capacity, transferrin has been used as a targeting moiety to deliver both small molecule chemotherapeutics and peptides/proteins. transferrin-drug conjugates have been employed to target chemotherapeutics to tumors delivery and reduce the off-target toxicity of the drug. szwed et al coupled dox to transferrin and showed that transferrin could overcome anthracycline resistance by impairing the pglycoprotein activity in several anthracycline-resistant cell lines such as dox resistant k cells [ ] . in a subsequent study, they demonstrated that a dox-transferrin conjugate successfully induces apoptosis in leukemia cells while avoiding toxicity to non-malignant cells [ ] . transferrin was also used to target cytochrome c to lung tumors by conjugating it to transferrin using sulfo-lc spdp as a crosslinker [ ] . this strategy resulted in nanocarriers ~ nm in diameter, allowing them to avoid rapid kidney filtration. upon endocytosis by cells, the redox-sensitive bond in the linker was cleaved, releasing cytochrome c from transferrin and initiating apoptosis [ ] . transferrin based nanoparticles have also been engineered to deliver photothermal and photodynamic agents for cancer therapy. wang et al treated transferrin with dtt, leading to exposure of hydrophobic residues that could interact with the near-infrared dye ir through hydrophobic interactions [ ] . these dye-loaded nanoparticles had a half-life of h and were j o u r n a l p r e -p r o o f readily endocytosed by malignant cells within h. they accumulated in which tumor with a high tumor-to-background ratio and, when pulsed with an nm laser, caused a temperature increase to . °c that caused tumor regression, whereas tumors treated with pbs only showed a temperature increase to . °c, which had no effect on tumor growth [ ] . this study demonstrates how transferrin could be used to enhance the pharmacokinetics and delivery of hydrophobic agents. transferrin has similarly been fused to glp- to improve the with improved pharmacokinetics of this peptide drug for treatment of type diabetes [ ] . this technology, developed by biorexis and later acquired by pfizer, consists of non-glycosylated transferrin genetically fused to glp- , creating a fusion protein that is expressed in yeast cells. fusion with transferrin did not affect the insulinotrophic properties of glp- and yielded a half-life of - days, compared to a half-life of minutes for native glp- [ ] . furthermore, the glp- transferrin fusion promoted greater β-cell proliferation and β-cell area compared to transferrin alone. two days after receiving an intraperitoneal injection of glp- -transferrin, treated mice demonstrated a significant increase in β-cell proliferation with nearly % of total islet cell possessing brdu+ nuclei. meanwhile, β-cells in animals treated with transferrin alone did not show signs of proliferation after two days [ ] . as such, glp- -transferrin may provide a novel strategy for restoring β-cell function in diabetics while maintaining blood glucose control. transferrin has also been exploited to treat autoimmune diseases such as myasthenia gravis (mg). in patients with mg, autoantibodies recognize and block nicotinic acetylcholine receptors (achr). by fusing the α-subunit of achr to transferrin, keefe et al created shg , a fusion protein that is recognized and bound by anti-achr autoantibodies and is internalized by cells via the transferrin receptor [ ] . results from in vitro binding and internalization studies show that anti-achr autoantibodies bind shg with an ic of . µm, while no binding to transferrin alone was observed [ ] . when the shg :antibody complex was added to hela cells in culture, the complex was readily internalized via the transferrin receptor, as seen by the j o u r n a l p r e -p r o o f negligible uptake by the cells in the presence of saturating levels of a soluble achr inhibitor. after internalization, the complex was trafficked to the lysosome and was degraded [ ] . transferrin delivery platforms were further optimized by chen et al, who evaluated the effect of various linkers on the binding affinity and pharmacokinetics of transferrin fusion proteins. in this work, growth hormone (gh) or granulocyte colony-stimulating factor (g-csf) were genetically linked to transferrin via a dipeptide linker, a helical peptide linker, or a thrombincleavable, disulfide cyclopeptide linker [ ] . for both gh-tf and g-csf-tf, the longer -helical or cyclopeptide linkers-linkers resulted in less steric hindrance and thus provided greater binding affinity between gh or g-csf and their receptors as well as transferrin and its receptor. fusion proteins with the highest affinity for their targets also had the greatest plasma half-life [ ] . the fusion containing the cyclopeptide linker had the greatest affinity for the transferrin receptor (ic of . nm) and achieved a half-life of . hr. in contrast, the fusion with the dipeptide linker had an -fold lower affinity for the transferrin receptor and exhibited a shorter half-life of . hr [ ] . this study demonstrates how delivery of a protein drug via a transferrin carrier can be modulated by tuning the length and type of linker between the transferrin and its cargo. like transferrin, ferritin has also been exploited to target and carry drugs. ferritin is an iron-storage protein composed of subunits that assemble into a spherical nanocage [ ] . while this nanocage naturally houses up to iron atoms, it can also be engineered to house a variety of therapeutics [ , ] . its loading capacity depends on several factors, including mw of the drug, the encapsulation strategy, and choice of co-solvent for entrapping drugs. metal salts achieve the highest encapsulation efficiency with nearly particles of agno fitting in a single cage, whereas small, hydrophobic moleculesincluding many chemotherapeutics, exhibit lower loading efficiency [ , ] . taking advantage of ferritin's natural ability to traverse the blood brain barrier, fan et al loaded a ferritin nanocage with an infrared dye and administered it to mice bearing u mb j o u r n a l p r e -p r o o f orthotopic gliomas [ ] . accumulation of the ferritin nanocage in the glioma-bearing mice was -fold greater than in the control group that received the dye alone, with maximum accumulation - hr after administration. interestingly, nanocage accumulation was limited to the tumor and no significant accumulation was observed in healthy brain tissue [ ] . fan et al then loaded the nanocages with dox. and administered them intravenously. the dox-loaded nanocages nearly doubled survival time compared to treatment with free dox and significantly reduced tumor volume. additionally, mice treated with the dox-loaded nanocages exhibited slowed weight loss, indicating that the formulation reduced toxicity compared to free dox [ ] . in a separate study, dox loaded ferritin nanocages increased the half-life of dox -fold and the auc by over -fold [ ] . ferritin nanocages have also been used to deliver the poly adp ribose polymerase inhibitor olaparib to treat multiple forms of breast cancer. a study conducted by mazzucchelli et al demonstrated that the encapsulation of the drug in a ferritin nanocage led to -fold greater uptake of the drug by triple negative breast cancer cells compared to the free drug [ ] . the nanocage enhanced the cytotoxicity of the drug in cancer cells and reduced off-target toxicity in healthy cells. these studies demonstrate that ferritin nanocages can be harnessed to improve the delivery of chemotherapeutics in a variety of cancers. in another application, jiang et al synthesized a cobalt nanozyme -a nanomaterial with intrinsic enzyme-like activity -within a ferritin nanocage [ ] . synthesis of the nanozyme within the confines of the nanocage provided better control over the size and structure of the nanozyme compared to nanozymes synthesized outside of the nanocage. the hepatocellular carcinoma targeting peptide sp was recombinantly fused to ferritin such that it was displayed on the nanocage exterior for optimal targeting [ ] . though this design was used for diagnostic purposes, the remarkable targeting specificity it achieved demonstrates how it can be readily adapted as a drug delivery vehicle. ferritin nanocages can also be employed to deliver nucleic acids. li et al successfully encapsulated sirna into the nanocage, protecting it from rapid in vivo degradation [ ] . this j o u r n a l p r e -p r o o f system exhibited highly efficient transfection into primary human tumorigenic cell lines, pmbcs, and mesenchymal stem cells. notably, up to % gene knockdown was achieved by the nanocage, while lipofectamine -the gold standard-only achieved % gene knockdown [ ] . at the low sirna concentration of nm used in this study, lipofectamine is relatively inefficient at transfection as it requires a higher sirna concentration for optimal activity, whereas the nanocage is more effective at gene knockdown at this low sirna concentration. because the biophysical properties and encapsulation of sirna are sequence-independent, this approach could be adapted for sirna delivery for numerous applications [ ] . the research discussed in this review highlights the versatility and multifunctionality of protein-based materials for drug delivery. these carriers benefit from exceptional biocompatibility and biodegradability and minimal toxicity. most importantly, protein-based materials are highly customizable. their chemical, physical, and biological properties can be tailored for a specific application with sequence-level precision, thus providing the researcher with full control over the formulation's pharmacokinetics, pharmacodynamics, biodistribution, and in vivo interactions. much of this work has been made possible by continuing advances in genetic engineering and synthetic biology, which have made it possible to incorporate new chemistries and structural motifs into the polypeptide sequence to improve drug conjugation, targeting, stimulus responsiveness, and self-assembly. researchers continue to explore new ways to engineer proteins to possess desirable chemical and mechanical properties. one such strategy is to create hybrid materials by combining a protein with other proteins or synthetic components to generate a material with the favorable physicochemical properties of its building blocks. another method focuses on conferring new functionalities onto proteins through chemical processing, post-translational modification, or the incorporation of unnatural amino acids into the protein sequence. this approach expands the range of drugs that can be recombinantly fused to, chemically conjugated to, or physically encapsulated by the material. new protein-based materials also continue to be developed from existing proteins and by de novo design of peptide sequences that can introduce new attributes to protein materials to improve drug delivery. as researchers continue to make progress in adapting existing and developing new protein materials, there is also great opportunity for biological and materials science research that will maximize the clinical success of protein drug carriers. despite the progress made in this field, formulations using protein-based materials rarely reach or survive clinical trials. they are hindered by poor in vivo stability and performance, which results from an insufficient understanding of how to create materials that remain stable and functional in the harsh environments present during storage, administration, and circulation. this is further highlighted by the fact that, even though the half-life of a drug can be significantly increased by its protein carrier, the half-life of the formulation still falls short of that of the native protein. alterations to the chemical and physical structure can change how a material and drug are endocytosed by their target, degraded and cleared from circulation, or recognized by immune cells. as new chemistries and hierarchical structures are explored, this knowledge becomes more critical. through increased understanding of these fundamental principles, we can better design and predict the in vivo behavior of protein-based drug delivery vehicles. as new materials are developed, enhanced biological and materials science research can improve tools used to predict their behavior and improve their design. for example, experimental results will boost theoretical models and molecular simulations, which will in turn enhance de novo design of protein architectures. this will inform our understanding of structurefunction relationships such that new morphologies can be engineered to control the delivery of therapeutics. it is also necessary to continue developing tools to improve the production of protein materials. current methods suffer from low yields, limited options for post-translational modification, or high endotoxin levels. by exploring alternate systemsincluding bacterial, yeast, mammalian, and plant cell-basedresearchers can address these challenges. advances in biomaterials for drug delivery recent progress in drug delivery recent advances in natural polymerbased drug delivery systems protein-based drug-delivery materials the past, present and future of protein-based materials engineering the architecture of elastin-like polypeptides: from unimers to hierarchical self-assembly protein-based nanocarriers as promising drug and gene delivery systems s magic bullet concept: years of progress controlled drug delivery: historical perspective for the next generation the origins and evolution of -controlled‖ drug delivery systems controlled drug delivery systems: past forward and future back advanced materials and processing for drug delivery: the past and the future the rise and rise of drug delivery polymeric systems for controlled drug release polymer-drug conjugate therapeutics: advances, insights and prospects polymeric carriers: role of geometry in drug delivery control of polymeric nanoparticle size to improve therapeutic delivery natural and synthetic polymers as drug carriers for delivery of therapeutic proteins bioengineering approaches to controlled protein delivery advances in protein-based materials: from origin to novel biomaterials bt -cutting-edge enabling technologies for regenerative medicine applications of de novo designed peptides current development of biomedical applications chemical modification of biomaterials from nature protein-engineered biomaterials: highly tunable tissue engineering scaffolds unnatural amino acid incorporation in e. coli: current and future applications in the design of therapeutic proteins the coming of age of de novo protein design designing smart materials with recombinant proteins nanostructures based on protein self-assembly: from hierarchical construction to bioinspired materials the drawbacks and advantages of vehicle selection for drug formulation transport and interactions of nanoparticles in the kidneys glomerular permselectivity: barrier function based on discrimination of molecular size and charge macrophage uptake of core-shell nanoparticles surface modified with poly(ethylene glycol) alliance with epr effect: combined strategies to improve the epr effect in the tumor microenvironment active targeting drug delivery nanocarriers: ligands, nano-structures and nano-objects an overview of active and passive targeting strategies to improve the nanocarriers efficiency to tumour sites vascular permeability and drug delivery in cancers challenges associated with penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects nanoparticles' interactions with vasculature in diseases drug delivery with polymeric nanocarriers-cellular uptake mechanisms improving cellular uptake of therapeutic entities through interaction with components of cell membrane subcellular targeting strategies for drug design and delivery mechanisms of drug release from advanced drug formulations such as polymeric-based drug-delivery systems and lipid nanoparticles key cost drivers of pharmaceutical clinical trials in the united states cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines the effects of ligand valency and density on the targeting ability of multivalent nanoparticles based on negatively charged chitosan nanoparticles heuristics for the optimal presentation of bioactive peptides on polypeptide micelles review of collagen i hydrogels for bioengineered tissue microenvironments: characterization of mechanics, structure, and transport increasing mechanical strength of gelatin hydrogels by divalent metal ion removal albumin-based nanoparticles as potential controlled release drug delivery systems clarithromycin-and omeprazole-containing gliadin nanoparticles for the treatment of helicobacter pylori the use of keratin in biomedical applications controlling silk fibroin particle features for drug delivery genetically encoded stealth nanoparticles of a zwitterionic polypeptide-paclitaxel conjugate have a wider therapeutic window than abraxane in multiple tumor models avidity and cell uptake of integrin-targeting polypeptide micelles is strongly shape-dependent genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly controlled drug release from pharmaceutical nanocarriers a noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins conjugate of doxorubicin to albumin-binding peptide outperforms aldoxorubicin sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the c terminus of a protein improved variants of srta for site-specific conjugation on antibodies and proteins with high efficiency dams-kozlowska, silk as an innovative biomaterial for cancer therapy silk as a leading-edge biological macromolecule for improved drug delivery silk fibroin nanoparticle as a novel drug delivery system silk-based biomaterials impact of sterilization on the enzymatic degradation and mechanical properties of silk biomaterials silk-based biomaterials for sustained drug delivery design, fabrication, and function of silk-based nanomaterials biomedical applications of chemically-modified silk fibroin silk-based delivery systems of bioactive molecules genetically engineered silk-elastinlike protein polymers for controlled drug delivery recombinant protein-based polymers for advanced drug delivery biomedical applications of recombinant silk-based materials silk fibroin-derived nanoparticles for biomedical applications silk nanospheres and microspheres from silk/pva blend films for drug delivery self-assembled silk fibroin particles: tunable size and appearance, powder technol release and cellular acceptance of multiple drugs loaded silk fibroin particles silk fibroin nanoparticles for celecoxib and curcumin delivery: ros-scavenging and anti-inflammatory activities in an in vitro model of osteoarthritis biosynthesis of insulin-silk fibroin nanoparticles conjugates and in vitro evaluation of a drug delivery system bioengineered silk protein-based gene delivery systems gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs silk-based gene carriers with cell membrane destabilizing peptides biomineralization-inspired crystallization of manganese oxide on silk fibroin nanoparticles for in vivo mr/fluorescence imaging-assisted tri-modal therapy of cancer cyclic crgdfk peptide and chlorin e functionalized silk fibroin nanoparticles for targeted drug delivery and photodynamic therapy multifunctional adhesive silk fibroin with blending of rgd-bioconjugated mussel adhesive protein injectable and ph-responsive silk nanofiber hydrogels for sustained anticancer drug delivery silk hydrogels for sustained ocular delivery of anti-vascular endothelial growth factor (anti-vegf) therapeutics injectable silk nanofiber hydrogels for sustained release of small-molecule drugs and vascularization injectable thixotropic hydrogel comprising regenerated silk fibroin and hydroxypropylcellulose, soft matter deciphering the mechanism of protein interaction with silk fibroin for drug delivery systems skin-penetrating polymeric nanoparticles incorporated in silk fibroin hydrogel for topical delivery of curcumin to improve its therapeutic effect on psoriasis mouse model novel silk fibroin nanoparticles incorporated silk fibroin hydrogel for inhibition of cancer stem cells and tumor growth injectable carbon nanotube impregnated silk based multifunctional hydrogel for localized targeted and on-demand anticancer drug delivery an injectable silk fibroin nanofiber hydrogel hybrid system for tumor upconversion luminescence imaging and photothermal therapy morphology and primary crystal structure of a silk-like protein polymer synthesized by genetically engineeredescherichia coli bacteria foams made of engineered recombinant spider silk proteins as d scaffolds for cell growth fabrication and characterization of silk fibroin-coated liposomes for ocular drug delivery fibrous protein-based biomaterials (silk, keratin, elastin, and resilin proteins) for tissue regeneration and repair, in: pept biologically derived scaffolds a review of keratin-based biomaterials for biomedical applications the use of keratin in biomedical applications preparation of keratin/chlorhexidine complex nanoparticles for long-term and dual stimuli-responsive release dual-sensitive hydrogel nanoparticles based on conjugated thermoresponsive copolymers and protein filaments for triggerable drug delivery the role of trypsin:chymotrypsin in tissue repair preparation and characterization of dox loaded keratin nanoparticles for ph/gsh dual responsive release triple stimuli-responsive keratin nanoparticles as carriers for drug and potential nitric oxide release biological stimuli responsive drug carriers based on keratin for triggerable drug delivery functionalized keratin as nanotechnology-based drug delivery system for the pharmacological treatment of osteosarcoma development of keratin nanoparticles for controlled gastric mucoadhesion and drug release convenient procedures for human hair protein films and properties of alkaline phosphatase incorporated in the film protein matrices for improved wound healing: elastase inhibition by a synthetic peptide model transglutaminase-modified wool keratin film and its potential application in tissue engineering keratin: dissolution, extraction and biomedical application keratin hydrogels support the sustained release of bioactive ciprofloxacin halofuginone infused keratin hydrogel attenuates adhesions in a rodent cecal abrasion model alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications tunable keratin hydrogel based on disulfide shuffling strategy for drug delivery and tissue engineering ph-sensitive keratin-based polymer hydrogel and its controllable drug-release behavior fabrication of dual-sensitive keratin-based polymer hydrogels and their controllable release behaviors enzymatic digestion of keratin for preparing a ph-sensitive biopolymer hydrogel sustainable and smart keratin hydrogel with ph-sensitive swelling and enhanced mechanical properties smart release of antimicrobial zno nanoplates from a ph-responsive keratin hydrogel three-dimensional structure of human serum albumin, science ( -. ) albumin as a versatile platform for drug half-life extension harnessing albumin as a carrier for cancer therapies genetically encoding albumin binding into chemotherapeutic-loaded polypeptide nanoparticles enhances their antitumor efficacy structure and properties of ovalbumin electrospinning of food-grade nanofibers from cellulose acetate and egg albumen blends alginate microsphere-collagen composite hydrogel for ocular drug delivery and implantation characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension binding of anti-inflammatory drug cromolyn sodium to bovine serum albumin bovine serum albumin nanoparticles as to the inner ear evaluation of berberine/bovine serum albumin nanoparticles for liver fibrosis therapy self-assembly of ibuprofen and bovine serum albumin-dextran conjugates leading to effective loading of the drug structure of serum albumin crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites crystal structure of human serum albumin at . Å resolution human serum albumin, systemic inflammation, and cirrhosis fatty acid distribution in systems modeling the normal and diabetic human circulation: a c nuclear magnetic resonance study review: modifications of human serum albumin and their binding effect unraveling the interaction between fcrn and albumin: opportunities for design of albumin-based therapeutics simple bioconjugate chemistry serves great clinical advances: albumin as a versatile platform for diagnosis and precision therapy human serum albumin ( hsa ) and its applications as a drug delivery vehicle abstract the reactivity of human serum albumin toward trans- -hydroxy- -nonenal lysine residue of human serum albumin is modified by acetylsalicylic acid the thiol pool in human plasma: the central contribution of albumin to redox processes, free radic gp is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis albondin-mediated capillary permeability to albumin. differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins sparc is a possible predictive marker for albumin-bound paclitaxel in non-small-cell lung cancer aldoxorubicin: a tumor-targeted doxorubicin refractory soft tissue sarcomas discovery of the once-weekly glucagon-like peptide- (glp- ) analogue semaglutide pharmacokinetics and clinical implications of semaglutide: a new glucagon-like peptide (glp)- receptor agonist a method for the determination of plasma and blood volume using evans blue dye to determine blood-brain barrier integrity in rodents albumin-binding evans blue derivatives for diagnostic imaging and production of long-acting therapeutics first functionalized mri contrast agent recognizing vascular lesions in vivo labeling of serum albumin for pet proc. natl. acad. sci. u combined ga-nota-evans blue lymphoscintigraphy and ga-nota-rm evaluation of sentinel lymph node metastasis in breast cancer patients a portable albumin binder from a dna-encoded chemical library isolation and characterization of a -kda albumin-binding fragment of streptococcal protein g the serum albumin-binding domain of streptococcal protein g is a three-helical bundle: a heteronuclear nmr study engineering of a bispecific affibody molecule towards her and her by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications extended in vivo half-life of human soluble complement receptor type fused to a serum albumin-binding receptor increased tumor penetration of singledomain antibody-drug conjugates improves in vivo efficacy in prostate cancer models dual affinity fusion approach and its use to express recombinant human insulinlike growth factor ii fusion of an albumin-binding domain extends the half-life of immunotoxins the serum albumin-binding region of streptococcal protein g (bb) potentiates the immunogenicity of the g - rsv-a protein albumin-polymer-drug conjugates: long circulating, high payload drug delivery vehicles phase i trial of methotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. phase i study group of the association for medical oncology of the german cancer society a phase ii trial of methotrexate-human serum albumin (mtx-hsa) in patients with metastatic renal cell carcinoma who progressed under immunotherapy phase ii study of mtx-hsa in combination with cisplatin as first line treatment in patients with advanced or metastatic transitional cell carcinoma increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, j o u r n a l p r e -p r o o f journal pre-proof albumin-bound paclitaxel, abi- , compared with cremophor-based paclitaxel impact of albumin on drug delivery -new applications on the horizon albumin-based cancer therapeutics for intraperitoneal drug delivery: a review blood-brain-barrier-penetrating albumin nanoparticles for biomimetic drug delivery via albumin-binding protein pathways for antiglioma therapy influencing selectivity to cancer cells with mixed nanoparticles prepared from albumin-polymer conjugates and block copolymers mannose -phosphatemodified bovine serum albumin nanoparticles for controlled and targeted delivery of sodium ferulate for treatment of hepatic fibrosis folic acid receptor-targeted human serum albumin nanoparticle formulation of cabazitaxel for tumor therapy targeted delivery of -fluorouracil with monoclonal antibody modified bovine serum albumin nanoparticles antibody-nanoparticle conjugate constructed with trastuzumab and nanoparticle albumin-bound paclitaxel for targeted therapy of human epidermal growth factor receptor -positive gastric cancer aptamer functionalized curcumin-loaded human serum albumin (hsa) nanoparticles for targeted delivery to her- positive breast cancer cells recombinant fusion protein linking coagulation factor ix with albumin (rix-fp) in previously treated children < years of age with hemophilia b: long-term efficacy and safety from an ongoing phase b extension clinical trial enediyne anticancer antibiotic lidamycin: chemistry, biology and pharmacology the recombinant and reconstituted novel albumin-lidamycin conjugate shows lasting tumor imaging and intensively enhanced therapeutic efficacy expression, purification and characterization of recombinant human interleukin- -serum albumin (rhil- -hsa) fusion protein in pichia pastoris pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin- -human serum albumin fusion protein in mice an ifn-βalbumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase collagens -structure, function, and biosynthesis the collagen family collagen as a carrier for on-site delivery of antibacterial drugs structure of collagen a review on collagen based drug delivery systems collagen as biomaterial for medical application-drug delivery and scaffolds for tissue regeneration: a review collagen-like peptides and peptide-polymer conjugates in the design of assembled materials collagen-like peptide bioconjugates amino acid propensities for the collagen triple-helix collagen mimetic peptides: progress towards functional applications isolation and characterization of the cyanogen bromide peptides from the α (iii)chain of human collagen conformational properties of (pro-hyp-gly)n model trimers with n-and c-terminal collagen type iii cystine knots, chem. -a eur folding mechanism of the triple helix in type-iii collagen and type-iii pn-collagen: role of disulfide bridges and peptide bond isomerization tren (tris( -aminoethyl)amine): an effective scaffold for the assembly of triple helical collagen mimetic structures acetyl-terminated and templateassembled collagen-based polypeptides composed of gly-pro-hyp sequences. . synthesis and conformational analysis by circular dichroism, ultraviolet absorbance, and optical rotation metal-assisted stabilization and probing of collagenous triple helices metal stabilization of collagen and de novo designed mimetic peptides self-assembling amphiphiles for construction of protein molecular architecture self-assembly of model collagen peptide amphiphiles minimal lipidation stabilizes protein-like molecular architecture effects of the stereo-configuration of the hydroxyl group in -hydroxyproline on the triple-helical structures formed by homogeneous peptides resembling collagen inductive effects on the energetics of prolyl peptide bond isomerization: implications for collagen folding and stability code for collagen's stability deciphered a hyperstable collagen mimic promoting self-assembly of collagen-related peptides into various higher-order structures by metal-histidine coordination self-assembly of collagen peptides into microflorettes via metal coordination how electrostatic networks modulate specificity and stability of collagen facile modification of collagen directed by collagen mimetic peptides targeting and mimicking collagens via triple helical peptide assembly high serum stability of collagen hybridizing peptides and their fluorophore conjugates detection and characterization of molecular-level collagen damage in overstretched cerebral arteries self-assembled water-soluble nanofibers displaying collagen hybridizing peptides molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide spatio-temporal modification of collagen scaffolds mediated by triple helical propensity direct detection of collagenous proteins by fluorescently labeled collagen mimetic peptides targeting collagen strands by photo-triggered triple-helix hybridization visualizing collagen proteolysis by peptide hybridization: from d cell culture to in vivo imaging immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis nanoparticle-assisted visualization of binding interactions between collagen mimetic peptide and collagen fibers conducting polymer nanoparticles decorated with collagen mimetic peptides for collagen targeting a cmp-based method for tunable, cell-mediated gene delivery from collagen scaffolds integration of growth factor gene delivery with collagen-triggered wound repair cascades using collagen-mimetic peptides collagen-binding il- enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours self-assembly of synthetic collagen triple helices self-complementary peptides for the formation of collagen-like triple helical supramolecules artificial collagen gels via self-assembly of de novo designed peptides a collagen-mimetic triple helical supramolecule that evokes integrindependent cell responses development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial collagen-related peptides: self-assembly of short, single strands into a functional biomaterial of micrometer scale thrombogenic collagenmimetic peptides: self-assembly of triple helix-based fibrils driven by hydrophobic interactions promotion of human platelet adhesion and aggregation by a synthetic, triple-helical -mini-collagen‖ aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures self-assembly of short collagen-related peptides into fibrils via cation-π interactions construction of biologically active protein molecular architecture using self-assembling peptideamphiphiles structure and dynamics of peptide-amphiphiles incorporating triple-helical proteinlike molecular architecture self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber d-periodic collagenmimetic microfibers multi-hierarchical self-assembly of a collagen mimetic peptide from triple helix to nanofibre and hydrogel peptide tessellation yields micrometrescale collagen triple helices investigation of ph-dependent collagen triple-helix formation metal-mediated tandem coassembly of collagen peptides into banded microstructures controlling the morphology of metal-promoted higher ordered assemblies of collagen peptides with varied core lengths a metal-collagen peptide framework for three-dimensional cell culture selective decoration and release of histagged proteins from metal-assembled collagen peptide microflorettes accessing three-dimensional crystals with incorporated guests through metal-directed coiled-coil peptide assembly advances in the design and higher-order assembly of collagen mimetic peptides for regenerative medicine thermoresponsive elastin-b-collagen-like peptide bioconjugate nanovesicles for targeted drug delivery to collagen-containing matrices noncovalent modulation of the inverse temperature transition and self-assembly of elastin-b-collagen-like peptide bioconjugates self-assembly of stable nanoscale platelets from designed elastin-like peptide-collagen-like peptide bioconjugates effect of peptide sequence on the lcst-like transition of elastin-like peptides and elastin-like peptide-collagen-like peptide conjugates: simulations and experiments noncovalent modulation of the inverse temperature transition and self-assembly of elastin-b-collagen-like peptide bioconjugates thermoresponsive self-assembly of nanostructures from a collagen-like peptide-containing diblock copolymer thermoresponsive elastin-b-collagen-like peptide bioconjugate nanovesicles for targeted drug delivery to collagen-containing matrices engineering and functionalization of gelatin biomaterials: from cell culture to medical applications gelatin: a valuable protein for food and pharmaceutical industries: review helix ⇆ coil transitions in dilute aqueous collagen solutions gelatin-based nanoparticles as drug and gene delivery systems: reviewing three decades of research gelatine-based biomaterials and their biocompatibility: review and perspectives recent advancement of gelatin nanoparticles in drug and vaccine delivery recent advances in the use of gelatin in biomedical research recombinant collagen and gelatin for drug delivery gelatin nanoparticles and their biofunctionalization crosslinking structures of gelatin hydrogels crosslinked with genipin or a water-soluble carbodiimide collagen structure in solution. i. kinetics of helix regeneration in single-chain gelatins mechanism of gelation of gelatin. influence of certain electrolytes on the melting points of gels of gelatin and chemically modified gelatins gelation of aqueous gelatin solutions. i. structural investigation gelatin nanoparticle preparation by nanoprecipitation effects of mass ratio, ph, temperature, and reaction time on fabrication of partially purified pomegranate ellagitannin-gelatin nanoparticles gelatin behaviour in dilute aqueous solution: designing a nanoparticulate formulation effect of molecular weight heterogeneity on drug encapsulation efficiency of gelatin nano-particles design of gelatin nanoparticles as swelling controlled delivery system for chloroquine phosphate a novel smart pegylated gelatin nanoparticle for co-delivery of doxorubicin and betanin: a strategy for enhancing the therapeutic efficacy of chemotherapy combined delivery of bmp- and bfgf from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo poly-(ethylene glycol) modified gelatin nanoparticles for sustained delivery of the anti-inflammatory drug ibuprofen-sodium: an in vitro and in vivo analysis functionalized photosensitive gelatin nanoparticles for drug delivery application development of gelatin nanoparticle-based biodegradable phototheranostic agents: advanced system to treat infectious diseases in vivo activity against hiv and favorable toxicity profile of ′, ′-dideoxyinosine stiffness of cationized gelatin nanoparticles is a key factor determining rnai efficiency in myeloid leukemia cells engineered cartilage via self-assembled hmsc sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-β in vitro and in vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein- fabrication and characterization of a novel microparticle with gyrus-patterned surface and growth factor delivery for cartilage tissue engineering mmp-mediated mesenchymal morphogenesis of pluripotent stem cell aggregates stimulated by gelatin methacrylate microparticle incorporation improving antioxidant and antimicrobial properties of curcumin by means of encapsulation in gelatin through electrohydrodynamic atomization, food hydrocoll sterically stabilized gelatin microassemblies of noscapine enhance cytotoxicity, apoptosis and drug delivery in lung cancer cells monodisperse microshell structured gelatin microparticles for temporary chemoembolization electrospun gelatin nanofibers loaded with vitamins a and e as antibacterial wound dressing materials induction of angiogenesis using vegf releasing genipin-crosslinked electrospun gelatin mats gelatin-based hydrogels for biomedical applications cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution a novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering a cisplatin slow-release hydrogel drug delivery system based on a formulation of the macrocycle cucurbit[ ]uril, gelatin and polyvinyl alcohol bioprintable alginate/gelatin hydrogel d in vitro model systems induce cell spheroid formation engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics directing the self-assembly of tumour spheroids by bioprinting cellular heterogeneous models within alginate/gelatin hydrogels in vitro microbial inhibition, bonding strength, and cellular response to novel gelatin-alginate antibioticreleasing soft tissue adhesives novel gelatin/alginate soft tissue adhesives loaded with drugs for pain management: structure and properties fibrous proteins: coiled-coils, collagen and elastomers elastin-based materials applications of elastin-like polypeptides in drug delivery elastin-like polypeptides: biomedical applications of tunable biopolymers molecular description of the lcst behavior of an elastin-like polypeptide ucst and lcst behavior in polymer blends a unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures temperature dependence of length of elastin and its polypentapeptide effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity? predicting transition temperatures of elastin-like polypeptide fusion proteins purification of recombinant proteins by fusion with thermallyresponsive polypeptides expression and purification of recombinant proteins from escherichia coli: comparison of an elastin-like polypeptide fusion with an oligohistidine fusion simple bioseparations using self-cleaving elastin-like polypeptide tags expression and purification of moricin cm and human β-defensins in escherichia coli using a new technology basics and recent advances in peptide and protein drug delivery macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution insoluble drug delivery strategies: review of recent advances and business prospects drug delivery systems: an updated review optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants elpylated anti-human tnf therapeutic singledomain antibodies for prevention of lethal septic shock influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants drug delivery in stealth mode stealth properties to improve therapeutic efficacy of drug nanocarriers long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery thermo-reversible self-assembly of nanoparticles derived from elastin-mimetic polypeptides self-assembly of block copolymers derived from elastinmimetic polypeptide sequences thermoplastic elastomer hydrogels via self-assembly of an elastin-mimetic triblock polypeptide temperature triggered self-assembly of polypeptides into multivalent spherical micelles a quantitative recipe for engineering protein polymer nanoparticles elastin-like polypeptide diblock copolymers self-assemble into weak micelles morphing low-affinity ligands into highavidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer digital switching of local arginine density in a genetically encoded self-assembled polypeptide nanoparticle controls cellular uptake j o u r n a l p r e -p r o o f journal pre-proof the design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters unexpected multivalent display of proteins by temperature triggered selfassembly of elastin-like polypeptide block copolymers design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain tear-mediated delivery of nanoparticles through transcytosis of the lacrimal gland triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution a rapamycin-binding protein polymer nanoparticle shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of sjögren's syndrome elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles a hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody an amphipathic alpha-helical peptide from apolipoprotein a stabilizes protein polymer vesicles self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation a paclitaxel-loaded recombinant polypeptide nanoparticle outperforms abraxane in multiple murine cancer models niclosamide-conjugated polypeptide nanoparticles inhibit wnt signaling and colon cancer growth encapsulating a hydrophilic chemotherapeutic into rod-like nanoparticles of a genetically encoded asymmetric triblock polypeptide improves its efficacy active targeting of cancer cells by nanobody decorated polypeptide micelle with bioorthogonally conjugated drug recombinant synthesis of hybrid lipid-peptide polymer fusions that self-assemble and encapsulate hydrophobic drugs silk-elastin-like protein biomaterials for the controlled delivery of therapeutics tunable self-assembly of genetically engineered silk--elastin-like protein polymers hydrophobic drug-triggered self-assembly of nanoparticles from silk-elastin-like protein polymers for drug delivery injectable intratumoral depot of thermally responsive polypeptide-radionuclide conjugates delays tumor progression in a mouse model brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides <em&gt injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy one-month zero-order sustained release and tumor eradication after a single subcutaneous injection of interferon alpha fused with a body-temperature-responsive polypeptide injectable protease-operated depots of glucagon-like peptide- provide extended and tunable glucose control one-week glucose control via zero-order release fusion of fibroblast growth factor to a thermally responsive biopolymer forms an injectable depot with sustained anti-diabetic action a thermo-responsive protein treatment for dry eyes sustained release of antibiotics from injectable and thermally responsive polypeptide depots development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin- receptor antagonist: sustained release of a local antiinflammatory therapeutic phasebio-announces-global-license-of-pb -forthe-treatment-of-sarcopenia-related-diseases-to-immunoforge.html phasebio announces case study highlighting pb hemodynamic data presented at the th pulmonary vascular research institute world congress mechanism of resilin elasticity resilin-like polypeptide hydrogels engineered for versatile biological functions resilin-based materials for biomedical applications tentative identification of a resilin gene in drosophila melanogaster synthesis and properties of crosslinked recombinant pro-resilin comparisons of recombinant resilin-like proteins: repetitive domains are sufficient to confer resilinlike properties structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein rec -resilin purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilininspired polypeptides a genetically engineered protein responsive to multiple stimuli a ph-responsive interface derived from resilin-mimetic protein rec -resilin temperature-triggered phase separation of a hydrophilic resilinlike polypeptide ph-responsive schizophrenic diblock copolymers hydration changes during thermosensitive association of a block copolymer consisting of lcst and ucst blocks switching the inside and the outside of aggregates of water-soluble block copolymers with double thermoresponsivity micellar self-assembly of recombinant resilin-/elastin-like block copolypeptides hydrophilic elastomeric biomaterials based on resilin-like polypeptides tunable mechanical stability and deformation response of a resilin-based elastomer characterization of resilin-based materials for tissue engineering applications targeting angiogenesis: structural characterization and biological properties of a de novo engineered vegf mimicking peptide expression, cross-linking, and characterization of recombinant chitin binding resilin redox-responsive resilin-like hydrogels for tissue engineering and drug delivery applications resilin-like polypeptide hydrogels engineered for versatile biological function design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide genetically engineered silk-elastinlike protein polymers for controlled drug delivery the enhanced permeability and retention (epr) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting design of silk-elastin-like protein nanoparticle systems with mucoadhesive properties in-situ self-assembling protein polymer gel systems for administration, delivery, and release of drugs solute diffusion in genetically engineered silk-elastinlike protein polymer hydrogels controlled release of plasmid dna from a genetically engineered silk-elastinlike hydrogel in vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers comparison of silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene delivery silk-elastin-like hydrogel improves the safety of adenovirus-mediated gene-directed enzyme−prodrug therapy direct observation of interactions of silk-elastinlike protein polymer with adenoviruses and elastase temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis a recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner multivalent antiviral xten-peptide conjugates with long in vivo half-life and enhanced solubility extension of in vivo half-life of biologically active molecules by xten protein polymers safety, pharmacokinetics, and pharmacodynamics of a single subcutaneous dose of vrs- in patients with type diabetes gcg-xten: an improved glucagon capable of preventing hypoglycemia without increasing baseline blood glucose glp - g-xten: a pharmaceutical protein with improved serum half-life and efficacy in a rat crohn's disease model xten-annexin a : xten allows complete expression of long-circulating protein-based imaging probes as recombinant alternative to pegylation vrs- ), a long-acting rhgh, in pediatric growth hormone deficiency safety and efficacy of somavaratan (vrs- ), a long-acting growth hormone, in children with growth hormone deficiency (ghd): . -year results from the vista trial prolonged half-life and improved recovery of recombinant factor ix-xten fusion proteins in hemophilia b mouse model safety and prolonged activity of recombinant factor viii fc fusion protein in hemophilia a patients long-term safety and efficacy of recombinant factor viii fc fusion protein (rfviiifc) in subjects with haemophilia a bivv , a new class of factor viii replacement for hemophilia a that is independent of von willebrand factor in primates and mice xten as biological alternative to pegylation allows complete expression of a protease-activatable killin-based cytostatic off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in nhp pasylation: a biological alternative to pegylation for extending the plasma halfactive proteins influence of size and charge of unstructured polypeptides on pharmacokinetics and biodistribution of targeted fusion proteins fusion proteins for half-life extension of biologics as a strategy to make biobetters enhanced in vivo efficacy of a type i interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis pasylation technology improves recombinant interferon-β b solubility, stability, and biological activity pasylation of murine leptin leads to extended plasma half-life and enhanced in vivo efficacy treatment of diet-induced lipodystrophic c bl/ j mice with long-acting pasylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation molecular design, expression and evaluation of pasylated human recombinant erythropoietin with enhanced functional properties pasylated coversin, a c -specific complement inhibitor with extended pharmacokinetics, shows enhanced anti-hemolytic activity in vitro pasylation®: a versatile technology to extend drug delivery improved doxorubicin encapsulation and pharmacokinetics of ferritin-fusion protein nanocarriers bearing proline, serine, and alanine elements selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness influence of defined hydrophilic blocks within oligoaminoamide copolymers: compaction versus shielding of pdna nanoparticles, polym comparison of peptides from wheat gliadin and glutenin bioadhesive potential of gliadin nanoparticulate systems evaluation of gliadins nanoparticles as drug delivery systems: a study of three different drugs gliadin nanoparticles as carriers for the oral administration of lipophilic drugs anticancer drug-loaded gliadin nanoparticles induce apoptosis in breast cancer cells anti-helicobacter pylori effect of mucoadhesive nanoparticles bearing amoxicilin in experimental gerbils model fractionation of soybean globulins using ca + and mg +: a comparative analysis effect of concentration, ionic strength and freeze-drying on the heat-induced aggregation of soy proteins development of a bio-based composite material from soybean oil and keratin fibers nanoparticles synthesized from soy protein: preparation, characterization, and application for nutraceutical encapsulation soy protein/soy polysaccharide complex nanogels: folic acid loading, protection, and controlled delivery in vitro study of the release properties of soy-zein protein microspheres with a dynamic artificial digestive system elaboration and characterization of soy/zein protein microspheres for controlled nutraceutical delivery lactobacillus casei loaded soy protein-alginate microparticles prepared by spray-drying soy protein films for wound-healing applications: antibiotic release, bacterial inhibition and cellular response gelation modification of soy protein isolate by a naturally occurring cross-linking agent and its potential biomedical application kinetics of the breakdown of crosslinked soy protein films for drug delivery soy matrix drug delivery systems obtained by melt-processing techniques drug sorption onto and release from soy protein fibers microspheres of corn protein, zein, for an ivermectin drug delivery system zein in controlled drug delivery and tissue engineering three-dimensional structure of maize α-zein proteins studied by small-angle x-ray scattering fabrication, gastromucoadhesivity, swelling, and degradation of zein-chitosan composite ultrafine fibers pectin/zein beads for potential colon-specific drug delivery: synthesis and in vitro evaluation study of a pingyangmycin delivery system: zein/zein-saib in situ gels doxorubicin-loaded zein in situ gel for interstitial chemotherapy novel preparation of zein microspheres conjugated with ps-k available for cancer immunotherapy preparation of zein microspheres conjugated with antitumor drugs available for selective cancer chemotherapy and development of a simple colorimetric determination of drugs in microspheres antibacterial activity of ciprofloxacin-loaded zein microsphere films preparation and in vitro release of zein microparticles loaded with prednisolone for oral delivery simple coacervates of zein to encapsulate gitoxin preparation of new -fluorouracil-loaded zein nanoparticles for liver targeting doxorubicin-loaded biodegradable self-assembly zein nanoparticle and its anti-cancer effect: preparation, in vitro evaluation, and cellular uptake encapsulation of lovastatin in zein nanoparticles exhibits enhanced apoptotic activity in hepg cells fabrication of plant protein microspheres for encapsulation, stabilization and in vitro release of multiple anti-tuberculosis drugs preparation and characterization of zein and zein-chitosan microspheres with great prospective of application in controlled drug release invited review: caseins and the casein micelle: their biological functions, structures, and behavior in foods potential of casein as a carrier for biologically active agents milk nutritional composition and its role in human health formation and potential uses of milk proteins as nano delivery vehicles for nutraceuticals: a review unique milk protein based nanotubes: food and nanotechnology meet preparation of nanoparticles from denatured whey protein by ph-cycling treatment heat stability of micellar casein concentrates as affected by temperature and ph casein micelle as a natural nanocapsular vehicle for nutraceuticals, food hydrocoll fluorescence study of the curcumin−casein micelle complexation and its application as a drug nanocarrier to cancer cells beta-casein nanovehicles for oral delivery of chemotherapeutic drugs livney, β-casein-based nanovehicles for oral delivery of chemotherapeutic drugs: drug-protein interactions and mitoxantrone loading capacity doxorubicin-loaded casein nanoparticles for drug delivery: preparation, characterization and in vitro evaluation casein nanoparticles as carriers for the oral delivery of folic acid, food hydrocoll novel ionically crosslinked casein nanoparticles for flutamide delivery: formulation, characterization, and in vivo pharmacokinetics nio, &alpha; s -casein film prepared using the manufacture and characterization of casein films as novel tablet coatings genipin-crosslinked casein hydrogels for controlled drug delivery novel casein hydrogels: formation, structure and controlled drug release influence of sodium caseinate on the dissolution rate of hydrochlorothiazide and chlorothiazide dissolution mechanism of ibuprofen-casein compacts the effects of casein on diclofenac release from hydroxypropylmethylcellulose (hpmc) compacts evolution of the transferrin family: conservation of residues associated with iron and anion binding transferrin receptor in cancer: a new sight for cancer therapy exploiting transferrin receptor for delivering drugs across the blood-brain barrier relationship between therapeutic efficacy of doxorubicin-transferrin conjugate and expression of p-glycoprotein in chronic resistant to doxorubicin efficacy of doxorubicin-transferrin conjugate in apoptosis induction in human leukemia cells through reactive oxygen species generation inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate self-assembled ir -loaded transferrin nanoparticles as an imaging, targeting and pdt/ptt agent for cancer therapy transferrin fusion technology: a novel approach to prolonging biological half-life of insulinotropic peptides fusion protein technologies for biopharmaceuticals : applications and challenges effects of receptor binding on plasma half-life of bifunctional transferrin fusion proteins exploiting iron-binding proteins for drug delivery reconstituted and native iron-cores of bacterioferritin and ferritin humanized archaeal ferritin as a tool for cell targeted delivery cisplatin encapsulation within a ferritin nanocage: a high-resolution crystallographic study high hydrostatic pressure encapsulation of doxorubicin in ferritin nanocages with enhanced efficiency ferritin nanocarrier traverses the blood brain barrier and kills glioma h-ferritinnanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection h-ferritin-nanocaged olaparib: a promising choice for both brca-mutated and sporadic triple negative breast cancer biomineralization synthesis of the cobalt nanozyme in sp -ferritin nanocages for prognostic diagnosis of hepatocellular carcinoma ferritin-mediated sirna delivery and gene silencing in human tumor and primary cells a.v. and s.s. contributed equally to this work. a.v. acknowledges the support of the nsf through a graduate research fellowship. [ ] key: cord- -c d nk x authors: mikasa, keiichi; aoki, nobuki; aoki, yosuke; abe, shuichi; iwata, satoshi; ouchi, kazunobu; kasahara, kei; kadota, junichi; kishida, naoki; kobayashi, osamu; sakata, hiroshi; seki, masahumi; tsukada, hiroki; tokue, yutaka; nakamura-uchiyama, fukumi; higa, futoshi; maeda, koichi; yanagihara, katsunori; yoshida, koichiro title: jaid/jsc guidelines for the treatment of respiratory infectious diseases: the japanese association for infectious diseases/japanese society of chemotherapy – the jaid/jsc guide to clinical management of infectious disease/guideline-preparing committee respiratory infectious disease wg date: - - journal: journal of infection and chemotherapy doi: . /j.jiac. . . sha: doc_id: cord_uid: c d nk x nan the japanese association for infectious diseases (jaid) and japanese society of chemotherapy (jsc) announced the "guide for the use of antimicrobial drugs" in and the "guidelines for the use of antimicrobial drugs" in . subsequently, the "the jaid/ jsc guide to clinical management of infectious diseases " was published. with its revision, guidelines were newly prepared. concerning respiratory infectious diseases, in japan, the japanese respiratory society published guidelines for the management of community-acquired pneumonia, hospital-acquired pneumonia, respiratory tract infection, and -/nursing and healthcare-associated pneumonia. furthermore, the japanese society of pediatric pulmonology and japanese society for pediatric infectious diseases announced the "guidelines for the management of respiratory infectious diseases in children in japan". internationally, many guidelines, including those established by the american thoracic society and infectious diseases society of america, have been published from various countries. thereafter, clinical research on respiratory infectious diseases has advanced, leading to the accumulation of many outcomes regarding epidemiology, clinical diagnosis, and treatment. however, the types of microorganisms that cause respiratory infectious diseases have increased with the number of resistant bacteria. in addition, conditions have also varied with causative microorganisms through the recent compromised host's severe status. the place of treatment varies: from the outpatient clinic to the icu. physicians responsible for treatment also vary: practitioners, hospital doctors, pulmonologists, emergency physicians, board certified member of jaid, japanese antimicrobial chemotherapy physician. there are a large number of options of antimicrobial drugs that are available, including new drugs; therapeutic strategies are confused. on the other hand, recently, the entity of pk-pd has been commonly recognized, and the importance of scientifically using antimicrobial drugs has been emphasized. in addition, the japanese society of chemotherapy established a system for antimicrobial chemotherapy-certified physicians, and promoted the widespread, adequate use of antimicrobial drugs. based on these, the two societies prepared the jaid/jsc guidelines for the treatment of respiratory infectious diseases. if specific treatment guidelines can be presented, this may contribute to an improvement in the treatment responses of respiratory infectious diseases, a reduction in health expenditure, and the prevention of resistant bacteria. the guidelines were prepared based on the ebm so that they reflected the management of respiratory infectious diseases in japan and covered all such diseases in adults and children. to prepare the guidelines, a committee was established in , and a draft was published on homepage based on an approval from the boards of directors at the two societies through a review-based consensus. opinions were collected from the two societies' members. in japan, there have been no such guidelines covering respiratory infectious diseases. in the future, with further advances in research, the contents of the guidelines must be revised. however, we successfully provided treatment guidelines that are the most advanced at present. the guidelines were prepared for all clinicians to understand the treatment of respiratory infectious diseases and manage them with antimicrobial drugs adequately. they do not limit treatment by individual physicians or affect their rights to select it. the guidelines may be commonly applied for respiratory infectious disease management/research/education in japan, improving the quality of respiratory infectious disease management, preventing an increase in the number of resistant bacteria, and contributing to national health. we hope that the guidelines will be utilized by a large number of clinicians in respiratory infectious disease management. lastly, we thank the committee members and secretariat staff for their cooperation. . descriptions on the recommendation grade and evidence level . definition of firstand second-choice drugs . precautions -in this article, with respect to the administration method (especially doses) of antimicrobial drugs, they are recommended based on sufficient doses. considering the products adopted at each medical institution, antibiograms, severity, underlying disease, age, and presence or absence of organ disorder, the dose should be increased or decreased if necessary. -the spectra of third-generation cephems for intravenous injection, ctx and ctrx, are similar, but ctx, which is excreted in the kidney, should be primarily used when liver dysfunction is present, and ctrx, which is excreted in bile, should be primarily used when renal dysfunction is present. -as quinolones exhibit antitubercular actions, patients with pulmonary tuberculosis should be excluded for use. . a list of antimicrobial drug abbreviations and doses for neonates are presented at the end of this volume. . . community-acquired pneumonia . . . empiric therapy ---executive summary---patient with bacterial pneumonia should be treated primarily with high-dose penicillin (aii). in elderly patients and those with underlying lung diseases, the use of respiratory quinolones may be considered positively (bii). in case of atypical pneumonia, a macrolide or tetracycline is the first choice. respiratory quinolones should be reserved as alternative drugs (bii), but may be used depending on local circumstances about drug resistance (ciii). in case of whether pneumonia or atypical pneumonia dose not diagnose, comobination with high-dose penicillin and a macrolide or tetracycline should be attempted first (bii). respiratory quinolones should be reserved as alternative drugs (bii). in severer cases requiring treatment in the icu, a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment (aii). ---explanation---community-acquired pneumonia refers to hospital-acquired pneumonia that develops h or more after admission or pneumonia that develops in healthy adults on social activities other than medical practice-/nursing-associated pneumonia [ e ] . as signs and symptoms, cough, sputum, thoracic pain, and dyspnea appear, and this disease acutely occurs with systemic symptoms such as fever and general malaise [ e ] . however, these symptoms are not marked in some elderly patients. furthermore, atypical pneumonia including mycoplasma is characterized by a small amount of sputum, and can be differentiated (tables and ) [ , ] . concerning examination, gram staining and culture of sputum are used to identify causative microorganisms and select subsequent treatment strategies [ , ] (aii). kits for rapid diagnosis with urine or nasal swab are also used for auxiliary diagnosis [ , ] (aii). a blood test shows inflammatory findings such as leukocytosis and an increase in the crp level, facilitating a certain assessment of the disease [ , ] . on thoracic imaging, consolidation or a ground glasslike shadow is observed [ e ] (ii). when patients are in an immunosuppressive state related to an underlying disease, a causative microorganism test should be performed, considering the possibility of opportunistic infection [ e , , ] (a). in elderly patients, aspiration pneumonia is frequently observed, and the management of this disorder is necessary (refer to the section " . aspiration pneumonia" on page. ). in the presence of renal dysfunction, the type and dose of an antimicrobial drug must be carefully selected [ , ] (aii). bacterial pneumonia should be differentiated from atypical pneumonia in accordance with "the jrs guidelines for the management of community-acquired pneumonia in adults in " (edited by the committee to prepare guidelines regarding respiratory infectious diseases, japanese respiratory society) (tables and ) [ ] . although legionella pneumonia is routinely classified as atypical pneumonia, various types of atypical pneumonia do not include legionella pneumonia in this differentiation method. a. bacterial pneumonia ( ) outpatient treatment bacterial pneumonia is primarily caused by sterptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis [ e , , ] (ii). basically, these types of pneumonia should be treated by orally administering high-dose penicillin [ e ] (aii). in japan, macrolide-resistant s. pneumoniae is detected in most cases; therefore, macrolides are not recommended as the first choice, differing form those in europe and the united states [ , , , , ] (aii). for outpatient treatment, b-lactamase inhibitorcontaining penicillin is commonly used. therapy with cva/ampc or sbtpc ( tablets/ e times a day) is recommended with respect to the efficacy and suppression of resistant bacteria [ , , ] (aii). however, such high-dose prescriptions are not always accepted by health insurance system in japan, and the following prescriptions (examples) should also be considered. in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [ , , ] (bii). however, many new quinolones also have antimicrobial activities against mycobacterium tuberculosis; therefore, the presence or absence of active tuberculosis must be strictly checked before administration [ ] (aii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. considering s. pneumoniae, h. influenzae, and m. catarrhalis, high-dose penicillin or cephems, which are effective for these microorganisms, should be selected [ e ] (aii). if more potent treatment is required, respiratory quinolone injection should be used [ , ] (bii). ---drugs to be recommended---( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day * concerning cva/ampc and sbtpc, up to mg of ampc or up to mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral ( / mg), tablet/ times a day þ ampc, oral ( mg), tablet/ times a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day -tflx, oral, mg/twice a day ( ) hospital treatment a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day <> second choice -lvfx, intravenous drip, mg/once a day b. atypical pneumonia ( ) outpatient treatment atypical pneumonia is primarily caused by mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila [ e , , , , ] (ii). the oral administration of a macrolide or tetracycline is the first choice [ , , , ] (aii). to suppress resistant bacteria, respiratory quinolones should be reserved as alternative drugs [ , , , , ] (bii). however, recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue in japan. respiratory quinolones must be used as the first choice depending on local circumstances about drug resistance [ ] (ciii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [ e , , , ] (bii). ---drugs to be recommended---( ) outpatient treatment a first choices -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day -mino, oral, mg twice a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day table items used to differentiate between bacterial and atypical pneumonia [ ] . . under years of age . no or minor underlying diseases . stubborn cough . poor chest auscultatory findings . no sputum, or no identified aetiological agent by rapid diagnosis . a peripheral white blood cell count below , /ml table criteria for differentiation [ ] . in cases using the items in -tflx, oral, mg/twice a day ( ) hospital treatment -azm, intravenous drip, mg/once a day -mino, intravenous drip, mg/twice a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day. -pzfx, intravenous drip, to mg/twice a day c. cases in which whether the disease is bacterial pneumonia or atypical pneumonia is unclear ( ) outpatient treatment in this case, combination therapy with high-dose penicillin and a macrolide or tetracycline should be selected as the first choice to cover both bacterial and atypical pneumonia [ e , , , , , ] (bii). as respiratory quinolones cover both bacterial and atypical pneumonia, they are convenient, but should be reserved as alternative drugs from the perspective of suppression of resistant bacteria [ e , , , , ] (bii). however, in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [ , , ] (bii). recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue. respiratory quinolones may be used as the first choice depending on local circumstances about drug resistance [ ] (ciii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [ e , , ] (bii). ( ) severer cases requiring treatment in the icu in severer cases requiring treatment in the icu, s. pneumoniae should be initially considered, and a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment primarily to cover latent atypical bacteria (in particular, when l. pneumophila is not covered, the condition may become fatal) [ e , , , ] (aii). in particular, combination therapy with a macrolide is recommended from immunological aspects to suppress excessive inflammation related to cytokines [ ] (cii). as the possibility that causative microorganisms may be enteric bacteria including esbl-producing bacteria cannot be ruled out, carbapenem injection should be used as a first-choice drug in patients with a background factor for which esbl-producing bacteria are frequently detected [ , ] (bii). the sensitivity of a urinary antigen kit to s. pneumoniae and legionella spp. is approximately %. therefore, even when the patient is negative for these bacteria in the initial phase, the possibility of pneumonia related to these bacteria should not be ruled out [ e , , ] (ii). ---drugs to be recommended---( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day * concerning cva/ampc and sbtpc, up to mg of ampc or up to mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral ( / mg), tablet/ times a day þ ampc, oral ( mg), tablet/ times a day þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day -mino, oral, mg/twice a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day -tflx, oral, mg/twice a day ( ) hospital treatment a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day þ one of the followings: -azm, intravenous drip, mg/once a day -mino, intravenous drip, mg/twice a day -cam, oral, mg/twice a day <> second choices -lvfx, intravenous drip, mg/once a day -pzfx, intravenous drip, to mg/twice a day ( ) severer cases requiring treatment in the icu -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -bipm, intravenous drip, . e . g/ e times a day -drpm, intravenous drip, . e g/ times a day þ one of the followings: -azm, intravenous drip, mg/once a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day -mino, intravenous drip, mg/twice a day ---executive summary----when causative microorganisms are identified based on the results of microbial examination of good-quality sputum, blood culture, and urinary antigen (s. pneumoniae, l. pneumophila) tests and drug susceptibility testing of the causative agents, definitive therapy should be performed if possible [ , ] (biii). -the place of treatment and drugs should be selected in accordance with the severity of the disease [ , ] (aii). -antimicrobial drugs should be selected in reference to the susceptibility of isolated bacteria to antimicrobial drugs or a drugsusceptibility tendency in the area [ , , , ] when these data are available (aii). -the administration period of an antimicrobial drug is determined in accordance with the improvement of symptoms and laboratory data, with a target of e days [ , ] (biii). -when the patient is infected with l. pneumophila or c. pneumoniae, the optimal administration period is about days [ ] (biv). a. streptococcus pneumoniae -the clinical and laboratory standards institute (clsi) has established higher criteria for breakpoints for penicillin susceptibility on the administration of parenteral antimicrobial drugs for s. pneumoniae infections other than meningitis [ ] , based on the following findings: patients with severe pneumonia due to s. pneumoniae with a low pcg susceptibility (mic: . e mg/ml) showed no difference in responses to pcg and outcome [ , ] (ii). for the treatment of pneumococcal pneumonia, the dose of penicillin should be increased [ , ] (a). -in japan, most s. pneumoniae isolates are macrolide resistant [ , ] . -respiratory quinolones have potent anti-pneumococcal activities (iii). the clinical effects of such quinolones are similar to those of high-dose ampc [ ] (ii). -in japan, quinolone-resistant s. pneumoniae is detected in e % of the isolates [ ] . as quinolone resistance may be readily induced by point mutations of dna gyrase or topoisomerase genes [ ] , quinolones must be used adequately (aiii). b. haemophilus influenzae -the abpc-resistant mechanism of h. influenzae involves blactamase production and/or pbp mutation. previously, blactamase production was primarily involved, but, recently, pbp mutation-mediated b-lactamase-negative abpc-resistant (blnar) strains have been increasingly detected. abpcresistant strains with both b-lactamase production and pbp mutation are classified as b-lactamase-positive cva/abpcresistant (blpacr) strains. -according to a national survey in japan, ( . %) and ( . %) of h. influenzae strains were blnar and b-lactamase-producing strains, respectively [ ] . -blnar strains are also resistant to firstand secondgeneration cephems. -pipc exhibits an antimicrobial activity against blnar strains. however, it is ineffective for blpacr strains. c. klebsiella spp., escherichia coli, proteus spp. -the proportion of extended spectrum b-lactamase (esbl)producing bacteria has slightly increased among isolates from respiratory samples. -according to a national survey in japan, esbl-producing bacteria account for . e . % of respiratory sample-derived klebsiella spp. strains [ , ] . -most esbl-producing strains are simultaneously resistant to quinolones [ ] . antimicrobials should be selected according to the drug susceptibility of isolated bacteria. -in japan, carbapenemase-producing strains are extremely rare. d. mycoplasma pneumoniae -in the field of pediatrics, the detection rate of macrolideresistant m. pneumoniae has markedly increased. in adults, that of macrolide-resistant m. pneumoniae also increase [ , ] . -tetracyclines exhibit potent clinical effects on macrolideresistant m. pneumoniae [ ] . -respiratory quinolones have good activities against m. pneumoniae [ , ] . e. legionella spp. -it should be noted that pneumonia related to legionella spp. other than l. pneumophila sg cannot be diagnosed using legionella urinary antigen testing. -as neither b-lactams nor aminoglycosides have antimicrobial activities against legionella spp., which proliferates within host cells, they are clinically ineffective. -quinolones, macrolides, and tetracyclines have been confirmed to exhibit clinical effects on legionella spp. previously, em was the first choice for this infection, but many recent studies have showed the clinical efficacies of lvfx and azm [ , ] . -rfp is effective when combined with em. the combination of em and rfp is useful. a study suggested the effects of combination therapy with lvfx and a macrolide [ ] (ciii). -although there are no marked differences in antimicrobial drug susceptibility among legionella spp., clinical reviews to verify this are limited [ ] . f. chlamydophila pneumoniae -only a few studies have supported the clinical effects of antimicrobial drugs against c. pneumoniae pulmonary infections. -tetracyclines, macrolides, and quinolones may be effective. these drugs are recommended primarily based on the results of basic studies [ , ] . g. staphylococcus aureus -with respect to staphylococcus aureus in japan, there has been an increase in the number of methicillin-resistant strains even in patients with community-acquired pneumonia. in particular, recently, municipal-onset-type mrsa (ca-mrsa) with panton-valentine-leucocidine (pvl) has been detected in japan, raising an issue [ ] . -in cases of mssa infection (bacteremia), the clinical effects of cez are superior to those of vcm [ ] . -as the susceptibility of mrsa to oral antimicrobial drugs differs among isolates, drugs should be selected according to its drug susceptibility results. h. streptococcus spp. -among various types of streptococcus, the streptococcus anginosus group is frequently detected, and characterized by strong abscess-forming features [ ] . streptococcus pyogenes and streptococcus agalactiae may also cause pneumonia. the former may lead to serious pulmonary infection [ ] (v). -there is no penicillin resistance, but macrolide resistance is observed at a low frequency [ ] . -the anti-streptococcus activities of quinolones vary. among quinolones, grnx, mflx, and stfx have relatively potent antimicrobial activities [ , ] . i. moraxella catarrhalis -the number of b-lactamaseproducing strains has increased since the 's. currently, most strains produce b-lactamase [ , ] . b-lactamase produced by m. catarrhalis decomposes penicillin. -in japan, macrolide-or quinolone-resistance have not been reported [ ] . j. anaerobes -most anaerobes that cause pneumonia exist in the oral cavity. peptostreptococcus spp., prevotella spp., and fusobacterium spp. are involved. mixed infection with microaerophilic streptococci is often observed. -in many cases, infection with anaerobes may be associated with aspiration. -most oral anaerobes (prevotella spp., fusobacterium spp., and porphyromonas spp.) are susceptible to combination drugs consisting of penicillin and a b-lactamase inhibitor, cldm and mnz [ ] . k. pseudomonas aeruginosa -in patients with chronic respiratory tract infection, pseudomonas aeruginosa colonizes in the airway, and may cause community-acquired pneumonia [ ] . -as the susceptibility of p. aeruginosa to antimicrobial drugs differs among clinical isolates, drugs should be selected according to its drug susceptibility results. ---drugs to be recommended----the drug susceptibility of each clinical isolate should be classified in accordance with the clsi criteria [ ] . -establishment of prescriptions recommended in this article * antimicrobials have been approved for specific diseases and specific causative agents by japanese ministry of health and welfare. the approvals are based on the results of clinical studies with good clinical practice. as a general rule, the recommendations in this section refer to this (aii). however, recent trends in drug susceptibility are also considered. * the recent drug susceptibility results of the nationwide studies in japan were referred [ , ] . * the recommendations without the approvals by japanese ministry are graded by evidence levels. [ -when the susceptibility of identified causative microorganisms is clarified, or after the treatment responsiveness is evaluated, whether or not de-escalation is possible should be reviewed [ e ] (aii). ---explanation---definition: hospital-acquired pneumonia is defined as "pneumonia that newly develops h or more after admission". in many cases, treatment is difficult due to unfavorable patient conditions such as the presence of an underlying disease, immune capacity, and general condition [ e ]. laboratory findings: patients meeting of items, fever, an abnormal leukocyte count, and purulent secretes, in addition to the appearance of an abnormal shadow of the chest should be diagnosed with hospital-acquired pneumonia [ e ]. ) ventilator-associated pneumonia (vap): vap refers to pneumonia that newly develops h or more after endotracheal intubation/ventilator initiation. its onset within e days after endotracheal intubation is classified as early-type, and its subsequent onset as late-type [ , , , ] . ) hospital-acquired pneumonia other than vap: several types of hospital-acquired pneumonia other than vap include ( ) immunodeficiency (for example, neutropenia during anticancer therapy, cell-mediated immunodeficiency related to the administration of steroids or immunosuppressive drugs) and ( ) aspiration pneumonia including latent aspiration (refer to the section " . aspiration pneumonia" on page ). appropriate management and selection of antimicrobial drugs in accordance with individual conditions are necessary [ ] . with respect to microorganisms that are expected, refer to the section " . hospital-acquired pneumonia--- . . empiric therapy: cases in which gram staining is available" (p. ). ---drugs to be recommended--a. cases in which there is no risk of resistant bacteria antimicrobial drugs should be selected, targeting streptococcus pneumoniae, h. influenzae, and klebsiella spp. as causative microorganisms [ e ] (biii). although it is difficult to estimate/identify causative microorganisms using sputum samples, bacteria that are not isolated/cultured from goodquality sputum may not be causative microorganisms. if resistant bacteria such as mrsa and p. aeruginosa are not detected on sputum culture and there is no deterioration of clinical symptoms, an initial drug should be continued [ ] (biii). in patients in whom aspiration episodes are clear, those in whom oral hygiene is not maintained, or those with consciousness disorder, drugs with anti-anaerobe activities should be selected, considering the involvement of anaerobes [ ] (biii). if an adequate antimicrobial drug is administered, the treatment period may be e days, excluding mrsa and p. aeruginosa [ , ] (bii). a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ times a day -ctrx, intravenous drip, g/once a day or g/twice a day * if the involvement of anaerobes is suspected, sbt/abpc should be selected. <> second choice -lvfx, intravenous drip, mg/once a day (as its antimicrobial activity against anaerobes is weak, monotherapy with this drug should be avoided in patients with aspiration pneumonia.). b. cases in which there is a risk of multi-drug-resistant bacteria (table ) [ ] to cover multi-drug-resistant bacteria including p. aeruginosa, broad-spectrum antimicrobial drugs with anti-p. aeruginosa activities should be selected [ e ] (aiii). considering the frequency of esbl in each institution, carbapenems should be considered even when enteric bacteria, including klebsiella spp. and escherichia coli, are suspected (biv). if p. aeruginosa is not isolated on good-quality sputum culture, a treatment option should be de-escalated to drugs for cases in which there is no risk of resistant bacteria [ e ] (aii). if aspiration is suspected, or if the involvement of grampositive bacteria is suggested, combination therapy with cldm must be considered (biv). if there is a risk of mrsa carrier (table ) , combination therapy with anti-mrsa drugs should also be considered. the mean administration period of antimicrobial drugs with respect to causative bacteria in patients with an improvement was approximately days. however, that for resistant bacteria such as p. aeruginosa and mrsa was approximately days [ ] (bii). if appropriate antimicrobial drugs can be administered after clarifying causative bacteria, a treatment period of approximately days is recommended [ , , ] (bii). a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . g/ times a day or g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day <> second choices -cfpm, intravenous drip, e g/ e times a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day if the involvement of anaerobes is suspected, one of the following options should be combined with one of the above regimens: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day c. severe cases one of the following options must be combined with one of the regimens for cases in which there is a risk of multi-drugresistant bacteria. when comparing the results between patients undergoing appropriate and inappropriate treatments, the prognosis of the latter was significantly poorer [ , ] (bii). however, a study reported that the prognosis in a group with compliance with recommended drug selection was significantly poorer than in a non-compliance group in patients in whom infection with drug-resistant bacteria in the icu was suspected even among those in whom the etiology was bacteriologically investigated [ ] (bii). therefore, it must be considered that, even when resistant bacteria are etiologically involved, the administration of an appropriate antimicrobial drug that covers them does not always improve the prognosis. -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . g/ times a day or g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day one of the following options should be combined with one of the above regimens: should be estimated based on the results of the clinical microbiological culture (cmc: gram staining and culture) of a lower airway sample immediately before the start of treatment, and not based on bacteria isolated on active surveillance culture (asc), which was conducted as a strategy to prevent/ control infection prior to onset [ ] . -microorganisms that cause pneumonia or (colonization of) the lower airway should be estimated based on the presence or absence of neutrophils or phagocytosis (excluding those patients with neutropenia or functional impairment of neutrophil) [ ] (bii). ---explanation---[gram staining] diagnostic accuracy of hospital-acquired pneumonia is improved by confirming neutrophils and bacterial cells using gram staining of airway samples. this observation has also been confirmed through an increase in the likelihood ratio of hospital-acquired pneumonia in patients with a clinical pulmonary infection score (cpis) of points or higher [ ] . as bacteria isolated from the lower airways of inpatients are common colonizers in many cases, gram staining is also useful for discerning colonization from infection by evaluating the presence or absence of neutrophil and phagocytosis. therefore, it is desirable to combine bacterial culture with gram staining [ , e ] . antimicrobial-drug selection based on gram staining findings leads to appropriate empiric therapy in two-thirds of patients with hospital-acquired pneumonia, and it can be continued as definitive therapy in many cases [ ] . if there are no bacterial cells on gram staining of lower airway sample in whom an antimicrobial regimen was not changed within the past h, it is unlikely that the focus of infection/inflammation is within the lungs (lower airway) [ ] . in this case, the possibility of pneumonia mimic, such as pleural effusion, atelectasis, and pulmonary edema, is suggested if the lung field opacity still remains in chest x-ray. if there is no other infectious focus, the discontinuation of an antimicrobial drug may be warranted [ , , ] . a study has reported that the culture results of asc performed as a strategy of routine infection control measure prior to the development of nosocomial pneumonia accurately predicted the causative pathogen in only % of cases [ ] . therefore, it is necessary to submit airway samples for clinical microbiological culture (cmc) immediately before the start of presumptive treatment. [causative microorganisms and their origin] microorganisms that cause hospital-acquired pneumonia are derived from the oropharynx, airway (including the nasal cavity and nasal sinus), digestive tract, and environment. gastrointestinal tract-derived causative microorganisms are enteric bacteria (primarily, klebsiella spp., e. coli and others such as proteus spp., enterobacter spp., serratia spp., morganella spp., and citrobacter spp.). those derived from the upper airway include s. pneumoniae, h. influenzae, moraxella catarrhalis, s. aureus (namely methicillin sensitive strain), and oral anaerobes. those derived from the environment include methicillin-sensitive s. aureus, pseudomonas spp., acinetobacter spp., and stenotrophomonas spp. [ , , , ] . as the above bacteria derived from the airway and gastrointestinal tract basically exert strong virulence to the airway, they can be considered a core pathogen group of hospital-acquired pneumonia. their potential for developing airway inflammatory response is generally believed stronger than those caused by environmental pathogen [ , ] . table risk factors for multi-drug-resistant bacteria. . previous use of antimicrobial drugs within days . interval of days or more from admission . admission from an area/hospital in which resistant bacteria are frequent . immunosuppressive state or treatment table risk factors for carrying mrsa [ ] . conditions under which anti-mrsa drug therapy should be considered (including gram stain) mrsa---" (p. ). ( ) diplococcus consisting of a pair of two cocci (gpdc: grampositive diplococci) s. pneumoniae should initially be suspected. enterococcus is also a gpdc in microscopic appearance, but is basically considered a non-pulmonary pathogen [ ] . <> cases in which there have been no previous treatment with antimicrobial drugs or risks of penicillin-resistant pneumococcus -pcg, intravenous drip, , , to , , units/ e times a day -abpc, intravenous drip, g/ e times a day <> cases in which previous treatment with antimicrobial drugs or a risk of prsp is present -ctrx, intravenous drip, g/twice a day or g/once a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day -vcm, intravenous drip, g/twice a day (tdm should be conducted so that a trough is e mg/ml [ ] .) ( ) gram-positive coccus in either short or long chain (gpc in chain) aor b-hemolytic streptococci is indicated. -pcg, intravenous drip, , , to , , units/ e times a day -abpc, intravenous drip, g/ e times a day ( ) gram-positive bacillus with a rod-like morphology (gpr: gram-positive rod) corynebacterium spp. may be indicated. -vcm, intravenous drip, g/twice a day (tdm should be conducted so that a trough is e mg/ml [ ] .) c. gram-negative bacteria ---executive summary----when gram-negative bacteria are observed, h. influenzae, m. catarrhalis, enterobacteriaceae, p. aeruginosa, acinetobacter spp., and stenotrophomonas spp. may be indicated [ e , , ] (bii). -it is difficult to estimate the type of bacteria based on the morphology on gram staining in comparison with grampositive bacteria. -gram-negative bacteria frequently detected as causative microorganisms include enteric bacteria and p. aeruginosa. -it is encouraged important to recognize the basic antimicrobial drug susceptibility pattern of each type (group) of bacteria to make sure that the empiric antimicrobial therapy is appropriate (table ). ---drugs to be recommended--- ( ) cases of early-onset hospital-acquired pneumonia in which there have been no previous administration of antimicrobial drugs or risk of resistant bacteria aero-respiratory pathogen, such as h. influenzae and m. catarrhalis, and enteric bacteria, such as klebsiella spp., are indicated. -sbt/abpc, intravenous drip, g/ e times a day -ctrx, intravenous drip, g/twice a day or g/once a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day ( ) cases of late-onset hospital-acquired pneumonia or ventilator-associated pneumonia in which the risk of resistant bacteria is high an antimicrobial drug with anti-pseudomonal activity that targets non-glucose-fermentative gram-negative rod should be administered [ , , ] -to treat polymicrobial infection, the administration of an antimicrobial drug with an activity against obligate anaerobe is not always necessary [ , ] . -in non-severe cases, the administration of antimicrobial agents with anti-mrsa activity may be withheld in the initial phase even when staphylococcus-like bacterial cells are observed [ ] . ---explanation---when several types of bacteria differing in gram staining and morphology are observed, the condition is commonly interpreted as aspiration pneumonia, suggesting the involvement of anaerobes. however, the number of hospital-acquired pneumonia (including vap) caused by anaerobes have been reported relatively smaller than generally anticipated. [ ] , polymicrobial infection does not always require the prompt antimicrobial therapy that covers anaerobes. even though when aspiration pneumonia is suspected, sbt/abpc is frequently prescribed assuming anaerobic infection, which actually works good on many occasions, it has to be acknowledged that sbt/abpc exert good antimicrobial activity not solely against anaerobes, but also aero-enteric pathogen of pneumonia such as streptococcus pneumonia, oral streptococci, h. influenzae, m. catarrhalis, and klebsiella pneumonia. inpatients are often exposed to gram-negative bacteria residing in the hospital environment. furthermore, there are many opportunities to undergo antimicrobial drug therapy that affects the indigenous microflora. for such reasons, gram-negative bacillus (enteric bacteria or p. aeruginosa) frequently colonize within the oropharyngeal region of the elderly patients or long-term bedbound patients, many of whom need airway suctioning or have tracheostomy that may serve as portal of entry of environmental pathogen. oropharyngeal microflora primarily consisting of these gram-negative bacteria can be aspirated into the airway after surgery requiring sedation or anesthesia, or during or after endoscopic examination [ , , ] . briefly, anaerobes may be an occasional pathoen in polymicrobial infection as seen on gram staining of patients with suspected aspiration pneumonia, but s. pneumoniae, h. influenzae, s. aureus, klebsiella spp., p. aeruginosa, and acinetobacter spp. are more commonly involved in many cases, being similar to the microorganisms that are thought to be the major pathogen of hospital-acquired pneumonia. this is in contrast with the community-onset aspiration pneumonia, represented by lung abscess, in that anaerobes are primarily involved [ , ] . anaerobes involved in hospital-acquired pneumonia include facultative anaerobic a-hemolytic streptococci in the oral cavity and obligate anaerobes. oral obligate anaerobes include grampositive coccus (peptostreptococcus sp.), gram-negative coccus (veillonella sp.), and gram-negative bacillus "oral pigmented" bacteroides (bacteroides melaninogenicus), prevotella sp., porphyromonas sp., and fusobacterium sp.). many of these types of bacteria are susceptible to b-lactams that do not contain a b-lactamase inhibitor, new quinolones, macrolides, and tetracyclines. therefore, patients with hospital-acquired pneumonia may be basically treated by standard empiric therapy for hospital-acquired pneumonia even when aspiration pneumonia related to several types of bacteria is suspected [ ] . ---drugs to be recommended---( ) cases in which it is not necessary to consider the involvement of multi-drug-resistant bacteria, or early hospitalacquired pneumonia the involvement of oral streptococcus, oral anaerobes, s. pneumoniae, h. influenzae, and enteric bacteria should be considered. -sbt/abpc, intravenous drip, g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day ( ) late-onset hospital-acquired pneumonia or cases in which there is a risk of multi-drug-resistant bacteria in addition to the above pathogens, the involvement of non-glucose-fermentative gram negative bacteria or esblproducing enteric bacteria must be considered. -cfpm, intravenous drip, e g/ e times a day -czop, intravenous drip, e g/ e times a day -taz/pipc, intravenous drip, . g/ e times a day table basic susceptibility of various pathogen groups to antimicrobial drugs. ---explanation---if drug susceptibility test is not conducted for some reasons after the identification of causative microorganisms, an antimicrobial drug should be selected with reference to the susceptibility pattern (local sensitivity) of the identified bacteria at each institution. if the local sensitivity is not obtained, a drug should be selected based on the basic susceptibility of various pathogens to antimicrobial drugs (table ) [ ] . in the treatment of hospital-acquired pneumonia, the duration of antimicrobial therapy generally tends to be longer than required for the following reasons: opacity on chest x-ray often remains for reasons other than pneumonia even after the start of antimicrobial drug treatment; and there may be a large number of latent nonpneumonia (or non-infectious-disease) factors that may cause increase in body temperature or crp level in inpatients [ ] . however, if appropriate antimicrobial drug treatment is performed, it is possible to complete treatment in week [ ] . in strains such as enterobacter spp., serratia spp., citrobacter spp., and morganella spp. (table gnr b ), the expression of intrinsic antimicrobial-drugresistance genes encoded in chromosome genes is induced during antimicrobial drug treatment, a phenomenon which is basically rarely seen in e. coli, klebsiella spp., h. influenzae, and m. catarrhalis (table gnr a ) ( table ) [ , , ] . therefore, if adequately chosen treatment parameters are improved, antimicrobial treatment could be completed with careful follow up of patients' condition. although it is useful to recognize these pathogens, abbreviated as space (serratia, pseudomonas, acinetobacter, citrobacter, and enterobacter), as a representative microorganism group that causes hospital-acquired pneumonia, the space group is essentially a common colonizer. therefore it is important to bear in mind that antimicrobial drug is not always indicated upon the isolation of space to avoid selection of antimicrobial resistant bacteria related to unnecessary or long-term antimicrobial therapy [ , ] . lzd should be selected [ , ] (ai). the therapeutic efficacy of lzd is similar to those of glycopeptides [ , ] . the penetration of lzd into the alveolar epitheliumlining fluid and intra-alveolar sputum is more favorable. therefore, use of lzd should be encouraged in cases of restricted sputum expectoration, such as vap [ ] (bii). exclusive use of a single drug may accelerate the acquisition of resistance to the agent [ e ] (ci). as dap is inactivated by pulmonary surfactants, its use should be avoided for mrsa pneumonia. -glycopeptides should be selected as first-line drug for pneumonia caused by corynebacterium sp [ ] (aii). ---explanation---there is no significant difference in the therapeutic efficacy for mrsa pneumonia between glycopeptides and lzd. several studies reported that the overall clinical efficacy of lzd, including the incidence of side effects, was superior to vcm in patients with hospital-acquired pneumonia caused by mrsa [ , ] . however, since the dosing of vcm in these studies have been considered suboptimal, further study is needed [ , ] . some investigators have recommended that, when mrsa is susceptible to cldm or mino on a susceptibility test, lzd, a protein synthesis inhibitor should be administered given the possible involvement of the panton-valentine leukocidin [ , ] . if a prompt improvement is achieved by the intravenous drip of lzd mg q h, or if the patient's condition is not critical, switch from the intravenous administration to an oral preparation of lzd, which shows high bioavailability [ ] , is encouraged. as dap is inactivated by pulmonary surfactants, it should not be used to treat mrsa pneumonia. this may not apply to the treatment of septic pulmonary embolism [ ] . ---drugs to be recommended---( ) mrsa a first choices -vcm, intravenous drip, g/twice a day -teic, intravenous drip, mg for the first days/ twice a day for loading, mg/once a day from day * tdm should be conducted so that the trough levels of vcm and teic range from to mg/ml [ ] . -lzd, intravenous drip or oral administration, mg/ twice a day <> second choices -abk, intravenous drip, mg/once a day (a trough level was established as mg/ml using tdm.) -st combination drug (smx at mg/tmp at mg), oral administration, tablets/twice a day or intravenous drip, mg/twice a day -cldm, intravenous drip, mg/ e times a day (the results of drug susceptibility testing must be confirmed). [non-extended-spectrum b-lactamase (esbl)-producing bacteria] ( ) hospital treatment " (p. ). ( ) e. coli, klebsiella spp., proteus mirabilis (esbl-producing bacteria) refer to the section " . community-acquired pneumonia--- . . definitive therapy--- [ ] klebsiella spp. [esbl-producing bacteria] ( ) hospital treatment" (p. ). ( ) enterobacter spp., serratia spp., citrobacter spp., morganella spp., proteus vulgaris a third-generation cephems or quinolones should be administered [ , , ] (aii). -ctrx, intravenous drip, g/once a day or g/twice a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, mg/twice a day <> if a strain is estimated to constantly express cephalosporinase (highly resistant to b-lactamase inhibitorcontaining b-lactams, oxyimino [¼ rd generation] cephalosporin and cephamycin, through plasmid genes) on an antimicrobial drug susceptibility test, fourthgeneration cephems or carbapenems should be administered. -cfpm, intravenous drip, e g/ times a day -czop, intravenous drip, e g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day ( ) p. aeruginosa anti-pseudomonal penicillins, third-generation or later cephems, carbapenems, or new quinolones should be administered [ , ] refer to the section " . community-acquired pneumonia--- . . definitive therapy--- [ ] h. influenza (abpcsusceptible), [ ] h. influenza (b-lactamase-producing), [ ] h. influenza (b-lactamase-negative ampicillin-resistant (blnar), and [ ] h. influenza (b-lactamase-positive amoxicillin clavulanate-resistant (blpacr)" (p. e ). ---explanation---according to some investigators, enteric bacteria are classified into types: sensitive, gram-negative rods, such as e. coli, klebsiella pneumoniae, and p. mirabilis, which are susceptible to first-generation cephalosporin, and resistant, gram-negative rods, such as enterobacter spp., serratia spp., and citrobacter spp., which show an intrinsic or inducible resistance to third-generation cephalosporin through chromosomal ampc genes [ , , ] . in addition, the number of extended spectrum of b-lactamase (esbl)-producing strains of e. coli, klebsiella, and proteus sp. that are resistant to all cephalosporin has increased. among resistant gnrs, such as enterobacter spp., strains that constantly produce ampctype b-lactamase (cephalosporinase) (plasmid type) must also be considered [ , ] . concerning non-fermentative bacteria, their intrinsic susceptibility to antimicrobial agents differs among p. aeruginosa, stenotrophomonas spp., and acinetobacter spp. a study indicated that, in patients with p. aeruginosa pneumonia, monotherapy with a new quinolone might show unfavorable bacteria-eradicating effects or lead to recrudescence [ ] . in some patients, combination therapy with a b-lactam (pipc, caz, cfpm, or carbapenems), which has an anti-pseudomonal activity, and aminoglycoside or new quinolone may be considered [ , , ] . most strains of stenotrophomonas spp. are susceptible to mino or an st combination drug. m. catarrhalis and acinetobacter spp. are the frequent types of gram-negative coccus detected in patients with early and late hospital-acquired pneumonia, respectively. many strains of the former produce b-lactamase. the latter is a gnr existing in the hospital environment, and may be resistant to many antimicrobial drugs. however, in japan, the multi-drug resistance of this type of bacteria has not widely distributed. carbapenems and new quinolones should be selected. however, the vast majority of acinetobaccter strains are susceptible to sbt/abpc. in particular, sbt has an antimicrobial activity against this type of bacteria, and their susceptibility to sbt/abpc should routinely be confirmed. primary test drugs for an antimicrobial susceptibility of this type of bacteria (drugs appropriate for a routine examination panel) are sbt/abpc, caz, ipm/cs, mepm, gm, tob, lvfx, and cpfx [ ] . pan-sensitive strains of h. influenzae are b-lactamase (bl)-negative, abpc-sensitive (blnas) strains. however, there are various resistance patterns: bl-producing, abpcresistant (blpar), bl-negative, abpc-resistant (blnar), and bl-producing, ampc/cva-resistant (blpacr) strains. blnas strains can be treated with abpc, but sbt/abpc therapy is required to control blpar strains. the administration of ctrx or new quinolones is necessary for blnar or blpacr. a randomized-controlled trial with multivariate analysis has shown that factors for favorable bacteriological effects included the absence of p. aeruginosa-related pneumonia (< . ), a higher body weight (< . ), a low apache ii score (severity) ( . ), and cpfx therapy ( . ) [ ] . conditions suggesting the use of new quinolones include allergy to b-lactams, the presence of or concern for nephropathy (an aminoglycoside cannot be combined with a b-lactam), necessity of covering obligate intracellular pathogen, or situations in which switching to an oral preparation is indicated [ ] . an in vitro study indicated that the alveolar epithelial lining fluid (elf) concentration of lvfx reached as high as its serum concentration. furthermore, a prospective open-label study reported that switching of intravenous drip to oral administration decreased the elf concentration, but it was within the range at which many pathogens are deemed sensitive based on the cumulative data of minimum inhibitory concentrations for causative microorganisms [ ] . ---executive summary----nursing and healthcare-associated pneumonia (nhcap) is a category independently defined in japan based on medical circumstances. -the attending physician proposes a treatment category (groups a to d) by evaluating what treatment is necessary as the most important item based on the patient's and his/her family's will ( fig. ) [ ] . -risk factors for resistant bacteria are categorized into two items, and initial treatment options are recommended, assuming target causative microorganisms (civ). -in patients in whom the general condition is unfavorable due to complications or in terminal-stage patients, initial treatment options are recommended considering side effects from the perspective of innocent properties (civ). -in group d, in which intensive care is required, combination therapy with broad-spectrum (involving resistant bacteria and legionella) and potent antimicrobial drugs is recommended (bi). ---explanation--- in , the japanese respiratory society issued the "guidelines for the management of nursing and healthcare-associated pneumonia (nhcap)" [ ] , considering medical circumstances in japan with reference to the entity of healthcare-associated pneumonia (hcap) proposed in the untied states [ ] . the definition of nhcap is shown in table . as this committee has no objection to the entity itself, the selection of drugs will be explained based on evidence to avoid duplications with the above guidelines. the mortality rate and frequency of resistant bacteria in patients with nhcap are intermediate between community-acquired pneumonia (cap) and hospital-acquired pneumonia (hap). however, nhcap may be primarily regarded as being similar to geriatric pneumonia [ , ] . there is no fact that the rate at which resistant bacteria are isolated increases with severity [ ] . even when pneumonia is not severe, the host's activities of daily living (adl) and an underlying disease/immunodeficiency reduce the prognosis in many cases [ ] . as this type of pneumonia develops in a variety of uneven populations, it is difficult to simply determine severity classification. therefore, considering various conditions, the entity of "treatment category", involving the ethical aspects of geriatric care, was introduced based on evaluation by the attending physician who knows the patient well ( fig. ). frequent basic conditions or concomitant diseases for nhcap in japan include an advanced age, central nervous diseases, aspiration, a reduction in adl, and tubal feeding. their factors are aspiration pneumonia itself or risk factors, and hcap in japan may overlap with aspiration pneumonia [ ] . on the other hand, in nhcap patients, mrsa, p. aeruginosa, and anaerobes are more frequently isolated in comparison with cap patients. it is necessary to switch therapeutic strategies, considering these causative microorganisms. refer to the next section " . aspiration pneumonia" (p. ). [type and frequency of causative microorganisms] concerning causative microorganisms in nhcap patients, resistant bacteria are frequently detected, differing from cap patients. however, with respect to microorganisms that cause hcap, the distribution and frequency of streptococcus pneumoniae and h. influenzae, which are frequently isolated in cap patients, as well as mrsa, p. aeruginosa, and gram-negative bacillus, which are frequently detected in hap patients, differ among countries, areas, and institutions due to their variety (iii). concerning causative microorganisms, a study reported that there was no marked difference between nhcap and cap [ ] . on the other hand, a study in the united states indicated that s. aureus was frequently detected [ ] , and another study in italy, where the rapid aging of society is advanced, as described for japan, reported that aspiration pneumonia, h. influenzae, s. aureus, and gram-negative bacillus were more frequent than in patients with cap [ ] . as a result, the rate of resistant bacteria increased, and inappropriate antimicrobial drugs were selected in a high proportion of patients. in addition, the mortality rate was higher than in cap patients, suggesting the association between the two factors. representative causative microorganisms with respect to the presence or absence of risk factors for resistant bacteria are presented in table [ ] . of these, resistant bacteria, which are not targeted in cap patients, were isolated in approximately %. however, the value was lower than in hap patients. this is a current status of japan (iii). however, we must consider that patients in whom isolated bacteria are unclear account for approximately %, with the involvement of aspiration as a background factor [ ] . in addition to bacteria commonly isolated in cap patients, the frequency of enteric bacteria and anaerobes has increased [ ] . [rules of antimicrobial drug therapy] risk factors for resistant bacteria in nhcap patients include "the previous use of antimicrobial drugs for days or more within days" and "tubal feeding" ( [ ] . risk factors for involvement by drug-resistant pathogens. -if no antibiotic therapy in the preceding days or current tube feeding, the patient can be assumed to have no risk of involvement by drug-resistant pathogens. -however, if past medical history indicates isolation of mrsa, the patient should be assumed to have risk of involvement by mrsa. * ) inappropriate when aspiration pneumonia is suspected, because it has insufficient activity against anaerobic bacteria. * ) because of insufficient activity against anaerobic bacteria, when used to treat suspected aspiration pneumonia, it should be used in combination with an antibiotic that has activity against anaerobic bacteria (e.g., mnz, cldm, sbt/abpc). definition of nhcap [ ] . . pneumonia diagnosed in a resident of an extended care facility or nursing home . pneumonia diagnosed in a person who has been discharged from a hospital within the preceding days . pneumonia diagnosed in an elderly or disable d person who is receiving nursing care . pneumonia diagnosed in a person who is receiving regular endovascular treatment as an outpatient (dialysis, antibiotic therapy, chemotherapy, immunosuppressant therapy) standards for nursing care patients whose performance statues is ps (capable of only limited self-care, confined to bed or a chair more than % of their waking hours) or more. item incudes patients on psychiatric wards. not isolated in those who had made favorable daily life activities without a history of antimicrobial drug therapy [ ] . another study indicated that tubal feeding was an independent risk factor for infection with p. aeruginosa (odds ratio: . ) [ ] (ii). this is the reason why treatment category c was established in the guidelines. briefly, patients who do not meet the above two items are regarded as having no risk factor for resistant bacteria, and assigned to group b. patients meeting or items or those in whom mrsa was previously isolated are assigned to group c. respective drugs to be recommended were separately established. patients in whom outpatient treatment is considered to be appropriate are assigned to group a, and those in whom the attending physician considers ventilator or icu management necessary to group d. drugs to be recommended were added, and a treatment category algorithm ( fig. ) [ ] was prepared. concerning hcap treatment in europe and the united states, there is a gap between drugs used in clinical practice and those recommended in guidelines [ ] (ii). therefore, treatment category-based empiric therapy in japan may be acceptable in clinical practice; future investigation is necessary. there is no evidence regarding the administration period of antimicrobial drugs. an administration period of e days, which is routinely adopted in the highest percentage of patients, is appropriate (biv). when administering antimicrobial drugs for a longer period, equivalent-spectrum antimicrobial drugs should be selected, or de-escalation of antimicrobial drugs should be performed. in this case, fever, crp, and leukocyte counts are often used as indices of the treatment response. in cases of aspiration pneumonia in which aspiration recurs during treatment despite the efficacy of antimicrobial drugs, it is necessary to evaluate whether the effects of antimicrobial drugs are not obtained or recurrence occurs. ---drugs to be recommended--a. empiric therapy ( fig. ) [ ] ( ) cases in which there is no risk of resistant bacteria and outpatient treatment is performed (group a) according to a study, chlamydophila spp. and m. pneumoniae accounted for . and . % of patients in whom the type of microorganisms that cause nhcap was clarified in japan, respectively [ ] . the results suggested that chlamydophila spp. is a target of treatment, as described for cap. therefore, in group a, combination therapy with a b-lactam and macrolide or monotherapy with a respiratory quinolone should be performed (bii). in group d, an anti-p. aeruginosa drug should be combined with cpfx, pzfx, or azm for injection, considering legionella or chlamydophila spp. pneumonia. however, concerning combination therapy with a macrolide (cii) in patients without "severe pneumonia requiring intensive care", as described below, the evidence level is not always high from the perspectives of medical economics, side effects, and resistant bacteria [ ] . some studies examined the mortality rate with respect to the presence or absence of treatment covering atypical pathogens, and reported that the mortality rate was significantly lower in the presence of such treatment [ ] . a recent meta-analysis also showed a difference [ ] . respiratory quinolones were established as an option (bii) based on many references describing that their effects are similar to or more potent than those of combination therapy with a b-lactam and macrolide. however, this must be further examined, considering factors such as severity and the presence or absence of concomitant sepsis [ ] . furthermore, the prevalence of penicillinresistant pneumococcus, which has been internationally emphasized as an issue, and macrolide-resistant pneumococcus, which has been markedly observed in japan, was also a background factor for establishing respiratory quinolones as an option [ ] . the previous use of antimicrobial drugs, which is often observed in patients with nhcap, is considered to be a risk factor for resistant pneumococcus [ ] . a study reported that penicillin or em resistance in hcap patients was more advanced than in cap patients [ ] . a study indicated that the efficacy of oral therapy with lvfx was similar to that of ctrx injection therapy in patients with cap [ ] . another study reported that, among patients with non-severe hcap, oral lvfx was useful in those with no description of causative microorganisms [ ] . however, when aspiration pneumonia is suspected, grnx or mflx should be selected, because the effects of lvfx on anaerobes are weak. furthermore, several studies suggested the usefulness of mflx, which is not influenced by the kidney function and does not require dose regulation, in elderly patients with nhcap [ , ] . as treatment is completed with a single, high dose, compliance is favorable. azm sustained-release preparations [ e ], which simultaneously cover bacteria and atypical pathogens, and stfx, which shows a favorable mic for anaerobes, may also be recommended [ ] . a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day table risk factors for involvement by drug-resistant pathogens a in nhcap [ ] . history of antibiotic therapy for or more days in the preceding days current tube feeding the risk of mrsa should be taken into account whenever there is past history of mrsa isolation. when attempting to predict the isolation of drug-resistant pathogens based on the presence of these risk factors, it should be borne in mind that their sensitivity and negative predictive value are high, but their specificity and positive predictive value are low. a drug-resistant pathogens include pseudomonas aeruginosa, mrsa, acinetobacter, esbl-producing enteric bacteria, and stenotrophomonas maltophilia. table possible pathogens isolated from nhcap patients [ ] . when an nhcap patient has no risk factors for involvement by drug-resistant pathogens pneumococcus mssa gram-negative enteric bacteria (including klebsiella and e. coli) haemophilus influenzae oral streptococci atypical pathogens (particularly chlamydophila) when an nhcap patient has a risk factor for involvement by drug-resistant pathogens (the following will be considered in addition to the above-mentioned pathogens) pseudomonas aeruginosa mrsa acinetobacter esbl-producing enteric bacteria þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day <> second choices -mflx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/l to times a day or -ctrx * , intravenous drip, g/once a day or g/twice a day -ctx * , intravenous drip, e g/ e times a day þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. ( ) cases in which there is no risk of resistant bacteria and hospital treatment is performed (group b) in this category, we recommend monotherapy from the perspectives of causative microorganisms resembling those for cap and side effect-based "innocent properties". a study reported that initial treatment with narrow-spectrum antimicrobial drugs did not always lead to a poor prognosis [ ] . in particular, according to two articles [ , ] , it is not necessary to consider resistant bacteria in hcap patients in whom causative microorganisms are unclear; treatment in accordance with cap treatment is sufficient (bii). however, an advanced age, central nervous diseases, aspiration, and a reduction in adl are the clinical characteristics of aspiration pneumonia. the condition of nhcap in japan overlaps with aspiration pneumonia [ ] . therefore, in group b, to which patients admitted for the first time, with no recent use of antimicrobial drugs, correspond, antimicrobial therapy with b-lactamase inhibitor-containing penicillins is appropriate, as described for cap. however, when aspiration pneumonia is suspected, ctrx and lvfx should be avoided (biv). for the management of enteric bacteria, candidate drugs for group b, papm/bp, may be selected. actually, it is not necessary to consider p. aeruginosa in patients with cap or non-icu hap. a study indicated that ertapenem, which is not effective for p. aeruginosa, was useful [ ] , as demonstrated for papm/bp. however, another study reported that the widespread use of ertapenem induced the cross resistance of p. aeruginosa to other carbapenems; the use of papm/bp alone should be avoided [ ] (biv). in elderly persons, with a high risk of aspiration, who are repeatedly admitted and discharged, klebsiella is often involved. taz/pipc is more useful according to a study [ ] (bii). in patients in whom gram-negative bacillus is detected on gram staining of sputum or those in whom the involvement of enteric bacteria is suspected, papm/bp or taz/pipc should be selected (biv). -sbt/abpc, intravenous drip, g/ e times a day -ctrx * , intravenous drip, g/once a day or g/twice a day -ctx * , intravenous drip, e g/ e times a day -lvfx * , intravenous drip, mg/once a day -papm/bp, intravenous drip, . e g/ e times a day * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. ( ) cases in which there is a risk of resistant bacteria and hospital treatment is performed (group c) the target microorganisms include p. aeruginosa, mrsa, and acinetobacter spp. in addition to frequent microorganisms that cause respiratory infection [ , , , ] . as antimicrobial drugs, taz/pipc, with an antimicrobial activity against p. aeruginosa, fourth-generation cephems, carbapenems, and quinolones (cpfx, pzfx) are recommended. taz/ pipc exhibits effects similar to those of ipm/cs and mepm in patients with nursing and healthcare-associated pneumonia [ ] (bii). pzfx also has an antimicrobial activity against s. pneumoniae when used at a high dose ( g/day). when pneumonia related to atypical pathogens such as chlamydophila spp. is suggested, quinolones should be selected. as the antimicrobial activities of fourth-generation cephems and quinolones against anaerobes are weak, these drugs should be combined with mnz, cldm, or sbt/abpc. recently, the resistance of the bacteroides fragilis group to cldm has advanced [ ] . therefore, in europe and the united states, mnz is selected as a first-choice antimicrobial drug against anaerobes. however, the rate at which the b. fragilis group is involved in oral anaerobes is low, and combination therapy with cldm may be selected [ , ] . therefore, for combination therapy with fourth-generation cephems, we recommend the two drugs. when there is a risk of mrsa, such as previous admission, they should be combined with vcm, teic, or lzd. if there is no abscess formation, abk is also effective. a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day <> second choices -cfpm* , intravenous drip, e g/ e times a day -cpr * , intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day or -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day *in addition to the above drugs, if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. ( ) severe cases requiring intensive care (group d) to cover l. pneumophila and atypical pathogens, which are rare as causative microorganisms but may cause severe conditions, group-c antimicrobial drugs should be combined with cpfx, pzfx, or azm injection (bi). concerning the usefulness of combination therapy with a b-lactam and macrolide injection for severe pneumonia, evidence has been accumulated [ ] . a study indicated that, in severe community-acquired pneumonia patients with sepsis or requiring icu management, combination therapy with a blactam and macrolide led to a more favorable prognosis compared to that with a quinolone (i), suggesting that antiinflammatory actions are involved in the mechanism [ ] . in addition, another study reported that, among pneumonia patients with acute pulmonary disorder, both the ventilator withdrawal and survival rates in a macrolide-treated group were higher than in a non-macrolide-treated group [ ] (i). several meta-analyses also support them [ , ] . a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day þ one of the followings: -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day -azm, intravenous drip, mg/once a day <> second choices -cfpm * , intravenous drip, e g/ e times a day -cpr * , intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day þ one of the followings: -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day -azm, intravenous drip, mg/once a day in addition to the above drugs, *if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. antimicrobial drugs against identified causative microorganisms should be selected in accordance with the section " . hospital-acquired pneumonia" (p. ). ---executive summary----as oral indigenous bacteria, including anaerobes, cause aspiration pneumonia, b-lactamase inhibitor-containing penicillins are appropriate (bii). -in cases of nosocomial onset, it is necessary to cover gramnegative bacillus, including p. aeruginosa. -in cases of severe ventilator-associated pneumonia (vap), the selection of broad-spectrum antimicrobial drugs or combination therapy with them should not be hesitated (ai). -the detection rate of esbl-producing gram-negative bacillus has increased, and antimicrobial drugs should be carefully selected. -it is important to prevent subclinical aspiration through oral care and the prevention of gastroesophageal reflux, such as head lifting (bii). -to prevent aspiration pneumonia, it is also important to improve the nutritional status and avoid the overuse of sleeping pills/ sedatives (bii). ---explanation---[characteristic and classification of diseases] aspiration pneumonia occurs with a background factor, dysphagia, which is frequently observed in the presence of a reduction in adl or systemic functions, especially cerebrovascular disorder. its onset is associated with dietary ingestion in elderly persons [ ] . currently, aspiration pneumonia is accurately defined only in the guidelines for the management of hospitalacquired pneumonia (hap) in adults, which were prepared by the japanese respiratory society [ ] . the guidelines present conditions that may cause dysphagia, which were proposed by the japanese study group on aspiration pulmonary disease (table , modified) [ ] . in our guidelines, we primarily explain antimicrobial drugs to be selected for patients with such conditions. the above definition is also adopted in the guidelines for the management of nursing and healthcare-associated pneumonia (nhcap) [ ] . in elderly persons admitted to long-term care beds or nursing homes, risk factors include dysphagia and tubal feeding according to international data on pneumonia that develops in nursing homes [ e ]. in japan, frequent underlying diseases in patients with nhcap also include central nervous diseases and dementia, which are closely associated with aspiration. the proportion of patients after percutaneous endoscopic gastrostomy (peg) is high [ ] . however, among various types of community-acquired pneumonia, a diagnosis of aspiration pneumonia is made based on onset factor-based classification, and is not equal to nhcap diagnosed primarily based on the place of onset or grade of nursing. according to data in spain, aspiration pneumonia accounts for . % of patients with healthcareassociated pneumonia (hcap) requiring admission. this percentage was markedly higher than in those with communityacquired pneumonia (cap) requiring admission ( . %), but corresponded to no more than / [ ] . on the other hand, a multicenter cooperative study involving inpatients with pneumonia in japan, where the rapid aging of society is advanced, reported that . % of patients who were admitted with cap had aspiration pneumonia. even in patients with cap, which is not classified as nhcap, the involvement of aspiration cannot be ignored [ ] . furthermore, the study indicated the involvement of aspiration in . % of patients, aged over years, with cap/ hap [ ] . in the future, the significance of distinguishing aspiration pneumonia among patients with nhcap or hap and changing therapeutic strategies should be examined. however, nhcap more markedly affects adl compared to cap, and the aspect of elderly pneumonia is emphasized; it may be significant to positively diagnose aspiration pneumonia and establish therapeutic strategies different from those for cap [ ] . concerning hap, a reduction in the immune function is a background factor. hap has two aspects: pneumonia with a high risk of resistant bacteria and that in which central nervous diseaserelated aspiration is involved. in the guidelines for the management of hospital-acquired pneumonia in adults, which were prepared by the japanese respiratory society, mendelson syndrome and vap are categorized as a group, and classifications, involving diffuse deglutition-related bronchiolitis, in which there are no findings of pneumonia, are proposed. in addition, a flow chart for diagnosis is presented [ ] (fig. ) . with respect to the condition and treatment of vap, refer to a review described by chastre et al. [ ] management other than antimicrobial drug therapy should also be considered, and bundle (table )-based prevention should be performed [ ] (aii). [type and frequency of causative microorganisms] streptococcus pneumoniae, s. aureus, and enterobacteriacae have been reported. a study indicated that k. pneumoniae was frequent [ ] . the involvement of oral indigenous bacteria, such as streptococcus anginosus group. and anaerobes, has been suggested [ , ] . in cases of nosocomial onset, gram-negative bacillus, including p. aeruginosa, must also be considered. concerning e. coli, klebsiella spp., and proteus spp., the number of esbl-producing strains may increase in the future. [rules of antimicrobial drug therapy] if appropriate antimicrobial drug therapy is not selected under a diagnosis of aspiration-related pneumonia, insufficient treatment may lead to a fatal condition, or excessive treatment may increase the number of resistant bacteria, showing negative effects. there may be differences in options for empiric therapy between patients with vap (most patients show severe conditions) and those with diffuse deglutition-related bronchiolitis, in whom the start of treatment is not accelerated. on the other hand, approaches to prevent pneumonia after aspiration or avoid aspiration are important. oral care, head lifting, and improvement in the nutritional status must be considered, and the overuse of sleeping pills/ sedatives should be avoided (bii). the best option for standard-type aspiration pneumonia is an antimicrobial drug that exists an antimicrobial activity against both aerobes and anaerobes. sbt/abpc and taz/pipc are effective for anaerobes frequently isolated in the respiratory system, such as fusobacterium spp., prevotella spp., and peptostreptococcus spp. [ , ] . as the resistance rates of these types of bacteria to the two regimens are low, these regimens are also recommended as first choices in the guidelines established by the japanese association for anaerobic infection research [ ] . however, a study reported that the previous administration of antimicrobial drugs and adl were correlated with the frequency of enterobacteriacae-or p. aeruginosa-related pneumonia [ ] . a retrospective study involving patients with aspiration pneumonia showed that the frequency of k. pneumoniae-related pneumonia was % [ ] . based on these studies, drugs to be selected should be changed in accordance with the previous administration of antimicrobial chemotherapeutic drugs in patients admitted to general or medical wards. among patients with hospital-acquired pneumonia, broad-spectrum drugs should be selected in those with severe aspiration pneumonia or vap (bii). when causative microorganisms are identified and an improvement in the condition is achieved, de-escalation should be performed. ---drugs to be recommended--a. empiric therapy ➀ no risk of resistant bacteria drugs with potent antimicrobial activities against oral anaerobes are presented. however, no article has provided high-level evidence regarding aspiration pneumonia. as the following drugs affect the intestinal flora, antimicrobial drugassociated diarrhea may occur. if symptom improvement is delayed, patients must be promptly admitted, and drip table conditions that may cause dysphagia [ ] [ ] . bundles for the prevention of ventilator-associated pneumonia. ( ) upper body lifting the head should be lifted at e . ( ) discontinuation of sedatives a sedative should be discontinued once a day to evaluate whether or not extubation is possible. ( in addition to the above items, methods to prevent aspiration pneumonia include oral care, the administration of drugs that improve the deglutition function, such as ace inhibitors and cilostazol, improvement in the nutritional status, eating/swallowing rehabilitation, and anti-pneumococcus vaccination. infusion therapy should be performed (outpatient treatment should not be prolonged). ( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day <> second choices -mflx, oral, mg/once a day -stfx, oral, mg/ e times a day -grnx, oral, mg/once a day ( ) hospital treatment when a diagnosis of aspiration pneumonia is made, sbt/abpc is most frequently used in japan [ ] . kaneko et al. reported that the sensitivity of oral anaerobes that may cause aspiration pneumonia, such as peptostreptococcus spp., prevotella spp., and fusobacterium spp., to sbt/abpc was %, similar to that to taz/pipc [ ] . the effects of cldm on aspiration pneumonia or a pulmonary abscess are similar to those of sbt/abpc (bi) [ ] . sbt/abpc and cldm showed similar effects and tolerance on aspiration pneumonia ( . and . %, respectively) [ ] . oral anaerobes, excluding bacteroides spp., are still susceptible to cldm. a randomized clinical trial (rct) indicated that cldm was more potent than cephems [ ] . a first choice -sbt/abpc, intravenous drip, g/ e times a day <> second choice -cldm, intravenous drip, mg/ e times a day ➁ cases in which there is a risk of resistant bacteria or severe cases in cases in which there is a risk of resistant bacteria or severe cases, drugs should be selected in accordance with options for group c for nhcap. tubal feeding is a risk factor for aspiration, and is also a risk factor for resistant bacteria [ ] . when the involvement of enterobacteriacae, such as k. pneumoniae and e. coli, is suggested, empiric therapy should be selected in accordance with cases in which there is a risk of resistant bacteria [ ] . in japan, the proportion of esbl-producing bacteria on sputum culture in patients with respiratory infectious diseases is % or less [ ] , but the number of esbl-producing bacteria has slightly increased; this must be considered in the future [ , ] . a study reported that the clinical effects of taz/pipc on non-esblproducing k. pneumoniae were more potent than those of sbt/abpc; caution is needed [ ] . it must be considered that, in cases of aspiration pneumonia classified as hcap, enterobacteriacae is isolated at a frequency that cannot be ignored. concerning aspiration pneumonia that occurs in hospitals, some reviews proposed that antimicrobial drugs should be selected, regarding the condition as hospitalacquired pneumonia; empiric therapy may be selected in accordance with the guidelines for the management of hospital-acquired pneumonia, which were published by the japanese respiratory society [ , ] . according to a study, bipm, which does not cause kidney dysfunction in elderly patients, is also effective (civ); therefore, it is presented as an option for cases in which there is a risk of resistant bacteria [ ] . the mortality rate in patients with vap is high. if causative microorganisms cannot be initially covered, the mortality rate may increase [ ] . therefore, drugs should be selected, regarding the condition as severe aspiration pneumonia. three studies reported that, in a group in which taz/pipc was selected as a drug to be combined with aminoglycosides for vap treatment, the mortality rate was lower than in a group in which caz was selected [ e ]. in particular, when pneumonia was caused by p. aeruginosa, the clinical effects of taz/pipc were more potent than those of ipm/cs (bii). for monotherapy, information on the culture of protected specimen brush (psb) samples or broncho-alveolar lavage (bal) fluid is strongly recommended [ , ] . on the other hand, an observational study indicated that threedrug therapy (two anti-p. aeruginosa drugs þ an anti-mrsa drug) deteriorated the prognosis; an rct should be conducted in the future [ ] . therefore, if there is a risk of resistant bacteria, at least broad-spectrum antimicrobial drugs must be used for empiric therapy. however, assuming causative microorganisms with reference to gram staining reactions, minimum necessary antimicrobial drugs should be selected based on local factors (antimicrobial drug susceptibility pattern of each type of bacteria in each hospital). recently, the entity of ventilator-associated tracheobronchitis (vat) was proposed, and the disadvantages of aggressive treatment have been discussed [ ] . in a multicenter cooperative study, patients with vat, which occurred in the icu, were divided into two groups with and without antimicrobial drug therapy, and the results were compared. in the former, the incidence of vap was significantly lower, and the mechanical ventilation-free period was significantly longer. in addition, the icu mortality rate was significantly lower. on the other hand, there was no significant difference in the appearance of resistant bacteria between the two groups [ ] . a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day <> second choices -cfpm, intravenous drip, e g/ e times a day -cpr, intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day or -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day -sbt/abpc, intravenous drip, . e g/ e times a day *if mrsa infection is suspected, antimicrobial drugs should be administered in accordance with the section "mrsa pneumonia" in addition to the above drugs. [administration period of antimicrobial drugs] it is recommended that the treatment period of hospitalacquired pneumonia should be e days. however, the treatment period should be days in patients with pneumonia related to non-glucose-fermenting bacteria such as p. aeruginosa [ ] (bii). concerning vap, a study reported that there was no difference in the clinical effects between and days [ ] . to control identified causative microorganisms, antimicrobial drugs should be selected in accordance with the section "hospital-acquired pneumonia". if mrsa infection is suspected, antimicrobial drugs should be selected in accordance with the section "mrsa pneumonia". (bii). -in severe or refractory cases in which the efficacy of initial treatment is not sufficient, combination therapy with an antifungal drug should also be considered [ e ] (bii). -as the effects of combination therapy with an azole and amph-b preparation antagonize in some strains, a combination of these drugs should be avoided [ ] (aiii). -for the target treatment of this disease, an antifungal drug of which the class is different from that of a drug used for preventive administration should be used (biii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in severe immunocompromised hosts such as patients received chemotherapy for leukemia or hematopoietic stem cell/organ transplantation. symptoms such as fever that does not respond to broadspectrum antimicrobial drugs, cough, dyspnea, sputum, and bloody sputum/hemoptysis are observed. -laboratory findings: chest x-ray shows an infiltrative shadow (typically, a wedge shadow involving the pleura as the base). on thoracic ct, infiltrative and nodular shadows (with a halo sign in some cases) are observed. in the recovery phase of neutrophils, an air crescent sign is noted. an increase in the inflammatory marker such as the crp level, aspergillus galactomannan antigen-positive reactions, and an increase in the ( / )-b-d-glucan level are useful for diagnosis. however, neither the sensitivity nor specificity is sufficient. the results should be carefully evaluated. -causative microorganisms: aspergillus fumigatus is frequently detected, but, recently, an increase in the number of patients with non-fumigatus aspergillus-related ipa has been indicated. -specific condition: lesions are sometimes formed in the nasal sinus and brain; caution is needed. -early diagnosis: early treatment is important for successful treatment for this disease. ---drugs to be recommended---a study reported that, in a group in which vrcz was used for the initial treatment of ipa, the results of treatment were more favorable than in a group in which d-amph was used [ ] . furthermore, another study indicated that therapy with l-amb at mg/kg/day was safer than that at mg/kg/ day, although there was no significant difference in the clinical efficacy [ ] . cpfg, mcfg, and itcz also have anti-aspergillus activities, and can be used. it is important to consider different strategies in accordance with the appearance of the host's allergy or adverse events and interactions with drugs used to treat an underlying disease. b. chronic progressive pulmonary aspergillosis (cppa) ---executive summary----in japan, various disease types such as aspergilloma with infiltration and enlargement of an existing cavity are included. cppa includes various diseases such as chronic necrotizing pulmonary aspergillosis (cnpa), chronic cavitary pulmonary aspergillosis (ccpa), and chronic fibrosing pulmonary aspergillosis (cfpa). it refers to a series of syndrome for which the administration of antifungal drugs is essential. -treatment should be started with injection. if symptoms and findings are stabilized, injection should be switched to oral drugs. -initial treatment with mcfg or cpfg should be performed [ , ] (ai). -initial treatment with itcz, vrcz, or l-amb can also be selected in accordance with the host's underlying disease or drugs used to treat the underlying disease. -for maintenance therapy, itcz and vrcz oral preparations are recommended (aiii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in hosts with organic diseases such as a cavity or cystic disease of the lung or bronchus. symptoms such as fever, sputum, bloody sputum/ hemoptysis, and dyspnea are observed. -laboratory findings: chest x-ray and ct show an infiltrative shadow, enlargement of a cavity, thickening of the cavity wall/pleura, and a niveau in the cavity. there is an increase in the crp level in many patients. most patients are positive for anti-aspergillus precipitating antibody. neither aspergillus galactomannan antigen nor b-d-glucan is a clue to diagnosis. -causative microorganisms: a. fumigatus is frequently detected. non-fumigatus aspergillus-related cppa is also often observed. ---drugs to be recommended---a clinical study in japan indicated that there was no marked difference in the efficacy of treatment between mcfg-and vrcz-treated groups, whereas mcfg was safer [ ] . another study reported that there was no difference in treatment results between mcfg and cpfg [ ] . in the phase of severe symptoms such as fever and bloody sputum, treatment should be started using these injections. -pulmonary aspergilloma is classified into two types: simple and complex aspergilloma based on the grade of difficulty in resection. the former refers to aspergilloma formation in a focus with a thin wall, such as a cyst, without accessory lesions at the periphery. the latter refers to aspergilloma formation in a cavity derived from a strongly destructed existing structure of the lung, such as old pulmonary tuberculosis and bronchiectasis, with marked destructive lesions or pleural adhesion at the periphery of the cavity. ---drugs to be recommended---resection should be selected as a first choice. when resection is impossible, medical treatment can be considered. in preparation, such as l-amb, and -fc should be performed for weeks or more. subsequently, treatment should be continued using flcz or f-flcz. -in japan, cryptococcus gattii infection has also been reported. if possible, causative fungus must be isolated/identified. ---explanation---[characteristics of the disease] -symptoms: this disease is often asymptomatic, and is detected on a health checkup in many cases. -laboratory findings: chest x-ray and ct show solitary or multiple nodular and infiltrative shadows. -some cavities are observed in a lot of cases. there is no enhancement of the inflammatory marker in many cases, but glucuronoxylomannan antigen-positive reactions are detected. -causative microorganisms: this disease is caused by cryptococcus neoformans. recently, infection with c. gattii has been reported in vancouver, canada and the north area of the west coast of the united states of america; caution is needed. -c. gattii is primarily distributed in the tropical and subtropical zones. infection in humans has been considered to be rare. however, since , patients infected with c. gattii have been reported in the pacific coast of north america. even healthy adults are infected with c. gattii, and the mortality rate is high. ---drugs to be recommended---although there is no evidence regarding pulmonary cryptococcosis, flcz tablets, which have a potent activity against cryptococcus, are frequently selected when the patient's condition is stable in the absence of an underlying disease. azoles other than this drug can also be selected. [ ] (aii). -if the lesion is localized, resection should be considered. -combination therapy with an iron chelating agent and l-amb should be avoided [ ] (ai). ---explanation--- recently, an increase in the incidence of infection with cunninghamella has also been indicated. -specific condition: nasal/brain-type, dermal, and disseminated zygomycosis is observed. ---drugs to be recommended---currently, only amph preparations may be clinically useful for treating zygomycosis among antifungal drugs that are available in clinical practice in japan. as high-dose therapy must be started as early as possible, not d-amph but l-amb should be selected. -l-amb, intravenous drip, mg/kg/once a day precautions for the use of antifungal drugs (confirm the package inserts.) ➀ vrcz vision disorder, liver dysfunction, and neurological/ mental adverse events may occur. combination therapy with rfp, rbt, efavirenz, ritonavir, carbamazepine, long-acting barbiturate, pimozide, quinidine sulfate, ergot alkaloid, or triazolam is contraindicated. this drug is also contraindicated for pregnant women. as a rule, it is contraindicated for patients with a ccr of < ml/ min (injection only). as the blood concentration of vrcz may vary, tdm should be conducted. in patients with mild to moderate liver dysfunction, the dose should be regulated. ➁ itcz hepatopathy and congestive heart failure may occur. combination therapy with pimozide, quinidine, bepridil, simvastatin, triazolam, azelnidipine, ergotamine, nisoldipine, dihydroergotamine, vardenafil, eplerenone, blonanserin, sildenafil, tadalafil, aliskiren, dabigatran(itcz oral only), rivaroxaban, ergometrine, or methylergometrine is contraindicated. this drug is contraindicated for patients with severe liver diseases, pregnant women. patients with a ccr of < ml/min are also contraindicated (injection only). ➂ flcz hepatopathy and a prolongation of qt may occur. combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➃ f-flcz combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➄ l-amb adverse events such as nephropathy, hypopotassiumemia, and fever may occur. this drug is contraindicated during leukocyte transfusion. ➅ cpfg this drug is safe, but hepatopathy may occur. caution is needed for combination therapy with cyclosporin, tacrolimus, rfp, efavirenz, nevirapine, phenytoin, dexamethasone, or carbamazepine. ➆ mcfg this drug is safe, but hepatopathy may occur. ➇ -fc anorexia and myelopathy may occur. combination therapy with tegafur-/gimeracil-/oteracil potassium-containing drugs is contraindicated. even after the discontinuation of tegafur-/gimeracil-/oteracil potassium-containing drugs, combination therapy should be avoided within days. this drug is also contraindicated for pregnant women. ---drugs to be recommended---st combination drugs are used as a gold standard of pcp treatment. however, there have been a large number of patients in whom treatment was discontinued due to their side effects. recently, atovaquone also became available in japan as a second-choice drug. in patients with mild pcp, atovaquone tablets were as effective as st combination drugs. in those with moderate pcp, st combination drugs were more effective, but there was no significant difference due to a small number of subjects. in atovaquone-treated patients, the incidence of adverse events for which administration was discontinued was lower than in st combination drug-treated patients, suggesting that the tolerance is high [ ] . the administration period should be days in hivinfected patients and days in non-hiv-infected patients. a target daily dose of trimethoprim should be e mg/kg. -st combination drug, oral, to tablets/ times a day -st combination drug, intravenous drip, e mg as trimethoprim/ times a day (infused over e h) <> second choices -pentamidine, intravenous drip, mg/kg/once a day (infused over e h) -atovaquone oral suspension, ml ( mg as atovaquone)/twice a day for days (orally administered after meals) * adjuvant therapy in patients with a pao of < mmhg or a-ado of > mmhg in room air, one of the above drugs should be combined with a corticosteroid from the start of treatment. however, the dose may be reduced or administration may be discontinued in the early phase in accordance with symptoms. when the respiratory state is extremely unfavorable, pulse therapy should also be considered. prednisolone days e : oral, e mg/twice a day days e : oral, e mg/twice a day days e : oral, . e mg/twice a day ---precautions for each drug---(confirm the package inserts.) ➀ st combination drug (baktar tablets) fever, exanthema, digestive symptoms, hepatopathy, nephropathy, and blood disorder may occur. this drug may interact with methotrexate, sulfadoxine, pyrimethamine, diaphenylsulfone, sulfonyl amide/sulfonylurea oral drugs for diabetes, warfarin, phenytoin, cyclosporin, zidovudine, digoxin, tricyclic antidepressants, and lamivudine. this drug is contraindicated for neonates, low-birthweight infants, pregnant women, and patients with g- -pd deficiency. in patients with renal dysfunction, dose reduction must be considered. side effects such as hypoglycemia, hypotension, nephropathy, taste disorder, numbness of the tongue/lips, ventricular arrhythmia, exanthema, and fever may occur. combination therapy with zalcitabine, pfa, or amiodarone is contraindicated. this drug is contraindicated for patients with severe ventilatory disturbance. ➂ atovaquone nausea/vomiting, exanthema, and diarrhea may occur. this drug should be carefully administered to patients with severe kidney or liver dysfunction. this drug may interact with rfp, rbt, tetracycline, metoclopramide, zidovudine, acetaminophen, benzodiazepines, aciclovir, opioid analgesic drugs, cephalosporin antibiotics, antidiarrheal drugs/laxatives, and indinavir. h. cytomegalovirus (cmv) pneumonia ---executive summary----in the field of transplantation, preemptive treatment with gcv should be conducted through cmv antigenemia test monitoring. -the efficacy of preemptive treatment with vgcv or pfa is similar to that of gcv. -if a diagnosis of cmv pneumonia is made, treatment with gcv should be promptly started [ ] (aii). -vgcv and pfa are recognized as alternative drugs for gcv [ , ] (bii). -combination therapy with an antiviral drug and high-dose immunoglobulin should be performed [ ] (aiii). ---explanation--- ---antimicrobial drugs to be recommended---a first-choice drug for cmv pneumonia treatment is gcv, which has been frequently used. pfa has been used to treat cmv infection in aids patients, but experience on its use is limited in patients after hematopoietic stem cell transplantation. ( ) initial administration a first choice -gcv, intravenous drip, mg/kg (over h or more)/ every h for e weeks þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days <> second choices -pfa, intravenous drip, mg/kg (over h or more)/ times a day, every h for e weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days or -pfa, intravenous drip, mg/kg (over h or more)/ twice a day, every h for e weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days ( ) maintenance administration a first choices -gcv, intravenous drip, mg/kg (over h or more)/once a day, days a week or -gcv, intravenous drip, mg/kg (over h or more)/once a day, days a week * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. <> second choice -pfa, intravenous drip, e mg/kg (over h or more)/once a day (in clinical practice in japan, there have been few case reports on once-a-day administration at mg/ kg as maintenance therapy. a dose exceeding mg/ kg should be avoided. for administration at mg/kg, twice-a-day administration at mg/kg is commonly selected.) * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. ---precautions for each drug---(confirm the package inserts.) ➀ gcv severe leukopenia, neutropenia, anemia, thrombopenia, pancytopenia, aplastic anemia, and bone marrow suppression may occur. an animal experiment showed that this drug induced transient or irreversible spermatogenic dysfunction and reduced fertility. in humans, this drug may cause spermatogenic dysfunction. an animal experiment demonstrated the teratogenicity, mutagenicity, and carcinogenicity of this drug. in the presence of renal hypofunction, it is necessary to regulate the dose. this drug is contraindicated for patients with marked bone marrow suppression (neutrophil count: < /mm or platelet count: < , /mm ) and pregnant women. it may interact with didanosine, zidovudine, ipm/cs, bone marrow-suppressing and kidney function-affecting drugs, zalcitabine, st combination drugs, cyclosporin, probenecid, and mycophenolate mofetil. ➁ vgcv this is a prodrug of gcv. ➂ pfa acute renal failure, shock, heart failure, thrombophlebitis, and convulsion may occur. it is necessary to regulate the dose in accordance with the kidney function. combination therapy with pentamidine is contraindicated. this drug is contraindicated for patients with a ccr of < . ml/min/kg. ---executive summary---for the treatment of community-acquired pneumonia in children, antimicrobial drugs should be selected, considering age and severity. ---explanation--- patients with acute respiratory infectious disease symptoms, such as fever, nasal discharge, pharyngeal pain, and cough, and the appearance of a new infiltrative shadow in the lung on imaging examinations such as chest x-ray and ct are regarded as having pneumonia [ ] . in patients with pneumonia, thoracic auscultation findings often include accessory murmurs and the attenuation of respiratory sounds. most patients with respiratory infectious diseases consult hospitals with fever and cough. the lesion site of the airway is estimated based on symptoms and physical findings (fig. ) [ ] . in addition to thoracic findings, it is necessary to check the presence or absence of dyspnea signs, such as tachypnea, nasal alar breathing, retractive breathing, shoulder breathing, orthopnea, groaning, and cyanosis. to consider the need of antimicrobial-drug administration and options of antimicrobial drugs, pneumonia is classified into three types: bacterial, viral, and atypical pneumonia based on causative microorganisms [ ] . [type and frequency of causative microorganisms] microorganisms that cause childhood community-acquired pneumonia differ among ages. according to the data on investigation of causative microorganisms based on lavage sputum culture in japan, bacterial and viral pneumonia is frequent in infants/children aged year or younger. in those aged e years, the incidences of bacterial, viral, and atypical pneumonia are similar. in those aged over years, the incidence of atypical pneumonia is the highest [ ] (fig. ) insufficient. however, a review reported similar findings [ ] ( table ) [ ] . in children, it is not easy to investigate causative microorganisms. in institutions in which it is impossible to investigate causative microorganisms, treatment must be performed based on the statistical frequency of causative microorganisms described below. however, in those in which investigation is possible, the etiology should be investigated if possible. [rules of antimicrobial drug therapy] it is important to improve the efficacy of treatment by accurately predicting causative microorganisms and selecting an appropriate antimicrobial drug and its administration method. whether or not an antimicrobial drug should be indicated must be comprehensively evaluated by differentiating bacterial, viral, and atypical pneumonia with reference to age, severity, clinical symptoms, physical findings, laboratory data, and x-ray findings [ ] . as a rule, a single antimicrobial drug should be selected. when the type of microorganisms that caused pneumonia is identified, an antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics. [clinical symptoms, physical findings] wet cough and tachypnea are frequently observed in children with bacterial pneumonia. the proportion of labored breathingfree patients is high in children with mycoplasma pneumonia [ , ] . auscultation findings include intermittent accessory murmurs (rales) regardless of the type of pneumonia. in children with mycoplasma pneumonia, the proportion of those in whom auscultation findings are not marked is significantly higher than in other groups. in children with chlamydia pneumonia, fever is mild, and cough is protracted. thus, clinical symptoms and physical findings show characteristics related to causative microorganisms, but it is difficult to identify causative microorganisms based on clinical symptoms and physical findings alone in individual patients [ e ] . concerning laboratory findings on admission in children with bacterial and viral pneumonia, there are significant differences in the leukocyte count, crp level, and erythrocyte sedimentation rate between the two groups (p < . ). however, measurements overlapping in about one-third of patients are presented [ ] (fig. ) . briefly, it is impossible to accurately differentiate bacterial from viral pneumonia based on inflammatory responses reflected by the leukocyte count, crp level, and erythrocyte sedimentation rate. mycoplasma pneumonia is characterized by increases in the crp level and erythrocyte sedimentation rate, but many patients show normal leukocyte counts or a slight decrease in this parameter. furthermore, it is difficult to differentiate mycoplasma from viral pneumonia based on laboratory data [ ] . [chest x-ray] chest x-ray findings show characteristics related to causative microorganisms to some degree, but it is difficult to identify causative microorganisms in individual patients [ ] . it is important to evaluate the severity of pneumonia, for selecting outpatient or hospital treatment and reviewing the necessity of antimicrobial drugs and route of administration (oral or intravenous). the classification of severity in the guidelines for the management of respiratory infectious diseases in children in japan is presented [ ] (table ) . causative microorganisms with respect to age in children with community-acquired pneumonia [ ] . the severity classification of childhood pneumonia has not been reached in japan or internationally. this should be examined in the future. [standards for outpatient/hospital treatment] as a rule, outpatient treatment should be performed in mildstatus patients evaluated according to the severity classification, and hospital treatment in mild-status patients with dehydration. in addition, it is necessary to determine admission when outpatient treatment does not lead to an improvement in symptoms or considering social adaptation [ ] (table ) . [initial antimicrobial drug therapy] when examining children with pneumonia, treatment must be started without any precise information about causative microorganism in many cases. basically, empiric therapy should be performed, considering the severity of pneumonia and causative microorganisms. -the type of causative microorganisms depends on age and severity. therefore, the necessity of antimicrobial drugs should be examined, and selected, considering age and severity. in addition, bacterial, viral, or atypical pneumonia should be differentiated, and comprehensively evaluated in reference to clinical symptoms, physical findings, laboratory findings, and xray findings [ ] . in particular, acute bronchitis/pneumonia related to intermediately susceptible h. influenzae (mic: mg/ml) can be managed with oral ampc or abpc intravenous injection therapies [ ] . recently, the number of abpc-susceptible strains has annually decreased [ , ] . the proportion of blnar strains (mic: mg/ table age-related distribution of microorganisms that cause pneumonia in children [ ] . immediately after birth to . inflammatory response on admission in children with pneumonia [ ] . ml or more) has increased, raising an issue with respect to drug selection. when the involvement of blnar strains is suspected, high-dose ampc or new oral cephems may be necessary at outpatient clinics [ ] . the efficacy of outpatient antimicrobial drugs for blnar strains, which will increase in the future, should be carefully monitored. concerning hospital treatment, the clinical effects of abpc intravenous injection for e days until the results of a susceptibility test were clarified were investigated, and approximately % of patients responded to this therapy. there was no exacerbation in any patient [ ] . in non-responders or patients in whom clinical effects are insufficient, it is necessary to switch the antimicrobial drug. pipc, ctx, and ctrx have stable antimicrobial activities. when reviewing the clinical effects of pipc on childhood bronchopulmonary infection, the response rate was %; the results were satisfactory [ ] . -concerning treatment for m. catarrhalis pneumonia, m. catarrhalis produces b-lactamase. however, when examining the clinical course, ampc is effective [ , ] . this is because the enzymatic activity of b-lactamase produced by m. catarrhalis is low [ ] . (table ) . [evaluation of the treatment response and administration period] the administration period of antimicrobial drugs is shown in table [ ] . classification of the severity of community-acquired pneumonia in children [ ] . moderate severe . increases in the number of resistant strains of s. pneumoniae and h. influenzae derived from respiratory infectious diseases [ ] . to treat community-acquired pneumonia, antimicrobial drugs should be administered for e days. the treatment response should be evaluated after e days. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days [ ] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients; therefore, it is difficult to establish standardized criteria. as m. pneumoniae and c. pneumoniae slowly proliferate, the treatment period is prolonged (table ). in patients infected with general bacteria, the administration of antimicrobial drugs can be discontinued days after pyretolysis [ ] . however, further antimicrobial drug therapy is necessary to treat s. aureus pneumonia. [management for non-responders to antimicrobial drugs] when there are no therapeutic effects of antimicrobial drugs on pneumonia, whether or not a diagnosis of pneumonia is correct should be initially investigated [ ] . the possibility of diseases other than pneumonia, with a pneumonia-like shadow, must be reviewed (table ). if it can be ruled out, whether or not the expected type of pathogenic microorganisms is correct should be examined. if the type of causative microorganisms is the same as expected, the possibility of resistant microorganisms should be considered. new therapeutic strategies should be devised carefully and promptly. when the condition further exacerbates despite treatment switching, additional examination should be conducted. [ ] . concepts regarding the diagnosis and treatment of childhood mycoplasma pneumonia [ ] . . as it is often difficult to make a diagnosis of mycoplasma pneumoniae infection based on serum antibody titer-positive findings in the acute stage alone, the mycoplasma pneumoniae nucleic acid identification test (lamp method) should be conducted to make a definitive diagnosis in the acute stage. . as first-choice drugs for mycoplasma pneumonia, macrolides should be used. . the effects of macrolides can be evaluated based on pyretolysis within e days after administration. . to treat pneumonia that does not respond to macrolides, the administration of tosufloxacin or tetracyclines should be considered if necessary. however, as a rule, tetracyclines are contraindicated for children aged years or younger. . these antimicrobial drugs should be administered in accordance with administration periods recommended for individual drugs. . in patients with severe pneumonia, systemic steroid therapy must be considered. however, easy steroid administration should be avoided. however, mycoplasma pneumoniae is not included in bacterial types for which this preparation may be indicated. ---drugs to be recommended--- a first choices ) cases in which there is no risk of resistant bacteria -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day ) cases in which infection with resistant bacteria is suspected i) two years or younger, ii) pretreatment with an antimicrobial drug (within weeks), iii) concomitant development of otitis media, iv) history of pneumonia/repeated otitis media -ampc, oral, e mg/kg/ times a day -cva/ampc ( : preparation), oral, . mg/ kg/twice a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day ) cases in which onset/recurrence/recrudescence is observed despite previous treatment ) -tbpm-pi, oral, e mg/kg/twice a day -tflx, oral, mg/kg/twice a day <> second choices -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day ( ) admission (moderate, general ward) a first choices -abpc, intravenous injection or drip, mg/kg/ times a day -pipc, intravenous injection or drip, mg/kg/ times a day -sbt/abpc, intravenous injection or drip, mg/kg/ times a day * when m. pneumoniae, chlamydia trachomatis, or c. pneumoniae infection is strongly suspected, one of the above regimens should be combined with a macrolide [with respect to the administration method/ dose, refer to the section "➁ six years or older---( ) outpatient clinic (mild)".]. <> second choices -ctx, intravenous injection or drip, mg/kg/ times a day -ctrx, intravenous injection or drip, e mg/kg/ once to twice a day ( e mg/kg/day) ( ) admission (severe, icu) -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -taz/pipc, intravenous drip, . mg/kg/ times a day * when legionellosis cannot be ruled out, one of the above regimens should be combined with a macrolide [with respect to the administration method/dose, refer to the section "➁ six years or older---( ) admission (moderate, general ward)".]. ➁ six years or older ( ) outpatient clinic (mild) a first choices -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day <> second choices -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day -mino, oral, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or nonresponders to other drugs.) ( a first choices ) cases in which bacterial pneumonia is suspected -abpc, intravenous injection or drip, mg/kg/ times a day -pipc, intravenous injection or drip, mg/kg/ times a day -sbt/abpc, intravenous injection or drip, mg/ kg/ times a day -ctx, intravenous injection or drip, mg/kg/ times a day -ctrx, intravenous injection or drip, e mg/ kg/once to twice a day ( e mg/kg/day) ) cases in which atypical pneumonia is suspected -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day -em, intravenous drip, mg/kg/ e times a day -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) ) cases in which it is impossible to differentiate bacterial from atypical pneumonia one drug each should be selected from choices ) and ), and combined. -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day -em, intravenous drip, mg/kg/ e times a day -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) ---executive summary---for the treatment of hospital-acquired pneumonia in children, antimicrobial drugs should be selected, considering severity and the involvement of resistant bacteria. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for communityacquired pneumonia (biii). hospital-acquired pneumonia is defined as pneumonia that newly develops h or more after admission. ventilator-associated pneumonia is defined as pneumonia that develops h or more after endotracheal intubation [ ] . these conditions may become severe due to an underlying disease, reduced immune capacity, or the deterioration of the general condition, and are caused by drugresistant microorganisms in many cases; treatment is difficult in most cases. not only microorganisms acquired in the community but also those existing in the hospital environment cause hospital-acquired pneumonia in children, as reported in the adult field. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause hospital-acquired pneumonia [ ] . in addition, not only general bacteria but also fungus and viruses sometimes cause hospital-acquired pneumonia in patients with immunodeficiency. among patients with nosocomial infection, causative microorganisms differ, and drug-resistant microorganisms are involved in many cases. in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, lavage or aspiration sputum culture should be conducted to investigate the etiology, if possible [ , ] . [rules of antimicrobial drug therapy] basically, empiric therapy should be performed, considering the severity of pneumonia, an underlying disease, and causative microorganisms. in particular, the involvement of drug-resistant microorganisms, such as mrsa, extended-spectrum b-lactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [ ] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. however, when complications such as severe immunodeficiency, pulmonary suppuration, lung abscess, and pleuritis are absent, antimicrobial drugs should be administered for days after pyretolysis ( e days) [ ] . considering an underlying disease or the immune state, flexible management must be performed. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days [ ] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . concerning multi-drug-resistant microorganisms, it is important to promote standard preventive strategies and those to control nosocomial infection, such as the prevention of droplet/contact infection, thoroughly. furthermore, oral care and devised postures (if there is no medical contraindication, the head should be lifted at e .) are necessary to prevent vap [ ] . in this article, the classification of severity (table , p. ) in the guidelines for the management of respiratory infectious diseases in children in japan was used [ ] (refer to the section " . pneumonia (children), . community-acquired pneumonia".). ---drugs to be recommended--- a first choices -caz, intravenous injection or drip, mg/kg/ times a day -czop, intravenous injection or drip, mg/kg/ e times a day -cpr, intravenous injection or drip, mg/kg/ e times a day if necessary, one of the above regimens should be combined with one of the following drugs: ) cases in which anaerobe infection is suspected (such as aspiration pneumonia) -cldm, intravenous drip, in children with immunodeficiency-/blood disease-related pneumonia, antimicrobial drugs should be selected, considering an underlying disease, the grade of immunodeficiency, and involvement of various causative microorganisms. initial antimicrobial drug therapy should be started by combining two drugs if necessary, considering various causative microorganisms, differing from that for community-acquired pneumonia (biii). [characteristics and classification of the disease] as immunodeficiency-/blood disease-related pneumonia in children often develops in hospitals, it has the characteristics of hospital-acquired pneumonia in many cases. it may become severe due to the patient's unfavorable conditions, such as an underlying disease, reduced immune capacity, and the deterioration of the general condition. even non-pathogenic microorganisms may cause pneumonia in many cases. furthermore, drug-resistant microorganisms often cause pneumonia; treatment is difficult in many cases [ , ] . to achieve multidisciplinary, comprehensive treatment to save children, it is necessary to cooperate with other special fields. [type and frequency of causative microorganisms] not only microorganisms acquired in the community in the presence of various immunodeficiency states but also nonpathogenic microorganisms existing in the hospital environment cause immunodeficiency-/blood disease-related pneumonia in children. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause this type of pneumonia. in addition, not only general bacteria but also fungus and viruses often cause this type of pneumonia. furthermore, drugresistant microorganisms are involved in many cases, as indicated for hospital-acquired pneumonia [ , ] . [type of immunodeficiency, causative microorganisms to be monitored, and precautions for diagnosis] ➀ humoral immunodeficiency: as bacterial opsonization and complement activation are affected, patients with humoral immunodeficiency are prone to be infected with general bacteria. among immunodeficiency patients with hyper-igm-emia, pneumocystis pneumonia should be considered in those with conditions related to cd ligand abnormalities. ➁ cellular immunodeficiency: in addition to infection with general bacteria, infection with intracellular parasitic bacteria, fungus, or protozoa may become severe, and be protracted. as the differentiation and induction of b and killer t cells by cd -positive lymphocytes are affected, the eradication of virus-infected cells is inhibited (table ) [ ] . ➂ neutrophil abnormalities: neutrophil abnormalities are classified into two types: neutropenia and neutrophil functional disorders. patients with a peripheral blood absolute neutrophil count (anc) of < /ml or those in whom the anc is estimated to reach < /ml within h are regarded as having neutropenia [ ] . of these, the risk is higher in patients with an anc of /ml or less in whom the period is estimated to exceed days. many patients with neutropenia do not show purulent sputum or abnormal findings on chest x-ray even in the presence of pneumonia. therefore, when fever persists, thoracic ct should be performed in the early stage. all microorganisms including general bacteria (gram-positive bacteria, gramnegative bacteria), fungus, and viruses may cause pneumonia. in particular, in addition to neutropenia early after homologous hematopoietic stem cell transplantation, infection-prone features associated with humoral/cellular immunodeficiency related to the administration of immunosuppressives persist over a long period. furthermore, the concomitant development of acute/chronic graft-versus-host disease (gvhd) is a risk factor for the onset of pneumonia. in addition, non-infectious pulmonary disorder related to drugs/radiation for pretreatment may occur, and it is important to differentiate it from infectious diseases (fig. ) [ , ] . a representative neutrophil functional disorder, chronic granulomatosis, induces active oxygen production disorder of neutrophils, affecting bactericidal actions. therefore, patients with this disorder are prone to be infected with non-h o -producing catalase-positive bacteria (s. aureus, k. pneumoniae, e. coli, candida spp., aspergillus spp.). ➃ complement deficiency: patients with complement deficiency are prone to be infected with bacteria with capsules, such as s. pneumoniae, h. influenzae (capsule strains), and neisseria meningitidis [ ] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, various cultures should be conducted to investigate the etiology, if possible. in addition, testing of various antigens, such as urinary s. pneumoniae/legionella antigens, b-dglucan, aspergillus antigen, cryptococcus antigen, candida antigen, and cytomegalovirus antigen, and tests using nucleic acid amplification methods, such as the pcr method, should be utilized, if possible. [rules of antimicrobial drug therapy] initial antimicrobial drug therapy should be performed, considering the severity of pneumonia and an underlying disease. for the treatment of immunodeficiency-/blood disease-related pneumonia in children, antimicrobial drug therapy should also be basically selected, considering causative microorganisms. as described for nosocomial infection, the involvement of drugresistant microorganisms, such as mrsa, extended-spectrum blactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. initial antimicrobial drug therapy should be started by combining two drugs, if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [ , ] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . monitoring culture of the airway is useful for treating immunodeficiency-/blood disease-related pneumonia in children [ ] . general preventive methods are presented in table [ ] . in addition, long-term low-dose macrolide therapy or the intermittent administration of b-lactams to prevent p. aeruginosa fixation is effective in some patients with chronic bronchitis [ , ] . in those with chronic granulomatosis, the oral administration of itcz ( e mg/kg/day, maximum: mg/day) or subcutaneous injection of ifn-g ( , domestic standard units/m , e times a week) is useful for preventing infection [ ] . in this article, the classification of severity (table , p. ) in the guidelines for the management of respiratory infectious diseases in children in japan was used (refer to the section " . pneumonia (children), . community-acquired pneumonia".). ---drugs to be recommended--- ( ) pneumonia related to mild immunodeficiency in the initial phase after admission -sbt/abpc, intravenous injection or drip, mg/kg/ e times a day -ctx, intravenous injection or drip, mg/kg/ e times a day -ctrx, intravenous injection or drip, mg/kg/twice a day ( ) pneumonia related to moderate/severe immunodeficiency -caz, intravenous injection or drip, mg/kg/ e times a day -czop, intravenous injection or drip, mg/kg/ e times a day -cpr, intravenous injection or drip, mg/kg/ e times a day in severe cases, -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -drpm, intravenous drip, mg/kg/ times a day -taz/pipc, intravenous drip, . mg/kg/ times a day -vcm, intravenous drip, mg/kg/ times a day -abk, intravenous drip, e mg/kg/once a day -teic, intravenous drip, mg/kg/every h, times, subsequently: e mg/kg/once a day when there is no response, -amk, intravenous drip, e . mg/kg/twice a day -tob, intravenous drip, . mg/kg/ times a day if necessary, an antifungal drug (mcfg) and st combination drug should be additionally administered. when aspergillus infection is suspected, treatment should be started with vrcz or l-amb instead of mcfg. in patients with cellular immunodeficiency, one of the above drugs should be combined with mcfg and an st combination drug in the early stage. in those with neutrophil abnormalities, one of the above drugs should be combined with mcfg in the early stage. when legionellosis cannot be ruled out in the severest cases, one of the above regimens should be combined with a macrolide (with respect to the administration method/dose, refer to the section " . community-acquired pneumonia---➁ six years or older---( ) admission (moderate, general ward)" (p. ).). in the presence of gcv resistance, the intravenous drip of pfa (foscarnet), mg/kg/ times a day should be performed. ---executive summary---in the treatment of neonatal pneumonia, antimicrobial drugs should be selected after differentiating congenital from acquired pneumonia. initial antimicrobial drug therapy should be started by combining two drugs, regarding the condition as severe systemic infection and considering various causative microorganisms. the administration method and dose should be selected based on the age (days) and birth weight (biii). [characteristics and classification of the disease] the incidence of neonatal pneumonia is not high. according to sakata, pneumonia occurred in only ( . %) of approximately , neonates who were admitted during a -year period [ ] . neonatal pneumonia is frequently observed as a portion of systemic infection represented by sepsis, and not as a single condition. it is classified into two types: congenital and acquired pneumonia [ ] . in most cases, neonatal pneumonia does not cause any typical symptoms such as fever or cough in comparison with infantile/ childhood pneumonia. the neonatal protective capacity against infection is physiologically immature, often leading to a severe condition. in the neonatal phase, artificial respiration management is performed in many cases, and ventilator-associated pneumonia (vap) is frequently observed [ e ]. in addition, many neonates are admitted to the neonatal intensive care unit (nicu) for a long period; therefore, the incidence of nosocomial infection is high. [type and frequency of causative microorganisms] several types of congenital pneumonia include transplacental infection, intrauterine infection related to aspiration of infected amniotic fluid on premature rupture, and birth canal infection with vaginal discharge on delivery. these types of congenital pneumonia are primarily caused by various viruses (cytomegalovirus, herpes simplex virus), bacteria (streptococcus agalactiae (gbs), e. coli, listeria monocytogenes), chlamydia, and fungus. the clinical onset of perinatal pneumonia is frequently observed immediately to days after birth [ ] . postnatal pneumonia usually develops weeks or more after birth. it is caused by viruses and bacteria. viral infection may occur when rs virus, parainfluenza virus, or adenovirus prevails in the ward. however, it rarely leads to the onset of pneumonia. frequent causative bacteria include s. aureus, e. coli, p. aeruginosa, acinetobacter spp., enterobacter spp., and legionella spp., which exist in the environment. these often cause nosocomial infection [ ] . in premature babies, gram staining of tracheal aspirate samples is useful for selecting antimicrobial drugs to be administered in the initial phase [ ] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, blood or bronchial lavage fluid culture should be conducted to investigate the etiology, if possible. [rules of antimicrobial drug therapy] as many neonates have systemic infection in an immunodeficiency state, the same administration method and dose as selected for the treatment of sepsis should be used [ ] . therefore, the severity of pneumonia is not considered. when bacterial pneumonia is suspected, combination therapy with abpc and ctx is commonly selected as initial treatment. considering l. monocytogenes infection, combination therapy with abpc is recommended. after causative microorganisms are identified, antimicrobial drugs should be selected with reference to their drug susceptibility. in cases of vap, antimicrobial drug options should be customized based on records on the drug susceptibility (antibiograms) of microorganisms that cause nosocomial infection at each institution. in neonates, there are marked age-related differences in the pharmacokinetics of antimicrobial drugs; therefore, these drugs must be carefully administered in the neonatal phase. concretely, the same dose as established in the field of pediatrics should be used, and the administration interval should be prolonged in accordance with age [ ] . in cases of severe asphyxia or acute renal failure, the administration interval must be further prolonged. it should be shortened with an improvement in the kidney function [ e ] . a consensus regarding the administration period of antimicrobial drugs has not been reached. however, the standard administration period is days. with respect to the administration method/dose, refer to the section "xi. doses for neonates". in neonates, disease progression is often prompt, and the treatment response should be evaluated after days, and not after days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . ---drugs to be recommended--- ---executive summary----pyothorax refers to a condition in which pus accumulates in the thoracic cavity. usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: < . (biii). -in patients with acute pyothorax related to community-acquired pneumonia, treatment should be performed in accordance with that for community-acquired pneumonia, considering microorganisms that cause community-acquired pneumonia, such as streptococcus pneumoniae (biii). -in patients with slowly progressing pyothorax, mixed infection with oral aerobes/anaerobes is frequently observed. combination therapy with pcg or abpc and cldm or mnz, which show anti-anaerobe activities, or therapy with a single antimicrobial drug with an anti-anaerobe activity, such as sbt/abpc, should be selected (biii). -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem, combination therapy with a fourthgeneration cephalosporin and cldm or mnz, and combination therapy with a quinolone and cldm or mnz should be considered, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, anaerobes, and acinetobacter (biii). -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them (aiii). -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, the use of aminoglycosides should be avoided as a rule (biii). -if a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be started, and drainage must be performed (aii). if possible, the attending physician should consult a surgeon in the early stage (aiii). -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary (biii). in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgical interventions such as thoracotomy or decortication is performed in some cases (biii). ---explanation--- [diagnosis] -pyothorax is defined as a condition in which pus accumulates in the thoracic cavity, but this definition has no diagnostic objectivity. for this reason, usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: < . [ , ] . [causative microorganisms] -pleural effusion appears in e % of patients with bacterial pneumonia, but leads to pyothorax in . e %. other etiological factors for pyothorax include surgery, trauma, and esophageal perforation. -microorganisms that cause pyothorax depend on its etiology and course. in the presence of bacterial pneumonia, pyothorax is caused by the same microorganisms as caused bacterial pneumonia. furthermore, acute pyothorax is frequently caused by s. pneumoniae and s. aureus. however, in many patients with chronic pyothorax, mixed infection primarily with anaerobes is involved. among anaerobes, fusobacterium spp. (especially fusobacterium nucleatum), prevotella spp., peptostreptococcus spp., and bacteroides spp. are frequently detected [ e ]. according to recent studies, the detection rate of the streptococcus anginosus group is high [ , ] . -in many cases, pyothorax with a relatively slow course is associated with m. tuberculosis. it must be considered that pulmonary lesions do not always concurrently exist with tuberculous pleuritis. [treatment] -no randomized comparative study regarding antimicrobial drug treatment for pyothorax has been conducted. an antimicrobial drug with an activity against microorganisms expected or obtained on culture should be selected and administered. in acuteonset patients in whom there is no risk of resistant bacteria, for example, those with pyothorax accompanying communityacquired pneumonia, antimicrobial drugs such as pcg and abpc should be initially selected, considering s. pneumoniae. these drugs simultaneously cover fusobacterium, peptostreptococcus, and the viridans streptococcus group. however, prevotella and bacteroides produce b-lactamase; therefore, when the results of culture are not obtained, combination therapy with antimicrobial drugs with activities against anaerobes, such as cldm and mnz, or therapy with such a single drug, such as sbt/abpc, should be selected. -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem should be selected as a first choice, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, and acinetobacter. a fourth-generation cephalosporin should be combined with cldm, or a quinolone should be combined with cldm or sbt/abpc. -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them. however, the culture of anaerobes is difficult, or is not conducted in some cases. therefore, this should be confirmed to the laboratory. when only one type of bacteria are detected on a culture test despite several types of bacteria detected on gram staining of pleural effusion, anaerobes must also be considered. -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, for pyothorax treatment, the use of aminoglycosides should be generally avoided [ e ]. [treatment period] -the pyothorax treatment period has not been established. however, when pneumonia promptly responds to treatment and thoracic drainage is successfully achieved in patients with pneumonia-related pyothorax, a treatment period of e days is required. in patients in whom drainage is unsuccessful, those with marked pleural thickening, or those with encapsulated/septum-like pyothorax, a treatment period of about weeks is often required. [treatments other than antimicrobial drug therapy] -after a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be promptly started, and drainage is necessary. when the pleural fluid is purulent and viscous in the absence of a multilocular pattern, chest-tube insertion is routinely performed. the position of insertion should be confirmed using thoracic ct within h after insertion. wozniak et al. performed multivariate analysis involving patients with pyothorax and indicated that failure in the first drainage was strongly correlated with mortality, suggesting the necessity of early consultation with surgeons [ ] . -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary. in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgerical interventions such as thoracotomy or decortication is performed in some cases [ ] . ---drugs to be recommended--- a first choice -sbt/abpc, intravenous drip, g/ e times a day <> second choices -pcg, intravenous drip, , , to , , units/ times a day -abpc, intravenous drip, g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day ➁ cases in which there is a risk of multi-drug-resistant bacteria a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day <> second choices (i) -cfpm, intravenous drip, e g/ e times a day -czop, intravenous drip, e g/ e times a day -cpr, intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day <> second choices (ii) -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day -mnz, intravenous drip, mg/ times a day * with respect to the risk of multi-drug-resistant bacteria, refer to the section " . hospital-acquired pneumonia" table (p. ). * in particular, mrsa infection must be considered in patients with nosocomial onset or the previous administration of antimicrobial drugs. when staphylococcus infection is suspected on gram staining of pleural effusion, an anti-mrsa drug should be used as an empiric therapy. if mssa is identified as causative bacteria, deescalation should be performed. . definitive therapy -antimicrobial drugs against causative microorganisms identified should be selected in accordance with the section " . ---executive summary---for the treatment of pyothorax in children, antimicrobial drugs should be administered after investigating the etiology using thoracentesis or blood culture, if possible. in addition to antimicrobial drug therapy, treatment for retention fluid (pleural effusion drainage, continuous drainage) must also be considered (biii). [characteristics and classification of the disease] pleuritis refers to inflammation of the pleura. fluid (pleural effusion) is retained in the pleural cavity. pleuritis is classified into types: fibrinous (dry) pleuritis, exudative (wet) pleuritis, and purulent pleuritis (pyothorax) based on conditions [ ] . on auscultation, the attenuation of respiratory sounds, as well as pleural friction rubs, are heard. percussion dullness is noted. in the chronic stage, localized pleural thickening in various shapes is observed. when pus is obtained on thoracentesis, a definitive diagnosis of pyothorax is made. when the protein level in nonpurulent pleural effusion is high, tuberculous pleuritis should be differentiated [ ] . [type and frequency of causative microorganisms] previously, pyothorax was associated with s. aureus in many cases. however, recently, such cases have been rare. according to data form a national survey in the former half of the 's, there were only a few patients with s. pneumoniae-or anaeroberelated pyothorax [ ] . in many cases, pleuritis follows m. pneumoniae-or virus-related pneumonia. decubitus-view imaging shows the retention of pleural effusion in approximately % of patients with mycoplasma pneumonia [ ] . if the drug susceptibility of causative microorganisms is clarified, it contributes to successful treatment. therefore, pleural effusion obtained by thoracentesis should be cultured to investigate the etiology, if possible. when bacterial infection-related pyothorax is suspected, the intravenous injection or drip of sbt/abpc, ctx, or ctrx should be selected in community-onset patients without an underlying disease. on the other hand, combination therapy with sbt/abpc (intravenous injection or drip) and cldm (intravenous drip) or carbapenem therapy (intravenous drip) should be started in those with an underlying disease or nosocomial onset, considering s. pneumoniae, anaerobes, h. influenzae, and s. aureus [ ] . based on the gram staining reactions of pleural effusion, causative microorganisms should be estimated, and antimicrobial drugs must be reviewed. if necessary, tuberculosis should also be investigated. the administration period of antimicrobial drugs must be longer than that for pneumonia. as the type of causative microorganisms, patient background, and state of retention-fluid drainage differ among individual patients, it is difficult to establish standardized criteria. however, target administration periods for s. pyogenes (gas)-, s. pneumoniae-, and s. aureus-related pyothorax are , , and days or more, respectively. the treatment response should be evaluated e days after the start of administration. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until causative microorganisms and their drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a targetfocused antimicrobial drug should be selected through deescalation, considering drug susceptibility and pharmacokinetics [ ] . for the treatment of pyothorax, treatment for retention fluid (pleural effusion drainage) is also a basic procedure in addition to antimicrobial drug therapy. if necessary, continuous drainage should be performed (table ) [ ] . if pleural thickening leads to underexpanded lung, decortication should be indicated. the widespread application of thoracoscopic surgery has facilitated minimally invasive surgery [ ] . ---drugs to be recommended--- ( ) community onset (without an underlying disease) -sbt/abpc, intravenous injection or drip, mg/kg/ times a day -ctx, intravenous injection or drip, mg/kg/ e times a day -ctrx, intravenous injection or drip, mg/kg/twice a day ( ) community-acquired infection (with an underlying disease), nosocomial onset -cldm, intravenous drip, mg/kg/ times a day þ -sbt/abpc, intravenous injection or drip, mg/kg/ times a day or one of the following drugs alone should be administered: -taz/pipc, intravenous drip, . mg/kg/ times a day -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -drpm, intravenous drip, mg/kg/ times a day . definitive therapy refer to the section " . community-acquired pneumonia--- . . definitive therapy" (p. ) or " . hospital-acquired pneumonia--- . . definitive therapy" (p. ). table indications for continuous drainage [ ] . ➀ cases in which pleural effusion obtained on thoracentesis is purulent ➁ cases in which clinical effects are not achieved by antimicrobial drug therapy alone (within h) ➂ cases in which retention fluid affects the respiratory function ---executive summary----as initial treatment, four drugs (inh, rfp, and pza þ eb or sm) should be administered for months. subsequently, as a rule, standard treatment (a), in which two drugs, inh and rfp, are administered for months, should be performed as maintenance treatment (ai). -when pza cannot be used for initial treatment for some reason, inh, rfp, and eb or sm, should be administered for months as initial treatment. subsequently, standard treatment (b), in which two drugs, inh and rfp, are administered for months, should be performed as maintenance treatment (aii). -in patients in whom chest x-ray shows a cavity on initial consultation to during initial treatment and the septum culture is still positive at the completion of initial treatment, maintenance treatment should be prolonged over months. in addition, the prolongation of maintenance treatment should also be considered in patients with severe tuberculosis, such as military tuberculosis and tuberculosis of the central nervous system, those with immune depression, and those with relapse of tuberculosis (aii). -for the treatment of latent tuberculosis infection, inh should be administered for or months (ai). when m. tuberculosis, as the source of infection, is resistant to inh, or when the oral administration of inh is difficult due to side effects, rfp should be used as a second-choice drug for or months (ai). ---explanation--- [ , ] . in particular, this method is named "standard treatment (a)" in the guidelines for the management of tuberculosis in in japan. in this article, this name is also used. -the secondary assessment of a study examining the efficacy of rifapentine and inh showed that factors for unsuccessful treatment/recurrence included a cavity on chest x-ray at the start of treatment and positive findings on culture at the completion of initial treatment for months [ ] . similarly, when the treatment period was extended from to months in patients with silicotuberculosis, in whom the unsuccessful treatment/recurrence rates are high, the recurrence rate decreased from to %. therefore, various guidelines recommend that maintenance treatment should be prolonged over months in patients with a cavity and those showing positive findings on septum culture at the completion of initial treatment [ , ] . -in , combs et al. compared the results of treatment between a group treated with inh, rfp, and pza for months and, then, with inh and rfp for months and that treated with inh and rfp for months, and reported that the efficacy and incidence of side effects were similar [ ] . based on such a study, the following regimen is recommended as standard treatment (b) in the guidelines for the management of tuberculosis in in japan: when pza cannot be used for some reason, inh, rfp, and eb or sm, are administered for the first months, and, subsequently, two drugs, inh and rfp, are administered for months. -patients who are infected with m. tuberculosis, but do not develop tuberculosis are regarded as having latent tuberculosis infection (ltbi). inh administration for ltbi decreases the incidence of tuberculosis by e % [ ] . previously, this was called preventive therapy, but is currently termed ltbi treatment. the decrease in the incidence of tuberculosis is correlated with compliance with inh, and more marked preventive effects may be achieved when compliance is higher [ e ]. in a study involving inh administration to ltbi patients with an old shadow on chest x-ray, this therapy inhibited the onset of tuberculosis in and % of patients treated for and months, respectively (there was no significant difference between the two groups) [ ] . based on the data, some studies recommended that the period of standard inh administration for ltbi should be months [ e ]. however, a consensus regarding an effective administration period ( or months) has not been reached from various aspects including the efficacy, compliance, expenses, and incidence of side effects. actually, the administration period should be determined based on compliance and the incidence of side effects. -in ltbi treatment, rfp should be used for or months as an alternative drug when the oral administration of inh is impossible for some reason. the preventive effects of rfp on the onset of tuberculosis in ltbi patients may be similar to those of inh. furthermore, the incidence of liver dysfunction is lower than that related to inh [ e ]. however, for the use of rfp, drug interactions must be considered. ---drugs to be recommended---a first choice -inh, oral, mg/kg/once a day (maximum: mg/ day) þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ pza, oral, mg/kg/once a day (maximum: mg/day) þ eb, oral, mg/kg/once a day (maximum: mg/day) or sm, intramuscular injection, mg/kg/once a day (maximum: mg/day)/ e times a week. * the above drugs should be administered for months, and, subsequently, two drugs, inh and rfp, should be administered for months. <> second choice -inh, oral, mg/kg/once a day (maximum: mg/ day) þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ eb, oral, mg/kg/once a day (maximum: mg/day) or sm, intramuscular injection, mg/kg/once a day (maximum: mg/day)/ e times a week. * the above drugs should be administered for months, and, subsequently, two drugs, inh and rfp, should be administered for months. [ ] . in japan, tanaka et al. conducted combination therapy with cam ( mg/kg) and eb/rfp/km in patients, and indicated that . % of those who underwent initial treatment became negative for mac [ ] . concerning cam, many studies have reported a correlation between the in vitro drug susceptibility and treatment response [ e ]. furthermore, a study indicated that azm was as effective as cam [ ] . -rbt, which was published in the drug price in nhi scheme in japan in , was also approved for tuberculosis and non-tuberculous mycobacterium infection, including pulmonary mac infection. the drug interactions of rbt are less marked than those of rfp, and rbt is used as a first-choice drug for disseminated mac infection in hiv-infected patients [ ] . on the other hand, rbt induces side effects such as uveitis. in elderly patients, in whom pulmonary mac infection frequently develops, various side effects, such as gastrointestinal disorder, make long-term therapy difficult [ ] . in addition, no study has indicated that rbt is more effective than rfp for pulmonary mac infection in non-hiv-infected patients. therefore, rfp should be selected as a first-choice drug for pulmonary mac infection in non-hiv-infected patients. -kobashi et al. divided patients with pulmonary mac infection into two groups: a group treated with cam, rfp, eb, and sm (intramuscularly injected at mg/kg times a week for months) and a group treated with saline, and conducted a randomized, double-blind, comparative study [ ] . in the sm-treated group, the rate at which the culture of sputum became negative was higher than in the salinetreated group ( . vs. . %, respectively). there has been no high-quality study demonstrating the usefulness of combination therapy with aminoglycosides other than their study. however, various guidelines recommend that combination therapy with sm, amk, or km should be performed for e months in the initial phase of treatment in patients with a cavity or severe nodular/bronchodilatation type infection based on experience [ , , ] . guidelines in the united states recommend sm or amk, and comment that no study has showed which of two drugs, sm and amk, is more effective, although sm has been more frequently used [ ] . in japan, sm or km is recommended [ ] . -based on the results of these studies, the non-tuberculous mycobacterium infection control committee, japanese society for tuberculosis recommends the following doses and administration methods for chemotherapy for pulmonary mac infection in the "opinions regarding chemotherapy for pulmonary non-tuberculous mycobacterium infection---revision in ": rfp: mg/kg (up to mg)/day, once a day, eb: mg/kg (up to mg)/day, once a day, cam: e mg/day ( e mg/kg), once a day or two divided doses ( mg: two divided doses), and sm or km: mg/kg or less (up to mg), intramuscularly injected or times a week [ ] . -a cooperative statement on non-tuberculous mycobacterium infection by the american thoracic society/infectious diseases society of america recommends therapy with cam at to mg/day or azm at mg/day, eb at mg/kg/day, and rfp at mg/kg/day (up to mg) for patients with a cavity or severe nodular/bronchodilatation type infection. in addition, it is recommended that combination therapy with sm or amk ( e mg/kg, e times a week, patients aged over years: mg or less) for e months in the initial phase should be considered [ , ] . -the treatment period is established as about year after the culture becomes negative in the above guidelines, but this is not based on evidence. in the guidelines for the management of non-tuberculous mycobacterium infection, which were published by the british thoracic society, the treatment period of pulmonary mac infection is established as years. in the future, an optimal treatment period should be investigated. ---drugs to be recommended----cam, oral, mg/ times a day or mg/twice a day þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ eb, oral, mg/kg/once a day (maximum: mg/day) * severe-status patients and those with cavity-type lesions in addition to the above regimen, the intramuscular injection of sm or km should be added. abscessus is sometimes susceptible to lzd, tgc (tigecycline), and ketolides, but it is unclear whether there is a correlation between the drug susceptibility and clinical effects [ , ] . -combination therapy with cam and several intravenous antimicrobial drugs (amk, cfx, and ipm/cs) may control the symptoms and progression of pulmonary infection with m. abscessus [ , ] . however, actually, hospitalization is required to administer these intravenous antimicrobials, and the administration period is limited to e months. subsequently, treatment with oral drugs is performed, but cam is the only reliable oral drug, as described above. on the other hand, monotherapy with cam should be avoided from the perspective of resistance induction. although some studies reported combination therapy with lzd or quinolones, its efficacy has not been established. -based on such a background, a combination of surgical resection of the lesion and combination chemotherapy is the only treatment that is expected to achieve the complete cure of pulmonary infection with m. abscessus in which the lesion is localized [ , , ] . ---drugs to be recommended--- a first choice based on the results of a drug susceptibility test, the following antimicrobial drugs should be combined: -cam, oral, mg/ times a day or mg/twice a day þ amk, intravenous drip, mg/kg/once a day þ ipm/cs, intravenous drip, . g/ times a day or g/ times a day * surgery must be considered. the treatment period should be at least months after culture becomes negative. ---executive summary----for the treatment of childhood tuberculosis, several drugs should be combined, and administered for a specific period (aii). -for the treatment of non-tuberculous mycobacterium infection, several drugs should be combined, and administered for a specific period. however, mycobacterium often resists treatment. if there is no treatment response, surgery must be considered (ciii). ---explanation--- [characteristics and classification of the disease] in japan, tuberculosis is still an important infectious disease. when encountering patients with chronic infection who do not respond to general antimicrobial drugs, tuberculosis should be considered for differential diagnosis. mycobacteria that can be cultured are classified into two types: m. tuberculosis complex and non-tuberculous mycobacteria (ntm) [ ] . m. tuberculosis is a major type of m. tuberculosis complex, and has a strong infectivity from humans to humans. childhood tuberculosis is classified into two types based on age [ ] . briefly, primary tuberculosis represented by hilar lymph node tuberculosis and meningitis, which develop following primary infection, is characteristic of infants and children. the interval from infection until onset is short, and the morbidity rate is high. in addition, this disease may lead to a severe condition. pulmonary/hilar lymph node tuberculosis in infants and children is asymptomatic, or the general condition is favorable even in the presence of fever or cough in many cases. when primary tuberculosis is detected based on dyspnea or an unfavorable general condition in addition to fever or cough, many infants/children have military tuberculosis or meningitis. on the other hand, secondary tuberculosis with a cavity lesion or nodular shadow in the lung field is frequent in junior high school students or older. symptoms such as cough, sputum, fever, and thoracic pain are often observed. in most children, the source of infection can be clarified through detailed peripheral contact screening at the time of onset. usually, the source of infection is clarified in / to / of children. it is often their fathers/mothers or grandfathers/grandmothers [ e ]. as childhood tuberculosis does not form a cavity in the lung field, the m. tuberculosis level in the focus is lower than in adults. in many cases, it is difficult to bacteriologically or histologically make a definitive diagnosis in comparison with adult tuberculosis. usually, it is possible to make a definitive diagnosis by comprehensively evaluating epidemiological/clinical information such as opportunities for the source of infection to contact with tuberculosis patients, tuberculin reaction-or quantiferon tb (qft)-based verification of infection, imaging findings suggestive of tuberculosis, such as chest x-ray findings, verification of m. tuberculosis from sputum or gastric juice, and individuals' resistance including the grade of bcg vaccination-acquired immunity and age, as well as by considering treatment responses in some cases. qft is a very useful testing method to quantitatively measure ifn-g and diagnose tuberculosis infection without being influenced by bcg. however, assessment in infants/children should be further examined in the future [ ] . when a definitive diagnosis of pulmonary tuberculosis cannot be made based on chest x-ray findings alone, thoracic ct, which facilitates the detailed evaluation of the presence or absence and extent of tuberculous lesions, is useful for diagnosis. furthermore, imaging findings of tuberculosis do not change in a short period in many cases. non-tuberculous mycobacterium belongs to mycobacterium, the same category as reported for m. tuberculosis. therefore, it is often detected as a mycobacterium-positive smear of sputum, that is, gaffky's positive reaction. initially, some patients are regarded as having infectious tuberculosis, and admitted to a tuberculosis ward. symptoms and imaging findings are also similar between nontuberculous mycobacterium-and m. tuberculosis-infected patients. unless detected bacteria are identified, or unless either gene is detected using the nucleic acid amplification method, it is difficult to differentiate the two types of bacteria. however, it is important to recognize that tuberculosis and non-tuberculous mycobacterium infection are different diseases [ , ] . the most important point is that non-tuberculous mycobacterium infection does not transmit from humans to humans, differing from tuberculosis, an infectious disease in humans. therefore, it is not necessary to isolate the patient, and, as a rule, patients requiring admission should be managed in a general ward. as there are no public hygieneassociated problems, it is not necessary to submit a report to a health center. [type and frequency of causative microorganisms] in japan, the number of patients with childhood tuberculosis has markedly decreased. the number of newly registered patients with tuberculosis decreased from , ( ) to ( ) in children aged e years [ e ]. however, a decrease in the incidence of smear-positive pulmonary tuberculosis, which is important as the source of infection, is not marked in great urban areas. we cannot conclude that the opportunity of infection in children is favorably decreasing; caution is needed. the number of patients who newly develop non-tuberculous mycobacterium infection in japan is estimated to be approximately . in the adult field, it accounts for about / of that of patients who newly develop tuberculosis. however, it is relatively low in children. approximately % of patients with non-tuberculous mycobacterium infection are infected with m. avium complex (m. avium and mycoboterium intracellulare, pulmonary mac infection), and approximately % are infected with m. cansasii. [rules of antimicrobial drug therapy] -the characteristics of antitubercular chemotherapy in children are that children are tolerable to a relatively high dose per body weight in comparison with adults with respect to pharmacokinetics, and that the incidence of side effects is low [ ] . in the pediatric field, -month treatment with inh, rfp, and pza for childhood pulmonary tuberculosis is internationally selected as standard chemotherapy: three drugs, inh, rfp, and pza, are administered every day for the first months, and inh and rfp every day for the subsequent months. when drug resistance is suspected, these drugs should be combined with sm or eb in the initial phase until the results of a resistance test are clarified. in patients with secondary tuberculosis, -drug combination therapy with inh, rfp, pza, and sm (or eb) should be initially performed. in addition, as a rule, follow-up must be continued for years after the completion of treatment [ e ]. on the other hand, drug resistance, referral to another hospital, and discontinued treatment are present among patients who drop out of treatment, although the number of such patients is small. it is necessary to support the resistance and continuation of treatment. in particular, recently, the number of patients in whom it is difficult to continue treatment has increased. potent compliance support must be considered in connection with direct observed therapy (dot) by health centers and welfare activities [ ] . side effects during treatment include liver dysfunction. however, if the maximum ast or alt levels are approximately , administration should be carefully continued without discontinuing treatment. if these levels exceed , treatment should be transiently discontinued, and additional administration at a low dose should be conducted after confirming the normalization of the liver function. the dose should be gradually increased. liver dysfunction requiring a change of treatment is not frequent. furthermore, there is an increase in the serum uric acid level, but continuous treatment leads to normalization. there have been few patients with arthralgia. -prevention of tuberculosis: to prevent the onset of tuberculosis in uninfected persons, bcg vaccination should be performed. concerning its efficacy, a consensus regarding its potent preventive effects on severe disseminated tuberculosis, such as tuberculous meningitis and military tuberculosis, has been reached. considering the importance of tuberculous meningitis prevention, bcg vaccination in the early phase of infancy ( e months after birth, or earlier in accordance with the state of peripheral tuberculosis prevalence) is still necessary in japan [ ] . -treatment for latent tuberculosis: to prevent the onset of tuberculosis in persons with a history of tuberculosis, treatment for latent tuberculosis (conventional chemoprevention) should be conducted. a large-scale controlled study reported that inh therapy decreased the incidence of tuberculosis by approximately e %. for drug administration, the risk of tuberculosis onset should be concretely and flexibly evaluated based on the tuberculin reaction, opportunity of infection, age, and state of bcg vaccination in individual patients [ ] . -treatment for non-tuberculous mycobacterium infection: nontuberculous mycobacterium infection is refractory despite combination therapy with antitubercular drugs. in particular, there is no evidence regarding treatment in children [ ] . the effects of monotherapy are weak, and monotherapy with cam may lead to the appearance of cam-resistant bacteria within a few months [ ] ; therefore, this therapy should be avoided. the responses of m. kansasii to antitubercular drugs are relatively favorable, and cure may be achieved. however, pulmonary mac infection is often resistant to treatment. if there is no response, surgery must be considered. recurrence after the completion of treatment is also often observed. ---drugs to be recommended--- * as the administration period is longer than that for tuberculosis patients, the development of vision disorder should be considered even at these doses. * if there is no response, surgery must be considered. ---explanation---acute bronchitis is characterized by cough that persists for days or more. in most cases, cough persists for e weeks, but spontaneously subsides [ , ] . sputum is present in some cases, but is absent in others. sputum may be purulent even when viral infection is etiologically involved. neither chest x-ray nor ct shows the appearance of a new abnormal shadow, differing from pneumonia. viruses . there is no evidence that infection with other bacteria directly causes acute bronchitis in adults without an underlying disease [ ] . however, in a study using the transtracheal aspiration method in japan, h. influenzae, s. pneumoniae, and m. catarrhalis were primarily isolated in patients diagnosed with bacterial acute bronchitis in the absence of a chronic lower respiratory infectious disease as an underlying disease [ ] . in cases of pertussis, cough persists particularly over a long period, and paroxysmal coughing, inspiratory whooping, and vomiting after coughing may occur [ ] . acute bronchitis caused by m. pneumoniae also induces severe, persistent cough. in cases of influenza, fever, headache, general malaise, and arthralgia are observed. furthermore, acute viral bronchitis may lead to acute bacterial exacerbation in patients with chronic respiratory lesions as underlying diseases; fever and an increase in the amount of purulent sputum are observed. as a rule, when an underlying disease or complication is absent, the routine administration of antimicrobial drugs for acute bronchitis is not recommended [ , ] . to control symptoms such as cough, symptomatic therapy should be performed if necessary. on the other hand, antimicrobial drug treatment with macrolides is indicated for patients with pertussis. treatment after the catarrhal period does not reduce the degree or duration of cough, but antimicrobial drugs are necessary to prevent infection to peripheral persons [ , ] . when performing antimicrobial drug treatment for acute bronchitis caused by m. pneumoniae or c. pneumoniae, macrolides should be selected as first-choice drugs. however, an increase in macrolide-resistant m. pneumoniae must be considered [ ] . in cases of influenza, anti-influenza therapy should be conducted within h after onset [ ] . in patients with underlying diseases or elderly persons with complications, bacterial (e.g., s. pneumoniae) infection may occur following viral infection, although this is not frequent in healthy adults. when acute bronchitis related to bacterial infection, including secondary infection, is strongly suspected based on cough/sputum, fever, leukocytosis, or findings suggestive of the presence of causative microorganisms on gram staining of sputum despite the absence of a new infiltrative shadow on chest x-ray, antimicrobial drug treatment is considered in accordance with treatment for community-acquired bacterial pneumonia [ , ] . ---drugs to be recommended--- when there are no complications such as chronic respiratory diseases, the administration of antimicrobial drugs for acute bronchitis are not recommended as a rule (with respect to the selection of antimicrobial drugs for acute bronchitis complicated by chronic respiratory diseases with secondary bacterial infection, refer to the section " . miller & jones classification of purulent sputum. m : saliva, complete mucous sputum m : mucous sputum containing a small volume of purulent sputum p : sputum in which the purulent area comprises / or smaller p : sputum in which the purulent area comprises / to / p : sputum in which the purulent area comprises / or greater fever and shortness of breath, in addition to bacterial infection-related respiratory symptoms, such as increases in the frequency of cough, volume of purulent sputum, and degree of purulence, from the chronic, stable conditions of underlying diseases, such as copd, bronchiectasis, and old pulmonary tuberculosis. concerning laboratory data, inflammatory responses involving the leukocyte count and crp level are enhanced, and pao is often reduced on blood gas analysis. [imaging findings] imaging findings are necessary to differentiate chronic respiratory disease-related airway infection from pneumonia. the absence of a shadow must be confirmed. ct should also be performed to evaluate underlying diseases such as pulmonary emphysema and bronchiectasis. [estimation of causative microorganisms and gram staining] it is possible to collect sputum in many patients. gram staining is useful for predicting causative microorganisms or differentiating respiratory tract infection in the presence of chronic respiratory disease from persistent infection. according to a study, the tone of sputum suggests the presence of pathogenic microorganisms rather than the degree of purulence; macroscopic examination is also necessary [ ] . sputum involves much information, and is the most important sample. samples should be collected before the administration of antimicrobial drugs. those collected on waking-up early in the morning are ideal. to evaluate the degree of sputum purulence, the miller & jones classification [ ] (table ) is used, but, if samples are evaluated as p or higher, causative microorganisms may be predicted using gram staining. on gram staining, an area where the number of inflammatory cells is large should be initially searched at a low magnification, and detailed observation should be conducted at a high magnification. before the administration of antimicrobial drugs, sputum should always be submitted for a susceptibility test. as causative microorganisms, h. influenzae, p. aeruginosa, m. catarrhalis, and s. pneumoniae are frequently detected. persistent infection with p. aeruginosa is often observed, but it must be differentiated from acute exacerbation based on clinical symptoms and laboratory data. in addition, s. aureus and k. pneumoniae should be considered [ ] . the involvement of atypical pathogens such as c. pneumoniae or mixed infection with viruses and bacteria must also be considered. [treatment] the purpose of treatment is to reduce clinical symptoms, prevent recurrence, prolong the interval until subsequent exacerbation, and inhibit lung tissue damage. the administration of appropriate antimicrobial drugs relieves clinical symptoms, and maintains the respiratory function [ ] . on the other hand, inappropriate antimicrobial drugs may deteriorate the prognosis, inducing recurrence. in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced [ , ] . several studies have reported that new quinolones are more useful than b-lactams [ , e ] . respiratory quinolones have potent antimicrobial activities against all types of causative microorganisms, and against resistant bacteria [ e ]. concerning the administration period, a study indicated that the efficacy of administration for days was similar to that for days, and that the former was safer than the latter. the administration period should be shortened [ ] . ---drugs to be recommended--a. empiric therapy internationally, some studies have supported the usefulness of b-lactams [ , ] . however, in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced, and p. aeruginosa is also sometimes isolated. therefore, the use of b-lactams and macrolides is limited to patients without risk factors. an international comparative study reported that the efficacy of azm sustained-release preparations was similar to that of new quinolones [ ] . however, in japan, the long-term administration of macrolides is performed in many patients; therefore, circumstances differ. beta-lactamase-producing strains are detected in approximately e % of h. influenzae strains. beta-lactamasenegative, ampicillin-resistant (blnar) strains account for approximately %. therefore, when drug susceptibility is unclear, new quinolones should be selected as first-choice oral antimicrobial drugs. if drug susceptibility is clarified, they should be switched to effective and narrow-spectrum drugs. as injection, penicillins should be initially selected, followed by b-lactamase inhibitor-containing penicillins, carbapenems, and new quinolones. ➁ m. catarrhalis beta-lactamase-producing strains account for % of m. catarrhalis strains. as oral antimicrobial drugs, macrolides should be initially selected, followed by b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, and new quinolones. as injection, b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, new quinolones, or carbapenems should be selected. ➂ p. aeruginosa as oral drugs, new quinolones should be selected. as injection, anti-p. aeruginosa penicillins, cephems, monobactums, carbapenems, or new quinolones should be selected. as the drug susceptibility of this type of bacteria markedly differs among strains, drugs should be selected based on the results of culture tests. ➃ s. pneumoniae as oral drugs, penicillins should be initially selected, followed by new quinolones. in patients with a risk of resistant bacteria, respiratory quinolones such as lvfx and grnx should be selected. as injection, penicillins or ctrx should be selected, but carbapenems must be considered in severestatus patients. ---explanation---[characteristics/classification of the disease] dpb is a chronic inflammatory disease of the respiratory tract, which is frequently observed in east asians including japanese. there is no gender difference, and this disease frequently develops in persons aged e years. it is often detected in patients with a history of chronic sinusitis or in those with the concomitant development of chronic sinusitis. this disease is classified as the category of sinobronchial syndrome. [symptoms] the most typical symptoms of dpb are persistent cough and purulent sputum. symptoms such as exertional shortness of breath and dyspnea appear in accordance with disease progression. in patients with a complication of chronic sinusitis, purulent nasal discharge and nasal obstruction are observed. chest x-ray shows pulmonary overexpansion or a diffuse scattered nodular shadow. thoracic hrct reveals a diffuse centrilobular nodular shadow. furthermore, obstructive respiratory dysfunction, hypoxemia, and an increase in the cold agglutinin value ( -fold or more on the hemagglutination method) are observed. [type and frequency of causative microorganisms] in patients with dpb, persistent respiratory tract infection with h. influenzae, s. pneumoniae, or m. catarrhalis is often observed. however, the incidence of persistent infection with p. aeruginosa increases with progression. [long-term macrolide therapy] previously, the prognosis of dpb was unfavorable; respiratory failure gradually progressed through repeated acute exacerbation related to respiratory tract infection, leading to a fatal outcome; however, the prognosis of dpb has been markedly improved since long-term macrolide therapy with low-dose administration of em or other -membered ring macrolides was established [ e ]. early diagnosis/treatment have facilitated the complete cure of dpb. therefore, if once a diagnosis of dpb is made, long-term macrolide therapy should be started promptly. ---drugs to be recommended---➀ persistent infection -the oral administration of em at e mg/day should be continued for months to be evaluated its clinical effects [ ] . -in many cases, an improvement in symptoms (such as a decrease in the volume of sputum) will be achieved within e months after the start of administration. -in addition, an improvement in imaging findings or the respiratory function will be achieved after e months of treatment. croup syndrome is characterized by acute laryngeal stenosisassociated respiratory disturbance such as barking cough, hoarseness, and inspiratory stridor. most lesions involve not only the larynx but also the trachea/bronchus. this disease is sometimes called laryngotracheobronchitis [ ] . etiological factors are classified into two types: infectious and non-infectious (allergy-/ foreign body-related) factors [ , ] . the incidence of infectious croup syndrome is high in infants/children aged monthse years [ , ] . [type and frequency of causative microorganisms] croup syndrome is primarily caused by viruses. parainfluenza virus type is the most common virus [ ] . in addition, parainfluenza virus type / , influenza a/b virus, rs virus, human metapneumovirus, coronavirus, adenovirus, and measles virus are relatively frequently isolated [ , ] . [rules of antimicrobial drug therapy] in most cases, croup syndrome is caused by viruses, and antimicrobial drugs are not necessary. therefore, no study has evaluated the efficacy of antimicrobial drugs in patients with croup syndrome. there are no treatment guidelines regarding croup syndrome in which antimicrobial drugs are recommended [ , ] . ---executive summary---bronchiolitis is caused by viruses, and the administration of antimicrobial drugs is not necessary (ai). ---explanation--- bronchiolitis is an acute, inflammatory, obstructive disease involving the bronchiole. narrowing of the bronchiolar lumen related to mucosal epithelial injury, inflammatory-cell infiltration, interstitial edema, or an increase in mucus secretion causes air trapping in the peripheral respiratory tract, leading to obstructive respiratory disorder. this disease frequently develops in children aged years or younger. however, infants aged months or younger account for % or more [ ] . [type and frequency of causative microorganisms] bronchiolitis is primarily caused by viruses. rs virus accounts for e %. in addition, parainfluenza virus, human metapneumovirus, adenovirus, and influenza virus are relatively frequently isolated [ e ]. [rules of antimicrobial drug therapy] in most cases, bronchiolitis is caused by viruses, and antimicrobial drugs are not necessary. basic treatment is symptomatic therapy. in double-blind comparative studies involving abpc and non-treated groups [ ] , azm and non-treated groups [ ] , and abpc intravenous injection/oral em and non-treated groups [ ] , respectively, there were no significant differences in the admission period or symptom improvement. however, a small-scale doubleblind comparative study reported that the interval until recovery in the cam-treated group was shorter than in the non-treated group [ ] . according to another study, the incidence of secondary bacterial infection during the course of rs virus-related bronchiolitis was . %, and there was no difference between antimicrobial drug-treated and non-treated groups [ ] . therefore, it is not necessary to administer antimicrobial drugs to children with bronchiolitis for routine treatment or the prevention of secondary bacterial infection. however, follow-up must be carefully continued during the course of bronchiolitis. when a diagnosis of secondary bacterial infection-related pneumonia or otitis media is made, antimicrobial drug therapy should be started. ---executive summary---bacterial tracheitis is a bacterial disease with the rapid progression of dyspnea. if symptoms are progressive, antimicrobial drugs should be used even when a definitive diagnosis is not made (aiii). ---explanation--- fever and croup syndrome-like cough/stridor initially appear, and respiratory disorder rapidly progresses, but there is no specific posture, salivation, or dysphagia, which are characteristic of acute epiglottitis. a definitive diagnosis can be made based on characteristic clinical features and purulent secretion in the respiratory tract. in some cases, a lateral view of the larynx on x-ray shows stenosis below the larynx [ ] . this disease frequently develops in children aged e years [ ] . [type and frequency of causative microorganisms] s. aureus-related tracheitis accounts for approximately %, followed by that related to m. catarrhalis, h. influenzae, streptococcus pneumoniae, and streptococcus pyogenes [ e ]. mixed infection with viruses and bacteria is frequent, and parainfluenza virus type i [ ] and influenza a virus [ ] are often detected. [rules of antimicrobial drug therapy] antimicrobial drugs should be intravenously administered for the following reasons: this disease rapidly progresses, and oral administration is difficult in many cases. as empiric therapy, combination therapy with vcm, which may be effective for infection with s. aureus (including mrsa), and third-generation cephems (ctrx, ctx), which have potent antimicrobial activities against m. catarrhalis, h. influenzae, s. pneumoniae, and s. pyogenes, should be performed. the administration period is e days [ ] . ---drugs to be recommended---refer to the section " . pneumonia (children)---drugs to be recommended . definitive therapy---" (p. ). ---executive summary---acute bronchitis is primarily caused by viruses, and the necessity of antimicrobial drug administration is low (ai). when acute bronchitis is caused by m. pneumoniae, c. pneumoniae, or b. pertussis, antimicrobial drugs should be administered if necessary (aiii). secondary infection with s. pneumoniae or h. influenzae may occur, although its incidence is unclear. therefore, when there is no improvement, the administration of antimicrobial drugs should be considered (aiii). ---explanation---[characteristics and classification of the disease] bronchitis causes symptoms such as cough, fever, and general malaise. various causative microorganisms induce inflammation of the epithelial tracheobronchial tissue, leading to the onset of bronchitis. clinically, there are no special findings on auscultation, or only rough respiratory sounds (intermittent accessory murmurs) are heard. chest x-ray does not also show any marked infiltrative shadow. usually, patients in whom the interval after onset is less than weeks are regarded as having acute bronchitis [ ] . however, bronchitis diagnosed in japan slightly differs from that in europe and the united states. the latter primarily causes persistent cough. in japan, patients in whom there are no findings on chest x-ray despite clinical signs of pneumonia or those in whom chest x-ray is not performed are often diagnosed with bronchitis; the disease entity must be arranged. [type and frequency of causative microorganisms] viruses, such as rhinovirus, influenza virus, rs virus, adenovirus, parainfluenza virus, human metapneumovirus, and human bocavirus, account for % of causative microorganisms. m. pneumoniae, c. pneumoniae, and b. pertussis also cause bronchitis, although such cases are relatively rare [ , ] . [rules of antimicrobial drug therapy] acute bronchitis is primarily caused by viruses, and the administration of antimicrobial drugs is not necessary. a metaanalysis compared adults to whom antimicrobial drugs were administered for bronchitis treatment with non-treated adults, and indicated that there was no difference in the efficacy [ ] . few reports on clinical studies involving children have been published, and the scale is small; objective data are insufficient, but no study has reported that antimicrobial drugs are effective [ e ]. however, if secondary bacterial infection following viral infection causes fever, purulent sputum, leukocytosis, or an increase in the crp level, antimicrobial drugs should be administered, considering s. pneumoniae and h. influenzae. other indications for antimicrobial drug administration include m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis with protracted cough. as m. pneumoniae-or c. pneumoniae-related bronchitis tends to show spontaneous cure, the administration of antimicrobial drugs is not always necessary, but the necessity of administration should be evaluated, considering the severity of symptoms and course (with respect to indications and administration methods, refer to the section " . pneumonia (children)".). first-choice drugs for m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis are macrolides. in patients with b. pertussis-related bronchitis, antimicrobial drugs relieve symptoms only during the catarrhal period, but, if b. pertussis-related bronchitis is suspected based on clinical symptoms, previous vaccination, lymphocyte-predominant leukocytosis, an anti-pt antibody titer, and lamp findings, antimicrobial drugs should be used. however, -membered ring macrolides are ineffective for b. pertussis-related bronchitis. symptoms are similar to those of pneumonia, but, when chest x-ray does not show any abnormalities, or, when it is impossible to strictly differentiate bronchitis from pneumonia due to difficulty in chest x-ray, treatment should be performed in accordance with pneumonia. ---drugs to be recommended--- secondary bacterial infection after viral infection (cases in which fever, purulent sputum, leukocytosis, or an increase in the crp level is observed) a first choices -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day <> second choices -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day . definitive therapy ➀ b. pertussis -em, oral, e mg/kg/ times a day -cam, oral, . mg/kg/twice a day -azm, oral, mg/kg/once a day, days ➁ m. pneumoniae ▪ macrolide-sensitive strains -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day ▪ macrolide-resistant strains -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders.) -tflx, oral, mg/kg/twice a day (administration is limited to children aged years or younger in whom mino cannot be used.) ➂ chlamydia (c. pneumoniae, c. psittaci, c. trachomatis) -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day . influenza ---executive summary----both m protein inhibitors and neuraminidase inhibitors (nais) are commercially available as anti-influenza drugs as of july . -influenza viruses a (h n ) and a (h n ) pdm (seasonal influenza) have been reported to be resistant to amantadine, an m protein inhibitor which can be used in japan. the use of this drug as an anti-influenza drug should be avoided for a while [ , ] . -during the influenza outbreak period, anti-influenza therapy should be promptly started based on a clinical diagnosis even when patients with influenza-like symptoms show negative results on a rapid diagnosis kits (because influenza cannot be completely ruled out) [ ] (ai). -nais significantly improve influenza survival, and nai administration within days after onset significantly reduces the rate at which the condition becomes severe [ , ] (ai). -currently, the following nais can be selected in japan. during the outbreak period, an appropriate drug should be selected based on the patient background and latest information on a prevalent influenza strain: -oseltamivir (oral), efficacy: a (h n ) pdm , a (h n ), b, resistance: h y mutant -zanamivir (inhalation), efficacy: type a/b -laninamivir (inhalation), efficacy: type a/b -peramivir (intravenous drip), efficacy: type a/b ---drugs to be recommended---there is no meta-analysis of anti-influenza drugs other than oseltamivir and zanamivir as of july . however, it has been shown that the early introduction of anti-influenza therapy for influenza significantly inhibits not only the mortality and admission rates but also the incidences of influenza-associated pneumonia, otitis media, and ischemic heart disease in comparison with symptomatic therapy. nais such as laninamivir and peramivir have also been confirmed to be as effective as oseltamivir at the time of development [ ] (ai). <>outpatient treatment -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -zanamivir, inhalation, mg/twice a day, days -laninamivir, inhalation, mg/single dose -peramivir, intravenous drip, mg/single dose <>hospital treatment ➀ patients with severe, life-threatening influenza in patients with severe, life-threatening influenza requiring admission, respiratory failure or encephalopathy is present. in either case, the complication must be treated, but, as a rule, nais should be introduced within h after the onset of influenza to obtain their effects. -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -peramivir, intravenous drip, mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➁ non-life-threatening influenza patients with pneumonia -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -peramivir, intravenous drip, mg ( mg for patients in whom the condition may become severe)/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➂ non-life-threatening influenza patients without pneumonia -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -zanamivir, inhalation, mg/twice a day, days -laninamivir, inhalation, mg/single dose -peramivir, intravenous drip, mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) * patients with a (h n ) -basic treatment is the early administration of anti-influenza drugs [ ] . -according to an article, there were no significant differences in the viral level or mortality rate days after the start of administration between double-and standard-dose oseltamivir therapies [ ] . however, treatment was not started within h after onset in all patients in the article. ---executive summary---influenza is caused by influenza virus. antimicrobial drugs are not necessary. it is recommended that neuraminidase inhibitors should be administered within h after onset (ai). if pneumonia or otitis media occurs through secondary bacterial infection, the administration of antimicrobial drugs must be considered (biii). ---explanation---[characteristics and classification of the disease] influenza often appears with sudden fever and shivering/ headache/general malaise/muscular pain/dry cough, followed by marked respiratory or digestive symptoms. in underlying diseasefree children, recovery is achieved after e days [ , ] . however, during the outbreak period, even underlying disease-free children are often admitted with serious symptoms such as encephalopathy, myocarditis, and pneumonia requiring artificial respiration. although the outbreak period is from december until march every year, outbreaks are observed in the summer in some areas [ , ] . [type and frequency of causative microorganisms] since influenza a (h n ) pdm prevailed in , the outbreaks of types of influenza, a (h n ) pdm , a (h n ), and b, have been repeated. their incidences differ among years. [rules of antimicrobial drug therapy] no antimicrobial drug is indicated for influenza. it is recommended that neuraminidase inhibitors should be administered within h after onset [ ] . the doses of neuraminidase inhibitors approved in japan are presented in table . during the course of influenza, bacterial infection such as pneumonia and otitis media may occur. previously, the incidence of secondary bacterial infection in children exceeded % [ ] , but it has been % or less since neuraminidase inhibitors were developed [ , ] . however, bacterial infection is observed in e % of severe-status patients requiring intensive care [ , ] . streptococcus pneumoniae, s. aureus, and h. influenzae are frequently detected as causative microorganisms [ , , , ] . no study has demonstrated that the prophylactic administration of antimicrobial drugs at the onset of influenza prevents secondary bacterial infection. among children with influenza, antimicrobial drug therapy should be considered in those in whom signs of pneumonia or otitis media do not subside despite the administration of a neuraminidase inhibitor. . parasitic diseases of the respiratory system ---executive summary----parasitic diseases are widely distributed throughout the world. in addition to domestic infection, japanese travelers may be infected in overseas endemic areas. furthermore, parasitic diseases always considered in foreign patients from endemic areas [ ] (biv). -when peripheral blood eosinophilia is observed in addition to respiratory symptoms and abnormal findings of the chest imaging, examinations should be performed to differentiate parasitic diseases [ ] (biv). -parasitic diseases of the respiratory system are caused by paragonimus spp., ascaris lumbricoides, ancylostoma duodenale, necator americanus, strongyloides stercoralis, toxocara canis, toxocara cati, wuchereria bancrofti, brugia malayi, or dirofilaria immitis. -there are two major diagnostic tests for parasitic diseases: ( ) detection of parasite eggs or larvae in sputum or stools, and ( ) detection of parasite-specific antibodies using serum or pleural effusion samples (immunodiagnosis). -an anti-parasite antibody screening test against species of parasite is commercially available; paragonimus spp., strongyloides sp., t. canis, d. immitis, ascaris suum, anisakis simplex, gnathostoma spp., fasciola hepatica, clonorchis sinensis, spirometra erinaceieuropaei, and cysticercus cellulosae. -diagnosis/treatment consultations regarding parasitic diseases by the japanese society of parasitology are available (as of ). refer to the homepage (http://jsp.tm.nagasaki-u.ac.jp). ---explanation----paragonimus spp. several paragonimus species are known to cause human infection. paragonimus westermani and p. miyazakii are distributed in japan. infection occurs through the consumption of freshwater crabs (intermediate host: eriocheir japonica, e. sinensis, geothelphusa dehaani) or wild boars (paratenic host:sus scrofa) contaminated with metacercaria (infective larvae) as a raw or insufficiently cooked food [ ] . typical symptoms are cough, sputum, thoracic pain, and exertional dyspnea. in patients with such symptoms, the presence of peripheral blood eosinophilia suggests this disease. in many cases, a diagnosis is made based on the peripheral blood eosinophilia, a history taking of food and table standard doses of neuraminidase inhibitors in children. oseltamivir a oral mg/kg, twice a day, days (the use of this drug should be avoided in children/adolescents aged e years.) zanamivir b inhalation mg, twice a day, days laninamivir inhalation years or older: mg years or younger: mg, single dose peramivir intravenous drip mg/kg, once a day a the preventive administration of this drug at mg/kg (once a day) for days was approved, but is not covered by health insurance. b the preventive administration of this drug at mg (once a day) for days was approved, but is not covered by health insurance. immunodiagnosis. some of paragonimiasis patients are asymptomatic, and the presence of lung lesions is detected on a health checkup. extrapulmonary paragonimiasis such as cutaneous and cerebral paragonimiasis are classically known form of the ectopic infection with paragonimus spp. in cases of cutaneous paragonimiasis, a slowly moving nodular lesion in the subcutaneous tissue is a characteristic symptom. the worms may migrate through mediastinal soft tissues to the brain, causing eosinophilic meningitis or cerebral paragonimiasis in some cases [ ] . since paragonimus spp. is widely distributed around the world, japanese travel to endemic area such as china, korea, thailand, and philippines possible to infect with paragonimus spp. as well as foreign patients from endemic areas, this disease always is considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia [ ] . chest x-ray findings vary: not only pulmonary parenchymal lesions, such as nodular (±cavity formation) and infiltrative shadows, but also pleural lesions, such as the retention of pleural effusion and pneumothorax, are sometimes observed [ ] . many patients show peripheral blood eosinophilia and/or elevated serum total ige. immunodiagnosis to detect parasite-specific antibody has been proven as the most useful and reliable tool. not only patient's serum but also pleural effusion could examine by immunological test. commercially available anti-parasite antibody screening test is including paragomimus spp. in japan, egg-detection rate among paragonimiasis patients nowadays is low; for example . % in sputum and . % in balf [ ] , . % in bronchoscopic aspirate [ , ] . after a definitive diagnosis is made, oral treatment should be started. the type of treatment to be selected, outpatient or hospital treatment, depends on the patient's general condition. however, usually, outpatient treatment is possible. in patients having pleural effusion, pleural fluid must be extensively drained off before starting chemotherapy [ ] (biii). in patients with chronically encapsulated pleural effusion, surgery is required [ ] (ciii). ---drugs to be recommended---a first choice (same dose for adults and children) -praziquantel, oral, mg/kg/ times a day, e days [ ] (aiii) -a. lumbricoides, a. duodenale, n. americanus the larvae of these parasites pass through the lung in the human body, causing asthma-or pneumonia-like symptoms such as transient fever, cough, and dyspnea. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or elevated serum total ige. previously, the condition was called loffler syndrome. currently, it is classified as simple pulmonary eosinophilia in the category of pie syndrome [ ] . symptoms appear e weeks after the oral ingestion of a. lumbricoides embryonated eggs. the latency period of percutaneous infection with n. americanus larvae or percutaneous/oral infection with a. duodenale larvae is approximately days. larvae appeared in the small intestine penetrate the intestinal wall, enter the portal vein, migrate through liver to heart/lung. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults [ ] . simple pulmonary eosinophilia related to parasites spontaneously resolved in a few weeks. in many cases, a definitive diagnosis is made based on parasite eggs detected on a stool examination, although larvae are sometimes detected in sputum [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -pyrantel pamoate, oral, mg/kg/single-dose administration * dry syrup for children is available. * this drug is effective for adult worms in the intestinal tract, but not for larvae migrating in the human body. therefore, a stool examination should be performed weeks after oral administration. if parasite eggs are detected, additional administration should be conducted. <> second choices (same dose for adults and children) -albendazol, oral, mg/single-dose administration -mebendazole, oral, mg/twice a day, days, or mg/single-dose administration -ivermectin, oral, e mg/kg/single-dose administration (after fasting) -s. stercoralis in japan, s. stercoralis is distributed in nansei islands, chain of islands extending from southwestern kyushu to northern taiwan. there are few young persons newly infected with s. stercoralis, but the incidence of infection is high in elderly persons [ ] . internationally, s. stercoralis is widely distributed in tropical/subtropical areas. not only domestic infection but also japanese travel to endemic area possible to infect with this parasite. foreign patients from endemic areas, strongyloidiasis always considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia. s. stercoralis percutaneously infects humans. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults. parasite eggs delivered by adults are hatched while descending the intestinal tract, and larvae excreted in stools. some larvae developed to infective form again invade/transfer through the mucosa around the anus, maintaining a life cycle in the human body. such a mode of infection is termed autoinfection. with the transfer of larvae to the lung, asthma-or pneumonia-like symptoms, such as transient fever, cough, and dyspnea, appear, as indicated for simple pulmonary eosinophilia related to a. lumbricoides, a. duodenale, or n. americanus. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or an increase in the total ige level. in immunocompromised patients, suppressing the cellmediated immunity such as atl patients, hiv/aids patients, those receiving immunosuppressive drugs, the number of s. stercoralis increases through acceleration of autoinfection, and larvae are disseminated in various organs, leading to a severe condition (disseminated strongyloidiasis). in such cases, stridor, bloody sputum, tachypnea, protein-losing gastroenteritis, ileus, and mobile exanthema are observed. furthermore, severe pneumonia related to enteric bacteria disseminated with larvae, lung abscess, or bacterial meningitis concomitantly occurs [ ] . a diagnosis is made based on s. stercoralis larvae detected in stools/sputum. immunological diagnosis is also useful [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -ivermectin, oral, mg/kg/single-dose administration (after fasting), additional administration at the same dose after weeks (ai) * repeated administration must be considered in cases of disseminated strongyloidiasis [ ] . * a study reported that transrectal or percutaneous administration was useful in patients in whom oral administration was difficult due to digestive symptoms [ ] . <> second choice (same dose for adults and children) -albendazol, oral, mg/twice a day, days (bi) -t. canis, t. cati infection to humans occurs through the oral ingestion of the embryonated eggs of t. canis or t. cati [ ] . it also occurs through the consumption of beef/chicken liver or meat contaminated with larvae as a raw or insufficiently heated food. this is considered to be a dominant route of infection in japan [ ] . as humans are not definitive host for t. canis and t. cati, they do not become adults in the human body [ ] . larvae invading the human body transfer to various organs with blood flow through the intestinal mucosa. target organs are the lung, liver, eyes, and central nervous system including spinal cord. toxocariasis is a typical larva migrans. lesions are often observed in the lung/liver (visceral larva migrans). there are few symptoms, or nonspecific symptoms are present. in many cases, a diagnosis is made based on peripheral blood eosinophilia and multiple nodular shadows of the lung or liver on ct. ocular or central nervous symptoms appear in some patients. it is impossible to make a diagnosis on a stool examination due to larva migrans. immunological diagnosis is useful. after a definitive diagnosis is made, treatment should be started. when visceral larva migrans is asymptomatic, follow-up may be continued. ---drugs to be recommended---a first choice (same dose for adults and children) -albendazol, oral, mg/kg/ or times a day, e weeks [ ] (biii) * this drug should be taken with meals for days. a -day period of discontinuation should be established and then restarted drug if required. <> second choices (same dose for adults and children) -albendazol, oral, mg/twice a day, days [ ] (biii) -mebendazole, oral, e mg/twice a day, days [ ] (ciii) -w. bancrofti, b. malayi w. bancrofti and b. malayi are called lymphatic filaria. infection occurs when these parasites are transmitted to humans through mosquitoes. the pathogenesis of lymphatic filariasis is relate to structural and functional abnormality of lymphatic channels induced by parasitized adult worms. infection with w. bancrofti is characterized by febrile attacks, lymphedema/elephantiasis, hydrocele, and chyluria. in patients infected with b. malayi, neither hydrocele nor chyluria is observed, and lymphedema of the lower limbs/ elephantiasis are localized in the lower thighs. microfilaria produced by adult worms is not pathogenic, but rarely induces allergic reactions in the lung, contributing to tropical pulmonary eosinophilia (tpe) [ ] . this condition shows a chronic course, differing from simple pulmonary eosinophilia. cough, dyspnea, and stridor with exacerbation at night are observed. fever, malaise, and weight loss are noted in some patients. on chest x-ray, bilateral reticular nodular lesions are detected. peripheral blood eosinophilia and an increase in the anti-filaria antibody titer are observed. in case of tpe, no microfilaria is detected [ ] . if treatment is not performed, the condition may gradually exacerbate. it is important to adequately make a diagnosis for the differentiation of this disease. in the world, , , persons are infected with lymphatic filaria, but tpe occurs in less than . % of these. the risk of this disease in travelers is unclear, and most patients consist of foreign persons from endemic areas [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -diethylcarbamazine, mg/kg/ times a day, days * in cases of malayan filariasis, marked side effects, such as digestive symptoms, fever, lymphangitis/ lymphadenitis, and orchitis/epididymitis, may appear. therefore, a half dose ( mg/kg) should be administered times a day for days [ ] . -d. immitis d. immitis parasitize in the dog right ventricle and pulmonary artery, where mature female worms produce microfilaria that circulate in peripheral blood. infection to humans is mediated by microfilaria-ingesting mosquitoes. d. immitis larvae in human subcutaneous tissue inserted by mosquito bite, some larvae migrate to the heart and die. dead worms produce infarcts then they lodge in pulmonary vessels. there are few symptoms [ ] . in patients with symptoms, thoracic pain, cough, bloody sputum, stridor, and fever have been reported. typical chest x-ray findings include solitary coin lesions. in many cases, an abnormal shadow of the chest x-ray is indicated on a health checkup, and d. immitis infection is pathologically diagnosed from a tissue specimen extirpated under a tentative diagnosis of lung cancer. at this point, peripheral blood eosinophilia is rarely observed [ ] . ---drugs to be recommended (same dose for adults and children)----as a diagnosis is made using pathological specimens in many patients, oral treatment is not necessary. -if necessary, diethylcarbamazine at mg/kg should be orally administered times a day for days. ---precautions for each drug--- in the japanese package inserts, it is described that this drug at mg/kg/day should be orally administered twice a day for days for the treatment of paragominiasis. however, we recommend administration at mg/kg/day ( times a day) for e days based on reference no. . the incidence of side effects is low, but fever, abdominal discomfort, nausea, diarrhea, and headache are sometimes observed. masahumi seki: speaker's honorarium from shionogi & co., ltd., and taisho toyama pharmaceutical co., ltd dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the committee for the japanese respiratory society guidelines for the management of respiratory infections. the japanese respiratory society guidelines for the management of community-acquired pneumonia in adults. tokyo: the japanese respiratory society nationwide survey on the guide-lines for the management of community-acquired adult pneumonia : validation of differentiation between bacterial pneumonia and a typical pneumonia evaluation of an assessment system for the jrs : a-drop for the management of cap in adults sputum gram stain assessment in community-acquired bacteremic pneumonia assessment of the usefulness of sputum culture for diagnosis of communityacquired pneumonia using the port predictive scoring system detection of legionella pneumophila antigen in urine samples by the binaxnow immunochromatographic assay and comparison with both binax legionella urinary enzyme immunoassay (eia) and biotest legionella urin antigen eia rapid urinary antigen test for diagnosis of pneumococcal community-acquired pneumonia in adults revision of the severity rating and classification of hospital-acquired pneumonia in the japanese respiratory society guidelines the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of nursing and healthcare-associated pneumonia. the japanese respiratory society the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. the japanese respiratory society nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of japanese society of chemotherapy, japanese association for infectious diseases, and japanese society for clinical microbiology in : general view of the pathogens' antibacterial susceptibility prospective multi center study of the causative organisms of communityacquired pneumonia in adults in japan clinical evaluation of levofloxacin versus oral b-lactams for acute exacerbation of copd fluoroquinolone exposure prior to tuberculosis diagnosis is associated with an increased risk of death a worldwide perspective of atypical pathogens in community-acquired pneumonia infectious diseases to be focused on mycoplasma pneumoniae combination anti-biotic therapy with macrorides improves survival in intubated patients with community-acquired pneumonia antibiotic therapy for klebsiella pneumoniae bacteremia: implications of production of extended-spectrum b-lactamases dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases the study group on antimicrobial susceptibility of pathogens isolated from respiratory infections. antimicrobial susceptibility of pathogens isolated from more than patients with infectious respiratory diseases: a -year longitudinal study performance standards for antimicrobial susceptibility testing. twenty-third informational supplement. clsi resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in rationale for revised penicillin susceptibility breakpoints versus streptococcus pneumoniae: coping with anti microbial susceptibility in an era of resistance bts guidelines for the management of community acquired pneumonia in adults: update cap moxifloxacin study group. oral moxifloxacin vs high-dosage amoxicillin in the treatment of mild-to-moderate, community-acquired, suspected pneumococcal pneumonia in adults spafloxacin resistance in clinical isolates of streptococcus pneumoniae: involvement of multiple mutations in gyra and parc genes nationwide surveillance of bacterial respiratory pathogens condected by japanese society of chemotherapy in : general view of the pathogens' antibacterial susceptibility molecular characteristics of extended-spectrum b-lactamase-producing escherichia coli in japan: emergence of ctx-m- -producing e. coli st macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of communityacquired pneumonia macrolide-resistant mycoplasma pneumoniae in adolescents with communityacquired pneumonia rapid effectiveness of minocycline or doxycycline against macrolide-resistant mycoplasma pneumoniae infection in a outbreak among japanese children in vitro and in vivo antibacterial activities of dc- a, a new fluoroquinolone in vitro activities of antimicrobial agents against macrolide-resistant mycoplasma pneumoniae isolates from pediatric patients: results from a multicenter surveillance study antimicrobial chemotherapy for legionnaires disease: levofloxacin versus macrolides macrolides versus quinolones in legionella pneumonia: results from the community-acquired pneumonia organization international study community-acquired legionella pneumonia in the intensive care unit: impact on survival of combined anti biotic therapy infection due to legionella species other than l. pneumophila in vitro activities of azithromycin, clarithromycin, and other antibiotics against chlamydia pneumoniae dissemination of panton-valentine leukocidine positive metchicillinresistant staphylococcus aureus in okinawa comparative effectiveness of nafcillin or cefazolin versus vancomycin in metchicillin susceptible staphylococcus aureus bacteremia the streptococcus milleri group as a cause of pulmonary infections review of cases of pneumonia caused by streptococcus pyogenes distribution of emm type and antibiotic susceptibility of group a streptococci causing invasive and non invasive diseases in vitro activities of sitafloxacin compared with several fluoroquinolones against streptococcus anginosus and streptococcus constellatus moraxella catarrhalis: from emerging to established pathogen antimicrobial susceptibility of anaerobic isolates from patients with dentoalveolar infection to oral antibiotics community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. tokyo: the japanese respiratory society infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia guidelines for the management of hospital-acquired pneumonia in the uk: report of the working party on hospital-acquired pneumonia of the british society for antimicrobial chemotherapy hap study group: multicenter survey on hospital-acquired pneumonia and the clinical efficacy of first-line antibiotics in japan international conference for the development of consensus on the diagnosis and treatment of ventilator-associated pneumonia ventilator-associated pneumonia short-course empiric antibiotic therapy for patients with pulmonary infiltrates in the intensive care unit. a proposed solution for indiscriminate antibiotic prescription comparison of vs days of antibiotic therapy for ventilator-associated pneumonia in adults: a randomized trial nosocomial pneumonia: a multivariate analysis of risk and prognosis study group of the italian society of internal medicine. outcomes of patients hospitalized with community-acquired, health care-associated and hospital-acquired pneumonia implementation of guidelines for management of possible multidrugresistant pneumonia in intensive care: an observational, multicentre cohort study diagnosing pneumonia during mechanical ventilation: the clinical pulmonary infection score revisited value of gram stain examination of lower respiratory tract secretions for early diagnosis of nosocomial pneumonia evaluation of bronchoscopic techniques for the diagnosis of nosocomial pneumonia invasive and noninvasive strategies for management of suspected ventilatorassociated pneumonia: a randomized trial hospital-acquired pneumonia in the st century: a review of existing treatment options and their impact on patient care role of serial routine microbio-logic culture results in the initial management of ventilator-associated pneumonia pneumonia essentials clinical practice guidelines for hospital-acquired pneumonia and ventilatorassociated pneumonia in adults the committee for the japanese society of chemotherapy guidelines for tdm of antimicrobial agents. guidelines for tdm of antimicrobial agents the role of an aerobes in patients with ventilatorassociated pneumonia and aspiration pneumonia: a prospective study mandell, douglas, and bennett's principles and practice of infectious diseases physiologic factors contributing to a transition in oral immunity among mechanically ventilated adults indigenous microflora and innate immunity of the head and neck antimicrobial treatment of hospital-acquired pneumonia nosocomial fever of unknown origin. fever of unknown origin. ny: informa health-care b-lactam resistance in the st century updated functional classification of b-lactamases. antimicrob mrsa working party of he british society for antimicrobial chemotherapy. guidelines ( ) for the prophylaxis and treatment of methicillin-resistant staphylococcus aureus (mrsa) infections in the united kingdom clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant staphylococcus aureus infections in adults and children linezolid versus vancomycin for the treatment of infections caused by methicillin-resistant staphylococcus aureus in japan response to emerging infection leading to outbreak of linezolid-resistant enterococci nosocomial spread of enterococcus faecium resistant to vancomycin and linezolid in a tertiary medical center emergence of linezolid resistance in the vancomycin-resistant enterococcus faecium multilocus sequence typing c epidemic lineage clinical microbiology of coryneform bacteria linezolid vs vancomycin: analysis of two double-blind studies of patients with methicillin-resistant staphylococcus aureus nosocomial pneumonia the linezolid nosocomial pneumonia study group. linezolid (pnu- ) versus vancomycin in the treatment of hospitalized patients with nosocomial pneumonia: a randomized, double-blind, multi center study guidelines for uk practice for the diagnosis and management of methicillin-resistant staphylococcus aureus (mrsa) infections presenting in the community oral antibiotic therapy of serious systemic infections daptomycin versus standard therapy for bacteremia and endocarditis caused by staphylococcus aureus treatment of severe pneumonia in hospitalized patients: results of a multicenter, randomized, double-blind trial comparing intra-venous ciprofloxacin with imipenem-cilastatin ventilator-associated pneumonia caused by potentially drug-resistant bacteria acinetobacter infection acinetobacter baumannii: emergence of a successful pathogen alvarez-beltr an m. levofloxacin in the treatment of ventilator-associated pneumonia performance standards for antimicrobial susceptibility testing; twentieth informational supplement. m es . wayne pa: clinical and laboratory standards institute the committee for the japanese respiratory society guidelines for the management of nursing and healthcare associated pneumonia. the japanese respiratory society guidelines for the management of nursing and healthcare associated pneumonia. tokyo: the japanese respiratory society health-care-associated pneumonia among hospitalizes patients in a japanese community hospital a prospective comparison of nursing homeacquired pneumonia with health care associated pneumonia in nonintubated elderly characteristics and disease severity of healthcare-associated pneumonia among patients in a hospital in kitakyushu health care-associated pneumonia requiring hospital admission: epidemiology, antibiotic therapy, and clinical outcomes epidemiology and outcomes of healthcare-associated pneumonia: results from a large us database of culture-positive pneumonia what is health-care associated pneumonia, and how should it be treated? indicators of potentially drug-resistant bacteria in severe nursing home acquired pneumonia community-acquired pneumonia through enterobacteriaceae and pseudomonas aeruginosa: diagnosis, incidence and predictors the hcap gap: differences between self-reported practice patterns and published guidelines for health care-associated pneumonia how good is the evidence for the recommended empirical antimicrobial treatment of patients hospitalized because of community-acquired pneumonia? a systematic review macroride-based regimens and mortality in hospitalized patients with communityacquired pneumonia: a systematic review and meta-analysis respiratory fluoroquinolones for the treatment of community-acquired pneumonia: a meta-analysis of randomized controlled trials combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia risk factors and response to antibiotic therapy in adults with bacteremic pneumonia caused by penicillin-resistant pneumococci a comparative study of levofloxacin and ceftriaxone in the treatment of hospitalized patients with pneumonia effect of a clinical pathway to reduce hospitalizations in nursing home residents with pneumonia: a randomized controlled trial randomized controlled trial of sequential intravenous (i.v.) and oral moxifloxacin compared with sequential i.v. and oral co-amoxiclav with or without clarithromycin in patients with community-acquired pneumonia requiring initial parenteral treatment moxifloxacin monotherapy is effective in hospitalized patients with community-acquired pneumonia: the motiv studyda randomized clinical trial azithromycin vs cefuroxime plus erythromycin for empirical treatment of communityacquired pneumonia in hospitalized patients: a prospective, randomized, multicenter trial meta-analysis of randomized controlled trials on the comparative efficacy and safety of azithromycin against other antibiotics for lower respiratory tract infections efficacy of azithromycin in the treatment of community-acquired pneumonia, including patients with macrolide-resistant streptococcus pneumoniae infection in vitro antibacterial activity of dx- , a novel des-fluoro ( ) quinolone management of patients with community-acquired pneumonia treated in hospital in sweden a comparison of culture-positive and culture-negative health-care-associated pneumonia effect of antibiotic guidelines on outcomes of hospitalized patients with nursing home-acquired pneumonia health-care-associated pneumonia is primarily due to aspiration pneumonia ertapenem versus cefepime for initial empirical treatment of pneumonia acquired in skilled-care facilities or in hospitals outside the intensive care unit selectivity of ertapnem for psudomonas aeruginosa mutants cross-resistant to other carbapenems retrospective investigation of the clinical effects of tazobactam/piperacillin and sulbactam/ampicillin on aspiration pneumonia caused by klebsiella pneumoniae japanese society of chemotherapy and journal of japanese association for anaerobic infection research. guidelines for treatment of anaerobic infections prospective randomized comparison study of piperacillin/tazobactam and meropenem for health care-associated pneumonia in japan clinical evaluation of bedridden patients with pneumonia receiving home health care national survey on the susceptibility of bacteroides fragilis group: report and analysis of trends for d examination of anaerobes in patients with respiratory infectious diseases using the transtracheal aspiration method impact of initial antibiotic choice on clinical outcomes in community-acquired pneumonia: analysis of a hospital claims-made database management of community-acquired pneumonia macrolide antibiotics and survival in patients with acute lung injury aspiration pneumonitis and aspiration pneumonia the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. tokyo: the japanese respiratory society japanese study group on aspiration pulmonary disease. diagnosis and treatment for aspiration pulmonary disease epidemiology of community-acquired pneumonia in the elderly risk factors for pneumonia and other lower respiratory tract infection in elderly residents of long term care facilities pneumonia in residents of long-term care facilities: epidemiology, management, and prevention hospitalized nursing home-acquired pneumonia: comparison with community-acquired pneumonia in older adults high incidence of aspiration pneumonia in community-and hospital-acquired pneumonia in hospitalized patients: a multi-center, prospective study in japan rethinking the concepts of communityacquired and health-care-associated pneumonia effectiveness of a multi dimensional approach for prevention of ventilator-associated pneumonia in adult intensive care units from developing countries of four continents: findings of the international nosocomial infection control consortium the sanford guide to antimicrobial therapy pathogenic bacteria in aspiration pneumonia microbiology of severe aspiration pneumonia in institutionalized elderly ampicillin þ sulbactam vs clindamysin ± cephalosporin for the treatment of aspiration pneumonia and primary lung abscess tazobactam/piperacillin for moderate-to-severe pneumonia in patients with risk for aspiration: comparison with imipenem/cilastatin indicators of potentially drug-resistant bacteria in severe nursing home-acquired pneumonia changing bacteriology of adult community-acquired lung abscess in taiwan: klebsiella pneumoniae versus anaerobes survey regarding antimicrobial drug therapy for aspiration pneumonia treatment of aspiration pneumonia based on the antimicrobial susceptibility pattern of oral bacteria pathogens reappraisal of clindamycin iv monotherapy for treatment of mild-tomoderate aspiration pneumonia in elderly patients regional variation in the prevalence of extended-spectrum b-lactamase-producing clinical isolates in the asia-pacific region (sentry d ) special issue, current topics on urological infectious diseases---refractory factors and management---, resistance of bacteria isolated from patients with urinary tract infection to antimicrobial drugs and methods to prevent drug resistance aspiration pneumonia in adults uptodate, topic version . . this topic last updated anaerobic bacterial infection of the lung evaluation of the efficacy and safety of biapenem against pneumonia in the elderly and a study on its pharmacokinetics treatment of ventilator-associated pneumonia with piperacillin-tazobactam/amikacin versus ceftazidime/amikacin: a multicenter, randomized controlled trial prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis ventilatorassociated pneumonia due to pseudomonas aeruginosa preemptive antibiotic treatment based on gram staining reduced the incidence of ards in mechanically ventilated patients ventilator-associated tracheobronchitis: the impact of targeted antibiotic therapy on patient outcomes antimicrobial treatment for ventilator-associated tracheobronchitis: a randomized, controlled, multicenter study imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign voriconazole versus amphotericin b for primary therapy of invasive aspergillosis liposomal amphotericin b as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (ambiload trial) caspofungin use in daily clinical practice for treatment of invasive aspergillosis: results of a prospective observational registry micafungin (fk ), alone or in combination with other systemic antifungal agents, for the treatment of acute invasive aspergillosis intravenous itraconazole followed by oral itraconazole in the treatment of invasive pulmonary aspergillosis in patients with hematologic malignancies, chronic granulomatous disease, or aids combination of voriconazole and caspofungin as primary therapy for invasive aspergillosis in solid organ transplant recipients: a prospective, multicenter, observational study combination antifungal therapy for invasive aspergillosis liposomal amphotericin b in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies. a randomized pilot study triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation intravenous micafungin versus voriconazole for chronic pulmonary aspergillosis: a multicenter trial in japan a doubleblind comparative study of the safety and efficacy of caspofungin versus micafungin in the treatment of candidiasis and aspergillosis aspergilloma: a series of surgical cases fluconazole monotherapy for cryptococcosis in non-aids patients cryptococcosis in human immunodeficiency virus-negative patients in the era of effective azole therapy primary treatment of zygomycosis with liposomal amphotericin b: analysis of cases the deferasirox-ambisome therapy for mucormycosis (defeat mucor) study: a randomized, double-blinded, placebo-controlled trial pneumocystis pneumonia the national institute of health-university of california expert panel for corticosteroids as adjunctive therapy for pneumocystis pneumonia. consensus statement on the use of corticosteroids as adjunctive therapy for pneumocystis pneumonia in the acquired immunodeficiency syndrome comparison of atovaquone ( c ) with trimethoprim-sulfamethoxazole to treat pneumocystis carinii pneumonia in patients with aids international consensus guidelines on the management of cytomegalovirus in solid organ transplantation oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients forscarnet vs ganciclovir for cytomegalovirus (cmv) antigenemia after allogeneic hemopoietic stem cell transplantation (hsct): a randomised study treatment of interstitial pneumonitis due to cytomegalovirus with ganciclovir and intravenous immune globulin: experience of european bone marrow transplant group committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia. the guidelines for the management of respiratory infectious diseases in children in japan limitations of the etiological diagnosis of bronchopulmonary infectious diseases---appearance of emb community-acquired pneumonia in children fifth pediatric respiration seminar. review of chest x-ray findings of pneumonia, childhood pneumonia with respect to causative pathogens world health organization: programme for the control of acute respiratory infections: technical bases for the who recommendations on the management of pneumonia in children at first-level health facilities pneumonia in pediatric outpatients: cause and clinical manifestations etiology of pediatric inpatients with pneumoniaeanalysis of clinical symptoms, physical examination and simple laboratory findings basic examination regarding "the guidelines for the management of pneumonia in children", selection of treatment differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital current status of pneumonia in children, review of chest x-ray findings problems associated with high prevalence of multidrug-resistant bacteria in patients with community-acquired infections nationwide survey of the development of drug-resistant pathogens in the pediatric field: drug sensitivity of streptococcus pneumonia in japan nationwide survey of the development of drug-resistance in the pediatric field: drug sensitivity of haemophilus influenzae in japan macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of communityacquired pneumonia th informational supplement. performance standards for antimicrobial susceptibility testing annual change of ampicillin susceptibility of h. influenzae and the clinical efficacy of penicillins in bronchopulmonary infections caused by b-lactamase negative h. influenzae with an ampicillin-minimum inhibitory concentration (mic) of mg/ml clinical effects of piperacillin and tazobactam/piperacillin on haemophilus influenzae lower respiratory tract infection in pediatric patients clinical studies on lower respiratory tract infections caused by moraxella catarrhalis in childhood ( st report) clinical studies on lower respiratory tract infections caused by moraxella catarrhalis in childhood ( nd report)-indirect pathogenicity of moraxella catarrhalis increased prevalence of penicillin-resistant viridans group streptococci in japanese children with upper respiratory infection treated by beta-lactam agents and in those with oncohematologic diseases strong macrolide resistance in children/ pandemic of mycoplasma pneumoniae infection macrolide resistance of mycoplasma pneumoniae in primary hospitals committee to prepare the guidelines for the management of respiratory infectious diseases in children, vaccination/infectious disease control committee of the japan pediatric society. supplement revision ( ) of the guidelines for the management of respiratory infectious diseases in children in regarding the diagnosis and treatment of pneumonia with mycoplasma pneumoniae in children ventilator-associated pneumonia in neonatal and pediatric intensive care unit patients committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease . blood disease. guidelines for the management of respiratory infectious diseases in children committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease . immunodeficiency. guidelines for the management of respiratory infectious diseases in children clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: update by the infectious diseases society of america indicative therapy during blood disease treatment experience on long-term therapy with macrolides in the field of pediatrics refractory respiratory infectious diseases. tokyo: van medical co itraconazole to prevent fungal infections in chronic granulomatous disease special issue---respiratory infectious disease---neonatal pneumonia committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease . neonates. guidelines for the management of respiratory infectious diseases in children nosocomial infection in a newborn intensive care unit (nicu), south korea a -year prospective surveillance of nosocomial infections in neonatal intensive care units risk factors and outcomes for ventilatorassociated pneumonia in neonatal intensive care unit patients usefulness of gram staining of tracheal aspirates in initial therapy for ventilatorassociated pneumonia in extremely preterm neonates childhood infectious diseases-i. review of childhood infectious diseases with respect to the type of infection, . neonatal infectious diseases chapter . various infectious diseases and how to select/use drugs, neonatal period, neonatal infectious diseases neonatal care/basic knowledge v. management method, use of antimicrobial drugs antimicrobial/antiviral drugs for severe infectious diseases iii. clues to diagnosis and selection of drugs in patients with various infectious diseases/conditions, infectious diseases in neonates medical and surgical treatment of parapneumonic effusions: an evidence-based guideline parapneumonic effusions and empyema aerobic and anaerobic microbiology of empyema. a retrospective review in two military hospitals a retrospective review of cases of anaerobic empyema and update of bacteriology anaerobic microbiology in cases of pleural empyema: a bulgarian study thoracic empyema in patients with community-acquired pneumonia controlled trial of intrapleural streptokinase for pleural infection antibiotic therapy of pleural empyema pharmacokinetics and therapeutic efficacy of gentamicin in an experimental pleural empyema rabbit model amikacin concentrations in uninfected post thoracotomy pleural fluid and in serum after intravenous and intrapleural injection penetration of aminoglycosides in uninfected pleural exudates and in pleural empyemas choice of first intervention is related to outcomes in the management of empyema; discussion d management of pleural infection in adults: british thoracic society pleural disease guideline committee to prepare the guidelines for the management of respiratory infectious diseases in children. pleuritis/pyothorax. guidelines for the management of respiratory infectious diseases in children pleural tuberculosis radiological study of mycoplasma pneumonia in children principle and practice of pediatric surgery committee to prepare the guidelines for the management of respiratory infectious diseases in children. primary diseases for which vaccination should be performed . tuberculosis. guidelines for the management of respiratory infectious diseases in children controlled clinical trial of four short-course ( -month) regimens of chemotherapy for treatment of pulmonary tuberculosis controlled clinical trial of four short-course ( -month) regimens of chemotherapy for treatment of pulmonary tuberculosis controlled trial of , , and months of pyrazinamide in -month, three-times-weekly regimens for smear-positive pulmonary tuberculosis, including an assessment of a combined preparation of isoniazid, rifampin, and pyrazinamide. results at months a controlled trial of months' chemotherapy in pulmonary tuberculosis. final report: results during the months after the end of chemotherapy and beyond short-course antituberculous chemotherapy for pulmonary and pleural disease: years' experience in clinical practice joint tuberculosis committee of the british thoracic society. chemotherapy and management of tuberculosis in the united kingdom: recommendations infectious diseases society of america: treatment of tuberculosis the japanese society for tuberculosis. guidelines for the management of tuberculosis rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in hiv-negative patients: a randomised clinical trial usphs tuberculosis short-course chemotherapy trial :effectiveness, toxicity, and acceptability. the report of final results controlled chemoprophylaxis trials in tuberculosis. a general review prophylactic effect of isoniazid in young tuberculin reactors isoniazid chemoprophylaxis of tuberculosis international union against tuberculosis committee on prophylaxis. efficacy of various durations of isoniazid preventive therapy for tuberculosis: five years of follow-up in the iuat trial clinical practice. latent tuberculosis infection how much isoniazid is needed for prevention of tuberculosis among immune competent adults? this statement was endorsed by the council of the infectious diseases society of america. (idsa) madras/british medical research council. a double-blind placebo-controlled clinical trial of three antituberculosis chemoprophylaxis regimens in patients with silicosis in hong kong rifampin preventive therapy for tuberculosis infection: experience with adolescents adverse events with months of rifampin therapy or months of isoniazid therapy for latent tuberculosis infection: a randomized trial the research committee of the british thoracic society. pulmonary disease caused by mycobacterium avium-intracellulare in hiv-negative patients: five-year follow-up of patients receiving standardized treatment clarithromycin regimens for pulmonary mycobacterium avium complex. the first patients effect of clarithromycin regimen for mycobacterium avium complex pulmonary disease clarithromycin in the treatment of mycobacterium avium lung infections in patients without aids initial clarithromycin monotherapy for mycobacterium avium-intracellulare complex lung disease clinical and molecular analysis of macrolide resistance in mycobacterium avium complex lung disease azithromycin activity against mycobacterium avium complex lung disease in patients who were not infected with human immunodeficiency virus two controlled trials of rifabutin prophylaxis against mycobacterium avium complex infection in aids infectious disease society of america. an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases a double-blind randomized study of aminoglycoside infusion with combined therapy for pulmonary mycobacterium avium complex disease sub committee of the joint tuberculosis committee of the british thoracic society. management of opportunist mycobacterial infections: joint tuberculosis committee guidelines the scientific assembly for infection and tuberculosis of the japanese respiratory society. guidelines for chemotherapy of pulmonary nontuberculous mycobacterial disease e revised version this official statement of the american thoracic society was approved by the board of directors treatment of pulmonary disease due to mycobacterium kansasii: recent experience with rifampin detection of rpob mutations in rifampicin-resistant mycobacterium kansasii treatment of pulmonary disease caused by mycobacterium kansasii: results of vs months' chemotherapy clinical features of pulmonary disease caused by rapidly growing mycobacteria. an analysis of patients clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria pulmonary disease caused by rapidly growing mycobacteria mycobacterium abscessus from respiratory isolates: activities of drug combinations antibiotic treatment of mycobacterium abscessus lung disease: a retrospective analysis of patients clinical and microbiologic outcomes in patients receiving treatment for mycobacterium abscessus pulmonary disease supervised by the tuberculosis and infectious diseases control division, health service bureau, ministry of health, labour and welfare. statistics of tuberculosis in special issue---new vaccination system of bcg-for the prevention of tuberculosis based on scientific grounds usefulness of interferon-gamma release assays for diagnosing tb infection and problems with these assays subsidy for scientific research by the ministry of health, labour and welfare in , research business for new/old infectious diseases such as new influenza "evaluation of tuberculosis control strategies and study regarding the development/application of new diagnostic/treatment techniques japanese new guidelines for nontuberculous mycobacterial pulmonary disease clinical practice. acute bronchitis uncomplicated acute bronchitis principles of appropriate antibiotic use for treatment of uncomplicated acute bronchitis: background chronic cough due to acute bronchitis: accp evidence-based clinical practice guidelines clinical study of acute bacterial bronchitis health burden of pertussis in adolescents and adults recommended antimicrobial agents for the treatment and post-exposure prophylaxis of pertussis: cdc guidelines antibiotics for whooping cough (pertussis). cochrane database syst rev increased macrolide resistance of mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia antivirals for treatment of influenza: a systematic review and meta-analysis of observational studies the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of respiratory tract infection. basic concept of respiratory tract infection in adults. tokyo: the japanese respiratory society antibiotic prescribing for self limiting respiratory tract infections in primary care: summary of nice guidance antibiotic therapy in exacerbation of chronic obstructive pulmonary disease characterisation of exacerbations of chronic bronchitis and copd in europe: the giant study indicators of bacterial infection in patients with acute exacerbation of chronic bronchitis for application in clinical trials of anti bacterial drugs infection in the pathogenesis and course of chronic obstructive pulmonary disease a multicentre surveillance study on the characteristics, bacterial aetiologies and in vitro antibiotic susceptibilities in patients with acute exacerbations of chronic bronchitis clinical response of levofloxacine mg qd to respiratory tract infection open study of sitafloxacin in patients with respiratory tract infections e pk/pd study efficacy and safety of moxifloxacin in acute exacerbations of chronic bronchitis: a prospective, multicenter, observational study (avanti) five-day moxifloxacin therapy compared with -day co-amoxiclav therapy for the treatment of acute exacerbation of chronic bronchitis levofloxacin mg qd for five days versus amoxicillin/clavulanate mg/ mg bid for ten days for treatment of acute bacterial exacerbation of chronic bronchitis: a post hoc analysis of data from severely ill patients shortcourse antibiotic treatment in acute exacerbations of chronic bronchitis and copd: a meta-analysis of double-blind studies shortversus long-duration antimicrobial treatment for exacerbations of chronic bronchitis: a meta-analysis sputum colour and bacteria in chronic bronchitis exacerbations: a pooled analysis antibiotics are associated with lower relapse rates in outpatients with acute exacerbations of copd in vitro antibacterial activity of garenoxacin molecular characterization of quinolone-insensitive streptococcus pneumonia isolates from japanese patients comparative mutant prevention concentration and mutant selection window of sitafloxacin versus other quinolones using strains of haemophilus influenzae with decreasing susceptibility to levofloxacin randomized, double-blind study comparing -and -day regimens of oral levofloxacin in patients with acute exacerbation of chronic bronchitis the efficacy of cefditoren pivoxil in the treatment of lower respiratory tract infections, with a focus on the per-pathogen bacteriologic response in infections caused by streptococcus pneumoniae and haemophilus influenzae: a pooled analysis of seven clinical trials role of oral extended-spectrum cephems in the treatment of acute exacerbation of chronic bronchitis efficacy and safety of -day azithromycin versus -day moxifloxacin for the treatment of acute bacterial exacerbations of chronic bronchitis final draft report of the treatment guideline of dpb. diffuse lung disease study group, scientific research specific disease study business by the ministry of health, labour and welfare. study report in macrolides for diffuse panbronchiolitis study regarding the clinical efficacy of long-term therapy with low-dose erythromycin for diffuse panbronchiolitis long-term therapeutic effects of erythromycin and newquinolone antibacterial agents on diffuse panbronchiolitis longterm low-dose administration of erythromycin to patients with diffuse panbronchiolitis report by the diffuse lung disease survey/study group of the ministry of health and welfare in diffuse panbronchiolitis: followup ct examination effects of long-term erythromycin therapy on diffuse panbronchiolitis leading to chronic respiratory failure long term effect of erythromycin therapy in patients with chronic pseudomonas aeruginosa infection improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin diffuse panbronchiolitis: role of macrolides in therapy efficacy of lomg-term macrolide antibiotic therapy in patients with diffuse panbronchiolitis: comparison between hla-b -positive and negative cases long-term macrolide antibiotic therapy in the treatment of chronic small airway disease clinically mimicking diffuse panbronchiolitis analysis of cases allowed to cease erythromycin therapy for diffuse panbronchiolitisecomparative study between patients with cessation of the therapy and patients continuing the therapy long-term efficacy and safety of clarithromycin treatment in patients with diffuse panbronchiolitis roxithromycin treatment in patients with chronic lower respiratory tract diseaseeits clinical efficacy and effect on cytokine clinical and immune regulatory effects of roxithromycin therapy for chronic respiratory tract infection study on azithromycin in treatment of diffuse panbronchiolitis effects of long-term low-dose azithromycin in patients with non-cf bronchiectasis effect of long-term, low-dose erythromycin on pulmonary exacerbations among patients with non-cystic fibrosis bronchiectasis: the bless randomized controlled trial effect of azithromycin maintenance treatment on infectious exacerbations among patients with non-cystic fibrosis bronchiectasis: the bat randomized controlled trial inhibitory effects of macrolide antibiotics on exacerbations and hospitalization in chronic obstructive pulmonary disease in japan: a retrospective multicenter analysis azithromycin for prevention of exacerbations of copd the guidelines for the management of respiratory tract infection" basic concept of respiratory tract infection management in adults acute inflammatory upper airway obstruction (croup, epiglottitis, laryngitis, and bacterial tracheitis) croup (laryngitis, laryngotracheitis, spasmodic croup, laryngotracheobronchitis, bacterial tracheitis) croup: an -year study in a pediatric practice croup hospitalizations in ontario: a -year time-series analysis a current perspective respiratory viruses in laryngeal croup of young children croup: an overview croup-assessment and management wheezing, bronchiolitis, and bronchitis feigin and cherry's textbook of pediatric infectious diseases bronchiolitis-associated hospitalizations among us children prospective multicenter study of the viral etiology of bronchiolitis in the emergency department antibiotic treatment of epidemic bronchiolitisea double-blind trial azithromycin does not improve disease course in hospitalized infants with respiratory syncytial virus (rsv) lower respiratory tract disease: a randomized equivalence trial management of bronchiolitis without antibiotics: a multicentre randomized control trial in bangladesh clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial risk of secondary bacterial infection in infants hospitalized with respiratory syncytial viral infection bacterial tracheitis in children: approach to diagnosis and treatment branhamella catarrhalis as a cause of bacterial tracheitis bacterial tracheitis: report of eight new cases and review is bacterial tracheitis changing? a -month experience in a pediatric intensive care unit feigin and cherry's textbook of pediatric infectious diseases antibiotics for acute bronchitis the value of antibiotics in minor respiratory illness in children. a controlled trial amoxycillin and cotrimoxazole in presumed viral respiratory infections of childhood: placebo-controlled trial cough illness/bronchitisdprinciples of judicious use of antimicrobial agents high levels of adamantane resistance among a (h n ) viruses and interim guidelines for use of anti-viral agentseunited states, d influenza season update: drug susceptibility of swineorigin influenza a (h n ) viruses accuracy of rapid influenza diagnostic tests. a meta-analysis critically ill patients with influenza a (h n ) in mexico enteric absorption and pharmaco-kinetics of oseltamivir in critically ill patients with pandemic (h n ) influenza antivials for treatment of influenza. a systemic review and meta-analysis of observation studies influenza committee of the japanese association for infectious diseases, proposal by the japanese association for infectious diseases effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double -blind randomised controlled trial influenza viruses feigin and cherry's textbook of pediatric infectious diseases ameican academy of peiatrics. haemophilus influenza infections a study of illness in a group of clevel and families. xvii. the occurrence of asian influenza clinical characteristics of pediatric hospitalizations associated with pandemic influenza a (h n ) in northern bavaria otolaryngological complications in patients infected with the influenza a (h n ) virus severe influenza cases in paediatric intensive care units in germany during the pre-pandemic seasons critically ill children during the d influenza pandemic in the united states the current status of parasitic diseases in japan tropical lung disease paragonimiasis cases recently found among immigrants in japan clinicoradiologic features of pleuropulmonary paragonimus westermani on kyusyu island efficacy of chemotherapy of paragonimiasis in southern kyushu, japan. in: the th asian-pacific congress for parasitic zoonosis a case of chronic pleural empyema by paragonimus westermani infection resistant to chemotherapy and cured by surgical decortication paragonimiasis: experimental and clinical experience with praziquantel in korea parasitic infections of the lung:a guide for the respiratory physician current status of strongyloides infection in okinawa pulmonary strongyloidiasis: the varied clinical presentations non-oral treatment with ivermectin for disseminated strongyloidiasis how common is human toxocariasis? towards standardizing our knowledge toxocariasis in japan lessons from eight cases of adult pulmonary toxocariasis :abridged republication thiabendazole vs. albendazole in treatment of toxocariasis: a clinical trial a case of adult hepatic toxocariasis tropical pulmonary eosinophilia: a case series in a setting of nonendemicity study group regarding the establishment of medical management by appropriate treatment with rare-disease drugs for imported tropical diseases/parasitosis. guidelines for drug therapy for parasitosis. study group regarding drugs for tropical diseases drug to be contraindicated for combination therapy: rfp precautions for combination therapy: decrease in the blood concentration: dexamethasone, phenytoin, carbamazepine, chloroquine increase in the blood concentration: cimetidine ➁ pyrantel pamoate patients can take this drug regardless of meals. there are few side effects. ➂ albendazol this drug should be taken with meals for days. a day period of discontinuation should be established and then restarted drug if required.as liver dysfunction is frequently observed, caution is needed during the administration period. bone marrow suppression and stevens-johnson syndrome must also be considered.precautions for combination therapy: decrease in the blood concentration: ritonavir, phenytoin, carbamazepine, phenobarbital increase in the blood concentration: praziquantel ➃ mebendazolealthough this drug has been used in few children, the same dose as established for adults is used. in children weighing kg or less, a half dose should be used. this drug is contraindicated for pregnant women or those who may be pregnant. combination therapy with cimetidine may increase the blood concentration of mebendazole. combination therapy with metronidazole may cause toxic epidermal necrolysis (ten) and stevens-johnson syndrome. after fasting, this drug should be taken with water. the incidence of side effects is low, but nausea/vomiting and mild hepatic disorder are sometimes observed. the safety of this drug in pregnant women or children weighing less than kg has not been established. to these patients, this drug should be administered only when its therapeutic advantage is considered to exceed its risk. ➅ diethylcarbamazine as side effects, fever, lymphangitis/lymphadenitis, and orchitis/epididymitis, which result from anti-parasitic actions, are observed in addition to abdominal discomfort, nausea, and abdominal pain. the safety of this drug in pregnant women has not been established. upper row: dose (mg/kg), lower row: frequency of administration per day key: cord- -spgidqh authors: das, shaoli; camphausen, kevin; shankavaram, uma title: in silico drug repurposing to combat covid- based on pharmacogenomics of patient transcriptomic data date: - - journal: res sq doi: . /rs. .rs- /v sha: doc_id: cord_uid: spgidqh the ongoing global pandemic of coronavirus disease (covid- ) continues to affect a growing number of populations in different parts of the world. in the current situation, drug repurposing is a viable strategy to combat covid- . the drugs targeting the host receptors that interact with sars-cov- are possible candidates. however, assessment of their effectiveness in covid- patients is necessary before prioritizing them for further study. we attempted to shortlist the candidate drugs using an in-silico approach. first, we analysed two published transcriptomic data sets of covid- - and sars-infected patients compared to healthy individuals to find the key pathways altered after infection. then, using publicly available drug perturbational data sets in human cell lines from the broad institute connectivity map (cmap), we assessed the effects of the approved drugs on the altered pathways. we also used the available pharmacogenomic data sets from the genomics of drug sensitivity in cancer (gdsc) portal to assess the effects of the altered pathways on resistance or sensitivity to the drugs in human cell lines. our analysis identified many candidate drugs, some of which are already being investigated for treatment of covid- and can serve as a basis for prioritizing additional viable candidate drugs for covid- . in december , outbreak of a new virus causing severe respiratory disease was reported in wuhan, china. the disease was attributed to infection by a novel member of the coronavirus family, named sars-cov- . in contrast with the two other highly pathogenic coronaviruses, sars-cov (outbreak in ) and mers-cov (outbreak in ), the novel virus sars-cov- had a lower fatality rate, but it could spread from person to person more e ciently . the outbreak spread worldwide , and by march , the world health organization declared the disease, by then named coronavirus disease (covid- ), a global pandemic. covid- is currently a leading public health concern in countries worldwide as the hospitalization rate and death toll increase daily (https://www.who.int/emergencies/diseases/novel-coronavirus- ). apart from mitigation efforts, there is an urgent need for effective treatments for patients showing severe symptoms. after chinese researchers published the rst sequence of sars-cov- in january , many groups worldwide began working to develop an effective vaccine. some of the candidates are undergoing clinical trials, among them two promising candidates from oxford university and moderna, inc., that are entering phase ii . however, clinical approval of the vaccines will take some time; consequently, the best solution in the interim is to look for drugs that have already been approved for humans and that may be repurposed for covid- treatment. the u.s. food and drug administration recently approved remdesivir, an antiviral drug targeting the viral rna-dependent rna polymerase, for covid- treatment after a clinical trial run by the national institute of allergy and infectious diseases found it to reduce the time to recovery in treated patients compared to the control group . however, the drug did not show signi cant improvement in patient survival. thus, there is still a persistent need to search for alternative candidate drugs that can be used as single or as combination therapy to bene t patients with severe illness. researchers are also exploring targeting of the host receptors of sars-cov- as another potential solution. inhibitors of the two main receptors for sars-cov- /sars-cov- entry in human cells, ace and tmprss , are being tested as potential treatment options . other host interactors have been curated from previously published literature in a recent study , and a list of high-con dence interactions between sars-cov- and human proteins was recently published by gordon et al., who used a nity puri cation mass spectrometry on a human cell line expressing the viral proteins . we collected the drugs targeting these sars-cov- /sars-cov- -interacting proteins from drugbank and the drug gene interaction database (dgidb) and assessed their effect in covid- -and sarsinfected patients in silico using published patient transcriptomic data and drug perturbational data sets. by combining recently published covid- -infected patient transcriptomic data and previous sarsinfected patient transcriptomic data , we found key pathways that are altered in infected patients compared to healthy individuals. the common altered pathways were related to androgen biosynthesis, steroid biosynthesis, gluconeogenesis, regulation of mitosis, the proteasome, and in ammatory pathways. next, using the available drug perturbational data sets from the broad institute connectivity map (cmap project , we assessed how the candidate drugs targeting sars-cov- -interacting proteins affect the pathways that are altered after covid- or sars infection in a time-dependent manner. using another available pharmacogenomic data resource, genomics of drug sensitivity in cancer (gdsc) , we assessed whether the altered pathways affect sensitivity or resistance to our candidate drugs in human cell lines. finally, we present a list of potential drugs for covid- treatment that affect the covid- altered pathways and incur less resistance in the presence of alteration in the pathways. figure summarizes our approach. to get an understanding of the transcriptomic alterations in covid- -infected patients, we analysed a published rna sequencing (rna-seq) data set of peripheral blood mononuclear cells (pbmc) of three covid- patients from china compared to three healthy individuals . as there were not enough patient transcriptomic data available for covid- -infected people, we searched the literature for transcriptomic data sets of sars-infected patients. we included a microarray-based transcriptomic data set of pbmcs of sars-infected and heathy individuals from a paper published in . for both of these transcriptomic data sets, we performed single-sample gene set enrichment analysis (ssgsea) for a set of curated pathways collected from the broad institute molecular signatures database (msigdb) and a set of immune function pathways collected from published resources [ ] [ ] [ ] . while checking the differential enrichment of immune functions between covid- vs. healthy as well as sars vs. healthy patients, we found that the macrophages, monocytes, dendritic cells (momadc), b cells, pathogen defence, and interferon signalling were the common immune functions with high enrichment in both covid- -and sars-infected patients compared to healthy patients ( figure ). on the other hand, among other pathways, androgen biosynthesis, steroid biosynthesis, potassium channel, proteasomes, glycolysis, gluconeogenesis, tryptophan metabolism, and p -dependent dna damage response were the common functions that were highly enriched in both covid- -and sars-infected patients compared to healthy patients ( figure ). some of the common pathways with less enrichment in the covid- -and sarsinfected patients are nk-cell-and t-cell-mediated responses (figure c, c) . assessing the effect of potential drug candidates on the pathways altered in covid- and sars infection in an attempt at nding already approved drugs that could be repurposed for covid- treatment, we explored the list of all human proteins interacting with sars-cov- that were collected from the a nity capture mass-spectrometry study by gordon et al. and the list of sars-cov- -interacting proteins curated from previous literature . we collected the list of drugs targeting these host proteins from drugbank and dgidb databases , . additionally, we procured the list of drugs currently in trial for covid- treatment from literature (https://www.excelra.com/covid- -drug-repurposingdatabase/data). the resulting list comprised potential drugs for covid- . using the drug perturbational data sets from cmap , we found the drugs that affected the gene expression of these targets (differences in gene expression at hours vs. hours of drug treatment). we mapped drugs to the drug perturbational data set from cmap. the network in figure a shows the drugs and sars-cov targets, which get signi cantly downregulated at hours compared to six hours of the respective drug treatments ( drugs) along with the drugs in clinical trial for covid- treatment (figure a , supplementary dataset shows the list of drugs and their targets). many of these drugs have been already reported as potential treatments for covid- and/or sars (table ) , including sirolimus, apicidin, mercaptopurine, midostaurin, camostat, metformin, fedratinib, ribavirin, entacapone, azacitidine, and chloroquine. next, we sought to check if the drugs are effective in the context of transcriptomic changes in covid- or sars patients after infection. we checked the effect of these drugs on the perturbation of the pathways upregulated or downregulated after sars and covid- infection ( figure b ). in cmap, gene expression is pro led in cell lines treated with drugs at different dose/time points, and we see how drugs change the expression of a given set of genes. to get a pathway-based estimation instead of individual genes, we calculated ssgsea scores for the differentially enriched pathways in covid- or sars infection for all drug-treated cell lines at different dose/time points. then the ssgsea scores of a pathway were compared between two time points ( hours vs. hours) in cells treated with a certain drug using student's t-test. among all drugs targeting sars-cov- -interacting proteins, drugs altered the covid- /sars differentially enriched pathways in a time-dependent manner. in figure b upregulate the pathways that get downregulated after covid- /sars infection (red cells on the right panel), and ) downregulate the pathways that get downregulated after covid- /sars infection (blue cells on the right panel). among these drugs, vorinostat, panobinostat, tacrolimus, lovastatin, midostaurin, daunorubicin, cytarabine, digitoxin, digoxin, mercaptopurine, bortezomib, azacitidine, and clorafarabine can suppress the pathways that were upregulated due to covid- /sars infection (p-value < . , t-test with alternative hypothesis = less), while they can also boost the pathways that were downregulated due to covid- /sars infection (p-value < . , t-test with alternative hypothesis = greater). as examples, figure c considering the time needed for developing a functional vaccine, the feasible way to ght the covid- pandemic is repurposing drugs already approved for clinical use. we tried to address this issue by identifying drugs that target potential host proteins interacting with sars-cov- and predicting their effect on the patient transcriptomic signatures of covid- -and/or sars-infected patients. we analyzed covid- -and sars-infected patients' and healthy individuals' transcriptomic data sets , to identify the pathways commonly altered due to infection with these viruses. thereafter, combining the potential human interactome of sars-cov- from a recently published study and sars-cov- -interacting proteins curated in another publication with drug target databases , , drug perturbational data sets , and drug sensitivity screening data sets , we propose a map of the drugs that can be effective in covid- treatment. our proposed framework for assessing drug e cacy is based on two approaches: ) whether the drugs can regulate the pathways that get altered due to covid- /sars infection and ) whether the sensitivity or resistance to the drugs depends on the alteration of these pathways. we acknowledge that there are some limitations to our study. first, we found only one publicly available data set for transcriptomic signatures of covid- -infected patients, and that study only included three patients and three healthy individuals . since it would be di cult to draw meaningful conclusions on the patient transcriptomic changes from so few patient samples, we included a previously published data set of sars-infected patient transcriptomes consisting of sars-infected patients and healthy individuals . we reasoned that the viruses sars-cov- (causing sars) and sars-cov- (causing covid- ) share many similarities, and the symptoms of the infected patients are also similar, though sars infection causes more severe symptoms and a higher fatality rate . another limitation of our study is the availability of drug perturbational and drug sensitivity screening data in human cell lines. many of the drugs targeting the potential sars-cov- -or sars-cov- -interacting proteins did not have drug perturbational data available in human cell lines from cmap, and only a few drugs have the drug sensitivity screening data available from gdsc, which is limited to drugs commonly used in cancer treatment. without the available drug perturbational data, we could not assess the effects of antiviral drugs like remdesivir, favipiravir, and oseltamivir in the human transcriptome and were limited to drugs targeting the potential human receptors of the viruses. our analysis of covid- -and sars-infected patient transcriptomic signatures identi ed the common pathways upregulated or downregulated after infection with these viruses. among the common pathways, androgen biosynthesis, steroid biosynthesis, proteasome, tryptophan metabolism, and mitotic cell cycle-related pathways were upregulated due to infection, while tcr, interleukin , tgf-beta receptor signaling, and beta-cell development pathways were downregulated. among the immune pathways, momadc, b cells, and interferon responses were elevated in patients with covid- and sars infection, while t-cell and nk cell functions were downregulated. many of these pathways have been previously implicated in viral infections. androgens have already been linked to the severity of covid- as possible mechanisms driving disease outcomes . steroid, proteasome, and tryptophan metabolisms are linked to viral infections and subsequent host immune response [ ] [ ] [ ] . moreover, monocyte and macrophage activation has been linked to the hyperin ammatory phenotypes in sars-cov- infection that have adverse clinical outcomes . by analysing the drug perturbational data sets from cmap, we identi ed drugs that can upregulate or downregulate the pathways that get altered due to covid- /sars infection. as at this point, it's unclear which pathways will be clinically bene cial for covid- treatment when inhibited or elevated. we provided the full map of all drugs that ) downregulate the pathways that get upregulated due to covid- /sars infection, ) upregulate the pathways that get upregulated due to covid- /sars infection, ) upregulate the pathways that get downregulated due to covid- /sars infection, and ) downregulate the pathways that get downregulated due to covid- /sars infection. some drugs that could downregulate the androgen biosynthesis pathway (upregulated due to covid- /sars infection), which is linked to severe outcomes, are ponatinib, amlexanox, lenalidomide, menadione, and decitabine. the drugs vorinostat, panobinostat, tacrolimus, lovastatin, midostaurin, daunorubicin, cytarabine, digitoxin, digoxin, mercaptopurine, bortezomib, azacitidine, and clofarabine could downregulate the mitotic cell cycle-related pathways and proteasome activity. the mitotic cell-cycle arrest and uniquitin-proteasome pathway systems play an important role in viral replication for coronaviruses , . drugs that could downregulate the momadc cell pathway potentially linked to severe symptoms in covid- patients are menadione, decitabine, amlexanox, mercaptopurine, nicotinamide, and pevonedistat. the drugs that could upregulate the ctl or interleukin pathways, which are important for antiviral immune responses, are: icosapent, lovastatin, azacitidine, decitabine, vorinostat, entinostat, digoxin, ponatinib, apicidin, daunorubicin, and metformin. many of these drugs have been recently reported as possible treatment options for covid- , including ponatinib, mercaptopurine, azacitidine, apicidin, daunorubicin, and metformin . considering that host transcriptomic features can affect the sensitivity or resistance to drugs , we checked whether the pathways that get altered after covid- /sars infection can reduce the resistance to the drugs under consideration using gdsc cell-line drug sensitivity screening data. we were limited to drugs approved for cancer treatment that have been screened in human cancer cell lines, so we could not test all potential drugs for covid- . however, among the drugs we tested, we found that there was reduced drug resistance (increased sensitivity) related to the pathway alteration signature due to covid- /sars infection with the drugs navitoclax, bortezomib, fedratinib, lenalidomide, cytarabine, temsirolimus, paclitaxel, docetaxel, vorinostat, belinostat, entinostat, dacinostat, and vinblastine. among these drugs, fedratinib is a potential treatment option for covid- . fedratinib is a semi-selective janus kinase inhibitor with anti-in ammatory properties that can be bene cial for treatment of severe covid- patients with hyperin ammatory symptoms that may lead to lung damage and death. among the other cancer drugs, the histone deacetylase inhibitors vorinostat, belinostat, entinostat, and dacinostat have anti-in ammatory properties, and the drug lenalidomide has immunomodulatory properties that may have treatment bene ts in covid- patients with severe symptoms, possibly when treated with antiviral agents. the chemotherapeutic drugs paclitaxel, docetaxel, and cytarabine may also have potential antiviral properties , ; however, their suitability for treating virus infection is not well established, and therefore, these drugs should be carefully examined before being considered as treatment options. our computational study provides an assessment of the potential effects of possible repurposable drugs targeting sars-cov- -and sars-cov- -interacting proteins in the context of transcriptomic signatures of covid- -or sars-infected patients. because we have limited understanding of the virus pathogenesis and the safety of targeting speci c pathways for treatment of this disease, we have included a complete map of the drug-pathway relationships that can be important in the context of covid- infection and treatment. due to the urgency of identifying effective drugs for treatment of covid- patients, many groups have been working on repurposing existing drugs that can be used for this disease . our study may be the rst that links potentially repurposable drugs to the pathway signatures of infected patients. the drugs we presented in this study as potentially repurposable for covid- treatment should be clinically tested for e cacy before use. the rna-seq transcriptomic data of the pbmcs from three covid- infected patients and three healthy individuals were obtained from the study by xiong et al. . the microarray transcriptomic data of the pbmcs from sars-infected and healthy patients were obtained from the study by cameron et al. . the rna-seq data from the covid- patients and healthy controls was normalized to fractions per kilobase per million (fpkm). the microarray data from sars patients and healthy controls was normalized with locally weighted scatterplot smoothing (lowess). transcriptomic data from both covid- and sars studies were log -transformed before further processing. the list of human proteins interacting with sars-cov- was collected from the study by gordon et al. . in addition, the human proteins interacting with sars-cov are collected from the article by zhou et al. . the drugs targeting the sars-cov- -interacting proteins were collected from drugbank and dgidb , . the drug perturbational data set containing dose-and time-dependent transcriptomic pro les upon drug treatments in human cell lines was collected from cmap . the drug-screening data measuring ic upon drug treatments in human cell lines and cell-line transcriptomic pro les are collected from gdsc . the general pathway gene sets were obtained from the c collection of msigdb . we curated immunologic signatures speci c to immune functions from four published resources: the lm immune in ltration signature , the lm immune in ltration signature , the imsig signature of solid tumor immune in ltration , and the nanostring immune signature panel (https://www.nanostring.com). the combined , curated gene sets and immune gene sets were used for a single-sample gene-set enrichment analysis on the rna-seq and microarray transcriptomic tumor data sets for covid- -and sars-infected patient transcriptomes as well as for cell-line transcriptomic data for the cmap drug perturbational studies and gdsc data sets. r-package gene-set variation analysis was used . differential enrichments of pathways between covid- -and sars-infected patients vs. healthy controls were calculated with limma r package using ssgsea scores. the pathways with adjusted p-value < . are identi ed as differentially enriched. a one-sided student's t-test was used for calculating the p-value of differences in pathway ssgsea scores between time-dependent drug treatment responses (from cmap) at hours vs. hours. for pathways that are differentially regulated due to covid- /sars infection, we checked whether a drug can signi cantly downregulate the pathway at hours compared to hours of transfection in human cell lines from cmap by performing t-tests on the pathway ssgsea scores between these two time points using an alternative hypothesis equal to less. similarly, to check whether a drug can signi cantly upregulate the pathway at hours compared to hours of transfection, we performed t-tests on the pathway ssgsea scores between these two time points using an alternative hypothesis equal to greater. the pathway-dependent drug sensitivity/resistance for each pathway and drug combination from gdsc is calculated using the following approach. first, ssgsea scores for the pathways are calculated for the gdsc cell lines. then, the cell lines are strati ed into two sets based on the ssgsea scores of the pathways. for upregulated pathways, cell lines with pathway ssgsea z-score > have high pathway enrichment, and for downregulated pathways, cell lines with pathway ssgsea z-score < - have low pathway enrichment. the difference between drug ic in gdsc cell lines with high ssgsea scores (zscore > ) for covid- /sars upregulated pathways and all other gdsc cell lines was calculated using a one-sided t-test with an alternative hypothesis equal to greater. the difference between drug ic in gdsc cell lines with low ssgsea scores (z-score < - ) for covid- /sars downregulated pathways and all other gdsc cell lines was calculated using a one-sided t-test with an alternative hypothesis equal to less. targets interacting with sars-cov- literature/clinical trial evidence sirolimus psma ,acsl ,scarb ,plod ,pola ,xpo ,comt,bcl l ,ikbkb,chek , graphical description of our approach to systematically assess candidate drugs that can be re-purposed for covid- (or sars) treatment, based on the transcriptomic changes of infected patients. we combined recently published covid- -infected patient transcriptomic data and previous sars-infected patient transcriptomic data, to nd key pathway alterations in infected patients. on the other hand, we collected the drugs targeting these sars-cov- /sars-cov- -interacting proteins from drugbank and dgidb to assess their effect in covid- -and sars-infected patients, in-silico. next, using the available drug perturbational data sets from the broad institute cmap project, we assessed how the candidate drugs targeting sars-cov- -interacting proteins affect the pathways that are altered after covid- or sars infection in a time-dependent manner. using another available pharmacogenomic data resource, genomics of drug sensitivity in cancer (gdsc), we assessed whether the altered pathways affect sensitivity or resistance to our candidate drugs in human cell lines. finally, we present a list of potential drugs for covid- treatment that affect the covid- -altered pathways and incur less resistance in the presence of alteration in the pathways. where the pathway has low enrichment (scaled ssgsea < - ) vs. other cells. this is a list of supplementary les associated with this preprint. click to download. supplementarydataset .xlsx supplementarydataset .xlsx a new coronavirus associated with human respiratory disease in china responding to covid- -a once-in-a-century pandemic? a novel coronavirus outbreak of global health concern genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan developing covid- vaccines at pandemic speed compassionate use of remdesivir for patients with severe covid- sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor network-based drug repurposing for novel coronavirus -ncov/sars-cov- a sars-cov- protein interaction map reveals targets for drug repurposing drugbank . : a major update to the drugbank database dgidb . : a redesign and expansion of the drug-gene interaction database transcriptomic characteristics of bronchoalveolar lavage uid and peripheral blood mononuclear cells in covid- patients interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease genomics of drug sensitivity in cancer (gdsc): a resource for therapeutic biomarker discovery in cancer cells robust enumeration of cell subsets from tissue expression pro les assessment of tumor-in ltrating tcrvgamma vdelta gammadelta lymphocyte abundance by deconvolution of human cancers microarrays immune cell gene signatures for pro ling the microenvironment of solid tumors from sars to covid- : a previously unknown sars-related coronavirus (sars-cov- ) of pandemic potential infecting humans -call for a one health approach androgen hazards with covid- steroid hormone synthesis by vaccinia virus suppresses the in ammatory response to infection the ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle activation of the tryptophan/serotonin pathway is associated with severity and predicts outcomes in pneumonia: results of a long-term cohort study pathological in ammation in patients with covid- : a key role for monocytes and macrophages cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication th responses in cytokine storm of covid- : an emerging target of jak inhibitor fedratinib anti-hiv, antitumor and immunomodulatory activities of paclitaxel from fermentation broth using molecular imprinting technique antiviral activity of cytarabine in herpesvirus-infected rats gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression pro les gsva: gene set variation analysis for microarray and rna-seq data tables table . literature evidence of drugs that can be repurposed to target sars-cov- -interacting proteins all analysis data generated during this study are included in this published article as supplementary datasets. key: cord- -mwu q w authors: dent, paul title: cell signaling and translational developmental therapeutics date: - - journal: reference module in chemistry, molecular sciences and chemical engineering doi: . /b - - - - . - sha: doc_id: cord_uid: mwu q w the relationships between drug pharmacodynamics and subsequent changes in cellular signaling processes are complex. many in vitro cell signaling studies often use drug concentrations above physiologically safe drug levels achievable in a patient's plasma. drug companies develop agents to inhibit or modify the activities of specific target enzymes, often without a full consideration that their compounds have additional unknown targets. these two negative sequelae, when published together, become impediments against successful developmental therapeutics and translation because this data distorts our understanding of signaling mechanisms and reduces the probability of successfully translating drug-based concepts from the bench to the bedside. this article will discuss cellular signaling in isolation and as it relates to extant single and combined therapeutic drug interventions. this will lead to a hypothetical series standardized sequential approaches describing a rigorous concept to drug development and clinical translation. the field of cell signaling and signal transduction dates back to the late th century. in , epinephrine (adrenaline) was discovered. by the s, insulin and glucagon had been discovered. , collectively, these discoveries paved the way for researchers to explore how these hormones acted to regulate glucose metabolism in the liver and skeletal muscle. the laboratory of dr. carl cori played a seminal role in partially unravelling how glycogen could be broken down by glycogen phosphorylase. he, his wife gerty and bernardo houssay received the nobel prize in physiology or medicine for their work. although the cori laboratory had discovered and described glycogen phosphorylase, it was not until that leloir discovered the enzyme that made glycogen, glycogen synthase. during the s, fischer, krebs and sutherland not only discovered and characterized the kinase which a dedicated to professor sir philip cohen on the occasion of his th birthday. comprehensive pharmacology https://doi.org/ . /b - - - - . - regulated glycogen phosphorylase, phosphorylase kinase, but defined for the first time that the phosphorylation of proteins could regulate enzyme activity. [ ] [ ] [ ] [ ] further development of signal transduction up until the late s, however, no-one had been able to elucidate how insulin signaled to make a cell store glucose as glycogen nor how epinephrine and glucagon activated phosphorylase kinase/glycogen phosphorylase to break down glycogen. sutherland and colleagues during their investigations into glycogen phosphorylase discovered a heat-stable factor in liver sections whose levels were regulated by epinephrine and glucagon: cyclic amp, the first second messenger. [ ] [ ] [ ] subsequently, fischer and krebs isolated the kinase regulated by camp, protein kinase a (pka). for these discoveries, sutherland, as well as fischer and krebs, received the nobel prize. sutherland, fischer, and krebs during their studies also discovered an enzyme activity which could remove phosphate from glycogen synthase, i.e. a protein phosphatase. a postdoctoral researcher from the laboratory of fischer in the late s, philip cohen, focused their independent career upon characterizing the many protein phosphatases in cells and above all understanding how phosphatases regulated glycogen metabolism, naming the ser/thr protein phosphatases. over the or so years after the discovery of pka, multiple additional small molecule second messengers were discovered including: calcium ions, diacyl glycerol and ip ; and nitric oxide and cyclic gmp (cgmp). [ ] [ ] [ ] [ ] signaling by cgmp in the eye was shown to be essential for the perception of light and cgmp as well as with nitric oxide in the regulation of smooth muscle contractility resulted in the nobel prize being awarded to murad in . , during the s and s work by lefkowitz, gilman and johnson led to the discovery of serpentine plasma membrane receptors for hormones, e.g. the beta-adrenergic receptor for epinephrine, as well as receptor-associated large gtp binding protein complexes on the inner leaflet of the plasma membrane which transduced receptor signals to intracellular effectors such as: ( ) adenylyl cyclase leading to the generation of camp; ( ) activation of phospholipases leading to the generation of diacyl glycerol and inositol , , -trisphosphate (ip ), with ip triggering the release of calcium ions into the cytosol. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diacyl glycerol and calcium ion then activated multiple protein kinase c (pkc) isoforms. this resulted in the award of additional nobel prizes. serpentine g-protein coupled receptor (gpcr) signaling can be down-regulated by proteins called arrestins. [ ] [ ] [ ] arrestin proteins prevent both the ga gbg proteins interacting with the gpcr and cause the gpcr to be internalized. internalization can result either in receptor degradation or recycling back to the plasma membrane. thus, by the mid-to late- s a large body of literature existed which argued that signal transduction pathways consisted of a receptor linked to a large gtp-binding protein which in turn regulated an enzyme that generated "second messengers;" the second messengers would then diffuse throughout the cytosol activating cellular processes, predominantly for metabolism. in parallel to the study of serpentine receptors, other investigators were focused on the relatively few proteins who became phosphorylated on tyrosine. studies in this field were focused on the insulin receptor (metabolism) and the epidermal growth factor receptor (egfr, erbb ) (growth, cancer). [ ] [ ] [ ] [ ] insulin caused the insulin receptor to become tyrosine phosphorylated, and a substrate for the receptor, insulin receptor substrate (irs ), was discovered. for many years prior to the s, diagrams of insulin receptor signaling would include the receptor and irs , together with downstream insulin targets such as glycogen synthase. in-between the receptor and synthase was drawn a "black box" as the pathway by which insulin regulated glycogen synthase appeared to be intractable to investigation. studies by the laboratory of larner and villar-palasi argued that insulin caused the generation of a "mediator" second messenger which was an inositol phospholipid, that regulated glycogen synthase. [ ] [ ] [ ] although at the time this concept was not widely supported, subsequent studies over the following years demonstrated that insulin activated phosphatidyl inositol -kinase whose product, phosphatidylinositol ( , , )-trisphosphate (pi( , , )p ), caused activation of the membrane-associated kinase, phosphoinositide-dependent kinase- (pdk ). [ ] [ ] [ ] [ ] [ ] pdk was shown to phosphorylate akt t causing enzyme activation, and akt to phosphorylate and inactivate glycogen synthase kinase (gsk ). reduced gsk activity results in reduced glycogen synthase phosphorylation, leading to activation of synthase activity. one additional component within this process was activation of protein phosphatases to facilitate the dephosphorylation and activation of glycogen synthase. thus, after years of research, by the mid- s, the regulation of glycogen metabolism by epinephrine and insulin had largely been elucidated. for the egfr and other subsequently discovered membrane associated tyrosine kinases, e.g. the non-receptor scr family and the fibroblast growth factor receptor (fgfr) family, understanding how these enzymes signaled into the cell again initially rested on studies using traditional biochemical methods. [ ] [ ] [ ] [ ] in the mid- s, a postdoctoral researcher in the laboratory of dr. ora rosen, thomas sturgill, was given a project in which he was to identify a kilo dalton protein whose tyrosine phosphorylation was increased after exposing cells to insulin. as an independent investigator sturgill continued his studies into the enzyme he called map -kinase, microtubule associate protein (map ) being the substrate used to measure its kinase activity. it was subsequently renamed to be "mitogen activated protein kinase" (mapk) after it was discovered to not only be regulated by many growth factors, but also that it was an intermediary kinase in the regulation of another insulin-activated kinase p ribosomal s kinase (p rsk). this enzyme should not be confused with p s kinase with is a component of the pi k pathway. , by the end of the s, it had been determined that there was another mapk isoform (p ) and that these kinases were regulated by tyrosine/threonine joint phosphorylation. , at that time, kinases were considered to be specific for either serine/threonine or for tyrosine. the discovery of mek and mek (mitogen/extracellular regulated kinase), kinases that phosphorylated the mapks on both tyrosine and threonine was considered biochemically novel. [ ] [ ] [ ] [ ] from work in yeast (cerevisiae, pombe), however, was in parallel demonstrating that they also expressed mapk-like and mek-like enzymes, and that their mek-like enzymes phosphorylated the mapk-like enzymes on tyrosine and threonine. [ ] [ ] [ ] the mammalian mapk/renamed erk / (extracellular regulated kinase) pathway in yeasts regulates the yeast response to pheromones. this understanding facilitated the further characterization of mek and mek . the next question in the development of the "mapk pathway" was to define the kinase(s) upstream of mek / . based on data from yeasts, this kinase should have been similar to the mammalian map k, known as mekk (mitogen/extracellular regulated kinase kinase). however, in , two groups linked c-raf- and its truncated oncogenic variant v-raf as the kinase activity which enhanced mek / phosphorylation and activity; there are no yeast homologues of the raf family proteins. , of note, prior to those studies it was believed that raf- was downstream of erk / . the function of mekk subsequently, and with its family members, was linked in mammalian cells to the regulation of the c-jun nh -terminal kinase (jnk / ) and p mapk pathways. [ ] [ ] [ ] [ ] [ ] [ ] contemporaneously with these studies, researchers were determining how receptor tyrosine kinases regulated ras family small gtp binding proteins, and other groups determining how ras proteins signaled downstream off the plasma membrane and into the cytosol. [ ] [ ] [ ] [ ] [ ] it was demonstrated that the proteins grb (growth factor receptor-bound protein ) and sos (son of sevenless homolog ) linked receptor tyrosine phosphorylation to the exchange of gtp for gdp in ras proteins. within months of these discoveries being published, it was shown that gtp-bound ras would associate with the nh -terminal domain of raf- . - gdp-bound ras proteins did not associate with raf- . thus, within the period between and , the first of the "map kinase pathways" had been delineated. because of extant data from yeasts, other parallel mammalian map kinase pathways were rapidly discovered and delineated. for example, as mentioned previously, the p mapk pathway in mammalian cells is a stress-induced signaling pathway and was the equivalent of the hog osmo-sensing pathway in yeasts. the jnk pathway has similarities to several yeast and mammalian mapks, but only a % best-fit to erk and erk . it was discovered as a uv-activated kinase that bound to the nh -terminus of the transcription factor c-jun. a parallel mapk pathway, the erk "big map kinase pathway" was discovered and inhibitors of mek / also inhibit mek , demonstrating the close functional alignment of both pathways. hence, by the mid- s the basic structures of multiple map kinase as well as the pi k pathway were in place. broadly, over the past years, signaling by erk / and erk were most often linked to tumor cell growth whereas signaling by p mapk and jnk were linked to cell death. [ ] [ ] [ ] however, coordinated erk/jnk signaling strongly promoted growth and under prolonged high activity erk / signaling would cause growth arrest via the induction of cyclin dependent kinase inhibitor proteins or tumor cell death. [ ] [ ] [ ] these were also reflected at the level of receptor tyrosine kinases, comparing different ligands for the same receptor with different on-/off-rates, e.g. egf and tgfa, as well as associated with ligand concentration. high ligand levels permanently downregulate the receptor, and ligands such as egf that remain with the receptor in endosomes cycle the receptor for degradation. [ ] [ ] [ ] [ ] signaling by p mapk regulated chaperone functions but also could cause cell cycle arrest and dna damage repair. , what also became readily apparent was that activation of the same pathway to the same extent in different tumor cells could result in different changes in tumor cell biology, with some cells exhibiting growth/growth arrest and other cells becoming moribund either through apoptosis, necrosis or autophagy. [ ] [ ] [ ] some of these behaviors could in part be explained due to the differential expression of driving oncogenes such as mutation of p , ras proteins, receptor tyrosine kinases or the lipid phosphatase: phosphatase and tensin homologue on chromosome ten (pten). - the cellular process of autophagy was discovered in the s. , the primary purpose of the process is to recycle cellular components into their elemental building blocks during times of metabolic stress, permitting the cell to survive. materials are first encapsulated in a double membrane, called an autophagosome. [ ] [ ] [ ] autophagosomes fuse with lysosomes, the interior acidifies, and they become autolysosomes where materials are digested, ready for recycling. the regulation of autophagy and with it the sensing of nutrient and atp energy levels within a cell are regulated by mammalian target of rapamycin (mtor) and the ampdependent protein kinase (ampk), respectively. [ ] [ ] [ ] [ ] [ ] the regulation of mtor is complex as it integrates upstream signaling from akt in the pi k pathway, together with other signals that sense amino acid, lipid and carbohydrate levels. there are two complexes of proteins which associate with mtor, with the kinase being termed mtorc or mtorc based on the members of the protein complex. [ ] [ ] [ ] the ampk senses amp levels, which are high when the cell is depleted of atp; high amp levels cause allosteric activation of the ampk, and activated ampk then acts to phosphorylate and inactivate mtor. [ ] [ ] [ ] the ampk is itself regulated by phosphorylation, with the most notable regulators being liver kinase b (lkb ) and ataxia-telangiectasia mutated (atm). [ ] [ ] [ ] [ ] [ ] [ ] lkb is often mutated in tumor cells, leading to dysregulation of energy sensing and autophagy regulation. in the nucleus atm senses dna damage and cytosolic atm senses the levels of reactive oxygen species; atm at both cellular locations phosphorylates and activates the ampk. the key regulatory target for both mtor and the ampk is the kinase unc- like autophagy activating kinase (ulk / ). - ulk is a classic example of a protein whose function is regulated by multi-site phosphorylation. phosphorylation of ulk at specific sites by mtor inactivates the kinase. phosphorylation of ulk at different specific sites by the ampk activates the kinase. [ ] [ ] [ ] the primary substrate of ulk is the gate-keeper protein for autophagosome formation, atg . phosphorylation of atg leads to the formation of multi-protein complexes which act to form a double membrane around the cellular materials that will be digested. autophagic flux occurs where a fully-formed autophagosome fuses with an endosome/lysosome to form an autolysosome. , autolysosomes acidify their interior, activating a variety of proteases and other enzymes required to break down the vesicle's contents. many tumor cells exquisitely rely on autophagy to survive, which explains why drugs such as chloroquine, which prevent autophagosome lysosome fusion, have been trialed as cancer therapeutics. , alternatively, as tumor cells utilize autophagy for survival, drugs which profoundly stimulate autophagosome formation and autophagic flux cause the over-digestion of cellular proteins and cause the cytosolic release from the autolysosome of active proteases, which collectively leads to a multi-factorial form of tumor cell death. using our understanding of autophagy and cell signaling to therapeutically kill tumor cells in all scientific studies, experiments should be performed from an agnostic standpoint. that is, follow the data wherever it may lead, regardless of prior opinions or perceptions. twenty years ago, in collaboration with dr. paul fisher, we began to investigate the molecular mechanisms by which the cytokine il- acted to kill tumor cells. [ ] [ ] [ ] at that time, the mechanisms by which tumor cells died were not particularly sophisticated, with death receptor signaling via caspases / (the extrinsic apoptosis pathway) and mitochondrial dysfunction via caspase (the intrinsic apoptosis pathway) being the two pathways then considered most important in the causation of tumor cell death. because we had observed the cytokine was inactivating mtor, studies were performed to define if "autophagy" played any role in the cytokine's biology. molecular knock down of key autophagy regulatory proteins, atg or beclin , profoundly suppressed il- lethality. our studies with autophagy and il- resulted in other laboratory projects exploring the role of autophagy in their biology and killing mechanisms. for example, in hepatoma cells, the combination of the multi-kinase inhibitor sorafenib with the histone deacetylase (hdac) inhibitor vorinostat killed cells by activating the death receptor cd , and in hepatoma cells, knock down of atg or beclin enhanced drug combination lethality, i.e. autophagy was acting as a protective cellular response. however, in pancreatic cancer cells, knock down of atg or beclin significantly reduced the ability of this drug combination to kill, i.e. autophagy played a role in the killing process. subsequent studies in the laboratory over the past decade have almost invariably discovered that autophagosome formation was playing an essential role in the tumor cell killing process. one consideration when discussing the role of autophagy in causing cell death is whether the autophagic process caused killing directly, or indirectly by causing, e.g. mitochondrial dysfunction, followed by release of cytochrome c and apoptosis inducing factor (aif) into the cytosol. aif moves to the nucleus to cause dna fragmentation in a fashion similar to necrosis. cytochrome c binds to apoptotic protease activating factor (apaf- ) which together with atp causes the cleavage of pro-caspase . activated caspase cleaves and activates caspase , which moves to the nucleus to cause apoptotic dna fragmentation, with dna fragments encapsulated in membranes. alongside the apoptotic processes, cathepsin proteases released from autolysosomes can cleave and activate the pro-apoptotic protein bid that is upstream of mitochondria, and which will lead to mitochondrial dysfunction and death. however, it is possible that release of activated proteases by themselves into the cytoplasm can also cause death, without involvement of the mitochondria. we will now illustrate in more detail the role of autophagy in the development of anti-cancer therapeutics and in the development of anti-viral therapeutics. the multi-kinase inhibitor drugs sorafenib and pazopanib are approved for the treatment of liver/kidney cancers and soft tissue sarcoma, respectively. , for both drugs, we demonstrated that they synergized with hdac inhibitors to kill liver, kidney, pancreatic and sarcoma tumor cells. [ ] [ ] [ ] [ ] contemporaneously with these studies, we were also studying the celecoxib derivative developmental drug, osu- . originally osu- was proposed to inhibit pdk within the pi k/akt pathway. , osu- has an order of magnitude anti-cancer efficacy than the parent compound. the key, arguably single, mechanism by which we found osu- acted to kill tumor cells was by causing the generation of autophagosomes followed by autophagic flux and the cytotoxic actions of autolysosome localized proteases such as cathepsin b. ultimately, we determined that osu- was an inhibitor of chaperone proteins, in particular grp . grp is an endoplasmic reticulum (er) localized chaperone that plays an essential role in regulating er stress signaling during times of protein overload and protein denaturation. as we compared the chemical structures of osu- , pazopanib and sorafenib we realized that had many similarities. compared to osu- which had ic values of inhibiting the atpase activities of hsp and hsp in the and nm range, respectively, the chaperone inhibitory activities of sorafenib were found to be similar, and the inhibitory activity of pazopanib significantly stronger with ic values of and nm, respectively. [ ] [ ] [ ] [ ] [ ] thus, drugs that had been developed and marketed as "multi-kinase inhibitors" for many years also had multiple unknown chaperone targets. hence, just because a drug company states on their packaging that a drug inhibits enzymes a, b and c to cause a therapeutic effect, does not mean that the drug also inhibits unknown enzymes y and z. furthermore, it is probable that without inhibition of y and z, the inhibition of a, b and c together will only have a modest therapeutic effect. in the case of osu- , despite a phase i trial in cancer patients (nct ), further studies with drug took an unexpected turn away from cancer therapeutics, and towards infectious disease and the development of the drug as an anti-viral agent. , all human pathogenic viruses require cells express functional grp . , in a virus-dependent manner, different viruses also recruit other additional chaperone proteins to facilitate their replication and life cycle. , osu- is not a highaffinity inhibitor of a single chaperone or chaperone family, unlike many chaperone inhibitors developed for use in the cancer therapeutics field. , however, because the drug inhibits multiple hsp family and hsp family chaperones within its clinically relevant safe concentration range, osu- could potentially become a broad spectrum anti-viral drug. osu- prevented the reproduction of viruses including mumps, influenza, measles, coxsackie virus b , junín, rubella, west nile, yellow fever, hiv (wild type and protease resistant), and ebola, effects that were replicated by molecular knock down of multiple chaperone proteins, alone or in combination. very recently we discovered, to some extent not surprisingly, that osu- could also prevent synthesis of the sars-cov- spike protein. in three separate animal model systems, rabbit hemorrhagic fever virus, zika and dengue osu- prolonged animal survival and significantly reduced the negative sequelae of virus infection. [ ] [ ] [ ] subsequent studies using the fda approved cancer therapeutic drugs sorafenib and pazopanib also demonstrated that these fda approved drugs also have potent anti-viral properties. thus, a project which began as development of an anticancer drug became a project developing broad spectrum anti-viral drugs. the role of an activating point mutant in the egf receptor was first demonstrated in non-small cell lung cancer (nsclc). [ ] [ ] [ ] [ ] subsequently, as patient tumors carrying the activated egfr were treated for prolonged periods with egfr inhibitors such as gefitinib, it became evident that drug resistance, when it eventually evolved, was mediated by the evolution of a second point mutation in the egfr. , [ ] [ ] [ ] second and third generation egfr inhibitory drugs such as afatinib and osimertinib potently inhibit double mutant egfr and are in first-line clinical use. [ ] [ ] [ ] at the time of these discoveries we had several research projects determining whether we could combine afatinib with other agents to kill nsclc cells. [ ] [ ] [ ] as part of this work, we generated afatinib-resistant h nsclc cells by treating tumors in mice until the tumor completely regressed and then had begun to regrow. h cells already express a double mutant egfr, so we were expecting to discover novel evolutionary survival signals. initial characterization of the resistant cells demonstrated they had permanently up-regulated signaling by the receptors c-kit, c-met and erbb to survive during exposure to afatinib. additional characterization studies then delivered unexpected data; whilst afatinib-resistant h cells were resistant to the irreversible erbb receptor inhibitor afatinib, they were not resistant to the irreversible erbb inhibitor neratinib. furthermore, the ability of neratinib as a single agent or when combined with other drugs, including afatinib, was enhanced in the afatinib-resistant cells. ostensibly, both drugs should mechanistically "do" exactly the same thing to a tumor cell. thus, by implication, in addition to erbb family receptors, neratinib had to have additional "targets" to cause killing in the resistant cells. two molecular modeling manuscripts had stated neratinib, in addition to inhibiting erbb family tyrosine kinases could also inhibit map k and map k serine/threonine kinases. , in parallel to the studies described above, from our loading control data, we observed that neratinib but not afatinib, could rapidly reduce the protein expression of erbb family receptors in a wide variety of tumor cell types. , , we also included negative controls in our studies; c-met and c-kit. to our surprise, neratinib also reduced c-met and c-kit levels, albeit in a delayed fashion. to down-regulate the egfr required a ubiquitination step whereas to down-regulate c-met did not. growth factor receptors localize in large quaternary structures in the plasma membrane and we hypothesized that if neratinib was reducing the levels of the egfr, c-met and c-kit, could it also reduce the levels of an important signal transducer on the inner leaflet of the plasma membrane: ras. in pancreatic cancer cells neratinib not only caused internalization and degradation of the egfr, it also caused the degradation of the key oncogenic driver in this disease, mutant k-ras. subsequently, in melanoma cells expressing a mutant n-ras, similar findings with neratinib were obtained. , the convergence of the afatinib-resistance studies and the ras down-regulation studies was a project to define the roles of map k and map k enzymes in the biological actions of neratinib. from the modeling studies, two potential neratinib targets were mst and mst . this caught our interest because the dose-limiting sequela for neratinib is diarrhea, and mst and mst play important roles in regulating the integrity of the epithelial brush boarder in the gut. , because we did not know what effects would be observed, we agnostically examined the activities of multiple map k enzymes, as well as associated chaperone/docking proteins following neratinib exposure. as map k/map k enzymes are expressed in carcinoma cells which express high levels of erbb family receptors as well as in blood cancer cells that express none or very low levels of that receptor family, we performed studies in both tumor cell types. regardless of erbb family receptor expression, neratinib reduced the expression of ras proteins and reduced tumor cell viability. neratinib reduced the phosphorylation of mst / , mst and mst in carcinoma and blood cancer cells; this would a priori predict that phosphorylation of their downstream substrates such as lats / or the cytoskeletal protein ezrin, would be reduced. , as was a priori expected, the phosphorylation of ezrin was reduced. however, the phosphorylation of lats / was enhanced, as were the downstream substrates of these enzymes, the co-transcription factors yap and taz. yap and taz are hippo pathway effectors and when phosphorylated leave the nucleus which is followed by degradation in the cytoplasm. , as yap and taz cooperate with mutant k-ras to drive pancreatic cancer growth and metastasis, our data suggest that neratinib could be a useful drug to employ in the treatment of this disease. , this data also suggests that inhibition of the mst "map k" kinases probably caused a compensatory activation of another "map k" kinase(s) which phosphorylated lats / . thus, the key take-home messages from this section are that without a full appreciation and understanding of all potential targets of a particular drug, its mechanisms of action cannot be properly understood. because neratinib inhibits map k/map k enzymes besides erbb family receptors and particularly her /erbb , very few pre-clinical studies were performed in cells that did not over-express her /erbb and none in cells that express mutant ras proteins or in blood cancer cells. these findings emphasize that in developmental drug and therapeutics studies, a broad agnostic approach is essential so as not to miss potential unknown off targets. this is diametrically different to almost all cell biology research projects where intense focus on a particular pathway, or even a component of a pathway is a standard approach. similarly, studying the mechanisms of cell killing by a drug by their nature have to be conceptually broad because very frequently drug-induced killing is not "pure" with only one pathway to tumor cell death being engaged. the drug-induced killing mechanism, for example, could include death receptor signaling, mitochondrial dysfunction and autophagosome formation, all interacting in a contemporaneous fashion. again, this approach is diametrically different to almost all basic science cell biology research projects. developing a compound into a putative drug and eventually into an agent that can be tested in humans is a long process that generally costs in the region of $ - million dollars. to some extent, the high cost of all prescription drugs to the consumer is influenced by this math. the screening of millions of compounds may result in the discovery of a new agent with anti-cancer, antiviral or anti-bacterial properties. alternatively, compounds are screened against a specific target until molecules are defined that potently act to inhibit the target's biological activity. optimization of these compounds, either by computer aided design, or by traditional organic chemistry methods, results, hopefully, in a series of compounds all with a low nanomolar ic inhibitory activity. drug development companies will then determine which of the drugs has the greatest apparent bioactivity in a range of tumor cell lines, alongside determination of in-animal stability and bioactivity against tumors. these studies collectively will deliver one or two compounds that are considered worthy of further investigation and development. it is at this point where drug companies will often seek outside academic collaborators to assist in their drug development studies. the first thing the independent academic collaborator needs to know is what was the highest safe dose of the compounds used in prior mouse studies? and, ideally, if pharmacodynamic and pharmacokinetic studies were performed, what was the safest peak plasma concentration of the compound, termed the c max and often listed as ng/ml (which requires conversion into a molar value). thus, if the highest safe dose of a compound is mg per kg of animal, with a plasma c max of mm, then all preliminary in vitro cell-based investigative studies must use the compound at concentrations well below mm. to further understand the biology of the compound, preliminary in vitro dose-response studies against tumor cells are most often performed on a log-scale, e.g. , , , , and nm. the first question the academic investigator should ask is, in their hands, does the dose-response effect on tumor cell growth/viability correspond to the claimed inhibitory ic of the compound against its purified specific target? i.e. if the protein target has an ic inhibition of nm and an ic for growth inhibition and cell killing of nm, it suggests the compound may be binding tightly to the serum in the culture media, resulting in a very low concentration of free "active" drug. on the other hand, if the target inhibition ic is nm but the ic for growth arrest/killing is nm, the data implies the compound may have additional unknown higher affinity targets in addition to its primary target which all collectively contribute to the biological efficacy of the agent. in this article we have discussed the fda approved drugs sorafenib and neratinib. sorafenib was originally developed to inhibit raf- and b-raf. prior to the discovery that raf- phosphorylated mek / , it was noted that the catalytic site of the raf- serine/ threonine kinase most closely resembled the active sites of src family non-receptor tyrosine kinases. hence, it was no surprise that within a few years sorafenib was also shown to also inhibit class iii receptor tyrosine kinases, and investigators now considered the biology of drug to be as an "anti-angiogenic" agent rather than per se an inhibitor of raf- . , finally, sorafenib was shown to be an inhibitor within its physiological range of hsp and hsp chaperone proteins. , similarly, neratinib was developed solely with the intention of inhibiting the receptor tyrosine kinase her (erbb ) as a putative therapeutic for her + breast cancer. yet, within several years of neratinib entering the clinic, two groups demonstrated it could inhibit map k and map k serine/threonine kinases with low nanomolar ic values. , so, if the compound under investigation is considered by a drug company to be a "specific" inhibitor of a particular protein kinase, regardless as to whether the agent is also fda approved, the in vitro studies the academic investigator should perform are an agnostic wide-ranging series of assessments, over a clinically-relevant drug dose-response range and over a time course. these studies will define, in your own hands, the changes in phosphorylation of the proposed target but also of multiple other cellular signaling pathways. this involves studying components of each specific pathway, e.g. the regulatory phosphorylation and total expression of erbb , erbb , erbb , erbb , raf- , b-raf, mek / and erk / , as well as of downstream nuclear transcription factors whose functions are controlled by each pathway, such as camp response element-binding protein (creb). such wide-ranging data-intense screening studies are difficult to perform using traditional sds page and western blotting approaches, and more advanced methods such as dot-blots or staining fixed cells in situ and measuring the intensity fluorescent staining, using validated antibodies, which permit a high-throughput approach, are required. an old phrase in science is: "the data is what it is." thus, if signaling from the primary target in one signaling pathway is only partially inhibited by the drug under examination, but signaling through an unrelated pathway is almost abolished, one would therefore tentatively conclude that the compound has an unknown target in a different signaling pathway. or, if at a low concentration of the drug no inhibition of the primary target is observed, but that this occurs alongside changes in the activities of other pathways, an effect which is also associated with significant levels of growth arrest and tumor cell death, one would conclude that the biological primary target is not the key functional target which regulates tumor cell biology. these examples for drug actions are binary, and in reality, the differential effects upon signaling and tumor cell biology of any drug are more subtle and nuanced. a different set of concepts come into play when rationally combining fda approved drugs to develop a novel anti-cancer therapeutic approach. first, the safe c max values for both agents in patients should be determined alongside their plasma half-lives, c min at h values and serum binding properties. the c max/c min values alongside the drug's half-life should inform the researcher that, for example during a h in vitro time course assay, a drug concentration considerably less than the c max but above the c min should be used to approximate for a physiologic treatment concentration. many clinically relevant drugs are stated to be % protein bound in % serum; most in vitro studies are performed using % (v/v) fetal calf serum. what is self-evident, however from extant data, is that for a drug such as sorafenib, with a safe c max of mm and a stated % plasma protein binding, is that a free sorafenib concentration of nm in vitro has a very modest impact on altering tumor cell biology, i.e. the partitioning on-off rate for drug association with plasma proteins and with tumor tissue must also be taken into consideration when deciding the most in vitro physiologic drug concentration. thus, taking all of these parameters into account, studies in the author's laboratory, in vitro with cells in % (v/v) serum, and in an attempt to remain within the physiologic range, do not use sorafenib above mm. an additional consideration for drug combination studies is to determine from the literature the doselimiting toxicities of each drug. regardless of excellent laboratory-based data, if the two drugs being combined both have doselimiting toxicities (dlts) in the same tissue, e.g. the gastrointestinal tract (gi), the likelihood that both agents can be safely and successfully combined in a patient is considerably reduced. studies to define hormonal signaling and intracellular signal transduction are almost years old. although much essential biological information was gleaned from work performed in the s to the late s, it was only with the widespread use of more modern molecular biology approaches combined alongside classic biochemical approaches that the signal transduction landscape of the last years evolved. today, essentially all of the building blocks of all signal transduction pathways are known. what is still under investigation are the complex protein-protein interactions which define nuanced signaling during growth, development, and various pathologies. small molecule therapeutic interventions have been and are being developed using ever more sophisticated technologies, of which some have shown considerable clinical utility. however, many of the newly developed "specific" targeted drugs have had little to no testing to fully define off-target effectors of their biology. it is very probable that this on-target/off-target issue for all drugs will never be fully resolved. hence, the step-wise approaches described in this article will still be required to fully understand the use and application of all new drugs. the samuri chemist, and his work on adrenalin observations with insulin on department of soldiers' civil re-establishment diabetics aqueous extracts of pancreas iii. some precipitation reactions of insulin the synthesis of a polysaccharide from glucose- -phosphate in muscle extract biosynthesis of glycogen from uridine diphosphate glucose conversion of phosphorylase b to phosphorylase a in muscle extracts the interconversion of phosphorylase a and phosphorylase b from dog heart muscle the relationship of epinephrine and glucagon to liver phosphorylase. i. liver phosphorylase; preparation and properties the relationship of epinephrine and glucagon to liver phosphorylase. ii. enzymatic inactivation of liver phosphorylase the role of cyclic adenylic acid in hormone action comparative studies on glycogen phosphorylase. iii. the phosphorylated site in human muscle phosphorylase alpha the role of cyclic- ', '-amp in responses to catecholamines and other hormones activation of skeletal muscle phosphorylase b kinase by ca the control of phosphorylase kinase phosphatase by "second site phosphorylation"; a new form of enzyme regulation elevation of guanosine ', '-cyclic phosphate in rat heart after perfusion with acetylcholine calcium regulation of phosphatidyl inositol turnover in macrophage activation by formyl peptides regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase c agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cgmp purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments properties of the nerve growth factor receptor. relationship between receptor structure and affinity receptor and g betagamma isoform-specific interactions with g protein-coupled receptor kinases regulation of adenosine ', '-cyclic monophosphate metabolism in cultured neuroblastoma cells purification from sf cells and characterization of recombinant gq alpha and g alpha. activation of purified phospholipase c isozymes by g alpha subunits protein kinase c inhibits epidermal growth factor-dependent tyrosine phosphorylation of phospholipase c gamma and activation of phosphoinositide hydrolysis identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking two types of protein kinase c with different functions in cultured rabbit aortic smooth muscle cells an agonist-specific effect of guanine nucleotides on binding to the beta-adrenergic receptor epitope-tagged gq alpha subunits: expression of gtpase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase c but not mitogen-activated protein kinase activity regulated by the m muscarinic acetylcholine receptor relationship between the beta-adrenergic receptor and adenylate cyclase receptor-tyrosine-kinase-and g beta gamma-mediated map kinase activation by a common signalling pathway beta-arrestin: a protein that regulates beta-adrenergic receptor function manifold roles of b-arrestins in gpcr signaling elucidated with sirna and crispr/cas the role of beta-arrestins in the termination and transduction of g-protein-coupled receptor signals insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. comparison to epidermal growth factor-stimulated phosphorylation epidermal growth factor induces rapid tyrosine phosphorylation of proteins in a human tumor cells insulin stimulates tyrosine phosphorylation of the insulin receptor in a cell-free system identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in a- cell membranes phosphorylation of exogenous substrates by the insulin receptor-associated protein kinase insulin-signaling mechanisms. lessons from the old testament of glycogen metabolism and the new testament of molecular biology d-chiro-inositol glycans in insulin signaling and insulin resistance insulin and a putative insulin metabolic mediator fraction from liver and muscle stimulate p messenger ribonucleic acid accumulation by apparently different mechanisms insulin-mediated effect on the activity of udpg-glycogen transglucosylase of muscle molecular basis for the substrate specificity of protein kinase b; comparison with mapkap kinase- and p s kinase mechanism of activation of protein kinase b by insulin and igf- characterization of a -phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase b alpha inhibition of glycogen synthase kinase- by insulin mediated by protein kinase b specific binding of the akt- protein kinase to phosphatidylinositol , , -trisphosphate without subsequent activation mechanism of activation and function of protein kinase b the molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle facts and new hopes on selective fgfr inhibitors in solid tumors membrane phosphorylation: a crucial role in the action of insulin, egf, and pp src? isolation of an additional member of the fibroblast growth factor receptor family, fgfr- characterization of sites for tyrosine phosphorylation in the transforming protein of rous sarcoma virus (pp v-src) and its normal cellular homologue (pp c-src) muscle proteins related to microtubule associated protein- are substrates for an insulin-stimulatable kinase rapid stimulation by insulin of a serine/threonine kinase in t -l adipocytes that phosphorylates microtubule-associated protein in vitro insulin-stimulated map- kinase phosphorylates and activates ribosomal protein s kinase ii associated protein kinases, mediate the phosphorylation of tyrosine hydroxylase at serine- in situ insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase renaturation and partial peptide sequencing of mitogen-activated protein kinase (map kinase) activator from rabbit skeletal muscle molecular structure of a protein-tyrosine/threonine kinase activating p mitogen-activated protein (map) kinase: map kinase kinase identification and characterization of a new mammalian mitogen-activated protein kinase kinase, mkk a conserved kinase cascade for map kinase activation in yeast the map kinase cascade is essential for diverse signal transduction pathways the pheromone response pathway in saccharomyces cerevisiae regulation of the map kinase cascade activation of mitogen-activated protein kinase kinase by v-raf in nih t cells and in vitro raf- activates map kinase-kinase raf- is a potential substrate for mitogen-activated protein kinase in vivo stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p /rk mitogen-activated protein kinases and c-jun n-terminal kinases are activated by ischemia/reperfusion induction of cyclooxygenase- by the activated mekk ! sek /mkk ! p mitogen-activated protein kinase pathway activation of the ikappab alpha kinase complex by mekk , a kinase of the jnk pathway identification of c-jun nh -terminal protein kinase (jnk)-activating kinase as an activator of jnk but not p the tao of mekk wiring diagrams of mapk regulation by mekk , , and association of sos ras exchange protein with grb is implicated in tyrosine kinase signal transduction and transformation bar-sagi, d. grb mediates the egf-dependent activation of guanine nucleotide exchange on ras guanine-nucleotide-releasing factor hsos binds to grb and links receptor tyrosine kinases to ras signalling the sh and sh domain-containing protein grb links receptor tyrosine kinases to ras signaling association of the shc and grb /sem sh -containing proteins is implicated in activation of the ras pathway by tyrosine kinases activation of (his) -raf- in vitro by partially purified plasma membranes from v-ras-transformed and serum-stimulated fibroblasts regulation of raf- and raf- mutants by ras-dependent and ras-independent mechanisms in vitro complexes of ras.gtp with raf- and mitogen-activated protein kinase kinase association of mek with p ras the mek kinases mekk /ssk p facilitate complexity in the stress signaling responses of diverse systems upstream regulators of the c-jun amino-terminal kinases? isolation of mek and differential expression of alternatively spliced forms the role of c-jun n-terminal kinase (jnk) in apoptosis induced by ultraviolet c and gamma radiation. duration of jnk activation may determine cell death and proliferation opposing effects of erk and jnk-p map kinases on apoptosis the ras/rac /cdc /sek/jnk/c-jun cascade is a key pathway by which agonists stimulate dna synthesis in primary cultures of rat hepatocytes prolonged activation of the mitogen-activated protein kinase pathway promotes dna synthesis in primary hepatocytes from p cip- /waf -null mice, but not in hepatocytes from p ink a-null mice bile acid regulation of c/ ebpbeta, creb, and c-jun function, via the extracellular signal-regulated kinase and c-jun nh -terminal kinase pathways, modulates the apoptotic response of hepatocytes ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo the endocytosis of epidermal growth factor in a cells: a ph of microenvironment and the dynamics of receptor complex dissociation recycling of epidermal growth factor-receptor complexes in a cells the role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor stress signaling and the shaping of the mammary tissue in development and cancer the role of p mapk pathway in p compromised state and telomere mediated dna damage response a systematic review of p regulation of oxidative stress in skeletal muscle activated forms of h-ras and k-ras differentially regulate membrane association of pi k, pdk- , and akt and the effect of therapeutic kinase inhibitors on cell survival role of pi k/akt pathway in cancer: the framework of malignant behavior egfrviii: an oncogene with ambiguous role functions of lysosomes autophagy wins the nobel prize in physiology or medicine: breakthroughs in baker's yeast fuel advances in biomedical research aminopeptidase i of saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction isolation and characterization of autophagy-defective mutants of saccharomyces cerevisiae so many roads: the multifaceted regulation of autophagy induction mtor at the nexus of nutrition, growth, ageing and disease two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury ampk: regulation of metabolic dynamics in the context of autophagy diverse signaling mechanisms of mtor complexes: mtorc and mtorc in forming a formidable relationship mtor as a central hub of nutrient signalling and cell growth distinct roles of mtor targets s k and s k in breast cancer allosteric regulation of amp-activated protein kinase by adenylate nucleotides and small-molecule drugs ampk and tor: the yin and yang of cellular nutrient sensing and growth control lkb /ampk pathway and drug response in cancer: a therapeutic perspective controlling the master-upstream regulation of the tumor suppressor lkb multifaceted roles of atm in autophagy: from nonselective autophagy to selective autophagy role of the lkb /ampk pathway in tumor invasion and metastasis of cancer cells dna-dependent protein kinase regulates lysosomal amp-dependent protein kinase activation and autophagy reactive nitrogen species regulate autophagy through atm-ampk-tsc -mediated suppression of mtorc a review of ulk -mediated autophagy in drug resistance of cancer recruitment and activation of the ulk /atg kinase complex in selective autophagy canonical and noncanonical functions of ulk/atg the mammalian ulk complex and autophagy initiation phosphorylation of ulk by ampk is essential for mouse embryonic stem cell self-renewal and pluripotency guidelines for the use and interpretation of assays for monitoring autophagy evolution of tools and methods for monitoring autophagic flux in mammalian cells metastatic cells are preferentially vulnerable to lysosomal inhibition regulation of apoptosis by autophagy to enhance cancer therapy osu- promotes caspase-independent but perk-, cathepsin b-, bid-, and aif-dependent killing of transformed cells mda- /il- -induced cell killing in malignant renal carcinoma cells occurs by a ceramide/cd /perk-dependent mechanism caspase-, cathepsin-, and perk-dependent regulation of mda- /il- -induced cell killing in primary human glioma cells regulation of gst-mda- toxicity in human glioblastoma cells by erbb , erk / , pi k, and jnk - pathway signaling vorinostat and sorafenib increase er stress, autophagy and apoptosis via ceramide-dependent cd and perk activation vorinostat and sorafenib increase cd activation in gastrointestinal tumor cells through a ca( +)-de novo ceramide-pp a-reactive oxygen species-dependent signaling pathway apoptosis-inducing factor (aif) in physiology and disease: the tale of a repented natural born killer osu- stimulates pkr-like endoplasmic reticulum-dependent increases in -kda heat shock protein expression, attenuating its lethal actions in transformed cells sorafenib: delivering a targeted drug to the right targets therapeutic developments osu- suppresses grp /bip expression that causes perk-dependent increases in tumor cell killing the role of cell signaling in the crosstalk between autophagy and apoptosis in the regulation of tumor cell survival in response to sorafenib and neratinib not the comfy chair! cancer drugs that act against multiple active sites signaling alterations caused by drugs and autophagy role of the unfolded protein response, grp and grp in organ homeostasis bip/hspa /dna k is a universal therapeutic target for human disease osu- and viagra treatment inhibits the activity of multiple chaperone proteins and disrupts the blood-brain barrier: implications for anti-cancer therapies multi-kinase inhibitors can associate with heat shock proteins through their nh -termini by which they suppress chaperone function ar- inhibits multiple chaperones concomitant with stimulating autophagosome formation collectively preventing virus replication grp /dna k is a target for nexavar/stivarga/votrient in the treatment of human malignancies, viral infections and bacterial diseases endoplasmic reticulum stress, and interferon responses master regulator of the unfolded protein response and crucial factor in flavivirus biology he, a. blockade of hsp by ver- synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma discovery of novel heat shock protein (hsp ) inhibitors based on luminespib with potent antitumor activity the celecoxib derivative kinase inhibitor ar- (osu- ) inhibits zika virus via down-regulation of the pi k/akt pathway and protects zika virus-infected a mice: a host-targeting treatment strategy ar- suppresses dengue virus replication by down-regulation of pi k/akt and grp exploring the in vitro potential of celecoxib derivative ar- as an effective antiviral compound against four dengue virus serotypes egfr mutation and resistance of non-small-cell lung cancer to gefitinib irreversible inhibitors of the egf receptor may circumvent acquired resistance to gefitinib activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib gefitinib-sensitizing egfr mutations in lung cancer activate anti-apoptotic pathways egf receptor mutations in lung cancer: from humans to mice and maybe back to humans presence of epidermal growth factor receptor gene t m mutation as a minor clone in non-small cell lung cancer epidermal growth factor receptor mutations in patients with non-small cell lung cancer third-generation epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer afatinib in locally advanced/metastatic nsclc harboring common egfr mutations, after chemotherapy: a phase iv study. lung cancer manag observational study of sequential afatinib and osimertinib in egfr mutation-positive nsclc: patients treated with a -mg starting dose of afatinib multi-kinase inhibitors interact with sildenafil and erbb / / inhibitors to kill tumor cells in vitro and in vivo the afatinib resistance of in vivo generated h lung cancer cell clones is mediated by src/erbb /c-kit/c-met compensatory survival signaling rationally repurposing ruxolitinib (jakafi ( w )) as a solid tumor therapeutic hdac inhibitors enhance neratinib activity and when combined enhance the actions of an anti-pd- immunomodulatory antibody in vivo neratinib inhibits hippo/yap signaling, reduces mutant k-ras expression, and kills pancreatic and blood cancer cells comprehensive analysis of kinase inhibitor selectivity the target landscape of clinical kinase drugs exposure permanently reduces erbb and ras expression in t mammary tumors and enhances m macrophage infiltration the levels of mutant k-ras and mutant n-ras are rapidly reduced in a beclin /atg -dependent fashion by the irreversible erbb / / inhibitor neratinib neratinib degrades mst via autophagy that reduces membrane stiffness and is essential for the inactivation of pi k, erk / , and yap/taz signaling risk of gastrointestinal complications in breast cancer patients treated with neratinib: a meta-analysis targeting neratinib-induced diarrhea with budesonide and colesevelam in a rat model yap/taz functions and their regulation at a glance yap/taz: drivers of tumor growth, metastasis, and resistance to therapy yap activation enables bypass of oncogenic kras addiction in pancreatic cancer downstream of mutant kras, the transcription regulator yap is essential for neoplastic progression to pancreatic ductal adenocarcinoma primary structure of v-raf: relatedness to the src family of oncogenes safety and pharmacokinetics of the dual action raf kinase and vascular endothelial growth factor receptor inhibitor, bay - , in patients with advanced, refractory solid tumors results of phase i pharmacokinetic and pharmacodynamic studies of the raf kinase inhibitor bay - in patients with solid tumors neratinib: an oral, irreversible dual egfr/her inhibitor for breast and non-small cell lung cancer bay - : early clinical data in patients with advanced solid malignancies key: cord- - pm l d authors: quinteros, daniela a.; bermúdez, josé m.; ravetti, soledad; cid, alicia; allemandi, daniel a.; palma, santiago d. title: therapeutic use of monoclonal antibodies: general aspects and challenges for drug delivery date: - - journal: nanostructures for drug delivery doi: . /b - - - - . - sha: doc_id: cord_uid: pm l d monoclonal antibodies are routinely used in several fields but the great challenge has been their use as therapeutic agents for the treatment of diseases, such as breast cancer, leukemia, asthma, macular degeneration, arthritis, crohn’s disease, and transplants, among others. monoclonal antibodies are protein molecules made in the laboratory from hybridoma cells by recombinant dna technology. important advances have been made over the past decade to improve some critical points, such as safety and efficacy of the first generation of therapeutic antibodies. this type of molecules presents a significant challenge from the pharmaceutical point of view due to their characteristics, such as molecular size, stability, and solubility. in this chapter we have attempted to identify the major issues associated with therapeutic approaches, formulating drawbacks and delivering antibody drugs, particularly focused on the challenges and opportunities that these present for the future. mabs are indispensable not only to health, but also to prevent food poisoning, and are used to investigate environmental pollution. however, despite their widespread distribution and significance, most people have never heard of mabs or how they have both transformed healthcare and spawned an entire new industry. produced in the laboratory, mabs are derived from the billions of tiny antibodies made every day by our immune systems to combat substances, known as antigens, which are regarded as foreign or potentially dangerous. millions of different types of antibodies can be found in the blood of humans and other mammals. made by white blood cells known as b lymphocytes, each antibody is highly specific-that is, it has the ability to bind to only one particular antigen, which may be derived from bacteria, viruses, fungi, parasites, pollen, or nonliving substances, such as toxins, chemicals, drugs, or foreign particles considered alien to the body. once antibodies have marked their antigen, they and other types of cells produced by the immune system can attack it. the field of genetically engineered therapeutic mabs has relied on many inventions during decades of research, but two key discoveries in the mid- s stand out as seminal events that laid the groundwork for this field to exist as we know it today. although mabs were first described in (kohler and milstein, ) , only when the original rodents forms were replaced by their human equivalents did their potential as therapeutic agents began to be properly appreciated (lonberg, ; winter et al., ) . the reasons for this are complex, but related to a combination of perceptions including patentability, immunogenicity, effector function, and a wish to avoid undesirable side effects. the increasing scientific interest on mabs can be clearly seen in fig. . , which shows the rising number of published articles using the keyword monoclonal antibodies in different databases (pubmed and scopus). athough fewer published articles, the number of publications is constant over the years to science direct and wiley. general introduction mabs are used not only as drugs for treating various diseases, but are also used as powerful tools for a wide range of medical applications. they are routinely used in hospitals for blood type and tissue, a vital process to ensure safe blood transfusion and organ transplantation. in other cases, they are employed as research probes to determine the pathological pathway and the cause of diseases, such as cancer, autoimmune diseases, and neurological disorders. on the diagnostic front, monoclonal antibodies are intrinsic components of test kits for the detection of ovulation, pregnancy, or menopause. they are also used for analyzing body fluids for medical diagnosis, and to determine whether there has been a heart attack. unlike polyclonal antibodies, mabs are identical antibodies because they are produced by one type of immune cell. using current hybridoma (mouse/human hybrid cells) technology originally developed by georges kohler, cesar milstein, and neils jerne, mabs can be produced to bind tightly to virtually any material or antigen, which is defined as a substance that prompts the generation of antibodies that specifically bind to it. antigens typically consist of proteins or polysaccharides. epitopes, also known as antigenic determinants, are the part of the antigens through which actual binding to antibodies occurs. the mabs technology allowed scientists to produce huge quantities of pure antibodies aimed at specific selected targets, leading to the design of new diagnostic tests and therapeutics. by injecting a payload of mabs into the bloodstream, the antibodies were headed straight to their disease target. today, the growth and profitability of mabs are outstripping those of earlier types of biotechnology drugs and more traditional pharmaceutical ones. indeed, their expansion is among the fastest in the global pharmaceutical world. in part, this reflects the sector's embrace of mabs as an answer to dwindling drugs in the pipeline and reduced revenue streams in the face of the expiration of key patents and the growth of generic medicines. drugs based on mabs technology have names ending in "mab." the first drug that received food and drug administration (fda) approval was rituxan (rituximab) (fig. . ) for the treatment of non-hodgkin's lymphoma, in . it was developed by idec, and it has since merged with biogen. rituximab is a mab against the protein cd , which is primarily found on the surface of immune system b cells. rituximab destroys b cells and is therefore used to treat diseases that are characterized by excessive numbers of b cells, overactive b cells, or dysfunctional b cells. this includes many lymphomas, leukemias, transplant rejection, and autoimmune disorders. idec teamed up with genentech to get fda approval and to comarket rituxan. in parallel, genentech developed herceptin (trastuzumab) for the treatment of breast cancer associated with the her /neu receptor. herceptin was approved by the fda in and is the first important example of personalized medicine requiring a diagnostic test to ensure efficacy, since only patients who express the her gene in their breast cancer tissue have a chance to benefit from treatment with this mab. today, six therapeutic mabs are among the best-selling drugs in the world. in it was estimated that the global sales of the more successful mabs were worth more than $ . billion. among these, adalimumab (humira) is used for the treatment of rheumatoid arthritis and other autoimmune diseases and was at the top of the selling drug list across the world in , reaching an annual income of $ . million. thus, mab sales are expected to be higher than those for lipitor, used for cholesterol reduction, which has been the largest prescribed drug historically (king, ) . since the s, the methodologies available for generating mabs have dramatically evolved from the hybridoma technology used to produce murine mabs (kohler and milstein, ) to sophisticated recombinant engineering technologies (brüggemann et al., a,b; steinitz et al., ) appropriate to design specific mabs of choice, thus producing humanized, chimeric, and completely (fully) human mabs (fig. . ) (brüggemann et al., a,b; jones et al., ; lonberg, ; riechmann et al., ) . these novel methods have had major consequences in the development of clinically applicable mabs against disease targets. the immunogenicity and safety of mabs, a substantial limitation of antibodies produced by the hybridoma technology, could be overcome, and mabs against any antigen specificity and with desirable physicochemical properties could be developed. high-scale production of mabs at the industrial level could further be achieved. this has sparked a race for the development of antibody-mediated therapeutics in the clinic with the investment of large amounts of money from the biopharmaceutical sector toward that end, which is already paying off. the major success of human mabs in immune and inflammatory conditions, such as rheumatoid arthritis (ra), crohn's disease, ulcerative colitis, spondyloarthropathies, juvenile arthritis, psoriasis, psoriatic arthritis, and others (andreakos et al., ) , marks merely the beginning of a rapidly evolving field with more than diverse mabs currently being evaluated in clinical trials or being candidates for approval by the fda in the usa. scaling up mabs production for therapeutics posed several challenges. the major issue was how to produce high quantities of drugs at a reasonable cost. most cell lines in the s yielded only half a gram of mabs per liter, so production was time consuming and expensive. the ideal was to develop figure . monoclonal antibody rituximab antibody-drug conjugates a hybrid cell line that could produce between and g of mabs per liter. this demanded several steps, and even more, each requiring skill and patience. first was the creation of a hybrid cell, after which a clone secreting mabs in high concentrations had to be selected. then a culture medium had to be developed to encourage the optimal growth of the hybridoma. scaling up such media was not easy in terms of quality control because they contained fifty or more ingredients, and it was important to determine how many nutrients to add. hybridomas stop secreting mabs, for example, if given too much glucose. antibody-drug conjugates (adcs) are mabs bearing cytotoxic drugs covalently bound via a chemical linker and can be defined as prodrugs. prodrugs are inactive or less active derivatives of drug molecules and undergo enzymatic or chemical transformation to regenerate the active forms. adcs are designed to be superior to either antibody therapeutics or chemotherapy alone by overcoming their limitations while preserving the merits from both. the antibody connected to the cytotoxic warhead (drug) via a linker serves as targeted delivery system to the tumor expressing the antigen/target recognized by the antibody. ideally, in blood, after systemic administration, this prodrug is nontoxic. upon binding of the antibody to the targeted tumor antigen and internalization of the complex into the cancer cell, the drug is then released in its active form and in sufficient quantity to kill the cell. on top of the careful choice of a target/antigen expressed in specific tumor indication, it requires finding the best combination between the antibody, the linker, and the drug, which, besides its own characteristics and constraints, are linked and impact each other (bander, ) . the combination of nanoparticles and antibodies can offer versatility together with specificity ( fig. . ) , allowing a huge potential market. the target/antigen is the starting point to build an adc. it first determines which tumor indication will be targeted by the adc and potentially impacts the choice of the conjugated drug. in addition, the target will also drive the criteria that will be defined for the selection of the targeted patient population within the tumor indication. mabs are typically susceptible to noncovalent aggregation upon storage. aggregation is driven by the minimization of free energy achieved when antibodies come into close contact-for instance, at relatively hydrophobic sites on the molecules. dimerization may be thought of as the first step in aggregation, and sometimes the process proceeds no further. dimers maybe reversible (moore et al., ) or not, but growth can lead to larger and larger oligomers and even potentially insoluble particulates, as time progresses (wang et al., ) . because adcs are decorated with one or more small molecule drug moieties, they have a different set of biophysical properties compared with the unconjugated antibody, leading to new or altered intermolecular interactions. an antibody that exhibits acceptable figure . conjugation of nanoparticles to antibodies and their advantages antibody-drug conjugates aggregation behavior in its unconjugated form may have different physical behavior in its conjugated form, either because of changes in surface properties, such as hydrophobicity due to drug attachment or because the drugs have altered the higher-order structure of the antibody such that new modes of antibody-antibody interactions are possible. these factors could contribute to a substantially different propensity for aggregation. this effect is most readily observed in a formulation screen designed to test both conjugated and unconjugated forms in parallel and subjected to the same assays. such a study does not automatically reveal the exact cause of the effect, only that it is related to the conjugated form or conjugation process. the degree to which the molecule has been physically altered by conjugation may be assessed by techniques such as differential scanning calorimetry (dsc) (wakankar et al., ) , with physical perturbations manifested as changes (likely decreases) in the onset of melting or melting temperature, tm, of the conjugated mab as compared to the unconjugated antibody. this decrease in thermodynamic stability may translate to a decreased colloidal stability of the conjugate compared to an unconjugated antibody. part of adc chemical stability is inherited from the unconjugated antibody. for instance, if the unconjugated antibody is susceptible to deamidation or isomerization with known ph dependence, this degradation mode is also likely to be present in the adc. these types of degradations may impact the product potency, especially if the affected residues are found in the complementarity-determining region. much has been learned through years of experience with unconjugated antibodies about the susceptibility of various amino acid residues to chemical degradations, allowing substantial insights to be gained from examination of the protein primary sequence alone, coupled to available information about local solvent exposure and flexibility (kosky et al., ; robinson and robinson, ) . it may be expected that most chemical modifications to the primary sequence of the unconjugated antibody will also occur in the adc form. fragmentation is another possible chemical degradation pathway for adcs, driven by the breakage of covalent bonds between chains or within the peptide backbone. the altered physical state of the molecule following addition of the small molecule drug may result in some differences in the susceptibility of the adc to fragmentation, but the fundamental susceptibility of the adc to fragmentation is likely derived mostly from the mab. a common unconjugated antibody degradation mode involves breaking the heavy chain peptide near the hinge region, leading to free fab and fab + fc products (cordoba et al., ) . this is still a feasible degradation pathway for adcs as well. comparison of fragmentation rates between conjugated and unconjugated antibodies can reveal whether the adc form has different stability toward fragmentation. the covalent bonds linking drugs to antibodies may be broken by a variety of mechanisms over the shelf life of the product and under stressed storage conditions. formulation development of adcs aims to ensure that stable, high-quality products are dosed to patients. though this goal is common to all pharmaceutical formulations, adcs present a unique set of physicochemical properties that can impact safety, quality, and efficacy as compared to traditional therapeutic proteins. although there is a body of literature that outlines which product attributes affect the safety, quality, and efficacy for conventional, unconjugated mabs (goetze et al., ) , an understanding of attributes important to adcs is only now emerging. the choice of formulation has the potential to affect all categories of adcs quality attributes. because conjugation of an adc requires an unconjugated antibody as an intermediate, the formulation development process for a conjugate necessarily involves formulating a mab at some stage, likely as an intermediate for manufacturing of the final adc form. the formulation optimization for the antibody portion of the adc follows a similar course to that of an unconjugated antibody and, therefore, can leverage the biotechnology industry's considerable experience developing this class of molecule. many examples of successful commercial antibody formulations, both liquid and lyophilized, have been reported (wang et al., ) . however, the complexity of the formulation-development process potentially increases when quality attributes of the small molecule drug and those of the conjugated form are taken into account. the antibody-conjugated nanoparticles can be used principally in two biomedical applications: diagnosis and therapy. in therapy, the development of targeted drug delivery represents, together with tissue repair, the main applications of antibody-conjugated nanoparticles. in diagnosis, the applications can be divided into those using in vivo and those using in vitro experimentation and include contrast agents for magnetic resonance imaging (mri), sensing, cell sorting, bioseparation, enzyme immobilization, immunoassays, transfection (gene delivery), and purification. today, one of the most interesting challenges posed by drug-delivery systems is the development of smart vectors, which are required to be safe, easily administered, and economic, and which allow simultaneous diagnosis and treatment. moreover, it is intended that the delivery is precisely controlled in term of dose and site of action to reduce adverse side effects. in this regard, there are many important site-selective drugs, such as highly toxic antitumor molecules, that must reach targeted cells or tissues without being released before. nanoscience and nanotechnology have found their way into the fields of biotechnology and medicine. nanoscience is the study of structures and materials on an atomic and molecular scale, in a level that ranges usually below nm. nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. antibodies are nanosize biological products that are part of the specific immune system. in addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc.), they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc.) and be used in several diagnostic procedures, or even in therapy to destroy a specific target. the conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. since the properties of materials differ significantly between atomic or subatomic level and larger scales, nanomaterials have attracted the attention of researchers for pharmaceutical application, especially in the area of drug delivery. this technology overcomes some of the drawbacks of large-size materials, such as poor bioavailability, in vivo stability, solubility, intestinal absorption, sustained and targeted delivery to site of action, and generalized side effects, among others. in this regard, nanostructures have been reported to protect drugs from the degradation in the gastrointestinal tract, and even more, to provide means of bypassing the liver, preventing the first pass metabolism. this technology also has the ability to target delivery of drugs to various areas of the body, and enables the delivery of drugs that are poorly water-soluble. moreover, nanotechnology improves drugs bioavailability and allows them to remain circulating in the blood for a long time, controlling their release. nanostructures can also be able to penetrate tissues and be easily taken up by cells, allowing for efficient delivery of drugs to target sites of action. nanostructures were reported to be taken up by the cells at rates - times greater than microparticles. nanotechnology improves performance and acceptability of dosage forms and even may enhance the performance of drugs that fail in clinical trial phases. nanotechnology will definitely revolutionize the science of drug delivery and help overcome the major challenges of conventional drugs used for the treatment and management of chronic diseases such as, cancer, asthma, hypertension, hiv, and diabetes. in this context, nanomedicine can be defined as the medical application of nanotechnology and involves research, and diagnostic and therapeutic applications related with nanotechnology. nanotechnology has been applied in medicine to develop strategies to manage, treat, or even cure virtually every type of human disease. mabs are not only successful drugs but also powerful tools for a wide range of medical applications. in this regard, over the last years several nanotechnological platforms for medical applications have converged upon a specialization toward diagnosis, therapy or even both, as in the case of magnetic nanoparticles. the study of nanoscience and the optimization of technologies, such as adcs and nanocarriers have driven the design of methodologies that are tailored to address specific diseases and medical anomalies. adcs, for example, represent excellent nanoscale-based drug delivery vehicles and thus tend to be focused on delivery to certain drugs, as in the treatment of cancer. the conjugation of different moieties to the nanoparticles widens their application fields and provides them with new or enhanced properties. a range of biomoieties can be conjugated to the nanoparticles including low molecular weight ligands (folic acid, thiamine, dimercaptosuccinic acid), peptides (rgd, lhrd, antigenic peptides, internalization peptides), proteins (bsa, transferrin, antibodies, lectins, cytokines, fibrinogen, thrombin), polysaccharides (hyaluronic acid, chitosan, dextran, oligosaccharides, heparin), polyunsaturated fatty acids (palmitic acid, phospholipids), dna, and plasmids. nanotemplates provide unique access to extremely high-throughput capabilities for rapidly defining the presence of a biomarker and thus lend themselves as excellent platforms for diagnostics applications. rapid progress is being made to drive these advances in nanomedicine, especially in the area of cancer treatment. although still in its infancy when compared to other medical technologies, nanomedicine is undoubtedly here to stay and will have an enormous impact on the future health and well-being of man. nanocarriers are nanometer-sized materials that have the capacity to deliver therapeutic agents at the disease site (arias, ; schroeder et al., ) . they are designed to possess unique physicochemical properties, aiming to improve the pharmacokinetic and biodistribution of a drug molecule (li and huang, ) and to deliver a significant amount of drug molecules. examples of some therapeutic nanocarriers are lipid-based particles (al-jamal and kostarelos, ), micelles (kedar et al., ) , nanoparticles (shi et al., ) , dendrimers (svenson, ) , and polymersomes (levine et al., ) . some of these have been proposed for the treatment of various diseases including cancer (dhar et al., ) , coronary artery diseases (chan et al., ) , and rheumatoid arthritis (thomas et al., ) . in the particular case of cancer, the unique anatomy-that is, the leakiness of the tumoral vasculatures-concedes a passive transport of the nanocarriers by enhanced permeability and retention (epr) effect (maeda et al., ) . however, the porosity of the tumor blood vessels may vary with the tumor type (bae, ) . even with a successful delivery by the epr effect, the nanocarriers must be able to internalize into the cancer cells (gradishar et al., ) . a new paradigm in drug delivery embroils a combination of active and passive targeting. targeting ligands, such as antibodies (mamot et al., ) , peptides (srinivasan et al., ) , small molecules (kukowska-latallo et al., ) , or aptamers (farokhzad et al., ) can be attached onto the surface of a nanocarrier. the carriers recognize and bind to the cell-surface receptors and are subsequently taken by the cells via receptor-mediated endocytosis for releasing the therapeutic payloads (shi et al., ) . the binding affinity of targeted nanocarriers can also be increased several orders of magnitude by the multivalent effect . among all the targeting ligands, antibodies are well known for their high binding affinity, specificity, and availability for a number of disease biomarkers (manjappa et al., ). an antibody can be simply absorbed on the surface of a nanocarrier via hydrophobic and/or electrostatic interaction (sokolov et al., ) . however, using this approach, the absorbed antibody may orient randomly on the surface and result in losing the binding affinity. furthermore, the antibody may exchange with other endogenous protein in vivo (nobs et al., ) . therefore, antibodies are generally preferred to attach to the nanocarriers covalently (arruebo et al., ). an antibody consists of a number of functional groups that provides many options for bioconjugation (manjappa et al., ) . bioconjugation can take place by means of adsorption (at the isoelectrical point of the antibody via electrostatic interaction), by direct covalent linkage between the surface of the nanoparticle and the antibody, or by using adapter molecules. the use of adapter molecules generally involves streptavidin and biotin for the formation of the complex. biotin-labeled polyclonal goat anti-escherichia coli antibodies have been attached to streptavidin-coated magnetic nanoparticles, and used for the separation and selective quantification of escherichia coli o :h in ground beef in the presence of other bacteria (varshney et al., ) . mabs are capable of specifically recognizing and binding to many molecules, mainly those of protein nature. this capability have been widely used in the field of diagnosis for the detection of hormones, vitamins, cytokines, allergens, numerous tumor markers, and a wide range of markers associated with many diseases, including microbial infections. for these properties, the mabs are the substances most commonly used in the field of clinical diagnosis and in identifying markers and therapeutic targets. besides applications in disease diagnosis and in drug discovery, therapeutic applications include mabs, such as vehicles or carriers of other drugs. current major therapeutic applications of mabs include cancer, chronic inflammatory disease, and infection, and they constitute the largest and fastest growing sector of the biological pharmaceutical industry. in addition to offering a host of new drugs to fight disease, mabs have provided the means to monitor a patient's response to therapy and helped lead the way in personalized medicine. mabs are now marketed not only for cancer and autoimmune disorders, but also for a range of other diseases, including allergic conditions, such as asthma, age-related macular degeneration (an eye disorder), multiple sclerosis (a neurological disorder), and osteoporosis (brittle bones). they are also being investigated for central nervous system disorders, such as alzheimer's disease (a degenerative brain disease), metabolic diseases such as diabetes, and the prevention of migraines. mabs used for therapeutic purposes may bind to a variety of antigens present on the surface of tumor and cancer cells. they may also be utilized to deliver a number of different types of payloads to destroy the targeted cells in question including radioactive ligands, cytokines, toxins, liposomes containing drugs, and specific killer cell types. these immunoconjugates, which are defined as antibodies linked to a second molecule, such as a toxin, radioisotope, or label may act at the cell surface as killing agents or be internalized to release payloads intracellularly. despite their high sensitivity and reproducibility, conventional diagnostic methods for a microbial infection require cumbersome sample preparation and long readout times (kaittanis et al., ) . unique electrical, magnetic, luminescent, and catalytic properties of nanomaterials enable fast, sensitive, and cost-effective diagnosis, as well as rapid determination of the susceptibility and resistance of antibacterial drugs (bruchez et al., ; edgar et al., ) . antibody-conjugated nanoparticles amplify the signals for bioanalysis and enumeration of highly pathogenic bacteria, such as e. coli o :h , resulting in highly selective, convenient, and rapid detection of single bacterium within min (look et al., ) . the rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology and medical diagnosis. current methods either lack ultrasensitivity or take a long time for analysis. the use of magnetic nanoparticles also could be a very sensitive and rapid strategy to detect microbial infection. magnetic nanoparticles, in particular superparamagnetic iron oxide nanoparticles (spion) with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications, such as magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery, in cell separation, and so forth (neuberger et al., ) . dextrancoated supermagnetic iron oxide nanoparticles were clustered by con-a treatment, or equipped with con a-conjugated nanosensors (zhao et al., ) . supermagnetic iron oxide nanoprobes greatly assisted the identification of mycobacterium avium spp. paratuberculosis, as well as the quick quantification of these bacteria in milk and blood with high sensitivity (basu et al., ) . a quick method for detecting infections in the urinary tract has also been developed using gold nano-wire arrays in conjunction with a linker arm attached to specific e. coli antibodies (de la escosura-muñiz and merkoçi, ). li and huang ( ) have shown that nanoparticles with specific raman spectroscopic fingerprints can distinguish antibiotic resistant bacteria, by detecting single-nucleotide polymorphisms in microarray-based systems . a bioassay based on bioconjugated nanoparticle was developed by zhao et al. ( ) for in situ pathogen quantification. the detection is carried out by a high fluorescent signal provided by the nanoparticle, which is easily attached to an antibody or other biorecognition molecule. these conjugated nanoparticles can be used in the specific identification of a wide range of bacteria, such as e. coli o :h , through antibody antigen interaction. these nanoparticles were able to assess the microbial metabolic activity and determine antimicrobial susceptibility in blood, by rapidly quantifying polysaccharides. the broad absorption spectra of quantum dots (qds) can be exploited to simultaneously excite qds emitting different colors using a single wavelength (cao et al., ) . these characters suggest that qds are a promising modality for the analysis of complex samples for histology, pathology and cytology, and can facilitate double or even triple immunostaining of bacterial cells (kaittanis et al., ) . studies have demonstrated that the use of nanotechnology feasibility of achieving fast and reliable pharmaceutical assays for microbial infections in opaque media (e.g., whole blood and milk), without any sample preparations (alper et al., ; tully et al., ) . the ability to track the distribution and differentiation of progenitor and stem cells by high resolution in vivo imaging techniques would have significant clinical and research implications. lewin et al. ( ) developed a cell labeling approach using short hiv-tat peptides to derivatize superparamagnetic nanoparticles. these particles were efficiently internalized into hematopoietic and neural progenitor cells in quantities up to - pg of superparamagnetic iron per cell. iron incorporation did not affect cell viability, differentiation, or proliferation of cd + cells. following intravenous injection into immunodeficient mice, % of magnetically cd + cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance imaging in tissue samples. in addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells (lewin et al., ) . nanomaterials with fluorescent properties or nanoparticles labeled/encapsulated with fluorescent dyes have also been applied for microbial detection. using antibody-conjugated silica nanoparticles, these materials can be used as a superior signaling element, to detect cells, proteins and bacteria in an immunoassay (grifantini et al., ; tan et al., ) . ultrasensitive methods for bioassays have been developed using fluorescent bioconjugated silica nanoparticles (santra et al., ; zhao et al., ) . thousands of fluorescent dye molecules are encapsulated in a protective silica matrix, resulting in a nanoparticle with an amplified and reproducible signal for fluorescence-based bioanalysis. whereas in conventional immunoassays only one or a few dye molecules are linked to an antibody molecule and then used to signal an antibody-antigen interaction event, the bioconjugated nanoparticles, which are attached to the antibody molecule, are enabled to carry many dye molecules inside, allowing a significant amplification of the analytical signal. furthermore, bacteria present many surface antigens available for antibody recognition, and, therefore, a greatly amplified signal can be achieved since thousands of nanoparticles can attach to each bacterium. these bioconjugated nanoparticles allow the detection of a bacterium cell per given sample just in min with a spectrofluorometer. santra and coworkers used covalent method to attach surface-modified, rubpy-doped silica nanoparticles to mouse antihuman cd antibody. this complex was then incubated with mononuclear lymphoid target cells. after washing away the unbound nanoparticles, target leukemia cells could be clearly detected. in comparison to control group, results of this experiment have shown that this technique was very effective to detect leukemia cells selectively (santra et al., ) . tan and coworkers developed an assay tool for in situ detection of single bacterium cells in less than min and developed multicolored fret (fluorescence resonance energy transfer) silica nanoparticles by coencapsulating three tandem dyes that emit unique colors upon excitation with a single wavelength . among therapeutic applications of antibody-conjugated nanoparticles, the use of gold nanoparticles was tested in killing staphyloccocus aureus (zharov et al., ) . the bacteria were killed by light-adsorbing gold nanoparticles conjugated to antiprotein a antibodies, using laser irradiation at nm. protein a was chosen because it interacts specifically with the fc fragment of the antibody. according to the authors, killing efficiency depends on the local overheating effects accompanied by bubble-formation phenomena. direct irradiation of bacteria with the laser did not damage the bacteria, because of low absorption by natural endogenous cytochromes. the diagnosis of cancer at early stages of growth is a critical factor for obtaining optimal results in therapy and for improving the chances of survival. several imaging techniques collaborate with physicians in the diagnosis, including magnetic resonance imaging (mri), positron emission tomography (pet), computed tomography (ct), ultrasound, radiography, photoacoustic imaging, fluoroscopy, and so forth. in some of these techniques, antibodyconjugated nanoparticles may offer increased selectivity and sensitivity. fluorescent semiconductor nanocrystal qds are a novel class of multifunctional inorganic fluorophores that are promising in biological imaging, including immunofluorescence imaging (wu, ) , and are useful for labeling molecules (lidke et al., ; wu, ) and cells in various materials (gao et al., ; jaiswal et al., ; kim, ) , even clinical human samples (tholouli et al., ) . the first generation of adcs presented limitations in relation to the linker instability because the early linkers were either too stable, resulting in low potency and reduced efficacy, or too unstable, resulting in poor targeting and high systemic toxicity. furthermore, cytotoxics without sufficient potency, inefficient internalization, limited expression of the target antigen and immunogenicity of mabs (alley et al., ; chari, ) . second-generation adcs were designed to deliver potent anticancer agents to tumors in a targeted manner to limit systemic exposure. the success of targeted adcs depends on antibody selection, potency of the cytotoxin and the method selected to link the antibody to the cytotoxin. nanotechnology is a disruptive technology that drives a new generation of cancer preventive diagnosis, and therapeutic products, resulting in dramatically improved cancer outcomes. nanotechnology in the field of cancer has the potential to improve the monitoring of therapeutic efficacy, provide novel methods for the detection and profiling of cancers at early stages, and allow surgeons to delineate tumor margins and sentinel lymph nodes. nanomaterials have unique features that are attractive and can be applied to biosensing. the development of various nanomaterials and nanotechnology has enabled detection of cancer biomarkers with great precision and sensitivity that could not be achieved before. many studies are being conducted on developing sensing mechanisms that will push down the detection limit as far down as possible (anajwala et al., ) . ligand-free nano-formulations generally possess passive targeting property with little tissue specificity. to address these limitations, research has been continued to advance active or specific targeting to enhance the efficacy of anticancer therapeutic agents, as well as to reduce the toxicity to nontargeted healthy tissues. nanoparticles containing the chemotherapeutic agents are specifically designed to target the cancerous cells either by ligand receptor interaction or antibody-antigen recognition. highly specific mabs are used to strengthen the immune response and to intensify the immune system's antitumor capacity. moreover, mabs are highly specific when attached to nanoparticles to aid in targeted delivery of various antitumor cytotoxic agents or function themselves as effective therapeutic agents (sutradhar and amin, ) . there are different ways of destroying cancer cells with mabs: directly by inducing apoptosis, blocking growth factor receptors or inducing the formation of antiidiotype, and indirectly by activating complement-mediated cellular cytotoxicity and antibody dependent cell-mediated cytotoxicity . the paramagnetic properties of iron-oxide nanoparticles have been harnessed for therapeutic and imaging applications (mccarthy et al., ) . chen et al. ( ) have reported that dextran-coated iron-oxide nanoparticles conjugated to radiolabeled (iodine isotope , i) anti-vegf mab significantly increased imaging resolution, as well as destruction of liver cancer in mice (chen et al., ) . srinivasan, lakshmikuttyamma, and shoyele (srinivasan et al., ) studied selective targeting of mabs to oncoproteins in cancer cells while avoiding their accumulation in normal cells. results of fluorescence microscopy, tem, and flow cytometry revealed that bevacizumab nanoparticles were internalized by a cells times more than by mrc- cells. macropinocytosis and energy-dependent pathways were elucidated to be involved in their uptake by a cells. this study presents the first evidence that uncoated mab nanoparticles can be selectively delivered to cancer cells while avoiding normal cells. mabs have been also employed as an effective active targeted therapy since these antibodies can specifically recognize the over-expressed her positive tumor cells and internalize through receptor mediated endocytosis. to treat her positive breast cancer, anti-her humanized mabs are commonly used, although advances can be made in targeted cellular localization via conjugation strategy through a nano-particulate system focusing on surface modified ligand/receptor-mediated nano-therapy to target the tumor cell at the molecular level. various nanoparticle systems, such as liposomes, micelles, and dendrimers, have been studied not only in vitro but also in vivo, concluding that the presence of receptor targeting antibody on the nanocarriers demonstrated a higher toxicity than the nano-carriers without an antibody after time dependent incubation (hamidreza montazeri et al., ; matsumura, ; yokoyama, ) . antibodies against cell surface biomarkers are the commonly used ligands for the development of targeted nanoparticles. although mouse mabs have been used for making targeted nanoparticles, strong cross-species immune responses limit their potential for future clinical translation. currently only a few types of humanized mabs, such as her- antibody (herceptin), are available for the production of targeted nanoparticles (slamon and pegram, ) . alternatively, high affinity recombinant antibody fragments have been developed as targeting ligands (yang et al., ) . for example, a human single chain antibody against the epidermal growth factor receptor (scfvegfr) that is highly expressed in the majority of epithelial tumors was conjugated to different types of nanoparticles. specificity of tumor imaging and targeted therapeutic effects of these nanoparticles have been demonstrated in several animal tumor models (yang et al., ) . the major advantages of using natural ligands for tumor targeting are their high binding affinity, specificity, and, most importantly, low immunogenicity. owen et al. ( ) reported higher antibody-conjugated lysosomal p (la-co-tmcc)-g-peg-furan micellar concentration in her positive cell lines with significant cellular distribution resulting in two-to threefold increase in lysosomal accumulation compared to using the antibody alone (owen et al., ) . in addition, nobs et al. ( ) reported that thiol functionalized anti-her mab can be conjugated with the maleimide containing liposomes. internalization of these antibody coupled immunoliposomes by her positive breast cancer cell line (sk-br- ) was observed with enhanced effects compared to the control liposomes (nobs et al., ) . many authors observed that the use of lisposomes was more effective in vitro than in vivo. to prove targeted drug-loaded liposome as an effective and safe treatment option for her positive breast cancer, more in vivo studies need to be conducted. when used, anti-her mab conjugated with dendrimers resulted in the effective delivery to target the overexpressed her receptors of tumor cells in animal models. more recently, miyano et al. ( ) developed biocompatible sixth generation (g ) anionic lysine (amino acid) dendrimer, on which the surface was modified with glutamate and, thereafter the glutamate modified g lysine dendrimer was coupled with trastuzumab mab and a fluorescent label. following this, the whole conjugated formulation was evaluated in both her positive and negative cell lines to assess the targeting efficiency and cellular internalization compared to the free antibody application. the results of this study revealed high binding affinity with low cytotoxicity; and cellular internalization or lysosomal trafficking in her positive cells with a dose-dependent profile compared to the her negative cells (miyano et al., ) . in subsequent studies by chen et al. ( ) and gao et al. ( ) , a model protein toxin (pe kdel) was used to develop anti-her antibody functionalized pe kdel loaded plga nanoparticles utilizing a two phase carbodiimide process. modified encapsulated nanoparticles showed higher in vitro cytotoxicity and more protective antitumor activity compared to the controls in her overexpressing cells and tumor-bearing mice, respectively. in addition, they reported well tolerability for maximum tolerated dose, as well as reduced systemic toxicity when modified nanoparticles were administered to mice model compared to control (chen et al., ; gao et al., ) . nanotechnology has promoted greater opportunities for higher specific drug delivery with minimum side effects. bio-conjugation strategies of therapeutic agents loaded nanoparticles with mabs have exhibited a targeted drug delivery approach both in vitro and in vivo. mabs can act as highly specific probes when they are attached to nanoparticles to aid in targeted delivery of various antitumor cytotoxic agents (sutradhar and amin, ) . the combination therapy of nanopartilces of mabs and other chemotherapeutic agents, such as doxorubicin, -fluorouracil, paclitaxel, docetaxel, campothecin, and topotecan, could bring about synergistic tumor growth inhibition. some nanoparticulate systems combining antibody with these chemotherapeutic agents are listed in table . . neovascularization is a major cause of visual loss in a number of ophthalmic diseases. it consists of new blood vessel growth from preexisting vascular structures. this process occurs in pathologies of the anterior and posterior segments of the eye, such as corneal neovascularization, age-related macular degeneration (amd), diabetic macular edema, viral retinitis, proliferative vitreoretinopathy, posterior uveitis, retinal vascular occlusions, choroid neovascularization (cnv), and diabetic retinopathy, to name a few. anti-vegf agents have demonstrated efficacy in reducing neovascularization in both animal models and clinical trials. specifically, anti-vegf antibodies have shown initial therapeutic success. bevacizumab is a full-length, humanized murine mab that recognizes all isoforms of vegf. bevacizumab was initially approved by the u.s. food and drug administration (fda) to treat metastatic colon cancer, but it has also shown efficacy in the treatment of various neovascular ocular diseases and is used off-label to treat neovascular age-related macular degeneration. topical delivery is a relatively easy and a less risky method of drug administration. however, delivery to the posterior segment via this route is considered inefficient and unsuccessful, since less than % of the topically applied dose enters the eye and an even smaller fraction of it ( . %) is expected to reach the posterior segment. intravitreal administration involves the direct administration of drug solution/suspension into vitreous humor via pars plana using a -g needle. in contrast to the topical roth et al. ( ) and systemic routes, intravitreal injection makes high concentrations of drug locally available to the internal eye tissue, including the choroid and the retina. similarly, the intravitreal administration of macugen (pegaptanib sodium; pfizer) and lucentis (ranibizumab; genentech/novartis), vascular endothelial growth factor (vegf) inhibitors, is highly successful for the control of amd. however, agents with molecular weight less than da when applied intravitreally tend to be drained off from the site of application with a half-life of less than days, indicating a need for repetitive injections. anyway, the period requiring a repeat dose may extend from a few days to several months for macromolecular antibodies. for example, three is the mean number of injections of bevacizumab (avastin; roche) required to be administered per year for the treatment of amd. on the other hand, the recommended dosing frequency of ranibizumab (lucentis) is once a month ( . mg; µl) for at least months (rosenfeld et al., ) , whereas pegaptanib sodium (macugen) needs to be injected intravitreally at -week intervals for year (kitagawa and yuzawa, ) . nevertheless, repetitive intravitreal injections, even if spaced widely, are invariably associated with complications, such as vitreous hemorrhage, retinal detachment, cataract, and endophthalmitis. the rate of endophthalmitis and retinal detachment being observed with intravitreal injection is . and . %, respectively (edelhauser et al., ) . moreover, patient compliance is lower with such regimens because of the painful and invasive procedures requiring hospitalization and specially trained physician for administration, in addition to the high cost of the medicine per se (kaur and kakkar, ) . biodegradable polymeric nanoparticles offer properties that make them suitable candidates to overcome these administration issues. in this regard, abrishami et al. ( ) studied encapsulated bevacizumab into liposomesand and found that the intravitreal injection of liposomes was well tolerated through days in rabbits. the clearance of this drug in vitreous from liposomal formulations was slower than the soluble form. the concentration level of bevacizumab after intravitreal injection suggested that intravitreal injection of drug by these carriers provide sufficient concentration of therapeutic drug for weeks for diabetic neovascularization and probably for other neovascular eye diseases (abrishami et al., ) . other authors developed nanoparticles of plga loaded with bevacizumab and stabilized whit albumin. they found that the vitreous concentration of bevacizumab was sustained for about weeks at values greater than ng ml − and that the nanoparticles presented a drug vitreous medium retention time (mrt) . times higher than the control. in addition, it was confirmed the nanoparticles persistence in ocular tissues for days (varshochian et al., (varshochian et al., , . recently lu et al. ( ) studied the effects of intravitreal injection of bevacizumab-chitosan nanoparticles on pathological morphology of retina and the expression of vascular endothelial growth factor (vegf) protein and vegf mrna in the retina of diabetic rats. the results have offered a new approach for inhibiting angiogenesis of diabetic retinopathy and indicated that the intravitreal injection of bevacizumab inhibited vegf expression in retina. in addition, bevacizumab-chitosan nanoparticles presented a longer period of action (zhou et al., ) . chronic respiratory diseases (crd) are chronic diseases of the airways and other structures of the lung. among the most common, asthma, chronic obstructive pulmonary disease (copd), and respiratory allergies can be mentioned. the airway inflammation in these diseases cannot always be controlled with conventional therapies. the critical role of the combination therapy of an inhaled corticosteroid (ics) and a long-acting β-adrenoceptor agonist (laba) in the treatment of patients also suffering from severe asthma and chronic obstructive pulmonary disease (copd) explains why there is a strong interest within the pharmaceutical industry in developing new pharmaceutical alternatives. biological therapies represented in particular by mabs against selective targets, could improve the outcome of these diseases. many current publications have shown the important role of nanomedicine in the treatment of respiratory diseases (card et al., ; mansour et al., ; smola et al., ) . systems releasing nanoparticles have several possibilities to improve treatment of upper respiratory tract since they can protect drugs from degradation by enzymes present in the epithelial lining fluid, the particle size may permit circumvent pass macrophages, and crossing the endothelium will allow systemic treatment of nanoparticles and pulmonary drug intravenously administration. identifying asthma phenotypes has given impetus to the search for biomarkers to help classify patients, pointing to new therapies and predicting different pathological mechanisms of disease progression with strong benefits for affected patients . an ideal biomarker is easy to detect and measure, noninvasive, and inexpensive, and it can be used to identify phenotypes either for clinical response or treatment, assessing changes in disease activity, or confirming a diagnosis. copd is currently the fourth leading cause of death in the world, and if current trends continue, it is likely to rank as the third leading cause of death by (world health organization, ) . both cytokines and chemokines play a fundamental role in the organization of copd, so the use of mabs in the treatment options is very valuable to provide more targeted therapies (li et al., ; yamagata and ichinose, ) . numerous antibodies directed to cytokines, chemokines, growth factors, and their receptors are considered for the treatment of copd and some have already been studied in clinical trials in patients with copd. the macrophage imaging using mri, together with the use of magnetic nanoparticles of iron oxide, has recently emerged as a promising noninvasive technique for preclinical and clinical studies of inflammatory diseases. however, limited research on inflammation and passage of macrophages in the lung, using imaging technology has been performed due to difficulties in imaging the body (i.e., the signal loss due to the pulsations and heart breathing, low proton density, and susceptibility artifacts). novel perspectives for imaging, diagnosis, and treatment of respiratory diseases, such as copd can appear due to technical improvements and the development of techniques of magnetic resonance pulse sequence detection. studies reported the possibility of noninvasive monitoring of subsets of macrophages in an inflammatory model using high resolution mri after intravenous administration. the possibility of noninvasive monitoring of subsets of macrophages was reported in an inflammatory model using high resolution mri after intravenous administration. furthermore, other studies have proven that polarization and proliferation was not affected using iron oxide nanoparticles with ex vivo labeled subpopulation of macrophages. the coupling of a specific antibody with the iron oxide nanoparticles directed to a particular subpopulation of macrophages may provide a promising strategy for early diagnosis and improved various inflammatory diseases noninvasively using mri. al faraj et al. ( ) evaluated the in vivo effect of intrapulmonary administration of spion on the profiles of alveolar macrophage polarization in a model of copd and developed a protocol mri noninvasive to specifically target and monitor a subpopulation of macrophages using specific antibodyconjugated spion. an interesting approach was performed to target a subpopulation of macrophages in the lung of a copd animal model using biocompatible antibody-conjugated iron oxide nanoparticles. this conjugation allowed noninvasive tracking using a free-breathing mri protocol. antibodies to il- abgenix improved dyspnea but not lung function. this limited clinical benefit is attributed to suboptimal dosing into the airways because of intravenous administration (mahler et al., ) . therefore, the use of nanoparticles may allow administration by inhalation for local concentrations and avoiding side effects. so far, clinical trials with anti-tnfalpha antibody infliximab have been carried out, but no effects in patients with copd were observed (dentener et al., ; rennard et al., ; van der vaart et al., ) . therefore, the release of cetuximab (currently undergoing clinical trials for cancer therapy) could be a valuable option for the treatment of copd. molecularly targeted nanoparticles were chosen as the best strategy for imaging inflammation to make progress in health care (mccarthy and weissleder, ; weissleder, ) . magnetic nanoparticle's versatility makes them well suited for applications to enable early detection and prevention, and so improving diagnosis, treatment, and follow-up of diseases (oghabian and farahbakhsh, ) . al faraj et al. ( ) developed an assay to quantify the antibodies cd or cd conjugated to spion, which involves the reduction of cu + to cu + by proteins and the appearance of a purple-blue copper-protein complex in alkaline medium. some significant trends that appear to be developing include bispecific antibodies, adcs, and companion diagnostics, which should help to identify the most appropriate patient populations for treatment. even though the health benefits of mab therapeutics are proven, controversies will continue about their economic viability because of their expensive price. high prices, however, are generally associated with early innovative treatments, and mabs are not the only drugs to have staggering prices. the cost of cancer treatments, for example, has more than doubled in the past two decades, leading to an outcry by many european and american cancer specialists. another reason why mabs are so expensive is the fact that many of them are still protected by patents. a drug's patent life is years from the date of filing, in order to help developers recover some of the research and development costs involved in getting a drug to market. some of the patent life is in fact reduced because some of that time, an average of years, is taken up by clinical trials and regulatory approval (siddiqui and rajkumar, ) . the slow progress of mab therapeutics for infectious diseases can be attributed in part to the large arsenal of other antiinfective drugs, such as vaccines and antibiotics. because they are specific to a single pathogen, mab drugs are also commercially less attractive than traditional drugs because they cover a narrower spectrum of patients. in addition, mabs need to be administered by intravenous or subcutaneous injection, unlike other antiinfectives that can be taken orally, so they are unsuitable for patients in developing countries who have limited access to healthcare. mabs are also more effective at preventing infection rather than treating established ones, and unlike vaccines provide only short-term prophylaxis. and finally, the high development and manufacturing costs associated with mabs and the poor record in winning approval for treating infectious diseases with mabs lessen their commercial appeal (reichert, ) . more promising are recent developments to enhance the potency and efficacy of mabs, so as to make it possible to prescribe lower doses and potentially reduce costs. a number of approaches have been adopted to augment the efficacy of mabs. one of the most encouraging is the use of genetic engineering to remove glycosylation sites from the variable domain of the antibody. this enhances the effector function of mabs, such as antibody-dependent cell-mediated cytotoxicity (adcc), which activates the patient's innate immune cells to kill a target cell like cancer. although the new generation of mabs may greatly improve the treatment of cancer and autoimmune disorders, which are well-established disease targets for mab therapeutics, it remains to be seen whether mab therapeutics will be effective in other areas. nowhere is this question more urgent than in the case of infectious diseases. the fact that mab therapeutics is pathogen-specific could hinder its use for treating mixed infections. one solution might lie in the use of a cocktail of mabs to target the diverse range of antigens that viruses carry. such a strategy would effectively mimic the natural immune response: once infected, the body tends to develop several antibodies in response to the antigens presented by a virus, each of which attaches to one of the different antigens. it is this diversity of antibodies that helps the immune system in fighting the invader. the use of a cocktail of mabs is already being investigated for the treatment for rabies recent advances in mab engineering have opened up new opportunities for serum therapy. importantly, mabs offer the means to prepare standardized agents, which, when combined in a cocktail, can yield a product that is more precise and more potent than traditional serum therapy. to date, the development of antiinfective mab products has attracted little commercial investment. in part this is because infectious diseases are short-lived and therefore have a limited market. this is in contrast to chronic conditions like cancer and autoimmune diseases, which require regular treatment and therefore have greater profit potential. nonetheless, the pharmaceutical climate is changing, fueled by concern over the rise in new pathogens (such as west nile and corona viruses); the reemergence of old pathogens (like tuberculosis), increasing antibiotic resistance among microorganisms; and the rise of superbugs like mrsa, as well as the growing epidemic of patients who are immunocompromised as a result of hiv infection, organ transplantation, chronic degenerative diseases, and improvements in cancer care. likewise, mabs have been used to investigate and treat cancer, providing powerful tools for identifying and targeting different antigens on tumors. although these applications did not quickly translate into successful mab therapeutics for cancer, in more recent years mabs therapeutics have offered alternatives to drugs with a broad spectrum and high toxicity. this has transformed the care of cancer patients, who no longer face the prospect of losing hair and other serious side effects associated with other cytotoxic drugs. the advantage of mabs is that they can be given as maintenance therapies. this is reshaping our perceptions of some cancers from what was once seen as inevitably fatal to a chronic condition. mabs have also enabled the prescription of specific therapeutics for particular tumor antigens in individual patients. this allows a greater degree of personalization in the management of cancer than in the past. indeed, mabs are expected to be an increasingly important component in personalized cancer therapies. in a world of antibiotic-resistant superbugs and an aging population grappling with autoimmune disorders and cancer, mabs offer the potential for new, targeted treatments and drugs that can offer personalized care-and a window into the complex, overlapping conditions that underlie human disease. preparation, characterization, and in vivo evaluation of nanoliposomes-encapsulated bevacizumab (avastin) for intravitreal administration mr imaging and targeting of a specific alveolar macrophage subpopulation in lps-induced copd animal model using antibody-conjugated magnetic nanoparticles liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine the pharmacologic basis for antibody-auristatin conjugate activity detection of bacteria in suspension by using a superconducting quantum interference device current trends of nanotechnology for cancer therapy monoclonal antibodies in immune and inflammatory diseases specific targeting of her overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified human serum albuminnanoparticles advanced methodologies to formulate nanotheragnostic agents for combined drug delivery and imaging antibody-conjugated nanoparticles for biomedical applications drug targeting and tumor heterogeneity antibody-drug conjugate target selection: critical factors nanotechnology-mediated targeting of tumor angiogenesis nano-biosensor development for bacterial detection during human kidney infection: use of glycoconjugate-specific antibody-bound gold nanowire arrays (gnwa) semiconductor nanocrystals as fluorescent biological labels the immunogenicity of chimeric antibodies a repertoire of monoclonal antibodies with human heavy chains from transgenic mice nanoparticles with raman spectroscopic fingerprints for dna and rna detection pulmonary applications and toxicity of engineered nanoparticles in vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipidpolymeric nanoparticles targeted cancer therapy: conferring specificity to cytotoxic drugs preparation and characterization of pe kdel-loaded anti-her nanoparticles for targeted cancer therapy using anti-vegf mcab and magnetic nanoparticles as double-targeting vector for the radioimmunotherapy of liver cancer non-enzymatic hinge region fragmentation of antibodies in solution a nanochannel/nanoparticle-based filtering and sensing platform for direct detection of a cancer biomarker in blood effect of infliximab on local and systemic inflammation in chronic obstructive pulmonary disease: a pilot study targeted delivery of a cisplatin prodrug for safer and more effective prostate cancer therapy in vivo ophthalmic drug delivery systems for the treatment of retinal diseases: basic research to clinical applications high-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo pe kdel-loaded anti-her nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity in vivo cancer targeting and imaging with semiconductor quantum dots assessing monoclonal antibody product quality attribute criticality through clinical studies phase iii trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer previously unrecognized vaccine candidates against group b meningococcus identified by dna microarrays disposition of drugs in block copolymer micelle delivery systems: from discovery to recovery tumor targeting of doxorubicin by anti-mt -mmp antibodymodified peg liposomes the development, characterization and in vivo anti-ovarian cancer activity of poly(propylene imine) (ppi)-antibody conjugates containing encapsulated paclitaxel long-term multiple color imaging of live cells using quantum dot bioconjugates replacing the complementarity-determining regions in a human antibody with those from a mouse rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation role of nanoparticle valency in the nondestructive magnetic-relaxationmediated detection and magnetic isolation of cells in complex media antibody conjugated plga nanoparticles for targeted delivery of paclitaxel palmitate: efficacy and biofate in a lung cancer mouse model nanotherapy for posterior eye diseases advances in polymeric micelles for drug delivery and tumor targeting near-infrared fluorescent type ii quantum dots for sentinel lymph node mapping the best-selling drugs of all time: humira joins the elite intravitreal pegaptanib sodium for myopic choroidal neovascularization: year results of a prospective pilot study continuous cultures of fused cells secreting antibody of predefined specificity multivariate analysis of the sequence dependence of asparagine deamidation rates in peptides nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer double-effective chitosan scaffold-plga nanoparticle system for brain tumour therapy: in vitro study polymersomes: a new multi-functional tool for cancer diagnosis and therapy tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells what's in the pipeline? prospects for monoclonal antibodies (mabs) as therapies for lung diseases extractive electrospray ionization mass spectrometry toward in situ analysis without sample pretreatment pharmacokinetics and biodistribution of nanoparticles quantum dot ligands provide new insights into erbb/her receptor-mediated signal transduction human antibodies from transgenic animals application of nanotechnologies for improved immune response against infectious diseases in the developing world effect of intravitreal injection of bevacizumab-chitosan nanoparticles on retina of diabetic rats vascular permeability enhancement in solid tumor: various factors, mechanisms involved and its implications efficacy and safety of a monoclonal antibody recognizing interleukin- in copd: a pilot study epider mal growth factor receptortargeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor nanomedicine in pulmonary delivery polymeric micellar delivery systems in oncology antibody targeting of camptothecin-loaded plga nanoparticles to tumor cells multifunctional magnetic nanoparticles for targeted imaging and therapy targeted delivery of multifunctional magnetic nanoparticles targeted co-delivery of docetaxel, cisplatin and herceptin by vitamin e tpgscisplatin prodrug nanoparticles for multimodality treatment of cancer anionic amino acid dendrimertrastuzumab conjugates for specific internalization in her -positive cancer cells characterization of the severe asthma phenotype by the national heart, lung, and blood institute's severe asthma research program kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor superparamagnetic nanoparticles for biomedical applications: possibilities and limitations of a new drug delivery system current methods for attaching targeting ligands to liposomes and nanoparticles potential use of nanoparticle based contrast agents in mri: a molecular imaging perspective targeting her + breast cancer cells: lysosomal accumulation of anti-her antibodies is influenced by antibody binding site and conjugation to polymeric nanoparticles engineered nanoparticles in cancer therapy engineered nanoparticles in cancer therapy. recent pat preparation of multi-functionalized fe o /au nanoparticles for medical purposes anti-infective monoclonal antibodies: perils and promise of development the safety and efficacy of infliximab in moderate to severe chronic obstructive pulmonary disease reshaping human antibodies for therapy molecular clocks ranibizumab for neovascular age-related macular degeneration anti-cd single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers treating metastatic cancer with nanotechnology nanotechnology in drug delivery and tissue engineering: from discovery to applications self-assembled targeted nanoparticles: evolution of technologies and bench to bedside translation the high costs of cancer drugs and what we can do about it rationale for trastuzumab (herceptin) in adjuvant breast cancer trials nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases real-time vital optical imaging of precancer using anti-epider mal growth factor receptor antibodies conjugated to gold nanoparticles investigation of stability and cellular uptake of self associated monoclonal antibody (mab) nanoparticles by non-small lung cancer cells in vitro and in vivo platelet targeting by cyclic rgd-modified liposomes eb virus-induced b lymphocyte cell lines producing specific antibody multifunctional poly (d,l-lactide-co-glycolide)/montmorillonite (plga/mmt) nanoparticles decorated by trastuzumab for targeted chemotherapy of breast cancer nanotechnology in cancer drug delivery and selective targeting review dendrimers as versatile platform in drug delivery applications development of functionalized fluorescent europium nanoparticles for biolabeling and time-resolved fluorometric applications imaging of multiple mrna targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies folatetargeted nanoparticles show efficacy in the treatment of inflammatory arthritis the development of rapid fluorescence-based immunoassays, using quantum dot-labelled antibodies for the detection of listeria monocytogenes cell surface proteins first study of infliximab treatment in patients with chronic obstructive pulmonary disease magnetic nanoparticle-antibody conjugates for the separation of escherichia coli o :h in ground beef the protective effect of albumin on bevacizumab activity and stability in plga nanoparticles intended for retinal and choroidal neovascularization treatments albuminated plga nanoparticles containing bevacizumab intended for ocular neovascularization treatment physicochemical stability of the antibody-drug conjugate trastuzumab-dm : changes due to modification and conjugation processes antibody structure, instability, and formulation the complex role of multivalency in nanoparticles targeting the transfer rin receptor for cancer therapies molecular imaging in cancer making antibodies by phage display technology world health organization immunofluorescent labeling of cancer marker her and other cellular targets with semiconductor quantum dots agents against cytokine synthesis or receptors theranostic nanoparticles carrying doxorubicin attenuate targeting ligand specific antibody responses following systemic delivery drug targeting with nano-sized carrier systems targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-her trastuzumab a rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles targeted co-delivery of docetaxel and siplk by herceptin-conjugated vitamin e tpgs based immuno-micelles quantitative control of targeting effect of anticancer drugs formulated by ligand-conjugated nanoparticles of biodegradable copolymer blend photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles beyond: a randomized, doubleblind, placebo-controlled, multicenter, phase iii study of first-line carboplatin/paclitaxel plus bevacizumab or placebo in chinese patients with advanced or recurrent nonsquamous non-small-cell lung cancer further reading formulation and delivery issues for monoclonal antibody therapeutics sterically stabilized anti-her immunoliposomes: design and targeting to human breast cancer cells in vitro anti-her immunoliposomes: enhanced efficacy attributable to targeted delivery key: cord- -v pf eg authors: el-ghiaty, mahmoud a.; shoieb, sherif m.; el-kadi, ayman o.s. title: cytochrome p -mediated drug interactions in covid- patients: current findings and possible mechanisms date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: v pf eg at the end of , the entire world has witnessed the birth of a new member of coronavirus family in wuhan, china. ever since, the severe acute respiratory syndrome coronavirus (sars-cov- ) has swiftly invaded every corner on the planet. by the end of april , almost . million cases have been reported worldwide, with a death toll of about deaths. it is currently well-recognized that patient’s immune response plays a pivotal role in the pathogenesis of coronavirus disease (covid- ). this inflammatory element was evidenced by its elevated mediators that, in severe cases, reach their peak in a cytokine storm. together with the reported markers of liver injury, such hyperinflammatory state may trigger significant derangements in hepatic cytochrome p metabolic machinery, and subsequent modulation of drug clearance that may result in unexpected therapeutic/toxic response. we hypothesize that covid- patients are potentially vulnerable to a significant disease-drug interaction, and therefore, suitable dosing guidelines with therapeutic drug monitoring should be implemented to assure optimal clinical outcomes. coronavirus disease (covid- ) is an infectious disease that was first reported as pneumonia of ambiguous etiology in a cluster of patients in the chinese city of wuhan by the end of december [ ] . the causative organism was identified several days later as a novel coronavirus ( -ncov). later, its name was changed to severe acute respiratory syndrome coronavirus (sars-cov- ) as it was found to be genetically related to the coronavirus responsible for the sars outbreak of [ ] . the infection broke out of china to spread to every continent around the world and was declared as an emerging pandemic by who in march [ ] . full-length genome sequences have revealed that sars-cov- is % identical to a bat coronavirus, thus providing a clue about its original reservoir host [ ] . human-to-human transmission of sars-cov- is evident and, as a respiratory infectious disease, covid- primarily spreads with close contact through respiratory droplets and secretions. controlling the disease is based mainly on directing the public towards reducing the transmission [ ] . the disease is contagious, but people are reacting differently upon exposure to the virus. the virus may get eliminated by immune system and the infection can pass unnoticed. however, after an asymptomatic incubation period of up to days, infected persons may develop mild flu-like symptoms, including fever, dry cough, fatigue, and shortness of breath [ ] . other reported manifestations include upper respiratory symptoms as sneezing, runny nose and sore throat, in addition to gastrointestinal symptoms as nausea, vomiting and diarrhea [ ] . anosmia or ageusia have been also reported as characteristic signs of infection [ ] . while most cases develop mild symptoms, some may progress rapidly to a more sever stage that necessitates admission to an intensive care unit and probably mechanical ventilation [ ] . the complications of this critical stage include severe pneumonia, acute respiratory distress syndrome (ards), respiratory failure, multiple organ failure, and ultimately death [ ] . in the absence of a proven therapy for covid- , scientists are currently endeavoring to find an effective drug capable of eradicating this infection. several agents are currently under extensive laboratory and clinical investigations. some of these agents are investigational new drugs while the others are repurposed drugs which are already approved for other ailments [ ] . when it comes to medications, pharmacokinetics should be strongly considered especially when dealing with such critical illnesses. drug metabolism is a highly important aspect of its pharmacokinetics that may significantly influence its clearance and, eventually, its efficacy and/or toxicity. cytochromes p (cyps) are a superfamily of heme-containing monooxygenase enzymes that have been identified in all kingdoms of life [ ] . they represent the major enzyme family involved in the oxidative biotransformation of most drugs and other lipophilic xenobiotics [ ] . in mammals, these enzymes are found primarily in microsomes of the liver, the dominant metabolizing organ, in addition to other extrahepatic tissues [ ] . in humans, there are functional members in cyps families, most of which have specific endogenous functions including the metabolism of arachidonic acid, cholesterol, bile-acids, steroid hormones, vitamin d, and others [ ] . the biotransformation of the majority of hepatically cleared drugs and other foreign chemicals involves members belonging to the cyp , cyp , and cyp families. pathways involving cyp a / , cyp c , cyp c and cyp d are the most common, and responsible for about % of the phase i oxidation system reactions [ , ] . through their profound contribution to xenobiotic biotransformation, cyps can significantly modulate the overall body exposure to a drug. the metabolic activity of the cyps may result in decreasing efficacy and/or toxicity of a drug by enhancing the clearance of its active form. for another drug, such metabolic activity may lead to increased efficacy or toxicity by activating its inert prodrug or generating toxic metabolites, respectively. understanding cyps activity in relation to the target drug is crucial in predicting its behavior inside the body and the consequences of its exposure [ ] . we hypothesize a pharmacokinetic disease-drug interaction in which hepatic cyps metabolizing capacity, and eventually drug response, is altered in covid- patients. based on the conclusions drawn from the currently rapidly evolving knowledge about covid- , our hypothesis is built on the potential modulation of cyps activity by the inflammatory environment provoked by sars-cov- infection, as well as the pathologic involvement of the liver which harbors the majority of the drug metabolizing enzymes (dmes). patient characteristics are also believed to increase the likelihood of the incidence of such interaction. systemic inflammation and immune response represent a substantial element in many acute and chronic diseases which is strongly implicated in altering drug pharmacokinetics through, mainly, modulating the expression and activity of dmes. as a main contributor to the metabolic biotransformation of most drugs, cyps are widely involved in such disease-drug interactions [ ] . regulation of cyps has been linked to inflammation in several disease states such as infectious diseases (including viral infections), cancer, type diabetes, rheumatoid arthritis, and inflammatory bowel disease, in addition to age-related disorders such as normal aging, metabolic disorders, and neurodegenerative diseases [ ] . alterations in hepatic cyps expression and activity are caused by the mediators produced during the inflammation process which are mainly cytokines [ ] . cytokines are a broad category of small cell signaling proteins responsible for keeping homeostasis of the immune system and its components. the immune response is driven by a complex interplay between pro-and anti-inflammatory signals mediated by different cytokines [ ] . it has been reported that different cyps are regulated differently, that is, multiple cytokines may participate in regulating single enzymes, while a subset of enzymes can be regulated by individual cytokines. this fact about specificity of regulation is highly important when considering drug interactions because drug pharmacokinetics will ultimately vary depending on disease type and its released cytokines, as well as the administered drug and its involved metabolizing enzymes [ , ] . since the s, cytokine-induced changes in hepatic cyps activity have been described using hepatocyte cultures from different mammals including rats [ ], rabbits [ ], pigs [ ] , and humans [ , ] . a strong evidence has been also provided through in vivo studies in mice [ ] , rats [ ] , and humans [ ] . some studies have also demonstrated the suppression of extrahepatic cyps by inflammatory mediators [ ] [ ] [ ] [ ] . interleukin (il- ) [ ] [ ] [ ] , interleukin (il- ), tumor necrosis factor-α (tnf-α), and interferon gamma (ifn-γ) [ ] [ ] [ ] are the most prominent proinflammatory cytokines that have exhibited suppression of cyps expression and activity, in addition to other cytokines such as interleukin (il- ) [ , ] and interleukin (il- ) [ ] . theophylline is metabolized by several cyps isoforms (including cyp a , cyp a , and cyp a) yielding different metabolites. serum treatment resulted in reducing the formation of all theophylline metabolites as well as the amount of total cyps, indicating a significant down-regulation of these metabolizing enzymes [ ] . in a later study using the same experimental model, the down-regulatory effect was mainly attributed to il- , interleukin β (il- β), and ifn-γ [ ] . cyps down-regulation was also proven to be subsequent to the reduction of their activity. additionally, the inflammatory mediators in human serum provoked varying degrees of suppression to different cyps with the cyp a being more vulnerable than cyp a to such effect [ ] . for decades, il- has been recognized as the major inflammatory element that provokes a significant repressive effect on the expression and activity of different cyps. human recombinant interleukin (rhil- ) has shown concentration-dependent blocking of phenobarbital-mediated induction of cyp b / mrna and activity in rat hepatocytes [ ] . in fischer rats, rhil- resulted in reducing different cyps activities with variable degrees [ ] . the mrna levels of cyp a , cyp a , and cyp a were markedly suppressed in three human hepatoma cell lines (hepg , hepg f, and hep f ) because of rhil- treatment [ ] . acute-phase inflammatory reaction provoked by turpentine or purified bacterial lipopolysaccharide (lps) administration to male rats resulted in significant suppression of hepatic cyp c [ ] , an effect that was later confirmed to be transcriptional, involving cyp c promoter sequences, using rhil- treatment [ , ] . il- -knockout (il- -/-) mouse was adopted in several studies as a model to assess the extent of il- contribution to the suppression of different cyp isoenzymes during inflammatory response generated by various stimuli. in turpentine-induced inflammation, the inhibitory effect observed in wild-type (wt) mice on cyp a , cyp a , and cyp a mrnas was revoked in il- -deficient mice [ ] . induction of immune response by bacillus calmette-guérin (bcg) has led to down-regulation of transcriptional expression of both cyp a and cyp c , but only cyp a in il- α/β-knockout mice and tnf-α-knockout mice, respectively. however, tuberculosis vaccine had non-significant effect on these enzymes in il- -knockout mice [ ] . in a disease model, citrobacter rodentium infection was used to elicit inflammatory response in il- -/mice, where cyp a mrna suppression seen in wt mice was abolished [ ] . the mentioned studies have shown that the role of il- is usually critical and can't be played by another cytokine or mediator, however, sometimes il- -/mice may react similarly as wt mice, i.e. show down-regulation too, for certain cyps in certain inflammation models. this can be explained by the functional redundancy existing among the released cytokines which, sometimes, may replace il- [ ] [ ] [ ] . interestingly, sometimes the suppression of cyps may happen only in il- -/mice, but not wt mice, thus indicating that il- may have an opposing signal with inductive effect [ , ] . several studies have evaluated the impact of il- triggered by malignancies. in an interesting study by charles et al, the hepatic levels of both human cyp a as well as its murine orthologue cyp a were simultaneously assessed in a tumor-derived inflammation model using transgenic mice loaded with genetic constructs containing the upstream regulatory elements of the human cyp a gene linked to the lacz reporter gene. the tumor generated a systemic inflammatory response with high levels of circulating il- resulting in down-regulation of cyp a orthologue at mrna, protein, and activity levels as well as cyp a transgene product which was assessed through measuring β-galactosidase enzyme activity [ , ] . different tumor types have also demonstrated a significant suppressive effect on hepatic cyp a with an accompanying increase in il- , and such effect was progressively promoted by tumor growth [ ] . the pivotal role of il- in cancer-mediated repression of hepatic cyp a has been further demonstrated by attenuation of such effect via anti-il- monoclonal antibody treatment [ ] , or interleukin- receptor blocking [ ] . anti-il- antibody intervention was also tested in il- -treated primary human hepatocytes. the inhibitory effect of il- affected cyp a , cyp a , cyp b , and cyp a ; and it was also capable of overriding cyp a and cyp a induction mediated by omeprazole and rifampicin, respectively. anti-il- antibody partially abrogated enzyme activity suppression [ ] . the basis of cytokine-induced down-regulation of cyps activity is not fully elucidated; however, the associated decrease in their respective mrnas strongly suggests a transcriptional mechanism involving a number of transcriptional factors [ , ] . nuclear factor kappa b (nf-κb), a pivotal regulatory transcription factor in the inflammatory and immune response, has been shown to regulate gene expression of many hepatic cyp enzymes in humans, rats, and mice [ , ] . the aryl hydrocarbon receptor (ahr) is a gene battery that regulates a group of dmes including cyp a , cyp a , and cyp b . both nf-κb and ahr have a mutual inhibitory effect thus repressing each other's functions [ , ] . for instance, pyrrolidine dithiocarbamate (pdtc), an inhibitor of nf-κb signal, has demonstrated partial blocking of the inflammatory reduction in cyp a activity [ , ] . the reported inhibitory effect of nf-κb activators on the pregnane x receptor (pxr) and its target genes (most notably of which is cyp a ) on one hand, and enhanced pxr activity by inhibiting nf-κb on the other hand, indicates how inflammatory stimuli can manipulate hepatic cyps expression [ ] [ ] [ ] . activation of nf-κb results in repressing glucocorticoid receptor (gr), thus down-regulating constitutive androstane receptor (car) expression and its associated genes such as cyp b, cyp c, and cyp a [ ] . other studies have shown that nf-κb can interfere with cyps expression, such as cyp c and cyp e , by binding directly to their respective genes [ , ] . . . immunopathological aspects of covid- : the immune system plays a crucial role in resolving covid- infection, but it also can go out of control and contribute to its progression. after invading the alveolar cells, the host body starts mounting an immune response during the incubation period and the mild stage to exterminate the virus and hamper disease progression to the severe stage. however, if body defenses are impaired, the suboptimal antiviral immune reaction will allow viral proliferation resulting in lung damage and dissemination to other angiotensin-converting enzyme (ace )-expressing cells such as enterocytes [ ] [ ] [ ] . the initial containment of the infection by innate immune response generates a strong local inflammatory reaction with a myriad of inflammatory cytokines. upon failure of this attempt, the highly specific adaptive immune cells are summoned, by systemically disseminated cytokines, to augment the immune response and clear the increasing viral load [ ] . if coordinated recruitment of innate and adaptive immunity fails to effectively control the pathogen, more immune cells will get activated, with subsequent release of more cytokines, in a positive feedback loop of pathogenic inflammation. this state of systemic inflammation is called cytokine storm syndrome (css) and it has been reported in severe cases as an important cause of ards and multiple organ failure [ ] . at this point, the immune system is doing more harm than good to the body and becoming a major cause of lung damage and subsequent mortality, thus suppressing such hyperinflammation is strongly recommended together with symptomatic treatment [ , ] . it is noteworthy that the immune system starts its response upon viral exposure and continues to reinforce it till, in most cases, it effectively diminishes viral burden. premature halting of immune response may delay virus clearance and perpetuate the infection, therefore the use of immunosuppressants should be the last resort and only for severe cases with profound inflammatory lung injury [ ] . it has been reported that a wide range of proinflammatory cytokines are elevated in peripheral blood of covid- patients such as interferon gamma (ifn-γ), tumor necrosis factor-α (tnf-α), granulocyte-macrophage colony stimulating factor (gm-csf), macrophage colony-stimulating factor (mcsf), and interleukins il- , il- , and il- ; and many more [ , ] . interfering with these inflammatory mediators can modulate the amplified immune response that ends up with lung damage. interleukin- is one of the important targets for anti-cytokine therapy as it is believed to be a key player in the process and its elevation is associated with poor prognosis [ ] [ ] [ ] . tocilizumab is a monoclonal antibody against the interleukin- receptor (il- r) approved for the treatment of rheumatoid arthritis. it has been reported that a single dose of tocilizumab significantly improved the clinical outcomes in covid- patients [ , ] . sarilumab is another il- r antagonist which is also approved for treating rheumatoid arthritis and is currently assessed for its efficacy against covid- [ ]. different organs, such as lung, kidney, and intestine, contribute to drug biotransformation in the body; however, the liver is generally regarded as the major metabolizing organ responsible for drug clearance [ ] . through several enzyme systems, the most prominent of which is cyps, the liver can drive numerous metabolic reactions. acute as well as chronic insults to the liver will eventually hamper its metabolic machinery resulting ultimately in inefficient drug clearance [ , ] . regardless of the cause, it is believed that the severity of liver disease is correlated to the extent of metabolism alteration [ , ] . lungs represent the hot spot for the pathogenesis of covid- , however, potential involvement of other organs, such as the liver, has been also reported [ , ] . the hepatic involvement can be tied to the incidence of other extrapulmonary, or more specifically gastrointestinal, manifestations [ , ] , and it has been reported in covid- patient with or without underlying liver disease [ ] . reported findings of hepatic dysfunction in covid- cases include elevated aminotransferases [ ] and bilirubin [ ] , hypoalbuminemia [ ] , in addition to microvesicular steatosis with mild lobular activity [ ] . it is worth mentioning that hepatic impairment has been also associated with severe acute respiratory syndrome coronavirus (sars-cov) [ ] , which is highly homologous to sars-cov- [ ] , as well as middle east respiratory syndrome coronavirus (mers-cov) [ ] . the exact mechanism behind liver function deterioration is not fully understood, however, it has been proposed that sars-cov- causes direct liver injury. viral hepato-tropism can be attributed to the relatively high expression of its cellular entry gate, i.e. ace , in cholangiocytes, rather than hepatocytes, rendering them vulnerable to viral attack [ , ] . this assumption is supported by the increase in circulating levels of serum gamma-glutamyl transferase (ggt) in covid- patients [ ] . hepatic abnormalities can be also a secondary effect induced after initiating disease management by consumption of hepatotoxic antipyretic agents (e.g. acetaminophen), antiviral medications (e.g. oseltamivir and lopinavir), antibiotics, and steroids [ , ] . despite the absence of viral antigens in the liver, the overwhelming systemic inflammatory reaction elicited by sars-cov- can be also a major cause of multiple organ dysfunction including liver damage as seen in other respiratory viral infections [ ] [ ] [ ] . . drug outcome alteration in a vulnerable patient population: cyps have been implicated in mediating clinically relevant drug interactions in several disease states [ ] . such metabolic interactions have been regarded as a major reason behind revising and updating safety profiles of pharmaceutical products [ ] . diseasedrug interactions are commonly observed in inflammatory conditions with drugs whose clearance is predominantly cyps-mediated [ ] . examples of such conditions include rheumatoid arthritis [ ] , hepatitis [ ] , crohn's disease [ ] , acquired immunodeficiency syndrome (aids) [ ] , influenza [ ] , congestive heart failure [ ] , and cancer [ ] . as a central inflammatory regulator, agents interfering with il- actions have led to regaining the inhibited cyps activity, thus normalizing drug disposition pattern. blocking il- receptor by the monoclonal antibodies; tocilizumab [ ] and sarilumab [ ] in rheumatoid arthritis patients co-administering simvastatin has reversed cyp a activity suppression as demonstrated by a clinically significant decrease in simvastatin exposure. halting il- signaling by direct binding with the monoclonal antibody sirukumab has also proven effective in rheumatoid arthritis patients who received a cyps probe cocktail consisting of midazolam (cyp a), omeprazole (cyp c ), and warfarin (cyp c ). area under the plasma concentration-time curve (auc) for probe substrates was reduced after sirukumab administration indicating that the activity of their respective metabolizing enzymes was recovered [ ] . as an attempt to predict the risk of such interactions, physiologically based pharmacokinetic (pbpk) models were developed to assess the impact of anti-il- agents on the metabolism of concomitant medications [ ] [ ] [ ] . the immune response, with il- in the core of its network of mediators, is a hallmark of covid- pathogenesis. based on the aforementioned examples, it is strongly postulated that cyps metabolic activity will be inevitably altered, mostly downregulated, during the course of sars-cov- infection in a similar manner, resulting in a clearance-related pharmacokinetic interaction with the administered drugs. additionally, liver involvement in covid- may further complicate the picture. in may , the investigational antiviral agent, remdesivir, has received u.s. food and drug administration (fda) emergency use authorization for the treatment of covid- . according to its manufacturer, remdesivir undergoes extensive metabolism by cyps especially cyp a [ ] . moreover, other potential candidates for treating covid- such as chloroquine [ ] and colchicine [ ] are also hepatically metabolized, so understanding the nature of such interaction is highly essential as it can influence the therapeutic/toxic response of patients to the agents intended for managing the disease [ , ] . full attention should be paid when anti-cytokine therapy comes into play. in this case, partial or complete regain of normal metabolic status can be achieved as a result of the immunomodulatory effect. in case of co-administration of multiple drugs, the risk of drug interactions increases, however, a more complex disease-drug-drug interaction is expected in covid- . sars-cov- infection has resulted in high rates of hospitalization and intensive care unit (icu) admission [ , ] . this can be regarded as a major clinical concern especially when considering that critically ill patients are more predisposed to drug interactions, and that cyps are involved in the metabolism of commonly prescribed drugs in the icus [ ] . moreover, demographic analysis of covid- patients has revealed that older people with comorbidities are the most affected group [ , ] who also represent the potential candidates for advancing to severe stages and inevitable icu admission [ ] . medical comorbidities in elderly patients warrant concomitant drug therapies, thus rendering them the most vulnerable group for both drug-drug and disease-drug interactions [ , ] . therefore, the utmost level of caution is required to deliver the optimal dose for these patients. clinical characteristics of coronavirus disease in china sars-cov- is an appropriate name for the new coronavirus who declares covid- a pandemic a pneumonia outbreak associated with a new coronavirus of probable bat origin down-regulation of multiple cytochrome p gene products by inflammatory mediators in vivo. independence from the hypothalamo-pituitary axis plasma levels of tnf-alpha and il- are inversely related to cytochrome p -dependent drug metabolism in patients with congestive heart failure modulation of cytochrome p by inflammation in astrocytes cytokines influence mrna expression of cytochrome p a and mdri in intestinal cells role of cytokines in the lipopolysaccharide-evoked depression of cytochrome p in the brain and liver the impact of cytokines on the expression of drug transporters, cytochrome p enzymes and chemokine receptors in human pbmc repression of cytochrome p by cytokines: il- beta counteracts clofibric acid induction of cyp a in cultured fetal rat hepatocytes suppression of cyp c gene transcription by interleukin- mediated by nf-kappab binding at the transcription start site effects of interleukin beta (il- beta) and il- beta/interleukin (il- ) combinations on drug metabolizing enzymes in human hepatocyte culture pretranslational down-regulation of cytochromes p c and a in male rat liver by tumor necrosis factor alpha nitric oxidemediated inhibition of cytochrome p by interferon-gamma in human hepatocytes inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines the interleukin- receptor downregulates the expression of cytochrome p in cultured rat hepatocytes decrease in hepatic cytochrome p after interleukin- immunotherapy in vivo effects of interleukin- on human cytochrome p activity down-regulation of the hepatic cytochrome p by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver cytochrome p down-regulation by serum from humans with a viral infection and from rabbits with an inflammatory reaction effect of interleukin on phenobarbital induction of cytochrome p- iib in cultured rat hepatocytes chauvelot-moachon l. effects of interleukin- on cytochrome p -dependent mixed-function oxidases in the rat interleukin- down regulates the expression of transcripts encoding cytochrome p ia , ia and iiia in human hepatoma cells suppression of constitutive cytochrome p- gene expression in livers of rats undergoing an acute phase response to endotoxin selective suppression of cytochrome p- gene expression by interleukins and in rat liver suppression of the constitutive expression of cytochrome p- c by cytokines and interferons in primary cultures of rat hepatocytes: comparison with induction of acute-phase genes and demonstration that cyp c promoter sequences are involved in the suppressive response to interleukins and hepatic cytochrome p down-regulation during aseptic inflammation in the mouse is interleukin dependent involvement of interleukin- and tumor necrosis factor alpha in cyp a and c down-regulation by bacillus calmette-guerin and lipopolysaccharide in mouse liver modulation of hepatic cytochrome p s by citrobacter rodentium infection in interleukin- -and interferon-{gamma}-null mice cytochrome p and antioxidant activity in interleukin- knockout mice after induction of the acute-phase response transgenic mouse models of human cyp a gene regulation transcriptional repression of hepatic cytochrome p a gene in the presence of cancer downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies extrahepatic cancer represses hepatic drug metabolism via interleukin (il)- signalling effects of cytokines on cyp a expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line flc- effects of interleukin- (il- ) and an anti-il- monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture gene transcription in hepatocytes during the acute phase of a systemic inflammation: from transcription factors to target genes regulation of cytochrome p by inflammatory mediators: why and how? nf-kappab and the immune response role of nf-kappab in the regulation of cytochrome p enzymes mechanism of suppression of cytochrome p- a expression by tumor necrosis factor-alpha and lipopolysaccharide ah receptor and nf-kappab interactions: mechanisms and physiological implications modulation of cyp a and cyp a catalytic activities by serum from rabbits with a turpentine-induced inflammatory reaction and interleukin inhibition of nuclear factor-kappab signal by pyrrolidine dithiocarbamate alleviates lipopolysaccharide-induced acute lung injury mutual repression between steroid and xenobiotic receptor and nf-kappab signaling pathways links xenobiotic metabolism and inflammation role of nf-kappab in regulation of pxr-mediated gene expression: a mechanism for the suppression of cytochrome p- a by proinflammatory agents pregnane x receptor is required for interleukin- -mediated down-regulation of cytochrome p a in human hepatocytes is nuclear factor kappa-b the missing link between inflammation, cancer and alteration in hepatic drug metabolism in patients with cancer? a novel lipopolysaccharide-modulated jun binding repressor in intron of cyp e pathological findings of covid- associated with acute respiratory distress syndrome genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding the digestive system is a potential route of -ncov infection: a bioinformatics analysis based on single-cell transcriptomes covid- infection: the perspectives on immune responses covid- : consider cytokine storm syndromes and immunosuppression induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies covid- : combining antiviral and antiinflammatory treatments immunosuppression for hyperinflammation in covid- : a double-edged sword? the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality cytokine release syndrome in severe covid- effective treatment of severe covid- patients with tocilizumab tocilizumab treatment in severe covid- patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices the effect of liver cirrhosis on the regulation and expression of drug metabolizing enzymes cytochrome p and liver diseases selective effect of liver disease on the activities of specific metabolizing enzymes: investigation of cytochromes p c and d liver disease selectively modulates cytochrome p --mediated metabolism covid- and the liver: little cause for concern liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor. archives of academic emergency medicine clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china first case of novel coronavirus in the united states coronavirus disease (covid- ) and prevalence of chronic liver disease: a meta-analysis. liver international evaluation of hepatic enzymes changes and association with prognosis in covid- patients bilirubin levels in patients with mild and severe covid- : a pooled analysis sars-associated viral hepatitis caused by a novel coronavirus: report of three cases covid- : gastrointestinal manifestations and potential fecal-oral transmission histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia. zhonghua gan zang bing za zhi = zhonghua ganzangbing zazhi = chinese journal of hepatology specific ace expression in cholangiocytes may cause liver damage after -ncov infection liver injury in covid- : management and challenges covid- and liver dysfunction: current insights and emergent therapeutic strategies covid- and liver disease systemic viral infections and collateral damage in the liver kupffer cell-dependent hepatitis occurs during influenza infection liver involvement during influenza infection: perspective on the influenza pandemic complex drug-drug interactions involving cytochromes p : systematic review of published case reports and clinical perspectives trends in new drug interactions for pharmaceutical products in japan infection and inflammation leading to clozapine toxicity and intensive care: a case series decreased dromotropic response to verapamil despite pronounced increased drug concentration in rheumatoid arthritis influence of chronic hepatitis c infection on cytochrome p a activity using midazolam as an in vivo probe substrate drug-disease interaction: crohn's disease elevates verapamil plasma concentrations but reduces response to the drug proportional to disease activity variability in drug metabolizing enzyme activity in hiv-infected patients altered theophylline clearance during an influenza b outbreak the use of imipramine in depressed patients with congestive heart failure understanding disease-drug interactions in cancer patients: implications for dosing within the therapeutic window disease-drug-drug interaction involving tocilizumab and simvastatin in patients with rheumatoid arthritis disease-drug interaction of sarilumab and simvastatin in patients with rheumatoid arthritis evaluation of disease-mediated therapeutic protein-drug interactions between an anti-lnterleukin- monoclonal antibody (sirukumab) and cytochrome p activities in a phase i study in patients with rheumatoid arthritis using a cocktail approach a physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of cyp a by il- development of a physiologically based pharmacokinetic model to predict disease-mediated therapeutic protein-drug interactions: modulation of multiple cytochrome physiologically based pharmacokinetic model to assess the influence of blinatumomab-mediated cytokine elevations on cytochrome p enzyme activity summary on compassionate use of remdesivir gilead breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies the greek study in the effects of colchicine in covid- complications prevention (grecco- study): rationale and study design in vitro metabolism of chloroquine: identification of cyp c , cyp a , and cyp d as the main isoforms catalyzing ndesethylchloroquine formation colchicine--update on mechanisms of action and therapeutic uses critical care utilization for the covid- early experience and forecast during an emergency response forecasting covid- impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next months drug-associated disease: cytochrome p interactions baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region clinical implications from drug-drug and drug-disease interactions in older people an update on the clinical consequences of polypharmacy in older adults: a narrative review key: cord- -imciixde authors: babayeva, mariana; loewy, zvi title: repurposing drugs for covid- : pharmacokinetics and pharmacogenomics of chloroquine and hydroxychloroquine date: - - journal: pharmgenomics pers med doi: . /pgpm.s sha: doc_id: cord_uid: imciixde background: a new coronavirus sars-cov- has been identified as the etiological agent of the severe acute respiratory syndrome, covid- , the source and cause of the – coronavirus pandemic. hydroxychloroquine and chloroquine have gathered extraordinary attention as therapeutic candidates against sars-cov- infections. while there is growing scientific data on the therapeutic effect, there is also concern for toxicity of the medications. the therapy of covid- by hydroxychloroquine and chloroquine is off-label. studies to analyze the personalized effect and safety are lacking. methods: a review of the literature was performed using medline/pubmed/embase database. a variety of keywords were employed in keyword/title/abstract searches. the electronic search was followed by extensive hand searching using reference lists from the identified articles. results: a total of results were obtained after screening all sources. mechanisms underlying variability in drug concentrations and therapeutic response with chloroquine and hydroxychloroquine in mediating beneficial and adverse effects of chloroquine and hydroxychloroquine were reviewed and analyzed. pharmacogenomic studies from various disease states were evaluated to elucidate the role of genetic variation in drug response and toxicity. conclusion: knowledge of the pharmacokinetics and pharmacogenomics of chloroquine and hydroxychloroquine is necessary for effective and safe dosing and to avoid treatment failure and severe complications. sars-cov- is a new coronavirus type that has not been previously identified in humans. little is known about the highly infectious virus or how to combat it. the current strategy considers two broad categories of therapies: antivirals, which may target the coronavirus directly, and host modifiers and immune-based medications, which may influence the immune response to the virus. currently, all the therapeutic agents are repurposed medications. for approximately identified medical conditions, only have approved therapies; a critical need currently exists for the availability of drug therapies. [ ] [ ] [ ] drug repurposing helps to minimize the deficiency and delivers a candidate at a shorter development time and a lesser cost. a key advantage of repurposed drugs is that safety has been established and only efficacy of the new indication needs to be assessed. many of the wellknown repurposed drugs, including sildenafil, minoxidil and aspirin emerged by chance from "unorganized" drug discovery processes. several diverse disease states have common transcriptional inflammatory and metabolic pathways, suggesting that drugs designed for treatment of one disease can potentially be used to treat other diseases. drug repurposing is being applied to finding a therapeutic approach for the covid- pandemic. thirty-one potential broad-spectrum antiviral agents (bsaas) were recently identified as having potential for treating sars-cov- /covid- . several existing bsaas have been initiated into clinical trials ( table ) . two of the drugs listed in table , hydroxychloroquine and chloroquine, have been proposed as treatments for covid- . chloroquine (aralen ® ) and hydroxychloroquine (plaquenil ® ) are -aminoquinoline medications used to treat several disease states. chloroquine (cq) was first developed for the treatment of malaria. hydroxychloroquine (hcq) is β-hydroxylated analogue of cq. both medications have been successfully used to treat extraintestinal amebiasis, several infectious (hiv, q fever, zika virus, fungal infections) and rheumatological (systemic lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis, sjögren's syndrome) diseases. [ ] [ ] [ ] in the therapy of malaria, the agents inhibit the action of heme polymerase, which causes the buildup of toxic heme in plasmodium species. , the antiviral activity is not fully understood. the drugs accumulate in human organelles, raise the endosomal ph and prevent viral activity. [ ] [ ] [ ] the elevated ph inhibits nucleic acid replication, glycosylation of viral proteins, viral fusion and entry into the cell, viral assembly and release. postmarketing cases of life-threatening and even fatal events have been reported for chloroquine and hydroxychloroquine. an overdose of cq can cause acute poisoning and death. hcq was demonstrated to be % less toxic than chloroquine, although prolonged and overdose administration can still cause poisoning. , patients may present with atrioventricular block, pulmonary hypertension, sick sinus syndrome or with cardiac complications. the most life-threatening adverse reaction is qtc prolongation with subsequent risk of ventricular arrhythmias. concomitant qtc-prolonging medications may increase the severity of the complication even more. the mechanism of qtc prolongation by chloroquine and hydroxychloroquine is unknown, largely depending on the cardio-vascular health of the patients. [ ] [ ] [ ] another complication of the two medications is retinopathy (chloroquine retinopathy), which can result in irreversible impairment of the retina. , high concentrations of the drugs in the retina, due to binding to retinal melanin, result in the damage of the tissue. hydroxychloroquine may also produce a severe cutaneous adverse effect such as a generalized pustular figurate erythema (gpfe). high concentrations of the medications in the skin and very slow cutaneous elimination (longer than months) may result in this severe cutaneous reaction. , the most common adverse effects of the drugs are nausea, vomiting, diarrhea. other complications include hypoglycemia in diabetics, hemolytic anemia in g pd deficiency patients, tinnitus and headache. , chloroquine and hydroxychloroquine for covid- therapy chloroquine and hydroxychloroquine have shown the ability to inhibit replication of multiple coronaviruses in vitro, [ ] [ ] [ ] including sars-cov- in concentration-dependent manner. , , , , the anti-sars-cov- activity of hcq seems to be less potent compared to cq. the ec for cq ( . μm) was significantly lower than that of hcq ( . μm). in contrast, hydroxychloroquine was found more potent than chloroquine against sars-cov when given post-infection and prophylactically. clinical evidence of the effectiveness of hcq or cq for the treatment of covid- is limited. some small clinical trials have shown therapeutic benefits of the drugs, while others have shown the opposite. in recent clinical trials, over people with covid- have been treated with pharmacogenomics and personalized medicine : chloroquine. these patients had less severe disease and a shorter illness duration compared to those who did not receive chloroquine. another open-label non-randomized clinical trial with covid- patients demonstrated that hydroxychloroquine treatment resulted in viral load reduction/disappearance in the patients. the effect was reinforced by azithromycin. contrasting results were reported in a small study with hospitalized patients; no difference in clinical outcomes was observed between patients treated with hcq and azithromycin and patients on standard care. in a randomized trial with hospitalized patients, patients on hcq had a more substantial proportion of clinical improvement of pneumonia ( % vs %) than patients with standard care. in another clinical trial with covidpatients, an increased overall mortality was observed in the patients treated with hydroxychloroquine. more clinical trials are going on. the fda has issued an emergency use authorization for cq and hcq to treat covid- infection, allowing the unapproved use of these medications in light of a public health emergency. , on april , the fda issued warning against hcq or cq unless the therapy is closely supervised by a healthcare professional. the caution was initiated after the agency received reports of serious adverse effects in covid- patients. these findings do not apply to the use or evaluation of hydroxychloroquine in pre-or post-exposure prophylaxis in patients exposed to covid- . these results bring forward the need for large controlled clinical trials to provide guidance on safe and effective dosing of cq/hcq for covid- therapy. furthermore, response to drugs is subject to interindividual variability. patients treated with the same dose of the same drug, may exhibit lack of efficacy, or adverse reactions. the variability, at least in part, is attributed to genetic polymorphisms. knowledge of pharmacogenomic (pgx) of the drugs is necessary to estimate effective and safe dosing and to avoid/minimize adverse reactions. no pgx studies have been conducted to investigate the interpatient variability of cq and hcq in covid- patients. the purpose of the study was to highlight the importance of large, randomized, controlled clinical trials and pharmacogenomic studies to assess the optimal dosing of cq and hcq in diverse populations as a treatment for covid- . a review of the literature was performed using medline/ pubmed/embase database resources for english language papers from up to july to identify appropriate articles that addressed the objectives of this review. a variety of keywords were employed in keyword/title/abstract searches that included: chloroquine, cq, hydroxychloroquine, hcq, pharmacokinetics, pharmacogenomics, covid- , sars-cov- . we obtained appropriate results after screening all sources; relevant and nonrelevant. the publications were reviewed independently by two investigators. the investigators extracted the data and inspected each reference identified by the search. in cases where the same studies were reported in more than one publication, the study's results were accounted for only once. limits to the search strategy were english language articles and human studies. the electronic search was followed by extensive hand searching using reference lists from the identified articles. the search method was used to strengthen existing concepts and to identify study designs for upcoming research studies. this paper presents the current knowledge on chloroquine (cq) and hydroxychloroquine (hcq) pharmacokinetics (pk) with a focus on stereoselectivity of their disposition. both drugs are racemic mixtures, consisting of equal amounts of r(-) and s(+)-enantiomers. the pharmacokinetics of these two -aminoquinolines are similar and regulate dosing of the drugs. cq is available as chloroquine phosphate, aralen ® . each -mg tablet of chloroquine phosphate contains mg of chloroquine. hcq is available as hydroxychloroquine sulfate, plaquenil ® . each -mg tablet of hydroxychloroquine sulfate contains mg of hydroxychloroquine. doses of both agents are based on ideal body weight (ibw). chloroquine doses are . - . mg/kg/day and produce plasma levels of to × − m/l. doses of hydroxychloroquine are . - . mg/kg/day and produce plasma concentrations of . to . × − m/l. an initial adult dose of chloroquine phosphate for malaria therapy is g followed by mg given at - hours, , and hours. lupus erythematosus and rheumatoid arthritis chloroquine phosphate dosage is mg daily with dose reduction after remission. initial adult dose of hydroxychloroquine sulfate is mg followed by mg given at - hours, , and pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress hours. lupus erythematosus and rheumatoid arthritis initial treatment is - mg of hydroxychloroquine sulfate daily for several weeks or months. pediatric dosage of chloroquine and hydroxychloroquine is based on body weight. an initial pediatric dose of chloroquine phosphate for treatment of malaria is . mg/kg followed by . mg/kg given at , , and hours after initial dose. maximum total dose is . g. initial pediatric dose of hydroxychloroquine sulfate for treatment of malaria is . mg/kg followed by . mg/ kg given at , , and hours after the first dose. maximum total dose is g. cq and hcq have been used for prophylaxis and treatment of malaria in pregnant women without evidence of adverse effects on the fetus. dosing for treatment and prophylaxis of uncomplicated malaria is the same in pregnant and nonpregnant adults. due to pregnancy-induced physiologic changes, some pharmacokinetic properties of the drugs may be altered, suggesting dose adjustments may be needed. but data are not sufficient to determine an appropriate dosing during pregnancy. , small amounts of chloroquine and hydroxychloroquine excrete into breast milk. the amounts of the drugs are not sufficient to harm the infant nor to protect the child from malaria. weekly cq/hcq of / mg may be given until breastfeeding is completed. , no information is available on the effect of chloroquine and hydroxychloroquine in geriatric patients. but because cq and hcq are mostly excreted in the urine, elderly patients with age-related kidney problems may require caution and a dose adjustment for the patients. the dose adjustment should be based on the kidney function. the optimal dosing of hcq and cq for treatment of covid- is unknown. most of the published clinical studies had hcq dosage of mg/ days or mg on the first day and mg for the next days. the latest regimen was supported by pharmacokinetic modelling, where an oral hcq sulfate loading dose of mg twice daily, followed by a maintenance dose of mg twice daily for days was able to achieve treatment efficacy and a good safety profile. this regimen reached three times the potency of cq phosphate given mg twice daily for days. however, more reliable information is required before it can be widely used to treat covid- . oral absorption of chloroquine and hydroxychloroquine in humans is efficient. both drugs have oral bioavailability of . - . . , although - -fold difference in the absorbed fraction of oral doses was reported. , maximum blood concentrations (cmax) for the oral doses showed significant differences between subjects (range - ng/ml), but not within subjects. , oral bioavailability of chloroquine is - %. cq oral tablets have slightly greater bioavailability than oral solutions, - % vs - %, respectively. hydroxychloroquine has oral bioavailability of - %. , antacids may decrease the bioavailability of both drugs. oral chloroquine reaches cmax faster than hydroxychloroquine. time to reach the maximum level (tmax) for cq was estimated at minutes, , - while tmax for hcq was estimated . hours. , absorption of the r and s enantiomers was not significantly different. distribution cq and hcq have multicompartment disposition in humans with wide distribution to the body tissues. highest concentrations were found in the melanin-containing cells, the retina and the skin. high levels were observed in the liver, spleen, kidney, and lung. in the blood, concentrations in erythrocytes were up to times higher than in plasma. reported volumes of distribution were , l and , l for hcq and cq, respectively. , , [ ] [ ] [ ] plasma protein binding of the drugs ranges between % and %. , cq and hcq are mostly bound to two plasma proteins, albumins and α- -acid glycoproteins. binding of both compounds to plasma proteins is stereoselective. chloroquine is approximately % bound to plasma proteins. , extend of s(+)-chloroquine plasma protein binding is greater than binding of r(-)-chloroquine ( % vs %). binding of hydroxychloroquine to plasma proteins is around %, which is less than chloroquine binding. the s-hydroxychloroquine is % bound to plasma proteins, while the r-hydroxychloroquine is only % protein bound. following separate administration of the individual enantiomers of both drugs, r(-)-isomers reach higher and more sustained plasma and ocular concentrations than s(+)-forms. , , metabolism chloroquine and hydroxychloroquine have long half-lives and low blood clearance. cq is rapidly n-desethylated into two major metabolites: desethylchloroquine ( %) and bisdesethylchloroquine ( %). , desethylchloroquine is the pharmacologically active metabolite and further metabolizes to bidesethylchloroquine. cq is metabolized primarily by cyp c and cyp a mediating % of the total submit your manuscript | www.dovepress.com pharmacogenomics and personalized medicine : metabolism of the drug. other enzymes, cyp a and cyp d break down chloroquine to a lesser extent. , [ ] [ ] [ ] metabolism of cq is stereoselective. after administration of the individual enantiomers, the concentration of (r)-chloroquine was . -fold higher compared to concentrations of (s)-chloroquine in plasma and . -fold higher in the blood in patients with rheumatoid arthritis. blood concentrations of the active metabolite s(+)desethylchloroquine exceeded those of the r(-)forms. , , hcq has similar to chloroquine biotransformation but breaks down into more metabolites. hcq is n-dealkylated by cyp a to the two active metabolites desethylhydroxychloroquine, desethylchloroquine and an inactive metabolite bidesethylchloroquine. other cytochrome p enzymes (cyp c , d , and a ) are involved in the metabolism to a lesser extent. , biotransformation of hcq is also stereoselective. several studies have reported faster hepatic metabolism of s(+)-enantiomers compared to metabolism of r(-)-enantiomers. , , [ ] [ ] [ ] the blood and plasma concentrations of r-hydroxychloroquine exceeded those of the s-hydroxychloroquine with the mean r/s ratio of . in the blood and . in the plasma. the mean blood concentration ratio r/s for desethylhydroxychloroquine was . and for desethylchloroquine was . , indicating stereoselective metabolism of the compound. similar doses of the two drugs produced -fold variations in the blood concentrations in patients with rheumatoid arthritis , , , and in healthy volunteers, , suggesting different extend of metabolism among individuals. moreover, chloroquine and hydroxychloroquine are involved in several metabolic drug-drug interactions (ddis). a cyp a inhibitor, cimetidine increased serum concentrations of cq by %. another cyp a inhibitor, ketoconazole reduced the formation of active metabolite desethylchloroquine. , excretion urinary excretion is the main route of elimination for chloroquine and hydroxychloroquine. the % of a chloroquine dose is recovered in the urine as unchanged drug, with % of the dose recovered in the urine as its active metabolite desethylchloroquine. the % of a cq dose is recovered in feces. small amounts ( %) of the drug eliminate through the skin and up to % stored in lean tissues. elimination from the skin is very slow. cq remains in the skin longer than months, a time when the drug is no longer detectable in the plasma. chloroquine and active metabolite desethylchloroquine have elimination half-lives of to days and may be detected in urine months after a single dose. chloroquine has a total clearance of . - l/h/kg. renal clearance accounts for half of the total systemic clearance and increases by acidification of the urine. the renal excretion accounts for - % of hcq elimination, where only - % is excreted as unchanged drug. the - % of absorbed dose is excreted in the feces, which is greater than cq feces excretion. the elimination through the skin and long-term storage in lean tissues is identical to those of chloroquine, % and %, respectively. , , the total clearance of hydroxychloroquine is ml/min. iv hydroxychloroquine has a halflife of days ( . days in blood, and . days in plasma). the elimination half-life of both drugs is significantly longer in patients with chronic renal disease. , in anuretic patients, the plasma levels were % and - % higher for cq and hcq, respectively, compared to concentrations in subjects with normal kidney function. enantioselective renal elimination of the medications has been demonstrated in patients. , (s)-hydroxychloroquine had a mean renal clearance approximately twice that of (r)hydroxychloroquine. in addition, the mean renal clearance of active (s)-metabolites was also higher than that of (r)metabolites. the main mechanism of renal elimination of the medications is tubular secretion as renal excretion -fold exceeds the glomerular filtration rate. , the tubular secretion is an active process mediated by membrane proteins. it was reported that chloroquine is a substrate and potent competitive inhibitor of multidrug and toxin extrusion protein (mate ). , as substrates and/or inhibitors of active transport and metabolism, cq and hcq may be involved in several drug-drug interactions. [ ] [ ] [ ] [ ] [ ] [ ] response to drugs is subject to inter-individual variability. - % of individuals that receive a drug, exhibit lack of efficacy, or adverse drug reactions. up to % of the variability is attributed to genetic polymorphisms. cytochrome p enzymes are major determinants of drug response. they are responsible for approximately % of phase i drug metabolism, and % of drug clearance. the human cyp supergene family includes genes, of pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress which are responsible for more than % of all drug oxidation reactions. the cyp genes are highly polymorphic composed of large numbers of single-nucleotide polymorphisms (snps) and copy number variations. the most studied are genes of cyp d , c , c , a , and a enzymes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, drug pharmacokinetics also depend on the renal excretion of the medications. mate , encoded by slc a gene, has been identified as a major efflux transporter involved in the renal excretion of many drugs including chloroquine. , [ ] [ ] [ ] [ ] [ ] pharmacogenomics informing chloroquine malaria pharmacotherapy individual variation in drug response is a critical challenge in effective drug pharmacotherapy. both the nature of the drug, as well as the dose of the drug, are subjected to vary on an individual basis. genetic polymorphisms in metabolizing enzymes influence the pharmacokinetics and drug response. plasmodium vivax is the major cause of malaria disease outside africa. the world health organization (who) recommends chloroquine as a component of the treatment protocol for uncomplicated p. vivax malaria. chloroquine is metabolized by the cyp isozymes c , a , a and d . the cyp c genes are located in a cluster on chromosome q , organized as cent-cyp c -cyp c -cyp c -cyp c -tel. the cyp c gene is approximately kb in size and includes nine exons. cyp c is the most divergent with respect to its protein sequence. interindividual variability in chloroquine efficacy was previously reported in africa and asia and attributed to: p. vivax resistance to chloroquine, noncompliance, suboptimum dose and drug-drug interactions. in a study reported in , assessment of genetic polymorphisms in chloroquine metabolizing enzymes was identified as a need. to that end, the investigators focused on a cohort consisting of p. vivax malaria patients followed during malaria treatment from to . the study reported for the first time the influence of the cyp c gene on gametocyte clearance rate with patients undergoing chloroquine/primaquine malaria treatment. from baseline until the first day of treatment, wild-type cyp c homozygous individuals achieved greater reduction in gametocytes as compared to individuals without this genotype. the results suggested that cyp c , cyp c and cyp a genetic variants influenced chloroquine malaria treatment. discoid lupus erythematosus is the most common form of cutaneous lupus. patients diagnosed with systemic lupus erythematosus and rheumatoid arthritis show a positive correlation between whole blood hydroxychloroquine levels and clinical response. hcq is metabolized to n-desethylhydroxychloroquine in the liver. the reaction is mediated by cyp a , cyp c , cyp d and cyp a isoforms. , in a study reported by lee et al, systemic lupus erythematosus patients were genotyped for snps in cyp a * b, cyp d * , cyp a * . the association of the respective genotypes with blood hydroxychloroquine and n-desethyl hydroxychloroquine was the focus of the investigation. the cyp d * allelic variants were found to be significantly associated with the n-desethylhydroxychloroquine/hydroxychloroquine ratio. the study demonstrated that this ratio is related to cyp d polymorphisms in systemic lupus erythematosus patients treated with hydroxychloroquine. a multicenter observational and pharmacogenetic study with discoid lupus erythematosus patients treated with hcq was reported by wahie et al. thirty-nine percent of the patients failed to respond to hydroxychloroquine, or developed toxicity. the study showed a trend for cyp c variants to be associated with better response. the pk and pd characteristics of chloroquine and hydroxychloroquine need to be evaluated in order to provide safe and effective covid- therapy. the pharmacokinetics of chloroquine and hydroxychloroquine are similar. oral absorption of the drugs is comparable with bioavailability values of . - . . , but the two medications have significant variations in bioavailability between individuals. , genetic polymorphism of cyp enzymes involved in the presystemic metabolism can explain at least in part the individual differences in the oral absorption of the drugs and may reflect the variations in blood and tissue concentrations of the drugs in covid- patients. both cq and hcq have wide distribution to the body tissues. plasma protein binding of the drugs varies between % and %. , cq and hcq are known to be enantioselective in their dispositions. both medications have similar stereoselective patterns of protein binding. s (+)-isomers are more bound to plasma proteins than r(-)isomers, suggesting that free plasma concentrations are higher for r(-)-forms. such differences in the plasma submit your manuscript | www.dovepress.com pharmacogenomics and personalized medicine : protein binding can be responsible in part for the variations in therapeutic response and toxicity of the isomers in covid- patients treated with cq or hcq, as only free drug can interact with receptors and produce therapeutic and/or side effects. chloroquine and hydroxychloroquine have long halflives. the long half-life can be attributed to extensive tissue uptake rather than decreased elimination. chloroquine is n-desethylated into two major metabolites largely by cyp a and cyp c , while hydroxychloroquine metabolizes into three metabolites primarily by cyp a . , metabolism of both drugs is stereoselective. the higher blood and plasma concentrations of (r)-forms confirm the stereoselective metabolism of the medications, where s-enantiomers metabolize faster than r-enantiomers. , as a result, s(+)-isomers have shorter half-life than r(-)isomers. similar doses of both medications produce large ( -fold) variations in the blood concentrations in patients with rheumatoid arthritis and in healthy volunteers. , , , comparable differences in drug levels may be expected in covid- patients. the variability can be explained by the stereoselective metabolism as well as genetic polymorphism of the p- enzymes involved in the biotransformation of the medications. individual variation in drug response is a critical challenge in effective drug pharmacotherapy. up to % of individuals that receive a drug, exhibit lack of efficacy or adverse drug reactions, at least partially, due to genetic polymorphisms. gene polymorphisms influence metabolism as well as active transport. cyp d the most extensively studied cyp gene metabolizes approximately % of all drugs. genetic polymorphisms resulting in increased cyp d metabolic capabilities have been linked to decreased treatment response with tricyclic antidepressants, increased occurrences of respiratory depression and opioid toxicity. , cyp c deficiency is related to bleeding complications with warfarin and other anticoagulants treatment. fifty percent of interindividual variability in dose requirements is observed in concert with age, body surface area and polymorphisms in vkorc . cyp c is involved in the metabolism of many medications including non-steroidal anti-inflammatory drugs, thiazolidinediones, chemotherapy agents, chloroquine and hydroxychloroquine. cyp c genotyping should be considered as a viable option in africans and europeans in which . % and . % of cyp c alleles, respectively, exhibit reduced functionality. the cyp a subfamily is the most abundant of the p- enzymes. cyp a and cyp a metabolize more than half of the marketed drugs. , the most common variant of cyp a enzyme, cyp a * b has been associated with reduced cyp a activity. the cyp a * b allelic frequency varies among different ethnic groups, ranging from % in chinese to % in african americans. [ ] [ ] [ ] is polymorphically expressed in - % in caucasians, % in japanese and % in african americans. the primary variant is cyp a * , which has been associated with low cyp a protein expression and reduced metabolic activity. the cyp a * allele frequency varies from approximately % in african americans to % in caucasians. other allelic variants have been reported for both cyp a and cyp a . however, the variants occur at relatively low allelic frequencies and their functional significance has not been verified and validated. determination of cyp a, cyp c and cyp d polymorphism and, therefore, activity is important to establish safe and efficient dosing of chloroquine and hydroxychloroquine for treatment of covid- patients. a recent study reported for the first time the influence of the cyp c gene on clearance in patients with chloroquine/primaquine therapy. wild-type cyp c homozygous individuals achieved greater reduction in gametocytes as compared to individuals without this genotype. another study demonstrated cyp d polymorphisms in systemic lupus erythematosus patients treated with hydroxychloroquine. cyp d * allelic variants were found to be significantly associated with altered metabolism of hcq. a study with lupus erythematosus patients treated with hydroxychloroquine demonstrated % of the patients failed to respond to the therapy or developed toxicity. similar trend was observed in a recent clinical trial with covid-patients treated with hydroxychloroquine. additionally, the study with lupus erythematosus patients showed a trend for cyp c variants to be associated with better response. overall, the results suggest that cyp c , cyp d and cyp a genetic polymorphisms may influence chloroquine and hydroxychloroquine pharmacokinetics and covid- patients treated with the same dose of cq or hcq may exhibit lack of efficacy or adverse reactions. the variability in therapeutic response may require dose adjustment of cq/ hcq in treatment of covid- patients. urinary excretion is the primary route of elimination for chloroquine and hydroxychloroquine. the % of a cq dose is recovered in the urine as unchanged drug, while only - % of an hcq dose is renally excreted as pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress unchanged drug. , however, a greater fraction of absorbed hcq dose excretes in the feces compared to the fraction of cq dose. the elimination half-lives of both medications are significantly longer in patients with chronic renal disease. , this finding recommends that the two drugs should be used with caution in patients with renal impairment, as kidney dysfunction may lead to greater drug retention and higher risk of adverse effects. renal elimination of both compounds is stereoselective, (s)-isomers have a mean renal clearance approximately twice that of (r)-isomers. the main mechanism of renal elimination of the medications is tubular secretion. , however, the molecular mechanisms of the renal tubular secretion remain mostly unidentified. it was reported that chloroquine is a substrate and potent inhibitor of the mate transporter. given the similarity in structure between cq and hcq, it is possible to propose that hcq is also a substrate for mate . mate is a proton-substrate antiporter expressed in the kidney and liver that facilitates the export of organic cations, such as metformin, paraquat, and oxaliplatin into urine and bile. genetic polymorphisms of mate may alter the pharmacokinetics and pharmacodynamics of the medications, as drug transporters are key determinants of elimination of the drugs. mate , encoded by the slc a gene, has been identified as a major efflux transporter involved in the renal excretion of chloroquine. functional snp of mate (rs g>a) was associated with increased glucose-lowering activity of metformin through slowing renal excretion of the anti-diabetic drug. , the allele frequency ranges from . % in african americans to % in mexican americans. other snps may also alter transport activity of mate and lead to changed elimination of the corresponding drugs. , genetic polymorphisms of mate can affect renal elimination of cq and hcq and, therefore, may require dose adjustment based on pharmacogenomic profiles of covid- patients. indeed, the active transporters and cyp enzyme polymorphisms may explain the variations in blood concentrations, therapeutic responses and severity of adverse effects of chloroquine and hydroxychloroquine. despite the evidence of the influence of genetic polymorphisms on the pharmacokinetics of chloroquine and hydroxychloroquine, no large pharmacogenomics studies have been conducted to provide guidance on the use, dosing, and duration of the therapy in covid- patients. additionally, chloroquine and hydroxychloroquine are involved in several ddis. cimetidine and ketoconazole, cyp a inhibitors increased serum concentrations of cq. , predictably, cimetidine and ketoconazole may also increase hcq blood concentrations by inhibition metabolism of the drug. co-administration of cq and hcq with moderate and strong cyp a (boceprevir, cobicistat, azole anti-fungal agents, macrolide antibiotics, etc.), cyp c (gemfibrozil, clopidogrel, deferasirox, teriflunomide) and mate inhibitors may result in increased plasma concentrations, longer half-life, exaggerated therapeutic effect and the toxicity of chloroquine and hydroxychloroquine. significant drug interactions with chloroquine and hydroxychloroquine that should be avoided or require additional monitoring include digoxin, antiepileptics, antacids, cyclosporine, amiodarone, azithromycin, moxifloxacin, insulin and other antidiabetic agents, tamoxifen, and praziquantel. , with the currently known or potential ddis, the use of chloroquine and hydroxychloroquine with other drug therapy requires consideration for patient safety in covid- patients. limited pharmacogenomic studies have been performed investigating the inter-patient variability of chloroquine and hydroxychloroquine in both malaria and lupus patient populations. moreover, data to support the use of hydroxychloroquine and chloroquine for covid- are limited and inconclusive. the off-label use of chloroquine and hydroxychloroquine to treat covid- must be used with caution given the toxicities: cardiac, retinal and cutaneous severe adverse effects. well-designed randomized trials incorporating pharmacogenomics need to be performed in a timely manner to achieve safe and effective dosing and to reduce severity of adverse effects. cq, chloroquine; ddi, drug-drug interaction; ec , % maximal effective concentration; fda, food and drug administration; hcq, hydroxychloroquine; ibw, ideal body weight; mate , multidrug and toxin extrusion protein ; pgx, pharmacogenomics; pk, pharmacokinetics; snp, single nucleotide polymorphism. the authors report no conflicts of interest in this work. omim gene map statistics diagnosing the decline in pharmaceutical r&d efficiency can you teach old drugs new tricks? challenges and opportunities with drug repurposing: finding strategies to find alternative uses of therapeutics drug repositioning: current approaches and their implications in the precision medicine era a transcriptional signature and common gene networks link cancer with lipid metabolism and diverse human diseases discovery and development of safe-in-man broad spectrum antiviral agents aralen phosphate. the american society of health-system pharmacists the american society of health-system pharmacists fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice repurposing of the anti-malaria drug chloroquine for zika virus treatment and prophylaxis current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review malarial hemozoin: from target to tool mechanism of action of hydroxychloroquine as an antirheumatic drug clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) review of side effects and toxicity of chloroquine animal toxicity and pharmacokinetics of hydroxychloroquine sulfate hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine cardiomyopathy -a review of the literature the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/azithromycin. medrxiv randomized dose-ranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine improving the risk-benefit relationship and informed consent for patients treated with hydroxychloroquine american academy of ophthalmology. revised recommendations on screening for chloroquine and hydroxychloroquine retinopathy chloroquine and hydroxychloroquine binding to melanin: some possible consequences for pathologies increased incidence of gastrointestinal side effects in patients taking hydroxychloroquine: a brand-related issue? eua chloroquine phosphate health care provider fact sheet eua hydroxychloroquine sulfate health care provider fact sheet effects of chloroquine on viral infections: an old drug against today's diseases? new insights into the antiviral effects of chloroquine design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities chloroquine and hydroxychloroquine as available weapons to fight covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies submit your manuscript | www hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- .medrxiv; covid- ) update: fda reiterates importance of close patient supervision for 'off-label' use of antimalarial drugs to mitigate known risks, including heart rhythm problems -update-fda-reiteratesimportance-close-patient-supervision-labeluse?utm_campaign= _pr_fda% notes% importance% of% patient% supervision% of% antimalarial% drug% use&utm_ medium=email&utm_source=eloqua determination of the stereoisomers of hydroxychloroquine and its major metabolites in plasma and urine following a single oral administration of racemic hydroxychloroquine pharmacologic actions of -aminoquinoline compounds pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic diseases recent developments in the understanding of the pharmacokinetics and mechanism of action of chloroquine clinical pharmacokinetics of slow-acting antirheumatic drugs antirheumatic therapy: actions and outcomes. progress in inflammation research bioavailability of hydroxychloroquine tablets in healthy volunteers hydroxychloroquine relative bioavailability: within subject reproducibility antimalarials in rheumatic diseases treatment of hydroxychloroquine overdose pharmacokinetic interactions and safety evaluations of coadministered tafenoquine and chloroquine in healthy subjects influence of route of administration on the pharmaco-kinetics of chloroquine and desethylchloroquine disposition of chloroquine in man after single intravenous and oral doses dose optimization of chloroquine by pharmacokinetic modeling during pregnancy for the treatment of zika virus infection choloroquine: a review pharmacogenetics of disease-modifying anti-rheumatic drugs therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases concentration-effect relationship of hydroxychloroquine in rheumatoid arthritis-a cross sectional study hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax characterization of chloroquine plasma protein binding in man protein binding of chloroquine enantiomers and desethylchloroquine stereoselective distribution of hydroxychloroquine in the rabbit following single and multiple oral doses of the racemate and the separate enantiomers drugs used in the chemotherapy of malaria new concepts in antimalarial use and mode of action in dermatology pharmacokinetic interaction of chloroquine and methylene blue combination against malaria cytochrome p c and cyp a / are involved in chloroquine metabolism in human liver microsomes high sensitivity methods to quantify chloroquine and its metabolite in human blood samples using lc-ms/ms in vitro metabolism of chloroquine: identification of cyp c , cyp a , and cyp d as the main isoforms catalyzing n-desethylchloroquine formation the pharmacokinetics of (+)-and (-)-chloroquine in patients with rheumatoid arthritis stereoselectivity in the disposition of chloroquine and desethylchloroquine in rabbits enantioselective disposition of hydroxychloroquine after a single oral dose of the racemate to healthy subjects hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation enantioselective analysis of the metabolites of hydroxychloroquine and application to an in vitro metabolic study capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates enantioselective metabolism of hydroxychloroquine employing rats and mice hepatic microsomes disposition of the enantiomers of hydroxychloroquine in patients with rheumatoid arthritis following multiple doses of the racemate pharmacokinetics and pharmacodynamics of hydroxychloroquine enantiomers in patients with rheumatoid arthritis receiving multiple doses of racemate dose-loading with hydroxychloroquine improves the rate of response in early, active rheumatoid arthritis: a randomized, double-blind six-week trial with eighteen-week extension chloroquine elimination in humans: effect of low-dose cimetidine fda. drug development and drug interactions the metabolism of chloroquine in man during and after repeated oral dosage the influence of orally administered alkali and acid on the renal excretion of quinacrine, chloroquine and santoquine the disposition of chloroquine in healthy nigerians after single intravenous and oral doses molecular mechanism of renal tubular secretion of the antimalarial drug chloroquine a human transporter protein that mediates the final excretion step for toxic organic cations chloroquine reduces the bioavailability of methotrexate in patients with rheumatoid arthritis. a possible mechanism of reduced hepatotoxicity chloroquine and hydroxychloroquine are novel inhibitors of human organic anion transporting polypeptide a the interactions of p-glycoprotein with antimalarial drugs, including substrate affinity, inhibition and regulation drug interaction of chloroquine with ciclosporin interaction between cyclosporin and chloroquine influence of hydroxychloroquine on the bioavailability of oral metoprolol pharmacogenomics: translating functional genomics into rational therapeutics increased incidence of cyp d gene duplication in patients with persistent mood disorders: ultrarapid metabolism of antidepressants as a cause of nonresponse. a pilot study respiratory depression with tramadol in a patient with renal impairment and cyp d gene duplication clinical pharmacogenetics implementation consortium guidelines for cyp c and vkorc genotypes and warfarin dosing worldwide distribution of cytochrome p alleles: a meta-analysis of population-scale sequencing projects summary of information on human cyp enzymes: human p metabolism data analysis of single-nucleotide polymorphisms (snps) in human cyp a and cyp a genes: potential implications for the metabolism of hiv drugs modification of clinical presentation of prostate tumors by a novel genetic variant in cyp a novel mutations of cyp a in chinese genetic contribution to variable human cyp a-mediated metabolism submit your manuscript | www association between a cyp a genetic variant and clinical presentation in african-american prostate cancer patients interaction between polymorphisms in the oct and mate transporter and metformin response slc a gene rs g>a variants enhance the glucose-lowering effect of metformin via delaying its excretion in chinese type diabetes patients genetic polymorphisms of multidrug and toxin extrusion proteins (mate and mate ) in south indian population identification of multidrug and toxin extrusion (mate and mate -k) variants with complete loss of transport activity genetic variants in multidrug and toxic compound extrusion- , hmate , alter transport function pharmacogenetics -five decades of therapeutic lessons from genetic diversity challenges for malaria elimination in brazil a . -megabase physical map spanning the cyp c gene cluster on chromosome q gene structure of cyp c and extrahepatic distribution of the human cyp cs effectiveness of antimalarial drugs the effect of snps in cyp in chloroquine/primaquine plasmodium vivax malaria treatment epidemiology and socioeconomic impact of skin disease in lupus erythematosus pharmacogenetics: implications for therapy in rheumatic diseases association of polymorphisms of cytochrome p d with blood hydroxychloroquine levels in patients with systemic lupus erythematosus clinical and pharmacogenetic influences on response to hydroxychloroquine in discoid lupus erythematosus: a retrospective cohort study generalized pustular figurate erythema: a newly delineated severe cutaneous drug reaction linked with hydroxychloroquine pharmacogenomics and personalized medicine dovepress publish your work in this journal pharmacogenomics and personalized medicine is an international, peer-reviewed, open access journal characterizing the influence of genotype on pharmacology leading to the development of personalized treatment programs and individualized drug selection for improved safety, efficacy and sustainability. this journal is indexed on the american chemical society's chemical abstracts service (cas). the manuscript management system is completely online and includes a very quick and fair peer-review system key: cord- -vd qr o authors: gianturco, stephanie l.; yoon, sejeong; yuen, melissa v.; mattingly, ashlee n. title: outsourcing facilities and their place in the u.s. drug supply chain date: - - journal: j am pharm assoc ( ) doi: . /j.japh. . . sha: doc_id: cord_uid: vd qr o objective: the purpose of this commentary is to describe the ideal role of b outsourcing facilities in the u.s. drug supply chain. we also address the challenges that b outsourcing facilities are facing that limit their utilization and offer possible solutions. summary: section b outsourcing facilities are emerging contributors in compounding owing to their ability to compound large quantities of medication without requiring patient-specific prescriptions. as such, they play a valuable role in the u.s. drug supply chain. the use of outsourcing facilities to compound ready-to-use drug products is gaining traction in hospitals and other health care systems. outsourcing facilities help hospitals that are facing time and cost constraints owing to the evolving regulatory landscape around compounding. although outsourcing facilities are assets to the drug supply chain, there are several challenges to their use. the lack of a finalized b bulks list has led to outsourcing facilities being overly cautious in compounding products using bulk drug substances. in addition, the time between food and drug administration (fda) inspections is undefined, and a lack of follow-up information regarding concerns identified during an inspection may result in uncertainties about the current state of the outsourcing facility. conclusions: health care providers, outsourcing facilities, and fda need to work together to ensure that patients are provided the drugs they need in a safe and effective way. the u.s. drug supply chain includes a variety of participants who ensure that the drug reaches the patient, while maintaining safety and efficacy. although it is preferable for providers to use food and drug administration (fda)eapproved, commercially available drug products, there are times when drug products need to be compounded to meet a patient-specific need. in , a meningitis outbreak linked to compounded steroid injections led to the passage of the drug quality and security act, which established distinct types of pharmaceutical compounders for human patients: a and b. compounding under section a is performed by a physician or a pharmacist in a state-licensed pharmacy or federal facility, pursuant to a patient-specific prescription. limited quantities may be compounded in anticipation of a prescription if the facility has an established relationship with the patient or the prescriber. outsourcing facilities, established under section b, may register with fda, and compounding is supervised by a pharmacist. outsourcing facilities are not required to receive a patient-specific prescription before compounding and therefore are able to compound drug products in larger quantities than a facilities. as a requirement, all drug products must be compounded following current good manufacturing practices, must be reported to fda every months, and a portion of these must be sterile preparations. any drug product that an outsourcing facility intends to compound from a bulk drug substance (or a pure active pharmaceutical ingredient) must be on the b bulks list that fda is currently developing; a list of substances for which there is a demonstrated clinical need. while the list is being developed, bulk drug substances were divided into categories. fda does not intend to take action against outsourcing facilities that compound drug products using bulk drug substances in category , in which information supports their clinical use or need, and fda was unable to identify significant safety risks. however, outsourcing facilities cannot compound drug products using substances listed in category (which have significant safety risks identified) and category (which were not nominated with sufficient supporting information). drug products compounded under sections a and b cannot be identical to commercially available products, appear on the list of drug products that have been withdrawn or removed from the market for reasons of safety or efficacy, or appear on the list of drug products that present demonstrable difficulties for compounding. - a commercially available drug product can be compounded if it is on the drug shortage list published by fda. , how outsourcing facilities can take part in the u.s. drug supply chain outsourcing facilities can provide hospitals and independent practices with ready-to-use sterile drug products because they can compound drug products on a larger scale and are not limited by the requirement of a patient-specific prescription. as some commercial products require preparation into unitdosed forms for hospital use, this can be especially useful for hospitals that do not have their own in-house sterile compounding pharmacy. in a january letter, fda encouraged hospitals to purchase compounded sterile drug products from fda-registered outsourcing facilities because these facilities are under increased federal oversight. the use of outsourcing facilities has gained traction, on the basis of a recent report from the office of inspector general, which surveyed a stratified random sample of medicare-participating hospitals. of the hospitals that responded, % exclusively obtained nonpatient specific compounded drug products from fdaregistered outsourcing facilities, % obtained some drug products from outsourcing facilities, and % exclusively obtained drug products from unregistered compounders. another role of outsourcing facilities is to ensure continued availability of drug products and to provide a bridge in the event of manufacturer discontinuation or other scenarios in which there is a shortage of a commercial drug products. , in the event of a commercial manufacturer electing to withdraw a drug product from the market for reasons not relating to safety or efficacy, a provider may use an outsourcing facility to obtain the withdrawn drug products. the same holds true for drug shortage events; outsourcing facilities can bridge the gap until the commercial product becomes accessible again or until the provider can find another therapy that works for the patient. finally, outsourcing facilities can provide stock for provider to use for in-office procedures in specialties such as dentistry, ophthalmology, and podiatry. having drug products readily available in their office is more efficient for some providers than the process of using a a compounding pharmacy, in which the patient must take the prescription to the pharmacy, have it compounded, and then bring it back to the prescriber before it can be used. by keeping the drug product stocked in their offices, providers have control of the storage conditions, assuring the providers of drug product quality and safety, which is especially important when using sterile drug products. the ideal role of outsourcing facilities in the u.s. drug supply chain we believe the ideal role for outsourcing facilities is to produce ready-to-use sterile drug products for hospitals. for some hospitals, implementing best practices guidelines such as united states pharmacopeia (usp) < > standards can be time-consuming and costly, as addressed in the public comments for the recent revision of the usp < >. commenters on the usp revision felt that identifying microorganisms when the sampling level exceeds a certain threshold, changing filters during batch processing, and compounding record requirements would be burdensome. hospitals can avoid these requirements if products are supplied by outsourcing facilities. another benefit of using outsourcing facilities for hospital supply is that they can provide aseptic drug products with longer beyond-use dates, which can help decrease waste, turnover, and cost for hospital pharmacies. in these scenarios, outsourcing facilities may end up a permanent part of the hospital's drug supply chain. outsourcing facilities can also be temporary suppliers for hospitals that require a longer implementation time to adhere to regulatory standards. in the summary of public comments received for the revised usp < >, there was a concern of not key points background: the drug quality and security act was passed in , leading to the establishment of distinct types of pharmaceutical compounders, a and b. compounding under section a is performed by a physician or a pharmacist in a licensed facility, pursuant to a patient-specific prescription. compounding under section b is performed by outsourcing facilities and supervised by a pharmacist; a patient-specific prescription is not required for b compounding. we believe that the ideal role for outsourcing facilities is to produce ready-to-use sterile drug products for hospitals. challenges that limit the use of outsourcing facilities in the u.s. drug supply chain include a lack of a finalized b bulks list, inconsistences in the inspection process, and lack of transparency in compliance status. when selecting outsourcing facilities, buyers should thoroughly research the facility before selecting it for their compounded drug needs. having enough time to implement changes, with several commenters requesting at least - months. there were also commenters who expressed concerns about remediation plans that involved closing the pharmacy, which would lead to the hospital not being able to continue compounding. although this revised chapter is on hold, there may still be delays in implementing future revisions. during states of emergency, hospitals in need of sterile drug products that are in high demand could consider using outsourcing facilities. for the coronavirus disease pandemic, fda has implemented a temporary policy for compounding facilities that allows them to take a more active role in compounding certain drug products for hospitalized patients. , challenges and potential solutions to participation in the u.s. drug supply chain despite the opportunities that outsourcing facilities have in the u.s. drug supply chain, there are multiple challenges that currently limit their use. although the b bulks list is in development, outsourcing facilities can compound using category nominated substances. however, there is no guarantee that these substances will be included on the final list, so resources invested in compounding them may be wasted. because the b bulks list is not finalized, outsourcing facilities remain concerned about using nominated drug substances still under review, especially because no nominated drug substance has been added to the list thus far. [ ] [ ] [ ] in a recent comment to fda, the outsourcing facilities association (ofa), the trade organization that represents outsourcing facilities, detailed their concerns about how the b bulks list is being developed. they suggested additional questions fda should consider when making decisions for the b bulks list and recommended consistent updates to the interim list and more transparency of the review process. similar challenges with using outsourcing facilities to provide compounded drug products were identified in a recent report from the pew charitable trusts. another challenge to the use of outsourcing facilities is inconsistencies in the inspection process. after an initial inspection, outsourcing facilities are inspected on a risk-based schedule. there is no specific time interval in which subsequent inspections take place, which can lead to a facility being left uninspected for a prolonged period of time. in ofa's comment to fda, the organization expressed concerns about the "pace at which inspections are classified and closed, [creating] an inconsistent enforcement environment." they noted that at some facilities, inspections remain open for years, with some of these facilities operating under a warning letter or other actions. ofa called for a clear distinction between outsourcing facilities that are compliant and those that are not. at the end of an inspection, the facility may be issued an fda form , which describes the investigator's concerns for conditions that the facility may be violating. the fda form is publicly available on the fda's website along with information on the dates of initial and most recent registration, the last fda inspection, and whether any fda action was taken on the basis of the inspection. however, the publicly available information does not mention whether the observed conditions have been resolved. moreover, the form may not provide a complete picture of the facility because it "does not include observations of questionable or unknown significance at the time of the inspection." these observations can include anything from lack of written procedures for production and process controls to deficiencies in aseptic processing areas, quality control units, and employee training. therefore, the observed objectionable conditions are important for potential buyers because they could affect product quality. for outsourcing facilities to be part of the u.s. supply chain, they need to proactively respond to the concerns outlined in fda form in a transparent and publicly accessible manner. this may include resolving the problems quickly and posting a notice about their action on their website so potential buyers can verify facility status. in addition, fda should provide the current compliance status of facilities on their website, along with a definitive time frame in which actions will next be taken by them. this will allow health professionals to know the status of an outsourcing facility before selecting one for use. when selecting an outsourcing facility for compounded drugs, buyers should first check the fda's list of registered outsourcing facilities, which is available on the fda website. most hospitals look at fda registration and the facility's history of federal enforcement actions, including product seizures or injunctions, when choosing an outsourcing facility. buyers should also review the outsourcing facility's inspection history and most recent results from fda and the state board of pharmacy. the fda website also has a list of products that outsourcing facilities have reported making in the past year. for more information about what products are available, buyers should review the outsourcing facility's website or reach out to the contact person provided on the fda's list of registered facilities. buyers can also use the american society of health system pharmacists contractor assessment tool to evaluate a facility's compliance with usp < >. although it may be logistically challenging, buyers should try to visit potential outsourcing facilities themselves whenever possible to assess if they meet their needs. buyers can also speak to other buyers who have previously ordered from the facility to have a better understanding of the environment and their compounding capabilities. outsourcing facilities can play an important role, especially for hospital supply of ready-to-use sterile drug products. however, health care providers should recognize that drug products from outsourcing facilities are not held to the same regulatory standards as commercially available drug products and thoroughly research a facility before deciding to use it for their compounded drug needs. fda and outsourcing facilities should consider being more transparent about compliance status. fda could also provide more frequent updates about the substance list and the outsourcing facilities' status on the fda webpage. health care providers, outsourcing facilities, and fda need to work together to ensure that patients are provided the drugs they need in a safe and effective way. silver spring, md: food & drug administration, center for drug evaluation and research, office of compliance/oudlc evaluation of bulk drug substances nominated for use in compounding under section b of the federal food, drug and cosmetic act: guidance for industry. silver spring, md: food and drug administration, center for drug evaluation and research interim policy on compounding using bulk drug substances under section b of the federal food, drug and cosmetic act: guidance for industry. silver spring, md: food and drug administration, center for drug evaluation and research, office of compliance/oudlc drug products withdrawn or removed from the market for reasons of safety or effectiveness, cfr, x drug products that present demonstrable difficulties for compounding under the federal food, drug, and cosmetic act; establishment of a public docket compounded drug products that are essentially copies of approved drug products under section b of the federal food, drug, and cosmetic act compounded drug products that are essentially copies of a commercially available drug product under section a of the federal food, drug, and cosmetic act: guidance for industry silver spring, md: u.s. department of health and human services, food & drug administration most hospitals obtain compounded drugs from outsourcing facilities, which must meet fda quality standards pharmaceutical compoundingsterile preparations temporary policy for compounding of certain drugs for hospitalized patients by outsourcing facilities during the covid- public health emergency: guidance for industry. silver spring, md: food and drug administration, center for drug evaluation and research temporary policy for compounding of certain drugs for hospitalized patients by pharmacy compounders not registered as outsourcing facilities during the covid- public health emergency (revised) fda- -n- : agency information collection activities; submission for office of management and budget review; comment request; obtaining information to understand challenges and opportunities encountered by compounding outsourcing facilities list of bulk drug substances for which there is a clinical need under section b of the federal food, drug, and cosmetic act list of bulk drug substances for which there is a clinical need under section b of the federal food, drug and cosmetic act market for compounded drugs needs greater transparency and regulatory certainty. philadelphia, pa: the pew charitable trusts fda form frequently asked questions. u.s. food and drug administration registered outsourcing facilities. u.s. food and drug administration ashp guidelines on outsourcing sterile compounding services key: cord- -xurmzmwj authors: lin, xiaoqian; li, xiu; lin, xubo title: a review on applications of computational methods in drug screening and design date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: xurmzmwj drug development is one of the most significant processes in the pharmaceutical industry. various computational methods have dramatically reduced the time and cost of drug discovery. in this review, we firstly discussed roles of multiscale biomolecular simulations in identifying drug binding sites on the target macromolecule and elucidating drug action mechanisms. then, virtual screening methods (e.g., molecular docking, pharmacophore modeling, and qsar) as well as structure- and ligand-based classical/de novo drug design were introduced and discussed. last, we explored the development of machine learning methods and their applications in aforementioned computational methods to speed up the drug discovery process. also, several application examples of combining various methods was discussed. a combination of different methods to jointly solve the tough problem at different scales and dimensions will be an inevitable trend in drug screening and design. with the rapid development of both computer hardware, software, and algorithms, drug screening and design have benefited much from various computational methods which greatly reduce the time and cost of drug development. in general, bioinformatics can help reveal the key genes from a massive amount of genomic data [ , ] and thus provide possible target proteins for drug screening and design. as a supplement to experiments, protein structure prediction methods can provide protein structures with reasonable precision [ ] . biomolecular simulations with multiscale models allow for investigations of both structural and thermodynamic features of target proteins on different levels [ ] , which are useful for identifying drug binding sites and elucidating drug action mechanisms. virtual screening then searches chemical libraries to provide possible drug candidates based on drug binding sites on target proteins [ ] [ ] [ ] . with greatly reduced amount of possible drug candidates, in-vitro cell experiments can further evaluate the efficacy of these molecules. in addition to virtual screening, de novo drug design methods [ ] , which generate synthesizable small molecules with high binding affinity, provide another type of computer-aided drug design direction. artificial intelligence, e.g., machine learning and deep learning, is playing more and more important roles in the aforementioned computational methods and thus drug development [ ] [ ] [ ] . in this review, we will focus on developments of the last four computational methods as well as their applications in drug screening and design. predicted by the transition state theory. hence, they used isomorphism between probabilities obtained from the mc process and probability factors obtained from transition state theory [ ] , and converted the mc process to a time-dependent simulation with additional simplified modifications [ ] . the design, discovery, and development of drugs are complex processes involving many different fields of knowledge and are considered a time-consuming and laborious inter-disciplinary work [ ] [ ] [ ] [ ] . different drug design methods and virtual screening will be very useful to design and find rational drug molecules based on the target macromolecule that interacts with the drug and thus speed up the whole drug discovery process. here, we will discuss structure-based drug design, ligand-based drug design, and virtual screening. structure-based drug design must be performed with available structural models of the target proteins, which are provided by x-ray diffraction, nuclear magnetic resonance (nmr) or molecular simulation (homologous protein modeling, etc.) [ ] [ ] [ ] [ ] [ ] . keeping in mind the complexity of cancers which show diverse phenotypes and multiple etiologies, a one-size-fits-all drug design strategy for the development of cancer chemotherapeutics does not yield successful results. lately, arjmand et al. [ ] adopted a series of methods, such as the combination of x-ray crystal structures and molecular docking, to design, synthesize, and characterize novel chromone based-copper(ii) antitumor inhibitors. in general, after obtaining the structure of the receptor macromolecule by x-crystal single-crystal diffraction technique or multi-dimensional nmr, molecular modeling software can be used to analyze the physicochemical properties of drug binding sites on the receptor, especially including electrostatic field, hydrophobic field, hydrogen bond, and key residues. then, the small molecule database is searched, or the drug design technique is used to identify the suitable molecules whose molecular shapes match the binding sites of the receptor and binding affinity is high. then, these molecules are synthesized and their biological activities will be tested for further drug development. in short, structure-based drug design plays an extremely important role in drug design. unlike structure-based drug design, ligand-based drug design doesn't search small molecule libraries. instead, it relies on knowledge of known molecules binding to the target macromolecule of interest. using these known molecules, a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target can be derived [ , ] . then, this model can be further used to design new molecular entities that interact with the target. on the other hand, ligand-based drug design can also use quantitative structure-activity relationships (qsar) [ , ] in which a correlation between calculated properties of molecules and their experimentally determined biological activity is derived, to predict the activity of new analogs. both the pharmacophore model and qsar model will be discussed in detail in the following sessions. in recent years, the rapid development of computational resources and small molecule databases have led to major breakthroughs in the development of lead compounds. as the number of new drug targets increases exponentially, computational methods are increasingly being used to accelerate the drug discovery process. this has led to the increased use of computer-assisted drug design and chemical bioinformatics techniques such as high-throughput docking, homology search and pharmacophore search in databases for virtual screening (vs) technology [ ] . virtual screening is an important part of computer-aided drug design methods. it may be the cheapest way to identify potential lead compounds, and many successful cases have proven successful using this technology. the primary technique for identifying new lead compounds in drug discovery is to physically screen large chemical libraries for biological targets. in experiments, high-throughput screening identifies active molecules by performing separate biochemical analysis of more than one million compounds. however, this technology involves significant costs and time. therefore, a cheaper and more efficient calculation method came into being, namely, virtual high-throughput screening. the method has been widely used in the early development of new drug. the main purpose is to determine the novel active small molecule structure from the large compound libraries. it is consistent with the purpose of high-throughput screening. the difference is that virtual screening can save a lot of experimental costs by significantly reducing the number of compounds for the measurement of the pharmacological activity, while high-throughput screening needs to perform experiments with all compounds in the database. here, we will discuss common methods of virtual screening. molecular docking, which predicts interaction patterns between proteins and small molecules as wel as proteins and proteins, to evaluate the binding between two molecules [ ] , is widely used in the field of drug screening and design. the theoretical basis is that the process of ligand and receptor recognition relies on spatial shape matching and energy matching, which is the theory of "inducing fit". determining the correct binding conformation of small molecule ligands and protein receptors in the formation of complex structures is the basis for drug design and studying its action mechanism. molecular docking can be roughly divided into rigid docking, semi-flexible docking and flexible docking. in rigid docking, the structure of molecules does not change. the calculation method is relatively simple, and mainly studies the degree of conformation matching, so it is more suitable for studying macromolecular systems, such as protein-protein, protein-nucleic acid systems. in semi-flexible docking, the conformation of molecules can be varied within a certain range, so it is more suitable to deal with the interaction between proteins and small molecules [ ] . in general, the structure of small molecules can be freely changed, while macromolecules remain rigid or retain some of the rotatable amino acid residues to ensure computational efficiency. in flexible docking, the simulated system conformation is free to change, thus consuming more computing resources while improving accuracy. what's more, the establishment of binding sites in molecular docking methods is very important. for the first time, collins [ ] successfully determined the binding sites on the surface of proteins using a multi-scale algorithm and performed flexible docking of molecules, which greatly promoted the development of molecular docking. a pharmacophore is an abstract description of molecular features necessary for molecular recognition of a ligand by a biological macromolecule, which explains how structurally diverse ligands can bind to a common receptor site. when a drug molecule interacts with a target macromolecule, it produces a geometrically and energetically matched active conformation with the target. medicinal chemists found that different chemical groups in drug molecules have different effects on activity, and changes to some groups have a great influence on the interaction between drugs and targets, while others have little effect [ ] . moreover, it was found that molecules with the same activity tend to have some of the same characteristics. therefore, in , ehrlich proposed the concept of pharmacophores, which referred to the molecular framework of atoms with active essential characteristics [ ] . in , gund further clarified the concept of pharmacophores as a group of molecules that recognize receptors and form structural features of molecular biological activity [ ] . there are two main methods for the identification of pharmacophores. on one hand, if the target structure is available, the possible pharmacophore structure can be inferred by analyzing the action mode of receptor and drug molecule. on the other hand, when the structure of the target is unknown or the action mechanism is still unclear, a series of compounds will be studied for pharmacophores, and information on some groups that play a key role in the activity of compound will be summarized by means of conformational analysis and molecular folding [ ] . active compound that is suitable for constructing the model will be selected in the pharmacophore recognition process. then, conformation analysis is used to find the binding conformation of molecule, and to determine the pharmacophore [ ] . in recent years, with the development of compound databases and computer technology, the virtual screening of databases using the pharmacophore model has been widely used, and has become one of the important means to discover lead compounds. qsar is a quantitative study of the interactions between small organic molecules and biological macromolecules. it contains a correlation between calculated properties of molecules (e.g., absorption, distribution, metabolism of small organic molecules in living organisms) and their experimentally determined biological activity [ ] . in the case of unknown receptor structure, the qsar method is the most accurate and effective method for drug design. drug discovery often involves the use of qsar to identify chemical structures that could have good inhibitory effects on specific targets and have low toxicity (non-specific activity). with the further development of structure-activity relationship theory and statistical methods, in the s, d structural information was introduced into the qsar method, namely d-qsar. since s, with the improvement of computing power and the accurate determination of d structure of many biomacromolecules, structure-based drug design has gradually replaced the dominant position of quantitative structure-activity relationship in the field of drug design, but qsar with the advantages of small amount of calculation and good predictive ability [ ] still plays an important role in pharmaceutical researches. based on d structural characteristics of ligands and targets, d-qsar explores the d conception of bioactive molecules, accurately reflects the energy changes and patterns of interactions between bioactive molecules and receptors, and reveals the drug-receiving mechanism of body interactions. the physicochemical parameters and d structural parameters of a series of drugs are fitted to the quantitative relationship. then, the structures of new compounds are predicted and optimized. in short, d-qsar is actually a research method combining qsar with computational chemistry and molecular graphics. it is a powerful tool for studying the interactions between drugs and target macromolecules, speculating the image of simulated targets, establishing the relationship of drug structure activity, and designing drugs. computer-based de novo design methods of drug-like molecules are mainly for generating small molecule compounds with ideal physicochemical and pharmacological properties. in the past decades, fragment-based drug discovery had appeared as a novel concept that has proved a good prospect for improving lead optimization, in order to decrease the clinical attrition rates in drug design. it is an approach that uses small molecular fragments to deduce the biomolecular targets [ ] . fragment-based de novo design has obtained the long-term clinical success [ ] . despite the fact that modern drug discovery has made some successes in offering effective drugs, drug design has been affected by several factors, such as the tremendous chemical space for exploring drug molecules [ ] . further, as a large number of data increase in biological, chemical, and clinical medicine, it is obvious that the drug design should be solved with multiscale optimization methods, and concentrate on the data beyond molecular levels [ ] . thus, it is essential to discuss the function of multiscale models in drug discovery, and how they have predicted multiple biological properties in different biological targets. accordingly, we discuss the combined application of both the concept of fragment-based on de novo design and multiscale modeling. the fragment-based de novo design method starts with small building blocks. the initial molecular building blocks with desired properties are either elaborated upon (growing), directly connected (joining), or connected by a linker (linking). this process can be iterated until one or more molecules with the desired properties are obtained. there are two methods, namely structure-based and ligand-based methods [ ] . structure-based de novo design method searches novel ligands by using the d structural information of the protein target, which are usually constructed directly in the target protein binding site and evaluated by calculating the interaction energy of the target protein with ligands. nevertheless, in de novo ligand design method, the molecule structure of protein target is unknown, and the new molecule is suggested based structure analogous to the known ligand molecule. nowadays, proteochemometrics has emerged as a relatively new discipline for drug discovery. in this filed, qsar analysis is a powerful tool for the efficient virtual screening, which shows physicochemical properties of various compounds. compared to the classical qsar, the qm calculations use reactivity descriptors in ligand-based qsars, which provides an implicit model and calculate an exact enthalpy contributions of protein-ligand interactions. however, for the ab initio fragment, molecular orbital calculation in the structure-based qsars, which obtains an explicit model and a clear enthalpy, changes the binding energy in different additional conditions. moreover, it also calculates the free energy contribution of ligand-target complexes formation in structure-based and ligand-based qsar models. using a large number of ligand-target complexes to discuss the change of their binding affinity, more accurate optimization steps can be conducted based on good prediction and interpretation models [ ] . the key of any qsar model is how to accurately describe the molecule, and qm approaches provides a better understanding to the molecular and structural characteristics of ligands and drugs, so as to solve the problems existed in drug discovery. the qsar models of different scales are built according to the different computational precision, multiscale-qsar research object mainly refers to the structure description of the training set, and involves small molecules and macromolecules [ , ] . in micro-, meso-and macroscopic scales, different molecular approaches will be used. qm approaches are often used to perform precise calculations at microscales, such as atom-based qsar. molecular force field focuses on mesoscopic-scale simulation, such as fragment-based qsar. and coarse-grained study mainly performed in the macroscopic scales, such as macromolecule-based qsar and cell-based qsar. moreover, multi-scale can also be reflected by different dimensions used in different qsar models. comfa is a technique of d fragment-based qsar, which can complete skeleton transition and r-groups substitution, providing different structures for new drug design. besides, derived from proteochemometrics (pcm) [ ] , the . d kinochemometrics (kcm) approach using d descriptor for protein kinases and d fingerprints for ligands can greatly increase the efficiency as well as the precision compared the traditional d qsar methods [ ] . the multiscale qsar provides effective predictions for drug design, which integrates qsar more systematically and applies all existing qsar methods effectively. multiscale de novo drug design is a novel concept that combines qsar models, qm calculations [ ] and fragment-based drug discovery (fbdd) [ ] . here, the importance of explicit molecular descriptors is shown in a model from a molecular structural point of view through qm calculations. with the assembly of reasonable molecular fragments, the objective of drug design method is to produce a certain novel molecule that display highest biological activity, absorption, metabolism, elimination (adme) and lowest toxicity properties at different environments, which belong to the application range of qsar models. the multiscale de novo drug design methods can efficiently handle a large amount of biochemical/clinical data and obtain the chemical characteristics in order to improve the properties of the drug molecule. it is considered to be a more effective and safer method to discover new therapeutic agents. in the process of drug discovery, machine intelligence methods have mostly been used in the above-mentioned computational methods over the past few decades [ ] . with the booming era of "big" data, machine learning methods have developed into deep learning approaches, which are a more efficient way for drug designers to deal with important biological properties from large amount of compound databases. here, we introduce applications of machine learning methods in qsar analysis as well as the recent advances in deep learning methods. decision trees (dts) are a simple, interpretable and predictive machine learning method. ordinarily, there are two fundamental steps, that is, selecting properties and pruning for the decision trees building. the selected properties are considered as internal nodes, the branch representing the test result on the molecule, and the leaf node as a classification label. in order to avoid the complexity of the decision tree, the pruning program is used to prune the established tree. the dt is a typical classification algorithm, which is widely used in the prediction and auxiliary diagnosis of the disease, such as management decision-making, the classification mode for creating the metabolic disorder, and data mining of diabetes etc. [ ] . abdul et al. [ ] developed a task-based chemical toxicity prediction framework, and used a decision tree to obtain an optimum number of features from a collection of thousands of them, which effectively help chemists perform prescreening of toxic compounds effectively. the artificial neural network (ann) achieves problem-solving by mimicking brain function. just as the brain applies information obtained from past experience to solve new problems, a neural network can construct a system of "neurons" that reaches new decisions, classifications, and prediction based on previous experiences. the processing element is similar to a neuron, and a massive processing element is organized by the layers. they include three types: input, hidden, and output layers. ann benefits from high self-organization, robustness, and fault tolerance, and has been widely applied in prognosis evaluation and early prevention of diseases. lorenzo et al. [ ] used the interpretable ann to predict biophysical properties of therapeutic monoclonal antibodies, include melting temperature, aggregation onset temperature and interaction parameters as a function of ph and salt concentration from the amino acid composition. artificial neural networks had their first heyday in molecular informatics and drug discovery approximately two decades ago. currently, we are witnessing renewed interest in adapting advanced neural network architectures for pharmaceutical research by borrowing ideas from the deep learning field. compared with some other fields in life sciences, their applications in drug discovery is still limited. the support vector machine (svm) is one of the most promising machine learning methods that can use molecular descriptors to construct a predictive qsar models and deal with high-dimensional datasets. ann and multiple linear regression analysis were used to construct linear and nonlinear models, which were then compared with the results gained by svm. for linear models, the svm approaches use space mapping points to separate different classification for maximizing the range between different categories of points [ ] . further, for the nonlinear models, svms use nucleus mapping to transform into a high-dimension space for linear classification. at present, the svm approach has been widely used in modeling at different scales for drug discovery [ ] . the k nearest neighbor (knn) is one of the simplest and most intuitive algorithms among all machine learning methods, and is usually jointly used with other selection algorithms in the feature space. further, it is used for classification and regression based on example learning. normally, molecules are classified by votes of its closest neighbors, resulting in the most common class that molecules are distributed to its closest neighbors. here, the value of k is the number of closest neighbors. based on ligand-based virtual screening, knn can be viewed as a prolongation form chemical similarity searching to supervised learning, and the top search results predicted the best bioactivities. weber et al. [ ] tried two machine learning algorithms of classification (knn and rf) to analyze genotype-phenotype datasets of hiv protease and reverse transcriptase (rt). as a result, both algorithms had high accuracies for predicting the drug resistance for protease and rt inhibitors. the random forest (rf) is an ensemble learning approach involving the building of multiple dts based on the training examples. similar to knn algorithms, it is also used to for classification and to predict regression [ ] . compared to dts, it is impossible that rf over-fits the data, and the rf has been used for bioactivity data classification [ ] , toxicity modeling [ ] , and drug target prediction [ ] , etc. wang et al. [ ] used the rf approach to model the binding affinity of protein-ligand on hiv- proteases complexes, trypsin complexes, and carbonic anhydrase complexes, which demonstrated that individual representation and model construction for each protein family is a more reasonable way in predicting the affinity of one particular protein family. currently, the multiscale models can predict toxicity, activity, and adme properties of different proteins and microbial targets by integrating different genomes and proteomics. cheminformatics has played an important role in rationalize drug discovery. the qsar model has become the main auxiliary tool which can achieve virtual screening of various pharmacological characteristics. although the qsar model has been widely used in the search and design of new drug, classical qsar models can only predict the activity and toxicity of one biomolecule against one certain target. however, the multi-target qsar (mt-qsar) can be used to carry out rational drug design at multiple targets, which provides a better way to understand various pharmacological characteristic molecules including antibacterial activity and toxicity. furthermore, uniform multitasking models based on quantitative structure biological effect relationship (mtk-qsber) have been used in a lot of researches. these models were built by ann and the topological indices, which can predict the biological activity and toxicity correctly and classify the compounds in experimental conditions. meanwhile, these models used perturbation models to form structural-activity relationships between the site of infection and the drug, such as the ptml model [ ] and the chembl model [ ] , which has been applied in infectious diseases [ ] , immunology [ ] , and cancer [ ] widely. currently, the mtk-qsber model has been able to carry out the in-silico design and virtual screening of an antibacterial drug efficiently, and these antibacterial drugs have good biosafety. these methods have provided a powerful tool for in silico screening reasonable drugs. the deep learning network is a concept closely related to ann, which are learning of the concept of layering. in other words, it is a multiple learning approaches ranging from low to high levels. just when the molecular descriptors are not selected, the deep learning method will automatically select representations from original data and high-dimensional data [ ] . therefore, it allows deep learning to be applied to the model building of drug discovery [ ] . the convolutional neural networks (cnn) are most commonly used, which have made great progresses in the computer vision community [ ] , and been applied in the drug design fields including de novo drug molecule identification, protein engineering and gene expression analysis. with the rapid development of deep learning concepts such as cnn, the molecular modeler's tool box has been equipped with potentially game-changing methods. judging from the success of recent pioneering studies, we are convinced that modern deep learning architecture will be useful for the coming age of big data analysis in pharmaceutical research, toxicity prediction, genome mining and chemogenomic applications, to name only some of the immediate areas of application. kiminori et al. [ ] developed a fundamental technology that can predict the resistance of free cancer cells to fluorinated pyrimidine anticancer drugs by deep learning from the morphological image data taken from images. cai et al. [ ] developed a deep learning approach, termed deep human ether-a-go-go-related gene (herg), for the prediction of herg blockers of small molecules in drug discovery and post-marketing surveillance. the group found that deepherg models built by a multitask deep neural network (dnn) algorithm outperformed those built by single-task dnn, svm and rf. now, the drug development technologies usually include artificial intelligence-based (ai-based) techniques. most ai applications only concentrate on limited tasks. moreover, current ai can only direct patients' specific problems, it cannot make subjective inferences like doctors with the overall physical context of a patient. as a subfield of ai, ml can be successfully used for training in the quality of examples. however, this process is very time-consuming and costly. the development of ml techniques and the application of existing algorithms to process massive amounts of digital data resulted in higher requirements for computer hardware, which also increases the clinical cost. dl, which is also a subset of ml, and can process big data and create patterns by layers of neurons. however, it is difficult to understand how each decision is obtained by algorithm. ml methods have achieved great successes in the field of chemoinformatics to design and discover new drugs. an important innovation is the combination of ml methods and big data analysis to predict more extensive biological features. it is vital to discover more secure and efficient drugs by integrating structural, genetic information and pharmacological data from the scale of molecular to organism [ ] . in addition, dl approaches have proven to be a promising way for efficiently learning from a large variety of datasets for modern novel drug discovery. multiscale modeling of the drugs in an excitable system is critical because experiments on a single system scale cannot reveal the underlying effects of multiple drug interactions. a computationally based approach to predict the emergency effects of drugs on excitatory rhythms may form an interactive technology-driven process for the drug and disease screening industry, research and development academia, and patient-oriented medical clinic. there are potentially far-reaching implications because millions of people affected by arrhythmia each year will benefit from improved risk stratification of drug-based interventions. much progress has been made in developing multiscale computational modeling and simulation approaches for predicting effects of cardiac ion channel blocking drugs. structural modeling of ion channel interactions with drugs is a critical approach for current and future drug discovery efforts. modeling of drug receptor sites within an ion channel structure can be useful to identify key drug-channel interaction sites. drug interactions with cardiac ion channels have been modeled at the atomic scale in simulated docking and md simulations, as well as at the level of channel function to simulate drug effects on channel behavior [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structural modeling of drug-channel interactions at the atomic scale may ultimately allow for the design of novel high-affinity and subtype selective drugs for specific targeting of receptors for cardiac and neurological disorders. the world health organization (who) stated that cancer remains one of the most dangerous diseases today. considering that cancer is a multifactorial disease, there is increasing interest in multi-target compounds that can target multiple intracellular pathways. however, the study of large data sets for the analysis of anticancer compounds is difficult, with a large amount of data and high data complexity. for example, the chembl database [ ] compiles big datasets of very heterogeneous preclinical assays. bediaga et al. [ ] have reported a ptml-lda model of the chembl dataset for the preclinical determination of anticancer compounds. ptml is a model that combines perturbation theory (pt) ideas and ml methods to solve similar problems. they compared this model with other ptml models which was reported by speck-planche et al. [ , [ ] [ ] [ ] and then concluded that this is the only one that can predict activity against multiple cancers. speck-planche et al. also derived a multi-task (mtk) chemical information model combining broto moreau autocorrelation with ann from a dataset containing peptide cases. this model is used to virtually design and screen peptides with potential anti-cancer activity against different cancer cell lines and low cytotoxicity to a variety of healthy mammalian cells, and the model shows greater than % in both training and prediction (test)accuracy. in addition, due to the inherent complexity of tumors, it is necessary to analyze their growth at different scales. it includes many phenomena that occur at various spatial scales from tissue to molecular length. the complexity of cancer development is manifested in at least three scales that can be distinguished and described by mathematical models, namely microscale, mesoscale, and macroscale. wang et al. conducted a number of studies on how to use multiscale models for the identification and combination therapy of drug targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this method is based on quantification of relationship between intracellular epidermal growth factor receptor (egfr) signaling kinetics, lung cancer extracellular epidermal growth factor (egf) stimulation and multicellular growth. the multiscale modeling of tumors combined with systemic pharmacology will contribute to the development of practical smart drugs. it will produce a comprehensive system-level approach to determine the dynamics and effects of existing and new drugs in preclinical trials, model organisms and individual patients. in addition, mathematical and computational studies will provide a better way to understand many factors that influence the effects of drugs, thus helping to uncover better ways to therapeutically interfere with disease. multiscale models can be also used to identify pathophysiological processes to allow disease staging. in many cases, like cancer, treatments vary depending on the stage of disease. the model can help determine prognosis, which is an important clinical determination that can help determine the right type of medication to be administered or discovered. several models focus on the neuronal network levels, including cutsuridis and moustafa for alzheimer's disease [ ] , and lytton for epilepsy [ ] . ann is a class of ml techniques that can be used for clinical analysis of big data including that related to drug testing, which is critical for drug discovery. in addition, anastasio [ ] introduced process algebra, a computer technology widely used to analyze complex computing systems, used here to calculate neurology. sirci et al. [ ] described how network (map) theory is used to identify similarities and differences between different pharmacological agents. in this type of study, each drug is a node, and the edges between drugs represent chemical and transcription-based interactions that characterize the drug. in addition, ferreira da costa et al. [ ] report the first ptml (pt + ml) study of a large number of chembl datasets for preclinical determinations of compounds for dopamine pathway proteins. molecular docking or ml models can be used to solve a specific protein, but these models cannot explain the large and complex large data sets of preclinical assays reported in public databases. pt models, on the other hand, allow us to predict the properties of a query compound or molecular system in an experimental analysis with multiple boundary conditions based on previously known reference cases. in their work, the best ptml model found in the training and external validation series has an accuracy of - %. hansch's model is a classic method for solving quantitative structural binding relationships (qsbr) in pharmacology and medicinal chemistry. abeijon et al. [ ] developed a new pt-qsbr hansch model based on pt and qsbr methods for a large number of drugs reported in chembl, focusing on a protein expressed in the hippocampus of the brain of alzheimer's disease (ad) patients. now, by decomposing how risks and causes are combined in complex systems to produce disease, and how to prevent or improve these diseases through multi-stage, multi-target, multi-drug techniques, multiscale modeling is gradually being grasped. from aids, hepatitis c, influenza, and other disease-related viruses to the current -ncov, we have been working hard to develop antiviral drugs targeting them. however, the unique structure and proliferation of the virus pose a natural challenge for drug development. viruses do not have their own cellular structure and metabolic system, and must replicate and proliferate in host cells. therefore, it is difficult to find compounds that target only viral targets without affecting the normal function of host cells. at present, the main way that some antiviral drugs work is to inhibit viral replication. however, many of the tools used for virus replication come from human cells, such as ribosomes, and the corresponding antiviral drugs will also bring great side effects to the human body. therefore, the discovery of drugs requires the introduction of a multi-scale model to screen out drugs that can inhibit viral replication while reducing the damage to the human body. so far, retroviral infections, such as hiv, are incurable diseases. chembl manages big data capabilities through complex datasets, which make the information difficult to analyze because these datasets describe numerous features for predicting new drugs for retroviral infections. without proper model, it is impossible to make full use of these features. hence, vásquez-domínguez et al. [ ] proposed a ptml model for the chembl dataset, which can be efficiently used for preclinical experimental analysis of antiretroviral compounds. the pt operator is based on a multi-conditional moving average, which combines different functions and simplifies the management of all data. the ptml model they proposed was the first to consider multiple features combined with preclinical experimental antiretroviral tests. in order to simultaneously explore antibacterial activity against gram-negative pathogens and in vitro safety related to absorption, distribution, metabolism, elimination, and toxicity (admet), the speck-planchee et al. [ ] further proposed the first mtk-qsber model. the accuracy of this model in both the training and prediction (test) sets is higher than %. they also have developed a chemoinformatic model for simultaneous prediction of anti-cocci activities and in vitro safety [ ] . the best model displayed accuracies around % in both training and prediction (test) sets. additionally, focusing on exploring anti-hepatitis c virus (hcv), the accuracy shown in the training and prediction (test) sets is higher than % using this model [ ] . cytotoxicity is one of the main concerns in the early development of peptide-based drugs. kleandrova et al. [ ] introduce the first multi-task processing (mtk) computational model focused on predicting both antibacterial activity and peptide cytotoxicity. gonzalez-diaz et al. [ ] developed a model called lnn-alma to generate complex networks of the aids prevalence with respect to the preclinical activity of anti-hiv drugs. multiscale models are also imperfect and have their limitations. models are expressions and simplifications of real life. no model can represent everything that can happen in the system. all models contain specific assumptions, and models vary widely in their comprehensiveness, quality, and utility. in other words, each model can only solve limited problems. hence, we need to integrate different computational models and data in order to make full use of these models. computational methods have come to play significant roles in drug screening and design. multiscale biomolecular simulations can help identify the drug binding sites on the target macromolecules and elucidate the drug action mechanisms. virtual screening can efficiently search massive chemical databases for lead compounds. de novo drug design provides alternative powerful way to design drug molecules from scratch using building blocks summarized and abstracted in previous successful drug discovery. ml is revolutionizing most computational methods in drug screening and design, which may greatly improve the efficiency and precision for the big data era. as we frequently emphasize, different models or efficient algorithms (e.g., dimensionality reduction) need to be integrated properly to achieve the comprehensive study of biological processes at multiple scales as well as accurate and effective drug screening and design. the integrated computational methods will accelerate drug development and help identify effective therapies with novel action mechanisms that can ultimately be applied to a variety of complex biological systems. the authors declare no conflict of interest. prediction of drug-target interaction networks from the integration of chemical and genomic spaces properties and identification of human protein drug targets critical assessment of methods of protein structure prediction (casp)-round xii multiscale modeling of biomolecular systems: in serial and in parallel virtual screening of chemical libraries computational protein-ligand docking and virtual drug screening with the autodock suite rapid virtual screening of enantioselective catalysts using catvs automated de novo drug design: are we nearly there yet? deep reinforcement learning for de novo drug design machine learning for molecular modelling in drug design. biomolecules machine learning based dimensionality reduction facilitates ligand diffusion paths assessment: a case of cytochrome p cam development of multiscale models for complex chemical systems: from h + h to biomolecules (nobel lecture) the many roles of computation in drug discovery role of molecular dynamics and related methods in drug discovery advancing drug discovery through enhanced free energy calculations excited state properties of non-doped thermally activated delayed fluorescence emitters with aggregation-induced emission: a qm/mm study exploring the dependence of qm/mm calculations of enzyme catalysis on the size of the qm region spectroscopy in complex environments from qm-mm simulations peptide folding kinetics from replica exchange molecular dynamics structural characterization of λ-repressor folding from all-atom molecular dynamics simulations macrolide antibiotics allosterically predispose the ribosome for translation arrest current tools and methods in molecular dynamics (md) simulations for drug design modeling structural dynamics of biomolecular complexes by coarse-grained molecular simulations the impact of molecular dynamics on drug design: applications for the characterization of ligand-macromolecule complexes molecular dynamics simulations and drug discovery the future of molecular dynamics simulations in drug discovery identification of drug binding sites and action mechanisms with molecular dynamics simulations assessing the performance of the mm/pbsa and mm/gbsa methods. . the accuracy of binding free energy calculations based on molecular dynamics simulations physical properties of the hiv- capsid from all-atom molecular dynamics simulations biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm online tools for protein ensemble pocket detection and tracking multiscale modeling in the clinic: drug design and development multiscale methods in drug design bridge chemical and biological complexity in the search for cures recent advances in fragment-based computational drug design: tackling simultaneous targets/biological effects monte carlo simulations of proton pumps: on the working principles of the biological valve that controls proton pumping in cytochrome c oxidase multiscale simulations of protein landscapes: using coarse-grained models as reference potentials to full explicit models realistic simulations of proton transport along the gramicidin channel: demonstrating the importance of solvation effects diverse strategies in drug discovery and development good practices in model-informed drug discovery and development: practice, application, and documentation advances in computational structure-based drug design and application in drug discovery expediting the design, discovery and development of anticancer drugs using computational approaches identification of potential crac channel inhibitors: pharmacophore mapping, d-qsar modelling, and molecular docking approach homology model versus x-ray structure in receptor-based drug design: a retrospective analysis with the dopamine d receptor new insights for drug design from the x-ray crystallographic structures of g-protein-coupled receptors an improved receptor-based pharmacophore generation algorithm guided by atomic chemical characteristics and hybridization types x-ray crystallographic structure of a teixobactin analogue reveals key interactions of the teixobactin pharmacophore design, synthesis and characterization of novel chromone based-copper (ii) antitumor agents with n, n-donor ligands: comparative dna/rna binding profile and cytotoxicity pharmacophore modeling and applications in drug discovery: challenges and recent advances searching for potential mtor inhibitors: ligand-based drug design, docking and molecular dynamics studies of rapamycin binding site best practices for qsar model development, validation, and exploitation rational drug design of antineoplastic agents using d-qsar, cheminformatic, and virtual screening approaches molecular docking and structure-based drug design strategies molecular docking as a popular tool in drug design, an in silico travel h-nmr of rh (nh ) phi + bound to d (tggcca) : classical intercalation by a nonclassical octahedral metallointercalator d pharmacophore modeling techniques in computer-aided molecular design using ligandscout Über den jetzigen stand der chemotherapie three-dimensional pharmacophoric pattern searching pharmacophore models and pharmacophore-based virtual screening: concepts and applications exemplified on hydroxysteroid dehydrogenases pharmacophore-based virtual screening identification of potential tumour-associated carbonic anhydrase isozyme ix inhibitors: atom-based d-qsar modelling, pharmacophore-based virtual screening and molecular docking studies revealing the macromolecular targets of complex natural products multi-objective molecular de novo design by adaptive fragment prioritization click chemistry for drug discovery legacy data sharing to improve drug safety assessment: the etox project multi-objective optimization methods in de novo drug design. mini rev using local models to improve (q) sar predictivity a multiscale simulation system for the prediction of drug-induced cardiotoxicity multiscale quantum chemical approaches to qsar modeling and drug design polypharmacology modelling using proteochemometrics (pcm): recent methodological developments, applications to target families, and future prospects prediction of protein kinase-ligand interactions through . d kinochemometrics chemoinformatics for medicinal chemistry: in silico model to enable the discovery of potent and safer anti-cocci agents fragment-based in silico modeling of multi-target inhibitors against breast cancer-related proteins quantitative structure-activity relationship: promising advances in drug discovery platforms machine learning method for knowledge discovery experimented with otoneurological data efficient toxicity prediction via simple features using shallow neural networks and decision trees application of interpretable artificial neural networks to early monoclonal antibodies development computational prediction of anti hiv- peptides and in vitro evaluation of anti hiv- activity of hiv- p -derived peptides in silico de novo design of novel nnrtis: a bio-molecular modelling approach automated prediction of hiv drug resistance from genotype data cct is a novel potent and selective chk inhibitor with oral efficacy alone and in combination with genotoxic anticancer drugs qsar based model for discriminating egfr inhibitors and non-inhibitors using random forest using random forest and decision tree models for a new vehicle prediction approach in computational toxicology identification of human drug targets using machine-learning algorithms a comparative study of family-specific protein-ligand complex affinity prediction based on random forest approach ptml model for proteome mining of b-cell epitopes and theoretical-experimental study of bm protein sequences from colima computational modeling in nanomedicine: prediction of multiple antibacterial profiles of nanoparticles using a quantitative structure-activity relationship perturbation model ptml combinatorial model of chembl compounds assays for multiple types of cancer big data deep learning: challenges and perspectives deep learning in neural networks: an overview in imagenet classification with deep convolutional neural networks deep learning recognizes ftd-resistant isolated cancer cells of colon cancer deep learning-based prediction of drug-induced cardiotoxicity data integration: challenges for drug discovery a computational model of induced pluripotent stem-cell derived cardiomyocytes incorporating experimental variability from multiple data sources multi-scale modeling of the cardiovascular system: disease development, progression, and clinical intervention binding pocket optimization by computational protein design molecular mechanism matters: benefits of mechanistic computational models for drug development parameterization for in-silico modeling of ion channel interactions with drugs a molecularly detailed nav . model reveals a new class i antiarrhythmic target a computational model to predict the effects of class i anti-arrhythmic drugs on ventricular rhythms cibrián-uhalte, e. the chembl database in chemoinformatics in anti-cancer chemotherapy: multi-target qsar model for the in silico discovery of anti-breast cancer agents rational drug design for anti-cancer chemotherapy: multi-target qsar models for the in silico discovery of anti-colorectal cancer agents unified multi-target approach for the rational in silico design of anti-bladder cancer agents. anti-cancer agent multiscale modeling of ductal carcinoma in situ a multiscale agent-based model of ductal carcinoma in situ mathematical modeling in cancer nanomedicine: a review mathematical modeling in cancer drug discovery mathematical modeling of tumor organoids: toward personalized medicine an in silico approach for assessing drug efficacy within a tumor tissue current advances in mathematical modeling of anti-cancer drug penetration into tumor tissues multiscale models of pharmacological, immunological and neurostimulation treatments in alzheimer's disease computer modeling of epilepsy: opportunities for drug discovery modeling neurological disease processes using process algebra computational drug networks: a computational approach to elucidate drug mode of action and to facilitate drug repositioning for neurodegenerative diseases gonzález-díaz, h. perturbation theory/machine learning model of chembl data for dopamine targets: docking, synthesis, and assay of new l-prolyl-l-leucyl-glycinamide peptidomimetics multi-target mining of alzheimer disease proteome with hansch's qsbr-perturbation theory and experimental-theoretic study of new thiophene isosters of rasagiline multioutput perturbation-theory machine learning (ptml) model of chembl data for antiretroviral compounds de novo computational design of compounds virtually displaying potent antibacterial activity and desirable in vitro admet profiles speeding up early drug discovery in antiviral research: a fragment-based in silico approach for the design of virtual anti-hepatitis c leads enabling the discovery and virtual screening of potent and safe antimicrobial peptides. simultaneous prediction of antibacterial activity and cytotoxicity ann multiscale model of anti-hiv drugs activity vs. aids prevalence in the us at county level based on information indices of molecular graphs and social networks key: cord- -xvba mqq authors: wang, l.-y.; cui, j.-j.; ouyang, q.-y.; zhan, y.; wang, y.-m.; xu, x.-y.; guo, c.-x.; yin, j. title: genetic profiles in pharmacogenes indicate personalized drug therapy for covid- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: xvba mqq background: the coronavirus disease (covid- ) has become a global pandemic currently. many drugs showed potential for covid- therapy. however, genetic factors which can lead to different drug efficiency and toxicity among populations are still undisclosed in covid- therapy. methods: we selected potential drugs for covid- therapy (dcts) from clinical guideline and clinical trials databases. pharmaco-genes related to these therapeutic drugs were included. variation information in , exomes were collected for racial differences analyses. the expression level of pharmaco-genes in single cell resolution was evaluated from single-cell rna sequencing (scrna-seq) data of healthy adults. results: pharmacogenes, including cyp a , abcb , slco b , alb, cyp a , were involved in the process of more than multi dcts. potential drug-drug interactions (ddi) of dcts were predicted, while of them have been reported. racial discrepancy of common nonsynonymous mutations was found in pharmacogenes including: vdr, itpa, g pd, cyp a and abcb which related to dcts including ribavirin, -interferon, chloroquine and lopinavir. moreover, ace , the target of -ncov, was only found in parts of lung cells, which makes drugs like chloroquine that prevent virus binding to ace more specific than other targeted drugs such as camostat mesylate. conclusions: at least drugs for covid- therapy with predictable pharmacogenes should be carefully utilized in risk races which are consisted of more risk allele carriers. at least drugs with potential of ddis are reported to be affected by other ddis, they should be replaced by similar drugs without interaction if it is possible. drugs which specifically targeted to infected cells with ace such as chloroquine are preferred in covid- therapy. in the end of last year, wuhan (the capital city of hubei province in china) reported a new unknown viral pneumonia [ ] . subsequent next generation sequencing identified that it was caused by a novel coronavirus which named as novel coronavirus ( -ncov) [ ] . clinical characteristics reporting in china revealed that most patients are mild and moderate [ , ] . however, severe cases progress rapidly to acute respiratory distress syndrome (ards), shock, multiple organ failure and even death. the national health commission of china released at least seven versions of guideline of diagnosis and treatment of pneumonitis caused by covid- [ ] . oxygen therapy, mechanical ventilation and drug therapy are recommended as major treatments, so that several drugs may be utilized simultaneously in such conditions. it is noteworthy that the individual difference of drug treatment is mentioned for a special in the guideline. this promotes the importance of personalized therapy for covid- patients. of life-threatening severe hemolysis after taking chloroquine [ ] . as one of the most common human enzyme defects, genetic variants affecting g pd activity show remarkably individual and ethnical differences [ ] . thus, it should be widely informed that chloroquine needs precision medicine. in addition to chloroquine, a number of other drugs are also used in the covid- therapy. with the rapid spread of this disease in the worldwide, these drugs will be used in patients with different ethnic backgrounds. however, the genetic variants potentially affect their effects and safety are not still available. their distributions in different populations in the world are also not indicated. this may constitute an obstacle for successful treatment and control of this disease. pharmacogenetics (pgx) investigates the affection of genetic variants on drug effects and safety. in the current study, we provide a pgx landscape of drugs with potential to be used in the covid- treatment. candidate choices from different drugs based on pharmacogenetic analyses were provided. drugs and their clinical trial information for covid- treatment were col lected from guideline of diagnosis and treatment of pneumonitis caused by c ovid- (version . ) (http://www.nhc.gov.cn/yzygj/s p/ / c a dfe cef dc f eb .shtml), clinical trails (https://clinicaltrials.gov) and chine se clinical trail registry (http://www.chictr.org.cn). the pharmacogenes related elines. genetic variation data on each of the genes were retrieved from gnom ad database (http://gnomad.broadinstitute.org/, version: . . ). finally, single-cel l rna sequencing (sc-rna seq) data from two organs (liver (gse , n= ) and lung (gse , n= )) of healthy adults was download from geo da tabase (https://www.ncbi.nlm.nih.gov/gds). the raw count matrix (unique molecular identifier counts per gene per cell) was processed by seurat v [ ] . firstly, low-quality data was filtered out as following: ( ) cells expressed less than genes; ( ) genes expressed in less than two percent of cells. secondly, we calculated log (count-per-million + ) expression, and followed by normalization and standardization. thirdly, the cells were clustered. in detail, the highly variable genes were identified and cells were then clustered through embedding them into a graph structure in principal component analysis space. the parameter resolution was set as . to identify only major cell types. the clustered cells were then projected onto a two-dimensional space and visualized. finally, the differentially expressed genes on each cluster were identified. they were used to annotate and merge the cell clusters according to curated known cell markers (http://biocc.hrbmu.edu.cn/cellmarker/index.jsp). all genetic variations were annotated by allele frequency, location and function in different populations using annovar (version: oct ) (table s ). in the current study, populations were divided into eight categories: african, latino, east asian, south asian, finnish, non-finnish european, ashkenazi jewish and other. based on the location and functions, all mutations were divided into categories: frameshift, non-frameshift, synonymous single nucleotide polymorphism (snp), all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . nonsynonymous snp, stop-gain, untranslated regions, intronic and others (including splicing, upstream of a gene, downstream of a gene, stop-loss and ncrna_exonic mutations). functional nonsynonymous mutations were predicted by provean (http://provean.jcvi.org/). the drug-gene network was constructed by cytoscape software (version: . . ) [ ] . in the current study, a total of drugs were collected in our study, including agents being used clinically, undergoing clinical trials, only confirmed in vitro experiments or simply with the therapeutic potential. they could be classified into nine categories according to their functions: viral toxicity ( . %) and their sensitizers ( . %), inhibiting virus invasion ( . %), improving lung function ( . %), maintaining tissue and organ function balance ( . %), anti-hypoxic ( . %), anti-septic shock ( . %), anti-secondary infection ( . %) and others ( . %) ( figure a ). among them, antiviral drugs were mostly important and account for . % in all drugs (including virus invasion inhibitors, viral toxicity and their sensitizers). as of march , , a total of clinical trials have been conducted to study the safety and efficacy of antiviral drugs. and most of these clinical trials are all ongoing and have not been completed. there are six clinical trials involving remdesivir, the earliest closed one involving participants will be completed on april , (nct ). as many as clinical trials related to chloroquine, and two clinical trials for mild and common patients and severe patients will be completed on april , (chictr , chictr ), each clinical trial includes a total of participants. as for hydroxychloroquine, two of the nine clinical trials have ended on february , (chictr , chictr ), and the results have not yet been available. in addition, the clinical trials of lopinavir ( ) and ritonavir ( ) will both be completed as early as march , . information of other categories of drugs can be found in figure s . genetic variation is one of the most powerful biomarkers to guide personalized therapy. thus, it is important to systematically identify pharmacogenes which could affect response and toxicity of these drugs. a drug-gene network which demonstrating the connection of each drug and pharmacogene was constructed ( figure a ). after excluding drugs without pharmacogene data, a total of drugs and genes were connected. based on the nodes in this network, we could easily find genes shared by multiple drugs. they could be the mediator of drug-drug interactions (ddis) ( figure b ). in addition, their genetic variations could potentially affect response and toxicity author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . another information obtained from this network was that there were actionable pgx biomarkers for three drugs based on cpic or dpwg guidelines. they were g pd and chloroquine hemolysis toxicity, vitamin d receptor (vdr) and ribavirin efficacy, inosine triphosphatase (itpa) and ribavirin/α-interferon anemia risk, angiotensin i converting enzyme (ace) and captopril response. we recommend that these genes should be preemptively tested before drug treatment. we next investigated the allelic frequency, location and function of genetic mutations for all pharmacogenes in different populations. a total number of , variations in these genes were found in , subjects from gnomad databases. although the mutations distributed in all gene regions, the most common mutation types were intronic, non-synonymous and synonymous mutations ( figure a ). it should be noteworthy that . % of the mutations were non-synonymous, which is the major types of functional pgx variants. they could be the explanation of individual and ethnic difference for covid- drug treatment. to learn the distribution of mutations in pharmacogenes in more detail. the fractions of different mutation types were provided for each gene. they were summarized based on the gene functions in figure s . the frequencies of these variations were indicated in figure b , they showed similar distributions. however, there were remarkably differences for the total number of mutations. then, we explored the allelic frequencies of all mutations. as indicated in figure b , . % of the mutations were rare genetic variants with minor allele frequency (maf) lower than %. this result emphasized again the importance of pharmacogenomic scale test before treatment. we further analyzed mutations with different maf stratified by populations. except african population, there was no all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . significant difference of maf distribution between the remained seven races ( figure c ). the african individuals were higher genetically variable for these pharmacogenes, since they showed more percentages of common mutations (maf> %). this result indicated that they had higher requirement for pgx test. taken together, these results indicated that covid- pharmacogenes were highly genetically variable, especially for african population. thus, personalized drug treatment and pgx test is needed for different patients. in addition, these mutations distributed in all gene regions and most of them were rare variations ( figure d ). therefore, sequencing technologies is recommended for the preemptive test. to provide clinical guide for covid- treatment, we analyzed the actionable and key pgx biomarkers in detail. there are four paired actionable pharmacogenes and drugs: vdr and ribavirin, itpa and ribavirin/α-interferon, g pd and chloroquine, ace and captopril. ribavirin is a classic antivirus drug which has been involved in the guideline for -ncov therapy as well. vdr polymorphism is associated with its efficacy ( figure a ). there is only a common non-synonymous polymorphism m t (rs ) that could hamper the vdr activity and the m t carriers showed lower efficacy. the frequency of this mutation is over . in all populations ( figure a ), which means more than half patients will be resistant to this drug. -interferon is usually utilized in combination with antivirus drugs such as ribavirin in covid- treatment. itpa polymorphism was reported to be associated with the anemia risk in ribavirin/α-interferon treatment ( figure b ). the functional mutation p t (rs ) reduced the activity of the enzyme encoded by itpa. the p t carriers showed lower anemia risk. another common non-synonymous all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . polymorphism e g (rs ) is also found in itpa ( figure b ), while its effect has not been revealed. the p t was more identified in asians (both of east asians and south asians, figure b ). therefore, asians may have a higher anemia risk in ribavirin/α-interferon treatment. chloroquine has been utilized for -ncov therapy as guideline referred. g pd polymorphisms has been reported to be associated with hemolysis risk ( figure c ). there are two important variants v m (rs ) and n d (rs ), which could decrease the function of g pd. the v m and n d carriers showed higher hemolysis risk for chloroquine treatment. in addition to these two mutations, we further identified three other common non-synonymous polymorphisms in all populations. among them, the mutation s f (rs ) was also reported to decrease the activity of g pd, which indicated that this mutation could increase the hemolysis risk as well. what's more, both v m and n d were more common in the african population ( figure c ), suggesting that chloroquine is not recommended in african patients. lopinavir and ritonavir are used together to reduce their metabolism in liver through inhibiting the activity of cyp a , a very important enzyme which could metabolize about half of dcts. the cyp a polymorphism l p (rs , cyp* b) was linked with increased cyp a activity, thus facilitate the metabolism of drugs like lopinavir. l p carriers may need to increase the dosage of these drugs compared with wild type patients ( figure d ). another common non-synonymous polymorphism r q in cyp a was still not be reported with cyp a phenotype now ( figure d ). l p was more in east asians ( figure d ), indicated that east asians may metabolize lopinavir and ritonavir more rapidly. as we mentioned above, abcb can pumping out many drugs across the all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . cellular membrane thus alleviate the efficacy. in our drug list, drugs like lopinavir can be transported out of cells by abcb , and its efficacy can be reduced ( figure e ). we found there are common non-synonymous polymorphisms in abcb ( figure e ). among them, s t, s a (rs ), n d (rs ) and s n (rs ) were reported as deleterious mutations, which can increase the drug concentration through decreasing the efflux of abcb . s a carriers were found in all populations with high frequency, which is up to % in africans. s t carriers were almost east asians, while n d carriers were europeans ( figure e ). these patients may be more response to these drugs transported by abcb . in addition, ace inhibitors (acei) can be used to improve the lung function in covid- treatment. it was widely reported that ace polymorphisms could affect acei therapeutic response ( figure f ). the most important variant was i/d (rs ) polymorphism, which was a low frequency indel mutation causing low enzyme expression. the d allele carriers showed better response for acei treatment. in addition to this mutation, we further identified common non-synonymous polymorphisms in all populations ( figure f ). although there is no related report about these variations, g v (rs ), a t (rs ), y c (rs ), r w (rs ) and r w (rs ) were predicted to be loss-of-function mutations by provean. thus, we speculated that patients harboring these single nucleotide polymorphisms (snps) were poor responders of this drug. based on their mafs in different populations, rs were more identified in the east asian population ( figure f ). thus, we concluded that east asian patients could be more resistance to this drug. in summary, african patients could be more hazardous to chloroquine, asian patients have a higher risk of anemia in ribavirin/α-interferon treatment and are more all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. recently, sc-rna seq technology was rapidly developed. it is capable to specifically profile cell populations at the single-cell resolution. thus, it transformed many fields of genomic research. here, we tried to explore the utility of sc-rna seq on personalized covid- treatment. we collected healthy adults sc-rna seq data from lung and liver tissues. lung is the major -ncov attacking organ. thus, we analyzed the expression of virus or drug target genes in different lung cells ( figure a ). based on the current findings, ace and transmembrane serine protease (tmprss ) were the two major targets of both virus and some drugs. as indicated in figure b , tmprss was almost expressed in all kinds of cells. in our enrolled drugs, camostat mesylate act as a trypsin like protease inhibitor and could be potentially used to anti-virus by inhibiting tmprss activity. due to the ubiquitous expression of its target, camostat mesylate could also cause potential toxicity when exerting therapeutic effect. on the other hand, ace was mainly expressed in type i pneumocyte, type ii pneumocyte, foxn + , club and mast cells. chloroquine phosphate can change the ace structure, or inhibit the binding of coronavirus s protein with ace . based on these data, chloroquine was more effectively targeting drugs with less side effects ( figure c ). sc-rna seq data in lung tissue suggested that ace targeting drugs (chloroquine) were superior for covid- treatment than tmprss targeting drugs (camostat mesylate). most drugs can be metabolized in the liver. we next analyzed the expression of all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint drug metabolic enzymes in different liver cells ( figure a ). cyp a , cyp e , cyp c , cyp c , cyp b , cyp d , cyp a and cyp a were the major cyp enzymes responsible for drugs metabolism. their expression was investigated ( figure b, d) . in addition to hepatocytes, it was unexpected to found that some enzymes were also found to be highly expressed in immune cells. based on the expression distribution, we categorized all enzymes into two groups: ( ) figure c ). therefore, leflunomide may be not recommended while other immunosuppressive drugs are available. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . since there are many candidate drugs for covid- therapy, it is important to make a better choice for each patient. can be found in table . as a large part of drugs are metabolized by cyp in liver, the activity of cyp family may impact many drugs metabolization rate. due to the activity difference exists among populations, the genotypes of important alleles in cyp family should be detected after diagnosis, then treatment strategies for these drugs can be suggested in these patients. meanwhile, ddi should also be considered as many drugs may be utilized together during covid- treatment as mentioned above. for instance, drugs like lopinavir and ritonavir can inhibit the activity of cyp a , thus the drugs dosage should be adjusted when combined with drugs metabolized by cyp a . drugs that were metabolized by cyp family in covid- therapy were listed in table . in addition, when chloroquine is utilized, drugs like losartan which can increase the concentration of chloroquine may be replaced by valsartan. validated ddis between drugs for covid- treatment are given in table . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint finally, based on current researches, -ncov can only infect cells with ace . as indicated above, ace is not expressed in all cells, while tmprss can be found in large part of cells. so that drugs like camostat mesylate is not recommended compared to ace specific inhibitors due to the side effect caused by tmprss in uninfected cells. this study firstly focused pharmacogenetics and provided clinical suggestions for precision medicine in covid- treatment systematically. dcts were showed to associated with at least one gene. and drug-gene network highlighted multi drug related genes in covid- treatment. meanwhile, inter-racial variances in drug efficacy were found related to nonsynonymous mutations among different races, which indicated that racial special strategy for covid- therapy should be considered during the outbreak of covid- worldwide. since nonsynonymous mutations and gene expression levels can both affect the dcts efficacy and toxicity as mentioned above, optimized therapy strategies are needed to benefit these affected patients all over the world. in this study, we give three suggestions for dcts as follows. firstly, drugs in cpic guideline including ribavirin, α -interferon, chloroquine and captopril should be utilized with genetic detections as guided. in this situation, adequate gene detection kits should be prepared in africa for covid- therapy, as related genetic variation frequency is higher in afrs than other countries. secondly, drug such as chloroquine whose efficacy and toxicity can be determined by nonsynonymous mutations, may be preferred in special population with lower frequency of risk alleles such as finnish and non-finnish european. otherwise, alternative drug for chloroquine can be used in high risk populations. thirdly, for drugs that can inhibit the activity of cyp , ddi should be prevented in covid- all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . treatment (table ) . when inhibitors of cyp were utilized, the dosage of these drugs should be adjudged carefully to avoid overdosage. although precision medicine should be considered in covid- treatment, there are still many challenges to face. the first challenge is that pharmacogenes of many drugs for covid- are still not clear, due to that many newly developed drugs are still under clinical trials, their pharmacogenes can hardly be fully identified currently. a potential solution is to predict candidate pharmacogenes according to the chemical structure of a new drug by the artificial intelligence and machine learning algorithm [ , ] . then related molecular biological studies can be conducted to validate these candidate pharmacogenes rapidly. then the period of pharmacogenes identification can be shorted. the second challenge is that some serious nonsynonymous mutations can be hardly identified in current pharmacogenetics studies, due to the frequencies of many nonsynonymous mutations are lower than %. that's why the nonsynonymous mutations we presented above are common variants. if there are only a few patients carried a serious nonsynonymous mutation, related study can hardly be conducted to identify this mutation because of the limited sample size. however, as we indicated in figure , most of nonsynonymous mutations are rare variations. therefore, a reliable way to predict potential pharmacogenetic variation is also important to improve the efficacy or avoid serious toxicity [ ] . the third challenge is that a suitable strategy for precision medicine in covid- treatment is still lack. a well-designed gene detection panel is important to fully characterized the patients' genotypes which relate to the drug effects [ ] . then the best treatment regimen can be utilized for this patient, and both of the efficacy and the safety can be guaranteed. in our study, feasible suggestions were given, and it still can be much improved with the development of some pharmacogenetic studies in the future. last but not least, the expression level of all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . pharmacogenes in patients can hardly be detected in single cell resolution during covid- treatment, although the expression level of these genes can be associated with drug efficacy or toxicity. a potential solution is to build a systematic pharmacogenes expression map in single cell level among different populations in different age stages [ ] . this map would be helpful for personalized medicine in the future. this work was supported by the national natural science foundation of china the authors declare no conflict of interest. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . the color of the column is as same as the color of the drug, which indicated the category of the drug. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan guidelines for the diagnosis and treatment of novel coronavirus ( -ncov) infection by the national health commission (trial version building evidence and measuring clinical outcomes for genomic medicine the shift to personalised and population medicine the haemolytic effect of various regimens of of the world health organization association of g pd with lower comprehensive integration of single-cell data therapeutic options for the novel coronavirus ( -ncov host genetic variants in the pathogenesis of hepatitis c demonstrating the pharmacogenetic effects of angiotensin-converting enzyme inhibitors on long-term prognosis of diastolic heart failure cyp a -mediated lopinavir bioactivation and its inhibition by ritonavir discovery and preclinical validation of drug indications using compendia of public gene expression data computational and analytical challenges in single-cell transcriptomics human immunodeficiency virus protease inhibitor ritonavir inhibits cholesterol efflux from human macrophage-derived foam cells structure analysis of the receptor binding of -ncov admetsar . : web-service for prediction and optimization of chemical admet properties applications of machine learning in drug discovery and development deep learning sequence-based ab initio prediction of variant effects on expression and disease risk complexity of genome sequencing key: cord- - nvi rq authors: arshad, usman; pertinez, henry; box, helen; tatham, lee; rajoli, rajith kr; curley, paul; neary, megan; sharp, joanne; liptrott, neill j; valentijn, anthony; david, christopher; rannard, steve p; o’neill, paul m; aljayyoussi, ghaith; pennington, shaun; ward, stephen a; hill, andrew; back, david j; khoo, saye h; bray, patrick g; biagini, giancarlo a; owen, andrew title: prioritisation of anti‐sars‐cov‐ drug repurposing opportunities based on plasma and target site concentrations derived from their established human pharmacokinetics date: - - journal: clin pharmacol ther doi: . /cpt. sha: doc_id: cord_uid: nvi rq there is a rapidly expanding literature on the in vitro antiviral activity of drugs that may be repurposed for therapy or chemoprophylaxis against sars‐cov‐ . however, this has not been accompanied by a comprehensive evaluation of the target plasma and lung concentrations of these drugs following approved dosing in humans. accordingly, ec( ) values recalculated from in vitro anti‐sars‐cov‐ activity data was expressed as a ratio to the achievable maximum plasma concentrations (cmax) at an approved dose in humans (cmax/ec( ) ratio). only of the analysed drugs achieved a cmax/ec( ) ratio above . a more in‐depth assessment demonstrated that only nitazoxanide, nelfinavir, tipranavir (ritonavir‐boosted) and sulfadoxine achieved plasma concentrations above their reported anti‐sars‐cov‐ activity across their entire approved dosing interval. an unbound lung to plasma tissue partition coefficient (k(p)u(lung)) was also simulated to derive a lung cmax/ec( ) as a better indicator of potential human efficacy. hydroxychloroquine, chloroquine, mefloquine, atazanavir (ritonavir‐boosted), tipranavir (ritonavir‐boosted), ivermectin, azithromycin and lopinavir (ritonavir‐boosted) were all predicted to achieve lung concentrations over ‐fold higher than their reported ec( ). nitazoxanide and sulfadoxine also exceeded their reported ec( ) by . ‐ and . ‐fold in lung, respectively. this analysis may be used to select potential candidates for further clinical testing, while deprioritising compounds unlikely to attain target concentrations for antiviral activity. future studies should focus on ec( ) values and discuss findings in the context of achievable exposures in humans, especially within target compartments such as the lung, in order to maximise the potential for success of proposed human clinical trials. coronavirus (covid- ) is a respiratory disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ) infection. fever, a persistent cough and respiratory symptoms are common, with some patients reporting vomiting, nausea, abdominal pains and diarrhoea ( ) . to date, no specific treatment is available, and this has resulted in significant morbidity and mortality globally. according to the international clinical trials registry platform search portal, clinical trials for covid- have been registered ( ) . this rapidly expanding pandemic warrants the urgent development of strategies, particularly to protect people at high risk of infection. repurposing currently available drugs that have been utilised clinically with a known safety profile, is the quickest way to address this serious unmet clinical need. antiviral drugs are urgently required for treatment of patients with mild/moderate disease to prevent the worsening of symptoms and reduce the burden upon healthcare systems. however, a different approach is likely to be needed for patients that are already in a critical state, due to the immune dysregulation which is so apparent in severe cases ( ) . previous investigations have shown that the entry by sars-cov- occurs via the angiotensin converting enzyme (ace ) receptor ( ). a study on normal lung tissue showed that % of ace -expressing cells were alveolar epithelial type ii cells ( ), highlighting the lungs as the primary target organ that facilitate viral invasion and replication. furthermore, the ace receptor is also highly expressed in gastrointestinal epithelial cells, with sars-cov- rna observed to be present in stool specimens of patients during infection ( , ) . a recent retrospective analysis of patients with laboratory-confirmed covid- also indicated that sars-cov- infects human kidney tubules and induces acute tubular damage in some patients ( ). furthermore, - % of patients with covid- exhibit liver comorbidities ( ). of note is an observation of sars and middle east respiratory syndrome (mers) having a tropism to the gastrointestinal tract ( ) and causing liver impairment in addition to respiratory disease. the genomic similarity between sars-cov- and sars-cov ( . % sequence identity) would imply that the current virus would act in a similar manner and be present within the body systemically ( - ). therefore, treatment options that provide therapeutic concentrations of drug(s) within the systemic circulation and other affected organs are likely to be required. in the absence of a vaccine, antiviral drugs could also be deployed as chemoprophylaxis to protect against infection and would present an essential tool for protecting healthcare staff and other key workers, as well as household contacts of those already infected. for chemoprevention drugs will need to penetrate into the multiple sites where sars-cov- infection occurs, and do so in sufficient concentrations to inhibit viral replication ( ). this may include the mucous membranes present in the nasal cavity and throat, the this article is protected by copyright. all rights reserved ocular surface, tears and the upper respiratory tract/lungs ( , ). however, therapeutic concentrations may not be needed in the systemic circulation for chemoprophylaxis, but this is yet to be determined. although difficult and scarcely studied, work in animals has shown that the size of the inoculum of other respiratory viruses such as influenza is associated with the severity of the resultant disease ( , ). reports with sars-cov- indicate that higher viral loads are indicative of poorer prognosis and correlate with the severity of symptoms, with viral load in severe cases reported to be times higher than that of mild cases ( , ) . in light of this, even if a chemoprophylactic drug reduced inoculum size without completely blocking transmission, major benefits for morbidity and mortality may still be achievable. many ongoing global research efforts are focussed on screening the activity of existing compounds in vitro in order to identify candidates to repurpose for sars-cov- . however, current data have not yet been systematically analysed in the context of the plasma and target site exposures that are achievable after administration of the approved doses to humans. the purpose of this work was to evaluate the existing in vitro anti-sars-cov- data to determine and prioritise drugs capable of reaching antiviral concentrations within the blood plasma. accepted physiologically based pharmacokinetic (pbpk) equation were also used to predict the expected concentration in lung ( - ), in order to assess the potential of these drugs for therapy in this key disease site and the potential for chemoprevention. to identify compounds and their relevant potency and pharmacokinetic data, we performed a literature search on pubmed, google scholar, biorxiv, medrxiv, and chemrxiv. the following search terms were used for in vitro activity data -(covid- or sars-cov- ) and (ec or ic or antiviral). for pharmacokinetic data (cmax or pharmacokinetics) was used along with the drug name for drugs with reported anti-sars-cov- activity (up to th april ). further clinical pharmacokinetic data were obtained from the food and drug administration (fda), the european medicines agency (ema) and through publications available online. inhaled medications were excluded from all analyses because the purpose was to assess systemically administered medicines. an indication of the degree to which candidate drugs are expected to accumulate in lung (a presumed site of primary efficacy and for prevention of sars-cov- infection) was provided by calculation of unbound accepted article lung to plasma tissue partition coefficient (k p u lung ) according to the methodology of rodgers and rowland ( - ). equations therein were implemented in the r programming environment (version . . ) and are replicated in the supplementary methods. briefly, the physicochemical properties of the drug (pka, log p, classification as acid/base/neutral) and in vitro drug binding information (fraction unbound in plasma, blood to plasma ratio), in combination with tissue-specific data (lipid content, volumes of intra/extracellular water etc.) were used to predict tissue k p u values. measured log p and pka values were used where available but substituted with calculated values where necessary and all parameter values used for the calculations for each drug, and their references/sources, are provided in supplementary table . k p u lung values were converted to k p_lung by multiplying by fraction unbound in plasma to allow estimation of lung exposure from in vivo measurements of plasma cmax concentration. a similar analysis was conducted to assess the tissue distribution into other tissues. in the absence of observed tissue distribution data, the rodgers and rowland method is an accepted means to provide initial estimates of tissue partitioning for pbpk modelling. however, there are known limits on accuracy with predicted k p u by the rodgers and rowland method generally reported to be within - fold of observed tissue k p u values ( - ). this was confirmed for a limited number of drugs within the current dataset for which measure k p values for lung were available from animal studies in the literature (see data analysis below). since in the majority of papers only an ec value was available, concentration-response data were digitised using the web plot digitizer® software. graphs were then replotted in sigmaplot . (systat software, inc.) and curves were fitted to confirm ec values and determine ec values. a cmax/ec and cmax/ec ratio was then calculated for each drug for which previous evidence of clinical use in humans and availability of human pharmacokinetic data were available. lung and other tissue k p u values were used in combination with reported cmax values to derive an estimate of lung exposure at cmax for each drug. for a subset of molecules, the absence of available physicochemical or plasma protein binding parameters prohibited derivation of a k p u estimate. for the remaining drugs, a lung (or other tissue) cmax/ec and lung cmax/ec was calculated. published plasma concentration-time data for the most promising candidates were then digitised (where available) and replotted to visually represent human pharmacokinetics relative to the calculated ec and ec data. equivalence between values for the predicted lung k p and those observed in vivo was undertaken for drugs with available animal lung and plasma concentration data. for this analysis, animal lung concentration data were available for anidulafungin (rat), bazedoxifene (rat), chloroquine (three albino rat studies), favipiravir (monkey), hydroxychloroquine (two albino rat studies), nitazoxanide (mouse), tamoxifen (rat), accepted article cyclosporine (rat), ritonavir (rat), azithromycin (mouse), dolutegravir (mouse), gilteritinib (albino rat), and lopinavir (rat) ( - ). agreement between the predicted and measured k p was assessed by simple linear regression and by constructing bland-altman plots, the limits of agreement (mean ± standard deviation) were included in these plots as previously described ( ). we identified key studies that detailed the antiviral activity of compounds ( nitazoxanide. however, it should be noted that for amodiaquine, atazanavir, chloroquine, hydroxychloroquine, lopinavir, mefloquine, nelfinavir, remdesivir and toremifene, more than one ec value had been reported across the available literature and these were not always in agreement ( figure a ). moreover, this variability in reported ec values sometimes resulted in cmax/ec ratios giving a different estimation of the likely value of the molecule. meaning that for the same drug, the cmax/ec ratio could be above or below ( figure b) table . this article is protected by copyright. all rights reserved simulated exposure relative to reported anti-sars-cov- activity in lung and other tissues lung k p u was simulated for all molecules for which the necessary physicochemical properties and in vitro drug binding information were available. regression and bland-altman plots were first used to assess the agreement between predicted lung k p and that observed in previously published animal studies for drugs with available prior data. good agreement was observed across the available drugs with the exception of choloroquine. an r = . was observed in linear regression when chloroquine was excluded, but decreased to r = . when included (supplementary figure a) . similarly, good agreement between measured and predicted kp was observed by bland-altman analysis for all data points with the exception of one chloroquine measurement (supplementary figure b ). k p u lung was then used along with fraction unbound in plasma (fu) and plasma cmax values to calculate a predicted cmax/ec ( figure ) and cmax/ec in lung (data not shown). tissue cmax/ec ratios are also shown for other tissues in figure . for drugs, ebselen, merimepodib, niclosamide and remdesivir, the fraction unbound data were unavailable. for other drugs, benztropine, indinavir, loperamide, nelfinavir, saquinavir and toremifene, the blood to plasma ratios were unavailable. for a further drugs, camostat, emetine, fluspirilene and umifenovir, both fraction unbound and blood to plasma ratios were unavailable. therefore, these drugs were excluded from the analysis. a total of drugs with available data were predicted to give concentrations in lung above at least one of their reported ec against sars-cov- ( figure ) and of these were predicted to exceed their ec by more than -fold. the rank order of lung cmax/ec ratio was chloroquine > atazanavir (ritonavir boosted) > tipranavir (ritonavir boosted) > hydroxychloroquine > mefloquine > ivermectin > lopinavir (ritonavir boosted) > azithromycin > nitazoxanide > ritonavir > gilteritinib > amodiaquine > imatinib > oxprenolol (data excluded due to this analysis only being possible for of the drugs). this article is protected by copyright. all rights reserved the systematic development of mechanism-based inhibitors for key targets involved in viral replication or pathogenesis is likely to result in highly effective and safe medicines in the coming years. however, the repurposing of already approved medicines in antiviral treatment or chemoprevention strategies is undoubtedly the fastest way to bring forward therapeutic options against the urgent unmet need posed by sars-cov- . a range of different drugs and drug classes have been demonstrated to display varying degrees of antiviral activity against sars-cov- in vitro, and many of these drugs are already licenced for use in humans for a range of indications. however, currently the data emerging from global screening efforts are not being routinely benchmarked and prioritised against achievable concentrations after administration of doses proven to have acceptable safety profiles in humans. the current analysis indicates that only drugs with reported antiviral activity are likely to achieve plasma exposures above that required for antiviral activity for at least some of their dosing interval. notably, neither chloroquine, hydroxychloroquine nor lopinavir/ritonavir exhibited a sustained plasma concentration above their reported sars-cov- ec across their reported dosing interval. ultimately, the implications of this for therapy will depend upon whether systemic suppression is a prerequisite for a reduction in morbidity or mortality, but this does raise some concern for ongoing trials with these drugs (chloroquine: nct ; nct , hydroxychloroquine: nct ; nct ; nct and lopinavir/ritonavir: nct ; nct ; nct ). however, the predicted lung accumulation rather than plasma exposure may provide some therapy advantage and/or give more reassurance for ongoing chemoprevention trials. this article is protected by copyright. all rights reserved plasma pharmacokinetic exposure above their lowest reported ec and derived ec (where available) for the duration of their approved dose and dosing interval. nitazoxanide is an antiprotozoal drug that has previously been demonstrated to display broad antiviral activity against human and animal coronaviruses ( ) as well as various strains of influenza ( , ). importantly, nitazoxanide is rapidly metabolised to tizoxanide in humans and this active metabolite is being investigated against sars-cov- (nct and nct ). tizoxanide has been reported to exhibit similar activities to nitazoxanide for other viruses as well as other pathogens ( - ). the mechanism of antiviral action is not fully understood for nitazoxanide, but it has been reported to affect viral genome synthesis, prevent viral entry and interfere with the n-glycosylation and maturation of the influenza hemagglutinin ( - ). notably, the sars-cov- spike protein is also highly n-glycosylated ( ). this drug has also been shown to elicit an innate immune response that potentiates the production of type interferons ( , ) and a phase b/ clinical trial demonstrated a reduction in symptoms and viral shedding in patients with uncomplicated influenza ( ). the safety of nitazoxanide is well understood, but it has not been fully investigated during renal or hepatic impairment. the antiviral activity of nitazoxanide for sars-cov- requires further study but the existing data for this drug are encouraging. niclosamide is another antiprotozoal drug that exhibits broad antiviral activity due to its ability to perturb the phdependent membrane fusion required for virus entry ( ), but it was reported to have no impact upon the attachment and entry of sars-cov- ( ). for mers-cov, niclosamide was observed to inhibit skp activity impairing viral replication ( ). niclosamide has been reported to be well tolerated and does not influence vital organ functions ( ). however, it has low aqueous solubility and poor oral bioavailability ( ) and, despite a higher reported sars-cov- potency ( ) than nitazoxanide ( ), the cmax/ec ratio was slightly lower. there is a paucity of published pharmacokinetic data for niclosamide and this prohibited a thorough investigation of exposures in relation to activity over its entire dosing interval. both nitazoxanide and niclosamide have also been reported to be potent antagonists of tmem a, calciumactivated chloride channels that modulate bronchodilation ( ). tipranavir and nelfinavir are hiv protease inhibitors ( ) and both drugs ranked highly in terms of their cmax/ec ratio. moreover, a more in-depth analysis demonstrated that the concentrations across the dosing interval for both these drugs remained above the calculated ec values at approved doses and schedules. unlike nelfinavir, tipranavir has to be co-administered with a low dose of ritonavir to boost its pharmacokinetics via cyp a inhibition ( ). since ritonavir itself has been reported to exert anti-sars-cov- activity, this could be advantageous, but would need to be balanced against the much higher risk of drug-drug interactions that could negatively impact patient management. the implications of drug this article is protected by copyright. all rights reserved interactions have already been raised for this reason with lopinavir/ritonavir use for covid- ( ) and are likely to be exacerbated with the higher ritonavir dose needed for tipranavir. moreover, tipranavir has a black box warning from the fda for fatal and nonfatal intracranial haemorrhage as well as severe hepatotoxicity ( - ). the major route of metabolic clearance for nelfinavir is via cyp c and this pathway generates the m metabolite that retains activity against the hiv protease ( ). no data are available for inhibition of sars-cov- replication by the m metabolite but if active, this could provide an advantage for nelfinavir over tipranavir for covid- . conversely, while the analysis of pharmacokinetics relative to potency of these molecules against sars-cov- is encouraging, it should be noted that the reported in vitro activity for hiv ( , ) is far higher than that against sars-cov- and both drugs are highly protein bound ( , ). given that tipranavir and nelfinavir are associated with long-term toxicities ( , - ), there will be concern over giving even short-term exposure for covid- . sulfadoxine is another antimalarial drug that is usually administered in combination with pyrimethamine as a folic acid antagonist combination ( ). sulfadoxine inhibits the activity of dihydropteroate synthase within the malaria parasite, but its mechanism of action for sars-cov- is unclear. it should also be noted that the authors can find no data describing antiviral activity of this drug against other viruses. also, the concentrations used in the in vitro activity used in this analysis ( ) were not high enough to reach or calculate an ec value. therefore, like other molecules described in this manuscript, in vitro anti-sars-cov- activity should be repeated. notwithstanding, sulfadoxine plasma concentrations far above the reported ec are maintained in patients receiving a single mg dose (with mg pyrimethamine) for over days ( ). compared to some other reported molecules, sulfadoxine is not expected to have as high an accumulation in the lungs, but concentrations higher than its ec are estimated from the analysis of its lung k p u. therefore, if the reported antiviral activity is confirmed, this drug may offer opportunities for therapy and/or chemoprophylaxis. indomethacin is a nonsteroidal anti-inflammatory drug that is indicated for rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, acute painful shoulder or acute gouty arthritis. the recommended dose for acute gouty arthritis is mg three times a day and the pharmacokinetic exposure for this is shown in figure relative to the reported ec . indomethacin mechanism of action for sars-cov- remains elusive, but it was shown to inhibit translation of the vesicular stomatitis virus by activating protein kinase r leading to the phosphorylation of eukaryotic initiation factor- α-subunit ( ). this abrogated viral protein translation, leading to a dramatic inhibition of viral replication and infectious viral particle production. the reported in vitro antiviral activity data for indomethacin were insufficient to calculate an ec and this activity requires confirmation in other studies ( ). furthermore, the drug has a accepted article black box warning for serious cardiovascular and gastrointestinal events from fda so its use should be managed with caution ( ). considering that most of the impact of severe disease occurs in the lung and that this tissue may be a key site for transmission, the potential of candidate drugs to accumulate in lung tissue was considered. the lung k p predictions were validated across drugs for which previously reported animal plasma and lung concentrations were available, and showed good agreement for all agents other than chloroquine. the poor fit for chloroquine does highlight that the predictions may not be accurate for all of the drugs listed and this should be considered in interpretation. notwithstanding, the analysis of predicted lung cmax/ec ratio revealed more candidates expected to exceed the concentrations needed for antiviral activity in this tissue. hydroxychloroquine, chloroquine, mefloquine, atazanavir (ritonavir boosted), tipranavir (ritonavir boosted), ivermectin and lopinavir were all predicted to achieve lung concentrations over -fold higher than their reported ec . all of these drugs were also predicted to exceed their ec in lung by at least . -fold (data not shown). the lung prediction was not possible for nelfinavir because insufficient data were available to calculate k p u lung , but nitazoxanide and sulfadoxine were also predicted to exceed their reported ec by . -and . -fold in lung, respectively. nitazoxanide was predicted to exceed its ec by . -fold in lung but an ec was not calculable from the available data for sulfadoxine. predictions for cmax/ec ratio were also made for other tissues, and were generally in agreement with observations in the lung with some important exceptions. gliteritinib, amodiaquine, imatinib, indomethacin, oxprenolol, and sulfadoxine were predicted to be subtherapeutic in brain and bone, with indomethacin and sulfadoxine being predicted to be subtherapeutic across most of the tissues in which cmax was estimated. during inflammation or injury, changes to the vascular microenvironment could have a profound effect on the ability of these drugs to accumulate in lung cells. due to the recruitment of neutrophils and leaky endothelial cells ( ), the lung inflammatory microenvironment is characterised by increased body temperature, excessive enzymatic activity and, most importantly, by a low interstitial ph ( ). in the case of chloroquine and hydroxychloroquine, these diprotic weak bases are exquisitely dependent on a ph gradient to drive lysosomal uptake as a mechanism of lung accumulation. it has been demonstrated that cellular chloroquine uptake is diminished -fold for every ph unit of external acidification ( ). this situation is likely to deteriorate further on mechanical ventilation, which also induces acidification of the lung tissue, independently of inflammation ( , ). therefore, the benefits of lung accumulation for many of these drugs may be lost during treatment of severe sars-cov- infection. conversely, mefloquine is monoprotic and more lipophilic than chloroquine, which may make it much less reliant on this article is protected by copyright. all rights reserved the ph gradient to drive cellular accumulation in lung. it is likely that the charged form of the drug is sufficiently lipophilic to allow movement across biological membranes along a concentration gradient ( ). only two studies have described mefloquine uptake into cells, one study suggested that mefloquine uptake is not energy dependent and the other suggested that mefloquine uptake is mediated by secondary active transport, rather than passive proton trapping ( , ). mefloquine is known to cause severe psychiatric side effects in some patients and so use of this drug should be managed with care ( ). therefore, mefloquine may offer opportunities for treatment during severe disease that are not available with other drugs currently being tested for covid- therapy. if the high lung exposures are proven empirically for the drugs on this list, then some may also prove to be valuable for chemoprevention strategies. this study represents the first holistic view of drugs with reported activity against sars-cov- in the context of their achievable pharmacokinetic exposure in humans. while the analysis does provide a basis to rationally selected candidates for further analysis, there are some important limitations. firstly, cmax was the only pharmacokinetic parameter that was universally available for all of the candidate drugs, but cmin values are generally accepted as a better marker of efficacy since they represent the lowest plasma concentration over the dosing interval. however, cmax was only used to assess whether plasma concentration would exceed those required at any point in the dosing interval, and this was followed by a more in-depth analysis of the most promising candidates. table ) and in several cases varied between the same molecule assessed in different studies ( figure ) . also, some of the studied drugs (e.g. nitazoxanide and amodiaquine) are rapidly metabolised such that the major species systemically is a metabolite that has not been investigated for anti-sars-cov- activity. no mitigation strategy was possible for these limitations and the data should be this article is protected by copyright. all rights reserved interpreted in the context that the quality of the available data may profoundly impact the conclusions. in vitro activity should be confirmed for the promising candidates and/or relevant metabolites. fourthly, plasma protein binding can be an important factor in determining whether sufficient free drug concentrations are available to exert antiviral activity ( ) and insufficient data were available across the dataset to determine protein binding-adjusted ec values. this is important because for highly protein bound drugs the antiviral activity in plasma may be lower than reported in vitro activity because protein concentrations used in culture media are lower than those in plasma. fifthly, robust pharmacokinetic data were not available for all the molecules and subtle differences have been reported in the pharmacokinetics in different studies. where possible, this analysis utilised the pharmacokinetics described at the highest doses approved for other indications and checked them to ensure that profound differences were not evident between different studies. however, in some cases, higher doses and/or more frequent dosing has been investigated for some of the drugs mentioned so higher exposures may be available for some drugs with off-label dosing. sixthly, the digitised pharmacokinetic plots presented in this manuscript represent the mean or median profiles depending on what was presented in the original manuscripts. many of the drugs presented are known to exhibit high inter-individual variability that is not captured within the presented analysis and it is possible that even for promising candidates, a significant proportion of patients may have sub-therapeutic concentrations despite population mean/median being higher than the cmax. advanced pharmacokinetics modelling approaches will be needed to unpick the exposure-response relationship and these studies are currently underway by the authors. seventhly, the presented predictions for lung accumulation may offer a basis for ranking molecules for expected accumulation in that organ, but ultimate effectiveness of a chemoprophylactic approach will likely depend upon penetration into other critical matrices in the upper airways, for which there are currently no robustly validated methods of prediction. also, while a generally accepted method for assessing kpu was employed, the predictions were only validated for a subset of drugs for which previous animal lung accumulation data were available. in addition, the kpu method assumes all the processes are passive and perfusion limited, and the complexity of pulmonary tissue pharmacokinetics is not captured in this analysis. the lungs include different structures including airways, bronchioles and alveoli with different blood flow perfusion and more detailed modelling validated through animal experiments will be required to capture this complexity. finally, this analysis assumes that drugs need to be active within the systemic compartment in order to have efficacy against sars-cov- . since current evidence suggests that the virus is widely disseminated this article is protected by copyright. all rights reserved throughout the body this is a logical assumption. however, ultimate efficacy of any drug can only be demonstrated with robust clinical trial designs. the current analysis reveals that many putative agents are never likely to achieve target concentrations necessary to adequately suppress sars-cov- under normal dosing conditions. it is critical that candidate this article is protected by copyright. all rights reserved what is the current knowledge on the topic? what question did this study address? the manuscript describes a comprehensive analysis of literature reported anti-sars-cov- activity for approved medicines in the context of their known pharmacokinetic exposure. a combination of physiochemical and pharmacological parameters was used to predict the accumulation of these drugs within lung tissues using a widely accepted modelling approach. plasma and lung pharmacokinetic parameters were then used to rank the reported molecules according to whether they would provide therapeutic or chemopreventative exposures with the plasma or lung tissue. of the identified molecules with reported anti-sars-cov- activity, the overwhelming majority are not expected to reach active concentrations within the key target compartments. however, a number of candidates were identified that are expected to exceed the concentrations necessary to provide viral suppression at doses approved for use in humans. the manuscript identifies key drug repurposing opportunities and dramatically highlights the importance of considering pharmacokinetic exposure when interpreting the emerging candidacy of drugs for covid- treatment and prevention. infect dis, doi: . /cid/ciaa ( ). ec in terms of the cmax/ec ratio is also provided (b). amodiaquine and toremifene were estimated to exhibit sub-therapeutic pharmacokinetics irrespective of which ec value was used. similarly, nelfinavir was estimated to have cmax value higher than its ec irrespective of which ec was used in the analysis. for the other drugs, interpretation was highly dependent upon which reported ec was utilised and this underscores the caution that should be taken in interpreting the available data. ( ), chloroquine mg administered over days ( ), mefloquine mg over days ( ) , anidulafungin mg qd ( ) . robust pharmacokinetic data were unavailable for niclosamide mg, ritonavir mg and merimepodib mg in order to conduct this digitised interrogation of these molecules. a bar chart displaying the simulated lung cmax/ec . drugs with a ratio below were deemed not to provide lung concentrations at their approved doses to exert sufficient pulmonary antiviral activity for treatment or prevention strategies. those drugs with a ratio above (shown in orange) were estimated to provide lung concentrations sufficient to exert at least some antiviral activity at their this article is protected by copyright. all rights reserved approved dose. drugs shown in green were predicted to exceed lung concentrations over their ec by more than -fold. a heatmap displaying the simulated tissue cmax/ec values for all drugs with available data. those drugs with a ratio above (shown in orange) were estimated to provide tissue concentrations sufficient to exert at least some antiviral activity at their approved dose. drugs shown in green were predicted to exceed tissue concentrations over their ec by more than -fold. figure s table s table s supplemental figure legends supplemental methods this article is protected by copyright. all rights reserved covid- : gastrointestinal manifestations and potential fecal-oral transmission world health organisation. covid- trials -international clinical trials registry platform (ictrp) dysregulation of immune response in patients with covid- in effect of rifampin on steady-state pharmacokinetics of atazanavir with ritonavir in healthy volunteers concentration-effect relationship of hydroxychloroquine in patients with rheumatoid arthritis--a prospective, dose ranging study pharmacokinetics and pharmacodynamics of eltrombopag in healthy japanese males. rinsho yakuri clinical pharmacology and biopharmaceutics review of kaletra oral solution (nda# ) the pharmacokinetics of chloroquine in healthy thai subjects and patients with plasmodium vivax malaria new fixed-dose artesunate-mefloquine formulation against multidrug-resistant <em>plasmodium falciparum</em> in adults: a comparative phase iib safety and pharmacokinetic study with standard-dose nonfixed artesunate plus mefloquine pharmacokinetics of anidulafungin in critically ill patients with candidemia/invasive candidiasis atazanavir sulphate statpearls (treasure island (fl) hydroxychloroquine: from malaria to autoimmunity pharmacokinetic overview of indomethacin and sustained-release indomethacin kaletra (lopinavir/ritonavir) artesunate/mefloquine treatment of multi-drug resistant falciparum malaria a randomized, double-blind, placebo-controlled dose-escalation trial of merimepodib (vx- ) and interferon-alpha in previously untreated patients with chronic hepatitis c viracept) approved: fourth protease inhibitor available niclosamide: beyond an antihelminthic drug ritonavir (norvir) severe cutaneous reactions among american travelers using pyrimethamine-sulfadoxine (fansidar) for malaria prophylaxis tipranavir: a review of its use in the management of hiv infection accepted article the authors thank nathan morin from alberta health services for being proactive in making them aware of previously published data for indomethacin. the authors also thank articulate science for publication support. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved ( ) bukreyeva, n., mantlo, e.k., sattler, r.a., huang, c., paessler, s. & zeldis, j. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -kzdp edn authors: domingo, esteban title: quasispecies dynamics in disease prevention and control date: - - journal: virus as populations doi: . /b - - - - . - sha: doc_id: cord_uid: kzdp edn medical interventions to prevent and treat viral disease constitute evolutionary forces that may modify the genetic composition of viral populations that replicate in an infected host and influence the genomic composition of those viruses that are transmitted and progress at the epidemiological level. given the adaptive potential of viruses in general and the rna viruses in particular, the selection of viral mutants that display some degree of resistance to inhibitors or vaccines is a tangible challenge. mutant selection may jeopardize control of the viral disease. strategies intended to minimize vaccination and treatment failures are proposed and justified based on fundamental features of viral dynamics explained in the preceding chapters. the recommended use of complex, multiepitopic vaccines, and combination therapies as early as possible after initiation of infection falls under the general concept that complexity cannot be combated with simplicity. it also follows that sociopolitical action to interrupt virus replication and spread as soon as possible is as important as scientifically sound treatment designs to control viral disease on a global scale. medical interventions have dramatically increased over the last century, and in the case of infectious diseases, the discovery and development of antibiotics and antiviral agents have represented a very powerful external selective constraint imposed upon replicating microbes. hundreds of antiviral agents have been developed since the second half of the th century, and viruses can generally find evolutionary pathways to continue replication in their presence. the same is true of antibiotics and replication of bacteria. the belief that bacterial diseases were on their way toward extinction was quite widespread in the middle of the th century. sir m. burnet wrote in his textbook: "and since bacterial infections are, with unimportant exceptions, amenable to treatment with one or other of the new drugs, our real problems are likely to be concerned with virus diseases" (burnet, ) . in , a prominent spanish medical doctor, g. marañ on, declared: "in the year cancer will be a historical disease. infections will be almost entirely absent as a cause of mortality." (on a personal note, when i joined the university of california, irvine in , to work as postdoctoral student with r.c. warner, i attended some biology courses in which the teachers expressed to students that infectious diseases would disappear in a few decades as a consequence of the use of antibiotics and antiviral agents). predictions in science tend to fail. the optimistic view was not unanimous. a. fleming, the discoverer of penicillin, recognized the adaptive capacity of bacteria and suggested virus as populations https://doi.org/ . /b - - - - . - that bacteria would inevitably find ways of resisting the damage to them caused by antimicrobial drugs [quoted from the document "new antimicrobial drugs" from the european academies science advisory council, november (www.easac.eu); see also chapter ]. furthermore, there was early evidence of selection of mycobacterium tuberculosis mutants resistant to streptomycin (mitchison, ) . antibiotic resistance in bacteria has similarities and differences with antiviral resistance in viruses, and they are compared in chapter . we are now very aware that one of the major problems in antiviral therapy is the nearly systematic selection of drug-resistant virus mutants, which is often associated with treatment failure. other external influences, such as vaccination or immunotherapy, particularly using monoclonal antibodies, can also evoke the selection of viral subpopulations capable of replicating in the presence of those components inherent to an immune response. thus, selective constraints intended to limit rna virus replication meet with the broad and dynamic repertoire of variants ingrained in quasispecies dynamics. two space-time levels of the effects of drugs or vaccines are distinguished in coming sections: (i) short-term consequences for the individual in the form of treatment or vaccination failure and (ii) long-term consequences at the population level in the field, or vaccine-driven evolution of the antigenic properties of viruses. there are other medical interventions that may alter virus survival. individuals who are immunocompromised as a consequence of treatment after organ transplantation or those subjected to anticancer chemotherapy become particularly vulnerable to viral infections. enhanced viral replication can favor pathological manifestations in the affected individual as well as the spread of a large number of viruses into the environment, with consequences for the emergence and reemergence of viral disease (section . in chapter ). viral diseases are an important burden for human health and agriculture (bloom and lambert, ) . virus evolution, through the basic mechanisms exposed in previous chapters, can influence the two major strategies to combat viral infections: prevention by vaccination and treatment by antiviral inhibitors. for the design of new antiviral vaccines, a critical issue is the diversity displayed in the field by the virus to be controlled. the natural evolution of the virus may result in the circulation of one major antigenic type or the cocirculation of multiple antigenic forms. the vaccine composition (independently of the type of vaccine; see section . . ) must match the antigenic composition of the virus to be controlled. hepatitis a virus (hav) circulates as a single serotype, while foot-and-mouth disease virus (fmdv) circulates as seven serotypes and diverse subtypes, and the antigenic types are unevenly distributed in different geographical locations. a monovalent vaccine made of the prevailing antigenic type of hav should be sufficient to confer protection, while a multivalent vaccine composed of several types or subtypes is required to confer protection against fmdv, and the antigenic composition of the vaccine should match the circulating viruses in each geographical region. this is why antifmd vaccines of different compositions are used in different world areas at a given time, and vaccine composition must be periodically updated to maintain its efficacy. thus, one effect of virus evolution relevant to vaccine design derives from the necessity to prepare a vaccine that mirrors the antigenic composition of the virus to be controlled. in the case of live-attenuated antiviral vaccines, the evolution of the vaccine virus while it replicates in the vaccinee is a risk factor to produce virulent derivatives. the invasion of a susceptible host by a virus and the ensuing viral replication can be regarded as a step-wise process during which the virus must adapt to a series of selective pressures presented by the host, notably the immune response. the outcome can be either viral clearance (elimination of the infection) or virus survival and progression toward an acute or a persistent infection. administration of antiviral agents is an additional selective constraint that limits viral replication. evolutionary mechanisms may either succeed in the selection of mutants resistant to the antiviral agent that will permit the infection to continue or fail in sustaining the infection, resulting in the clearing of the virus from the organism. treatment planning, one of the aims of the new antiviral pharmacological interventions, based on information of viral genomic sequences present in each infected patient, has parallels with vaccine composition design. for vaccines, the information comes from the analyses of antigenic composition of circulating viruses, and for antiviral agents, the information comes from the mutant spectrum composition of the virus to be controlled in the infected patient. world-wide vaccination campaigns made possible the eradication of human smallpox [with the official declaration by the world health organization (who) in ] and animal rinderpest [with the official declaration by the world organization for animal health, office international des epizooties (oie) in ]. the number of new cases has decreased as a result of vaccination programs against several viral diseases, including measles or hepatitis b (bloom and lambert, ) , and substantial progress has been made toward the eradication of poliomyelitis (chumakov and kew, ; himman, ) . only the deliberate decision not to vaccinate (for religious reasons or misinformation campaigns) or lack of vaccine accessibility (for socioeconomic circumstances) jeopardizes vaccine efficacy. these facts demonstrate that at least some viral diseases can be controlled on a global basis by vaccination, an unprecedented achievement of human and animal health. despite the huge economic investment, however, there are important viral diseases such as acquired immunodeficiency syndrome (aids), hepatitis c, or viral hemorrhagic fevers for which no effective vaccines are available. for some diseases such as human influenza or animal fmd, vaccines are accessible, but they require periodic updating to approximate the antigenic composition of the vaccine to that of the circulating virus (section . ). in the case of influenza virus (iv), a major change in antigenic composition can occur through antigenic shift, in which the virus acquires new hemagglutinin and neuraminidase genes by genome segment reassortment (section . in chapter ), with the first evidence obtained by g. laver as early as (for the early history of influenza, its causative virus, and vaccine designs, see beveridge, ; kilbourne, ) . antigenic variation of viruses, whatever the mechanism might be, can affect vaccine efficacy and in some cases, the extreme rapid intra and interhost evolution of a virus may render an effective vaccine unfeasible at least with the current tools of vaccinology. cd þ t cell responses may act soon after infection and promote the selection of escape mutants (bull et al., ) . the difficulties for the control of virus disease derived from the adaptive potential of viruses (domingo, ; domingo and holland, ; bailey et al., ; hamelaar et al., ) require the judicious application of existing tools and innovative approaches that are still in their infancy. a first basic requisite for the preparation of a vaccine against a viral agent is the understanding . antiviral vaccines and the adaptive potential of viruses of the immune response evoked by the virus when it infects the organism to be protected (activation of b and t lymphocytes for antibody production, cellular responses, and generation of memory cells) and correlates of protection (bloom and lambert, ; van regenmortel, ; hagan et al., ; cunningham et al., ; rolland, ) . despite social pressure to rapidly obtain a vaccine, for each virus-host system, well-designed experiments are necessary to try to establish the determinants of protection, which is not a simple issue. the discussions in coming paragraphs are focused on the relevance of virus evolution in vaccine efficacy, irrespective of the type of protection afforded by the vaccine. what we term "protection" may mean the total absence of replication of the infecting virus (termed "sterilizing" immunity) or absence of disease manifestations despite infection and virus replication. as a general initial statement, which is widely accepted by vaccinologists, a vaccine is likely to be effective when it evokes an immune response that is similar to the response elicited by the authentic viral pathogen when it produces disease successfully overcome by the infected organism (evans and kaslow, ; bloom and lambert, ) . we refer to this as the basic principle of vaccinology. when infection by an antigenically constant virus produces lifelong immunity (i.e., measles virus infection), a vaccine is likely to evoke longlasting protection. in contrast, if a patient cured of a virus can be reinfected by the same (or a closely related) virus (i.e., hepatitis c virus infection) a vaccinedat least one prepared by standard methodologydis unlikely to evoke protection. some points to be considered in the design of antiviral vaccines are listed in box . . they are intended to minimize the selection of vaccineescape mutants and favor the success of vaccination campaigns. some of the recommendations deserve further comment. first, a basic knowledge of virus evolutionary dynamics and how it affects virus antigenic stability (or lack of) is essential. the fact that a methodology is available (i.e., vectors that can express large amounts of based on domingo and holland ( ) . . quasispecies dynamics in disease prevention and control antigens displaying good immunogenicity) does not guarantee vaccine efficacy, and even less if correlates of protection are not understood. the order of efficacy of different vaccine designs proposed in box . is justified both by the basic principle of vaccinology and by the mechanisms of selection of antibody (ab)-and cytotoxic tcell (ctl)-escape mutants by viruses. single amino acid substitutions at b-and t-cell epitopes in viral proteins are often sufficient to elude neutralization by the corresponding cognatespecific antibody or to escape recognition by a clonal ctl population. for many viruses, the frequency of monoclonal antibody-escape mutants has been measured in À to À , even in clonal populations obtained under controlled laboratory conditions and that have undergone a limited number of replication rounds (section . . in chapter ). generation of immune-escape variants can result in lack of vaccine efficacy, contribute to viral persistence (pircher et al., ; weidt et al., ; ciurea et al., ciurea et al., , richman et al., ; pawlotsky, ) , and provoke vaccination-induced virus evolution (section . . ). in human immunodeficiency virus type (hiv- ), antibody-escape variants are incessantly being produced in vivo to the point that virus replication continues despite the antibody response (richman et al., ; bailey et al., ) . the frequency of selection of mutants that can escape a number (n) of components in which we could hypothetically separate a global immune response is far lower than the frequency of escape to a single (a, b, c, etc.) of the i components of the response. making a simple mathematical abstraction that is applicable also to antiviral-escape mutants (section . ), the frequency of mutants that escape n components of an immune response is the product of frequencies of escape to each individual component [ Àa  Àb  Àc  . . this is an oversimplification because it is not realistic to dissect the selective impact of a complex immune response into discrete components. a virus generally includes multiple antigenic sites and each of them is often composed of several overlapping or nonoverlapping epitopes; in addition, a virus has several t-cell epitopes in different structural and nonstructural proteins, and each epitope displays a different degree of relative dominance. however, the above abstraction reflects the advantage of stimulating the host immune system with a sufficiently broad array of b-and t-cell epitopes to prevent selection of vaccineescape mutants due to a high genetic and phenotypic barrier (compare with the barrier to drug resistance described in section . . ). therefore, selection of vaccine-escape viral mutants is more likely with synthetic peptidic vaccines, than with whole virus-attenuated or inactivated vaccines because the latter present a broad epitope repertoire to the immune system. selection of escape-mutants by peptidic vaccines that evoked partial protection of cattle was documented with fmdv (taboga et al., ; tami et al., ) . the arguments in favor of multiepitopic presentation are also endorsed by a notorious scarcity of licensed peptidic vaccines for viral diseases despite horrendous economic investments (orders of magnitude greater than investments in quasispecies research!). use of a complex, multiepitopic vaccine, however, need not prevent long-term selection of antigenic virus variants as a result of vaccine usage, an important still largely underexplored topic discussed in section . . . experimental evolution with fmdv has opened the way to a new generation of antiviral vaccines that share features of attenuated and inactivated vaccines. this new design is based on the conversion of the monopartite fmdv genome into a segmented genome version that dominated the population after extensive high moi passages (section . in chapter and section . in chapter ). for the segmented virus version to be able to produce progeny, the two genome classes (which are encapsidated into separate particles) must reach the same cell. therefore, administration of the segmented virus preparation should lead to an . antiviral vaccines and the adaptive potential of viruses immune response akin to that evoked by inactivated viral particles, followed by a self-limiting infection. the vaccine was tested successfully in mice and swine, the authentic host of the parental virus (rodriguez-calvo et al., ) . potential concerns with this type of vaccine are that the standard (monopartite) genome can be reconstructed by recombination in the early stages of replication in the animal. a safety level is included in that particular fmd vaccine because of multiple mutations that accumulated during the transition toward genome segmentation, which deviated the genome sequence from the one present in the original swine isolate . exploration of evolutionary mechanisms that result in altered forms of viruses may open new possibilities for vaccine design. new prospects for attenuated vaccines have been opened with the engineering of viruses with suboptimal replication fidelity or deoptimized codon or codon pair usage (coleman et al., ; vignuzzi et al., ; cheng et al., ) . altered polymerase copying fidelity often leads to virus attenuation (gnadig et al., ; graham et al., ; korbouk et al., ; rozen-gagnon et al., ; van slyke et al., ) . a critical issue with live-attenuated vaccines based on deviation from the standard mutation rate is the stability of the attenuation trait. not only true revertants or other site revertants of the polymerase may arise and displace the vaccine virus, but other viral proteins may affect nucleotide incorporation (smith et al., stapleford et al., ; agudo et al., ). when a virus circulates in a population where vaccinated and unvaccinated host individuals coexist, and the vaccine does not induce sterilizing immunity, viruses with an altered antigenic profile might be selected. the larger the overall effective population size of the circulating virus, and the longer the virus is allowed to replicate in such a scenario, the higher the probability of incorporation of compensatory mutations that yield high-fitness antigenic variants. these events in the case of vaccines used in veterinary medicine are particularly significant because they may alter the cell tropism and host range of viruses, thus increasing the possibilities of their zoonotic transmission into humans (schat and baranowski, ) . evidence of vaccination-induced dna and rna virus evolution is increasing, and it has been documented with bovine respiratory syncytial virus, bovine herpesvirus- , marek's disease virus, porcine circovirus , and classical swine fever virus, among others [ (valarcher et al., ; muylkens et al., ; ji et al., ; kekarainen et al., ; constans et al., ; yoo et al., ) ; reviews in gandon et al., ; schat and baranowski, ) ]. the timing of dominance of ctl-escape mutants of the simian immunodeficiency virus (siv) was influenced by vaccination, and the process could be analyzed by penetration into the mutant spectra of the relevant viral populations (loh et al., ) . for human viruses, evidence of vaccineescape mutants has been obtained for hepatitis a and b viruses. vaccination-associated escape mutants of hav with substitutions around the immunodominant site of the virus were identified in a cohort of hiv- , hav doubly infected individuals (perez-sautu et al., ) . the study suggested that an incomplete vaccination schedule, combined with the hiv- -produced immunosuppression might have contributed to high-hav loads, thus facilitating the generation and dominance of antigenic variants. in taiwan, the prevalence of mutants at a major antigenic determinant of the surface antigen of hepatitis b virus (hbv) tripled in decade, and it has been suggested that this increase of prevalence might be due to the ample vaccination coverage in the region (hsu et al., ) . other studies also suggest the circulation of hbv mutants . quasispecies dynamics in disease prevention and control associated with vaccine escape and diagnosis failures (di lello et al., ) . vaccines can rarely afford protection to all vaccinated individuals due to many factors that include variations in vaccine receptivity factors due to polymorphisms in genes involved in the adaptive immune response, immunosuppression of the vaccine recipient, the insufficient time between vaccination and exposure to the viral pathogen, and antigenic differences between the vaccine strain and circulating viruses. in addition, for massive vaccinations in veterinary medicine, damage to the vaccine (during transport, storage, etc.) and improper administration are additional problems. vaccination may occasionally promote the selection not only of antigenic variants but also host cell tropism, host range, or virulent variants (swayne and kapczynski, ; kirkwood, ; read et al., ; rolland, ) . it is not known to what extent the widespread use of vaccination can contribute to antigenic variation relative to other factors (persistence of antibodies from previous infections, genetic drift due to genetic bottlenecks, etc.). however, our current understanding of virus dynamics should encourage investigations on the genetic and antigenic modifications of breakthrough viruses that arise from vaccinated individuals as compared with changes in viruses from unvaccinated host populations. reversion of live-attenuated vaccine viruses into virulent forms is a cause of disease derived from the evolutionary potential of viruses. in the case of attenuated sabin poliovirus vaccine, the rate of vaccine-associated poliomyelitis among those vaccinated for the first time was one per , to , vaccinees, and the rate of those receiving the second vaccine dose was about one in million (reviewed in rowlands and minor, ) . attenuated antifmd vaccines were used in some countries during the second half of the th century, but a reversion to virulence forced the halting of the vaccination programs. vaccine-escape mutants may arise due to ineffective vaccines, and concomitant factors, such as immunosuppression. the escape mutants may remain confined to the unsuccessfully vaccinated host or may spread to other susceptible individuals, and attain different degrees of epidemiological relevance. escape mutants may be direct mutants of the infecting virus or may originate by recombination between the infecting virus and other coinfecting related viruses, as observed with poliovirus and bovine herpesvirus- [kew et al., ; thiry et al., ; among other studies]. reiteration of vaccine selection and fitness increase processes over many generations of vaccinees (be it humans or animals) may result in accelerated virus evolution. since systematic use of vaccines for humans and animals in intensive production units is relatively recent in terms of evolutionary time (less than years, and in some cases even only a few decades), it is still premature to evaluate whether vaccination is a significant factor in promoting long-term virus evolution. the first description of virus resistant to an antiviral inhibitor was by j. barrera-oro, h.j. eggers, i. tamm, and colleagues working with enteroviruses and guanidine hydrochloride and -(alpha-hydroxybenzyl)-benzimidazole as inhibitors (eggers and tamm, ; melnick et al., ) . these early results that suggested that antiviral-resistant mutants could be readily selected have been amply confirmed with many viruses and inhibitors in cell culture and in vivo. indeed, the selection of viral mutants resistant to antiviral agents is an extremely frequent occurrence that has been known for decades, although it became widely recognized in the course of development and clinical use of antiretroviral agents to treat hiv- infections and aids. the description of drug-escape mutants has been based on three main groups of observations: • detection of antiviral-resistant mutants in patients during treatment. when a reverse genetics system is available, the suspected mutation should be introduced in an infectious clone and resistance ascertained and quantified in cell culture or in vitro enzyme assays. • selection of resistant mutants in cell culture, by subjecting the viruses to passages in the presence of inhibitors. the viral population size is an important variable in this type of experiment (section . . ). • calculation of the frequency of resistant mutants by plating a virus in the absence and presence of the antiviral agent similar to the assays to calculate the frequency of monoclonal antibody (mab)-resistant mutants (described in chapter , section . . ). in the three groups of observations, the frequency at which a specific escape mutant is found depends on a number of barriers to resistance (section . . ). traditionally, the fact that a drug can select virus-resistant mutants is regarded as a proof of the selectivity of the drug, as opposed to unspecific or toxic effects on the host cell that indirectly impair virus replication (herrmann and herrmann, ; golan and tashjian, ) . selection of viral mutants resistant to antiviral inhibitors is a major problem for the control of viral disease for two main reasons: (i) because it often results in virus breakthrough (increase of viral load) resulting in treatment failure and (ii) because resistant virus variants may become epidemiologically relevant, with the consequent decrease of inhibitor efficacy at the population level (domingo and holland, ; huang et al., ) . increasing numbers of antiviral agents have been developed based on the three-dimensional structure of viral proteins and their complexes with natural and synthetic ligands, in efforts that have engaged academic institutions and pharmaceutical companies. others have been incorporated as a result of drug repositioning, that is, the discovery of antiviral activity of compounds licensed for other medical purposes, often with the help of computational tools (ab ghani et al., ) . antiviral agents may target viral or cellular proteins involved in any step of the virus life cycle. they may interact with virions and inhibit an early step of infection, such as the attachment to the host cell, penetration into the cell, or uncoating to liberate the genetic material of the virus inside the cell. other agents interfere with the synthesis of viral nucleic acids or viral protein processing, particle assembly, or virus release from cells. selection of resistant mutants has been described for virtually any chemical type of antiviral agent directed to any step of the infectious cycle of dna or rna viruses, including important pathogens, such as herpesviruses, picornaviruses, iv, hbv, and hepatitis c virus (hcv). several reviews and articles have covered the theoretical basis of drug resistance, and consequences for treatment management [as examples see (domingo et al., b) and previous versions in progress in drug research (richman, (richman, , ribeiro and bonhoeffer, ; domingo et al., a domingo et al., , menendez-arias, ; perales, ; mokaya et al., ; nitta et al., ; pawlotsky, ) , and the articles in the current opinion of virology volume edited by l. menendez-arias and d. richman (menendez-arias and richman, ) ]. therefore, the general mechanisms that confer adaptability to viruses are very effective in finding drug-escape pathways through molecular mechanisms that are summarized in section . . considering the implications of quasispecies dynamics explained in previous chapters, the . quasispecies dynamics in disease prevention and control following statement will be obvious to the reader: "if a single mutation is able to confer resistance to an antiviral agent, and the mutation does not cause a significant selective disadvantage to the virus (fitness decrease) in the considered environment, a drug-resistant virus mutant will be present in most, if not all, virus populations" (domingo, ) . if a virus replicates in such a way that a population size of can never be achieved in a single population, it is extremely unlikely that any drug-resistance mutation (or any mutation associated with a phenotypic change) that is generated at a frequency of À or lower will be propagated from that viral population (perales et al., ) . selection of escape mutants depends on the replicative load, and the concentration of inhibitor attained at the sites of virus replication. consider different cell or tissue compartments in which an antiviral inhibitor reaches different concentrations (exerts different intensity of selection) ( fig. . ). in each compartment, there are multiple replication complexes. a mutation conferring resistance to the inhibitor will occur at the same rate in each of them, assuming that the mutation rate is independent of the presence of the inhibitor. however, after its occurrence, the proportion of viral rnas harboring the mutation will decrease depending on the inhibitor concentration. the time at which the effect of the inhibitor will be manifested depends on the inhibitor target. in the example of fig. . we assume that the concentration of inhibitorresistant mutants will decrease in the replication complexes, reaching resistant mutant frequencies of À , À , and À in compartments , , and , respectively. the frequency of inhibitor-resistant mutants in the entire cell, tissue, or organism at that time will be given by the weighted average of mutation frequencies at the individual compartments. in the case of a virus-producing viremia, assuming no bottleneck effects or differential selection for other figure . frequency of a drug-resistant mutant in different compartments (subcellular site, cell, tissue, or organ) of the same organism. three compartments labeled , , and three are drawn. replication complexes are depicted as ellipses and replicating genomes as lines. a replicative unit is defined here as a set of replication complexes. the inhibitor-resistant mutant is represented by a red circle in a genome. compartments , , and reach increasing concentration of the inhibitor, rendering inhibitor-resistant mutant frequencies of À , À , and À , respectively. see text for the difference between occurrence and presence of the resistant mutant, and implications of compartmentalization. . resistance to antiviral inhibitors traits, the frequency of resistant mutants calculated for the virus in blood should reflect the average frequency in all compartments that supply virus to blood. low inhibitor concentration in a compartment will favor the selection of the resistant mutant that can either be archived as an adaptive reservoir or penetrate other compartments, depending on the sequence of events of virus spread. if two or more independent mutations can confer resistance to an inhibitor, the probability of occurrence of an inhibitor-resistant mutant is equal to the sum of probabilities of occurrence of each mutation. for multiple mutations, the probability will be the sum of probabilities of the different mutations, a frequent case in viruses since they often display several evolutionary pathways to drug resistance. the probability of finding a viral genome resistant to two or more inhibitors directed to different targets is given by the product of probabilities of resistance to each of the individual inhibitors. the basic probability considerations regarding the frequency of occurrence of inhibitor-resistant viral mutants are summarized in box . . when two or more mutations occur in the same genome, they may be subjected to epistatic effects, meaning either increase (positive epistasis) or decrease (negative epistasis) of viral fitness (see section . of chapter for the concept of epistasis). the diversity of chemical structures of the antiviral compounds that can select for escape mutants is illustrated in figs. . and . with the formulae of some antiviral agents in current or historical use. they include relatively simple organic molecules, nucleoside analogs, and complex heterocyclic compounds with a variety of residues (ch -, c═ o, nh, nh , f, and cl) that may contribute to interactions with viral proteins or alter the electronic structure of neighbor bonds thus modifying the interaction behavior of some atoms. for all of them, resistant viral mutants have been identified, despite barriers imposed upon the virus to reach a drug-resistance phenotype. • if there are two or more different mutations that produce the same inhibitor-resistance phenotype, and once one of the mutations is present additional mutations are no longer necessary to produce the phenotype, the probability of achieving the phenotypic change is equal to the sum of probabilities of finding each mutation individually. • if two or more independent mutations must happen to produce resistance to an inhibitor, the probability of occurrence of the necessary mutations is equal to the product of probabilities of occurrence of each mutation individually. • if a virus is inhibited by an inhibitor combination, and the mutations that confer resistance to each inhibitor are independent (no cross-resistance is involved), the probability of a combination-resistant mutant to arise is equal to the product of probabilities of resistance to the individual mutations. these probability calculations are applicable to other mutation-dependent virus variations. the impediments for a virus to attain resistance to an inhibitor are divided into genetic, phenotypic, and mutant swarm (population) barriers to resistance (box . ). the genetic barrier to resistance to a specific inhibitor is not a universal value for a virus group, since it may be affected by genetic differences among natural viral isolates. the diversification of hcv into genotypes a and b influenced the genetic barrier to resistance to the ns / a protease inhibitor telaprevir [formula ( ) in fig. . ]. one of the amino acid substitutions that confer resistance to telaprevir is r k in ns . in genotype a, the triplet encoding r is aga; therefore, a single nucleotide transition g / a can yield the triplet aaa, which encodes k. in genotype b, the triplet encoding r- is cga; therefore, two nucleotide changes (transversion c / a and transition g / a) are required to reach aaa, the triplet encoding k. reaching the alternative aag codon for k would require the same or a larger number of mutations (see section . . in chapter for another example of how the synonymous codon usage can influence an ) rimantadine, (a-methyl- -adamantane methylamine). ( ) oseltamivir (trade name tamiflu), ethyl ( r, r, s)- -amino- -acetamido- -(pentan- -yloxy)-cyclohex- -ene- -carboxylate. ( ) zanamivir (trade name relenza), ( r, r, s)- -guanidino- -(prop- -en- -ylamino)- -(( r, r)- , , -trihydroxypropyl)- , -dihydro- h-pyran- -carboxylic acid. . resistance to antiviral inhibitors figure . some inhibitors of human immunodeficiency virus type (antiretroviral agents) and hepatitis c virus. the inhibitors are ( ) zidovudine (azt), -[( r, s, s)- -azido- (hydroxymethyl)oxolan- -yl]- -methylpyrimidine- , -dione. ( ) zalcitabine (ddc), -amino- -(( r, s)- -(hydroxymethyl)tetrahydrofuran- -yl)pyrimidin- ( h)-one. ( ) r, r , r, r)- -( , -dioxopyrimidin- -yl)- -fluoro- -hydroxy- methyl-tetrahydrofuran- -yl]methoxy-phenoxy-phosphoryl]amino]propanoate. additional drugs currently used in antiviral therapy can be found in the references quoted in the text and in chapter . . quasispecies dynamics in disease prevention and control evolutionary outcome). the requirement of transitions versus transversions to reach the resistant phenotype will affect the genetic barrier to resistance. most viral polymerases tend to produce transition mutations more readily than transversions presumably because in the course of rna elongation it is easier to misincorporate a purine by another purine than by a pyrimidine, and the same for pyrimidine misincorporations (chapter ). mutation preference is one of several factors that determine the frequency of drug-escape mutants. thus, evolution may diversify viruses to display different genetic barriers to the same drugs. since in many cases, several independent mutations may confer resistance to the same drug to complicate matters even more, it also has to be considered that the genetic barrier to one inhibitor may be affected by the presence of other inhibitors (beerenwinkel et al., ) . the phenotypic barrier to drug resistance is equivalent to the fitness cost inflicted upon the virus by the mutations and corresponding amino acid substitution(s) required for resistance [fitness cost is treated in chapter (section . ) and in chapter (section . . ) in connection with the frequency of monoclonal antibody-or cytotoxic t-cell-escape mutants in viral populations]. when a drug-resistance mutation inflicts a high fitness cost, a likely result is a reversion of the mutation when the virus replicates in the absence of the drug. an alternative outcome is that compensatory mutations are introduced in the genome so that viral fitness increases while maintaining the inhibitor-resistance mutation. the two outcomes are not mutually exclusive and may contribute to the multiple, transient selection pathways observed by the application of deep sequencing to monitor the response of a viral population to specific selective force (tsibris et al., ; fischer et al., ; cale et al., ; kortenhoeven et al., ) (see section . in chapter ). a high fitness cost may prevent or delay the selection of escape mutants. sofosbuvir [formula ( ) in fig. . ] is a very effective ns b (viral polymerase) inhibitor of hcv. amino acid substitution s t in ns b has been associated with sofosbuvir resistance, and the substitution has been detected in patients and in some natural isolates of hcv. in one of several clinical studies on sofosbuvir efficacy, the mutant spectrum composition of hcv genotype b in an infected patient treated with the drug was followed by deep sequencing of the virus at baseline (prior to initiation of treatment), in the course of treatment, and posttreatment. the frequency of s t was . % at baseline, indicating preexistence of resistance mutations despite no exposure of the virus to the drug (section . ). two days after initiation of sofosbuvir treatment, the level of s t decreased to . %, and viral breakthrough was detected weeks later when . % of the viral population included s t. during the posttreatment period, genomes with the wild-type s amino acid regained dominance that was attributed to the true reversion of mutant genomes rather than the outgrowth of baseline wild-type genomes (hedskog et al., ) . this result suggests a high phenotypic barrier for sofosbuvir, and that hcv has mechanisms to overcome the barrier. the complexities of virus-host interactions render the elucidation of the pathways exploited by a virus to overcome the phenotypic barrier to a drug a highly empirical endeavor. the hope is that a combination of inhibitors that display a high barrier to resistance may impede escape and drive the virus to extinction (chapter ). the mutant swarm barrier to resistance is a consequence of the interfering interactions that operate within quasispecies, and that are described in chapter (section . ). it is a particular case of interference that can delay or impede the increase of frequency of a resistance mutation (crowder and kirkegaard, ; kirkegaard et al., ) . the possible contribution of mutant swarms to facilitate or prevent the dominance of drug-resistant mutants in infected patients is still largely unexplored. it is difficult to anticipate how the three types of barrier listed in box . may result in a level of drug resistance for a particular virus, in a particular host individual, in a specific target organ, at a given time. additional influences are drug pharmacokinetics, drug penetration into different cells, tissues, and organs where the virus replicates (see fig. . ), and prior history of virus replication in the infected host. it is not surprising that the study of drug resistance in viruses remains fundamentally descriptive. when independent amino acid substitutions can lead to resistance to the same drug, alternative evolutionary pathways may be followed depending on trna abundances, mutational preferences, and relative nucleotide substrate concentrations at the virus replication sites. if resistance requires two or more amino acid substitutions, the genetic barrier will be correspondingly increased (section . . and box . ). quantification of barriers to resistance in experiments in cell culture requires a prior characterization of the drug to be tested when acting on the cell culture-adapted virus as it infects a specific cell line. the two basic parameters to be determined are the toxicity of the drug for the host cell, and its capacity to inhibit the production of infectious virus. toxicity is quantified by the concentration of drug that kills a given percentage (generally %, but sometimes another value) of cells under the conditions used in the infection. it is expressed as the cytotoxic concentration (cc ), as depicted in fig. . . toxicity may depend on the cell concentration, the extent of confluence in a cell monolayer, and the metabolic state of the cell (resting vs. actively dividing). the capacity of inhibition is quantified by the concentration of inhibitor that reduces the infectious progeny production by a given percentage (generally %, but sometimes another value) under the defined conditions of the infection, including a multiplicity of infection (moi). it is expressed as the inhibitory concentration (ic ), as depicted in fig. . . the therapeutic index (ti) is given by the quotient cc /ic , and although generally used for in vivo experiments of drug efficacy testing, it can also be applied to cell culture measurements. the three parameters, cc , ic , and ti, are not universal for a virus and a drug since they may be influenced by the composition of the viral population and environmental factors, as repeatedly expressed for other features of viruses in the present book. as a guide, ti values of or more suggest the excellent performance of an antiviral agent; values higher than ten are acceptable, but values lower than ten predict limited efficacy. the quantitative effects of a drug may vary when analyzing a single round of infection versus multiple rounds in serial passages, or when comparing in vivo versus cell culture experiments. cc and ic values serve as a guide to decide the range of the drug concentration to be used in serial passage experiments to evaluate the possible selection of inhibitor-resistant mutants and to estimate the genetic barrier. the possibility to overcome a genetic barrier depends on the virus population size. for viruses that replicate in cell culture, it is possible to estimate the minimal viral population size needed to select a drug-resistant mutant which is generally positively correlated with the genetic barrier ( fig. . ). in the hypothetical example of the figure, a viral population is composed of inhibitor-sensitive viruses (blue spheres), and a low level of inhibitor-resistant viruses (red spheres). the proportion of inhibitorresistant viruses is given by the mutational pressure (e.g., at a frequency of À , which is increased in the picture for clarity). passage of a small amount of virus (e.g., infectious virus in the small circle at the upper part of the figure) will exclude the mutant virus (red spheres) that will be maintained at the basal level dictated by mutational pressure in the course of passages (limited to two in the figure for simplicity). selection of escape mutants is precluded by the limited population size at each transfer. in contrast, if the population size used for the successive infections is sufficiently large (> , larger circles at the bottom that surround both sensitive and resistant viruses), the resistant mutant can become figure . schematic representation of two experiments to determine the concentration of an inhibitor needed to kill % of cells in culture (cc value, left) and the concentration of inhibitor that reduces the viral production to % (ic value, right). the therapeutic index is the quotient between cc and ic (box at the bottom). similar tests can be performed with tissue explants or animals, under controlled environmental conditions. see text for pharmacological implications. . resistance to antiviral inhibitors gradually dominant and can be isolated for further studies. to give another example, a single amino acid replacement that requires two mutations (the change from cag to aug to attain substitution q m in hiv- reverse transcriptase, associated with resistance to multiple antiretroviral nucleosides) will occur at a lower frequency than replacements that require a single mutation. if each of the two mutations reaches a frequency of  À , the expected frequency of the drug-resistant genomes (ignoring fitness effects) will be (  À )  (  À ) ¼  À . thus, at least  viral genomes must undergo one round of copying (or a lower number of genomes a proportionally higher number of rounds of copying) to approach a good probability to obtain a drug-resistant genome in that viral population. population size limitation of a drug selection event is a specific example of how random events may intervene in the process of positive selection (compare with section . and fig. . in chapter ; in that figure, the random event that excludes the positively selected population is conceptually equivalent to the insufficient population size depicted in the upper infection series of fig. . ). when two or more mutations are needed to confer the resistance phenotype, drug resistance will be less likely not only due to the lower probability of generating the two required mutations but also because of the increased chances of two mutations in the same genome entailing a fitness cost. a virus that requires three or more mutations to overcome a selective constraint may occur at a frequency in the range of À or lower which will often be insufficient for the mutant to be present in the mutant spectrum of the infected host ( fig. . ). failure to select for a drug-resistant mutant in cell culture does not necessarily mean that the resistant mutant is not present in the population. it may mean that due to a high genetic barrier, the selection experiment was designed to infect with an insufficient amount of virus. similar and even more accentuated problems are encountered in selection experiments in vivo, since not only the phenotypic barrier or fitness cost inflicted by a drug-resistance mutation (box . ) is often estimated empirically from the frequency of the relevant substitution in patients treated with the drug or in cell culture assays. an adequate procedure to quantify the phenotypic barrier to resistance is to determine the fitness of the virus expressing the protein with the wild-type amino acid (the one that confers drug sensitivity) relative to the virus expressing the protein with the substituted amino acid that confers resistance; fitness is measured in the absence and presence of the drug (double assay). this is an extension of the determination of fitness vectors described in section . . of chapter , as depicted in fig. . ; the assays are best performed in cell culture, although the use of explants or in vivo assays is also feasible. two parameters can be calculated: the fitness cost inflicted by the amino acid substitution associated with resistance, in the absence of the drug (reflected in a fitness value f À drug relative to the wild type), and the selective advantage conferred by the substitution in the presence of the drug (reflected in a fitness value f þ drug > relative to the wild type). the lower the value of f À drug , the higher the fitness cost. we define the selective strength of the resistance mutation as f þ drug /f À drug . for example, if we put arbitrary numbers (unrelated to values shown in ordinate) to the fitness values in the first graph of fig. . , figure . decreased frequency and fitness of mutant genomes resistant to one, two, or three inhibitors. the genome frequency level decreases by several orders of magnitude when resistance to one inhibitor (red asterisk in the upper mutant spectrum), or two inhibitors (red and green asterisks in the middle-mutant spectrum), or three inhibitors (red, green, and blue asterisk in the bottom mutant spectrum) must occur in the same genome (left part of the figure and numerical values at the center). increased number of mutations generally implies fitness decrease (right part of the figure) . see text for implications. . resistance to antiviral inhibitors f þ drug ¼ . and f À drug ¼ . , we obtain a selective strength of . . for the vectors in the second graph, if f þ drug ¼ . and f À drug ¼ . , the selective strength is . high selective strength means an important selective advantage conferred by the amino acid substitution for virus replication in the presence of the drug despite a high fitness cost inflicted by the substitution (compare with section . in chapter for the trade-off and "no free lunch" concepts). if the substitution does not entail any fitness cost (f À drug ¼ ), the fitness value in the presence of the drug equals the selective strength. selective strength can be calculated for a mutation or group of mutations that confer resistance to a drug used at a given concentration in a defined environment [example in (de la higuera et al., ) ]. the limitations of fitness measurements (environment dependence, etc.) described in section . . of chapter apply here. since viral genomic sequences may vary in the course of fitness assays, a limited number of passages and triplicate parallel assays are recommended. if a substitution entails a high fitness cost, direct reversion of the substitution or incorporation of compensatory mutations may occur. nucleotide sequence monitoring in the course of the assay should reinforce the conclusions. most drug-resistance mutations inflict a fitness cost upon the virus and yet very rarely drug resistance represents an unsurmountable barrier to maintain viral infectivity. several possibilities can account for the pertinacious occurrence and selection of drug-resistant, viable viral mutants. one possibility, supported by some experimental and clinical observations, is that a drug-resistant phenotype may be achieved through a number of alternative genetic modifications. even if a specific amino acid substitutiondthat would serve as the most direct and effective determinant of drug resistancedwere highly detrimental or lethal for the virus, alternative mutations can often be found that lead to a similar resistance phenotype, or at least a sufficient resistance to permit virus replication and exploration of sequence space to find compensatory mutations. the connectivity among points of sequence space and the fact that several space positions map into the same (or similar) drug-resistance phenotype contribute to the extended occurrence of drug resistance. this phenotypic redundancy applies to both standard nonmutagenic inhibitors and to mutagenic inhibitors. the cascade of mutations that confer resistance of picornaviruses to the mutagenic purine analog ribavirin illustrates how alternative amino acid substitutions in the viral polymerase (some being genuine resistance mutations and others acting as compensatory substitutions to maintain polymerase function) can lead to the ribavirin-resistance phenotype (discussed in chapter ). a speculative interpretation of the systematic occurrence of drug-resistant viral mutants is that the majority of the chemicals used in antiviral therapy (figs. . and . ) have a structure which may be related to natural compounds that viruses and their ancestral replicative machineries encountered in their continuous struggle to survive. in this view, drug resistance would have been gradually built as a consequence of coevolution (section . of chapter ) between virus replicative and gene expression machineries and the "space" of chemical compounds that interacted with them. mechanisms of drug resistance might have had their roots in molecular events repeatedly experienced as viruses evolved in an interactive manner with protocellular and cellular metabolites in our biosphere. discrimination in favor of small molecule substrates compatible with a flow of genome replication and gene expression and avoidance of perturbing intruders that could alter catalytic activities should have been positively selected. unfortunately, this is a possibility we will never be able to test. whatever the reasons behind, the unfortunate reality is that drug resistance is an extremely frequent event that complicates enormously the control of the viral disease. multiple drug evasion mechanisms have been identified or proposed for rna and dna viruses, and they can operate depending on basic replicative parameters in connection with drug levels and their variation with time. delayed lysis of bacteriophage vx that reduced the replication rounds in the presence of -fluorouracil (fu) produced resistance to this analog (pereira-g omez and sanju an, ). in this line, synchronization of a virus life cycle so that replication occurs when drug levels are minimal has been proposed as a potential resistance mechanism (neagu et al., ) . virus plasticity favors modification of life cycle parameters for virus survival in the presence of drugs, provided time for the relevant selection events is allowed once the treatment has been implemented. the great majority of inhibitor-resistance mechanisms involve amino acid substitutions in viral proteins that directly or indirectly diminish the binding of the drug to the viral target. the following major mechanisms have been documented: • substitutions in the protein targeted by the drug that decreases the affinity of the protein for the drug. mutations that modify nucleotide selectivity in viral polymerases belong to this group. substitutions may affect the neighborhood of the polymerase catalytic site, other polymerase domains, or even nonstructural proteins that interact with the polymerase. • if the inhibitor acts on a viral protein that itself has some other viral protein or genomic structure as a target, amino acid substitutions, or mutations that affect that target may also contribute to resistance. correlated mutations . molecular mechanisms of antiviral resistance in the first and second target may also yield the resistance phenotype. this is the case of some protease inhibitor-resistance mutations in hiv- . • in the case of viral polymerases, some mutations permit the excision of a chainterminating nucleotide at the -end of the primer. it is achieved through phosphorolysis mediated by a pyrophosphate donor, probably atp. resistance to nucleoside/nucleotide reverse transcriptase (rt) inhibitors (nrtis) is achieved by one of at least two mechanisms: (i) discrimination against the incorporation of the triphosphate form of the nrti and (ii) excision of the chain-terminating nucleotide once incorporated at the -end of the growing dna chain. this occurs with thymidine analog resistance mutation (or tams) that are typically selected under treatment with azt or d t [formulae ( ) and ( ) in fig. . ]. groups of amino acid substitutions may yield a multidrugresistance phenotype. a well-studied example in hiv- is the q m complex in the reverse transcriptase, which includes substitutions a v, v i, f l, f y, and q m. the phenotype consists of the limitation of incorporation of several nucleotide analogs. these and other mutants are characterized by a decrease in the catalytic rate constant (k pol ) of incorporation of the analog or analogs relative to standard nucleotides (see section . in chapter for the basic kinetic parameters for polymerase activity). the combination of enzymological and structural studies has provided a molecular interpretation of the mechanism of inhibition of hiv- by nrtis (reviews in men endez-arias, ; men endez-arias and alvarez, ; clutter et al., ; g€ unthard et al., ) ; for a predictive model that includes nucleotide levels, see von kleist et al. ( ) . nonnucleoside rt inhibitors (nnrtis) bind to an rt pocket Å away (a considerable distance) from the catalytic site composed of residues of the p and p subunits of the enzyme. the hydrophobic nature of nnrtis is illustrated in fig. . with the structures of nevirapine, efavirenz, and delavirdine [formulae ( ), ( ), and ( ), respectively]. several mechanisms have been proposed to explain their inhibitory activity, including alteration of the catalytic amino acids ymdd at the rt active site, distortion of the nucleotidebinding site, or modification of the position of the primer that receives the incoming nucleotides (men endez-arias, ). amino acid substitutions that confer resistance to nnrtis block the access of the inhibitors to their binding sites or alter the conformation and volume of the binding pocket. the essential viral proteases are a target for the development of specific antiviral inhibitors. examples are saquinavir and ritonavir for the hiv- [formulae ( ) and ( ), respectively, in fig. . ]. multiple resistance mutations have been described for protease inhibitors. they can affect the substrate-binding site or neighbor positions, often accompanied of compensatory substitutions at distant positions including sites of the target viral protein [e.g., the gag cleavage sites in hiv- (fun et al., ; flynn et al., ) ]. some hiv- protease inhibitor combinations display a high genetic barrier to resistance but, significantly for the capacity of viruses to explore sequence space, resistant mutants with e amino acid substitutions in the proteasecoding region have been isolated (rhee et al., ) . the reviews by men endez-arias ( ) and men endez-arias and alvarez ( ) provide excellent and detailed accounts of mechanisms of resistance of hiv- and hiv- to inhibitors that include virus entry and integrase inhibitors, in addition to the rt and protease inhibitor briefly described here. . quasispecies dynamics in disease prevention and control because of the distance effects that can be exerted among amino acids on the structure of proteins, substitutions that confer resistance to inhibitors of viral enzymes may lie far from the catalytic site. the effect of drug-resistance mutations on the general catalytic efficiency of a viral enzyme is one of the determinants of the functional barrier to resistance, since it may diminish replicative fitness. when an enzyme activity assay in vitro is available, the effects of specific drug-resistance amino acid substitutions on enzyme activity can be tested, although the observed alteration may not be the only influence on the fitness modification of the corresponding mutant virus. the reason is that most viral proteins (including viral enzymes) are multifunctional, and an enzyme activity assay may not capture the range of influences exerted by the enzyme. regarding direct-acting antiviral (daa) agents for hcv, the inhibitors that target the hcv polymerase (ns b) generally display a higher functional barrier to resistance than the protease inhibitors and manifest a broader genotype coverage. fitness decreases entail reductions in viral load and, consequently, lower probability of viral breakthrough (treatment failure). nucleotide analogs that bind to conserved residues at or near the active site of the viral polymerase tend to show subtypeindependent antiviral activity. because amino acid substitutions at or near the active site of viral enzymes are likely to inflict a fitness cost, such substitutions may not preexist in treatment-naive patients (margeridon-thermet and shafer, ; sarrazin and zeuzem, ) . interestingly, in the case of hiv- , mutations conferring resistance to rt inhibitors inflict a lower fitness cost than mutations that confer resistance to protease inhibitors (reviewed in martinez-picado and martinez, ) . thus, enzymes that perform similar functions for different viruses may have evolved to display different tolerance to amino acid substitutions. it is not possible to generalize which types of resistance mutations will display high or low functional barriers. there is a broad range of frequencies of antibody-and drug-escape mutants in viral populations, although values of À to À mutants per infectious unit are frequent for many rna and dna viruses (see table . in chapter for antibody-escape mutants). a few estimates for drug-escape mutants are listed in table . ; several observations and characterization of escape mutants have not been accompanied by frequency measurements. a point worth emphasizing is that quantification of viruses harboring biologically relevant mutations has been possible because an adequate biological assay is available. an antiviral agent or a neutralizing antibody measures the proportion of infectious viral particles that differ from the majority of the population in the relevant resistance trait. there is no reason to suspect that the viral amino acid residues (that are the target of an inhibitor or an antibody) that are substituted to confer the resistance phenotype are more prone to accept variations than many other amino acids in viral proteins. if we had additional selective agents to probe other viral sites, we expect a similar range of variant amino acids than using inhibitors or antibodies. this quite straightforward prediction is another way to state that there is general agreement in the mutation rates and frequencies for viruses being in the range of À to À substitutions per nucleotide (s/nt), calculated using a variety of biochemical and genetic methods (chapter ). a quite general observation is that in antibody neutralization experiments, a fraction of the virus population remains infectious despite the . molecular mechanisms of antiviral resistance addition of high antibody concentrations. although the resistance mechanism is unclear, a possibility is that the heterogeneous viral population includes a small proportion of antigenic variants with decreased affinity for antibodies. incomplete neutralization with the nonsigmoidal slope in the neutralization curves has been characterized for broadly neutralizing antibodies directed to hiv- (mccoy et al., ) . this general observation is an added complication to preventive designs that consider administration of neutralizing antibodies either as vaccine additives or in combination with antiviral inhibitors (section . in chapter ). the calculated frequencies of antibody-or drug-resistant mutants in viral populations may be lower than the rate at which they originate by mutation due to the fitness cost of the mutation (section . . ) . the argument is parallel to that used to justify why mutation rates and frequencies differ due to the fitness effects of mutations (chapter ). another prediction derived from the above considerations is that mutations conferring resistance to antiviral agents are expected to be detected in viral populations never exposed to the relevant drugs. all forms of genetic variation of viruses can contribute to dominance and spread of drugresistant mutants, including the combined effect of mutation, recombination, and genome segment reassortment (richman, ; neher and leitner, ; rogers et al., ) . the basal level of mutational pressure may be sufficient to provide a detectable proportion of escape mutants without the need for selection by the selective agent. this is an important aspect of antiviral therapy that is addressed next. the first demonstration that the baseline mutation level in viral quasispecies can include a detectable level of mutations that confer resistance to inhibitors in the absence of selection by the inhibitors, was obtained by d.d. ho, i. n ajera, c. l opez-galíndez, and their colleagues working with hiv- (mohri et al., ; n ajera et al., , . one of the studies examined the pol gene of hiv- genomes obtained directly from lymphocytes of infected patients. mutation frequencies for independent viral isolates were in eggers and tamm ( ) amantadine and rimantadine iv  À to  À measurements in cell culture appleyard ( ) , lubeck et al. ( ) rimantadine a iv % percentage of children treated with rimantadine that shed resistant iv belshe et al. ( ) disoxaril a hrv  À to  À low-level resistance in cell culture heinz et al. ( )  À high-level resistance in cell culture guanidine d pv .  À to  À measurements in cell culture pincus and wimmer ( ) a the formula of these drugs is included in figure . with the following number in parenthesis: amantadine ( ); rimantadine ( ); disoxaril ( ) the range of . . À to . . À s/nt, while for mutant spectrum components of individual isolates the values were . . À to . . À s/nt. in the virus from these patients, mutation frequencies at the codons for amino acids involved in antiretroviral resistance were very similar to the average mutation frequency for the entire pol gene. consistently with the mutation frequency values, several mutations that led to amino acid substitutions that conferred resistance to reverse transcriptase inhibitors were identified in patients not subjected to therapy. at the time of the study, the number of antiretroviral agents was still limited, and a considerable number of patients were not treated. the authors gave convincing epidemiological arguments that the background of mutations related to antiretroviral resistance was a consequence of high mutation rates and quasispecies dynamics, and not due to the transmission of resistant virus from individuals that had been subjected to therapy (primary resistance) (n ajera et al., ) . the presence of inhibitor-resistance mutations in viral populations never exposed to the corresponding inhibitor has been confirmed for hiv- and for several other viruses, including hcv, and it is supported by the calculated mutant frequencies in viral quasispecies (havlir et al., ; lech et al., ; ribeiro et al., ; ribeiro and bonhoeffer, ; cubero et al., ; johnson et al., ; toni et al., ; tsibris et al., ; peres-da-silva et al., ) . ample support has also come from deep sequencing analyses of mutant spectra, opening a point of debate on the basal frequency of inhibitor-resistance mutations that constitutes an indication to avoid the use of the corresponding inhibitors in therapy. at least in the case of hcv (and probably applicable to other viruses), there is no basis to suggest that the presence of a drug resistance mutation below a certain level in a patient will not have relevance for treatment failure when the inhibitor is administered. the understanding of quasispecies dynamics cautions against such reductionist arguments as evidenced by clinical cases (perales et al., ) . the data underline the relevance of mutant spectra as phenotypic reservoirs to confront selective constraints before constraints are in operation. for treatments, including a drug that has already been administered to a patient in the past, the influence of quasispecies memory should also be considered (section . . in chapter ). mutant spectra can be viewed as an anticipatory reservoir of phenotypes. in addition to the presence of drug resistance mutations in viral populations due to mutant frequency levels, the transmission of drug-resistant mutants from treated to na € ive patients may contribute to epidemiological relevance of resistance mutations. such primary resistance has been amply documented with hiv- , and it appears to increase in the case of hcv (franco et al., ; echeverría et al., ; huang et al., ) . higher levels of resistance mutations as a function of time in untreated patients is an indication that the mutations are not due to basal mutant frequencies but to the epidemiological expansion of virus mutants that originated in treated patients. from the clinical data available, the rate of expansion of virus harboring resistance mutations may vary depending on transmission and epidemiological features of each pathogen, but it seems unavoidable in the face of extended treatments for genetically variable pathogenic viruses. we confront a situation with parallels with antibiotic resistance in bacteria (chapter ). the major mechanism of drug resistance in viruses is based on amino acid substitutions that render the drug ineffective through the several molecular mechanisms summarized in section . . application of the cell culture system of hcv replication in human hepatoma cells (lindenbach et al., ; wakita et al., ; zhong et al., ) to examine the effects of . fitness or a fitness-associated trait as a multidrug-resistance mechanism long-term evolution has indicated that viral fitness can be an additional mechanism of drug resistance. the evidence was obtained when addressing the important issue of hcv resistance to interferon-alpha (ifn-a). ifn-a and ribavirin were the two components of the standard of care treatment against hcv infections until the advent of new therapies based on daa agents in . natural hcv isolates differ in ifn-a sensitivity, and the molecular basis of the difference is largely unknown. the study in cell culture consisted in subjecting a clonal population of hcv (termed hcvp , prepared by electroporation of hepatoma cells with rna encoding the viral genome, transcribed from a plasmid) to serial passages (of the type described in section . of chapter ) in the absence or presence of increasing concentrations of ifn-a added to the culture medium. several mutations scattered throughout the hcv genome were associated with ifn-a resistance (perales et al., ) . the selection of multiple alternative mutations is most likely influenced by the fact that ifn-a evokes a multicomponent antiviral response, which is not focused toward a single viral protein (perales et al., ) . unexpectedly, even the control hcv populations (those passaged times in the absence of ifn-a) displayed a partial (but statistically significant) resistance to ifn-a that could not be attributed to endogenous ifn production by the hepatoma cells (perales et al., ) . in view of this intriguing result, the initial hcvp population and the hcv population passaged and times in the absence of ifn-a (termed hcvp and hcvp , respectively) were tested for their resistance to other inhibitors of hcv replication: the protease inhibitor telaprevir [formula ( ) in fig. . ], the ns a inhibitor daclatasvir, the cellular protein cyclophilin a inhibitor cyclosporin a, the mutagenic purine nucleoside ribavirin, and the high barrier inhibitor sofosbuvir [formula ( ) in fig. . ]. hcvp and hcvp displayed significantly increased resistance to all inhibitors tested, as compared with the parental population hcvp gallego et al., ) (fig. . ) . passage of hcv entailed a -to -fold increase of viral fitness and a broadening of the mutant spectrum that might have increased the frequency of mutations associated with drug resistance, thus explaining the behavior of the multiply passaged hcv populations. the search for the resistant mutations was easier for telaprevir, daclatasvir, and cyclosporin a than for the other drugs because amino acid substitutions in the target protein had been previously identified as responsible for drug resistance. (in the case of cyclosporin a resistance, substitutions map in ns a and ns b, because the drug binds to cyclophilin a, which in turn interacts with ns a). analysis of the mutant spectra of hcvp and hcvp by molecular cloning and sanger sequencing and by deep sequencing failed to identify specific drugresistance mutations. since it could not be excluded that the broadening of the mutant spectrum might have increased the frequency of resistance mutations still to be characterized, two additional tests were performed. one was to determine the kinetics of viral production over a -fold range of moi in the absence and presence of telaprevir. both the unpassaged and multiply passaged hcv displayed parallel kinetics at the different mois, which excludes that drug resistance was due to the presence of resistance mutations in minority components of the mutant spectrum ( fig. . ). to further substantiate the findings, biological clones obtained by end-point dilution of the corresponding hcvp and hcvp populations were tested regarding drug resistance. a biological clone should have eliminated the minority genomes that harbored drug-resistance mutations since biological cloning is the most severe form of bottleneck event (sections . and . in chapter ). the biological clones did not display any decrease in drug resistance as compared . quasispecies dynamics in disease prevention and control moreno, e., gallego, i., pineiro, d., et al., . increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus. j. virol. , e , with permission from the american society for microbiology, washington dc, usa. . fitness or a fitness-associated trait as a multidrug-resistance mechanism with their corresponding parental, uncloned populations . the above observations have established viral fitness as a multidrug-resistance determinant in hcv that may also apply to other viruses. one possible molecular mechanism may consist of competition between replicative complexes and inhibitory molecules inside the infected cells. this model implies that fitness increase is reflected either in more replicating molecules per each replicative unit or in an increase in the number of replicative units per cell, without any influence on the number of inhibitor molecules that reach the replication sites. exploration of this competition model and alternative models and the extension to other viral-host systems are important challenges in the field of antiviral research. to sum up, mutant spectra and quasispecies dynamics can mediate antiviral resistance by at least two mechanisms: (i) by increases in the proportion of resistance mutations in the mutant spectra and (ii) by a fitness increase promoted by continued viral replication in the same environment. both mechanisms may act conjointly during viral infections in vivo. some studies with hcv have documented drugresistance phenotypes in infected patients, in the absence of specific drug-resistance mutations (sullivan et al., ; svarovskaia et al., ; sato et al., ; stross et al., ; di maio et al., ; dietz et al., ) . in fact, prolonged chronic hcv infections represent an adequate scenario for fitness increase due to extended rounds of infections in the same host liver. as a consequence, chronic infections may be prone to display fitness-associated multidrug-resistance phenotypes in the absence of drug-resistance mutations. the multiple mechanisms of drug resistance related to quasispecies dynamics justify even further the need for new antiviral strategies, as presented in chapter . high viral loads are predictors of disease progression. for hiv- and other lentiviruses, efficient early control of virus replication by the host immune response is generally associated with limited disease severity. the viral load that follows after the initial immune response to hiv- is referred to as the "viral set point." in the absence of early therapy, low set points in hiv- are generally attributed to a strong cellular immune response, likely influenced by additional host and viral factors. [this and other aspects of hiv- replication and pathogenesis have been reviewed in excellent monographs by levy ( levy ( , ]. a low set point predicts an asymptomatic outcome, and this is generally the case for viruses that establish persistent rather than acute infections. high viral fitness during the early stages of viral replication can promote disease manifestations. this was suggested by the progression toward the disease of a cohort of individuals that were infected during blood transfusion with an hiv- containing a large deletion in nef, an adaptor protein that mediates replication and pathogenesis (reviewed in arien and verhasselt, ) . after more than years, some of the infected individuals showed clinical signs, probably as a result of the accumulation of mutations in the hiv- genome that compensated for the lack of nef. more generally, fitness-decreasing (but not lethal) genetic lesions in a viral genome may be compensated by additional genomic mutations that become increasingly dominant in the course of further viral replication. the kinetics of fitness gain will depend on the nature of the lesion and the functional implications of the altered protein or genomic regulatory region (chapter ). fitness, replicative capacity, and viral load are directly interconnected parameters, and they . quasispecies dynamics in disease prevention and control affect disease progression (domingo et al., ) ( fig. . ). fitness gain will be more effective with a high load of actively replicating virus in the infected organism. elevated replicative capacity and fitness sustain high viral loads. the reason for this basic feature of viral population dynamics is that given a basal mutation rate, a large number of replicating genomes entails a correspondingly higher probability that a required mutation for fitness gain can be produced. the events involved are a specific case of search for adaptive mutations in terms of exploration of sequence space, as discussed in section . of chapter . while active viral replication, high load, and high fitness favor progression of the infection and disease manifestations, the fourth parameter included in the large arrow of fig. . , mutant spectrum diversity, has an optimal range. too low or too high intrapopulation diversity is detrimental to virus adaptability. insufficient diversity limits adaptability to complex environments (pfeiffer and kirkegaard, ; vignuzzi et al., ) , while excess diversity may lead the virus to cross an extinction threshold, and this is the basis of lethal mutagenesis as an antiviral therapy (chapter ). an additional implication of the parameters shown in fig. . for antiviral interventions is that fitness decrease is recognized as an alternative to inhibition of viral replication to control viral infections[ (clementi, ; clementi and lazzarin, ) ; reviewed in (domingo et al. ) ]. thus, key features of quasispecies dynamics have a direct implication on the management of viral infections. external interventions that have been applied or have been envisaged to limit or suppress virus infection include not only vaccination and administration of antiviral agents as described in previous sections, but also passive immunotherapy, antisense rnas, or oligonucleotides with various chemical modifications, interfering rnas, ribozymes, or their combinations. biotechnological developments have favored the design of chemically defined vaccines (consisting of expressed immunogenic proteins, synthetic peptides, or peptide arrays), without the need to handle or administer live virus. one of the most na € ive manifestations of trust in biotechnology in the middle of the th century was the belief that a catalog of plasmids encoding the antigenic proteins of the circulating types of pathogenic viruses would suffice to prepare the required vaccine as needed. concerning influenza vaccines, w.i. beveridge wrote the following: "the first objective would be to capture the full range of influenza a subtypes. their antigens would be studied by specialists at central laboratories and made available for the preparation of particular vaccines if and when required. it might be feasible to stockpile some vaccine against all the principal hemagglutinin antigens to be used in a figure . a schematic representation of interconnected parameters of viral replication that often relate to disease progression. an understanding of quasispecies dynamics has made it evident that the aim of antiviral therapy need be not only to directly diminish the viral load but also to affect other parameters that can then reduce the viral load. see text for justification and references and chapter for new antiviral strategies that follow the concept expressed in this figure. . limitations of simplified reagents and small molecules as antiviral agents fire brigade type of action as soon as an incipient pandemic is spotted" (beveridge, ) . naivety is also perceived in current designs of universal vaccines based on conserved antigens, considering the different escape mechanisms that operate in viruses to elude neutralization by antibodies (chai et al., ) . it is remarkable how our present understanding of viral populations renders obsolete the views expressed in the w.i. beveridge book, and in other writings at the boom of implementation of dna recombinant techniques. yet, attempts to produce vaccines against highly variable viruses, based on antigenic structures or some viral isolates, are ongoing (christiansen et al., ) . a critical issue is if the host immune response against a vaccine engineered with "universal" (conserved) antigenic motifs will be sufficient to prevent disease upon infection by other forms of the same pathogen (freeman and cox, ) . from our current knowledge of viral population dynamics, it seems unlikely but time will tell, since efforts toward the manufacturing of universal antiviral vaccines are under way. with a conceptual similarity to vaccines, in medical practice, monotherapy with an antiviral agent was traditionally preferred over drug mixtures. (the change of paradigm was largely a consequence of the aids epidemic, and it was publicly expressed by the pioneer hepatologist s. sherlock in a summary address of an international symposium on viral hepatitis held in madrid in ). the change of perspective is clear. some antiviral strategies, such as antisense nucleic acids or virus-directed ribozymes, were intensely investigated decades ago. it is unlikely that when used in isolation, they can be converted into useful antiviral therapies because resistant mutants are likely to be selected. yet, they could be part of combinations with other antiviral inhibitors to provide a larger antiviral barrier (chapter ). a similar fate is likely for interfering rnas (boden et al., ; gitlin et al., ; herrera-carrillo and berkhout, ; mcdonagh et al., ) . to a large extent, the failures of defined chemical entities (oligonucleotides, ribozymes, small molecule inhibitors, etc.) to control virus replication and spread are a consequence of their targeting a very defined viral genomic sequence combined with the adaptability of viral populations. combination of such multiple elements have been envisaged and tested, but off-target effects and the adaptive potential of viruses are likely to limit their efficacy. unfortunately, biotechnological developments that have been so positive for many research areas and practical applications tend to simplify the types of agents to prevent disease or inhibit virus replication, ignoring the inherent complexity of the object to be controlled. success is unlikely when "complexity" is combated with "simplicity." an increased understanding of viral population dynamics over the last decades has changed the picture dramatically by providing an interpretation of "virus escape" as a general and largely unavoidable phenomenon. such awareness has pushed the development of new antiviral designs, which are fundamentally centered in two strategies: a combination of multiple, independently acting elements or fitness decrease through excess mutations (chapter ). a pronouncement by p. ehrlich at the international congress of medicine held in london in , reflected old traditions on how to treat microbial infections ["here, therefore, the old therapeutic motto is applicable: frapper fort et frapper vitte" and "therefore, it is in my opinion necessary to allow the therapeutic treatment to come into action as early as possible, ..."; sentences taken from (ehrlich, ) , with emphases as in the original text]. this pronouncement fits our current understanding of viral populations. following ehrlich, the full implications of quasispecies-mediated adaptation of viruses for . quasispecies dynamics in disease prevention and control antiviral therapy were expressed by d.d. ho in an influential article entitled "time to hit hiv, early and hard" (ho, ) . the article title captures what is needed to prevent adaptation of a virus in the infected host. any opportunity to replicate is exploited by the virus to increase its fitness and to become less vulnerable to internal (intrahost) or external interventions such as antiviral therapy. treatment interruptions during chronic infections, such as "drug holidays" that in the case of hiv- -infected patients were justified to alleviate side effects associated with administration of antiretroviral agents, provided an opportunity for the virus to gain fitness. in principle, given our current understanding of viral quasispecies dynamics, the proposal of p. ehrlich and d.d. ho is applicable to other viral pathogens. one argument that tones down the strength of the "hit early and hit hard" proposal is that some infected patients may not progress to disease, but maintain an asymptomatic lifelong persistent infection. this is the case with elite controllers in the case of hiv- infection, and individuals infected with hcv who will not progress toward liver disease. in cases in which such nonprogression can be anticipated by viral and host parameters, it may be justified to exclude some patients from aggressive interventions (suthar and harries, ; casado et al., ) . as a general rule, however, the potential benefits of early treatment are obvious not only to avoid disease on an individual basis, but also to diminish the chances of virus transmission (reviewed in suthar and harries, ; see also sections . and . in chapter regarding the relevance of viral population numbers in transmission). restricting the number of treated patients for economic reasons will result in more expensive public health interventions when infected individuals develop the disease. box . includes recommendations for the use of antiviral agents and recapitulates concepts explained in this and preceding sections. despite the emphasis on evolutionary aspects, prevention and treatment of viral disease have many other angles some of which were box . • avoid monotherapy. ideally, use two or more antiviral agents which do not share a mechanism of action ( fig. . ). • treat as soon as possible after virus diagnosis, to avoid virus adaptability associated with high virus population size and to minimize transmission of inhibitor-resistant mutants. • individual patients should be treated only during the time at which the drug proves effective. when viral load rebounds, treatment should be discontinued. • use deep sequencing methodology to determine mutant spectrum composition for an adequate choice of inhibitors. the aim is to design personalized treatments that consider the probability of drug combination efficacy with minimal side effects. • consider temporary shelving of effective drugs when resistant mutants acquire epidemiological relevance. considered in chapter in connection with factors of disease emergence (smolinski et al., ) . three of them should be mentioned here because they are as important as the adequate treatment designs described in this chapter: (i) adequacy of public health measures, (ii) public information about virus sources and means of contagion and (iii) need of global political action. information to the public should aim at limiting the spread of disease that is, undertaking personal and collective actions to reduce the r value for a given virus (chapter , section . ). as an illustration of this key point, there was a quite extensive information campaign on hiv- and aids in developed countries during the early decades of hiv- spread, while the information about other potentially threatening viruses such as ebola or the severe acute or the middle east respiratory syndrome (sars and mers, respectively) coronaviruses was more limited. the need of a global response to limit the extension of disease episodes at the sites where they are initiated has been recognized for a long time, but it became obvious with the e west african ebola epidemic (see siedner et al., ) . there is a need for international organizations and governments of developed countries to provide the health-care workforce to assist low-and middle-income countries to control viral episodes at an early stage. "help early, help effectively" is the recognized need at a global scale, which is the parallel to "hit early, hit hard" for the treatment of infected patients. global early action and adequate information can be as important as an adequate treatment design to control viral disease. it can restrict viral replication rounds and consequent adaptability. information is thought to have been critical for the control of ebola epidemic in nigeria (siedner et al., ) . however, information must also be planned to reach the target population in a convincing manner, as learned from the poliovirus vaccination and eradication campaign (renne, ) . the uncertainties regarding whether an initial, limited episode of viral disease will expand or die out do not help in decision making. however, the best choice in the case of emerging and reemerging infections is to act assuming the worst scenario. medical interventions represent a totally new set of selective constraints that viruses are facing only since decades ago, an infinitesimal time of their existence as biological entities. however, the evolutionary mechanisms available to viruses have successfully coped with many selective pressures, notably the effect of vaccination when a broad immune response is not evoked, or treatment with antiviral agents. a common way to proceed is to test a new vaccine with an animal host, be it the authentic host or an animal model, and obtain full protection when the animal is challenged with a virus that matches the antigenic composition of the vaccine. following the initial excitement, very often the vaccine displays only partial protection when tested in the natural environment. somewhat parallel arguments can be made about clinical trials for antiviral agents, usually performed initially with selected groups of patients. clinicians have coined the term "reallife" treatment studies when patients are not selected for optimal results (often pushed by commercial and political interests). this chapter has emphasized how the complexity of viral populations is a serious (often underestimated) difficulty to prevent and treat viral disease. examination of the molecular mechanisms exploited by viruses to survive despite antiviral interventions suggests two major lines of action: first, more judicious use of existing tools that should consider the complexity of viral populations and their dynamics; complexity . quasispecies dynamics in disease prevention and control cannot be combated with simplicity. second, the need to design new antiviral strategies, a topic addressed in chapter . several interconnected parameters determine the probability of success of an antiviral intervention. most of them follow from the general concepts of darwinian evolution explained in preceding chapters. it is important, however, to quantify as much as possible the evolutionary events that determine therapy success or failure. for this reason, the importance of viral population size, basic probability calculations of developing resistance, and the selective strength of mutations, have been explained with numerical examples. hopefully, these simple quantifications will permit a higher awareness of when and why treatment may succeed or fail. we live in a very unequal society. the chapter closes with the recognition that there are many social economic issues that are as important as scientific planning to combat the pathogenic viruses around us (see summary box). • medical interventions represent a new class of selective constraints acting on viral populations. • viral evolution affects antiviral preventive and treatment strategies in two different ways: (i) through the molecular mechanisms of short-term response in treated individuals that select escape mutants, and (ii) through the epidemiological impact of viruses that have acquired escape mutations. • ineffective vaccines can contribute to the selection of antigenic viral variants. • selection of viral mutants resistant to antiviral agents is a general phenomenon. selection is favored by suboptimal treatments and is delayed by the combined administration of multiple inhibitors. resistance may also occur in the absence of specific resistance mutations, and it is associated with viral fitness. • the aim of therapy should be to increase the functional barrier to resistance and to give no opportunity to the virus to pursue replication that increases its replicative fitness. • replication rate, viral load, fitness, and mutant spectrum complexity are interconnected parameters that may tip the balance toward either control of the infection or disease progression. each of these parameters can be targeted in an antiviral design. • health care resources, adequate public information, and firm political action are as important as antiviral designs to control virus infections at a global level. drug reposer: a web server for predicting similar amino acid arrangements to know drug binding interfaces for potential drug repositioning involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism amantadine-resistance as a genetic marker for influenza viruses hiv nef: role in pathogenesis and viral fitness mechanisms of hiv- escape from immune responses and antiretroviral drugs estimating hiv evolutionary pathways and the genetic barrier to drug resistance genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection influenza: the last great plague; and unfinished story of discovery the vaccine book transmitted/founder viruses rapidly escape from cd þ t cell responses in acute hepatitis c virus infection natural history of infections disease epitope-specific cd þ t lymphocytes cross-recognize mutant simian immunodeficiency virus (siv) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during siv infection two escape mechanisms of influenza a virus to a broadly neutralizing stalk-binding antibody development of live-attenuated arenavirus vaccines based on codon deoptimization immunological responses following administration of a genotype a/ b/ / a quadrivalent hcv vlp vaccine the poliovirus eradication initiative viral persistence in vivo through selection of neutralizing antibody-escape variants cd þ t-cell-epitope escape mutant virus selected in vivo perspectives and opportunities for novel antiviral treatments targeting virus fitness human immunodeficiency virus type fitness and tropism: concept, quantification, and clinical relevance hiv- drug resistance and resistance testing antigenic determinants of possible vaccine escape by porcine circovirus subtype b viruses trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses naturally occurring ns -protease-inhibitor resistant mutant a t in the liver of an untreated chronic hepatitis c patient vaccine development: from concept to early clinical testing molecular and functional bases of selection against a mutation bias in an rna virus molecular epidemiology of hepatitis b virus mutants associated with vaccine escape, drug resistance and diagnosis failure patterns of resistanceassociated substitutions in patients with chronic hcv infection following treatment with direct-acting antivirals multiclass hcv resistance to direct-acting antiviral failure in real-life patients advocates for tailored secondline therapies rna virus evolution and the control of viral disease complications of rna heterogeneity for the engineering of virus vaccines and antiviral agents quasispecies dynamics in disease prevention and control quasispecies and rna virus evolution: principles and consequences virus population dynamics, fitness variations and the control of viral disease: an update viral quasispecies evolution. microbiol naturally occurring ns resistance-associated variants in hepatitis c virus genotype : their relevance for developing countries. virus res spectrum and characteristics of the virus inhibitory action of -(alpha-hydroxybenzyl)-benzimidazole coxsackie a virus: mutation from drug dependence to drug independence address in pathology on chemotherapy viral infections of humans. epidemiology and control. plenum medical book company transmission of single hiv- genomes and dynamics of early immune escape revealed by ultra-deep sequencing deep sequencing of protease inhibitor resistant hiv patient isolates reveals patterns of correlated mutations in gag and protease detection of a sexually transmitted hepatitis c virus protease inhibitor-resistance variant in a human immunodeficiency virus-infected homosexual man lessons from nature: understanding immunity to hcv to guide vaccine design human immunodeficiency virus gag and protease: partners in resistance barrier-independent, fitness-associated differences in sofosbuvir efficacy against hepatitis c virus imperfect vaccination: some epidemiological and evolutionary consequences poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches coxsackievirus b mutator strains are attenuated in vivo principles of pharmacology: the pathophysiologic basis of drug therapy a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease systems vaccinology: enabling rational vaccine design with systems biological approaches global and regional molecular epidemiology of hiv- , - : a systematic review, global survey, and trend analysis nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients evolution of the hcv viral population from a patient with s t detected at relapse after sofosbuvir monotherapy genetic and molecular analyses of spontaneous mutants of human rhinovirus that are resistant to an antiviral compound the impact of hiv- genetic diversity on the efficacy of a combinatorial rnaibased gene therapy a working hypothesisevirus resistance development as an indicator of specific antiviral activity time to hit hiv, early and hard changes of hepatitis b surface antigen variants in carrier children before and after universal vaccination in taiwan comparison of naturally occurring resistanceassociated substitutions between and in chinese patients with chronic hepatitis c virus infection vaccination influences the evolution of classical swine fever virus minority hiv- drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy genetic variability of porcine circovirus in vaccinating and non-vaccinating commercial farms outbreak of poliomyelitis in hispaniola associated with circulating type vaccinederived poliovirus influenza. plenum medical book company my cousin, my enemy: quasispecies suppression of drug resistance genetic and antigenic diversity of human rotaviruses: potential impact on vaccination programs rna virus population diversity, an optimum for maximal fitness and virulence in vivo sequence diversity of the protease of human immunodeficiency virus type : presence of protease inhibitor-resistant variants in untreated subjects hiv and the pathogenesis of aids dispelling myths and focusing on notable concepts in hiv pathogenesis vaccination and timing influence siv immune escape viral dynamics in vivo susceptibility of influenza a viruses to amantadine is influenced by the gene coding for m protein comparison of the mechanisms of drug resistance among hiv, hepatitis b, and hepatitis c hiv- reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo incomplete neutralization and deviation from sigmoidal neutralization curves for hiv broadly neutralizing monoclonal antibodies combination sirna therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro rapid development of drug-resistant mutants of poliovirus molecular basis of human immunodeficiency virus type drug resistance: overview and recent developments antiretroviral therapy and drug resistance in human immunodeficiency virus type infection development of streptomycin resistant strains of tubercle bacilli in pulmonary tuberculosis; results of simultaneous sensitivity tests in liquid and on solid media quantitation of zidovudine-resistant human immunodeficiency virus type in the blood of treated and untreated patients a systematic review of hepatitis b virus (hbv) drug and vaccine escape mutations in africa: a call for urgent action quasispecies dynamics in disease prevention and control exploration of sequence space as the basis of viral rna genome segmentation pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy impact of novel ns a resistance-associated substitutions of hepatitis c virus detected in treatment-experienced patients hepatitis c virus population dynamics during infection retreatment of hepatitis c virus infected patients with direct-acting antiviral failures influence of mutagenesis and viral load on the sustained low-level replication of an rna virus response of hepatitis c virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype molecular basis of interferon resistance in hepatitis c virus baseline hepatitis c virus resistance-associated substitutions present at frequencies lower than % may be clinically significant delayed lysis confers resistance to the nucleoside analogue -fluorouracil and alleviates mutation accumulation in the single-stranded dna bacteriophage vx hepatitis a virus vaccine escape variants and potential new serotype emergence increased fidelity reduces poliovirus fitness under selective pressure in mice production of guanidineresistant and -dependent poliovirus mutants from cloned cdna: mutations in polypeptide c are directly responsible for altered guanidine sensitivity imperfect vaccination can enhance the transmission of highly virulent pathogens the politics of polio in northern nigeria hiv- protease mutations and protease inhibitor cross-resistance production of resistant hiv mutants during antiretroviral therapy resistance, drug failure, and disease progression antiviral drug resistance rapid evolution of the neutralizing antibody response to hiv type infection new vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment and natural selection hiv- phylogenetics and vaccines vaccine strategies alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models resistance to direct antiviral agents in patients with hepatitis c virus infection deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype- b hepatitis c virus animal vaccination and the evolution of viral pathogens increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics mutations in coronavirus nonstructural protein decrease virus replication fidelity microbial threats to health. emergence, detection and response natural hcv variants with increased replicative fitness due to ns helicase mutations in the c-terminal helix a evolution of treatmentemergent resistant variants in telaprevir phase clinical trials a public health approach to hepatitis c control in low-and middle-income countries infrequent development of resistance to genotype - hepatitis c virus-infected subjects treated with sofosbuvir in phase and clinical trials strategies and challenges for eliciting immunity against avian influenza virus in birds a large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo recombination in the alphaherpesvirus bovine herpesvirus detection of human immunodeficiency virus (hiv) type m v and k n minority variants in patients with primary hiv infection quantitative deep sequencing reveals dynamic hiv- escape and large population shifts during ccr antagonist therapy in vivo basic research in hiv vaccinology is hampered by reductionist thinking sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes quasispecies diversity determines pathogenesis through cooperative interactions in a viral population hiv- polymerase inhibition by nucleoside analogs: cellular-and kinetic parameters of efficacy, susceptibility and resistance selection production of infectious hepatitis c virus in tissue culture from a cloned viral genome emergence of virus escape mutants after immunization with epitope vaccine genetic evolution of classical swine fever virus under immune environments conditioned by genotype -based modified live virus vaccine robust hepatitis c virus infection in vitro key: cord- -yqr e authors: attia, yasmeen m.; ewida, heba; ahmed, mahmoud salama title: successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma date: - - journal: drug repurposing in cancer therapy doi: . /b - - - - . - sha: doc_id: cord_uid: yqr e this chapter offers comprehensive overview for successful stories for drug repurposing/repositioning for affiliated molecular targets related to the progression of hepatocellular carcinoma (hcc). there are detailed sections about the etiology, pathogenesis, current therapeutic regimen, and affiliated molecular targets contributing to hcc. drug repositioning has been an emerging successful tool for the drug discovery process within the last decade to repurpose new clinical indications for already existing drugs with established safety and pharmacokinetics profiles. in this chapter, we focused on correlating the adopted strategies for drug repositioning targeting molecular targets affiliated with hcc along with different drugs preidentified for other clinical indication. hepatocellular carcinoma (hcc) is a major health problem with devastating consequences associated with treatment failure. it has an estimated global incidence of more than , new cases annually [ ] . hcc is currently the second leading cause of cancer-related death worldwide and accounts for % of cases with primary liver cancer [ ] . several risk factors contribute to hcc development such as liver cirrhosis, viral hepatitis including hepatitis b virus (hbv) and hepatitis c virus (hcv) infections, fatty liver, and alcohol abuse. smoking and the fungal carcinogen, aflatoxin b , are also well-known contributors to hcc [ ] . recent advances in hbv vaccination and antiviral therapeutics have remarkably contributed to a decrease in incidence. however, nonalcoholic fatty liver disease (nafld) and its progressive form, nonalcoholic steatohepatitis (nash), at its current pace of growing prevalence approaching epidemic proportions is projecting as the most common underlying etiology of hcc, presented in almost % of cases, which made nafld-associated hcc an emerging indication for liver transplantation [ e ] . additionally, hcc has a notable gender predilection where incidence in men is threefold that in women [ , ] . over the past decade, substantial progress has been achieved in understanding how hcc develops and progresses in an attempt to improve treatment options [ ] . hcc is a very heterogeneous disease in terms of both phenotype and genotype. this heterogeneity could be attributed, in part, to the divergent nature of the contributing factors, the complexity of the liver microenvironment, and the stage at which hcc turns to be clinically evident/detectable. malignant transformations in liver cells are driven by several factors such as chronic injury or inflammation due to oxidative stress that may lead to genetic and epigenetic modification. these modifications consequently lead to disrupted cellular signaling pathways leading to an overexpression in several growth factors and their receptors. this inevitably results in cell resistance to apoptotic signals, stimulation of angiogenesis, and uncontrollable proliferation besides the acquisition of a metastatic phenotype [ , ] . since hcc almost exclusively develops in patients with chronic liver diseases, injury of liver cells can promote the progression to hcc over a long period of time [ ] driven by a number of cytokines and inflammatory mediators along with aberrant activity of several signaling pathways, as shown in fig. . (will be discussed later). hcc mostly exhibits resistance to conventional chemotherapy. besides, patients with hcc are usually intolerant to treatment due to an underlying hepatic dysfunction. . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma however, almost half of hcc patients still receive chemotherapy at some point during the course of the disease [ , ] . chemotherapeutic intervention mainly relies on the multitargeted tyrosine kinase, sorafenib, that acts by effectively blocking the ras/raf/mapk pathway impeding cancer cell ability to circumvent apoptotic signals and induce angiogenesis, proliferation, and invasion. sorafenib was found to extend the median survival in advanced hcc patients for up to months with manageable adverse effects; however, it lacks predictive biomarkers to reflect on responsiveness [ ] . sorafenib remained the only approved systemic treatment for hcc between and . yet, promising outcomes were reported in randomized phase iii trials using other multiple kinase inhibitors such as lenvatinib [ ] , regorafenib [ ] , cabozantinib [ ] , and ramucirumab [ , ] , where regorafenib has received fda approval in the second-line setting. moreover, nivolumab, a monoclonal immunotherapy-based antibody targeting the immune checkpoint programmed cell death protein , showed also positive response rates and mean overall survival durations in a phase ieii trials performed on patients who were formerly treated with sorafenib [ ] for which it has been successfully granted an accelerated fda approval. on the other hand, and c viruses (hbv and hcv), genetic and epigenetic alterations, dysbiosis and leaky gut, role of innate immunity through toll-like receptors- (tlr ) and kupffer cells (kcs), metabolic syndrome and its hepatic manifestation (nonalcoholic fatty liver disease), and adaptive immune cells (cd þ and cd þ t cells) whose role is still quite debatable. also, dendritic cells (dcs) and natural killer t cells (nk t cells) bridge innate with adaptive immune systems. several other kinase inhibitors such as sunitinib and erlotinib failed to show comparable improved survival rates especially in unrespectable hcc patients [ ] . sorafenib efficacy over other proposed interventions is most likely attributed to its ability to target several molecules and pathways in tumor cells as well as the microenvironment. also, the heterogeneous nature of hcc may limit the efficacy of other targeted therapeutic approaches possessing higher selectivity [ ] . hcc is undoubtedly a resistant type of cancer making treatment more challenging. despite the fact that systemic therapy enhanced survival rates in hcc patients, therapeutic outcomes are still incremental and inadequate, especially if compared to other types of cancer. sorafenib was the first fda-approved drug for treatment of patients with advanced hcc for its ability to increase the median overall survival. however, with the use of the newly developed and approved multiple kinase inhibitors, the median overall survival still remains almost year. moreover, de novo resistance developing to sorafenib has been recently heavily reported impeding its beneficial clinical applications [ e ]. resistance to sorafenib involves a cross talk between several pathways such as janus kinase/signal transducer and activator of transcription (stat ), phosphatidylinositol- -kinase (pi k)/akt/mammalian target of rapamycin (mtor), and hypoxiainducible pathways beside others [ ] . providing new treatment options for hcc, therefore, still remains an unmet medical need. accordingly, further insights into the molecular targets affiliated with the pathophysiology of hcc will be dissected in the following section. to date, we still have very few identified "druggable" drivers for hcc. however, most of the preclinical and clinical attempts to identify pharmacological interventions, whether new or repositioned ones, focused on signaling pathways implicated in disease progression. hence, investigating effect on oncogenic drivers for the identification of new molecular targeted therapies in hcc needs to be revisited. in order to provide pharmacological interventions for hcc, the main drivers and pathways involved have to be identified first. as it is the case with solid tumors, a simultaneous alteration in at least three signaling pathways and five to eight driver genes should take place for hcc to develop [ ] . the main drivers and pathways contributing to hcc development and progression will be discussed in the following section in more details. these include, but not limited to, ras/ raf/mapk, pi k/akt/mtor, wnt/b-catenin, hedgehog (hh), and il- /stat pathways. moreover, the role of the following key players will be explored: tumor necrosis factor-alpha (tnf-a), nuclear factor kappa-b (nf-kb), c-jun n-terminal kinase (jnk), gut microbiome, and toll-like receptors and also adaptive immunity. all these molecular targets and signaling pathways represent potential candidates for targeting hcc, as shown in fig. . . among the most extensively investigated pathways implicated in hcc development and progression is the ras/raf/mapk pathway. cell surface receptor tyrosine kinases such as insulin-like growth factor (igf) receptor, endothelial and vascular epidermal growth factor receptor (egfr), c-met, and platelet-derived growth factor receptor send signals that are transmitted to the nucleus via this pathway to control cell survival, growth, and differentiation. upregulation of the ras/raf/mapk pathway in liver cells induces cell growth augmenting antiapoptotic signals leading ultimately to hcc [ ] . aberrant upstream igf and egfr signaling, suppression of raf kinase inhibitor protein, . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma and hbv-and hcv-related proteins induction are among the mechanisms by which the ras/ raf/mapk pathway can be activated toward hcc development [ ] . mounting evidence over the past years suggested the aberrant upregulation of pi k/ akt/mtor pathway in hcc where mtor phosphorylation and a subsequent upregulation of its downstream effector, p s k, were reported in almost % of hcc patients [ ] . likewise, almost % of patients encountered an activated mtor [ ] . aberrant pi k/akt/ mtor pathway was also correlated with poor prognosis, especially in advanced-stage hcc [ ] . the underlying mechanism by which pi k/akt/mtor pathway is activated in hcc is not yet fully deciphered, yet overly expressed upstream igf, c-met, or egfr is likely [ e ]. hepatitis viral infections are also capable of activating pi k/akt/mtor pathway in liver [ ] , where hcv infection was found to increase neuroblastoma (n)-ras expression in hcc, which turns on the pi k/akt/mtor pathway [ ] . loss of pten, a tumor suppressor gene, by mutations was also evident in hcc patients [ ] , contributing to akt activation. apart from pten, mutations in pi k catalytic subunits were also reported [ ] . based on the previously presented sound evidence from previous studies, different agents that target the main key players of this pathway such as pi k, akt, or mtor are being tested on hcc animal models and others currently enrolled in clinical trials [ , ] . the wnt pathway comprises a canonical or b-cateninedependent pathway as well as a noncanonical or b-catenineindependent one. the classic or canonical pathway consists of the wnt protein and wnt protein ligand, also known as frizzled protein, besides other proteins such as b-catenin and glycogen synthase kinase [ , ] . aberrant activation of wnt/b-catenin pathway was found to support tumor growth. when the upstream wnt protein complexes with its ligand, b-catenin builds up in cells and gets transferred to the nucleus where it dimerizes with lef/tcf, a downstream transcription factor that regulates the transcription of other genes such as cyclin d [ ] . abnormal upregulation of wnt/b-catenin pathway was linked to hcc development and cancer stem cell maintenance. aberrant b-catenin was detected in almost % of liver cancers [ ] . hh signaling pathway is among the most important pathways implicated in hcc. it consists of hh ligand, ptch and smo (two transmembrane receptors), and glib nuclear transcription factor along with downstream genes. once activated, hh ligands bind to ptch receptors blocking ptch inhibitory effect on smo. smo then moves to the cytoplasm activating gli, to induce specific genes upregulation, thereby controlling growth and division. the hh pathway is rarely activated in normal liver cells; however, in hcc, it is abnormally active [ , ] . in hcc, the inhibition of the gli gene can also cause bcl- and c-myc downregulation, while increasing the expression of p , turning off the cell cycle, and thus impeding tumor growth [ ] . tnf-a is a pivotal protumorigenic cytokine as it is capable of stimulating both nf-kb and jnk signaling pathways [ , ] . it is currently widely believed that inflammation is the fuel that feeds the genetic aberration sparks that inaugurate the tumorigenic process. moreover, the wide spectrum of chronic injury etiologies in the liver can sufficiently act as both initiators and promoters of hcc. in this last decade, several inflammatory mediators were identified and their roles were extensively elucidated in the pathogenesis of chronic liver disease [ ] . most of these inflammatory mediators act as activators or targets for nf-kb [ , ] . nf-kb is considered a key transcriptional factor for almost all chronic liver diseases such as viral hepatitis, alcoholic liver disease, and nash [ e ]. it also finely tunes crucial functions in liver cells such as kupffer cells (kcs; hepatic resident macrophages) and hepatic stellate cells (hscs). inhibition of nf-kberelated signaling cascades, however, may lead to liver fibrosis and tumorigenesis and that is why nf-kb is considered very essential in maintaining homeostasis and wound-healing processes in the liver [ ] . accordingly, nf-kb is thought of as a twoedged sword since inhibition may be sometimes beneficial; however, it may also influence other essential processes pertaining to liver homeostasis. regarding jnk, two isoforms are expressed in liver cells, namely, jnk and jnk , where the former being linked to carcinogenesis. jnk protumorigenic potential is defined by its ability to induce the proliferative potential of hcc [ ] . it also regulates the proliferation of hcc cells via downregulation of p and upregulation.of c-myc [ ] . moreover, apoptosis induced by caspase- was found to activate jnk and hence . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma promote proliferation of liver cells [ ] . these findings propose that hepatocyte apoptosis is what triggers jnk activation. although, the role of jnk in hcc is controversial, jnk and knockout attenuated hcc in an animal model of den, exhibited p upregulation, and decreased c-myc [ ] . overall, tnfa, nf-kb, and jnk pathways can either have prosurvival or proapoptotic potentials augmenting hcc proliferation and growth. thus, treatment approaches involving nf-kb and jnk inhibition should act moderately to avoid a complete shutdown in hepatocytes leading to liver injury and subsequent hcc. il- /stat pathway il- , as a mediator of stat activation, is an essential driver of liver cell proliferation, which may consequently lead to hcc [ ] . moreover, overactive stat coinciding with high levels of il- was found in patients with hcc [ ] . likewise, stat upregulation had promoted den-induced hcc experimentally [ ] . il- autocrine production is essential for malignant transformations in hcc, which once develops, il- paracrine production from kcs start initiating growth and proliferation of hcc cells [ ] . activation of stat may also take place via il- , produced by th cells, that is overly expressed in hcc patients. similar to il- , il- also plays a role in promoting den-induced hcc in mice via stat pathway [ ] . besides il- and il- , il- can also activate stat . this action, however, is il- dependent [ ] collectively, these findings propose a role for il- /stat pathway in promoting hepatic carcinogenesis suggesting it as a potential therapeutic target for treatment of hcc. overwhelming evidence from the past few decades have underlined the role of dysbiosis in the development of chronic liver disease and hcc. moreover, the role of activating the innate immune systemerelated receptors in general and toll-like receptors (tlrs) in particular, in hcc development, and progression was also explored [ ] . due to its anatomical location, the liver is considered the first organ exposed to gut-derived microbial products translocated through the portal vein that in turn leads to tlr activation in the liver [ ] . tlr- is expressed in different liver cells such as kcs, hscs, and hepatocytes. it was previously reported that activation of tlr- on liver cells by bacterial lipopolysaccharides, a gramnegative cell wall component, leads to subsequent fibrotic and carcinogenic events [ ] . the immune surveillance hypothesis suggests that the immune system may protect against nascent tumors by destroying malignant cells early on, before they develop into detectable tumors. the t-cell antitumor role is facilitated through immune surveillance by cd þ and cd þ t cells. interestingly, an animal model of nash-associated hcc was found to cause a depletion of cd þ t cells impairing immune surveillance [ ] . other studies, however, reported a protumorigenic potential of cd þ t, natural killer t cells, and t-helper (th) cells in an animal model of choline-deficient high fat diet [ ] . yet still debatable, these findings suggest a role for adaptive immunity in hcc development and progression. thus, targeting it in the appropriate context may offer a chemopreventive strategy for hcc patients. drug discovery process involves multidisciplinary integrating experiences starting with in silico computational modeling to design scaffolds of interest based on the affiliated therapeutic molecular targets to different clinical diseases. typically, this is followed by synthetic methodologies optimization to assemble final ligands ready for biological evaluation [ ] . preclinical biological evaluations involving in vitro and in vivo assessments create significant portion for getting the potential ligands to be considered as potential drug candidates or active pharmaceutical ingredients; however, there are different discovery stories that failed to make it into the market due to intellectual property, efficacy, safety, toxicity, biopharmaceutical compatibility, cost/benefit feasibility, regulatory affairs, etc. the whole discovery process typically takes e years to get a drug into the market at a cost close to billion us dollars. in the last decade ( e ), drug repurposing/repositioning approach has been an emerging drug discovery tool to overcome the associated challenges faced by the pharmaceutical industry. conceptually, the whole druge receptor binding theory focuses on two approaches, whether key-lock theory or induced-fit theory [ ] , where most of the pharmaceutical ingredients exhibit on-target effects targeting receptors/proteins of interest in a drug positioning fashion, as shown in fig. . . therefore, existing drugs might show potential binding interactions along with other molecular targets, where drug repositioning shows off-target effects to pronounce newly alternative therapeutic applications. drug repositioning offered excellent opportunity to offer more pharmaceutical candidates to the market with established postmarketing surveillance safety data, toxicity, and pharmacokinetics profile [ ] . this approach was adopted by the industrial, funding, and academic bodies to evaluate assets on the shelves by having a trilateral funding mechanism integrating universities along with pharmaceutical industry through national institute of health to support preclinical/clinical investigations for already existing drugs/assets with established efficacy and safety for novel clinical applications. most recently, the outbreak of the new strain of covid- virus has prompted the researchers to adopt the repurposing strategy to offer rapid therapeutic regimens targeting polymerase, protease, or spike protein. epositioning approach, where already existing molecule is repurposed for alternative molecular target (off-target mechanism) whether structurally or biochemically to reveal alternative therapeutic application. . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma the biological system is complicated enough to repurpose different pharmaceutical drugs for various clinical indications blindly based on off-targets screening, adverse effects reporting, serendipity, or clinical trials. in this section, there is a general chronological highlight for the drug development strategies development, as shown in fig. . . the whole arise of the drug repositioning integration started with blinded serendipityebased repositioning stories, such as sildenafil, minoxidil, and everolimus [ , ] . consequently, the strategic progress led to disease networkebased repositioning where certain diseases share same histological, pathological, pharmacological, or biochemical phenotypes; this certainly could be potential opportunity for testing the already established drugs for identified diseases to be evaluated for other different diseases having the same histopathological/biochemical features. the advancement of biological sciences made a huge transition from -ology era to -omics era; consequently, this showed huge impact on the transformation of the drug repositioning strategy; where the integration of proteomics, metabolomics, transcriptomics, and genomics heavily participated in the evolution of the drug repositioning. ultimately, this gave the opportunity for the high-throughput screeningebased repositioning to evolve; where the phenotypic assessment using cell lines, fluorescence assays, or enzyme kinetics and the genomic/proteomic-based assays by analyzing multigenomics profiling, mrna, and protein sequencing offered molecular signature for different molecular targets [ ] . this strategy helped potential drugs, such as metformin, digoxin, and statins to be repurposed for different types of cancer. the integration of computational tools has the adequate share for the advancement and validation of the drug repositioning strategy to evolve in silico computational screeningebased repositioning [ ] . this strategy helped the repositioning of nonsteroidal antiinflammatory drugs and proton-pump inhibitors for different types of cancer [ , ] . the main in silico screening involves different tactics: ( ) molecular structure similarity assessment using jaccard index or tanimoto coefficient calculations. ( ) molecular docking to validate the drugereceptor(s) interactions with respect to binding mode/energy with respect to enthalpy and entropy. ( ) dealing with huge consortium of data on the molecular and structural levels applying artificial intelligence network to create algorithm connecting the structural input along with molecular output. the association of omics-based repositioning coupled with computational-based repositioning led to the appearance of systems biologye based repositioning, for instance, digoxin was repurposed for medulloblastoma [ ] . in this chapter, the authors will focus on drug repositioning stories to target hcc, showing the affiliated molecular targets identifying the repurposing strategy, as shown in table . . the complexity of hcc leads to multiple mutations or malfunction of different receptors, proteins, and molecular targets. pimozide is an antipsychotic agent that showed potential inhibitory in vitro effect against cell proliferation of hcc cell lines via induction of apoptosis at g /g phase. in addition, pimozide exhibited inhibitory profile for hcc stem-like cells, particularly the cd positive cells side population. pimozide was found to target stat expression via luciferase assay activity along with downregulation of the transcription levels of downstream oncogenes for stat signaling. the antitumor activity for pimozide was further validated in vivo in nude mice [ ] . valproic acid (vpa), a potent and specific histone deacetylase (hdac) inhibitor, is widely used antiepileptic drug. hdac has been recognized for its significant role as identified target with known molecular signature in the progression of different types of cancer. the mono-and adjuvant therapeutic in vitro effects of vpa and doxorubicin (dox) managed to identify specific, efficient, and antiproliferative profiles of vpa and dox combination against hepg cell line in a synergistic manner. molecular levels of caspase- and poly (adp-ribose) polymerase (parp) activation validated the synergism caused by vpa and dox to induce apoptosis. dual treatment of vpa and dox increased the levels of reactive oxygen species (ros) [ ] . the antitumor activity of niclosamide and its ethanolamine salt (nen) were discovered by computational identification of drugs that can increase the mrna expression of downregulated genes in hcc and reduce the mrna expression of the upregulated ones. this was followed by in vitro evaluation to show the antiproliferative activity of niclosamide and nen in various hcc cell lines and primary human hepatocytes. this was further validated by in vivo evaluation against two mouse models . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma (genetically induced liver tumors and patientderived xenografts [pdxs]) for hcc to show significant reduction in the tumor growth after oral administration of nen compared to niclosamide. the dual administration of nen and sorafenib also showed significant improvement to reduce the progression of pdx compared to either sole drug treatment. in hepg cell lines and pdx models, administration of niclosamide or nen showed unique molecular signature in terms of gene expression compared to hcc molecular signature. administration of nen to pdx model reduced expression of proteins in the wnt/b-catenin, stat , akt/mtor egfr/ras/raf signaling pathways, and disruptive interactions along with heat shock protein [ ] . amiodarone, a class iii antiarrhythmic agent and a potent mtor inhibitor, was found to suppress liver tumor formation through induction of autophagy activity in the rat orthotropic model and in the mouse xenograft model. furthermore, a big data analysis of , caseecontrol provided by taiwan's national health insurance program revealed that long-term regular amiodarone usage significantly decreases the risk of hcc. amiodarone, as a repurposed drug, has antitumor potential to suppress liver tumor formation and prevent hcc incidence through induction of the autophagy activity [ ] . lanatoside c lanatoside c is an antiarrhythmic agent, naturally occurring compound extracted from digitalis lanata. integration of systems biology repositioning approach showed the potential of lanatoside c to tackle hcc at the in vitro and in vivo levels with significant reduction in tumor growth. the molecular mechanistic investigation revealed the ability of lanatoside c to trigger mitochondrial membrane potential (mmp) loss, followed by activation of apoptotic markers to induce cell death. inhibition of thr phosphorylation for protein kinase delta (pkcd) reversed lanatoside ceinduced mmp loss and apoptosis validating the molecular mechanism for lanatoside c, where akt/mtor pathway is involved via modulation of pkcd activation [ ] . statins are hmg-coa reductase inhibitors, to intervene with the cholesterol synthesis via inhibiting mevalonate pathways targeting different cardiovascular system complications. statins were identified as potential drugs tackling various types of cancer, including breast cancer, prostate, head, and hcc using highthroughput activityebased screens of disease phenotypes and in silico data-driven algorithms. simvastatin induced cell cycle arrest at g /g phase accompanied by series of molecular changes via promoting amp-activated protein kinase (ampk), leading to induction of p and p accumulation. using the transcriptomics, simvastatin showed significant reduction in the skp expression, resulting in p accumulation by preventing proteasomal degradation, mediated by stat inhibition. in addition, simvastatin significantly decreased tumor growth in hepg xenograft mice [ ] . guanabenz acetate, an antihypertensive drug, was screened as a potential candidate for hcc via a phenotypic screening assay by in vitro antiproliferative activity against hcc cell lines using high-throughput screeningebased repositioning approach. guanabenz acetate reduced hcc cell viability via inhibition of growth and induction of dna damage, leading to increased phosphorylation of eukaryotic initiation factor a, increased activation of transcription factor , and induction of apoptosis [ ] . fenofibrate is known for its lipid lowering capability; however, it has shown huge potential to target various types of cancer recently. fenofibrate showed significant reduction in the viability of human hepg cells via necrosis, but not apoptosis. the mechanistic investigation for the fenofibrate revealed an increase in the levels of ros and a decrease in glutathione (gsh, an important cellular antioxidant) accompanied with impairment for the mitochondrial function in hepg cell lines. in other studies, fenofibrate showed cell cycle arrest for huh- cell lines at g /m phase through downregulation of cyclins group with upregulation of p . fenofibrate also activated endogenous peroxisome proliferator-activated receptor (ppar)a in huh , hepg , and li cell lines, but the antiproliferative activity induced by fenofibrate was not affected by the ppara inhibitor gw or the knockdown of the expression of ppara by sirna. moreover, fenofibrate suppressed akt phosphorylation and increased the expression of c-terminal modulator protein, which binds specifically to akt [ , ] . metformin is the drug of choice toward treatment of type ii diabetes. the in vitro antiproliferative activity for metformin showed multiple . successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma phenotypic events starting with induction of cell cycle arrest at g /g phase accompanied with significant reduction in cell growth with elevated levels of klf /p protein content in hepg- cell line. metformin played a unique role via modulating the microenvironment of tumors by reducing cellular lipid accumulation and promoting ampk activity. metformin downregulated the expression of igf i and ii [ ] . canagliflozin, a sodiumeglucose cotransporter inhibitor (sglt -i), is known as an antidiabetic agent. canagliflozin was selected for the phenotypic screening based on the given fact of high expression levels of sglt in hcc cell lines. initially, the antiproliferative ability of canagliflozin against hcc cell lines was assessed to show inhibitory profile in a dosedependent manner along with induction of apoptosis at g /m phase with elevated levels of caspase- and inhibition of erk. this suggested that canagliflozin can inhibit glycolytic metabolism including glucose uptake, lactate, and intracellular atp production. this was validated by in vivo screening for tumor growth assay after oral administration of canagliflozin ( mg/kg/day) to show significant reduction in the tumor size and attenuated intratumor vascularization in hepg -derived xenograft tumors in balb/c nude mice [ ] . linagliptin is known as antidiabetic class targeting dipeptidyl peptidase- , where the scaffold of , dihydro- h-purine- , -dione functional group was an attractive scaffold to be structurally repositioned in a trial to target hcc by modulation of adenosine receptors (adora ) via in silico molecular modeling simulations coupled with in vitro analysis. linagliptin and its degradation product showed antiproliferative activity against hepg- and huh- cell lines inducing cell cycle arrest at g /m phase with elevated levels of apoptotic marker, caspase- . this was validated by monitoring the expression levels of adora [ ] . atovaquone, an fda-approved drug for pneumocystis pneumonia, significantly inhibited hepatoma cell proliferation via s phase cell cycle arrest and induced both extrinsic and intrinsic apoptotic pathways associated with upregulation of p and p . molecular investigations demonstrated that atovaquone inhibits hepatoma cell proliferation by inducing double-stranded dna breaks, leading to sustained activation of ataxia telangiectasia mutated and its downstream molecules such as cell cycle checkpoint kinase- and h ax. in addition, atovaquone also induced apoptosis, inhibited both cell proliferation and angiogenesis in vivo, and prolonged the survival time of tumor-bearing mice, without any obvious side [ ] . ketoconazole is a broad-spectrum antifungal agent, exhibiting antiproliferative activity against hcc cell lines by worsening mitophagy in vitro and in vivo. ketoconazole-induced suppression of prostaglandin-endoperoxide synthase led to pink -prknemediated mitophagy, resulting in mitochondrial dysfunction and induction of apoptosis [ ] . obeticholic acid (oca) was granted accelerated approval from the fda in for the treatment of primary biliary cholangitis. oca is a synthetically modified bile acid agonist for the nuclear farnesoid x receptor (fxr). a cross talk between il- /stat pathway and the hepatic fxr in hcc was previously reported [ ] . in this study, activating fxr using oca interfered with hcc cell growth by downregulating il- /stat pathway in vitro. these effects were hampered in the presence of an fxr antagonist. drug repositioning has become a significant emerging shortcut approach to deliver chemical entities into the pharmaceutical market cutting cost with an eye on the established toxicity profile, pharmacokinetics behavior, and postmarketing safety surveillance. however, repurposing novel clinical indications for already existing molecules should not grab our attention from investing in assembly of novel chemical entities to different molecular targets for different pathological conditions. conceptually, the repositioning approach should be good platform for novel scaffold repurposing to synthesize novel ligands and pharmacophores. in this chapter, we have outlined the repositioning as an excellent opportunity to bypass the toxicity issues, where the fda-approved drugs show clinical safety profiles including cardiac, hepatic, and metabolic safety profiles. the authors tried to cover the serious repurposing trials in that field, where most of the repositioned drugs for hcc were revealed via phenotypic screening or systems biology without rational approaches with respect to structural or ligand-based drug design. however, the literature is full of random repositioning studies without solid understanding for fundamental basics for in vitro/in vivo biological assessment coupled with in silico molecular modeling simulations with no revealing for molecular target validation. the authors believe that despite the huge efforts for drug repositioning targeting hcc, amiodarone can be considered as a potential therapeutic drug for hcc due to its repositioning strategy counting on disease network, where data analysis of , caseecontrol provided by taiwan's nhi program revealed that long-term regular amiodarone usage significantly decreases the risk of hcc. technically, the integration of artificial intelligence to get business solutions, servers, and healthcare platforms, such as watson developed by ibm, can create potential opportunity for drug repositioning to offer robust methodology driving more drugs to clinical trials for novel therapeutic applications. global cancer statistics global and country underestimation of hepatocellular carcinoma (hcc) in and its implications nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace population-based risk factors and resource utilization for hcc: us perspective impact of sex on the survival of patients with hepatocellular carcinoma: a surveillance, epidemiology, and end results analysis genetic landscape and biomarkers of hepatocellular carcinoma novel advancements in the management of hepatocellular carcinoma in environmental factors and risk for hepatocellular carcinoma immune pathogenesis of hepatocellular carcinoma management of hepatocellular carcinoma: an update hepatocellular carcinoma successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma molecular therapies and precision medicine for hepatocellular carcinoma lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase non-inferiority trial regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (resorce): a randomised, double-blind, placebocontrolled, phase trial cabozantinib in patients with advanced and progressing hepatocellular carcinoma reach- : a randomized, double-blind, placebo-controlled phase study of ramucirumab versus placebo as second-line treatment in patients with advanced hepatocellular carcinoma (hcc) and elevated baseline alpha-fetoprotein (afp) following first-line sorafenib ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased a-fetoprotein concentrations (reach- ): a randomised, double-blind, placebo-controlled, phase trial nivolumab in patients with advanced hepatocellular carcinoma (checkmate ): an open-label, non-comparative, phase / dose escalation and expansion trial hepatocellular carcinoma: reasons for phase iii failure and novel perspectives on trial design advances in targeted therapies for hepatocellular carcinoma in the genomic era sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors dnmt b/oct expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through il- /stat regulation treatment with a new benzimidazole derivative bearing a pyrrolidine side chain overcomes sorafenib resistance in hepatocellular carcinoma non-coding rnas: emerging regulators of sorafenib resistance in hepatocellular carcinoma new knowledge of the mechanisms of sorafenib resistance in liver cancer loss of hepatic nf-kb activity enhances chemical hepatocarcinogenesis through sustained c-jun n-terminal kinase activation molecular therapies in hepatocellular carcinoma: what can we target? synergistic inhibition of hcc and liver cancer stem cell proliferation by targeting ras/raf/mapk and wnt/b-catenin pathways mtor and p s kinase expression in primary liver neoplasms mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation the mtor pathway is associated with the poor prognosis of human hepatocellular carcinoma u-pa and c-met mrna expression is co-ordinately enhanced while hepatocyte growth factor mrna is down-regulated in human hepatocellular carcinoma hepatocyte growth factor, transforming growth factor a, and their receptors as combined markers of prognosis in hepatocellular carcinoma igf activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of igf- r blockage the hepatitis b virus-x protein activates a phosphatidylinositol -kinase-dependent survival signaling cascade activation of the n-ras-pi k-akt-mtor pathway by hepatitis c virus: control of cell survival and viral replication associations of components of pten/ akt/mtor pathway with cancer stem cell markers and prognostic value of these biomarkers in hepatocellular carcinoma functional role of sgk in pi k/ pten driven liver tumor development microrna- , which is regulated by lncrna duxap , inhibits cell proliferation by targeting the gpr -mediated pi k/akt/mtor pathway in hcc capsaicin and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma activity by suppressing egfr and pi k/akt/mtor signaling activation of beta-catenin during hepatocarcinogenesis in transgenic mouse models: relationship to phenotype and tumor grade wnt/b-catenin signaling in liver health and disease hedgehog signaling blockade delays hepatocarcinogenesis induced by hepatitis b virus x protein hedgehog signaling pathway regulates autophagy in human hepatocellular carcinoma cells selective down-regulation of gliomaassociated oncogene inhibits the proliferation of hepatocellular carcinoma cells the immunology of hepatocellular carcinoma review-article inflammation, immunity and cancer: coming of age inflammation and liver cancer: molecular mechanisms and therapeutic targets nf-kappab, an evolutionarily conserved mediator of immune and inflammatory responses missing pieces in the nf-kappab puzzle not interferon, but interleukin- controls early gene expression in hepatitis b virus infection nuclear factor-kb in the liver of patients with chronic hepatitis c: decreased rela expression is associated with enhanced fibrosis progression the role of inflammation in cholestasis: clinical and basic aspects signalling pathways in alcoholinduced liver inflammation lipid metabolism and liver inflammation ii tak suppresses a nemodependent but nf-kb-independent pathway to liver cancer proliferation of human hcc cells and chemically induced mouse liver cancers requires jnk -dependent p downregulation rip inhibits inflammatory hepatocarcinogenesis but promotes cholestasis by controlling caspase- -and jnk-dependent compensatory cell proliferation the role of jnk in the development of hepatocellular carcinoma il- , through p-stat rather than p-stat , activates hepatocarcinogenesis and affects survival of hepatocellular carcinoma patients: a cohort study noncell-autonomous activation of il- /stat signaling mediates fgf -driven hepatocarcinogenesis hepatocyte ikkb/nf-kb inhibits tumor promotion and progression by preventing oxidative stress-driven stat activation xidentification of liver cancer progenitors whose malignant progression depends on autocrine il- signaling interleukin- promotes human hepatocellular carcinoma by activation of stat il- activates the il- /stat signal pathway in the proliferation of hepatitis b virusrelated hepatocellular carcinoma toll-like receptors in alcoholic liver disease, non-alcoholic steatohepatitis and carcinogenesis promotion of hepatocellular carcinoma by the intestinal microbiota and tlr nafld causes selective cd þ t lymphocyte loss and promotes hepatocarcinogenesis successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma metabolic inflammation-associated il- a causes non-alcoholic steatohepatitis and hepatocellular carcinoma the assessment of efficient representation of drug features using deep learning for drug repositioning protein flexibility and ligand recognition: challenges for molecular modeling computational drug repositioning. a lateral approach to traditional drug discovery? exploring the potential of minoxidil tretinoin liposomal based hydrogel for topical delivery in the treatment of androgenic alopecia agingrelated repositioned drugs, donepezil and sildenafil citrate, increase apoptosis of anti-mitotic drug-resistant kbv c cells through different molecular mechanisms screening-based approach to discover effective platinum-based chemotherapies for cancers with poor prognosis proton pump inhibitors can reverse the yap mediated paclitaxel resistance in epithelial ovarian cancer a journey of celecoxib from pain to cancer the neuroleptic drug pimozide inhibits stem-like cell maintenance and tumorigenicity in hepatocellular carcinoma valproic acid induces endocytosismediated doxorubicin internalization and shows synergistic cytotoxic effects in hepatocellular carcinoma cells computational discovery of niclosamide ethanolamine, a repurposed drug candidate that reduces growth of hepatocellular carcinoma cells in vitro and in mice by inhibiting cell division cycle signaling the prophylactic effect of amiodarone on hcc occurrence lanatoside c, a cardiac glycoside, acts through protein kinase cd to cause apoptosis of human hepatocellular carcinoma cells simvastatin-induced cell cycle arrest through inhibition of stat /skp axis and activation of ampk to promote p and p accumulation in hepatocellular carcinoma cells guanabenz acetate induces endoplasmic reticulum stress-related cell death in hepatocellular carcinoma cells cytotoxic effect of peroxisome proliferator fenofibrate on human hepg hepatoma cell line and relevant mechanisms fenofibrate suppresses growth of the human hepatocellular carcinoma cell via ppara-independent mechanisms metformin counteracts hcc progression and metastasis enhancing klf /p expression and downregulating the igf axis sodium glucose cotransporter inhibitor canagliflozin attenuates liver cancer cell growth and angiogenic activity by inhibiting glucose uptake structural re-positioning, in silico molecular modelling, oxidative degradation, and biological screening of linagliptin as adenosine receptor (adora ) modulators targeting hepatocellular carcinoma anti-malarial atovaquone exhibits anti-tumor effects by inducing dna damage in hepatocellular carcinoma a novel role for ketoconazole in hepatocellular carcinoma treatment: linking ptgs to mitophagy machinery the fxr agonist, obeticholic acid, suppresses hcc proliferation & metastasis: role of il- /stat signalling pathway key: cord- - yuw jo authors: zhou, yadi; hou, yuan; shen, jiayu; huang, yin; martin, william; cheng, feixiong title: network-based drug repurposing for novel coronavirus -ncov/sars-cov- date: - - journal: cell discov doi: . /s - - - sha: doc_id: cord_uid: yuw jo human coronaviruses (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov) and novel coronavirus ( -ncov, also known as sars-cov- ), lead global epidemics with high morbidity and mortality. however, there are currently no effective drugs targeting -ncov/sars-cov- . drug repurposing, representing as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. in this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the hcov–host interactome and drug targets in the human protein–protein interaction network. phylogenetic analyses of hcov whole genomes reveal that -ncov/sars-cov- shares the highest nucleotide sequence identity with sars-cov ( . %). specifically, the envelope and nucleocapsid proteins of -ncov/sars-cov- are two evolutionarily conserved regions, having the sequence identities of % and . %, respectively, compared to sars-cov. using network proximity analyses of drug targets and hcov–host interactions in the human interactome, we prioritize potential anti-hcov repurposable drugs (e.g., melatonin, mercaptopurine, and sirolimus) that are further validated by enrichment analyses of drug-gene signatures and hcov-induced transcriptomics data in human cell lines. we further identify three potential drug combinations (e.g., sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the “complementary exposure” pattern: the targets of the drugs both hit the hcov–host subnetwork, but target separate neighborhoods in the human interactome network. in summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations targeting -ncov/sars-cov- . coronaviruses (covs) typically affect the respiratory tract of mammals, including humans, and lead to mild to severe respiratory tract infections . in the past two decades, two highly pathogenic human covs (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), emerging from animal reservoirs, have led to global epidemics with high morbidity and mortality . for example, individuals were infected and died in the sars-cov pandemic, which cost the global economy with an estimated $ to $ billion , . according to the world health organization (who), as of november , mers-cov has had a total of diagnosed cases causing deaths, the majority in saudi arabia . in december , the third pathogenic hcov, named novel coronavirus ( -ncov/sars-cov- ), as the cause of coronavirus disease (abbreviated as covid- ) , was found in wuhan, china. as of february , there have been over , cases with over deaths for the -ncov/sars-cov- outbreak worldwide; furthermore, human-to-human transmission has occurred among close contacts . however, there are currently no effective medications against -ncov/sars-cov- . several national and international research groups are working on the development of vaccines to prevent and treat the -ncov/sars-cov- , but effective vaccines are not available yet. there is an urgent need for the development of effective prevention and treatment strategies for -ncov/sars-cov- outbreak. although investment in biomedical and pharmaceutical research and development has increased significantly over the past two decades, the annual number of new treatments approved by the u.s. food and drug administration (fda) has remained relatively constant and limited . a recent study estimated that pharmaceutical companies spent $ . billion in , up from $ million in , in the development of an fda-approved new chemical entity drug . drug repurposing, represented as an effective drug discovery strategy from existing drugs, could significantly shorten the time and reduce the cost compared to de novo drug discovery and randomized clinical trials [ ] [ ] [ ] . however, experimental approaches for drug repurposing is costly and time-consuming . computational approaches offer novel testable hypotheses for systematic drug repositioning [ ] [ ] [ ] , . however, traditional structure-based methods are limited when threedimensional ( d) structures of proteins are unavailable, which, unfortunately, is the case for the majority of human and viral targets. in addition, targeting single virus proteins often has high risk of drug resistance by the rapid evolution of virus genomes . viruses (including hcov) require host cellular factors for successful replication during infection . systematic identification of virus-host protein-protein interactions (ppis) offers an effective way toward elucidating the mechanisms of viral infection , . subsequently, targeting cellular antiviral targets, such as virus-host interactome, may offer a novel strategy for the development of effective treatments for viral infections , including sars-cov , mers-cov , ebola virus , and zika virus , [ ] [ ] [ ] . we recently presented an integrated antiviral drug discovery pipeline that incorporated gene-trap insertional mutagenesis, known functional drug-gene network, and bioinformatics analyses . this methodology allows to identify several candidate repurposable drugs for ebola virus , . our work over the last decade has demonstrated how network strategies can, for example, be used to identify effective repurposable drugs , [ ] [ ] [ ] [ ] [ ] [ ] and drug combinations for multiple human diseases. for example, network-based drug-disease proximity sheds light on the relationship between drugs (e.g., drug targets) and disease modules (molecular determinants in disease pathobiology modules within the ppis), and can serve as a useful tool for efficient screening of potentially new indications for approved drugs, as well as drug combinations, as demonstrated in our recent studies , , , . in this study, we present an integrative antiviral drug repurposing methodology, which combines a systems pharmacology-based network medicine platform that quantifies the interplay between the virus-host interactome and drug targets in the human ppi network. the basis for these experiments rests on the notions that (i) the proteins that functionally associate with viral infection (including hcov) are localized in the corresponding subnetwork within the comprehensive human ppi network and (ii) proteins that serve as drug targets for a specific disease may also be suitable drug targets for potential antiviral infection owing to common ppis and functional pathways elucidated by the human interactome ( fig. ) . we follow this analysis with bioinformatics validation of drug-induced gene signatures and hcovinduced transcriptomics in human cell lines to inspect the postulated mechanism-of-action in a specific hcov for which we propose repurposing (fig. ). to date, seven pathogenic hcovs (fig. a, b) have been found: , (i) -ncov/sars-cov- , sars-cov, mers-cov, hcov-oc , and hcov-hku are β genera, and (ii) hcov-nl and hcov- e are α genera. we performed the phylogenetic analyses using the wholegenome sequence data from hcovs to inspect the evolutionary relationship of -ncov/sars-cov- with other hcovs. we found that the whole genomes of -ncov/sars-cov- had~ . % nucleotide sequence identity across three diagnosed patients (supplementary table s ). the -ncov/sars-cov- shares the highest nucleotide sequence identity ( . %) with sars-cov among the six other known pathogenic hcovs, revealing conserved evolutionary relationship between -ncov/sars-cov- and sars-cov (fig. a) . hcovs have five major protein regions for virus structure assembly and viral replications , including replicase complex (orf ab), spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (fig. b) . the orf ab gene encodes the non-structural proteins (nsp) of viral rna synthesis complex through proteolytic processing . the nsp is a viral rna-dependent rna polymerase, together with co-factors nsp and nsp possessing high polymerase activity. from the protein d structure view of sars-cov nsp , it contains a larger n-terminal extension (which binds to nsp and nsp ) and polymerase domain (fig. c) . the spike is a transmembrane glycoprotein that plays a pivotal role in mediating viral infection through binding the host receptor , . figure d shows the d structure of the spike protein bound with the host receptor angiotensin converting enznyme (ace ) in sars-cov (pdb id: ack). a recent study showed that -ncov/sars-cov- is able to utilize ace as an entry receptor in ace -expressing cells , suggesting potential drug targets for therapeutic development. furthermore, cryo-em structure of the spike and biophysical assays reveal that the -ncov/sars-cov- spike binds ace with higher affinity than sars-cov . in addition, the nucleocapsid is also an important subunit for packaging the viral genome through protein oligomerization , and the single nucleocapsid structure is shown in fig. e . protein sequence alignment analyses indicated that the -ncov/sars-cov- was most evolutionarily conserved with sars-cov (supplementary table s ). specifically, the envelope and nucleocapsid proteins of -ncov/sars-cov- are two evolutionarily conserved regions, with sequence identities of % and . %, respectively, compared to sars-cov (supplementary table s ). however, the spike protein exhibited the lowest sequence conservation (sequence identity of %) between -ncov/sars-cov- and sars-cov. meanwhile, the spike protein of -ncov/sars-cov- only has . % sequence identity compared to mers-cov. fig. overall workflow of this study. our network-based methodology combines a systems pharmacology-based network medicine platform that quantifies the interplay between the virus-host interactome and drug targets in the human ppi network. a human coronavirus (hcov)-associated host proteins were collected from literatures and pooled to generate a pan-hcov protein subnetwork. b network proximity between drug targets and hcov-associated proteins was calculated to screen for candidate repurposable drugs for hcovs under the human protein interactome model. c, d gene set enrichment analysis was utilized to validate the network-based prediction. e top candidates were further prioritized for drug combinations using network-based method captured by the "complementary exposure" pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human interactome network. f overall hypothesis of the network-based methodology: (i) the proteins that functionally associate with hcovs are localized in the corresponding subnetwork within the comprehensive human interactome network; and (ii) proteins that serve as drug targets for a specific disease may also be suitable drug targets for potential antiviral infection owing to common protein-protein interactions elucidated by the human interactome. to depict the hcov-host interactome network, we assembled the cov-associated host proteins from four known hcovs (sars-cov, mers-cov, hcov- e, and hcov-nl ), one mouse mhv, and one avian ibv (n protein) (supplementary table s ). in total, we obtained host proteins associated with covs with various experimental evidence. specifically, these host proteins are either the direct targets of hcov proteins or are involved in crucial pathways of hcov infection. the hcov-host interactome network is shown in fig. a . we identified several hub proteins including jun, xpo , npm , and hnrnpa , with the highest number of connections within the proteins. kegg pathway enrichment analysis revealed multiple significant biological pathways (adjusted p value < . ), including measles, rna transport, nf-kappa b signaling, epstein-barr virus infection, and influenza (fig. b ). gene ontology (go) biological process enrichment analysis further confirmed multiple viral infection-related processes (adjusted p value < . ), including viral life cycle, modulation by virus of host morphology or physiology, viral process, positive regulation of viral life cycle, transport of virus, and virion attachment to host cell (fig. c ). we then mapped the known drug-target network (see materials and methods) into the hcov-host interactome to search for druggable, cellular targets. we found that human proteins ( %, blue nodes in fig. a) can be targeted by at least one approved drug or experimental drug under clinical trials. for example, gsk b, dpp , smad , parp , and ikbkb are the most targetable proteins. the high druggability of hcov-host interactome motivates us to develop a drug repurposing strategy by specifically targeting cellular proteins associated with hcovs for potential treatment of -ncov/sars-cov- . the basis for the proposed network-based drug repurposing methodologies rests on the notions that the proteins that associate with and functionally govern viral infection are localized in the corresponding subnetwork ( fig. a) within the comprehensive human interactome network. for a drug with multiple targets to be effective against an hcov, its target proteins should be within or in the immediate vicinity of the corresponding subnetwork in the human protein-protein interactome ( fig. ), as we demonstrated in multiple diseases , , , using this network-based strategy. we used a state-of-theart network proximity measure to quantify the relationship between hcov-specific subnetwork (fig. a) and drug targets in the human interactome. we constructed a drug-target network by assembling target information for more than fda-approved or experimental drugs (see materials and methods). to improve the quality and completeness of the human protein interactome network, we integrated ppis with five types of experimental data: ( ) binary ppis from d protein structures; ( ) binary ppis from unbiased high-throughput yeast-two-hybrid assays; ( ) experimentally identified kinase-substrate interactions; ( ) signaling networks derived from experimental data; and ( ) literature-derived ppis with various experimental evidence (see materials and methods). we used a z-score (z) measure and permutation test to reduce the study bias in network proximity analyses (including hub nodes in the human interactome network by literature-derived ppi data bias) as described in our recent studies , . in total, we computationally identified drugs that were associated (z < − . and p < . , permutation test) with the hcov-host interactome (fig. a , supplementary tables s and ). to validate bias of the pooled cellular proteins from six covs, we further calculated the network proximities of all the drugs for four covs with a large number of know host proteins, including sars-cov, mers-cov, ibv, and mhv, separately. we found that the z-scores showed consistency among the pooled hcov-associated proteins and other four individual covs (fig. b) . the pearson correlation coefficients of the proximities of all the drugs for the pooled hcov are . vs. sars-cov (p < . , t distribution), . vs. mers-cov (p < . ), . vs. ibv (p < . ), and . vs. mhv (p < . ). these network proximity analyses offer putative repurposable candidates for potential prevention and treatment of hcovs. to further validate the repurposable drugs against hcovs, we first performed gene set enrichment analysis (gsea) using transcriptome data of mers-cov and sars-cov infected host cells (see methods). these transcriptome data were used as gene signatures for hcovs. additionally, we downloaded the gene expression data of drug-treated human cell lines from the connectivity map (cmap) database to obtain drug-gene signatures. we calculated a gsea score (see methods) for each drug and used this score as an indication of bioinformatics validation of the drugs. specifically, an enrichment score (es) was calculated for each hcov data set, and es > and p < . (permutation test) was used as cut-off for a significant association of gene signatures between a drug and a specific hcov data set. the gsea score, ranging from to , is the number of data sets that met these criteria for a specific drug. mesalazine (an fig. a discovered drug-hcov network. a a subnetwork highlighting network-predicted drug-hcov associations connecting drugs and hcovs. from the drugs evaluated, ones achieved significant proximities between drug targets and the hcov-associated proteins in the human interactome network. drugs are colored by their first-level of the anatomical therapeutic chemical (atc) classification system code. b a heatmap highlighting network proximity values for sars-cov, mers-cov, ibv, and mhv, respectively. color key denotes network proximity (z-score) between drug targets and the hcov-associated proteins in the human interactome network. p value was computed by permutation test. approved drug for inflammatory bowel disease), sirolimus (an approved immunosuppressive drug), and equilin (an approved agonist of the estrogen receptor for menopausal symptoms) achieved the highest gsea scores of , followed by paroxetine and melatonin with gsea scores of . we next selected high-confidence repurposable drugs ( fig. a and table ) against hcovs using subject matter expertise based on a combination of factors: (i) strength of the network-predicted associations (a smaller network proximity score in supplementary table s ); (ii) validation by gsea analyses; (iii) literature-reported antiviral evidence, and (iv) fewer clinically reported side effects. specifically, we showcased several selected repurposable drugs with literature-reported antiviral evidence as below. an overexpression of estrogen receptor has been shown to play a crucial role in inhibiting viral replication . selective estrogen receptor modulators (serms) have been reported to play a broader role in inhibiting viral replication through the non-classical pathways associated with estrogen receptor . serms interfere at the post viral entry step and affect the triggering of fusion, as the serms' antiviral activity still can be observed in the absence of detectable estrogen receptor expression . toremifene (z = - . , fig. a ), the first generation of nonsteroidal serm, exhibits potential effects in blocking various viral infections, including mers-cov, sars-cov, and ebola virus in established cell lines , . compared to the classical esr -related antiviral pathway, toremifene prevents fusion between the viral and endosomal membrane by interacting with and destabilizing the virus membrane glycoprotein, and eventually inhibiting viral replication . as shown in fig. b , toremifene potentially affects several key host proteins associated with hcov, such as rpl , hnrnpa , npm , eif i, eif f, and eif e , . equilin (z = - . and gsea score = ), an estrogenic steroid produced by horses, also has been proven to have moderate activity in inhibiting the entry of zaire ebola virus glycoprotein and human immunodeficiency virus (zebov-gp/hiv) . altogether, network-predicted serms (such as toremifene and equilin) offer candidate repurposable drugs for -ncov/sars-cov- . angiotensin receptor blockers (arbs) have been reported to associate with viral infection, including hcovs [ ] [ ] [ ] . irbesartan (z = - . ), a typical arb, was approved by the fda for treatment of hypertension and diabetic nephropathy. here, network proximity analysis shows a significant association between irbesartan's targets and hcov-associated host proteins in the human interactome. as shown in fig. c , irbesartan targets slc a , encoding the sodium/bile acid cotransporter (ntcp) protein that has been identified as a functional pres -specific receptor for the hepatitis b virus (hbv) and the hepatitis delta virus (hdv). irbesartan can inhibit ntcp, thus inhibiting viral entry , . slc a interacts with c orf , a potential transcriptional repressor that interacts with nsp- of sars-cov . there are several other arbs (such as eletriptan, frovatriptan, and zolmitriptan) in which their targets are potentially associated with hcov-associated host proteins in the human interactome. previous studies have confirmed the mammalian target of rapamycin complex (mtorc ) as the key factor in regulating various viruses' replications, including andes orthohantavirus and coronavirus , . sirolimus (z = - . and gsea score = ), an inhibitor of mammalian target of rapamycin (mtor), was reported to effectively block viral protein expression and virion release effectively . indeed, the latest study revealed the clinical application: sirolimus reduced mers-cov infection by over % . moreover, sirolimus usage in managing patients with severe h n pneumonia and acute respiratory failure can improve those patients' prognosis significantly . mercaptopurine (z = - . and gsea score = ), an antineoplastic agent with immunosuppressant property, has been used to treat cancer since the s and expanded its application to several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and crohn's disease . (see figure on previous page) fig. a discovered drug-protein-hcov network for candidate repurposable drugs. a network-predicted evidence and gene set enrichment analysis (gsea) scores for potential repurposable drugs for hcovs. the overall connectivity of the top drug candidates to the hcovassociated proteins was examined. most of these drugs indirectly target hcov-associated proteins via the human protein-protein interaction networks. all the drug-target-hcov-associated protein connections were examined, and those proteins with at least five connections are shown. the box heights for the proteins indicate the number of connections. gsea scores for eight drugs were not available (na) due to the lack of transcriptome profiles for the drugs. b-e inferred mechanism-of-action networks for four selected drugs: b toremifene (first-generation nonsteroidalselective estrogen receptor modulator), c irbesartan (an angiotensin receptor blocker), d mercaptopurine (an antimetabolite antineoplastic agent with immunosuppressant properties), and e melatonin (a biogenic amine for treating circadian rhythm sleep disorders). , . mechanistically, mercaptopurine potentially target several host proteins in hcovs, such as jun, pabpc , npm , and ncl , (fig. d) . inflammatory pathways play essential roles in viral infections , . as a biogenic amine, melatonin (n-acetyl- -methoxytryptamine) (z = - . and gsea score = ) plays a key role in various biological processes, and offers a potential strategy in the management of viral infections , . viral infections are often associated with immune-inflammatory injury, in which the level of oxidative stress increases significantly and leaves negative effects on the function of multiple organs . the antioxidant effect of melatonin makes it a putative candidate drug to relieve patients' clinical symptoms in antiviral treatment, even though melatonin cannot eradicate or even curb the viral replication or transcription , . in addition, the application of melatonin may prolong patients' survival time, which may provide a chance for patients' immune systems to recover and eventually eradicate the virus. as shown in fig. e , melatonin indirectly targets several hcov cellular targets, including ace , bcl l , jun, and ikbkb. eplerenone (z = - . ), an aldosterone receptor antagonist, is reported to have a similar anti-inflammatory effect as melatonin. by inhibiting mast-cell-derived proteinases and suppressing fibrosis, eplerenone can improve survival of mice infected with encephalomyocarditis virus . in summary, our network proximity analyses offer multiple candidate repurposable drugs that target diverse cellular pathways for potential prevention and treatment of -ncov/sars-cov- . however, further preclinical experiments and clinical trials are required to verify the clinical benefits of these network-predicted candidates before clinical use. drug combinations, offering increased therapeutic efficacy and reduced toxicity, play an important role in treating various viral infections . however, our ability to identify and validate effective combinations is limited by a combinatorial explosion, driven by both the large number of drug pairs and dosage combinations. in our recent study, we proposed a novel network-based methodology to identify clinically efficacious drug combinations . relying on approved drug combinations for hypertension and cancer, we found that a drug combination was therapeutically effective only if it was captured by the "complementary exposure" pattern: the targets of the drugs both hit the disease module, but target separate neighborhoods (fig. a) . here we sought to identify drug combinations that may provide a synergistic effect in potentially treating -ncov/sars-cov- with welldefined mechanism-of-action by network analysis. for the potential repurposable drugs (fig. a, table ), we showcased three network-predicted candidate drug combinations for -ncov/sars-cov- . all predicted possible combinations can be found in supplementary table s . sirolimus, an inhibitor of mtor with both antifungal and antineoplastic properties, has demonstrated to improve outcomes in patients with severe h n pneumonia and acute respiratory failure . the mtor signaling plays an essential role for mers-cov infection . dactinomycin, also known actinomycin d, is an approved rna synthesis inhibitor for treatment of various cancer types. an early study showed that dactinomycin ( μg/ml) inhibited the growth of feline enteric cov . as shown in fig. b , our network analysis shows that sirolimus and dactinomycin synergistically target hcov-associated host protein subnetwork by "complementary exposure" pattern, offering potential combination regimens for treatment of hcov. specifically, sirolimus and dactinomycin may inhibit both mtor signaling and rna synthesis pathway (including dna topoisomerase -alpha (top a) and dna topoisomerase -beta (top b)) in hcov-infected cells (fig. b) . toremifene is among the approved first-generation nonsteroidal serms for the treatment of metastatic breast cancer . serms (including toremifene) inhibited ebola virus infection by interacting with and destabilizing the ebola virus glycoprotein . in vitro assays have demonstrated that toremifene inhibited growth of mers-cov , and sara-cov (table ) . emodin, an anthraquinone derivative extracted from the roots of rheum tanguticum, has been reported to have various anti-virus effects. specifically, emdoin inhibited sars-cov-associated a protein , and blocked an interaction between the sars-cov spike protein and ace (ref. ). altogether, network analyses and published experimental data suggested that combining toremifene and emdoin offered a potential therapeutic approach for -ncov/ sars-cov- (fig. c) . as shown in fig. a , targets of both mercaptopurine and melatonin showed strong network proximity with hcovassociated host proteins in the human interactome network. recent in vitro and in vivo studies identified mercaptopurine as a selective inhibitor of both sars-cov and mers-cov by targeting papain-like protease , . melatonin was reported in potential antiviral infection via its anti-inflammatory and antioxidant effects [ ] [ ] [ ] [ ] [ ] . melatonin indirectly regulates ace expression, a key entry receptor involved in viral infection of hcovs, including -ncov/sars-cov- (ref. ). specifically, melatonin was reported to inhibit calmodulin and calmodulin interacts with ace by inhibiting shedding of its ectodomain, a key infectious process of sars-cov , . jun, also known as c-jun, is a key host protein involving in hcov infectious bronchitis virus . as shown in fig. d , mercaptopurine and melatonin may synergistically block c-jun signaling by targeting multiple cellular targets. in summary, combination of mercaptopurine and melatonin may offer a potential combination therapy for -ncov/sars-cov- by synergistically targeting papainlike protease, ace , c-jun signaling, and antiinflammatory pathways (fig. d) . however, further experimental observations on ace pathways by melatonin in -ncov/sars-cov- are highly warranted. in this study, we presented a network-based methodology for systematic identification of putative repurposable drugs and drug combinations for potential treatment of -ncov/sars-cov- . integration of drug-target networks, hcov-host interactions, hcovinduced transcriptome in human cell lines, and human protein-protein interactome network are essential for such identification. based on comprehensive evaluation, we prioritized candidate repurposable drugs (fig. ) and potential drug combinations (fig. ) for targeting -ncov/sars-cov- . however, although the majority of predictions have been validated by various literature data (table ) , all network-predicted repurposable drugs and drug combinations must be validated in various -ncov/sars-cov- experimental assays and randomized clinical trials before being used in patients. we acknowledge several limitations in the current study. although -ncov/sars-cov- shared high nucleotide sequence identity with other hcovs (fig. ) , our predictions are not -ncov/sars-cov- specific by lack of the known host proteins on -ncov/sars-cov- . we used a low binding affinity value of μm as a threshold to define a physical drug-target interaction. however, a stronger binding affinity threshold (e.g., μm) may be a more suitable cut-off in drug discovery, although it will generate a smaller drug-target network. although sizeable efforts were made for assembling large scale, experimentally reported drug-target networks from publicly available databases, the network data may be incomplete and some drug-target interactions may be functional associations, instead of physical bindings. for example, silvestrol, a natural product from the flavagline, was found to have antiviral activity against ebola and coronaviruses . after adding its target, an rna helicase enzyme eif a , silvestrol was predicted to be significantly associated with hcovs (z = - . , p = . ) by network proximity analysis. to increase coverage of drug-target networks, we may use computational approaches to systematically predict the drug-target interactions further , . in addition, the collected virus-host interactions are far from completeness and the quality can be influenced by multiple factors, including different experimental assays and human cell line models. we may computationally predict a new virus-host interactome for -ncov/sars-cov- using sequence-based and structure-based approaches . drug targets representing nodes within cellular networks are often intrinsically coupled with both therapeutic and adverse profiles , as drugs can inhibit or activate protein functions (including antagonists vs. agonists). the current systems pharmacology model cannot separate therapeutic (antiviral) effects from those predictions due to lack of detailed pharmacological effects of drug targets and unknown functional consequences of virus-host interactions. comprehensive identification of the virus-host interactome for -ncov/sars-cov- , with specific biological effects using functional genomics assays , , will significantly improve the accuracy of the proposed network-based methodologies further. owing to a lack of the complete drug-target information (such as the molecular "promiscuity" of drugs), the dose-response and dose-toxicity effects for both (see figure on previous page) fig. network-based rational design of drug combinations for -ncov/sars-cov- . a the possible exposure mode of the hcovassociated protein module to the pairwise drug combinations. an effective drug combination will be captured by the "complementary exposure" pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human interactome network. z ca and z cb denote the network proximity (z-score) between targets (drugs a and b) and a specific hcov. s ab denotes separation score (see materials and methods) of targets between drug a and drug b. b-d inferred mechanism-of-action networks for three selected pairwise drug combinations: b sirolimus (a potent immunosuppressant with both antifungal and antineoplastic properties) plus dactinomycin (an rna synthesis inhibitor for treatment of various tumors), c toremifene (first-generation nonsteroidal-selective estrogen receptor modulator) plus emodin (an experimental drug for the treatment of polycystic kidney), and d melatonin (a biogenic amine for treating circadian rhythm sleep disorders) plus mercaptopurine (an antimetabolite antineoplastic agent with immunosuppressant properties). repurposable drugs and drug combinations cannot be identified in the current network models. for example, mesalazine, an approved drug for inflammatory bowel disease, is a top network-predicted repurposable drug associated with hcovs (fig. a ). yet, several clinical studies showed the potential pulmonary toxicities (including pneumonia) associated with mesalazine usage , . integration of lung-specific gene expression of -ncov/sars-cov- host proteins and physiologically based pharmacokinetic modeling may reduce side effects of repurposable drugs or drug combinations. preclinical studies are warranted to evaluate in vivo efficiency and side effects before clinical trials. furthermore, we only limited to predict pairwise drug combinations based on our previous network-based framework . however, we expect that our methodology remain to be a useful network-based tool for prediction of combining multiple drugs toward exploring network relationships of multiple drugs' targets with the hcov-host subnetwork in the human interactome. finally, we aimed to systematically identify repurposable drugs by specifically targeting ncov host proteins only. thus, our current network models cannot predict repurposable drugs from the existing anti-virus drugs that target virus proteins only. thus, combination of the existing anti-virus drugs (such as remdesivir ) with the network-predicted repurposable drugs (fig. ) or drug combinations (fig. ) may improve coverage of current network-based methodologies by utilizing multi-layer network framework . in conclusion, this study offers a powerful, integrative network-based systems pharmacology methodology for rapid identification of repurposable drugs and drug combinations for the potential treatment of -ncov/ sars-cov- . our approach can minimize the translational gap between preclinical testing results and clinical outcomes, which is a significant problem in the rapid development of efficient treatment strategies for the emerging -ncov/sars-cov- outbreak. from a translational perspective, if broadly applied, the network tools developed here could help develop effective treatment strategies for other emerging viral infections and other human complex diseases as well. in total, we collected dna sequences and protein sequences for hcovs, including three most recent -ncov/sars-cov- genomes, from the ncbi genbank database ( january , supplementary table s ). whole-genome alignment and protein sequence identity calculation were performed by multiple sequence alignment in embl-ebi database (https:// www.ebi.ac.uk/) with default parameters. the neighbor joining (nj) tree was computed from the pairwise phylogenetic distance matrix using mega x with bootstrap replicates. the protein alignment and phylogenetic tree of hcovs were constructed by mega x . we collected hcov-host protein interactions from various literatures based on our sizeable efforts. the hcov-associated host proteins of several hcovs, including sars-cov, mers-cov, ibv, mhv, hcov- e, and hcov-nl were pooled. these proteins were either the direct targets of hcov proteins or were involved in critical pathways of hcov infection identified by multiple experimental sources, including highthroughput yeast-two-hybrid (y h) systems, viral protein pull-down assay, in vitro co-immunoprecipitation and rna knock down experiment. in total, the virus-host interaction network included hcovs with host proteins (supplementary table s ). next, we performed kyoto encyclopedia of genes and genomes (kegg) and gene ontology (go) enrichment analyses to evaluate the biological relevance and functional pathways of the hcov-associated proteins. all functional analyses were performed using enrichr . here, we collected drug-target interaction information from the drugbank database (v . ) , therapeutic target database (ttd) , pharmgkb database, chembl (v ) , bindingdb , and iuphar/bps guide to pharmacology . the chemical structure of each drug with smiles format was extracted from drug-bank . here, drug-target interactions meeting the following three criteria were used: (i) binding affinities, including k i , k d , ic , or ec each ≤ μm; (ii) the target was marked as "reviewed" in the uniprot database ; and (iii) the human target was represented by a unique uni-prot accession number. the details for building the experimentally validated drug-target network are provided in our recent studies , , . to build a comprehensive list of human ppis, we assembled data from a total of bioinformatics and systems biology databases with five types of experimental evidence: (i) binary ppis tested by high-throughput yeasttwo-hybrid (y h) systems; (ii) binary, physical ppis from protein d structures; (iii) kinase-substrate interactions by literature-derived low-throughput or high-throughput experiments; (iv) signaling network by literature-derived low-throughput experiments; and (v) literature-curated ppis identified by affinity purification followed by mass spectrometry (ap-ms), y h, or by literature-derived low-throughput experiments. all inferred data, including evolutionary analysis, gene expression data, and metabolic associations, were excluded. the genes were mapped to their entrez id based on the ncbi database as well as their official gene symbols based on genecards (https:// www.genecards.org/). in total, the resulting human protein-protein interactome used in this study includes , unique ppis (edges or links) connecting , proteins (nodes), representing a % increase in the number of the ppis we have used previously. detailed descriptions for building the human protein-protein interactome are provided in our previous studies , , , . we posit that the human ppis provide an unbiased, rational roadmap for repurposing drugs for potential treatment of hcovs in which they were not originally approved. given c, the set of host genes associated with a specific hcov, and t, the set of drug targets, we computed the network proximity of c with the target set t of each drug using the "closest" method: where d(c, t) is the shortest distance between gene c and t in the human protein interactome. the network proximity was converted to z-score based on permutation tests: where d r and σ r were the mean and standard deviation of the permutation test repeated times, each time with two randomly selected gene lists with similar degree distributions to those of c and t. the corresponding p value was calculated based on the permutation test results. z-score < − . and p < . were considered significantly proximal drug-hcov associations. all networks were visualized using gephi . . (https://gephi.org/). for this network-based approach for drug combinations to be effective, we need to establish if the topological relationship between two drug-target modules reflects biological and pharmacological relationships, while also quantifying their network-based relationship between drug targets and hcov-associated host proteins (drug-drug-hcov combinations). to identify potential drug combinations, we combined the top lists of drugs. then, "separation" measure s ab was calculated for each pair of drugs a and b using the following method: where d Á h i was calculated based on the "closest" method. our key methodology is that a drug combination is therapeutically effective only if it follows a specific relationship to the disease module, as captured by complementary exposure patterns in targets' modules of both drugs without overlapping toxic mechanisms . we performed the gene set enrichment analysis as an additional prioritization method. we first collected three differential gene expression data sets of hosts infected by hcovs from the ncbi gene expression omnibus (geo). among them, two transcriptome data sets were sars-cov-infected samples from patient's peripheral blood (gse ) and calu- cells (gse ), respectively. one transcriptome data set was mers-cov-infected calu- cells (gse ). adjusted p value less than . was defined as differentially expressed genes. these data sets were used as hcov-host signatures to evaluate the treatment effects of drugs. differential gene expression in cells treated with various drugs were retrieved from the connectivity map (cmap) database , and were used as gene profiles for the drugs. for each drug that was in both the cmap data set and our drug-target network, we calculated an enrichment score (es) for each hcov signature data set based on previously described methods where j = , , …, s were the genes of hcov signature data set sorted in ascending order by their rank in the gene profiles of the drug being evaluated. the rank of gene j is denoted by v(j), where ≤ v(j) ≤ r, with r being the number of genes ( , ) from the drug profile. then, es up/down was set to a up/down if a up/down > b up/down , and was set to −b up/down if b up/down > a up/down . permutation tests repeated times using randomly generated gene lists with the same number of up-and down-regulated genes as the hcov signature data set were performed to measure the significance of the es scores. drugs were considered to have potential treatment effect if es > and p < . , and the number of such hcov signature data sets were used as the final gsea score that ranges from to . coronaviruses-drug discovery and therapeutic options coronavirus infections-more than just the common cold sars and mers: recent insights into emerging coronaviruses host factors in coronavirus replication epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia putting the patient back together-social medicine, network medicine, and the limits of reductionism the $ . billion pill-methodologic and policy considerations silico oncology drug repositioning and polypharmacology individualized network-based drug repositioning infrastructure for precision oncology in the panomics era drug repurposing: new treatments for zika virus infection? a comprehensive map of molecular drug targets network-based approach to prediction and population-based validation of in silico drug repurposing systems biology-based investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis understanding human-virus protein-protein interactions using a human protein complex-based analysis framework. msystems computational network biology: data, models, and applications repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis c virus infection a screen of fda-approved drugs for inhibitors of zika virus infection identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen prediction of drug-target interactions and drug repositioning via network-based inference a genome-wide positioning systems network algorithm for in silico drug repurposing deepdr: a network-based deep learning approach to in silico drug repositioning target identification among known drugs by deep learning from heterogeneous networks network-based prediction of drug-target interactions using an arbitrary-order proximity embedded deep forest network-based translation of gwas findings to pathobiology and drug repurposing for alzheimer's disease network-based prediction of drug combinations molecular evolution of human coronavirus genomes structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors structure of sars coronavirus spike receptor-binding domain complexed with receptor genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the -ncov spike in the prefusion conformation transient oligomerization of the sars-cov n protein-implication for virus ribonucleoprotein packaging the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease a structure-informed atlas of human-virus interactions screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture toremifene interacts with and destabilizes the ebola virus glycoprotein the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling the central role of angiotensin i-converting enzyme in vertebrate pathophysiology effect of the angiotensin ii receptor blocker olmesartan on the development of murine acute myocarditis caused by coxsackievirus b the impact of statin and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker therapy on cognitive function in adults with human immunodeficiency virus infection irbesartan, an fda approved drug for hypertension and diabetic nephropathy, is a potent inhibitor for hepatitis b virus entry by disturbing na(+)-dependent taurocholate cotransporting polypeptide activity the fda-approved drug irbesartan inhibits hbv-infection in hepg cells stably expressing sodium taurocholate co-transporting polypeptide identification of a novel transcriptional repressor (hepis) that interacts with nsp- of sars coronavirus host mtorc signaling regulates andes virus replication host cell mtorc is required for hcv rna replication adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h n pneumonia and acute respiratory failure middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell bird flu"), inflammation and anti-inflammatory/ analgesic drugs the development of anti-inflammatory drugs for infectious diseases melatonin: its possible role in the management of viral infections-a brief review melatonin in bacterial and viral infections with focus on sepsis: a review ebola virus disease: potential use of melatonin as a treatment one molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? on the free radical scavenging activities of melatonin's metabolites, afmk and amk anti-inflammatory effects of eplerenone on viral myocarditis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro systematic identification of synergistic drug pairs targeting hiv antiviral potential of erk/mapk and pi k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d toremifene is an effective and safe alternative to tamoxifen in adjuvant endocrine therapy for breast cancer: results of four randomized trials mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells emodin inhibits current through sars-associated coronavirus a protein emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction calmodulin interacts with angiotensin-converting enzyme- (ace ) and inhibits shedding of its ectodomain modulation of intracellular calcium and calmodulin by melatonin in mcf- human breast cancer cells activation of the c-jun nh -terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-jun the natural compound silvestrol is a potent inhibitor of ebola virus replication broad-spectrum antiviral activity of the eif a inhibitor silvestrol against corona-and picornaviruses review of computational methods for virus-host protein interaction prediction: a case study on novel ebola-human interactions pleiotropic effects of statins: new therapeutic targets in drug design integrative functional genomics of hepatitis c virus infection identifies host dependencies in complete viral replication cycle crispr-cas genetic analysis of virushost interactions acute eosinophilic pneumonia related to a mesalazine suppository mesalamine induced eosinophilic pneumonia translational high-dimensional drug interaction discovery and validation using health record databases and pharmacokinetics models mega x: molecular evolutionary genetics analysis across computing platforms enrichr: a comprehensive gene set enrichment analysis web server update drugbank . : shedding new light on drug metabolism therapeutic target database update : enriched resource for bench to clinical drug target and targeted pathway information chembl: a large-scale bioactivity database for drug discovery bindingdb: a webaccessible database of experimentally determined protein-ligand binding affinities the iuphar/bps guide to pharmacology: an expertdriven knowledgebase of drug targets and their ligands uniprot: the universal protein knowledgebase database resources of the national center for biotechnology information conformational dynamics and allosteric regulation landscapes of germline pten mutations associated with autism compared to those associated with cancer expression profile of immune response genes in patients with severe acute respiratory syndrome cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus srebp-dependent lipidomic reprogramming as a broadspectrum antiviral target discovery and preclinical validation of drug indications using compendia of public gene expression data this work was supported by the national heart, lung, and blood institute of the national institutes of health (nih) under award number k hl and r hl to f.c. the content of this publication does not necessarily reflect the views of the cleveland clinic. key: cord- - yoilsho authors: nan title: abstracts of the (nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the (th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - -y sha: doc_id: cord_uid: yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise % of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only . % of the volume, but % of the total cell number (kmiec ) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. ) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr breast cancer cells were exposed to the anti-cancer drug herceptin ( μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm- cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around % of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm- cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu th health program grant agreement no . deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases and (erk / ) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk / activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine as a prerequisite for nuclear erk / signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk / signaling: since activation of erk / triggers erk dimerization, a prerequisite for erk t autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t -phosphorylation. first, we investigated the impact of monomeric erk on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk ∆ - and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk ∆ - overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆ - in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk and prevents its dimerization. moreover, the peptide effectively inhibited erk t -phosphorylation and nuclear translocation of yfp-tagged wild-type erk after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype (aav )-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i knockout on cardiac function and survival in a murine heart-failure model of cardiac β adrenoceptor overexpression. methods and results: β -adrenoceptor overexpressing mice lacking gα i (β -tg/gα i -/-) were compared to wild-type (c bl/ ) mice and littermates either overexpressing cardiac β -adrenoceptors (β -tg) or lacking gα i (gα i -/-). at days of age mortality of mice only lacking gα i was higher compared to wild-type or β -tg, but similar to β tg/gα i -/mice. beyond days mortality of β -tg/gα i -/mice was enhanced compared to all other genotypes (mean survival time: ± days). echocardiography revealed similar cardiac function of wild-type, β -tg and gα i -/mice, but significant impairment for β -tg/gα i -/mice (e.g. ejection fraction ± % versus ± % in wild-type mice). significantly increased ventricle-to body-weight ratio ( . ± . % versus . ± . % in wild types), left-ventricular size (length . ± . cm versus . ± . cm in wild types) and anp and bnp expression (mrna: % and % of wild type, respectively) clearly indicated hypertrophy. gα i was significantly upregulated in gα i -knockouts (protein compared to wild-type mice: ± % in gα i -/and ± % in β -tg/gα i -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β adrenoceptors in β -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than -fold higher (b max : ± fmol/mg) than on the otherwise used c bl/ background. accordingly, fvb/n-based β -tg mice showed a significantly impaired cardiac function at an age of d while c bl/ -based β -tg mice did not. conclusions: gα i -deficiency combined with cardiac β -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c bl/ background β adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day while there was overt cardiomyopathy in mice additionally lacking gα i . we propose an enhanced effect of increased β -adrenergic drive by lack of protection via gα i . the observed gα i upregulation was not sufficient to compensate for gα i deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i is currently addressed in a subsequent study using β -tg and gα i deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; mg/kg per d) for and hours and days. overall days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; d iso ± ) vs. wt cardiomyocytes ( d iso ± ). at this time point a % decrease of cardiac output and a % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after h ( -fold), and declined after h ( -fold) to . fold increase after days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: regulated genes (up-regulated: %) in untreated; altered genes (up %) after h; changed genes (up %) after h and altered genes (up %) after days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m receptors, while m receptors appear to exert a heart-specific function only at later stages. continuous m blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m inhibition. taken together, we postulate that muscarinic m receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir- has proved effective against fibrosis of the myocardium and in other organs. mir- has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir- in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir- fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir- in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav -icre vector allowed for specific and almost complete deletion of mir- in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir- in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir- gene in mice. aav -mediated deletion of mir- , albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir- demonstrated that mir- exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir- therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c acts as a multikinase inhibitor. results of a thermal shift assay revealed that c shows by far the highest binding affinity to the bmp- -inducible kinase (bmp k/bike). bmp k represents an as yet largely uncharacterized protein, which is not regulated by bmp- in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c and (ii) the role of bmp k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c affected the viability of human umbilical vein endothelial cells (huvecs). both c and the knock-down of bm k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec- ). also the proliferation of hmec- was reduced by c treatment (ic : µm). a tube formation assay on matrigel demonstrated that c significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c treatment nor by bmp k gene silencing. in regard to inflammatory processes, c treatment or bmp k silencing of huvecs decreased the adhesion of thp- cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c or bmp k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c treatment, the knock-down of bmp k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c in vitro. the precise role of bmp k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp k silencing and c treatment will be further elucidated. moreover, since the inhibition of bmp k seems not to be responsible for all actions of c , we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). cxcr seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr agonists or antagonists could result in reduced tlr signaling. however, the role of cxcr and the influence of different cxcr ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose ( mg/body weight) in mice. the plasma stable cxcl analog ctce- d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr , cxcl and tlr expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce- d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately %, % or %, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce- d diminished the lps induced expression of cxcr , cxcl , tlr , nf-κb, cleaved caspase- and gp phox, whereas heme oxygenase expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce- d in all organs. the cxcr is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce- d attenuated tlr signaling, induced heme oxygenase activity and mitigated apoptosis. thus, the administration of cxcl analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c bl/ j mouse strains with a loxp flanked exon of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef - (fxn -/-) and fx-mef - (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over days, with medium and substance changes at day and , which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day , , and and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor which leads to disruption of protein synthesis and finally causes cell death [ ] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [ , ] . in detail, we confirmed the involvement of the host cell chaperone hsp and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [ , , ] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp and fk -binding protein (fkbp) as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp , cypa, cyp , fkbp and fkbp . the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [ ] murphy ( ) toxins , - . [ ] barth ( ) naunyn-schmied arch pharmacol , - . [ ] kaiser et al. ( ) cell. microbiol. , - . [ ] dmochewitz et al. ( ) cell. microbiol. , - . [ ] ratts et al. ( ) j. cell biol. , - . [ ] schnell et al. ( ) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins ( ). prompted by these result, we found that only human α-defensin- (hnp- ) but not human β-defensin- (hbd- ), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin- prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp- causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy -asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a . % knockdown (p< . student's t-test, n= ) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n= ). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s mix), as was the synthesized ba-epoxide (-s mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr -positive colon stem cells, which coincided with atr-chk -p signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h ax, suggesting the usefulness of γ-h ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [ ] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines ( ). in particular, the chemokines monocyte chemoattractant protein (mcp- , ccl ), interleukin- (il- /cxcl ) and stromal cell-derived factor (sdf- a/cxcl ) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl was shown to attenuate obesity in mice ( ) . the cc chemokine ccl is known to stimulate ccr receptors, and the cxc chemokines cxcl and cxcl to activate cxcr and cxcr or cxcr and cxcr , respectively. the receptor(s) activated by cxcl is currently unknown. a role of cxcl in modulating cxcr signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr and the related receptor fprl , and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr receptors were absent ( ) . yet, the analysis revealed appreciable levels of mrna encoding ccl , cxcl , and cxcl , and ccr , cxcr , cxcr , fpr , and fprl , and cxcr . of interest, cxcr -and cxcr -mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl , cxcl , cxcl . while sgbspreadipocytes and adipocytes did not accumulate ccl -mcherry upon stimulation, they showed a small accumulation of cxcl -mcherry, and a strong accumulation of cxcl -mcherry in the endosomal compartment. similar results were obtained in murine t l- preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl -binding putative receptor protein(s) in murine t l- preadipocytes. ( ) makki, k. et al., ( ) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic ', and to some extent of the corresponding cytidylyl analogue ccmp , . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac -camps in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell , - ( ) . koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. ( ): p. - ( ) . nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem. , - ( ) cardiovascular pharmacology hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel ( %: %: . mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology ( % strain, hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen . ; collagen + cms . ; collagen + cms + cfbs . , collagen + cms + ffbs . . we next generated ehm with cms and ffbs or cfbs. after weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p< . ) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm : . ± . , n= vs. . ± . , n= ). cross sectional area (csa) of tissues was greatly increased (p< . ) in cfb-ehms (csa in mm : . ± . , n= vs. . ± . , n= ) and nonmyocyte content was higher in cfb-ehms ( . ± . , n= vs. . ± . , n= ; x cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase (has ) enzyme was significantly (p< . ) upregulated. based on these findings, we pharmacologically intervened with has mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p< . ) reduced csa (csa in mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ) with a concurrent, statistically significant (p< . ), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type receptor (vegfr ), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with µm of the camp analog -pcpt- '-o-camp ( ) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac in huvec decreased the basal and vegf-induced sprouting. surprisingly, µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than , indicating an additional role of pka. in accordance, µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh cre ) were created using the cre/loxp system. mr cdh cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received % nacl with drinking water for weeks. the mineralocorticoid deoxycorticosterone acetate (doca, . mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype ( . ± . vs. ctrl . ± . mmhg, p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . mmhg, p< . ) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh cre mice (ventricle weight . ± . mg vs. mr wildtype . ± . mg, p< . ). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam ) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: . ± . vs. ctrl . ± . , p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . , p< . ) mice. mrna expression of the fibrosis marker gene col a (mr wildtype . ± . -fold; mr cdh cre . ± . -fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype . ± . fold; mr cdh cre . ± . -fold vs. ctrl). in the kidney vcam mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca + concentration due to ca + entry from the extracellular space is critical for platelet activation and aggregation. ca + entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a (txa ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca + entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca + entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc and trpc in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca + signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc /c proteins for procoagulant activity. in addition, we detected the expression of s trpc transcripts in mouse platelets. therefore, we tested trpc /c /c -deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc /c /c inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc /c /c -deficient mice and we observed that the aggregation was reduced after adp ( µm) or txa -analogue ( µm) stimulation, but not after collagen stimulation ( µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca + response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/ mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/ mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/ mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/ mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/ mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp b ( -fold in females, -fold in males) in -day old offspring of adult female mice exposed one week prior mating to tcpobop ( mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop ( mg/kg) prior to mating. the analysis of hepatic cyp b expression in -day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a -fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e . followed by crossfostering proved transmission to be mediated predominantly via lactation (f hepatic cyp b induction -fold) and only to a minor part via intra-uterine exposure ( fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f and f generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp b catalytic activity. in both tcpobop-pretreated f and in their f descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by > %. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by % in -weeks-old offspring. in summary, the car-mediated cyp b induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with alzheimer´s disease patients indicated that the combined treatment with donepezil and egb ® had less side effects than donepezil alone (yancheva et al., ) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment ( days of pretreatment) with donepezil ( , mg/kg p. o.), egb ® ( mg/kg p. o.), or the combination of the two drugs, or vehicle in - month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. ( ) . ginkgo biloba extract egb (r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, ( ) , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations ( - µm; just et al. expert opin emerging drugs : - , ) observed in patients treated with dantrolene. although not yet proven directly, oat may be involved in renal secretion of dantrolene and -oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp . since oat was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. : - , ) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr , by influx through oat , where it inhibits calcium efflux by ryr thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat . in addition, we identified, besides dantrolene and -oh dantrolene, several further fdaapproved drugs such as tyrphostin ag , ceefourin , glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat . controls with other defects and controls with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥ mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with % confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls and additionally adjusted for maternal age in controls . results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were / ( . %) for cases, / ( . %) for controls and / ( . %) for controls and adjusted ors ( %cis) were . ( . - . ) and . ( . - . ), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-( . vs. . for controls , and . vs. . for controls ). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being and . times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac protects non-transformed cells from topoisomerase ii-(top )-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac -mediated regulation of topoisomerase ii activity. however, the link between rac and top -poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top -poison-induced cytotoxicity. here, we investigated the relevance of rac -regulated actin cytoskeleton integrity as well as mitochondrial integrity in top -poison-induced dna damage responses as well as cytotoxicity under situation of rac inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h v) as well as cardiomyocytes (h c ) were chosen as in vitro model systems for top -poisoning. the cells were pre-treated with rac inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top -poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc -assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac -inhibitor. top -poison-induced raise in mitochondrial mass was slightly enhanced by the rac -inhibitor and statins. interestingly, inhibition of rac counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top -poisoninduced cytotoxicity in h c and h v cells. the data point to a role of rac -regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top -poisons. poly(adp-ribose) polymerase (parp ) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [ ] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp as well as by non-covalent poly(adp-ribose)-wrn interaction [ ] [ ] [ ] . furthermore, pharmacological parp inhibition as well as a genetic parp ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [ ] . here we report a novel role for parp and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h o -induced oxidative stress, parp is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp -dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [ , ] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [ ] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [ ] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [ - ] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases methylcytosine and -hydroxymethylcytosine as well as their derivatives ( formylcytosine and -carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene , , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb , xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle , mangerich and bürkle ) . here we generated a genetic knock out of parp in one of the most widely used human cell systems, i.e. hela parp ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp ko cells with a series of artificial and natural parp variants, we analyzed structure-function relationships of parp in a cellular environment without interfering with endogenously expressed wt-parp . we confirmed that the parp e k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp l f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp -par interaction via at least one specific par binding motif located in zinc finger of parp . expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp l f itself triggered apoptosis, parp e k expression led to a strong g -arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt mitigated effects of the e k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp ko cells with a natural cancer-associated parp snp variant (v a), as well as a newly identified parp mutant present in a patient of pediatric colorectal carcinoma (f l-v a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp -dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle ( ) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer ( ): - . mangerich, a. and a. bürkle ( ) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: - . leukemic cells frequently overexpress the transcription factor wilms tumor (wt ) and the persistence of wt expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt and cell death. in addition, we show that an rna interference-mediated elimination of wt sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt is a novel biological target of hydroxyurea and they suggest that wt has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res : , ) and stably transfected to hek cells (hekß -fret). to monitor the camp response the epac -based fret sensor of camp, was stably transfected alone (hekwt-e ) and together with a moderate level of native ß ar to hek cells (hekß -e ; nikolaev et al. jacc : , ) . fret-activity was measured with recently developed fluorescence detectors ( channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using nm for excitation and / nm for the emmission ratio. cells were cultivated in -format -well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß -fret cells exhibited ec -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: . ± . , adrenaline . ± . , noradrenaline . ± . ). ßar-expression levels were controlled by radioligand binding with [ h]-(-)-cgp , resulting in different densities of ~ x and ~ . x receptors/cell in hekß -fret and hekß -e , respectively, whereas in hekwt-e cells only ~ ß ar were found. surprisingly, the low level of ßar in hekwt-e cells allowed the measurement of the action of ß -sympathomimetics (ß sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~ . ) to an effective regulation of fret activity in the presence of . mm ibmx (pec ~ . ± . ), nearly matching the ~ -fold amplification of iso (pk d~ . ; pec ~ . ). in order to determine ß ar-mediated side effects of ß sym, hekß -e cells characterized by a -fold higher level of ß ar over ß ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec ~ . whereas the high affinity ß sym salmeterol acted a partial agonist (~ % of iso maximum, pec ~ . ), both compounds being rather insensitive against the highly effective ß ar-blockade with µm cgp , a. for that reason hekß -fret cells were used characterizing fenoterol as partial agonist (~ %) whereas salmeterol activated less than % of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß ar present in hekß -e cells precludes the exact determination of ß ar-mediated side effects of ß sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin- affinity and trafficking. the affinity of arrestin- to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin- and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin- dissociation from the β ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin- into the bleached region was prolonged between two-and fourfold for the β ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek t cells transfected with the appropriate cdnas. here we focused on muscarinic m -, m -, and m -receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/ -proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m -and m -achrs was found to be -fold faster in comparison to gq. similarly, we observed a -fold right shift of the concentration-response curves of go proteins binding to m -achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m -achr by using a fret-based assay. our results showed that gq activation required -fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m -achr for gi family members by comparison of dissociation kinetics of gi -, gi , gi -, and go-proteins from m -achr under nucleotide depleted conditions. k off of gi and gi were found to be two-fold higher in comparison to gi and go proteins, indicating the higher affinity of the latter ones to m -achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine that is predominantly catalyzed by g protein-coupled receptor kinase . as a consequence, the selective morphine-induced s phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s but also drive higher order phosphorylation on the flanking residues threonine , threonine , and threonine in a hierarchical phosphorylation cascade that specifically requires grk and grk isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels hcn in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn and hcn isoforms for brain function and epilepsy has been demonstrated, however the role of hcn , the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn in controlling oscillations in the thalamocortical network. hcn is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn -deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc and channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc / / channels are directly activated by diacylglycerol (dag), trpc and channels are commonly regarded as daginsensitive. in contrast to trpc / / channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) and . interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc confers dag-sensitivity on trpc channels. trpc channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf and proteins and overexpression of a nherf mutant incapable of trpc binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip depletion itself caused slight trpc current increases while during pip depletion, the membrane permeable dag analogue oag evoked even higher trpc currents suggesting that pip depletion induces an active and dag-sensitive channel conformation. receptor mediated pip depletion also resulted in dissociation of nherf and from the c-terminus of trpc thereby eliciting a dag-sensitive trpc channel conformation. thus, our findings suggest that dag-sensitivity of trpc is the result of an activation cascade starting with pip depletion and subsequent dynamic dissociation of nherf and from the c-terminus of trpc . altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm -deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm may exert antinociceptive properties. fluorometric ca + assays and a compound library containing approved drugs were used to identify trpm inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura- -loaded single cells was applied to monitor [ca + ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm inhibitors. the compounds exhibited half-maximal inhibitory concentrations of . - µm. the selectivity profiles of maprotiline and primidone for trpm were promising with no inhibitory effects on trpm , trpm , trpa , trpv , trpc , trpc and p x receptor channels. primidone inhibited pregs-induced [ca + ] i signals in rat drg neurones, indicating a block of native trpm channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm -related effects by acutely challenging the channel´s function. pharmacological interference with trpm applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il- ) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e /k can be crosslinked efficiently in both states, the closed-and open state of the p x r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e /r , e /k , e /r and e /k and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p x r. the comparison of the closed-state and open-state model of the rat p x r showed that atp promotes a marked rearrangement of the side chains of the residues r and k to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r and k with atp competitively releases e , e and e from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem , tmem and tmem . expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem proteins exhibit to plasma-membrane spanning domains, but fluorescently labeled tmem fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem -eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem -deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura- or indo- indicate higher resting ca ++ concentrations in tmem -/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem -yfp-knock-add-on mice we identified tmem in organelles of the apical acinar cell pole and a partial colocalisation with lamp proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh cl application. the role of tmem for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck- stimulation indicating that tmem inactivation leads to an enhancement of the globalization of cck- evoked ca + release from intracellular organelles. taken together, our study identifies tmem as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [ ] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [ ] gundert-remy, u. et al. ( ) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol : - . during the past decades, considerable progress has been made in implementing r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes ( x gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed h after the last irradiation by γh ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht , a small molecule inhibitor of the small rho-gtpase rac , was applied in vivo, pointing to an involvement of rac in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh -interacting domain death agonist) knockout cell line in neuronal ht- cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas -knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht- cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin- and the bid inhibitor bi- c on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas -bid-knockout cells. bi- c inhibited glutamate-induced morphological changes of ht- cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin- in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi- c abolishes lipid peroxide formation in the erastin model and ferrostatin- in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi- c and ferrostatin- in the respective model systems. investigating the crispr/cas -bid-knockout ht- cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type (aav ) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange at the azo-and other weak bonds in our experimental set-up (fig. a) . amongst other substances, the carcinogens aniline (max. conc. . ± . µg/ml) and , -dichlorobenzidine (max. conc. . ± . µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. b) . here, , -benzenedicarbonitrile (max. conc. . ± . µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. . ± . µg/ml) and the human carcinogen benzene (max. conc. . ± . µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥ µg/ml evolving upon ruby laser irradiation of > . mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep , ( ) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco- model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). ; ( ) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [ , ] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > °c [ ] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [ ] . we here present results of a nine-day human intervention study with healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [ ] . the study design included washout periods with an aa-minimized diet ( - ng /kg bw), a low aa intake day ( . - . µg /kg bw) as well as a high aa intake day ( . - . µg /kg bw). after a three-day washout period an aama baseline level of ± nmol/d was determined. low aa intake led to an aama excretion within h of ± nmol/d, high intake to ± nmol/d corresponding to an aama excretion rate of about % of the ingested aa dose within h, whereas aama output within h corresponded to % of the respective aa intake, the aama baseline after days washout corresponds to a net exposure level of . - . μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [ ] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste , doi: . iarc, iarc monographs on the evaluations of carcinogenic risks to humans , . [ ] tareke et al., j. agric. food chem. , , - . [ ] efsa panel on contaminants in the food chain (contam), efsa journal ; ( ): [ pp.] . [ ] ruenz et al., arch. toxicol. doi: . /s - - heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf /skn- , daf ), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + %). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn- (homolog to mammalian nrf ), but not daf- (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct cells, baicalein activates nrf ; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf . the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf- transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn- but not daf- . experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf /skn- activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [ ] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in healthy postmenopausal women ( - yrs).in a randomized doubleblind protocol each subject received four treatments, doses of intranasal progesterone ( . mg mpp ; . mg mpp ), mg of zolpidem and placebo. the conditions consisted of experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from : to : . simultaneously blood was collected every min between : and : by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after . and . mg mpp . univariate f-tests in the manovas pointed to the following results (significant p-values at α= . ). sei was higher after zolpidem than after the other three treatments. after . mg mpp sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after . mg mpp and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after . mg mpp and after zolpidem. after all active treatments with mpp and zolpidem the time spent in sleep stage was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after . mg mpp . no other changes of hormone secretion were found. our study show sleep promoting effects of . mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after . mg mpp . no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january and june from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of or and possible anticholinergics with a score of ). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of . a patient's individual score of or higher is considered to be clinically relevant. in total, , geriatric patients (median age years, . % female, median no. of drugs ) were evaluated. of these, , ( . %) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in , ( . % of the patients taking anticholinergic drugs) patients. , ( . % of the patients taking anticholinergic drugs) patients had a score of or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine ( ), amitriptyline/quetiapine ( ) and amitriptyline/carbamazepine ( ). , ( . %) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct and mate expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. , although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct (encoded by slc a ), oct (slc a ), oct (slc a ) and mate (slc a ). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek stably expressing the organic cation transporters oct , oct , oct or mate . intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct and mate in a time-and concentration-dependent manner, but not by oct or oct . timolol is only transported by mate , but not by the octs. as shown by immunolocalization studies, the oct and mate transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct and mate may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: . zhang et al., nat rev drug discov. jun ; ( ) : - . . lavik et al., eye (lond). may; ( ): - . . gaudana et al., pharm res. may; ( ): - . . kraft et al., invest ophthalmol vis sci. ( ): - . . nies et al., plos one. ( ) :e . supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel mg, prasugrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s = . ; p= . ), hipf (r s = . ; p= . ), ipc r s = . ; p< . ). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p< . ). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p c polymorphisms (* and * ), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s was the strongest predictor of on-treatment platelet reactivity (partial η = . ; p< . ) followed by intrinsic platelet reactivity (partial η = . ; p < . ). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp c * polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [ ] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [ ] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [ ] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki- index -fold in the cscc model (p£ . ). while the presence of claudin- and occludin were distinctly reduced, zonula occludens protein- was more wide-spread, and claudin- was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [ ] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for . -fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [ ] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb r with integrated graduate education was launched in . the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the rs and to find alternatives to animal experimentation within the next years. a panel of r experts has been set up to provide advice and assistance and to raise awareness in society for r-related issues. research in bb r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on r including modules on ethics and legislation. currently, phd students are qualified for management positions in professional areas related to the rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd c and d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz- -derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer , -dinitrochlorobenzene (dncb) induced il- and il- secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd and pd-l in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine ( rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than , compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. %) fail in the late drug development due to toxicity and adverse effects [ ] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about , compounds and cancer cell lines from the national cancer institute's (nci) nci- growth inhibition data [ ] with focus on five immune cell lines (rpmi- , ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [ ] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [ ] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [ ] , and encourage the reduction of in vivo testing according to the principles of the rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype e activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult e ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for d pharmacophore development. eight specific d pharmacophores were created that allow identification of sult e substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over , experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult e inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult e for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. ( ) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. ( ) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p= . ). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p= . ). % of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in . by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the -organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the -organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused -organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd -values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the -organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [ , ] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck , which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il- alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [ ] vávrová, k., henkes, d., strüver, k., sochorová, m., Školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a d skin construct. the journal of investigative dermatology. , - ( bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive / has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the -rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the -r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c /bl j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored and minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb r is funded by bmbf. in the r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the r-concept is the overall goal of scientific animal welfare. article of the 'directive / /eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [ ] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately % [ ] [ ] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [ ] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from . to . mg/kg per bodyweight [ ] [ ] [ ] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [ ] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [ ] rl / /eu. [ ] bmel ( ) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [ ] eu commission ( ) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [ ] carbone l ( ) pain in laboratory animals: the ethical and regulatory imperatives. plos one , e . [ ] gv-solas, a.f. a.d. ( ) . empfehlung zur schmerztherapie bei versuchstieren. [ ] carpenter, j.w., t.y. mashima, and d.j. rupiper ( ) . exotic animal formulary, nd edition, w.b. saunders co., phila. [ ] flecknell, p. ( ) . laboratory animal anaesthesia, nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a -slice scanner were collected from female minipigs, consisting of animals aged months (group , n= ) and animals (group ; n= ) examined at the age of and months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to % where the largest volume was , ml and the lowest , ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between and months of age. comparing and months old minipigs, no significant difference in distance could be observed. from the age of months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between and months of age indicates ongoing anatomical changes of this parameter until the age of months. therefore animals should be older than months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [ ]. worldwide it is estimated that over million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [ ] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [ ] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals ( r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin ( -ht) system, in particular the -ht a receptor, playing a major role. activating -ht a receptors by the -ht a receptor agonist -hydroxy- -(dipropylamino)tetralin ( -oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic -ht a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the -ht a receptor in serotonergic projection areas, bert et al. ( , pmid: ) revealed exaggerated -oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic -ht a receptors to thermoregulation, more precisely to the hypothermic effect of -oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of -oh-dpat ( . mg/kg - mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the -ht synthesis inhibitor parachlorophenylalanine (pcpa; mg/kg, i. p. on four consecutive days) would alter the effects of -oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the -ht a receptor. results: -ht a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: . °c; nmri wild-type mice: . °c). in both genotypes, systemic administration of -oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (- . °c compared to - . °c in nmri wild types). dose response curves of -oh-dpat revealed an ed = . mg/kg in transgenic and an ed = . mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to -oh-dpat in mice. the dose-response curves indicate a higher potency of -oh-dpat in transgenic mice. the exaggerated hypothermic response to -oh-dpat in mutant mice implies that postsynaptic -ht a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that -oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing -ht a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg gly in the human beta -adrenoceptor (adrb ). the adrb carrying arginine at position in helix in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta -adrenoceptor variants arg -adrb and gly -adrb is poorly understood. despite its hyperfunctionality, we found the arg -variant of the adrb to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly -variant. using adrb sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg -adrb exerted faster activation speed and arrestin recruitment than the gly -variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved -residue sequence, trehpstant , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine (s ) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase (grk ). by contrast, high-efficacy opioids not only induce phosphorylation of s but also drive higher-order phosphorylation on the flanking residues threonine (t ), threonine (t ), and threonine (t ) in a hierarchical phosphorylation cascade that specifically requires grk / isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk and grk to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk / . in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk / isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk or grk . conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk or grk . mutation of s to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/ , and g / , which interact with distinct cellular effectors to shape the final cellular response . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/ / inhibitor fr on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m acetylcholine receptor (cho-m ). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor , . we show that fr can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m membranes. in contrast to this, bim- , a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation . interestingly, inhibition of high affinity agonist binding by fr was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr , combined uncoupling of both gi and gs proteins from m was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [ ] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [ ] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [ ] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m machr which preferentially activates g proteins of the g q/ type but can also promiscuously stimulate g s proteins. g q/ -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m machr in ip and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper- -naph completely lost intrinsic activity for both g q/ and g s activation at the m machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper- -phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper- -phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/ -mediated signaling, activation of the g s protein in m machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades -( h-imidazol- -yl)propylguanidine (sk&f- , ( ) [ ] ) is known as the prototypic pharmacophore of highly potent histamine h -receptor (h r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [ ] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f- , , we characterized newly synthesized derivatives including several bivalent compounds (e.g., ) by using different pharmacological in-vitro methods. [ ] the potential h r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf cells [ ] (hh , , , r). compounds were also functionally characterized in the [ s]gtpgs assay (hh r, sf cell membranes). [ ] selectivity vs. hh , , r was determined for selected derivatives also using this technique. organ bath studies (gph r (ileum), gph r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f- , analogues displayed partial or full agonism via hh r and gph r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine , was a partial agonist (e max = %) and -times as potent as histamine vis-à-vis the gph r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa values for the antagonist ( . - . ) which are in accordance with literature data. however, in the functional in-vitro assay on gph r, the positive chronotropic response evoked by sk&f- , was surprisingly resistant to antagonism by cimetidine and other typical h r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h r agonist. this behaviour is unique within the large series of sk&f- , analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [ ] parsons, m.e. et al.; agents actions , , . [ ] buschauer, a.; j. med. chem. , , - [ ] pockes, s.; dissertation, univ. regensburg . [ ] seifert, r. ; j. pharmacol. exp. ther. , ( ) , - . the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine (s ) and threonine /serine (t /s ), which enabled us to selectively detect either the s -or the t /s -phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch and ro - induce a stably phosphorylation at s and t /s followed by a profound internalization of the receptor. the nociceptin-induced s and t /s phosphorylation can be blocked by selective nop receptor antagonists such as j or sb , . nnc - , buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s phosphorylation occurred at a faster rate than phosphorylation at t /s indicating that s is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol -myristate -acetate facilitated receptor phosphorylation only at s but not at t /s , indicating that s can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t /s in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling ( ). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation ( ) . in man, two cc chemokine receptors ccr a and ccr b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin ( ) . interested in differential signaling of ccr a and ccr b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk / ) in the presence of ccl , ccl , ccl , and ccl . all these chemokines have been shown to interact with human ccr receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr b receptors by ccl induced both rho-dependent srf activation and receptor internalization, while ccl -stimulation resulted in srf but little if any receptor internalization. in contrast, ccr a-expressing cells showed ccl dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr receptors for coupling to g proteins revealed that arginine within the putative 'eighth helix' of the carboxyl-terminal portions of ccr a and ccr b is not involved in ga i -mediated induction of erk / and plays a minor role in ccr b receptor internalization, but is specifically required for the ccr a/ and ccr b/ga q -mediated activation of srf. serotonin -ht c receptors ( -ht c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). -ht c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express -ht c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa - cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where -ht c r in vivo expression has been detected. thus, we tested mhypoa - cells for putative -ht c r expression by performing single cell calcium imaging. we observed that serotonin and the specific -ht c r agonist, way , induced robust calcium transients, which were strongly inhibited by two unrelated -ht c r-selective antagonist (sb , , rs , ). serotonin and way , also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases- / in a sb , and rs , sensitive manner, providing further evidence for functional -ht c r expression in mhypoa- / cells. intrinsic activity of way , ranged between . and . compared to serotonin in all assays, defining way , as a partial -ht c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa- / cells endogenously express -ht c r and thus represent the first cell line to analyse -ht c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with h-adenine and calculation of the conversion rate to h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α a or µ opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc or heteromeric kir . /kir . or kir . /kir . channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp a mice leading to double transgenic mice (h xpp a = dt). pp a mice (j biol chem ; : ) overexpress the catalytic subunit of protein phosphatase a (pp a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about days we noted reduced ef in pp a ( . ± . %, n= ) compared to wt ( . ± . %, n= ) and tg ( . ± . %, n= ). interestingly, in dt the ef ( . ± . %, n= ) was higher than in pp a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp a is involved in h -receptor signaling and we tentatively conclude that the h -receptor is able to ameliorate systolic but not diastolic cardiac function of pp a mice. serotonin ( -ht) can exert positive inotropic and chronotropic effects in humans via -ht -receptors. we have generated transgenic mice (tg) which overexpress the human -ht -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and , and hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to . ± . % seven hours after lps in wt and to . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] p< . vs pre-lps value). however, hours after lps, diastolic function, measured as e/a, amounted to . ± . in p< . tg vs. wt). moreover, after hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after and hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from . ± . to . ± . in wt and from . ± . to . ± . in tg (tg vs. wt, p> . , n= - ; after hours). we tentatively conclude: -ht -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec = nm) or amthamine (ec = nm), a more selective and potent h -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to mm potassium chloride. thereafter, histamine ( µm) induced a pie ( . ± . % of control, n= ) in tg but not in wt preparations whereas isoprenaline ( µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound / , a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine ( µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk , a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek t cells transfected with yfp-tagged µor and mturquoise-tagged grk as well as non-fluorescent gα i , gβ and gγ subunits showed a robust increase in fret upon superfusion with µm [d-ala , n-mephe , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine ( µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately - % of that of the corresponding damgo signal. grk binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk interaction with that of the µor-gβγ interaction in the presence of grk revealed similar time constants both for the on-and off-kinetics (grk : k on . s ). this suggests that, in the absence of grk , the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk , however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk -gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor in the absence and presence of grk and compared the kinetics with the kinetics of grk binding and unbinding to these receptors. in both cases, we found that grk and gβγ in the presence of grk associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c bl/ mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin- (et- ) for sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et- treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et- had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin- was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn and ccn to be moderately up-regulated after h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et- treatment. finally, we could show that the perinuclear localization of cav and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production ( ). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs ( ) ( ) ( ) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac -camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine -phosphate (s p) is generated by sphingosine kinase (sk)- and - and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s p - . after activation, s p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s p mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru- . in addition, in vivo studies showed that dexamethasone downregulated s p expression in glomeruli isolated from c bl/ mice treated with dexamethasone ( mg/kg body weight). functionally, we identified s p as a key player mediating s p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s p-induced migration of mesangial cells. this effect was again reversed in the presence of ru- . conclusion: we suggest that dexamethasone inhibits s p-induced mesangial cell migration via downregulation of s p . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. ; : - ) . the g protein-coupled receptor mrgd is a receptor for angiotensin-( - ) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-( - ) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-( - ) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-( - ) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-( - ). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek cells, we provided final pharmacological proof for mas to be a receptor for ang-( - ). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-( - ). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type receptor at as a receptor for the heptapeptide, but discovered that the at blocker pd can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-( - ) but excluding the at receptor, and by enforcing the revisit of such publications which concluded at function by using pd as a specific at blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within ms . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between - ms . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor (mglur ) sensors by fret after uncaging of an inert glutamate derivative. -methoxy- -nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α aadrenergic receptor, among others receptors, has been extensively studied in single cell assays ( ) ( ) . the activation kinetics of the labeled α a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology ( ) . additionally, we used halotag in combination with the nanoluc ( ) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec value in accordance with the previously published data. this suggests the functionality of the fret-based α a -halo/cfp sensor. similar results were obtained with the α a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin biosensors. upon stimulation, β -adrenergic receptors bound β-arrestin with a time constant τ = . ± . s, indicating that β-arrestin binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin with a τ = . ± . s. stimulation of β -adrenergic vs. m muscarinic or ffa receptors resulted in different patterns of conformational changes in the various β-arrestin sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin activation. upon agonist removal, first the interaction (delay = . ± . s) and only then the active state of β-arrestin (delay = . ± . s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β -adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about , fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi (historical strain), strain (serotype f, variant tcdbf), strain r (hypervirulent strain, ribotype o ), and strain r (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain ) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi . in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in d and d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p rhogef-knockout mice were adenovirally transduced for to h with recombinant adenoviruses or directly used. for d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p rhogef implies that p rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il- and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/ , g / and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr (r h), a bmp (bone morphogenic protein) type receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c c cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp -induced osteoblast differentiation in c c cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr r h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for h in culture. for activation of the g q/ -signaling the cells were treated with endothelin- (et- ), angiotensin ii (ang ii) or phenylephrine (pe) for s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p rhogef is involved in the regulation of the et- -induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor (kdelr ) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin- the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p rhogef in the transport of membrane proteins, we overexpressed p rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr and its co-localization with p rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr was found in control-transduced cells. further, p rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p dn and detected similar changes with respect to kdelr localization and golgi structure suggesting that this regulatory function of p rhogef is not dependent on its activity. conclusion: p rhogef mediates the activation of rhoa from gpcrs coupled to g q/ proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/ , g / ) exist in mammals and these must suffice to convey signals from about g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr , a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/ / over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek cells that lack functional gq/ alleles by crispr-cas genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/ using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept , , and accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc , borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [ ] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [ ] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [ ] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at hours post fertilization, larvae at days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [ ] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac ). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [ ] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [ ] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences , - ( ) . ccmp, ', '-cump and ', '-cimp were ineffective. to further characterize the action of ', '-cgmp on hut- cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). ', '-cgmp significantly increased apoptosis (ec = µm) and inhibited proliferation (ec = µm) of okt -activated hut- cells. interestingly, also ', '-cgmp exhibited comparable effects on apoptosis and proliferation with ec values of µm and µm, respectively, while ', '-camp, ', '-ccmp and ', '-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested ', '-cgmp degradation products under the same experimental conditions and found that both '-gmp and guanosine increased apoptosis and inhibited proliferation with ec -values between and µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of ', '-cgmp, ', '-cgmp, '-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [ ]. ', '-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut- cells (e.g. enpp ), yielding '-gmp, which could be further degraded to guanosine by the '-ekto-nukleotidase cd . a similar pathway may lead from ', '-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms a and b as well as the cgmp-selective pde a also degrade the emerging second messenger cump [ , ] . we analyzed the enzyme kinetics of pde b-mediated cump-hydrolysis using recombinant gst-tagged pde b and a highly sensitive and specific hplc-ms/ms method. our data show that pde b is a low-affinity enzyme for cump with a cump k m -value of > µm. the pde -selective competitive inhibitor milrinone inhibited pde b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde b is highly expressed in adipose tissue [ , ] . thus, we differentiated murine t -l -mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde b expression and basal cnmp-concentrations. in both differentiated and undifferentiated t -l cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by % and %, respectively, while ccmp was reduced by % and cump even by %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde b was upregulated ~ -fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde a-mediated cump hydrolysis. pde a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi . they comprise a "green" membrane-targeted version (mcgi ) and a "red" variant (red cgi ) that contains the fluorophores tsapphire and dimer . methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi sensor showed a good signal-to-noise ratio, an ec value of ≈ . µm for cgmp, and a high selectivity for cgmp over camp (> -fold). flincg , a non-fret-based cgmp sensor, showed similar properties as cgi . the new membrane-targeted mcgi and the new red cgi displayed ec values of ≈ . µm cgmp and a high selectivity for cgmp over camp (> -fold). in vsmcs, the red cgi showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de . the "green" fret-based camp sensor, epac -camps, showed a signal-to-noise ratio comparable to that of cgi , an ec value of ≈ µm for camp and a selectivity for camp over cgmp of ≈ -fold. finally, imaging of cells expressing both the epac -camps and the red cgi demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi would be useful to investigate membrane-associated cgmp compartments and red cgi to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at -receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [ ] . whether this causes an activation of cgks is not known. these enzymes exist in different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [ ] , [ ] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet ( . % na + ) and enalapril ( mg/kg/d) for weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay - , a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw /lrmp (lymphoid-restricted membrane protein) is a type membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [ ] , [ ] . jaw and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw and irag share the highest homology [ ] , [ ] . the coiled coil domains of irag and jaw are important for the interaction with ip rs. as already known, irag forms a trimeric complex with cgkiβ and ip r and gets phosphorylated by cgkiβ [ ] . hence we examined if jaw is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw possible phosphorylation sites can be found. our in vitro studies with jaw and cgki showed that jaw gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw and cgkiβ was detected. to examine the importance of jaw in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw . mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw . hence, the function of jaw in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine ', '-cyclic monophosphate (camp) and guanosine ', ' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp and , hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine ', '-cyclic monophosphate (ccmp) and uridine ', '-cyclic monophosphate (cump) also fulfill the criteria of second messengers [ , ] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [ ] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [ ] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap ) were examined as potential ccmp and cump target proteins. hek cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor (npr , also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase a (pde a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor (npr ) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e . embryos. because npr binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr could lower the activity of the cnp-npr -cgmp signaling cascade in drg neurons. in the absence of npr , the majority of sensory axons showed normal bifurcation, but » % of the axons turned only in rostral or caudal direction. this study shows ( ) that pde a is important for the degradation of cgmp in embryonic drg neurons, ( ) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde a, and ( ) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi ). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d- -amino- -phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac -camps fused to a phosphodiesterase (pde a ). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families , and ) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde . however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde a (v max = µmol/min/mg) to a camp fret sensor (epac -camps) we monitor camp concentrations in direct vicinity of pde a . in comparison to pde , we show that pde a , albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j macrophages, arglabin strongly induced accumulation of lc type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc -ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp and pro-interleukin (il)- β, we further analyzed whether arglabin inhibits the nlrp inflammasome. arglabin reduced expression of nlrp and pro-il- β, inhibited activation of caspase- , and release of mature il- β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp inflammasome. intraperitoneal injection of arglabin into female apoe .ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il- β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par activating peptide sligrl ( µg/kg), hydralazine ( mg/kg), isoproterenol ( mg/kg), losartan ( mg/kg) and furosemide ( mg/kg) in conscious wild-type (wt) and par -deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet ( . % nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par -deficient mice than in wild-type mice ( ± versus ± ). renin mrna expression was ± % of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par -deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par (-/-) and wt mice. administration of sligrl increased renin secretion about -fold (p< . ). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par (-/-) and wt mice. the absolute changes (delta prc) were similar ( ± , ± , ± , ± in wt, and ± , ± , ± , ± in par (-/-)). in conclusion, chronic absence of par reduces basal renin expression and renin release. however, par -deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca + sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin- -gallate (egcg), which has been shown to decrease myofilament ca + sensitivity, on cardiac myocyte contractility and force-ca + relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc -targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca + transients at °c under hz pacing using the ionoptix system in the absence or presence of egcg ( . µm). furthermore, force-ca + relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg ( µm). results: in baseline settings and absence of fura- , ki cardiomyocytes displayed higher sarcomere shortening ( type- serine/threonine protein phosphatase (pp ) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca + handling and contractility. all pp heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor (i- ). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i- resulted in an improved basal ca + handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i- , regulated by gsk -dependent phosphorylation at thr , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with . % nacl (n= ). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca + transients were measured in isolated, indo- -loaded myocytes. the peak amplitude of [ca] i was reduced by % in tg nacl compared to wt nacl (p< . ), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by % in tg iso compared to wt iso (p< . ). consistently, sr ca + load was diminished by % in tg nacl compared to wt nacl (p< . ). chronic iso stimulation led to an unchanged sr ca + content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than -fold higher phospholamban phosphorylation at ser in tg (p< . ). thus, these findings suggest that overexpression of i- is able to reduce the progression of heart failure by an improvement of myocyte ca + handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp- macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a (abca ) expression in human thp- macrophages by this leoligin derivative. the transporters abcg and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock and rock are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock and rock are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock and rock by % was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with μm fasudil or µm h p for general rock inhibition and µm slx- for inhibition of rock . protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock and rock influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock and rock in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock , but not of rock led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h p and qpcr analysis of cf with a downregulation of rock and rock showed that both rocks are involved in the regulation of ecm proteins, such as collagens a , a, and a , biglycan, decorin, elastin and its respective degrading enzyme mmp . conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, ] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, ] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm- as a nucleus-specific marker was used to enrich cm nuclei to > %. proteins were extracted from nuclear lysate with % sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from murine cell lines served as silac (lys /arg )-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified proteins, of which are annotated to the nucleus. %/ % (adult / p ) of nuclear proteins are annotated as dna-binding. . %/ . % belong to transcription factor complexes; . %/ . % are able to bind transcription factors. . %/ . % have chromatin modification functions; . %/ . % modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. % of proteins are shared between p and adult nuclei. proteins are exclusive to p , are only found in adult. proteins are enriched ( . -fold increase in abundance) in p hearts, proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p cip /waf , senescence-associated ß-galactosidase (sa-ß-gal) and p ink a were increased -, -and -fold (n= - ; p < . ), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β -adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin ( ± . %), platelet derived growth factor receptor α ( ± . %) and α-smooth muscle actin ( ± . %), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor (cb ) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, , mg/kg/day) with osmotic minipumps for days. treatment with cb antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna- a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb antagonist treatment and foxb (a direct target of mir- a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir- a/b family seems to be sensitive to cb antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type diabetes mellitus (t dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: -week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing mg/g dabigatran or respective placebo for weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in t -l cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il- from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using t l cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca + -atpase (serca a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca + cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca + kinetics were measured in isolated, fura -loaded and electrically paced ( . hz) cardiomyocytes as the time to % decay of the ca + signal (t % ). the time to % baseline of sarcomere length (t % baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed . -fold stronger phosphorylation signals if pentamers were absent (p< . ). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t % [ms]: ± and ± , respectively, p< . ). notably, t % of pln-ko myocytes was ± ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca + clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t % baseline [ms]: ± in tgpln versus ± in tgafa-pln and ± in pln-ko, p< . ). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca + kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the ' end of mrnas ( ' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous ' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih- t fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor- (par ) regulates inflammatory responses including monocyte migration and cytokine release. par is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine- -phosphate (s p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s p on the expression of par , tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp and u cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par , tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with ng/ml pma (phorbol -myristate -acetate) over h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp and u cells s p induced a time-( to h) and concentration-dependent ( . to µm) significant upregulation of par mrna and total protein expression. par total protein was upregulated maximally (about . -fold, n= ) with µm s p after h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp monocytes and tm was not regulated by s p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s p resulted in a time-( to h) and concentration-dependent ( . to µm) significant upregulation of tf expression within to h of incubation. conversely, par total protein expression was reduced by about % after h s p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp cells as tf source. timedependent incubation with s p ( µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from . ± . to . ± . min and elevated total the thrombin generation capacity from ± to ± nm. peak thrombin formation was elevated from ± to ± nm/min (control versus s p for h, mean±sd, n= , respectively). these data suggest that s p induces an enhanced expression of par in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s p does upregulate tf expression but attenuates par levels. since par is involved in regulation cell migration, s p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin- (il- ) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il- expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il- mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp- cells was determined by western blot. immunostainings for il- and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa ( nm) induced a time-dependent ( - h) increase in il- mrna expression. maximum expression was observed within h to a . ± . fold increase (mean±sd, n= , p< . ). incubation with an inhibitor of p map kinase (sb , µm) or pi k (ly , µm) significantly attenuated fxa-induced il- mrna expression (n= ). inhibition of p / mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for h resulted in a markedly increased il- secretion into the smc culture media from . ± . to . ± . ng/ml (p< . , n= ). stimulation of thp- cells with il- ( ng/ml) induced a time dependent ( - h) increase of up to . ± . fold (p< . , n= ) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il- expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il- regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il- expression and secretion in venous smc which may be regulated via p and pi k signaling. il- enhanced tf expression in thp- monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il- release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p ). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week - with vpa ( . % in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week and ). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week : tg veh : ± %, wt veh : ± %; week : tg veh : ± %, wt veh : ± %, p< . ). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week : ± %, tg vpa at week : ± %, p< . vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p< . ) proteins were significantly changed while proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p< . ). proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p< . ), whereas vpa affected proteins in tg atria (tg vpa vs. tg veh , p< . ), out of which proteins were common. prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine ( mm), shifted the concentration response curves for the pie of histamine from ec = nm to nm (p< . ). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine ( µm), shifted the pie of histamine from ec = nm to nm and increased the efficacy of histamine for the pie (p< . ). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp , pp a, pp b (calcineurin) and pp in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase cβ (pp cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased ( . ± . mg/g) compared to wild type (wt) littermates ( . ± . mg/g; p< . ) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg ( . ± . mg/g) vs. wt ( . ± . mg/g; n= - ; - months of age; p< . ). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . and fractional shortening: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . ). the left ventricle was dilated (systolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ; diastolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein (igfbp ) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein (igfbp ) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp in murine cardiac tissue at different developmental stages by qpcr normalized to tpt (tumor protein, translationally-controlled ). this analysis showed temporal changes of cardiac igfbp expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp expression in different heart cells showed a very low igfbp in adult cardiomyocytes in contrast to a high expression in undifferentiated sca- positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp was found up-regulated (p< . ). further we found an increased igfbp expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p< . ). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p< . ). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp in angiotensin or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin , showed the opposite results, namely a reduction in its expression (p< . ; p< . , respectively). summary and conclusion: our results show active igfbp transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp in a conserved manner. we hypothesize that igfbp may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c bl/ j mice fed a high-fat diet (hfd) for weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last weeks with crataegus extract ws® ( mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® , neither in pvat nor in aorta. phosphorylation at serine is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine in pvat, but not in aorta. ws® treatment significantly improved enos serine phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® treatment. in addition, ws® treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin- (sirt ), known also as a longevity gene. the activity of sirt depends, among others, on the intracellular content of its cofactor nad. ws® treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt is enos. deacetylation of enos at lysine residues and by sirt enhances the activity of the enos enzyme. currently, we are studying the effect of ws® on nad synthesis, sirt activity, and enos (de)acetylation. in conclusion, crataegus extract ws® reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine and upregulation of sirt . the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk ) and the raf-erk / pathway. these two functions of rkip could counteract each other. while grk inhibition is cardio-protective, inhibition of the pro-survival erk / axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. ). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp ) under control of the alpha-mhc promoter. two different rkip-transgenic lines with . -fold and . -fold increased cardiac rkip protein level were generated (ref. , and jax id number ). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of . ± . % (n= ; ±s.d.) were documented at an age of months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk and raf-erk / axis by the human rkip gene (pebp ) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp ), which does not seem to inhibit the raf-erk / axis in vivo but instead confers grk inhibition- heterodimerization between the at receptor (at r) for the vasopressor peptide, angiotensin ii, and the b receptor (b r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at r--b r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at r--b r heterodimer in vivo, we generated a transgenic model of at r--b r heterodimerization (tg-b r+) by transgenic expression of the b r gene (bdkrb ) in the b r-deficient tg-b r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at r and b r at a distance of less than nm in tg-b r+ mice whereas the at r--b r heterodimer was absent in tg-b r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b r in the aorta of tg-b r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b r. the efficient at r--b r heterodimerization in tg-b r+ mice was accompanied by an enhanced angiotensin ii at r-stimulated vasopressor response relative to that of tg-b -/-mice, which lack the at r--b r heterodimer. as a control, the endothelin- -stimulated vasopressor response mediated by the etar, which did not dimerize with b r, was not significantly different between tg-b r+ and tg-b r-/-mice. together these findings provide strong evidence that at r--b r heterodimerization occurs in vivo and enhances the angiotensin ii at r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase (grk ) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk , grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk / axis and involved the erk / -dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine- . as a consequence of erk-dependent phosphorylation of pparg on serine- , the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine- phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine- a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as weeks of age in homozygous mice and - weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for weeks with vpa ( . mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of . ± v/s (tg: n= cells) vs. . ± . v/s (wt: n= , p= . ). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at % (tg: . ± . ms, n= vs. wt: . ± . ms, n= , p< . ), at % (in ms: . ± . vs. . ± , p< . ) and at % (in ms: . ± . vs. ± . , p< . ) repolarization. vpa treatment reduced ap amplitude in wt mice by mv (n= , p< . ) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced ( %: . ± . ms, %: . ± . ms, %: . ± . ms, n= , p< . vs. untreated tg), the amplitude was increased by mv (n.s.) and the slope of depolarization was increased by % (p= . , n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: ± . pf, n= vs. wt: ± . pf, n= , p< . ), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: . ± pf, n= n.s. vs. wt; tg-vpa: ± pf, n= , p< . vs. wt-vpa; p< . vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca + exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca + imaging revealed that the transduction rate of spontaneous sub-threshold ca + -waves into supra-threshold transient-like ca + -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca + transport rate (r serca ) was enhanced in tg vcms potentially limiting ca + extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during - m isoproterenol challenge, tg: . *, ctl: . ; n= /condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx , serca a and decrease of kchip protein levels. however, the respective mrna levels (slc a , atp a and kcnip ) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir- (fold change in tg: . *) and mir- (fold change in tg: . *) (n= /condition). atp a is a predicted target of mir- and mir- has recently been shown to regulate ncx and i to related potassium channel subunits. (*p< . vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from . (mg/g) in untreated control animals to . (mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of . (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr ( . mg/g) compared to saline treated animals ( . mg/g) and pka mutants ( . mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: . vs . mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh / ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged ( . mg/g) or a strongly elevated hbr ( . mg/g). however, additional hypertrophic factors including anp, myh / ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of µm after and h (ctb assay) nor increase in the apoptosis rate after , , or h up to µm. measuring adenosine triphosphate (atp) levels revealed that µm soraphen a does not alter the atp levels in huvecs after h treatment. in contrast, a h treatment significantly lowered the atp levels by %. also gene silencing of acc in huvecs attenuated the atp levels by %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels ( %) in soraphen a-treated cells after h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic value of µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g /g phase by % and increases the number of cells in the g /m phase by %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, µm soraphen a lowered the migration of endothelial cells by %. gene silencing of acc in huvecs strongly decreased endothelial migration, whereas a knockdown of acc had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for , fu / - ) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type (pcsk ) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october . we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor (klf ) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w domain containing protein (bzw ), a phylogenetically conserved protein, as a β-catenin and klf interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p< . , cm n= , cfb n= , cpc n= ), and skeletal muscle. bzw was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw , containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf knockout (ko) mice **p< . n= , and in a cardiac specific β-catenin stabilized mouse model, **p< . n= ). a mouse model with constitutively bzw loss of function (bzw ko) showed cardiac specific upregulation of β-catenin on rna level (***p< . , p< . , ctrl n= , bzw ko n= ) and on protein level (**p< . , ctrl n= , bzw ko n= ). echocardiography analysis in eight-weeks-old bzw ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p< . ctrl n= , angii n= ) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p< . ctrl n= , dcm n= , icm n= ). conclusion: these data demonstrated that bzw is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine- -phosphate (s p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s p lyase [ ] . mice that lack s p lyase are characterized by the accumulation of s p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [ ] [ ] [ ] . on the other hand, embryonic fibroblasts from s p lyase-deficient mice (sgpl -/--mefs) are resistant to chemotherapy-induced apoptosis [ ] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [ ] . interestingly, s p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [ ] . the aim of the present study was to analyze the link between s p lyase deficiency and altered cholesterol homeostasis using sgpl background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine- (p-ser ) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser -pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> . fpkm, gene expression rank < , ) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser -pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type (tnni ) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin (acta ) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt fl/fl mice with heterozygous dnmt fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl -cre) resulted in embryonic deletion of dnmt (ko). embryos without myl -cre served as controls (ctl). embryos were dissected and genotyped at e . , e . , e . and e . . rna-seq and pyrosequencing of genomic dna was performed on e . hearts, histology on e . hearts and electron microscopic imaging on e . hearts. for deletion of dnmt in adult mice, homozygous dnmt fl/fl mice expressing an inducible cre recombinase (myh -mcm) were given tamoxifen i.p. over days. homozygous dnmt fl/fl mice not carrying myh -mcm as well as myh -mcm carrying mice without dnmt fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl -cre mediated loss of dnmt resulted in progressive embryonic lethality with absence of living ko embryos after e . . ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet . methods: female mice homozygous for tet flox and male mice heterozygous for tet flox and heterozygous for myl -cre were mated and offspring were genotyped after weaning. mice homozygous for tet flox and heterozygous for myl -cre (ko) or homozygous for tet flox without myl -cre (controls) were sacrificed at weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet and tet . cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp a gene and at the binding site of the transcription factor gata but not near the promotor and the binding site of the transcription factor tbx . higher methylation levels were not accompanied by changes in atp a expression. however, both myh and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet is involved in dna demethylation in cardiomyocytes. loss of tet expression resulted in embryonic lethality. compensatory upregulation of tet and tet isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor (pi ). a recent gwas analysis showed an association of a genetic variant in the pi genomic locus (rs ) with chemerin plasma levels. here we tested the hypothesis that pi determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi , which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi -deficient animals compared to littermate controls. to investigate whether pi and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi . when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi to potently inhibit cathepsin k activity. taken together, we propose pi to act as a regulator of chemerin processing. the transient receptor potential canonical (trpc ) is a second messenger-gated cation channel, which mediates depolarization and ca + entry. it is known to be activated by diacylglycerol derivatives (dag, -oleoyl- -acetyl-sn-glycerol oag) [ ] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [ ] thus, the discovery of potent inhibitors of trpc may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc . [ ] larixyl acetate, another component of the resin showed an even higher potency in trpc inhibition (ic = . µm) and a -fold selectivity compared to trpc . these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc . hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc with similarly low ic values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c position, however, seems to play no important role for the inhibition of trpc currents. larixyl methylcarbamate lead to trpc inhibition with an ic value of . ± . µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc , this compound bears the benefit of high subtype selectivity towards trpc . even with concentrations up to µm of larixyl methylcarbamate, no complete inhibition of the ca + influx via trpc channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α nachr using whole-cell patch clamping under voltage-clamping conditions (- mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, -tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes ( ). among the mammalian trp channels, the canonical channel (trpc ) is a ca + -permeable ion channel, which is predominantly expressed in the brain ( ) . many aspects of trpc function are still elusive although behavioral experiments with trpc -knock-out mice suggest a role in innate fear-response ( ) and some studies indicate a trpc -mediated down regulation of neurite outgrowth in nerve cells ( , ) . to elucidate trpc function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo ( , ) . thus, there is still a need for identifying novel and efficient trpc modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc activator. hek cells heterologously expressing trpc upon tetracycline induction (hek trpc ) show a btd-induced concentration-dependent activation in both ca + assays (ec = . µm) and in electrophysiological whole cell patch clamp recordings (ec = . µm). btd elicits currents with an n-shaped i/v curve, typical for trpc . the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc inhibitor ( ) . mtt assays revealed that incubating hek trpc cells for h with btd concentrations above µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca + -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to µm. ca + assays showed no influence of btd on closely related trpc channels, as well as trpc / / at concentrations up to µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc subunits can also assemble to heteromeric channel complexes with their closest relatives trpc and trpc ( ) . trpc / and trpc / heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc subunit. transient receptor potential canonical channels / and are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc , based on the recently available structural information on trpv , we performed structure-guided mutagenesis and identified a single residue in transmembrane domain (g ), which is conserved within the canonical family of trp channels. single point mutations at position in trpc largely eliminated lipid sensitivity. trpc g a expressed in hek cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc / / channels (gsk a) activated wild-type trpc and trpc channels as well as the respective lipid insensitive mutants (trpc g a, trpc g a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g to gsk recognition by trpc . our results demonstrate the existence of two different mechanisms of trpc / activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc / involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s provided by g . lipids and synthetic activators of trpc / may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb ( , '-(propane- , -diyl) bis ( -tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c -c ) of mb were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of µm carbamoylcholine in the functional assay. mb and several bispyridinium structure analogues (mainly c -c linker) exhibited no regular displacement curves at [ h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the -tert-butyl-substituted bispyridinium compound mb showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb or mb , could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations ( - µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field ( , , hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb ( , ′-(propan- , -diyl) bis ( -tertbutylpyridinium) di(iodide)) and mb ( , ′-(propan- , -diyl) bis ( -ethylpyridinium) di(iodide)) in the tested concentration range ( - µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low ( hz) and high ( and hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p x receptor-induced current by the opening of asic channels g. stephan , p. illes universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p x receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel (asic ) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p x and asic subunits has to be taken into consideration. we transfected rat (r)p x r and rasic constructs individually or together in a ratio of : into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p x r-and asic -constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from . to . caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to . . the opening of asic channels failed to facilitate the p x r current when -guanidine- -methylquinazoline was used to stimulate a non-proton ligand-sensor of asic . then, we substituted ca + in the extracellular medium by ba + or decreased the intrapipette concentration of egta, to modify the free intracellular ca + concentration. in cells individually transfected with the receptor-channels, external ba + increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p x r-and asic -specific currents in a similar manner as external ba + . in cells co-transfected with p x r/asic , the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic activation. taken together, our results suggest that p x r and asic interact with each other, since the activation of asic had a marked impact on the p x r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca + and the proton sensor of asic appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned- . in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned- support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv has been identified in endothelial cells ( ), epithelial cells ( ) and arterial smooth muscle cells ( ) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in ). it is a matter of debate however if trpv activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc ( ) which is also expressed in both cell types. to analyze the potential role of trpv on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after minutes of ischemia in trpv -deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv /trpc double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc deficiency protected lungs from iri-induced edema ( ) . moreover, we identified reduced expression levels of aquaporin in trpv -deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily (trpm ) has been shown to be expressed in human kidney , . using newly developed anti-trpm antibodies, we are able to visualize trpm protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm (trpm the atp-gated p x receptor (p x r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p x -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p x r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p x r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p x r are overexpressed under the control of the endogenous p x promoter. the egfp-tagged p x rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p x -egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map -and neun-positive cells indicating that under non-pathological conditions, the p x receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd protein was observed in the p x r-overexpressing mice. these results suggests that overexpression of p x rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd primarily localizes to lysosomes and endosomes, this further supports a role of the p x r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc and tpc -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca + channels or such as trp channels. due to their biophysical properties, tpc and tpc are assumed to be involved in the efflux of ca + from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc for vesicle fusion & fission processes we investigated a potential impact of tpc on the uptake of bacterial toxins. first we determined the precise localization of tpc in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc -deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland , . the trpm gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists , . variations include the presence or absence of five stretches of to amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini . furthermore, three different aminotermini of human and mouse proteins have been described , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified variants that differ in exons , , , and . however, only variants bearing the short, ca + permeable pore loop were detected. ´ rapid amplification of cdna ends ( ´race) revealed that trpm proteins of the pituitary gland carry truncated c-termini exclusively. ´race identified five independent regions of transcription initiation within the trpm gene implying the presence of five independent promotors. the different transcripts encode four trpm amino termini α, β, γ and δ of - amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~ %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to different bilayers at a time. here we utilize the "orbit" multichannel system with a meca chip by ionera to measure tpc channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav ) and potassium channels (like kv , kv ). the voltage-gated na + channel nav . initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv . is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav . and kv . have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) is known to interact with the kv . channel complex, modulating kinetics and voltage dependence. the overexpression of dpp in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv channels. to study the potential influence of dpp on nav . channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k cells, expressing scn a alone or with dpp . surprisingly, we observed significant effects of dpp on nav . channel voltage dependence and kinetics. thus, the co-expression of dpp significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav . channels alone. in addition we analysed the effects of dpp on the kinetics nav . currents. while time to current peak was not affected in cells co-expressing nav . and dpp compared to nav . alone, dpp slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav . and dpp compared to nav . alone. in summary, we provide first evidence that dpp not only interacts with kv channels, but also influences nav . channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv . and nav . behave very differently depending on the expression of its associated proteins like dpp . cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc /c -deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca + -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc and trpc (camacho londoño et al., ) . nevertheless, the role of trpc /c for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal days. our results show that there is no difference in nm at-ii induced ca + -release or ca + -entry in trpc /c -deficient cf compared to wt. to evaluate whether the lack of trpc /c can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca + -release and ca + -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca + ] i like isoproterenol, -ht, thrombin and endothelin- . cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv . channels, which are known to be expressed in vascular smooth muscle cells. k v . voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l (k v . opener), hmr , chromanol- b (k v . inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq -/-mice to determine whether k v . plays a role in the regulation of arterial tone. we found that r-l produces similar concentration-dependent relaxations (ec ~ , µm) of wild-type (kcnq +/+) and kcnq -/-arteries pre-contracted with either phenylephrine or mm kcl. this relaxation was not affected by µm chromanol- b, µm hmr or µm xe (pan-k v blocker). the anti-contractile effects of pvat were normal in kcnq -/-arteries. chromanol- b and hmr did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq -/-mice exhibited normal peak k v currents. the k v . - opener retigabine caused similar relaxations in kcnq -/-and wild-type vessels. we conclude that k v . channels are apparently not involved in the control of arterial tone by alpha adrenergic vasoconstrictors and pvat. in addition, r-l is an inappropriate pharmacological tool for studying the function of native vascular k v . channels in mice. introduction: according to international guidelines [ ] [ ] [ ] [ ] [ ] [ ] [ ] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [ ] [ ] . material and methods: tissue the skin samples are descended from female pigs of german landrace ( kg weight, approx. month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from different skin area (fig. ) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of . µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of mm were punched out from the frozen skin stripes and stored at - °c. hplc and skin absorption waters corporation hplc containing sample manager, binary gradient pump, pda detector (optional: electron spray mass spectrometer); column: nucleodur® - c ec mm x . mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, -cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of h at °c. the dosage compartments of each cell were filled with approximately µl of the caffeine solution ( mg/ml). samples of ml are taken after , , , and hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. ) . thickness was in the order of magnitude between to . µm and intra variations were less than %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [ ], demonstrating intact barrier properties of the membranes after month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in , the mak commission derived a new threshold value of . mg/m for gbs with a density of , recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day , and ) and the lung toxicity after intratracheal instillation in rats (day and ): tio (rutile, micro), tio (anatase, nano), eu o (micro-nano mixed), baso (micro), zro (micro) and amorphous sio (nano). two doses of . and . µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio (rutile) and baso (pmn < % after days in the low dose group; < % in the high dose group; full recovery after days). in contrast, the tio (anatase) showed a stronger response (pmn > % after and days). the rare earth eu o (micro-nano) dust showed the strongest effect (approx. % pmn after and days) including a red-coloured lung lavage fluid. µ-zro and amorphous sio showed a strong acute response after days, however, mostly complete recovery after days. the low solubility criterion was met by the following dusts: tio (both) and zro . -similar volumetric lung burdens were deposited in a parallel validation experiment ( -day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, µm of pyr and µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp a gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp b in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from ) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and ) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of µm (bpa), µm (fen) and . µm (keto), for concentrations in target organs resulted in estimated oral loels of , and . mg/kg. when calculations were made for plasma levels oral loels were estimated to be , and mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of mg/kg bw day for bpa ( ), mg/kg day for fen ( ) and mg/kg day for keto ( ) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal /uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp a promotor activity was investigated using a pxr-dependent cyp a reporter gene assay. cyp a induction was analyzed at the mrna and protein levels in hepg cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp a promotor activity could only be detected for echimidine. cyp a induction by echimidine was verified at the mrna and protein level in hepg wildtype and hepg pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases ( isoforms are known, nox - , duox & ) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox -, nox -and nox -deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg during days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox and nox could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox -knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox -knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox -knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox . conclusion: separate eliminations of nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last years about drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin , podocin, synaptopodin, retinol-binding protein , clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of , -intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c exoenyzme (c ) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c . knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c . moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c . proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c -induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c compared to wild type cells. as vimentin knock out did not completely inhibit c uptake into cells, an additional uptake mechanism or additional receptor for c is likely. nevertheless, our data reveals that c employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for h before toxicants were added to the cells. migration and viability of the cells were then measured after another h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein (rip ) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht- cells erastin promotes activation of rip and subsequent rip -rip necrosome formation which has been investigated as a hallmark of regulated necrosis . in fact, silencing of rip by sirna or by the rip inhibitor necrostatin- prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin- fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein- (rip ) and rip kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) , . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip /rip -dependent necroptosis and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip -rip complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi- , was from medchem express. murine embryonic stem cells (es-d ) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of . % in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between . and mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in , and wells out of wells in total at concentrations of , and mg/l. at mg/l no differentiation was observed. effects on cell viability were observed at mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of mg/l this nsa -inibitor showed a clear impact on differentiation with out of wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi- at concentrations up to mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg ) and developmental toxicity studies (oecd tg ) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the -generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some -days old pups. for all effects clear noaels were determined. in the -generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies ( -generation toxicity study and a -week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in , dimoxystrobin was classified with r (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r (which has been translated into the ghs classification repr. , h d) was based on offspring body weight and heart effects seen in the -generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in and a restriction program for pfoa was initiated in . therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and , , , -tetrafluoro- -(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek- t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a -fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a -fold repeated estrogen response element (ere). both constructs were stably transfected into t d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone- and -mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of betaestradiol (e ) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) . furthermore, rats fed idd until pnd were fed ird for days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e and estrone (e ) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e and diet (idd vs ird and idd vs idd+ird). e levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e and ratios e /e in mg and ratio of e levels in pl/mg were not affected by diet. in contrast, diet tended to affect e concentrations in pl (p= . ) due to an increase in the ird group (p= . ) whereas e levels in the idd+ird group only tended to be elevated (p= . ). in mg, ird and idd+ird increased e levels only weakly (p= . each). likewise, besides significant changes in transcript levels of cyp a and a , putatively decreasing oxidation of e to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp , mt a) was observed in the idd+ird group. e mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e levels but not diet were significantly associated with gata transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt ) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e (increase in gdf , hgf, igf r, wnt a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e and even decreased not significantly by idd+ird. taken together, despite an increase in e levels in pl, less er activation was observed after dietary exposure to if. whereas e and transcript levels of enzymes involved in e (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le / - . august copenhagen irish (aci) rats with β-estradiol (e )-releasing implants are an accepted model to study the etiology of breast cancer, but neither e (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) and , placebo (-e ) or silastic implants containing mg e were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd . palpable mg tumors (pt) and - mg per animal without pt were characterized histologically and categorized into normal (-e group, n= ), hyperplasia and non-pt and pt with and without solid tumors (+e group, n= ). e , estrone (e ), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e and e in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of transcripts involved in (biotrans)formation of e and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e , plasma e as well as e and (borderline) e levels in mg were higher in ird. plasma e as well as e and e levels in mg were lower in the -e group than that in the +e group. e levels as well as e /e and e mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki ). ird increased e /e ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata ) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd ) tissues. levels of e and -meo-e were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd b (conversion e to e ) and cyp a . transcript levels of gstm and gstm were decreased in the whole +e group and of gstt and gstt in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp and mt a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of -meo-e nor cellular stress (gclc, mt a, tp ). of note, neither -meo-e , nor e catechols, nor e catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e or e were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e or e . taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e and cellular (oxidative) stress, pt exhibited highest e levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e /e ratio in pt. supported by dfg le- / - . level of beta-estradiol (e ) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from mammoplasty specimen obtained from women (age - years old) not taking estrogen active drugs. of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n= ) and bacterial metabolites (n= ) as well as the estrogens estrone (e )-sulfate, e , e and -methoxy-e were determined by uhplcand gc-ms/ms, respectively and transcript levels of enzymes involved in e (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e /e ratio with agl(+con) was observed in glandular tissues (r= . , p= . ), accompanied by a significant negative correlation of e levels with agl (r=- . /p= . ), possibly due to reduction of beta-hydroxysteroid dehydrogenase (conversion of e to e ) expression as indicated by a weak negative correlation of transcript levels of beta-hydroxysteroid dehydrogenase with agl+con (r=- . , p= . ). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e /e ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le- / - . allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp- cells were used as a model system and treated with the sensitizer , dinitrochlorobenzene (dncb; , and µm) and the irritant sodium dodecyl sulfate (sds; µm). samples were taken after , and hours. thp- activation was analyzed by measuring the established activation markers cd and cd after hours. a targeted lc-ms/ms approach was used to analyze metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., ) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp- were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp- -macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp- cells. evaluation of hepatic cell stress showed the strongest effect on thp- -macrophages when phh were incubated with apap or dic for h.treatment of kc and thp- -macrophages with supernatants of phh challenged for h with hepatotoxic compounds indicates that thp- -derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp- derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to , . , . , and mg/cm³ ceo as well as mg/m³ baso nanoparticles ( h/day, days/week, weeks). animal dissections were conducted at five time points (exposure day and ; recovery day , and ) aiming for endpoints mandatory according to oecd guideline . additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo and baso retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to days was detected by balf and histopathology examination. based on our current results effects of ceo nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt- project is funded by the german federal ministry of education and research (bmbf) - x a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco- model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco- cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana . expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana . project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana . provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana . is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around % of total pm emissions are emitted by wood burners (uba ) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a ) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted : and directly delivered to the exposure chamber. after h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. part fibre toxicol : ), or amosite asbestos at µg/cm for h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed significantly differentially regulated genes for mwcnt a, for mwcnt d, for amosite, and for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht ). for this purpose ht cells were exposed for hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of - µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of . pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about %; control about %), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about %; control about %) for concentrations above µg/ml. induction of apoptosis was determined for silver particles (up to % early apoptotic and % late apoptotic cells) as well as for titanium dioxide and zinc oxide ( % each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to %). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [ ] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of nm and µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in -well plates and exposed to doses of , , and µg/cm² growth area of the respective cnt type for h. the wst- assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h so /hno mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [ ] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union th framework program (grant agreement no. ), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of µg/cm ( h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il- liberation (elisa) as primary endpoints, and quartz dq and al o as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) nanoplatelets (mg ; mean lateral size: , - µm; mean thickness: - nm) represented the least, and pristine few layers graphene nanoplatelets (gr ; mean lateral size: µm; mean thickness: nm; graphene layers: ± . ) the most biologically active nm. clear concentration dependencies were detected for gr in follow-up experiments. mg and gr were further tested in other lung-relevant cell types, i.e. mrc- primary human lung fibroblasts and a lung adenocarcinoma epithelial cells. interestingly, mrc- cells were less sensitive towards biological effects of gr , compared to am, whereas a cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for h to . or µg/cm of gr on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr , i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc- cells and limited uptake in a cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc- cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) for two years; a control group was exposed to clean air. after one year exposure, µg/lung was found in animals exposed to . mg/m³ and . mg/lung in animals exposed to mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of . and . mg/m³ ceo . neither pre-neoplastic nor neoplastic changes were observed after -months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) and barium sulfate (nm- ; mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo lung burdens were maximally . mg/g lung tissue at the highest exposure concentration of mg/m³. in comparison, highest ceo burdens in organs remote to exposure were liver and spleen with maximally roughly x - g/g tissue. in brain, maximum ceo levels were x - mg/g lung tissue. baso lung burdens were comparatively low ( mg/g) within the first weeks of exposure and steeply increased to mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df nano) that consists of tiers to assign nanomaterials to main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass ( ) soluble nanomaterials, ( ) biopersistent high aspect ratio nanomaterials, ( ) passive nanomaterials, and ( ) active nanomaterials. the df nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df nano for nanomaterials were validated against grouping by results of existing in vivo data and demonstrated concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo np with an average diameter of - nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations ( . , . , and %) and were used to expose human lung adenocarcinoma cells (a ) for up to hours. at µg/ml ceo np showed little to no toxic effecton growth arrested a cells at fcs concentrations of % or below, but cell viability was decreased to about % in proliferating cells in cultures with % fcs. the proinflammatory effect of ceo np was investigated through measurement of il- mrna expression after hours and cellular il- secretion after hours. the qrt-pcr showed that the expression of il- mrna in cells treated with µg/ml ceo np in % fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il- secretion, which showed a stronger increase by cells treated at % fcs. differences in cellular uptake of ceo np was determined by fluorescence-activated cell sorting (facs) after and hours of exposition. after hours, the cells treated with ceo np in % fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about nm ( % fcs) to nm ( . % fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase- (ho- ) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)- β, il- , il- and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il- β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio np by wst- assay overall revealed only minimal toxicity in comparison to sio np which were used as reference material. ho- and g-gcs mrna analyses indicated that specific tio np triggered a moderate induction of oxidative stress. il- was only induced after sio treatment, whereas il- was not affected by any of the tested np. in contrast, various tio np caused a significant induction of il- β mrna expression. however, no significant induction of il- β and tnf-α protein secretion was observed for any of the tio np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp programme. specific types of tio particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase (nat ) activity j. lichter , b. blömeke trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase (nat ), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat has not been analyzed yet. to address whether human nat is a target of silver nanoparticles and released irons on protein, purified nat was exposed to silver ions (agno ) and silver nanoparticles (ag -cooh, average size nm, carboxyl functionalized), and nat enzyme activity was analyzing the n-acetylation of the nat substrate para-aminobenzoic acid (paba). therefore, purified nat ( ng/µl) was co-exposed for min to paba ( mm) and agno or ag -cooh ( . , . , , and µg/ml each), resulting in a nat :silver relation of : . - (w/w). both, agno and ag -cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat (w/w, µg/ml) enzyme activity was reduced about ± . % (agno ) and ± . % (ag -cooh). the lowest concentration analyzed ( . µg/ml) reduced nat activity about ± % (agno ) and ± % (ag -cooh). fifty percent activity reduction was caused by . ± . µg/ml of agno and . ± . µg/ml of ag -cooh, which is -fold lower compared to the published ic values for other metal oxide nanoparticles ( - µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat -days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of and mg/kg body weight for nanosilver and corresponding amounts of silver acetate ( . and . mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by -d gel electrophoresis and differentially expressed spots were identified by maldi-ms. unique proteins, showing a log ratio of ≤ - . for downregulation and ≥ . for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf -mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated - - protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below % for mg/ml after h). in general the effects of both nps were almost similar. additionally the effects of the baso -nps were compared more in detail. uptake of the baso was quantified by icp-ms after h and h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso of plc and nhbec to . mg/ml over h and h leads to ~ µg baso / mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il- and il- could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after h. summing up baso -nps showed toxic effects in primary human lung cell cultures after h for concentrations under mg/ml. c exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht revealed a c -mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., ) . former results of studies on map kinase signalling indicated c -induced modulations of downstream signalling modules. therefore, ht cells treated with nm c for h were applied for screening of the activity of various transcription factors followed by luciferase reporter assays. five transcription factors namely sp , atf , e f- , cbf, stat were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp target genes p and cox- as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd , a target gene of atf , was decreased. our results suggest that c is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of . g/cm , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from to xg resulted in higher yields. in general, large variations in yield ( . % ± . %) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl- -naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or -tert-butyl- -methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system ( qtrap) and a hybrid quadrupole time-of-flight instrument (tt + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. ( ) chem res toxicol , resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of fmol per injection ( pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and -monochloropropane- , -diol ( -mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and -mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and -mcpd. the metabolite , -dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is -mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary -mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound c -dhpma. the limit of quantification is µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = ) of one male volunteer collected over ten days was ± µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing . mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b c f mice and nasal neoplasms in male f /n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic -sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n -[(furan- -yl)methyl]- ′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase (nat ) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n= ), dortmund (n= ) and neuss (n= ). nat was genotyped using seven characteristic polymorphisms (rs , rs , rs , rs , rs , rs , rs ). haplotypes were reconstructed using phase v. . . . we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat * b/* b and * b/other slow haplotypes as well as between the ultra-slow * a/* a and * a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of ( %) patients showed a relapse within the first yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators ( % vs. %, p= . ). this frequency is even higher in ultra-slow acetylators ( %, or= . , p= . ) but not in slow * b/* b genotypes ( %, p= . ). ultra-slow acetylators had a significantly shorter relapse free time within yr after bc diagnosis than rapid acetylators (median . vs. . yr, hr= . , p= . ). this trend was not that pronounced in all slow acetylators combined ( . yr, hr= . , p= . ) nor in the subgroup of nat * b/* b genotypes ( . yr, hr= . , p= . ). the effect of ultraslow nat is even more pronounced in smokers (hr= . , p= . ) but absent nonsmokers (hr= . , p= . ). ultra-slow nat seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand β-estradiol (e ). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf- cells treated with e or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [ ]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; / ). urine specimens from children aged from to months from different day-care centers were analyzed. in total, preschool children were recruited with mean age of months. our study results show that nearly all children (> %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, oh-mehp, oxo-mehp, oxo-minp), for bisphenol a and methylparaben. triclosan was detected in % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about - µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about µg/l. median urinary concentration for methylparaben and bisphenol a were about µg/l and µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were µg/l, . µg/l and , µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study ( / ) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of µg/kg benzo[a]pyrene and µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [ ]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl ). first, the time-dependent internalization of hemin and fecl into hct cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco- and hct -cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion -oxoguanine ( -oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue (mth ), which protects the nucleotide pool by hydrolysis of -oxogtp, -oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct and caco- cells using western blot analysis, which was followed by a reduction in cell viability at high doses after h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between . % and %. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [ , ] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [ ] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [ ] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [ ] göggmann, schimmer ( ) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. , [ ] [ ] [ ] [ ] wiseman et al. fatty acid esters of -chloro- , and of -chloro- , are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free -mcpd and -mcpd. -mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which mg/kg b.w./day of either -mcpd or -mcpd were orally administered to rats over a period of days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis ( de). among others, the redox sensor protein dj- was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with -mcpd or -mcpd. up to six different isoforms of dj- were identified by de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either -mcpd or -mcpd predominantly led to a shift of the abundance between two dj- isoforms in the rat tissues. this effect was more pronounced with -mcpd compared to -mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys ) of dj- to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either -mcpd or -mcpd. dj- is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys was shown to be crucial for the activity of dj- , and the irreversible oxidation of cys to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of -mcpd and -mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. , -unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure ( h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure ( d) comparatively to short-term exposure ( h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp a (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp , which results in cell stress and caspase / dependent cell death. recently, we discovered that higher concentrations of suba ( mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [ ] . in contrast to hela and caco- cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of -mcpd and its esters. high amounts of -mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related -mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of -mcpd, pointing to kidney and testes as main target organs. -mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for -mcpd, showing similar organ toxicity compared to -mcpd and identifying heart as additional target organ. no such data exist for -mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for -mcpd, -mcpd and their esters. hence, an oral -days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by -mcpd and -mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of -mcpd + -mcpd ( mg/kg body weight), and -mcpd-dipalmitate ( + . mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by -d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of unique proteins deregulated at a log ratio of ≤ - . for downregulation and ≥ . for upregulation at p ≤ . were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by -mcpd versus -mcpd. dipalmitate treatment even caused more deregulation than -mcpd. upregulated cytochrome b-c complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after -mcpd and -mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after -mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after -mcpd treatment. protein dj- (park ), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with -mcpd and the higher dose of the -mcpd diester. tropomyosin beta chain (tpm ) was commonly upregulated after -mcpd and -mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of -mcpd-and -mcpd dipalmitate-induced proteomic changes exist compared to -mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as α-hydroxy-chol ( α-ho-chol), β-hydroxy-chol ( β-ho-chol), -keto-chol ( -o-chol), , α-epoxy-chol (α-epoxy-chol) and , β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where β-ho-chol (milk) and -o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: α-cholestan- β-ol), respectively in breast adipose tissues of healthy women undergoing mammoplasty. furthermore, tissue levels of fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: . nmol/g tissue, range: . - . nmol/g), followed by α-epoxy-chol > -o-chol > α-ho-chol> β-ho-chol ( . nmol/g, range: . - . nmol/g). tissue levels of chol ( . micro mol/g, range: . - . micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of α-ho-, β-ho-, and -o-chol (r = - . , p= . - . ) median oxychol/chol ratios ranged from . ( , to . ( β-ho-chol). -o-chol and -ho-chols correlated strongly with each other (r= . - . , p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r= . , . , and . , respectively, p= . , . , . , respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products ( ) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin ( ) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them ( ) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation ( ) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument ( ). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently ( , ). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points ( ). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas - - ), icaridine (cas - - ) and ebaap (cas - - ) ( ). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than % as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than , international publications have been transferred to a database, with more than , entries. according to the database, pharmaceuticals have been found in the environment of countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, chemicals (ca. %) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a and days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet ( - mg/l) and els study ( . - . mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat- (slc a ) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat- for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat- expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat- in human ea.hy endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat- mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb ( , -dichloro- -β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat- gene responsible for this regulation. to our surprise and in contrast to data published by others the chromosomal region up to kb upstream of the first hcat- exon exhibited no promoter activity in either endothelial or dld- colon carcinoma cells that exhibit a very strong endogenous hcat- expression. we could however identify a promoter element within the first intron of the hcat- gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat- expression. our results indicate starvation-induced transcriptional regulation of hcat- through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc -family consisting of the two multidrug and toxin extrusion transporters mate- (slc a ) and mate -k (slc a ). while mate- is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate -k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with different anti-neoplastic agents comparative transport experiments with the model substrate -methyl- -phenylpyridinium (mpp) have been carried out. therefore stably transfected hek -cells expressing mate- or mate -k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek -cells were used. the interaction analyses were carried out by determining the uptake of [ c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) b that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp b substrate estrone sulfate (e s), and observed a significantly lower k m for mdck-hoatp b compared to . ± . µm, *p< . student´s t-test) whereas there was no difference in v max (mdck-hoatp b . ± . fmol/min/µg protein; mdck-roatp b . ± . fmol/min/µg protein). the human oatp b exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [ ] . to identify whether rat oatp b owns the same characteristics, the cellular accumulation of µm e s (low affinity site) or . µm e s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp b . as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e s in mdck-hoatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein, *p< . student´s t-test), there was no inhibition of e s accumulation in mdck-roatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp b while human oatp b is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per ng of total rna) in different tissues of rat revealed a high expression of oatp b in liver ( . ± . ), a moderate expression in small intestine ( . ± . ) and colon ( . ± . ), and a low expression level in kidney ( . ± . ) . the latter is in accordance with previous findings showing that oatp b is abundant in rat kidney as quantified by absolute proteomics technics [ ] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v. , targetscan . ) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of mirnas, were detected in all tissue types. out of ten transporters five showed significant inverse correlations with putatively targeting mirnas (e.g. pept and hsa-mir- a, r= - . , p= . ). reporter gene assays indicated interactions of mir- a/b with pept '-utr (p = . and p = . ) as well as of mir- a with abcb '-utr (p = . ). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept and abcb . background: the human uptake transporter nact [gene symbol slc a ; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc a gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc a gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g r, -p.t m and -p.l p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of tested) inhibited nact-mediated citrate transport at the tested drug concentration of µm. from these, benzbromarone shows the strongest inhibition with an ic value of . µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g r, -p.g e,p.t m, -p.l p and -p.l p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc a gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member (abcb ) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol- , -bisphosphate (pip ) is a known regulator of abcb . the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht- served as a model. we employed various in-vitro methods for the measurement of abcb activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb activity and re-sensitize our cell model against chemotherapeutic agents like -fluorouracil which are known to be substrates of abcb . taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb might be a new approach to target resistant tumors without interfering with the natural function of abcb in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ h]-labeled homoarginine as substrate and human embryonic kidney (hek ) cells stably overexpressing human cat [gene: slc a (solute carrier family )], cat a (slc a a) or cat b (slc a b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat ( -fold), cat a ( . -fold) or cat b ( . -fold) after . min using µm substrate (each p< . ). apparent k m values for cellular net uptake of homoarginine were . µm for cat , µm for cat a and . µm for cat b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat , cat a and cat b. compared to other cat substrates, the cat -and cat b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter (oct , alternative name slcc a ) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b ), known oct substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct has been shown to have strong inter-species differences. in rodents oct is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct orthologs. methods: overexpression of oct orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ . the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct . human oct showed a fold higher v max for the uptake of asp + and -fold increase of v max for sumatriptan in comparison to the rodent oct orthologs. conversely, human oct showed a -fold lower v max for the uptake of fenoterol compared to rodent oct s. there was no difference between rodent and human oct in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct constructs and by comparing the effects of key point mutations in mouse and human oct orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct orthologs. therefore oct pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct . ranitidine is a histamine h -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately % of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct [ ]. oct is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct alleles are known [ ] . thereof alleles confer partial or complete loss of oct activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct on the uptake of ranitidine. we used hek cells stably transfected to overexpress wild type or variant oct isoforms. the variant oct alleles oct * a (met val), oct * c (phe leu), oct * d (pro leu/met val), oct * (met del), oct * (arg cys), oct * (gly ser), oct * (gly arg/met del), oct * (cys arg/met del), oct * (ser phe), oct * (arg met), oct * (pro leu), oct * (ser leu), oct * (ile thr), oct * (ser leu) and oct * (thr met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct isoforms. wild type oct showed a time and concentration dependent uptake of ranitidine with a k m of . ± . µm and a v max of . ± . pmol/min/mg protein. variants oct * , oct * , oct * and oct * completely lacked ranitidine transport activity. variants oct * , oct * , oct * and oct * showed v max values decreased by , , and %, respectively. oct * variant showed an increase of v max by %. there was no significant changes in ranitidine uptake by variants oct * a, oct * c, oct * d, oct * , oct * and oct * compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct substrate and demonstrated that genetic polymorphisms in oct can strongly affect ranitidine uptake. the effects of oct polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of -ht a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate -ht a receptors, we here compare lsd and dmt with regard to processes of -ht a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the -ht selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective -ht a antagonists ketanserin and mdl . after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ s]gtpуs binding) is paralleled by a desensitisation of frontocortical -ht receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha- -ht a transfected) hek and (endogenously -ht a expressing) c glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise -ht a receptors, fails to activate the phospholipase d (which is needed for -ht a internalisation), and fails to inhibit the synthesis of -ht a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of -ht a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of -ht a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., ) and sunset (schmidt et al., ) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div . in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor- kinase inhibitor a and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a and spermidine had no effect on de novo protein synthesis in juvenile neurons (div ) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div ). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin a receptors ( -ht a r) apparently play a pivotal role in the development of depression. the involvement of an altered, -ht a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located -ht a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic -ht a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic -ht a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment ( days) with the selective -ht a r agonist -oh-dpat ( , mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; mg/kg/day). mice are sacrificed either one day (proliferation) or days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic -ht a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia ( days), but not after spontaneous remission (age days). as indicated by unaltered homeoboxprotein nkx . (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc and cavii mrna, used as markers for the gaba-switch, were unchanged in and day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha subunit). by stereological determination, we found a % decrease in alpha subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in ( %) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem ( . and . mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam ( . and . mg/kg i.p.) in the dt sz hamster. examinations of αlpha gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha gaba a r subunits. changes in αlpha gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. ) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh- ( x - m) and jwh- ( x - m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh- was not affected by pregnenolone ( - m), the inhibition by jwh- was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone ( - m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ -tetrahydrocannabinol ( x - m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ tetrahydrocannabinol were not changed by pregnenolone ( - m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, - m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol : - , ) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β -antagonist sr a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor (trh-r ) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par- , par- and par- . in human adipose tissue, par- is found in vascular stromal cells and par- in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in t l cells. results: differentiation of t l preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par- is time-dependently upregulated in maturing cells while par- expression is detectable but not altered. in preadipocytes, thrombin ( u/ml) activates the mitogenic kinase erk / , promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki and the inflammatory cytokine interleukin (il)- are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par -activating peptide (gypgkf, µm) and cathepsin g, a proteolytic par- activator released from neutrophils and mast cells. repeated exposure of maturing t l cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il- expression. pretreatment of mccm with a par- inhibitor further drives lipid accumulation, the induction of il- by contrast is suppressed. conclusion: par- activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par- expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr antagonist amd exerts deleterious effects in endotoxemia in vivo s. seemann , a. lupp the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). although a blockade of the cxcr /cxcl axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr seems to be part of the lipopolysaccharide sensing complex, cxcr agonists or antagonists may have a positive impact on tlr signaling. additionally, cxcr is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr blockade on endotoxemia by applying a sublethal lps dose ( mg/body weight) in mice. the selective cxcr inhibitor amd was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp phox as well as of heme oxygenase in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than % or % when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about % in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp phox to occur above average, whereas heme oxygenase expression was distinctly decreased. our results indicate that a blockade of the cxcr in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr antagonist cannot be recommended, hence indicating the activation of cxcr to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr , tlr and tlr mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr expression in a time-dependent and concentration-dependent manner and reduced tlr / expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il- β further increased tlr mrna levels. gene expression of mapk phosphatase- (mkp- ) was also upregulated by dexamethasone. glucocorticoid-induced tlr expression was negatively regulated by p mapk signalling under inflammatory conditions through mkp- induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr signalling as demonstrated by reduced il- , il- β, mmp- and mcp- levels. however, the expression of traf , a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il- cytokines and t helper (th ) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il- β, propranolol induced the th priming cytokines il- and il- in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il- γ and lps failed to increase il- release in molc and modc in the presence of propranolol but further induced secretion of il- β. autophagy has been linked with the secretion of il- family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p and lc -i to lc -ii conversion, induced accumulation of lc positive vesicles, as well as expression of il- signalling downstream adapter molecule traf , indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz- (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr expression and responded robustly to tlr engagement, confirmed by increased cd and cd expression, upregulated il- , il- p and il- p mrna levels and il- release. tlr activation reduced ccr and elevated ccr mrna expression and induced migration of mutz-lcs towards ccl . similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il β whereas ligands of tlr and tlr failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr -activated mutz-lcs, co culture with naive cd + t cells led to significantly increased ifn-γ and il- levels indicating th differentiation independent of il- . tlr -mediated effects were blocked by the putative tlr / antagonist cu-cpt , however, no selectivity for either tlr / or tlr / was observed. computer-aided docking studies confirmed non-selective binding of the tlr antagonist. taken together, our results indicate a critical role for tlr signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep - . , a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep - . and the structurally related compound pep - lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr (fsl- ) but not tlr (lps) activation by increased il- production, as determined by elisa. western blot analysis showed that both salps inhibited fsl- -induced phosphorylation of nf-κb p and p mapk. furthermore, the peptides significantly reduced il- release and gene expression of il- β, ccl (mcp- ) and hbd- as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il- secretion, downregulated expression of the maturation markers cd and cd , as analysed by flow cytometry, and inhibited ccr -dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il- levels and cd /cd expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep - . affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid -related factor (nrf ), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf -deficient (nrf -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for min (v t = ml/kg, f= min - , peep= cmh o, fio = . ), before µl hydrochloric acid (hcl) with ph= . or ph= . were instilled intratracheally, controls received nacl. mice were then ventilated for further h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph= . induced mild lung injury, indicated by hypoxemia (po /fio ~ mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf -/mice. in contrast, hcl ph= . caused more severe lung injury (po /fio ~ mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf -/mice. conclusion: we conclude that the presence of nrf augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [ ] [ ] [ ] [ ] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [ ] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [ ] . adma is degraded by dimethylarginine dimethylaminohydrolase and (ddah) enzymes [ ] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide- kinase (pi k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh ] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah and ddah enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah and enzymes. the pi k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-( - ) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-( - ) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-( - ) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-( - ) infusion provided best protection from experimental ali when administered by continuous infusion starting min after oa infusion till the end of the experiment ( - min). both pretreatment (- - min before oa) and short-term therapy ( - min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-( - ) min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-( - ) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y- and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of µm y- under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens- (zo- ). under conventional culture, y- induced higher growth rate of primary ebec and increased the passage number up to passages with retained epithelial cell behavior. in the presence of y- , ebecs under ali showed higher teer values. expression of zo- correlated with the increase in teer, but in y- -treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y- facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic values for the viability in the low micromolar range [ ] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe + . the radicals were trapped by -methyl- -nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = . g, and a h = . g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic , -dimethyl- pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe + a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = . and a h = . g. according to previously published data [ ] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [ ] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine : - , . nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca + -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the ' untranslated region ( 'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld . using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c a-enos). normal enos (enos-tg) or a novel dimer-destabilized c a-enos described previously (antioxid redox signal. sep ; ( ) : - ) were introduced in c bl/ in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for weeks or treatment with sod mimetic tempol. results: c a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos / phosphorylation and amp kinase (ampkα) phosphorylation at thr in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd . exercise training increased phosphorylation of enos at ser / and of ampkα at thr in wt but not in c a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp- cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka ( . mg/ml) and mya ( mg/ml) treatment down to % and % resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka ( mg/ml) treatment by % and % resp.; after mya ( . - mg/ml) treatment by % and % resp. lps-induced tnfα release from thp- cells was inhibited concentrationdependently by my (ic = . μg/ml; % inhibition), ka (ic = μg/ml; % inhibition) and cc (ic = μg/ml; % inhibition). furthermore, ka ( µg/ml) and cc ( µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to -fold (for cxcl ). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a a receptors (a ar) and adenosine a b receptors (a br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a ar agonist (cgs ) and an a br antagonist (psb- ) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw with a ar as a possible mechanism of the protective effect of stw in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of , , -trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a , a b, and a receptor with specific agonists reduced the acetylcholine (ach, µm)-induced contractions, while activation of a br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs ( µm) or with psb ( µm). the same effects with smaller intensity were found for stw ( µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw interact with the a ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw as a therapeutic approach in ibs. (allescher ) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw (ottillinger et al. ) . stw (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw ) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than pharmacological tests, is needed. all data from studies including stw alone, or stw and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of d histograms (lorkowski et al. ) . results: more than pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw contribute differently to the overall effect of stw . altogether, the evaluation of the data shows that stw is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw consists of a mixture of standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw ii lacks the last components. stw was shown to be effective clinically to treat functional dyspepsia ( ) and irritable bowel syndrome ( ) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis ( ) and in the management of ulcerative colitis ( ) . the present study was initiated to show whether stw ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with % dextran sodium sulfate in drinking water for week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw in doses of and ml/kg or stw ii in a dose of ml/kg for week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of mg/kg. conclusions: the modified preparation, stw ii thus proved to be as effective as stw , thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. ( ) schmulson mj ( ) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor (maropitant) and serotonin receptor (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk ) and channel opening (activation of ht ) on the intracellular calcium homeostasis (using fluorometric ca + analysis) and cell proliferation rates in endogenously nk and ht receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk or ht cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk and ht receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every - days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh promoter, further corroborated that rasal expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa- ht], urb ) on a lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, -ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases- (timp- ). using sirna approaches, a causal link between the timp- -upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp- expression. inhibitor experiments point toward a decisive role of cb and transient receptor potential vanilloid in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa- ht and urb . as underlying mechanism of its anti-invasive properties an upregulation of timp- was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, -arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb and arachidonoyl serotonin (aa- ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p / mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p / mapk activation, a blockade of pparα left p / mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p / mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes and leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k . an inverse correlation of mir- expression and protein levels of the efflux transporter atp-binding cassette transporter g (abcg ) was observed in cells resistant to different im-concentrations, pointing to a relation of mir- to im-resistance. hence, we investigate in current studies, how the influence of mir- on im-sensitivity could be explained. methods: we transfected k cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir- or inhibitory anti-mir- , challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst- -, caspase glo -assay and cell counting. in addition, we analyzed changes in abcg expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k showed an effect of mir- -inhibition using anti-mir- . this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p< . ) and cell membrane integrity and reduced capase- activity (p< . ). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg expression, we found that µm im-resistant k do not express higher amounts of abcg , but showed higher transport rates of im or the abcg -substrate hoechst . conclusions: overall, these experiments indicate that mir- does not only affect abcg -expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg . in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe( %)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of - gy using a kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [ ] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [ ] to % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to % of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h s production in the nociceptive system are only poorly understood. here we investigated the expression of the h s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h s. this work was supported by the deutsche forschungsgemeinschaft (sfb -a ) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt- ) and mesenchymal-like (j- ) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt- r , j- r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh ax and bp foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j- and j- r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt- cells showed an about - -fold higher resistance to cispt than j- cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt- r and j- r revealed a - -fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt- r cells disclosed a reduced apoptosis and enhanced g /m arrest following cispt exposure as compared to rt- wild-type cells. by contrast, induction of cell death was similar in j- and j- r cells. notably, j- r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j- r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt- r and rt- cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j- r and rt- r . unexpectedly, j- r and rt- r shared the upregulation of xaf- . treatment of j- r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk- and, moreover, re-sensitization to cispt by chk- inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt- cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j- cells. furthermore, our findings indicate that chk- might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein (satb ) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb in the upregulation of her after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb expression in gastric cancer cells, and the downregulation of satb by sirnas completely prevented the induction of her upon c-met inhibition. in contrast, her or her expression levels were not affected by satb -specific sirnas. the function of satb as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her expression after c-met inhibition. interestingly, the upregulation of her in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb and pkc are critically involved in the regulation of her expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb- breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki- proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb- breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for , pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb- ) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam- , vcam- , e-selectin, n-cadherin, and galectin- . moreover, the chemokine system cxcl /cxcr was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl could be excluded, since blocking the cxcl receptor cxcr on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β -integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β -integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for , fu / - ). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse ( ). based on an evolutionary cancer model ( ) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc and hcc cell lines; both erlotinib sensitive) ( ). plasma concentrations from two single-dose groups, mg/kg and mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens ( mg/kg daily, mg/kg daily, mg/kg every days, mg/kg every days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem . . results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the mg/kg dose. a -compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: and days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec = . μm for both cell lines), and on the partially sensitive transit phase (ec = . μm and . μm, for hcc and pc cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and -lipoxygenase ( -lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of min whereas the increase in vocalisation threshold persisted over a period of min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox- and -lo but not of cox- . we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox- and -lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. , million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., and buchner et al., documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > , . in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over purified compounds and over extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of , %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d receptor partial agonist bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d receptor partial agonist -bromoterguride ( -bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [ ]. to determine its atypical character in vivo, the ability of -bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of -bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: week old male sprague-dawley rats were injected with -bt ( . or . mg/kg; i.p.) followed by pcp ( . mg/kg; s.c.) or apomorphine ( . mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of -bt ( . or . mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp ( . mg/kg; i.p. twice daily for days) was assessed in the nor task consisting of a min acquisition trial and a min retention trial separated by a h inter-trial interval. clozapine ( . mg/kg; i.p) or haloperidol ( . mg/kg; i.p) were used as positive controls. results: the dopamine d receptor partial agonist -bt ( . mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly -bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that -btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that -bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of -bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of -bromoterguride [ ], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of -bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid- receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in . - . year old cb -/and control mice, fed with a standard diet and assessed effects of the cb agonist, hu during high fat diet in - week old mice. results: the cb -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb agonist hu prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m -like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb agonists may fortify cb -mediated anti-obesity signaling without the risk of anti-cb mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic : xan µm, res µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic : xan µm, res µm) while had less influence on the viability of pmm (ic : xan µm, res > µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin ( -ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph ), or the raphe nuclei of the brainstem (tph ) and the respective -ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral -ht is mainly produced by tph -expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, -ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral -ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg as a hcc tumor cell line and the fibroblast cell line fi as stroma simulation. additionally caco cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of . - µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: . µg/ml -cr: µg/ml -es: µg/ml. performance of the caco -transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with -fluorouracil ( fu), which is used as a chemotherapeutic agent, caused a reduction of its ic values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin- , - , - , - . claudin- is known to be the key tj protein tightening the bbb. therefore, claudin- has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin- and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin- containing barriers. the claudin- targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin- and immortalized brain endothelial cells (bend. ). the peptides decreased the amount of claudin- and zo- at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein ( da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin- (cldn ) which tightens the bbb for molecules < da. consequently, cldn is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn assay, the barrier opener (bo ) was identified as a cldn modulator. initially, a significant removal of cldn from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw da) demonstrated the effect of bo . concentration dependent treatment ( - µm) of cell monolayers with bo reduced tightness of the tjs between some hours and h. applying -hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo became detectable in mice. due to short stability (< h) of bo in the bloodplasma repeated administration ( . mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw da). the small molecule bo is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about h of incubation with concentrations varying from to µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow ( da) and fitc-dextran ( kda); the localization of tric elongated from ttjs towards btjs and cldn was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c bl/ mice. c bl/ mice without transplantation were used as normal controls. donor c bl/ mice to recipient c bl/ mice were served as the isograft group. days after transplantation, mice were scanned using an in vivo small animal µct (skyscan ). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf- pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf- (igf r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf- pathway is the transcription factor daf- (hfoxo a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf- pathway. recently, a cohort study showed that a decreased mortality rate in humans between age - correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about %) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf- overactivity. consequently we found an increased nuclear localization of daf- in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf- -ko mutant background. rt-qpcr brought jnk- , a known activator of daf- , into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf- dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf- pathway and particularly the transcription factor daf- . the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. ( ) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance ( ). in hfd/stz model, the rats are fed with high-fat diet for - weeks or for a relatively long time (≥ months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz ( - mg/kg), some of the diabetic rats receive treatment ( ) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats ( . ± . mg/dl vs. . ± . mg/dl, p < . ). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats ( ± . gvs. . ± . g, p < . ). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for weeks. further details can be found in table . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency ( ) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type diabetes ( ). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: . srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. ( ) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type diabetes and pharmacological screening. pharmacol res ( ): - . . oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. ( ) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem ( ): - . . franz, m. j. ( ) . the dilemma of weight loss in diabetes. diabetes spectr ( ) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt dystonia, which is caused by a gag deletion in tor a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d receptor mediated acetylcholine release has not been examined yet. the aim of present study was to ( ) generate more powerful tests to detect behavioural alterations in the dyt knock-in mouse and to ( ) examine the behavioral effects of the d receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole ( . - mg/kg i.p.) were tested in dyt knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt knock-in mice showed increased thigmotaxis at a dose of . mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay - after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay - was ( mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay - administration influenced the activity of the ecm degrading matrix metalloproteases (mmp and mmp ) and their inhibitor timp- , the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor- (pai- )) and the secretion of cytokines, e.g. il- . thereby, bay - increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay - might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly (sh -domain protein expressed inlymphocytes ) is essential for the generation of a fully functional t-cell response. the lack of sly leads to reduced survival rates of infected mice. the increased susceptibility of sly ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly ko t cells displayed a dysregulation of forkhead box protein o (foxo ) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo shuttles to the cytoplasm after phosphorylation in a protein complex including - - proteins. interestingly, we observed a similar regulation for the adapter protein sly , where tcr stimulation results in sly phosphorylation and sly export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly to - - proteins. altogether, this study describes sly as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly regulates t-cell proliferation (schäll et al., eur j immunol. ) . the catalytical isoforms p γ and p δ of phosphatidylinositol- , -bisphosphate kinase γ (pi kγ) and pi kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p γ and p δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi kγ and pi kδ in p γ ko δ d a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p γ and p δ might exacerbate asthma. here we analysed p γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il- and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p γ and p δ causes eosinophilia and ige hyperproduction, p γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type receptors (b ) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med : ; - ) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually - min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a , kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately µm below the surface. after stabilization of the green fluorescent signal, a , kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b changes physiologic endothelial permeability. this newly developed transgenic mouse line (b tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment. we observed that b tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the , kda dextran at each time point (fig. ) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type (b ). using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b was generated and backcrossed to c bl/ for more than times (b tg ). lung mrna using primers specific for the human or the mouse b cdna revealed a . -fold stronger expression of human b in b tg (n= ), while the expression of murine b mrna was unchanged and similar to transgene negative littermates (b n ). we have evaluated the specificity of several antibodies directed against b and found that a rabbit monoclonal anti b antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b -/mice. however, this antibody primarily stains rodent b and has only little cross-reactivity to human b . hence, we were not able to detect a significant increase of b protein in tissues of b tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b n , we found a significant icatibant sensitive aortic dilation in b tg following preincubation with diclofenac which indicates functional overexpression of b in conductance vessels of b tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching . ± . fold at . nmol bradykinin in c bl/ (n= each, p< . vs. vehicle). a similar increase was found in b n ( . ± . fold, n= , p< . vs. vehicle), while there was a stronger response in b tg ( . ± . fold, n= , p< . vs. vehicle) which was significantly different to b n (p< . ) and c bl/ (p< . ). in another set of experiments the specific and ace-resistant b agonist labradimil ( . nmol) was used instead of bradykinin. labradimil increased extravasation by . ± . fold in c bl/ (n= each, p< . vs. vehicle), by . ± . fold in br n (n= each, p< . vs. vehicle) and . ± , fold in b tg (n= , p< . vs. vehicle) which was significantly different to c bl/ (p< . ) but not to b n (p> . ). the effects of bradykinin and labradimil were largely blocked by nmol icatibant (i.v.) in c bl/ , b n and b tg mice (p< . ) and hence mediated by activation of b . these data suggest that overexpression of b in b tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller universitätsklinikum, innere , nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = . …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd + and cd + b-cells completely persists for month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = mcg/ml and the infusion time t = hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t / = hours (not shorter than the time t of infusion). with the terminal half-life of hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t / < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: ) are the observed metabolites ruminant specific and formed directly in the rumen? ) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro > days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph . ) and redox potential (~- mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within h to . . .-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides ( c-polydatin, octyl- c-β-d-glucopyranosid) were cleaved within hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd and contribute significantly to animal welfare ( r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. ( ) and including an empiric correction factor, mrd for the training set was . whereas bias was . µmol/l. by applying the developed pbpk model to the test set the respective values were mrd= . and bias= . µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco- cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco- cell system under different experimental conditions. the permeation characteristics of hypericin ( µm) in absence or presence of hypericum extract , . µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from to . %). the majority of hypericin was found after cell extraction ( % in absence and % in presence of the extract). the recovery was in the range of %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in healthy foals ( ♂ and ♀, age: - days, body weight: - kg) which were treated once with rif alone ( mg/kg s.i.d., p.o., a) followed by the administration of gam ( mg/kg once weekly, i.v., b) for weeks. study period ("rif-gam acute", c) includes the administration of gam and rif for days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for weeks. all periods were completed with blood sampling for pharmacokinetic analysis for ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c h : . ± . ; balc/c h : . ± . ). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c h -ratio was unaffected (peters et al. ) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp d . direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp d . however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about % of caucasians are compound heterozygous carriers of loss-of-function oct polymorphisms. therefore, oct polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct alleles. methods: mg desvenlafaxine (pristiq®) was orally administered to healthy subjects preselected according to their oct genotypes. oct * allele was regarded full active, oct * to * alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the subjects carried two active, one active, and zero active oct alleles. age, height and weight were . ± . years (mean ± standard deviation), . ± . m and . ± . kg with no significant differences among the oct genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc -infinity of desvenlafaxine varied between . and . min*mg/l and auc -infinity of didesmethylvenlafaxine between . and . mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average % greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct genotype groups. conclusions: our results suggest that oct genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly ( ). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression ( ) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september and december using the medical records of elderly patients (≥ years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss . statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n= ). median age was (min: , max: ), patients were female ( . %). according to medical records of patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug ( . % vs. % . ) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics ( . % vs. . %) and angiotensin receptor blockers ( . % vs. . %) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women ( . % vs. %, . % vs. . %, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over % of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e guideline [ ], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [ ] the ability to detect qtc-prolongations of about ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [ , ] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like- (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin- , - and - . apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in patients with nstemi, stemi patients and controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap ) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for sec to min reduced platelet aggregation in lta studies by up to % for apelin- . this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name ( µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group ( , % ± , % in mi versus % ± , %in controls; p = . ). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin- in patients with mi ( . ± . pg/ml versus . ± . pg/ml; p = . ). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = - . ; p = . ). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin- . upon platelet activation, apj is internalized and surface expression is reduced by about %. in mi patients, plasma levels of apelin- and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin- levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from to . inclusion criteria comprised the initial absence of dementia and age of years or older in . anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn , , s. schmiedl , , k. janhsen , , p. thuermann , , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of ( ) drugs with a known association to a certain adverse event (=positive controls) and ( ) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for % of the positive control drugs, and for none of the negative controls. ryan et al. developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. ; suppl :s - ) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in to were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of % (relative risk . ) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with % statistical power. results: from a total of index drugs in the reference sets of ryan et al., ( . %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of , , index drug initiators were included in the analysis. for a total of index drugs, a relative risk of . could be detected with % power. the numbers of index drugs for each of the outcomes of interest were: ami ( positive controls; negative controls); aki ( positive controls; negative control); ugib ( positive controls; negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with % power to detect a relative risk of . was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of -nor- -carboxy- administration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study -nor- -carboxy-∆ -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > . ). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term / ) at technische universität münchen (tum). the course consisted of a -day teaching period, followed by a -day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of out of ( %) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. ). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n= rd -year medical students, n= th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the items were presented as statements for restudy, items were tested using single-choice questions, and items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of single-choice questions, questions of which had not been used before (as a standard of comparison). the remaining questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of of these reworded questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [ ] . in order to address the persisting prescribing deficiencies [ ] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [ ] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least . % of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [ ] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising indications, drugs representing mechanisms, and up to adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. / chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, % to % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., ) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category . this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of - t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to ml/m³ and in mice in response to ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category with a mak-value of ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl- cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm ( mhz) and umts ( mhz). additionally, lte ( . mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from to w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ % (m) in % (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in ( ) a simple non-probabilistic way, ( ) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or ( ) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector ( ) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between . and . µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox , which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s assays in addition to the h r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than control groups with each males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., ) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = . , the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately to mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca + -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of and mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca +/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: , mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to mg per day over the period of up to weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of mg of hca for weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include and , respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg and especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt cells were either continuously incubated for h (approximately . cell cycles duration) with methyl methanesulfonate (mms; - µm), benzo[a]pyrene (b[a]p; . - µm), vincristine ( - nm) , and colcemid ( - nm) or cells were irradiated with xrays ( . - sv) and then cultured for h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation ( w, min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for out of the treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤ -fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk- e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s phosphorylated h ax (γh ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk- e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p , p-chk , p-chk and p-kap . furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk- e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann , g. fritz institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a ´- ´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic ) and moderate toxic (ic ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic and ic were determined after h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, h after permanent tbhq treatment. pictures were taken at the time points h, h and h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht- cell line on the ea.hy monolayer was investigated. wrn-deficient or non-deficient cells were treated with µm and µm tbhq or with tnfα. results: for ea.hy cells, µm and µm tbhq were determined as ic and ic , respectively. performing the migration assay, ea.hy cells showed % gap closure, whereas wrn-deficient cells showed a closure of only % after h. the gap was closed of % and % after µm and µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after µm tbhq treatment, whereas the treatment with µm tbhq showed a slight decrease in migration of about % compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after µm tbhq treatment a slight increase of adhesion was detected in ea.hy cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht- adhesion to ea.hy , which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl , jurkat and tk ) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk cells showed similar increasing trends, whereas hl cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration ( nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk cells. the results for the highest investigated concentrations ( . nm and nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk cells. in contrast the hl cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p status of hl (null), jurkat (multiple mutations), tk (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc- were applied. accumulation of p and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p and pakt analysis, and quantification of the oxidized dna base -oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than bacterial species and population densities of up to cfu per cm . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., ) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of - hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and - photoproducts ( - pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [ ] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct . similar results were obtained in sw and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p as observed by flow cytometry and western blot analysis in hct , sw and hcec. cdt-and irinduced autophagy rates in hct increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein (lamp- ) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser . pharmacological inhibition of akt in hct cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc b and lamp after h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after h and significantly lower apoptosis/necrosis rates after h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n -guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n -ethylthioethyl- ´desoxyguanosine (n -ete-dg) and n -ethylthioethyl- ´desoxyadenosine (n -ete-da) and their thermal hydrolysis products n ethylthioethyl-guanine (n -ete-gua) and n -ethylthioethyl-adenine (n -ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone ax (gh ax), which can be visualised as gh ax foci by immunocytochemistry. in the present study, we tried to assess if gh ax is a reliable biomarker for detecting the cellular response to dna damage. we selected well-characterised genotoxic compounds and compared them with non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh ax by manual and automatic scoring of γh ax foci, and by flow cytometry counting of γh ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh ax positive cells showed a significant correlation. further we compared dna damage induced by selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh ax assay detects a specific response to dna damage which can persist longer. the γh ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh ax), nuclear division (phospho-h , dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for and hrs in triplicates on a microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide -chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h events at and hrs and polyploidy at hrs time point. eto induced a clear increase of yh ax with a simultaneous reduction of phospho-h at and hrs. finally, cccp caused a reduction of phospho-h events, increased cleaved parp events and did not influence γh ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh ax and phsopho-h pilot study results as well as cleaved parp with caspase / . in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage , additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih t , mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g /g = ~ %) and semi-confluent cultures, similarly arrested in the g /g phase by serum-starvation or the mek inhibitor u , ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk . however, inhibition of atm, the kinase responsible for chk activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip , since the chemical rip kinase inhibitor necrostatin- (nec- ) blocked cell death up to averagely %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp- and p are probably not involved. deeper analyses could give evidence that nec- did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec- , possibly rip , acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca + signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc and mtorc ) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c t cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for days with the tumour initiator mca ( -methylcholanthrene) followed by days with the promotor tpa ( -o-tetradecanoylphorbol- -acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi- . surprisingly, an incubation with µm osi- led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s . our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi- , which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi- by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c t cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator -methylcholanthrene (mca) and the tumour promotor -otetradecanoylphorbol- -acetate (tpa). in the first experiment several metformin concentrations ( . - mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using mm metformin. this effect was not caused by growth inhibition of metformin itself since mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to , , and ppm for years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [ ]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: . ppm) after whole body exposure to vapor [ ] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of , , , ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, -pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [ ] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day (e ) at . °c and % humidity. on e , the eggs were cracked open and transferred into petri dishes. on e , cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with -methylcholanthrene as a tumour initiator and -o-tetradecanoylphorbol- -acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in -well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march ). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between - %. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's ( r), described by russel and burch in . in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches , resulting in a refinement of in vitro testing. in this study, human tk cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of . (± . ) h was observed while fbs supplemented cells showed a doubling time of . (± . ) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by ) determining reactivity class cut offs based on published data on substances for the dpra performed according to oecd c to predict un ghs sensitizer classes, ) a variant of the dpra assessing reaction kinetics (time and concentration) for substances or ) an extended protocol testing several test substance concentrations for reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of . % (ec . %). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes a and b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between min and hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. , , and mm) indicated that potency classes could be assigned using the ec . % value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec . % value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase (nat ) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u and thp- ) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p -dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or -amino- -methylphenol, respectively. in contrast, covalent peptide adducts were identified for putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a ' out of ' prediction model provided a high sensitivity of % for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the ' out of ' prediction model in a set of direct haptens was comparable (sensitivity = % when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and two-test and three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. ) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [ ] [ ] [ ] [ ] . in recent supplement, -methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [ , ] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [ ] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of mg/ml ( %), finite dosing and min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [ ] and electrostatic potentials using gamess [ ] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. ). toluene- , -diamine shows a higher accumulated mass due to increased lipophilicity (fig. ) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [ ] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [ ] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba ] contains data for about substances on dermal absorption which has been found through the echemportal [ ] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. chemicals have been investigated in a comparable test system; from these were shown to have a low dermal absorption of less than % and compounds showed a high absorption rate of more than %. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than % to more than %. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in [ ] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [ ]who ( ) ehc dermal absorption [ ] scholz et al ( ) naunyn-schmiedeberg´s arch pharmacol (suppl ):s [ ] www.echemportal.org [ ] http://edetox.ncl.ac.uk [ ] jung et al ( ) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett : - , ) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology : - , ) . we modelled the concentration time profile exploring the dose which would lead to a concentration at hour and at hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between and µm /sec (truisi et al., ) and an absorption of % and an absorption rate of /h (cristofoletti and dressman, j pharm sci : - , ). results: the data of the in vitro study with µm ibuprofen could well be modelled. when assuming a clearance of µm /sec the dose of mg resulted in an hour concentration of . µm in the hepatic vein of pbhkm equal to . nmol/well (volume of the well = ml) in the in vitro study in which the measured concentration was . nmol/well. the concentration at hours of . µm (equal to . nmol/well) corresponded with the in vitro concentration ( . nmol/well). the modelling approach was less successful with in vitro dosing of µm. the fold higher dose of mg lead to nearly double the concentration at hour than measured in vitro. with a dose of mg/kg an approximation was feasible resulting in . µm in the hepatic vein at hour which is equal to . nmol/well whereas the measured concentration in vitro was . nmol/well. even with a clearance value as low as the . percentile ( µm /sec), the concentration at hours was modelled to be lower than the in vitro measured value (in vivo model: . µm which corresponds to . nmol/well; measured in vitro concentration: . nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro ( µm) corresponds to a dose of mg (note: the highest approved daily dose is , mg). for the high in vitro dose modelling was successful only for the concentration hour after dosing and a dose of , mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and . fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score ( positive out of possible genotoxicity categories) was assigned to of the tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [ ], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin (trpa ) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk / , are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk / and increased protein biosynthesis of sre-regulated genes in hek cells overexpressing htrpa . cees increased perk / levels already after . min which could be prevented by the trpa blocker ap . activation of target genes through perk / signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa channel activation. thus, trpa might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst- ) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort (" rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a ), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg is often used as a model for liver toxicity studies. however, under two-dimensional ( d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for days in a three-dimensional ( d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg cells. in our present study we extended previous studies and compared hepg cell cultivation in three different d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after , , , and days of cultivation. our results show that the previously reported increase of metabolic competency of hepg cells is not primarily the result of d culture but a consequence of the duration of cultivation. hepg cells grown for days in d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg is not due to d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [ ]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [ ] , whereas asfas were less toxic [ ] . a substantial transfer across an intestinal barrier model (caco- ) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [ ] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [ ] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic ~ - . µm) in a concentration range comparable to that of arsenite (ic ~ . µm), whereas asfas were less cytotoxic (ic > µm). after incubation with ashcs the cellular arsenic concentrations increased - fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [ , ] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic values of . ± . µm for hache and . ± . µm for hbche. ic values with chloroquine were . ± . µm for hache and . ± . µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after minutes, only paraoxon-inhibited hache ( %) and cyclosarin-inhibited hbche ( %) were reactivated by µm chloroquine. on the contrary, µm amodiaquine reactivated all tested ops after minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [ ] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., , , - . [ ] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., , , - bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional ( d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a d model was used consisting of the permanent breast epithelial cell lines mcf a (er -, estrogen receptor negative) and mcf a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the d culture of mcf a and mcf a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf a) and er-independent (mcf a and mcf a) effects. the d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg ) cells and (b) co-culture systems of human monocytic or macrophage-like (thp- ) and hepg cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg and thp- monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about % cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg . we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator ( -methylcholanthrene) and promotor ( -o-tetradecanoylphorbol- -acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day , morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that ( ) several substances have a different impact on the transformation process, ( ) it is possible to identify for each substance the phase with the greatest effectiveness and ( ) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c t assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg ) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg ) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg ) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et (exposure time reducing viability of treated tissue to %) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg , one of the five severe ocular irritant formulations (un ghs cat ) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat formulations was just above the un ghs cat classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins- e) and exocrine (ar j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, . ( ): p. . briefly, pancreas was infused and embedded with agarose. tissue sections of app. µm were prepared using a vibratome and maintained in cell culture medium for up to days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins- e cells) or amylase/lipase (ar j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins- e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers ( , -dinitrochlorobenzene, -mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology ; : - ) . dendritic cells generated in fcs supplemented medium were cd a+/cd c+, whereas dendritic cells from serum free culture conditions were cd a−/cd c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd a+/cd c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp- cells, as model for dc, enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd on thp- cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp- cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp- cells by microarray technology. we identified differentially expressed mirnas in cocultured thp- cells compared to monocultured thp- cells irrespective of the treatment (medium, . % dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after h) additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp- cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp- cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp- and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm and . trpa has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in ). although recently two reports identified trpa in pulmonary epithelial and endothelial cells ( , ) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa -deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa on a cellular level we cultured a hek cell line stably expressing trpa ( ) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca + -influx in this cell line, which was blocked by the specific inhibitor a . in the future, we will utilize the isolated perfused lung model ( ) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa -deficient mice after exposure to potential toxic inhalation hazards (tih see ) to challenge the hypothesis of trpa as an important toxin sensor in the lung. by this strategy we hope to understand trpa function in lung cells and to evaluate trpa proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for days to male rats including blood sampling for metabolic profiling after , and days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole- -acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole- -propionic acid for aminoglycosides, taurine for fluoroquinolones, indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., ; luo, schomaker, houle, aubrecht, & colangelo, ; yamazaki et al., ) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low ( mg/kg) and a high ( mg/kg) dose. rats were sacrificed following , , or consecutive daily doses, or after recovery days following consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) and days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide (oatp ) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/ proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann background: in a -day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp r , gc, cyp a, ugt a) in liver was also affected, while initial indications for modulation of renal cyp a and renal and hepatic cyp b could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n= for single substances, n= for combinations) were treated for days at dose levels tested based on noaels from -day subchronic feeding studies and ranged from noael/ to noaelx . quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total ( -oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n= ) were . ± . ng/ml (min/max: / ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c ( ppm in food, n= ), there was a statistically nonsignificant reduction of vitd levels to . ± . % of the concurrent control (n= ). however, for of animals of this group, measured vitd level were below the range observed in pooled controls (n= ). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p for c, e and pz; cdk and gadd a for e; cdkn c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp a / and ugt a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with ppm cyproconazole, there were indications for a moderate reduction of -oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a -year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the th week, and she has been taking a multidrug therapy (dapsone mg/day, rifampicin mg/month, clofazimine mg /day and clofazimine mg/month) for the past months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about weeks. in the th, nd, th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the th week of gestation without perinatal complications. the baby was also healthy (apgar - , g, cm), and its growth and development were normal during a -month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in in berlin, germany, to evaluate kidney function in people ≥ years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january until april ). the probability of dabigatran use rose with increasing age (+ %), and the probability of phenprocoumon use rose in case of egfr < ml/min/ . m (+ %) or male sex (+ %). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ years in nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > mmhg and/or dbp decrease of > mmhg within min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > mmhg and/or mean dbp > mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with nhr (mean age = . ± . years). in total, subjects ( . %) had oh. the mean change in sbp from sitting to standing was . ± mmhg (range + . to - . mmhg) in patients with oh and . ± . mmhg (range + . to - mmhg) in patients without oh. mean sbp was significantly higher ( . ± . mmhg) in people with oh than in those without ( . ± . mmhg). all of the nhr with oh were hypertensive compared to % of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p> . ). medication data was available for patients. all individuals with oh and nhr without oh ( %) had antihypertensive medication. more than different antihypertensive drugs were present in patients with oh ( . %) and in patients without oh ( . %). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than % of patients in both groups reached points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around % of patients with oh as well as patients without oh needed more than sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer ( : ) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from mg/d clom citrate to up to mg/d in case of non-ovulation. however, therapy outcome is variable and approximately - % of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via -hydroxylation of trans-clom by the highly polymorphic cytochrome p (cyp) d leading to the major active metabolite trans- hydroxyclomiphene (trans- -oh-clom) [ ] . recently, we identified a less active trans- -oh-clom which is also formed by cyp d . besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of mg clom citrate. results: incubations with human liver microsomes revealed an almost -fold higher glucuronidation rate for trans- -oh-clom, which is exclusively catalyzed by ugt b , compared to the more potent trans- -oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans- -oh-clom to its sulfate is -fold higher compared to -oh-clom. for both metabolites a participation of sult a and sult e was identified. these results were in line with previous studies, which identified the same sults [ ] and ugts [ ] responsible for the conjugation of the structurally related trans- -hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans- -oh-clom and trans- -oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom- -o-glucuronide. conclusion: our results suggest a new metabolic route via trans- -oh-clom which appears to be a potential inactivation pathway of clom. institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of . µm sm (ic ). today the cells are able to tolerate a concentration of . µm sm (ic ), which reflects to a concentration of which % of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h o , glutathione, ca + -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening significant up-regulated and significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp a expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm -mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in . the program was accredited in by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates places per year to an average of applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about % of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler , m. hermanns-clausen uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from to in total cases of human exposures to chlorine developed from mixtures of hypochlorite and acids ( . of cases) were registered. in cases the exposure was due to mixtures of products intended for domestic use. % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough ( %), dyspnea ( %), irritated upper airway ( %), abdominal discomfort (pain, nausea, vomiting) ( %), thoracic pain ( %), irritated eyes ( %), dizziness ( %), and bronchospasm ( %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in patients ( %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( % ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were patients in the same period exposed to hypochlorite evaporation alone. of them ( %) had symptoms of which in cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway ( %), nausea or vomiting ( %), cough ( %), irritated eyes ( %). dyspneoa was less fequent than in the mixture group ( %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh ax assay for genotoxic and nongenotoxic agents: comparison of h ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant - is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [ ] mürdter t, et al. hum mol genet, , : - [ ] nishiyama t. et al., biochemical pharmacology, , : - [ ] sun d. et al., drug metabolism and disposition, , : background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: bd patients ( / m/f, . ± . y, tbsa . ± . %) and patients ( . ± . y) from the icu were included in this observational study. blood samples were taken within the rd interval of the h-lasting dosing period of piperacillin/tazobactam ( / . g within . h) at , and . h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least xmic but preferably xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at , , and . h, were as follows: c max . ± . vs. . ± . mg/l, p< . , half-life . ± . vs. . ± . h, p> . , clearance . ± . vs. . ± . l/h, p< . , volume of distribution . ± . vs. . ± . l, p< . . free concentrations (which were included in tdmx calculations) were ± vs. ± % (p< . ) of total concentrations. duration per day while concentrations exceeded xmic ( . ± . vs. . ± . h, p< . ) or xmic ( . ± . vs. . ± . h, p< . ) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for xmic could be enhanced to . ± . h if the piperacillin amount will be increased to x g/d and the infusion duration to h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as > mg mtx/m bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years - were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. patients aged - years (male = , female = ) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, mtx treatment cycles corresponding to mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph < . , trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [ ] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [ ] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [ ] , we speculated that repression of gene expression as the predominant transcriptional response to ota [ , ] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [ ] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk- ) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h a, h b, h and h . we further selected acetylation at histone h lysine (h k ), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [ ] and which was reduced from ~ % in controls to < . % in response to ota, to establish a link between loss of h k acetylation and expression of genes consistently shown to be down-regulated in response to ota [ , ] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h k acetylation at promoter regions of the selected genes (% of controls: amigo : %, clasp : %, ctnnd : %). overall, these data provide first evidence for a mechanistic link between h k hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays ( most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to - % (average prediction accuracy was %). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [ ] [ ] started in and is a public-private partnership project within the european innovative medicines initiative (imi) [ ] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to week) repeated dose studies, mostly in rodent. overall more than study reports for approximately investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [ ] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [ ] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than active substances approved for being used in pesticides and over chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg , heparg, rat: h iie) and adrenal gland (human: h r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a -day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg , heparg, hek ). cytoxicity has been analysed by wst- and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp- as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd on thp- cells after incubation with these prohaptens (hennen et al., ) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp- with b[a]p and eugenol in coculture with hacat cells augmented cyp a and/or cyp b mrna levels, while this was not found for thp- monoculture. augmentation of cyp a mrna needed continuous presence of hacat cells. in coculture, levels of -oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of -oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp- cells by , -dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp- cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci : - .toxicology -toxic pathway analysis/aop background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry to minutes and day after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at min (r= . ; p< . ). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations min/day : p= . for clopidogrel, p= . for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so ) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc- and miapaca- cells in comparison to the non-target cell line nih t with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc- cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so by subcutaneous injection at the neck. after the tumor reached a diameter of to mm in size treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k ). complete blood count analysis was done by labor gmbh, berlin. results: the adenine release mediated by dianthin-egf was . pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic value of approximately nm for egf receptor expressing miapaca- and bxpc- cells as compared to nm for non-target nih t cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from pm to µm. toxicity studies in balb/c mice revealed . µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be % less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet % less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc- cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp a and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp a at different concentrations with and without nadph to determine the ic shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version . and admetsar. results: fractions (f -f ) indicated ic shift reduction ≥ post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = . ) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic = . µg/ml) across mdr -mdckii cells. niaziminin indicated . % bioavailablity via the human intestinal membrane with % chance of inhibiting cyp a . βsitostenone showed strong p-gp inhibition ( . %) with % absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp a or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than departments of all berlin hospitals from october until december . through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases ( . %). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient , pelargonium sidoides in patient ), and eight times as possible (valeriana in patients , , , , , mentha piperita in patient , hypericum perforatum in patient , eucalyptus globulus in patient ). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient ). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. ). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. , fig. ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil for one of our patients ( fig. & ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for % of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, ) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n= per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at °c throughout the experiment and electrical resistance barrier integrity testing was performed at the start ( h) and end of the experiment ( h). four test preparations at final bpa concentrations of . , , , and mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca %, v/v), uridine '-diphosphoglucuronic acid (udpga, mm) and '-phosphoadenosine- '-phosphosulfate (paps, µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration ( mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between . and . % of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between and % of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z and instead of the molecule ions (m/z and ) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were . ng/ml and . ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter (mct ), encoded by the slc a gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct and its epigenetic regulation by slc a promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct /slc a might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct protein expression was analysed in paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v. . (definiens ag). dna methylation in the slc a promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in paraffin embedded metastasis samples by maldi tof-ms. mct protein expression data and dna methylation at the specific cpg site in the slc a promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately % and % in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc a promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the '-regulatory region of mct may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) -hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in postmenopausal breast cancer patients of european origin who were treated with mg/day of tamoxifen for at least months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of premenopausal breast cancer patients mainly of european descent , . twelve snps close to or above genome-wide significance (p < e- ) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes , and for (e) metabolite e and to chromosomes and for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < % were confirmed as putative candidates: rs was associated with a - % variant allele-dependent increase of (e) and (z) metabolite e isomers (p< . ), and rs , mapping to a gene encoding zinc finger protein znf , was associated with increased risk of reccurrence or death (hr carriers . , % ci: . - . ; p < . ). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water ( : ) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from . nm to nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the % tolerance range for all analytes in all matrices. matrix effects were between - % (plasma) and - % (urine), apparent recoveries in plasma were above %. basal plasma ba level (mean ± sd) in fasting healthy subjects were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord- -j pj d authors: norinder, ulf; tuck, astrud; norgren, kalle; kos, vesna munic title: existing highly accumulating lysosomotropic drugs with potential for repurposing to target covid- date: - - journal: biomed pharmacother doi: . /j.biopha. . sha: doc_id: cord_uid: j pj d given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing covid- pandemic. only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. with the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on existing drugs. in addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. by this approach we have identified compounds with possible antiviral effects, also against coronaviruses. for of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target covid- , as well as open doors to finding more potent and safer alternatives. currently available antiviral drugs were mainly designed to target a specific viral or human protein crucial for infection with a specific type of virus (de clercq and li, ) . considering the diversity and constant mutations of viruses, it will not come as a surprise if these antiviral drugs are shown to be inefficient against the new sars-cov- virus and thus not adequate for repurposing. certain marketed drugs seem to exhibit modest antiviral activity via the mechanism that is not specific for a single protein, but rather for a general mechanism of viral infection. for instance, chloroquine and hydroxychloroquine have been reported to affect endosomes, the critical point of viral entry into the cell, resulting in antiviral effect (al-bari, ) . various steps in the viral infection have been examined to understand the mechanism of (hydroxy)chloroquine's activity, but it has remained not entirely clear. it has been reported that the drug's effect of increasing the endosomal ph results in reduced capacity of the virus to infect the cell (savarino et al., ) . it has also been proposed that the antimalarial drug reduces the level of glycosylation of ace in endosome critical for recognition of the sars virus and the endosomal membrane (vincent et al., ) . recent studies indicate possible interaction with sigma receptors as important for their antiviral activity (gordon et al., ) . regardless, in a recent small clinical trial hydroxychloroquine demonstrated moderate effectiveness in treatment of covid- patients, and the effect was even more pronounced in co-treatment with antibacterial azithromycin (gautret et al., ) . this led both drugs to numerous currently ongoing larger clinical trials as well as to heated debates in science and public on their potential benefits, mechanisms and safety. the mentioned drugs, chloroquine, hydroxychloroquine and azithromycin, highly accumulate in lysosomes/endosomes and their membranes, reaching about -fold higher intracellular than their extracellular concentration (easwaranathan et al., , de duve et al., , and thus belong to a group of lysosomotropic compounds (compounds that accumulate in lysosomes). we propose that the antiviral activity of (hydroxy)chloroquine and azithromycin is shared among all strong lysosomotropic drugs and is a consequence of their extremely high accumulation in cells and membranes, and subsequently of all the processes affected by this pharmacokinetic property. j o u r n a l p r e -p r o o f lysosomotropic compounds or cationic amphiphilic drugs (cads) belong to various pharmacological classes but share the same physicochemical properties that enable them to accumulate in acidic compartments of cells (figure ) . such compounds can pass the membrane in neutral form but as they come to a more acidic environment, they become protonated and as such cannot diffuse back through the membrane. consequently, as the drug moves into more acidic compartment, the equilibrium between protonated and neutral forms is shifted towards the protonated form and a higher fraction of drug molecules become trapped inside the cell (de duve et al., ) . the highest concentration of lysosomotropic drugs is reached in lysosomes with ph of - , and in late endosomes with ph of - (schmitt et al., ) . for strong lysosomotropic drugs it was estimated that - % of intracellularly accumulated compound is stored in lysosomes and endosomes (togami et al., ) , leading to extreme concentrations in these compartments. while accumulating, such drugs are heavily loading in lysosomal and other biological membranes inside the cell due to their amphiphilic nature (sanchez garcia et al., ) , meaning that the overall molecule is relatively lipophilic (often with logp ranging from to ) but also bears positive charge, which enables the molecule to bind close to the surface of the phospholipid bilayer (kosol et al., , gh et al., . the impact of lysosomotropic drug accumulation on cells is obvious: lysosomes increase in volume , lysosomal function is impaired leading to downregulation of autophagy (ashoor et al., , mauthe et al., , endocytosis and the entire membrane trafficking in the cell is reduced (nujić et al., ) . lysosomal and endosomal ph increases as a consequence of the overload of basic compounds (logan et al., , schmitt et al., . due to the cationic drug binding to phospholipid bilayer and change of the surface bilayer charge, the degradation of phospholipids is slowed (kosol et al., ) resulting in the accumulation of excess phospholipid membrane and vesicles inside the cell, which may lead to an adverse effect known as phospholipidosis (shayman and abe, ) . if not too extreme, all these effects on cells are reversible upon the cessation of drug treatment (munić et al., ) . in our previous studies on a set of compounds we have shown that the extent of lysosomotropic accumulation in cells correlates with the compound's extent of induction of phospholipidosis and lysosomal swelling (easwaranathan et al., ) . the correlation is so apparent that it is even possible to determine the level of accumulation from the intensity of j o u r n a l p r e -p r o o f either of these processes caused by a drug. strong correlation with accumulation was also shown for the inhibition of autophagy on a smaller set of compounds. finally, accumulation in cells was also shown to correlate with their extent of binding to layers of phosphatidyl choline, the most abundant phospholipid in biological membranes (sanchez garcia et al., ) . the importance of intracellular membranes in coronavirus infection is immense. firstly, viruses enter the cell by formation of membrane vesiclesendosomes, and subsequently enter cytoplasm through the endosomal membrane (figure ) . during the assembly of new virion particles, the virus is taking a portion of the membrane of endoplasmic reticulum (er) to pack the rna in a lipid envelope enriched with transmembrane proteins. new virions are then packed in an exocytic membrane vesicle, which ultimately fuses with the plasma membrane and releases virions outside of cells (j alsaadi and jones, ). therefore, it is conceivable that a drug that is heavily bound to cellular membranes, negatively affects the viral life cycle, if not even the structure of its lipid envelope itself. since the assembly of virions relies on electrostatic forces between the lipid envelope, their proteins, and rna inside it, it is likely that the overload of cationic drug in the er membrane, that viral lipid envelope is made of, leads to serious disturbances of virion structure. the evidence supporting antiviral activity of lysosomotropic cationic amphiphilic drugs (cads) are described in various reports on individual drugs and have been extensively summarized by salata et al. (salata et al., ) . chloroquine and amiodarone have so far been amongst the most widely studied cads for their antiviral effects. a recent review on broad spectrum antiviral agents, summarized the data on antiviral activity of various drugs in the drugvirus database (andersen et al., ) ( https://drugvirus.info/ ). even though the database is logically enriched in antiviral drugs, there are more than cads registered for various non-viral indications listed in the database with collected evidence of their broad antiviral activity in vitro, in vivo and in clinical studies, including activities on coronaviruses, influenza, zika and ebola virus. among listed drugs with antiviral effects, the cads include: psychoactive drugs (chlorpromazine, fluoxetine, clomipramine); antiarrhythmics (amiodarone); antimalarials (chloroquine, hydroxychloroquine, amodiaquine, mefloquine, quinacrine); channel blockers (amiodarone, verapamil, manidipine); antibacterial (azithromycin); estrogen receptor modulators (tamoxifen, raloxifene, toremifene); two antivirals, the only ones known to utilize cads mechanism of j o u r n a l p r e -p r o o f antiviral action via the inhibition of endosomal pathway (arbidol (umifenovir) and tilorone (with additional activity of interferon induction)) (boriskin et al., , ekins et al., . it is noteworthy that there are more than marketed and investigational drugs known to induce phospholipidosis (orogo et al., ) , and a majority, but not all of them classify as lysosomotropic cationic amphiphilic drugs. phospholipidosis is also the most common side effect of lysosomotropic compounds and a direct consequence of their binding to membranes (kosol et al., ) and reduction of vesicle trafficking in the cell (ashoor et al., , nujić et al., . therefore, we anticipate that, among phospholipidosis inducers there are many registered drugs that may have intrinsic antiviral activity via the same mechanism as described for a handful of lysosomotropic compounds so far; by inhibiting endocytosis, increasing endosomal ph, and slowing down total intracellular membrane trafficking (salata et al., ) . their potency may be moderate but in combination with other treatments they can likely show synergistic effects. these drugs thus represent a pool of potential existing modest antiviral drugs that may prove useful as the first line of defence in current and possible new viral epidemic outbreaks. another possible positive contribution of cads in covid- could be their potential antiinflammatory effects, which was for azithromycin and chloroquine as well as for a number of tool drugs found linked to their accumulation properties (nujić et al., , munić et al., . apart from these potential beneficial antiviral and anti-inflammatory effects, it is important to be aware of potential safety issues linked to lysosomotropic drugs. cads are often, but not always, linked to liver-and cardiotoxic effects (dragovic et al., , sun et al., . sar studies on herg channel indicate that increasing basicity of many compounds increases the chance to block the channel resulting in cardiotoxicity . despite these effects, these drugs are still widely used, and several of them were true blockbusters not so long ago, for example antibacterial azithromycin, selective serotonin reuptake inhibitors (ssri) antidepressants fluoxetine, citalopram and sertraline, as well as well-known antimalarial drug chloroquine. in repurposing for viral infections, additional types of toxicity may arise from their primary pharmacological targets, as they were developed to treat completely different diseases. we believe, however, that by careful analysis of pharmacological and toxicological properties of lysosomotropic cads, new drugs among existing ones could be found that may be reasonably j o u r n a l p r e -p r o o f safe during short term treatment, at least for a group of covid- patients that do not have preexisting heart and liver conditions. we have now applied our in silico model for the prediction of lysosomotropic accumulation on all marketed and investigational drugs that are known inducers of phospholipidosis (orogo et al., , goracci et al., , and that according to their physicochemical properties have the ability to get trapped by protonation in lysosomes (the strongest basic ionisation constant, pka> . ) (nadanaciva et al., ) . the aim of this analysis was to find other strong lysosomotropic cads which have a high likelihood of interacting with cell compartments and processes critical for viral infection cycle, such as endosomes, lysosomes and membranes in general. as additional proofs of their lysosomotropic nature and possibility of high accumulation in cells in vivo, we have considered the presence of tertiary nitrogen atoms in their structure, lipophilicity (logp), volume of distribution and half-life in plasma or blood in humans. all filtering criteria are listed in table and the results of this analysis are presented in table . from the compounds with experimental or clinical proofs of induction of phospholipidosis, we have selected those that according to our in silico prediction model for cellular accumulation are predicted to accumulate moderately to extremely high (accumulation (acc) levels - ) for compounds within and at the borderline of the applicability domain for the model; and high to extremely high (acc levels - ) for compounds outside the domain i.e. with less reliable prediction of accumulation intensity . for several molecules in table the data on experimentally determined cellular accumulation is also provided (measured acc) (easwaranathan et al., ) . third inclusion criterion was the strongest basic dissociation constant, pka, higher than . which is an indication that the molecule can become protonated, and thus trapped, inside the acidic compartment in addition to eventually increasing lysosomal/endosomal ph (nadanaciva et al., , logan et al., . in table , all data marked green are in favour of the compound having lysosomotropic behaviour which may result in antiviral effects. in addition to the initial three inclusion criteria, we have considered beneficial and marked compounds which have physiological charge higher or equal , and one or more tertiary nitrogen atoms, which are the most common structural features of cads (rorig et al., ) . also marked were data on logp between and , which indicates the capability of the compound to enter cells via diffusion through the membrane in neutral form, in addition to the ability of binding to the membrane (nadanaciva et al., , lipinski et al., . pharmacokinetic parameters, volume of distribution higher than l kg - and half-life in blood/plasma equal or higher than h, are also marked green as they indicate that the compound in its original form accumulates in tissues in vivo, and remains in the organism for a longer period (hanumegowda et al., ) . due to their strong primary pharmacology we have excluded psychoactive compounds and antiarrhythmics from our further analyses, as we think that these compounds, with strong effects on brain and hearth, are not suitable for repurposing for fighting viral infections. there is a strong possibility that they may cause rather specific and possibly dangerous side effects if needed to be given at higher doses than what is needed for their primary indication. it would, however, be important to know whether chronic use of antidepressants, or any other cads, can in a positive or negative manner affect the activity of antiviral cads. it is possible they may function as protection against viral infection, but conversely, in chronic use the body may adapt to constant exposure to lysosomotropic drugs, rendering cad antivirals less effective. more research is needed on the effects of chronic usage of lysosomotropic drugs on antiviral activity of drugs exploiting the cad mode of action. in addition, although still in use, many cads are associated with liver toxicity and cardiotoxicity in terms of arrythmia caused by qt interval prolongation (dragovic et al., , sun et al., , and thus we have added to our analysis known data on these adverse effects. liver toxicity evaluation is taken from the livertox database (where a levels indicates the highest likelihood of liver toxicity, and e the lowest) ((niddk), ). for cardiotoxicity, we have marked positive (orange) if there is a reported effect of the compound on either qt interval prolongation/arrhythmia in humans or a positive result in in vitro herg assay. finally, the existing information about broad spectrum antiviral activity of selected drugs has been taken from the drugvirus database (andersen et al., ) , including number of viruses that the drug was shown effective against, as well as whether there are also supporting in vivo and clinical data on their antiviral activity, and specifically data on activity against sars-cov, sars-cov- and mers viruses. in addition to looking at compounds passing both phospholipidosis and cell accumulation criteria we have also analysed compounds which failed one of these criteria, but still had pka values higher than . . these drugs, shown in table , are considered possible to exert cad's mode of antiviral action, but with less certainty than those in table . drugs and compounds have been included in the analysis: compounds with known potential to induce phospholipidosis (either positive or negative), drugs with unknown or unclear phospholipidosis results and additional antiviral drugs without phospholipidosis data ( figure ) . all compounds were analysed by our in silico qsar model for prediction of cellular accumulation, which than resulted in compounds passing both phospholipidosis and accumulation criteria, compounds passing acc criterion with unknown phospholipidosis data, compounds passing acc criterion but not phospholipidosis, and passing phosholipidosis criterion but not accumulation prediction. compounds were then additionally filtered according to their pka values and primary pharmacology, to find compounds less likely to induce serious side effects when used for a different indication than what they were developed for. this resulted in total of drugs with potential for repurposing for viral infections due to the high likelihood of exhibiting antiviral effects as lysosomotropic drugs. among drugs that failed in either phospholipidosis or accumulation criterion there are compounds which passed other criteria and can be considered as additional drugs that may possibly induce the same effects. drugs identified with high likelihood for antiviral effects due to cad properties mainly belong to macrocyclic antibacterials, antimalarials, antihistaminics, antivirals and antiparasitic drugs. since most, except antihistaminics, have non-human targets, they are among the drugs most acceptable for repurposing since their primary pharmacological activity will likely not pose a safety issue. it is important to consider that out of identified drugs were found either to induce qt prolongation/arrhythmia or interact with herg channel in vitro, or both. this indicates that, even if proven effective against the virus, these drugs will likely need to be avoided in patients with heart conditions. cautious approach is needed for six drugs that were found negative on qt prolongation and arrhythmia, as the lack of effect may still be linked to applied doses for their primary indication. in addition, higher likelihood of liver toxicity was found in six out of drugs. care should also be taken with identified antihistaminics in the group, as they share structural features with some lysosomotropic antidepressants, and some still have a mild sedative effect. out of the four antiviral drugs that may show cad's mode of action, two drugs have been claimed to utilize this mechanism: tilorone and arbidol (umifenovir). even though tilorone was invented in the us as an interferon inducing broad spectrum antiviral, additional studies showed that its lysosomotropic potential is comparable to chloroquine, which likely contributes to its antiviral properties (ekins et al., ) . tilorone is currently registered and used only in russia and some neighbouring countries, where it is approved for indications such as influenza, acute respiratory viral infection, viral hepatitis, viral encephalitis and myelitis. arbidol is registered in russia and china for the treatment of influenza (boriskin et al., ) . it is claimed to inhibit the membrane fusion of virus with the endosomal membrane, which possibly occurs via increasing the endosomal ph, and thereby preventing infection of the cell. the third antiviral, nelfinavir is a protease inhibitor drug approved for treatment of hiv infection, but also reported effective against a wide variety of viruses: herpes simplex, sars-cov, hepatitis c, dengue and j o u r n a l p r e -p r o o f chikungunya (andersen et al., ) (drugvirus database). it is likely that its lysosomotropic properties may be responsible for this broad-spectrum activity. the last identified antiviral drug is vicriviroc, which was developed as nanomolar ccr antagonist for hiv infections and came to phase iii clinical trials where it did not meet primary efficacy endpoints. when analysing the potential of repurposing such highly specific and potent antiviral drugs for fighting a different virus, it should be kept in mind that they would likely need a much higher concentration since their original target that they are specifically acting upon is not present in other viruses. impact of strong lysosomotropic compounds on membranes and membrane trafficking often needs low micromolar concentrations (easwaranathan et al., ) . it remains to be seen whether efficacious levels of any of cads can be achieved in covid- patients without trespassing into toxicity levels. in the case of cad's antiviral activity, it is worth investigating whether stereoselectivity could help to further separate activity and toxicity dose ranges. it has been shown previously that enantiomers (mirror image compounds) have different affinities for a range protein targets, including herg , grilo et al., . if the antiviral effect of cads is linked only to their physico-chemical properties, which are the same for both enantiomers, they would have the same activity, but may differ in the affinity for herg. this would result in one enantiomer having a better safety profile than the other, with unchanged antiviral activity. / compounds from table possess at least one chiral centre, some even more than , meaning that there are many potential enantiomer pairs that could be examined from the perspective of obtaining increased safety margins. table are less likely to show antiviral effects caused by lysosmotropic behaviour (compared to those in table ) as they failed in one of the primary inclusion criteria. three of them target respiratory system (bromhexine, ambroxol and formoterol), but should be cautiously assessed at which stages of the disease they may be most effective. interestingly, in this group of compounds, we have also identified imatinib, which was proven effective against sars-cov and mers-cov viruses by inhibiting viral fusion and entry into the cells (dyall et al., , sisk et al., and is currently being tested in a clinical trial on covid- patients (medicine) . its efficacy is speculated to be linked to its primary target abl kinase, but it is conceivable that the observed effects are the consequence of its already proven lysosomotropic behaviour (fu et al., ) . at the moment, there are more than cads currently being tested in numerous ongoing clinical trials on covid- , with chloroquine, hydroxychloroquine and azithromycin being most widely studied. fda has recently revoked the emergency use authorization (eua) to use hydroxychloroquine and chloroquine to treat covid- outside clinical trials due to the lack of efficacy, and due to marked toxicity findings in patient populations treated in clinical trials so far (fda). nevertheless, other clinical trials with these drugs are still ongoing (medicine) . in addition, other well known cads are also being clinically tested, such as amiodarone, fluoxetine, chlorpromazine and fluvoxamine, but also other drugs with possible cad properties such as imatinib, bromhexine, formoterol (medicine) . the number of clinical trials and diversity of test protocols will hopefully make it feasible to get a better picture in near future on potential clinical value of these compounds as broad spectrum antivirals and open door to the development of new safer cads for antiviral purpose. in this study, we have analysed compounds with the aim to find existing registered drugs which may be useful in combating current covid- pandemics. specifically, we were looking for drugs capable of accumulating in endosomes and membranes and thereby possessing inherent antiviral activity. the drugs were analysed based on their known physicochemical and pharmacokinetic data, in addition to our in silico prediction of accumulation in lysosomes/endosomes. we have identified drugs with high and drugs with lower probability of showing antiviral effects due to their lysosomotropic behaviour. broad spectrum antiviral activity has already been reported for some of these drugs and therefore, it is crucial to investigate their antiviral properties on sars-cov- . although they were not designed to be specific and may thus lack potency, these drugs may, either in combination or alone, be capable of reducing the extent of infection and helping patients avoid serious or long-term illness. keeping in mind the low probability of finding a highly potent and specific antiviral drug for the ongoing pandemic, these drugs may represent at least a partial, but possibly one of the best antiviral pharmacotherapeutic solutions currently at hand. it will be critical in further investigations of lysosomotropic drugs to find out which administration regimens would be useful. although evidence of their antiviral activities is mounting, it is still unclear at which stage of disease development endosomal pathway disruption could play the most important role and whether the efficacious level of any of these drugs can be achieved in the body without significant toxic effects. moreover, it should be determined whether prior exposure to lysosomotropic drugs could help prevent the disease or, on the contrary, induce adaptations in the body and reduce the efficacy of antivirals with lysosomotropic mechanism. defining target patient populations, based on disease status, drug safety profiles and other factors need to be carefully investigated. answers to these crucial questions are required for assessing the potential of these drugs as broad-spectrum antivirals, suitable for repurposing to treat covid- or any future viral infection epidemics. authors declare no conflicts of interest. tables and footnotes table . criteria for the selection of drugs with high likelihood of lysosomotropic effects. livertox clinical and research information on drug-induced liver injury drugs@fda: fda-approved drugs targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases discovery and development of safe-in-man broad-spectrum antiviral agents the contribution of lysosomotropism to autophagy perturbation infective hepatitis and toxic jaundice in a municipal hospital during a five-year period. incidence and prognosis arbidol: a broad-spectrum antiviral compound that blocks viral fusion human ether-à-go-go-related potassium channel: exploring sar to improve drug design chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia pharmacokinetic/pharmacodynamic modeling of the antiretroviral activity of the ccr antagonist vicriviroc in treatment experienced hiv-infected subjects (actg protocol ) approved antiviral drugs over the past years evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection quantification of intracellular accumulation and retention of lysosomotropic macrocyclic compounds by high-throughput imaging of lysosomal changes efficacy of tilorone dihydrochloride against ebola virus infection fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated raman scattering cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: implications for an intracellular distribution-based drug interaction hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial azithromycin-induced changes to bacterial membrane properties monitored modeling phospholipidosis induction: reliability and warnings stereoselective inhibition of the herg potassium channel azithromycin, cardiovascular risks, qtc interval prolongation, torsade de pointes, and regulatory issues: a narrative review based on the study of case reports phospholipidosis as a function of basicity, lipophilicity, and volume of distribution of compounds membrane binding proteins of coronaviruses therapeutic areas ii: cancer, infectious diseases, inflammation & immunology and dermatology probing the interactions of macrolide antibiotics with membrane-mimetics by nmr spectroscopy pharmacokinetics of quinine, chloroquine and amodiaquine. clinical implications covid- experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings drug-drug interactions involving lysosomes: mechanisms and potential clinical implications chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion intensity of macrolide anti-inflammatory activity in j a. cells positively correlates with cellular accumulation and phospholipidosis a high content screening assay for identifying lysosomotropic compounds qsar models for predicting five levels of cellular accumulation of lysosomotropic macrocycles impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype effect of vicriviroc on the qt/corrected qt interval and central nervous system in healthy subjects construction and consensus performance of (q)sar models for predicting phospholipidosis using a dataset of compounds clemastine, a conventional antihistamine, is a high potency inhibitor of the herg k+ channel structural determinants of cationic amphiphilic amines which induce clear cytoplasmic vacuoles in cultured cells antiviral activity of cationic amphiphilic drugs cellular accumulation and lipid binding of perfluorinated alkylated substances (pfass) -a comparison with lysosomotropic drugs effects of chloroquine on viral infections: an old drug against today's diseases? quantitation of lysosomal trapping of basic lipophilic compounds using in vitro assays and in silico predictions based on the determination of the full ph profile of the endo-/lysosomal system in rat hepatocytes the pharmacokinetics and bioavailability of clemastine and phenylpropanolamine in single-component and combination formulations mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology drug induced phospholipidosis: an acquired lysosomal storage disorder clinical pharmacokinetics of telithromycin, the first ketolide antibacterial coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors characterization of the inhibitory effects of erythromycin and clarithromycin on the herg potassium channel are herg channel blockers also phospholipidosis inducers pharmacokinetics of promethazine and its sulphoxide metabolite after intravenous and oral administration to man subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (nr ) in vitro antimalarial drugs: qt prolongation and cardiac arrhythmias the influence of cisapride and clarithromycin on qt intervals in healthy volunteers chloroquine is a potent inhibitor of sars coronavirus infection and spread pharmacokinetics of cyclizine following intravenous administration to human volunteers this study has been funded by karolinska institutet, stockholm, sweden.j o u r n a l p r e -p r o o f (goracci et al., , orogo et al., , accumulation in cells calculated according to , molecular and phys-chem properties from pubchem and drugbank (wishart et al., ) (https://www.drugbank.ca/), hepatotoxicity from livertox ((niddk), ), broad spectrum antiviral activity (andersen et al., ) (https://drugvirus.info/), pharmacokinetics and cardiotoxicity (hancox et al., , van haarst et al., , stanat et al., , kaneko et al., , taylor et al., , walker and kanfer, , krishna and white, , traebert and dumotier, , shi et al., , bjorneboe et al., , ducharme and farinotti, , schran et al., , ridley et al., , crawford et al., , o'mara et al., , schrezenmeier and dörner, , chen et al., . green footnote: data sources: phospholipidosis (goracci et al., , orogo et al., , accumulation in cells calculated according to , molecular and phys-chem properties from pubchem and drugbank (wishart et al., )(https://www.drugbank.ca/), hepatotoxicity from livertox ((niddk), ), broad spectrum antiviral activity (andersen et al., ) (https://drugvirus.info/), pharmacokinetics and cardiotoxicity (hancox et al., , van haarst et al., , stanat et al., , kaneko et al., , taylor et al., , walker and kanfer, , krishna and white, , traebert and dumotier, , shi et al., , bjorneboe et al., , ducharme and farinotti, , schran et al., , ridley et al., , crawford et al., , o'mara et al., , schrezenmeier and dörner, , chen et al., . for colour legend see table . key: cord- -jo rqy y authors: maurya, priyanka; singh, samipta; saraf, shubhini a. title: inhalable hybrid nanocarriers for respiratory disorders date: - - journal: targeting chronic inflammatory lung diseases using advanced drug delivery systems doi: . /b - - - - . - sha: doc_id: cord_uid: jo rqy y rapid advancements in the field of drug delivery lead to increased use of inhalable formulations as they are cost effective, noninvasive, and targeted and have less systemic side effects and above all better patient compliance. development of inhalable hybrid systems has offered manifold advantages to this area of drug delivery. inclusion of polymer and lipid, inorganic and organic substances, and metallic nanoparticles all of them aim to achieve codelivery of drugs which are incompatible in single phase systems. the recent progress in nanotechnology has gained momentum toward delivery of sirna and mirna and vaccines to the targeted site. the present work is an attempt to compile all the hybrid and inhalable systems to give readers an overview toward this delivery system as much more work is needed in this field to achieve better resolution of inflammatory disorders. pulmonary route offers various outstanding features when studied in comparison with the oral and parenteral routes. it offers a highly vascularized area with a thin blood-alveolar barrier. the route also possesses increased selectivity, reduced systemic side effects, and low administered dose [ ] . inhalation drug delivery has long been used in the treatment of tuberculosis, asthma, bronchitis, cystic fibrosis (cf), chronic obstructive pulmonary disease (copd) [ ] , and systemic diseases like osteoporosis, pain, and diabetes. it is particularly useful in drugs with low bioavailability and in reducing the dose with reference to oral administration. it offers a noninvasive route for drug delivery increasing patient acceptability. one of the major challenges in oral systems is achieving delivery to the site desired and avoiding its side effects. inhalable delivery overcomes these challenges by achieving site specific delivery and overcoming side effects such as decline in lung and kidney functions due to prolonged exposure subsequent to oral therapy. the inhalable formulations are delivered as a solution, suspension, or dry powder [ ] . nanoparticles (np) possess promising targeting ability and have high cell uptake characteristics when administered through vascular routes. nanoparticles show rapid clearance as they are coughed out after pulmonary administration. to overcome this problem, they are incorporated in large microparticles. microparticles with appropriate aerodynamic characters ( - μm) allow drug delivery to lung tumors [ ] . the different anatomical barriers to successful pulmonary delivery are as follows: (i) the mucus layer/surfactant layer (~ - μm thick) poses a barrier to the deposited drug. it has to be quickly dissolved into this mucus layer before enzymatic degradation of the drug or else there would be rapid mucociliary clearance; hence, permeation enhancers must act rapidly to improve the bioavailability of the active ingredient. (ii) the epithelial layer consists of pseudo-stratified columnar cells interconnected via tight junctions. most of the molecules are absorbed through transcellular diffusion (small lipophilic molecules partition between the membranes via concentration gradient), and polar molecules follow paracellular diffusion, but the tight junction poses a barrier to the absorption of drug molecules. (iii) capillary endothelium is of importance to systemically targeted drugs but does not favor locally acting drugs [ , ] . the different barriers to pulmonary delivery are listed in fig. . lungs achieve slow clearance of nanoparticles. the np escape mucociliary and phagocytic inactivation. they achieve controlled release. the major hurdles in delivery through nanoparticle are poor particle size distribution, instability, and notso-good loading efficiency. hybrid nanoparticles are comparatively new class of nanoparticles with the beneficial effects of both lipids and polymers. hybrid nanoparticles possess good loading efficacy, structural integrity, cell targeting properties, and better cellular affinity. it is also beneficial in drug delivery of both hydrophilic and hydrophobic drugs [ ] . pulmonary administration of nanoparticles also reduces the drug dose to about % of the initial dose [ ] . drug release from these hybrid systems is affected by the thickness of the lipid layer in the case of polymeric cores and lipidic shell-type hybrid systems. as the thickness of the lipid layer decreases, the size is reduced, but the release increases as the water permeation is increased and the dissolution, in turn, increases for hydrophilic drugs. when hybrid systems are formulated with a polymeric shell and lipid core, the clearance from the lung decreases as the polymeric shell protects the core from macrophage uptake, thereby increasing their residence time [ ] . particles inhaled through inhalation devices are deposited in the airways through sedimentation under gravity, brownian diffusion, and inertial impaction. large particles more than μm deposit in the throat and upper respiratory tract due to high mass and inertia and are eliminated through spitting or swallowing. particles between and μm travel up to the lower airways by gravitational sedimentation. particles smaller than μm are deposited in the alveoli by brownian motion but are mostly eliminated as they do not get sufficient time to adhere leading to exhalation [ ] . inhalable micro-and nanocarriers deliver the drug or active ingredient to the lung epithelium and basal layers for quicker onset of action. np that is below nm is delivered directly to the basal epithelium and avoids phagocytosis. endocytosis of np occurs between a range of - nm. dry nebulized nanoparticles have low deposition values. to achieve better results, they can be nebulized by dispersing them in normal saline. nowadays, apart from delivering drugs, research is undergoing to deliver mirna and antisense oligonucleotides that work by inhibiting protein expression of various inflammatory conditions. inflammatory lung disorders can be described as copd, cf, and asthma. bronchial epithelium, the first in line of defense as presented by the airway, secretes il- in response to toll-like receptor- activation. as these proinflammatory cytokines are released, they start neutrophil infiltration at the site of release and all of proteases and oxidants start damaging the epithelium and begin losing in tissue function and start tissue transformation. mir- downregulates the secretion of il- secreted in response to lipopolysaccharide by more than % [ ] [ ] [ ] . vencken et al. designed and prepared hybrid nanoparticles for targeting bronchial epithelial cells loading them with mir- to downregulate proinflammatory chemokine il- . mirna delivery through nanoparticles which opens up various avenues for disease targeting [ ] . it has been reported that mirna are involved in various pulmonary disorders such as copd, asthma, and lung cancer [ ] . targets for mirna involved in lung diseases are il- , il- , il- a, il- , ifnb , tlr , tgf-β pathway, phosphoinositide -kinase (pi k)/akt pathway, and glycogen synthase kinase beta (gsk b). the mirna involved are mir- , mir- , mir- a, mir- a, mir- , mir- , mir- a, mir- , mir- a, mir- , mir- a, mir- , and mir- [ ] . mirna possess a low molecular weight and small size that enable them to be efficiently given as drugs. the mirna targeting is done by two mechanisms, by direct downregulation using mirna antagonists or by using mirna sponges to sequester misexpressed mirna. the second approach is used in mirna targeting [ ] . the various challenges and approaches to overcome mirna targeting are given in fig. . antigen-presenting cells are commonly called dendritic cells that are of immense importance in immune response related to its regulation and initiation. the dendritic cells (dc) continuously sample the inhaled air for antigens and present them to the specific t cells in the regional lymph nodes. thereby, they activate the t cells and present them the processed peptides. they also activate memory t-cell responses in the lungs. they are quite sensitive to environmental factors such as allergens, pollutants, microbes, and tissue damage products [ ] . the mirna is nowadays utilized as biomarker. dc hold popularity as antitumor agents. dc possess antitumor effects . mirna mimics and antagonists are degraded in the blood circulation. . optimize the particle size, surface charge and chemical modification of the mirnas or delivering them locally. challenges and approaches to overcome mirna targeting. that are affected by trivial dc count, low antigen presentation efficacy, and reduced ability of dc to migrate into the tumor mass. apart from improving the antigenpresenting efficacy of dc, blocking the signal between dc and tumor cells is also beneficial in developing antitumor therapy. wand et al. reported that to support tumor growth, lung cancers restrict functional dcs from that region. dc-derived exosomes are efficiently used in immunotherapy in nonsmall cell lung cancer [ ] . when a np utilizes the multifunctionality given to it by way of different materials of construction, it becomes hybrid. depending upon the properties required for a system, a suitable selection can be made. hybrid nanoparticles of the core-shell type in the nanorange are composed of the polymeric layer and a lipidic layer. hybrid nanoparticles were prepared to reduce the limitations of lipidic and polymeric systems. these are solid submicron particles composed of the lipid and the polymer containing various bioactives such as proteins, genes, targeting ligands, and drugs, which can be encapsulated, covalently attached, and entrapped. hybrid nanoparticles are used to overcome the biobarriers limiting oral drug absorption, that is, acid hydrolysis, ph differences, enzymatic degradation, mucosal barriers, and variable transit time. lipids provide an endless opportunity as an excipient enabling formulation of solid lipid nanoparticle, self-emulsifying systems, nanoemulsions, nanostructured lipid carriers, and liposomes. lipidic systems can be used efficiently in formulating poorly water-soluble drugs as they provide a lipophilic microenvironment for enhancing drug solubilization and preventing p-glycoprotein (p-gp) efflux, and avoid the first-pass metabolism, reduces hepatic metabolism by promoting lymphatic drug transport and lipidic systems, and protect encapsulated drugs from digestive enzymes in git. inclusion of a polymeric nanocarrier can be utilized to deliver compounds not easily delivered via lipidic systems. polymeric systems are more stable in gi transit and provide the versatility of a natural, synthetic, or semisynthetic alternative. an advance in polymer sciences enables superior control over the properties and architecture of these carriers facilitating controlled delivery although issues regarding their inadequate encapsulation and burst release still pop up from time to time [ ] . hybrid nanoparticles of core-shell type can be classified as. lipid-core/polymer-shell (lc-ps) systems include a lipid core (cholesterol, dppc, dspe-peg ) enclosed by one or multiple polymeric coats (peg, chitosan, polyacrylic acid, and polyallylamine). the core holds the active ingredient, while the coating stabilizes the lipid core to overcome biological obstacles. significant work has been done on linking nanocarriers such as liposomes, sln or nlc with chitosan, and peg like multifunctional polymers [ ] [ ] [ ] [ ] . incubation method is the preferred method for preparing chitosan-coated lipid nanoparticles, that is, uncoated lipid nanoparticles are prepared first, and then the prepared nanoparticles are incubated in an aqueous chitosan solution. the chitosan self-assembles at the interface of oil and water (o/w) through electrostatic interactions during incubation. alternatively, glutaraldehyde can be utilized as the crosslinking agent for stabilization. chitosan-coated systems are beneficial in terms of efficiently loading hydrophobic drugs in nanocolloidal systems. the chitosan coated formulation due to its positive charge possessed better uptake and targeting ability [ ] . garcia et al. developed chitosan-coated lipidic nanoparticles of tripalmitin and lecithin and isolated them using ultracentrifugation. the negatively charged nanoparticles possessed a positive charge after being coated by chitosan. the chitosan-coated np remained unaltered in simulated gastric and intestinal media and proved that it is suitable for oral use [ ] . pegylated nanoparticles possess a longer circulation half-life because they escape capture and phagocytosis by various organs. pegylated lipid nanoparticles are prepared by adding peg esters in the lipid phase. thin-film hydration method is frequently used in its preparation. dipalmitoyl phosphatidylcholine (dppc), distearoyl phosphatidylethanolamine-polyethylene glycol (dspe-peg ), and cholesterol are the lipids of choice for pegylated lipid nanoparticles. pegylated sln are prepared by emulsification and can be purified by ultracentrifugation. a blend of peg-stearic acid and tripalmitin is solubilized in an organic solvent, followed by sonication, in an aqueous surfactant solution. the organic solvent gets evaporated during stirring, and the final peg-sln is collected and purified by ultracentrifugation [ ] . pegylated nlc (peg-nlc) can also be prepared by substituting tripalmitin with liquid medium-chain triglyceride (miglyol ) [ , ] . polyelectrolyte-stabilized liposomes or layerosomes are prepared by a multiple coatings of polyelectrolyte (positive and negative charged), utilizing the layer-by-layer approach. the coatings so applied on the liposomal surface preserve the original structure and protect it from the physiological damage. layering helps in incorporating various materials in the system and in its overall stabilization through ionic interactions [ ] . the liposomal core consisting of cholesterol and phospholipid core achieve a positive charge on inclusion of stearyl amine. anionic polymers, namely, polyacrylic acid (paa) are then made use of in coating liposomes followed by another coat of polyallylamine hydrochloride (pah) cationic polymer. transmission electron microscopy is used to characterize layerosomes. layerosomes range from to nm, and it depends on the size of crude or uncoated liposomes and the mass ratio amidst polymer and lipids [ ] . in the polymer-core/lipid-shell systems, polymer core is delimited by unit coat or several coats of lipid. the hydrophobic nature of the shell inhibits water infiltration toward the core. core-shell system modifies polymer degradation, prevents drug movement, and sustains drug release, as the polymer provides structural integrity to the shell. the drug may be captured within the core or simultaneously in core and in the adjacent lipid layer, depending upon the nature of drug(s) [ ] [ ] [ ] [ ] [ ] . lipidic carriers are reported in the literature to augment the oral administration of biomacromolecules to the systemic circulation via intestinal lymphatic system, thereby evading degradation by first-pass metabolism. enclosing hydrophilic drugs in the lipid system promotes transport via the lymphatic system. a hybrid nanoparticle is utilized for various purposes. the use of cationic polymer increases the loading efficiency of drug (heparin) in the lipid carrier, and chitosan is recognized to improve the passage of heparin across the intestinal membrane through tight junctions. chitosan when used as a constituent of the hybrid system increases the entrapment efficiency of lipid nanoparticle [ ] . the hydrophilic peg shell improves the steadiness of np in physiological environment and minimizes the mucin adhesive interfaces [ , [ ] [ ] [ ] ]. polymerized liposomes (pl) possess features similar to liposomes, wherein the covalent bonds link the phospholipid bilayers. pl are classically created from lipids, which can be polymerized by the thin-film hydration method. polymerization is used to stabilize the lipid bilayer [ , ] . o'brein et al., in , prepared polymerized liposomes from reactive lipids containing diacetylene, methacryloyl, and dieneoyl groups. complete polymerization was not observed in diacetylene and methacryloyl lipids while moderate decrease in permeability of water-soluble compound (glucose) was observed in methacryloyl groups. dienoyl lipids can be efficiently polymerized, and maximal reduction in membrane permeability is achieved as assessed by glucose permeation [ ] . a lipidic nanocarrier composed of solid-state medium enhances the formulation stability and facilitates various desirable biopharmaceutical functions. nano-inmicro-type silica-lipid hybrid microparticles have been reported in the literature. foreseeable and precise lipid digestion can be achieved by altering the internal nanostructures including surface area and porosity. lately, spray drying and incubation methods have been used in plga-lipid hybrid (plh) systems. they are prepared from miglyol and plga np. chitosan-zein nano-in-micro-type formulations have also been reported for gene delivery [ ] . hybrid nanoparticles have been used to overcome the bio barriers limiting oral drug absorption, that is, acid and enzymatic degradation, ph differences, mucosal barricades, and variable transit time. lipids provide an endless opportunity as an excipient enabling formulation of sln, self-emulsifying systems, nanoemulsions, nlc, and liposomes. lipidic systems can be used efficiently in formulation of hydrophobic drugs as they provide a lipophilic micro-environment for enhancing drug solubilization, averting p-glycoprotein (p-gp) efflux and dodging the first-pass metabolism as well as reducing hepatic metabolism by promoting lymphatic drug transport. lipidic systems also protect encapsulated drugs from the degradative effect of the digestive enzymes in git. inclusion of a polymeric nanocarrier can be used to deliver compounds not delivered via lipidic systems. compared with their lipidic counterparts, polymeric systems are more stable in gi transit and provide the versatility of a natural, synthetic, or semisynthetic alternative. developments in polymer sciences enable good control over the properties and architecture of these carriers, facilitating controlled delivery. however, issues regarding their adequate encapsulation and burst release still surface from time to time [ ] . the preparation of hybrid nanoparticles falls under two broad categories: the twostep method and the single-step method. this involves independent preparation of core and shell components, which may then be combined by either direct hydration, extrusion, or sonication. positively charged lipid vesicles and negatively charged polymeric nanoparticles fuse by electrostatic magnetisms. dry lipid films are hydrated by polymeric dispersions or the dispersions can be added to preformed lipid vesicles, followed by vortexing and subsequent heating to arrange lipids onto the particle surface. the formulation is finally centrifuged to remove nonadsorbed lipids, micelles, or free polymeric nanoparticles, as the case may be [ , ] . this method involves synchronizing nanoprecipitation with self-assembly, simultaneously. modifications to this single-step method have also been reported, which include modified solvent extraction/evaporation or nanoprecipitation method respectively [ ] . firstly the polymer and drug are dissolved in a water-immiscible solvent (chloroform, ethyl acetate, dichloromethane, etc.) followed by mixing lipid in aqueous phase by sonication. rotary evaporator can be utilized for removal of organic solvents. the product is further centrifuged followed by controlled washing. the product is dried by a suitable method like lyophilization to obtain a free-flowing dry powder [ , , ] . a water-miscible organic solvent (acetone and acetonitrile) is used to solubilize the polymer and hydrophobic drugs. dropwise addition of organic solution to aqueous dispersion of lipid or peg-lipid conjugate is carried out. the blend is then vortexed and sonicated to achieve nanosized particles [ , ] . pulmonary drug delivery is mainly aimed at passive targeting of tumor sites via enhanced permeation and retention (epr) effect rather than active targeting via overexpressed receptors. particles in the range of - nm are internalized better than microparticles of size - μm. therefore size uniformity is a major concern in passive targeting as it plays an important role in deep lung deposition and internalization of the delivery system [ ] . pulmonary delivery of drugs via active targeting is achieved by the coupling of the drug to carrier molecules through a bioreceptive bond. extensive studies are however required to establish the efficacy of these targeting strategies. the size and shape of the np formulation plays a significant part in pulmonary drug targeting. the anatomy of the airways and ventilation parameters also determine the drug delivery and its time of residence in the respiratory tract [ ] . very small particles are exhaled, and very large particles are removed by mucociliary machineries. therefore there is a size range that determines the extent to which a formulation may reach the desired site of action. drug delivery to deep lung tissues is dependent on size [ , ] . respirable particles between and μm are suitable for achieving significant drug concentrations. small size and hydrophilic molecules are passively taken up by epithelial cells, and insoluble vectors are taken up by macrophages and cleared. alveolar macrophages do not recognize particles less than nm on the surface of the lungs and allow them access to pulmonary interstitium and further to the capillary blood flow [ , ] . calcium carbonate microparticles prepared from vaterite polymorph possess suitable properties for drug delivery such as uniform size, and narrow size distribution, and spherical geometry as assessed by bet, sem, and tem in various studies. alginate, when used in conjunction with calcium carbonate, reduces its toxicity and aids in the formation of a biocompatible formulation. these hybrid microstructures are capable of further modification through changes in polymer content and microsurface changes in porosity. these microsystems possess good drug loading capability. islan et al. prepared spherical microparticles in the range of - μm, which is ideal for pulmonary delivery. as evidenced from the in vivo data, the microparticles reached . times the concentration of pure drug solution in the lung at a dose of . mg and at . mg, the lung levels were . times higher than pure drug [ ] . hybrid systems can be efficiently used to incorporate nuclease that helps in the breakdown of dna chains, which are products of cell lysis and lead to mucus accumulation. the reduction in viscoelasticity imparted by nuclease improves the antimicrobial efficacy of drugs [ ] . hybrid nanoparticles containing lipids have been prepared in various studies for their diagnostic and colloidal stabilization properties. localized laser heat generation can be utilized efficiently as absorption of light leads to the oscillation of surface electrons and generation of heat. this simple mechanism can be used in hybrid systems that are effectively made with the purpose of releasing the drug with respect to change in temperature. hybrid nanoparticles releasing the drug in response to external stimuli were successfully prepared by ahmady et al. various formulations were prepared to contain gold, silver, and iron oxide in the hybrid formulation containing thermosensitive liposomes. the presence of metals in the formulation makes it stimulus responsive. the controlled drug release from the formulation continued for min at mild heating conditions while maintaining good drug retention. the authors also provide support to the use of these np in the theranostic application [ ] . it has been reported that mirna are involved in gene regulation in eukaryotes, and they are a group of small noncoding rnas. they can target multiple sites and multiple genes within a signaling network. they also function as regulators, in the advancement and expansion of lung diseases. variations in their number in blood, inflammatory cells and tissues act as disease modifiers and drivers. they act as biomarker tools as well, for the detection of diseases. in some diseases, mirna act as disease drivers, but in the majority, they only maintain signaling pathway and gene expression. knockout models for mirna do not exist or are not published [ ] . proinflammatory chemokines such as interleukin (il)- are secreted by human bronchial epithelial cells to retort lipopolysaccharide as a stimulant. to study its effect on the treatment of respiratory disorders, modulation of mirnas was studied by sebastian et al. they studied the role of mirna- in secretion of il- . nebulized hybrid particles containing mir- down regulated lps-induced il- secretion by % in epithelial cells. hybrid nanocarriers containing dotap-plga are efficient carriers for mirna delivery for inflammatory conditions [ ] . the mirna delivery to cancer cells via aptamer-functionalized hybrid nanoparticles for lung cancer was successfully achieved by perepelyuk et al. to efficiently carry mirna- b to muc -expressing cancer cells, bioconjugate system was prepared. a cells showed significant downregulation of oncoproteins dnmt b and mcl , and this further led to apoptosis and better antiproliferative effect [ ] . novel calcium phosphate-polymer hybrid nanoparticle systems were reported by zhou et al. hydrophilic mirna and hydrophobic drugs were encapsulated in this system by coprecipitation in water-in-oil emulsion followed by coating with anionic lipid. the system delivered both the drug and mirna to the target site and inhibited the proliferative effect of mir / and enhanced the therapeutic effect of paclitaxel [ ] . dendritic cell targeting is nowadays being used in monovalent and polyvalent vaccine delivery. the current approaches use bifunctional fusion protein (truncated core streptavidin fused to anti-dec- -single chain antibody) in conjunction with biotinylated plga nanoparticles (containing antigen) as the delivery system is biodegradable and can be used without adjuvants also. it also dodges a postformulation modification that has a detrimental effect on the antigen. the process involved is also quite simple as it requires a simple mixing of the two components (targeting ligand with nanoparticles). it utilizes the dec- as the target moiety. in vitro studies showed a twofold increase in receptor-mediated uptake when compared with nontargeted nanoparticles [ ] . migdal et al., prepared hybrid nanoparticles containing para amino benzoic acid, and titanium dioxide internalized in dendritic cells was found to be noncytotoxic. cellular uptake occurs through macro pinocytosis. np were not present in the nucleus as seen through confocal microscopy. the np remained in contact with cytoplasmic vesicles and cellular membrane without colocalizing with clathrin-coated vesicles. the nanoparticles are nonimmunogenic and do not induce oxidative stress [ ] . raghuvanshi et al. targeted plasmid dna, through loaded biotinylated nanoparticles containing chitosan for immunization against sars-cov as antigen. the nanoparticles were prepared by the coacervation process and evaluated for nuclease digestion and plasmid dna loading. the formulation being noninvasive is efficiently delivered via receptor mediation through the nasal route. the dendrimer targeting ability toward dec- is imparted by the bifunctional fusion protein. intranasal administration enhanced igg and nasal iga antibodies. intramuscular route was also evaluated for delivery of vaccines, which revealed its superiority for igg response over the intranasal route [ ] . inhalable hybrid nanocarriers have varied applications in drug delivery for respiratory disorders. the inhalable nanocarriers that are below nm are rapidly deposited to the basal epithelial cells and provide for site-specific delivery and avoid phagocytosis. hybrid microcarriers ( - μm) allow for passive targeting to lungs. hybrid nanocarriers can effectively deliver mirna for treatment of inflammatory lung conditions and in lung cancer [ , ] . dendritic cell targeting (in inflammation) can be achieved by a preparing a hybrid between targeting ligand and plga np. cystic fibrosis can be targeted by silica-coated silver np containing antibiotic. colloidal hybrid systems can be used for imaging, diagnostics, and drug delivery. hybrid nanocarriers are mostly useful in treating lung cancers [ ] . hybrid nanoparticles are nowadays used in various fields of pharmacotherapy and theranostics, tumor being the front-runner followed by gene delivery [ ] , peptide [ , ] , protein [ ] , vaccines [ ] , diagnostics [ ] [ ] [ ] [ ] , and materials science [ ] [ ] [ ] [ ] . the different applications of hybrid formulations are listed in table . a hybrid nanoparticle with a suitable target offers a novel approach for a rational design for effective therapeutic drug delivery. in inflammatory disorders, oral delivery of drugs leads to poor patient compliance and more of dose dumping. the route that is mostly preferred for inflammatory conditions is the pulmonary route as it offers the advantage of immediate delivery of the drug to the site and a lesser systemic side effect. the only concern in pulmonary delivery is that the drug once delivered cannot be reversed. hybrid nanoformulations are nowadays preferred due to the flexibility in formulation procedures and the advantage of codrug delivery. this system also reduces the concern for interaction in terms of codelivery as both the drugs are present in their respective phases. the added benefits now are advances in drug delivery with novel systems that aim to deliver si rna, mirna, vaccines, and antivirals. these normally require sophisticated carriers that deliver these agents directly to the target site, preventing the degradation of gene/vaccine. the hybrid carriers can be suitably modulated to keep all these properties intact. as with core-shell systems, the formulator can efficiently select the core and shell material to either actively target the formulation or avoid the res uptake mechanism and aim to amplify the circulation time of the nanoformulation so that it keeps releasing the drug at a predetermined rate without being taken up by the immune system. as discussed in the previous sections, these systems efficiently target and treat various therapeutic indications including tumors, colorectal disorders, alzheimer's disease, psoriasis, lung disorders, and atherosclerosis. they are now also used in diagnostics and theranostics for the effective delivery of contrast agents and fluorescent dyes. the future scope for this formulation should aim at delivering the therapeutics to tubercle nodules and chronic inflammatory disorders, which occur as a result of antibiotic resistance developed due to misuse/overuse of antibiotics. the pulmonary route and the complications offered in the way can be very easily modulated by the hybrid systems to overcome and heal the damaged tissue in affected areas, without being concerned about the excessive dose and related side effects. recent advances in controlled pulmonary drug delivery pulmonary drug delivery systems for tuberculosis treatment a review on recent technologies for the manufacture of pulmonary drugs development of an inhalable, stimuli-responsive particulate system for delivery to deep lung tissue strategies to enhance drug absorption via nasal and pulmonary routes delivering drugs to the lungs: the history of repurposing in the treatment of respiratory diseases pulmonary delivery of antitubercular drugs using spray-dried lipid-polymer hybrid nanoparticles a comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid-polymer hybrid nanoparticles nebulised lipid-polymer hybrid nanoparticles for the delivery of a therapeutic anti-inflammatory microrna to bronchial epithelial cells micrornas as therapeutics for future drug delivery systems in treatment of lung diseases the role of micrornas in chronic respiratory disease: recent insights dendritic cells in human lung disease: recent advances impaired dendritic cell functions in lung cancer: a review of recent advances and future perspectives coreshell-type lipid-polymer hybrid nanoparticles as a drug delivery platform development and evaluation of artesunate-loaded chitosan-coated lipid nanocapsule as a potential drug delivery system against breast cancer new surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin studies on peg-modified slns loading vinorelbine bitartrate (i): preparation and evaluation in vitro rgd modified and pegylated lipid nanoparticles loaded with puerarin: formulation, characterization and protective effects on acute myocardial ischemia model polyelectrolyte stabilized multilayered liposomes for oral delivery of paclitaxel lipid nanoformulations for oral delivery of bioactives: an overview dual-ligand modified polymerlipid hybrid nanoparticles for docetaxel targeting delivery to her /neu overexpressed human breast cancer cells evaluation of a polymer-lipid-polymer system utilising hybrid nanoparticles of dapsone as a novel antiacne agent folate-modified lipidpolymer hybrid nanoparticles for targeted paclitaxel delivery synthesis and characterization of hybrid polymer/lipid expansile nanoparticles: imparting surface functionality for targeting and stability quick synthesis of lipid-polymer hybrid nanoparticles with low polydispersity using a single-step sonication method development and characterization of polymer lipid hybrid nanoparticles for oral delivery of lmwh peg-detachable lipid-polymer hybrid nanoparticle for delivery of chemotherapy drugs to cancer cells hybrid poly(lactic-co-glycolic acid) nanoparticles: design and delivery prospectives plga/liposome hybrid nanoparticles for short-chain ceramide delivery preparation and characterization of polymerized liposomes phosphoinositide-containing polymerized liposomes: stable membrane-mimetic vesicles for protein-lipid binding analysis chitosan-zein nano-in-microparticles capable of mediating in vivo transgene expression following oral delivery polymer-lipid hybrid systems: merging the benefits of polymeric and lipid-based nanocarriers to improve oral drug delivery design of lipid-polymer hybrid nanoparticles for therapy of bph: part i. formulation optimization using a design of experiment approach oxaliplatin immuno hybrid nanoparticles for active targeting: an approach for enhanced apoptotic activity and drug delivery to colorectal tumors one-step synthesis of hybrid nanocrystals with rational tuning of the morphology dry powder pulmonary delivery of cationic pga-co-pdl nanoparticles with surface adsorbed model protein lipomer of doxorubicin hydrochloride for enhanced oral bioavailability inhalable particulate drug delivery systems for lung cancer therapy: nanoparticles, microparticles, nanocomposites and nanoaggregates role of size of drug delivery carriers for pulmonary and intravenous administration with emphasis on cancer therapeutics and lung-targeted drug delivery preparation, optimization, and in vitro simulated inhalation delivery of carvedilol nanoparticles loaded on a coarse carrier intended for pulmonary administration hybrid inhalable microparticles for dual controlled release of levofloxacin and dnase: physicochemical characterization and in vivo targeted delivery to the lungs fabrication of inhaled hybrid silver/ciprofloxacin nanoparticles with synergetic effect against pseudomonas aeruginosa engineering thermosensitive liposome-nanoparticle hybrids loaded with doxorubicin for heat-triggered drug release aptamer-hybrid nanoparticle bioconjugate efficiently delivers mirna- b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins calcium phosphate-polymer hybrid nanoparticles for enhanced triple negative breast cancer treatment via co-delivery of paclitaxel and mir- / inhibitors a simple approach for enhanced immune response using engineered dendritic cell targeted nanoparticles internalisation of hybrid titanium dioxide/para-amino benzoic acid nanoparticles in human dendritic cells did not induce toxicity and changes in their functions dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein fabrication of liposomal doxorubicin exhibiting ultrasensitivity against phospholipase a for efficient pulmonary drug delivery to lung cancers stable and efficient transfection of sirna for mutated kras silencing using novel hybrid nanoparticles hybrid protein-synthetic polymer nanoparticles for drug delivery arginine-glycine-aspartic acidmodified lipid-polymer hybrid nanoparticles for docetaxel delivery in glioblastoma multiforme lipid-polymer hybrid nanoparticles as a new generation therapeutic delivery platform: a review polymer-peptide hybrids as a highly immunogenic single-dose nanovaccine engineered hybrid nanoparticles for on-demand diagnostics and therapeutics amphiphilic dna block copolymers: nucleic acid-polymer hybrid materials for diagnostics and biomedicine hybrid fe o -poly(acrylic acid) nanogels for theranostic cancer treatment synthesis of magnetic nanoparticles and magnetic fluids for biomedical applications towards the development of a novel bioinspired functional material: synthesis and characterization of hybrid tio /dhica-melanin nanoparticles design of antimicrobial membrane based on polymer colloids/multiwall carbon nanotubes hybrid material with silver nanoparticles immobilizing gold nanoparticles in mesoporous silica covered reduced graphene oxide: a hybrid material for cancer cell detection through hydrogen peroxide sensing hybrid films from blends of chitosan and egg phosphatidylcholine for localized delivery of paclitaxel aptamerfunctionalized hybrid nanoparticle for the treatment of breast cancer hydrophobic superparamagnetic fept nanoparticles in hydrophilic poly(n-vinylcaprolactam) microgels: a new multifunctional hybrid system rna nanoparticles harboring annexin a aptamer can target ovarian cancer for tumor-specific doxorubicin delivery rgd conjugated liposome-hollow silica hybrid nanovehicles for targeted and controlled delivery of arsenic trioxide against hepatic carcinoma nipam-co-mps)-grafted multimodal porous silica nanoparticles as reverse thermoresponsive drug delivery system effects of nanoparticles with hydrotropic nicotinamide on tacrolimus: permeability through psoriatic skin and antipsoriatic and antiproliferative activities surfactant protein b (sp-b) enhances the cellular sirna delivery of proteolipid coated nanogels for inhalation therapy in situ hybrid nano drug delivery system (ihn-dds) of antiretroviral drug for simultaneous targeting to multiple viral reservoirs: an in vivo proof of concept targeted delivery of glucocorticoids to macrophages in a mouse model of multiple sclerosis using inorganic-organic hybrid nanoparticles ultra-small lipid-dendrimer hybrid nanoparticles as a promising strategy for antibiotic delivery: in vitro and in silico studies hybrid lipid/ polymer nanoparticles for pulmonary delivery of sirna: development and fate upon in vitro deposition on the human epithelial airway barrier local anesthetic effects of bupivacaine loaded lipid-polymer hybrid nanoparticles: in vitro and in vivo evaluation co-delivery of pemetrexed and mir- antisense oligonucleotide by lipid-polymer hybrid nanoparticles and effects on glioblastoma cells development and in vitro evaluation of core-shell type lipid-polymer hybrid nanoparticles for the delivery of erlotinib in non-small cell lung cancer construction and comparison of different nanocarriers for codelivery of cisplatin and curcumin: a synergistic combination nanotherapy for cervical cancer influence of cationic lipid concentration on properties of lipid-polymer hybrid nanospheres for gene delivery core-shell lipid polymer nanoparticles for combined chemo and gene therapy of childhood head and neck cancers engineering of a hybrid polymer-lipid nanocarrier for the nasal delivery of tenofovir disoproxil fumarate: physicochemical, molecular, microstructural, and stability evaluation lipid polymer-based hybrid nanoparticles as a novel delivery system for methotrexate to treat ovarian cancer targeted nanomedicine for prostate cancer therapy: docetaxel and curcumin co-encapsulated lipid-polymer hybrid nanoparticles for the enhanced anti-tumor activity in vitro and in vivo codelivery of doxorubicin and triptolide with reduction-sensitive lipid-polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment a nanoparticle-based nicotine vaccine and the influence of particle size on its immunogenicity and efficacy lysozyme-loaded lipid-polymer hybrid nanoparticles: preparation, characterization and colloidal stability evaluation molecularly targeted co-delivery of a histone deacetylase inhibitor and paclitaxel by lipid-protein hybrid nanoparticles for synergistic combinational chemotherapy a smart ph-responsive nano-carrier as a drug delivery system: a hybrid system comprised of mesoporous nanosilica mcm- (as a nano-container) & a ph-sensitive polymer (as smart reversible gatekeepers): preparation, characterization and in vitro release studies of an anti-cancer drug paliperidone-loaded nanolipomer system for sustained delivery and enhanced intestinal permeation: superiority to polymeric and solid lipid nanoparticles multifunctional organic-inorganic hybrid nanoparticles and nanosheets based on chitosan derivative and layered double hydroxide: cellular uptake mechanism and application for topical ocular drug delivery screening of lipid carriers and characterization of drug-polymer-lipid interactions for the rational design of polymer-lipid hybrid nanoparticles (pln) polymer-lipid hybrid nanoparticles synchronize pharmacokinetics of co-encapsulated doxorubicin-mitomycin c and enable their spatiotemporal co-delivery and local bioavailability in breast tumor a new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells simultaneous delivery of doxorubicin and gg (elacridar) by new polymer-lipid hybrid nanoparticles (pln) for enhanced treatment of multidrug-resistant breast cancer in vivo evaluation of a new polymer-lipid hybrid nanoparticle (pln) formulation of doxorubicin in a murine solid tumor model lipid-polymer nanoparticles for folate-receptor targeting delivery of doxorubicin polymer-lipid hybrid theranostic nanoparticles co-delivering ultrasmall superparamagnetic iron oxide and paclitaxel for targeted magnetic resonance imaging and therapy in atherosclerotic plaque lecithmer: preparation, characterization, and pharmacokinetic/pharmacodynamic evaluation a novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery synthesis of polymer-lipid nanoparticles by microfluidic focusing for sirna delivery development of novel self-assembled es-plga hybrid nanoparticles for improving oral absorption of doxorubicin hydrochloride by p-gp inhibition: in vitro and in vivo evaluation mechanically tuned nanocomposite coating on titanium metal with integrated properties of biofilm inhibition, cell proliferation, and sustained drug delivery key: cord- - hx kc authors: movia, dania; prina-mello, adriele title: preclinical development of orally inhaled drugs (oids)—are animal models predictive or shall we move towards in vitro non-animal models? date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: hx kc simple summary: this commentary focuses on the methods currently available to test the efficacy and safety of new orally inhaled drugs for the treatment of uncurable respiratory diseases, such as chronic obstructive pulmonary disease (copd), cystic fibrosis or lung cancer, prior to entering human experimentation. the key question that the authors try to address in this manuscript is whether there is value in using and refining current animal models for this pre-clinical testing, or whether these should be relinquished in favor of new, more human-relevant non-animal methods. abstract: respiratory diseases constitute a huge burden in our society, and the global respiratory drug market currently grows at an annual rate between % and %. inhalation is the preferred administration method for treating respiratory diseases, as it: (i) delivers the drug directly at the site of action, resulting in a rapid onset; (ii) is painless, thus improving patients’ compliance; and (iii) avoids first-pass metabolism reducing systemic side effects. inhalation occurs through the mouth, with the drug generally exerting its therapeutic action in the lungs. in the most recent years, orally inhaled drugs (oids) have found application also in the treatment of systemic diseases. oids development, however, currently suffers of an overall attrition rate of around %, meaning that seven out of new drug candidates fail to reach the clinic. our commentary focuses on the reasons behind the poor oids translation into clinical products for the treatment of respiratory and systemic diseases, with particular emphasis on the parameters affecting the predictive value of animal preclinical tests. we then review the current advances in overcoming the limitation of animal animal-based studies through the development and adoption of in vitro, cell-based new approach methodologies (nams). respiratory diseases constitute a huge burden in our society. it has been calculated that, worldwide, around million people are living with asthma [ ], million with chronic obstructive pulmonary disease (copd) [ ] , and more than , people with cystic fibrosis [ ] . furthermore, million people are affected by idiopathic pulmonary fibrosis (ipf) [ ] , and million people contract tuberculosis (tb) annually [ ] . in addition to this, lung cancer continues to be the leading cause of cancer death worldwide, accounting for . million deaths in [ ] ; whereas, pneumonia still constitutes the inhalation is the preferred administration method for treating respiratory diseases [ ] , as: (i) it delivers the drug directly at the site of action, resulting in a rapid therapeutic onset with considerably lower drug doses, (ii) it is painless and minimally invasive thus improving patients' compliance, and (iii) it avoids first-pass metabolism, providing optimal pharmacokinetic conditions for drug absorption and reducing systemic side effects [ ] [ ] [ ] . it should be noted here, inhalation differs from intranasal administration for the drug portal-of-entry (poe) and targeted site of action. intranasal drugs are sprayed into the nostrils, producing a local effect in the nasal mucosa; whereas, inhalation occurs through the mouth, with the oids, also referred to as orally inhaled drug products (oips), having their efficacy in the lungs. notably, attempts have been made to develop oids that exert their therapeutic action outside the lung, for the treatment of systemic diseases [ ] . the latter include, for example, migraine headaches, treated with aerosols of ergotamine or hydroxyergotamine, and type /type diabetes, for which inhaled insulin products have been developed (e.g., exubera-withdrawn in due to poor revenue-and afrezza-the uptake of which has also been impacted by socio-economic issues). oid therapeutic categories currently approved for the clinical treatment of respiratory diseases include drugs for the treatment of asthma and copd, such as β adrenergic agonists (e.g., albuterol, formoterol) and muscarinic antagonists (e.g., ipratropium, tiotropium) inducing bronchodilation, or glucocorticosteroids (e.g., fluticasone and budesonide) reducing inflammation. oids for the treatment of cystic fibrosis are also available for clinical use, with most of them falling into the therapeutic categories of mucolytics (e.g., saline and acetyl choline), aiming at thinning the mucus for facilitating its clearance from the patient's lungs. alternatively, leukocyte dnase, reducing inflammation, and antimicrobial agents (e.g., tobramycin), treating the bacterial infection characteristic of this disease, are also administered as oids. various devices can be used to administer oids to patients, including dry-powder inhalers (dpis), pressurized metered-dose inhalers (pmdis) and nebulizers. these devices have been extensively discussed in several recent works [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, dpis deliver powder particles carrying the drug; pmdis and nebulizers generate liquid droplets containing the drug. to be effective, an inhalation device must be easy to use and forgiving of poor patient's compliance, while providing reproducible effective dosing. thus, a through characterization of the performance of the inhalation device is required at regulatory level, when developing an oid. such characterization is based on in vitro, ex vivo and in vivo (on human volunteers) tests, as extensively described in the scientific literature [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] animal models are not used in the characterization of the efficiency and reproducibility of inhalation delivery devices. this is due to the fact that, dpis and pmdis are breath-actuated and therefore not compatible with animal exposure; whereas for nebulizers modifications are needed in line with the animal model adopted. thus, our manuscript, which focuses on the potential reduction and replacement of animals studies in oid development, does not discuss the impact of inhalers' performance on the effectiveness of inhalation therapies [ ] , a current challenge discussed in detail elsewhere [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite the major advantages over i.v. administration of drugs, inhalation therapy encounters several obstacles in achieving an effective therapeutic dose for the successful treatment of respiratory and/or systemic diseases. below, we describe the journey of an oid once administered and the human-specific features that, in the authors' opinion, strongly impact on the current low translation rate of oids, as these are poorly replicated in the current preclinical models. when an oid is administered to a patient, its liquid or powder aerosol enters the human respiratory system via the oropharynx. oid deposition in the oropharynx is invariably wasteful, reducing the oid dose reaching the lungs. this indeed constitutes the first feature to keep into account for developing an effective inhalation therapy [ ] . rodent models cannot reproduce this feature, as they are obliged nose-breathers. however, other animal models (e.g., dogs) can be used to overcome the limitations posed by rodents. also, oid deposition in the oropharynx must be minimized in clinics to avoid severe side-effects in the patients. side-effects can be due to both local and systemic toxicity, as oids accumulating in the mouth and throat enter the body through swallowing. achieving an optimal oid deposition pattern in the patients' lung is the second feature to keep into account for an effective inhalation therapy [ ] . to reach its site of action and/or absorption, the oid needs to pass through the so-called extrathoracic (or et) region of the larynx, enter the tracheobronchial region and reach the small and/or peripheral (alveoli) airways. drug absorption and translocation into the blood flow can in fact occur from all parts of the lung, but it occurs more readily in the alveoli [ ] , where there is a large surface area and a relatively thin layer of epithelial and endothelial cells separating the inhaled drug from the blood flow. the oid journey within the complex, branched structure of the human lung is influenced by two main parameters of the particles/droplets carrying the drug [ ] : (i) velocity [ ] ; and (ii) aerodynamic size distribution (the so-called apsd) [ ] . both parameters strongly impact on the drug deposition pattern and, subsequently, on the effectiveness of the inhalation therapy. velocity is defined by the delivery system employed in the oid administration. generally, high velocity results in increased deposition in the oropharynx and tracheobronchial regions; whereas, low velocity generates a peripheral deposition pattern [ ] . it goes without saying that oids cannot reach those part of the respiratory tract where velocity is null, i.e., those parts of the lung that are not ventilated. this is particularly relevant to consider when developing oids against respiratory diseases [ ] , which are characterized by the partial or full obstruction of the respiratory tract (e.g., asthma, copd, cystic fibrosis and lung cancer). combination of drugs where bronchodilators or mucolytics are used in a synergistic manner with other drug therapies, can be used to modulate oid velocity and increase the efficacy of the inhalation therapy. in parallel, the deposition mechanism of the aerosol particle/droplets in the bronchial tree changes depending on their apsd [ ] . droplets/particles with large aerodynamic size deposit by impaction or interception mechanisms in the oropharynx or just beyond the trachea bifurcation. the smaller droplets/particles deposit in the smaller airways by sedimentation, subject to gravity. among those, the droplets/particles with aerodynamic size below µm further move to the alveoli by diffusion or brownian motion. it should be noted here, droplets/particles deposition follows stokes' law [ ] . the consequence is that, since most of the droplets/particles are near spherical, their aerodynamic size can be small despite being geometrically large. this happens when particles/droplets have low density, which is determined by the composition of the oid formulation. oid deposition pattern is currently evaluated in in vitro, cell-free experiments, achieving good predictive value [ ] . once the oid deposits on the airways, removal mechanisms, such as mucociliary clearance in the conducting airways and macrophage clearance in the alveolar space, can be responsible for the drug elimination and/or degradation [ ] , thus hindering the local efficacy and/or the systemic absorption of the oid. mucociliary clearance is the upward movement of mucus driven by beating cilia towards the pharynx, where mucus is subsequently swallowed and pass into the gastrointestinal tract [ ] . in macrophage clearance, the oid is phagocytosed by alveolar macrophages and cleared by transport to the lung-draining lymph nodes [ , ] . compared with mucociliary clearance, macrophage clearance is far slower [ ] and, therefore, its action is typically assumed to be negligible for oids, unless the drug is known to be degraded by alveolar macrophages [ ] . absorptive drug clearance is yet another clearance mechanism by which an oid is cleared from the lung through the blood circulation, a mechanism that is heavily dependent on perfusion. perfusion levels, however, vary between the different lung regions. in the alveoli, perfusion levels are the highest and drugs have a very short half-life; by contrast, in the tracheobronchial region, the perfusion rate is lower, thus offering a longer drug bioavailability [ ] . removal mechanisms constitute the third feature to keep into account for developing an effective inhalation therapy. as described in detail in section . . , this feature is species-specific [ ] and, therefore, human-specific removal mechanisms are not replicated by animal models. notably, human-specific removal mechanisms can be reproduced by in vitro, cell-based nams [ ] [ ] [ ] [ ] , as discussed in detail in section . . to exert local or systemic efficacy, oid dissolution and absorption are indeed necessary [ ] . the thickness and constitution of the pulmonary lining fluid, which can be modified by lung diseased states [ ] , influence oid dissolution and, subsequently, absorption [ ] , constituting the fourth feature to keep into account for developing an effective inhalation therapy. while the mucus layer (produced by goblet cells in the bronchial region) acts as a physical barrier, surfactants produced by alveolar cells in the peripheral airways reduce surface tension and facilitate drug dissolution [ ] . noteworthy, oid dissolution rates strongly depend on disease-specific airway characteristics (e.g., copd is characterized by a thick mucus, hindering oid efficacy), which are not replicated by conventional preclinical models. noteworthy, in vitro, cell-based nams have the potential to reproduce the disease-specific composition of pulmonary lining fluid [ ] . finally, the multicellular composition of the lung is the fifth feature to keep into account for developing an effective inhalation therapy, by playing an important role in defining oid delivery efficiency. for example, mast cells have protective functions against inhaled drugs; dendritic cells, together with macrophages, are the first line of defense of the lung immune system, sampling for and removing constantly any exogenous material such as drugs. clara cells are involved in oid metabolism. interestingly, the human lung has relatively low metabolic activity as compared to the gastro-intestinal tract or the liver [ , ] . this constitutes a distinct advantage for inhalation therapy over oral drug administration. however, protease activity is generally increased in lung diseases as a result of chronic inflammation (e.g., enhanced activity of cytochrome p in patients affected by lung cancer [ , ] or copd [ ] ); this can indeed reduce the biopersistence and bioavailability of some oids (e.g., insulin [ ] ). protection against metabolic activity has been achieved in inhalation therapy by drug encapsulation into carriers (e.g., liposomes [ ] [ ] [ ] [ ] [ ] ). animal models and humans differ in the metabolism and distribution/types of cell populations lining the airways. for example, it has been shown that the average number of cells per alveolus for rats versus humans is: vs. , for endothelial cells, vs. for interstitial cells, vs. for epithelial type ii cells, vs. for epithelial type i cells, and . vs. for alveolar macrophages [ ] . this has important clinical implications during the oid development. notably, the human-specific composition and metabolism of the lung can indeed be replicated more closely by adopting in vitro, cell-based nams, as described in the following sections. based on the multiple mechanisms and processes described above, it is evident that oid development is not an easy task. overall, a sound understanding of the features involved in the oid journey is necessary to use the most predictive preclinical models to overcome the complex, intrinsic challenges associated with inhalation therapy. interestingly, such challenges have certainly not hindered the interest of the pharmaceutical industry in inhalation therapy. based on a search carried out by the authors in july , inhalation clinical trials for new, combination, and existing products, encompassing drug interventions, different conditions and rare diseases, have been logged on clinicaltrials.gov in the last four years (search terms: interventional studies; inhalation; start date from / / to / / ). to put this into context, a total of , interventional studies, comprising drug interventions, have been registered on clinicaltrials.gov in the same time period. consequently, inhalation clinical trials make for the . % of the total number of interventional studies registered in the time period under consideration ( - ), and . % of the total drug interventions examined. it is important to observe that more than half of these inhalation studies are for systemic conditions, thus demonstrating an interest that expands beyond the domain of respiratory diseases. preclinical studies of new oid candidates generally start from compound profiling in high-throughput in vitro studies [ ] . compounds with promising efficacy results progress to in vivo studies. three preclinical animal-based studies are currently required by regulatory authorities before approving the request of clinical study for a novel oid. these are: (i) the range finding study, (ii) the repeat dose study, and (iii) the carcinogenicity study. other specialized studies can be necessary, such as safety pharmacology studies, reproductive studies, and neonatal/juvenile studies for pediatric oids. animal-based inhalation studies are carried out mainly in rats, mice or rabbits by exposure in restraint tubes [ ] . dogs and primates can also be used for testing oids in more realistic settings, via facemasks or helmets [ ] . although high-throughput cell-based assays can provide insightful information at the early stages of preclinical development, the cell models used fall short in recapitulating the complex interactions between different cell types and tissues/organs occurring in human. conventional in vitro models are in fact formed by one cell type grown as a flat, two-dimensional culture; thus, they are a simplistic representation of the human lung tissue [ ] . furthermore, many in vitro assays use transformed cell lines that exhibit gene and protein expression that strongly differ from their primary counterpart [ ] . on the other hand, various uncertainties characterize the animal-based preclinical studies currently required for regulatory purposes. the first level of uncertainty is associated with the type of devices used to administer the oid to the animal. while clinical nebulizers can be used in the preclinical environment (upon small modifications), dpis and pmdis cannot be employed to expose animal models at the preclinical screening level, as these devices are breath actuated. to overcome this issue, specialized equipment is used to expose the animal to an aerosol in a restrained environment. aerosol of powders is achieved via, for example, rotating brush generators or wright dust feed. an algorithm-based extrapolation [ ] is then applied to define dose ranges to be used in clinical trials. the delivered dose is calculated as the amount of oid per unit of body weight that is presented to the animal. due to the two parameters (velocity and aerodynamic size distribution) affecting oid deposition patterns in the lungs, as discussed in the section above, and to the species of the animal model used, the deposited dose is only a fraction of the delivered dose. the fda assumes % deposition in humans, % in rats and % in dogs or non-human primates, irrespective of any information that has been produced by the submitting company [ ] . this indeed generates uncertainties when calculating clinical overages. the second level of uncertainty in in vivo studies is posed by the animal model itself [ ] . for example, rodents are obligate nose breathers; this strongly influences how inhaled compounds deposit in the respiratory tract. this and other interspecies differences have been extensively discussed by the authors in a recent perspective [ ] . preclinical studies during oid development requires a clear understanding of such interspecies differences and their impact on the screening outcomes in terms of oid efficacy, toxicity and recovery from adverse effects. although not required at regulatory level, disease animal models are also used in preclinical research, particularly in the oncological field, as proof of concept for demonstrating oid efficacy. the authors have performed a literature search on pubmed using the searching terms "(inhaled drug) and (in vivo) and (efficacy)". the search results showed that, in the last five years, articles used disease animal models to test the efficacy of oids. however, animal use as disease models needs to be viewed cautiously. in animal models, disease features are reproduced by applying exogeneous stimuli (e.g., allergens, irritant gas exposures, cigarette smoke, etc.) [ ] . this modelling process is however incomplete, as the use of single stimuli does not mimic the disease etiology and chronicity observed in patients. the next section of this commentary focuses on this specific aspect, complementing the authors' previous publication [ ] and further discussing if and how new approach methodologies (nams) could become useful in the attempt to overcome the limitations of current animal models and increase oid translation rate. for completeness, it should be mentioned here that the abbreviation "nams" is often used in toxicology to refer broadly to any non-animal technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment. examples of nams include non-mammalian model systems, (e.g., caenorhabditis elegans [ ] [ ] [ ] , drosophila melanogaster [ ] [ ] [ ] , zebrafish [ ] [ ] [ ] and dictyostelium [ ] ) and computational (in silico) approaches [ ] , which indeed offer opportunities for mimicking human respiratory diseases in a predictive manner. however, the scope of the nams considered in our commentary includes only in vitro, non-animal cell models for the testing of oids. based on the most recent advances in tissue-engineering technologies, in vitro cell-based nams for screening the efficacy of oids can be classified in three main categories [ ] : (i) tissue-mimetic lung cultures grown at the air-liquid interface (ali); (ii) lung organoids; and (iii) lung-on-chip. ali cultures mimic one of the main properties of the lung epithelium, i.e., the direct contact with the gas phase (air). this provides a tissue-mimetic environment that makes it possible for airway epithelial cells to proliferate and differentiate in vitro into a pseudostratified, ciliated epithelium that produces mucus. thus, ali cultures provide an excellent method for testing oid dissolution and absorption, while enabling testing of the drug in its aerosol form. whitcutt et al., were among the first research groups to report mucociliary differentiation in ali cultures [ ] . today, ali cultures are known to be particularly useful in understanding the mechanisms of respiratory diseases, including the cell-cell and cell-extracellular matrix interactions during airways remodeling [ ] [ ] [ ] . also, they can replicate some of the key features that need to be kept into account when developing an inhalation therapy, namely (i) the constitution and thickness of the pulmonary lining fluid [ ] and (ii) mucociliary clearance [ ] [ ] [ ] . for example, ali cultures have been used to model the effects of smoke exposure on epithelial cells [ ] and the authors have created a complex, diseased ali culture model capable of reproducing the chemoresistance mechanisms observed in patients affected by non-small-cell lung cancer [ , ] . also, culturing human airway epithelial cells isolated from patients, makes it possible to conduct patient-specific research and drug-screening, for example in cystic fibrosis, asthma and copd [ ] [ ] [ ] [ ] . with the aim of further increasing the predictive value of this in vitro nam, ali co-cultures have also been developed. in ali co-cultures, the lung cell populations are mixed or partially separated, depending on the experimental set-up. in general, the immune cells are cultured in direct contact with the epithelial cells; whereas, fibroblasts and endothelial cells are separated from the epithelial cells by the transwell permeable membrane. cell separation is due to the relative difference in the culturing conditions of the various cell types and the consequent need to separate them. this constitutes one of the main limitations of ali models, as separated cells cannot establish physical (cell-to-cell) interactions as per in vivo conditions. this indeed affects the detected responses during oid preclinical testing. the second type of in vitro, cell-based nams currently available for oid testing are lung organoids. these are grown from human induced pluripotent stem cells (ipscs) cultured within a natural or synthetic extracellular matrix to form three-dimensional ( d), hollow cell spheroids of basal, ciliated and secretory cells [ ] . through differentiation and self-organization of the ipscs, an in vitro culture with lung tissue-specific morphogenetic and histological properties is formed [ ] . to date, several organoids representative of the various human lung regions [ ] and assessing a variety of pulmonary diseases [ , , ] have been developed. in the context of oid preclinical testing, lung organoids can be used for modeling respiratory diseases and, therefore, as a platform for screening the efficacy of inhalation therapies [ , ] . indeed, technical limitations are inherent with the use of lung organoids. lungs are in fact subjected to mechanical deformation during breathing cycles, a deformation that is currently hard to model in organoids. furthermore, there is still a lack of established in vitro lung organoids with a functional representation of the vasculature network. most importantly, lung organoids lack an important feature for oid testing, i.e., the direct contact of epithelial cells with the air. as mentioned above, lung organoids are spherical cultures. they present an interiorized lumen, with epithelial cells facing inwards rather than outwards; this makes drug administration extremely difficult and reduces the application of organoids in the screening of oid absorption. microfluidic technologies allow to add further complexity and functionality to the in vitro ali models described above. the so-called "lung-on-chip" is a microfluidic-based in vitro system in which lung epithelial cells are grown on one side of a membrane, and stromal cells on the other surface. liquid and air are circulated through the system to mimic air and blood flow in the lung. the applications of lung-on-chip range from basic research to drug discovery [ ] , where the oid can be introduced in the air flow as per in vivo conditions. probably the most famous example of this in vitro, cell-based nam is the breathing lung-on-chip developed by huh and co-workers at the wyss institute of harvard university (usa), capable of reproducing both the physiological and pathological responses of the human lung, a rudimentary circulatory system and the mechanical stress associated with breathing [ ] [ ] [ ] . the immediate application of lung-on-chip has been for toxicity testing [ , ] ; more recently, this model has been exploited for improving understanding of the complex lung disease processes and their responses to therapeutics [ ] [ ] [ ] , with applications extending even to the most recent need of a fast drug discovery for covid- treatment [ ] . lung-on-chip systems allow, in fact, the in vitro creation of highly tissue-mimetic lung disease models [ , ] , thus allowing, for example, to model the human response and the effects of existing and novel therapeutics when the lung is infected by the influenza virus or by viral pseudoparticles expressing spike protein of sars-cov- , the virus responsible for covid- development [ ] . the clear advantage of lung-on-chip systems over ali cultures or lung organoids is the possibility of mimicking the pulmonary mechanical stretch during in-and exhalation, while replicating the air-blood barrier for studying oid absorption. furthermore, lung-on-chip models allow evaluating the impact of the mucociliary clearance mechanism overcoming the lack of directionality in cilia beating function characteristic of fully-differentiated in vitro ali models [ ] . nevertheless, the lung-on-chip models share some of the limitations of ali cultures, i.e., the impairment of physical crosstalk among different cell types. in fact, even in the most recent and advanced developments in "tumor-on-a-chip" cell culture technology, successfully used to create in vitro human orthotopic models of non-small-cell lung cancer [ ] , the lung cancer cells (cultured under ali conditions) are physically separated from the lung endothelial cells by a porous, permeable membrane [ ] . it is noteworthy to mention that, in the respiratory disease field, two additional categories of in vitro, cell-based nams exists, although these have not been used for oid testing to date. the first category is constituted by explant or ex vivo cultures, namely isolated perfused lungs and precision cut lung slices. these are better representations of the in vivo situation than any of the previous three nam types mentioned above. the use of ex vivo cultures in oid testing is however hindered by the hurdles associated with their manipulation, and by donor-specific differences that make the oid screening outcomes often not significant or difficult to interpret [ ] . the second category includes the engineered, reconstructed lung organs [ ] . these are formed from several cell types co-cultured within scaffolds that aim at replicating the composition and architecture of the human lung acellular stroma [ ] . mechanical or biochemical stimuli can be added to tailor the properties of the scaffold and increase the similarity to the lung stroma in vivo. the first engineered lung organ was built from a decellularized lung matrix used as scaffold [ ] . more recently, d bioprinting techniques have been used to produce the lung organs in vitro. for d bioprinting, cells are combined with bioactive hydrogels composed of synthetic (e.g., polyethylene glycol, pluronic) or natural (collagen, chitosan, fibrin, gelatin, matrigel, alginate) polymers [ ] . the use of reconstructed lung organs in oid preclinical screening is currently hampered by the low throughput of these methods. to summarize, in this commentary we have presented an overview of the in vitro, cell-based nam systems that, to date, have been successfully employed to fill the technological gap that is believed to hindering the effective oid translation from the lab bench to the clinic. in the past, oid failure at clinical trial stage was mainly due to poor pharmacokinetics and bioavailability. today, these are rarely a cause of failure, as the pharmaceutical industry greatly invested in the development and application of much more accurate prediction and modelling approaches. lack of efficacy is now the most common cause of oid attrition [ ] ; this appears to be associated to the fact that preclinical animal models are poorly representative of human respiratory diseases [ ] . improved in vitro non-animal methods could provide a more human-relevant predictive value so that compounds would fail earlier in their course of development [ ] . furthermore, we have provided a brief overview of those in vitro, cell-based nams that, in the future, we believe they could be adapted towards oid testing. although in vitro, cell-based nams still have limitations, the advantages associated with their use is evident and future efforts should aim at validating these systems for regulatory acceptance [ ] . in the development of oids, we should therefore invest in moving away from animal studies. in the last decades, significant funding and precious time have been spent on developing animal models, despite the known species differences that make the results obtained from such models often unreliable when translated to humans. as dr. francois busquet and colleagues from the center for alternatives to animal testing-europe state for covid- , human-relevant approaches offer crucial advantages of speed and "much more robust and exacting data than any animal experiment could deliver" [ ] . in this instance, we believe it is important to highlight that directive / /eu on the protection of animals used for scientific purposes aims non only at reducing but at the "full replacement of procedures on live animals for scientific and educational purposes, as soon as it is scientifically possible to do so" [ ] . consistently with this aim, in the netherlands has been the first eu member state to present a roadmap for phasing out animal testing in the safety research on chemical substances, food ingredients, pesticides and medicines (including veterinary medicines) [ ] . the recent advances in tissue engineering, microfluidic and organ-on-chip technologies are providing researchers with tools for the development of human-relevant, in vitro nams. thus, it is essential now that the respiratory disease research community embraces these tools, bringing them forward towards regulatory validation. chronic obstructive pulmonary disease (copd) global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review world cancer report-cancer research for cancer prevention who. coronavirus disease (covid- ) situation reports. available online global respiratory drugs market to -a changing therapeutic landscape as key patents expire and biologics, targeted therapies and cftr modulators for asthma and cystic fibrosis treatment emerge as market growth drivers global respiratory drugs market barriers to new drug development in respiratory disease the r&d cost of a new medicine inhaled therapy in respiratory disease: the complex interplay of pulmonary kinetic processes heuze-vourc'h, n. in a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration heuze-vourc'h, n. inhalation of immuno-therapeutics/-prophylactics to fight respiratory tract infections: an appropriate drug at the right place! front pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications will pulmonary drug delivery for systemic application ever fulfill its rich promise? recent advances in aerosolised drug delivery years of drug delivery to the lungs understanding dry powder inhalers: key technical and patient preference attributes delivery technologies for orally inhaled products: an update inhalation devices, delivery systems, and patient technique inhalation devices: from basic science to practical use, innovative vs generic products optimizing drug delivery in copd: the role of inhaler devices inhalation therapy devices for the treatment of obstructive lung diseases: the history of inhalers towards the ideal inhaler drug delivery devices: issues in drug development developing ways to evaluate in the laboratory how inhalation devices will be used by patients and care-givers: the need for clinically appropriate testing metered dose inhaler (mdi) and dry powder inhaler (dpi) products-quality considerations guidance for industry inhaled formulation and device selection: bridging the gap between preclinical species and first-in-human studies in vitro testing for orally inhaled products: developments in science-based regulatory approaches pulmonary drug delivery. part ii: the role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications in vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals validation of a general in vitro approach for prediction of total lung deposition in healthy adults for pharmaceutical inhalation products biological obstacles for identifying in vitro-in vivo correlations of orally inhaled formulations patient education and adherence to aerosol therapy trying, but failing" -the role of inhaler technique and mode of delivery in respiratory medication adherence the role of inhalation delivery devices in copd: perspectives of patients and health care providers matching inhaler devices with patients: the role of the primary care physician organoids as a model system for studying human lung development and disease device use errors with soft mist inhalers: a global systematic literature review and meta-analysis irregular and ineffective: a quantitative observational study of the time and technique of inhaler use problems with inhaler use: a call for improved clinician and patient education inhalers: to switch or not to switch? that is the question inhaler competence in asthma: common errors, barriers to use and recommended solutions particle transport and deposition: basic physics of particle kinetics pulmonary drug delivery: from generating aerosols to overcoming biological barriers-therapeutic possibilities and technological challenges inhaling medicines: delivering drugs to the body through the lungs guideline on the pharmaceutical quality of inhalation and nasal products drug delivery to the small airways the impact of pulmonary diseases on the fate of inhaled medicines-a review models of deposition, pharmacokinetics, and intersubject variability in respiratory drug delivery overcoming lung clearance mechanisms for controlled release drug delivery mucociliary clearance in the airways challenges for inhaled drug discovery and development: induced alveolar macrophage responses alveolar macrophages transport pathogens to lung draining lymph nodes report no -deposition, retention and dosimetry of inhaled radioactive substances pharmacometric models for characterizing the pharmacokinetics of orally inhaled drugs systems pharmacology approach for prediction of pulmonary and systemic pharmacokinetics and receptor occupancy of inhaled drugs in vitro alternatives to acute inhalation toxicity studies in animal models-a perspective air liquid interface culture can alter ciliary beat pattern in epithelium from primary ciliary dyskinesia patients characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling responses of well-differentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to burkholderia cenocepacia infection mucociliary defense: emerging cellular, molecular, and animal models pulmonary drug metabolism, clearance, and absorption pulmonary surfactants and their role in pathophysiology of lung disorders measurements of deposition, lung surface area and lung fluid for simulation of inhaled compounds human cellular models for the investigation of lung inflammation and mucus production in cystic fibrosis expression and localization of cyp a and cyp a in human lung expression and regulation of xenobiotic-metabolizing cytochrome p (cyp) enzymes in human lung smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome p ia in lung cancer patients cytochrome p -mediated pulmonary metabolism of carcinogens: regulation and cross-talk in lung carcinogenesis expression of cytochrome p mrnas in type ii alveolar cells from subjects with chronic obstructive pulmonary disease proteolytic enzymes as a limitation for pulmonary absorption of insulin: in vitro and in vivo investigations development of liposomal ciprofloxacin to treat lung infections liposomal formulations for inhalation the rationale and evidence for use of inhaled antibiotics to control pseudomonas aeruginosa infection in non-cystic fibrosis bronchiectasis liposomes for pulmonary drug delivery: the role of formulation and inhalation device design amikacin liposome inhalation suspension for treatment-refractory lung disease caused by mycobacterium avium complex (convert). a prospective, open-label, randomized study lower respiratory-tract structure of laboratory-animals and humans-dosimetry implications in vitro cell culture models for evaluating controlled release pulmonary drug delivery in vivo animal models for controlled-release pulmonary drug delivery preclinical models for pulmonary drug delivery reconstituted d cell and tissue models association of inhalation toxicologists (ait) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals toxicologic testing of inhaled pharmaceutical aerosols species comparison of drug absorption from the lung after aerosol inhalation or intratracheal injection translational models of lung disease modeling molecular and cellular aspects of human disease using the nematode caenorhabditis elegans modeling human diseases in caenorhabditis elegans elegans as a model organism for in vivo screening in cancer: effects of human c-met in lung cancer affect c. elegans vulva phenotypes a drosophila model of cigarette smoke induced copd identifies nrf signaling as an expedient target for intervention drosophila in asthma research a drosophila asthma model-what the fly tells us about inflammatory diseases of the lung zebrafish: model for the study of inflammation and the innate immune response to infectious diseases using in vivo zebrafish models to understand the biochemical basis of neutrophilic respiratory disease modeling inflammation in the zebrafish: how a fish can help us understand lung disease what can dictyostelium bring to the study of pseudomonas infections? semin heuze-vourc'h, n. innovative preclinical models for pulmonary drug delivery research options for modeling the respiratory system: inserts, scaffolds and microfluidic chips a biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells adapting the electrospinning process to provide three unique environments for a tri-layered in vitro model of the airway wall use of porous membranes in tissue barrier and co-culture models a novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture ali multilayered co-cultures mimic biochemical mechanisms of the cancer cell-fibroblast cross-talk involved in nsclc multidrug resistance multilayered cultures of nsclc cells grown at the air-liquid interface allow the efficacy testing of inhaled anti-cancer drugs antibacterial defense of human airway epithelial cells from chronic obstructive pulmonary disease patients induced by acute exposure to nontypeable haemophilus influenzae: modulation by cigarette smoke altered generation of ciliated cells in chronic obstructive pulmonary disease primary epithelial cell models for cystic fibrosis research asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus regeneration of the lung: lung stem cells and the development of lung mimicking devices in vitro generation of human pluripotent stem cell derived lung organoids a three-dimensional model of human lung development and disease from pluripotent stem cells modelling cryptosporidium infection in human small intestinal and lung organoids use of three-dimensional organoids and lung-on-a-chip methods to study lung development, regeneration and disease organoids as a powerful model for respiratory diseases lung-on-a-chip technologies for disease modeling and drug development reconstituting organ-level lung functions on a chip a human breathing lung-on-a-chip a human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice a lung/liver-on-a-chip platform for acute and chronic toxicity studies a d human lung-on-a-chip model for nanotoxicity testing multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development microphysiological lung models to evaluate the safety of new pharmaceutical modalities: a biopharmaceutical perspective impaired wound healing of alveolar lung epithelial cells in a breathing lung-on-a-chip human organs-on-chips as tools for repurposing approved drugs as potential influenza and covid therapeutics in viral pandemics biomimetic human lung-on-a-chip for modeling disease investigation small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro human organ chip models recapitulate orthotopic lung cancer growth, therapeutic responses, and tumor dormancy in vitro microengineered cancer-on-a-chip platforms to study the metastatic microenvironment bridging the gap between science and clinical efficacy: physiology, imaging, and modeling of aerosols in the lung modeling the lung: design and development of tissue engineered macro-and micro-physiologic lung models for research use tissue-informed engineering strategies for modeling human pulmonary diseases three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration d in vitro/ex vivo systems animal models of asthma: value, limitations and opportunities for alternative approaches human tissue models for a human disease: what are the barriers? thorax harnessing the power of novel animal-free test methods for the development of covid- drugs and vaccines on the protection of animals used for scientific purposes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank moreno carrer for the technical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -qy lccjq authors: mubagwa, kanigula title: chloroquine cardiac effects and toxicity.a short update. date: - - journal: int j antimicrob agents doi: . /j.ijantimicag. . sha: doc_id: cord_uid: qy lccjq there is currently an increased interest in using the antimalarials chloroquine and hydroxychloroquine for the treatment of other diseases, including cancer and viral infections such as covid- . however, risks of cardiotoxic effects tend to limit their use. the effects of these drugs on the electrical and mechanical activities of the heart, as well as on the remodeling of the cardiac tissue are presented, and the underlying molecular and cellular mechanisms discussed. the drugs can have proarrhythmic as well as antiarrhythmic actions, resulting from their inhibition of ion channels, including voltage-dependent na(+) and ca( +) channels, background and voltage-dependent k(+) channels, and pacemaker channels. the drugs also exert a vagolytic effect, due at least in part to a muscarinic receptor antagonist action. they also interfere with the normal autophagy flux, an effect which could aggravate ischemia/reperfusion injury or post-infarct remodeling. most of the toxic effects occur at high concentrations, following prolonged drug administration or in the context of drug associations. -the antimalarials chloroquine and hydroxychloroquine have been proposed for antiviral therapy, including for covid- . -their use is limited for fear of cardiotoxic effects. -the mechanisms underlying cardiac chloroquine effects include direct actions on ion channels and receptors, while others involve an inhibition of autophagy. -despite the effects on many ion channels, the drugs are not associated with prominent qt prolongation or life-threatening arrhythmias when used for a short duration and at low concentrations. -side effects are usually associated with long-term treatments or with very large doses. -the drugs are relatively safe, but care should be taken to avoid association with other drugs with known toxic effects on the myocardium. chloroquine (and its related drug hydroxychloroquine), introduced more than years ago and used traditionally to treat malaria or chronic inflammatory diseases, has re-emerged as possible therapeutic agent in viral diseases including covid- [ ] . renewed interest in this drug is not novel, as there have also been assessments of its potential effects as anti-cancer [ , ] , anti-arrhythmic [ ] or pulmonary vasodilator [ ] agent. whenever considering to (re)use a drug, one should weigh the benefits against the risks (mainly the side effects) associated with the drug use. the usefulness of chloroquine/hydroxychloroquine in the treatment of covid- is still being currently debated [ , ] . whereas on the one hand there is little doubt that chloroquine and hydroxychloroquine can display antiviral action in vitro [ , [ ] [ ] [ ] and in vivo [ ] , on the other hand controversy exists on whether this effect is of use in covid- patients. clinical studies are under way to answer the question concerning an anti-covid- protective action in humans [ ] . while waiting for a clear answer to this issue, it is necessary to reflect on risks of using the drug. the effects of chloroquine/hydroxychloroquine, especially those related to the immunomodulatory action have been reviewed very recently [ ] . among side effects of chloroquine/hydroxychloroquine, those involving the heart are frequently invoked as being life-threatening, and cardiotoxicity is a major concern for many other antimalarial drugs [ , ] . cardiotoxic effects have been presented as arguments against recent trials to repurpose these drugs for the treatment of covid- [ ] . in the present note, we describe the major known cardiac effects of the drugs, with the aim to determine which effects can be expected to occur at usual therapeutic concentrations and account for observed beneficial or side effects. in the presentation we do not make a distinction between the effects of chloroquine versus those of hydroxychloroquine, the underlying assumption being that, despite potential pharmacokinetic and potency differences, their pharmacological actions are essentially identical. we suggest ) that major side effects occur at high concentrations (e.g. following poisoning), after long-term treatment or in the context of drug associations, and ) that common effects after short-term treatment with low clinical doses are generally well tolerated. we also comment on the cellular or molecular mechanisms underlying the effects. chloroquine/hydroxychloroquine can be administered via parenteral or oral routes. acute toxic effects were recorded more frequently when the drugs were given via the parenteral (especially the intravenous) route [ ] . drug absorption is practically complete even when administered via the oral route, but the concentration profile reaches lower peak levels post enteral than post parenteral administration [ ] . the drugs accumulate into cells, due to ion trapping in more acidic environments, especially in lysosomes [ , ] , where the concentration can be many orders of magnitude higher than in the blood. the biological half-life is as long as - days [ , ] . hence there is possibility for progressive drug accumulation on repeated administration even of small doses, and the drug effects also develop following different time scales: from seconds or minutes, to hours or days, and to many months or years. there are many case reports of rapidly developing cardiovascular effects following intoxication by chloroquine/ hydroxychloroquine, ranging from bradycardia and hypotension to eventual cardiac arrest [ ] . as mentioned, fear or real risks of cardiotoxicity has limited the use of these drugs. cardiotoxic effects associated with chloroquine/hydroxychloroquine use in clinical conditions have been recently reviewed [ , ] . here we summarize major effects detected under either clinical or experimental conditions, and have grouped them into ) purely functional (electrophysiological and mechanical) effects, and ) those associated with structural changes. electrophysiological and mechanical effects effects on the cardiac rhythm and on the conduction and duration of the electrical activity electrocardiographic (ecg) changes associated with chloroquine/hydroxychloroquine treatment have been reported as proarrhythmic in some studies, or as antiarrhythmic in others. rhythm and electrophysiological effects of chloroquine/hydroxychloroquine include bradycardia as well as tachycardia, flattening of the t wave, prolongation of the qt interval, and various forms of conduction blocks. for example, sinus dysfunction with bradycardia [ ] and atrioventricular (av) or intraventricular conduction abnormalities [ ] were noted to be frequent in rheumatoid arthritis and lupus erythematosus patients treated with chloroquine/hydroxychloroquine. in a review of case reports till , conduction disturbances were the most prevalent among all side effects [ ] . however, although arrhythmias in such patients have been traditionally attributed to the drugs, a role of the underlying diseases (as direct cause or as favoring factor) cannot be excluded. indeed, in one study, ventricular conduction blocks observed in chloroquine/hydroxychloroquinetreated patients had a prevalence that was not different from the one in those not receiving these drugs [ ] . given the role played by the duration of ventricular depolarization (measured as qt interval on the ecg) in arrhythmogenesis, it has been of interest to examine whether chloroquine/hydroxychloroquine increase the qt duration. qt prolongations have been noted under clinical conditions with high chloroquine concentrations ( mg base.kg - .day - ; [ ] ) or in experimental conditions using parenteral administration [ ] . the qt prolongation is dominated by the effect on the qrs duration, and marginal dose-dependent prolongation is seen on the j-t interval [ ] , which measures more accurately the duration of depolarization. in most studies no or only marginal qt prolongation is induced by clinical doses (< mg.kg - .day - ) of chloroquine/hydroxychloroquine, especially using the oral route [ , ] , suggesting that chloroquine/hydroxychloroquine are in this respect safer than other quinoline-based antimalarial drugs (e.g. halofantrine [ ] ). clinically, low chloroquine doses ( - mg.kg - .day - ) were associated with significant qt prolongation when given over many months [ ] , suggesting a role of drug accumulation or of myocardial remodeling. in contrast to the pro-arrhythmic actions of chloroquine/ hydroxychloroquine, an antiarrhythmic action, known since the middle of the past century [ , ] , can be further demonstrated from experimental animal data [ ] and from clinical data of patients treated for chronic inflammation [ ] . recently, it could be shown that chloroquine ( - mg.day - ) can cause conversion to sinus rhythm in a patient with established atrial fibrillation [ ] . such an antiarrhythmic action is not unexpected given the drug action on voltage-dependent na + channels (see below). it should be noted that the "janus-faced" (pro-and anti-) arrhythmic action is also known for classic antiarrhythmics [ ] . the effect of chloroquine/ hydroxychloroquine on cardiac contraction remains unclear. early studies in amphibian hearts suggested that chloroquine exerts a positive inotropic effect, attributed to an indirect action due to the activation of brain sympathetic centers [ ] . similarly, a transient positive inotropic action was obtained in guinea-pig atria [ ] . it was suppressed by pretreatment with reserpine or propranolol, indicating that it was due to a release of catecholamines, which then acted on cardiomyocyte beta-adrenergic receptors [ ] . most subsequent studies in mammalian hearts either failed to show any inotropic action or demonstrated a negative action. no change of cardiac mechanical function assessed using echocardiography was observed in rats injected with mg.kg - .day - chloroquine for weeks [ ] . in contrast, negative inotropism was evident in atrial muscles, but only at concentrations above µm [ , ] . similarly, in guinea-pig cardiac muscle chloroquine decreased the contraction at - µm in isolated atria [ ] . in rat isolated left ventricular papillary muscles, chloroquine ( µm) was found either to have no effect [ ] or to decrease contractile force and the rates of shortening and relaxation [ ] . in langendorff-perfused guinea-pig ventricles chloroquine decreased the contraction at concentrations above µm [ ] , but this apparently higher potency in ventricular muscle could be an artifact due to a time-dependent rundown of the contraction in this preparation. in rat isolated perfused hearts, systolic function was decreased with chloroquine concentrations > µm and was completely suppressed at µm [ ] . this negative inotropic action was associated with a decrease in heart rate. unless heart rate is controlled it is difficult to accurately determine the inotropic effect since part of the decrease in contraction may be related to a positive force-frequency relation prevailing in the preparation. in addition, at high chloroquine concentrations loss of excitability and suppressed conduction (due to chloroquine effects on the upstroke of the action potential) might account for an apparent loss of contraction. thus, negative inotropic of chloroquine/hydroxychloroquine is typically obtained at high drug concentrations, above those reached by usual therapeutic regimens. the mechanism underlying the reduction of contraction could be related to the inhibition of i ca-l discussed below. it has been noted that the effects of chloroquine ( . mg. ml - , i.e. about µm) to reduce contractility in turtle hearts is similar to those of quinine, and that for both drugs, the effect is reversed by raising the external ca + concentration [ ] . this points to a role of decreased excitability and/or of decreased ca + influx, and makes less likely an effect on myofilament ca + sensitivity. however, in guinea-pig hearts [ ] , increasing external ca + was not able to fully reverse the chloroquine-induced contraction decrease. contractions obtained while inactivating na + channels with high external k + were as sensitive to chloroquine as those recorded in normal conditions, whereas post-rest potentiation of contraction was not affected by the drug. these data point to a role of diminished ca + influx in the negative inotropism of chloroquine. cardiovascular shock is common following chloroquine poisoning [ ] , but it is not clear whether the condition is mainly due to vascular or to cardiogenic mechanisms. since cardiogenic mechanisms can be primarily arrhythmic and not necessarily primarily inotropic, the contribution of decreased inotropy is unclear. it is of note that, beside studies of the mechanical dysfunction related to chloroquine/hydroxychloroquine cardiomyopathy, there are no studies that report on eventual acute inotropic effects in human patients or volunteers. a dose-dependent decrease of systolic pressure can also be demonstrated in experimental animals intoxicated with intravenous chloroquine [ ] . the hemodynamic failure was associated with decrease of maximum rates of pressure development or relaxation, bradycardia and qrs widening. this suggests the participation of an important non-vascular, cardiac component. chronic treatment (usually over many months or years) with chloroquine/ hydroxychloroquine can result in cardiomyopathy [ ] , characterized by wall thickening and microscopic structural changes. cardiomyopathy seems to occur more with chloroquine than with hydroxychloroquine, and involves at the microscopic level an enlargement and vacuolization of cardiac myocytes, with deposition of myeloid and curvilinear bodies. the cardiomyopathy is accompanied by functional changes, including av or bundle-branch conduction block [ ] as well as mechanical dysfunction, with diffusive hypokinesia and decreased ejection fraction [ ] . chloroquine cardiomyopathy can develop in the absence of skeletal myopathy or of retinal toxicity, although similar cellular mechanisms might be involved. in the context of short-term treatment, these effects are not expected. other structural changes occurring in the myocardium are not primarily due to chloroquine/hydroxychloroquine but are modified by these drugs. myocardial remodeling involves structural and functional changes triggered by many stress factors, including physiological conditions such as physical exercise, pregnancy or fetal body growth, as well as pathological conditions such as ischemia/reperfusion (infarction), pressure or volume overload, inflammation, metabolic factors (e.g., hyperglycemia and hyperlipidemia), reactive oxygen radicals [ ] . physiological remodeling is adaptive and lead to hypertrophy to increase function in athletes, pregnant women and growing fetus. in contrast, remodeling following pathological triggers is usually maladaptive, as it evolves with time towards heart failure and increased propensity to arrhythmias. characteristic structural changes during pathological remodeling include increase in cardiomyocyte size (causing hypertrophy or dilatation) and ultramicroscopic structure (e.g. loss of t-tubules; [ ] ), modifications of the extracellular matrix, proliferation of myofibroblasts with development of fibrosis. after myocardial infarction, concomitantly with the development of fibrosis and formation of a scar in the infarcted zone there are changes in the infarct border and in the remote non-infarcted myocardium. with time the heart undergoes chamber dilatation. functional changes in remodeled myocardium include modifications in the electrical properties (called electrical remodeling), as a result of altered expression of ion channels (e.g., kir k + channels, l-type ca + channels, connexins, ryanodine receptors, etc.), transporters (e.g., na + -ca + -and exchangers, sarcoplasmic reticulum atpases, etc.) and other proteins, which impact on the action potential and its conduction within the cardiac tissue, on the excitation-contraction coupling, and on the contractile properties. electrical remodeling is responsible for the increased propensity to arrhythmias. many weeks following myocardial infarction, there is progressive deterioration of myocardial function with increased left ventricular end-diastolic volume, diastolic dysfunction and decreased systolic function. many processes and cell types are implicated in the remodeling process [ , ] . inflammation, with the associated release of mediators, cytokines and chemokines by inflammatory cells, play an important role in the remodeling process, and an experimental model of inflammation-induced remodeling, resulting in dilated cardiomyopathy, is obtained in mice overexpressing tumor necrosis factor, tnf [ ] . the effect of chloroquine on remodeling does not necessarily involve a direct action on cardiomyocytes, and may involve primary effects on other cardiac cells such as fibroblasts. during post-infarct remodeling cardiac fibroblasts are activated and converted to myofibroblasts, which display a higher proliferative and secretory capacity. in the fibroblasts chloroquine significantly reduced the expression of proteins such alpha smooth muscle actinin (α-sma) and fibronectin. in myofibroblasts chloroquine reduced cell migration and contractility [ ] . chloroquine/hydroxychloroquine may also affect the electrical remodeling. as far as ion channel remodeling is concerned, reduced amplitude and slightly modified kinetics of voltage-dependent sodium current were evident in epicardial cells of the infarct border zone, and these changes were supposed to play a role in decreased conduction underlying reentrant arrhythmias [ ] . chloroquine ( µm), in addition to displaying its inhibitory action on the sodium current, was shown to completely suppress the camp-dependent kinase (pka)-mediated restauration of the ion current in the cells from the infarct border zone [ ] . remodeling in infarcted hearts can be reduced by calorie restriction or pharmacologic agents such as resveratrol or rapamycin, which probably act by promoting autophagy [ ] . chloroquine suppressed the anti-remodeling effects of food restriction and of resveratrol. this was shown in mice that developed ventricular dilation and dysfunction a few days/weeks after induction of myocardial infarction by left coronary artery ligation. restriction by - % of the food intake, started week after infarction and maintained for weeks, was accompanied by an improvement of the cardiac chamber size and of most contractile parameters [ ] . in addition the increase in cell size was also attenuated. treatment with chloroquine ( mg/kg - .day - , given by continuous subcutaneous injection) for weeks reversed the improvement afforded by food restriction. like the effect of food restriction, a two-week treatment with resveratrol significantly attenuated the ventricular dilation and improved cardiac function in mice that underwent left coronary artery ligation. this protective effect was absent in mice treated with chloroquine ( mg.kg - .day - ) simultaneously with resveratrol [ ] . as discussed below, these effects of chloroquine are apparently due to a suppression of observed increases in autophagy by food restriction or drugs in the cells. while chloroquine appears to have deleterious effect in the remodeling induced by primary cardiac lesions, the drug may have a protective action on remodeling of the right heart associated with pulmonary arterial hypertension. rats in which this condition was induced (by monocrotaline) and which were intraperitoneally injected with either chloroquine ( or mg.kg - .day - ) or hydroxychloroquine ( mg.kg - .day - ) for weeks displayed less right ventricular hypertrophy and improved function due to a decrease of pulmonary vascular resistance. in this case the remodeling in the pulmonary vasculature was suppressed by chloroquine/hydroxychloroquine [ ] . exploring the cellular and molecular mechanisms of chloroquine/hydroxychloroquine effects allows to understand possible synergy or antagonism with other drugs administered simultaneously or the interaction with changes already induced by pathological conditions. inactivation of voltage-dependent na + channels: local anesthetic effect chloroquine is structurally close to quinine and quinidine, which act on voltage-dependent na + (na v ) channels: they favor an inactivated state of the channels, therefore making them less available for opening upon depolarization (excitation). chloroquine exerts similar "local anesthetic" effects [ ] . given the involvement of na v channels in the genesis and transmission of the electrical impulse in nerves, chloroquine can suppress pain sensation as do classic local anesthetics, but the drug is not used for this effect because of its anticoagulant effect. na v channels are present in other excitable cells, including cardiac atrial contracting cells and all ventricular cell types, where they are responsible for the upstroke (phase ) of the action potential. chloroquine decreases all indices of na v channel contribution to the cardiac action potential: the maximal rate of depolarization during phase and the action potential amplitude [ ] , both of which determine the rate of conduction within the tissue. side effects concerning intraventricular conduction (qrs prolongation, bundle branch block) observed with the drugs [ , ] are likely explained on this basis. a persisting, late na + current is among determinants of the action potential duration and favors arrhythmogenesis [ ] . it contributes to intracellular na + overload and the subsequent na + -ca + exchange-mediated ca + overload during ischemia. suppression of this current by chloroquine [ ] may counterbalance the effect of blocking k + currents on the qt duration, and partly account for antiarrhythmic and anti-ischemic protective actions. decrease of the l-type ca + current chloroquine/hydroxychloroquine inhibit l-type ca + channels, since they decrease the amplitude of i ca-l in sino-atrial (sa) [ ] and in working myocardial cells [ ] . the effect in sa cells likely contributes to the slowing of the pacemaker activity. similar effects are expected in av nodal cells and can explain av conduction block. given the role of i ca-l in cardiac excitation-contraction coupling, negative inotropic action of the drugs [ , ] might also be explained on this basis, although most studies report a lack of significant inotropic effect at clinically useful concentrations. cardiac automatic activity is due to the presence of a spontaneous depolarization of nodal and conduction cells during the diastolic time. many processes contribute to this diastolic depolarization [ , ] , among them the opening of hyperpolarization-activated channels [ ] [ ] [ ] . these channels are called "funny channels" and the corresponding current "funny current, i f ", because of the apparently strange behavior in comparison with other channels, which usually open upon depolarization. i f amplitude (measured during hyperpolarizing steps under voltage-clamp) was decreased by hydroxychloroquine [ ] . there was no change in voltage-dependent activation, suggesting that the drug acts on the channel conductance, but the underlying molecular mechanism is unknown. this inhibitory action on i f was translated into a decrease of the rate of diastolic depolarization and of the beating rate of sa isolated cells or of atrial multicellular preparations [ , ] . based on its effect to decrease i f , hydroxychloroquine was proposed as a relatively safe bradycardic agent [ ] , and bradycardia was obtained with intravenous injections in anesthetized animals, without negative inotropic or hemodynamic action. therefore, the bradycardic effect was proposed to be of potential value in decreasing oxygen demand, of benefit in conditions such as angina. it should be noted that bradycardia occurred within a few (typically ) minutes using hydroxychloroquine in anesthetized animals, while tachycardia is observed for hours after a single dose of chloroquine in awake human volunteers (see below). it is likely that in humans the fast developing rate-slowing action on pacemaker tissue is followed and opposed by a slowly developing effect due to an interaction with the autonomic nervous system (see below). chloroquine also acts on various k + channels [ , [ ] [ ] [ ] [ ] [ ] [ ] . chloroquine blocks kir . inward-rectifier channels, largely responsible for the background conductance and i k current [ , ] that set the resting potential but also contribute to the repolarization and the duration of the action potential. the underlying mechanism involves an accession of chloroquine from the intracellular side and its binding near the intracellular mouth of the pore [ ] . there appears to be a dual action of chloroquine on kir . channels. in contrast to the acute decrease of channel conductance, an increase in the plasmalemmal expression of the channel protein has been reported in hek cells used as expression system [ ] . this effect developed over hours and was related to the inhibition of the lysosomal degradation of the channels. to our knowledge no study has tested this effect in cardiomyocytes, but such an increase in channel expression could have a corrective action against the decrease due to block, and partly contribute to limit qt prolongation. chloroquine/hydroxychloroquine also block the g-protein-activated inward rectifier k + (girk) channel present in cardiac supraventricular (atrial contracting and nodal) cells. the channel is also present in ventricular cells of some species but practically absent in human ventricular contracting cells. it is activated following acetylcholine (ach, the neurotransmitter of the parasympathetic system) binding on m muscarinic receptors, or adenosine binding on a receptors, hence the names i k-ach or i k-ado for the corresponding ion currents. the blocking action of chloroquine is also observed in hek cells expressing kir . and kir . channels, which contribute subunits forming girk channels [ , ] . the underlying mechanism is similar to that of kir . (i k ) channels described above: it involves the binding from the intracellular side to a site within the internal mouth of the channel. the ic of chloroquine for both channels was µm, a concentration similar to the plasma concentrations following oral doses of about mg.kg - . the blocking effect on i k , i k-ach or both should result in a lengthening of the action potential duration. as mentioned earlier no or only marginal qt prolongation is induced by low doses of chloroquine/hydroxychloroquine given via the oral route [ , ] . given that action potential shortening is associated with arrhythmogenesis, the action potential lengthening effect of chloroquine may be antiarrhythmic under pathological conditions which markedly shorten the action potential duration. such an antiarrhythmic effect is mostly obvious in case of short qt syndrome due to mutations of kir . channels [ ] , but it was also shown to occur in sheep hearts with sustained atrial fibrillation induced by chronic pacing [ ] . under the latter condition i k-ach is spontaneously activated in the absence of agonist binding on g-protein coupled receptors. chloroquine decreased the dominant fibrillation frequency and finally induced a conversion to sinus rhythm [ ] , an effect that was associated with the prolongation of the atrial action potential and could be reproduced by a toxin (tertiapin) known to suppress i k-ach . chloroquine/hydroxychloroquine also act on atp-sensitive k + channels. these channels monitor the cell metabolism and open following a decrease of intracellular atp and an increase of adp, as occur in conditions such as ischemia. in the heart the channels are formed by a pore unit made of kir . and a sulfonylurea-binding regulatory unit, sur a. chloroquine blocks the atp-sensitive current (i k-atp ) activated pharmacologically (by pinacidil) in ventricular cardiomyocytes [ ] and the corresponding channels units expressed in hek cells [ ] . the suppression involved two mechanisms: a pore block by chloroquine entering from the intracellular side and occluding ion movement within the channel, and an inhibition, given the amphiphilic character of chloroquine, of the channelactivating interaction between kir . and the membrane phospholipid phosphatidyl-inositol bisphosphate (pip ). i k-atp inhibition in cardiac myocytes occurred at micromolar concentrations (ic : . µm), indicating that chloroquine at therapeutic doses could also act on the heart through mechanisms involving atp-sensitive channels, e.g. to antagonize fibrillation following action potential shortening and inhomogeneity during ischemia. in sa node cells, purkinje fibers and ventricular myocytes, hydroxychloroquine was shown to decrease the amplitude of the rapid delayed rectifier i kr by % [ ] . the drug did not affect the slow component i ks . the effect on i kr is consistent with the inhibition of herg channels in expression systems (ic : . - . m) and could contribute to qt prolongation, especially at high concentrations [ , ] . channels carrying the transient outward current, i to , and other channels a transient outward current, i to , underlies phase- early repolarization in cardiac cells. it is a mixed current, made of a ca + -independent but voltage-dependent k + component, and a ca + -activated clcomponent [ , ] . chloroquine does not seem to have potent effect on i to [ ] . inhibitory effects occurred only at very high concentrations (k d : µm) [ ] ), which are not reached under therapeutic conditions. the effect on other (chloride-selective and non-selective sarcolemmal, sarcoplasmic reticulum, and mitochondrial) cardiac channels have not, to our knowledge, been investigated. in contrast to the slowing of the heart rate by chloroquine observed under experimental conditions as expected from the suppression of i f and i ca-l [ ] , the drug increases heart rate during more than hours after a single per os dose of . - mg.kg - in healthy human volunteers [ ] . this latter effect may be attributed to a dual effect on the autonomic nervous system. on the one hand there might be an activation of heart accelerating mechanisms resulting from a stimulation of the sympathetic system [ ] , but such a mechanism has not been demonstrated in humans. on the other hand there is inactivation of heart decelerating mechanisms resulting from an inhibition of the parasympathetic (or vagal) system. this latter effect has been more thoroughly investigated by the author. the heart rate increase was associated with a decrease of cardiovascular reflexes used as indices of the vagal influence on the heart. one of the most readily assessable parameters of the autonomic nervous control of the heart is the beat-to-beat variation of the heart cycle. chloroquine caused a narrowing of the average dispersion between heart cycle durations. other reflexes, such as the cardiac cycle increase during a valsalva maneuver or post deep inspiration, were also decreased, indicating that chloroquine exerts a vagolytic action on the heart. this effect is dose-dependent and its underlying mechanism seems to involve, at least in part, a direct atropine-like, antagonist action of chloroquine on muscarinic receptors. chloroquine antagonized the negative chronotropic effect of muscarinic receptor activation under experimental conditions using animal isolated hearts [ ] . chloroquine is known to bind on muscarinic receptors (k d : - µm [ ] ). its binding on cardiac muscarinic receptors could be demonstrated by its displacement of saturation binding curves of a radioactive label of these receptors [ ] . however, this interaction with receptors occurred at rather higher concentrations than those interfering with the negative chronotropic effect of muscarinic agonists. thus, despite the demonstrated interaction of chloroquine with muscarinic receptors, the mechanism of chloroquine effect on the heart rate appears to be complex. as discussed above, effects on channels, including a block of i k and i k-ach , which contribute to the regulation of the heart rate, could play a role and partly account for the increase of heart rate caused by chloroquine. besides the effects described above, which basically occur at the surface membrane, chloroquine also targets intracellular processes, mainly (but not exclusively) those involving the intracellular processing of proteins by autophagy. autophagy is an important homeostatic mechanism, which is responsible for the clearance of damaged intracellular protein complexes and organelles [ , ] , including in the heart. it plays an important role in the response to stress and in disease conditions such as ischemia/reperfusion [ , ] , hypertrophy [ ] , heart failure [ , ] , etc. drug-induced changes in autophagy may also underlie therapeutic or toxic effects. macro-autophagy involves the fusion of autophagosomes with lysosomes, to constitute autolysosomes, where cargo brought by the phagosomes is digested. chloroquine, which accumulates into lysosomes by ion trapping [ , ] and causes alkalization of the intralysosomal ph, inhibits the fusion between autophagosomes and lysosomes and the basic macro-autophagy flux [ ] . hearts pre-treated with chloroquine ( mg.kg - .day - , a dose within clinical range) for days show a decreased autophagy flux, with accumulation of autophagosomes. another type of autophagy, which involves chaperone-mediated transport of abnormal proteins into lysosomes, also appears to be inhibited by chloroquine [ ] . given the importance of ischemic diseases in cardiac pathology, it is of interest that ischemia/reperfusion is reported to modify autophagy, and that chloroquine has a potential to modulate the changes occurring under these conditions. an excessive decrease or increase of autophagy beyond its basal level may be expected to have harmful consequences. autophagy is upregulated during myocardial ischemia and reperfusion [ ] . whereas during the ischemia phase activation is adenosine monophosphate-activated kinase (ampk)-dependent, adaptive and protective, during reperfusion it is ampkindependent, excessive and it enhances myocardial death. protection during ischemia is likely due to the ability of autophagy to deliver metabolites and to clear damaged organelles such as mitochondria (mitophagy). excessive autophagy and cell death during reperfusion are attributed to the formation of reactive oxygen radicals. autophagy inhibition by chloroquine could limit the adaptive increase during ischemia and aggravate the condition. this is the case in isolated rats submitted to episodes of intermittent hypoxia for many weeks. in this experimental model set to mimic the condition of sleep apnea, inhibition of autophagy with chloroquine was associated with a development of myocardial dysfunction, while no dysfunction was present with intermittent hypoxia alone [ ] , suggesting that autophagy provided cardiac adaptive mechanisms to hypoxia. in contrast, attenuation of the excessive increase of autophagy during reperfusion could a priori be expected to confer protection. however, reperfusion by itself, despite upregulating autophagy, is associated with decreased clearance of autophagosomes [ ] . further suppression of autophagosome clearance by chloroquine can therefore aggravate this process. increased cell death caused by chloroquine during ischemia/reperfusion has been linked to a decrease of oxygen radical buffering action of mitochondria [ ] . chloroquine could be expected to be protective in other conditions that enhance autophagy flux, such as cardiac hypertrophy. the drug reversed the myocardial wall thickening caused by pressure overload in rats that underwent ascending aorta banding. it also appears to confer protection against ischemia/reperfusion in diabetic cardiomyopathy [ , ] . modification of autophagy is at least in part responsible for the effect of chloroquine/hydroxychloroquine on the cardiac remodeling induced by stress conditions such as myocardial ischemia/reperfusion. autophagy is decreased during post-infarction remodeling in rabbit heart [ ] . in the days following myocardial infarction in rats there was maintained cell death by controlled necrosis (necroptosis), including in the noninfarcted border zone. autophagy flux was increased during the first day but, despite maintained increase in autophagosome formation, with time autophagy flux was decreased at the level of autophagolysosome processing and there was an associated increase in heart dysfunction [ ] . the impaired autophagy appeared to be responsible for the necroptosis since the latter could be attenuated by genetic overexpression of beclin, an initiator of autophagy. in this model, chloroquine caused an aggravation of the structural and functional deterioration. as already mentioned, because of reduced autophagy during myocardial remodeling, various strategies, including food restriction and pharmacological agents, can be used to stimulate autophagy. a similar protection can also be demonstrated by targeting autophagy in fibroblasts using micro-rna [ ] . the cellular signaling involved in some of these strategies may be complex. for example, the effect of food restriction and resveratrol may implicate an increased ampk activity, and a downregulation of the mtor signaling [ ] . chloroquine seem to suppress the protective effect of these strategies by modifying the effects of these signaling pathways on autophagy. it is not known whether there are additional inhibitory effects at more proximal steps. the scope of effects due a modulation of autophagy may be larger than heretofore known. it is plausible that the inhibition of autophagy affects the expression of many proteins (e.g. of kir . channels mentioned earlier) and impact on function. chloroquine has been reported to enhance the loss of t-tubules observed in cultured ventricular cardiomyocytes from failing human and from healthy adult rat hearts cultured in the absence of glucocorticoids [ ] . chloroquine, by inhibiting lysosome function in cultured mouse neonatal cardiomyocytes, was shown to cause an accumulation of phospholamban [ ] , a negative regulator of the sarco-endoplasmic reticulum ca + atpase (serca; responsible for storing ca + needed for contraction in the lumen of the sarcoplasmic reticulum). it is not known to which extent this effect occurs in adult cardiomyocytes and impacts on contractility. reports of neurotoxic effects of chloroquine include the development of extrapyramidal symptoms (rolling of the eyeballs, involuntary movements, trismus) following a few hoursdays of treatment with low doses in children [ ] as well as in adults. these effects were reversible within - hours after stopping the treatment. behavior disturbances have also been described [ ] [ ] [ ] . whether they can be related to actions on ion channels and receptors, or to rapid consequences of the interference with autophagy is not clear. retinotoxicity, a noted frequent side effect of treatment with chloroquine/ hydroxychloroquine [ ] , and skeletal muscle myopathy, which is less frequent [ ] [ ] [ ] [ ] , are largely results of changes in autophagy. administration of chloroquine can cause a degeneration of skeletal muscle cells resulting in myopathy. chloroquine myopathy can be reproduced experimentally by high doses ( mg.kg - .day - ) of the drug over at least weeks. the degenerated muscle fibers contain numerous autophagic vacuoles [ ] , into which various proteins accumulate [ , ] . as mentioned earlier, a similar accumulation can occur in cardiomyocytes and is responsible for cardiotoxicity following long-term treatment [ , ] . such effects are unlikely to complicate treatments limited to a few days and involving low to moderate doses (≤ mg.kg - .day - ). chloroquine poisoning is known to induce hypokalemia [ ] . the degree of hypokalemia is largest the higher the dose ingested and the blood concentration. hypokalemia in patients is systematic at chloroquine concentrations of µm and above, but in a few cases it could be obtained at much lower concentrations [ ] . the condition can develop within two hours or less following large doses and its underlying mechanism is not very clear but increased renal loss has been excluded. it has been proposed to involve reduced cellular k + efflux due to channel block by chloroquine (described above). however, in the absence of an increased k + influx pathway, suppression of outflux is not a satisfactory explanation for the cellular uptake of k + that causes hypokalemia. since the cellular membrane potential of resting or active cells is positive to the k + equilibrium (nernst) potential, only outward k + movements can occur via channels. one should therefore explore the role of a non-channel influx pathway to account for the increased transport of k + into the cell. the effect of chloroquine on the na + -k + pump is unclear. chloroquine has been shown to inhibit the na + -k + atpase in vitro [ ] , but a transient stimulation could be obtained in vivo at low concentration [ ] . it is conceivable that the hypokalemia is triggered by conditions such as stress and exercise that stimulate the na + -k + atpase by enhancing the sympathetic tone, as is the case in hypokalemic periodic paralysis [ ] or by an eventual increase of insulin secretion. in addition, the vagolytic action of chloroquine (described above) could remove the known "accentuated antagonism" exerted by the parasympathetic system towards the sympathetic system. increased na + -k + pump activity could then result from increased -adrenergic receptor activation [ , ] . this or other possible mechanisms have not been examined, and the main basis for the hypokalemia thus remains unclear. low extracellular k + concentration, largely because of the decrease in conductance of inward rectifier channels and of the corresponding outward currents, is associated with a lengthening of cardiomyocyte action potential duration and of the qt interval, and is arrhythmogenic. indeed, ventricular tachyarrhythmias were the immediate cause of death in about a third of patients admitted for chloroquine poisoning [ ] . one should be careful when attributing any clinically observed qt lengthening to a direct effect of chloroquine, without excluding the eventual contribution of concurrent hypokalemia. the possible efficacy of chloroquine/hydroxychloroquine in the treatment of various diseases, including viral infections such as covid- [ , , , ] but also cancer [ , ] , has received increasing interest. beside the drug efficacy in patients, the issue concerning the safety is also important. evidently, if the side effects were to be such marked that they put the life of treated patients in danger, then, as long as beneficial effects have been confirmed, the drug use could be restricted to only very severe cases, when there is no other choice than to try everything that can potentially save the patients. such use can only be done with close monitoring of the patient. in contrast, if side effects are mild and tolerable, such that they do not jeopardize the patient's life, then one can or should consider using the drug for its proven or potential benefit. the side effects of chloroquine/hydroxychloroquine on the heart are mainly of two types: those involving changes in electrical conduction, heart rate and rhythm [ , , , , , , ] , and those involving structural changes associated with cardiomyopathy [ , ] or myocardial remodeling. conduction and rhythm disturbances can be seen under acute conditions before any structural change, but they can also be favored by the cardiomyopathy [ ] . on a time frame, the rhythm changes are acute and reversible upon drug withdrawal, whereas cardiomyopathy is induced after treatment for months and is largely irreversible. all effects are also dose-dependent, and cardiomyopathy [ ] and interference with remodeling can be experimentally induced over shorter periods (weeks) with high doses. the mechanisms underlying the conduction and rhythm changes mainly involve inhibitory actions on various ion channels. block of na + channels (local anesthetic action) is likely responsible for observed bundle branch and intraventricular block [ , , ] . block of k + channels will tend to lengthen the action potential duration, whereas the inhibition of na + and ca + channels with have opposite effects. that the drugs given alone at low or moderate concentrations and for a short duration produce only limited prolongation of the qt interval is likely due to this balanced contribution of opposite effects [ ] . heart rate changes also result from a balance between a bradycardic action of i f and i ca-l inhibition [ , ] and a tachycardic effect of i k and i k-ach inhibition [ , , ] , in addition to the suppression of the vagal tone on the heart [ , ] . the resultant action of chloroquine can be pro-or antiarrhythmic, depending on the underlying condition [ , , , , , , ] . the mechanism underlying chloroquine cardiomyopathy involves an inhibition of autophagy [ , , ] . even in the absence of cardiomyopathic changes, such an inhibition can aggravate cell damage under ischemia-reperfusion, which by itself causes a change in the autophagic flux and recruits additional cell death mechanisms [ ] [ ] [ ] [ ] [ ] ] . the trend to favor myocardial death may be opposed by protective mechanisms due to an inhibition of intracellular na + and ca + load and of arrhythmias resulting from the effects on ion channels or due to inhibition of apoptosis [ ] . it should be underlined that all cardiac effects of chloroquine/hydroxychloroquine could not be covered in this brief review. for example, metabolic effects due to phospholipase a inhibition have been proposed to mediate an anti-ischemic action of chloroquine [ ] . in addition, other effects may not yet be known, and the consequences of autophagy inhibition on the expression of various proteins could be more complex. finally the effect of the drugs and their mechanisms may depend on coexisting non cardiac (e.g. electrolyte) changes or cardiac diseases (e.g. the presence of myocarditis). the side effects of chloroquine/hydroxychloroquine are generally mild, but they can be exacerbated by drug associations. many macrolide antibiotics can cause a qt prolongation when used in isolation [ ] . if co-administered with chloroquine/hydroxychloroquine, the effects on the qt interval might add and become marked, even if the effect of every drug taken separately was mild. use of high doses and association with other potential qt-prolonging drugs are likely the cause of the marked qt prolongation and occurrence of arrhythmias that have recently led to interrupt clinical trials testing the benefit of chloroquine in the treatment of covid- [ ] . even when apparently moderate mean doses are reported to have been used in large registries of patient groups, there will be large variations among different patient groups and a significant number of them might have been exposed to high drug doses. in any case, drug associations and the role of complicating conditions such as hypokalemia that are themselves drug dose-dependent, should also be analyzed before concluding on the direct cardiotoxic effect of chloroquine/hydroxychloroquine. the antimalarial drugs chloroquine and hydroxychloroquine have been proposed for antiviral therapy, including for covid- . the drugs are relatively safe when used at low or moderate doses, with blood concentrations ≤ µm. at such doses they can display beneficial, e.g. antiarrhythmic, actions, and despite the many effects on k + channels, they do not seem to be associated with prominent qt prolongation acute life-threatening cardiac effects, including ventricular tachyarrhythmias, are obtained with high doses. given the long half-life of the drug, other side effects are usually associated with long-term treatments. the mechanisms underlying chloroquine effects include direct actions on ion channels and receptors, while others (especially a cardiomyopathy developing following long-term treatment) involve the inhibition of autophagy. care should therefore be taken to limit the dose and the duration of treatment with the drugs, as well as to avoid association with other drugs with known toxic effects on the myocardium. declarations funding: work of the author is supported by funding from vlir-uos in the frame of an institutional university cooperation between flemish universities and the université catholique de bukavu. new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? recycling antimalarial leads for cancer: antiproliferative properties of n-cinnamoyl chloroquine analogues current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a minireview evidence for cardiac safety and antiarrhythmic potential of chloroquine in systemic lupus erythematosus chloroquine is a potent pulmonary vasodilator that attenuates hypoxia-induced pulmonary hypertension chloroquine and hydroxychloroquine as available weapons to fight covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread a call for randomized controlled trials to test the efficacy of chloroquine and hydroxychloroquine as therapeutics against novel coronavirus disease (covid- ) mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology the arrhythmogenic cardiotoxicity of the quinoline and structurally related antimalarial drugs: a systematic review cardiotoxicity of antimalarial drugs effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial parenteral chloroquine for treating falciparum malaria the disposition of chloroquine in healthy nigerians after single intravenous and oral doses the contribution of lysosomal trapping in the uptake of desipramine and chloroquine by different tissues excessive lysosomal ion-trapping of hydroxychloroquine and azithromycin clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements pharmacokinetics of quinine, chloroquine and amodiaquine. clinical implications cardiac arrest after intravenous chloroquine injection cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature sinus node dysfunction in adult systemic lupus erythematosus flare: a case report hydroxychloroquineinduced cardiotoxicity in a -year-old woman with systemic lupus erythematosus and systolic dysfunction heart conduction disorders related to antimalarials toxicity: an analysis of electrocardiograms in patients treated with hydroxychloroquine for connective tissue diseases the pharmacokinetics and electrocardiographic effects of chloroquine in healthy subjects azithromycin/chloroquine combination does not increase cardiac instability despite an increase in monophasic action potential duration in the anesthetized guinea pig assessment of multi-ion channel block in a phase i randomized study design: results of the cipa phase i ecg biomarker validation study the effects of antimalarial drugs on ventricular repolarization chloroquine and qtc interval comparison of antifibrillatory potency of certain antimalarial drugs with quinidine and procaine amide chloroquine and hydroxychloroquine in the treatment of cardiac arrhythmias chloroquine terminates stretch-induced atrial fibrillation more effectively than flecainide in the sheep heart the antimalarial chloroquine reduces the burden of persistent atrial fibrillation antiarrhythmic drugs and cardiac ion channels: mechanisms of action increase of electrocardiogram voltage and contractile force of the frog's heart induced by chloroquine comparative effects of some antimalarial drugs on isolated cardiac muscle of the guinea pig highdose chloroquine is metabolically cardiotoxic by inducing lysosomes and mitochondria dysfunction in a rat model of pressure overload hypertrophy acute chloroquine poisoning: a comprehensive experimental toxicology assessment of the role of diazepam depressant effects of chloroquine on the isolated guinea-pig heart diazepam does not improve the mechanical performance of rat cardiac papillary muscle exposed to chloroquine in vitro revisiting the cardiotoxic effect of chloroquine the effects of calcium ions on the depression of cardiac contractility by chloroquine and quinine treatment of severe chloroquine poisoning chloroquine cardiomyopathy -a review of the literature chloroquine cardiomyopathy with conduction disorders molecular mechanisms of myocardial remodeling reduced synchrony of ca + release with loss of t-tubules-a comparison to ca + release in human failing cardiomyocytes molecular and cellular mechanisms involved in cardiac remodeling after acute myocardial infarction impaired protein quality control during left ventricular remodeling in mice with cardiac restricted overexpression of tumor necrosis factor inhibition of autophagy inhibits the conversion of cardiac fibroblasts to cardiac myofibroblasts alterations of na + currents in myocytes from epicardial border zone of the infarcted heart. a possible ionic mechanism for reduced excitability and postrepolarization refractoriness can pka activators rescue na + channel function in epicardial border zone cells that survive in the infarcted canine heart? the role of autophagy emerging in postinfarction cardiac remodelling restriction of food intake prevents postinfarction heart failure by enhancing autophagy in the surviving cardiomyocytes resveratrol reverses remodeling in hearts with large, old myocardial infarctions through enhanced autophagy-activating amp kinase pathway chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type ii receptor degradation a new local anesthetic with anticoagulant properties, chloroquine (aralen) dihydrochloride blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes late sodium current: a mechanism for angina, heart failure, and arrhythmia drug potency on inhibiting late na + current is sensitive to gating modifier and current region where drug effects were measured hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current i f : novel electrophysiological insights and therapeutic potential pacemaking in cardiac tissue. from i k to a coupled-clock system a novel quantitative explanation for the autonomic modulation of cardiac pacemaker cell automaticity via a dynamic system of sarcolemmal and intracellular proteins pacemaker mechanisms in cardiac tissue a brief history of pacemaking the funny current has a major pacemaking role in the sinus node chloroquine blocks the kir . channels by an openpore blocking mechanism molecular mechanisms of chloroquine inhibition of heterologously expressed kir . /sur a channels structural bases for the different anti-fibrillatory effects of chloroquine and quinidine chloroquine blocks a mutant kir . channel responsible for short qt syndrome and normalizes repolarization properties in silico the molecular basis of chloroquine block of the inward rectifier kir . channel chloroquine blocks the background potassium current in guinea pig atrial myocytes lysosome mediated kir . breakdown directly influences inward rectifier current density structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule action potential clamp and chloroquine sensitivity of mutant kir . channels responsible for variant short qt syndrome specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets antimalarial drugs: qt prolongation and cardiac arrhythmias inhibition of herg k + currents by antimalarial drugs in stably transfected hek cells calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes two components of transient outward current in canine ventricular myocytes open channel block of the fast transient outward k + current by primaquine and chloroquine in rat left ventricular cardiomyocytes muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers chloroquine interacts with muscarinic receptors and inhibits cholinergic effects in the heart binding and doseresponse studies with chick embryo cells the role of autophagy in the heart the role of autophagy in the heart is autophagy in response to ischemia and reperfusion protective or detrimental for the heart? the role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart distinct roles of autophagy in the heart during ischemia and reperfusion: roles of amp-activated protein kinase and beclin in mediating autophagy the altered expression of autophagy-related genes participates in heart failure: nrbp and calcoco are associated with left ventricular dysfunction parameters in human dilated cardiomyopathy autophagy-inflammasome interplay in heart failure: a systematic review on basics, pathways, and therapeutic perspectives chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice chloroquine and amodiaquine enhance ampk phosphorylation and improve mitochondrial fragmentation in diabetic tubulopathy progressive reduction in myocyte autophagy after myocardial infarction in rabbits: association with oxidative stress and left ventricular remodeling necroptosis mediated by impaired autophagy flux contributes to adverse ventricular remodeling after myocardial infarction microrna- repairs infarcted myocardium by accelerating cardiomyocyte autophagy glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux metformin increases degradation of phospholamban via autophagy in cardiomyocytes extrapyramidal syndrome following chloroquine therapy toxic psychosis: a complication of antimalarial therapy lethality and behavioral side effects of chloroquine behavioral toxicity and equivocal suicide associated with chloroquine and its derivatives hydroxychloroquine retinopathyimplications of research advances for rheumatology care hydroxychloroquine-induced autophagic vacuolar myopathy with mitochondrial abnormalities experimental chloroquine myopathy immunohistochemical evidence for amyloid beta in rat soleus muscle in chloroquineinduced myopathy snake coiled fibres in rat soleus muscle in chloroquine induced myopathy share immunohistochemical characteristics with amyloid depositions in alzheimer's disease brain tissue hypokalaemia related to acute chloroquine ingestion the in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible the interaction of chloroquine with transport atpase and acetylcholine esterase in microsomal membranes of rat in vitro and in vivo extracellular potassium homeostasis: insights from hypokalemic periodic paralysis regulation of the beta-stimulation of the na + -k + pump current in guinea-pig ventricular myocytes by a camp-dependent pka pathway effects of alpha-adrenergic stimulation on intracellular sodium activity and automaticity in canine purkinje fibers antimicrobials and qt prolongation hydroxychloroquine cardiotoxicity in systemic lupus erythematosus: a report of cases and review of the literature chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p independent hydroxychloroquine protects against cardiac ischaemia/reperfusion injury in vivo via enhancement of erk / phosphorylation effect of chloroquine in experimental myocardial ischaemia ethical approval: not required. key: cord- - sukdb authors: quade, bianca n.; parker, mark d.; occhipinti, rossana title: the therapeutic importance of acid-base balance date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: sukdb baking soda and vinegar have been used as home remedies for generations and today we are only a mouse-click away from claims that baking soda, lemon juice, and apple cider vinegar are miracles cures for everything from cancer to covid- . despite these specious claims, the therapeutic value of controlling acid-base balance is indisputable and is the basis of food and drug administration-approved treatments for constipation, epilepsy, metabolic acidosis, and peptic ulcers. in this narrative review, we present evidence in support of the current and potential therapeutic value of countering local and systemic acid-base imbalances, several of which do in fact involve the administration of baking soda (sodium bicarbonate). furthermore, we discuss the side effects of pharmaceuticals on acid-base balance as well as the influence of acid-base status on pharmacokinetic properties of drugs. our review considers all major organs systems as well as information relevant to several clinical specialties such as anesthesiology, infectious disease, oncology, dentistry, and surgery. the normal function of nearly all physiological processes in the body depends on maintenance of appropriate acid-base balance. the value of intracellular ph and interstitial ph strongly depends on the value of arterial blood ph, which ranges between . and . under normal physiological conditions. when ph deviates from its normal range, ph-dependent enzymes and membrane transport proteins may not work properly and metabolic pathways can be negatively affected. acidemia, which is defined as arterial ph lower than . , can cause a variety of disturbances including arterial vasodilation, insulin resistance, compromised immune function, and reduced neuronal excitability. alkalemia, which is defined as arterial ph greater than . , can also cause many disturbances including reduced myocardial blood flow and seizures. thus, it is imperative that the value of blood ph is tightly controlled. therapies for acid-base disturbances are not new. infusion of sodium carbonate (na co ) into cholera patients to compensate for loss of serum alkali in diarrhea was recorded in the s [ ] and the commercial production of sodium bicarbonate (nahco ) for use as an antacid (brioschi®) apparently dates back to the s. since then, decades of research advances have led to a broad appreciation of the importance of acid-base balance in health and disease. this research is now coming to fruition in the form of inspired and effective medical advances. at the same time, some in the alternative medical community have seized on anecdotes and the results of limited trials to generate ubiquitous clickbait headlines about the miraculous properties of household acids and bases such as baking soda and, in some cases, propagate conspiracy theories about suppression of this information. in the first major section of our review ( acid-base homeostasis) we discuss how the body controls the abundance and distribution of acids and bases in order to achieve acid base homeostasis. we describe the importance of the powerful co /hco buffer system, the vital functions of the lungs and kidneys in excreting excess acids and bases, the role of membrane transport proteins and carbonic anhydrases in the local redistribution of acids and bases, and the drugs that can be harnessed to control these processes. in the second major section of our review ( systemic acid-base disturbances) we discuss the causes and consequences of generic acidbase imbalance caused by disturbances in co and hco levels and how our knowledge of their etiology has informed therapeutic strategies. our third and fourth major sections ( applications by organ system and other applications by clinical specialty) bring together a wealth of information from in vitro, in vivo, and clinical studies that demonstrate the current and potential utility of acid-base-balance correcting therapies. for each organ system or clinical specialization, as appropriate, we provide the fundamental physiological aspects of normal acid-base balance, the pathological consequences of systemic and local acid-base disturbances, as well as considerations of corrective therapies based on restoring (or harnessing the agents of) acid-base balance. in our fifth and final major section ( ph-dependent aspects of pharmaceutical therapy) we discuss how acid-base chemistry can influence drug pharmacokinetic properties and how this phenomenon can be advantageous for optimizing therapeutic interventions. our review highlights an emerging and dynamic field of research that is in the process of translating numerous basic scientific findings into clinical therapies. these findings appear to touch on nearly all aspects of health. we note a wide array of therapeutic paradigms developed around the control of acid-base balance including numerous reports of the successful application of nahco , the so-called 'enemy of the pharmaceutical industry,' to the amelioration of disease signs in animal models and in limited clinical trials. although studies of the role of acid-base balance in health and disease have resulted in the generation of several fda-approved pharmaceuticals such as contraceptive gels and gastric-acid suppressors, systematic reviews of random trials of the clinical effectiveness of nahco itself tend to be circumspect in their conclusions. a note to the reader: we, the authors, are basic scientists and do not intend this review to serve as a diagnostic or therapeutic guide. in many cases, a lack of consistency among study methods and subject demographics makes it difficult to draw firm conclusions regarding outcomes. for these reasons it is also impossible to extrapolate findings of therapeutic effectiveness in animal models and limited trials into an assessment of general clinical utility. but, in as much as we are reporting potential, we have not discounted any positive outcomes. finally, we note that the scope of this narrative review is extremely broad and the literature is extensive. for this reason, we have often cited reviews instead of primary literature in order to simplify the document and provide a cue to further reading. we apologize in advance to any authors whose work we have omitted. the maintenance of blood ph in the face of a ~ - meq h + /day acid load imposed by diet and metabolism (net endogenous acid production: neap [ , ] ), requires robust homeostatic mechanisms. regulation of blood ph and, by extension, the entire extracellular fluid compartment depends on the interplay between (i) the urinary system, which controls the blood bicarbonate concentration ([hco − ]), and (ii) the neuro-respiratory systems, which control the partial pressure of co (pco , see figure ). the kidneys perform the tasks of generating hco -, depositing it into circulation, and recycling hco from filtered plasma back into circulation (see figure ). the lungs exhale co , with respiratory drive being controlled by chemosensitive neural circuitry [ ] . a third mechanism of defense, which does not exactly regulate ph but only tends to minimize its changes, is provided by the multitude of buffer systems present in the extracellular fluid compartment. among these, the most powerful is the co /hco − buffer system, the efficacy of which is conferred by the body behaving as an open system, with respect to co , from which co can escape [ ] . a feature of the co /hco − buffer system is that the first of the two-step reactions that describe the interconversion between co and hco − (co +h o ⇌ h co ⇌ hco − + h + ) is very slow unless catalyzed by a carbonic anhydrase (ca) enzyme. thus, efficient buffering requires the presence of a ca. the henderson-hasselbalch equation [ ] describes how [hco − ] and pco determine ph:   co [hco ] ph = p + log . pco k s four of the members of the monocarboxylate transporter family (mcts, slc a - [ ] ) are h + -coupled lactate transporters (mct - ). mct and mct , being expressed in tumors, are of main therapeutic interest. the directionality of their transport process is determined by kinetic, thermodynamic, and situational considerations; for example the widely expressed isoform mct typically mediates h + /lacimport, while mct , which is abundantly expressed in glycolytic (i.e., lactate-producing) cells such as cancer cells and astrocytes, mediates h + /lacexport [ ] . members of several other solute carrier families also cotransport h + with their substrates such as the h + -coupled oligopeptide transporters of the slc family (e.g., peptt [ ]) or the h + -coupled neurotransmitter transporters of the slc family (e.g., the excitatory amino acid transporter eaat [ ]). the mct inhibitor azd [ ] is currently in phase i clinical trial for its effectiveness in treating cancer (clinicaltrials.gov identifier: nct ). there are several membrane proteins that conduct h + but share no obvious commonality in protein sequence. one group act as h + -selective channels and include the voltage-gated h +channel hv (hvcn [ ] ) and the voltage-independent h + conductors slc a [ ] and otopetrin (otop [ ]). all permit the movement of h + down their transmembrane electrochemical gradient, but each differ in their regulation of gating. hv only opens when the gradient favors h + efflux, slc a favors h + influx (particularly at elevated phi [ ] , perhaps to defend phi), and otop also favors h + influx (particularly at low phe, perhaps consistent with its sensory role). in acidotic conditions, the transient receptor potential cation channel subfamily v member (trpv ) can also mediate a significant, but non-canonical, acid-loading h + conductance [ ] . we are unaware of any fda-approvals for inhibition of this class of proteins, with the exception of trpv agonists whose influence on h + conductance is untested. although there is no description of a hco --specific ion channel, several anion channels have significant hco permeability. the electrochemical gradient for hco typically favors hco -efflux. these channels include the cystic fibrosis transmembrane regulator (cftr [ ]), the ca +activated cl --channel anoctamin (ano [ ]), as well gaba-and glycine-activated cl -channels (gabr and glr families [ ] ). drugs such as ivacaftor (kalydeco®, increases cftr channel open probability), or cocktails that include ivacaftor and one or more of the cftr folding chaperone drugs elexacaftor/lumacaftor/tezacaftor (e.g., orkambi®, symdeko®, trikafta®) are indicated for the treatment of cystic fibrosis by the restoration of certain defective cftr channels. both ivacaftor and tezacaftor rescue the hco permeability of the Δ -cftr mutant. in fact the rescued mutant has a greater hco -:clpermeability ratio than wild-type cftr, which may be therapeutically valuable. the importance of cftr-mediated hco secretion is discussed in sections . the respiratory system, . the lower digestive system, and . the urinary system). five of the ten members of the slc family of proteins mediate some form of na + -coupled hco --transport [ ]. nbce and nbce (slc a and slc a ) are electrogenic na + / hco cotransporters that may either act as acid-extruders or acid-loaders, depending on the electrochemical gradient. for example, nbce mediates hco efflux in renal proximal tubule epithelia, but hco influx in pancreatic duct epithelia. the remaining three are all acid extruders. nbcn (slc a ) is an electroneutral na + -hco cotransporter, while ndcbe (slc a ) is an electroneutral na + -driven cl -/hco exchanger. nbcn /ncbe (slc a ) has been described as being capable of both actions. we are unaware of any fda-approvals for inhibition of this class of proteins, nor of any drug that is specific for this class of protein. we note however that the nonsteroidal anti-inflammatory drug tenidap, which failed clinical trials for the treatment of rheumatoid arthritis due to renal and hepatic toxicity, is an effective blocker of nbce and nbce [ , ]. equation ( ) and figure show that, provided that pco remains constant, (i) a fall in extracellular [hco − ] causes ph to decrease whereas (ii) a rise in extracellular [hco − ] causes blood ph to increase. these two cases describe states of metabolic acidosis (mac) and metabolic alkalosis (malk), respectively. equation ( ) also indicates that ph can return towards its normal physiological value by a decrease in extracellular pco (in case 'i') or an increase in extracellular pco (in case 'ii'). this compensatory normalization of the [hco -]:pco ratio to restore ph, describes the physiological response of the neuro-respiratory system to mac and malk. moreover, equation ( ) and figure show that, provided that [hco -] remains constant, (iii) a rise in extracellular pco causes blood ph to decrease whereas (iv) a decrease in extracellular pco causes blood ph to rise. these two cases describe states of respiratory acidosis (rac) and respiratory alkalosis (ralk), respectively. again, equation ( ) indicates that ph can return towards its normal value by an increase (case 'iii') or decrease (case 'iv') in extracellular [hco − ], describing the compensatory physiological response of the urinary system to rac and ralk. mac, malk, rac, and ralk are usually referred to as the four classic/simple acid-base disturbances. respiratory compensations usually occur quite rapidly, within an hour of the appearance of the metabolic disorders and are fully resolved within to hours. in contrast, metabolic responses to respiratory disorders occur more slowly and may take up to several days to fully resolve as they require remodeling of acid-base handling mechanisms in the urinary system. the most rapid response-and the first line of defense of our body-to an acid-base disorder is given by chemical buffering which usually occurs within minutes. in the following four sub-sections we will review the causes, consequences, and therapeutic paradigms for each of the four systemic acid-base disturbances. several of these considerations are also relevant to the resolution of local acid-base disturbances and will be revisited in sections and . for a more complete and clinical perspective on these disturbances in isolation, and in combination, we refer the reader to reference [ ] . metabolic acidosis is defined as acidemia due to a primary pathological deficit in [hco -] rather than a physiological, compensatory lowering of [hco -] in response to respiratory alkalosis [ ] . the body can counter acid shifts in plasma ph in the short term by increasing respiratory drive to lower co and, in the longer term, by increasing renal h + excretion/hco generation. however, if these compensatory systems are defective or overwhelmed, mac will result. for example: mac can result from diet, chronic kidney disease, and diabetes (diabetic ketoacidosis) or can follow acute myocardial infarction (lactic acidosis), mutations in renal acid-base transporters (renal tubular acidosis, see section . ), intoxication with compounds (e.g., aspirin), and diarrhea (loss of hco --rich secretions) [ ] [ ] [ ] [ ] [ ] . clinical manifestations vary depending on underlying cause, but generally include weakness, nausea, and flushed skin [ ] . as we shall see, chronic mac has severe consequences for long-term health. in cases where mac is secondary to another disturbance such as in diabetes or diarrhea, treatment of the underlying disorder is the ultimate goal. however, for short term remediation of mac, or for situations in which the primary defect is with acid-base homeostatic mechanisms, the typical course of action is 'alkali therapy' to address mac by normalizing plasma ph. this can be achieved via two mechanisms. the following paragraph describes therapies that increase base load while the final paragraph describes therapies that lower acid load. increasing base load. the simplest paradigm is administration of hco salts. a direct rise in plasma [hco -] can be achieved either intravenously or by peritoneal dialysis. an indirect rise in plasma [hco -] can be achieved by oral dosing; as the parietal cells of the stomach replace neutralized stomach acid, they also generate new hco -, which is absorbed into circulation [ ] . there are however a number of caveats associated with hco administration [ ] . one caveat is that the counter anion (usually na + or k + ) may contribute to fluid retention or k + imbalance. a second caveat is that the treatment has the potential to rapidly generate co . with oral administration this can manifest as bloating or even gastric rupture, whereas with intravenous administration, the co , if not effectively eliminated by the lungs, can enter cells causing a paradoxical intracellular acidification. a third caveat is that ph overshoot (i.e., overcompensation that creates its own ph disturbance) is possible if the dose is not well titrated. however, in practice, manifestation of the side effects associated with nahco administration is not a foregone conclusion [ , ] . alternative vehicles for intravenous alkali delivery such as na co and caco produce less co per neutralized h + and impose less of an osmotic stress [ ] . carbicarb is a mixture of nahco and na co that does not cause intracellular acidification [ , ] . citrate salts provide a gentler, indirect mean of raising hco as citrate is converted into hco in the liver. alternative buffers such as tham (tris-hydroxymethyl aminomethane aka tris-base aka tromethamine aka trometamol) bind h + without generating co and the protonated product is readily cleared by the kidneys [ ] . furthermore, because a certain proportion of tham is uncharged at physiological ph, it is cell permeable and can counter intracellular acidosis. other hco --replacing bases include lactate and acetate [ , ] . finally, potential side effects can be ameliorated by administering buffers at a lower dose as part of an intravenous cocktail of buffers. for example, tribonat is a mixture that includes nahco , na hpo , and sodium acetate [ , ] . an added bonus of that mixture is that the inclusion of phosphate counters the hypophosphatemia associated with mac. dietary acid load is associated with lower serum hco - [ , , ] and thus there is scope for dietary correction of mac by, for example, adherence to a very low protein [ ] or otherwise "alkaline" diet [ ] . ph imbalance in mac can also be redressed by increasing h + excretion. the thiazide diuretic hydrochlorothiazide increases h + secretion by the renal collecting duct and has been used as an adjunct therapy with nahco for mac [ ] . its role as a diuretic ought also to assist with excretion of the na + load associated with nahco treatment. veverimer is an orally dosed h + binding polymer that is in phase iii clinical trials at the time of writing for the treatment of mac in the context of chronic kidney disease (clinicaltrials.gov identifier: nct ). it binds h + in the stomach for eventual excretion in the feces [ , , ] . moreover, the raising of gastric ph by veverimer prompts parietal cells to deposit hco into circulation, mimicking the alkaline tide associated with feeding. another approach to counter mac, is to increase cellular h + consumption (and/or decrease lactic acid production) by metabolic means either by pyruvate administration or by stimulating pyruvate dehydrogenase using dichloroacetate (dca) [ , ] . malk is defined as alkalemia caused by a primary excess of hco -. malk may follow volume depletion or hyperaldosteronism (promotes renal h + secretion), vomiting (eliminate gastric acid, stimulating an alkaline tide), or the use of certain pharmaceuticals that mimic those responses (loop diuretics, antacids). clinical manifestations can include confusion and tetany [ ] . malk can also have a genetic cause. for example, liddle syndrome is associated with hyperactivity of the epithelial na + channel enac, the action of which promotes renal h + secretion [ ] . besides treatment of the underlying conditions, correction of malk has been achieved using the ca inhibitor acz, which by itself results in mac [ ] , by intravenous infusion of hcl [ ] , or (if malk follows loss of gastric acid) the use of h -receptor agonists to prevent alkaline tide [ ] (see section . . ). rac is defined as acidemia with a plasma pco > mmhg at rest and at sea level [ ] . it usually occurs when there is a disruption in the ventilatory system that causes a mismatch between the rate of co removal and the rate of co production, with consequent accumulation of co into the blood (i.e., co retention). this disruption can be caused by (i) inability of the lungs to remove the metabolically produced co (i.e., reduced ventilation), (ii) defects in co transport from tissue to lungs and (iii) overproduction of co . reduced ventilation can result from a depression of the respiratory center (e.g., due to sedative overdose or brain injury), airway obstruction (e.g., due to vomit aspiration or laryngospasm), neuromuscular disorders (e.g., due to guillain-barré syndrome) or restrictive defects of the chest (e.g., due to impaired functioning of the diaphragm) [ ] . defects of co transport that lead to hypercapnia are less common and usually the result of reduced pulmonary perfusion in response, for example, to cardiac arrest or pulmonary embolism. overproduction of co is rarely the sole cause of rac. in fact, under normal circumstances the body responds to increases in co production by appropriately increasing ventilation in order to remove the excess co and prevent hypercapnia. situations in which the lungs are unable to match the increased co production can occur in patients undergoing mechanical ventilation or with reduced respiratory reserve [ ] . in fact, for therapeutic reasons, individuals on mechanical ventilation are often deliberately maintained in a state of ''permissive hypercapnia'' (see section . ). as for metabolic disturbances, rac can be either acute or chronic. acute rac occurs when pco rises very rapidly and the kidneys are unable to adequately increase hco production to compensate in such a short amount of time. thus, only a very modest renal compensation occurs. on the contrary, during the longer timespan of chronic rac (such as with chronic obstructive pulmonary disease, copd), the kidneys are able to restore the acid-base balance by increasing acid excretion and hco production [ ] . treatment is usually directed towards reversing the underlying cause and also at restoring adequate alveolar ventilation, which can be accomplished by endotracheal intubation with mechanical ventilation or positive pressure ventilation [ ] . because the sum of pco and po must be constant in the alveolar gas of patients breathing room air, hypercapnia leads to hypoxemia, a condition that can have consequences far more dangerous than those caused by hypercapnia [ ] . consequently, management of acute respiratory acidosis is often also directed towards ensuring adequate oxygenation. administration of o must be performed carefully because it may lead to increased co retention, especially in patients with copd [ ] . correction of hypercapnia in chronic rac usually occurs slowly because rapid reduction of pco can lead to overshoot alkalosis due to the renal compensation that increases [hco -]. in the central nervous system (cns), rapid alkalinization of the cerebrospinal fluid (csf) can cause seizures and even coma [ ] . the use of alkali therapy in rac is controversial and indicated only in patients with acute hypercapnia and concurrent metabolic acidosis [ ] . administration of nahco is contraindicated because it may increase co production, reduce alveolar ventilation as well as cause a paradoxical acidosis in the cns. as noted above for mac, alterative alkali therapies such as carbicarb that do not generate as much co as nahco alone (see section . . ) may be preferable to correct ph in rac. in patients with copd, the ca inhibitor acz is sometimes used to stimulate respiration in order to improve oxygenation, reduce co retention and possibly remove the need for mechanical ventilation [ ] . however, because ca is ubiquitous, the inhibitory effect of acz may impact a variety of tissues and have potential negative consequences on patients with pulmonary diseases. for this reason the role of acz as a respiratory stimulant is controversial, especially in patients with severe copd with or without hypercapnia [ ] . finally, co can be de-gassed from blood using an extracorporeal co removal (ecco r) device or [ ] lowered by dialysis using a dialysate that has a low [hco -] [ ] . ralk refers to alkalemia with a plasma pco < mmhg at rest and sea level [ ] . it occurs when the ventilatory system does not work properly causing an increase in alveolar ventilation and/or reduced co production with consequent co depletion in the blood. hyperventilation can result from stimulation of the respiratory centers (e.g., due to drugs or disorders of the cns), hypoxemia or tissue hypoxia (e.g., due to high altitude), lung diseases (e.g., pneumonia). reduced co production can result from a decrease in the basal metabolic rate (e.g., due to hypothermia) or in physical activity (e.g., due to muscle paralysis). clinical manifestations can include rapid breathing and dizziness [ ] . although usually considered not life-threatening, severe ralk can have serious consequences on the brain, lungs and the heart. treatment is usually directed towards correcting the underlying disorders. hormone replacement therapy caused ralk in a study of postmenopausal women [ ] . abrupt correction of severe ralk should be avoided because of the risks of cerebral and pulmonary reperfusion injury. acz is used in the prevention and treatment of ralk associated with hyperventilation at high altitude (acute mountain sickness: ams) in part because it enhances hco excretion in the urine, providing a compensatory lower of ph [ ] . neuronal activity presents a substantial challenge to local acid-base balance. neurotransmitterfilled vesicles release h + into the synaptic cleft [ , ] (h + themselves may be considered to be neurotransmitters [ ] ) and are removed from the synaptic cleft by h + -coupled neurotransmitter transporters such as the excitatory amino acid transporter eaat . gaba-activated anion channels in neurons and astrocytes release hco - [ , ] , and the ca + /h + exchange activity of the plasma membrane ca + -atpase (pmca) in neurons causes a rise in extracellular ph as it restores intracellular ca + following an action potential [ ] . the acid load that results from intensive neuronal firing can result in a drop in phi that dampens neuronal activity: a mechanism that prevents excessive firing via effects upon ph-sensitive channels such as asic a and nmda receptors [ ] [ ] [ ] . conversely, alkalosis is associated with an increase in neuronal activity and seizures [ ] . neurons and astrocytes express numerous abts and cas to maintain ph homeostasis and their importance is highlighted by the effects of their disruption [ ] . for example, genetic disruptions in ae or nbcn are associated with epilepsy [ , ] , although the mechanism is not simply related to effects of neuronal phi on excitability as ae is an acidloader while nbcn is an acid extruder and may depend on whether the neurons in question are excitatory or inhibitory. several abts and cas are expressed in the choroid plexus epithelia where their action supports the secretion of cerebrospinal fluid (csf). genetic ablation of these transporters (e.g., nbcn , nbce ) in rodents is linked to reductions in ventricle fluid volume [ ] while pharmacological inhibition of cas results in reduction of intracranial pressure [ ] . however, it is unclear whether these changes are accompanied by a fall in ph of the csf. besides its role in determining ph, hco plays an important role in neuronal plasticity because the transmembrane gradients of cland hco determine the reversal potential of gabaactivated channels and consequently whether gabaergic signals are depolarizing and excitatory or hyperpolarizing and inhibitory [ ] . changes in these gradients are important in two ways. firstly, developmental changes in the gradient during central nervous system maturation promote the switch to inhibitory gaba signaling [ ] . secondly, activity-dependent changes in the gradient contribute to the pathophysiology of epilepsy by promoting a pathological switch to excitatory gaba signaling [ ] . neuronal cl − -hco − exchangers such as ae and ndcbe are likely to contribute to the status of these gradients [ ] . finally, mutations in endosomal nhe cause intellectual disability and are associated with defective synaptic remodeling [ ] . acidosis has a number of other consequences. for example in stroke, lactic acidosis is linked to ischemic damage [ ] . in protein-aggregating neurodegenerative diseases, acidic ph promotes the aggregation of alzheimer's amyloid proteins [ ] . a major genetic risk factor for alzheimer's disease is incidence of the apolipoprotein e allelic variant apoe , which causes the epigenetic downregulation of nhe [ ] . loss of nhe from endosomes causes aberrant acidification and defective clearance of amyloid deposits [ ] . brain acid-base status also has consequences for mental health (see mental health). the role of ph in the retina is considered in a later section (see the sensory systems). the link between ph and neuronal excitability is exploited in the anticonvulsant value of inhaled % co to induce hypercapnic acidosis [ ] . hypercapnia also has a neuroprotective role in stroke, by inhibiting caspase and other cytotoxic activities [ ] , and during reperfusion [ ] . ca inhibitors are used as adjunct therapies for epilepsy [ ] and have potential application for treatment of neuropathic pain [ ] , alzheimer's disease [ ] , and cognitive disorders [ ] . however, mac is a side effect of systemic ca inhibition [ ] . lowered seizure thresholds in some strains of abt-null mice suggest that abts may be potential targets for anticonvulsant therapy. however, the need for caution is shown by the observation that, at least in the case of nbcn -null mice, a reduced seizure-threshold does not mean reduced neuronal excitability [ ] . the role of abts and cas in csf secretion hints at the potential for targeting of these proteins to lower intracranial pressure in idiopathic intracranial hypertension (iih). the use of ca inhibitors in patients with iih produces some symptom relief, but the mechanism of action is uncertain [ ] . regarding therapies for neurodegenerative diseases, histone deacetylase inhibitors have shown potential to release nhe from its epigenetic restraints to restore amyloid protein processing in apoe mice [ ] . another strategy that has been proposed to have potential to reverse amyloid deposition in alzheimer's disease is the raising of brain ph [ ] . therapies that target the peripheral nervous system are discussed in the following section. sight. most ocular tissues express one or more abt or ca for the purpose of maintaining fluid and ph balance. perhaps the most therapeutically tractable tissue is the ciliary body that employs caii and a range of abts to secrete hco --containing aqueous humor into the anterior chamber [ , ] ( figure ). this fluid leaks into the corneal stroma to flush out metabolic wastes and is returned to the anterior chamber by corneal endothelial cells which express a similar array of abts including nbce , mct , and the h + channel slc a [ , ] . finally, the fluid is drained from the anterior chamber via the trabecular meshwork. individuals with mutations in nbce have band keratopathy, glaucoma, cataracts, and corneal edema linked to fluid/ph imbalance in the cornea, lens, and elsewhere [ ] . abts and hco are also important for retinal function [ ] [ ] [ ] , as suggested by the link between nbcn mutation and progressive rod-cone dystrophy [ ] , or retinal degeneration in mice with defective expression of nbce and mcts [ , ] . hearing loss is a symptom of several systemic diseases linked to defects in abts, including pendrin (pendred syndrome [ ] ), the h + /k + -atpase (distal renal tubular acidosis [ ] ), and slc a (harboyan syndrome [ ] ). all of these abts are expressed in the inner ear where they help to maintain inner ear fluid ph and endocochlear potential [ ] . although a human correlate has not yet been reported, progressive hearing loss is also a feature of nbcn null mice [ ] . disruption of the h + -channel otop and the anion exchanger pendrin in mice is associated with malformation of the caco crystals (otoconia) that are essential for maintenance of balance [ , ] . taste. in addition to its role in the inner ear, otop is required for sour taste sensation [ ]. pain sensation. it is generally recommended to keep the ph of injected formulation close to physiological ph to avoid injection-site pain, with the added note that the inclusion of certain buffers may increase pain (hence new citrate-free formulations of adalimubab aka humira®) [ ] . low phe exacerbates sensation of pain due to its effects on trpv channel activation in nociceptive neurons. furthermore, activation of these channels under acidotic conditions is associated with a drop in neuronal phi that is mediated in part by a trpv -mediated h + sight. ca inhibitors applied as eye drops have long been used to treat glaucoma by virtue of their ability to reduce the production of aqueous humor, although even their localized ophthalmic use has been documented to lead to the side-effect of systemic mac in some prone individuals [ , ] . corneal edema that results from the expression of mutant misfolded slc a may be amenable to correction by small molecule folding chaperones [ ] . nhe blockers are cytoprotective in a rat model of diabetic cataract formation and retinopathy [ ] . hypercapnia is protective against ischemia-reperfusion injury in the retina [ ] , as it is elsewhere in the central nervous system (see section . ). nahco solution is useful for softening and dispersing hardened ear wax [ ] . however, we are unaware of any therapies specifically targeted to restoring the acid-base chemistry necessary for correct generation of endolymph or ostoconia. on a related topic there is one side effect of ear drops that pertains to acid-base balance. the acetic acid in some ear drops used to treat outer ear infection can be ototoxic because acetic acid can move across the round window into the inner ear, resulting in a drop in endocochlear potential (perhaps by acid inhibition of the na + /k + -atpase) and endolymph and perilymph ph [ ] . we are unaware of any demonstrations of the usefulness of otop modulation in this area, but inhibitors of proteins that mediate bitter taste sensation have been used to mask bitter tastes, suggesting potential utility of otop block for masking sour tastes and increasing the palatability of sour-tasting medications [ , ] . pain sensation. adjuvant nahco raises the ph of an injectable lidocaine solution and lowers perception of pain associated with lidocaine injection in one study, but the mechanism of the effect is uncertain [ ] . see also sections . (anesthesiology). besides the increased respiratory drive to exhale co in response to rac [ ] and the bohr effect (see section . the circulatory system) the highest profile link between ph and respiration relates to the role of cftr. defects in cftr are devastating because the cland hco secretion that this channel normally mediates is a fundamental part of the mechanisms that drive fluid secretion in our bodies [ ] ( figure ). the majority of deaths associated with cf are caused by respiratory failure [ ] . in the lungs, secretions are required to provide a moist surface for gas exchange, to liquefy mucus, and to flush inhaled particles and pathogens out towards the throat (mucociliary clearance). besides the general importance of anion secretion, cftr-mediated hco secretion plays a further role in ph homeostasis in the airway surface liquid (asl); hco helps to unfold and hydrate mucus [ ] and, by defending airway ph, has been hypothesized to promote a healthy local immune response to airway bacteria [ , ] . hco secretion is modulated by epithelial h + secretion mediated by a host of acid-extruding transporters [ ] ( figure ). airway acidification is a feature of individuals with cystic fibrosis, as well as those with asthma and tuberculosis [ ] and is exacerbated by lactic acid production by airway pathogens and airway epithelia [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] , but are targeted to individuals with specific cf genotypes and thus alternative general therapies are still required. strategies specifically focused on correcting asl ph include inhalation of nebulized bases such as nahco [ , ] and tham [ ] as well as block of airway h + secretion using h + /k + -atpase inhibitors [ ] . all these strategies result in improvements in asl ph and some also improve mucus viscosity and/or pathogen clearance. an in vitro study suggests that mct blockade could also be protective of asl ph in individuals with cf [ ] by reducing epithelial h + secretion. a newly described paracellular pathway for hco secretion by cf airway epithelia might also be amenable to therapeutic modulation [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] (e.g. lumafactor/ivacaftor, see section . . ), but are targeted. heart. mac is associated with reduced cardiac contractility. this phenomenon is explained by diverse mechanistic elements such as the ph-dependence of the channels and transporters that regulate ca + handling in myocytes as well as the dampening effect of acidosis on the responsiveness of the contractile apparatus to ca + [ ] . whether the heart rate is lowered by acidosis is harder to predict because of the complex effects of acidosis upon the sympathoadrenal system [ ] . intracellular acidosis in myocardial infarction after a period of ischemia, is countered, during reperfusion, by the action of acid-extruders such as ncbts and nhes [ ] . however, the accompanying na + load can be sufficient to reverse the action of the na + / ca + exchanger, raising [ca + ]i and increasing susceptibility to ventricular arrhythmias [ , ] . paradoxically, the loss of nbce function can also result in ca + overload because compensatory acid-extrusion mediated by nbcn and nhe imposes double the na + load per hco equivalent; a mechanism proposed to promote hypertrophy of cardiomyocytes in spontaneously hypertensive rats [ ] . ca activity is also pro-hypertrophic [ ] . on the other hand, the action of the acidloading anion exchange ae is considered to be protective against hypertrophy [ ] . finally, nhe action in the mitochondria is proposed to contribute to mitochondrial damage in the diseased heart [ ] . vasculature. typically, acidosis causes arterial vessels to dilate resulting in a fall in peripheral resistance, while veins may constrict [ ] . it is perhaps then no surprise that numerous blood pressure traits are linked to polymorphisms in abt genes [ ] . at least at the level of the vascular response of arteries, nbcs and nhes are required for normal vascular smooth muscle contractility and sensitivity to vasodilators [ ] . however, blood pressure is a complex trait that is not determined by vascular response alone, so explanation of these linkages is not simple. another important aspect is that mac inhibits progression of vascular calcification [ ] . the bohr effect describes the influence of ph and pco upon the oxygen carrying capacity of hemoglobin. in systemic capillaries, metabolically produced co enters the red blood cells (rbcs) where it is hydrolyzed into hco and h + by the action of caii. the newly produced hco is then extruded by ae , causing a fall in rbc phi which, by the bohr effect, reduces the hb-o binding affinity, promoting o release from hb to tissue ( figure b ). the reverse process occurs in the pulmonary capillaries. here, as co leaves rbc phi rises thereby favoring o binding to hb ( figure b ). thus acidosis enhances o delivery into tissues, but diminishes o loading in the lungs [ ] . this relationship between ph and gas exchange is partly sensitized by the content of the hemoglobin-regulating molecule , -dpg (diphosphoglyceric acid) in rbcs, a parameter which itself is ph-dependent; , -dpg levels increase with chronic acidosis promoting o release [ ] . heart. exogenous expression of skeletal muscle caiii in mouse cardiomyocytes enhances defense of phi and preserves cardiac function during mac [ ] . nahco is used to counter lactic acidosis in cardiac arrest and during prolonged cardiopulmonary resuscitation, but aside from its value at normalizing pre-existing mac or hyperkalemia (acidosis promotes cellular k + release), compelling data that this treatment improves outcomes are lacking [ ] [ ] [ ] . nhe blockers have shown promise as cardioprotective agents in reperfusion injury [ ] and likely act by targeting both plasma membrane and mitochondrial nhe [ , ] . although the nhe blocker cariporide caused serious side-effects in clinical trials (see section . . ), alternative approaches are available. for example, a microrna that lowers nhe expression protects cardiomyocytes from apoptosis during prolonged endoplasmic reticulum stress [ ] . in addition, antibodies and drugs that block nbcs have also demonstrated cardioprotective properties in animal models of ischemia reperfusion injury [ , ] . just as blockade of acid-extruders is cardioprotective, so too is the stimulation of the acid loader ae . this has been achieved in cell models using the glycoside sasanqua saponin [ ] , an extract from a herb used in traditional chinese medicine. we are unaware of any reports of acid-base based therapies for blood pressure that directly target the vasculature, but a discussion of diuretics for lowering blood pressure in congestive heart failure is provided in the urinary system section. some alkali-containing therapies may enhance progression of vascular calcification [ ] while use of the ca blocker acz has therapeutic value in calcifying disease [ , ] , perhaps by lowering ph. one study has cautioned the use of nahco in congestive heart failure because, in the face of adaptively elevated , -dpg levels, a sudden rise in ph could result in a maladaptive increase in hb-o affinity and risk of myocardial ischemia [ ] . in skeletal muscles, the build-up of lactic acid during intense exercise correlates with muscle weakness and self-limiting fatigue. however, the contribution of lactic acidosis to those symptoms may not be as direct or major as once thought [ , ] . generalized acidosis may contribute to weakness via alterations in neuromuscular drive [ ] and/or a decreased driving force for lactate efflux [ ] . regardless, acidosis promotes degradation of muscle protein [ ] . a high estimated dietary acid load has been associated with frailty in elderly japanese women [ ] recovery from lactic acidosis is mediated by mcts, nbcs, and nhes [ ] , while caiii specifically has been shown to plays a role in defense from muscle fatigue [ ] . many studies suggest the utility of nahco for improving exercise performance. for example, induction of malk by ingestion of oral nahco solutions has been shown to improve exercise endurance [ ] and reduce perception of effort [ ] in limited trials. however, taking a broader view of the field, the results of trials that link ph and exercise performance are deemed inconclusive due to inconsistent methodology and subgroup effects [ , ] . it has also been suggested that any competitive benefits that could be gained from nahco administration, from an athletic viewpoint, may be outweighed by gastrointestinal side effects such as bloating [ ] . away from the arena, hco administration or a reduced dietary acid load could have value in maintaining muscle mass in older adults [ , ] . mineralized material is eroded by acids as is evident in the case of tooth enamel, which is subject to demineralization by dietary acids (see oral health). however acidosis also inhibits bone growth by inhibiting osteoblasts, stimulating the activity of bone-resorbing osteoclasts [ ] , and influencing hormonal axes [ , ] (see also section . about the effects of ph on the endocrine system). accordingly, serum [hco -] positively correlates with bone mineral density (bmd) [ ] and negatively correlates with levels of serum parathyroid hormone (which promotes bone resorption) [ ] . at a local level, the process of bone remodeling, as well as the hormonal mobilization of ca + and pi from bone, requires that osteoclasts secrete h + onto the bone surface. these cells express intracellular caii to generate h + and hco -, an apical v-type h + -atpase to secrete h + onto the bone surface, and basolateral ae to export hco and defend osteoclast phi from alkalosis during h + secretion ( figure ). mutations in the v-type atpase and caii disable bone resorption by osteoclasts and are associated with increased bone density and osteopetrosis in humans [ , ] . the acid-base regulating proteins of osteoclasts are amenable to pharmaceutical modulation and their blockade ought to be protective of osteoporosis. for example, ae may be a useful target for increasing bmd because bmd is elevated in ae -null mice and cattle (reviewed in ref. [ ] ). regarding caii, one study showed a fortuitous bone-sparing effect in post-menopausal women were chronic users of ca-inhibitors for glaucoma treatment, [ ] . another study showed a paradoxical, but therapeutically valuable, bmd-lowering effect of ca inhibition in three children with sclerosing bone dysplasias. in these children, osteoclasts are already defective so the predominant effect of ca-inhibition is induction of chronic mac which promotes bone resorption [ ] . the therapeutic utility of proton pump inhibitors (ppis) to treat osteoporosis is negated by their negative influence on intestinal ca + absorption [ ] . in fact, several studies link ppi use with fracture susceptibility and low bmd (reviewed in refs. [ , ] ). because of these side effects, ppis are used with caution in some groups who may take them as antacid therapy [ , ] (and see next section). the three major health-related aspects of acid-base in this system are the roles of salivary hco in defense of enamel (which are discussed in section . oral health), gastric acid secretion, and peptic ulceration with helicobacter pylori. gastric acid is required to activate digestive enzymes, stimulate downstream secretory processes, and to kill ingested pathogens. it is secreted by parietal cells using similar transport mechanisms employed by osteoclasts (described in the previous section). thus, the secretion of acid across the apical membrane is mediated by a h + /k + -atpase and is balanced by the extrusion of hco into the plasma via ae (the alkaline tide associated with feeding [ , ] , see figure ). stomach epithelia are protected from acid injury by a mucus lining. the pathogenic bacterium h.pylori is able to survive in gastric acid because it can take up urea from its environment, via a h + -gated urea channel, and convert it into nh to neutralize acid in its immediate environment [ ] . in the vicinity of the mucus layer, this action causes h.pylori to raise mucus ph, lowering its viscoelasticity, promoting bacterial infiltration, and ultimately resulting in inflammation, ulceration [ ] , and risk of gastric cancer [ ] . another condition, gastroesophageal reflux disease, is caused by reflux of gastric acid into the esophagus and can cause heartburn and, in severe cases, can lead to esophageal damage. salivary hco plays an important role in esophageal acid defense [ ] [ ] [ ] by neutralizing gastric acid [ ] . conversely, the action of nhe in esophageal epithelia may exacerbate the damage, perhaps by indirectly stimulating pro-apoptotic pathways [ ] . acid reflux symptoms can be relieved by neutralizing gastric acid with antacids, which at their simplest are just bicarbonate or carbonate salts (e.g., tums® is calcium carbonate). however, an early antacid regimen for peptic ulcers, based on administration of milk and caco and still observed in the modern age in self-medicating individuals, results in adverse outcomes: the so-called 'milk-alkali syndrome' characterized by malk and hypercalcemia [ , ] . an alternative approach to lowering gastric ph is to use ppis or h -receptor agonists which dampen the signaling pathways that stimulate h + secretion. h agonists, in addition, therapeutically lower the activity of esophageal nhe [ ] . some over-the-counter formulations combine these drugs with an antacid to lower the dose of each and minimize side effects of each such as bloating (from gastric co generation) and osteoporosis (from chronic inhibition of intestinal ca + reabsorption, see section . ). orally-dosed acid-chelators such as veverimer, also raise gastric acid ph [ ] but have not been tested as a therapy for heartburn. the achlorhydric phenotype of ae -null mice suggests that ae blockage may have potential as a therapeutic target [ ] . ppis in combination with antibiotics are used to treat h.pylori infections: it has been proposed that raising stomach ph permits faster bacterial growth, potentiating the effects of antibiotics that act on dividing bacteria [ ] . inhibitors of the urease and h + -gated urea transporter of h.pylori are potential therapeutic modalities that remain in development [ ] . the exocrine pancreas secretes a hco --rich fluid that is vital for neutralizing gastric juices passing into the duodenum. the alkaline ph of pancreatic juice holds digestive enzymes such as amylase and lipase in an inactive state until the secreted fluid is neutralized in the duodenal lumen by chyme, preventing damage to the pancreatic ducts. in cf, duodenal hyperacidity also holds pancreatic enzymes in an inactive state, but without the neutralization of chyme, they are not even active in the duodenum leading to malabsorption of nutrients such as lipids [ ] . all along the intestine, hco --containing fluid secretions are required to promote gastric motility. the loss of this fluid in feces represents a substantial acid load. consequently, cftr mutations result in intestinal blockage [ ] and secretory diarrhea can result in mac [ ] . balancing secretory processes, nhe and slc a promote fluid reabsorption ( figure ). accordingly, downregulation of intestinal nhe by the enterotoxigenic bacteria (e.coli and c.difficile) and inactivating genetic defects in nhe and slc a are all associated with hypersecretion and diarrhea [ ] [ ] [ ] [ ] . on the subject of gut microbiota, intestinal ph can both influence and be influenced by the composition of gut microbiome [ ] . in individuals with insufficient intestine to absorb nutrients (short bowel syndrome), unabsorbed carbohydrates promote the growth of lactic acid-producing bacteria, which can lead to d-lactic acidosis [ ] . finally, the absorption of many nutrients depends on the action of h + -coupled abts (e.g. the h + -coupled oligopeptide transporters of the slc family: [ ] ), which in turn require the presence of acid extruders such as nbce to maintain epithelial ph during nutrient absorption. indeed, nbce -null mice exhibit defective nutrient absorption, which contributes to their general failure to thrive [ ] . the ability of small molecule inhibitors of nhe (tenapanor) and slc a (the , dimethylcoumarin drug "drainh-a ") to reduce intestinal fluid absorption makes them valuable therapies for irritable bowel syndrome with constipation and for relief of constipation in cf [ , ] . the cftr corrector ivacaftor improves intestinal hco secretion and nutrient absorption in individuals with cf [ ] . the mode of action of the anti-constipation drug linaclotide (linzess®: a guanylate cyclase c receptor agonist) encompasses both paradigms by the cgmpmediated reduction of nhe [ ] and activation of cftr activities [ ] . nephron function. the kidneys are vital to whole body ph balance (see acid-base homeostasis). it is the kidneys that generate hco -, reabsorb hco from the glomerular filtrate to prevent its loss in urine, and excrete h + in the form of nh + or titratable acids such as phosphate [ ] ( figure ). thus, it is no surprise that defects in renal transport mechanisms result in mac. these acidifying diseases can be acquired or genetic. fanconi syndrome is a degeneration of the proximal tubule, while renal tubular acidosis (rta [ ] ) can result from mutations in acid-base transporters such as nbce (type ii proximal rta: prta), h + -atpase or ae (type i distal rta: drta), caii (type iii rta), or disruption of h + secretion due to hypoaldosteronism (type iv drta). chronic kidney disease (ckd) is also associated with mac [ ] , and mac itself promotes progression of ckd (see below). malk is a common finding in cf patients. cf-model mice are less capable of defending against hco loads than their wild-type counterparts, due to downregulation of the renal hco -secreting anion exchanger pendrin [ ] and presumably loss of direct cftr-mediated hco secretion. concurrently, acid-base status has profound influence on kidney function. the very mechanisms that allow the kidneys to increase acid excretion in response to acute increases in acid load (e.g., ammoniagenesis and the renal endocrine response to acidosis) can be maladaptive in chronic mac, leading to inflammation and fibrosis [ ] . it is perhaps then not coincidental that low serum [hco -] is linked to a higher risk of chronic kidney disease in both adults and children [ , ] . an additional set of renal pathologies follows the integration of acid/base and salt/water handling by the nephron. for example, states and conditions of increased sodium reabsorption by the proximal tubule (e.g., volume contraction) or collecting duct (e.g., hyperactivity of the epithelial na + channel enac in liddle's syndrome) result in malk (see metabolic alkalosis) while hyperkalemia can cause mac [ ] . stone formation. urinary ph can influence stone formation which can lead to inflammation and obstructive kidney injury. a high urinary ph can cause the formation of calcium oxalate or calcium phosphate crystals, while a low urinary ph promotes uric acid crystallization [ ] . urinary ph can be modified by uropathogenic bacteria. urease-expressing bacteria generate nh , which can substantially raise urinary ph, promoting deposition of struvite and apatite crystals [ ] . besides the consequences of stone formation in the urinary tract, these deposits can cause the encrustation and blockage of indwelling catheters [ ] . it is interesting to recall that the pathogenic action of another bacterium, h.pylori, in the stomach also depends on urease action (see section . ). nephron function. many studies point to the value of correcting mac for preserving the function of the failing kidney and slowing ckd progression [ ] [ ] [ ] . we outlined corrective strategies based around alkali therapy in section . . ), but there is an additional prophylactic value in emergency settings. nahco infusion is protective against the kidney damage that can result from traumatic rhabdomyolysis (due to a crush injury), preventing development of mac and tempering the renal toxicity of myoglobin [ , ] . another consideration related to therapies is that a number of drugs cause metabolic acid-base disturbances because they are nephrotoxic [ ] or incidentally interfere with the kidneys ability to excrete acid [ ] . for example, ca inhibitors such as acz that are used as diuretics, due to their ability to interfere with fluid reabsorption, also cause mac [ ] . on the other hand, loop diuretics use can cause malk [ ] . another example are penicillin antibiotics which, acting as significant non-reabsorbed anions in the collecting duct lumen, promote hypersecretion of k + and h + , resulting in hypokalemia and malk [ , ] . finally in this section, the ability of cf kidneys to secrete excess hco is restored by treatment with the cftr restoring drug cocktail lumacaftor/ivacaftor (orkambi®) [ ] . stone formation. both citrate and low ph discourage the formation of calcium precipitates [ ] . thus, ingesting lemon juice, which raises urinary citrate while lowering urinary ph, decreases the propensity to form kidney stones and catheter-blocking deposits [ ] . dietary supplementation with citrate salts is also effective for this purpose because, despite resulting in a rise in urinary ph, the accompanying rise in urinary citrate increases the ph of crystal nucleation to an even higher value [ , ] . urinary ph can also exert a meaningful influence on drug excretion as discussed later (see section . ). at this point in our review we have presented ample evidence that abts are necessary to sustain life, and now we will see that they are also necessary to create new life. in the male reproductive tract, h + secretion by clear cells in the tail of the epididymis is required to maintain an acidic luminal ph for storage of sperm [ ] . hco secretion along the length of epididymis is necessary to functionally activate sperm before ejaculation [ ] and prevent their inactivation by the acidic vaginal environment (discussed later). indeed low levels of hco are associated with lowered sperm motility [ ] . in the female reproductive tract, endometrial epithelial cells further secrete a hco --rich fluid that is necessary for sperm capacitation and fertilization [ ] . furthermore, the secretory phase of the uterine cycle is associated with a dramatic rise in the ph of the oviduct lumen, corresponding with a level of hco that is sufficient to promote thinning of mucus during ovulation to promote sperm mobility [ ] and to promote dispersal of the egg-surrounding corona cells to allow the sperm access for fertilization [ ] . ultimately hco is even a prerequisite for the acrosome reaction [ ] , by virtue of its ability to stimulate soluble adenylyl cyclase to produce camp and initiate requisite signaling cascades [ ] . finally, once fertilization has occurred, acidification of uterine fluid is a necessary prerequisite for embryo implantation [ ] . numerous abts are involved in these processes; for example, loss of ae , slc a , cftr, or nhe are all associated with infertility or reduced fertility in male mice [ , [ ] [ ] [ ] . the acidic ph of the vagina noted earlier is caused by the metabolic activity of lactobacilli and serves to defend against sexually transmitted disease pathogens. loss of the acidity in bacterial vaginosis is associated with increased susceptibility to std infection [ ] . with regard to ultimate reproductive success, maternal-fetal acid-base balance is an important determinant of perinatal outcomes [ ] . for example, obstructed labor has poor outcomes due to intermittent hypoxia and lactic acidosis [ ] . because vaginal acidity tends to dampen sperm motility, vaginal douching with nahco improves fertility [ , ] . conversely, vaginal acidification is contraceptive and prophylactic. the spermicidal properties of lemon juice have long been appreciated [ ] . phexxi™ (formerly known as acidform: [ ] ), is the most recent of a series of acidic contraceptive gels. phexxi™ is a vaginally-applied gel of lactic acid, citric acid, and potassium bitartrate that is indicated by the fda to prevent pregnancy [ ] . an alternate approach 'buffergel' utilizes an acidic polymer for the same purpose [ ] . by lowering vaginal ph these products also confer microbicidal benefits [ ] . finally, some abt-targeted drugs interfere with fertility: ca inhibitors, for example, prevent dispersal of corona cells [ ] and ppis inhibit sperm motility [ ] . finally, with regard to childbirth, peri-operative nahco infusion has been proposed as a possible measure to improve outcomes in obstructed labor [ ] . many metabolic reactions that consume or generate acids and bases can, in disease, result in mac. the liver makes important contributions to acid-base balance by consuming lactate (countering lactic acidosis), generating albumin (a weak acid: hypoalbuminemia is associated with malk) and producing keto acids (contributing to diabetic ketoacidosis) [ ] . furthermore, acidbase status impacts the activity of the enzymes that constitute several key metabolic pathways. for example, acidosis inhibits glycolysis [ ] and lipolysis [ ] but stimulates gluconeogenesis [ , ] . the ability of disturbed acid-base balance to interfere with glycemic control is further evidenced by the following observations: (i) acidosis and alkalosis both lower glucose-stimulated insulin release from pancreatic islets [ ] ; (ii) the hco content of plasma correlates with insulin solubility [ ] , (iii) acidosis is associated with decreased insulin sensitivity [ , ] . this latter observation is due in part to the ph-sensitivity of the interaction between insulin and its receptor: a bell-shaped ph-dependence that exhibits strongest binding at ph ~ . [ ] . in fact, insulin can also influence abt action. for example, insulin promotes renal nbce activity. in type diabetes the resulting pathological increase in renal fluid absorption caused by nbce upregulation is thought to contribute to hypertension [ ] . other hormones that influence acid-base balance include secretin (increases pancreatic hco secretion in response to duodenal acidity [ ] ), angiotensin ii and aldosterone (increases renal acid excretion in acidosis [ , ] ), and parathyroid hormone (increases renal acid excretion and excretion of urinary-buffer phosphate in acidosis: [ ] ). interestingly, licorice can cause malk because one of its constituent compounds (glycyrrhizic acid) indirectly causes overstimulation of the aldosterone receptor [ ] . hormones whose levels are pathologically altered in acidosis include aldosterone and endothelin (increased: [ , ] ) cortisone (increased: [ ] ), and igf- (decreased: [ ] ). metabolic reprogramming of cancer cells in the hypoxic and acidotic tumor environment is discussed further in a later section (see . oncology.) the influence and therapeutic relevance of ph upon endocrine and metabolic aspects of heart function, muscle mass, and bone growth have been discussed in earlier sections ( . , . , and . ). the link between dietary acid load and development of insulin sensitivity has made dietary control an appealing target for lowering the incidence of type diabetes, although overwhelming evidence of efficacy is currently lacking [ ] . because of the ph-dependence of insulin solubility, the dissolution time of administered insulin is slower in plasma from diabetics with dka than in otherwise normal plasma, suggesting at face value that combined insulin-bicarbonate therapy might be valuable [ ] . however, such treatment in practice may be of limited value and has been linked to development of cerebral edema in children [ ] . the influence of ph on drug solubility is discussed in more detail in section . . ph plays a role in all aspects of the immune response. first we will enumerate the subcellular effects: (i) the cytosolic alkalinization that promotes cytoskeletal rearrangement during neutrophil spreading (the morphological change that is important for capillary adhesion and extravasation) requires nhe action [ ] . (ii) inflammatory sites are usually acidic due to the metabolic activities of invading bacteria and neutrophils, an environment that promotes the production of proinflammatory cytokines [ ] (see also section . ). (iii) during bacterial killing, h + efflux into the phagosome is necessary for charge compensation during the respiratory burst that produces cytotoxic superoxide anions. this action is mediated by the voltage-gated h + channel hv [ ] . (iv) some of the generated superoxide is converted into cytotoxic hypochlorous acid (hocl) which is able to diffuse back into the neutrophil cytoplasm. thus the action of phagosomal hv , together with the action of nhe in the plasma membrane, defends the neutrophil cytoplasm from acidosis which would otherwise dampen nadph oxidase activity [ , , ] . secondly we can consider the effect of acid-base disturbance on the whole cell. although the ph sensitivity of individual immune cell types are well characterized in vitro (e.g., acidosis decreases leukocyte and neutrophil mobility [ ] , promotes complement activation [ ] , modulates expression of inflammatory mediators [ ] ), there are many subtleties to these effects. for example the type of acidosis, type of cell, activation state of the cell, and effects on phagocytic activity versus migration may all influence the effect of acid-base disturbance on the immune response mediated by a given cell type [ ] . thus, for example, acidosis enhances bacterial killing by neutrophils [ ] and leukocytes [ ] but not by macrophages [ ] . although it is complicated to tease out a set of concerted mechanisms, in general, systemic acidosis is associated with compromised immune function [ , ] . [ ] . the link between ph and disturbed immune response is demonstrated by the following example. single nucleotide polymorphisms in the acid-loading protein ae are linked to progression of primary biliary cholangitis. a mechanism is suggested by studies of ae -null mice in which loss of ae from cytotoxic t-cells causes a phi increase that promotes their proliferation, activation and survival, amplifying the autoimmune response against damaged liver cells. furthermore, type iv drta is linked to systemic lupus erythematosus, although the causal relationship is unclear [ , ] . several paradigms have been proposed to suppress a pathological immune response. oral nahco dosing in rats stimulates an anti-inflammatory response; this effect could be harnessed to prevent tissue damage in autoimmune diseases such as rheumatoid arthritis [ ] . one preliminary study in mice even suggests that oral nahco dosing could be useful to suppress peanut allergy [ ] . regarding the pharmacological aspect, mct inhibitors act as immunosuppressors by interfering with the disposal of lactate during t-cell activation [ ] . finally, the nhe blocker cariporide suppresses the systemic immune response to burn injury in rats, although the mechanism is unclear [ ] . on the other hand, knowledge of acid-base balance can be exploited to promote an immune response. it has been suggested that blocking ae to promote a stronger cytotoxic t-cell response could be useful for treatment of chronic infections [ ] . considerations about the role of ph in cancer immunotherapy are included in section . . neuronal activity is ph dependent (see section . ) and there is emerging evidence that agents of acid-base balance can influence behavior and progression of neuropsychiatric disorders. inhalation of co (despite its general dampening action on neuronal excitability, see section . . ) invokes anxiety and panic, and the influence of co is exacerbated in individuals with panic disorders [ ] . studies in mice indicate that lowering brain ph triggers the action of the acidsensing ion channel asic a in the regions of the brain responsible for stress, fear, and social behavior [ , ] . the acquisition of fear-related freezing behavior in mice is enhanced by asic a overexpression [ ] and dampened by asic a disruption [ ] . in humans, decreased brain ph is also associated with schizophrenia, bipolar disorder, and autism spectrum disorder [ ] . one study even suggests that a high dietary acid load correlates with incidence of emotional problems and hyperactivity in young children, but causality could not be established as could not be discounted that behavior influences dietary habits [ ] . in terms of the linkage between abts and neuropsychiatric disorders, slc a is a biomarker (both in terms of incidence of a specific single nucleotide polymorphism and in terms of reduced expression determined by microarray) of suicide ideation and completion, especially with bipolar disorder [ ] . although the mechanistic details of the linkage are unknown, the role of nbce in control of brain ph is likely to be relevant. perhaps also of tangential relevance, due to its impact upon systemic acid-base balance [ ] , is that estimated glomerular filtration rate in ckd, which typically correlates with ability to excrete acid, is inversely correlated to depressive symptoms and suicide ideation [ ] . the anxiety response to co inhalation is a useful clinical test to follow the effectiveness of treatments for panic disorders [ ] . the response itself can be quelled by acz [ ] but, as this is just a model of panic disorder, the use of ca blockers to treat actual panic disorders is unclear. the above-mentioned studies of asic a-null mice suggest that asic a inhibition could be therapeutic for panic disorder. regarding the link between slc a and suicide, it is unclear how modulating nbce activity might influence depression, although detection of the biomarker could help to identify high risk individuals and thereby inform therapeutic strategies [ ] . finally, it has been suggested that antipsychotic medications could contribute to lactic acidosis and be partly responsible for decreased ph in the brains of individuals with schizophrenia [ ] . as mentioned previously, plasma ph depends on adequate ventilation to exhaust co thus mechanical ventilation can induce respiratory acid-base disturbances. another important aspect for consideration in this section is that the bioavailability of anesthetic agents can be influenced by acid-base status. when using mechanical ventilation, two important considerations, related to acid-base balance, must be raised. the first consideration relates to low-flow anesthesia or closed-circuit rebreathing systems that return exhaled anesthetic gas mixtures. in these systems co must be removed from the recirculated air. for this purpose, co scrubbers are used to adsorb co . the archetypal scrubber is soda lime (a mixture of naoh and ca(oh) , which reacts with carbonic acid to form an insoluble caco precipitate), although a number of other technologies are available [ , ] (see also section . . ). the choice of technology can be important as many co scrubbers can have undesirable reactions with anesthetic gases, producing toxins such as carbon monoxide [ ] . the second consideration is for individuals in which low-tidal-volume ventilation is indicated, such as those with acute respiratory distress syndrome or copd. these individuals may be deliberately under-ventilated to prevent mechanical stress on the lungs. thus, patients are maintained in a state of compensated rac called "permissive hypercapnia". under some circumstances, such as in critically ill patients, this hypercapnic state may be protective due to its anti-inflammatory influence [ ] (see also the immune system). acid-base balance concerns are not limited to inhaled anesthetics. prolonged infusion with the intravenously administered anesthetic propofol can cause severe lactic acidosis [ ] . the influence of ph on the pharmacokinetics of drugs in general is discussed in section . perioperative interventions have the potential to disturb acid-base homeostasis with negative consequences for outcome. post-operative metabolic acidosis is a well described but complex phenomenon related to issues including lactate accumulation in poorly perfused tissues and hyperchloremic acidosis due to dilution/displacement of hco --containing plasma by infused saline [ ] [ ] [ ] [ ] . pre-operative acidosis has also been described and has been linked to stress and fasting [ ] . post-operative malk has also been described following general surgery and has been linked to the infusion of citrate-buffered plasma [ , ] . peri-operative malk is linked to the removal of stomach acid by nasogastric suction. poor outcomes have been associated with both post-operative mac [ ] and malk [ ] , although this is not a universal finding [ ] . on a related theme, the usefulness of stored blood for transfusion can be compromised by numerous storage lesions including low ph that follows anaerobic metabolism by stored cells [ , ] . finally, an acidic tissue environment appears to favor natural wound healing, yet alkaline ph favors the success of skin grafts [ ] . altering the chemistry of infused fluids is the obvious strategy to counter post-operative acid-base disturbances. with specific regard to post-operative mac, dichloroacetate treatment tempered the pathological rise in lactate following liver transplantation, although no effect on outcome was observed [ ] . other treatments for mac are discussed in section . . . treatment for malk are discussed in section . . . finally, a study in mice suggests that raising the ph of stored blood enhances red blood survival after transfusion [ ] . the ph-dependence of wound healing suggests that therapeutic maintenance of the ph of the wound or graft could aid healing [ ] . hco is a major buffer in stimulated saliva [ ] , defending oral ph against acidic foods and drinks and against those acids produced from sugars by acidophilic bacteria in the oral cavity. acid defense protects enamel from erosion and oral ph is also an important determinant of a healthy oral microbiome; low salivary flow and ph generally encourage the presence of pathogenic and cariogenic bacteria (e.g., [ , ] ). as a consequence, low salivary ph, [hco -], and/or buffer capacity are predictors of cavity formation [ ] [ ] [ ] . salivary ph is lowered in many groups of individuals such as patients undergoing chemotherapy for head/neck cancer [ ] , cocaine users [ ] , or tobacco smokers [ ] . low salivary ph is also described in individuals with diseases such as cf [ ] , sjögren's syndrome [ ] , and juvenile idiopathic arthritis [ ] . not all studies report a major impact on dental health in these cases as compensating factors may be in play [ ] . abts and cas play important roles in salivary secretion and well as in enamel formation [ , ] . for example, defective dentition is a feature of some individuals with nbce mutations [ ] and appears not to be a secondary consequence of acidemia [ ] . salivary ph is a useful biomarker for oral health [ ] and the usefulness of baking soda in oral hygiene was first suggested as long ago as [ ] . nahco delivery either as a mouth wash [ ] , mucoadhesive spray [ ] or sugar-free gum [ ] raises salivary ph and in some cases may lower colonization of acidophilic bacteria. in individuals undergoing chemotherapy for leukemia, use of a nahco mouthwash lowered susceptibility to mouth ulcers [ ] . in smokers the similar treatment reduced levels of the inflammatory biomarker il- β [ ] . nahco containing dentifrices have been shown to be effective at neutralizing plaque ph [ ] , enhancing plaque removal [ ] , and inhibiting formation of caries [ ] . ph-stabilizing resins used in restorative dentistry have also been suggested to be useful cariostatic by releasing oh - [ ] . finally, reducing dietary intake of sugars is also beneficial to oral ph because it limits the acidification that can be caused by acidophilic bacteria. hence sugar-free gum is effective at raising plaque ph [ ] . we have dealt with various aspects of acid-base-related medical microbiology in earlier sections (see sections on the respiratory system, the upper digestive system, the lower digestive system, the reproductive system, the immune system and oral health). in this section we will confine our considerations to viruses and parasites, using influenza and malaria as examples. . both of these mechanisms are necessary to support viral replication. in defending phi against the increased acid load, infected cells extrude h + , creating a concomitant acidification of phe at the cell surface [ ] . there are multiple acid-base-related aspects to the malarial lifecycle and its pathological impact. first of all, mosquitos are attracted by co and co sensitizes them to human odors [ ] . secondly, the erythrocytic phase of malaria infection requires that plasmodium invades red blood cells. one of the surface antigens that plasmodium exploits for host-cell recognition and invasion is the ae -glycophorin a complex. consequently, ae -null mice are immune to infection [ ] as are individuals with an ae defect that causes the abnormal red cell morphology (south east asian ovalocytosis, sao) [ ] . finally, malarial infection causes numerous metabolic disturbances, such as mac that, in part, follows the tissue hypoxia and hyperlactemia caused by blocked microvasculature. mac is a strong prognosticator of fatal outcome in infected individuals [ , ] . influenza. m h + -channel blockers have potential to target influenza strains that are resistant to currently available antiviral treatments [ ] . on the other hand, the use of ppis may paradoxically increase susceptibility to viral infection in the gastrointestinal tract by neutralizing the stomach acidity that typically destroys viral particles [ ] . lactic acidosis in malaria can be ameliorated by dichloroacetate treatment [ ] . malarial resistance in individuals with sao suggests that transfusions with sao blood may be a useful therapy for individuals infected with drug-resistant plasmodium [ ] . finally, the parasite's own abts may be a target for antimalarial action [ ] . the rapid proliferation of cancerous cells is associated with the warburg effect (also known as 'aerobic glycolysis'): a shift from aerobic to anaerobic metabolism even in the presence of oxygen [ , ] . in this state, cells increase their atp production by increasing their glucose uptake with the consequence of increased lactate and h + production. because of their high metabolic rates, such cells would tend to have a much lower phi than normal cells. however, cancer cells are able to maintain near-normal phi by upregulating acid-extruding abts such as nbce , nbcn , nhe , h + -atpase, and mct to facilitate the removal of h + , as well as extracellular cas (caix, caxii) to facilitate the removal of co [ ] [ ] [ ] [ ] [ ] . aquaporins (aqps)-which can serve as a conduit for transmembrane movements of co [ ] -also promote tumor growth and survival, but it is not clear that promotion of co removal is a major part of their pathological importance [ , , ] . as shown in cancer cell-lines [ ] , acidosis can also increase the drive on the tca cycle (promoting atp production for h + extrusion) and the pentose phosphate pathway (promoting nadph production to counter ros, promoting cell survival) [ ] . the combined action of these processes results in a drastic reduction in local phe. these changes allow cancer cells both to outcompete neighboring non-cancer cells and to mobilize and spread to other parts of the body. metastasis and tumor survival are further enhanced by acid-dependent remodeling of the extracellular matrix [ ] and suppression of anti-tumor immune responses [ ] (see also the immune system). it is noteworthy that a high level of net endogenous acid production was associated with higher mortality in breast-cancer recurrence in a cohort of early stage breast cancer survivors [ ] . moreover, acid-treatment of melanoma cells selects for more invasive phenotypes [ ] and the extent of upregulation of various abts and cas can be an adverse prognostic factor (e.g., [ ] [ ] [ ] ). the importance of acid-base balance in cancer has suggested that targeting tumor ph could be a valuable adjuvant therapy. the three main therapeutic modalities are (i) interfering with the ability of cancer cells to defend their phi, (ii) interfering with the ability of cancer cells to create an acidic extracellular environment, and (iii) bolstering the immune response in an acidic tumor microenvironment. interfering with phi defense. blocking the defense of phi by cancer cells can be achieved by inhibiting acid-extruding abts and/or cas [ , ] . the value of these approaches is demonstrated in diverse studies that report anything from delayed tumor-growth in nbcn -null mice [ ] to positive clinical outcomes with adjuvant use of proton pump inhibitors [ ] . however, reports from clinical trials are currently sparse. given the potential for side effects due to the physiological importance of these proteins in all organ systems, much attention has focused on inhibiting those proteins, such as caix, which are specifically upregulated in cancer cells (in response to hypoxia) and not highly expressed elsewhere [ ] . another approach, the combinatorial use of blockers, allows for a lower dose of each and thus fewer undesired effects on non-tumor cells. the use of a combination of five compounds, each of which targets a different abt/ca, revealed effectiveness at reducing intracellular brain tumor acidification in mice and the consequent activation of the pro-apoptotic marker caspase- in tumor cells, with little negative effect on non-tumor cells [ ] . interestingly, the potency of such approaches is enhanced by glucose loading, which increases the acid-load in these glycolytic tumor cells [ ] . finally, as different abts/cas are upregulated in different tumor types, generic approaches are also valuable to consider. for example, a number of anticancer drugs, such as salinomycin, that are not known to specifically target abts or cas, also promote cellular acidification [ ] . interfering with extracellular acidification. anecdotal evidence suggests that interfering with extracellular acidification might be achieved by dietary means [ ] . indeed, oral nahco supplementation can raise tumor phe and inhibit metastasis in mice [ ] , but the overall consequence is complex, with one recent study suggesting that such therapy may confoundingly promote tumor proliferation [ ] . however, the addition of adjuvant nahco to a chemotherapeutic agent that was fed directly into a hepatic tumor via catherization of tumorfeeding arteries (tila-tace: targeting intra-tumoral lactic acidosis with trans-arterial chemoembolization) was associated with markedly improved outcomes [ ] . in short, there is currently insufficient data to show that lowering dietary acid load improves outcomes, except by virtue of the association of such diets with general well-being [ ] and the role of alkalinization in enhancing the safety of certain chemotherapeutic drugs ( [ ] ). the acidic tumor microenvironment can also be exploited to promote local drug delivery as discussed in section . . bolstering antitumor immune response. the acidic tumor microenvironment promotes an antiinflammatory t-cell response (see section . . ), but the deletion of ae promotes a proinflammatory t-cell response (see section . . ). therefore inhibition of t-cell ae could be a valuable paradigm for enhancing a cytotoxic immune response [ ] . ph-dependent aspects of pharmaceutical therapy in this section, we will focus on the influence of ph upon pharmacokinetic properties of drugs. we will also consider how all of these ph-related properties can be harnessed to therapeutic advantage, particularly in diseases associated with acid-base imbalance. oral drug delivery is the most common and convenient method used to administer drugs whereby they can either directly access their targets or be absorbed into circulation to reach their intended targets. thus, oral drug delivery will serve as our paradigm for discussion. however, these concepts are also relevant to other, parenteral, methods of drug delivery such as inhalation of nebulized substances and injection into subcutaneous, intramuscular, or intravenous compartments. gastrointestinal (gi) ph is one of the major determinants of oral drug bioavailability as it affects various properties including solubility and dissolution rate, stability, and absorbability. ph varies drastically along the gi tract [ ] [ ] [ ] . starting from a value of ~ . in the stomach, it rises to ~ . in the duodenum, reaches ~ . in the terminal ileum, and falls again to ~ . in the colon [ ] . an additional consideration is that these values can vary with age, presence of food, diseases, and also by the co-administration of other drugs [ ] [ ] [ ] [ ] . finally, we note that the influence of ph on bioavailability could, in ph-disturbed states, result in underdose or overdose. most drugs are weak acids and bases. the relationship between the drug ionization constant (pka) and the ph of the environment to which the drug is exposed (hereafter referred to as the environmental ph) is a critical determinant of drug solubility and dissolution rate in aqueous compartments [ ] . this relationship determines the ratio of the concentration of the unionized (i.e., uncharged) form (u) to the concentration of the ionized (i.e., charged) form (i) of the drug and is described by the henderson-hasselbalch equation. now, according to equation ( ), . if ph<pka (i.e., ph−pka < ), the ionized form i (i.e., the acidic conjugate weak base bh + ) prevails; . if ph>pka (i.e., pka−ph > ) , the unionized form u (i.e., the basic neutral for b) prevails. thus, because the ionized form i is more water-soluble than the unionized form (due to better solvation between the ionized form i and the dipole of the water molecule), weakly acidic drugs have higher solubility at high ph whereas weakly basic drugs have higher solubility at low ph. this means that weakly acidic drugs tend to dissolve more easily in the intestine whereas weakly basic drugs tend to dissolve more easily in the stomach. because drug solubility is ph-dependent, the dissolution profile of the drug (i.e., the process by which a solid drug dissolves into solution) may also be ph-dependent [ , ] . drug solubility and dissolution may be enhanced by several techniques including (i) chemical derivatization to alter drug pka, (ii) administration of the drug in ionic form (as a salt) rather than as a free acid or base, or (iii) alteration of environmental ph by the adjuvant use of acids or bases to better match the drug's pka [ ] [ ] [ ] . in some cases, it may be desirable to lower the solubility and dissolution of parenterally-administered drugs in order to prolong their half-life. as we discuss in the next section, the influence of drug pka and environmental ph on drug ionization has important ramifications for drug absorption and distribution. besides solubility in aqueous solvent, the ionization state of a drug can influence its solubility in the lipid phase (e.g., drug pka can be modified to increase drug polarity and therefore reduce drug lipophilicity) and therefore affect its ability to permeate cell membranes [ , ] . assuming that passive diffusion (i.e., transmembrane concentration gradient is the driving force) is the mechanism by which a drug moves across a membrane and that only uncharged ions can freely diffuse across the membrane, we can turn again to the henderson-hasselbalch equation to describe the importance of drug pka and environmental ph to drug absorption and distribution between body compartments. we note that, according to point ) above, the henderson-hasselbalch equation predicts that weak acids tend to be absorbed in an acidic environment (e.g., in the stomach). similarly, according to point ) above, the henderson-hasselbalch equation predicts that weak bases tend to be absorbed in a basic environment (e.g., in the small intestine). in , shore and coworkers proposed the ph partition hypothesis (first described by jacobs in [ ] ) to describe the influence of pka and ph upon the gastric secretion of a variety of intravenously-administered weakly acidic and basic drugs [ ] . the results of their experiments could be explained with their theoretical model of drug absorption across a lipoid barrier (i.e., the gastric mucosa) that separates two compartments (i.e., the stomach lumen and the blood) with different ph and permeable only to the unionized form, which was assumed at equilibrium. they found that the extent to which a drug moves between compartments (the gi tract and blood) depends on the value of the drug pka and the environmental ph values of the two compartments such that (using equations ( ) and ( ) that is to say that weakly acidic drugs such as aspirin (pka= . ) can be effectively absorbed from the stomach. in the case of a weakly basic drug. in practice, the model has several limitations because the ph partition hypothesis ignores other substantial influences upon drug absorption. for example, the assumption of equilibrium is unrealistic in such a dynamic system. furthermore, even weakly acidic drugs can be substantially absorbed in the small intestine because of the large luminal surface area that it presents [ ] . finally, the ph-partition hypothesis does not consider the mechanism by which drugs can move across epithelial layers. today we know that both the transcellular and paracellular pathways play important roles in the absorption and elimination of both charged and uncharged drug forms [ ] . low-specificity membrane transporter proteins that contribute to transcellular drug transport around the body include organic anion transporters (oats), organic cation transporters (octs), some mcts, and members of the abc transporter superfamily, such as the p-glycoprotein transporters (p-gp) [ ] [ ] [ ] [ ] [ ] . in some cases, the transporters themselves may be ph-sensitive or coupled to the transport of acids and bases. the importance of such considerations is exemplified by lowered absorption of weakly basic drugs in individuals with an unusually high stomach ph such as those taking ppis or individuals with achlorhydria [ ] . for example, the dissolution and absorption rate of the weakly basic antifungal agent ketoconazole, which is soluble only at ph lower than , can be enhanced by coadministration of an acidic, carbonated beverage [ ] . a related example, albeit related to absorption of drugs by bacteria, is that the adjuvant use of nahco enhances the in vitro potency of antibiotics by interfering with the proton motive force that drives antibiotic efflux from bacteria [ ] . as we will see in section . , considerations of drug solubility and transepithelial movement also influence drug elimination in urine by the kidneys. when developing drugs for oral delivery it is important to account for the adverse acidic environment of the stomach, which can cause instability and rapid degradation of drugs before they reach the small intestine for absorption. this could happen because the polymers used for the tablet coating may be susceptible to ph. for this reason, carriers for oral drug delivery are tested for ph-sensitivity and endurance in acidic environment. acid-resistant polymers that only dissolve above certain ph values are sometimes used as enteric or gastro-resistant coatings [ ] [ ] [ ] [ ] . this is the case, for example, for oral delivery of insulin [ , ] . the ph partition hypothesis provides the theoretical framework for understanding how urinary ph influences renal drug excretion; acidification of urine favors elimination of weakly basic drugs (because their absorption is reduced) whereas alkalinization of urine favors elimination of weakly acidic drugs. for example, urine acidification via administration of ammonium chloride increases elimination of the weakly basic drug amphetamine [ ] , whereas urine alkalinization via intravenous administration of nahco enhances elimination of acidic drugs like salicylic acid (i.e., aspirin) and can be helpful in the management of drug poisoning like aspirin intoxication [ ] [ ] [ ] . as we considered earlier, partitioning is just one aspect of drug distribution. many oats and octs [ ] are expressed in nephron epithelia where their action is vital for delivering drugs from the peritubular capillaries into the nephron lumen for excretion in urine and for delivering diuretics (e.g., furosemide) to their therapeutic targets in the nephron lumen. the direct secretion of these drugs into the nephron lumen is necessary because many such drugs are substantially bound to albumin in circulation and are not effectively filtered into the nephron lumen at the glomerulus. . exploiting ph for targeted drug delivery acidity can be harnessed to target drug release to acidic environments such as the stomach or pathological acidotic microenvironments such as tumors. one example under development is a gastro-floating matrix tablet that contains adjuvant nahco with the drug, produces co upon reaction with gastric acid causing the dosage form to remain buoyant in the stomach for prolonged release [ ] . another example is the use of ph-sensitive vehicles such as micelles that could release cytotoxic chemotherapeutic agents only in the acidic tumor environment [ ] . similarly, as suggested by the disparity between the usefulness of an anticancer drug that was identified in an in vitro screening performed at neutral ph and its value in vivo in the acidic tumor microenvironment [ ] , it is possible that some drugs may be inactive in circulation and may not be activated until they reach an acidic environment. abts and cas play major roles in a variety of pathologies and provide an array of potential therapeutic targets, but many are currently lacking specific or safe drugs. the application of epigenetic modulators and other genetic tools to alter their expression is an underexplored area of research. it is interesting to note that, among the many treatment paradigms for diverse acid disturbances, nahco administration is a common thread. its low cost and ready availability have prompted its nickname "enemy of the pharmaceutical industry." however, despite the promise of numerous limited trials, robust evidence in favor of its broad effectiveness in many fields is currently lacking. this is perhaps due to a lack of appreciation of subgroup effects or a lack of standardization among trails. nonetheless, research into the therapeutic importance of balancing ph remains robust and promises the delivery of many more effective treatments in the coming years. figure . targeting carbonic anhydrase to treat glaucoma. glaucoma is retinal degeneration caused by increased intralocular pressure. eye drops containing ca-inhibitors such as acetazolamide (acz) target cas in the ciliary body and reduce the production of aqueous humor, lowering intraocular pressure. the ciliary body is a complex epithelial tissue comprised of two cell layers joined by gap junctions. a variety of abts and other transporters are required to move nacl, which is followed by water, from the interstitial fluid into the anterior chamber of the eye. figure . enhancing fluid secretion in the lungs. cftr promotes the movement of hco -containing fluid onto the airway surface to promote mucociliary clearance and lung health. drugs such as lumacaftor/ivacaftor rescue this function in some individuals with cf by helping misfolded cftr molecules to function normally. alternative ph-based strategies have been suggested as adjunct cf therapy, such as blockade of the many h + secreting abts. figure . the bohr effect. the action of ae and cas promote o release in systemic capillaries (panel a) and co release in pulmonary capillaries because of the ph-dependence of the affinity of hemoglobin for o (the bohr effect). figure . the role of abts and cas in bone remodeling. osteoclasts secrete acid onto the bone surface to resorb minerals during bone growth/remodeling and in response to hormonal requirements for release of mineralized ca + and phosphate. one report suggested an off-target bone-sparing effect of ca inhibitors, used to treat glaucoma, in a group of post-menopausal women. figure . targeting the stomach h + /k + -atpase to treat acid-reflux disease. proton pump inhibitors are widely used to reduce gastric acid secretion, as an alternative or adjunct strategy to neutralizing stomach acid with an antacid such as caco . figure . targeting intestinal abts to treat constipation. intestinal fluid absorption is promoted by the combined action of nhe and slc a , which perform the net uptake of nacl, and therefore water. inhibition of either, to reduce fluid absorption from the intestinal lumen is a useful therapy for irritable bowel syndrome with constipation. ivacaftor is similarly useful in cf by restoring intestinal fluid secretion. figure . the role of abts and cas in cancer. numerous acid-base handling proteins are upregulated in rapidly proliferating tumor calls to help them dispose of metabolic acids and create an acidic microenvironment that disadvantages non-tumor cells in their vicinity. as most of these abts and cas are gainfully expressed elsewhere in the body, therapies in development are focused on blocking those rarer targets that are preferentially expressed in the hypoxic tumor environment such as mct and caix. other approached include exploiting the acidic milieu of the tumor for the targeted delivery of chemotherapeutic drugs (see section . ). figure . the influence of ph on drug distribution. theoretical distribution of a hypothetical weakly acidic drug (pka = . , panel 'a') and a hypothetical weakly basic drug (pka = . , panel 'b') between two aqueous compartments with different ph (gi tract at ph = . and blood at ph = . ). assuming that only the unionized form u (ha in panel 'a' and b in panel 'b') can cross the membrane and that u is equilibrated across the plasma membrane, panel 'a' shows that a weakly acidic drug is more concentrated in the alkaline compartment. this result suggests that weakly acidic drugs tend to be absorbed from the more acidic compartment to the more basic compartment (blue arrows). panel 'b' shows that a weakly basic drug is more concentrated in the acidic compartment, indicating that weakly basic drugs are poorly absorbed in an acidic compartment (blue arrows). weakly basic drugs are in fact poorly absorbed from the stomach. [ the history of blood gases, acids and bases dietary acid, age, and serum bicarbonate levels among adults in the united states higher estimates of daily dietary net endogenous acid production (neap) in the elderly as compared to the young in a healthy, free-living elderly population of pakistan cellular mechanisms involved in co( ) and acid signaling in chemosensitive neurons mathematical modeling of acid-base physiology acid base physiology racial/ethnic-specific reference intervals for common laboratory tests: a comparison among asians, blacks, hispanics, and white centers for disease control and prevention chronic kidney disease surveillance team, race/ethnicity, dietary acid load, and risk of end-stage renal disease among us adults with chronic kidney disease nhanes iii: influence of race on gfr thresholds and detection of metabolic abnormalities ethnicity and young age influence the frequency of diabetic ketoacidosis at the onset of type diabetes admissions for diabetic ketoacidosis in ethnic minority groups in a city hospital differences in acidosisstimulated renal ammonia metabolism in the male and female kidney effect of age on blood acid-base composition in adult humans: role of age-related renal functional decline chapter -human carbonic anhydrases: catalytic properties role of carbonic anhydrases and inhibitors in acid-base physiology: insights from mathematical modeling statpearls, statpearls publishing, treasure island (fl), clinical physiology of acid-base and electrolyte disorders metabolic acidosis in ckd: core curriculum renal pathophysiology: the essentials renal tubular acidosis: developments in our understanding of the molecular basis drug-induced metabolic acidosis, f res metabolic acidosis: pathophysiology, diagnosis and management diet-induced low-grade metabolic acidosis and clinical outcomes: a review handbook of pathophysiology the alkaline tide phenomenon tribonat--a comprehensive summary of its properties bicarbonate therapy and intracellular acidosis short-and long-term effects of alkali therapy in chronic kidney disease: a systematic review treatment of acidified blood using reduced osmolarity mixed-base solutions brain ph effects of nahco and carbicarb in lactic acidosis carbicarb: an effective substitute for nahco for the treatment of acidosis the treatment of acidosis in acute lung injury with tris-hydroxymethyl aminomethane (tham) sodium acetate as a replacement for sodium bicarbonate in medical toxicology: a review lactate versus bicarbonate. a reconsideration of the therapy of metabolic acidosis estimated net endogenous acid production and serum bicarbonate in african americans with chronic kidney disease dietary acid load is associated with serum bicarbonate but not insulin sensitivity in chronic kidney disease very low-protein diet (vlpd) reduces metabolic acidosis in subjects with chronic kidney disease: the "nutritional light signal" of the renal acid load fruit and vegetable treatment of chronic kidney disease-related metabolic acidosis reduces cardiovascular risk better than sodium bicarbonate therapy of bicarbonate-losing renal tubular acidosis long-term safety and efficacy of veverimer in patients with metabolic acidosis in chronic kidney disease: a multicentre, randomised, blinded, placebo-controlled, -week extension veverimer: an advance in base therapy for metabolic acidosis pyruvate in the correction of intracellular acidosis: a metabolic basis as a novel superior buffer a controlled clinical trial of dichloroacetate for treatment of lactic acidosis in adults. the dichloroacetate-lactic acidosis study group pathophysiology of metabolic alkalosis: a new classification based on the centrality of stimulated collecting duct ion transport acetazolamide-mediated decrease in strong ion difference accounts for the correction of metabolic alkalosis in critically ill patients hydrochloric acid infusion for the treatment of metabolic alkalosis in surgical intensive care unit patients cimetidine in the management of metabolic alkalosis induced by nasogastric drainage respiratory acid-base disorders national kidney foundation primer on kidney diseases acid-base transport by the renal proximal tubule statpearls, statpearls publishing alkali therapy for respiratory acidosis: a medical controversy acetazolamide use in severe chronic obstructive pulmonary disease. pros and cons acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates co removal from blood a proof of concept study, demonstrating extracorporeal carbon dioxide removal using hemodialysis with a low bicarbonate dialysate hormone replacement therapy causes a respiratory alkalosis in normal postmenopausal women mechanisms of action of acetazolamide in the prophylaxis and treatment of acute mountain sickness minireview: ph and synaptic transmission protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala role of hco -ions in depolarizing gabaa receptor-mediated responses in pyramidal cells of rat hippocampus bicarbonate efflux via gabaa receptors depolarizes membrane potential and inhibits two-pore domain potassium channels of astrocytes in rat hippocampal slices rapid rise of extracellular ph evoked by neural activity is generated by the plasma membrane calcium atpase regulation and modulation of ph in the brain seizure termination by acidosis depends on asic a cull-candy, proton inhibition of n-methyl-d-aspartate receptors in cerebellar neurons brain alkalosis causes birth asphyxia seizures, suggesting therapeutic strategy emerging roles of na+/h+ exchangers in epilepsy and developmental brain disorders disruption of sodium bicarbonate transporter slc a in a patient with complex partial epilepsy and mental retardation association of the asp variant of the human anion exchanger gene with common subtypes of idiopathic generalized epilepsy na + dependent acid-base transporters in the choroid plexus; insights from slc and slc gene deletion studies acetazolamide lowers intracranial pressure and modulates the cerebrospinal fluid secretion pathway in healthy rats ionic mechanisms of neuronal excitation by inhibitory gabaa receptors the gaba excitatory/inhibitory developmental sequence: a personal journey chloride concentration in cultured hippocampal neurons increases during long-term exposure to ammonia through enhanced expression of an anion exchanger a christianson syndromelinked deletion mutation (Δ es ) in slc a impairs hippocampal neuronal plasticity cerebral acidosis in focal ischemia dramatic aggregation of alzheimer abeta by cu(ii) is induced by conditions representing physiological acidosis amyloid clearance defect in apoe astrocytes is reversed by epigenetic correction of endosomal ph % co is a potent, fast acting inhalation anticonvulsant the role of carbon dioxide in acute brain injury a novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by co inhalation at reperfusion acetazolamide in the treatment of seizures a class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects a new kid on the block? carbonic anhydrases as possible new targets in alzheimer's disease carbonic anhydrase modulation of emotional memory. implications for the treatment of cognitive disorders type ii renal tubular acidosis secondary to topiramate: a review disruption of slc a augments neuronal excitability and modulates synaptic short-term plasticity preclinical update on regulation of intracranial pressure in relation to idiopathic intracranial hypertension, fluids and barriers of the cns studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium na+/h+ and ci-/hco -antiporters of bovine pigmented ciliary epithelial cells slc a and the pathophysiology of congenital hereditary endothelial dystrophy h(oh): a holiday perspective. focus on "mouse slc a expressed in xenopus oocytes is an ideally selective h + /oh -conductance pathway that is stimulated by rises in intracellular and extracellular ph functional roles of electrogenic sodium bicarbonate cotransporter nbce in ocular tissues retinal ph and acid regulation during metabolic acidosis sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in ambystoma tigrinum retina bicarbonate modulates photoreceptor guanylate cyclase (ros-gc) catalytic activity novel mutation in slc a gene causing autosomal recessive progressive rod-cone dystrophy severe neurologic impairment in mice with targeted disruption of the electrogenic sodium bicarbonate cotransporter nbce (slc a gene) role of monocarboxylate transporters in regulating metabolic homeostasis in the outer retina: insight gained from cell-specific bsg deletion genetics and phenomics of pendred syndrome novel atp v b and atp v a mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss congenital hereditary endothelial dystrophy with progressive sensorineural deafness (harboyan syndrome) genetic disorders of transporters/channels in the inner ear and their relation to the kidney early hearing loss upon disruption of slc a in c bl/ mice non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin calcium oxalate stone formation in the inner ear as a result of an slc a mutation subcutaneous injection of drugs: literature review of factors influencing pain sensation at the injection site loss of slc a /nhe impairs nociception in a mouse model of christianson syndrome metabolic acidosis and anaemia associated with dorzolamide in a patient with impaired renal function metabolic acidosis with ophthalmic dorzolamide in a neonate ophthalmic nonsteroidal anti-inflammatory drugs as a therapy for corneal dystrophies caused by slc a mutation na+/h+-exchanger- inhibition counteracts diabetic cataract formation and retinal oxidative-nitrative stress and apoptosis protective effects of hypercapnic acidosis on ischemia-reperfusion-induced retinal injury cerumenolytic efficacy of . % sodium bicarbonate versus docusate sodium: a randomized, controlled trial the preparation of acetic acid for use in otic drops and its effect on endocochlear potential and ph in inner ear fluid discovery and development of s and s as potent tas r antagonists modulation of bitter taste perception by a small molecule htas r antagonist effect of ph modification by bicarbonate on pain after subcutaneous lidocaine injection role of cftr in epithelial physiology pulmonary complications of cystic fibrosis a new role for bicarbonate in mucus formation extracellular ph and lung infections in cystic fibrosis airway acidification initiates host defense abnormalities in cystic fibrosis mice mechanisms of acid and base secretion by the airway epithelium airway ph homeostasis in asthma and other inflammatory lung diseases hyperglycaemia and pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter dependent lactate-h+ secretion cftr modulator theratyping: current status, gaps and future directions rheological properties of cystic fibrosis bronchial secretion and in vitro drug permeation study: the effect of sodium bicarbonate safety, tolerability, and effects of sodium bicarbonate inhalation in cystic fibrosis repurposing tromethamine as inhaled therapy to treat cf airway disease esomeprazole increases airway surface liquid ph in primary cystic fibrosis epithelial cells paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid ph effects of changes of ph on the contractile function of cardiac muscle the effects of acid-base disturbances on cardiovascular and pulmonary function relative contributions of na+/h+ exchange and na+/hco -cotransport to ischemic nai+ overload in isolated rat hearts impact of systemic acidosis on the development of malignant ventricular arrhythmias after reperfusion therapy for st-elevation myocardial infarction mechanisms underlying the emergence of post-acidosis arrhythmia at the tissue level: a theoretical study reduced sarcolemmal expression and function of the nbce isoform of the na + -hco₃ − cotransporter in hypertrophied cardiomyocytes of spontaneously hypertensive rats: role of the reninangiotensin system carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy loss of the ae anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure mitochondrial nhe : a newly identified target to prevent heart disease disturbed acid-base transport: an emerging cause of hypertension extracellular acidosis suppresses calcification of vascular smooth muscle cells by inhibiting calcium influx via ltype calcium channels regulatory mechanisms of hemoglobin oxygen affinity in acidosis and alkalosis transgenic expression of carbonic anhydrase iii in cardiac muscle demonstrates a mechanism to tolerate acidosis sodium bicarbonate on severe metabolic acidosis during prolonged cardiopulmonary resuscitation: a double-blind, randomized, placebo-controlled pilot study an evidence-based evaluation of the use of sodium bicarbonate during cardiopulmonary resuscitation sodium bicarbonate: basically useless therapy nhe- : still a viable therapeutic target cariporide (hoe ), a selective na+-h+ exchange inhibitor, inhibits the mitochondrial death pathway na+/h+ exchanger inhibitor cariporide attenuates the mitochondrial ca + overload and ptp opening inhibits endoplasmic reticulum stress-induced apoptosis by targeting na+/h+ exchanger- in the heart inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury antibodies against the cardiac sodium/bicarbonate co-transporter (nbce ) as pharmacological tools sasanquasaponin induces increase of cl-/hco -exchange of anion exchanger and promotes intracellular cl-efflux in hypoxia/reoxygenation cardiomyocytes bicarbonate therapy, phosphate binders, and risk for vascular calcification hyperphosphatemic familial tumoral calcinosis: response to acetazolamide and postulated mechanisms metabolic and hemodynamic consequences of sodium bicarbonate administration in patients with heart disease cellular mechanisms of muscle fatigue challenging the role of ph in skeletal muscle fatigue lactic acid and exercise performance : culprit or friend? k+ and lac-distribution in humans during and after high-intensity exercise: role in muscle fatigue attenuation? metabolic and clinical consequences of metabolic acidosis three-generation study of women on diets and health study group, higher dietary acid load is associated with a higher prevalence of frailty, particularly slowness/weakness and low physical activity, in elderly japanese women regulation of ph in human skeletal muscle: adaptations to physical activity carbonic anhydrase iii is expressed in mouse skeletal muscles independent of fiber type-specific myofilament protein isoforms and plays a role in fatigue resistance effects of sodium bicarbonate on high-intensity endurance performance in cyclists: a double-blind, randomized cross-over trial sodium bicarbonate supplementation does not improve elite women's team sport running or field hockey skill performance the impact of sodium bicarbonate on performance in response to exercise duration in athletes: a systematic review mechanistic insights into the efficacy of sodium bicarbonate supplementation to improve athletic performance effect of sodium bicarbonate on prolonged running performance: a randomized, double-blind, cross-over study potassium bicarbonate reduces urinary nitrogen excretion in postmenopausal women dietary patterns, skeletal muscle health, and sarcopenia in older adults the effects of acid on bone metabolic acidosis inhibits growth hormone secretion in rats: mechanism of growth retardation serum bicarbonate and bone mineral density in us adults acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat defects in tcirg subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis carbonic anhydrase ii deficiency in families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification mouse models of slc -linked disorders of hco -transporter dysfunction effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women can acetazolamide be used to treat diseases involving increased bone mineral density? chronic proton pump inihibitor therapy and calcium metabolism proton-pump inhibitor use and fracture risk: an updated systematic review and meta-analysis proton pump inhibitors therapy and risk of bone diseases: an update meta-analysis revisiting the parietal cell a h+-gated urea channel: the link between helicobacter pylori urease and gastric colonization helicobacter pylori moves through mucus by reducing mucin viscoelasticity helicobacter pylori and gastric cancer: factors that modulate disease risk salivary bicarbonate as a major factor in the prevention of upper esophageal mucosal injury in gastroesophageal reflux disease analysis of bicarbonate, phosphate and other anions in saliva by capillary electrophoresis with capacitively coupled contactless conductivity detection in diagnostics of gastroesophageal reflux disease role of saliva in esophageal defense: implications in patients with nonerosive reflux disease acid neutralizing capacity of human saliva physiology of na+/h+ exchangers (nhes) in the digestive system milk-alkali syndrome health-behavior induced disease: return of the milk-alkali syndrome regulation of na/h exchanger- in gastroesophageal reflux disease: possible interaction of histamine receptor mice with a targeted disruption of the ae cl -/hco -exchanger are achlorhydric the role of acid inhibition in helicobacter pylori eradication the development of urease inhibitors: what opportunities exist for better treatment of helicobacter pylori infection in children? the cystic fibrosis of exocrine pancreas intestinal obstruction syndromes in cystic fibrosis: meconium ileus, distal intestinal obstruction syndrome, and constipation acid-base disturbances in gastrointestinal disease update on slc a mutations in congenital chloride diarrhea differential regulation of na+/h+ exchange isoform activities by enteropathogenic e. coli in human intestinal epithelial cells inhibition and redistribution of nhe , the apical na+/h+ exchanger, by clostridium difficile toxin b reduced sodium/proton exchanger nhe activity causes congenital sodium diarrhea ph and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon d-lactic acidosis: an underrecognized complication of short bowel syndrome h+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine defective small intestinal anion secretion, dipeptide absorption, and intestinal failure in suckling nbce -deficient mice slc a inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation efficacy of tenapanor in treating patients with irritable bowel syndrome with constipation: a -week, placebo-controlled phase trial (t mpo- ) impact of cftr modulation on intestinal ph, motility, and clinical outcomes in patients with cystic fibrosis and the g d mutation linaclotide improves gastrointestinal transit in cystic fibrosis mice by inhibiting sodium/hydrogen exchanger linaclotide activates guanylate cyclase-c/cgmp/protein kinase-ii-dependent trafficking of cftr in the intestine medical physiology. a cellular and molecular approach renal tubular acidosis metabolic acidosis and subclinical metabolic acidosis in ckd downregulation of the cl-/hco -exchanger pendrin in kidneys of mice with cystic fibrosis: role in the pathogenesis of metabolic alkalosis mechanisms of metabolic acidosis-induced kidney injury in chronic kidney disease low serum bicarbonate and ckd progression in children serum bicarbonate levels and the progression of kidney disease: a cohort study mechanism of hyperkalemia-induced metabolic acidosis kidney stones: pathophysiology and medical management the primary cause of infection-induced urinary stones urinary catheter blockage depends on urine ph, calcium and rate of flow daily oral sodium bicarbonate preserves glomerular filtration rate by slowing its decline in early hypertensive nephropathy bicarbonate supplementation slows progression of ckd and improves nutritional status clinical evidence that treatment of metabolic acidosis slows the progression of chronic kidney disease a causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure drug-induced renal fanconi syndrome diuretics: a review metabolic alkalosis the behavior of carbenicillin as a nonreabsorbable anion hypokalaemia, metabolic alkalosis, and hypernatraemia due to "massive" sodium penicillin therapy impaired renal hco -excretion in cystic fibrosis citrate and renal calculi: an update lemonade therapy increases urinary citrate and urine volumes in patients with recurrent calcium oxalate stone formation long-term treatment of calcium nephrolithiasis with potassium citrate strategy to control catheter encrustation with citrated drinks: a randomized crossover study role of v-atpase-rich cells in acidification of the male reproductive tract the activating effects of bicarbonate on sperm motility and respiration at ejaculation lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients critical role of cftr in uterine bicarbonate secretion and the fertilizing capacity of sperm a new role for bicarbonate secretion in cervico-uterine mucus release hydrogen ion and carbon dioxide content of the oviductal fluid of the rhesus monkey (macaca mulatta) bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction factors and pathways involved in capacitation: how are they regulated? acidification of uterine epithelium during embryo implantation in mice loss of the na+/h+ exchanger nhe causes male infertility in mice by disrupting acrosome formation disruption of the slc a -mediated anion transport is associated with male subfertility anion exchanger is essential for spermiogenesis in mice tests of buffergel for contraception and prevention of sexually transmitted diseases in animal models maternal and fetal acid-base chemistry: a major determinant of perinatal outcome effect of preoperative bicarbonate infusion on maternal and perinatal outcomes of obstructed labour in mbale regional referral hospital: a study protocol for a randomised controlled trial improvement of cervical mucus viscoelasticity and sperm penetration with sodium bicarbonate douching sodium bicarbonate douching for improvement of the postcoital test spermicidal effects of lemon juice and juices from other natural products, agriculture and natural resources acidform: a review of the evidence a novel vaginal ph regulator: results from the phase ampower contraception clinical trial bicarbonate ion; the corona cell dispersing factor of rabbit tubal fluid pantoprazole, a proton-pump inhibitor, impairs human sperm motility and capacitation in vitro acid-base disorders in liver disease effect of ph on the kinetics of frog muscle phosphofructokinase inhibition by acidosis of adenosine ', '-cyclic monophosphate accumulation and lipolysis in isolated rat fat cells bicarbonate in the treatment of metabolic acidosis: effects on hepatic intracellular ph, gluconeogenesis, and lactate disposal in rats renal gluconeogenesis in acidosis, alkalosis, and potassium deficiency: its possible role in regulation of renal ammonia production effect of extracellular ph on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets a physiological solvent for crystalline insulin insulin sensitivity and glucose homeostasis can be influenced by metabolic acid load dietary acid load, metabolic acidosis and insulin resistance -lessons from cross-sectional and overfeeding studies in humans the ph dependence of insulin binding. a quantitative study stimulatory effect of insulin on renal proximal tubule sodium transport is preserved in type diabetes with nephropathy molecular mechanism of pancreatic and salivary glands fluid and hco − secretion effect of mineralocorticoids on acid-base balance bartter or gitelman-how to differentiate? acid retention during kidney failure induces endothelin and aldosterone production which lead to progressive gfr decline, a situation ameliorated by alkali diet role of endothelin- in renal regulation of acidbase equilibrium in acidotic humans higher dietdependent renal acid load associates with higher glucocorticoid secretion and potentially bioactive free glucocorticoids in healthy children effect of chronic metabolic acidosis on the growth hormone/igf- endocrine axis: new cause of growth hormone insensitivity in humans in vivo bicarbonate deficiency and insulin dissolution combined insulin and bicarbonate therapy elicits cerebral edema in a juvenile mouse model of diabetic ketoacidosis intracellular ph regulation during spreading of human neutrophils extracellular acidosis is a novel danger signal alerting innate immunity via the nlrp inflammasome the intimate and controversial relationship between voltage-gated proton channels and the phagocyte nadph oxidase voltage-gated proton channels maintain ph in human neutrophils during phagocytosis na+/h+ exchange activity during phagocytosis in human neutrophils: role of fcgamma receptors and tyrosine kinases science review: extracellular acidosis and the immune response: clinical and physiologic implications the effect of ph and nucleophiles on complement activation by human proximal tubular epithelial cells unravelling the interplay between extracellular acidosis and immune cells acute asphyxia affects neutrophil number and function in the rat ph changes observed in the inflamed gingival crevice modulate human polymorphonuclear leukocyte activation in vitro acidosis differently modulates the inflammatory program in monocytes and macrophages the effects of extracellular ph on immune function anion exchanger is critical for cd (+) t cells to maintain phi homeostasis and modulate immune responses systemic lupus erythematosus associated with type renal tubular acidosis: a case report and review of the literature systemic lupus erythematosus with distal renal tubular acidosis presenting as hypokalemic paralysis with respiratory failure oral nahco activates a splenic anti-inflammatory pathway: evidence that cholinergic signals are transmitted via mesothelial cells sodium bicarbonate facilitates low-dose oral tolerance to peanut in mice monocarboxylate transporter mct is a target for immunosuppression inhibition of na+/h+ exchanger by cariporide alleviates burn-induced multiple organ injury a. van den hout, co vulnerability in panic disorder overshadowed by the amygdala: the bed nucleus of the stria terminalis emerges as key to psychiatric disorders acidsensing ion channel is localized in brain regions with high synaptic density and contributes to fear conditioning overexpression of acid-sensing ion channel a in transgenic mice increases acquired fear-related behavior decreased brain ph as a shared endophenotype of psychiatric disorders dietary acid load and mental health outcomes in children and adolescents: results from the giniplus and lisa birth cohort studies understanding and predicting suicidality using a combined genomic and clinical risk assessment approach metabolic acidosis of ckd: an update prevalence of depression and suicidal ideation increases proportionally with renal function decline, beginning from early stages of chronic kidney disease carbon dioxide test as an additional clinical measure of treatment response in panic disorder responses to hypercarbia induced by acetazolamide in panic disorder patients increased lactate levels and reduced ph in postmortem brains of schizophrenics: medication confounds possible alternatives to soda lime absorption of carbon dioxide interaction of inhalational anaesthetics with co absorbents hypercapnia and acidosis in sepsis: a double-edged sword? propofol infusion associated metabolic acidosis in patients undergoing neurosurgical anesthesia: a retrospective study perioperative metabolic acidosis: the bradford anaesthetic department acidosis study chasing the base deficit: hyperchloraemic acidosis following . % saline fluid resuscitation factors related to postoperative metabolic acidosis following major abdominal surgery cause of metabolic acidosis in prolonged surgery postoperative metabolic alkalosis following general surgery: its incidence and possible etiology citrate metabolism and its complications in non-massive blood transfusions: association with decompensated metabolic alkalosis+respiratory acidosis and serum electrolyte levels outcome of surgical patients who present acidosis postoperatively biochemical changes in stored donor units: implications on the efficacy of blood transfusion clinical impact of blood storage lesions influence of ph on wound-healing: a new perspective for wound-therapy? dichloroacetate stabilizes the intraoperative acid-base balance during liver transplantation ph modulation ameliorates the red blood cell storage lesion in a murine model of transfusion an analysis of the buffer systems in saliva change of saliva composition with radiotherapy effect of decreased salivation and ph on the adherence of klebsiella species to human buccal epithelial cells evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries salivary parameters and oral health status amongst adolescents in mexico salivary flow rate, ph, buffering capacity, total protein, oxidative stress and antioxidant capacity in children with and without dental caries salivary buffer capacity, ph, and stimulated flow rate of crack cocaine users effect of sodium bicarbonate mouth wash on salivary ph and interleukin- β levels among smokers salivary biomarkers and oral microbial load in relation to the dental status of adults with cystic fibrosis salivary changes and dental caries as potential oral markers of autoimmune salivary gland dysfunction in primary sjögren's syndrome unstimulated salivary flow, ph, proteins and oral health in patients with juvenile idiopathic arthritis new paradigms on the transport functions of maturation-stage ameloblasts a novel mutant na + /hco -cotransporter nbce in a case of compound-heterozygous inheritance of proximal renal tubular acidosis extrarenal signs of proximal renal tubular acidosis persist in nonacidemic nbce b/c-null mice salivary ph: a diagnostic biomarker baking soda dentifrices and oral health the effect of sodium bicarbonate oral rinse on salivary ph and oral microflora: a prospective cohort study salivary ph after a glucose rinse: effect of a new mucoadhesive spray (cariex) based on sodium bicarbonate and xylitol effect of chewing bicarbonate-containing sugar-free gum on the salivary ph: an in vivo study sodium bicarbonate solution versus chlorhexidine mouthwash in oral care of acute leukemia patients undergoing induction chemotherapy: a randomized controlled trial the effect of bicarbonate/fluoride dentifrices on human plaque ph enhancement of plaque removal efficacy by tooth brushing with baking soda dentifrices: results of five clinical studies bicarbonate-based powder and paste dentifrice effects on caries ph stabilizing properties of a posterior light cured resin composite: an in vivo study six months of daily high-dose xylitol in high-risk schoolchildren: a randomized clinical trial on plaque ph and salivary mutans streptococci the influence of virus infection on the extracellular ph of the host carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours merozoite surface protein recognition of host glycophorin a mediates malaria parasite invasion of red blood cells reduced risk of plasmodium vivax malaria in papua new guinean children with southeast asian ovalocytosis in two cohorts and a case-control study the pathophysiologic and prognostic significance of acidosis in severe adult malaria unidentified acids of strong prognostic significance in severe malaria put a cork in it: plugging the m viral ion channel to sink influenza proton pump inhibitors are risk factors for viral infections: even for covid- ? pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria can exchange transfusions using red blood cells from donors with southeast asian ovalocytosis prevent or ameliorate cerebral malaria in patients with multi-drug resistant plasmodium falciparum? na(+) regulation in the malaria parasite plasmodium falciparum involves the cation atpase pfatp and is a target of the spiroindolone antimalarials the warburg effect: how does it benefit cancer cells? we need to talk about the warburg effect ph control mechanisms of tumor survival and growth regulation and roles of bicarbonate transporters in cancer role of ph regulatory proteins and dysregulation of ph in prostate cancer disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth carbonic anhydrases: role in ph control and cancer sharpey-schafer lecture: gas channels rapid co permeation across biological membranes: implications for co venting from tissue water transport proteins-aquaporins (aqps) in cancer biology acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress the acidic tumor microenvironment as a driver of cancer tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages increased acid-producing diet and past smoking intensity are associated with worse prognoses among breast cancer survivors: a prospective cohort study acid treatment of melanoma cells selects for invasive phenotypes the hypoxic response expression as a survival biomarkers in treatment-naive advanced breast cancer, asian pac increased expression of na+/h+ exchanger isoform predicts tumor aggressiveness and unfavorable prognosis in epithelial ovarian cancer monocarboxylate transporters in breast cancer and adipose tissue are novel biomarkers and potential therapeutic targets cellular acidification as a new approach to cancer treatment and to the understanding and therapeutics of neurodegenerative diseases disrupting na + , hco₃ − -cotransporter nbcn (slc a ) delays murine breast cancer development intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer development of a small molecule tubulysin b conjugate for treatment of carbonic anhydrase ix receptor expressing cancers brain tumor acidification using drugs simultaneously targeting multiple ph regulatory mechanisms glucose-dependent growth arrest of leukemia cells by mct inhibition: feeding warburg's sweet tooth and blocking acid export as an anticancer strategy manipulating ph in cancer treatment: alkalizing drugs and alkaline diet bicarbonate increases tumor ph and inhibits spontaneous metastases targeting the acidic tumor microenvironment: unexpected pro-neoplastic effects of oral nahco therapy in murine breast tissue a nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis pros and cons of dietary strategies popular among cancer patients irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database martínez-climent, targeting the anion exchanger with specific peptides as a new therapeutic approach in b lymphoid neoplasms intestinal luminal ph in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs measurement of gastrointestinal ph profiles in normal ambulant human subjects advances in oral drug delivery for regional targeting in the gastrointestinal tract -influence of physiological, pathophysiological and pharmaceutical factors impact of gastrointestinal disease states on oral drug absorption -implications for formulation design -a pearrl review food, gastrointestinal ph, and models of oral drug absorption literature review of gastrointestinal physiology in the elderly prediction of ph-dependent drug-drug interactions for basic drugs using physiologically based biopharmaceutics modeling: industry case studies study of ph-dependent drugs solubility in water acidic and basic drugs in medicinal chemistry: a perspective drug solubility: importance and enhancement techniques, isrn pharm physicochemical properties, formulation, and drug delivery a quantitative assessment of herg liability as a function of lipophilicity some aspects of cell permeability to weak electrolytes the gastric secretion of drugs: a ph partition hypothesis surface area of the digestive tract -revisited tight junction modulation and its relationship to drug delivery polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics the human organic cation transporter oct mediates high affinity uptake of the anticancer drug daunorubicin the organic anion transporter (oat) family: a systems biology perspective the p-glycoprotein transport system and cardiovascular drugs drugs as p-glycoprotein substrates, inhibitors, and inducers impaired drug absorption due to high stomach ph: a review of strategies for mitigation of such effect to enable pharmaceutical product development bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force ionotropically cross-linked ph-sensitive ipn hydrogel matrices as potential carriers for intestine-specific oral delivery of protein drugs peppas, ph-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: effects of protein size, crosslinking density, and hydrogel degradation on protein delivery nanotechnology for protein delivery: overview and perspectives eudragit: a technology evaluation preparation of layer-by-layer thin films containing insulin and its ph-sensitive decomposition oral delivery of insulin using ph-responsive complexation gels fundaments of toxicology-approach to the poisoned patient the role of sodium bicarbonate in the management of some toxic ingestions acid-alkaline balance: role in chronic disease and detoxification., alternative therapies in health and medicine relationship between the urinary excretion mechanisms of drugs and their physicochemical properties gastro-floating matrices designed for simultaneous improvement of floating and drug release capabilities: an in vitro case study of matrices loaded with ciprofloxacin hydrochloride environmental ph-sensitive polymeric micelles for cancer diagnosis and targeted therapy a drug screening assay on cancer cells chronically adapted to acidosis key: cord- - nljzm a authors: tang, zhongmin; zhang, xingcai; shu, yiqing; guo, ming; zhang, han; tao, wei title: insights from nanotechnology in covid- treatment date: - - journal: nano today doi: . /j.nantod. . sha: doc_id: cord_uid: nljzm a in just a few months, sars-cov- and the disease it causes, covid- , created a worldwide pandemic. virologists, biologists, pharmacists, materials scientists, and clinicians are collaborating to develop efficient treatment strategies. overall, in addition to the use of clinical equipment to assist patient rehabilitation, antiviral drugs and vaccines are the areas of greatest focus. given the physical size of sars-cov- and the vaccine delivery platforms currently in clinical trials, the relevance of nanotechnology is clear, and previous antiviral research using nanomaterials also supports this connection. herein we briefly summarize current representative strategies regarding nanomaterials in antiviral research. we focus specifically on sars-cov- and the detailed role that nanotechnology can play in addressing this pandemic, including i) using fda-approved nanomaterials for drug/vaccine delivery, including further exploration of the inhalation pathway; ii) introducing promising nanomaterials currently in clinical trials for drug/vaccine delivery; iii) designing novel biocompatible nanomaterials to combat the virus via interfering in its life cycle; and iv) promoting the utilization of nanomaterials in pneumonia treatment. the covid- pandemic is not a unique case in the history of viruses but one representative of the new century, which is sobering news for humans. at this time of writing, over , , patients have been infected sars-cov- , with over , , deaths, according to the world health organization (who). [ ] with an in-depth understanding of the sars-cov- , including knowing its gene sequence, [ ] analyzing the structure of key viral proteins by cryogenic electron microscopy (cryo-em), [ , ] undertaking pathway analysis of its variation [ ] and transmission, [ ] as well as exploring the origin of the virus, [ ] potential antiviral drugs and vaccines should advance rapidly. however, the ever-increasing pandemic, in terms of numbers of people and geographic areas, rapid transmission, repeated infections , and genomic variation in sars-cov- [ ] , presents difficult obstacles to the development of antiviral drugs and vaccines, although they can also be seen as stimulating challenges for researchers. generally, current trials relevant to covid- encompass antiviral drugs, drugs to modulate the immune response, neutralizing antibodies, and vaccines. based on the life cycle of sars-cov- and its structure, antiviral drugs work through inhibiting rna polymerase (remdesivir, [ ] favipiravir with tocilizumab [ ] ), inhibiting viral protease (ivermectin, [ ] lopinavir/ritonavir, [ ] ), blocking membrane fusion (leronlimab, [ ] rhace , [ ] hydroxychloroquine [ ] and arbidol hydrochloride [ ] ), and broad-spectrum antiviral effects (eidd- [ ] ). however, vaccines are probably a stronger weapon in this war. examples currently include the bnt mrna from biontech. inc., [ ] mrna- from moderna. inc., [ ] ino- from inovio. inc., [ ] , and chadox ncov- [ ] from the university of oxford. despite the differences in working principles, the success of any vaccine in terms of stability and efficacy depends on the delivery platform: mrna- needs lipid particles, ino- requires the cellectra® portable device, and chadox ncov- requires its non-replicating adenovirus vector. among vaccine delivery platforms, lipid particles and non-replicating adenovirus vectors have similar nano size, attracting the attention of nanotechnologists. not only that, but the latest clinical results on antiviral drugs such as remdesivir [ ] and hydroxychloroquine [ ] are not very satisfactory, while the results of mrna vaccine trials are much more exciting; phase / clinical trial data on bnt mrna and phase clinical trial data on mrna- has proven the safety and efficacy of these agents. [ , ] some other drugs also being utilized to mitigate sars-cov- -induced lung injury include dexamethasone, [ ] ruxolitinib, [ ] fostamatinib, [ ] and nebulized heparin [ ] . scientists have also proposed the use of dexamethasone nano-formulations in the treatment of covid- via targeting hyper-activated immune cells through inhalation or intravenous injection. [ ] not only the above technologies, but also neutralizing antibodies like bgb-dxp [ ] , ly [ ] , regn /regn monoclonal antibodies [ ] and antibody fragments (inosars) [ ] etc. were developed for covid- treatment and vaccination. the antibody fragments called nanobodies (for their nano-scale size) are yet another connection to nanotechnology. as the clinical trial results of the first wave of covid drugs have yet to be been announced, now would be an appropriate time to push forward on other technologies, including nanotechnology. [ ] [ ] [ ] we recently shared our views on tackling covid- from a materials science perspective. [ ] in the current opinion we specifically highlight the potential role of nanotechnology in treatments for covid- . although nanotechnology has been introduced into antiviral research before, its prospects for success with sars-cov- are better than one might imagine, considering the following factors: ) sars-cov- and nanomaterials are similar in size, setting the stage for direct contact/"combat"; ) sars-cov- ( - nm) [ ] is also near in size to most current fda-approved nanomaterials; ) nanomaterials can decrease the side effects of current antiviral drugs; ) nanomaterials can co-deliver multiple drugs; ) nanomaterials can enhance the stability of mrna vaccines; and ) nanotechnology facilitates controlled/targeted drug j o u r n a l p r e -p r o o f release. in this perspective, we focus on the most promising antiviral research involving nanotechnology and offer relevant paths forward in the current "war" against sars-cov- and future threats posed by other infectious viruses . decades of rapid development have witnessed the widespread application of nanotechnology in the biomedical field. [ , ] although it is not being widely applied in current antiviral research, its potential is unquestionable. to summarize, the advantages of nanotechnology in antiviral research include the following: ) promotes the delivery of water-insoluble drugs; [ ] ) enhances the circulation time of drugs in vivo; [ ] ) achieves co-delivery of drugs; [ ] ) improves drug utilization efficiency and reduce side effects through targeting antibody modification; [ ] ) protects dna and mrna vaccines, overcoming bottlenecks for in vivo applications; [ ] and ) the physicochemical properties of nanomaterials can also be employed directly against viruses. [ ] in this section, we will focus on the aspects of nanotechnology that have the greatest potential for clinical transformation or that have already been applied in the clinic, including in the delivery of antiviral drugs and vaccines, and the design of nanomaterials directly effective against viruses. the fda-approved nanomaterials liposomes [ ] and polylactic-co-glycolic acid (plga) [ ] are already in mature use in the delivery of drugs (and not only antiviral drugs). as described before, fda-approved nanomaterials offer unique advantages for antiviral drug delivery, and potential nanomaterial candidates are still emerging. for example, to guarantee high loading efficiency, biocompatible porous metal-organic-framework nanomaterials were utilized as nanocarriers to deliver drugs including retrovirals to treat acquired immune deficiency syndrome (aids), [ ] releasing the drugs gradually. through chemical bioconjugation, lipid-coated plga nanomaterials first target t helper cells, which express the cd + (cluster of differentiation ) protein on their surface. [ ] subsequent nanomaterials were designed to release encapsulated latency-reversing agents (lras) sustainably, ensuring synergistic effects against human immunodeficiency virus type (hiv- ). in addition, nanoparticles were engineered to offer the controlled release of anti-hiv drugs when triggered by external energy. owing to the magnetoelectricity capability of these nanoparticles, the bond between anti-hiv drugs and the nanoparticles can be broken as required with the application of a uniform magnetic field, releasing the drugs in a controlled manner. [ ] three antiretroviral drugs were also chemically conjugated to human vault nanoparticles , which are intracellular ribonucleoprotein particle complexes that can be easily engulfed by immune-related cells, ensuring the targeted delivery of antiretroviral drugs against hiv type (fig. a) . [ ] moving into the era of "universal" influenza vaccines, pulmonary surfactant (ps)-biomimetic liposomes were designed for heterosubtypic immunity potentiation regulated by cytotoxic t lymphocytes that express the cd + protein. [ ] moreover, j o u r n a l p r e -p r o o f the ps assisted in the delivery of liposomes to alveolar epithelial cells and achieved further immune activation, which ensured good treatment results in vivo. to improve immunogenicity and prevent the humoral immunity of dna vaccines, dual-functional fullerene nanomaterials with an hiv virus-like morphology were fabricated to deliver the vaccine to efficiently stimulate immune response. [ ] since the rna interference (rnai) strategy was proposed, a number of sirna-or mrna-based vaccines have been carefully designed. combining them with nanomaterials improves the stability of rna to the levels necessary for in vivo applications. for instance, adjuvant-free dendrimer nanomaterials were created for mrna vaccine delivery; only one dose was necessary to sustain immunity and combat not only h n influenza but also ebola virus (fig. b) . [ ] in response to the lethal zika virus, mrna vaccines were mentioned in many research projects and some clinical trials. as an example, scientists first embedded the mrna vaccines designed to protect against the zika virus into lipidnanomaterials, and then evaluated the immune response in vivo. [ ] the exciting results drew global attention. coincidentally, the positive results of recent clinical trials by moderna. inc. using an anti-zika mrna vaccine encapsulated in lipid-nanomaterials [ ] provide strong encouragement for the advancement of mrna vaccines against sars-cov- . recent data from pfizer inc. and biontech se has confirmed improvements in the safety and dosedependent efficiency of bnt b mrna vaccines, supporting their potential for ongoing phase / large-scale evaluation. [ , ] the mrna- vaccine against sars-cov- is in phase clinical trials to determine its safety and efficiency for covid- prevention up to years after the second dose. [ ] in other work, the endogenous untranslated regions (utrs) of mrnas were further engineered to enhance the generation of sars-cov- antigens. in addition, using tt nanoparticles for the delivery of mrna vaccine improved performance over fda-approved lipid nanoparticles. [ ] besides these vaccine examples, nanotechnology also shows potential in developing adjuvants for vaccines that promote antibody expression. as traditional aluminum hydroxide (alum) adjuvants , presenting as plate-like microgels with a positive charge, are likely to attach to the membrane rather than be internalized by the dendritic cells, an oil/water interphase of particulate alum via pickering emulsion (rather than a surfactant-stabilized emulsion) was generated, which not only absorbed plenty of antigens but also promoted the uptake of loaded antigens by dendritic cells, improving the immune response induced by vaccines while maintaining safety. [ ] in addition to serving as a delivery platform for antiviral drugs or vaccines, carefully designed nanomaterials themselves can also directly fight viruses. [ , ] first, understanding the virus replication cycle is central to a suitable antiviral j o u r n a l p r e -p r o o f strategy. although we may eventually come to understand viral life cycles differently, the current model includes attachment, entry, biosynthesis, assembly of new viruses , and release; viral inhibition is possible within each of those steps. in this area, the exciting potential of nanomaterials has just begun to be tapped. the advent of dna origami technology has also enriched the original nanomaterial library, while its application in antiviral research is still in its early stages. by designing the dna nanoarchitecture to have a specific star shape, the activity of the dengue virus can be inhibited through spatial pattern interaction. [ ] specifically, the nanoarchitecture was modified with ed -targeting aptamers for recognizing ed clusters on the viral surface. moreover, using heparan sulfate proteoglycan (hspg), gold nanomaterials were employed as broad-spectrum viral inhibitors. unlike previous hspg substances, newly synthesized mercaptoundecane sulfonic (mus) acid-containing ligands are capable of binding viral attachment ligands even after dilution, producing forces that deform viruses. [ ] also, hspg was incorporated into nanogels with various degrees of flexibility to help prevent virus entry. [ ] furthermore, mus acid-modified cyclodextrins have also been the subject of broad-spectrum antiviral research, [ ] as the modified mus acid can mimic hspg to generate a virucidal reaction. in addition to the above nanomaterials, mercaptobenzoic acid-modified gold nanomaterials were also used for hiv inhibition via the multivalent binding approach. [ ] based on the same antiviral strategy, new types of giant globular glycofullerenes were designed against artificial ebola virus infection. [ ] another candidate using nanostructured glycan architecture with suitable space between ligands , composed of multivalent ′-sialyllactose-polyamidoamine ( sl-pamam) conjugates, was designed for influenza a virus infection inhibition. [ ] most recently, bacteriophage capsids carrying ligands that tightly bind the influenza virus were also introduced to prevent its entry through a defined multivalent strategy. [ ] similarly, the glycodendrinanoparticles assembled by high-valency glycodendrimeric components were generated to mimic pathogens and block viral infection (fig. c) . [ ] the good polyvalency effects on the nanoparticles ' surface area suggest a universal strategy to inhibit viral infection. via cell membrane technology, [ ] zika virus host cells' membrane-derived vesicles were added to the surface of gelatin nanomaterials. [ ] these "nanodecoys" capture the zika virus from intended targeting sites and silence viral infection . more recently, plasma membranes derived from human lung epithelial type ii cells or human macrophage cell membrane-derived nanosponges were utilized to mimic the host cell surface and capture the sars-cov- for neutralization (fig. d) . the maturity of antiviral research has always depended on time, effort, and following the right direction. based on our j o u r n a l p r e -p r o o f deepening understanding of sars-cov- acquired just over the last few months, we have witnessed the continuous emergence of clinical trials against sars-cov- . predictably, some are exciting while others are unsatisfactory. throughout this process, nanotechnology has consistently demonstrated its potential, and we can look forward to its bright future (fig. ) . even with the similarities between sars-cov- and other viruses and our accumulated understanding of antiviral mechanisms, the existing drugs used to treat covid- currently have unsatisfactory efficiency, as well as side effects. we anticipate that fda-approved nanomaterials including liposomes, plga nanoparticles , etc. can be used to encapsulate antiviral drugs to achieve long-term circulation, sustained release, and co-delivery of multiple drugs for better treatment efficiency. modified with antibodies that target the proteins in sars-cov- , nanomaterials can simultaneously enhance drug utilization while reducing side effects, thus ensuring better treatment outcomes. the cryo-em structure of potential drugs and the mechanism of their interaction with the life cycle of the virus (e.g. their channels into the rna polymerase that the sars-cov- needs for replication) can be visualized with Å resolution for better understanding to improve drug design. [ ] drug combinations can be further tes ted and co-delivered by nanomaterials. besides, it is well-known that in patients with severe covid- , the hypoxia caused by pneumonia [ ] suppresses the delivery efficiency of antiviral drugs. perhaps nanomaterials will soon be used to co-deliver angiogenesis factors (or other drugs to help relieve hypoxia) and antiviral drugs to generate better outcomes. the inhalation of antiviral drug-loaded nanomaterials is another promising direction. for the delivery of vaccines, current fda-approved nanomaterials can also be modified with targeting antibodies or bionic molecules such as ps for superior immune activation. when deciding whether to introduce potential new candidates for drug and vaccine delivery, it is wise to investigate the nanomaterials currently employed in clinical trials. [ ] since the discovery of exosomes, their potential has been demonstrated in the field of biomedicine. exosomes with nano -size can be derived from cells, have unparalleled biocompatibility, and also carry out some of the same functions as cells. [ ] one promising direction would be to apply exosomes to the delivery of antiviral drugs or vaccines. with features similar to exosomes, nanomaterials mimicking host cell membranes, bacterial membranes, and inactivated viral envelopes may become the next candidate delivery platform. in addition, self-assembly proteins and peptide-based nanomaterials are also promising delivery candidates. based on the life cycle of sars-cov- , and inspired by previous research, we put forward the following possibilities: i) j o u r n a l p r e -p r o o f modified dna or rna origami nanostructures could be designed to inhibit attachment on host cells ; ii) hspg-mimicking nanomaterials or nanomaterials modified with the multivalent ligands could be utilized for inhibition of the infusion process; iii) external zn-based biocompatible nanomaterials may inhibit rna polymerase through zinc ion release; [ ] iv) membrane-targeting photosensitizers/nanomaterials may induce lipid oxidation to prohibit viral fusion via o generation; [ ] and v) viral membrane-targeting photothermal nanomaterials (such as gold) may also destroy virus via local hyperthermia, [ ] as heat has been employed for virus inactivation for many years. in addition, nanomaterials with the antiviral capabilities mentioned above could also be embedded in personal protective equipment (ppe), yielding antiviral ppe. for instance, we could combine existing eco-friendly and mass-production methods [ ] to synthesize nanofibrous materials with the above antiviral capabilities as components of ppe. [ ] introducing nanomaterials to relieve pneumonia symptoms addressing pneumonia, the main symptom of covid- infection, urgently requires the incorporation of nanotechnology. from using nanomaterials to deliver anti-inflammatory drugs, introducing antioxidant nanomaterials, providing inhalation methods, fabricating platelet-derived nanomaterials that are actively targeted to inflammatory sites, and offering the capability for controlled drug release, to utilizing oxygen-generation nanomaterials, such strategies may not only provide timely solutions but also inspire future explorations. for example, platelet-derived nanomaterials can be introduced to encapsulate [ -(p-fluorophenyl)- -ureido]thiophene- -carboxamide (tpca- ) to target the pneumonia site and calm cytokine storm. [ ] moreover, antioxidant nanomaterials such as cerium dioxide nanoparticles can also be utilized to eliminate reactive oxygen species (ros) in the inflammatory site. [ ] other technologies could also be combined with nanotechnology to facilitate and promote its application in covid- treatments. for example, artificial intelligence, which has been explored for covid- diagnosis [ ] and the identification of drugs that could be repurposed to treat covid- , [ ] should also be considered to help isolate (or find) suitable drugs/vaccines-loaded nanomaterials, therapeutic nanomaterials , and nanomaterials that could provide pneumonia relief. with the deepening of our understanding of sars-cov- and related research advancements, we hope and believe that nanotechnology can offer timelier and more effective approaches to dealing with sars-cov- , not to mention other emerging viruses in the future. snapgene fda fda during the ph.d. study, he was focusing on the development of various nanomaterials with energy conversion properties and their corresponding biomedical applications including cancer theranostics and bacterial killing. currently, dr. tang is a postdoctoral fellow at brigham and women's hospital nature nano ambassador with stem degrees/strong background in sustainable naturederived/inspired/mimetic materials for biomed/sensing/catalysis/energy/environment applications, with more than high-impact journal publications in now she is a ph where he holds class ' career development professorship. before joining mit, ming obtained his ph.d. in applied physics at harvard university, and b.s. in engineering mechanics at tsinghua university. his group works on developing tools to probe and understand how mechanics and biology impact each other in a multicellular system and how these properties coordinate in space and time to together sculpt the evolution of multicellular physiological and pathological processes j o u r n a l p r e -p r o o f discussed the content, and made some edits before submission. all authors contributed to the discussion and writing of this manuscript. the authors declare no conflict of interest. key: cord- -ihd qi authors: daughton, christian g. title: natural experiment concept to accelerate the re-purposing of existing therapeutics for covid- date: - - journal: glob epidemiol doi: . /j.gloepi. . sha: doc_id: cord_uid: ihd qi one of the many questions with respect to controlling the novel coronavirus pandemic is whether existing drugs can be re-purposed (re-positioned) for the prevention or treatment of covid- - or for any future epidemic. the usefulness of existing approaches for re-purposing range from computational modeling to clinical trials. these are often time-consuming, resource intensive, and prone to failure. proposed here is a new but simple concept that would capitalize on the opportunity presented by the on-going natural experiment involving the collection of data from epidemiological surveillance screening and diagnostic testing for clinical treatment. the objective would be to also collect for each covid- case the patient's prior usage of existing therapeutic drugs. these drug usage data would be collected for several major test groups - those who test positive for active sars-cov- infection (using molecular methods) and those who test negative for current infection but also test positive for past infection (using serologic antibody tests). patients from each of these groups would also be categorized with respect to where they resided on the spectrum of morbidities (from no or mild symptomology to severe). by comparing the distribution of normalized usage data for each drug within each group, drugs that are more associated with particular test groups could be revealed as having potential prophylactic, therapeutic, or contraindicated effects with respect to disease progression. these drugs could then be selected as candidates for further evaluation in fighting covid- . also summarized are some of the numerous attributes, advantages, and limitations of the proposed concept, all pointing to the need for further discussion and evaluation. drug treatments for covid- are urgently needed. discovery and development of new molecular entities (us fda a) ultimately requires long and costly clinical trials to gain regulatory approval. the shortest route to potential treatments is the repurposing (repositioning) of existing approved drugs (both legend and otc) for pre-and post-exposure prophylaxis to sars-cov- or for treatment of covid- . the concept presented here is intended to rapidly reveal if any existing drugs have therapeutic potential (or those that could be contraindicated) in treating covid- . drug "repurposing" in this article refers to the use of existing approved drugs for the treatment of a never-considered therapeutic indication -in this case, covid- . just considering unique small-molecule drug entities, the universe of those already approved in the us -for multitudes of different medical conditions -might number several thousand (kinch et al. ) ; the numbers of biologics and biosimilars expand this universe considerably (kinch ) . april (revised may) draft for global epidemiology christian g. daughton drug repurposing has proved to be a successful enterprise over the years (baker ; pizzorno et al. ; shameer et al. ) . it is often guided by virtual screening methods such as computational approaches and bioinformatics to reveal possible druggable pathways (e.g., see: smith and smith ; pizzorno ) or even by traditional literature-mining and synthesis (e.g., cahill et al. ) . but even these approaches often end up selecting potential drug candidates that eventually prove ineffective in subsequent clinical trials. and these approaches are seldom used for predicting drugs that would be contraindicated -in this case, those that could adversely interact with covid- or with co-administered drugs. computational approaches are also unable to easily identify multiple drugs that might be effective as combination therapies. proposed here is a simple, straightforward approach to possibly accelerate the identification of existing drugs for potential repurposing and to quickly identify candidates more likely to succeed in phase - clinical trials. these candidates should also have better safety profiles than by trialing candidates selected by other means, especially by avoiding the selection of drugs that adversely interact with other drugs. at the same time, it could also potentially reveal drug combinations that provide enhanced clinical outcomes. a method that could rapidly narrow the initial selection of potential candidates (including combinations) for repurposing with covid- (and also avoiding contraindications) could greatly streamline the conventional approaches for repurposing. it could reduce the probability of candidate drugs failing trials and thereby reduce the wasting of time and resources. an additional important point is that data mined by this approach during an epidemic could also have the potential to short-circuit imprudent decisions to investigate whether drugs rumored to be effective treatments actually have any potential. during the covid- epidemic, one of the best examples was hydroxychloroquine and chloroquine. rumors of effectiveness caused shortages for their approved critical therapeutic uses as anti-malarials and anti-rheumatics (yazdany and kim ) . this proposed approach would capitalize on a unique opportunity presented by a "natural experiment" inherent in the ongoing clinical treatment and epidemiological examination of the covid- epidemic. this approach would also be applicable to any future epidemic, including the possible seasonal resurgence in covid- . opportunities for natural experiments present themselves without active human intervention. natural experiments can reside in an event where different subpopulations are suspected of experiencing a spectrum of exposures, ranging from none to extensive. the archetype example is the historic london cholera outbreak, where dr. john snow was able to identify the route of transmission and propose the solution for ending the epidemic. the proposed approach would identify all drugs used by each of two groups of patient cases defined by whether they test positive or negative for exposure to sars-cov- and/or test positive or negative for active disease. each potential drug candidate could be revealed by whether its per capita usage rate for test each group is significantly higher or lower than the usage rates for the other groups. usage rates that have significant associations with a particular j o u r n a l p r e -p r o o f page of april (revised may) draft for global epidemiology christian g. daughton group may have either increased or reduced potential for providing protection or ameliorating adverse therapeutic outcomes in treating covid- . below is an outline of this proposed approach, termed "natural experiment for repurposing drugs for covid- therapy" -or "nerd" as shorthand. nerd would essentially serve as a continually ongoing, multivariate retrospective observational study. nerd would attempt to guide the repurposing of currently used drugs for pre-and postprophylaxis or therapeutic treatment of covid- . nerd would examine the prior and current drug usage among several combinations of covid- case groups, primarily: ( ) those known to have been exposed or infected but successfully cleared the virus and ( ) those who required extensive medical interventions or died. an excess prevalence of usage of specific drugs within one group versus another group may reveal those drugs having increased potential to be therapeutic or contraindicated (meriting avoidance in prevention or therapy). important to emphasize is that nerd might prove useful not just for revealing potential therapeutics, but also reveal those drugs that might be contraindicated or actually potentiate the progression of adverse covid- outcomes. many different scenarios can be hypothesized for how drug usage might be associated with covid- clinical outcomes. consider the two bookend case outcomes for covid- : ( ) the vast majority that resolve with little to no morbidity and ( ) the minority that become acutely serious. the most general hypothesis is that there are drugs (or combinations of drugs) that have protective effects and perhaps others that have adverse effects. to explain the lower incidence of serious covid- outcomes compared with favorable outcomes, perhaps it is the drugs with the lowest overall clinical usage rates that could explain at least a portion of the serious outcomes -especially among clinical cases having no apparent underlying risk factors. it is likely that widespread covid- testing among the general asymptomatic population will reveal that its infected subpopulation is even larger than currently believed for the subpopulation that progresses to serious outcomes. this would point even further to the possibility that perhaps it is the seldom-used drugs that play a role in serious outcomes. a major obstacle to successfully implementing nerd also needs to be stressed. initially, the only type of therapeutic drug usage data that would be straightforward to collect would be for drugs used in long-term maintenance therapies (both small-molecule and biologics); vaccines and hormonal agents should also be included. therapy would needed to have begun several weeks before exposure but exposure dates would almost always have to be inferred. in contrast, drugs used in short-term therapy could be problematic to collect at first because many additional details would be needed: daily dose, duration of therapy, and time-frame of therapy. many patients may be unable to supply this level of detail. moreover, the fact that the short-term therapy might overlap with different intervals of the disease progression would make interpretation of collected data much more challenging. ( ) as covid- epidemiological surveillance screening and diagnostic testing proceeds, a national database would be continually populated with drug usage data collected from each case among three different combinations of sub-groups based upon whether they tested positive or negative for active sars-cov- infection or tested positive for past sars-cov- infection (see table ). patients would provide a list of all drugs they have used over a time period that safely encompasses the earliest possible date at which they could have been exposed to sars-cov- (for example, months prior). data for all types of drugs would be collected: legend, otc, and recreational/illicit drugs -both small-molecule drugs and biologics, as well as vaccines. drug metadata to collect would include the route of administration, especially inhalation via the nose or mouth, as these might have a greater chance for adversely affecting the pulmonary system. it might seem that drugs of obvious interest during data collection might be immunosuppressants or antivirals, especially those to be effective against enveloped viruses. but many other drug classes might also be important. a major advantage of nerd would be that the screening process is not guided by known or postulated pathways of exposure, infection, or disease progression, as nerd would essentially test all drugs for which usage data is collected. the importance of avoiding cognitive bias is shown by the evolving knowledge of sars-cov- infection, where a major route of infection is believed to be pulmonary (via angiotensin converting enzyme -ace -receptors), leading to severe respiratory inflammation. but ace receptors not only populate alveolar epithelial cells, but also endothelial cells, which means that various organs and blood vessels are vulnerable to inflammatory damage and clotting (phend ) . the collected data would be entered into a computer app that transmits to a central, national relational database pre-populated with generic and trade drug names cross-referenced to their respective unique molecular entities. this would speed data entry, ensure consistent spelling, and account for multiple drug names that correspond to the same molecular entity. note that it can be very challenging to compile a comprehensive list of unique molecular entities just for smallmolecule drugs (kinch et al. ) . biologics and biosimilars would present their own challenges (e.g., see: dabrowska ). ( ) for each of the three test groups, the collective percent-usage rate of each drug (d x ur) would be continually updated in real-time. percentages could also be calculated for sub-groups, determined by the spectrum of severity of clinical status or outcomes -ranging, for example, from asymptomatic, mild, moderate, severe, critical, and death. note that the population-wide incidence of "asymptomatic" covid- cases (where no symptoms are noted over the entire course of the disease) has yet to be determined (e.g., see: lippi et al. ) ; also note that "asymptomatic" is used in this presentation to encompass those who have never been infected as well as those with undetectable subclinical infections. but asymptomatic cases often progress to being pre-symptomatic, where symptoms become evident days after testing positive. this distinction, however, does not matter for nerd, as all of j o u r n a l p r e -p r o o f page of april (revised may) draft for global epidemiology christian g. daughton these cases can be considered positive for infection. so the term "asymptomatic" will be used here. ( ) the d x ur for each group (and subgroups) would then be statistically compared with each other; alternative comparisons might be with the known rates of general population use of each legend drug, or the estimated population-wide use of each non-legend drug. ( ) potential drug candidates would be revealed by whether d x ur distributions are significantly skewed within the major case test groups (see "options" in table ). normalized usage rates that have significant associations with a particular group may have potential for enhancing protection or therapeutic outcomes or, alternatively, the potential for increasing the risk for developing overt signs of infection or exacerbating clinical outcomes. the database would have to be coded to account for patients who receive multiple tests. because of the natural heightened rate of mutation with rna viruses, it would be prove important to constantly re-screen the same universe of existing drugs for therapeutic efficacy. this would occur automatically as the database is updated in real time. ( ) interpretation of nerd data and defining the covid- test groups of most use to nerd. there are currently two general categories of tests for covid- : tests that indicate active infection and tests that primarily indicate past infection that has resolved. the former currently relies on rt-pcr or antigen testing, and the latter relies on serum testing for antibodies to sars-cov- ; conceivable future tests would be those predicting vulnerability to disease progression. these two groups provide complementary information for clinical, epidemiological, and public health purposes. for nerd, however, the drug usage data collected from these two approaches would yield insights for the screening of drugs with a higher probability of success in repurposing. a very limited number of possible combinations exist for how case groups can be defined for use in nerd. these groups are determined by the results for each clinical case and the type of testing used: ( ) positive or negative for active infection, ( ) positive or negative for past infection (i.e., past infections that resolved via immune response with display of little symptomology), and ( ) where the case resides on a morbidity spectrum ranging from asymptomatic, to mild signs and symptoms, to severe complications, to death (refer to table ). while there are two overarching test groups with respect to all suspected cases (positive and negative), there are three major types of tests: molecular nucleic acid (e.g., rt-pcr), antigen, and serologic antibody (isotypes igm and/or especially igg) (see: find ; sheridan ; nas a). rt-pcr is the current gold standard for revealing whether an active infection is underway, as it reveals viable, replicating virus; less costly nucleic acid tests with higher throughput are emerging (broughton et al. ; suo et al. in contrast, antibody tests (in asymptomatic and mild cases) can indicate a graduated spectrum of possibilities, including whether an infection had been cleared sometime in the past or that an active infection is currently being resolved (assuming that more serious morbidities do not develop). important to recognize, however, is that nerd cannot be fully implemented until reliable (low rates of false-negative and false-positive), accurate, standardized, and inexpensive antibody testing becomes widely available in order to perform random serological surveys. not until april was the first emergency use authorization (eua) for a covid- serological test (for use in clinical laboratories) issued by the us fda (https://www.fda.gov/media/ /download); unknown is when any serological test will receive full approval in the us, where validation for specificity and sensitivity are critical. antibody testing would also have to assume that pre-immunity was not possible (e.g., from exposure to other coronaviruses). there are many unknowns and problems surrounding serologic tests for covid- (gronvall et al. ; nas b) . a positive test using rt-pcr or antigen testing would yield a relatively definitive indication for an active infection (especially with the presence of key signs or symptoms). in contrast, a negative test for active disease (without any other complementary test) would not be useful for nerd. this is because negative-test cases would aggregate two primary subgroups: (i) those who have never been exposed to sars-cov- and (ii) those who were infected but who resolved the infection via a successful immune response (being asymptomatic or experiencing only mild infection). both of these subgroups can be disaggregated only by igm/igg antibody testing (option # or # ); the course of the disease can transition from infection-positive to infection-negative after testing immune-positive. it is only this second negative-test subgroup (i.e., positive serum antibody test) that would be useful to nerd. antibody tests provide additional opportunity for interpretation in nerd, as they would represent a portion of the population that was most successful in recovering from self-limited asymptomatic or mild infection. but antibody testing could also prove problematic for interpretation because the patient would need to know when they had probably become infected. if this is not known, then the patient may not know what drug usage they had experienced over the time period encompasses pre-exposure and infection. in table , the group "negative test for past infection" (absence of any immune response), whether used alone (option # ) or even if it were used in concert with a test of active infection (option # ), would not provide any useful information for nerd. note, however that there are two additional sub-groups among the negative test group, which creates two more groups: (iii) those who are infected but their virus titers are too low to yet detect (these could become positive with subsequent testing), and (iv) those with an active infection but the test yielded a false negative (because of sampling errors or test problems). these two groups would serve as confounders for interpreting negative-test data. could point to: (i) potential contraindications and warn of those drugs to avoid for covid- treatment, (ii) potential for increasing the risk of developing more serious infection or exacerbating clinical outcomes, and (iii) drugs that might also make exposed individuals more susceptible to developing serious disease (thereby allowing for more vigilance in avoiding exposure). a positive correlation with severe, critical, or especially fatal outcomes could point to negative interactions of a drug with the disease, and thereby inform recommendations to avoid taking the drug during an epidemic (possible examples being the use of il- inhibitors and steroids; see: kalil ) . conversely, also for the positive-test group, a lower d x ur for a particular drug among severe and critical cases (or higher d x ur among negative cases) could also indicate a protective or prophylactic effect of the drug. while the usefulness of positive cases for nerd is straightforward (indicating drugs to possibly avoid), the predictive power of nerd would be maximized by actively seeking out and testing asymptomatic cases from the general population that never displayed signs and symptoms and therefore would have otherwise possibly escaped diagnostic testing. it is these very cases (along with a subgroup of the clinical diagnostic negatives (see option # or # in table ) that could most likely reveal potential therapeutic drug candidates. indeed, widespread epidemiologic surveillance screening of the general population for asymptomatic covid- has been done in very few countries -iceland being one example (iceland, minister of health ). a major limitation is inherent with nerd with its reliance on the binary choice of infected cases versus uninfected. the limitation results from oversimplifying the actual process of transmission, which is undoubtedly a function of the magnitude of the delivered dose of viral particles. this exposure "intensity" likely results in a continuum of severity of symptoms, rate of disease progression, and modulation of disease outcomes. but this important aspect of covid- is ignored in the concept of nerd developed here simply because there is no current way to easily determine exposure intensity. it is important to recognize that it is within the actual complexities of the disease, such as exposure intensity, that nerd attempts to reveal drugs that impact the disease. so any apparent indication that a drug might correlate with positive or negative disease outcomes might instead simply be a confounding reflection of the type of exposure and not the drug itself. additional attributes, advantages, and limitations of the nerd concept are compiled in table . worth noting is that additional opportunities with the natural experiment presented by the covid- epidemic (as well as any future epidemic) include epidemiologic surveys regarding not just common risk factors such as smoking, but also occupational exposures and real-world inadvertent human exposures to known environmental hazards (e.g., volatile algal toxins and pulmonary toxicants such as atmospheric particulate matter and synthetic chemicals, asbestos, silica, and beryllium). in particular, the possibility exists for discovering whether such non-drug exposures increase the susceptibility or vulnerability to more serious covid- outcomes. this might be especially germane to explaining cases of serious covid- disease among healthy younger adults and reported gender and racial disparities. this would entail a more ambitious data collection effort involving collaboration with industrial and environmental toxicologists to extend the utility beyond drug usage by broadening the exposome and incorporating known occupational and environmental hazards. finally, another unique possibility may exist for verifying the findings of nerd. the relatively new monitoring approach called wastewater-based epidemiology (wbe) has been in use for identifying drugs that are collectively consumed across entire communities. one of the primary applications of wbe has been the monitoring of sewage for drugs (or their metabolites) and back-calculating per capita consumption (sims and kasprzyk-hordern ) . wbe has also been used for monitoring the presence of pathogens such as viruses (to provide early warnings for emerging outbreaks) and more recently proposed for gauging community-wide overall health (daughton ; choi ) or for gauging community-wide status and trends of infection (daughton ) . wbe could therefore possibly be used to complement and verify the findings of nerd. for example, drugs found by nerd to be predominantly associated with asymptomatic or mild cases of covid- could be targeted for wbe monitoring in communities suspected as having low infection rates; note that the overall rates of community infection could also be gauged by wbe (e.g., see: medema et al ; wu et al. ) . conversely, drugs found to be predominantly associated with severe or critical cases of covid- could be targeted for wbe monitoring in communities suspected as having high infection rates. likewise, wbe could be used to quickly assess which communities may have high infection rates, and then target these communities for testing of asymptomatic individuals. surveillance testing and diagnostic testing are critical tools for timely detection, control, and mitigation of any disease epidemic. testing during epidemics provides rare opportunities to capitalize on natural experiments for expanding what is known about exposure, transmission, disease progression, and therapeutic treatment. presented here is a concept for a potential natural experiment that could reveal existing drugs (or multi-drug combinations) that may have higher probabilities of success in their re-purposing (re-positioning) for disease prevention or clinical therapy -or conversely those drugs whose use should be avoided during infection. this concept (natural experiment for re-purposing of drugs -nerd) would require considerable additional discussion and examination before reaching any decision to develop pilot studies to assess its potential viability or usefulness. if successful, however, the concept could prove invaluable in fighting future epidemics involving novel pathogens or mutated strains of prior pathogens for which past immunity or previously effective therapies are no longer effective. the author declares no conflict of interest. the work had no affiliation with any public or private agency. the concept and ideas presented did not originate from any prior published works. could reveal potential contraindications and warn of those drugs to avoid during or for covid- treatment. any knowledge of the pathogen itself or disease progression would not be needed because nerd would rely solely on real-time, real-world evidence for selecting drug candidates. because of this natural process of drug selection, any selected drug candidates for repurposing would have a higher probability of success in phase - clinical trials or for compassionate use. as testing of a population expands, the test population size could be orders of magnitude larger than possible in any clinical trial. the predictive power of nerd would increase as the incidence of testing covers an increasingly larger percentage of the randomly selected general population. the approach naturally incorporates its own inherent natural control group as a result of the diagnostic testing -namely, the negative-test group with positive antibody test. a major advantage might be the ability to reveal combinations of drugs that could possess synergistic therapeutic potential. potential combination drug treatments are extremely difficult to predict with virtual screening approaches. association with the positive-test group might indicate a drug that makes covid- -exposed individuals more susceptible to developing serious morbidities. this would allow for more vigilance against pathogen exposure if the drug is medically necessary or in cessation of the drug if possible. unproven off-label drug therapies that could cause harm (such as during compassionate use) could be potentially avoided; some known examples include the use of il- inhibitors and steroids for end-stage covid- (e.g., see: kalil ) . furthermore, by revealing combinations of drugs that significantly correlate with the positive-test group, further harmful experimental therapies could be avoided. one of the major obstacles to implementing nerd would be the practical difficulties in collecting the needed drug usage information from each patient during or after covid- testing. collecting drug usage information has long been the initial step in the conduct of "medication reconciliation" reviews between the physician and patient during normal visits. the collection step has been problematic (psnet ). one possible means of addressing this weakness would be public service announcements urging everyone to maintain a list of all medications used in the preceding months during epidemics. as with many epidemics, a major problem is inaccurate case counts and under-reporting (weinberger et al. ) . with overwhelmed health care systems, an unknown but possibly very large percentage of positive cases will go undetected. many will die from unknown causes (because of unavailable testing) and many cases will be endured solely at home, with only mild symptoms. very valuable data are therefore lost simply because of incomplete medical records and lack of widespread testing (kliff and bosman ). many mild cases can be missed when they are advised to avoid testing when testing is in short supply. in the absence of widespread randomized testing of the asymptomatic population, drug usage data from a large percentage of the population belonging to test option # would never be acquired. this reduces the predictive value of nerd for potentially beneficial drugs. nerd as presented here makes a liberal assumption that the total population has full access to rx and otc drugs. this is overly simplistic for populations under-served by healthcare. for some pathogens, specific sub-groups of the population can be more susceptible or less susceptible to serious complications or higher death rates, often because of added risk factors. this means that those drugs having higher usage rates among those vulnerable groups could bias the results for negative-and positive-test groups unless the drug usage data collected from patients were also coded for the known risk factors. nerd would not be suitable for screening of those drugs where efficacy is determined by the timing of administration. examples for covid- are drugs used for reducing lung inflammation and damage, such as those required to fight cytokine storm (e.g., geraci and hawill ) . this is especially true if premature timing could worsen clinical outcomes. this could confound nerd's usefulness for these types of drugs. the collection of usage data on biologics and biosimilars, which continue to be a rapidly growing class of therapeutics, could prove difficult, if not just for the fact that the naming conventions are complex (jordan for drugs associated with the positive-test group, they might simply be serving as proxies for co-morbidities that make patients more vulnerable to covid- . a very important factor that nerd would not be able to address is whether a drug might have an impact on increasing or decreasing the overall level or rate of virus shedding, which is a key parameter in inter-individual transmission. because the more limited classes of drugs used in pediatric populations, it would probably be more representative to normalize group drug-usage rates against pediatric and non-pediatric populations. as the ranked, overall nation-wide usage rate for each drug trends downward, the confidence intervals for distinguishing significant differences between the tested groups will increase accordingly, simply because there would be less collected drug usage data. so it would become increasingly more difficult to detect associations for drugs with less market penetration. all types of diagnostic tests have a range of rates for false-negatives and false-positives. this could be a major contributor of error in the confidence of any nerd conclusions regarding drug usage rates. among the active infection test group, a very small portion of the negative-test group may actually comprise newly infected cases (covert virus) whose viral titers are insufficient to meet the minimum sensitivity for a positive test (resulting in a false-negative test); re-testing these cases days later can yield a positive test. crispr-cas -based detection of sars-cov- wastewater-based epidemiology biomarkers: past, present and future biologics and biosimilars: background and key issues monitoring wastewater for assessing community health: sewage chemical-information mining (scim) the international imperative to rapidly and inexpensively monitor communitywide covid- infection status and trends a bibliometric review of drug repurposing scientists to stop covid- diagnostics resource centre. foundation for innovative new diagnostics. geneva switzerland candidate drug, dupilumab, to mitigate covid- patients with severe acute respiratory syndrome by mitigating cytokine storm developing a national strategy for serology (antibody testing) in the united states. the johns hopkins center for health security large scale testing of general population in iceland underway biologics and biosimilars need distinguishable names. stat news treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics an overview of fda-approved new molecular entities an overview of fda-approved biologics medicines official counts understate the u.s. coronavirus death toll coronavirus disease (covid- ): the portrait of a perfect storm presence of sars-coronavirus- in sewage rapid expert consultation on sars-cov- laboratory testing for the covid- expert consultation on sars-cov- viral shedding and antibody response for the covid- pandemic covid- : abnormal clotting common in more severe disease drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy agency for healthcare research and quality (ahrq) patient safety network systematic analyses of drugs and disease indications in repurpose db reveal pharmacological, biological and epidemiological factors influencing drug repositioning fast, portable tests come online to curb coronavirus pandemic future perspectives of wastewater-based epidemiology: monitoring infectious disease spread and resistance to the community level repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface ddpcr: a more sensitive and accurate tool for sars-cov- detection in low viral load specimens covid- ) update: fda continues to accelerate development of novel therapies for covid- covid- ) update: fda authorizes first antigen test to help in the rapid detection of the virus that causes covid- in patients. us food & drug administration, b estimating the early death toll of covid- in the united states sars-cov- titers in wastewater are higher than expected from clinically confirmed cases. medrxiv use of hydroxychloroquine and chloroquine during the covid- pandemic: what every clinician should know. annals of internal medicine this work was unfunded. key: cord- -bajpr a authors: watson, c. james; whitledge, james d.; siani, alicia m.; burns, michele m. title: pharmaceutical compounding: a history, regulatory overview, and systematic review of compounding errors date: - - journal: j med toxicol doi: . /s - - - sha: doc_id: cord_uid: bajpr a introduction: medications are compounded when a formulation of a medication is needed but not commercially available. regulatory oversight of compounding is piecemeal and compounding errors have resulted in patient harm. we review compounding in the united states (us), including a history of compounding, a critique of current regulatory oversight, and a systematic review of compounding errors recorded in the literature. methods: we gathered reports of compounding errors occurring in the us from to from pubmed, embase, several relevant conference abstracts, and the us food and drug administration “drug alerts and statements” repository. we categorized reports into errors of “contamination,” suprapotency,” and “subpotency.” errors were also subdivided by whether they resulted in morbidity and mortality. we reported demographic, medication, and outcome data where available. results: we screened reports and identified errors. twenty-one of were errors of concentration, harming patients. twenty-seven of were contamination errors, harming patients. fifteen errors did not result in any identified harm. discussion: compounding errors are attributed to contamination or concentration. concentration errors predominantly result from compounding a prescription for a single patient, and disproportionately affect children. contamination errors largely occur during bulk distribution of compounded medications for parenteral use, and affect more patients. the burden falls on the government, pharmacy industry, and medical providers to reduce the risk of patient harm caused by compounding errors. conclusion: in the us, drug compounding is important in ensuring access to vital medications, but has the potential to cause patient harm without adequate safeguards. in the modern-day united states (us), medications are by-inlarge manufactured in commercial facilities, and this production is regulated and overseen by the us food and drug administration (fda). historically, however, medications were mixed-or compounded-by independent pharmacists for use by individual patients. while traditional compounding is becoming less prevalent, it still occurs in instances where a particular patient may require a formulation of a medication that is not otherwise available. furthermore, a new form of large-scale compounding has become commonplace, whereby pharmacies produce bulk volumes of medications which are not available commercially, and broadly distribute them to healthcare practices and individual patients. compounding does not traditionally fall under the purview of fda oversight, instead being regulated by individual states' boards of pharmacy. this approach has resulted in a patchwork and oftentimes underfunded regulatory framework, which has subsequently harmed patients [ ] [ ] [ ] [ ] . morbidity and mortality frequently result either from a compounded medication that is contaminated with bacteria, fungi, or another medication during production, or from an error whereby the concentration of the drug dispensed is not as intended, which can lead to inadvertent over-or underdosing. patient harm caused by compounded medications has been the focus of media, medical, and legislative attention in recent years, especially following a multistate, multi-fatality outbreak of fungal meningitis caused by contaminated steroid injections compounded at a pharmacy in framingham, ma [ , , , ] . this article seeks to provide a comprehensive review of the state of outpatient compounding in the us. compounding performed by hospital pharmacies for inpatient use is beyond the scope of this paper. much has been written on compounding pharmaceuticals; this paper is an effort to succinctly address the history, purpose, and regulatory framework in a unified location, as well as to perform a systematic review of all us compounding errors over the past years. to our knowledge, no systematic review of both contamination and non-contamination errors has to this point been undertaken. we will first explore the definition and modern role of compounding. then, we will briefly discuss the modern us history of compounding, with a particular focus on factors influencing the current state of compounding. next, we will examine compounding through a legislative and regulatory lens, to better decipher how governmental oversight-or a lack thereof-may contribute to errors in compounding resulting in patient harm. understanding the interventions being made on a federal level can help improve the safety of compounding. finally, we have performed a systematic review of documented compounding errors and categorized those errors by type and patient outcome. whereby, we elucidate just how and with what frequency patients are harmed by compounding errors, with the ultimate aim of identifying potential strategies for reducing these adverse events. compounding is defined by the fda as the combination, mixing, or alteration of drug ingredients to create medications tailored to individual patient needs [ ] . the united states pharmacopeia (usp), which sets quality standards for drugs, describes compounding as "the preparation, mixing, assembling, altering, packaging, and labeling of a drug … in accordance with a licensed practitioner's prescription …" [ ] put simply, it is the creation of a medication that is not commercially available. in the us, compounding is performed in both the inpatient hospital setting and in outpatient pharmacies, with a trend in recent decades towards larger scale outpatient production [ ] . as will be discussed later in this paper, compounding may now occur in newly defined "outsourcing facilities," which are designed to compound in bulk; some examples of these facilities include quva pharma and leiters [ ] . there are many indications for compounding. some patients may not tolerate pills and require a compounded liquid drug formulation; examples include young children taking antibiotics, feeding tube-dependent patients, or patients with dysphagia from neurologic compromise such as a stroke [ , ] . patients may be allergic to binding agents, dyes, diluents, or other inactive ingredients in commercially available formulations. dietary restrictions, such as a ketogenic diet in pediatric epilepsy patients, may necessitate compounding of sugar-free medications [ ] . refractory neuropathic pain may benefit from compounded analgesic topical creams containing multiple medications not commercially available in combination; examples include ketamine, baclofen, gabapentin, amitriptyline, bupivacaine, and clonidine [ ] . painful oral lesions can be treated with "magic mouthwash" and dyspepsia can be treated with a "gastrointestinal (gi) cocktail"; these are terms that actually encompass a range of compounded preparations [ ] . total parenteral nutrition (tpn) is needed for patients unable to take in sufficient oral nutrition, and numerous chemotherapy regimens must be compounded for cancer treatment [ , ] . healthcare providers may need compounded medications to perform specialized procedures such as intraarticular or intravitreal injections. in some instances, commercial preparations may be available but expensive, and a compounded equivalent is more affordable [ ] . drug shortages, a longstanding healthcare problem exacerbated by crises such as the covid- pandemic and the devastation of puerto rico by hurricane maria, may be addressed by compounding as well [ , ] . the fda has responded to significant shortages during the covid- pandemic by temporarily relaxing restrictions on compounding of commercially available drugs [ , ] . when a compounded medication is prescribed or administered, patient safety depends on adherence to current good manufacturing practices (cgmp), which are outlined in chapter of the usp for non-sterile preparations and chapter for sterile preparations. appropriateness of the prescription indication, safety, and dosing should be assessed by the pharmacist. ingredient quantities must be meticulously calculated, and the source quality of those ingredients assured. compounding facilities and equipment must be clean and monitored continuously. staff must routinely practice and be assessed for competency in proper hygienic measures. sterile preparations, by definition, require a higher level of care to prevent contamination than do non-sterile preparations, including differences in staff training and personal protective equipment (ppe), environment and air quality monitoring, and disinfection. compounded sterile preparations are further subdivided into low-, medium-, and high-risk depending upon the quantity of ingredients, number of manipulations required during compounding, and whether nonsterile ingredients requiring subsequent sterilization are incorporated. multiple medications must not be simultaneously compounded in the same workspace. the compounding process must be reproducible such that medication quality is consistent throughout many production cycles. finally, prescriptions must be correctly labeled and patients instructed in appropriate use [ , [ ] [ ] [ ] . failure to adhere to these standards has the potential to result in patient harm through multiple mechanisms including medication suprapotency, subpotency, contamination, and consumer misuse. throughout pre-industrial history, pharmacists played the critical role of admixing various materials to produce a finished therapeutic substance. this role was, in essence, one of compounding [ , ] . however, the industrial revolution and the resultant mass production of pharmaceuticalscoupled with the increasing presence of synthetic proprietary medications-led to a change in pharmacists' primary role. instead of compounding, community pharmacists in the early s turned their focus to the dispensing of previously manufactured medications as well as to general retail, including operating the soda fountains which came into vogue with the prohibition of alcoholic beverages. in fact, by the s, fewer than % of pharmacies in the us made a majority of their income from pharmaceutical sales [ ] . the decline in community pharmacy compounding was precipitous through the mid- s. in the s, % of prescriptions required some sort of in-pharmacy compounding. that number fell to % by the s, less than % by , and to % by [ ] . interestingly, there was a concurrent increase in the need for hospital pharmacy compounding during the same period; largely due to the advent of chemotherapy, tpn, and cardiac surgery which necessitated the administration of complex cardioplegic regimens. by the s, these advanced therapeutics began to spill into the outpatient setting, generating a novel home infusion industry for treatments such as tpn, antibiotics, and chemotherapeutics [ ] . as a result, the s and s yielded further diversification within the compounding industry as pharmacies began to compound in bulk. this development was brought about by expanding home infusion programs, the more frequent outsourcing of hospital compounding to the outpatient setting, and the rise of hormone replacement therapy. large volume compounding blurs the line between traditional compounding which has state-based regulatory oversight, and the mass manufacture of pharmaceuticals which falls under wellestablished federal fda regulations [ ] [ ] [ ] . the inspiration for this article is a well-documented history of medication errors attributable to pharmaceutical compounding, for which a lack of regulatory oversight persists as a common thread [ , , , ] . the most lethal and infamous of these cases occurred in , when an outbreak of fungal meningitis occurred amongst patients who had received epidural spinal injections. the outbreak affected patients across states, killing [ , , , ] . ultimately, the outbreak was linked to a compounding pharmacy, the new england compounding center (necc, located in framingham, ma). amongst other pharmaceuticals, necc produced injectable sterile methylprednisolone acetate for epidural injections, which it then distributed nationally. following the outbreak (hereafter "framingham"), the fda determined that the pharmacy had disregarded basic sanitary standards and had not taken corrective measures despite internal knowledge of potential contamination [ , , , [ ] [ ] [ ] . as with many compounding pharmacies, necc operated in a historically murky regulatory space, producing medications in bulk as would a commercial pharmaceutical manufacturer, while only being subjected to reduced state oversight given to compounding pharmacies. in fact, in the years preceding the outbreak, the fda had thrice investigated necc and found sterility violations, but they were unable to enforce any interventions or penalties due to the fda's contested regulatory jurisdiction [ ] . both preceding and following framingham, efforts have been made at the federal level to improve oversight of compounding; these are reviewed in depth later in this article. currently, there is incomplete tracking of compounded pharmaceuticals in the us, though they are estimated to comprise - % of all prescriptions [ , , , ] . ultimately, compounding is highly prevalent, and so clinicians must be familiar with the risks associated with compounded medications as they care for patients who may be suffering from a related adverse event. prior to framingham, modern compounding pharmacies evolved within a regulatory framework that lacked distinct federal or state oversight roles. in , the federal food, drug, and cosmetic act (fdca) authorized fda oversight of pharmaceutical manufacturing [ ] . however, because compounders traditionally produced drugs in response to individual prescriptions and on a much smaller scale than conventional drug manufacturers, pharmaceutical compounding developed and remained under the regulatory purview of individual state boards of pharmacy [ , ] . towards the end of the twentieth century, pharmacies began bulk compounding in response to ( ) the home infusion industry and ( ) hospitals' financial interest in outsourcing compounding from their inpatient pharmacies to the outpatient setting [ ] . concerned that bulk compounders were self-classifying as pharmacies to avoid the rigorous federal oversight required of drug manufacturers under the fdca, congress passed the food and drug administration modernization act (fdama) [ ] . fdama addressed the changing nature of compounding pharmacies by creating a "safe harbor" exempting pharmacies from the more stringent fdca regulations so long as compounders refrained from advertising their product and abided by requirements designed to increase drug safety [ , ] . despite congress's attempt to strengthen oversight of compounding pharmacies, litigation challenging fdama tempered the fda's authority to regulate compounders. in , a narrowly divided us supreme court ruled in thompson v. western states medical center that the fdama advertising prohibition was unconstitutional on first amendment free speech grounds [ ] . the ensuing regulatory confusion is well described in the literature, and the details are beyond the scope of this review [ , , , , , ] . for reference, a summary of the relevant legislation and litigation is provided in fig. . decades of regulatory uncertainty culminated in the framingham incident, which revived congressional efforts to address pharmaceutical compounding industry safety concerns. in response to framingham, congress passed and president barack obama signed into law the bipartisan-supported compounding quality act (cqa) as part of a broader legislative package (the drug quality and security act) [ ] . the cqa delineated state and federal oversight authority by defining two distinct categories of compounding pharmacies. the first category is traditional compounding pharmacies, or " a" pharmacies [ ] . a pharmacies under the cqa may compound only in response to individual prescriptions. importantly, a pharmacies may not compound bulk medications either in anticipation of receiving prescriptions or with plans to distribute broadly to healthcare facilities [ , ] . in exchange for complying with these limitations, a pharmacies largely avoid the more burdensome regulations required of drug manufacturers under the fdca, including adhering to cgmp [ , , [ ] [ ] [ ] . accordingly, state boards of pharmacy continue to serve as the primary regulators of a pharmacies [ ] . the cqa created a second category of compounding pharmacy, called an "outsourcing facility." [ ] unlike a pharmacies, outsourcing facilities voluntarily opt-in to this category by paying the fda a user fee (approximately $ , in fy ) [ ] , and comply with stringent cgmp standards as well as reporting requirements [ , ] . because they submit to more robust fda oversight, outsourcing facilities are permitted to compound in bulk in advance of receiving a prescription, and may distribute their products across state lines [ , ] . though the fda enjoys primary regulatory authority over outsourcing facilities, states are not precluded from imposing additional requirements [ ] . should a compounding pharmacy fail to comply with the a criteria or voluntarily register as an outsourcing facility, it is subject to the full breadth of regulations required of drug manufacturers under the fdca [ ] . the distinctions between a pharmacies and outsourcing facilities are illustrated in fig. . following enactment of the cqa, states have taken numerous steps to further develop their respective oversight structures under the new framework. a majority of states have strengthened regulations empowering state boards of pharmacy to hold a pharmacies accountable to higher safety practices, such as requiring conformation with recognized sterile compounding guidelines. however, state oversight of a pharmacies continues to vary, with fewer than half of all states reporting annual inspections of a pharmacies in [ ] . the fda has similarly adjusted its enforcement priorities [ , ] . for example, the cqa permits a a pharmacy to distribute no more than % of its total prescriptions out of state . the goal of this provision is to avoid another national outbreak by reducing the likelihood that contaminated drugs cross state lines. if a given state enters into a mou with the fda, a pharmacies in that state may distribute a higher percentage of prescriptions (now up to %) across state lines in exchange for that state's board of pharmacy agreeing to identify, investigate, and report associated adverse events [ ] . importantly, the mou standardizes procedures for state boards of pharmacy to report concerns to the fda and other states; however, the agreement also grants states significant discretion in how states conduct investigations [ ] . in short, states that participate in the mou, rather than the fda, will undertake primary responsibility for detecting poor quality or dangerous compounded medications distributed by a pharmacies from their state. the fda also announced an effort to entice more compounding pharmacies to register as outsourcing facilities by embracing a risk-based approach [ ] . since enactment of the cqa, far fewer pharmacies have registered as outsourcing facilities than the fda had expected. in fact, the fda anticipated pharmacies to register per year, but only total were registered as of may (even fewer than the registered in ) [ , , ] . to attract compounding pharmacies-some of which have cited cost of compliance with cgmp as a prohibitively expensive barrier to registering as an outsourcing facility-the fda plans to reduce cgmp requirements for compounding pharmacies it deems as "lower risk." [ ] though the fda published draft guidance in describing how the agency may tailor cgmp requirements for outsourcing facilities, the fda has yet to issue final guidance on this matter [ , ] . critics warn that fda and state efforts to implement the cqa regulatory scheme excludes compounding pharmacies from the decision-making process and may limit patients' access to compounded medications. for example, the preserving patient access to compounded medications act (h.r. ) introduced in the us house of representatives attempts to address complaints expressed by compounders [ ] . the proposed legislation seeks to ensure that compounders and other interested parties have an opportunity to comment on (and influence) fda compounding regulations. furthermore, the proposed legislation would explicitly allow physicians who engage in in-office sterile compounding, or who otherwise maintain a supply of compounded medications for "office use," to avoid complying (where state law permits) with outsourcing facility regulations [ ] . meanwhile, the sterility practices of some compounding pharmacies continue to raise alarm: between and , the fda issued more than warning letters to compounding pharmacies, resulting in approximately recalls. as acknowledged by the agency, the fda's transition to a risk-based approach may assist the agency in more efficiently targeting its limited resources, but it could also increase the likelihood of compounders engaging in unsafe practices that elude regulators [ ] . in sum, the cqa and subsequent state and fda actions have somewhat clarified oversight roles after framingham, largely by defining separate a pharmacies and outsourcing facilities. seven years after its enactment, however, uncertainty regarding the relative strength and consistency of said regulatory framework remains. we performed a systematic review of compounding errors, including both errors that resulted in patient harm and those that did not, as reported in the academic literature. we searched the national center for biotechnology information (pubmed; u.s. national library of medicine: bethesda, m a r y l a n d ) a n d e m b a s e ( e l s e v i e r : amsterdam, the netherlands) using the following search criteria: "'compounding and pharmacy' and 'error,' 'overdos*,' 'toxicol*,' 'infect*,' 'death,' 'outbreak,' 'injur*,' or 'case report.'" this search was limited to january through march . additionally, we reviewed abstracts for years - for the following conferences using keyword searches for "compound" and "compounding": american college of medical toxicology (acmt) annual scientific meeting, north american congress of clinical toxicology (nacct), american college of emergency physicians (acep) scientific assembly, society for academic emergency medicine (saem) annual meeting, american academy of pediatrics (aap) national conference & exhibition, and the pediatric academic societies (pas) meeting. we also reviewed the fda's online "drug alerts and statements" repository for alerts regarding compounding pharmacies' failures in sterility and potency standards. authors cjw and jdw screened reports by title and, when necessary for clarification, by abstract. under manual review, articles were excluded if they were obviously irrelevant, consisted of research comparing samples of compounded and commercial pharmaceuticals, were in a non-english language, regarded medications compounded outside of the us, were redundant with another included report, represented misuse of properly compounded medications, regarded veterinary patients, were compounded by an inpatient hospital pharmacy (including chemotherapeutics and parenteral nutrition), were published prior to , or if the report lacked sufficient information to provide substantive value. redundant reports of the same error were included for analysis only once, but efforts were made to reference all identified reporting sources. for included reports, cjw and jdw extracted information including date, type of error, cause of error, number of patients affected, age of patients affected, and clinical course of patients affected. incomplete data was acknowledged and by-inlarge was not grounds for exclusion from the study. we categorized errors under the conceptual framework described by sarah sellers, pharmd, mph, former board member for the fda's advisory committee on pharmacy compounding, in testimony to the us senate committee on health, education, labor, and pensions, namely, that "suprapotency," "subpotency," and "contamination" are the primary risks associated with pharmaceutical compounding [ ] . we further broke down "contamination" into subgroups of "microbiologic contamination" for cases of bacterial, viral, or fungal contamination and "toxic contamination" for noninfectious contaminants. when available, we documented patient age and outcome, route of administration, and medication-in-question, so as to better characterize the types of medications, errors, and patients most associated with adverse events. we referenced and applied the principles for authoring review articles delineated within the journal of medical toxicology when feasible and appropriate during the review process [ ] . our search terms identified potential articles in pubmed and potential articles in embase. the review of conference abstracts yielded additional potential cases as follows: [ , ] . in total, a total of articles, statements, and reports were identified and underwent our manual review (performed by cjw and jdw). after the application of our exclusion criteria, a total of errors were included. these errors are documented as harming patients. when broken down by type, contamination accounted for errors adversely affecting patients (appendix table ) and errors in concentration accounted for events adversely affecting patients (appendix table ). there were reports of identified compounding errors which potentially exposed innumerable patients but did not end up causing any known harm; these were predominantly errors of contamination (appendix table ). the number of patients exposed to potential harm cannot be calculated based on the available data, but reaches at least the several thousands (framingham alone exposed , patients with documented instances of patient harm). table is a summary of the included contamination errors. with patients over errors, the median number of patients affected per error is . the mean number of patients affected per error is ; however, by excluding framingham, that number is . with deaths over errors, the mean number of fatalities per error is ; however, excluding framingham drops that number to less than ( . ). the median number of deaths per error is . five out of the contamination errors were from intraarticular (including epidural) steroids, and eight of were from medications injected intravitreally. a total of of the errors were from medications administered parenterally, in healthcare settings. three of were from toxic contamination rather than microbiologic contamination. interestingly, six of errors with documented adverse outcomes occurred following the cqa. table is a summary of the included sub-and supratherapeutic errors. one report describes a subtherapeutic error affecting pediatric patients who were on posttransplant immunosuppression with tacrolimus. the remaining reports involved errors of supratherapeutic drug concentrations; they affected a total of patients, of which ( %) were pediatric. of the total patients affected by concentration errors, ( %) were pediatric and ( %) were over the age of years. three patients died, all of whom received supratherapeutic intravenous colchicine at an alternative medicine infusion clinic for chronic back pain [ ] . appended to this article are appendix tables , , , which respectively catalog all contamination errors causing patient harm, all sub-and suprapotency errors causing patient harm, and all potential compounding errors identified and rectified before patient harm occurred. of the potential errors identified before patient harm occurred, came after the enactment of the cqa. in this study, we separated compounding errors into the categories of contamination, suprapotency, and subpotency. we found that medications with contamination errors are frequently ( ) bulkproduced and distributed, ( ) used parenterally, and ( ) administered by physicians. because of their parenteral administration, medications contaminated with otherwise benign environmental flora are able to disseminate throughout the body and cause the devastating outcomes documented here. furthermore, because contamination errors are often associated with larger-even multistate-distribution networks, the reach of their impact is large. framingham was the archetypal contamination error. it woke much of the medical and lay communities to the potential dangers of compounding. it inspired the federal government to enact the cqa in and create an entirely new form of compounding facility-the outsourcing facility-to attempt to regulate the subsection of pharmacies who were bulk-compounding medications not available (or not available cheaply) through commercial channels, and who exported [ ] certainly, contamination has persisted despite the cqa and the fda's efforts to oversee outsourcing facilities. given this ongoing concern, the medical community must bear some of the responsibility for reducing the number of medications manufactured in substandard environments. it should be the expected standard for healthcare practices to purchase exclusively from compounding pharmacies strictly adherent to cgmp standards and formally approved as outsourcing facilities by the fda. leading expert outterson referenced the potential for this approach in [ ] , and it is unclear how purchasers have responded. while these policies may be more expensive; the physical, ethical, and even financial [ ] consequences of purchasing compounded medications from organizations not sufficiently invested in safety are clearly documented here. subpotency and suprapotency can be considered as the single category of errors of concentration, as the sources and scope of concentration errors are largely similar. our findings demonstrate that subpotency is largely not a reportable issue, but that does not mean it is not a danger. as an example, beyond the cited series of subtherapeutic tacrolimus concentrations [ ] , another case series (excluded for location outside the us) identifies dozens of patients who received subtherapeutic chemotherapy treatments [ ] . these subtherapeutic errors are difficult to capture. identification must be done during routine serum testing, as occurred with the tacrolimus series; or on the supply side, as occurred with the chemotherapy series. when considering subtherapeutic and supratherapeutic errors together as errors of concentration, we found a somewhat different pattern than that which we identified amongst errors of contamination. the concentration errors we were able to identify, with a few notable exceptions [ , ] , were caused by traditional compounding pharmacies. these pharmacies, labeled as a pharmacies under the cqa, are limited in their scope to producing compounded medications only after an individual prescription is in-hand. per the cqa, a pharmacies are still solely regulated by state boards of pharmacy, meaning that oversight is patchwork across the us. many concentration errors are of orders of magnitude, suggesting that simple mathematical and measurement mistakes are to blame. in addition to hoping that states will implement greater oversight of these a pharmacies, we call on the pharmacy industry to emphasize and standardize compounding training amongst its students and even consider a mandatory credential before allowing a pharmacist or pharmacy technician to compound a medication [ , [ ] [ ] [ ] . it is worth noting that -aminopyridine and liothyronine are fairly uncommon medications, however they accounted for a large number of compounding concentration errors. there is nothing particularly special about these medications which make them prone to concentration errors, except for the fact that they are not readily commercially available, and so they must be compounded. the prescribers of these medications need to carefully consider the benefits and risks of prescribing a treatment which requires compounding; especially when the risks are so great (status epilepticus with -aminopyridine and thyrotoxicosis with liothyronine). liothyronine, in particular, has had its clinical utility recently questioned. for example, the national health service (uk) has recently called on general practitioners to stop prescribing liothyronine without specialist consultation, as most patients benefit equally from commercially prepared levothyroxine [ ]. given the risks of inadvertent overdose due to compounding errors, providers must consider commercially available alternatives whenever able. in fact, it has been questioned w h e t h e r p r o v i d e r s w h o k n o w i n g l y p r e s c r i b e a compounded medication despite commercially available alternatives might be legally liable for any harm resulting from compounding errors [ ] . at the very least, it is incumbent on prescribers as well as pharmacists to educate their patients on the risks of taking a compounded medication-both from errors in concentration and contamination-and to instruct them on when to present to a healthcare provider. additionally, practitioners must be aware of their patients' medication lists, and consider a possible compounding error as a cause of medical illness. notably, we found that the majority of concentration errors were made in pediatric and geriatric patients, vulnerable populations who are already at increased risk of providers failing to diagnose toxicity from prescription medications. in and beyond, we anticipate the demand for compounding to only increase. the number of novel therapeutics continues to rise rapidly, as do their approved routes of administration. the anti-angiogenesis medication bevacizumab is a classic example; it is commercially manufactured but is frequently compounded into smaller aliquots for intravitreal administration. as we have seen, this process has unfortunately resulted in multiple outbreaks of endophthalmitis [ ] [ ] [ ] [ ] [ ] . furthermore, regional and global disasters have recently resulted in significant pharmaceutical supply chain issues. examples of this phenomenon include hurricane maria's impact on puerto rican manufacturers in and the covid- pandemic [ ] [ ] [ ] [ ] . these disruptions place increased demand on alternative means of supply, including via pharmaceutical compounding. in fact, the covid- pandemic and its associated drug shortages has already resulted in the loosening of fda restrictions, including allowing outsourcing facilities to compound copies of commercially available drugs for hospital use [ ] . our study has its limitations. while we made every effort to capture published cases of compounding errors, it is possible that our search criteria missed some cases that would have impacted our analyses. while we also strove to review less-traditional sources, including conference abstracts and fda alerts, we are not free of publication bias and are at risk for having excluded compounding errors not associated with adverse events, or with very small numbers of patients affected. similarly, it must be noted that compounding errors can only be identified following adverse events, laboratory screening, or industry or governmental report. even once identified, we were dependent on the publication of the error in order to capture it here. as such, we are likely underreporting the frequency with which compounding errors occur. compounding is more relevant than ever. appreciating that the need for compounding is unlikely to diminish in the near future, we can only re-emphasize the critical nature of our recommendations for the federal and state governments to fully fund the oversight of outsourcing facilities, for healthcare practices to refuse medications compounded without strict adherence to cgmp and fda regulations, for pharmacy schools to expand compounding training and certification, and for physicians to think critically about the risks of prescribing medications that are not commercially produced. conversely, we must remain aware that compounding pharmacies frequently provide an essential service and poorly calibrated regulations may contribute to issues of access. ultimately, medical providers must remain vigilant, especially when caring for members of vulnerable populations, and consider the possibility that a new-onset illness may very well be the result of a compounding error. supplemental outsourced compounding can be problematic meningitis outbreak reveals gaps in us drug regulation regulating compounding pharmacies after necc how gaps in regulation of compounding pharmacy set the stage for a multistate fungal meningitis outbreak fungal infections associated with contaminated methylprednisolone in tennessee fungal infections associated with contaminated methylprednisolone injections human drug compounding. u.s. food & drug administration pharmaceutical compounding -nonsterile preparations. united states pharmacopeia potential risks of pharmacy compounding guidance for entities considering whether to register as outsourcing facilities under section b of the federal food, drug, and cosmetic act. u.s. food and drug administration medication administration through enteral feeding tubes penicillin vk oral suspension the use of ketogenic diet in pediatric patients with epilepsy. isrn pediatr topical treatments for diabetic neuropathic pain (review) characterization of tetracycline hydrochloride compounded in a miracle mouthwash formulation parenteral nutrition therapy over the next - years: where are we heading? intravenous chemotherapy compounding errors in a follow-up pan-canadian observational study compounded preparations with nystatin for oral and oromucosal administration compounding pharmacy conundrum temporary policy for compounding of certain drugs for hospitalized patients by pharmacy compounders not registered as outsourcing facilities during the covid- public health emergency (revised): guidance for industry. united states food and drug administration pharmaceutical compounding -sterile preparations. united states pharmacopeia guidelines for compounding practices pharmacy compounding of high pharmaceutical compounding: the oldest, most symbolic, and still vital part of pharmacy towards a greater professional standing: evolution of pharmacy practice and education the continuing evolution of american pharmacy practice improving the quality of compounded sterile drug products: a historical perspective accuracy of testosterone concentrations in compounded testosterone products update on medical and regulatory issues pertaining to compounded and fda-approved drugs, including hormone therapy compounding problems and compounding confusion: federal regulation of compounded drug products and the fdama circuit split compounding errors multistate outbreak of fungal infection associated with injection of methylprednisolone acetate solution from a single compounding pharmacy -united states multistate outbreak of fungal meningitis and other infections clinical response, outbreak investigation, and epidemiology of the fungal meningitis epidemic in the united states: systematic review health policy brief: regulating compounding pharmacies. health aff may. ; . . drug compounding: fda has taken steps to implement compounding law, but some states and stakeholders reported challenges. united states government accountability office federal food, drug, and cosmetic act, usc § § - i federal authority to regulate the compounding of human drugs food and drug administration modernization act of , usc § a the drug quality and security act the proposed drug quality and security act (h.r. ). congressional research service examining implementation of the compounding quality act, hearing before the subcommittee on health of the house committee on energy and commerce. th cong the drug quality and security act -mind the gaps usc § a(a)-(b) human drug compounding outsourcing facility fees navigating through a complex and inconsistent regulatory framework: section b of the federal food drug and cosmetic act outsourcing facilities engaged in clinical investigation state oversight of drug compounding. pew charitable trust compounding policies priorities plan agency-informationcollection-activities-submission-for-office-of-management-andbudget-review b)(i)-(ii) registered outsourcing facilities. united states food and drug administration current good manufacturing practice -guidance for human drug compounding outsourcing facilities under section b of the fd&c act: guidance for industry. united states food and drug administration preserving patient access to compounded medications act of , hr , th cong federal and state role in pharmacy compounding and reconstitution: exploring the right mix to protect patients: hearing before the senate committee on health, education, labor, and pensions. th cong, st sess ( ) (testimony of sarah sellers, pharmd, mph, executive director writing an effective review article description of outbreaks of health-care-associated infections related to compounding pharmacies, - compounding pharmacy-related outbreaks, - : public health and patient safety lessons learned outbreak of serratia marcescens infections following injection of betamethasone compounded at a community pharmacy exophiala infection from contaminated injectable steroids prepared by a compounding pharmacy -united states north carolina department of health and human services estimated deaths and illnesses averted during fungal meningitis outbreak associated with contaminated steroid injections multistate investigation of suspected infections following steroid injections neurologic complications including paralysis after a medication error involving implanted intrathecal catheters sphingomonas paucimobilis bloodstream infections associated with contaminated intravenous fentanyl outbreak of bacteremia due to burkholderia contaminans linked to intravenous fentanyl from an institutional compounding pharmacy an outbreak of postoperative gram-negative bacterial endophthalmitis associated with contaminated trypan blue ophthalmic solution eye opening: are compounded drugs causing harm? in: society of healthcare epidemiology of america annual scientific meeting an outbreak of streptococcus endophthalmitis after intravitreal injection of bevacizumab an outbreak of fungal endophthalmitis after intravitreal injection of compounded combined bevacizumab and triamcinolone endophthalmitis outbreak associated with repackaged bevacizumab management of a cluster of endophthalmitis cases due to nutritionally variant streptococcus following intravitreal bevacizumab fungal endophthalmitis associated with compounded products fda alerts health care professionals of adverse events associated with guardian's compounded triamcinolone and moxifloxacin product for intravitreal injection. united states food and drug administration life-threatening sepsis caused by burkholderia cepacia from contaminated intravenous flush solutions prepared by a compounding pharmacy in another state in the matter of central admixture pharmacy services, inc. -summary suspension. maryland state board of pharmacy outbreak of systemic inflammatory response syndrome linked to a compounding pharmacy -virginia multistate outbreak of pseudomonas fluorescens bloodstream infection after exposure to contaminated heparinized saline flush prepared by a compounding pharmacy a multistate outbreak of serratia marcescens bloodstream infection associated with contaminated intravenous magnesium sulfate from a compounding pharmacy outbreak of serratia marcescens bloodstream infections in patients receiving parenteral nutrition prepared by a compounding pharmacy compounding: inspections, recalls, and other actions against nuvision pharmacy united states food and drug administration compounding: inspections, recalls, and other actions against specialty compounding, llc; cedar park, tx. united states food and drug administration how a drug shortage contributed to a medication error leading to baclofen toxicity in an infant fda announces voluntary recall of all unexpired human and animal compounded drug products produced by reliable drug pharmacy outbreak of mycobacterium chelonae skin infections associated with human chorionic gonadotropin injections at weight loss clinics. open forum infectious diseases: in fda warns compounders not to use glutathione from letco medical to compound sterile drugs. united states food and drug administration deaths from intravenous colchicine resulting from a compounding pharmacy error -oregon and washington response to mold contamination of intravenous magnesium sulfate produced by a compounding pharmacy the risk of using compounded immunosuppressants in children. in: th congress of the international pediatric transplant association effect of unintentional cyclophosphamide underdosing on diffuse large b-cell lymphoma response to chemotherapy: a retrospective review fda announces pharmakon pharmaceuticals voluntary recall of morphine sulfate . mg/ml preservative free in . % sodium chloride. united states food and drug administration united states food and drug administration compounded drugs: are customized prescription drugs a salvation, snake oil, or both? compounding pharmacies: a viable option, or merely a liability? time for compounding certification? drug topics risk and liabilities of prescribing compounded medications hospitals wrestle with shortages of drug supplies made in puerto rico. the new york times update on recovery efforts in puerto rico, and continued efforts to mitigate iv saline and amino acid drug shortages. united states food and drug administration covid- lockdown: reports indicate shortage in antidepressants hospitals see shortages of a cheap steroid that one study says helps covid- patients a -fold overdose of clonidine caused by a compounding error in a -year-old child with attention-deficit/hyperactivity disorder pediatric clonidine poisoning as a result of pharmacy compounding error toxicity from a clonidine suspension prolonged hypertension from a , fold clonidine compounding error clonidine compounding error: bradycardia and sedation in a pediatric patient akathisia in two patients following newly compounded -aminopyridine severe accidental overdose of -aminopyridine due to a compounding pharmacy error unintentional -aminopyridine overdose in a multiple sclerosis patient: case presentation with a focus on intervention thyroid storm from a liothyronine compounding error a case of thyrotoxicosis due to a compounding error iatrogenic thyrotoxicosis and the role of therapeutic plasma exchange atropine overdosage with a suppository formulation containing atropine sulfate beware of what is in the mixture: calculation error in compounded gi cocktail atenolol compounding and atrioventricular block: a case report flecainide toxicity in a pediatric patient due to differences in pharmacy compounding iatrogenic cushing syndrome in a child with congenital adrenal hyperplasia: erroneous compounding of hydrocortisone pyrimethamine-induced seizure caused by compounding error unusual presentation of iatrogenic phenytoin toxicity in a newborn fda alerts patients and health care providers not to use budesonide solution from the compounding shop. united states food and drug administration fda alerts health care professionals not to use sterile drugs from downing labs (aka nuvision pharmacy) fda announces medistat rx's nationwide voluntary recall of sterile drug products. united states food and drug administration fda alerts health care professionals not to use sterile drug products from qualgen. united states food and drug administration united states food and drug administration fda alerts health care professionals and patients not to use sterile drug products from vital rx, dba atlantic pharmacy and compounding. united states food and drug administration fda alerts health care professionals to voluntary nationwide recall of all sterile products from coastal meds. united states food and drug administration fda announces ranier's rx laboratory's voluntary recall of all sterile compounded drugs. united states food and drug administration fda alerts health care professionals and patients not to use sterile drug products from pharm d solutions. u.s. food & drug administration fda alerts health care professionals and patients not to use drug products intended to be sterile from promise pharmacy. united states food and drug administration fda alerts patients and healthcare professionals to infusion options' voluntary recall due to quality issues. united states food and drug administration dba amex pharmacy, voluntary recall of all sterile compounded drugs. united states food and drug administration publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - rbbe c authors: mukherjee, pulok k. title: antiviral evaluation of herbal drugs date: - - journal: quality control and evaluation of herbal drugs doi: . /b - - - - . - sha: doc_id: cord_uid: rbbe c the viral infection and resistance to the existing antiviral drugs are alarming, which is a serious public health concern. medicinal plants are valuable resources for treatment of viral infections and can be used for the management of infections like herpes simplex virus (hsv), human immunodeficiency virus (hiv), influenza, etc. the antiviral screening of plant extracts should be highly selective, specific, and sensitive for bioactivity guided isolation of the active compounds from the plant extracts. the antiviral screening system should be validated for accuracy, reproducibility, simplicity, and cost effectiveness. this chapter highlights on various aspects for screening and evaluation of antiviral natural components including factors affecting antiviral in vivo studies, host cells, organisms, and culture media followed by different virus-specific assays for antiviral screening of natural products. although vaccines have been very successful in controlling many viral diseases, some diseases are likely to be controlled only by antiviral chemotherapy. the concept of antiviral drugs has only been accepted slowly, partly because of the toxicity of many of the earlier antiviral agents. in contrast to the development of antibiotics, attempts to develop antiviral drugs have indeed met a variety of problems. being strictly dependent on cellular metabolic processes, viruses possess only limited intrinsic enzyme systems and building blocks that may serve as specific targets for a drug. moreover, contrary to a bacteriostatic compound, an effective antiviral drug should not only display considerable specificity in its antiviral action, but should also irreversibly block viral synthesis in order to stop cell suicide due to the viral infection and restore normal cell synthesis (vanden berghe et al., ) . in addition to this inhibition, the antiviral agent must have a broad spectrum of activity, favorable pharmacodynamic properties, and not be immunosuppressive. in the ideal situation, the antiviral drug checks the infection while the immune system prepares to destroy the last virus particles (munro et al., ) . this point is critical for those immune-compromised by illness (aids, cancer) or drug therapy (transplants, cancer). a frequent cause of death in these instances is from viral infections, so that adjuvant antiviral chemotherapy is vital in these circumstances (shannon and schabel, ) . many viral infectious diseases still cause high mortality. although antiviral chemotherapy has shown outstanding progress, antiviral agents are still required. the emergence of drug-resistant viruses during treatment raises a potential problem for effective therapy. furthermore, new viral pathogens may be discovered. biologically active substances of plant origin have long been known as viral inhibitors. these antiviral compounds may be extracted from sources, such as higher plants, which have, for various reasons, been explored considerably less than the traditional ones. the first clinically useful antiviral drug was methylisatin-thiosemicarbazone (methisazone), which was active against pox viruses, especially smallpox virus. methisazone ( mg/kg) was found to inhibit variola virus in mice and was later used successfully to treat cases of eczema vaccinatum and to treat and prevent smallpox. kaufman et al. ( ) , an ophthalmologist, successfully treated a herpes eye infection with an antineoplastic drug, idoxuridine. at the same time, a group of chemists at the du pont chemical company in the united states investigated the antiviral activity of a molecule called amantadine. it was active against influenza virus type a. in rapid succession, further nucleoside analogs, cytosine arabinoside, trifluorothymidine, and adenine arabinoside, were found to inhibit herpes virus. in the s, the antiherpes activity of a new compound, -( -hydroxyethoxymethyl) guanine (acyclovir), was detected by j. bauer at the wellcome research labs. within a decade, the same group was to discover azidothymidine (zidovudine), the first effective inhibitor of the newly emerged hiv. the alkaloid from the australian horse chestnut (castanospermine australe), isolated in the s, was also found to be active against hiv. a research program to detect and isolate antiviral compounds from higher plants is best carried out by a multidisciplinary team, consisting of at least a pharmacognosist and a virologist. the antiviral screening system should meet all requirements of any good assay, including validity, lack of ambiguity, accuracy, reproducibility, simplicity, and reasonable cost. moreover, because we are dealing with plant extracts, the antiviral screen should be highly selective, specific, and sensitive; it is advisable to discriminate a true antiviral activity from a viricidal one at this stage. because most of the aforementioned requirements are better met by in vitro testing, we not only prefer in vitro screening of the plant extracts, but also the use of the same bioassay to guide the isolation of the antivirally active compounds from the plant extracts. the antiviral activity of the pure compounds then has to be confirmed in a later stage by in vivo assays (leven et al., ; vanden berghe et al., ) . one of the possible strategies for finding new antiinfective drugs may involve the search for compounds with chemotherapeutic activities supplementary to, and structures widely different from, those in current use. these compounds could be extracted from sources that have, for various reasons, been explored considerably less than the traditional microorganisms, including, among others, higher plants (mitscher and rao, ) . considering the enormous number and the amazing structural diversity of the currently available antimicrobially and antivirally active plant constituents, one might hope that promising systemic and/or locally acting antiinfective agents might be discovered in the plant kingdom (vanden berghe et al., ; vlientinck et al., ) . the increase of drug-resistant viruses in treatment raises an important issue for effective treatment. moreover, new viral pathogens might be found. along these lines, there is a strong requirement for promptly accessible antiviral medications at moderate costs with minimal side effects. henceforth, traditional medicines ought to be investigated as novel antiviral agents, as many of these ancient medicaments, containing different plant metabolites, have potent antiviral activities (chattopadhyay and khan, ) . the design of new antiviral drugs potentially focuses on the structural dynamics and replication cycles of the various infections causing viruses. a suitable example is the invention of acyclovir, which hinders certain proteins of herpes infections responsible for the triggering of disease. ethnomedicines are a vast repository of structural diversities and extensive bioactivities that can serve as a huge source of potential antiviral drugs. a significant number of medicinal plants from ayurveda and the traditional chinese system of medicine serve as potential remedies to decrease the severity of illness caused by viruses (chattopadhyay et al., ; khan et al., ; jadhav et al., ) . research on the antiviral potentials of plants was first started in , and out of plants were found to be effective against influenza (chattopadhyay and naik, ) . numerous screening studies have been conducted in the last few years to determine the antiviral efficacy of medicinal plants using in vitro and in vivo assays. a few out of a british colombian medicinal plants showed antiviral efficacy against respiratory syncytial virus (rsv), corona virus, influenza virus, and herpes simplex virus (hsv) (mccutcheon et al., ) , while the marine algae spirulina showed antimutagenic, immunomodulatory, and antiviral activities (chamorro et al., ) . interestingly, cyanovirin n, an -kda protein of blue green algae, inactivates hiv- by binding with its glycoprotein (clercq , while sulfated polysaccharides of seaweeds and algae showed anti-hiv and anti-hsv activities (schaeffer and krylov, ) . the plant kingdom is highly diverse and ranges from unicellular microscopic plants to long lived, huge trees. to screen each and every plant or their individual parts for the identification of antiviral components is a huge task. several examples of plants having antiviral properties and newly identified active compounds from them are reported in various journals. one of the examples that can be cited here is cyanovirin n (cv-n), an -kda protein isolated from the cyanobacterium nostoc ellipsosporum, which exhibits the property of inhibiting hiv- infection and also possesses broad-spectrum activity. the phytochemical, podophyllotoxin, isolated from the aqueous extract of podophyllum peltatum l., inhibited hsv type (hsv- ) (bedows and hatfield, ) . the acetone extract of another plant, phyllanthus urinaria, also suppressed hsv- and hsv- (yang et al., ) . bessong et al. ( ) reported a comparison of various plants and their individual parts (stem, leaves, roots, and so forth.) in repressing viral reverse transcriptase (rt) and integrase, the two basic enzymes in hiv disease. after comparing all the extracts and fractions of the various plants, it was found that the n-butanol fraction of bridelia micrantha (hochst) exhibited the highest anti-rt activity. it has also been reported that the aqueous extract from the roots of carissa edulis (forssk.) vahl, a plant grown in kenya, displayed noteworthy activity against hsv for both wild type and resistant strains (tolo et al., ) . polyphenol-rich extract from the medicinal plant geranium sanguineum l. has been reported to show a strong antiinfluenza virus activity, as well as antioxidant and radical scavenging capacities (sokmen et al., ) hepatitis a, b, c, d, and e viruses are the leading causes for the prevalence of viral hepatitis and liver inflammation. despite the fact that presentation to any of these infections prompts intense disease, in any case, types b, c, and d are unique in causing chronic infection. plants belonging to the genus phyllanthus of the euphorbiaceae family were extensively used as a traditional remedy against these infections. clinical investigations were additionally intended to look at the inhibitory effects of different species of phyllanthus, that is, p. amarus (l.), p. niruri (l.), and p. urinaria (l.) (wang et al., ) . the screening of different chinese medicinal herbs led to the identification of two potent plant extracts against duck hepatitis b virus, namely, ardisia chinensis and pithecellobium clypearia . similarly, this also led to the identification of oenanthe javanica (blume) dc flavones (ojf). they acted as a strong inhibitor of hbsag and hbeag secretion (involved in viral pathogenesis) in . . cells and also reduced dhbv-dna levels in the hbv-infected duck model (wang et al., ) . because of the strong prevalence of hcv infection in poor countries, screening for the identification of anti-hcv potentials from medicinal plants are still ongoing. according to hussein et al. ( ) various plant extracts, such as methanol extracts acacia nilotica l. willd ex delile, boswellia carterii, embelia schimperi, quercus infectoria, trachyspermum ammi l., and aqueous extracts of piper cubeba l., q. infectoria, and syzygium aromaticum l., were found to possess significant inhibitory activity against hsv. combination therapy for treating diseases is an age-old practice of traditional medicine in which several plants are mixed together to develop an effective formulation for a particular disease. such combination therapies have also been tried for the inhibition of viral hepatitis. as an example, a chinese herbal medicine, prepared by liquid fermentation broth of ganoderma lucidum supplemented with an aqueous extract of radix sophorae flavescentis, was potent against hepatitis b virus activity in vitro and in vivo. viral infections are a matter of great concern for this planet. plants having broad-spectrum activity against many viruses could be evaluated for isolation and identification of molecules for treating viral infections. as an example, glycyrrhizin, a bioactive component of licorice (glycyrrhiza uralensis fisch), and lycorine, isolated from lycoris radiata l., showed strong anti-sars-cov activity, and was initially used for treating other indications (li et al., a, b) . a variety of herbal preparations have shown potentials for inhibiting viruses that cause serious infections among humans, such as measles viruses (olila et al., ) , human rotaviruses (hrv) (husson et al., ; takahashi et al., ) , respiratory syncytial virus (rsv), human rhinoviruses (glatthaar-saalmuller et al., ) , the coxsackie group of viruses (evstropov et al., ; su et al., ) , neurotropic sindbis virus (nsv) (paredes et al., ) , and various strains of polio virus (andrighetti-frohner et al., ; melo et al., ) . one such illustration is the atomic investigation of the heated water concentrates of stevia rebaudiana l., which blocked a section of different irresistible serotypes of hrv into permissive cells by an anionic polysaccharide having a molecular weight of with uronic acid as a noteworthy sugar constituent (takahashi et al., ) . thus, an alkaloid concentrate of haemanthus albiflos globules repressed rna amalgamation of hrv spread in ma- cells (husson et al., ) . in contrast to the many publications on antibacterial and antifungal screening of plant extracts that have appeared in the last decades, far fewer antiviral screening studies of plant extracts have been reported. this is due chiefly to the complexity of the different techniques involved in such research, which consequently requires the know-how and dedication of a multidisciplinary team. nevertheless, many antiviral agents have been isolated from natural sources and partly or completely characterized. from these studies, several substances have emerged as true antivirals having a good chemotherapeutic index based on the viability of infected cells and on in vivo tests. thus, different -methoxy flavones and synthetic derivatives have shown to be promising leads for the development of antirhinovirus drugs (van hoof et al., ; vlientinck et al., ) , whereas the spanonin, glycyrrhizic acid, was found to be highly active in vitro against herpes simplex (pompei et al., ) , varizella-zoster (baba and shijeta, ) , and human immunodeficiency viruses (ito et al., ) . whether these compounds have any clinical potential, that is, in the therapy of the corresponding viral diseases, remains to be determined. there, in vivo bioavailability and other pharmacokinetic parameters are subjects of future study. because of the problems of drug resistance stated earlier and of side effects, the pharmaceutical industry is looking forward toward natural products (mainly medicinal plant extracts) as a source of possible antiviral agents. approximately medicinal plant species have been recorded globally to treat a myriad of inflictions and diseases. polyphenols, alkaloids, flavonoids, saponins, quinones, terpenes, proanthocyanidins, lignins, tannins, polysaccharides, steroids, thiosulfonates, and coumarins are prominent bioactive phytochemicals that have been observed to combat viral infections, as they are harmless to the systems of the human body. some selected anthraquinones and anthraquinone derivatives are noted for their dammarane saponins, ginsenoside rb (grb ), and chikusetsusaponin (chi-iii) have been found to possess antiviral activity against hsv-i using an in vitro plaque elimination assay. chi-iii prevented plaque formation at half the concentration of grb (fukushima et al., (hudson and towers, ) . the inhibitory effect of ferulic acid and isoferulic acid on murine interleukin- production in response to influenza virus infections in vitro has been reported (hirabayashi et al., ) and the effect of isoferulic acid was found to be greater than that of ferulic acid. hayashi et al. ( ) found a direct inhibitory activity of the steam distillate prepared from fresh plants of houttuynia cordata (saururaceae) against hsv- , influenza virus, and hiv- , without showing cytotoxicity, but not against poliovirus and coxsackie virus. montanha et al. ( a) evaluated the action of a series of aporphine alkaloids against hsv- in cell cultures. on the basis of viral titer reduction, six alkaloids were found to be active. ellagitannins isolated from phyllanthus myrtifolius and p. urinaria (euphorbiaceae) showed activity against epstein-barr virus dna polymerase. flavonoidal constituents, such as biflavonoids and robustaflavones, exhibited strong inhibitory effects against influenza a and b viruses. the antiviral potential of the flavonoids of chamaesyce thymifolia has been reported; they showed high cytotoxicity on hep- cells and moderate inhibitory activity against hsv- and bovine viral diarrhea virus (amaral et al., ) . sotanaphun et al. ( ) isolated sclerocarpic acid from the stem bark of glyptopetalum sclerocarpum, which exhibited antiviral activity against herpes simplex virus types and . rhamnan sulfate, a natural sulfated polysaccharide isolated from monostroma latissimum, showed potent inhibitory effects on the virus replication of hsv- , hcmv, and hiv- in vitro . matsuse et al. ( ) tested aqueous and methanolic extracts of panamanian medicinal plants for anti-hiv effects. seven of these were found to be moderate inhibitors of hiv-protease enzyme. serkedjieva and ivancheva ( ) investigated the inhibitory effect of five extracts from the bulgarian medicinal plant g. sanguineum on herpes simplex virus types and . yoosook et al. ( ) evaluated the anti-hsv- activities of barleria lupulina and clinacanthus nutans. the results suggested a therapeutic potential against hsv- for b. lupulina, but not for c. nutans. the antiviral activities of various water and methanol soluble substances isolated from g. lucidum against hsv types and , influenza a virus, and vesicular stomatitis virus were studied using cytopathic effect inhibition assay and plaque reduction assay (eo et al., ) . kudi and myint ( ) investigated the antiviral activity of medicinal plant extracts against poliovirus, astrovirus, hsv, and parvovirus. most of the extracts showed activity against more than one virus at a dose rate of between and μg/ μl (eo et al., ) . bourne et al. ( ) assessed plant products in vitro by plaque reduction assay to determine their activity against a commonly transmitted pathogen, herpes simplex virus type . docherty et al. ( ) found that resveratrol, a phytoalexin, inhibited hsv- and hsv- replication in a dose-dependent reversible manner. anani et al. ( ) prepared methanol extracts from medicinal plants of togo and analyzed them for antiviral and antibiotic activities. ten of the showed significant antiviral activity against one or another of the test viruses (herpes simplex, sindbis, and poliovirus). hudson et al. ( a, b) evaluated ethanolic extracts of plants, endemic to madagascar, in order to determine the potential of malagasy plants as sources of antiviral activities. nine of the extracts had significant activity against hsv, whereas only four were active against the sindbis virus. a bioactive flavonoid, "baicalein," isolated from the chinese medicinal plant scutellaria baicalensis georgi showed antiviral properties using the high-speed countercurrent chromatography (hsccc) technique (li and chen, ) . many other substances, including flavonoids, phenolics, tannins, triterpenes, and alkaloids, interfere with host cell replication at their antivirally active concentrations or only exhibit extracellular viricidal activities. some of these viricides, including flavonoids and tannins present in foodstuffs, might be important, because they can inhibit virus replication of picorna-, rota-, and arena viruses in the gastrointestinal tract of humans and animals. artemisia capillaris was found to possess an active , -dimethylesculetine having potent cytotoxic potential. in the fruits of schisandra chinensis (schizandraceae) used in oriental medicine, lignans were identified, some of which, such as wuweizisu c and gomisine n, are very active. the same method threw light on the mechanism of the antihepatotoxic action of such well-known compounds as glycyrrhizin and its intestinal metabolites, which are protective against the first stages preceding hepatic lesions. other tests of this type are used to track down active substances. from taxus baccata, potier's group isolated new analogs of taxol, a terpenic compound displaying very good antileukemic and antitumor activities. taxol promotes the polymerization of tubulin into microtubules and inhibits their depolymerization. the choice among various fractions obtained by extraction was guided by the antitubulin activity in an in vitro test. many substances that are present in only trace quantities and are difficult to purify have been studied chemically; for example, the demonstration of xylose-bearing derivatives is new in this series of compounds. regarding structure activity relationships, in vitro cytotoxicity tests have shown that the activity is mainly related to the presence of a complex ester function in the compound. a list of plant extracts and their phytoconstituents that have antiviral potentials are listed in table . . in recent years, a lot of development has taken place regarding the identification of antiviral molecules from plant sources and the molecular mechanistic approach. compounds, such as spiroketalenol ether derivatives isolated from rhizome extract of tanacetum vulgare, have been reported to block virus entry and also arrest the synthesis of hsv- gc and hsv- gg glycoproteins (fernandes et al., ) . samarangenin b from the roots of limonium sinense exhibited inhibition of hsv- α gene expression (kuo et al., ) , whereas oxyresveratrol from artocarpus lakoocha plant was found to inhibit the early and late phases of viral replication of hsv- and hsv- , respectively (chuanasa et al., ) . also, pterocarnin a compound isolated from pterocarya stenoptera inhibited hsv- from binding and penetrating to the host cells (cos et al., ) . the structures of some of the potential phytoconstituents having significant antiviral activity are depicted in fig. . . many of the antiviral drugs now known have been discovered by random search in the laboratory. most labs use a biological test system in which new molecules are added to tissue culture cells at a range of concentrations (e.g., - μg/ ml); the drug-treated cells (and untreated cells as control) are then infected with a known multiplicity of infective virus particles. thousands of compounds can inhibit viral replication in a cell culture. in general, the more complex the regulatory mechanisms of a virus, the easier it is to find molecules that inhibit it. a broad estimate of the ratios of the activity of antiviral compounds in a cell culture, animal models, and humans is : : . during the evaluation of antiviral agents, many test conditions, such as the cell culture system, virus strain, virus challenge dose, virus input multiplicity of infection, and time of harvesting, can affect or even alter the test results. to test the inhibitory activity of a new antiviral agent, it is first necessary to select the host cell system(s) in which the virus replication can be measured. viruses vary considerably in their ability to replicate in cultured cells. many viruses can cause cpe while some of them can form plaques. others can produce some specialized functions, such as hemagglutination, hemadsorption, or cell transformation. virus replication in cell cultures can also be detected by the presence of viral products, namely, viral dna, rna, or polypeptides. the antiviral tests selected may be based on: (a) inhibition of the virus-induced cytopathic effect (cpe) in which the % effective dose (ed ) of the antiviral agent is expressed as the concentration that inhibits cpe in half of the quadruplicate cultures. (b) plaque reduction assay in which the dose of the drug required to reduce the plaque number by %, that is, ed is calculated. (c) virus yield reduction assay in which the drug concentration required to reduce % ( log reduction), or % ( log reduction) of the virus yield as compared with the virus control (infected cultures without drug) are determined from the dose-response curves and are expressed as ed or ed of the antiviral agent. (d) assay systems based on the measurement of specialized functions and viral products; a number of viruses do not produce plaques nor do they cause cpe readily, but they may be quantified by certain specialized functions based on their unique properties, for example, hemagglutination and hemadsorption tests used to study the antiviral activity against myxoviruses and elisa, used to determine the extent of virus replication and, thus, obtain a measure of the inhibitory effect of various antiviral agents on virus replication, etc. (hu and hsiung, ). colorimetric assays quantify cell viability through enzyme-mediated biochemical reactions owing to ingress of certain dyes inside living cells. mosmann, borenfreund, and puerner first advocated the application of tetrazolium (mtt) and neutral red (nr) assay, respectively, to quantitate cell viability and the cytotoxicity to cells in vitro. parida et al. ( ) compared the efficacy of two colorimetric assays (mtt and neutral red) to determine viral infectivity in microculture virus titration employing polio virus type- and dengue virus type- . mtt assay, also known as tetrazolium assay, has been exploited extensively to reveal the protective efficacy of therapeutic agents and plant extracts against cancer, hiv- , hsv, polio virus type , den- virus, and to the determination of neutralizing antibody levels to hiv and respiratory syncytial virus. mtt assay using microculture virus titration (mcvt) was applied for the determination of infectivity titers of influenza viruses and was found to be compatible with the well-established procedure of egg infectivity assay. unlike mtt, neutral red dye uptake assay has not been substantially exploited in virologic research. nr dye assay was earlier performed for the study of the antiviral efficacy of basil leaves extract against polio virus type . mtt, a tetrazolium salt, is a yellow-colored dye [ -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide] which gets cleared by mitochondrial succinate dehydrogenase enzyme into a blue-colored formazan in active cells. this product does not form crystals when it interacts with isopropyl alcohol and thus can be accurately measured. there is no need to harvest the viable cells, wherein the cell viability can be directly measured by a spectrophotometer (parida et al., ) . the assay procedure has been discussed in section . . . a colorimetric assay based on the cleavage of the tetrazolium salt wst- has been developed for human cytomegalovirus (hcmv) antiviral susceptibility testing and adapted to a microtiter plate format. bedard et al. ( ) developed a colorimetric assay based on the cleavage of the tetrazolium salt wst- for human cytomegalovirus antiviral susceptibility testing. the response of different cell cultures to a given antiviral agent, including the drug metabolism and toxicity, among other factors, may vary greatly. to perform antiviral testing against a particular virus infection, it is necessary to obtain a suitable host cell system for that virus infection. the same antiviral agent may behave very differently in different cell culture systems, although the same virus strain is used. (ii) virus strain and passage history the variability of sensitivity to a given antiviral agent has been noted among different strains of hsv and cmv. drugresistant strains have emerged to some antiviral agents, especially in the herpes virus group. the passage history of a virus strain may also affect its sensitivity to some antiviral agents. moi can influence substantially the evaluation of antiviral activity by the plaque reduction method or the virus yield reduction assay. high moi will decrease the sensitivity of the virus to an antiviral agent. this may also contribute to the disparity among antiviral evaluation results, even when the virus moi is kept constant (hu and hsiung, ) . ( the activities of several known antiviral phytochemicals are profoundly affected by the presence of serum components. for example, the terthiophene, α-terthienyl (α-t) was inhibited in a concentration-dependent manner by serum. in the case of the carboxylic acid derivative of α-t, the compound appeared to have no antiviral activity at all in the presence of serum, yet in its absence this compound was as effective as α-t. in contrast, the complex anthraquinone hypericin required a small amount of serum for maximal antiviral activity. the reactions were also strongly affected by the order of incubation of the components: virus, compound, serum, and light (hudson and towers, ) . the viruses to be selected for initial evaluation of plant extracts are obviously of major importance. they must be chosen to represent the different groups of viruses according to their morphology and various multiplication mechanisms and a range of virus diseases for which chemical control would be useful. besides the need for control, also the prevalence of the viral diseases and the resulting projection of the market potential, which are determined by the antigenic abundance of the causative viruses and the problems this represents for vaccine control, are important selection criteria (grunert, ) . in vitro methods are therefore more appropriate, because they allow simultaneous screening of a battery of viruses. in vivo screening of extracts against a broad array of viruses, in contrast, is not only very expensive but also extremely laborious. in vitro antiviral bioassays utilize thinly confluent monolayers of tissue culture cells with sufficient susceptibility to the infecting viruses that a visibly cytopathogenic effect (cpe) occurs, for example, rounding up. shrinking or detaching of cells from the monolayer can be produced and readily observed microscopically. a monolayer of cells consists of animal or human cells, such as chick embryo, rabbit, or green monkey kidney cells (vero cells), or human skin fibroblasts and carcinoma cells (hela cells), grown in a culture medium. such continuous cell lines used in virology are mostly "transformed" cells that can be maintained for an indefinite number of generations. the host cells require an appropriate tissue culture medium in which they can survive for at least week without having to renew the medium. renewal of the medium causes changes in intra-and extracellular products and alters the virus concentration. the medium must have a stable ph during the whole incubation time and may contain only a small amount of serum, because blood products tend to absorb many compounds. mostly, a defined synthetic medium, supplemented by some type of serum (such as fetal bovine, calf, or horse), a buffer on sometimes bacterial and fungal inhibitory antibiotics, is used. according to experience, vero cells, which allow the growth of many human viruses with visible cpe, grown in the medium described by hronovosky et al. ( ) , and slightly modified by lowering the pyruvate concentration, are most suitable for antiviral screening of plant extracts (van den berghe et al., ) . many combinations of test viruses are possible, but a battery of six viruses seems to be quite acceptable. virus types and strains may vary in sensitivity, but have to be selected as a function of their ability to multiply in the same tissue culture when cell culture models are used as screening systems. in this way, an objective comparison of antiviral activities is possible, whereas toxicity tests are minimized. moreover, virus multiplication must cause a visible cpe within a reasonable period of time, preferably within a week after infection. which are expressed as the number of infectious units per volume. an infectious unit is defined as the smallest amount of virus capable of producing a reaction after virus inoculation and can be carried out by two generally applied methods, namely, the plaque test (pt) and the % endpoint titration technique (eptt). in the plaque test, monolayers of cells grown in plastic or glass petri dishes are inoculated with dilutions of a viral suspension. after adsorption of infectious virus particles to the host cell, the monolayers are overlaid with an agarosecontaining medium so that the newly formed virus particles are localized by the solid agar over layer. these newly formed infectious particles spread from the initially infected cell to the adjacent cells and develop well-circumscribed foci of cellular degeneration. these areas of dead cells are called "plaques" and are visualized by staining the cell monolayer with a vital dye, such as neutral red. the plaques may also be detected microscopically by determining the multinucleated cells (e.g., measles) or by immunofluorescence. the concentrations of viral suspensions measured by counting the plaques are expressed as plaque-forming units per ml (pfu ml − ). in the endpoint titration technique, the concentration of infectivity is measured by determining the highest dilution of the suspension, which produces cpe in % of the cell cultures inoculated. this dilution is called the % tissue culture dose endpoint (tcd ). dilutions are therefore made in a maintenance medium and a certain volume of each of them ( . - . ml) is added to four or more tube cultures. the proportion of positive cultures is registered for each dilution and the precise dilution at which % of the inoculated tube cultures are infected is calculated. at this dilution, the inoculum contains, on average, one tcd or one tissue culture (infectious) dose for % of the tissue culture tubes. the influence of a plant extract on virus multiplication can be determined as a viricidal or an antiviral activity. the viricidal activity is measured by titration of the residual infectious virus after incubation of the plant extract with a virus suspension of at least tcd ml − . on the other hand, the antiviral activity is determined by comparing the virus titers of infected cells, which have been cultured with a maintenance medium containing plant extracts or test substances and a maintenance medium without test material (colegate and molyneux, ) . in contrast with antibacterial screening, no solvents, other than physiological buffer solutions, should ideally be used in the in vitro antiviral screening of plant extracts because the samples have to be added to tissue culture cells. it has been observed that many nonpolar plant extracts, prepared and evaporated, are reasonably soluble in dimethyl sulfoxide (dmso), especially if little or no water is present in the sample and the dissolving sample is heated on a water bath. viricidal and antiviral determinations may then be carried out on test solutions containing not more than % and % dmso, respectively. therefore, dissolved samples of nonpolar plant extracts in dmso are added dropwise to the maintenance medium in a ratio of : or : under stirring. as already mentioned, the maintenance medium may contain antibiotics, such as penicillin g ( μg ml − ), neomycin ( μg ml − ), and amphotericin b ( μg ml − ), in order to avoid sterilization of the test solutions. any contamination by bacteria or fungi would indeed ruin the in vitro antiviral bioassay (colegate and molyneux, ) . there are various methods for validation of antiviral activity. the major techniques have been highlighted in the preceding sections and in fig. . . most currently used antiseptics and disinfectants kill pathogenic bacteria and fungi at °c within min when present in a concentration of about . % ( -log titer reduction). because it has been noticed that most of these preparations fail to kill all pathogenic viruses under these circumstances, a method has been developed for testing the in vitro viricidal effect of plant extracts, as will be described in the following section. thoroughly mix the preincubated ( °c) plant extracts, dissolved in a physiological buffer, or their twofold dilutions (e.g., / to / ), with the same volume of a preincubated ( °c) virus suspension of, for example, pfu ml − or tcd ml − in physiological buffer. incubate the mixture at °c for min. stop the incubation by the addition of a fold volume of ice-cold maintenance medium and filter the mixture immediately through a . μm filter to eliminate all possible precipitate. then, filter the ice-cold filtrate through a . μm filter to concentrate residual virus on the filter and separate the virus from possibly cytotoxic plant components, which pass the filter. remove the residual virus from the filter with maintenance medium, supplement with % serum ( ml), sonicate in an ice-bath for s, and titrate in -fold dilutions at °c by plaque formation or in microtiter plates according to the eptt. carry out a virus control in a physiological buffer containing no plant extract simultaneously. an essential step of this methodology is the separation of all cytotoxic plant components from the residual virus, which has to be measured at °c. cytotoxic substances have a greater influence on the activity of an extracellular virus at °c than at °c. this step, however, can be omitted when the plant extracts to be tested are not toxic to the host cells under the conditions of the evaluation procedure (colegate and molyneux, ) . the eptt technique is performed on preemptied confluent monolayers of vero or other cells, grown in the holes of microtiter plates, which are infected with serial -fold dilutions of a virus suspension ( μl). starting with monolayers containing cells per hole and a virus suspension of, for example, tcid ml − or pfu ml − , infect the first monolayers of cells with a multiplicity of infection (moi). the antiviral activity is expressed as the virus titer reduction at the maximal nontoxic dose (mntd) of the test substance, that is, the highest concentration (μg ml − ) that does not affect the monolayers under the antiviral test condition. viral titer reduction factors, that is, the ratio of the viral titer reduction in the absence (virus control) and presence of the mntd of the test sample of × to × indicate a pronounced antiviral activity and are suitable as selection criteria for further investigation of plant extracts (colegate and molyneux, ) . the eptt is more suitable for testing complex samples, such as plant extracts, for many reasons. (i) first, the concentration of many compounds in the extract remains constant, and consequently the proportion of toxic versus active compounds does not change. (ii) second, the exact duration of the antiviral action can be determined by using the eptt, because the action starts from the moment the extract is added to the cells. (iii) third, the eptt using serial diluted extracts deals with a dynamic process, because the cells are subsequently infected with different moi. (iv) this system allows the correlation of all possible moi values in the same microtiter plate with decreasing amounts of plant extracts, so that the noncytotoxic concentration of plant extracts can be determined. at the same time, a correlation between extract toxicity and antiviral activity according to the corresponding moi can be determined in the same microtiter plate. (v) it can be stated as a general rule that the detected antiviral activity should be stable in at least two subsequent dilutions of nontoxic concentration of the extract; otherwise the activity is directly correlated with the toxicity of the extract or is only viricidal. moreover, a true antiviral product has to protect the cells, which have been infected with low virus dilutions (starting from . pfu per cell onward). (vi) finally, it should be stressed that all possible steps of virus manipulation are to be completed before the plant extracts are added. this means that an antiviral product tcd ml − ), under nontoxic conditions, must act on virus replication steps after uncoating. when the cells are completely protected only in the lower moi ( . tcd ml − ), replication processes before uncoating may be involved. an important aspect of the inhibition of viral cytopathic effect (cpe) is the determination of tcid ( % tissue culture infective dose). after harvesting, dilute the virus stock -fold. add . ml of each dilution in wells each of a -well microtiter plate. add . ml of cell suspension of , cells/well, incubate at °c with % co atmosphere, and observe for vial cpe on alternate days. take a final reading on the fifth day and calculate tcid as per the method of reed and muench ( ) , from which tcid can be further calculated from the log value. an antiviral drug will inhibit the cpe of a virus. therefore, for detecting an antiviral agent, the amount of inhibition of cpe of a virus can be observed microscopically (kenny et al., ) . for this purpose, trypsinise the monolayer and allow to seed in -well microtiter plates. after a -h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after h, wash the cell culture and inoculate with . ml of tcid , tcid , and tcid of the virus suspensions in different wells. incubate them for h at °c in an incubator for the adsorption of the virus onto the cells. after incubation, remove excess virus suspension by washing with rpmi. add . ml of selected concentration of the test compound and keep both virus and cell control wells with . ml of rpmi containing % sheep serum. observe the plates under a microscope every h until the virus control shows % cpe and tabulate the results (hu and hsiung, ) . trypsinize the cell monolayer, allow to seed in a -well microtiter plate and incubate for h at °c with % co atmosphere. remove the medium, wash the cell monolayer with rpmi without serum, and add . ml of different virus suspensions in different wells containing the cell layer and incubate for h for virus adsorption; wash off the excess virus suspension after adsorption. to each well, add the selected concentration of the test drug diluted in rpmi containing % sheep serum. to the virus control and cell control, rpmi is added ( % serum) and incubated for h. freeze the plates at − °c and thaw at room temperature a couple of times to liberate the associated virus. determine the virus titer by the end point dilution method and express as tcid (cinatl et al., ) . trypsinize the monolayer culture and allow to seed in a -well microtiter plate. after a -h incubation, wash the monolayer in each well and add different test drug dilutions and incubate. after h, wash the cell culture and inoculate with . ml of tcid , tcid , and tcid of the virus suspensions in different wells. incubate for h at °c in a co incubator for adsorption of the virus onto the cell. after incubation, remove excess virus suspension by washing with rpmi without serum. add . ml of the selected concentrations of the test compound and keep both the virus and cell control wells with . ml of rpmi containing % sheep serum. incubate the plates at °c for h. after h, discard the old media from the cell cultures and add μl of mg/ml of mtt ( -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide) to each well and incubate for h. after h of incubation, discard the mtt and add μl of isopropyl alcohol to each culture and set aside for min. record the absorbance using an elisa plate reader at nm. in this experiment, the effect of the test drug on mitochondrial synthesis due to viral infection is studied. the "plaque" is a confined region of contaminated cells formed by multiplying virus particles. the plaques are revealed either as regions of dead/decimated cells recognized by cell stains or as zones of polluted cells by immunostaining. the blended cell monolayer is infected with a log dilution of viral plaque-forming unit (pfu) in the absence or presence of the test drug and permitted to adsorb ( h at °c in % co ); afterward, the cells should be washed twice with prewarmed tcid cpe at dilution next above cpe at dilution nex % % % t t above cpe at dilution next below % % % § © · minimum essential medium (mem). the overlaid drug dilutions are arranged in the overlay medium on the contaminated culture, without the test drug. then, ml of carboxy methyl cellulose is added to ml of the medium; the plates are incubated for - days, then settled with % formalin or % formaldehyde for min. the cells are stained with methylene blue ( ml/well) or % gem violet (w/v), washed, and dried to check the plaques (dark areas) by low-power amplification of a binocular microscope. the antiviral impact should be measured as the percentage inhibition of plaque formation: the concentration of the test drug required to exert % of virus inhibition (ic or ec ), as compared with the infection control, is evaluated from the graphical plot as dose-response curves by regression analysis . viruses, for example, influenza, can agglutinate rbcs due to surface ha proteins, which can be analyzed visually by blending viral dilutions with rbc. this can be utilized to inspect the inhibitory impact of any medication on ha. the -overlay serially diluted ( - x) test drug is used alongside the diluted viral stocks ( : to : ). this dilution ( ml/well) is added to drug-containing wells. it should be preincubated for min and mixed with rbc ( / in pbs) sample solution. here, up to a specific dilution, the viral particles possibly lose their capacity to agglutinate rbcs, which shows a linkage of the drug with the viral ha. known quantities of virus (moi - ) are used for infecting both untreated or drug-treated cells and allowed to adsorb for - min. the unabsorbed virus particles are washed and fresh media is added to incubate for - h. then, cells are washed with pbs, fixed with paraformaldehyde ( %- %), and permeabilized with acetone or triton x- . the cells are again washed and blocked with % bovine serum albumin (bsa) in pbs for min. then, the cells are incubated with mouse or rabbit antibody against a specific viral protein for - h at °c. after that, the cells are subjected to repeated washing and incubated with a fluorescent-tagged secondary antibody for h and washed again. after washing, the cells are visualized under a fluorescent microscope and compared with the fluorescence of untreated and drug-treated cells. on the other hand, for quantitation, the cells are trypsinized after treatment and fixed with % paraformaldehyde. the cells are then washed, permeabilized, and labeled with a fluorescent-tagged antibody, followed by propidium iodide (pi: mg/ml in pbs). the cells are then counted in a fluorescent-activated cell counter to quantitate the fluorescence percentage. known quantities of virus (moi - ) are used for infecting both untreated and drug-treated cells, adsorbed for h, washed, and incubated ( - days) for evaluation of the inhibition of the virus-induced cytopathic effect (cpe). the virus stock is centrifuged after freeze thawing and diluted for elisa. each well of a plate coated with a virus-specific antibody is mixed with ml of controls or test drug and incubated at room temperature ( h) with horseradish peroxidase conjugate, alkaline phosphatase, or b-d-galactosidase-labeled virus-specific antibody. the wells are washed five times and the substrate ( ml) is added and incubated in the dark for min. the reaction is stopped by adding % h so solution and the absorbance is read at nm. the -well plates are seeded with a quadruplicate cell monolayer having a log dilution of the test drug followed by infection with the virus. after - h of incubation at °c, the monolayers are fixed with . % glutaraldehyde and examined for virus-specific protein(s) on the cell surface. the elisa should be performed with a monoclonal antibody to the specific protein of the corresponding virus and protein and the od (optical density) is measured at nm. the concentration is calculated by % reduction in od values (ec ) from extrapolating graphical plots followed by the determination of si (ratio of cc :ec ) in which the results are expressed as a percentage of virusinfected cells (virus control) . the test drug and the virus ( pfu/ml) are mixed to incubate for h at °c. then, immediately dilute the virus-drug mixture to -fold (final inoculums pfu/well) with media containing % fbs to get a subtherapeutic concentration of mean number of plaques in control mean number of plaques in sample m ean number of plaque in control the test drug. following that, mix the monolayer, with the virus inoculums seeded using a -well plate. alternately, add the virus-test drug mix diluted to -fold, with no incubation period, with the respective cells for infection. allow to adsorb for h at °c, discard the diluted inoculums, and wash the cells with pbs. pour an overlay medium (with % fbs), and incubate at °c for h, followed by plaque reduction assay. count the viral plaque numbers obtained from infections set in the presence of the test drug and compare it with the control. viral attachment to the cell surface is carried out at °c, as it permits binding, but stops viral entry, by elisa. briefly, incubate susceptible cells ( × cells/well) in -well plates and allow growth overnight. the cell monolayers are cooled at °c. the cells are infected with the virus (moi ) using heparin in presence of the test drug for h at °c as a control. after washing the wells with ice-cold pbs, fix with prechilled % paraformaldehyde in pbs for h on ice, blocked with % bsa at °c. the samples are incubated at °c for h with a primary antibody in pbst pbs with . % tween along with % bsa. the wells are washed twice with pbst, % bsa, and again only with pbst twice, at -min intervals on a shaker. this is mixed with secondary antibody in pbst with % bsa and incubated at °c for h. the reaction is developed with a , ′, , ′-tetramethyl-benzidine two component microwell peroxidase substrate for min; the reaction is stopped with m phosphoric acid. the absorbance is measured at nm, and the values are expressed as the fold change of absorbance relative to the mock infection control (lin et al., ) . the cell monolayers are cooled and grown in -well plates at °c for h. subsequently, infect the prechilled cells with hsv- ( pfu/well) and incubate for h at °c to allow for viral adsorption. incubate the infected cell monolayers in the presence of test drug or heparin ( mg/ml) for an additional min at °c to facilitate hsv- penetration. the extracellular virus is inactivated by citrate buffer (ph . ) for min, and the cells are washed with pbs followed by overlay with dmem containing % fbs. after h of incubation at °c, the viral plaques are stained and counted (lin et al., ) . add the plated cells ( . × cells/well for a -well plate) grown overnight at % confluence ( ml) with virus dilution and deae dextran at a final concentration of mg/ml. after adsorption ( h at °c in co incubator), place the plates in a rocker to prevent the cells from drying and add fresh medium ( - ml) containing the test drug to each well and incubate for - h in % co at °c for subconfluent growth. after removing the media, add fixing solution ( - ml) to each well and incubate for min at room temperature (β-galactosidase activity decreases dramatically if the fixing solution is left for > min). then, discard the fixing solution, wash the cells twice with pbs, stain, and incubate at °c for min. finally, stain the plates to count the number of blue syncytia and express the titration values as the number of stained cells multiplied by the viral dilution . this assay is applicable for hiv- , simian immunodeficiency virus (siv), and simian-hiv and is carried out in tzm-bl cells as it reveal the reduction in tat-induced luciferase (luc) reporter gene expression after a single round of virus infection. place the tzm-bl cells ( × /well) in a -well plate and incubate overnight. in a separate vial, mix the hiv- nl . (moi . ) virion with the test drug or vehicle for h at °c, then add to tzm-bl cells and incubate for h. wash the cells (with cold pbs), and add fresh media with the test drug to culture for h, using untreated hiv- -infected cells (negative) and azidothymidine (azt)-treated cells (positive) as controls. wash the cells twice with pbs, lyse with x lysis buffer, and add the supernatant with the substrate, which should then be analyzed for luciferase activity in an optiplate using a fluorimeter. the results are expressed as percentage inhibition: and the percent inhibition is calculated by subtracting the above value from (wan et al., ) . luminescence in the experimental group test drug or azt lu / m minescence of infected cells without the drug u (b) cem-green fluorescent protein (gpt) cell-based assay cem-gfp is a stable t-cell line-containing a plasmid encoding gfp and is suitable for hiv- nl . (moi . ) culture. for postinfection, incubate the cells ( . × /well) with the test drug up to days, using azt and solvents (used to prepare the test drug) as control(s). lyse the virus-infected cells with x promega cell lysis buffer ( ml), and transfer to a culture plate to read the absorbance at nm (excitation) and nm (emission) by a fluorimeter. the results can be expressed as percentage inhibition: with the he percent inhibition calculated by subtracting the above value from . place hep ad cell suspension ( × viable cells/ml of hep ad seeding medium) in a -well microtiter plate, and incubate at °c for days. discard the medium and wash the cell monolayers with warm ( °c) dpbs. to the proper wells, add μl of hepad assay medium that contains either test or control compounds at the desired concentrations. also include wells with hep ad assay medium alone as "virus only" controls. incubate at °c for days. on day , wash the cells once with warm dpbs and add fresh medium containing the appropriate compound to the wells. after h, transfer the supernatants to v-bottomed -well plates and remove cellular debris by centrifugation ( min, rpm at °c). transfer μl of the clarified supernatants to new v-bottomed plates and store at − °c for quantification of hbv dna. thaw the supernatants that were collected and add μl of x denaturation solution to each well and mix. incubate at °c for min. cut the nylon membrane to size and prepare it for blotting by wetting it first with distilled water and then x hybridization buffer, ssc (saline sodium-citrate). dot-blot the denatured supernatants on to the nylon membrane as directed by the manufacturer. wash the blot with μl of neutralization solution followed by μl of x ssc. remove the blot, rinse it briefly in x ssc, and then crosslink the dna to the nylon filter by uv irradiation. prehybridize the blot at °c for h in μl of hybridization solution. prepare a p-labeled probe by random priming using a portion of the hbv genome as a template. purify the probe through a clontech chroma spin column. denature the probe by boiling for min and add it immediately to the hybridization solution. hybridize the nylon filter overnight at °c. wash the nylon filters twice with ml of x ssc, . % sds (sodium dodecyl sulfate) at room temperature for min and twice with ml of . x ssc, . % sds at °c for min. expose the nylon filters to a molecular imager screen for h and scan on a phosphor imager to obtain the data. to determine the percent inhibition of hbv replication, subtract the background value (counts of radiation detected from the nylon filter itself) from all control and experimental values. divide the average values of the experimental wells (cells treated with test compounds) by the average value for the "virus only" control (cells not treated with compound or tetracycline during the experiment) and multiply this number by (king and ladner, ) . the above mentioned in vitro studies are listed in table . . drug development programs include preclinical screening of immense quantities of chemicals for specific and nonspecific cytotoxicity against numerous sorts of cells, which is imperative to show the potential therapeutic target and safety evaluation. the screening of plant extracts or pure compounds for investigating their antiviral properties can be more significant with cytotoxicity measures (meyer et al., ) . it is essential for an investigational item to establish the antiviral activity at concentrations that can be accomplished in vivo without inducing toxicity to cells. moreover, in a cell culture display, antiviral activity of an investigational item can be the aftereffect of host cell death after exposure to the item. cytotoxicity tests utilize a series of increasing concentrations of the antiviral product to determine what concentration results in the death of % of the host cells. this value is referred to as the median cellular cytotoxicity concentration (cc or ctc or ccic ). the relative effectiveness of the investigational product in inhibiting viral replication compared with inducing cell death is defined as the therapeutic or selectivity index (cc value/ec value). it is desirable to have a high therapeutic index giving maximum antiviral activity with minimal cell toxicity. according to us fda guidelines, it is recommended to determine cc values in both stationary and dividing cells from multiple relevant human cell types and tissues to establish the potential for cell cycle, species, or tissue-specific toxicities. studies determining cytotoxicity and therapeutic indexes should be conducted before the initiation of phase clinical studies. there are a number of advantages for in vitro testing using cell cultures, which include gfp fluorescence in the experimental group fluorescence in / i infected cells without the test drug × analysis of species specificity, feasibility of using only small amounts of test substances, and facility to do mechanistic studies (guidance for industry, ). after confirming the cytotoxic concentration, the drug concentrations are selected for antiviral studies based on the percentage viability of cells and are used to study the antiviral activity by cpe inhibition assay, virus yield assay, followed by mtt assay. any compound that is cytotoxic to cells inhibits the cell proliferation and kills the cells. trypan blue is a dye that is capable of penetrating dead cells; therefore, the dead cells take up the blue stain whereas the viable cells do not. this method gives an exact number of dead and viable cells (strober, ) . protein content is widely used for estimating total cellular material and can be used in growth experiments. the colorimetric method of estimating protein is more sensitive. the cell pellets are treated with % cold trichloroacetic acid to remove amino acid pools and dissolved in alkaline cupric sulfate and folin ciocalteau phenolic reagent. folin's reagent and cupric sulfate together react with amino acid to give a blue color and this color intensity is proportional to the protein concentration, which can be measured colorimetrically (maya et al., ) . to proceed with the same technique, the cells from the wells were trypsinized using μl trypsin and transferred into eppendorf tubes and centrifuged at rpm for min to obtain pellets. the cell pellets are dissolved in naoh and diluted , tzm-bl cell-based assay (hiv), cem-green fluorescent protein cell-based assay (hiv), hep ad assay (hbv), immunofluorescence assay, enzyme-linked immunosorbent assay (elisa) • reduction or inhibition of virus-specific polypeptides synthesis in infected cell cultures, e.g., viral nucleic acids, determination of the uptake of radioactive isotope labeled precursors or viral genome copy numbers other assays for validation of antiviral activity • virus inactivation assay, virus adsorption assay, virus attachment, and penetration assay to . n. the test drug is to be added to μl of protein sample, mixed, and left for min. to this, μl of test reagent is added with constant mixing and left for min in incubator. the absorbance was read at nm using an elisa reader (bio-rad). the color development was correlated with the cell number as follows: the cytotoxic concentration found by dye exclusion techniques gives superficial data. the selected concentrations from a trypan blue dye exclusion study are used further for estimating proteins. mtt ( -( , -dimethylthiazol- yl)- , diphenyl tetrazolium bromide) in live cells enters the cells and enzyme succinate dehydrogenase present in mitochondria reduces it to formazan blue product. the color intensity is directly proportional to the number of live cells. to perform this process, the plates were seeded with hep- cells at , cells/well. they are incubated for h. after h, the medium is discarded and drug concentrations were added and incubated for h. then, μl of mg/ml of mtt is to be added and incubated for h and then μl of isopropyl alcohol is added and absorbance is read at nm in an elisa plate reader (bio-rad). the results are tabulated and percentage growth inhibition is calculated using the following formula: the concentrations of the test drug used in the previous experiments can be further confirmed by studying the mitochondrial synthesis by mtt assay. the formazan blue color formation is directly proportional to the number of viable cells and therefore the absorbance is to be read at nm. in this test, brine shrimp (artemia salina) eggs are hatched in artificial sea water ( g/l of sea salt). the brine shrimp test (bst) bioassay experiment is performed according to the procedure described by meyer et al. ( ) . generally, samples of the test drugs for the experiment are prepared in methanol solution, which acts as control vehicle. after h of incubation, brine shrimps are transferred to each sample vial using a pasteur pipette and artificial sea water is added to make ml. sample vials are previously prepared by dissolving specific concentrations of test drugs with different dilutions. the solvent is then evaporated overnight. survivors are counted after h and the lc values, with % confidence intervals are determined using probit analysis, as described by finney ( ) . control vials are prepared using methanol only. three replicates are prepared for each concentration of the test drugs. control disks are prepared using only methanol. replicates are prepared for each dose level. to begin the bioassay, brine shrimp eggs are hatched in a shallow rectangular dish ( × cm ) under the same conditions described in the literature except that natural instead of artificial seawater is used. ten shrimps are selected and transferred into each sample vial by means of a -cm disposable pasteur pipette and the final volume in each vial is adjusted to ml using natural seawater. a drop of dry yeast suspension ( mg in ml seawater) is added as food to each vial. the vials are maintained under illumination. survivors are counted with the aid of a stereomicroscope, after , , and h, and the deaths at each dose level and control are determined. no deaths are usually observed to occur in the control after h. the brine shrimp test (bst) represents a rapid, inexpensive, and simple bioassay for testing plant extract lethality, which, in most cases, correlates reasonably well with cytotoxic properties (mclaughlin, ) . following the procedure of meyer et al. ( ) , the lethality of the test drugs/plant extracts to brine shrimp is determined. the lethal concentrations of test drugs/plant extract resulting in % mortality of the brine shrimp (lc ) and % confidence intervals are determined from the and -h counts and the dose-response data are transformed into a straight line by means of a trend line fit linear regression analysis; the lc is derived from the best-fit line obtained. caffeine (lc = μg/ml) (meyer et al., ) is used as a positive control and methanol ( μl) as a solvent and a negative control in the bioassay experiments. the current scenario of viral diseases is lethal and there is an upsurge in new viral diseases and resistance to existing viral infections worldwide. the currently accessible antivirals, however effective, are exorbitant and past the reach of a majority of individuals. along these lines, the advancement of safe, effective, and low-cost antiviral medications, for example, rt inhibitors, is among the top priorities, as many viral infections are not yet treatable and have high death rates. for the past few years, substantial work has been carried out regarding the effectiveness of medicinal plants on hiv infection (premanathan et al., ; calabrese et al., ; asres et al., ) and an increasing popularity of over-the-counter plant products containing orthodox drugs has been observed. the main focus is to lower the adverse effects associated with viral infections and an inclination toward synergistic interactions of multiple molecules present in plant extracts. be that as it may, because mostly pharmacological mechanisms of the combinations are not studied, antagonistic impacts or remedial disappointments have been seen (chan et al., ) . a prerequisite that should be considered significant for medicinal plants is to identify and standardize the method of extract preparation, the suitable season for collecting plant material, and the details of its administration (chattopadhyay et al., ) . as a lot of plant extracts and subsequent formulations have shown significant outcomes, it seems to be rational to endorse the idea of the study of medicinal plants as a quest to find potential antivirals. antiviral investigation on the flavonoids of chamaesyce thymifolia investigation of medicinal plants of togo for antiviral and antimicrobial activities in vitro virucidal activity of selected anthraquinones and anthraquinone derivatives antiviral evaluation of plants from brazilian atlantic tropical forest antiviral activity against human immunodeficiency virus type (hiv- ) and type (hiv- ) of ethnobotanically selected ethiopian medicinal plants suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns protease activity anti-herpes virus activities of bioactive fraction and isolated pure constituent of mallotus peltatus: an ethnomedicine from andaman islands the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine) n -specific plant lactin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro -cinnamoyl- , -dihydroxymeliacarpin is a natural bioactive compound with antiviral and nuclear factor-kappa b modulating properties a high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors an investigation of the antiviral activity of podophyllum peltatum in vitro anti-hiv- activities of kaempferol and kaempferol- -o-glucoside isolated from securigera securidaca further screening of venda medicinal plants for activity against hiv type reverse transcriptase and integrase the natural compound silvestrolis a potent inhibitor of ebola virus replication plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type a phase i trial of andrographolide in hiv positive patients and normal volunteers comparative study of propofol versus midazolam in the sedation of critically ill patients: results of a prospective, randomized, multicenter trial survey for the presence and distribution of human herpesvirus in healthy brain ethnomedicines and ethnomedicinal phytophores against herpes viruses antivirals of ethnomedicinal origin: structure-activity relationship and scope dose dependent therapeutic antiinfectives from ethnomedicines of bay islands recent advancements for the evaluation of anti-viral activities of natural products validation of antiviral potential of herbal ethnomedicine inhibitory effects of quercetin -rhamnoside on influenza a virus replication anti-herpes simplex virus (hsv- ) activity of oxyresveratrol derived from thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous hsv- infection in mice chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and hiv infection antiviral effects of -diazo- -oxo- -nor-leucine on replication of herpes simplex type- novel compounds in preclinical/early clinical development for the treatment of hiv infections molyneux bioactive natural products plant substances as antiviral agents: an update potential antiviral lignans from the roots of saururus chinensis with activity against epstein-barr virus lytic replication drug screening for autophagy inhibitors based on the dissociation of beclin -bcl complex using bifc technique and mechanism of eugenol on anti-influenza a virus activity structure and in vitro antiviral activity of triterpenoid saponins from calendula arvensis antiviral activity of chlorogenic acid against influenza a (h n /h n ) virus and its inhibition of neuraminidase resveratrol inhibition of herpes simplex virus replication antiviral activities of various water and methanol soluble substances isolated from ganoderma lucidum sennoside a, derived from the traditional chinese medicine plant rheum l., is a new dual hiv- inhibitor effective on hiv- replication anti-enterovirus and immunostimulating activity of the polyphenol complex extracted from pethaphylloides fruticosa (l.) o. schwarz honokiol, a lignanbiphenol derived from the magnolia tree, inhibits dengue virus type infection screening of brazilian plants for antiviral activity against animal herpes viruses probit analysis antiviral effects of glycyrrhiza species biotransformation of ursolic acid by syncephalastrum racemosum cgmcc . and anti-hcv activity anti-aids agents . betulinic acid and platanic acid as anti-hiv principles from syzygium claviflorum, and the anti-hiv activity of structurally related triterpenoids antiviral activity of dammarane saponins against herpes simplex virus type activity of melaleuca alternifolia (tea tree) oil on influenza virus a/pr : study on the mechanism of action antiviral activity of an extract derived from roots of eleutherococcus senticosus search for antiviral agents antiviral product development-conducting and submitting virology studies to the agency. u.s. department of health and human services, food and drug administration michellamines d-f, new hiv-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine e, a new antimalarial monomer, from ancistrocladus korupensis virucidal effects of the steam distillate from houttuynia cordata and its components on hsv- , influenza virus, and hiv inhibitory effect of ferulic acid and isoferalic acid on murine interleukin- production in response to influenza virus infections in vitro and in vivo a modified plaque method for arboviruses on plastic panels evaluation of new antiviral agents: i, in vitro perspectives anti-viral effect of a compound isolated from liriope platyphylla against hepatitis b virus in vitro isolation of the anthropogenic compound fluoranthene in a screening of chinese medicinal plants for antiviral compounds further investigations on the antiviral activities of medicinal plants of togo antiviral activities in plants endemic to madagascar inhibitory effects of sudanese medicinal plant extracts on hepatitis c virus (hcv) protease study of antiviral action of total alkaloids from haemanthus albiflos inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus antiviral potential of selected indian medicinal (ayurvedic) plants against herpes simplex virus and . n anti-hbv active constituents from piper longum the flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis b virus-e antigen comparison of specific antiviral agents in herpes simplex keratitis in vitro and in vivo anti picorna virus activity of some phenoxypyridine carbonitriles extracts and molecules from medicinal plants against herpes simplex viruses hep ad assay a high-throughput, cell-based screen for the evaluation of compounds against hepatitis b virus samarangenin b from limonium sinense suppress herpes simplex virus type replication in vero cells by regulation of viral macromolecular synthesis antiviral activities against hsv- , hcmv, and hiv- of rhamnan sulfate from monostroma latissimum antiviral effects of black raspberry (rubus coreanus) seed and its gallic acid against influenza virus infection in vitro antiviral activities of chinese medicinal herbs against duck hepatitis b virus plant antiviral agents iii. isolation of alkaloids from clivia miniata regel (amaryllidaceae) isolation and purification of baicalein, wogonin and oroxylin a from the medicinal plant scutellaria baicalensis by high-speed counter-current chromatography antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla (l) identification of natural compounds with antiviral activities against sarsassociated coronavirus mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoproteine glycosaminoglycan interactions to inhibit herpes simplex virus entry and cell-to-cell spread saikosaponin b is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry ethnopharmacology in overdrive: the remarkable anti-hiv activity of artemisia annua the alkaloids. vol antiviral potential of curcumin a search for anti-viral properties in panamian medicinal plants, the effects on hiv and its essential enzymes interaction of filacial proteins on growth regulation of normal lung epithelial cells in vitro antiviral screening of british columbian medicinal plants crown-gall tumors in potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation the in vitro antivirial activity of an aliphatic nitro compound from heteropteris aphrodisiaca brine shrimp: a convenient general bioassay for active plant constituents natural products and drug development. munksgaard anti-herpes virus activity of apporphine alkaloids anti-herpes virus activities of achyranthes aspera: an indian ethnomedicine, and its triterpene acid bio organic marine chemistry pedilanthus tithymaloides inhibits hsv infection by modulating nf-κb signalling screening extracts of zanthoxylum chalybeum and warburgia ugandensis for activity against measles virus (swartz and edmonston strains) in vitro biological activity of guatteria cardoniana fractions comparison of two colorimetric assays to determine viral infectivity in microculture virus titration characteristic of alkylated chalcones from angelica keiskei on influenza virus neuraminidase inhibition glycyrrhizic acid inhibits virus growth and inactivates virus particles in vitro anti-human immunodeficiency virus activity of polysaccharide from rhizophora mucronata poir maslinic acid, a natural triterpenoid compound from olea europaea, protects cortical neurons against oxygen-glucose deprivation induced injury in vitro study of the antiviral activity of some b-carboline alkaloids a simple method of estimating fifty percent endpoints anti-hiv activity of extracts and compounds from algae and cyanobacteria antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral agents as adjuncts in cancer chemotherapy effects of phyllanthus plant extracts on duck hepatitis b virus in vitro and in vivo antiviral activity of carnosic acid against respiratory syncytial virus in vitro antioxidant activity of polyphenol extracts with antiviral properties from geranium sanguineum l a new antiviral and antimicrobial sesquiterpene from glyptopetalum sclerocarpum trypan blue exclusion test of cell viability antiviral activity and constituent of ardisia chinensis benth against coxsackie b virus analysis of anti-rotavirus activity of extract from stevia rebaudiana proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis c virus rna anti-viral activity of the extracts of a kenyan medicinal plant carissa edulis against herpes simplex virus screening of higher plants for biological activities. ii. antiviral activity plant products as potential antiviral agents plant antiviral agents: v. -methoxyflavones as potent inhibitors of viral-induced block of cell synthesis in vitro antiviral activity of plant extracts from asteraceae medicinal plants current organic chemistry, natural product chemistry issue plant flavonoids in biology and medicine ii: biochemical, cellular and medicinal properties inhibition of hepatitis c virus replication by chalepin and pseudane ix isolated from ruta angustifolia leaves fangchinoline inhibits human immunodeficiency virus type replication by interfering with gp proteolytic processing herbs of the genus phyllanthus in the treatment of chronic hepatitis b: observations with three preparations from different geographic sites effect of oenanthe javanica flavone on human and duck hepatitis b virus infection antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus three new secoiridoids, swermacrolactones a-c and anti-hepatitis b virus activity from swertia macrosperma lignans with antihepatitis b virus activities from phyllanthus niruri l antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata 'chachi the in vitro activity of geraniin and , , , -tetra-o-galloyl-beta-d-glucose isolated from phyllanthus urinaria against herpes simplex virus type and type infection the protective effect of -deoxysappanchalcone on in vitro influenza virusinduced apoptosis and inflammation mechanism of inhibition of hiv- infection in vitro by purified extracts of prunella vulgaris evaluation of anti-hsv- activities of barleria lupulina and clinacanthus nutans novel antiviral activity of baicalein against dengue virus a dihydro-pyrido-indole potently inhibits hsv- infection by interfering the viral immediate early transcriptional events comparative inhibitory effects of suramin and other selected compounds on the infectivity and replication of human t-cell lymphotropic virus (htlv-iii)/lymphadenopathy-associated virus (lav) discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development emerging anti-hiv drugs agar diffusion method for detection and bioassay of antiviral antibiotics anti-herpes virus activity of aporphine alkaloids antiviral potentials of medicinal plants evaluation of anti-infective potential of a tribal folklore odina wodier roxb against some selected microbes and herpes simplex virus associated with skin infection sensitive and rapid assay on mt- cells for the detection of antiviral compounds against the aids virus a rapid and simple colorimetric test for the study of anti-hiv agents plant-derived leading compounds for chemotherapy of human immunodeficiency virus (hiv) infection resistance of human immunodeficiency virus type to the high-mannose binding agents cyanovirin n and concanavalin a key: cord- - ozf lg authors: al-khafaji, khattab; al-duhaidahawil, dunya; taskin tok, tugba title: using integrated computational approaches to identify safe and rapid treatment for sars-cov- date: - - journal: j biomol struct dyn doi: . / . . sha: doc_id: cord_uid: ozf lg sars-cov- is a new generation of coronavirus, which was first determined in wuhan, china, in december . so far, however, there no effective treatment has been found to stop this new generation of coronavirus but discovering of the crystal structure of sars-cov- main protease (sars-cov- mpro) may facilitate searching for new therapies for sars-cov- . the aim was to assess the effectiveness of available fda approved drugs which can construct a covalent bond with cys inside binding site sars-cov- main protease by using covalent docking screening. we conducted the covdock module mmgbsa module in the schrodinger suite - , to examine the covalent bonding utilizing. besides, we submitted the top three drugs to molecular dynamics simulations via gromacs . . the covalent docking showed that saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir and prevacid have the highest binding energies mmgbsa of – . , − . , − . , − . , − . , − . , and − . kcal/mol, respectively. the ns molecular dynamics simulation was conducted for saquinavir, ritonavir and remdesivir to evaluate the stability of these drugs inside the binding pocket of sars-cov- main protease. the current study provides a powerful in silico results, means for rapid screening of drugs as anti-protease medications and recommend that the above-mentioned drugs can be used in the treatment of sars-cov- in combined or sole therapy. communicated by ramaswamy h. sarma sars-cov- also called 'severe acute respiratory syndrome coronavirus ' abbreviated sars-cov- was recognized to be the causative of atypical pneumonia (joshi, ; pant et al., ) outbreak in wuhan, china (hasan, ; s. a. khan et al., ) . the virus belongs to the family known as 'coronaviruses' because of the crown-like appearance of spikes glycoproteins on the envelope under an electron microscope . world health organization (who) recently announced that the virus transforms from epidemic to pandemic, which requires urgent intervention to prevent the growing spread of the virus across the globe (chan et al., ) . the total confirmed cases , , with , cases in the united state of america (usa) alone and the total death of , (as of april ), with mortality estimated within % and about . %, according to estimates of approved cases and death worldwide (n. . the most familiar is a virus that arose from the rhinolophus bat which is > % homologous with the modern sars-cov- virus and it is just % homologous with the initial sars-cov (fisher & heymann, ) . the fast-growing number of infected cases globally urged the world health organization to announce a state of global health emergency to correlate scientific and medical disciplines to develop rapidly an effective treatment for patients, (morse et al., ; sarma et al., ) elderly patients and people with severe underlying health diseases like heart diseases, lung illness, and diabetic patients, for instance, appear to be at greater risk of revealing severe sars-cov- requires immediate intervention rather than waiting virus vaccine which may require year to be available (enayatkhani, ) . while drug repurposing could be a short-term and fast resolution to handle sars-cov- patients (elfiky, ; r. j. kumar et al., ) , repurposing existing drugs can offer a good choice to overcome the virus and offer better risk-versus trade-off as compared with discovering new drug and can help overcome time waiting for new therapy rather than use the available resources (elmezayen, ; muralidharan, ) one successful repurposing drug story includes duloxetine which originally developed for depression and fda approved as the first-inclass choice for stress urinary incontinence (sweeney & chancellor, ) , duloxetine initially created as antidepressant also is now passed to phase iii clinical trials as a first-in-class treatment for premature ejaculation (mcmahon, ) and thalidomide, which had a tragic start as an over-the-counter sedative for morning sickness in pregnancy is now being applied to manage leprosy and multiple myeloma (hideshima & anderson, ) . as a result of, a newly issued x-ray crystal of sars-cov- main protein (mpro), we planning to use computational approaches (cameron et al., ) to contribute to find an effective treatment for sars-cov- .thereby, computational analyses speed up these approaches since they allow to handle millions of data simultaneously (gupta et al., ) . molecular docking includes a set of computational methods and algorithms that aimed to identify novel relationships between chemical ligands and targets through using the modelling of their direct physical interaction (aanouz, ; ekins et al., ) . in present study, we attend to evaluate some of approved drugs to be as covalent binders, irreversible interactions, which can provide a powerful strategy to fight against epidemic viruses. and molecular dynamics simulations can give a more detail for the image which got from molecular covalent docking. the crystal structure of fda-available covalent drugs which available in table were selected based on the review of kumalo et al. ( ) and some of the antiviral drugs that can form a covalent bond to the target protein. and we aim also here to redirect them for other indications specially to see their possibility to fight against sars-cov- . thence, we searched about the chosen drugs in pubchem (https://pubchem.ncbi.nlm.nih.gov/) to identify the possibility of the selected drugs to be as covalent binders toward sars-cov- mpro. where, pubchem provides detailed information about the selected drugs rather than other repositories, especially, the property of drug to form covalent bond. before starting covalent docking, we downloaded the selected drugs one by one and optimize them by using the ligprep (lim et al., ) based on the opls_ force field and generated possible state employing epik in the schrodinger (elfiky ; s. a. khan et al., ) . in the next step, the structure of sars-cov- mpro ( lu ) was downloaded from protein data bank (http://www.rcsb.org/pdb) (jin et al. , ) . the protease structure was optimized by adding hydrogens, removing water molecules and optimizing charge using the protein preparation wizard module (kumalo et al., ) in schrodinger suite - . the covalent docking protocol was preferred since the cysteine residue which is available in the binding site of sars-cov- mpro considered as a vital residue that can form a covalent bond with the drug if it can interact covalently. the different mechanisms for the cys -fda drug were mentioned in table based on the nature of the drug so that the reactive functional group on the ligand and receptor residue are identified and the bond is formed between the correct atoms. these covalently docked complexes were created using covdock in schrodinger suite - . finally, we selected the lowest the mmgbsa value for each drug as a propriate conformation of the drug inside the binding pocket. the chosen drugs binding energies were calculated using prime mm-gbsa modules (vijayakumar et al., ) in the schr€ odinger ( ). the best poses of selected drugs-sars-cov- mpro-were chosen to obtain the binding free energy calculation. prime mmgbsa is a method that combines optimized potential for liquid simulations-all atoms (oplsaa) force field, molecular mechanics energies (emm), an sgb solvation model for polar solvation (gsgb), and a non-polar solvation term (gnp) composed of the non-polar solvent accessible surface area and van der waals interactions. the total binding free energy: molecular dynamics simulations are a decision-making process for inspections of protein-drug complexes' stabilities (al-khafaji & taskin tok, b) . it is used to clarify the dynamic behavior at an atomic level of biological systems, which is hard to handle in labs (shukla et al., ) . in the current study, we conducted molecular dynamics simulations for the top three drugs based on mmgbsa values. the got protein-drug complex structures from covalent docking were submitted to md simulations (saquinavir, ritonavir, and remdesivir with sars-cov- mpro). we hired gromacs . to run ns md simulations (abraham et al., ) . charmm force field for all atoms were chosen to run md simulation (bjelkmar et al., ) . we used swiss param to produce the topologies of drugs (zoete et al., ) . all protein-drug systems were solvated with three-point transferable intermolecular potential (tip p) and their charges were neutralized via adding na or cl ions. in the following step, the protein-drugs systems were energetically minimized through the steepest descent algorithm at a tolerance value of kj/mol.nm. then the equilibration with position restraint on the protein molecules for . ns using nvt and npt ensembles were done. electrostatic interactions were evaluated by particle mesh ewald summation (darden et al., ) . we performed the molecular dynamics simulation with no restraint on the protein molecules or ligand to determine the stability in the final step (time step of . ns). rmsd, rmsf, rg, and number of hydrogen bonds were chosen to analyze md trajectories by using gromacs utilities. the principal component analysis (pca) approach was employed to calculate eigenvectors and eigenvalues and their projection along with the first two principal components (al-khafaji & taskin tok, a). this approach is based on the protocol of gromacs . (abraham et al., ) . it is used to simplify the effect of drugs on the dynamic motion of the targeted protein where it extracts the dynamic motions in simulations that are required for their biological function (amadei et al., ) . we got the principal component analysis from the md trajectories. a series of eigenvectors and eigenvalues were generated by diagonalizing the matrix. we chose trajectories of the protein backbone of the complexes to get d-projection of motion of trajectory. to assess the possibility of selected fda drugs to work as treatment of sars-cov- , the covalent docking was utilized to screen the selected library and rank them according to their binding affinities. the calculated binding free energies of some available drugs using docking score, glide gscore, and ensemble-average mm/gbsa are shown in table . to validate our covalent docking results, the correlation between mmgbsa and docking score was constructed ( figure ). the binding energy mmgbsa-docking score correlation shows a good correlation (r ¼ . ). based on this correlation, we chose the top three ranked-mmgbsa and docking score values of fda drugs for dissection their binding modes inside the binding site. covalent docking showed that saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir, and prevacid have À . , À . , À . , À . , À . , À . , and À . , respectively. where all the topeight fda drugs show a higher affinity to form a covalent, irreversible bond with cys of sars-cov- mpro. here we investigated the role of molecular weight upon the affinity of selected drugs to bind covalently to cys , whereas % of selected drugs which have molecular weight over than g/ mol ( figure ). and have higher free binding energy mmgbsa than À kcal/mol. while the ratio of selected drugs decreased to be % of drugs which can form covalent bonding with over than À kcal/mol. surprisingly, none of the selected drugs which have a molecular weight below than g/mol can form a good affinity of binding energy. this indicates that higher molecular weight covalent warheads can form stable and efficient binding energy. what stands out in the table is saquinavir has the highest binding affinity (lowest binding energy mmgbsa of . kcal/mol). therefore, the deep examination of saquinavir is needed. figure shows that saquinavir not only formed covalent bond of . Å with cys but also formed five hydrogen bonds inside the pocket of sars-cov- mpro. in the second rank ritonavir as presented in table has À . kcal/mol. this high affinity resulted from covalent bond between ritonavir and cys of . Å (figure ) through nucleophilic addition to double bond reaction besides it interacted within binding site by forming three hydrogen bonds. despite remdesivir formed covalent bond with cys of . Å and three hydrogen bonds ( figure ) and this is similar to ritonavir, but the mmgbsa value is lower than that of ritonavir this may due to nature of reaction which in remdesivir nucleophilic addition to triple bond. the effect of drug-protein interactions upon dynamics of biological system is a fundamental in drug discovery thereby we used rmsd to investigate the influence of saquinavir, ritonavir, and remdesivir upon the stability of sars-cov- mpro. we utilized gromacs to execute the md simulations of ns for three drug-protein systems besides of apo protein. rmsd fluctuations for both apo and hollo forms were measured and presented. the rmsd was calculated to assess the overall dynamics, stability, and convergence of the various systems and the results are presented in figure (a). figure (a) shows there is a significant decrease in the rmsd value when sars-cov- main protease whether it bound to saquinavir, ritonavir, or remedisivir. further analysis revealed that the rmsd average of apo sars-cov- main protease was . nm but when it bound to saquinavir, ritonavir, and remedisivir the rmsd averages were. , . , and . nm, respectively. another significant aspect of md simulation is the flexibility of protein's backbone which can be assessed through measuring rmsf value. the results of the comparative analysis between these drugs and their effects upon sars-cov- main protease are illustrated in figure (b). closer scrutiny of figure (b) exhibits the binding of saquinavir diminished the fluctuations of the protein's backbone. and behavior is also can be seen from figure (b) where the binding of both ritonavir and remedisivir led to reducing the flexibility of the protein. the radius of gyration (rg) is a definition of system's density, and substantially influences the folding rate and stability of proteins. rg was employed to assess the compactness of all complexes. in this work, rg values are in agreement with rmsf values where there are no significant differences between apo form and hollo forms as presented in figure (c). this reveals that protein remained stable and compact all through the ns time. the number of intermolecular hydrogen bonds is a vital aspect to give an impression about the stability of drug and protein. also, the number of hydrogen bonds is relevant to the binding scores of molecular docking process. we calculated the hydrogen bonds over time to validate the stable interactions between top three drugs and their corresponding target. as seen in the figure (d) the ritonavir has highest order of hydrogen bonding (the average ¼ . ), while saquinavir has an average of . . the essential dynamic method is a tool to explore the dynamical behavior in the space of sars-cov- main protease combined with saquinavir, ritonavir, and remedisivir. basically, the comparison of the drug-bound sars-cov- mpro and drug-unbound was made as reference. in order to further understand the configurational space, we selected the first two principal components (pc , pc ) to analyze their projection of trajectories during the simulations of ligand free and ligand bound sars-cov- mpro of the phase space (shown in figure ) . during the four system simulations, the results clearly show that the unbound ligand protein covered a wider region of phase space, while all three drug-protein system occupied a smaller region of phase space. especially, saquinavir reduced the essential dynamics to lowest degree of functional motions as compared with another drugs. moreover, the pca results suggest that the drug-bound sars-cov- mpro is more stable than ligandunbound sars-cov- mpro form of sars-cov- mpro. in short, the pca results are also in agreement with the rmsd and rmsf results, which enhance the validity of the performed analysis. sars-cov- causes major pandemic health issue since its spread across the world and can infect humans mainly respiratory system causing severe pneumonia with no vaccine and drug treatment available. prior study that have referred to the significance of molecular docking to determine effective treatment in short time (wu, et al., ) . in reviewing the literature, we took the advantage of possibility of fda available drugs that can be as a covalent warhead to inhibit the sars-cov- mpro with cys . an initial objective of the project was to identify effective and applicable treatment. the present study focused on the main protease (mpro), especially pdb id ( lu ) as a potential target for several marketed drugs as possible therapeutic option to combat the virus to see the capability of these drugs to bind with the cysteine residue which is available in the binding site of sars-cov- main protease. the mpro in sars-cov- is necessary for the proteolytic maturation of the virus targeting this protein to limit the expansion of infection by hindering the cleavage of the viral polyprotein. the most interesting finding was that the both saquinavir and ritonavir have the same affinity (binding energy mmgbsa % À kcal/mol) to block binding site of sars-cov- with irreversible interactions. this study supports evidence from previous observations that lopinavir/ritonavir can inhibit sars-cov- (lim et al., ) . another important outcome was that the remdesivir comes in second rank with binding affinity (mmgbsa ¼ À kcal/mol ). whereas previous research has established that remdesivir can inhibit sars-cov- m proenzyme through docking results. another significant finding, delavirdine computationally showed the ability to form irreversible covalent bond of À . kcal/mol mmgbsa binding energy. moreover, indinavir and cefuroxime axetil exhibited the possibility to halt the pocket of sars-cov- mpro by forming stable interaction through covalent bonding and hydrogen bonding (mmgbsa binding energies % À kcal/ mol). furthermore, oseltamivir and prevacid exhibited same affinity to bind with sars-cov- protease through one covalent bond and one hydrogen bond (% À kcal/mol), which is a good explanation for the activity of oseltamivir (peeri et al., ) . in this study, results obtained show the binding affinity of drugs to an active site depends on several factors mainly the ability of a compound to form a covalent bond with amino acid residues of the mpro (cys ) and length of a covalent bond, the number of h-bonds that can form with a pocket of the active site and type of nucleophilic addition of unsaturated bonds. in this concept, the structural features in saquinavir and ritonavir like free amine group (-nh ), hydroxyl groups (-oh), carbonyl groups (c¼o) in addition to ether group play key structural feature to form h-bond. results show the promising activities for antiretroviral drugs saquinavir that used for hiv/aids more than ritonavir and remdesivir followed by lopinavir as the best drugs to bind covalently toward sars-cov- mpro with lowest energy of binding. as discussed above, saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir, and prevacid showed the ability to block the binding site of sars-cov- mpro by forming covalent bond and stabilized with hydrogen bonding. these outcomes are contrary to that of kandeel and al-nazawi ( ), they found that ribavirin, telbivudine, vitamin b , and nicotinamide can be can form non-bonding interactions. whilst our results showed that, saquinavir, ritonavir, and remdesivir can form irreversible interactions, which are considered an effective way for viral infections. in the present work, comparative molecular dynamics simulations were conducted to evaluate the effects of saquinavir, ritonavir, and remdesivir on the conformational and dynamical demeanor in either apo or hollo forms to understand the inhibitory possibility at atomic level. an initial mission of the project was to identify rapid, effective, safe, and available for large proportions of people, so we run covalent docking and mmgbsa to sift the possibility of these drug to form irreversible interactions. therefore, we had chosen these drugs because they carry covalent warheads that can bind covalently to the target. but the docking results are not adequate, so we run md simulations examine how much these drugs are able to form stable interactions with targeted protein. one interesting finding is the rmsd of protein backbone in apo status has higher average value, whereas the binding with top three drugs diminished these rmsd average. another important finding was that the rmsf of protein's backbone be less flexible when it compared to apo form of sars-cov- main protease. it is somewhat surprising that the binding of three drugs have not noted in rg values. it is not yet clear whether the top three drugs can show an evidence to inhibit the targeted protein, thereby we lean on the principal component analysis to analyze the md trajectories to judge without dispute. the most obvious finding to emerge from d pca analysis is that the binding of under investigated drugs caused stately impact on essential dynamics of protein by reducing its essential dynamics to its least possible motions. these results seem to be consistent with rmsd results. a possible explanation for this might be that binding of the drugs make the binding site much narrower due to hydrogen bindings this makes the d structure of targeted protein more rigid so it will lose its biological functions. it is possible, therefore, that using these available drugs in two ways: either alone or in combined way. these findings suggest the possible use of nominated drugs against sars-cov- in short time as approved by covalent docking screening. also, the present results are significant in at least directing the clinicians to use these safe drugs to stop development of corona virus and second giving hope that available drugs that can be efficient treatment. coronavirus today emphasizes as a potential threat to all people worldwide. although extensive researches have been directed to stop sars-cov- , but till now there is no medication. meanwhile, the spreading with complicated crisis requires immediate therapy to overcome the spread and minimize mortality of sars-cov- . the aim of the present study was to discover effective treatment through repurposing some of available fda-approved drugs against sars-cov- mpro. where, they can provide covalent warheads in virtual screening. the most prominent finding to emerge from this study is that the affinity of covalent binder toward sars-cov- mpro is ranked: saquinavir > ritonavir > remdesivir > delavirdine > cefuroxime axetil > oseltamivir ¼ prevacid. one of the more noteworthy findings in this study is that md simulation analysis that saquinavir, ritonavir, and remdesivir can form stable interaction inside the binding site of sars-cov- mpro. also, they restricted the essential motions of protein. overall, the results of screening toward mpret encourage for further clinical evaluations. they can be easy to reach and exploit as persuasive treatments for sars-cov- . moroccan medicinal plants as inhibitors of covid- : computational investigations gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers amygdalin as multi-target anticancer drug against targets of cell division cycle: double docking and molecular dynamics simulation understanding the mechanism of amygdalin's multifunctional anti-cancer action using computational approach essential dynamics of proteins implementation of the charmm force field in gromacs: analysis of protein stability effects from correction maps, virtual interaction sites, and water models a graph-based recovery and decomposition of swanson's hypothesis using semantic predications a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study emerging coronaviruses: genome structure, replication, and pathogenesis particle mesh ewald: an nÁlog(n) method for ewald sums in large systems in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling anti-hcv, nucleotide inhibitors, repurposing against covid- drug repurposing for coronavirus (covid- ): in silico screening of known drugs against coronavirus cl hydrolase and protease enzymes reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid- : an in silico study two-dimensional projection of motion of trajectory of sars-cov- mpro bound with drugs over the pc and pc q&a: the novel coronavirus outbreak causing covid- in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme- and furin molecular mechanisms of novel therapeutic approaches for multiple myeloma structure of mpro from covid- virus and discovery of its inhibitors discovery of potential multi-target-directed ligands by targeting host-specific sars-cov- structurally conserved main protease virtual screening and repurposing of fda approved drugs against covid- main protease identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and -o-ribose methyltransferase theory and applications of covalent docking in drug discovery: merits and pitfalls extrapolation of phenolic compounds as multi-target agents against cancer and inflammation case of the index patient who caused tertiary transmission of coronavirus disease in korea: the application of lopinavir/ritonavir for the treatment of covid- pneumonia monitored by quantitative rt-pcr dapoxetine: a new option in the medical management of premature ejaculation learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- peptide-like and small-molecule inhibitors against covid- the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? in-silico homology assisted identification of inhibitor of rna binding against -ncov n-protein (n terminal domain) identification of novel small molecules against gsk b for alzheimer's disease using chemoinformatics approach treatment of stress urinary incontinence with duloxetine hydrochloride identification of natural inhibitors against angiotensin i converting enzyme for cardiac safety using induced fit docking and mm-gbsa studies analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods swissparam: a fast force field generation tool for small organic molecules authors thankful for staff membered of gaziantep university institute of health sciences, department of bioinformatics and computational biology. the authors declare no conflict of interest. key: cord- - f g m authors: herzig, volker; cristofori-armstrong, ben; israel, mathilde r.; nixon, samantha a.; vetter, irina; king, glenn f. title: animal toxins — nature’s evolutionary-refined toolkit for basic research and drug discovery date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: f g m venomous animals have evolved toxins that interfere with specific components of their victim’s core physiological systems, thereby causing biological dysfunction that aids in prey capture, defense against predators, or other roles such as intraspecific competition. many animal lineages evolved venom systems independently, highlighting the success of this strategy. over the course of evolution, toxins with exceptional specificity and high potency for their intended molecular targets have prevailed, making venoms an invaluable and almost inexhaustible source of bioactive molecules, some of which have found use as pharmacological tools, human therapeutics, and bioinsecticides. current biomedically-focused research on venoms is directed towards their use in delineating the physiological role of toxin molecular targets such as ion channels and receptors, studying or treating human diseases, targeting vectors of human diseases, and treating microbial and parasitic infections. we provide examples of each of these areas of venom research, highlighting the potential that venom molecules hold for basic research and drug development. venomous animals have taken advantage of the inherent biological complexity of their victims by developing toxins that target one or all of the nervous, musculoskeletal, or cardiovascular systems in order to facilitate prey capture or defense against predators. for example, many predatory venoms contain neurotoxins that disrupt neuromuscular transmission in order to rapidly paralyse envenomated prey [ ] . due to the inherent specificity and potency of most venom molecules, venoms have become an invaluable source of modulators to study a wide range of molecular targets, especially ligand-activated [ ] [ ] [ ] and voltage-gated ion channels [ ] [ ] [ ] . while research on many potential drug targets is hampered by the lack of selective modulators, our increasing knowledge of venom pharmacology is beginning to shed light on some of these poorly studied targets (e.g. [ ] ). here we provide examples of how venom compounds have been used to study diverse molecular targets and aid our understanding of their role in human disease. we use knowledge gained from approved venom-derived drugs [ , ] to illustrate how basic research can lead all the way from venom to a human therapeutic. finally, we touch on other potential uses of toxins for human health, such as targeting vectors of human diseases, and development of antimicrobial and antiparasitic drugs (fig. ). during the course of evolution, venom has evolved independently at least times across eight different phyla [ ] . it has been estimated that there are more than , venomous species on the planet, representing ~ % of extant animal biodiversity [ ] , spanning invertebrates such as annelids, arthropods, cnidarians, molluscs, nematodes, sea urchins, star fish, and vertebrates such as snakes, lizards, fish, shrews, and platypus [ , [ ] [ ] [ ] . chemically, venoms are complex mixtures of salts, small molecules, peptides, and proteins [ , ] , with an estimated million compounds in spider venoms alone [ ] . most of these compounds are toxins, which are defined as substances that cause dose-dependent pathophysiological injury to a living organism, thereby reducing their viability [ ] . the high metabolic cost of venom production [ ] has ensured that over the course of evolution only the most potent toxins have prevailed. venoms can therefore be considered natural libraries of highly optimised bioactive compounds [ , ] . given the vastly different phyletic origins of venomous species and the even greater diversity of their target organisms, it is expected that venom toxins modulate an extremely diverse range of molecular targets (fig. ) . the dominant components of most venoms are small peptides. the high potency, selectivity and biological stability of many venom-derived peptides has made them particularly attractive from a drug discovery perspective. there are already two fda-approved venom-derived peptides in clinical use -ziconotide, an analgesic drug from the venom of a marine cone snail [ ] , and exenatide, an anti-diabetic drug from a venomous lizard [ ] -plus numerous others in clinical trials [ , ] or preclinical development [ , ] . the larger size of peptides compared to small molecules provides a larger binding surface with their molecular target, typically yielding higher potency and greater target specificity, thereby minimising unwanted side-effects. however, the larger size and polar nature of peptides translates into poor oral bioavailability. other disadvantages of peptides can be poor membrane permeability and low metabolic stability [ ] , although these disadvantages can often be overcome by chemical modification [ ] [ ] [ ] . in addition, progress in the chemical synthesis [ ] [ ] [ ] and recombinant production [ , ] of complex peptides has facilitated cost-effective venom-peptide manufacture. venomous animals have evolved peptide toxins with a huge diversity of molecular frameworks, most of them with tertiary structures stabilised by disulfide bonds [ ] . the most well-known of these disulfide-rich frameworks is the ubiquitous inhibitor cystine knot (ick) or knottin motif [ , ] , which is defined as an antiparallel -sheet stabilised by a cystine knot [ ] . a major advantage of ick peptides for drug development is their resistance to proteases, high temperature, and harsh chemicals, which has been attributed to their knotted structure [ ] [ ] [ ] . however, despite our extensive knowledge about a limited number of disulfide frameworks present in animal venoms, there are many families of disulfide-rich venom peptides that remain completely unexplored (e.g. [ ] [ ] [ ] [ ] ). further research on these novel disulfide-rich frameworks is therefore required to unleash any potential they might hold for drug development. recent advances in proteomics and transcriptomics have now made it possible to rapidly elucidate the venom proteome of even miniature venomous animals [ ] . while these developments have greatly accelerated our understanding of the structure, function and evolution of venom proteins, they have led to a general neglect of non-proteinaceous venom molecules. a wide variety of small molecules have been found in venoms including salts, amino acids, hormones, nucleosides, neurotransmitters, and polyamines [ ] [ ] [ ] . in addition to playing important roles in envenomation [ ] , some of these molecules are valuable pharmacological tools; for example, the polyamine toxins found in spider and wasp venoms are potent modulators of glutamate and nicotinic acetylcholine receptors [ , ] . early toxinological research focused on study of venoms that adversely affect humans, with the aim of understanding their mechanism of action. the molecular targets of these venoms were typically delineated using low-throughput approaches such as in vivo and ex vivo tissue studies, manual patch-clamp recording, or biochemical approaches such as radioligand binding studies [ ] . these bioassays can be highly informative because they provide information on integrated tissue or cellular responses that can be used to provide target information [ ] . however, they require a priori knowledge about well-defined receptor populations and signalling pathways in target tissues, as well as the availability of selective agonists or antagonists. in addition to providing fundamental insights into the molecular targets and mode of action of venoms and toxins, these traditional approaches yielded key insights into mammalian physiology, as exemplified below. however, these approaches neglected toxins that are of no consequence to human health, those that are present in low abundance in venom, and toxins that act on novel targets that are not represented in conventional bioassays. these neglected toxins may nonetheless offer fundamental insights into human disease, and some may even have therapeutic benefit. accordingly, high-throughput screening approaches have been developed over the past decade to exploit the exceptional biodiversity of animal toxins for drug discovery. since many venoms and toxins exert these biological effects through actions on cell membranes, receptors and ion channels, high-throughput techniques assessing changes in cellular signalling have proven particularly insightful. these include electrophysiological techniques [ ] , fluorescent imaging of na + , ca + , k + or clflux [ , ] , enzyme-linked immunosorbent assays (elisas), as well as assays based on detection of bioluminescence, fluorescence polarisation, fluorescence-resonance energy transfer (fret), bioluminescence resonance energy transfer (bret), and scintillation proximity [ ] . invariably, these assays are designed to determine pharmacological activity at specific targets of interest, although each technique has limitations with regard to assay flexibility, throughput, amount of toxin required, and specificity (for a review see [ ] ). thus, the choice of assay will depend on whether the intention is to identify the biological target of venom components that exert effects on biological processes either at the level of the cell or organism, or whether the screen is aimed at discovery of novel bioactivity at specific molecular targets or processes of interest. it has been known since the early s that the specific and virtually irreversible blocking action of -bungarotoxin, a venom peptide from the banded krait bungarus multicinctus, at the vertebrate neuromuscular junction is due to functional antagonism of the depolarising effects of acetylcholine [ ] . however, the identity of the presumed target of -bungarotoxin, the cholinergic receptor protein, was unclear until a single toxin-bound membrane protein was isolated from electric tissues of the marbled electric ray torpedo marmorata [ ] and the electric eel electrophorus electricus [ ] . it is now known that acetylcholine receptors (achrs) can be classified into muscarinic (machr) and nicotinic (nachr) subtypes, with the latter subtype being the target of bungarotoxin and numerous other venom peptides. machrs are metabotropic receptors whereas nachrs are pentameric ligand-gated ion channels. the profound impact of -neurotoxins on various biological processes highlighted the importance of nachrs in both physiology and pathology. early medical applications included the surgical use of tubocurarine, a plant-derived toxin that potently inhibits muscle nachrs and is famous for its use as a paralysing agent in poison arrows employed by south american indians [ ] . the use of as a tubocurarine as a muscle relaxant allowed patients undergoing surgery to be paralysed without using dangerously high doses of general anaesthetics [ ] . more recently, nachrs have gained attention as putative therapeutic targets for a range of human disorders. for example, a number of nachr subtypes are thought to play key roles in chronic pain and inflammation [ ] , and several α-conotoxins with good selectivity for these subtypes are being developed as drug leads [ , ] . the high affinity interaction of α-neurotoxins with nachrs also provided key insights into the structure and mode of action of these receptors [ ] , and they remain important pharmacological tools due to their exquisite selectivity for specific achr subtypes. for example, fluorescent derivatives of α-bungarotoxin are excellent probes for identifying α nachrs [ ] , while subtypeselective α-conotoxins from the venom of marine cone snails can distinguish between nachr subtypes containing common subunits [ , ] . since ablation of nachr subunits using gene editing approaches would lead to complete loss of all nachr isoforms containing the deleted subunit, α-neurotoxins remain invaluable tools for dissecting the physiological roles of specific nachr subtypes and their contribution to human disease. in addition to being a valuable source of molecules that modulate known drug targets, venoms can also be used to discover new therapeutic targets. for example, the centre of excellence for new target discovery in brazil, which is co-funded by glaxosmithkline, is focused on using venoms to identify and validate new drug targets. one therapeutic area in which venom peptides have been successfully used to identify new drug targets is chronic pain [ , ] . although the primary role of venom for most animals that possess a venom system is predation, most venomous animals also use venom for defense. some animals, such as bees, caterpillars, stonefish, and stingrays, use venom exclusively for this purpose. in contrast to the diverse mechanisms by which predatory venoms debilitate prey, defensive venoms are largely algogenic, that is they are designed to cause intense pain (as most people that have been stung by a bee or wasp can testify). unlike death, pain induces a lasting memory that can be conveyed to conspecific predators. since pronociceptive venom toxins have evolved to target vertebrate sensory neurons they have the potential to uncover new components of mammalian pain signalling pathways and to serve as pharmacological tools for studying these components [ , ] . from a screen of venoms that activate mouse sensory neurons, osteen et al. recently isolated two homologous peptides (hm a and hm b) from venom of the african tarantula heteroscodra maculata that activated only a subset of sensory neurons [ ] . the effect of these peptides was blocked by tetrodotoxin (ttx), a non-selective inhibitor of voltage-gated sodium (na v ) channels. this ultimately led to the discovery that these peptides are highly potent and selective agonists of na v . channels [ ] , which were not previously thought to be involved in pain signalling. na v . was shown to regulate the excitability of a nerve fibres that transmit mechanical pain signals to the spinal cord, thereby highlighting this ion channel for the first time as a potential analgesic target [ ] . moreover, na v . was shown to be present in sensory neurons that innervate the gut, and its expression was upregulated in a mouse model of irritable bowel syndrome (ibs) [ ] . this suggests that compounds that specifically inhibit na v . might be useful for treatment of ibsrelated gut pain, and indeed na v . inhibitors were recently shown to reduce mechanical hypersensitivity in several models of chronic visceral pain [ ] . an unexpected consequence of the discovery of hm a and hm b was their application to dravet syndrome (ds) epilepsy [ , ] , which is caused by heterozygous loss-of-function mutations in the gene encoding na v . . ds is a pharmacoresistant epileptic encephalopathy characterised by childhood-onset polymorphic seizures, cognitive impairment, psychomotor regression, autistic traits, and increased risk of sudden death [ , ] . in the central nervous system, na v . is found in the axon initial segments of gabaergic inhibitory interneurons, and consequently epileptic seizures in ds are thought to result from the reduced inhibitory activity of these neurons [ ] . remarkably, in a mouse model of ds, intracerebroventricular infusion of hm a restored the function of inhibitory interneurons and led to almost complete abolition of seizures within days, which rescued the mice from premature death [ ] . in a similar approach to that described above, a screen of snake venoms on somatosensory neurons revealed robust activation of trigeminal ganglia by texas coral snake venom [ ] . this activity could only be reproduced when two toxin components were combined to form the high affinity heteromeric mittx complex. subsequent patch-clamp studies revealed the target of mittx to be acid-sensing ion channels (asics). asics are unusual homo-or heterotrimeric channels that are activated by protons and expressed throughout the nervous system [ , ] . although mittx promotes activation of several asic subtypes, it is ~ -fold more potent at the splice-isoforms asic a and asic b compared with other subtypes. texas coral snake envenomation causes intense pain in humans, and indeed mittx elicited robust nocifensive behaviour when injected into the hindpaw of mice. this response was absent in pan-asic knockout mice, but present in an asic knockout, revealing the contribution of asic in peripheral pain sensation. a subsequent study further delineated the specific roles of asic a and asic b in different pain pathways using mambalgin toxins isolated from black mamba venom [ ] . mambalgins were found to block asics expressed in the central (asic a, asic a/ a, and asic a/ b) and peripheral (asic b and asic a/ b) nervous system. intrathecal and intracerebroventricular injection of mambalgins in mice produced potent analgesia that was naloxone resistant and dependent on the presence of asic a, suggesting an opioid independent mode of action proposed to be via central inhibition of asic a/ a channels. strikingly, peripheral intraplantar injection of mambalgins in mice produced analgesic effects in wild-type and asic a knockout mice. this demonstrated for the first time that asic b-containing channels in rodent nociceptors, as opposed to the splice isoform asic a, are important for peripheral analgesia. more recent work using an asic b knockout animal confirmed the role of this subtype in peripheral pain sensing [ ] . in this model, peripheral injection of mambalgin had no effect on acid-induced hyperalgesia in asic b knockout mice. thus, the combined use of genetic techniques and venom toxins (mittx and mambalgins) enabled dissection of the roles of asic splice isoforms in pain signalling. in addition to venom peptides, non-peptidic marine toxins have proven to be invaluable tools for elucidating diverse pain pathways. ciguatoxins (ctx) are temperature-stable polyethers produced by various species of gambierdiscus, a benthic dinoflagellate [ ] . the consumption of fish with high levels of ciguatoxins present in their flesh and viscera, a result of bioaccumulation, causes ciguatera fish poisoning [ ] . these effects are due the action of ctx on ion channels including na v channels [ ] . the most potent congener p-ctx- has been used to pharmacologically probe neuropathic pain (fig. ). ciguatera is characterised by both gastrointestinal and neurological symptoms including paresthesia, pruritus and cold allodynia, and it can even lead to death in severe cases [ ] . the pathognomonic cold allodynia observed in ciguatera patients is also reported in a broad range of clinical indications including in chemotherapy patients, complex regional pain syndrome, and fibromyalgia [ ] [ ] [ ] . defined by sensitivity to innocuous cooling temperatures, cold allodynia is a poorly understood phenomenon. subcutaneous injection of p-ctx- into the rodent hindpaw causes robust and concentration-dependent cold allodynia [ ] , and therefore it is uniquely positioned to elucidate the molecular basis of cold allodynia. the large family of thermosensitive transient receptor potential (trp) channels plays a critical role in temperature sensation [ ] . interestingly, trpm , the canonical cold sensing channel that is sensitive to menthol, is not associated with p-ctx- -induced cold allodynia, as trpm null mice administered p-ctx- display robust pain behaviours in response to innocuous cooling [ ] . rather, the combined findings from ex vivo skin-saphenous nerve recordings, in vivo studies and in vitro assays highlighted a role for trpa in p-ctx- -induced cold allodynia [ ] . however, trpa is not directly activated by p-ctx- . both ttx-sensitive and ttx-resistant na v channels were responsible for the altered neuronal excitability, which drives a trpa -dependent change in cold sensing [ ] . although the precise role of different trp channel isoforms in cold sensing remains contentious, the use of this marine toxin has proved valuable in delineating these complex mechanisms. p-ctx- was also utilised to assess a range of analgesic treatments for cold allodynia. using a suite of selective pharmacological agents including venom-derived peptides (such as analgesic conotoxins; recently reviewed in [ ] ), p-ctx- -induced cold allodynia was found to be dependent on na v . and na v . [ ] . oxaliplatin-induced cold allodynia was completely blocked by -conotoxin giiia, a na v . inhibitor from cone snail venom [ ] , although intraplantar administration of cn , a na v . -selective agonist from scorpion venom [ ] , elicited spontaneous pain and mechanical allodynia but did not enhance cold sensitivity. the mechanisms underlying neuronal cold sensitivity were further delineated using a class of presynaptic neurotoxins produced by mamba snakes (dendroaspis sp.) (fig. ) . specifically, in trigeminal neurons, application of αdendrotoxin-k, a blocker of kv . channels, elicited a reversible, concentration-dependent shift in the cold threshold [ ] . subtype-selective na v channel toxins have confirmed a crucial role for both na v . and na v . in pain signalling (fig. ) . the na v . subtype is highly expressed in the central and peripheral nervous system, in particular at the nodes of ranvier [ ] . due to the expression of na v . on motor neurons, na v . null mice are not viable beyond post-natal day (p ) [ ] . this greatly impeded the study of na v . in peripheral sensory neurons, where its role in excitability was less well defined. the subtype-selective scorpion toxin cn was therefore used to probe the role of this channel in nociception. cn causes na v . to open at hyperpolarised (more negative) potentials, and it enhances resurgent currents in large diameter sensory neurons in vitro and increases excitability of medium-diameter a fibres ex vivo [ , ] . therefore, it follows that in vivo nocifensive behaviours elicited by cn administration are primarily mediated by medium-diameter rather than smaller sensory neurons. critically, this demonstrates how toxins can be used to pharmacologically isolate a target of interest (in this case na v . ) in the presence of highly similar homologs of the target (na v . , na v . , na v . and na v . ). the toxin od , isolated from venom of the iranian yellow scorpion odonthobuthus doriae, potently enhances the activity of na v . by inducing a hyperpolarising shift in the voltagedependence of activation, inhibiting channel fast inactivation, and increasing peak current [ ] . injection of od into the footpad of mice elicits pain behaviours including licking, lifting and flinching of the injected paw that can be reversed by local or systemic treatment with selective na v . inhibitors. thus, the analgesic efficacy of na v . inhibitors can be rapidly profiled in vivo using od -induced pain behaviour, demonstrating that even toxins with no therapeutic potential can be effective tools for understanding the complexities of peripheral pain sensing [ ] (fig. ). snakes are the most well-studied venomous animal, and hundreds of snake-venom proteins have been isolated over several decades. these venom components have improved our understanding of venom toxicity, significantly advanced our the knowledge of hemostatic disease mechanisms, and led to the development of diagnostic agents and life-saving drugs [ ] [ ] [ ] . due to the immense amount of research on snake venoms, we focus here on just a few examples where studies of snake venom have advanced our understanding of hemostasis and associated diseases. studies into the lethal effects of envenomation by the burrowing asp atractaspis engaddensis led to the discovery of a novel peptide named sarafotoxin (sftx). sftx is a potent vasoconstrictor of coronary blood vessels that can induce cardiac arrest [ ] [ ] [ ] . several sftx isoforms were soon isolated from other stiletto snakes [ , ] , and it was subsequently discovered that the sftxs are in fact orthologs of mammalian vasoconstrictor peptides known as endothelins (ets) [ ] . however, a major difference between these peptides is that a protease cleavage site involved in et inactivation is not present in the sftxs [ , ] , making them more stable in vivo. this allowed the sftxs to be used as molecular tools to study vasoconstriction and expanded the available pharmacological toolbox beyond the ets. the sftxs and ets have both been used to characterise et receptors and downstream signalling pathways [ , ] . von willebrand factor (vwf) is important for platelet adhesion, and its deficiency in humans leads to a common blood coagulation disorder known as von willebrand disease (vwd). botrocetin is a heterodimeric protein found in venom of the south american pit viper bothrops jararaca and it functions as a platelet-aggregating protein. it was initially named venom coagglutinin [ ] as it leads to agglutination and aggregation of platelets by forming a complex with vwf and enhancing its affinity for the platelet receptor glycoprotein ib (gpib). this effect makes botrocetin an excellent diagnostic tool to assay for vwf binding to gpib in diseases such as vwd, or thrombopathies such as bernard-souler disease [ , ] . botrocetin has also been used as a tool to study the vwf-platelet interaction and its role in disease [ , ] . unlike other vwf binding proteins such as the antibiotic ristocetin, botrocetin-induced platelet agglutination activity is independent of the vwf multimer size. this is advantageous as it facilitates quantitation of vwf and allows assessment of vwd severity. the combination of botrocetin and ristocetin has allowed identification of missense mutations that cause of type b vwd [ ] . a fortuitous observation using viper venom to study neurite outgrowth in healthy and tumorous cells led to the nobel prize in . in studies of the regulation of cell growth, a fraction from mouse sarcoma extracts was shown to induce nerve cell growth in chick embryos [ ] . this fraction was termed nerve growth factor (ngf) [ ] , and it was shown to be composed of both nucleic acid and protein. snake venom from the cottonmouth viper agkistrodon piscivorus was known to contain phosphodiesterase activity, and thus it was proposed that this venom could degrade the nucleic acid component (which was thought to be a contaminant) and reveal the active ingredient. surprisingly, minute amounts of crude snake venom strongly promoted nerve growth, approximately - times more potently than the ngf-containing sarcoma fraction [ ] . comparison of the sarcoma extract and venom contents ultimately led to isolation and characterisation of the proteinaceous ngf. ngf has since been found in several snake venoms from the viperidae, crotalidae, and elapidae families and it has been used widely to study cell growth regulation [ , ] . the use of snake venom in this case not only led to a serendipitous finding with widespread importance to basic science, but ngf has also been important for elucidating the etiology of diseases such as developmental/growth deformations, tumours, and delayed wound healing. cobra venom factor (cvf) from the venom of cobra (naja sp.) snakes has the intriguing property of disrupting activation of the complement component of the innate immune system. the complement system is comprised of a set of interacting proteins, and it can be activated in three different ways for it to play a role in pathogen clearance. the alternative pathway of activation involves spontaneous hydrolysis of complement factor c to c b, which allows the plasma protein complement factors b and d to bind to c b and transform it to c -convertase on the external surface of an invading pathogen membrane. subsequent recruitment of additional components of the complement pathway system leads to assembly of the membrane attack complex, which forms pores in the target cell membrane and that kill or damage the pathogen [ , ] . cvf is a structural and functional analogue of the human complement factor c , and it acts in a similar manner to human factors c b and c c to activate and deplete the complement cascade [ , ] . these properties of cvf have made it possible to independently study the role of complement in host defense, immune responses, and pathogenesis of disease. cvf was in fact pivotal to the discovery of complement factor b itself, which facilitated further characterisation of this complex pathway [ , [ ] [ ] [ ] [ ] . cvf is not only an immensely useful research tool, but it has recently been used as an immunosuppressant in tissue and organ transplants and cancer therapies [ , ] , and cvf derivatives are being designed to activate the complement system in disease states where it has been depleted [ , ] . stroke is the second leading cause of death worldwide [ ] , yet therapeutic options are limited to dissolution of blood clots with tissue plasminogen activator (tpa). unfortunately, due to the risk of inducing an intracranial haemorrhage, tpa is used in < % of stroke patients [ ] , and consequently there is an unmet need for compounds that can protect the brain from stroke-induced injury. clinical trials of numerous agents, especially glutamate receptor antagonists, have failed to provide effective neuroprotection in stroke patients, which has spurred interest in better understanding the etiology of ischemia-induced brain injury [ ] . spider-venom peptides have been crucial for uncovering the key role of asics in stroke-induced brain damage, and validating these channels as a target for neuroprotective drugs [ ] [ ] [ ] [ ] . during ischemic conditions the brain switches from oxidative metabolism of glucose, its primary fuel, to anaerobic glycolysis, which results in lactic acidosis. the brain extracellular ph can fall from ~ . to as low as . in the ischemic core [ , , ] , which is sufficiently acidic to robustly activate proton-gated asics. the asic a isoform is the dominant subtype in rodent and human brain [ , ] and it primarily conducts sodium ions, although it is the only asic subtype that is also permeable to calcium (p na /p ca ~ ) [ ] . it has been proposed that asic a activation contributes to toxic intracellular calcium overload during stroke via both direct calcium permeability and downstream activation of voltage-gated calcium (ca v ) channels and nmda receptor-gated channels via asic-induced membrane depolarisation [ ] . however, the primary mechanism by which asic a activation leads to neuronal cell death appears to be recruitment and activation of ripk [ , ] , a master regulator of necroptosis. although several small molecules and endogenous ligands of asic a have been discovered to date, their lack of potency and selectivity limits their use as pharmacological tools for dissecting the role of asics in disease. the first potent and moderately selective asic a ligand discovered is pctx , a -residue peptide from venom of the trinidad chevron tarantula psalmopoeus cambridgei [ ] . pctx inhibits homomeric asic a (ic ~ nm), asic a/ b heteromers (ic ~ nm), and asic a/ a ( - % inhibition at nm) channels, and potentiates proton-evoked asic b currents by over -fold control (ec ~ nm) [ ] [ ] [ ] [ ] [ ] . the key role of asic a in stroke-induced brain damage was first highlighted in a seminal study which showed that genetic ablation of the asic a-encoding gene reduced brain infarct size by ~ % in a mouse model of ischemic stroke [ ] . however, since genetic ablation can give rise to compensatory upregulation of related channels with unknown consequences [ , ] , these researchers also used "pctx venom" (i.e., crude p. cambridgei venom) as a pharmacological tool to validate the genetic studies. they showed that "pctx venom" administered min before and after stroke onset reduced infarct size by the same magnitude as genetic ablation of asic a [ ] . however, spider venoms are complex molecular cocktails that modulate an array of ion channels, and the venom of p. cambridgei is no exception. pctx constitutes only ~ . % of total p. cambridgei venom [ ] , and other components in this venom are known to target trpv and voltage-gated potassium (k v ) channels [ , ] . this left open the question of whether the observed neuroprotective effects were induced by pctx , other components in the venom, or a combination of both. to address this question a more recent study used pure recombinant pctx to confirm the involvement of asic a in stroke [ ] . structure-activity relationship (sar) studies of pctx [ , ] facilitated design of a weakly active pctx mutant, which was not neuroprotective in the same rodent stroke model, thereby strengthening the evidence that asic a inhibition reduces stroke-induced brain damage. hadronyche infensa proved to be highly neuroprotective in a focal model of ischemic stroke, even when administered h after the stroke onset [ ] . in comparison to pctx , hi a is a slightly more potent inhibitor of asic a (ic . nm) and it is more selective, with > -fold higher potency for asic a than homomeric asic b, asic a, and asic channels. hi a inhibition of asic a is much less reversible than pctx [ ] , and these venom peptides have a different mechanism of action [ , ] . the different properties of pctx and hi a broaden the pharmacological toolkit for studying asic a and asic a-related pathologies. together these two venom peptides have provided striking pharmacological evidence that asic a is involved in ischemic injuries of not just the brain, but also the heart [ ] and kidney [ ] . the evolution of drug resistance across bacteria, fungi and parasites has created an unmet need for novel anti-infectives. the united nations report on antimicrobial resistance estimated that > , people die annually due to drug-resistant infections [ ] . while most focus has been on human antibiotic resistance, control of parasitic nematodes in both livestock [ , ] and companion animals [ , ] is also seriously threatened by resistance. despite the clear need for new anti-infectives, there is a diminishing pipeline of anti-infective drug candidates in both the human and veterinary spaces [ ] [ ] [ ] , which has created interest in venoms as a potential source of novel anti-infectives. antimicrobial peptides (amps) are found in many animal venoms, and hundreds of examples have been reported with varying activity against fungi and both gram-positive and gram-negative bacteria [ ] [ ] [ ] [ ] [ ] [ ] . venom-derived amps (vamps) are typically small, linear peptides that are rich in cationic residues (lys, his and arg), which facilitates their interaction with anionic membranes [ , ] . they are often unstructured in aqueous solution but form ordered -helices upon interaction with lipid membranes [ , ] . many vamps exhibit broad-spectrum cytolytic effects. this can be a double-edged sword for antimicrobial development as it often confers desirable broad-spectrum activity against a wide range of bacterial and fungal pathogens but also cytotoxicity against host cells. for example, the ponericin peptides isolated from the amazonian stinging ant neoponera goeldii have broadspectrum activity against bacteria and fungi, but they are potently hemolytic [ ] . likewise, cupiennin a from venom of the wandering spider cupiennius salei exhibits non-specific cytolytic activity against bacteria, insects, protozoan parasites, and human cells [ ] . mammalian cytotoxicity is typically assessed by screens against red blood cells or mammalian cell lines, but an often overlooked property of cytolytic vamps is that they can also cause pain. for example, venom-peptide ∆-myrtoxin-mp a from the australian ant myrmecia pilosula has submicromolar activity against bacterial pathogens but it causes mechanical allodynia when injected into mice [ ] . melittin, the primary component of honeybee venom, is potently antimicrobial but it induces pain through direct and indirect activation of sensory neurons [ ] . hence, future research on vamps should place greater emphasis on screening across a broad range of cell lines and phenotypic readouts, particularly for cytolytic peptides. a small number of vamps show some degree of inherent taxonomic selectivity. for example, bicarinalin from the ant tetramorium bicarinatum is highly active against bacterial and fungal pathogens (minimal inhibitory concentration (mic) of . - µm, which is comparable to commercial anti-infectives), with at least -fold selectivity over human lymphocytes [ ] . it also has similar potency to commercial antibiotics against clinical isolates of the helicobacter pylori, a pathogen that causes stomach ulcers [ ] . however, most vamps will require further engineering of their selectivity in order for them to be therapeutically useful [ ] , and several strategies have been employed successfully for this purpose. for example, shorter analogues of the scorpion-venom peptides hadrurin and vejovine were engineered with -fold improvement in selectivity for escherichia coli over red blood cells [ ] . modification of the hydrophobicity and net charge of the wasp-venom peptide mastoparan through selective amino acid substitutions [ ] and n-terminal acylation [ ] increased its affinity for negatively-charged bacterial membranes and decreased its cytotoxicity towards mammalian cells. although most vamps have not progressed beyond basic in vitro studies, some have proven effective in in vivo infection models. zy is a -residue cyclised derivative of cathelicidin-bf from venom of the snake bungarus fasciatus with improved potency, selectivity and stability over the parent toxin [ ] . in vitro, zy kills multidrug-resistant isolates of the human pathogens pseudomonas aeruginosa and acinetobacter baumannii at lower concentrations than commercial antibiotics, and it reduces bacterial load and inflammation in the lungs of mice in a p. aeruginosa infection model, with no reported side-effects [ ] . lyetxi-b, an n-terminal acetylated derivative of lyetxi from venom of the wolf spider lycosa erythrognatha, has improved potency and selectivity against e. coli and also reduces both bacteraemia and inflammation in a mouse model of septic arthritis when administered intraarticularly [ ] . route of administration is a key issue that must be considered for therapeutic deployment of vamps; traditionally, peptides have poor oral bioavailability [ ] , and hence alternative methods of vamp administration such as topical application should be considered. the effectiveness of this approach was demonstrated for kn - , a derivative of the peptide bmkn from venom of the scorpion mesobuthus martensii, which protected the skin of mice in a staphylococcus aureus in vivo infection model [ ] . topical administration can also circumvent or alleviate off-target effects that are evident when the vamp is present systemically. finally, it might be possible to achieve additive or synergistic effects by combining vamps with existing antibiotics by utilising the pore-forming activity of the vamp to enhance antibiotic delivery. for example, garcia et al. found an additive effect when pathogen-specific antibiotics were combined with vamps isolated from the venom of arachnids [ ] . viruses continue to pose an enormous threat to global health, as exemplified by the current global pandemic caused by sars-cov- [ ] . despite intensive intervention efforts, an estimated , people continue to die annually from human immunodeficiency virus acquired immunodeficiency syndrome (hiv-aids) [ ] . many serious viral diseases such as severe acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers), dengue, zika, and ebola lack vaccines or efficacious therapeutic treatments, highlighting the need for novel antivirals. most reported venom toxins with antiviral activity are vamps with broad-spectrum activity; examples include melittin, snake venom pla and l-amino oxidases, and mastoparan derivatives [ , ] . thus, a key challenge will be engineering these compounds to have selectivity for either the viral particles or the process by which they infect host cells. mastoparan- , derived from wasp-venom mastoparan, has broad-spectrum activity against lipid-enveloped viruses (including influenza and flaviviruses) through direct insertion and disruption of the envelope, but it remains highly cytotoxic to host cells in vitro [ ] . peptides with activity on specific viral protein targets rather than lipids may provide better selectivity. lactarcin- from the central asian spider lachesana tarabaevi inhibits replication of dengue virus- (ic ~ µm) by virtue of its ability to inhibit the viral protease ns b-ns , but it has low selectivity over mammalian cells [ ] . the antibacterial activity of mucroporin, a vamp from the scorpion lychas mucronatus, was improved through histidine and arginine substitutions to create the analog mucroporin-m with increased net charge. mucroporin-m has improved activity against both gram-positive and gram-negative bacteria [ ] , and these modifications also conferred micromolar antiviral activity in vitro (ec - µm) against measles virus and pseudovirus models of the highly pathogenic sars-cov and avian influenza h n viruses [ ] . a key limitation of current research on venom-derived antiviral toxins is that few studies have progressed to in vivo testing. one example found that pre-treating vesicular stomatitis virus, a serious pathogen of cattle, with mastoparan- significantly improved clinical outcomes in a mouse challenge model through presumed inactivation of viral particles [ ] . however, this needs to be confirmed in a more clinically relevant challenge model of viral infection followed by toxin administration to probe efficacy. overall, further research is required to ascertain the potential of venom-derived peptides as viable antiviral drugs. parasites remain one of the most serious threats to human and veterinary medicine, with current treatment options limited by drug resistance, side-effects and limited efficacy [ ] . this has stimulated interest in venoms as a source of novel antiparasitic compounds (for a review see [ ] ). most such studies have focused on protozoan parasites, which are responsible for a range of human diseases. the most problematic parasitic disease is malaria, which is caused by plasmodium parasites; despite intensive intervention efforts, malaria kills an estimated , people each year and control is threatened by drug resistance [ ] . a further million people worldwide are infected by parasitic protozoans of the genera leishmania and trypanosoma, with approximately million new infections per year [ ] . most antiparasitic compounds isolated from animal venoms are small cationic vamps, which again raises the issue of taxonomic selectivity. the vamps mastoparan [ ] and melittin [ ] inhibit development of trypanosoma cruzi (the causative agent of chagas disease), but selectivity over host cells is low. however, bicarinalin potently inhibits growth of leishmania infantum (mic . µm), the causative agent of infantile visceral leishmaniasis, with good selectivity over human lymphocytes [ ] . remarkably, crotamine, a peptide from venom of the south american rattlesnake crotalus durissus terrificus, crosses infected red blood cell membranes to lyse the plasmodium parasitophorous vacuole, thereby killing the parasite [ ] . this may present an opportunity for combination therapy with commercial antimalarials, facilitating drug transport to the parasite. further examples of venom components active against protozoan parasites include trimorphin, a pla from venom of the north american rear-fanged snake trimorphodon biscutatus lambda that inhibits growth of leishmania major [ ] , and crovirin, a cysteine-rich secretory protein (crisp) from the rattlesnake crotalus viridis viridis that is active against protozoan parasites at concentrations that are not toxic to host cells [ ] . the protozoan toxoplasma gondii, which causes the key zoonotic disease toxoplasmosis, is susceptible to a venom peptide from the marine cone snail conus californicus [ ] , while a c-type lectin and a pla from b. pauloensis snake venom impair toxoplasma adhesion and replication [ , ] . the greatest burden of disability-adjusted life years in the neglected tropical diseases comes from helminth infections, including roundworms (nematoda) and flatworms (platyhelminthes) [ ] . these worms cause important parasitic diseases in humans, including onchocerciasis, lymphatic filariasis, and schistosomiasis. about million people worldwide suffer from schistosomiasis, and it is considered the second most socioeconomically devastating parasitic disease after malaria [ ] . the burden of parasitic disease is also a key concern in animal health and production, with the worldwide cost of controlling livestock parasites amounting to tens of billions of dollars [ ] . despite this clear need for novel anthelmintics, it is an underexplored area of venom research with very few reports of anthelmintic venom toxins. crotamine [ ] and ε-latroinsectotoxin from venom of the black widow spider latrodectus mactans tedecimgutattatus [ ] both impair growth of the non-parasitic nematode caenorhabditis elegans, but anthelmintic activity has not been reported against parasitic species. more recently, the spider-venom peptide hi a was shown to inhibit larval development of the barber's pole worm haemonchus contortus, a parasite of sheep production [ ] . gomesin, a peptide found in the venom [ ] and hemocytes [ ] of spiders, has broad-spectrum activity against bacteria, fungi, parasites and tumor cells [ , ] , and a cyclized version was recently shown to have improved stability and activity against plasmodium in vitro [ ] . to our knowledge, no venom-derived antiparasitics have yet been demonstrated to have in vivo efficacy. as for antibacterials, antifungals and antivirals, most venom-derived antiparasitics will likely require engineering to enhance their selectivity over host cells. an alternative strategy to developing venom compounds as anti-parasitic drugs is to instead impair plasmodium development and transmission via transgenic mosquitoes engineered to express the anti-parasitic compound in the mosquito midgut, as recently demonstrated with scorpion-venom peptides [ ] . as most research on venom compounds with antibacterial, antiparasitic, or antifungal activity has been performed in the last decade, we expect that more toxins with such activities will be discovered in the near future. most work to date has focussed on human pathogens, but there is a clear need for novel veterinary anti-infectives which could be further explored in future. due to the commonly found cytolytic nature of these peptides, a key challenge across all of these diseases will be engineering analogs with selectivity for the targeted pathogen. arthropods, and in particular hematophagous (blood-sucking) species, are vectors for a wide-range of organisms that can cause human disease [ ] . the most pernicious vectors are undoubtedly mosquitoes, which transmit protozoans (e.g. plasmodium species that cause malaria), parasitic nematodes (e.g. brugia species that cause filariasis), parasitic platyhelminths (e.g. trematodes that cause schistosomiases), and viruses (e.g. flaviviruses that cause yellow fever, west nile fever, and dengue) [ , , ] . other important hematophagous disease vectors include triatomine bugs which transmit trypanosomes that cause chagas disease, fleas which transmit the bacterium yersinia pestis that causes bubonic plague, and ticks which transmit a wide variety of viral, bacterial and protozoan pathogens [ , ] . rather than directly targeting disease-causing organisms, venom toxins could be used instead to target the vectors of these diseases. insects are the dominant vectors of human diseases, and chemical insecticides are the primary means of controlling these insect pests [ , ] . unfortunately, all chemical insecticides target one of only a handful of molecular targets, and consequently their indiscriminate use has led to widespread resistance [ ] . thus, new insecticides with novel modes of action are much sought after. spiders are professional insect killers, and they are the most speciose venomous animal, and consequently their venoms have been intensively studied as a potential source of novel bioinsecticides [ , [ ] [ ] [ ] [ ] [ ] . as a result, many insecticidal toxins have been isolated from spider venoms, including peptides, proteins and polyamines, but they mostly lack the necessary taxonomic selectivity to be useful as insecticides. an ideal insecticide should be inactive on humans and other vertebrates, as well as beneficial insects such as pollinators and natural predators of the targeted insect pest [ ] . this narrow target-organism profile makes insecticide development particularly difficult. however, spidervenom peptides have been isolated with desirable taxonomic selectivity. for example, by carefully counter-screening insect toxins from the venom of australian funnel-web spiders in vertebrate assays, a number of insect-selective neurotoxins were isolated [ ] . one of these peptide toxins (gs-/-hexatoxin-hv a), which has complex polymodal pharmacology, has been developed commercially as a bioinsecticide by vestaron corporation; this peptide is active against a broad range of insect pests but is inactive against bees and vertebrates [ ] . in order to target anopheline mosquitoes that transmit malaria, a gene encoding gs-/-hexatoxin-hv a was engineered into the entomopathogenic fungus metarhizium pingshaense [ ] , and this weaponised entomopathogen proved highly successful in controlling malaria vectors in a field trial in burkina faso [ ] . an advantage of this approach is that the range of susceptible species is determined by the entomopathogen rather than the venom peptide, thereby allowing deployment of insecticidal venom toxins that would otherwise lack the desired taxonomic selectivity [ , ] . some insecticidal venom peptides have exceptional taxonomic selectivity. for example, -diguetoxin-dc a (dc a), a -residue knottin from venom of the desert bush spider diguetia canities [ ] , and -theraphotoxin-ae a, a -residue knottin from venom of african tarantula augacephalus ezendami [ ] , have potent insecticidal effects on german cockroaches (blattella germanica), without significantly affecting american cockroaches (periplaneta americana). both knottins target insect na v channels, and mutational analyses revealed that the susceptibility of insects to these toxins is determined by small sequence differences in their proposed binding site on the extracellular surface of the domain ii voltage sensor (vsd ii ) [ , ] . a high-resolution structure of dc a bound to a cockroach nav channel was recently determined using cryoelectron microscopy [ ] , and it not only confirmed that vsd ii is the binding site for dc a but revealed that the toxin's inactivity against vertebrates [ ] can be explained by sequence variations in the vsd ii region of each of the human na v channels. the dc a-na v channel structure allows predictions to be made about which insects will be susceptible to dc a based on their na v channel vsd ii sequences, and it should allow use of structure-guided protein engineering to develop toxin analogs with improved potency and/or altered taxonomic selectivity. in addition to highlighting how venom compounds can be used to elucidate disease mechanisms and uncover new drug targets, we want to provide examples of venom compounds that have progressed all the way from basic research to marketed drugs. there are currently six venomderived drugs approved by the fda, plus a snake-venom-derived serine protease (batroxobin; marketed as defibrase®) that is approved for clinical use outside of the usa [ , ] . two further venom-derived drugs -dalazatide for treatment of autoimmune diseases [ ] and tozuleristide for intraoperative imaging of brain tumours [ ] -are currently undergoing clinical trials. the examples below demonstrate the potential for developing venom compounds into drugs [ ] but, like any class of therapeutics, there are many challenges and the success rate is low [ ] . the first venom-derived drug was captopril, an angiotensin-converting enzyme (ace) inhibitor that is used to treat hypertension. the observation that patients envenomated by the lethal brazilian viper bothrops jararaca suffered from hypotension and severe intestinal contractions led to the discovery that the venom inhibits ace [ ] [ ] [ ] . inhibition of ace lowers blood pressure by preventing conversion of angiotensin i to the vasoconstrictive hormone angiotensin ii, and by decreasing degradation of the vasodilatory peptide bradykinin. researchers at squibb isolated six ace inhibitory peptides from b. jararaca venom, one of which (teprotide, residues) was both potent and stable in vivo [ ] . detailed sar studies of teprotide ultimately led to a smaller, orally active peptidomimetic known as captopril (d- -methyl- -mercaptopropanoyl-l-proline) [ , ] . captopril was approved by the fda for treatment of hypertension in , and by the mid s it had become a blockbuster drug, reaching peak annual sales of more than usd $ . billion [ ] . another example of a reptile peptide that was developed into a human drug is exendin- , a residue peptide isolated from venom of the gila monster heloderma suspectum. in striking contrast to captopril, the marketed drug (exenatide) is identical to the peptide found in the lizard's venom. exenatide mirrors the effects of human hormone glucagon-like peptide- (glp- ), an incretin that stimulates glucose-dependent insulin secretion, suppresses glucagon secretion, slows gastric emptying, and promotes satiety [ ] , but it is much more stable in vivo than human glp- with a plasma half-life of . h [ ] . byetta®, a formulation of exenatide that requires twice daily subcutaneous injections, was approved by the fda in for control of type diabetes, and it achieved worldwide sales of usd $ million in fiscal year [ ] . in order to improve patient acceptance and compliance, a new formulation was developed in which the peptide is embedded in biodegradable polymeric microspheres that extends the duration of exenatide release to such an extent that therapeutic levels can be maintained with weekly injections [ ] . this new formulation (bydureon®) was approved by the fda in , and annual sales have remained steady at usd $ - million despite the introduction of competing incretin mimetics. another drug that is an exact copy of a venom peptide is ziconotide, a synthetic version of ωconotoxin mviia, a -residue knottin peptide found in venom of the marine cone snail conus magus. ziconotide is used to selectively inhibit ca v . channels in the spinal cord, thereby preventing the propagation of pain signals from the spinal cord to processing centres in the brain [ ] . it was approved by the fda in for treating patients with chronic pain. ziconotide is administered by intrathecal infusion, which limits its use to patients with intractable chronic pain that have failed on frontline analgesic drugs such as morphine. while ziconotide set a landmark as the first venom-derived drug with a neuronal target, its limited market acceptance due to its mode of administration and neuropsychiatric side-effects [ ] has caused venom researchers to focus attention on analgesic targets in peripheral nociceptors rather than central neurons, as outlined in section . . the integration of advanced venom proteomics with next-generation sequencing of venom-gland transcriptomes has made it possible to rapidly determine, at only moderate cost, the complete venom proteome of even miniature venomous animals [ ] . as a result, novel venom peptides and proteins, often with unique three-dimensional folds, are being discovered at an unprecedented rate. the coupling of these omics workflows with high-throughput screening technologies [ , ] is allowing the pharmacology of these compounds to be explored with unprecedented efficiency, often leading to the discovery of toxins with novel pharmacology [ , , , , ] . all of these advances have combined to create tremendous interest in venoms as a potential source of drugs and bioinsecticides [ , ] . while there have been some notable successes -there are seven venom-derived drugs in clinical use and several in phase / clinical trials -the triumphs are scarcer than many predicted. no novel venom-derived drug has been approved by the fda since [ , ] , and only one venomderived compound has been commercialised as a bioinsecticide [ ] . the reasons for this are manifold. drug development is inherently difficult and the overall success rate is low [ ] ; venomderived drug candidates are no different than any other class of therapeutic in this respect, although the overall approval rate for venom-derived drugs entering clinical trials is higher than other classes of therapeutics. most venom researchers are not trained in the nuances of drug development, and this has led to over-hyped claims about the therapeutic potential of venom compounds, often in the absence of in vivo data. we encourage collaboration between venom researchers and those with experience in drug development so that, at a minimum, in vivo efficacy and toxicity can be assessed in validated disease models, using clinically viable routes of administration, before making claims about therapeutic potential. renewed interest from pharmaceutical companies in venoms as source of drug leads and pharmacological tools [ ] [ ] [ ] [ ] will help to drive these collaborations. notwithstanding these caveats, there is little doubt that the field of venoms-based drug discovery has rapidly matured over the past decade and is now producing drug leads at an unprecedented rate. advances in cryoelectron microscopy have facilitated determination of the three-dimensional structure of venom compounds bound to large membrane receptors [ , , [ ] [ ] [ ] [ ] , and this should facilitate the rational design of drug candidates with improved potency and selectivity for their cognate molecular target. we predict that these advances this will lead to significant impact in some disease areas. for example, ion channels are now the third most common human drug target after kinases and g-protein-coupled receptors [ ] ; venoms are unquestionably the best natural source of potent ion channel modulators and venom-derived ion channel modulators have already attracted significant interest from pharmaceutical companies [ , [ ] [ ] [ ] [ ] . the authors declare that they have no conflicts of interest. figure : fields of application in basic research and human health to which venom compounds have been applied. venom compounds have the potential to be used as pharmacological tools, therapeutics, insecticides, or for targeting vectors of human disease or disease-inducing organisms. botrocetin bound von willebrand factor (vwf) and platelet glycoprotein ib (gpib) ( u n) [ ] ; cobra venom factor (cvf) bound complement factor b ( hrz) [ ] ; captopril bound angiotensin converting enzyme (ace) ( uzf) [ ] ; triflin bound small serum protein (ssp ) ( imf) [ ] ; mittx-α and -β bound acid sensing ion channel (asic ) ( ntw) [ ] . (b) cone snail toxin complexes: α-conotoxin imi bound acetylcholine binding protein (achbp) ( byp) [ ] ; μconotoxin kiiia bound voltage-gated sodium channel (na v ) . /auxiliary β subunit ( j e) [ ] . (c) spider toxin complexes: pctx bound asic ( fz ) [ ] ; dc a and tetrodotoxin (ttx) bound na v pas ( a ) [ ] ; dktx and resiniferatoxin (rtx) bound transient receptor potential cation channel subfamily v member (trpv ) ( irx) [ ] ; protx-ii bound na v . voltage sensor domain ii/na v ab chimera ( n i) [ ] . (d) gila monster toxin exendin- bound glucagon-like peptide receptor (glp- r) ( c t) [ ] . (e) scorpion toxin complexes: charybdotoxin (ctx) bound voltage-gated potassium channel (k v ) . - . paddle chimera ( jta) [ ] ; α-mammal toxin aah bound human-cockroach hybrid na v channel ( nt ) [ ] . structures are shown as a cartoon representation and from two angles ( o rotation). toxin components are shown in purple and with a transparent surface representation where possible (note: ttx and rtx are shown as red sticks). where applicable for the target structure, protein subunits or domains are colored differently. structures are not to scale. toxins providing insight into the pathophysiological role of specific ion channels in sensory neurons. subtype-selective na v channel activators, including δ-trtx-hm a and the scorpion toxins cn and od , have highlighted modality-specific nociceptive roles for na v . , na v . and na v . , respectively, in sensory neurons. activation of na v . by the scorpion toxin od leads to spontaneous action potential firing in myelinated a-fibres and unmyelinated cfibres, while activation of na v . leads to symptoms of mechanical allodynia and increased mechanical sensitivity. cn , a selective na v . activator, elicits pain behaviour and mechanical allodynia after local administration consistent with effects on myelinated a-fibres. similarly, cold pain induced by ciguatoxin is elicited by selective activation of unmyelinated peripheral sensory neurons expressing the cold-sensitive trpa and na v . channels, while α-dendrotoxin revealed a crucial role for k v . in setting the activation threshold of cold-sensitive c-fibre neurons. the toxicogenomic multiverse: convergent recruitment of proteins into animal venoms venom toxins in the exploration of molecular, physiological and pathophysiological functions of acid-sensing ion channels conotoxins targeting nicotinic acetylcholine receptors: an overview nicotinic acetylcholine receptor inhibitors derived from snake and snail venoms calcium channel diversity and neurotransmitter release: the ω-conotoxins and ω-agatoxins peptide toxins that selectively target insect na v and ca v channels from foe to friend: using animal toxins to investigate ion channel function selective spider toxins reveal a role for na v . channel in mechanical pain venoms as a platform for human drugs: translating toxins into therapheutics peptide therapeutics from venom: current status and potential the diversity of venom: the importance of behavior and venom system morphology in understanding its ecology and evolution venoms to the rescue arthropod assassins: crawling biochemists with diverse toxin pharmacopeias entomo-venomics: the evolution, biology and biochemistry of insect venoms pharmacology and biochemistry of spider venoms venom composition and strategies in spiders: is everything possible? advances in insect physiology spider-venom peptides: structure, pharmacology, and potential for control of insect pests poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them the venom optimization hypothesis revisited combinatorial peptide libraries in drug design: lessons from venomous cone snails were arachnids the first to use combinatorial peptide libraries? intrathecal ziconotide: a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics exenatide: from the gila monster to the pharmacy safety and pharmacodynamics of dalazatide, a kv . channel inhibitor, in the treatment of plaque psoriasis: a randomized phase b trial phase safety, pharmacokinetics, and fluorescence imaging study of tozuleristide (blz- ) in adults with newly diagnosed or recurrent gliomas the future of peptide-based drugs fusion proteins containing neuropeptides as novel insect contol agents: snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion engineering stable peptide toxins by means of backbone cyclization: stabilization of the α-conotoxin mii optimization of physicochemical and pharmacological properties of peptide drugs by glycosylation recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins venoms to drugs: venoms as a source for the development of human therapeutics modern tools for the chemical ligation and synthesis of modified peptides and proteins production of recombinant disulfiderich venom peptides for structural and functional analysis via expression in the periplasm of e. coli high-throughput expression of animal venom toxins in escherichia coli to generate a large library of oxidized disulphidereticulated peptides for drug discovery the structural universe of disulfide-rich venom peptides toxin structures as evolutionary tools: using conserved d folds to study the evolution of rapidly evolving peptides knottin: the database of inhibitor cystine knot scaffold after years, toward a systematic structure modeling a common structural motif incorporating a cystine knot and a triple-stranded β-sheet in toxic and inhibitory polypeptides thermal, chemical, and enzymatic stability of the cyclotide kalata b : the importance of the cyclic cystine knot spider-venom peptides as therapeutics the cystine knot is responsible for the exceptional stability of the insecticidal spider toxin ω-hexatoxin-hv a a bioinformatics survey for conotoxin-like sequences in three turrid snail venom duct transcriptomes centipede venom: recent discoveries and current state of knowledge a dipteran's novel sucker punch: evolution of arthropod atypical venom with a neurotoxic component in robber flies venom peptide repertoire of the european myrmicine ant manica rubida: identification of insecticidal toxins venom collection and analysis in the pseudoscorpion chelifer cancroides (pseudoscorpiones: cheliferidae) one scorpion, two venoms: prevenom of parabuthus transvaalicus acts as an alternative type of venom with distinct mechanism of action nmr-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species giant fish-killing water bug reveals ancient and dynamic venom evolution in heteroptera targeting ionotropic receptors with polyamine-containing toxins polyamine toxins: development of selective ligands for ionotropic receptors venoms-based drug discovery: bioassays, electrophysiology, high-throughput screens and target identification ion channel screening development of a high-throughput fluorescent nowash sodium influx assay high-throughput fluorescence assays for ion channels and gpcrs pharmacological screening technologies for venom peptide discovery isolation of neurotoxins from the venom of bungarus multicinctus and their modes of neuromuscular blocking action isolation of the cholinergic receptor protein of torpedo electric tissue use of a snake venom toxin to characterize the cholinergic receptor protein from arrow poison to neuromuscular blockers toxins and drug discovery nicotinic acetylcholine receptors in neuropathic and inflammatory pain α -containing nicotinic acetylcholine receptors and the modulation of pain the α α nicotinic acetylcholine receptor antagonist αo-conotoxin gexiva[ , ] alleviates and reverses chemotherapy-induced neuropathic pain crystal structure of the extracellular domain of nachr α bound to α-bungarotoxin at . Å resolution moon is, carbofuran induces apoptosis of rat cortical neurons and downregulates surface ⍺ subunit of acetylcholine receptors conus peptides targeted to specific nicotinic acetylcholine receptor subtypes conus venom peptide pharmacology pain-causing venom peptides: insights into sensory neuron pharmacology receptor-targeting mechanisms of pain-causing toxins: how ow? nav . inhibition can reduce visceral hypersensitivity selective na v . activation rescues dravet syndrome mice from seizures and premature death a selective na v . activator with potential for treatment of dravet syndrome epilepsy a heteromeric texas coral snake toxin targets acid-sensing ion channels to produce pain structure, function, and pharmacology of acid-sensing ion channels (asics): focus on asic a acid-sensing ion channel (asic) structure and function: insights from spider, snake and sea anemone venoms black mamba venom peptides target acid-sensing ion channels to abolish pain involvement of acid-sensing ion channel b in the development of acid-induced chronic muscle pain differential toxin profiles of ciguatoxins in marine organisms: chemistry, fate and global distribution australian perspectives on a global problem clinical observations on , cases of ciguatera (fish poisoning) in the south pacific quantitative and qualitative perceptual analysis of cold dysesthesia and hyperalgesia in fibromyalgia chemotherapy-induced neuropathy reduction of allodynia in patients with complex regional pain syndrome: a double-blind placebo-controlled trial of topical ketamine ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling an introduction to trp channels conotoxins that could provide analgesia through voltage gated sodium channel inhibition analgesic treatment of ciguatoxin-induced cold allodynia μ-conotoxin giiia, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization resurgent current and voltage sensor trapping enhanced activation by a β-scorpion toxin solely in nav . channel. significance in mice purkinje neurons variable threshold of trigeminal cold-thermosensitive neurons is determined by a balance between trpm and kv potassium channels sodium channel na v . is localized at nodes of ranvier, dendrites, and synapses molecular and pathological effects of a modifier gene on deficiency of the sodium channel scn a (na v . ) na v . regulates excitability of mechanosensitive sensory neurons analgesic effects of gptx- , pf- and cnv in a mouse model of na v . -mediated pain toxins in thrombosis and haemostasis: potential beyond imagination from snake venom toxins to therapeutics-cardiovascular examples non-enzymatic proteins from snake venoms: a gold mine of pharmacological tools and drug leads a new type of toxin in the venom of snakes of the genus atractaspis (atractaspidinae) cardiotoxic effects of the venom of the burrowing asp coronary vasospasm as the primary cause of death due to the venom of the burrowing asp, atractaspis engaddensis sarafotoxins s : several isotoxins from atractaspis engaddensis (burrowing asp) venom that affect the heart a novel potent vasoconstrictor peptide produced by vascular endothelial cells endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance the hydrolysis of endothelins by neutral endopeptidase . (enkephalinase) three apparent receptor subtypes for the endothelin/sarafotoxin family endothelin-like peptides venom coagglutinin: an activator of platelet aggregation dependent on von willebrand factor structure and function of snake venom toxins interacting with human von willebrand factor role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von willebrand factor practical applications of snake venom toxins in haemostasis snake c-type lectin-like proteins and platelet receptors von willebrand disease type b: a missense mutation selectively abolishes ristocetin-induced von willebrand factor binding to platelet glycoprotein ib in vitro experiments on the effects of mouse sarcomas and on the spinal and sympathetic ganglia of the chick embryo nerve growth factors from snake venoms: chemical properties, mode of action and biological significance a nerve growth-stimulating factor isolated from snake venom nerve growth factor from cobra venom molecular diversity of snake venom nerve growth factors a molecular concept of immune cytolysis alternative pathway for the activation of complement in human serum. formation and composition of the complex with cobra venom factor that cleaves the third component of complement molecular characterization of the complement activating protein in the venom of the indian cobra (naja n. siamensis) cobra venom factor: structural homology with the third component of human complement the c -activator system: an alternate pathway of complement activation isolation of the anticomplementary protein from cobra venom and its mode of action on c alternate complement pathway: factors involved in cobra venom factor (covf) activation of the third component of complement (c ) the relationship of glycine-rich -glycoprotein to factor b in the properdin system and to the cobra factor-binding protein of huan serum inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model snake venom: from fieldwork to the clinic functional characterization of human c /cobra venom factor hybrid proteins for therapeutic complement depletion cobra venom factor: structure, function, and humanization for therapeutic complement depletion arumugam tv, pathophysiology, treatment, and animal and cellular models of human ischemic stroke beyond nmda and ampa glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels prolonged activation of asic a and the time window for neuroprotection in cerebral ischaemia pctx affords neuroprotection in a conscious model of stroke in hypertensive rats via selective inhibition of asic a potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acidsensing ion channel a translational strategies for neuroprotection in ischemic strokefocusing on acid-sensing ion channel a identification of a calcium permeable human acid-sensing ion channel transcript variant acid-sensing ion channels in acidosis-induced injury of human brain neurons tissue acidosis induces neuronal necroptosis via asic a channel independent of its ionic conduction disruption of auto-inhibition underlies conformational signaling of asic a to induce neuronal necroptosis isolation of a tarantula toxin specific for a class of proton-gated na + channels interaction of acid-sensing ion channel (asic) with the tarantula toxin psalmotoxin is state dependent heteromeric acid-sensing ion channels (asics) composed of asic b and asic a display novel channel properties and contribute to acidosisinduced neuronal death functional and pharmacological characterization of two different asic a/ a heteromers reveals their sensitivity to the spider toxin pctx the modulation of acid-sensing ion channel by pctx is ph-, subtype-and species-dependent: importance of interactions at the channel subunit interface and potential for engineering selective analogues genetic compensation triggered by mutant mrna degradation ptc-bearing mrna elicits a genetic compensation response via upf a and compass components isolation and characterization of psalmopeotoxin i and ii: two novel antimalarial peptides from the venom of the tarantula psalmopoeus cambridgei spider toxins activate the capsaicin receptor to produce inflammatory pain a dynamic pharmacophore drives the interaction between psalmotoxin- and the putative drug target acidsensing ion channel a molecular dynamics and functional studies define a hot spot of crystal contacts essential for pctx inhibition of acid-sensing ion channel a the tarantula toxin psalmotoxin inhibits acid-sensing ion channel (asic) a by increasing its apparent h + affinity enhanced donor heart preservation by therapeutic inhibition of acid sensing ion channel a, biorxiv acid-sensing ion channel a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis no time to wait: securing the future from drug-resistant infections anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in europe anthelmintic resistance in haemonchus contortus: history, mechanisms and diagnosis the emergence of macrocyclic lactone resistance in the canine heartworm, dirofilaria immitis multiple drug resistance in the canine hookworm ancylostoma caninum: an emerging threat? repurposing drugs for the treatment and control of helminth infections polishing the tarnished silver bullet: the quest for new antibiotics anthelmintic discovery and development in the animal health industry antimicrobial and cytolytic peptides of venomous arthropods antimicrobial peptides from scorpion venoms insects, arachnids and centipedes venom: a powerful weapon against bacteria. a literature review animal venoms as antimicrobial agents animal venom peptides: potential for new antimicrobial agents hitchhiking with nature: snake venom peptides to fight cancer and superbugs toxins and antimicrobial peptides: interactions with membranes latarcins: versatile spider venom peptides ponericins, new antibacterial and insecticidal peptides from the venom of the ant pachycondyla goeldii cupiennin a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells △-myrtoxin-mp a is a helical heterodimer from the venom of the jack jumper ant that has antimicrobial, membrane-disrupting, and nociceptive activities melittin, the major pain-producing substance of bee venom biochemical and biophysical combined study of bicarinalin, an ant venom antimicrobial peptide anti-helicobacter pylori properties of the ant-venom peptide bicarinalin enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin selective amino acid substitution reduces cytotoxicity of the antimicrobial peptide mastoparan selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x the antimicrobial peptide zy combats multidrug-resistant pseudomonas aeruginosa and acinetobacter baumannii infection lyetxi-b, a synthetic peptide derived from lycosa erythrognatha spider venom, shows potent antibiotic activity in vitro and in vivo current challenges in peptide-based drug discovery antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo antimicrobial peptides from arachnid venoms and their microbicidal activity in the presence of commercial antibiotics the sars-cov- outbreak: what we know global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries and territories, - : a systematic analysis for the global burden of disease study antiviral activity of animal venom peptides and related compounds antiviral peptides as promising therapeutic drugs a mastoparanderived peptide has broad-spectrum antiviral activity against enveloped viruses identification of natural antimicrobial agents to treat dengue infection: in vitro analysis of latarcin peptide activity against dengue virus mucroporin, the first cationic host defense peptide from the venom of lychas mucronatus virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses antiparasitic agents: new drugs on the horizon venoms as sources of novel anti-parasitic agents souto-padron t, melittin peptide kills trypanosoma cruzi parasites by inducing different cell death pathways trypanocidal activity of mastoparan from polybia paulista wasp venom by interaction with tcgapdh inhibition of malaria parasite plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom a comparative study of the effects of venoms from five rear-fanged snake species on the growth of leishmania major: identification of a protein with inhibitory activity against the parasite in vitro effect of the synthetic cal . a conotoxin, derived from conus californicus, on the human parasite toxoplasma gondii anti-parasitic effect on toxoplasma gondii induced by bnsp- , a lys -phospholipase a homologue from bothrops pauloensis venom insights into antiparasitism induced by a c-type lectin from bothrops pauloensis venom on toxoplasma gondii water-related diseases. world health organization impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance -an australian perspective anthelmintic effects of a cationic toxin from a south american rattlesnake venom latrophilin is required for toxicity of black widow spider venom in caenorhabditis elegans the antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against haemonchus contortus in vitro structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene isolation and characterization of gomesin, an -residue cysteine-rich defense peptide from the spider acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family the biological and biophysical properties of the spider peptide gomesin gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell death and proliferation cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity from noxiustoxin to scorpine and possible transgenic mosquitoes resistant to malaria spider-venom peptides as bioinsecticides australian funnel-web spiders: master insecticide chemists the insecticidal potential of venom peptides versatile spider venom peptides and their medical and agricultural applications tying pest insects in knots: the deployment of spider-venom-derived knottins as bioinsecticides improved efficacy of an arthropod toxin expressing fungus against insecticide-resistant malaria-vector mosquitoes transgenic metarhizium rapidly kills mosquitoes in a malaria-endemic region of burkina faso construction of a hypervirulent and specific mycoinsecticide for locust control characterization and cloning of insecticidal peptides from the primitive weaving spider diguetia canities molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the african spider augacephalus ezendami a distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin dc a structural basis for the modulation of voltage-gated sodium channels by animal toxins venoms to drugs: venom as a source for the development of human therapeutics london history of the design of captopril and related inhibitors of angiotensin converting enzyme the discovery of captopril: from large animals to small molecules design of angiotensin converting enzyme inhibitors blockbuster drugs: the rise and decline of the pharmaceutical industry exenatide once weekly for the treatment of type diabetes ziconotide: neuronal calcium channel blocker for treating severe chronic pain do the potential benefits outweigh the risks? an update on the use of ziconotide in clinical practice venomics: a new paradigm for natural products-based drug discovery a distinct three-helix centipede toxin ssd inhibits i ks channels by interacting with the kcne auxiliary subunit novel venom-derived inhibitors of the human eag channel, a putative antiepileptic drug target potency optimization of huwentoxin-iv on hna v . : a neurotoxin ttx-s sodium-channel antagonist from the venom of the chinese bird-eating spider selenocosmia huwena engineering potent and selective analogues of gptx- , a tarantula venom peptide antagonist of the na v . sodium channel structural basis of na v . inhibition by a gating-modifier spider toxin comprehensive engineering of the tarantula venom peptide huwentoxin-iv to inhibit the human voltage-gated sodium channel hna v . mechanisms of channel block in calcium-permeable ampa receptors structural basis of ⍺-scorpion toxin action on na v channels structures of human na v . channel in complex with auxiliary subunits and animal toxins molecular basis for pore blockade of human na + channel na v . by the μ-conotoxin kiiia a comprehensive map of molecular drug targets the snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation insights into complement convertase formation based on the structure of the factor b-cobra venom factor complex structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme crystal structure of the complex between venom toxin and serum inhibitor from viperidae snake x-ray structure of acid-sensing ion channel -snake toxin complex reveals open state of a na + -selective channel structures of aplysia achbp complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations molecular basis for pore blockade of human na(+) channel nav . by the μ-conotoxin kiiia structural plasticity and dynamic selectivity of acid-sensing ion channelspider toxin complexes trpv structures in nanodiscs reveal mechanisms of ligand and lipid action crystal structure of the ligand-bound glucagonlike peptide- receptor extracellular domain structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent k + channel king: conceptualization, supervision king: writing, review & editing, funding acquisition key: cord- -z c ex authors: brunsdon, priya; saluja, bhawana; sahajwalla, chandrahas title: clinical pharmacology considerations for developing small‐molecule treatments for covid‐ date: - - journal: j clin pharmacol doi: . /jcph. sha: doc_id: cord_uid: z c ex numerous drugs are being investigated for the treatment of covid‐ , including antivirals and therapies targeting complications related to covid‐ . the clinical presentation of covid‐ varies from mild fever, cough, and dyspnea in the early stages of disease to severe complications such as acute respiratory distress syndrome, systemic hyperinflammation, and sepsis. a thorough understanding of the disease pathogenesis and the disease complications is essential to developing effective therapies to treat this potentially life‐threatening disease. this review offers key clinical pharmacology considerations involved in the development of small molecules for the treatment of covid‐ . they are based on the major observed disease complications that impact drug absorption, distribution, metabolism, and elimination. we also address considerations regarding potential drug interactions, alternative routes and methods of administration, and dosing in patients on hemodialysis. the respiratory disease (covid- ) that was first reported in wuhan, china, in december is caused by severe acute respiratory syndrome coronavirus (sars-cov- ). , which was declared a pandemic by the world health organization in march , has been characterized by a rapid spread and profound impact to public health worldwide. as of june , , more than reported cases and deaths globally were attributed to the covid- outbreak. mild cases of the disease may present only with cough, fever, myalgia, and malaise. gastrointestinal symptoms include nausea, diarrhea, and anorexia. abnormal symptoms such as total loss of taste and smell also have been reported. in severe cases of covid- , complications can include sepsis and septic shock, multiorgan failure, and acute kidney injury. in addition, observational studies detailing neurological symptoms of the disease, along with reports of large-vessel stroke in young patients indicate that the disease manifests throughout the body. , infection with sars-cov- begins with viral binding to airway epithelial cells that express angiotensinconverting enzyme (ace ). one proposed mechanism for antiviral drugs involves blocking viral binding to ace , thereby blocking viral entry into host cells. following viral infection, a local immune response is triggered, which can be beneficial to clearing the infection. however, the response can become dysregulated, resulting in cytokine release syndrome (or "cytokine storm"), which is an overproduction of proinflammatory cytokines such as interleukin (il)- , il- , and tumor necrosis factor. numerous biologics and small molecules with immunomodulatory effects have been proposed as investigational therapies targeting the inflammatory response or cytokine storm. therapies for other complications associated with covid- , such as thrombosis, also have been proposed. at the time of writing, no specific treatment for covid- had been approved by the u.s. food and drug administration (fda), and most drugs sought for evaluation of efficacy are being repurposed from other clinical indications. this review will offer key clinical pharmacology considerations for developing small molecules for the treatment of covid- based on the major disease complications that impact drug absorption, distribution, metabolism, and elimination (adme). we also will address special scenarios such as changing the method of drug administration because of intubation/ventilation and hemodialysis. each stage of covid- presents unique clinical pharmacology considerations for developing covid- therapeutics. the major symptoms and clinical progression of covid- can be described within the framework of a proposed staging scheme by siddiqi et al. the first stage of early infection occurs imme-diately after inoculation and is characterized by mild, nonspecific symptoms such as fever and cough. at this stage, patients normally are treated on an outpatient basis with supportive care. antiviral therapy may be most useful at this stage to diminish the escalation of viral replication. no specific antivirals have been approved for covid- ; however, remdesivir received an emergency use authorization (eua) by the fda for the treatment of covid- in adults and children who are hospitalized with severe disease. oral formulations of chloroquine phosphate and hydroxychloroquine sulfate also received an eua for the treatment of covid- on march , , which was later revoked on june , , because of new information indicating the drugs may not be effective in treating covid- . as the viral load in the body increases, pulmonary symptoms worsen, and viral pneumonia develops. stage , or moderate disease severity, can be identified by an increase in pulmonary inflammation and usually results in hospitalization. although most people with covid- experience mild or uncomplicated illness, approximately % of patients develop severe disease, and % require admission to an intensive care unit. in the later parts of this stage, development of hypoxia can result in the need for mechanical ventilation. patients who develop severe cases of covid- can develop respiratory infections associated with acute lung injury and acute respiratory distress syndrome (ards). management of ards can require supplemental oxygen, mechanical ventilation, and endotracheal intubation. stage , the most severe form of covid- , is marked by systemic hyperinflammation and often the elevation of inflammatory cytokines. possible complications include cytokine storm, septic shock, acute kidney injury, and multiorgan failure. of major concern is sepsis, defined as "life-threatening organ dysfunction caused by a dysregulated host response to infection." in study, septic shock, which is distinguished by persistent hypotension, elevated serum lactate levels, and increased mortality, was a complication in about % of severely ill covid- patients. both sepsis and septic shock result in a complex set of physiological changes that can impact the adme of drugs. most repurposed investigational drugs that target complications associated with the severe stages of disease, such as hyperinflammation, have not previously been evaluated in patient populations that routinely are septic or critically ill. the remainder of this review will cover the physiological changes and clinical pharmacology considerations for selection of dosing in severe covid- patients and will address special dosing situations. critically ill covid- patients may have altered rates and/or extents of drug absorption through the gastrointestinal (gi) tract because of hypoperfusion, delayed gastric emptying, and use of vasopressors. in patients with sepsis or septic shock, blood is shunted away from peripheral tissues toward vital organs in response to pronounced systemic hypotension. poor blood flow to the gut combined with delayed gastric emptying can slow or allow for incomplete absorption of oral drugs. delayed absorption consequently can slow a drug's expected onset of action. vasopressors are routinely initiated in septic shock patients with hypotension that is unresponsive to fluid resuscitation. in a study of consecutive covid- patients admitted to new york hospitals, % of all patients (and % of subjects who required mechanical ventilation) received vasopressor support. vasopressors, such as norepinephrine, increase vasoconstriction to produce an increase in mean arterial pressure and improve perfusion to organs. several studies report improved splanchnic blood flow following vasopres-sor therapy. whether these changes in splanchnic blood flow will translate to meaningful increases in oral drug absorption, however, is not entirely clear in light of conflicting results from previous studies. drugs administered intramuscularly or subcutaneously also can produce unpredictable alterations in absorption because of a combination of decreased perfusion of the muscle tissue and possible vasopressor therapy. the potential for decreased or delayed absorption and variability associated with vasopressor therapy should be considered during the drug development process, especially with drugs for which the time to onset of action is of critical importance. the possibility of intravenous administration should be examined to reduce the variability associated with altered absorption of oral, intramuscular, and subcutaneous drugs. the safety margin and expected exposure following conversion to the intravenous route also should be considered when selecting doses for intravenous bolus or infusion. intravenous fluid resuscitation is a first-line therapy for the treatment of sepsis-induced hypoperfusion. infused fluids can increase the volume of distribution for water-soluble drugs, such as beta-lactams and aminoglycosides, leading to unexpectedly lower serum drug concentrations. for example, beta-lactam antibiotics administered in patients with increased volumes of distribution may not achieve therapeutic plasma concentrations, and consequently, risk treatment failure. in a pharmacokinetic study of critically-ill patients during and after fluid resuscitation therapy while receiving treatment with gentamicin, the apparent volume of distribution was % greater during initial fluid resuscitation therapy, therefore necessitating higher doses of gentamicin. therapeutic drug monitoring has been employed with antibacterial agents to address complications posed by increased volumes of distribution, although that approach likely is not feasible in studies with investigational drugs. for water-soluble investigational therapies that are intended for administration in the severely ill covid- population, thought should be given to targeting serum drug concentrations and the drug's exposure-response profile when determining if increased doses would be beneficial for patients receiving intravenous fluids. hypoperfusion of tissues in septic patients can decrease the efficacy of hydrophilic medications at the site of action. under septic conditions, reduced perfusion of peripheral tissues can be caused by an array of hemodynamic changes. reduced perfusion and therefore delivery of the drug via the blood, can impede the ability of water-soluble medications to reach the desired site of action. in such cases, systemic drug concentrations may not be indicative of the drug concentration at the site of action. higher systemic plasma concentrations of hydrophilic medications should be considered to achieve target concentrations in the targeted tissues. hyperinflammatory and septic conditions can increase or decrease acute-phase plasma protein levels, impacting drug binding, volumes of distribution, and free drug fractions. albumin and a -acid-glycoprotein (aag) are major drug-binding plasma proteins. levels of negative acute-phase proteins such as albumin decrease in conditions of inflammation, possibly leading to initial increases in free drug plasma fractions for drugs extensively bound to albumin. positive acutephase proteins such as aag and c-reactive protein increase during situations of infection or inflammation. hundreds of drugs, including propranolol, imatinib, and vinblastine, have been identified as binding to aag. increase in production of these proteins will similarly increase drug binding and decrease free drug fractions. these changes in protein levels were observed in a retrospective analysis of covid- patients in wuhan, china, where % of admitted patients had decreased albumin levels and % had increased c-reactive protein levels. the clinical impact of these potential changes in free drug fractions on investigational therapies that are highly proteinbound is an important consideration when empirically selecting doses for critically ill covid- patients. septic conditions and the development of organ dysfunction in covid- patients can affect the metabolic capacity of the liver, leading to alterations in drug exposure. drugs that are inactivated through metabolism can have prolonged exposure because of lowered enzymatic capacity of the liver and decreased hepatic blood flow in septic conditions. prodrugs that are metabolized by cytochrome p enzymes to active metabolites may conversely experience slowed or incomplete conversion, lessening the therapeutic exposure. the hepatic expression and activity of cytochrome p enzymes is decreased in situations of acute inflammation, possibly because of their direct downregulation mediated by proinflammatory cytokines. in addition, decreased hepatic blood flow reduces the clearance of drugs with high hepatic extraction ratios. use of vasopressors to increase mean arterial pressure in sepsis further potentiates the decrease in hepatic blood flow. in addition, because in most cases only unbound drug is capable of being metabolized, the changes in protein binding described previously can also alter the free drug concentration. renal filtration is the primary route of excretion for many drugs. these drugs carry a risk of accumulation in patients with renal impairment and often require dose reductions. drug exposure of investigational covid- therapies should be estimated in subjects with renal impairment based on available data to inform dosing in this population. in a survey of labels for drugs approved between and , dosing information for patients with severe renal impairment or kidney failure was only available for about % of the drugs. if a change in exposure is unknown because of lack of clinical data and cannot reliably be estimated based on the drug's elimination pathway, subjects with renal impairment may be restricted from enrollment in studies based on the severity of impairment. they may even be excluded if higher or prolonged drug concentrations pose significant safety concerns. excluding these subjects from studies, however, only addresses those with prior known renal impairment on enrollment and not those who develop impairment as part of the covid- disease course. in a study of covid- patients in new york, % developed acute kidney injury (aki). increased age and comorbidities such as diabetes mellitus and hypertension were determined to be predictors of aki. proposed mechanisms for the development of aki in covid- patients include cytokine damage and intrarenal inflammation, renal hypoperfusion, fluid expansion leading to renal vein congestion, and direct cytopathic effects of the virus on kidney cells. , sufficient monitoring plans for assessing renal function throughout the treatment period of a clinical trial are needed to detect the development of aki and implement any required dose adjustments or dose discontinuation based on the drug's toxicity profile. the standard therapies indicated for patients with aki, namely, continuous renal replacement therapy (crrt) and intermittent hemodialysis, can pose obstacles for the administration of investigational therapies. in an observational study from china, % of severe covid- patients required the use of crrt. hemodialysis removes waste, toxins, and drugs from the bloodstream by diffusion through a dialyzer membrane. drugs with low molecular weight, low volume of distribution, and high solubility carry more risk of being cleared by hemodialysis. one possible dosing strategy for these drugs is to administer the dose following the end of hemodialysis. drugs with higher molecular weight diffuse more slowly and in the case of therapeutic proteins are too large to pass through the dialyzer membrane. these drugs may be dosed irrespective of dialysis because drug exposure would not be impacted by a dialysis session. furthermore, only unbound drug fractions are capable of being dialyzed; therefore, drugs with low protein binding will undergo greater clearance from dialysis. the impact of hemodialysis on drug exposure is assessed infrequently during the drug development process, and clinical data for dosing during hemodialysis is unlikely to exist prior to the initiation of studies in covid- patients. however, if an investigational therapy is at risk of clearance from hemodialysis based on its physiochemical properties and drug binding, timing of drug administration and hemodialysis needs to be spaced appropriately. if hemodialysis must resume following drug administration, the time required for absorption and distribution of the drug to the site of action and the expected time course of pharmacologic effects must be weighed. drugs under investigation for the treatment of covid- will need to be administered on top of the standard of care at study sites. for hospitalized covid- patients, this may include antivirals, antibacterial agents, acid-reducing agents (as indicated for stress ulcer prophylaxis), deep vein thrombosis prophylaxis, and other supportive care agents such as vasopressors or sedatives. other investigational therapies such as remdesivir also may be included in the standard of care. in addition, medications for preexisting comorbidities also may require continued dosing in the hospital setting. pharmacokinetic (pk) drug interactions could impact the efficacy either of the standard of care or of the investigational drug. for example, routine use of proton pump inhibitors in intubated patients, as indicated for stress ulcer prophylaxis, increases gastric ph and can reduce the solubility of drugs that require an acidic environment for absorption. in addition to pharmacokinetic interactions, potential pharmacodynamic (pd) interactions need to be assessed. pharmacodynamic interactions can cause additive or opposing physiological effects to occur. for instance, azithromycin is a qt-prolonging drug that has been previously studied for treatment of covid- . the possibility for additive qt prolongation with investigational drugs requires assessment through adequate electrocardiogram-monitoring plans. other types of pd interactions can include competition or agonism at the target receptors through competitive and noncompetitive inhibition or allosteric modulation. additional possibilities include synergistic or antagonistic effects related to the second-messenger systems or downstream effects. a rapidly changing clinical landscape for the treatment of covid- means that drug interaction potential needs to be assessed using the most up-to-date standards of care. however, background medications or standards of care that include other investigational therapies may create difficulty when interpreting efficacy data, so their concomitant use should be carefully considered. most drugs proposed for the treatment of covid- are administered through the oral or intravenous route. given the sites of viral proliferation, however, delivery of antiviral drugs via inhalation may provide increased efficacy while reducing systemic risks. in an analysis of covid- patients with mild symptoms, high concentrations of sars-cov- rna were detected in the upper respiratory tract and lungs, as measured by pharyngeal swabs and sputum samples. live virus was isolated from the pharyngeal swabs, signifying active viral replication in the upper respiratory tract during the first week following the onset of symptoms. the virus was not detected at any time in blood or urine, and live virus was not detected in stool. drug delivery via inhalation, especially early in the disease course, could therefore confer the benefit of targeted antiviral activity at the site of viral replication, namely, the upper respiratory tract, while reducing systemic exposure. investigational antivirals such as remdesivir may potentially benefit patients in inhaled dosage forms. remdesivir is currently administered via intravenous infusion, restricting its use to hospitalized patients. other constraints for remdesivir based on renal impairment are detailed in the eua. an inhaled formulation of remdesivir could expand its availability to patients who have less severe symptoms, are early in the disease course, or are otherwise ineligible for treatment with intravenous remdesivir. lower systemic exposure after drug inhalation may also eliminate the need to exclude patients with renal impairment. commonly used inhalation devices include metereddose inhalers, dry powder inhalers, and nebulizers. in covid- patients, drug delivery by metered-dose inhalers or dry powder inhalers could be negatively affected by pulmonary symptoms that limit the force of inhalation, improper patient technique, and an inability to coordinate breaths with actuation. for these reasons, nebulizers may be the most practical choice for drug delivery via inhalation in covid- patients, as it only requires tidal breathing without coordinated inhalation. in studies of chronic obstructive pulmonary disease patients, nebulizers were found to have similar efficacy to metered-dose and dry powder inhalers when properly used. drawbacks of treatment of nebulizers include high intersubject variability, which may be because of differences in breathing pattern or nebulizer device type. in addition, nebulizers can increase the risk of sars-cov- transmission to study investigators and staff through the generation of aerosols during exhalation. developers of inhaled formulations that are intended to reach the site of action should consider the drug's individual characteristics, such as particle size. in addition, considerations in determining an appropriate inhalation device could include patients' ability to use proper inhalation technique, to effectively inhale given their progression of pulmonary symptoms, and the added risk to health care providers. progression of covid- can result in respiratory deterioration, leading to ventilator use. from a study of covid- patients, % of patients with severe disease required mechanical ventilation. mechanical ventilation and endotracheal intubation often prevent drug administration through the typical oral route. enteral feeding tubes such as nasogastric (ng) tubes must be employed for oral medications if another method or route of drug administration is not available. crushing or dissolving of solid oral dosage forms can result in issues with safety/toxicity or efficacy. increased drug absorption can put the patient at risk for toxicity-related adverse events. a common medication error involves improper crushing or dissolving of modified-release formulations, leading to dose dumping. decreased first-pass metabolism can occur when feeding tubes bypass a portion of the gastrointestinal tract and terminate in the jejunum. in this case, drugs with high first-pass metabolism, such as opioids and beta-blockers, would have increased bioavailability. decreased drug absorption, resulting in decreased efficacy, also may occur when modifying an oral drug for administration via feeding tube. enteric coatings of tablets protect the drug substance from breakdown by stomach acids, allow for absorption in the small intestine, and/or prevent gastric irritation. crushing or dissolving these tablets for tube administration removes the protective coating, likely reducing its systemic absorption and increasing the potential for gastric adverse events. in addition, enteral feeding solutions increase the gastric ph, which can reduce the absorption of drugs with ph-dependent solubility. small-bore feeding tubes are prone to obstruction or clogging by powdered tablets or capsules and also can result in decreased drug absorption. adsorption or adherence of the drug to the feeding tube also can reduce the administered dose. flushing feeding tube lines before and after drug administration may prevent clogging, but study protocols need to provide detailed instructions on volume and timing to ensure consistency in administration. feeding tubes can terminate in the stomach, duodenum, or jejunum. their placement in the jejunum can decrease overall absorption time in the gi tract or impact the absorption of drugs that require an acidic gastric environment. investigational study protocols for covid- oral therapies should assess alternative routes or methods of administration if medications cannot be taken by mouth. if administration via feeding tube is proposed, detailed instructions to ensure consistent drug administration should be established. dosing plans need to be based on the physiochemical properties of the drug (eg, solubility), site of absorption, and formulation. severe cases of covid- are associated with numerous complications and physiological changes that have the potential to alter investigational drug adme. when investigating new therapies to treat covid- , these alterations need to be evaluated carefully to determine initial dosing in covid- patients, as well as the need for dose modifications or drug discontinuation. the average time from covid- symptom onset to death is estimated to be days. most therapeutic agents are proposed for investigation in moderate to severe covid- patients, leaving a limited window of time to determine appropriate dosing based on the most impactful physiological alterations in a patient. assessing potential drug interactions in covid- patients is an especially arduous task, given rapidly evolving therapeutic knowledge and treatments. concomitant administration of other investigational therapies may create difficulty in interpreting efficacy data; therefore, permitted and prohibited background therapies should be selected carefully based on potential pk and pd interactions with the investigational drug. existing population pharmacokinetic models can be used to simulate exposure of new doses and/or regimens and support initial dosing. however, many drugs proposed for evaluation in covid- patients are being repurposed from indications in which the patient population is not critically ill. some information can be extrapolated from other patient populations for empiric dosing, but blood sample collection for analysis of pk and pd end points in covid- patients is critical to characterizing the drug exposure in that population and optimizing dosing in future studies. a joint statement from major pharmacologic societies also describes this need to measure drug concentrations to develop pk models in covid- patients as part of a core clinical pharmacology principle guiding development of covid- treatments. numerous drugs have been proposed to treat covid- , addressing both complications and direct antiviral activity. the many clinical pharmacology considerations discussed in this review, such as alterations in absorption and distribution, relate to initial dose selection in the covid- population. other issues, such as the development of aki or ng tube administration of drugs, need to be specified in proposed protocols for investigational drugs. the fda's office of clinical pharmacology has frequently commented on certain protocol areas that need to be addressed, such as the lack of a dosing plan for hemodialysis. although by no means all-encompassing, following are samples of our common advice regarding investigational protocols: • "the development of acute kidney injury requiring hemodialysis has been reported as a possible complication during the disease progression of covid- . address how the dose and regimen will be modified in the event that patients require hemodialysis (eg, dosing after hemodialysis, treatment discontinuation)." • "conditions permitting, you should collect blood samples for pk assessment in the proposed study in order to characterize pk in the covid- population and better inform dose selection in future studies." • "detailed dosing instructions for the scenario that patients develop the need for intubation should be included in the protocol." • "all likely concomitant medications or medications administered as part of the standard of care should be assessed for drug interaction potential with the investigational drug. dose adjustments or appropriate monitoring plans should be based on the known drug interaction potential and toxicity profile of the drug." the totality of evidence should be considered for inclusion of patients with compromised organ impairment and other comorbidities. hospitalization, criticalillness, and mortality are higher in patients with various comorbidities. most notably, diabetes, obesity, older age, and hypertension are the risk factors most strongly associated with covid- hospitalization. exclusion of these subjects from clinical trials may delay availability of drugs to vulnerable populations. reports from new york city and chicago also show that black and latino covid- patients have death rates that are to times higher than white patients. , current fda recommendations propose including subjects with high-risk comorbidities, older adults, and racial and ethnic minorities in clinical trials. existing clinical and nonclinical adme data along with the safety profile of the drug should be leveraged to select populations for inclusion. a suitable approach for inclusion of subjects with organ impairment or other risk factors must be determined on a case-by-case basis. one possible method is to include these patients in a stepwise manner with appropriate stopping criteria for adverse events or toxicity. early collection of pk/pd data in these patients could then inform dosing in more severe patients. the nature and severity of anticipated safety events, whether they are monitorable, and whether dose reductions are possible also need to be weighed in the decision-making process. covid- has proven to be a unique drug development scenario because the rapid spread of severe illness has resulted in an urgent compression of the drug development timeline. clinical pharmacologists now have an added responsibility to make rapid, informed decisions, often based only on preliminary or incomplete knowledge of the drug and pathophysiology. however, this scenario also creates an opportunity for the clinical pharmacology community to positively influence the strategic, expedited development of drugs that are vital to combating a public health crisis. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- who-director-general-s-opening-remarks-at-the-mediabriefing-on-covid mild or moderate covid- neurologic manifestations of hospitalized patients with coronavirus disease in wuhan, china large-vessel stroke as a presenting feature of covid- in the young the trinity of covid- : immunity, inflammation and intervention covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal emergency use authorization (eua) for remdesivir. silver spring, md: u.s. food and drug administration letter revoking eua for chloroquine phosphate and hydroxychloroquine sulfate. silver spring, md: u.s. food and drug administration clinical characteristics of coronavirus disease in china the third international consensus definitions for sepsis and septic shock (sepsis- ) introduction to drug pharmacokinetics in the critically ill patient clinical characteristics of covid- in new york city pharmacokinetic and pharmacodynamic considerations when treating patients with sepsis and septic shock pharmacokinetics in sepsis does beta-lactam pharmacokinetic variability in critically ill patients justify therapeutic drug monitoring? a systematic review gentamicin volume of distribution in critically ill septic patients antimicrobial pharmacokinetic and pharmacodynamic issues in the critically ill with severe sepsis and septic shock effect of alpha- -acid glycoprotein binding on pharmacokinetics and pharmacodynamics epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study impact of infectious and inflammatory disease on cytochrome p -mediated drug metabolism and pharmacokinetics the impact of renal impairment on patient drug response -assessing the need for a consensus approach acute kidney injury in patients hospitalized with covid- kidney involvement in covid- and rationale for extracorporeal therapies covid- and kidney failure in the acute care setting: our experience from seattle pharmacokinetic principles during continuous renal replacement therapy: drugs and dosage assessment of the impact of renal impairment on systemic exposure of new molecular entities: evaluation of recent new drug applications hydroxychloroquine as an aerosol might markedly reduce and even prevent severe clinical symptoms after sars-cov- infection virological assessment of hospitalized patients with covid- working to supply remdesivir for covid- inhalation drug delivery devices: technology update a review of nebulized drug delivery in copd determination of the relative bioavailability of salbutamol to the lungs and systemic circulation following nebulization practical strategies for a safe and effective delivery of aerosolized medications to patients with covid- a guide to drug therapy in patients with enteral feeding tubes: dosage form selection and administration methods therapeutic concerns when oral medications are administered nasogastrically clogged feeding tubes: a clinician's thorn medication administration through enteral feeding tubes estimates of the severity of coronavirus disease : a model-based analysis a call for the appropriate application of clinical pharmacological principles in the search for safe and efficacious covid- (sars-cov- ) treatments factors associated with hospital admission and critical illness among people with coronavirus disease rates of cases, hospitalizations and deaths by race/ethnicity group covid- : developing drugs and biological products for treatment or prevention guidance for industry. silver spring, md: u.s. food and drug administration the authors thank joanne berger, fda library, and karen valentine, fda center for devices and radiological health, for manuscript editing assistance. the authors have declared no conflicts of interest for this article. key: cord- -kuwargnp authors: opatz, till; senn‐bilfinger, joerg; richert, clemens title: thoughts on what chemists can contribute to fighting sars‐cov‐ – a short note on hand sanitizers, drug candidates and outreach date: - - journal: angew chem int ed engl doi: . /anie. sha: doc_id: cord_uid: kuwargnp the sars‐cov‐ outbreak causing the respiratory disease covid‐ has left many chemists in academia without an obvious option to contribute to fighting the pandemic. some of our recent experiences indicate that there are ways to overcome this dilemma. a three‐pronged approach is proposed. thei nterruption of infection chains through appropriate hand sanitization was pioneered in by the hungarian physician semmelweis ignµcf ülçp (ignaz philipp semmelweis) and became what is probably the first example of evidence-based medicine. [ ] isolation or quarantine,s ocial distancing and physical barriers like masks,a re additional measures to slow the transmission of respiratory viruses. other measures,l ike prophylactic coating of airways with polymers,a re under development. [ ] frequent handwashing proved effective against the spread of the first severe acute respiratory syndrome pandemic in / [ ] and of the middle east respiratory syndrome (mers) in . [ ] however,t his hygienic measure is not easy to implement for individuals with frequent customer or patient contact or travelers.h and sanitizer solutions or gels are recommended as an alternative in these cases.a mong the most popular sanitizers are alcoholic hand rub solutions. while non-enveloped viruses like norovirus or rhinoviruses have al ow sensitivity towards alcoholic denaturing agents,members of the (enveloped) coronaviridae family are highly sensitive towards ethanol or isopropanol ( -propanol) . chlorhexidine gluconate and sodium hypochlorite were found to be surprisingly inefficient. [ ] repeated use of alcohols dries out the skin, and small quantities of the alcohols may be absorbed, but they are effective. [ ] the literature on the inactivation of previously known coronaviruses by surface disinfection procedures suggested - % ethanol to be effective against sars-cov- within min. [ ] experimental work by several laboratories on the virus itself then showed that it is even less stable toward denaturation by alcoholic solutions. [ ] exposure to concentrations of just % of either ethanol or isopropanol for seconds fully suppressed viral infectivity.likewise,the virucidal activity of the hand rub solutions known as who formulation , with % ethanol, and who formulation , with %i sopropanol, against sars-cov- was found to be excellent, with full inactivation of the coronavirus at %or %concentration, respectively.w hile the alcohol component is the main virucide, . % v/v h o is added to kill bacterial spores that may be present in the raw materials or the container.the addition of . % v/v glycerol as ah umectant improves the dermatological properties and thus the acceptance of the product. [ ] fors urface disinfection, the same formulation may be used, with no additives required, and surgical hand preparation may be achieved by three applications for aduration of - min. unfortunately,c ommercial sanitizer solutions containing either alcohol can be difficult to obtain in the current health crisis.w ea re unaware of data for other countries,b ut data published by the german federal statistical office (destatis) show the severity of the problem. during the week of march - , , sales of disinfectants were . -fold higher than usual, and in the subsequent weeks,they dropped to half the normal sales,a st he products were largely sold out. [ ] by comparison, the increase in sales of soap reached ap eak of . -fold of the average and that of toilet paper just . -fold of the sales averaged over the preceding six months. ethanol is produced on al arge scale on our planet. the u.s. renewable fuels association reported that million metric tons were produced in worldwide. [ ] thee uropean renewable ethanol association (epure)stated that the production of the european union in the same year was . million metric tons,while an additional . million tons were imported. [ ] besides minor usage by the chemical industry and food and beverages production ( %e ach), the lions share ( %) of the production was used as af uel additive. against the backdrop of reduced individual mobility during the crisis in most countries,i ta ppears likely that enough (bio)ethanol should currently be available for the health sector. several factors appeared to contribute to the supply problems.a sm entioned above,o ne factor was hoarding by consumers who bought unreasonable quantities of sanitizers while they were still available,q uickly depleting what pharmacies and drug stores had in stock. furthermore,t he supply chain was partially disrupted. while truckloads of ethanol were available in europe,t he logistics of getting trucks to sites that can fill ld rums became more challenging due to closed or tightly controlled borders. drums of this size then had to be transported to facilities that can either produce sanitizers or fill the smaller containers commonly used by pharmacists.f urthermore,t he containers used in medical practices,a sw ell as the dispensers used by physicians,health care workers,and consumers,became ever more difficult to obtain, prompting unconventional measures, such as refills. on march , the bundesanstalt füra rbeitsschutz und arbeitsmedizin (baua), [ ] the relevant regulatory body in germany,issued adecree that allowed pharmacies to produce hand sanitizer solutions locally.t he decree and its update of march c onfirmed that aqueous solutions of ethanol do not require regulatory approval to be produced for sale as biocidical agents.t he first decree also explicitly mentioned aqueous -propanol ( %) and who formulation a s biocidal agents for hand hygiene. unfortunately,medical grade alcohol as astarting material for the local production of disinfectant solutions,including hand rub formulations,was largely sold out in germany in the second week of march. theu sual suppliers of pharmacists typically gave delivery times of several weeks at the peak of the supply crunch. this left technical grade ethanol or isopropanol of unknown purity as the only available starting material for the preparation of hand rub formulations. without analysis,t his source was considered problematic by many.s olving the analytical problem is where organic chemists in academia were able to contribute. theb auad ecree mentions ap urity of %( w/w)o r . %( v/v)f or isopropanol to be in accordance with eu law.t he who guideline states that analysis should be made when an analysis certificate is not available to verify the alcohol concentration and to adjust the volume used to obtain the final recommended concentration. what local pharmacists use for quality control and what the who guideline mentions is an alcoholmeter that measures the density of the liquid, but does not provide information on its chemical composition. gas chromatography is mentioned in the who guideline for higher level quality control,b ut this option is largely unavailable to pharmacists in the field. we and others then analyzed technical grade isopropanol and ethanol by hn mr spectroscopy.a % solution of either alcohol in cdcl gave well-resolved spectra with excellent signal-to-noise ratio,s howing minimal levels of impurities.for isopropanol, acetone was detected as the most prominent trace impurity by spiking with authentic material. due to its low toxicity and its traditional use in nail polish remover,t his impurity was considered unproblematic for hand rubs,a nd the purity of our technical grade isopropanol was declared sufficient for producing hand sanitizer locally together with ac ollaborating pharmacist. as ac onsequence, substantial supplies of the alcohol could be freed up for local production quickly.a so fm id april, over m etric tons of who formulation have been delivered to physicians in the state of baden-württemberg. another area where chemists may contribute is the development of antiviral drugs.s mall molecule drugs have been game changers in the fight against other viruses.among them are azt (zdv, zidovudin, retrovir), [ ] an ucleoside reverse transcriptase inhibitor and the first compound approved to treat aids.a nother break-through antiviral drug is sofosbuvir ( ,trade name sovaldi)shown in figure , [ ] an inhibitor of the rna-dependent rnap olymerase of hepatitis cvirus (hcv) that is able to cure patients of the vicious disease caused by this virus.l ike sars-cov- , hcv is an enveloped, single-stranded, positive-sense rnavirus. while sars-cov- is commonly described as a" novel" virus,genetically,itisclosely related to the sars coronavirus (sars-cov or sars-cov- ). [ ] ther na-dependent rna polymerase (rdrp) is %h omologous for the two viruses. this polymerase is ap rime target for antiviral therapy,a s human cells do not contain such an enzyme that copies rna sequences into rna. tw oo ther drug targets are the main proteinase ( cl pro )a nd the spike protein of the viral envelope,w ith am uch lower level of sequence identity.a review of this and the avenues it offers for drug intervention can be found in arecent paper by liu and colleagues. [ ] in this context, it is worth mentioning that opportunities for therapy were identified for sars-cov- more than years ago. [ ] [ ] [ ] another betacoronavirus that is related to sars-cov- is the virus causing mers.again, this virus has been studied in detail, and options for therapeutic intervention have been identified. [ , ] successful rnap olymerase inhibitors,s uch as sofosbuvir, [ ] work by incorporating modified nucleosides into the viral genome that halt viral replication. this approach has been very successful in the treatment of hepatitis c, which can be cured within ashort treatment period. thedrug sofosbuvir is ap hosphoramidate prodrug or protide,atype of compound pioneered by chris mcguigan. [ ] it is metabolized in the liver in several steps into its active triphosphate form, which is recognized and incorporated as an analog of uridine- '-triphosphate by the hepatitis-c virus polymerase.o ne of the frontrunner drug candidates for the treatment of covid- , remdesivir ( ), is as imilar prodrug construct. it is a cnucleoside mimic of adenosine monophosphate [ ] in protide phenyl phosphoramidate form. remdesivir was developed by gilead sciences to combat ebola and related filoviruses,b ut did not have sufficient activity against these targets.in , it was shown in vitro and in animals that it can inhibit mers-cov and sars-cov- replication. [ ] in the replication processes,remdesivir, in its triphosphate form, competes with its natural counterpart, adenosine-triphosphate (atp), inducing rnac hain termination. based on the high sequence similarity between sars-cov- and the causal agent of the covid- pandemic, homology modeling was used to predict the d structures of the rdrp and other important viral proteins of the latter.out of the more than viral proteins encoded in the sars-cov- genome,p roteases cl pro ,m entioned above, and pl pro represent potential targets for antiviral drugs.t he -chymotrypsin-like protease ( cl pro )ofsars-cov- differs in only out its amino acid residues from its ortholog in sars-cov- . its structure was quickly predicted in silico and this was used for the virtual docking of ac ollection of commercial drugs in the search for candidates for drug repurposing. [ ] among the top-scoring candidates,f or which the highest binding affinities were predicted, were the two antivirals velpatasvir and ledipasvir. both compounds are fda-approved inhibitors of the ns a protein of the hepatitis cv irus,b ut so far, the predicted action against the sars-cov- cl pro has not been confirmed experimentally.a similar approach on the same enzyme was employed by another team, and led to different candidate structures, [ ] while ateam from china and saudi arabia chose to identify phytochemicals as potential inhibitors in similar fashion using in silico methods. [ ] while modeling is fast, it does not produce an ew drug, and experimental work is needed to validate those predictions.arecent crystal structure of cl pro ,a lso referred to as m pro ,c omplexed with an aketoamide inhibitor shows how quickly encouraging results can be obtained experimentally. [ ] ac rystal structure of the rdrp of sars-cov- has also been published in the last few days. [ ] one of the most detailed theoretical studies thus far used homology modeling on different sars-cov-proteins and on the human ace receptor protein, the putative molecular entry vehicle for the virus into human cells,f or virtual drug screening. [ ] fore ach viral protein and for the ace receptor,s everal drugs or natural products were identified as potential binders.n otably,r emdesivir was also found to bind to the rdrp enzyme of sars-cov- in silico.e xperimentally,activity against sars-cov- was also demonstrated in vitro. [ ] in the same experimental study,a ntiviral activity was found for the antimalarial drug chloroquine, which is unlikely to act on either rdrp or proteases. chloroquine and hydroxychloroquine [ ] act on several targets and can have severe side effects.t heir anti-inflammatory effect may reduce the level of pro-inflammatory cytokine il- , mobilized by the immune system of covid- patients in order to kill infected cells before too many copies of the virus are made.s ometimes this defense mechanism overreacts, resulting in al ife-threatening condition. [ ] both remdesivir and chloroquine,are currently among the compounds undergoing clinical trials with covid- patients. thes ynthesis of compounds like remdesivir is far from trivial, though, not least because the amino acid ester and aryloxy group make the phosphorus of the phosphoramidate astereogenic center.synthesizing chiral phosphoramidates of this level of complexity is as ynthetic challenge, [ ] and so is the synthesis of other drug candidates for treating the virus that will be identified by homology modeling and targetbased virtual ligand screening or conventional medicinal chemistry approaches.developing efficient syntheses for such compounds and making them available to laboratories that test them is as econd opportunity for chemists who wish to contribute to fighting sars-cov- . there is at hird opportunity for chemists in the current crisis:r eaching out to others through our scientific societies. when av irus spreads and employees stay home,r unning an efficient operation becomes difficult. as soon as an employee gets infected and obtains apositive test result, entire divisions may be shut down to prevent the spreading of the virus. colleagues in quarantine must be provided with food and other supplies,a nd homeschooling ties up significant resour- angewandte chemie viewpoint ces.a saconsequence,m any members of our societies are experiencing unprecedented levels of stress or are too overwhelmed by emails and calls to remain fully operational. this problem affects the supply chain from drivers to executives, but it is not limited to logistics and production. it also affects authorities.t he apparent suicide of dr. thomas schäfer,the minister of finance of the german state of hesse,i satragic example of what can happen to those put under ever increasing pressure in the current pandemic.t rying to spread useful information through governmental channels can thus add to the stress level of administrators. we feel that the network our scientific societies,s uch as the german chemical society (gdch), offer can and should be used to reach out to each other.social media can be very useful, but are also the source of unfiltered anger and accusations.emails to aselect group of colleagues,such as the members of the liebig-vereinigung, the organic division of the gdch, well maintained and checked by the officers of the society,can be away to disseminate information that does not suffer from said disadvantages.a nother option is posting useful information on the newly installed corona webpage of the gdch. [ ] chemists have contributed very significantly already,n ot least by synthesizing the necessary amounts of primers and dntp'sfor test kits,but there are countless challenges that lie ahead. broader testing for viral infections, [ ] as well as serological testing to confirm immunity,atask that proved difficult, even for the relatively confined mers outbreak, [ , ] is just one of them. improving the social acceptance of face masks in some western countries, [ ] and producing such masks in sufficient quantities,i sanother. whether it is instructions for producing buffers required for test kits,o rs imple face masks for protection against infectious droplets,u seful information can be shared. the same is true for funding opportunities or legal issues that affect society members.chemists are resourceful people,and scientific societies are exceptionally experienced in sorting through information submitted to their journals.h ere lies athird opportunity for chemists to contribute. when we almost ran out of isopropanol for our hand sanitizer solutions,anon-public email call led to acompany in the area offering larger quantities of the solvent. the company was willing to offer it well below its current inflated market price,and our university was willing to support us with logistics and the free use of facilities.atthe end of abusy day, one of the clinical pharmacologists who helped with the local production of who formulation l ooked at our less than attractive facility and said:"there is still an important role for good old chemistry in our society." progress in molecular and environmental bioengineering renewable fuels association proc.natl. acad. sci -x. manuscript received:m arch key: cord- -y puao authors: scherrmann, jm. title: intracellular abcb as a possible mechanism to explain the synergistic effect of hydroxychloroquine-azithromycin combination in covid- therapy date: - - journal: aaps j doi: . /s - - -w sha: doc_id: cord_uid: y puao the co-administration of hydroxychloroquine with azithromycin is proposed in covid- therapy. we hypothesize a new mechanism supporting the synergistic interaction between these drugs. azithromycin is a substrate of abcb (p-glycoprotein) which is localized in endosomes and lysosomes with a polarized substrate transport from the cell cytosol into the vesicle interior. sars-cov- and drugs meet in these acidic organelles and both basic drugs, which are potent lysosomotropic compounds, will become protonated and trapped within these vesicles. consequently, their intra-vesicular concentrations can attain low micromolar effective cytotoxic concentrations on sars-cov- while concomitantly increase the intra-vesicular ph up to around neutrality. this last effect inhibits lysosomal enzyme activities responsible in virus entry and replication cycle. based on these considerations, we hypothesize that abcb could be a possible enhancer by confining azithromycin more extensively than expected when the trapping is solely dependent on the passive diffusion. this additional mechanism may therefore explain the synergistic effect when azithromycin is added to hydroxychloroquine, leading to apparently more rapid virus clearance and better clinical benefit, when compared to monotherapy with hydroxychloroquine alone. the co-administration of the antimalarial chloroquine or the anti-rheumatic hydroxychloroquine with the antibacterial azithromycin is presently proposed as a therapy amidst the covid- pandemic ( ). these aminoquinoline drugs and azithromycin are being repurposed for their clinical use in covid- , and they should be evaluated according to current practices of drug clinical trials for this indication. although this process is normally time-consuming, population, media, and political pressures have propelled the use of this combined therapy on an emergency basis, thus opening controversial pro and con debates. moreover, most of the different aminoquinoline clinical trials over the world are not comparable as they differ in terms of mono or bi-therapy choice, dosage regimen, and optimal time of administration versus the disease time course and severity. several chinese clinical trials have been based on the sole chloroquine or hydroxychloroquine administration and their clinical efficacies remain uncertain ( ) ( ) ( ) . additionally, most of these methodological protocols suffer from major concerns in term of trial design quality such as inappropriateness in randomization, patient population size, and other limiting factors ( , ) . the purpose of this commentary is to discuss the possible rationale for co-administering the antibiotic azithromycin with hydroxychloroquine, recognizing that azithromycin is not being used for its antibacterial activity but for its additive or synergistic effect on the antiviral action of the aminoquinolines ( ) . moreover, several in vitro and in vivo studies support additional antiviral properties of azithromycin although the drug is not approved for the treatment of viral infections ( ) . here, we provide comments and hypotheses to develop a rationale for this drug combination based upon sub-cellular pharmacokinetic and pharmacodynamic arguments. two recent works have detailed the targets and cellular pathways supporting the antiviral activity of chloroquine and hydroxychloroquine ( , ) . in fact, the attempt to combine chloroquine and azithromycin started with the observation of resistance of plasmodium falciparum to chloroquine. gingras and jensen were the first in to demonstrate in vitro that the association of azithromycin to chloroquine reversed the resistance of chloroquine-resistant strains of p. falciparum ( ) . subsequent in vivo and clinical studies have validated this dual administration in malaria chemotherapy ( , ) . an inverted association was also found effective to kill intracellular bacteria, such as staphylococcus aureus when chloroquine was used to synergize the effect of the antibiotic azithromycin ( ) . moreover, this drug combination was also extended to virus diseases with many clinical studies but most of them were moderately conclusive ( , ) . nevertheless, for each recent and new virus infections, the chloroquineazithromycin combination strategy reappears as a possible miraculous solution such as in ebola, sars and mers, hiv diseases, and now with sars-cov- in covid- ( ) ( ) ( ) ( ) . in this last indication, hydroxychloroquine, mostly used for treating severe rheumatic diseases, is preferred to chloroquine as more effective in vitro and clinically safer than chloroquine ( ) . moreover, a recent physiologically based pharmacokinetic (pbpk) model, combining in vitro data with published drug pharmacokinetics, was interestingly developed to predict the optimal dosage regimen of hydroxychloroquine in covid- ( ) . another recent mechanistic pk modeling study including efficacy with historical data on viral replication and the cardiotoxicity risk of qtc interval prolongation attempted to predict the highest hydroxychloroquine doses which are needed to achieve both safety and cure within days ( ) . why could this couple of antimalarial and antibiotic drugs be efficient for such a broad number of infectious diseases caused by so various pathogens such as parasites and intracellular bacteria or viruses? that is the fundamental question. besides their anti-infectious properties, the aminoquinoline compounds are immunomodulators that could counteract the cytokine storm observed in the severe form of covid- ( , , ) . also, they are known to stop virus replication as in most of the clinical trials with both aminoquinolines; the virus load is considered as a critical biomarker endpoint ( ) . this direct effect on virus replication seems to be dependent on the fact that these drugs and viruses share common sub-cellular organelles trafficking routes involving either the trans-golgi secretory pathway or the endosomal/lysosomal endocytotic pathway. on the one hand, the virus uses endosome formation as a cellular entry mechanism. thus, following the binding of the cov- spike protein to the angiotensin-converting enzyme (ace ) virus-receptor, sars-cov- travels inside the vesicular network including endosomes and lysosomes. on the other hand, several processes occur within the organellar lumen, like the glycosylation of cov- proteins during their biogenesis and assembly as well as the action of several endosomal proteases able to cleave cov- spike proteins for virus entry ( , , , ) . therefore, all these life-virus steps are dependent on the remarkable intra-organelle acidic conditions, with ph around - . a vacuolar-type proton adenosine triphosphatase membrane (v-atpase), pumping protons towards the vesicle interior, contributes to maintain this optimal ph medium for nearly different hydrolytic lysosomal enzymes, such as some acid proteases which are implicated in the virus replication cycle ( ) (fig. ) . in fact, viruses and drugs together meet in this common biophase of these sub-cellular organelles during their respective time-dependent distribution. these drugs share similar pharmacokinetic profiles with extensive tissue distributions characterized by elevated volumes of distribution, moderate plasma protein binding, and high intracellular distribution with tropism for lysosome-rich organs, such as liver and lung. they also exhibit low total body clearances which contribute to long body exposure ( ) ( ) ( ) . more interestingly, chloroquine, hydroxychloroquine, and azithromycin have also been described as potent lysosomotropic compounds as they are diprotic weak bases with pka at . - . , . - . , and . - . , respectively ( , , ) . these physicochemical criteria associated with elevated log p, ranged from . to . , meet those defining a subgroup of lysosomotropic amines, known as cationic amphiphilic drugs ( ) . thus, they will become protonated and trapped within the endosomal vesicles under these acidic conditions and their backward diffusion into the cytosol was hindered ( , ) . consequently, their intravesicular concentrations can reach hundredfold that of the cytosolic concentrations and attain or even surpass the low micromolar effective concentrations that are able to inhibit sars-cov- virus cycle ( , , ) . concomitantly, the drug-induced lysosomotropic effect is completed by buffering the intravesicular acidity with ph increasing up to around neutrality. this last effect on the luminal ph is the most critical on virus development by decreasing several lysosomal enzyme activities responsible for either glycosylation of both ace receptor and cov- proteins or cleavage of cov- spike proteins. presently, this intracellular trapping mechanism is largely reported for sustaining the additive or synergistic effect on various pathogens following this drug co-administration ( , , ) . nevertheless, i believe that a supplementary mechanistic explanation may be missing in the literature for supporting the additive or synergistic interaction when these drugs are combined. the first reason is that azithromycin has been shown, both in vitro and in vivo, to be a substrate of abcb (p-glycoprotein) ( ) ( ) ( ) ( ) ( ) ( ) . abcb , a member of the atpbinding cassette (abc) transporter superfamily, is mainly known as being expressed on the cellular plasma membrane in various tissues and can limit the cellular uptake of a large number of drug substrates like azithromycin ( ) . a second reason is linked to the abcb localization in intracellular compartments, such as the endoplasmic reticulum and golgi (site of abcb synthesis), the endosomes for abcb trafficking and recycling, and lysosomes for its degradation ( , ) . finally, the abcb substrate transport direction on endosome and lysosome membranes is a very critical property. the polarized transport direction occurs from the cell cytosol, where the neutral ph allows the atp catalytic cycle of abcb , into the interior of the vesicle. this transport direction is the opposite from that mediated at the plasma membrane surface (fig. ) . based on these overall information, we might hypothesize that abcb could play a possible role for the additive or most likely, the synergistic effect of azithromycin on hydroxychloroquine. at this stage, the question on how the abcb -dependent synergy might occur is still open. based on known pharmacological modulations mediated by abcb , we could imagine two possible scenarios. the first one is related to the inverted influx transport of abcb , meaning that the azithromycin molecules, in addition to their passive diffusional uptake, are actively captured and trapped inside the vesicles, even if the vesicular intraluminal ph increases and the percentage of protonic drug molecules decreases enabling the backward diffusion of neutral species to the cytosol (fig. ) . therefore, this could represent an explanation for the synergistic effect when azithromycin is added to hydroxychloroquine or chloroquine. at all these intracellular stages, abcb can be a possible enhancer by confining the substrate azithromycin more extensively as expected when the trapping mechanism is solely limited to the protonated-diffusion explanation. this first hypothesis could be supported by results obtained from a clinical study where increasing amounts of azithromycin (from mg to g) combined with a fixed dose of mg chloroquine showed a clear dose-response relationship with the maximum clearance rates of p. falciparum observed with the highest dose of azithromycin ( ) . moreover, in vitro experiments using vero e cells infected with sars-cov- virus have also reported concentration and time-dependent effects of hydroxychloroquine on the virus cytotoxic ec values, which decreased with longer incubation times ( , ) . this suggests that time is required for drug accumulation or/ and drug cytotoxicity kinetics. these observations could be supported by the long-lasting abcb -mediated exposure effect of azithromycin. this azithromycin trapping effect induced by abcb could also be extended to the aminoquinoline compounds to the extent that they could interact with abcb . this could most likely occur with chloroquine which was classified in several vesicular and cellular in vitro models as a moderate inhibitor and a nons u b s t r a t e o f a b c b ( - ) . u n f o r t u n a t e l y, hydroxychloroquine has been demonstrated neither as a substrate nor as inhibitor of abcb with the usual in vitro models. nevertheless, this aminoquinoline has been reported in human pharmacokinetics to interact with digoxin and nelfinavir, two known abcb substrates, by moderately increasing their oral bioavailability ( , ) . thus, all these data suggest that hydroxychloroquine and chloroquine could have a moderate inhibition effect on the azithromycin abcb -mediated intravesicular influx. they also point to the need to further investigate the inhibitor/substrate status of hydroxychloroquine towards abcb . additionally, it is noteworthy that the antibiotic and fluoroquinolone ciprofloxacin which is also both a lysosomotropic compound with pka . and substrate of abcb behaves like azithromycin to synergize the anti-infectious effect of chloroquine ( ) . this last observation appears to demonstrate that the lysosomotropic drug property is certainly a key factor in the synergistic effect of this drug combination. the second scenario could be similar to the well known reversal of multidrug resistance, the mdr effect, observed with anticancer drugs such as doxorubicin or daunomycin which are also actively trapped inside lysosomes under abcb active influx. the administration of abcb inhibitors, like valspodar or elacridar, tends to displace the anthracyclines from the lysosomal lumen into the cytosol making doxorubicin available for its nucleus targets ( ) . the translation of this mdr reversal to azithromycin might suggest its ability to inhibit abcb , and therefore to increase aminoquinoline cytosolic concentrations which could be active for other biological targets and cellular pathways impacting the virus development ( , , ) . therefore, this second scenario seems unlikely as hydroxychloroquine and chloroquine are not substrates of abcb . the diagram shows how the unionized drugs can readily diffuse across lysosomal membranes. due to the acidic environment of the lysosome, the equilibrium between charged and uncharged drug species shifts in favor of the ionized species, thus limiting their backward diffusion into the cytosol. as a substrate of abcb , the active uptake of azithromycin contributes to the enhancement of this trapping effect and to the neutralization of the acidic ph. this last effect contributes to a cascade of antiinflammatory and antiviral activities nevertheless, this drug release from the vesicle lumen to the cytosol could proceed from the deleterious effects of these cationic amphiphilic drugs on lysosomal viability. as a result of their over-accumulation in the endolysosomal lumen which could be exacerbated by the azithromycin effect on abcb , they could permeabilize the lysosomal membranes to protons, cl − and water causing enlargement of the lysosomes and phospholipidosis ( ) . interestingly, azithromycin, chloroquine, and hydroxychloroquine have been all reported as responsible of these types of endolysosomal damages which can affect vesicular trafficking by inhibiting the surface expression of receptors, the fusion to autophagosomes, and lipid metabolism which indirectly contributes to the inhibition of the cytokine production. all these impairments could contribute to the panel of cytotoxic mechanisms affecting the sars-cov- replication cycle as well as the risks of organtoxicity like cardiomyopathy following this drug combination ( , , ) . they also highlight a critical need for optimal dosing of both drugs using pbpk and pkpd modelings to achieve both safety and efficacy ( , ) . to sum up, the intracellular abcb could represent a potent target for enhancing the antiviral and antiinflammatory activities of the aminoquinolines when lysosomotropic abcb substrates like azithromycin or ciprofloxacin are combined. this could explain why the association strategy leads to apparent rapid virus clearance and better clinical benefit vs. aminoquinoline use alone ( ). moreover, these observations justify the bi-therapy administration in the early stage of the disease or for prophylactic use, i.e., when the virus distributes within the disease target cells, such as the pulmonary epithelial cells. experimental assessments of this hypothetical abcb role could be easily investigated using both in vitro cellular models currently used with sars-cov- and widely developed for abcb transport ( , , , ) . moreover, the reported co-localization of abcb with other abcs on the lysosomal membranes, such as abcg , abcc , and abcc could also expand this experimental field of investigation as to their possible involvement in the activity of these lysosomotropic drugs ( ) . by taking into consideration this possible role of abcb in the synergistic combination of these two drugs, the question of the modulation of abcb transport could be raised for preventing risks of a lysosomotropic effect decrease on virus dynamics and of interindividual variability in the clinical use of this drug combination and with possible additional drug co-therapy. these risks should be considered at both the intracellular level and at the drug disposition level where abcb has been extensively reported as mediating drug-drug interactions (ddi) ( ) . first, the intracellular ddi risk can be anticipated from previous studies showing that lysosomal abcb can be a ddi target ( , ) . in addition to the previous mdr reversal example, abcb mediated lysosomal competition between two weak bases has been elegantly shown in a study using positron emission tomography imaging where the radiotracer [ c] ndesmethyl-loperamide was displaced from lysosomes by tariquidar and other abcb competitors like cyclosporine or verapamil ( ) . finally, current pharmacokinetic considerations indicate that these drugs are characterized by a low safety margin and by cardiotoxicity risks ( , ) . therefore, risks of inter and intra-individual variabilities have to be considered. presently, no systemic pharmacokinetic interactions have been observed between chloroquine and azithromycin ( ) . azithromycin is known for interindividual variabilities in drug response and among ethnic groups and could be a ddi perpetrator candidate. azithromycin is not metabolized by cytochromes p and not an inducer/inhibitor of these hepatic enzymes. the dominant azithromycin excretion pathway occurs at the biliary and intestinal levels via abcb and abcc (mrp ) active transport which could represent ddi targets ( ) . however, a few and insignificant clinical ddis have been observed between azithromycin and co-prescribed drugs ( ) . lastly, the most intriguing interpopulation variability could result from the azithromycin sensitivity to abcb genetic polymorphism. two clinical studies have described that heterozygous and mutant abcb genes in chinese han and pakistani subjects have decreased rate and extent of azithromycin absorption when compared to the wild-type subjects ( , ) . this suggests that the abcb -dependent effectiveness of azithromycin at the intracellular level could be variable according to the patient abcb genetic status. in conclusion, we hypothesize that the intracellular abcb may serve as a possible new target for improving the effects of the aminoquinolines when co-administered with a lysosomotropic drug, such as azithromycin or ciprofloxacin in covid- chemotherapy. in vitro experiments could readily confirm or contradict this hypothesis. the proposed hypothesis might stimulate further investigations and experimental validation for the clinical relevance of this possible treatment. moreover, this case example highlights that molecular and cellular pharmacokinetics should be considered in the future for the understanding of drug interaction at the intracellular level. postscript. while this manuscript was under review, a paper describing similar lysosomal accumulation of the two drugs via ion-trapping mechanism was published ( ) . the author is very grateful to dr. fanchon bourasset and to mrs. marie-christine scherrmann for editing help. conflict of interest the author declares that he has no conflict of interest. hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression covid- ? a rapid review. bjgp open. a call for randomized controlled trials to test the efficacy of chloroquine and hydroxychloroquine as therapeutics against novel coronavirus disease (covid- ) macrolide treatment for covid- : will this be the way forward? clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid- new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? activity of azithromycin (cp- , ) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of plasmodium falciparum in vitro treatment of adults with acute uncomplicated malaria with azithromycin and chloroquine in india, colombia, and suriname in vivo and in vitro antimalarial properties of azithromycin-chloroquine combinations that include the resistance reversal agent amlodipine killing of staphylococcus aureus in murine macrophages by chloroquine used alone and in combination with ciprofloxacin or azithromycin targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases use of chloroquine in viral diseases evaluation of ebola virus inhibitors for drug repurposing effects of chloroquine on viral infections: an old drug against today's diseases? an evaluation of chloroquine as a broad-acting antiviral against hand, foot and mouth disease remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) optimizing hydroxychloroquine dosing for patients with covid- : an integrative modeling approach for effective drug repurposing lysosomes as a therapeutic target steady-state pharmacokinetics of hydroxychloroquine in patients with cutaneous lupus erythematosus azithromycin plus chloroquine: combination therapy for protection against malaria and sexually transmitted infections in pregnancy development of a population pharmacokinetic model to describe azithromycin whole-blood and plasma concentrations over time in healthy subjects azithromycin and ciprofloxacin have a chloroquine-like effect on respiratory epithelial cells cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: implications for an intracellular distribution-based drug interaction lysosome membrane permeabilization and disruption of the molecular target of rapamycin (mtor)-lysosome interaction are associated with the inhibition of lung cancer cell proliferation by a chloroquinoline analog subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (nr ) in vitro differences in assessment of macrolide interaction with human mdr (abcb , p-gp) using rhodamine- efflux, atpase activity and cellular accumulation assays quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (ly ) activities against intracellular staphylococcus aureus in mouse j macrophages the effect of azithromycin on ivermectin pharmacokinetics-a population pharmacokinetic model analysis influence of abcb gene polymorphisms on the pharmacokinetics of azithromycin among healthy chinese han ethnic subjects the effect of gender and abcb gene polymorphism on the pharmacokinetics of azithromycin in healthy male and female pakistani subjects possible involvement of the drug transporters p glycoprotein and multidrug resistance-associated protein mrp in disposition of azithromycin p-glycoprotein abcb : a major player in drug handling by mammals intracellular trafficking of p-glycoprotein revealing the fate of cell surface human p-glycoprotein (abcb ): the lysosomal degradation pathway comparison of mdck-mdr and caco- cell based permeability assays for anti-malarial drug screening and drug investigations ranking of p-glycoprotein substrates and inhibitors by a calcein-am fluorometry screening assay. anti-cancer drugs atovaquone and quinine antimalarials inhibit atp binding cassette transporter activity the potential inhibitory effect of antiparasitic drugs and natural products on p-glycoprotein mediated efflux digoxin-hydroxychloroquine interaction? a study of the pharmacokinetics of azithromycin and nelfinavir when coadministered in healthy volunteers p-glycoprotein mediates drug resistance via a novel mechanism involving lysosomal sequestration lysosomal membrane permeabilization and cell death lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (fa n- cells) impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype cellular models and in vitro assays for the screening of modulators of p-gp, mrp and bcrp subcellular localization and activity of multidrug resistance proteins drugs as p-glycoprotein substrates, inhibitors, and inducers lysosomal p-gp-mdr confers drug resistance of brentuximab vedotin and its cytotoxic payload monomethyl auristatin e in tumor cells lysosomal trapping of a radiolabeled substrate of p-glycoprotein as a mechanism for signal amplification in pet in vitro activity and pharmacodynamic/ pharmacokinetic parameters of clarithromycin and azithromycin: why they matter in the treatment of respiratory tract infections chloroquine and hydroxychloroquine are associated with reduced cardiovascular risk: a systematic review and meta-analysis. drug des dev ther excessive lysosomal ion-trapping of hydroxychloroquine and azithromycin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ob euspo authors: durdagi, serdar; dag, cagdas; dogan, berna; yigin, merve; avsar, timucin; buyukdag, cengizhan; erol, ismail; ertem, betul; calis, seyma; yildirim, gunseli; orhan, muge d.; guven, omur; aksoydan, busecan; destan, ebru; sahin, kader; besler, sabri o.; oktay, lalehan; shafiei, alaleh; tolu, ilayda; ayan, esra; yuksel, busra; peksen, ayse b.; gocenler, oktay; yucel, ali d.; can, ozgur; ozabrahamyan, serena; olkan, alpsu; erdemoglu, ece; aksit, fulya; tanisali, gokhan; yefanov, oleksandr m.; barty, anton; tolstikova, alexandra; ketawala, gihan k.; botha, sabine; dao, e. han; hayes, brandon; liang, mengning; seaberg, matthew h.; hunter, mark s.; batyuk, alex; mariani, valerio; su, zhen; poitevin, frederic; yoon, chun hong; kupitz, christopher; sierra, raymond g.; snell, edward; demirci, hasan title: near-physiological-temperature serial femtosecond x-ray crystallography reveals novel conformations of sars-cov- main protease active site for improved drug repurposing date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ob euspo the covid pandemic has resulted in + million reported infections and nearly . deaths. research to identify effective therapies for covid includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. to assist in drug repurposing and design, we determined two apo structures of severe acute respiratory syndrome coronavirus- main protease at ambienttemperature by serial femtosecond x-ray crystallography. we employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. the combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against sars-cov- . one sentence summary radiation-damage-free high-resolution sars-cov- main protease sfx structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of covid . in late , after the first patient was diagnosed with pneumonia of unknown etiology reported to the world health organization (who) from china, millions of cases followed in a short span of four months (who). on th of march, ; who declared covid outbreak as a pandemic, which originated from severe acute respiratory syndrome corona virus- (sars-cov- ) infection. sars-cov- has a high spread rate (rₒ) value, deeming the pandemic difficult to control (petersen et al., ) . moreover, the absence of a vaccine to provide immunity and the lack of effective treatments to control the infection in high comorbidity groups make this pandemic a major threat to global health (ahn et al., ) . the first human coronavirus to cause a variety of human diseases, such as common cold, gastroenteritis, and respiratory tract diseases was identified in the s (tyrrell et al., ) . in , a deadly version of coronavirus responsible for sars-cov was identified in china (heymann et al., ) . sars-cov- , the most recent member of the coronavirus family to be encountered, is a close relative of sars-cov and causes many systemic diseases (andersen et al., ; dutta and sengupta., ; braun et al., ) . covid patients exhibit (i) high c-reactive protein (crp) and pro-inflammatory cytokine levels; (ii) macrophage and monocyte infiltration to the lung tissue; (iii) atrophy of spleen and lymph nodes which weakens the immune system; (iv) lymphopenia; and (v) vasculitis . release of a large amount of cytokines results in acute respiratory distress syndrome (ards) aggravation and widespread tissue injury leading to multi-organ failure and death. therefore, mortality in many severe cases of covid patients has been linked to the presence of the cytokine storm evoked by the virus (ragab et al., ) . the sars-cov- genome encodes structural proteins including surface/spike glycoprotein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins; and the main reading frames named orf a and orf b that contain non-structural proteins (nsp) (gordon et al., ; chen, liu & guo., ) . among these, orf a/b encodes papain-like protease (plpro), main protease (mpro), a chymotrypsin-like cysteine protease, along with polyproteins named polyprotein a (pp a) and polyprotein b (pp b) (astel et al., ) . encoded polyproteins are then proteolyzed to nsps by precise mpro and plpro cleavages of the internal scissile bonds. nsps are vital for viral replication, such as rna-dependent rna polymerase (rdrp) and nsp , which are used for the expression of structural proteins of the virus thiel et al., ; ullrich & nitsche., ; ziebuhr et al., ) . sars-cov- mpro has no homologous human protease that recognizes the same cleavage site (pillaiyar et al., ) . therefore, drugs that target its active site are predicted to be less toxic and harmful to humans (l. . high sequence conservation of mpro provides minimized mutation-caused drug resistance . given its essential role in the viral life cycle, the sars-cov- mpro presents a major drug target requiring a detailed structural study. drug repurposing is a rapid method of identifying potential therapies that could be effective against covid compared to continuous investigative efforts (e.g identification of new drugs and development of preventive vaccine therapies). the well-studied properties of food and drug administration (fda) approved drugs means such molecules are better understood compared to their counterparts designed de novo. a putative drug candidate identified by drug-repurposing studies could make use of existing pharmaceutical supply chains for formulation and distribution, an advantage over developing new therapies pushpakom et al., ; jarada et al., ) . in typical drug repurposing studies, approved drug libraries are screened against the active site or an allosteric site of target protein structures obtained by methods that have limitations in revealing the enzyme structure, such as cryogenic temperature or radiation damage and investigations that only involve screening of drugs currently on the market (zhou et al., ; choudhary et al., ; beck et al., ; wang, ) . current structural biology-oriented studies that display a repurposing approach to sars-cov- research focused on target-driven drug design, virtual screening or a wide spectrum of inhibitor recommendations (rathnayake et al., ; chauhan and kalra, ; muralidharan et al., ; khan et al., ; kumar et al., ; joshi et al., ) . it is critical to interfere with the ongoing pandemic, instead of time and resource-consuming drug design studies, drug repurposing studies with the existing drugs are crucial in short terms should be a priority. the main purpose of our study is to reveal the conformational dynamics of mpro, which plays a central role in the viral life-cycle of sars-cov- . linac coherent light source (lcls) with its ultrafast and ultrabright pulses enables outrunning secondary radiation damage. the updated highthroughput macromolecular femtosecond crystallography (mfx) instrument of lcls that is equipped with the new autoranging epix k detector that provides a dynamic range of eleven thousand kev photons in fixed low gain mode to collect secondary radiation-damage-free structural data from mpro microcrystals remotely at ambient-temperature (sierra et al., ; blaj et al., ; . here we present two sfx structures of sars-cov- mpro which provide structural dynamics information of its active site and their deep in silico analysis. our results emphasize the importance of structure-based drug design and drug repurposing by using the xfel structures. we obtained more relevant results of flexible areas over two different structures. radiation-damage-free sfx method which enables obtaining the novel high-resolution ambient-temperature structures of the binding pocket of mpro provides an unprecedented opportunity for identification of highly effective inhibitors for drug repurposing by using a hybrid approach that combines structural and in silico methods. besides, structure-based drug design studies will be more accurate based on these novel atomic details on the enzyme's active site. conformations of the drug-binding pocket. we determined two radiation-damage-free sfx crystal structures of sars-cov- mpro in two crystal forms at . Å and . Å resolutions with the following pdb ids: cwb and cwc, respectively (fig. a, b) (supplementary table & the diffraction data collected remotely at the mfx instrument of the lcls at slac national laboratory, menlo park, ca (sierra et al., analysis for sfx studies at lcls). we used an mpro structure determined at ambient-temperature using a rotating anode home x-ray source (pdb id: wqf; as our initial molecular replacement search model for structure determination. two high-resolution sfx structures obtained in different space groups were superposed with an overall rmsd of . Å (fig. s ). they reveal novel active site residue conformations and dynamics at atomic level, revealing several differences compared to the prior ambient-temperature structure of sars-cov- mpro that was obtained at a home x-ray source (fig. a, b ). mpro has a unique n-terminal sequence that affects enzyme's catalytic activity (chang, ; l. zhang et al., ) . besides the native monoclinic form of mpro at . Å (fig. a , pdb id: cwb) we also determined the structure of the mpro with additional extra n-terminal amino acids (generated by thrombin specific n-terminal cleavage) at . Å resolution (fig. b , pdb id: cwc). the structure obtained from this modified version of mpro reveals how the minor changes introduced at the n-terminus affects both the three-dimensional structure of the mpro and promotes the formation of a new orthorhombic crystal form. biologically relevant dimeric structure of native monomeric mpro can be generated by adding the symmetry-related chain b ( fig. a) . each protomer of sars-cov- mpro is formed by three major domains s ). domain i starts from the n-terminal of protein and includes anti-parallel beta-sheet structure. this beta-sheet forms a beta-barrel fold which ends at residue . the domain ii of mpro resides between residues through and mostly consists of anti-parallel beta-sheets. the third domain of the mpro is located between residues through and consists of mostly alpha helices and has a more . previous biochemical studies of mpro suggested there is a competition for dimerization surface between domains i and iii. in the absence of domain i, mpro undergoes a new type of dimerization through the domain iii (zhong et al., ) . during our purifications, we repeatedly observed a combination of monomeric and dimeric forms of mpro on the size exclusion chromatography steps which may be caused by this dynamic compositional and conformational equilibrium. the two sars-cov- mpro sfx crystal structures reveal a non-flexible core active site and the catalytic amino acid cys fig. s . temperature factor analysis revealed that the active site is surrounded by mobile regions (fig. s ). the presence of these mobile regions was observed in both sfx crystal structures, suggesting an intrinsic plasticity rather than an artifactual finding that could have arisen based on the crystal lattice contacts ( fig. s ; indicated with red circles). further, this plasticity suggests that molecules that interact via non-covalent bonds to the mpro binding pocket would do so weakly. our investigation of pdb structures with available electron densities identified that the majority of the mpro inhibitors formed covalent bonds with the active site residue cys . additionally, few non-covalent inhibitors were identified and exhibited weak electron densities (fig. we compared our radiation-damage-free ambient-temperature sfx structure (pdb id: cwb) with ambient-temperature x-ray structure (pdb id: wqf). structures were similar with an rmsd value of . Å (fig. a) . however, we observed significant conformational differences especially in the side chains of thr , ser , glu , leu , asn , cys , met , and gln residues (fig. b & fig. s ). the calculated bias-free composite omit map that covers the active region has been shown ( fig. b & fig. s ). this structure offers previously unobserved new insights on the active site of mpro in addition to wqf structure which is important for the future in silico modeling studies. all three domains contribute to the formation of the active site of the protein. the intersection part of the domain i residue his , domain ii residues cys and his interact via a coordinated water molecule with asp located at the n-terminal loop region of domain iii to form the active site, that is an important drug target area (fig. c & fig. s ). the n-terminal loop of domain iii is suggested to be involved in enzyme activity (ma et al., ) . the distance between cys s and his nε is . Å, very similar to the ambient-temperature structure ( wqf) . oδ -oδ atoms of asp and nh -nε atoms of arg contribute to a salt bridge between these two residues and stabilize the positions of each other. the w water molecule (indicated with a red sphere at the figure) in the active site plays crucial roles for catalysis. w forms triple h-bonds with his , his , and asp side chains with the distances of . Å, . Å, and . Å, respectively (fig. c & fig. s ). when compared to the room temperature structure of mpro ( wqf) our structure displays additional active site residue dynamics while it has an overall high similarity (fig. d) . canonical chymotrypsin-like proteases contain a catalytic triad composed of ser(cys)-his-asp(glu) in their catalytic region, however, sars-cov- mpro possesses a cys and his catalytic dyad which distinguishes the sars-cov- mpro from canonical chymotrypsin-like enzymes (gorbalenya & snijder., ; . during the catalysis, the thiol group of cys is deprotonated by the imidazole of his and the resulting anionic sulfur nucleophilically attacks the carbonyl carbon of the substrate. after this initial attack, an n-terminal peptide product is released by abstracting the proton from the his , resulting in the his to become deprotonated again and a thioester is formed as a result. in the final step, the thioester is hydrolyzed which results in a release of a carboxylic acid and the free enzyme; therefore, restoring the catalytic dyad (pillaiyar et al., ; ullrich & nitsche., ) . catalytic residue conformations of sfx structures are consistent with wqf and support the proposed mpro catalytic mechanism (fig. c, d) . the crystal contact of the symmetry-related molecule with the n-terminal region of the mpro is essential for the formation of the crystal lattice in the c space group ( cwb) (fig. a& fig. s ). & s for cwb and cwc, respectively) to determine the flexible regions. in simulations for both cwb and cwc, the protomers exhibit non-identical behavior as also observed by others in apo form of mpro (suarez and diaz., ; amamuddy et al., ) though as expected, higher rmsf values were observed for the loop regions of protomers. additionally, the protomers of cwc display higher fluctuations compared to cwb around the loops covering the active site (such as loops containing residues - and - ) which could affect the accessibility of the active site by inhibitor compounds. on the drug-binding pocket dynamics. the trajectory frames obtained from md simulations were used to perform pca to determine the variations of conformers of protein structures, i.e. to observe the slowest motions during md simulations. as pca and pca-based methods are useful to reveal intrinsically accessible movements such as domain motions (bahar et al., ) , we have performed pca for backbone atoms of dimeric units for the structures belonging to different space groups. we focused on the first three pcs, that show around % of the total variance in md trajectories to determine the regions of protein structures that display the highest variation (fig. s & s for cwb and cwc, respectively). the first three pcs were projected onto the protein structures to determine the contributions of each residue to specified pcs which displays the motions of specific regions with blue regions with higher thickness representing to more mobile structural parts of the protein along the specified pcs. it can be seen that both in c and p space group structures, again protomers a and b display asymmetric behavior and domain movements along all considered pcs ( fig. a-c for cwb and fig. d -f for cwc) which was also observed in residue dynamics for the same domain between alternate chains (amamuddy et al., ) . when the motions displayed by two dimeric forms are compared to each other cwb (supplementary movies m - for cwb and m -m for cwc), we observe that domain iii is more mobile in cwb (the crystals in the c space group) compared to cwb (the crystals in the p space group). however, the loop region containing residues - around the catalytic site is more mobile for both protomers in cwc while it is only mobile for the chain b of cwb. interestingly, protomer a of cwc is more mobile compared to protomer a of cwb which could reflect the differences in dimeric interface due to the additional n-terminal amino acids and missing interactions with chain b n-terminus residues in the dimer interface. however, the loop regions surrounding the binding pockets with residues - and - display higher flexibility in chain a of cwb while its mobility is somewhat restricted in cwc. we have also performed cross correlation analysis of residues along the pc space to understand the correlation between motions especially for different domains. the correlation between the motions along the first three pcs were plotted as dynamical cross correlation maps displayed in in which motions of some residues are along the same directions, others are in opposite directions within both protomers a and b of cwb while domain ii has limited mobility as being more buried than other domains (suarez and diaz., ) except for the β-strand segment of residues - of protomer a. surprisingly, this segment also has some mobility albeit limited in protomer b of cwc instead of protomer a (supplementary movies m , fig. h ). glu of this segment is actually an important residue that plays a role in stabilizing the substrate binding site s by interacting with ser of the alternate protomer along with phe (ghahremanpour et al., ; and we observed that along pc , for protomer a of cwb and protomer b of cwc, the motions of these residues are correlated. this could have implications about information transfer from one protomer to the other though other mpro structures of sars-cov- needs to be studied in atomistic details. in addition, we observe that correlations between domains of protomers a and b for cwb and cwc are dissimilar. for instance, domains i of cwb have defined anti-correlated motions as can be seen from the purple areas in . space groups of these structures are c , p , p and i respectively. the completeness of the structures was also evaluated, wqf, and y e crystallized in full-length sequence ( - ), however, w only lacks gln at both c-terminal ends of its chains. among compared structures, c y, which has the same space group as cwc has, and lacks amino acids (chain a, - , - , - , and ; chain b, - , - , - , and - ) . in our structures cwb has a full-length sequence, however, cwc lacks its last amino acid residues from c-terminal in chain a, and starts with phe (lacks ser and gly ), and ends at ser , lacks the last residues. when six structures ( w , y e, wqf, c y, cwb, and cwc) were compared based on hydrogen bonding interactions at the dimerization interfaces, more similarities were observed for the latter three ( c y, cwb, and cwc). the only difference between our structures and the other four is the hydrogen bond between ser (chain a) and gln (chain b) (fig. s ). although this hydrogen bond is not observed in other structures, the corresponding residues are close to each other, however they are not within hydrogen bonding distance we also monitored all interface interactions throughout the md simulations and compared cwb and cwc. interestingly, the hydrogen bond between ser (chain a) and gln (chain b) was lost and the interaction was turned into a van der waals interaction in cwb, and conserved as by % of the simulation time. in cwc structure, ser (chain a) was not in the vicinity of gln (chain b) (fig. s ). more interestingly, the same interaction but this time between gln (chain a) and ser (chain b), that is present at the cwb crystal structure, was only observed % of the simulation time ( fig. s ). in the cwc crystal structure, there was no hydrogen bonding interaction between gln (chain a) and ser (chain b). however during simulation, this bond was formed and retained in % of the simulation time (fig. s ). when static structures were compared based on hydrogen bond formation analysis, c y, cwb, and cwc clustered together, having the same hydrogen bonding network at the dimerization interface. the explanation for the hydrogen bond differences observed at the interface is the lack of amino acids at the n-terminal and c-terminal ends of the c y and cwc structures, compared to the cwb. simulation trajectories of cwb and cwc were compared based on hydrogen bond occupancies. the major differences were lys (chain a) and arg (chain b), asn (chain a) and ser (chain b), and arg (chain a) and tyr (chain b). the first interaction was not observed in cwc, however the latter two interactions were not presented in cwb. lys (chain a) and arg (chain b) was conserved % of the simulation time; asn (chain a) and ser (chain b); and arg (chain a) and tyr (chain b) retained % and % of the obtained trajectory frames, respectively (figs. s and s ). we also observed that during md simulations, in the case of cwc, gln (chain a) and phe (chain b) come closer to each other, and this van der waals contact was conserved % of the simulation time and not observed in the case of cwb (fig. s ). mpro. we used three well-known sars-cov- mpro inhibitors (i.e., ebselen, tideglusib, and carmofur) at the investigation of ligand-target interactions. these compounds were docked to the cwb and cwc structures and all-atom md simulations were performed for the top-docking poses. these three compounds were also docked to structures with the following pdb ids: w and y e, for comparison. md simulations were performed using the same md protocol. results showed that especially for apo form dimer targets, ebselen and carmofur are quite flexible at the binding pocket throughout the simulations and in most of the cases, they do not form a stable complex structure ( fig. s ). tideglusib has a more stable structure at the binding pocket of mpro, however, its binding modes are different (fig. ) . in the following section, details of md simulations results of tideglusib at the binding site of the mpro will be discussed. comparison of binding pocket volumes of cwc and ye shows that latter has a bigger average binding pocket volume. average binding pocket volumes were and Å , for the tideglusib bound ye and cwc structures, respectively. corresponding solvent accessible surface area (sasa) values throughout the md simulations also support this result. the sasa, which is the surface area of a molecule accessible by a water molecule, of cwc is smaller than y e ( fig. s ). tideglusib. tideglusib initially docked to the binding pockets of y e and cwc structures using induced-fit docking (ifd) approach. top-docking poses of these compounds were then used in allatom md simulations using the same md protocols. while tideglusib was structurally very stable at the binding pocket of the cwc during the simulations, it was not so stable at the y e the binding site (fig. a ). representative trajectory frames (i.e., the frame that has the lowest rmsd to the average structures) were used in the comparison of binding modes (fig. b) . results showed that while the binding mode of tideglusib forms hydrogen bonds and pi-pi stacking interactions with glu , gln , and his , respectively at the cwc; its corresponding binding mode at the y e only forms van der waals type interactions with hydrophobic moieties. a timeline protein-ligand contacts were visualized throughout the simulations (fig. c ). results showed that thr , leu , met , glu , and gln form stable interactions with the ligand. d ligand atom interactions with protein residues are also represented (fig. d ). interactions that occur more than % of the simulation time are shown. however, corresponding interactions of the ligand at the y e were not stable ( fig. s ). forms of mpro, we applied ifd protocol to predict the binding mode of tideglusib at the binding pocket of wqf and cwb. carbonyl oxygens of the thiadiazolidine ring of the tideglusib formed hydrogen bonds between asn , gly , and glu from their backbone atoms. the naphthalene ring of the ligand formed a pi-pi stacking interaction with the his in the cwb structure. a similar binding mode of tideglusib was observed when wqf was used. however, in this binding mode, his and gly are found to be important. a backbone hydrogen bond was observed between one of the carbonyl oxygen of the thiadiazolidine ring and gly . his formed two pi-pi interactions between the benzyl and the naphthalene rings of the tideglusib (fig. s a, b) . these two binding modes predicted by ifd were used in ns classical all-atom md simulations. each mpro-tideglusib system is evaluated based on rmsd changes from the average structure and we obtained representative structures from corresponding trajectories. in the representative structure of cwb-tideglusib complex, we observed van der waals interactions between surrounding residues, thr , his , cys , thr , ser , met , asn , cys , his , his , met , glu , and gln . in the representative structure of wqf-tideglusib complex, in addition to the similar van der waals sfx utilizes micro-focused, ultrabright and ultrafast x-ray pulses to probe small crystals in a serial fashion. structural information is obtained from individual snapshots; capturing bragg diffraction of single crystals in random orientations (martin-garcia et al., ) . the main advantages of sfx over its counterparts are the capability of working with micron to nanometer sized crystals which does not necessitate the lengthy and laborious optimization steps and enables working with multiple crystal forms and space groups. it enables obtaining high resolution structures at physiologically meaningful temperature and confirms the dynamic regions of the active site without secondary radiation damage. sfx offers great potential and provides much needed critical information for future high-throughput structural drug screening and computational modeling studies with sensitivity to dynamics, insensitivity to potential radiation-induced structural artifacts resulting in production of detailed structural information. sars-cov- mpro catalyzes the precise cleavage events responsible for activation of viral replication and structural protein expression . it has been the focus of several structural and biochemical studies, many of which have been performed at cryogenic temperatures, aiming to provide better understanding of the active site dynamics and reveal an inhibitor that affects the enzyme based on structural information. two crystal forms of mpro, native and modified, were determined at ambient-temperature with resolutions of . Å and . Å respectively. the two forms produced are optimal for co-crystallization and soaking respectively. co-crystallization experiments provide efficient interaction in the binding pocket as both drug and protein are stabilized before the formation of crystals. due to the close crystal lattice contacts, co-crystallization seems to be the preferred method for the native form of mpro (c ). the n-terminal of mpro which plays a critical role in crystal packing. elimination of the h-bonding network produced orthorhombic crystals in the p space group yielding a wider binding pocket, increasing the probability of capturing an expanded number of protein-drug complexes by soaking. an added advantage of two different crystal forms was the elimination of the artifacts introduced by specific lattice packing restraints of each crystal form in the dynamics analysis. the high-resolution mpro sfx structures presented here in two different crystal forms collectively revealed the intrinsic plasticity and dynamics around the enzyme's active site. due to the anionic nature of cys , it seems challenging to design molecules that interact with the active site only through non-covalent bonds as it has a very flexible environment. these findings provide a structural basis for and is consistent with studies claiming that the majority of inhibitors form covalent bonds with the active site of mpro , mainly through cys (figs. especially, unlike some studies, ebselen does not form a stable complex structure (sies et al., ; menendez et al., ; zmudzinski et al., ; węglarz-tomczak et al., ) . in addition to the importance of cys residue in our two different structures, a coordinated w molecule, regulating the catalytic reaction via triple hydrogen-bonding interactions with the his , and the asp that stabilizing the positive charge of his residue . this active site residue conformations of sfx structures are consistent with previous ambienttemperature structure ( wqf) (fig. c, d) . gln contributes to the stability of the ligands , along with asn and ser , are the active site residues forming the flank of the cavity (fig. ) . in a recent study, asn and gln have been indicated to interact with a and b inhibitors that have proven in vitro effectiveness . along with these amino acids, thr , which makes van der waals interaction with the n inhibitor, and ser which undergoes conformational changes in the presence of this inhibitor (pdb id: lu , and leu binding to n , gave different side-chain conformations with sfx, according to wqf structure (fig. b ). there were differences in the crystallization conditions between the sfx and wqf studies, the former making use of charge effects and the latter molecular crowding. the sfx study is also secondary radiation damage-free, eliminating potential artifacts to examine crucial amino acids at the atomic level, especially in terms of catalytic and inhibitor binding sites (figs. s -s ). these two highresolution sfx structures in different space groups reveal new active site residue conformations and intra-and inter-domain network and their dynamics at the atomic level, which helps us to better understand any related structural allosteric transitions of mpro structure interacting with the inhibitors (fig. & figs. s - ) . therefore, considering the importance of the required sensitivity in drug design or the use of natural compounds studies, these active site residue conformations reveal the critical importance of our study more clearly. there are many in silico docking studies performed based on cryogenic mpro protein structures of sars-cov- ton et al., ; pillaiyar et al., ; durdagi et al., ) . although potent antiviral drug candidates are identified and a vaccine research is still ongoing, they have not yielded a desirable final treatment/cure yet. in pursuit of effective drugs against covid , the key role of mpro in viral replication of sars-cov- , highly conserved structure, and the low toxicity of the antiviral molecules targeting this protein due to the absence of homolog of this protease in humans made mpro the target of our study. having access to the alternative ambienttemperature structures of mpro and observed conformational changes on active site residues will be a significant boon for the development of therapeutics and provide better understanding on ligand and inhibitor binding. at this point, our work has two original aspects. firstly, we used a comprehensive platform, sfx, which helps to deeply understand the complexity of sars-cov- to gain access to the high-resolution and radiation damage-free structure and the structural dynamics of the target protein mpro at an unprecedented level at near-physiological-temperatures. secondly, we determined two high-resolution sfx structures of sars-cov- mpro in two different space groups due to the new high-throughput data collection setup offered by the mfx instrument of the lcls-ii. drug repurposing has been the preferred area of research in the insufficiency of time and resources in emergency cases such as a novel pandemic. the most important advantage of the repurposing research is the bypass of several lengthy early stages in drug development. also, knowing the beneficial and detrimental effects of targeted drugs, along with well-established precautions will help with time limitations. this procedure has emerged as a fundamental and very strategic approach not only for prospective cohort design but also for many types of clinical trials, particularly crosssectional studies because as the molecules considered in repurposing studies passed through several stages, have well-defined profiles, they would not require prolonged pre-clinical studies, and hence they are important candidates to consider in case of disease emergencies or outbreaks (su et al., ; cavalla et al., ; choo et al., ; aguila et al., ) . therefore, enormous contribution to clinical studies, repurposing is a rapid step towards the conclusion of not only randomized controlled trials but also critical structural biology investigations (bumb et al., ; pihan et al., ; choudhary et al., ) . considering all this, adopting the drug repurposing approach and using known inhibitors ebselen, tideglusib, and carmofur to carry out mpro-based in silico molecular docking, md simulations and post-md analyses make our combined study more specific. a grave issue with drug research is the variability of the target proteins as the mutations and modifications may render the found drugs ineffective (dinesh et al., ) . for this reason, it is only sensible to work on a protein, whose biochemical properties are conserved over time and among different strains. evolutionarily, viruses try to hide by mimicking the proteins involved in the functioning of the host organism. cytomegalovirus (hcmv) can mimic a common host protein to hijack normal cell growth machinery or human immunodeficiency virus (hiv) can mimic a high percentage of human t cell receptors (bernstein, ; robertson, ) . sars-cov- virus contains the plpro enzyme, which is highly similar to the deubiquitinating enzymes (usp , usp ) in human metabolism (ratia et al., ) . that's why targeting this enzyme carries a high risk. in addition, spike protein, one of the other targeted proteins, also has a high mutation rate . also, spike protein includes a similar restriction site with epithelial channel protein (anand p et al., ) . besides that, inhibitors which target spike protein may act only for preventive aims (i.e., before the virus infection), if the virus already infected the host cell, targeting this region may not be useful. there is no mpro homolog in the human genome, targeting this protease is therefore safer and harmless to humans with reduced cross reactivity and side effects making it an ideal candidate for drug therapy. contributions of residues to the first three pcs for a-c) cwb and d-f) cwc with chain a displayed on the right and chain b on the left side. the aligned trajectory frames were generated to interpolate between the most dissimilar structures in the distribution along specified pcs. color scale from red to blue represents low to high displacements along specified pcs with broadening of the tubes depict the trajectory movements. the cα atoms of catalytic residues his and cys are displayed as spheres with cyan and lime colors, respectively. g-h) dynamic cross correlation matrix generated from the motions observed in pc space with values ranging from - (complete anticorrelation) to + (complete correlation) g) for cwb and h) for cwc. the boundary between chain a and b is denoted by dashed lines. bacterial vector by using ndei and bamhi restriction cleavage sites at ' and ' ends respectively. nterminal canonical sars-cov- mpro autocleavage cut site is indicated by green and purple which generates the native n-terminus. c-terminus has the prescision tm restriction site shown in red which is used to generate the native c-terminus after ni-nta hexa-histidine affinity purification chromatography. in-frame hexa-histidine tag and stop codon is shown in blue color. standard chromatography purification methods were applied to both constructs with slight modifications as described below. soluble mpro proteins were purified by first dissolving the bacterial cells in the lysis buffer containing mm tris ph . , mm nacl, % v/v glycerol supplemented with . % triton x- followed by sonication (branson w sonifier, usa). after sonication step, cell lysate was centrifuged by using the beckman optima™ l- xp ultracentrifuge at rpm for minutes at °c by using ti rotor (beckman, usa). after ultracentrifugation the pellet which contains membranes and insoluble debris was discarded and clear supernatant applied to nickel affinity chromatography by using a ni-nta agarose resin (qiagen, usa). to purify the mpro protein, first the chromatography column was equilibrated by flowing column volume of the loading buffer containing mm tris-hac ph . , mm imidazole, mm nacl. after equilibration, the supernatant containing the overexpressed mpro protein was loaded into the ni-nta agarose column at ml/minute flow rate. unbound proteins were removed by washing with column volumes of the loading buffer to clear the non-specific binding. after washing, hexa-histidine tagged mpro proteins were eluted from the column with the elution buffer containing mm tris-hac ph . , mm nacl, mm imidazole in ml of total volume. after elution, purified protein was placed in a kda cut off dialysis membrane and dialyzed against the buffer containing mm tris-hac ph . , mm nacl overnight to get rid of the excess imidazole. after the dialysis step we applied : stoichiometric molar ratio c protease (prescission protease, genscript, usa) to cleave the c- for initial crystallization screening, we employed sitting-drop microbatch under oil screening method by using well terasaki crystallization plates (greiner-bio, germany). purified mpro protein at mg/ml mixed with : volumetric ratio with ~ commercially available sparse matrix crystallization screening conditions. the sitting drop solutions were then covered with μl of % paraffin oil (tekkim kimya, turkey). all the crystallization experiments were performed at ambienttemperature. for our native construct- we were able to obtain multiple hit conditions and among them the best crystals were obtained at pact premier tm crystallization screen condition # from molecular dimensions, uk. the best crystallization condition has contained mm mmt buffer ph . and % w/v peg [mmt buffer; dl-malic acid, -morpholine ethane sulfonicacid (mes) monohydrate, -amino- -(hydroxymethyl)- , -propanediol (tris)-hcl]. for the modified construct only one crystallization condition yield the macrocrystals. after multiple optimization of the seeding protocol by using crystals obtained by microbatch under oil, we scaled up the batch crystallization volume to total of ml for native construct- and total volume of ml for modified construct- . microcrystals - × - × - μm in size were passed through micron plastic mesh filters (millipore, usa) in the same mother liquor composition to eliminate the large single crystals and other impurities before the data collection. crystal concentration was approximated to be - particles per ml based on light microscopy. due to covid travel restrictions none of the initial crystals or the batched crystalline slurry were able to be pretested for their diffraction quality before the scheduled xfel beamtime. . ml total volume of crystal slurry was transferred to ml screw top cryovial (wuxi nest biotechnology, china cat# ). to absorb the mechanical shocks during transport from istanbul to menlo park, ca these vials were wrapped loosely by kimwipes (kimberly-clark, usa) and placed in ml screw top glass vials and tightly closed to provide insulation during transport via air. the vials were wrapped with excess amounts of cotton (ipek, turkey) and placed in a ziploc tm bag (sc johnson, usa) to provide both added layer of insulation and mechanical shock absorption. the ziploc tm bags were placed in a styrofoam box that was padded with ~ kg of cotton to provide more insulation and mechanical shock absorption during the transport. the styrofoam box was sealed and wrapped with an additional layer of cm thick loose cotton layer and duck taped all around to further insulate the delicate mpro crystals during ambient-temperature transport. all these packing materials and techniques provided us with crystals diffracting to . Å - . Å resolution as described below. the . ml sample reservoir was loaded with mpro crystal slurry in their unaltered mother liquor as described above. we used standard microfluidic electrokinetic sample holder (mesh) (sierra et al., ; sierra et al., ) injector for our sample injection. the sample capillary was a μm id × μm od × . m long fused silica capillary. the applied voltage on the sample liquid was typically - v, and the counter electrode was grounded. the sample ran typically between . and μl/min. the sfx experiments with native mpro microcrystals were carried out at the lcls beamtime id: mfx at the slac national accelerator laboratory (menlo park, ca). the lcls x-ray beam with a vertically polarized pulse with duration of fs was focused using compound refractive beryllium lenses to a beam size of ~ × μm full width at half maximum (fwhm) at a pulse energy of . mj, a photon energy of . kev ( . Å) and a repetition rate of hz. om monitor and psocake (damiani et al., , thayer et al., were used to monitor crystal hit rates, analyze the gain switching modes and determine the initial diffraction geometry of the new epix k m detector . a total of , , detector frames were collected in h m s continuously with the new from native (construct- ) mpro microcrystals. a total of , detector frames were collected in h m s continuously with the new epix k m pixel array detector from modified (construct- ) mpro microcrystals. the total beamtime needed for native (construct- ) and modified (construct- ) datasets were h m s and h m s respectively, which shows the efficiency of the mfx beamline installed with the new epix k m detector and robust injector system, as due to lack of blockages no dead time was accumulated. individual diffraction pattern hits were defined as frames containing more than bragg peaks with a minimum signal-tonoise ratio larger than . , which were a total of , and , images for native and modified respectively. the detector distance was set to mm, with an achievable resolution of . Å at the edge of the detector ( . Å in the corner). an example diffraction pattern is shown in fig. s . the diffraction patterns were collected at the mfx instrument at the lcls using the epix k m detector . the raw data images were subjected to detector corrections with cheetah (barty et al., ) , as well as for hit finding based on bragg reflections. the hitfinding parameters for all datasets classifying a hit were as follows (using peakfinder ): a minimum pixel count of above an adc-threshold of with a minimum signal to noise ratio of was considered a peak, and an image containing at least peaks was classified as a crystal hit. the crystal hits were then indexed using the software package crystfel version . (white et al., ) using the peaks found by cheetah. indexing was attempted using the indexing algorithms from xgandalf (gevorkov et al., ) , dirax (duisenberg et al., ) , mosflm (powell et al., ) and xds (kabsch et al., ) , in this order. after an approximate cell was found, the data was indexed using cell axis tolerances of Å and angle tolerances of º (--tolerance option in crystfel). the integration radii were set to , , and the "multi" option was switched on to enable indexing of multiple crystal lattices in a single image. the indexed reflections were subsequently integrated and merged using partialator ) applying the unity model over iterations and the max-adu set to . the complete reflection intensity list from crystfel was then scaled and cut using the truncate program from the ccp suite (winn et al., ) prior to further processing. for the native mpro protein crystals the final set of indexed patterns, containing , frames ( . % indexing rate), was merged into a final dataset (overall cc* = . ; . Å cutoff) for further analysis (c , unit cell: a = . Å, b = . Å, c = . Å; α = °, β = °, γ = °). the final resolution cutoff was estimated to be . Å using a combination of cc* (karplus & diederichs, ) and other refinement parameters. the final dataset had overall rsplit = . %, and cc* = . in the highest resolution shell. for the n-terminally modified mpro protein crystals the final set of indexed patterns, containing , frames ( . % indexing rate), was merged into a final dataset (overall cc* = . ; . Å cutoff) for further analysis (p , unit cell: a = . Å, b = . Å, c = . Å; α = β = γ = °). the final resolution cutoff was estimated to be . Å using a combination of cc* and other refinement parameters. the final dataset had overall rsplit = . %, and cc* = . in the highest resolution shell. we determined two ambient-temperature mpro structures by using two crystal forms in space group c and p structures using the automated molecular replacement program phaser (mccoy et al., ) implemented in phenix (adams et al., ) with the previously published ambienttemperature structure as a search model (pdb id: wqf) . this choice of starting search model minimized experimental temperature variations between the two structures. coordinates of the wqf were used for initial rigid body refinement with the phenix software package. after simulated-annealing refinement, individual coordinates and tls parameters were refined. we also performed composite omit map refinement implemented in phenix to identify potential positions of altered side chains and water molecules were checked in program coot (emsley & cowtan, ) , and positions with strong difference density were retained. water molecules located outside of significant electron density were manually removed. the ramachandran statistics for native monoclinic mpro structure (pdb id: cwb) (most favored / additionally allowed / disallowed) are . / . / . % respectively. ramachandran statistics for orthorhombic mpro structure (pdb id: cwc) (most favored / additionally allowed / disallowed) are . / . / . % respectively. the structure refinement statistics are summarized in supplementary table s . structure alignments were performed using the alignment algorithm of pymol (www.schrodinger.com/pymol) with the default σ rejection criterion and five iterative alignment cycles. all x-ray crystal structure figures were generated with pymol. the two ambient-temperature mpro in space group c and p were examined to generate ellipsoid structures based on b-factor with pymol and these two structures were compared with the mpro structures at k (pdb id: xkh) to provide better understanding on the flexibility of atoms, side chains and domains. the all ellipsoid structures were colored with rainbow selection on pymol. we have used different crystal structures of mpro available in literature as well as the obtained crystal structures in this study (pdb ids: cwb and cwc) as target structures for molecular docking and md simulations. the biologically-relevant dimeric form of cwb is generated by application of a symmetry operator. as the crystal structures in this study were obtained at ambient-temperature, for comparison another ambient-temperature structure of mpro (pdb id: wqf) in apo form was also selected as target structure. another apo form structure of mpro (pdb id: y e) in dimeric form was also chosen for comparison. additionally, mpro structure bound to a non-covalent inhibitor (pdb id: w ) in both monomeric and dimeric forms was utilized as target structure. for ligands, we have considered three compounds that have shown promising inhibitory activity based on the highthroughput screening of over , compounds by jin et al., namely; ebselen (ic = . ± . μm), tideglusib (ic = . ± . μm) and carmofur (ic = . ± . μm) . all the target structures considered in this study were firstly prepared using protein preparation module of maestro modeling program in which missing atoms were added, water molecules not in the vicinity of co-crystallized ligands were removed and bond orders were assigned (madhavi sastry et al., ) . the protonation states of amino acids at physiological ph were adjusted using propka (bas et al., ) to optimize the hydrogen binding and charge interactions. as a final step of preparation, a restrained minimization was performed with opls e force field parameters (harder et al., ) . the structures of three compounds were taken from pubchem; ebselen (pubchem id, ), tideglusib (pubchem id: ) and carmofur (pubchem id: ). the compounds also needed preparation hence, ligprep module (schrödinger release - , ) of maestro modeling program was employed with opls e force field parameters (harder et al., ) . the ionization states of the molecules were predicted by epik module (shelley et al., ) at physiological ph of . . the prepared target protein and ligand structures were used for molecular docking studies. we have employed a grid-based docking method, induced fit docking (ifd) protocol of maestro (sherman et al., a,b) which uses glide halgren et al., ; friesner et al., ) and prime (jacobson et al., ) the selected docking poses at each considered target structure of mpro with the three compounds were subjected to md studies. the apo form structures obtained in this study were also subjected to md simulations. for comparison reasons, md simulations were also performed for the holo form structure (pdb id: w ) with its co-crystallized ligand, x . the target protein-ligand complexes were placed in simulation boxes with orthorhombic shape in which box sizes were calculated based on buffer distance of . Å along all three dimensions and solvated with explicit water molecules of spc (berendsen et al., ) model. the simulation systems were neutralized by the addition of counter ions (na+ or cl− depending on the charge of the systems) and . m nacl solution was added to adjust concentration of the solvent systems. all atom md simulations package desmond (bowers et al., ) was employed. proceeding the production md simulations, the systems were equilibrated using relaxation protocols of desmond package in which a series of minimizations and short md simulations which are performed with small time-steps at lower temperature and restrains on the nonhydrogen solute atoms in the initial stages and slowly time-steps are increased as well as simulation temperature and restrains on solute atoms are released. the production simulations were performed under constant pressure and temperature conditions, i.e. npt ensemble. temperature was set as k while being controlled by nose-hoover thermostat (nosé, ; hoover, ) . the pressure was set as atmospheric pressure of . bar with isotropic pressure coupling and controlled by martyna-tobias-klein barostat (martyna et al., ) . smooth particle mesh ewald method (essmann et al., ) was utilized to calculate long range electrostatic interactions with periodic boundary conditions (pbc). for short range electrostatics and lennard-jones interactions, the cut-off distance was set as . Å. the multi-step integrator respa was employed in which the time steps were varied for interaction types as followed in fs for: bonded, . ; near . and far . . principal components analysis (pca), a statistical data processing method, were performed to reduce the large-dimensional data by extracting large amplitude motions onto collective sets. a covariance matrix were generated from md trajectory data for backbone atoms of protein structures as follow here, i and j represent the backbone atom number, i.e. residue numbers of proteins while n is the number of backbone atoms considered in analysis. the cartesian coordinates of atoms are denoted by and for ith and jth atom, respectively with and representing the time-averaged values over md simulations. by diagonalization of covariance matrix, a collection of eigenvectors and corresponding eigenvalues were obtained. the eigenvectors of the diagonalized matrix are referred as principal components (pcs) and constitute a linear basis set that matches the distribution of observed structures. the corresponding eigenvalues of the diagonalized matrix display the variance of the distribution along each pcs. in this study, we have utilized the bio d package (grant et al., ; yao et al., ) , a platform independent r package to perform pca for considered simulation systems. the trajectories obtained from independent md simulations were concatenated and frames were aligned with respect to the initial (reference) frame before pca. the correlation of atomic displacements is evaluated by cross-correlation analysis to appreciate the coupling of motions. the magnitudes of all pairwise cross-correlation coefficients were investigated to assess the extent of atomic displacement correlations for each simulation system in principal component space. the normalized covariance matrix of atomic fluctuations was calculated as where and are the displacements of residues i and j, i.e., mean square atomic fluctuations. the values of varies between - to with representing completely correlated motions (same period and same phase), representing completely anticorrelated motions (same period and opposite phase) while value indicates motions are uncorrelated. (ichiye et al., ; mccammon et al., ) . bio d package (grant et al., ; yao et al., ) in r environment was employed to generate atom-wise crosscorrelations of motions observed in pcs to and dynamical cross-correlation map, or dccm were generated and displayed as a graphical representation of cross-correlation coefficients. interface analysis of crystal structures and md trajectories were carried out with the getcontacts python scripts (https://getcontacts.github.io/). two different approaches were followed, in the first one only hydrogen bonds at the dimerization interface were taken into account, and in the second approach all possible interactions, namely; salt bridges, pi-cation, pi-pi stacking, t-stacking, van der waals (vdw), and hydrogen bonds were calculated. if the distance between the acceptor and the donor atoms is < . Å and the angle < ° hydrogen bond is defined between atom groups. salt bridges were defined between atoms of negatively charged [asp (od , od ) and glu (oe , oe )] and positively charged [lys (nz) and arg (nh , nh )], where distances were < . Å. t-stacking, pi-cation, and pi-stacking distance criteria were . , . , and . Å, respectively. hydrophobic and vdw interactions were calculated based on atom r (radii), if the distance between atoms is less than the sum of r of atom a, r of atom b, and . Å. one frame and trajectory-based calculations were performed. one frame calculations were applied for the crystal structures, and means the corresponding residues are in contact, and means no interaction. however, in trajectory-based calculations, we used all available frames from our md trajectories. for the interaction frequencies, we applied a . threshold to only take into account the contacts that are occurred at least % of the simulations. fig. s . superposition of two crystal forms. native mpro in space group c colored in darksalmon and its symmetry mate in green. modified mpro in space group p is colored in palecyan and light blue. two crystal structures align with an overall rmsd of . Å. domain i is colored in light blue, domain ii is colored in pale cyan and domain iii is colored in dark salmon. active site formed by residues from all three subdomains such as critical cys is shown as spheres, his , his and asp shown in sticks. red sphere labeled as w represents the water molecule in the binding pocket. s . projection of md trajectory frames onto subspaces defined by the first three largest pcs as well as distribution of variance observed for eigenvalues for cwc. instantaneous conformations (i.e. trajectory frames) colored from blue to red in order of trajectory time ( ns). the black triangle represents the initial crystal structure conformation projected onto specified pcs. domain i residues depicted in light blue, domain ii and domain iii depicted in pale cyan and dark salmon, respectively. blue color shows hydrogen bonding, lines with red ones lack hydrogen bonding. tables supplementary table s . data collection and refinement statistics for x-ray crystallography datasets are collected by serial crystallography the highest resolution shell is shown in parentheses identification of potent covid- main protease (mpro) inhibitors from flavonoids cultivation of a novel type of common-cold virus in organ cultures genomic characterization of a novel sars-cov- order -nidovirales" in virus taxonomy middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir susanna lau predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov- ) through a drug-target interaction deep learning model molecular characterization of ebselen binding activity to sars-cov- main protease gc- , and calpain inhibitors ii, xii inhibit sars-cov- viral replication by targeting the viral main protease genome organization and structural aspects of the sars-related virus cytokine release syndrome in severe covid- : interleukin- receptor antagonist tocilizumab may be the key to reduce mortality antiviral drug targets of single-stranded rna viruses causing chronic human diseases predictive methods in drug repurposing: gold mine or just a bigger haystack? a sars-cov- protein interaction map reveals targets for drug repurposing drug repurposing for coronavirus (covid- ): in silico screening of known drugs against coronavirus cl hydrolase and protease enzymes current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease (covid- ) the architecture of sars-cov immune mechanisms of pulmonary intravascular coagulopathy in covid- pneumonia interleukin- use in covid- pneumonia related macrophage activation syndrome the covid- cytokine storm c-like protease inhibitors block coronavirus replication in vitro and improve survival in mers-cov-infected mice us fda approves new class of hiv therapeutics sars-cov main protease: a molecular dynamics study structural plasticity of sars-cov- cl mpro active site cavity revealed by room temperature x-ray crystallography sars-cov- infection: response of human immune system and possible implications for the rapid test and treatment viral cysteine proteinases repurposed gi drugs in the treatment of covid- comparing sars-cov- with sars-cov and influenza pandemics d structure collections dedicated to drug repurposing and fragment-based drug design systematic drug repositioning through mining adverse event data in clinicaltrials. gov ebselen as a highly active inhibitor of plprocov . biorxiv ( ) sars-cov- renal tropism associates with acute kidney injury the protein expression profile of ace in human tissues identification of amitriptyline hcl, flavin adenine dinucleotide, azacitidine and calcitriol as repurposing drugs for influenza a h n virus-induced lung injury a new coronavirus associated with human respiratory disease in china the proximal origin of sars-cov- performance of epix k, a high dynamic range, gain auto-ranging pixel detector for fels quaternary structure of the sars coronavirus main protease the macromolecular femtosecond crystallography instrument at the linac coherent light source idrug: integration of drug repositioning and drug-target prediction via cross-network embedding designing of improved drugs for covid- : crystal structure of sars-cov- main protease mpro potential therapeutic use of ebselen for covid- and other respiratory viral infections. free radic comprehensive insights into the catalytic mechanism of middle east respiratory syndrome c-like protease and severe acute respiratory syndrome c-like protease normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins cytokines and autoimmunity drug repurposing and emerging adjunctive treatments for schizophrenia history and recent advances in coronavirus discovery fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study virus-encoded proteinases and proteolytic processing in the nidovirales efficacy and safety of lianhuaqingwen capsules, a repurposed chinese herb structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease crystal structure of sars-cov- main protease provides a basis for design of improved aketoamide inhibitors potential effects of coronaviruses on the cardiovascular system: a review serial femtosecond crystallography: a revolution in structural biology isolation of a novel coronavirus from a man with pneumonia in saudi arabia ebselen derivatives are very potent dual inhibitors of sars-cov- proteases -plpro and mpro in vitro studies computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- without its n-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its c-terminal domain drugs for hyperlipidaemia may slow down the progression of hearing loss in the elderly: a drug repurposing study sars-cov- strategically mimics proteolytic activation of human enac targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and ′-o-ribose methyltransferase human immunodeficiency virus proteins mimic human t cell receptors inducing cross-reactive antibodies understanding human coronavirus hcov-nl ali identification of potent covid- main protease (mpro) inhibitors from natural polyphenols: an in silico strategy unveils a hope against corona impact of emerging mutations on the dynamic properties the sars-cov- main protease: an in silico investigation mechanisms of coronavirus cell entry mediated by the viral spike protein identification of sars-cov- cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach screening of clinically approved and investigation drugs as potential inhibitors of covid- main protease: a virtual drug repurposing study sars-cov- and male infertility: possible multifaceted pathology clinical trials on drug repositioning for covid- treatment evolutionary and structural analyses of sars-cov- d g spike protein mutation now documented worldwide coronavirus hku and other coronavirus infections in hong kong main protease ( m pro ) from several medicinal plant compounds by molecular docking study managing cytokine release syndrome associated with novel t cell-engaging therapies characterizations of sars-cov- mutational profile, spike protein stability and viral transmission overcoming drug development bottlenecks with repurposing: old drugs learn new tricks drug repurposing: progress, challenges and recommendations the sars-cov- main protease as drug target the epix k -megapixel hard x-ray detector at lcls in silico screening of natural compounds against covid- by targeting mpro and ace using molecular docking a review of computational drug repositioning: strategies, approaches, opportunities, challenges, and directions an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy drug repositioning: identifying and developing new uses for existing drugs rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds mechanisms and enzymes involved in sars coronavirus genome expression structure-based design of antiviral drug candidates targeting the sars-cov- main protease. science the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease (covid- ): the experience of clinical immunologists from china timeline: who's covid- response emerging coronaviruses: genome structure, replication, and pathogenesis analysis of the mutation dynamics of sars-cov- reveals the spread history and emergence of rbd mutant with lower ace binding affinity in silico prediction of potential inhibitors for the main protease of sars-cov- using molecular docking and dynamics simulation based drug-repurposing cheetah: software for high-throughput reduction and analysis of serial femtosecond x-ray diffraction data phaser crystallographic software very fast prediction and rationalization of pka values for protein-ligand complexes linac coherent light source data analysis using psana structural plasticity of sars-cov- cl mpro active site cavity revealed by room temperature x-ray crystallography opls : a force field providing broad coverage of drug-like small molecules and proteins constant pressure molecular dynamics algorithms protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments the missing term in effective pair potentials autoindexing diffraction images with imosflm dynamics of proteins and nucleic acids epik: a software program for pka prediction and protonation state generation for drug-like molecules bio d: an r package for the comparative analysis of protein structures indexing in single-crystal diffractometry with an obstinate list of reflections data systems for the linac coherent light source proceedings of the acm/ieee conference on supercomputing overview of the ccp suite and current developments a hierarchical approach to all-atom protein loop prediction linking crystallographic model and data quality phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics glide: a new approach for rapid, accurate docking and scoring. . method and assessment of docking accuracy extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem ii nanoflow electrospinning serial femtosecond crystallography a unified formulation of the constant temperature molecular dynamics methods glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening processing serial crystallography data with crystfel: a step-by-step guide crystfel: a software suite for snapshot serial crystallography recent developments in crystfel collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations the epix k -megapixel hard x-ray detector at a smooth particle mesh ewald method onda: online data analysis and feedback for serial x-ray imaging canonical dynamics: equilibrium phase-space distributions domain-opening and dynamic coupling in the α-subunit of heterotrimeric g proteins structure of mpro from sars-cov- and discovery of its inhibitors hd acknowledges support from national science foundation (nsf) science and technology centers grant nsf- (biology with x-ray lasers, bioxfel) and the scientific and technological research council of turkey (tubitak) grant ( c ). hd would like to thank michelle young, ritu khurana, lori anne love and tracy chou for their invaluable support and discussions. use of key: cord- - vax m authors: becker, richard c. title: covid- treatment update: follow the scientific evidence date: - - journal: j thromb thrombolysis doi: . /s - - - sha: doc_id: cord_uid: vax m nan there is not an fda-approved treatment for covid- at this time; however, high-level effort and investigations remain and are in progress, respectively. taking a sars-cov- specific approach to drug development is logical and was recently summarized by kupferschmidt et al. [ ] ( on march , the united states food and drug administration (fda issued) an emergency use authorization (eua) permitting chloroquine phosphate (medical grade) and hydroxychloroquine sulfate to be added to the strategic national stockpile (sns) (fig. ) . the sns exists under the authority of the united states department of health and human services (hhs) and accepted million doses of hydroxychloroquine sulfate donated by sandoz™, the novartis™ generics and biosimilars division, and one million doses of chloroquine phosphate donated by bayer pharmaceuticals™ for potential use in treating patients who were hospitalized with covid- or for use in clinical trials. in addition to the sns donations, these companies and potentially others could offer additional doses (www. fda.gov). there was an expectation that companies would increase production for the commercial market as well to avoid shortages for patients with autoimmune disease who rely on hydroxychloroquine sulfate (plaquenil®) for routine treatment [ ] . the coordinated use of chloroquine phosphate and hydroxychloroquine sulfate is governed by the hhs office of the assistant secretary of preparedness and response (aspr). in turn, apsr's biomedical advanced research and development authority (barda) works collaboratively with the national institutes of health (nih) and fda to assure safety and the drugs appropriate use to include well-designed clinical trials (fig. ) . the eua grants permission to barda allowing chloroquine phosphate and hydroxychloroquine sulfate to be distributed and prescribed by physicians to hospitalized teen and adult patients with covid- , when deemed appropriate, if a clinical trial is either not available or feasible. the eua mandates that fact sheets summarizing important information about the drugs and their administration are made availability to patients and prescribers, including potential risk and drug interactions. drug shipments to individual states is overseen by the federal emergency management agency (fema) who works closely with the department of state and department of homeland security to secure donated shipments. what is chloroquine, how does it work and why might it be effective in the treatment of covid- ? chloroquine was discovered in the early ′s by hans andersag, a scientists working for bayer ag™ on compounds with anti-malarial properties. it is a -aminoquinoline compound taken by mouth and has a long track record of mass administration for the prevention of malaria to include plasmodium vivax, plasmodium ovale and plasmodium malariae. it is typically not used for plasmodium falciparum given well-documented resistance [ ] . chloroquine is absorbed rapidly and has a wide volume of distribution and undergoes hepatic metabolism giving rise to its primary metabolite desethylchloroquine. approximately, % of the drug is excreted unchanged in the urine. chloroquine accumulates within lysosomes where it alters cellular ph and is stored in a protonated form. the antiviral effect is believed to be from chloroquine's ability to increase endosomal and lysosomal ph thereby attenuating the ability of the virus to release its genetic material into the cell and replicate. the drug also inhibits important post-translational steps in newly formed proteins, acts as a zinc ionophore that increases intracellular zinc concentration and inhibits rnadependent polymerases, decreases endosomal release of iron required for the replication of dna and inhibits glycosylation of envelope glycoproteins (reviewed in savarino [ ] ). there has been interest in the potential antithrombotic effects of chloroquine. while the contribution to its overall benefit may be modest and largely unknown in patients with covid- , chloroquine reduces neutrophil extracellular trap (net) formation, platelet aggregation and circulating tissue factor in mice [ ] . chloroquine has been shown to differentially modulate coronary arterial vasodilation in diabetic mice [ ] . in humans, it decreases nitric oxide production in coronary artery endothelial cells. the potential clinical impact in fig. targeted approach to drug development for sars-cov- . the lines of attack against sars-cov- , numbered - , include inhibition of: ( ) fusion; ( ) translation; ( ) proteolysis; ( ) translation and rna replication; ( ) packaging and; ( ) virion release. from kupferschmidt and cohen [ ] . with permission patients with coronary artery disease is unknown. chloroquine has been shown in animal models to inhibit autophagy with a resulting improvement in diastolic performance in diabetic mice [ ] . the potential mechanism(s) cardiovascular effects, including vasodilation, hypotension, decreased myocardial performance and arrhythmias have been reported following chloroquine administration at high doses (reviewed in ben-zvi [ ] ). the potential cardiotoxic effects of chloroquine have been summarized by several groups. blignaut and colleagues investigated several specific functional effects, including ex vivo myocardial performance and glucose uptake, mitochondrial function and in vivo heart function [ ] . control or obese male wister rats were used in the experiments and exposed to varying concentrations of chloroquine. doses that achieved a concentration of um or higher decreased heart function. ex vivo exposure did not affect mitochondrial function, but chronic exposure decreased cardiac output. the centers for disease control and prevention (cdc) posted a warning on their website about non-medicinal chloroquine phosphate products and their potential risks to humans after ingestion, including death (www.cdc.com). it is also important to be mindful of and attentive to the potential risk of medicinal chloroquine and hydroxychloroquine (to be discussed) when taken in doses higher that recommended. cardiac arrhythmias, including torsades de pointe ventricular tachycardia stemming from q-tc prolongation and often, but not always concomitant hypokalemia can occur (figs. and ). additional electrocardiographic effects include bundle branch block and atrioventricular (av) block. non-cardiovascular adverse effects that have been reported range from nausea, vomiting and diarrhea to thrombocytopenia, aplastic anemia, shock, seizures, coma and death. there are in vitro data for chloroquine and sars-cov replication [ ] . kayaerts et al. tested its antiviral potential against sars-cov-induced cytopathicity in vero e cell culture. the ic of chloroquine for antiviral activity ( . ± . μm) was significantly lower than its cytostatic activity; cc ( . ± . μm) and approximated the plasma concentrations commonly achieved during treatment of malaria. the same group [ ] investigated whether chloroquine, provided either transplacentally or via maternal milk could prevent electrocardiographic qtc interval prolongation. the qt interval represents the period of ventricular depolarization (qrs interval) and repolarization (st interval). it is corrected for the heart rate and referred to as the qtc interval. prolongation of repolarization is caused by an increase of inward current through sodium or calcium channels or a decrease in outward current through potassium channels. the normal value in adults is ms in men and ms in women. the normal qtc interval is shorter in children. a sudden prolongation of qtc interval (greater than - % above baseline) or an absolute value > ms increases the risk for ventricular tachycardia that can be life-threatening. other intervals, including the pr (atrial depolarization and conduction leading up to ventricular depolarization) are shown hcov-oc -induced death in newborn mice. the highest survival rate ( . %) occurred when mother mice received mg of chloroquine per kg of body weight daily. wang and colleagues [ ] tested the in vitro antiviral activity of chloroquine in vero e cells infected with ncov- betacov/wuhan/wiv / at a multiplicity of infection (moi) of . . chloroquine (ec = . μm; cc > μm, si > . ) blocked virus infection at lowmicromolar concentrations at both entry, and at post-entry stages. huang and colleagues [ ] conducted a small pilot study in patients with covid- . a total of patients were divided into two groups: one (n = ) treated with chloroquine ( mg, oral administration, twice daily) and another (n = ) with lopinavir/ritonavir ( / mg, oral administration, twice daily) for days and monitored for a total of days. chloroquine treated patients were more likely to turn negative for viral rna. in fact, by day , all the chloroquine-treated patients became negative for viral rna test. in the lopinavir/ritonavir treated patients, out of turned negative by day . improvement in pulmonary ct scans occurred at twice the rate in chloroquine-treated patients compared to those receiving lopinavar/ritonavir and duration of hospitalization was shorter in the former group as well. serious adverse events were not reported in either treatment group. hydroxychloroquine was developed and subsequently approved for the treatment of malaria in (reviewed in al-bari; [ ] ). like chloroquine, but with a better safety profile, particularly with prolonged use, it is included on the world health organization's list of essential medications with indications for administration that have expanded over the years to include autoimmune diseases such as rheumatoid arthritis, sjogren's syndrome, systemic lupus erythematosus and post-lyme's disease arthritis (reviewed in plantone; [ ] ). hydroxychloroquine exhibits anti-inflammatory, immunomodulating, anti-infective, antithrombotic, and metabolic effects. hydroxychloroquine is absorbed rapidly after oral administration, metabolized by hepatic p enzymes and cleared by the kidneys. as a result of its lipophilic properties and relatively high ph, hydroxychloroquine enters cell readily and torsades de pointes or "twisting of the points or peaks" ventricular tachycardia occurs in the setting of a prolonged qtc interval that is either inherited or acquired or both. some individuals with familial long qtc syndrome (lqts) are not aware of their condition and symptoms may not occur until a medication with qtc-prolonging capabilities is administered. the ventricular rate is fast and almost always causes low blood pressure and hemodynamic compromise. it is a cause of sudden cardiac death accumulates within lysosomes. there is a resulting increase in ph from to that attenuates glycosylation and release of antigenic proteins, interference of lysosomal acidification, decreased macrophage cytokine production to include interleukins and tumor necrosis factor (tnf)-α, antagonist effects on prostaglandins, binding and stabilizing of nucleic acids, inhibition of t and b-cell signaling, inhibition of matrix metalloproteinases [ ] [ ] [ ] , chemotaxis, superoxide production and phagocytosis of neutrophils, a reduction in toll-like receptor signaling and attenuated activation of dendritic cells. hydroxychloroquine has also been shown to bind cell surface sialic acid and gangliosides with high affinity, thereby impairing sars-cov- spike protein recognition and binding to host cell angiotensin converting enzyme (ace)- receptors [ ] . hydroxychloroquine's blockade of tnf-α has been associated with a reduction of atherosclerotic cardiovascular disease-associated events among patients with autoimmune rheumatologic disease [ ] (age adjusted rate ratio . , % ci . - . ). interleukin (il)- inhibition has also been shown to reduce cardiovascular events (reviewed in ali and becker [ , ] ) and reduced synthesis of mmp- may lessen atherosclerotic plaque disruption. hydroxychloroquine's ability to lower cholesterol and hemoglobin aic in diabetes may not contribute to benefit with short courses of treatment [ ] . however, if ongoing studies show that it offers protection against sars-cov- infection, then longer courses of drug administration may be recommended. the platelet inhibiting properties of hydroxychloroquine, either alone or in combination with aspirin [ ] may be attractive with either short or more prolonged courses of treatment [ ] . chatre et al. [ ] performed a systematic review of cardiac complications attributable to hydroxychloroquine (and chloroquine). there were a total of patients from individual case reports and case series. the median duration of treatment was years, with a range from days to years. the cumulative doses were g or more. the most comment adverse cardiovascular effect was av conduction abnormalities ( % of patients). reduced ventricular performance was observed as was heart failure with the former recovering in nearly half of patients after drug withdrawal. it is important to acknowledge that may of the patients included in the systematic review were being treated for autoimmune disease. the adverse effects associated with taking hydroxychloroquine are similar to those observed with chloroquine and include nausea, vomiting, diarrhea, av conduction defects, a prolonged qtc interval with torsades de pointe ventricular tachycardia, hypokalemia, hypotension and circulatory collapse. the hemodynamic complications are much more likely to occur with excessively large doses; however, qtc prolongation can be seen with standard recommended doses even in the absence of concomitant electrolyte abnormalities (low potassium, magnesium or calcium). drug-drug interactions must be considered in patients taking hydroxychloroquine and include digoxin (increased levels), insulin and oral hypoglycemic agents (heighted responses and hypoglycemia), antiepileptic drugs, methotrexate, cyclosporine and drugs that prolong the qtc. the antiviral properties of hydroxychloroquine have been summarized and provide a foundation for additional formative discussion. yao and colleagues [ ] determined the pharmacological activity of hydroxychloroquine employing sars-cov- infected vero cells. pharmacokinetic models were constructed by integrating vitro data. hydroxychloroquine concentrations in lung fluid were simulated. hydroxychloroquine (ec = . μm) was found to be more potent than chloroquine (ec = . μm). based on modeling, a loading dose of mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of mg given twice daily for days may be recommended for sars-cov- infection. the in vitro cytotoxicity of hydroxychloroquine was determined in veroe cells and showed that the % cytotoxic concentration (cc ) value for hcq was . μm [ ] . there are patient demographics and characteristics that are associated with a higher likelihood of developing drugrelated qtc prolongation (table ) . all clinicians and prescribing health providers should be familiar with each. the number of drugs that can prolong the qtc is substantial (− known) and typically divided into those with a known risk of qtc prolongation or torsades de pointes ventricular tachycardia, possible risk and conditional risk (i.e. if administered with another qtc prolonging drug; in the setting of hypokalemia or in patients with predisposing conditions such as familial long qt syndrome). (crediblemeds.org) [ ] in the context of current treatment for covid- , including empiric regimens being employed by clinicians as they await the results of clinical trials, azithromycin's effect on qtc must be recognized and included in the equation. the fda issued a warning on may , on the risk of potentially fatal heart rhythm abnormalities associated with the drug. employing a large claims database of over million patients with greater than million azithromycin prescriptions, patel and colleagues [ ] identified nearly % of patients were receiving another drug with qtc producing effects. the presence of pre-existing cardiac conditions was ~ . %. neither concomitant qtc prolonging drugs nor cardiac conditions differed after as compared with before the fda warning. the clinical community to include emergency room physicians, nurse practitioners, physician assistants and pharmacists, as well as others employing a tele-health platform must adopt a well-informed and consistent approach to safely prescribe drugs with qtc prolonging or proarrhythmic potential [ ] . a management algorithm must include a comprehensive understanding of drug pharmacology, risk-benefit relationships and a track record of safety. in addition, there must be a thorough evaluation of patient history focusing on conditions known to be associated with arrhythmias, as well as a detailed family history of cardiomyopathy, arrhythmias, sudden cardiac death and internal cardioverter-defibrillator (icd) insertion (see table ). last, dose adjustments, monitoring of electrolytes and serial electrocardiograms should be included in the management plan when indicated. consensus recommendations from the internal council for harmonization of technical requirements of pharmaceuticals for human use (ich) should be followed (www.ich.org). vandael et al. [ ] developed a risk score for qtc prolongation following treatment with a drug known to have this potential effect. in a majority of patients ( of ), the risq-path score was ≥ ( table ) . the score had a sensitivity of . % ( % ci . - . %) and a negative predictive value of % ( % ci . - . %). the brazilian study of chloroquine underscores the importance of cardiovascular safety when using or studying antimalarial drugs [ ] . the study included hospitalized covid- confirmed patients of whom approximately half received mg chloroquine twice daily for five days and the remainder received mg twice daily for days. the clorocovid- study was performed on a background of treatment with azithromycin and ceftriaxone. arrhythmias were observed within three days of study initiation and by six days patients had died. there was a greater likelihood of having a qtc interval > ms ( %) in patients receiving high-dose chloroquine. accordingly, this arm of the study was terminated. while more detailed information is needed, high-dose chloroquine may not be safe in the treatment of covid- . the safety of lower doses must be established in covid- . it is vital to educate the lay community about the potential risks associated with non-medicinal chloroquine products. similarly, patients with covid- for whom a clinician believes that either chloroquine or hydroxychloroquine is indicated must receive information, preferably in the form of a fact sheet that clearly summarized the dose, duration of treatment, potential risks, side-effects and drug-drug interactions. the number of clinical trials in covid- patients, either planned or ongoing is increasing on a daily basis. the reader is referred to clinicaltrial.gov for regular updates. the world health organization (who) working with the global research collaborative for infectious disease preparedness (glopid-r) established a covid- research roadmap (www.glopi d-r.org) in january . the design is tailored for a global research response that embraces cross-border collaborations and partnerships across professional disciplines worldwide. a major focus of glopid-r is data sharing based on filling knowledge gaps, establishing research priorities and accelerating the generation of scientific information required to address the covid- pandemic. an initial evaluation by the who called for priority to embark upon global research built on common platforms for standardized processes, protocols and tools. it also emphasized several knowledge gaps about sars-cov- to include compartments of replication, the prognostic importance of viral load, immune-biomarkers, genotype-phenotype relationships, genotype drifts that might impact diagnostic assays and technical limitations for serologic assay development (simple immunofluorescence assay [ifa], differential ifa, enzyme linked immunoabsorbance assay [elisa] and neutralization assays). the group exhibited substantial foresight and raised questions about the development of point-of-care testing, the identification of prognostic markers and digital solutions for field laboratory needs. they also emphasized the importance of ethical considerations as being crucial to governance, transparency, accountability, equity and trust. last, the group underscored the importance of establishing a framework for social science research, global social science researchers and understanding social and behavioral dynamics during the covid- pandemic ( table ) . the medical and scientific community's collective response to the covid- pandemic is predicated on obtaining the best available evidence from prevention, treatment and overall management. the evidence will come from well-designed and conducted clinical trials. it is particularly important in the context of current events to employ a framework that can be applied and adapted to environments and conditions, maintain operational rigor and interpret the results according to the data. while all investigators strive to answer complex questions about covid- and make major contributions, reporting and publishing negative trials are equally, and not infrequently, more important than positive trials. the r & d blueprint working group has advocated for the use of "core protocols" when designing clinical trials during public health emergencies like covid- [ , ] . in addition to providing a strong research platform in the midst of highly dynamic settings encountered during public health emergencies, core or master protocols, inclusion of independent monitoring committees, "pause or stopping" rules, publication metric requirements and clear ground rules for public messaging are needed [ ] . among the many attractive features of core protocols, their inherent design is collaborative in nature, engaging investigators and stakeholders early in the public health emergency to determine the most important research question(s), optimal study design, ethic and regulatory needs, funding sources, country health authority and governmental integration, communications, data security, oversight and clinical operations. an ability to conduct high quality research that is valuable and worthy of serious consideration by the scientific community requires a research quality framework (rqf). while experienced investigators have been using rqf's for many years, a reminder of its vital role and component parts is fitting given the covid- pandemic and desire for study results to emerge as rapidly as possible. the key processes, metrics and tenets of a rqf are as follows: • data provenance (internal and external reproducible of the findings) • quantitative expertise (applied under proper standards to the design, conduct analysis and presentation of the results) • governance and oversight • special review of potential conflicts of interests • comprehensive vetting, implementation and assessment of research practices based on instrumental, research network or organizational standards • transparency of processes. . mobilize research on rapid point of care diagnostics for use at the community level-this is critical to be able to quickly identify sick people, treat them and better estimate how widely the virus has spread . immediately assess available data to learn what standard of care approaches from china and elsewhere are the most effective-there is an imperative to optimize standard of care given to patients at different stages of the disease and take advantages of all available technological innovations to improve survival and recovery . evaluate as fast as possible the effect of adjunctive and supportive therapies-the global research community need to understand what other adjunctive treatments than currently being used are at our disposal that may help with the standard of care provided to patients, including the quick evaluation of interventions such as steroids and high flow oxygen . optimize use of protective equipment and other infection prevention and control measures in health care and community settings-it is critical to protect health care workers and the community from transmission and create a safe working environment . review all evidence available to identify animal host(s), to prevent continued spill over and to better understand the virus transmissibility in different contexts over time, the severity of disease and who is more susceptible to infection-understanding transmission dynamics would help us appreciate the full spectrum of the disease, in terms of at risk groups, and conditions that make the disease more severe as well as the effectiveness of certain public health interventions . accelerate the evaluation of investigational therapeutics and vaccines by using "master protocols"-rapidly developing master protocols for clinical trials will accelerate the potential to assess what works and what does not, improve collaboration and comparison across different studies, streamline ethics review and optimize the evaluation of new investigational drugs, vaccines and diagnostics . maintain a high degree of communication and interaction among funders so that critical research is implemented-funders reiterated their current financial commitments to tackling this outbreak and agreed that the priorities agreed at the forum would help to coordinate existing investments and inform mobilization of additional resources in the coming days, weeks and months . broadly and rapidly share virus materials, clinical samples and data for immediate public health purposes-it was agreed that virus materials, clinical samples and associated data should be rapidly shared for immediate public health purposes and that fair and equitable access to any medical products or innovations that are developed using the materials must be part of such sharing the covid- pandemic has touched everyone on earth in many ways. its scope and impact on health, well-being, economic stability and daily life is tragic, heart-breaking and quite personal. despite an honest and natural human response to a clear threat, this is not a time for reactions; this is not a time for assumptions; this is not a time for desperate measures; this is not a time for guessing what might be a safe and effective treatment. this is a time for facts, measured steps, thoughtful responses and collaborations without borders. this is without question a time to follow the scientific evidence. escaping pandora's box-another novel coronavirus characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention race to find covid- treatments accelerates potential of chloroquine and hydroxychloroquine to treat covid- causes fears of shortages among people with systemic lupus erythematosus antimalarial drug resistance in africa: strategies for monitoring and deterrence effects of chloroquine on viral infections: an old drug against today's diseases chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps chloroquine differentially modulates coronary vasodilation in control and diabetic mice chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice hydroxychloroquine: from malaria to autoimmunity revisiting the cardiotoxic effect of chloroquine in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine antiviral activity of chloroquine against human coronavirus oc infection in newborn mice remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro treating covid- with chloroquine chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin alpha (il- -alpha) and il- in human monocytes and t cells hydroxychloroquine inhibits calcium signals in t cells: a new mechanism to explain its immunomodulatory properties structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection treatment with tumor necrosis factor blockers is associated with a lower incidence of first cardiovascular events in patients with rheumatoid arthritis inflammation and coronary artery disease: from pathophysiology to canakinumab anti-inflammatory thrombosis outcomes study (cantos) metalloproteinases promote plaque rupture and myocardial infarction: a persuasive concept waiting for clinical translation cholesterol-lowering effect of hydroxychloroquine in patients with rheumatic disease: reversal of deleterious effects of steroids on lipids hydroxychloroquine's efficacy as an antiplatelet agent study in healthy volunteers: a proof of concept study the protective effect of antimalarial drugs on thrombovascular events in systemic lupus erythematosus cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial credible-meds.org: what does it offer? impact of the fda warning for azithromycin and risk for qt prolongation on utilization at an academic medical center how to prescribe drugs with an identified proarrhythmic liability development of a risk score for qtc-prolongation: the risq-path study chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov- ) infection: preliminary safety results of a randomized, double-blinded who r&d blueprint: a global coordination mechanism for r&d preparedness master protocols to study multiple therapies, multiple diseases, or both key: cord- - bpl n j authors: singh, sweta; florez, hector title: coronavirus disease drug discovery through molecular docking date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: bpl n j background: the dawn of the year witnessed the spread of the highly infectious and communicable disease coronavirus disease (covid- ) globally since it was first reported in . severe acute respiratory syndrome coronavirus- is the main causative agent. in total, , , cases and , deaths owing to covid- were reported by th april, by the world health organization. this means infection and deaths show an exponential growth globally. in order to tackle this pandemic, it is necessary to find possible easily accessible therapeutic agents till an effective vaccine is developed. methods: in this study, we present the results of molecular docking processes through high throughput virtual screening to analyze drugs recommended for the treatment of covid- . results: atovaquone, fexofenadine acetate (allegra), ethamidindole, baicalin, glycyrrhetic acid, justicidin d, euphol, and curine are few of the lead molecules found after docking known antivirals, antimalarial, antiparasitic drugs and natural products. conclusions: these molecules could act as an effective inhibitory drug against covid- . coronavirus disease (covid- ) is caused by the severe acute respiratory syndrome coronavirus- (sars-cov- ), that is responsible for respiratory illness and probably many more is yet to be discovered. this novel virus was first identified on th december, , with its first infection case infecting a human, which was reported in wuhan city located in hubei, china . coronaviruses are mainly zoonotic, and are present amongst birds and mammals, causing respiratory, neurological, hepatic and enteric diseases as well as comprises of enveloped rna. the world health organization (who) declared this disease as a pandemic on th march, and sars-cov- as the deadliest virus till date on earth claiming , deaths till th april, . then, it is necessary for the rapid development and approval of a vaccine, which is not yet available . nevertheless, chang et al., have suggested that some drugs against same type of viruses approved by the us food and drug administration (fda) might offer promising results. hydroxychloroquine is one such drug that is used worldwide whereas remdesivir and ivermectin have been reported to work against covid- in silico by others. the transmission of this coronavirus occurs due to the binding of the cov spike protein to the angiotensin converting enzyme (ace ) receptor present on the cell surface of the human host. the ace receptor is present in the respiratory organs, kidneys, gastrointestinal tract (at high levels in the esophagus, colon, and small intestine, but low in the stomach), and testes. virulence of this novel virus is due to the presence of main protease responsible for virus replication along with many major functions . therefore, we have employed the main protease structure m as the target protein to identify the best inhibitory drugs in silico for our study. sars-cov- (negatively stained) when observed under the electron micrograph was found to be spherical in shape with some pleiomorphic characteristic. the epithelial sections of human airway when observed, viruses were found in membrane bound vesicles in cytoplasm along with inclusion bodies. the virions appear similar to solar corona due to -to -nm distinctive spikes and the virions are to nm in diameter. thus, it was established due to these morphological characteristics that this virus belongs to the coronaviridae family along with its genome having more than % identity with a bat sars-like cov (bat-sl-covzc , mg . ) genome as previously assessed via genome sequencing . sars-cov- initially infects lower airways, binds to ace receptor on cells activating immune cells, thus, inducing the secretion of inflammatory cytokines and chemokines in human pulmonary system . most covid- patients exhibit flu-like symptoms within a span of two weeks from the exposure to the virus whereas there have been a majority rise in the asymptomatic covid- patients. in this work, we have performed high throughput virtual screening since it is the fastest approach in finding the probable drug against the target. high-throughput virtual screening (htvs) of two databases was carried out via pyrx (python prescription) software, which uses dock, vina and autodock as the docking tool. autodock itself uses mgltools comprising of computer aided drug discovery (cadd) pipeline for high throughput virtual screening of large databases for probable hits as target drugs. htvs enables docking of multiple ligands on a single protein. pyrx is a freely available htvs software. docking results are based on the identification of pose visually and quantitatively using a scoring algorithm. docking calculates the free binding energy (∆g) between the ligands and the protein. the free binding energy, thus calculated, is fundamental to the formation of complex systems in biochemistry and molecular biology. lower free binding energy corresponds to a more favorable ligand binding affinity between a receptor and a ligand . molecular docking is a bioinformatics method that allows predicting the orientation of a molecule, when it is bounded to another molecule , . there are two main approaches for molecular docking. the first approach describes the protein and the ligand as complementary surfaces . the second approach simulates the docking process calculating the ligand protein interaction based on the free binding energy ∆g . selection of database and the covid- main protease structure in this study, we have docked the x-ray crystal structure of main covid protease protein (pdb id: m , resolution: Å) with molecules obtained from drugbank and molecules from the zinc natural product database. the list of molecules are provided along with the link for zinc natural database in the extended data . these molecules chosen were either antimalarial, antiparasitic, antibiotics, or antivirals, since hydroxychloroquine, remdesivir and ivermectin are antimalarial, antiviral and antiparasitic drugs, respectively. the zinc natural product database was chosen since most of the drugs are natural derivatives used against various diseases at present and it is a freely available database. similarity search could not be undertaken since there is no known drug to function % against this novel disease at time of publication. docking requires processing of the macromolecules and the ligands , . water molecules were removed, polar hydrogen bonds were inserted into the crystal structure of m and it was converted to pdbqt format using autodock version . the energy of all ligands were minimized and they were converted into pdbqt files using open babel version . . in pyrx version . . the grid box was determined as center the coordinates x: . , y: . , and z: . , while as dimension the coordinates x: . , y: . , and z: . . the docking was done using vina version . in pyrx. after the run, the out files stored in the user folder where the path run was specified in the edit preference. these output files were stored in pdbqt files, each having nine poses. the autodock application file was launched which then showed the empty dashboard along with "file" on the left hand corner of the page. the out file models were loaded using the "read molecule" application from the selected out file folder. different poses were analyzed in the autodock tool. the pose with the lowest binding energy in kcal/mol was selected for further analysis. the docked molecules were then further converted into pdb format in pymol and their interaction was studied using the software discovery studio version . . the interaction can also be studied with pymol but a better quality picture is obtained via discovery studio. three known drugs (hydroxychloroquine, remdesivir and ivermectin) were first docked against the virus main protease to check their binding energy. the interaction of these three drugs with the covid- main protease could later be utilised for getting the hits. docking results for reference molecules the free binding energy for drugs known to act against covid- , which are hydroxychloroquine, remdesivir, and ivermectin, were found to be - . kcal/mol, - . kcal/mol, and - . kcal/mol respectively as indicated in table , which describes: a) pubchem compound id (cid), which is the compound identifier in the pubchem database from where the d mol files of molecules were downloaded; b) common drug name; and c) the free binding energy obtained after docking. these drugs are known to improve the condition to some extent and yet their functions against covid- are under study [ ] [ ] [ ] [ ] . therefore, we used these three molecules as our reference drugs. the interaction of these drugs with the virus main protease can be seen in the figure . we could observe the interaction of these reference molecules as hydroxychloroquine forms a hydrogen bond with tyr residue of the m main protease with a distance of . Å. it also interacts with leu and leu . remdisivir forms six hydrogen bonds with lys , thr , and tyr along with interacting with leu , leu , tyr , and asn residues of the m main protease. ivermectin interacts with leu , tyr , leu , leu , gly , asn , and met residues of the m main protease. docking results for additional molecules keeping the free binding energy of our reference molecules in mind, we shortlisted molecules from the database of molecules with a cut of - kcal/mol free binding energy. eprinomectin, artefenomel, doramectin, betulinic acid, atovaquone, and tetrandrine showed the lowest binding energies, at - kcal/mol, - . kcal/mol, - . kcal/mol, - . kcal/mol, - . kcal/mol and - kcal/mol, respectively. table presents the details of the best performing of the molecules along with their cid. these molecules have been considered due to the lowest free binding energy between the ligand and protein. furthermore, their interactions with the virus main protease m was studied. artefenomel interacts with pro , val , ile , pro , and phe residues of the m main protease. eprinomectin forms a hydrogen bond ( . Å) with lys residue of the m main protease and also interacts with leu , leu , and asn . tetrandrine forms a hydrogen bond with arg and also interacts with leu , leu and leu . betulinic acid forms a hydrogen bond with arg with . Å distance and also interacts with leu , leu , leu , tyr , and tyr . doramectin forms three hydrogen bonds with thr , lys , and gly as well as interacts with leu and asp . atovoquone forms a hydrogen bond with the thr residue of the m main protease with a distance of . Å and also interacts with lys , leu , leu , leu , and tyr . these interactions can be observed in figure . full results are available in the extended data . docking results for natural molecules a total of molecules of natural origin were chosen from the datasets of molecules with a cut off - . kcal/mol. table presents the details of the best molecules along with their common name and zinc id sorted by ascending order of the free binding energy of the top molecules from natural products database. the interaction of various molecules with the m main protease was studied. allegra forms three hydrogen bonds with thr , asn , and asp as well as interacts with arg , val , and phe residues of m main protease. baicalin forms four hydrogen bonds with thr , thr , ile , and arg along with interacting with asp , asn , and val residues of the main protease m . curine forms three hydrogen bonds with arg , thr , and leu along with having interactions with asp , leu , and tyr residues of m main protease. etamidindole forms two hydrogen bonds with thr and asp , it also interacts with phe , phe , arg , arg , and pro residues of main protease m . glycyrrhetic acid forms a hydrogen bond with lys ( . Å) as well as interacts with tyr , tyr , leu , leu , and leu residues of the main protease m . euphol interacts with phe , val , arg , and val residues of the main protease. the interaction of few of the molecules with the protein can be seen in figure . full results are available in the extended data . htvs is one of the best methods for identifying molecules acting against drug targets in a very short time period, when compared to traditional drug identification strategies. remdesivir was detected as covid- inhibitory drug via virtual screening method . therefore, keeping the present scenario in our mind, we undertook this study to provide individuals with covid- with drugs already available. surprisingly, we have found good hits from the databases with medicinal properties. artefenomel is known to treat malaria and other parasitic diseases. betulinic acid is under trial for the treatment of dysplastic nevus syndrome. atovaquone is an approved drug for the treatment of pneumocystis carinii pneumonia and malaria. tetrandrine is in the experimental stage for anticancer, antimalarial, antiparasitic category. eprinomectin and doramectin are veterinary antiparasitic drugs. many of the natural compounds identified have medicinal properties. taraxerone has allelopathic and antifungal effect , morusin has anti-oxidant and anticancer properties , ra vii compound is an antitumor agent , and neoruscogenin is used against chronic venous disorders . justicidin d exhibits anti-inflammatory properties , licoricidin is an antimetastatic molecule whereas euphol is used against asthma and cancer along with syphilis, and rheumatism . schisandrene has anti-oxidant activity , curine is reported as a vasodilator , angoluvarin has antimicrobial activity , baicalin is used to treat cardiovascular diseases, inflammation and hypertension . glycyrrhetic acid shows anti-inflammatory, anti-ulcer, hepatoprotective, anti-allergic, anti-tumor, antioxidant and anti-diabetic activity . isomitraphylline has an antioxidant properties , bikaverin and rutarensin has anti-tumour activities , . jolkinol b has anticancer properties and ethamidindole exhibits antihistamine properties . fexofenadine acetate, commonly known as allegra, is an antihistamine pharmaceutical drug presently used in the treatment of allergy symptoms such as urticaria and hay fever . when we compared our two datasets, we found that majority of the molecules showed lesser free binding energy as compared to the reference molecules as in table , table , and table . surprisingly, we obtained good hits from our natural database which is good news since after observing their interaction and biotherapeutic functions, we might have achieved our covid- inhibitory drugs. one such drug from natural database is fexofenadine acetate (allegra) which is pres- ently in use as anti-allergic medicine. we also have hits from drugs but there is one, atovaquone, which is presently used against pneumonia and malaria and also a very good candidate for covid- treatment. the best therapeutic drugs inferred from our studies are atovaquone, fexofenadine acetate (allegra), justicidin d, baicalin, glycyrrhetic acid and ethamidindole based on their docking score, interaction studies and their present applications for probable covid- treatment. the rest of the molecules could also be used as covid- inhibitory drugs after further evaluation. when we compared our data with reference molecules score of currently in use drug against covid- , we found that atovaquone showed better binding energy than hydroxychloroquine and remdesivir. it is one of the best drug candidate for covid- treatment since it is already in use for treating pneumocystis carinii pneumonia and malaria. fexofenadine acetate is another good target drug for covid- treatment since it is naturally derived and presently used for its anti-histamine properties. ethamidindole could possibly act as covid- inhibitor since it is reported as anti-histamine and this novel virus activates cytokine secretion in human body. part from these, anti-inflammatory natural molecules such as justicidin d, baicalin, and glycyrrhetic acid could work against covid- since sars-cov- virus induces inflammation. the rest of the top molecules could also be considered since they all have some medicinal properties as explained above. this project contains the following extended data: • drugs repurposing list (pdf). (pubchem cid of each ligand along with the minimized energy of each molecule and binding affinity results) original> the transmission of this coronavirus occurs due to the binding of the cov spike protein to the angiotensin converting enzyme (ace ) receptor present on the cell surface of the human host. suggestion>the transmission of this coronavirus occurs due to the binding of the cov spike protein to the angiotensin-converting enzyme (ace ) receptor present on the human host's cell surface. i suggest the authors to focus in the evidences showed by chang we know that paragraph : et al. vaccines are not available for covid yet but your aim is to identify drugs for treatment. potential move this paragraph below " paragraph : the world health organization (who) declared this disease as a pandemic on th march, and sars-cov- as the deadliest virus till date on earth claiming , deaths till th april, ". please remove this paragraph because is not discussed in the corresponding section. i recommend the authors to rewrite this paragraph. authors performed an approach for paragraph : searching drugs candidates based on databases. please, focus in the main objective of the work. in my opinion authors could highlight the importance of bioinformatic tools (docking as the first approach) using available databases. i suggest to rewrite this paragraph because most of it content correspond to the methodology section. in my opinion authors should be more clear with the methodology. you could cite a work in the introduction section where docking was the better strategy for drugs candidates search. in this section, you should explain your criteria of protein selection, provide a list and finally, the docking process. i suggest to reorganize this section as: "molecule selection and processing (or edition)" and "molecular docking". in my opinion it is not necessary to split this information. for a better understanding, i strongly recommend to use a general graphical scheme explaining the selection, the macromolecules edition, and the pipe used for the docking analysis. i suggest to focus only in the docking results against different ligands (from databases, natural etc). some sentences correspond to the discussion section. it is not clear if you work with information from databases or with clinical samples. please, rewrite this sentence. the paragraph underline ( nd) is disconnected from this section, please review. in my opinion the third paragraph of this section is not necessary. authors could write in a sentence all this information and cite. i also suggest to discuss better your results and make a contrast with the available bibliography; authors repeated in this section the results. i understand that your contribution is relevant but from other authors repeated in this section the results. i understand that your contribution is relevant but from other perspective: bioinformatic tools for a quick detection of potential antivirals or drugs, using as an example the covid. several papers cited in the introduction section are not discussed. i suggest the integration of this section with the discussion. authors proposed several treatments, inferred from htvs, however, you should be careful with your statements. i recommend to discuss and compare the variables or data as indicators for potential treatments. the manuscript is written with understandable english, though some english revision is necessary. in my opinion, after several major revisions and reorganization of the text, the study will be acceptable for indexing on f research. if applicable, is the statistical analysis and its interpretation appropriate? i cannot comment. a qualified statistician is required. are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. reviewer expertise: evolutionary biology, genomics i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however i have significant reservations, as outlined above. evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission molecular docking covid- bioinformatics methods to discover antivirals against zika virus bioinformatics study of mutations of resistance to antivirals in the ns a gen of hcv. international information institute (tokyo) virtual screening for potential inhibitors of ctx-m- protein of klebsiella pneumoniae hydroxychloroquine prophylaxis for covid- contacts in india peptide-like and small-molecule inhibitors against covid- . . . - | j biomol struct dyn pubmed abstract publisher full text is the work clearly and accurately presented and does it cite the current literature? partly is the study design appropriate and is the work technically sound? yes are sufficient details of methods and analysis provided to allow replication by others? yes if applicable fceqyn, conicet-unam, posadas, argentina i think that all the data is a quite good contribution, but in my opinion, text should be deeply modified to be more attractive to the readers and to point more directly their main objective the authors would like to thank members of the research group iti for their valuable feedback.i might suggest the authors remark the differences between their approach and the one presented by: suyash pant, peptide-like and small-molecule inhibitors against covid- it could be included in the discussion. . . version july reviewer report https://doi.org/ . /f research. .r © ghosh p. this is an open access peer review report distributed under the terms of the creative commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original attribution license work is properly cited. laboratory of bioinformatics and protein engineering, international institute of molecular and cell biology in warsaw, warsaw, - , polandthe manuscript by singh and florez presents the discovery of drugs against covid using molecular docking methods. the authors must discuss in further detail as to how this work compares with other published reports of drugs targeted against sars-cov- .the authors should cite references for the results mentioned in the introduction section of the manuscript.how was the - kcal/mol free binding energy cut-off selected? as i understand this is not the lowest, the highest or the average of the reference molecule binding energies. then what is the basis for the selection of this cut-off value?the authors have discussed the docking results for the reference and the test molecules independently, but have not provided a comprehensive comparison of the observations. in my opinion, such a comparison is necessary for assessing the effectivity of the new drugs as compared to the existing drugs. the authors have also not discussed the importance of the residues tyr , leu , leu etc. are these known to be critical residues in the functioning of the main protease? the statistics on the number of cases and deaths need to be updated before the indexing.the authors should be more careful about the technicalities mentioned in the paper. for example, " m " has been mentioned loosely in the introduction section without pointing to the fact that it is a pdb structure; the htvs abbreviation has not been introduced in the manuscript before the first instance of its use. the paper requires english editing by a professional and/or a native english speaker. are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. competing interests: the paper is of interest under the current cov pandemic. simulation of the strength of the bonds between molecules can identify inhibitors without human subjects' need to test. this paper did not focus on finding new compounds but in examining those present in existing medicine. this approach expedites the process of finding a cure, and the suggestions done are well-founded. overall, the materials and methods section is exhaustive and asserts reproducibility. there are some wordy paragraphs with redundancies. please take a look at the following one and the replacement that i suggest.original> the transmission of this coronavirus occurs due to the binding of the cov spike protein to the the benefits of publishing with f research:your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and morethe peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f .com key: cord- -hznnoxw authors: benavides-cordoba, vicente title: drug repositioning for covid- date: - - journal: colombia medica doi: . /cm.v i . sha: doc_id: cord_uid: hznnoxw drug repositioning is a strategy that identifies new uses of approved drugs to treat conditions different from their original purpose. with the advance of covid- and the pandemic declaration; it has become the closest alternative to reduce the advance of the virus. antimalarial, antiviral drugs, antibiotics, glucocorticoids, monoclonal antibodies, among others, are being studied; their findings, although preliminary, could establish a starting point in the search for a solution. in this review, we present a selection of drugs, of different classes and with potential activity against covid- , whose trials are ongoing; and as proofs of concept, double blind, add-on event-driven, would allow proposing research that generates results in less time and preserving quality criteria for drug development and approval by regulatory agencies. the new disease caused by the coronavirus (covid- ) was reported in the city of wuhan, hubei province in china, and has spread rapidly around the world. infected patients have been reported in countries. this disease was declared as pandemic by the world health organization on march th (who) . sars-cov- is a positive single-stranded rna beta-coronavirus. like sars and mers, the genome encodes non-structural proteins such as chymotrypsin type protease, papain-like protease, helicase, and rna-dependent rna polymerase; and structural proteins such as spike glycoprotein and accessory proteins . so far, there are no vaccines that had been approved. however, just a few weeks after the first reports of the disease, several laboratories began investigating for a vaccine that immunizes against this virus, and approximately research laboratories and academic centres are currently doing so . vaccine development already has a starting point from the knowledge gained from the sars-cov and mers epidemics and the selection of antigens based on them. however, it would take several months for a vaccine to have a consistent safety profile across populations and to mimic the immune response . another option would be to develop new molecules that allow treating the disease according to its stage; however, this process could be even slower. furthermore, de novo drug development takes place in an environment where preclinical research findings may not be replicated . likewise, when researching new molecules in humans, it is necessary to ask several questions that could improve the designs, and avoid some failures, such as, for example, did the drug hit the target?, did the medication change the target?, what was the dose response?, and what are the characteristics of the study patients?. to answer these questions, you need financial resources and time, which in many occasions must be abundant . according to the advances in technology and knowledge of human disease, the transfer of these benefits in advance has great expectations, and moreover, the challenges of the pharmaceutical industry are great and exhausting . the costs and time required for the development of a medicine have made working with the industry less attractive to researchers , which is accentuated by the global economic crisis secondary to the pandemic. due to this, the strategy that could offer results in less time, with better levels of safety and at a lower cost is known as "drug repurposing". this strategy consists of identifying new uses of approved drugs different from the original therapeutic indication. . this strategy differs from "off label" use in that it requires research and development to gain regulatory agency approval . it has several advantages; first, the risk of toxicity failure is low because the drug is safe enough and has been tested in preclinical and early human studies. second, the time is reduced since preclinical and safety studies have already been carried out, and third, the necessary resources are less . these advantages can generate a quick return on investment ( million dollars in repositioning against - trillion dollars in the study of a new molecule) and in case of failure there is less loss . about medicines that have been described in human clinical trials are in the marketing phase around the world, thus, the possibility of selection is wide . three actors have been identified in the field of repurposing: the academy, the research institutes and, of course, the pharmaceutical industry, each with its own characteristics. in academia and research institutes, there is less need for economic or commercial success, but they depend on securing resources from the state or other funder. furthermore, the pharmaceutical industry has the complete platform to do the trials, but they are not always motivated because many countries do not recognize second-use patents . among the diseases in which researchers are using repositioning are cancer (thalidomide, metformin, chlorpromazine, digoxin, doxycycline) [ ] [ ] [ ] , neurodegenerative diseases such as parkinson's (ambroxol, amantadine) , viral and infectious diseases (azithromycin, mycophenolic acid) , , asthma and allergies (ruxolitinib, imatinib, metformin) , neuropathic pain (gabapentin, amitriptyline) , kidney disease (levosimendan, allopurinol) and cardiovascular-pulmonary disease (tadalafil, fasudil) [ ] [ ] [ ] . the best examples of drug repurposing are in the cardiovascular system, from the use of aspirin as a platelet antiplatelet to the approval of sildenafil, a drug previously used for erectile dysfunction that is now the first-line treatment in pulmonary hypertension. , . since the first reports of coronavirus's infections began to be known in late in china, studies have been started with drugs approved for other diseases. to date, there are studies registered at www.clinicaltrials.gov, of which are intervention studies that include clinical trials of repositioning in covid- , mostly from china. . chloroquine, which was initially approved for the treatment of malaria , and which has been studied in approximately diseases (with the first trial being carried out in ) , was evaluated for the treatment of sars-cov infection in and now in the sars-cov- pandemic . hydroxychloroquine, a chloroquine analog, is a medicine widely used in the treatment of systemic autoimmune diseases , being currently the most studied drug for treating covid- . hydroxychloroquine is metabolized in the liver and is eliminated via the kidneys after to days. it is a -aminoquinolone with immunosuppressive, anti-autophagic and antimalarial properties. although its mechanism of action is unknown, it can suppress immune function by interfering with antigen processing and presentation , . it is a medicine with a higher risk / benefit than chloroquine and allows higher doses to be used. in covid , clinical trials are registered, taking different degrees of severity, ranging from prophylactic use in the general population and in health workers to patients with severe acute respiratory syndrome (sars). it is believed that, as happened with sars-cov, it could reduce the viral load of patients in pre and post infection stages, in addition to reducing the expression of cd in t cells . the results obtained so far are controversial, since on the one hand it has been reported that treatment with hydroxychloroquine did not generate additional effects to standard treatment and instead increased the risk of ventilatory support , and on the other, french researchers documented that the addition of azithromycin to hydroxychloroquine treatment can generate synergistic effects by improving the elimination of the virus. it is necessary to clarify that the previously described are preliminary studies and that they require large sample sizes to generate a definitive conclusion . this mixture is being studied for treating patients with and without complications (table ) . antivirals are other group of drugs being tested to combat the pandemic. in wuhan, more than % of patients received medications such as oseltamivir, remdesivir, ganciclovir, lopinavir/ritonavir and ribavirin , . these antivirals have been combined with systemic corticosteroids and inhalation of interferon in the treatment of patients with severe complications . satisfactory results were achieved using remdesivir, obtaining clinical improvement in % of patients with sars, with a significant reduction in mortality, days of hospitalization and days of mechanical ventilation. this study was developed with patients from the united states, japan, italy, austria, france, germany, the netherlands, spain and canada , however, just as it happens with hydroxychloroquine, its effectiveness should be contrasted with more clinical trials and in more patients. immunotherapy has shown effectiveness in the treatment of infectious diseases; thus, the use of monoclonal antibodies constitutes a new stage in their prevention, due to its versatility and specificity. , . therapy with antibodies that bind to the angiotensin -converting enzyme , . currently there are trials registered in clinical trials that are testing this hypothesis, using monoclonal antibodies, which are being performed only in complicated patients, none in stable patients or in prophylaxis. famotidine, a histamine h receptor antagonist; have shown effects on inmune system ( ) it is currently being studied and results obtained in patients in china can generate positive expectations, currently in the united states, they are in the recruitment phase; the reduction of the mortality and its low probability of adverse effects generates an attractiveness for researchers ( , ) . glucocorticoids (methylprednisolone and dexamethasone) as monotherapy with discrete results , or accompanying other medications such as thalidomide ; antiinflammatories such as colchicine , or drugs that do not yet have a clear mechanism of action such as pirfenidone, are being studied to be repositioned in covid- . china and the united states lead most of the trials, however, countries such as italy, spain, the united kingdom, canada, and denmark also participate in the search for a medicine that could change the course of a crisis that can only be compared to the world wars of the early twentieth century. with all the limitations, repositioning tests are an attractive strategy, providing the possibility of combinations that seek synergism . the search for these treatments is an obligation acquired by science and public health. there are controversies about the maximum and minimum recommendations and the essential requirements to conduct a good clinical trial of drug repositioning. the infrastructure, the preparation of products, materials, protocols, forms of reporting, databases, statistics, monitoring and reports, as well as laboratory evaluations are often insufficient and their availability is reserved, a situation that is accentuated with the pandemic , therefore, the maximum recommendations should be flexible when approving studies. the covid- clinical research coalition initiative establishes flexible guidelines that allow research to be carried out. this initiative shows that the research agenda should not be controlled, but facilitated, for which objectives are proposed: first, to facilitate rapid reviews by ethics committees and regulatory agencies; second, to facilitate permits for the importation of medicines and materials, as was done with the ebola vaccine trials; third, to ensure simple data collection strategies sufficient to strengthen efficacy analyses; and fourth, to provide a reference framework for governments to share the results obtained before publishing in scientific journals . compliance with these objectives will allow studies to be carried out in shorter times and with enough resources to ensure quality. thus, the food and drug administration (fda) in the united states , and the invima in colombia, approved the use of hydroxychloroquine in hospitalized patients via fast track. to move forward and generate fast and reliable results, it might be considered doing a proof of concept, double blind, add-on event-driven. in add-on studies, one group receives the standard treatment, while the other group receive the drug to be tested in addition to the standard treatment. using these studies, the comparison of relative or absolute efficacy could be obtained in short periods of time . the fda recognizes add-on designs in cancer, behavioural disorders, hiv, and heart failure studies . in this methodology processes are developed over time and results are produced that then turn into events that ultimately lead to additional results as the narrative unfolds. . unlike most diseases, there are currently no proven treatments to treat covid- . therefore, there are registered studies that use placebo as a comparator of the drug to be repurposed [ ] [ ] [ ] . the ethics committees are expected to be willing to meet the needs that the ethical conflict the placebo group generates, with enough updating to control the ethical aspects without preventing the execution of the projects. probably there will be problems, one of them would be the entry of participants linked to the health system who could "sabotage" the study, taking drug repositioning for covid - the drug without belonging to the experimental group or trying another drug at the same time in the hope of avoiding a contagion or treating the illness. the dissemination of these findings should be responsible, avoiding anecdotal evidence . differences in plasma concentrations of medicines from person to person could generate more adverse reactions, and therefore, rapid clinical trials but with high scientific quality should be carried out . while the vaccine arrives, the repositioning of medicines will continue to be the first research strategy against this crisis that has changed the rhythm of life of humanity. covid- ) -events as they happen therapeutic options for the novel coronavirus ( -ncov) immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic challenges in early clinical development of adjuvanted vaccines organs-on-chips at the frontiers of drug discovery why drugs fail in clinical trials in pulmonary arterial hypertension, and strategies to succeed in the future an analysis of the attrition of drug candidates from four major pharmaceutical companies drug repurposing: progress, challenges and recommendations drug repositioning: identifying and developing new uses for existing drugs from off-label to repurposed drug in nononcological rare diseases: definition and state of the art in selected eu countries overcoming the legal and regulatory barriers to drug repurposing can you teach old drugs new tricks? the drug repurposing hub: a nextgeneration drug library and information resource drug repositioning for covid- drug repurposing from the perspective of pharmaceutical companies drug repurposing in cancer drug repurposing in anticancer reagent development repurposing vitamin d as an anticancer drug drug repurposing in parkinson's disease drug repurposing for viral infectious diseases: how far are we? recent drug-repurposing-driven advances in the discovery of novel antibiotics drug repurposing to treat asthma and allergic disorders: progress and prospects drug repurposing for the development of novel analgesics drug repurposing in kidney disease repurposing treatments to enhance innate immunity. can statins improve neutrophil functions and clinical outcomes in copd? pleiotropic mechanisms of action of perhexiline in heart failure targeting phosphodiesterase as a therapeutic option against myocardial ischaemia/reperfusion injury and for treating heart failure aspirin for primary prevention of coronary disease sildenafil in the treatment of pulmonary hypertension. vasc health risk manag a bibliometric review of drug repurposing covid -list results -clinicaltrials.gov malaria chemotherapeutics part i: history of antimalarial drug development, currently used therapeutics, and drugs in clinical development activity of a new antimalarial agent, chloroquine (sn ) drug repositioning for covid- in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine: a multifaceted treatment in lupus hydroxychloroquine compound summary hydroxychloroquine: an update use of hydroxychloroquine and chloroquine during the covid- pandemic: what every clinician should know covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression clinical outcomes of hydroxychloroquine in hospitalized patients hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial treatment of covid- : old tricks for new challenges covid- ): the epidemic and the challenges critical care response to a hospital outbreak of the -ncov infection in shenzhen compassionate use of remdesivir for patients with severe covid- evaluation of human monoclonal antibody r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants monoclonal antibodies for prophylactic and therapeutic use against viral infections the molecular biology of coronaviruses perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease- (covid- ) immunomodulatory properties of cimetidine: its therapeutic potentials for treatment of immune-related diseases delivering benefits at speed through real-world repurposing of off-patent drugs: the covid- pandemic as a case in point new york clinical trial quietly tests heartburn remedy against coronavirus. science; effectiveness of glucocorticoid therapy in patients with severe novel coronavirus pneumonia: protocol of a randomized controlled trial thalidomide combined with low-dose glucocorticoid in the treatment of covid- pneumonia the greek study in the effects of colchicine in covid- complications prevention (grecco- study): rationale and study design race to find covid- treatments accelerates clinical trials on drug repositioning of covid- access to pathology and laboratory medicine services: a crucial gap covid- : what science advisers must do now global coalition to accelerate covid- clinical research in resource-limited settings fact sheet for health care providers emergency use authorization (eua) of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid- in certain hospitalized patients statistical approaches to analysis of small clinical trials event-driven research transplantation of ace -mesenchymal stem cells improves the outcome of patients with covid- pneumonia drug repositioning for covid- hydroxychloroquine versus placebo in covid- patients at risk for severe disease university hospital, angers evaluation of the efficacy and safety of sarilumab in hospitalized patients with covid- scientists identify drugs to test against the coronavirus key: cord- -aq s x authors: andersen, petter i.; ianevski, aleksandr; lysvand, hilde; vitkauskiene, astra; oksenych, valentyn; bjørås, magnar; telling, kaidi; lutsar, irja; dumpis, uga; irie, yasuhiko; tenson, tanel; kantele, anu; kainov, denis e. title: discovery and development of safe-in-man broad-spectrum antiviral agents date: - - journal: international journal of infectious diseases doi: . /j.ijid. . . sha: doc_id: cord_uid: aq s x abstract viral diseases are one of the leading causes of morbidity and mortality in the world. virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. however, broad-spectrum antiviral agents (bsaas, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. here, we review discovery and development of bsaas and summarize the information on safe-in-man agents in a freely accessible database (https://drugvirus.info/). future and ongoing pre-clinical and clinical studies will increase the number of bsaas, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections. viruses are one of the major causes of morbidity and mortality in the world (dalys and collaborators, ; disease et al., ; howard and fletcher, ; who, ) . antiviral drugs and vaccines are used to fight viral infections in human (de clercq and li, ; marston et al., ) . previously, there has been a focus on "one drug, one virus" dogma, which relied on targeting virusspecific factors. a counterpoint to this is the "one drug, multiple viruses" paradigm, which came with the discovery of broadspectrum antiviral agents (bsaas), small-molecules that inhibit a wide range of human viruses (bekerman and einav, ; de clercq and montgomery, ; debing et al., ; ianevski et al., ; rada and dragun, ; sidwell et al., ) . this paradigm was based on the observation that different viruses utilize similar pathways and host factors to replicate inside a cell (bosl et al., ) . although the concept of bsaas has been around for almost years, the field received a new impetus with recent outbreaks of ebola, zika, dengue, influenza and other viral infections, the discovery of novel host-directed agents, as well as development of drug repositioning methodology. drug repurposing, also called repositioning, redirecting, reprofiling, is a strategy for generating additional value from an existing drug by targeting disease other than that for which it was originally intended (nishimura and hara, ; pushpakom et al., ) . this has significant advantages over new drug discovery since chemical synthesis steps, manufacturing processes, reliable safety, and pharmacokinetic properties in pre-clinical (animal model) and early clinical developmental phases (phase , i and iia) are already available (figure ) . therefore, repositioning of launched or even failed drugs to viral diseases provides unique translational opportunities, including a substantially higher probability of success to market as compared with developing new virus-specific drugs and vaccines, and a significantly reduced cost and timeline to clinical availability pizzorno et al., ; zheng et al., ) . here, we detail the steps of bsaa repurposing, from discovery of novel antiviral activities in cell culture to post-market studies. moreover, we summarized currently available information on bsaas in freely available database, focusing on those antivirals, which have been already tested in human as antivirals, antibacterials, antiprotozoals, anthelmintics, etc. finally, we discuss future perspectives of using safe-in-man bsaas for treatment of emerging and re-emerging viral infections as well as viral and bacterial co-infections. discovery of novel bsaa activities in immortalized cell cultures and co-cultures the discovery of novel activities of bsaas starts with exposing cells to the candidate antiviral agent at different concentrations and infecting the cells with a virus or mock. immortalized cancerous cell cultures and co-cultures, which express appropriate viral receptors, are most commonly used in this first step. the halfmaximal cytotoxic concentrations (cc ) for a compound are calculated based on their dose-response curves obtained on mockinfected cells. the half-maximal effective concentrations (ec ) are calculated based on the analysis of curves obtained on infected cells. statistical analyses can help to determine if the differences between cc and ec are significant, given the inherent variability of the experiment (meneghini and hamasaki, ) . a relative effectiveness of a drug is defined as selectivity index (si = cc /ec ). cell viability assays and cell death assays are commonly used to assess the cytotoxicity and efficacy of bsaas ( figure a ). cell viability assays include mtt, mts, resazurin or similar assays, mitochondrial membrane potential-dependent dyes-based assays, esterase cleaved dye-based assays, atp-adp assays, and assays that measure glycolytic flux and oxygen consumption. other cell death assays include ldh enzyme leakage assays, membrane impermeable dye-based assays, and apoptosis assays, such as annexin v, tunel, and caspase assays (shen et al., ) . for example, the cell titer glo (ctg) assay quantifies atp, an indicator of metabolically active living cells, whereas cell tox green assay uses fluorescent asymmetric cyanine dye that stains the dna of dead cells (bosl et al., ; bulanova et al., ; ianevski et al., ; muller et al., ) . viral strains or cell lines expressing reporter proteins are also used to assess the efficacy of bsaas in infected cells. for example, tzm-bl cells expressing firefly luciferase under control of hiv- ltr promoter allowed quantitation of bsaa action on hiv- infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay (sarzotti-kelsoe et al., ; xing et al., ) . rfp-expressing rvfv, nanoluc-expressing chikv and rrv, as well as gfp-expressing fluav, hcv and hmpv also allowed identification of novel activities of several bsaas (andersen et al., b; bosl et al., ; de graaf et al., ; habjan et al., ; ianevski et al., ; jupille et al., ; kittel et al., ; lee et al., ; utt et al., ) . in addition, qpcr/rt-qpcr, rna/dna sequencing, rna/dna hybridization, immuno-and plaque assays as well as crispr-cas systems could be used for detection of inhibitory effects of bsaas (boonham et al., ; fischer et al., ; konig et al., ; laamiri et al., ; landry, ; perez et al., ; sashital, ; zhou et al., ) . interestingly, crispr-cas , sirna and shrna approaches were used for identification of bsaa targets (deans et al., ; puschnik et al., ) . novel anti-hsv- and anti-ev activities of emetine were discovered recently using ctg/plaque assays in human nonmalignant rpe cells. moreover, novel antiviral activities of the drug were identified using rfp-expressing rvfv, and gfp-expressing hmpv or fluav strains in rpe cells (andersen et al., a) . given that emetine also inhibits zikv, ebov, rabv, cmv, hcov-oc and hiv- infections (chaves valadao et al., ; macgibeny et al., ; mukhopadhyay et al., ; shen et al., ; yang et al., ) , and that it is an fda-approved anti-protozoal drug, it may represent a promising safe-in-man bsaa candidate. immortalized cell cultures/co-cultures and reporter viral strains represent excellent model systems for the discovery of novel activities of safe-in-man bsaas. however, these genetically modified systems have certain limitations (attenuated or incomplete virus replication cycle, accumulation of mutations during repeated cell and virus passaging, defective innate immune responses and viral counter-responses, etc.) (carter and shieh, ) . thereby, novel antiviral activities of bsaas should be further validated in primary human cells using different viral strains (including wild-type viruses), different viral loads, different times of compound addition, different endpoint measurements and compound concentration range. primary cell cultures give more accurate images of drug responses (alves et al., ; denisova et al., ; koban et al., ; postnikova et al., ) . they have a low population doubling level and therefore more closely recapitulate the physiological conditions observed in vivo. primary cells are cells isolated directly from tissues or blood using enzymatic or mechanical methods. the cells are characterized by their high degrees of specialization, are often fully differentiated and thus require defined culture conditions (serum-free media) in order to preserve their original phenotype. peripheral blood mononuclear (pbmc), placental, amniotic and fetal primary cultures as well as vaginal/cervical epithelial and male germ cells have been used intensively to validate bsaa activity (barrows et al., ; denisova et al., ; fink et al., ; rausch et al., ; robinson et al., ) . although primary cell cultures are relevant systems for validation of bsaas, there are technical difficulties limiting their use, such as ethical issues, purity of population of primary cells, and limited shelf life of the cells. in addition, age, race, sex and other genetic and epigenetic factors of donor cells should be considered to determine common biological effect across a significant number of donors thereby avoiding minor variants (lee et al., ; zhang et al., ) . the obstacles associated with use of human primary cell cultures can be bypassed using human embryonic stem cells (escs) and human induced pluripotent stem cells (ipscs). escs are isolated from surplus human embryos, whereas ipscs are obtained by reprogramming somatic cells. these cells proliferate extensively and retain multi-lineage activity, which allows them to generate virtually any cell type of the body. the escs-and ipsc-derived cells have been used successfully to investigate the efficacy of several bsaas against hbv, zikv, chikv and hsv- infections (table s ) (ferreira et al., ; iwasawa et al., ; lanko et al., ; simonin et al., ; xia et al., ; zhou et al., ) . ipscs, escs and primary tissue cells can be used to generate complex cultures termed organoids. organoids are miniature and simplified version of organs. establishing human airway, gut, skin, cerebral, liver, kidney, breast, retina and brain organoids allowed researchers to study toxicity and efficacy of several safe-in-man bsaas against coronaviruses, influenza, enteroviruses, rotaviruses and flaviviruses sacramento et al., ; watanabe et al., ; xu et al., ; yin et al., ; yin et al., ; yin et al., ; zhou et al., ) . however, ipscs, escs and ipscs/escsderived organoids have the same disadvantages as human primary cells (genetic differences, line-to-line and organoid batch-to-batch variability). on the other hand, these models allow researchers to predict the behavior of viruses in vivo and, therefore, to reduce animal use and in cases where animal models are unavailable to initiate clinical trials. for example, novel anti-zikv activities of enoxacin, amodiaquine and niclosamide were discovered recently using human neural progenitor cells, human pluripotent stem cell-derived cortical neural progenitor cells, and human induced neural stem cells, respectively (cairns et al., ; xu et al., ; zhou et al., ) . enoxacin is an oral broad-spectrum fluoroquinolone antibiotic, which also possesses anti-hcv and anti-hiv- activities in immortalized cell cultures (kashiwase et al., ; young et al., ) . amodiaquine is an anti-malaria drug, which also possesses antiviral activities against denv, hcv, rrv, sinv, wnv, efv, ebov, lasv, rabv, vzv, and hsv- in immortalized cell cultures (boonyasuppayakorn et al., ; hulseberg et al., ; mazzon et al., ) . niclosamide is an orally bioavailable anthelmintic drug which inhibits the broadest range of viruses in vitro and, in if the drug is repositioned from another disease (i.e. its safety profile in man is available) it could bypass the pk and safety studies in man. some cases, in vivo (cairns et al., ; fang et al., ; huang et al., ; hulseberg et al., ; jurgeit et al., ; kao et al., ; mazzon et al., ; stachulski et al., ; wang et al., ; wu et al., ) . these safe-in-man bsaas represent promising drug candidates. in vitro and ex vivo models do not fully reflect the complexity and physiology of living organisms. therefore, several in vivo models have been developed to test novel antiviral activities of bsaas. these include immunocompetent and genetically or chemically immunocompromised mice, guinea pigs, hamsters, ferrets, pigs, macaques and other animals ( figure b ) (alves et al., ; haese et al., ; louz et al., ; morrison and diamond, ; taylor, ; thangavel and bouvier, ) . pk/pd studies determine drug absorption, dosage and half-life of bsaas. toxicological studies determine if the drugs have any adverse effects on the tissues and organs of the animals and define the dosage of adverse effects (alabaster and in vivo pharmacology training group, ; parasuraman, ; rizk et al., ) . studying the efficacy of bsaas is generally done by treating the animal with the drug or vehicle and infecting it with a virus of interest. endpoints are usually body weight/ mortality (depending on the virus), histopathology, virus titers in organs, presence of clinical signs and development of immunity (oh and hurt, ; smee and barnard, ) . although animal models can give the initial characterization of bsaa, it is important to keep in mind that they differ significantly from humans, with respect to symptoms, disease manifestation, susceptibility, immune responses, pathogenesis, and pharmacokinetics (barré-sinoussi and montagutelli, ; shanks et al., ) . often animals require higher concentration of an experimental antiviral as compared to effective in vitro concentrations. moreover, it is relatively difficult to achieve micromolar ec in vivo. for example, aminoglycoside antibiotics, kasugamycin and neomycin were successfully tested against zikv, fluav, and hsv- infections in mice (gopinath et al., ) . polyether antibiotic salinomycin also showed anti-fluav effect in mice (jang et al., ) . in addition, investigational anticancer agent, flavopiridol, was effective against fluav in mice . these findings support further development of these and other bsaas. clinical trials are the most critical and time-consuming step of a drug candidates' journey to being approved ( figure c ). however, safe-in-man bsaas make this journey relatively short, because they have been already at phase , i and, sometime, at iia of clinical trials as antibacterial, antiprotozoal, anticancer, etc. agent; i.e. they have been administered at sub-therapeutic doses to healthy volunteers to ensure the drugs are not harmful to the participants. thus, safe-in-man bsaas enter phase ii and iii, which assess the efficacy, effectiveness, safety and side effects of the drugs in clinic. it is important, however, to differentiate acute and chronic viral infections when repurposing bsaas, given that drug concentrations and duration of the treatment could be different, and therefore, drug safety issues should be considered. for phase ii, patients with the viral disease in question are invited to join the study, where they are administered the bsaas at the ideal therapeutic doses. phase iii is the longest of the phases, and includes multiple levels of securities to the studies, such as the use of placebos and double-blinded studies, to ensure the data is as unbiased as possible. upon completing phase iii, depending on its performance and efficacy, bsaas may end either being approved or dropped. the u.s food and drug administration (fda) estimates that only - % bsaa candidates that enter phase iii are approved for use in the public (u.s food and drug administration, ). after approval and marketing of the drug, phase iv may be initiated to follow up on the use of the drug in public, to surveil for rare effects (u.s food and drug administration, ; umscheid et al., ) . forty-eight safe-in-man bsaas undergo clinical studies as antivirals. there are currently compounds in phase i, agents in phase ii and compounds in phase iii clinical trials. for example, nitazoxanide, remdesivir and brincidofovir are under clinical investigations against different viral infections (nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct ). twenty-one bsaas were approved by fda, ema or other agencies. these bsaas altogether target viruses. for example, favipiravir, also known as t- , was approved against fluav in japan; cidofovir is an injectable antiviral medication used as a treatment for cmv retinitis in people with aids; ribavirin, also known as tribavirin, is used for treatment of rsv and hcv infections; pleconaril is used against viruses in the picornaviridae family, including enterovirus and rhinovirus; and valacyclovir is used against cmv, ebv, hbv, hsv- , hsv- and vzv infections. twenty bsaas are undergoing surveillance studies (phase iv). azithromycin, chloroquine, cyclosporine, ezetimibe, mycophenolic acid, nitazoxanide and rapamycin progressed to phase iv studies without approvals from national or international authorities (nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct , nct ). we have developed a database for safe-in-man bsaas, which is available at https://drugvirus.info/ (figure ) . the drug annotations were obtained from pubchem, drugbank, drugcentral, pubmed and clinicaltrials.gov databases (table s ) ursu et al., ; wishart et al., ) . the information on virus families was exported from virus pathogen database and analysis resource (table s ) (pickett et al., ) . the database summarizes activities and developmental status of bsaas. we decided to set no limits for ec , si and statistical significance because the studies were not harmonized (different cell lines, assays, end-point measurements, time of compound addition, etc.). the database allows interactive exploration of virus-bsaa interactions. it also includes information on bsa targets. a feedback form is available on the website. the database will be updated upon request or as soon as a new safe-in-man bsaa emerges or novel activity for an existing bsaa is reported. altogether, the database contains approved, investigational and experimental safe-in-man bsaas, which inhibit human viruses, belonging to viral families. the bsaas inhibit viral or host factors and block viral replication, reduce the viral burden to a level at which host immune responses can deal with it or facilitate apoptosis of infected cells (table s ). analysis of bsaa targets and structures (figure ) revealed that the most abundant are nucleotide and nucleoside analogues which inhibit viral rna and dna polymerases. imatinib, erlotinib, gefitinib, and dasatinib, which inhibit tyrosine kinases, are the most abundant hostdirected bsaas. most of the host targets (except bcl-xl protein) are essential for viral replication but redundant for the cell, which is critical for reducing putative toxicities associated with blocking cellular pathways. the limited diversity of the targets and scaffolds could slow down the development of bsaa concept. emerging bsaas, such as , -dimethoxyindan- -one, saliphenylhalamide, and gs- (denisova et al., ; kuivanen et al., ; muller et al., ; muller et al., ; patil et al., ; sheahan et al., ) , whose safety profiles in humans are not yet available, are not included in the database. however, they could serve as valuable antivirals in the future, pending the results of further pre-clinical and clinical investigations. sars-cov- is a novel strain of coronaviruses which is associated with a cluster of cases of pneumonia in china (zhou et al., ) . coronaviruses (cov) are a broad family of virusesthat includes sars-cov, mers-cov, hcov- e, hcov-oc , hcov-nl and hcov-hku strains. hcov- e, hcov-oc , hcov-nl and hcov-hku strains are usually associated with mild, self-limiting upper respiratory tract infections, such as the common cold. by contrast, people infected with mers-cov, sasrs-cov or sars-cov- could develop severe respiratory illness, and many of the infected have died. no vaccines and drugs are available for prevention, prophylaxis and treatment of coronavirus infections in humans (eurosurveillance editorial, ) . however, safe-in-man bsaas could be effective against sars-cov- and other coronaviruses ( figure ). for example, teicoplanin, oritavancin, dalbavancin, monensin and emetine could be repurposed for treatment of covid- . teicoplanin, ritavancin, dalbavancin and monensin are approved antibiotics, whereas emetine is an anti-protozoal drug. these drugs have been shown to inhibit several corona-as well as some other viral infections . moreover, chloroquine and remdesivir were shown to effectively inhibit sars-cov- infection in vitro. in addition, clinical investigations into the effectiveness of lopinavir, ritonavir, remdesivir, hydroxychloroquine and arbidol against covid- have started recently (nct ; nct ; nct ; nct , nct ). bsaas could be combined with other antiviral agents to obtain synergistic or additive effects against certain viruses zheng et al., ) . several combination therapies which include bsaas (such as abacavir/dolutegravir/lamivudine (triumeq), darunavir/cobicistat/emtricitabine/tenofovir (symtuza), lopinavir/ritonavir (kaletra), ledipasvir/sofosbuvir and sofosbuvir/velpatasvir) became a standard for the treatment of hiv or hcv infections. several synergistic drug combinations, such as obatoclax/saliphenylhalamide and gemcitabine/pimodivir, could enter clinical studies and become effective treatment of zikv and fluav infections (fu et al., ; kuivanen et al., ) . by contrast to individual drugs, combinations of - bsaas could be used to target an even broader range of viruses (foucquier and guedj, ; zheng et al., ) . such combinations could serve as front line therapeutics against poorly characterized emerging viruses or re-emerging drug-resistant viral strains. for example, a cocktail of nitazoxanide, favipiravir, and niclosamide could be developed for the treatment of viruses belonging to families. fifty bsaas possess not only antiviral but also antibacterial activity (figure ; table s ) (schor and einav, ) . moreover, of the agents are approved as antibiotics ( withdrawn). these agents with dual activity could be used for treatment of viral and bacterial co-infections or for the protection of patients from the secondary infections. for example, azithromycin could be used against fluav and chlamydophila pneumoniae, haemophilus influenzae, mycoplasma pneumoniae or streptococcus pneumoniae infections (nct ) (mandell et al., ) . in addition, bsaas showed activity against a wide range of other medically important human pathogens, including fungi, protozoa and parasites (table s ) (montoya and krysan, ) , pointing out that some pathogens utilize common mechanisms to infect hosts. moreover, structure-activity relationship analysis of bsaas suggests that some agents, such as doxycycline, artesunate, omeprazole, nitazoxanide, suramin, azithromycin, minocycline and chloroquine, could have novel antibacterial, antiprotozoal, antifungal or anthelmintic activities (figure ). if confirmed, this could lead to development of broad-spectrum anti-infective drugs. bsaas could also serve as treatment of other co-morbidities, therefore, simplifying the therapy and lowering its cost (table s ). for example, the concomitant actions of ezetimibe and statins could be beneficial for treatment of both hypertension and several viral infections in patients with these co-morbidities (nct , nct , nct , nct , nct , nct ). here we reviewed a process of bsaa development and summarized information on safe-in-man agents in freely available database. we hope that further pre-clinical and clinical studies on bsaas will be harmonized, and data collection will be standardized. furthermore, the follow-up studies as well as the results of on-going, finalized or terminated clinical trials will be made publicly available to allow prioritization and translation of emerging and existing bsaas into clinical practice. this would allow bsaas to play a pivotal role in the battle against emerging and re-emerging viral diseases. discovery of novel as well as repositioning existing safe-in-man bsaas may shorten time and resources needed for development of virus-specific drugs and vaccines. in the future, bsaas will have a global impact by decreasing morbidity and mortality from viral and other diseases, maximizing the number of healthy life years, improving the quality of life of infected patients and decreasing the costs of patient care. the authors declare no conflict of interest. this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to d.k.). approval was not required. we thank katarzyna kolasa for illustrations. this manuscript has been released as a pre-print (doi: https:// . /pre-prints . .v ). we thank the european regional development fund, the mobilitas pluss project mobtt . figure . safe-in-man broad-spectrum antiviral agents and coronaviruses they inhibit. a snapshot is taken from https://drugvirus.info/ website. different shadings indicate different development status of bsaas. grey shading indicates that the antiviral activity has not been either studied or reported. the fall and rise of in vivo pharmacology research models and tools for the identification of antivirals and therapeutics against zika virus infection novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine animal models are essential to biological research: issues and perspectives a screen of fda-approved drugs for inhibitors of zika virus infection infectious disease. combating emerging viral threats methods in virus diagnostics: from elisa to next generation sequencing amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity common nodes of virus-host interaction revealed through an integrated network analysis antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl- proteins niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells chapter -cell culture techniques natural plant alkaloid (emetine) inhibits hiv- replication by interfering with reverse transcriptase activity improving therapy of severe infections through drug repurposing of synergistic combinations global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study approved antiviral drugs over the past years broad-spectrum antiviral activity of the carbocyclic analog of -deazaadenosine an improved plaque reduction virus neutralization assay for human metapneumovirus parallel shrna and crispr-cas screens enable antiviral drug target identification the future of antivirals: broad-spectrum inhibitors obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries and territories, - : a systematic analysis for the global burden of disease study note from the editors: world health organization declares novel coronavirus ( -ncov) sixth public health emergency of international concern identification of three antiviral inhibitors against japanese encephalitis virus from library of pharmacologically active compounds beyond members of the flaviviridae family, sofosbuvir also inhibits chikungunya virus replication lineagespecific real-time rt-pcr for yellow fever virus outbreak surveillance analysis of drug combinations: current methodological landscape jnj inhibits influenza a virus replication without altering cellular antiviral responses topical application of aminoglycoside antibiotics enhances host resistance to viral infections in a microbiota-independent manner t rna polymerase-dependent and -independent systems for cdna-based rescue of rift valley fever virus animal models of chikungunya virus infection and disease emerging virus diseases: can we ever expect the unexpected? niclosamide inhibits lytic replication of epstein-barr virus by disrupting mtor activation arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses expanding the activity spectrum of antiviral agents novel activities of safe-in-human broad-spectrum antiviral agents increased cytotoxicity of herpes simplex virus thymidine kinase expression in human induced pluripotent stem cells salinomycin inhibits influenza virus infection by disrupting endosomal acidification and viral matrix protein function mutations in nsp and pe are critical determinants of ross river virus-induced musculoskeletal inflammatory disease in a mouse model niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects the antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mtor a new fluoroquinolone derivative exhibits inhibitory activity against human immunodeficiency virus type replication pubchem update: improved access to chemical data rescue of influenza virus expressing gfp from the ns reading frame a novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells efficient long-term amplification of hepatitis b virus isolates after infection of slow proliferating hepg -ntcp cells obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism a multiplex real-time rt-pcr for simultaneous detection of four most common avian respiratory viruses nucleic acid hybridization in viral diagnosis replication of the zika virus in different ipsc-derived neuronal cells and implications to assess efficacy of antivirals a novel inhibitor idpp interferes with entry and egress of hcv by targeting glycoprotein e in a genotype-specific manner common genetic variants modulate pathogen-sensing responses in human dendritic cells -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model animal models in virus research: their utility and limitations retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults emerging viral diseases: confronting threats with new technologies identification of broad-spectrum antiviral compounds by targeting viral entry the electroretinogram of the iguana and tokay gecko repurposing estrogen receptor antagonists for the treatment of infectious disease animal models of zika virus infection, pathogenesis, and immunity efficacy and mechanism of action of low dose emetine against human cytomegalovirus the proton translocation domain of cellular vacuolar atpase provides a target for the treatment of influenza a virus infections inhibition by cellular vacuolar atpase impairs human papillomavirus uncoating and infection editorial: drug repositioning: current advances and future perspectives using the ferret as an animal model for investigating influenza antiviral effectiveness toxicological screening identification of novel , -dimethoxyindan- -one derivatives as antiviral agents insertion of a gfp reporter gene in influenza virus virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs a crispr toolbox to study virus-host interactions drug repurposing: progress, challenges and recommendations antiviral action and selectivity of -azauridine screening bioactives reveals nanchangmycin as a broad spectrum antiviral active against zika virus importance of drug pharmacokinetics at the site of action male germ cells support long-term propagation of zika virus the clinically approved antiviral drug sofosbuvir inhibits zika virus replication optimization and validation of the tzm-bl assay for standardized assessments of neutralizing antibodies against hiv- pathogen detection in the crispr-cas era combating intracellular pathogens with repurposed host-targeted drugs are animal models predictive for humans broadspectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses broadspectrum antiviral activity of virazole: -beta-d-ribofuranosyl- , , -triazole- -carboxamide zika virus induces strong inflammatory responses and impairs homeostasis and function of the human retinal pigment epithelium methods for evaluation of antiviral efficacy against influenza virus infections in animal models phosphoproteomics to characterize host response during influenza a virus infection of human macrophages thiazolides as novel antiviral agents. . inhibition of hepatitis c virus replication animal models of respiratory syncytial virus infection animal models for influenza virus pathogenesis, transmission, and immunology food and drug administration. the drug development process: step key concepts of clinical trials: a narrative review drugcentral : an update versatile trans-replication systems for chikungunya virus allow functional analysis and tagging of every replicase protein antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission self-organized cerebral organoids with human-specific features predict effective drugs to combat zika virus infection who publishes list of top emerging diseases likely to cause major epidemics drugbank . : a major update to the drugbank database inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide human stem cellderived hepatocytes as a model for hepatitis b virus infection, spreading and virus-host interactions comparison of three quantification methods for the tzm-bl pseudovirus assay for screening of anti-hiv- agents identification of smallmolecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry modeling rotavirus infection and antiviral therapy using primary intestinal organoids -thioguanine inhibits rotavirus replication through suppression of rac gdp/gtp cycling mycophenolic acid potently inhibits rotavirus infection with a high barrier to resistance development small molecule modifiers of microrna mir- function for the treatment of hepatitis c virus infection and hepatocellular carcinoma interferon-induced transmembrane protein- genetic variant rs -c is associated with severe influenza in chinese individuals drug repurposing screens and synergistic drugcombinations for infectious diseases the applications of crispr/cas system in molecular detection a pneumonia outbreak associated with a new coronavirus of probable bat origin glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat originnature highcontent screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult braincell stem cell supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.ijid. . . . key: cord- -pn j authors: sahakijpijarn, sawittree; moon, chaeho; koleng, john j.; christensen, dale j.; williams, robert o. title: development of remdesivir as a dry powder for inhalation by thin film freezing date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: pn j remdesivir exhibits in vitro activity against sars-cov- and was granted approval for emergency use. to maximize delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (tff). tff produces brittle matrix nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. in vitro aerodynamic testing demonstrated that drug loading and excipient type affected the aerosol performance of remdesivir. remdesivir combined with optimal excipients exhibited desirable aerosol performance (up to . % fpf; . μm mmad). remdesivir was amorphous after the tff process, which benefitted drug dissolution in simulated lung fluid. tff remdesivir formulations are stable after one-month storage at °c/ %rh. in vivo pharmacokinetic evaluation showed that tff-remdesivir-leucine was poorly absorbed into systemic circulation while tff-remdesivir-captisol® demonstrated increased systemic uptake compared to leucine. remdesivir was hydrolyzed to the nucleoside analog gs- in lung, and levels of gs- were greater in lung with the leucine formulation compared to captisol®. in conclusion, tff technology produces high potency remdesivir dry powder formulations for inhalation suitable to treat patients with covid- on an outpatient basis and earlier in the disease course where effective antiviral therapy can reduce related morbidity and mortality. the coronavirus disease (covid- ) is an ongoing worldwide pandemic. as of september , laboratory-confirmed cases have been reported in countries and territories with more than million reported cases and close to million reported deaths [ ] . although this disease of antiviral activity in the lungs, and limit the potential for systemic side effects [ ] . in addition, the cost of the drug can be reduced, and the supplies of the drug can be maximized, thus treating more patients due to less dose required by inhalation as compared to injectable forms. the treatment cost can also be decreased when administered by inhalation, since patients may not need to visit hospitals as is required to administer the iv injectable dose. therefore, more affordable and early stage treatment can be provided to patients with inhaled remdesivir. nebulization of the current iv formulation in a diluted form is a potential method of pulmonary administration; however, the drug is prone to degrade by hydrolysis in aqueous solution to form the nucleoside monophosphate, which has difficulty penetrating cell membranes, thereby minimizing the antiviral activity in the lung cells [ ] . another concern is the use of sbecd as an excipient in although several techniques have been used to prepare inhalable powders, including mechanical milling and spray drying, the advantages of thin film freezing (tff) over other techniques of rely on the ability to produce aerosolizable particles composed of brittle matrix, nanostructured aggregates. these are high surface area powders that are ideally suited for dry powder inhalation. tff employs ultra-rapid freezing (on the order of - , k/sec) such that precipitation (either as a crystalline nanoaggregate or amorphous solid dispersion) and particle growth of the dissolved solute can be prevented [ ] . subsequently, nanostructured aggregates are formed as a low-density brittle matrix powder [ ] , which is efficiently sheared into respirable low-density microparticles by a the ef was calculated as the total amount of remdesivir emitted from the device as a percentage of the pan with a t-zero hermetic lid were crimped, and a hole was drilled in the lid before placing the the dispersion was adjusted to ml by adding m hbss). the dispersion was frozen at - c for intratracheal administration was carried out using the dry powder insufflator (dp- m model, penn-century inc., philadelphia, pa) connected to the air pump (ap- model, penn-century inc., philadelphia, pa the aerodynamic particle size distribution of tff remdesivir formulations was evaluated using a plastiape® rs high resistance dpi and ngi apparatus. figure and h-nmr was performed to identify interactions between remdesivir and excipients. figure b demonstrates an expansion of h-nmr spectra for selected tff remdesivir powder formulations and remdesivir unprocessed powder. while the peak at . ppm is sharp and does not show any differences from the presented samples, the peaks at . and . ppm of f exhibited broader peak. also, these peaks were slightly shifted to downfield. figure c shows the rs high resistance monodose dpi is a capsule-based dpi device that is available for commercial product development, and it functions to disperse the powder based on impaction force. a previous study confirmed that this impact-based dpi can disperse low-density brittle matrix powders made by tff process into respirable particles better than a shear-based dpi (e.g., handihaler®) [ ] . another study also evaluated the performance of different models of the monodose dpi (rs and rs ) on the aerosol performance of brittle matrix powders containing voriconazole nanoaggregates prepared by tff [ ] . it was shown that the rs device exhibited better powder shearing and deaggregation through smaller holes of the capsule created by the piercing system of the rs device [ ]. therefore, the rs high resistance plastiape® dpi was selected for this study. we found that excipient type and drug loading affect the aerosol performance of tff remdesivir powder compositions. overall, the aerosol performance of tff remdesivir powders increased as the drug loading was increased, a highly desirable feature. this trend is obvious for the captisol®-, lactose-, and mannitol-based formulations when the drug loading was increased from % to %. furthermore, high potency tff remdesivir powder without excipients (f and f ) also exhibited high fpf and small mmad, which indicates remdesivir itself has a good dispersing ability without the need of a dispersing excipient when prepared using the tff process. this shows that the tff technology can be used to minimize the need of excipient(s) in the formulation, thus maximizing the amount of remdesivir being delivered to the lungs by dry powder inhalation. the aerosol performance of the leucine-based formulations did not significantly change when the drug loading was increased from % to %, and these formulations exhibited superior aerosol performance compared to the other excipient-based formulations studied in this paper. this is likely one potential concern related to the amorphous drug is physical instability due to its high energy state. according to criteria described by wyttenbach in addition to the physical stability, remdesivir, as a prodrug, is prone to degrade by hydrolysis in aqueous solution. since an organic/water co-solvent system is required to dissolve the drug and excipients in the tff process, chemical stability is another concern during preparation. nmr spectra demonstrated that remdesivir did not chemically degrade as a result of the tff process. even though remdesivir was exposed to binary co-solvent systems consisting of water during the process, the entire tff process used to produce remdesivir dry powder inhalation formulations did not induce chemical degradation of the parent prodrug. furthermore, remdesivir was chemically stable by hplc (data not shown) and nmr after one-month storage at c/ %rh. since remdesivir is a poorly water-soluble drug, its dissolution may be a critical factor of drug release in the lung fluid, especially in high drug load formulations. undissolved particles can be cleared by mucociliary clearance or macrophage uptake, causing lower drug concentration, lung irritation, and inflammatory response [ ] . therefore, a dissolution test was evaluated in this study. the dissolution profile demonstrated that physical form of drug appears to have a significant effect generally, even though leucine is a hydrophobic amino acid, it is a water-soluble excipient that has higher solubility than poorly water-soluble drugs like remdesivir. therefore, the interactions between drug and leucine at the molecular level can increase the dissolution rate of poorly water-soluble drug [ , ] , like that observed in our study . significantly higher plasma levels of gs- than remdesivir were observed in our rat pharmacokinetic study. the half-life of remdesivir is reportedly much shorter than that of gs- . while half-life of gs- is approximately . hours, for remdesivir it is only about hour in humans following multiple once-daily iv administrations [ ] . in rats, the half-life of remdesivir in plasma is reported to be less than . minute, which is much shorter than that in humans, due to we have demonstrated that low density, highly porous brittle matrix particles of remdesivir covid- dashboard by the center for systems science and engineering (csse) at johns a review of coronavirus disease- (covid- ) summary on compassionate use: remdesivir gilead arguments in favour of remdesivir for treating sars-cov- infections current knowledge about the antivirals remdesivir (gs- ) and gs- as therapeutic options for coronaviruses therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus clinical benefit of remdesivir in rhesus macaques infected with sars-cov- gs- and its parent nucleoside analog inhibit filo-, pneumo- , and paramyxoviruses coronavirus susceptibility to the antiviral remdesivir mediated by the viral polymerase and the proofreading exoribonuclease compassionate use of remdesivir for patients with severe remdesivir in covid- mild/moderate -ncov remdesivir rct -full text view -clinicaltrials severe -ncov remdesivir rct -full text view -clinicaltrials sulfobutylether-beta-cyclodextrin physicochemical characterization, molecular docking, and in vitro dissolution of glimepiride- the use of captisol (sbe -beta-cd) in oral solubility-enabling formulations: comparison to hpbetacd and the solubility-permeability interplay pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications remdesivir for treatment of covid- : combination of pulmonary and iv administration may offer aditional benefit inactive ingredient search for approved drug products. u.s. food and drug administration characterization and pharmacokinetic analysis of aerosolized aqueous voriconazole solution dose tolerability of chronically inhaled voriconazole solution in rodents dry powder insufflation of crystalline and amorphous voriconazole formulations produced by thin film freezing to mice a guide to aerosol delivery devices for respiratory therapists use of thin film freezing to enable drug delivery: a review effect of processing parameters on the physicochemical and aerodynamic properties of respirable brittle matrix powders respirable low-density microparticles formed in situ from aerosolized brittle matrices in vitro and in vivo performance of dry powder inhalation formulations: comparison of particles prepared by thin film freezing and micronization small airway absorption and microdosimetry of inhaled corticosteroid particles after deposition novel ultra-rapid freezing particle engineering process for enhancement of dissolution rates of poorly water-soluble drugs physicochemical characterization of d-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region agglomerated novel spray- dried lactose-leucine tailored as a carrier to enhance the aerosolization performance of salbutamol sulfate from dpi formulations formulation strategy and use of excipients in pulmonary drug delivery. int j pharm (d) of the securities exchange act of for the fiscal year ended december , form -k savara inc: united states securities and exchange commission a study of aerovanc for the treatment of mrsa infection in cf patients using thin film freezing to minimize excipients in inhalable tacrolimus dry powder formulations processing design space is critical for voriconazole nanoaggregates for dry powder inhalation produced by thin film freezing direct evidence on reduced adhesion of salbutamol sulphate particles due to l-leucine coating correlations between surface composition and aerosolization of jet-milled dry powder inhaler formulations with pharmaceutical lubricants viscosity measurements of methanol-water and acetonitrile-water mixtures at pressures up to bar using a novel capillary time-of-flight viscometer density, dynamic viscosity, and derived properties of binary mixtures of , dioxane with water at t= . k effect of process variables on morphology and aerodynamic properties of voriconazole formulations produced by thin film freezing glass-forming ability of compounds in marketed amorphous drug products glass forming ability on the physical stability of supersaturated amorphous solid dispersions myth or truth: the glass forming ability class iii drugs will always form single-phase homogenous amorphous solid dispersion formulations interpretation and prediction of inhaled drug particle accumulation in the lung and its associated toxicity nebulization of nanoparticulate amorphous or crystalline tacrolimus--single-dose pharmacokinetics study in mice in vitro and in vivo evaluation of porous lactose/mannitol carriers for solubility enhancement of poorly water-soluble drugs the role of functional excipients in solid oral dosage forms to overcome poor drug dissolution and bioavailability nmr methods to characterize protein-ligand interactions hydrogen bonding and chemical reactivity nmr techniques to study hydrogen bonding in aqueous solution. monatshefte fur chemie chemical properties, aerosolization and dissolution of co-spray dried azithromycin particles with l- amino acids as co-amorphous stabilizers for poorly water soluble drugs improved aqueous solubility of crystalline astaxanthin ( , '-dihydroxy-beta, beta-carotene- , '-dione) by captisol (sulfobutyl ether beta- cyclodextrin) what do we know about remdesivir drug interactions? pulmonary dissolution of poorly soluble compounds studied in an ex vivo rat lung model remdesivir inhibits sars-cov- in human lung cells and chimeric sars-cov expressing the sars-cov- rna polymerase in mice advantages of the parent nucleoside gs- over remdesivir for covid- treatment key: cord- -b a z w authors: samji, hasina; yu, amanda; wong, stanley; wilton, james; binka, mawuena; alvarez, maria; bartlett, sofia; pearce, margo; adu, prince; jeong, dahn; clementi, emilia; butt, zahid; buxton, jane; gilbert, mark; krajden, mel; janjua, naveed z. title: drug-related deaths in a population-level cohort of people living with and without hepatitis c virus in british columbia, canada date: - - journal: int j drug policy doi: . /j.drugpo. . sha: doc_id: cord_uid: b a z w background: the majority of new hcv infections in canada occur in people who inject drugs. thus, while curative direct antiviral agents (daas) herald a promising new era in hepatitis c virus (hcv) treatment, improving the lives and wellbeing of people living with hcv (plhcv) must be considered in the context of reducing overdose-related harms and with a syndemic lens. we measure drug-related deaths (drds) among hcv-negative people and plhcv in british columbia (bc), canada, and the impact of potent contaminants like fentanyl on deaths. methods: we identified drds among plhcv and hcv-negative individuals from to in the bc hepatitis testers cohort, a population-based dataset of ~ . million british columbians comprising comprehensive administrative and clinical data. we estimated annual standardized liver- and drug-related mortality rates per , person-years (py) and described the contribution of specific drugs, including fentanyl and its analogues, implicated in drds over time. results: drds constituted . % of deaths among plhcv and . % of deaths among hcv-negative individuals; a . -fold ( % confidence interval: . - . ) difference. drug-related mortality overtook liver-related mortality for plhcv in and hcv-negative individuals in and rose from . to . per , py from to amongplhcv and from . to . per , py for hcv-negative individuals over the same period. the proportion of deaths attributable to drugs among plhcv and hcv-negative individuals increased from . % to . % and . % to . %, in and , respectively. the proportion of drds attributed solely to synthetic opioids such as fentanyl averaged across both groups increased from . % in to . % in . conclusion: steep drug-related mortality increases among plhcv and hcv-negative individuals over the last decade highlight the urgent need to address overdose-related drivers and harms in these populations using an integrated care approach. the advent of direct acting antiviral (daa) short-course, well-tolerated therapy has dramatically altered the hepatitis c virus (hcv) treatment landscape. response rates over % are leading to dramatic reductions in liver-related mortality as well as new global targets to eliminate hcv as a public health threat by (n. world health organization, ) . however, increasing injecting drug use and the epidemic of drug overdose-related deaths in north america threaten to arrest or even reverse the progress heralded by the daa era among people who use drugs with hcv and the morbidity, mortality and well-being of all people who use drugs, regardless of hcv status. in canada, new hcv infections occur mainly among people who inject drugs (pwid) (n. z. . moreover, syndemic conditions of co-occurring mental illness, hiv co-infection and higher material deprivation are more common among people living with hcv (plhcv) compared to hcv-negative individuals (mckee et al., ) , prompting a need to examine data by hcv status. in british columbia (bc), an estimated . to . % of the population injects drugs and more than % of those will have a lifetime exposure to hcv (jacka et al., ; public health agency of canada, ) . increasing rates of injecting drug use have been observed in canada and the us since , likely exacerbated by policies to reduce prescription opioid overprescribing, diversion and misuse (powell et al., ) . other policies (e.g. prescription drug monitoring programs, more stringent guidelines) have also been implicated . a national us study observed a % increase in drug treatment admissions (from % to %) due to injecting drug use between and ; these increases were particularly pronounced among individuals to years of age (zibbell et al., ) . acute hcv infections subsequently tripled in the us between and (centers for disease control and prevention, ; gonzalez & trotter, ; zibbell et al., ) , representing a reversal in progress towards reducing rates of acute hcv in the us, which declined by almost % between and . similarly, hcv incidence in bc decreased between and and then began rising thereafter (li y et al., ) . in both canada and the us, these "dual epidemics" of injecting drug use and hcv transmission have the potential to derail years of sustained progress in reversing the hcv epidemic, as pwid are at high risk of drug overdose-related mortality. while the rate of drug-related deaths (drds) in canada has nearly doubled from . deaths per , population in to . deaths per , population in , the situation in bc is particularly concerning with % of canadian drds recorded in that province (statistics canada, a) . in , more than , people died of a drug overdose in bc -approximately . times the number of deaths due to motor vehicle accidents within the province (bc coroners service, a). the proportion of drds in which synthetic opioids such as fentanyl and its analogs have been detected increased more than six fold between and , prompting the bc provincial health officer to declare a state of emergency under the public health act in april, for the first time (bc gov news, ) . national and provincial gains in life expectancy have also been halted and reversed for the first time in four decades due to the overdose epidemic; men in bc were the most affected, losing . years between (statistics canada, b ye et al., ) . as noted above, plhcv have a higher proportion of injecting drug use in canada and plhcv who inject drugs also have higher proportion of co-occurring mental illness, hiv co-infection, hbv co-infection and material and social deprivation (mckee et al., ) . it is hypothesized that as plhcv have higher drug use and are more marginalized, drds will be higher among them. however, there is no data assessing if the rate of drds is higher among plhcv compared to hcv-negative individuals. we use a population-based database of all individuals tested for hcv in bc to compare rates and trends over time in drd and liverrelated deaths (lrds) and identify whether there are differences in drds among plhcv and hcv-negative individuals. we also quantify the impact of fentanyl on drds in our study population over the last decade. the bc hepatitis testers cohort (bc-htc) includes more than . million individuals tested for hcv or hiv at the bc centre for disease control public health laboratory and all confirmed cases of hbv, hcv, hiv, and active tb reported by the public health system in bc from to . individuals in bc are generally tested due to hcv risk, investigation of liver disease, routine screening in pregnancy and if they are undergoing procedures such as renal dialysis, and those tested during the mid- s as part of blood system lookbacks . these data were integrated with data on prescription drug dispensations, physician diagnostic and billing data, hospitalization records, emergency department visits, cancers, and deaths and are described in more detail in supplemental table s . additional information about the bc-htc, including methodology, data linkages, and population characteristics by hcv status has been published previously (n. z. mckee et al., ) . the dynamic, retrospective cohort was formed under a public health mandate of the bc centre for disease control. all residents in bc are registered in the publicly funded insurance plan that acts as a single payer system and covers services provided by fee-for-service practitioners, allowing for comprehensive population-level monitoring. eligible individuals for this study had at least one hcv test (i.e., anti-hcv or hcv rna) performed or were a reported hcv case from april to december . characterization of drd was restricted to onwards to highlight the surge of drds among people with hcv over the last decade. drds were identified using bc vital statistics agency (bcvsa) death registry, which includes all deaths registered in the province of bc, underlying and/or contributing cause of death mortality data, and categorized using the international classification of diseases, tenth revision (icd- ) codes (supplemental table s ). any deaths not deemed "natural" (a death primarily resulting from a disease or progressive fatigue of the bodily systems) are investigated by the bc coroners service (bccs). the finalization of the medical certificates of death can take anywhere from several months to several years from date of death. bcvsa processes information received from the bccs on the finalized medical certificate of death and applies these icd- codes using an internationally standardized coding software provided by statistics canada. a broad definition specifying both acute and chronic poisonings was adapted from consensus recommendations for national and state poisoning surveillance developed by the safe states alliance (the safe states alliance, ) and which has also been used by the us centres for disease control and prevention. in addition, icd- multiple-cause codes provide information on the types of drugs or drug classes involved in the death ("t-codes"); of these, heroin (t . ); natural/semisynthetic opioids (t . ); methadone (t . ); synthetic opioids other than methadone (including fentanyl and fentanyl analogues) (t . ); cocaine (t . ); other and unspecified narcotics ( . ); and psychostimulants with abuse potential (t . ) were used. deaths involving methadone (t . ) and natural/ semisynthetic opioids such as hydrocodone and oxycodone (t . ) are grouped together as prescription opioid deaths (jalal et al., ; jones et al., ) , a category which includes opioids prescribed to the deceased person or prescribed to someone else (diverted). in , in response to the drd public health emergency, supplementary codes to identify deaths where fentanyl ("z ") or carfetanil ("z ") were specifically implicated in finalized death investigations were added to the "lifestyle / environmental" field in bcvsa mortality data files; thus, we also included these codes in our categorization of "synthetic opioids other than methadone." drug-related t-codes are attached to a parent "external cause" icd code that signals the circumstances or intent under which the substance was used (accidental, intentional / suicide, homicide, undetermined intent or adverse reaction in therapeutic use) and is assigned during drds investigations. bccs investigations include toxicology results, scene evidence and death investigation information (such as existing prescription information). these investigations also help determine drug origin; for instance, whether a drug is pharmaceutically-derived (including prescribed to the deceased or diverted), non-pharmaceutical, or undetermined (e.g. fentanyl identified through toxicology but without prescription or evidence suggesting non-pharmaceutical fentanyl use) (onatrio agency for health protection and promotion (public health ontario) ). similarly, illicit fentanyl is differentiated from prescribed fentanyl through investigation of evidence of prescription/patch versussuggestion of a non-pharmaceutical origin (e.g., other illicit substances detected on toxicology or drug paraphernalia on the scene). heroin ( -monoacetyl morphine), which is rapidly metabolized into morphine and therefore may be underestimated, also relies on investigation to finalize classification (onatrio agency for health protection and promotion (public health ontario) ). most deaths involved more than one type of drug; thus, categories are not mutually exclusive. lastly, if an underlying cause of death code was recorded as icd- r (other ill-defined and unspecified causes, usually assigned when cause of death is still under investigation), and manner of death was listed as "pending," "accident," or "undetermined," we re-categorized the death as drug-related if the death met the following criteria: attributable to a person aged to years who also injects drugs (defined as any drug-related diagnoses, use of injection drugs, record of opioid agonist therapy dispensation or injection related infection using physician diagnostic and billing data, hospitalizations, prescription data, vital statistics and emergency room data), based on validation studies in the us and in the bc-htc (centers for disease control and prevention, ; to mitigate the impact of missing data on the analysis. as the majority of r codes are resolved within a few years as the bccs completes investigations of sudden deaths prior to death categorization, we excluded deaths occurring after december st , to reduce the impact of open investigations and the need to impute missing data by re-categorizing deaths. while we were able include many drds with r codes in the analysis through application of our algorithm described above, it was not possible to assign t-codes to these deaths as these codes are only assigned in concluded investigations bc coroners service, ) . as such, analyses involving t-codes were limited to data collected on or before december st , . we used deidentified linked databases to identify other important variables for analyses. lrds included deaths due to decompensated liver disease, liver cancer, hcv, other types of viral hepatitis, and alcoholic or non-alcoholic liver disease. we assessed baseline participant characteristics such as age, sex and material and social deprivation (québec index of material and social deprivation) (pampalon et al., ) . we also evaluated concurrent conditions including hiv and hbv co-infection, major mental illness (hospitalization or at least two visits to a psychiatrist for disorders including schizophrenia, bipolar, major depressive and personality), and psychosis diagnosis as well as access to opioid agonist therapy (oat) and hcv treatment. diagnostic codes and variable definitions are presented in more detail in supplemental table s . we compared plhcv and hcv-negative individuals who died of drds, estimated annual age and sex-adjusted drug-related mortality rates per , person-years (py) for these groups and examined trends over time by sex. rates are age and sex-standardized to the canadian population. py were calculated from either first hcv negative or first hcv positive date, censored at death or hcv sero-conversion. secondly, we compared demographic and risk factor characteristics of people who died of liver-vs. drug-related causes and estimated annual age and sex-adjusted liver-and drug-related mortality rates per , py for these groups. lastly, we described the contribution or "co-involvement" of specific drugs, including fentanyl and its analogues, implicated in drds overall and stratified by hcv status over time. for instance, for deaths involving heroin, we identify what proportion also included fentanyl involvement each year using the t-codes described above assigned through coroner's investigation of each drug-related death. as more than one drug can contribute to a drd, categories representing specific drug involvement in a drd are not mutually exclusive. for descriptive analyses comparing groups, we used wilcoxon's rank sum test for continuous variables and the chi test for categorical data. sas version . was used for all analyses. the study was approved by the university of british columbia clinical research ethics board (h - ). there were , , people who were hcv tested or reported as an hcv case in the cohort as of december , , of whom , ( . %) were plhcv and , , ( . %) were hcv-negative individuals. a total of , deaths were recorded from to of which . % were among plhcv. drds constituted . % ( , / , ) of deaths among plhcv and . % ( , / , ) of deaths among hcv-negative individuals; a . -fold ( % confidence interval [ci]: . - . ) difference. the majority of drds among plhcv occurred among those with a history of injecting drug use ( . %). similarly, a majority of hcvnegative individuals individuals dying of a drd had a history of injection drug use ( . %) (tables and ) . the population of plhcv dying of drds was also older (born prior to : . % vs. . %), more likely to have co-infections such as hiv ( . % vs. . %), and hbv ( . % vs. . %), to have a record of oat ( . % vs. . %), and be more materially (most deprived quintile: . % vs. . %) and socially (most deprived quintile: . % vs. . %) deprived compared to the hcv-negative individuals ( table ) . more than half of plhcv and hcv-negative groups had a major mental illness diagnosis and more than % in each group reported a diagnosis of psychosis. annual drug-related standardized mortality rates among plhcv and hcv-negative individuals by sex are presented in figure . drugrelated mortality was . ( % ci: . - . ) to . ( % ci: . - . ) times higher in men compared to women living with hcv and from . ( % ci: . - . ) to . ( % ci: . - . ) times higher among hcv negative men compared to women. increases over time were observed for all groups, with rates among men without hcv increasing the most dramatically, from . per , py in to . per , py in ; a . -fold increase ( % ci: . - . ). increases over the same time period were also observed for men living with hcv ( . to . per , py; . fold increase [ % ci: . - . ]) and women without hcv ( . to . per , py; . -fold increase [ % ci: . - . ]) and women living with hcv ( . to . per , py; . -fold increase [ % ci: . - . ]). the proportion of deaths attributable to drugs among plhcv increased from . % ( / ) in to . % ( / ) in . similarly, the proportion of deaths attributable to drugs among hcvnegative individuals more than doubled from to , increasing from . % ( / ) to . % ( / ). annual liver-and drug-related standardized mortality rates among plhcv and hcv-negative individuals and overall deaths are presented in figure . drug-related mortality rate rose from . per , py in to . per , py (irr: . ; % ci: . - . ) in for plhcv and from . to . per , py (irr: . ; % ci: . - . ) for hcv-negative individuals with the majority of the increase after . from to , liver-related mortality rates among plhcv almost halved from . to . per , py (irr: . ; % ci: . - . ); similarly, liver-related mortality rates among hcv-negative individuals decreased from . to . per , py (irr: . ; % ci: . - . ) over the same period. drug-related mortality overtook liver-related mortality for plhcv in and hcv-negative individuals in . from to , drds increased from to , , totaling , over that period. . % ( , / , ) of drds had an assigned t-code specifying at least one drug, and . % of these had more than one drug recorded. since , the proportion of drds attributed solely to synthetic opioids such as fentanyl has increased in our cohort from . % in to . % in (figure ) . in , synthetic opioids were co-involved in . % of all opioid-related deaths, . % of prescription opioid deaths, . % of heroin-related deaths, . % of cocaine-related deaths, and . % of deaths involving psychostimulants. results stratified by hcv status are included in supplemental table s . we examined drds in a population-based cohort of hcv testers in bc. we found that drds were higher among plhcv compared to hcvnegative individuals. drug-related causes have overtaken liver-related causes of death among plhcv, accounting for more than a quarter of deaths in this population in , with similar trends observed among hcv-negative individuals. drug-related mortality has increased among men and women living with and without hcv since ; the steepness of the curves for each group has increased every year, indicating a worsening problem, with absolute rates several fold higher for plhcv compared to hcv-negative individuals. our data suggest that synthetic opioids such as fentanyl are ubiquitous in the drug supply, contributing to more than three quarters of deaths involving opioids in . while injecting drug use was pervasive among both plhcv and hcv-negative individuals dying of drug-related causes in bc, we found differences in group characteristics. plhcv were older, had a higher proportion of co-occurring hiv, hbv infections and social and material deprivation and may therefore represent pwid with a longer duration of drug use. the median age of drd in our study among hcv-negative individuals was years (vs. years among plhcv); similarly, provincial data on overdose deaths illustrates that people between the ages of and years had the highest rate of illicit drug overdose deaths ( . per , person years) in (bc coroners service, a). these younger hcv-negative individuals could represent pwid with shorter duration of drug use who are dying before they have a chance to access drug-related treatment services or to acquire a blood-borne pathogen. another explanation is that practices protecting people from acquiring blood-borne pathogens such as using drugs through non-injecting pathways do not protect against fentanyl exposure. more than half of overdose deaths in bc were not injecting-related in (bc coroners service, ). these data underscore the need to prevent initiation of injecting drug use, to promote reversal to non-injecting use, and to minimize exposure to fentanyl to reduce drds (des jarlais et al., ) . indeed, increasing rates of injecting drug use, acute hcv infection, and overdose deaths signal a crisis for public health in north america and elsewhere. recent outbreaks of hiv following changing drug use h. samji, et al. international journal of drug policy ( ) practices and trends have been observed among pwid in both rural and urban settings. a rapid increase in new cases of hiv infection among pwid in indiana (peters et al., ) led the us centers for disease control to identify regions at high risk of hiv transmission due to the opioid epidemic (van handel et al., ) ; subsequent hiv outbreaks in washington, massachusetts and ontario among pwid have also been described (alpren et al., ; ball et al., ; golden et al., ) . structural inequalities such as poor economic prospects, unaddressed trauma, stigma, and unequal access to healthcare are driving this syndemic and must be addressed to reduce drug-related harms (parker et al., ) . overdose deaths and sequalae from drug use such as infectious disease acquisition are preventable; developing a comprehensive suite of services, such as integrating hiv pre-exposure prophylaxis services for pwid at risk of acquiring hiv as well as infectious disease treatment (antiretroviral treatment for hiv and daa therapy for hcv) and medication-assisted treatment for opioid use disorder (singer, ) should be explored in these contexts. our results indicate that potent synthetic opioids such as fentanyl have increasingly contaminated the illicit drug supply in bc, with the proportion of deaths with co-involvement of synthetic opioids increasing sharply across all drug categories including cocaine and stimulant-related deaths since . substantiating these findings are drug testing results in bc, which found that % of drugs tested positive for fentanyl, including % of heroin samples, and % of nonopiate drugs such as crystal meth, ecstasy/mdma, and cocaine (vancouver coast health, ) . the strong association between the number of fentanyl-positive samples seized by canadian law enforcement agencies and overdose deaths in bc (karamouzian et al., ) also supports fentanyl's direct impact on mortality rates. while there was a decrease in overdose deaths in bc in , the number of deaths and non-fatal overdoses remain at epidemic levels. moreover, the covid- pandemic era appears to have precipitated a striking spike in deaths, with june recording the highest number of illicit drug toxicity deaths observed in bc to date (bc coroners service, b). there is thus renewed urgency to develop and implement strategies to reduce overdose deaths in bc and elsewhere. novel proximal level interventions such as take-home drug checking test strips may be explored to reduce the impact of the toxic drug supply on pwid. existing harm reduction interventions such as supervised consumption sites and take-home naloxone kits are operational in bc and have averted an estimated deaths over a -month period during which , deaths were observed (irvine et al., ) but can be expanded and implemented elsewhere. furthermore, distal interventions should address the underlying motives propelling the syndemic of mental health, blood-borne infections, and injecting drug use and other "diseases of despair" such as suicide and problematic alcohol use including unaddressed antecedent physical and psychological trauma, economic disadvantage, social isolation, and hopelessness (dasgupta et al., ) to create a durable impact on drug-related mortality. for instance, using "life projects" approaches that de-stigmatize and humanize pwid engaging in hcv treatment as well as culturally-safe, healing-centered approaches that acknowledge histories of trauma (e. williams et al., ; pearce et al., ) are paramount for addressing barriers to care and treatment and promoting long term wellness beyond hcv or drug treatment. notably, we found that more than half of plhcv and hcv-negative individuals were diagnosed with a major mental illness and more than a fifth of each group with psychosis. severe and persistent mental illness has been associated with an increased risk of acquiring sexually transmitted and blood-borne infections (stbbis); a recent meta-analysis observed an hcv prevalence of . % among individuals with severe and persistent mental illness compared to % of those in the general population (hughes et al., ) . this increased risk is primarily due to co-morbid substance use, though a national u.s. study of veterans found a % increased odds of having an hcv diagnosis among veterans with severe and persistent mental illness even after controlling for substance use (himelhoch et al., ). as such, strategies to improve screening for individuals with severe and persistent mental illness for hcv, drug use and other stbbis and providing risk reduction counseling in this population are warranted. there are several limitations to consider in our study. recent deaths of undetermined intent (icd- code r ) pending a coroner's investigation, with manner of death reported "pending," "accident," or "undetermined," and classified as drds in our study based on age and pwid criteria may be misclassified, possibly leading to an over-estimation of drd, though age restriction and history of idu decreases figure . synthetic opioid co-involvement in drug-related deaths among individuals in the bc hepatitis testers cohort, - ^(n= )^categories are not mutually exclusive; many deaths involve multiple drugs *proportion of total drug-related deaths with synthetic opioid co-involvement likelihood of misclassification. an estimated . % ( / , ) of all deaths in were assigned an icd- r code. however, not including any of these deaths as drug-related would certainly under-estimate drds; we chose to be inclusive. similarly, using administrative data to identify pwid, while a useful strategy for large population-level datasets, may also lead to misclassification. another important consideration is that there may be temporal trends in the classification of deaths whereby investigation practices and therefore classification of deaths differed; for instance, as the overdose epidemic grew, it is possible that more deaths were investigated as drug-related. next, although our inclusion criteria specify that everyone is tested for hcv on entry, drds among undiagnosed hcv seroconverters who do not have a positive test will be misclassified. lastly, we see a decline in drug related mortality among men living with hcv in which preceded a decline in overall drds in bc surveillance data (bc coroners service, a) in . this decline in men living with hcv may be real, related to improvements in healthcare and hcv treatment access, or could be a data artifact which may become clear as additional data become available. in summary, fentanyl has significantly impacted mortality rates among pwid with and without hcv; widespread interventions to reduce harms and address the toxic drug supply are urgently needed. for plhcv, drds represent a failure of engagement in care and a missed opportunity to deliver highly effective daa treatment and resulting quality of life improvements. thus, integration of overdose prevention and hcv services is necessary to improve survivability as well as quality of life of these populations and to realize the benefit of curative hcv therapy. this work was supported by the bc centre for disease control and the canadian institutes of health research (grant numbers nhc- , pjt- , phe- ). all inferences, opinions, and conclusions drawn in this publication are those of the authors, and do not reflect the opinions or policies of the data steward(s). mk has received research grant funding via his institution from roche molecular systems, boehringer ingelheim, merck, siemens healthcare diagnostics and hologic inc. srb has received speakers' honoraria and participated in a medical advisory board program with gilead sciences (personal payments given as unrestricted donation to bccdc foundation). all other co-authors have no potential conflict of interest to declare. opioid use fueling hiv transmission in an urban setting: an outbreak of hiv infection among people who inject drugs-massachusetts sharing of injection drug preparation equipment is associated with hiv infection: a cross-sectional study illicit drug overdose deaths in b illicit drug toxicity deaths in bc illicit drug toxicity deaths in bc excerpts from: issues to consider when analyzing icd- coded data on drug poisoning (overdose) deaths a surveillance data for viral hepatitis opioid crisis: no easy fix to its social and economic determinants hepatitis c virus prevalence and estimated incidence among new injectors during the opioid epidemic life projects: the transformative potential of direct-acting antiviral treatment for hepatitis c among people who inject drugs the rise of the opioid epidemic and hepatitis c-positive organs: a new era in liver transplantation understanding associations between serious mental illness and hepatitis c virus among veterans: a national multivariate analysis prevalence of hiv, hepatitis b, and hepatitis c in people with severe mental illness: a systematic review and meta-analysis. the lancet psychiatry modelling the combined impact of interventions in averting deaths during a synthetic-opioid overdose epidemic prevalence of injecting drug use and coverage of interventions to prevent hiv and hepatitis c virus infection among people who inject drugs in canada changing dynamics of the drug overdose epidemic in the united states from through identifying injection drug use and estimating population size of people who inject drugs using healthcare administrative datasets assessing hepatitis c burden and treatment effectiveness through the british columbia hepatitis testers cohort (bc-htc): design and characteristics of linked and unlinked participants twin epidemics of new and prevalent hepatitis c infections in canada: bc hepatitis testers cohort the impact of hcv svr from direct acting antiviral and interferon-based treatments on mortality in a large population based cohort study changes in synthetic opioid involvement in drug overdose deaths in the united states known fentanyl use among clients of harm reduction sites in british columbia what is killing people with hepatitis c virus infection? analysis of a population-based cohort in canada hepatitis c incidence rate among people who inject drugs (pwid) in british columbia from prescription drug monitoring programs operational characteristics and fatal heroin poisoning syndemic characterization of hcv, hbv, and hiv co-infections in a large population based cohort study. eclinicalmedicine onatrio agency for health protection and promotion (public health ontario); office of the chief coroner; ontario forensic pathology service; ontario drug policy research network an area-based material and social deprivation index for public health in québec and canada facing opioids in the shadow of the hiv epidemic another thing to live for": supporting hcv treatment and cure among indigenous people impacted by substance use in canadian cities hiv infection linked to injection use of oxymorphone in indiana a transitioning epidemic: how the opioid crisis is driving the rise in hepatitis c provincial health officer declares public health emergency |bc gov news i-track: enhanced surveillance of hiv, hepatitis c, and associated risk behaviours among people who inject drugs in canada -phase the daily -causes of death b). the daily -changes in life expectancy by selected causes of death consensus recommendations for national and state poisoning surveillance county-level vulnerability assessment for rapid dissemination of hiv or hcv infections among persons who inject drugs, united states fentanyl checking expanding to overdose prevention sites global health sector strategy on viral hepatitis impact of drug overdose-related deaths on life expectancy at birth in british columbia. health promotion and chronic disease prevention in canada increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states we thank the british columbia centre for disease control (bccdc), bccdc provincial health lab, provincial health services authority, bc ministry of health, bc cancer and their respective program staff involved in data access, procurement and data management for their assistance. we also appreciate the thorough review of the paper by rosemary armour at the bc vital statistics agency and tej sidhu from the bc coroner's service. supplementary material associated with this article can be found, in the online version, at doi: . /j.drugpo. . . key: cord- - ydn bvy authors: kumar, neeraj; awasthi, amardeep; kumari, anchala; sood, damini; jain, pallavi; singh, taru; sharma, neera; grover, abhinav; chandra, ramesh title: antitussive noscapine and antiviral drug conjugates as arsenal against covid- : a comprehensive chemoinformatics analysis date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: ydn bvy coronavirus pandemic has caused a vast number of deaths worldwide. thus creating an urgent need to develop effective counteragents against novel coronavirus disease (covid- ). many antiviral drugs have been repurposed for treatment but implicated minimal recovery, which further advanced the need for clearer insights and innovation to derive effective therapeutics. strategically, noscapine, an approved antitussive drug with positive effects on lung linings may show favorable outcomes synergistically with antiviral drugs in trials. hence, we have theoretically examined the combinatorial drug therapy by culminating the existing experimental results with in silico analyses. we employed the antitussive noscapine in conjugation with antiviral drugs (chloroquine, umifenovir, hydroxychloroquine, favlplravir and galidesivir). we found that noscapine-hydroxychloroquine (nos-hcq) conjugate has strong binding affinity for the main protease (mpro) of sars-cov- , which performs key biological function in virus infection and progression. nos-hcq was analyzed through molecular dynamics simulation. the md simulation for ns affirmed the stable binding of conjugation unprecedentedly through rmsd and radius of gyration plots along with critical reaction coordinate binding free energy profile. also, dynamical residue cross-correlation map with principal component analysis depicted the stable binding of nos-hcq conjugate to mpro domains with optimal secondary structure statistics of complex dynamics. also, we reveal the drugs with stable binding to major domains of mpro can significantly improve the work profile of reaction coordinates, drug accession and inhibitory regulation of mpro. the designed combinatorial therapy paves way for further prioritized in vitro and in vivo investigations for drug with robust binding against mpro of sars-cov- . the recent outbreak of the novel severe acute respiratory syndrome coronavirus- (sars-cov- ) is unprecedented. the world health organization (who) has given the guidelines that the current pandemic might go long, and the virus may become endemic in our society. as per the statistics of who, seven million people (as of th june ) have already been infected with coronavirus disease- and expected to increase exponentially in some of the regions (who, ) . the coronavirus is a single-stranded rna virus that typically affects the vertebrates and causes lethal effects (fung & liu, ) . the transmission of the sars-cov- virus between different host species is generally through respiratory droplets, in aerosol form, and fomites . due to the genetic proximity of this novel betacoronavirus, it is similar to the previous outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (zhu et al., ) . the diverse spike protein of sars-cov- makes it more lethal than previous sars and mers family viruses. among the various routes for its treatment/ prevention by the genetic code information of coronavirus, reported by the chinese research group in march (ren et al., ) provided an early opportunity for scientists across the globe to develop the vaccine for the covid- disease at earliest. however, it is too early to assume success for the covid- vaccine because the development of vaccination is tricky; it may take time to manufacture an effective and safe vaccine on a large scale. alternatively, to the current situation, drug repurposing seems to be an effective drug discovery strategy by utilizing the existing drugs and natural plant products for the treatment of the sars-cov- outbreak. drug repurposing strategy can potentially shorten the time and as well as reduce the cost compared to develop novel drug discovery and their clinical trials (cheng, ; cheng et al., cheng et al., , , . overall, the world research community is repurposing known fda approved drugs and improving them by conjugation or synthesizing potential analogs. remdesivir (beigel et al., ) , hydroxychloroquine (colson et al., ) , chloroquine , favipiravir (t- ) (coomes & haghbayan, ) , and galidesivir (bcx ) (li & de clercq, ) are some of the most successful repurposed small molecule drugs which has shown broad-spectrum activity against coronavirus. these repurposed drugs had initially shown good results; however, there were some major limitations as well, likewise potency, effectiveness, specificity to viral targets, and in some cases, side effects due to non-specificity, etc. interestingly, combination therapy of approved drugs suggested for being an effective strategy to treat corona infection. recently, hydroxychloroquine (hcq), a well-known drug for malaria, has been repurposed, which brought attention to potentially helping patients to recover from covid- . also, it has been used in the combination therapy and reaches to the trials such as; hydroxychloroquine with mivermectin (patr ı & fabbrocini, ), hydroxychloroquine with azithromycin (magagnoli et al., ) , hydroxychloroquine with nitazoxanide (amawi et al., ) , etc. however, these have not shown expected outcomes and associated with issues like high mortality rates and increased frequency of irregular heartbeat. as per who reports, these hcq based combination therapies took major setbacks after not performing significantly in clinical trials. hence in present work, we propose a drug conjugate of noscapine along with major antiviral drugs in repurposing trials (chloroquine, hydroxychloroquine, umifenovir, favlplravir, and galidesivir) . noscapine is an approved antitussive drug for the treatment of cough through simulating the lung linings. it is known to inhibit bradykinin enhanced cough response (ebrahimi et al., ) . we are aware of the fact that the primary target for sars-cov is the lungs, and noscapine has been widely used to inhibit bradykinin, eventually inhibiting inflammation and lung damage (ebrahimi, ) . hence it makes it sense to conjugate noscapine with antiviral drugs, which can decrease the severe symptoms due to the lung damage; this can overcome the setbacks of previous combinations therapies, including hcq conjugations, by attenuating the high mortality rates due to covid- . noscapine is known to obtain from the naturally occurring opium poppy (papaver sominiferum) medicinal plant, and it is characteristically a non-toxic phthalide-isoquinoline alkaloid (aneja et al., ) . we aimed to design the combinatorial therapy against the main protease (mpro or clpro) enzyme, a key regulator of the life cycle of the coronavirus. mpro is essential for the replication of the virus as it processes the polyproteins that are translated from the viral rna to yield functional viral proteins (chen et al., ) . this makes the mpro enzyme a potential drug target to combat coronaviruses. this fact has led to some of the research groups to develop a potential inhibitor to the protease (mengist et al., ; zhang et al., ) , . these inhibitors prevent the replication of the virus after entering into the host cells. mpro protease inhibitors are reported to modify the polypeptides essential for sars-cov- infection and progression in the host. also, our group has recently reported the potent interaction of noscapine analogs and subsequent inhibition of the mpro protease of the sars-cov- (kumar, kumari, et al., ) . with detailed literature, we designed and analyzed the noscapine conjugates with antiviral drugs (chloroquine, hydroxychloroquine, favipiravir, and galidesivir that are currently being clinically tested (table ). the molecular binding affinity of conjugates to inhibit the mpro has been evaluated using high throughput computational assays. the molecular dynamics studies have been widely used in modern drug discovery and development. computer-aided drug design has been employed to annotate the molecular binding mechanism of drugs to their specific targets (singh et al., ; , . in many studies, advanced molecular docking approach has been harnessed to screen an extensive library, define the binding affinity of drugs and to elucidate the involved molecular interactions in the complexation of the ligand with receptor protein chaudhary et al., ) , . molecular dynamics (md) analysis helps to insight into the binding mechanism of the drugs through the root-mean-square deviation (rmsd), radius of gyration binding energy calculations, and relevant calculations (kumar, sood, sharma, et al., ; kumar, sood, tomar et al., ; singh et al., ; ., . with employing these advanced computational approaches, deigned noscapine conjugations with antiviral drugs were examined to derive the highly efficient combination against the mpro of sars-cov- for further validation with experimental lab assays. . . drug target main protease (mpro) of sars-cov- retrieval and structural assessments the main protease regulatory enzyme of sars-cov- has been reported to play an essential role in coronavirus infection and progression in the host (xue et al., ) . to design the drug therapy against mpro, a three-dimensional crystal structure of mpro was retrieved from the protein data bank (pdb id lu we have designed the noscapine based antiviral cognates with antiviral drugs in trials (chloroquine, umifenovir, hydroxychloroquine, favlplravir, and galidesivir) . noscapine is reported possess the appeased effects on lung and respiratory infection with its antitussive properties (kim et al., ) . noscapine conjugates were drawn using the chemdraw software and assessed for their compatibility and deformation. the molecular interaction analyses of all conjugates were performed with the mpro to illustrate their binding affinity. to perform the docking calculations, the target mpro was prepared and optimized by the whatif server (vriend, ) . mpro was confined to add missing hydrogen atom, amino acid chain, any other unwanted chain and removing the water molecules. similarly, ligand files of noscapine-antiviral conjugates were prepared and optimized for any missing atom and chain. the molecular docking was performed using the hex . fast fourier algorithm-based module (macindoe et al., ) . by incorporating the binding sites, grid box in all axes to mpro was formed and ligand files were inserted to the server. the receptor grid was set to . Å for x, y, and z coordinates of mpro structure. the translational step, twist range of and protein flip ranges were set as per the molecular docking manual. moreover, to strengthen our results, we redocked the high-rank noscapine conjugate with another molecular docking tool with the swissdock server. swissdock works on the basis of the eadock dss algorithm with curated scripts of receptors and ligands (grosdidier et al., ) . from the obtained various docked conformation of mpro-conjugates, the model with the lowest energy was analyzed for the binding mechanism by protein-ligand profiles (laskowski & swindells, ; salentin et al., ) , . the model with the lowest docked energy score in all drug conjugates was selected for molecular dynamics simulation analysis. to mechanistic insights into the binding of the conjugate (nos-hcq), the molecular dynamics simulation analysis was performed. md simulations were implemented through gromacs v . under the force field gromos a having water model spc along with the time step of fs for ns . we investigated the atomic motions, conformational changes, structural stability, and binding energy contributing to the binding of the ligand to the target molecule. in an explicit water solvent, internal atomic motion simulation was initiated. the binding complex of nos-hcq conjugate was solvated in the octahedron system and stretched to Å in all directions. the complex operation was minimized through four steps, and all atoms were fixed. in the minimization process steepest descent steps were executed, and all atoms were relaxed at k with one atmospheric pressure in boundary states. npt ensembles, along with periodic boundary conditions, were utilized to carry out md simulations. a cut-off of about Å was used in order to manage the vander waals forces. the particle mesh ewald model having a cut-off of Å was further utilized to calculate the electrostatic interactions. the obtained trajectory for ns md simulation run was analyzed for root mean square deviation (rmsd), root mean square fluctuations (rmsf), hydrogen bond analyses, solvent accessible surface area analysis (sasa) and radius of gyration (rg) plot analysis (grant et al., ) . also, the native contacts, along with the principal component analysis (pca), cross-correlation map analyses were performed throughout the simulation run (mcgibbon et al., ) . moreover, the binding energy calculation lead conjugate with mpro was investigated through the reaction coordinates analysis. it determined the ligand compatibility with threshold energy and the progression of ligand for stabilized binding with the target receptor. a total of frames of interactions complex trajectory was analyzed for binding energy computation. the equation employed for the calculation is dgcomplex, dgreceptor, dgligand represents the binding energy of complex, binding energy receptor, binding energy of ligand. . . sars cov- mpro enzyme as a potential target sars coronavirus is an enveloped positive-stranded rna virus, which majorly affects the respiratory system and enteric system (graham et al., ) . sars-cov- infection and progression in the host are regulated by multiple structural proteins. among various structural proteins, the main protease enzyme of coronavirus is reported to play significant roles in viral replication through proteolytic machinery and involved in transcription, translation, and amplification of viral proteins (paules et al., ) . mpro enzyme is of size amino acids and possesses high similarity with protease enzyme from different human and animal by sequence analysis. the protein annotation results depicted mpro has various roles in coronavirus viz; a nucleic acid-binding domain of sars coronavirus, polyprotein cleavage domain, coronavirus endopeptidase, corona nsp , nsp and nsp replication implication regulatory roles, reported through pfam annotation database. moreover, d structural analysis of mpro by the cath server showed it consisted of beta-barrel and alpha domain (figure ). the secondary structural analysis by the dssp server showed it comprised of % alpha-helices ( helices, residues) and % beta-barrel sheets ( strands). these significant analyses along with the literature survey, suggested the mpro enzyme as a potential target to design the covid- therapeutic drugs. moreover, prior to molecular binding analyses the d structure of the mpro enzyme was assessed for structural conformations and physicochemical by ramachandran plot and saves server. the ramachandran plot analyses depicted . % residues are in the favorable region, and . % residues and . % residues are in the outlier region of the plot. the local quality assessment showed good structural quality factors with high homology ( . to . Å) with the native structures in database of , known protein structures. with noscapine significant roles in lung linings and dilation and coronavirus movements into the lung cells evokes the cytokine storm and leads to neutrophilic infiltration and other implication (martin & ernst, ) . hence, it was essential to investigate the effectiveness of antitussive noscapine in conjugation with antiviral drugs against mpro. we chose and designed the noscapine conjugation with potential antiviral drugs (chloroquine, umifenovir, hydroxychloroquine, favlplravir and galidesivir) using the chemdraw and simultaneously assessed their applicability and conformations. for molecular docking, both receptor mpro and ligand files of noscapine conjugates were prepared for molecular docking using the whatif prepdock module and ligprep server. for performing the molecular docking, binding site of mpro enzyme-n inhibitor crystal structure, recently submitted to the protein data bank, was analyzed for binding sites and insights into the molecular mechanism of inhibition by drugs . similar binding sites were employed for performing the molecular docking of the noscapine conjugations with the target mpro of coronavirus using the hex . and swissdock servers. the molecular docking was performed using hex . , which works based on a fast fourier transform by spf shape-density correlations, and results were recalculated with electrostatic association additionally. first, we have performed the molecular docking analysis of drug alones which depicted the high binding score for hydroxychloroquine with À . kj/mol, among all drugs. further we assessed their drug likeliness properties of all drugs, none of them violated lipinski rule of five except galidesivir which has six hydrogen donor groups (table ) . taking these results altogether, we performed the docking for all conjugates. interestingly, obtained results showed the strong binding of noscapine conjugates with mpro of coronavirus. the nos-hcq conjugate depicted the most substantial binding with an energy score of À . kj/mol, among all conjugates. besides, to assess the effectivity of noscapine (singlet state) against the mpro, it was also docked using the same grid, which showed the binding score of À . kj/mol, larger than the known molecule n molecule bound to mpro with binding score of À . kj/mol. it can be seen from the molecular binding analyses that hydroxychloroquine depicted the high binding score in drug alone analyses, whose binding efficiency was enhanced by conjugation of noscapine by about double with a docking score of À . kj/mol to mpro (table ) . moreover, lead conjugation of nos-hcq does not found to follow the drug likeliness lipinski rule of five for high molecular weight and hydrogen bond acceptor groups with combinations of two drugs. in addition, to strengthen our results, we redocked the all conjugates with mpro using the swissdock module. the outcomes affirmed the highest efficacy of nos-hcq conjugate with binding dg energy score of À . kcal/mol and combined full fitness score of À . kcal/mol, among all noscapine conjugates (figure ). with these significant results, it can be attributed that nos-hcq conjugate has a high potential to bind the target mpro enzyme and further can be used as effective therapeutics for sars-cov- . the detailed characterization of contacts of noscapine conjugates is vital to recognize the interaction pattern of ligands for stringent binding to target mpro. the protein-ligand profiler was employed to determine the non-covalent contacts of complex systems. the mpro structural domain analysis by the cath server showed it forms majorly three ligand-binding domains and one additional domain. the globular structure analysis by chimera modeling application suggested the plausibly first domain from ser -pro , second binding remained confined to domain- and domain- ( figure ). from these contact analyses of all conjugates, it can be attributed that nos-hcq conjugate has high affinity and wide molecular contacts for major domains of mpro, among all conjugates. moreover, it was further studied through molecular dynamics to get insight into inhibitory patterns and efficacy to combat the mpro enzyme of coronavirus. the binding affinity of the nos-hcq conjugate was investigated by the molecular dynamics simulation analyses for ns. the stable binding of nos-hcq along with protein dynamics in the target mpro was assessed through the gromos a force field with water model spc consisting time step of fs. the obtained trajectory was solvated in the octahedron box in an explicit water state to provide sustainable conformations. the system minimization was done for steps by the steepest descent program and further steps by conjugated gradient method through inserted force fields. the root means square deviation (rmsd), root means square fluctuation (rmsf), and radius of gyration (rg) were computed for the resulting trajectory to assess the stable binding of lead combination (nos-hcq) with mpro of coronavirus. moreover, to compare and determine the boosting power and synergetic contribution, md simulation was also performed for the singlet state of mpro. the steep rmsd variation can be considered an implication of a malleable and instinctive protein. the rmsd (average backbone range of complex system) for the frames of native contacts with target mpro depicted the stable binding of lead nos-hcq conjugate to mpro with fluctuation in the range of . - . nm and for mpro singlet state fluctuation in the range of . - . nm ( figure ) . interestingly, rmsd of the complex system was more stabilized than the native form of target receptor mpro. moreover, the secondary structure analysis showed the compelling flexibility in terminal residues and other loops of beta linker segments of the mpro (residues - ) to about . nm and mpro-conjugate (residues - and - ) to about . - . nm. also, we found the residues to have comparatively lesser discrepancies in rmsf statistics, which are corresponding to the interacting segment of the mpro-conjugate complex after md simulation. interestingly, the binding region for lead conjugation before md simulation was also within the residues to , which remains the same after simulation, suggesting a strong binding of nos-hcq towards the mpro and provides stability to the mpro-conjugate. after that, the obtained trajectory was computed to evaluate the radius of gyration of the mpro and compared with the noscapine singly. the lead combination with the mpro protein showed the rg fluctuation in the range of . - . nm and for mpro in range of . - . nm and demonstrated the stable and robust binding of nos-hcq with mpro ( figure ) . furthermore, similar outcomes were obtained by sasa computation, which represented the solvent-accessible protein surface, and various orientations through the folding and proceeding to alterations in buried and exposed regions of the surface area of the protein complex. similar observations were determined through sasa analysis representing the solvent defined protein surface and its orientation through folding, making the alterations in the exposed and remains same as before till ns ( figure ). here, mpro-noshcq solvation profile shows a convincing sasa value suggesting a stable structure and sturdy binding. also, the secondary structure analysis of complex trajectory and native mpro trajectory was performed by employing the dssp tool of gromacs. the cluster analyses of both depicted the conformational changes before and after the simulation run. mpro-noshcq and mpro were consisted of mainly conserved b-sheets along with loops of b linker segments and other random coil regions infused with the various bend, a-helices, turn and b bridges. the observations of both plot analyses showed the mpro rationale unfolded and implemented surface with b-sheets to bind stably with lead conjugation nos-hcq. binding energy landscape mm/pb (gb) surface area (sa) calculations were performed to estimate the lead conjugation binding affinities to mpro protein. the system was found to be the most favorable in terms of the sum of various binding energy computations. mm/gbsa free binding energy calculations were executed with two fifty frames dynamically equilibrated for the stabilized trajectory of the mpro-noshcq complex system between ns to ns. the electrostatic energy distribution in molecular mechanics was obtained through the high vander waal energy (vdw) À . ± . kj/mol, electrostatic energy À . ± . kj/mol and total binding energy of À . ± . kj/mol, which are considered as most significant contributor in the binding of ligand (nos-hcq conjugation) to the target protein. the polar solvation energy (pbsol) was calculated to be À . ± . kj/ mol, which added a vital contribution to the stable binding of the ligand to target protein. further, the stability and flexibility of the binding of nos-hcq with mpro were strengthening with the stabilized run of trajectory with set nvt parameters. the average temperature for both native mpro and in conjugation was close to kelvin; average pressure was found to be close to . bars and an average density of . kg/m ( figure ). these outcomes through ns md simulation suggested the stable binding of lead combination with significant contribution of binding energy to the target mpro of the coronavirus. also, the energy work profile of ligand binding and mpro protein dynamics was investigated by smd trajectory based on jarzynski relationships in molecular dynamics simulation. the reaction coordinate work profile demonstrated the contribution of binding energies in potential interaction. the calculated work profile for ligand binding through the reaction coordinates stated the stabilized coupling with energy contribution reached a free energy profile of kcal/mol at an rc value of Å and sustained throughout binding with the less flexible atomic motions. in the early stages, it reached to the maximum value and stabilized at kcal/mol with hydrogen bonds and reached to work profile of kcal/mol at rc Å in later stages. the atomic energy profiles for mpro-nos-hcq reaction coordinates showed an average radius of . Å, with overall priority of conjugated reaction progression about . %. along with the binding energy calculations, hydrogen bond consistency and dynamic contribution of bound nos-hcq revealed the involvement of a high number of hydrogen bonds throughout the simulation run for both native mpro and complex system ( figure ). mpro formed an average of hydrogen bonds with the lead combination during the simulation and played an essential role in the selectivity of intermolecular interactions. these outcomes through the md simulation signified for the stable binding of nos-hcq conjugation in correlation with molecular docking results. the principal component analysis (pca) was performed to insight into the binding clusters variances analyses of the complex system. the binding cluster frames were categorized into two coordinate clusters in black and red color. pca calculations of the atomic backbone of the complex system were computed by three conformations pc , pc , and pc by normal mode molecular dynamics. obtained results showed that the pca cluster possesses the highest variability of . % by binding of lead combination to mpro in terms of internal motions of trajectory. after that, pc statistics depicted the minimal variability with . %, and consequently, pc calculations . % and suggested the stabilized binding with eigen scores and minimal atomic motions variability of nos-hcq in complexation with mpro (figure ) . both the conformations in two groups in different pca analyses were superimposed and aligned, as shown in figure d . the pca trajectory analyses showed a conformational change in clusters from the first blue cluster to white cluster progressively to red color cluster, similarly recovered from first cluster (black color) to second cluster (red color) through simple clustering in pc subspace (figure ) . furthermore, the dynamical residue cross-correlation map (dccm) analysis was performed, which depicted the pairwise correlation of atomic coordinates of nos-hcq to the mpro through the magnitude of pairwise cross-correlation coefficient. the map showed the correlation residues (> . ) of the complex system in blue color, and anti-correlated residues (<- . ) depicted in red color. the highly pairwise correlated residues (light blue color) of complex trajectory in the map showed the stable binding of ligand file nos-hcq. the outcomes were also found in correlation with optimal secondary structures of complex protein dynamics during the md simulation run. the binding ability of nos-hcq towards the mpro and its essence for coronavirus for replication and regulation suggests the potency of nos-hcq and possible role in covid- clinical drug development. with the computational rationalization of lead conjugate nos-hcq against the mpro, we propose the plausible scheme of chemical synthesis. the drug conjugates of noscapine and selected re-purposed small molecules drugs depict the feasible tentative synthetic methodology (scheme ). earlier in , our research group developed a series of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death. iodo-nos was one of the crucial analogs of halogenated noscapine. -iodo-nos was synthesized by the iodination of noscapine using pyridine-iodine chloride in acetonitrile solvent with a % yield (aneja et al., ) . the target compound can be achieved by the application of a well-known stille metal cross-coupling reaction. compound , organostannanes, which can act as a precursor for the stille cross-coupling reaction, can be achieved via radical pathways in the presence of snme , dipea (base) and mecn (solvent) (chen et al., ) . the presence of the chloro group at hydroxychloroquine facile stille coupling reaction to take place at ease. the pd(pph ) catalytic system with copper(i) iodide and cesium fluoride in dmf is most effective for coupling iodides. copper(i) iodide and cesium fluoride combination facile the synthesis of sterically hindered systems such as (figure ). we report the efficient combinatorial therapy by conjugating the noscapine (antitussive drug) with potential hydroxychloroquine (nos-hcq) against the sars-cov- , through the computational assays with insights into the experimental results. the conjugate nos-hcq showed the most definite binding affinity towards the mpro, among the various noscapine based conjugates with (chloroquine, umifenovir, favlplravir, and galidesivir). noscapine (natural source opium poppy) is deemed to form the conjugation with antiviral drugs in trials against sars-cov- . the molecular docking assays depicted that by conjugation binding affinity score almost doubled with nos-hcq than with noscapine in the singlet state. the binding mechanism analyses of all conjugations illustrated that the lead conjugation (nos-hcq) has strong binding to three major domains of mpro. moreover, md simulation analyses of the trajectory (mpro-nos-hcq) for ns in explicit solvent depicted the stable binding of nos-hcq conjugate to mpro with minimal conformational variations in rmsd and rg plots. we reveal the drug binding with three major domains of mpro has high reaction coordinate completion efficacy (energy work profiles) with a significant contribution of immense binding energies and meticulous atomic level stabilization. the dccp and pca analyses confirmed the pivotal interactions and stabilize the binding of lead conjugation with mpro. besides rationalizing the combinatorial drug therapy, our study provides the fundamental plausible route of chemical synthesis of nos-hcq conjugate. this work paves the way for boosting the current medications into the effective combinational strategy to bind target mpro strongly, which can enhance the inhibitory action of drugs and contributes to fighting against the pandemic covid- with saving time, cost with minimal time screening in the current emergency. further, the warranted lead drug conjugation needs to be validated experimentally in laboratories and clinical trials. the authors declare no competing financial interest. covid- pandemic: an overview of epidemiology, parthenogenesis, diagnostics and potential vaccines and therapeutics synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death remdesivir for the treatment of covid- -preliminary report -bromo- '-chloro pyrazoline analog of curcumin augmented anticancer activity against human cervical cancer, hela cells: in silicoguided analysis, synthesis, and in vitro cytotoxicity synthesis of aryl trimethylstannanes from aryl halides: an efficient photochemical method emerging coronaviruses: genome structure, replication, and pathogenesis in silico oncology drug repositioning and polypharmacology individualized networkbased drug repositioning infrastructure for precision oncology in the panomics era drug repurposing: new treatments for zika virus infection? chloroquine and hydroxychloroquine as available weapons to fight covid- favipiravir, an antiviral for covid- noscapine, a possible drug candidate for attenuation of cytokine release associated with sars-cov- . drug development research interaction of noscapine with the bradykinin mediation of the cough response human coronavirus: host-pathogen interaction breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies a decade after sars: strategies for controlling emerging coronaviruses bio d: an r package for the comparative analysis of protein structures swissdock, a proteinsmall molecule docking web service based on eadock dss structure of mpro from covid virus and discovery of its inhibitors natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc infection of mrc- human lung cells understanding the binding affinity of noscapine with protease of covid- using md simulation at different temperature multiepitope subunit vaccine to evoke immune response against acute encephalitis antimicrobial peptide designing and optimization employing large-scale flexibility analysis of protein-peptide fragments preclinical evaluation and molecular docking of , -benzodioxole propargyl ether derivatives as novel inhibitor for combating the histone deacetylase enzyme in cancer ligplot þ : multiple ligand-protein interaction diagrams for drug discovery procheck: a program to check the stereochemical quality of protein structures therapeutic options for the novel coronavirus ( -ncov) research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases hexserver: an fft-based protein docking server powered by graphics processors outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- antiviral agents from plants and herbs: a systematic review mdtraj: a modern open library for the analysis of molecular dynamics trajectories designing of improved drugs for covid- : crystal structure of sars-cov- main protease m pro stereochemical quality of protein structure coordinates hydroxychloroquine and ivermectin: a synergistic combination for covid- chemoprophylaxis and treatment? coronavirus infections-more than just the common cold identification of a novel coronavirus causing severe pneumonia in human: a descriptive study plip: fully automated protein-ligand interaction profiler designing of a novel indoline scaffold based antibacterial compound and pharmacological evaluation using chemoinformatics approach a novel peptide thrombopoietin mimetic designing and optimization using computational approach mechanistic interaction study of bromo-noscapine with bovine serum albumin employing spectroscopic and chemoinformatics approaches antibacterial and pharmacological evaluation of fluoroquinolones: a chemoinformatics approach what if: a molecular modeling and drug design program structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design larmd: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors network-based drug repurposing for novel coronavirus -ncov/ sars-cov- a novel coronavirus from patients with pneumonia in china £-nk and aa contributed equally. n.k, a.a., d.s., r.c., and n.s. designed the studies. n.k., d.s., and a.a, a.k., carried out the in silico experiments. n.k, a.a., a.g., and d.s. wrote the manuscript. no potential conflict of interest was reported by the authors. key: cord- -m do t j authors: rossi, carlo maria; beretta, flavio niccolò; traverso, grazia; mancarella, sandro; zenoni, davide title: a case report of toxic epidermal necrolysis (ten) in a patient with covid- treated with hydroxychloroquine: are these two partners in crime? date: - - journal: clin mol allergy doi: . /s - - - sha: doc_id: cord_uid: m do t j background: stevens-johnson syndrome/toxic epidermal necrolysis (sjs/ten) is the most serious cutaneous adverse reaction (scar) often with a fatal outcome. coronavirus disease (covid- ) is caused by severe acute respiratory syndrome–coronavirus— (sars-cov ) and is an emergent pandemic for which no cure exist at the moment. several drugs have been tried often with scant clinical evidence and safety. case presentation: here we report the case of -years-old woman with cardiometabolic syndrome and covid- . a multidrug regimen including others hydroxychloroquine, antibiotics, dexamethasone and paracetamol, low-molecular-weight-heparin and potassium canrenoate was started. after almost weeks, the patient started to display a violaceous rash initially involving the flexural folds atypical targetoid lesions and showing a very fast extension, blister formation and skin detachments of approximately % of the total body surface area and mucous membranes involvement consistent with toxic epidermal necrolysis (ten). the alden algorithm was calculated inserting all drugs given to the patient in the days preceding the onset of the skin manifestations. the highest score retrieved was for hydroxychloroquine. other less suspicious drugs were piperacillin/tazobactam, ceftriaxone and levofloxacin. conclusions: to our knowledge, this is the first case of ten in a patient suffering from covid- probably associated with hydroxychloroquine. given the activation of the immune system syndrome induced by the virus and the widespread off-label use of this drug, we suggest a careful monitoring of skin and mucous membranes in all covid- positive patients treated with hydroxychloroquine in order to early detect early signs of toxicities. toxic epidermal necrolysis (ten) is a rare serious cutaneous adverse reaction (scar). it displays an acute onset and is characterized by erythematous or violaceous patches, atypical targetoid lesions, bullae, erosions and skin detachment. it differs from stevens-johnson syndrome (sjs) only in the percentage of skin involvement, which in ten is greater than % of the body surface [ ] . etiopathogenetically, it results from the combination of drug and host genetic factors (such as drug metabolism and t cell clonotypes) resulting in a delayed-type hypersensitivity reaction, where drug or drug-peptide complexes are recognized by t cell receptors [ ] . clinical and molecular allergy *correspondence: flavioniccolo.beretta@gmail.com scuola di specializzazione in farmacia ospedaliera, università degli studi di milano, via l. mangiagalli , milan, mi, italy full list of author information is available at the end of the article coronavirus disease (covid- ) is caused by severe acute respiratory syndrome-coronavirus- (sars-cov- ) and can be potentially fatal disease [ ] . the most common symptoms at onset of covid- illness are fever, cough, and fatigue, followed by dyspnea and diarrhea [ ] . lymphopenia, elevation of creatinine kinase (ck), lactate dehydrogenase (ldh) are also frequently observed. the lung involvement is frequently that of an interstitial pneumonia, covid- later shows an over exuberant inflammatory response with a no correlation between viral load and the worsening of symptoms [ ] . at the moment there is no effective cure for covid- , several drugs often in combination have been used, often with scant clinical evidence or conflicting results, with the aim of targeting the virus replication and/or the inflammatory process, such as anti-retrovirals, macrolides, hydroxychloroquine and monoclonal antibodies targeted on inflammatory cytokines [ ] [ ] [ ] [ ] . there is limited data establishing their safety profile during sars-cov- infection. here we report the case of -year old woman with several comorbidities (hypertension, obesity, unstable angina, type diabetes) admitted to our hospital for respiratory insufficiency with fever requiring non-invasive ventilation and a diagnosis of covid- lung infection with bacterial superinfection was formulated given the radiological evidence of a bilateral interstitial pneumonia, the positivity of nasopharyngeal swab for sars-cov- and (fig. ) . lymphopenia ( . /µl [ . - . ]), elevation of ck ( u/l ) and ldh ( u/l ) were also observed. a multidrug regimen with hydroxychloroquine mg twice a day, sodium enoxaparin and dexamethasone were started. besides, an antibiotic treatment with ceftriaxone for day, followed by piperacillin/tazobactam for days was given. the patient was initially treated in a sub-intensive care unit and after the favorable clinical evolution was transferred to our general medicine division, days after the admission. at the arrival in our unit, the patient started to display a violaceous erythematous rash mainly involving the flexural folds. the patient was still taking hydroxychloroquine, along with levofloxacin (started the day before); other drugs were reported in table . the rash presented in the course of days a rapid extension with the involvement of the whole trunk and buttocks, reaching approximately % of the total body surface area, with the appearance of atypical targetoid lesions and the formation of blisters, with subsequent skin detachment (figs. , and ). nikolsky's sign was present. a severe desquamation of the buccal and nasal mucosa was also observed. the patient referred severe skin pain requiring morphine. blood tests did not show eosinophilia or alterations of liver and renal function tests. a clinical diagnosis of toxic epidermal necrolysis (ten) also confirmed by the dermatologist was formulated. methylprednisone mg/kg, as an intravenous bolus regiment was administred along with intravenous immunoglobulin (ivig) mg/kg for days. then oral prednisone mg/kg daily was given and subsequently tapered in month. to identify the culprit drug, the alden algorithm was calculated (table ) [ ] (additional file ). the highest score retrieved was for hydroxychloroquine, with a possible correlation (+ score). other less suspicious drugs were piperacillin/tazobactam, ceftriaxone the patient was cared for by the wound care service of the hospital with topical therapy, a marked improvement in the skin conditions was observed progressively over a period of weeks, until the complete resolution (figs. and ) . paracetamol, pantoprazole, enoxaparin were tolerated after the reaction, ruling out their correlation with the skin reaction. due to the covid- epidemic, no skin biopsies were technically feasible for logistical problems. however, the clinical manifestations were highly compelling for sjs/ ten. other differential diagnosis included drug-reaction with eosinophilia and systemic symptoms (dress), which was excluded given the absence of eosinophilia and internal organ involvement, staphylococcal scalded skin syndrome (ssss), which is a pediatric form, and bullous dermatoses that were ruled out in our case given the minor mucous involvement and intense skin inflammation. sjs/ten is the most severe scar being associated with a high mortality rate [ ] . it is a delayed reaction occurring after - days from drug exposure, therefore it is of paramount importance to acquire precise retrospective pharmacological information for a long period of time preceding the onset of skin manifestations. sts/ten has an approximate incidence of or cases/ , , annually. it results from the clonal expansion of cd + cytotoxic t lymphocytes (ctls) and natural killer cell (nk),which are major histo-compatibility complex (mhc)-restricted and induce epidermal apoptosis [ , , ] . recently, it has been shown that low molecular weight drugs can directly bind the t cell receptor (tcr) of t cells or the human leukocyte antigen (hla) of antigen-presenting cells [ ] [ ] [ ] [ ] . usually implicated drugs are allopurinol, anticonvulsant drugs nonsteroidal anti-inflammatory drugs (nsaids), antibiotics such as cephalosporins, aminopenicillins and rarely macrolides, as shown in the euroscar study, together with the react project and the regis-car project [ ] [ ] [ ] [ ] [ ] . the alden algorithm represents the gold standard tool in sjs/ten to identify the culprit drug and to discriminate it from the "innocent" drugs which can be safely administered. it gives to each drug taken by patient a score, ranging from − to + , which corresponds to the probability of having caused the reaction, ranging from very unlikely to very probable [ ] . besides, from a recent analysis a significant statistic for agreement (kappa . ) was found between alden score for related-drugs (alden ≥ ) and lymphocyte transformation test (ltt) results performed after recovery [ ] . according to the alden algorithm results, the most likely implicated drug in our case is hydroxychloroquine. hydroxychloroquine is an -aminoquinoline with a low molecular weight ( , da) [ ] [ ] [ ] [ ] . it has been used for decades to treat rheumatologic conditions (with a dose of mg/kg) with a general good safety, even though in last years cases of skin reactions, also severe, have increasingly been reported [ ] . we searched the eudravigilance database and found cases of tens related to the use of hydroxychloroquine [ ] . hydroxychloroquine is being profusely used to treat sars-cov- infection with an unconventionally high dosage (from mg twice a day to mg three times a day, which is independent of body weight sometimes with a loading dose, due to its high volume of distribution). interestingly, some new severe skin manifestation to hydroxychloroquine have also been reported during the treatment of sars-cov- positive patient [ ] [ ] [ ] . in this instance, it is not to be discounted that a rare side effect, such as ten, occurring with a not commonly associated drug, hydroxychloroquine, could have been favored by the particular immune stimulation induced by the virus sars-cov- [ ] . sjs/ten, and scar more generally, have been classically demonstrated to be associated with viral replication [ , [ ] [ ] [ ] . to our knowledge, this is the first case of ten in a covid- positive patient. another salient aspect of the case is the favorable evolution of the patient given that this type of scar is typically associated with a bad prognosis [ ] [ ] [ ] , even more so because the patient displayed all the negative typical prognostic factors also for covid- [ ] [ ] [ ] [ ] , indeed the calculation of the severity-of-illness score for toxic epidermal necrolysis (scorten) in our patient led to an estimated mortality rate of . % (ci , - , ) ( table ) [ , ] . it is possible that the prompt diagnosis of ten, the suspension of all the suspected drugs (in particularly hydroxychloroquine) and the administration of steroid and ivig, both targeting the inflammatory syndrome initially triggered by the virus, may have had contributed to a better prognosis, [ , ] . further studies are needed to assess the safety profile of hydroxychloroquine in patients with covid- . to our knowledge, this is the first case of ten associated with hydroxychloroquine in patient suffering from covid- . given the widespread off-label use of this drug, the high degree of immune activation induced by the virus and the recent increase of hydroxychloroquine-related scars, we believe that it is necessary to adequately monitor the skin in all covid- positive patients treated with hydroxychloroquine in order to early detect early signs of toxicities. more risk factors result in a higher score and a higher mortality rate (%) as follows: toxic epidermal necrolysis and stevens-johnson syndrome cytotoxic proteins and therapeutic targets in severe cutaneous adverse reactions the epidemeology and pathogensis of coronavirus (covid- ) outbreak clinical features of patients infected with novel coronavirus in wuhan covid- : combining antiviral and anti-inflammatory treatments sars cov- : recent reports on antiviral therapies based on lopinavir/ritonavir, darunavir/umifenovir, hydroxychloroquine, remdesivir, favipiravir and other drugs for the treatment of the new coronavirus in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) a trial of lopinavir-ritonavir in adults hospitalized with severe covid- tocilizumab in severe covid- pneumonia and concomitant cytokine release syndrome an algorithm for assessment of drug causality in stevens-johnson syndrome and toxic epidermal necrolysis: comparison with case-control analysis toxic epidermal necrolysis with prominent facial pustules: a case with reactivation of human herpesvirus toxic epidermal necrolysis and stevens-johnson syndrome are induced by soluble fas ligand immune pathomechanism and classification of drug hypersensitivity classification of drug hypersensitivity into allergic, p-i, and pseudo-allergic forms. int arch allergy immunol cutaneous t-cell recruitment in toxic epidermal necrolysis stevensjohnson syndrome and toxic epidermal necrolysis: an update tolerated drugs in subjects with severe cutaneous adverse reactions (scars) induced by anticonvulsants and review of the literature drug-induced stevens-johnson syndrome and toxic epidermal necrolysis in children: years study in a tertiary care hospital stevensjohnson syndrome and toxic epidermal necrolysis in association with commonly prescribed drugs in outpatient care other than anti-epileptic drugs and antibiotics: a population-based case-control study incidence, causative factors and mortality rates of stevens-johnson syndrome (sjs) and toxic epidermal necrolysis (ten) in northern italy: data from the react registry regiscar project assessment of drug causality in stevens-johnson syndrome/toxic epidermal necrolysis: concordance between lymphocyte transformation test and alden pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research ready to submit your research ? choose bmc and benefit from fda approved drug products: hydroxychloroquine oral tablets hydroxychloroquine efficiently suppresses inflammatory responses of human class-switched memory b cells via toll-like receptor inhibition characterizing the adverse dermatologic effects of hydroxychloroquine: a systematic review european medicines agency. eudravigilance oped data adr hydroxychloroquine drug reaction with eosinophilia and systemic symptoms syndrome to hydroxychloroquine, an old drug in the spotlight generalized pustular figurate erythema: a newly delineated severe cutaneous drug reaction linked with hydroxychloroquine cutaneous side-effects of the potential covid- drugs drug hypersensitivity in hiv infection the interferon-γinduced protein /cxcr axis is associated with human herpesvirus- reactivation and the development of sequelae in drug reaction with eosinophilia and systemic symptoms reactivation of cytomegalovirus in a patient with stevens-johnson syndrometoxic epidermal necrolysis toxic epidermal necrolysis: part ii. prognosis, sequelae, diagnosis, differential diagnosis, prevention, and treatment treatment of toxic epidermal necrolysis by a multidisciplinary team a review of literature and treatment results stevens-johnson syndrome and toxic epidermal necrolysis: an analysis of triggers and implications for improving prevention mortality rate and gender differences in covid- patients dying in italy a comparison with other countries istituto nazionale di statistica. impatto dell'epidemia covid- sulla mortalità totale della popolazione residente primo trimestre covid- : monitoraggio della situazione clinical characteristics of coronavirus disease in china performance of the scorten during the first five days of hospitalization to predict the prognosis of epidermal necrolysis scorten: a severity-of-illness score for toxic epidermal necrolysis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions cmr and fnb collected data. cmr and fnb wrote the draft paper and analyzed the data. cmr, gt and sm followed clinical course of the patient. dz revised the draft paper. all authors read and approved the final manuscript. the authors declare that they have not received any funding. not applicable. not applicable. informed consent to clinical data collection and publication was obtained from the patient. the authors declare that they have no competing interests. key: cord- -slouuryl authors: baker, jeremy d.; uhrich, rikki l.; kraemer, gerald c.; love, jason e.; kraemer, brian c. title: a drug repurposing screen identifies hepatitis c antivirals as inhibitors of the sars-cov- main protease date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: slouuryl the sars coronavirus type (sars-cov- ) emerged in late as a zoonotic virus highly transmissible between humans that has caused the covid- pandemic , . this pandemic has the potential to disrupt healthcare globally and has already caused high levels of mortality, especially amongst the elderly. the overall case fatality rate for covid- is estimated to be ∼ . % overall and . % in hospitalized patients age - years . therapeutic options for treating the underlying viremia in covid- are presently limited by a lack of effective sars-cov- antiviral drugs, although steroidal anti-inflammatory treatment can be helpful. a variety of potential antiviral targets for sars-cov- have been considered including the spike protein and replicase. based upon previous successful antiviral drug development for hiv- and hepatitis c, the sars-cov- main protease (mpro) appears an attractive target for drug development. here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of sars-cov- mpro. we screened a collection of ∼ , drugs with a previous history of use in humans for compounds that inhibit the activity of mpro in vitro. in our primary screen we found ∼ compounds with activity against mpro (overall hit rate < . %). subsequent dose validation studies demonstrated dose responsive hits with an ic ≤ μm. hits from our screen are enriched with hepatitis c ns / a protease targeting drugs including boceprevir (ic = . μm), ciluprevir ( . μm). narlaprevir (ic = . μm), and telaprevir ( . μm). these results demonstrate that some existing approved drugs can inhibit sars-cov- mpro and that screen saturation of all approved drugs is both feasible and warranted. taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis c ns / a viral protease should be revisited because some previous lead compounds may be more potent against sars-cov- mpro than boceprevir and suitable for rapid repurposing. limited by a lack of effective sars-cov- antiviral drugs, although steroidal anti-inflammatory treatment can be helpful. a variety of potential antiviral targets for sars-cov- have been considered including the spike protein and replicase. based upon previous successful antiviral drug development for hiv- and hepatitis c, the sars-cov- main protease (mpro) appears an attractive target for drug development. here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of sars-cov- mpro. we screened a collection of ~ , drugs with a previous history of use in humans for compounds that inhibit the activity of mpro in vitro. in our primary screen we found ~ compounds with activity against mpro (overall hit rate < . %). subsequent dose validation studies demonstrated dose responsive hits with an ic < m. hits from our screen are enriched with hepatitis c ns / a protease targeting drugs including boceprevir (ic = . m), ciluprevir ( . m). narlaprevir (ic = . m), and telaprevir ( . m). these results demonstrate that some existing approved drugs can inhibit sars-cov- mpro and that screen saturation of all approved drugs is both feasible and warranted. taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis c ns / a viral protease should be revisited because some previous lead compounds may be more potent against sars-cov- mpro than boceprevir and suitable for rapid repurposing. the sars virus and sars-cov- , the cause of the covid- pandemic, are zoonotic coronaviruses found in bats that can infect humans. initial symptoms of sars-cov- infection include fever, myalgia, cough, and headache. infection usually resolves without active medical intervention, but for a subset of cases infection can progress to viral pneumonia and a variety of complications including acute lung targetable activities for covid- , the coronavirus mpro seems a likely choice for rapid drug to accelerate drug development we employed a drug repurposing strategy, an approach of utilizing previously approved drugs for new indications , . previous work suggests libraries enriched with known bioactive drug-like compounds provide the best opportunity for finding new lead compounds , . thus we attempted the selective optimization of side activities (sosa) approach as a rapid and cost effective means to identify candidate hits while minimizing the number of compounds screened. the sosa approach proceeds by two steps. first a limited set of carefully chosen, structurally diverse, well- characterized drug molecules are screened; as approved drugs, their bioavailability, toxicity and efficacy in human therapy has already been demonstrated , . to screen as much of the available approved drug space as possible in an easily accessible format we chose to screen the broad institute drug repurposing library ( compounds, see table s ) . this represents about half of the approximately , approved or experimental drugs known to human clinical medicine . there are significant cost and time advantages realized by drug repurposing as it can accelerate the preclinical phase of development and streamline clinical trials to focus on efficacy rather than safety. repositioning existing approved drugs with the capacity to inhibit covid- virus replication and infection would be of profound utility and immediately impact health care in the current pandemic. there are no drugs in clinical use specifically targeting coronavirus replication. the major advantage of the approach taken here is that by screening drugs with a history of previous clinical use, we will be focusing on compounds with known properties in terms of pharmacokinetics (pk), pharmacodynamics (pd) and toxicity. thus, the broad repurposing library we screened consists of compounds suitable for rapid translation to human efficacy trials. we began assay development by selecting potentially suitable synthetic mpro substrates and compared catalyzed hydrolysis curves between fluorescently labeled substrates (ac-abu-tle-leu-gln-afc , dabcyl-vklq-edans, ac-vklq-afc, dabcyl-tsavlqsgfrkm-edans , and mca- . we chose to use the recently published ac-abu-tle-leu-gln-afc (abu= -aminobutyrate, tle=tbutylglycine) synthetic non-canonical amino-acid containing peptide as mpro more readily cleaves this preferred sequence as compared to the native vklq sequence (fig a) . substrates dabcyl-tsavlqsgfrkm-edans and mca-avlqsgfr-k(dnp)-k-nh had drastically lower rates of mpro catalyzed hydrolysis and were not considered further in our assay development ( fig a) . to determine concentration ratios between mpro and substrate, we next preformed a two-dimensional titration and chose nm mpro and µm substrate for a balance of relatively modest mpro protein requirement and a robust fluorescence intensity ( fig b) . before screening the broad library, we piloted our assay conditions against the nih clinical collections library (~ compounds) and calculated our z'-factor for each plate at . and . (fig c and d) . z'-factor is a score of suitability of assays for high-throughput screening and is derived from the equation z ' -factor = − ( + ) | − | , where σ = standard deviation, µ=mean, p=positive controls, and n=negative controls. a score greater than . indicates a screenable assay. although no promising compounds were identified from this smaller library, it demonstrated that our assay was sufficiently robust for screening the much larger broad repurposing library. window was considered at z-score ≤ - and was calculated as the z-score of Δrfu at minutes corresponding to the linear portion of the curve. x-axis indicates arbitrary compound number arranged by increasing z-score. (d) z'-factor for the two nih clinical collection -well plates. pink circles indicate negative control (dmso) and black circles represent positive controls (no protein). z'factor calculated at . and . for plates and respectively. y axis represents change in rfu over minutes. the concept of drug repurposing is to utilize existing therapeutic drugs to treat a new disease indication. this approach is particularly relevant for covid- because of the potential for an accelerated clinical impact as compared to de novo drug development. a systematic approach to facilitate drug repurposing has recently been described ( , http:// www.broadinstitute.org/repurposing) and has made a large collection of drugs with previous history of use in humans available for high throughput screening. we acquired this at -well density using the optimized kinetic mpro assay described in fig . our overall repurposing strategy is described in fig a. we conducted a single point screen at m compound concentration and observed ~ compounds with activity against sars-cov- mpro for an overall hit rate < . %. these compounds were screened in parallel against the natural amino acid substrate (ac-vklq-afc) as well as a kinetically preferred substrate (ac-abu-tle-leu-gln-afc) ( fig b) . individual compounds are shown in table . virtually. any hit from the broad library (z-score ≤ - ) was validated for dose-responsiveness. all suitable compounds passing this filter with satisfactory curve fitting and potency were ordered as powder and re- validated. future efforts will test for selectivity and in orthogonal assays for suitability. although outside the scope of this report, determination of viral anti-replicative properties as well as toxic profile at required dosage will be determined. the goal of this paradigm is to find suitable candidates for development both as tools for probing underlying mechanisms of sars-cov- as well as for translational potential. (b) screen of the broad repurposing library. library was screened at a concentration of µm against both ac-vklq-afc (black) and ac-abu-tle-leu-gln-afc (purple). hit window was considered for compounds falling below z-score ≤ - against both substrates and consisted of compounds. compounds ordered by average z-score. we validated the hits from the primary screen by conducting a -point dose-response analysis with a drug concentration range from m down to . nm ( -fold dilution series). from this dose-response analysis, repurposing library. using this approach, we derived a docking score for each compound (see table s for broad repurposing library with docking scores). we observe a poor correlation (pearson r= . ) between mpro docking score and z-score in the protease inhibition assay (fig a) . furthermore, top hits from the screen also exhibit a weak correlation (pearson r=- . ) between compound potency and docking score (fig b) . is to complete a survey of approved drugs to identify therapies that can block covid- viral replication by inhibiting the main viral protease. the advantage of this approach is that any approved drug identified can be advanced rapidly to clinical trials without extensive multi-year preclinical development efforts. this is also particularly germane given the limitations of animal models of covid- infection and a diverse variety of initial hits were identified in our high throughput screen of the broad library. of these, the most potent hits are all known protease inhibitors and there is strong representation from protease inhibitors developed to inhibit hcv protease ns / a (boceprevir, ciluprevir, narlaprevir, and telaprevir). clearly as approved or well-developed clinical candidates, these drugs exhibit pharmacological and pharmacodynamic properties well suited to repurposing as a covid- antiviral therapy. boceprevir and narlaprevir appear the most potent against mpro and may be suitable for repurposing. ( nm final concentration in reaction buffer detailed above) was added with a multiflo fx liquid dispenser using a µl cassette. compounds were incubated with mpro for minutes at rt after which ul of substrate ( um final concentration of either ac-vklq-afc or ac-abu-tle-leu-gln-afc) was dispensed into the plate and read using a cytation multi-mode reader immediately at / nm excitation and / nm emission wavelengths every minutes for minutes. data was analyzed using biotek gen software, microsoft excel, and graphpad prism . hit compounds were ordered from the broad institute pre-plated in -well format (greiner ) as -point serial dilutions ( -fold) at nl per well. mpro ( nm final concentration) and substrate (ac- abu-tle-leu-gln-afc at µm final concentration) were dispensed in the same manner described above. graphs were generated using graphpad prism . ic calculations were performed using graphpad prism the novel coronavirus originating in wuhan china: challenges for global health governance epidemiologic and clinical characteristics of novel coronavirus infections involving patients outside wuhan cov- and coronavirus disease : what we know so far mortality in older patients with covid- clinical conditions of coronavirus disease (covid- ) pneumonia: a multicenter sars and mers: recent insights into emerging coronaviruses viral and cellular proteins involved in coronavirus replication crystal structure of sars-cov- main protease provides a basis for design of improved alpha-ketoamide inhibitors structure of m(pro) from covid- virus and discovery of its inhibitors atazanavir: a novel hiv- protease inhibitor. expert opinion on investigational drugs simeprevir for the treatment of chronic hepatitis c. expert opinion on pharmacotherapy drug repositioning: identifying and developing new uses for existing drugs a small-molecule screen in c. elegans yields a new calcium channel antagonist an antidepressant that extends lifespan in adult caenorhabditis elegans selective optimization of side activities: the sosa approach selective optimization of side activities: another way for drug discovery the drug repurposing hub: a next-generation drug library and information resource drug bank: open data drug & drug target database profiling of substrate specificity of sars-cov cl structure-based design of antiviral drug candidates targeting the sars-cov- main protease challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection the discovery and development of boceprevir discovery of narlaprevir (sch ): a potent sustained virologic response after therapy with the hcv protease inhibitor narlaprevir in combination with peginterferon and ribavirin is durable through long-term follow-up rapid decline of viral rna in hepatitis c patients treated with vx- : a phase ib, placebo-controlled, randomized study combination therapy with telaprevir and pegylated interferon suppresses both wild-type and resistant hepatitis c virus accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field prospective evaluation of free energy calculations for the prioritization of recent progress in the discovery of inhibitors targeting coronavirus proteases recent developments on coronavirus main protease/ c like protease inhibitors. recent patents on anti-infective drug discovery role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings prediction of the sars-cov- ( -ncov) c- like protease ( cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates glecaprevir and maraviroc are high-affinity inhibitors of sars-cov- main protease: possible implication in covid- therapy boceprevir for untreated chronic hcv genotype infection antiviral activity of narlaprevir combined with ritonavir and pegylated interferon in chronic hepatitis c patients preclinical characterization of the antiviral activity of sch (narlaprevir), a novel mechanism-based inhibitor of hepatitis c virus ns protease broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro glide: a new approach for rapid, accurate docking and scoring method and assessment of docking accuracy glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening key: cord- - s npse authors: du, sean quan; yuan, weiming title: mathematical modeling of interaction between innate and adaptive immune responses in covid‐ and implications for viral pathogenesis date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: s npse we have applied mathematical modeling to investigate the infections of the ongoing coronavirus disease‐ (covid‐ ) pandemic caused by sars‐cov‐ virus. we first validated our model using the well‐studied influenza viruses and then compared the pathogenesis processes between the two viruses. the interaction between host innate and adaptive immune responses was found to be a potential cause for the higher severity and mortality in covid‐ patients. specifically, the timing mismatch between the two immune responses has a major impact on disease progression. the adaptive immune response of the covid‐ patients is more likely to come before the peak of viral load, while the opposite is true for influenza patients. this difference in timing causes delayed depletion of vulnerable epithelial cells in the lungs in covid‐ patients while enhancing viral clearance in influenza patients. stronger adaptive immunity in covid‐ patients can potentially lead to longer recovery time and more severe secondary complications. based on our analysis, delaying the onset of adaptive immune responses during the early phase of infections may be a potential treatment option for high‐risk covid‐ patients. suppressing the adaptive immune response temporarily and avoiding its interference with the innate immune response may allow the innate immunity to more efficiently clear the virus. vere illness tend to have severe pneumonia, sometimes acute respiratory distress, which could lead to multiple organ failures and death. the hospital stay can be weeks for such patients. [ ] [ ] [ ] [ ] there is no proven cure or antiviral drugs available yet. the influenza virus typically binds to the sialic acid receptors on the surface of epithelial cells in the upper respiratory tracks and the upper divisions of bronchi. in very severe, and often fatal cases of influenza, the infection will spread to the lower lungs. , similarly, the sars-cov- virus binds to the angiotensin-converting enzyme (ace ) receptors on the surface epithelial cells. these cells with ace expression are hence vulnerable to virus attack and are called target cells in mathematical models. the density of the target cells has been found to vary significantly in different regions of the respiratory tracks, with the highest in the lungs, followed by the nose, and lastly, the trachea/bronchi tissues. [ ] [ ] [ ] as such pneumonia is relatively common among covid- patients. the incubation time for influenza is typically hours or less. the eclipse phase is reported to be around hours, with viral load peaks around to days postinfection (dpi). the infection typically resolves in to days. in typical influenza patients, the adaptive immune responses (airs) including pathogen-specific antibodies (abs) and cd + cytotoxic t lymphocytes (ctl) are first observed around dpi, peaking around dpi. the mean incubation period for covid- patients is estimated to be days, with a range distribution of days. , the median time from symptoms to discharge from the hospital was about days, whereas the median time to death was days. in this paper, we used mathematical modeling to investigate the dynamics of the viral infection/replication inside a human host, in particular, the influenza and the sars-cov- virus, as well as the interactions of target cells with the innate and airs. our model suggests that most of the differences between the two types of infections can potentially be attributed to the timing mismatch between the two immune responses. more specifically, influenza is a very acute infection; all vulnerable cells are completely depleted and viruses are more or less cleared by the innate immune response, before the adaptive immune response (air), which has a transient nature, reaches a significant level. the arrival of air may seem futile but it helps to completely clear the remaining viruses. on the other hand, the peak level of the air of the covid- patients is more likely to come before the number of infected cells reaches its peak, due to the slower disease progression outlined above. as such the air helps to eliminate more viruses in the rapid growth phase and slow down the infection and depletion of vulnerable epithelial cells in the lungs of covid- patients. a strong air, in this case, may be bad for the patients, as the disease progression is extended longer while a weaker air can be beneficial for the patients due to the rapid depletion of the target cells. one recent observation puzzling for many is the apparent resurgence of the disease for some "cured" patients. based on our theory and modeling, this phenomenon can be explained by that of a strong interaction of innate and airs in a host, which can sometimes create double peaks in viral load, separated by weeks. in the second part of our study, we applied our modeling to the current bedside treatment regimen. antiviral drugs and immunosuppressive drugs have been evaluated as potential treatments for high-risk patients. we proposed an immune-suppressing treatment based on the leanings of our modeling study, which is to apply immunosuppressive drugs during the early phase of infection to reduce the airs to a level low enough not to interfere with the innate immune response. once the vulnerable cells are depleted, the drug should be removed to let the air run its course to clear the remaining virus. this new approach can potentially lead to fast and complete recovery. this counter-intuitive approach aims to mimic the excellent job human immune systems have done with the influenza infection. further studies on immune responses of covid- patients and validation of our core assumptions and theory are needed before this can be applied in real patients' situations. mathematical models have been developed to understand the dynamics of viral infections. [ ] [ ] [ ] [ ] [ ] most of them fall into the category of the target cell-limited model with some variations. the simplest version includes three species: uninfected susceptible target cells (t), which in our case is the surface epithelial cells with ace receptors, located at the respiratory tracks including lungs, nasal and trachea/ bronchial tissues, infected virus-producing cells (i), and the virus particles (v). they can be described by the following set of differential equations: the ability of a virus to grow the infection or to be cleared is determined by the basic reproductive ratio r: where r represents the number of infected cells newly generated by one infected cell. with r> the infection grows, while with r< infected cells decrease and the virus is cleared out. here it is important to note that t is a variable. when the target cells are depleted, t goes down, and so does r. this is a very simple yet critically important concept for understanding the virus clearance. complete depletion of the target cells, namely = t , leads to = r , hence achieving the best virus clearing ability. r is the native ( ). in their experiment, the authors found that the viral load peaked at to dpi. the level of + cd cells and igm data shows a very narrow peak at about days dpi, while the level of igg antibody shows a relatively broad peak lasting well over days. we seek to mimic these temporal profiles, using Δ ( ′) t e as an example: , . with such a profile, we can choose the peak position ′ t c , and the rising and falling width w r and w f in our simulation to see the effects of these parameters on the viral infection dynamics. our main assumption is that the air is a short term effect, which assumes that the virus is successfully cleared timely and the patient survived. as such, it is not our goal to develop a detailed model for airs, but rather to understand the necessary conditions that are needed for a successful outcome for the patients. other components like iga, cd + t cells, and a number of other components of the air could also play important roles here. to analyze the effect of antiviral drugs that either block infection ϵ k and/or production of viral particles ϵ p , the target cell-limited model is modified as follows: refers to a drug that is % effective. with the action of the drug, the reproduction number r is revised as follows: finally, we want to add another component to describe the effect of a drug that suppresses the innate immune response, for reasons that will be discussed in the section of treatments by immunosuppressive drugs. equations ( ), ( ), and ( ) are changed to the following: c describes no effect of immunesuppressing drugs while δ ϵ = , c refers to complete suppression. mathematical models of influenza infections have been studied extensively, , - our interest is to use it as a starting point before we embark on more challenging work on sars-cov- infections. we have carefully evaluated the parameter values tabulated by beauchemin et al and decided to use the values shown in table . the choice of the parameters is based on cross-checking multiple sources, and testing many simulation runs with variations of parameters to ensure that the output agrees with what is known about influenza infections. it is not our intent to fit the model to any specific set of data, but rather to be able to reproduce the general features of the influenza infections. the result is shown at figure . with these parameters, the infected cell count peaks at . dpi; viral load peaks at . dpi, and and the r is about . γτ , which describes an exponential growth rate (shown in appendix) is . . as we should expect, the target cells are depleted shortly following the peak of the infected cells, after which the virus count decays exponentially. the situation with regard to the decay portion is more complicated, and the consideration of the air is warranted if one is to understand it beyond dpi. table shows the parameters for the three components we considered in equations ( ). these values are chosen so that the temporal profiles resemble what was presented by miao et al the choice of the peak time is based on beauchemin et al we should clarify that the temporal profiles are not completely the same as in reference, as some other literature has shown broader profiles. we have done sensitivity analysis by doing multiple simulations, and find that as long as the width is not too narrow, the results are not sensitive to the width or shape of the peak. on the other hand, the location of the peak time is very important for the complete clearing of the virus. the result is shown in figure . in figure , we included the same graph for virus count without the air, as shown in figure as long as the native r is greater than , this pattern is the universal behavior of the equations ( ), namely, a rapid exponential rise followed by an exponential decay, then eventually reaching an equilibrium at a low level of infection. changing parameters in table will change the time scale, the peak/trough levels and locations of virus count etc., but the general pattern is the same. when the air is included, the virus clears out much faster. by day , the virus is completely gone. mathematically virus and infected cells count never actually go to zero. however, we know that the infected cell count has to be an integer. therefore when this number is below , one can safely consider it game-over for the virus. in our modeling, we set this cutoff to be . infected cell to be conservative. the temporal profile of the three components of the air are also shown in figure so that the readers can see their peak positions as compared to the actual virus counts over time. after the air becomes active, the decay of virus count becomes much faster. we also tried to vary the parameters around what is listed in due to the lack of experimental data, the discussions below are based on our best assumptions and observations. when shifting gears from the influenza virus to the novel corona virus, the most important difference we noticed is the difference in the pace of infection progression. multiple reports suggested that the viral counts of sars-cov- do not reach a peak until to weeks postinoculation. [ ] [ ] [ ] here we will make a bold assumption that, if without the air, the peak time is around to days, if not more. as most of the severe cases result in pneumonia and the lungs are significantly larger than the nose, t is estimated to be higher by a factor of . table lists the only two parameters that are changed from tables and , together with the three dependent variables as outputs from the model. here we aimed to have a peak time for infected cells at around dpi if considering the effect of innate immune response only. together with a times increase of t , these constraints essentially limited our choice for the only other variable to a very narrow range. the result is shown in figure . as we can see in figure , the curve of virus count for an innate immune response only (no air) is similar to that of influenza infec- our initial choice of air activity of . is purely artificial at this point, so we want to explore the effect of different levels of air activity. the result is shown in figure . as we can see, with the air activity level of . - . , the virus count reaches a high level, and then the growth is slowed with the activation of the air at around dpi. after the effect of the air wanes, the virus count continues its ascent, reaching its peak at a later date. at a higher air activity level, we start to see an apparent drop in virus count after the onset of air, reaching a trough then rising to its peak at a much later day. overall, an increase in the air activity tends to increase the duration of viral activity extensively in this case. this is because early activation of the air helps to reduce infection and depletion of target cells before it reaches a peak level, which saves a large number of uninfected target cells for later infection. when the effect of the air wanes, the virus takes control again when the effective r rises above . whether the virus count grows or drops during the peak air activity is dependent on how strong the air effect is, and whether the effective reproductive ratio r at the time is greater or less in figure with the air activity level greater or equal to . offers a ready explanation for some perplexing clinical observations that some patients can appear to be recovered, but have the virus level resurges at a later time. what's even more interesting is when we shift the day that the air activity peaks, as shown in figure . to show the air effect on viral replication in a more pronounced fashion, we fix the air activity level at . , and vary the day of its peak with additional simulations, we have found that if the air peak is after the viral peak, a higher level of air activity always helps to clear the virus faster. contrarily, if the air peaks before the viral peak, higher air activity can increase the duration of viral activity. we also found that if the air level is low enough, for example below . , its effect becomes negligible and the virus replication and clearing process is in essence, our modeling aims to explore potential means to manage the covid- infections so that it can be similar to influenza infections. when the virus can be quickly and completely cleared, the air should be transient, similar to the influenza infections. our modeling then establishes that one of the requirements is that the peak of air needs to come after the peak of infected cells, or equivalently, the peak of viral load. this can be viewed as a necessary condition for fast and complete clearing of virus. to achieve rapid clearing of the virus, it is also desirable to have the target cells more or less completely depleted. in reality, when the virus cannot be cleared timely, it is likely that the air will also be extended longer, so the conclusions from our modeling related to the later stage development may need to be modified. when searching for a cure for covid- patients, an effective antiviral drug is highly anticipated, so we decided to model the effect of the antiviral drug on the corona virus infection. we started our base case with a peak of the air at dpi, and air activity level of . , as shown before. from equations ( ), the expression for r is revised as follows: here r is the native reproductive ratio; t t is the percentage of the target cells that are not depleted. the first two brackets show the effect of the antiviral drugs on the reproductive ratio r, which shows that the drug has an equivalent effect regardless of whether it is acting on k or p. next, we tried to simulate the effect of an antiviral f i g u r e effect of adaptive immuneresponse (air) peak day on viral dynamics: data shows the viral load over time when the day of air peak is changed from to days drug with % efficacy on k, namely, ϵ = . k , taken for days consecutively. taking this drug for a longer time does not necessarily make it more beneficial in this particular scenario. as a comparison, the base scenario is also included with a native r of . , and air activity of . peaking at dpi. the base scenario is shown as a blue line in figure . when the drug is applied, the effective r is reduced to . , according to the formula above, not considering the effect of target cell depletion. similar to the air effects shown in figure , figure shows that additional drug-induced antiviral activity has a pronounced effect on the duration of viral activity within the host for the three scenarios when the antiviral drug is started on dpi, dpi, or dpi. during the days when the antiviral drug is effective, the virus growth rate is reduced. after the drug is removed, the virus either resumes its growth or remains at a plateau. in all three scenarios, the duration of virus activity is extended substantially, which can lead to detrimental consequences for the patients. on the other hand, if the drug is started or dpi, after the peak of infected cells, the effect of the antiviral drug is to speed up the virus clearing, which is beneficial for the patient. in figure , the depletion of target cells is also shown for the scenario of the drug started at dpi. as we can see, the depletion is not complete ( % target cells remain), which explains why the decay is slow even with the additional drug activity. the situation is very different if the antiviral drug efficacy is raised to %, namely, ϵ = . k . when the drug is applied, the effective r becomes . , which leads to virus clearance. we ran the same set of scenarios as above and the results are shown in figure . the blue line again shows the base scenario with no drug applied. if the drug is applied early, as shown for the scenario with a start date of dpi, the infection can be stopped completely before it makes any real progress. when the drug is started at or dpi, double peaks of viral count are observed. the reason is that when the drug is stopped, r jumps back to , so that virus resumes its rapid growth until the target cells are depleted, causing a second peak in virus count. therefore if the drug is stopped before the virus is cleared out completely, the infection will resurge. this is similar to what we have seen in figure with the transient antiviral activity of strong air, except the peaktrough-peak curve is smoother there. when the drug is applied after the viral peak is reached, it has a small but positive effect of faster recovery, similar to the scenario shown in figure . the level of target cell depletion is also shown for the scenario of the drug started at dpi. at the first peak of viral load, less than % of the target cells are depleted by the infection. this is why a second exponential growth starts after the drug is removed. to avoid this kind of unwanted effect, the antiviral drug needs to be maintained until the virus is completely cleared. in the appendix, we showed the relationship between the duration the drug needs to be administered, and the time the drug is started. the simple rule of thumb is that the later the drug is started, the longer it is required to be applied to ensure complete clearing of the virus. it is interesting to note that γ-immunoglobulin has been widely used to treat covid- patients in china. in a way, injection of γ-immunoglobulin can be modeled similarly as antiviral drugs f i g u r e effect of antiviral drug on viral dynamics: the drug is assumed to be % effective on k, and applied to a host with r of . for days consecutively with various starting dates; viral counts to the left with exponential scale; target cell ∕ t t is shown for the scenario of the drug applied at dpi, to the right with linear scale du and yuan | (assuming that it is able to deliver some antiviral function). it will be interesting to know if some researchers have compiled data to correlate the efficacy of this treatment with the timing of the injections. our theory predicts that if the γ-immunoglobulin is used early on, it will most likely delay the depletion of target cells so the duration of the disease will be extended for the patients, hence with poor prognosis. on the other hand, if it is applied after the peak of infected cells, it should be helpful to some extent. in summary, for an antiviral drug to be useful, the drug needs to be effective enough so that the effective reproductive ratio r falls below (see equation ( ) for the factors affecting r). under such a condition, it is desirable to apply it early on and must be maintained until the viral load is completely cleared (in the appendix we provide an estimate for the duration of the regimen). when a drug cannot meet this criterion, applying it before the target cells are fully depleted is typically not beneficial to the patient, and likely will make things worse for the patients. this should have some significant implications for drug companies when they design the clinical trials. at this point, as we do not know much about the potential level of the air, r , and the efficacy of the antiviral drug, it is difficult to identify a risk-free way of applying the antiviral drug. a prudent approach is to measure the viral loads frequently post-drug administration. if the viral load does not drop rapidly shortly after, it may indicate that the effective r is not below , and the drug should be stopped. if the drug is known to be effective but not effective enough to cause r to go below , it might still be useful to apply it after a large percentage of the target cells have been depleted. as a safety measure, it may be desirable to continue the regimen for a few more days after the viral load drops below the detection limit to ensure complete clearance. currently, there is already a class of drugs called immunosuppressive drugs (drugs to suppress airs). a large body of research has been done on the effects of the drugs on different parts of immune response. , the goal of investigating immunosuppressive drugs is to explore their possible applications in covid- disease management. immunosuppressants such as corticosteroids have been applied to sars and covid- patients, with positive results. , from the previous discussions, we propose a potential treatment plan by applying immunosuppressive drugs as soon as an infection is detected in the lungs, and remove the drug as soon as the target cells are fully depleted. this approach is simple and only requires the immunosuppressive drugs to be able to reduce the air activity to a level low enough not to interfere with the innate immunity. from the discussions around figure , this approach should clear out the virus quickly if the air activity level is below . . if the drug effect is not removed quickly, and the air does not rise after the drug is removed, the patients may not be able to completely clear the residue level of the virus. we projected that a short regimen of antiviral drugs to days after the peak of virus counts may help to completely clear the remaining low level of virus load. the purpose of the antiviral drug f i g u r e effect of antiviral drug on viral dynamics: the drug is assumed to be % effective on k, and is applied for days consecutively with various starting dates; viral counts to the left with exponential scale; target cell ∕ t t is for drug started on dpi only, shown with linear scale to the right regimen is to mimic the effect of the air in patients with influenza infections. if the drug effect can be removed immediately after the peak infection, so that the air level increases after the drug is removed, we then have a situation that the air peaks after the target cells are depleted. the discussions around figure established that with this scenario, the virus can always be cleared completely, regardless of the level of the air. with this approach, the novel coronavirus infections are essentially reduced to influenza-like infections in adults, except with slower disease progression. one possible concern with our proposed approach is that the immunosuppressive drugs may inevitably have some limited side effects on the innate immune response, in addition to the intended effect on the air. this is considered by equations ( ), which gives us the following (see appendix): to address this possible concern, we did some simulations with the following: the immunosuppressive drug is assumed to have either a % or % effect on ϵ δ , the drug with % effect on ϵ δ is applied from dpi up to dpi, while the second drug is applied from dpi up to dpi. the choice to apply the drug from dpi is merely to simplify our simulation, and may not be necessary, as our main goal is to suppress the air. the air peak day is assumed to be the same as the base scenario at dpi, and the air activity level is reduced to . . the same base scenario without drug is also shown as the blue line in figure . with both scenarios, we observed a big reduction of the duration of viral activity. not only the date of viral peak is moved from to dpi, but the decay from the peak is also much faster. this rapid decay is beneficial for the patients as well, and it's due to the complete depletion of the target cells. the overall virus replication curve with the immunosuppressive drug resembles closely the curve for adult influenza infections (shown in figure ) except with a longer time scale. when compared to the base scenario with air (the blue line in figure ), we find that the maximum viral load is increased by a factor of . - . . however, if we compare it to the base scenario of innate response alone without air, the maximum viral load varies by less than %. in summary, we think that the benefit of shortening the disease duration outweighs the cost of increased viral load for a short period. we see multiple potential advantages of this novel approach. there are a number of well-studied, fda-approved drug f i g u r e effect of immunosuppressive drug on viral dynamics: the drug is assumed to be or % effective so ϵ δ = . or . ; drug is applied from dpi until or dpi (with the end day shown on legend); viral counts shown to the left with exponential scale; target cell ∕ t t is shown for dpi for both % and % drugs, with linear scale to the right du and yuan | candidates to choose from. animal models can help to define the range for the main parameters quickly. finally, as long as the drug is applied for enough days so that the suppression of the air is beyond the peak of virus load, this approach always resulted in a quick clearance of the virus. removing the drug in time should enable the air to be fully stimulated, so the patient most likely can have antibody-based immunity for the virus in the near future, as in the case of patients recovered from influenza infections. in our discussion so far, we have assumed that the drug effect is immediate, namely, the drug effect shows up immediately after its application and the effect goes away immediately after the drug is removed. this is clearly not realistic, so one of the key considerations when selecting the right immunosuppressive drugs is its pharmacokinetics; a drug with short elimination and absorption half time is more desirable, considering the time-sensitive nature of the effect. in a recent study by wang et al, the authors reviewed the effect of a low-dose and short-term application of corticosteroid to covid- patients at an early stage, and found some clear benefits of for the patients as compared to the patients without the treatment, including the shortening the disease course. this is consistent with our findings. in their study, the drug corticosteroid was applied for days or more, which according to our theory, might be a little too long for optimal results, considering its half time of to days. previous studies have investigated the rna expression of ace in human tissues. two more recent studies have shown that the percentage of epithelial cells with ace receptors in bronchial is much lower than in turbinate and lung tissues ( . % vs - %). , considering the difference in surface area between the lungs and the nasal cavity, [ ] [ ] [ ] we arrived at a rough estimate of as the ratio of target cells in these two organs. a quick simulation using the much lower t suggests that the peak of infected cells is changed from to dpi in the nose with the innate immune response alone. if infection is localized at the nasal cavity only, it is then much more likely for the peak of the air to come after the peak of infected cells. in this case, most likely the infection will resolve itself just like influenza infections, except the disease may take a few more days. this may explain why a lot of people do not develop severe symptoms or remain asymptomatic if the infection is localized in the nasal area without reaching and propagating in the lungs. if our analysis can be verified clinically, methods and techniques (for example antiviral nasal sprays) to enhance trachea/ bronchial tubes as a barrier to stop the spread of the virus from upper respiratory tracks to the lungs can be effective in reducing the percentage of patients with pneumonia. another important parameter we have not discussed is the cell regeneration rate, τ d , of the epithelial cells. in table , we have listed it as . × − , which corresponds to − day − for d. this is based on a study by rawlins and hogan on the half-life of epithelial cells in the mouse lung. this parameter, if it is higher, will make the depletion of the target cells more difficult. this is because as the target cells are depleted, new target cells are generated. the higher the regeneration rate, the more new target cells are available for viruses to infect and stay alive. our simulation results remain essentially the same if this parameter is lower or increased by no more than a factor of . beyond that, depletion of the target cells becomes substantially more difficult to achieve. thereby, clearing the virus also becomes harder to accomplish. could this be the reason for the observed higher severity rate among older patients? we reason that this is not likely, as the cell regeneration rate should be higher for younger patients, while older people are known to have more difficulty repairing lung damage. in this article, we have applied the target cell-limited model to study the novel corona virus infections mostly in human lungs. through comparison between sars-cov- and influenza viruses, and by analyzing the interactions among various players (target cells, innate and airs, and different types of drugs), we propose that the main reason for the higher probability of severe symptoms, prolonged hospital stays, and even fatal outcomes for covid- patients , may be that the peak of infected cells and virus counts often comes after the peak of the air in these patients. this mismatch of timing and the resulted interference with innate immunity by adaptive immunity, leads to incomplete depletion of the target cells, thus providing uninfected target cells for continued infection. prolonged infection can induce overactive immune responses, secondary complications, and sometimes fatal outcomes. we also discussed the prospects of antiviral drugs and immunosuppressive drugs in combating covid- infections. our proposal to apply immunosuppressive drugs at an early stage to reduce the interference from adaptive immunity so that the innate immunity and the depletion of target cells can together achieve fast elimination of the virus seems to be unconventional. however, it is particularly interesting to note that, as we were working on our mathematical modeling, the recent study by wang et al demonstrated that an early stage low-dose and short-term application of corticosteroid treatment in patients with severe covid- pneumonia was beneficial and essentially validated our findings. we envision that new insights from our analysis and modeling will encourage more work in this direction. clinical features of patients infected with novel coronavirus in wuhan clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a review of mathematical models of influenza a infections within a host or cell culture: lessons learned and challenges ahead h n virus attachment to lower respiratory tract tropism of avian influenza a (h n ) in the upper and lower respiratory tract evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis single-cell rna expression profiling of ace , the putative receptor of wuhan -ncov single-cell rna expression profiling of ace , the putative receptor of wuhan -ncov principles and practice of clinical virology defense mechanisms against influenza virus infection in the respiratory tract mucosa incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia mathematical analysis of viral replication dynamics and antiviral treatment strategies: from basic models to age-based multi-scale modeling population dynamics of immune responses to persistent viruses viral dynamics in hepatitis b virus infection virus dynamics and drug therapy mathematical models of hiv pathogenesis and treatment virus dynamics: mathematical principles of immunology and virology influenza and the challenge for immunology cell-mediated protection in influenza infection pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response quantifying the early immune response and adaptive immune response kinetics in mice infected with influenza a virus hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy effects of antirejection drugs on innate immune cells after kidney transplantation immunobiology: the immune system in health and disease early, low-dose and short-term application of corticosteroid treatment in patients with severe covid- pneumonia: single-center experience from wuhan, china. medrxiv description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china histology a text & atlas anatomical and histological factors affecting intranasal drug and vaccine delivery ciliated epithelial cell lifespan in the mouse trachea and lung regeneration of the aging lung: a mini-review the authors would like to express their appreciation for professor yong-zhong qian for some productive discussions. weiming yuan is supported by the nih grant (r ai ) and partially supported by nih grant p ca to the university of southern california norris comprehensive cancer center from the national cancer center. the authors declare that there are no conflict of interests. http://orcid.org/ - - - solving the above equation, we find the following:the time it takes for the infected cells to reach the peak, t r , is as follows, assuming complete depletion of the target cells: notice that here we used i max and i min , because to clear virus completely we need to get to a level lower than the initial level of infected cells. immediately we can see that when = r , we have the lowest t c , which means the fastest clearing of virus. if we assume that, the above result can be further simplified: key: cord- - el ryl authors: watkins, laura c.; degrado, william f.; voth, gregory a. title: influenza a m inhibitor binding understood through mechanisms of excess proton stabilization and channel dynamics date: - - journal: j am chem soc doi: . /jacs. c sha: doc_id: cord_uid: el ryl [image: see text] prevalent resistance to inhibitors that target the influenza a m proton channel has necessitated a continued drug design effort, supported by a sustained study of the mechanism of channel function and inhibition. recent high-resolution x-ray crystal structures present the first opportunity to see how the adamantyl amine class of inhibitors bind to m and disrupt and interact with the channel’s water network, providing insight into the critical properties that enable their effective inhibition in wild-type m . in this work, we examine the hypothesis that these drugs act primarily as mechanism-based inhibitors by comparing hydrated excess proton stabilization during proton transport in m with the interactions revealed in the crystal structures, using the multiscale reactive molecular dynamics (ms-rmd) methodology. ms-rmd, unlike classical molecular dynamics, models the hydrated proton (hydronium-like cation) as a dynamic excess charge defect and allows bonds to break and form, capturing the intricate interactions between the hydrated excess proton, protein atoms, and water. through this, we show that the ammonium group of the inhibitors is effectively positioned to take advantage of the channel’s natural ability to stabilize an excess protonic charge and act as a hydronium mimic. additionally, we show that the channel is especially stable in the drug binding region, highlighting the importance of this property for binding the adamantane group. finally, we characterize an additional hinge point near val , which dynamically responds to charge and inhibitor binding. altogether, this work further illuminates a dynamic understanding of the mechanism of drug inhibition in m , grounded in the fundamental properties that enable the channel to transport and stabilize excess protons, with critical implications for future drug design efforts. proton transport (pt) across cellular membranes is a critical component of many biomolecular systems, necessary, for example, to maintain ph gradients, , to drive atp synthesis, and to facilitate the co-or antitransport of other small molecules. − because of their essential role in such systems, channels and transporters with pt functionality are often targets for drug design to inhibit or control pt: in the case of viruses and bacteria, to slow or prevent infection, but there are myriad other disease applications. − drug design is notoriously challenging, as both thermodynamic and kinetic factors must be considered but are difficult to predict and control, and its success depends on high quality structures, an understanding of structural dynamics, and a knowledge of the protein's function and its mechanism. thus, beyond elucidating mechanisms of pt in order to understand how a specific channel or transporter works, studying the detailed interactions that facilitate pt can provide valuable insights to help guide drug design efforts. the influenza virus kills up to people each year, and the impact of the recent global coronavirus pandemic emphasizes how critical it is to maintain our focus on understanding and treating viral infections. the influenza a virus matrix (m ) proton channel is a homotetrameric protein responsible for the acidification of the viral interior, a critical step in the influenza infection process. − it is the target of two of the three currently available oral antivirals, amantadine and rimantadine. , while these are effective at blocking pt in wild-type m , drug-resistant mutants have become the predominant strains, the majority of which contain an s n mutation. , this widespread resistance requires a continued drug design effort informed by a deeper understanding of the pt and drug inhibition mechanisms. additionally, m is considered an archetype for the viroporin family, a class of viral channels considered ideal drug targets. the sars-cov- virus responsible for the covid- pandemic contains two viroporins: protein e and . − thus, viroporins are a critical class of proteins to study as potential therapeutic targets. m is located in the viral capsid and is acid-activated: as the ph of the endosome encapsulating the virus is lowered, the m channel becomes activated and facilitates unidirectional proton flow to the viral interior, allowing the virus to escape the endosome and infect the cell. the key residue that controls activation is his , − which can bind one additional proton and take on a + charge. one histidine from each helix forms the his tetrad, which can collectively hold a + to + excess charge, dependent on ph. the channel becomes activated and the c-terminal portion opens (adopting the inward open conformation) upon reaching the + state, and pt occurs as the channel cycles through a transporter-like mechanism. − amantadine and rimantadine belong to the adamantyl amine class of inhibitors, binding in the upper-middle portion of the channel. these drugs were the predecessors of many related adamantane-based compounds featuring a relatively rigid, apolar group and an attached charged group. − recently, thomaston et al. published several high-resolution x-ray crystal structures of m with amantadine, rimantadine, and a novel spiro-adamantyl amine bound. these structures provided the first opportunity to see the specific interactions that facilitate stable inhibitor binding and the disruption of the hydrogen bonded water network otherwise present. along with an earlier qualitative md simulation study that guided the design of the spiro-adamantyl amine inhibitors, the crystallographic analysis provided potential insights into the mechanism of inhibition, suggesting that the backbone carbonyls of pore-lining residues act as "physiochemical chameleons", able to engage in both hydrophobic and hydrophilic interactions, and that the drug is tilted off the channel's axis and interacts with waters in the ala layer. taken together, it is hypothesized that amantadine acts as a mechanism-based inhibitor, with the ammonium group functioning as a hydronium mimic. computational studies to date have primarily focused on the means of entry into the channel and location of binding, − but have not deduced specific interactions between the drug, channel, and channel water involved in binding as they relate specifically to similar interactions seen in the pt mechanism. proton transport is an inherently quantum mechanical process, as the hydrated proton structure (hydronium-like) exists in a complex hydrogen bonded network that rearranges dynamically as bonds break and form according to the grotthuss shuttling mechanism. − thus, classical molecular dynamics (md) with fixed bonding topology cannot be used to study pt; moreover, ab initio methods are not efficient enough to reach the many nanosecond time scales necessary to obtain sufficient sampling in biomolecular systems that may have important degrees of freedom several orders of magnitude slower than proton shuttling. multiscale reactive molecular dynamics (ms-rmd) − (and multistate empirical valence bond, ms-evb, before it) was developed to efficiently and accurately capture the solvation and delocalization of an excess proton in water, such that the quantum-chemical nature of the hydrated proton can be studied in the context of membrane proteins over the long time scales needed for accurate simulation of such systems. ms-rmd has been successfully applied in several protein systems to predict and explain mechanisms of pt. , , − in previous work, , quantum mechanics/molecular mechanics (qm/mm) and ms-rmd were used to calculate potentials of mean force (pmfs; i.e., free energy profiles) of pt through the m channel in the + to + states, providing critical insight into the ph-dependent activation behavior and the role of the his tetrad in pt. most recently, we further analyzed the ms-rmd simulations to explore the detailed interactions between the hydrated excess proton and the channel and found that the proton dynamically, as a function of its position, alters several properties of the protein and pore waters, including the hydrogen bonding network and the protein structure. this latter work illustrates how ms-rmd can be used successfully to investigate explicit, dynamic interactions between a hydrated proton and its immediate environment, as well as its indirect effects on other parts of the system. here, we employ an approach similar to that in this previous work to focus specifically on properties related to drug binding and how the position of the bound drug relates to the overall pt mechanism. through this analysis, we examine the hypothesis that the adamantyl amine drugs act as mechanism-based inhibitorsby identifying stabilizing interactions between the excess proton and the channel, we show how the drug may similarly be stabilized in support of this hypothesis. additionally, by examining conformational fluctuations, we show that the drug binding pocket is an especially stable and symmetrical portion of the channel, conducive to binding a roughly spherical drug, and we reveal an additional minor hinge point toward the top of the channel which may be a relevant feature for future drug design efforts. simulations for calculating properties as the proton moves through the top of the channel were run as follows. starting structures were taken from previous simulations, which were initiated from a crystal structure of the transmembrane portion of the m channel (this construct is referred to as m tm) resolved at room temperature and high ph (pdb: qkl ) embedded in a -palmitoyl- -oleoyl-snglycero- -phosphocholine (popc) bilayer solvated with water. m tm is the minimum construct necessary to retain proton conduction similar to full-length m , and it has been shown that the presence of amphipathic helices, included in the full-length m protein, do not significantly influence the pt mechanism. the water and excess proton in the system were modeled using the ms-rmd method, which allows bonds to break and form by taking a linear combination of different bonding topology states at each time step. the ms-evb version . parameters were used to describe the hydrated excess proton. we refer the reader to previous work for a full description of the method. − the excess proton center of excess charge (cec) is defined as where r i coc is the center of charge of the ith diabatic ms-rmd state and c i is the amplitude of that state. the sum is over all n states. the interactions in the remainder of the system were defined by the charmm force field. simulations were run in the nvt ensemble at . k using lammps (http://lammps.sandia.gov) with the ms-rmd package. the collective variable (cv) defined for umbrella sampling (us) is the z-coordinate of the vector between the excess proton cec and the center of mass of the four gly α-carbons, as in our previous work, such that the cv has negative values at the top (n-terminal end) of the protein and progresses to positive values at the bottom (c-terminal end). simulations were run with the excess proton at every . Å along the cv coordinate between − . and . , generating windows. to ensure that the proton remained in the channel, a cylindrical restraint was added at Å with a force constant of kcal/mol·Å using the open-source, communitydeveloped plumed library. , after a ps ms-rmd equilibration, the replica exchange umbrella sampling technique was used to facilitate convergence. production simulations were run for ∼ − ns with frames saved every ps. for calculating hydrogen bond residence times, longer independent trajectories were run with the cec restrained in five different journal of the american chemical society pubs.acs.org/jacs article positions using us as described above. each trajectory was run for . − ps with frames saved every fs. simulation frames were binned by excess proton cec value for subsequent analyses, which were performed in python using the scipy, numpy, and pandas libraries. for hydrogen bond analysis, values were averaged over the four helices. hydrogen bonds were defined by the following criteria: the donor−acceptor distance must be less than . Å and the donor−hydrogen−acceptor angle must be greater than °. several hydrogen bond definitions were tested and did not affect the conclusions (not shown). for calculating residence times, a hydrogen bond was considered in place as long as the particular water molecule remained the closest water to the protein atom and the hydrogen bond criteria were met. images of molecular structures were rendered in visual molecular dynamics (vmd), while other figures were generated using matplotlib. if the adamantyl amine drugs are acting as mechanism-based inhibitors as hypothesized, we would expect to see specific aspects of the pt mechanism taken advantage of or replicated by the drug upon drug binding. to test this, we performed ms-rmd simulations of m in the + his charge state with an explicit excess proton to evaluate the hydrogen bonding networks, pore shape, and protein fluctuations throughout pt that relate to drug binding. by focusing on pt in the + state, we are studying the process of proton entry and diffusion to his in the first key step of channel activation, paralleling inhibitor entry into the channel. we additionally expect this to be a prevalent charge state in drug-bound structures due to the lowered his pk a 's. replica exchange umbrella sampling was used to obtain sufficient sampling of proton positions throughout the top portion of the channel, with windows from cec z = − . to . Å where the coordinate origin is defined as the center of mass of the gly α-carbons. the channel is aligned along the z-axis for all subsequent analyses. to understand how properties of pt may provide insight into drug binding, we primarily examine variations dependent on proton position. we compared the values of each property when the proton is at the drugs' ammonium group positions versus other parts of the channel to determine if the drugs could be taking advantage of the channel's natural ability to stabilize a proton. this idea is highlighted in figure , which shows both the drug-bound crystal structure and a snapshot of a hydrated excess proton in the channel from our simulations. we refer to the drugs' ammonium nitrogen position along the z-axis in the crystal structure as ammn z . this value is − . and − . Å for the inward closed amantadine and rimantadine bound structures, respectively (averaged over the two tetramers in each crystal structure). flexible hydrogen bonds stabilize the excess proton near ammn z . it has been shown in our previous work that hydrogen bonds within the channel, including those between water and protein atoms, help facilitate proton transport by altering their direction and frequency of interaction as the proton moves through the channel. here, we focus specifically on water interactions that may help account for excess charge stabilization near ammn z . in figure , we calculate the occupancy of three different hydrogen bonds between protein atoms and water as a function of the excess proton position in the channel. while the ala hydrogen bond occupancy is constant as the excess proton enters and moves through the top of the channel, as it approaches the ala carbonyls, the occupancy decreases ∼ %. this dip indicates the ala hydrogen bonded waters can flexibly reduce their interaction with the protein as a result of an excess charge in their vicinity. additionally, this dip is centered at − . Å, near ammn z . at this point, the role of the waters near ala carbonyls in hydrating the proton is maximized. in a previously published pmf of the + his charge state, there is notably a local minimum near this point, further indicating that an excess charge is relatively stable here. this supports the hypothesis that amantadine and rimantadine are mechanism-based inhibitors and take advantage of the channel's natural ability to stabilize a hydrated excess proton in order to stabilize the drug's ammonium group. the hydrogen bond occupancy of waters with the gly carbonyls increases once the excess proton passes through the val gate and remains fairly consistent across proton positions thereafter, exhibiting little dependence on the hydrated proton position once it is in the channel. the ser side chain water occupancies are shown for comparison, which do not show a noticeable trend based on proton position. thus, this change in interactions is not a universal effect throughout the channel, but the ala waters seem to be uniquely flexible in this manner. these differences are consistent with drug design studies: while compounds such as spiro-adamantyl amine have been able to displace the water in the ala layer, no designed inhibitors have displaced the water around gly . to further understand how the dynamics of the hydrogen bond network may show how these drugs benefit from the channel's inherent excess-charge stabilization used in proton transport, we examined the average residence times of hydrogen bonds between water and several important protein atoms. to do this, independent trajectories were run for five different excess proton positions, including two trajectories with the proton completely outside the channel (cec z = − . , . Å) and three when the proton is near ammn z . these results are shown in figure . the ala water residence times slightly increase when the excess proton is near ammn z , the ser water residence times do not show any significant difference between the proton outside the channel and at ammn z , and those of gly waters decrease at ammn z . the waters hydrogen bonded to his imidazole nitrogens show the greatest change in residence times and are shown to highlight the ability of this method to describe such differences. with the above results for ala , this may indicate that several waters remain tightly hydrogen bonded to the ala backbone carbonyls, while one or more are bonded less frequently. while the gly hydrogen bonds do not form less frequently (as indicated in figure ) with an excess charge in this region, they do exhibit greater dynamics and flexibility. this change indicates an increase in water dynamics when the excess proton is near, which could help stabilize and solvate the excess charge in the ala water layer. taken together, these results further support the hypothesis that amantadine and rimantadine act as mechanism-based inhibitors: the channel acts as a scaffold to facilitate pt by harboring flexible protein−water interactions that can adapt and respond to a positive excess charge, with the specific ability to stabilize an excess proton near ammn z . drug tilt positions ammonium group in highest cec density. one prominent characteristic of the amantadine and rimantadine bound structures is the drug's tilt within the pore. this tilted conformation is also seen in solid-state nmr studies. given the drug's threefold symmetry in a fourfold symmetric channel, the ammonium group cannot form hydrogen bonds with all four waters hydrogen bonded to ala , leading in part to this tilt. based on our previous work examining the proton's path through the channel, we used a similar analysis to examine the density of cec positions when the excess proton is near the ammonium position in drugbound structures. figure a shows the difference in cec density when the proton is near ammn z compared with the average over all proton positions through the top portion of the channel. this two-dimensional histogram of cec positions in the xy-plane is calculated for cec z = − . ± . Å, minus the average over all normalized histograms for cec z positions [− . , . ] Å binned every . Å. interestingly, in this portion of the channel the excess proton prefers to be near the edge of the pore, unlike the predominant preference for the center of the pore throughout the rest of the channel, as indicated by the positive values around the edge and negative values in the center. figure b shows the radial density of the cec in this same region of the channel. possible positions of amantadine's ammonium group nitrogens were calculated based on the drug's position and tilt in the crystal structure, and their radii are included as dashed lines (these positions were also used to generate the image in figure ). these hydrogens can extend to a radius of ∼ . Å in this static crystal structure, which means that the slightly off-centered ammonium group directly positions one to two of its hydrogens in the region of the cec's highest density. the cec's propensity for the edge of the pore indicates that the drug's tilt in the channel may not only be a necessary component of its binding, but also a thermodynamic advantage. this tilt further allows the drug to the analysis of hydrogen bonding changes and proton densities indicates how the ammonium group is a functional addition to the adamantane scaffold, as the charged group is positioned in a region where the channel is especially adept to stabilize an excess charge. this stabilization relies on flexible water structures and hydrogen bond interactions that can undergo minor changes to accommodate the proton, suggesting that the adamantyl amine inhibitors are acting as hydronium mimics: they take advantage of these inherent features to help solvate the charged ammonium group. the identification of other regions of the channel with increased ability to stabilize an excess charge, such as areas of increased proton density or significantly flexible water interactions, could help provide new targets for drugs to act as hydronium mimics. pore shape and stability near ser are ideal for adamantane binding. another hypothesis about the adamantyl amine class of inhibitors is that adamantane is effectively spherical and can freely rotate within the channel, but has no rotatable bonds, which minimizes the entropy lost upon binding. this rapid rotation can be seen on the nmr time scale and is consistent with the recent thomaston et al. crystallographic studies, in which the motion was indirectly inferred. nevertheless, its significance depends on the dynamic nature of the channel: if the protein exhibits great structural fluctuations in the region where the drug binds, then drug binding may induce changes that greatly decrease the entropy and this hypothesis would not fully explain the drugs' efficacy. to better understand how the channel's natural dynamics may lend itself to favorable drug binding, we examined the pore shape throughout our trajectories. as an estimate of the asymmetry of the channel, we calculated the eccentricity, which is essentially a measure of how "circular" a given oval is. the eccentricity is defined as where a and b are the semimajor and semiminor axes, respectively, which we approximate by the distance between αcarbons on opposing helices. a schematic of this is shown in si these results are shown in figure . eccentricity maximum, minimum, and root-mean-square-deviation (rmsd) values are calculated in si table . the pore-lining residues in the bottom half of the channel, gly , his , and trp , all show a greater degree of asymmetry and a wider range of eccentricity values, dependent on the excess proton position, than the porelining residues in the top part of the channel. interestingly, the proton entry at cec z = − Å has a pronounced effect on the channel near trp , greater than that when the proton nears the center of the channel. ser , however, has overall the smallest average eccentricity and the lowest minimum value than the other pore-lining residues during pt in this portion of the channel. additionally, ser and val have smaller proton position dependent changes in eccentricity than the pore-lining residues in the bottom half of the channel. this result indicates that the ser region is the most symmetrical and stable in the channel. while analyzing the α-carbon distances and eccentricity, we also examined the correlation between these α-carbon distances on opposing helices, shown in figure , to gain further insight into protein motion and conformational fluctuations on the nanosecond time scale. these motions captured here are equilibrium fluctuations in the + , inward closed state, not necessarily motions driving the transition between inward open and inward closed . the calculated correlations indicate that the channel's equilibrium structural fluctuations are dominated by alternating inward−outward motions of opposing helices. at each pore-lining residue, the distances are negatively correlated: that is, when helices a and c move farther apart, helices b and d move closer together, and vice versa. gly is known to be the hinge point whose kinking controls the large structural change between inward-open and inward-closed conformations, which may falsely lead to the conclusion that the conformational fluctuations at equilibrium above and below gly are decorrelated, with a stable core centered at gly . interestingly, however, the motions at gly are strongly and similarly correlated with the motions at both ser and his . this correlation indicates that, in this fixed charge state, the gly kink is relatively rigid. instead, there is a noticeable lack of correlation between val and the other pore-lining residues, suggesting that there is a secondary, minor "hinge" between val and ser that decorrelates the inward−outward motions between the helices above and below this point. this natural hinge observed near val furthers our understanding of val acting as a secondary gate that opens to allow proton and water entry into the channel. , in our simulations, this valve can readily hydrate, particularly in the presence of a nearby excess proton. moreover, it is frequently closed, which may make passage of a hydrated sodium or chloride ion more difficult. this aspect of the val gate and its relevance for pt and proton selectivity is likely an important feature of the m channel and could be further explored in future work. given that the adamantane group of the drug is centered in the ser tetrad plane, we hypothesize that these facets of the channel's dynamics are critical for fully explaining the drugs' favorable binding. because of the more circular shape of the pore at the ser tetrad, the spherical adamantane group can fit snugly under the hydrophobic val cleft and block pt. additionally, the relative stability of the pore in the region of drug binding helps explain why drug binding is thermodynamically favorable. because the channel exhibits smaller structural fluctuations here than in other regions of the channel, the adamantane-based drugs are able to bind with minimal loss of entropy as the channel does not need to lose flexibility to create a stable drug-binding interface. this has important implications for designing drugs that use scaffolds different from the adamantane group , − or that interact with drugresistant mutants such as s n. while drug binding to more flexible regions of the channel is possible, only modest changes in potency are often observed despite large changes in the size of the drugs. this is likely because of the need to counter the greater loss of entropy resultant from structural changes and reduced fluctuations. one limitation of this study, however, is the homogeneous popc bilayer, which is commonly used in experiments and is standard in computational studies but does not capture the complexity of the viral membrane and may influence channel dynamics. thus, these features need to be examined in the more complex membrane to fully understand their physiological relevance. altogether, we have shown how the adamantyl amine inhibitors of m are suited to exploit various inherent features of the m channel that naturally facilitate proton transport, further supporting the claim that they function as mechanismbased inhibitors. the flexible hydrogen bond interactions, measured in both hydrogen bond occupancy and residence times, indicate how the channel is suited to stabilizing an excess charge near ammn z . thus, the ammonium group of these inhibitors can act as a hydronium mimic by binding in this region. we also analyzed the pore shape throughout the channel by calculating the eccentricity of the pore based on αcarbon distances. the results from these calculations indicate that the drug binding pocket is an especially stable and figure . pearson correlation coefficients of the distances between αcarbons on opposing helices, for all pore-lining residues, when excess proton cec z = − . Å. each row and column correspond to a specific residue and distance, as labeled, where "a−c" is the distance between the α-carbons of helices and and "b−d" is that of helices and . only those values with a p-value of < . are shown; any other values are set to . . journal of the american chemical society pubs.acs.org/jacs article symmetrical portion of the channel, conducive to binding a roughly spherical drug. finally, by examining the correlations between these distances, we found an additional minor hinge point toward the top of the channel which may be a relevant feature for future drug design efforts. understanding these features as they relate to drug binding gives further insight into the specific interactions that stabilize the adamantyl amine inhibitors in wild-type m , and these results suggests that this approach could be used to aid future drug-design efforts to methodically create new inhibitors for s n mutants. with the recent publication of high resolution influenza b m (bm ) structures, it is possible to conduct similar studies to elucidate the detailed pt mechanism in bm to guide drug design in this functionally similar protein. this work also shows how similar analyses to understand the details of explicit proton transport mechanisms (not those inferred by water structures alone) could be used in other systems and extended to ion transporters such as the sars-cov- viroporins, to help inform mechanism-based inhibitor design. elucidating the inherent features of drug-targetable proton transporters, such as flexible water and hydrogen bonding interactions, preferred proton positions, dynamic pore shapes, and structural fluctuations, can help guide the design of drug scaffolds and added substituents. the ms-rmd simulation methodology utilized in this work has made these studies possible both for m and for other important drug targets. the supporting information is available free of charge at https://pubs.acs.org/doi/ . /jacs. c . schematic of eccentricity calculations; average, maximum, minimum, and rmsd values for eccentricity (pdf) voltage-gated proton channels and other proton transfer pathways proton permeation of lipid bilayers a protonmotive force drives atp synthesis in bacteria coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism chance and design-proton transfer in water, channels and bioenergetic proteins importance of ph homeostasis in metabolic health and diseases: crucial role of membrane proton transport proton pump inhibitors and acid-related diseases lysosomal storage disease upon disruption of the neuronal chloride transport protein clc- the m proton channels of influenza a and b viruses influenza virus m protein has ion channel activity selective proton permeability and ph regulation of the influenza virus m channel expressed in mouse erythroleukaemia cells ion channel activity of influenza a virus m protein: characterization of the amantadine block the next wave of influenza drugs adamantane-resistant influenza a viruses in the world ( − ): frequency and distribution of m gene mutations discovery of m channel blockers targeting the drug-resistant double mutants m -s n/l i and m -s n/v a from the influenza a viruses prospects for specific influenza treatment viroporins: structure, function and potential as antiviral targets sars coronavirus e protein forms cation-selective ion channels severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release sars-cov- and orf a: nonsynonymous mutations, functional domains, and viral pathogenesis activation of the m ion channel of influenza virus: a role for the transmembrane domain histidine residue histidines, heart of the hydrogen ion channel from influenza a virus: toward an understanding of conductance and proton selectivity chemical rescue of histidine selectivity filter mutants of the m ion channel of influenza a virus the gate of the influenza virus m proton channel is formed by a single tryptophan residue solid-state nmr characterization of conformational plasticity within the transmembrane domain of the influenza a m proton channel structure and mechanism of the m proton channel of influenza a virus mechanisms of proton conduction and gating in influenza m proton channels from solid-state nmr conformational plasticity of the influenza a m transmembrane helix in lipid bilayers under varying ph, drug binding, and membrane thickness effect of cytosolic ph on inward currents reveals structural characteristics of the proton transport cycle in the influenza a protein m in cell-free membrane patches of xenopus oocytes multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza a m proton channel acid activation mechanism of the influenza a m proton channel put a cork in it: plugging the m viral ion channel to sink influenza in vitro pharmacokinetic optimizations of am -s n channel blockers led to the discovery of slow-binding inhibitors with potent antiviral activity against drug-resistant influenza a viruses slow but steady wins the race: dissimilarities among new dual inhibitors of the wild-type and the v a mutant m channels of influenza a virus divalent copper complexes as influenza a m inhibitors inhibitors of the m channel of influenza a virus exploring the requirements for the hydrophobic scaffold and polar amine in inhibitors of m from influenza a virus identification of hits as matrix- protein inhibitors through the focused screening of a small primary amine library unraveling the binding, proton blockage, and inhibition of influenza m wt and s n by rimantadine variants inhibitors of the m proton channel engage and disrupt transmembrane networks of hydrogen-bonded waters molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza a virus m investigation of the free energy profiles of amantadine and rimantadine in the am binding pocket mechanism of the pseudoirreversible binding of amantadine to the m proton channel interpreting thermodynamic profiles of aminoadamantane compounds inhibiting the m proton channel of influenza a by free energy calculations hydrogen-bonded water molecules in the m channel of the influenza a virus guide the binding preferences of ammonium-based inhibitors how do aminoadamantanes block the influenza m channel, and how does resistance develop? alchemical free energy calculations and isothermal titration calorimetry measurements of aminoadamantanes bound to the closed state of influenza a/ m tm the curious case of the hydrated proton sur la dećomposition de l'eau et des corps qu'elle tient en dissolution ál'aide de l'eĺectricitégalvanique the grotthuss mechanism computationally efficient multiconfigurational reactive molecular dynamics multiscale reactive molecular dynamics for absolute pka predictions and amino acid deprotonation computationally efficient multiscale reactive molecular dynamics to describe amino acid deprotonation in proteins multiscale simulations reveal key aspects of the proton transport mechanism in the clc-ec antiporter multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase the origin of coupled chloride and proton understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase proton movement and coupling in the pot family of peptide transporters modulating the chemical transport properties of a transmembrane antiporter via alternative anion flux kinetic modeling reveals an ensemble of cl(−)/h(+) exchange pathways in clc-ec antiporter proton-induced conformational and hydration dynamics in the influenza a m channel high-resolution structures of the m channel from influenza a virus reveal dynamic pathways for proton stabilization and transduction identification of the functional core of the influenza a virus a/m proton-selective ion channel role of presolvation and anharmonicity in aqueous phase hydrated proton solvation and transport a second generation multistate empirical valence bond model for proton transport in aqueous systems fast parallel algorithms for short-range molecular dynamics promoting transparency and reproducibility in enhanced molecular simulations plumed : new feathers for an old bird multidimensional replicaexchange method for free-energy calculations python reference manual guide to numpy data structures for statistical computing in python {vmd} -{v}isual {m}olecular {d}ynamics matplotlib: a d graphics environment the chemical and dynamical influence of the anti-viral drug amantadine on the m proton channel transmembrane domain structure of the amantadine binding site of influenza m proton channels in lipid bilayers computational study of drug binding to the membrane-bound tetrameric m peptide bundle from influenza a virus a secondary gate as a mechanism for inhibition of the m proton channel by amantadine viral m ion channel protein: a promising target for anti-influenza drug discovery exploring the size limit of templates for inhibitors of the m ion channel of influenza a virus identification of camphor derivatives as novel m ion channel inhibitors of influenza a virus design and synthesis of pinanamine derivatives as anti-influenza a m ion channel inhibitors undecane derivatives: from wild-type inhibitors of the m ion channel of influenza a virus to derivatives with potent activity against the v a mutant expeditious lead optimization of isoxazole-containing influenza a virus m -s n inhibitors using the suzuki-miyaura cross-coupling reaction the conformation of the pore region of the m proton channel depends on lipid bilayer environment atomic structures of closed and open influenza b m proton channel reveal the conduction mechanism key: cord- -hrdanbn authors: ghahremanpour, mohammad m.; tirado-rives, julian; deshmukh, maya; ippolito, joseph a.; zhang, chun-hui; de vaca, israel cabeza; liosi, maria-elena; anderson, karen s.; jorgensen, william l. title: identification of known drugs as inhibitors of the main protease of sars-cov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: hrdanbn a consensus virtual screening protocol has been applied to ca. approved drugs to seek inhibitors of the main protease (mpro) of sars-cov- , the virus responsible for covid- . drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with mpro, were chosen for evaluation in a kinetic assay for mpro inhibition. remarkably of the compounds at -μm concentration were found to reduce the enzymatic activity and provided ic values below μm: manidipine ( . μm), boceprevir ( . μm), lercanidipine ( . μm), bedaquiline ( . μm), and efonidipine ( . μm). structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the p , p ’, and p pockets of mpro. further study of the most active compounds in the context of covid- therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic. sars-cov- , the cause of the covid- pandemic, is a coronavirus (cov) from the coronaviridae family. its rna genome is ~ % identical to that of sars-cov, which was responsible for the severe acute respiratory syndrome (sars) pandemic in . sars-cov- encodes two cysteine proteases: the chymotrypsin-like cysteine or main protease, known as cl pro or m pro , and the papain-like cysteine protease, pl pro . they catalyze the proteolysis of polyproteins translated from the viral genome into non-structural proteins essential for packaging the nascent virion and viral repication. therefore, inhibiting the activity of these proteases would impede the replication of the virus. m pro processes the polyprotein ab at multiple cleavage sites. it hydrolyzes the gln-ser peptide bond in the leu-gln-ser-ala-gly recognition sequence. this cleavage site in the substrate is distinct from the peptide sequence recognized by other human cysteine proteases known to date. thus, m pro is viewed as a promising target for anti sars-cov- drug design; it has been the focus of several studies since the pandemic has emerged. , [ ] [ ] [ ] [ ] an x-ray crystal structure of m pro reveals that it forms a homodimer with a fold crystallographic symmetry axis. , each protomer, with a length of residues, is made of three domains (i-iii). domains ii and i fold into a six-stranded β-barrel that harbors the active site. , , domain iii forms a cluster of five antiparallel αhelices that regulates the dimerization of the protease. a flexible loop connects domain ii to domain iii. the m pro active site contains a cys-his catalytic dyad and canonical binding pockets that are denoted p , p , p , p , and p . the amino acid sequence of the active site is highly conserved among coronaviruses. the catalytic dyad residues are his and cys and the residues playing key roles in the binding (figure ). these residues have been found to interact with the ligands co-crystallized with m pro in different studies. , , crystallographic data also suggested that ser of one protomer interacts with phe and glu of the other as the result of dimerization. , these interactions stabilize the p binding pocket, thereby, dimerization of the main protease is likely for its catalytic activity. , drug repurposing is an important strategy for immediate response to the covid- pandemic. in this approach, the main goal of computational and experimental studies has been to find existing drugs that might be effective against sars-cov- . for instance, a molecular docking study suggested remdesivir as a potential therapeutic that could be used against sars-cov- , which was supported experimentally by an ec value of μm in an infected-cell assay. however, a clinical trial showed no statistically significant clinical benefits of remdesivir on adult patients hospitalized for severe covid- . nonetheless, patients who were administered remdesivir in the same trial showed a faster time to clinical improvement in comparison to the placebo-control group. in another clinical trial, only patients on mechanical ventilation benefitted from remdesivir. an ec value of μm was also reported for lopinavir , suggesting it may have beneficial activity against sars-cov- . however, neither lopinavir nor the lopinavir/ritonavir combination has thus far shown any significant benefits against covid- in clinical trials. chloroquine, hydroxychloroquine, and favipiravir have also been explored for repurposing against covid- ; however, clinical studies with them have been controversial. [ ] [ ] [ ] [ ] these studies reflect the urgent need for systematic drug discovery efforts for therapies effective against sars-cov- . thus, we decided to pursue discovery of small-molecule inhibitors of m pro . the aim of this initial work was two-fold: to identify known drugs that may show some activity, but also to identify structurally promising, synthetically-accessible substructures suitable for subsequent lead optimization. our expectation was that existing drugs may show activity but not at the low-nanomolar levels that are typical of effective therapies. this report provides results for the first goal. the work began by designing and executing a consensus molecular docking protocol to virtual screen were predicted using the propka and the h++ severs. , accordingly, lysines and arginines were positively charged, aspartic and glutamic acids were negatively charged, and all histidines were neutral. all histidines were built with the proton on nε except for his , which was protonated at nδ. the resulting m pro structure has a net charge of - e. extensive visual inspection was carried out using ucsf chimera. consensus molecular docking. most docking programs apply methods to generate an initial set of conformations, and tautomeric and protonation states for each ligand. this is followed by application of search algorithms and scoring functions to generate and score the poses of the ligand in the binding site of a protein. scoring functions have been trained to reproduce a finite set of experimental ligand-binding affinities that are generally a mix of activity data converted to a free-energy scale. therefore, the accuracy of the scores is dependent on multiple factors including the compounds that were part of the training set. to mitigate the biases, we performed four independent runs of protein-ligand docking with a library of ca. approved, oral drugs using glide, autodock vina, and two protocols with autodock . . the results were compiled and further consideration focused on those compounds that ranked among the top % percent in at least out of the runs. glide. schrödinger's protein prep wizard utility was used for preparing the protein. a -Å grid was then generated and centered on the co-crystallized ligand, which was subsequently removed. the drug library members were neutralized and/or ionized via schrödinger's ligprep. the epik program was used for estimating the pka values of each compound. plausible tautomers and stereoisomers within the ph range of ± were generated for each compound using the opls force field. these conditions resulted in a total of structures, which were then docked into m pro using schrödinger's standard-precision (sp) glide. , autodock. the autodocktools (adt) software was used for creating pdbqt files from sdf and pdb files of compounds and the protein, respectively. non-polar hydrogen atoms were removed and gasteiger-marsili charges were assigned for both the protein and the ligands using adt. the autogrid . simulations. the protonated m pro dimer, with a net charge of - e, was represented by the opls-aa/m force field. tip p water was used as the solvent. sodium counterions were added to neutralize the net charge of each system. the selected ligand candidates were represented by the opls/cm a force field, as assigned by the boss software (version . ) and the ligpargen python code. the parameters were converted to gromacs format using ligpargen. for neutral ligands, the cm a partial atomic charges were scaled by a factor of . . each m pro -ligand complex was put at the center of a triclinic simulation box with -Å padding. an energy minimization was then performed until the steepest descent algorithm converged to a maximum force smaller than . kcal mol - Å - . a cutoff radius of Å was used to explicitly calculate non-bonded interactions. long-range electrostatic interactions were treated using the particle mesh ewald (pme) algorithm. the pme was used with an interpolation order of , a fourier spacing of . Å, and a relative tolerance of - . the van der waals forces were smoothly switched to zero between and Å. analytical corrections to the long-range effect of dispersion interactions were applied to both energy and pressure. all covalent bonds to hydrogen atoms were constrained at their equilibrium lengths using the lincs algorithm with the order of in the expansion of the constraint-coupling matrix. each system was subsequently simulated for ns in the canonical ensemble (nvt) in order for the solvent to relax and the temperature of the system to equilibrate. initial velocities were sampled from a maxwell-boltzmann distribution at k. the v-rescale thermostat with a stochastic term was used for keeping the temperature at k. the stochastic term ensured that the sampled ensemble was canonical. the coupling constant of the thermostat was set to . ps. table , and the structures of some of the ones that turned out to be most interesting are shown in figure . the primary indications include bacterial and viral infections, hypertension, psychosis, inflammation, and cancer. their mechanisms of action are also broad ranging from kinase and protease inhibitors to dopamine receptors agonists/antagonists, and calcium channel blockers. it is not surprising that peptidic protease inhibitors are well-represented in view of the peptide substrate and prior discovery of peptidic inhibitors for m pro and its sars-cov relative. , , in almost all cases the predicted poses for the compounds from the different docking programs agreed well. the poses from glide were then subjected to extensive visual scrutiny to check for unsatisfied hydrogen-bonding sites, electrostatic mismatches, and unlikely conformation of the ligand. about half of the compounds were ruled out for further study due to the occurrence of such liabilities and the presence of multiple ester groups (e.g., methoserpidine and nicomol) or overall size and complexity (e.g., bromocriptine and benzquercin). a repeated motif was apparent with high-scoring ligands having a cloverleaf pattern with occupancy of the p , p ', and p pockets, as illustrated in figure for the complex of azelastine. other common elements are an edge-to-face aryl-aryl interaction with his and placement of a positively-charged group in the p pocket in proximity to glu , e.g., the methylazepanium group of azelastine, the protonated trialkylamino group of bedaquiline, and protonated piperazine of periciazine. however, glu forms a saltbridge with the terminal ammonium group ser b (figure ). the electrostatic balance seems unclear in this region, so our final selections included a mix of neutral and positively-charged groups for the p site. the analysis of the high-scoring compounds also considered structural variety and potential synthesis of analogs. in the end, we settled on compounds, which are highlighted in table , for purchase and assaying. sixteen were commercially available, mostly from sigma-aldrich. the seventeenth, cinnoxicam, was not available, but it was readily prepared in a one-step synthesis from the commercially-available ester components. it may be noted that three calcium channel blockers, efonidipine, lercanidipine, and manidipine were purchased ( figure ). this was not done owing to the characteristic dihydropyridine substructure, since this end of the molecule protrudes out of the p ' site in the docked poses. it was for the variety in the left-sides of the molecules in figure , which form the cloverleaf that binds in the p , p ', and p pockets, as illustrated in figure for manidipine. the steric fit in this region appears good, though the only potential hydrogen bond is between the nitro group and the catalytic cys . remarkably, fourteen of the drugs at μm decreased m pro activity ( nm), as shown in figure and table . five drugs decreased m pro activity to below %. the top five hits from the kinetic assay were manidipine, boceprevir, efonidipine, lercanidipine, and bedaquiline. dose-response curves were obtained to determine ic values, when possible, as shown in figure for the five most potent inhibitors, with the raw data as a function of time and concentration given in figure s . the computed structures for the complexes of boceprevir and bedaquiline are illustrated in figure . for boceprevir, the dimethylcyclopropyl subunit is predicted to μm. further study of these compounds in the context of covid- therapy is warranted, while all of the active compounds reported here may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic. the supporting information is available free of charge on the acs publications website. an excel file with the names and docking scores for the full drug library, a the authors have no competing interests. gratitude is expressed for support to the u. s. national institutes of health (gm ) and to the yale university school of medicine for a corect pilot grant. the m pro plasmid was kindly provided by the hilgenfeld lab. a new coronavirus associated with human respiratory disease in china structure of mpro from sars-cov- and discovery of its inhibitors identification of a novel coronavirus in patients with severe acute respiratory syndrome structural basis for the inhibition of sars-cov- main protease by antineoplastic drug carmofur crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors prediction of novel inhibitors of the main protease (m-pro) of sars-cov- through consensus docking and drug reposition gc- , and calpain inhibitors ii, xii inhibit sars-cov- viral replication by targeting the viral main protease learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov drug repurposing: progress, challenges and recommendations repurposing against covid- lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro favipiravir and other drugs for the treatment of the new coronavirus chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) observational study of hydroxychloroquine in hospitalized patients with covid- pandda analysis of covid- main protease against the dsi-poised fragment library asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation propka : consistent treatment of internal and surface residues in empirical p k a predictions h++: a server for estimating pkas and adding missing hydrogens to macromolecules h++ . : automating pk prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations a visualization system for exploratory research and analysis towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution epik: a software program for pka prediction and protonation state generation for drug-like molecules opls : a force field providing broad coverage of drug-like small molecules and proteins a new approach for rapid, accurate docking and scoring. . method and assessment of docking accuracy glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening autodock and autodocktools : automated docking with selective receptor flexibility autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading : a high-throughput and highly parallel open source molecular simulation toolkit improved peptide and protein torsional energetics with the opls-aa force field comparison of simple potential functions for simulating liquid water potential energy functions for atomic-level simulations of water and organic and biomolecular systems molecular modeling of organic and biomolecular systems using boss and mcpro ligpargen web server: an automatic opls-aa parameter generator for organic ligands a smooth particle mesh ewald method lincs: a linear constraint solver for molecular simulations canonical sampling through velocity rescaling molecular dynamics with coupling to an external bath polymorphic transitions in single crystals: a new molecular dynamics method structure-based design of antiviral drug candidates targeting the sars-cov- main protease an overview of severe acute respiratory syndrome -coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy calcium channel blockers: a possible potential therapeutic strategy for the treatment of alzheimer's dementia patients with sars-cov- infection targeting the sars-cov- main protease to repurpose drugs for single-molecule pulling simulations can discern active from inactive inhibitors investigating drug-target association and dissociation mechanisms using metadynamics-based algorithms estimation of drug-target residence times by τ-random accelerated molecular dynamics simulations dynamic undocking and the quasi-bound state as tools for drug discovery key: cord- - qvxw authors: lesaint, kathy t.; snyder, hannah r. title: impact of social distancing on individuals who use drugs: considerations for emergency department providers date: - - journal: west j emerg med doi: . /westjem. . . sha: doc_id: cord_uid: qvxw the isolation that comes from social distancing during the covid- pandemic can be particularly detrimental to the united states’ population of people who use drugs. people with substance use disorders may be at risk for return to use, exacerbation of existing mental health disorders, and risky drug practices. in this commentary, we review the risk to people who use drugs and how emergency department providers can best support these individuals during the unprecedented time of social distancing. the isolation that comes from social distancing during the covid- pandemic can be particularly detrimental to the united states' population of people who use drugs. people with substance use disorders may be at risk for return to use, exacerbation of existing mental health disorders, and risky drug practices. in this commentary, we review the risk to people who use drugs and how emergency department providers can best support these individuals during the unprecedented time of social distancing. [west j emerg med. ; ( ) - .] approximately one third of pwud seeking treatment for substance use disorders are unhoused or live in congregate settings including residential treatment facilities, shelters, and single-room occupancy hotels. in these settings, following the cdc guidance on social distancing may be difficult or impossible. this issue has led to covid- clusters in some homeless shelters and has led several communities to seek alternative housing in hotels for people experiencing homelessness. , those pwud who have the ability to practice social distancing may face an increased risk of drug-related harms. coping with isolation and health threats may lead to increased stress and anxiety in a vulnerable population already stricken with trauma and mental health issues. social isolation can act as a trigger and is strongly correlative with mood and substance use disorders. these factors can exacerbate existing substance use as patients self-treat psychiatric symptoms or lead people with a history of substance use disorder to return to use. for those who are actively using drugs, practicing harm reduction can be difficult in the setting of covid- . traditional guidance is for pwud to use with another person so that if they overdose, can be called and naloxone can be used, but by following social distancing pwud are unable to do this. as borders close and supply chains are disrupted, impact of social distancing on individuals who use drugs pwud may seek out drugs from places other than their usual, trusted sources and thereby be at greater risk of exposure to an adulterated or contaminated supply. in addition, many needle and syringe exchange programs changed their models in response to the covid- pandemic, either closing down completely or being unable to provide their typical services and programs (eg, referral to treatment programs, harm reduction education, naloxone distribution and education, etc). such changes place pwud at greater risk for unsafe practices and increased risk of communicable infectious diseases, skin and soft tissue infections, and drug overdose and death. furthermore, maintaining access to treatment and recovery services during a time of social distancing mandates is difficult. throughout the country, support groups have been cancelled, treatment programs are limiting new patients, and inpatient treatment centers have limited visits. [ ] [ ] [ ] opioid treatment programs, in which most patients rely on daily dispensing of medications to treat opioid use disorder, may have reduced access as well. such changes make it harder for patients to newly access treatment and present challenges for those who are already in treatment. without ease of access to places of recovery and medication-assisted treatment, patients are at risk of serious medical and psychological complications. fortunately, the federal government has recently made several changes to increase access to life-saving addiction treatment. the centers of medicare & medicaid services has loosened regulations and is compensating for telemedicine services, the drug enforcement agency now supports telephone and audiovisual buprenorphine prescribing, and the office of civil rights at the department of health and human services approved usage of popular apps to provide telehealth without risk of penalties for noncompliance with hipaa. [ ] [ ] [ ] in addition, opioid treatment programs are providing longer durations of take-home doses of medications for treatment of opioid use disorder. it is likely that emergency departments (ed) across the us will see an increase in the number of pwud experiencing withdrawal, experiencing overdose, or seeking treatment for their substance use disorder. preliminary data from the ed at san francisco's only public hospital revealed a near twofold increase in the number of patients presenting with the chief complaint of "drug overdose" in march ( patients/month; . % of all ed encounters) when compared to averaged data from the prior six months ( patients/month; . % of all ed encounters). in addition, more than states have reported increases in cases of opioid-related overdose and mortality. therefore, in this unprecedented time of social distancing, emergency providers are placed in an additional frontline role of delivering patient-centered care for a highly at-risk population of pwud. emergency clinicians should provide compassionate, evidence-based care to pwud. establishing rapport and motivational interviewing can be difficult in a time of enhanced precautions and extra personal protective equipment. however, continuing to take the time to speak in a non-stigmatizing way is vital in the therapeutic process and is the start to effective treatment for pwud. in recent years, the practice of ed initiation of buprenorphine has rapidly become the standard of care. we encourage emergency clinicians to offer buprenorphine to any patients presenting with opioid use disorder. data waivers are not required to administer buprenorphine in the ed. first doses of buprenorphine can be rapidly administered in the ed, and patients should be linked to ongoing treatment. in addition, while it is not the usual practice of the emergency clinician to provide long-term medication prescriptions, in this unique time we encourage providers with data waivers to offer longer durations of buprenorphine prescriptions (up to days) to appropriate patients. during the covid- pandemic, it is more essential than ever that emergency clinicians provide this service and while doing so, receive institutional support that is much needed to overcome barriers to buprenorphine administration. individual institutions and departmental leadership can best support their clinicians by providing adequate training and resources regarding buprenorphine use, as well as assisting providers in coordination of outpatient linkage to care. on march , , the substance abuse and mental health services administration provided additional guidance for managing the treatment of alcohol or benzodiazepine withdrawal in acute settings. providing buprenorphine to treat patients with opioid use disorder and medication treatment for alcohol withdrawal is particularly essential for those patients who are diagnosed with covid and entering quarantine. adequately treated withdrawal and compassionate care will support them in staying for the duration of their quarantine period. as much as possible, emergency care providers must continue to offer harm reduction strategies to pwud. strategies of harm reduction include supporting drug use hygiene (eg, giving education on safe consumption, distributing pipes or syringes), providing overdose prevention supplies (eg, take-home or prescriptions of naloxone, fentanyl test strips), and encouraging patients to not use drugs in isolation (eg, video-chatting with a buddy, contacting support at www.neverusealone.com). involving an ed social worker or substance use navigator who is familiar with local outpatient resources and/or changes to the outpatient landscape during this time can help facilitate linkage to care. finally, continuing to address other social determinants of health (eg, housing insecurity, psychiatric illnesses) is paramount to providing safe discharge to the community. the isolation that comes from social distancing during the covid- pandemic can be particularly detrimental to the volume , no. : september impact of social distancing on individuals who use drugs lesaint et al. population of people who use drugs. people with substance use disorders may be at risk for return to use, exacerbation of existing mental health disorders, and risky drug practices. in this time, emergency care providers have a vital role in supporting this vulnerable population of people who use drugs by establishing rapport, encouraging best practices in harm reduction, providing medication treatment, and connecting patients to outpatient resources. individual characteristics of the literally homeless, marginally housed, and impoverished in a us substance abuse treatment-seeking sample assessment of sars-cov- infection prevalence in homeless shelters -four us cities coronavirus forces san francisco to put homeless into hotels the association between social isolation and dsm-iv mood, anxiety, and substance use disorders: wave of the national epidemiologic survey on alcohol and related conditions people in addiction treatment are losing crucial support during coronavirus pandemic opioid addiction is "a disease of isolation medicare telemedicine health care provider fact sheet dea- letter notification of enforcement discretion for telehealth remote communications during the covid- nationwide public health emergency issue brief: reports of increases in opioid-related overdose and other concerns during covid pandemic what is the impact of mental health-related stigma on help-seeking? a systematic review of quantitative and qualitative studies management of opioid use disorder in the emergency department: a white paper prepared for the american academy of emergency medicine initiating buprenorphine treatment in the emergency department emergency department clinicians' attitudes toward opioid use disorder and emergency department-initiated buprenorphine treatment: a mixed-methods study considerations for crisis centers and clinicians in managing the treatment of alcohol or benzodiazepine withdrawal during the covid- epidemic key: cord- - i balq authors: darvishi, behrad; manoochehri, saeed; kamalinia, golnaz; samadi, nasrin; amini, mohsen; mostafavi, seyyed hossein; maghazei, shahab; atyabi, fatemeh; dinarvand, rassoul title: preparation and antibacterial activity evaluation of -β-glycyrrhetinic acid loaded plga nanoparticles date: journal: iran j pharm res doi: nan sha: doc_id: cord_uid: i balq the aim of the present study was to formulate poly (lactide-co-glycolide) (plga) nanoparticles loaded with -β-glycyrrhetinic acid (gla) with appropriate physicochemical properties and antimicrobial activity. gla loaded plga nanoparticles were prepared with different drug to polymer ratios, acetone contents and sonication times and the antibacterial activity of the developed nanoparticles was examined against different gram-negative and gram-positive bacteria. the antibacterial effect was studied using serial dilution technique to determine the minimum inhibitory concentration of nanoparticles. results demonstrated that physicochemical properties of nanoparticles were affected by the above mentioned parameters where nanoscale size particles ranging from to nm were achieved. the highest encapsulation efficiency ( . ± . %) was obtained when the ratio of drug to polymer was : . zeta potential of the developed nanoparticles was fairly negative (- ± . ). in-vitro release profile of nanoparticles showed two phases: an initial phase of burst release for h followed by a slow release pattern up to the end. the antimicrobial results revealed that the nanoparticles were more effective than pure gla against p. aeuroginosa, s. aureus and s. epidermidis. this improvement in antibacterial activity of gla loaded nanoparticles when compared to pure gla may be related to higher nanoparticles penetration into infected cells and a higher amount of gla delivery in its site of action. herein, it was shown that gla loaded plga nanoparticles displayed appropriate physicochemical properties as well as an improved antimicrobial effect. discovery and development of methods for targeted delivery of antibiotics to infected cells and the foci of bacterial infections has received a great deal of attention during the recent years. this is especially important regarding the fact that intracellular infections are difficult to eradicate ( ) and infected cells and tissues are considered as microorganisms reservoirs, which may release them from time to time resulting in the recurrence of various systemic infections ( ). various colloidal systems such as niosomes, ethambutol encapsulated plga nps where used orally against mycobacterium tuberculosis and results showed enhanced bioavailability and improved pharmacodynamics when compared to the related free drug counterparts ( ) . parenteral administration of plga nps loaded phosphorothioate antisense oligonucleotide nanoparticles for hiv treatment was also found to be successful and protected oligonucleotides from degradation ( ). antimicrobial agents delivery to bacteria by nps may be based on two different approaches. in the first approach, nanoparticles are fused with the bacterial cell wall or membrane and the carried antimicrobial agent is subsequently released across the cell wall or cell membrane. in the second approach, nanoparticles are bound to cell wall and serve as a drug depot which continuously releases the anti-microbial drug molecules. the released anti-microbial agent will diffuse into the interior compartments of the bacteria ( ). -β-glycyrrhetinic acid (gla) is a pentacyclictriterpenoid derivative of betaamyrin which is present in highly pure extracts of rhizomes of glycyrrhiza glabra. glycyrrhetinic acid has a range of pharmacological properties including calmative, anti-inflammatory and antiallergic effects as well as antitumor properties. moreover antibacterial and antifungal activities have been reported for this naturally occurring substance. it is shown that gla can hinder dna replication and may result in an inhibition in microbial toxins and enzymes production ( ) . it has been demonstrated that high acute exposure to gla would cause brief systemic changes, including an increased level of potassium excretion along with an enhanced sodium and water retention. furthermore, following the administration of gla, various side effects such as weight gain, alkalosis, renin-angiotensinaldosterone system suppression, hypertension and muscular paralysis may occur. by physical encapsulation of the drug molecules inside the nanoparticulate systems, pharmacokinetics and therapeutic index of drugs significantly improve. furthermore, a lower in-vivo toxicity is achieved due to reduced accumulation of gla in kidney and liver ( ). to our best knowledge, there are currently liposomes, microemulsions and micro and nanoparticles are amongst the most important drug delivery systems which are utilized for the preparation of such targeted carriers ( , ). for instance lutwyche et al. achieved a higher intracellular antibiotic delivery by gentamicin encapsulation in a liposomal drug delivery system and an increased therapeutic activity against intracellular pathogens was achieved ( ). to efficiently eradicate pathogens, an antibiotic should be able to be systemically transported in a form that can be endocytosed by phagocytes and the antimicrobial agent should be released inside the cells ( ). in this respect, nanoparticles have a great value as they are rapidly picked up by mononuclear phagocytic cells when administered intravenously. mononuclear phagocytic cells are kept responsible for providing a sanctuary and shelter for the intracellular bacteria ( ). therefore antibiotic encapsulation inside the nanoparticles has been suggested as a potential strategy for the management of various intracellular infections ( ). polymeric nanoparticles (nps) have several advantages which make them distinctive among various nanoparticulate systems. these advantages include their structural stability, narrow size distribution and the possibility of their functionalization for targeted drug delivery ( ). various polymers may be utilized for polymeric nps preparation, among which plga or poly (lactic-co-glycolic acid) has a special place. plga has received fda approval as a biocompatible and also biodegradable polymer and is commonly used in pharmaceutical industry and research as a desirable drug carrier ( , ). an advantage of plga copolymers is that their degradation rate varies from months to years. there have been several reports of antimicrobial agents loaded plga nps in the literature indicating their improved pharmacological and pharmacokinetic behavior when compared to their untreated counterparts ( ). plga nps may also have the potential to be utilized for oral and parenteral administrations. for instance oral administration of plga loaded curcumin nps increased drug bioavailability at different levels ( ). in another study, rifampicin, isoniazid, pyrazinamide, and no studies available on the formulation of gla containing polymeric nanoparticles with the intention of enhancing gla antibacterial activity. the aim of the present study was to develop a plga based nanocarrier system for gla to enhance the antibacterial effects of gla and to minimize its side effects. the developed nanoparticles have the potential to target infected tissues and phagocytic cells and exert their antibacterial effect on various microorganisms. staphylococcus aureus (s. aureus), staphylococcus epidermidis (s. epidermidis) and pseudomonas aeruginosa (p. aeruginosa) were chosen as model bacteria for evaluating the effect of the developed nanoparticles on microbial organisms as they are considered as the most significant bacteria involved in various infectious diseases in human being and are internalized in phagocytic cells ( ) ( ) ( ) ( ) ( ) . s. aureus is a widespread pathogen which is considered to be responsible for food poisoning and nosocomial infections ( ). s. epidermidis is another pathogen which is involved in extraneous devices and implants related infections due to its particular capability of growing biofilms on polymer surfaces ( ). p. aeruginosa is a gram negative bacterium which is another common cause of nosocomial infections, where only a limited number of antibacterial agents show an acceptable defense against this organism ( ). the antimicrobial activity of the nanoparticles was further compared with pure gla against different gram-negative and grampositive bacteria. preparation of gla loaded plga nanoparticles plga nps were fabricated using a sonication/ solvent evaporation method ( ). in the first step, pva ( . % w/w) was dissolved in deionized water forming the aqueous phase and different ratios of gla to plga were dissolved in acetone at room temperature ( °c) to prepare organic phase (oil phase). the prepared oil phase was added drop wise to the aqueous phase while sonicating using an ultrasonic probe (misonix, farmingdale, ny, usa) equipped with a microtip (power: watt). the entire system was located in an ice bath to avoid any loss of oil phase as a result of organic solvent evaporation during sonication. the above mixture was subsequently magnetically stirred overnight at room temperature where acetone was removed under constant stirring condition. finally the solution was centrifuged three times (sigma k , osterode, germany) at rpm for min in order to remove any extra pva and was then lyophilized (christ alpha - , osterode, germany). the final dry powder was used for physicochemical and antibacterial investigations. for measuring the size, polydispersity index and zeta potential of nanoparticles, lyophilized nanoparticles were dissolved in deionized water and were assessed by photon correlation spectroscopy (pcs) using a zeta sizer nano zs (malvern instruments, worcestershire, uk) at °c. all the experiments were accomplished in triplicate and mean±sd was reported. surface morphology and shape of nanoparticles were investigated by scanning electron microscopy (sem, philips xl , philips, the netherlands). a small amount of freeze dried powder of nps was fixed on a double-sided tape attached to a metallic sample stand, and was gold coated before sem ( ). encapsulation efficiency mg lyophilized nps were dispersed in ml acetonitrile and were shaken for seconds and were further sonicated for approximately min. ml methanol was then added to the mixture in order to precipitate the polymer. the sample was then centrifuged at rpm for min and the free gla containing supernatant was separated for further analysis. drug quantity in the supernatant was determined by a uv spectrophotometry method. drug loading was calculated as the gla content in nps divided by the total weight of nps. the encapsulation efficiency was obtained by calculating the ratio of gla amount incorporated inside the nps to the gla amount which was used for nps preparation ( ). each experiment was repeated three times. differential scanning calorimetry (dsc) was performed for plga, gla, their physical mixture and gla loaded plga nanoparticles with a mettler dsc unit (mettler toledo, greifensee, switzerland) and a julabo thermocryostate model ft y (julabo labor technik, seelbach, germany) with indium used for instrument calibration. mg of samples were placed into dsc aluminum pans and sealed. pure water with the same amount was used as the reference. sample scanning was performed with a temperature range of to °c and with a rate of °c min - under a kpa nitrogen atmosphere (n= ) ( ). drug release evaluation was performed according to a previously published method for piroxicam nanoparticles ( ) with some modifications. mg of lyophilized gla loaded plga nps was dispersed in ml phosphate buffer saline (pbs, ph= . ) and placed in a dialysis tube (cut-off: kda), and was floated in a flask containing ml phosphate buffered saline (pbs) medium. the entire system was located at °c in a beaker ( rpm). after a predetermined period, the old medium was fully removed and fresh medium was replaced each time. ml of the old medium was analyzed by uv spectrophotometry for its gla content. the release profile of free gla was also evaluated by a dialysis bag under the same conditions. minimum inhibitory concentration (mic) or the lowest concentration of gla which is able to inhibit microorganisms outgrowth was determined for gla loaded nps using broth micro dilution method. gla nps suspensions with equivalent concentration of gla based on their encapsulation efficiency were used against s. aureus, s. epidermidis and p. aeroginosa. furthermore drug free nps were utilized to see if the carrier or other formulation components exert any antibacterial activity which may interfere with the results. it is noteworthy that positive and negative control groups were also considered in this mic determination study, where positive control groups did not contain any anti-microbial agent and negative control groups were not inoculated with any bacteria. briefly, the lyophilized bacteria were first suspended in sterile water for injection and were then cultured in luria bertuni agar mediums (scharlau, spain) and were incubated for h at °c. a single colony from the plates was then transferred into ml fluid of luria bertuni agar medium and was incubated at °c and rpm in a shaking incubator for an overnight period. in the next step centrifugation at rpm for min at °c was used for cellular harvesting. the supernatant was finally removed and the cellular sediment was washed two times with ringer solution and was dispersed in the same medium in a concentration range of about to cfu/ml to be exploited for broth dilution method ( ). for each sample and each bacterial strain, eight tubes were employed and autoclaved at °c for min. the samples were then serially diluted in the tubes and ml of inoculums of tested bacterium (final concentration of - cfu/ml) was introduced into the tubes. after h of incubation at °c, the tubes were evaluated according to their bacterial growth related turbidity and mic was determined as the minimum gla concentration without any visible bacterial growth ( ). preparation and characterization of gla loaded nanoparticles gla loaded nps were prepared by sonication/ solvent evaporation method. encapsulation efficiency, drug loading, size, polydispersity index and zeta potential of gla loaded nps are reported in table . according to table , nps size was in the range of to nm and the prepared nanoparticles were highly uniform and monodispersed (figures a and b) . by increasing the drug to polymer ratio from : to : , size was increased from to nm. this result may be related to an increase in viscous forces of droplets which resists against droplets break down by sonication especially in higher polymer concentrations. these resisting forces stand against the shear forces in the oil phase and determine the final size and particle size distribution in nanoparticles ( ). furthermore, table shows that by increasing the amount of polymer, higher encapsulation efficiencies were achieved. maximum encapsulation efficiency was attained when the amount of drug to polymer ratio was : . as it is observed higher polymer concentration is related to a higher encapsulation efficiency which may be explained in two different ways. first, it can be proposed that when the polymer concentration is high, the polymer molecules may precipitate on the surface of the dispersed phase droplets. as a result drug molecules diffusion through the two phases boundary is restricted and a higher encapsulation efficiency is achieved ( ). alternatively, it may be proposed that a higher polymer concentration can increase the viscosity of the system and make a barrier toward the free diffusion of drug molecules through the boundary phase of polymeric droplets ( ). table also shows the influence of the acetone content on the size of nps. the ratio between continuous and dispersed phases may have an effect on the stability and size of nanoparticles. as shown in table by increasing the ratio of the oil phase, the size of nanoparticles is slightly decreased and their encapsulation efficiency and drug loading is slightly increased. it has been suggested that by increasing the ratio of aqueous phase, the external surface energy of oil droplets, will be dispersed in a higher volume and results in a lower droplet breakage and subsequently the particle size will be increased. it should be mentioned that gla is poorly soluble in water and has a higher affinity for the oil phase in an oil in water emulsion which will lead to a higher entrapment efficiency and drug loading with increasing the oil phase ratio ( ). another factor which was evaluated for its effects on the size of nanoparticles was sonication time which was between and min. the results shown in table suggest that an increase in the sonication time would result in a reduction in the size of nanoparticles. when a higher sonication time ( min) is utilized, the high energy results in a rapid dispersion of oil droplets with a smaller size and a monomodal profile of distribution. the emulsification step is one of the most vital steps in the nanoparticles formation process. inappropriate phase dispersion will result in the formation of larger particles with a wider particle size distribution. the nps size is dependent on the size of droplets which are formed during the emulsification process. as a result by reducing the size of emulsion droplets, smaller nanoparticles can be achieved. nanoparticles stabilization phase is a very significant step in the process of emulsification. protection of nanodroplets is usually achieved by surfactant molecules utilization. surfactant molecules prevent the occurrence of coalescence in nanodroplets and produce nanoparticles with a smaller particle size. herein, a . % concentration of pva was used to stabilize the formed nanoparticles and develop some sort of coverage between the phases interface in oil in water emulsion. other than size, zeta potential is another major characteristic of nanoparticles which may have impacts on both nanoparticles stability and cellular adhesion. zeta potential was also measured in plga nps and as shown in table , the values of negative zeta potential on the nanoparticles were reduced as the polymer concentration decreased from : to : drug to polymer ratio. this reduced negative charge may be attributed to the higher amount of unentrapped drug molecules with lower polymer concentrations and their shielding effect on the carboxylic moieties of plga molecules on the particle surface ( ). it has been shown that the extent of phagocytosis is increased with an increase in nps zeta potential where the minimum phagocytosis is related to the instance in which zeta potential is approximately zero ( ). on the other hand, due to the negative surface charge of endothelial cells and their repulsive forces toward the negatively charged nps, the half life of these nps may increase in blood systemic circulation keeping the nps more available to the phagocytic cells ( ). scanning electron microscopy sem micrograph of gla loaded nps showed that nps are roughly spherical and have smooth surfaces (figure ). all formulations appeared to be monodispersed and homogenous irrespective of their compositions. the nps size obtained by photon correlation spectroscopy were larger than those observed by sem which may be related to hydrodynamic diameter of swollen and inflated polymeric nps in water ( ). differential scanning calorimetry dsc was used to investigate the thermal behavior of formulations. figure shows that pure plga exhibits an endothermic peak at °c which can be related to its relaxation peak following the glass transition phase. due to the amorphous nature of plga, no melting point was observed in plga thermograms. the pure gla demonstrated a sharp peak at °c which can be related to its melting point. the dsc thermograms of the physical mixture of gla and plga showed peaks which were the result of plain superposition of each of the two substances dsc thermograms. the enthalpy reduction for the physical mixture of drug and polymer can be explained by the presence of smaller amount of drug molecules in the mixture in comparison with pure drug. the dsc curve of gla containing nps did not show the endothermic peak of gla which suggests that the drug is incorporated into the nanoparticles in a disordered and amorphous shape. any severe alteration in the thermal behavior of either the polymer or the drug may be related to drug polymer interaction ( ). in the current study no formation of any new peak and no shifting of any peak in the dsc thermogram was observed. the release profiles of pure gla and gla loaded nps with different drug to polymer ratios are shown in figure . t % is the time needed for dissolving % of drug and is inversely related to the dissolution rate. t % was h and h for nps with : and : drug to polymer ratios, respectively. figure also demonstrates that gla release from nps was slower and more sustained than that of pure gla. drug release rate from various drug delivery systems is usually controlled by dissolution and/or diffusion ( ). in this study the presence of water insoluble polymer in nps matrix composition may interfere with the drug release from nps where it can reduce the amount of water penetration in nps and subsequently affect drug dissolution and diffusion. plga microparticles drug release is usually composed of two phases of initial burst release followed by a slower release phase. this burst release is of special importance as it affects microparticles toxicity and their therapeutic efficacy. the burst release is generally defined as the total amount of released drug from the particulate system through the first h. this total amount of drug release is normally about to % of the total drug loading in such particles ( , ) . in the present study, the release profiles of gla from np formulations followed a two phase pattern: in the first phase, an initial burst release occurs which takes about h and is followed by the second slow release phase. the release curves demonstrated that the initial burst release level is dependent on the amount of plga polymer in nps samples. the results showed that during the first h, an initial burst release led to an early release of %, % and % of drug from pure gla, gla loaded nps with : drug to polymer ratio and gla loaded nps with : drug to polymer ratio, respectively. the burst release of gla may be related to the drug molecules that are poorly entrapped in the polymer matrix. it is noteworthy that similar observations were reported by other researchers working on paclitaxel and azithromycin plga nps ( ). the mic of gla loaded plga nps as well as free gla was reported for s. aureus, s. epidermidis and p. aeruginosa ( figure ). drug free plga nanoparticles displayed no antibacterial effect which suggests that nps components and ingredients did not have any considerable antibacterial activity. the mic of gla loaded nps was approximately times lower for s. aureus, times lower for s. epidermidis, and times lower for p. aeroginosa compared to free gla solution regardless of drug to polymer ratio. this indicates that the effective dose of this antibiotic can be reduced through the administration of gla loaded plga nps against the above mentioned bacteria which will subsequently result in lower side effects. consequently, in-vitro antimicrobial activity of gla loaded plga nps was better than the free drug. it is noteworthy that an enhanced antimicrobial activity with antibiotics loaded plga nps has been previously reported ( ). this better antibacterial activity may be attributed to the improved penetration of nps from biological membranes and its better access to the bacteria internalized within the phagocytes. it has been revealed that phagocytic cells can endocytose nps and can release the drug inside these cells ( ). the gla loaded nps could be useful in drug targeting to phagocytic cells and can improve the treatment and management of intracellular infections compared to free antibiotics. other kinds of nps have been successfully evaluated for carious other routes of administration including ocular ( ) and oral ( ), and the formulated gla loaded plga nps may have the potential to be used for these routes of administration as well. gla loaded plga nanoparticles were successfully prepared with an appropriate size and zeta potential. the antimicrobial activity evaluation showed that nps were more effective than pure gla against s. aureus, s. epidermidis and p. aeroginosa. it can be proposed that the clinical activity of gla can be enhanced if a gla nanoparticle formulation is used and this allows for a more efficient therapy compared to the conventional formulation of free antibiotics present in the market. inhibitory concentration (mic) of free -β-glycyrrhetinic acid (gla) and gla loaded poly (lactide-co-glycolide) (plga) nanoparticles with different drug to polymer ratios in three different bacterial strain. encapsulated in ph-sensitive liposomes development of azithromycin-plga nanoparticles: physicochemical characterization and antibacterial effect against salmonella typhi treatment of experimental salmonellosis in mice with ampicillin-bound nanoparticles plga-based nanoparticles: an overview of biomedical applications surface modification of plga nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel plga-and pla-based polymeric nanoparticles for antimicrobial drug delivery anticancer activity of nanoparticles based on plga and its copolymer: in-vitro evaluation formulation, characterization and evaluation of curcumin-loaded plga nanospheres for cancer therapy development of nanoparticles for antimicrobial drug delivery antibacterial effect of glycyrrhetinic acid on hospital strains of staphylococcus aureus and actinobacillus actinomycetemcomitans glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus response to staphylococcus aureus requires cd -mediated phagocytosis triggered by the coohterminal cytoplasmic domain phagocytosis and intracellular killing of staphylococcus aureus by normal mouse peritoneal macrophages tnf-α reduces the level of staphylococcus epidermidis internalization by bovine endothelial cells nonopsonic phagocytosis of pseudomonas aeruginosa by macrophages and polymorphonuclear leukocytes requires the presence of the bacterial flagellum antibacterial activities of essential oils from eight greek aromatic plants against clinical isolates of staphylococcus aureus upregulation of icaa, atle and aap genes by linezolid but not vancomycin in staphylococcus epidermidis rp a biofilms emergence of antibiotic-resistant pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents inhibition of endotoxin-induced uveitis by methylprednisolone acetate nanosuspension in rabbits improved drug loading and antibacterial activity of minocycline-loaded plga nanoparticles prepared by solid/oil/water ion pairing method protein-loaded poly (dl-lactide-co-glycolide) microparticles for oral administration: formulation, structural and release characteristics solvent selection in the preparation of poly (dl-lactide) microspheres prepared by the solvent evaporation method paclitaxel-loaded plga nanoparticles surface modified with transferrin and pluronic® p , an in vitro cell line and in vivo biodistribution studies on rat model targeting to macrophages: role of physicochemical properties of particulate carriers-liposomes and microspheres-on the phagocytosis by macrophages this work was financially supported by a grant from tehran university of medical sciences. ( ) key: cord- - vnz zzg authors: garcia, sònia title: pandemics and traditional plant-based remedies. a historical-botanical review in the era of covid date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: vnz zzg pandemics are as old as humanity and since ancient times we have turned to plants to find solutions to health-related problems. traditional medicines based mostly on plants are still the only therapeutic possibility in many developing countries, but even in the richest ones, herbal formulation currently receives increased attention. plants are natural laboratories whose complex secondary metabolism produces a wealth of chemical compounds, leading to drug discovery – % of widespread use drugs are indeed of plant origin. their therapeutic potential is even bigger: although many plant-based compounds show inhibitory effects against a myriad of pathogens, few reach the stage of clinical trials. their mechanism of action is often unknown, yet traditional plant-based remedies have the advantage of a long-term experience in their use, usually of hundreds to thousands of years, and thus a precious experience on their safety and effects. here i am providing a non-systematic historical-botanical review of some of the most devastating pandemics that humanity has faced, with a focus on plant therapeutic uses. i will revisit the middle ages black death, in which a plant-based lotion (the four thieves vinegar) showed some effectiveness; the smallpox, a viral disease that lead to the discovery of vaccination but for which the native americans had a plant ally, an interesting carnivorous plant species; tuberculosis and the use of garlic; the spanish flu and the widespread recommendation of eating onions, among other plant-based treatments; and malaria, whose first effective treatment, quinine, came from the bark of a peruvian tree, properties already known by the quechua people. synthetic analogues of quinine such as chloroquine or hydroxychloroquine are now being revisited for the treatment of covid symptoms, as they are artemisinin and derivatives, other plant-based compounds effective against malaria. finally, i will give some hints on another facet of plants to aid us in the prevention of infectious diseases: the production of biotechnological plant-based vaccines. altogether, my aim is to stress the significant role of plants in global health (past, present and future) and the need of enhancing and protecting the botanical knowledge, from systematics to conservation, from ecology to ethnobotany. pandemics have shaped the history of mankind, and plants were usually the first available therapeutic choice. there is evidence of herbal preparations by egyptians around bc, later improved by greeks and romans, and widely documented in official drug books known as pharmacopoeias. still in our days, traditional medicines based mostly on plants are the only therapeutic possibility for many people in developing countries (akerele, ) . but, also in the first world, with wide access to the most modern drugs, the use of plant-based traditional medicine is experiencing a revival, as it is seen as safer and healthier than synthetic drugs. indeed, one advantage of traditional remedies over modern drugs is that their effects and margin of safety have been known for long. there is also a renewed scientific interest on plant-derived drug discovery, according to the current increasing publication trend on the topic (atanasov et al., ) . the rich secondary metabolism that characterises plants make them a source of compounds that may have a yet unknown therapeutic potential, only limited by the availability of resources to perform clinical trials. it is claimed that natural products (mostly from plant origin) will be the most important source of new drugs in the future (atanasov et al., ) . a recent editorial (nature plants, ) highlighted the need of funding and understanding botanical knowledge in the context of the current, and possibly future, pandemics. it is urgent to develop therapeutic tools to protect from high risk of infection (mitjà and clotet, ) and plant-based remedies with proven safety profiles could be one of the faster solutions. here i present a non-systematic review with a historical-botanical perspective on some of the most important pandemics that humanity has faced, and in some cases is still facing, and how certain plants or plantbased remedies have been used, and may continue being used, to treat these diseases, possibly including covid . the black death or black plague took place in the middle ages ( ) ( ) ( ) ( ) ( ) in eurasia, and still is the deadliest pandemic ever, with an estimated loss of million of human lives wiping out to % of european population in roughly four years (deleo and hinnebusch, ) . although this is the most know outbreak of the bacterium yersinia pestis, the much earlier -and longer-plague of justinian (from to ad) was also caused by the same pathogen (harbeck et al., ) , killing about - million people during two centuries. there have been other less spectacular, but still important plague outbreaks, arriving to the most recent ones in madagascar during the present decade. originated in china, the plague was usually spread by trade boats, whose rats carried fleas with the bacterium, which was transmitted to humans directly by the bite of the flea, and then between humans by contact or aerosol inhalation. there are several forms of the disease, the most common being the bubonic plague, which provokes the inflammation of the lymph nodes (buboes) as its most recognizable sign; a second form is the pneumonic plague which affects the respiratory system and is more deadly; the third form, the septicaemic plague, is the least common but has a mortality ca. % (byrne, ) . the antibiotic treatment, starting in early xxth century, reduced the death rate to about %- % which previously was between %- %; however, little is known on the remedies used before antibiotics were a reality and the major plague outbreaks occurred much earlier. in the middle ages, some preventive measures included, among others, carrying sweet smelling herbs to clear "the evil air" (which was believed to carry the pathogen) around the person (jones, ) , garlic for cleaning kidneys and liver, and lavender or chamomile teas to calm the stomach bile (khaytin, ) . a remedy named "the four thieves vinegar" was very popular: it consisted in several herbs, such as angelica (angelica archangelica), camphor (cinnamomum camphora), cloves (syzygium aromaticum), garlic (allium sativum), marjoram (origanum majorana), meadowsweet (filipendula ulmaria), wormwood (artemisia absinthium), and sage (salvia officinalis), brewed in vinegar (gattefosse, ) . before going out, people should apply it on hands and face for avoiding to contract the plague. some of these plants are well known flea repellents, so this may be one of the reasons for its efficacy. other herbs such as meadowsweet might have been included to release pain (as it contains salicylic acid, a precursor of aspirin) and to mask odours, a very helpful property considering that decomposing bodies were usually encounteredthe legend states that the name of the remedy might refer to thieves using it to rob the plague dead or sick (lucas, ) . another treatment coming from ancient greeks also gained popularity: the king mithridates antidote (totelin, ) , an extract of about fifty plants in a mixture with opium (papaver somniferum) paste, which if any, at least eased the pain or promoted a peaceful death. other prescriptions included lavender or rosewater baths, probably due to their antimicrobial and buboes healing properties. willow bark (another source of salicylates) was also given as a painkiller (khaytin, ) . a curious "prophylactic" plant-based remedy was recommended by the napolitan doctor angelerio during a plague outbreak in alguero in the xvi century ( - ) ( figure ): "any person going out from home must carry a cane (note: probably from the species arundo donax) six spans long, and as long as the cane is, one must not approach other people" (bianucci et al., ) . the origin of smallpox, a viral infectious disease caused by variola virus (two variants: v. major and v. minor) is unknown, but it dates back at least to the ancient egypt (third century bc), since some mummies showed smallpox-like eruption, the characteristic macules of the disease. as with the plague and other pandemics, the disease has occurred in several outbreaks around the world, the most recent in the late s. this virus has killed between and million people during the th century (koplow, ) until the global eradication campaign by the world health organisation (who) in . smallpox was the first infectious disease to have been eradicated ( ), the (only) second one being rinderpest, a viral illness of cattle. the smallpox vaccine (the first ever) was based on edward jenner's demonstration, by the end of the xviii th century, that inoculation with cowpox (a variant of the smallpox virus infecting cows) protected against the disease. actually, jenner's contribution popularized the practice of vaccination, a word coined by himself coming from the latin word vaccinus (i.e., or/from the cow) for the prevention of several other infectious diseases. however, before vaccination was discovered, how did people deal with the illness? particularly interesting was the approach of the native americans, which were deeply affected by the disease. by the end of xix th century several surgeons and practitioners related to the us army, as well as the prestigious botanist charles f. millspaugh ( ) , described the use of poultices and infusions from the indigenous medical flora based on the plant sarracenia purpurea (family sarraceniaceae) to be effective for treating smallpox, in a likely case of medical appropriation of the indigenous therapeutic knowledge (lawrence-mackey, ). known by native americans (mi'kmaq people) as mqo'oqewi'k, also named purple pitcher plant, it belongs to a genus of carnivorous species that use modified pitcher-shaped leaves to trap insects. possibly, the spotted appearance of the plant (figure ), resembling one of the main clinical signs of the disease (clarke, ) , inspired its use to the indigenous people. this may be another example of the doctrine of signatures, an ancient concept by which god somehow indicated to men what plants would be useful for, by certain signs (coles, ) , a pseudoscience which has caused more harm than good in general, although exceptions appear. compelling descriptions of their effectiveness were recorded, such as ''the greatest remedy known for the dreadful scourge'' or "'it seemed to arrest the development of the pustules, killing, as it were, the virus from within" (clarke, ) . the advent of vaccination put forward the botanical remedy, but the antiviral properties of sarracenia purpurea have been later demonstrated in vitro (arndt et al., ) . the authors showed that the plant extract was not only active against smallpox, but also against other poxviruses, papovirus sv- and various herpes viruses, including papillomavirus and epstein-barr virus-associated carcinomas, usually by inhibiting the virus replication at the level of early transcription (moore and langland, ) . another global and persistent pandemic is tuberculosis, caused by mycobacterium tuberculosis. together with smallpox, it is one of the oldest known diseases, since molecular data and archaeological evidence support that it coexisted with humans from the neolithic (gutierrez et al., ; nicklisch et al., ) . relevant figures in the history of medicine such as hippocrates or avicenna identified the disease, which involved coughing blood and fever and which was often lethal. actually, avicenna detected the infectious nature of the illness and based on tuberculosis, was probably the first to come up with the idea of quarantine to stop the spread of infectious diseases (shephard, ) . tuberculosis infected million people only in , of which . million died (unaids, ). it is found in every country, being the first infectious cause of death worldwide. from the hiv/aids outbreak, the combination of both is usually fatal where tuberculosis is endemic (mainly developing countries), as the immune weakening caused by hiv facilitates the onset of tuberculosis. this disease is indeed the final cause of death of many hiv infected people; paradoxically, while many could live now with aids they are dying from tuberculosis. the main reason is that the bacterium has developed resistance to most antibiotics, which usually have to be taken during long and tedious treatments. this is why researchers have turned to the search of effective alternatives also among medicinal plants, as some of them have already demonstrated anti-tuberculosis activity. besides, an effective plant-based treatment would be more affordable in poor countries which are those more affected by the disease. among the plants that are being investigated, garlic (allium sativum, family alliaceae), a former remedy already used to treat the plague (see above) stands out for its renowned properties, although it still far from being an alternative. the benefits of garlic (of which there are about varieties) are well-known already from the ayurvedic and unani medicine systems (raghunandana et al., ) as well as from the chinese traditional medicine, but also ancient greeks, romans and even egyptians used it to treat illnesses. it has been used as a food and folk medicine for centuries by many cultures. garlic has a variety of pharmacological virtues, including antimicrobial, anticancer, antioxidant (dini et al., ) , fungicidal and as a cure for heart diseases, among others (majewski, ) . the antitubercular and other antimicrobial activities of garlic, however, have been demonstrated in vitro but still, seldom in vivo. although garlic has more than biologically active compounds, allicin is the most relevant, albeit highly unstable; therefore, depending on the preparation of the garlic-based remedy the efficacy may not be as high as expected (majewski, ) . the wide antimicrobial and even antifungal spectrum of allicin is explained by its inhibitory effects on sulfhydryl metabolic enzymes. by interacting with these enzymes, allicin induces thiol stress in bacteria, which, among others, inhibits the growth of the microorganisms (müller et al., ) . malaria, caused by protozoan species of the plasmodium group, is an infectious disease coming from the bite of a mosquito, usually an infected anopheles sp. female. the current name of the illness was given by the italians around xix th century, as a contraction of the words "mal aria" (i.e., bad air) from the belief that the disease was transmitted by the "miasma" coming from marshes (macip, ) . the most typical symptom is fever, together with nausea and vomiting, tiredness, headache, occasionally yellow skin and in severe cases it can lead to seizures, coma and death. it is another ancient disease, spanning from the neolithic to our days, and currently found in all intertropical continents. recent studies detected the parasite in african monkeys, probably being the source of the disease, although it is still debated how it spread worldwide (molina-cruz and barillas-mury, ) . it is known that malaria arrived to europe by the first century ad, probably coming from the african rainforests and travelling by the nile to the mediterranean, where it spread to the middle east and from there to greece, italyhistorians hypothesize on the triggering role of malaria in the fall of the roman empire -and the rest of europe, even as far as england and denmark (karlen, ) . between the xvi th -xix th centuries the disease crossed the atlantic ocean probably on slave ships to reach the american continent (yalcindag et al., ) . it was suffered by presidents of north america such as g. washington or a. lincoln and it raged specially with native americans, taking thousands of their lives. the centers for disease control and prevention, the leading national public health institute of the united states, was founded because of malaria in . in the last century, probably - million people have died from the disease, accounting for %- % of deaths (carter and mendis, ) . at present malaria is mostworrying in sub-saharan africa, accounting for ca. % of current cases, although there is also a resurge in southern asia (arrow, ) . the most well-known and one of the most effective historical treatments against malaria is quinine, an alkaloid extracted from the bark of the cinchona tree (cinchona officinalis) belonging to family rubiaceae, the same of coffee. it is original from peru (where it is the national tree, although currently it is considered an endangered species) and the quechua traditionally used the ground bark of these trees to stop shivering because of cold, not for malaria treatment per se. most likely, spanish jesuits missionaries brought cinchona to europe for the first time, having observed how the quechuas used it to threat shivering, by the end of xvi th century -a second case of medical appropriation in this story. the tree was named (by c. linnaeus) after the spanish countess of chinchon, who was treated with its bark in peru back in the early xvi th -linneaus misspelled the name of the countess, omitting the first "h" in the name (meshnick, ) . it is also said that the countess may have introduced the curative bark to europe when she returned to spain, but it is currently considered that this a legend rather than what actually happened (haggis, ) . quinine is effective on the "cessation of febrile paroxysms" (stephens et al., ) , one of the main symptoms of malaria, and which has given its popular name to the species (fever tree). malaria outbreaks, however, continued to appear during centuries with no alternative to quinine. during world war i the german army was strongly affected by the parasite in the troops of east africa, as the allies controlled java, the main worldwide quinine producer. in an interesting historical moment that impelled science, the german government commissioned a search for a substitute to quinine, determined not to suffer again from its shortage. in chloroquine, a synthetic compound similar to quinine was synthesized at the bayer laboratories. much later ( ) another very similar derivative, hydroxychloroquine, was produced in the us. both chloroquine and hydroxychloroquine are used to prevent and treat malaria, being some of the antimalarial drugs of choice in areas where the disease is not resistant to them (a recurrent problem in this disease); they were preferred over quinine because of much less severe adverse effects, although at present there are many other even safer alternative drugs for the treatment of malaria. in recent years, these antimalarials have shown several immunomodulatory effects and they currently treat, mostly, diseases such as lupus erythematosus or rheumatoid arthritis. as with tuberculosis, the parasite tended to develop resistance to these treatments sooner or later, and researchers were urged to look for alternatives. a very popular one was found in the plant artemisia annua (sweet wormwood, from family asteraceae) (figure ) a remedy known in chinese traditional medicine as qing-hao for more than years (klayman, ) . chinese herbalists had been using it for treating haemorrhoids, chills, and fevers (trigg and kondrachine, ) . the species, as other members of genus artemisia such as absinthe or tarragon, is aromatic and bitter. one of the compounds responsible for its bitterness, a sesquiterpene lactone extracted from the glandular trichomes named artemisinin, is the active compound against malaria. the discovery of artemisinin is also very remarkable from the historical point of view. in , during the chinese cultural revolution under mao zedong's mandate, the secret "project " was a plant screening research program to find an alternative treatment for malaria, which was ravaging vietnamese army during vietnam war. in , dr. youyou tu a researcher of that program, isolated artemisinin, "rediscovering" the ancient remedy qing-hao. the drug started to be used in , a relatively short-period to establish a new medicine in the market, but it was also based on thousands of years of experience by the chinese traditional practitioners. nowadays artemisinin and its synthetic derivatives are one of the main defences against drug-resistant malaria in the asiatic southeast. however, who recommends it in combined therapy with other drugs, in part to avoid the development of resistance and in part to counteract the short half-life of artemisinin in plasma, leading to the artemisinin combination therapies (acts) which include companion drugs such as some cloroquine derivatives (e.g., mefloquine). in tu was awarded the nobel prize for the discovery of artemisinin, which represents an important contribution of china to the global health, as well as the first and awaited nobel prize in the sciences for china. it is considered the most significant milestone of tropical medicine of the last century, contributing to a better health and saving tens of thousands of lives every year in tropical developing countries of south asia, south america and africa. in the spring of started one of the deadliest pandemics in recent history, caused by one aggressive strain of the h n influenza virus (the same virus that caused the swine flu pandemic). it was popularly known as the spanish flu because in the context of world war i censorship minimized the effects of the pandemic to keep people's morale in the countries involved in the conflict, while in the neutral spain newspapers were free to report its effects, giving the impression that this country had been particularly devastated by the disease. during months, until summer , there were three waves of the pandemic, being the second the worst. a fourth, much fainter wave, took place in the spring of and after this one, the virus disappeared as it had arrived. it infected about half a billion people (ca. / of the world's population) and killed about million (with some estimates as high as million); for a reference world war i estimates range from to million deaths. the origin of the virus is unclear but it is thought that it started as a zoonosis from birds to humans which later was transmitted from humans to swine. the symptoms were an amplified version of those of normal flu, but typically deaths were caused by complications derived from a secondary pneumonia. contrary to other h n flu strains, this one was unusually lethal among young people, and almost one century later its high-virulence is only partly understood (tumpey et al., ) . since at that time there was neither vaccine, nor antibiotics to treat secondary pneumonia, the main prophylactic options were, as with the covid pandemic, to avoid contact through lockdown and quarantines, to increase personal hygiene and to use disinfectants widely. also, people turned to folk remedies and some recommendations got popular, such as the widespread advice "eat more onions!" (figure ) (arnold, ) . as with garlic, onion (allium cepa) has certain compounds (particularly a polyphenol named quercetin) which have demonstrated antiviral properties (lee et al., ; sharma, ) but still more research and clinical trials are needed. besides, in the usa a group of doctors known as "the eclectics" got positive results by treating the flu symptoms with plant remedies, together with other measures that included exercise. they reported a fatality rate ca. . % for their patients while the average in that pandemic was ca. % (abascal, ) . by selecting the herbs to match the symptoms, they used a wide variety of species. the most remarkable among them, and that have later proved therapeutic, were: gelsemium sempervirens (known as yellow yasmine, with antipyretic properties), eupatorium perfoliatum (boneset, already known by native americans to treat cold-related symptoms), actea racemosa (black cohosh, also used by native americans as a painkiller, probably due to the content in salicylic acids of its roots) and asclepias tuberosa (pleurisy root, used to treat respiratory problems and with expectorating properties) (abascal, ) . however, despite their long history of use, again there is little applied research on these plants. currently, flu is partly under control by the release of annual vaccination campaigns with newly synthesized vaccines that collect most of the virus' seasonal variability. in the latter most important flu pandemic ( ) besides the vaccine, oseltamivir (tamiflu ® ) a drug derived from the species ilicium verum (star anise, from family schisandraceae) was also crucial to treat most severe cases, although the production of this compound is limited by the low productivity of the tree, and synthetic derivatives are being developed (macip, ) . finding adequate treatments for flu is still and urgent task as the fear of a pandemic similar to the one in is a still a sword of damocles in the concerns of most epidemiologists. from the s, the science of "molecular farming" gives another potential role to plants on the prevention of infectious diseases, involving plants or plant cell cultures to produce recombinant proteins (rybicki, ) . the first steps of this approach were the "manufacturing" of the human growth hormone, monoclonal antibodies or human serum albumin in transgenic tobacco or sunflower plants ( barta et al., ; hiatt et al., ; sijmons et al., ) . other recombinant proteins more recently produced in plants -shifted from bacterial, mammalian or fungal cell to plants and plant cell cultures-and commercialized, include human type collagen i manufactured in tobacco, bovine trypsin in maize or human lysozyme and lactoferrin in rice (yang et al., ; hennegan et al., ; shoseyov et al., ; takeyama et al., ) . as with the mentioned proteins, the vaccine production would follow similar steps: isolation of a specific antigen protein, the one that triggers an immune response from the virus; the gene(s) encoding that protein is transferred to bacteria and these bacteria are used to infect plants, so the plant will in turn produce the antigen proteinthe vaccine. plants would provide a flexible, cheap and easily scalable method to manufacture vaccines. they would also be safer than traditional vaccines, because of the absence of pathogens of animal origin. plant-based vaccines for humans are not yet in the market, although some candidates have entered clinical trials (rosales-mendoza et al., ) . it is likely, however, that they will start being approved in the mid-term, at least in the cases of ebola or rabies, or in a longer term for seasonal influenza (rybicki, ) . the outbreak of covid caused by the coronavirus sars-cov- , originated in china (province of wuhan) in december and has caused , , infections and , deaths worldwide (updated th june ). even earlier than the pandemic status was declared by who ( th march ) researchers across the world engaged in hundreds of clinical trials, in an unprecedented quest for a cure to the disease, vaccine, drug or both. given the long time-frames that usually imply finding a good candidate, many research groups have turned to repurpose other drugs. the reasons are that the effects (including adverse or side effects) of these drugs are well known and have been used in broad population groups with different ages and idiosyncrasies, so the security margin is increased, allowing to save precious time in long trials. in this regard, antimalarials are potential candidates (schlagenhauf et al., ) and both chloroquine but particularly hydroxychloroquine (as explained above, synthetic derivatives of quinine, the antimalarial alkaloid coming from the bark of the fever tree) are being studied to fight covid , although it is perhaps too soon to draw conclusions on their efficacy. several studies have reported their utility for some patients and some national guidelines have recommended both drugs for treatment of covid (see colson et al., and singh et al., ) despite there has been certain controversy. the who halted studies on these drugs by the end of may prompted by an observational study reporting that hydroxychloroquine produced a higher mortality rate in hospitalised patients, but the study was soon retracted on the basis of questionable veracity of data and analysis (mehra et al., ) and trials on the drug have been resumed shortly after. the effectiveness of hydroxycloroquine taken at initial stages of the disease was recently tested in a multicentre randomised controlled trial based on previous experiences of post exposure prophylaxis (pep) drugs to prevent infections (mitjà and clotet, ) , but no benefit was observed beyond the usual care . other plant-based antimalarials, artemisinin and derivatives, are also being tested against sars-cov- , again not without controversy. in many african countries, an elixir based on artemisia annua extract, "covid-organics" is being distributed as a cure against covid . however, there is little scientific evidence of the effectiveness of such elixir and its extended consumption can have associated problems, the most important the development of resistance to the drug by the malaria parasites in a continent particularly sensitive to the disease. nevertheless, there is evidence that the extract of artemisia annua has antiviral properties, being active against sars-cov- (li et al., ) , herpes simplex (karamoddini et al., ) , hepatitis a (seo et al., ) , hepatitis b, bovine viral diarrhoea, and epstein-barr (haq et al., ) . this has stimulated the research of the potential use of artemisinin and derivatives (such as artesunate) to treat covid , which is now being conducted by several biotech companies (e.g., mateon therapeutics, artemilife) as well as by public research institutions (e.g., the liverpool school of tropical medicine, the max planck institute of colloids and interfaces). traditional chinese medicine has also had a say in the cure of covid : the national administration of traditional chinese medicine (natcm) organized a study in late january to identify potential treatments, and the lung cleansing and detoxifying decoction (lcdd) was widely used and studied through clinical trials; its apparently high effectiveness made that the natcm officially recommended lcdd as a treatment for covid (weng, ) . among the lcdd ingredients, there were species such as ephedra sinica (well known as decongestant and bronchodilator through the active compound ephedrine), atractylodes macrocephala (showing antiviral activity against influenza viruses in experimental assays) or scutellaria baicalensis (containing anti-inflammatory flavonoids), and the combination of these effects and others from the remaining ingredients likely counteracts covid by their synergistic activities. however, as weng ( ) points out, it is difficult to transfer the success of the lcdd treatment to other countries, both because the cultural acceptance of tcm is not present outsides china, and due to the lack of knowledge on the precise chemical composition and mechanism of action, which are required in modern therapy. once the first vaccine for covid is finally developed (with estimates ranging from september to several years) another plant may also play an important role in order to produce it in large amounts: nicotiana benthamiana, a close relative to tobacco. this species is the focus of the eu project newcotiana, coordinated by researcher dr. diego orzaéz at the csic, the leading public research body in spain. in this project, genome editing practises (e.g., crispr) will be used to transform the plant in a biofactory for the large-scale production of the vaccine once it is available. moreover, one company may be currently developing a covid vaccine based on the expression of a sars-cov- protein in tobacco -kentuchy bioprocessing, a biotechnological branch of british american tobacco (capell et al., ) . as rosales-mendoza et al. ( ) stated in a recent review, the production of a plant-based vaccine in the context of the current pandemic would have the advantages of low cost, fast and escalable production, easy administration, and safety. beyond plant-based vaccines, molecular farming through plants, usually by transient expression of target proteins, can also be deployed to produce diagnostic reagents, as well as antibodies and antiviral proteins for therapeutic use. the italian biotechnology company diamante is generating antigens, to use in elisa tests (serological), based on a sars-cov- protein also in tobacco plants (capell et al., ) . another eu consortium, pharma-factory is also developing plant-based platforms to produce medical, veterinary and diagnostic products, for dealing with covid and also other diseases. i hope that the reader finds this review useful to call for the important role that plants have played and still play in human health. as schlagenhauf et al. ( ) commented, plant-based remedies are, more than an "alternative medicine", the organisms to which we owe some of the most useful therapeutic tools. still in the era of wide implementation of (synthetic) drug treatments, we turn to plants in many cases when resistances appear, as shown. paradoxically, there is a human tendency to ignore plants, a form of cognitive bias known as "plant blindness" (allen, ) that should be opposed, perhaps by enhancing and implementing more widely the botanical education. in this context, it is also essential not only to maintain but to increase societal funding into basic sciences such as botany, as well as to foster collaboration between scientists from different disciplines, whose interaction may open new therapeutic possibilities. finally, i would like that this review serves as a little recognition to the usually ignored ethnobotanical traditional knowledge of many indigenous peoples across the world of which the so-called western culture has in most occasions, illegitimately appropriated. the author confirms being the sole contributor of this work and has approved it for publication. i am thankful to roi rodriguez (institut botànic de barcelona) who kindly revised this paper and contributed interesting observations, as well as to the reviewer. herbs and influenza: how herbs used in the flu pandemic can be effective in the next nature's medicinal bounty: don't throw it away plant blindness in vitro characterization of a nineteenth-century therapy for smallpox pandemic : the story of the deadliest influenza in history the locus of decisions about aids/hiv, malaria treatment: what does welfare economics say? a question discovery and resupply of pharmacologically active plantderived natural products: a review the expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue quinto tiberio angelerio and new measures for controlling plague in th-century alghero the black death potential applications of plant biotechnology against sars-cov- evolutionary and historical aspects of the burden of malaria dictionary of practical materia medica in three volumes adam in eden, or nature's paradise chloroquine and hydroxychloroquine as available weapons to fight covid- a plague upon the phagocytes the potential role of garlic (allium sativum) against the multi-drug resistant tuberculosis pandemic: a review aromatheŕapie -les huiles essentielles hormones veǵetales ancient origin and gene mosaicism of the progenitor of mycobacterium tuberculosis fundamental errors in the early history of cinchona artemisia annua: trials are needed for covid- yersinia pestis dna from skeletal remains from the th century ad reveals insights into justinianic plague improvement of human lysozyme expression in transgenic rice grain by combining wheat (triticum aestivum) puroindoline b and rice (oryza sativa) gt promoters and signal peptides production of antibodies in transgenic plants the new cambridge medieval history: c. -c. antiviral activities of aerial subsets of artemisia species against herpes simplex virus type (hsv ) in vitro man and microbes: disease and plagues in history and modern times arcgis storymaps. herbal medicine in the black plague qinghaosu (artemisinin): an antimalarial drug from china smallpox: the fight to eradicate a global scourge medical appropriation in the 'red'atlantic: translating a mi'kmaq smallpox cure in the mid-nineteenth century anti-influenza a virus effects of fructan from welsh onion (allium fistulosum l.) identification of natural compounds with antiviral activities against sarsassociated coronavirus nature's medicines; the folklore, romance, and value of herbal remedies les grans epidèmies modernes (edicióactualitzada): la lluita de l'home contra els enemics invisibles allium sativum: facts and myths regarding human health retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis from quinine to qinghaosu: historical perspectives medicinal plants: an illustrated and descriptive guide to plants indigenous to and naturalized in the united states which are used in medicine, their description, origin, history, preparation, chemistry and physiological effects fully described use of antiviral drugs to reduce covid- transmission hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial the remarkable journey of adaptation of the plasmodium falciparum malaria parasite to new world anopheline mosquitoes sarracenia purpurea: a botanical extract with anti-papilloma virus and oncolytic activity allicin induces thiol stress in bacteria through s-allylmercapto modification of protein cysteines rib lesions in skeletons from early neolithic sites in central germany: on the trail of tuberculosis at the onset of agriculture redeploying plant defences investigations on plant antibiotics; studies on allicin, the antibacterial principle of allium sativum (garlic) what does plant-based vaccine technology offer to the fight against covid- ? vaccines plant-based vaccines against viruses repurposing antimalarials and other drugs for covid- antiviral activity of herbal extracts against the hepatitis a virus efficacy of garlic and onion against virus the middle-ages: monasteries, medical schools and the dawn of state health care," in an illustrated history of health and fitness, from pre-history to our post-modern world human collagen produced in plants: more than just another molecule production of correctly processed human serum albumin in transgenic plants chloroquine or hydroxychloroquine for prevention and treatment of covid- syst. rev. , cd studies in the treatment of malaria: v. intramuscular injections of quinine alkaloid in simple tertian malaria plant-based vaccines for animals and humans: recent advances in technology and clinical trials mithradates' antidote-a pharmacological ghost commentary: malaria control in the s characterization of the reconstructed spanish influenza pandemic virus tuberculosis-good progress, but not enough plant solutions for the covid- pandemic and beyond: historical reflections and future perspectives multiple independent introductions of plasmodium falciparum in south america expression and localization of human lysozyme in the endosperm of transgenic rice key: cord- - x vmyi authors: masoudi-sobhanzadeh, yosef; masoudi-nejad, ali title: synthetic repurposing of drugs against hypertension: a datamining method based on association rules and a novel discrete algorithm date: - - journal: bmc bioinformatics doi: . /s - - -w sha: doc_id: cord_uid: x vmyi background: drug repurposing aims to detect the new therapeutic benefits of the existing drugs and reduce the spent time and cost of the drug development projects. the synthetic repurposing of drugs may prove to be more useful than the single repurposing in terms of reducing toxicity and enhancing efficacy. however, the researchers have not given it serious consideration. to address the issue, a novel datamining method is introduced and applied to repositioning of drugs for hypertension (ht) which is a serious medical condition and needs some improved treatment plans to help treat it. results: a novel two-step data mining method, which is based on the if-then association rules as well as a novel discrete optimization algorithm, was introduced and applied to the synthetic repurposing of drugs for ht. the required data were also extracted from drugbank, kegg, and drugr+ databases. the findings indicated that based on the different statistical criteria, the proposed method outperformed the other state-of-the-art approaches. in contrast to the previously proposed methods which had failed to discover a list on some datasets, our method could find a combination list for all of them. conclusion: since the proposed synthetic method uses medications in small dosages, it might revive some failed drug development projects and put forward a suitable plan for treating different diseases such as covid- and ht. it is also worth noting that applying efficient computational methods helps to produce better results. hypertension (ht) is a long-term medical condition, in which blood circulates anomalously through the vessels. in terms of the nature of ht, the patients are divided into two groups, which are as follows [ ] : i) the primary ht: some genetic factors as well as unhealthy lifestyles such as having a salty diet, smoking, drinking alcohol, stresses and strains, overweight etc. [ ] all have an important role in inducing ht. more than % of the hypertensive patients who are mostly adults, are placed in this category. ii) the secondary ht: some medical conditions such as the chronic kidney illnesses might also give rise to ht [ ] . less than % of the patients, whose ht may be reduced through treating the main condition [ ] , are placed in this group. according to the world health organization, about . billion people around the world, which is a remarkable number, suffer from ht [ ] . most of these patients who are from the low and middle-paid countries [ ] , need some proper therapeutic plans such as drug repurposing methods which could yield the desired effect [ ] . drug repurposing or drug repositioning process, which may be the best option to treat diseases, brings about some big advantages as follows: first, it may make the finding treatment plan against ht a cost-effective and time-saving process. second, it might be useful in treating both the orphan and rare ht diseases such as covid- , considering the fact that drug companies cannot afford to develop new molecular entities or cannot develop a suitable medication in a reasonable time order. third, it might prove that the existing drugs have many hidden medical benefits in treating ht, in view of the fact that such advantages have not been detected before. beside taking advantage of drug repurposing, the synthetic repurposing of drugs, in which instead of recommending a specific drug, a combination of two or more medicines is prescribed, might yield various advantages as follows: i) it might lessen the medicines' toxicity through applying them in small doses [ ] , which in turn can lead to reviving the previous drug development projects which have failed due to facing some biological problems such as bioavailability or large amounts of toxicity. ii) it may increase the effectualness of drugs and yield some better treatment methods. in contrast to a single therapy, a proper combination of drugs can produce much better synergic effect and have more control over the disease. however, the drug-drug adverse reactions are considered to be the main challenge in front of the researchers [ ] . iii) it may pave the way for developing a new research branch of drug repurposing and expanding the drug usages in a wide range of diseases [ ] , which in turn can help pharmaceutical companies or pharmacologists determine the doses of drugs and address their technical issues. iv) the results obtained from the synthetic repurposing of drugs may also be applied to a combination therapy, in which a drug can increase the effectualness of one particular drug when combined with it [ ] . there is a marked difference between the synthetic treatment and combination therapy concepts. the first one is based on a synthesis of different drugs instead of a given medication, whereas the second one combines one or more medications with a given drug [ ] . in other words, in the synthetic repurposing method, a combination of two drugs (a and b) is used instead of a given drug (c), whereas in the combination therapy, a combination of a given drug (c) and another drug (e) is being used. the present study aims to introduce the synthetic repurposing of drugs as a useful approach to treat various diseases such as ht. for this purpose, a novel datamining method, which is based on our proposed algorithm and the association rules, is presented. the approach employed to conducting the research, consists of two main parts as follows: first, the if-then association rules are applied to a large volume of data to extract information about the drug-target interactions, the drug-drug adverse reactions, and the drug-diseases data. second, the discrete version of the proposed discrete algorithm (trader) [ ] is introduced and used to discover the synthetic lists which might be useful in controlling ht. in the present study, the related works on the repurposing of drugs are examined and categorized from a computational perspective, which are listed as follows: i) the machine learning-based researches: in these researches, the existing data are examined to discover the relationship between inputs and outputs [ ] . overall, until recently, three types of machine learning methods, including supervised, semi-supervised, and unsupervised have been applied to the scope of drug discovery processes [ ] . it has been also shown that some modified and improved versions of the present approaches such as deep neural networks could yield better predictive models [ ] , and overfitting and insufficient amounts of data have been the main challenges in front of the researchers who have been engaged in generating an appropriate predictive model [ ] [ ] [ ] . ii) the theory-based researches: normally, researchers formulate the relationship among the biological entities based on the numerical and experimental experiences [ , ] . for this purpose, they have proposed many mathematical equations to calculate the drugs' structural similarities [ , ] and based on the similarity score, they have examined the role of the analogous drugs in treating diseases [ ] . the fact that the drugs with similar structures (scores) might be replaced with one another, is of the utmost importance. however, it has been reported that the theorybased methods are not applicable to the most projects and to qualify, they need to meet some other criteria [ ] . iii) the graph and network theory-based researches: these studies employ a graph or a network to show the communication among the biological components [ ] . for this purpose, first the graph algorithms are applied to the generated network [ , ] , and then the hidden relations are detected [ ] . although these techniques are confronted with minimal validity challenges and can produce significant results, they are not applicable to the most drug repurposing cases because the biological elements act according to the hypergraph concepts [ , ] . iv) the text mining-based researches: in these studies, different algorithms are applied to delve into a massive volume of raw data and gather the desired ones [ ] . the prevalent drug repositioning strategies which are employed to apply this approach, are as follows: k-means, knn, the association rules, and optimization algorithms [ , ] . for best results, it is essential to organize the data effectively and apply the state-of-the-art algorithms. our proposed method, which employs both the association rules (if-then) [ ] and the novel discrete algorithm (trader), fits into this category of the related works. v) the ensemble method-based researches: in such research projects, various techniques are combined in several different ways [ ] to generate an efficient tool for predicting and discovering the hidden benefits of the drugs [ ] . for example, some researchers have mixed different aspects of the computational methods to obtain a suitable predictive model [ ] [ ] [ ] . however, from the biological perspective, it has been shown that the simple techniques are sometimes better than the complex ones [ ] , and the ensemble methods' overfitting possibilities might be more than the other approaches'. the newly proposed method for the synthetic repurposing of drugs was implemented in the matlab programming language and then compared with the four state-of-theart algorithms, including the discrete symbiotic optimization search (dsos) [ ] , the forest optimization algorithm (foa) [ ] , the world competitive contests algorithm (wcc) [ ] , and the cuckoo optimization (cuk) algorithm [ ] . afterwards, the above-mentioned state-of-the-art algorithms were applied to the generated datasets which have been presented in table . next, some candidate medicines were selected from medications which have been suggested for use in controlling ht by the different proposed treatment methods. in the datasets, there exist three pieces of information about every drug, which are as follows: i) the total number of a drug's targets, including the main targets and the side effects, ii) the total number of a drug's targets which are effective in controlling hypertension, and (iii) the total number of the drugs which have a target in common with one of the main targets of the specified drug. for instance, in the case of nicardipine which interacts with targets, only targets play a key role in treating hypertension, and the remaining targets are regarded as just some side effects. there are also drugs which have at least an interaction with the main targets of nicardipine. the present study aims to substitute a specified drug such as nicardipine with an optimal subset of drugs which help control hypertension. the results obtained from applying the algorithms to the datasets have been divided into two categories, which are as follows: i) in the first class of evaluations, the performance of the algorithms was examined in terms of convergence, stability, and some statistical criteria such as the p-value, the standard deviation (std). the stochastic operations of the optimization algorithms generate various results in their different runs. for this reason, each algorithm was individually executed times, and then the generated data were analyzed. each algorithm was executed under similar circumstances and invoked the same number of the score functions. figure represents the convergence of the algorithms on the generated datasets. the horizontal and vertical axes show the iteration number and the best-obtained score, respectively. when the size of the problem or the candidate drugs is small, most of the algorithms can choose the tnt the total number of targets, tnmt the total number of main targets, tnc the total number of candidates fig. the stability behavior of the algorithms on the generated dataset. the stability of the algorithms on the a trandolapril, b atenolol, c carteolol, d nicardipine, e felodipine, f nifedipine, g nisoldipine, h doxazosin, and i prazosin datasets. trader is remarkably more stable than the others and delivers better results on the datasets except for trandolapril and nicardipine. moreover, trader's performance is better than the others' because the performance of the other algorithms lowers through increasing the total number of candidate drugs best possible subset of medicines to cure ht. however, both their performance and their convergence are reduced when the total number of the candidate drugs rises, which in turn can lead to their failure to acquire the best answer to the synthetic repurposing of drugs. for instance, although the foa algorithm has acquired the best solution to the trandolapril dataset, it does not produce the best synthetic medicines on the remaining datasets and therefore falls into the local optima solutions. the principles of the meta-heuristic algorithms are approximately the same. for instance, these algorithms generate some random potential answers to a problem and, then, try to improve them based on some random operations [ ] . therefore, they must be executed at least times, and their performance should be evaluated based on the produced data [ ] . an algorithm which can yield very similar results in disparate runs, is more stable than the others and consequently, its generated outcomes should be better than the others'. figure demonstrates the algorithms' stability on the created datasets in distinct executions. in fig. , the horizontal and vertical axes present the number of runs and the bestobtained score values, respectively. since all of the algorithms produce the best possible results on trandolapril in all of their executions, the generated results overlap. the stability of each algorithm will differ from that of the other ones as the number of the candidate drugs increases. overall, trader, wcc, dsos, foa, and cuk, respectively are the best algorithms in terms of the stability power. table , which shows the algorithms' performance on the generated datasets in distinct executions, has been provided to examine the functionality of the algorithms accurately. for different executions, this table presents the following information: (i) the worst results of the algorithms, (ii) the best results of the algorithms, (iii) the average value of the results, (iv) the p-value which indicates how much the algorithms' results is produced by chance, (v) the standard deviation (std) of the results, and (vi) the confidence interval (ci) which indicates a range in which the outcomes of the algorithms are expected to be obtained. due to falling into the local optima solutions, the value of std is higher than the other algorithms' value on some datasets. for instance, the dsos algorithm does not show stable behavior in finding a potential answer to the problem as the size of the problem increases. every algorithm's performance on the trandolapril dataset is the same as the other algorithms'. also, the best results obtained from applying both trader and the wcc algorithm on the atenolol and nicardipine datasets are alike. the wcc algorithm's best-obtained score on the felodipine dataset is better than the other algorithms'. in the case of the newly introduced algorithm (trader), its best-acquired outcomes are very similar to the wcc algorithm's on the felodipine dataset. compared to the other algorithms, trader has better performance on the remaining datasets. table summarizes the data presented in table and makes a comprehensive comparison among the algorithms. this table shows that compared to the other methods, the newly proposed method is the most suitable option for the synthetic repositioning of drugs. to make a comparison among the algorithms, their performance was examined based on the wilcoxon statistical test [ ] . for the purpose of this comparison, the results of the proposed algorithm (trader) which were regarded as the test base, were compared with the other algorithms' outcomes ( table ) . as mentioned earlier, the p-value in table shows what amount of the results of the algorithms is produced randomly, but the p-value in table demonstrates whether the proposed algorithm's performance is the same as or more efficient than the others' performance. for the purpose of comparing the proposed algorithm with an algorithm, named a, we considered the following hypotheses: h : the performance of the proposed algorithm is the same as the performance of a. h : the performance of the proposed algorithm is more efficient than the performance of a. if the p-value is less than . , h will be rejected and h will be accepted. otherwise, h will be accepted, and h will be rejected. ii) in the second part of the results, the outcomes of the algorithm are discussed from the drug synthetic repurposing aspect. for the selected drugs against ht, table demonstrates a list of drugs which may be replaced with a given drug. trandolapril which is used for treating the minor ht, is a molecule which blocks the angiotensin-converting enzyme's activity (ace) [ ] . this medication can interact with table the synthetic repurposing of drugs for the ht ace whose involvement in regulating the rate of fluids in the body makes it capable of controlling ht. as shown in table , all of the algorithms have proposed a drug with a few side effects, which may be replaced with trandolapril. atenolol, which is used to cure the abnormal rhythm of heartbeats, is a beta-blocker. like trandolapril, it interacts with ace [ ]. once again, all of the algorithms have suggested a drug which can be replaced with atenolol, as in trandolapril's case. however, the proposed drugs have a number of side effects and may produce some undesired effects on the body. trader, wcc, and dsos managed to find some substitutes without side effects and therefore are better than foa and cuk. since carteolol acts against the beta- adrenergic receptor and agonists the beta- adrenergic receptor [ ] it can reduce the blood pressure; for this reason, it is regarded as a candidate for treating ht. all of the algorithms propose a list of drugs, but both their lists and their costs differ. compared to the other algorithms, both trader and wcc algorithm have produced a proper result based on the score function. nicardipine, which is a calcium channel blocker, controls the blood pressure [ ] . cuk and foa could not propose some candidate drugs which can be replaced with nicardipine. while trader yielded two lists which can be replaced with nicardipine, wcc and dsos proposed just a synthetic list instead of nicardipine. based on the score function, all of the lists have an identical value and may be a suitable option for treating ht. felodipine is capable of controlling the blood pressure by blocking the calcium channels, so it can be a proper option for curing both the mild and minor ht [ ] . to treat ht, instead of proposing felodipine, algorithms have acquired a list of medications which include pinaverium. trader's suggested list has the best score value compared to the lists proposed by the other ones. nifedipine and nisoldipine slow down the penetration of calcium into the heart cells and vessel walls [ ] . as a result, heart can pump blood around the body and widen the blood vessels efficiently. all of the algorithms except trader have failed to discover candidates which can be used instead of nifedipine and nisoldipine. in this case, trader puts forward one or two possible lists. doxazosin blocks the alpha- a, alpha- b, and alpha- d adrenergic receptors and smooths the growth of muscle cells [ ] . in contrast to cuk and foa which did not manage to detect any drug substitutes for doxazosin, trader, wcc, and dsos succeeded in discovering a single-member list. based on the score value, trader, wcc, and dsos, respectively can be ranked among the most functional algorithms. prazosin works against the alpha- a, alpha- b, and alpha- d adrenergic receptors. in a comparison made among the results obtained from different algorithms, it is shown that to control ht, trader has substituted three different lists for prazosin, whereas the other algorithms have replaced prazosin with just a two-member list. moreover, from the score value viewpoint, trader's proposed lists are more suitable than those suggested by the other algorithms. based on the obtained results, it can be concluded that trader (the newly introduced algorithm) is more efficient than the other state-of-the-art algorithms and proposes some better synthetic drug lists to treat ht. in addition, the results of the newly proposed drug lists by trader have been presented in detail in table and demonstrate how similar the suggested drugs are. to discover the hidden applications of the existing drugs, a drug repositioning method, which is based on the newly introduced discrete optimization algorithm (trader) and if-then association rules, was proposed. the proposed method may reduce the toxicity of drugs and enhance their effectualness in curing diseases. to investigate the applicability of the suggested method, it was applied to the nine hypertension-related datasets, and the results were analyzed and examined from two perspectives. from the first viewpoint, it was shown that the state-of-the-art algorithms yield better outcomes than the others. the average score value and the confidence interval of the proposed algorithm (trader) were − . and [− . - . ], respectively, which were remarkably better than those obtained from the other algorithms. besides, while the proposed algorithm could detect some candidate lists for all of the datasets, the others failed to do so. for example, the other algorithms except for trader failed to explore for the potential drugs which can be replaced with nifedipine and nisoldipine. from the second viewpoint, the application of the suggested drugs was examined in treating hypertension. a detailed examination of the data yielded some important results as follows: first, the proposed method succeeded in suggesting some proper substitutes for the selected drugs, which may be more useful than the current therapeutic methods in treating ht. for example, the proposed approach managed to replace atenolol with esmolol which belongs to a family of beta-blocker drugs and does not have any side effects [ ] . as mentioned before, atenolol can interact with two targets [ ] , one of which is responsible for ht, and the next one is a side effect. the small doses of the above-mentioned drugs may combine with their substitutes and produce the desired effect. for instance, in databases, it has been reported that a combination of trandolapril and cilazapril might prove more useful in controlling ht [ ] . second, the proposed approach can introduce the novel applications of some drugs. for example, pinaverium which is a first-line option for curing bowl dysfunctionality, operates as an inhibitor and antagonist of the voltage-dependent calcium channel protein [ ] . moreover, more than countries are exploiting pinaverium to treat gastrointestinal disorders. however, it still has not won approval from fda. pinaverium's application in controlling ht may be based on the kind of method employed in this research. therefore, the introduced approach might especially revitalize the projects which have already failed due to the various undesired biological effects of drugs [ ] . besides, this method might be a suitable therapeutic plan for treating orphans or diseases because developing an efficient drug is both costly and time-consuming and therefore not affordable for drug companies [ ] . another introduced medication for managing ht is dapiprazole which is an alpha-blocker agent. dapiprazol helps to reduce the size of the pupils of the eyes in patients who suffer from mydriasis [ ] . although different studies have reported many undesired side effects of dapiprazole and some other drugs such as pinaverium [ ] , the proposed approach aims to employ these drugs in an appropriate manner. to this end, such medications are used in small doses to decrease their undesired effects, [ ] . furthermore, a proper combination of drugs might produce a synergistic effect on treating ht [ ] . a few studies have investigated the synergic effects of drugs and for this purpose, have examined some drug-drug adverse reactions. however, most of them have not been specified, yet. the machine learning approaches can help predict the drug-drug adverse reactions and introduce the most reliable synthetic candidates. a novel discrete algorithm, named trader, was introduced for the synthetic repurposing of drugs. this method can resume most of the failed drug discovery projects and might be the most suitable option for treating the orphan and rare diseases. the proposed approach takes account of the various aspects of the synthetic repositioning of medications, including the drugs' mechanism of action on targets, the drug-drug adverse reactions, and the total number of side effects. based on the obtained results, it can be concluded that the-state-of-the-art algorithms yield better results and show more suitable performance in comparison to the others. furthermore, the literature findings validate the functionality of the proposed method and suggest several synthetic repurposing lists to reduce hypertension. a two-step datamining method was proposed to discover drugs that might be useful in treating ht. in the first step, based on the if-then rules, it was determined which drugs may inhibit or prevent the targets inducing ht. in the second step, an optimal subset of the candidate drugs whose combinations may be helpful to treat ht, was selected by the proposed discrete optimization algorithm (trader). figure presents the framework of the proposed method. the first part of the proposed approach includes a number of steps which are as follows: i) obtaining the data: data on the drugs and their different targets were extracted from drugr+ (version . ) [ ] , which is a relational database and integrates drugbank (version . ) [ ] and kegg (version september , ) [ ] databases. drugr+ database provides an online tool that takes a drug and suggests some potential drugs which can be substituted for the first one [ ] . meanwhile, drugr+ has an advanced search section in which users can state their sql queries and download the results immediately. in selecting the datasets, the size of the data and the various treatment properties of drugs have been considered. ii) constituting the drug-disease (d-di) matrix: based on the downloaded data, a binary matrix, named ddi, and incorporated drugs-diseases relationships, was formed using eq. ( ). where i and j indicate a drug and a disease, respectively. the total number of drugs and diseases are , and , respectively. fig. the framework of the proposed method. in the first step, the desired data are obtained from drugr+ database. next, drug-disease (d-di), drug-target (d-t), and drug-drug-adverse reaction (d-d ar) matrices are formed. drugs which can affect the ht inducing targets, are acquired based on the if-then rules. in the second step, the proposed optimization algorithm (trader) is used to select a combination of drugs for the repurposing of medications for ht iii) constituting the drug-target (d-t) matrix: this matrix shows whether a drug affects a target or not. the extracted data show that there are four classes of drugtarget effects, including (a) agonizing, (b) antagonizing, (c) inhibiting, and (d) inducing. the targets whose total numbers were , consisted of both proteins and enzymes. the dt matrix was formed using eq. ( ) . v) mining: during every stage of the process, we employed the dt and ddi matrices to extract some information which were used as the input of the discrete optimization algorithm (trader). in the first part of the proposed method, different targets of blood pressure along with their effects (inhibiting, inducing, agonizing, and antagonizing) are determined and placed in a set named ht_targets. equation ( ) presents the mentioned set as follow: where n, t i , and r i are the total number of the obtained targets, the i th target, and the effect which leads to high blood pressure, respectively. furthermore, another set, named drugs, was created for drugs that directly interact with the ht_targets collection and exert exactly the same impact on the targets. for instance, both the angiotensin ii and candesartan drugs interact with the type- angiotensin ii receptor. however, angiotensin ii affects the mentioned target as an agonist whereas candesartan affects it as an antagonist. therefore, angiotensin ii is ignored because its function is not exactly the same as candesartan's. in this study, the interaction between a drug such as d and a target such as t (protein (p) and an enzyme (e)) is presented by "➔". the effect of d on t and the cause of hypertension due to t are shown by f(d,t) and f(ht,t), respectively. next, the following rules are applied to all of the existing drugs, and the candidate drugs are added to the drugs set. a) if d t && f(d,t) = f(ht,t) then d may be useful for controlling ht b) if e p && f(e,p) = f(ht,p) then the drug, which interacts with e, may be useful for controlling ht c) if p e && f(e,p) = f(ht,e) then the drug, which interacts with p, may be useful for controlling ht d) if p p && f(p ,p ) = f(ht,p ) then the drug, which interacts with p , may be useful for controlling ht e) if e e && f(e ,e ) = f(ht,e ) then the drug, which interacts with e , may be useful for controlling ht. in the second part of the proposed method, the discrete optimization algorithm (trader) was applied to select the optimal subsets of the obtained drugs which may reduce the pressure of blood. for this purpose, a number of steps were followed: i) creating the first population of the candidate solutions (css): trader begins with randomly created potential answers, which are presented by an array shown in eq. ( ). cs ¼ v ; v ; …; v m ; g; score ½ ð Þ where v i , m, g, and score are the i th variable, the total number of variables, the group, and the score or fitness of the cs. every variable shows a drug whose value is set or for the selected and unselected drugs, respectively. ii) calculating the score of the css: the css which are answers to the mentioned problem, differ widely in terms of how much they are worthy of consideration. in this study, the score is calculated using eq. ( ). where m, v i , se, and at represent the length of a cs, the value of the i th variable ( or ), the total number of side effects related to the i th drug, and the total number of the covered targets which are correspondent with ht, respectively. iii) grouping css: the total number of the groups and the total number of the traders are the same concepts, and they show a group. at the beginning of the algorithm, the total number of the members in the groups is the same, and in the next iterations, they are updated using eq. ( ). where tm i , c, and t present the total number of the members in the i th group, the total number of css, and the total number of the traders or groups, respectively. m is a constant value ( ) and guarantees that none of the groups will be eliminated during the iterations of the algorithm in each stage. the property of the i th trader is calculated using eq. ( ) . where m and score show the total number of css in the i th group and the score of the related cs, respectively. in other words, the property of a group is equivalent to the sum of its css' scores. iv) changing the css: there are three operators who change both the master and the slave css. as shown in eqs. ( , , and ), these operators, named retailing, distributing, and importing-exporting, try to improve the css. when the retailing operator is employed, the minimum number of changes will be applied to a slave-cs. the distributing operator obtains some values from the best cs of the group (the master-cs) and then, assigns them to the other css of the group (slave_cs). while both the distributing and retailing operators change the slave-css, the importing-exporting operator brings about changes in the master-css. the changes can be accepted in all of the operators, provided that they improve the value of a cs's score. where j and m are the importer and exporter css, respectively. also, the values of k and r can be measured using eq. ( ) . an instance of the mentioned operators has been illustrated in fig. . p t cell subset imbalance in hypertension is not associated with angiotensin ii levels in patients with primary and secondary hypertension hypertension in high school students: genetic and environmental factors: the hygef study acute and chronic kidney disease, hypertension, and cardiovascular disease: report of a scientific workshop organized by the national kidney foundation can treatments for hypertension be repurposed for the treatment of dementia? comparative safety of drugs targeting the nitric oxide pathway in pulmonary hypertension: a mixed approach combining a meta-analysis of clinical trials and a disproportionality analysis from the world health organization pharmacovigilance database improving hypertension outcome measurement in low-and middle-income countries new drug repurposing opportunities for hypertension uncovered by the convergence of genomics with kidney transcriptomics and epigenomics drug combination therapy increases successful drug repositioning therapeutic drug monitoring in ibd: the new standard-of-care for anti-tnf therapy off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia mek inhibitors and their potential in the treatment of advanced melanoma: the advantages of combination therapy synthesis of polyphosphazene and preparation of microspheres from polyphosphazene blends with pmma for drug combination therapy trader as a new optimization algorithm predicts drug-target interactions efficiently exploiting machine learning for end-to-end drug discovery and development applications of machine learning in drug discovery and development deep transferable compound representation across domains and tasks for low data drug discovery the advances and challenges of deep learning application in biological big data processing proteinnet: a standardized data set for machine learning of protein structure deepcda: deep cross-domain compoundprotein affinity prediction through lstm and convolutional neural networks current mathematical models for cancer drug discovery rpinbase: an online toolbox to extract features for predicting rna-protein interactions tcm-admepred: a novel strategy for poly-pharmacokinetics prediction of traditional chinese medicine based on single constituent pharmacokinetics, structural similarity, and mathematical modeling a probabilistic approach for collective similarity-based drug--drug interaction prediction design of efficient computational workflows for in silico drug repurposing systematic drug repositioning for a wide range of diseases with integrative analyses of phenotypic and molecular data network-based approach to prediction and population-based validation of in silico drug repurposing drug repurposing: progress, challenges and recommendations masoudi-nejad a. mrna--mirna bipartite network reconstruction to predict prognostic module biomarkers in colorectal cancer stage differentiation measuring phenotype-phenotype similarity through the interactome hypergraph-based connectivity measures for signaling pathway topologies -way networks: application of hypergraphs for modelling increased complexity in comparative genomics literature-based discovery of new candidates for drug repurposing a review on naive baye's (nb), j and k-means based mining algorithms for medical data mining recent advances and emerging applications in text and data mining for biomedical discovery early anomaly detection in smart home: a causal association rule-based approach computational prediction of drug-target interactions via ensemble learning computational model development of drug-target interaction prediction: a review prediction of drug-disease associations based on ensemble meta paths and singular value decomposition laying in silico pipelines for drug repositioning: a paradigm in ensemble analysis for neurodegenerative diseases featureselect: a software for feature selection based on machine learning approaches detection of novel biomarkers for early detection of nonmuscle-invasive bladder cancer using competing endogenous rna network analysis discrete symbiotic organisms search algorithm for travelling salesman problem forest optimization algorithm world competitive contests (wcc) algorithm: a novel intelligent optimization algorithm for biological and non-biological problems cuckoo optimization algorithm hybrid multi-objective evolutionary algorithm based on search manager framework for big data optimization problems tuning multi-objective evolutionary algorithms on different sized problem sets prognostic value of ct radiomic features in resectable pancreatic ductal adenocarcinoma pharmacological management of stable angina pectoris aims to relieve symptoms and maximize survival beta-adrenergic receptor blockers in hypertension: alive and well hypertensive crisis in children and adolescents potential protective role of blood pressure-lowering drugs on the balance between hemostasis and fibrinolysis in hypertensive patients at rest and during exercise therapeutic effect of nifedipine combined with enalapril on elderly patients with coronary heart disease complicated with hypertension drugs for systemic hypertension and angina early reversal cardiac with esmolol in hypertensive rats: the role of subcellular organelle phenotype intolerable side effects during propranolol therapy for infantile hemangioma: frequency, risk factors and management drugbank . : a major update to the drugbank database otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro high-dose rifampicin, moxifloxacin, and sq for treating tuberculosis: a multi-arm, multi-stage randomised controlled trial rare genetic diseases: update on diagnosis, treatment and online resources recovery dynamics of multifocal pupillographic objective perimetry from tropicamide dilation effect of topical \% pilocarpine on the ocular tear film ph attenuation of hypertensive response with esmolol and labetalol in low doses in orotracheal intubation: a comparative study synergistic interaction between a pde inhibitor (sildenafil) and a new adenosine a a receptor agonist (lassbio- ) improves pulmonary hypertension in rats drugr+: a comprehensive relational database for drug repurposing, combination therapy, and replacement therapy drugbank: a knowledgebase for drugs, drug actions and drug targets kyoto encyclopedia of genes and genomes drug databases and their contributions to drug repurposing predicting inhibitory and activatory drug targets by chemically and genetically perturbed transcriptome signatures publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to appreciate iranian national science founding (insf) for their supports. calculating drug-drug similarity score equation ( ) was employed to calculate the similarity score between drugs [ ] :where d i , d j , n, c i,r , and c j,r are the i th drug, the j th drug, the total number of the chemical components, the total number of the r th chemical component in i th , and the total number of the r th chemical component in the j th chemical component, respectively. w r , which is calculated by eq. ( ) , is the weight of the r th chemical component.where d r shows the data frequency of the r th chemical component. the changes can be accepted, provided that the new score is better than the previous one. in contrast, the changes will be ignored, if the previous values are retrieved from the memory authors' contributions yms: conceptualization, implementation, formal analysis, investigation, writing, editing, and revising the manuscript. am: conceptualization, supervision, project administration, writing, editing, and revising the manuscript. all authors have read and approved the manuscript. not applicable. all the source codes are available in the following link: https://github.com/lbbsoft/trader. in addition, data are accessible using our developed database drugr+ (http://drugr.ir/). not applicable. not applicable.competing interests not applicable.received: april accepted: july key: cord- -w jgw p authors: guharoy, roy title: medication shortages: a matter of national security—time for action date: - - journal: mayo clin proc doi: . /j.mayocp. . . sha: doc_id: cord_uid: w jgw p nan to the editor: as noted by choo and rajkumar in the june issue of mayo clinic proceedings, the covid- (coronavirus disease ) pandemic has exposed extreme vulnerabilities in our nation's drug supply chain. the fragility of the drug supply chain was not created overnight and has been brewing for over a decade. frequent generic drug shortages and quality deficiencies are compromising standard of care, producing waste, and increasing costs. for example, antibiotic shortages contribute to resistance, when clinicians are forced to use broad-spectrum agents. today, % of active product ingredient (api) manufacturers are located overseas, with the majority being obtained from india and china. india, a large manufacturer of finished products, obtains % of apis from china. china has a virtual monopoly on the apis required to make critical drugs such as antibiotics, antihypertensives, and many others. although the united states remains a global leader in drug discovery, we have almost completely transitioned manufacturing abilities to overseas because of lower production and labor costs. the united states' overreliance on importing apis from overseas has created a devastating impact on our public health with a potential for catastrophic events in the event of a war, trade conflict, or pandemic such as covid- and impacting our national security. as expected, the united states is waiting in line with every other country for essential drugs during the current pandemic. although there are no easy or fast solutions to this problem, we must act now. in addition to choo and rajkumar's recommendations, the following steps may be considered with the ultimate goal of domestic manufacturing of most of the essential lifesaving drugs to protect our patients from harm. d designate the pharmaceutical industry as a high-priority infrastructure critical to national security (like the aviation and energy sectors) to allow the federal government to coordinate efforts during shortages or national crises. d develop an essential national security drug supply list that includes medications for which a supply interruption could cause an immediate risk to public health. d create a national agency to monitor, plan, and identify potential manufacturers in the event of a shortage. d manufacture products in multiple locations to protect the supply chain in the event of a natural disaster or other threats. d strengthen federal oversight of manufacturers to ensure the highest product integrity and require manufacturers to provide transparency related to source of apis and quality issues. d develop a partnership between the private and public sectors acting as a united cohort to uncover incentives to drive actions to ensure domestic production of essential drugs. d require manufacturers to report to the us food and drug administration (fda) immediately on discovery of an interruption, disclosing the reason and the expected time to resolution. d create a national database for tracking of essential drug supplies and use predictive analytics to identify surge, production problems, and future shortages. editor's note: when publishing a letter that comments on an article published previously in mayo clinic proceedings, it is the journal's policy to invite the author(s) of the referenced article to publish a response. drs choo and rajkumar were fully supportive of the letter by guharoy and did not feel any response from them was necessary. to the editor: we read the recent commentary medication shortages during the covid- crisis by drs choo and rajkumar with great interest. as supply chain management leaders at mayo clinic, we appreciate the attention these authors draw towards the issue of drug shortages and drug costs. one of the interesting observations we have made from the supply chain perspective while navigating the coronavirus disease (covid- ) pandemic is that the impacts on drug cost from covid- have been minimal in the retail segment and are more significant in the hospital sector. this phenomenon is a result of costly inpatient care for covid- and there are currently no effective commercially available drug therapies that are being routinely used in the ambulatory setting. due to the high cost of covid- inpatient care within health care, we project that the cost containment recommendations of medicare price negotiations and controls of deductibles/rebates suggested by drs choo and kumar will have minimal impact on drug costs at this time (there could be a benefit if new therapeutic agents emerge in the future or if brand name drugs are used for covid- patients). one potentially highly impactful and easy to implement public policy solution we call attention to is the need to remedy unintended effects of the drug efficacy study implementation and unapproved drugs initiative programs. these programs were implemented to provide review of drugs that have been available for decades and that had not completed formal food and drug administration review. because of these programs and abuses by manufacturers, dramatic escalation of cost and expense for certain older drugs that were once generic continue to occur. , the most egregious example in the hospital sector over the past decade is the dramatic rise in the cost of vasopressin, a drug that has been around for over years and that is now among the top drugs expenditures in hospitals as of and has patent protection until . because of the high use of vasopressin in critically ill covid- patients and the greater than % price increase that has occurred after completing the unapproved drugs initiative process, vasopressin will likely become a top drug expense within the hospital sector by the end of . inflation for vasopressin since undergoing approval through the unapproved drugs initiative has been and continues to be dramatic. as result of covid- , the manufacturer parr pharmaceuticals will experience further massive windfall profits and inflict billions of dollars of unreasonable costs for managing critically ill patients over the next decade. policy adjustments that address unintended consequences of the drug efficacy study implementation and unapproved drugs initiative programs could prevent further economic harm related to vasopressin pricing and avoid similar abuses in the future. medication shortages during the covid- crisis: what we must do treatment considerations for covid- : a critical review of the evidence (or lack thereof) the fda unapproved drugs initiative: an observational study of the consequences for drug prices and shortages in the united states estimating the impact of food and drug administration's unapproved drug initiative on drug prices and sales national trends in prescription drug expenditures and projections for mayo clinic proceedings key: cord- -j zyodw authors: zeng, xiangxiang; song, xiang; ma, tengfei; pan, xiaoqin; zhou, yadi; hou, yuan; zhang, zheng; li, kenli; karypis, george; cheng, feixiong title: repurpose open data to discover therapeutics for covid- using deep learning date: - - journal: j proteome res doi: . /acs.jproteome. c sha: doc_id: cord_uid: j zyodw [image: see text] there have been more than . million confirmed cases and over deaths from the human coronavirus disease (covid- ) pandemic, caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ), in the united states alone. however, there is currently a lack of proven effective medications against covid- . drug repurposing offers a promising route for the development of prevention and treatment strategies for covid- . this study reports an integrative, network-based deep-learning methodology to identify repurposable drugs for covid- (termed cov-kge). specifically, we built a comprehensive knowledge graph that includes million edges across types of relationships connecting drugs, diseases, proteins/genes, pathways, and expression from a large scientific corpus of million pubmed publications. using amazon’s aws computing resources and a network-based, deep-learning framework, we identified repurposable drugs (including dexamethasone, indomethacin, niclosamide, and toremifene) whose therapeutic associations with covid- were validated by transcriptomic and proteomics data in sars-cov- -infected human cells and data from ongoing clinical trials. whereas this study by no means recommends specific drugs, it demonstrates a powerful deep-learning methodology to prioritize existing drugs for further investigation, which holds the potential to accelerate therapeutic development for covid- . as of june , , in the united states alone, more than . million cases and over deaths from coronavirus disease (covid- ) , the disease caused by the virus sars-cov- , have been confirmed. however, there are currently no proven effective antiviral medications against covid- . there is an urgent need for the development of effective treatment strategies for covid- . it was estimated that in , pharmaceutical companies spent $ . billion for the development of an fda-approved new chemical entity drugs using traditional de novo drug discovery. drug repurposing, a drug-discovery strategy using existing drugs, offers a promising route for the development of prevention and treatment strategies for covid- . in a randomized, controlled, open-label trial, lopinavir and ritonavir combination therapy did not show a clinical benefit compared with standard care for hospitalized adult patients with severe covid- , limiting the traditional antiviral treatment for covid- . sars-cov- replication and infection depend on the host cellular factors (including angiotensin-converting enzyme (ace )) for entry into cells. the systematic identification of virus−host protein− protein interactions (ppis) offers an effective way toward the elucidation of the mechanisms of viral infection; furthermore, targeting the cellular virus−host interactome offers a promising strategy for the development of effective drug repurposing for covid- , as demonstrated in previous studies. − we recently demonstrated that network-based methodologies leveraging the relationship between drug targets and diseases can serve as a useful tool for the efficient screening of potentially new indications of fda-approved drugs with wellestablished pharmacokinetic/pharmacodynamic, safety, and tolerability profiles. − deep learning has also recently demonstrated its better performance than classic machine learning methods to assist drug repurposing, − yet without foreknowledge of the complex networks connecting drugs, targets, sars-cov- , and diseases, the development of affordable approaches for the effective treatment of covid- is challenging. prior knowledge of networks from the large scientific corpus of publications offers a deep biological perspective for capturing the relationships between drugs, genes, and diseases (including covid- ), yet extracting connections from a large-scale repository of structured medical information is challenging. in this study, we present the state-of-the-art knowledge-graph-based, deep-learning methodologies for the rapid discovery of drug candidates to treat covid- from million pubmed publications ( figure ). via systematic validation using transcriptomics and proteomics data generated from sars-cov- -infected human cells and the ongoing clinical trial data, we successfully identified drug candidates that can be further tested in large-scale randomized control trials for the potential treatment of covid- . specifically, a comprehensive knowledge graph that contains million edges across types of relationships connecting drugs, diseases, genes, pathways, expressions, and others by incorporating data from million pubmed publications and drugbank (table s ) . subsequently, a deeplearning approach (rotate in dgl-ke) was used to prioritize high-confidence candidate drugs for covid- under amazon supercomputing resources (cf. methods and materials). finally, all cov-kge predicted drug candidates were future-validated by three gene expression data sets in sars-cov- -infected human cells and one proteomics data set in sars-cov- infected human cells. tables s and s ). in this kg, we represent the coronaviruses (covs) by assembling multiple types of known covs, including sars-cov- and mers-cov, as described in our recent study. we next utilized dgl-ke's knowledge graph embedding (kge) model, rotate, to learn representations of the entities (e.g., drugs and targets) and relationships (e.g., inhibition relation between drugs and targets) in an informative, lowdimensional vector space. in this space, each relationship type (e.g., antagonists or agonists) is defined as a rotation from the source entity (e.g., hydroxychloroquine) to the target entity (e.g., toll-like receptor / (tlr / )). in this study, we constructed a comprehensive kg from global network of biomedical relationships (gnbr) and drugbank. first, from gnbr, we included in the kg relations corresponding to drug−gene interactions, gene−gene interactions, drug−disease associations, and gene−disease associations. second, from the drugbank database, we selected the drugs whose molecular mass is > da and also exist in gnbr, resulting in fda-approved and clinically investigational drugs. for these drugs, we included in the kg relationships corresponding to the drug−drug interactions and the drug side-effects, drug anatomical therapeutic chemical (atc) codes, drug mechanisms of action, drug pharmacodynamics, and drug-toxicity associations. third, we included the experimentally discovered cov−gene relationships from our recent work in the kg. fourth, we treated the covid- context by assembling known genes/ proteins associated with covs (including sars-cov and mers-cov) as a comprehensive node of covs and rewired the connections (edges) from genes and drugs. the resulting kg contains four types of entities (drug, gene, disease, and drug side information), types of relationships (table s ) , nodes, and edges (table s ) . models for computing kges learn vectors for each of the entities and each of the relation types so that they satisfy certain properties. in our work, we learned these vectors using the rotate model. given an edge in the kg represented by the triplet (head entity, relation type, and tail entity), rotate defines each relation type as a rotation from the head entity to the tail entity in the complex vector space. specifically, if h and t are the vectors corresponding to the head and tail entities, respectively, and r is the vector corresponding to the relation type, then rotate tries to minimize the distance where ⊗ denotes the hadamard (element-wise) product. to minimize the distance between the head and the tail entities of the existing triplets (positive examples) and maximize the distance among the nonexisting triplets (negative examples), we use the loss function where σ is sigmoid function, γ is a margin hyperparameter with γ > , (h i , r, t i ) is a negative triplet, and p(h i , r, t i ) is the probability of occurrence of the corresponding negative sample. dgl-ke is a high-performance, easy-to-use, and scalable package for learning large-scale kges with a set of popular models including transe, distmult, complex, and rotate. it includes various optimizations that accelerate training on kgs with millions of nodes and billions of edges using multiprocessing, multi-gpu (graphics processor unit), and distributed parallelism. dgl-ke is able to compute the rotatebased embeddings of our kg in ∼ mins on an ec instance with gpus under amazon's aws computing resources. we divide the triplets (e.g., a relationship among drug, treatment, and disease) into a training set, validation set, and test set in a : : manner. we selected the embedding dimensionality of dim = for nodes and relations. the rotate is trained for epochs with a batch size and . as the learning rate. we choose γ = as the margin of the optimization function. gene set enrichment analysis was performed to further validate the predicted drug candidates from cov-kge. the goal of the gene set enrichment analysis was to identify drugs that can reverse the cellular changes (transcriptome or proteome levels) that result from virus infection. four differential expression data sets were collected, including two transcriptome data sets from sars-cov patients' peripheral blood (gse ) and calu- cells (gse ), one transcriptome data set of calu- cells infected by mers-cov (gse ), and one proteome data set of human caco- cells infected with sars-cov- . these four data sets were used as the gene signatures for the viral infections. for the drugs, we retrieved the connectivity map (cmap) database journal of proteome research pubs.acs.org/jpr article where j = , , ..., s are the genes from the cov signature data set sorted in ascending order using the gene profiles of the drug being computed. v(j) denotes the rank of j, where ≤ v(j) ≤ r, with r being the total number of genes ( ) from the cmap database. next, es up/down is set to a up/down if a up/down > b up/down and is set to −b up/down if b up/down > a up/down . permutation tests are repeated times to quantify the significance of the es score. in each repeat, the same number of up-and down-expressed genes as the cov signature data set was randomly generated. es > and p < . are considered significantly enriched. the number of significantly enriched data sets is used as the final result for a certain drug. we introduced the area under the receiver operating characteristic (roc) curve (auroc) and several evaluation metrics for evaluating the performance of drug−target interaction prediction. the auroc is the global prediction performance. the roc curve is obtained by calculating the true-positive rate (tpr) and the false-positive rate (fpr) via varying cutoffs. ■ results after mapping drugs, covs, and the treatment relationships to a complex vector space using rotate, the top most relevant drugs were selected as candidates for covs in the treatment relation space ( figure s ). using the ongoing covid- trial data (https://covid -trials.com/) as a validation set, cov-kge has a larger auroc (auroc = . , figure ) for identifying repurposable drugs for covid- . we next employ t-sne (t-distributed stochastic neighbor embedding algorithm ) to further investigate the lowdimensional node representation learned by cov-kge. specifically, we projected drugs grouped by the first level of the anatomical therapeutic chemical (atc) classification systems code onto a d space. figure a indicates that cov-kge is able to distinguish types of drugs grouped by atc codes, which is consistent with a high auroc value of . ( figure ) . we further validated the top candidate drugs using an enrichment analysis of drug−gene signatures and sars-covinduced transcriptomics and proteomics data in human cell lines (cf. methods and materials). specifically, we analyzed three transcriptomic data sets in sars-cov- -infected human cell lines and one proteomics data set in sars-cov- -infected human cell lines. in total, we obtained repositioned drug candidates (table ) using subject-matter expertise based on a combination of factors: (i) the strength of the cov-kge predicted score, (ii) the availability of clinical evidence from ongoing covid- trials, and (iii) the availability and strength of enrichment analyses from sars-cov- / -affected human cell lines. among the candidate drugs, drugs are or have been under clinical trials for covid- , including thalidomide, methylprednisolone, ribavirin, umifenovir, tetrandrine, suramin, dexamethasone, lopinavir, and azithromycin ( figure a and table ). we excluded chloroquine and hydroxychloroquine from our ongoing clinical trial list based on recently controversial reports. , discovery of drug candidates for covid- using cov-kge we next turned to highlight three types of predicted drugs for covid- , including anti-inflammatory agents (dexamethasone, indomethacin, and melatonin), selective estrogen receptor modulators (serms), and antiparasitics ( figure ) . anti-inflammatory agents. given the well-described lung pathophysiological characteristics and immune responses (cytokine storms) of severe covid- patients, drugs that dampen the immune responses may offer effective treatment approaches for covid- . , as shown in figure a , we computationally identified multiple anti-inflammatory agents for covid- , including dexamethasone, indomethacin, and melatonin. indomethacin, an approved cyclooxygenase (cox) inhibitor, has been widely used for its potent anti-inflammatory and analgesic properties. indomethacin has been reported to have antiviral properties, including sars-cov- and sars-cov- . importantly, a preliminary in vivo observation showed that oral indomethacin ( mg/kg body weight daily) reduced the recovery time of sars-cov- -infected dogs. melatonin plays a key role in the regulation of the human circadian rhythm that alters the translation of thousands of genes, including melatonin-mediated anti-inflammatory and immune-related effects for covid- . melatonin has various antiviral activities by suppressing multiple inflammatory pathways , (i.e., il and il- β); these inflammatory effects are directly relevant given the well-described lung pathophysiological characteristics of severe covid- patients. melatonin's mechanism of action may also help to explain the epidemiologic observation that children, who have naturally high melatonin levels, are relatively resistant to covid- disease manifestations, whereas older individuals, who have decreasing melatonin levels with age, are a very highrisk population. in addition, exogenous melatonin administration may be of particular benefit to older patients given the aging-related reduction of endogenous melatonin levels and the vulnerability of older individuals to the lethality of sars-cov- . dexamethasone is a u.s. fda-approved glucocorticoid receptor (gr) agonist for a variety of inflammatory and autoimmune conditions, including rheumatoid arthritis, severe allergies, asthma, chronic obstructive lung disease, and others. glucocorticoid medications have been used in patients with mers-cov and sars-cov- infections. as shown in figure a , dexamethasone is the fourth predicted drug among candidates. the randomized evaluation of covid- therapy (recovery, clinicaltrials.gov identifier: nct ) trial showed that dexamethasone reduced mortality by one-third in patients requiring ventilation and by one-fifth in individuals requiring oxygen, yet dexamethasone did not reduce death in covid- patients not receiving respiratory support. selective estrogen receptor modulators. an overexpression of the estrogen receptor has played a crucial role in inhibiting viral replication and infection. several serms, including clomifene, bazedoxifene, and toremifene, are identified as promising candidate drugs for covid- ( figure a and table ). toremifene, the first generation of the nonsteroidal serm, was reported to block various viral infections at low micromolar concentration, including ebola virus, , mres-cov, sars-cov- , and sars-cov- ( figure b ). toremifene prevents fusion between the viral and endosomal membranes by interacting with and destabilizing the virus glycoprotein and eventually blocking replications of the ebola virus. the underlying antiviral mechanisms of sars-cov- and sars-cov- for toremifene remain unclear and are currently being investigated. toremifene has been approved for the treatment of advanced breast cancer and has also been studied in men with prostate cancer (∼ subjects) with reasonable tolerability. toremifene is % bound to plasma protein with good bioavailability and typically orally administered at a dosage of mg. in summary, toremifene is a promising candidate drug with ideal pharmacokinetics properties to be directly tested in covid- clinical trials. antiparasitics. despite the lack of strong clinical evidence, hydroxychloroquine and chloroquine phosphate, two approved antimalarial drugs, were authorized by the u.s. fda for the treatment of covid- patients using emergency use authorizations (euas). in this study, we identified that both hydroxychloroquine and chloroquine are among the predicted candidates for covid- ( figure a and table ). between the two, hydroxychloroquine's in vitro antiviral activity against sars-cov- is stronger than that of chloroquine (hydroxychloroquine: % effective concentration (ec ) = . μm, whereas for chloroquine: ec = . μm). hydroxychloroquine and chloroquine are known to increase the ph of endosomes, which inhibits membrane fusion, a required mechanism for viral entry (including sars-cov- ) into the cell. although chloroquine and hydroxychloroquine are relatively well tolerated, several adverse effects as june , , the national institutes of health halted the clinical trial of hydroxychloroquine owing to the lack of clinical benefits. thus further niclosamide, an fda-approved drug for the treatment of tapeworm infestation, was recently identified to have a stronger inhibitory activity on sars-cov- at the submicromolar level (ic = . μm). gassen et al. showed that niclosamide inhibited skp activity by enhancing autophagy and reducing mers-cov replication as well. altogether, niclosamide may be another drug candidate for covid- , which is warranted to be investigated experimentally and further tested in randomized controlled trials. given the up-regulation of systemic inflammationin some cases, culminating to a cytokine storm observed in severe covid- patients combination therapy with an agent targeting inflammation (melatonin, dexamethasone, or indomethacin) and with direct antiviral effects (toremifene and niclosamide) has the potential to lead to successful treatments ( figure ) . because of the aging-related reduction of endogenous melatonin levels and the vulnerability of older individuals to the lethality of sars-cov- , combining exogenous melatonin administration and antiviral agents (such as toremifene or niclosamide) may be of particular benefit to older patients with covid- . yet all computationally predicted drug candidates (table ) and proposed drug combinations (figure ) must be validated experimentally and be tested in randomized controlled trials. several combination antiviral and anti-inflammatory treatment trials (remdesivir plus baricitinib) are underway for patients with covid- (clinicaltrials.gov identifier: nct ), indicating the proof-of-concept of this combination therapy for covid- . as covid- patients flood hospitals worldwide, physicians are trying to search for effective antiviral therapies to save lives. multiple covid- vaccine trials are underway, yet it might not be physically possible to make enough vaccines for everyone in a short period of time. furthermore, sars-cov- replicates poorly in multiple animals, including dogs, pigs, chickens, and ducks, which limits preclinical animal studies. to fight the emerging covid- pandemic, we introduced an integrative, network-based, deep-learning methodology to discover candidate drugs for covid- , named cov-kge. via cov-kge, we built a comprehensive kg that includes million edges across types of relationships connecting drugs, diseases, proteins/genes, pathways, and expressions from a large scientific corpus of million pubmed publications. using the ongoing covid- trial data as a validation set, we demonstrated that cov-kge had high performance in identifying repurposable drugs for covid- , indicated by the larger auroc (auroc = . ). using amazon's aws computing resources, we identified high-confidence repurposed drug candidates (including dexamethasone, indomethacin, niclosamide, and toremifene) for covid- , which were validated by an enrichment analysis of gene expression and proteomics data in sars-cov- infected human cells. altogether, this study offers a powerful, integrated deep-learning methodology for the rapid identification of repurposable drugs for the potential treatment of covid- . we acknowledge several potential limitations in the current study. potential data noises generated from different experimental approaches in large-scale publications may influence the performance of the current cov-kge models. the original data of gnbr contain the confidence values of the relations between entities. however, we ignored the weights so that we could directly apply the rotate algorithm because we tried to obtain the prediction result in a cheap computing-cost way. in our future work, we will take these confidence values into account and try to design a knowledgegraph-embedding algorithm that can be used for a kg with weighted relationships. the lack of dose-dependent profiles and the biological perturbation of sars-cov- virus−host interactions may generate a coupled interplay between adverse and therapeutic effects. the integration of pharmacokinetics data from animal models and clinical trials into our cov-kge methodology could establish the causal mechanism and patient evidence through which predicted drugs would have high clinical benefits for covid- patients without obvious adverse effects in a specific dosage. in summary, we presented cov-kge, a powerful, integrated ai methodology that can be used to quickly identify drugs that can be repurposed for the potential treatment of covid- . our approach can minimize the translational gap between preclinical testing results and clinical outcomes, which is a significant problem in the rapid development of efficient treatment strategies for the covid- pandemic. from a translational perspective, if broadly applied, the network tools developed here could help develop effective treatment strategies for other emerging infectious diseases and other emerging complex diseases as well. however, all predicted drugs not used in clinical trials must be tested in randomized clinical trials before being used in covid- patients. table . details of the five categories of relationships in our kg. supplementary table . statistics of nodes (entity) and edges (relation) in our kg (pdf) an interactive web-based dashboard to track covid- in real time pharmacologic treatments for coronavirus disease (covid- ): a review the $ . billion pill-methodologic and policy considerations coronavirus puts drug repurposing on the fast track sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor systems biology-based investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis network-based drug repurposing for novel coronavirus -ncov/ sars-cov- network-based approach to prediction and population-based validation of in silico drug repurposing a genome-wide positioning systems network algorithm for in silico drug repurposing c.; et al. a deep learning approach to antibiotic discovery deepdr: a network-based deep learning approach to in silico drug repositioning predicting drug− protein interaction using quasi-visual question answering system target identification among known drugs by deep learning from heterogeneous networks deep graph library: towards efficient and scalable deep learning on graphs a global network of biomedical relationships derived from text knowledge graph embedding by relational rotation in complex space expression profile of immune response genes in patients with severe acute respiratory syndrome cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target proteomics of sars-cov- -infected host cells reveals therapy targets the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease discovery and preclinical validation of drug indications using compendia of public gene expression data visualizing data using t-sne covid- ) update: fda revokes emergency use authorization for chloroquine and hydroxychloroquine halts clinical trial of hydroxychloroquine clinical and immunologic features in severe and moderate coronavirus disease sars-cov- infects t lymphocytes through its spike protein-mediated membrane fusion indomethacin has a potent antiviral activity against sars coronavirus as adjuvant treatment for coronavirus disease pneumonia patients requiring hospitalization (mac- pro): a case series beneficial actions of melatonin in the management of viral infections: a new use for this ″molecular handyman″? corticosteroids: mechanisms of action in health and disease impact of corticosteroid therapy on outcomes of persons with sars-cov- , sars-cov, or mers-cov infection: a systematic review and meta-analysis dexamethasone in hospitalized patients with covid- -preliminary report a structure-informed atlas of human-virus interactions toremifene interacts with and destabilizes the ebola virus glycoprotein mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection infection and rapid transmission of sars-cov- in ferrets toremifene, a new antiestrogenic compound, for treatment of advanced breast cancer. phase ii study prostate cancer and prostatic intraepithelial neoplasia: true, true, and unrelated? effect of toremifene on clinical chemistry, hematology and hormone levels at different doses in healthy postmenopausal volunteers: phase i study hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/azithromycin fatal toxicity of chloroquine or hydroxychloroquine with metformin in mice susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus key: cord- -n n ii k authors: singh, thakur uttam; parida, subhashree; lingaraju, madhu cholenahalli; kesavan, manickam; kumar, dinesh; singh, raj kumar title: drug repurposing approach to fight covid- date: - - journal: pharmacol rep doi: . /s - - - sha: doc_id: cord_uid: n n ii k currently, there are no treatment options available for the deadly contagious disease, coronavirus disease (covid- ). drug repurposing is a process of identifying new uses for approved or investigational drugs and it is considered as a very effective strategy for drug discovery as it involves less time and cost to find a therapeutic agent in comparison to the de novo drug discovery process. the present review will focus on the repurposing efficacy of the currently used drugs against covid- and their mechanisms of action, pharmacokinetics, dosing, safety, and their future perspective. relevant articles with experimental studies conducted in-silico, in-vitro, in-vivo, clinical trials in humans, case reports, and news archives were selected for the review. number of drugs such as remdesivir, favipiravir, ribavirin, lopinavir, ritonavir, darunavir, arbidol, chloroquine, hydroxychloroquine, tocilizumab and interferons have shown inhibitory effects against the sars-cov in-vitro as well as in clinical conditions. these drugs either act through virus-related targets such as rna genome, polypeptide packing and uptake pathways or target host-related pathways involving angiotensin-converting enzyme- (ace ) receptors and inflammatory pathways. using the basic knowledge of viral pathogenesis and pharmacodynamics of drugs as well as using computational tools, many drugs are currently in pipeline to be repurposed. in the current scenario, repositioning of the drugs could be considered the new avenue for the treatment of covid- . drug repurposing is the process to identify the new indications for existing drugs and considered as an efficient and economical approach [ ] . it is also known as repositioning, re-profiling, re-tasking and rescue of drugs [ ] . it has been considered that % of known drugs could be repositioned for various diseases [ ] . outbreaks of novel emerging infections such as coronavirus disease (covid- ) have unique challenges in front of the health professionals to select appropriate therapeutics/pharmacological treatments in the clinical setup with very little time available for the new drug discovery [ ] . further, development of a vaccine for any disease including covid- takes time and even if the process is put on accelerated mode it would take - months to introduce it as ready-to-use product. currently, no specific treatment is available against the new virus severe acute respiratory syndrome coronavirus (sars-cov ). hence, the search for effective therapeutic agents to tackle covid- is vital and urgent. the discovery and licensed use of a drug come with a long-gestation period. the cost of the new drug development process amounts to more than a billion dollars extending for a period of - years [ ] with the success rate of only . % [ ] . this creates a lag in the productivity of pharmaceutical research to develop a new drug which results in a persistent gap between therapeutic needs and available treatments [ ] . considering the time and cost required for coming up with new therapies, probing the existing antiviral and other drugs against sars-cov is cost-effective. in recent times, repurposing of available drugs for the management of several disease conditions is increasingly becoming a popular strategy as it uses de-risked compounds with known preclinical, pharmacokinetic, pharmacodynamic profiles which can directly enter phase iii or iv clinical trial making the drug development process potentially a low-cost and relatively rapid [ ] . therefore, reassessing the efficacy of licensed and experimental drugs has become go to choice of world health organization (who) and other health agencies to treat emerging health problems. drug repurposing follows mainly two concepts. one is that a single drug interacts with multiple targets, which paves the way for searching new target sites of action for the known compound [ , ] . the other concept is that targets associated with a disease are often relevant to a number of biological processes of pathogenesis [ , ] which paves the way for designation of a new indication for the known target. notionally, a drug that acts on these common elements can, in principle, be useful for several disorders. broadly, there are three kinds of approaches which are widely used in drug repositioning: computational approaches, biological experimental approaches, and mixed approaches. data such as gene expression, drug-target interactions, protein networks, electronic health records, clinical trial reports, and drug adverse event reports has become accessible in standardized forms. the repository of knowledge and omics data available in pharmaceutical research leads to the rise of some computational methods which are novel and exciting in the field of drug repositioning. these computational methods are capable to make a high-level integration of all the knowledge and data and help in understanding the new signaling pathways and generate novel insights into drug mechanisms, side effects, and interactions which further speed up drug discovery. a recent study presented integrative network-based systems pharmacology, methodology which quantifies the interplay between the coronavirus-human cell interactome and drug targets in the human protein-protein interaction network which help in rapid identification of repurposable drugs against sars-cov . a study using this kind of approach was able to identify potential repurposable drugs against covid- [ , ] . the sars-cov mainly spreads through the respiratory tract [ ] and affects alveolar cells. unlike, middle east respiratory syndrome coronavirus (mers-cov) which employs dipeptidyl peptidase (dpp ) [ ] , sars-cov and sars-cov adopt angiotensin-converting enzyme (ace ) as receptor for entering the cell. attachment, fusion and entry of the virus are aided by spike protein which makes it an interesting target for the development of antibodies, entry inhibitors and vaccines [ ] . anti-viral agents including ritonavir, lopinavir, ribavirin, interferons have been used against sars-cov and mers-cov clinically, however, clinical data is still limited [ ] . currently, with the lack of effective agents against sars-cov as well as public-health emergency, who has identified some therapies which doctors and researchers believe are the most promising, such as a combination of two hiv drugs (lopinavir and ritonavir), anti-malarial drugs (chloroquine and hydroxychloroquine), and an experimental antiviral compound remdesivir. further, many available drugs with the intention of repurposing against covid- have been subjected to clinical trials [ ] ( table ) . however, the search for other agents cannot pause while waiting for the results because the demand for new effective agents is huge. human population of the twenty-first century is suffering a large-scale epidemic of highly pathogenic coronaviruses such as severe acute respiratory syndrome coronavirus (sars-cov) in - [ ] , middle east respiratory syndrome coronavirus (mers-cov) in [ ] and severe acute respiratory syndrome coronavirus (sars-cov ) in - . coronaviruses are pleomorphic, enveloped, positive sense, - kb large rna viruses with typical crown-shape glycoprotein spikes (peplomers) that cause respiratory and enteric diseases in humans and other animals [ ] . very high recombination rates lead to constant transcription errors and rna-dependent rna polymerase (rdrp) jumps in coronaviruses [ ] give them a chance to develop into diverse zoonotic pathogens such as sars-cov . sars-cov is a positive-sense single-stranded rna virus classified as a strain of the species sars-cov, (genus beta-coronavirus; subgenus sarbecoronavirus; subfamily orthocoronavirinae; family coronaviridae) which earlier in - caused an outbreak of sars in guangdong, china [ ] . overall genome sequence of sars-cov showed . % similarity with sars-cov and interestingly, . % similarity with bat coronavirus ratg , suggesting its origin from a bat virus [ , ] . though not certain, yet it is suspected that the introduction of this new coronavirus to humans might have been facilitated by an intermediate host, the pangolins [ ] . as per who report, a case of pneumonia of unknown causative pathology from wuhan city of china was first time reported to the who country office on december . the outbreak of the coronavirus-associated acute respiratory disease throughout the world was named as covid- and it was classified as -ncov (sars-cov ) [ ] . the classification and naming of the virus were done by the coronaviridae study group of the international committee on taxonomy of viruses. novel coronavirus genome sequencing by china was publicly available on the global in the current scenario of lacking efficient and specific treatments for sars-cov infections and the urgent need to restrain the pandemic, drug re-tasking appears to be the most suitable tool to find out the best therapeutic option for covid- [ ] . the mechanisms of infection by the sars-cov are not clear yet, however, it is genetically similar to sars-cov and other coronaviruses [ ] . hence the common mode of pathogenicity is being explored for targets of treatment. the therapeutic targets depending on the common mode of pathogenicity can be classified as virus-related targets and host-related targets. replication of sars-cov depends directly on the key enzyme rdrp. it is interesting to note that potential drug targets protease and polymerase for sars-cov and sars-cov are highly conserved with % and % overall identity. therefore, blockers developed against the sars may act as good therapeutic candidates to bind protease or polymerase sites against sars-cov (gisaid, ) ( fig. ). on the basis of current knowledge about the use of remdesivir in sars-cov infection, remdesivir is considered as the potential drug candidate for repurposing against the covid- . remdesivir (gs- ) is a nucleoside analog originally developed against ebola viruses by gilead sciences inc, a usa based biopharmaceutical company ( table ) . though the drug failed to show effect against ebola, the preliminary results from in-vitro and in-vivo preclinical studies as well as case reports indicate its efficacy against sars-cov . recently, in vero e cells, remdesivir has been shown to block the viral infection at low concentration (ec = . μm) with high selectivity index (si > . ). the ec value ( . µm) was also low enough to be achieved in non-human primate models [ ] . it also efficiently inhibited sars-cov infection in human liver cancer huh- cells [ ] . in rhesus macaques, the drug showed therapeutic and prophylactic efficacy against both sars and mers coronaviruses, indicating its potential against diverse coronaviruses including sars-cov [ ] . similarly, in a ces c-knockout mouse model of sars-cov infection, remdesivir in both prophylactic and therapeutic use significantly reduced lung viral titers, though the survival and lung pathology were not improved [ ] . treatment with intravenous remdesivir on day of hospital admission, clearly improved the clinical condition from the next day in a -year-old covid- patient in washington, usa [ ] . in italy, remdesivir has been used ( mg every h as a loading dose followed mg every h for days thorough intravenous route) with other supportive therapy in the clinical management of covid- patients presented with a range of modified early warning score (mews) from less than to more than [ ] . in a recent report, clinical improvement in of patients ( %) of severe covid- patients has been observed with the compassionate-use of remdesivir in a cohort study [ ] . according to another study remdesivir was superior over the placebo in shortening the time to recovery in adult patients hospitalized with covid- . in the trial that was conducted at sites and subsites in different parts of the world, mortality rate was . % with remdesivir and . % with placebo by days in patients in which were in remdesivir group and were in the placebo group [ ] . another trial, however, suggests differently, that remdesivir has no such statistically significant clinical benefits, although, patients who were administered with remdesivir showed a faster clinical improvement in numerical terms, with symptom duration of days or less in a study of patients in which patients received remdesivir and (one withdrew) were on placebo in a randomised, double-blind, placebocontrolled, multicentre trial at ten hospitals in hubei, china [ ] . another clinical trial done with remdesivir in severe covid- patients without mechanical ventilation support indicated no significant difference between -day and -day course of remdesivir therapy, however, by day , a clinical improvement was noticed in % of patients in the -day group and in % in the -day group at points or more on the ordinal scale out of -point ordinal scale [ ] . being a nucleotide (adenosine) analog, remdesivir gets incorporated into the replicating genome of the virus after being converted into its triphosphate form. the triphosphate forms compete with adenosine triphosphate (atp) to act as a substrate of rdrp and have been found to cause significantly efficient incorporation as compared to atp. remdesivir adds three more nucleotides before terminating the growing rna chain [ ] . the extra three nucleotides may protect the inhibitor from removal by the viral ′- ′ exonuclease activity [ ] , contributing to the lack of acquiring resistance. the pharmacokinetics, metabolism and distribution of remdesivir have been studied in non-human primates, previously. in rhesus monkeys, mg/kg dose yields a half life (t / ) of . h with fast systemic elimination. the active metabolite of remdesivir, ′-cyano-substituted adenine c-nucleoside ribose analogue (nuc) then appears which produces antiviral activity [ ] . however, the drug is parenterally administered through intravenous route and is not expected to be given to mildly infected patients, though potent action is expected in mild infection. it is under clinical trial in china and usa against covid- by th of april, (nct and nct ). however, seven clinical trials are underway on remdesivir to evaluate its efficacy on sars-cov infection (https ://www.gilea d.com/purpo se/advan cing-globa l-healt h/covid - /remde sivir -clini cal-trial s). the results of nct clinical trial showed that intravenous remdesivir was adequately tolerated by covid- patients [ ] . in the third trial (nct ) with the severely infected covid- patients, with the use of remdesivir, common adverse effects were observed such as nausea, acute respiratory failure, increased alanine aminotransferase (alt) and constipation [ ] . in all these studies ramdesivir was administered intravenously as mg sid on day and followed by mg sid for the subsequent period. virus enters the cell by fusion of the viral spike proteins with cellular ace receptor, followed by ace downregulation. angiotensin receptor blockers (arbs), angiotensin-converting enzyme inhibitors (aceis) and statins increase ace expression, hence may have efficacy in this condition. . fusion is followed by endocytosis of the virus, where low endosomal ph helps in lysis of the viral structural proteins. disruption of this acidic environment by diprotic bases like chloroquine and hydroxychloroquine may produce an antiviral effect. , . release of nucleic acid (na) into the cytoplasm and translation of the viral proteins using host ribosomes, . proteolysis by viral main protease enzyme makes functional proteins e.g. rna dependent rna polymerase (rdrp). thus, inhibition of the main protease enzyme by inhibitors like lopinavir, ritonavir and darunavir, may have efficacy against the virus. . rdrp is essential for replication and transcription of the virus. rdrp inhibitors, remdesivir, favipiravir, ribavirin and arbidol may be effective against coronaviruses. , . subsequent translation and proteolysis into structural proteins followed by packaging makes intact virions, which get exocytosed ( ) from the cell as per the information obtained from the chinese news channel by dong and co-workers, this drug got an approval against the sars-cov infected patients in china on th february, since it had drastically attenuated the illness of the sars-cov infected patients [ ] . favipiravir ( -fluoro- -hydroxy- -pyrazinecarboxamide, t- also table chemical structure of pharmacological agents reviewed marketed as avigan) was developed by a japanese firm, toyama chemical co., ltd. and has been approved in japan for influenza since ( table ). the drug effectively inhibits the enzyme rdrp. in vero e cells this drug showed an ec of . µm and selectivity index of more than . against sars-cov [ ] . though this concentration against sars-cov is very high, the drug may be tested in an animal model based on its performance against ebola virus. remarkably this drug had shown % efficacy against ebola in a mice model although it had a very high ec in vero cells [ ] . randomized trials are being conducted for this drug in combination with other drugs against coronaviruses (favipiravir + interferon-α, chictr , favipiravir + baloxavir, chictr ). favipiravir is a nucleoside precursor which inhibits the broad range of influenza virus strains, however, it shows antiviral activity through its ntp form (converted into an active phosphoribosylated form, t- rtp) via direct inhibition of the rdrp activity of influenza a virus polymerase and it has also shown lethal mutations within the viral genome [ ] . nevertheless, the exact mode of action, precise molecular mechanism and its interaction between the nucleotide and the viral polymerase could be investigated in the sars-cov infection. favipiravir exerts its antiviral action in a dose-dependent manner [ ] . its oral bioavailability is close to % and has a short half life of - . h [ , ] . in humans, the plasma protein binding of favipiravir is %. favipiravir undergoes metabolism in the liver mainly by aldehyde oxidase (ao) and partially by xanthine oxidase (xo) and the inactive oxidative metabolite (t- m ) is excreted by kidneys. a study reported that in pichindé arenavirus infection the kinetics of absorption, elimination and time to maximum drug concentration is altered. the t- m levels were higher in the infected animals [ ] . further, favipiravir concentration may be increased by drugs those undergo metabolism through ao such as raloxifene, tamoxifen, estradiol, cimetidine, felodipine, amlodipine, verapamil, propafenone, amitriptyline, zaleplon, citalopram, sulindac and famciclovir. concomitant administration of acetaminophen and favipiravir showed increased area under curve (auc) of former drug possibly due to inhibition of the sulfate transferase by the latter drug [ ] . the drug did not show any toxicity at the oral dose of mg/kg/day for days in guinea pigs [ ] . this can be converted to mg/kg of human equivalent dose [ ] which is far greater than the dose prescribed for covid- on first day i.e. mg/kg/day ( mg bid on the first day) (https ://www.clini caltr ialsa rena.com/comme nt/influ enza-favip iravi r-covid - /). however, use of this drug may be approved with the availability of more number of clinical results. ribavirin, a broad-spectrum antiviral drug, is a guanosine analog approved for treating hepatitis c virus in combination, and respiratory syncytial virus as monotherapy. effect of this drug has been assessed in patients with sars [ ] and mers [ ] (table ) . against sars-cov , it has very high ec of . μm and selectivity index more than . in vero cells. ribavirin in its monophosphate form inhibits host inosine monophosphate dehydrogenase (impdh) enzyme that controls intracellular guanosine triphosphate (gtp) pools [ ] . exhaustion of intracellular gtp pool indirectly inhibits viral rdrp enzyme. it also interferes with mrna capping. ribavirin at a dose rate of mg - times/day in combination with other drugs such as lopinavir/ritonavir or interferon (ifn)-α through intravenous route for not more than days made the sars-cov infected patients more resistant to respiratory distress syndrome as well as death [ ] . the oral bioavailability of ribavirin is %, which is due to modest first-pass metabolism in liver [ ] . estimated halflife is . h [ ] . ribavirin-induced hemolytic anemia is a most commonly reported adverse effect and frequently it requires dose reduction [ ] . further, close monitoring of renal impairment in terms of creatinine clearance during therapy is required. old age, decreased renal function, low body weight and female gender are other risk factors to be considered during ribavirin therapy [ , ] . animal studies have shown teratogenic potential of ribavirin. therefore, exposure during pregnancy should be avoided. other less common yet pertinent adverse effects are bronchospasm and pulmonary edema on inhalation [ , ] . neutropenia, thrombocytopenia, skin rashes, anorexia and depression are some minor adverse effects of ribavirin that need to be monitored in the sensitive patients [ ] . in a recent clinical trial with combinations of ribavirin + interferon-alpha, lopinavir/ritonavir + interferon-alpha and ribavirin + lopinavir/ritonavir + interferon-alpha in patients with mild to moderate covid- showed that ribavirin plus lopinavir/ritonavir combination showed a significant increase in gastrointestinal adverse effects [ ] . the viral rnas get translated into polypeptide chains which get cleaved into functional proteins before packing into virions. the viral main proteases are responsible for the cleavage of these polypeptide chains [ ] . sars-cov protease has % overall similarity with sars-cov [ ] . protease inhibitors used in hiv- therapy are shown to be effective against sars-cov [ ] . in-silico and in-vitro approaches have been used to validate the inhibition of sars-cov protease by hiv- protease inhibitors [ , ] . numerous protease inhibitors are approved by fda for use in hiv therapy. the agents available from this class include saquinavir, amprenavir, indinavir, nelfinavir, ritonavir, and lopinavir. few of them are being considered against sars-cov (fig. ). this drug combination is available under brand name kaletra and was developed by abbott laboratories, usa. this drug was approved by fda in the year as an anti-retroviral for the treatment of hiv patients. lopinavir is rapidly degraded in the human body by the host proteases, hence is given with ritonavir (another protease inhibitor) at a lower dose, which helps lopinavir remain active for a longer time by inhibiting the metabolizing enzyme cytochrome p [ ] (table ) . coronavirus main proteases are cysteine proteases whereas hiv main proteases are aspartic proteases. nonspecific protease inhibition by protease inhibitors used in hiv therapy has been found effective against sars-cov. lopinavir has comparable binding energies against the sars-cov and hiv- proteases [ ] . in sars-cov patients treated with lopinavir-ritonavir, a significant virus clearance has been achieved [ , ] . a -year old patient, who failed to respond to methylprednisolone and interferon therapy, got quick improvement with additional lopinavir and ritonavir tablets therapy [ ] . in paediatric patients between the age of - years with confirmed covid- from three hospitals in zhejiang, china, use of lopinavir-ritonavir (syrup twice a day) along with interferon α to patients ( patients needed oxygen inhalation but not sure whether it included lopinavir-ritonavir group) showed a mean of hospital stay time of days and all were cured [ ] . based on these initial successful reports, sars-cov main protease has been docked for . billion protease inhibitor compounds [ ] . also, lopinavir-ritonavir has been included in solidarity trial (nct ). however, in a recent clinical trial with severely ill patients with confirmed sars-cov infections, lopinavirritonavir treatment did not improve the time to clinical improvement and mortality rate beyond standard care and further, some patients also showed adverse drug effects such as gastrointestinal disturbances [ ] . in a study on four covid- patients treated with western (lopinavir mg/ ritonavir in a ratio of mg to mg for q h through oral route), arbidol ( . g, three times in a day through oral route), and chinese traditional medicine shufeng jiedu capsule (sfjdc, . g, three times in a day through oral route) combination for - days, it has been observed that out of the two mild and two severe sars-cov -infected pneumonic patients, three patients showed significant improvement in pneumonia associated symptoms and the remaining patient with severe pneumonia has also shown signs of improvement till the date of reporting [ ] . most hiv protease inhibitors show poor bioavailability. they are extensively metabolized by microsomal cyp a enzymes. other agents that induce or inhibit these metabolizing enzymes influence their effectiveness. side effects associated with the use of protease inhibitors include diarrhea, vomiting, diabetes, hypertriglyceridemia, and hypercholesterolemia. it also could cause severe hepatic damage [ ] . therefore, a lot of clinical and experimental study regarding the use of lopinavir-ritonavir is required to reach a conclusive statement. the most common adverse effect of ritonavir/lopinavir is diarrhea and gastrointestinal disturbance. elevated liver enzymes, dyslipidemia, asthenia, headache and skin rashes are some minor side effects [ ] [ ] [ ] . ritonavir/lopinavir coadministration causes mild hepatotoxicity (elevated alanine aminotransferase, alt) [ ] . however, sole use of ritonavir at a higher dose ( mg/bid) may increase the risk of severe hepatotoxicity [ ] . a rarely observed adverse effect with ritonavir is retinal pigment epitheliopathy. the risk factors for the retinopathy are high dose and hepatic impairment [ ] . darunavir is a second-generation non-peptide protease inhibitor effective against hiv- . it has a distinct chemical structure that enhances binding affinity and reduces dissociation rate, making it more potent than the other protease inhibitors [ ] (table ). using computational drug design methods, darunavir was identified as one of the promising hits for inhibition of chymotrypsin-like protease of sars-cov [ ] . results of a structural analysis showed no binding of darunavir to sars-cov protease (https ://www.eacso ciety .org/home/covid - -and-hiv.html). anyhow, recently in shanghai, in-silico and an enzyme activity test based drug screening revealed agents with potential antiviral activity against sars-cov including darunavir (https ://www. simm.ac.cn/xwzx/kydt/ /t _ .html). interestingly, therapeutic doses of darunavir are reported to be too low to cause cytotoxic effects, affording a wide margin of safety [ ] . in an in-vitro study, darunavir at micromolar concentration was found to inhibit sars-cov virus replication by times in comparison to the untreated group (https ://www.sd.china news.com/ / / / .html). further, darunavir has been used ( mg tablet every h) along with other anti-viral drugs and supportive therapy in the clinical management of covid- patients presented with a range of mews from less than to more than in italy [ ] . darunavir is rapidly absorbed after oral administration and has a terminal elimination half-life of h. approximately % of the drug is plasma protein bound and metabolized exclusively by cyp a . therefore, co-administration of small doses of ritonavir (cyp a inhibitor) increases the bioavailability of darunavir. combination therapy with other cyp a inhibitors (e.g. statins) with darunavir/ritonavir requires caution or is even contraindicated [ , ] . a recent study correlated the daurnavir use with increased risk of myocardial infarction in hiv patients and concludes that cardiovascular disease (cvd) risk increases with darunavir exposure. therefore, it may be used cautiously in patients with underlying cardiac diseases [ ] . before, regular use of this drug, more pharmacological profile may further be investigated during the use in covid- patients. the pattern recognition receptors in immune cells recognize the viral pathogen-associated molecular pattern (pamps) to stimulate antiviral interferon responses in the host [ ] . the secreted interferons activate hundreds of interferon-stimulated genes which encode proteins with profound effects against the virus (fig. ) . the interferons (ifns) are antiviral molecules classified into type i (ifnα, ifnβ, ifnω, and ifnτ) and type ii (ifnγ). ifnα plays a critical role in innate immunity against viral infection which prompts its use in the treatment of many clinical viral infections. the recombinant ifnα is interferon alfacon- . the pegylated types are pegylated ifnα- a and pegylated ifnα- b [ ] . pegylated interferon alfa- b acts to target b cells through host interferon receptor, ifnar signalling and enhances immune response against viral infections [ ] . recombinant human ifnα- b has been shown to possess wide antiviral spectrum, low toxicity and high therapeutic index in vitro. quantitative reverse transcriptasepolymerase chain reaction (rt-pcr) results revealed antiviral effect of recombinant human ifnα- b on respiratory viruses such as influenza b virus, parainfluenza virus, respiratory syncytial virus and coronavirus which was stronger in comparison to the effect of ribavirin [ ] . in another study, ifnα- b administered by nasal spray reduced positive rates of immunoglobulin m (igm) antibody against all four respiratory viruses (parainfluenza virus, influenza b, adenovirus and respiratory syncytial virus) possibly suggesting the lower virus titre due to ifnα- b [ ] . in the rhesus macaque, ifnα- b with ribavirin showed very good effect against beta-coronavirus emerged in saudi [ ] , however, was unsatisfactory in humans [ ] . in china, intranasal ifnα ( × u) twice a day in combination with ribavirin is one of the guidelines for the treatment of covid- patients [ , ] . it was further showed that the infection rate of sars-cov was decreased by ifnα- b sprays [ ] . ifnα- b has demonstrated potent anti-viral activities against respiratory viruses and may serve for the prevention and treatment of sars-cov as well. nevertheless, thorough evaluation in appropriately designed randomized trials is recommended before forging on. peg-ifnα- b is a linear kda molecule and susceptible to hydrolysis. it is absorbed quickly and distributed widely. after administration peg-ifnα- b gets hydrolysed and the free ifnα- b circulates in the body. most of the free ifnα- b excreted solely through kidneys. therefore, it requires dose adjustment in patients with renal impairment. further, the clearance of peg-ifnα- b decreases after repeated dosing. therefore, it is administered according to body weight. conversely, the peg-ifnα - a is a kda branched chain molecule which is absorbed more slowly and has poor tissue distribution. peg-ifnα - a has long half-life. therefore, it is used at a fixed dose of μg per week for the treatment of hepatitis c virus infection. peg-ifnα- a is metabolized by both the kidneys and liver, therefore this drug does not require a major dose modification [ ] . number of adverse events was recorded with peg-ifnα therapy. dizziness, headache, depression, fatigue, insomnia, alopecia, myalgia, arthralgia, pyrexia, anorexia are the most common constitutional symptoms observed with peg-ifnα- a and peg-ifnα- b therapy in various other diseases. hematologic adverse events such as leucopoenia, thrombocytopenia and myelosuppression, thyroid disease, lung disease and retinopathy are the other adverse events recorded in the patients receiving peg-ifnα- a/ peg-ifnα- b [ ] [ ] [ ] . dose limiting, mild injection site reactions/inflammations were observed commonly in peg-ifnα - b therapy [ ] . most of the adverse events are well tolerated and become less severe during the progression of therapy. the neuropsychiatric adverse effects were observed in patients undergoing interferon-α therapy and may require timely medical interventions [ ] . in the case of severe events, discontinuation and dose reduction will help to overcome these adverse events [ ] . lopinavir-ritonavir along with interferon beta is undergoing clinical trial (miracle) against mers in saudi arabia wherein a total of patients have enrolled till jan [ ] . interferons are the antiviral weapons of the body and coronaviruses are known to reduce the host antiviral immunity by suppressing the production of interferons. so, replacement therapy with interferons or interferon inducers has the potential to reduce the viral load in the body. they augment the host response to the viral infection. lopinavir-ritonavir-interferon-β b treatment improved the clinical findings and lowered the lung viral load in mers-affected common marmosets [ ] . results of a recent clinical trial (nct ) on covid- patients showed the safety and efficacy of this combination. adverse events like selflimited nausea and diarrhoea were recorded in this study. however, there was no significant difference in the adverse effects between the combination group and ritonovir/lopinavir alone (control group) [ ] . the coronaviruses enter the cell by two ways, ( ) endocytosis, virus is taken up into the cell along with the endosomes, ( ) fusion of the viral spike protein with the cell surface receptor ace [ ] , the latter being the predominant pathway of virus entry [ ] . blockade of entry pathways may be effective targets for treatment (fig. ). chloroquine, primarily known for its anti-plasmodium actions, has antiviral activity as well. this drug originally derived from cinchona plant is now largely a synthetic drug ( -amino quinoline) discovered by bayer laboratories. chloroquine and its analogs are potent inhibitors of most coronaviruses [ ] (table ) . chloroquine and hydroxychloroquine are weak diprotic bases. these drugs take a similar pathway like the virus and concentrate in the endosomes increasing the ph of the endosomal fluid. the acidic ph of the endosomes is necessary for the optimal activity of viral enzymes responsible for proteolysis and post-translational modification of nascent proteins. disruption of acidic ph thus blocks the replication and lifecycle of the virus [ ] . in addition, the drug is known to interfere with the glycosylation of the host receptor for the virus, ace . faulty terminal glycosylation may affect the binding and subsequent entry of the virus into host cells [ ] . cell culture studies in african green monkey vero e cells indicate significantly higher potency for chloroquine compared to hydroxychloroquine [ ] . chloroquine has ec value of . μm against the sars-cov in vero e cells, which can be clinically achievable after administration of mg as shown in rheumatoid arthritis patients [ ] . a safe dosage of - . mg/kg per day of hydroxychloroquine could yield serum levels of . - . μm in humans [ ] . it is speculated that with a safe dosage, hydroxychloroquine could achieve concentration in the above tissues to inhibit sars-cov infection. hydroxychloroquine phosphate ( mg tablet every h as a loading dose followed mg tablet every h for days) or chloroquine phosphate ( mg of two tablet every h for days) along with other anti-viral drugs and supportive therapy have been used in the clinical management of covid- patients presented with a range of mews from less than to more than in italy [ ] . chloroquine and hydroxychloroquine have been considered for solidarity trial (nct ). hydroxychloroquine has shown side effects such as prolonged qt interval and heart failure, though controversy exists. recently, who stopped hydroxychloroquine arm of the solidarity trial to find an effective covid- treatment. this recommendation was developed on the basis of data obtained from solidarity trial (including the french discovery trial data and uk's recovery trial data). these trials showed that hydroxychloroquine did not result in the reduction of mortality of covid- patients who were hospitalized when compared with standard care (briefed on june by who). use of chloroquine for sars-cov asks for a high dose but an overdose of chloroquine has been reported to cause poisoning and death [ ] . in comparison, hydroxychloroquine is safer with % less toxicity in animals [ ] . oral absorption of chloroquine and hydroxychloroquine in humans is efficient. both the drugs distribute similarly in different tissues with high concentrations in the liver, kidney, lungs and spleen [ ] . chloroquine is two to three times as toxic in animals as hydroxychloroquine [ ] . the acute toxicity of chloroquine causes death due to cardiac and respiratory arrest [ ] . adverse effects of chloroquine/hydroxychloroquine therapy at the therapeutic doses include retinopathy, myopathy, electrocardiographic changes, bleaching of hair, pruritus, headaches, dizziness and gastrointestinal upset [ ] [ ] [ ] . hydroxychloroquine retinal toxicity in the patients is far more common in patients taking this drug for greater than years with the overall prevalence of . % [ ] . a study showed even with the recommended therapeutic dose of hydroxychloroquine produced bilateral maculopathy and it was attributed to the differential susceptibility of the retinal epithelium to hydroxychloroquine [ ] . hydroxychloroquine and chloroquine are orally well absorbed and show - % oral bioavailability. chloroquine is % bound to plasma proteins and distributed extensively. following administration, chloroquine is rapidly dealkylated to pharmacologically active desethylchloroquine and bisdesethylchloroquine. metabolism of hydroxychloroquine is similar to chloroquine except for a third metabolite desethylhydroxychloroquine which is also produced during metabolism. both hydroxychloroquine and chloroquine have prolonged half-lives, between and days, and low blood clearance by kidneys and liver. for both chloroquine and hydroxychloroquine, approximately - % is excreted as an unchanged or metabolized drug through the kidneys, - % is excreted in an unchanged or changed form in the feces, % is sloughed off through the skin, and - % is stored long term in lean body tissues [ ] [ ] [ ] [ ] . hydroxychloroquine should be cautiously used in patients with known hepatic or renal dysfunction. the co-administered drugs with hydroxychloroquine excreted via liver/ kidney may interact with hydroxychloroquine and modulates its pharmacokinetics and toxicity. hydroxychloroquine has been reported to cause severe hypoglycaemia when coadministered with oral hypoglycaemic drugs. there have been few reports of mild to severe hepatic failure in patients with hydroxychloroquine treatment [ ] . racial difference in toxicity has also been reported wherein the incidence of pericentral maculopathy was common in asian ( %) than caucasian ( %) patients [ ] . further, the use of chloroquine and hydroxychloroquine may be warranted in prophylaxis strategy as well as in covid- patients with more number of clinical trials. arbidol, an indole-derivative, also known as umifenovir is a potent broad-spectrum antiviral agent. this drug has shown activity against a wide range of enveloped and nonenveloped viruses [ ] (table ) . it is effective against numerous pathogenic respiratory viruses and relatively very safe for use [ , ] . arbidol is an approved therapeutic agent against influenza in russia and china [ ] . arbidol and arbidol mesylate were reported to act directly on viral replication of sars-cov at an early stage in vitro [ , ] . the anti-viral mechanism of arbidol against influenza a and b involves viral fusion inhibition by hindering the hemagglutinin fusion machinery with the membrane, thus blocks virus entry into the cell [ ] . arbidol is one of the drugs in clinical trial phase for pharmaceutical interventions of covid- and treatment of covid- patients with arbidol leads to a reduction in the mortality rate and increase in the recovery rate [ , ] . arbidol treatment coupled with lopinavir/ritonavir reckoned to retard the development of pulmonary lesions concurrently reducing the respiratory and gastrointestinal covid- viral load thus lowering the transmission [ ] . encouragingly, seasonal prophylaxis with arbidol reduced influenza morbidity in patients with asthma and copd during epidemic [ ] . four cases with mild to severe sars-cov pulmonary symptoms were subjected to antiviral therapy consisting of arbidol, lopinavir/ritonavir, and shufeng jiedu (traditional chinese medicine) along with supportive care. following medication, three patients showed a notable reduction in pneumonia-related manifestations and two of them were discharged after testing negative for sars-cov [ ] . in covid- patients, reduction in fever and cough; improvement in chest computed tomography (ct) and clinical status among the arbidol, lopinavir/ritonavir and control group remained statistically indifferent. however, arbidol was better tolerated by patients whereas test subjects administered with lopinavir/ritonavir experienced adverse events during the follow-up period [ ] . the half-life of arbidol is between and h and % of the total drug excreted unchanged in feces. arbdiol conjugation with glucuronide and sulfonation is evident. the major phase i enzyme involved in its metabolism was cyp a , therefore, a possible interaction with cyp a inducers/ inhibitors may happen [ ] . the drug has oral lethal dose (ld s) of - mg/kg in mice, and > mg/kg in rats and guinea pigs. intravenous ld s are mg/kg in mice and mg/kg in rats. in long term animal studies, it showed no adverse effects with - times of human dose and no developmental toxicities were observed [ ] . it has been noticed that sars-cov binds the ace receptors similar to the sars coronavirus [ ] . covid- is associated with the acute respiratory distress syndrome (ards) and higher activity of ace leads to attenuation in ards [ , ] . ace expression is downregulated in covid- patients [ ] . it is a general observation throughout the world that aged persons affected with covid- suffer from high mortality. association between covid- infection and the process of chronological aging has been understood with the presence of two host receptors cd and ace which are associated with senescence. the major pathway of virus entry into the host in case of sars-cov and sars-cov is through the target cell ace . spike proteins of the virus attach to the cell surface ace expressed in epithelial cells of the oral mucosa, lungs, intestine, blood vessels and kidney. angiotensin receptor blockers (arbs) which inhibit the action of ace, an isoform of ace , are known to increase the expression of ace [ ] (fig. ; table ). the patients with diabetes and cardiovascular diseases regularly take ace inhibitors and arbs. hence, they remain under continuous risk of sars-cov infection. however, experimental studies with ards indicate a higher level of ace reduces the severity of the disease [ ] . increased ace /ace ratio may improve the host response to viral infection by correcting the endothelial dysfunction common to most viral infections [ ] . experimental and clinical studies are needed urgently on this aspect for repurposing of commonly used arbs or ace inhibitors (aceis). arbs are well-tolerated drugs with minimal side effects in the population. however, a lot of patients from south america, central america and spain, had stopped or intended to interrupt their treatments with anti-hypertensive drugs like aceis such as enalapril or arbs such as losartan fearing the risk of sars-cov infection [ ] . however, another school of thought provided alternative suggestions about the use of renin-angiotensin-aldosterone system (raas) inhibitors. they suggested that though ace expression may be enhanced with the use of raas inhibitors, sudden withdrawal of these drugs may be responsible for higher risk in patients of covid- already affected with cardiovascular illnesses. therefore, the use of raas inhibitors should continue in patients with covid- under continuous observation [ , ] . out of covid- patients taking antihypertensive drugs, patients who were treated with aceis/arbs showed less mortality in comparison to patients taking drugs other than aceis/arbs in shenzhen third people's hospital, china [ ] . aceis and arbs may improve the clinical condition of the covid- patients in a clinical setting by maintaining a low level of il- level in peripheral blood and through increment in a cluster of differentiation cd and cd t cell counts in peripheral blood and decreased the peak viral load [ ] . contrary to the previous report, another retrospective study from the union hospital of wuhan, china done on patients suggested that acei/arbs medication has no difference in the critical group and general group and further, did not show any effect on the morbidity and mortality of covid- patients associated with cardiovascular diseases [ ] . it is reported that ace inhibits the formation of ang ii in the presence of aceis; therefore, these prevent the negative effects induced due to at r as well as positive effects derived from the ang ii binding with at r and further, its transformation into ang-( - ) [ ] . therefore, it could be assumed that the use of aceis leads to the protective effects in the lung in covid- patients. angiotensin receptor blockers are the most commonly used drugs for hypertension and related cardiac problems. in general, aceis and arbs have few interactions with ritonavir [ , ] . generally, arbs are well tolerated and the frequency of adverse reactions is less than %. there are no specific considerations for use in patients with hepatic and renal impairment [ ] . arbs are structurally related to losartan (except eprosartan) but the individual members exhibit diverse pharmacokinetic properties. their bioavailabilities vary from - % and half-lives are between and h. they are highly plasma protein bound ( - %) and mostly metabolized and eliminated by the liver and, therefore, can be safely used in mild to moderate renal failure without any dose adjustment. however, losartan should be started with a lower dose in hepatic impairment and telmisartan should be avoided in case of renal impairment and congestive heart failure/digoxin therapy [ , ] . statins, the lipid-lowering drugs, have shown pleiotropic activity through anti-inflammatory and immunomodulatory properties to prevent acute lung injury in different experimental and clinical conditions; therefore, it may be used as re-tasking drug for the covid- patients [ ] (table ) . statins are commonly taken for a lifetime to prevent coronary artery diseases. studies have reported that these drugs increase the ace expression [ ] . ace cleaves ang ii, a peptide which promotes endothelial dysfunction and cardiovascular diseases, and produces ang-( - ) which counters the effects of ang ii [ ] . stimulation of ace /angiotensin-( - )/mas and angiopoietin/tie- signaling axes help restoring viral infection-induced endothelial dysfunction and maintain the homeostasis of the patients [ ] . ace activity has shown to be up-regulated with the use of arbs and atorvastatin [ , ] (fig. ) . statins being immunomodulatory have been hypothesized to work against mers coronaviruses [ ] . however, in animal models of sars and mers infections, reduced expression of the adapters, tirdomain-containing adapter-inducing interferon-β (trif) or myeloid differentiation primary response protein (myd) causes severe respiratory disease leading to death. hence, the use of statins, which can further suppress myd signaling, may exacerbate the disease condition [ ] . fungus derived statins (lovastatin, pravastatin and simvastatin) have elimination half-lives of - h and other synthetic compounds have elimination half-lives ranging from h for fluvastatin to h for rosuvastatin. bioavailability of statins varies from - % and - % of the absorbed statins. majority of satins are metabolized by the liver and the biliary excretion is the predominant route of excretion. pravastaitn and rosuvastatin are excreted mostly unchanged by liver and kidneys. all statins are extensively bound to plasma proteins (> %) with the exception to pravastatin which is only % bound to plasma proteins [ ] . myopathy and rhabdomyolysis are the most frequent adverse effect of statins. other common adverse effects include hepatotoxicity, peripheral neuropathy, cardiac toxicity, cognitive dysfunction, cataracts, diabetes mellitus and autoimmune necrotizing myopathy [ , ] . most of the statins are cyp a substrates and the inhibitors of this enzyme may increase blood levels of statins and therefore associated with adverse effects [ ] . statins have potential interactions with the protease inhibitor drugs and they are contraindicated in combination therapy [ ] . usfda warns using atorvastatin, lovastain, rosuvsatin, pravstatin, simvastatin with antiviral protease inhibitor drugs (for list please see https ://www.fda.gov/drugs /drug-safet y-and-avail abili ty/fda-drug-safet y-commu nicat ion-inter actio ns-betwe en-certa in-hiv-or-hepat itis-c-drugs -and-chole stero l#dose). in this case dose reduction and therapeutic drug monitoring will reduce the occurrence of adverse effects. tocilizumab is a drug used against rheumatoid arthritis as well as cytokine release syndrome/systemic inflammatory response syndrome. this drug was introduced in japan in the year and subsequently in the european union (trade name roactemra) in the year . later in the year , it was used in the usa (actemra) and this drug is an antihuman monoclonal antibody of the immunoglobulin g k subclass [ ] . this drug does not act on the virus but it acts to reduce the cytokine response of the host. tocilizumab is a recombinant humanized monoclonal antibody which is responsible to bind the human il- receptor and inhibiting its signal transduction pathway [ ] (fig. ) . a -year old man of wuhan, china developed symptoms of chest tightness without fever and cough on st february and confirmed to be the first case of covid- with multiple myeloma. the case was successfully treated with humanized anti-il- receptor antibody, tocilizumab. the patient was administered mg/kg tocilizumab through iv route for one time from day to day upon hospital admission and a decrease in il- level was recorded, however, a rebound phenomenon of il- level was observed, may be due to the recovery of the normal t cells and the patient was cured on day . the patient, however, had also received other therapies like mg dose of umifenovir (arbidol) tablets orally for three times daily as antiviral treatment and mg of moxifloxacin iv daily for three days [ ] . however, in another years old covid- patient of end-stage renal disease (esrd) in the usa treated with tocilizumab, hydroxychloroquine and broad spectrum antibiotics, the patient remained in critical condition throughout the study period [ ] . a years old woman from switzerland suffering from systemic sclerosis (ssc) was on tocilizumab treatment for arthritis and ssc-associated interstitial lung disease (ssc-ild) @ mg/kg body weight every weeks intravenously. on the basis of observation it has been reported that tocilizumab treatment given for chronic autoimmune diseases may prevent the development of severe covid- [ ] . a case of years old patient with a respiratory failure linked to covid- from france showed a rapid favourable outcome after two infusions of the tocilizumab at a dose rate of mg/kg through intravenous route along with other therapy like lopinavir-ritonavir [ ] . tocilizumab treatment in of the patients showed improvement in terms of oxygen intake, lung lesion opacity (in patients), lymphocytes in peripheral blood ( patients), c-reactive protein ( patients) and further, all patients were discharged on average . d after giving tocilizumab in a study conducted in china between and february [ ] . a study on covid- patients with severe pneumonia treated with tocilizumab and other supportive therapy from - march in italy, the respiratory condition was improved or stabilized in patients in days. of these patients had shown clearing of diffuse bilateral opacities on chest x-ray and were discharged from the hospital; however, respiratory condition of patients was worse during the treatment and died out of patients [ ] . in another clinical study, tocilizumab administration showed a reduction in serum il- level in patients out of patients, however, it could not decrease il- level in four patients. still, observations suggest that tocilizumab is a good treatment option in patients who have a risk of cytokine storms [ ] . however, it is difficult to reach on a conclusion regarding the use of tocilizumab in covid- patients with the limited number of clinical sample size. the absolute bioavailability of tocilizumab following subcutaneous administration was estimated to be . % [ ] . tocilizumab is eliminated from the body in a concentrationdependent manner. therefore, its half-life is directly proportional to its serum concentration. at higher serum concentration, it shows linear elimination and has the terminal half-life of . days (https ://www.acces sdata .fda.gov/drugs atfda _docs/label / / s , s lbl.pdf). the notable adverse effect of tocilizumab is liver toxicity. steatosis, steatohepatitis and focal hepatocellular necrosis were seen in this drug-induced hepatotoxicity. other common adverse reactions observed were skin and soft tissue infections, dyslipidemia, transient neutropenia, headache, nausea and flu-like symptoms. therefore, a full assessment for liver injury and dose adjustment is required in patients administered with tocilizumab [ , ] . tocilizumab also showed good efficacy in long term dosing for rheumatoid arthritis with similar adverse reactions and few malignancies also were reported [ ] . in a recent study, it has been demonstrated that an antiprotozoal agent nitazoxanide has shown antiviral activity against a number of viruses which include human and animal coronaviruses. it is used orally and chemically, this drug is nitrothiazoly-salicylamide, a broad-spectrum anthelmintic and antiviral prodrug which is metabolized to an active compound tizoxanide [ , ] (table ). it had shown inhibitory potential against sars-cov at a low-concentration with % effective concentration of . μm in vero e cells. it has been observed that antiviral activity of this compound may be due to interference with host-regulated pathways which are involved in viral replication rather than virus-specific pathways [ , ] . nafamostat is an anticoagulant in nature and further, its activity was also evaluated in the vero e cells infected with sars-cov . this drug is a synthetic serine protease inhibitor and inhibited the sars-cov at % effective concentration of . μm in vero e cells. further, it is speculated that it may act through the inhibition of membrane fusion by reducing the release of cathepsin b. as this agent has previously shown potential against the mers-cov through prevention of membrane fusion, it could also be recommended for further in-vivo evaluation against covid- [ ] (fig. ; table ). ivermectin is a broad-spectrum anti-parasitic agent and has also shown efficacy against some viral infections. this drug is basically a macrolide endectocide macrocyclic lactone derived from streptomyces avermitilis with anthelmintic activity (table ) . recently, this drug has been investigated against sars-cov in in-vitro cell culture system in australia. it reduced the viral rna of sars-cov at two hours post infection in vero-hslam cells with the addition of single dose and its reduction intensity was approximately -fold. this study hypothesized that ivermectin may act through inhibition of importin (imp) α/β -mediated nuclear import of viral proteins similar to other rna viruses, however; further investigation is required to understand the precise mechanism of action in sars-cov [ ] . it is also important to conduct the trials in in-vivo animal models and further, the pre-clinical trials in human beings are also required with fast pace if in-vivo models results have potential to mitigate the covid- infection (fig. ) . cepharanthine, a naturally occurring plant alkaloid, is an anti-inflammatory and anti-neoplastic and is approved for leukopenia treatment (table ). cepharanthine showed the most potent inhibition of gx_p v infection and decreased the viral rna yield in the pangolin coronavirus gx_p v workable model, therefore, this therapeutic agent may also be a potential candidate for repurposing against covid- [ ] . further, it has been speculated that this drug may target host cell pathways [ ] . a gold-containing triethyl phosphine coated drug, auranofin, is used as therapy for the arthritis and approved from fda for this treatment (table ) . previously, this drug has shown effects against viral, bacterial and parasitic infections. recently, auranofin has shown inhibitory effect against viral rna in huh cells infected with sars- cov fig. overview of the drugs with potential for repurposing against covid- . the drugs being considered for primary therapy of covid either acts on targets of the virus or on the targets of the host as well as a reduction in proinflammatory cytokines in an in-vitro study [ ] . endoplasmic reticulum (er) stress and unfolded protein response (upr) activation are responsible for the viral replication and pathogenesis in coronavirus infection [ , ] and high levels of upr activation has been recorded in the cells which have higher expression of sars-cov spike protein [ , , ] . therefore, it is speculated that auranofin may act through the inhibition of redox enzymes such as thioredoxin reductase and induction of er stress, however, its confirmatory investigation is required [ , ] . janus kinase inhibitors have anti-inflammatory and antiviral effects and may have a purported advantage over other immunomodulatory strategies in covid- [ ] . jak / inhibitor ruxolitinib, baricitinib, jak / inhibitor tofacitinib and pan-jak inhibitor td- are presently under different phases of clinical trial and being investigated for covid- treatment (https ://www.pharm aceut ical-techn ology .com/comme nt/incyt e-eli-lilly -jak-inhib itor-covid - /). a phase-i clinical study showed improvement in covid- inflammation score with clinical improvement by ruxolitinib. however, it has toxicity on the liver and hematopoietic system. but in this study with the short term dosage, it did not show any major adverse events [ ] . baricitinib is presently under phase-iii clinical trial (https ://www.clini caltr ialsa rena.com/news/lilly -baric itini b-covid - -trial /). initial studies showed the drug baricitinib corrected the immune dysregulation and improved clinical disease outcome with no serious adverse events [ , ] . all the above drugs are under different phases of clinical trials and none have been approved for the treatment of covid- . dexamethasone is a corticosteroid and it has been used in some clinical trials. the preliminary results of recovery trial conducted in patients showed dexamethasone @ mg once daily for up to days showed improvement in recovery with . % mortality in the dexamethasone group compared to the . % mortality in the usual care group [ ] . however, few concerns have been raised on recov-ery trial outcome in choosing the correct steroid and dose. in another study, methylprednisolone reduced the mortality rates in covid- patients with ards. further, the effect of dexamethasone in geriatric patients and effects on viral load are not mentioned in the recovery trial [ ] . theoharides and conti have also suggested restricting the use of dexamethasone during the initial phase of disease in severely ill patients for shorter period. using this drug in recovery phase may lead to reduced viral clearance and nosocomial infections [ ] . as it suppresses immunity and it may aggravate some latent infections whose occurrence is of no importance to the developed countries. therefore, region-specific study is required before incorporating into covid- therapy [ ] . a chemotherapeutic fluoroquinolone antibiotic, prulifloxacin, with broad-spectrum activity, and some anti-hiv drugs such as tegobuvir, (a novel non-nucleoside inhibitor of human coronavirus rna replication), nelfinavir (a protease inhibitor which inhibits the cleavage of the polyprotein gag-pol) and bictegravir (hiv- integrase inhibitor) have protein binding sites of the proteases which have been shown using the high throughput screening as molecular docking with bioinformatics analysis and would be considered the potential candidates for re-tasking against covid- in future [ ] and in-vitro and in-vivo animal models may provide a lead against this disease. according to another recent molecular docking study based on rdrp modelling and multiple sequence alignment (msa) showed a binding capacity to rdrp against sars-cov by various antiviral agents such as sofosbuvir (fda approved against hepatitis c virus), ribavirin, remdesivir and idx- ; (under clinical trial against hepatitis c virus) [ ] . elbasvir, an antiviral drug, (approved for the treatment of hepatitis c) has shown predicted multiple binding sites at rdrp, papain-like proteinase and helicase of sars-cov using the docking simulations and computational modelling [ ] (fig. ; table ). in a recent review, it has been stated that azithromycin, quercetin, rapamycin and doxycycline may have senolytic activity and azithromycin and doxycycline are used to inhibit viral replication and il- production, therefore, these therapeutic agents may be potential re-tasking agents against covid- in future with proof of sufficient invitro and in-vivo studies [ ] (fig. ; table ). peptide (ek ), tdf, tc (rna synthesis inhibitors), shufeng jiedu and lianhuaqingwen capsules (chinese traditional medicine) are screened using standard bioassays, chemical screening and genome and biophysical understanding of the targeted virus for being the potential drug candidates against sars-cov infection as described in a recent review. however, the efficacy and safety of these drugs for sars-cov still need to be further confirmed by clinical experiments [ ] (fig. ) . covid- is a pandemic and has no treatment till date including vaccine and drugs. however, there are number of available drugs with approval of fda for treatment of other diseases which could be used on the basis of the trial against covid- and considered as the repurposed drugs. these are antivirals, antimalarials, aceis, arbs, statins and monoclonal antibodies. it has been noticed that remdesivir, favipiravir, ribavirin, lopinavir-ritonavir combination, arbidol, tocilizumab have shown benefits in various clinical studies done on the basis of compassionate use to save the life of covid- patients; therefore, in future, these drugs could be the potential drug therapy against this deadly disease. however, the use of chloroquine and hydroxychloroquine has shown the controversial results in the different trials, therefore, who also stopped their solidarity trials recently. in future, chloroquine and hydroxychloroquine require a large number of research studies to reach a conclusion for its use in covid- patients. further, aceis and arbs could be the potential supportive therapy against this infection. some drugs are in the early phase of investigation like ivermectin and auranofin to be used against the covid- and these agents could be potential therapeutic agents in future. molecular docking would be the central technique to identify the probable therapeutic agents against covid- patients and the screened agents, thereby, could be verified for their effectiveness in in-vitro and in-vivo studies. identification of amitriptyline hcl, flavin adenine dinucleotide, azacitidine and calcitriol as repurposing drugs for influenza a h n virus-induced lung injury. plos pathog drug repositioning for rare diseases: knowledge-based success stories repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection examination of clinical trial costs and barriers for drug development. us department of health and human services, office of the assistant secretary for planning and evaluation report protein localization vector propagation: a method for improving the accuracy of drug repositioning in silico methods for drug repurposing and pharmacology drug repurposing: progress, challenges and recommendations global mapping of pharmacological space cheminformatics at the interface of medicinal chemistry and proteomics network medicine: linking disorders network-based drug repurposing for novel coronavirus -ncov/ sars-cov- network bioinformatics analysis provides insight into drug repurposing for covid- characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine structure of mers-cov spike receptor-binding domain complexed with human receptor dpp the spike protein of sars-cov-a target for vaccine and therapeutic development coronavirusesdrug discovery and therapeutic options coronavirus puts drug repurposing on the fast track severe acute respiratory syndrome (sars) isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronavirus genomics and bioinformatics analysis genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences sars, the first pandemic of the st century a pneumonia outbreak associated with a new coronavirus of probable bat origin the -new coronavirus epidemic: evidence for virus evolution from bats to pangolins, how do viruses reach us the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- teicoplanin: an alternative drug for the treatment of coronavirus covid- ? preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses first case of novel coronavirus in the united states national institute for the infectious diseases "l. spallanzani", irccs. recommendations for covid- clinical management compassionate use of remdesivir for patients with severe covid- remdesivir for the treatment of covid- -preliminary report remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for or days in patients with severe covid- the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys discovering drugs to treat coronavirus disease (covid- ) successful treatment of advanced ebola virus infection with t- (favipiravir) in a small animal model. antiviral res nucleosides for the treatment of respiratory rna virus infections clinical regimens of favipiravir inhibit zika virus replication in the hollow-fiber infection model. antimicrob agents chemother favipiravir pharmacokinetics in ebola-infected patients of the jiki trial reveals concentrations lower than targeted favipiravir: pharmacokinetics and concerns about clinical trials for -ncov infection alterations in favipiravir (t- ) pharmacokinetics and biodistribution in a hamster model of viral hemorrhagic fever effective oral favipiravir (t- ) therapy initiated after the onset of clinical disease in a model of arenavirus hemorrhagic fever dose translation from animal to human studies revisited role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with interferon-α b and ribavirin improves outcome in mers-cov-infected rhesus macaques mechanisms of action of ribavirin against distinct viruses pharmacokinetics and absolute bioavailability of ribavirin in healthy volunteers as determined by stable-isotope methodology optimizing ribavirin dosage: a new challenge to improve treatment efficacy in genotype hepatitis c patients anaemia predictors in patients with chronic hepatitis c treated with ribavirin and direct-acting antiviral agents ribavirin-induced anemia: mechanisms, risk factors and related targets for future research a pharmacological profile of ribavirin and monitoring of its plasma concentration in chronic hepatitis c infection usefulness of monitoring ribavirin plasma concentrations to improve treatment response in patients with chronic hepatitis c comparative effectiveness and safety of ribavirin plus interferonalpha, lopinavir/ritonavir plus interferon-alpha and ribavirin plus lopinavir/ritonavir plus interferon-alpha in patients with mild to moderate novel coronavirus pneumonia: results of a randomized analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods. version prediction of the sars-cov- ( -ncov) c-like protease ( cl pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates expanding the frontiers of existing antiviral drugs: possible effects of hiv- protease inhibitors against sars and avian influenza computational determination of potential inhibitors of sars-cov- main protease compounds with therapeutic potential against novel respiratory coronavirus lopinavir/ritonavir in the treatment of hiv- infection: a review unrevealing sequence and structural features of novel coronavirus using in silico approaches: the main protease as molecular target case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatmentof covid- infected pneumonia monitored by quantitative rt-pcr management of corona virus disease- (covid- ): the zhejiang experience. zhejiang da xue xue bao yi xue ban the course of clinical diagnosis and treatment of a case infected with coronavirus disease clinical and epidemiological features of children with coronavirus disease (covid- ) in zhejiang, china: an observational cohort study rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds. mol inform a trial of lopinavir-ritonavir in adults hospitalized with severe covid- clinical characteristics and therapeutic procedure for four cases with novel coronavirus pneumonia receiving combined chinese and western medicine treatment hivantiretroviral therapy induced liver, gastrointestinal, and pancreatic injury lopinavir/ritonavir: a review of its use in the management of hiv infection predictive factors of lopinavir/ritonavir discontinuation for drug-related toxicity: results from a cohort of multi-experienced hiv-infected individuals pharmacokinetics of high-dose lopinavir-ritonavir with and without saquinavir or nonnucleoside reverse transcriptase inhibitors in human immunodeficiency virus-infected pediatric and adolescent patients previously treated with protease inhibitors pharmacokinetics and hepatotoxicity of lopinavir/ritonavir in non-cirrhotic hiv and hepatitis c virus (hcv) co-infected patients hepatotoxicity associated with antiretroviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis c or b virus infection retinal toxicity with ritonavir chemical characteristics, mechanism of action and antiviral activity of darunavir identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach clinical pharmacokinetics of darunavir darunavir: pharmacokinetics and drug interactions darunavir and cardiovascular risk: evaluating the data to inform clinical care identification of the rna pseudoknot within the ' end of the porcine reproductive and respiratory syndrome virus genome as a pathogenassociated molecular pattern to activate antiviral signaling via rig-i and toll-like receptor total chemical synthesis of human interferon alpha- b via native chemical ligation mechanisms of action of interferon and nucleoside analogues recombinant human interferon alpha b broad-spectrum anti-respiratory viruses pharmacodynamics study in vitro a field trial of recombinant human interferon alpha- b for nasal spray to prevent sars and other respiratory viral infections inhibition of novel β coronavirus replication by a combination of interferon-α b and ribavirin ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study drug treatment options for the -new coronavirus ( -ncov) diagnosis and treatment of, novel coronavirus infection in children: a pressing issue review article: pegylated interferons: chemical and clinical differences side effects of therapy for chronic hepatitis c a pharmacokinetic and pharmacodynamic comparison of a novel pegylated recombinant consensus interferon-α variant with peginterferon-α- a in healthy subjects reversible bone marrow aplasia induced by pegylated interferon-α- a therapy in a patient with primary myelofibrosis neuropsychiatric adverse effects of interferon-alpha: recognition and management treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β b (miracle trial): study protocol for a randomized controlled trial treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial mechanisms of coronavirus cell entry mediated by the viral spike protein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro dose refinements in long-term therapy of rheumatoid arthritis with antimalarials dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. a clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine world health organization. review of side effects and toxicity of chloroquine (no. who/mal/ . ). . geneva: world health organization animal toxicity and pharmacokinetics of hydroxychloroquine sulfate chloroquine: a review studies on the chronic toxicity of chloroquine cardiovascular toxicity and distribution kinetics of intravenous chloroquine chloroquine and hydroxychloroquine toxicity hydroxychloroquine retinopathy hydroxychloroquine toxicity despite normal dose therapy pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic diseases clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax pharmacology of chloroquine and hydroxychloroquine pericentral retinopathy and racial differences in hydroxychloroquine toxicity the antiinfluenza virus drug, arbidol is an efficient inhibitor of sars-cov- in vitro. version sensitivity of various influenza virus strains to arbidol. influence of arbidol combination with different antiviral drugs on reproduction of influenza virus a antiviral activity of arbidol against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo arbidol as a broad-spectrum antiviral: an update antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures recent developments in anti-severe acute respiratory syndrome coronavirus chemotherapy clinical features of cases with coronavirus disease clinical trials on drug repositioning for covid- treatment arbidol combined with lpv/r versus lpv/r alone against corona virus disease : a retrospective cohort study efficacy of arbidol in the prevention of virus-induced exacerbations of bronchial asthma and chronic obstructive pulmonary disease an exploratory randomized, controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid- (elacoi) pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans hiding in plain sight: an approach to treating patients with severe covid- infection imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme activity in acute respiratory distress syndrome. pediatr renin-angiotensin-aldosterone system inhibitors in patients with covid- receptor recognition by novel coronavirus fromwuhan: an analysis based on decade-long structural studies of sars do viral infections mimic bacterial sepsis? the role of microvascular permeability: a review of mechanisms and methods facts and reflections on covid- and anti-hypertensives drugs inhibitors of ras might be a good choice for the therapy of covid- pneumonia reninangiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension clinical characteristics and outcomes of cardiovascular disease patients infected by -ncov covid- and ras: unravelling an unclear relationship antihypertensive drugs in patients treated with antiretrovirals drug interactions and antiretroviral drug monitoring angiotensin ii receptor blockers clinical pharmacokinetics of angiotensin ii (at ) receptor blockers in hypertension pharmacology and clinical efficacy of angiotensin receptor blockers covid- treatment by repurposing drugs until the vaccine is in sight tissue specific up regulation of ace in rabbit model of atherosclerosis by atorvastatin: role of epigenetic histone modifications ace : more of ang - or less ang ii? treating the host response to emerging virus diseases:lessons learned from sepsis, pneumonia, influenza and ebola acute respiratory distress syndrome leads to reduced ratio of ace/ace activities and is prevented by angiotensin-( - ) or an angiotensin ii receptor antagonist statins may decrease the fatality rate of middle east respiratory syndrome infection reply to "statins may decrease the fatality rate of mers infection pharmacokinetic and pharmacodynamic properties of statins: an update adverse effects of statins-mechanisms and consequences diagnosis, prevention, and management of statin adverse effects and intolerance: canadian consensus working group update clinical management of drug-drug interactions in hcv therapy: challenges and solutions drugdrug interactions between hmg-coa reductase inhibitors (statins) and antiviral protease inhibitors tocilizumab (actemra) controversies about covid- and anticancer treatment with immune checkpoint inhibitors first case of covid- in a patient with multiple myeloma successfully treated with tocilizumab case of novel coronavirus disease in a chronic hemodialysis patient presenting with gastroenteritis and developing severe pulmonary disease covid- in a patient with systemic sclerosis treated with tocilizumab for ssc-ild tocilizumab, an anti-il receptor antibody, to treat covid- -related respiratory failure: a case report effective treatment of severe covid- patients with tocilizumab tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in brescia tocilizumab treatment in covid- : a single center experience pharmacokinetic and pharmacodynamic analysis of subcutaneous tocilizumab in patients with rheumatoid arthritis from randomized, controlled trials: summacta and brevacta tocilizumab: a review of its safety and efficacy in rheumatoid arthritis hepatotoxicity of tocilizumab and anakinra in rheumatoid arthritis: management decisions long-term safety and efficacy of tocilizumab, an anti-il- receptor monoclonal antibody, in monotherapy, in patients with rheumatoid arthritis (the stream study): evidence of safety and efficacy in a -year extension study basic & clinical pharmacology, e. mcgraw-hill education nitazoxanide/azithromycin combination for covid- : a suggested new protocol for early management nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus antiviral treatment of covid- the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro repurposing of clinically approved drugs for treatment of coronavirus disease in a -novel coronavirus ( -ncov) related coronavirus model the fda-approved gold drug auranofin inhibits novel coronavirus (sarscov- ) replication and attenuates inflammation in human cells coronavirus infection, er stress, apoptosis and innate immunity comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku the ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf role of endoplasmic reticulum-associated proteins in flavivirus replication and assembly complexes. pathogens jak inhibitors in covid- : need for vigilance regarding increased inherent thrombotic risk the janus kinase / inhibitor ruxolitinib in covid- with severe systemic hyperinflammation baricitinib therapy in covid- : a pilot study on safety and clinical impact baricitinib restrains the immune dysregulation in covid- patients dexamethasone in hospitalized patients with covid- -preliminary report dexamethasone in the management of covid - dexamethasone for covid- ? not so fast dexamethasone for covid- : data needed from randomised clinical trials in africa. lancet glob health therapeutic drugs targeting -ncov main protease by high-throughput screening ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study computational target-based drug repurposing of elbasvir, an antiviral drug predicted to bind multiple sars-cov- proteins covid- and chronological aging: senolytics and other anti-aging drugs for the treatment or prevention of corona virus infection? aging (albany ny) sars-cov- rna dependent rna polymerase (rdrp) targeting: an in silico perspective experimental treatment with favipiravir for covid- : an open-label control study. engineering (beijing) favipiravir versus arbidol for covid- : a randomized clinical trial fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study sars-cov- and sars-cov: virtual screening of potential inhibitors targeting rna-dependent rna polymerase activity (nsp ) virtual screening of potential inhibitors for sars-cov- main protease multidrug treatment with nelfinavir and cepharanthine against covid- sofosbuvir as repurposed antiviral drug against covid- : why were we convinced to evaluate the drug in a registered/approved clinical trial nucleotide analogues as inhibitors of sars-cov- polymerase remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro potential inhibitors against -ncov coronavirus m protease from clinically approved medicines lack of antiviral activity of darunavir against sars-cov- arbidol: a potential antiviral drug for the treatment of sars-cov- by blocking trimerization of the spike glycoprotein arbidol monotherapy is superior to lopinavir/ritonavir in treating covid- nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- three cases of treatment with nafamostat in elderly patients with covid- pneumonia who need oxygen therapy in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) potential drugs targeting early innate immune evasion of sars-coronavirus via '-o-methylation of viral rna the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sarscov- spike (s) glycoprotein warranting further evaluation as an antiviral against covid- infections targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and '-o-ribose methyltransferase azithromycin and ciprofloxacin have a chloroquine-like effect on respiratory epithelial cells hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial virtual screening based on molecular docking of possible inhibitors of covid- main protease ruxolitinib in treatment of severe coronavirus disease (covid- ): a multicenter, single-blind, randomized controlled trial beneficial impact of baricitinib in covid- moderate pneumonia; multicentre study deciphering the binding mechanism of dexamethasone against sars-cov- main protease: computational molecular modelling approach in silico studies on therapeutic agents for covid- : drug repurposing approach repurposing ivermectin to inhibit the activity of sars cov helicase: possible implications for covid therapeutics tentolouris n. in vitro data of current therapies for sars-cov- treatment with hydroxychloroquine vs hydroxychloroquine + nitazoxanide in covid- patients with risk factors for poor prognosis: a structured summary of a study protocol for a randomised controlled trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ix un c authors: teixeira, maria c.; carbone, claudia; sousa, maria c.; espina, marta; garcia, maria l.; sanchez-lopez, elena; souto, eliana b. title: nanomedicines for the delivery of antimicrobial peptides (amps) date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: ix un c microbial infections are still among the major public health concerns since several yeasts and fungi, and other pathogenic microorganisms, are responsible for continuous growth of infections and drug resistance against bacteria. antimicrobial resistance rate is fostering the need to develop new strategies against drug-resistant superbugs. antimicrobial peptides (amps) are small peptide-based molecules of – amino acids in length, with potent and broad-spectrum antimicrobial properties. they are part of the innate immune system, which can represent a minimal risk of resistance development. these characteristics contribute to the description of these molecules as promising new molecules in the development of new antimicrobial drugs. however, efforts in developing new medicines have not resulted in any decrease of drug resistance yet. thus, a technological approach on improving existing drugs is gaining special interest. nanomedicine provides easy access to innovative carriers, which ultimately enable the design and development of targeted delivery systems of the most efficient drugs with increased efficacy and reduced toxicity. based on performance, successful experiments, and considerable market prospects, nanotechnology will undoubtedly lead a breakthrough in biomedical field also for infectious diseases, as there are several nanotechnological approaches that exhibit important roles in restoring antibiotic activity against resistant bacteria. the discovery of new antimicrobial molecules in the early s was a landmark in the field of pharmacology allowing the reduction of morbidity and mortality from infectious diseases, which were among the main causes of death worldwide [ ] . however, the widespread and indiscriminate use of nanomaterials , , of burrer et al. have used several strains of murine coronavirus in cell culture and in vivo in mouse models for the assessment of the antiviral properties of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (p-pmos) [ ] . the authors have reported the targeting effect of p-pmos against various target sites in the viral genome in cell culture and protected mice against virus-induced tissue damage. ykeda et al. have used an antimicrobial peptide isolated from the skin of xenopus tropicalis (pxt- ), and its modified peptide (modify-pxt- ) to produce self-assembled discoidal nanoparticles composed of amphiphilic alpha-helical scaffold proteins or peptides organised in a lipid bilayer [ ] . both the peptides pxt- , having hydrophobic and hydrophilic faces, behaved like general surfactants and can be used as carriers. bacteriocins, while very active at low concentrations, usually have low in vivo stability, being susceptible to degradation by proteolytic enzymes [ ] . another major limitation on the use of these peptides is the difficulty encountered in their large-scale production, compromising altogether their clinical application. an approach to overcome their limited stability in vivo is their loading into nanoparticles. nanomedicine is currently a well-established approach intimately related to the design and development of nanomaterials with unique therapeutic and diagnostic properties [ ] . nanotechnologies have also shown great potential in almost every aspect of the management of microbial infection with more than ten nanoparticles (nps)-based products marketed for bacterial diagnosis, antibiotic delivery and medical devices in . with unique physicochemical characteristics, nanomaterials are sensitive and selective in the detection of bacterial signalling and may also exbibit intrinsic antimicrobial properties. furthermore, nanomaterials can be used for antimicrobial drug delivery, and the incorporation of antimicrobial nanomaterials in medical devices and implants can prevent microbial adhesion and infection [ , ] . all these facts are instrumental against antimicrobial resistance by compromising bacterial mechanisms of resistance [ ] . focusing on nanomaterials-based drug delivery systems, these offer an improved strategy to increase the therapeutic index, by decreasing the dosage and frequency of administration. besides, nanomaterials promote intracellular drug delivery, mitigating the development of drug-resistant bacteria and also allowing targeted organ accumulation by functionalized surface modifications, thus limiting systemic side effects and immunosuppression [ ] . despite these promising outcomes, the main challenge of establishing clinical use is related to the evaluation of interactions of nano-antibiotics with cells, tissues and organs achieving information about their possible toxic effects, together with the production on a large scale [ , , ] . several types of nanomaterials have shown potential in the pharmaceutical field. they have also been studied as potential drug carriers with applications in the delivery of amps, promising antimicrobial molecules which, due to their nature and physicochemical characteristics, have limited bioavailability. the aim of this work is to revise the state-of-the-art on the approach that combines the advantages of the design of new drug delivery systems for the improvement of antimicrobial bioavailability, taking into account the recent developments in nanomaterials for antimicrobial peptide delivery. amps are small natural oligopeptides that have recently showed a potential activity against antibiotics resistance mechanisms, due to their ability in lysing bacterial membranes, thus providing broad-spectrum effects, targeting microorganisms from viruses to parasites. the main interesting property of amps is their ability to selectively disrupt bacterial membranes without affecting mammalian cells, thus being safe. in addition, amps are referred to in the literature as host-defence peptides with anti-inflammatory and anticancer activities, because they are synthesized molecules that take action on the defence mechanisms against biological threats of the living organism of origin [ ] . the discovery of amps dates back to the first half of the th century when in , dubos extracted an antimicrobial agent from a soil bacillus strain, proven to be effective in mice pneumococci infection. this extract was then fractioned allowing the identification of gramicidin. despite some systemic toxicity, gramicidin has shown to be effective in the topical treatment of wounds and ulcers. the first animal-originated amp to be reported is defensin, isolated from rabbit leukocytes in [ ] . nowadays, more than amps have been described and current molecular developments can be consulted in a series of databases available on the web (table ) , including natural identified molecules, as well as peptidomimetic molecules and analogues that are pharmacologically designed thanks to the use of bioinformatics [ ] . current food and drug administration (fda)-approved amps with well-established use include bacitracin, colistin and polymyxin b (although only for topical administration) [ ] . despite the lack of consensus on the influence of peptide sequence in biologic activity of amps, some common characteristics seem to be important and fairly related to their antimicrobial property. the main one is primarily charge, with % of amps being cationic, and secondly, hydrophobicity or amphipathicity, influencing solubility profiles and consequently bioavailability [ ] . associated with structural characteristics, these are also the main physicochemical properties that should also be taken into account in the design of new synthetic amps [ ] . compared to conventional antibiotics, amps demonstrate significant advantages such as potency and board spectrum of activity, as well as an additional activity to modulate the immune system responses and low resistance rates. they also show some limitations that impair their safe therapeutic use, such as sensitivity to proteolysis, influencing stability and undefined toxicological data for systemic use [ ] . in addition, their cationic and amphiphilic nature lead to high binding to serum proteins after parenteral administration, with consequent rapid elimination from bloodstream circulation and accumulation in the reticuloendothelial system, thus resulting in toxic effects and reduced activity [ ] . to overcome these limitations, some strategies have been developed, maximizing the proven therapeutic potential of amps. there are numerous methods for obtaining amp delivery systems. amps could be immobilized onto a variety of materials or onto a variety of surfaces and still retain their antibacterial activity. also, amps can be targeted through loading them in nanoparticulate systems with selective delivery capacities, including polymers, liposomes, hydrogels, nanospheres, nano-capsules and carbon nanotubes [ ] . as practical examples on of these of strategies, the studies of mishra et al. were focused on the development of an amp-coated surface, specifically immobilizing lassioglossin-iii onto silicone-based catheters and its activity against urinary tract infections [ ] . at the same time, water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). nanomaterials , , x for peer review of water et al. developed the possibility of encapsulating plectasin, a cationic amp, into polymeric nps and evaluated their efficacy on s. aureus strains [ ] . bacteria show resistance to antibiotics drugs through a variety of mechanisms. moreover, the development of even new mechanisms of resistance has resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (mdr) bacterial strains [ , ] . however, when bacteria are drug-resistant it does not mean that they stop responding to an antibiotic, but that occurs only at higher concentrations [ , ] . of greater concern are cases of acquired resistance, where initially susceptible populations of bacteria become resistant to an antibacterial agent, in particular antibiotics, and proliferate and spread under the selective pressure of use of that drug. one approach to address this challenge is to design analogues of drugs [ , ] that are already in clinical use and that have activity against resistant organisms. however, bacteria are constantly succeeding to develop a resistant mechanism to new antibiotic drugs as well as to their analogues [ , ] . nanotechnology offers opportunities to re-explore the biological properties of already known antimicrobial compounds such as antibiotics by manipulating their size to modify their effect. the combination of amps with nanomaterials is not new. out of the available , papers indexed in the web of science, a total of , combining amps with nanoparticles or nanomaterials have been published over the last years in a range of disciplines ( figure ). evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever evidence collected in the review work of huh and kwon [ ] , pelgrift and friedman [ ] , brooks and brooks [ ] , diab et al. [ ] and shimanovich and gedanken [ ] clarifies microbial drug resistance mechanisms and how nanotechnology may be considered a tool against this issue. development of drug resistance occurs in (at least) three steps: (i) acquisition by microbes of resistance genes; (ii) expression of those resistance genes; (iii) selection for microbes expressing those resistance genes. first, bacteria acquire resistance to single and multiple drugs through horizontal gene transfer by transformation, conjugation and transduction [ ] . bacteria can also acquire resistance genes by spontaneous mutation of existing genes [ ] . mdr is acquired when a bacterial cell already containing one type of drug resistance gene acquires another type of drug resistance gene [ , ] . second, in response to exposure to an antimicrobial drug, microbes express the resistance gene [ ] . third, resistance becomes widespread when there is selection for microbes that express resistance genes against the antimicrobial drug. this selective pressure in favour of resistance occurs whenever microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug nanomaterials , , of followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: (i) controllable and relatively uniform distribution in the target tissue; (ii) improved solubility; (iii) sustained and controlled release; (iv) improved patient-compliance; (v) minimized side effects; and (vi) enhanced cellular internalization [ , ] . nanomaterials , , x for peer review of microbes are exposed to the drug but not eradicated (either by the microbicidal effects of the drug itself or by microbiostatic effects of the drug followed by killing by the host's immune system) [ ] . a schematic representation of some specific mechanisms of antimicrobial drug resistance is shown in figure . nanomaterials ( figure ), which either show antimicrobial activity by themselves or elevate the effectiveness and safety of antibiotics administration [ , ] , are called "nano-antibiotics" and their capability of controlling infections in vitro and in vivo has been explored and demonstrated. unlike many antimicrobial agents currently being used in the clinic, antimicrobial nps may not pose direct and acute adverse effects, although potential toxicity upon long-term exposure is questionable. most importantly, antimicrobial nps tackle multiple biological pathways found in broad species of microbes and many concurrent mutations would have to occur to develop resistance against nps' antimicrobial activities. preparation of antimicrobial nps could be cost-effective, compared to antibiotics synthesis, furthermore they are stable enough for long-term storage with a prolonged shelf-life [ ] . in addition, some nps can withstand harsh conditions, such as high-temperature sterilization, under which conventional antibiotics are inactivated. antibiotics delivery using nanomaterials offer multiple advantages: i) controllable and relatively uniform distribution in the target tissue; ii) improved solubility; iii) sustained and controlled release; iv) improved patientcompliance; v) minimized side effects; and vi) enhanced cellular internalization [ , ] . current advances in the use of inorganic nanoparticles for amp delivery involve essentially the development of silver nanoparticles (agnps) and gold nanoparticles (aunps), as well as silicon derivates nano-systems [ ] . the studies revised in this work are summarized in table . the development of polymeric nanoparticles for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety, avoiding drug chemical and enzymatic degradation, preventing aggregation, enhancing controlled release. chitosan (cs) nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in recent years, an increasing number of papers reporting on a new generation of antimicrobial metallic nps has been published [ ] . consequently, many of the information on the application of nanotechnology in the infectious disease field regards the use of silver (ag) and gold (au) nps [ , , ] . recently, derivatives of other metals have been studied for antimicrobial applications, and the antibacterial effects of zero-valent bismuth nps and uncoated au, nickel (ni) and silicon (si) nps were reported [ , ] . despite the demonstrated intrinsic antimicrobial properties, dispersed metallic nps tend to aggregate and separate in solution, resulting in a decrease in their antimicrobial efficiency. with the aim of improving antibacterial properties, functionalization of nps has been attempted with surfactants, polymers or antibiotics resulting in more stable, less aggregated nps suspension and innovative, synergistic antibacterial agents. for instance, silver nps stabilized by polymers(polyvinylpyrrolidone) and surfactants (sds and tween ) exhibit enhanced antibacterial activities [ ] . nps can also act as drug-carriers able to pass through cell membranes [ , ] . widely used antibiotics such as ciprofloxacin may benefit from the association with nps, and conjugation may result in an antibacterial effect also against micro-organisms resistant to the same molecule in the naturally occurring form [ ] . when the antimicrobial agents are covalently linked to or contained within, nps, a higher drug concentration is attained in the area of interest, resulting in better efficacy at comparable doses and in slower release over time that may be exploited for preventing bacterial colonization [ , ] . moreover, specific biological sites can be attacked after modification of nps with target molecules [ , ] . as the nps themselves may have antibacterial properties, the combination of nps and loaded drugs exert a synergistic action [ ] . current advances in the use of inorganic nanostructures for amp delivery involve essentially the development of ag and aunps, as well as silicon derivate nano-systems. the studies revised in this work are summarized in table . research on gold nps is increasing thanks to their many advantages, such as their ease of synthesis and conjugation to biomolecules, their capability to maintain their own structure when in circulation and their improved effectiveness against bacteria, thus demonstrating their high potential in the field of nanomedicine. even if at the beginning, the research in this field was focused on the possibility to exploit gold nps in combination to laser radiation, thus significantly reducing bacteria viability due to cell lysis and mechanical disruption [ ] . currently, two recent studies have been reported exploring the use of au nanostructures for amp delivery. photoluminescent au nanodots (aunds) were prepared by chen et al. [ ] . these aunds were functionalized with hybridized ligands, an antimicrobial peptide (surfactin; sft), and -dodecanethiol (dt), on aunps. ultrasmall sft/dt-au nds (size ≈ . nm) were achieved and exhibited highly efficient antimicrobial activity. the photoluminescence properties and stability as well as the antimicrobial activity of sft/dt-au nds were also studied, and it was shown that these characteristics are highly dependent on the density of sft on au nds. relative to sft, sft/dt-aunds exhibited greater antimicrobial activity, not only to non-multidrug-resistant bacteria but also to the multidrug-resistant bacteria. the minimal inhibitory concentration values of sft/dt-aunds were much lower (> -fold) than that of sft. the authors considered that the antimicrobial activity of sft/dt-aunds was mainly achieved by the synergistic effect of sft and dt-aunds on the disruption of the bacterial membrane. in vitro cytotoxicity and hemolysis, analyses were also performed and had revealed superior biocompatibility of sft/dt-aunds than that of sft. moreover, in vivo methicillin-resistant s. aureus infected wound healing studies in rats showed faster healing, better epithelialization. this study suggested that the sft/dt-aunds system may be a promising antimicrobial candidate for preclinical applications in treating wounds and skin infections [ ] . rai et al. also reported a one-step methodology to generate amp-conjugated (aunps) [ ] . the amp-conjugated aunps prepared showed controlled size ( nm) and low polydispersity and allowed the inclusion of high concentration of amps. further, these systems demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble amp, as well as low cytotoxicity against human cells [ ] . interestingly, akrami et al. showed the possibility to exploit gold nps as a novel anticancer platform [ ] . results of this research confirm the improvement of cell internalization of gold nps, with higher cytotoxicity and cellular uptake for smaller nps compared to larger nanospheres and nanorods, suggesting that anticancer effects of the selected peptides were modulated by the size and shape of the gold nanoparticles [ ] . geilich et al. developed novel delivery systems by combining silver nps and ampicillin so to achieve a synergistic dose-dependent effect on bacterial cells [ ] . the obtained polymersomes were safe on human fibroblasts and more effective in inhibiting bacterial cells with a silver-to-ampicillin ratio of one to . , respectively. recent advances in amp delivery by ag nanostructures, pazos et al. [ ] reported on supramolecular assemblies of novel peptide amphiphiles (pas) containing aldehyde functionality in order to reduce ag ions and subsequently nucleate ag metal nps in water. this proposed system spontaneously generates monodisperse ag particles at regular distances along the length of the filamentous organic assemblies. the metal−organic hybrid structures exhibited antimicrobial activity and significantly less toxicity toward eukaryotic cells. metallized organic nanofibers of the type described offer the possibility to create other structures. for instance, hydrogels, that can be potentially applied in wound dressing development [ ] . also addressing the wound infection problem, salouti et al. investigated the synergistic antibacterial effect of plant peptide mbp- and agnps on infected wounds caused by s. aureus [ ] . the mic and mbc of mbp- and agnps both on their own and in combination form were determined against s. aureus via macrodilution and microdilution methods. the mic and mbc of mbp- were found to be . and . mg/ml, respectively. mic and mbc of agnps were determined to be . and . mg/l, respectively. mic and mbc of the agnps and mbp- combination were found to be . mg/ml, . mg/l; and . mg/ml, . mg/l, respectively. the synergistic antibacterial effect of ag nps and mbp- was investigated on infected wounds caused by s. aureus in a mouse model, and the infected wound healed properly after the combined use of mbp- and agnps [ ] . it is worth to note recent findings by chaudhari et al. concerning the conjugation of silver-coated carbon nanotubes with the antimicrobial peptide tp . herein, authors underline the occurrence of an additive effect of silver-coated nanotubes and tp regarding antibacterial activity. the innovative nano-system was found to be safe to murine macrophages and hep cells at the mic concentrations [ ] . as delivery systems for amps, silicon and silicon derivates nanostructures have also been investigated recently. membrane interactions are critical for the successful use of mesoporous sinps. in order to elucidate these, braun et al. have studied the effects of np charge and porosity on amp loading and release, as well as consequences of this for membrane interactions and antimicrobial effects [ ] . anionic mesoporous sinps were found to incorporate considerable amounts of the camp ll- , whereas loading was found to be much lower for non-porous or positively charged sinps. the results also demonstrated that due to preferential pore localization, anionic mesoporous particles, but not the other particles, protects ll- from degradation by infection-related proteases. for anionic sinps, membrane disruption is mediated almost exclusively by peptide release. in contrast, non-porous sinps built up a resilient ll- surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. for positively charged mesoporous sinps, ll- incorporation promoted the membrane binding and disruption displayed by the particles in the absence of peptide, but also caused toxicity against human erythrocytes. thus, the use of mesoporous sinps as amp delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nps [ ] . the properties of amps adsorbed on inorganic or organic surfaces are of interest for their potential applications in intracellular drug delivery. in the work of syryamina et al., continuous-wave (cw) electron paramagnetic resonance (epr) and pulsed electron-electron double resonance (peldor) techniques were applied to study the adsorption of the amps trichogin ga iv and ampullosporin a on monodisperse colloidal silica nanospheres (sins) of nm diameter [ ] . the results obtained by cw epr supported the view that the adsorbed peptides form close-packed clusters. peldor data show that both trichogin and ampullosporin adsorbed on the silica surface possess a more disordered conformation as compared to that in solution. for ampullosporin, disordering is much more pronounced than for trichogin. after desorption, the peptides restored their conformations; upon adsorption, the peptides in some cases may lose partly their biradical character [ ] . these results may be of interest as the antimicrobial activity is often related to peptide conformation. nano-clays or layered silicates are an interesting nanostructure that has been used for remediation of environmental contaminants, delivery of drugs and various active molecules, and to enhance polymer mechanical and barrier properties in packaging films. they typically present a stacked arrangement of silicate layers with a nanometric thickness [ ] . meira et al. studied three different nano-clays (bentonite, octadecylamine-modified montmorillonite, and halloysite) as potential carriers for the amps nisin and pediocin, known bacteriocins, the first referred above as having application as a food preservative. higher adsorption at room temperature of nisin and pediocin was obtained on bentonite. the antimicrobial activity of the resultant bacteriocin-nano-clay systems was analysed using skimmed milk agar as food simulant, and the largest inhibition zones were observed against gram-positive bacteria for halloysite samples. bacteriocins were intercalated into the interlayer space of montmorillonites as deduced from the increase of the basal spacing measured by x-ray diffraction (xrd) assay. these results indicate that nano-clays, especially halloysite, are suitable nano-carriers for nisin and pediocin adsorption, and the results may be considered interesting for the food industry [ ] . different biodegradable polymeric nano-systems have been explored as carriers for antimicrobial agents that exhibit a high bactericidal activity. the efficacy of this strategy is well proven, highlighting polymeric nano-systems can effectively improve the cellular penetration, intracellular retention and specific subcellular distribution of antimicrobial agents, and even evade intracellular inactivation of antimicrobial agents [ ] . controlled drug release using biocompatible and biodegradable polymers further emerged in the s [ ] . after the first polymer-based delivery of macromolecules using poly[ethylene vinyl acetate] polymer was demonstrated in [ , ] . antimicrobial drug delivery using polymeric nps offers several advantages: (i) structural stability in biological fluids and under harsh and various conditions for preparation; (ii) precisely tuneable properties, such as size, zeta-potentials, and drug release profiles, by manipulating polymer lengths, surfactants, and organic solvents used for np preparation [ ] , and (iii) facile and versatile surface functionalization for conjugating drugs and targeting ligands [ ] . concerning antimicrobial nanoparticulate delivery systems, two major types of polymers have been explored: linear polymers (e.g., polyalkyl acrylates and polymethyl methacrylate) and amphiphilic block copolymers. the majority of polymeric nps prepared with linear polymers are nano-capsules or solid nanospheres [ ] . in polymeric nano-capsules, a polymeric membrane that controls the release rate surrounds the drugs that are solubilized in aqueous or oily solvents. in solid nanospheres, drugs are homogeneously distributed in the polymeric matrices of variable porosities [ , ] . amphiphilic block copolymers form self-assemble micellar nps with the drug being encapsulated in the hydrophobic core and surrounded by a hydrophilic shield. this shied allows the core to be protected from degradation [ ] . several biodegradable polymers, including poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(lactide-co-glycolide) (plga), poly(caprolactone) (pcl), and poly(cyanoacrylate) (pca), have been used as the hydrophobic core of the amphiphilic copolymers, whereas peg has been the most commonly used hydrophilic segment [ , [ ] [ ] [ ] [ ] . targeting ligands can also be conjugated on the termini of peg for targeted and selective delivery [ , ] . polymeric nps have been explored to deliver various antimicrobial agents and greatly enhanced therapeutic efficacy in treating many types of infectious diseases has been reported. for instance, encapsulated ampicillin in polymeric nps was effective for s. typhimurium infection treatment [ ] and intracellular l. monocytogenes infection in mouse peritoneal macrophages [ ] . the use of polymeric nps can overcome the limited oral administration of unstable or inadequately absorbed drugs, [ ] and, in addition, pegylation of nps, can increase drug half-life in serum, and improve mucoadhesive capabilities by reducing phagocytosis [ ] thus, among nanoparticle platforms, polymer nps may be the most suitable system that can be used for antimicrobial drug delivery. biodegradable polymers and bioorganic polymers are also promising materials in the delivery of peptide-based drugs due to their compatibility, degradation behaviour, and nontoxic nature of administration [ ] . the development of polymeric therapeutic nanostructures for amp delivery may offer an excellent technological strategy to improve drug bioavailability and safety. cs-based nps (csnps) are particularly interesting as the broad spectrum of antibacterial activity of cs is well known and documented, offering the possibility of synergistic effects with antimicrobial molecules. moreover, due to its biocompatibility properties, cs nanostructures have been extensively studied for drug delivery, and that is no different for amp delivery. in fact, the majority of the studies performed so far involves cs nanostructures and was already revised in previous work [ ] from which we reproduce table . research has been covering, the technological development of new carrier systems and their full characterization, and the evaluation of their efficacy as drug delivery improvers. in addition to cs-based nanostructures, recent studies on other polymeric nanostructures for amp delivery have been developed (table ). cs-based nanoparticles vancomycin cs particles were prepared by ionic gelation and freeze-drying or spray-drying as recovery methods. antibacterial activity against s. aureus. cerchiara et al. [ ] cs-based nanoparticles lysozyme as model development of a nanoparticle model with commercially available cs loaded with lysozyme as antimicrobial protein drug model. piras et al. [ ] cs and poly-gammaglutamic acid composites ll- the results indicated that both ll- and no were co-loaded successfully in micro particles, and the composite particles could sustain ll- and no release at physiological ph, in vitro. cryptdin- preparation of cs tripolyphosphate (cs-tpp) nps by ionotropic gelation. the formulation was then characterized on the basis of particle size, zeta potential and polydispersity, and antimicrobial in vivo assays against salmonella enterica were performed. cs-based nanoparticles temporin b cs-nps were prepared based on the ionotropic gelation between cs and sodium tripolyphosphate. the nano-carrier evidenced a sustained antibacterial action against various strains of s. epidermidis. nanogel composites -prepation of agnps embedded in a biocompatible nanogel comprising degradable, natural polymers. in this study, hybrid nanogels were prepared with varying polymer content and their potential by determining their antibacterial properties against e. coliand s. aureus strains. coll ferrer et al. [ ] glycol-cs nanogels -study of the biocompatibility of a glycol cs nanogel by evaluation of effects on metabolic activity, cell cycles blood compatibility. overall, the results demonstrated the safety of the use of the gc nanogel as drug delivery system. nanofibers and films cs thin films hlf - immobilization performed onto cs thin films as a model for an implant coating due to its reported osteogenic and antibacterial properties. cs thin films were produced by spin-coating on au surfaces. activity against methicillin-resistant s. aureus (mrsa). nanofibers defensin- , dermaseptin ll- magainin alternate deposition of polycation (cs) and polyanion over cotton gauzes. antimicrobial assays were performed with two strains: s. aureus and k. pneumonia. food packaging systems study of the efficiency as antimicrobial carriers of hydroxypropyl methylcellulose [ ] , chitosan (cs), sodium caseinate (sc) and polylactic acid [ ] films, in the release rates of fluorescently labeled nisin z, evaluating their potential as food packaging polymers. imran et al. [ ] nanomaterials , , of plla-l -pllain situ gel ap- potential application of amps in wound healing, by developing a biodegradable poly (l-lactic acid)-pluronic l -poly (l-lactic acid) (plla-l -plla) in situ gel-forming system li et al. [ ] food preservation systems nisin study of a safe suitable antimicrobial system to be used in food industry. influence of pectin degree of acetylation on np properties. krivorotova et al. [ ] chitosan/pga nanoparticles nisin influence of chitosan coating on colloidal stability, loading capacity and encapsulation efficiency wu et al. [ ] d'angelo et al. designed and developed a system of nano-embedded microparticles (nem) for sustained delivery of cationic amps (camps) [ ] in the lung, studying its effect on p. aeruginosa, a known lung infection pathogen. to this purpose, plga nps containing a model camp, colistin (col), were produced by the emulsion/solvent diffusion technique, and then spray-dried in different carriers (lactose or mannitol), thus producing nem. the most promising nem formulations were selected from bulk and flow properties, distribution of nps in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. col-loaded nem were found to kill p. aeruginosa biofilms and to display a prolonged efficacy compared to the free col. [ ] . another camp, plectasin, was encapsulated into plga nps using the double emulsion solvent evaporation method, in the work of water et al. [ ] the plectasin-loaded nps displayed a high encapsulation efficiency ( - %) and mediated release of the peptide over h. the antimicrobial efficacy was investigated using bronchial epithelial calu- cell monolayers infected with s. aureus, and encapsulated plectasin displayed improved efficacy as compared to non-encapsulated plectasin. the author also assessed the subcellular localization of the prepared nps in different relevant cell lines: calu- epithelial cells, thp- macrophages and a epithelial cells. here the results have shown good patterns of penetration on calu- epithelial cell lines, as well as in thp- macrophages [ ] . hydrogels and nanogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. the biological performance of these systems, particularly those used as wound dressing; can be complemented with antimicrobial activity capable of preventing colonization of the wound site by opportunistic bacterial pathogens [ , , ] . these types of structures have also been studied recently for amp delivery. continuing their study of the antimicrobial activity of multi-domain camps (md-camps) in solution, jiang et al. investigated the same effect of self-assembled -d hydrogels supramolecular nanostructures and its rheological properties [ ] . among the studied md-camps solutions, the bactericidal activity of peptide hydrogels was found to be improved. the improved antimicrobial activity of the self-assembled peptide hydrogels was found to be related to the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. thus, the structure-property-activity relationship developed through this study may provide important knowledge for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications [ ] . the water et al. group also designed novel nanogel-based novicidin delivery system. the peptide novicidin was self-assembled with a nano-ctenyl succinic anhydride-modified analogue of hyaluronic acid, and this formulation was optimized using a microfluidics-based quality-by-design approach. the encapsulation efficiency of novicidin ( - %) and the zeta potential (− to − mv) of the nanogels could be tailored by changing the preparation process parameters, with a maximum peptide loading of ± %. the nanogels exhibited good colloidal stability under different ionic strength conditions and allowed complete release of the peptide over days. furthermore, self-assembly of novicidin with hyaluronic acid into nanogels significantly improved the safety profile at least five-fold and six-fold when tested in huvecs and nih t cells, respectively, while showing no loss of antimicrobial activity against e. coli and s. aureus [ ] . li et al. explored the potential application of amps in wound healing, by developing a biodegradable poly(l-lactic acid)-pluronic l -poly(l-lactic acid) (plla-l -plla) in situ gel-forming system [ ] . an injectable formulation composed of human amps (ap- ) loaded nps, and thermosensitive hydrogel was prepared. ap- peptides were enclosed with biocompatible nps (ap- -nps) with high drug loading and encapsulation efficiency. ap- -nps were further encapsulated in a thermosensitive hydrogel (ap- -nps-h) to facilitate its application in cutaneous wound repair. as a result, ap- -nps-h released ap- in an extended period and exhibited quite low cytotoxicity and high anti-oxidant activity in vitro. the in vivo wound healing assay using a full-thickness dermal defect model of sd rats indicated that ap- -nps-h could significantly promote wound healing. at day after an operation, the treated group showed nearly complete wound closure of , ± , % [ ] . other studies of nisin nanoencapsulation were performed, with the purpose of protection to ensure the stability of this amp during food processing and storage period. nisin-loaded pectin nps (nlp-nps) were prepared and analysed by krivirotova et al. by a simple complexation method [ ] . three types of pectin biopolymer were tested and it was found that the methoxylation degree of pectin influenced nisin loading efficiency and particle size. for the complex formation, both electrostatic and hydrophobic interactions were important. nlp-nps exhibited antimicrobial activity that was dependent on the type of biopolymer. overall, the results indicated that nlp-nps might be a suitable antimicrobial system to be used in the food industry [ ] . nisin nanoencapsulation in self-assembly chitosan-and poly-glutamic acid, was recently reported by wu et al. [ ] . herein, the authors showed that the use of a coating layer of chitosan improved colloidal stability, loading capacity and encapsulation efficiency. furthermore, chitosan layer composite nanoparticles were more effective compared to uncoated nps and nisin in inhibiting the growth of escherichia coli and listeria monocytogenes. solid lipid nanoparticles (sln) and nanostructured lipid carriers (nlc) stand for nanoparticles composed of lipid materials that melt at temperatures above • c [ ] . their main purpose is to modify the release profile of the payload attributed to their solid core. while sln are composed of solid lipids only, nlc matrix is a blend of solid and liquid lipids (which also melts above the body temperature) to improve the solubility of lipophilic compounds. both sln and nlc are biodegradable and non-toxic [ ] [ ] [ ] , and their versatility of is also attributed to their capacity to load a range of chemically different bioactives, including peptides and proteins [ ] [ ] [ ] [ ] , and be modified on their surface [ , ] . fumakia et al. have developed sln for the simultaneous delivery of an endogenous host defence peptide (ll ) with antimicrobial activity and an elastase inhibitor serpin a (a ) for the treatment of wound infections [ ] . the authors reported that sln could modify the release profile of simultaneous delivery of both bioactives, accelerate wound healing in bj fibroblast cells and keratinocytes and promote antibacterial activity against staphylococcus aureus and escherichia coli in comparison to ll or a alone. ll loaded into nlc have also been proposed by garcia-orue for the topical treatment of chronic wounds [ ] . ll -loaded nlc did not affect cell viability tested against human foreskin fibroblasts while the in vitro bioactivity assay showed that the peptide remained active after the loading into lipid nanoparticles as the system reversed the macrophages activation as happened with the ll in solution. ll -loaded nlc were also active against escherichia coli. the in vivo testing in mice also demonstrated the systems' capacity to improve healing by promoting wound closure, reepithelization grade and restoration of the inflammatory process. lewies et al. demonstrated that biodegradable nlcs enhance the antibacterial activity of antimicrobial peptides [ ] . the authors have studied the effect of nisin z when loaded into nanostructured lipid carriers on staphylococcus aureus, staphylococcus epidermidis and escherichia coli. nisin z exhibited additive interactions with numerous conventional antibiotics, in particular, with novobiocin. the presence of edta improved the antimicrobial activity of free nisin z towards escherichia coli significantly. nisin z-loaded nlcs were effective against gram-positive species at physiological ph, also showing an increased efficacy when adding edta. nisin z-loaded nlcs were shown potential to enhance the antibacterial activity of nisin z towards gram-positive bacteria commonly found in skin infections. other types of nanomaterials, such as dendrimers and carbon nanodots, have also been successfully proposed for the delivery of amps. due to their ease of synthesis and low manufacturing costs, antimicrobial polymers including dendrimers have been exploited to mimic the antibacterial mechanism host defence peptides, by compromising bacterial cell membranes [ ] . gide et al. developed nanomaterials-lipidated amphiphilic dendrimers which displayed potent and selective antimicrobial activity against both gram-positive and gram-negative bacteria, including multidrug-resistant strains [ ] . these dendrimers are also shown to inhibit bacterial biofilms effectively. carbon nanodots or carbon quantum dots were originally discovered due to their bright fluorescence emissions as those found in conventional semiconductor quantum dots, and can also be used for drug delivery [ ] . carbon dots modified with vancomycin were proposed for the treatment of gram-positive bacterial infections [ ] . zhong et al. synthesized surface-modified carbon dots with vancomycin and successfully tested them against bacillus subtilis, listeria monocytogenes, salmonella, pseudomonas aeruginosa and escherichia coli. the modified carbon dots showed affinity to the tested gram-positive bacteria owing to the ligand-receptor interactions between vancomycin and the cell walls. pramanik et al. synthesis red/blue fluorescent carbon dot-attached magnetic nanoparticles for selective separation and identification of superbugs from infected blood samples [ ] . the nanoparticles were capable of isolating methicillin-resistant staphylococcus aureus (mrsa) and salmonella dt superbug from whole blood samples, followed by accurate identification via multicolor fluorescence imaging. the authors also described the design of antimicrobial peptide-conjugated multicolor fluorescent magneto-carbon dots for separation, identification and disinfection of multidrug resistance superbugs from infected blood. amps are antimicrobial compounds recognized as among the most promising drug candidates against infections. they exhibit distinct mechanisms of action and may show additional biological activities, e.g., as signalling molecules and biomarkers, and even as tumoricidal agents. amps, however, show low stability and bioavailability. the formulation of amps into nanomaterials seems to offer a very appealing and effective manner to improve these limitations, and several systems have been designed and studied for this purpose (e.g., inorganic, polymeric and lipid nanoparticles, carbon dots and dendrimers). however, concerns on how to regulate the distribution of nanomaterials in the body or specific organs are also raised. nano-drugs are foreign substances to the body and may produce inflammation. therefore, safety data for long-term therapy or repeated dosage are needed to circumvent the potential risk. biodegradable nanomaterials have been proposed to reduce the risk of toxicological events both in vitro and in vivo. to date, few studies have investigated the toxicological and environmental effects of direct and indirect exposure to nanomaterials, and no clear guidelines exist to quantify these effects. therefore, there is an urgent need for developing guidelines, which can assure the safe use of nanomaterials. moreover, more powerful ex vivo models or animal models are needed to assess the safety issues and to comply with government regulations. how to extend the shelf life of nanodrugs is also a problem due to their agglomeration also being a problem. the production methods for nanostructures should also be improved, and scalable studies for industrial production are also of great importance in order to promote cost effectiveness of these new formulations. the cost and production of nanomaterials on a large scale is one of the hurdles in effective implementation of these products. hence, the scientific community should also pay attention to developing affordable methodologies so that nanotechnology can reach patients. in conclusion, it seems that, although the promising research results in this area are rising, it is also urgent to start directing efforts in making these new drug formulations a reality as therapeutic agents. nanoantibiotics": a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era therapeutic strategies to combat antibiotic resistance nanotechnology as a therapeutic tool to combat microbial resistance potent antimicrobial peptides and the fight against multi drug resistant species: resistance is futile? antibiotics antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and nanosystems as vehicles for the delivery of antimicrobial peptides (amps) antiviral effects of antisense morpholino oligomers in murine coronavirus infection models lipid nanodisc formation using pxt- peptide isolated from amphibian (xenopus tropicalis) skin, and its altered form, modify-pxt- nanomedicines for antimicrobial interventions silver nanoparticles-composing alginate/gelatin hydrogel improves wound healing in vivo development of chitosan/silver sulfadiazine/zeolite composite films for wound dressing nanomedicine in the management of microbial infection-overview and perspectives nanopharmaceutics: part ii-production scales and clinically compliant production methods nanopharmaceutics: part i-clinical trials legislation and good manufacturing practices (gmp) of nanotherapeutics in the eu delivery systems for antimicrobial peptides antimicrobial peptides antimicrobial peptides: an alternative for innovative medicines? antimicrobial peptides: possible anti-infective agents therapeutic peptides: new arsenal against drug resistant pathogens effects of pegylation on membrane and lipopolysaccharide interactions of host defense peptides nanomaterials site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens nanoparticle-mediated delivery of the antimicrobial peptide plectasin against staphylococcus aureus in infected epithelial cells the rise of the enterococcus: beyond vancomycin resistance the future of antibiotics and resistance selection of resistant bacteria at very low antibiotic concentrations a self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics sampling the antibiotic resistome bacteria on the rampage resistance to antibiotics mediated by target alterations tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. microbiol insights in nanoparticle-bacterium interactions: new frontiers to bypass bacterial resistance to antibiotics nanotechnology solutions to restore antibiotic activity antibacterial properties of nanoparticles effects of salt stress on the antimicrobial drug resistance and protein profile of staphylococcus aureus antibiotic resistance: an overview of mechanisms and a paradigm shift nanotechnology based anti-infectives to fight microbial intrusions metal-based nanoparticles as antimicrobial agents: an overview self-assembly of antimicrobial peptides on gold nanodots: against multidrug-resistant bacteria and wound-healing application one-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model haririan, i. tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms silver nanoparticle-embedded polymersome nanocarriers for the treatment of antibiotic-resistant infections nucleation and growth of ordered arrays of silver nanoparticles on peptide nanofibers: hybrid nanostructures with antimicrobial properties nanomaterials synergistic antibacterial activity of plant peptide mbp- and silver nanoparticles combination on healing of infected wound due to staphylococcus aureus a novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides peptides on the surface: spin-label epr and peldor study of adsorption of the antimicrobial peptides trichogin ga iv and ampullosporin a on the silica nanoparticles adsorption of nisin and pediocin on nanoclays design and characterization of chitosan/zeolite composite films-effect of zeolite type and zeolite dose on the film properties zerovalent bismuth nanoparticles inhibit streptococcus mutans growth and formation of biofilm photogeneration of reactive oxygen species on uncoated silver, gold, nickel, and silicon nanoparticles and their antibacterial effects aggregation kinetics of citrate and polyvinylpyrrolidone coated silver nanoparticles in monovalent and divalent electrolyte solutions oligonucleotide-modified gold nanoparticles for intracellular gene regulation the effects of size, shape, and surface functional group of gold nanostructures on their adsorption and internalization by cells investigations of the antibacterial properties of ciprofloxacin@sio in vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the escherichia coli o ab toxins gold nanocages covered by smart polymers for controlled release with near-infrared light selective binding of mannose-encapsulated gold nanoparticles to type pili in escherichia coli role of size scale of zno nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells silver-nanoparticle-embedded antimicrobial paints based on vegetable oil photothermal killing of staphylococcus aureus using antibody-targeted gold nanoparticles antimicrobial nanostructures in food packaging biodegradable nanoparticles for intracellular delivery of antimicrobial agents biodegradable polymeric nanoparticles as drug delivery devices polymers for the sustained release of proteins and other macromolecules controlled release of microquantities of macromolecules development of nanoparticles for antimicrobial drug delivery nanotechnology applied to the treatment of malaria new old challenges in tuberculosis: potentially effective nanotechnologies in drug delivery poly(alkylcyanoacrylates) as biodegradable materials for biomedical applications drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles innovative hybrid vs polymeric nanocapsules: the influence of the cationic lipid coating on the " s eco-friendly aqueous core surface-modified nanocapsules polymeric nanoparticles augment the ocular hypotensive effect of melatonin in rabbits revisiting the role of sucrose in plga-peg nanocarrier for potential intranasal delivery formulation of functionalized plga-peg nanoparticles for in vivo targeted drug delivery presenting vancomycin on nanoparticles to enhance antimicrobial activities improved oral therapeutic potential of nanoencapsulated cryptdin formulation against salmonella infection effect of nanoparticle-bound ampicillin on the survival of listeria-monocytogenes in mouse peritoneal-macrophages preparation, characterization and in vitro antimicrobial activity of ampicillin-loaded polyethylcyanoacrylate nanoparticles amoxicillin-loaded polyethylcyanoacrylate nanoparticles: influence of peg coating on the particle size, drug release rate and phagocytic uptake chitosan and its derivatives for drug delivery perspective chapter -delivery of antimicrobials by chitosan-composed therapeutic nanostructures a -ficai hollow chitosan/alginate nanocapsules for bioactive compound delivery chitosan based micro-and nanoparticles for colon-targeted delivery of vancomycin prepared by alternative processing methods preparation, physical-chemical and biological characterization of chitosan nanoparticles loaded with lysozyme chitosan microparticles ionically cross-linked with poly(gamma-glutamic acid) as antimicrobial peptides and nitric oxide delivery systems chitosan nanoparticles loaded with the antimicrobial peptide temporin b exert a long-term antibacterial activity in vitro against clinical isolates of staphylococcus epidermidis designing nanogel carriers for antibacterial applications biocompatibility of a self-assembled glycol chitosan nanogel incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applications controlled release of nisin from hpmc, sodium caseinate, poly-lactic acid and chitosan for active packaging applications overcoming barriers in pseudomonas aeruginosa lung infections: engineered nanoparticles for local delivery of a cationic antimicrobial peptide self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity hyaluronic acid-based nano gels produced by microfluidics-facilitated self-assembly improves the safety profile of the cationic host defense peptide novicidin in situ gel-forming ap- peptide delivery system for cutaneous wound healing nisin-loaded pectin nanoparticles for food preservation. food hydrocoll formation, characterization and release kinetics of chitosan/gamma-pga encapsulated nisin nanoparticles the chemistry and engineering of polymeric hydrogel adhesives for wound closure: a tutorial antimicrobial polymeric hydrogels chapter -solid lipid nanoparticle formulations pharmacokinetic and biopharmaceutical aspects in drug delivery preclinical safety of solid lipid nanoparticles and nanostructured lipid carriers: current evidence from in vitro and in vivo evaluation comet assay reveals no genotoxicity risk of cationic solid lipid nanoparticles nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers-a systematic review of in vitro data lipid nanoparticles (sln ® , nlc ® ) for cutaneous drug delivery:structure, protection and skin effects solid lipid nanoparticles as a drug delivery system for peptides and proteins oral bioavailability of cyclosporine: solid lipid nanoparticles (sln) versus drug nanocrystals cyclosporine-loaded solid lipid nanoparticles (sln): drug-lipid physicochemical interactions and characterization of drug incorporation perillaldehyde , -epoxide loaded sln-tailored mab: production, physicochemical characterization and in vitro cytotoxicity profile in mcf- cell lines surface-tailored anti-her /neu-solid lipid nanoparticles for site-specific targeting mcf- and bt- breast cancer cells nanoparticles encapsulated with ll and serpin a promotes wound healing and synergistically enhances antibacterial activity ll loaded nanostructured lipid carriers (nlc): a new strategy for the topical treatment of chronic wounds interactions of the antimicrobial peptide nisin z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity nano-sized lipidated dendrimers as potent and broad-spectrum antibacterial agents employing carbon dots modified with vancomycin for assaying gram-positive bacteria like staphylococcus aureus magnetic multifunctional carbon dots for selective separation, identification, and eradication of drug-resistant superbugs this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -x nxxled authors: di lorenzo, giuseppe; di trolio, rossella; kozlakidis, zisis; busto, giuseppina; ingenito, concetta; buonerba, luciana; ferrara, claudia; libroia, annamaria; ragone, gianluca; ioio, concetta dello; savastano, beatrice; polverino, mario; de falco, ferdinando; iaccarino, simona; leo, emilio title: covid therapies and anti-cancer drugs: a systematic review of recent literature date: - - journal: crit rev oncol hematol doi: . /j.critrevonc. . sha: doc_id: cord_uid: x nxxled background: it is reasonable to think that cancer patients undergoing chemotherapy, targeted therapy or immunotherapy could have a more aggressive course if positive for coronavirus disease cov- (covid- ). methods: we conducted a literature review on https://www.ncbi.nlm.nih.gov/pubmed/, https://scholar.google.com, www.arxiv.org, www.biorxiv.org, of all articles published using the keywords covid- therapy or treatment and cancer until may , . a total of articles were identified and were included in this review. results: we describe the ongoing covid- therapies that should be known by oncologists and highlight the potential interactions with antineoplastic drugs, commonly used in clinical practice. the main drug interactions were found with tocilizumab, ruxolitinib and colchicine. conclusions. the literature provides an inconclusive picture on potential preferred treatments for covid- and their interactions with antineoplastic agents. future clinical trials are needed to better understand the interactions between different drugs in the context of covid- pandemic. in our recent history there have been three epidemics related to coronavirus infections: the sars-cov (severe acute respiratory syndrome), - ; the mers-cov (middle-east-respiratory-syndrome), ; and currently the sars-cov- , ( - ) ( ) . until may , , there were approximately confirmed cases, with deaths due to coronavirus disease cov- (covid- ) in italy, according to the italian civil protection bulletin ( ). according to the world health organization (who) as of may due to covid- , in spain there were confirmed cases and deaths; in the united states, milion confirmed cases and deaths; france has confirmed cases with deaths while united kingdom has cases and deaths ( ). from the available scientific literature it is evident that about . % of the deaths with the coranavirus had an oncological pathology as comorbidity ( , ) . thus, during this covid- crisis, cancer patients are regarded as a highly vulnerable group. it was found that within days, anti-cancer treatments were significantly associated with occurrence of severe clinical events in j o u r n a l p r e -p r o o f covid- infection ( ) . the cancer population subjected to chemotherapy and/or radiotherapy is more exposed to infections in general and, therefore, also to that from coronavirus primarily due to the effect of the cytotoxic action on the hematopoietic and immune systems with a reduction in the number of neutrophils, the first bulwark of infections, and decreased immune capacity ( ) . although there is no data yet on the risks of contracting coronavirus infection or on the clinical course of the infection during immunotherapy and/or immunosuppressive treatment with chemotherapy. it is reasonable to think, by analogy of what happens in the case of seasonal flu, due to the presence of immunosuppression, that in treated cancer patients, there may be a greater number of complications and the clinical course to be more serious ( ) . therefore there remains an urgent need to answer whether covid- -positive cancer patients will have worse outcomes, such as death, from the coronavirus-induced pneumonia for example, and whether cancer patients should receive anti-cancer treatments. additionally, oncologists are required to know the toxic effects of the drugs used in the experimental therapy of covid- and the possible interactions of these drugs with the commonly used antineoplastic drugs. the systematic review followed the prisma guidelines (figure ) ( ) . two investigators (el and rdt) independently conducted literature search using as combined keywords covid- therapy or treatment and cancer on https://www.ncbi.nlm.nih.gov/pubmed/, www.arxiv.org ( ), www.biorxiv.org ( ) and https://scholar.google.com ( ) . the database search was run of all the published articles from database inception until may , . in pubmed the following strategy was used: (covid- or novel coronavirus-infected pneumonia or novel coronavirus or -ncov or sars-cov- therapy or treatment) and cancer. the strategy was then adapted for the other databases, including website of italian medicines agency (aifa) for ongoing trials ( https://www.aifa.gov.it/emergenza-covid- ) ( ). all studies reporting information on both covid- therapy/treatment and cancer were included. articles were identified and reviewed independently by two authors (el and rdt) and articles were considered relevant to the scope of the current review, as described in figure ( , . any inconsistencies were resolved by consensus with a third author (gdl). all health outcomes were included, due to the anticipated scarcity of data. the lists of the drugs being tested and their side-effects are listed in table . they are presented below in more detail. considering the etiological therapy of sars-cov- , antiviral agents, already on the market and in use for other viral pathologies, in monotherapy or in combination are currently being tested. it has a mechanism of action related to the selective inhibition of the viral polymerase-rnadependent rna and is used as a backup drug when other therapies do not work ( ) . commonly used for the treatment of hepatitis c and for inflammatory lung diseases, such as bronchiolitis, this antiviral drug is always used in combination with other medicines, such as interferon alfa and peginterferon alfa and can be used both in the treatment of adult patients and in the treatment of pediatric patients ( ). it is an antiviral drug in the class of nucleotide analogues. it was developed as a treatment for ebola virus disease and marburg virus infections. it has also been shown to have antiviral activity against rna viruses such as human respiratory syncytial virus and coronaviruses, including viruses that cause the mers and sars ( ) . on april, the director of the us national institute of allergy and infectious diseases (niaid), announced the results of a study in which patients taking remdesivir recovered in days compared with days for those on a placebo. the shortened recovery time was so significant that investigators decided to stop the trial ( ). this news contradicts previous mixed results on the drug. gilead sciences announced that in a non-randomized trial, more than half of participants with severe covid- had recovered from their disease within two weeks of receiving treatment. another smaller trial in china announced that it had found no benefits from remdesivir when compared with a placebo. the niaid did not release informations on safety data ( ). lopinavir and ritonavir is indicated, in combination with other antiretroviral medicines, for the treatment of adults, and children over the age of years with human immunodeficiency virus (hiv- ) infection ( ) . lopinavir-ritonavir in association with ribavirin is being evaluated. it is a drug that counteracts the over-reaction of the immune system, at the origin of some of the most serious complications of covid- and which in these days seems to offer hope to patients with coronavirus-induced pneumonia. in addition to rheumatoid arthritis therapy, the medicine is also used to treat cytokine release syndrome, a side effect of car-t anticancer therapy which consists of a massive release of inflammatory molecules in response to the immune cells used in the treatment ( ). it is an anti-interferon gamma. this drug keeps children with hemophagocytic lymphohistiocytosis (hlh) alive, awaiting transplantation. the administration of emapalumab is able to "turn off" the abnormal and excessive inflammatory response in patients with hlh, neutralizing the effects deriving from the excessive production of interferon-gamma ( ). sarilumab is an interleukin- (il- ) receptor antagonist indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis who have had an inadequate response or intolerance to one or more disease-modifying antirheumatic drugs (dmards) ( ). it is a member of the drug class selective immunosuppressants and is used to treat hemolytic uremic syndrome, myasthenia gravis, neuromyelitis optica and paroxysmal nocturnal hemoglobinuria ( ). it inhibits the activity of interleukin- (il- ), and has already been approved for the treatment of siltuximab like tocilizumab, binds to interleukin- and is indicated for the treatment of adult patients with castleman disease ( ) . the sisco study (siltuximab in serious covid- ) is configured as an observational study conducted both on hospitalized patients and those already in intensive care ( ). table shows italian medicines agency (aifa) approved experimental studies in covid- therapy ( - ). the main currently ongoing studies are: ) tocivid- , multicenter study on the efficacy and tolerability of tocilizumab in the treatment of patients with covid- pneumonia; ) sobi study, immuno- , a randomized, open, parallel, multicenter, phase / groups, that evaluates the efficacy and safety of intravenous doses of emapalumab, antiinterferon gamma monoclonal antibody (anti-ifnγ), and anakinra, interleukin receptor antagonist- (il- ), compared to standard therapy; ) sarilumab covid- study: a randomized, double-blind, placebo-controlled phase / study evaluating the efficacy and safety of intravenous administration of sarilumab, an interleukin receptor antagonist- (il- ). other potential therapeutic agents. the required urgency for the identification of potential treatments has necessitated the further investigation of a wide-range of potential therapeutic agents. the ones identified in this review are listed below in some detail. ruxolitinib is an inhibitor of the signal transmission pathway mediated by janus kinase (jak), with anti-inflammatory effects related to the inhibition of the release of cytokines. normally ruxolitinib is used in the hematology field and is indicated for the treatment of splenomegaly or disease-related symptoms in adult patients with primary myelofibrosis. it is also approved for the treatment of adult patients with polycythemia vera who are resistant or intolerant to hydroxyurea ( ) . a compassionate use of ruxolitinib has also been approved by aifa in covid- patients with respiratory failure who do not require invasive assisted ventilation. baricitinib is another jak inhibitor indicated for the treatment of active rheumatoid arthritis ( ) . aifa has licensed a randomized phase trial to evaluate the efficacy, safety and tolerability of j o u r n a l p r e -p r o o f baricitinib in addition to the usual treatment in patients with pneumonia in covid- (barcivid study) ( ). a new line of research comes from diabetologists. according to a study, in addition to the main entrance door of virus, the angiotensin-converting enzyme (ace ) receptor, the dpp receptor must be evaluated. the dipeptidyl peptidase (dpp ) receptor is present on all human cell types (bronchi and hearth) and is the same on which many medicines for diabetes work ( ) . colchicine is a molecule, capable of interfering with the inflammatory immune response observed in subjects with covid- . colchicine is a drug that has been used for a long time and is effective for the treatment of acute attack of gouty arthritis. specifically, it manages to reduce the release of cytokines, molecules that, like il- , are responsible for the inflammatory ( , ). a study has recently started in italy ( ). a reduction of up to % in mortality among covid- patients with a marked increase in an indicator of the presence of blood clots, was achieve due to the use of an old drug, heparin ( ). this was reported by a study according to which the use of heparin in covid- patients could have anticoagulant effects, as well as anti-inflammatory and potentially even antiviral ones. it has been shown that new coronavirus infections are associated with a high mortality in the presence of high value of d-dimer, a particularly important marker for coagulopathy ( ) . however, it will be necessary to study in depth at what dosage the drug could have such potential antiviral properties. a recent study has been approved by aifa with enoxeparin (inhixacovid) ( ). table table shows interferences with tocilizumab, ruxolitinib and colchicine. tocilizumab increases immunosuppressive action and inhibits fluouracil-resistance ( ) ; it interferes with pharmacodynamic activity and the therapeutic efficacy of durvalumab, nivolumab, atezolizumab ( , , ) . moreover it is important to emphasize that tocilizumab decreases the concentration of several medications as a cytochrome p (cyp), isoenzyme cyp a inducer ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) . therefore if drugs in lung cancer are used, such as ceritinib, crizotinib, brigatinib, gefitinib, docetaxel, a reduction in the anticancer drug may become noticeable, as well as paying attention in the case of concomitant use of tyrosine kinase inhibitors. anakinra and ruxolitinib could interfere with checkpoint inhibitors (cpi) in terms of antagonisms or synergy but this is not yet entirely clear ( , , ) . several drugs, such as cyp a inhibitors, increase concentration of colchicine and attention should be paid when the latter is used with cisplatin or vinca alkaloids ( ). furthermore, one should not forget a concomitant use of antidiabetic drugs with antineoplastics that require cortisone ( ) or cytostatic drugs that reduce the value of platelets with the concomitant use of heparin ( ) . based on the increased risk of disease aggression of covid- in patients with cancer it is important to describe which anti covid- drugs could be administered and which not in cancer patients. the current review attempts to provide a first systematic glance at this issue. there are certain emergent points of attention that have already been described. specifically, particular attention to the potential cardiotoxicity of chloroquine with anthracyclines is necessary. hydroxychloroquine is an antimalarial that has become a mainstay in the management of systemic lupus erythematosus and rheumatoid arthritis. similar to chloroquine in structure, hydroxychloroquine is used more frequently because it confers lower toxicity. cardiotoxicity manifests as restrictive or dilated cardiomyopathy or with conduction system abnormalities such as atrioventricular and bundle-branch block ( ) . recently, some doubts it is important also to note that some chemotherapeutic agents or hormonal therapies such as abiraterone require concomitant steroids that are immunosuppressive agents and could weaken anticitokines drugs such as tocilizumab ( ) . however in some cases, the use of steroids could be reduced without interfering with the efficacy and toxicity profile. tocilizumab decreases concentrations of many anticancer drugs due to interference with cyp ( ). deserving to underline that today many patients carry out cpi, currently used in daily practice for the treatment of solid tumors. cpi, activating t-lymphocytes, increase the risk of autoimmune disease such as pneumoniae ( ). few case-reports have described significant reduction in il- and cpi immunorelated toxicity after tocilizumab administration ( ). to date the interaction between cpi and anti covid- agents is not well-described. consideration could be given to either extending the timing of the doses or delaying pretreated patients. thirdly the data are based on retrospective findings, while the results of prospective cohorts are being awaited and expected to shed more light. moreover considering the rapid evolution of information related to the pandemic we conducted a search on arxiv and biorxiv databases indentifying articles of interest; these are preliminary reports that have not been peer-reviewed. among the articles only were considered as relevant ( , ) and subsequently has been published in peer-reviewed journal ( ) , testifying to the current urgent need of relevant, well-designed studies. the literature so far available provides an inconclusive picture on potential preferred treatments for covid- and their interactions with antineoplastic agents. in order to continue treating cancer patients likely to be infected with covid- , we believe that it is appropriate to modify available therapeutic choices and arrive at a new therapeutic protocol that provides: ) where there are no interactions to continue or delay antineoplastic therapy by - weeks; ) where there are interactions and the patient has already done many cycles to stop the administration of the anticancer drug; ) for newly diagnosed cancer patients to prefer antineoplastic drugs without interactions; ) where further attention is given to cpi, the choice of which may vary from case to case. this new therapeutic proposal awaits the constructive suggestions of the oncological community in order to make improvements, such as to better protect our patients, already covid global consequences for oncology lancet oncology covid and italy: whatnext? lancet cancer patients in sars-cov- infection: a nation wide analysis in china clinical characteristics of covid- -infected cancer patients: a retrospective case study in three hospitals within wuhan preferred reporting items for a systematic review and meta-analysis of individual participant data: the prisma-ipd statement urgent considerations for the neuro-oncologic treatment of patients the possible of immunotherapy for covid- : a systematic review upheaval in cancer care during the covid- outbreak. cancermedicalscience covid- and treatment with nsaids and corticosteroids: should we be limiting their use in the clinical setting?. ecancermedicalscience the impact of coronavirus (covid- ) on head and neck cancer patients' care touboul recommendations for the surgical management of gynecological cancers during the covid- pandemic -francogyn group for the cngof how we treat patients with lung cancer during the sars-cov- pandemic: primum non nocere. esmo open a practical approach to the management of cancer patients during the novel coronavirus disease (covid- ) pandemic: an international collaborative group first case of covid- in a patient with multiple myeloma successfully treated with tocilizumab cancer, covid- and the precautionary principle: prioritizing treatment during a global pandemic the effectiveness of an anti-human il- receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells international guidelines on radiation therapy for breast cancer during the covid- pandemic nivolumab combined with ruxolitinib: antagonism or synergy? coronavirus pandemic and colorectal surgery: practical advice based on the italian experience safety recommendations for evaluation and surgery of the head and neck during the covid- pandemic covid- in a patient with chronic lymphocytic leukaemia controversies about covid- and anticancer treatment with immune checkpoint inhibitors managing cancer care during the covid- pandemic: agility and collaboration toward a common goal arbidol combined with lpv/r versus lpv/r alone against corona virus disease : a retrospective cohort study cancer patients in sars-cov- infection: a nationwide analysis in china first statement on preparation for the covid- pandemic in large german speaking university-based radiation oncology departments clinical features of cases with coronavirus disease prostate cancer radiotherapy recommendations in response to covid- case of the index patient who caused tertiary transmission of coronavirus disease in korea: the application of lopinavir/ritonavir for the treatment of j o u r n a l p r e -p r o o f covid- pneumonia monitored by quantitative rt-pcr covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics novel coronavirus (covid- ) based on current evidence covi d : combining antiviral and anti inflammatory disease challenges and countermeasures of integrative cancer therapy in the epidemic of covid- . integr cancer ther sars-cov- transmission in cancer patients of a tertiary hospital in wuhan pakdzad-shahabi l. estimating the risks from covid- infection in adult chemotherapy patients. medrxiv, -medrxiv comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial fda approval: siltuximab for the treatment of patients with multicentric castleman disease prospects in connective tissue diseases covid- and diabetes: can dpp inhibition play a role? the versatile heparin in covid- abiraterone and increased survival in metastatic prostate cancer zip a phase / , randomized, openlabel, parallel, -arm, multicenter study investigating the efficacy and safety of intravenous doses of emapalumab, a monoclonal antiinterferon gamma (anti-ifnγ) and anakinra antibody all authors declare no conflict of interest a randomized phase study to evaluate the safety and antiviral activity of remdesivir key: cord- - y k authors: rahman, md. mahbubur; saha, titon; islam, kazi jahidul; suman, rasel hosen; biswas, sourav; rahat, emon uddin; hossen, md. rubel; islam, rajib; hossain, md nayeem; mamun, abdulla al; khan, maksud; ali, md ackas; halim, mohammad a. title: virtual screening, molecular dynamics and structure–activity relationship studies to identify potent approved drugs for covid- treatment date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: y k computer-aided drug screening by molecular docking, molecular dynamics (md) and structural–activity relationship (sar) can offer an efficient approach to identify promising drug repurposing candidates for covid- treatment. in this study, computational screening is performed by molecular docking of food and drug administration (fda) approved drugs against the main protease (mpro) of sars-cov- . several promising approved drugs, including simeprevir, ergotamine, bromocriptine and tadalafil, stand out as the best candidates based on their binding energy, fitting score and noncovalent interactions at the binding sites of the receptor. all selected drugs interact with the key active site residues, including his and cys . various noncovalent interactions including hydrogen bonding, hydrophobic interactions, pi–sulfur and pi–pi interactions appear to be dominant in drug–mpro complexes. md simulations are applied for the most promising drugs. structural stability and compactness are observed for the drug–mpro complexes. the protein shows low flexibility in both apo and holo form during md simulations. the mm/pbsa binding free energies are also measured for the selected drugs. for pattern recognition, structural similarity and binding energy prediction, multiple linear regression (mlr) models are used for the quantitative structural–activity relationship. the binding energy predicted by mlr model shows an % accuracy with the binding energy determined by molecular docking. our details results can facilitate rational drug design targeting the sars-cov- main protease. communicated by ramaswamy h. sarma a novel coronavirus was identified in wuhan, china in december spreading an infectious pneumonia-like disease . the international committee on taxonomy of viruses (ictv) named the novel coronavirus ( -ncov) as sars-cov- (gorbalenya et al., ) . the world health organization (who) officially named the disease caused by sars-cov- as coronavirus disease (covid- ) on february . on the basis of 'alarming levels of spread and severity, and by the alarming levels of inaction', on march , the director-general of who characterized the covid- situation as a pandemic (bedford et al., ) . to date, the total numbers of reported cases have reached over million and the number of death is more than thousand (worldometer, ) . earlier, epidemics of severe acute respiratory syndrome coronavirus (sars-cov) in guangdong, china in and middle east respiratory syndrome coronavirus (mers-cov) in have shown a high fatality rate (hilgenfeld & peiris, ; ton et al., ) . sars-cov- was found to be an enveloped positive-sense, single-strained rna virus belonging to the genus betacoronavirus (beta-cov) (chan et al., ; lu et al., ) . phylogenetic analysis showed that sars-cov- is closely related to (with % identity) two bat-derived severe acute respiratory syndrome (sars)-like coronaviruses, bat-sl-covzc and bat-sl-covzxc , collected in in zhoushan, eastern china, but are more distant from sars-cov (about %) and mers-cov (about %) (lu et al., ) . covs have the largest known rna virus genomes, ranging from to kb (sexton et al., ) . inside the host cell, coronavirus genomes are translated into two groups of proteins; structural proteins, such as spike (s), envelope (e), matrix (m) and nucleocapsid (n) and nonstructural proteins, such as c-like protease ( cl-pro, nsp ), rna dependent rna polymerase (rdrp, nsp ) (elfiky, ) . coronavirus genome replication and transcription takes place at cytoplasmic membranes and involves a coordinated process of both continuous and discontinuous rna synthesis that is mediated by the viral replicate (sola et al., ) . the main protease (mpro) of sars-cov- plays an important role in cutting down polyproteins into functional pieces (chen et al., ; shaghaghi, ) . thus, the main protease plays a vital role in viral replication. the main protease (also known as c-like protease) can be an attractive drug target to inhibit coronavirus. inhibiting the activity of the main protease could block the replication of the virus inside infected cells. the crystallized structure of the main protease (mpro) or chymotrypsin-like protease ( cl-pro) of sars-cov- was identified and repositioned at the rcsb protein data bank (pdb) (jin et al., ) . the crystallized structure (pdb id: lu ) contains two chains, a and b, which form a homodimer. chain a was used for macromolecule preparation. the protein chain is composed of residues. his and cys form an uncharged catalytic dyad (paasche et al., ) . the active site of the protease is activated by a protonation reaction in the catalytic site (paasche et al., ) . inhibiting the activity of the catalytic dyad will ultimately help to inhibit the activity of the main protease. currently, no effective drugs targeting sars-cov- are available. drug repurposing, a strategy to identify new uses of approved drugs, could shorten the time and reduce the cost for identifying effective drugs against covid- . we have selected fda approved drugs from the zinc database (irwin & shoichet, ) . the focus of this study is to identify the binding affinities and molecular interactions of these drugs against the main protease using computational and statistical tools. molecular docking, molecular dynamics (md), principal components analysis (pca) and quantitative structure-activity relationships (qsar) were executed to evaluate the performance of the drugs. the structures of fda approved drugs are obtained from the zinc database. the database was then checked for redundant molecules. the three-dimensional crystal structure of sars-cov- main protease (mpro, cl pro ) was collected from the rcsb protein data bank (pdb) database (pdb-id: lu , resolution ¼ . Å, method: x-ray diffraction, organism: bat-sars like coronavirus) . the crystal structure of mpro was prepared according to previously published methods for molecular docking . potential active site residues were specified by their vicinity to the ligand, n (sekhar, ) . pyrx virtual screening tools incorporated with autodock vina was used for virtual screening (dallakyan & olson, ) . structural optimization of the drugs was carried out with universal force field (uff) using the steepest descent optimization algorithm, a total of minimization steps (ahmed et al., ) . these drugs were converted to autodock ligand (pdbqt format) for docking. the grid center points were set to x ¼ À . , y ¼ . , z ¼ . and box dimensions (angstrom) x ¼ . , y ¼ . , z ¼ . . grid box center points and dimensions were set to target the substrate binding-binding pocket of the protein (chen et al., ) . the binding affinities of the drugs were measured in kcal/mol unit and sorted according to the higher negative values, which imply the best binding affinities (de sousa et al., ) . based on calculated binding affinities, the top-ranked potential drugs were chosen for further analysis. the top-ranked drugs were then optimized by gaussian software using semi-empirical pm method (frisch, ). these optimized drug structures are then docked again using autodock vina software and gold (jones et al., ) docking programs. the molecular interactions of the drugs as predicted by docking simulation were analyzed in biovia discovery studio visualizer (studio, ) . the md simulations for the main protease were conducted ( lu ) in apo-form (protein without ligand) and in holo-form (protein-drug complex) to assess any probable conformational changes to and interactions with their structures over the nanoseconds (ns). amber force field (dickson et al., ) was used on yasara dynamics (krieger et al., ) for the simulation. water molecules were added and the system was neutralized by the addition of nacl salt of . % concentration at a temperature of k (krieger et al., ) . the particle-mesh ewald method was used for calculating long-range electrostatic interactions. a periodic boundary condition was utilized to carry out the simulation. in all cases, the cell size was larger than the protein by Å. the temperature of the simulation was controlled by the berendsen thermostat. the initial energy minimization process of each simulation was performed by the simulated annealing method, using the steepest gradient approach ( cycles). normal simulation speed ( . fs time step) was maintained during all the simulations (krieger & vriend, ) . a snapshot was saved in every ps during simulations. after completion of ns md simulation for all systems, the results were analyzed in yasara. during the analysis of the simulations, time, energy, bond distances, bond angles, dihedral angles, solvent-accessible surface area (sasa), molecular surface area (molsa), coulombic and van der waals interactions, root mean square fluctuation (rmsf) and root mean square deviation (rmsd) values for backbone, alpha carbon and heavy atoms were recorded. for more accuracy, the ns md simulation was conducted twice for every complex and the average result was used for analysis. for binding free energy calculations, molecular mechanics/ poisson-boltzmann surface area (mm/pbsa) method was used in yasara dynamics software (baker et al., ; chirag et al., ; massova & kollman, ) . the amber force field was used during the binding free energy calculation. the default macro file was modified for the calculation. the following equation was used for the binding free energy calculation: here, dg mm is the molecular mechanics interaction (sum of electrostatic and van der waals interaction), dg pb and dg sa correspond to polar and nonpolar solvation energyies, respectively. tds is the entropic contribution. due to the high computational time, the entropy contribution was not considered. the final ns data of md simulation was used for binding free energy calculations. principal component analysis (pca) is applied to identify similarities and dissimilarities among the collected energy profiles of md trajectory data (martens & naes, ) . the important components of a pca model are highlighted in the following equation: expresses a product of two new matrices, i.e. t k , p k , t k is the matrix of scores that shows how samples relate to each other, p k is the matrix of loadings, which carries information about how the variables relate to each other, k represents the number of factors involved in the model and e is the matrix of residuals. the selected four drug-main protease complexes may have dissimilarities with the apo-mpro during md simulation regarding the energy profile. these dissimilarities can be identified using the pca algorithm (islam et al., . all calculations were carried out on r platform employing inhouse developed codes and plots were produced using the package ggplot (kassambara, ) . data were preprocessed employing autoscale function before applying the pca algorithm (martens & naes, ) . the last ns data of the md trajectory was utilized for pca. structure-activity relationship (sar) is a method for establishing computational or mathematical models that aim to detect a statistically significant correlation between structure and function employing chemometric technique (verma et al., ) . initially selected drugs are explored in this study. topological polar surface area (tpsa, Å ), molecular weight (mw, gmol À ), xlogp -aa, hydrogen bond donor (hbd), hydrogen bond acceptor (hba), number of the rotatable bonds (rotb), number of h, c, o, cl, f atoms, single bonds (sb) count, number of double bonds (db) and number of benzene rings of the drug candidates were studied as variables. along with calculated binding energies, these variables were applied to correlate with the sar by multiple linear regression (mlr) ( the structure of fda approved drugs is considered for virtual screening using autodock vina. considering À . kcal/ mol as a cutoff value, the binding affinities of the drugs are distributed between À . to À . , À . to À . , À . to À . , À . to À . and À . to À . kcal/mol (figure ). most drugs are found to have binding affinities between À . to À . kcal/mol. the top candidates are selected for further analysis according to their binding affinities. the binding affinity of two already reported drugs remdesivir and lopinavir were observed À . and À . kcal/mol, respectively. after optimizing by pm level of theory, the selected drugs are screened again in autodock vina. gold suit was also employed to understand the binding fitness using the chemplp fitness score. here, a higher fitness score suggests better docking interaction between protein and ligand. along with previously suggested fda approved drugs (contini, ) , the binding affinities and gold fitting scores of drugs are summarized in table . according to best binding affinities, simeprevir, ergotamine, bromocriptine and tadalafil are selected for further analysis (figure ). remdesivir and lopinavir are also considered as control drugs for md simulations. noncovalent interactions of the selected drugs which are detected by autodock vina reveals that all of them interact with either both catalytic residues (his and cys ) or at least one of them, as shown in figure . simeprevir, ergotamine, bromocriptine and tadalafil form a number of hydrogen bonds and hydrophobic interactions with the protein (noncovalent interactions detected by gold are reported in (table s , supporting information). among them, most of the interactions with the active site residues are categorized as hydrophobic (table ) . both simeprevir and ergotamine form a hydrogen bond with at least one of the catalytic residues. simeprevir is observed to show three hydrophobic interactions with pro , two with leu and one with each of the thr and ala . ergotamine exhibits one pi-alkyl and one pi-pi t-shaped hydrophobic interaction with met and his , respectively. among all the protein-drug complexes, bromocriptine demonstrates the most noncovalent interactions with the main protease. it shows two hydrophobic interactions with his , two with leu , three with met and one with each of the met , cys and gln residues. tadalafil exhibits hydrophobic interactions with both catalytic residues. both ergotamine and tadalafil show one pi-sulfur interaction with met and met residues, respectively. md simulation for all complexes of the main protease ( lu ) with the top four selected drugs and apo-form is performed for ns. rmsd of alpha carbon atoms, rmsf of all amino acid residues, solvent accessible surface area, radius of gyration and the number of hydrogen bonds of all the drug-protein complexes and apo-mpro are investigated. the rmsd of simeprevir-mpro and ergotamine-mpro are stabilized after ns of simulation, with an average value of . ± . Å and . ± . Å, respectively (table ). figure a reveals that bromocriptine-mpro follows a similar trend in md trajectory like apo-mpro. tadalafil-mpro complex shows fluctuation until ns, while it reaches a peak of . Å at . ns. it exhibits structural stability after ns of md simulation. these drug-mpro complexes exhibit better stability in the rmsd trajectory than the control drugs. unstable nature is observed for the rmsd of remdesivir-mpro after ns of md simulation, while the lopinavir-mpro complex followed the similar trend like the bromocriptine-mpro complex. figure b displays root-mean-square fluctuation as a function of the residue number of the apo-mpro and drug-mpro complexes. this is explored to understand how the binding of drug molecules changes the behavior of the amino acid residues of the protein. a low rmsf is observed for the apo-mpro which suggests low flexibility of protein even without a ligand. it also shows flexibility at ser to asn , tyr , val , thr , phe and gln . the residues arg to asp show high flexibility in tadalafil-mpro complex. rmsf of simeprevir-mpro, ergotamine-mpro, bromocriptine-mpro, remdesivir-mpro and lopinavir-mpro complexes are almost similar in most areas. the radius of gyration (r g ) is the root mean square distance of various atoms of a protein from the axis of rotation, and it illustrates structural compactness. a greater change in r g value may occur due to the folding or conformational changes of protein structure, while a lower degree of fluctuation over the simulation period indicates rigidity and higher compactness of a structure. according to the observed r g value, the apo-mpro and simeprevir-mpro complex showed almost similar structural compactness over the phase of the ns simulation. the apo-mpro complex experienced a fluctuation in r g value during - ns. ergotamine showed structural compactness all over the simulation time with an average of . ± . Å. bromocriptine-mpro followed a similar trend like the remdesivir-mpro complex after ns. a loose packing is observed for tadalafil-mpro during ns to ns. except for this time frame, tadalafil-mpro and lopinavir-mpro followed a similar trend in md trajectory. based on the overall r g , the average values are . ± . Å, . ± . Å, . ± . Å, . ± . Å, . ± . Å and . ± . Å for apo-mpro, simeprevir-mpro, bromocriptine-mpro, tadalafil-mpro, remdesivir-mpro and lopinavir-mpro complexes, respectively. to anticipate the solvent accessibility of all complexes, the solvent-accessible surface area (sasa) of apo-mpro and complexes are calculated (figure (c) ). binding of a drug to the protein may impact its structural properties, thus, sasa may also change. a higher sasa value suggests the expansion of protein structure. a low fluctuation of sasa value is expected during the simulation. all the examined complexes exhibit a similar trend until ns of md trajectory. simeprevir-mpro and ergotamine-mpro showed lower sasa value than the control drug-mpro complexes. the overall average sasa value suggests lopinavir could induce protein expansion, and thus, increase the solvent accessible surface of the protein (table ) . since intermolecular hydrogen bonds between protein-drug complexes have a significant contribution to the conformational stability of the complex, the total number of hydrogen bonds is calculated (figure (e) ). the average number of hydrogen bonds observed in apo-mpro is . ± . . the bromocriptine-mpro complex has the most hydrogen bonds over the ns simulation time (table ) . binding free energy calculations for each complex were conducted by employing the mm/pbsa method. the simeprevir-mpro shows the highest average binding free energy of À . ± . kcal/mol. all our selected drug-mpro complexes demonstrate better binding free energy than the remdesivir-mpro complex (figure (f) ). the binding free energy of the remdesivir-mpro complex persisted as positive most of the time, which indicates loose binding between the drug and the protein. the binding free energy of ergotamine-mpro, bromocriptine-mpro, tadalafil-mpro and lopinavir-mpro complexes remained negative most of the time, which also indicates good binding ( figure s , supporting information). the average binding free energy was observed À . ± . , À . ± . , À . ± . , À . ± . and À . ± . kcal/mol for ergotamine, bromocriptine, tadalafil, remdesivir and lopinavir, respectively. the snapshots of mpro-drug complexes are visualized at different time intervals to explore the drugs pose during md simulation. all our selected drugs stayed at the binding pocket of the protein over the phase of ns md simulation ( figure ) . additionally, all drugs interact with at least one of the catalytic residues during most of the time of md simulation (table s , supporting information). pca is applied to observe the structural properties and energy profiles of apo-mpro and the selected four drug-mpro complexes. a single pca model consisting of five training sets (mpro and four drug-mpro complexes) was constructed for analysis. the first two pcs explained . % of the total variance, where pc contributed . % and pc contributed . % of the variance. it is observed from the scores plot (figure (a) ) that the apo-mpro (violet) and simeprevir-mpro complex (cyan) overlap with each other, thus, indicating similarity in energy and structural profile. moreover, ergotamine-mpro (green) and tadalafil-mpro (orange-red) shifted slightly toward the right in the pc (figure (b)) revealed that the variables are separated in the pc direction, which explains different cluster formation in the pc direction. therefore, it is evident from the pca scoring plot that simeprevir has the best binding stability with mpro, while ergotamine and tadalafil also demonstrate almost similar behavior in md simulation. sar has been used for decades by researchers as a cheaper way to develop relationships between physiochemical properties of chemicals and their bioactivities (santos-filho et al., ; verma et al., ) . it is a widely used tool in bioinformatics, drug discovery for clinical research, pharmaceutical industry, petrochemical and agrochemical sectors for modeling and predictive pattern analysis (alam & khan, ; fakayode et al., ; funar-timofei et al., ) . for further analysis, mlr has been employed. for instance, tpsa (Å ), molecular weight, xlogp , h-bond donor count, h-bond acceptor count of the drugs are the most significant variables on qsar contributors to the mlr model (table s , supporting information). the drugs with similar functional groups are found to stay side by side on the score plot (figure ) . for example, drugs (d , d and d ) containing indole ring, amide group and alcohol group are clustered together on the fourth quadrant of the score plot. on the other hand, drugs (d , d , d , d , d and d ) with diazole ring attached to the benzene ring are clustered on the second and third quadrants. the grouping on the score plot was highly significant. the mlr model is generated and employed to predict the binding energy obtained from molecular docking. additionally, a test set is utilized for the validation of drug candidates. the observed results of validation reveal similarity in the binding energies (table ). the ability of the mlr model to accurately anticipate the binding energy of the validated drugs is evaluated by the root-mean-square-relative percent-error (rms%re). the result obtained from mlr is % accurate while comparing to the binding energy. yet, findings from this analysis can help researchers predict the drug performances against the main protease considering the binding energy, chemical behavior and pharmacological efficacy of upcoming drug candidates. in this study, several computational and statistical tools such as molecular docking, md simulation, sar and pca are used to detect the best existing drugs against the main protease of sars-cov- . among the studied drugs, simeprevir, ergotamine, bromocriptine and tadalafil are found to have the highest binding affinity. as these drugs are already certified for human use, they do not require long-term clinical trials. these drugs show a significant number of noncovalent interactions including hydrogen bond, hydrophobic interaction and electrostatic interaction with the binding site residues of mpro. all the selected drugs interact either with both (his and cys ) or at least one of the catalytic residues. furthermore, md simulations exhibit that simeprevir, ergotamine and bromocriptine drugs with mpro protein preserve the structural stability over the simulation period. additionally, the mm/pbsa free energy profiles of these drugs suggest strong binding with the receptor. sar pattern recognition reveals that drugs containing an indole ring, amide group and alcohol group are clustered together in the fourth quadrant and drugs containing an azole ring attached to benzene ring are assembled together in the second and third quadrant of the scoring plot. the predicted binding affinity values from mlr shows % accuracy compared to values obtained from molecular docking. it can be concluded that the selected drugs are promising and can be used to design effective drugs against the sars-cov- . no potential conflict of interest was reported by the authors. xlstat-software, version virtual screening, molecular dynamics, density functional theory and quantitative structure activity relationship studies to design peroxisome proliferator-activated receptor-c agonists as anti-diabetic drugs d-qsar studies on maslinic acid analogs for anticancer activity against breast cancer cell line electrostatics of nanosystems: application to microtubules and the ribosome covid- : towards controlling of a pandemic genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan only one protomer is active in the dimer of sars c-like proteinase emerging coronaviruses: genome structure, replication, and pathogenesis identification of potential binders of the sars-cov- spike protein via molecular docking, dynamics simulation and binding free energy calculation virtual screening of an fda approved drugs database on two covid- coronavirus proteins small-molecule library screening by docking with pyrx virtual screening as a tool to discover new b-haematin inhibitors with activity against malaria parasites lipid : the amber lipid force field anti-hcv, nucleotide inhibitors, repurposing against covid- chemometric approach to chiral recognition using molecular spectroscopy. encyclopedia of analytical chemistry determination of boiling point of petrochemicals by gas chromatography-mass spectrometry and multivariate regression analysis of structural activity relationship combined molecular docking and qsar study of fused heterocyclic herbicide inhibitors of d protein in photosystem ii of plants the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- from sars to mers: years of research on highly pathogenic human coronaviruses clinical features of patients infected with novel coronavirus in wuhan. the lancet zinc-a free database of commercially available compounds for virtual screening prediction of deleterious non-synonymous snps of human stk gene by combining algorithms, molecular docking, and molecular dynamics simulation a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov- structure of m pro from covid- virus and discovery of its inhibitors development and validation of a genetic algorithm for flexible docking edited by f. e. cohen practical guide to principal component methods in r. sthda, , - yasara-yet another scientific artificial reality application fast empirical pka prediction by ewald summation new ways to boost molecular dynamics simulations genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding chemometrics in spectroscopy multivariate calibration combined molecular mechanical and continuum solvent approach (mm-pbsa/gbsa) to predict ligand binding evidence for substrate binding-induced zwitterion formation in the catalytic cys-his dyad of the sars-cov main protease the receptor-dependent qsar paradigm: an overview of the current state of the art virtual screening based prediction of potential drugs for covid- homology-based identification of a mutation in the coronavirus rna-dependent rna polymerase that confers resistance to multiple mutagens molecular docking study of novel covid- protease with low risk terpenoides compounds of plants continuous and discontinuous rna synthesis in coronaviruses dassault systemes biovia, discovery studio modelling environment rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds d-qsar in drug design-a review coronavirus cases crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors network-based drug repurposing for novel coronavirus -ncov/ sars-cov- we are grateful to our donors (http://grc-bd.org/donate/) who supported the building of the computational platform in bangladesh. the authors acknowledge the world academy of science (twas) for purchasing the high-performance computer for the md simulations. key: cord- - w ycx authors: dömling, alexander; gao, li title: chemistry and biology of sars-cov- date: - - journal: chem doi: . /j.chempr. . . sha: doc_id: cord_uid: w ycx sars-cov- (previously -ncov or wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world’s traffic highways. the coronavirus (cov) family members were responsible for several deadly outbreaks and epidemics during the last decade. not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. for example, the genetic fingerprint was shared, and the d structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. an overview is given on the current knowledge of the spread, disease course, and molecular biology of sars-cov- . we discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines. sars-cov- (previously -ncov or wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world's traffic highways. the coronavirus (cov) family members were responsible for several deadly outbreaks and epidemics during the last decade. not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. for example, the genetic fingerprint was shared, and the d structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. an overview is given on the current knowledge of the spread, disease course, and molecular biology of sars-cov- . we discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines. in december , an outbreak of pneumonia of an unknown cause was reported in wuhan, in hubei province, china. it was speculated that the first patient caught the infection from a seafood market that also traded wild animals. the causing agent was quickly identified as a novel coronavirus (cov) . the cov responsible for the outbreak is now called sars-cov- . the respiratory illness caused by sars-cov- is called covid- . the symptoms of the sars-cov- infection range from asymptomatic to mild to severe to death. it soon became clear that person-to-person transmission was also occurring, as was the case with the previous human cov. in an unprecedented documented speed, the sars-cov- travels around the globe, and as of april th led to > . million infections and , fatalities. based on the previous experience with the sars-cov outbreak at the beginning of this century, very stringent measures were taken by the chinese government, and several multimillioninhabitant cities were isolated and put under quarantine in order to slow the pandemic spread. different hosts of the sars-cov- are proposed, including snails, bats, and pangolins. covs are a large family of zoonotic viruses and their outbreaks are common to humans, although major outbreaks have been experienced in animals, especially in cattle. under the electron microscope, they exhibit formations that are reminiscent of the solar corona. the common cold is often caused by human covs. they are single-stranded enveloped positive rna viruses and stand out because of their rather large genome. as with viruses in general, the structure is rather simple. sars-cov- is generally less pathogenic than sars-cov, much less pathogenic than the middle east respiratory syndrome mers-cov, but more pathogenic than practically harmless hcov-oc , hcov-hku , hcov- e, and hcov-nl . the reported case-fatality rate of covid- is % % and is thus rather low as compared with sars ( % , table ). however, the transmission rate (tr) (number of newly infected people per infected person) of . to is high and accounts for the danger of the current pandemic. for comparison, the tr of the yearly common cold is less than . . challenges and opportunities: the sars-cov- pandemic has changed our world in just half a year: a large number of people have died from the virusinduced pneumonia covid- , and the global economy is at an unprecedented low with unknown near-and long-term consequences. the coronavirus pandemic needs a two-pronged approach: a short-term solution to find a drug to treat the massive number of seriously ill patients, and long-term development of preventive and curative drugs for future corona outbreaks. de novo discovery of any therapeutic takes years to move from idea/ q pre-clinic to market release and is not a short-term solution for the current sars-cov- pandemic. drug repurposing is perhaps the only short-term solution, and vaccination is a middle-term solution. advice guidelines for diagnosis and treatment of sars-cov- infected pneumonia have been shared rapidly. what are the issues and chances for a rapid approval of a new medicine to treat covid- ? in principle, there are several potential strategies to pharmacologically fight covid- : vaccines, monoclonal antibodies, oligonucleotide-based therapies, peptides, interferon therapies, small-molecule drugs, or natural medicines (e.g., traditional chinese medicine [tcm]). the timelines for de novo development of a small-molecule drug are historically > - years, and in the best case less than years. vaccines can be developed much faster, but rapid development in the range of - years is very challenging. antibodies to support the body's immune system are also a strategy to combat viral diseases. again, the typical development timelines are several years. therefore, is there a hope for a q rapid drug to come to the market? a strategy that is promising in the current situation is drug repurposing. drug repurposing aims to discover novel indication areas for already approved drugs. the overwhelming advantage of drug repurposing is the potential for much faster approval because of the already extensive knowledge of the drug's behavior in humans. an expert opinion on the potential for repurposing existing antiviral agents to treat covid- , some of which are already clinically evaluated, was recently given. here, we discuss molecular targets of the sars-cov- , some of the known small molecules, and the potential for repurposing existing drugs. despite the rather large size of the rna virus genome of $ , bases, the sars-cov- genome encodes for few proteins (figure ): the structural spike (s) protein, nucleocapsid (n) protein, membrane (m) protein, and the envelope (e) protein, which are needed to produce a structurally complete viral particle. additionally, the sars-cov- genome encodes - non-structural proteins (ns to ns ), such as -chymotrypsin-like protease ( clpro q ), papain-like protease (plpro), helicase, and rna-dependent rna polymerase (rdrp). both the virus-encoded proteases q are involved in the processing of the two viral polyproteins in a coordinated manner, and thus comprise important drug targets. the structure, function, and inhibition of cov clpro (also called mpro) has been recently comprehensively reviewed. the clpro is a cysteine protease that cleaves and processes the viral polyproteins. sars-cov- and sars-cov share % sequence identity in their clpro. on the basis of the rapidly published virus sequence data, a homology model was created. moreover, an x-ray structure of the c lpro covalently bound to a peptidomimetic acrylester ( ) is now available ( figure , pdb id lu ). because of the high sequence similarity of different cov clpros, a lot of previously described inhibitors can be considered to be of great use in the current sars-cov- . a majority of inhibitors of the clpro are covalent in nature, binding to the activesite cysteine (scheme ). different electrophilic warheads are known, including a-halocarbonyl, acrylamides, sulfonyl chlorides, aldehydes ( ), isatines ( ), or a-ketoheteraromates ( ). many of the molecules are rather large and are based on extensive amide chemistry, mimicking part of the peptide substrate of the protease. moreover, their selectivity toward other potential targets in the human body has not been established. interestingly, some compounds binding to the active site of the clpro-although using a noncovalent mechanism-have been established . a high-throughput screening (hts) identified , -dihydropyrazolidine ( ), which displayed , , -triaryl substitution patterns, as sars-cov clpro inhibitors. nitroanilides ( ), derived from the drug niclosamide were also found to inhibit clpro. a-aminoacylamides were identified by an hts, and a strong stereochemical effect was noted. the simple one-pot accessible scaffold by an ugi-four component condensation was the key to rapidly generate structure activity relationship (sar) for the putative p -p and p subgroups. an optimized version ml ( ) was designated as the probe status ( figure ) . a p truncated version of allowing for significant molecular weight (mw) reduction without diminishing potency was developed as a second probe ml ( ) with potent enzyme inhibition and cellular activity. these compounds comprise rare examples of a noncovalent sars-cov clpro inhibitor of moderate mw with good enzyme and antiviral inhibitory activity. however, these molecules suffer from extensive metabolism and rapid clearance. nonetheless, they are a promising starting point for further drug development. even if such compounds cannot be rapidly developed to cope with the current situation, their development is highly warranted to be prepared for likely future cov outbreaks. it is noteworthy that different computational approaches, including machine learning, have been published to propose approved drugs potentially binding to clpro (drug repurposing). , one such approach virtually screened commercial medicines in the drugbank database for binding into the active site of mpro. ten different commercial medicines were proposed that might form hydrogen bonds to key residues within the binding pocket of sars-cov- clpro, which might have higher mutation tolerance than lopinavir or ritonavir. flavonoids ( ) ( ) ( ) ( ) ( ) are plant-derived natural products with diverse reported biological activities, and they have been shown to be also able to inhibit the clpro (figure ) . , the broad-spectrum and established use of plant-based medicines to combat infectious diseases in tcm is the basis of several currently ongoing clinical trials in china. one of the largest among them assesses shuanghuanglian, a chinese herbal medicine that contains extracts from the dried fruit lianqiao (forsythiae fructus), which is purported to have been used for treating infections for more than , years. several approved hiv protease inhibitors ( , , and ) were previously repurposed for the treatment of sars (scheme ). [ ] [ ] [ ] they were hypothesized to inhibit the sars-cov clpro: hiv protease is a asp protease and differs considerably from the cys protease clpro, but it also shares some common elements, such as a tetrahedral transition state and receptor pockets to recognize the amino acid side chains of the substrates. given that sars-cov- and sars-cov share very high identical sequence in their clpro, these hiv protease inhibitors are currently again repurposed for the treatment of covid- (chinese clinical trial registry: chictr ). [ ] [ ] [ ] plpro the coronaviral plpro is another attractive antiviral drug target because it is essential for cov replication. the structure, function, and inhibition of the sars-cov plpro has been extensively reviewed. although the primary function of plpro and clpro is to process the viral polyprotein in a coordinated manner, plpro has the additional function of stripping ubiquitin and isg from host-cell proteins to help cov to evade the host innate immune responses. therefore, it was recently argued that targeting plpro with antiviral drugs might have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that might lead to cell death in surrounding, uninfected cells. different compounds forming a covalent bond to the active-site cys have been described, including epoxyketones, a-halo-ketones, alkynes, aldehydes, trifluoromethyl ketones, a,b-unsaturated ketone, activated esters, or vinyl sulfones. disulfiram ( ) , an approved drug for the treatment of chronic alcohol dependence, has a great potential for drug repurposing because it has been shown to inhibit plpro of mers-cov and sars-cov. the antimetabolites -mercaptopurine ( ) and -thioguanine ( ) are additional drugs inhibiting plpro. potent naphthyl methylamine q hits ( ) which have been structurally characterized and have been subsequently optimized toward better metabolic stability were identified from an hts campaign, . ( ) the naphthyl methylamines work through a noncovalent mechanism and show a rather drug-like appearance ( figure ). the envelope-anchored s protein mediates cov entry into host cells by first binding to a host receptor and then fusing viral and host membranes. the sars-cov- s protein was solved by cryoelectron microscopy and just released; knowing the Å -resolution structure of the sars-cov- spike will allow for additional protein engineering efforts that could improve antigenicity and protein expression for vaccine development. moreover, the Å -resolution detail will enable the design and screening of small molecules with fusion-inhibiting potential. it is the affinity between the viral receptor-binding domain (rbd) and the host receptor in the initial viral attachment step that primarily determines which host is susceptible to sars-cov infection. the sars-cov- entry through receptor binding was elucidated independently by several groups. , on the basis of the rich knowledge about sars-cov and the sequence homology, it was suggested that sars-cov- uses angiotensin-converting enzyme (ace ) as its receptor ( figure ). it uses the sars-cov receptor ace and the cellular protease tmprss for entry into target cells. , the interplay of the ace receptor in cardiovascular diseases (with the well-known drug class of ace inhibitors) and as the docking point for sars-cov- cellular infection is a current point of intense debate and research. it was found that several critical residues in sars-cov- rbm (particularly gln ) provide favorable interactions with human ace , consistent with sars-cov- 's capacity for human cell infection. on the basis of the extensive modeling of the virus-human receptor interactions, it was also predicted that a single n t mutation might significantly enhance the binding affinity between sars-cov- rbd and human ace and thus potentially lead to much more virulent bodies. , the emergency and evolution of novel mutations at the position in sars-cov- -infected and covid- patients have to be closely monitored. mutations in the s protein are the ones causing the zoonosis, and as a result, not all of them will lead to their binding to ace . therefore, drugs targeting the s protein-ace interaction might not apply to some of the future covs. on the other hand, the spike-shaped protein on the surface of the viruses causing sars, mers, and covid- provides a tantalizing target for antibodies or other compounds, which could prevent covs from invading human cells. the virus' s protein seems to emerge as the consensus target antigen. the rdrp enzyme allows the viral genome to be transcribed into new rna copies by using the host cell's machinery. rdrp inhibitors are emerging as a new strategy to fight viral infections. the chemistry and biology of rdrp have been extensively reviewed. rna polymerase inhibitors are promising agents to fight covid- . rdrp proteins of sars-cov- and sars-cov share % sequence identity. favipiravir ( ) is an approved rna polymerase inhibitor for the treatment of the influenza pandemic. it has been shown not only to be active in influenza but also active against other rna viruses (figure ) . favipiravir is a prodrug , which is metabolized in cells into the active purine nucleotide that mimics favipiravir-ribofuranosyl- -triphosphate that inhibits the rna replication and thus the viral progression. interestingly, it does not inhibit host dna and rna synthesis and inosine -monophosphate dehydrogenase (impdh) activity. favipiravir reportedly demonstrated efficacy with minor side effects in an ongoing -patient clinical trial in shenzhen, guangdong province. the drug's generic version received approval by the health authorities in china. the broad-spectrum antiviral drug remdesivir together with chloroquine effectively inhibit the recently emerged novel cov (sars-cov- ) in vitro. , remdesivir ( ) reduced sars-cov- infection of monkey kidney cells with an ec of . mm. the compound is in late-stage clinical development and has been recently described to inhibit multiple rna viruses on a cellular level, including ebola and sars. the presumed mode of action of the adenosine analog prodrug remdesivir is pre-mature rna synthesis termination by incorporation into nascent viral rna chains. galidesivir ( ) is another antiviral drug under clinical development with potential in covid- treatment (figure ). it is an adenosine analog and is currently developed for ebola virus disease and marburg virus disease. it also shows broadspectrum antiviral activity against rna virus families including covs. multiple other rdrp inhibitors are described in the literature, for example, broadspectrum antiviral -bis fluorinated aristeromycin analog ( ). chloroquine ( ) is an existing anti-malaria medicine also used to treat several other diseases. it blocked virus infection with an ec of . mm. , its mode of action is unclear. however, chloroquine inhibits endosomal acidification and thus could stop the release of viral dna into the cytoplasm. it is under assessment in more than patients at over ten hospitals in beijing and guangdong province. plans for an additional study in hunan province are underway. the role of the other sars-cov- n proteins as drug-discovery targets is less clear. for the assembly of the replication/ q transcription complexes, there is a vast interaction network described between the non-structural proteins. similarly, viral particle assembly requires orchestrated interaction between n, s, m, and e proteins. all these interactions can be potential targets, but the structural information is currently minimal. resolution of the protein and complex structures will provide new unique drug targets. for example, the crystal structure of sars-cov- n protein rna-binding domain was just published and will give structural insight as a potential drug target. it is rapidly detected by antibodies in serum, plasma, and peripheral blood, and might therefore serve to develop specific diagnostics. using methods of machine learning-enabled scientific literature analysis, the biotech company benevolentai q proposed the ap -associated protein kinase (aak ) as a host target to fight sars-cov- . aak is the key enzyme of receptor-mediated endocytosis, which is the major mechanism of most viruses to enter their host cells. thus, they predict the approved (for rheumatoid arthritis) kinase inhibitor baricitinib to reduce the ability of the virus to infect lung cells (figure ). the recent emergence of the wuhan cov (sars-cov- ) has put the world on alert. the rapid worldwide spread and the high human-to-human transmissibility, combined with the inability to contain the pandemic, is causing an increasing death toll and also considerable paralysis of the world economy (table q ). the covid- could decrease and disappear or could be established worldwide in the human population and reoccur seasonally in future mutations through zoonosis from one of the animal reservoirs. however, it is very likely that in the upcoming years, we will see more outbreaks from cov and other viruses. the basic, translational, and public health research communities have to prepare for this much better. the outbreak has emphasized the urgent need for renewed efforts to develop broad-spectrum antiviral agents to combat covs. on the positive side, much new information of the virus biology and the spread was immediately shared, whereas, on the negative side, many past opportunities to develop antivirals against covs were not taken, despite a large number of promising approaches and compounds. the past decade has shown that cov outbreaks are regularly reoccurring with more or less health effect on human and livestock. it remains to be hoped that the current pandemic will slow down and end as predicted in summer. furthermore, it turns out that containment measures are not effective to avoid more severe spread. it remains to be seen whether efficient and long-lasting immunity will develop in the infected population with regard to future outbreaks and whether pharmacological measures can be rapidly developed to be able to treat severely sick people. a.d. and l.g. wrote the manuscript. severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges viral metagenomics revealed sendai virus and coronavirus infection of malayan pangolins (manis javanica) a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) drug repurposing: progress, challenges and recommendations therapeutic options for the novel coronavirus ( -ncov) an overview of severe acute respiratory syndromecoronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy homology models of wuhan coronavirus clpro protease structure of m pro from covid- virus and discovery of its inhibitors synthesis, crystal structure, structureÀactivity relationships, and antiviral activity of a potent sars coronavirus cl protease inhibitor synthesis and evaluation of isatin derivatives as effective sars coronavirus cl protease inhibitors synthesis and evaluation of pyrazolone compounds as sars-coronavirus c-like protease inhibitors discovery of potent anilide inhibitors against the severe acute respiratory syndrome cl protease potential broad spectrum inhibitors of the coronavirus clpro: a virtual screening and structure-based drug design study gliptin repurposing for covid- potential inhibitors for -ncov coronavirus m protease from clinically approved medicines characteristics of flavonoids as potent mers-cov c-like protease inhibitors inhibition of sars-cov cl protease by flavonoids role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of severe acute respiratory syndrome with lopinavir/ ritonavir: a multicentre retrospective matched cohort study sars: systematic review of treatment effects molecular modeling evaluation of the binding effect of ritonavir, lopinavir and darunavir to severe acute respiratory syndrome coronavirus proteases a trial of lopinavir-ritonavir in adults hospitalized with severe covid- sars-cov- and coronavirus disease : what we know so far. pathogens the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand x-ray structure and biological evaluation a noncovalent class of papain-like protease/ deubiquitinase inhibitors blocks sars virus replication xray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases cryo-em structure of the -ncov spike in the prefusion conformation the novel coronavirus ( -ncov) uses the sars-coronavirus receptor ace and the cellular protease tmprss for entry into target cells receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus covid- and the cardiovascular system we shouldn't worry when a virus mutates during disease outbreaks a pan-coronavirus fusion inhibitor targeting the hr domain of human coronavirus spike nucleosides for the treatment of respiratory rna virus infections insights into rna-dependent rna polymerase inhibitors as antiinfluenza virus agents t- (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections potential coronavirus drug approved for marketing. china daily remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro insights from nanomedicine into chloroquine efficacy against covid- therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys galidesivir limits rift valley fever virus infection and disease in syrian golden hamsters design, synthesis, and anti-rna virus activity of -fluorinated-aristeromycin analogues crystal structure of sars-cov- nucleocapsid protein rna binding domain reveals potential unique drug targeting sites baricitinib as potential treatment for -ncov acute respiratory disease coronaviruses -drug discovery and therapeutic options key: cord- - esn t b authors: tyndall, mark title: safer opioid distribution in response to the covid- pandemic date: - - journal: int j drug policy doi: . /j.drugpo. . sha: doc_id: cord_uid: esn t b covid- has turned the world upside down in a very short period of time. the impact of covid- will disproportionately effect people who are least able to protect themselves and this will include people who use drugs. the arrival of the covid- pandemic comes at time when north america is in the midst of a protracted overdose epidemic caused by a toxic illegal drug supply. overdose deaths are likely to rise when people are isolated, social support programs are cut back, and the illicit drug supply is further compromised. safer opioid distribution in response to a toxic street drug supply is a pragmatic and effective way to reduce overdose deaths. covid- makes such an approach even more urgent and compelling. covid- has turned the world upside down in a very short period of time. without effective treatments or a vaccine, the primary approach to limiting the transmission of covid- has been to reduce person-to-person contact. this has resulted in many communities locking downpeople staying at home, businesses shutting down, schools closing, and social gatherings cancelled. modelling suggests that this has been an effective strategy with regards to reducing covid- transmission (kent, ) , although the unintended social and economic consequences are massive and are still playing out (undp, ; van lancker & parolin, ) . like other natural disasters the people who suffer the most are those who are least able to protect themselves. covid- has shown that pre-existing health and social inequality create huge vulnerabilities in certain communities (abedi et al., ; dorn, cooney & sabin, ) . this has played out directly with high death rates from covid- infection, as well as indirectly through the interventions set up to control the spread. one group who are especially at risk are people who use drugs (pwud). the impact of covid- is expected to be severe in communities where illegal drug use is common due to the convergence of multiple factors (volkow, ) . the actual environment that many people live in is not conducive to physical distancing. scenes of tented encampments, emergency shelters, and people sleeping on the streets is one of the greatest challenges facing cities across north america. the need to address the homeless crisis has received a lot of media attention during the covid- response (fuller t, ; picard, ) . some see this as an opportunity to deal with two crises at once by opening up unoccupied hotel rooms (osman l, ) . the use of drugs among those who are unhoused is high, as chronic alcohol and drug use is tightly intertwined with homelessness (doran et al., ) . the emergency use of shelters is also problematic as crowding within a closed indoor space is set-up for covid- transmission and is not a substitute for housing (tobolowsky et al., ) . even if emergency housing can be found and mitigation strategies to reduce covid- transmission are put in place, the need to access an illegal drug supply makes staying in place extremely unlikely (bodkin et al., ) . it is not reasonable to expect that people who are dependent on drugs will stay in isolation (jenkins et al, ) the drugs that they need are from illegal sources and the ways that people generate money to purchase drugs are generally not home based. even for people on opioid agonist therapy (oat), restrictive rules with regards to prescriptions and daily observed pick-ups makes physical distancing extremely challenging. revising these restrictive regulations around oat in response to covid- must be a priority (brico, ) the prison system is also ill-equipped to deal with covid- and many people who use drugs are in and out of the criminal justice system (akiyama & spaulding & rich, ) . this sets up a continuous loop of new infections from those leaving prison who introduce covid- into the community and those entering prison, who bring it back into the prison facilities. there have already been serious outbreaks of covid- infection in correctional institutions like rikers island jail complex in new york, and this is likely to continue (bryant, ) . in addition to a range of prison mitigation strategies, some inmates have been discharged in order to reduce their chances of contracting the infection, but often with little attention to where they will go following discharge (wurcel et al., ; bbc, ) . while discharge from prison may help to reduce covid- transmission, it should be recognized that this can be a hazardous event for people who have been forced to reduce or stop their opioid consumption. a waning tolerance to opioids means that the first exposure to street drugs can be deadly and it is critical that proper discharge planning and supports are in place (bukten et al., ) many people who use drugs rely on public services for medical care, harm reduction supplies, street outreach, and food distribution. all of these critical supports have been disrupted by covid- with either reduction in services or complete shutdowns (khatri & perrone, ) . this includes restrictions to hospital emergency departments where many pwud receive their primary medical care (dunlop et al., ) . in fact, the messaging around overdose prevention is entirely opposite to what has been recommended in order to reduce covid- exposure. for years we have been warning people about the risk of using drugs alone and now we are asking people to self-isolate and avoid unnecessary contact. unless there are options to reduce the risk of overdosing when using alonesuch as access to a safer drug supplythis will result in more deaths. in efforts to reduce contact, many supportive housing programs have restricted guests, and this again increases the likelihood that people will be using alone (st. denis, ) . another often overlooked impact of the covid- pandemic is that the entire illegal drug market is disrupted and more unpredictable. a report by the canadian community epidemiology network on drug use highlights a number of converging issues that have made the illegal drug supply so unstable including disruption of the precursor supply chains, shut down of overseas synthesis facilities and disruption of drug shipping routes due to border closures (ccendu, ) . this creates further uncertainties in supply and the quality of the drugs being sold on the street deteriorates (smith, ) . these factors precipitated by covid- will all combine to make the ongoing overdose epidemic even worse (becker & fiellin, ) . there is no exact day or month or even year when the overdose epidemic started in north america. drug overdose has always been a risk for people who relied on an unregulated street market for their drugs. however, beginning around the opioid drug market shifted in many communities. what had a been a mixture of diverted pharmaceutical medications and imported heroin was replaced by fentanyl and other synthetic opioids (scholl, seth, kariisa et al., ) . the shift was gradual and uneven at first but by most street-based opioids in major cities across north america contained fentanyl or similar synthetic opioids (colon-berezin, nolan, blachman-forshay & paone, ). between and the drug enforcement association reported a -fold increase in synthetic fentanyl seizures (zibbell, ) . this shift to fentanyl and fentanyl analogues has proven to be deadly. the potency of these drugs is extremely high and there is very little room for error when mixing and producing them. people with long histories of opioid use have taken these very potent drugs and overdosed. in the canadian province of british columbia, where data is readily available, the number of illicit drug toxicity deaths went from in to in . of note, in the number of deaths went down to but monthly rates from show increasing trends returning. over % of the toxicology reports show that fentanyl is responsible for the deaths (b.c. coroner service, ). in , the centers for disease control attributed over , overdose deaths to synthetic opioids (cdc, ). the deeply entrenched stigma and indifference shown towards people who overdose is a major political and societal failing (drug policy alliance, ). deaths of this scale due to anything else would have demanded a response. the reasoning goes that there is really no justification or mechanism to regulate these drugs because they are illegal. so instead of viewing the overdose epidemic as a protracted mass poisoning event, it is viewed as a collection of self-inflicted harms or unintentional suicides. based on this rationale, people should know that these drugs can be lethal and it is their fault for using them. this victim blaming goes against everything that we know about addiction, trauma, and the structural barriers that perpetuate the epidemic. while the response to the overdose crisis has been entirely inadequate, harm reduction interventions have played an important role in reducing overdose deaths. harm reduction programs act as critical first steps to connecting with health and social services and provide a lifeline to treatment and other supports . in british columbia it was estimated through a modeling exercise, that the combined impact of naloxone, supervised injection sites and new starts on opioid agonist therapy (oat) reduced overdose deaths by over % from april through to december (irvine et al., ) . it is unfortunate that many jurisdictions across north america are still embroiled in debates that should have been settled long ago around the role of harm reduction and the merits of methadone programs, safe needle distribution, and supervised injection sites (nadelmann & lasalle, ) . despite the important role that harm reduction programs play, the overdose epidemic has brought to light the limitations of existing interventions. while supervised injection sites and communitycentred naloxone programs can save lives through intervening in overdoses once they occur, these interventions are not able to reduce the chances of overdosing in the first place nor do they reduce the harms, violence and interaction with policing that is associated with acquiring the drugs (cooper, ) . the idea that people who are dependent on illegal drugs should be given access to a safer, regulated supply is both controversial and long overdue (tyndall, ) . it has become obvious to those who are most impacted by the overdose crisis that nothing short of providing access to safer opioid drugs will reduce overdoses (fischer & pang & tyndall, ) . in fact, we have always known that people who acquire drugs through an illegal street market put themselves at great risk. from the international drug cartels to the street-based dealer, the motive to sell these drugs is profit driven and the well-being of the customers along with the quality and content of the drugs are secondary. this drive for profit has led to a tragic but predictable place where prohibition has filled the streets with fentanylcheap, concentrated, and highly addictive (beletsky & davis, ) . fentanyl is the predictable outcome of opioid prohibition and the main victims are the people who are dying of overdoses. from a purely pragmatic perspective it only makes sense to offer people a regulated and safer drug source. if drugs were regulated and accessible, then much of the violence and harms associated with drug acquisition through criminal sources would be eliminated. people would not be forced into desperate situations. from the person who collects discarded bottles, to those involved in low level drug dealing, to those involved in sex work, providing a regulated supply of drugs would reduce these activities and their risk. it would also greatly reduce the cost to the health care system and to the criminal justice system who are responsible for enforcing drug laws. on a global scale, the united nations office on drugs and crime (unodc) estimates that the cost of enforcing current drug laws is in excess of $ billion per year (unodc, ). even more incredible than the cost to society is just how ineffective enforcement is in relation to reducing the trafficking and uptake of illegal drugs. even re-distributing percent of the resources from law enforcement to health and harm reduction, as called for by harm reduction international, could have a dramatic impact on the health and safety of pwud (hri, ). a safer opioid supply is an especially difficult concept to promote when drugs are illegal and so much media attention has been given to the role of prescription opioids in the current overdose epidemic (griesler et al., ) . there are in fact a long list of barriers that will make providing a safe drug supply difficult. while it is clear that the majority of overdoses are now due to fentanyl and other synthetic opioids, there is still a reluctance to prescribe pharmaceutical opioids because of the potential for overdose. there are also concerns around diversion of the prescribed medications as this could contribute to more people becoming dependent. like many harm reduction programs, opposition will come from those who feel that all our efforts and resources should be put towards addiction treatment rather than harm reduction. there is also the cost of medications themselves and some will feel that it is not the responsibility of the taxpayer to pay for a safer opioid supply. finally, the regulations around controlled drugs and substances are complex and any regulatory changes must pass through a large number of organizations and bureaucratic bodies including multiple levels of government, colleges of physicians, colleges of pharmacists, and law enforcement associations. navigating through this bureaucracy can be a slow and arduous process. prior to covid- , the call to provide a safer opioid supply was gaining momentum. the theme of the canadian national day of action on the overdose crisis on april , was "safe supply" (capud, ). in a document produced by the canadian association of people who use drugs (capud) the concept of a safe supply is described including what a safe supply means, how it can be dispensed, and models of successful programs. it also makes clear that a safe supply does not include methadone, buprenorphine/suboxone and slow release morphine as they are not intended to provide the mind/body altering properties that people are looking for. the question remained however around how to do this. how to provide a regulated supply of pharmaceutical-grade opioids that is accessible, scalable and will attract people who are most at risk of overdose ? the most recognized and researched form of a safe opioid supply is heroin assisted treatment (hat) designed for chronic heroin dependent individuals. this is an evidenced-based response to people who have tried and failed other forms of addiction treatment (ferri, davoli & perucci, ) . one of the larger clinical trials was conducted in vancouver and montreal and found that compared to standard methadone maintenance treatment, the provision of heroin was associated with better retention in care, reduced illicit drug use and less illegal activities (oviedo-joekes, et al., ) . a subsequent study in vancouver found that injectable hydromorphone produced similar positive outcomes when compared directly to heroin maintenance (oviedo-joekes, et al., ) . these clinical trials have resulted in the formation of a permanent clinic site to administer these protocols. despite the positive outcomes, these programs have not been scaled up due to the costs of the facilities, the drugs, the security, the need for a highly skilled staff and political will. it is also designed specifically for people who have been refractory to other forms of treatment. in response to covid- there has been renewed interest around providing a safer drug supply to people who are required to isolate (bach, robinson, sutherland & brar, ) . the bc centre for substance use released a set of prescription guidelines designed to support physicians who were willing to provide pharmaceutical-grade opioids to those who were diagnosed with covid- or who were at risk of exposure (bccsu, ). these guidelines included hydromorphone tablets or long-acting morphine (m-eslon) capsules for opioid dependency, dextroamphetamine or methylphenidate for stimulant dependency and nicotine patches for nicotine dependency. the unique aspect of the safe supply guidelines is that the drugs can be consumed unwitnessed and that the length of the prescription is up to the prescribing physician. while an extremely important step in opening up the possibility of a safe supply, the uptake has been slow. one of the main barriers is the lack of physicians willing to prescribe and ultimately take on the responsibility and liability for the medications. physicians are reluctant to act as the gate keepers for a public health response to the overdose crisis (woo, ) . it may indeed be necessary to establish a regulated dispensing program that does not require individual physician prescribing, but rather a public health model whereby eligible participants are given direct access to a regulated supply of opioids. the participants could still be closely monitored but the goal would be to provide an alternative to the toxic street drug supply rather than embedding the program in a physician-led primary care model. this harm reduction approach would greatly reduce the risk of overdose, help break the cycle of poverty and despair, and increase the opportunity to engage in health care and social services. one way to provide a low-barrier, scalable, distribution model for a safer drug supply is through a technology that can dispense and monitor the medications. the mysafe machine is a biometric storage locker where people can pick-up their prescribed medications. this technology has been piloted in vancouver, canada since december (turvill, ) . mysafe is an -pound secure metal case, much like a large atm, where medications can be dispensed using a nontouch biometric scanner. the biometrics reads the vein pattern of the palm and can only be used by the machine to identify the participant. unlike fingerprints, for example, the biometrics is not linkable to any other identification systems and could not be used for surveillance. the main advantage of the biometrics, beyond security, is that it avoids the use of cards and pin numbers that may be lost, forgotten or shared. it can be placed in a variety of locations including supervised injection sites, supportive housing buildings, pharmacies, or at a stand-alone location. once enrolled in the program, the machine can deliver the prescribed medication in under seconds and every transaction is tracked in real time. the mysafe machine is equipped with a video screen that can display messages to the participants and future modifications will include full telemedicine capabilities. initial observations with mysafe has identified a number of important advantages when providing a safer opioid supply. firstly, the machine is entirely non-judgemental and provides people some autonomy when picking up their medications. this can be a big advantage over clinic-based or pharmacy-based programs where participants must follow more rigid protocols. participants can still receive assistance and referrals, but this can happen through their own initiative. secondly, pick-ups can happen over a -hour period and this provides maximum flexibility. clinic and pharmacy-based programs often require participants to attend during specified times. thirdly, the machine can be programmed to dispense medications on an individual schedule from multiple times per day to daily or even weekly. some people who are using opioids may benefit from dispensing smaller quantities at each pick-up to be successful. fourthly, there is real-time tracking for every transaction on a secure web-based dashboard that can be accessed by pharmacists and physicians. this essentially eliminates medication recording errors, tracks inventory and can send alerts if participants do not pick up doses. this is not used punitively but can add a level of safety if normal patterns of pick-ups are interrupted and some follow-up is required. the data collected on the machine is encrypted and kept on a secure server and follows security protocols that govern all medically sensitive information. fifthly, the machine requires minimal staffing other than registering participants and loading the medications, so that it is cost-effective compared with programs that require person-to-person dispensing. sixthly, the machine can dispense whatever medication is required. for the pilot program, hydromorphone tablets were used as they were legal, available, cheap and acceptable to a majority of opioid users. however, other formulations of opioids or even stimulant drugs could be dispensed as part of a safe supply program (fleming et al. ) . and finally, in an environment where physical distancing is required as an infection control measure during covid- , the machine allows quick pick-ups that avoids any line-ups and does not require any person-to-person contact. the covid- pandemic will have a major impact on the overdose crisis. not only will covid- contribute to even higher rates of drug overdoses, it will erode some of the progress that has been made to reduce overdose deaths. drugs that were toxic have become more toxic, harm reduction interventions have been stalled, treatment programs have been reduced, many critical social services have contracted, and isolation has increased. overall, the lives of pwud have become even more precarious. at the root of this response is stigma and indifference supported by global drug policies that are ineffective in curbing drug use and extremely destructive for people who use drugs (csete et al., ; coyne & hall, ) . while we advocate to reduce stigma, improve care and treatment, and change drug policy, we cannot simply wait as people continue to buy toxic street drugs. with any mass poisoning the urgent response would be provide non-toxic alternatives until a safer supply can be restored. with fentanyl on the streets, many people will not get a second chance. covid- may not have changed the need for regulated and safer opioid distribution but it has made it much more compelling and urgent. racial, economic and health inequality and covid- infection in the united states flattening the curve for incarcerated populations -covid- in jails and prisons innovative strategies to support physical distancing among individuals with active addiction. the lancet. psychiatry us jails begin releasing prisoners to stem covid- infections illicit drug toxicity deaths risk mitigation guidelines in the context of dual public health emergencies when epidemics collide: coronavirus disease (covid- ) and the opioid crisis today's fentanyl crisis: prohibition's iron law, revisited. the international journal on drug policy pandemic planning in homeless shelters: a pilot study of a covid- testing and support program to mitigate the risk of covid- outbreaks in congregate settings addiction treatment clinics struggle to keep up with covid- coronavirus spread at rikers is a 'public health disaster', says jail's top doctor. the guardian high risk of overdose death following release from prison: variations in mortality during a -year observation period canadian union of people who use drugs (capud) changes related to covid- in the illegal drug supply and access to services and resulting health harms war on drugs policing and police brutality overdose deaths involving fentanyl and fentanyl analogs four decades and counting: the continued failure of the war on drugs public health and international drug policy substance use and homelessness among emergency department patients. drug and alcohol dependence covid- exacerbating inequalities in the us no shortcuts to safer opioid prescribing drug policy alliance. stigma and people who use drugs challenges in maintaining treatment services for people who use drugs during the covid- pandemic heroin maintenance for chronic heroindependent individuals. the cochrane database of systematic reviews stimulant safe supply: a potential opportunity to respond to the overdose epidemic the opioid death crisis in canada: crucial lessons for public health california governor declares homeless crisis 'a disgrace' medical use and misuse of prescription opioids in the us adult population harm reduction international modelling the combined impact of interventions in averting deaths during a synthetic-opioid overdose epidemic tackling the overdose crisis: the role of safe supply covid- during the opioid epidemic -exacerbation of stigma and vulnerabilities covid- data shows how social distancing impacts virus spread. health it analytics opioid use disorder and covid- : crashing of the crises two steps forward, one step back: current harm reduction policy and politics in the united states hotels for homeless people could tackle two problems at once: advocates diacetylmorphine versus methadone for the treatment of opioid addiction hydromorphone compared with diacetylmorphine for long-term opioid dependence: a randomized clinical trial first and foremost, the homeless need housing downtown eastside residents still waiting for visitors to be allowed drug and opioid-involved overdose deaths -united states drug supply potentially more dangerous amid covid- pandemic. the calgary herald methadone maintenance treatment among patients exposed to illicit fentanyl in rhode island: safety, dose, retention, and relapse at months evaluation of the southern harm reduction coalition for hiv prevention: advocacy accomplishments evaluation of the southern harm reduction coalition for hiv prevention: advocacy accomplishments. health promotion practice covid- outbreak among three affiliated homeless service sites opioid vending machine opens in vancouver. the guardian an emergency response to the opioid overdose crisis in canada: a regulated opioid distribution program socio-economic impact of covid- covid- , school closures, and child poverty: a social crisis in the making collision of the covid- and addiction epidemics .'s move to safe supply has a bumpy roll-out clinical infectious diseases : an official publication of the infectious diseases society of america, ciaa association of law enforcement seizures of heroin, fentanyl, and carfentanil with opioid overdose deaths in ohio key: cord- -zrjx ev authors: demain, arnold l; sanchez, sergio title: microbial drug discovery: years of progress date: - - journal: j antibiot (tokyo) doi: . /ja. . sha: doc_id: cord_uid: zrjx ev microbes have made a phenomenal contribution to the health and well-being of people throughout the world. in addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products. this review centers on these beneficial secondary metabolites, the discovery of which goes back years to the time when penicillin was discovered by alexander fleming. back in , alexander fleming began the microbial drug era when he discovered in a petri dish seeded with staphylococcus aureus that a compound produced by a mold killed the bacteria. the mold, identified as penicillium notatum, produced an active agent that was named penicillin. later, penicillin was isolated as a yellow powder and used as a potent antibacterial compound during world war ii. by using fleming's method, other naturally occurring substances, such as chloramphenicol and streptomycin, were isolated. naturally occurring antibiotics are produced by fermentation, an old technique that can be traced back almost years, initially for beverages and food production. beer is one of the world's oldest beverages, produced from barley by fermentation, possibly dating back to the sixth millennium bc and recorded in the written history of ancient egypt and mesopotamia. another old fermentation, used to initiate the koji process, was that of rice by aspergillus oryzae. during the past years, penicillium roqueforti has been utilized for cheese production, and for the past years soy sauce in asia and bread in egypt has represented examples of traditional fermentations. natural products with industrial applications can be produced from primary or secondary metabolism of living organisms (plants, animals or microorganisms). owing to technical improvements in screening programs, and separation and isolation techniques, the number of natural compounds discovered exceeds million. among them, - % are produced by plants (alkaloids, flavonoids, terpenoids, steroids, carbohydrates, etc.) and % have a microbial origin. of all the reported natural products, approximately - % show biological activity, and of these approximately % have been obtained from microbes. furthermore, from the biologically active compounds that have been obtained so far from microbes, % are produced by actinomycetes, % by fungi and % by unicellular bacteria. the increasing role of microorganisms in the production of antibiotics and other drugs for treatment of serious diseases has been dramatic. however, the development of resistance in microbes and tumor cells has become a major problem and requires much research effort to combat it. drugs of natural origin have been classified as (i) original natural products, (ii) products derived or chemically synthesized from natural products or (iii) synthetic products based on natural product structures. evidence of the importance of natural products in the discovery of leads for the development of drugs for the treatment of human diseases is provided by the fact that close to half of the best selling pharmaceuticals in were either natural products or their derivatives. in this regard, of the top-selling drugs reported in , % were natural products or their derivatives and of these, % were antibiotics. today, the structures of around secondary metabolites have been elucidated. it is important to understand that many chemically synthesized drugs owe their origin to natural sources. applications of chemically synthesized natural metabolites include the use of a natural product derived from plant salicyclic acid derivatives present in white willow, wintergreen and meadowsweet to relieve pain and suffering. concoctions of these plants were administered by hippocrates back in the year bc, and even earlier in egypt and babylonia, for fever, pain and childbirth. synthetic salicylates were produced initially by bayer in , and later in , arthur eichengrun at bayer discovered that an acetyl derivative (aspirin), reduced acidity, bad taste and stomach irritation. these plant-based systems continue to play an essential role in health care, and it has been estimated by the world health organization (who) that approximately % of the world's inhabitants rely mainly on traditional medicines for their primary health care. other synthesized compounds originating from natural products include a nonapeptide, designated teprotide, which was isolated from the venom of the brazilian pit viper bothrops jararaca. this led to the design and synthesis of angiotensin-converting enzyme (ace) inhibitors such as captopril, which was the first marketed, orally active ace inhibitor. enalapril, another ace inhibitor used in the treatment of cardiovascular disease, was approved for marketing by the food and drug administration (fda) in . the alkaloid quinine, the active constituent of the 'fever tree' cinchona succirubra, has been known for centuries by south american indians to control malaria. during the twentieth century, massive programs to synthesize quinoline derivatives, based on the quinine prototype, were carried out. the first of the new quinolones to be used clinically as an antibacterial agent was nalidixic acid, which emerged as part of a large chemical synthesis program developed at the sterling winthrop research institute. , the program was begun when -chloro- , -dihydro- -ethyl- -oxoquinolone- -carboxylic acid was obtained as a side product during purification of chloroquine and found to have antibacterial activity. the best compound found in the program was nalidixic acid, which had remarkable activity against gram-negative bacteria and was shown to be an inhibitor of dna gyrase. its discovery led to a whole series of synthetic quinolone and fluoroquinolone antibiotics (pefloxacin, norfloxacin, ciprofloxacin, levofloxacin, ofloxacin, lomefloxacin, sparfloxacin, etc.), which have been very successful in medicine and have achieved major commercial success (table ) . it is important to appreciate that all quinolones, though synthetic, are based on the structure of the natural plant product quinine. secondary metabolites have exerted a major impact on the control of infectious diseases and other medical conditions, and the development of pharmaceutical industry. their use has contributed to an increase in the average life expectancy in the usa, which increased from years in to years (in men) and years (in women) in . probably, the most important use of secondary metabolites has been as anti-infective drugs. in , the market for such antiinfectives was us$ billion (table ) and in it was us$ billion. table shows that, among the anti-infective drugs, antivirals represent more than % of the market. two antivirals that are chemically synthesized today were originally isolated from marine organisms. they are acyclovir (active against the herpes virus by inhibition and inactivation of dna polymerase) and cytarabine (active against non-hodgkin's lymphoma). both compounds are nucleoside analog drugs, originally isolated from sponges. other antiviral applications of natural compounds are related to human immunodeficiency virus (hiv) treatment. in the pathogenesis of this disease, hiv- , similar to other retroviruses, depends on its stable integration into the host genome to facilitate efficient replication of the viral rna and maintenance of the infected state. therefore, de novo viral dna synthesized during reverse transcription is immediately integrated into the host cell dna (through the integration step), allowing for further transcription of viral rna. in the late phase of hiv viral replication, the large precursor polyprotein (gag-pol precursor, pr ) must be appropriately cleaved by a viral protease. the cleavage of the gag precursor protein of hiv is critical for the maturation and infectivity of the viral particle. without the appropriate cleavage of the precursor polyproteins, non-infectious viral particles are generally produced. to confront this problem, a tremendous effort has been made at the us national cancer institute (nci), in search of natural metabolites capable of inhibiting hiv reverse transcriptase and hiv protease. chemically synthesized derivatives of these compounds are the main agents now used against hiv. furthermore, reports have been published on natural product inhibitors of hiv integrase obtained from among the marine ascidian alkaloids; that is, the lamellarins (produced by the mollusk lamellaria sp.), and from terrestrial plants (baccharis genistelloides and achyrocline satureioides). the most consistent anti-hiv activity was observed with extracts prepared from several baccharis species. in addition, nci has been evaluating the hiv- inhibitory activity of pepstatin a, a small pentapeptide produced by several streptomyces species. it contains a unique hydroxyamino acid, statine, that sterically blocks the active site of hiv- protease. , reasons for developing new antibiotics new antibiotics that are active against resistant bacteria are required. bacteria have lived on the earth for several billion years. during this time, they encountered in nature a wide range of naturally occurring antibiotics. to survive, bacteria developed antibiotic resistance mechanisms. therefore, it is not surprising that they have become resistant to most of the natural antimicrobial agents that have been developed over the past years. this resistance increasingly limits the effectiveness of current antimicrobial drugs. the problem is not just antibiotic resistance but also multidrug resistance. in , more than % of pathogenic bacteria were estimated to be resistant to at least one of the currently available antibiotics. the so-called 'superbugs' (organisms that are resistant to most of the clinically used antibiotics) are emerging at a rapid rate. s. aureus, which is resistant to methicillin, is responsible for many cases of infections each year. the incidence of multidrug-resistant pathogenic bacteria is increasing. the infectious disease society of america (idsa) reported in that in us hospitals alone, around million people acquire bacterial infections each year (http://www.idsociety.org/content.aspx?id¼ ). s. aureus is responsible for half of the hospital-associated infections and takes the lives of approximately patients each year in the usa alone. the bacteria produce a biofilm in which they are encased and protected from the environment. biofilms can grow on wounds, scar tissues and medical implants or devices, such as joint prostheses, spinal instrumentations, catheters, vascular prosthetic grafts and heart valves. more than % of the bacterial species producing such biofilms are likely to be resistant to at least one of the drugs commonly used in anti-infectious therapy. called 'nosocomial bacteria.' more than % of sepsis cases in hospitals are caused by gram-negative bacteria. among them, pseudomonas aeruginosa accounts for almost % of these opportunistic infections. they represent a serious problem in patients hospitalized with cancer, cystic fibrosis and burns, causing death in % of cases. other infections caused by pseudomonas species include endocarditis, pneumonia and infections of the urinary tract, central nervous system, wounds, eyes, ears, skin and musculoskeletal system. this bacterium is another example of a natural multidrug-resistant microorganism. although many strains are susceptible to gentamicin, tobramycin and amikacin, resistant forms have also developed. these multidrug-resistant bacteria make hospitals ''dangerous places to be, especially if you are sick, but even if not.'' although we are seeing a steady increase in resistance in almost every pathogen to most of the current antibiotics over time, not all the antibacterial agents show the same rate of resistance development. for example, antimicrobials such as rifampicin, which targets single enzymes, are most susceptible to the development of resistance, whereas agents that inactivate several targets irreversibly generate resistance more slowly. in addition to the antibiotic-resistance problem, new families of anti-infective compounds are needed to enter the marketplace at regular intervals to tackle the new diseases caused by evolving pathogens. at least new diseases emerged in the s and s and they are growing in incidence. emerging infectious organisms often encounter hosts with no prior exposure to them and thus represent a novel challenge to the host's immune system. several viruses responsible for human epidemics have made a transition from animal host to humans and are now transmitted from human to human. hiv, responsible for the acquired immunodeficiency syndrome (aids) epidemic, is one example. although it has not been proven, it is suspected that severe acute respiratory syndrome (sars), caused by the sars coronavirus, also evolved from a different species. in the early s, after decades of decline, the incidence of tuberculosis began to increase. the epidemic took place owing to inadequate treatment regimens, a diminished public health system and the onset of the hiv/aids epidemic. the who has predicted that between and , nearly billion people will become infected with mycobacterium tuberculosis and that this disease will cost the lives of million people. sexually transmitted diseases have also increased during these decades, especially in young people (aged - years). the human papillomavirus, chlamydia, genital herpes, gonorrhea and hiv/aids are examples. hiv/aids has infected more than million people in the world. together with other diseases such as tuberculosis and malaria, hiv/aids accounts for over million illnesses and more than million deaths each year. additional evolving pathogens include the (i) ebola virus, which causes the viral hemorrhagic fever syndrome with a resultant mortality rate of %; (ii) the bacterium legionella pneumophila, a ubiquitous aquatic organism that lives in warm environments, which causes legionnaire's disease, a pulmonary infection; (iii) the hantavirus, which can infect humans with two serious illnesses: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome; (iv) at least three species of bacteria from the genus borrelia, which cause lyme disease, an emerging infection. in this case, the infection is acquired from the bite of ticks belonging to several species of the genus ixodes. borrelia burgdorferi is the predominant cause of lyme disease in the us, whereas borrelia afzelii and borrelia garinii are implicated in most european cases. the disease presentation varies widely, and may include a rash and flu-like symptoms in its initial stage, followed by musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations. in the majority of cases, symptoms can be eliminated with antibiotics, especially if the treatment begins early in the course of illness. however, late or inadequate treatment can lead to 'late-stage' lyme disease that can be disabling and difficult to treat. (v) other evolving pathogens include the escherichia coli :h (enterohemorrhagic e. coli), a strain that causes colitis and bloody diarrhea by producing a toxin called shiga toxin, which damages the intestines. it is estimated that this bacterium causes infection in more than patients a year in the usa. another example is (vi) cryptosporidium, an obligate intracellular parasite commonly found in lakes and rivers. cryptosporidium parvum is one of the common species affecting the digestive and respiratory organs. intestinal cryptosporidiosis is characterized by severe watery diarrhea. pulmonary and tracheal cryptosporidiosis in humans is associated with coughing and is frequently a low-grade fever. people with severely weakened immune systems are likely to have more severe and more persistent symptoms than healthy individuals. in the developing world, nearly % of the infectious disease deaths are caused by six diseases or disease processes: acute respiratory infections, diarrhea, tuberculosis, hiv, measles and malaria. in both the developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease. in the developing world, acute respiratory infections are attributed primarily to seven bacteria: bordetella pertussis, streptococcus pneumoniae, haemophilus influenzae, staphylococcus aureus, mycoplasma pneumoniae, chlamydophila pneumoniae and chlamydia trachomatis. in addition, the major viral causes of respiratory infections include respiratory syncytial virus, human parainfluenza viruses and , influenza viruses a and b, as well as some adenoviruses. these diseases are highly destructive in economic and social as well as in human terms and cause approximately million deaths per year, and innumerable serious illnesses besides affecting the economic growth, development and prosperity of human societies. morse identified six general factors in the emergence of infectious diseases: ecological changes, human demographics and behavior, international travel, technology and industry, microbial adaptation and change, and breakdown in public health measures. one additional reason for developing new antibiotics is related to their own toxicity. as with other therapeutic agents, the use of antibiotics may also cause side effects in patients. these include mild reactions such as upset stomach, vomiting and diarrhea (cephalosporins, macrolides, penicillins and tetracyclines), rash and other mild and severe allergic reactions (cephalosporins and penicillins), sensitivity to sunlight (tetracyclines), nervousness, tremors and seizures (quinolones). some side effects are more severe and, depending on the antibiotic, may disrupt the hearing function (aminoglycosides), kidneys (aminoglycosides and polypeptides) or liver (rifampin). during recent decades, we have seen an increasing number of reports on the progressive development of bacterial resistance to almost all available antimicrobial agents. in the s, the major problem was the multidrug resistance of gram-negative bacteria, but later in the s the gram-positive bacteria became important, including methicillin-resistant staphylococci, penicillin-resistant pneumococci and vancomycin-resistant enterococci. in the past, the solution to the problem has depended primarily on the development of novel antimicrobial agents. however, the number of new classes of antimicrobial agents being developed has decreased dramatically in recent years. the advent of resistant gram-positive bacteria has been noticed by the pharmaceutical, biotechnology and academic communities. some of these groups are making concerted efforts to find novel antimicrobial agents to meet this need. a new glycopeptide antibiotic, teicoplanin, was developed against infections with resistant gram-positive bacteria, especially bacteria resistant to the glycopeptide vancomycin. in another instance, the approach involved the redesign of a mixture of two compounds, called streptogramin, into a new mixture, called pristinamycin, to allow administration of the drug parenterally and in higher doses than the earlier oral preparation. the two components of streptogramin, quinupristin and dalfopristin, were chemically modified to allow intravenous administration. the new combination, pristinamycin, was approved by the fda for use against infections caused by vancomycin-resistant enterococcus faecium. additional moves against resistant microorganisms are the glycylcyclines developed to treat tetracycline-resistant bacteria. these modified tetracyclines show potent activity against a broad spectrum of gram-positive and gram-negative bacteria, including strains that carry the two major tetracycline-resistance determinants, involving efflux and ribosomal protection. two of the glycylcyline derivatives, dmg-mino and dmg-dmdot, have been tested against a large number of clinical pathogens isolated from various sources. the spectrum of activity of these compounds includes organisms with resistance to antibiotics other than tetracyclines; for example, methicillin-resistant staphylococci, penicillin-resistant s. pneumoniae and vancomycin-resistant enterococci. tigecycline was approved by the fda in as an injectable antibiotic. among the novel class of antimicrobial agents used in treating resistance to gram-positive infections, we can also mention the cyclic lipopeptide antibiotic daptomycin produced by streptomyces roseosporus. this compound was approved in by the fda for skin infections resulting from complications following surgery, diabetic foot ulcers and burns. it represents the first new natural antibiotic approved in many years. its mode of action is distinct from any other approved antibiotic: it rapidly kills gram-positive bacteria by disrupting multiple aspects of bacterial membrane function (by binding irreversibly to the bacterial cell membrane, causing membrane depolarization, destroying the ion concentration gradient and provoking the efflux of k + ). it acts against most clinically relevant gram-positive bacteria (staphylococcus aureus, streptococcus pyogenes, streptococcus agalactiae, streptococcus dysgalactiae subsp. equisimilis and enterococcus faecalis), and retains in vitro potency against isolates resistant to methicillin, vancomycin and linezolid. traditionally, these infections were treated with penicillin and cephalosporins, but resistance to these agents became widespread. [ ] [ ] [ ] [ ] daptomycin seems to have a favorable side effect profile, and it might be used to treat patients who cannot tolerate other antibiotics. telithromycin, a macrolide antibiotic, is the first orally active compound of a new family of antibacterials named the ketolides. it shows potent activity against pathogens implicated in communityacquired respiratory tract infections, irrespective of their b-lactam, macrolide or fluoroquinolone susceptibility. some of the microorganisms susceptible to this antibiotic are pneumococci, h. influenzae and moraxella catarrhalis, including b-lactamase-positive strains. in addition, telithromycin has a very low potential for selection of resistant isolates or induction of cross-resistance found with other macrolides. clavulanic acid, first detected in streptomyces clavuligerus, contains a bicyclic b-lactam ring fused to an oxazolidine ring with an oxygen in place of a sulfur, a b-hydroxyethylidene substituent at c- and no acylamino group at c- . it was first described in and shown to be a potent inhibitor of the b-lactamases produced by staphylococci and plasmid-mediated b-lactamases of e. coli, klebsiella, proteus, shigella, pseudomonas and haemophilus. although it is a broad-spectrum antibiotic, clavulanic acid possesses only very low antibacterial activity. therefore, the molecule has been combined, as a b-lactamase inhibitor, with a variety of broad-spectrum semisynthetic penicillins. for example, when administered with amoxicillin, it is used for the treatment of infections caused by b-lactamase-producing pathogenic bacteria. it has world sales of over us$ billion, and in it was the second largest selling antibacterial drug. clavulanic acid can also be combined with ticarcillin, which is a penicillin effective against organisms such as e. coli, proteus, salmonella, haemophilus, pseudomonas and s. aureus. it is normally used in hospitals for treating severe infections affecting blood or internal organs, bones and joints, upper or lower airways or skin and soft tissue. the combination extends ticarcillin antimicrobial activity by inhibiting the action of the b-lactamases produced by certain bacteria. mycosis is a condition in which fungi pass the resistance barriers of the human or animal body and establish infections. these organisms are harmless most of the time, but sometimes they can cause fungal infections. in most cases, these infections are not life threatening. however, when they are deeply invasive and disseminated, they lead to more serious infections, particularly in critically ill patients, elderly people and those who have conditions that affect the immune system (by disease or through the use of immunosuppressive agents). in addition, the use of antineoplastic and broad-spectrum antibiotics, prosthetic devices and grafts, and more aggressive surgery has increased invasive fungal infections. patients with burns, neutropenia, pancreatitis or after organ transplantation ( % of liver transplants, - % of heart transplants and % of kidney transplants) are also predisposed to fungal infection. approximately % of death from nosocomial infections are caused by fungi, and % of these are caused by candida and aspergillus, although cryptococcus spp., fusarium spp., scedosporium spp., penicillium spp. and zygomycetes are increasingly involved. pulmonary aspergillosis is the main factor involved in the death of recipients of bone marrow transplants, and pneumocystis carinii is the leading cause of death in aids patients from europe and north america. the rising incidence of invasive fungal infections and the emergence of broader fungal resistance have led to the need for novel antifungal agents. amphotericin b is the first-line therapy for systemic infection because of its broad spectrum and fungicidal activity. however, considerable side effects limit its clinical utility. echinocandins are large lipopeptide molecules that inhibit the synthesis of , -b-dglucan, a key component of the fungal cell wall. three echinocandins (caspofungin, micafungin and anidulafungin) have reached the market. caspofungin is also known as pneumocandin or mk- . this compound was the first cell-wall-active antifungal approved as a new injectable antifungal; this was in . it irreversibly inhibits , -b-d-glucan synthase, preventing the formation of glucan polymers and disrupting the integrity of fungal cell walls. it is more active and less toxic than amphotericin b and shows a broad spectrum of activity against candida (including fluconozole resistance), aspergillus, histoplasma and p. carinii, the major cause of hiv death. micafungin is licensed for clinical use in asian countries and in the us. this compound exhibits extremely potent antifungal activity against clinically important fungi, including aspergillus and azole-resistant strains of candida. in animal studies, micafungin is as efficacious as amphotericin b with respect to improvement of survival rate. it is characterized by a linear pharmacokinetic profile and substantially fewer toxic effects. anidulafungin is currently licensed in the us. although several new antifungal drugs have been developed in the past years, some patients remain resistant to treatments. the main reasons for this include intrinsic or acquired antifungal resistance, organ dysfunction preventing the use of some agents and drug interactions. in addition, some drugs penetrate poorly into sanctuary sites, including the eye and urine, and others are associated with considerable adverse events. however, there has been some progress. posaconazole is a new member of the triazole class of antifungals. it has shown clinical efficacy in the treatment of oropharyngeal candidiasis and has potential as a salvage therapy for invasive aspergillosis, zygomycosis, cryptococcal meningitis and a variety of other fungal infections. it is available as an oral suspension and has a favorable toxicity profile. the wide spectrum of posaconazole activity in in vitro studies, animal models and preliminary clinical studies suggests that it represents an important addition to the antifungal armamentarium. in addition to the screening programs for antibacterial activity, the pharmaceutical industry has extended these programs to other disease areas. , microorganisms are a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important products of the pharmaceutical industry. microbial secondary metabolites are now being used for applications other than antibacterial, antifungal and antiviral infections. for example, immunosuppressants have revolutionized medicine by facilitating organ transplantation. other applications include antitumor drugs, enzyme inhibitors, gastrointestinal motor stimulator agents, hypocholesterolemic drugs, ruminant growth stimulants, insecticides, herbicides, coccidiostats, antiparasitics vs coccidia, helminths and other pharmacological activities. further applications are possible in various areas of pharmacology and agriculture, developments catalyzed by the use of simple enzyme assays for screening before testing in intact animals or in the field. in the year , approximately million new cases of cancer were diagnosed in the world, resulting in million cancer-related deaths. the tumor types with the highest incidence were lung ( . %), breast ( . %) and colorectal ( . %). microbial metabolites are among the most important of the cancer chemotherapeutic agents. they started to appear around with the discovery of actinomycin and since then many compounds with anticancer properties have been isolated from natural sources. more than % of the current compounds with antineoplasic activity were originally isolated as natural products or are their derivatives. among the approved products deserving special attention are actinomycin d, anthracyclines (daunorubicin, doxorubicin, epirubicin, pirirubicin and valrubicin), bleomycin, mitosanes (mitomycin c), anthracenones (mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin), taxol and epothilones. actinomycin d is the oldest microbial metabolite used in cancer therapy. its relative, actinomycin a, was the first antibiotic isolated from actinomycetes. the latter was obtained from actinomyces antibioticus (now streptomyces antibioticus) by waksman and woodruff. as it binds dna at the transcription initiation complex, it prevents elongation by rna polymerase. this property, however, confers some human toxicity and it has been used primarily as an investigative tool in the development of molecular biology. despite the toxicity, however, it has served well against wilms tumor in children. the anthracyclines are some of the most effective antitumor compounds developed, and are effective against more types of cancer than any other class of chemotherapy agents. they are used to treat a wide range of cancers, including leukemias, lymphomas, and breast, uterine, ovarian and lung cancers. anthracyclines act by intercalating dna strands, which result in a complex formation that inhibits the synthesis of dna and rna. it also triggers dna cleavage by topoisomerase ii, resulting in mechanisms that lead to cell death. in their cytotoxic effects, the binding to cell membranes and plasma proteins plays an important role. their main adverse effects are heart damage (cardiotoxicity), which considerably limits their usefulness, and vomiting. the first anthracycline discovered was daunorubicin (daunomycin) in , which is produced naturally by streptomyces peucetius. doxorubicin (adriamycin) was developed in . another anthracycline is epirubicin. this compound, approved by the fda in , is favored over doxorubicin in some chemotherapy regimens as it appears to cause fewer side effects. epirubicin has a different spatial orientation of the hydroxyl group at the ¢ carbon of the sugar, which may account for its faster elimination and reduced toxicity. epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas. valrubicin is a semisynthetic analog of doxorubicin approved as a chemotherapeutic drug in and used to treat bladder cancer. bleomycin is a non-ribosomal glycopeptide microbial metabolite produced as a family of structurally related compounds by the bacterium streptomyces verticillus. first reported by umezawa et al. in , bleomycin obtained fda approval in . when used as an anticancer agent (inducing dna strand breaks), the chemotherapeutic forms are primarily bleomycins a and b . mitosanes are composed of several mitomycins that are formed during the cultivation of streptomyces caespitosus. although the mitosanes are excellent antitumor agents, they have limited utility owing to their toxicity. mitomycin c was approved by the fda in , showing activity against several types of cancer (lung, breast, bladder, anal, colorectal, head and neck), including melanomas and gastric or pancreatic neoplasms. recently, mitomycin dimers have been explored as potential alternatives for lowering toxicity and increasing efficiency. mithramycin (plicamycin) is an antitumor aromatic polyketide produced by streptomyces argillaceous that shows antibacterial and antitumor activity. it is one of the older chemotherapy drugs used in the treatment of testicular cancer, disseminated neoplasms and hypercalcemia. it binds to g-c-rich dna sequences, inhibiting the binding of transcription factors such as sp , which is believed to affect neuronal survival/death pathways. it may also indirectly regulate gene transcription by altering histone methylation. with repeated use, organotoxicity (kidney, liver and hematopoietic system) can become a problem. streptozotocin is a microbial metabolite with antitumor properties, produced by streptomyces achromogenes. chemically, it is a glucosamine-nitroso-urea compound. as with other alkylating agents in the nitroso-urea class, it is toxic to cells by causing damage to dna, although other mechanisms may also contribute. the compound is selectively toxic to the b-cells of the pancreatic islets. it is similar enough to glucose to be transported into the cell by the glucose transport protein of these cells, but it is not recognized by the other glucose transporters. as b-cells have relatively high levels of glucose permease, the relative streptozotocin toxicity for these b-cells can be explained. in , fda granted approval for streptozotocin as a treatment for pancreatic islet cell cancer. pentostatin (deoxycoformycin) is an anticancer chemotherapeutic drug produced by s. antibioticus. it is classified as a purine analog, which mimics the nucleoside adenosine and thus tightly binds and inhibits adenosine deaminase (k i of . Â À m). it interferes with the cell's ability to process dna. pentostatin is commonly used to treat hairy cell leukemia, acute lymphocytic leukemia, prolymphocytic leukemia (b-and t-cell origin), t-cell leukemia and lymphoma. however, it can cause kidney, liver, lung and neurological toxicity. the fda granted approval for pentostatin in . calicheamicins are highly potent antitumor microbial metabolites of the enediyne family produced by micromonospora echinospora. their antitumor activity is apparently due to the cleavage of double-stranded dna. they are highly toxic, but it was possible to introduce one such compound into the clinic by attaching it to an antibody that delivered it to certain cancer types selectively. this ingenious idea of the wyeth laboratories avoided the side effects of calicheamicin. in this regard, gemtuzumab is effective against acute myelogenous leukemia (aml). calicheamicin is bound to a monoclonal antibody against a transmembrane receptor (cd ) expressed on cells of monocytic/myeloid lineage. cd is expressed in most leukemic blast cells, but in normal hematopoietic cells the intensity diminishes with maturation. it was approved by the fda for use in patients over the age of years with relapsed aml who are not considered candidates for standard chemotherapy. a successful non-actinomycete molecule is taxol (paclitaxel), which was first isolated from the pacific yew tree, taxus brevifolia, but is also produced by the endophytic fungi taxomyces andreanae and nodulisporium sylviforme. this compound inhibits rapidly dividing mammalian cancer cells by promoting tubulin polymerization and interfering with normal microtubule breakdown during cell division. the drug also inhibits several fungi (pythium, phytophthora and aphanomyces) by the same mechanism. in , taxol was approved for refractory ovarian cancer, and today it is used against breast and advanced forms of kaposi's sarcoma. a new formulation is available in which paclitaxel is bound to albumin. taxol sales amounted to us$ . billion in for bristol myers-squibb, representing % of the company's pharmaceutical sales and its third largest selling product. currently, taxol production uses plant cell fermentation technology. the epothilones (a name derived from its molecular features: epoxide, thiazole and ketone) are macrolides originally isolated from the broth of the soil myxobacterium sorangium cellulosum as weak agents against rust fungi. they were identified as microtubulestabilizing drugs, acting in a similar manner to taxol. , however, they are generally - times more potent than taxol in inhibiting cell growth in cultures. five analogs are now undergoing investigation as candidate anticancer drugs, and their preclinical studies have indicated a broad spectrum of antitumor activity, including taxol-resistant tumor cells. with the best currently available therapies, the median survival time for patients with metastatic breast cancer is only - years, and many patients develop resistance to taxanes or other chemotherapy drugs. one epothilone, ixabepilone, was approved in october by the fda for use in the treatment of aggressive metastatic or locally advanced breast cancer no longer responding to currently available chemotherapies. in tumor cells, p-glycoprotein reduces intracellular antitumor drug concentrations, thereby limiting access of chemotherapeutic substrates to the site of action. the epothilones are attractive because they are active against p-glycoprotein-producing tumors and have good solubility. epothilone b is a -membered polyketide macrolactone with a methylthiazole group connected to the macrocycle by an olefinic bond. testicular cancer is the most common cancer diagnosis in men between the ages of and years, with approximately cases detected in the united states annually. the majority ( %) of testicular neoplasms are germ cell tumors, which are relatively uncommon carcinomas, accounting for only % of all male malignancies. remarkable progress has been made in the medical treatment of advanced testicular cancer, with a substantial increase in cure rates from approximately % in the early s to almost % today. , this cure rate is the highest of any solid tumor, and improved survival is primarily due to effective chemotherapy. a major advance in chemotherapy for testicular germ cell tumors was the introduction of cisplatin in the mid- s. two chemotherapy regimens are effective for patients with a good testicular germ cell tumor prognosis: four cycles of etoposide and cisplatin or three cycles of bleomycin, etoposide and cisplatin. of the latter three agents, bleomycin and etoposide are natural products. enzyme inhibitors have received increasing attention as useful tools, not only for the study of enzyme structures and reaction mechanisms but also for potential utilization in medicine and agriculture. several enzyme inhibitors with various industrial uses have been isolated from microbes. the most important are ( ) clavulanic acid, the inhibitor of b-lactamases discussed above in the section 'moves against antibiotic resistance development in bacteria,' and the statins, hypocholesterolemic drugs presented below in the section 'hypocholesterolemic drugs.' some of the common targets for other inhibitors are glucosidases, amylases, lipases, proteases and xanthine oxidase (xo). acarbose is a pseudotetrasaccharide made by actinoplanes sp. se . it contains an aminocyclitol moiety, valienamine, which inhibits intestinal a-glucosidase and sucrase. this results in a decrease in starch breakdown in the intestine, which is useful in combating diabetes in humans. amylase inhibitors are useful for the control of carbohydratedependent diseases, such as diabetes, obesity and hyperlipemia. , amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. the inhibitors may also be useful for weight loss, as some versions of amylase inhibitors do show potential for reducing carbohydrate absorption in humans. , the use of amylase inhibitors for the treatment of rumen acidosis has also been reported. examples of microbial a-amylase inhibitors are paim, obtained from culture filtrates of streptomyces corchorushii, and tai-a, tai-b, oligosaccharide compounds from streptomyces calvus tm- . lipstatin is a pancreatic lipase inhibitor produced by streptomyces toxytricini that is used to combat obesity and diabetes. it interferes with the gastrointestinal absorption of fat. the commercial product is tetrahydrolipstatin, which is also known as orlistat. in the pathogenic processes of some diseases, such as emphysema, arthritis, pancreatitis, cancer and aids, protease inhibitors are potentially powerful tools for inactivating target proteases. examples of microbial products include antipain, produced by streptomyces yokosukaensis, leupeptin from streptomyces roseochromogenes and chymostatin from streptomyces hygroscopicus. leupeptin is produced by more than species of actinomycetes. xo catalyzes the oxidation of hypoxanthine to uric acid through xanthine. an excessive accumulation of uric acid in the blood, called hyperuricemia, causes gout. the inhibitors of xo decrease the uric acid levels, which result in an antihyperuricemic effect. a potent inhibitor of xo, hydroxyakalone, was purified from the fermentation broth of agrobacterium aurantiacum sp. nov., a marine bacterial strain. fungal products are also used as enzyme inhibitors against cancer, diabetes, poisonings, alzheimer's disease, etc. the enzymes inhibited include acetylcholinesterase, protein kinase, tyrosine kinase, glycosidases and others. immunosuppresants suppressor cells are critical in the regulation of the normal immune response. an individual's immune system is capable of distinguishing between native and foreign antigens and of mounting a response only against the latter. a major role has been established for suppressor t lymphocytes in this phenomenon. suppressor cells also play a role in regulating the magnitude and duration of the specific antibody response to an antigenic challenge. suppression of the immune response either by drugs or by radiation, to prevent the rejection of grafts or transplants or to control autoimmune diseases, is called immunosuppression. a number of microbial compounds capable of suppressing the immune response have been discovered. cyclosporin a was originally introduced as a narrow-spectrum antifungal peptide produced by the mold, tolypocladium nivenum (originally classified as trichoderma polysporum and later as tolypocladium inflatum), by aerobic fermentation. cyclosporins are a family of neutral, highly lipophilic, cyclic undecapeptides containing some unusual amino acids, synthesized by a non-ribosomal peptide synthetase, cyclosporin synthetase. discovery of the immunosuppressive activity led to its use in heart, liver and kidney transplants and to the overwhelming success of the organ transplant field. cyclosporin was approved for use in . it is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially t lymphocytes. this complex of cyclosporin and cyclophilin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin- . it also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector t cells. sales of cyclosporin a have reached us$ . billion per year. other important transplant agents include sirolimus (rapamycin) and tacrolimus (fk ), which are produced by actinomycetes. rapamycin is especially useful in kidney transplants as it lacks the nephrotoxicity seen with cyclosporin a and tacrolimus. it is a macrolide, first discovered in as a product of s. hygroscopicus, and was initially proposed as an antifungal agent. however, this was abandoned when it was discovered that it had potent immunosuppressive and antiproliferative properties. this compound binds to the immunophilin fk -binding protein (fkbp ), and this binary complex interacts with the rapamycin-binding domain and inactivates a serine-threonine kinase termed the mammalian target of rapamycin. the latter is known to control proteins that regulate mrna translation initiation and g progression. the antiproliferative effect of rapamycin has also been used in conjunction with coronary stents to prevent restenosis, which usually occurs after the treatment of coronary artery disease by balloon angioplasty. rapamycin also shows promise in treating tuberous sclerosis complex (tsc), a congenital disorder that leaves sufferers prone to benign tumor growth in the brain, heart, kidneys, skin and other organs. in a study of rapamycin as a treatment for tsc, university of california, los angeles (ucla) researchers observed a major improvement in mice regarding retardation related to autism. as rapamycin has poor aqueous solubility, some of its analogs, rad (everolimus), cci- (tensirolimus) and ap (ariad), have been developed with improved pharmaceutical properties. everolimus is currently used as an immunosuppressant to prevent the rejection of organ transplants. although it does not have fda approval in the usa, it is approved for use in europe and australia, and phase iii trials are being conducted in the us. everolimus may have a role in heart transplantation as it has been shown to reduce chronic allograft vasculopathy in such transplants. everolimus is also used in drug-eluting coronary stents as an immunosuppressant to prevent rejection. cci- is a rapamycin ester that can be converted to rapamycin in vivo. rad is a rapamycin analog currently being investigated in phase ii trials for recurrent endometrial cancer as a single agent, and in phase i/ii trials for the treatment of glioblastoma in combination with the inhibitor of certain epidermal growth factor receptor and vascular endothelial growth factor receptor family members. ap is a novel non-prodrug rapamycin analog with a nonlinear pharmacokinetic behavior that has demonstrated antiproliferative activity against several human tumor cell lines in vitro and against experimental tumors in vivo. this agent is currently under evaluation in phase i-ii trials, including patients with different tumors. two additional small-molecule rapamycin analogs, ap and ap , are currently in preclinical development for the treatment of bone metastases and primary bone cancer. tacrolimus (fk ) was discovered in in japan. it is produced by streptomyces tsukubaensis. however, its use was almost abandoned because of dose-associated toxicity. dr thomas starzl (university of pittsburgh) rescued it by using lower doses, realizing that it was approximately times more active as an immunosuppressive than cyclosporin a. it was introduced in japan in , and in it was approved by the fda for use as an immunosuppressant in liver transplantation. furthermore, its use has been extended to include bone marrow, cornea, heart, intestines, kidney, lung, pancreas, trachea, small bowel, skin and limb transplants, and for the prevention of graft-vs-host disease. topically, it is also used against atopic dermatitis, a widespread skin disease. in the laboratory, tacrolimus inhibits the mixed lymphocyte reaction, the formation of interleukin- by t lymphocytes, and the formation of other soluble mediators, including interleukin- and interferon g. recently, it has been reported that tacrolimus inhibits tumor growth factor-b-induced signaling and collagen synthesis in human lung fibroblastic cells. this factor plays a pivotal role in tissue fibrosis, including pulmonary fibrosis. therefore, tacrolimus may be useful for the treatment of pulmonary fibrosis, although its use in the acute inflammatory phase may exacerbate lung injury. hypocholesterolemic drugs atherosclerosis is generally viewed as a chronic, progressive disease characterized by the continuous accumulation of atheromatous plaque within the arterial wall. the past two decades have witnessed the introduction of a variety of anti-atherosclerotic therapies. the statins form a class of hypolipidemic drugs used to lower cholesterol by inhibiting the enzyme hmg-coa reductase, the rate-limiting enzyme of the mevalonate pathway of cholesterol biosynthesis. inhibition of this enzyme in the liver stimulates low-density lipoprotein (ldl) receptors, resulting in an increased clearance of ldl from the bloodstream and a decrease in blood cholesterol levels. through their cholesterol-lowering effect, they reduce the risk of cardiovascular disease, prevent stroke and reduce the development of peripheral vascular disease. in addition, they are anti-thrombotic and antiinflammatory. currently there are a number of statins in clinical use. the entire group of statins reached an annual market of nearly us$ billion before it became a generic pharmaceutical. the first member of the group (compactin; mevastatin) was isolated as an antibiotic product of penicillium brevicompactum and later from penicillium citrinum. although not of commercial importance, compactin's derivatives achieved overwhelming medical and commercial success. an ethylated form, known as lovastatin (monacolin k; mevinolin), was isolated in the s in the broths of monascus ruber and aspergillus terreus. lovastatin, the first commercially marketed statin, was approved by the fda in . a semisynthetic derivative of lovastatin is simvastatin, a major hypocholesterolemic drug, selling for us$ billion per year before becoming generic. another statin, pravastatin (us$ . billion per year), is made through different biotransformation processes from compactin by streptomyces carbophilus and actinomadura sp. other genera involved in the production of statins are doratomyces, eupenicillium, gymnoascus, hypomyces, paecilomyces, phoma, trichoderma and pleurotus. a synthetic compound, modeled from the structure of the natural statins, is atorvastin, which has been the leading drug of the entire pharmaceutical industry in terms of market share (approximately us$ billion per year) for many years. an insecticide is a pesticide used against insects in all developmental forms. they include ovicides and larvicides used against the eggs and larvae of insects, respectively. insecticides are used in agriculture, medicine, industry and households. the use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity in the twentieth century. synthetic insecticides pose some hazards, whereas natural insecticides offer adequate levels of pest control and pose fewer hazards. microbially produced insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low. compared with other commonly used insecticides, they are safe for both the pesticide users and consumers of treated crops. the action of microbial insecticides is often specific to a single group or species of insects, and this specificity means that most microbial insecticides do not naturally affect beneficial insects (including predators or parasites of pests) in treated areas. the spinosyns (a group) are a group of natural products produced by saccharopolyspora spinosa that were discovered in . the researchers isolated spinosyn a and d, as well as minor analogs. they are active on a wide variety of insect pests, especially lepidopterans and dipterans, but do not have antibiotic activity. the compounds attack the nervous system of insects by targeting two key neurotransmitter receptors, with no cross-resistance to other known insecticides. the spinosyns are a family of macrolides with carbon atoms, containing four connected rings of carbon atoms at their core to which two deoxysugars (forosamine and , , , tri-o-methylrhamnose, which are required for bioactivity) are attached. novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of saccharopolyspora erythraea. a mixture of spinosyn a ( %) and d ( %) (spinosad) is being produced through fermentation and was introduced to the market in for the control of chewing insects on a variety of crops. spinosyn formulations were recently approved for use on organic crops and for animal health applications. recently, a new naturally occurring series of insect-active compounds was discovered from a novel soil isolate, saccharopolyspora pogona nrrl . the culture produced a unique family of over new spinosyns. they have a butenyl substitution at the position on the spinosyn lactone and are named butenyl-spinosyns or pogonins. herbicides are chemicals marketed to inhibit or interrupt normal plant growth and development. they are widely used in agriculture, industry and urban areas for weed management. approximately kinds of weeds are widely distributed in the world; yield losses caused by kinds of weeds are approximately . % of total crop production every year. herbicides provide cost-effective weed control with a minimum of labor. most are used on crops planted in large acreages, such as soy, cotton, corn and canola. there are numerous classes of herbicides with different modes of action, as well as different potentials for adverse effects on health and the environment. over the past century, chemical herbicides, used to control various weeds, may have caused many serious side effects, such as injured crops, threat to the applicator and others exposed to the chemicals, herbicide-resistant weed populations, reduction of soil and water quality, herbicide residues and detrimental effects on non-target organisms. for example, alachlor and atrazine were reported to cause cancer in animal tests. with increasing global environmental consciousness, bioherbicides, which are highly effective for weed control and environmentally friendly as well, are very attractive both for research and for application. microbial herbicides can be divided into microbial preparations (microorganisms that control weeds) and microbially derived herbicides. the first microbial herbicide was independently discovered in germany and japan. in , the zähner group in germany isolated phosphinothricin tripeptide, a peptide antibiotic consisting of two molecules of l-alanine and one molecule of the unusual amino acid l-phosphinothricin; that is, n( [hydroxyl(methyl)phosphinoyl]homoalanyl)alanylalanine. they isolated it from streptomyces viridochromogenes as a broad-spectrum antibacterial including activity against botrytis cinerea. in japan, it was discovered at the meiji seiki laboratories in from s. hygroscopicus and named bialaphos. the bioactive l-phosphinothricin is a structural analog of glutamic acid, acting as a competitive inhibitor of glutamine synthetase, and has bactericidal (gram-positive and gram-negative bacteria), fungicidal (b. cinerea) and herbicidal properties. glufosinate (dl-phosphinothricin) (without ala-ala) was developed as a herbicide. therefore, the agent acts as a herbicide with or without ala-ala. bialaphos has no influence on microorganisms in the soil and is easily degraded in the environment, having a half-life of only h. this low level of environmental impact is of great interest to environmentalists. in , the global animal health market was valued at us$ billion, of which % was derived from parasiticides. parasites are organisms that inhabit the body and benefit from a prolonged, close association with the host. antiparasitics are compounds that inhibit the growth or reproduction of a parasite; some antiparasitics directly kill parasites. in general, parasites are much smaller than their hosts, show a high degree of specialization for their mode of life and reproduce more quickly and in greater numbers than their hosts. classic examples of parasitism include the interactions between vertebrate hosts and such diverse animals as tapeworms, flukes, plasmodium species and fleas. parasitic infections can cause potentially serious health problems and even kill the host. parasites mainly enter the body through the mouth, usually through ingestion of tainted food or drink. this is a very common problem in tropical areas, but is not limited to those regions. there are varieties of parasites in four major categories: protozoa, trematoda, cestoda and nematoda. the major groups include protozoans (organisms having only one cell) and parasitic worms (helminths). each of these can infect the digestive tract, and sometimes two or more can cause infection at the same time. the who reported that approximately % of the world's population is infected with roundworms. in addition, a major agricultural problem has been the infection of farm animals by worms. the predominant type of antiparasitic screening effort over the years was the testing of synthetic compounds against nematodes, and some commercial products did result. certain antibiotics were also shown to possess antihelmintic activity against nematodes or cestodes, but these failed to compete with the synthetic compounds. although merck had earlier developed a commercially useful synthetic product, thiabendazole, they had enough foresight to examine microbial broths for antihelmintic activity, and found a non-toxic fermentation broth that killed the intestinal nematode nematosporoides dubius in mice. the streptomyces avermitilis culture, isolated by Ō mura and coworkers at the kitasato institute in japan, produced a family of secondary metabolites (eight compounds) with both antihelmintic and insecticidal activities. these compounds, named 'avermectins,' are pentacyclic, -membered macrocyclic lactones, that harbor a disaccharide of the methylated sugar, oleandrose, with exceptional activity against parasites, especially nemathelminthes (nematodes) and arthropod parasites ( times higher than any known synthetic antihelmintic agent). surprisingly, avermectins lack activity against bacteria and fungi, do not inhibit protein synthesis and are not ionophores. instead, they interfere with neurotransmission in many invertebrates, causing paralysis and death by neuromuscular attacks. the annual market for avermectins surpasses us$ billion. they are used against both nematode and arthropod parasites in sheep, cattle, dogs, horses and swine. a semisynthetic derivative, , -dihydroavermectin b ('ivermectin') is times more active than thiabendazole and is a commercial veterinary product. the efficacy of ivermectin has made it a promising candidate for the control of human onchocerciasis and human strongyloidiasis. another avermectin, called doramectin (or cyclohexyl avermectin b ), produced by 'mutational biosynthesis' was commercialized for use by food animals. a semisynthetic monosaccharide derivative of doramectin called selamectin is the most recently commercialized avermectin, and is active against heartworms (dirofilaria immitis) and fleas in companion animals. although the macrocyclic backbone of each of these molecules (ivermectin, doramectin and selamectin) is identical, there are different substitutions at pharmacologically relevant sites such as c- , c- , c- , and c- . the avermectins are closely related to the milbemycins, a group of non-glycosidated macrolides produced by s. hygroscopicus subsp. aureolacrimosus. these compounds possess activity against worms and insects. coccidiostats are used for the prevention of coccidiosis in both extensively and intensively reared poultry. coccidiosis is the name given to a common intestinal disease caused by the invading protozoan parasites of the genus eimeria that affects several different animal species (cattle, dogs, cats, poultry, etc.). the major damage is caused by the rapid multiplication of the parasite in the intestinal wall and the subsequent rupture of the cells of the intestinal lining, leading to high mortality and severe loss of productivity. coccidia are obligate intracellular parasites that show host specificity; only cattle coccidia will cause disease in cattle; other species-specific coccidia will not. for many years, synthetic compounds were used to combat coccidiosis in poultry; however, resistance developed rapidly. a solution came on the scene with the discovery of the narrow-spectrum polyether antibiotic monensin, which had extreme potency against the coccidian. made by streptomyces cinnamonensis, monensin led the way for additional microbial ionophoric antibiotics, such as lasalocid, narasin and salinomycin. all are produced by various streptomyces species. they form complexes with the polar cations k + , na + , ca + and mg + , severely affecting the osmotic balance in the parasitic cells and thus causing their death. the widespread use of anticoccidials has revolutionized the poultry industry by reducing the mortality and production losses caused by coccidiosis. of great interest was another extremely valuable application of monensin; that is, growth promotion in ruminants. synthetic chemicals had been tested for years to inhibit wasteful methane production by cattle and sheep and increase fatty acid formation (especially propionate) to improve feed efficiency; however, they failed. the solution was monensin, which became a major success as a ruminant growth enhancer. for more than years, certain antibiotics have been used in foodanimal production to enhance feed utilization and weight gain. from a production standpoint, feed antibiotics have been consistently shown to improve animal weight gain and feed efficiency, especially in younger animals. these responses are probably derived from an inhibitory effect on the normal microbiota, which can lead to reduced intestinal inflammation and improved nutrient utilization. pigs in the usa are exposed to a great variety of antibiotics. these include b-lactam antibiotics (including penicillins), lincosamides and macrolides (including erythromycin and tetracyclines). all these groups have members that are used to treat infections in humans. in addition, bacitracin, flavophospholipol, pleuromutilins, quinoxalines and virginiamycin are utilized as growth stimulants. flavophospholipol and virginiamycin are also used as growth promoters in poultry. as described above, cattle are also exposed to ionophores such as monensin to promote growth. the animal health institute of america has estimated that without the use of growth-promoting antibiotics, the usa would require an additional million chickens, million more cattle and million more pigs to reach the levels of production attained by the current practices. considering that animal health research and the development of new anti-infective product discovery have decreased, the discovery of new antibiotics has decreased over the past years, with few new drug approvals. therefore, it will be incumbent on veterinary practitioners to use the existing products in a responsible manner to ensure their longevity. it remains to be seen what effects the dearth of new antibiotics for veterinary medicine will have on the future practice of veterinary medicine, production agriculture, food safety and public health. since the eu decision to prohibit antibiotic use for foodanimal growth promotion, four antibiotic growth promoters have been banned, including the macrolide drugs tylosin and spiramycin. although macrolides are no longer formally used as 'growth promoters,' their use under veterinary prescription has risen from tons in to tons in , which suggests that more of them are being used now than before the prohibition. it is well known that the most effective route for feeding is via the gastrointestinal tract. many critically ill patients who accept early feeding improve their health. in some post-operative patients, gastric stasis and excessive volumes in the stomach increase the risk of aspiration and subsequent pneumonia. on account of the importance of achieving early and adequate nutritional intake, it is common practice in many intensive care units to use drugs to improve gastrointestinal motility. erythromycin is a macrolide antibiotic with a broad spectrum of activity. it is well recognized that when prescribed, either intravenously or orally, it causes side effects, such as diarrhea, nausea and vomiting. these side effects are, in part, due to the action of erythromycin at motilin receptors in the gut. this makes this antibiotic very attractive to be used in ill patients with gastrointestinal motility problems. there have been some developments on erythromycin analogs that lack antibiotic action but retain action at motilin receptors. these have been named 'motilides.' , recently, an orally active erythromycinderived motilin receptor agonist (mitemcinal) has been tested in patients with idiopathic and diabetic gastroparesis. in both cases, an improvement of gastroparetic symptoms was observed. the -year contribution of microorganisms to medicine and agriculture has been overwhelming. however, antibiotic resistance in microbes has created a dangerous situation and the need for new antibiotics is clear. unfortunately, most of the large pharmaceutical companies have abandoned the search for new antimicrobial compounds. owing to the economics, they have concluded that drugs directed against chronic diseases offer a better revenue stream than do antimicrobial agents, as for the latter the length of treatment is short and government restriction is likely. some small pharmaceutical and biotechnology companies are developing antibiotics, but most depend on venture capital rather than sales income, and with the present regulations, they face huge barriers to enter the market. these barriers were raised with the best intentions of ensuring public safety, but will have the opposite effect if they terminate antibiotic development while resistance continues to increase. however, there are some bright possibilities. one of the most promising is the utilization of uncultivated microorganisms. considering that % of the bacteria and % of the fungi have not been cultivated in the laboratory, putting efforts into finding means to grow such microorganisms are proceeding and succeeding. furthermore, researchers are now extracting bacterial dna from soil and marine habitats, cloning large fragments into, for example, bacterial artificial chromosomes, expressing in a host bacterium and screening the library for new antibiotics. this metagenomic effort is allowing access to a vast untapped reservoir of genetic and metabolic diversity, , which could result in the discovery of new and useful natural products. in addition to these two relatively new techniques, the chemical and biological modification of old antibiotics could still supply new and powerful drugs. these comments also apply to non-antibiotics such as antitumor agents and other microbial products. bioactive microbial metabolites. a personal view natural products in drug discovery and development natural products and design: interrelated approaches in drug discovery design of angiotensin converting enzyme inhibitors antibiotics: where did we go wrong? natural and synthetic substances related to human health the future of cephalosporins business infectious history oceans: medicine chests of the future? inhibitors of hiv- replication that inhibit hiv integrase medicinals for the millennia. the historical record natural products based anti-hiv drug discovery and development facilitated by the nci developmental therapeutics program the end of an era? where have all the antibiotic patents gone? barriers on the road to new antibiotics cycling may be bad for your health the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents sherris medical microbiology th edn health of nations: combating infectious diseases the public health threat of emerging viral disease the future challenges facing the development of new antimicrobial drugs jr problems with antimicrobial resistance in gram-positive cocci recent progress in the field of antibacterial pristinamycins recent developments in tetracycline antibiotics case studies in current drug development: 'glycylcyclines' development of daptomycin for gram-positive infections lipopeptides, focusing on daptomycin, for the treatment of gram-positive infections daptomycin-a novel antibiotic against gram-positive pathogens overcoming antimicrobial resistance: profile of a new ketolide antibacterial, telithromycin biosynthesis and molecular genetics of clavulanic acid invasive aspergillosis in organ transplant recipients: new issues in epidemiologic characteristics, diagnosis, and management invasive fungal infections: a review of epidemiology and management options antifungal resistance trends towards the year . implications for therapy and new approaches caspofungin acetate: an antifungal agent antifungals targeted to cell wall: focus on b- , -glucan synthase role of micafungin in the antifungal armamentarium posaconazole: a new broad-spectrum antifungal agent signal-transduction cascades as targets for therapeutic intervention by natural products thiolactomycin and related analogues as novel anti-mycobacterial agents targeting kasa and kasb condensing enzymes in mycobacterium tuberculosis the combinatorial chemistry of nature anticancer drug discovery and development throughout the world actinomyces antibioticus, a new soil organism antagonistic to pathogenic and non-pathogenic bacteria anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity new antibiotics, bleomycin a and b the fam ( -fluorouracil, adriamycin, mitomycin c) and smf (streptozotocin, mitomycin c, -fluorouracil) chemotherapy regimens mitomycin dimers: polyfunctional cross-linkers of dna identification of two genes from streptomyces argillaceus encoding glycosyltransferases involved in transfer of a disaccharide during biosynthesis of the antitumor drug mithramycin glut in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice improved production of pentostatin and identification of fermentation cometabolites pentostatin in t-non-hodgkin's lymphomas: efficacy and effect on cd + t lymphocytes cleavage behavior of calicheamicin gamma and calicheamicin t approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia study on the preparation and regeneration of protoplast from taxol-producing fungus nodulisporium sylviforme natural products as sources of new drugs over the last years epothilons a and b: antifungal and cytotoxic compounds from sorangium cellulosum (myxobacteria): production, physico-chemical and biological properties epothilones, a new class of microtubulestabilizing agents with a taxol-like mechanism of action activities of the microtubule-stabilizing agents epothilones a and b with purified tubulin and in cells resistant to paclitaxel (taxol) epothilones: mechanism of action and biologic activity curing metastatic testicular cancer medical treatment of advanced testicular cancer refining the optimal chemotherapy regimen for good-risk metastatic nonseminomatous germ-cell tumors: a randomized trial of the genito-urinary group of the french federation of cancer centers (getug t bp) enzyme inhibitors of microbial origin chemistry and biochemistry of microbial a-glucosidase inhibitors gastrointestinal and metabolic effects of amylase inhibition in diabetics enzyme inhibitors of marine microbial origin with pharmaceutical importance effect of an a-amylase inhibitor on body weight reduction in obese women effect of a purified amylase inhibitor on carbohydrate metabolism after a mixed meal in healthy humans treatment of rumen acidosis with alpha-amylase inhibitors european patent inhibitory properties of an a-amylase inhibitor, paim, from streptomyces corchorushii novel amylase inhibitors united states patent lipstatin, an inhibitor of pancreatic lipase, produced by streptomyces toxytricini progress towards the discovery of xanthine oxidase inhibitors agar plate method, a new assay for chitinase inhibitors using a chitin-degrading bacterium fungal enzyme inhibitors as pharmaceuticals, toxins and scourge of pcr history of the discovery of cyclosporin and of its early pharmacological development rapamycin, an mtor inhibitor, disrupts triglyceride metabolism in guinea pigs reversal of learning deficits in a tsc +/À mouse model of tuberous sclerosis everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients targeting mtor signaling for cancer therapy therapeutic targets: mtor and related pathways targeted mtor in human gynecologic cancers fk- , a novel immunosuppressant isolated from a streptomyces. . fermentation, isolation, and physico-chemical and biological characteristics correlation of rejection episodes with fk dosage, fk level and steroid following primary orthotopic liver transplant use of tacrolimus, a potent antifibrotic agent, in bleomycin-induced lung fibrosis statins, high-density lipoprotein cholesterol, and regression of coronary atherosclerosis mevinolin, a highly potent competitive inhibitor of hydroxylmethylglutaryl-coenzyme a reductase and a cholesterol-lowering agent a two-component-type cytochrome p- monooxygenase system in a prokaryote that catalyzes hydroxylation of ml- b to pravastatin, a tissue-selective inhibitor of -hydroxy- -methylglutaryl coenzyme a reductase a new hydroxylase system in actinomadura sp. cells converting compactin to pravastatin production and purification of statins from pleurotus ostreatus (basidiomycetes) strains evaluation and development of spinosyns to control ectoparasites on cattle and sheep engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes research progress on microbial herbicides weed control for the preservation of biological diversity control of problem vegetation: a key to ecosystem management metabolic products of microorganisms. . phosphinothricin and phosphinothricyl-alanyl-analine studies on a new antibiotic, sf- . i. isolation and physicochemical and biological characterization of sf- substances biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from streptomyces viridochromogenes tu avermectins, antiparasitic lactones produced by streptomyces avermitilis isolated from a soil in japan avr- encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in caenorhabditis elegans efficacy of ivermectin against strongyloides stercoralis in humans doramectin-a potent novel endectocide comparison of ivermectin, doramectin, selamectin, and eleven intermediates in a nematode larval development assay a new family of macrolide antibiotics. structure determination of milbemycins d polyether antibiotics: versatile carboxylic acid ionophores produced by streptomyces the determination of anticoccidial drugs (nicarbazin, lasalocid, monensin, salinomycin and narasin) in animal livers and eggs by liquid chromatography linked with tandem mass spectrometry (lc-ms-ms) the role of enteric antibiotics in livestock production (national association for crop production and animal health antibiotics as growth promotants: mode of action antibiotic resistance back in the news antibiotic development pipeline runs dry the future of anti-infective products in animal health the european ban on growth-promoting antibiotics and emerging consequences for human and animal health bioactive metabolites of em and em , erythromycin derivatives having strong gastrointestinal motor stimulating activity erythromycin as a gastrointestinal prokinetic agent clinical trial: effect of mitemcinal (a motilin agonist) on gastric emptying in patients with gastroparesis-a randomized, multicentre, placebocontrolled study the need for new antibiotics isolating 'uncultivable' microorganisms in pure culture in a simulated natural environment fulfilling the promise of biotechnology new antibiotics from bacterial natural products we thank beatriz ruiz and marco a ortiz for their assistance during the development of this review. key: cord- -pf mlzrw authors: moura-neto, josé a.; misael, ana maria; da silva, dirceu reis; d’avila, ronaldo; andreoli, maria claudia cruz; kraychete, angiolina; bastos, kleyton; do nascimento, marcelo mazza title: position statement from the brazilian society of nephrology regarding chloroquine and hydroxychloroquine drug dose adjustment according to renal function date: - - journal: j bras nefrol doi: . / - -jbn- -s sha: doc_id: cord_uid: pf mlzrw chloroquine and hydroxychloroquine have shown promising preliminary results and have been discussed as therapeutic options for patients with covid- . despite the lack of robust evidence demonstrating the benefits and justifying the use of one of these drugs, the final decision is the responsibility of the attending physician and should be individualized and shared, whenever possible. this position statement recommends dosage adjustment for these drugs in the context of renal impairment. em razão de resultados preliminares promissores, a hidroxicloroquina e a cloroquina têm sido discutidas como opção terapêutica para pacientes com covid- . apesar da ausência de estudos robustos que evidenciem o benefício e justifiquem o uso de uma dessas drogas, a decisão final compete ao médico assistente, devendo ser individualizada e, sempre que possível, compartilhada. a presente nota pretende orientar o ajuste posológico dessas drogas no contexto da disfunção renal. cloroquina; hidroxicloroquina; antimaláricos; posologia; cálculos da dosagem de medicamento; insuficiência renal. chloroquine and hydroxychloroquine have shown promising preliminary results and have been discussed as therapeutic options for patients with covid- . despite the lack of robust evidence demonstrating the benefits and justifying the use of one of these drugs, the final decision is the responsibility of the attending physician and should be individualized and shared, whenever possible. this position statement recommends dosage adjustment for these drugs in the context of renal impairment. keywords: chloroquine; hydroxychloroquine; antimalarials; posology; drug dosage calculation; renal failure. on the first day of april , informative note nº / -daf/sctie/ms was published, establishing that the brazilian ministry of health (ms) would make the medications available for use, in confirmed cases and at medical criteria, chloroquine and hydroxychloroquine as adjunctive therapy in the treatment of severe forms in hospitalized patients, without other supportive measures being neglected in their favor . on april , the ms published "guidelines for the diagnosis and treatment of covid- ", in which it also instructed on the use of chloroquine and hydroxychloroquine as adjuvant therapy in severe forms of the disease, in confirmed cases and upon medical discretion . on april , the federal board of medicine (cfm) published its note no. / , which establishes criteria and conditions for the prescription of chloroquine and hydroxychloroquine for patients with a confirmed diagnosis of covid- . the cfm, in its understanding, concluded that there is no solid evidence that these drugs have a confirmed effect on the prevention and treatment of this disease. however, considering the exceptional nature of the situation, and during the declared period of the covid- pandemic, cfm considered it possible to prescribe these drugs in three specific situations: . the use of chloroquine or hydroxychloroquine in patients with mild symptoms, in the onset of the clinical picture, after other viruses (such as influenza, h n , dengue) and a confirmed diagnosis of covid- can be considered. . patients with important symptoms, but still without the need for intensive care, with or without a recommendation for hospitalization. . patients in critical condition receiving intensive care, including mechanical ventilation. in these situations, the principle that must, mandatorily, guide the treatment of the patient is that of the physician's autonomy, as well as upholding the doctorpatient relationship, "this being the closest possible, with the objective of offering the best treatment currently available to the patient". in all contexts, the prescription of drugs will be the responsibility of the attending physician, in a decision shared with the patient . the brazilian society of nephrology understands that there is no solid evidence that these drugs have a confirmed effect on the prevention and treatment of covid- . if the doctor chooses to use one of these drugs in the population with chronic kidney disease, especially in dialysis patients, he/she must consider its long half-life (up to - days). both are not excreted by dialysis and have renal excretion of around - %, with % protein binding [ ] [ ] [ ] [ ] . therefore, chloroquine and hydroxychloroquine should be used with extreme caution in patients with chronic or acute renal dysfunction, especially due to the arrhythmogenic potential of the drug. while the manufacturer and some sources do not advise on dose adjustment for renal function, other recommendations suggest a % dose reduction in patients with glomerular filtration rate < ml/min/ . m on hemodialysis, hemodiafiltration, peritoneal dialysis or under conservative treatment , . given the above and the associated risks, the brazilian society of nephrology advises its associate doctors to prescribe one of these drugs according to the recommendations established by cfm and ms, which recommend a % reduction in the recommended dose of chloroquine and hydroxychloroquine in patients with glomerular filtration rate < ml/min/ . m , in dialysis or conservative treatment. the doctor should also note that, regardless of the modality of renal replacement therapy, no additional dose of the drug is required after dialysis. secretaria de ciência, tecnologia, inovação e insumos estratégicos em saúde secretaria de ciência, tecnologia, inovação e insumos estratégicos em saúde. diretrizes para o diagnóstico e tratamento da covid- tratamento de pacientes portadores de covid- com cloroquina e hidroxicloroquina therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases gestione dei farmaci antireumatici nell'insufficienza renale drug prescribing in real failure: dosing guidelines for adults and children chloroquine for sars-cov- : implications of its unique pharmacokinetic and safety properties the renal drug handbook. the ultimate prescribing guide for renal practitioners key: cord- -wz o tt authors: liu, shao; luo, ping; tang, mimi; hu, qin; polidoro, joseph p.; sun, shusen; gong, zhicheng title: providing pharmacy services during the coronavirus pandemic date: - - journal: int j clin pharm doi: . /s - - - sha: doc_id: cord_uid: wz o tt the coronavirus disease (covid- ) is quickly spreading across china and globally. pharmacy services are an important pillar in public health to prevent and contain the covid- pandemic. chinese pharmacists have acted swiftly in the public health response in china, such as drafting professional service guidance to pharmacists and pharmacies, establishing emergency drug formularies, monitoring and resolving drug shortages, establishing remote pharmacy services to prevent human-to-human infections, providing event-driven pharmaceutical care, educating the public on infection prevention and disease management, and participating in clinical trials and drug evaluation. this commentary reviews the unique needs of pharmacy services in the covid- pandemic, and shares our experiences with the international pharmacy community in the response to these needs. an outbreak of coronavirus disease (covid- ) caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ) began in wuhan, hubei province, china in december [ ] . the genetic characteristics of sars-cov- have proven to be significantly different from human sars cov and middle eastern respiratory syndrome (mers) cov [ ] . common signs of sars-cov- infection include respiratory symptoms, fever, cough, shortness of breath, and breathing difficulties. in more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, organ failure and even death [ ] . the virus is highly infectious, spreading rapidly via human-to-human transmission. as of march , , there were , confirmed cases in china ( deaths) and , cases in countries/territories/areas outside china ( deaths) [ ] . currently there are no approved antiviral therapies for covid- . the world health organization (who) has now declared the coronavirus a global pandemic. when responding to the covid- pandemic, medical services are under great pressure. an effective coordinated pharmacy support system and event-driven pharmaceutical care activities are needed. mobile cabin hospitals, the "square cabin hospitals" have been built in wuhan, china (the epidemic epicenter in china) to treat patients with mild symptoms of the virus. pharmacists are actively engaging shao liu and ping luo contributed equally, should be considered co-first authors. in the public health operation in china since the outbreak, either at their home hospitals and community pharmacies or at the square cabin hospitals in wuhan. the chinese pharmacy profession has gained valuable experiences through providing essential support to the public health operation. in this commentary, we share our experiences of providing pharmacy services combating coronavirus in china to disseminate what we have learned to the international pharmacy society. the timely supply of preventive and treatment medications, and the provision of event-driven pharmaceutical care are critical to support and enhance public health emergency operations during the coronavirus pandemic. specifically, pharmacy needs to work with other healthcare organizations, professionals, and government agencies to address the following seven service needs: ( ) drafting professional service guidances to pharmacists and pharmacies, ( ) establishing emergency drug formularies based on treatment guidelines, ( ) coordinating with drug companies and distributors to ensure adequate supply, storage and transport of identified formulary drugs, ( ) providing event-driven pharmaceutical care, ( ) establishing remote pharmacy services to reduce the incidence of human-to-human infections, ( ) educating the public with a focus on infection prevention and disease management, and ( ) involving in clinical trial research to screen, evaluate and develop antiviral medications in line with national and international guidelines [ ] . additionally, pharmacists must learn how to protect themselves from getting infected as well. in the next section, we share the pharmacy's responses in meeting the needs of pharmacy services in china during the epidemic. to outline the roles that pharmacists in community, hospital and other diverse healthcare settings can play in preventing the spread of covid- and supporting the efficient management of infection by healthcare systems, the chinese pharmaceutical association (cpa) published a separate expert consensus, the "coronavirus sars-cov- infection: expert consensus on hospital pharmaceutical work guidance and prevention and control strategies", and the "coronavirus sars-cov- infection: expert consensus on work guidance and prevention and control strategies for retail pharmacies" [ , ] . based on these two consensus documents, the "coronavirus -ncov outbreak: information and interim guidelines for pharmacists and the pharmacy workforce" international guidelines have been drafted by the international pharmaceutical federation (fip) with the participation of the cpa. the document gives reliable information on -ncov and covers preventive measures (from how to wear a mask to effective disinfection), what equipment to stock, advice that pharmacists can give, and appropriate laboratory testing. the document is downloadable in the six official united nations languages on the fip website [ ] . the department of pharmacy at xiangya hospital, central south university, proposed to develop a "pharmacy emergency support guarantee system" within a public health response operation, a novel initiative in chinese public health (fig. ). the pharmacy system aims to establish mechanisms to address drug shortages through surveillance, early warnings, drug emergency supply and distribution, monitoring the safe use of medications, and event-driven pharmaceutical care during the coronavirus epidemic [ ] . concurrently, the xiangya hospital pharmacy department also published a reference book "pharmacy service guarantee support for the prevention and control of the new coronavirus pneumonia". the book provides pharmacists with evidence-based information and standardized operation protocols during the prevention and control of the epidemic. there is currently no specific vaccine and no effective antiviral therapy against sars-cov- . the china national health commission (cnhc) has released the "diagnosis and treatment guidelines for the new coronavirus infected pneumonia" (the cnhc guidelines), currently in its sixth update [ ] . the cnhc guidelines suggest the use of antiviral drugs (α-interferon, lopinavir/ritonavir, ribavirin, chloroquine, umifenovir) and traditional chinese medicines (tcms) for treatment. although these therapies may not be recommended by the who, their treatment roles are based on past experiences during the sars outbreak in china in with the consideration of lack of drug therapies to treat the coronavirus. the cnhc guidelines recommend eight tcm injections for severe and critical cases. for severe cases, xiyanping, xuebijing, reduning, tanreqing and xingnaojing injections are recommended. for critical cases, three additional tcm injections can be considered, shenfu, shengmai and shenmai. to assist clinicians in better understanding and prescribing these drugs, pharmacists applied evidence-based medication use evaluation approaches to collect and summarize drug information with these recommended drugs. the hunan pharmaceutical association and the chinese hospital association pharmaceutical committee each separately compiled the "drug information for the diagnosis and treatment of new coronavirus pneumonia" and the "drug formulary in square cabin hospitals". during the coronavirus pandemic, both prescription (including resuscitation drugs) and over-the-counter (otc) medication shortages may occur. drugs may also be in short supply due to production disruptions for various reasons. pharmacists and pharmacies must conduct active surveillance and establish early warning mechanisms to address drug shortages in a timely manner. the american society of health-system pharmacists (ashp) guidelines on managing drug product shortages and a published provincial drug shortage surveillance and early warning platform in hunan, china, can be referenced and adapted to create local, regional and national platforms to manage prescription drug shortages during a pandemic [ , ] . figure shows the proposed prescription drug shortage surveillance and early warning mechanism. community pharmacies and pharmacists should pay equal attention to potential shortages of otc drugs. this is especially important due to an increase in the public buying otcs to treat the symptoms of the illness (fever, cough and shortness of breath) from the advice of healthcare professionals and public agencies. event-driven pharmaceutical care is provided to covid- patients in china during the epidemic. the event-driven pharmaceutical care goes beyond routine activities (medication review, prescribing support and patient counseling). three characteristics of the event-driven pharmaceutical care are ( ) ensuring the appropriate use of off-label drugs: many drugs recommended in the cnhc guidelines are used offlabel. pharmacists conduct literature evaluations and publish guidances for the off-label use of these drugs. adverse drug reactions of some of these drugs are similar to the symptoms of the coronavirus disease (such as fatigue, fever, and flu-like symptoms with α-interferon). it is necessary for pharmacists to assist in evaluating the efficacy and safety of these drugs and to monitor adverse drug reactions. the pathological anatomy of the first patient with a covid- related death fig. pharmacy emergency support guarantee system within a public health response showed liver damage [ ] , which was either drug-induced or caused by the sars-cov- . pharmacists are reminded to monitor drugs that may cause liver damage and to adjust drug dosing based on the patient's liver function, ( ) ensuring the rational use of tcms: the cnhc guidelines recommend the use of tcms for the coronavirus treatment. preliminary evidence shows that integrating tcms and western medicines for covid- can improve the clinical cure rate and reduce the rate of disease progression from common infection type to severe type [ ] . however, it is worth noting that the current research of tcms is based on clinical observations and is mostly used for patients with mild symptoms. pharmacists with specialized training in tcm are confronted with preparing tcm formulations, providing drug information to physicians, and tcm pharmaceutical care for patients. pharmacists should warn physicians about any interactions between tcms and western medicines when these drugs are prescribed. for example, the combination of hanshi yufei decoction and antipyretic analgesics can cause excessive sweating and even dehydration. some proprietary tcms contain western medicine ingredients, such as pipa lu, a strong antitussive, contains . mg of morphine per milliliter. serotonin syndrome may occur when pipa lu is administered with monoamine oxidase inhibitors such as linezolid, and ( ) providing online real-time clinical support to frontline physicians and pharmacists working at the epidemic epi-center: wechat ® (the largest social communication mobile platform in china) groups are established by pharmacy experts and pharmacists in china to provide clinical support for front-line pharmacists in hubei, the epi-center of the coronavirus outbreak. these chat groups create accessible drug use platforms by engaging in pharmacotherapy consultation through information and resource sharing in real-time. in order to effectively reduce overcrowding and block the spread of the virus through person-to-person transmission during the coronavirus epidemic, medical institutions across china have launched remote pharmacy services such as online drug prescribing, drug consultation, and drug delivery services. for example, the pharmacy department of xiangya hospital, central south university, created an online remote pharmacy service via wechat ® . pharmacists provide patients with free medication consultation and education, allowing patients to purchase medications online and arrange for home drug delivery. the implementation of these remote pharmacy services provides out-of-hospital pharmaceutical care access and helps to reduce the risk of cross-infection during unnecessary hospital visits. the positive effects of these remote services on patient care during the epidemic is helpful for pharmacy to further establish its professional role in china. during an epidemic, false information and rumors can generate serious negative effects when the public cannot access timely, authoritative, and scientific information regarding the epidemic [ ] . for example, after the media in china reported that shuanghuanglian, a tcm that contains extracts from the dried fruit of forsythiae fructus, inhibits sars-cov- in vitro, it sparked panic buying of this medication online and offline. the mass of people assembling to purchase this oral solution increased the risk of human-to-human disease transmission. similarly, after the media reported that chloroquine phosphate can inhibit sars-cov- in vitro, there were reports of a female patient who was not infected with sars-cov- but self-medicated with hydroxychloroquine sulfate tablets ( . g, h) . the patient subsequently developed mental disorders and cardiac arrhythmias requiring hospitalization in an intensive care unit. these incidences demonstrate that it is necessary for pharmacists and pharmacy associations to engage in public education to disseminate reliable authoritative information to the public and guide the public's rational thinking and behavior during the coronavirus epidemic. to this end, the chinese pharmacists association and the chinese pharmacological society released the "expert consensus guidance on the rational drug administration and home drug therapy during an epidemic" [ ] . a large number of popular science articles on coronavirus infection prevention and treatment and on home medication use during an epidemic have been written by pharmacists and published over the internet with the hope of decreasing drug-related problems. the need to provide education to particular population subgroups (homeless, prisoners, etc.) should be addressed. it is important to note that the impact of these public education activities depends on the public trust of pharmacists and pharmacy as a profession. currently, more than clinical trials have been registered in the chinese clinical registry to test for a coronavirus treatment. during the first week of february, china launched two placebo-controlled trials of remdesivir, slated to include people with covid- . the studies should be completed by the end of april, . a few trials have started to test chloroquine, an antimalarial drug that killed off the sars-cov- in cell cultures. researchers are studying whether steroids diminish inflammation in people with severe covid- , or cause harm. another -person controlled trial is aimed to test whether serum antibodies from covid- survivors could rapidly help someone newly infected with the sars-cov- virus. fifteen trials listed in china's registry expect to enroll a total of more than , people in studies on a variety of tcms, including the largest trial assessing shuanghuanglian with participants [ ] . pharmacists are actively cooperating with research sponsors in the management of clinical trials including the proper supply, use, storage and disposal of experimental drugs in compliance with relevant clinical trial regulations. pharmacists are also actively conducting pharmaceutical evaluations on the efficacy and safety of related trial drugs. in summary, during the coronavirus epidemic in china, the chinese pharmacy profession has acted swiftly and forcefully with the above seven responses. the impact of these measures needs to be further evaluated, especially the several responses that rely on the public trust of pharmacy as a profession in china. the epidemic of covid- is quickly evolving, and the prevention and treatment pose great challenges to pharmacy services. although chinese pharmacists have made significant contributions to the public health operation in fighting the epidemic, the epidemic exposes a series of threats that we should face directly. the first is that the number of frontline pharmacists is inadequate. for example, there are nearly beds in a square cabin hospital, equipped with nearly drugs, including antiviral drugs, rescue drugs, and drugs used in the respiratory, digestive and cardiovascular systems. however, only three to five pharmacists are stationed at each square cabin hospital. pharmacists are undertaking dual responsibilities in the process of drug supply and management, and pharmaceutical care. it is not possible for a pharmacist to be able to provide quality, essential pharmacy services with these heavy workloads. second, the lack of pharmacist consultations can lead to an overutilization and inappropriate use of antibacterial medications. some doctors routinely prescribe covid- patients antibiotics such as quinolones or cephalosporins in the absence of a diagnosed bacterial infection. this prescribing behavior increases the risk of microbial resistance and adverse reactions. third, most research manuscripts related to the coronavirus pharmacy services were written in chinese and published in chinese journals, thus they cannot easily be shared with the international pharmacy community. fourth, although many drugs are being studied in clinical trials, the design and quality of these clinical trials should be properly monitored. many trials contain levels of bias such as small sample size and lack of control, randomization, and objective outcome evaluation criteria. as a result, these clinical trials are wasting valuable resources, and will be unable to provide high-quality evidence of treatment efficacy and safety. in response, the china state council issued the "notice on standardizing medical institutions to conduct clinical research on new coronavirus pneumonia drug therapy" [ ] . the notice aimed to screen more effective drugs through standardized, scientific, orderly, and efficient clinical drug research. the first point is to further clarify the conditions for conducting research. for example, the drugs used for clinical research should be a marketed drug that has been validated by in vitro and animal experiments. the second point is to further standardize the research process with regards to ethical review and record-keeping, and hospitals should perform quality control and risk management throughout the process. the third point is to speed up the application in an orderly manner. for example, the scientific research group recommends experimental drugs to the joint prevention and control medical treatment group, and the medical treatment group decides whether to expand the scope of their use or include them in the treatment plan. two final reflections: professional guidances should be established not only to pharmacists and pharmacies but also to the general population and to particular population subgroups (e.g. homeless, prisoners, etc.). the role of the community pharmacists in preventing the spread of covid- virus should be strengthened. community pharmacists are charged with key responsibilities including informing, advising, and educating the community; maintaining a stable supply of pharmaceuticals and personal hygiene products; and screening suspected cases and making appropriate referrals as necessary [ ] . in summary, chinese pharmacists and pharmacy associations have responded forcefully to the covid- epidemic. the experiences and lessons learned in china not only allow the chinese pharmacy profession to improve pharmacy operations, but also provide insight to the international pharmacy community to plan and operate pharmacy services to combat current and future epidemics. recent insights into -ncov: a brief but comprehensive review a novel coronavirus from patients with pneumonia in china who coronavirus disease (covid- ) situation report- pharmaceutical emergency guarantee difficulties and countermeasures for the prevention and control of outbreak of novel coronavirus pneumonia (ncp) the chinese pharmaceutical association. the coronavirus sars-cov- infection: expert consensus on hospital pharmaceutical work guidance and prevention and control strategies the chinese pharmaceutical association. the coronavirus sars-cov- infection: expert consensus on work guidance and prevention and control strategies for retail pharmacies international pharmaceutical federation (fip) diagnosis and treatment guidelines for the new coronavirus infected pneumonia ashp guidelines on managing drug product shortages combating drug shortages in china: surveillance warning and practice standardization pathological findings of covid- associated with acute respiratory distress syndrome thinking on clinical rational use of tcm injection in the treatment of novel coronavirus pneumonia (covid- ) covid- : fighting panic with information. lancet expert consensus guidance on the rational drug administration and home drug therapy during an epidemic slew of trials launch to test coronavirus treatments in china notice on standardizing medical institutions to conduct clinical research on new coronavirus pneumonia drug therapy community pharmacist in public health emergencies: quick to action against the coronavirus -ncov outbreak acknowledgements authors would like to thank emily m. ciamaricone and jessica a. breitweg, doctor of pharmacy candidates at western new england university college of pharmacy and health sciences, for proof-reading this manuscript.funding none. the authors declare that they have no conflict of interest. key: cord- - qck j w authors: martin, jennifer h.; clark, julian; head, richard title: buying time: drug repurposing to treat the host in covid‐ h date: - - journal: pharmacol res perspect doi: . /prp . sha: doc_id: cord_uid: qck j w in fedson stated …. “for almost two decades, leading scientists and health officials have warned that we must prepare for a potentially devastating global pandemic of an infectious disease. initial concern was focused on …h n …. more recently…a devastating outbreak of ebola virus..(and) several other emerging viruses are believed to seriously threaten global health and global security. to prepare, scientists have been urged to discover new vaccines and treatments for these emerging viruses. at the same time, political leaders have been urged by global health experts to invest millions in a “top down” restructuring of the global health system. this article takes a different view. it focuses on an alternative approach to the scientific discovery of treatments for individual patients, reviews the mechanisms of action and clinical experience with specific drugs that might be useful, and considers whether or not recent lessons regarding this “bottom up” approach to treatment have been learned”. now with a new virus and pandemic upon us, fedson's comments appear chilling, are cause for reflection on what we have learnt and importantly offer focus on an immediate opportunity in the area of treating the host (fedson ds, ann transl med, ; : ). has driven huge morbidity and mortality globally, destroying much of the social and economic fabric of societies. the immediate application of evidence-based research and its strategic timing (distinct from evidence-based medicine) is essential to enhance patient survival. for this to occur, a strategy must correctly be aligned with unmet need and this is where an international research strategy can build on success. currently there is discussion on two key issues in this covid pandemic; first, physical isolation, testing (current), and barrier isolation, which is fundamental; second, vaccine and/or antiviral development (possible long-term future). in physical isolation and impact, some countries appear to be doing very well. likewise, there is outstanding global research capability and scientists in epidemiology, modeling, immunology, antiviral development, and diagnostics. within the urgency of a pandemic we are still not achieving treatments with actual efficacy data for prevention, treatment or cure. realistically, a vaccine may never be developed as planned, or potentially take in fedson stated …. "for almost two decades, leading scientists and health officials have warned that we must prepare for a potentially devastating global pandemic of an infectious disease. initial concern was focused on …h n …. more recently…a devastating outbreak of ebola virus..(and) several other emerging viruses are believed to seriously threaten global health and global security. to prepare, scientists have been urged to discover new vaccines and treatments for these emerging viruses. at the same time, political leaders have been urged by global health experts to invest millions in a "top down" restructuring of the global health system. this article takes a different view. it focuses on an alternative approach to the scientific discovery of treatments for individual patients, reviews the mechanisms of action and clinical experience with specific drugs that might be useful, and considers whether or not recent lessons regarding this "bottom up" approach to treatment have been learned". covid , drug repurposing, global collaboration, host response, renin-angiotensin of | commentary a further - years to complete necessary studies, and finalise the regulatory pharmaceutics dossier, but even then, time is still needed to find funding to manufacture, upscale, and develop supply lines to roll it out globally. these future hopes are happening against a backdrop of the extremely urgent need to have effective treatments on the wards here and now. we also need an approach to provide a therapeutic insurance with the inevitable easing of social isolation in the absence of vaccines and antivirals. finally, in seeking effective antivirals one of the barriers to a rational drugs design program for humans is the disconnect between the time and resources required to conduct well-controlled preclinical and clinical studies and those of biomedical researchers dealing with a global healthcare crisis in real time. the arguments for buying time by utilizing the principles of treating the host are compelling. in the first instance, we would suggest the world needs to have a much more effective approach to using well-tested drugs and their dosing schedules that can block a dysregulated innate immune response at clinically tolerated doses. an approach based on treating the host built on sound physiology and pathophysiology, together with thorough administrative data input and accepted principles of drug repurposing based upon pharmacology and clinical pharmacology is needed. it follows that the immediate unmet need and opportunity sits between these two domains of social isolation and antiviral/vaccine development. it involves pharmacological-based approaches to treating the host (not necessarily the virus) in an acute setting. treating the host in a viral pandemic context has been highlighted at least years ago. , a "treating the host" approach has the potential to enable infected people to survive an acute pulmonary/vascular inflammatory dysregulation with a decreased call on high end health resource. this approach is increasingly recognized as being important to patient care. a critical aspect of covid- is the constellation of inflammatory processes that become grossly distorted, particularly in the lung, that can be life threatening. the unmet need and opportunity involve the identification of pharmaceuticals to dampen the inflammatory storm in infected individuals with a view to lessen the acute need for intubation and ventilation. it is this need that is displayed vividly in some infected societies across the globe. the key is therefore not to get overly distracted by the virus, rather the acknowledgment of collateral immediate immune exuberance that is the mortality and health care resourcing problem. and knowing that cytokine storms are quick and overwhelming, we must also focus on therapeutic repurposing for immune modulation. repurposing itself is a broad discipline; for covid, drug repurposing to treat the host is the focus of this commentary; in distinction, repurposing to identify a novel antiviral effect requires a much longer time frame which will reach fruition outside of this pandemic. drug repurposing to treat the host includes re-reviews of previously repurposed drugs in sars or other treatments for adult respiratory distress syndrome, for example. in this context, the details of repurposing and its limitations in the current era been comprehensively discussed recently. in order to convert to reality, the opportunity of repurposing drugs to treat the host, we suggest an urgent international drug repurposing clinical research program, governed from a central site, and which in the time of covid- is based on treating the host. the features of such a program would be: • an international approach to rapidly identify drugs that treat the host in a pandemic to permit time for vaccine and antiviral development. • an international approach to coordinating the repurposing of existing drugs in a pandemic based on the principles of treating the host and repurposing existing drugs for new indications. • focusing on treating the host, utilizing existing pharmacology and physiology knowledge and observational administrative data, as is in several countries currently but is unlinked to other data and other countries, and needs to be at scale. • a fast-moving program driven by a sense of urgency with the ability to disseminate information to minimize unwanted duplication. overall then we would thus urge attention be given immediately to coordinated clinical evaluation of existing pharmaceuticals in an international repurposing program targeted toward managing the host, via antagonizing the innate immune pathway or inhibition of the ras at appropriate dose and timing, in this pandemic. such a program can provide adequate time for the development of vaccines, serum-based approaches or antiviral drugs, and separately will provide a therapeutic insurance with the inevitable easing of social isolation. there are no conflicts of interest to declare. treating the host response to emerging virus diseases: lessons learned from sepsis, pneumonia, influenza and ebola clinician-initiated research on treating the host response to pandemic influenza drug repurposing in the era of covid -a market failure needing leadership and government investment a sars-cov- protein interaction map reveals targets for drug repurposing renin-angiotensin system inhibitors improve the clinical outcomes of covid- patients with hypertension treatment with ace-inhibitors is associated with less severe disease with sars-covid- infection in a multi-site uk acute hospital trust association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid- a call for the appropriate application of clinical pharmacological principles in the search for safe and efficacious covid- (sars-cov- ) treatments rapid repurposing of drugs for covid- buying time: drug repurposing to treat the host in covid- h key: cord- -ubazgvov authors: cafiero, concetta; re, agnese; micera, alessandra; palmirotta, raffaele; monaco, delio; romano, francesca; fabrizio, claudia; di francia, raffaele; cacciamani, andrea; surico, pier luigi; d’amato, gerardo; pisconti, salvatore title: pharmacogenomics and pharmacogenetics: in silico prediction of drug effects in treatments for novel coronavirus sars-cov disease date: - - journal: pharmgenomics pers med doi: . /pgpm.s sha: doc_id: cord_uid: ubazgvov the latest developments in precision medicine allow the modulation of therapeutic approaches in different pathologies on the basis of the specific molecular characterization of the patient. this review of the literature coupled with in silico analysis was to provide a selected screening of interactions between single-nucleotide polymorphisms (snps) and drugs (repurposed, investigational, and biological agents) showing efficacy and toxicityin counteracting covid- infection. in silico analysis of genetic variants related to each drug was performed on such databases as pharmgkb, ensembl genome browser, www.drugs.com, and snpedia, with an extensive literature review of papers (to may , ) on covid- treatments using medline, embase, international pharmaceutical abstracts, pharmgkb, and google scholar. the clinical relevance of snps, known as both drug targets and markers, considering genetic variations with known drug responses, and the therapeutic consequences are discussed. in the context of clinical treatment of covid- , including infection prevention, control measures, and supportive care, this review highlights the importance of a personalized approach in the final selection of therapy, which is probably essential in the management of the covid- pandemic. covid- , an emergency all over the world, is caused by severe acute respiratory syndrome coronavirus (sars-cov ), a positive-sense rna virus with higher mutation rates than dna viruses. , laboratory tests on respiratory tract specimens (swab/saliva/tears pcr and serological tests) and computed tomography (ct; for chest or other tissue) should stage as soon as possible the presence of disease and its evolution/progression. , sars-cov binds principally to the cell-membrane ace receptor through the viral structural spike (s) protein, although other "virus doors" have been suggested (integrins and toll-like receptors) (figure ) . at the intracellular level, the virus activates cellular processes to produce viral proteins that replicate the virus's genetic material, providing potential targets for drug therapy. , , covid- features, diagnostic route, and early, mild, and severe covid- symptoms are summarized in figure . virus entry and multiple signaling pathways (ca + release, csrc, fak, mapk, and pi k) occur upon spike -rgd-integrin interaction. a "cytokine storm" is locally released (il β, il , il , tnfα, mip α, and vegf), as first observed in patients showing fatal complications. this specific panel was used to screen patients requiring prompt intervention to avoid acute respiratory distress syndrome (ards). in a recent study, candidate drug targets for nonstructural proteins ( -chymotrypsin-like protease, papain-like protease, rnadependent rna polymerase), viral access, and related immuno regulatory pathways have been prospected for evaluation. , , a wide range of pulmonary manifestations can be observed, varying from mild respiratory symptoms (cough or sore throat) to severe pneumonia, up to a sudden development of respiratory failure. silent hypoxemia is a peculiar aspect frequent in frail and elderly patients that usually precedes the onset of an overt acute and severe respiratory syndrome. , chest ct imaging is strongly recommended for an early diagnosis of novel coronavirus pneumonia. , ct findings, including patterns associated with less frequent signs, eg, lobular, bronchial, pleural, and subpleural involvement, as well as pleural lymphadenopathy and pericardial effusion, have been described. sars-cov initially causes airspace exudates, interstitial edema, hyaline membranes, and inflammatory infiltrates in the alveoli. other than the deleterious lung effects, systemic sars-cov has a significant impact on the hematopoietic system and tissue homeostasis. initially, lymphopenia showed interesting prognostic value, for both neutrophil:lymphocyte and platelet:lymphocyte ratios in severe sars-cov cases. lymphocyte parameters (count dynamics), some inflammatory indices (ldh, crp, and il ), and some recent circulating biomarkers (high serum procalcitonin and ferritin) have been suggested for identifying cases with poor prognosis (prompt intervention and improved outcomes). as observed, blood hypercoagulability, high d-dimer levels (particularly associated with disease worsening), protraction of both prothrombin (pt) time, activated partial thromboplastin time (aptt), and disseminated intravascular coagulation overview of covid- infection: sars-cov -host interaction and tissue manifestation. schematic representation of sars-cov virus interacting with mucosa epithelia (framed) and gradual sprouting to gut, kidney, brain, and lung tissue. "virus doors" reportedwere ace , integrin, tlr , or tmprss . overall symptoms are listed. briefly, most common covid- symptoms occur at the upper respiratory tract, together with asthenia and general malaise. early, less common clinical features include gastrointestinal symptoms (nausea, anorexia, diarrhea), anosmia and dysgeusia. severe cases encompass pneumonia or bronchopneumonia, severe acute respiratory syndrome, renal failure, up to death. the lower respiratory tract participation and complications are more frequent in people aged over with pre-existing chronic diseases of cardiovascular and/or respiratory system, as well as some autoimmune diseases (diabetes). cardiac involvement represents another potentially life-threatening complication, caused by direct viral damage, hypoxic injury, microvascular dysfunction and disclosing with diverse clinical pictures (arrhythmias, myocarditis, and heart failure). finally, virus can reach central nervous system causing central (acute cerebrovascular diseases, impairment of consciousness) and peripheral complications. the evolution of infection from early detection severe complications and death (shaded arrow). as reported, molecular and serological, as well as imaging tests, are useful depending on disease evolution: real-time pcr applied to nasopharyngeal swabs, venous blood as well as conjunctival swabs can track disease ongoing, while early-released igm and late-released igg might assess for immune response, in line with cytokine storm panel (il , tnfα, ifnγ, and il ) typifying tissue/organ complications. require constant surveillance to allow prompt intervention. in addition to upper-airway contact, conjunctivitis may be an ocular manifestation of sars-cov infection, if quickly detected. in december , a chinese ophthalmologist reported an unusual viral conjunctivitis. to date, several internet articles and scientific reports have documented the potential use of eye swabs as a tool to screen virus infection, although contrasting data are available, most probably related to progression of infection and virus detection. in january , a worldwide expert in infectious diseases referred to conjunctivitis during an inspection of wuhan and tested positive for sars-cov , but quickly recovered from infection. this led to the idea of "eye infection" as a possible alternative route of sars-cov transmission, alternative to the respiratory one. ocular secretion might represent a reservoir of virus at the early stages of contagious. when pinkeye occurs as a sign of conjunctivitis, a differential diagnosis can be of great utility to screen symptomatic and even presymptomatic individuals. recently, pharmacogenomics (the effects of a single genetic marker) and pharmacogenetics (the collective influence of variability across the genome to modulate an individual's drug response) have received great attention for their abilities to provide a new way to select drugs for personalized therapy (optimal dosing for maximizing drug efficacy or minimizing the risk of toxicity). , through drug therapy, pharmacogenetics might influence both pharmacokinetics and pharmacodynamics with respect to dosing, formulation sensitivity, and adverse drug reactions (adrs), as well as drug-hypersensitivity reactions (allergic, pseudoallergic, and exaggerated pharmacological reactions to medications), determining an enhanced immunologic reaction or inflammatory response (side effects). genetic assessment can predict the occurrence of drug-related toxic effects. single-nucleotide polymorphisms (snps) are the most common type of polymorphism found in the human genome, and represent the main reason for % of all types of genetic variations among individuals. an example of an adr is the hypersensitivity reaction occurring in snp carriers of the hla-b* allele who receive the antiviral abacavir for treatment of hiv. , covid- therapy is wide-ranging and multiple, and risk factors for adrs can occur. therefore, optimal doses, duration of treatment, side effects. and long-term outcomes are critical aspects of covid- therapy. cov-mediated inflammation may be counteracted by anti-inflammatory cytokines, including il family members, il , and tnfα. since no specific drug/therapy for covid- treatment has been us food and drug administration (fda)-approved, an array of drugs approved for other diseases and under investigation (including off-label and biological drugs) have been included in clinical trials. , as observed, the metabolic pathways of these drugs included various polymorphic cytochrome p enzymes, strongly suggesting genotyping for the cyp d * , cyp d * , cyp d * , and cyp d * alleles. , as an example, no predictability in the metabolic function of the cyp a * allele can result in alternative mrna splicing with a trunked protein, due to the formation of an untimely stop codon. moreover, haplotype cyp a * has been related to a reduced clearance of both ritonavir and lopinavir (substrates) ( table ) . of interest is the association between polymorphisms inabcc and therapy efficacy, as the drug transporters are one of the primary mechanisms related to subtherapeutic antiretroviral-drug concentrations. prominent research has highlighted the relationship between the abcb polymorphism ( c>t) and hepatotoxicity risk after antiviral treatments, and few additional studies were found regarding other nucleoside analogues. more recently, the presence of grade - hyperbilirubinemia is directly proportional to the homozygosity, heterozygosity, and wild-type genotyping for the ugt a * allele in patients who receive ritonavir and lopinavir. therefore, the attempt of this literature review coupled with in silico analysis was to provide a selected screening of drugs showing efficacy and toxicity effects useful for counteracting covid- infection. an extensive literature review of papers published until may , was performed based on a standard procedure (medline, embase, international pharmaceutical abstracts, pharmgkb. and google scholar). search terms were "covid- ", "novel coronavirus", "sars-cov ", "pharmacogenetics", "treatment/s", "adverse side effects", "therapy", "lung", "ocular", "pulmonary infection", "drugs", "drug response", "virus", "candidate drugs", "potential inhibitors", "protease inhibitors", "personalized medicine", "individual therapy", "pneumonia", "ace", "heparin", "vasculitis", "conjunctivitis", "rhinitis", "hematological complication" and "main metabolic routes", either alone or in combination. from this manually performed analysis, drugs reported in at least two studies or in a clinical trial were included. ongoing clinical trials and the index of studies of covid- were identified using the search term "coronavirus infection" on clinicaltrials.gov and the chinese clinical trial registry (http://www/chictr.org/enindex.aspx). in silico analysis of genetic variants related to each drug was performed on dedicated databases, such as pharmgkb (table ) , ensembl genome browser, www.drugs.com, and snpedia. the allelic frequency of each variant average in all populations was based on data from the genomes project phase . we considered snps related to both efficacy and toxicity response with allele frequencies ≥ %, as already described in previous studies. , the quality of each study was assured, and resulting information included study design, baseline characteristics of disease, treatment regimens, and allelic frequencies of the genetic variant. drugs in use as routine therapy or in clinical trials for covid- include steroids and antiviral and biological humanized neutralizing antibodies against some proinflammatory cytokines, such as il , il , ifn, and tnfα, in addition to supportive measures and symptomatic treatment, according to the severity of the disease. data analysis on registered clinical trials of covid- in the table overview of pharmgkb • pharmgkb is one of the foremost worldwide resources for pgx knowledge, and the organization has been adapting and refocusing its mission along with the current revolution in genomic medicine. annotates drug labels containing pharmacogenetic information approved by the us food and drug administration, european medicines agency swiss agency for therapeutic products, pharmaceuticals and medical devices agency (japan) and health canada. attempts to interpret the level of action implied in each label with the "pgx level". • pharmgkb website provides a diverse array of pgx information, from annotations of the primary literature to guidelines for adjusting drug treatment based on genetic information. • pharmgkb online pharmacogenomics knowledge resource maintains a list of very important pharmacogene summaries (www. pharmgkb.org/view/vips.jsp), which (until march ) describes , allelic variant annotation in which allelic variation has been shown to significantly influence drug metabolism, drug transport, drug-target response, expression of genes encoding drug-metabolizing enzymes, or risk of adverse drug reactions. us is available at https://clinicaltrials.gov. nowadays, a great number of agents have been evaluated for potential use in covid- management, and only a fewhave been included within international and/or local protocols. according to the us national institutes of health, no pharmacological agent has earned approval for safe and effective use yet, mainly because of the lack of evidence in favor or against these agents (https://covid treatmentgui delines.nih.gov). one important aspect of a therapeutic approach consists in the choice of the right drugs throughout the natural history of disease. indeed, a three-phase pathogenetic model has been proposed, with different clinical and laboratory features, each requiring a specific treatment based on the changing role over time,of direct viral damage and host inflammatory response in the disease course. this model describes an early stage (stage i), coinciding with incubation and/or mild flulike symptoms, during which antiviral drugs might reach high effectiveness, a moderate stage (stage ii) characterized by pulmonary involvement without iia or with iib hypoxia, which may benefit from the use of antivirals and anti-inflammatory therapy (including steroids), and lastly a severe stage (stage iii), in which a dysregulated, systemic hyperinflammatory response takes place, thus requiring the administration of immunomodulating agents, several of which are currently under investigation, and in some cases already in use within management protocols. gene variants associated with pharmacological responses to drugs are reported in a dedicated database -pharmgkb (https://www.pharmgkb.org) -allowing the identification of relationships between genetic variations (eg, snps, indels, repeats, haplotypes) and individual drug responsiveness. , herein, in silico pharmacogenetic analysis shows the potential clinical efficacy and/or toxicity of the major drugs selected for covid- treatment. the main drugs proposed for covid- treatments and reference sequence (rs) related to adrs and efficacy are shown in table . genes and related snps (rs) associated with drug effects and their study annotation are summarized in table . major clinical information regarding "variant drug" responsiveness (clinical annotations) is highlighted. bloodstream fluidity requires a tidy balance among factors favoring flow of blood and thrombosis. any modification in this physiological balance triggers pathological conditions: deficit of coagulation factors favor bleeding, while genetic mutations of coagulation factors can determine a thrombotic risk framework characterized by differences in genetic variants of coagulation (table ). all drugs used as therapeutic options and their study annotation based on pharmacogenetic data are discussed. chloroquine (cq) and hydroxychloroquine (hcq) cq is used for chemoprophylaxis of malaria and amebiasis, while hcq is used to treat autoimmune disease. the mechanism of action includes the blocking of viral entry through the inhibition of glycosylation of host receptors, proteolytic processing, and endosomal acidification. effects of immunomodulation on cytokine production and inhibition of autophagy/lysosomal activity have been also reported. [ ] [ ] [ ] several randomized controlled trials (rcts) are currently investigating cq and hcq in covid- treatment (https:// clinicaltrials.gov). based on current data, both drugs are recommended for treatment of unhospitalized covid- patients, and the effects of cardiotoxicity in immunosuppressed subjects or patients with kidney or liver problems are known. recommendations for oral administration in covid- treatment are mg twice a day and mg twice a day, followed by mg twice a day for cq and hcq, respectively. blood monitoring is required, as hemolytic anemia might occur, particularly when the drug is delivered in association with other drugs that cause hemolysis. it should not to be underestimated the extent to which both drugs may cause hemolysis in glucose- phosphate dehydrogenase (g pd)-deficient individuals. of note, about % of covid- patients suffering from lupus have failed to respond or were even intolerant to hcq. moreover, long-term cq-hcq treatment induces retinal toxicity, and particularly when hcq-related retinopathy is diagnosed, the retinal damage endures, even after cessation of therapy. high doses or prolonged administration of hcq, even at recommended doses, may increase the risk of ocular toxicity (paracentral scotomas, color-vision changes, corneal/ciliary body/conjunctival as well as retinal abnormalities), and visual disturbances (retinal and macular toxicity). this would imply that accurate eye screening for confirming sars-cov presence in early conjunctivitis or monitoring ocular structure during covid- therapy is recommended to counteract early or even prevent eventual signs of ocular drug toxicity, including the retinopathy. the oral combination of these two agents (fda-approved and currently authorised as anti-hiv medicine) demonstrate activity against other novel coronaviruses. , several rcts examining lopinavir/ritonavir in covid- are in progress. recommendations for administration in covid- treatment is mg/ mg twice daily for up to days. adverse effects of lopinavir/ritonavir include several gastrointestinal complains, such as nausea, vomiting, and diarrhea, and hepatotoxicity, pancreatitis, and cardiac conduction abnormalities. these adverse effects are increased in %- % of covid- patients with elevated transaminases. previous studies have shown that these adrs with lopinavir-ritonavir combination are related to several polymorphisms present in the genes ugt a , ugt a , apoe, and apoc (table ) . this antiviral drug (guanine analogue) inhibits viral rnadependent rna polymerase. ribavirin activity against covid- disease is limited and requires high doses or combination therapy to be effective in humans. no evidence exists for inhaled ribavirin for covid- treatment -no benefit over enteral or intravenous administration though several studies have demonstrated possible harm due to adverse effects (hematologic and liver toxicity). ribavirin causes severe dose-dependent hematologic toxicity. the inconclusive efficacy and toxicity results suggest that ribavirin has limited value in treatment of covid- . case-control pharmacogenetic association studies indicate different polymorphisms in the itpa, vdr, and slc a genes that are related to toxicity and adverse effects, while an increased pharmacological response is associated when some variants of the vdr, slc a , ifnl , and micb-oasl genes are present. it must be noted that numerous studies have undertaken pharmacogenetic evaluations on populations undergoing ribavirin therapy combined with peg-ifnα. this is an antiviral agent that exerts a mechanism of action targeting the s protein-ace interaction, inhibiting membrane fusion of the viral envelope. the recommendation for umifenovir oral dose ( mg every hours, - days) for influenza treatment was studied for covid- therapy (nct ). in covid- patients treated with umifenovir, adverse effects include allergic-reaction gastrointestinal upset and elevated transaminases, although there are no studies that describe an association of these episodes with genetic variants. , miscellaneous agents ifnα and -β have been studied for novel covs. several studies reported clinical outcomes in combination with ribavirin and/or lopinavir/ritonavir. adverse effects have been described in almost every organ, clearly dosedependent. , several toxic mechanisms for ifnα/β have each variant report show the reference snp (refsnp) cluster id numbers (rs#), a code by which each snp can be queried from the dbsnp website (https://www.ncbi.nlm.nih.gov/ snp) and through which further information is available (such as functional analysis, population-specific allele frequencies, validation information, or clinical significance). furthermore, for each variant, a summary of pharmgkb clinical annotations with respective identification codes (phenotype for any given genotype) is reported. using the access codes for dbsnp and pharmgkb, it is thus possible to find more information for the sequence variants and the respective variant-drug responsiveness associations. finally, the last columnshows the most significant bibliographic reference concerning the association between the genetic variant reported and individual drug responsiveness. pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress been investigated in recent years. much remains still to be elucidated, although most side effects disappear on dose reduction or interruption of treatment. previous genomewide association studies on the response to ifnβ in neurological degenerative diseases suggest an increase in therapeutic response with the presence of several snps in the genes cd , znf , and fhit. in parallel, adrs have been observed with the presence of variants of the gapvd and irf genes. as of now, the use of interferons to treat covid- disease is controversial. although an antihelminthic agent, nitazoxanide has shown antiviral activity with a favorable safety profile. , camostat mesylate prevents novel cov-cell entry through inhibition of the host surface transmembrane protease/serine (tmprss ), as observed by in vitro studies. neither drug has pharmacogenetic studies regarding efficacy or toxicity effects associated with genetic variants. this macrolide antibiotic is used extensively in patients with viral infections to prevent severe respiratory tract infections. azithromycin-hcq combination shows a synergistic effect on covid- disease in vitro at concentrations comparable with that observed in human lung, and has been found useful in combination with remdesivir, lopinavir/ritonavir and ifnβ. to date, the interaction between this macrolide and genetic factors is unknown. however, due to the potential risk of prolonged qt interval, remarkably increased by the mutual interaction between these two agents, their concomitant use is not recommended by several guidelines, unless within the setting of controlled trials (https://covid treatmentguidelines.nih.gov). this anticoagulant is used to reduce thromboembolic events in patients with covid- infection. as known, the sars-cov pandemic is characterized by the development of ards that results from acute inflammation within the alveolar space and prevention of normal gas exchange. indeed, huge deposits of fibrin in the lung parenchyma and air spaces have been reported. the raising of proinflammatory cytokines within the lung leads to recruitment of leukocytes, increasing the local inflammatory response, and since the coagulation route contributes to reducing pathogen invasion by improving compartmentalization, this anticoagulant treatment has potential risk in those covid- patients with no significant coagulopathy. the drug enoxaparin can be started as soon as the day of covid- diagnosis and continued over days, after baseline assessment and monitoring of pt, aptt, complete blood-cell count, and creatinine levels. clinical aggravation causing worsening and death in covid- inpatients appears to be thrombotic. therefore, special attention should be devoted to both risks and benefits of using heparin in covid- patients. prescreening is essential to highlight genetic predisposition related to thrombophilia, as these subjects with thrombophilic mutations (based on the degree found) are at greater risk of thrombotic complications. remdesivir this is an rna-polymerase inhibitor (gs- ), a retroviral drug belonging to the class of nucleotide analogues, and a monophosphate prodrug that was discovered during a screening study of antimicrobials with antiviral activity, showing promise against the ebola virus. , recently approved by the fda for the treatment of patients with covid- , remdesivir is recommended as a single mg dose, followed by mg daily infusion. besides the drug's clinical use in the treatment of ebola, several clinical trials are ongoing to evaluate the antiviral activity of remdesivir in patients with covid- (nct , nct , nct , nct , and nct ). no pharmacogenetic indications are present, allowing modulation of dosage for therapy. due to its toxicity, remdesivir is not currently fda-approved, and must be obtained via compassionate use, expanded access, or enrollment in a clinical trial. favipiravir ribofuranosyl- ′-triphosphate is a retroviral drug acting as an inhibitor of viral replication (rna-polymerase inhibitor t ). this agent demonstrates broad activity against ebola infection and other rna viruses. favipiravir is recommended at a dose of , - , mg every hours (two doses) followed by maintenance doses of , - , mg every hours. favipiravir is overall well tolerated, although a mild adverse-event profile for higher-dose regimens has been observed. [ ] [ ] [ ] the high doses used result in hyperuricemia, diarrhea, elevated transaminases, and reduction in neutrophil count, but nothing is known about any pharmacogenetic correlations. favipiravir presents efficacy in the treatment of covid- disease, but to date limited clinical experience has reported support for its use. no pharmacogenetic indications are present, allowing modulation of dosage for therapy using this drug. these drugs are well-known protease inhibitors used for the treatment of aids, with good efficacy and safety profiles. while atazanavir is combined with other antiretrovirals, darunavir is used in a fixed-dose combination with cobicistat, a new pharmacokinetic enhancer. with respect to the other protease inhibitors, atazanavir is less likely to cause lipodystrophy and is preferentially used in combination with other antiviral agents (ritonavir), providing antiviral potency equivalent to lopinavir, although concomitant use of ritonavir decreases the efficacy of atazanavir. valid pharmacogenetic correlations of the efficacy of these antivirals can be deduced from studies carried out on hiv patients in which the efficacy of polymorphisms in the ugt a , cyp a , cyp a , and slco a genes has been demonstrated. in contrast, cases of toxicity and adrs are related to sequence variants in the ugt a , ugt a , ugt a , apoe, and apoc genes. [ ] [ ] [ ] [ ] [ ] , , , in some settings, darunavir-cobicistat may be used in the presence of tolerability or availability of the lopinavir-ritonavir combination (https://covid treatmentguidelines.nih.gov). concerning the metabolism of azatavir, the variants cyp a * , cyp a * , cyp a * , cyp a * in cyp a (rs ) have been reported. briefly, individuals carrying one or two copies of the * allele may metabolize atazanavir more rapidly than individuals with one or more copies of the * , * , or * alleles. anticytokine or immunomodulatory agents (tocilizumab, sarilumab, and bevacizumab) monoclonal antibodies directed against key inflammatory cytokines represent another potential class of adjunctive anti-covid- therapies. , il is a key driver of this dysregulated inflammation. the use of monoclonal antibodies against il shows a dampening of this process and improves clinical outcomes. tocilizumab, a monoclonal antibody il -receptor antagonist, is used in severe covid- cases with success. adult doses are mg or corticosteroids are used to reduce lung inflammatory responses that in many cases evolve into acute lung injury and ards. at present, the effects of corticosteroids in patients affected by covid- have been poorly described, so our considerations are based on observations in other viral pneumonia types, such as sars and mers. these previous studies do not indicate any association between corticosteroid use and increased survival, but slower viral clearance in the respiratory and blood tracts, hyperglycemia (high blood sugar), vascular necrosis, and psychotic episodes. as stated, sars-cov has the ability to introduce and infect the host cell using the ace receptor. this has alarmed several researchers, who hypothesized how ace inhibitors and angiotensin-receptor blockers could interfere negatively or positively with the viral infection process. , since ace inhibitors upregulate ace receptors, possible worsening of disease under ace inhibitor therapy has been hypothesized. by contrary, angiotensin-receptor blockers could theoretically prevent the entry of the virus at the cellular level. some rcts specific for captopril used alone or in combination in patients with covid- with severe pneumonia are currently under investigation in order to understand ards (nct , https://clinicaltrials.gov). since ace inhibitors are among the most frequently used drugs in medical practice, consequent knowledge on related pharmacogenetic aspects is now widely known. the polymorphisms that modulate the activity of these drugs in ace, ace , and agtr genes have been widely studied and described in several recent studies. the covid- pandemic is due to the novel pathogenic coronavirus sars-cov , which emerged in china and spread quickly worldwide. , therapeutic options for covid- are wide-ranging, and some drugs have gained emergency-use authorization from the fda and/or european medicines agency. at present, patients are treated with symptomatic therapy and vital support in severe cases. several international efforts are aimed at the investigation and development of antiviral agents, other immunotherapies, and vaccine strategies. pharmacogenomics might be a promising tool in the development of more appropriate therapies (including drug management) and the prevention of fatal-complication onset due to adrs. pharmacogenomics can predict from the beginning the effect of a specific drug formulation in terms of efficacy/ toxicity with respect to individual genetic background and minimize exposure to drugs potentially less/ineffective other than toxic (precision medicine). , as known, drug formulations can elicit different cell/tissue responses depending on individual genetic background. this is possible because each subject can have variations in nucleotide sequences belonging to genes encoding for enzymes involved in the activity of the drug that has been considered for therapy. [ ] [ ] [ ] this aspect appears of great importance in patients having several comorbidities or simply "fragile" old patients. as there is no specific cure for covid- , we reviewed all the therapies (drugs) actually in use to counteract sars-cov effects and the related adverse effects to certain drugs (antiviral, antimalarial, and several biological humanized agents able to reduce the levels of some cytokines belonging to the cytokine storm), as reported by rcts. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] response to these drugs was extremely complex, and numerous cases of toxicities and antiretroviral drug resistance (viral mutations) have been reported. a possible explanation of this variability might be the presence of factors that modify pharmacokinetics/pharmacodynamics and the activity of the virus itself (viral pharmacodynamics). in addition, viral pharmacodynamics and mutagenesis are still unclear with regard to covid- drug resistance. genetic factors might account at least in part for the unpredictability of therapy among covid- patients. merely, a significant number of snps in genes encoding proteins implicated in the transport and metabolization of drugs may be responsible for the wide variability in drug pharmacokinetics and toxicity. consistently, this review, coupled with a wide in silico analysis on the relevance of identification of snps involved in drug metabolism, provides a list of specific drug-associated snps associated with efficacy (green) or toxicity (red), useful in predicting individual response to therapy in covid- patients. this would imply the possibility of checking variants in biological samples collected and evaluated before the beginning of therapy with the aim of predicting the outcome of a single or combined covid- therapy. to our knowledge, although a considerable number of adr episodes in covid- patients have to date been described in the literature, there has been no pharmacogenetic study attempting to correlate the clinical outcomes of drug treatment with gene variants. the identification of gene variants is only the first step in a complex process prior to applicability into clinical practice. in fact, the clinical application of pharmacogenetic analysis requires previous studies confirming its validity and usefulness. to support this, essential measures applied to genetic tests are required: analytical and clinical validity and clinical utility. while analytical validity defines test accuracy, sensitivity, and specificity, guaranteeing that a "positive" or "negative" result corresponds with the real presence or absence of the sequence variant investigated, clinical validity represents the ability to identify the clinical phenotype of interest, evaluating clinical sensitivity and specificity, and positive or negative predictive values, or in other cases, the association measured as a risk or odds ratio. , of note, clinical utility is related to the evidence that genetic testing can provide useful information for the diagnostic process, better measures for clinical outcomes, and odds ratios for patient-management decision-making (precision medicine). , as such, a pharmacogenetic testpotentially useful for patient treatment -must improve clinical outcomes. evidence on the clinical utility of a pharmacogenetic assay is obtained by experimental studies, preferably rcts. a valid method to evaluate clinical utility is the use of prospective trials on randomized subjects undergoing genetic testing or not to compare the same treatment between the two groups. similarly, prospective trials on genetically stratified groups are also mandatory to comparing treatment outcomes between different groups. , in other cases, clinical utility is determined by a "chain of indirect evidence" linking the results of a genetic test to intermediate data that are associated with improved clinical outcomes. , however, pharmacogenetic research remains an expanding field, and to date there is no unanimous consensus on the best appropriate study designs to uniquely evaluate drug-response variability related to genetic variations. finally, several national drug agencies are carefully evaluating risk:benefit ratios in individual cases, carefully considering the concomitant pathologies (long qt syndrome, major arrhythmias, liver or kidney failure, electrolyte disorders), pharmacological associations (in particular for drugs that increase the qt), and above all the clinical anamnesis and identification with genetic diagnosis of favism (g pd deficiency). in our opinion, this is the first study to suggest the application of personalized medicine tools during the treatment of sars-cov infection. all identified snps, including allelic efficacy/toxicity, selected from an accurate in silico analysis have been identified for the most promising and repurposed drugs, and the investigational and adjunctive experimental drugs have been reported in both tables and figures. particularly, in figure we graphically represent the frequency of snp alleles related to an efficacy response (green bars) or toxicity (red bars), useful in predicting individual response to therapy with the main therapies used for covid- patients. in this study, tocilizumab showed varying efficacy, while inside the antiviral group a divergent response was observed. therefore, to understand from the beginning specific susceptibility (efficacy/toxicity) to a drug, as displayed by the presence of specific functional clusters in the genetic background of the patient under treatment, might assist the specialist toward a more "specific" selection of therapeutic agent. this would result in a more appropriate therapeutic response, with fewer adrs. as reported in other therapies, this individualized approach dovepress appears of great utility, and particularly for the covid- pandemic could improve the choice of more efficient therapy. the screening is less invasive for the patient, as it is possible by venous blood or buccal-cell swab using realtime pcr analysis. any attempt to find more suitable tests, identify asymptomatic/presymptomatic and/or confirm symptomatic subjects, and therapeutic agents/strategies to sustain therapeutic decisions in covid- -affected patients appears mandatory. herein, we performed a wide in silico study of genetic variants associated with the main drugs in use for covid- therapy, providing a list of genetic variants of efficacy/toxicity. this study highlights the clinical utility of a pharmacogenetic tool in planning personalized treatments that are likely to become essential in the pharmacological management of covid- patients. journal to which the article has been submitted, and agree to be accountable for all aspects of the work. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest and report no conflicts of interest for this work. pharmacogenomics and personalized medicine is an international, peer-reviewed, open access journal characterizing the influence of genotype on pharmacology leading to the development of personalized treatment programs and individualized drug selection for improved safety, efficacy and sustainability. this journal is indexed on the american chemical society's chemical abstracts service (cas). the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. emerging coronaviruses: genome structure, replication, and pathogenesis the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak iga-ab response to spike glycoprotein of sars-cov- in patients with covid- : a longitudinal study interpreting diagnostic tests for sars-cov- the trinity of covid- : immunity, inflammation and intervention the pathogenesis and treatment of the `cytokine storm' in covid- the eye as the discrete but defensible portal of coronavirus infection effects of chloroquine on viral infections: an old drug against today's diseases? targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases severe acute respiratory syndrome: radiographic and ct findings middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings coronavirus disease (covid- ): a systematic review of imaging findings in patients early clinical and ct manifestations of coronavirus disease (covid- ) pneumonia correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases chest ct findings in patients with coronavirus disease and its relationship with clinical features pathological findings of covid- associated with acute respiratory distress syndrome hematological findings and complications of covid- ophthalmic manifestations of coronavirus (covid- ) the prevalence of ophthalmic manifestations in covid- and the diagnostic value of ocular tissue/fluid characteristics of ocular findings of patients with coronavirus disease facing covid- in ophthalmology department navagate: a rubric to move from pharmacogenomics science to pharmacogenomics practice translational approach for pharmacogenomics and personalized medicine pharmacogenomics and adverse drug reactions: primetime and not ready for primetime tests association between presence of hla-b* , hla-dr , and hla-dq and hypersensitivity to hiv- reverse-transcriptase inhibitor abacavir genetic variations in hla-b region and hypersensitivity reactions to abacavir incidence of adverse drug reactions in covid- patients in china: an active monitoring study by hospital pharmacovigilance system induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies recent progress and challenges in drug development against covid- coronavirus (sars-cov- ) -an update on the status coronavirus disease treatment: a review of early and emerging options pharmacogenetics of long-term responses to antiretroviral regimens containing efavirenz and/or nelfinavir: an adult aids clinical trials group study selected pharmacogenetic panel test for toxicity prevention of drug-drug interactions between highly active antiretroviral therapy (haart) and antiblastic chemotherapy drug transporter and metabolizing enzyme gene variants and nonnucleoside reversetranscriptase inhibitor hepatotoxicity genetic factors influencing atazanavir plasma concentrations and the risk of severe hyperbilirubinemia comparison of pubmed, scopus, web of science, and google scholar: strengths and weaknesses pharmacogenomics and individualized medicine: translating science into practice heterogeneity in the distribution of drug-response related snps in world populations and their genetic relatedness pharmacologic treatments for coronavirus disease (covid- ): a review covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal pharmgkb: the pharmacogenomics knowledge base pharmgkb summary: lamotrigine pathway, pharmacokinetics and pharmacodynamics hgvs recommendations for the description of sequence variants: update the single nucleotide polymorphism database (dbsnp) of nucleotide sequence variation. mcentyre j, ostell j editors, the ncbi handbook effect of the ugt a * allele on unconjugated hyperbilirubinemia in hivpositive patients receiving atazanavir: a systematic review single-nucleotide polymorphisms in the udp-glucuronosyltransferase a- ʹ untranslated region are associated with atazanavir-induced nephrolithiasis in patients with hiv- infection: a pharmacogenetic study genomewide association study of atazanavir pharmacokinetics and hyperbilirubinemia in aids clinical trials group protocol a genetic variation among pharmacogenes: the pgrnseq data from the emerge network gilbert's disease and atazanavir: from phenotype to udp-glucuronosyltransferase haplotype swiss hiv cohort study. modeling the influence of apoc , apoe, and tnf polymorphisms on the risk of antiretroviral therapy-associated lipid disorders associations among race/ ethnicity, apoc-iii genotypes, and lipids in hiv- -infected individuals on antiretroviral therapy polymorphisms associated with renal adverse effects of antiretroviral therapy in a southern brazilian hiv cohort functional defect caused by the g>a snp in abcc : potential impact for drug cellular disposition abcb and abcc variants associated with virological failure of first-line protease inhibitors antiretroviral regimens in northeast brazil patients genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c differences in genetic variants in lopinavir disposition among hiv-infected bantu africans simultaneous pharmacogenetics-based population pharmacokinetic analysis of darunavir and ritonavir in hiv-infected patients response to interferon-beta treatment in multiple sclerosis patients: a genome-wide association study common variation near irf is associated with ifn-β-induced liver injury in multiple sclerosis association of cd polymorphism with multiple sclerosis and response to interferon ß therapy in a subset of iranian population identification of a genomic region between slc a and hsp ab associated with risk of bevacizumab-induced hypertension: calgb (alliance) involvement of mirna polymorphism in mucositis development in childhood acute lymphoblastic leukemia treatment genetic variation associated with bortezomib-induced peripheral neuropathy pnpla rs and hepatotoxicity in children with b-cell acute lymphoblastic leukemia: a validation study in a spanish cohort pharmgkb summary: very important pharmacogene information for angiotensin-converting enzyme new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies chloroquine and hydroxychloroquine as available weapons to fight covid- examination of hydroxychloroquine use and hemolytic anemia in g pdh-deficient patients clinical and pharmacogenetic influences on response to hydroxychloroquine in discoid lupus erythematosus: a retrospective cohort study hydroxychloroquine and chloroquine retinopathy: screening for drug toxicity rapid onset of retinal toxicity from high-dose hydroxychloroquine given for cancer therapy hydroxychloroquine and ritonavir for covid- infection: a possible synergic toxicity for retinal pigmented epithelium role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study a trial of lopinavir-ritonavir in adults hospitalized with severe covid- risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease oral versus aerosolized ribavirin for the treatment of respiratory syncytial virus infections in hematopoietic cell transplant recipients sars: systematic review of treatment effects adverse events associated with high-dose ribavirin: evidence from the toronto outbreak of severe acute respiratory syndrome peginterferon-alpha a and ribavirin combination therapy in chronic hepatitis c: a randomized study of treatment duration and ribavirin dose umifenovir treatment is not associated with improved outcomes in patients with coronavirus disease : a retrospective study triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial. the lancet broad-spectrum coronavirus antiviral drug discovery remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov- shows synergistic effect remdesivir as a possible therapeutic option for the covid- discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[ , -f][triazin- -amino] adenine c-nucleoside (gs- ) for the treatment of ebola and emerging viruses remdesivir for severe acute respiratory syndrome coronavirus causing covid- : an evaluation of the evidence favipiravir (t- ), a broad spectrum inhibitor of viral rna polymerase discovering drugs to treat coronavirus disease (covid- ) inmi ebola team. qtc interval prolongation during favipiravir therapy in an ebolavirusinfected patient lack of effect of favipiravir, a novel antiviral agent, on qt interval in healthy japanese adults anrs -cophar study group. effect of adherence as measured by mems, ritonavir boosting, and cyp a genotype on atazanavir pharmacokinetics in treatment-naive hiv-infected patients clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study hlh across speciality collaboration, uk. covid- : consider cytokine storm syndromes and immunosuppression. the lancet effective treatment of severe covid- patients with tocilizumab clinical evidence does not support corticosteroid treatment for -ncov lung injury. the lancet saudi critical care trial group. corticosteroid therapy for critically ill patients with middle east respiratory syndrome research progress of drug treatment in novel coronavirus pneumonia angiotensin receptor blockers as tentative sars-cov- therapeutics angiotensin receptor blockers and covid- egapp working group. the evaluation of genomic applications in practice and prevention (egapp) initiative: methods of the egapp working group board on the health of select populations; committee on the evidence base for genetic testing. an evidence framework for genetic testing a critical appraisal of pharmacogenetic inference clinical utility of genetic and genomic services: context matters am and ac thank the italian ministry of health and fondazione roma (italy). cc, ar, am, rp, and sp conceived the study and participated in its design and coordination. cc, ar, am, and rp contributed to data collection and analysis. all authors made a significant contribution to the work reported, whether in its conception, study design, execution, acquisition of data, analysis and interpretation, or all these areas, took part in drafting, revising, or critically reviewing the article, gave final approval to the version to be published, have agreed on the key: cord- -icyut xa authors: pillaiyar, thanigaimalai; meenakshisundaram, sangeetha; manickam, manoj; sankaranarayanan, murugesan title: a medicinal chemistry perspective of drug repositioning: recent advances and challenges in drug discovery date: - - journal: eur j med chem doi: . /j.ejmech. . sha: doc_id: cord_uid: icyut xa drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs. recently, it emerges as an alternative approach for the rapid identification and development of new pharmaceuticals for various rare and complex diseases for which lack the effective drug treatments. the success rate of drugs repurposing approach accounts for approximately % of new fda approved drugs and vaccines in recent years. this review focuses on the status of drugs repurposing approach for various diseases including skin diseases, infective, inflammatory, cancer, and neurodegenerative diseases. efforts have been made to provide structural features and mode of actions of drugs. sir james whyte black, winner of the nobel prize in medicine [ ] it is reported that the food and drug administration (fda) has approved agents against about human proteins [ ] , % of which comes under the classification of enzymes, transporters, g protein-coupled receptors (gpcrs), cluster of differentiation (cd) markers, voltage-gated ion channels, and nuclear receptors. it is a fact that the typical de novo drug discovery program takes to years [ , ] from the identification of lead molecule to market the drug and the probability of success rate is less than % [ ] . over five years, the number of new drugs approved by fda has been around per year, although billions of us dollars spent by various pharma industries on the research and development [ ] [ ] [ ] . the success rate is less than % of new drug discovery and development, which is far away from addressing an unmet clinical need for disease treatments. because, the effective therapeutics for complex diseases like alzheimer's disease (ad), parkinson's disease (pd), cardiovascular diseases, and neglected diseases are still lacking. this outcome strongly suggests that new strategies, approaches, and technologies are needed to accelerate drug discovery to advance the success rate of drug development. drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs [ ] . it is a drug discovery program, which is faster and safer to develop medications against diseases/disorders for which no potential treatment is available. in recent years, the success rate of drug repurposing approach accounts for approximately % of the newly fda approved drugs, and vaccines. this is one of the main reasons for pharmaceutical companies to show their interest in drug repurposing approach. this approach does not require the initial six to years typically needed for the development of new drugs. additionally, many phases of de novo drug discovery and development can be by-passed, melanin protects human skin from the radiation, continuing irradiation can result in the risk of skin damage and malignant melanoma, a cancer of melanocytes. besides, the abnormal production of melanin leads to a serious of dermatological disorders including melasma [ ] [ ] [ ] [ ] , freckles, age spots, and post-inflammatory melanoderma. [ , ] . melanogenesis, a process of synthesis of melanin, is a complex enzymatic and biochemical catalyzed reactions, in which tyrosinase plays a rate-limiting step: hydroxylation of l-tyrosine to l- , -dihydroxyphenylalanine (l-dopa) followed by the oxidation of l-dopa to ldopaquinone, which serves as a substrate for the production of melanin [ ] . therefore, targeting tyrosinase has been recognized as a potential approach for controlling the abnormal production of melanin. tyrosinase is also an important target in the food industry as inhibition of tyrosinase can prevent the enzymatic browning of fruits and vegetables. besides, it essential for wound healing process and immune responses in many plants, sponges and some invertebrates. abnormal activities of tyrosinase have been linked to neurodegenerative disorders including parkinson's [ ] and huntington's diseases [ ] [ ] [ ] [ ] [ ] . thiourea and analogs. tyrosinase inhibitors can be broadly classified into two categories: (a) polyphenols, which are mostly natural products such as arbutin, hydroquinone and kojic acids, and b) thiourea and its derivatives. since the 's, phenylthiourea (ptu, ) has been well known as a tyrosinase inhibitor [ , ] . many research groups including ours have extensively studied the structure-activity relationships for ptu as tyrosinase inhibitors [ , ] . choi et al. screened the fda approved drug library that has closed structural similarity to ptu (see figure ) [ ] . for example, ethionamide ( ), a second-line antituberculosis drug used for the treatment of multi-drug resistant tuberculosis, shares a chemical similarity that led to the discovery of a new mushroom tyrosinase inhibitor with an ic value of . µm. other commercially available analogs of including, prothionamide ( ), thioisonicotinamide ( ), pyridine- -carbothiomide ( ), pyridine- -carbothiomide ( ) , and thiobenzamide ( ) were identified as potent tyrosinase inhibitors. in particular, compound had a strong inhibitory activity than other molecules, which suggests that the pyridine ring in compound can be replaced by other aromatic moieties, including a benzene ring. however, the poor inhibitory activity of isoniazid ( ), a first-line antituberculosis drug, suggests that carbothiomide group was crucial for tyrosinase inhibitory activity. moreover, comparing the inhibitory activities of drugs and implies that the additional aliphatic tail was not required for inhibiting the tyrosinase. inhibitory kinetic studies suggest that drug , and its analogs - were reversible and non-competitive. in cellular assay, drugs and markedly decreased the melanin content in b cells to the values of % and %, respectively, without inducing any cytotoxicity up to µm concentration. further studies suggest that the drug had strong inhibition of mammalian tyrosinase. however, the inhibition exhibited by drug , and its analogs were weaker than those obtained by ptu observed in enzyme, and melanin content assays. the same research group continued to search for molecules that are in clinical usage, and contain thiourea moiety [ ] . as a result, they could retrieve some thiourea containing drugs such as thioacetazone ( ) , ambazone ( ) , methimazole ( ) , carbimazole ( ) , thiouracil ( ) , methylthiouracil ( ) , and propylthiouracil ( ) . thioacetazone ( ) (also called as thiacetazone) is an anti-tuberculosis drug [ ] . ambazone ( ) is an oral antiseptic drug used in europe [ ] . the other five molecules ( ) ( ) ( ) ( ) ( ) are antithyroid drugs [ ] . these drugs, except , exhibited remarkable inhibitory activities against mushroom tyrosinase (see ic values of each in figure ), although they were comparatively weaker than ( µm) . kinetics studies of tyrosinase inhibition assigned these thiourea-containing drugs as non-competitive inhibitors. in cellular assay, using b cells, drug among other thioureas, significantly decreased the melanin content by % without inducing any cytotoxicity up to µm. further, enzymatic studies with cell extracts of b f cells confirmed that thioureacontaining drugs affected the function of mammalian tyrosinase. in an extended study, the same group repositioned thiopurine drugs - as tyrosinase inhibitors [ ] . thioguanine ( ) , a drug used for the treatment of leukemia, is one of the essential medicines required for a basic health system, recommended by the world health organization (who). it inhibited tyrosinase activity with a k i value of µm. in addition to that, two other thiopurine drugs such as mercaptopurine ( ) and azathioprine ( ) were discovered as tyrosinase inhibitors, and among them, drug showed stronger k i value (k i µm) than the drug . azathioprine ( ) exhibited a poor tyrosinase inhibition. these results suggest that the sulfur atom possibly plays an important role in the interaction of tyrosinase. interestingly, thioinosine ( ) , a metabolic product of mercaptopurine through the attachment of sugar moiety exhibited an excellent tyrosinase inhibitory activity with a k i value of . µm. further, kinetics studies classified the drugs , , and as competitive inhibitors. in the cellular assay, these drugs inhibited the melanin content without cytotoxicity up to µm. in particular, drug at -µm concentration, remarkably reduced the melanin content by %, without any apparent cytotoxicity. the thiopurine drugs were docked into four different crystal structures complexed with inhibitors tropolone, kojic acid, hydroquinone and ptu ( figure ) [ ] [ ] [ ] . in the molecular it also predicted that the intramolecular hydrophilic contacts with the residue of e- disappeared in mercaptopurine ( figure c ), whereas thioguanine ( figure b ) and thioinosine possess the contacts within . Å. thioinosine ( ) are represented. this figure was adopted from the publication of choi, j. et al [ ] . drugs repositioning for cancer therapy. as cancer is one of the leading cause of death worldwide [ ] , pharmaceutical companies invest billions of dollars in developing new anticancer drugs. the drug discovery and development process for the cancer treatment takes an average of years at a cost of approximately $ . billion [ ] . however, only % of the drugs that enter clinical trials are approved. this prolonged duration for the drug development and enormous cost of the (pre)clinical trials for their approval emphasizes the need for a drug repurposing approach (see figure for drugs repurposing in cancer therapy). aspirin ( ) , also known as acetylsalicylic acid, is one of the non-steroidal anti-inflammatory drugs (nsaids), which has been widely used as an analgesic and an antipyretic to prevent the heart attack and stroke. for the first time, gasic and co-workers reported the possible role of aspirin in cancer therapy. they discovered that the antiplatelet activity of in tumor-bearing mice was associated with a % reduction in lung metastasis [ ] . a recent study also indicated that the daily intake of the drug ( mg) produced a significant beneficial effect against gastrointestinal, esophageal, pancreatic, brain, prostate, and lung cancer [ ] . the mode of action aspirin is reported to modulate numerous molecules, which are associated with the tumorigenesis process [ ] . preclinical studies revealed that the anticancer activity of drug has been attributed by its inhibition of cyclooxygenase (cox) enzymes that promotes carcinogenesis through the synthesis of prostaglandins (pg), including pge [ ] . besides, drug was reported to inhibit the activation of transcription factor nf-κb, which is critical to regulating the expression of genes involved in apoptosis [ ] . in a study reported by li ling et al. compound not only inhibited proliferations and promoted apoptosis of cancer cells, but also delayed and overcame acquired resistance to targeted therapy. the underlying mechanism could be attributed to enhanced cancer stemness and activated nf-kb signaling in acquired resistant tumors were suppressed by aspirin and rendered resistant tumors more sensitive to aspirin than parental, sensitive cells in terms of proliferation, apoptosis and cancer stemness. on the contrary, aspirin has no effects on normal lung and mammary epithelial cell proliferation at concentrations used on lung and breast cancer cells. hence, aspirin could be a potential candidate for combination therapy for lung and breast cancers [ ] . other studies suggest that the anticancer property of aspirin ( ) has been linked to the phosphatidylinositol- -kinase (pi k) pathway, and ras-raf-mek-erk signaling cascade. while numerous studies suggest the potent anticancer activities of drug , the overall benefit is limited as it is associated with serious side effects including the gastrointestinal and renal toxicities. therefore, there is no clear recommendation to take for population-wide use. on the other hand, as a primary cancer prevention tool, many reports revealed using would have a greater benefit in the population at age - . thus, u.s. preventive services task force recommendation statement (uspstf), recommended for daily intake of for patients above years with increased risk of cardiovascular disease and colorectal cancer. celecoxib ( ) belongs to the family of nsaid, which has been used to treat pain and inflammation associated with rheumatoid arthritis (ra) and osteoarthritis (oa) [ ] . in , the fda approved celecoxib, which is a highly selective reversible inhibitor of cox- , a well-known inflammatory cancer target. because of its cox- inhibitory activity, the antitumor activities of drug have been extensively studied and shown to have chemopreventive activities against various cancer types. other than cox- , celecoxib targets glycogen synthase kinase (gsk) β, β-catenin, nf-κb, akt virus oncogene cellular homolog (akt), and b cell lymphoma (bcl)- families [ ] . in people with familial adenomatous polyposis (fap), a daily dosage of drug ( mg) significantly reduced the risk of colorectal adenomas. fda approved this compound to reduce colon and rectal polyps in people with familial adenomatous polyposis [ ] . however, it was associated with some drawbacks including gastrointestinal, renal, and cardiotoxic effects. ibuprofen ( ) is an nsaid, which has been primarily used to treat fever, pain, and inflammation. at the molecular level, ibuprofen inhibits cox, which converts arachidonic acid to prostaglandin. however, it is not selective towards any isoform of cox. the drug was marketed for the treatment of rheumatoid arthritis in the united kingdom ( ) , and in the united states ( ) as well. the anticancer activity of drug has been investigated in various cancer cell types. ibuprofen has been shown to inhibit the growth of prostate cancer cells [ ] . in adenocarcinoma gastric cells, drug showed antitumor effects, which have been mediated by the anti-angiogenesis, induction of apoptosis, and reduction of cell proliferation [ ] . the administration of drug induces apoptosis in metastatic melanoma cell lines [ ] . ibuprofen also reported to increases the chemosensitivity of cisplatin by decreasing the levels of heat shock protein s (hsp s) in lung cancer cells. hsp s are an important part of the cell's machinery for protein folding, and their function was associated with resistance to apoptosis [ ] . therefore, by blocking hsp s, ibuprofen increased the apoptosis by increasing the sensitivity of cisplatin. thalidomide ( ) is an immunomodulatory drug, which was originally developed as a sedative-hypnotic for the treatment of nausea during pregnancy. however, it was withdrawn due to its teratogenic effects. the drug was demonstrated whether it could be used for treating patients with refractory myeloma, because of its anti-angiogenic activity. after successful clinical evaluations, fda approved the drug for treating multiple myeloma. additionally, molecule also showed efficacy against several malignancies, including acute myeloid leukemia [ ] , myelodysplasia [ ] , and myelodysplastic syndrome [ ] . mechanistically, thalidomide binds to cereblon, which forms an e ubiquitin ligase complex, resulting in the rapid ubiquitination and proteasomal degradation of transcription factors, ikaros and aiolos [ ] . these two transcription factors are transcriptional regulators of b and t cell development [ ] . metformin ( ) is an orally available first-line drug and has been widely used for the treatment of type diabetes. the molecular mechanism of metformin involves the activation of adenosine monophosphate (amp)-induced protein kinase (ampk), a key enzyme regulating cellular metabolism. rapamycin (mtor), a gene that is involved in the survival of cancer cells is negatively regulated by ampk. metformin is also able to reduce the signals of mtor by inhibiting rag-mediated activation of mtor [ ] . in general, diabetic patients have an increased risk of several cancer types; especially diabetic women have a % risk of developing breast cancer. several studies suggested the anticancer property of drug . the drug at the dose of - mg/day has been shown to reduce the risk of cancer as well as its daily dose reduces the incidence of gastrological cancer in patients with diabetes [ , ] . several reviews and meta-analysis suggests that taking drug was associated with reduced risk of all cancer mortality in patients with diabetes [ ] . in a recent meta-analysis of several anti-diabetic drugs, it was found that the patients using drug had an overall reduced risk of cancer and decreased mortality rate by and %, respectively. whereas, the use of insulin was associated with an increased risk of cancer and mortality. the recent phase clinical trial studies using the occurrence of colorectal adenomas as a biomarker for cancer as a primary endpoint at year after intervention revealed that metformin reduced both occurrence and number of adenomas/polyps in the patients at low dosage level. methotrexate ( ) is a competitive inhibitor of dihydrofolate reductase (dhfr); a critical enzyme that involved in the synthesis of dna, rna, thymidylates, and proteins. the hydrofolate inhibitory activity of drug was responsible for its anti-leukemia activity. besides, drug was effective against a wide range of malignancies including breast, head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, and trophoblastic neoplasms [ ] . fda approved this compound in for the treatment of osteosarcoma, breast cancer, acute lymphoblastic leukemia, and hodgkin lymphoma. several reports suggest that the antitumor activity of methotrexate is also due to its ability to target inflammatory pathways. for instance, methotrexate was reported to suppress the nf-κb through the release of adenosine in cancer cells [ ] . rapamycin ( ) , also known as sirolimus, which was originally developed as an anti-fungal agent. however, drug was withdrawn due to its potent immunosuppressant and antitumor activities. mechanistically, the drug inhibits t cells and b cells by decreasing their sensitivity to il- through inhibition of mtor, which is highly upregulated in many tumor cells [ ] . in the year , fda, approved rapamycin for the prevention of allograft rejection. after that, this drug has been investigated for its anticancer properties. in recent studies, drug was reported to reduce the colony formation of leukemia progenitor cells in patients with acute myeloid leukemia [ ] . in addition to that, drug also showed efficacious in patients with imatinib-resistant chronic myelogenous leukemia through the suppression of vascular endothelial growth factor (vegf) mrna levels in leukemia cells with mild side effects [ ] . diclofenac ( ) is an acetic acid derivative of nsaid class, which has been used to treat pain and inflammatory diseases such as gout. its mode of action was believed to suppress the [ ] . in the phase ii clinical trial, it has been investigated in combination with calcitriol for recurrent prostate cancer. the results showed that the combination was well tolerated. statin family of drugs are lipid-lowering agents that inhibit the rate-limiting -hydroxy- methylglutaryl-coenzyme a (hmg-coa) reductase in the cholesterol biosynthesis pathway. statins are commonly prescribed to reduce cholesterol synthesis in patients with a high risk of cardiovascular disease. in addition to that, statins inhibit the mevalonate pathway that provides mevalonate, farnesyl, and geranyl pyrophosphate. these molecules are important for the cell cycle progression and cell proliferation, and therefore statins represent promising candidates in cancer therapeutics. in chronic myeloid leukemia cells, simvastatin ( ) [ ] . and other natural statins including mevastatin ( ) , lovastatin ( ) , and pravastatin ( ) displayed tnf-induced apoptosis through the downregulation of nf-κb mediated antiapoptotic gene products [ ] . the anti-cancer activity of statins was also investigated in animal studies, in which statins were effective in reducing the incidence and growth of tumors [ ] . several observational studies and meta-analysis supports the positive correlation of using statins with their chemopreventive effect in humans. meta-analyses revealed that the use of statins reduced the risk of patients with gastric cancer [ ] . as well as esophageal [ ] , and hepatocarcinoma cancer types [ ] . in a case-control study, drug with a dosage of mg/day for - years significantly reduced the incidence of colorectal cancer [ ] . depakine ( ) or valproic acid (vpa) is a short-chain free fatty acid mainly used to treat epilepsy, bipolar disorders, and migraine. its anticonvulsant activity has been attributed to the blockade of voltage-gated sodium channels and increased levels of gamma-aminobutyric acid (gaba) in the brain. anti-cancer activity of drug was first established in leukemia cells, in which the drug was shown to inhibit histone deacetylase (hdac) [ ] . depakine has also been found to suppress the production of cytokine and to modulate inflammatory signaling cascade. in human leukemia and human glioma cells, drug was able to suppress the production of il- and tnf-α [ ] . in prostate cancer cells, drug suppressed the il- through inhibition of nf-κb activity [ ] . some of the clinical trials of drug have advanced to phase ii for sarcomas, thyroid cancers, acute myelogenous leukemia, b cell lymphoma, breast cancer, melanoma, non-small, and small-cell lung cancers, prostate cancer, recurrent glioblastoma, and relapsed/refractory leukemia (www.clinicaltrials.gov.) recently, abdullah et al [ ] has shown that pitavastatin ( ) antagonizing the prc catalytic activity [ ] . treatment for acute myeloid leukemia (aml) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates [ ] . bromocriptine ( ) is an ergoline derivative and dopamine agonist that is used in the treatment of parkinson's disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus [ , ] . repurposing strategy handled by lara-castillo, maría carmen, et al has shown bromocriptine as a potent anti-leukemia drug that mainly targets leukemia stem cells [ ] . [ ] [ ] [ ] . typically, the drug-discovery program to develop new potent anti-viral agents and to obtain approval for clinical use takes more than years. until now, no effective vaccines or drugs are approved treat these infections, although many are in (pre)clinical development. hence, the drugs that have been used for the treatment of other diseases or disorders that may inhibit the replication of coronaviruses (covs) might be useful in an attempt to save the life of several affected patients. in a search of potential anti-viral agents against mers-cov, de wilde et al identified four drugs such as chloroquine ( ) , chlorpromazine ( ) , loperamide ( ) , and lopinavir ( ) from the screening of fda approved drugs library ( figure ) [ ] . they all were able to inhibit the replication of mers-cov in the low micromolar range. in addition to that, all four drugs inhibited sars-cov as well as human coronavirus (hcov)- e, which suggests that they could be used for broad-spectral anti-viral activity. as a mode of action, compounds [ ] . this yielded a series of hit compounds, primarily categorized as, anticancer ( , ) , antipsychotics ( ) , antidepressent ( ) and antipsychotic ( ) pathways. interestingly, co-treatment of the drug with gemcitabine, a deoxycytidine analog that is commonly used for the treatment of cancers [ , ] , showed a synergistic anti-viral effect with a minimal cytotoxic effect. this supports the hypothesis of using them in a combination therapy to treat cov diseases. dengue fever is a life-threatening disease caused by four antigenically distinct dengue virus serotypes. it became a global burden, which causes approximately million infections each year, of which around , to , people die. even though a vaccine against dengue is available, its long-term protective action against each of the serotypes of dengue virus remains yet to be determined. besides, currently, no clinically approved antiviral therapy is available to combat this virus. among the many approaches applied to identify novel drugs for dengue fever, drug repurposing gained much attention to the scientific community. several antiviral, antimalarial, antidiabetic, antihistamine, anticancer, antipsychotic, antiparasite, and anticholesteremic drugs have been repurposed to combat dengue virus infection. a recent publication by botta et al. discussed each class of drugs and its repositioning in detail [ ] . to identify novel and potent drug candidates, approved drugs such as lovastatin ( ), chloroquine ( ) , prednisolone ( ), balapiravir ( ) , and celgosivir ( ) were investigated for the proof-off concept clinical trials for dengue viral infection ( figure ). although the results showed that they were safe in patients with acute dengue, drugs failed to meet prior-defined trial endpoints [ ] [ ] [ ] [ ] [ ] . besides, two clinical trials conducted in thailand and singapore involving ivermectin ( ) and ketotifen ( ), the preliminary result was quite promising as phase study of the drug suggested a reduction in serum ns levels and body temperature [ ] . recently, malakar et al. screened various classes of fda approved drugs including aminolevulinic acid ( ) , azelaic acid ( ), mitoxantrone hydrochloride ( ), quinine sulfate ( ) and tested their ability to inhibit dengue virus (denv) replication [ , ] . figure . representative example of drugs repurposed for dengue infections. aminolevulinic acid ( ), an endogenous non-proteinogenic amino acid, and azelaic acid ( ) are used to treat skin diseases [ , ] , while mitoxantrone hydrochloride ( ) , an anthracenedione antineoplastic agent used for the treatment of leukemia [ ] . quinine sulfate ( ) is a natural compound extracted from cinchona bark, which is commercially available in -mg tablets under the brand name qualaquin. it has been widely used to treat chloroquine-resistant plasmodium falciparum [ ] . its ability to reduce the viral replication was also demonstrated against herpes simplex virus (hsv) [ ] , and influenza virus [ ] . among these four drugs investigated, the drug was found to be very effective in inhibiting the replication of denv by about % compared to untreated controls, while the others showed only moderate reduction of about %. it was very impressive that drug was able to reduce virus replication of all four serotypes of denv in three different cell lines of human origin. at the molecular level, it inhibited denv replication by reducing viral protein and rna synthesis in a dose-dependent manner. moreover, drug enhanced the expression of genes related to innate immune response. these findings suggest that the efficacy of drug for stimulating antiviral genes, which led to reduce denv replication. from another set of prescribed drug candidates ( - ) were tested against blood-stage p. falciparum cultures ( figure ) and liver-stage p. berghei [ , ] . they all showed promising antimalarial activity with ic values ranged from . µm to . nm. drugs raloxifene hydrochloride ( ) salirasib ( , figure ) is a promising cancer drug candidate inhibits isoprenylcysteine carboxyl methyltransferase (icmt), a validated target for cancer drug development. recently, salirasib and its analogs with , , -triazole were repuposed for their pontential antimalarial activity [ ] . in general, triazole derivartives are known to have potent antimalarial activity [ ] . compound were investigated the in vitro toxicity to p. falciparum in the asexual stages and in vero cells. an antiplasmodial activity assay was performed using a simple, highsensitivity methodology based on nanoluciferase (nluc)-transfected p. falciparum parasites. the results showed that some of the analogs were active at low micromolar concentration, alzheimer's is the most common type of dementia. it affects around % of people over the age of , % over the age of , and more than a third of those over the age of [ ] . in the year , there were approximately . million people worldwide with ad of which dementia resulted in about . million deaths. it is estimated that there will be more than million people with dementia worldwide by . therefore, ad represents a major and rising public health concern, and there is an urgent need to develop more therapies that are effective. ad refers to a devastating condition leading to progressive cognitive decline, functional impairment, and loss of independence. accumulation of amyloid-β peptide (aβ)-enriched neuritic plaques, and neurofibrillary tangles, synaptic, and neuronal dysfunction, as well as loss in combination with the associated neurochemical changes in the brain, are the crucial pathological event for ad [ ] . recent work suggests that higher levels of total tau may potentiate the toxic effects of aβ [ ] . other factors such as inflammatory processes and mitochondrial function are also likely to have an important role [ ] . three acetylcholinesterase inhibitors including donepezil ( ), rivastigmine ( ) and are cost-effective [ ] . for example, acetylcholinesterase inhibitors improve cognition to above pre-treatment performance for ~ - months. the availability of these drugs has substantially advanced the treatment of patients with ad, but there is a persistent need to build on our increasing understanding of disease pathogenesis to develop more effective symptomatic treatments and disease-modifying therapies. drugs. the md simulation results exposed that these five drugs (risperidone, domperidone, verapamil, tamsulosin and cinitapride) showed better profiles with respect to their rmsd, rmsf, sasa and rg evaluations graphs and steady stable behavior in all docking complexes. in-vitro ache inhibition assay of the above best-screened drugs were performed by spectrophotometric method using acetylthiocholine iodide as substrate. the enzyme inhibition and kinetic mechanism of these drugs showed that cinitapride ( ) has good therapeutic potential with respect to standard and other drugs. it is well known that, cintapride ( ) is a gastroprokinetic agent and antiulcer agent of the benzamide class [ ] . it is an agonist of -ht and -ht receptors and as an antagonist of the -ht receptors [ ] . based on aforementioned results, it is justified that cinitapride has better repositioning profile which may be used in the treatment of ad after clinical assessment. efforts to develop more effective therapies have so far been unsuccessful with several highprofile clinical trial fails to demonstrate the benefit. the reasons for this are probably multifactorial. the majority of putative disease-modifying therapies that have been evaluated have targeted amyloid pathology. this lack of breadth in treatment approaches has been criticized, and some commentators have argued that a more sophisticated knowledge of disease pathways is needed before we can develop more effective candidate therapies. for example, phase ii trial of tarenflurbil ( , figure ) only provided a suggestion of benefit in a posthoc subgroup analysis [ , ] ; the putative mechanism of action via γ-secretase modulation and its related impact on amyloid pathology was never confirmed in patients with ad. subsequent phase iii trial had negative outcomes. although, the results of a phase ii trial of dimebon ( ) were much more favorable than of analog (figure ). it seems that the significant benefit seen in the treatment group was driven by a larger-than-expected deterioration in the group receiving placebo treatment, and the mechanism of action was not well characterized. in the central nervous system (cns), angiotensin ii mediates key processes including, the release of inflammatory mediators, vasoconstriction, mitochondrial dysfunction, and inhibition of acetylcholine release at central synapses. all of which are proposed to be relevant to ad and are potential targets for therapeutic intervention [ , ] . based on this background, it has been proposed that angiotensin receptor blockers (arbs) may confer symptomatic benefits on cognition. a large-scale screen of antihypertensive drugs by want et al. [ ] , identified the arb valsartan ( , figure ) as the only compound that was able to reduce aβ accumulation in cultured neurons and inhibit aβ aggregation in vitro. the same group was demonstrated that the reduced plaque burden as well as the improved learning and memory in cognitive tests (including the morris water maze task) following months of treatment with valsartan in month-old tg transgenic mice. as a result, the greatest benefits were seen at a dose of mg/ kg/day, which is equivalent to . times the maximum recommended dose for treating patients with hypertension. using another arb, olmesartan ( , figure ) by takeda et al. [ ] , demonstrated that daily treatment of young app mice for one month improved cerebral blood flow without affecting aβ - and aβ - levels. in the aβ - -injected mouse model, in which aβ fragments are injected intracranially to generate deficits, pre-treatment with telmisartan ( , figure ) increased cerebral blood flow and inhibited the plaque deposition [ ] . however, the physiological relevance of this model is unclear. perhaps, the most striking preclinical evidence comes from a study in which losartan ( , figure ) was administered intranasally to app/psen mice, at a dose much lower than that mediating hypotensive effects; drug led to a . -fold reduction in aβ plaques compared to vehicle-treated mice but also reduced the levels of pro-inflammatory mediators and increased the levels of anti-inflammatory mediator il- in the serum of these animals [ , ] . overall, there was significant reduction in the incidence of dementia in patients taking arbs compared to those taking the comparator cardiovascular drugs (hazard ratio: . ; % confidence interval: . - . ) and those taking angiotensin-converting enzyme (ace) inhibitor lisinopril (hazard ratio: . ; % confidence interval: . - . ). although the evidence for the potential of arbs in ad is conflicting, and difficult to interpret, in our view, there is a sufficient indication of potential benefit to merit further in vivo work to clarify the relative importance of different mechanisms, the optimal dose, and the optimal agent, which could lead to proof-of-concept study in patients with ad. the best evidence from in vitro and in vivo studies points to either drug or as a preferred arb, with some animal studies also highlighting drug as a potential candidate. however, there is an unfortunate disconnect between in vivo and clinical studies, as no formal studies to provide direct clinical evidence have so far been conducted on any of the most promising candidates. calcium channel blockers (ccbs) of the dihydropyridine class are widely used to treat hypertension and angina through their vasodilatory activity on smooth muscle vasculature. most of the drugs in this class have good blood-brain barrier penetration and induces cerebral vasodilatation, increased cerebral blood flow in animals and humans [ ] . in vitro studies have revealed that certain ccbs reduce aβ production, oligomerization, and accumulation, rescue aβ-induced neurotoxicity, and improve cell survival in the presence of aβ [ ] [ ] [ ] . ccbs have also been shown to reduce glutamate-induced cell death and levels of intracellular calcium [ ] . the ability of ccbs to prevent aβ - and aβ - production was investigated in chinese hamster ovary cells (cho) by paris et al. and iwasaki et al. in that study, amlodipine ( ) and nilvadipine ( ) (figure ) were identified as the only agents that inhibited aβ production [ ] . however, the concentrations studied were several-fold higher than can be achieved therapeutically. isradipine ( , figure ) was shown to have a neuroprotective effect against aβ-induced apoptosis in neuroblastoma mg cell lines. a protective effect in a drosophila melanogaster model of aβ-induced neurotoxicity and it's brain-penetrant in the xtg-ad mouse model of ad was also reported [ , ] . the differential effects of ccbs indicate that their potential benefits in ad are probably independent of their anti-hypertensive activity and may be specific to individual drugs within this class. dihydropyridines seem to be more effective than compounds with different chemical structures (such as verapamil or diltiazem), with evidence from preclinical studies that highlighted nilvadipine ( ) as the best therapeutic candidate. there was some clinical evidence regarding the potential benefit of the ccb nimodipine ( , figure ) in patients with clinically significant dementia. the evidence has been summarized in a cochrane review of nimodipine trials in more than , patients with dementia. the review reported that the treatment showed efficacy in improving cognition, but not activities of daily living, at doses of mg/day [ ] . however, the evidence was limited by small size and duration (mostly weeks) of trials as well as the lack of operational diagnostic criteria for ad or vascular dementia. tetracycline antibiotics are widely used to treat bacterial infections and are well tolerated in older people, but most of the treatment data pertain to short-term periods of exposure. studies related to ad have predominantly focused on minocycline ( , figure ) because it is the most lipophilic tetracycline, with greater blood-brain barrier penetration than other agents in this class. concerning preclinical studies, the drug has been shown to reduce aβ - aggregation, and promote disassembly of pre-formed fibrils in vitro studies. various groups have independently shown that drug reduces the levels of proinflammatory mediators and microglial activation in a range of mouse models of ad [ ] [ ] [ ] [ ] [ ] . besides, two of the three studies using transgenic mouse models of ad for days or more of treatment have shown a significant decrease in cerebral aβ accumulation and improvements in behavioral outcomes as well [ ] . one of these studies used -month-old xtg-ad mice and reported reductions in cortical amyloid levels following months of minocycline treatment, but no changes in tau pathology was observed. the third study reported that no benefit concerning amyloid accumulation or behavior over a treatment period of months. an additional study in a rat model of diabetes demonstrated a reduction in aβ - and aβ - levels and associated improvements in behavioral outcomes over weeks of treatment [ ] . doxycycline ( ) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. compound is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in ad mice. balducci, claudia, et al. showed that -to month-old app/ps de (app/ps ) ad mice receiving under different treatment regimens recovered their memory without plaque reduction. an acute treatment was, also, sufficient to improve app/ps mouse memory, suggesting an action against soluble abos. this was confirmed in an abo-induced mouse model, where the abo-mediated memory impairment was abolished by a its pretreatment. although abos induce memory impairment through glial activation, assessing the anti-inflammatory action of , we found that in both the abotreated and app/ps mice, the memory recovery was associated with a lower neuroinflammation. our data promote as a hopeful repositioned drug counteracting crucial neuropathological ad targets [ ] . drugs that activate retinoic acid receptors (rars) are used to treat several skin-related conditions such as acne and psoriasis. retinoic acid is also vital for normal nerve function and repair. there is genomic and epidemiological evidence suggest that impaired retinoic acid signaling may contribute to the etiology of ad [ ] . chronic deprivation of retinoic acid in rats leads to deposition of aβ in the vasculature [ ] and dysregulation of amyloid processing in the cortex [ ] . it has been shown that treatment with retinoid x receptor (rxr) agonist bexarotene ( , figure ) , which is approved for the treatment of cutaneous t cell lymphoma, leads to pathological and behavioral improvements in transgenic mouse models of ad. acute treatment with drug (lasting less than days) caused a rapid reduction ( %) in aβ - and aβ - levels and aβ plaque burden in both young and old mice [ ] . chronic -day treatment resulted in a sustained reduction ( %) insoluble aβ levels. mechanistically, drug resulted in the upregulation of components of the high-density lipoprotein (hdl) pathway such as apolipoprotein e (apoe), which promotes the proteolytic degradation of aβ [ ] . further, potential mechanism of action of retinoids may include the upregulation of enzymes involved in amyloid clearance such as insulin-degrading enzyme [ ] and components of the apoe pathway [ ] . retinoids may also induce potentially beneficial changes related to insulin signaling and increased neurogenesis and promote neuronal differentiation of progenitor cells [ , ] . retinoids also act as antioxidants (by regulating sod and inhibiting glutathione depletion to reduce mitochondrial damage) as well as anti-inflammatory agents (by reducing the production of il- ) in vitro. both of these activities have potential importance in ad pathology [ , ] . therefore, there was a strong mechanistic rationale for the potential benefit of retinoid therapies beyond amyloid modulation, but there is a need to further clarify the impact of treatment on these pathways through in vivo studies. overall, studies in the literature indicate that retinoids have strong potential mechanistic plausibility as therapies for ad owing to their effects on app processing, aβ clearance, insulin signaling, and neurogenesis. out of the approved drugs, data for bexarotene have provided proof of concept as potential candidate for the treatment of alzheimer's disease as noted above, whereas acitretin ( , figure ) , which is known to penetrate tissues including brain may also be a promising candidate for ad [ ] . it is a long-term degenerative disorder of the cns, which mainly affects the motor system. as of , pd affected . million individuals, of which people died. it usually occurs people over the age of , of whom about one percent are affected. in addition to the classic motor symptoms caused by the death of dopaminergic neurons, parkinson's disease encompasses a wide range of nonmotor symptoms. although novel disease-modifying medications that slow or stop parkinson's disease progression are being developed, drug repurposing, which is the use of existing drugs that have passed numerous toxicity and clinical safety tests for new indications, can be used to identify treatment compounds. this strategy has revealed that tetracyclines ( ) are promising candidates for the treatment of parkinson's disease [ ] . tetracyclines, which are neuroprotective, inhibit proinflammatory molecule production, matrix metalloproteinase activity, mitochondrial dysfunction, protein misfolding/aggregation, and microglial activation. two commonly used semisynthetic second-generation tetracycline derivatives, minocycline ( ) and doxycycline ( ) , exhibit effective neuroprotective activity in experimental models of neurodegenerative/ neuropsychiatric diseases and no substantial toxicity. moreover, novel synthetic tetracyclines with different biological properties due to chemical tuning are now available. in this review, we discuss the multiple effects and clinical properties of tetracyclines and their potential use in parkinson's disease treatment. in addition, we examine the hypothesis that the anti-inflammatory activities of tetracyclines regulate inflammasome signaling. based on their excellent safety profiles in humans from their use for over years as antibiotics, we propose the repurposing of tetracyclines, a multitarget antibiotic, to treat parkinson's disease. isradipine ( ) is an l-type calcium channel blocker of the dihydropyridine class, which has been widely used for the treatment of high blood pressure to reduce the risk of heart attack and stroke. this drug came into medical use in the year . epidemiological data support that calcium channel blockers may have the potential to reduce the risk of developing pd. among dihydropyridines, drug has attracted very much as it inhibits the subtype cav ca + channels cav . and cav . , which are most likely mediate the risk in pd. moreover, the good brain bioavailability of drug has made it the most promising candidate for repurposing [ , ] . studies have shown that drug dose repentantly protect the dopaminergic neurons from -methyl- -phenyl- , , , -tetrahydropyridine mptp) and hydroxydopamine ( -ohda)-induced toxicity by reverting dopaminergic neurons to a latent juvenile pacemaking mechanism independent of calcium [ , ] . an open-label, doseescalation study assessing safety (steady-pd) of drug controlled release - mg/day in patients with early pd suggested that the acceptable tolerability at doses of ≤ mg per day; however at higher doses caused leg edema and dizziness [ ] . isradipine ( ) with mg being the highest dosage was again confirmed in another steady-pd-ii, a randomized, double-blinded trial, which was undertaken in subjects with early pd not requiring dopaminergic therapy [ ] . results suggest that the most common adverse effect was peripheral edema, which occurred in % of patients receiving drug ( mg). placebocontrolled phase iii clinical study to assess the efficacy of drug ( inosine ( , figure ) is a purine nucleoside, which has been used as a dietary supplement by athletes for improving aerobic performance. it has been shown to have neuroprotective roles by elevating the level of serum urate, a natural antioxidant and peroxynitrite scavenging property with potential benefits to patients with multiple sclerosis. many studies suggest that individuals with increased levels of urate in serum have a reduced risk of developing pd, as well as in patients with pd are associated with a reduced rate of disease progression. moreover, in toxin-based models of pd, increased urate levels have conferred protection against dopaminergic cell death stimulated by mptp, -ohda, and rotenone. akt-gsk- b signaling and nuclear factor (erythroid-derived- )-like (nrf ) were thought to involve in these effects. therefore, given the data supporting a neuroprotective role of urate, inosine has been repurposed in the pathogenesis of pd. the ability to raise urate level of drug in serum was demonstrated in sure-pd, a randomized, double blind, placebo-controlled in patients with early pd not yet requiring any medication. the results showed that inosine raised the mild urate level ( . - . mg/dl) or moderate urate elevation ( . - . mg/dl) after months and well-tolerated with favorable progression rate in updrs score, which amounted to ~ point per year on the total updrs scale. on the other hand, the elevated level of urate in serum have the risk of hypertension, coronary heart disease, and stroke over the long term. these side effects are potentially limiting its utility in older patients with pd. however, in patients of asian origin with pd, inosine elevated urate levels ( . - . mg/dl) without side effects after year of treatment was reported. the multicenter sure-pd trial, which involves a large number of patients ( patients), is currently underway intending to elevate the urate level to . - . mg/dl. the result of the study will be expected in the year . simvastatin ( ), since statins are also known to modulate various biological processes relevant to the pathogenesis of pd [ , ] . simvastatin has been repurposed for treating this disease. pretreatment with simvastatin preserved dopaminergic cells and motor behavior in rodents treated with -hydroxydopamine ( -ohda), promoting antioxidant protein expression or via modulation of nmda receptor and pro-inflammatory cytokine expression [ / ] . similarly, in -methyl- -phenyl- , , , -tetrahydropyridine (mptp) models, pretreatment with simvastatin suppressed activation of nf-κb, protected dopaminergic neurons, and improved the motor function [ , ] . although, stains, in general, have given encouraging preclinical studies, epidemiological data regarding the association between usage of statins and the risk of pd are unclear. moreover, the modest protective effect of statins that disappeared when adjusted for cholesterol level [ ] . however, due to the promising biochemical and pharmacological properties of simvastatin, it is currently being examined with patients having moderate-stage pd in phase ii double-blind, randomized, controlled, multicenter trial. nilotinib ( , figure ) is a selective c-abl tyrosine kinase inhibitor approved for the treatment of imatinib-resistant chronic myelogenous leukemia (cml). accumulating evidence suggests that c-abl activation has been linked in the pathogenesis of pd and other synucleinopathies. activated (phosphorylated) c-abl has been found in a high level in postmortem studies of patients with pd [ , ] . it was also reported that activation of c-abl in mice induces neurodegeneration in the hippocampal and striatal brain areas [ ] . continuous work has demonstrated that c-abl phosphorylation occurs as a result of mitochondrial dysfunction, and oxidative stress [ ] , which can promote the accumulation of α-synuclein in through effects on autophagy mechanisms [ ] and can further promote phosphorylation of parkin, causing inhibition of its ubiquitin e ligase activity, inducing mitochondrial dysfunction and dopaminergic neuronal death [ ] . all the above evidences propose that c-abl may be a promising therapeutic target in the management of pd. the cns penetration of nilotinib over other abl inhibitors has favored it to fetch more data in pd related studies. indeed, in preclinical models of pd, drug has been shown to cross the blood-brain barrier and reduces c-abl activity, ameliorating autophagic clearance of α-synuclein in transgenic and lentiviral gene-transfer models [ ] . more importantly, these effects were seen at doses far lower ( - mg/kg/day) than those used to treat cml, which is considered as the key characteristics for potential drug repurposing. furthermore, nilotinib prevented dopaminergic cell loss and motor impairments induced by mptp in mice, which were associated with inhibition of parkin phosphorylation and reduced accumulation of parkin substrate paris, thus hinting at another potential mechanism of action [ , ] . based on these preclinical data, a small, open-label proof-of-concept study was recently conducted to evaluate the safety and tolerability of nilotinib in patients with pd dementia or dementia with lewy bodies followed-up for weeks, followed by a final assessment weeks later [ ] . the necessary lowest choice of dose for the clinical study was taken as - mg. the authors reported that nilotinib was well tolerated, though one patient receiving mg was diagnosed with myocardial infarction, and two had transient qtc prolongations. there was also evidence of cns penetration with nilotinib csf plasma ratio of and % with and mg, respectively. due to numerous methodological limitations, these findings should be interpreted with caution [ ] . due to unwanted off-target nonselective tyrosine kinase inhibition; side effects of nilotinib at doses used to treat cml include cardiac conduction abnormalities. therefore, claims of tolerability should be interpreted with caution. indeed, the effects on efficacy was also highly impossible to be concluded. besides, it was also reported in the csf, none of the markers used were validated as biomarkers in pd; they can also vary greatly between patients and track poorly with disease stage and progression. this situation again raises questions about the optimum dose of nilotinib, its brain penetrance, assessments of cardiovascular effects in patients. parkinson's disease (pd) is a neurodegenerative disorder for which a greater prevalence and incidence is described in men. this suggests a protective effect of sex hormones in the brain. intellectual property related issues hinder the commercialization of repositioned molecule. in conclusion, repurposing of drugs has the potential to find and improve treatments for various diseases. the drugs listed above are only a few of plenty drugs that can be repurposed in various therapies. the drugs discussed in the perspective are summarized in table and compared to the previous uses of each drug with the repurposed use. the opportunities for drug repurposing are diverse, but a lot still has to be done for its exploration. drug repurposing or drug repositioning offers a new strategy to academic centers, research council programs, and not-for-profit organizations, as well as pharmaceutical and biotechnology companies for drug development. the main advantage of drug repurposing is the established safety of the known candidate compounds when compared to that of the development of novel therapeutic compounds. the time and cost required to advance a candidate into clinical trials can be substantially reduced because in vitro and in vivo screening, chemical optimization, toxicity studies, bulk manufacturing, and formulation development have already been completed in many cases, and can, therefore, be bypassed. moreover, these drugs have been on the market for many years, and consequently, the side effects are already known, and safety is therefore very high. for medicinal chemists, the repurposing of drugs on cancer is a rewarding job for the global healthcare system and possibly a step forward for people to get cheaper and safer medicines rather than afford for cancer therapy. however, time and minimum investment have to be spent to conduct further studies to improve the safety and success of the repurposed drugs. drug repurposing also offers the possibility to develop multi-target drugs that can interact with more than one pathway/protein at the same time. for complex diseases such as (neuro) inflammatory and degenerative disorders, the development of multi-target drugs will be an emerging area for the treatment having multiple advantages such as i) synergistic effect ii) reduced drug-resistance iii) better compliance iv) simplified pharmacokinetic and pharmacodynamic profile and a reduced risk of drug-drug interactions. on the other hand, the limitations of drug repurposing should also be considered. they involve a) technical challenges and legal requirements such as intellectual property rights which could hamper the whole process and are often hard to overcome b) serious problem on the development of resistant germs on account of consuming a drug for a variety of diseases c) owing to target selectivity, it is difficult to identify a drug that can cure or treat two different diseases on its own. nevertheless, drug repurposing is only a part of the solution for the sinking numbers of new diseases and its treatment. however, it is quite hard to replace the common way of identifying new drug candidates. in furuter, this approach could be improved in many differents ways: integrating data about repositioning drugs which are available in many public platforms, such as pubchem), the nobel chronicles a comprehensive map of molecular drug targets trends in development and approval times for new therapeutics in the united states drug repositioning identifying and developing new uses for existing drugs rebuilding big pharma's business model fda drug approvals genetic and rare diseases information centre drug repositioning: bringing new life to shelved assets and existing drugs drug repurposing: progress, challenges and recommendations a review of computational drug repurposing cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells melanins and melanosomes: biosynthesis, biogenesis, physiological, and pathological functions skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors melanogenesis-inhibitory and cytotoxic activities of triterpene glycoside constituents from the bark of albiziaprocera inhibitors of melanogenesis: a patent review recent updates in melanocyte function: the use of promising bioactive compounds for the treatment of hypopigmentary disorders inhibitors of melanogenesis: an updated review brain tyrosinase overexpression implicates age-dependent neuromelanin production in parkinson's disease pathogenesis novel tyrosinase inhibitory peptide with free radical scavenging ability tyrosinase-expressing neuronal cell line as in vitro model of parkinson's disease the reaction of alpha-synuclein with tyrosinase: possible implications for parkinson disease skaltsounis, design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors tyrosinase exacerbates dopamine toxicity but is not genetically associated with parkinson's disease degradation of tyrosinase induced by phenylthiourea occurs following golgi maturation inhibition of l-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase structural requirement(s) of n-phenylthioureas and benzaldehyde thiosemicarbazones as inhibitors of melanogenesis in melanoma b cells analogues of ethionamide, a drug used for multi drug resistant tuberculosis, exhibit potent inhibition of tyrosinase repositioning of thiourea-containing drugs as tyrosinase inhibitors pharmacovigilance and tuberculosis: applying the lessons of thioacetazone antithyroid drugs thiopurine drugs repositioned as tyrosinase inhibitors crystal structure of agaricusbisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone the unravelling of the complex pattern of tyrosinase inhibition exploring the interaction of n/s compounds with a dicopper center: tyrosinase inhibition and model studies united states cancer statistics: - incidence and mortality web based report cancer drug discovery by repurposing: teaching new tricks to old dogs systematic review update of observational studies further supports aspirin role in cancer treatment: time to share evidence and decision-making with patients? effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials aspirin, salicylates, and cancer the biology of prostaglandin synthesis and inhibition nonsteroidal antiinflammatory agents differ in their ability to suppress nf-kappa b activation, inhibition of expression of cyclooxygenase- and cyclin d and abrogation of tumor cell proliferation repositioning aspirin to treat lung and breast cancers and overcome acquired resistance to targeted therapy american society of health-system pharmacists targeting apoptosis pathways by celecoxib in cancer an international randomised trial of celecoxib versus celecoxib plus difluoromethylornithine in patients with familial adenomatous polyposis molecular mechanisms and bioavailability of polyphenols in prostate cancer inhibitory effect of ibuprofen on tumor survival and angiogenesis in gastric cancer cell ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation induced migration in melanoma cells ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein single-agent lenalidomide induces complete remission of acute myeloid leukemia in patients with isolated trisomy thalidomide potentiates etoposide-induced apoptosis in murine neuroblastoma through suppression of nf-κb activation a combination of thalidomide and arsenic trioxideis effective and well tolerated in patients with myelodysplastic syndromes how thalidomide works against cancer suppression of aiolos and ikaros expression by lenalidomide reduces human ilc -ilc /nk cell transdifferentiation metformin, independent of ampk, inhibits mtorc in a rag gtpase-dependent manner type diabetes increases and metformin reducestotal, colorectal, liver and pancreatic cancer incidences in taiwanese: a representative population prospective cohort study of , individuals the potential effect of metformin on cancer: an umbrella review cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis mir- - p modulates resistance to methotrexate in human colorectal adenocarcinoma cells by downregulating hipk anti-inflammatory therapy in chronic disease: challenges and opportunities clinical activity of mammalian target of rapamycin inhibitors in solid tumors evaluation of combination of atorvastatin with sulindac or naproxen profoundly inhibits colonic adenocarcinomas by suppressing the p /beta-catenin/cyclin d signaling pathway in rats simvastatin potentiates tnf-alpha-induced apoptosis through the down-regulation of nf-kappab-dependent antiapoptotic gene products: role of ikappabalpha kinase and tgfbeta-activated kinase- role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements statin uses and mortality in colorectal cancer patients: an updated systematic review and meta-analysis statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study statin use and the risk of colorectal cancer in a population-based electronic health records study the role of statins for primary prevention in non-elderly colorectal cancer patients statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study sodium valproate and -aza- '-deoxycytidine differentially modulate dna demethylation in g phase-arrested and proliferative hela cells exploring the drug repurposing versatility of valproic acid as a multifunctional regulator of innate and adaptive immune cells therapeutic value of voltage-gated sodium channel inhibitors in breast, colorectal, and prostate cancer: a systematic review screening a library of approved drugs reveals that prednisolone synergizes with pitavastatin to induce ovarian cancer cell death towards the first targeted therapy for triplenegative breast cancer: repositioning of clofazimine as a chemotherapy-compatible selective wnt pathway inhibitor, cancer letters a drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine acute myeloid leukaemia in adults drug reformulations and repositioning in pharmaceutical industry and its impact on market access: reassessment of nomenclature differential actions of antiparkinson agents at multiple classes of mono-aminergic receptor i. a multivariate analysis of the binding profiles of drugs at native and cloned human receptor subtypes repositioning of bromocriptine for treatment of acute myeloid leukemia the genome sequence of the sars-associated coronavirus severe acute respiratory syndrome an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy middle east respiratory syndrome-coronavirus (mers-cov): an updated overview and pharmacotherapeutics mers: south korea closes schools after third death severe respiratory disease associated with middle east respiratory syndrome coronavirus interaction of ethambutol with human organic cation transporters of the slc family indicates potential for drug-drug interactions during antituberculosis therapy screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome corona virus replication in cell culture effects of chloroquine on viral infections: an old drug against today's diseases? a systematic screen of fda-approved drugs for inhibitors of biological threat agents simulating henipa virus multi cycle replication in a screening assay leads to identification of a promising candidate for therapy pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents clathrin mediates infectious hepatitis c virus particle egress inhibitors of alpha virus entry and replication identified with as table chikungunya replicon cell line and virus-based assays mouse hepatitis virus type enters cells through a clathrin-mediated endocytic pathway independent of eps clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace with the cytoplasmic tail deleted small molecules targeting severe acute respiratory syndrome human coronavirus saracatinib inhibits middle east respiratory syndrome-coronavirus replication invitro gemcitabine: a critical nucleoside for cancer therapy synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses drug repurposing approaches to fight dengue virus infection and related diseases drug repositioning for dengue haemorrhagic fever by integrating multiple omics analyses effects of short-course oral corticosteroid therapy in early dengue infection in vietnamese patients: a randomized, placebo-controlled trial efficacy and safety of celgosivir in patients with dengue fever (celaden): a phase b, randomised, double-blind, placebo-controlled, proof-of-concept trial lovastatin for the treatment of adult patients with dengue: a randomized, double-blind, placebo-controlled trial ivermectin: a promising anti-dengue replication treatment pesented at th european congress of clinical microbiology and infectious diseases current status of dengue therapeutics research and development repurposing approved drugs on the pathway tonovel therapies. schein update on the management of rosacea: a status report on the current role and new horizons with topical azelaic acid cure of condylomaacuminata covering the glans penis using aminolevulinic acid/photodynamic therapy a phase study of azacitidine with high-dose cytarabine and mitoxantrone in high-risk acute myeloid leukemia quinine an old anti-malarial drug in a modern world: role in the treatment of malaria antiviral effects of quinine sulfate on hsv- hacat cells infected: analysis of the molecular mechanisms involved inhibition of influenza virus replication by targeting broad host cell pathways quinoline hybrids and their antiplasmodial and antimalarial activities evaluation of antiplasmodial potential of c and c modified quinolines: in vitro and in silico astemizole analogues with reduced herg inhibition as potent antimalarial compounds liver-stage malaria parasites vulnerable to diverse chemical scaffolds new leads for drug repurposing against malaria repurposing drugs to target the malaria parasite unfolding protein response repositioning salirasib as a new antimalarial agent triazole derivatives and their antiplasmodial and antimalarial activities antimalarial agents against both sexual and asexual parasites stages: structure-activity relationships and biological studies of the malaria box compound -[ -( -bromo- -chlorophenyl)furan- -yl]-n-[(piperidin- -yl)methyl]methanamine (mmv ) and analogues world alzheimer report : the global economic impact of dementia (alzheimer's disease international the amyloid cascade hypothesis for alzheimer's disease: an appraisal for the development of therapeutics dendritic function of tau mediates amyloid-β toxicity in alzheimer's disease mouse models alzheimer's disease aligning the evidence with practice: nice guidelines for drug treatment of alzheimer's disease the exploration of novel alzheimer's therapeutic agents from the pool of fda approved medicines using drug repositioning, enzyme inhibition and kinetic mechanism approaches the prokinetic cinitapride has no clinically relevant pharmacokinetic interaction and effect on qt during coadministration with ketoconazole cinitapride protects against ethanol-induced gastric mucosal injury in rats: role of -hydroxytryptamine, prostaglandins and sulfhydryl compounds treating alzheimer's disease by targeting iron tarenflurbil phase study group. effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild alzheimer disease: a randomized controlled trial peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate alzheimer-related disease the renin-angiotensin system and antihypertensive drugs in alzheimer's disease: current standing of the angiotensin hypothesis? valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of alzheimer disease angiotensin receptor blocker prevented β-amyloid-induced cognitive impairment associated with recovery of neurovascular coupling telmisartan prevented cognitive decline partly due to ppar-γ activation protective effects of intranasal losartan in the app/ps transgenic mouse model of alzheimer disease use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis favourable effects of nilvadipine on cognitive function and regional cerebral blood flow on spect in hypertensive patients with mild cognitive impairment identification of antihypertensive drugs, which inhibit amyloid-β protein oligomerization selective dihydropyiridine compounds facilitate the clearance of β-amyloid across the blood-brain barrier l-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for alzheimer's disease protective effects of ginsenoside rg against glutamate-induced neurotoxicity in pc cells selective antihypertensive dihydropyridines lower aβ accumulation by targeting both the production and the clearance of aβ across the blood-brain barrier a translational continuum of model systems for evaluating treatment strategies in alzheimer's disease: isradipine as a candidate drug nimodipine for primary degenerative, mixed and vascular dementia minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathyin vivo in transgenic mice minocycline as a potential therapeutic agent in neurodegenerative disorders characterised by protein misfolding reductions in amyloid-β-derived neuroinflammation, with minocycline, restore cognition but do not significantly affect tau hyperphosphorylation increases in β-amyloid protein in the hippocampus caused by diabetic metabolic disorder are blocked by minocycline through inhibition of nf-κb pathway activation doxycycline counteracts neuroinflammation restoring memory in alzheimer's disease mouse models evidence for defective retinoid transport and function in late onset alzheimer's disease disruption of the retinoid signalling pathway causes a deposition of amyloid β in the adult rat brain retinoic acid normalizes nuclear receptor mediated hypo-expression of proteins involved in β-amyloid deposits in the cerebral cortex of vitamin a deprived rats apoedirected therapeutics rapidly clear β-amyloid and reverse deficits in ad mouse models human apoe isoforms differentially regulate brain amyloid-β peptide clearance regulation by retinoic acid of insulin degrading enzyme and of a related endoprotease in human neuroblastoma cell lines interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth sequential rarβ and α signallingin vivo can induce adult forebrain neural progenitor cells to differentiate into neurons through shh and fgf signalling pathways all-trans retinoic acid as a novel therapeutic strategy for alzheimer's disease towards retinoid therapy for alzheimer's disease kinetics of tissue distribution and elimination of retinoid drugs in the rat tetracycline repurposing in neurodegeneration: focus on parkinson's disease expression and , -dihydropyridine binding properties of brain l-type calcium channel isoforms rejuvenation protects neurons in mouse models of parkinson's disease the l-type channel antagonist isradipine is neuroprotective in a mouse model of parkinson'sdisease tolerability of isradipine in early parkinson's disease: a pilot dose escalation study phase ii safety, tolerability, and dose selection study of isradipine as a potential disease-modifying intervention in early parkinson's disease (steady-pd) simvastatin as a potential disease modifying therapy for patients with parkinson's disease: rationale for clinical trial, and current progress prospects of statins in parkinson disease simvastatin inhibits activation of nadph oxidase/p mapk pathway and enhances expression of antioxidant protein in parkinson disease models simvastatin prevents neuroinflammation by inhibiting n-methyl-d-aspartic acid receptor in -hydroxydopamine-treated pc cells neuroprotective potential of atorvastatin and simvastatin (hmg-coa reductase inhibitors) against -hydroxydopamine ( -ohda) induced parkinson-like symptoms simvastatin inhibits the activation of p ras and prevents the loss of dopaminergic neurons in a mouse model of parkinson's disease simvastatin prevents -methyl- -phenyl- , , , -tetrahydropyridineinduced striatal dopamine depletion and protein tyrosine nitration in mice confounding of the association between statins and parkinson disease: systematic review and meta-analysis novel regulation of parkin function through c-abl-mediated tyrosine phosphorylation: implications for parkinson's disease phosphorylation by the c-abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic degradation of -synuclein in parkinson's disease models regulation of the c-abl and bcrabl tyrosine kinases a novel tyrosine kinase inhibitor amn (nilotinib) normalizes striatal motor behaviors in a mouse model of parkinson's disease, front the c-abl inhibitor, nilotinib, protects dopaminergic neurons in a preclinical animal model of parkinson's disease nilotinib effects in parkinson's disease and dementia with lewy bodies nilotinib-differentiating the hope from the hype repurposing sex steroids and related drugs as potential treatment for parkinson's disease drug repurposing: a promising tool to accelerate the drug discovery process the drug repurposing landscape from to : evolution, challenges, and possible solutions • repositioning is a promising approach for the rapid identification and development of new pharmaceuticals for rare and complex diseases key: cord- -wnbyy gk authors: liu, tingting; lu, dong; zhang, hao; zheng, mingyue; yang, huaiyu; xu, yechun; luo, cheng; zhu, weiliang; yu, kunqian; jiang, hualiang title: applying high-performance computing in drug discovery and molecular simulation date: - - journal: natl sci rev doi: . /nsr/nww sha: doc_id: cord_uid: wnbyy gk in recent decades, high-performance computing (hpc) technologies and supercomputers in china have significantly advanced, resulting in remarkable achievements. computational drug discovery and design, which is based on hpc and combines pharmaceutical chemistry and computational biology, has become a critical approach in drug research and development and is financially supported by the chinese government. this approach has yielded a series of new algorithms in drug design, as well as new software and databases. this review mainly focuses on the application of hpc to the fields of drug discovery and molecular simulation at the chinese academy of sciences, including virtual drug screening, molecular dynamics simulation, and protein folding. in addition, the potential future application of hpc in precision medicine is briefly discussed. according to moore's law [ ] , the transistor count of central processing units (cpus) increases -fold every - months. the performance of supercomputers has developed even more rapidly, and high-performance computing (hpc) has become a strategic technology worldwide [ ] . china has made remarkable achievements in the development of supercomputers, under the support of the chinese government, to which the tianhe series of supercomputers have recently made significant contributions. milky way- was designed by the national university of defense technology (nudt) in ; its appearance made china the third country to design such supercomputers. in , dawning- was developed by the institute of computing technology of the chinese academy of sciences (cas), which accelerated the independent development of chinese supercomputers. in november , tianhe- , developed by the nudt, has retained its position as the world's fastest supercomputer at the international supercomputing conference in austin, america. according to the th edition of the twice-yearly top list of the world's most powerful supercomputers, it has kept the first-place position for six consecutive times since its appearance. the national hpc environment (nhpce) has played an important role in the development of hpc applications in china. the supercomputing center of the chinese academy of sciences (sc-cas), one of china's earliest established supercomputing centres, is the main node and operating centre of the china national grid (cngrid) service environment and supercomputing environment of cas (scgrid). currently, sccas, equipped with the supercomputer era with . pflops capacity, offers excellent supercomputing services for users in china. other supercomputing centres, such as in shanghai, shenzhen, tianjin, and guangzhou, have been supported by national programmes since the th five-year plan. these supercomputer centres have more than users from all over the country, who are not only from basic review research fields such as mathematics, physics, chemistry, astronomy, mechanics, biology, medicine, and earth sciences, but are also from important industrial sectors including automotive, aviation, aerospace, nuclear power, steel, shipbuilding, civil engineering, and cartoon rendering. with the improvement of high-performance technologies, hpc and supercomputers are being applied to an increasing number of emerging fields including deep learning, big data mining, computational finance, and precision medicine, with the expectation that it will accelerate innovation. computational drug discovery and design (cddd), also known as computer-assisted drug design, has undergone significant improvements since the s. with the rapid development of molecular and structural biology, the d structures and functions of biological macromolecules have been determined. meanwhile, the application of computational methods to drug discovery and molecular simulation has increased together with the rapid advancement of computer science, powerful functions of graphic workstations, and high-performance computers. quantum mechanics, molecular mechanics, molecular dynamics (md), and combinations of these methods have been widely used in drug development. traditionally, marketing a new drug in the pharmaceutical industry is a difficult process that often costs more than one billion us dollars and takes approximately years. a series of cddd approaches based on the d structures of biological macromolecules (e.g. proteins and nucleic acids), such as the highthroughput virtual screening method, have greatly improved the efficiency of drug discovery. according to a united states government report, because of the application of cddd, drug development costs are reduced by around million us dollars and research time is shortened by a year [ ] . cddd based on hpc is a combination of pharmaceutical chemistry, computational chemistry, and biology using supercomputers, and has become a critical technology in drug research and development. many cddd techniques, such as drug virtual screening, md simulations, and molecular modelling, can be accelerated by recent technologies including grid computing, cloud computing, and accelerating hardware such as graphics processing units [ ] . grid computing, which is generally composed of several geographically distributed supercomputers, can handle massive computationally intensive tasks [ , ] , and is widely used in highthroughput virtual drug screening, thereby reducing time and cost. the drug discovery grid, a project supported by cngrid, can support massive virtual drug screening tasks [ ] . in recent decades, computational drug discovery and molecular simulation in china have significantly advanced. researchers have developed many new drug design algorithms, as well as drug design software and databases with independent intellectual property, thereby ending a situation of complete dependence on foreign commercial software. currently, the cddd field receives consistent financial support from the chinese government [ ] . in addition, more than universities and research institutions all over the country have established departments for computational drug discovery. the shanghai institute of materia medica (simm) at cas has developed and compiled a series of drug design algorithms and programs, some of which possess thousands of registered users that are currently implanted on supercomputers and are widely used worldwide. for example, the molecular docking software groupdock, developed by simm, has completed parallelization on domestic supercomputers, which has reached hundreds of thousands of cpu cores, allowing virtual screening of large-scale databases in a short time. besides, the development of groupdock is based on the ucsf dock. it mainly deals with the massive docking tasks and optimization of parallelization on china-made hpc systems such as shenwei. it otherwise performs nearly the same as ucsf dock. the software has become a drug research and development platform based on nhpce, which promotes innovational drug discovery and development. with the help of chinese supercomputers, simm has achieved many things. simm/cas has established the advanced platforms of innovative drug research and development ( fig. ) . during the implementation of china's th and th five-year plans, biomedical information and diverse key technology platforms were preliminarily constructed and perfected. these platforms included drug design and screening, preclinical pharmacodynamics of novel drugs, and pharmacokinetics and drug safety evaluations. in addition, computational genomics, computational proteomics, computational regulation networks, and computational drug design are playing an increasing number of important roles in the preclinical stages of the drug development pipeline. most such computation is computationally intensive and requires the support of hpc supercomputers. by applying hpc, these technology platforms are able to produce biomedical big data that include massive amounts of pharmacological, pharmacokinetic, and pharmacogenomic information with increasing speed. understanding how to better utilize these data (e.g. huge amounts of public information including patents) and improving our understanding of the complex biological processes underlying drug action and the efficiency of drug discovery are important issues to address for the future of drug development. currently, simulation and d structure prediction of protein folding, as well as active compound discovery against targets from current compound libraries, have become important directions for drug discovery. the simulation accuracy and enrichment rate of virtual drug screening, to a large extent, depend on computational performance. a supercomputer on the exascale is required for a microsecond simulation of a system containing millions of atoms. innovative drug-related computational genomics, proteomics, network interaction, and drug design technologies demand mass hpc resources. these critical technologies rely on the development of the next generation of exascale high-performance computers. it is exascale computing that will be in urgent demand for personalized drug development in the next - years. this review mainly focuses on the application of hpc to the field of drug discovery and molecular simulation at cas in recent years, including several cases involving virtual screening (molecular docking), md simulation, and protein folding. potential prospects for the application of hpc to precision medicine are briefly discussed. in most cases, the goal of drug discovery is to search for potent inhibitors against a target, using high-throughput screening or virtual screening. the former approach is more reliable, but is expensive and time consuming, whereas the latter is a trade-off between cost and accuracy via the technology of high-performance computers. thus, highthroughput screening is widely used against wellestablished targets, mainly in the laboratories of pharmaceutical companies, whereas virtual screening is primarily applied against new or unconfirmed targets, mainly in the laboratories of academic institutions, to discover potent inhibitors and to explore the mechanism of these unconfirmed targets. within a typical workflow of virtual screening, as shown in fig. , a compound database including hundreds of thousands or even millions of small molecule entries is first screened against the designated target protein, via the method of molecular docking. usually, molecular docking takes a few seconds to minutes on modern cpus considering only the flexibility of small molecules and keeping the protein rigid. thus, virtual screening against a target consumes a great amount of computational resources that only supercomputers can provide. next, thousands of compounds with the best docking scores are kept for structural clustering. within the clustered compound list, about candidate compounds are finally selected for subsequent validation. the criteria for manual selection are drug-like properties and binding modes generated by molecular docking. to validate the potency of these candidate compounds, various methods are employed, including enzymatic activity assays, enzymatic binding assays, enzymatic selectivity assays, structureactivity relationship studies, and cell assays. recent review successful applications of virtual screening in our lab are briefly introduced below. protein arginine methyltransferases (prmts) are important for chromatin remodelling, signal transduction, and rna metabolism. various diseases, such as cancers and cardiovascular disorders, are associated with the aberrant activity of prmts. through the combination of virtual screening and radioactive methylation assays, several hits were identified as inhibitors with micromolar potency against prmt . among these hits, two compounds showed even higher potency than the well-known prmt inhibitor ami- . they achieved inhibition by directly targeting substrate h rather than prmt . one of these two compounds also significantly inhibited the proliferation of castrate-resistant prostate cancer cells [ ] . the dna methyltransferases (dnmts) found in mammals are dnmt , dnmt a, and dnmt b, which are attractive targets in cancer chemotherapy. the first dnmt to be characterized, dnmt , is responsible for maintaining the dna methylation pattern of the genome. most of the existing dnmt inhibitors are nucleoside analogues that can cause toxic side effects. through a combination of docking-based screening and biochemical assays, a novel non-nucleoside dnmt inhibitor named dc was identified, which inhibits dnmt at low micromolar concentrations and has remarkable selectivity against other similar methyltransferases. after similarity-based searching, analogues dc and dc showed even higher potency than dc . all three compounds significantly inhibited the proliferation of two cancer cell lines [ ] . polycomb repressive complex (prc ) modulates the structure of chromatin and transcriptional repression by trimethylating lysine of histone h (h k me ), which is necessary for the proteinprotein interaction between the catalytic subunits ezh and eed. aberrant prc activity has been broadly implicated in cancer initiation and progression, making it a promising target for cancer therapy. through a combination of docking-based virtual screening and biochemical assays, an fdaapproved drug, astemizole, was identified as an inhibitor against the ezh -eed interaction of prc . this work highlighted the therapeutic promise of treating prc -driven human cancers via targeted disruption of the ezh -eed complex [ ] . methicillin-resistant staphylococcus aureus (mrsa) is the principal cause of hospital-acquired infection, which manifests as an infection at the site of surgery, bacteraemia, and sepsis. frequently, prophylaxis of mrsa infection with antibiotics fails or leads to nosocomial diseases such as clostridium difficile infection, due to drug resistance. sortase a is a transpeptidase that anchors surface proteins in the envelope of s. aureus, and mutant sortases are unable to cause bacteraemia or sepsis in mice. through virtual screening and structural optimization, a series of inhibitors were identified that blocked the activity of sortase in vitro and in vivo. these inhibitors protected mice against lethal s. aureus bacteraemia without affecting staphylococcal growth in vitro. thus, these sortase inhibitors may act as an anti-infection therapy to protect high-risk patients from hospital-acquired s. aureus infection, without incurring the side effects of antibiotics [ ] . as a rich source of compounds for drug discovery [ ] , natural products, especially traditional chinese medicines (tcm), have been attracting attention in recent years. various in silico methods have been applied in tcm studies to identify potential natural ligands for targets [ ] . as an example, the case of molecular docking discovered that sieboldigenin can bind to the pocket of soybean lipoxygenase (slox) with an ic of μm [ ] . artemisia annua (qinghao) was discovered as an inhibitor of sars-covm pro (coronavirus main proteinase) by the combination of molecular docking and fingerprint studies [ ] . besides, several natural product databases based on tcm are available for virtual-screening campaigns, such as universal natural product database, chinese natural product database and ismart [ ] . as the central technology of computer-aided drug design, molecular docking is not only applied in virtual screening for drug discovery, but also aids with target identification. over the past few years, simm has participated in research into target identification with international colleagues using this technology. the pleiotropic lipid mediator sphingosine- phosphate (s p) can act independently of its membrane receptors through an intracellular mechanism. sphingosine kinase (sphk ), one of the isoenzymes that produce s p, is associated with histone h and regulates histone acetylation via s p, which specifically binds to the histone deacetylases hdac and hdac to inhibit their enzymatic activity [ ] . on the other side, fty (fingolimod), an fda-approved drug for the treatment of multiple sclerosis, has beneficial effects in the central nervous system that are independent of its effects on immune cell trafficking, although the mechanism is not currently well understood. fty is phosphorylated by sphk in the nucleus, after which it binds and inhibits hdacs, enhancing specific histone acetylation. having identified the possible binding mode of fty within the pocket of hdac via molecular docking, as shown in fig. , it was concluded that fty may be a useful adjuvant therapy to facilitate the extinction of aversive memories by targeting hdacs [ ] . irf is essential for the il- -induced expression of the chemokines cxcl and ccl , which recruit mononuclear cells to the sites of sterile inflammation. once produced, irf acquires lys (k )-linked polyubiquitination mediated by the inhibitor of apoptosis ciap , which is enhanced by the bioactive lipid s p. in response to the stimulation of il- , ciap and the sphingosine kinase sphk (which are responsible for the production of s p) form a complex with irf , leading to its activation. after confirmation that s p directly binds to ciap , molecular docking was used to explore the binding mode of s p with ciap [ ] . tumour necrosis factor (tnf) receptorassociated factor (traf ) is a key component in the nf-κb signalling cascade triggered by tnf-α. traf binds to sphk , which is one of the isoenzymes that produce s p, a prosurvival mediator in cells. sphk and the production of s p are necessary for lysine- -linked polyubiquitination of rip and nf-κb activation. with the molecular docking of s p into the pocket of traf , this work highlighted the important role of sphk and its product s p in tnf-α signalling and the canonical nf-κb pathway in inflammatory, anti-apoptotic, and immune processes [ ] . drug targets are commonly associated with a desirable therapeutic effect or an unwanted adverse effect [ ] . the identification of potential ligand-protein interactions is helpful for predicting therapeutic benefits or the side effects of drug-like molecules. target prediction and identification is of high significance to drug discovery and research. however, it is a laborious and time-consuming task if undertaken by experimental approaches alone. thus, there is an urgent need to develop efficient computational methods that can detect the targets of chemical compounds. recently, li et al. [ ] developed a reverse ligand-protein docking programme, tarfisdock, to search for potential ligand-protein interactions through the screening of the potential drug target database. the identification of the enzyme peptide deformylase (the target of natural product n-trans-caffeoyltyramine) in helicobacter is regarded as a successful application of tarfisdock [ ] . liu et al. proposed a reverse pharmacophore mapping strategy, via which a web server called pharmmapper was established to predict potential drug targets [ ] . with the exponential growth of bioactivity data, it has become increasingly difficult to handle the large-scale interaction data of small molecules and their targets via traditional computational strategies. thus, target prediction can be considered a big data problem. liu et al. [ ] developed a ligandbased target fishing method based on d fingerprint similarity rankings with data fusion, which is suitable for large-scale drug target prediction because of its simple algorithm and fast calculation speed. a ligand-target reference library was established that could be updated over time with the increasing availability of bioactivity data. this library currently contains drug targets and active ligands, of which each target has its own ligand set. based on the large reference library, the similarity fusion scores between a query molecule and each ligand set were calculated as the corresponding target scores of this molecule, using the tanimoto coefficient [ ] of ecfp fingerprints [ ] . finally, the top targets ranked by fusion scores were considered as the potential targets of the query. a -fold cross validation was adopted to evaluate the performance of the similarity fusion schemes or knn fusion. similarity ensemble approach (sea) [ ] , a statisticsbased chemoinformatics approach, was also tested in parallel with our fusion schemes for comparison. it was demonstrated that knn fusion performed better than sea on a reference set and that nn outperformed the most. the target prediction accuracy of the nn fusion strategy would be improved with the increasing size of the reference set, which shows that the strategy could be applied to better deal with the problem of big data on active ligands. on the two external test sets compiled from drugbank and in order to further verify the effectiveness of the approach on practical cases, target prediction was made for nine drugs withdrawn from the market because of herg toxicity [ ] using the nn fusion scheme. in this test, herg was identified in the top target lists of of drugs, and for terfenadine, sparfloxacin, and droperidol, their interactions with herg were ranked in first place. furthermore, the therapeutic targets of nine drugs were all in the top . these results demonstrate that the nn fusion scheme performs well on the prediction of both therapeutic targets and off-targets. to make this method more convenient, a userfriendly web server was developed, called tarpred [ ] , for the prediction of therapeutic benefits and side effects of query molecules. tarpred is freely accessible at http://www.dddc.ac.cn/tarpred. for chemicals queried by users, tarpred accepts both graphical and smiles format input. after the calculation, which takes only - minutes, the top interacting targets ranked by nn score were listed. tarpred also provides detailed information, such as bindingdb and drugbank names, gene ids and predicted diseases. the disease information associated with targets was extracted from the comparative toxicogenomics database and integrated into the web application. the information provided by tarpred is useful for understanding the functional mechanisms and safety profiles of bioactive ligands. entecavir and salvianolic acid b were selected as case studies to validate the effectiveness of tarpred, and the results produced successfully predicted their targets. as a quick and low-cost method, computer-aided target identification plays an important role in the prediction of new therapeutic targets and potential toxicity for biologically active compounds, which has drawn an increasing amount of attention in recent years. the in silico tool tarpred is useful and meaningful for target prediction in several cases. moreover, it can be applied as a prefilter for drug development and could provide some crucial guidance to drug repurposing. several online web services for target prediction in china are summarized in table . several of these web services are supported by hpc servers, which can make the computation time to reduce radically. in addition, on the platform of the tianhe- supercomputer, simm has performed a number of calculations regarding drug repositioning (old drugs for new uses) using large-scale virtual screening. early warning and response to newly emerging infectious diseases before they become pandemic, which is an important part of the national emergency response system, requires the support of hpc. the period of a week to a month after the emergence of a new infectious disease is the most critical time, during which there is usually an absence of effective drugs and vaccines. however, the development of new vaccines takes at least to months, and the traditional process of developing new drugs requires - years. fortunately, drug discovery based on hpc systems can reduce these time periods and boost social confidence in the handling of epidemic situations. for example, in a situation where only small molecule flexibility is considered alongside lower calculation precision, and by employing all the computational resources of tianhe- , virtual screening of almost all known compounds on the world can be finished within day. if protein flexibility is review liu et al. considered with higher calculation precision, the current computational power should be elevated by one or two orders of magnitude. that is, exascale calculation can meet the demand. in the research and development of drugs against major malignant infectious diseases, drug virtual screening becomes more useful due to time limitations and the fact that related experiments cannot be carried out directly. therefore, upon the sudden outbreak of a new pandemic, the discovery of effective drugs against new diseases from a list of current drugs by applying high-performance computers is an efficacious way of responding to new infectious diseases. we have successfully fought against the sars [ ] [ ] [ ] [ ] [ ] [ ] and influenza viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] by employing cddd approaches and hpc. during the discovery of anti-sars agents, an in silico virtual screening approach was applied to against the small molecules database. as a result, several compounds were discovered as potential drug leads, which offered positive measures against sars [ , , ] . cddd approaches also played an important role in the development of the famous drug tamiflu, which is now used against the avian influenza viruses. in the design of drugs against avian influenza viruses, virtual screening was performed by using multiple compound libraries (acd, cnpd, specs) with more than compounds, predicting more than potential neuraminidase inhibitors. computer-aided structural modification and drug synthesis was performed, and nearly analogues of oseltamivir and zanamivir were designed and synthesised. a total of neuraminidase inhibitors of avian influenza were discovered with enzyme activity similar to that of zanamivir and effective in vivo. these inhibitors possess independent intellectual property rights, of which have satisfactory activity. several further lead compounds with innovative structures, independent intellectual property rights, and activities similar to zanamivir (which can act against the virus at a viral level) have been acquired. furthermore, drug candidates for preclinical studies have been determined. therefore, it is evident that virtual screening based on hpc systems can remarkably improved the efficiency of drug discovery and drug compound optimisation. recently, the virtual screening platform md dockxb [ ] was developed by the combination of nudt, simm, and the national supercomputer center in guangzhou. it is a many integrated core enabled version of d dockxb [ , ] . the authors accelerated the lamarckian genetic algorithm deeply, and achieved × to × sppdup. based on such platform, simm carried out a virtual screening project on the ebola virus on tianhe- . using all of tianhe- 's resources, a virtual screening task with more than small chemicals can be completed within day. the combination of hpc and high-efficiency platforms that enabled our country has the ability to deal with the super-high-throughput screening tasks of handling the acute infectious diseases. with constant improvements in both computer power and algorithm design, md simulation has become a powerful research tool for drug discovery over the last few decades. md simulation calculates the time-dependent behaviour of a molecular system and examines the dynamics of atomic-level phenomena that cannot be observed directly. by exploiting the full power of current hpc platforms on a given scientific problem through effective utilisation of all available resources (cpus, memory, and in many cases, input/output (i/o)), md simulation exerts an ever growing role in the life sciences and drug discovery. in the following section, we will discuss our application of md simulation with the help of three case studies. just as a single photograph of a runner tells little about her stride, the static models produced by nmr and x-ray crystallography provide limited information. meanwhile, md simulation can deliver an ensemble of conformations, which promote understanding of the dynamic processes underlying receptor activation [ , ] . through μs md simulations on the apo glucagon receptor (gcgr) and glucagon-bound gcgr performed using gromacs . . package [ ] , we recently discovered that full-length gcgr, a potential drug target for type diabetes [ ] , has two conformations: an open conformation stabilized by the peptide glucagon and a closed conformation in apo-gcgr, in which the extracellular domain (ecd) forms extensive contacts with the seven transmembrane ( tm) domain (fig. a) . the two conformations are very different from the full-length gcgr-glucagon model constructed using crystal structures [ ] (fig. a) . this was the first report of the putative closed state of gcgr at the atomic level, consistent with hydrogen/deuterium exchange (hdx) results and validated by disulphide cross-linking studies. gcgr belongs to the secretinlike (class b ) family of g-protein-coupled receptors (gpcrs) [ ] in humans, which are activated by endogenous hormonal peptides and play causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. as there are several conserved structural features and ligand interaction hotspots in secretin-like class b gpcrs, the relative movements and interaction dynamics of the ecd and tm domain of full-length gcgr provide useful information to guide the design of new experiments to investigate class b gpcr structure-function relationships. in addition, the protein motions offered by md simulation provide essential information for the identification of druggable allosteric sites that are not visible in static structures. previous studies have shown that the voltage-sensor domain (vsd) of shaker and kv . - . chimaeric channels, which are members of voltage-gated potassium (kv) channels, is occluded [ ] [ ] [ ] [ ] [ ] , as shown in our simulation on the kv . that there were internal and external water crevices (fig. b) . however, in the simulation of the kcnq channel, another member of kv channels and an anti-epilepsy target, a cavity filled with water molecules appeared in the vsd (fig. b) . as the volume of this space is ∼ Å , we guessed that a potential-binding pocket was located in the kcnq vsd. based on the docking model of ztz (a kcnq activator reported previously [ ] ) to the kcnq homology model, one md simulation on the ztz /kcnq complex was conducted by using gromacs . . package [ ] , which provided a structural binding model for ztz . further mutagenesis and electrophysiological studies validated this activator-binding pocket in the vsd of kcnq [ ] (fig. b) . subsequent structure-based virtual screening targeting the identified pocket discovered nine activators with five new chemotypes, two of which exhibited significant anti-epilepsy activity [ ] (fig. b) . these results demonstrate the capability of the kcnq vsd to accommodate small chemical ligands. the new functionality of the gating charge pathway offers new insights into the therapeutically relevant kcnq channel. biomacromolecules sharing high-sequence identity, as well as similar structures, play distinct roles in pathology. beside subtle differences in themselves, the environment they belong to and their interactions with their surroundings, such as small molecules, contribute to their different functions. because md simulation enables us to investigate atomic interactions at a very detailed level, it can be successfully applied to identify and predict the differential regulation of highly homologous receptors. as an example, we will first show its successful application to predict interactions of phosphatidylinositol- , -bisphosphate (pip ) with the kcnq channel. pip is a phospholipid component that is enriched at the plasma membrane, and a substrate for a number of important signalling proteins including the kv channels [ ] [ ] [ ] [ ] . though pip alters the physiological function and pharmacological selectivity of kcnq channels [ ] [ ] [ ] , the effects and interactions of pip on kcnq channels are not well understood. to predict the interactions of pip with the kcnq channel, ns allatom md simulations on both the open and closed conformations of the kcnq channel were performed using gromacs . . package [ , ] . liu et al. simulations with two different force fields yielded similar pip interactions: pip preferentially interacted with k in the s -s linker of the openstate kcnq channel, whereas it contacted k and other positive residues in the s -s loop of the closed state (fig. c) . these interactions were different from the pip -shaker and pip -kv . interactions [ ] . consistently, the effects of pip on kcnq were also different relative to the shaker and kv . channels. the results indicate the diversity of pip effects on kv channels. the mechanism we proposed for the action of pip on the kcnq channel is consistent with the reported subtype sensitivity of pip to kv channels [ , ] and indicates that pip may exert specific effects on kcnq channels [ ] . thus, md simulation is an effective method with which to analyse protein-lipid interaction. md simulation has also been successfully applied to identify cholesterol binding for a variety of gpcrs [ ] [ ] [ ] [ ] . of interest are the two major β-adrenergic receptor subtypes β ar and β ar, which share % sequence identity and are highly similar in structure but have distinct and sometimes opposing effects in both cardiovascular physiology and the pathogenesis of heart failure [ , ] . because β ar and β ar are subtypes specifically compartmentalized on cholesterolenriched caveolae or lipid rafts, two μs md simulations of β ar and β ar embedded in a : palmitoyl- -oleoyl phosphatidylcholine-cholesterol mixture membrane were performed using the gro-macs . . package [ ] to compare the potential cholesterol-binding sites for β ar and β ar. big differences were observed in the cholesterol interaction sites between β ar and β ar, which are relevant to cholesterol-mediated dimerisation and the allosteric regulation of receptor activity [ ] . our md simulations provide valuable clues regarding cholesterol binding to β ar and β ar and shed light on general determinants of cholesterol binding to gpcrs. thanks to the efficient computational resources of the national supercomputing center in jinan, the national supercomputing center in tianjin (tianhe- a) and the tianhe research and development team of the nudt (tianhe- ), the abovementioned studies have been accomplished. because the biological systems studied using all-atom md simulations can be very large, comprising millions of atoms, the calculations required are often too complex and computationally intensive for even the best supercomputers. it is clear that there is room for improvement. steady growth in hpc has made md simulation spans wider spatial and temporal ranges and resolutions, which are likely to play an increas-ingly important role in the development of novel pharmacological therapeutics. large-scale conformational changes of proteins are not only related to the biological functions of the proteins, but are also associated with the binding of ligands or drugs. md simulation has emerged as a practical approach for exploring protein dynamics but is still unequal as a generally applicable strategy to explore large-scale protein conformational transitions. considering the structure of the new generation supercomputer, we developed a new approach, called numd, to deal with largescale protein conformational movement, which is a combination of normal mode analysis and umbrella sampling md simulation. this new approach can produce thousands of the protein conformations along the conformational transition pathway, and each conformation can be simulated simultaneously with thousands of cpus. therefore, compared with other widely used md approaches including namd, gromacs, and amber, the new code is specifically suitable for hpc to delineate atomically detailed conformational transition pathways and the associated free energy landscapes. its application on three well-known protein systems (adenylate kinase (adk), calmodulin (cam), and p α kinase) in the absence and presence of their respective ligands revealed that the predicted conformational transition pathway and thermodynamic observables were in good agreement with experimentally and computationally determined ones (fig. ) . thus, this simple approach offers a generally applicable strategy by using hpc for probe intrinsic or ligand-induced largescale protein conformational transitions and the associated free energy landscape [ , ] . protein folding is the physical process by which a polypeptide folds into its d structure, from a random coil to its characteristic protein functional shape or conformation. the correctly folded protein structure or conformation is essential to the protein's biological function. a failure to fold into the native structure may produce inactive proteins, but misfolded proteins may also have modified or toxic functionality. for example, aggregated proteins are associated with illnesses such as alzheimer's disease (ad) and parkinson's disease. md simulation is a good tool for the study of protein folding and dynamics in silico. ad is a progressive, irreversible review figure . schematic representations of numd application on three well-known protein systems (adenylate kinase (adk), calmodulin (cam), and p kinase). the 'apo' represents the ligand-free conformation, 'complex' means ligand-bound state. the order parameter d rmsd is defined as the difference in rmsd values of each structure from the reference starting and final states [ ] . neurological disorder and the most frequent cause of senile dementia. two pathologically characterized hallmarks of ad are the appearance of extracellular senile plaques and intracellular neurofibrillary tangles [ , ] . the major constituents of the plaques are amyloid β-peptides (aβ) of - amino acids, which are produced by the sequential action of βand γ -secretases on the amyloid precursor protein [ ] . aβ and aβ are the two most abundant aβ isoforms, with the only difference found at their c-termini, where the latter has two more hydrophobic residues. it has been demonstrated that aβ is more neurotoxic than aβ , and that an increased aβ /aβ concentration ratio correlates with the onset of ad [ , ] . extensive experimental and theoretical studies exploring the structures of monomeric as well as aggregated aβ have revealed remarkable molecularlevel insights, including a key point at which the structural feature associated with different morphologies of aβ are distinct [ ] [ ] [ ] [ ] . this implies that the peptides are able to adopt a diverse set of conformations in which the anti-parallel or parallel β-sheet is often the key structural element. there is a dire need for structural studies of aβ monomers and aggregates at the atomic level, to answer crucial questions such as when and how the β-sheet structure is formed and why aβ aggregates more rapidly and severely than aβ . in view of the high propensity for self-assembly, md simulations, complementary to the standard tools of structural biology such as nmr and x-ray diffraction, appear to be a useful tool with which to obtain atomic level insights into the structures as well as the conformational changes of aβ, a prerequisite to the development of more specific drugs with optimal affinities for aβ monomers or toxic oligomers. we performed multiple parallel md simulations with a total of ns on both the wild-type peptide and its mutant [ ] . in aqueous solution, an αhelix to β-sheet conformational transition of aβ was observed and a completed unfolding process of the peptide from helix to coil was traced by our md simulations. structures with few short β-sheets and helices were identified as intermediate states in the unfolding pathway of aβ (fig. ) . four tandem glycines (g , g , g , and g ) at the cterminal of aβ were found to account for the short β-sheet formation. further mutation of these glycines to alanines almost abolished the β-sheet formation and increased the content of the helix component instead (fig. ) . in the lipid bilayer of dipalmitoyl phosphatidylcholine, the major secondary structure of aβ is helical; however, the peptide tends to exit from the hydrophobic region and lie down on the surface of the bilayer. overall, these results provide an annotation of conformational transition in relation to the dynamic process of aβ exiting from the membrane and entering into aqueous solution. it is beneficial to understand the underlying molecular mechanism of aβ aggregation and design compounds that can inhibit the formation of oligomers as well as amyloid fibrils (fig. ) . the α-helix/β-sheet intermediate structure revealed by our simulations has a core domain constituted by the segment of residues - , of which four residues (gly , gly , gly , and gly ) are essential to β-sheet formation. chemical compounds that lock the structure of aβ into the αhelix/β-sheet intermediate state could possibly inhibit aβ fibrillation. targeting this intermediate structure, a virtual screening based on molecular docking was performed with the goal of discovering small molecule inhibitors that interrupt the formation of the pleated β-sheet structures involved in amyloid fibrils via binding to the intermediate structure [ ] . one of the docked compounds, dc-ab , was shown to effectively inhibit the aggregation and fibrillation of aβ by thioflavin t fluorescence assay, atomic force microscopy, and polyacrylamide gel electrophoresis analysis. meanwhile, cd spectroscopy measurement revealed that the βsheet component of aβ increased in the presence of dc-ab , suggesting that the binding of dc-ab to aβ stabilized the β-sheet structure of the peptide. taken together, our study not only demonstrated that the α-helix/β-sheet intermediate structures revealed by the aforementioned simulations could be used as a binding target for inhibitor design, but also provided new insights into the molecular events involved in the conformational transition of aβ peptides in fibrillogenesis. subsequent structure optimization of dc-ab was performed and nine compounds with higher potency were obtained [ ] . in particular, one of the nine new compounds not only suppressed the aggregation of aβ but also dissolved the preformed fibrils in vitro. cellular assays revealed that it has no toxicity to neuronal cells. moreover, it can effectively inhibit aβ -induced neurotoxicity and increase cell viability. therefore, based upon the intermediate structure of aβ revealed by md simulations, virtual screening together with further structure optimization has resulted in a lead compound for the development of a drug against ad. characterizing the conformation dynamics of monomeric aβ and aβ is a prerequisite to comprehending the aβ self-assembly pathway and the differences between the two peptides. in a recent study, we set out to investigate the conformation dynamics of aβ and aβ by exploring the impact of intramolecular interactions on conformation dynamics using equilibrium md simulations [ ] . our μs-scale simulations revealed heterogeneous conformation ensembles of aβ and aβ that encompassed ∼ % β-strand and ∼ % unstructured coils. two conformational states were identified in both isoforms: a collapsed state (cs) that resembles the structural motif of face-to-face hydrophobic clustering in amyloid fibrils, and an extended state (es) that carries the structural feature of anti-parallel βsheets observed in amyloid oligomers. this observation is in line with previous nmr studies. in aβ , the c-terminus remains unstructured and rarely interacts with other parts, therefore, the hydrophobic clustering is loosely structured and the peptide assumes an es with high probability. in contrast, the c-terminus of aβ adopts a β-strand structure that strongly interacts with segments e -r and v -a . the active associations of this extra β-strand lead to a more compact hydrophobic collapse and prevent the isoform from forming the es. based on our structural characterization, we propose that the aggregation begins in the cs, and that the propensity of the various isoforms to self-associate into a particular form is encoded in their equilibrium conformational states. as our simulation results demonstrate that the probability of aβ staying in cs is significantly higher than that of aβ , the enhanced aggregation propensity of the former could result from the consequent lowered energy barrier for nucleate formation. besides, a greater resemblance to fibril structure is observed in the cs of aβ , including increased occupancy of the β-strand at v -a and i -m and compact hydrophobic collapse. these structural advantages could also contribute to the effectiveness of nucleation. precision medicine is also known as 'p medicine' [ ] , which refers to predictive, personalized, preventive, and participatory medicine. owing to the rapid pace of human genome research and highthroughput technologies, personalized medicine is expected to become a new paradigm of future healthcare [ ] . personalized medicine will be likely to affect society in many aspects [ ] . radical changes to medical practice and the pharmaceutical industry will be involved. currently, personalized medicine is leading the third wave of drug discovery. the united states introduced a 'precision medicine initiative' in early , in order to promote the development of personalized medicine, hoping to lead a new era of medication and healthcare. the rapid development of supercomputers will play a significant role in the development of 'precision medicine', and will greatly strengthen the computation and data analysis ability of genomics. under the vigorous promotion of highperformance computers, precision medicine based on genomics and personalized drug research and discovery should greatly improve therapeutic effects, by employing personalized accurate treatment of 'the remedy to the case'. the discovery and validation of functional genes and targets in the early stages of drug research and development lead discovery and optimization, and the platform of drug virtual screening and design must be based on hpc infrastructure. with rapid improvement of the computing technologies, the expense of genome sequencing has been dropping [ ] . besides, more and more information technology (it) has been applying to biomedicine, which makes patients achieve more health information. the process of personalized drug development requires the integration of medical data, as well as biological information, from diverse omics levels including dna, rna, proteins, networks, cells, metabolites, and tissues. more than just the integration of biomedical information, it also involves the regulation networks of multiple genes and targets. in other words, computational genomics, computational proteomics, computational regulation networks, and computational drug design play crucial and indispensable roles in the process of personalized medicine discovery. however, significant computation resources are required for the process. hpc infrastructure will play an increasingly important role in dealing with such tasks. big data in biomedicine, which refers to the increasing availability and explosive growth of medi-cal data and biological information, provides an opportunity for future biomedical research and personalized medicine studies [ ] . it also requires a new generation of hpc architectures and diverse technologies, data mining systems including omics information, inscription data, clinical data, and even financial data. in conclusion, in the age of precision medicine and big data, opportunities and challenges exist together. hpc systems are increasingly essential components, which can compute, analyse, and integrate raw data efficiently, because of the large size of most biomedical data. in order to make personalized medicine more efficient and accurate, the tools and algorithms for integrating and analysing biomedical data need to be developed and improved [ ] . cramming more components onto integrated circuits molecular dynamics-based virtual screening: accelerating the drug discovery process by highperformance computing new drug development: science, business, regulatory, and intellectual property issues cited as hampering drug development efforts the cloud and other new computational methods to improve molecular modelling the emerging role of cloud computing in molecular modelling ddgrid: a grid with massive drug virtual-screening support computational methods for drug design and discovery: focus on china pharmacophore-based virtual screening and biological evaluation of small molecule inhibitors for protein arginine methylation identifying novel selective non-nucleoside dna methyltransferase inhibitors through docking-based virtual screening astemizole arrests the proliferation of cancer cells by disrupting the ezh -eed interaction of polycomb repressive complex enabling the 'host jump': structural determinants of receptor-binding specificity in influenza a viruses the re-emergence of natural products for drug discovery in the genomics era in-silico studies in chinese herbal medicines' research: evaluation of in-silico methodologies and phytochemical data sources, and a review of research to date anti-inflammatory activities of sieboldogenin from smilax china linn.: experimental and computational studies virtual screening for finding natural inhibitor against cathepsin-l for sars therapy regulation of histone acetylation in the nucleus by sphingosine- -phosphate active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory k -linked polyubiquitination of transcription factor irf is essential for il- -induced production of chemokines cxcl and ccl sphingosine- -phosphate is a missing cofactor for the e ubiquitin ligase traf drug repositioning for personalized medicine tarfisdock: a web server for identifying drug targets with docking approach pharmmapper server: a web server for potential drug target identification using pharmacophore mapping approach silico target fishing: addressing a "big data" problem by ligand-based similarity rankings with data fusion on the properties of bit string-based measures of chemical similarity extended-connectivity fingerprints relating protein pharmacology by ligand chemistry drug therapy: drug-induced prolongation of the qt interval tarpred: a web application for predicting therapeutic and side effect targets of chemical compounds drar-cpi: a server for identifying drug repositioning potential and adverse drug reactions via the chemical-protein interactome idrug: a web-accessible and interactive drug discovery and design platform a d model of sars cov cl proteinase and its inhibitors design by virtual screening putative hapn receptor binding sites in sars cov spike protein nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a in vitro biochemical and thermodynamic characterization of nucleocapsid protein of sars severe acute respiratory syndrome coronavirus c-like proteinase n terminus is indispensable for proteolytic activity but not for enzyme dimerization. biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations techniques used for the discovery of therapeutic compounds: the case of sars neuraminidase pharmacophore model derived from diverse classes of inhibitors qsar analyses on avian influenza virus neuraminidase inhibitors using comfa, comsia, and hqsar why are oseltamivir and zanamivir effective against the newly emerged influenza a virus (a/h n )? recent advances in neuraminidase inhibitor development as anti-influenza drugs synthesis of c- -modified zanamivir analogs as neuraminidase inhibitors and their anti-aiv activities structure-based design and synthesis of c- -and c- -modified analogs of zanamivir as neuraminidase inhibitors computational assay of h n influenza neuraminidase reveals r k mutation reduces drug binding affinity induced opening of influenza virus neuraminidase n -loop suggests an important role in inhibitor binding binding interaction of quercetin- -betagalactoside and its synthetic derivatives with sars-cov cl(pro): structureactivity relationship studies reveal salient pharmacophore features md dockxb: a deep parallel optimized software for molecular docking with intel xeon phi coprocessors a quantum mechanics-based halogen bonding scoring function for protein-ligand interactions target flexibility: an emerging consideration in drug discovery and design exploring the role of receptor flexibility in structure-based drug discovery gromacs : algorithms for highly efficient, load-balanced, and scalable molecular simulation targeting the glucagon receptor family for diabetes and obesity therapy structure of the human glucagon class b g-protein-coupled receptor structural diversity of g protein-coupled receptors and significance for drug discovery mechanism of voltage gating in potassium channels histidine scanning mutagenesis of basic residues of the s segment of the shaker k+ channel a proton pore in a potassium channel voltage sensor reveals a focused electric field transmembrane movement of the shaker k+ channel s the orientation and molecular movement of a k(+) channel voltage-sensing domain isoform-specific prolongation of kv (kcnq) potassium channel opening mediated by new molecular determinants for drugchannel interactions the gating charge pathway of an epilepsyassociated potassium channel accommodates chemical ligands crystal structure of the mammalian girk k+ channel and gating regulation by g proteins, pip , and sodium structural basis of pip activation of the classical inward rectifier k+ channel kir . pip is a necessary cofactor for ion channel function: how and why? regulation of ion transport proteins by membrane phosphoinositides impaired kcnq -kcne and phosphatidylinositol- , -bisphosphate interaction underlies the long qt syndrome channelopathies linked to plasma membrane phosphoinositides phosphatidylinositol , -bisphosphate alters pharmacological selectivity for epilepsy-causing kcnq potassium channels dynamic pip interactions with voltage sensor elements contribute to kcnq channel gating pip controls voltagesensor movement and pore opening of kv channels through the s -s linker molecular analysis of pip regulation of herg and ikr pip( ) activates kcnq channels, and its hydrolysis underlies receptor-mediated inhibition of m currents identification of cholesterol binding sites in the serotonin a receptor predictions for cholesterol interaction sites on the a a adenosine receptor structural and dynamic effects of cholesterol at preferred sites of interaction with rhodopsin identified from microsecond length molecular dynamics simulations a role for direct interactions in the modulation of rhodopsin by omega- polyunsaturated lipids opposing effects of beta( )-and beta( )-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensitive g protein subtype-specific beta-adrenoceptor signaling pathways in the heart and their potential clinical implications cholesterol-beta ar interaction versus cholesterol-beta ar interaction mapping central alpha-helix linker mediated conformational transition pathway of calmodulin via simple computational approach exploring transition pathway and free-energy profile of large-scale protein conformational change by combining normal mode analysis and umbrella sampling molecular dynamics alzheimer's disease: the amyloid cascade hypothesis the amyloid hypothesis of alzheimer's disease: progress and problems on the road to therapeutics a portrait of alzheimer secretases-new features and familiar faces an increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta app ) mutants visualization of a beta ( ) and a beta in senile plaques with end-specific a beta monoclonals: evidence that an initially deposited species is a beta ( ) amyloid beta protein and alzheimer's disease: when computer simulations complement experimental studies recent progress in understanding alzheimer's beta-amyloid structures solid-state nmr studies of amyloid fibril structure polymorphism in alzheimer abeta amyloid organization reflects conformational selection in a rugged energy landscape conformational transition of amyloid betapeptide inhibitor discovery targeting the intermediate structure of beta-amyloid peptide on the conformational transition pathway: implications in the aggregation mechanism of beta-amyloid peptide structural optimization and biological evaluation of substituted bisphenol a derivatives as betaamyloid peptide aggregation inhibitors varied probability of staying collapsed/extended at the conformational equilibrium of monomeric abeta and abeta predictive, personalized, preventive, participatory (p ) cancer medicine promise of personalized omics to precision medicine personalized medicine: revolutionizing drug discovery and patient care big data in biomedicine applications of high performance computing in bioinformatics, computational biology and computational chemistry we thank the sccas, the shanghai supercomputer center, the national supercomputer center in tianjin, and the national supercomputer center in guangzhou for supporting our virtual screening and dynamic simulation projects. key: cord- -aqgauumt authors: csete, joanne title: united states drug courts and opioid agonist therapy: missing the target of overdose reduction date: - - journal: nan doi: . /j.fsiml. . sha: doc_id: cord_uid: aqgauumt • the united states (us) has witnessed dramatic increases in opioid-overdose mortality since . the need for ready access to scientifically sound treatment for opioid use disorder (oud) is urgent, but gold-standard agonist therapy is often inaccessible. • drug courts should help to reduce barriers to oud therapy by offering court-supervised treatment as an alternative to criminal prosecution for certain categories of drug offenses. • many drug courts, however, reject including the opioid agonist methadone and partial agonist buprenorphine as treatment options, often because these medicines are seen as “another form of addiction”. • many us drug courts have steered participants to extended-release naltrexone (xr-ntx), an opioid antagonist, sometimes offering only xr-ntx and no agonist therapies. xr-ntx is very expensive in the us market and also requires a period of abstinence before it can be initiated. • drug courts overall have had limited success in improving access to oud treatment and other health and social services needed to achieve significant reduction in overdose death. • the united states (us) has witnessed dramatic increases in opioid-overdose mortality since . the need for ready access to scientifically sound treatment for opioid use disorder (oud) is urgent, but gold-standard agonist therapy is often inaccessible. • drug courts should help to reduce barriers to oud therapy by offering court-supervised treatment as an alternative to criminal prosecution for certain categories of drug offenses. • many drug courts, however, reject including the opioid agonist methadone and partial agonist buprenorphine as treatment options, often because these medicines are seen as "another form of addiction". • many us drug courts have steered participants to extended-release naltrexone (xr-ntx), an opioid antagonist, sometimes offering only xr-ntx and no agonist therapies. xr-ntx is very expensive in the us market and also requires a period of abstinence before it can be initiated. • drug courts overall have had limited success in improving access to oud treatment and other health and social services needed to achieve significant reduction in overdose death. the united states witnessed a six-fold increase in overdose deaths linked to opioids from to (scholl, seth, kariisa, wilson & baldwin, ) . from to inclusive, there were over , overdose deaths (all drugs) in the us (national institute on drug abuse, ). in and , almost % of overdose deaths were linked to opioids of some kind, with fentanyl more and more present in us drug markets since (ibid.). in , drug-related mortality per capita in the us was about times that of the european union (european monitoring centre for drugs and drug addiction, ). drugand alcohol-related deaths and suicides, characterized collectively by case and deaton ( ) as "deaths of despair", increased so markedly since that the decades-long upward trend of life expectancy in the us was interrupted. covid- has also obviously upset historical mortality trends in the us, but drug overdoses quietly claimed lives over a longer period and often in parts of the country not in the media spotlight. the lamentable story of how the us came to this level of drug-related mortality has been well told elsewhere (mcgreal, ; beletsky, ; president's commission on combating drug addiction, ) . at this writing, litigation continues against opioid manufacturers and distributors accused of having distorted science to persuade physicians that opioids prescribed for chronic pain would not be addictive if used properly (leung, macdonald, dhalla & juurlink, ) . government regulation to limit this deceptive marketing was largely not to be found. extraordinary profits were made by unscrupulous "pill mills" often run by persons with no medical training, where people could get lengthy opioid prescriptions without even having to see a doctor (mcgreal, ) . when these operations were finally shut down, it was often without any measures to ensure that those living with opioid dependence would have access to treatment. it is not surprising that many of these persons found their only recourse in unregulated street drugs with high overdose risk. in the face of such a crisis, it might be supposed that policy-makers would do everything possible to reduce overdose risk, including improving access to the several authorized forms of proven treatment for opioid use disorder (oud). this strategy indeed was among those recommended by a federal commission on the overdose problem, which highlighted the need particularly to remove policy and health insurance barriers to treatment services (president's commission on combating drug addiction, ). but, as of , it was estimated that only about % of people with oud in the us received any treatment for that condition in the previous months (substance abuse and mental health services administration, ). methadone maintenance, used to treat oud in the us since the s, remains out of reach to many who might benefit from it due to historical entrenchment of an array of barriers (mcelrath, ) . methadone is extremely heavily regulated in the us and administered almost exclusively in stigmatizing stand-alone opioid treatment programs (otp) where people are required to queue daily to be observed swallowing their medicine. take-home doses are relatively rare and mobile programs practically nonexistent, though that may be changing in a limited way because of covid- (substance abuse and mental health services administration, ). in addition, many of the parts of the us most heavily affected by overdose mortality have few otps as local residents have often objected to having these services "in our backyard". buprenorphine, especially combined with naloxone, is in theory more available as take-home doses from the doctor's office, but there are too few physicians who choose to prescribe it, and it is often not covered by the health insurance that is available to people with oud (mcelrath, ; breen & fiellin, ) . thus, people in much of the us who seek agonist therapies that are the gold standard for treatment of opioid use disorders encounter very high thresholds at the door. the period of dramatic increase in overdose mortality in the us coincides with a period of equally dramatic expansion of an important institution on the drug scene -specialized drug treatment courts (hereinafter "drug courts"). drug courts are meant to offer some persons accused of drug offenses an alternative to criminal prosecution (and incarceration) in the form of an extended period of courtsupervised treatment for drug use disorders. the first us drug courts appeared in the late s. by there were drug courts, by there were , and as of january there were over drug courts in the country, present in all states (franco, ; us department of justice, ). the non-partisan congressional research service noted that the rapid expansion of drug courts may be seen as a "movement" in that it happened largely in the absence of empirical evidence of the effectiveness of the courts (franco, ) . drug courts were intended as a response to high rates of federal and state-level incarceration, including for non-violent drug offenses, especially in the s and early s with the passage of laws imposing "mandatory minimum" prison sentences for a wide range of drug infractions (ibid.). the idea was to select as drug court participants persons accused of non-violent offenses where it was judged that drug dependence was an underlying factor in the offense, though the selection criteria of the courts vary considerably. the federal department of justice notes that in contrast to the adversarial "defense vs. prosecution" approach in regular us courts, drug court proceedings are meant to be managed by a nonadversarial team of "judges, prosecutors, defense attorneys, community corrections officers, social workers and treatment service professionals" (us department of justice, ). drug court participants agree to abide by the rules of the court, which normally include frequent drug testing by urinalysis and sanctions for failure to adhere to treatment (us department of justice, ). in most us drug courts, participants are required to plead guilty to the charges before them as a condition of drug court participation. the guilty plea may be removed from their records if they complete the drug court program, but it also may work against them if they fail to "graduate" from drug court (franco, ) . drug courts are not uniform in their policies or practices. judges and their management staff have a great deal of discretion, including about the types of drug treatment on offer. the federal department of justice (doj) in collaboration with the national association of drug court professionals (nadcp), an ngo, has established some standards for the functioning of the courts, but they are voluntary as the federal government has no direct authority over county and municipal courts (us department of justice, ). among the "key components" set out by the doj are that the courts "provide access to a continuum of alcohol, drug, and other related treatment and rehabilitation services" (ibid.). there is no way to know whether there is a true "continuum" of services offered in the drug courts across the country at a given moment. the congressional research service estimated in that drug courts required from to months (average about year) of court supervision with greatly varying options of in-patient and out-patient treatments on offer (franco, ) . drug courts in theory should be exactly what is needed to help reduce barriers to treatment and thus to reduce overdose risk. among other things, diversion from prison in the us means diversion from a setting where evidence-based treatment of oud is unlikely. unfortunately, in the midst of an overdose crisis, many drug courts systematically reject the gold standard of agonist therapy with methadone or buprenorphine as an option for court-supervised treatment. a national survey of drug courts found that although virtually all of the courts reported having participants with opioid use disorders, only % offered agonist therapy as an option for court-supervised treatment (matusow, dickman, rich, fong, dumont, hardin et al., ) . of the judges and drug court managers surveyed, % said, with respect to methadone, that it was not allowed simply as a policy of the court. the lack of knowledge of the science of agonist therapy among respondents to this survey was striking. for example, in spite of decades of evidence from all over the world that well-supervised methadone maintenance reduces relapses, % of the respondents said that it does not reduce relapses or they were not sure it would do so (ibid.). only % of respondents could state with certainty that methadone maintenance therapy should not interfere with the ability to drive a car. but perhaps more telling were the attitudes reflecting moral judgments about people with oud. only % of respondents disagreed with the statement that methadone "rewards criminals for being drug users". attitudes about buprenorphine were slightly less judgmental but still reflected a poor understanding of the clinical importance of this therapy. there has not been a more recent nationwide survey of the drug courts on this subject, but there have been numerous ngo and media reports of persistent refusals of many drug courts to offer agonist therapy. an investigation in three states by the ngo physicians for human rights ( ) found that in addition to shortages of methadone and buprenorphine providers in some jurisdictions, the rejection of agonist therapy in some courts reflected the judge's attitude that people should not be dependent on these medications -that they should "start clean and stay clean" in their court-supervised therapy. this study also found, as others have (csete & catania, ) , that drug court judges often approved treatment plans that did not accord with recommendations of health professionals. in pennsylvania, the policy of the drug court serving one of the largest cities, pittsburgh, is that the court is meant to help people "live as sober and clean a lifestyle as they can," which means no methadone (melamed & purcell, ) . the rejection by drug court judges and administrators of agonist therapies has a long history, dotted with tragic human stories. in , a drug court judge in california ordered a participant, bradley moore, to stop methadone abruptly, saying that anyone who wanted to stop being a drug addict should "choose not to be addicted to methadone" (hora, ) . two months later, mr. moore died from a heroin overdose. the case led to an amendment of the state penal code, specifying that people receiving methadone from a licensed otp could not be excluded from drug court only on those grounds (ibid.). at the annual conference of the nadcp, a large majority of those in attendance registered their agreement to the statement "taking methadone is trading one addiction for another" (ibid.). arbitrary rejections of gold-standard treatment by the drug courts have not escaped the notice of the federal drug authorities. the federal substance abuse and mental health services administration (samsha) announced in that federal grants for drug courts would no longer be available to courts that deny participation to eligible persons because of their use of medication-assisted treatment for oud where that treatment is authorized by a physician (samhsa, ) . for a sense of the resources behind this policy, it may be noted that in fiscal year , an estimated us $ million was granted by the federal government to drug courts (sacco, ) . the samhsa order was a step forward for agonist therapy availability in the drug courts, but many drug courts receive significant state, county and municipal funding and are thus not reliant on federal support. since , however, several states have passed laws meant to ban arbitrary or blanket exclusion of people in agonist therapy from drug court participation [physicians for human rights, ; kitchenman, ) . another barrier to opioid agonist therapy in many drug courts has been the pushing aside of methadone and buprenorphine in favor of the opioid antagonist naltrexone and especially the injectable extendedrelease naltrexone (xr-ntx) marketed under the brand name vivitrol®. since it is not an opioid and has no euphoric effect, xr-ntx appeals to drug court personnel who see methadone as "just another addiction". alkermes, the us-based firm that manufactures vivitrol, has successfully pursued the unusual strategy of marketing this product directly to drug court judges and managers, bypassing medical professionals (macgillis, ) . thanks partly to having found a receptive market in drug courts, the value of alkermes' stock went from $ . billion in to over $ billion in (ibid.). many people can benefit from an antagonist such as xr-ntx, which should be part of the "continuum" of treatment that drug courts are meant to offer. but it should not be offered at the expense of proven agonist therapies. and there are other concerns for those steered to xr-ntx by drug courts, including cost. the price tag of $ per monthly injection of vivitrol is many times the cost of agonist therapy. in addition, xr-ntx requires a period of complete opioid abstinence before treatment can be initiated, which one set of experts called a significant limitation to the clinical utility of the treatment (jarvis, holtyn, subramaniam, tompkins, oga, bigelow et al., ) . peer-reviewed studies have moreover generally reported poor adherence -on average about half of patients dropping off the treatment in the first six months (ibid.). compared to agonist therapies, the evidence base for xr-ntx is quite thin. alkermes was widely criticized for seeking regulatory approval of vivitrol based on one study conducted in the russian federation, where comparisons with agonist treatments were not possible since they are illegal, and for not following patients for overdose risk in that study, given that overdoses after detoxification with other forms of naltrexone have been well documented (wolfe, carrieri, dasgupta, wodak, newman & bruce, ) . later work has suggested elevated overdose risk associated with xr-ntx and the need for more research on this point (binswanger & glanz, ) . the warm reception that vivitrol has had among drug courts underscores the prejudice against agonist therapy harbored by some drug court personnel and the inclination of some to ignore science in favor of moral judgments. one pro-xr-ntx drug court judge expressed his delight at having been approached by the alkermes salesperson, noting, "i'm certainly not going to do a medication-assisted program with drugs that get people high" (harper, ) . the investigative media outlet propublica reported that in hundreds of drug courts across the country, vivitrol is the only medication on offer and even some where those who refuse to take it are excluded from drug court (macgillis, ) . thanks to aggressive marketing and lobbying by alkermes, some states have written vivitrol into their drug court statutes or regulations, creating "vivitrol courts" (wolfe, ) . as noted by daniel wolfe of the open society foundations, these decisions show ideological rather than science-based decision-making: "anyone who says there is one answer to addiction treatment and one medicine is probably not motivated by the evidence" (macgillis, ) . in contrast to the national survey results noted above in which % of drug court personnel believed that methadone "rewards" people for drug use or were not sure whether it does so, in a more recent study in the state of indiana only % of drug court personnel believed that xr-ntx could be seen as a reward for drug use (andraka-christou, nguyen, bradford, & simon, ) . even if an evidence-based continuum of therapies for opioid use disorder were offered by drug courts, there is good reason to question whether these courts could usefully address the overdose crisis. another example of drug courts' rejection of scientifically sound approaches is the practice of many courts to punish drug relapse with jail time. a meta-analysis found that courts included in the study reduced the number of times people were incarcerated compared to controls but did not significantly reduce overall time in custody, largely because of jail time imposed as a punishment for "failing" treatment (sevigny, fuleihan & ferdik, ) . given that people are required to plead guilty, being returned to the regular adversarial criminal justice system after failing drug court may mean that they receive a harsher sentence than if they never had been in drug court (melamed & purcell, ) . drug courts are criticized on many other grounds as well. in some jurisdictions it is not clear that everyone in drug court is actually in need of treatment, or at least not in need of the treatments on offer. treatment providers in new york told physicians for human rights ( ) that people brought into drug court because of cannabis offenses would be placed into extended residential treatment that they did not need, taking up rare treatment beds. in the state of delaware, an investigation using urinalysis in addition to self-reports found that about one third of the drug court participants in the study were not drug-dependent and not in need of treatment (dematteo, marlowe, festinger & arabia, ). drug courts have often been accused of selecting participants to favor those most likely to succeed in treatment or those most able to pay for the lengthy therapy (sevigny, pollack & reuter, ; tyler, ) . drug court premises typically display dramatic stories of individuals whose lives are transformed for the better through court-supervised drug treatment, complete with striking "before and after" photographs. some people plainly benefit from the discipline or structure of treatment provided through the courts, and some courts provide a reasonable range of proven treatment options. in united nations meetings, the us points to its drug courts as proof that the country is treating drug dependence primarily as a public health problem rather than as a crime (csete & wolfe, ) . but the public health benefit of these courts is questionable, especially with regard to the urgent need in the country for access to scientifically sound treatment for opioid use disorders. the drug policy alliance, a prominent ngo in the us drug policy reform scene, concludes that drug courts make the criminal justice system more punitive, not less: "drug courts have adopted the disease model of addiction but continue to penalize relapse with incarceration…." (drug policy alliance, ). advocates have decried drug courts as a "softer form of criminalization" at a time when it is imperative to find ways to deal with drug dependence through the health and social sectors (tyler, ) . expansion of drug courts may simply "widen the net" of the criminal justice system and not facilitate access to health and social services. what has occurred in the drug courts mirrors what occurred in the larger society to make the us an outlier among nations in drug-related mortality. that is, one sees in the drug courts the results of aggressive and scientifically unsound marketing of a profitable product, as well as failure of regulatory and health authorities to ensure that proven treatments are made available to all who need them and are overseen by persons with medical credentials. it is unlikely that the us will make significant inroads in reducing overdose mortality without finding more truly public health-based measures to get people with drug use disorders out of the criminal legal system and rather on a path to the health and social support they may need. the author has no competing interests to declare. assessing the impact of drug courts on provider-directed marketing efforts by manufactures of medications for the treatment of opioid use disorder america's favorite antidote: drug-induced homicide in the age of the overdose crisis potential risk window for opioid overdose related to treatment with extended-release injectable naltrexone buprenorphine supply, access and quality: where we have come and the path forward deaths of despair and the future of capitalism methadone treatment providers' views of drug court policy and practice: a case study of new york state seeing through the public health smoke-screen in drug policy outcome trajectories in drug court: do all participants have drug problems? drug courts are not the answer: toward a health-centered approach to drug use drug-related deaths and mortality in europe drug courts: background, effectiveness, and policy issues for congress to grow market share, a drug maker pitches its product to judges trading one drug for another? extended-release injectable naltrexone for opioid use disorder: a systematic review letting drug court participants use methadone reflects changing approach a letter on the risk of opioid addiction the last shot. propublica medication assisted treatment in us drug courts: results from a nationwide survey of availability, barriers and attitudes medication-assisted treatment for opioid addiction in the united states: critique and commentary. substance use and misuse american overdose: the opioid tragedy in three acts the probation trap. philadelphia inquirer number of national drug overdose deaths involving select prescription and illicit drugs (excel document) president's commission on combating drug addiction and the opioid crisis federal support for drug courts: in brief drug and opioid-involved overdose deaths -united states do drug courts reduce the use of incarceration? a meta-analysis can drug courts help to reduce prison and jail populations? grants to expand substance abuse treatment capacity in adult and family drug courts key substance use and mental health indicators in the united states: results from the national survey on drug use and health (hhs publication no. pep - , nsduh series h- ) covid- and opioid treatment programs criminal justice reformers are hooked on drug courts; they should kick the habit. the hill drug courts (fact sheet) defining drug courts: the key components concerns about injectable naltrexone for opioid dependence vivitrol offers the fantasy of being drug-free, but that's the not most important thing in tackling addiction • the united states (us) has witnessed dramatic increases in opioid-overdose mortality since .the need for ready access to scientifically sound treatment for opioid use disorder (oud) is urgent, but gold-standard agonist therapy is often inaccessible.• drug courts should help to reduce barriers to oud therapy by offering court-supervised treatment as an alternative to criminal prosecution for certain categories of drug offenses.• many drug courts, however, reject including the opioid agonist methadone and partial agonist buprenorphine as treatment options, often because these medicines are seen as "another form of addiction". • many us drug courts have steered participants to extended-release naltrexone (xr-ntx), an opioid antagonist, sometimes offering only xr-ntx and no agonist therapies. xr-ntx is very expensive in the us market and also requires a period of abstinence before it can be initiated.• drug courts overall have had limited success in improving access to oud treatment and other health and social services needed to achieve significant reduction in overdose death. key: cord- -j y oxu authors: ozdemir, vural; williams-jones, bryn; glatt, stephen j; tsuang, ming t; lohr, james b; reist, christopher title: shifting emphasis from pharmacogenomics to theragnostics date: journal: nat biotechnol doi: . /nbt - sha: doc_id: cord_uid: j y oxu what will be the role of theragnostic patents in upstream and downstream biomarker research? what will be the role of theragnostic patents in upstream and downstream biomarker research? p harmacogenomics aims to identify the genetic basis of variability in drug efficacy and safety, and ultimately develop diagnostics that can individualize pharmacotherapy. theragnostics, a term denoting the fusion of therapeutics and diagnostics, is receiving increasing attention as pharmacogenomics moves to applications at point of patient care. in contrast to pharmacogenomics, theragnostic tests focus not on a singular marker set, such as genetic polymorphisms, but rather on the integration of information from a diverse set of biomarkers (e.g., genomic, proteomic, metabolomic). although it remains to be seen whether theragnostics reflects a form of hyperbole in biomarker research, it is grounded in both established (e.g., genomics) and exploratory (e.g., metabolomics) technologies that can offer, respectively, mechanistic and heuristic insights for therapeutics ( fig. ) . recent social science analyses suggest that in some cases, bio-hype can be an integral component or driver for the establishment of biotechnologies, particularly in the early stages of development of a new concept or idea [ ] [ ] [ ] [ ] [ ] . the synthesis of both types of technologies (established and exploratory) will likely have a differential impact on the regulation and economic promise of pharmaceuticals developed under the overarching theme of theragnostics. moreover, we suggest that advances in this field may shape, in potentially unexpected ways, the pursuit of theragnostic patents depending on whether the research is conducted in an upstream drug discovery-oriented context or towards downstream point-of-care applications , . as biomarker applications move towards point-of-care to individualize drug therapy, a number of qualitatively different concerns arise relating to gene patents and ethical and therapeutic policy aspects of theragnostic testing , , . for example, a fear is that theragnostic tests could be adopted without regard for the particular research context in which they are being applied, or be understood as a homogeneous category, a ubiquitous set of biotechnologies with similar implications for therapeutic policy , [ ] [ ] [ ] . in effect, this may result in a predicament where the patents on biomarkers are conceptualized in a one-size-fits-all manner (as in drug prescriptions) thereby precluding the equitable implementation of emerging biomarker technologies and informed critique of their societal implications. additionally, the effects of theragnostic patents on 'knowledge commons'-that is, a space (usually in universities) where knowledge is shared without undue restriction-have not been adequately evaluated - . in the present analyses, we 'unpack' and contrast the motivations at play that are driving the pursuit for theragnostic patents and its bioethical corollaries in: ( ) fundamental upstream basic research oriented to the discovery of genes for human diseases; and ( ) downstream clinical applications at point-of-care as theragnostic tests to stratify patient populations for individualization of pharmacotherapy. we emphasize the need for integration, as well as the risk for excessive compartmentalization, of various biomarker technologies, and evaluate the subtle distinctions between dna-and protein-based theragnostic tests. the search for genetic determinants of common complex human diseases reflects one of the pivotal upstream applications of pharmacogenomics. as dna samples are being increasingly archived in pharmaceutical clinical trials, a sizable proportion of research resources are devoted to identifying the genes causally related to human diseases . for many rare congenital monogenic human diseases (e.g., duchenne muscular dystrophy), pre-or postnatal cytogenetic analysis of chromosomes or conventional clinical chemistry tests have existed for several decades. more recently, highthroughput genomic technologies spun off from the human genome project (hgp) and the decreasing cost of genotyping have made possible the adoption of molecular genetic tests that can pinpoint the precise etiology or predisposition for a few adult-onset human diseases such as huntington disease, certain familial forms of breast cancer, and alzheimer disease . while genetic tests hold the promise of a more rational clinical forecast and management of disease risk in the future, they are also raising concerns about the provision of public healthcare services (reduced access) and the impact of market forces on the products of research (commercialization of technologies) and academic freedom. some of these concerns have crystallized around the issue of commercial genetic testing for disease risk, as exemplified by the case of testing for hereditary breast cancer (brca testing) . in the mid- s, two genes (brca & brca ) that greatly increase the risk for hereditary breast cancer were identified and sequenced. the brca genes were given broad patent protection in the us (and subsequently internationally in canada, europe, australia, etc.), and granted to the biopharmaceutical company myriad genetics (salt lake city, ut), which had been involved in much of the initial research. this strong intellectual property protection has allowed myriad to effectively control the brca testing market in the us; the commercial bracanalysis test is available only from myriad or their licensees. most recently, myriad has licensed their test to san franciscobased dnadirect, in order to provide services direct to consumers . basic research into the function of the brca genes or resulting proteins would be permissible without infringing on myriad's patent rights, although there is still contention about what precisely constitutes basic research exclusions . myriad has, for example, signed agreements with the us national institutes of health and national cancer institute to provide sequencing at cost (us $ , ) for research purposes . however, research that results in a commercial or clinical service, defined by myriad as any research in which a fee is charged for testing or in which results are provided to patients, would infringe on their patents. notably, technology assessment research by third parties, for example to evaluate test performance metrics such as sensitivity, specificity, or positive predictive value, is particularly jeopardized. moreover, research aimed at comparing the bracanalysis test against other testing methodologies would prove difficult, as the clinical nature of such a trial would constitute an infringement on myriad's patents. hence, the brca patents give myriad the ability to constrain research-oriented applications of brca patents and particularly head-to-head comparisons of which genotyping methodology or test product is most informative for clinical management of the susceptibility to breast cancer. in part due to myriad's broad patent rights and the attendant concerns to ensure patients' access to affordable genetic testing for breast cancer risk, the european brca patents have been constrained or not enforced in recent years , . in some sense the myriad case may be thought of as an extreme scenario, and one from which industry and governments have learned. one specific consequence is that the initial enthusiasm for granting such broadly defined upstream patents on any and all forms of biological material has waned due to concerns for public good and scientific progress. more generally, there is a growing awareness that patents on genes and other biological materials can have an unfavorable effect on downstream genetics research and knowledge commons , . thus, without an adequate conceptual framework on patents relating to theragnostic technologies, upstream patents on potential drug target genes or genetic methodologies for molecular definitions of human diseases (as in familial breast cancer) may lead to a monopoly on theragnostic tests. this may also dissuade some research laboratories from investigating otherwise potentially promising lines of inquiry for technology transfer towards downstream theragnostic products in the clinic . the myriad patent case remains relevant since, in part, the current trends at the us patent and trademark office (uspto), and in patent offices in other countries, to grant more narrowly defined patents on biological materials were driven by this case . according to some commentators, "no other event has had as big an impact on the human gene patent debate…and the case [myriad] has thus become a 'harbinger' of the policy challenges created by gene patents" . more recent examples also support the idea that upstream theragnostic patents can limit translational applied clinical research. for instance, the sars-associated coronavirus genome patents were filed by the us centers for disease control (cdc) and the british columbia cancer agency (bcca) [ ] [ ] [ ] , officially to ensure continued public access to the viral genome and pre-empt other entities from exerting restrictive or controlling rights. notably, it is suggested that this type of patent is symptomatic of what is wrong with the [gene patent] system, when pre-emptive patents have to be filed to protect science and the public good , . a more worrying example is illustrated by the australian company genetic technologies' patents on the non-coding regions of the human genome. formerly conceived to be 'junk dna' , the biological role of non-genic segments of the genome is receiving increasing attention, and their patenting may significantly constrain the free design of primers for pcr analysis of coding regions of the genome , . a common thread in these two recent examples, however, is that broad theragnostic patents, particularly those granted on dna and other biological materials, may serve as tollbooths that impede downstream research or discourage competition and innovation due to the broad exclusive rights granted to an individual scientist, institution or company (e.g., as in the case of myriad). this 'anticommons' effect of upstream theragnostic patents is now increasingly being recognized. patents with a broad scope may actually enclose the 'knowledge-commons' and inhibit technology transfer and development at a societal or macro level (i.e., as a contrast from the viewpoint of an individual investigator), such that the promises of new diagnostics and therapeutics are not realized , , . pharmacogenomic-guided drug development represents a fundamental conceptual departure from conventional 'one-size-fits-all' clinical tri- figure hierarchy of biomarkers and their integration into theragnostic tests, from gene sequence (upstream or static marker) to downstream (dynamic) markers on gene and protein expression or cellular metabolites. a theragnostic profile is depicted as a synthesis of various biomarker tests that characterize an individual patient and her/his drug treatment outcome. the theragnostic profile may be heuristic in nature when only a singular biomarker is associated with treatment outcomes while more mechanistic insights can be achieved when biomarkers from different levels of the biological hierarchy corroborate and complement each other. . this is a favorable advance for rational therapeutics and optimal patient care but it also engenders varying degrees of trepidation among pharmaceutical companies and financial investors about proactive implementation in the clinic: pharmacogenomics may inevitably result in smaller economic markets for drugs introduced with an attendant genetic test predictive of drug efficacy or toxicity , , . the pharma companies fervently respond that only drugs with large-scale markets allow recovery of the r&d costs for new medications , , which can range from $ - million according to different estimates . while r&d costs per se are not necessarily prohibitive to pursue targeted therapies, the pharmaceutical industry will still need to find mechanisms to maintain their growth rates in such niche markets defined by theragnostic tests. the ways in which upstream and downstream theragnostic patents are sought may play a decisive role in the development of focused therapeutic interventions in smaller markets that can benefit public good and industry growth equally. as a contrast to arguments of market fragmentation, pharmacogenomics and related theragnostic technologies may enhance therapeutic differentiation and market penetration of new medicines [ ] [ ] [ ] . many of the currently marketed drugs, however, fall under the 'metoo' designation with comparable efficacy and safety profiles differing only in terms of slight changes in their chemical structures or pharmacophore composition . hence, in diseases or therapeutic areas characterized by me-too drugs, the diagnostic companies without a pharmaceutical pipeline may be more inclined to develop theragnostic tests that can impact more than one drug by virtue of being in the same therapeutic or chemical class. conversely, in the case of large drug manufacturers, a theragnostic test for a me-too drug may be equally predictive of treatment outcomes for most if not all drugs within the same me-too category, redistributing the financial gains on the theragnostic test from an individual pharma company holding the theragnostic patent to multiple firms who manufacture similar me-too drugs. consider, for example, a patient with major depression receiving the result of a theragnostic test on the serotonin transporter gene in relation to antidepressant response to paroxetine, a selective serotonin reuptake inhibitor (ssri). in this case, the patient has the freedom afterwards to choose from among a host of comparable ssri drugs without necessarily having to commit to paroxetine co-developed with the hypothetical theragnostic test. therefore, the pursuit of theragnostic patents can also be shaped by the type of industry setting (e.g., diagnostic sector versus large pharma) as well as the type of pharmaceutical (e.g., me-too drugs) associated with theragnostic tests. regardless of the 'true' cost of drug development or the varied perceptions of the impact of pharmacogenomics or theragnostic tests on the economic promise of pharmaceuticals, the fact is that the blockbuster model of drug development with large-scale markets is increasingly less viable , . when new technologies such as pharmacogenomics and theragnostics enter the market, they can become 'paradigm-disruptive' forces that significantly undermine the traditional broadly defined market model of drug development and commercialization. diverse and divergent diagnostic tests, multiple actors (e.g., biotech diagnostic companies, small and large pharma companies) seeking to create and protect their intellectual property, and changing social and political contexts (global demands for patent reform and licensing of drugs in the developing world) create an unstable environment for drug manufacturers. despite the often very public proclamations about an interest in integrating pharmacogenomic research into drug development strategies, prospective stratification of patients using genetic tests in advanced stages of drug development with a view to proactive incorporation of pharmacogenomic data into drug labels is still rare , . this illustrates that there is a great uncertainty about how pharmacogenomic and theragnostic tests ought to be developed as functional commercial products. it is noteworthy that multiplicity of diagnostic patents anticipated by the introduction of theragnostic technologies may also result in an 'anticommons effect' since most pharmacotherapeutic outcomes are polygenic or multifactorial in nature. if each segment of this expanding sphere of patentable biological elements along the biological dogma is held by different individuals, academic researchers or commercial firms, scientific advances in theragnostics can again be stifled, as in the case of broadly defined upstream gene patents. this is further supported by at least two seemingly divergent but complementary lines of evidence. first, the uspto and patent offices in other countries increasingly favor narrowly defined gene patents, in part as a response to the myriad case. secondly, theragnostics is now introducing the need to characterize (and motivations to patent) downstream gene products such as mrna, proteins or cellular metabolites to individualize drug therapy. because time-dependent changes in gene expression or encoded proteins cannot always be accurately inferred from the upstream gene sequence, it is conceivable that there will be many more narrowly defined patents granted in the near future along the biological dogma from gene sequence to proteins and metabolites. coupled with trends in patent offices in favor of narrowly defined gene patents, there will likely be a fragmentation of the diagnostic sector, as in the case of the blockbuster drugs and the niche therapies guided by theragnostic tests. many of today's most common diseases (including most forms of cancer, heart disease and psychiatric disorders) are known to arise not exclusively from either genes or environmental factors, but through a combination of the two (along with a significant amount of incalculable stochastic factors). moreover, certain environmental exposures may only evoke illness when experienced during a critical period and in concert with a high-risk genetic background. therefore, theragnostic tests predicated on genetic information alone would have a low a priori likelihood of capturing all of the predictable variance in a particular response outcome. ideally, genetic tests could be fashioned to capture the entire heritable portion of a response variable (distributed across one or many genetic polymorphisms). in this context, genetic polymorphisms impart a constant or 'static' state of responsiveness that can be assayed once in each individual and presumed not to change over the course of the lifespan (barring de novo somatic mutations). the residual non-heritable portion of a given response phenotype must then be assayed by other means. to the extent that environmental (i.e., non-heritable) exposure influences response phenotypes by impinging on biological systems, this additional proportion of variance can be assayed through more 'dynamic' biomarker platforms, such as transcriptomics, proteomics, and metabolomics, each of which may be influenced by both genetic and environmental factors (fig. ) . thus, through a combination of static genetic and other dynamic biological '-omic' technologies (i.e., theragnostics), the potential to identify a more comprehensive set of predictors is maximized. there are certain unique aspects of pharmacogenomic (and theragnostic) tests that differ from genetic testing for disease susceptibility. for all the parallels between genetic tests for disease risk and drug response, the latter are applied in reference to a drug that will be administered to patients in the immediate foreseeable future, while genetic testing for disease susceptibility usually predicts a risk in the distant future, often several years or decades away. this 'temporal dissociation' between the genetic test and the future disease occurrence may permit the estimation of the attendant cumulative disease risk with use of genetic data only; there is also a functional disconnect because the disease susceptibility test provides information which is rarely accompanied by effective treatment options. in contrast to genetic testing for disease risk, pharmacogenomic tests are envisioned as being both temporally and functionally proximal. the purpose of a pharmacogenomic test is not to provide risk information as such, but to aid in the individualized prescription of a particular drug. further, most drug effects are elicited within a matter of minutes, hours or days which may require a more precise estimation of the present or acute state of the pathophysiological pathway whose function is inferred through a genetic test. hence, because the only barrier between the patient and drug safety or efficacy may be reliance on the accuracy of a pharmacogenomic test, clinicians need to know both the genetic variants in patients' dna as well as the corresponding proteins encoded by the same genes. this is essential because ( ) proteins are responsible for the eventual functional or clinical significance of genes and, ( ) there may be marked differences or fluctuations in protein function (than what is predicted solely by gene structure) due to environmental factors or physiological feedback mechanisms that may influence posttranscriptional/posttranslational modification of gene products and proteins. further, an accurate prediction of drug effects may require a two-step complementary strategy involving, for example, both genetic and proteomic tests for the same gene and its protein product. this may create unprecedented challenges for patents and their legal defense. for instance, what are the implications of a biotechnology company attempting to develop a metabolomics-based, non-genetic 'dynamic phenotyping biomarker' for a gene patented hitherto for a static genotyping test to predict drug response or toxicity? there are presently no definitive answers to such emerging novel intellectual property issues associated with theragnostic tests. since its first appearance in the research literature in september (refs. , ) , the term 'pharmacogenomics' has been hailed as a revolutionary enabling technology that can deliver highly customized drug therapies in the short term. now, nearly a decade after its introduction, the more realistic expectation is that pharmacogenomics will complement efforts for rational individualization of drug therapy in conjunction with existing therapeutic monitoring tools and other novel biotech-nologies (e.g., proteomics and metabolomics). there is increasing support for the view that the human genome is highly dynamic and that gene expression, as well as the regulation of gene function, is subject to poorly understood plasticity. to achieve the much hoped for provision of personalized medicines, the role of environmental and social factors on both drug response and the human genome (and its expressed products, mrna and proteins) requires detailed consideration. there is also growing recognition that the search for genetic biomarkers of outcomes associated with therapeutic interventions may carry the risk for compartmentalization among biomarkers through excessive reliance on a singular biotechnology. it is against this background that theragnostics is slowly emerging as a new concept to synthesize information from various biotechnologies directed at different levels of the biological dogma ranging from dna (genomics), mrna (transcriptomics), proteins (proteomics) or cellular metabolites (metabolomics). unlike mainstream genetic tests for disease susceptibility, the commercialization of theragnostic tests is inextricably linked with the deployment of patented pharmaceuticals. we suggest that theragnostic patents, particularly in the case of downstream applications at point-of-care, can be at variance with the traditional blockbuster model of drug development which stipulates the development of drugs for the entire population even though this approach yields modest therapeutic response and suboptimal drug safety , , . hence, a very different and unprecedented story is evolving for theragnostic patents at point-of-care: the traditional tight 'coupling' between patents and their subsequent commercialization may not always occur in clinical trials designed for the registration of new therapeutic candidates under the blockbuster model . experts in biotechnology patent law have thus slowly begun to point to this potential 'uncoupling' between the discovery of biomarkers on treatment outcomes and the necessary technology transfer to develop theragnostic products in the clinic. instead, the downstream theragnostic patents on biomarker discoveries may remain primarily as in-house discoveries within the pharmaceutical industry to benefit future drug discovery efforts but without the accompanying translational clinical research for their development as a diagnostic kit for prediction of treatment response, failure or drug toxicity , . this uncoupling of biomarker discovery and necessary technology transfer towards their clinical application poses a threat to theragnostic product development. it is thus con-ceivable that academic research initiatives for biomarker discovery that consider both drug efficacy and broader functional treatment outcomes , will play a critical role in the development of theragnostic-guided personalized medicine. in contrast to downstream patents on biomarkers associated with drug efficacy and safety, upstream patents in drug discovery and the identification of novel drug targets may be particularly welcomed by the pharmaceutical industry. this raises concerns over such patents becoming tollbooths that can increase costs for theragnostic tests in the clinic and slow or block downstream applied research. these nuanced contextual differences in applications of theragnostic patents in upstream research or at point-of-patient-care can shape the motivations at play, the strategies behind the patenting of genes, and the subsequent commercialization into theragnostic tests that may (or may not) become available to patients and consumers. the theoretical and practical framework on patents needs to incorporate implications of theragnostics on both upstream and downstream biomarker research while ensuring technology transfer by more than one stakeholder, to prevent future market monopoly and excessive premium pricing of theragnostic tests . as the pharmaceutical industry transitions from the blockbuster model towards targeted therapies with market shares that resemble orphan drugs, there is a parallel need to offer incentives to stakeholders who pursue theragnostic-guided drug development. the discipline of science and technology studies (sts) is already focused on the complex issues at the intersection of emerging biotechnologies, genetic research, bioethics, market forces and the pharmaceutical industry , , . unfortunately, the expertise in the sts research community does not always find its way into the mainstream medical research literature , . such collaboration among geneticists, ethicists, applied pharmacologists and social scientists is essential for the equitable implementation of commercial theragnostic testing in the clinic, but also to prevent the risk of bioethics being used as a 'rubber stamp' that can 'deal with the issues' prior to the development of anticipated theragnostic-guided customized therapies. as witnessed during the initial planning and implementation stages of the hgp, we suggest that adequate attention and research resources should be made available to resolve these and similar policy and patent issues associated with theragnostic testing at the point-of-care. additionally, these efforts should parallel the development of much needed prospective clinical investigations, designed primarily for the purpose of biomarker discovery, which can importantly contribute to development of targeted therapeutic interventions in the near future. we thus believe there is reason for guarded optimism that theragnostics may allow the synthesis of different types of biomarker data, dna, protein or metabolomicbased, to achieve individualized therapeutics in medicine. the politics of personalised medicine-pharmacogenetics in the clinic ethics and law of intellectual property: current problems in politics world health organization resource on patentability of the sars virus genome we have never been modern science in action: how to follow scientists and engineers through society all authors contributed to the ideas, critique and synthesis of the data discussed in the present review, as well as the specific considerations involving the role of gene patents on theragnostic tests and nuanced distinctions among different types of biomarkers. nature biotechnology volume number august key: cord- -eo auo f authors: gusarov, sergey; stoyanov, stanislav r. title: cosmo-rs-based descriptors for the machine learning-enabled screening of nucleotide analogue drugs against sars-cov- date: - - journal: j phys chem lett doi: . /acs.jpclett. c sha: doc_id: cord_uid: eo auo f [image: see text] chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as covid- , correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. we propose novel descriptors based on a cosmo-rs (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ml). the descriptors’ performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. the cosmo-rs-based descriptors account for both chemical reactivity and structure, and are more effective for ml-based screening than fingerprints based on molecular structure and simple physical/chemical properties. the descriptors are evaluated using principal component analysis, an unsupervised ml technique. our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug eidd- with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. the cosmo-rs-based descriptors could help accelerate drug discovery for the treatment of emerging diseases. o n march , , the world health organization declared the disease covid- , caused by the new virus severe acute respiratory syndrome coronavirus (sars-cov- ), as a pandemic. sars-cov- is a positive, nonsegmented, single-stranded ribonucleic acid (rna) β-coronavirus that belongs to the large family of human coronaviruses, along with sars-cov- and middle east respiratory syndrome coronavirus (mers-cov). , to date, a vaccine and effective therapies for the prevention or treatment of covid- are not available. the research community has vigorously launched or planned numerous covid- research projects and clinical trials. considering the limited knowledge of the sars-cov- infection process, researchers are currently developing treatments based on the knowledge of other sars viruses. the covid- drug discovery effort is to a large extent focused on the inhibition of the angiotensin converting enzyme ii (ace ), assisting in viral entry, and rna-dependent rna polymerase (rdrp), catalyzing viral transcription. − currently, the leading anti-sars-cov- drug remdesivir and highly promising eidd- with active forms designed to inhibit the rdrp-catalyzed rna replication by mimicking nucleotides are under intensive clinical studies. , drug development faces multifold challenges, such as extended time to bring new drugs to the market, high attrition rates, and changing regulatory requirements. , the repurposing of market-available or investigational drugs for the treatment of new or rare diseases is increasingly becoming attractive, because it reduces the risk, cost, and timeline for drug development. , computational data-driven approaches for drug screening are particularly useful for the identification of drug candidates. the initial identification step is typically followed by a mechanistic assessment in preclinical models and ultimately by clinical trials. among the computational approaches, signature matching and molecular docking are based on chemical structure analysis, while genome association, pathway mapping, and retrospective clinical analysis involve genome data and electronic health records screening. chemical and structural similarity-based methods rely on the hypothesis that structural similarity implies correlation at the therapeutic effect or drug-target level. molecular docking and chemical similarity-based strategies have been employed to study the pharmacological activity and screen antiviral drugs against key enzymes of sars-cov- . − however, in some cases, computational screening methods are not in full agreement with in vitro, preclinical, and clinical studies, and even the clinical studies of remdesivir have different conclusions. , due to the global urgency and large number of possible drugs, more accurate and enhanced computational methods are needed. one of the potentially promising ways to improve predictability is through the use of artificial intelligence (ai) and machine learning (ml). increasingly popular in drug development, ml techniques allow the automation of multidimensional data analysis, and have shown great promise for new drug development and repositioning. , , based on a training set, ml approaches enable the screening of efficient drug candidates without having a deep knowledge of the underlying processes. the key to the efficiency of ml methods is the use of representative descriptors or molecular fingerprints to translate the molecular system to a machine-understandable language. a number of molecular descriptors have been developed and implemented into the leading software packages, such as biovia discovery studio and schrodinger suite, which provide good results for drug screening. , the majority of these descriptors are based on the molecular structure, such as the number of rings, hydrogen bond donors and acceptors, induced charges, etc. it is worth noting that such descriptors are important but not sufficient for the chemical similarity analysis of ligands and its extension to ml, as these yield mixed results regarding the ability to enhance the accuracy of ensembleaveraged quantity prediction. typically, small drug molecules interact with a protein active site localized on a small surface area in the presence of a complex environment. to capture the full picture of interactions, the ideal drug screening descriptors should combine both structural and chemical thermodynamicsbased features. methods based on ml have successfully been employed to replace the computationally expensive docking and binding energy calculations. , in order to better represent the ligand and its interaction with the active site of a protein, the list of descriptors should be kept short, and strong correlations among them should be avoided to maintain low degeneracy. in addition, some descriptors might be chosen to avoid overlap dependence from the local structure of the binding site. in many cases, ml techniques can identify internal correlations among variables and reduce the number of descriptors. one such technique is the principal component analysis (pca), a nonparametric statistical ml technique, which is a very important hypothesis-generating tool for approaching drug discovery by a systemic perspective. the contributions of hydrophobic and aromatic character, and molecular shape to the chemical similarity of small molecule drugs have been analyzed using pca to establish correlations of drug structures with transcriptional responses. the pca approach has been employed to reduce the dimensions of molecular descriptors and construct a chemical space to choose descriptors which are presumed best for the particular effect. , the molecular property distributions of drug-like, non-drug-like, and natural compounds from traditional chinese medicines are analyzed by pca. the translation of molecular properties into a format suitable for ml requires an appropriate combination of structural and chemical data. however, while the geometrical properties are well represented by molecular fingerprints, the characterization of chemical reactivity is a more challenging task. typically, the number of hydrogen bond donors or acceptors, functional groups, heats of formation, polarizabilities, etc. are used to represent the ligand's ability to interact with a protein's active site. however, in many cases, the chemical reactivity information is incomplete and/or overlaps with the molecular structure, making it insufficient to thoroughly represent the interaction. as a possible alternative to the predictive methods based on the group contribution approach, the conductor-like screening model for real solvents (cosmo-rs) type models, originally developed by dr. a. klamt, has attracted substantial interest in the recent years. for the first time, the efficiency of cosmo-rs σ-profiles for the detection of new bioisosteric drug candidates has been studied by thormann and klamt. our idea is to further combine the ability of σ-profiles to represent the chemical potentials of different compounds in almost arbitrary phases with the strength of ml in order to enhance σ-profiles' drug screening and design capabilities. the cosmo-rs is a fast and efficient tool for molecular similarity screening and the study of physicochemical and physiological properties originating from solvation thermodynamics and computational quantum mechanics. these methods rely on solvent-accessible surfaces with induced charges screening the electrostatic potential produced by nuclear charges and electronic density. the resulting surfaces are specific to each molecule and projected further to the onedimensional ( d) function called the σ-profile, which represents the probability distribution of a molecular surface segment having a specific charge density. the cosmo-rs σ-profiles capture in a d format the entire set of chemical properties calculated from quantum chemistry, including hydrogen bond donor and acceptor, and hydrophobic (lipophilic) interactions. the σ-profiles have been employed to calculate phase diagrams, azeotropes, and the solubility of drugs and pesticides in water. the calculation of drug absorption, distribution, metabolism, and excretion properties has been demonstrated for high-throughput screening by using the molecular fragmentbased approach cosmof rag. despite this potential, cosmo-rs σ-profiles have rarely been employed for drug similarity screening, and not at all for sars-cov- inhibitor screening to date. in this study, we propose a novel set of drug screening descriptors based on cosmo-rs σ-profiles, augmented by dipole moment and induced charge of the phosphorus atom, to evaluate the chemical similarity of the drugs with nucleotides, as rna replication transcription initiation activators. the cosmo-rs-based descriptors are more suitable for ml because these effectively account for the chemistry, as expressed by chemical thermodynamics and interaction preferences, and are not strongly correlated. these advantages are highlighted in comparison with standard molecular structure-based descriptors. we illustrate the proposed approach, using a series of active compounds of market-available drugs built to mimic the chemical structures of adenosine, guanosine, uridine, and cytidine monophosphates (amp, gmp, ump, and cmp) that bind to messenger rna (mrna), when catalyzed in the central region of rdrp. the drug series includes the pyrimidine and purine nucleoside monophosphate analogues in the form that inhibits mrna replication from pubchem.ncbi.nlm.nih.gov and recent publications (supporting information table s ). the most stable conformations of these drugs and nucleotides are determined using biovia discovery studio . the geometries of these conformations, generated using the discovery studio visualizer, are optimized using becke's three-parameter hybrid functional with the local term of lee, yang, and parr (b lyp), and double-numerical basis set with polarization functions on h atoms (dnp) in dmol . the cosmo-rs calculations, , parametrized to optimally the journal of physical chemistry letters pubs.acs.org/jpcl letter reproduce thermodynamic properties, are conducted by using dmol , as implemented in biovia discovery studio . to identify the most important hydrogen bond donor and acceptor regions in the σ-profile, the geometry optimization of remdesivir monophosphate interacting with rdrp (pdb id: bv ) is conducted using quantum mechanics/molecular mechanics (qm/mm), as implemented in dmol in biovia discovery studio . the b lyp/dnp , approach and charmm force field with momany-rone charges are applied for the qm (f , ala , u , u , a ) and mm system (remaining), respectively. the qm/mm interactions are treated using electronic embedding with dispersed boundary charges. the molecular docking analysis is conducted using the site-features docking algorithm libdock implemented in biovia discovery studio . in figure a , we present the qm/mm optimized model system of the leading drug remdesivir in its monophosphate form integrated into the primer strand mrna interacting with rdrp (protein data bank entry bv ). the hydrogen bonding network with the target u is shown in dashed green lines pointing up from the remdesivir monophosphate molecule and with the pattern u pointing down from the remdesivir monophosphate molecule. the remdesivir monophosphate-rdrp interactions also include attractive van der waals, π− cation, π−π stacking, π−alkyl, and covalent as well as repulsive electrostatic interactions with rdrp functional groups, as shown in figure b the journal of physical chemistry letters pubs.acs.org/jpcl letter atom is selected as a precursor of the chemical activity of the phosphate group toward covalent bonding to mrna. the performance of the descriptor set i, made up of eight descriptors, is compared to descriptor set ii, made up of all ten drug screening descriptors (nine for molecular structure and one for hydrophobicity, listed in table ) implemented in biovia discovery studio . to select the most important combinations of descriptors for the series of nucleotides and active compounds of nucleotide drug analogues, we employed the pca implemented in matlab to the descriptor sets i and ii, without any preprocessing. the pca linear combination coefficients listed in the table give the loadings of each of the four principal components (pc). of these, the most important pc −pc for each descriptor set are presented in the score plot in figure . from the score plot (figure a) , it is clear that the reference group of conformers (marked by green color) represented by amp and the active monophosphate forms of the drugs remdesivir and eidd- known to exhibit some level of efficiency in the inhibition of sars-cov- mrna replication , form a cluster within the calculated pca. the similar but sparser distribution could be seen for descriptor set ii (figure b ). this shows that the similarity of these compounds is clear and could be captured with both sets. however, on a closer look, one can see that for descriptors set ii the conformers for each component are closer together than for the proposed new descriptor set i. this could be explained by the structural effects and degeneracy of descriptor set ii, as the molecular weight, number of hydrogen bonds, number of donors and acceptors, number of rotatable bonds, number of rings, and number of aromatic rings (listed in table ) are (nearly) identical for different conformers of the same compound. another important feature of the proposed descriptor set i is that the other drugs, idx- and mizobirine in monophosphate forms (colored in yellow, figure a) , which have been identified to potentially inhibit the sars-cov- mrna replication, , are better correlated with the reference conformers than those of descriptor set ii (figure b) . in a yield reduction assay, mizoribine has inhibited the replication of sars-cov- more strongly than ribavirin, reducing the virus titers to one-tenth or less. the anti-hepatitis c virus replication inhibitor drug irx- has shown promising results as sars-cov- rdrp inhibitor based on molecular docking that is yet to be confirmed experimentally. moreover, the extent of correlation among the conformers of the same drug is enhanced when descriptor set i is used (figure a) . the monophosphate forms of the drugs ribavirin, penciclovir, and acyclovir, shown in blue squares, are also anticipated to have some rdrp replication inhibition activity. the herpesvirus rna and deoxyribonucleic acid (dna) replication inhibitors ribavirin and penciclovir have been shown to reduce the sars-cov- viral infection against a clinical isolate in vitro at high concentrations. acyclovir treatment has been associated with significantly extending the life and improving the circulatory and pulmonary function in patients with ventilator-associated pneumonia and high levels of herpesvirus replication, making it promising for the treatment of covid- symptoms. currently, proven drug efficiency results are scarce, making further descriptor set evaluation challenging. this analysis could be extended in the future when more results become available in the literature. the pc loadings listed in table represent the normalized coefficients of linear combinations for transformation properties represented by descriptors to the new pcs. the pc loadings also indicate the weight of the descriptors, while the pc latency indicates its strength or order of importance of pca components. for descriptor set i, the cosmo-rs solvation energy and σ-profile projections w and w have the largest weights in pc (with the highest latency), followed by the dipole moment, accessible surface, and phosphorus atom charge. the largest weights in pc are calculated for the σ-profile projections w and w , and the phosphorus atom charge. in pc , the largest weights are for σ-profile projection w and the solvent accessible surface. for descriptor set ii, the molecular polar surface area and fractional polar surface area as well as the number of hydrogen bond donors and acceptors and hydrophobicity are the most important for pc , based on the weights. in pc , the molecular surface area and number of aromatic rings have the largest weights. the large weights of the molecular surface area, hydrogen bonding, and hydrophobicity descriptors of set ii effectively justify the use of the more advanced descriptors of the proposed cosmo-rs-based set i. the polar surface area descriptors of set ii have close physical meaning to the cosmo-rs σ-profiles, as both describe the "polarization" of the molecular surface. however, the advantage of the proposed descriptors is that the cosmo-rs σ-profiles are based on more accurate dft calculations, while polar surface area descriptors are calculated from molecular mechanics force fields and cannot fully account for the correlation of atoms within a molecule. this advantage is particularly important to separate the different conformers that are treated as distinct molecules with calculated atomic charges and cosmo-rs σ- the journal of physical chemistry letters pubs.acs.org/jpcl letter profiles rather than with the same set of force field parameters using descriptor set ii. the superior performance of proposed descriptor set i exhibited by the previously noted enhanced clustering and correlation with the reference conformers is attributed to the statistical thermodynamics the cosmo-rs chemical potential accounts for. this effectively highlights the importance of the cosmo-rs σ-profiles that are calculated by integration over the molecular surface, account for hydrogen bonding and hydrophobic interactions, and are parametrized to reproduce chemical thermodynamic properties. in order to enhance comparison and compensate for the lack of clinical trial results, we also perform libdock analysis for the pyrimidine and purine nucleotides and analogue drugs. the site-features docking algorithm libdock performs as well as docking programs based on genetic/growing and monte carlo driven algorithms. of course, a good docking score does not necessarily mean that the ligand is a good inhibitor, but only a good candidate for potential drug selection. in figure , we (table ) . (green squares, remdesivir; green diamonds, eidd- ; green circles, amp; yellow squares, gmp; yello triangles, idx- ; yello circles, mizobirine; blue squares, ribavirin, penciclovir, acyclovir; red circles, remaining drugs and nucleotides.) figure a ) dock around an active site of rdrp and have high libdock scores (figure b ). the results also show some agreement with the pca plot ( figure ) . penciclovir, ribavirin, and the nucleotide gmp have the highest libdock scores, followed by acyclovir and the nucleotide cmp. among these, only the nucleotide gmp is correlated well with the reference conformers including amp, based on the pca in figure a , but the libdock score of amp is low. the drugs ribavirin, penciclovir, and acyclovir, anticipated to have some inhibition activity based on the libdock score, are weakly correlated with the reference conformers group, based on the pca (figure a) . clearly, the difference between pca and docking results could be attributed to the chemical thermodynamics properties accounted for by descriptor set i. the ml results include additional information not only about complex processes but also about the interactions in the real system. a novel set of descriptors based on cosmo-rs σ-profiles and chemical thermodynamics is proposed and evaluated using pca for the initial screening of a series of nucleotides and nucleotide-analog rdrp replication inhibitor drugs to help accelerate the discovery of covid- treatments. the proposed descriptor set that accounts for chemical thermodynamics in addition to molecular structure and features descriptors with low degeneracy is evaluated with respect to a commonly used descriptor set based on molecular structure alone and a molecular docking algorithm. the pca results show that the novel σ-profile-based descriptor set i clearly correlates the leading covid- drugs remdesivir and eidd- in monophosphate forms and highlights weaker correlations with drugs that have been reported to exhibit anti-sars-cov- activity. the comparison shows that the proposed descriptor set is superior to both the commonly used descriptor set ii and molecular docking scores. the presented approach for conducting drug screening by using cosmo-rs-based descriptors and ml can be further augmented by taking into account (pre)clinical trial result-based descriptors. in addition, the proposed set of descriptors can be further enhanced based on the target, and combinations of antiviral drugs can be evaluated and optimized to continue improving covid- treatments. timeline -covid- origin and evolution of pathogenic coronaviruses the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak − an update on the status global coalition to accelerate covid- clinical research in resource-limited settings while we wait for a vaccine against sars-cov- , why not think about available drugs? structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir a pneumonia outbreak associated with a new coronavirus of probable bat origin treatment options for covid- : the reality and challenges remdesivir for the treatment of covid-  preliminary report ridgeback begins enrolment for phase covid- trial of eidd- an analysis of the attrition of drug candidates from four major pharmaceutical companies drug repurposing: progress, challenges and recommendations overcoming the legal and regulatory barriers to drug repurposing computational drug repositioning: from data to therapeutics thalidomide-induced teratogenesis: history and mechanisms sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study. life sci. , , . ( ) bouachrine, m. molecular docking analysis of n-substituted oseltamivir derivatives with the sars-cov- main protease evaluating the potential of different inhibitors on rna-dependent rna polymerase of severe acute respiratory syndrome coronavirus : a molecular modeling approach remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial delahaye-duriez, a. machine learning applications in drug development drug repositioning: a machine-learning approach through data integration concepts of artificial intelligence for computer-assisted drug discovery ) canvas, ver. . ; schrodinger l development of fukui function based descriptors for a machine learning study of co reduction predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov- ) through a drug-target interaction deep learning model the application of principal component analysis to drug discovery and biomedical data. drug discovery today comparing structural and transcriptional drug networks reveals signatures of drug activity and toxicity in transcriptional responses aicd: an integrated antiinfammatory compounds database for drug discovery structure−activity relationship for fda approved drugs as inhibitors of the human sodium taurocholate cotransporting polypeptide (ntcp) drug-likeness analysis of traditional chinese medicines: . property distributions of drug-like compounds, non-drug-like compounds and natural compounds from traditional chinese medicines conductor-like screening model for real solvents: a new approach to the quantitative calculation of solvation phenomena bioisosteric similarity based on cosmo-rs σ profiles cosmof rag: a novel tool for high-throughput adme property prediction and similarity screening based on quantum chemistry prediction of aqueous solubility of drugs and pesticides with cosmo-rs density-functional thermochemistry. iii. the role of exact exchange an all-electron numerical method for solving the local density functional for polyatomic molecules cosmo: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient refinement and parametrization of cosmo-rs validation of the general purpose quanta ® . /charmm® force field high throughput docking for library design and library prioritization kurane, i. inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus anti-hcv, nucleotide inhibitors, repurposing against covid- impact of acyclovir use on survival of patients with ventilator-associated pneumonia and high load herpes simplex virus replication validation studies of the site-directed docking program libdock the authors declare no competing financial interest. key: cord- -t hojshj authors: bayoumy, a. b.; de boer, n. k. h.; ansari, a. r.; crouwel, f.; mulder, c. j. j. title: unrealized potential of drug repositioning in europe during covid- and beyond: a physcian’s perspective date: - - journal: j pharm policy pract doi: . /s - - - sha: doc_id: cord_uid: t hojshj drug repositioning is the scientific strategy of investigating existing drugs for additional clinical indications. the advantages of drug repositioning are that it benefits patients and that it adds new indications to existing drugs for lower costs compared to de novo drug development. clinical research groups recognizing efficacy of these “old” drugs for a new indications often face an uphill struggle due to a lack of funding and support because of poor structural and regulatory support for clinical drug development. the current framework for drug repositioning allows “venture capital” companies to abuse loopholes in the legislation to gain long-term market authorization among with excessive high pricing. a new regulatory framework is needed to prevent abuse of the legislation and promote clinical investigator-driven drug repositioning. the covid- pandemic has boosted funding and regulatory support for drug repositioning. the lessons learned from the covid- pandemic should be implemented in a new clear blueprint for drug repositioning. this blueprint should guide clinicians through legislation for drug repositioning in the eu. this review summarizes the routes for registration and discusses the current state of drug repositioning in europe. drug discovery marked important events in the history of medicine. the discovery of anesthesia made it possible to perform complicated operations, and the discovery of penicillin by fleming has led to a breakthrough in modern medicine [ ] . in spite of the common interest of the whole society, today, drug development is almost exclusively pharma led. the reason for this includes prohibitory costs on the journey from drug development, data authorities need for registration, and results of preclinical tests and clinical trials. the resulting near monopolization of this entire process by pharma means that it is almost impossible to find the regulatory expertise to comply with the legal requirements for marketing authorization. this classical drug development is now such a costly enterprise which is difficult for noncommercial organizations such as university research institutes or academic hospitals to perform. unlike drug development, drug repositioning is the scientific strategy of investigating existing drugs for new therapeutic purposes [ ] . because the drugs' toxicity and pharmacokinetic properties are already understood the route to authorization via drug repositioning can be less costly than de novo drug development. on top of the advantage of cost savings, the clinical safety profile of the drug can be based on "real-world" clinical data and on the post-marketing pharmacovigilance reporting. the costs for authorization may therefore be reduced significantly and may provide clinicians a wide-array of new treatment modalities for their patients. however, successful drug repositioning also depends on additional factors including clinical resemblance of the disease (e.g., antiinflammatory drug for auto-immune disease), patient demographics, and other features of therapy (e.g., dosage and combination with other drugs). if these conditions are different, it can increase the costs of drug repositioning because of the need of additional data. clinical research groups recognizing the efficacy of these old and often off patent generic drugs need funding and regulatory support for patient benefit through the process of clinical drug development. however, pharmaceutical firms are not obliged and often not inclined to facilitate or support non-commercial efforts for drug repositioning. therefore, unlocking the potential of drug repositioning is lost due to lack of support for these clinical groups. our review summarizes the routes for registration and discusses the current state of drug repositioning in europe. first, it is important to understand how the procedures for drug registration work in europe. the main legal basis for drug applications for drug repositioning in the european union (eu) can be found in the directive / /ec. market access of medicinal products (any substance or combination of substances presented as having properties for treating or preventing disease in human beings) in the eu is regulated through legislation by setting standards for safety, quality, and efficacy. the main objective of the pharmaceutical legislation in the eu is to protect uncontrolled movement of medicinal products and to safeguard public health. a marketing authorization is required for all medicinal products before entering the eu market. during the marketing authorization procedure, the regulatory authority assesses the data provided by the applicant, in which the product satisfies these criteria of quality, safety, and efficacy. the application process for drug repositioning in the eu is based on three different procedures for submitting a marketing authorization application (maa) in the eu: . the centralized procedure (cp) . the decentralized procedure (dcp) . the mutual recognition procedure (mrp) the summary of the three application procedures can be found in table . the european medicines agency (ema) is responsible for execution of the centralized procedure, which allows to put a product on the market in all eu member states and in norway, iceland, and liechtenstein. submission to one maa thus leads to one assessment process and one authorization that allows access of the market of the entire eea (european economic area). the centralized procedure is mandatory for certain products as mentioned in the regulation / : . high technology medicinal products, particularly those resulting from biotechnological processes. . orphan medicinal products, these are products registered for orphan diseases. an orphan disease is defined as any disease affecting fewer than people in , . furthermore, the centralized procedure is also mandatory for any medicinal product for human use containing an entirely new active substance, i.e., one that has not yet been authorized in the eu. in the decentralized procedure, applicants can apply for simultaneous authorization of a drug in more than one if an applicant company already has applied for, or obtained market authorization in another eu member state, the national and decentralized procedures are not available. for this instance, the mutual-recognition procedure is required. the rms can apply for this authorization to be recognized in a chosen concerned member states (cms). this process allows member states to rely on each other's scientific assessments and could lead to authorization in the cms. however, this route cannot be used for drug repositioning because the drug has not yet been authorized for the new indication. applicants for a new active substance need to submit the latest safety and efficacy data, while companies, organizations, or individuals producing known active substance are allowed to submit "dossiers" that contain previous data. this can be done without the need for a new clinical trial by referring to previous or other "dossiers." the two important pathways for drug repositioning are the complete dossier pathway and the well-established use pathway. the complete dossier pathway, which can be found in article ( ) of directive / /ec, can be considered the standard application pathway. it is composed of general administrative information, complete (non)-clinical data based on the applicants' tests and studies, and/or bibliographic literature substituting/supporting certain tests or studies. the well-established use application pathway, which is provided in article a of directive / /ec, can be used for drugs that have been used for at least years in the eu, with recognized efficacy and an acceptable safety level. the applicant is not required to provide the (non)-clinical data in the dossier but may use existing literature. in addition, applicant's own data may be used to prove similarity of the applicant's product to the product used in the literature. a generic medicine has the same active ingredients as the authorized reference medicine; it may only be produced after expiry of the data exclusivity period. when a manufacturer aims to develop a generic drug but it differs in dosage strength, indication, or pharmaceutical form, then it must follow the hybrid application pathway. the authorization of these hybrid drugs may refer to the results of the authorized reference medicine but may also provide new data from clinical studies. the legal basis of the hybrid application can be found in article ( ) of directive / /ec. the european legislation provides the holders of a marketing authorization with regulatory protection in the form of data exclusivity and market protection. data exclusivity is the period of time during which a generic (or hybrid or biosimilar) application cannot cross-refer to the data in support of marketing authorization for the reference medicinal product (i.e., generic, hybrid, and biosimilar). such applications will be refused by regulatory authorities during period of data exclusivity. market protection refers to the period of time during which generic, hybrid, or biosimilar cannot be placed on the market, even if the medicinal product has already received marketing authorization before. the european medicines agency (ema) follows the + (+ ) exclusivity formula, in which the first years are data exclusivity, followed by years of market protection. there are also provisions available that could extend market protection and data exclusivity: the algorithm for drug repositioning pathways can be found in fig. . although history has many successful examples of drug repositioning, there is the important cautionary example of thalidomide, which as an anti-emetic in pregnancy, caused congenital malformations. the thalidomide disaster resulted in approximately deaths and serious birth defects in over , children leading to its withdrawal in [ ] . as a consequence, stricter regulations were initiated, and all new drugs had to be properly "authorized" before being placed on the market. in , thalidomide was prescribed as sedative in a patient who also had erythema nodosum leprosum (enl). in this patient, the enl skin eruption disappeared within h [ ] and led to positive clinical studies for this new indication. thalidomide was reintroduced for this complication of leprosy and supported by the world health organization [ ] . by , there were additional benefits of thalidomide discovered for several hiv complications such as oral ulceration, wasting syndrome, and kaposi's sarcoma [ ] . due to the continued interests in thalidomide, the drug remained available for controlled clinical trials and compassionate use treatments. in , the food and drug administration (fda) approved thalidomide for the treatment of enl, which contributed to the availability of the drug for other diseases [ ] . in , anti-angiogenic properties were first described using a rabbit cornea model of fgfinduced neovascularization [ ] . these anti-angiogenic properties were later confirmed in other studies [ , ] . the discovery of the anti-angiogenic properties has boosted interested in thalidomide as an anti-cancer therapy. plasma cell myeloma is associated with high serum vegf concentrations and increased bone marrow microvascular density [ ] . several clinical studies were performed to assess the clinical effectiveness of thalidomide in plasma cell myeloma [ , ] . thalidomide, in combination with dexamethasone, was approved as orphan drug treatment for plasma cell myeloma in may . thalidomide can be seen to have found multiple useful indications in the past six decades and was designated as orphan drug then approved through the centralized procedure [ ] . a more recent example is thioguanine, which was originally developed and licensed for the treatment of leukemia in the s [ ] . in the following decades, it has been investigated for a wide-range of inflammatory diseases such as psoriasis and systemic lupus erythematosus [ ] . in , thioguanine was found to be a promising rescue therapy for patients with inflammatory bowel disease (ibd) who failed mercaptopurine or azathioprine treatment [ ] . in the netherlands, thioguanine has been conditionally registered as rescue therapy for ibd after failure of conventional thiopurines since . however, authorizing this drug was complicated mainly because a blueprint for authorizing an off-patent drug for a new indication was lacking. there was also little interest from the pharmaceutical industry to invest in authorizing thioguanine due to an unattractive costrecovery (only year of market exclusivity when licensed). thioguanine was approved through the well-established use pathway in the netherlands [ ] . in clinical practice, there are many examples of off-label drug use [ ] , especially in the field of oncology or for special populations (e.g., pediatric patients and geriatric patients) [ ] . off-label use can be defined as any intentional use of an authorized product not covered by the terms of its marketing authorization and therewith not in accordance with the smpc. whereas market approval of medicinal products is the subject to eu legislation and falls under the responsibility of the national competent authorities or, in case of a centralized procedure, the european commission (ec), eu legislation does not regulate the use of medicinal products in daily clinical practice. off-label prescription can offer important advantages for the individual patients: it provides new treatment options, especially when approved treatment options have failed. furthermore, it also gives patients and clinicians earlier access to drugs and the adoption of new practices based on newly obtained evidence. however, a major disadvantage of off-label uses the generally low level of evidence regarding safety and efficacy. it was found that of all off-label drugs only approximately % is supported by strong evidence [ ] . other limitations of off-label use are the increased responsibility [ , ] (e.g., legal claims, risk-benefit analysis of evidence, and implicit informed consent) put on the prescribers and pharmacists, issues with reimbursement (i.e., special application or no reimbursement), as well as undermining of the drug authorization system of prior approval before marketing. there is a need for labelling these off-label drugs for the sake of patient safety. as these drugs are not formally licensed for the indication, there is no safety surveillance for the drug on that indication. lack of reimbursement for off-label drugs for new treatment indications vary from country to country in europe. often, off-label use of drugs is not reimbursed. however in daily clinical practice, especially for cancer and hiv drugs, in contrast to auto-immune diseases, reimbursement of these off-label drugs is possible due to strong media and political support. in major markets such as europe and the usa, a clinician should preferably prescribe a drug by reference to the generic name. however, due to strong pharmaceutical marketing, physicians usually refer to brand names. the prescription will be filled by a pharmacist without knowledge about its medical indication. the drug will then be reimbursed at a fixed level regardless of whether a branded or generic version is dispensed. thus, most reimbursement systems nearly always provide a financial incentive to the pharmacist to dispense, when available, a (cheaper) generic drug. this is also called the preference policy, which is mandatory in some major markets such as germany [ ] . for pharmaceutical companies, there are so far limited attractive financial incentives for drug repositioning. legislation only allows for year extra market protection if a new indication is added in the first years after a marketing authorization has been granted, and the new indication must have significant clinical benefit over existing therapies. however, off-label prescription will continue to occur without good incentives for the drug repositioning of "older" drugs, especially in off-patent products for which drug repositioning will not likely result in return for on investment. in recent years, drug development has shifted from blockbuster to "niche buster" due to improved incentives for orphan drugs [ ] . the regulation particularly designed for these orphan drugs is the orphan regulation / ec. to receive an orphan drug designation, the drug must meet three criteria: . the drug must be intended for the treatment, prevention, or diagnosis of a disease that is lifethreatening or chronically debilitating; . the prevalence of the condition in the eu must not be more than in , , or it must be unlikely that marketing of the drug would generate sufficient returns to justify the investment needed for its development; . no satisfactory method of treatment, prevention, or diagnosis of the condition concerned can be authorized, or, if such a method exists, the drug must possess a significant benefit to those affected by the condition. the orphan regulation declares that a drug that has been labeled as an orphan drug by the committee for medicinal products for human use receives market exclusivity for a period of years. also, there is an opportunity for a -year extension if a pediatric investigation plan is included. one medicinal product may have more than one orphan disease designation. the orphan drug designation can be allocated to products approved through either the . , . , or (a) pathway. all orphan drugs must follow the centralized procedure (ec regulation no / ) [ ] . orphan drugs can also be conditionally approved; in this case, market exclusivity is granted only for the duration of year. the price of (orphan) drugs is based on market-behavior, and therefore, the prices will reach to what the market is willing to pay for the drug [ ] . due to the relatively low amount of patients with an orphan disease, the price of orphan drugs is generally high [ ] [ ] [ ] . in , seven out of the top best-selling drugs in the world had an orphan indication in the usa [ ] . considering that in certain chronic orphan diseases like cystic fibrosis, patients could use these expensive drugs for many decades. the total costs per patient could therefore become as high as several millions of euros. an example of such a high priced drug is eculizumab, a drug that treats paroxysmal nocturnal hemoglobinuria, which can cost up to $ , per patient per year [ ] . recently, the public debate was excited by a price increase of a drug called mexiletine. this drug was originally developed years ago for the treatment of arrhythmias. however, it was recently granted marketing authorization for years after it was designated as orphan drug for non-dystrophic myotonia [ ] . the price of mexiletine used to be € . per patient per year but was increased to € . per patient per year. the estimated costs for drug repositioning of mexiletine are clearly disproportional to the new price. this example shows that drug repositioning in orphan disease can result in disproportional and undesirable high prices. the primary incentives for drug development in orphan disease were not intended to serve drug repositioning and lead to overpricing of drugs. thus, the legislation is not fitted for drug repositioning and should be adapted. pharmaceutical compounding is the production of a medicinal product adapted to a specific need of a patient. although authorized medicinal products are preferred, pharmacists can compound suitable medicinal products when the adequate commercial form or dosage is not available. however, the directive / /ec states in article ( ) that no medicinal product may be placed on the market of a member state unless a marketing authorization has been issued by the competent authorities of that member state or of the european commission (ec). in article ( ) of the directive, it was established that manufacturing of the medicinal products is subject to the holding of license issued by the member states. furthermore, the directive states in article ( ) that the wholesale distribution and storage are covered by an authorization granted by the member state according with this directive. the exceptions to the above mentioned regulations can be found in article ( ) of directive / /ec and shall not apply to: . magistral formula: any medicinal product prepared in a pharmacy in accordance with a medical prescription for an individual patient [ ] . . officinal formula: any medicinal product which is prepared in a pharmacy in accordance with the prescriptions of a pharmacopoeia and which is intended to be supplied directly to the patients served by the pharmacy in question [ ] . these exceptions can only be applied when they are all in compliance with the requirements. in this case, then the marketing authorization, manufacturing, and wholesale licenses, as mentioned in the directive, are not compulsory. the first requirement is that the medicinal product needs to be prepared in a pharmacy. second, it needs to be prepared according to the clinician's prescription. lastly, the prescription must be made for an individual patient. in case the directive does not require a marketing authorization for the medicinal product, eu member states are allowed to establish national regulations for pharmacy preparations. thus, the directive allows exceptions such as pharmaceutical compounding of drugs for which an alternative medicinal product with a marketing authorization is available on the market [ ] . pharmaceutical compounding; the case of chenodeoxycholic acid more than years ago, originally developed and registered for treatment of gallstones, chenodeoxycholic acid (cdca) was found to be also effective treatment for cerebrotendinous xanthomatosis (ctx) [ , ] . from until , cdca was available on the dutch market for the treatment of gallstones under the brand name chenofalk® which price was € . per capsule. chenofalk® has been used as off-label treatment for ctx since , which costed at that time € per patient per treatment year. after cdca was developed as xenbilox® by sigma-tau, this company acquired the rights for chenofalk® and later of chenix®, a belgian firm. by creating this monopoly position, sigma-tau gradually increased the price to around € per capsule. in , cdca was removed from the dutch market for the treatment of gallstones [ , ] . subsequently, sigma-tau adopted a new name, leadiant, and registered cdca as an orphan drug for the treatment of ctx in . the registration was based on two retrospective cohort studies [ ] . subsequently, the price of cdca was increased to approximately € . per patient per year on the dutch market, a price increase of times (see table ) [ , ] . so, the company exploited the orphan drugs regulation (eu regulation / ) to raise the price and gain years of market exclusivity [ ] . this regulation for orphan drugs is meant to stimulate drug development for orphan diseases that would otherwise have no economic incentives to develop drugs for. however, cdca was already being used for ctx for many years, and the estimated costs for preparing the registration file did not justify the steep raise in the price of ca. in april , the amsterdam umc took the initiative to start magistral preparation of cdca for its patient using raw materials imported from china. the costs were roughly € . per patient per year, a fraction of the price of the drug sold by the pharmaceutical company [ ] . because of the high price [ ] of the raw material, this magistral preparation is still times higher than the original price of chenofalk®. the spread of the novel coronavirus, sars-cov , has caused a worldwide pandemic of the coronavirus disease . since the start of the pandemic, drug repositioning research has been booming [ ] . it has been found that the coronavirus has more than druggable proteins that can be targeted by currently or previously approved drugs [ ] . it is no doubt that drug repositioning will play an important role in the current covid- pandemic [ ] . the fda has allowed the emergency use authorization for the unapproved product, remdesivir, for the treatment of severe hospitalized covid- patients. beigel et al. [ ] reported superiority of remdesivir over placebo in shortening time to recovery in hospitalized patients. however, one chinese study showed no clinical benefits of remdesivir [ ] . remdesivir was originally developed for hepatitis c and tested for the ebola and marburg viruses. it did not show to effective in any of these viral diseases [ , ] . so, by definition, the case of remdesivir is not drug repositioning but shows that an earlier developed drug can be repurposed for a new indication. more approved drugs have been identified to be potential candidates for drug repositioning for covid- [ ] , and dozens of randomized clinical trials are currently conducted [ , ] . a list of examples of these drugs can be found in table . however, it is not clear yet whether these drugs with repositioning potential will be successful candidates for repositioning. the ema has already announced that it will provide regulatory support to stimulate drug repositioning for covid- [ ] . it is necessary to provide such support to accelerate drug development for covid- ; however, it should be a standard procedure to provide regulatory support to researchers that conduct drug repositioning for any other disease because otherwise unrealized potential of many future drugs is lost. drug repositioning has the potential to offer benefits and new treatment options to patients. however, its potential has not been fully realized yet due to lack of incentives in the drug repositioning process. therefore, patients might not have access to a beneficial repositioned drug or only through off-label prescriptions with its potential safety concerns and lack of availability for individual patients all over the world. in some cases of drug repositioning, pharmaceutical companies abused loopholes in the regulations to benefit from long-term regulatory exclusivity. this came hand-in-hand with extreme high pricing of repositioned drugs without the justifiable investments. the high pricing became subject of public debate, and for a well-balanced discussion, it is necessary that key players (e.g., policymakers, researchers) have insights into drug repositioning. the current lack of adequate national and european legislation causes therapeutic chances to be missed, not only in europe, but also for the rest of the world. to improve the drug repositioning process, cooperation between the pharmaceutical industry and clinical researchers is necessary for the efficient generation of data to determine the risks and benefits to register a new indication. for clinical researchers, it can be difficult to gain access to historical (non)-clinical data of the generic drugs; therefore, support of the original manufacturer is urged. in some instances, it would even require the researcher to repeat costly experiments or clinical studies again. furthermore, clinicians should also collaborate together (e.g., by centers of expertise) to obtain adequate datasets for drug repositioning. this would ensure that these clinicians can operate more independently. clinical research groups may also have limited experience conducting trials to ensure that they meet regulatory requirements. this limited regulatory awareness and interest can hinder drug repositioning and can result in regulatory failures. well-balanced regulatory support from national market authority agencies or the ema is urgently needed for clinical research groups that intend to repurpose older drugs for new indications. these clinical research groups usually have extensive "real world" datasets that are valuable for regulatory authorities. the ema and national regulatory authorities have shown to be dedicated to support (e.g., free rapid scientific advice and rapid compliance check) drug repositioning for covid- . the same level of support should be given to clinical research groups who intent drug repositioning. there needs to be a formal blueprint to bring this data into the regulatory process with an identified marketing authorization holder. the covid- pandemic has boosted worldwide drug repositioning initiatives. rapid public and private funding became available for clinical groups to perform drug repositioning, and global data sharing initiatives were founded [ ] . the covid- pandemic has shown that it is possible to establish initiatives that stimulate drug repositioning and improve benefits for patients. the lessons learned from the covid- pandemic should be implemented in a new standard blueprint for drug repositioning for all diseases. drug repositioning has the potential to reregister older drugs for novel indications for the benefits of patients. it may provide new treatment opportunities for lower costs compared to de novo drug development. the current framework for drug repositioning allows "venture capital" companies to abuse loopholes in the legislation to gain long-term market authorization among with excessive high pricing. a new regulatory framework is needed to prevent abuse of the legislation and promote clinical investigator-driven drug repositioning. the covid- pandemic has boosted funding and regulatory support for drug repositioning. the lessons learned from the covid- pandemic should be implemented in a new standard blueprint for drug repositioning. the role of serendipity in drug discovery drug rediscovery to prevent offlabel prescription reduces health care costs: the case of tioguanine in the netherlands thalidomide regains respectability as new benefits are discovered thalidomide in the treatment of lepra reactions who co-ordinated short-term double-blind trial with thalidomide in the treatment of acute lepra reactions in male lepromatous patients effects of thalidomide on hiv-associated wasting syndrome: a randomized, doubleblind, placebo-controlled clinical trial the rise, fall and subsequent triumph of thalidomide: lessons learned in drug development thalidomide is an inhibitor of angiogenesis effects of thalidomide and related metabolites in a mouse corneal model of neovascularization identification of a primary target of thalidomide teratogenicity expression of vegf and its receptors by myeloma cells phase ii study of thalidomide plus dexamethasone induction followed by tandem melphalan-based autotransplantation and thalidomide-plus-prednisone maintenance for untreated multiple myeloma: a southwest oncology group trial (s ) thalidomide alone or with dexamethasone for previously untreated multiple myeloma the continuous rediscovery and the benefit-risk ratio of thioguanine, a comprehensive review an open-label pilot study using thioguanine as a therapeutic alternative in crohn's disease patients resistant to -mercaptopurine therapy finding hidden treasures in old drugs: the challenges and importance of licensing generics. drug discovery today off-label prescriptions of drugs used for the treatment of crohn's disease or ulcerative colitis off-label prescribing: a call for heightened professional and government oversight drug, patient, and physician characteristics associated with off-label prescribing in primary care ten common questions (and their answers) about off-label drug use overcoming the legal and regulatory barriers to drug repurposing the evolving drug development landscape: from blockbusters to niche busters in the orphan drug space the european union policy in the field of rare diseases. advances in experimental medicine and biology outrageous prices of orphan drugs: a call for collaboration orphan drugs for rare diseases: is it time to revisit their special market access status? the lancet. the balancing act of orphan drug pricing the orphan drug act: restoring the mission to rare diseases estimating the clinical cost of drug development for orphan versus nonorphan drugs radboud umc concerned about drug accessbility for patients with rare neuromuscular disease compounded medication for patients with rare diseases legislation on the preparation of medicinal products in european pharmacies and the council of europe resolution chenodeoxycholic acid therapy for gallstones: effectiveness, toxicity and influence on bile acid metabolism long-term treatment of cerebrotendinous xanthomatosis with chenodeoxycholic acid if a medicine is too expensive, should a hospital make its own? intramuscular glucocorticoid injection versus placebo injection in hip osteoarthritis: a -week blinded randomised controlled trial the safety and effectiveness of chenodeoxycholic acid treatment in patients with cerebrotendinous xanthomatosis: two retrospective cohort studies dutch hospital makes own drug for rare condition after manufacturer hikes price to euro gvs-advies chenodeoxycholzuur (chenodeoxycholic acid leadiant®) bij de behandeling van cerebrotendineuze xanthomatose (ctx) perception, price and preference: consumption and protection of wild animals used in traditional medicine drug repositioning is an alternative for the treatment of coronavirus covid- a sars-cov- protein interaction map reveals targets for drug repurposing coronavirus puts drug repurposing on the fast track remdesivir for the treatment of covid- -preliminary report united states food & drug administration -remdesivir eua letter of authorization current pharmacological treatments for covid- : what's next? identification of fda approved drugs targeting covid- virus by structure-based drug repositioning clinical trials on drug repositioning for covid- treatment ongoing clinical trials for the management of the covid- pandemic drug repurposing against covid- : focus on anticancer agents european medicines agency -eu actions to support availability of medicines during covid- pandemic -update # european commission -coronavirus: commission launches data sharing platform for researchers springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable amsterdam, the netherlands. authors' contributions abb, nkhb, fc, and cjjm conceptualized, wrote, and reviewed the manuscript. ara reviewed the manuscript. all authors read and approved the final manuscript. no funding was acquired for the preparation of this manuscript. no data was used to write this manuscript.competing interests ab bayoumy declares that he has no competing interests. nkhb de boer has served as a speaker for abbvie and msd and as a consultant and principal investigator for teva pharma bv and takeda. he has received unrestricted research grants from dr. falk, teva pharma bv, and takeda. f crouwel declares that she has no competing interests. ar ansari declares that he has no competing interests. cjj mulder has served as consultant for hlw pharma bv and teva pharma bv.author details faculty of medicine, amsterdam umc, university of amsterdam, key: cord- -lx vy authors: emwas, abdul-hamid; szczepski, kacper; poulson, benjamin gabriel; chandra, kousik; mckay, ryan t.; dhahri, manel; alahmari, fatimah; jaremko, lukasz; lachowicz, joanna izabela; jaremko, mariusz title: nmr as a “gold standard” method in drug design and discovery date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: lx vy studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. the pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of sars-cov- , which originated in wuhan, china, illustrates the critical and immediate need to improve drug design and development techniques. modern day drug discovery is a time-consuming, expensive process. each new drug takes in excess of years to develop and costs on average more than a billion us dollars. this demonstrates the need of a complete redesign or novel strategies. nuclear magnetic resonance (nmr) has played a critical role in drug discovery ever since its introduction several decades ago. in just three decades, nmr has become a “gold standard” platform technology in medical and pharmacology studies. in this review, we present the major applications of nmr spectroscopy in medical drug discovery and development. the basic concepts, theories, and applications of the most commonly used nmr techniques are presented. we also summarize the advantages and limitations of the primary nmr methods in drug development. the unexpected sars-cov- /covid- outbreak, with over million confirmed cases globally (oct. ) and the struggle for survival in the absence of a proven and efficient treatments, emphasizes molecules , the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $ . billion. the typical development cost is usually spread out over the course of years [ ] [ ] [ ] , making investment even more difficult (i.e., cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme ), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. emphasizes the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $ . billion. the typical development cost is usually spread out over the course of years [ ] [ ] [ ] , making investment even more difficult (i.e. cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme ), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. the emergence of a pandemic and the emergencies it creates worldwide understandably drive and motivate the rapid development and/or optimization of drugs. however, patient safety and subsequent earned public trust is a primary requirement. drug redirecting/repurposing (scheme ) is an efficient short-cut method in disease treatment that utilizes existing tools, and combines artificial intelligence, machine learning algorithms, and experimental nmr techniques (i.e. "from bench to bedside"). this process must be relatively rapid and efficient to have any benefit to patients and the health-care system. scheme . schematic representation of drug discovery [ ] . numerical data were reported from meigs et al. [ ] . compared to mass spectrometry and high-performance liquid chromatography (hplc), nuclear magnetic resonance (nmr) is another powerful technique with several unique advantages [ ] [ ] [ ] [ ] . nmr is intrinsically quantitative, and it provides several different approaches that are routinely utilized to identify and structurally elucidate molecules of interest [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast to mass spectrometry, nmr is non-destructive, non-invasive, has extremely high reproducibility permitting in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [ ] . an example of this is found in work by wyss et al. [ ] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace- in alzheimer's disease. bace- is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [ , ] . using nmr fragmentbased screening, wyss et al. identified isothiourea as binding to bace- and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [ ] . an example of this is found in work by wyss et al. [ ] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace- in alzheimer's disease. bace- is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [ , ] . using nmr fragment-based screening, wyss et al. identified isothiourea as binding to bace- and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection of iminopyrimidinones as bace- inhibitors [ ] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id jtl) with a total mass of kda using nmr [ ] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [ ] [ ] [ ] [ ] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e., spins) which vary because of different atomic, electronic, and chemical environments (i.e., structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. molecules , , x for peer review of of iminopyrimidinones as bace- inhibitors [ ] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id jtl) with a total mass of kda using nmr [ ] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [ ] [ ] [ ] [ ] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e. spins) which vary because of different atomic, electronic, and chemical environments (i.e. structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. table . h chemical shift assignments is shown as an example. the chemical structure and simulated h-nmr spectrum were created using chemdraw . . this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [ ] [ ] [ ] [ ] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the table . h chemical shift assignments is shown as an example. the chemical structure and simulated h-nmr spectrum were created using chemdraw . . this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [ ] [ ] [ ] [ ] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the signal contains precise details about the chemical environment of the involved and interacting spins in the structure of the molecule, dynamics of the spins in various timescales, conformational exchange, etc. [ ] [ ] [ ] . any change in the environment of the associated spin can be observed. these changes include molecules , , of molecular binding, interactions, and/or exchange between different conformations [ , [ ] [ ] [ ] . thus, nmr has been used to study a wide range of functional molecules such as natural products [ ] [ ] [ ] , saccharides [ , ] , metabolites [ , ] , dna [ , ] , and proteins [ ] , and its use as an analytical tool in drug design research has increased immensely in recent years (see figure ). as nmr is non-destructive in nature, the same sample can be analyzed repeatedly. nmr can be performed first and then submitted to mass spectrometry (ms); however, the addition of common deuterated nmr solvents (such as d o) can perturb ms results and should be avoided (e.g., tube-in-tube or by using non-deuterated solvent and running the nmr unlocked). in fact, high-performance liquid chromatography (hplc), ion-trap ms and nmr have been combined to detect the effects of drugs demonstration in urine and blood serum samples [ , , ] . corcoran and spraul [ ] emphasize that liquid chromatography (lc), ms, and nmr utilized in parallel give comprehensive structural data on molecules of novel drugs in development. in the following subsections we briefly describe nmr methods that have been used in drug design, and then discuss how nmr principles are used in drug discovery research. the one-dimensional ( d) experiment is by far the most common nmr experiment used for drug studies. the d acquisition takes the least amount of time, has one of the simplest hardware requirements, and therefore, in most cases, d-nmr is more attractive for high throughput studies. one dimensional nmr spectroscopy normally incorporates a preparation period, some form of induced excitation to form coherence, and lastly, a signal "read" detection period. the preparation period can be modified according to the needs of the experiment or the specifics of the sample. simple d nmr is capable of rapidly producing high-quality spectra of drugs and their targets while revealing how the drugs and targets may interact at the atomic level. d h-nmr is extremely effective in drug design studies because it has a (relatively) high sensitivity, it is non-destructive, and because hydrogen atoms are extremely abundant in most molecules of interest. therefore the resulting spectra usually contains a large amount of relevant information and this wealth of data can be acquired in a relatively short period of time. the basic d h-nmr, along with d c-nmr, d n-nmr, and d p-nmr, and their respective uses in drug design/discovery are briefly discussed below. the h hydrogen isotope is nmr visible, has the highest gyromagnetic ratio (apart from tritium) of all of nmr active nuclei, and is combined with a vast natural abundance in organic chemical compounds. this makes the d- h-nmr experiment the most commonly applied nmr approach. moreover, many software databases [ ] [ ] [ ] [ ] are well established for h-nmr spectra therefore assisting with processing, analyzing, and identifying the detected molecules automatically. since almost all drug discovery and drug development studies are performed on samples dissolved in water, many different solvent suppression methods have been applied. the most common is presaturation [ , ] . the key point of this method is to use a low power induced field at the specific frequency of water. this effectively averages out any coherence of the water resonance. the experiment is simple for common hardware to perform and easy to set up; however, presaturation has a substantial disadvantage in that signals resonating close to the solvent signal will show decreased intensity [ , ] or may be lost entirely. this is due to the fact the even selective pulses or very low power pulses also excite some area around the water signal. also suppressed hydrogen from h o in solution can exchange with atoms of interest in the molecule and effectively bleed the suppressive spin state to any neighboring atoms. the water signal itself is usually broad, so a wider area of suppression is not necessarily undesirable but affects more of the molecule(s) of interest. more recent water suppression techniques have been developed such as those based on a scheme known as excitation sculpting [ , ] . the basic pulse sequence consists of a double pulsed field gradient echo (dpfge) in each of which a selective component pulse is flanked by two pulsed field gradients [ ] . the particular elements differ for different applications. in the case of water suppression known as water suppression by gradient tailored excitation (watergate), this involves an initial encoding gradient along with the middle element; a combination of two selective • rotations on the water along with a central non-selective • excitation of all resonances [ ] . this is predicated in that water experiences a • rotation (effectively nothing) while all other spins experience • rotation. the application of the second refocusing gradient does not rephase the water and therefore removes the signal. the reader is referred to the detailed literature [ , , ] for further information. in principle, a water suppression element (or many elements combined) can be incorporated in any existing pulse sequence to enhance the performance, and it has been implemented in various d, d, and triple resonance d/ d experiments. although h is the most sensitive nucleus for nmr yielding strong, sharp signals within a few minutes [ ] , chemical shift dispersion of h is quite narrow (only around ppm). this has prompted the consideration of other nuclei such as c, n, or p for resolution improvements. compared with h, c has a much higher chemical shift dispersion (~ ppm), however the natural abundance of c is low ( . %). additionally, the gyromagnetic ratio is~ times weaker than h and therefore c spectra are far more difficult to obtain especially for less concentrated samples. there are some polarization transfer techniques such as distortionless enhancement by polarization transfer (dept) or insensitive nuclei enhanced by polarization transfer (inept), which can enhance signal intensity by starting the magnetization on a higher sensitivity and abundance proton and then transferring magnetization to the less sensitive carbon nuclei for subsequent direct detection [ ] , but this requires additional hardware and acquisition times. the use of d c in drug design studies was illustrated by tsujimoto et al. [ ] . the goal of the study was to examine if a metabolomics approach based on h and c offers significant improvements when comparing potential drugs. the authors prepared a total of samples with five different citrus-type crude drugs (kijitsu, tohi, chimpi, kippi and seihi) and measured d h and d c for each sample. while h-nmr spectra allowed the identification of three compounds (naringin, sucrose, and β-glucose), using c-nmr allowed unambiguous identification of eight additional compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). the added signal resolution from c-nmr spectra allowed researchers to obtain better structural information about the compounds than from h-nmr spectra alone. in comparison to the previous example, n has a lower shift dispersion (~ ppm) than c, but higher than that of h. here, the situation is unfortunately severely limited due to an even lower natural abundance ( . %) and a gyromagnetic ratio~ times smaller than h. this means that n's combined sensitivity is around , times lower than h. as a result, isotopic enrichment of n combined with h-mediated enhancement via indirect detection is often needed in order to obtain a satisfactory d n spectra. similar to c, a few methods are available to overcome such low sensitivity. one of them focuses on tagging molecules with carboxyl groups using n-ethanolamine and later detecting the signal using a d heteronuclear correlation nmr experiment [ ] . currently, novel approaches such as "smart isotope labeling" have been developed [ ] . also, the sofast (band-selective optimized flip angle short transient) technique can help but results in substantial hardware considerations/drawbacks and often increased concentrations, and/or dramatically longer experiments are still required [ ] [ ] [ ] . promising methods are on the horizon. these methods include n heteronuclear signal enhancement via signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (sabre-sheath); however, more work and research are required before such methods can be applied for biomedical purposes [ ] . with a natural abundance of % and a gyromagnetic ratio of about . times smaller than h, one may think that phosphorus could be broadly used for nmr experiments regarding the drug discovery and development. however, the application of p is limited due to the fact that most of the molecules of interest simply do not contain a phosphorus atom. therefore p-nmr is usually applicable for studies related to energy, phospholipid metabolism (atp, nadp), and/or characterization of changes in dna [ , , ] . for example, overall et al. conducted an experiment in which they showed that p solid-state nmr can be used for quantitative analysis of dna dynamics within live bacteria [ ] . for that, the researchers first prepared untreated cultures of e. coli, and measured them using a hartmann-hahn h to p cross-polarization ( p cp) experiment. afterwards, they measured e. coli treated with ampicillin and maculatin . (mac . ) in a similar manner. spectra obtained from treated bacteria compared to those obtained from untreated bacteria showed alterations in the lineshape, reduced signal intensity at the spectrum's edges, and a shift in spectral density towards ppm which indicated the increased dynamics of the phosphorus from nucleic acids [ ] . over time, several innovations have been applied to expand the usage of p. like in c and n labeling of specific biological compounds, incorporation of p can also be used. in order to achieve that, -chloro- , , , -tetramethyldioxaphospholane (ctmdp) can be used for tagging lipids containing hydroxyl, aldehyde, and carboxyl groups that can later be detected with better resolution [ ] . another fairly recent method enables toxicological screening of p in living cells for several hours without affecting cell viability [ ] . this specific method can be used to observe the changes in energy metabolism in real-time while enabling the evaluation of the effects of administered drugs. nmr experiments are not limited to one-dimensional direct acquisition; they can be extended to multidimensional methods including d, d, d, and even higher dimensionality. the focus of this section is common d nmr experiments that have been used in drug design and drug development. a brief description of correlation spectroscopy (cosy), total correlation spectroscopy (tocsy), and heteronuclear multiple bond correlation (hmbc), along with their uses in drug design and discovery will be presented. cosy is one of the simplest and most frequently used d nmr experiment [ ] . it shows the homonuclear coupling of nuclei (i.e., h- h) separated by up to several covalent bonds. the pulse sequence consists of a • excitation pulse followed by a specific evolution time (t ), a second pulse, and finally a measurement period (t , not to be confused with relaxation rates or times). the second pulse can be • or • or • , depending upon the specific requirements, and respectively yield cosy [ ] , cosy- or cosy- functionality (see [ ] [ ] [ ] ). a two-dimensional fourier transform (ft) yields the final spectra and shows the frequencies for proton ( h) or carbon (in the case of carbon detection) along both axes. there are two types of peaks; (i) diagonal peaks, which represent the peaks of the conventional d spectra, and (ii) cross-peaks, which have different values in the two frequency axes and are therefore off the diagonal. these off diagonal cross-peaks are the most important pieces of information as they mark correlations between pairs of nuclei due to through bond magnetization transfer. this helps in identifying which atoms are connected [ ] , critical for structural elucidation of both known molecules and unknown molecules in solution [ ] . by implementing phase-cycling [ , ] , it is also possible to distinguish different types of coupling and yields further helpful information about the chemical structure of a molecule [ ] . as an example, the use of the cosy experiment was presented in the work of zheng et al. [ ] . the main goal of their work was to investigate potential biological differences and compare the pharmacological effects between danggui (an herbal drug used in traditional chinese medicine) and european danggui. the difference between the two experiments is that a tocsy experiment will show multiple cross-peaks including indirectly coupled nuclei (i.e., longer range via scalar coupling) throughout the j-coupled spin system of a chemical compound. the basic pulse sequence of the tocsy consists of excitation by a • pulse, followed by a free variable evolution period which encodes the indirect dimension. this is normally followed by an isotropic mixing sequence to transfer magnetization between spins via the strong scalar coupling. the mixing generates in-phase magnetization throughout a spin coupled network of the associated nuclei during the mixing time. lastly, a direct detection is performed. a major advantage of the tocsy experiment is that it detects in-phase magnetization (i.e., pure absorptive line-shape) which is far easier to analyze compared to the anti-phase information in the phase sensitivity cosy-type experiment. the isotropic mixing is usually performed using a composite pulse scheme such as waltz, mlev or dipsi [ , ] pulse train, and can be sandwiched between two z-filters [ ] where isotropic mixing is performed on the longitudinal magnetization. the most obvious advantage of tocsy is that all cross-peaks of the same spin system can be observed for whole spin system at once. this is useful for identifying the complete network of spins and reducing the ambiguity of any spectral overlap. the tocsy experiment can be produced as d with a relatively shorter time and easier analysis compared to d but lacks the benefit of multi-dimensional resolution. the d tocsy is usually done to resolve spectra overlap [ ] when first identifying molecules [ ] [ ] [ ] . for example, jiang et al. used this to predict the response to gemcitabine-carboplatin (gc) chemotherapy in patients with metastatic breast cancer who were previously exposed to treatment with both anthracyclines and taxanes [ ] . for that, researchers collected serum samples from patients prior to treatment and measured them using d h-nmr. additionally, they conducted d nmr experiments such as the h, h-cosy, , h-tocsy, h, c-hsqc, and h, c-hmbc to help assign serum metabolites. after receiving the treatment with gemcitabine-carboplatin, patients were divided into four groups based on the results from the computed tomography: complete response (cr), partial response (pr), stable disease (sd), or progressive disease (pd). after comparing nmr results prior to the treatment with the outcome of chemotherapy, the researchers observed lower baseline levels of serum format and acetate in breast cancer patients who progressed with the disease than in those who achieved a clinical benefit from therapy, indicating that those two biomarkers could be used to distinguish between patients who will benefit from gc treatment from those who do not [ ] . d-heteronuclear single quantum coherence (hsqc) experiments are commonly used to help resolve spectral overlap [ ] while providing c information without the inherent sensitivity losses involved in c direct detection (see below). hsqc shows the correlations between directly coupled nuclei [ ] , e.g., h- c or h- n [ ] . as such, an hsqc spectrum will show clean peaks for each unique proton directly connected to the heteronuclear nuclear atom of interest [ , ] . in h, c/ n hsqc experiments, the magnetization is transferred from the more sensitive nucleus (i: h) to the less sensitive nucleus (s: c/ n) [ ] [ ] [ ] (figure ). this is especially useful when applying nmr spectroscopy to drug design, as most drugs are organic (i.e., contain carbon atoms), and the relative abundance of c ( . %) is quite low [ ] . by transferring sensitivity from h to c, one can circumvent the long experimental time required for d c experiments [ ] . molecules , , x for peer review of abundance of c ( . %) is quite low [ ] . by transferring sensitivity from h to c, one can circumvent the long experimental time required for d c experiments [ ] . for example, de castro et al. [ ] studied ptac s and its related cytotoxicity to the cisplatinresistant epithelial ovarian carcinoma (eoc), skov- cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov- cells reacted to treatment with ptac s. in particular, they used h, c-hsqc along with h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov- cells treated with ptac s produced more pyruvate than skov- cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac s and those treated with cisplatin. these results provide a possible explanation for how ptac s is able to overcome cisplatin resistance in skov- cells [ ] . heteronuclear d experiments are useful for transferring magnetization from sensitive nuclei (i.e. h) to less sensitive nuclei (i.e. c) [ ] thereby reducing the time needed for the acquisition of spectra [ ] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [ , ] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [ ] and in metabolites of complex biofluids [ ] , whose d h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [ , , , ] . as an example, hmbc was used in a recent study by xu et al. [ ] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)- . mg/kg/day, middle dose group (md)- . mg/kg/day, high dose group (hd)- . mg/kg/day and a control group (nc). after days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional days of recovery before the same samples were acquired. the samples were measured using h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [ ] [ ] [ ] along with h, h-cosy, h, c-hsqc, and h, c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. for example, de castro et al. [ ] studied ptac s and its related cytotoxicity to the cisplatin-resistant epithelial ovarian carcinoma (eoc), skov- cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov- cells reacted to treatment with ptac s. in particular, they used h, c-hsqc along with h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov- cells treated with ptac s produced more pyruvate than skov- cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac s and those treated with cisplatin. these results provide a possible explanation for how ptac s is able to overcome cisplatin resistance in skov- cells [ ] . heteronuclear d experiments are useful for transferring magnetization from sensitive nuclei (i.e., h) to less sensitive nuclei (i.e., c) [ ] thereby reducing the time needed for the acquisition of spectra [ ] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [ , ] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [ ] and in metabolites of complex biofluids [ ] , whose d h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [ , , , ] . as an example, hmbc was used in a recent study by xu et al. [ ] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)- . mg/kg/day, middle dose group (md)- . mg/kg/day, high dose group (hd)- . mg/kg/day and a control group (nc). after days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional days of recovery before the same samples were acquired. the samples were measured using h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [ ] [ ] [ ] along with h, h-cosy, h, c-hsqc, and h, c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [ ] . after application of a • rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [ ] . this process is described in figure , and is termed t relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [ ] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t and t . t refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e., t relaxation and refers to the rate of transverse (or spin-spin) relaxation [ ] which occurs in the xy plane. t is independent of the longitudinal relaxation (t ) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t and/or t [ ] , and is collectively referred to as "relaxation edited nmr" [ ] . molecules , , x for peer review of relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [ ] . after application of a ° rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [ ] . this process is described in figure , and is termed t relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [ ] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t and t . t refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e. t relaxation and refers to the rate of transverse (or spin-spin) relaxation [ ] which occurs in the xy plane. t is independent of the longitudinal relaxation (t ) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t and/or t [ ] , and is collectively referred to as "relaxation edited nmr" [ ] . over time (ms to seconds, and in extreme cases, minutes [ ] ), the bulk magnetization will decrease in the transverse plane, and increase in the longitudinal axis, returning to its original, equilibrium value. (a) represents t relaxation and (b) represents t relaxation. t -based methods typically measure and compare the t times of the free and bound ligands. a common way to measure the t value of a small molecule is the inversion recovery experiment [ , ] , although other experiments are also available such as ultrafast nmr t [ ] and saturation inversion recovery [ ] . in general, the shorter t the relaxation time the less intense the peak signal will be and the broader the signal linewidth [ ] . the t values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [ ] . bound ligands will have smaller t values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [ ] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [ ] , whereas non-binding ligands normally show small-positive noes [ ] . for binding ligands to display negative noes, their t values must be comparatively longer than the /koff value of the target [ ] . t -based methods typically measure and compare the t times of the free and bound ligands. a common way to measure the t value of a small molecule is the inversion recovery experiment [ , ] , although other experiments are also available such as ultrafast nmr t [ ] and saturation inversion recovery [ ] . in general, the shorter t the relaxation time the less intense the peak signal will be and the broader the signal linewidth [ ] . the t values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [ ] . bound ligands will have smaller t values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [ ] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [ ] , whereas non-binding ligands normally show small-positive noes [ ] . for binding ligands to display negative noes, their t values must be comparatively longer than the /k off value of the target [ ] . t relaxation times can be easily used to screen small molecules as ligands for dna [ ] and serve as a basis for hts experiments [ ] . an experiment related to drug design that utilized d and d relaxation edited nmr was done by hajduk et al. [ ] in which he and others used d and d relaxation edited nmr techniques to detect ligands that bind to fk binding protein and stromelysin. one year earlier, liu et al. [ ] used relaxation edited one-and two-dimensional h-nmr spectroscopy to characterize biological fluids. tang et al. [ ] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret n protein relaxation data [ ] , and even used deficient n relaxation data to rapidly calculate the dynamics of proteins [ ] . the t relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure ). molecules , , x for peer review of t relaxation times can be easily used to screen small molecules as ligands for dna [ ] and serve as a basis for hts experiments [ ] . an experiment related to drug design that utilized d and d relaxation edited nmr was done by hajduk et al. [ ] in which he and others used d and d relaxation edited nmr techniques to detect ligands that bind to fk binding protein and stromelysin. one year earlier, liu et al. [ ] used relaxation edited one-and two-dimensional h-nmr spectroscopy to characterize biological fluids. tang et al. [ ] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret n protein relaxation data [ ] , and even used deficient n relaxation data to rapidly calculate the dynamics of proteins [ ] . the t relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure ). figure . cpmg pulse sequence. first, a ° rf pulse is applied and results in transverse magnetization in the xy plane. then a °y pulse is applied to re-phase the magnetization vectors. after °y, the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a ° rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay- °-delay block) is responsible for mx/y magnetization decay. this period is repeated "n'' times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [ ] by changing the phase of the applied ° pulses from x to y [ ] . this procedure can be used to measure t relaxation times of any type of nuclei. for instance, in the case of c, all pulses and acquisitions are applied on c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [ ] . in a typical cpmg experiment, the effective transverse relaxation rate, r ,eff, is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [ ] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t * (also called as 'effective' or "observed'') by the following equation: ( ) figure . cpmg pulse sequence. first, a • rf pulse is applied and results in transverse magnetization in the xy plane. then a • y pulse is applied to re-phase the magnetization vectors. after • y , the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a • rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay- • -delay block) is responsible for mx/y magnetization decay. this period is repeated "n" times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [ ] by changing the phase of the applied • pulses from x to y [ ] . this procedure can be used to measure t relaxation times of any type of nuclei. for instance, in the case of c, all pulses and acquisitions are applied on c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [ ] . in a typical cpmg experiment, the effective transverse relaxation rate, r ,eff , is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [ ] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t * (also called as 'effective' or "observed") by the following equation: t represents the transverse relaxation times, and additional broadening comes from the magnetic field inhomogeneities (t inh ), which must be taken into account. t measurements of ligands are also useful for determining the binding nature of a small molecule. the t values of small molecules are quite large compared to those of bigger molecules (i.e., proteins) mostly because macromolecules have more spin-spin diffusion [ ] . bound ligands will, therefore, display shorter t values than non-binding ligands because they interact with the target (i.e., protein), adopting similar vibrational and rotational energies to the target [ ] . this interaction is represented by the resonance line broadening in the binding ligand's spectrum when a receptor is introduced into the sample [ ] . given the sizable difference of t values of binding and non-binding ligands, one can utilize d relaxation-edited experiments to distinguish the binding ligands from the non-binding ligands efficiently and effectively based on the differences in the t values [ ] . these and other related relaxation edited experiments prove useful in drug design. relaxation edited nmr spectroscopy takes advantage of an inherent atomic property (i.e., the return of bulk magnetization back to equilibrium [ ] ), so no molecular enrichment (e.g., n isotopic enrichment of protein targets) is required [ ] . furthermore, the slow time scale of nmr relaxation allows the user to manipulate the external conditions (i.e., length and power of pulse) to increase the resolution of targets and potential drugs [ ] in nmr drug design experiments. however, this slow timescale also sets the lower limit at which nmr drug design experiments can be performed [ ] , meaning that any external manipulations cannot decrease experimental time below a certain threshold. this varies based on the drugs and targets used in the experiment. low drug solubility is also a challenge, as the ligands must be at a sufficiently high concentration to allow detection via nmr, although the use of organic solvents has helped to attenuate this effect in relaxation edited nmr spectroscopy [ ] . for examples of experiments that use different nmr techniques mentioned above, see table . emodin can affect the immune response and interrupt energy metabolism (citric acid cycle) along with glutathione synthesis, which can lead to oxidative stress. dox decreases atp production and induces oxidative stress in h c cells. dex counteracts those changes, having a cardioprotective effect on h c cell lines. [ ] curcumin shows antihyperlipidemic effects on c bl/ slac mice by partially restoring metabolic defects induced by a high-fat diet. affected metabolic pathways include the citric acid cycle, glycolysis and gluconeogenesis, ketogenesis of bcaa, synthesis of ketone bodies and cholesterol, and choline and fatty acid metabolism. formosanin c shows the ability to inhibit synthesis and methylation of dna as well as reducing the activity of the citric acid cycle and energy metabolism in the mitochondria of hepg cells. melamine melamine disrupts metabolism of glucose, nitrogen, and protein in the liver of wistar rat. h-nmr cpmg [ ] aristolochic acid (aa) aristolochic acid causes renal lesions and a disorder in tubular reabsorption in wistar rats. evaluating response outcome for patients with rheumatoid arthritis, treated with rituximab. identification of metabolites changes between responders and non-responders such as succinate, taurine, lactate, pyruvate and aspartate. no distinction between metabolite profiles of serum from patients treated with levetiracetam, lamotrigine and topiramate. could not evaluate response of initial treatment of epilepsy. [ ] metallaprism mainly affects lipid metabolism in a (human ovarian cancer) and hek- (human embryonic kidney) cells, and increases gsh levels in all cell lines. in a cisr (cisplatin resistant a ) cells, lipid biogenesis and glycosylation are affected by treatment with metallaprism. extract from centella asiatica promotes glycolysis, boosts the citric acid cycle and decreases gluconeogenesis and lipid metabolism in t dm sprague-dawley rats. vr and vr improve glycerol metabolism, decrease betaine levels, and normalize the altered level of myoinositol in serum of dmh-induced crc (colorectal cancer) wistar rats. evaluating the efficacy (determined by the concentration of a drug able to reach the therapeutic site) of four drugs in cerebrospinal fluid of tuberculous meningitis patients. genipin can recover energy metabolism to normal levels, and regulate methylamine and amino acid metabolisms of diabetic sprague dawley rats. h-noesy- d [ ] adriamycin (adr) identification of seven biomarkers: trimethylamine oxide (tmao), taurine, trimethylamine (tma), hippurate, trigonelline, citrate and -oxoglutarate that can predict tumor's (gastric adenocarcinoma) response to adr treatment in balb/c-nu/nu mice. h-noesypresat- d [ ] danggui/european danggui fuzi causes a shift in energy metabolism (from aerobic respiration to anaerobic), induces membrane toxicity, and disrupts the balance of gut microbiota of wistar rats. administrating fuzi with gancao diminishes the toxic effects of fuzi. h-nmr spectra enabled the identification of three compounds (naringin, sucrose, and β-glucose), and c-nmr enabled the identification of eight compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). as stated, nmr spectroscopy can be fundamental in studying how drugs interact with their targets. this has been done mainly via the fragment based drug design (fbdd) approach, which has two sub-approaches: target-(i.e., protein) based, or ligand-(drug) based. target based screening monitors how the target responds to binding molecules in a method called structure activity relationship ("sar") by nmr. ligand (drug)-based screening methods provide ways to observe the binding/non-binding behavior of the drug in approaches such as saturation transfer difference (std) and other nuclear overhauser effect (noe) type methods, diffusion-based methods, relaxation-based methods (i.e., t and t ). target based screening, ligand (drug) based screening, and their respective methods, are discussed in detail below. nmr-based drug discovery can be broadly classified into two groups: chemical and biological (in-cell) categories. one of the principal methods of drug discovery using nmr spectroscopy is called fragment-based drug design (fbdd) [ ] . in-cell nmr (biological) based drug discovery techniques will be discussed later in this review. fbdd was first reported in [ ] and used throughout the late s as evidenced by the use of keywords related to fbdd in papers published during this time [ ] . the use of fbdd as a viable drug screening technique began to be widely adopted in the mid- s [ ] . high throughput screening (hts) is another technique widely used in drug discovery [ ] . hts analyzes molecules from a chemical library to see which ones are suitable leads [ ] [ ] [ ] [ ] (see figure ) . fbdd techniques will screen against a carefully designed fragment library composed of a few thousand molecules (for details on the choice of compounds and design of fragment libraries, see [ , ] ) and identified hits are further developed via fragment growing, fragment merging, or fragment linking [ ] . for examples of drugs derived from the fbdd approach that are currently in clinical trials, refer to table . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [ ] , and over are in clinical trials [ ] . the first marketed drug derived via the fbdd approach is vemurafenib [ ] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [ ] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [ ] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [ ] , is considered the second drug to be discovered using the fbdd approach [ ] , and ribociclib, a cdk inhibitor, the third [ ] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table . hts has been productive in drug design [ , ] , but the method is time and resource intensive [ ] and expensive [ ] because of the numerous molecules to be examined (~ million) [ ] . furthermore, the success rate is only estimated to be at~ % [ , ] . unlike traditional hts, which can survey a large number of molecules ranging from a few hundred thousand to a few million [ ] , fbdd usually surveys a few thousand molecules (~ - ) from libraries with greater chemical diversity [ , ] . fbdd is a main-stream screening technique for drug discovery [ , , [ ] [ ] [ ] [ ] [ ] [ ] and nmr is standard for many fbdd studies [ ] . additional methods and techniques such as spr, x-ray crystallography [ , [ ] [ ] [ ] [ ] etc. have also been used in fbdd studies, accompanied or unaccompanied by nmr experiments. for examples of fbdd derived drugs using methods besides nmr, refer to table . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [ ] , and over are in clinical trials [ ] . the first marketed drug derived via the fbdd approach is vemurafenib [ ] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [ ] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [ ] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [ ] , is considered the second drug to be discovered using the fbdd approach [ ] , and ribociclib, a cdk inhibitor, the third [ ] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table . nmr, x-ray crystallography, inhibition of cathepsin d as mentioned, nmr spectroscopy can be used in fbdd in two different ways: ( ) target (or receptor) based screening, and ( ) ligand-based screening. with the stated advantages and disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [ ] . sar is primarily used to identify and develop extremely tight-binding ligands [ ] . the ligand to target binding is traditionally monitored via chemical shift changes [ ] using a correlation spectroscopy such as h- n hsqc starting with the target and no ligand present [ ] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [ ] . for example hajduk et al. investigated the binding interactions of -phenylimidazole with the fkbp protein as shown in figure [ ] . disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [ ] . sar is primarily used to identify and develop extremely tight-binding ligands [ ] . the ligand to target binding is traditionally monitored via chemical shift changes [ ] using a correlation spectroscopy such as h- n hsqc starting with the target and no ligand present [ ] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [ ] . for example hajduk et al. investigated the binding interactions of -phenylimidazole with the fkbp protein as shown in figure [ ] . from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [ ] . then, by following a procedure completely analogous to that of fbdd (see figure ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [ , , ] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [ ] in both solution state and solid-state spectra [ ] , increasing the method's versatility [ ] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [ ] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [ ] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [ ] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e. cleavage from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [ ] . then, by following a procedure completely analogous to that of fbdd (see figure ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [ , , ] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [ ] in both solution state and solid-state spectra [ ] , increasing the method's versatility [ ] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [ ] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [ ] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [ ] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e., cleavage of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [ ] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: ) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on ) diffusion methods, or ) relaxation-based methods (i.e., t and t ). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as c and n [ , ] . this increase in sensitivity is possible because of dipolar coupling (i.e., through space interactions of separate nuclei) [ ] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [ ] of a specific chemical group (i.e., the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [ ] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [ ] (see figure ). of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [ ] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: ) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on ) diffusion methods, or ) relaxation-based methods (i.e. t and t ). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as c and n [ , ] . this increase in sensitivity is possible because of dipolar coupling (i.e. through space interactions of separate nuclei) [ ] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [ ] of a specific chemical group (i.e. the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [ ] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [ ] (see figure ). std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [ ] . a generic example of detecting ligand binding via std is presented in figure a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e. methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [ ] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure b ). the main difference with normal std nmr is that water is the saturated signal [ ] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [ ] . std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [ ] . a generic example of detecting ligand binding via std is presented in figure a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e., methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [ ] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure b ). the main difference with normal std nmr is that water is the saturated signal [ ] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [ ] . for std nmr to work properly, the ligand concentration must be in large excess (often - fold) over the receptor so that effective saturation transfer can take place [ ] . this means that for std nmr, and waterlogsy, only small amounts (µg) of protein are required to get results [ ] [ ] [ ] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure . waterlogsy has been extended to study ligand interactions with dna and rna [ ] . for std nmr to work properly, the ligand concentration must be in large excess (often - fold) over the receptor so that effective saturation transfer can take place [ ] . this means that for std nmr, and waterlogsy, only small amounts (µ g) of protein are required to get results [ ] [ ] [ ] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure . waterlogsy has been extended to study ligand interactions with dna and rna [ ] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [ ] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [ ] described the use of saturation transfer difference (std) nmr spectroscopy [ , [ ] [ ] [ ] [ ] [ ] [ ] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype (hrv ). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [ , ] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [ , ] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [ ] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [ ] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [ ] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [ ] described the use of saturation transfer difference (std) nmr spectroscopy [ , [ ] [ ] [ ] [ ] [ ] [ ] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype (hrv ). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [ , ] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [ , ] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [ ] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [ ] . the very large size of viruses makes them particularly attractive for studies by std nmr, as they inherently yield large line widths allowing for easy irradiation of the virus without affecting the ligand protons. furthermore, because of the larger correlation time of a virus in comparison to an average-sized protein, spin diffusion, and thus saturation transfer, is very efficient. the large line width has additional benefits not just for std-based nmr methods but also for transfer noesy spectra, as protons from the virus capsid are invisible in the nmr spectra (for an example of a transfer noesy spectrum, see [ ] ). moreover, competitive std titration experiments can be used to determine the k d value of a ligand [ ] . analysis of the std spectra using the group epitope mapping method [ ] allows for the determination of the binding epitope. std nmr methods can considerably speed up the determination of the binding epitope for potential antiviral lead compounds. simple std nmr experiments provide substantial information on the binding of ligands to native viruses and require very small amounts of the virus with measurement times in the range of tens of minutes. this allows for a high throughput of ligand samples without significant consumption of viral material because it remains unaffected by the experiments and is easily separated from the low molecular weight ligands by ultra-filtration subsequently. in addition to the detection of binding, a complete mapping of the ligand-binding epitope can be achieved [ ] . noroviruses (nv) are non-enveloped, single-stranded, positive-sense rna viruses that are the major cause of epidemic outbreaks of gastroenteritis worldwide [ ] [ ] [ ] . the viral coat consists of a single protein, vp , which assembles into a capsid with overall icosahedral symmetry [ ] [ ] [ ] . attachment of human noroviruses to histo-blood group antigens (hbgas) is thought to be critical for the infection process [ ] . the protruding domains of the vp proteins, called p-domains, harbor highly conserved binding sites for hbgas. std nmr-based epitope mapping was used [ , ] to identify structural features of different core types critical for the binding of synthetic a-and b-tetrasaccharides [ ] to virus-like particles (vlps) of a highly homologous gii. strain (ast ). std nmr experiments provide a robust and straightforward technique for obtaining ligand binding epitopes at atomic resolution. comparing binding epitopes of related ligands then delivers critical information about structural requirements for ligand recognition. conversely, comparison of binding epitopes of a given ligand binding to wild type, and to mutant proteins reveals the importance of individual amino acids for binding. std nmr experiments with l-fuc and b-trisaccharide in the presence of wild type and mutant vlps yield virtually identical binding epitopes and suggest that these two mutations do not significantly alter hbga recognition. the std nmr approach to characterize binding of hbga ligands to noroviruses has employed vlps as targets and thus taken advantage of the large size of vlps yielding excellent signal-to-noise ratios of the corresponding std nmr spectra, as demonstrated previously [ ] . the application of the transferred noe (tr-noe) effect was first demonstrated by bothner-by [ ] . the tr-noe is the nuclear overhauser effect between ligand spins, which are in chemical exchange between the bound and unbound form with the protein or receptor. ligands, which are a mixture of target molecules, are small in size (below - da). since they are usually low molecular weight molecules, they exhibit much shorter correlation times when compared to the receptor and have slow noe build-ups with no spin diffusion. this is the reason they show small positive noes in the free form. when binding to a protein receptor, the situation changes, where the ligand acquires large correlation times in the bound state with rapid noe build-up. then they show spin diffusion and a strong negative noe, which is termed the transferred noe. signals arising from the protein are usually not observed for large proteins as they are generally kept low in concentration, with ligands in a high excess concentration. in addition, most of the time protein signals are suppressed by their very short t period. it is worthwhile to mention that ligands that are in fast exchange between the bound and the free form (dissociation constants ranging from µm to mm) get enough bound time to transfer the negative noe from the protein complex to the population of the free molecules, yet usually retain the chemical shift of the free molecule along with the relaxation characteristics. in order to observe tr-noes, the following condition have to be fulfilled: where n and ∂ represent the number of molecules and the cross-relaxation rate, respectively. the subscript b and f represent the bound and free form, respectively. therefore, to observe the tr-noes, a high excess concentration of ligands over protein is maintained. on the other hand, if the ligand concentration is kept too high, the excess free ligand in solution will exhibit positive noe, which can result in a significant reduction of the tr-noesy enhancements due to negative noe developed by the very small concentration of bound ligand. hence, the preparation of the sample becomes tricky and an optimum ratio between - to is maintained while considering the dissociation constant values. the binding of a ligand to a receptor protein can easily be identified by observing the sign and size of the noes. there are some distinct experimental features for the discrimination between tr-noes from the bound state and noes of the ligand in free states like the build-up rate. for tr-noes, this is in the range of to ms, whereas for small ligands it is much longer. there have been various instances of experimental implementations to quickly determine the binding activity of ligand libraries. one example was to find the ligand molecule among a library of similar structure polysaccharides that is bioactive in binding with recombinant e-selectin [ ] . this is a protein present in an igg chimera with a molecular weight of about kda. in this case, two d noesy spectra were recorded. the noesy spectra for the ligand library was measured at several temperatures and it was found that most of the compounds exhibited the weak positive noes at k, which was then chosen to differentiate between trnoes showing large negative values. the trnoesy spectra of the ligand library in the presence of protein was recorded at different ratios, such as : , : , : , : , and : , at k. in all the ratios, trnoes were observed; however, the ratio of : represented the best-case scenario. the inpharma method (see figure ) was designed to determine the relative orientation between two competitive ligands in the receptor-binding pocket through the observation of inter-ligand noe between the two ligands. it is a tr-noe in nature as it is mediated by the bound conformation of the competing ligands and in exchange with the receptor protein. the first example was competitive binding and observation of inter-ligand noe between baccatin iii and epothilone a in the presence of tubulin, which acts as a receptor [ ] . since the observation is on the ligand site, it provides unique advantages. the detailed conformation of a ligand-protein complex can be addressed by conventional nmr. however, it is time-consuming and demands full solving of the structure and there is also a size limitation. from that aspect, ligand-based methods are more useful. the only limiting fact is that it should fulfill all the conditions of tr-noe explained previously in terms of dissociation constant (k d ), fast exchange regime, and proper ligand to protein ratio. then, information on the ligand structure can be derived from tr-noe build up as a function of mixing time. this can be readily explained using the originally proposed schematics [ ] . the noesy spectrum of a mixture of the two ligands a and b in the presence of the common receptor (t) is recorded. under the situation that each of a and b exhibit competitive binding in a fast exchange regime with the receptor t, intermolecular tr-noe peaks between the two ligands a and b can then be observed in the noesy spectrum due to extensive spin diffusion. during the noesy mixing time, the first proton of ligand a (h a ) binds to receptor t, which results in transfers of magnetization from h a to h t . subsequently, the complex at dissociates as they fulfill the dissociation constant range, which creates the opportunity for ligand b to bind to the receptor t at the same binding site. this results in the transfer of the magnetization of h t , which had been originally coming from h a , to h b . as a result, an inter-molecular correlation h a -h b can be seen, and this inter-molecular noe will be a function of mixing time as described above. the detailed analysis of such intermolecular noe peaks helps in assessing the relative orientation of each ligand in the binding pocket. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [ ] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [ ] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [ ] after a period of time, t, as shown in figure . fick's law can be used to model this type of movement [ ] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [ ] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [ ] . in homogeneous isotropic solutions, the root mean square distance (zrms) traveled by a molecule is given by following equation [ , ] : where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation ( )): where kb is the boltzmann's constant ( . × − j/k), t is the absolute temperature, η is the solution viscosity, and rs is the hydrodynamic radius of the molecule [ ] . equation ( ) and equation ( ), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [ ] . different molecular geometries and additional modes of diffusion (i.e. restricted and anisotropic) require more advanced mathematics and theory [ , ] , but the essential concepts of diffusion remain the same. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [ ] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [ ] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [ ] after a period of time, t, as shown in figure . fick's law can be used to model this type of movement [ ] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [ ] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [ ] . in homogeneous isotropic solutions, the root mean square distance (z rms ) traveled by a molecule is given by following equation [ , ] : z rms = ( dt) where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation ( )): where k b is the boltzmann's constant ( . × − j/k), t is the absolute temperature, η is the solution viscosity, and r s is the hydrodynamic radius of the molecule [ ] . equation ( ) and equation ( ), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [ ] . different molecular geometries and additional modes of diffusion (i.e., restricted and anisotropic) require more advanced mathematics and theory [ , ] , but the essential concepts of diffusion remain the same. molecules , , x for peer review of figure . visual representation of how molecules diffuse in solution from a high concentration to a low concentration across an arbitrarily defined point (x = ) after some period of time (t = ∞) [ ] . the red arrow indicates the direction of translational motion as the molecules (green) move from an area of higher concentration (left) to an area of lower concentration (right) [ ] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [ ] . the se pulse sequence is shown in figure . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the o pulse, and the other after the o refocusing pulse. the first gradient pulse (g ) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [ ] . additional nmr sequences are available for diffusion experiments [ ] , and are detailed in more comprehensive reviews dealing with the subject [ , ] . the signal intensity of the diffusing molecules depends on three factors, as described by equation where i is the observed intensity, i the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [ ] . from equation ( ), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [ ] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [ ] . the se pulse sequence is shown in figure . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the • pulse, and the other after the • refocusing pulse. the first gradient pulse (g ) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [ ] . additional nmr sequences are available for diffusion experiments [ ] , and are detailed in more comprehensive reviews dealing with the subject [ , ] . molecules , , x for peer review of figure . spin echo (se) sequence, as discovered by stejskal et al. [ ] . g is the first gradient pulse applied after the first ° pulse, and g is the second gradient pulse applied after the first ° pulse. δ and ∆ are the gradient length, and diffusion time, respectively. in silico (virtual) screening is now a standard technique in drug design and discovery [ ] that has been in use since at least [ ] , though the exact origin of the phrase "in silico" is not clear [ ] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [ ] that inhibit protein-protein interactions [ ] , and its ability to help identity ligand (drug) binding sites on the target of interest [ ] to lend insight to the mechanisms of action for lead compounds [ , ] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [ ] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [ ] . virtual screening has also been used to identify candidates for anti-viral drugs [ ] and anticancer drugs [ ] . several chemical databases are available both for public and academic use [ ] . virtual screening is properly identified as a high-throughput screening (hts) technique [ ] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, ( ) a three-dimensional model of the ligand (drug), and ( ) a three-dimensional model of the receptor (protein) [ ] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [ ] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [ ] , such as differential scanning fluorimetry [ ] , fluorescence polarization, and surface plasmon resonance [ ] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how [ ] . g is the first gradient pulse applied after the first • pulse, and g is the second gradient pulse applied after the first • pulse. δ and ∆ are the gradient length, and diffusion time, respectively. the signal intensity of the diffusing molecules depends on three factors, as described by equation ( ) [ ] : where i is the observed intensity, i the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [ ] . from equation ( ), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [ ] . the drug design approach based on diffusion nmr is basically a screening technique used to differentiate the binding ligands (drug) from non-binding components [ ] . ligands able to bind should have significantly different diffusion coefficients (d) compared to non-binding ligands [ ] , i.e., the diffusion coefficients of binding ligands will be smaller than those of non-binding ligands [ ] . thus, diffusion-based nmr is a way of effectively "filtering" and identifying which ligands are binding [ ] . diffusion-based nmr spectroscopy has advantages in ligand based screening applied to drug discovery. for example, diffusion ordered spectroscopy (dosy) does not require prior separation/purification of the ligand/target solution [ ] . diffusion based nmr allows simultaneous determination of diffusion coefficients in multicomponent systems containing large molecules (i.e., proteins) and possible binding partners (i.e., small drug compounds) [ ] , and no special labeling or contrasting agents are required, though their use is not exclusively inhibited (for an example of the use of labeled compounds in diffusion nmr spectroscopy, see [ ] ). a problem occurs when there is significant chemical shift overlap between the binding molecule signals and the target. this situation makes it hard to distinguish the nmr signals [ ] , and the calculations typically assign an intermediate value to the diffusion rate (i.e., one gets a smear). multidimensional diffusion nmr pulse sequences are available [ ] , which may help resolve spectral overlap in d experiments [ ] . another issue is that molecules in chemical databases may have generally low solubility [ , ] . low solubility decreases the overall signal intensity and therefore makes accurately measuring diffusion experiments far more difficult [ ] . there are many examples demonstrating the successful application of diffusion nmr in examining drugs of pharmaceutical interest [ ] , and ligand-target interactions [ ] . hajduk et al. [ ] exploited the changes in diffusion rates to detect ligands that bind to the fk binding protein and the catalytic domain of stromelysin. nishimura et al. [ ] utilized dosy, in combination with noesy to determine the orientation of two guest molecules, p-ethoxyiodobenzene and -iodo- -methoxynaphthalene, within a host composed of a tetrakis( -hydroxyphenyl)-cavitand and a tetra( -pyridyl)-cavitand. furthermore, matthias et al. [ ] used h molecular diffusion and f spin diffusion to probe the drug loading properties of the rf-peg hydrogel for -fluorouracil (fu) and , -dimethyl- -fluorouracil (dmfu), two anticancer drugs. dosy can be combined with saturation transfer difference (std, discussed earlier in this review) to yield new insights about ligand-target interactions. kramer et al. [ ] combined std with dosy to analyze a mixture composed of wheat germ agglutinin and two derivatives of n-acetyl glucosamine (ligands). using this new technique they were able to obtain high quality spectra of the components in the mixture. tanoli et al. [ ] also combined std and dosy to explore the interactions of smaller molecules with bovine serum albumin. these are just a few examples to show that diffusion nmr spectroscopy has played, and will continue to play, a prominent role in drug design. in silico (virtual) screening is now a standard technique in drug design and discovery [ ] that has been in use since at least [ ] , though the exact origin of the phrase "in silico" is not clear [ ] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [ ] that inhibit protein-protein interactions [ ] , and its ability to help identity ligand (drug) binding sites on the target of interest [ ] to lend insight to the mechanisms of action for lead compounds [ , ] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [ ] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [ ] . virtual screening has also been used to identify candidates for anti-viral drugs [ ] and anticancer drugs [ ] . several chemical databases are available both for public and academic use [ ] . virtual screening is properly identified as a high-throughput screening (hts) technique [ ] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, ( ) a three-dimensional model of the ligand (drug), and ( ) a three-dimensional model of the receptor (protein) [ ] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [ ] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [ ] , such as differential scanning fluorimetry [ ] , fluorescence polarization, and surface plasmon resonance [ ] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how virtual screening works, and how it applies to drug design outside of its combination with nmr is well documented in additional reviews [ , , [ ] [ ] [ ] [ ] [ ] . a prime example of the complementarity between nmr screening and virtual docking is found in the work of chen et al. [ ] , in which the authors sought to target the a a adenosine receptor (a a ar) protein, a drug target for the treatment of parkinson's disease [ ] . they used virtual screening and an nmr-based screening method against the same molecules in a fragment library so they could compare the results of both methods. the virtual screen successfully predicted (based on calculated binding affinities) four out of the five orthosteric ligands discovered by nmr that were within the top % of the fragment library, showing that the two separate methods can give similar and reliable results. later on, chen et al. discovered that virtual screening picked up three additional fragments that remained undetected by the nmr-based method, and were, in fact, a a ar ligands; this shows that though neither method is flawless, they are still perfectly complementary approaches for drug design [ , ] . in another scientific work that integrated nmr with virtual screening, di lello et al. [ ] found small molecular inhibitors of the enzyme ubiquitin specific protease (usp ), a key regulator of the tumor suppressor protein, p [ ] . a fragment screen by nmr revealed a series of small molecules that bind in the active site of usp near the catalytic cysteine (amino acid ). a ligand-based virtual screen utilizing the fastrocs program identified~ hit molecules, several of which were further characterized by h- n trosy chemical shift perturbation and line broadening to probe the binding site of the active hits. di lello. also tested the active compounds against eol- cells to verify the hits as identified by virtual screening and further characterized by nmr, showing that the active compounds do indeed inhibit usp activity. through additional study of the active molecules and further optimization of their structures, they eventually discovered a series of ligands that bind in the "palm" region of the catalytic domain of usp , inhibiting its catalytic activity [ ] . this study clearly demonstrates that nmr screening-based techniques can be combined with virtual screening to find viable drugs for targets of interest. additional examples of the successful integration of nmr and virtual screening as applied to protein targets are also found in the literature, further demonstrating the practicality and complementarity of virtual screening and nmr [ , [ ] [ ] [ ] . for example, li et al. [ ] used virtual screening filtered by nmr to identify and characterize non-metal chelating metallo-β-lactamase (mbl) inhibitors, and in particular, verona integron-encoded mbl (vim)- , when previously there were no clinically significant inhibitors of mbl, since mbl enzymes hydrolyse many, if not all, β-lactam antibacterials compounds specifically designed to inhibit their activity [ ] . furthermore, shan et al. [ ] and bertini et al. [ ] both used virtual screening and nmr, in their respective studies. through the combined use of nmr and virtual screening, shan et al. was able to identify, design, and synthesize novel pdz domain inhibitors, which are proteins implicated in tumorigenesis [ ] . bertini et al. was able to combine nmr to study the interaction of ligands with metalloproteinases, using known inhibitors of metalloproteinases as a starting point [ ] . while hsqc noesy nmr data provided structural and spatial constraints for the proposed d models, virtual screening was used to refine the models, and to probe the ligand-protein interaction. in each case (i.e., ligand-protein interaction), bertini et al. was able to obtain a well-defined ligand conformation in the protein binding site, thus offering a viable alternative to other approaches described in the literature [ ] . clearly, combining virtual screening with nmr-based methods is advantageous in studying how ligands (drugs) bind and interact with targets (proteins) of interest. paramagnetic nmr (pnmr) can also play a prominent role in drug discovery [ ] , as pnmr can provide key structural information in situations where crystal structures cannot due to the weak binding of ligands [ ] . pnmr can be used to quantify the binding between ligands and large biomolecules such as proteins, dna, and rna [ ] . pnmr depends on the presence of a group (called the paramagnetic center) with an unpaired electron [ ] , and since many naturally occurring biomolecules and organic compounds lack a paramagnetic center, one such as caged lanthanide (clanp) [ ] , must be introduced artificially [ ] . once the paramagnetic center (often a metal ion) is present, paramagnetic effects can be used to measure the distance and the relative orientation (i.e., angle) between molecules [ ] . this information is crucial when it comes to determining how ligands and substrates bind. thus, pnmr is quite a useful technique for drug discovery when a paramagnetic center is present. the most relevant consequence of pnmr for drug discovery is paramagnetic relaxation enhancement (pre), although there are a number of studies demonstrating the use of pseudocontact shift (pcs) effect in drug discovery research [ ] . paramagnetic relaxation enhancement (pre) is proportional to the inverse sixth power of the distance between the paramagnetic center and the nucleus of interest (i.e., h), although it does not reveal anything about relative orientation [ ] . pre can give quantitative information in the range of - angstroms [ ] . several researchers have taken advantage of this outstanding property to study the structural and dynamic properties of complex biomolecular machineries in their native environment [ ] . for example, iwahara et al. ( ) demonstrated that a protein's binding polarity to dna can be determined by pre, using edta-derivatized deoxythymidine (dt-edta) with a chelated metal ion (such as cu + or mn + ) as a probe. dt-edta with a chelated metal ion is a convenient choice, as it can be inserted into any position of a synthesized oligonucleotide. with data derived from the pre effect, one can easily determine the polarity of the protein (or drug) binding to dna [ ] . several researchers have investigated dna as a drug target [ ] , and the study of iwahara et al. clearly demonstrates, and even indicates, that pre can potentially be used to study the interactions between a drug and dna [ ] , provided that a paramagnetic center such as dt-edta or a metal ion is present. brasuń et al. [ ] also used pre derived distances between a paramagnetic center and a nucleus of interest. they replaced the cys-s-s-cys bridge found in oxytocin and vasopressin with the his-cu + -his motif to investigate if doing so would alter the stability of oxytocin and vasopressin. they determined the distances between the cu + ion and h nuclei (possible because of pre), and used these values to generate three-dimensional models of the his-cu + -his motifs in both oxytocin and vasopressin. in doing so, they indicated that such an approach using pre can help in designing new biologically active compounds [ ] , and hence in drug discovery research, as many drug discovery studies require a reliable models for the successful generations of hit-lead molecules, especially in the case of in silico docking [ ] . this study again proves the usefulness of pre, and therefore, pnmr, in drug discovery research. in two additional studies, huang et al. [ , ] used pre in their individual studies of protein binding and protein dynamics, respectively. in the huang et al. case [ ] , these authors used pre to establish a model of the binding between the g-actin protein, and thymosin β , an actinbinding protein. using pre determined constraints (distances) and h- n hsqc, they were able to establish a well-converging docking structure of the g-actin/thymonsin β complex [ ] . on the other hand huang et al. [ ] did not measure protein binding, but studied the conformational changes and dynamics of select large membrane proteins utilizing f-nmr spectroscopy, and ni + as the paramagnetic center. through a series of extensive experiments, they showed that conformational exchange rates of membrane proteins can be determined from measurements of the metal-enhanced longitudinal relaxation (i.e., pre) of the f nuclei [ ] , thus yielding additional information (i.e., protein conformation dynamics) that could be utilized in drug discovery projects targeting proteins (i.e., understanding how the protein changes shape based on its environment can be used to find potential binding sites for drug candidates). all these examples prove that pnmr is powerful approach in drug discovery research, given that pre can aid in generating trustworthy models of interacting molecules, and that it can help researchers understand better how the molecules interact in the first place. since the late s solid state nmr (ssnmr) has demonstrated its usefulness in complex biomolecular systems such as collagen or lipid bilayers [ ] . however, over the past years ssnmr has gained attention in the field of drug design and is slowly becoming a commonly used technique as its proving to be a powerful tool for structural analysis of membrane proteins and amyloid fibrils [ ] [ ] [ ] . ssnmr is becoming a more attractive alternative for several different reasons. one of them is the fact that it enables the characterization of a chemical compound in a solid-state form such as in a tablet/pill [ ] [ ] [ ] . moreover, ssnmr is not only restricted to analyzing the chemical structure but it can also provide insight into the physical properties of a compound such as polymorphism (different crystalline structures of the same compound), disorder (crystal defects and amorphous solids in the compound) or the presence of cocrystals (multicomponent crystal made of a compound and one or more small organic molecules) [ , ] . ssnmr can also be used to quantify the amount of crystalline against the amount of amorphous material in the sample to establish phase purity (the amount of desired phase separated from other, undesirable phase) [ ] [ ] [ ] . ssnmr differs from liquid state nmr by the presence of anisotropic interactions. in liquids nmr these effects are averaged to zero as a consequence of rapid molecular tumbling. in solid state however, the molecules are not tumbling rapidly and the residual effects of anisotropic (orientation depended) interactions such as anisotropic chemical shift, magnetic dipolar coupling, and quadrupolar coupling could be observed in the form of broad peaks, with could be much wider than the chemical shift range of the nucleus [ , , ] . as a results, there has been a constant effort to improve the sensitivity and resolution of solid state nmr spectra, which increased the potential of ssnmr in future applications [ ] . one of the methods that works for nuclei with spin value of i = / is called magic-angle spinning (mas). it increases the resolution by rapidly rotating the sample around a fixed (or so-called magic) angle of . • [ ] . this method can be combined with decoupling, to remove the dipolar couplings between spins. this is done by applying radiofrequency pulses or cross-polarization (cp) transfer of magnetization from abundant and sensitive nuclei such as h to less sensitive such as c [ , ] . a broader comparison between ssnmr and liquid state nmr is provided in [ ] . as mentioned before, ssnmr can provide information about membranes and membrane proteins. for this reason, ssnmr can be used to detect interactions of ligands with receptors embedded to the membrane which enables the mapping of binding site of a receptor by utilizing cp-mas (cross-polarization magic-angle spinning) nmr and site specific mutagenesis [ ] . ssnmr can provide the conformation of ligands bound to the receptor which can then be used to optimize future drug in terms of better affinity and efficiency [ ] . since ssnmr is also applicable to amyloid research, it can be used for probing polypeptide structures of amyloid and intermolecular contacts between fibrils. the potential is for the design a drug that will inhibit the process of aggregation of proteins and peptides [ ] . lastly, since ssnmr gains insight into physical properties of a chemical compound it can be used for control of the process of formulation and processing of a drug to help assess the purity of a compound [ ] . an example of ssnmr application related to drug design is the work of callari et al., who monitored the effect of drug loading on the properties of micelles [ ] . polymer micelles are widely used as nano-carries for drug delivery, but so far the effects of drug loading on the morphology of a drug carrier had not been thoroughly investigated [ ] . they created a model consisting of a fructose hydrophilic block and a pmaa block (micelle), to which a different amount of platinum complex was anchored. the results from this experiment showed that micelles loaded with a higher amount of platinum complex had reduced cellular uptake, release, and cytotoxicity. the micelles with a lower load (ll) of platinum complex were more effective at targeting cancer cells (of cell lines mda-mb- (breast cancer) and a (lung cancer) than the micelles with a higher load (hl) of the platinum complex. this is evidenced by the lower ic (half maximal inhibitory concentration) values of the ll micelles as compared to the hl micelles. both of those results could be related to the micellar structure and their potential for interaction between the sugar moieties and the cell wall [ ] . another example of practical application of ssnmr is the work of lee and colleagues [ ] in which they investigated the structure of a designed zinc-binding amyloid fibril that catalyzed ester hydrolysis. metals ions such as zinc where found to affect the process of protein aggregation which resulted in arise of amyloid like structures. therefore, understanding the processes of aggregation and the factors related to them is crucial for creation of new drugs for amyloid related diseases [ ] . in the experiment lee et al. used ac-ihvhlqi-conh peptide (referred as hhq) to form fibrils with varying zn + :hhq molar ratios. the results showed that zn + -bound hhq fibrils form parallel-in-register form of packing β-strand in each sheet and his residues are coordinated to zn + via nδ , while half of the his residues are also coordinated to zn + via nε . additionally, zn + binds in a : metal ion/peptide ratio. after further analysis using structural bioinformatics, it was concluded that each zinc ion was coordinated by three histidine nitrogens from two adjacent strands. half of all histidines bridged to zn + ions forming a metal-imidazolate chain [ ] . a "hit" is a molecule identified from a screening technique (hts, fbdd, etc) as having a desirable effect (i.e., decreased cellular growth, high affinity score) on a target [ , ] . however, the question of whether the activity is related to actual binding to the target, or to interference with one of the components of the assay readout mechanism, is uncertain. thus, a validation step is required. hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [ , ] . one of the highest-impacts of nmr on drug discovery is the use as a hit-validation tool. though the hit-validation or confirmation of drugs is mostly limited to the solution state [ ] , this aspect of nmr truly is a "gold standard" technique in drug discovery. nmr by itself is a powerful tool for drug validation as in the case of sharma et al. ( ) [ ] who sought to identify potential drug-like inhibitors against l-aspartate α-decarboxylase (adc) an enzyme responsible for the decarboxylation of l-aspartate in order to generate β-alanine and carbon dioxide [ ] , in mycobacterium tuberculosis. they began with known inhibitors of adc, and developed a protocol to measure the enzymatic activity of adc. upon addition of adc to a solution of l-aspartate, l-aspartate gradually disappeared because adc was converted to l-aspartate to β-alanine; therefore the peak intensity of l-aspartate decreased, and the peak intensity of β-alanine increased in the presence of adc (no inhibitor drug present). using this newly developed nmr-based protocol allowed direct measurement of adc enzymatic activity, and sharma et al. were able to confirm the enzymatic inhibiting activity of seven previously discovered inhibitors of adc [ ] . this study demonstrated that nmr can be an effective validation tool for known drugs and for new drugs generated by a screening approach. nmr is also able to remove false positives that emerge from biochemical screens [ ] . for example, an aptly named technique called a la assay to detect reactive molecules by nuclear magnetic resonance (alarm nmr) is able to eliminate false positives from hts methods [ ] , and in the presence of a test compound or mixture, measures dithiothreitol (dtt)-dependent c chemical shift changes of the human la antigen [ ] . dahlin et al. provided an updated protocol of alarm nmr to aid researchers in the production of the c-labeled la antigen reporter protein, in testing compounds with the la protein, and in the analysis of obtained nmr spectra. using alarm nmr prioritized hits identified from hts screening [ ] . an example of alarm nmr is found in the work of dahlin et al., where they used this technique to test molecules that were assumed to be inhibitors of histone acetyltransferase (hat) inhibitors, and from their studies, actually discovered that % ( out of ) of the most commonly reported hat inhibitors were actually faulty. they were actually nonselective interference compounds, not necessarily specific to the inhibition of hat [ ] . thus, alarm nmr (and nmr in general) served as a useful validation method, especially for unvalidated hits identified from biochemical screens [ ] or other screening techniques. the last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits. of course, nmr is not the sole technique used for drug validation. most often, nmr drug validation is coupled with additional methods [ ] such as surface plasmon resonance (spr) [ , ] x-ray crystallography [ ] [ ] [ ] , isothermal calorimetry (itc) [ ] , uv-vis and/or fluorescence spectroscopy [ ] . the work of goudreau et al. is an excellent example of combining nmr with another biophysical technique, in this case x-ray crystallography, for drug validation [ ] . a series of benzodiazepine inhibitors of human immunodeficiency virus (hiv- ) was identified using an in vitro capsid assembly assay, and further characterized by f-nmr. analysis of the chemical shift perturbation and line broadening effect on the f-nmr spectra of the benzodiazepine inhibitors revealed the specificity and reversibility of the binding inhibitors. the same set of f-nmr spectra were used to identify the n-terminal domain of the capsid as the binding site of the benzodiazepine inhibitors. the specific amino acids involved in the binding of the benzodiazepine inhibitors were identified from the chemical shift perturbation of h, n-trosy nmr spectra. later, use of x-ray co-crystallography confirmed binding locations of the benzodiazepine inhibitors and their binding modes, which was useful for further development and optimization of the benzodiazepine inhibitors [ ] . the work of goudreau et al. therefore showed how nmr could be used as a co-validation technique with another biophysical method [ ] . nmr can be also coupled with multiple biophysical techniques to validate a molecule's ability to inhibit protein-protein interactions (ppis) [ ] . an example of the combination of nmr with spr and x-ray crystallography can be found in the work of fry et al., where the authors sought to understand how the nutlin molecule inhibits mdm -p , a protein-protein interaction that has been an important cancer therapy target for several years [ ] [ ] [ ] . fry et al. [ ] gradually deconstructed rg , the first nutlin molecule to enter clinical trials [ ] , into fragments so they could study the inhibitory effect of rg on the mdm -p interaction by spr, nmr, and x-ray crystallography. spr was used to determine the k d values of the rg fragments and confirmed that rg and some of its fragments do bind to mdm , inhibiting the mdm -p interaction. h, n-hsqc nmr chemical shift perturbation was also used to assess and verify binding identified by spr. of the six fragments of rg confirmed by h, n-hsqc nmr as binding to mdm , spr showed binding for five of them; thus, the two separate techniques were in good agreement with each other. the fragments of rg that were confirmed to bind by both spr and h, n-hsqc nmr were further studied with x-ray crystallography, which can tell precisely where and how the molecules bind to the protein. using co-crystallization, fry et al. were able to obtain structures for several of the verified binding fragments in complex with mdm and were able to visualize the binding of the fragments to the mdm protein [ ] . nmr is obviously a powerful drug binding validation tool, but it becomes much more powerful when coupled with additional biophysical techniques, as seen in the work of fry et al. [ ] . dias et al. [ ] took a similar approach as fry et al. [ ] in that they took known inhibitors of a protein-protein interaction, and dissected them into individual fragments to assess a protein's drug-ability. the interaction studied was that between the proteins von hippel-lindau (vhl), and the alpha subunit of hypoxia-inducible factor (hif- α). twelve compounds (known inhibitors and derived fragments) were developed using a crystal structure of hif- α peptide bound to the stable multiprotein complex pvhl-elongin c:elongin b (vcb). each of these compounds was screened using three separate nmr techniques, saturation transfer difference (std), carr-purcell-meiboom-gill (cpmg) relaxation experiments, and waterlogsy (to assess drug binding and to predict drug binding mode. each compound that was unambiguously detected (i.e., the molecule was identified as successfully binding by at least two of the three nmr methods of std, cpmg, and waterlogsy) was subjected to further analysis by itc and x-ray crystallography. itc was used to determine the dissociation constants of binding molecules, and x-ray crystallography was used to confirm the binding mode predicted by the nmr studies. generally speaking, the designed fragments had similar ligands efficacies compared to the parent molecules but had much higher dissociation constants (k d values), meaning that the fragments bound less tightly than the original parent molecule [ ] . with this example, it is possible to see the strength of using nmr as its own hit-validation tool (i.e., three different nmr techniques were used for screening compounds [ ] ), and yet, the follow-up of nmr studies with itc and x-ray crystallography was useful in providing a basis for assessing the drug-ability of a protein-protein interaction [ ] [ ] [ ] [ ] . thus, it is clear to see that nmr is a prominent method of hit-validation in drug discovery research, especially in combination with other biophysical techniques. substantial progress has been made in the nmr field over the past - years, and various methods were established to determine the drug-target complexes. most of them utilize either noe or chemical shift perturbations (csp) although in silico models/programs, using nmr-derivate data also exist. one of the methods called difference of inversion recovery rate with and without target irradiation (direction) is used to map pharmacophores and can be an alternative to std experiments. this method uses the difference between longitudinal relaxation rates of ligand protons with-and with-out irradiation of the protons of the target protein. the direction approach, however cannot be used for slowly exchanging (strong binding) ligands. the practical approach of this method was demonstrated on the experiment when analyzed the interactions between p mapk (p a mitogen-activated protein kinase) and its inhibitor-sb [ , ] . the results from this experiment showed that protons h , h , h , and h of sb , are in close neighborhood with the protons of p mapk when compared with h , h , and methyl protons. it indicates that two aromatic rings (a pyridine ring and fluorophenyl ring) of sb interact tightly with p mapk. the results were later confirmed with proton density map of each ligand's proton, based on the crystal structure of sb -p mapk complex [ ] . moreover, the same authors already created a new and improved protein-ligand docking method by combining the direction obtained nmr data with docking software. [ ] . a second method that can be used to map pharmacophores is called inter-ligand nuclear overhauser effect (iloe). this d nmr experiment detects when two ligands bind simultaneously to adjacent sites on a protein surface although both of the ligands do not have to bind to the same binding pocket (opposite to inpharma, see above) [ , ] . a negative ligand−ligand noe signal will be created when ligands bind in close proximity to each other whereas ligands that do not bind will show no noes, or at most very weak positive ones [ , ] . iloe also enables determination of the ligand orientations with respect to one another [ ] . as in the case of inpharma, iloe can be utilized even in the absence of a d protein structure and used with large proteins. additionally, iloe differs from inpharma in mixing times-for iloe the mixing times are typically in the range of - ms [ ] . application of iloe was first shown on glycolate+nad + in the presence of porcine heart lactatedehydrogenase, and by glucose- -phosphate+nadph in the presence of l. mesenteroides glucose- -phosphatedehydrogenase and from that time it has been widely used [ , , ] . a third method called structural information using overhauser effects and selective labeling (sos-nmr), relies of std experiments performed on ligand complexes with different protein samples that have been fully deuterated excluding a specific type of amino acid. in other words, the data obtained by sos-nmr gives insight into the ligand-binding amino acid composition and when taken into consideration the d structure of targeted protein can be used to establish the structure of protein-ligand complex. this approach has been demonstrated using two complexes-fkbp complexed to -( -pyridyl)-benzimidazole and mura complexed to uridine diphosphaten-acetylglucosamine (udp-glcnac). the results showed that for fkbp and mura, only four and three amino acids (fkbp: ile, val, leu, met; mura: trp, phe, his) were needed to be selectively protonated in perdeuterated samples to establish the ligand-binding site. additionally, on average only amino acids were required for accurate identification of ligand-binding surface. according to authors sos-nmr can greatly improve the early stages of the drug discovery process [ ] . moreover, combining sos-nmr with other methods can even further increase chances for a positive outcome of an experiment [ ] . a completely different approach to this topic was taken by chen et al. [ , ] . instead of using isotope labeling, chen's group decided to use a tert-butyl group contained within ligand- to obtain structural information about the protein-ligand complex [ ] . the tert-butyl group formed an intense singlet in . to . ppm range thanks to rapid methyl rotation and methyl reorientation within that group. when compared with the protein's h-nmr signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as bacillus stearothermophilus dnab hexamer ( kda) [ ] . additionally, the tert-butyl group produces intense noesy cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable. this is partially because the signal corresponded to nine protons within tert-butyl group. those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein. as a result, it allows positioning of the ligand on the protein. an example of this approach, is dengue virus ns b-ns protease from serotype (referred as denpro) in complexed with ligand containing a tert-butyl group. the result of this experiment showed noes between the tert-butyl group of ligand- and residue val from denpro [ ] . solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy (salmon) is another method based on the data obtained via nuclear overhauser effect. this method utilizes waterlogsy [ ] to probe for solvent accessibility to the ligand and determine the orientation of the ligand by analyzing signal changes in waterlogsy spectra (positive signal from unbound ligand vs. negative for protein-bound ligands). this method was first used to determine the orientation of prodrug called tretazicar (( -(aziridin- -yl)- , -dinitrobenzamide) known as cb in nqo (quinone oxidoreductase ) binding site. previous attempts had been made to obtain the orientation of tretazicar bounded to nqo , however the results obtained from x-ray crystallography were inconclusive as two orientations of tretazicar could be possible. the information obtained via salmon showed that the side chain of asparagine at position formed a hydrogen bond with -nitrogroup of tretazicar, and that the aziridine moiety of tretazicar pointed toward the solvent [ ] . another variant of waterlogsy method called logsy utilizes the titration slopes as a measure of solvent accessibility. the titration slopes are created by a constant increase of protein concentrations. this method also provides more insight into the process of ligand solvation by checking the influence of protein concentration onto the process. this approach was used on the bromodomain of protein (brd -bd ) by mapping epitopes of two ligands interacting with brd -bd and predicting ligands position. the results showed that the triazolopyridazine moiety of both ligands was implanted into the binding pocket of the brd . additionally, the results from logsy titration showed that methyl-group of ligand , aromatic proton of ligand and aromatic proton of ligand exhibit strong water noe. this information enabled researchers to utilize a chemical replacement strategy (substitute bound water molecules by suitable functional groups) for aromatic proton in a series of ligands containing the triazolopyridazine ring. those protons were replaced with an amino or aminomethyl groups and as a result, the binding affinity of those ligands increased -fold. finally, the results obtained from x-ray crystallography for ligands with such modifications allowed to find the binding mode of the triazolopyridazine ring of ligand (with methyl group pointing internally) and the substituted amino group was found to create hydrogen bond to the side chain of asn of brd -bd [ ] . the most recent approach called nuclear magnetic resonance molecular replacement (nmr ) utilizes spatial data obtained through solution-state nmr in order to locate the binding pocket of a complex structure. for that, it uses a receptor model, e.g., a x-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment. to conduct an experiment using such an approach requires a few steps. first, either the protein or ligand used in the complex must be uniformly c and n labeled. then, an experiment to assign the ligand is needed such as d c, h-hmqc or c, h-hmbc. the next step is the evaluation of ligand intra-and ligand-protein intermolecular distances through noe cross peaks obtained from f - n, c-filtered h, h-noesy. lastly, choosing a proper input structure is required which can be either x-ray or nmr structures in apo form, with another bound ligand, or a homolog to the protein of interest. then the nmr program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [ , ] . this method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [ ] [ ] [ ] [ ] . in silico methods combined with nmr derived information can also be used to determine accurate drug-target complexes. . . . samplex another program that can utilize csp called smoothed automatic mapping of protein from listed extremes (samplex) can help to determine the interaction surface of proteins complexes. samplex takes the chemical shifts of the protein of interests in both the free and bound state and corresponding d structure of a protein in the free state. the programs returns a confidence value for each residue to be in a perturbed or unperturbed state ( . as being in a perturbed state, − . as remaining in their unperturbed state). this approach was tested on five examples, one of which was subtilisin bpn' (serine protease) complexed with its inhibitor-chymotrypsin inhibitor . the results showed that residue , and residues - of chymotrypsin inhibitor- were perturbed and residue was in an ambiguous state. to compare, the x-ray crystallography data showed residues and - to be involved in the interaction. for subtilisin bpn' the program predicted residues , , - , - , , - , - and - to perturbed and residues , and to be in ambiguous state. that information was also confronted with the x-ray crystallography data which shown residues - , - , - , , - to be perturbed [ ] . the interactions between targets (proteins) and ligands (small molecules) can be analyzed independently of the biological systems by using 'cell-based' nmr drug design approaches. three basic approaches [ ] are as follows: ( ) compound-detected in-cell nmr, ( ) target-detected in-cell nmr, and ( ) reporter-detected in-cell nmr. these methods, with the exception of compound detected in-cell nmr, differ according to the isotopically labeled structure (protein, cell structure, etc.), which enables nmr detection. a cartoon representation of each of these methods is given in figure . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e. not just a snapshot [ , ] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [ ] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [ ] [ ] [ ] . h d-nmr is used particularly in the analysis of metabolites, while the strength and std nmr is a technique that lies within the compound-detected in-cell nmr method but does not require isotope-labelling of the studied compound. however isotopic labelling of the compound may be used to enhance the quality of the spectra. in the target-detected in-cell nmr only the target of interest is isotopically labeled (i.e., n labeled protein). for instance, target proteins can be isotopically labeled during cell growth in isotopically enriched ( c, n, or both n/ c) media [ ] . the cell type and the labeling method may vary across experiments. different cell types, including bacteria [ ] , oocytes [ ] , yeast cells [ ] , mammalian cells [ ] , hela cells [ ] and even insect cells [ ] have been reported in the literature. the fact that in-cell nmr applies to more than one cell type testifies of the versatility and potential application of this technique. in terms of labeling, n is one of the most commonly used approaches [ ] when the targets of interest are proteins. recently, f labeling has been reported as a useful probe for protein-ligand interactions [ ] . it was shown that f can reveal information about the dynamics of protein-ligand interactions [ ] . methyl groups [ ] have also been used as probes for proteins and complexes in vivo [ ] , proving that labeling specific chemical groups instead of the entire biomolecule (i.e., protein) is feasible. in-cell nmr extends beyond proteins, and has been applied successfully to dna [ , ] and rna molecules [ , ] . telomeric repeats have also been studied using target detected in-cell nmr [ ] . the reporter-detected in-cell nmr technique isotopically labels neither the ligand nor the target, but rather a receptor that indirectly measures the effects of ligand-target binding [ ] . the "reporter" varies according to the experimental context. for instance, dose et al. [ ] used acetylation-and deacetylation-based assays to monitor the activity of histone deacetylase and acetyl-transferase. thongwichian et al. [ ] used peptide-based reporters to identify active kinases and phosphatases in cellular conditions. lastly, doura et al. [ ] designed a f probe that operates in biological conditions in order to study the adherence and dynamics of proteins found in human blood. in many viruses and phages, scaffolding proteins (sps) are required to ensure the correct organization of coat proteins (cps) and other minor capsid proteins into a precursor structure, called a procapsid [ , ] . although sps are critical for viral assembly and therefore potential therapeutic targets their structural properties (with only a few exceptions [ , ] ) are poorly understood. the size limitation of nmr can be used advantageously as a filter to identify disordered segments even in very large supramolecular protein complexes. in this way, nmr can provide a unique perspective on the dynamic and disordered elements of macromolecules not accessible by other techniques. the procapsid encapsulation experiments described by whitehead et al. [ ] were conceptually analogous to in-cell nmr experiments [ ] [ ] [ ] in which signals from small proteins, or flexible segments of proteins, can be observed when they are incorporated inside living cells, as long as the isotope-labeled proteins of interest do not interact strongly with other large cellular components [ ] [ ] [ ] . the so called "in-virus" nmr strategy applied by whitehead et al. [ ] could be more generally used to study the dynamic properties of macromolecules encapsulated into virus particles, including cargo molecules encased in viral capsids for nanotechnology applications. additionally, such studies could assess the level of interaction of cargo molecules with the virus and probe the release properties of cargo nmr [ ] . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e., not just a snapshot [ , ] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [ ] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [ ] [ ] [ ] . h d-nmr is used particularly in the analysis of metabolites, while the strength and intensity of the recorded signals is directly proportional to the concentration of the sample [ , , ] . h d-nmr can also be used to follow "real-time" analysis of different molecules [ ] . in the d h-nmr experiment, there are no polarization transfer techniques required (the h atom is already highly sensitive) and covers a spread of interesting nuclei in the molecule(s) being studied [ ] . assuming that the sample can be stored stably for extended periods of time, the non-destructive nature of nmr permits the re-use on the sample for different experiments [ , ] . aiding in the reproducibility of nmr [ ] , sample-recycling offers a significant advantage of nmr [ , ] . while always important in drug design, sensitivity and resolution of nmr are two major factors that need special consideration. since both factors improve with increasing magnetic field strength, we have seen a spike in the demand for ultra-high-field nmr spectrometers. recently, . t (i.e., . ghz for h) magnets have become commercially available, and with recent advances in magnet technology, such as liquid helium recycling and magnetic field shielding [ ] , nmr has begun to offer far better resolution and higher sensitivity while reducing the substantial costs of maintaining the instruments compared to past decades. other steps have been taken to enhance the sensitivity of nmr including: the development of cryoprobes increasing the signal to noise ratio to times, and micro-coil probes that not only increased sensitivity but also reduced the amount of sample required for the measurements [ ] . from another perspective, one can further optimize the process of obtaining spectra by using different methods of measurements. one of these, called sofast, helps to reduce the delay between scans resulting in lowering acquisition time for d experiments such as hmqc utilizing h, n or h, c [ , , ] . the basic principle of this method is to use selective h pulses that will excite only a small portion of the available nuclei pool, while the unperturbed spins provide a magnetization "heat sink" thus improving the spin-lattice relaxation (t ) rate via dipolar interactions [ ] . these methods can be highly efficient when studying drug binding and molecular interactions [ ] . another method, called ultrafast d nmr, enables obtaining a d spectra within a single scan but with the associated cost of reduced sensitivity [ , ] . the principle of this method is to divide the sample into n number of fractions, and apply the appropriate incremental aspect to each fraction, while recording them all simultaneously within the one scan [ ] . this method has been applied to many metabolomic studies [ , , ] as well as in the analysis of natural products [ ] . the difficulty is that the effective concentration of the sample is lowered by the fractionation level. the more slices the sample is split into, the greater the reduction of combined signal that is obtained. lastly, a method termed non-uniform sampling (nus) may provide an advantage by reducing the total time for measurement while maintaining the same resolution of spectra [ ] . nus effectively skips over parts of the total dataset, collecting only around to % of the total. usually sections containing higher concentrations of signal (over noise) are emphasized in the selection scheme, known as non-linear data acquisition. these methods reconstruct the complete data subset by applying various algorithms such as multidimensional decomposition (mdd), which essentially separates the sets of multidimensional data into one dimensional problems that are much easier to solve given the common process of signal overlapping in multidimensional nmr spectra [ ] . other algorithms such as compressed sensing (cs), maximum entropy method (max ent) and iterative soft threshold (ist) each have their own advantages but all focus on decreasing the time needed for collection of spectra [ , ] . the cost is in signal to noise, as no gain is ever absolutely free. the inherent advantages of nmr do not eliminate the disadvantages (table ) , which are namely being limited for some nuclei, and inherent insensitivity for many types of experiments. nmr can provide high quality resolution and sensitivity for some experiments [ , ] but the application can be challenging. when the experiments become multidimensional, there is often a tradeoff between the improved higher resolution and/or the resulting sensitivity and/or the amount of time an experiment takes, i.e., high-quality spectra for multidimensional experiments take much longer than their simple d counterparts [ ] (scheme ). one must always choose between resolution, sensitivity, and the amount time; as you can only ever have two out of the three. advantages disadvantages ability to identify simple chemical compounds. high quality resolution and sensitivity for many d experiments. less time consuming compared to d nmr. compared to d nmr less details can be obtained for more complex molecules. for nuclei other than h and f, relative sensitivity is fairly low-requires extra labeling to obtain better spectra. ability to identify complex molecules and observe different interactions between the nuclei, e.g., correlations between all spins in one spin system using tocsy experiment. requires long times to obtain a proper spectra (up to days). greatly reduces time to obtain d spectra. reduced sensitivity. significantly reduces acquisition time of hmqc. relatively low resolution lowers the time of measurement while keeping the same level of resolution. requires use of reconstruction algorithms since missing data points can lead to artifacts in spectra. multidimensional data are "broken" to one dimensional, which are easier to analyze. ability to resolve overlapping resonances. data must be (approximately) symmetrical (lorentzian shape) to obtain good spectra. good reconstruction of weaker peaks. large computational costs, low performance on noisy data. significant reduction in acquisition time. nominally lorentzian peak shapes may be distorted, and peak intensities may be altered. greatly reduced time to obtain nmr spectra. requires a grid of uniformly sampled data points. often makes stronger binding ligands from weakly binding fragments. less time and resource intensive. can be used only for small fragments of compound of interest. direct observation of target binding to ligand. several types of nmr experiments are possible. inability to distinguish binding modes, difficult to gage the "true" binding site of ligand to protein. only requires a small amount of sample. highly reproducible. allows direct observations of ligand binding. only works for ligands with low binding affinity (fast chemical exchange). inability to distinguish binding modes. in vivo studies are possible, can focus on specific cell parts. special labeling techniques may be required. spectra may be more challenging to interpret. can model protein drug interactions, helps speed up and reduce cost of drug delivery protein models need to be validated through experimental approach. can observe proteins interacting with metal ions, long observation distance ( - angstroms) between paramagnetic left and nearby atoms. paramagnetic left required in the system. enables the characterization of a chemical compound in a solid-state form such as a tablet/pill. provides insight into the physical properties of a compound. significant broadening of the spectral lineshapes due to anisotropic spin interactions. noticeable difference in spectra of binding and non-binding ligands. sets the lower limit of time for which experiments can be performed. unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme ) with fixed available instrument times. scheme can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in costtime matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g. concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme , the d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for d nmr, and still obtain high quality spectra (table ) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [ ] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( c, n) with an inherently low sensitivity [ , ] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [ ] . unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme ) with fixed available instrument times. scheme can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in cost-time matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g., concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme , the d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for d nmr, and still obtain high quality spectra (table ) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [ ] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( c, n) with an inherently low sensitivity [ , ] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [ ] . nmr has become a "gold standard" method in drug design due to its speed, simplicity, and reproducibility. the standardized ppm scale allows one to compare all nmr results and gather them in databases for the common use of researchers. although sample labeling was limiting in the beginning, it has now become a strength of nmr that permits the observation of big molecules and/or biomolecular processes "through the door lock". nmr is the only analytical technique that permits qualitative and quantitative analysis without previous sample purification or separation. high precision nmr data still requires long experiment times and has elevated costs; however, these will no doubt be alleviated in the future. estimating the cost of new drug development: is it really $ million? health aff. (millwood) the price of innovation: new estimates of drug development costs animal testing and its alternatives-the most important omics is economics what is the question? nmr in drug design evaluation of ligand-based nmr screening methods to characterize small molecule binding to hiv- glycoprotein- ramped-up nmr: multiplexed nmr-based screening for drug discovery extraction, characterization, and anticoagulant activity of a sulfated polysaccharide from bursatella leachii viscera ultra-low thermal conductivity in na/sb chalcobismuthates: synthesis, crystal structures, optical properties and na nmr spectroscopy layered copper thioaluminate k cu als : synthesis, crystal structure, characterization and solid-state al and k nmr studies identification and characterization of sse , a microtubule depolymerizing agent that overcomes multidrug resistance amide versus amine ratio in the discrimination layer of reverse osmosis membrane by solid state n nmr and dnp nmr current and future perspectives on the structural identification of small molecules in biological systems nmr experiments for the rapid identification of p=o···h-x type hydrogen bonds in nucleic acids separation and structural elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement by liquid chromatography-electrospray ionization mass spectrometry, infrared spectroscopy and nuclear magnetic resonance spectroscopy three-dimensional nmr spectroscopy of fluorinated pharmaceutical solids under ultrafast magic angle spinning essential parameters for structural analysis and dereplication by h-nmr spectroscopy uplc-hrms and nmr applied in the evaluation of solid-phase extraction methods as a rational strategy of dereplication of phyllanthus spp. aiming at the discovery of cytotoxic metabolites a light responsive two-component supramolecular hydrogel: a sensitive platform for the fabrication of humidity sensors nuclear magnetic resonance study of nanoscale ionic materials variable-temperature nmr and conformational analysis of oenothein, b nmr at low and ultralow temperatures h-nmr study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin spectroscopic studies of the intermediates in the conversion of , , , -tetrahydro- , -anthraquinone to , -anthraquinone by reaction with oxygen under basic conditions heterogeneous intraand intermolecular hydroamination catalysts -(phosphonomethoxy)ethyl]adenine (pmea) and of its n , n , and n deaza derivatives with copper(ii) in aqueous solution online reaction monitoring by single-scan d nmr under flow conditions solvent-and anion-dependent li+-o -coupling strength and implications on the thermodynamics and kinetics of li-o batteries nmr approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes screening protein-single stranded rna complexes by nmr spectroscopy for structure determination intramolecularly stapled amphipathic peptides via a boron-sugar interaction structure determination using solution nmr: is it worth the effort? nmr spectroscopy brings invisible protein states into focus fragment-based screening using x-ray crystallography and nmr spectroscopy utilizing nmr and epr spectroscopy to probe the role of copper in prion diseases combining nmr and x-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective bace- inhibitors. in fragment-based drug discovery and x-ray crystallography real-time structure-based drug development-tutorial: triad's nmr-based structural determinations are smart chemistry method development and validation: quantitation of telmisartan bulk drug and its tablet formulation by h-nmr spectroscopy p and n solid-state nmr study for the development of a novel membrane protein drug-screening methodology functional analyses of target proteins for drug development by nmr band-selective excitation nmr spectroscopy and quantitative time-domain analysis using complete reduction to amplitude-frequency table (craft) to determine distribution coefficients during drug development aggregation ability of three phylogenetically distant anammox bacterial species host-dependent nitrogen recycling as a mechanism of symbiont control in aiptasia recommended strategies for spectral processing and post-processing of d h-nmr data of biofluids with a particular focus on urine anti-cancer agents in saudi arabian herbals revealed by automated high-content imaging synthesis and evaluation of modified chalcone based p stabilizing agents chapter three-paclitaxel synthesis and characterization of a homogeneous and silica supported homoleptic cationic tungsten(vi) methyl complex: application in olefin metathesis ft-icr mass spectrometry, and nmr spectroscopy study of heavy fuel oil recommendations and standardization of biomarker quantification using nmr-based metabolomics with particular focus on urinary analysis marine bacterial transparent exopolymer particles (tep) and tep precursors: characterization and ro fouling potential active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase synthesis, characterization and conformational study of new α,β-unsaturated acylhydrazones based on calix [ ] arene backbone poly-γ-p-biphenylmethyl-glutamate as enantiodifferentiating alignment medium for nmr spectroscopy with temperature-tunable properties elucidation and partial nmr assignment of monosulfated maitotoxins from the caribbean elucidation of the structure of the -amino- , -dibromochalcone epoxides in solution and solid state synthesis, spectroscopy, and theoretical calculations of some -thiohydantoin derivatives as possible new fungicides identification of polyunsaturated triacylglycerols and cc location isomers in sacha inchi oil by photochemical reaction mass spectrometry combined with nuclear magnetic resonance spectroscopy coordination geometry determination of stannane compounds with phosphinoyldithioformate ligands using multinuclear nmr, sn mössbauer and dft methods clathrate structure determination by combining crystal structure prediction with computational and experimental xe nmr spectroscopy synthesis and biological activity of n-(arylsulfonyl) valine hydrazones and assistance of nmr spectroscopy for definitive d structure sández macho, i. use of h-nmr std, waterlogsy, and langmuir monolayer techniques for characterization of drug-zein protein complexes nmr spectroscopy techniques for screening and identifying ligand binding to protein receptors sequential h-nmr assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site nmr-based metabolomic techniques identify the toxicity of emodin in hepg cells h-nmr-based metabolomics study of the toxicological effects in rats induced by new insights about doxorubicin-induced toxicity to cardiomyoblast-derived h c cells and dexrazoxane cytoprotective effect: contribution of in vitro h-nmr metabonomics nmr-based metabonomic study on the subacute toxicity of aristolochic acid in rats solid state nmr and bioequivalence comparison of the pharmacokinetic parameters of two formulations of clindamycin bace is the major β-secretase for generation of aβ peptides by neurons structural basis for the antifolding activity of a molecular chaperone chapter ten-binding site identification and structure determination of protein-ligand complexes by nmr: a semiautomated approach fragment-based drug design nmr in target driven drug discovery: why not? nmr in integrated biophysical drug discovery for ras: past, present, and future nmr in pharmaceutical discovery and development clinical magnetic resonance spectroscopy principles of pulse nmr spectroscopy spin dynamics: basics of nuclear magnetic resonance nmr spectroscopy explained: simplified theory, applications and examples for organic chemistry and structural biology solid-state nmr paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers protein dynamics from nmr an introduction to nmr-based approaches for measuring protein dynamics using nmr spectroscopy to investigate the role played by copper in prion diseases monitoring dendrimer conformational transition using f and h o nmr retention of strong intramolecular hydrogen bonds in high polarity solvents in binaphthalene-benzamide derivatives: extensive nmr studies potential hepatoxicity risk of the shell of herpetospermum caudigerum wall in rats based on h-nmr metabonomics metabolomic analysis of anti-hypoxia and anti-anxiety effects of fu fang jin jing oral liquid integrative drug efficacy assessment of danggui and european danggui using nmr-based metabolomics ardá, a. nmr and molecular recognition of n-glycans: remote modifications of the saccharide chain modulate binding features obtaining d atomistic structure of saccharides from raman/roa/nmr spectroscopic techniques the strengths and weaknesses of nmr spectroscopy and mass spectrometry with particular focus on metabolomics research monitoring dna-ligand interactions in living human cells using nmr spectroscopy situ monitoring of bacteria under antimicrobial stress using p solid-state nmr nmr studies of protein structure and dynamics combined hplc, nmr spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine bioequivalence assessment of two formulations of ibuprofen lc-nmr-ms in drug discovery asics: an automatic method for identification and quantification of metabolites in complex d h-nmr spectra metabolite identification via the madison metabolomics consortium database a gui r package for proficient automatic profiling of d h-nmr spectra of study datasets open source software for identifying and quantifying metabolites in nmr spectra chapter -experimental aspects of nmr spectroscopy practical aspects of n-dimensional data acquisition and processing water suppression that works. excitation sculpting using arbitrary waveforms and pulsed field gradients distortion-free homonuclear spectra of peptides and nucleic acids in water using excitation sculpting water suppression that works. excitation sculpting using arbitrary wave-forms and pulsed-field gradients gradient-tailored excitation for single-quantum nmr spectroscopy of aqueous solutions solvent signal suppression in nmr experiments on ax systems distortionless enhancement of nmr signals by polarization transfer c-nmr-based metabolic fingerprinting of citrus-type crude drugs chemoselective n tag for sensitive and high-resolution nuclear magnetic resonance profiling of the carboxyl-containing metabolome n-cholamine-a smart isotope tag for combining nmrand ms-based metabolite profiling fast reconstruction of non-uniform sampling multidimensional nmr spectroscopy via a deep neural network n and c-sofast-hmqc editing enhances d-noesy sensitivity in highly deuterated, selectively [ h, c]-labeled proteins chapter -new advances in fast methods of d nmr experiments heterogeneous microtesla sabre enhancement of n nmr signals information from combined h and p nmr studies of cell extracts: differences in metabolism between drug-sensitive and drug-resistant mcf- human breast cancer cells assessment of pharmacological treatment of myocardial infarction by phosphorus- nmr with surface coils application of p nmr spectroscopy and chemical derivatization for metabolite profiling of lipophilic compounds in human serum development of a bioreactor system for cytotoxic evaluation of pharmacological compounds in living cells using nmr spectroscopy the organic set of nmr spectra. in and more essential nmr experiments: a detailed guide pulse pair technique in high resolution nmr a reprint of the historical lecture notes on two-dimensional spectroscopy choosing the best pulse sequences, acquisition parameters, postacquisition processing strategies, and probes for natural product structure elucidation by nmr spectroscopy the phase transfer catalysed synthesis of isoflavone-o-glucosides determination of the relative signs of j( p- p) in complexes of tungsten( ) and molybdenum( ) using two-dimensional [ p- p]-cosy- nuclear magnetic resonance chemical shift correlation two dimentional nmr knowns and unknowns in metabolomics identified by multidimensional nmr and hybrid ms/nmr methods product operators, coherence pathways, and phase cycling. part iii: phase cycling artifacts in two-dimensional nmr chapter -correlations through the chemical bond i: homonuclear shift correlation analytical optimization of active bandwidth and quality factor for tocsy experiments in nmr spectroscopy novel selective tocsy method enables nmr spectral elucidation of metabolomic mixtures elimination of zero-quantum interference in two-dimensional nmr spectra h-nmr and ms based metabolomics study of the intervention effect of curcumin on hyperlipidemia mice induced by high-fat diet pharmacometabonomics analysis reveals serum formate and acetate potentially associated with varying response to gemcitabine-carboplatin chemotherapy in metastatic breast cancer patients based metabolic profiling of cells in response to treatment with a hexacationic ruthenium metallaprism as potential anticancer drug the use of coupled hsqc spectra to aid in stereochemical assignments of molecules with severe proton spectral overlap chapter -nmr spectroscopy methods in metabolic phenotyping. in the handbook of metabolic phenotyping high-resolution nmr techniques in organic chemistry doubly compensated multiplicity-edited hsqc experiments utilizing broadband inversion pulses product operator formalism in nmr acac)(γ-acac)(dms)] treatment revealed by a metabolomic h-nmr study a comprehensive discussion of hsqc and hmqc pulse sequences evaluation of band-selective hsqc and hmbc: methodological validation on the cyclosporin cyclic peptide and application for poly( -hydroxyalkanoate)s stereoregularity determination chapter -nmr molecular recognition studies for the elucidation of protein and nucleic acid structure and function metabominer-semi-automated identification of metabolites from d nmr spectra of complex biofluids an improved n relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins improved hsqc experiments for the observation of exchange broadened signals quantification of metabolites by nmr spectroscopy in the presence of chapter -relaxation and dynamic processes basic principles of nmr a complete introduction to modern nmr spectroscopy nmr in drug discovery high-resolution diffusion and relaxation edited one-and two-dimensional h-nmr spectroscopy of biological fluids measurement of spin relaxation in complex systems -multiple-pulse nmr experiments ultrafast nmr t relaxation measurements: probing molecular properties in real time saturation-inversion-recovery: a method for t measurement chapter -the physical basis of the nuclear magnetic resonance experiment. part ii: pulse and fourier-transform nmr spin-lattice relaxation time in drug discovery and design nmr methods to characterize protein-ligand interactions the interaction of dna with intercalating agents probed by sodium- nmr relaxation rates nmr screening techniques in drug discovery and drug design one-dimensional relaxation-and diffusion-edited nmr methods for screening compounds that bind to macromolecules use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma the quest for simplicity: remarks on the free-approach models fast evaluation of protein dynamics from deficient n relaxation data effects of diffusion on free precession in nuclear magnetic resonance experiments modified spin-echo method for measuring nuclear relaxation times measuring t and t relaxation rates an exact solution for r , eff in cpmg experiments in the case of two site chemical exchange nmr-based screening in drug discovery extraction of t from nmr linewidths in simple spin systems by use of reference deconvolution quantitative metabolic profiling of nmr spectral signatures of branched chain amino acids in blood serum isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by h-nmr based metabolomics approach h-nmr-based metabolic profiling of naproxen-induced toxicity in rats h-nmr toxicometabolomics following cisplatin-induced nephrotoxicity in male rats the antitumor effect of formosanin c on hepg cell as revealed by h-nmr based metabolic profiling an nmr-based metabonomic investigation of the subacute effects of melamine in rats metabolomic profiling predicts outcome of rituximab therapy in rheumatoid arthritis response to drug treatment in newly diagnosed epilepsy: a pilot study of h-nmr-and ms-based metabonomic analysis metabolic alteration in obese diabetes rats upon treatment with centella asiatica extract novel , , -thiadiazoles inhibit colorectal cancer via blockade of il- /cox- mediated jak /stat signals as evidenced through data-based mathematical modeling plasma-metabolite-biomarkers for the therapeutic response in depressed patients by the traditional chinese medicine formula xiaoyaosan: a h-nmr-based metabolomics approach metabonomic profiles delineate the effect of traditional chinese medicine sini decoction on myocardial infarction in rats h-nmr spectral identification of medication in cerebrospinal fluid of pediatric meningitis h-nmr-based metabolomics approach to investigating the renal protective effects of genipin in diabetic rats potential metabolomic biomarkers for evaluation of adriamycin efficacy using a urinary h-nmr spectroscopy h-nmr-based serum metabolomics reveals erythromycin-induced liver toxicity in albino wistar rats nmr-based metabonomics study on the effect of gancao in the attenuation of toxicity in rats induced by fuzi nmr-fragment based virtual screening: a brief overview discovering high-affinity ligands for proteins: sar by nmr when fragments link: a bibliometric perspective on the development of fragment-based drug discovery current perspectives in fragment-based lead discovery (fbld) adaptation of high-throughput screening in drug discovery-toxicological screening tests high-throughput screening: the hits and leads of drug discovery-an overview review of applications of high-throughput sequencing in personalized medicine: barriers and facilitators of future progress in research and clinical application review article: high-throughput affinity-based technologies for small-molecule drug discovery design principles for fragment libraries: maximizing the value of learnings from pharma fragment-based drug discovery (fbdd) programs for use in academia how size matters: diversity for fragment library design high-throughput screening: update on practices and success industrial scale high-throughput screening delivers multiple fast acting macrofilaricides virtual high throughput screening using machine learning methods introduction to fragment-based drug discovery. in fragment-based drug discovery and x-ray crystallography the influence of lead discovery strategies on the properties of drug candidates biophysical screening in fragment-based drug design: a brief overview high-throughput screening and fragment-based design: general considerations for lead discovery and optimization nmr screening and hit validation in fragment based drug discovery structural biology in fragment-based drug design fragment-based drug discovery using nmr spectroscopy chapter nine-practical aspects of nmr-based fragment screening fragment-based drug design nmr-based screening: a powerful tool in fragment-based drug discovery nmr in drug discovery: a practical guide to identification and validation of ligands interacting with biological macromolecules applied biophysical methods in fragment-based drug discovery concepts and core principles of fragment-based drug design application of fragment-based drug discovery to versatile targets a three-stage biophysical screening cascade for fragment-based drug discovery protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry twenty years on: the impact of fragments on drug discovery churcher, i. fragment-based drug discovery-from hit discovery to fda approval: lessons learned and future challenge the first drug approved for braf -mutant cancer venetoclax: evidence to date and clinical potential fragment-based drug design: strategic advances and lessons learned krimm, i. fragment linking strategies for structure-based drug design discovery and development of venetoclax, a selective antagonist of bcl- an inhibitor of bcl- family proteins induces regression of solid tumours x-ray and nmr structure of human bcl-x l, an inhibitor of programmed cell death ribociclib for the first-line treatment of advanced hormone receptor-positive breast cancer: a review of subgroup analyses from the monaleesa- trial the csf receptor inhibitor pexidartinib (plx ) reduces tissue macrophage levels without affecting glucose homeostasis in mice structure-guided blockade of csf r kinase in tenosynovial giant-cell tumor characterizing and overriding the structural mechanism of the quizartinib-resistant flt "gatekeeper" f l mutation with plx discovery and chemical development of verubecestat, a bace inhibitor for the treatment of alzheimer's disease discovery of the -imino- , , -thiadiazinane , -dioxide derivative verubecestat (mk- )-a β-site amyloid precursor protein cleaving enzyme inhibitor for the treatment of alzheimer's disease fragment-based drug discovery applied to hsp . discovery of two lead series with high ligand efficiency discovery of ( , -dihydroxy- -isopropylphenyl)-[ -( -methylpiperazin - -ylmethyl)- , -dihydroisoindol- -yl]methanone (at ), a novel inhibitor of the molecular chaperone hsp by fragment based drug design optimization of pyrrolamide topoisomerase ii inhibitors toward identification of an antibacterial clinical candidate (azd ) using fragment-based approaches to identification of n-( -piperidinyl)- -( , -dichlorobenzoylamino)- h-pyrazole- -carboxamide (at ), a novel cyclin dependent kinase inhibitor using fragment-based x-ray crystallography and structure based drug design cyclin-dependent kinase inhibitor at as a potential drug for mycn-dependent neuroblastoma biological characterization of at , a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines development of second-generation cdk inhibitors for the prevention of cisplatin-induced hearing loss practical fragments: fragments in the clinic: edition sar by nmr: putting the pieces together encyclopedia nmr-based approaches for the identification and optimization of inhibitors of protein-protein interactions nmr-based screening of proteins containing c-labeled methyl groups estimating protein−ligand binding affinity using high-throughput screening by nmr exploring the binding of peptidic west nile virus ns b-ns protease inhibitors by nmr blocking the interactions between calcium-bound s a protein and the v domain of rage using tranilast characterization of protein−ligand interactions by high-resolution solid-state nmr spectroscopy using chemical shift perturbation to characterise ligand binding combining automated peak tracking in sar by nmr with structure-based backbone assignment from n-noesy atia-tul-wahab chapter -nuclear overhauser effect accuracy in determining interproton distances using nuclear overhauser effect data from a flexible molecule chapter -more d and d nmr experiments: the nuclear overhauser effect-polarization transfer-spin lock experiments- d nmr use of nmr saturation transfer difference spectroscopy to study ligand binding to membrane proteins waterlogsy as a method for primary nmr screening: practical aspects and range of applicability characterization of ligand binding by saturation transfer difference nmr spectroscopy detecting binding affinity to immobilized receptor proteins in compound libraries by hr-mas std nmr ligand based nmr methods for drug discovery virus-ligand interactions: identification and characterization of ligand binding by nmr spectroscopy method for detecting biologically active compounds from compound libraries evolutionary change of the heme c electronic structure: ferricytochrome c- from pseudomonas aeruginosa and horse heart ferricytochrome c nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase application of nmr based binding assays to identify key hydroxy groups for intermolecular recognition determining binding sites in protein-nucleic acid complexes by cross-saturation group epitope mapping by saturation transfer difference nmr to identify segments of a ligand in direct contact with a protein receptor synthetic derivatives of pyrrole and pyrrolidine suitable for the therapy of infections caused by rhinoviruses global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis economic impact of outbreaks of norovirus infection in hospitals norovirus-host interaction: implications for disease control and prevention x-ray crystallographic structure of the norwalk virus capsid structural insights into antigenic diversity and host specificity. proc. natl. acad. sci structural basis for the recognition of blood group trisaccharides by norovirus epitope mapping of histo blood group antigens bound to norovirus vlps using std nmr experiments reveals fine details of molecular recognition synthesis and nmr studies on the abo histo-blood group antigens: synthesis of type iii and iv structures and nmr characterization of type i-vi antigens nmr experiments reveal the molecular basis of receptor recognition by a calicivirus negative nuclear overhuaser effects as probes of macromolecular structure identification of an e-selectin antagonist in a substance mixture by transfer noe the inpharma method: protein-mediated interligand noes for pharmacophore mapping diffusion nmr spectroscopy in supramolecular and combinatorial chemistry: an old parameter-new insights beyond passive: chaotic transport in stirred fluids . passive transport chapter -momentum, heat and mass transfer in boundary layers diffusion nmr spectroscopy chapter -diffusion nmr spectroscopy the diffusion equation models for diffusion spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient diffusion by nmr the stejskal-tanner equation generalized for any gradient shape-an overview of most pulse sequences measuring free diffusion diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications measuring ligand-protein binding using nmr diffusion experiments chapter -dosy nmr for drug analysis diffusion-weighted nmr spectroscopy allows probing of c labeling of glutamate inside distinct metabolic compartments in the brain spatial encoding and spatial selection methods in high-resolution nmr spectroscopy spatially encoded d and d diffusion-ordered nmr spectroscopy enumeration of billion organic small molecules in the chemical universe database gdb- exploring chemical space for drug discovery using the chemical universe database f dosy diffusion-nmr spectroscopy of fluoropolymers pulsed-field gradient nuclear magnetic resonance measurements (pfg nmr) for diffusion ordered spectroscopy (dosy) mapping guest-encapsulation properties of a self-assembled capsule by dynamic boronic ester bonds properties of small molecular drug loading and diffusion in a fluorinated peg hydrogel studied by h molecular diffusion nmr and f spin diffusion nmr a new nmr method to analyze multi-component enzyme/substrate systems the exploration of interaction studies of smaller size, mostly ignored yet intrinsically inestimable molecules towards bsa; an example of std and dosy nmr. open chem structure-based virtual screening for drug discovery: principles, applications and recent advances from data banks to data bases in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling ultra-high-throughput structure-based virtual screening for small-molecule inhibitors of protein-protein interactions in silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen in silico target prediction for elucidating the mode of action of herbicides including prospective validation docking, virtual high throughput screening and in silico fragment-based drug design in silico virtual screening approaches for anti-viral drug discovery virtual screening strategies: recent advances in the identification and design of anti-cancer agents a review of ligand-based virtual screening web tools and screening algorithms in large molecular databases in the age of big data virtual high throughput screening (vhts)-a perspective application of nmr and molecular docking in structure-based drug discovery virtual ligand screening against comparative protein structure models biophysical screening for the discovery of small-molecule ligands the use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against mek the holistic integration of virtual screening in drug discovery retrospect and prospect of virtual screening in drug discovery docking and scoring in virtual screening for drug discovery: methods and applications nmr and in silico screening acceleration of the drug discovery process: a combinatorial approach using nmr spectroscopy and virtual screening complementarity between in silico and biophysical screening approaches in fragment-based lead discovery against the a a adenosine receptor adenosine a a receptors in parkinson's disease treatment discovery of small-molecule inhibitors of ubiquitin specific protease (usp ) using integrated nmr and in silico techniques molecular recognition of p and mdm by usp /hausp design and characterization of libraries of molecular fragments for use in nmr screening against protein targets antihypertensive drug valsartan in solution and at the at receptor: conformational analysis, dynamic nmr spectroscopy, in silico docking, and molecular dynamics simulations combining in silico tools and nmr data to validate protein−ligand structural models: application to matrix metalloproteinases nmr-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors metallo-β-lactamase inhibitors inspired on snapshots from the catalytic mechanism synthesis of potent dishevelled pdz domain inhibitors guided by virtual screening and nmr studies paramagnetic nmr in drug discovery paramagnetic nmr in drug discovery paramagnetic nmr in solution and the solid state clanp-an artificial paramagnetic centre to study proteins by nmr current nmr techniques for structure-based drug discovery requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by nmr paramagnetic nmr as a new tool in structural biology edta-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to dna by intermolecular paramagnetic relaxation enhancement dna as a target for drug action the structural effects of the cys-s-s-cys bridge exchange by the his-cu(ii)-his motif studied on natural peptides-a promising tool for natural compounds-based design towards reproducible computational drug discovery utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin monitoring dynamics of large membrane proteins by f paramagnetic longitudinal relaxation: domain movement in a glutamate transporter homolog solid-state nmr spectroscopy on complex biomolecules solid-state nmr spectroscopy as a tool for drug design: from membrane-embedded targets to amyloid fibrils new approaches to the characterization of drug candidates by solid-state nmr saikiran solid state nuclear magnetic resonance spectroscopy-a review solid-state nmr spectroscopy in pharmaceutical research and analysis solid-state nuclear magnetic resonance spectroscopy: theory and pharmaceutical applications recent advances in solid-state nuclear magnetic resonance spectroscopy solid-state nmr in drug design and discovery for membrane-embedded targets the effect of drug loading on micelle properties: solid-state nmr as a tool to gain structural insight zinc-binding structure of a catalytic amyloid from solid-state nmr aggregation of biologically important peptides and proteins: inhibition or acceleration depending on protein and metal ion concentrations principles of early drug discovery hit discovery and hit-to-lead approaches chapter twelve-hit-to-lead: hit validation and assessment biophysics: for hts hit validation, chemical lead optimization, and beyond advances in nuclear magnetic resonance for drug discovery validation of drug-like inhibitors against mycobacterium tuberculosis l-aspartate α-decarboxylase using nuclear magnetic resonance expression of bacterial l-aspartate-alpha-decarboxylase in tobacco increases beta-alanine and pantothenate levels and improves thermotolerance methods for identification of false positives in biochemical screens alarm nmr: a rapid and robust experimental method to detect reactive false positives in biochemical screens alarm nmr for hts triage and chemical probe validation assay interference and off-target liabilities of reported histone acetyltransferase inhibitors a combination of f nmr and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the atp-binding site of a kinase deconstruction of a nutlin: dissecting the binding determinants of a potent protein-protein interaction inhibitor monitoring binding of hiv- capsid assembly inhibitors using f ligand-and n protein-based nmr and x-ray crystallography: early hit validation of a benzodiazepine series is nmr fragment screening fine-tuned to assess druggability of protein-protein interactions de novo design of chiral organotin cancer drug candidates: validation of enantiopreferential binding to molecular target dna and -gmp by uv-visible, fluorescence, h and p nmr inhibiting the p -mdm interaction: an important target for cancer therapy inhibiting mdm -p interaction suppresses tumor growth in patient-derived non-small cell lung cancer xenograft models targeting p -mdm -mdmx loop for cancer therapy. in mutant p and mdm in cancer mdm small-molecule antagonist rg activates p signaling and regresses human tumors in preclinical cancer models small molecules, big targets: drug discovery faces the protein-protein interaction challenge exploring protein-protein interactions as drug targets for anti-cancer therapy with in silico workflows targeting protein-protein interactions for drug discovery protein-protein interaction modulators: advances, successes and remaining challenges sb is a specific inhibitor of a map kinase homologue which is stimulated by cellular stresses and interleukin- novel homologues of csbp/p map kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles an accurate pharmacophore mapping method by nmr spectroscopy protein-ligand docking guided by ligand pharmacophore-mapping experiment by nmr the inter-ligand overhauser effect: a powerful new nmr approach for mapping structural relationships of macromolecular ligands sar by iloes: an nmr-based approach to reverse chemical genetics targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules epitope mapping and competitive binding of hsa drug site ii ligands by nmr diffusion measurements a saturation transfer nmr-based method for determining the structures of protein−ligand complexes how much nmr data is required to determine a protein-ligand complex structure? o-tert-butyltyrosine, an nmr tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities sensitive nmr approach for determining the binding mode of tightly binding ligand molecules to protein targets solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy direct nmr probing of hydration shells of protein ligand interfaces and its application to drug design protein-ligand structure determination with the nmr molecular replacement tool, nmr nmr-based determination of the d structure of the ligand-protein interaction site without protein resonance assignment the nmr method to determine rapidly the structure of the binding pocket of a protein-ligand complex with high accuracy protein-fragment complex structures derived by nmr molecular replacement using ligand-induced protein chemical shift perturbations to determine protein-ligand structures automatic mapping of perturbed and unperturbed regions of proteins and complexes applications of in-cell nmr in structural biology and drug discovery protein f nmr in escherichia coli d structure determination of a protein in living cells using paramagnetic nmr spectroscopy structure of proteins in eukaryotic compartments direct structural evidence of protein redox regulation obtained by in-cell nmr cell nmr spectroscopy in protein chemistry and drug discovery high-resolution heteronuclear multidimensional nmr of proteins in living insect cells using a baculovirus protein expression system isotope labeling for solution and solid-state nmr spectroscopy of membrane proteins. in isotope labeling in biomolecular nmr f-nmr in target-based drug discovery applications of f-nmr in fragment-based drug discovery methyl groups as probes for proteins and complexes in in-cell nmr experiments nmr-based investigations into target dna search processes of proteins isotope labeling strategies for nmr studies of rna the first successful observation of in-cell nmr signals of dna and rna in living human cells g-quadruplex dna and ligand interaction in living cells using nmr spectroscopy nmr profiling of histone deacetylase and acetyl-transferase activities in real time a multiplexed nmr-reporter approach to measure cellular kinase and phosphatase activities in real-time an adhesive f mri chemical probe allows signal off-to-on-type molecular sensing in a biological environment scaffolding proteins and their role in viral assembly mechanism of scaffolding-assisted viral assembly structure of the coat protein-binding domain of the scaffolding protein from a double-stranded dna virus edited by m. summers structural assembly of the tailed bacteriophage φ nmr mapping of disordered segments from a viral scaffolding protein enclosed in a mda procapsid hydrogen exchange of monomeric α-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in escherichia coli investigating macromolecules inside cultured and injected cells by in-cell nmr spectroscopy physicochemical properties of cells and their effects on intrinsically disordered proteins (idps) beyond the paradigm: combining mass spectrometry and nuclear magnetic resonance for metabolomics sample collection and preparation of biofluids and extracts for gas chromatography-mass spectrometry gas chromatography-mass spectrometry of biofluids and extracts application of nmr metabolomics to search for human disease biomarkers optimized metabolite extraction from blood serum for h nuclear magnetic resonance spectroscopy molecular thermodynamics using nuclear magnetic resonance (nmr) spectroscopy. inventions a review of nondestructive characterization of composites using nmr chapter -mass spectrometry dehydrodimerization of pterostilbene during electrospray ionization mass spectrometry cryogenic probe c nmr spectroscopy of urine for metabonomic studies sofast-hmqc experiments for recording two-dimensional deteronuclear correlation spectra of proteins within a few seconds sofast-hmqc-an efficient tool for metabolomics ultrafast d nmr: an emerging tool in analytical spectroscopy evaluation of fast d nmr for metabolomics real-time separation of natural products by ultrafast d nmr coupled to on-line hplc compressed sensing and the reconstruction of ultrafast d nmr data: principles and biomolecular applications application of ex situ dynamic nuclear polarization in studying small molecules dynamic nuclear polarization for sensitivity enhancement in modern solid-state nmr determinants for optimal enhancement in ex situ dnp experiments hyperpolarized nmr spectroscopy: d-dnp, phip, and sabre techniques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank king abdullah university of science and technology (kaust) for financial support. the authors declare no conflict of interest. key: cord- -qw pf r authors: greaves, peter title: vii digestive system date: - - journal: histopathology of preclinical toxicity studies doi: . /b - - / - sha: doc_id: cord_uid: qw pf r publisher summary this chapter deals with the digestive system. the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity, and derangement of saliva production may lead to loss of integrity of the oral mucosa. drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. inflammation of the oral cavity may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis), and the peridontal tissues (peridontitis). therapeutic agents can induce inflammatory lesions in the tongue. moreover, a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species. the oral mucosa can be damaged by excessive local trauma from foreign materials, hard fragments in food and damaged teeth may produce ulceration of the mucosa with subsequent infection. however, the oral mucosa may show manifestations of local or systemic disease or derangement produced by therapeutic agents. excessive contact by therapeutic agents such as aspirin, potassium supplements and corticosteroids have been reported to produce local ulceration in the mouth (zentler-monro and northfield, ) . the increased use of mouthwashes over the last years has resulted in a number of reported adverse effects to the buccal mucosa in people (gargari and kabani, ) . systemic disorders produced by anticoagulants or chemotherapeutic drugs may also be evident by bleeding or ulceration in the oral cavity (goepp, ) . buccal ulceration is also described as part of a generalised hypersensitivity reaction to drugs (zentler-monro and northfield, ) . the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity and derangement of saliva production may lead to loss of integrity of the oral mucosa. drugs that effect motor co-ordination can give rise to drooling and disruption of cricopharyngeal co-ordination (wyllie et al., ) . drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. indeed, many alterations in the oral mucosa are those that are more readily detected by careful clinical and macroscopic observation rather than exhaustive histopathological examination of the buccal mucosa in laboratory animals -provided the basic toxicity profile of a novel agent is adequately assessed in the usual preclinical studies. oral irritation studies are used in the testing of products for use in the oral cavity, mainly for surgical, dental and hygiene purposes but also therapeutic agents administered by the sublingual route. this route may be selected for substances that are broken down in the stomach or show a rapid first pass effect. as it is technically not feasible to perform full preclinical toxicity studies by the sublingual route, conventional oral or parenteral routes are preferred for systemic toxicity studies on such compounds. the choice of the best route will to a large extent be dictated by pharmacokinetic considerations. however, it is necessary to assess local irritancy potential to oral mucosa using a laboratory animal model. test species for oral irritation studies are usually rats, hamsters (cheek pouch), guinea pigs, dogs or primates using gross and histopathological assessment. a similar scheme to that employed in the histological assessment of skin irritancy is appropriate. inflammation of the oral cavity (stomatitis) may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis) and the peridontal tissues (peridontitis). although inflammatory lesions are found sporadically in untreated laboratory rodents, dogs and primates, stomatitis can be induced by systemic administration of high doses of therapeutic agents. anticancer and antimitotic agents are particularly liable to induce stomatitis. a notable example is bleomycin that is capable of producing stomatitis as part of its general effect on squamous cells (thompson et al., ) . in humans, the adverse effects on therapeutic ionising radiation on the salivary glands may also give rise to inflammatory changes in the oral cavity (fox, ) diuretics and other agents, which are capable of producing severe electrolyte disturbances and uraemia at excessive doses, can also produce stomatitis when then are administered in high doses to laboratory animals (garthoff et al., ) . these lesions may be analogous to the well-described association of ulcerative stomatitis and uraemia in man and laboratory animals (boyd, ; barker and van dreumel, ) . the dog appears very sensitive to the ulcerogenic effects of uraemia in the oral cavity, although as there is a poor correlation between actual levels of blood urea and stomatitis, other biochemical factors are undoubtedly involved. compounds, which effect pigmentation of the skin, can produce similar changes in pigmented oral mucosa. a number of drugs including chlorpromazine, quinacrine, chloroquine, amodiaquine and pyrimethamine cause pigmentation of the oral mucosa in man notably over the hard palate. chloroquine and pyrimethamine have also been shown to significantly increase numbers of active melano-cytes within the palatal mucosa of pigmented da rats when treated orally for weeks (savage et al., ) . melanocytes in treated rats were shown to be enlarged and packed with melanin pigment and to possess extensive arborisation of cell processes between squamous cells. an experimental inhibitor of platelet aggregation, which produced pigment loss in the dark hair of long-evans rats and the skin of beagle dogs, also induced pallor of the normally pigmented oral mucous membranes in dogs (gracon et al., ; walsh and gough, ) . apart from loss of pigment, the histology of the mucous membranes and skin was normal. the tongue is conveniently sectioned for histological study, although reliance is often placed on careful visual inspection, because the usefulness of systematic histological examination of the tongue in routine preclinical safety studies has not been clearly established. a few lesions occur which are fairly specific to the tongue. amyloid may become deposited in the muscular and connective tissue of the tongue in amyloid-prone species, particularly mice (dunn, ) . mice, especially dba and dba/ ncrj strains, are liable to develop calcification in the lingual muscle spontaneously, even at a young age (imaoka et al., ) . calcified lesions are seen in the longitudinal muscle under the dorsolateral epithelium and the central part of the tongue, which, when severe, are associated with inflammation, granulation tissue, polypoid change, hyperplasia of the overlying squamous epithelium and ulceration. the histogenesis of this lesion is uncertain. in the dba/ ncrj mice, mineralisation of the tongue is associated with myocardial and aortic mineralisation (doi et al., ) . therapeutic agents can induce inflammatory lesions in the tongue. an example is provided by the investigational anticancer immunotoxin, zd , a mouse monoclonal antibody (c ) against colorectal carcinoma antigen conjugated to recombinant ricin a-chain. when administered to wistar-derived rats, this agent produced myocyte necrosis and inflammation specifically located below the ventral subepithelial surface of the tongue (westwood et al., ) . as the changes were different to the low grade myositis seen elsewhere in treated animals, these authors speculated that the changes in the tongue may have been related to the particular receptor profile of this area targeted by the monoclonal antibody. in common with other changes induced in the digestive tract of rats and cynomolgus monkeys by the administration of recombinant human epidermal growth factor, the tongue showed squamous epithelial hyperplasia characterised by a uniform increase in the thickness of the squamous epithelium in both species (breider et al., ; reindel et al., ) . at high doses, the squamous epithelium of the tongue of the primates was twice the thickness of control mucosa associated with elongation of rete pegs. teeth are usually only inspected by naked eye in conventional toxicity studies and this is appropriate for the assessment of a mature dentition. however, there has been an increasing awareness of dental lesions in toxicity studies, particularly as the teeth are visualised when the maxilla is examined histologically in inhalation studies. study of the rodent dentition in inhalation studies has shown that spontaneous lesions of the dentition are quite common. in one laboratory, malformations (dental dysplasia) of the maxillary incisors were observed in % of female and % of male cd- mice and . % female and . % sprague-dawley rats in and month inhalation studies respectively (losco, ) . the rat incisor and its pathology has been the subject of an excellent review (kuijpers et al., ) . unlike in humans, the rodent incisor continues to grow and differentiate throughout life and is renewed every - days. located at the centre of the tooth is the vascular pulp. this is surrounded by proliferating ondotoblasts which form predentin which when calcified becomes dentin. surrounding ameloblasts when induced by the presence of dentin produce the overlying enamel layer. it is these active cellular layers, which can be modified or damaged by xenobiotics, vitamin deficiencies, calcium, phosphate or magnesium deficiency, parathyroidectomy, hypophysectomy, hyperparathyroidism, adrenal insufficiency and fluorosis (kuijpers et al., ) . although in humans the mature dentition is no longer growing, in children the dentition is in a growth phase that starts in utero and lasts into the second decade. as increasing numbers of children survive malignant disease, damage to the mature dentition can occur as a result of cytotoxic therapy during childhood. clinical study of the teeth of children treated for malignancy have shown increased incidence of enamel hypoplasia and missing teeth (welbury et al., ) . histological examination of teeth from children treated with vincristine or combination chemotherapy for malignant disease has demonstrated prominent incremental lines in dentine correlating with the number of times the intravenous cytotoxic agents were administered (macleod et al., ) . it has also been shown that vincristine, a drug which interferes with the assembly of microtubules and reduces secretory activity in a number of cells including osteoblasts and chondroblasts, also effects dentine formation in the rat incisor (stene and koppang, ) . two weeks following a single intravenous dose of vincristine to young adult rats, a faint incremental line in the dentine was observed, probably a reflection of a direct effect of the drug on the dentinogenic tissue at the time of injection. at higher doses, focal niche-like or punched out defects in dentine were observed, expression of more severe injury to highly sensitive dentinogenic populations at the time of injection (stene and koppang, ). the precise mechanism of damage is not fully understood although decreased secretion of dentine matrix by odontoblasts has been demonstrated. calcification appears unaltered (stene and koppang, ) . administration of the alkylating anticancer agent cyclophosphamide or a sin-gle exposure to ionising radiation, produces localised niche-like or punched out defects in the rat incisor, rather than the more diffuse changes induced by vincristine. this presumably reflects more localised injury to a sensitive subpopulation of dentinogenic cells (koppang, ; vahlsing et al., ) . anticonvulsant drugs also produce changes in the dentition of man and experimental animals. in humans, reported alterations include tooth root resorption, small teeth, delayed shedding of deciduous teeth and retarded eruption of permanent teeth, features similar to those found in hypoparathyroid or pseudohypoparathyroid conditions (robinson et al., ) . tooth root alterations were also reported in a study in which young male wistar rats were treated with diphenylhyantoin for month. treated rats showed evidence of molar root resorption lacunae that penetrated the cementum and involved the dentine. the lacunae contained a dense infiltrate of cells contiguous with similar cells in the surrounding periodontal ligament. robinson and harvey ( ) showed that the changes were similar to those occurring in parathyroidectomized rats but not those in rats made hypocalcaemic with a calcium deficient diet. they suggested that the changes induced by diphenylhydantoin in rats were similar to those in pseudohypoparathyroidism in which resistance of tooth roots to resorption is reduced. discoloration of teeth and bone is a well-described side effect of tetracycline administration and it has also been reported in patients treated with the semisynthetic derivative, minocycline (cale et al., ) . interestingly the ameloblastic epithelium of the enamel forming tissues of growing incisors in wistar rats treated with high doses of human recombinant epidermal growth factor showed hyperplasia characterised by pseudostratification, increased nuclear-cytoplasmic ratio and increased cytoplasmic eosinophilia (breider et al., ) . this finding is consistent with the presence of epidermal growth factor receptors in the cells of the enamel organ (martineau et al., ) . periodontitis is a common and important disease in man and animals although overt cases are not usually seen in toxicity studies. however, periodontitis of a degree sufficient to disrupt chronic rat toxicity and carcinogenicity studies has been reported. robinson ( ) described periodontitis in alpk/ap rats in which there were erosive granulomatous cavities adjacent to molar teeth with fistulas opening into the nasal cavity. these changes were associated with penetrating food fibres in the gingival sulcus and it was suggested that the presence of long pointed food fibres in the powdered diet was the main reason for occurrence of periodontitis. peridontitis in rodents also results from the effects of dental pathology such as fractures, malformation or malposition of incisors (losco, ) . drug-induced overgrowth of the gingival tissues is a well-described phenomenon in both humans and laboratory animals including dogs, cats, and rats. in man, these changes have been associated with diphenylhydantoin (phenytoin) (beghi et al., ) nifedipine, calcium channel blockers (ledermann et al., ) , cyclosporin a (barthold, ) and valproic acid (syrjamen and syrjamen, ) . cyclosporin a, diphenylhydantoin and calcium channel blockers have been associated with similar changes in laboratory animals (do'nascimento et al., ; latimer et al., ; waner et al., ) . in most instances there is swelling of the gingiva by firm nodular overgrowths around the teeth. histologically, these overgrowths are characterised by marked acanthosis of the squamous epithelium overlying connective tissue that is infiltrated by large numbers of chronic inflammatory cells. fibrovascular proliferation may be marked. in patients treated with cyclosporin, myxomatous degeneration is described in association with dense infiltration of plasma cells and lymphocytes (barthold, ) . secondary acute inflammation in association with food debris and hair shafts is described in dogs treated with oxodipine (waner et al., ) . the forces behind these changes are unclear. studies of changed induced by nifedipine and hydantoin have shown increases in extracellular ground substance and increased numbers of fibroblasts containing sulphated acid mucopolysaccharides (kantor and hassel, ; lucas et al., ) . these drugs may alter fibroblastic proliferative and synthetic activity, possibly by selection of a subpopulation of fibroblasts (hassel et al., ) . it has also been suggested that an underlying mechanism in phenytoin-induced gingival hyperplasia involves the decrease in salivary iga that develops in some patients (beghi et al., ) . study of cyclosporin a-induced changes have suggested that impairment of t lymphocyte function may permit overgrowth of oral bacteria and bacterial products which may influence fibroblast function (barthold, ) . a spontaneous form of gingival hyperplasia has been described in non-human primates (macaca mulata). this is characterised by an enlargement of the marginal and alveolar gingiva by connective tissue consisting of relatively poorly cellular bundles of collagen fibres. the lesions show little inflammatory alterations and the overlying squamous epithelium shows mild hyperkeratosis only (schiødt et al., ) . this pathology is similar to hereditary gingival fibromatosis in humans. sessile or pedunculated squamous papillomas and infiltrating squamous carcinomas are occasionally found in the oral cavity of most laboratory animals including rodents (odashima, ; emminger and mohr, ; leiniger and jokinen, ; takahashi and okamiya, ; mohr, ) , rabbits (sundberg et al., ; sundberg and everitt, ) , and beagle dogs (watrach et al., ) . the microscopic structure of these neoplasms in rodents resembles those occurring in squamous epithelium in other sites. although a number of agents induce squamous neoplasms in the oral cavity, spontaneous squamous carcinomas are generally uncommon spontaneous lesions in laboratory animals. however, some strains of rodent may develop squamous neoplasms more commonly. for instance, in life time studies ad libitum fed brown-norway rats, % of males and % females developed oral squamous cell carcinomas although only % and % in food-restricted animals respectively (thurman et al., ) . it was suggested that certain pedigrees possessed a genetic predisposition to these neoplasms. papillomas occurring in rabbits and dogs are of note because they can occur in quite young animals, apparently as a result of infection with viruses of the papilloma group. viral inclusions may be seen in histological sections. the implications of papilloma viruses in laboratory species are that the progression of virally induced papillomas to malignant squamous carcinomas can be potentiated by nonviral factors including application of xenobiotics (howley et al., ) . in rabbits, the prevalence of oral papillomas varies considerably but they are quite common in some laboratory strains. they are overlooked because of their small size and a distribution limited to the ventral surface of the tongue (sundberg et al., ) . microscopically, they are typical squamous papillomas composed of irregular acanthotic squamous epithelium and a fibrovascular stalk of variable size. squamous cells at the margins of papillomas at the junction with normal mucosa, often show large, oval nuclei, marginated chromatin and central, basophilic, intranuclear inclusions, which electron microscopic examination shows to contain viral particles. oral papillomas in dogs develop as multiple growths, regressing spontaneously after a few months. they are also caused by a virus of the papilloma group, which possesses a high degree of specificity for the mucosa of the oral cavity and adjacent skin (watrach et al., ) . histologically, they are composed of proliferative masses of epithelial cells, keratinised on the surface and resting on an irregular connective tissue stroma or pedicule. large vesicular cells with basophilic intranuclear inclusions are also found in the granular cell layer, identifiable as virus arrays by electron microscopy (cheville and olson, ) . malignant change has been described in these canine lesions and this can occur in young beagle dogs (watrach et al., ) . although many types of papilloma viruses have been identified in both man and animals (pfister, ) , common antigenic determinants exist between viruses in different species. this immunological cross-reactivity can be exploited in the immunocytochemical localisation of papilloma viruses in epithelial lesions of many animal species. papilloma virus antigen has been demonstrated in oral papilloma of dogs and rabbits using antisera to bovine papilloma virus type i (sundberg et al., ) . cells positive for virus and viral inclusions are located in the upper layers of the epithelium, especially within cells of the granular layer. spontaneously developing odontogenic tumours are rare in rodents but they have been induced in laboratory animals given carcinogens such as nitrosoureas or exposed to ionising radiation (gössner and luz, ) . a range of tumours originating from dental tissues with epithelial, mesenchymal or mixed appearances has been reported in rodents (kuijpers et al., ) . the classification of odontogenic tumours is complex and confusing. they range from benign anomalies and cystic structures through to malignant neoplasms. the ameloblastoma comprises cords, nests, anastomosing strands or islands of ondontogenic epithelial cells within a fibrous stroma. the tumour cells resemble ameloblasts with the cords of spindle shaped cells similar to the stellate epithelium bounded by a peripheral layer of cuboidal or columnar cells resembling the inner enamel epithelium. other tumours of the odontogenic epithelium show induction of the mesenchymal elements or develop a complete sequence of odontogenic epithelium, odontogenic mesenchyme and dental hard tissues including dentine, enamel and cementum. in the rat these have been classified as odontoma characterised by the presence of all dental hard tissues and odontogenic fibroma composed of undifferentiated or primitive mesenchymal cells of developing dental tissue (mohr, ) . odontogenic tumours developing in fischer rats treated with aflatoxin were located in the upper jaw associated with the incisor teeth and were composed of proliferating fibroblast-like cells within which ovoid calcified bodies resembling cementum were seen (cullen et al., ) . occasional inclusions of solid epithelial nests were also seen. no metastatic deposits were found although the neoplasms were locally aggressive. in addition, squamous tumours and neoplasms of mesenchymal origin typical of other organs, bones and soft tissues are found in this region. although salivary glands may not represent vital organs in the same sense as the kidneys or heart, severe derangement of their secretions can alter both the quality and quantity of saliva. depending on the particular glands and cells affected, dry mouth, mucositis, and dental caries may develop . the severe oral complications of irreversible salivary damage and dysfunction, which can occur patients with head and neck cancer as a consequence of local irradiation, may have a significant impact on the efficacy of therapy, quality of life and survival (fox, ) . a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. these mucins usually contain more than % carbohydrate in the form of neutral and acidic oligosaccharide chains, o-glycosidically linked to threonine or serine. mucins possess several roles including mechanical flushing of the oral cavity, protection and lubrication of soft and hard tissues, modulation of oral microbial flora, buffering activity, regulation of calcium/phosphate equilibrium, digestion and extracellular post translation processing of molecules present in saliva . the heterogeneity of salivary glycoproteins suggests that they act as a defence against pathogenic microorganisms by competing with microbial binding sites of similar structure on the surface of cells lining the digestive tract (schulte, ) . minor salivary glands may also play an important part in the local immunosurveillance of the oral cavity for their ducts are anatomically closely associated with lymphoid tissue (nair and schroeder, ; nair et al., ) . salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species (junqueira et al., ) . the phylogenetic association of the salivary glands with the thyroid gland is evident functionally because salivary glands are capable of concentrating iodide in their secretions, although this is not under control of thyroid stimulating hormone (ingbar, ) . it has been shown that thyroxine accelerates the differentiation of the granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse (chabot et al., ) . the structure of the salivary glands differs among laboratory species, between different glands in the same species and between sexes. it is usually considered that there are three major salivary glands, the parotid, the sublingual and the submandibular (submaxillary) glands. minor salivary glands are scattered in other locations throughout the mouth and oropharynx. in dogs and other carnivores, the zygomatic (infra-orbital) gland, located just below the zygomatic arch and the buccal (molar) gland are also often referred to as major salivary glands. microscopically, salivary glands are composed of secretory glands or 'endpieces' attached to a connecting system of intralobular and extralobular (secretory) ducts. secretory endpieces may be acinar or tubulo-acinar in nature. the secretory cells have been subdivided into serous, mucous, seromucous and special serous types. controversy remains about the precise nature of the secretory cells found in the various salivary glands of different species and this makes critical interspecies comparisons difficult (see detailed discussion of this problem by pinkstaff, ). the duct system is less complex. this comprises an intercalated duct which leads from the secretory endpiece into a striated (secretory or intralobular) duct, so termed because their lining cells are striated by delicate eosinophilic cytoplasmic rods. the striated ducts converge into interlobular ducts and a main excretory duct system. in rats, mice and hamsters, an overall similarity in gross and microscopic anatomy of the various salivary glands exists although there are histochemical differences (munhoz, ; glucksmann and cherry, ; dawe, ; pinkstaff, ; emmiger and mohr, ) . moreover, it has been demonstrated that salivary glands in rodents as well as a number of other species show morphological and histochemical sexual dimorphism (pinkstaff, ) . the sublingual gland in rats, mice and hamsters is composed principally of mucous acini, with indistinct serous demilunes. acini open into fairly long intercalated ducts lined by flat or cuboidal cells devoid of granules. the parotid gland is composed of serous-type secretory cells containing zymogen granules and prominent hyperchromatic basal cytoplasmic poles. the submandibular gland is anatomically the most complex salivary gland in rodents. secretory endpieces are composed of small or moderately sized cells with foamy cytoplasm and basophilic basal poles. the most striking feature is the presence of an additional duct segment interposed between the intercalated and striated ducts. this segment is lined by cylindrical epithelium with basal nuclei and eosinophilic cytoplasm containing secretory granules. this duct segment is termed the granular duct or granular convoluted tubule. these granular cells are of special interest because they contain a large number of heterologous biologically active peptides including nerve growth factor, epidermal growth factor, renin, and kallikrinins (barka, ; mori et al., ). the precise physiological role of many of these peptides in salivary gland remains uncertain. epidermal growth factor was originally isolated from the mouse salivary gland. it initiates premature eyelid opening and incisor eruption when injected into the neonatal mouse (cohen, ) . study of the mouse submandibular gland has shown that both epidermal growth factor and nerve growth factor are released into saliva following the administration of phenylephrine, sympathomimetic amine acting mainly on αreceptors and isoprenaline (isoproterenol), a β-adrenergic agent (murphy et al., ) . immunohistochemical study also demonstrates that epidermal growth factor becomes depleted in mouse salivary tissue following administration of phenylephrine and similar agents (tsukitani and mori, ) . phenylephrine has been shown to cause marked secretory activity accompanied by loss of granules from granular cells, as well as loss of immune reactive carbonic anhydrase, an enzyme which participates both in membrane transport of bicarbonate ions into saliva and glandular secretion (noda, ) . morphological studies have shown that both acinar and granular tubular cells participate in this response to adrenergic agents (murphy et al., ) . this is in contrast to the effects of pilocarpine, a cholinergic agent, which elicits the secretion of saliva deficient in serous proteins with little or none of the growth factors, as its effects are more limited to acinar cells. glycoprotein secretion of rodent salivary glands has stimulated histochemical study using both conventional mucin histochemical techniques and labelled lectins which possess affinity for specific sugars or sugar sequences (tables and , pages and ). studies of rat, mouse and hamster salivary glands using batteries of labelled lectins have shown a greater heterogeneity of oligosaccharides in salivary glands than seen by classical histochemical techniques. there are considerable species differences and variations between murine strains and sexes of the same strain as well as heterogeneity among morphologically similar cells within one gland (schulte and spicer, , ; schulte, ) . the results of histochemical studies are in excellent agreement with studies using biochemical methods but suggest a significant influence of genetic and hormonal factors on the synthesis of salivary glycoproteins. less attention has been paid to the structure and cytochemistry of the dog salivary glands. there appears to be little variation between the structure of salivary tissues between beagles and other strains although variation with age has been reported (reifel and travill, ; nagoyo and tandler, ) . munhoz ( ) has described the histochemical features of the dog parotid gland. the dog parotid is of seromucinous type secreting both acidic and neutral mucosubstances, in contrast to the more neutral mucosubstances secreted by rodent glands. the salivary glands of non-human primates are similar to those in man. they possess parotid glands of serous or seromucous type, submandibular glands with both serous and mucous acini and sublingual glands of mainly mucous type. the salivary glands of the non-human primate react to adverse stimuli such as ionising radiation in a similar manner to human salivary tissue . focal chronic inflammation of the salivary glands occurs sporadically in untreated rats, mice, hamsters, dogs or primates employed in toxicology although severity and prevalence is variable. sialoadenitis as a result of a corona virus, the sialodacryoadenitis virus, is a well-known and fairly ubiquitous condition in rats, first described by innes and stanton ( ) . the condition is characterised histologically by oedema and congestion of submandibular and parotid salivary glands as well as extra-orbital lachrymal and harderian glands. it is accompanied by inflammation of variable severity and chronicity in both glandular and connective tissue as well as degeneration and necrosis of duct epithelium (fig. ) . the regenerative hyperplasia of the duct epithelium may be quite intense about a week after infection but all changes regress after about weeks and glands are essentially normal after or weeks (carthew and slinger, ; percy and wojcinski, ) . there may be a delay in the appearance of inflammatory cells and the onset of repair in rats immunosuppressed with cyclophosphamide (hanna et al., ) . depletion of salivary gland epidermal growth factor also occurs during the infection (percy et al., ) . suppurative infections in the neck region of the rat such as those produced by klebsiella aerogenes also cause acute and chronic inflammation of salivary glands with fibrosis and glandular proliferation of salivary tissue (arseculeratne et al., ) . sialadenitis occurs spontaneously in autoimmune-prone strains of mice such as the nzb/nzw and sl/ni strains and it has been reported in ageing female, but not male bdf mice (hayashi et al., ) . the non-obese diabetic mouse known for its spontaneous insulin-dependent diabetes mellitus also develops immune mediated damage to submandibular glands (fujino-kurihara et al., ; törnwall et al., ) . in ageing bdf females the submandibular gland was shown to be involved by a destructive inflammatory process characterised by an intense infiltration by small and medium sized lymphocytes, associated with mild inflammation in other organs such as the parotid and sublingual glands, pancreas and kidney. immunocytochemistry showed that most of the lymphocytes were t cells (thy- . and lyt- positive) of the helper/inducer subset (l t or cd positive) and less than % were of suppresser/cytotoxic (lyt- or cd fig. . section from salivary tissue from a sprague-dawley rat during an infection with the sialodacryoadenitis virus showing intense ductular inflammation. (he, × .) positive) type (see haemopoietic and lymphatic systems, chapter iii). circulating anti-salivary duct antibody of igg was also detected in afflicted mice. it was suggested that helper/inducer t cells played a key role in the production of this change, unlike induced autoimmune sialoadenitis in which cytoxic t-cell subsets may directly destroy glandular tissue. it has been suggested that this process in ageing females is related to the decline in the number of splenic lyt- cells in mice with advancing age (hayashi et al., ) . these cells are believe to be the most susceptible to ageing (see haemopoietic and lymphatic systems, chapter iii) in the non-obese diabetic strain of mouse derived from jcl-icr mice, a periductal chronic inflammatory infiltrate is found in the submandibular gland at about the same time that immune-mediated insulitis is most marked. this suggests that there is an extension of the autoimmune process to salivary tissue (fujino-kurihara et al., ) . it is probable that helper/inducer cd t cells are essential components of this infiltrate and a number of cytokines and their receptors such as ip- (interferon-γ inducible protein ) and rantes (regulated upon activation normal t cell expressed and secreted) may have an important role (törnwall et al., ) . an autoimmune type of sialoadenitis can also be experimentally induced certain strains of mice. crj:cd mice, thymectomized at days, a time point at which lyt- positive cells (cd suppresser t lymphocytes) can be maximally reduced, followed by immunisation at and days after birth with homogenates of salivary gland and complete freund's adjuvent, develop a distinctive sialoadenitis in the submandibular and to some extent the parotid glands (hayashi et al., ) . this sialoadenitis is characterised by degenerative changes in salivary glandular tissue associated with an extensive and intense infiltrate of small and medium sized lymphocytes. these cells appear shortly after immunisation but increase in number with time. immunocytochemical study has shown that many of these cells are reactive to antisera to thy- . and lyt- (cd ) features of suppresser/cytotoxic t lymphocytes. later appearing cells demonstrate features of plasmacytoid lymphocytes and contain immunoglobulin of mainly igg class (hayashi et al., ) . these authors therefore suggested the sialoadenitis appeared as both a result of cytotoxic/suppresser t-cell activity and an antibody-dependent cell-mediated cytotoxicity. in the hamster salivary glands, interstitial infiltrates of lymphocytes and plasma cell are quite common and may become more marked with advancing age (mcmartin, ) . whereas necrosis of the parotid gland of uncertain aetiology sometimes occurs in the dog, mild focal chronic inflammation is quite a common incidental finding in canine salivary glands and has been reported in about % of normal beagle dogs (kelly et al., ) . although the inflammation in salivary tissue which results from ionising radiation is only indirectly relevant to drug safety evaluation, it is of interest in view of the notable species differences in sensitivity to this form of insult. serous acinar cells in man and rhesus monkey appear least resistant to the effects of ionising radiation, where damage is characterised by widespread degranulation and degeneration of acini, infiltration by polymorphonuclear cells followed by lymphocytes, plasma cells and subsequent atrophy and fibrosis . these changes contrast with the lesser effects of ionisation radiation on the rodent salivary glands in which there is little or no acute inflammatory response. lymphoid bodies are sharply circumscribed collections of lymphoid cells generally located between the parotid and sublingual glands close to a cervical lymph node in mice. they are apparently normal aggregates of lymphoid tissue. like many other glandular organs, the size of the secretory tissue of the salivary gland is responsive to functional demand and is subject to age-related changes. in man, the gland parenchyma frequently becomes atrophic and replaced by connective tissue or fat with advancing age, possibly partly related to vascular changes (waterhouse et al., ; scott, ) . in ageing rats, the extent and height of granular ducts and their content of mature secretory granules has also been shown to decrease with age (sashima, ) . dietary factors influence salivary gland size. decreased food consumption or protein starvation can reduce the weight of salivary glands in rats. there is shrinking of mucous and serous glands and loss of zymogen granules associated with decreased rna but unchanged dna content, attributable to the reduced requirements for protein synthesis , mcbride et al., . as salivary gland function is responsive to adrenergic stimulation, it is not surprising that atrophy occurs following adrenergic blockade. the weights of the submandibular gland in mice were shown to decrease following administration of the β-adrenergic blocking agent, propranolol (smith and butler, ) . this was associated with a reduction in stainable neutral mucins and a decrease in the thickness of the acinar cells making the gland lumens appear larger than normal. the cytotoxic agent, alloxan, known primarily for its specific effect on pancreatic b cells, has also been shown to produce weight loss of the rat submandibular gland, associated with lipid inclusions in the acinar cells, capillary basement membrane thickening and reduced salivary flow (reuterving et al., ) . it is probable that alloxan exerts a cytotoxic effect on the acinar cells of the rat submandibular gland (sagström et al., ) . methotrexate, a folic acid antagonist, has also been reported to cause vacuolization of acinar and ductular cells with reduction of secretory granules in rat salivary glands (mcbride et al., ) . ligation of the main excretory ducts has frequently been used as an ex-perimental model for study of salivary gland atrophy as well as the regeneration that follows removal of the ligature. there is marked atrophy of all cell types but most markedly the acinar cells through apoptosis. although overt necrosis has been reported following ligation of the excretory duct, it appears that this may have been the result of constriction of the vasculature for acinar cells are relatively intolerant to a decrease in oxygen and nutrient (denny et al., ) . a number of therapeutic agents increase salivary gland size in man, although the scarcity of biopsy data precludes a critical assessment of the precise mechanism in many cases. drugs reported to produce salivary gland enlargement in man include iodide-containing radiological contrast media, isoprenaline and anti-inflammatory agents phenylbutazone and oxyphenbutazone. enlargement may also occur after endotracheal anaesthesia and upper gastrointestinal tract endoscopy in man (riddell, ) . some of these agents and procedures may produce spasm of large salivary ducts and retention of secretions. several pharmacological agents, particularly sympathomimetic amines, have been shown to produce increases in salivary gland size in rodents following repeated dosing (brenner and stanton, ) . there is an intimate relationship of sympathomimetic amines with the control of the secretory process in salivary tissue. whereas a single injection of isoprenaline (isoprotorenol) in the range of - mg/kg induces discharge of preformed secretory granules followed by gradual re-synthesis and reconstitution, repeated injections produces an increase in the size of salivary glands (simson et al., ) . histologically, the enlarged glands are composed of secretory cells of increased size that contain increased amounts of secretory substances in the cytoplasm (simson et al., ) . although these histological features are principally those of diffuse cellular hypertrophy, the increase in dna content and radioactive thymidine uptake described in the salivary tissue following repeated administration of isoprenaline suggests that a degree of hyperplasia also occurs (barka et al., ) . these effects do not depend on the integrity of the autonomic nerves because they occur after ablation of the autonomic ganglia (barka et al., ) . they appear to be mediated by an effect on adrenergic β-receptors. the effects can be blocked by propranalol, a β-receptor antagonist but not by phenoxybenzamine, an α-receptor antagonist (brenner and stanton, ) . as theophylline and caffeine also elicit salivary gland enlargement in rats, a role for cyclic ', -adenosine monophosphate (camp) in salivary gland enlargement has been postulated (brenner and stanton, ) . detailed study of hypertrophy, protein synthesis, and intracellular camp activity in the salivary glands of rats treated for days with isoprenaline (isoproterenol), a series of β-adrenergic receptor agonists and the phosphodiesterase inhibitors, theophylline and caffeine, showed that similar effects occurred with all agents although differences in the degree of hypertrophy, the nature of pro-tein and glycoprotein synthesis and golgi membrane enzyme activity were recorded (wells and humphreys-beher, ) . the parotoid gland showed the most pronounced hypertrophy followed by the submandibular gland but the sublingual gland appeared to be unaffected by treatment. the degree and nature of the changes induced by the various β /β receptor agonists suggested that most of these effects were mediated through β receptors which are present in greatest numbers on the parotid and salivary cells. it was suggested that the effects of β-adrenergic agonists on salivary gland are produced by a receptor-mediated stimulation of adenylate cyclase activity causing an increase in levels of intracellular camp. however, other factors may be important for wells and humphreys-beher ( ) also showed that although isoproterenol and caffeine increased salivary cell camp to comparable levels, the hypertrophy was greater with isoproterenol. cardioactive phosphodiesterase inhibitors were shown to produce submaxillary hypertrophy in rat subacute toxicity studies (rogers et al., ; jayasekara et al., ; smith et al., ) . parotid and submaxillary glands were those most affected by the inotropic phosphodiesterase inhibitor ici , (westwood et al., ) . as the agents produced their positive inotropic action via selective inhibition of the cardiac phosphodiesterase subfraction iii specifically requiring camp as its substrate, it was suggested that the salivary gland hypertrophy was a result of phosphodiesterase inhibition (smith et al., ) . other classes of drugs can also produce salivary gland enlargement in rats in repeated dose studies. doxylamine, a representative of the widely used ethanolamine group of antihistamines, has been reported to produce marked cytomegaly in the fischer rat parotid gland. enlarged cells were characterised by a basophilic and coarsely granular or vacuolated cytoplasm (jackson and blackwell, ) . the b c f mouse did not develop these changes after a similar treatment schedule. in view of the presence of considerable amount of epidermal growth factor in salivary glands, it is of interest to note the effects of its administration to laboratory animals. salivary gland weights were increased in rats and cynomolgus monkeys infused with high doses of recombinant human epidermal growth factor (breider et al., ; reindel et al., ) . however histological features seen are primarily those of ductular epithelial hyperplasia (see below under hyperplasia). epithelial cells characterised by abundant granular eosinophilic cytoplasm as a result of the accumulation of mitochondria are often referred to as oncocytes, a term used by hamperl ( ) to describe similar cells in hürthle tumours of the thyroid gland. they may be found in various focal nodular and neoplastic states of the salivary glands in both man and laboratory animals. the precise significance of these cells is uncertain. the mitochondria usually appear unremarkable except for lack of dense granules and it has been suggested that the mito-chondrial changes represent an adaptive phenomenon or compensatory hyperplasia (ghadially, ) . in human salivary tissue their prevalence seems to increase with advancing age and they can be associated with hyperplastic lesions or neoplasms such as oxyphil adenomas and adenolymphomas. eosinophilic cells also occur in the salivary glands of certain strains of aged rats (bogart, ) and in mice with experimentally induced autoallergic sialoadenitis (takeda et al., ) . in the study of takeda et al. ( ) the eosinophilic cells appeared to arise predominantly in the secretory (glandular) ducts of the submandibular glands, although eosinophilic cells can apparently develop from either duct or acinar cells. well-defined, unencapsulated foci of enlarged acinar cells occur spontaneously in the salivary glands, particularly the parotid of rats, mice (chiu and chen, ) , and hamsters although their reported incidence varies between laboratory. the enlarged cells possess greatly expanded cytoplasmic volume that retains a vesicular, vacuolated or foamy appearance or possesses a pale eosinophilic granular texture. the basal parts of the cells usually stain intensely blue in haematoxylin and eosin stained sections and contain large, dense, irregular hyperchromatic or pyknotic nuclei showing little evidence of mitotic activity. although there has been little ultrastructural study of these foci, the cytoplasmic alterations appear to be distinct from those of so-called oncocytes that characterised by granular eosinophilic cytoplasm packed with mitochondria. the biological nature of these foci is uncertain. the lack of any prominent mitotic activity, cell proliferation or expansive growth suggests that they are most aptly regarded as hypertrophic lesions (chiu and chen, ). although they increase in prevalence with increasing age in certain strains of rat, there is no evidence to suggest that they represent pre-neoplastic lesions or possess any relationship with development of neoplasia in salivary tissue (dawe, ) . hyperplasia and squamous metaplasia of the salivary ducts are common features of many inflammatory and reactive conditions in the salivary glands of rodents, dogs, non-human primates and man and can be associated with the presence of stones and calculi with the duct system. squamous metaplasia and regenerative change in the ducts occurs in rats afflicted with sialodacryoadenitis (carthew and slinger, ) . it is also described specifically located in the ducts of the sublingual glands in the wistar rat in the absence of obvious sialodacryoadenitis or evidence of any specific disease. similar regenerative hyperplastic duct changes are also seen in necrotic and inflammatory conditions in the dog salivary gland . detailed morphological examination with immunocytochemical study of epi-dermal cytokeratins of the rat salivary gland after arterial ligation has shown that the acinar units can also undergo squamous metaplasia (dardick et al., ) . it appears that the acinar-intercalated duct complexes can rapidly reprogram to produce epidermal cytokeratin filaments in ischaemic or inflammatory states. hyperplasia of the ductular epithelium appears to be the principle result of the administration of epidermal growth factor to rats and cynomolgus monkeys. in rats histological features were primarily of ductular epithelial hyperplasia without evidence of significant acinar hyperplasia (breider et al., ; reindel et al., ) . in primates the changes were most striking in the interlobular and large intralobular ducts where the epithelium showed multilayered and papilliform projections. however, mitotic activity was evident throughout the duct epithelia and acinar cells showed hypertrophy with depletion secretory granules and the presence of large vesicular nuclei . focal duct and acinar hyperplasia, showing minimal compression of the surrounding parenchyma and distinct from focal hypertrophy is also described in the classification of rat salivary lesions (mohr, ) . primary neoplasms of salivary glands are uncommon in the usual strain of rats and mice employed in carcinogenicity bioassays (haseman et al., ) . acinar and tubular adenomas and adenocarcinomas as well as squamous carcinomas are reported in rats (mohr, ) , mice (frith and heath, ) and hamsters (takahashi and okamiya, ) . some carcinomas showing squamous or glandular differentiation may be observed infiltrating the salivary gland that originate in other local structures of the head and neck region. occasionally, salivary gland neoplasms show adenomyomatous differentiation. mixed glandular and lymphoid tissue patterns resembling wartin's tumour in man are also sometimes seen. neoplasms of soft tissues also develop in and around the major salivary gland in rodents (see integumentary system chapter i). in humans the oesophagus is not considered a common site for drug-induced injury although some studies have suggested that medication-induced changes are more prevalent than previously supposed (bonavina et al., ) . severe damage can occur following prolonged contact between mucosa and ingested tablets or capsules which results in local high concentrations of potentially irritant substances (bott and mccallum, ; brors, ; kikendall, ; levine, ) . damage as a result of local contact may be more common in elderly subjects as the amplitude of oesophageal contractions decrease with age and capsules more liable to lodge in the lumen of the oesophagus (bonavina et al., ) . however patients of all ages may be affected. women have been injured more frequently than men probably because of the greater likelihood of their being treated with potentially injurious drugs (kikendall, ) . the shape and surface coating of tablets may influence their tendency to adhere to the mucosa and lodge in the oesophagus (marvola et al., ) . a wide variety of drugs have been implicated. in the united states the majority of cases appear to be caused by ingestion of tetracycline or doxacycline (levine, ) . some of the causative agents such as potassium chloride, aspirin and other non-steroidal anti-inflammatory drugs are also implicated in ulceration lower in the gastrointestinal tract. over recent years the bisphosphonate, alendronate has been one of the most commonly reported causes of adverse effects in the oesophagus with severe injury being reported. although injury is linked to ingestion without water or failing to remain upright after swallowing the medication, alendronate is particularly caustic (kikendall, ) . oesophagitis due to candida albicans is a well-described complication of antibiotic therapy. administration of immunosuppressive drugs may predispose to viral infections in the oesophagus. a number of agents affecting neuromuscular co-ordination may also predispose to gastro-oesophageal regurgitation and reflux oesophagitis (bott and mccallum, ) . in laboratory rodents spontaneous lesions of the oesophagus are occasionally seen. oesophageal impaction has been described in untreated srl:bhe rats. this is characterised by massive dilatation of the oesophagus with food or bedding (ruben et al., ) . histologically, the muscle fibres in the wall of the oesophagus show varying degrees of degeneration including swelling or shrinking of fibres, myofibrillar fragmentation, cytoplasmic vacuolation and mineralisation. so-called megaoesophagus, characterised by enlargement of the oesophagus, degeneration of muscle fibres and ganglion cells in the myenteric plexus has also been described in certain strains of rats and mice (harkness and ferguson, ; randelia and lalitha, ) . its cause is unknown. a commonly occurring lesion reported in fischer rats is oesophageal hyperkeratosis, which occurs at all ages . in the study by maeda et al. ( ) , it occurred more commonly in rats fed a protein-restricted, calorie unrestricted diet than in rats fed ad libitum with normal diet. it was suggested that the particular high prevalence of oesophageal hyperkeratosis observed in all groups in this particular study was related to acidification of drinking water . another pathological findings in rodents is perforation of the oesophagus as a result of a gavage accident. under these circumstances there is a variable inflammatory and purulent exudate localised around the perforation or spread within the pleural or occasionally the pericardial cavities. the oesophagus and surrounding tissues need careful examination by the pathologist for it is not always clear from clinical findings that oesophageal damage has occurred. spontaneous oesophageal lesions are uncommon in laboratory beagles, even though emesis and vomiting are frequent responses of this species following dosing in toxicity studies. local oesophageal irritancy potential of drugs has been assessed in a number of animal models, notably the cat and pig (carlborg and densert, ; olovson et al., ) . in these models, the test drugs are placed in the upper oesophagus using endoscopic techniques for periods of several hours to allow dissolution of the preparation. subsequently, the animals are followed for - days and histopathological assessment performed on the oesophagus. the degree of inflammation, erosion of mucosa or deep ulceration is recorded in a semiquantitative manner. the degree of ulcerogenic activity of drugs in these models seems to correlate with reported ulcerogenic activity in the human oesophagus (carlborg et al., ) . systemic administration of drugs with radiomimetic or antimitotic activity can cause hypoplastic changes in the oesophageal mucosa as well as the remaining gastrointestinal tract mucosa (tucker et al., ) . conversely, hyperplasia with increased keratinization has been reported in the oesophagus of the rat following chronic high dose administration of alcohol (mascrès et al., ) . acanthosis with hyperkeratosis and parakeratosis has been reported in the oesophagus but not stomach of rats treated for up to months with mesuprine hydrochloride, a β-adrenergic receptor stimulator (nelson et al., ) . as part of its effects on the gastrointestinal tract, the oesophagus in rats and primates has been reported to develop uniform hyperplasia of the squamous epithelium following infusion of recombinant epidermal growth factor (breider et al., ; reindel et al., ; vinter-jensen, ) . in the rat, mouse and hamster the forestomach occupies about two-thirds of the proximal stomach area and is lined by cornified stratified squamous epithelium. the limiting ridge is a distinct elevated mucosal fold at the junction between the forestomach and the mucosa of the glandular part of the stomach. as humans lack a forestomach, the relevance of changes produced by drugs and chemicals in the rodent forestomach is disputed. studies in rats in which the forestomach has been removed have suggested that the forestomach acts as a storage organ releasing relatively undigested food into glandular stomach in response to energy demand (gärtner and pfaff, ) . hence, the forestomach mucosa may be exposed to xenobiotics mixed in undigested food for far longer periods than elsewhere in the gastrointestinal tract. the interpretation of forestomach changes should take into account physiological factors, residence time and exposure differences to drugs between the rodent forestomach and human oesophagus. however the squamous mucosa lining the oesophagus in species without a forestomach may react to xenobiotics in a similar way to the forestomach mucosa of rodents if equivalent exposure levels are attained. inflammation and ulceration of the forestomach mucosa are some of the commonest spontaneous gastrointestinal lesions in laboratory rats, mice and hamsters. the prevalence of these gastric lesions varies between species, strains of laboratory rodents as well as between different laboratories. the precise causes of forestomach ulceration remain unclear although a variety of factors have been associated with its development including advanced age, infection, parasitism, diet, feeding regimens and stress. in rats, conflict-induced ulceration occurs in the forestomach and there is an age-related susceptibility, older rats developing more ulcers than younger rats (sawrey and sawrey, ) . in rats and mice dying of spontaneous disease, ulceration of the forestomach is also quite frequently observed. protein restriction or starvation has also been shown to produce forestomach ulceration in rats . ulceration of the forestomach in rodent toxicology studies may be incidental, particularly if the lesions are few and show no clear relationship to dose. if limited to high dose groups, ulceration may be a result of non-specific toxicity and stress-related. however, administered chemicals may have direct local effects of sufficient severity to cause focal damage to the forestomach mucosa. histological features of ulcers and inflammatory lesions of the forestomach are similar in rats, mice and hamsters. in mild cases, a scattering of acute inflammatory cells is seen in the intact squamous mucosa. ulcers can be single or multiple and are characterised by loss of squamous epithelium with a variable accumulation of neutrophils, mononuclear cells, cellular debris, fibrin and hair fragments in the ulcer crater. the inflammatory process may extend deeply into the stomach wall and be associated with intramural inflammation, oedema, endarteritis and fibrosis. haemosiderin pigment is also found in the ulcer margins. profuse haemorrhage may follow erosion of large blood vessels and complete perforation of the stomach wall with peritoneal involvement also occurs (greaves and faccini, ) . in long-standing cases of ulceration, hyperplasia of the adjoining squamous epithelium occurs, characterised by irregular acanthosis and down-growths of squamous epithelium into the submucosa . xenobiotics may produce inflammatory changes in the forestomach mucosa following initial dosing but subsequently, repair occurs even though treatment continues. an example of this phenomenon is illustrated by butylated hydroxyanisole. after week of administration of this agent in a % mixture in diet to rats, a vesicular inflammatory reaction characterised histologically by the presence of subepithelial vesicles containing inflammatory cells and exudate was seen (altmann et al., ) . after further treatment, only hyperplasia of the squamous epithelium was evident, presumably as an adaptive response to the effects of the continued insult. hyperkeratosis associated with hyperplasia of the squamous epithelium is seen sporadically in untreated aged rodents. these changes may be localised to the margins of chronic forestomach ulcers or they can be associated with diffuse inflammation of the mucosa. occasionally, the forestomach mucosa of untreated, aged rodents exhibits hyperkeratosis with hyperplasia without inflammation (fig. ). such changes may be diffuse or focal, but they are often localised to the zone adjoining the glandular stomach mucosa. there may be evidence of basal cell proliferation and downgrowth of the epithelium into the underlying stroma. dietary factors also influence the thickness of the forestomach mucosa. vitamin a deficiency, known to produce squamous metaplasia in glandular tissues may produce forestomach hyperplasia and hyperkeratosis in rats. when spf fischer rats were maintained in a vitamin a deficient state for over months, hyperplasia with hyperkeratosis, not unlike that produced by known carcinogens was reported (klein-szanto et al., ) . administration of a wide range of both industrial chemicals, therapeutic agents including both genotoxic and non-genotoxic carcinogens produces hyperkeratosis and hyperplasia of the forestomach epithelium which may be followed by preneoplastic lesions and squamous carcinoma (see below). histologically, the changes are characterised by hyperkeratosis, parakeratosis with varying degrees of acanthosis and papillomatosis (greaves and faccini, ) . the changes can be florid and it may be difficult to make a clear distinction between severe hyperplasia and neoplasia. nevertheless, it has been shown that the florid hyperplasia of the forestomach epithelium without evidence of cellular atypia can be completely reversible following the withdrawal of an inciting stimulus, ethyl acrylate (ghanayem et al., ) . hence, a critical feature may be the presence of cellular atypia in view of its association with agents with potent (genotoxic) carcinogenic activity. neoplasms arising in the forestomach of rodents are usually squamous carcinomas although basaloid features are also seen (fukushima and leiniger and jokinen, ; mohr, ; tatematsu, ) . squamous carcinomas, as at other sites, show variable differentiation being composed of proliferating squamous epithelium with moderate to marked cellular atypia, pleomorphism and mitotic activity with clear evidence of invasion into the muscularis. although they are relatively uncommon spontaneous lesions in aged rodents, they can be induced in rodents by administration of nitroso compounds (tatematsu ) as well as a range of non-genotoxic agents (see below). some authors report basal cell carcinoma when basaloid features are pronounced (tatematsu, ) . a wide range of agents is capable of producing squamous hyperplasia of the rodent forestomach and a number of these also induce squamous carcinomas. in kroes and wester reviewed over genotoxic and non-genotoxic compounds that were reported to produce hyperplasia and carcinoma in the forestomach of rats, mice or hamsters. a well-studied example is butylated hydroxyanisole (bha) an important food antioxidant (reviewed by whysner and williams, ) . structurally related phenols and acids produce similar changes (rodrigues et al., ) . ethyl acrylate, used in the production of materials for dental and medical devices is also capable of inducing marked squamous hyperplasia, papillomas and carcinomas after long-term treatment of f rats and b c f mice (ntp, ; ghanayem et al., ) . sk&f , an experimental histamine h receptor antagonist produced atypical forestomach hyperplasia in rats following administration by gavage for year by a mechanism which appeared unrelated to the inhibition of the h receptor (betton and salmon, ) . other therapeutic agents associated with squamous hyperplasia and neoplasia include the -hydroxy- -methylglutaryl coenzyme a (hmg-coa) reductase inhibitors (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) and aristolochic acid (göggelmann et al., ; schmeiser et al., ) . some cytoprotective prostaglandins appear capable of inducing hyperkeratosis and hyperplasia without neoplasia presumably through a mechanism related to their pharmacological activity (levin, ) . this occurs in rats treated with misprostol, a synthetic prostaglandin e methyl ester analogue with gastric anti-secretory and anti-ulcer activity (kotsonis et al., ) , cl , , a synthetic analogue of prostaglandin e type (kramer et al., ) and , -dimethyl prostaglandin e (reinhart et al., ) . even the extensively used antibiotic, ampicillin has been associated inflammation, ulceration with acanthosis and hyperkeratosis in mice but not rats treated for years (national toxicology program technical report, ) . sodium saccharin is also reported to produce hyperplasia without neoplasia of the forestomach in f rats (hibino et al., ) . among its wide range of pharmacological effects on the gastrointestinal tract, the forestomach has also responds to recombinant epidermal growth factor when infused into rats (breider et al., ; vinter-jensen, ) . histological examination showed hyperkeratosis and hyperplasia of the squamous epithelium. the large body of studies performed with butylated hydroxyanisole illustrates the various factors that can influence the development of treatment-induced hyperplasia and neoplasia of the rodent forestomach and subsequent assessment of human risk. butylated hydroxyanisole possesses little or no mutagenic activity in vitro but when administered to rats for years as a % mixture in the diet, it produced squamous hyperplasia, squamous papillomas and squamous carcinomas of the forestomach. at . % in the diet butylated hydroxyanisole induced only hyperplasia . it also produces proliferative lesions in the forestomach of both mouse and hamster (ito et al., ) . studies in which butylated hydroxyanisole was fed in the diet to rats for shorter periods have shown that squamous epithelial hyperplasia occurs after only week of treatment preferentially over the lesser curvature, the site at which carcinomas developed in the -year studies (altmann et al., ) . after weeks' treatment, mucosal hyperplasia characterised by pronounced hyperkeratosis, parakeratosis and acanthosis most pronounced over the lesser curvature, was present in rats given % butylated hydroxyanisole in diet but not in rats given . , . and . % mixtures . abundant mitoses were found in the basal cells layers and tritiated-thymidine labelling confirmed that the changes were accompanied by a high rate of cell proliferation. following cessation of administration of butylated hydroxyanisole after weeks, the tritiatedthymidine labelling index rapidly reverted to control levels within about week although hyperplasia took longer to regress. nearly complete regression of the hyperplasia occurred after about weeks of normal diet . the distribution of squamous hyperplasia induced in the rodent stomach by butylated hydroxyanisole is influenced by the mode of administration. whereas following feeding of rats with butylated hydroxyanisole mixed in the diet lesions tended to be located near the limiting ridge, altmann et al. ( ) showed that gavage of butylated hydroxyanisole in corn oil produced similar changes at the apex of the forestomach. it was suggested that this difference was due to incomplete mixing of butylated hydroxyanisole in the stomach lumen when given by gavage and prolonged contact of the gavage mixture with the upper segment of the forestomach (altmann et al., ) . more recently, it was shown that fischer , shr, lewis and sprague-dawley rats differ in their response to the hyperplastic and carcinogenic effects of % butylated hydroxyanisole in pelleted diet. the most sensitive was the shr strain followed by the f rats and the differences correlated with the cytotoxic effects of butylated hydroxyanisole in the different strains (tamano et al., ) . it was suggested that the presence of vascular damage in the stomachs of the shr rats might have contributed to the response to cytotoxicity and subsequent carcinogenicity. residence time of administered compounds in the forestomach may influence the development of lesions. although it has been demonstrated that butylated hydroxyanisole does not produce hyperplasia in the oesophagus of animals without a forestomach, have shown that high-doses given to primates are capable of producing an increase in mitotic activity in the lower end of the oesophagus similar to that occurring at equivalent exposure levels in the rat. the implication is that these interspecies differences may simply be a question of differences in exposure of the squamous mucosa to compound. this underlines the fact that mechanisms of action and exposure levels of xenobiotics attained in the gastrointestinal tract of rodent and non-rodent species as well as of man need to be carefully assessed when hyperplastic changes are induced in the forestomach mucosa of rodents. such information can be helpful in facilitating regulatory decisions in this area (moch, ) . on balance, the evidence suggests that the tumour development by butylated hydroxyanisole in rodents represents an epigenetic phenomenon related to largely reversible cytotoxicity and increased cell proliferation (whysner and williams, ) . in view of the low levels of exposure to butylated hydroxyanisole that occurs with the usual use of this agent, carcinogenic hazard for the human stomach is therefore probably very small. similar phenomena have also been reported in studies of phenols and acids that are structurally related to butylated hydroxyanisole (rodrigues et al., ) . these agents include n-butyl and n-propyl- -hydroxybenzoic acid esters, propionic acid and -methoxyphenol. however, these studies suggested that certain areas of the forestomach epithelium react differently to structurally related chemicals, possibly due to the variable levels of activating enzymes within different zones of the forestomach epithelium. co-administration of acetylsalicylic acid was shown to abrogate some of these effects, suggesting that prostaglandin synthetase may be involved in the hyperplastic response (rodrigues et al., ) . a number of hmg-coa reductase inhibitors with different chemical structures including marketed products such as lovastatin, simvastatin and fluvastatin are also associated with the development of squamous hyperplasia of the rodent forestomach. the hyperplasia is time and dose dependent and may be associated with oedema and some inflammation of the submucosa. some, but not all of these agents are also capable of producing squamous neoplasia of the forestomach mucosa of rats, or mice or both after long-term treatment (kloss et al., ; bueld et al., ; akiba et al., ; physicians' desk reference, ) . the mechanism of action remains unclear although the degree of hyperplasia seems related to pharmacological potency. their carcinogenic potential in rodent bioassays does not seem to relate to the degree of hyperplasia in shortterm studies. moreover, the development of hyperplasia depends on local high concentrations of drug because when administered by non-oral routes, hyperplasia does not occur (kloss et al., ) . as most of these drugs are non-mutagenic, these findings are presumably also epigenetic in origin and possess relatively little risk for humans when given in the usual therapeutic doses. a contrasting example is provided by aristolochic acid, a nitrophenanthrene derivative of the ancient medicinal plant aristolochia clematis which was used as an anti-inflammatory component in a number of medicinal preparations in germany until (göggelmann et al., ; schmeiser et al., ) . aristolochic acid is a direct acting mutagen in salmonella typhimurium. when fed to rats at doses of . and mg/kg/day, aristolochic acid produced severe papillomatosis of the entire forestomach within a period of months. this was characterised histologically by the presence of branched squamous papillomas up to mm high with focal dysplastic features. invasive squamous carcinomas with metastases were found subsequently, or months later without further treatment (mengs et al., ) . even at a low dose of . mg/kg/day papillomas and squamous carcinomas developed months after a -month period of treatment. quite clearly the complexity of the hyperplastic response of the rodent stomach to xenobiotics, the association of hyperplasia induced by non-mutagenic compounds with the development of forestomach carcinomas, and the similarity of response in the forestomach to that of the oesophagus, dictates the need for a careful analysis of hyperplasia induced by novel drugs in the forestomach. the prelude to this assessment is careful histopathological characterisation of the changes. unlike the mouth and oesophagus through which tablets, capsules, gavage fluids and drug/diet mixtures pass relatively rapidly, the human stomach mucosa remains in contact with high local concentrations of administered compounds for much longer periods of time. administration of compounds in liquid or solid form, particle size, fasting and feeding all affect the gastric motility pattern. in the fasted state there is a cyclical pattern of motility consisting of three main phases. the first is a quiescent phase, followed by a phase of irregular contractions that increase in amplitude and frequency to reach a maximum in a third phase. feeding results in the replacement of this cyclic pattern by regular tonic contractions that move food towards the antrum and mix it with gastric secretions. these patterns have been well studied in both dog and man and appear to be qualitatively similar in the two species (sarna, ) . these motility patterns may have an impact on the length of time drugs remain in contact with stomach mucosa. for instance, the residence time of large non-disintegrating capsules or tablets administered in the fasting state is more dependent on the frequency of powerful phase iii contraction than if drugs are given as fluids or mixed with diet. for dosage forms released in the stomach, gastric residence time will influence drug supply to the main absorptive surfaces in the small intestine, which in turn may affect drug absorption (dressman, ) . gastric acid is also important in making ingested salts soluble. although the presence of food in the stomach is a stimulus of acid production, the ph in the forestomach of rats is highest in full stomachs and lowest when empty, presumably as a consequence of the buffering action of food (ward and coates, ) . the glandular stomach is conveniently divided into the fundus characterised by mucosal folds or rugae and the smoother antrum, which opens into the pylorus and duodenum. in species devoid of a forestomach, the proximal stomach mucosa or cardia is also lined by glandular mucosa. the glandular mucosa is covered by surface epithelium of regular columnar cells that extends downwards to form small gastric pits or foveolae. the gastric glands are simple tubular structures usually considered to comprise three segments. the base is the deepest part, the neck the mid-region, and the most superficial is the isthmus, continuous with the gastric pit. the upper part of the gastric gland contains mucous neck cells. small cuboidal chief or zymogenic cells, which secrete pepsinogen and stain blue or purple in haematoxylin and eosin sections, are located in deeper parts of the gland. the eosinophilic-staining parietal (oxyntic) cells, which produce hydrochloric acid, are distributed more randomly throughout the gastric glands. parietal cells can also be visualised by immunocytochemical staining with antibodies directed at h + k + -atpase (canfield et al., ) . the gastric glands situated near the limiting ridge in rodents, show a modified structure. in species not endowed with a forestomach, the mucosa near the cardia is composed of simplified branched glands lined by columnar epithelium. the antral mucosa is covered by a surface epithelium with gastric pits similar to that of the fundus but mucous secreting columnar glands line the glands. the stomach mucosa is richly endowed with endocrine cells, not all of which have been well characterised. enterochromaffin cells are quite numerous in the basal parts of the gastric glands of the fundus, particularly in the rat . they are generally argyrophilic, staining with silver staining techniques such as that of grimelius (grimelius, ; grimelius and willander, ) that utilise exogenous reducing agents. these cells contain histamine and histamine-related enzymes such as histidine decarboxylase in the rat and other species . endocrine cells which are argentaffin in type stain with silver stains such as that of masson ( ) because of the presence of endogenous reducing substances including -hydroxytryptamine and catechol-amines are also reported in the mucosa of the fundus of some species including man but apparently not in the rat . enterochromaffin cells are characterised ultrastructurally by the presence of numerous rounded or oval, vesicular, electron-lucent granules frequently containing a small eccentric electron dense core. gastric enterochromaffin cells can also be stained by immunocytochemical techniques using antisera to histamine and histidine decarboxylase as well as to non-specific enolase and chromogranin a betton et al., ) . immunocytochemical study of the rat fundus using a battery of antisera to a variety of gastrointestinal peptides has shown somatostatin containing (d) cells and glucagon staining cells but no cells with gastrin (g cells) or serotonin reactivity (bishop et al., ) . gastrin or g cells possess apical processes reaching the stomach lumen believed to be important in stimulation of gastrin release as a result in increases in antral lumen ph or the presence of amino acids or peptides . glucagon and serotonin containing endocrine cells have also been located in the rat antral mucosa (bishop et al., ) . increased gastric acid secretion is initiated by activation of central vagal efferent pathways but acid secretion is maintained by both neural and endocrine reflexes activated by the presence of food in the stomach. gastrin secreted from the g cells of antrum is the main stimulant of acid secretion. somatostatin is secreted from antral d cells when the luminal ph falls to below . to act by a paracrine mechanism to suppress g cell function thus forming a negative feedback loop (dockray, ) . the two main endocrine cell types from the body mucosa integrate neurohumoral stimuli rather than respond to luminal chemicals. although gastrin is capable of stimulating parietal cells directly, it has an even greater effect through stimulation of enterochromaffin cells to release histamine, a potent paracrine stimulator of parietal cells (hinkle and samuelson, ) . gastrin stimulates release of histamine from enterochromaffin cells of the body mucosa, which increases acid secretion through activation of parietal cell histamine-h receptors. both parietal and enterochromaffin cells are inhibited by somatostatin released from the d cells of the body mucosa in response to a variety of neurohumoral stimuli such as noradrenaline, vasoactive intestinal peptide, calcitonin gene-related peptide, and cholecystokinin. it should also be noted that gastrin has a role in kinetics and differentiation of both parietal and enterochromaffin cells (dockray, ) . gastrin acts at the gastrin/cholecystokin-b receptor that is expressed by gastric epithelial cells and by neurones in the central nervous system (kopin et al., ; wank, ) . the gastrin receptor is simply the cholecystokinin-b receptor located in the stomach. the other cholecystokinin receptor, cholecystokin-a has high affinity for cholecystokinin. stimulation of this receptor in the stomach mediates secretion of pepsin from gastric chief cells and release of somatostatin from d cells resulting in inhibition of acid secretion. in the central nervous system cholecystokinin and its receptors contribute to the regulation of satiety, anxiety, analgesia and dopamine-related behaviour (wank, ) . finally, it is worth recording that both progesterone and oestrogen receptors have been identified in both normal and pathological gastric tissues of humans (wu et al., ) . generative cells in the gastric mucosa as shown by uptake of tritiated thymidine for dna synthesis are distributed principally in the isthmus (inokuchi et al., ) . tracing of cells using thymidine labelling have shown that most of the cells in the generative zone migrate in a successive manner to the mucosal surface to form columnar epithelium. the life span of surface epithelium in the stomach of rats, mice and hamsters has been calculated to be about - days. studies of cell cycle and dna synthesis time in the proliferative zones in the stomach of rat, hamster and man have suggested that the generative cells in the isthmus undergo mitoses at about -hour intervals in rodents and -hour intervals in man (inokuchi et al., ) . although this process of migration from the proliferating cell zone of the isthmus renews surface epithelial cells rapidly, cell migration to the lower parts of the gastric glands is much slower and more complex. detailed studies have show that undifferentiated cells in the region of the isthmus represent a common source for surface mucous cells and mucous neck cells (karam and leblond, ) . electron microscopic and ultrastructural cytochemistry has suggested note: saccharide binding specifications are much more complex than the inhibition by simpler sugars outlined above suggests. see review by nicholson ( ) . *source: nicholson, ; goldstein and hayes, ; schulte and spicer, ; giannasca et al., . that chief cells in the adult rat stomach develop from undifferentiated stem cells in the isthmus (suzuki et al., ) . graft experiments in mice have also suggested that immature cells of the isthmus differentiate into chief cells as well as parietal cells (matsuyama and suzuki, ) . studies in transgenic mice have shown that mature parietal cells influence the fate of other gastric epithelial cells because targeted degeneration of parietal cells is associated with loss of chief cells suggesting interactions between these cell populations in determining their differentiation (li et al., ; canfield et al., ) . gastrin is also an important regulator of parietal cell and enterochromaffin differentiation and number (dockray, ; montgomery et al., ) . labelling experiments in the hamster stomach have shown that both chief and parietal cells possess a similar but quite long life span of about days (hattori, ; hattori and fujita, ) . it has been suggested that the relative distribution of chief and parietal cells in the gastric gland represents an expression of their different migration patterns downwards from the proliferative zones in the isthmus. this type of migration pattern in which cells are able to overtake each other has been termed a 'stochastic flow system' (inokuchi et al., ) . the origin and kinetics of endocrine cells of the stomach has also been the subject of debate but the available morphological, cytochemical and kinetic evidence suggests that the majority of these cells develop from the same stem cells as the other non-endocrine cells of the gastric mucosa, although self replication also occurs (matsuyama and suzuki, ; inokuchi et al., ; solcia et al., ) . much of our knowledge about mucins produced by the epithelial cells lining the gastrointestinal tract has been obtained using histochemical techniques and these approaches continue to be helpful in the understanding of spontaneous and drug-induced gastrointestinal disease (sheahan and jarvis, ; filipe, ; tsiftsis et al., ; jass and roberton, ) . for these reasons, mucin histochemical techniques represent useful tools for the characterisation and elucidation of experimentally or drug-induced changes in the glandular mucosa of gastrointestinal tract. techniques commonly employed are presented in table . the physiochemical properties of gastrointestinal mucins are dependent on their glycoprotein constituents. these glycoproteins are high molecular weight compounds with large numbers of sugar chains attached to a polypeptide backbone by o-glycosidic linkages between n-acetylgalactosamine and serine or threonine (berger et al., ) . the principle monosaccharides present are fucose, galactose, n-acetylgalactosamine, n-acetylglucosamine and sialic acid. traces of mannose may be present and ester sulphate residues are common (filipe, ) . due to this extensive glycosylation, mucins have a filamentous conformation, which is often negatively charged. this is believed to be important in forming a protective barrier to the cell. however, this property is a two-edged sword because when opposing cells have specific receptors for mucins, adhesion may become the predominant factor (van klinken et al., ) . although mucins are important in the gastrointestinal tract, it should be remembered that other products secreted by goblet cells might be important in mucosal defence. it has been recently recognised that trefoil proteins, a family of small proteins secreted by goblet cells and present on the mucosal cell surface, can also protect against a variety of deleterious agents, including bacteria, toxins and drugs (podolsky, ) . there are considerable regional variations in glycoprotein constituents in the gastrointestinal tract and these differences are probably related to physiological and functional factors. furthermore, synthesis and secretion of glycoproteins alter with changes in cell differentiation. alterations also occur in mucins in various inflammatory and neoplastic disease states as well as following administration of certain drugs and chemicals (ishihara et al., ) . terminal sugars or sugar sequences can be demonstrated histochemically by the use of labelled lectins, mostly plant proteins which combine non-enzymatically with particular sugar molecules, see table (goldstein and hayes, ; debray et al., ; rudiger, ) . magenta: all mucosubstances containing hexoses schiff d-pas (pearse, ) and deoxyhexoses with vicinal glycol groups. some non-sulphated acid mucosubstances. neutral mucosubstances. periodate-borohydride/ magenta: pas activity following periodate borosaponification/pas, hydride/potassium hydroxide indicates pb/koh/pas presence of o-acylated sialic acids. (reid et al., ; periodate borohydride reduces periodate culling et al., ; generated aldehydes. potassium hydroxide removes o-acylesters from potential vicinoldiols and sialic residues linked glycosidically to a potential vicinoldiol. alcian blue ph . basophilia: weakly sulphated mucins. carboxyl (pearse, ) groups of sialomucins alcian blue ph . basophilia: sulphated mucins (lev & spicer, ) alcian blue ph . -magenta: neutral mucins periodic acid schiff, ab/pas basophilia: acid mucins (mowry & morard, ) purple-blue: neutral and periodate reactive acid mucins high iron-diamine, brown-black: sulphated mucins hid (spicer, ) unstained: sialomucins high iron-diamine-alcian brown-black: sulphated mucins blue ph . , hid/ab basophilia: non-sulphated acid mucins (sialomucins) (spicer, ) source: adapted from filipe, . when gastrointestinal mucins were studied in several species using histochemical techniques under uniform conditions, species differences were most obvious in the stomach and duodenum (sheahan and jarvis, ) . neutral mucins generally predominate in the stomach, contrasting with acid mucins in the small intestine, and sulphated mucins in the colon. in the stomach neutral mucins staining purple with the pas/alcian blue stain, predominate in the surface and foveolar mucosa, whereas mucous neck cells and antral glands contain acidic mucins that stain blue with pas/alcian blue procedure. sulphated mucins, as shown by the high iron diamine technique (hid) are also found in the deep glandular mucosa of the antrum in rat, mouse and man (filipe, ; jass, ; greaves and boiziau, ) . extremely heterologous staining patterns are seen in the gastric mucosa with labelled lectins, each lectin staining quite different cell populations. there are considerable interspecies differences in staining patterns with the same lectins (kuhlmann et al., ; suganuma et al., ) . the so-called paradoxical concanavalin a stain, in which conjugated concanavalin a is used to label mucins before and after periodate oxidation, has also been used to classify the alterations in mucins in proliferative and neoplastic conditions of the rat stomach mucosa (kobayasi et al., ; tatematsu ). although gastric erosions and ulcers in the glandular mucosa occur quite commonly in laboratory animals in toxicity studies, it is often difficult to determine whether such lesions in treated animals indicate a real ulcerogenic risk for the test compound. there is little that is histologically specific to drug-induced ulceration of the gastric glandular mucosa. mucosal haemorrhage, depletion of mucin, erosions and ulcers with or without inflammation may all be found. erosions represent mucosal breaks superficial to the muscularis mucosa. ulcers are lesions that extend through the muscularis mucosa. whilst the histopathological features of gastric erosions and ulcers are themselves relatively non-specific, it is important to look for any associated pathology in the stomach such as mucus depletion, epithelial hyperplasia or dysplasia, intestinal metaplasia and vascular lesions (see below). in humans, biopsy data suggests that drug-induced ulceration is characteristically devoid of an inflammatory component, but the most usual histological appearances are those of underlying gastric pathology (riddell, ) . formation of gastric and duodenal ulcers is dependent on the presence of both acid and peptic activity in gastric juice because acid without pepsin appears to have little digestive power. important predisposing factors in human patients with peptic ulceration include helicobacter pylori (campylobacter pylori) infection of the antrum, cigarette smoking and ingestion of non-steroidal anti-inflammatory drugs (soll, ) . helicobacter pylori is believed to infect over half the human population and its presence in the gastric mucosa is associated with chronic atrophic gastritis and peptic ulceration (cover and blaser, ) . it is a microaerophilic, gram-negative organism that possesses potent urease activity crucial for its survival at acidic ph. genome sequence analysis has shown that helicobacter pylori has well developed sequences for motility, scavenging iron and dna restriction and modification systems used by bacteria to degrade foreign dna. the link between helicobacter pylori infection and peptic ulceration is related to increases in gastrin release, perhaps through bacterial products or cytokines released from activated lymphocytes (richter-dahlfors et al., ) . although helicobacter pylori can infect other species, apart from non-human primates, the usual laboratory animal models do not appear to develop the inflammatory disease seen in humans (nedrud, ) . erosions and ulcers also develop following stress, reflux of intestinal contents and bile, changes in acid secretion and hypoxia, all of which may develop under the conditions occurring in high-dose toxicity studies. the requirement to give the test compound in high doses may also dictate the need to administer exceedingly high concentrations of test agent. this may produce damaging high local concentrations on the mucosa not relevant to therapeutic doses used in clinical practice. it has been demonstrated that hyperosmolar solutions of quite innocuous substances such as glucose can cause haemorrhage, erosions and ulcers of the rat gastric mucosa (puurunen et al., ) . the well-known association of gastric erosions and haemorrhage with uraemia may also be manifest following administration of high doses of drugs such as diuretics which severely derange fluid and electrolyte balance (garthoff et al., ) . stress ulceration may be linked to temporary ischaemia of the mucosa (dubois, ) . synergism between the ulcerogenic action of drugs and stress is a well-described phenomenon (rainsford, ; beattie, ) . protein depletion and starvation is also capable of inducing gastric ulceration in rats . although gastric pathology represents the largest cause of morbidity and mortality in man following therapy with non-steroidal inflammatory agents (fowler, ) , the reasons for this are probably multifactorial. the acidic properties of some of these drugs may cause direct local damage of gastric epithelial cells, demonstrable by the fact that appropriate formulation can reduce gastric toxicity of these agents in man (brors, ) . anti-inflammatory agents are capable of decreasing synthesis of glycoproteins and this may adversely influence protective mucus production of gastric mucosa (azuumi et al., ; ishihara et al., ) . it has also been suggested that non-steroidal anti-inflammatory agents cause cellular damage to the gastric mucosa by back-diffusion of gastric acid into mucosal tissues (davenport, ) or by causing damage to the gastric capillary bed with subsequent mucosal infarction (robins, ) . the theory that has gained widespread acceptance is that the ulcerogenic potential of non-steroidal anti-inflammatory drugs is related to their pharma-cological activity. vane ( ) proposed that the ulcerogenic potential of these agents was largely a result of their ability to inhibit prostaglandin synthetase, thereby reducing the protective effects of prostaglandins. pharmacokinetic factors may also be important. lipid solubility in the low ph environment of the stomach may influence local penetration into the mucosa (mccormack and brune, ) . moreover, it has been proposed that certain anti-inflammatory agents may possess lesser ulcerogenic potential in man because their inhibition of prostaglandin production is more limited to sites of inflammation, sparing gastric mucosa (whittle et al., ; whittle and vane, ) . it has also been demonstrated that factors altering the enterohepatic circulation of drugs can influence the expression of gastric damage (overvold et al., ) . comparative studies of the ulcerogenic activity of indomethacin in beagle dogs and domestic pigs has suggested that the dog may be an excessively sensitive species as a result of extensive enterohepatic circulation of indomethacin in this species (hanhijarvi et al., ) . prediction of ulcerogenic potential for man based on data from animal models is clouded by the lack of good comparative data on the relative ulcerogenic potential of non-steroidal anti-inflammatory agents in man because of extensive differences in side effect reporting (fowler, ) . moreover proper comparisons in man require not only equivalent therapeutic doses but also comparable dosage forms (brors, ) . in laboratory animals, a variety of different patterns of drug-induced gastric damage have been described. the study by shriver et al. ( ) in which a wide variety of different anti-inflammatory drugs were administered to fasted sprague-dawley rats under identical conditions, suggested the drugs could be divided into three groups based on their profiles of gastrointestinal toxicity. immunological agents such as azathiaprine, cyclophosphamide, methotrexate and d-penicillamine produced gastric mucosal haemorrhage whereas aspirin and related agents produced gastric mucosal haemorrhage and ulcers. the powerful nonsteroidal anti-inflammatory drugs indomethacin and phenybutazone produced gastric mucosal erosion and ulcers as well as small intestinal damage. comparative single oral dose studies of several different non-steroidal antiinflammatory agents at three different dose levels by suwa et al. ( ) in the rat using histology and measurement of faecal blood loss with cr-labelled blood cells have also shown that different patterns of ulceration can be produced by different agents when administered under identical conditions. single oral doses of some non-steroidal anti-inflammatory drugs including aspirin produced widespread superficial damage and desquamation of gastric epithelium with little or no inflammation at hours following dosing which completely healed weeks later. this damage was associated with transient faecal blood loss. by contrast, indomethacin and ibuprofen produced both gastric damage and circumscribed, penetrating ulcers along the mesenteric border of the jejunum and ileum. furthermore, ulcers were still present after weeks and were associated with prolonged or biphasic blood loss (see small intestine). in addition feeding conditions can influence the distribution of erosions and ulcers in laboratory animals. in fasted rats, erosions due to indomethacin treatment are found in the body of the stomach whilst in conventionally fed rats they are most prominent in the small intestine. detailed studies by satoh et al. ( ) showed that rats fed for hour after a hour-fast and given a single dose of indomethacin within hours of re-feeding developed erosions and ulcers in the antrum primarily along the lesser curvature. indomethacin given to fasted rats produced erosions in the body mucosa. a further factor that needs to be kept in mind is that chronic administration of ulcerogenic compounds may produce quite different pathological appearances to those found following single dose administration. administration of aspirin to rats for weeks has been shown to stimulate epithelial proliferation of the gastric body but not antral mucosa, possibly by an effect on cyclic adenosine ', ' monophosphate (cyclic amp) or though increasing the rate of epithelial exfoliation (eastwood and quimby, ) . such a response may be the basis for increased resistance of the gastric mucosa to the chronic affects of these agents. it also may explain the tendency for ulcers to occur in the antrum following chronic administration of aspirin-like drugs as the proliferative response and presumably the adaptive potential appears less in this part of the gastric mucosa. both interspecies variations and strain differences have been reported in the response to ulcerogenic compounds. rainsford et al. ( ) showed that extravasation of red blood cells and greater vascular damage was observed in rats treated with aspirin or benoxprofen than in pigs given similar doses. sprague-dawley rats appear less susceptible to the ulcerogenic effects of cold-restraint stress than wistar rats (goldenberg, ) . diuretics and some angiotensin converting enzyme (ace) inhibitors and angiotensin ii antagonists have been associated with the development of gastric erosions and ulceration when administered in high doses to laboratory animals ( fig. ) (imai et al., ; garthoff et al., ) . however, these effects appear related to the severe electrolyte disturbances produced by excessive doses of these drugs. this is perhaps analogous to the well-known association of gastrointestinal tract erosion and haemorrhage with uraemia. dogs appear to have a particular predisposition to this effect where it may be associated with deposition of basophilic ground substance and mineral in connective tissues and blood vessels in the mucosa (barker and van dreumel, ) . although inflammatory conditions due to microorganisms are generally uncommon in the stomach, gastritis is reported in laboratory rhesus monkeys in association with the presence of helicobacter organisms (reed and berridge, ) . as in the analogous condition in man, the stomach of affected animals shows an infiltration of the central mucosa by small lymphocytes and plasma cells, associated with reactive or atrophic changes in the mucosa and the presence of small curved bacteria in glands, visualised best with the warthin-starry stain. infiltration of the stomach by lymphocytes in rats treated with human recombinant interleukin- without ulceration was reported as part of a multisystem involvement induced by this agent (anderson and hayes, ). decrease in gastric mucus secretion may accompany both spontaneous inflammatory conditions and drug-induced lesions in the stomach of man and experimental animals. mucus depletion is characterised histologically by the presence of an intact epithelial layer in which cells show loss of the normal clear cytoplasm replete with mucous substances by more basophilic cells that contain little or no mucin. qualitative changes in mucus composition can also accompany mucus depletion. gastric epithelium in man may show decreases in sulphated mucosubstances following stress, high alcohol consumption or after aspirin administration (filipe, ) . similar changes occur in laboratory animals subjected to ulcerogenic regimens. stress ulceration in the rat is accompanied by decreased sulphation of gastric glycoproteins, presumably an expression of the changes in gastric cellular activity accompanying stress (lambert et al., ) . administration of aspirin and other anti-inflammatory agents including adrenocortical steroids to laboratory animals also reduces the content of sulphomucins in the gastric mucosa, probably by reducing their synthesis (denko, ; gerard, ; ishihara et al., ) . rather surprisingly, administration of histamine h -receptor antagonists and fig. . section from the glandular stomach from a rat treated with ah high dose of an angiotensin ii antagonist that shows superficial degeneration and ulceration (erosion) of the mucosa. (he, × .) proton pump inhibitors associated with reduction of gastric acid output and increases in gastrin secretion have also been associated with alterations in gastric mucus. administration of omeprazole or famotidine to rats for weeks was shown to inhibit prostaglandins pge as well as the synthesis of both total and sulphated glycoprotein synthesis along with histochemical evidence of reduction in pas staining of the surface mucus (yoshimura et al., ) . although the mechanism for this change is unclear, the reduction in mucus, particularly sulphated mucus that is believed to be particularly resistant to peptic digestion, may have implications for mucosal defence. intestinal metaplasia of the stomach is characterised by the presence of differentiated epithelium, which resembles small intestine on the basis of light microscopic and ultrastructural morphology, mucin patterns and enzyme histochemistry (morson, ; planteydt and willighagen, ; lev, ; goldman and ming, ; . it develops in man in gastric mucosa altered by chronic atrophic gastritis and its significance is due to the fact that a link exists between intestinal metaplasia and gastric cancer. although intestinal metaplasia is found much less commonly in laboratory animals, it has also been reported to occur in association with gastric cancer induced by polychlorinated biphenyls (ward, ) . in view of this association with gastric cancer, it has been suggested that intestinal metaplasia represents a pre-neoplastic lesion. however, over recent years prospective clinical studies and experimental data have suggested that it is an epiphenomenon, coexisting with, but unrelated to the development of cancer. in man, several forms of intestinal metaplasia have been described. these variants fall into two main groups, an incomplete type and a complete form (teglbjaerg and nielson, ; jass and filipe, ; jass, ) . complete intestinal metaplasia is characterised by the presence of goblet cells, paneth cells and absorptive cells with brush borders and variably developed intestinal villi. incomplete forms are more heterogeneous characterised by goblet and mucous columnar cells but no absorptive cells and variable patterns of mucin. the routine alcian blue: ph . , periodic acid-schiff stain (ab/pas) ( table ) distinguishes between the intestinal acid mucins (blue) from the neutral mucins of gastric type. however, variable sialomucin and sulphomucin staining patterns are seen in intestinal metaplasia in man with the high iron-diamine/alcian blue stain (hid/ab) (jass, ) . the incomplete form of intestinal metaplasia, showing marked sulphomucin secretion, has been found more commonly in association with gastric cancer in man (jass and filipe, ; jass, ; wells et al., ) . however, prospective studies have tended to indicate that intestinal metaplasia with sulphomucin secretion may be an age-related form of chronic atrophic gastritis and not a premalignant lesion (ectors and dixon, ) . it has been suggested that intestinal metaplasia represents an adaptive response to long-standing chronic inflammation and reduced acid secretion. it may also represent an adaptive defensive response to long-standing helicobacter pylori infection because intestinal mucosa is more resistant to these organisms (steer, ; ectors and dixon, ) . intestinal metaplasia has been found in association with gastric cancer in laboratory animals. fischer rats treated with the polychlorinated biphenyl, aroclor , mixed in the diet for years developed foci of intestinal metaplasia in the stomach epithelium in association with gastric adenocarcinomas (ward, ) . these lesions were characterised by abundant mucin-containing cells and alkaline phosphatase activity typical of the small intestine (morgan et al., ; ward, ) . similar, but more diffuse intestinal metaplasia was reported in the stomach of primates treated with polychlorinated biphenyls, although unassociated with gastric neoplasia (allen, ; mcconnell et al., ) . intestinal metaplasia is also found in the stomach of laboratory animals treated with powerful genotoxic gastric carcinogens. although tsiftsis et al. ( ) showed hyperplasia and foci of atypical changes (dysplasia) but little or no intestinal metaplasia in rats following administration of n-methyl-n´-nitro-n-nitroguanidine, tatematsu et al. ( ) were able to show intestinal metaplasia in rats treated with the same agent. however, intestinal metaplasia can be induced in rodents by a variety of different procedures that are not associated with the development of gastric cancer. intestinal metaplasia can be induced in the glandular stomach of rodents by fractionated, localised, ionising radiation (watanabe, ; watanabe et al., ) , injection of xenogenic stomach antigens as well as propantheline bromide and the non-carcinogen, iodoacetamine shirai et al., ) . the characteristics of intestinal metaplasia in laboratory rodents are similar to those seen in man with early increases in intestinal enzyme activity (alkaline phosphatase, lactase, trehalase, sucrose and maltase), development of goblet cells containing neutral, sialo-, or sulphomucins, and intestinal crypts with or without paneth cells. both the fundus and antrum can show changes although as in man, males appear more prone to develop intestinal metaplasia than females . based on these experimental findings, have also proposed that intestinal metaplasia is not a precancerous condition but an adaptive response to a chronic elevation in ph in gastric secretion due to the early loss of parietal cell mass brought about by these various procedures. on balance therefore, the evidence to date suggests that although intestinal metaplasia is associated with cancer and may consequently be considered a helpful morphological feature in the evaluation of human gastric biopsies, the finding of isolated intestinal metaplasia in safety studies does not indicate a preneoplastic state. finally, a form of metaplasia in which hepatocytes have been found has been reported as rare incidental findings in the glandular stomach of mice sacrificed at the end of -year carcinogenicity bioassays (leiniger et al., ) . histologically, focal accumulation of well-differentiated hepatocytes were found in the submucosa and lamina propria adjacent to the limiting ridge with dilated adjacent gastric glands showing epithelial hyperplasia and mineralisation with herniation into the submusosa. whilst these foci were not believed to be related to treatment with xenobiotics, it is not clear whether represented metaplasia or congenital ectopia. the gastric glandular epithelium is predisposed to the deposition of calcium possibly as it is a site at which marked ion exchange normally takes place. focal aggregates or concretions of densely blue-staining mineral are fairly commonly observed in haematoxylin-stained sections from the stomachs of aged rats where they are associated with cystic dilatation of the gastric glands (greaves and faccini, ) . mice and hamsters occasionally show similar changes. small concretions are also observed in gastric glands in the beagle dog. these appear to represent aggregates of calcium around mucoid material. gastric mineralisation may become marked in rodents and dogs when there is disturbance of mineral metabolism, particularly in association with renal pathology. this has been well described in rats with severe renal disease (glomerulosclerosis) and parathyroid hyperplasia (snell, ) . a similar phenomenon has been described in the stomach of dogs in uraemic states (cheville, ) . identical changes result from the administration of drugs that induce prolonged azotemia or electrolyte disturbances. these changes are characterised by diffuse deposition of mineral in the intestinal tissue of the mucosa of the gastric body but not cardia, antrum or pylorus. mineral deposits develop around basement membranes surrounding epithelium and blood vessels. the lamina propria becomes expanded by oedema and fibroplasia of the interstitium also develops. the gastric glands themselves become distorted with swelling and degeneration of parietal cells and atrophy of chief cells. erosion of the glandular epithelium with haemorrhage occurs presumably as a result of the ischaemia caused by diffuse vascular injury and altered parietal cell function. focal atrophy of the gastric glandular mucosa is a sporadic occurrence in laboratory rodents, usually as a result of previous focal gastric inflammation, ulceration, mineralisation or vascular occlusion. these changes, characterised histologically by focal fibrosis of the mucosa, gastric glandular dilatation and atrophy variably accompanied by polymorphonuclear cells and mast cells are common in certain strains of rats when years or more in age (anver et al., ) . whereas diffuse mucosal atrophy occurs following severe inflammatory insult, diffuse atrophy of the stomach glandular mucosa without inflammation can be a result of surgically or drug-induced reduction in trophic factors necessary for the maintenance of normal gastric morphology and function. this is observed in man and experimental animals following antrectomy because this removes the peptide-producing cells of the antrum (gjurldsen et al., ; neilsen et al., ) . in the rat, antrectomy is accompanied by hypogastrinaemia, reduced weight and height of the oxyntic mucosa and a reduced number of argyrophil cells (håkanson et al., . this is in contrast to procedures such as antral exclusion that lead to hypergastrinemia and increased thickness of the oxyntic mucosa. mice with genetic deletion of the gastrin gene also show reduction in the thickness of the gastric mucosa. whilst all cell types are present, there is a most pronounced decrease in the numbers of parietal cells as well as enterochromaffin cells associated with an increase in surface mucous cells. these changes are linked to a profound decrease in acid secretion, which becomes unresponsive to histaminergic, cholinergic and gastrinergic stimulation (hinkle and samuelson, ) . analogous atrophic changes have been reported following pharmacological removal of trophic stimuli. for instance, administration of the cholecystokinin-b/gastrin receptor antagonist, ci- to cynomolgus monkeys for periods of up to weeks was associated with an initial phase of multifocal degeneration of gastric glands primarily in the fundus followed by diffuse reduction in the thickness of the glandular mucosa with little or no qualitative changes to the cell populations (dethloff et al., ) . although bilateral vagotomy produces profound functional changes in the stomach, notably reduction of gastric acid secretion, morphological changes in the fundal mucosa are not marked either in experimental animals or in man (crean et al., ; aase and roland, ) . studies in the rat have shown that diffuse atrophy of the gastric glands characterised by a decrease in the number and size of parietal, chief and mucous cells occurs transiently following truncal vagotomy but histological features return to normal by about month after surgery (nakamura, ) . by contrast, unilateral vagotomy in the rat leads to marked and persistent atrophy of the oxyntic zone on the denervated side. this is characterised histologically by reduced height of the mucosa and reduced numbers and staining intensity of argyrophil cells (hakanson et al., ) . håkanson and his colleagues argued that this unilateral atrophy was due to the removal of the trophic action of the vagus. the lack of lasting atrophy after bilateral but not unilateral vagotomy was explained by the subsequent rise in gastrin that occurs after bilateral vagotomy as a result of lack of acid feedback inhibition of gastrin release (håkanson et al., ) . removal or reduction in extra-gastric trophic factors or hormones may also reduce the thickness of the gastric mucosa. this is has been demonstrated in the rat by hypophysectomy which causes a reduction in thickness of oxyntic and antral mucosa, compared with pair-fed controls. although there was little or no change in peptic:parietal cell ratios, a significant decrease in cell volume and secretory activity of gastric glandular cells were demonstrated which suggested a widespread disturbance of synthesis and secretory mechanisms (bastie et al., ) . atrophic changes in the chief cells were observed in rats treated for months with high doses of omeprazole, an inhibitor of acid secretion. the findings were considered to represent disuse atrophy secondary to the inhibition of acid secretion (hansson et al., ) . another inhibitor of gastric acid secretion, the tricyclic agent pirenzepin, also produced atrophy of the fundic mucosa of rats following months but not month of treatment (lehy et al., ) . the atrophy was characterised by reduction in parietal cell numbers associated with lower numbers of gastrin-containing cells in the antrum, features unlike those following prolonged treatment with histamine h -receptor antagonists. an increase in the thickness of the gastric mucosa can be the result of hypertrophy or hyperplasia of the mucosal cells and this occurs both spontaneously or following administration of drugs and chemicals. in view of the different cell populations in the gastric mucosa and the variety of morphological alterations that occur, it is difficult to make a clear distinction between hypertrophy and hyperplasia without morphometric techniques. morphometric techniques have shown that hypertrophy of some mucosal cells can coexist with hyperplasia of other gastric cell populations. a distinction also needs to be made between diffuse or uniform hyperplasia involving one or more of the cell populations from the hyperplasia associated with proliferative or adenomatous overgrowth. adenomatous hyperplasia also needs to be evaluated for atypical cytological features (dysplasia), which are linked to development of gastric carcinoma (see below). cells of gastric glandular mucosa undergo increases in size or number in response to the effects of gastrointestinal trophic hormones or their synthetic analogues. similar changes also follow administration of compounds that inhibit gastric acid secretion or modify other trophic hormones or growth factors. when gastrin or its synthetic analogue, pentagastrin is administered subcutaneously to rats and mice for several weeks, there is both an increase in the number and size of parietal cells without concomitant increase in zymogenic chief cells (willems and lehy, ; crean et al., ; balas et al., ) . in addition, diffuse hyperplasia of enterochromaffin cells also occurs. by contrast, cholecystokinin, a trophic peptide found in the duodenum and sharing the same c-terminal tetrapeptide sequence as gastrin, increases in the number of chief cells but not parietal cells when administered to mice under similar conditions . drugs which inhibit or neutralise gastric acid secretion such as histamine h antagonists, proton pump inhibitors and antacids also induce hypertrophy or hyperplasia of the parietal cell population (witzel et al., ; crean et al., ; mazzacca et al., ; kaduk and hauser, ; betton et al., ; white et al., ) . these agents are associated with a rise in serum gastrin levels, probably as a result of loss of feedback inhibition of low antral ph on gastrin-producing g cells (witzel et al., ) . not all histamine h antagonists produce identical effects. other cytological changes have been reported with famotidine, another h -receptor antagonist. this agent produced a dose-related increase in the prevalence and degree of eosinophilic granularity in chief cells of the stomach in toxicity studies in rats but not dogs (burek et al., ) . electron microscopy showed an increase in electron density of zymogen granules and it was argued that these effects were the result of secondary inhibition of pepsin secretion or turnover due to inhibition of acid secretion. cytoprotective agents of prostaglandin type produce different forms of diffuse gastric hyperplasia. rats treated with , -dimethyl prostaglandin e hourly for weeks, not only developed forestomach alterations (see above) but also thickening of both the body and antral mucosa. in the body mucosa, these changes were the result of a proportional increase in the total mass of surface and foveolar mucous cells, mucous neck cells, chief cells, parietal and endocrine cells as well as connective tissue. this was largely as a result of increase in cell number, although parietal cells also increased in size (reinhart et al., ) . unlike treatment with gastrin and gastrin analogues there was an increase in number of surface and foveolar mucous cells associated with increase in mucus content. misprostal, a synthetic prostaglandin e methyl ester analogue also produced diffuse glandular hyperplasia, characterised by lengthening of gastric pits and increased mucous secretion in the preclinical safety studies in dogs and rats (kotsonis et al., ) . this glandular hyperplasia not only affected the body but also the antral mucosal. studies with tritiated thymidine showed that the labelling index was reduced in rats treated with misprostal, suggesting hyperplasia following administration of prostanoids is a result of an increase in cell survival and decrease in cell shedding rather than an increase in cell proliferation (fich et al., ) . levin ( ) has reviewed the effects of prostaglandins of the e series on the gastrointestinal tract of dogs and rodents. a dose-related diffuse hyperplasia of the gastric glandular mucosa has been reported in both rats and cynomolgus monkeys given human recombinant epidermal growth factor. the gastric mucosa was thickened and there was a increase in the number of undifferentiated cells particularly in the neck region and upper part of the gastric glands (breider et al., ; reindel et al., ) . mitotic figures were also numerous in the upper reaches of the mucosa. the lower parts of the gastric glands were generally less affected. the large increase in the number of undifferentiated cells may have a functional effect on gastric acidity and function (vinter-jensen, ) . administration of recombinant growth hormone has also been reported to induce thickening of the gastric glandular mucosa in dog toxicity studies along with typical growth hormone-induced changes in other organs, body weight increases and insulin-like growth factor (prahalada et al., ) . the pyloric and fundic mucosa showed histological evidence of hyperplasia of the mucous neck cells. thickening of the gastric glandular mucosa as a result of an irregular proliferation and cystic dilatation of gastric glands associated with inflammation characterises a number of non-neoplastic conditions in the stomach of man and laboratory animals. cystic change with chronic inflammation and foveolar hyperplasia is observed in biopsies taken from the edge of chronic gastric ulcers in man (franzin and novelli, ) . ménétrier's disease ('polydadenomes en nappes'), a rare disease found primarily in middle-aged men is also characterised by enlarged gastric folds, foveolar hyperplasia and gastric glandular cystic dilation (berenson et al., ; wilkerson et al., ) . although its pathogenesis remains elusive, increased expression of transforming growth factor α (tgfα) and the epidermal growth factor receptor has been described (demsey et al., ) . as tgfα is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin, and transgenic mice that overexpress tgfα in gastric mucosa develop a similar condition (see below), it was suggested by demsey et al. ( ) that tgfα might have an important role in this condition. similar changes have been observed in animals in association with infestation of the gastrointestinal tract by parasites (jubb and kennedy, ; cook et al., ) . laboratory rodents may develop a similar pattern of changes spontaneously with advancing age, although the cause of this change remains uncertain. the distinction between adenomatous hyperplasia and adenoma is not clear cut. nevertheless, adenomas are usually defined as localised or focal proliferative lesions with well-ordered glandular patterns with a clear boundary with the surrounding normal mucosa. they are usually exophytic or polypoid in nature but adenomas with localised downward growth are described (mohr, ) . proliferation of the gastric glandular mucosa has been well characterised in the laboratory mouse because certain strains have a particular tendency to develop this condition spontaneously with advancing age (stewart and andervont, ; rowlatt et al., ) . hyperplasia also occurs spontaneously in conventional laboratory strains employed in carcinogenicity bioassays. its prevalence can be influenced by environmental factors such as housing (chvédoff et al., ) , food restriction (rehm et al., ) and the administration of xenobiotics (poynter et al., ; betton et al., ) . similar gastric changes have also been reported to occur in mice thymectomised shortly after birth (suzuki et al., ) . histologically, these changes in mice are characterised by hyperplasia of the foveolar and neck regions of the body mucosa (fig. ) . in advanced cases this is accompanied by elongated, tortuous, or dilated glands lined by simple columnar or cuboidal epithelium, devoid of parietal or chief cells. the abnormal cells show only mild cellular pleomorphism and mitotic activity. the abnormal glands dis-place normal glandular tissue and may penetrate through the muscularis mucosa to reach the muscularis externa and serosa. step sections demonstrate continuity between these glandular elements and a total absence of metastatic spread in the adjacent tissues and lymph nodes. the lamina propria also shows increased amounts of smooth muscle and collagen accompanied by variable numbers of lymphocytes and other chronic inflammatory cells. oedema may be observed and blood vessels are often dilated. the antral mucosa remains relatively unaffected. histochemistry has shown variable mucin secretion of the altered glands. some glands are devoid of mucin, others show an increase in sulphomucin as revealed by the high-iron diamine technique (greaves and boiziau, ) . it has been suggested that these features are similar to ménétrier's disease in man and might have a similar pathogenesis (dempsey et al., ; takagi et al., ) . the aetiology of the spontaneous condition in the mouse is uncertain. the occurrence of similar lesions in thymectomized mice has given rise to the suggestion that autoimmune damage to the gastric mucosa may be responsible (kojima et al., ) . the presence of circulating anti-parietal antibodies and the decrease in the number of parietal cells in thymectomized mice suggested that autoimmune damage can occur to parietal cells with compensatory chronic stimulation and proliferation of the generative zones (suzuki et al., ) . however, based on findings in female han nmri mice, rehm et al. ( ) showed that this proliferative condition can develop in mice in the absence of antiparietal antibodies. they demonstrated that this change is associated with an increase in the number of antral gastrin cells, raising the possibility of a hormone or paracrine mechanism. a similar proliferative form of gastropathy has been reported in mice which overexpression transforming growth factor α (tgfα), a potent mitogen and member of the epidermal growth factor family of peptides. tgfα acts by binding to and activating the tyrosine kinase of the epidermal growth factor receptor. transgenic mice overexpressing tgfα develop severe adenomatous and cystic hyperplasia of the gastric glandular mucosa starting from about months of age along with loss of mature parietal cell numbers and a diminution in gastric acid production (takagi et al., ) . the degree of change was to some extent dependent on the genetic background on which the transgene operated. an increased prevalence of similar changes have been reported in cd- mice treated with the novel histamine h -receptor antagonist sk&f for months (betton et al., ; . although treated mice developed hyperplasia of gastric neuroendocrine cells similar to that observed in rodents treated with other antisecretory agents, they also showed an increase in the severity of glandular hyperplasia. like the spontaneous condition, these changes were characterised by thickening of the mucosa by hyperplasia of the foveolar and neck regions, and downward proliferation of glandular elements into gastric glands (betton et al., ) . poynter et al. ( ) have also reported similar glandular hyperplasia in the mouse stomach associated with histamine h -blockade with the agent ioxtidine. these findings were similarly associated with hyperplasia of neuroendocrine cells. adenomatous polyps have also been reported in the pyloric antrum of c bl/ j mice treated for weeks with the synthetic progestin, cyproterone acetate (tucker et al., ) . these were single, pedunculated and well-differentiated lesions showing little evidence of dysplasia. the mechanism for the induction of these polyps is unclear although they may have been hormonally mediated as progesterone receptors have been identified in gastric tissue (wu et al., ) . although usually less prevalent and less exuberant than in mice, the aged rat also develops proliferative gastric glandular changes spontaneously. these changes are characterised by hyperplasia of the foveolar and mucin-secreting cells of the body mucosa, development of cystic glands lined by simple mucous or flattened cells, accompanied by chronic inflammatory cells, prominent blood vessels and smooth muscle in the lamina propria (greaves and faccini, ) . the antrum remains relatively unaffected. proliferative alterations can be induced by administration variety of xenobiotics as well as following surgical procedures that induce chronic reflux of normal intestinal contents. for instance, hyperplasia of the gastric mucosa, notably over the lesser curvature has been described in rats following the so-called bilroth ii gastrectomy that allows reflux of intestinal and biliary secretions into the stomach (kobayashi et al., ) . a proliferative condition of the gastric mucosa has been shown to develop following long-term treatment of rats with an ulcerogenic regimen of aspirin. female sprague-dawley rats given mg/kg of aspirin in % methylcellulose once daily orally by gavage for months followed for periods of up to months without treatment, developed focal proliferative changes at the sites of healed ulcers, mainly in the mucosa of antrum or antral-body junction (st john et al., ) . these lesions were characterised by the presence of proliferating gastric glands lined by columnar, cuboidal or flattened epithelial cells in the mucosa, which also extended through the muscularis mucosa. mucus content of these glands was variable but when present was principally acidic in type, as shown by staining with alcian blue at ph . . the lesions were accompanied by increased collagen in the lamina propria, endarteritis and an infiltration of lymphocytes, plasma cells and mast cells. the lesions were not associated with the development of carcinoma following months' observation and it is probable that they were the result of the chronic damage and repair induced by aspirin treatment. hyperplasia of the gastric glandular mucosa also occurs in rats following the administration of powerful genotoxic carcinogens, although characteristically in association with of atypical histological changes and ultimately carcinoma. these changes have been best characterised in sequential studies with the rat using the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine at doses low enough to avoid overt gastric ulceration and regenerative hyperplasia. it was shown that hyperplasia developing under these conditions occurs diffusely both in the body and antral mucosa. furthermore, the changes occurred earlier in the antrum than in the body and were focal or polypoid in character (tsiftsis et al., ) . involvement of the antrum in this way is quite unlike the spontaneous hyperplasia of the rat gastric mucosa. histologically, this form of hyperplasia is characterised by lengthening of the foveolae and neck regions both in the antrum and body. hyperplastic pits or foveolae show increased secretion of sialomucins and sulphomucins with a concomitant loss of neutral mucins. polychlorinated biphenyls such as arochlor , which produce intestinal metaplasia and adenocarcinoma in the stomach of rats, also induce proliferative alterations characterised by proliferative cystic lesions in the mucosa associated with inflammation and fibrosis (morgan et al., ) . in common with lesions induced by genotoxic agents, these changes are found primarily in the antrum and pyloric regions, zones of predilection for the development of gastric carcinoma in man and experimental animals. it is important to distinguish between the various hyperplastic and adenomatous conditions found in the gastric glandular mucosa in laboratory animals that are not associated with neoplasia from those which precede the development of carcinoma. this distinction is complicated by the fact that proliferative changes associated with the development of cancer both in man and laboratory animals possess features in common with lesions not associated with neoplasia. however, a key distinctive feature is the degree of epithelial dysplasia. dysplasia is considered to be the lesion common to gastric conditions in man such as atrophic gastritis and gastric polyps that have been linked with a significantly increased risk of gastric cancer. although the term dysplasia may be less widely employed in experimental pathology, similar dysplastic changes to those occurring in man have been characterised in laboratory animals in which precancerous gastric lesions have been studied (tsiftsis et al., ) . it therefore represents a unifying concept in the assessment of proliferative changes in the gastric glandular mucosa of laboratory animals. as defined by an international group concerned with the diagnosis of preneoplastic conditions in the stomach of man, the principle features of dysplasia are (i) cellular atypia; (ii) abnormal differentiation; and (iii) disorganised mucosal architecture (morson et al., ; nagayo, ) . cellular atypia is characterised by nuclear pleomorphism, hyperchromasia and stratification of nuclei, increased nuclear-cytoplasmic ratio and loss of cellular and nuclear polarity. abnormal differentiation is shown by reduction or alteration in the normal secretory products of the mucosa. disorganised mucosal architecture is shown by irregularity of crypt structure, back-to-back glands, budding and branching of crypts and intraluminal and surface papillary growths. it is important to assess gastric mucosa very carefully for the features of dysplasia when hyperplastic gastric changes are found in treated animals. in the rat gastric cancer model employing the carcinogen n-methyl-n´-nitro-n-nitrosoguanidine, dysplastic changes were shown to start in the proliferating neck region of hyperplastic zones (tsiftsis et al., ) . these changes were characterised histologically by irregular growth patterns of glandular cells showing reduced mucin secretion, numerous mitoses and enlarged pleomorphic nuclei. these atypical glands were observed to extend downwards, eventually replacing normal gastric glands and ultimately penetrating the muscularis mucosa forming infiltrating adenocarcinomas of variable differentiation. the antrum developed these changes earlier than the body mucosa (tsiftsis et al., ) . these considerations were important in the safety evaluation of the histamine h receptor antagonist, tiotidine (ici , ) a guanidino-thiazole derivative that also produced proliferative gastric lesions in the stomach of rats in a month carcinogenicity study (streett et al., ; . these changes were found mainly in the pyloric region and were characterised histologically by superficial erosions and irregular pyloric glands lined by cells with basophilic cytoplasm and enlarged hyperchromatic nuclei. some atypical glands penetrated the muscularis mucosae. dysplastic lesions situated primarily in the pyloric region were also associated with the development of invasive carcinoma in some rats. extensive histological sectioning of the stomach in rats treated with tiotidine for only months also revealed evidence of early proliferative changes (streett et al., ) . therefore, these lesions produced by tiotidine possessed more in common with those induced by powerful carcinogens such as n-methyl-n´-nitro-n-nitrosoguanidine than the benign, species-specific proliferative change of little or no relevance for human safety. interestingly, mice treated for months with tiotidine were devoid of dysplastic changes in the gastric mucosa (streett et al., ) . one of the most remarkable examples of drug-induced gastric alterations in rodent bioassays is the hyperplasia of enterochromaffin cells and development of carcinoid-like neoplasms in the stomach of rats treated with omeprazole (havu, ) . omeprazole is a substituted benzimidazole which inhibits gastric acid secretion by blocking the enzyme h + , k + -atpase, the proton pump of the parietal cells, in a specific and dose-dependent manner (fellenius et al., ) . although in rats there is a increase in number of gastric argyophilic cells with increasing age, rats treated with omeprazole for weeks showed a marked, dose-related and diffuse increase of argyophilic, non-argentaffin cells in the basal half of the oxyntic fundal mucosa (havu, ) . these changes were more marked in female than in male rats but were not observed in the bioassay in which cd- mice were treated with similar doses of omeprazole for weeks. these diffuse changes in the rat stomach were associated with focal hyperplasia of argyrophilic cells. these focal lesions were also associated with a doserelated increase in larger focal nodular lesions of argyrophilic cells, some of which were undoubtedly locally infiltrating carcinoid tumours. these nodular argyrophil lesions posed the usual problems of differential diagnosis of endocrine hyperplasia and neoplasia (see endocrine system, chapter xiii) and distinction of hyperplasia from neoplasia was uncertain. histologically, nodular lesions were composed of multifocal anastomosing solid or pseudoacinar cords of proliferating, regular cells with uniform nuclei and moderately abundant fine granular pale cytoplasm. these nodules showed little or no cellular pleomorphism or mitotic activity but clear evidence of submucosal infiltration without involvement of the muscularis externa was observed in some cases. the overall light microscopic features were similar to those of gastrointestinal carcinoid tumours reported in man. the incidence of gastric carcinoids was reported to be as high as % in females in the high dose group but only a few cases observed in similarly treated males (ekman et al., ; havu, ) . electron microscopy of the altered argyophil cells confirmed the presence of electron-lucent, vesicular granules, frequently with small irregular dense cores characteristic of enterochromaffin cells of the stomach. immunocytochemical study showed that these cells contained histidine decarboxylase which is found normally in gastric enterochromaffin cells which produce and store histamine . other findings reported in rats treated with omeprazole have been a proportional increase in the number and size of non-endocrine cells of the fundus (blom, ) , an increase in the number and immunostaining properties of the antral gastrin-containing g cells and hypergastrinaemia (bishop et al., ; creutzfeldt et al., ) . all functional and morphological changes following treatment for days were fully reversible after days' drug withdrawal (creutzfeldt et al., ) . as a result of these treatment-related increases of these normally rare gastric carcinoids in the rat bioassay with omeprazole, clinical trials with this agent were suspended until it was agreed that the endocrine alterations were a result of prolonged drug-induced achlorhydria. it was postulated that omeprazole causes a prolonged inhibition of acid secretion in the rat, which causes activation, and subsequently hyperplasia of antral gastrin cells and marked hypergastrinaemia. hypergastrinaemia in turn stimulates enterochromaffin cells of the fundus, which in time results in enterochromaffin hyperplasia . this argument is supported by the fact that similar morphological findings are reported in chronic atrophic gastritis and other achlorhydric sites in man (solchia et al., ; müller et al., ) and that antrectomy in the rat prevents the appearance of enterochromaffin hyperplasia following treatment with omeprazole . although mild dose-related gastric argyrophil cell hyperplasia was noted in dogs treated with omeprazole for year, neoplasms of the stomach were not observed during this time period. why mice neither developed neither argyrophil hyperplasia nor gastric carcinoids with a similar treatment regimen is not clear, as the mechanism of action of omeprazole is similar in rat, dog and mouse. however, as the duration of action of omeprazole is shorter in the mouse, it was postulated that sustained inhibition of gastric acid secretion over hours is necessary to activate increased gastrin secretion from antral cells (havu, ) . it has also been suggested that the mouse possesses fewer gastric enterochromaffin cells than the rat and shows a much lower serum gastrin response to omeprazole treatment (ekman et al., ) . duration of action or potency may also be the explanation for the lack of reports of carcinoid neoplasms in rats following inhibition of gastric acid secretion by the histamine h -receptor blockers cimetidine and ranitidine. neither of these drugs completely inhibits gastric acid secretion in the rat for hours (leslie and walker, ; larsson et al., ) , although mild gastric neuroendocrine hyperplasia has been recently described in cimetidine-treated rats (hirth et al., ) . however, the long-acting h -receptor antagonist sk&f produced gastric carcinoid neoplasms when administered at a high dose ( mg/kg) to rats for years (figs. and ) (betton et al., ; . although this dose level of sk&f did not entirely suppress gastric acid secretion and control gastric ph over hours, plasma gastrin levels remained elevated at - times control values over this period. in a -month oral carcinogenicity study in cd- mice at the same dose level ( mg/kg), a diffuse neuroendocrine cell hyperplasia and multifocal glandular hyperplasia or neoplasia was also observed (betton et al., ) . similarly, loxitidine, a potent, non-competitive, insurmountable histamine h -antagonist produced hyperplasia of neuroendocrine cells and carcinoid tumours in the gastric fundus of both rats and mice after years' treatment in diet and drinking water, respectively (poynter et al., (poynter et al., , . other histamine antagonists bl- and ici have been reported to produce neuroendocrine neoplasms in the stomach of rats and rats and mice, respectively (hirth et al., ; streett et al., ) . wormsley ( ) reviewed the considerations in the risk-benefit analysis of agents intended for long-term administration for peptic disease. drugs of other classes also cause hyperplasia of gastrin-containing cells. immunocytochemical study using antigastrin antibody revealed increased gastrin cell numbers in the antral mucosa of dogs given high doses of adrenocorticosteroids for weeks and these changes were accompanied by enhanced serum and tissue gastrin levels (delaney et al., ) . these results suggest that adrenocorticosteroids have a trophic effect on gastrin-containing cells. in human patients hypergastrinaemia is also produced by pharmacologically induced hypochlorhydria although this is usually only slight and hyperplasia of enterochromaffin cells has not been observed (soll, ) . a complicating factor in drug safety evaluation is the association of gastric cancer in both man and laboratory animals with n-nitroso compounds. some of the most effective stomach carcinogens in laboratory animals have proved to be nnitroso compounds particularly since sugimura and fujimura ( ) induced gastric adenocarcinomas in rats with n-methyl-n´-nitro-n-nitrosoguanidine dissolved in drinking water. furthermore, epidemiological evidence associating nnitroso compounds with human cancer is also fairly strong for the stomach (corea et al., ; pocock, ) . the formation of n-nitroso compounds is theoretically possible with a variety of compounds that contain amino groups. it has been suggested that the formation of nitrosamines occurs in vivo under the acidic conditions in the stomach following dietary ingestion of nitrite, nitrates and secondary amines (mirvish, (mirvish, , . low levels of preformed nitrosamines are also present in some commercial pelleted diets for laboratory animals, principally derived from fishmeal (edwards et al., ) . calculations based on dietary intake and nitrosatability of precursors and carcinogenicity of derivatives have suggested that the risk that arises from endogeneous nitrosation is highly variable but highest from ureas and aromatic amines (shephard et al., ) . a number of drugs in widespread clinical use have been shown to produce nnitroso products in acidic aqueous media, although the extent to which this occurs in actual therapeutic use is unclear (gillatt et al., ) . some clinical evidence suggests that nitrosation of therapeutic agents can occur in clinical practice. for instance piperazine, a cyclic secondary amine, widely used as an antihelmintic drug, has been shown to form small quantities of n-mononitrosopiperazine in the human stomach as measured by gas chromatography-thermal energy analysis (bellander et al., ) . however, n-mononitrosopiperazine has not been shown to be carcinogenic in rodents (love et al., ) . n,n´-dinitrosopiperazine, carcinogenic to the upper gastrointestinal tract in rodents (lijinsky and taylor, ) , was not detected in man after administration of piperazine under the same circumstances (bellander et al., ) . the possibility of nitrosation is not usually taken into account in the testing of carcinogenic potential of novel drugs as bioassays are usually only performed with parent compound. however, concerns about nitrosation have arisen in subsequent clinical practice. an example of this was the proposal that a few gastric cancers found in patients whilst being treated with the histamine h receptor antagonist cimetidine, were the result of treatment (elder et al., ; reed et al., ; hawker et al., ) . it now seems likely that all those observed cancers associated with cimetidine were incidental (penston and wormsley, ) . however, at that time concerns were increased by the theoretical possibility that cimetidine has the potential be nitrosated in vivo (elder et al., ) . a further factor was the concept that the treatment-induced gastric secretory inhibition with subsequent bacterial colonisation of the stomach rendered the conditions conducive to the generation of n-nitroso compounds from normal dietary constituents (reed et al., ; penston and wormsley, ) . these concerns appear to be unfounded. long-term surveillance studies with cimetidine have shown no causal link between its clinical usage and gastric malignancy (colin-jones et al., ; langman, ) . in addition, carcinogenicity bioassays performed with cimetidine, cimetidine plus nitrite and nitrosocimetidine have not shown any tumorigenic effect in the gastric mucosa (anderson et al., ) . a -year study in dogs in which multiple gastric biopsies were taken at intervals of approximately months have also shown no indication of gastric hyperplasia, dysplasia, intestinal metaplasia or neoplastic change . although complacency is certainly not warranted with respect to the nitrosation of therapeutic agents in vivo, the risks of development of gastric malignancy from such drugs when administered on a short-term basis are probably very small (world health organisation, ) . even for gastric antisecretory agents which may be administered for longer periods of time, the balanced view would also permit development of novel agents provided they are not obviously mutagenic or carcinogenic in the usual preclinical studies and are not particularly liable to undergo rapid nitrosation. most carcinomas of the glandular mucosa are adenocarcinomas, whether induced by the potent genotoxic carcinogens or therapeutic agents. these tend to develop in the antral region in rodents (streett et al., ; szentirmay and sugar, ) . they range from those with well differentiated tubular or papillary features to poorly differentiated forms with trabecular, mucoid or signet ring features (tatematsu, ) . squamous metaplasia within adenocarcinoma can also be observed. stroma may be abundant with pronounced chronic inflammatory infiltration and hyalinisation. metaplastic cartilage and bone has also been described (szentirmay and sugar, ; mohr, ) . gastric adenocarcinomas induced in dogs by n-methyl-n´-nitro-n-nitrosoguamide show similar histological features although their reported distribution in the stomach appears more variable (fujita et al., ) . histological criteria for the diagnosis of invasive adenocarcinoma in experimental animals may vary between individual pathologists. some retain the old criteria of stewart and co-workers ( ) who defined invasive cancer as a neoplastic growth reaching the serosa. it is now considered more appropriate to apply criteria of use in human diagnostic pathology (see mohr, ) . unequivocal invasion of the submucosa is sufficient evidence of an invasive and therefore malignant process (greaves and faccini, ) . the small intestine is of major importance in drug safety evaluation for it represents the primary site of drug absorption. in view of its length and the presence of villi, it possesses an enormous surface area of specialised absorptive epithelium. furthermore, ingested substances have an extended residence time in this part of the gastrointestinal tract. the canine model has been one of the most popular for the study of drug absorption because the dimensions of the canine gastrointestinal tract permit administration of dosage forms intended for clinical use in humans. for this reason, factors, which influence drug absorption, have been better studied in dog and man than many other species. however, data from dog and man not only suggest that diverse factors influence drug absorption from the small intestine but that there are considerable species differences. residence time is of particular importance for drugs, which are incompletely absorbed because differences in mucosal contact time can be expected to result in differences in the fraction absorbed. dressman ( ) has shown using the heidelberg capsule technique that small intestine transit time in dogs is varies from between and over minutes whereas in man equivalent times are between and minutes. these results suggest that absorption of poorly absorbable drugs is likely to be quantitatively less although more variable in dogs than in man. however, these differences do not explain why some poorly lipophilic drugs such as chlorothiazine, acyclovir and phosphalinic acid are more extensively absorbed in dogs than in man. intestinal ph is consistently higher in dogs than in man so that drugs with half maximal absorption ph in the range ph - may also be expected to be absorbed at different rates in man and dog (dressman, ) . physiological and anatomical differences in the small intestine of other test species particularly rodents and humans are also likely to have an impact on drug absorption although many of these factors are still poorly understood. in addition to the small intestine acting as an absorptive surface, it is becoming increasingly obvious that it plays an important part in the metabolism of drugs (breckenridge, ) . although monoxygenase activity is relatively low in the gut compared with the liver, conjugation mechanisms are efficient and activity of udp-glucuronosyltransferase and glutathione-s-transferase are as high or even higher than in the liver (hänninen et al., ) . in addition, the gastrointestinal microflora not only possesses metabolic capacity itself but also can influence the turnover rate of mucosal cells and subsequent exfoliation and release of enzymes into the lumen (hänninen et al., ) . gastrointestinal metabolising activity is important because that the mucosa is exposed to high concentrations of xenobiotics in toxicity studies, and this can influence their overall bioavailability (chhabra and eastin, ) . studies in untreated rats have shown that the concentration of total cytochrome p in small intestinal microsomes is only about % of that found in liver microsomes (bonkovsky et al., ) . however, it exists in at least two forms and as in the liver, its activity can be induced by xenobiotics. it has been shown that in the rat, the concentration of cytochrome p and drug metabolising enzyme activity increases in intestinal epithelial cells as they move from crypt to villous tips and they are found in greater concentration in the proximal two-thirds of the small intestine than in the distal third (hoensch et al., ; bonkovsky et al., ) . bonkovsky et al. ( ) also showed that the phenobarbital-inducible form of cytochrome p represents less than % of total p in the small bowel, but as in the liver, phenobarbital treatment can increase this form to about % of total cytochrome p in small intestine cells. furthermore, it has been shown that drug metabolising activity in the tips of the villi in the duodenum is greater in rats fed a conventional diet than a semisynthetic diet and that the activity depends critically on the absorption of iron from the intestine (hoensch et al., ) . glutathione is also present throughout the entire mucosa, although in rats, cells at the tips of the villi contain less than cells located more basally, whereas related enzymes γ-glutamyl transpeptidase and glutathione-s-transferase show highest activity in the villous tip region (ogasawara et al., ) . the fact that these enzyme activities are highest in the duodenum and lowest in the terminal ileum suggests that detoxification systems for exogenous compounds are greater in the proximal small intestine. the small intestinal mucosa is constructed not only to act as an absorptive surface but also as a barrier to potentially pathogenic substances and microorganisms. although the main cell population of the epithelium is composed of absorptive cells, other major epithelial cell types, the mucous (goblet) cells, paneth cells and endocrine cells have important protective functions. in addition, specialised epithelial cells, the microfold (membranous or m) cells are located in the epithelium over peyer's patches. these cells form part of the other important protective system of the intestine, the gut associated lymphoid tissue (galt) or mucosal associated lymphoid tissue (malt). the mucosal lining is in a constant state of renewal. enteric epithelium possesses the fastest rate of turnover of any tissue exceeded only by a few rapidly growing neoplasms . in normal circumstances, the constant turnover of small bowel mucosa is maintained by equilibrium between cell production in the crypts and cell loss at the tips of the villi. there are intrinsic controls within the mucosa itself. exogeneous substances, intraluminal secretions, mechanical and neural factors as well as alterations in blood flow all possess potential to influence mucosal cell kinetics . all main epithelial cell types are believed to arise from undifferentiation columnar cells at the crypt base (cheng and leblond, ) , although mucous cells may also arise by proliferation of partly differentiated mucous cells in the crypts. as cell division is limited to crypts, the cell population in the crypts have high activities of enzymes such as thymidine kinase that are involved in nucleic acid synthesis (imondi et al., ) . the complete cell cycle lasts about - hours in rodents and at least hours in man. enteric epithelium is completely replaced within - days in mice and rats and within - days in man . after two or more divisions in the crypt cells migrate to the villus, lose ability to incorporate thymidine and differentiate into mature cells equipped with enzymes associated with nutrient absorption (imondi et al., ) . cell migration in the rat is completed more rapidly in the ileum than in the jejunum principally as a result of the lower villous height in the ileal mucosa (altman and enesco, ). migration terminates by loss of cells from the tip of the villi. surrounding the crypt is a sheath of fibroblastic cells. these cells also undergo synchronous division and migration with the epithelial cells, maintaining the intimate relationship between the epithelium and supporting tissues (parker et al., ) . mature absorptive cells are important in the active and passive transport of nutrients as well as in the endocytosis of macromolecules. they are characterised by the presence of a striated or brush border which is seen in haematoxylin and eosin-stained sections as a refractile bi-laminar band. the inner, wider lamina corresponds to the microvillous region that is associated with the presence of neutral mucosubstances in most species. the outer, thinner band corresponds to the glycocalyx, which is composed principally of acidic mucosubstances (sheahan and jarvis, ) . this outer band of the brush border shows histochemical staining predominantly for sulphomucins in most species including mouse, hamster, dog and rhesus monkey, although in the duodenum of the rats and in the entire human small bowel sialomucins predominate in this layer. electron microscopy of the absorptive cells shows that the surface of absorptive cells is covered by tightly packed and well developed microvilli approximately µm long and . µm wide. these are considered the first site of entry of food substances into the cell. the plasma membrane of microvilli is associated with fine filamentous projections, which probably represent the polysaccharide chains of the glycocalyx (bennett, ) . as the glycocalyx is composed of a network of polysaccharides, it has been suggested that it may behave like an ionexchange resin, be able to bind certain lectin-like molecules or trap substances in its matrix so providing a site for efficient intraluminal digestion (bennett, ; goldberg et al., ; king et al., ). the plasma membrane shows a trilamina structure at ultrastructural level. freeze fracture replicas from the microvilli which cleave this membrane through the plane of apposed non-polar groups of the lipid bilayer, demonstrate smooth complementary surfaces studded with small particles. these particles represent integral globular proteins of the plasma membrane. some of these intramembranous particles, mostly those of nm diameter show irregular outlines with a central pit and are believed to represent gap junctions or transport channels (yamamoto, ) . a particularly important aspect of the absorptive cell membrane is its high concentration of disaccharidases such as sucrase, maltase and lactase, related to the absorption of sugars. alkaline phosphatase activity is also abundant on the surface of absorptive cells, although its precise role here uncertain (owen and bhalla, ) . immunocytochemical demonstration of alkaline phosphatase provides a useful tool to examine the effects of xenobiotics on intrinsic membrane glycoproteins in the small intestine (hasegawa et al., ) . enterokinase, the glycoprotein enzyme, which initiates the activation of pancreatic zymogens by converting trypsinogen to trypsin, is also present in the brush border and glycocalyx of the small intestinal epithelium, both in man and animals. immunocytochemical studies have demonstrated that in man this enzyme is located in the duodenum and proximal jejunum but not ileum, colon and stomach (hermon-taylor et al., ) . these enzymes constitute integral structural proteins of the cell membrane with active sites protruding from the cell surface. they are synthesised by the absorptive cells (blok et al., ) . the lateral surfaces of absorptive cells are in direct contact with neighbouring cells and firmly attached to each other by terminal bars or junctional complexes. a terminal bar comprises an apically situated tight junction or zonula accludens, a central zone, the zonula adherens, below which is situated a desmosome or macula adherens. the junctional complexes are relatively impermeable to macromolecules. studies with labelled tracers in the rat jejunum have shown that horseradish peroxidase (molecular weight , , diameter nm) and ferri-tin (molecular weight , , diameter nm) do not penetrate junctional complexes (yamamoto, ) . below the junctional complexes, the cell membranes interdigitate and the intercellular space widens towards the cell base, a feature that may be important in the movement of electrolytes and water across the intestinal epithelium (rhodin, ) . the cytoplasm of absorptive cells contains smooth and granular endoplasmic reticulum, free ribosomes and mitochondria. the golgi apparatus is located above the nucleus. the apical part of the cytoplasm is devoid of organelles except for a tight meshwork of filaments called the terminal web. filaments of actin within the microvilli are linked to the terminal web and this is believed to be important in the movement of microvilli (moosker and tilney, ; moosker et al., ) . goblet cells are much fewer in number than absorptive cells in the small intestine but they increase in number from the duodenum to the lower ileum. they are important in the production of mucus, which remains on the surface of the mucosa as a viscous layer and acts as the first line of defence against intestinal pathogens. goblet cells are characterised by the presence of abundant mucous droplets formed by the golgi complex and which accumulate in the apical part of the cell cytoplasm. histochemical study shows that neutral mucosubstances are present in the goblet cells found in crypts and on the villi in the entire small bowel mucosa of most species including man but there is an interspecies variation in the population of sialo-and sulphomucins (sheahan and jarvis, ) . in the mouse, sulphomucins predominate but among rats considerable individual variation in the proportion of sialo-and sulphomucins is reported. in the hamster, sulphomucins are more prominent in the proximal and sialomucins in the distal small bowel. in the dog, both sulphomucins and sialomucins are found with predominance of one or other in individual animals. staining for acidic mucins is less intense in the goblet cells of the small intestine in man compared with non-human primates but sialomucins are predominant in both species. a few goblet cells in the distal ileum in man also contain sulphated mucins (sheahan and jarvis, ) . paneth cells remain located near the crypt base throughout the small intestine (sandow and whitehead, ) . they are found in rodents and man but typically not in carnivores such as dog and cat (rhodin, ; . they are characterised by the presence of numerous eosinophilic cytoplasmic secretory granules between about . and µm diameter that contain various enzymes and mucosubstances. particular care is needed in fixation and staining for optimal demonstration of paneth cells for they rapidly degranulate after death and granules are destroyed by acetic acid fixation. formalin and mercuric fixatives appear appropriate methods and they permit staining with methylene blue, lendrum's phloxine-tartrazine and masson's trichrome (lewin, ) . the apical parts of paneth cells show glucose- -phosphatase, carbonic anhydrase and monoamine oxidase activity and they have been shown to contain lysozyme and immunoglobulins, particularly iga (speece, ; riecken and pearse, ; ghoos and vantrappen, ; . staining for lysozyme appears to be the most practical immunocytochemical stain for the detection of paneth cells in formalin fixed paraffin wax embedded tissue sections. however, other immunocytochemical reagents have been employed in human and rodent tissues. these include antibodies to the cd antigen (ariza et al., ) and to a rat paneth cell zinc binding protein (sawada et al., ) as well as the use of pokeweed lectin for murine paneth cells (evans et al., ) . the paneth cell granules not only contain lysozyme but also a range of antimicrobial peptides such as secretory phospholipid a , α-defensins, also called cryptins. these are believed not only to possess antimicrobial activity but also important in the regulation of cell volume, chemotaxis, mitogenesis and inhibition of natural killer cell activity (ouellette, ) . it has been shown that these substances are released from paneth cells when germ free rats are dosed with the intestinal flora from specific pathogen free rats (satoh and vollrath, ; . however, it has been shown that paneth cells develop under germ free conditions and do not require luminal bacteria or dietary material for their development (ouellette, ) . endocrine cells are also scattered throughout the small intestinal mucosa. they are of both argentaffin and argyrophil types and are situated predominately in crypts. immunocytochemical study shows that they contain a variety of different peptides although gastrin, secretion and serotonin-containing cells have been those most extensively studied (inokuchi et al., ) . in addition to the barrier formed by mucus and epithelial cells, lymphocytes, plasma cells, macrophages, dendritic cells and mast cells also form part of the protective function of the small intestine. some lymphocytes are located within the epithelium mostly, above the basal lamina but below epithelial nuclei (pabst, ) . these lymphocytes are termed intraepithelial lymphocytes and are predominantly of t-suppresser/cytotoxic type in man and laboratory animals (selby, ; martin et al., ; pabst, ) . most lymphocytes in the lamina propria are also t cells but t-helper (cd positive) cells outnumber the t-suppresser/cytotoxic or cd positive phenotype (hirata et al., ; pabst, ; bruder et al., ) . intraepithelial lymphocytes are also mainly t cells (bruder et al., ) . many plasma cells present in the lamina propria produce iga, the major immunoglobulin of mucosal secretions (michalek et al., ) . iga represents another important component of the mucosal barrier between the gastrointestinal mucosa and intraluminal antigens. the main function of iga is to effect immune exclusion by intimate co-operation with non-specific defence mechanisms (brandtzaeg et al., ) . plasma cells in the lamina propria produce dimeric iga with two dimeric molecules joined by a joint (j) piece. a secretory component (sc), a glycoprotein expressed on the basolateral surface of epithelial cells acts as a receptor for dimeric iga and as a transport system for iga to the gut lumen where monomeric iga is secreted (brandtzaeg et al., ) . morphometric analysis of iga-containing immunocytes in the rat ileal mucosa using immunocytochemical staining has shown that the number of these cells varies with alterations in the microbiological status of intestinal contents (rodning et al., ) . a significant reduction in iga-containing lymphocytes and plasma cells was observed following microbial reduction associated with gnotobiosis, probably reflecting decreased microbial antigenic stimulation. experimental studies using labelled mesenteric lymphocytes also suggests that local microenvironments are important in the distribution of there cells in the intestinal wall (mcdermott et al., ) . peyer's patches are the most prominent aggregates of lymphoid tissue in the gastrointestinal tract and constitute important sites at which antigens from the gut lumen encounter immune competent cells which are responsible for the initiation of immune responses. peyer's patches are located on the ante-mesenteric wall of the small bowel and consist principally of collections of lymphoid follicles. in man, peyer's patches are more common in the ileum (cornes, ) but in mice they are more uniformly distributed (owen and neumanic, ) . in rats they are also more numerous in the distal than in the proximal small intestine and the number of follicles in patches usually varies from to but sometimes many more may be seen in any particular section (martin et al., ) . peyer's patches vary between rat strains. a comparative study showed that in fischer rats they are smaller than those in wistar rats (bruder et al., ) . particular care in selection and orientation of tissue blocks is therefore essential for any form of quantitative or semiquantitative assessment of peyer's patches. peyer's patches are more than simple aggregates of lymphoid follicles. they consist of lymphoid follicles surrounded by a corona of small lymphocytes principally of b-cell type. the interfollicular area contains post-capillary venules with specialised cobblestone type epithelium (yamaguchi and schoefl, ) and many t lymphocytes. beneath the epithelium, over the bulging follicles (dome area), mixtures of t and b lymphocytes, plasma cells and macrophages can be seen (pabst, ) . immunohistochemical study in rats demonstrates the presence of w / -positive t (cd ) cells in the interfollicular area. in the rat, many cells with macrophage morphology also stain with the w / (cd ) monoclonal antibody in the interfollicular zone (bland and warren, ; martin et al., ) . immunocytochemical study of the peyer's patches in the mouse has also shown considerable heterogeneity of staining patterns, particularly under the dome epithelium (ermak and owen, ) . similar patterns have been observed following immunohistochemical study of peyer's patches in man (spencer et al., ) . the epithelium overlying the peyer's patch follicles (dome area) contains specialised epithelial cells, called microfold, membranous or simply m cells. these cells have been identified in many species including rats, mice, hamsters, dogs, monkeys and man (owen and bhalla, ; wolf and bye, ) . these cells differ functionally from other enterocytes by their ability to transport large molecules such as ferritin and horseradish peroxidase and particulate matter from the lumen to the underlying lymphoid tissue (owen, ; jeurissen et al., ) . they have also been shown to be the site of penetration of reoviruses into the epithelium and they can transport vibrio cholerae and other organisms (wolf and bye, ; smith et al., ) . m cells therefore form weak points in the intestinal wall which transport intact antigen and macromolecules to the follicles where they can be processed and be transported to lymph nodes with consequent iga immune responses. this contrasts with the uptake of soluble antigens, which can be taken up by ordinary epithelial cells and transported in the circulation of the villi to be ultimately trapped in the spleen possibly to evoke an igm/ igg response (jeurissen et al., ) . understanding of these cellular and molecular characteristics is critical for the design of mucosal vaccines for pathogens that exploit this pathway (neutra, ) . no simple, specific histological markers for m cells have been identified for use in routine histological sections although many specialist techniques can be applied (reviewed by gebert et al., ) . this has hampered their study because they are still best identified on the basis of their ultrastructural characteristics or ability to capture and transport macromolecules (hamzaoui and pringault, ) . the m cell shares tight junctions and desmosomes with adjacent epithelial cells but it has fewer and shorter microvilli than absorptive cells. there is lack of a well organised terminal web. vesicles are abundant in the apical cytoplasm but lysosomes are reduced in number. attenuated cytoplasmic processes may be seen embracing lymphocytes (owen and nemanic, ; wolf and bye, ) . a cardinal feature is the presence of a intraepithelial pocket on the basal membrane which acts as an internal docking site for lymphocytes, such that they are filled by b and cd positive lymphocytes, macrophages and dendritic cells that move between underlying follicles and the epithelium (ermak et al., ; farstad et al., ) . with care, these attenuated microvilli can be seen at light microscopy in semithin plastic sections (wolf and bye, ) . cytochemical analysis has also demonstrated that they can be distinguished at light microscopic level by markedly reduced alkaline phosphatase activity on their terminal surface, in contrast to the dense reaction product produced by other enterocytes (owen and bhalla, ) . as the glycoproteins on the surface of m cells are different to those on surrounding cells, they can be selectively stained by labelled lectins, although these patterns are different in different species. for instance, mouse m cells are labelled by the fucose-specific probe ulex europaeus and to some extent anguilla anguilla (giannasca et al., ) . mucosal mast cells also appear to be involved in the immunological defence of the gastrointestinal tract. they respond by proliferation, migration and discharge of granules during nematode infestations (miller, ) . it has been shown in the rat that mucosal mast cells of the gut differ in several ways from connective tissue mast cells. these differences result in poor preservation of mast cells of the gut if the usual metachromatic staining techniques employed for the demonstration of mast cells in tissue sections (wingren and enerbäck, ) . histochemical study suggests that mucosal mast cells differ from connective tissue mast cells by a lower degree of sulphation of glycosaminoglycans and different spatial relationships of protein and glycosaminoglycans in their granules. these cross link following formalin fixation in a way, which is sufficient to prohibit cationic dye binding. wingren and enerbäck ( ) showed that these staining difficulties can be surmounted in tissues fixed in formaldehyde by staining in toluidine blue for prolonged periods of time ( - days), a procedure which allows adequate penetration of the toluidine blue molecule. optimal histopathological study of the small intestine is complicated by its length and mucosal fragility. it is important to avoid vigorous washing procedures or any form of excessive manipulation of the unfixed bowel, as artefact caused by washing may confound interpretation of changes induced by xenobiotics (roe, ) . combination of artefact due to washing, autolysis and the presence of neutrophils can produce a histological appearance that mimics in vivo damage. although careful visual inspection of the intestine and sampling of appropriate segments for histological examination is usually sufficient for routine examination, various forms of 'swiss roll' techniques are helpful for more complete study. rolling the unfixed, opened rodent intestine around a wooden stick prior to freezing or fixation is one proposed method (moolenbeck and ruitenberg, ) , although this method risks undue manipulation of the unfixed tissue. another more versatile technique applicable to rodent, large animal and human intestine can be performed after fixation. the unfixed opened bowel is pinned flat on a cork or board and fixed in a bath of formal saline. after fixation, the full thickness of rodent intestine can be rolled, transfixed by a pin and embedded in paraffin wax. likewise the mucosa of the intestine of large animal species or humans can be rolled after fixation by separating it from the muscularis externa (filipe and branfoot, ) . inflammation and ulceration of the mucosa occurs as a result of stress, infection with bacteria, viruses, and infestation by parasites or as a direct result of the effects of xenobiotics or ionising radiation. antimitotic or radiomimetic agents as well as ionising radiation are liable to adversely affect the rapidly dividing cells of the small intestine with resulting breakdown of the mucosal barrier. the ulcerogenic activity of non-steroidal anti-inflammatory drugs may also be expressed in the small bowel mucosa. different agents also act in synergy to enhance damage to the small bowel mucosa. an important example is the effect of drugs that depress the immune system and permit the development of pathological infections by microorganisms of the opportunistic type in the small intestine. the histological features of the inflammatory process in the small intestine are not usually specific for a particular agent. it is important to search for evi-dence of microbiological organisms and viral inclusions, which can indicate the cause of intestinal inflammation and ulceration. associated features in non-ulcerated mucosa such as morphology of the villi, accumulation of abnormal cells or foreign substances and changes in lymphoid cells or blood vessels are also important in the assessment of these changes. a number of organisms including those, which are normal residents of the gastrointestinal tract, can cause inflammatory changes in the intestinal mucosa of laboratory animals. with the notable exception of non-human primates, inflammatory bowel disease caused by microbiological organisms is not usually evident or of concern in most toxicity or carcinogenicity studies. however, when animals are treated with antibiotics, immunosuppressive agents or other drugs, which alter the normal intestinal flora, conditions may favour the proliferation of potentially pathogenic organisms in sufficient quantities to cause overt damage to the mucosa. certain bacterial flora may also act synergistically with intestinal protozoans to produce pathological changes (boorman et al., ) . in non-human primate colonies, gastrointestinal disease remains one of the most important causes of death . in contrast to other laboratory species, histological evidence of intestinal infectious disease is relatively common and may confound the interpretation of gastrointestinal alterations occurring in toxicity studies. although the majority of potentially pathogenic organisms affect the primate colon, a number of bacteria, protozoa and metazoa occur in the small intestine. a detailed review of the protozoa and metazoa occurring in the primate gastrointestinal tract has been published (toft, ) . owen ( ) has reviewed the parasites of laboratory animals including those of the gastrointestinal tract and the principle reference for identification of metazoa in tissue sections remains that of chitwood and lichtenfels ( ) . organisms which can cause inflammatory disease in the small bowel but which are primarily agents that produce inflammatory disease in large bowel are reviewed under 'colon' (see below). bacillus piliformis is the agent responsible for tyzzer's disease produces intestinal inflammation and ulcers in rats, mice and hamsters. susceptibility of different species and strains to experimental infection with bacillus piliformis is variable. for instance, c bl, balb mice and f rats appear more resistant to infection than outbred syrian hamsters (waggie et al., ) . lesions of variable severity usually occur in the ileum but may also extend into the caecum and colon. severe infections are characterised histologically by ulceration of the mucosa, oedema and acute inflammation of the submucosa and muscle coats. muscle may also show focal necrosis. non-ulcerated mucosa is typically infiltrated by polymorphonuclear cells and crypt abscesses form. there is blunting and fusion of villi and reactive hyperplasia and mucin depletion of the overlying epithelium . mucosal lymphoid tissue may also show reactive changes or hyperplasia. filamentous bundles of bacillus piliformis can usually be found in the cytoplasm of both epithelial cells and smooth muscle cells at the edges of necrotic zones. methylene blue, giemsa or silver impregnation techniques such as warthin-starry or levaditi stains are the best stains for the demonstration of these organisms although with care they can be visualised in haematoxylin and eosin stained sections (weisbroth, ) . they are gram negative and pas positive. intestinal infections due to salmonella species are relatively common in the mouse but also occur in the hamster and rat . salmonella typhimurium and salmonella enteritidis are regarded as the organisms typical of murine salmonellosis (weisbroth, ) . lesions occur in the ileum and may extend into the jejunum and caecum. they are characterised by the presence of ulcers covered by fibrinous exudate and associated with diffuse infiltration of the adjacent mucosa by macrophages, neutrophils and lymphocytes. intact crypt epithelium shows mucin loss and reactive proliferative changes. a characteristic feature is the presence of poorly defined granulomatous lesions composed of macrophages mainly in associated lymphoid tissue or peyer's patches. clostridia species, especially clostridia difficile which cause a pseudomembranous colitis in man and laboratory animals (especially hamsters) may also produce inflammation and ulceration in the terminal ileum with histological features similar to those found in the colon (see following). proliferative ileitis (transmissible ileal hyperplasia) is a striking lesion of hamsters affecting the distal segment of the ileum that is associated with intracellular invasion of the intestine mucosa epithelium by bacteria. the organism has not been cultivated, but has been suggested that it is a campylobacter (jacoby, ) although other organisms have been identified in this condition (fox et al., ; peace et al., ) . although it is characterised by hyperplasia of the ileal mucosa in its early stages, an inflammatory phase intervenes in which there is focal necrosis and haemorrhage of the mucosa, crypt abscesses and infiltration of the lamina propria by acute inflammatory cells and macrophages. the histological features of the associated hyperplasia are characteristic. the mucosa is covered by immature, mucin-depleted pseudostratified hyperchromatic epithelium with mitoses extending to the tips of villi and densely basophilic intracytoplasmic inclusions (jacoby, ) . helicobacter jejuni (campylobacter jejuni) is a common cause of diarrhoea in man and may be the causative agent in small intestinal inflammation in laboratory dogs and primates. campylobacter species may be more prevalent in beagle dogs and primates than commonly appreciated. it is important to recognise that dogs colonised with these agents may be susceptible to stress-induced, acute onset gastroenteritis (fox et al., ; reed and berridge, ) . in man this form of bacterial disease is characterised histologically by mucin-depletion, flat-tening and reactive changes in the small bowel epithelium, crypt abscesses, oedema and infiltration of the mucosa by neutrophils, lymphocytes and plasma cells. similar histological findings have been reported in dogs infected with this organism (prescott and monroe, ) . the organisms are gram-negative curved, slender rods, which can be visualised in tissue sections with the warthin-starry stain, a recognised technique for spiral bacteria. the carbol fuchsin technique of gimenez ( ) , first used for the identification of ricketsiae in yolk sac culture and a cresyl fast violet technique are also useful methods for the identification of campylobacter species in paraffin sections (burnett et al., ; mcmullen et al., ) . another campylobacter-like organism, helicobacter pylori (campylobacter pylori) has been identified in human patients with gastritis, gastric and duodenal ulcers. it is an aetiological or predisposing factor in these forms of gastrointestinal disease (marshall and warren, ; rouvroy et al., ; richter-dahlfors et al., ) . spironucleus muris (hexamitis muris) is also a cause of inflammation in the small bowel of rats, mice and hamsters. during overt infestation, organisms are seen extracellularly in crypts and intervillous spaces associated with blunting of intestinal villi, epithelial degeneration and mucin-depletion, reactive epithelial hyperplasia, oedema and leucocyte infiltration (boorman et al., ; wagner et al., ) . the morphological expression of damage is accompanied by decreased levels of disaccharidases such as maltase, sucrase and lactase, which may represent a direct effect of the trophozoites on the brush border enzymes (gillon et al., ) . trophozoites are characteristically elongated symmetrical flagellates approximately - µm wide, - µm long. giardia species represent marginally pathogenic flagellates, which are found in the upper gastrointestinal tract. they are opportunistic agents that can become important in both animals and man with depressed immune function. studies in mice infected experimentally with giardia muris have shown that an early response is an increased infiltration of the epithelium by lymphocytes, predominantly t cells (gillon et al., ) . this has led to the suggestion that the response to infection by these parasite is primarily a cell-mediated immune reaction similar to experimental graft versus host reaction in the small intestine of mice (mowat and furguson, ; gillon et al., ) . depression of the immune response by treatment with corticosteroids has been shown not only to increase parasite numbers in murine giardiasis but also cause recrudescence of occult infections (nair et al., ) . it has also been suggested that decreased gastric acidity can predispose to giardiasis in man (nalin et al., ) . giardia muris (lamblia muris) is sometimes found in the small intestine of rat and mouse but it is common in the hamster (fig. ) . trophozoites appear in histological sections as crescent-shaped structures on the brush border of the intestinal mucosa or in the adjacent lumen. mucosal lesions may be totally ab-sent or there may be blunting of villi, reactive epithelial hyperplasia. a typical feature is increased infiltration of the epithelium and lamina propria by mononuclear cells (boorman et al., ) . another important finding is that lactase, sucrase and maltase levels have been shown to decrease in the small intestine in mice infested with giardia muris (gillon et al., ) . giardia lamblia may colonise the small intestine of non-human primates and of man and produce similar morphological appearances to those found with infestations in rodents by giardia muris. other flagellates such as tritrichomonas muris are also be found in the small intestine of mice, rats and hamsters. the coccidian protozoan parasite cryptosporidium represents a striking example of the close relationship between some human and animal diseases. this organism was first recognised in the gastric glands of mice by tyzzer in and has since been confirmed as a cause of diarrhoea in animals and as a human pathogen. it causes mild diarrhoea in normal subjects especially children and young adults but it can produce severe intestinal disease in immunocompromised individuals (casemore et al., ) . histological examination of the small intestine of laboratory animals infested by cryptosporidium reveals the presence of organisms attached to the mucosal surface, often associated, as in man, with other parasites or infections. they are rounded, weakly basophilic structures - µm diameter in haematoxylin and eosin stained sections but are strongly basophilic following romanowsky staining. transmission electron microscopy reveals the detailed internal structure of cryptosporidium attached to the microvillous surface of the epithelial cells. the various stages in the life cycle have been visualised by light and electron microscopy. infection starts with ingestion of an oocyst containing four sporozoites, which are probably released by the action of digestive enzymes. these attach themselves to the intestinal mucosa and undertake their life cycle attached to the epithelial cells (casemore et al., ) . these organisms have been demonstrated in laboratory species including mice, hamsters, rabbits, dogs and nonhuman primates (cockrell et al., ; rehg et al., ; toft, ; fukushima and helman, ; davis and jenkins, ) . hymenolepis nana (dwarf tapeworm) and hymenolepis diminuta (rat tapeworn) are described in the intestine of rats, mice, hamsters, non-human primates and man (hsu, ) . a variety of other metazoan patients are found in the small intestine of non-human primates, see review by toft ( ) . certain viruses produce inflammatory small bowel changes in mice. mouse hepatitis virus (lethal intestinal virus of infant mice) can cause mucosal epithelial necrosis and inflammation with characteristic compensatory epithelial hyperplasia and the formation of epithelial syncytia . murine rotavirus (epidemic diarrhoea of infant mice) produces swollen enterocytes of small and large bowel with fine cytoplasmic vesiculation with little or no inflammation but dilated lymphatics and vascular congestion. cytoplasmic acidophilic inclusions, - µm diameter, are characteristic findings . electron microscopic examination shows cytoplasmic vesicles arising from the rough endoplasmic reticulum, which contain virus particles and electron dense granular material . a mouse adenovirus may also produce large basophilic intranuclear inclusions in the epithelial cells of the small intestine and caecum. k virus infection produces lesions in the jejunal and ileal mucosa of mice. histological features are characterised by mild polymorphonuclear infiltration, with ballooning of occasional endothelial cells within intestinal villi. intranuclear inclusions can be demonstrated in these endothelial cells by light microscopy using appropriate fixation (greenlee, ) . a variety of viruses have been isolated from the gastrointestinal tract of nonhuman primates including viruses of man (kalter, ) . however, they appear to be relatively infrequent causes of gastrointestinal disease and when disease is caused by these viruses it usually affects other organs. not only can non-steroidal anti-inflammatory agents such as indomethacin and phenylbutazone produce gastric ulceration but also penetrating ulcers of the small bowel of laboratory animals (shriver et al., ) . imaging studies with indium-labelled leukocytes in man have also suggested that subclinical intestinal inflammation is associated with long-term therapy with non-steroidal anti-inflammatory drugs (bjarnason et al., ) . it has been postulated that indomethacin-induced intestinal ulcers in rats and dogs are produced by a prostaglandin-independent mechanism, different from the manner in which gastric ulceration is induced (tabata and okabe, ; whittle, ) . conversely, satoh et al. ( ) have suggested that similar mechanisms are responsible for both gastric and intestinal ulceration. they showed that indomethacin-induced gastric ulceration developed in the body mucosa in fasted rats but in the antrum and small intestine in rats given indomethacin minutes after a -hour period of refeeding following a -hour fast. there was good temporal correlation between the development of intestinal ulcers and inhibition of prostaglandin synthesis (satoh et al., ) . the ulcers in the small intestine were morphologically similar to those occurring in the stomach and they were distributed mainly in the mucosa on the mesenteric aspect of the bowel wall. single-dose studies with indomethacin and ibuprofen in rats conducted by suwa et al. ( ) have demonstrated differences between pathology of induced gastric and intestinal damage. gastric damage was superficial, occurred within hours and was fully repaired weeks after dosing. ulcers in the jejunum and ileum reached a maximum area at - hours after dosing, occurred on the mesenteric border, penetrated through the muscularis mucosa and were accompanied by inflammation and oedema. ulcers were still present weeks later. rainsford ( ) has shown that potent intestinal ulcerogens such as indomethacin inhibit the incorporation of radioactive s sulphate into glycoproteins of the upper intestinal mucosa as well as the stomach of rats. this may decrease the capacity of the mucus in the intestine to act as a buffer for hydrogen ions. indomethacin given orally to dogs in doses of . mg/kg/day for - days was also shown to produce intestinal ulceration. these ulcers were deep, punchedout lesions, many of which were lying over peyer's patches (stewart et al., ) . some ulcers involved the whole circumference of the small intestine wall. histologically, the ulcers were associated with an intense inflammatory response principally of mononuclear cells, which infiltrated the bowel wall to the serosa particularly adjacent to peyer's patches. it was suggested that this distribution of ulcers was a result of an exaggerated immune response to normal intestinal antigens. these antigens may have been produced by inhibition of suppresser cells in peyer's patches, following depression of prostaglandin synthetase by indomethacin (stewart et al., ) . special dye techniques, scanning and transmission electron microscopy have also shown that non-steroidal anti-inflammatory drugs also produce adverse effects on the small intestine mucosa without overt pathological changes being evident by light microscopy (brodie et al., ; djaldetti and fishman, ) . following administration of aspirin to mice for weeks, shortening and erosion of microvilli and increased numbers of goblet cells were only demonstrated in the duodenum and jejunum by scanning and transmission electron microscopy (djaldetti and fishman, ) . morphometric studies of the intestinal mucosa of indomethacin-treated mice have also shown widespread alterations to columnar cells, goblet cells and paneth cells suggesting generalised effects on mitotic activity and crypt loss (ettarh and carr, ) . such submicroscopic findings support the idea that non-steroidal anti-inflammatory agents may induce damage to the small intestine more commonly than supposed. although non-steroidal, anti-inflammatory drugs are the best-known drugs with adverse effects on gastrointestinal mucosa, small intestinal inflammation and ulceration can also produced by other agents through different mechanisms. anticancer drugs and other therapeutic agents, which affect cell proliferation, depress the bone marrow or the immune system can also produce intestinal mucosal necrosis, haemorrhage, inflammation and opportunistic gastrointestinal overgrowth when administered to dogs or rodents in high doses (fig. ) bregman et al., ) . lymphoid infiltrates without tissue fig. . section of small intestine from a wistar rat treated with a -day course of an antiproliferative anticancer drug. the mucosa shows mucus-depletion, loss of villous architecture as well as a reparative and inflammatory response. (he, × .) damage were also reported in the small intestine of rats treated with human recombinant interleukin- (anderson and hayes, ) . agents of particular toxicological interest are cysteamine, propionitrile and their structural analogues as well as -methyl- -phenyl- , , , -tetrahydropyridine (mptp) which are capable of producing ulcers of chronic type in the duodenum of rats and mice (szabo and cho, ) . these compounds vary in their ulcerogenic capacity but they are all able to produce ulcers of chronic type with crater formation, granulation tissue and reactive changes in adjacent mucosa in the anterior and posterior wall of the proximal segment of the duodenum of rodents. although these different agents influence gastric acid secretion in different ways, structure-activity relationships suggest that they produce duodenal dysmotility, decrease bicarbonate production and reduce its delivery from the distal to proximal duodenum. these factors decrease the neutralisation of gastric acid in the first part of the duodenum and this may contribute to the development of ulceration (szabo and cho, ) . furthermore, these effects can be attenuated or prevented by dopamine agonists or their precursors whereas dopamine antagonists can potentiate their effects. this suggests that the central or peripheral dopamine-mediated actions of these agents may be involved in the pathogenesis of duodenal ulceration (szabo, ; . using appropriate fixation and staining procedures, fine granular lipid droplets can be normally visualised in the apical parts of epithelial cells covering the upper third of small intestinal villi. administration of drugs and chemicals may produce an excessive accumulation of lipid through specific effects on lipid metabolism or as part of general cellular toxicity. in the preclinical toxicity studies with , -di-tert-butylamino- -acetyl- -methylpyridine (sa h - ), an inhibitor of glucose transport intended for use as an anti-obesity drug, lipid accumulation occurred in the lamina propria of the small intestinal villi of sprague-dawley rats and guinea pigs but not in dogs or primates (visscher et al., ) . after administration of sa h - to rats, there was progressive accumulation of lipid droplets in the epithelial cells over the tips of the duodenal villi demonstrable by osmium tetroxide staining. ultrastructural examination revealed uniform electron-lucent droplets within profiles of the smooth endoplasmic reticulum and golgi apparatus. lipid droplets increased with time, accumulated and coalesced to form large droplets in the lamina propria. larger droplets were phagocytosed by macrophages in the lamina propria but there was no evidence of epithelial damage or necrosis. changes were most pronounced in the duodenum but were also noted to a lesser extent in jejunum and ileum but not in colon or stomach. sequential studies using electron microscopy showed that lipid rapidly accumulated within several hours in the profiles of smooth endoplasmic reticulum and golgi apparatus of the epithelial cells and formed droplets or chylomicra in the intercellular space. the absence of any other subcellular changes or evidence of derangement of protein synthesis suggested that sa h - altered the pathways of lipid resynthesis or transport. this was consistent with the distribution of the lipid in the upper third of the jejunal villous epithelium, a zone most active in lipid absorption, resynthesis and transport (dobbins, ) . it was suggested fatty change might have taken place because of alterations in the sugar moiety of chylomicra brought about by interference with glucose transport (visscher et al., ) . lipid droplets which stained with oil-red-o in formalin-fixed frozen sections and showed uniform electron density characteristic of neutral lipid were also observed in the epithelial cells and macrophages in the lamina propria of jejunum and duodenum and mesenteric lymph nodes in rats given a synthetic '-dodecyl glutaramide ester of erythromycin (gray et al., ) . unlike the erythromycin base, rats poorly tolerated this ester. it appeared that the ester was absorbed unhydrolyzed and converted to chylomicron-like droplets, which then accumulated in the macrophages of the lamina propria and local mesenteric lymph nodes, without overt damage to epithelial cells. accumulation of lipid in epithelial cells of intestinal villi has been observed in rats following administration of puromycin (friedman and cardell, ) and ethionine (hyams et al., ) , agents which have inhibitory effects on protein synthesis. detailed morphological study of the intestinal epithelial cell in rats treated with puromycin have shown that there is concomitant accumulation of lipid with a decrease in the quantity of rough endoplasmic reticulum and golgi membranes (friedman and cardell, ) . these changes were in keeping with the concept that lipid accumulates as a result of inhibition of the synthesis of membrane components of the golgi by the rough endoplasmic reticulum which are important for the transport of lipid. in addition to lipid droplets forming as a result of altered lipid metabolism, they may form in the epithelial cells of the intestinal mucosal as a result of a direct toxic effect of the ingested drugs on the small intestinal mucosa. in such instances atrophy of villi and degenerative changes in the epithelial cells may also be observed (see following). the small intestinal mucosa is also one of the many sites at which drug-induced accumulation of polar lipids form laminated structures (myeloid bodies) or crystalloid structures within lysosomes. this form of lipid storage disorder is produced by diverse amphiphilic cationic drugs in both man and laboratory animals probably as a result of drug interaction with polar lipids rendering them difficult to digest (lüllmann-rauch, ) . species differences in susceptibility and tissue distribution of phospholipid are probably not only related to physiochemical characteristics of the inducing drugs which influences their ability to permeate selective biomembranes and react with different lipids, but also to tissue concentrations of drugs achieved and the ability of organs to metabolise parent drug to less amphiphilic products. in general terms, this form of disorder is characterised by membrane-bound, acid phosphatase-positive cytoplasmic inclusions, which on ultra-structural study are seen as lamellated or crystalloid structures in lysosomes. these appearances are characteristically reversible on cessation of treatment with the inciting agent. an example of this phenomenon occurring in the small intestine is provided by the iodinated amphiphilic drug, amiodarone, which has been used clinically in europe for the past years in the treatment of angina and more recently in the control of supraventricular cardiac arrhythmias. its adverse effects in man are believed to be the result of accumulation of drug in lysosomes particularly in liver, skin and eye (d'amico et al., ; shepherd et al., ) . when high doses of amiodarone were administered orally to rats and beagle dogs, multilamellated lysosomal inclusion bodies accumulated first in the jejunal mucosa and mesenteric lymph nodes before becoming widely distributed in other organs particularly in the lungs (mazué et al., ) . in both rats and dogs the small intestinal lesions were characterised by the presence of foamy macrophages with pale finely vacuolated cytoplasm and condensed eccentric nuclei within the lamina propria of the jejunal villi (mazué et al., ) . mesenteric lymph nodes were also involved early after the onset of treatment. in the dog, jejunal villi were somewhat flattened and widened or showed a variable degree of villous atrophy, most marked in the proximal and middle jejunum (vic et al., ) . electron microscopy confirmed the presence of lamellated lysosomal bodies distending macrophages. the early accumulation of foam cells in the jejunal macrophages was probably a reflection of the disposition of drug following oral absorption. although similar lipidosis was seen in many organs following intravenous administration in dogs, more lipidosis was seen in the jejunum after oral dosing. moreover, there were species differences in sensitivity to these changes, baboons being relatively insensitive compared to dogs. fischer rats were very sensitive to these changes compared to sprague-dawley rats and wistar rats were almost completely resistant to lipidosis induced by amiodarone under similar conditions (mazué et al., ) . similar cytological changes have been reported in cells of some organs in patients treated with amiodarone (d'amico et al., ; shepherd et al., ) . villous shortening or stunting results when the proliferative activity of the crypt epithelium is reduced or under circumstances in which crypt cell proliferation is insufficient to compensate for increased cell loss as a result of mucosal cell damage. decreased cell proliferation can be seen segments, which are surgically bypassed, or following decreased food intake, parenteral nutrition, hypophysectomy or thyroidectomy bastie et al., ) . as adrenergic factors are important in the control of small intestinal epithelial cell division, drugs that alter α or β adrenoreceptor activity may influence the proliferative capacity of the epithelium. in mice, increased α or β receptor stimulation by appropriate agonists (e.g. phenylephrine) diminishes proliferation of crypt cells but proliferation is increased by stimulation of α receptor activity (kennedy et al., ) . yohimbine, a α -antagonist also reduces cell proliferation in the same animal model. some of the effects of these agents may be mediated by changes in splanchnic blood flow . the detailed morphological study of the small intestinal mucosa in the rat following hypophysectomy by bastie et al. ( ) has shown a reduction in the height of the small intestinal villi associated with reduction in mitoses in the crypt epithelium. the number of goblet cells was shown to fall particularly in the jejunum and the number of paneth cells increase in the ileum. ultrastructural examination showed decreased height of the microvilli of absorptive cells and a lower number of their intracytoplasmic organelles and ribosomes. there were also significant decreases in brush border enzyme activities of alkaline phosphatase, aminopeptidase, maltase and lactase reported about week following hypophysectomy. substances which reduce mitotic activity and therefore lower regenerative capacity of the intestinal epithelium also produce shortening or stunting of small intestinal villi and eventually flattening of the mucosa. a wide variety of anticancer agents and antiviral drugs with radiomimetic properties interfere with cell division in the crypts thereby reducing the number of epithelial cells produced. histologically, the effects of such agents are characterised by blunting, shortening or complete atrophy of villi. mitotic activity is reduced in the crypts and the crypts become dilated and lined by flattened cells. the overlying epithelium loses its normal regular arrangement and cells show pleomorphic nuclei with irregular chromatin patterns. increased numbers of inflammatory cells may infiltrate the lamina propria and epithelium. ulceration, haemorrhage and secondary infection of the gut wall ensue if there is overwhelming cell damage. comparison of the gastrointestinal toxicity expressed by antimitotic anticancer drugs of different classes in rodents, dogs, non-human primates and man have suggested that there is a higher degree of correspondence between effects in man and dog than between man and other species (owens, ; schein et al., ) . in studies with the antiviral agent acyclovir, a radiomimetic effect was noted in the gastrointestinal tract of dogs at high doses but not rodents (tucker et al., ) . another example is the villous atrophy described in rats following treatment with an antibacterial agent ici , . this was believed to arise as a result of both interference with cell division and a direct effect on the surface epithelial cells (murgatroyd, ) . the effects of ici , were characterised by atrophy of villi with dilatation of crypts and atypical features in the crypt epithelium suggestive of an effect on mitotic activity. in addition, vacuolated lipid-laden epithelial cells were observed over the tips of villi accompanied by reductions in the numbers of goblet cells and reduced activity of acid and alkaline phosphatase, esterase, adenosine triphosphatase, glucose- -phosphatase and succinic dehydrogenase, compatible with a direct adverse effect on superficial mature epithelial cells. a variety of factors stimulate cell proliferation in the small intestinal epithelium. these include enterectomy, increased feeding and stimulation of autonomic nerves. administration of neurotransmitters, thyroxine, growth hormone, corticosteroids, testosterone, gastrin, glucagon and recombinant epidermal growth factor may also stimulate epithelial cell proliferation breider et al., ; reindel et al., ; vinter-jensen, ) . in rats hypothalamic damage, hyperthyroidism, tube feeding, diabetes mellitus and insulin injections have been shown to produce intestinal hyperplasia (mackay et al., ; levin and smyth, ; jarvis and levin, ; forrester, ) . most causes of greater cell production lead to increased villous height and mucosal hyperplasia, although intense crypt cell proliferation as a compensatory regenerative response can be associated with villous atrophy (see previous). the compensatory response to the surgically resected or bypassed intestine has been the focus of the most detailed studies of increased cell renewal in the small intestine. partial resection in both rats and man is accompanied by increased villous height and crypt length (hanson et al., ) . this is primarily the result of hyperplasia for it has been shown that the numbers of cells per unit length of villus remains unchanged (hanson et al., ) but there is an overall increase in the cell population of villus and crypt (hanson and osborne, ) . dna/rna ratios also remain largely unaltered . no gross changes in villous shape have been reported after resection and the total number of crypts remains constant. although increased intestinal uptake of substances from the bowel lumen occurs in hypertrophied segments per unit length of bowel, disaccharide and dipeptidase activities are normal or even decreased after resection suggesting a comparative immaturity of cells in the residual mucosa. functional adaptation therefore is achieved by a larger number of cells, the individual absorptive capacity of which is not increased . increased numbers of specific goblet cell populations are also seen in hyperfunctional states. following jejuno-ileal bypass operations in rats, increased numbers of pas-positive goblet cells develop in the villi and crypts of the hyperfunctional segments of the duodenum, jejunum and ileum (olubuyide et al., ) . mucin histochemistry using the high-iron diamine and alcian blue techniques have shown that the goblet cells in the hyperfunctional segments contain increased sialomucins in the villi and crypts of the jejunum and ileum but not in the duodenum and increased sulphomucins in the distal ileal segment. sialomucin production may reflect relative cellular immaturity of the more rapidly proliferating cells under these circumstances. however, as sialic acid conveys more viscoelastic properties to mucin, it has been suggested that the goblet cells change following intestinal bypass fulfils a protective function against the increased flow of gastrointestinal contents (olubuyide et al., ) . a number of nutritional factors, particularly dietary fibre, can influence the proliferative characteristics of the small bowel mucosa. carefully controlled studies in rats given different forms of dietary fibre have shown that the prolif-erative characteristics of the small intestine can be modified by both the quantity and the quality of the fibre. one study has shown a decrease in the length of villi, crypt cell hyperplasia and shorter transit times in rats fed pectin-supplemented diet but an increase in mucosal growth without alteration in relative differences in crypt and villous length with guar supplementation compared with rats fed fibre-free diet (jacobs, ) . another rat study has shown that pectin feeding leads to increased mucosal area and height associated with an increase muscle mass (stark et al., ) . these different effects may be the result of differences in solubility, gel formation, water holding capacity, effect on transit time and ion exchange activity or bile acid adsorption of the different fibres. interactions between dietary constituents are complex. for instance, in rats the effects of % dietary cholestyramine, a non-absorbable ion exchange resin, on small intestinal histomorphology have be shown to depend on interaction of dietary factors (burkhardt et al., ) . administration of an inhibitor of cholesterol biosynthesis, α-cholest- -( )en- β-ol- -one, to rats for up to days was also shown to produce enlargement of the small intestine in a way which was morphologically similar to the changes found following intestinal bypass (smith et al., ) . the enlargement was most marked in the proximal segment of the small intestine and progressively diminished towards the ileocaecal junction, sparing the stomach, caecum and colon. histological examination and morphometric analysis revealed an increase in smooth muscle mass, lengthening of the villi as well as an increase in the depth and cellular proliferation in the crypts of lieberkuhn without evidence of cell damage or fatty change. like the changes following jejunal bypass procedures, there was also an increase in acid mucosubstances in the goblet cell population overlying the villous mucosa (smith et al., ) . the mechanism for this change in the rat was not clear, particularly as intestinal hyperplasia was not seen in baboons treated with this -ketosterol for long periods. however, it was suggested that it was an adaptive response, possibly related to inhibition of cholesterol metabolism and cholesterol absorption from the diet, particularly as the laboratory diet employed in the rat study was particularly low in cholesterol. local and systemic changes in hormones and various transmitter substances also influence the number of cells in the small intestinal epithelium. morphometric studies of the small intestinal mucosa in mice following gastrin administration have shown increases in villous area associated with decreases in microvillous area, increased number of goblet cells and paneth cells . studies in which rats were treated with the prolactin-inhibitor, ergocryptine, have shown that the total number of mucous cells and the number staining with alcian blue at ph . increase in the ileal crypts, possibly as a result of increased synthesis of sulphated mucosubstances (gona, ) . in this context, it is of interest that chronic treatment with the rauwolfia neuroleptic, reserpine, causes an increase in the sulphation of goblet cell mucin in the small intestine as demonstrated by alcian blue staining at ph . and the high iron diamine technique without changes in the goblet cell numbers (park et al., ) . agents which affect activity of the sympathetic nervous system can also alter epithelial cell proliferation in the small (and large) intestine. treatment of rats with adrenaline, isoprenaline, phenylephrine, phentolamine and yohimbine all result in decreased mitotic activity of jejunal and colonic crypt cells (tutton and helme, ; kennedy et al., ) . by contrast, administration of metaraminol, clonidine, propranolol, prazosin and labetolol as well as simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation (kennedy et al., ) . these results suggest that agents that stimulate α adrenergic receptor activity and those that are α -antagonists and β-adrenergic receptor antagonists increase proliferative activity in the rodent intestinal mucosa. caffeine is also reported to produce an increase in thickness of the intestinal mucosa when administered in high doses to rats (lachance, ) . this raises the possibility that intestinal mucosal hyperplasia can be produced by phosphodiesterase inhibition and resultant increases in intracellular camp in a similar way to the hypertrophy induced in salivary tissue. this is supported by recent findings in rats treated for periods of up to months with the inotropic vasodilator, ici , , a phosphodiesterase inhibitor intended for treatment of congestive cardiac failure. administration of high doses not only produced salivary gland hypertrophy but also marked thickening of the small and large intestinal mucosa. this was characterised histologically by an increase in villous length and deepening of intestinal glands, with a relatively unchanged number of epithelial cells per unit length of gland or villus (westwood et al., ) . although prostaglandin e analogues produce most of their effects in the stomach, increased thickness of the small intestine characterised by longer villi, deeper crypts and increase in cell size have been reported in rats treated with these agents (levin, ) . focal hyperplasia, focal avillous hyperplasia, focal atypical hyperplasia, duodenal plaque, polypoid hyperplasia, polyp -mouse irregular, atypical single or multiple foci of glandular hyperplasia may be found in the small intestinal mucosa of several strains of aged, untreated mice. the lesions are usually located in the first part of the duodenum where they form discrete, raised plaques composed of elongated, irregular or branched glands which replace the normal villous structure of the mucosa (rowlatt and chesterman, ) . the glands are lined by hyperchromatic columnar cells, which show marked pseudostratification and proliferative activity. paneth cells and mucinsecreting goblet cells may also be prominent. some glands are cystic and the stroma is fibrous and infiltrated by chronic inflammatory cells. the lesions become pedunculated or polypoid in appearance and show a fibrovascular core that is infiltrated by inflammatory cells. they resemble adenomatous polyps described in man. the cause of these changes in the mouse small intestine is unknown but their prevalence can be altered by dietary fibre and panthothenic acid deficiency as well as by administration of drugs and chemicals (hare and stewart, ; seronde, ; ito et al., ) . in their study of dba mice, hare and stewart ( ) considered that the lesions were not genuine neoplasms since they were composed of a mixture of cell types, which normally populate the mucosa. furthermore, they suggested that the presence of an inflammatory component in the stroma and the fact that the prevalence of these lesions was increased in mice fed a high roughage diet were consistent with the concept that they represent an inflammatory adenomatoid hyperplasia. seronde ( seronde ( , reported these lesions in mice fed purified diets, particularly when deficient in panthothenic acid. panthothenic acid deficiency was also associated with inflammation and deep penetrating chronic ulcers of the duodenum in affected mice, compatible with an inflammatory aetiology of the lesions. an increase in the prevalence of these duodenal changes was described in cd- mice treated with the synthetic prostaglandin e analogue, misoprostol for months . these authors suggested that the findings posed no real concerns for the safety of patients treated with misoprostol on the grounds that the mouse was unique in this aspect of the response to misoprostol because the mouse had a particular liability to develop such changes in the small intestine. the proliferative lesions were found in a few control cd- mice in the same study. in addition, it was also argued that the lesions were neither neoplastic nor preneoplastic in nature . they were not seen in rats treated with misoprostol for years . lesions characterised by such intense proliferative activity may be difficult to distinguish from neoplastic lesion. indeed chronic administration of hydrogen peroxide to c bl/ j mice in drinking water was not only shown to potentiate the development of a similar type of duodenal hyperplasia but also to produce frankly invasive adenocarcinomas (ito et al., ) . anatomically the large intestine is broadly similar in man and laboratory animals but there are significant functional differences. the rat colon is probably one of the best studied of the laboratory animal species because the rat is widely used for experimental work on colon carcinogenesis . as the canine model is popular for oral dosage-form testing, differences in colonic physiology between dog and man may be better understood than between man and many other species (dressman, ) . in man, as well as in the non-human primates, the large intestine can be divided anatomically into caecum, appendix, ascending colon, transverse colon, sigmoid colon rectum and anal canal. like the small bowel, the colon comprises mucosa, submucosa, muscularis mucosa and serosa. mucosal plicae are only found in the rectum although plicae semilumaris, formed by folds of the entire thickness of the bowel wall, are found in the colon. the large intestine of the dog resembles that of man more than that of most other domestic species. it is a simplified tubular structure only slightly larger in diameter than the small intestine. the colon of the dog is divided anatomically into ascending, transverse and descending parts, but there is no well-defined sigmoid segment. the caecum in dog is a small diverticulum, similar to that found in other carnivorous species and it communicates directly with the colon. the colon of the rat and mouse is shaped like an inverted v that can be divided into ascending and descending segments. there is no clearly defined transverse colon. a characteristic feature in both rat and mouse is the presence of a curved kidney-shaped caecum. its size is intermediate between the large and anatomically complex caecum of herbivores such as the rabbit and the small caecum of carnivorous species. this probably reflects the omnivorous nature and flexibility of the rat and mouse in their dietary habits, particularly their ability to breakdown cellulose (rerat, ) . the caecum of the rat and mouse is a blind pouch from which the colon and ileum exit in close proximity and in which antiperistaltic movements occur. this structure and the presence of bacteria undoubtedly contribute to its ability to function as a fermentation organ in which breakdown of substances can occur in a reasonably controlled milieu (snipes, ) . in rat and mouse, the caecum is the site of absorption of many substances including calcium, magnesium, water and electrolytes vitamin k and fatty acids (snipes, ) . caecectomy has been shown to decrease digestion of carbohydrates and protein and increase loss of faecal water in these species (ambuhl et al., ) . the activity of intestinal microflora in the metabolism of both endogenous and exogenous substances has been demonstrated in the rodent caecum rowland, ) . the usual stock diets for rodents contain abundant plant fibre which provides bulk and fermentable carbohydrate for the microbial population in the caecum. rats fed stock diets have been shown to possess high levels of reductive and hydrolytic enzyme activity (e.g. azoreductase, nitroreductase, nitrate reductase, β-glucosidase and β-glucuronidase) in their caecal contents compared with rats fed purified fibre-free diets . intestines of germ free animals have thinner lamina propria, lower cell turnover, enlarged caecum, altered metabolism of cholesterol, bilirubin and bile salts and larger amounts of mucin in faeces compared with animals possessing normal gastrointestinal microflora (midtvedt, ) . species differences in microflora are also reported. comparative studies with human and rat intestinal microflora have suggested that each population of organisms possesses a degree of autonomous self-regulation and capable of responding quite differently to dietary changes (mallett et al., ) . comparative studies have shown large differences in the numbers of facultative anaerobic gram-negative bacteria in the gastro-intestinal tract of mice from three, major specific pathogen free units in australian laboratories. it was shown that these differences could influence the immune system, susceptibility to infection and experimental results (o'rourke et al., ) . the colon and caecum in man and laboratory animals are lined by a fairly uniform mucosa devoid of villi. columnar cells of two main types cover the surface epithelium. these are absorptive and mucous cells similar to those found in the small intestine. intestinal glands or crypts of lieberkuhn extend downwards from the surface generally as simple, unbranched tubules lined principally by mucous cells with smaller populations of absorptive, endocrine and undifferentiated cells. the mucosa in man and laboratory animals is not entirely flat but shows a slightly corrugated or uneven pattern, which varies with the particular site within the colon. in histological sections of the colon in man, this corrugated pattern is seen as an anthemion-like structure of crypts reminiscent of a greek architectural feature (filipe and branfoot, ) . this is also seen in larger laboratory animal species. in rats and mice the crypts of the caecal mucosa are wider near the lumen than in the crypt base and crypts may be branched, features which may be related to the absorptive function of this zone (snipes, ) . the proliferative zone in the large bowel is found in the lower part of the gland and mitotic figures are normally limited to this zone. as in the small bowel, multipotent, undifferentiated stem cells situated in the gland base give rise to the principle cell types which migrate to the cell surface with subsequent differentiation and alteration of their enzyme activities and morphological features (chang and leblond, ). in studies with mouse aggregation chimaeras in which mosaic cell populations of the intestinal epithelium were localised immunocytochemically, it was demonstrated that the entire epithelium of each adult gland descended from a single progenitor cell (ponder et al., ) . the single progenitor may itself give rise to several stem cells responsible for the cell renewal in the complete crypt. absorptive cells are found most commonly in the surface epithelium but also to a lesser extent in the glandular epithelium. they are morphologically similar to those in the small intestine each possessing apical plasma membranes with uniform microvilli and a well-formed glycocalyx. microvilli of absorptive cells have been shown by electron microscopic study to become longer and denser with increasing distance distally in the gastro-intestinal tract. thus, they are longer and more dense in the ileum than in the caecum and least dense and shortest in the colon, perhaps reflecting their relative absorptive capacity (snipes, ) . this is reflected at light microscopic level by the less conspicuous brush border in the large intestine compared with that in the small intestine. there are species and regional differences in the glycoconjugates found in the brush border of the large intestine, although they generally contain predominantly acidic mucosubstances. in the mouse and rat, sialomucins with some neutral mucins are found. in hamsters, dogs, non-human primates and man both sulphomucins and sialomucins may be seen in the brush border (sheahan and jervis, ) . the glycocalyx is important in the protective function of the colonic mucosa for its disruption by agents such as salicylates has been shown to increase absorption of xenobiotics from the rat rectal mucosa (sithigorngul et al., ) . although there has been some debate about the precise nature of the mucous cell populations based on structural studies in mice and rats (chang, ; , for practical purposes two principle types of mucous cell can be defined. one of these is the typical goblet cell with cytoplasmic mucous droplets forming a goblet shape, which is found both in glandular and surface epithelium. the other type, the so-called vacuolated cell or mucous cell, is found only in the crypts (chang and leblond, ; thomopoulos et al., ) . these vacuolated or mucous cells show empty-appearing vacuoles in the cytoplasm which rather than being empty contain abundant sialomucins of a type different from those in goblet cells (spicer, ; wetzel et al., ) . detailed structural studies and cytochemistry using lectin probes have suggested that these vacuolated cells are able to differentiate into absorptive cells with the cellular apparatus to produce the cell surface glycoconjugates of the glycocalyx (thomopoulos et al., ) . sulphomucins, as demonstrated by the high-iron diamine technique (table ) , generally predominate in the distal colonic segment. in both rat and mouse, goblet cells of the proximal colonic mucosa contain largely sialomucins in the lower parts of the crypts with sulphomucins predominating in the upper parts of the crypt. the distal colon contains largely sulphomucins. the only difference between rat and mouse appears to be the fact that the mouse caecum contains almost exclusively sulphomucins but sulphomucins and sialomucins are found in the rat caecal mucosa. in hamsters, the entire colon contains predominantly sulphomucins. neutral mucins and sulphomucins predominate throughout the dog colon with occasional goblet cells containing sialomucin. in non-human primates, neutral mucins, sialomucins and sulphomucins are seen throughout the colon with sialomucins generally more prominent in the proximal colon and sulphomucins in the distal segment. in man, neutral mucins are found mostly in the caecum. in the caecum and ascending colon, sulphomucins are found in the upper crypts and sialomucins in the crypt base. the converse occurs in the distal colon where sulphomucins predominate in the lower two-thirds of the glands and sialomucins in the upper third of the glands and in the surface epithelium. lectin-labelling (table ) shows even greater heterogeneity of mucins in the colonic mucosal cell population, probably reflecting differentiation patterns and changes in glycosyltransferase activity as cells migrate upwards (freeman et al., ; thomopoulos et al., ) . it has been suggested by jass and roberton ( ) that the two principle changes in pathological processes in the human colonic mucins are loss of oacylation substituents at sialic acid c and c , , and increased sialylation, neither being specific for neoplasia. the colon, like many other tissues also possess drug metabolising activity, although less than in the liver. it has been shown that the activity of cytochromes p involved in hydroxylation of benzo[a]pyrene in microsomes prepared from the colons of sprague-dawley rats retain their activity and responsiveness to inducers better than those in the liver with advancing age (sun and strobel, ) . the lamina propria of the large bowel is arranged in a similar way to that of the small bowel. by virtue of the presence of lymphocytes, plasma cells, macrophages and dendritic cells as well as scattered small lymphoid aggregates or patches, it forms an integral and important part in the mucosal immune defence system. most of the lymphocytes in the lamina propria of the human colonic mucosa, like that of the ileum, have been shown to be t cells with helper t cells out numbering the t-suppresser phenotype (hirata et al., ; pabst, ) . this contrasts with intra-epithelial lymphocytes of the human colonic mucosa which are also t cells but more than % of which possess characteristics of the suppresser/cytotoxic phenotype and only - % being helper t cells. this distribution of lymphocyte subsets is seen immunocytochemically in the rat colon using the monoclonal antibodies w / , w / and mrc ox (see haemopoietic and lymphatic systems, chapter iii). the pan t-cell marker w / shows the presence of t lymphocytes in the lamina propria and most of these are labelled by w / demonstrating their cd helper phenotype (bland and warren, ) . mrc ox demonstrates that few lymphocytes in the lamina propria are of suppresser/cytotoxic (cd ) type, which contrasts, with the high proportion of mrc ox positive lymphocytes in the colonic epithelium (bland and warren, ) . the monoclonal antibodies mrc ox and mrc ox , specific for the rat ia antigen, also label numerous cells with macrophage and dendritic cell morphology in the large bowel of the rat (bland and warren, ; martin et al., ) . mature, small b lymphocytes are relatively uncommon in the colonic lamina propria of man and laboratory animals. however, the lamina propria contains large numbers of plasma cells, which are mainly of iga type, followed by smaller numbers of igm an igg subtypes (pabst, ) . a feature of the colonic mucosa is the presence of lymphoid aggregates also called lymphoid nodules, patches, lymphoid-glandular complexes or microbursa. these are similar to peyer's patches of the small intestine as they are composed principally of lymphoid cells of the b-cell series arranged in follicles with germinal centres with interfollicular and perifollicular zones composed of t cells (pabst, ) . they are distributed along the entire length of the colonic mucosa although they are generally smaller than peyer's patches. in sprague-dawley rats, lymphoid aggregates are usually about mm diameter except in the distal colon where they attain sizes of up to mm in maximum diameter (martin et al., ) . unlike peyer's patches which are characteristically not associated with crypts or villi, the colonic lymphoid aggregates frequently contain irregular atypical mucosal glands which may enter deeply in the lymphoid tissue and penetrate below the muscularis mucosa both in man and laboratory animals scott, ; martin et al., ) . in some strains of rat, cells in these glands express the ia antigen, unlike the other parts of the colonic epithelium (martin et al., ) . these glandular structures, which are intimately associated with lymphoid tissue, may be important in the immune protection of the colonic mucosa, perhaps by acting as a special local receptor for antigens . it has been proposed that these glandular structures represent sites of predilection for the spread of inflammatory disease to the submucosa by allowing microorganisms to pass through the muscularis mucosa (scott, ) . it has also been suggested that they constitute physical weak points in the bowel wall and may play a part in the pathogenesis of diverticular disease of the colon in man . colonic carcinomas induced by dimethylhydrazine in the rat also appear to develop more commonly in the lymphoid aggregates than in other zones (martin et al., ) . m cells have been described over the lymphoid aggregates in the caecum of the mouse (owen and nemanic, ) , see small intestine. although microorganisms are important causes of inflammatory disease in the large intestine of man and animals, among laboratory animals they are usually only significant problems in non-human primates and hamsters. in the strains of rats and mice and in beagle dogs commonly employed in drug safety evaluation, spontaneous disease of the colon as a result of infectious agents is uncommon. nevertheless, treatment with some therapeutic agents may alter the normal bacterial flora to permit overgrowth of pathogenic organisms or disturb the normal balance between antigens in the lumen or control mechanism to evoke inflammation. inflammation induced by organisms may also confound the histological assessment of drug-induced changes in the colon. ulceration and inflammation of the colon as a direct result of administration of potential therapeutic agents is reported in humans although less commonly that in the small intestine. it has been suggested from studies of the effects of anticancer compounds on neoplastic colonic cells that intestinal cells may possess inherent protective properties in the form of an accelerated efflux pump which can serve to protect them from potentially damaging agents (klohs and steinkampf, ) . ulceration and inflammation can be induced by the local application of drugs and vehicles to the rectal mucosa. assessment of these effects in an appropriate animal model is important in the safety evaluation of preparations designed for use in man as rectal suppositories. although inflammatory conditions of the large bowel may possess morphological features typical for some inducing agents, inflammation of the large intestinal mucosa is usually characterised by non-specific histological features. in early or mild inflammation, the surface and glandular mucosa remains intact but shows mucin depletion. this is characterised by reduction in the mucus in goblet cells and increased cytoplasmic basophilia. scattered neutrophils may be seen in the epithelium and adjacent lamina propria. in more severe cases, crypts become filled or distended with acute inflammatory cells (crypt abscesses). the lamina propria is variably hyperaemic and congested and contains increased numbers of mononuclear cells. severe changes are characterised by attenuation or frank erosion of the epithelium and the formation of penetrating ulcers filled with fibrinous exudate and surrounded by intense inflammation, granulation tissue and eventually fibrosis. residual glands may be dilated and lined by flattened epithelium or show reactive changes and mitotic activity. regenerative hyperplasia, which can become florid in chronic ulcerative conditions, is characterised by lengthening, irregularity and cystic dilatation of glands which are often lined by hyperplastic epithelial cells and goblet cells distended with mucin. where ulcerative damage has destroyed glands and supporting stroma, regeneration of glands may not occur in the normal regular fashion and branching of crypts may be evident. clostridium difficile may cause inflammatory changes in the colon of laboratory animals, particularly hamsters, and this may extend into the distal ileum. as in man this form of colitis, often referred to as pseudomembranous colitis, is usually associated with antibiotic therapy. in man it was originally associated with lincomycin and clindamycin therapy but other antibiotics have been implicated. it has been shown that both in man and the hamster experimental model that the enteritis is the result of the toxin produced by clostridium difficile (bartlett et al., ; milligan and kelly, ) . in man this condition is histologically characterised by the presence of plaques or pseudomembranes on the colonic mucosal surface. the pseudomembrane is composed of mucus, fibrin, blood cells, inflammatory cells and cell debris, which has an appearance of streaming from the underlying mucosa. the mucosa may be partly necrotic or mucosal glands are dilated and lined by flattened or hyperplastic cells. the ileal mucosa may show similar changes (milligan and kelly, ) . similar features are observed in the antibiotic-treated hamster although the pseudomembrane is less prominent and it may be distributed more proximally with involvement of the terminal ileum (rehg, ) . in the hamster, the condition is characterised histologically by erosion of the colonic epithelium and the variable presence of a pseudomembranous plaque of mucin and cell debris. intact but affected mucosa is thickened with reactive changes accompanied by mucin loss in the epithelium and infiltration of a hyperaemic and oedematous lamina propria and submucosa by polymorphonuclear cells (rehg, ) . although most instances of this form of clostridia colitis in the hamster have been associated with antibacterial therapy, it has also been reported in untreated hamsters (rehg and lu, ) and those treated with antineoplastic drugs (cudmore et al., ) . similar changes have been reported in antibiotic-treated guinea pigs and rabbits (rehg and lu, ; rehg and pakes, ) . guinea pigs are particularly sensitive to antibiotics especially those active against gram-positive organisms (young et al., ) . as in man, these drugs are believed to alter the intestinal flora, permitting overgrowth of clostridium difficile as well as gram-negative organisms, resulting in a severe and frequently fatal enterocolitis. a study of the disposition of ampicillin administered parenterally to guinea pigs showed that this drug was rapidly eliminated from the systemic circulation and excreted in urine and bile, possibly favouring this effect on flora in the colon (young et al., ) . citrobacter freundii, a gram-negative, short, plump rod and member of the family of enterobacteriaceae, is the causative agent of naturally occurring transmissible colonic hyperplasia of mice. this agent usually produces a mild or even asymptomatic enteritis in susceptible mouse populations, although it is a cause of rectal prolapse in mice (ediger et al., ) . marked strain differences have been noted in mice infected with this organism. nih swiss mice show the most severe histological changes and c bl/ j mice appear the least affected (barthold et al., ) . rats and hamsters seem to be unaffected by citrobacter freundii (barthold et al., ) . microscopic changes are found primarily in the descending colon, although proximal segments of the colon and the caecum may also be affected. an important morphological feature is epithelial hyperplasia, which occurs maximally - weeks after experimental inoculation with citrobacter freundii (barthold et al., ) . the colonic glands are elongated and lined by cells that show mucin depletion or loss of goblet cells, considerable immaturity and mitotic activity. the surface epithelium may be covered with numerous coccobacilli, which can be visualised in routine haematoxylin, and eosin stained sections. crypt abscesses, inflammatory cells in the lamina propria, mucosal erosions and ulceration are also features (barthold et al., ; . in regressing lesions there is a rebound increase in goblet cells, which are often distended with mucin. the colonic glands may be branched or irregular (barthold et al., ) . most laboratory animals are naturally resistant to shigella infections but this is not the case for most non-human primates (takeuchi, ) . in infections with shigella, the colon shows a superficial acute inflammatory reaction comprising oedema, congestion, haemorrhage and infiltration by acute inflammatory cells. the surface epithelium shows mucin loss and formation of microulcers where total destruction of the epithelium has occurred. ulcers can extend into the lamina propria but in general terms the inflammatory process remains relatively superficial (takeuchi, ) . organisms are also located predominantly in the superficial epithelium. another bacterial infection of the gastrointestinal tract, which affects the colon in primates, is that produced by non-tuberculous mycobacteria . large intestinal lesions are characterised by massive accumulation of epitheloid macrophages in the lamina propria, which may extend into the submucosa and muscular layers and along lymphatics to involve mesenteric lymph nodes. small intestinal lesions may also occur, characterised by the presence of similar large macrophages in the lamina propria of villus tips. superficial ulcers may occur in severely affected segments of intestine . acid-fast bacteria are typically found within macrophages. other organs, including spleen, liver, bone marrow and lungs, may also be involved by focal accumulations of bacteria-laden macrophages or occasionally discrete granulomas with multinucleated giant cells. numerous protozoa and metazoa have been described as inhabitants of the caecum and colon of the non-human primate (toft, ) . far fewer are observed in the usual laboratory rodents and beagle dogs. amoebiasis caused by entamoeba histolytica is a widespread disease among non-human primates. it is characterised histologically by the presence of necrotizing ulcers, which reach the muscularis mucosa to form typical flank-shaped ulcers containing or surrounded by trophozoites. extensive haemorrhage may be seen as well as an inflammatory infiltrate composed of neutrophils and mononuclear cells (toft, ) . the ciliate, balantidium coli, can also cause an ulcerative process in the colon of primates, characterised by ulcers which extend down to the muscularis mucosa accompanied by lymphocytic infiltrate and balantidium coli trophozoites of up to µm in greatest diameter (toft, ) . a variety of metazoan parasites can be observed in the primate colon and usually can be reasonably well identified in tissue sections (see review by chitwood and lichtenfels, ) . the nematode of species strongyloides is an important parasite, which may be observed in the intestinal mucosa of primates. oxyurids commonly known as pinworms are essentially innocuous parasites seen in man, non-human primates and rodents. enterobius vermicularis is found in the large intestine and appendix of man and non-human primates, syphacia muris and syphacia obvelata in rodents. oesophagostomum species (nodular worms) are especially common nematode parasites of non-human primates forming characteristic nodules up to mm diameter most frequently on the serosal surface of the large intestine and caecum and adjoining mesentery as well as in other sites in the peritoneal cavity. histologically, the nodules are composed of parasite cell debris surrounded by fibrous tissue and a variable mantle of chronic inflammatory cells and occasional foreign-body giant cells. they are frequently found in close proximity to small arteries and arterioles in the submucosa and subserosa of the colon and may be associated with a local granulomatous arteritis (lumb et al., ) . the inflammatory process may spread to surrounding or draining tissues, particularly if nodules rupture. mild periportal hepatic chronic inflammation is sometimes associated with the presence of this parasite in the mesentery, which may confound interpretation of drug-induced hepatic changes in the non-human primate. although the stomach and to a certain extent the small intestine remain the primary sites of predilection for the ulcerogenic action of non-steroidal anti-inflammatory, the colonic mucosa may become involved under certain conditions. less common complications of non-steroidal anti-inflammatory drugs and potassium chloride therapy in humans are colonic strictures. it appears that nonsteroidal anti-inflammatory drugs produce local inflammation followed by focal scarring of the submucosa with constriction and formation of a mucosal diaphragm whereas potassium causes segmental full thickness scarring and constriction (fellows et al., ; haque et al., ; van velzen et al., ; wolfe et al., ) . another form of induced colon damage has been reported in children with cystic fibrosis, the majority of who take high strength pancreatic-enzyme supplements to control malabsorption (smyth et al., ; fitzsimmons et al., ) . this condition has distinctive pathological features. there is involvement a long segment of ascending colon by a fusiform stenosis primarily as a result of submucosal thickening by deposition of mature collagen. the mucosa appears relatively spared but shows some ulceration and reparative changes (van velzen et al., ) . although it has been suggested that the changes may have been linked to the methylacrylate copolymer used for enteric coating of the high-strength preparations, a case-control study showed a strong relation between high daily doses of the enzyme supplements, accentuated by more recent availability of high dose forms (fitzsimmons et al., ) . in view of their usage for over years, preclinical data on this material is scarce. colonic damage can be induced experimentally by administration of therapeutic agents. dogs administered . mg/kg indomethecin orally each day for periods of up to days developed not only gastric and small intestinal ulceration but also scattered haemorrhagic erosions in the colon and rectum. histologically, these lesions were characterised by loss of superficial epithelial cells, mucus-depletion of glandular epithelium, crypt abscesses, frequently with acute inflammation in adjacent lymphoid aggregates in the submucosa (stewart et al., ) . an example of chemically induced colitis of relevance to safety assessment of therapeutic agents is that induced by degraded carrageenans or synthetic sulphated dextrans. carrageenans are a heterogeneous group of sulphated polysaccharides composed mainly of long chains of d-galactose subunits (d-galactan) derived from red seaweed species which are widely used as food emulsifiers, stabilisers, thickeners and gelling agents (ishioka et al., ) . when carrageenans are degraded by acid hydrolysis into smaller molecular weight fragments of about , - , and administered orally in high doses (e.g. % of diet) to rats, mice, guinea pigs, rabbits and rhesus monkeys, colitis results (sharratt et al., ; marcus and watt, ; benitz et al., ; fath et al., ; kitano et al., ) . similarly, colitis has been induced in rats following administration of a % dietary admixture of dextran sulphate sodium, a sulphated polymer of glucose (a d-glucose) of molecular weight of , (hirono et al., ) and a very high molecular weight d-glucan, amylopectin sulphate (ishioka et al., ) . although histological features of this form of induced colitis vary between study, species and strain, the colitis is generally characterised mucosal ulceration mainly in the caecum but also in the distal ileum, distal colon and rectum. there is mucus-depletion with variable acute inflammatory infiltrate of the in-tact epithelium, crypt abscesses, inflammatory infiltrate of the lamina propria with oedema, hyperaemia and even vascular thrombosis in the submucosa (hirono et al., ; fath et al., ) . increased proliferative activity of the mucosa is confirmed by an increase in the tritiated thymidine index compared with controls (fath et al., ) . in the caecum of rats, ulcers are linear but often circulating the entire circumference of the intestinal wall with subsequent scarring and stricture formation (oohashi et al., ) . ulcerating lesions in the rectum and at the anal margin are associated with squamous metaplasia. both the squamous metaplasia and the regenerative hyperplasia of the columnar epithelium have been shown to progress even after cessation of treatment (oohashi et al., ) . foamy macrophages containing metachromatic material, presumably polysaccharide, are also seen in the lamina propria, submucosa, regional lymph nodes, liver and spleen (hirono et al., ; oohashi et al., ) . the cause of this colitis is unclear. low dose levels, which may be expected to mimic human exposure, do not produce colitis. dextrans, carrageenans and other polysaccharides of molecular weights outside the range , - , tend not to incite colitis. an exception to this is the agent amylopectin sulphate, which has a far higher molecular weight. however, amylopectin is composed of polysaccharide chains, which can be degraded by amylase, and therefore smaller molecular weight fragments may be formed in vivo (ishioka et al., ) . it has been suggested that colonic disease produced by these agents is in some way linked to induced changes in intestinal microflora (marcus and watt, ) although the evidence for this is conflicting (ishioka et al., ) . a recent study in guinea pigs and rats using permeability markers of different molecular weights has suggested that degraded carrageenans enhance intestinal permeability in the absence of overt ulceration (delahunty et al., ) . it was therefore proposed that carrageenan-induced colitis could be the result of increased intestinal permeability to antigenic or inflammatory substances normally resident in the large intestine. moreover, long-term administration of high doses of these agents to rats leads to the development of colorectal cancer despite their being devoid of any mutagenic potential (see below). the only obvious features, which are common to a number of these non-genotoxic agents, is chronic inflammation and increased proliferative activity. the rectal administration of therapeutic agents and surfactants may also induce similar ulcerative and inflammatory changes. chemical colitis resembling pseudomembranous colitis has been reported in man as a result of chemical cleaning agents accidentally induced by endoscopic examination (jonas et al., ) . cellular degeneration, with loss of mitotic activity and mucin depletion can also occur in the colon following treatment with antimitotic drugs. lymphoid infiltrates without tissue damage were reported in the large bowel of rats treated with human recombinant interleukin- (anderson & hayes ) . melanosis coli is a well-described phenomenon in man associated with chronic ingestion of anthraquinone purgatives. it is considered to be due to the excessive accumulation of lipofuscin-like pigment in the macrophages of the colonic lamina propria (schrodt, ; ghadially and parry, ; steer and colin-jones, ) . this pigment probably originates from organelles of epithelial cells or macrophages, which are damaged by treatment. similar morphological changes have been induced in laboratory animals (guinea pigs) by treatment with anthraquinones (walker et al., ) . as a result of these animal studies, walker et al. ( ) suggested that the primary process is a treatment-induced increase in apoptotic bodies in the surface colonic epithelium that are phagocytosed by intraepithelial macrophages and transported to the lamina propria. lipofuscin and iron pigment is occasionally observed in the lamina propria of untreated rodents, notably hamsters, presumably a result of ageing, previous inflammatory processes and haemorrhage. as in other glandular epithelial tissues, hyperplasia may be focal or diffuse with or without atypical cellular features. the term used for hyperplasia with atypical features is atypical hyperplasia in the iarc classification (mohr, ) although others use the term dysplasia. like small intestine, cell proliferation in the large intestinal mucosa can be stimulated by a variety of different factors although these functional adaptive responses have been less well studied. physical stimulation by distension or increased dietary bulk is sufficient to initiate hyperplasia including thickening of the muscle coats (dowling et al., ; stragand and hagemann, ) . one of the most clearly documented forms of compensatory hyperplasia is that which occurs as a response to surgical resection or bypass of a segment of the colon. following resection of a segment of colon in rats, barkla and tutton ( ) showed that the remaining proximal segment of the right side of the colon showed an increase in the thickness of the mucosa and the muscularis externa as well as enlargement of lymphoid aggregates. histologically, the mucosa of the right side of the colon was uniformly thickened showing accentuated folds, elongated mucosal glands with increased height of the surface columnar cells. the changes were most marked up to days following surgery but were less pronounced after days. there was also a significant increase in the mitotic index in the proximal segment at days although at days and later the mitotic index had returned to normal. the distal, down-stream segment showed little or no morphological change but rather a long-lived increase in mitotic activity. it was suggested that these differences were related to the different embryological origin of the segments (barkla and tutton, ) . a similar form of uniform colonic hyperplasia affecting principally the caecal and right-sided colonic mucosa also occurs in rats following oral administration of sulphated dextrans of molecular weight of approximately , (figs and ). oral administration of a wide range of compounds such as raw and chemically modified starches, various dietary fibres, caramels, sugar alcohols (lactitol, sorbitol, mannitol, xylilol), lactose, a synthetic polydextrose, polyethylene glycol and magnesium sulphate to rats or hamsters has also been linked to an increase caecal size and colonic mucosal hyperplasia (leegwater et al., ; roe and bär, ; newberne et al., ; stark et al., ) . the characteristic histological appearance of the caecum following administration of these agents is lengthening of the mucosal glands which are lined by epithelium composed of increased numbers of enlarged epithelial cells (i.e. hypertrophy and hyperplasia) showing increased proliferative activity and more rapid incorporation of tritiated thymidine (newberne et al., ) . in addition, mucosal and submucosal oedema has been reported in association with the administration of lactose and increased mucosal lymphoid aggregates following lactose or xylitol feeding (newberne et al., ) . changes in the colon due to fibre are complex. morphometric analysis has shown that changes to the mucosa depend on the fibre type (stark et al., ) . there may also be an interaction between dietary fibre content and colonic microflora that influences mucosal growth, although the mechanism is unclear (whiteley et al., ) . hypertrophy of the muscularis external is also reported in rats fed high fibre diets (stark et al., ) . as the increase in caecal size and mucosal hypertrophy appears generally related to the osmotic activity of the caecal contents in rodents treated with these agents, it has been postulated that the changes represent a physiological adaptation to increased osmotic forces, irrespective of the contributing compounds (leegwater et al., ) . treatment of rodents with antibiotics also causes caecal enlargement or dilatation without significant histopathological changes, probably as a result of changes in caecal microflora. it has been suggested that the enlargement relates to accumulation of urea as a result of inhibition of bacterial ureases (juhr and ladeburg, ) . however, histochemical studies of the intestinal mucosa of rats treated with neomycin have also shown treatment-related reductions in activities of nad tetrazolium reductase, succinate dehydrogenase, esterase, alkaline and acid phosphatase in the distal ileum, suggesting that some antibiotics also posses the potential to directly influence absorption and metabolic functions of mucosal cells (van leeuwen et al., ) . long-term administration of , -dimethyl prostaglandin e to rats also produced thickening of the proximal colonic mucosa, although this was less fig. . similar area of colonic mucosa to that seen in fig. at the same magnification but from a rat treated with % dextran (molecular weight , ) in the diet for weeks. this shows diffuse hyperplasia of the mucosa with elongation of colonic glands that are lined by relatively normal epithelial cells with abundant mucin and prominent vesicular nuclei. (he, × .) marked than in the stomach and duodenum (reinhart et al., ) and similar changes have been reported in rats treated with other prostaglandin e analogues (levin, ) . as in the small intestine, administration of epidermal growth factor to rats and cynomolgus monkeys induces hyperplasia of the colonic mucosa characterised histologically by hyperplasia and increased mitotic activity of crypt cells and reduction in goblet cell numbers with an increase in crypt depth and a slight increase in the numbers of infiltrating neutrophils (breider et al., ; reindel et al., ) . in common with other epithelial surfaces, atypical hyperplasia is associated with the development of colonic cancer in both man and laboratory animals. the early alterations observed in rats treated with colonic carcinogens are similar to those found in the immediate vicinity of human colorectal carcinomas. the changes are characterised by lengthening, dilatation and branching of glands. the epithelium lining these glands shows mucous cell hyperplasia (goblet cell hyperplasia, see fig. ), goblet cells containing predominantly sialomucin instead of the normal sulphomucin (filipe and branfoot, ; filipe, ; olubuyide et al., ) . despite mucin alterations, activities of glucose- -phosphatase, glucose- -phosphate dehydrogenase and gly- fig. . section from the colon of an aged hamster from a colony that developed intestinal inflammation and neoplasia spontaneously. this shows focal mucous cell hyperplasia characterised by enlargement and lengthening of the colonic glands with lining cells replete with mucins. (he, × .) ceraldehyde- -phosphate dehydrogenase were shown to be normal in this epithelium in rats treated with , -dimethylhydrazine (mayer et al., ) . in man, this form of hyperplastic mucosa associated with cancer, has been termed 'transitional mucosa' (filipe and branfoot, ) . as lesions become more atypical, these dilated, branched glands become more complex and lined by epithelium that shows increasing pseudostratification and vesicular cell nuclei. for example in rats treated with the carcinogens azoxymethane or , -dimethylhydrazine, crypts show diminution of mucus secretion, increased cytoplasmic basophilia, prominent, rounded or enlarged nuclei which show variable degrees of pseudostratification and which eventually develop into frankly invasive glands . in contrast to goblet cell hyperplasia, these atypical zones show increased activity of glucose- -phosphate, glucose- -phosphate dehydrogenase and glyceraldehyde- -phosphate dehydrogenase (mayer et al., ) . described similar alterations in rats treated with azoxymethane and the non-genotoxic agent dextran sulphate. these authors also demonstrated that these atypical foci could be identified by low power microscopy as aberrant crypt foci by translumination of the whole mounts of the fixed mucosa stained with methylene blue. note: some compounds may induce qualitative and quantitative changes in mucin content in the colonic mucosa without marked morphological alterations. an example of this phenomenon was described in rats treated with reserpine for days. the colonic mucosa showed an increase in sulphomucin (high-iron diamine positive) containing goblet cells in the surface epithelium (park et al., ) . adenomas and adenocarcinomas of the small and large intestine are infrequent spontaneous neoplasms in laboratory animals compared with man where colorectal carcinoma is one of the most prevalent neoplasms in the western world. adenomas and adenocarcinomas probably occur spontaneously in older dogs more than in any other animal species and as in man these are located most frequently in the distal colon and rectum (lingeman and garner, ) . in nonhuman primates glandular neoplasms of the intestine occur with increasing age in the ileum and in the colon with a predilection for the zones near the ileocaecal valve (depaoli and mcclure, ) . in rats and mice, spontaneous intestinal neoplasms are uncommon although adenocarcinomas are occasionally observed in the ileum or colon in mice and rats used in chronic toxicity studies and carcinogenicity bioassays (burn et al., ; wells, ; maeda et al., ; greaves and faccini, ; zwicker et al., ) . most of these arise in the small intestine and appear to originate in the distal part of the small intestine, caecum and right side of the colon. they may produce metastases, mostly to liver and lungs. in a review of spontaneous adeno-carcinomas developing over a -year period in wistar rats, vandenberghe et al. ( ) identified adenocarcinomas, all in ascending colon. in of these cases there appeared to be an intimate relationship with campylobacter-like organisms together with diverticulae and chronic inflammation. these authors suggested that the organisms and the associated inflammation were involved in the pathogenesis of these cancers. in view of the importance of colon cancer in humans, a number of new genetic mouse models predisposed to colon cancer have been developed over recent years (heyer et al., ) . some hamster colonies, liable to develop inflammatory bowel disease (see above), also have a high incidence of small and large intestinal polyps, adenomas and adenocarcinomas (fortner, ; van hoosier et al., ; personal observations) . poorly differentiated carcinomas may infiltrate local lymph nodes and it may be difficult to locate the primary neoplasm. polyps are predominantly adenomatous in nature although inflammatory or regenerative polyps are observed (van hoosier et al., ) . adenocarcinomas are induced experimentally in the rodent intestine by the carcinogens , -dimethylhydrazine and azoxymethane. the histogenesis of these induced carcinomas has been extensively studied and it is generally accepted that they resemble human colorectal cancer (ward, ; freeman, ) . however, there are differences between reported studies. some have shown that these experimental carcinomas arise from pre-existing adenomas consistent with the 'adenoma-carcinoma sequence' theory (ward, ; ward et al., ) . others suggest that they arise 'de novo' from altered mucosa as microinvasive carcinomas (sunter et al., ; maskens and dujardin-loits, ; rubio et al., ) . these differences may be partly the result of different dosage schedules. rubio et al. ( ) have shown that a single dose of , -dimethyhydrazine produces non-polypoid, micro-invasive carcinomas, particularly in the mucosa overlying lymphoid aggregates, whereas in their earlier studies using multiple doses in the same strain of rat, an adenoma-carcinoma sequence was more evident. in addition, there are undoubtedly species and strain differences in the response to these agents. teague et al. ( ) demonstrated clear differences in the distribution and both macroscopic and histological types of adenomas and adenocarcinomas between three different inbred strains of rat given a similar dosage regimen of , -dimethylhydrazine. in general, many of these carcinomas develop in the distal colonic segments similar to the distribution of human colorectal cancer, although tumours also develop in the proximal colon and in the ileum in rats treated with this agent (ward, ; teague et al., ) . neoplasms occurring in the rat colon following administration of high doses of degraded carrageenans and sulphated dextran also commonly occur in the distal colon and rectum and are commonly polypoid adenomas and adenocarcinomas (hirono et al., ; oohashi et al., ; ishioka et al., ) . however, in these models adenomas and adenocarcinomas also occur in the caecum and proximal colon and squamous carcinomas are sometimes seen in association with squamous metaplasia at the colorectal junction (oohashi et al., ) . the pathogenesis of neoplasms induced by carrageenans and dextrans remains unexplained. although they are biologically active compounds, they are non-mutagenic in the usual short-term tests (ishioka et al., ) . it has been suggested that carrageenans act as tumour promoters as they potentiate the appearance of carcinomas in rats treated with standard intestinal carcinogens hirono et al., ) . conversely it has been proposed that these agents are tumour initiators based on the development of carcinomas in rats treated with degraded carrageenans for only months (oohashi et al., ) . however, despite only a short period of treatment, inflammation, regenerative changes and squamous metaplasia persisted throughout a period of months after treatment was withdrawn before development of cancer in these rats. the only consistent association of carrageenans with development of carcinoma in rats is that of chronic inflammation and increased cell proliferation. although dose levels needed to produce inflammation are far higher than any exposure likely to be achieved in man, interpretation of this inflammation-cancer sequence in rats remains a challenge in safety assessment for similar xenobiotics. this situation is interest in view of the unquestionable risk of carcinogenesis in ulcerative colitis in man (riddell et al., ) . a similar range of neoplasms can be defined histologically in both human and experimental pathology. it is appropriate, to use the same classification for all species including man. lingeman and garner ( ) who reviewed a range of tumours from domestic and laboratory animals were able to employ the classification for human gastrointestinal neoplasms. a similar approach has been used in the iarc classification of rat intestinal tumours (mohr, ) . this classification can be summarised as follows: these represent localised, sessile or polypoid neoplasms composed of proliferating tubular glands, which show varying degrees of nuclear hyperchromatism, pseudostratification and cellular pleomorphism. a useful scheme for grading the carcinogenic potential of hyperplastic mucosa and adenomatous polyps in man based on the degree of epithelial pseudostratification has been proposed by kozuka ( ) . although experimental neoplasms may not always show the full spectrum of these changes reported in man, this grading provides a useful baseline concept for the assessment of these non-invasive proliferative lesions. with increased nuclear pseudostratification and atypical branching of the glandular structures of these polyps becomes more prominent. if neoplastic cells or glands are seen in the stroma of the stalk or base the diagnosis of carcinoma is made. villous adenoma is a form of adenoma in which the epithelial proliferation takes the form of elongated villi with a sparse fibrovascular stroma. they can be graded in a similar way to other adenomas. these are glandular neoplasms of variable differentiation, sometimes originating in adenomatous polyps or villous adenomas but which show infiltration of the intestinal wall, i.e. beyond the boundary of the muscularis mucosa. squamous carcinomas also occur in the anorectal zone but are similar to those which occur in squamous epithelium elsewhere. similarly, mesenchymal neoplasms also are found in the small and large intestinal wall (see integumentary system, chapter i). light and electron microscopical studies of parietal cells before and one year after proximal vagotomy in duodenal ulcer patients six-month repeated oral toxicity study of nk- in rats response of the non-human primate to polychlorinated biphenyl exposure cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats induction of early lesions in the forestomach of rats by -tert-butyl- -hydroxyamisole (bha) effects of caecetomy in the young adult female rat on digestibility of food offered and libitum and in restricted amounts toxicity of human recombinant interleukin- in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement effects of cimetidine, cimetidine plus nitrite, and nitrosocimetidine on tumors in mice following transplancental chronic lifetime exposure age-associated lesions in barrier-reared male sprague-dawley rats: a comparison between hap: (sd) and crl:cobs[r] cd[r] (sd) stocks expression of cd in normal and metaplastic paneth cells of the digestive tract an epizootic of klebsiella aerogenes infection in laboratory rats correlation of quantitative changes of gastric mucosal glycoproteins with aspirin-induced gastric damage in rats long-term comparative effect cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas the effect of -hydro-xydopamine on rat salivary glands and on their response to isoproterenol biologically active peptides in submandibular glands the alimentary system proliferative and morphologic changes in rat colon following bypass surgery cyclosporin and gingival overgrowth the etiology of transmissible murine colonic hyperplasia dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia transmissible murine colonic hyperplasia mouse hepatitis virus infection, intestine, mouse murine rotavirus infection, intestine, mouse adenovirus infection, intestine, mouse clindamycinassociated colitis due to a toxin-producing species of clostridium in hamsters antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia histological variations jejunal and ileal mucosa on days and after hypophysectomy in rat: morphometric analysis in light and electron microscopy comparative study of histological and kinetic variations of the digestive mucosa and pancreatic parenchyma after hypophysectomy in the rat effect of drugs on rats exposed to cold-restraint stress adverse effects of anticonvulsant drugs: a critical review formation of n-mono-nitrosopiperazine in the stomach and its secretion in the urine after oral intake of piperazine intestinal effects of carrageenans in the rhesus monkey (macaca mulatta) the cell surface: components and configurations ménétrier's disease. serial morphological, secretory, and serological observations structure, biosynthesis and functions of glycoprotein glycans. experientia pathology of the forestomach in rats treated for year with a new histamine h -receptor antagonist, sk&f trihydrochloride fundic mucosal ecl cell hyperplasia and carcinoids in rodents following chronic administration of the histamine h -receptor antagonist sk&f and other antisecretory agents gastric ecl-cell hyperplasia and carinoids in rodents following chronic administration of the h antagonist sk&f and oxmetidine and omeprazole gastric regulatory peptides in rats with reduced acid secretion non-steroidal anti-inflammation in humans immunohistologic analysis of the t-cell and macrophage infiltrate in , -dimethylhydrazine-induced colon tumors in the rat turnover of brush-border glycoproteins in human intestinal absorptive cells: do lysosomes have regulatory function? alterations in gastric mucosal morphology induced by long-term treatment with omeprazole in rats the effect of aging on the rat submandibular gland. an ultrastructural, cytochemical and biochemical study drug-induced esophageal strictures cytochrome p of small intestinal epithelial cells. immunocytochemical characterization of the increase in cytochrome p caused by phenobarbital synergistic role of intestinal flagellates and normal intestinal bacteria in a post-weaning mortality of mice medication-induced oesophageal injury. survey of the literature resistance to starvation in albino rats fed from weaning on diets containing from to % of protein as casein diseases of the kidney the human gastrointestinal secretory immune system in health and disease clinical aspects: an overview single-dose and multiple-dose intravenous toxicity studies of bmy- in rats cellular hyperplasia in rats following continuous intravenous infusion of recombinant human epidermal growth factor adrenergic mechanisms responsible for submandibular salivary glandular hypertrophy in the rat aspirin: intestinal damage in rats gastrointestinal mucosal lesions: a drug formulation problem intestinal t lymphocytes of different rats strains in immunotoxicity effects of propionic acid and pravastatin on hmg-coa reductase activity in relation to forestomach lesions in the rat famotidine: summary of preclinical safety assessment effects of cholestyramine and diet on small intestinal histomorphology in rats spontaneous carcinoma of the colon of the rat cresyl fast violet staining method for campylobacter-like organisms pigmentation of the jawbone and teeth secondary to minocycline hydrochloride therapy genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa esophageal lesions caused by orally administered drugs. an experimental study in the cat tetracycline induced esophageal ulcers. a clinical and experimental study diagnosis of silodacryoadenitis virus infection of rats in a virulent enzootic outbreak cryptosporidium species a 'new' human pathogen thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse. a fine structural immunocytochemical study renewal of the epithelium in the descending colon of the mouse. i. presence of three cell populations: vaculated-columnar, mucous and argentaffin two types of mucous cells in the colon crypt origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. iii entero-endocrine cells cytology of the canine oral papilloma uremic gastropathy in the dog intestinal absorption and metabolism of xenobiotics in laboratory animals parasitological review. identification of parasitic metazoa in tissue section spontaneous basophilic hypertrophic foci of the parotid glands in rats and mice effects of housing conditions on food intake, body weight and spontaneous lesions in mice. a review of the literature and results of an -month study cryptosporidiosis in the intestines of rhesus monkeys (macaca mulatta) isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal post marketing surveillance of the safety of cimetidine: mortality during second, third, and fourth years of follow-up hyperplastic gastropathy in the rat due to taenia taeniaeformis infection: parabiotic transfer and hypergastrinaemia. gastroenterology number, size and distribution of peyer's patches in the human small intestine a model for gastric cancer epidemiology helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention the effects of vagotomy on the gastric mucosa of the rat the effect of prolonged administration of large doses of cimetidine on the gastric mucosa of rats effect of short-and long-term feeding of omeprazole on rat gastric endocrine cells clostridial enterocolitis produced by antineoplastic agents in hamsters and humans odontogenic tumours in fischer rats the histo-chemical demonstration of o-acylated sialic acid in gastrointestinal mucins: their association with the potassium hydroxide-periodic acid-schiff effect a new histochemical method for the identification and visualization of both side chain acylated and non-acylated sialic acids amiodarone keratopathy, drug-induced lipid storage disease salivary gland components involved in the formation of squamous metaplasia gastric mucosal injury by fatty and acetylsalicylic acids cryptosporidosis and proliferative ileitis in a hamster specificity of twelve lectins towards oligosaccharides and glycopeptides related to n-glycosylproteins intestinal permeability changes in rodents. a possible mechanism for degraded carageenan-induced colitis adrenal corticosteroids cause gastrin cell hyperplasia possible role of transforming growth factor alpha in the pathogenesis of menetrier's disease: supporting evidence from humans and transgenic mice the effect of hydrocortisone and cortisone on fixation of s in the stomach the effect of phenylbutazone and its derivatives, oxyphenbutazone and sulfinpyrazole, on s sulfate incorporation in cartilage and stomach salivary glands: a paradigm for diversity of gland development gastrointestinal neoplasms in non-human primates: a review and report of new cases gastric gland degeneration induced in monkeys by the cck-b/gastrin receptor antagonist ci- the effect of aspirin on small intestinal mucosa morphologic aspects of lipid absorption gastric and gastric epithelial physiology two-year evaluation of misprostol for carcinogenicity in cd sprague-dawley rats distribution and incidences of calcified lesions in dba/ ncrj and balb/canncrj mice interaction of phenytoin and inflammation induces gingival overgrowth in rats the intestinal response to high bulk feeding in the rat comparison of canine and human gastrointestinal physiology stress ulceration-clinical relevance of animal and human studies pathology of laboratory rats and mice effect of chronic aspirin ingestion on epithelial proliferation in rat fundus, antrum and duodenum the prognostic value of sulphomucin positive intestinal metaplasia in the the development of gastric cancer colitis in mice with high incidence of rectal prolapse volatile nitrosamine contamination of laboratory animal diets toxicological studies on omeprazole possible role of cimetidine and its nitrostated products in human stomach cancer cimetidine and gastric cancer tumours of the oral cavity, check pouch, salivary glands, oesophagus, stomach and intestines differential distribution of lymphocytes and accessory cells in mouse peyer's patches phenotypically distinct subpopulations of t cells in domes and m-cell pockets of rabbit gut-associated lymphoid tissues morphometric analysis of the small intestinal epithelium in the indomethacin-treated mouse expression of pokeweed lectin binding in murine intestinal paneth cells heterogeneity of m-cell-associated b and t cells in human payer's patches degraded carrageenan-induced colitis in cf mice. a clinical, histo-pathological and kinetic analysis substituted benzimidazoles inhibit acid secretion by blocking (h ++ k + ) atpase nonsteroidal anti-inflammatory drug induced jejunal and colonic diaphragm disease: a report of two cases effect of chronic misoprostol ingestion on rat gastric morphology and turnover abnormal patterns of mucous secretion in apparently normal mucosa of large intestine with carcinoma mucous secretion in rat colonic mucosa during carcinogenesis induced by dimethylhydrazine. a morphological and histo-chemical study mucins in the human gastrointestinal epithelium: a review. invest high-dose pancreatic-enzyme supplements and fibrosing colonopathy in children with cystic fibrosis the number of villi in rat's jejunum and ileum: effect of normal growth, partial enterectomy and tube feeding spontaneous tumors including gastrointestinal neoplasms and malignant melanoma, in syrian hamster aspirin, paracetamol and non-steroidal anti-inflammatory drugs. a comparative review of side effects campylobacter jejuni/coli in commercially reared beagles. prevalance and serotypes antigen specificity and morphological characteristics of chlamydia trachomatis, strain sfpd, isolated from hamsters with proliferative ileitis acquired salivary dysfunction. drugs and radiation gastritis cystica profunda application of lectins for detection of goblet cell glycoconjugate differences in proximal and distal colon of the rat lectin histochemistry of , -dimethylhydrazine-induced rat colon neoplasia effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption morphological aspects on pancreatic islets of non-obese diabetic (nod) mice carcinoma and related lesions in dog stomach induced by oral administration of n-methyl-n´-nitro-n-nitrosoguanidine cryptosporidiosis in a pup with distemper. vet.pathol squamous cell carcinoma, forestomach, rat adverse effects of mouthwash use. a review. oral surg.oral med.oral pathol tyzzer's disease, intestine, mouse, rat, hamster salmonellosis, intestine, mouse, rat, hamster adequate substitution with electrolytes in toxicological testing of 'loop' diuretics in the dog the forestomach in rats and mice, a food store without bacterial protein digestion m cells in peyer's patches of the intestine histochemie de la muqueuse gastrique fundique du chien traité par des drogues ulcérigène an electron-microscope and histo-chemical study of melanoisis coli mitochondria. in: ultrastructural pathology of the cell and matrix sustainability of forestomach hyperplasia treated with ethyl acrylate for weeks and regression after cessation of dosing the cytochemical localization of lysozyme in paneth cell granules regional differences in glycoconjugates of intestinal m cells in mice: potential targets for mucosa vaccines susceptibilities of drug to nitrosation under simulated gastric conditions features of small intestinal pathology (epithelial cell kinetics, intra-epithelial lymphocytes, disaccharidases) in a primary giardia muris infection staining rickettsiae in yolk sac cultures alterations of gastric mucosa following a graded partial gastrectomy tumours of the salivary glands the oral cavity aristolochic acid is a direct mutagen in s. typhimurim studies of the binding of trypsin and chymotrypsin by human intestinal mucosa study of cold plus restraint stress gastric lesions in spontaneously hypertensive, wistar and sprague-dawley rats mucins in normal and neoplastic gastrointestinal epithelium the lectins: carbohydrate-binding proteins of plants and animals prolactin and ergocryptine effects mucus glycoproteins of the rat ileum tumours of the jaws hypopigmentary changes with a platelet aggregation inhibitor (abstract no. ). fed.proc drug induced enteropathy characterized by lipid in macrophages altered patterns of mucin secretion in gastric hyperplasia in mice digestive system. in: rat histopathology. a glossary for use in toxicity and carcinogenicity studies k virus infection, intestinem mouse a silver nitrate stain for alpha- cells in human pancratic islets silver stains in the study of endocrine cells of the gut and pancreas effects of antrectomy or porta-aval shunting on the histamine-storing endocrine-like cells in oxyntic mucosa of rat stomach. a fluorescence histochemical, electron microscopic and chemical study the vagus exerts trophic control of the stomach in the rat activation and hyperplasia of gastrin and enterochromaffin-like cells in the stomach gastrin and the trophic control of gastric mucosa onkocytes and so-called hürthle cell tumor interaction of microorganisms, epithelium, and lymphoid cells of the mucosa-associated lymphoid tissue dog and swine as models for testing indomethacin-induced gastrointestinal irritation sialodacryoadenitis in the rat: effects of immunosuppression on the course of the disease role of gut in zenobiotic metabolism epithelial cell kinetics in the small intestine of the rat days after resection of percent of the ileum and jejunum compensation by the residual intestine after intestinal resection in the rat proceedings of the lst international symposium on omeprazole a cecal diaphragm associated with the use of nonsteroidal anti-inflammatory drugs chronic gastritis of the glandular stomach, adenomatous polyps of the duodenum, and calcareous pericarditis in strain dba mice idiopathic megaoesophagus in rat immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment spontaneous neoplasm incidences in fischer rats and b c f mice in two-year carcinogenicity studies: a national toxicology program update diphenyldydantoin (dilantin) gingival hyperplasia: drug-induced abnormality of connective tissue on cell proliferation and differentiation of the fundic mucosa of the golden hamster. fractographic study combines microscopy and h-thymidine autoradiography tritiated thymidine autoradiographic study on cellular migration in the gastric gland of the golden hamster enterochromaffin-like cell carcinoids of gastric mucosa in rats after life long inhibition of gastric secretion gastric cancer after cimetidine in a patient with two negative pre-treatment biopsies induction of experimental allergi sialadenitis in mice spontaneous development of auto-immune sialodenitis in aging bdf mice i mmunofluorescent localization of enterokinase in human small intestine mouse models for colorectal cancer morphologic changes in the urinary bladder and stomach after long-term administration of sodium saccharin in f rats lessons from genetically engineered animal models iii. lessons learned from gastrin gene deletion in mice immunohistological characterization of intra-epithelial and lamina propria lymphocytes in control ileum and colon and inflammatory bowel disease induction of intestinal tumors in rats by dextran sulphate sodium gastric enterochromaffin-like hyperplasia and neoplasia in the rat: an indirect effect of the histamine h -receptor antagonist bl- oxidative metabolism of foreign compounds in rats small intestine: cellular localization and dependence on dietary iron clinicopathological studies of gastrointestinal disease in macaques non-tuberculous myobacterial disease in rhesus monkeys on human papillomaviruses the laboratory rat. biology, diseases inhibition of intestinal protein synthesis and lipid transport by ethionine experimental toxicity studies with captopril, an inhibitor of angiotesin -converting enzymes . one month studies of chronic toxicity of captopril in rats development of spontaneous tongue calcification and polypoid lesions in dba/ ncrj mice changes in enzyme levels accompanying differentiation of intestinal epithelial cells textbook of endocrinology acute disease of the submaxillary and harderian glands (sialodacryoadenitis) of rats with cytomegaly and no inclusion bodies cellular kinetics of gastrointestinal mucosa, with special reference of gut endocrinecells changes of gastric mucus glycoproteins with aspirin administration in rats induction of colorectal tumours in rats by sulpated polysaccharides induction of duodenal tumors in mice by oral administration of hydrogen peroxide carcinogencity of butylated hydroxyanisole in f rats modifying effects anti-oxidants on chemical carcinogenesis a week feeding study of butylated hydroxyanisole: the subsequent regression of the induced lesions in male fischer rat forestomach epithelium an -day study of butylated ydroxyanisole in the cynomolgus monkey subchronic studies of doxylamine in fischer rats effects of dietary fiber on mucosal growth and cell proliferation in the small intestine of the rat: a comparison of oat, bran, pectin, and guar with total fiber deprevation transmissible ileal hyperplasia, hamster anatomic adaption of the alimentary tract of the rat to the hyperphagia of chronic alloxan-diabetes a variant of intestinal metaplasia associated with gastric carcinoma: a histochemical study role of intestinal metaplasia in the histogenesis of gastric carcinoma colorectal mucin histochemistry in health and disease: a critical review uptake of particulate and soluble antigens in the small intestines of the rat chemical colitis due to endoscopic cleaning solutions: a mimic of pseudomembranous colitis pathology of domestic animals intestinal accumulation of urea in germ-free animals: a factor in caecal enlargement digestive enzymes in the parotid and submandibular glands of mammals morphologishe veränderungen der magenmukosa von ratten nach chronischer antazidagabe enteric viruses of non human primates increased accumulation of sulfated glycoaminoglycans in cultures of human fibroblasts from phenytoin-induced gingival overgrowth new insights into the stem cells and the precursors of gastric epithelium colonic lymphoid-glandular complex (microbursa): nature and morphology lymphoid tissue and lymphoid-glandular complexes of the colon: relation to diverticulosis histology of salivary gland infarction in the dog adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse pill esophagitis immunogold localization of ingested kidney bean (phaseolus vugaris) lectins in epithelial cells of the rat small intestine epithelial dysplasia of the rabbit colon induced by degraded carrageenan hyperkeratinization and hyperplasia of the forestomach epithelium in vitamin a deficient rats intrinsic resistance of colon tumors to anthrapyrazoles and antracyclines may be linked with a detoxification mechanism of intestinal cells (abstract no. ) studies on the effects of -hydroxy- -methylglutaryl coenzyme a reductase inhibitors on the rodent forestomach reversibility of adenomatous hyperplasia in the gastric stump after diversion of bile reflux in rats experimental production of possible autoimmune gastritis followed by macrocytic anemia in athymic mice expression cloning and characterization of the canine parietal cell gastrin receptor histomorphologic investigations on the effect of cyclophosphamide on dentinogenesis of the rat incisor. scand preclinical toxicology profile of misoprostol premalignancy of the mucosal polyp in the large intestine: i. histologic gradation of the polyp on the basis of epithelial pseudostratification and glandular branching morphologic changes in the gastric mucosa of rats and dogs treated with an analog of prostaglandin e forestomach carcinogens: possible mechanisms of action lectin-peroxidase conjugates in histopathology of gastrointestinal mucosa the rat incisor in toxicologic pathology the pharmacology and toxicology of caffeine incorporation of radiosulfate in the gastric mucosa of the rat subjected to restraint antisecretory drugs and gastric cancer plasma gastrin and gastric enterochromaffin-like cell activation and proliferation. studies with omeprazole and ranitidine in intact and antrectomized rats effects of cyclosporin a administration in cats gingival hyperplasia associated with nifedipine therapy the aetiology of caecal enlargement in the rat effect de l'administration prolongée d'un antisécrétoire gastrique, le pirenzepin, sur les populations cellulaires de l'estomac de rat hepatocytes in the mouse stomach tumours of the oral cavity, pharynx, oesophagus and stomach a toxicological profile of cimetidine specific staining of sulphate groups with alcian blue at low ph the mucin histochemistry of normal and neoplastic gastric mucosa the effect of the thyroid gland on intestinal absorption of hexoses structural changes of the gastrointestinal mucosa induced by prostaglandins structural aspects of salivary glycoproteins drug-induced disorders of the esophagus histochemical observations on paneth cells diphtheria toxin-mediated ablation of parietal cells in the stomach of transgenic mice carcinogencity of methylated dinitro-sopiperazines in rats comparative study of intestinal adenocarcinoma of animals and man dental dysplasia in rats and mice chronic oral administration of -nitrosopiperazine at high doses to mrc rats nifedipine-induced gingival hyperplasia: a histochemical and ultrastructural study drug-induced lysosomal storage disorders oesophagostomiasis in feral monkeys (macaca mulatta) hyperalimentation in normal animals produced by protamine insulin effects of cytotoxic chemotherapy on dental development nutritional influences on aging of fischer rats: ii the use of rats associated with human faecal flora as a model for studying the effects of diet on the human gut microflora colonic ulceration in young rats fed degraded carrageenan unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration gut-associated lymphoid tissue and , -demethylhydrazine intestinal tumors in the rat: a histological and immunoenzymatic study leucocyte and bone marrow effects of a thiomorpholine quninazosin antihypertensive agent localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor effect of dosage form and formulation factors on the adherence of drugs to the esophagus morphologic changes of esophageal mucosa in the rat after chronic alcohol ingestion experimental adenomas and carcinomas of the large intestine behave as distinct entities: most carcinomas arise de novo in flat mucosa la glande endocrine de l'intestin chez l'homme differentiation of immature mucous cells into parietal, argryophil, and chief cells in stomach grafs sequential histochemical and morphometric studies on preneoplastic and neoplastic lesions induced in rat colon by , -dimethylhydrazine recovery from amiodarone-induced lipidosis in laboratory animals: a toxicological study parietal cell hyperplasia indued by long-term administration of antacids to rats methotrexate-induced changes in rat parotid and submandibular gland function a spontaneous outbreak of polychlorinated biphenyl (pcb) toxicity in rhesus monkeys (macaca mulatta): toxicopathology classical absorption theory and the development of gastric mucosal damage associated with non-steroidal anti-inflammatory drugs mesenteric lymphoblast localization throughout the murine small intestine: temporal analysis relating intestinal length and lymphoblast division morphologic lesions in ageing syrian hamsters histological identification of campylobacter using gimenez technique in gastric antral mucosal the carcinogenic action of aristolochic acid in rats rat immunoglobulins in serum and secretions: comparison of iga and igg in serum, colostrum, milk and saliva of protein malnourished and normal rats influence of ofloxacin on the faecal flora the structure, origin and function of mucosal mast cells. a brief review pseudomembranous colitis in a leukaemia unit: a report of five fatal cases formulation of n-nitroso compounds: chemistry kinetics, and in vivo occurrence the etiology of gastric cancer: intragastric nitro-samide formation and other theories forestomach lesions induced by butylated hydroxyanisole and ethylene dibromide: a scientific and regulatory perspective international classification of rodent tumours. part , the rat the 'swiss roll'. a simple technique for histological studies of the rodent intestine organization of an actin filament-membrane attachment in the microvilli of intestinal epithelial cells charaterization and localization of myosin in the brush border of intestinal epithelial cells development of the human gastrointestinal tract: twenty years of progress aroclor -induced intestinal metaplasia and adenocarcinoma in the glandular stomach of f rats immuno-histochemical localization of epidermal growth factor in rodent submandibular glands intestinal metaplasia of the gastric mucosa precancerous conditions and epithelial dysplasia in the stomach hypersensitivity reactions in the small intestine. . pathogenesis of the graft-versus-host reaction in the small intestinal mucosa of the mouse the distribution of acid mucopoly-saccharides in normal kidneys as shown by the alcian blue feulgan (ab-f) and alcian blue-periodic acid-schiff (ab-pas) stains gastric endocrine cell hyperplasia and carcinoid tumors in atrophic gastritis type a histochemical classification of acini and ducts of parotid glands from artiodactyles, carnivores and rodents a morphological and histrochemical study of a drug-induced enteropathy in the alderley park rat the mouse submandibular gland: an exocrine organ for growth factors dysplasia of the gastric mucosa and its relation to the precancerous state ultrastructure of dog parotid gland corticosteroid treatment increases parasite numbers in murine giardiasis duct-associated lymphoid tissue (dalt) of minor salivary glands and mucosal immunity minor salivary gland duct-associated lymphoid tissues (dalt) in monkeys, changes with age quantitative light and electron microscopic studies on the effect of vagotomy on parietal cells in rats toxicology and carcinogenesis studies of ampicillin trihydrate in f /n rats and b c f mice animal models for gastric helicobacter immunology and vaccine studies fundal gastritis after billroth-ii type resection in patients with duodenal ulcer mesovarial leiomyomas in rats in a chronic toxicity study of musuprine hydrochloride current concepts in mucosal immunity v. role of m cells in transepithelial transport of antigens and pathogens to the mucosal immune system the influence of food additives and related materials on lower bowel structure and function the interactions of lectins with animal cell surfaces immunohistochemical localization of carbonic anhydrase in submandibular salivary glands of mice and hamsters treated with phenylephrine, testosterone or duct ligation tumours of the oral cavity, pharynx, oesophagus and stomach distribution of glutathione and its related enzymes in small intestinal mucosa of rats the ulcerogenic effect on the oesophagus of three β-adrenoceptor antagonists, investigated in a new porcine oesophagus test model goblet cell hyperplasia is a feature of the adaptive response to jejunoileal bypass in rats a study on carcinogenesis induced by degraded carrageenan arising from squamous metaplasia of the rat coloretum differences in thegastro-intestinal mirobiota of specific pathogen free mice: an often unknown variable in biomedical research paneth cells and innate immunity in the crypt microenvironment. gastroenterology mucosal immunity and inflammation iv. paneth cell antimicrobial peptides and the biology of the mucosal barrier entero-hepatic cycling in rats plays a major role in fatal nsaid intestinal ulcerogenicity sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of peyer's patches in the normal unobstructed mouse intestine: an ultrastructural study antigen processing structures of the mammalian intestinal tract: an sem study of lymphoepithelial organs cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse peyer's patch m cells predicting anticancer drug effects in man from laboratory animal studies the anatomical basis for the immune function of the gut morphological and histochemical changes in intestinal mucosa in the reserpine-treated rat model of cystic fibrosis the pericryptal fibroblast sheath. iv. replication, migration and differentiation of the subepithelial fibroblasts of the crypts and villus of the rabbit jejunum comparative analysis of the s rna gene sequence of the putative agent of proliferative ileitis of hamsters histochemistry: theoretical and applied h -receptor antagonists and gastric cancer diagnostic exercise: inter-mandibular swelling in rats depletion of salivary gland epidermal growth factor by silodacryoadenitis virus infection in the wistar rat biology and biochemistry of papillomaviruses lescol (fluvastatin sodium) the cytology of salivary glands salivary gland sexual dimorphism: a brief review enzyme histochemistry of the human stomach with special reference to intestinal metaplasia nitrates and gastric cancer mucosal immunity and inflammation v. innate mechanisms of mucosal defense and repair: the best offense is the best defense derivation of mouse intestinal rypts from single progenitor cells twenty-one month evaluation of misoprostol for carcinogenicity in cd- mice association of long lasting unsurmountable histamine h blockade and gastric carcinoid tumours in the rat changes in the gastric mucosa of the mouse associated with long lasting unsurmountable histamine h blockade pharmacological and toxicological effects of chronic porcine growth hormone administration in dogs campylobacter jejuni enteritis in man and domestic animals is ethanol-induced damage of the gastric muosa a hyperosmotic effect? comparative studies on the effects of ethanol, some other hypperosmotic solutions and acetyl-salicylic acid on rat gastric mucosa synergistic interaction between aspirin, or other non-steroidal antiinflammatory drugs, and stress which produces severe gastric mucosal damage in rats and pigs the effects of aspirin and other non-steroid anti-inflammatory/analgesic drugs on gastrointestinal mucus glycoprotein biosynthesis in vivo: relationship to ulcerogenic actions electron microscopic observations comparing the gastric mucosal damage induced in rats and pigs by benoxaprofen and aspirin, reflecting their differing actions as prostaglandin-synthesis-inhibitors megaoesophagus in icrc mice compylobacter-like organisms in the gastric mucosa of rhesus monkeys gastric cancer in patients who have taken cimetidine effect of cimetidine on gastric juice n-nitrosamine concentration cryptosporidium cuniculus in the rabbit clostridium difficile colitis in a rabbit following antibiotic therapy for pasteurellosis clostridium difficile antitoxin neutralization of cecal toxin(s) from guinea pigs with penicillin-associated colitis clostridium difficile typhlitis in hamsters not associated with antibiotic therapy clostridial enteropathies, hamster spontaneous non-neoplastic gastric lesions in female han: nmri mice, and influence of food restriction throughout life saponification-induced increase in the periodic-acid-schiff reaction in the gastrointestinal tract. mechanism and distribution of reactive substance structure and carbohydrate histo-chemistry of postnatal canine salivary glands recombinant human epidermal growth factor - -induced structural changes in the digestive tract of cynomolgus monkeys (macaca fascicularis) influence of long-term , -dimethyl prostaglandin e treatment on the rat gastrointestinal mucosa digestion and absorption of carbohydrate and nitrogeneous matter in hindgut of the omnivorous non-reminant animal salivary glands in longterm alloxan-diabetic rats. a quantitative light and electron-microscopic study digestive system: intestines. in: histology. a text and atlas helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function the gastrointestinal tract dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications histochemical study on the paneth cell in the rat ultrastructural observations on the pathogenesis of aspirin-induced gastric erosions dietary related periodontitis and oro-nasal fistulation in rats abnormal skeletal and dental growth in epileptic children tooth root resorption induced in rats by diphenylhydantoin and parathyroidecotomy light microscopic morphometric analysis of rat ileal mucosa. i. component quantitation if iga-containing immunocytes short-term effects of various phenols and acids on the fischer male forestomach epithelium enzootic and epizootic adrenal medullary proliferative diseases of rats: influence of dietary factors which affect calcium absorption campylobacter pylori, gastritis, and peptic ulcer disease in central africa the effect of diet on the mammalian gut flora and its metabolic activities interactions of the gut microflora and the host in toxicology naturally occurring tumors and other lesions of the digestive tract in untreated c bl mice pathology of tumours in laboratory animals. tumours of the mouse esophageal impaction in the bhe rats minimal invasive carcinoma of the colon in rats early effects of alloxan on rat submandibular gland cyclic motor activity; migrating motor complex age-related changes of rat submandibular gland: a morphometric and ultrastructural study indomethacin produces gastric antral ulcers in the refed rat quantitative electron microscopic observations on paneth cells of germ-free and ex-germ-free wistar rats immunohistochemical observations of immunoglobin a in the paneth cells of germ-free and formerly-germ-free rats pigmentary changes in the rat oral mucosa following antimalaria therapy monoclonal antibodies to a zinc-binding protein of rat paneth cells age, weight and social effects on ulceration in rats the evaluation of anticancer drugs in dogs and monkeys for the prediction of qualitative toxicities in man gingival fibromatosis, macaca mulatta identification and mutagenicity of metabolites of aristolochic acid formed by rat liver melanosis coli: a study with the electron microscope light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. i. mouse light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates genetic and sex-related differences in the structure of subnmandibular glycoconjugates mucosal microhernias in the nonhuman primate colon: their role in the pathogenesis of colonic diseases quantitative age changes in the histological structure of human submandibular salivary glands immunohistological characterization of intra-epithelial lymphocytes of the human gastrointestinal tract chronic duodenal ulcers in pantothenate deficient mice focal avillous hyperplasia of the mouse duodenum colon epithelium. i. light microscopic, histochemical, and ultrastructural features of normal colon epithelium of male fischer rats colon epithelium. ii. in vivo studies of colon carcinogenesis. light microscopic, histrochemical, and ultrastructural studies of histogenesis of azoxymethane-induced colon carcinomas in fischer rats two types of mucous cells in the colon crypt carrageenan ulceration as a model for human ulcerative colitis. lancet, ii comparative histochemistry of gastro-intestinal mucosubstances assessment of risk of formulation of carcinogenic n-nitroso compounds from dietary precursors in the stomach granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study marked epithelial hyperplasia of the rat glandular stomach induced by long-term administration of iodoacetamide a profile of the gastrointestinal toxicity of drugs used to treat inflammatory diseases morphology and cyto-chemistry of rat salivary gland acinar secretory granules and their alteration by isoproterenol. i. parotid gland effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: a light and electron microscopic study the effects of long-term propranolol on the salivary glands and intestinal mucosa of the mouse effects of cardiotonic phosphodiesterase inhibitors on rat salivary gland. presented at the th annual acvp meeting inhibitors of sterol synthesis. morphological studies in rats after dietary administration, administration of α-cholest- ( )-en- β-ol- -one, a potent hypocholesterolemic compound m cell numbers increase after transfer of spf mice to a normal animal house environment strictures of ascending colon in cystic fibrosis and high-strength pancreatic enzymes pathology of laboratory rats and mice anatomy of the cecum of the laboratory mouse and rat gastric carcinoids and related endocrine growths pathogenesis of peptic ulcer and implications for therapy histochemical distribution of lysozyme activity in organs of normal mice and radiation chimeras human peyer's patches: an immunohistochemical study diamine methods for differentiating mucosubstances histochemically metabolic and morphometric changes in small and large intestine in rats fed high-fiber diets melanosis coli: studies of the toxic effects of irritant purgatives surface morphology of the gastroduodenal mucosa in duodenal ulceration the effect of vincristine on dentino-genesis in the rat incisor. scand autoradiographic investigation of dentine production in rats incisosrs after vincristine administration. scand acute and late radiation injury in rhesus monkey parotid glands. evidence of interphase death unique radiosensitivity of serous cells in rhesus monkey submandibular glands pathologic observations on the adenomatous lesions of the stomach in mice of strain i carcinoma of the glandular stomach of rats ingesting n,n´ ulcerative enterocolitis in dogs induced by drugs aspirininduced glandular dysplasia of the stomach. histologic and histochemical studies in rats effect of lumenal contents on colonic cell replacement pathologic findings in the stomach of rats treated with the h -receptor antagonist tiotidine morphologic stomach findings in rats and mice treated with the h -receptor antagonists, ici and ici lectin-peroxidase reactivity in rat gastric mucosa tumour production in glandular stomach of rat by nmethyl-n´nitro-n-nitrosoguanidine ageing affects the drug metabolism systems of rat liver, kidney, colon and lung in a differential fashion oral papillomatosis in new zealand white rabbits diagnostic exercise: lingual growths in rabbits immunoperoxidase localization of papillomaviruses in hyperplastic and neoplastic epithelial lesions of animals hypergastrinemia after blockade of acid secretion in the rat. trophic effects pathological features of the colonic tumors induced in rats by the administration of , -dimethylhydrazine comparative studies on the gastrointestinal lesions caused by serveral non-steroidal anti-inflammatory agents in the rats cells intermediate between mucous neck cells and chief cells in rat stomach fine structure of giant hypertrophic gastritis developed in thymectomized mice hyperplastic gingivitis in a child receiving sodium valproate treatment dopamine disorderin dodenal ulceration biology of disease. pathogenesis of duodenal ulcer disease from cysteamine to mptp: structure-activity studies with duodenal ulcerogens digestive system. monographs on pathology of laboratory animals role of salivary mucins in the protection of the oral cavity effects of , -dimethyl prostaglandin e -methyl ester on aspirin-and indomethacin-induced gastrointestinal lesions in dogs hypertrophic gastropathy resembling menetrier's disease in transgenic mice overexpressing transforming growth factor α in the stomach tumours of the oral cavity, buccal pouch, oesophagus, forestomach and salivary glands nodular hyperplasia of oncocytes in mouse submandibular glands early colonic lesions in experimental shigella infections in rhesus monkeys: revisited variation in susceptibility to the induction of forestomach tumours by butylated hydroxyanisole among rats of different strains pathology of neoplasia and preneoplasia in rodents independent induction of intestinal metaplasia and gastric cancer in rats treated with n-methyl-n´-nitro-n-nitrosoguanidine the response of three inbred strains of rat to the carcinogen , -dimethylhydrazine small intestinal type' and 'colonic type' intestinal metaplasia of the human stomach and their relationship to the histogenetic types of gastric adenocarcinoma light and electron microscopic cytochemistry of glycoconjugates in the recto-sigmoid colonic epithelim of the mouse and rat preclinical toxicologic evaluation of bleomycin (nsc ), a new anti-tumor antibiotic oral squamous cell carcinoma in ad libitum-fed and food restricted brown-norway rats the pathoparasitology of the alimentary tract and pancreas of non-human primates: a review t cell attractant chemokine expression initiates lacrimal gland destruction in nonobese diabetic mice altered patterns of mucin secretion in the precancerous lesions induced in the glandular part of the rat stomach by the carcinogen n-methyl-n´nitro-n-nitrosogaunidine immunohistochemistry and radioimmunoassay of egf in submanidbular glands of mice treated with secretogogues carcinogenicity of cyproterone acetate preclinical toxicology studies with acylovir: acute and sub-chronic tests the influence of adrenoreceptor activity on crypt cell proliferation in rat jejunum a sporozoan found in the peptic glands of the common mouse cyclophosphamide-induced abnormalities in the incisors of the rat spontaneous adencarcinoma of the ascending colon in wistar rats: the intracytoplasmic presence of a campylobacter-like bacterium inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs spontaneous hyperplastic and metaplastic duct epithelium in the sublingual salivary glands of wistar rats spontaneous tuors of the syrian hamster: observations in a closed breeding colony and a review of the literature mucin gene structure and expression: protection vs. adhesion morphological effects of high dose neomycin sulphate on the small and large intestine comparative and experimental pathology of fibrosing colonopathy intestinal pathology in the dog induced by sublethal doses of amiodarone pharmacological effects of epidermal growth factor (egf) with focus on the urinary and gastrointestinal tracts chemically induced lipidosis of the small intestinal villi in the rat lesions of experimentally induced tyzzer's disease in syrian hamsters, guinea pigs, mice and rats hexamitis in laboratory mice, hamsters, and rats melanosis coli: a consequence of anthraquinone-induced apoptosis of colonic epithelial cells safety evaluation of cimetidine: report at the termination of a seven-year study in dogs hypopigmentation in dogs treated with an inhibitor of platelet aggregation gingival hyperplasia in dogs induced by oxodipine, a calcium channel blocking agent cholecystokinin receptors gastrointestinal ph measurement in rats: influence of microbial flora, diet and fasting morphogenesis of chemically induced neoplasms of the colon and small intestine in rats natural history of intestinal neoplasms induced in rats by a single injection of methyl (acetoxymethyl) nitrosamine proliferative lesions of the glandular stomach and liver in f rats fed diets containing aroclor experimentally induced intestinal metaplasia in wistar rats by x-ray irradition. gastroenterolgy effect of dietary undegraded carrageenan on colon carcinogenesis in f treated with azoxymethane or methyl-nitrosourea induction of intestinal metaplasia in the rat gastric mucosa by local x-irradiation the effect of sex difference on induction of intestinal metaplasia in rats distribution of the histaminergic neuron system in the central nervous system of rats; a fluorescent immunohistochemical analysis with histidine decarboylase as a marker replacement of functional parenchymal cells by fat and connective tissue in human submandibular salivary glands. an age related change canine papilloma: progression of oral papilloma to carcinoma bacterial and mycotic disease dental health of survivors of malignant disease analysis of protein synthesis in rat salivary glands after chronic treatment with β-receptor agonists and phosphodiesterase inhibitors mucinous carcinoma of the ileum in the rat mucin histochemistry of gastric intestinal metaplasia long-term effects of an inotropic phosphodiesterase inhibitor (ici , ) on rat salivary gland, hardarian gland and intestinal mucosa the synovial membrane, liver, and tongue: target organs for a ricin a-chain immunotoxicin (zd ) ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains quantitation of glandular gastric changes in rats given a proton pump inhibitor for months with emphasis on sampling scheme selection aberrant crypt foci in the colonic mucosa of rats treated with genotoxic and nongenotoxic colon carcinogen the interactions of diet and colonic microflora regulating colonic mucosal growth selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastro-intestinal damage iinduced by indomethacin in the rat a biochemical basis for the gastro-intestinal toxicity of non-steroid antirheumatoid drugs butylated hydroxianisole mechanistic data and risk assessment: conditional species-specific cytotoxicity, enhanced cell proliferation, and tumor promotion menetrier's disease presenting as iron deficiency anaemia radioautographic and quantitative studies on parietal and peptic cell kinetics in the mouse: a selective effect of gastrin on parietal cell proliferation intestinal adaptation. structural, functionaland cytokinetic changes instestinal adaptation. mechanisms of control mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties with special reference to protein blocking and solubility of the granular glycosaminoglycan effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora effect of prolonged metiamide medication on the fundic mucosa the membraneous epithelial (m) cell and the mucosal immune system medical progress: gastrointestinal toxicity of nonsteroidal antiinflammatory drugs nitrosatable drugs: an assessment of the risks assessing the safety of drugs for the long-term treatment of peptic ulcers the mechanism of mitrazepam-induced drooling and aspiration blood vessels of the peyer's patch in the mouse. iii high endothelial venules ultrastructural basis of intestinal absorption the effects of omeprazole and famotidine on mucin and pge release in the rat stomach forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice forestomach ulcers in crj:b c (c bl/ ncrj × c h/hencrj) f mice an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs drug-induced gastro-intestinal disease naturally occurring intestinal neoplasms in aged crl:cd ® br rats key: cord- - bfchw u authors: rollinger, judith m.; stuppner, hermann; langer, thierry title: virtual screening for the discovery of bioactive natural products date: journal: natural compounds as drugs volume i doi: . / - - - - _ sha: doc_id: cord_uid: bfchw u in this survey the impact of the virtual screening concept is discussed in the field of drug discovery from nature. confronted by a steadily increasing number of secondary metabolites and a growing number of molecular targets relevant in the therapy of human disorders, the huge amount of information needs to be handled. virtual screening filtering experiments already showed great promise for dealing with large libraries of potential bioactive molecules. it can be utilized for browsing databases for molecules fitting either an established pharmacophore model or a three dimensional ( d) structure of a macromolecular target. however, for the discovery of natural lead candidates the application of this in silico tool has so far almost been neglected. there are several reasons for that. one concerns the scarce availability of natural product (np) d databases in contrast to synthetic libraries; another reason is the problematic compatibility of nps with modern robotized high throughput screening (hts) technologies. further arguments deal with the incalculable availability of pure natural compounds and their often too complex chemistry. thus research in this field is time-consuming, highly complex, expensive and ineffective. nevertheless, naturally derived compounds are among the most favorable source of drug candidates. a more rational and economic search for new lead structures from nature must therefore be a priority in order to overcome these problems. here we demonstrate some basic principles, requirements and limitations of virtual screening strategies and support their applicability in np research with already performed studies. a sensible exploitation of the molecular diversity of secondary metabolites however asks for virtual screening concepts that are interfaced with well-established strategies from classical pharmacognosy that are used in an effort to maximize their efficacy in drug discovery. such integrated virtual screening workflows are outlined here and shall help to motivate np researchers to dare a step towards this powerful in silico tool. in the field of drug discovery we are confronted by a paradox situation: highly efficient tools and advanced technological and molecular knowhow, e.g., in the area of genomics, combinatorial chemistry, high throughput screening (hts), robotized and miniaturized process cycles, could find entrance in big pharmaceutical industries. these costly procedures were expected to raise the number of launched drug substances; however the results were disappointing [ , ] . in , adam smith, the chief-editor of nature presented the sobering data of research and development expenses of the leading pharma companies versus new drugs on the market. they have steadily fallen in recent years despite the increasing financial efforts [ ] . on the other side we are faced by a high traditional impact of naturally derived medicines and incredible success stories of natural products (nps) as potent remedies from the beginnings of human therapeutic activity to modern research and drug development. nevertheless, most large pharmaceutical companies scaled down or terminated their work in nps operations. the reasons behind this are that the drug discovery process starting from natural sources is hardly compatible with the today's highly automated drug discovery technologies. thus, the pre-eminence of combinatorial chemistry as the preferred method for generating new drug leads has led to the comparative neglect of this valuable resource. william strohl from merck research laboratories summarized the difficulties of np programs versus synthetic chemicals in his editorial remarks in drug discovery today [ ] . these include (i) the existence of already found potent antimicrobic and antitumor nps and the lack of sufficient dereplication programs which prevent their repeated discovery; (ii) the fact that -in contrast to the highly sophisticated molecular targets -np extracts are generally regarded as too 'dirty', too difficult to assay and too time-consuming; (iii) obtaining an assay hit resulting from a bio-guided fractionation, the nps' structure still has to be elucidated compared with synthetic chemicals; (iv) nps are often deemed as too structurally complex, possessing multiple hydroxyl moieties, ketones and chiral centers. strohl nevertheless concluded by listing a number of advantages applying an active np program, which he finally described as an 'expensive endeavor' which, however, is 'well worth the cost'. the use of nps has been the single-most successful strategy for the discovery of new drug leads, which is clearly shown by different statistics [ , ] . with increased calls in recent years for further research on nps [ , ] there are again signs that they may play a more active role in the future drug discovery process, since their reintroducing may help to re-discover the sweet spot in drug discovery [ ] . to date some , natural compounds [ - ] have been published. the terrestrial flora has been intensively investigated over the last decades; the potential in finding new nps slumbering in untapped biota is however nearly inconceivable. it is estimated that only - % of the approximately , described high plant species have ever been in the focus of phytochemical and pharmacological investigations [ ] . more sobering is the percentage in the field of bacterial (less than %) and fungal species (less than %) [ ] . the main part of known nps belongs to secondary metabolites. these compounds provide living systems with their characteristic features mandatory for surviving. they contain an inherently large-scale of structural diversity. about % of the chemical scaffolds of published nps are unique and have not been made by any chemist [ ] . in the past years researchers have discovered many potential therapeutic targets. since the completion of the human genome, , to , genes and at least the same number of proteins are assumed [ ] . thus, we are up against an increasing number of macromolecular targets, like proteins, receptors, enzymes, and ion channels -that might be of pathological concern for humankind. among them, proteins continue to attract significant attention from pharmaceutical technology as a valuable source of drugable targets [ ] . proteins provide the critical link between genes and disease, and thus are the key to understanding the basic biological processes. up to now drug discovery has been performed against only approximately targets [ ] , though the number of potential targets are estimated to be in the range of , to , [ , ] . taken together, it can be assumed that a large number of drug leads and hits are conserved in the inexhaustible pool of nps pre-screened by evolution. but how to dig out and to recognize the respective drug leads is a challenging task for both industry and academia, for medicinal chemists, pharmacognosists and pharmacologists. np research is affected with a wealth of time-consuming and cost intensive investigations. collection of the natural material, phytochemical analysis, isolation and identification of the constituents is just the basic procedure. a biological screening of extracts or even the arbitrary testing of isolated metabolites is feasible and often performed, though is not at all a focused procedure, thus unpractical and too expensive. the nps' diversity has to be accessed in a more rational way. holistic versus molecular approaches in drug discovery from nature during the last century and even today the discovery of bioactive nps and their development into potential drug candidates are mainly covered by a holistic approach. a characteristic workflow of this procedure is given in figure . starting from the knowledge or assumption about a biological effect the natural material is selected and adequately extracted. if a positive effect in the obtained multi-component extract is recorded, it is attempted to trace it back to the active principle/s by intense phytochemical and analytical investigations (fig. ). this can for instance be achieved by a bioactivity-guided fractionation. a more targeted approach focuses on innovative technological tools combining analytical and biological information. an overview of recent developments in this area and successful examples thereof are presented by potterat and hamburger [ , ] . as soon as the constituent regarded to be responsible for the overall effect is isolated, further research focuses on a molecular level including structure elucidation and pharmacological profiling. synthesis and testing of series of derivatives enable an insight into a structure-activity-relationship and pharmacokinetic aspects. finally, potential drug leads become drug candidates after some intense toxicological studies and after the verified effectiveness in vivo (fig. ) . recent advances in lead identification from nature work on a molecular base more than on a holistic one. a first prerequisite for that is on bioin- traditional early drug development of a nature based drug candidate formatics comprising d structures from genes and proteins (bioinformatics), substantial knowledge on molecular target functions with accurate structural information and protein-ligand interactions. secondly, it is essential to refer to unambiguously characterized structures of secondary metabolites preferably with some information to their biological effect. based on available structural as well as biological knowledge from both sides, information can be deduced from chemoinformatics to bridge the gap between known ligands and the discovery of new lead structures (fig. ). the increasing understanding of fundamental principles of protein-ligand interactions and the steadily growing number of d-structures of potential and experimentally proved ligands provide undreamed of possibilities towards more rationalized concepts in drug discovery. however, too much is expected of the human brain to profit from the already published information. thus, efficient and effective approaches benefit from today's knowledge about nps. in the area of medicinal chemistry, computational methods, like virtual screening experiments, have already proved to satisfy these requirements. they are needed to exploit the available structural information, to understand specific molecular recognition events, and to clarify the function of the target macromolecule. though rationalized procedures in the search for bioactive natural products are in great demand to find the 'needles in a haystack', computational assistance could hardly break into natural product research. the common idea of all computational approaches within the early drug discovery process is to mine more or less large compound databases in silico and to select a limited number of candidates proposed to have the desired biological activity. for this process the term 'data mining' was coined in [ ] , which was concisely defined by gasteiger and coauthors: 'to extract knowledge from a large set of data in order to make predictions of new events' [ ] . within the lead discovery process, virtual screening technologies have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in early phase drug research [ , ] . the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug, by identifying the most promising candidates to focus the experimental efforts on. recently published books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts [ ] [ ] [ ] [ ] . if the d structure of the biological target is known, high throughput docking turned out to be a valuable structure-based virtual screening method to be used [ ] [ ] [ ] [ ] . within this context, the scoring of hits retrieved still remains a question that is often discussed. in fact, currently the major weakness of docking programs lies not in the docking algorithms themselves but still in the inaccuracy of the functions that are used to estimate the affinity between ligand and target, the so-called scoring functions. previously, stahl and rarey analyzed scoring functions for virtual screening [ ] , giving valuable insight into strengths and weaknesses of currently used models for affinity estimation. the combination of several different scoring functions termed as consensus scoring turns out to be one of the possible answers to the question raised previously. in fact, several authors recently described their efforts in this area; an example is given in reference [ ] . in a theoretical study, other authors demonstrate that consensus scoring outperforms any single scoring for simple statistical reasons and that a moderate number of scoring functions (i.e., three or four) are sufficient for the purpose of consensus scoring [ ] . however, it has been shown that consensus scoring alone is not suitable for all cases of docking, and, as highlighted in a recent review by krovat and co-authors, considerable efforts are still devoted to the optimization of scoring functions [ ] . because of the restricted free access to np d libraries (see below), the number of virtual screening studies published for the rational access to bioactive nps is limited. some examples using high throughput docking as a structure-based virtual screening tool will be given here: liu and zhou applied a theoretical approach to find natural ligands as potential inhibitors of the sars-cov protease, a virus target of the severe acute respiratory syndrome [ ] . they used a docking-based virtual screening cycle and applied drug-like filters to finally propose drug candidates out of two d databases comprising metabolites from marine organism and compounds from traditional chinese medicine. the same virus organism was the main interest in the study performed by toney et al., who focused on its main proteinase, clpro. the crystal structure of this attractive target was used as the starting point for the virtual docking screening of the nci database. searching for non-peptidyl inhibitors, the authors identified the naturally occurring terpenoid alkaloid sabadinine (i.e., cevine; ) as potential anti-sars agent [ ] . the author group around stefano moro could identify ellagic acid ( ) as inhibitor of the protein kinase ck screening an in-house generated database with almost , structures of natural compounds [ ] . a combination of four docking protocols and five scoring functions has been utilized to dock and rank the molecules in the database. the consensus docking suggested ellagic acid to be one of the most promising candidates. this assumption could be verified by experimental studies revealing this np as highly potent ck inhibitor (k i = nm). estrogen receptor-plays a key role in regulating brain development and estrogen-induced promotion of neurogenesis and memory. using the d coordinates of the co-crystal structure of human estrogen receptorbound with genistein as starting point, zhao and brinton pursued a receptor-based molecular docking approach [ ] . they focused on the search for natural estrogen receptor--selective ligands. twelve candidate molecules, which had been suggested by the database screening, were selected. the authors determined their binding affinity and selectivity; three of the com-pounds belonging to the flavanoid family ( ) ( ) ( ) displayed over -fold binding selectivity to the estrogen receptor-over . a similar approach was employed by liu and co-authors. applying a docking virtual screening filtering experiment, the authors discovered potent inhibitors of the potassium ion channel from a chinese np database [ ] . the pharmacophore concept has proven to be extremely successful, not only in rationalizing structure-activity relationships, but also by its large impact in developing the appropriate d-tools for efficient virtual screening [ ] . profiling of combinatorial libraries and compound classification are other often-used applications of this concept. although well established in combinatorial chemistry, it has to be pointed out that the tools described in this section have likewise a considerable impact on the rational finding of new potential lead compounds originating from the immense source of secondary metabolites. the prior use of pharmacophore models in biological screening of nps is an efficient procedure since it quickly eliminates molecules that do not possess the required features thus leading to a dramatic increase of enrichment, when compared to a purely random screening experiment. in a previous study conducted by doman and co-authors [ ] , only molecules or . % revealed as protein tyrosine phosphatase- b inhibitors (ic < µm) by a hts of approximately , compounds. on the other hand, of molecules suggested by molecular docking, or . % were found to be active. thus, dockingbased virtual screening enriched the hit rate by almost , -fold over random screening. one should not forget, however, that additional molecular characteristics not reflected by pharmacophore models (physicochemical properties relevant for adme and toxicological properties) must be taken into account when deciding upon which compounds should be further developed [ ] . a rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties is a pivotal step in the early drug discovery process [ , ] . they can be evaluated traditionally or by high throughput screening, which are discussed in detail by avdeef and testa [ ] . this must be considered for synthetics as well as nps, though studies revealed secondary metabolites not only high scaffold diversity; biosynthesized molecules also show structural and spatial characteristics that are closer to drug leads than those of synthetic molecules [ , ] . typically, nps include more chiral centers and their stereochemical architecture is much more complex than that of synthetic molecules. furthermore, they usually contain more carbons, hydrogen and oxygen, however, less nitrogen and other atoms compared to synthetics. surprisingly, nps often show a molecular weight higher than da combined with a high polarity [ ] , which is in clear contrast to lipinski's rule of five [ ] . nevertheless only about % of nps contain two or more violations of lipinski's rules [ ] . in summary, natural chemistry can be seen as highly diverse scaffolds endowed with potential drugable pharmacophores. structure-based pharmacophore model an inevitable prerequisite for generating a structure-based model is the knowledge about the ligand-target interaction [ ] including the availability of the d structure of the target either by x-ray crystallography or nmr or constructed on the basis of the structure of homologous proteins. a unique platform containing d coordinates of experimentally solved protein structures is the brookhaven protein data bank (pdb [ ] ). a crystalline complex with a ligand bound to a protein's active site is the best requirement to start the construction of a structure-based d model. in this case, one may profit from the exact information of the ligand's bioactive conformation which is preserved in the binding site of the crystalline complex. the building of a structure-based pharmacophore is depicted in a step by step way in figure . a new software tool has recently been described for the successful generation of such chemical features-based models: the software ligand-scout [ ] is a program for ligand interpretation and data mining in the pdb. the performance of this program allows the detection of relevant interaction points between ligand and protein. the binding mode of the ligand in the active site of a protein can be visualized in a sophisticated way. ligandscout's algorithms perform a stepwise interpretation of the ligand molecules: planar ring detection, assignment of functional group patterns, determination of the hybridization state and finally the assignment of kekulé pattern. the interpretation of the ligand molecules is the basis for the next step, an automated generation of pharmacophore models, derived from the data provided by a crystalline complex of the pdb. an automatic detection and classification of protein-ligand interactions into hydrogen bonds, charge transfer, and lipophilic regions leads to a collection of chemical features in a pharmacophore model. the graphical user-interface can provide an integrated view of protein, ligand, pharmacophore model, and interaction lines. in a previously published study, ligandscout was used for the detection and interpretation of crucial interaction patterns between ligands and the factor xa protein structure [ ] . in a second step, the program catalyst, a state of the art virtual screening platform, was used for rapid virtual screening of multiconformational d structure databases. the information for the pharmacophore pattern (i.e., d coordinates of interaction points) was obtained by the interpretation of ligandscout pharmacophore definitions and resulted in specific interaction models that were able to map the ligand in their bioactive conformation and to retrieve selectively a % fraction of the known factor xa inhibitors within a small subset of the large derwent world drug index library. a further application of the ligandscout pharmacophore definitions covers the rationalized search for angiotensin converting enzyme (ace)- inhibitors by virtual screening of approximately . million compounds from various commercial databases [ ] . hit reduction and selection was achieved using a five feature hypothesis based on a recently resolved inhibitor-bound ace crystal structure. seventeen virtual hits were selected for their experimental validation in a bioassay; the concept was confirmed since all of them were revealed as ace- inhibitors. barreca and co-authors developed a d structure-based pharmacophore model with ligandscout for the discovery of new scaffolds acting as hiv- non-nucleoside reverse transcriptase inhibitors by virtual screening of large chemical databases. six virtual hits were finally selected for determination of their inhibitory effects. those belonging to the new scaffold class of the quinolin- ( h)-one family exhibited reverse transcriptase inhibitory activity at sub-micromolar concentrations [ ] . in a recently published work, schuster et al. presented a so-called cytochrome p profiler [ ] . several structure-based (generated with ligandscout) and ligand-based pharmacophore models (using cata-lyst) for substrates and inhibitors of five cytochrome p isoenzymes ( a , p c , p c , p d , and p a ) were created and validated by the authors' group. their results showed that the models were suitable for fast pharmacokinetic profiling of large drug-like databases. in this context the parallel screening is of particular interest. whereas in usual virtual screening cycles interactions of thousands or even millions of d database entries are browsed against one pharmacophore model, it is contrary in the case of parallel screening; low-energetic conformers of one structure are screened for their potential interactions against numerous models. the basics of parallel screening have just recently been presented by steindl and co-authors [ , ] . furthermore, the authors exemplified this strategy for the activity profiling using a set of hiv protease pharmacophore models [ ] . this in silico concept is of particular interest to virtually scrutinize drug candidates for their preliminary activity profiling relevant to putative side effects and toxicity [ ] . according to the obtained interactions to virtually screened antitargets (e.g., herg, sigma- , sigma- , alpha- a, alpha- b, alpha- d, alpha- a, alpha- b, alpha- c, d l, d , d . , -ht a , -ht a , -ht , h , i , a a, a b, cytochrome p ) a first insight to potentially risky affinities is provided before time and cost intensive toxicological studies are performed. the virtual screening approach using a structure-based pharmacophore model has revealed some first application examples in np research: niko-lovska-coleska and co-authors successfully pursued this in silico strategy in the area of x-linked inhibitors of apoptosis (xiap) [ ] . a high resolution d structure of the xiap bir domain complexed with the n-terminal end of the smac/diablo protein [ ] , which is an endogenous ligand of the respective xiap binding pocket, was used as the starting point to virtually screen an in-house d-np database. embelin ( ) from the japanese ardisia herb emerged as virtual small molecule weight hit, which was found to be a fairly potent inhibitor of xiap using a fluorescence polarization binding assay. in our group, we previously focused on acetylcholinesterase (ache) [ ] ; according to the cholinergic hypothesis of the pathogenesis of alzheimer's disease, inhibitors of the ache are successfully used as therapeutic strategy. based on the co-crystal structure of ache with its ligand galanthamine, a structure-based pharmacophore model was generated and used for an in silico screening of a multi-conformational database consisting of more than , nps. from the obtained hit list, promising, virtually active candidates were selected, namely scopoletin ( ) and its glucoside scopolin ( ) . their ache inhibitory effect was first verified from the crude extract of scopolia carniolica roots using a bioautographic tlc assay. the isolated coumarins showed a significant and dose-dependent inhibition of the ache in the microplate enzyme assay as well as in the in vivo test. the i.c.v. application of both coumarins on rats resulted in a long-lasting, pronounced and -in case of the glucoside -even in a two-fold higher increase of the neurotransmitter's concentration than the one caused by the positive control galanthamine. ligand-based pharmacophore model very often, however, lead discovery projects have reached a well-advanced stage before detailed structural data on the protein target has become available, even though it is well recognized that modern methods of molecular biology together with biophysics and computational approaches enhance the likelihood of successfully obtaining detailed atomic structure information. a possible consequence is that often scientists identify and develop novel compounds for a target using preliminary structure-activity information, together with theoretical models of interaction. only responses that are consistent with the working hypotheses contribute to an evolution of the used models. within this framework, the chemical feature-based pharmacophore approach has proven to be successful [ ] allowing the perception and understanding of key interactions between a receptor and a ligand on a generalized level. a function-based pharmacophore represents the common ensemble of steric and electrostatic features of different compounds which are necessary for their interaction with a specific biological target structure (fig. ) . such pharmacophore models together with large d structure databases originating either from in-house compound collections, from commercial vendors, or from natural products databases have proven to be extremely useful in silico screening experiments. when using ligand-based pharmacophore models as screening filters instead of protein d structures, affinity estimation is only based on geometric fit of compound atoms or groups to features of the model. in these cases, the values calculated are often far away from reality, however, still are useful for filtering possible hits from non-binding molecules. additionally, in pharmacophore fitting procedures, calculation demands are considerably lower than in docking algorithms allowing the number of compounds to be processed in the same time to be by far higher than even in high throughput docking. since in most of the studies no experimental information on either the biological conformation of the ligand or the target protein are currently available, the ligand-based chemical feature pharmacophore approach can provide essential information for medicinal chemists. several successful applications within this subject have been performed using the cata-lyst program, one of the leading software packages in chemical featurebased pharmacophore modeling. schuster and co-workers succeeded in the identification of -hydroxysteroid dehydrogenase type inhibitors applying a common feature-based pharmacophore model for their virtual screening filtering experiments [ ] . similarly, the authors preceded by suggesting compounds with a proposed inhibition to the cytochrome p isoenzyme [ ] . several reviews covering successful applications of such feature-based methods have been published by kurogi et al. [ ] , by krovat et al. [ ] and by güner et al. [ ] . they outline the theoretical background and describe several significant studies including d database search strategies. in the field of nps only a very limited number of studies report from the rationalized access to bioactive compounds via ligand-based virtual screening. for example, this method was pursued for the discovery of inhibitors of the cop signalosome (cns) associated kinases ck and pkd [ ] . using nps curcumin and emodin as lead structures, a virtual screening of an in-house database was carried out. among the virtual hits seven nps, e.g., anthraquinone ( ) and piceatannol ( ), were found to significantly induce apoptosis by inhibition of the csn-associated kinases using in vitro and cell culture experiments. a further study has demonstrated the power of the ligand-based approach applied to pharmacophore modeling of sigma- ligands [ ] . therein, some reliable pharmacophore models could be extracted solely from ligand structure information. compounds with potent affinities to the sigma- receptor known from literature were structurally aligned to derive distinct common features. their d arrangement in combination with a spatial restriction was then used for the generation of a pharmacophore model, which was able to retrieve compounds with high affinity values, among them also nps, like solanidine ( ). further ligand-based approaches use various forms of discriminant analysis, e.g., artificial neural network simulations. they are based on collections of mathematical models that are interconnected and organized in different layers. they are analogous to an adaptive human learning process and usually trained with learning sets applying one or more molecular descriptors in order to form clusters that enable to distinguish between different objects and their properties. the resulting models are then applied to make predictions on test sets, until the validated models may be used to derive a qsar of chemically related structures or to mine larger datasets. one may distinguish between supervised and unsupervised (e.g., kohonen network) learning methods as discussed in detail by zupan and gasteiger [ ] . a successful application example within the field of nps was published by wagner et al. [ ] . the authors used a dataset of structurally diverse sesquiterpene lactones with known nf-b inhibitory activity to derive a qsar. by the application of multiple d structure representations as descriptors, a single model was achieved which provided detailed information on the structural influence of the investigated biological activity. sangma and co-authors pursued a combination of two approaches to predict new inhibitors of the hiv- rt and hiv- pr from a np database comprising metabolites from thai medicinal plants. after a high throughput docking of the molecules into the target enzymes, self-organizing maps were generated to reduce the number of promising candidates to be tested [ ] . a set of different in silico methodologies was previously applied by cherkasov and co-authors to aid in the discovery of natural non-steroidal ligands for human sex hormone binding globulin [ ] . therein, a rigorously cross-validated neural network based qsar model identified prospective compounds from a structure collection of , commercial natural substances. this stringent qsar ranking was combined with docking studies and pharmacophore-aided database search. the integrated computational methods resulted in a convincing predictive tool which identified a set of structurally diverse nps, of which every fourth compound was able to inhibit the target protein in a micromolar range. compounds of arbitrary structural diversity and with known activity against a target are particularly suitable not only for generating a ligand-based pharmacophore model (as described before), but also for structure similarity studies using a decision tree. the object is to find as good a distinction as possible on the basis of a set of molecular descriptors, which identify molecular features shared by different subsets of active compounds and accordingly filter out compounds within the dataset in which these combinations are lacking. using not only a simple logical description of one model, but an ensemble of decision trees tend to be the preferred option, since the consensus voting among trees give the approach higher predictive accuracy. one form of multiple decision trees well performed to virtually screen large d databases is random forest [ ] . this chemoinformatic method was recently applied in a theoretical work performed by ehrman and co-workers to predict ligands of multiple targets, like cyclooxygenase (cox), lipoxygenase (lox), aldose reductase, hiv- enzymes etc., from a large dataset of chinese herbs [ ] . the advent of structure databases has provided a basis for the development and feasibility of automatic methods for the search of new lead structures. conceptually, all the virtual screening concepts presented above have their origins in synthetic chemistry. their application, however, is just as well adaptable to nps' chemistry. prior to the in silico filtering experiment, a d structure database requires an efficient generation of reasonable, energetically minimized conformations assumed to meet approximately those conformations that might be of biological relevance [ ] . the underlying algorithms for d structure generation and conformation analysis are implemented in commercial software tools, e.g., in corina [ ] or the catalyst program (catalyst, available from accelrys inc., san diego, ca, usa; www.accelrys.com). in the field of nps the virtual screening application is mainly restricted due to the lack of searchable resources for structurally well defined natural compounds. in general, molecular databases with free access on the internet may comprise a high number of molecules, e.g., chembank (> , , , http://chembank.broad.harvard.edu) or pubchem (> , , ; http:// pubchem.ncbi.nlm.nih.gov); however, information about the number of contained natural molecules is rarely available. the library of the national cancer institute (nci) stores structural information of more than half a million compounds from both synthetic and natural origin that have been collected and tested by the nci since . about half of the synthetic compounds, which represent the large majority of the samples, may be used for free and are thus in the public domain. it is called the 'open nci database' (development therapeutics program nci/nih; http://dtp.nci. nih.gov/webdata.html). an interesting property prediction approach to the more than , compounds contained in this open database was provided by poroikov and co-authors [ ] . by use of the program pass (prediction of activity spectra for substances) an in silico tool for complex searches of different types of activities is provided; e.g., in the case of antineoplastic effects, the authors could demonstrate a substantial dataset enrichment over random selection by the use of pass-predicted probabilities. libraries covering a major part of entities from nature (at least some thousands) or consisting of structural information exclusively from natural origin are not free of charge, e.g., the traditional chinese medicinal database (tcmd; http://tcm d.com/services.htm [ ] ) or the dictionary of natural product database launched by chapman & hall (dnp; http:// www.chemnetbase.com) providing chemical and physical data on some , natural compounds gathered from the world's chemical literature. an excellent survey of public and commercial databases focusing on nps has recently been published by füllbeck and co-authors [ ] . the authors provide information as to storing characteristics of the databases, web-addresses, total number of compounds and -if given -number of natural ones. in addition, a selection of suppliers and manufacturers of natural compounds and extracts are given. a new database is introduced by the authors (super natural database [ ] ) storing information on available nps, thus allowing the selection of compounds that can be purchased. moreover a number of non-commercial in-house created databases have been used from different groups for their virtual screening studies on nps, e.g., a marine natural product database (mndp [ ] ), a natural product database (npd [ , ] ), a database based on the antique source 'de materia medica' by pedanius dioscurides (dios [ ] ), or a database fed with metabolites of ethnopharmacologically known plants [ ] . recently, ehrman and co-authors generated a d multiconformational database of chinese herbal constituents containing a total of more than , compounds from chinese herbs [ ] . integrated strategies for the discovery of bioactive nps the more or less accurate prediction of potentially active compounds by virtual screening has doubtlessly rationalized the early drug discovery process. these filtering experiments definitely assist in saving costly and time-intensive pharmacological assays, since the pool of predicted ligands (i.e., virtual hits) is usually drastically reduced compared to the initial amount of compounds (i.e., d-database). demands to be made on a good model are selectivity and target-specificity on the one hand, but it is also seminal not to lose too many valuable ligands during the filtering process. how far all of these demands can be fulfilled strongly depends on the quality of information used as the basis for generating the model and the algorithm underlying the virtual screening process. in medicinal chemistry, an activity prediction of - % is usually regarded as satisfying enrichment. in np research, however, this percentage may be too scarce. it is rarely found that a large set of natural compounds can be acquired so easily. only a minority of secondary metabolites are commercially available -usually at incredibly high prices. thus, extraordinary charges and efforts are typically necessary before a virtual hit from nature is available for pharmacological testing. this process embraces the acquisition of the natural material described to contain the desired metabolite to the point of phytochemical analysis and isolation. though advanced separation techniques, analytical instrumentation, and innovative tools for structure identification are at the phytochemists' disposal, it remains a complex and sometimes uncertain endeavor. this is why the results obtained from in silico predictions may nevertheless be too vague for a np researcher. methods are asked to further increase the probability of following the straight tip. there is the possibility to hyphenate sundry computational approaches, e.g., pharmacophore-based virtual screening combined with docking of the resulting virtual hits, or to consider only the consensus hits applying two or more screening concepts. nevertheless all these strate-gies remain virtual and speculative. the combination of two approaches, which are completely divergent in nature, like a computational and an empirical one may however offer a more deepened access to bioactive nps and may sometimes help to avoid a distorted view. thus, the computer-aided molecular selection is best combined with further discovery methods, labeled as integrated approaches, to increase the probability in finding a real hit. in traditional pharmacognosy there are some well established methods in targeting this aim starting from a holistic level. these include (i) hints from ethnopharmacology, (ii) phenomenological effects registered after application of naturally derived preparations, (iii) guidance of chemotaxonomy, (iv) phylogenetic selection criteria, or (v) simply information gathered from a high/medium throughput screening of extracts. in a recently published review from our group, different strategies in the field of nps have been presented with special emphasis on anti-inflammatory nps interacting within the arachidonic cascade [ ] . integrated computational strategies for the discovery of natural bioactive compounds have been introduced elsewhere concentrating on their scope, strengths and limits [ ] . some strategies and examples from literature combining virtual screening approaches and classical methods for activity exploitation are outlined below. as soon as a sensitive data-mining tool has been developed and has proved itself by more or less selectively finding the active compounds within a test set, it can be applied for screening a d multi-conformational database. the subsequent procedure consists of the evaluation of the virtual hits considering physicochemical properties, toxicity and pharmacokinetics. in this stage additional virtual filtering tools for the profiling of adme parameters [ ] might have an invaluable impact to aid a refined selection of compounds. then, a sensible choice of natural materials known to contain the focused metabolites and worth investigating in detail is a crucial step. it requires a comprehensive study in literature considering the hit content in the natural source, its availability and maybe hints from ethnopharmacology. once some natural materials are selected, it is advisable to perform a preliminary assay with those crude extracts and fractions assumed to contain the promising metabolite/s. though being aware that in case of small hit amounts present in the natural material the activity may be overseen. therefore, it is advisable to first identify the promising constituent and to possibly enrich it in the extract to be tested. those samples that scored well are then subjected to phytochemical investigations. in this way, the tricky selection of the natural material turns from a bold venture to a more rationalized endeavor. as soon as a promising (i.e., active) starting material is found, there are in principle two possible strategies to embark on: the first one relies more on the in silico approach and focuses directly on . strategy a for the discovery of bioactive nps using an integrated virtual screening approach the identification of the initially obtained virtual hits within the natural matrix applying analytical tools, like lc-ms or lc-nmr, gc-ms etc. in a straightforward manner the hits are isolated using different chromatographic separation steps. after structural confirmation the compounds are then tested to hopefully verify the predicted activities. this strategy is very goal-oriented, since only pharmacological assays for the finally isolated virtual hits are necessary. on the other side, one may run the risk of ignoring further active nps not necessarily fitting into the pharmacophore model. the second strategy focuses on a bioactivity-guided fractionation irrespective of the virtual hits used for the selection of the starting material. following the concept, the finally isolated active ingredients should correspond to the predicted virtual hits. this approach is usually associated with higher phytochemical efforts and costs, because it requires an iterative testing of all arising fractions and sub-fractions. for the evaluation of all the bioactive constituents in detail and for the discovery of possibly unknown metabolites this procedure is however indispensable. the decision, which of the presented ways is the more appropriate for the investigation at hand, strongly depends on the reliability and selectivity of the used pharmacophore model, and the costliness of the used assay. the strategy schematized in figure was recently applied to a medicinal plant with anti-inflammatory potential known from ethnopharmacological sources [ ] . from the pharmacophore based virtual screening filtering experiment a number of secondary metabolites known from the mulberry tree complied with all the models' requirements, thus revealed as virtual hits. indeed, in vitro tests attested extracts of morus root bark a distinct cox inhibitory potential. the objective was to find the active principles from this plant material applying both different methods for their discovery. first, the computer-aided approach was used to identify the virtually active compounds able to interact with the pharmacophore models for cox- and - . second, the bioactivity-guided fractionation was conducted for the isolation of the cox-inhibiting constituents. this resulted in the isolation of nine compounds belonging to the chemical classes of sanggenons and moracins. in the enzyme assay, all the isolates showed moderate to potent inhibitory effects on cox- and - . when comparing the hits of the virtual screening with the experimental data, a good correlation between predictions provided by the computer assisted method and in vitro data could be obtained in the case of the isolated sanggenons (e.g., sanggenon c; ). however, this agreement could not be achieved with the moracins (e.g., moracin m; ). in any case the virtual screening was particularly helpful for the decision regarding which plant material is worth extensive study. furthermore, the disclosed interactions of the sanggenons with the pharmacophore model -miming the binding site of the target -provided us with some essential information about the molecular requirements of cox-ligands. a different integrated procedure is schematized in figure . applying this approach, the pre-selection of the natural material is not guided by virtual prediction; but a number of extracts is roughly screened with a bioassay to identify the active ones. a similar strategy is to collect information about the traditional application of natural preparations in the field of the focused pharmacological target. a d database is then generated consisting of all the metabolites known from literature to be included in that extract/s that came off well. likewise, ethnopharmacological knowledge about useful preparations from nature may guide the selection of nps. the resulting biased database is virtually screened with an established pharmacophore model of the aiming target. the impact of ethnopharmacology has been analyzed in a previous study from our group; there we investigated the statistical evidence considering hints from folk medicine for the discovery of anti-inflammatory nps utilizing pharmacophore-based virtual screening techniques [ ] . cox- and - were used as preferential targets, since they are key enzymes in the inflammation process. dioscorides' de materia medica, which was written in the st century ad, was used as the ethno-pharmacological source. secondary metabolites of those medicinal plants, which dioscorides described as active against fever, rheumatism, pain and pus were stored in a multiconformational d database. this was virtually screened against the validated pharmacophore models. the resulted hit list was analyzed and compared with those obtained by screening unbiased databases of natural as well as of synthetic origin. the effectiveness of an ethnopharmacological approach could be statistically demonstrated by obtaining a significantly higher hit rate compared to the hit rates of the unbiased natural as well as synthetic databases. following this strategy the putative hits may then be identified by modern analytical tools like lc-ms or lc-nmr to isolate them from the natural matrix in a target-oriented way for pharmacological testing. this approach is especially helpful for intricate pharmacological assays, which would turn a bioguided fractionation into an unrealistic endeavor. a combination of an ethnopharmacologically based pre-selection of plant material and a computational approach was reported by bernard and co-workers, who used this strategy to rationalize a phytochemical lead discovery [ ] . starting with an in vitro screening on phospholipase a performed with traditionally used anti-inflammatory plant extracts, a focused structural database was generated and virtually screened on an established ligand-based pharmacophore model for human non-pancreatic phospholipase a . the combination of experimental data with database exploitation and molecular modeling resulted in the efficient identification of betulin ( ) and betulinic acid ( ) as extract ingredients with distinct anti-inflammatory in vitro effects. the combination of the two different, but complementary strategies consisting of in vitro screens and in silico assessment has recently been described by van de waterbeemd [ ] . he labeled this method as 'in combo' approach and used it for the straight forward access of various adme properties. the application of the 'in combo' approach for the discovery of nps has recently been tested in our group by the search of natural acetylcholinesterase inhibitors [ ] . in a medium-sized throughput screening about plant extracts were investigated using an acetylcholinesterase enzyme test. from the sample showing the best inhibitory activity, all the known secondary metabolites were fed into a small d multiconformational database and subsequently subjected to a virtual screening on a generated pharmacophore model. the efficacy of this procedure could be confirmed by the isolation of the obtained virtual hits, i.e., -deoxylactucin ( ) and lactucopicrine ( ) . they showed a significant and dose-dependent inhibitory effect in the enzyme assay. methods and expectations of this integrated virtual screening concept have previously been discussed in detail by j. bajorath [ , ] with the author's final statement that 'a meaningful integration of virtual and experimental screening programs, together with lessons to be learned from structural genomics, holds great promise for more rapid and consistent identification of high quality hits or leads across divers classes of therapeutic targets'. though this conclusion was not particularly coined to nps, it comes especially true in the rich world of secondary metabolites. further hybridized computational strategies are quite sensible to get an improved understanding of ligand-target interactions. in the following two examples docking protocols helped enlighten the molecular mechanism of bioactive natural compounds. chimenti and co-authors isolated quercetin ( ) among other secondary metabolites from the mediterra-nean shrub hypericum hircinum and identified this flavonol as selective inhibitor of the mao-a with an activity in the nanomolar range (ic = nm) [ ] . for a more comprehensive understanding of the underlying molecular selectivity, conformation analysis and docking simulations were performed using the most recent crystallographic structures of both human isoforms mao-a and mao-b. this enabled the authors to identify the most important interactions between the residues and the cofactor within the enzymatic cleft. the estimated free energies of complexation were in agreement with experimental data and confirmed the distinct preference for the mao-a cleft with more intermolecular hydrogen bonds and -interactions. the goal of a recent in-house study was to rationalize the binding interaction of the protoalkaloid taspine ( ) within acetylcholinesterase. taspine was isolated in a bioactivity-guided manner from magnolia x soulangiana and revealed as selective inhibitor of acetylcholinesterase with a significantly higher effect than the positive control galanthamine ( ; ic = . ± . µm). extensive molecular docking studies were performed with human and torpedo californica-acetylcholinesterase employing gold software (vers. . ; www.ccdc.cam.ac.uk/products/life_sciences/gold/). the results suggested taspine to bind in an alternative binding orientation than galanthamine [ ] . while this is located in close vicinity to the catalytic amino acid triad, taspine was found to be mainly stabilized by sandwichlike -stacking interactions in the aromatic gorge of the enzyme. in both case studies the active natural compound was already identified. thus, the in silico tool was not employed for data mining, but to elicit the putative binding mode in the macromolecular target. docking simulations turned out to be excellent tools to get an idea about the assumed molecular ligand target interaction. another approach capitalizes exactly on the just-mentioned observation that computational predictions may reveal an idea about the interaction to a specific target's binding site. thus, it is possible to start with one compound of unknown activity and to mine it against a number of structurally disclosed targets in terms of elaborated pharmacophore models (fig. ) , i.e., parallel in silico screening (see previous). as soon as the orphaned molecule is able to comply with all the requirements and restrictions imposed by any model, it can be assessed as rational hint. consequently, the focused compound will be subjected to a pharmacological testing on the predicted target/s. in this way, the parallel screening is not only helpful to estimate the interactions of a drug candidate with diverse antitargets; or to canvass its interactions to related targets as is performed for an activity profiling. in this approach, the parallel screening is a computational tool for target fishing to get a rational idea about any potential target interaction and to prioritize a few targets for experimental evaluation by applying simple ligand-based or target-based queries. the potential of virtual screening of target libraries was recently discussed by didier rognan [ ] . in his group a structure-based method for target screening was pursued applying inverse docking [ ] . the authors used , structurally well-defined pdb entries to build a d protein library. the virtual screening of this protein library with four unrelated ligands was suitable for recovering the true targets of specific ligands and may as well be used for virtual selectivity profiling of any ligand of interest. nettles and co-authors performed the target fishing approach using a ligand-based procedure [ ] . the potential of both d and d chemical descriptors were compared as tools for predicting the biological targets of ligand probes on the basis of their similarity to reference molecules in a chemical database comprising , biologically annotated compounds. the ligand-based d tool fepops (feature point pharmacophores), which provides pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/receptor complexes, was used for scaffold jumping within the screened database. using atp the authors were able to identify the natural compound balanol ( ) as ligand of cdk . the highest effort applying this strategy is the availability of a representative amount of reliable pharmacophore models covering a wide range of relevant targets (fig. ) . thus, it may be of particular interest to focus on one pathological syndrome, e.g., obesity, inflammation, apoptosis etc., where a phenomenological activity of a np is already evident. applying this approach the disposition of pharmacophore models for targets involved in the respective pathological complex is easier to manage. in this way, a goal-oriented strategy may help to bridge the gap between a phenomenological effect and the underlying molecular mode of action. pertaining to the drug discovery from nature we are facing two facts: (i) statistics show that the myriad of structurally diverse natural compounds are the most favored source of new drugs for clinical use [ ] ; (ii) the drug discovery process has moved towards more rational concepts based on the increasing understanding of the molecular principles of protein-ligand interactions. spurred on by economic interest fundamental advances have been made in research applying data mining strategies, like virtual screening. though being aware of both potentials, their combined benefit could only rudimentary be savored. only limited attempts applying innovative in silico tools in np research are pursued so far, because the search for bioactive compounds is a complex and multidisciplinary challenge. thus, a sensible adaptation of computational strategies is in demand to profit in an economic way from the unique chemical and biological diversity associated with nps. virtual screening techniques, however, must not be used exclusively as activity-predicting tools, since the results provide merely an indication for a putative activity: it is only by the creation of interfaces between computational tools and well-established methods from pharmacognosy that a reasonable standard of success can be achieved. the search for the most effective strategy is best performed by a drug discovery process that involves the exploitation of all the information which can be gathered from bioactivity-guided fractionation, on-line analytical activity profiling, ethnopharmacological screening, chemoinformatics, virtual and in vitro screening studies. in the first instance it behoves modern pharmacognosy to skillfully exploit knowledge from all these fields because it is of paramount importance to sift through the enormous wealth of nps. examples underlining the impact of virtual screening on the identification of active nps have been presented in this survey. though the full potential in this field is by far untapped, these early results indicate that the integrated virtual screening approaches are target-oriented and trendsetting strategies. however, as any computer-based technique, the successful use of virtual screening will entirely depend on the way it is utilized and the quality of its underlying experimental data. the advantages implemented to a virtual screening cycle compared to a conventional in vitro screening are obvious: (i) higher capacity, (ii) no need for isolated compounds, (iii) less experimental efforts for testing; (iv) theoretically, interactions of all known nps to all structurally defined targets can be calculated and predicted, (v) the quality of hit compounds can be increased by additional drug-like filters and virtually restricted adme properties; thus diminishing failures in the early drug development. nevertheless experimental investigations are seminal, but can be focused in a more effective fashion. a cautious handling of virtual hits together with lessons learned from traditional pharmacognosy seems to be crucial for a successful exploitation of treasures from nature. in this area, virtual screening will most likely play an essential role in accelerating the early stage of drug discovery by efficiently digging out lead compounds from nature. rediscovering the sweet spot in drug discovery strategic trends in the drug industry screening for drug discovery: the leading question the role of natural products in a modern drug discovery program natural products as sources of new drugs over the last years strategies for discovering drugs from previously unexplored natural products lessons from natural molecules the role of pharmacognosy in modern medicine and pharmacy rediscovery of known natural compounds: nuisance or goldmine strasburger -lehrbuch der botanik, . auflage, spektrum akademischer verlag plant-derived natural products in drug discovery and development: an overview natural products in drug discovery and development statistical investigation into the structural complementarity of natural products and synthetic compounds initial sequencing and analysis of the human genome recent developments in computational proteomics natural products in drug discovery -concepts and approaches for tracking bioactivity targeted approaches in natural product lead discovery mining information from databases for drug discovery neural networks as data mining tools in drug design chemoinformatics and drug discovery virtual screening in drug discovery the impact of informatics and computational chemistry on synthesis and screening virtual screening: an effective tool for lead structure discovery? virtual screening for bioactive molecules recent advances in docking and scoring high-throughput docking for lead generation virtual screening and fast automated docking methods lead discovery using molecular docking detailed analysis of scoring functions for virtual screening consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins how does consensus scoring work for virtual library screening? an idealized computer experiment sars-cov protease inhibitors design using virtual screening method from natural products libraries sabadinine: a potential nonpeptide anti-severe acute-respiratory-syndrome agent identified using structure-aided design identification of ellagic acid as potent inhibitor of protein kinase ck : a successful example of a virtual screening application structure-based virtual screening for plant-based er -selective ligands as potential preventative therapy against age-related neuro-degenerative diseases structure-based discovery of potassium channel blockers from natural products virtual screening and electrophysiological assay testing pharmacophore modelling: applications in drug discovery molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase- b rational selection of structurally diverse natural product scaffolds with favorable adme properties for drug discovery drugs? drug research? advances in drug research? musings of a medicinal chemist pharmacokinetics and metabolism in drug design physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry distinguishing between natural products and synthetic molecules by descriptor shannon entropy analysis and binary qsar calculations experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings structural biology and drug discovery the protein data bank ligandscout: d pharmacophores derived from proteinbound ligands and their use as virtual screening filters pharmacophore identification, in silico screening, and virtual library design for inhibitors of the human factor xa structure-based pharmacophore design and virtual screening for novel angiotensin converting enzyme inhibitors structure-based pharmacophore identification of new chemical scaffolds as non-nucleoside reverse transcriptase inhibitors development and validation of an in silico p profiler based on pharmacophore models parallel screening: a novel concept in pharmacophore modelling and virtual screening high throughput structure-based pharmacophore modeling as a basis for successful parallel virtual screening parallel screening and activity profiling with hiv protease inhibitor pharmacophore models discovery of embelin as a cell-permeable, small-molecular weight inhibitor of xiap through structure-based computational screening of a traditional herbal medicine three-dimensional structure database structural basis of iap recognition by smac/diablo acetylcholinesterase inhibitory activity of scopolin and scopoletin discovered by virtual screening of natural products chemical feature-based pharmacophores and virtual library screening for discovery of new leads the discovery of new -hydroxysteroid dehydrogenase type inhibitors by common feature pharmacophore modeling and virtual screening pharmacophore modeling and in silico screening for new p (aromatase) inhibitors pharmacophore modeling and three-dimensional database searching for drug design using catalyst pharmacophore modeling and three dimensional database searching for drug design using catalyst: recent advances novel curcumin-and emodin-related compounds identified by in silico d/ d conformer screening induce apoptosis in tumor cells feature based pharmacophore models for sigma receptor, erg and ebp neural networks in chemistry and drug design development of a structural model for nf-b inhibition of sesquiterpene lactones using self-organizing neural networks virtual screening for anti-hiv- rt and anti-hiv- pr inhibitors from the thai medicinal plants database: a combined docking with neural networks approach successful in silico discovery of novel nonsteroidal ligands for human sex hormone binding globulin random forest: a classification and regression tool for compound classification and qsar modeling virtual screening of chinese herbs with random forest comparative analysis of proteinbound ligand conformations with respect to catalyst's conformational space subsampling algorithms pass biological activity predictions in the enhanced open nci database browser a traditional chinese medicine plant-compound database aid its application for searching natural products: sources and databases supernatural: a searchable database of available natural compounds a marine natural product database combining ethnopharmacology and virtual screening for lead structure discovery: cox-inhibitors as application example computer-aided molecular selection and design of natural bioactive molecules phytochemical databases of chinese herbal constituents and bioactive plant compounds with known target specifities strategies for efficient lead structure discovery from natural products integrated in silico tools to exploit the natural products' bioactivity admet in silico modelling: towards prediction paradise? discovering cox-inhibiting constituents of morus root bark: activity-guided versus computer-aided methods ethnopharmacology and bioinformatic combination for leads discovery: application to phospholipase a inhibitors which in vitro screens guide the prediction of oral absorption and volume of distribution? application of the in combo screening approach for the discovery of non-alkaloid acetylcholinesterase inhibitors from cichorium intybus virtual screening in drug discovery: methods, expectations and reality integration of virtual and high-throughput screening quercetin as the active principle of hypericum hircinum exerts a selective inhibitory activity against mao-a: extraction, biological analysis, and computational study taspine: bioactivity-guided isolation and molecular ligand-target insight of a potent acetylcholinesterase inhibitor from magnolia x soulangiana in silico screening of the protein structure repertoire and of protein families recovering the true targets of specific ligands by virtual screening of the protein data bank bridging chemical and biological space: "target fishing" using d and d molecular descriptors key: cord- -k s j sz authors: silvestris, nicola; munafò, antonio; brunetti, oronzo; burgaletto, chiara; scucces, luisa; bernardini, renato title: on the management of drug interactions in the course of concomitant treatments for covid- and antineoplastic agents date: - - journal: front oncol doi: . /fonc. . sha: doc_id: cord_uid: k s j sz nan the unprecedented global pandemic caused by coronavirus (sars-cov ) drew medical attention toward patients with co-morbid disorders, who are more exposed to prognostically unfavorable outcomes ( ) . a peculiar clinical scenario is represented by oncologic patients, who appear more susceptible to covid- infection, as they may develop more severe symptoms than those observed in individuals with different comorbidity profiles ( ). comorbid patients require multiple pharmacological therapies, which may, in turn, result in issues that clinicians are asked to address quickly by considering the possible drug-drug interactions that may occur, with the aim of preventing reduced effectiveness or increased burden of adverse events ( ) . generally speaking, the question of whether concomitant pharmacological therapies may threaten the safety of patients is usually answered in a context that acknowledges the treatment options for each single disease, allowing fair management of interactions on the basis of robust clinical evidence ( ). on the other hand, in case of comorbidities occurring in covid- patients, physicians are now asked to answer the challenging question of whether interactions are possible between pharmacological treatments for covid- , which are not well-defined yet, and antineoplastic agents ( ) . in fact, whilst awaiting results from over clinical trials currently underway, which aim to identify effective therapies against the covid- syndrome, how drugs used for covid- patients (e.g., hydroxychloroquine, antiviral agents, monoclonal antibodies) ( ) may redundantly influence the pharmacokinetics and pharmacodynamics of cancer drugs (e.g., chemotherapy, hormonotherapy, targeted therapy, and immunotherapy) remains an object of investigation ( ) . this issue is crucial interest in patients bearing a cancer who are co-morbid for covid- , while remaining asymptomatic or paucisymptomatic. in these patients, a delay in the administration of a scheduled oncological treatment may have a meaningful impact on both survival and quality of life outcomes ( ) . attention should thus be focused on the interactions of the drugs most commonly used for covid- with different classes of antineoplastic drugs ( table ) . interactions, which require major attention (red color), may occur between antiretroviral agents and chloroquine/hydroxychloroquine with vinca alcaloids, taxanes, anti-estrogens, as well as cdk and tk inhibitors. abbreviations and explanations in the lower part of the table. all checker's matches between drugs were verified in the current literature ( ) ( ) ( ) . in light of the array of possible interactions with systems of hepatic metabolism, such as cytochrome p (cyp ), as most of the actual antiviral agents used in covid- infection are likely to impact on different cyp isozymes, dose adjustments of various drugs may be required for either cancer or sars-cov infection treatments. in fact, respective areas under the curve may be significantly different than expected in case of concomitant administration ( , ) . for example, either atazanavir or the combination lopinavir/ritonavir requires significant dose reduction of cdk / inhibitors, and a full dose may be re-administered after - half-lives ( ) . with the same agents, administration of chloroquine should be intensely monitored because of the increased risk of qtc prolongation ( , ) . also p-glycoprotein, a member of the abc superfamily, regulating the efflux of drugs from cells, and similarly the multidrug and toxin extrusion protein- (mate- ) are involved as targets for significant drug-drug interactions related to the administration of platinum compounds ( , ) . thus, decreased efficacy and increased rates of resulting grade / adverse events (e.g., peripheral neuropathy, qt prolongation, increased risk of infection) need to be constantly and carefully addressed during concomitant anti-covid- -tumor treatments ( , ) . thus, measurement of plasma/serum drug concentrations in the course of treatment appears an opportune procedure to avoid penalizing the efficacy of antiblastic treatments or exacerbation of toxicity. interactions appear more likely to happen when using chemotherapics and/or hormonal therapy and may jeopardize the relatively satisfying results obtained, for example, in diseases such as the hormone dependent breast cancer or prostate cancer, which highly benefit of actually available treatments in terms of survival and quality of life. in order to achieve a better knowledge on the management of different cancer treatment schedules in covid- patients without compromising efficacy and safety, ad hoc observational clinical studies should be implemented with proper clinical endpoints pointing to the dose adjustments needed in case of emerging interactions. ns and rb: manuscript conception, writing, and revision. ob, cb, ls, and am: manuscript elaboration and writing. all authors contributed to the article and approved the submitted version. a novel coronavirus from patients with pneumonia in china covid- infection in cancer patients: how can oncologists deal with these patients? front oncol a global view of drug-therapy interactions pharmacokinetic drug-drug interaction and their implication in clinical management covid therapies and anti-cancer drugs: a systematic review of recent literature pharmacologic treatments for coronavirus disease (covid- ): a review covid- treatment in patients with comorbidities: awareness of drug-drug interactions considerations for interactions of drugs used for the treatment of covid- with anti-cancer treatments drug-drug interaction checker comparison of nine tools for screening drug-drug interactions of oral oncolytics cancer drug interaction information from radboud umc and the university of liverpool the effect of cytochrome p metabolism on drug response, interactions, and adverse effects important drug-drug interactions in hiv-infected persons on antiretroviral therapy: an update on new interactions between hiv and non-hiv drugs ribociclib + letrozole for first-line treatment of hr+, her -abc: efficacy, safety, and pharmacokinetics management of adverse events during cyclin-dependent kinase / (cdk / ) inhibitor-based treatment in breast cancer p-glycoprotein limits ribociclib brain exposure and cyp a restricts its oral bioavailability role of transporters in the distribution of platinumbased drugs p-glycoprotein mediated drug interactions in animals and humans with cancer drug interaction database sensitivity with oral antineoplastics: an exploratory analysis systematic review of the efficacy and safety of antiretroviral drugs against sars, mers or covid- : initial assessment key: cord- -xw o s authors: blasiak, a.; lim, j. j.; seah, s. g. k.; kee, t.; remus, a.; chye, d. h.; wong, p. s.; hooi, l.; truong, a. t. l.; le, n.; chan, c. e. z.; desai, r.; ding, x.; hanson, b. j.; chow, e. k.-h.; ho, d. title: identif.ai: artificial intelligence pinpoints remdesivir in combination with ritonavir and lopinavir as an optimal regimen against severe acute respiratory syndrome coronavirus (sars-cov- ) date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: xw o s the emergence of severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease (covid- ) has led to the rapid initiation of urgently needed clinical trials of repurposed drug combinations and monotherapies. these regimens were primarily relying on mechanism-of-action based selection of drugs, many of which have yielded positive in vitro but largely negative clinical outcomes. to overcome this challenge, we report the use of identif.ai, a platform that rapidly optimizes infectious disease (id) combination therapy design using artificial intelligence (ai). in this study, identif.ai was implemented on a -drug candidate therapy search set representing over , possible drug combinations. identif.ai demonstrated that the optimal combination therapy against sars-cov- was comprised of remdesivir, ritonavir, and lopinavir, which mediated a . -fold improvement in efficacy over remdesivir alone. additionally, identif.ai showed hydroxychloroquine and azithromycin to be relatively ineffective. the identification of a clinically actionable optimal drug combination was completed within two weeks, with a -order of magnitude reduction in the number of tests typically needed. identif.ai analysis was also able to independently confirm clinical trial outcomes to date without requiring any data from these trials. the robustness of the identif.ai platform suggests that it may be applicable towards rapid development of optimal drug regimens to address current and future outbreaks. identif.ai showed hydroxychloroquine and azithromycin to be relatively ineffective. the identification of a clinically actionable optimal drug combination was completed within two weeks, with a -order of magnitude reduction in the number of tests typically needed. identif.ai analysis was also able to independently confirm clinical trial outcomes to date without requiring any data from these trials. the robustness of the identif.ai platform suggests that it may be applicable towards rapid development of optimal drug regimens to address current and future outbreaks. drug repurposing, or the use of approved and investigational therapies for other indications, has been a widely implemented strategy towards treating covid- . examples include clinical studies of ritonavir and lopinavir ( ); hydroxychloroquine in combination with azithromycin ( ) ; favipiravir in combination with tocilizumab (nct ); remdesivir ( ); and losartan (nct ), among others. in one trial, remdesivir met trial endpoints, reducing the median time to recovery from days to days (p < . ), and has ultimately received united states food and drug administration (fda) authorization for emergency use in severe covid- patients ( ) . the majority of trial outcomes are either pending or have not shown clinical benefit over standard of care or placebo. as such, while drug repurposing enables rapid intervention against covid- , there is still a lack of clarity with regards to how to best treat this disease. traditional methods for implementing combination therapy and monotherapy based on drug repurposing rely on mechanism of action (moa)-based drug selection and standard clinical dosing guidelines to achieve drug synergy and therapeutic efficacy. for example, a recent preclinical study showed that remdesivir as well as high-dose chloroquine were efficacious towards sars-cov- in vitro. while this is an established approach that has led to promising candidate therapies, many of these regimens were not able to translate their in vitro outcomes into successful clinical results. therefore, optimal efficacy that is clinically relevant is a different objective that presents substantial challenges to traditional drug screening and repurposing all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint methods. for example, if candidate effective drugs are given in combination at suboptimal respective doses, resulting efficacy is moderate or even absent. at the same time, the relative doses between drugs within a combination can substantially impact treatment efficacy and toxicity due to unpredictable drug interactions. therefore, drug dosing has a critical role in identifying which drugs belong in the optimal combination in the first place. therefore, optimizing treatment outcomes, particularly in combination therapy, ultimately relies on selecting the right drugs at the right respective doses ( , ) . reconciling drug-dose parameters also requires leveraging unpredictable drug interactions in order to mediate maximal efficacy of combination therapies. unfortunately, simultaneously pinpointing these parameters is an extraordinarily complicated task. for example, a parameter space of trillion ( ) possible combinations would be created from a pool of only candidate therapies interrogated at dose levels. this is an insurmountable barrier for traditional drug screening. important studies have previously sought to leverage drug synergy interactions to predict multi-drug combinations ( ) . other strategies have investigated higher order drug interactions to develop antimicrobial drug combinations ( ) . bridging these findings with clinical validation remains a challenge due to the size of the experimental search space. in this study, we sought to overcome these challenges in developing effective combination therapies against sars-cov- infection using the identif.ai platform. identif.ai harnesses a quadratic relationship between therapeutic inputs (e.g. drug and dose) and biological outputs (e.g. quantifiable measurements of efficacy, safety) to experimentally pinpoint optimal combinations from large parameter spaces with a marked reduction in the number of required experiments (fig. ). identif.ai does not use pre-existing training datasets, but rather uses an orthogonally-designed set of calibrating regimens to simultaneously identify effective drugs and corresponding doses that optimize treatment outcomes from prohibitively large drug-dose parameter spaces that cannot be reconciled by brute force drug screening ( , ) . in effect, identif.ai leverages these calibrating regimens to crowdsource sars-cov- live virus responses to experimentally drive all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the efficacy towards an optimal outcome. in this study, identif.ai was applied to a -drug set of candidate therapies to pinpoint clinically actionable combination therapy regimens against the live sars-cov- virus isolated from a nasopharyngeal swab of a patient in singapore ( ) . the drug set included a broad spectrum of repurposed agents that are currently being evaluated in clinical studies for treatment of covid- or being administered in conjunction with these therapies, including remdesivir (rdv), favipiravir (fpv), ritonavir (rtv), lopinavir (lpv), oseltamivir (osv-p), dexamethasone (dex), ribavirin (rbv), teicoplanin (tec), losartan (lst), azithromycin (azt), chloroquine (cq), and hydroxychloroquine (hcq). based on prior studies of minimal resolution experimental design, dosing levels were employed with these drugs, creating a combinatorial space of , regimens ( ) . with a -order of magnitude reduction in required tests, we identified a clinically actionable list of -, -, and -drug combinations ranked based on viral inhibition efficacy with accompanying safety data against kidney epithelial cells (vero e ), liver epithelial cells (thle- ) and cardiomyocytes (ac ). the top-ranked combination was comprised of remdesivir, ritonavir, and lopinavir, which mediated a . -fold increase in efficacy (viral inhibition %) compared to remdesivir alone. further demonstrating the clinical actionability of identif.ai, hydroxychloroquine and azithromycin combination was shown to be a relatively ineffective regimen, mirroring recent clinical results. importantly, the identif.aipinpointed relative efficacy of the combinations and monotherapies was independently confirmatory of many of the clinical trial endpoints to date. these outcomes, coupled with the fact that foundational precursors to identif.ai have been clinically validated for infectious disease, oncology, and organ transplantation human studies, support the potential application of identif.ai as a clinical decision support platform for the optimized design of actionable combination therapy regimens ( ) ( ) ( ) . all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. identif.ai, a dynamic optimization ai-based platform, identifies the drug-dose parameter space by harnessing the quadratic relationship between biological responses to external perturbations, such as drug/dose inputs ( ) . identif.ai analysis of the drug-dose parameter space identifies drug-drug interactions and ranks optimal drug-dosage combinations. this study aimed to use identif.ai to determine effective optimal drug-dosage combinations from a diverse all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. identif.ai analysis correlated drug combinations experimental results into a second-order quadratic series. each independent drug combination inhibition and monotherapy inhibition replicate was used in the optimization process. the second-order quadratic model is as follows: where y represents the desired biological response output (%inhibition), xn is the n-th drug concentration, β is the intercept term, β n is the single-drug coefficient of the n-th drug, β mn is the interaction coefficient between the m-th and n-th drugs and β nn is the second-order coefficient for the n-th drug, while m ≠ n. this second-order quadratic analysis and parabolic response surface plot analysis were conducted using the built-in "stepwiselm" function in matlab r a (mathworks, inc.) with custom-written code. identif.ai derived four quadratic series using bidirectional elimination approach with the p value from the f-statistic as the selection criterion for the experimental results: %inhibition, %cytotoxicity, %cytotoxicity ac , and %cytotoxicity all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint thle- . residual-based outlier analysis was performed for all four identif.ai series. single replicates identified as outliers remained in the data set to account for biological variation. the combinations with all replicates identified as outliers were excluded from the data set and the identif.ai analysis was repeated. identif.ai analysis yielded both drug-drug interaction plots and optimized drug combinations. the optimized drug combinations were ranked according to corresponding %inhibition from the correlated second-order quadratic series with the %cytotoxicity of the celllines (vero e , ac , and thle- ) serving as qualitative indicators for consideration. the predictive power was also calculated via adjusted r to establish the robustness of identif.ai optimization considering the number of drug and drug-drug interaction terms. correlation coefficients were derived from the experimental output values and projected output values for the corresponding drug combinations. all experiments were performed in at least triplicate biological repeats with data presented as means ± standard deviation (sd), unless otherwise stated. shapiro-wilk normality test was used to determine if samples were from normally distributed populations. variance equality was tested with bartlett's test. the kruskal-wallis test by ranks was used for multiple comparisons, followed by dunn's post hoc test for pairwise comparisons. student's two-tailed t test and wilcoxon rank sum test were used for comparing individual samples from normally and nonnormally distributed populations, respectively. bonferroni post-hoc correction was applied to account for multiple comparisons. statistical analyses for coefficient estimation in the identif.ai analyses were performed using sum of squares f-test. alongside the p-values, the results were interpreted in the light of logic, background knowledge and the specifics of the experimental design ( ) . all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. only high concentrations (> µm) of rdv, lpv, cq and hcq achieved half maximal absolute effective concentration (ec ) for the viral inhibition within the tested concentration ranges. high concentrations (> µm) of rtv, lpv and cq led to half maximal absolute cytotoxic all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint concentration (cc ) within the tested concentration ranges (table ) . these results indicated low cellular effects of the selected monotherapies at the tested concentrations. no effect of the maximum vehicle concentration ( . % dmso) was detected on viral inhibition or on cytotoxicity (student's t-test, n = , p > . ). the ec and cc of hcq, cq, rdv, fpv, and rbv were different from previously reported values, attributable to differences in the experimental conditions (e.g. sars-cov- strain, assays, incubation periods) ( , ) . because a common source of failure in translating in vitro results to clinical trials, including for coronaviruses, is the high ratio of ec to maximum plasma concentration (cmax) achieved in the human body, cmax was included as a crucial consideration for selecting drug concentrations at level and level for each drug that ensure none of the drugs were overrepresented in relation to other drugs and to human pharmacokinetics (table , supplemental results) ( ) . regardless of the monotherapy antiviral activity, all drugs were considered for the combinatorial optimization process in order to identify possible unpredictable drug interactions that could markedly impact treatment efficacy and safety. in order for identif.ai to determine optimized drug combinations from this -drug set, drug-dose combinations were generated according to oacd (table s ) and, together with drug monotherapies at concentration level and level , were evaluated for their antiviral and all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. utilizing the single drug and oacd drug treatment data, identif.ai analysis determined rdv/rtv/lpv to be the most efficacious -drug combination. it was also present in all top ranked -drug combinations. rdv/lpv was the top ranked -drug combination ( table ) . while rdv was identified as the most efficacious single drug, in line with current clinical trial outcomes, identif.ai analysis determined that the -drug combination of rdv/rtv/lpv is critical for achieving maximal therapeutic efficacy. identif.ai analysis allows for comparative ranking of all possible combinations within the -drug set, including analysis of regimens currently being clinically investigated but that are not observed as top ranked optimized drug combinations. both lpv/rtv (kaletra) and hcq/azt have been clinically evaluated as potential treatments against sars-cov- infection with discouraging outcomes. identif.ai analysis of these combinations revealed that they were identified to be sub-optimal -lpv/rtv ranked and hcq/azt ranked amongst all drug combinations that include up to -drugs, and predicted viral inhibition efficacies of % and %, respectively. the aforementioned findings were based on the identif.ai quadratic series assessing the %inhibition experimental data with a close proximity as indicated by adjusted r of . (table s ) . multi-parameter identif.ai analysis allowed cytotoxicity of ranked combinations to be interrogated as well via deriving %cytotoxicity quadratic series (table s - was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint and higher %cytotoxicity in the thle- cells. this identif.ai-derived thle- %cytotoxicity was predicted to decrease with the addition of dex in the top -drug combination ( table ) . outlier analysis performed for each identif.ai quadratic series (fig. s -s ) identified and excluded oacd combination from the ac %cytotoxicity data set (fig. s ) and combination from the thle- %cytotoxicity data set (fig. s ) . these data sets were subsequently re-analyzed ( fig. s -s ) . taken together, identif.ai identified rdv-based treatments as likely the most effective therapies against sars-cov- infections, with rdv/rtv/lpv capable of achieving maximal efficacy with potential reductions in overall toxicity if complemented with the fourth drug. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. validation results were interpreted considering not only the p-values, but also the logic, background knowledge and specifics of the experimental design ( ) . the studies confirmed identif.ai ranking of rdv in combination with lpv and rtv as the optimal combination of the study, resulting in complete viral inhibition ( fig. a, table s ). this combination resulted in a . fold increase in efficacy compared to rdv alone. rdv was confirmed as an essential driver of was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . (nct ) did not induce as much viral inhibition as compared to rdv alone. these data confirm that identif.ai can accurately reflect the unsatisfactory outcomes observed in those clinical trials, without incorporating any prior clinical data or drug mechanism assumptions as input. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint drug-drug interaction analysis of identif.ai results were also compared to experimental observations and known clinical investigations. %inhibition identif.ai response surface plot mirrored well-documented and experimentally confirmed synergy between rtv and lpv (fig. a ). in contrast, identif.ai identified an antagonistic interaction between rtv and osv-p (fig. b ), a combination that is currently being investigated in clinical trials (nct ). combining rdv with lpv only, which to our knowledge has not been explored clinically as a registered trial, doubled their individual viral inhibition when added together. accordingly, the corresponding %inhibition identif.ai response surface plot identified a previously unknown synergistic interaction between rdv and lpv (fig. c) . further confirming identif.ai rankings and validation experiments, the rdv/rtv interaction was not significant, but when given in drug combination, rtv boosted the rdv/lpv interaction almost two times (fig. d) . these results further highlight the ability of identif.ai to leverage unexpected drug-dose interactions to identify optimal drug combinations from a massive drug-dose search space. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . this study harnessed the identif.ai platform to interrogate a drug-dose parameter space against the sars-cov- live virus to develop actionable and optimized combination therapy regimens. identif.ai implementation addresses several important factors when designing multi-drug regimens that are best suited for clinical translation from in vitro validation, especially under urgent scenarios like covid- . importantly, identif.ai considers the critical need for simultaneous reconciliation of drug composition and dosing within combination therapy design. moa-based drug selection alone followed by dose finding, while an established method of combination therapy design, presents substantial barriers to the optimization process since drug dosing also plays a role in determining which drugs belong in an ideal combination. in lieu of validating a small number of moa-based potential drug combinations for efficacy which is all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint commonly observed in traditional workflows, identif.ai takes an moa-agnostic approach to efficiently analyze crowdsourced therapeutic responses to the live virus following expansive drugdose exposure to both outline the drug-dose space and define resulting drug-dose compositions of the optimal regimens ( ) . with this data, identif.ai is able to leverage on unexpected dosedependent drug interactions to mediate improved treatment outcomes over moa-based drug selection followed by dose finding. another critical aspect of identif.ai is that the in vitro drug dosing parameter space interrogated in this study is a departure from traditional drug screening approaches. in traditional drug screening, compounds that do not elicit at least a low micromolar ec treatment response during drug dose-response evaluations are typically removed from further consideration, thereby markedly reducing the number of candidate therapies and possible drug combinations. the removal of these drug candidates is a key driver of sub-optimal treatment responses as it ignores a broad spectrum of potential combinations that can be assessed. lack of monotherapy efficacy does not preclude the use of these drug candidates from identif.ai's combinatorial search space. instead, identif.ai's approach allows for continued evaluation of these drugs to determine if they are vital toward driving previously unknown drug interactions that optimize combinatorial treatment outcomes. in addition to being observed in this study, this phenomenon has also been observed with our prior clinical studies in chronic infectious diseases and blood and solid cancers, among other indications ( , , , ) . the outcome of applying identif.ai towards combating sars-cov- infection is an extensive list of combinations ranked by efficacy and/or safety that can be queried by a clinician based on clinically actionable criteria. these include, but are not limited to: highest ranked -, -, -drug combinations by efficacy; highest ranked combinations that do not contain certain drugs due to supply shortages; highest ranked combinations that do not contain certain drugs or contain lower dosages of certain drugs due to patient co-morbidities; and highest ranked combination comprised of only approved therapies, among others. in the context of optimized regimen design, all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . which assesses regimen performance from the entire landscape of possible drug/dose parameters, identif.ai-enabled comparative evaluation of the relative efficacy of a broad spectrum of optimized regimens and clinically investigated regimens also independently confirmed the reported outcomes of clinical trials. this provides additional support for the potential application of identif.ai as a clinical decision support platform. for example, the relatively low efficacy exhibited hcq alone ( . %) or by hcq and azt ( % inhibition) in this study aligned with recent reporting of clinical outcomes for this drug given in mono-and combinatory therapy ( , ). the relatively low efficacy ( . % and . %) of rtv and lpv combination when assessed by identif.ai at two different dosing ratios also aligned with recently reported outcomes showing no benefit over standard care ( ). identif.ai also revealed a relatively low efficacy of fpv monotherapy ( . % inhibition) and various combinations. this was consistent with clinical findings of fpv being potentially clinically effective only when administered with interferon-alpha, not included within our drug library ( ) . of note, rdv alone resulted in the highest relative efficacy for monotherapy ( . %) in this study. to date, compassionate use of rdv resulted in clinical improvement of % of the patients, and a larger study resulted in a statistically significant improvement in median time to recovery from days to days ( ). at the same time, an rdv study in severe covid- patients was also recently terminated early (nct ). nonetheless, it has received fda authorization for emergency use in severe covid- patients. the substantial difference in efficacy observed between sub-optimal and optimal regimens highlights the importance of leveraging platforms such as identif.ai to systematically design combination therapies. this capability, along with the potentially predictive capacity of identif.ai for clinical trial outcomes could provide clinicians with an expanded arsenal of evidence-based candidate treatments and important insights into which potential treatments to further evaluate or potentially avoid under time-sensitive circumstances. it is important to note that the results reported here are derived from primarily an in vitro sars-cov- study. further clinical validation of the outlined combinations in randomized all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint controlled trials will be needed. it should also be noted that, while rdv did not mediate a significant clinical benefit in severe covid- patients, its efficacy in patients with varying disease burden severities should be evaluated further. furthermore, the mixed reported clinical outcomes support the need for improved regimen design of rdv-based treatment. in the event of downstream clinical validation of identif.ai-designed combinations, the drug dosage ratios within the combination may vary from those pinpointed by identif.ai. in addition, it is possible that the optimal drug combinations may vary between patients due to their severity of infection, comorbidities, and other factors. it is for these reasons that potential downstream trials may be effective at determining the potential clinical benefit of the identif.ai-designed combinations if the enrolled patients are stratified by these aforementioned clinical parameters. the -drug search set used in this study did not include every therapeutic option currently under clinical investigation. additional studies, including other repurposed compounds, may yield additional highly ranked and effective combination regimens. also, as identif.ai can be applied to novel small molecules and antibody therapies, their inclusion into the drug pool would add further insight into other potentially actionable regimens. furthermore, given the rapid mutagenicity of rna viruses like sars-cov- , future studies with different drug candidates and different sars-cov- strains may yield different combinations. however, the efficiency and deterministic nature of identif.ai allows it to derive a ranked list of optimal regimens from a given set of drug candidates against a defined in vitro infectious disease model within two weeks. this further supports its potential application as a clinical decision support platform for the optimized design of combination therapy regimens against multiple sars-cov- strains as well as future unknown pathogens that will again require rapid mobilization and clinical guidance for effective treatment options. a.b., t.k., l.h. x.d., e.k.c., and d.h. are co-inventors or previously filed pending patents on artificial intelligence-based therapy development. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial compassionate use of remdesivir for patients with severe covid- nih clinical trial shows remdesivir accelerates recovery from advanced covid- , (nih), access date project identif.ai: harnessing artificial intelligence to rapidly optimize combination therapy development for infectious disease intervention artificial intelligence in cancer therapy prediction of multidimensional drug dose responses based on measurements of drug pairs enhanced identification of synergistic and antagonistic emergent interactions among three or more drugs optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (qpop) epidemiologic features and clinical course of patients infected with sars-cov- in singapore maximizing efficiency of artificial intelligence-driven drug combination optimization through minimal resolution experimental design modulating bet bromodomain inhibitor zen- and enzalutamide combination dosing in a metastatic prostate cancer patient using curate.ai, an individualizing liver transplant immunosuppression using a phenotypic personalized medicine platform application of an ex-vivo drug sensitivity platform towards achieving complete remission in a refractory t-cell lymphoma systematic quantitative characterization of cellular responses induced by multiple signals it's time to talk about ditching statistical significance coronavirus puts drug repurposing on the fast track clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid- pharmacologic treatments for coronavirus disease glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) on the use of corticosteroids for -ncov pneumonia considerations for certain concomitant medications in patients with covid- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) coronaviruses -drug discovery and therapeutic options remdesivir: summary on compassionate use norvir: epar -scientific discussion pharmacokinetics of adjusted-dose lopinavir-ritonavir combined with rifampin in healthy volunteers impact of ribavirin plasma level on sustained virological response in patients treated with pegylated interferon and ribavirin for chronic hepatitis c safety, efficacy and pharmacokinetic evaluations of a new coated chloroquine tablet in a single-arm open-label non-comparative trial in brazil: a step towards a user-friendly malaria vivax treatment food and drug administration, (fda) food and drug administration, (fda) pharmacokinetics of orally administered oseltamivir in healthy obese and nonobese thai subjects pharmacokinetics and biochemical efficacy after single and multiple oral administration of losartan, an orally active nonpeptide angiotensin ii receptor antagonist, in humans therapeutic drug concentrations of teicoplanin in clinical settings hemady: highlights of prescribing information no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection addressing covid- drug development with artificial intelligence output-driven feedback system control platform optimizes combinatorial therapy of tuberculosis using a macrophage cell culture model experimental treatment with favipiravir for covid- : an open-label control study. engineering (beijing) figs. s -s tables s -s references - key: cord- -b mdiont authors: zhou, yadi; hou, yuan; shen, jiayu; huang, yin; martin, william; cheng, feixiong title: network-based drug repurposing for human coronavirus date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: b mdiont human coronaviruses (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and novel coronavirus ( -ncov), lead global epidemics with high morbidity and mortality. however, there are currently no effective drugs targeting -ncov. drug repurposing, represented as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. in this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the hcov-host interactome and drug targets in the human protein-protein interaction network. phylogenetic analyses of hcov whole genomes reveal that -ncov has the highest nucleotide sequence identity with sars-cov ( . %) among the six other known pathogenic hcovs. specifically, the envelope and nucleocapsid proteins of -ncov are two evolutionarily conserved regions, having the sequence identities of % and . %, respectively, compared to sars-cov. using network proximity analyses of drug targets and known hcov-host interactions in the human protein-protein interactome, we computationally identified putative repurposable drugs for the potential prevention and treatment of hcovs. in addition, we prioritized potential anti-hcov repurposable drugs (including melatonin, mercaptopurine, and sirolimus) that were further validated by enrichment analyses of drug-gene signatures and hcov-induced transcriptomics data in human cell lines. finally, we showcased three potential drug combinations (including sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the complementary exposure pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human protein-protein interactome network. in summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations toward future clinical trials for hcovs. coronaviruses (covs) typically affect the respiratory tract of mammals, including humans, and lead to mild to severe respiratory tract infections [ ] . in the past decades, two highly pathogenic human covs (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), emerging from animal reservoirs, have led to global epidemics with high morbidity and mortality [ ] . for example, , individuals were infected and died in the sars-cov pandemic, which cost the global economy with an estimated $ to $ billion [ , ] . according to the world health organization (who), as of november , mers-cov has had a total of , diagnosed cases causing deaths, the majority in saudi arabia [ ] . in december , the third pathogenic hcov, named novel coronavirus ( -ncov), was found in wuhan, china. as of february , , there have been over , cases with ~ deaths for the -ncov pandemic (https://www.cdc.gov/coronavirus/ -ncov/index.html); furthermore, human-to-human transmission has occurred among close contacts [ ] . however, there are currently no effective medications against -ncov. several national and international research . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint groups are working on the development of vaccines to prevent and treat the -ncov, but effective vaccines are not available yet. there is an urgent need for the development of effective prevention and treatment strategies for -ncov outbreak. although investment in biomedical and pharmaceutical research and development has increased significantly over the past two decades, the annual number of new treatments approved by the u.s. food and drug administration (fda) has remained relatively constant and limited [ ] . a recent study estimated that pharmaceutical companies spent $ . billion in , up from $ million in , in the development of an fda-approved new chemical entity drug [ ] . drug repurposing, represented as an effective drug discovery strategy from existing drugs, could significantly shorten the time and reduce the cost compared to de novo drug discovery and randomized clinical trials [ ] [ ] [ ] . however, experimental approaches for drug repurposing is costly and timeconsuming. [ ] computational approaches offer novel testable hypotheses for systematic drug repositioning [ - , , ] . however, traditional structure-based methods are limited when three-dimensional ( d) structures of proteins are unavailable, which, unfortunately, is the case for the majority of human and viral targets. in addition, targeting single virus proteins often have high risk of drug resistance by the rapid evolution of virus genomes [ ] . viruses (including hcov) require host cellular factors for successful replication during infections [ ] . systematic identification of virus-host protein-protein interactions (ppis) offers an effective way toward elucidation of the mechanisms of viral infection [ , infections [ ] , including sars-cov [ ] , mers-cov [ ] , ebola virus [ ] , and zika virus [ , [ ] [ ] [ ] . we recently presented an integrated antiviral drug discovery pipeline that incorporated gene-trap insertional mutagenesis, known functional drug-gene network, and bioinformatics analyses [ ] . this methodology allows to identify several candidate repurposable drugs for ebola virus [ , ] . our work over the last decade has demonstrated how network strategies can, for example, be used to identify effective repurposable drugs [ , [ ] [ ] [ ] [ ] and drug combinations [ ] for multiple human diseases. for example, network-based drug-disease proximity that sheds light on the relationship between drugs (e.g., drug targets) and disease modules (molecular determinants in disease pathobiology modules within the ppis), and can serve as a useful tool for efficient screening of potentially new indications for approved drugs, as well as drug combinations, as demonstrated in our recent studies [ , , ] . in this study, we present an integrative, antiviral drug repurposing methodology that combines a systems pharmacology-based network medicine platform that quantifies the interplay between the virus-host interactome and drug targets in the human ppi network. the basis for these experiments rests on the notions that (i) the proteins that functionally associate with viral infection (including hcov) are localized in the corresponding subnetwork within the comprehensive human ppi network [ ] ; and (ii) proteins that serve as drug targets for a specific disease may also be suitable drug targets for potential antiviral infection owing to common ppis and functional pathways elucidated by the human interactome (figure ) . we follow this analysis with bioinformatics validation of drug-induced gene signatures and hcov-induced . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint transcriptomics in human cell lines to inspect the postulated mechanism-of-action in a specific hcov for which we propose repurposing (figure ). to date, pathogenic hcovs (figure a and b) have been found [ , ] : (i) -ncov, sars-cov, mers-cov, hcov-oc , and hcov-hku are b genera, and (ii) hcov-nl and hcov- e are a genera. we performed the phylogenetic analyses using the whole genome sequence data from hcovs to inspect the evolutionary relationship of -ncov with other hcovs. we found that the whole genomes of -ncov had ~ . % nucleotide sequence identity across three diagnosed patients (supplementary table s ). the -ncov shares the highest nucleotide sequence identity ( . %) with sars-cov among the other known pathogenic hcovs, revealing conserved evolutionary relationship between -ncov and sars-cov (figure a ). hcovs have five major protein regions for virus structure assembly and viral replications [ ] , including replicase complex (orf ab), spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins ( figure b) . the orf ab gene encodes the non-structural proteins (nsp) of viral rna synthesis complex through proteolytic processing [ ] . the nsp is a viral rna-dependent rna polymerase, together with cofactors nsp and nsp possessing high polymerase activity. from the protein threedimensional ( d) structure view of sars-cov nsp , it contains a larger n-terminal extension (which binds to nsp and nsp ) and polymerase domain ( figure c ). the . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint spike is a transmembrane glycoprotein that plays a pivotal role in mediating viral infection through binding the host receptor [ , ] . figure d shows the d structure of the spike protein bound with the host receptor angiotensin converting enznyme (ace ) in sars-cov (pdb id: ack). a recent study showed that -ncov is able to utilize ace as an entry receptor in ace -expressing cells [ ] , suggesting potential drug targets for therapeutic development. in addition, the nucleocapsid is also an important subunit for packaging the viral genome through protein oligomerization [ ] , and the single nucleocapsid structure was shown in figure e . protein sequence alignment analyses indicated that the -ncov was most evolutionarily conserved with sars-cov (supplementary table s ). specifically, the envelope and nucleocapsid proteins of -ncov are two evolutionarily conserved regions, with sequence identities of % and . %, respectively, compared to sars- table s ). however, the spike protein exhibited the lowest sequence conservation (sequence identity of %) between -ncov and sars-cov. meanwhile, the spike protein of -ncov only has . % sequence identity compared to mers-cov. first, we assembled the cov-associated host proteins from known hcovs (sars-cov, mers-cov, hcov- e, and hcov-nl ), one mouse mhv, and one avian ibv (n protein) (supplementary table s ). in total, we obtained host proteins associated with covs with various experimental evidences. specifically, these host . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint proteins are either the direct targets of hcov proteins or are involved in crucial pathways of hcov infection. the hcov-host interactome network is shown in figure a . we identified several hub proteins including jun, xpo , npm , and hnrnpa , with the highest number of connections within the proteins. kegg pathway enrichment analysis revealed multiple significant biological pathways (adjusted p value < . ), including measles, rna transport, nf-kappa b signaling, epstein-barr virus infection, and influenza ( figure b ). gene ontology (go) biological process enrichment analyses further confirmed multiple viral infection-related processes (adjusted p value < . ), including viral life cycle, modulation by virus of host morphology or physiology, viral process, positive regulation of viral life cycle, transport of virus, and virion attachment to host cell ( figure c ). we then mapped the known drug-target network (see methods) into the hcov-host interactome to search for druggable, cellular targets. we found that human proteins ( %, blue nodes in figure a ) can be targeted by at least one approved drug or experimental drug under clinical trial. for example, gsk b, dpp , smad , parp , and ikbkb are the most targetable proteins. the high druggability of hcov-host interactome motivates us to develop a therapeutic strategy by specifically targeting cellular proteins associated with hcovs, such as drug repurposing. the basis for the proposed network-based drug repurposing methodologies rests on the notions that the proteins that associate with and functionally govern a viral infection are localized in the corresponding subnetwork ( figure a ) within the comprehensive human interactome network. for a drug with multiple targets to be effective against an . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint hcov, its target proteins should be within or in the immediate vicinity of the corresponding subnetwork in the human interactome (figure ) , as we demonstrated in multiple diseases [ , , , ] using this drug repurposing strategy. we used a state-ofthe-art network proximity measure to quantify the relationship between hcov-specific subnetwork ( figure a ) and drug targets in the human protein-protein interactome. we constructed a drug-target network by assembling target information for more than , fda-approved or experimental drugs (see methods). to improve the quality and completeness of the human protein interactome network, we integrated ppis with five types of experimental data: ( ) binary ppis from d protein structures; ( ) binary ppis from unbiased high-throughput yeast-two-hybrid assays; ( ) experimentally identified kinase-substrate interactions; ( ) signaling networks derived from experimental data; and ( ) literature-derived ppis with various experimental evidences (see methods). we used a z-score (z) measure and permutation test to reduce the study bias in network proximity analyses (including hub nodes in the human interactome network by literaturederived ppi data bias) as described in our recent studies [ , ] . in total, we computationally identified drugs that were associated (z < - . and p < . , permutation test) with the hcov-host interactome ( figure a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint vs. sars-cov (p < . , t distribution), . vs. mers-cov (p < . ), . vs. ibv (p < . ), and . vs. mhv (p < . ). these network proximity analyses offer putative repurposable candidates for potential prevention and treatment of hcovs. to further validate the repurposable drugs against hcovs, we first performed gene set enrichment analysis (gsea) using transcriptome data of mers-cov and sars-cov infected host cells (see methods). these transcriptome data were used as gene signatures for hcovs. additionally, we downloaded the expression data of drug-treated human cell lines from the connectivity map (cmap) database [ ] to obtain drug-gene signatures. we calculated a gsea score (see methods) for each drug and used this score as an indication of bioinformatics validation of the drugs. specifically, an enrichment score (es) was calculated for each hcov data set, and es > and p < . (permutation test) was used as cut-off for a significant association of gene signatures between a drug and a specific hcov. the gsea score, ranging from to , is the number of data sets that met these criteria for a specific drug. mesalazine (an approved drug for inflammatory bowel disease), sirolimus (an approved immunosuppressive drug), and equilin (an approved agonist of the estrogen receptor for menopausal symptoms) achieved the highest gsea scores of , followed by paroxetine and melatonin with gsea scores of . we next selected potential repurposable drugs ( figure a and table ) against hcovs using subject matter expertise based on a combination of factors: (i) strength of the network-predicted associations (a smaller network proximity score in supplementary table s ); (ii) validation by gsea analyses; . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint (iii) literature-reported antiviral evidences, and (iv) fewer clinically reported side effects. specifically, we showcased several selected repurposable drugs with literature-reported antiviral evidences as below. selective estrogen receptor modulators (serms). an overexpression of estrogen receptor has been shown to play a crucial role in inhibiting viral replication [ ] . serms have been reported to play a broader role in inhibiting viral replication through the non-classical pathways associated with estrogen receptor [ ] . serms interfere at the post viral entry step and affect the triggering of fusion, as the serms' antiviral activity still can be observed in the absence of detectable estrogen receptor expression [ ] . toremifene (z = - . , figure a ), the first generation of nonsteroidal serm, exhibits potential effects in blocking various viral infections, including mers-cov, sars-cov, and ebola virus in established cell lines [ , ] . interestingly, different from the classical esr -related antiviral pathway, toremifene prevents fusion between the viral and endosomal membrane by interacting with and destabilizing the virus membrane glycoprotein, and eventually inhibiting viral replication [ ] . as shown in figure b , toremifene potentially affects several key host proteins associated with hcov, such as rpl , hnrnpa , npm , eif i, eif f, and eif e [ , ] . equilin (z = - . and gsea score = ), an estrogenic steroid produced by horses, also has been proven to have moderate activity in inhibiting the entry of zaire ebola virus-glycoprotein and human immunodeficiency virus (zebov-gp/hiv) [ ] . altogether, network-predicted serms (such as toremifene and equilin) offer potential repurposable candidates for hcovs. with viral infection, including hcovs [ ] [ ] [ ] . irbesartan (z = - . ), a typical arb, was . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint approved by the fda for treatment of hypertension and diabetic nephropathy. here, network proximity analysis shows a significant association between irbesartan's targets and hcov-associated host proteins in the human interactome. as shown in figure c , irbesartan targets slc a , encoding the sodium/bile acid cotransporter (ntcp) protein that has been identified as a functional pres -specific receptor for the hepatitis b virus (hbv) and the hepatitis delta virus (hdv). irbesartan can inhibit ntcp, thus inhibiting viral entry [ , ] . slc a interacts with c orf , a potential transcriptional repressor that interacts with nsp- of sars-cov [ ] . there are several other arbs (such as eletriptan, frovatriptan, and zolmitriptan) in which their targets are potentially associated with hcov-associated host proteins in the human interactome. confirmed the mammalian target of rapamycin complex (mtorc ) as the key factor in regulating various viruses' replications, including andes orthohantavirus and coronavirus [ , ] . sirolimus (z = - . and gsea score = ), an inhibitor of mammalian target of rapamycin (mtor), was reported to effectively block viral protein expression and virion release effectively [ ] . indeed, the latest study revealed the clinical application: sirolimus reduced mers-cov infection by over % [ ] . moreover, sirolimus usage in managing patients with severe h n pneumonia and acute respiratory failure can improve those patients' prognosis significantly [ ] . mercaptopurine (z = - . and gsea score = ), an antineoplastic agent with immunosuppressant property, has been used to treat cancer since the s and expanded its application to several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and crohn's disease [ ] . mercaptopurine has been reported as a selective inhibitor of both . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint sars-cov and mers-cov by targeting papain-like protease which plays key roles in viral maturation and antagonism to interferon stimulation [ , ] . mechanistically, mercaptopurine potentially target several host proteins in hcovs, such as jun, pabpc , npm , and ncl [ , ] (figure d ). anti-inflammatory agents. inflammatory pathways play essential roles in viral infections [ , ] . as a biogenic amine, melatonin (n-acetyl- -methoxytryptamine) (z = - . and gsea score = ) plays a key role in various biological processes, and offers a potential strategy in the management of viral infections [ , ] . viral infections are often associated with immune-inflammatory injury, in which the level of oxidative stress increases significantly and leaves negative effects on multiple organ functions [ ] . the antioxidant effect of melatonin makes it a putative candidate drug to relieve patients' clinical symptoms in antiviral treatment, even though melatonin cannot eradicate or even curb the viral replication or transcription [ , ] . in addition, the application of melatonin may prolong patients' survival time, which may provide a chance for patients' immune systems to recover and eventually eradicate the virus. as shown in figure e , melatonin indirectly targets several hcov cellular targets, including ace , bcl l , jun, and ikbkb. eplerenone (z = - . ), an aldosterone receptor antagonist, is reported to have a similar anti-inflammatory effect as melatonin. by inhibiting mast-cellderived proteinases and suppressing fibrosis, eplerenone can improve survival of mice infected with encephalomyocarditis virus [ ] . in summary, our network proximity analyses offer multiple putative repurposable drugs that target diverse cellular pathways for potential prevention and treatment of . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint hcovs. however, further preclinical experiments and clinical trials are required to verify the clinical benefits of these network-predicted candidates before clinical use. drug combinations, offering increased therapeutic efficacy and reduced toxicity, play an important role in treating various viral infections [ ] . however, our ability to identify and validate effective combinations is limited by a combinatorial explosion, driven by both the large number of drug pairs and dosage combinations. in our recent study, we proposed a novel network-based methodology to identify clinically efficacious drug combinations [ ] . relying on approved drug combinations for hypertension and cancer, we found that a drug combination was therapeutically effective only if it was captured by the 'complementary exposure' pattern: the targets of the drugs both hit the disease module, but target separate neighborhoods ( figure a ). here we sought to identify drug combinations that may provide a synergistic effect in treating hcovs with welldefined mechanism-of-action by network analysis. for the potential repurposable drugs ( figure a) , we showcased three network-predicted candidate drug combinations in the potential treatment of hcovs. sirolimus, an inhibitor of mtor with both antifungal and antineoplastic properties, has demonstrated to improves outcomes in patients with severe h n pneumonia and acute respiratory failure [ ] . the mtor signaling plays an essential role for mers-cov infection [ ] . dactinomycin, also known actinomycin d, is an approved rna synthesis inhibitor for treatment of various cancer . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint types. an early study showed that dactinomycin ( microgram/ml) inhibited the growth of feline enteric cov [ ] . as shown in figure b , our network analysis shows that sirolimus and dactinomycin synergistically target hcov-associated host protein subnetwork by 'complementary exposure' pattern, offering potential combination regimens for treatment of hcov. specifically, sirolimus and dactinomycin may inhibit both mtor signaling and rna synthesis pathway (including dna topoisomerase -alpha (top a) and dna topoisomerase -beta (top b)) in hcov infected cells ( figure b ). toremifene is the approved first generation nonsteroidal serms for the treatment of metastatic breast cancer [ ] . serms (including toremifene) inhibited ebola virus infection [ ] by interacting with and destabilizing the ebola virus glycoprotein [ ] . in vitro assays have demonstrated that toremifene inhibited growth of mers-cov [ , ] and sara-cov [ ] (table ) . emodin, an anthraquinone derivative extracted from the roots of rheum tanguticum, have been reported to have various anti-virus effects. specifically, emdoin inhibited sars-cov associated a protein [ ] , and blocked an interaction between the sars-cov spike protein and ace [ ] . altogether, network analyses and published experimental data suggested that combining toremifene and emdoin offered a potential therapeutic approach for hcovs ( figure c ). figure a , targets of both mercaptopurine and melatonin showed strong network proximity with hcov-associated host proteins in the human interactome network. recent in vitro and in vivo studies identified mercaptopurine as a selective inhibitor of both sars-cov and mers-cov by targeting papain-like protease [ , ] . melatonin was reported in potential treatment of . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint viral infection via its anti-inflammatory and antioxidant effects [ ] [ ] [ ] [ ] [ ] . melatonin indirectly regulates ace expression, a key entry receptor involved in viral infection of hcovs, including -ncov [ ] . jun, also known as c-jun, is a key host protein involving in hcov infectious bronchitis virus [ ] . as shown in figure d , mercaptopurine and melatonin may synergistically block c-jun signaling by targeting multiple cellular targets. in summary, combination of mercaptopurine and melatonin may offer a potential combination therapy for -ncov by synergistically targeting papain-like protease, ace , c-jun signaling, and anti-inflammatory pathways ( figure d) . however, further experimental and clinical validations are highly warranted. in this study, we presented a network-based methodology for systematic identification of putative repurposable drugs and drug combinations for potential treatment of hcov. integration of drug-target networks, hcov-host interactions, hcov-induced transcriptome in human cell lines, and human protein-protein interactome network are essential for such identification. based on comprehensive evaluation, we prioritized putative repurposable drugs ( figure ) and putative drug combinations (figure ) for the potential treatment of hcovs, including -ncov. however, all network-predicted repurposable drugs and drug combinations must be validated in preclinical models and randomized clinical trials before being used in patients. we acknowledge several limitations in our current study. in this study, we used a low binding affinity value of µm as a threshold to define a physical drug-target . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint interaction. however, a stronger binding affinity threshold (e.g., µm) may be a more suitable cut-off in drug discovery, although it will generate a smaller drug-target network. although sizeable efforts were made for assembling large-scale, experimentally reported drug-target networks from publicly available databases, the network data may be incomplete and some drug-protein interactions may be functional associations, instead of physical bindings. we may use computational approaches to systematically predict the drug-target interactions further [ , ] . in addition, the collected virus-host interactions are far from complete and the quality can be influenced by multiple factors, including different experimental assays and human cell line models. we may computationally predict a new virus-host interactome for hcovs using sequence-based and structure-based approaches [ ] . the current systems pharmacology model cannot separate therapeutic antiviral effects from those predictions due to lack of detailed pharmacological effects of drug targets and unknown functional consequences of virushost interactions. drug targets representing nodes within cellular networks are often intrinsically coupled with both therapeutic and adverse profiles [ ] , as drugs can inhibit or activate protein functions (including antagonists versus agonists). comprehensive identification of the virus-host interactome for -ncov, with specific biological effects using functional genomics assays [ , ] , will significantly improve the accuracy of current network-based methodologies. owing to lack of the complete drug target information (such as the molecular 'promiscuity' of drugs), the dose-response and dose-toxicity effects for both repurposable drugs and drug combinations cannot be identified in current network models. for example, mesalazine, an approved drug for inflammatory bowel disease, is . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint a top network-predicted candidate drug ( figure a ) associated with hcovs. yet, several clinical studies showed the potential pulmonary toxicities (including pneumonia) associated with mesalazine usage [ , ] . preclinical studies are warranted to evaluate in vivo efficiency and side effects before clinical trials. furthermore, we only limited to predict pairwise drug combinations based on our previous network-based framework [ ] . however, we expect that our methodology reminds to be a useful network-based tools for prediction of combining multiple drugs toward exploring network relationships of multiple drugs' targets with the hcov-host subnetwork in the human interactome. finally, we aimed to systematically identify repurposable drugs by specifically targeting ncov host proteins only. thus, our current network models cannot predict repurposable drugs from the existing anti-virus drugs that target virus proteins only. thus, combination of the existing anti-virus drugs (such as remdesivir [ ] ) with the networkpredicted repurposable drugs (figure ) or drug combinations (figure ) may improve coverage of current network-based methodologies by utilizing multi-layer network framework. in conclusion, this study offers a powerful, integrated network-based systems pharmacology methodology for rapid identification of repurposable drugs and drug combinations for the potential treatment of hcov. our approach can minimize the translational gap between preclinical testing results and clinical outcomes, which is a significant problem in the rapid development of efficient treatment strategies for the emerging -ncov outbreak. from a translational perspective, if broadly applied, the network tools developed here could help develop effective treatment strategies for other types of virus and human diseases as well. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint in total, we collected dna sequences and protein sequences for hcovs, including three most recent -ncov genomes, from the ncbi genbank database (january , , supplementary table s ). whole genome alignment and protein sequence identity calculation were performed by multiple sequence alignment in embl-ebi database with default parameters. the neighbor joining (nj) tree was computed from the pairwise phylogenetic distance matrix using mega x [ ] with bootstrap replicates. the protein alignment and phylogenetic tree of hcovs were constructed by mega x. we collected hcov-host protein interactions from various literatures based on our sizeable efforts. the hcov-associated host proteins of several hcovs, including sars-cov, mers-cov, ibv, mhv, hcov- e, and hcov-nl were pooled. these proteins were either the direct targets of hcov proteins or were involved in critical pathways of hcov infection identified by multiple experimental sources, including high throughput yeast-two-hybrid (y h) systems, viral protein pull-down assay, in vitro coimmunoprecipitation and rna knock down experiment. in total, the virus-host interaction network included hcovs with host proteins (supplementary table s ). . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint next, we performed kyoto encyclopedia of genes and genomes (kegg) and gene ontology (go) enrichment analyses to evaluate the biological relevance and functional pathways of the hcov-associated proteins. all functional analyses were performed using enrichr [ ] . here, we collected drug-target interaction information from the drugbank database (v . ) [ ] , therapeutic target database (ttd) [ ] , pharmgkb database, chembl (v ) [ ] , bindingdb [ ] , and iuphar/bps guide to pharmacology [ ] . the chemical structure of each drug with smiles format was extracted from drugbank [ ] . here, only drug-target interactions meeting the following three criteria were used: (i) binding affinities, including ki, kd, ic or ec each ≤ μm; (ii) the target was marked as 'reviewed' in the uniprot database [ ] ; and (iii) the human target was represented by a unique uniprot accession number. the details for building the experimentally validated drug-target network are provided in our recent study [ ] . to build a comprehensive list of human ppis, we assembled data from a total of bioinformatics and systems biology databases with five types of experimental . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint evidences: (i) binary ppis tested by high-throughput yeast-two-hybrid (y h) systems; (ii) binary, physical ppis from protein three-dimensional ( d) structures; (iii) kinasesubstrate interactions by literature-derived low-throughput or high-throughput experiments; (iv) signaling network by literature-derived low-throughput experiments; and (v) literature-curated ppis identified by affinity purification followed by mass spectrometry (ap-ms), y h, or by literature-derived low-throughput experiments. all inferred data, including evolutionary analysis, gene expression data, and metabolic associations, were excluded. the genes were mapped to their entrez id based on the ncbi database [ ] as well as their official gene symbols based on genecards (https://www.genecards.org/). in total, the resulting human protein-protein interactome used in this study includes , unique ppis (edges or links) connecting , proteins (nodes), representing a % increase in the number of the ppis we have used previously. detailed descriptions for building the human protein-protein interactome are provided in our previous studies [ , , ] . we posit that the human ppis provide an unbiased, rational roadmap for repurposing drugs for potential treatment of hcovs in which they were not originally approved. given , the set of host genes associated with a specific hcov, and , the set of drug targets, we computed the network proximity of with the target set of each drug using the "closest" method: . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint where ( , ) is the shortest distance between gene and in the human protein interactome. the network proximity was converted to z-score based on permutation tests: where e ccc and e were the mean and standard deviation of the permutation test repeated , times, each time with two randomly selected gene lists with similar degree distributions to those of and . the corresponding p value was calculated based on the permutation test results. z-score < - . and p < . were considered significantly proximal drug-hcov associations. all networks were visualized using gephi . . (https://gephi.org/). for this network-based approach for drug combinations to be effective, we need to establish if the topological relationship between two drug-target modules reflects biological and pharmacological relationships, while also quantifying their network-based relationship between drug-targets and hcov-associated host proteins (drug-drug-hcov combinations). to identify potential drug combinations, we combined the top lists of drugs. then, "separation" measure hi was calculated for each pair of drugs and using the following method: . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint where 〈 • 〉 was calculated based on the "closest" method. our key methodology is that a drug combination is therapeutically effective only if it follows a specific relationship to the disease module, as captured by complementary exposure patterns in targets' modules of both drugs without overlapping toxic mechanisms. [ ] we performed the gene set enrichment analysis as an additional prioritization method. we first collected three differential gene expression data sets of hosts infected by hcovs from the ncbi gene expression omnibus (geo). among them, two transcriptome data sets were sars-cov infected samples from patient's peripheral blood [ ] (gse ) and calu- cells [ ] (gse ) respectively. one transcriptome data set was mers-cov infected calu- cells [ ] (gse ). adjusted p value less than . was defined as differentially expressed genes. these data sets were used as hcov host signatures to evaluate the treatment effects of drugs. differential gene expression in cells treated with various drugs were retrieved from the connectivity map (cmap) database [ ] , and were used as gene profiles for the drugs. for each drug that was in both the cmap data set and our drug-target network, we calculated an enrichment score (es) for each hcov signature data set based on previously described methods [ ] as follows: . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint combinations. an effective drug combination will be captured by the 'complementary exposure' pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human interactome network. zca and zcb denote the network proximity (z-score) between targets (drugs a and b) and a specific hcov. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint coronaviruses -drug discovery and therapeutic options coronavirus infections-more than just the common cold sars and mers: recent insights into emerging coronaviruses early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia putting the patient back together -social medicine, network medicine, and the limits of reductionism the $ . billion pill--methodologic and policy considerations silico oncology drug repositioning and polypharmacology individualized network-based drug repositioning infrastructure for precision oncology in the panomics era drug repurposing: new treatments for zika virus infection? a comprehensive map of molecular drug targets network-based approach to prediction and population-based validation of in silico drug repurposing systems biology-based investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis understanding human-virus protein-protein interactions using a human protein complex-based analysis framework. msystems east respiratory syndrome coronavirus infection fdaapproved selective estrogen receptor modulators inhibit ebola virus infection repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis c virus infection identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen prediction of drug-target interactions and drug repositioning via network-based inference a genome-wide positioning systems network algorithm for in silico drug repurposing deepdr: a network-based deep learning approach to in silico drug repositioning identification of a novel transcriptional repressor (hepis) that interacts with nsp- of sars coronavirus host mtorc signaling regulates andes virus replication host cell mtorc is required for hcv rna replication adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h n pneumonia and acute respiratory failure middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell bird flu"), inflammation and anti-inflammatory/analgesic emodin inhibits current through sarsassociated coronavirus a protein emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction activation of the c-jun nh -terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-jun network-based prediction of drug-target interactions using an arbitrary-order proximity embedded deep forest review of computational methods for virus-host protein interaction prediction: a case study on novel ebola-human interactions pleiotropic effects of statins: new therapeutic targets in drug design integrative functional genomics of hepatitis c virus infection identifies host dependencies in complete viral replication cycle crispr-cas genetic analysis of virus-host interactions acute eosinophilic pneumonia related to a mesalazine suppository mesalamine induced eosinophilic pneumonia molecular evolutionary genetics analysis across computing platforms enrichr: a comprehensive gene set enrichment analysis web server update drugbank . : shedding new light on drug metabolism therapeutic target database update : enriched resource for bench to clinical drug target and targeted pathway information bindingdb: a web-accessible database of experimentally determined protein-ligand binding affinities the iuphar/bps guide to pharmacology: an expert-driven knowledgebase of drug targets and their ligands uniprot: the universal protein knowledgebase studying tumorigenesis through network evolution and somatic mutational perturbations in the cancer interactome a gene gravity model for the evolution of cancer genomes: a study of , cancer genomes across cancer types expression profile of immune response genes in patients with severe acute respiratory syndrome cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target key: cord- -vrqltz s authors: nan title: isar news date: - - journal: antiviral research doi: . /s - ( ) - sha: doc_id: cord_uid: vrqltz s nan as i enter the last six months of my presidency, i'm amazed how fast time has moved. soon we'll convene in la jolla for the th icar, and i'll hand over the reins to josé esté as our next president. we'll also know the results of the election for president-elect, for which we have two excellent candidates in rhonda cardin and johan neyts. after many years of participation and service to isar, it's great to know that we continue to attract such high quality people, who will lead our society forward. the program for the th icar is coming together nicely under the leadership of mark prichard, and we're just entering the abstract submission phase, using our new electronic submission system. i hope that everyone will find the site easy to use, and i look forward to your feedback. the best part of the new submission website is that we can modify many of its features, to provide a much better experience for both abstract submitters and reviewers. as this issue of isar news goes to press, i hope that everyone will have taken a moment to vote for our new officers and board members. for many years, voting has unfortunately remained fairly constant at around % of our membership. this year phil furman and the nominations committee have solicited an excellent slate of candidates, who have all been heavily involved with isar and represent the future of our society. i hope that we'll double the turn-out this year, especially since voting is now easy, using the system set up by our webmaster andrea brancale. the past year has also seen some new developments in isar that i hope will produce lasting benefits. first, raj kalkeri has formed an initial team of isar ambassadors around the world, who are now hard at work soliciting new sponsors and members. the ambassador program utilizes the isar website, facebook and other social media to spread the word about the society and our annual meeting. if you would like to get more involved in isar, i strongly recommend that you join our ambassador program and help bring more people to the meeting in california. we all know scientists from our local geographic areas and countries who would benefit from attending icar, and it will greatly benefit from their new ideas and vision. in another important development, the women in science committee is now soliciting applications for the second wis scholarship program -now proudly known as the chu family foundation women in science scholarships. thanks to generous funding from the chu family, up to five scholarships will be awarded for . applications are due by december st , so please encourage women students and postdocs to submit their applications. unfortunately, one of my goals as president that has not yet materialized is my desire to use the wis program as a model for developing ways to ensure that all young scientists are able to attend icar and contribute to the society. during my last six months in office, i would like to develop a committee of young researchers that would meet at icar and have regular conference calls between meetings, to discuss how the society could promote more participation from graduate students, postdocs and other young investigators. over the years, we've had special sessions at icar featuring talks by our young members, such as the shotgun poster presentations and a fantastic euvirna session at raleigh. we've also held young investigator receptions during the meeting (always one of my favorites!). i hope that many young scientists from around the world will want to get involved in the society through membership on this new committee, and will work with me to promote participation in icar. if you're interested, please email me, so that we can get started! i hope everyone will enjoy this informationpacked issue of isar news, and appreciates the efforts of our publications committee to expand it to four issues per year. it takes plenty of work to pull these issues together in a timely fashion, and i know that the guest editors spend a lot of time sending out reminders for submission of articles. i also hope that our newsletter will begin to feature more news items submitted directly by our membership. please let us know what's going on with you and your research, such as new grants, publications, and products. to summarize: submit abstracts, apply for chu family scholarships and volunteer for the ambassador or young investigator programs! i look forward to seeing everyone in la jolla in april. bob buckheit, isar president the th international conference on antiviral research will be held at the hilton la jolla torrey pines hotel in la jolla, california from sunday, april th through thursday, april st , . this venue will take advantage of the vibrant research community in the san diego area. while the meeting will focus on the latest scientific developments in antiviral research, it will also emphasize the interdisciplinary nature of the field and will foster collaborations by providing dedicated time for networking with colleagues. icar is designed to provide opportunities for virologists, chemists, pharmacologists and clinicians to establish and maintain the close collaborative relationships that are needed for the discovery and development of effective antiviral therapies. it also serves to stimulate innovative thinking on the drug development process, and provides specific events to welcome new scientists to our ranks to help them to establish successful careers. the breadth of viruses discussed and topics covered provide a rich environment to learn how scientists in different fields approach problems common to the development of all antiviral therapies. the conference will begin with a session on drug discovery and development at pm on sunday, april th . rich whitley, a past president of isar, will introduce the new "antiviral drug discovery and development center" funded by niaid/nih. this consortium illustrates how the cooperation of us academic institutions with a not-for-profit research organization and a commercial partner can lead to the rapid development of therapies for emerging infections. this interactive session will feature presentations by maaike everts, mark denison, bob bostwick and rob jordan. the th icar will begin at pm on april th , with presentations by two keynote speakers: ♦ heinz feldmann, m.d., ph.d. (niaid/nih) will speak on "ebola virus: past, present, future", reviewing research from the original discovery of the filoviruses in marburg, germany through the present west african epidemic. he will also provide a look ahead: what research is needed, where do we go from here? ♦ richard h. scheuermann, ph.d. (j. craig venter institute) will speak on: "decoding viral genomics in the next generation era". the availability of whole-genome sequence data, combined with standard representations of virus phenotypic characteristics from large numbers of viral isolates is allowing for extensive genotype-phenotype association studies that go well beyond traditional phylogenetic lineage tracing. his lecture will demonstrate the use of statistical genomics analysis to predict influenza virus evolution in the face of adaptive immunity and to identify novel genetic determinants of disease severity in enterovirus d . the first of two symposia will take place on the morning of monday, april th and will highlight recent developments in the use of structural biology to discover and develop antiviral drugs. the session is being organized by a trio of prusoff young investigator award winners: andrea brancale, bruno canard, and erica ollman saphire. the second symposium, focusing on dna viruses, is being organized by rhonda cardin and graciela andrei and will be held on the morning of wednesday, april th . it will feature recent advances in therapies and will included presentations by thomas lion on adenovirus infections, margaret a. stanley on papillomavirus, david bernstein on herpesvirus vaccines, and timothy kowalik on the evolution of human cytomegalovirus. each year icar features a poster awards competition, and the tradition will continue in . the committee, chaired by kathie seley-radke will review the candidates for awards. in past years, the competition has been intense and the program committee is fortunate to have dedicated members who are willing to serve on this important subcommittee. cash prizes of up to $ will be awarded in the categories of graduate student, postdoctoral fellow and young investigator. awardees will also have the opportunity to present their work in the shotgun presentation session. the prominence of the poster presentations at icar reflects their excellent quality and offers both new and experienced investigators a high-profile venue to present their work. the icar program, the posters presented and many of the posters will be placed on the isar website, so that members can review the material before and after the meeting. the program committee and the society are committed to bringing you the most rewarding scientific experience. in response to feedback from our membership, the committee has worked diligently this year to make changes to the meeting format, keeping the best features of icar, while adding scientific sessions and events which we believe will heighten the experience for all attendees. following the success of the session on women in science at the rome icar, it will be held again in , and will include panel discussions to provide advice to women scientists as their careers progress. the society will also maintain its commitment to the newest isar members by once again sponsoring an interactive career forum, in which attendees meet with established scientists and other professionals in the pharmaceutical, biotech, academic and government sectors to discuss various career options. there is no additional fee for the career forum, but space is limited, so attendees should indicate their interest when registering for the meeting. on thanks to a generous donation from the chu family, as many as five young women with the potential to make significant contributions to the field of antiviral research will receive $ scholarships in . the funds may be used to attend a conference, visit a laboratory, take a course or acquire specialized training. in general, the career development activity should not be one for which the applicant's advisor is already funded. the awards will also include a -year membership in isar and a commemorative certificate. to be eligible, an applicant must be working in an area of antiviral research and either be an undergraduate or graduate student, or have no more than five years of cumulative postdoctoral experience. graduate students and postdocs must be a member of isar at the time of application. each applicant must submit: • a cv and a statement, not exceeding two pages, describing her academic achievements and goals, including an explanation of how the award will help her career. • a letter of support from her research project director, department chair or center director. • if the funds will be used to visit another scientist's laboratory, the candidate must submit a letter from the head of the lab. • if the funds will be used to attend a meeting or take a course, the candidate must provide a description of the activity, including a link to online information. successful candidates will have demonstrated their ability to do independent scientific work, their potential for a high level of scientific endeavor and their leadership skills. for more information, and to apply online for a scholarship, go to http://www.isar-icar.com/?page=wiscda applications must be submitted by december st. winners will be selected by the cff award committee by march st , , and the awards will be presented at the th icar in la jolla. ilane (pronounced "elaine") studied veterinary medicine in her home country of mexico. in her first research project, she joined a group developing a dna vaccine against salmonella infections in chickens, and her thesis won the award for excellence in veterinary public health from paho in . however, when the novel swine-origin h n virus emerged in mexico that year, she switched her focus to influenza diagnostics and epidemiology. working jointly for the ministry of agriculture and the fao, ilane and her colleagues validated new diagnostic tests and screened specimens from hundreds of pig farms. she notes, "i quickly realized that i preferred virology to bacteriology." in , ilane moved to the netherlands to enter a masters program at wageningen university in cellular and molecular biotechnology, focusing on development of next-generation viral vaccines. when it came time for further training, her supervisor drew her attention to the european training network ilane hernández morales on (+)rna virus replication and antiviral drug development (euvirna). she applied for a ph.d. position in the consortium, and in became a euvirna fellow, conducting research on dengue virus in a project that reflects a strong interaction between academic science and the pharmaceutical industry. her day-to-day work is at janssen infection diseases and vaccines, part of j&j belgium, under the supervision of dr. marnix van loock, in collaboration with prof. johan neyts at the rega institute. she is studying the response of primary human mononuclear cells to dengue virus infection, aiming to establish a more relevant in vitro model for antiviral drug testing and to identify potential new antiviral targets, using genome-wide transcriptomic analysis. my primary interest is the study of virus-host interactions. i would like to contribute to the field of antiviral drug development by identifying new targets for prevention or treatment. i'm also strongly committed to bringing new knowledge and research collaborations back to mexico and to learn linking basic research to the "real world." i hope to make a positive impact as an individual -and specifically as a woman scientist -in under-represented groups, developing societies and in science itself. i attended the icar in raleigh, where the emphasis given to connecting with young scientists impressed me. networking was a priority, not only among experienced researchers, but more importantly with young ones. i was very motivated by all the members who invested so much time, effort and "heart" in sessions such as women in science and career discussion and networking. at that time, i already knew andrea brancale, who was my mentor in euvirna. i told him the great joy i felt meeting and learning from all the people attending icar, and asked how i could become part of such a great team. he suggested that i join the isar communications committee and help manage the isar facebook page and twitter media. i could only say "yes!" it was the right moment of inspiration, not just because of the friendly environment, but also to match my long-standing wish to work in science communications. what is your personal experience with social networking and science? i started following virology news by listening to vincent racaniello's podcast, "this week in virology," while on the bus going from antwerp to the j&j lab. it's been extremely valuable -like reading three scientific papers in one hour! also, in the euvirna training program, i attended a workshop on science dissemination and society outreach through social media. i got some "tips and tricks" from professors and experienced teams on blogging, facebook and twitter. together with euvirna fellows, we prepared a blog about viruses and antiviral research, which was reviewed by professor racaniello. he gave us serious feedback that helped me to get some experience in this kind of communication. one of my main activities is to follow science news sources such as science, nature and cdc for items of interest to our members, which i then post on isar's facebook page or send out on twitter. for each item, i include a link to the source and a short comment or paragraph explaining the information. i also contact scientists to ask permission to use figures from their publications. this has been very rewarding, because they typically are willing to share, and they also get to know isar. it's incredible how much networking a simple image can bring. for example, a picture that i posted from the "new attendees" reception at the rome icar reached almost people in less than a week -more than the total attendees! how can isar members make use of our social media sites? first, go to facebook, twitter or linkedin and sign up for an account, then look for isar in the network and like the page (facebook) or follow (on twitter). once you've signed up, then every time isar posts something new, you'll receive a paragraph called a "post" from facebook or a character "tweets" from twitter, and if you're interested, you can follow the link to the complete story. the isar linkedin page is mostly managed by andrea brancale, for sharing information about isar activities. once you have a facebook account, you can post information on the isar page by using the "post" space. you may respond to posts using the "comment" space. once you've signed up for twitter, you'll see the "tweets" automatically in your notification page. you may respond to a new tweet in two ways: "re-tweet," to share it with your followers; and "reply," to post your comments. members can make use of these sites in many ways, such as: • use isar social media to enhance communication with isar members and promote society activities; • network with isar members and with others doing antiviral research; • find reminders for important isar deadlines, such as nominations for the wis award and icar abstract submission; • promote the ambassador program. • find photos from past icars, including the poster sessions and the banquet; • share a link to a recent publication; • exchange information from other webpages and sites, such as news from research groups, job openings, etc. i encourage you to give it a try -there's a big benefit that you can only see once you've tried! the best thing is that social media is flexible and offers different resources for all personalities. other agencies and communities, such as cdc, who, nasa, etc., have had great success using social media. if you would like more information, or have a news item that we could publicize through the isar social network, email me at ilane.hernandezm@gmail.com those of us who follow news of the ebola epidemic are aware of the case of the scottish nurse who was accidentally infected in late while working as a volunteer in sierra leone, was treated at the royal free hospital in london and discharged this past january, but was readmitted to the same hospital in early october, when her ebola virus infection relapsed in the form of acute meningitis (see link below). this recurrent infection is an example of the increasingly recognized ability of ebola virus to persist in anatomical sites that are relatively inaccessible to the immune system, such as the chambers of the eye, the central nervous system and the seminal vesicles. fortunately, it's the only known example to date of a recrudescence involving the central nervous system. another unusual feature of this case is the antiviral medication used to treat the nurse, a new gilead compound, gs- (above) (gilead sciences press release, oct , ). according to isar member robert jordan, who heads the gilead team developing antivirals against respiratory viruses, the parent compound was originally discovered as part of the hepatitis c program, targeting the hcv polymerase, but the strong clinical efficacy of sofosbuvir, especially in combination with ledipasvir, resulted in the molecule being evaluated for other indications, including respiratory viruses such as respiratory syncytial virus (rsv). the story of how gs- moved from in vitro testing against respiratory viruses in california to an ebola patient in a high-security unit in london provides a good example of how a promising drug can be fast-tracked in an emergency. it began in , when robert decided to expand the testing of gilead nucleosides and nucleotides from rsv to two other members of the paramyxovirus family, the highly virulent nipah and hendra viruses, by establishing a collaboration with michael lo in the viral special pathogens branch at the cdc in atlanta. by may , when the ebola epidemic in west africa began to spread, the cdc researchers began testing the gilead compounds against filoviruses and identified a modified ′-cyano-substituted adenine cnucleoside ribose analogue gs- as a novel inhibitor of the west african ebola strain and other ebola species (below). the promising in vitro data led to further collaboration between gilead and usamriid to evaluate gs- in a macaque model of lethal ebola virus disease. a team headed by travis warren tested multiple different treatment doses and regimens, and obtained complete protection when they gave a daily iv dose of mg/kg, beginning on day postinfection. as some of the animals were already viremic when treatment began, the result was very impressive: usamriid science director sina bavari stated in a press release "this is the first example of a small molecule which can be easily prepared and made on a large scale, that shows substantive postexposure protection against ebola virus in nonhuman primates." as a consequence of the successful in vivo testing, gilead filed an ind this past july for the use of gs- to treat ebola virus disease, and phase i testing in healthy human volunteers began in august. when the scottish nurse was hospitalized in early october, sufficient data had been obtained to support compassionate use of the drug. as described by robert and tomas, gs- employs a phosphoramidate prodrug strategy that is similar to sofosbuvir and the recently approved tenofovir alafenamide. the prodrug structure enables the molecule to permeate the cell membrane and rapidly enter the cytoplasm. the ester bond is then cleaved by a cellular hydrolase, followed by chemically catalyzed release of phenol to yield the negatively charged ala-nucleotide conjugate, which is cleaved by an amidase to release the monophosphate nucleotide. cellular kinases then quickly produce the triphosphate molecule, which is incorporated into viral rna, terminating its synthesis. the prodrug strategy thus avoids the slow first phosphorylation kinetics of nucleoside analogues and greatly enhances intracellular concentration of the active triphosphate metabolite. additional in vitro testing at usamriid and at the university of north carolina, chapel hill has found that gs- is also active against the mers coronavirus. it will be interesting to see if gs- will have as great an impact on the treatment of ebola, mers and other severe rna viral infections as sofosbuvir has had on the therapy of chronic hepatitis c. (thanks to travis warren, robert jordan, tomas cihlar and michael lo for information and editing of this article.) http://www.nytimes.com/ / / /world/europe/ new-clues-into-ebola-as-ill-nurseimproves.html?_r= following the success of euvirna (www.euvirna.eu), whose participants received many prizes at icar , a new training network has been funded by the european union: antivirals. its mission is to prepare talented young researchers for leading roles in antiviral drug discovery in european industry or academia, by providing them with a multi-disciplinary, intersectoral training program that may lead to a ph.d. degree. the antivirals partnership includes seven outstanding european academic partners and four industrial partners (see map and logo above). all are leaders in their field of expertise, ensuring state-ofthe-art training, and their skills are highly complementary. three of the academic partners (utrecht university, leiden university medical center, university hospital of heidelberg) specialise in various aspects of molecular virology, while the other four (cardiff university, université d'aix marseille, university of leuven, university of vienna) focus on the identification and development of novel antiviral compounds and strategies. the four industrial partners include a pharmaceutical r&d company specialising in antiviral drug discovery and development (aicuris), another large company specialised in medicinal chemistry (prestwick chemical), a small company that pioneers an exciting novel class of biopharmaceuticals (complix), and a small business that develops antiviral drugs for animals (virovet). a company specialising in training (virology education) will contribute its expertise to the programme. to make a serious impact, the next generation of scientists must do more than good research. the antivirals network will therefore organise training both on relevant research topics, such as virus replication, structural biology and medicinal chemistry, and on a wide range of transferable skills, including teamwork, science communication, dissemination and societal outreach, ethics, biosafety, innovation and entrepreneurship and intellectual property rights. the selected group of young researchers consists of fourteen women and one man from different countries. they were chosen from hundreds of applicants from all over the world, and they excel in enthusiasm, laboratory skills and their potential to become researchers with a broad view and the desire to make a societal impact. they will each work on their own projects, which aim to gain insight into antiviral drug development for a selected group of viruses. in three years, you'll have the opportunity to meet these young interdisciplinary, intersectorial researchers at their final conference, hopefully at isar ! for more information, go to www.antiviralsetn.eu or contact the project manager antivirals-etn@uu.nl. this project has received funding from the european union's horizon research and innovation programme under the marie skłodowska-curie grant agreement . for more than years, scientists at the rega institute have made important contributions to the development and clinical application of antivirals against herpesviruses. that tradition continues with the creation of regavir, the research group for antiviral resistance. graciela andrei and robert snoeck, the program directors, would like to make sure that isar members are aware of what this reference center has to offer. regavir provides clinicians with rapid genotyping and and/or phenotyping of clinical isolates of human cytomegalovirus, herpes simplex virus and , varicella-zoster virus and human herpesvirus . the staff also provide assistance with the interpretation of results and treatment options. since , regavir has performed about , herpesvirus drug-resistance tests, and is now recognized as the belgian reference center for drugresistant dna viruses. thanks to funding from the belgian national cancer plan, an interactive network of hospitals in belgium and abroad has been established and is continuously growing. regavir carries out herpesvirus drug-resistant tests, assists clinicians in the choice of the adapted therapy and promotes scientific interactions in this network. the rationale for the establishment of regavir lies in the increasing importance of antiviral therapy for herpesviruses, which cause significant morbidity and mortality among cancer and leukemia patients, recipients of stem cell and solid organ transplants, neonates, patients with genetic immune deficiencies and hiv+ individuals. these viruses often reactivate and cause persistent infections that require prolonged treatment, increasing the risks for selection of drug resistant mutants. it is remarkable that the antiviralresistance of hsv has remained at a low level (< %) for three decades in immunocompetent individuals, but they are also susceptible to herpesvirus infections in "immune-privileged" sites, such as the eye and the central nervous system; the latter is life-threatening, and requires rapid therapeutic decisions. except for acyclovir, its oral prodrug valacyclovir and famciclovir, current fda-approved drugs for herpesviruses are associated with some toxicity. ganciclovir, its oral prodrug valganciclovir, foscarnet and cidofovir may produce hematologic abnormalities (primarily neutropenia, anaemia, and thrombocytopenia). ganciclovir has also caused long term-reproductive toxicity, and cidofovir and foscarnet are nephrotoxic. alternative antiviral regimens are therefore preferred, to avoid cumulative toxicity and the selection of multidrug-resistant viruses. physicians typically recognize drug-resistant herpesviruses based on signs of infection in patients. regavir aims to support clinical decision-making by providing prompt characterization of the virus. correct use of the few available drugs is necessary to avoid selection of multiple-resistant viruses. simultaneous adjustment of antiviral and immunosuppressive therapy enhances the success of treatment, and also decreases health care costs. for more information, please go to www.regavir.org or contact us at robert.snoeck@rega.kuleuven.be, graciela.andrei@rega.kuleuven regavir@rega.kuleuven.be in march, , antiviral research published two reports of the efficacy of the novel antiviral favipiravir (t- ) against ebola virus infection in ifn receptor-knockout mice. one study, by steve lever's group at the dstl at porton down in the uk showed that aerosol-infected mice were protected by treatment beginning an hour after virus challenge [ ] . the other, from stephan günther's team at the bernhard nocht institute of tropical medicine in hamburg, found that intranasally-infected mice were protected against death when treatment was started as late as days postinfection, when the mice were viremic and showed biochemical evidence of liver injury [ ] . probably because it demonstrated the efficacy of favipiravir late in the course of infection, the paper from the hamburg bsl- lab attracted wide interest, and it has been downloaded some , times, more than any other article in the history of avr. the two simultaneous reports on the anti-ebola activity of favipiravir may have helped to jump-start efforts to deliver the drug to patients in the west african epidemic: an open-label phase ii trial began months later at two treatment centers in guinea, as a collaboration between inserm and médecins sans frontières (see nct on www.clinicaltrials.gov). the first report of antiviral drug testing by the hamburg lab appeared in , when stephan and his colleagues described the utility of rt-pcr in drug screening [ ] . however, antiviral testing has accelerated in the past two years, as postdoc lisa oestereich (below) has taken the lead in evaluating antivirals against several highly pathogenic viruses. she was first author on the study of favipiravir treatment of ebola-infected mice, and in a follow-up paper, she and colleagues in hamburg and in france performed mathematical modeling of the effect of favipiravir therapy on the kinetics of ebola virus replication, using data from the earlier paper [ ] . for her doctoral thesis at the bni, lisa developed a new mouse model of lassa fever, and in a recently published paper, she and coworkers describe the use of that model to demonstrate favipiravir's activity against lassa virus, alone or in combination with ribavirin [ ] . lisa and her colleagues have also examined the efficacy of favipiravir, ribavirin and arbidol in mice infected with crimean-congo hemorrhagic fever virus [ ] . stephan's research in hamburg dates from , when he began a postdoctoral fellowship at the heinrich pette institute, studying hepatitis b virus. the direction of his career underwent a major change in , when he accepted a position as a research group leader at the bni and shifted his focus to tropical medicine and viral hemorrhagic fevers. in , he published his first paper on lassa fever, a report of a case imported into germany [ ] . he has since become a world authority on the disease, as author or co-author of more than articles. in , stephan became head of the department of virology at the bni, director of the bsl- lab and of the who collabor-ating centre. in addition to antiviral drug testing, stephan's group performs routine diagnostic services for more than different tropical and emerging viruses, testing samples from about patients each year, including cases imported into europe. they also routinely carry out isolation and biobanking of lassa, ebola and other bsl- viruses, providing them to academia and companies within the european virus archive (http://www.european-virus-archive.com/). since , the laboratory of virology has had a very successful collaboration with the irrua specialist teaching hospital in nigeria, focusing on the development of rapid diagnostics and research on the pathogenesis and immunology of lassa fever. other collaborative research projects are under way in guinea and ghana. any isar member interested in influenza and ebola virus infections should know about two websites that provide access to a wealth of scientific resources on those diseases. at www.filovir.com and www.influenzavir.com you'll find links to recent news items, scientific publications, official reports, notices of upcoming conferences and other useful information. both sites interface with the worlds of social networking and micro-blogging via twitter, which through manually curated annotation and filtering delivers timely, updated scientific news. in particular, filovir has provided daily coverage of the ebola epidemic in west africa, with tweets of news alerts almost in real time. luca zinzula at the max planck institute, munich the websites are the brainchild of luca zinzula (above), who created them in - , while he was a ph.d. candidate in the lab of enzo tramontano in cagliari, sardinia. the concept of filovir was born when luca found that, despite the existence of numerous virus-related databases on the internet, the information they presented was highly disparate, and it was limited to specific sub-fields and disciplinary areas. especially for ebola and marburg viruses, there was no middle ground between the extreme level of detail of repositories intended for specialists and the superficial (and frequently incorrect) information on non-scientific websites. luca's concept was to bring together information from a wide range of authoritative third-party resources in real time in a single web environment, in which every tool would be readily accessible and navigable. his goal was to create a virtual space where a researcher could find abstracts of the latest publications, pin-point the location of the latest outbreak, retrieve a genome sequence or a protein structure, or hear about upcoming conferences, with everything "just a click away". filovir went live in january , and one year later, influenzavir went online as a twin site focused on influenza viruses. once he had created the two websites, luca realized that he would need help to maintain them, and he was fortunate to obtain the support of two colleagues, massimiliano orsini, a bioinformatician expert in genomic databases, and cristian romagnani, a biologist who has moved to the field of network systems and big data analysis. luca presented the websites in a poster at the th icar in rome. in recognition of his efforts for the benefit of the virology community, he was recognized by antiviral research as the first recipient of an annual award for "most promising antiviral researcher." luca is a native of sardinia. before beginning his scientific studies, he spent several years working as a professional scuba diver, and was frequently involved in rescue and conservation activities for endangered marine species, with a special focus on monitoring infectious disease casualties. after hanging up his fins, he did undergraduate work in biochemistry and virology at the university of cagliari, then continued there for his doctoral research with enzo tramontano. he received his ph.d. from the university of rome tor vergata in for studies characterizing the dsrna-binding activity of the zaire ebolavirus vp protein. since the beginning of , luca has been a postdoctoral fellow in the lab of wolfgang baumeister at the max-planck institute of biochemistry in martinsried, munich, learning cryoelectron micro-scopy and using the technique to further investigate the role of the vp protein in filovirus replication and innate immune antagonism. in addition to his current work with cryo-em, luca's wide-ranging scientific interests include the expression, purification and characterization of recombinant viral proteins, in vitro assays of protein-rna binding and immune evasion by highly pathogenic rna viruses. in , he and enzo published of a remarkably thorough review article in avr on the latter topic: zinzula l, tramontano e. strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit. antiviral res. : - . lieve is an associate professor in the laboratory of virology and chemotherapy at the rega institute, katholieke universiteit leuven, belgium. she is clearly very happy at the university of leuven, as she began her undergraduate studies there in , and has spent her entire career in the rega institute. my passion for virology began during my masters studies, when i did my thesis on the recombinant hepatitis b vaccine. i then by chance was invited for an interview with erik de clercq, and was charmed by his positive and enthusiastic attitude towards young researchers. i immediately decided to do my phd work in his lab. at that time, the acyclic nucleoside phosphonates (anps) had just been discovered, and i was very lucky to be able to work on their preclinical development. my doctoral project focused on the pharmacokinetics and efficacy of antiretroviral anps in animal models, and my direct mentor was jan balzarini. i also had close interactions with the team of antonín holý in prague. in collaboration with gilead, i was the first to demonstrate the in vivo efficacy of the oral prodrug of tenofovir ( ), soon before it became a leading hiv blocker. at the end of my ph.d. period, i got the chance to compile my insights into this successful drug class in a highly cited review article in antiviral chemistry & chemotherapy ( ) . at the start of my postdoctoral period, i left the field of antiretrovirals, but not the anps. having worked with adefovir and tenofovir, it was easy to move to anti-dna virus anps like cidofovir. collaborating with my colleagues graciela andrei, robert snoeck and johan neyts, my new research focused on two dna viruses: adenovirus and hhv- . both are important pathogens, especially for recipients of organ or stem cell transplants, but unfortunately they are very much neglected in antiviral drug development. i also became involved in the hhv- foundation, which aims to keep this neglected virus on the research agenda. for many years, i have been a member of their scientific board. during this "dna virus period", i was assisted by my first two graduate students; our focus was on the virus-host interactions for hhv- and adenovirus, and how this can be combated with novel inhibitors ( , , ) . it was also during this period that i was appointed for academic teaching (i teach virology and immunology). as i just mentioned, i'm on the scientific board of the hhv- foundation, where my main role is to give advice on antiviral therapy. at the moment, however, i'm not performing any drug development for hhv- , since the need for new therapies is not clear. in the clinic, patients are treated with the classical anti-herpetic agents, such as ganciclovir and foscarnet. brincidofovir also appears to be a promising new drug for hhv- infections. after working for several years on dna viruses, i decided to shift my focus to influenza, because it was becoming evident that the current antivirals, which are neuraminidase inhibitors, are not sufficient, and new drugs are clearly needed. from a virological viewpoint, this was a new virus, but from a technical/experimental standpoint, i could rely on expertise i had acquired with other viruses. my group now has two main topics for influenza antivirals: inhibitors of the polymerase complex and inhibitors of viral entry. the first encompasses inhibitors of pa, pb or pb , or a cellular factor associated with the viral polymerase. regarding the influenza pa endonuclease, i am the driving force behind a network of academic researchers with expertise in medicinal chemistry, structural biology, enzymology and virology ( , ) . our cell culture and biochemical data are of high and direct relevance for the development of pa inhibitors, a drug class that several pharmaceutical companies are working on. inhibitors of viral polymerases are a good fit for our lab, because this concept has been a continuous line of research in my career. the second topic includes inhibitors of the hemagglutinin, such as fusion inhibitors ( ), or compounds that interfere with a cellular factor involved in virus entry, such as compounds that inhibit endocytosis or the proteolytic activation of ha ( ). the influenza virus hemagglutinin is an amazing research topic. even though this protein has been studied extensively, there are still knowledge gaps regarding its link to receptor signaling and the biochemical details of its proteolytic cleavage or species adaptation. hence, we use hemagglutinin inhibitors as tools to resolve these basic scientific questions. as for drug development, the hemagglutinin is not an easy target, given its subtypedependent and variable structure. because influenza viruses evolve very rapidly and easily develop drug resistance, i believe that inhibitors of a cellular factor will have high clinical relevance. some of these strategies are explained in our recent review article on influenza virus entry inhibitors ( ) . my influenza team now has seven coworkers. as the pi, i'm responsible for project design, mentoring graduate students, grant applications, writing papers, etc… i also have a network of medicinal chemists, mainly at european universities, with whom i collaborate to design and synthesize inhibitors. this is a very interesting translational aspect of my research. as an expert on anps, i hypothesized that these compounds may act like product inhibitors of hypoxanthine-guanine phosphoribosyltransferase, which is a crucial enzyme in plasmodium parasites and mycobacteria. the validity of the concept was proven in collaboration with the teams of luke guddat in brisbane, australia and the late antonín holý in prague. since our first joint publication in ( ), this has become a totally new concept in antiparasitic drug design. the project has been very successful, with many high-impact papers and citations. my current role in this network is the pharmacological aspect, relating to prodrug design, cell permeability and cytotoxicity. i strongly believe that pharmacologists should look at potentially new applications of inhibitor concepts. for instance, nucleoside analogues have been very successful as anticancer and antiviral drugs, and they can be equally relevant in the fields of antiparasitic or antibacterial therapy. i consider myself a pharmacologist-virologist. i don't do medicinal chemistry, but to reveal the interaction of a molecule with its viral target, i need to understand the sar of inhibitors, so some basic knowledge of chemistry is required. my goals are to find innovative drugs that inhibit viruses and elucidate their mechanism of action. the aim is not only to discover new inhibitors, which may have only short-term clinical relevance, but to use the inhibitors to understand viruses. it's a nice balance between applied and basic research. isar members know andrea (standing, above, with cardiff colleague salvatore ferla) as the winner of the prusoff young investigator award, but not all of us are familiar with the direction of his career, which has increasingly explored the use of computer-based methods to design new antivirals and anticancer drugs. andrea's involvement with antiviral research began in - , when he did an undergraduate project in romano silvestri's lab at the universita "la sapienza" in rome, investigating novel nonnucleoside rt inhibitors. after receiving his degree, he did his doctoral work in chris mcguigan's lab at the welsh school of pharmacy, focusing on the design, synthesis and evaluation of novel antiviral nucleosides. he became a research associate in , and has continued as a staff member. after finishing my undergraduate degree and military service, i wanted to do doctoral research in medicinal chemistry, possibly abroad. i applied to several places in the uk and the us, and chris mcguigan replied right away. it was very exciting to join chris's lab and continue to work in the antiviral field. in particular, i was very happy to have the opportunity to work on nucleoside analogues. difficult chemistry sometimes, but very satisfying when it works as planned! what aspects have you found most satisfying? i always wanted to have a career as an active, independent researcher, and i consider myself very lucky to have achieved it. the best part of my job is the possibility of interacting with a wide variety of collaborators in different fields. it's never boring, and i'm always learning something new. cardiff university, and my school in particular, is a dynamic and stimulating place, always committed to excellence. in particular, it's an ideal place to foster collaborations and develop new ideas: in the time i've been there, seven patents have been filed based on projects i've contributed to, and ten students have received their ph.d. under my supervision. my current group of three postdocs and five graduate students is involved in a variety of collaborative projects, and we receive funding from both the public and the private sector. to a certain extent, chemists like to work in specific fields, especially if they have solid collaborations with biologists, but they're also happy to move in a different direction if they have an indication that their beloved compounds could be interesting in another therapeutic context. from the point of view of computational chemistry, fidelity to a specific research area is even more tenuous, as the methods are "neutral," and can be applied to almost any drug design project [ ] . for example, i've collaborated on projects spanning all the way from designing antivirals against chikungunya virus [ ] to modeling simulations on small peptides for treatment of gastrointestinal diseases. what new approaches are you using in your research? i'm applying several different structure-based methodologies, including molecular docking, homology modeling and molecular dynamics. of course, we also make use of traditional organic synthetic methods and classical medicinal chemistry approaches to design and synthesize novel potential drugs. more recently, i've made a strategic decision to expand my research into the developing area of molecular modeling software. in particular, i've become interested in how researchers relate with the computer and how an interactive interface between the human and the computer would improve the quality of in silico drug design. this has led our group to develop a haptic-driven molecular modeling simulator, which we presented at icar in miami. coming from a medicinal chemistry background, this was a completely new research field for me, but i quickly become fascinated by it. recently, i attended a very interesting faraday discussions meeting on "molecular simulations and visualization." anyone who is interested in understanding where new software and methods are heading should look at the conference papers: http://pubs.rsc.org/en/journals/journalissues/fd#!issue id=fd &type=current&issnprint= - the development of antivirals shares some common ground with the anticancer field, and nucleoside analogues are very important drugs in both fields. in fact, this is not the only common aspect. we are increasingly seeing how some drugs designed to hit target host cell pathways are finding new applications in the antiviral field. some of the clearest examples are cyclin-dependent kinase inhibitors, such as roscovitine, that were developed as anti-cancer agents, but have also shown antiviral activity. efforts to develop new drugs often lead to disappointment, but are sometimes unexpectedly successful. what has been your own experience? disappointment is the most common emotion a researcher faces in his career, so we have to learn to forget failures quickly and use them to move forward with more impetus. i've had several small and some big disappointments in the last few years, but i tend to remember only the successes! potential interaction of a virtual screening hit compound with the bcl- protein, leading to reduced motility of malignant cells, blocking metastasis. at the moment, our most exciting project focuses on the development of an anti-metastatic agent, based on the recently discovered role of the bcl protein in cancer cell motility, especially for mammary cancer (above). it began with a virtual screening simulation just four years ago, but has since become the foundation stone of a spin-off company, tiziana life sciences (http://www.tizianalifesciences.com) which is now listed in the aim section of the london stock exchange. the major focus of your career has been drug discovery for flaviviruses. what are the principal diseases for which antivirals are needed? although vaccines are the best countermeasures against flaviviruses, approved vaccines are currently available only for yellow fever, japanese encephalitis and some tick-borne encephalitis viruses. antiviral drugs are needed for agents lacking approved vaccines, and dengue virus (denv) is the top priority within this category. even for those diseases for which vaccines are available, such as yellow fever, the low vaccination rates in many endemic regions mean that effective therapies are still needed. yes, many lessons can be learned from hcv drug discovery. for example, the pan-genotype coverage and high resistance barrier of sofosbuvir show a clear advantage of the nucleotide analog approach. it would be ideal if a single compound could be identified that inhibits a broad spectrum of flaviviruses. compared to hepatitis c, a chronic disease that is treated for at least weeks, the duration of therapy for dengue or other flaviviral diseases is expected to be no more than a week, so that the toxicity barrier will be much lower. over the past decade, both academia and industry have made effort to develop inhibitors of flaviviruses (especially denv). various approaches have been taken; each approach has proven to have its own challenges (lim et al., ) . nucleoside/nucleotide analogs exhibit the attractive features of broad-spectrum coverage and a high resistance barrier. these advantages have already been demonstrated by an adenosine analog (nitd- ) in both denv cell culture and mouse model (yin et al., ). however, a number of scientific hurdles still need to be addressed. for example, for an anti-denv drug, in what cells and tissues does the virus replicate during the acute phase of infection in humans? how could we engineer compounds to be selectively loaded onto these sites? for those who are interested in the nucleoside/nucleotide approach, please refer to our recent review on this topic (chen et al, ) . protease inhibitors appear to be less promising. in contrast to the hcv protease, for which peptidomimetic drugs have been successful, the two positively-charged amino acid residues located immediately upstream of the flavivirus protease cleavage site make this approach challenging. more effort is needed to develop a denv protease inhibitor. compounds targeting the denv envelope and capsid proteins have also been reported, but the physical/chemical properties of these inhibitors prohibit further development. other types of compounds, such as ns b inhibitors (xie et al., ) , remain to be explored. drugs that target host factors required for viral replication, such as alpha glucosidase inhibitors, or that play a critical role in disease development, such as mast cell stabilizers, are worth investigating. in addition to small-molecule drugs, therapeutic antibodies are being explored for denv and other flaviviruses, and antibodies active across serotypes have recently been reported in cell culture and in mouse models. potent serotype-specific antibodies have also been reported, but this approach will require at least four antibodies, each inhibiting one denv serotype. some of them are expected to enter clinical trials soon. vaccines and antivirals complement each other for disease control and intervention. recent results for front-runner dengue vaccines have shown great promise, despite certain limitations (e.g., weak efficacy against denv serotype for the cyd-tdv vaccine). the ongoing vaccine trials will continue to unravel many questions about dengue diseases and immune response in humans; such information will facilitate antiviral development, especially when antiviral candidates reach clinical trials. i'm really excited to witness and participate in the effort to translate our knowledge about dengue disease to benefit patients. safety and efficacy are the two most important parameters for any therapeutics. for travelers to a dengue-endemic area, prophylactic dosing may be sufficient to prevent disease during a short visit. for people living in endemic areas, it is likely that multiple treatment courses (either prophylactic or therapeutic) will be needed, making drug safety even more important. just as success in developing hcv drugs should benefit dengue antiviral development, success in therapeutic development against denv is expected to facilitate discovery for other flaviviruses. due to similarities among flavivirus proteins, it would be ideal if an inhibitor of denv would cross-inhibit other viruses, even if at a lower efficacy. such inhibitors would then be good starting points for chemical modifications to improve their efficacy against other flaviviruses. this strategy remains to be experimentally investigated. rapid point-of-care diagnostics will be essential, especially for recruitment of dengue patients with early, acute infection for clinical trials. like other acute viral infections, the treatment window for dengue patients is short. the feasibility of such short treatment window can only be experimentally addressed using a safe and efficacious compound in a clinical setting. once efficacy and safety have been established, one could conceive of treating febrile patients in areas with ongoing dengue outbreaks without a confirmed diagnosis. an opportunity that i find especially exciting is to help build partnerships between utmb and leading research institutes in china, especially the wuhan institute of virology of the academy of sciences, which is opening china's first bsl- laboratory. we hope to establish working relationships for the exchange of scientists and collaborative research projects. this year marks the th anniversary of the discovery of the australia antigen, subsequently identified as the hepatitis b virus surface antigen, by baruch blumberg and his colleagues at the national institutes of health and the fox chase cancer center. by providing a specific serum marker of hepatitis b, the discovery led to rapid progress in multiple areas, including the development of diagnostic tests, improved safety of blood transfusion and production of the first hepatitis b vaccine. the ability to detect asymptomatic infections also revealed the huge numbers of people around the world with chronic hepatitis b and its relationship to terminal cirrhosis and hepatocellular carcinoma. this th anniversary is a particularly special event for the hepatitis b foundation and its director, isar member tim block. tim and his wife joan established the foundation in as a public health, advocacy and outreach organization committed to helping people with hepatitis b. in , they created the institute for hepatitis and virus research, as an independent, non-profit organization dedicated to finding therapies "to improve the quality of lives of those affected by hepatitis b and liver cancer." dr. blumberg (below) maintained an office at the institute until his death in . after his passing, the institute was renamed in his honor. the blumberg institute is located within the pennsylvania biotechnology center, a life sciences "incubator" in doylestown, pa, which is home to in addition to hepatitis b, the blumberg institute also has strong programs in antivirals against rna viruses. its filovirus antiviral program has produced novel imino sugars shown to have efficacy in animal models. in , the merck company donated its collection of natural products, including the schering plough legacy, to the institute, which now has one of the most diverse and "druggable" libraries of natural products in the world. institute scientists screen them for antiviral and anticancer leads, and make the collection available to other researchers and institutions. other blumberg institute activities of potential interest to isar members include: --"out and up": established professionals from universities and the pharmaceutical and biotech industry may be offered faculty appointments, funding or space to develop their ideas; --recent college graduates may perform -or -year research fellowships, working with mentors on assigned projects; --graduate students in masters or ph.d. programs may obtain adjunct appointments at the university of pennsylvania, drexel university and other nearby institutions. to mark the th anniversary of the discovery of the australia antigen, the blumberg institute is sponsoring a collection of invited articles in antiviral research. the symposium papers review a range of novel therapies for hepatitis b that are now on the horizon. they can be accessed at http://www.journals.elsevier.com/antiviralresearch/symposia/symposium-hepatitis-b/ isar member bart tarbet reports two new projects at utah state university. the first is a response to recent outbreaks of enterovirus d in the midwestern usa and of ev- in china and other areas: the iar has received a contract from extramural niaid/nih to develop mouse models of enterovirus infections and use them to evaluate therapeutics. bart will direct the study. funding for the first two years will support model development, and if that's successful, additional funds will cover drug testing. the other project is something new for the iar. in october, , bart met dr. emmanuel assana, a professor of veterinary medicine in cameroon in central africa, at the annual meeting of the international society for vaccines. after a discussion of possible research collaborations, they decided to apply for a grant to establish a laboratory in ngaoundéré, cameroon for the surveillance and detection of emerging and re-emerging zoonoses. the government's minister of livestock, fisheries and animal industries has supported the proposal, recommending that the lab be included in the national program for the prevention and fight against emerging and re-emerging zoonoses, part of the usaid program on global health security and emerging pandemic threats. although the award is not yet official, recent word from usaid is that the proposal has been submitted to its implementing partners in one health central and eastern africa. if the grant is approved, bart will help set up the lab and provide training in biosafety and biosecurity, and personnel from cameroon will train in virology and diagnostics in the iar and the utah veterinary diagnostic laboratory. bart would enjoy hearing from isar members who have suggestions for a larger scope of activities for this project, such as the study of infectious disease transmission at the wildlife-livestock interface and environmental drivers of emerging infectious diseases. this year the nominations committee was charged with finding two candidates for the position of president-elect, two for the post of secretary, and six candidates for three positions on the board of directors. they have all given of their time and talents to the society and would make excellent board members. the election was held through the isar website beginning on november th , and has just concluded. we hope that all members voted, and were able to make the difficult choice among this slate of excellent candidates! in , rhonda joined parke-davis pharmaceuticals as a senior scientist, where she supervised the evaluation of anti-herpesvirus compounds in animal models and was also part of the hiv entry inhibitor program. after the pfizer merger, she joined chemocentryx, inc. to work on chemokine therapeutics for diseases and vaccine strategies. in , she joined the faculty of cincinnati children's hospital medical center. her research focuses on murine cytomegalovirus pathogenesis and latency as a model for human cytomegalovirus and is currently nih-funded. since , she has served as co-pi on a nih contract for evaluating novel antivirals and vaccine strategies in cmv and hsv animal models. she joined isar in and has actively attended and presented at icar and has served as a poster judge and herpesvirus co-chair. she is currently a member of the finance, membership, and publications committees and has served on the isar executive board for the past four years. she is also a member of the women in science committee and has led efforts to develop the women in science mentoring program. johan neyts is full professor of virology at the faculty of medicine of the university of leuven (ku leuven) in belgium, where he teaches medical virology at the school of medicine and dentistry. the focus of his laboratory is the development of novel antiviral and vaccination strategies. he is author of ~ peer reviewed papers and holds several patents. sixteen people have obtained their ph.d. degree under his guidance and bachelor or master students have been trained in his laboratory. he is an editor of antiviral research and is on the editorial board of several other journals. johan was co-founder and cso of the ku leuven spin-off okapi sciences nv a biotech company that developed antivirals for veterinary use. he is cso of virovet (www.virovet.com ), a new ku leuven spin-off that is currently being incorporated. research topics covered in his laboratory at the university in leuven include the development of novel antiviral strategies against a number of rna viruses, including flaviviruses (hepatitis c, dengue and others), picornaviruses (entero-and rhinoviruses), alphaviruses (chikungunya and others), paramyxoviruses (rsv and others), rabies, noroviruses and the hepatitis e virus, as well as a novel thermostable dna vaccine technology. johan has attended icars since . in , he received the william prusoff young investigator award. he has also served as a board member and chair of the membership committee. graciela's main scientific activities include the unraveling of the mode of action of novel antivirals, the molecular mechanisms of drug resistance in herpesviruses and poxviruses, the molecular anticancer mechanism of action of nucleotide analogues and the development of organotypic epithelial raft cultures for the study of epitheliotropic viruses. in , she participated in the set-up of the regavir, a translational research platform for typing drug-resistant herpesviruses in immuno-compromised patients who fail antiviral therapy. she has been a member of isar since , and in , she was elected secretary. she is a member of the editorial board of antiviral research and plos one. kara carter has years of experience in virology research and the discovery and development of antiviral agents. following undergraduate research at stanford university, in collaboration with chiron to develop an hsv- -specific diagnostic, she received her ph.d. in virology at university of chicago in the laboratory of dr. bernard roizman, identifying novel genes of hsv- and elucidating the mechanisms of their protein products. she performed postdoctoral studies at harvard university and brigham and women's hospital in the laboratory of dr. elliiott kieff, focusing on ebv-induced transcriptional changes of infected b cells and their effect on transformation. she then moved to praecis pharmaceuticals, where she led drug discovery post-exposure efficacy of oral t- (favipiravir) against inhalational ebola virus infection in a mouse model application of real-time pcr for testing antiviral compounds against lassa virus, sars coronavirus and ebola virus in vitro evaluation of antiviral efficacy of ribavirin, arbidol, and t- (favipiravir) in a mouse model for crimean-congo hemorrhagic fever imported lassa fever in germany: molecular characterization of a new lassa virus strain update on human herpesvirus biology, clinical features, and therapy inhibition of hypoxanthine-guanine phosphoribosyl transferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics pmea (adefovir) and related acyclic nucleoside phosphonate analogues: a review of their pharmacology and clinical potential in the treatment of viral infections antiretroviral efficacy and pharmacokinetics of oral bis(isopropyloxy carbonyloxymethyl)- -( -phosphonylmethoxy propyl) adenine in mice antiadenovirus activities of several classes of nucleoside and nucleotide analogues mutational analysis of the binding pockets of the diketo acid inhibitor l- , in the influenza virus pa endonuclease an integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus pa endonuclease antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin emerging antiviral strategies to interfere with influenza virus entry the search for nucleoside/nucleotide analog inhibitors of dengue virus ten years of dengue drug discovery: progress and prospects targeting dengue virus ns b protein for drug discovery an adenosine nucleoside inhibitor of dengue virus programs in rsv, influenza, hsv and hcv, focusing on cellular targets.in , kara moved to the genzyme corporation, where she led drug discovery programs in virology, immunology and oncology, as well as supporting the transplant business unit in the evaluation and acquisition of antiviral products to complement their portfolio. she is currently head of antiviral research at sanofi. she is an isar member and has served as a member of the women in science committee. since his appointment as lecturer, andrea's research has focused on the use of computer-aided techniques in the design and discovery of novel antiviral and anticancer compounds. in the antiviral field, his research has focused on the in silico design of rna virus inhibitors, including dengue, wnv, hcv, chikungunya virus and coxsackie virus. he is the chemistry editor for antiviral chemistry and chemotherapy. he has been a member of isar since and was elected a board member in . he has also been a member and chair of the website committee since and the publications committee since . he has been the isar webmaster since , and has implemented several technical developments, including online registration and membership management and enhancement of isar's social profile on facebook, twitter and linkedin.randall lanier is vice president for biology at chimerix, inc. before joining chimerix in , he contributed to the hiv and cancer programs at burroughs wellcome, glaxowellcome and glaxosmithkline, where he supervised a clinical virology/immunology laboratory, led teams for drug discovery and was involved in preclinical and clinical development, product differentiation, post-marketing support and licensing opportunity evaluation.randall has over years of experience in the discovery and development of antivirals, and has focused much of his career on understanding the activity, mechanism, and resistance profiles of nucleoside analogs used for prevention and treatment of viral disease caused by hiv, cmv, adenovirus, and poxviruses. his received his undergraduate degree in biology from new college and ph.d. in cellular and molecular biology from the university of texas health science center in san antonio. professor of chemistry and biochemistry at the university of maryland, baltimore county. she earned her ph.d. in organic chemistry from auburn university.her research involves a synthetic organic/medicinal chemistry approach to nucleoside and heterocyclic drug discovery and development. current projects include the investigation of flexible nucleosides/nucleobases "fleximers" for use against sars, mers-cov, ebola, hcv, hiv and other viruses. she has published over peer-reviewed papers, and is the president of the international society of nucleosides, nucleotides and nucleic acids (is na).kathy has chaired the icar poster awards for a number of years. notably, she initiated a new program for is na, the women's career development scholarships, as well as to obtain funding for the women in science scholarships for isar, both of which have been generously funded by the chu family foundation. she is a member of the acs medicinal chemistry division awards committee and is an associate editor for current protocols in chemical biology. karen's interests have also expanded into other areas of women's health, and she is involved in the continuing development of imquest's programs to identify prevention and treatment agents for hiv, hsv- , hsv- , hpv and various bacterial and fungal organisms. she has been first author or coauthor of more than papers on topical microbicide development. she has assisted with the development of isar programs for drug discovery and development and serves on the women in science committee. bill initially worked as a clinical virologist in support of the approved hbv nucleotide prodrugs adefovir dipivoxil and tenofovir disoproxil fumarate, which included characterization of the first clinical adefovir resistance mutations. following their approval, he focused on hcv, which included leading discovery efforts on ns protease inhibitors and overseeing biological support for other programs, including the ns a inhibitor program that led to ledipasvir. he is currently senior director of biology and heads gilead's viral hepatitis discovery biology group. he has been an isar member since , received the william prusoff young investigator award from the society in and was elected to the isar board of directors in . simon tucker is updating the calendar of future conferences on antiviral therapy, medicinal chemistry and other topics of interest that he posted on the isar website last year. isar members can access the calendar by logging in and downloading the pdf. key: cord- -ds hm authors: lee, paul j.; krilov, leonard r. title: developing infectious disease strategies for the developing world date: - - journal: annu rep med chem doi: . /s - ( ) - sha: doc_id: cord_uid: ds hm this chapter discusses various drugs for human influenza a (h n ) and multidrug-resistant mycobacterium tuberculosis (mdr-tb). the h n avian influenza does not presently meet the criteria of an antigenically shifted strain. it is presently an avian strain that has not undergone reassortment with a human strain and is not well adapted to humans. h n isolates are resistant to the m inhibitors amantadine and rimantadine; these antivirals do not have a role for the treatment or prophylaxis against the strain. the neuraminidase inhibitors, oseltamivir and zanamivir, have in vitro activities against the human h n isolates; however, recent data suggest that higher doses for longer periods may be required to be effective. oseltamivir is an oral agent approved for prophylaxis and the treatment of influenza infections. zanamivir is delivered topically to the respiratory tract with similar indications. the drugs discussed in the chapter for mdr-tb fall into three categories—quinolones, nitroimidazoles, and pyrroles. drugs such as moxifloxacin are methoxyfluoroquinolones, which are already available and approved for the treatment of acute respiratory infections, such as community-acquired pneumonia, intra-abdominal infections, acute sinusitis, and skin infections. gatifloxacin , is another methoxyfluoroquinolone that is in clinical development for tuberculosis treatment. new infectious diseases continue to emerge and old ones have re-emerged in recent decades. often these events begin and take root in developing countries. infectious diseases of the developing world or emerging infections are defined by the institute of medicine as an infectious disease that has come to medical attention within the past two decades, or for which there is a potential that its prevalence will increase in the near future. frequently, these diseases exist in nature as zoonoses and cross over to humans when people come into contact with formerly isolated animal population, such as monkeys in a rain forest that become less isolated with deforestation. drug resistant organisms may also be considered as emerging infections since they result from human influence. human immunodeficiency virus (hiv), ebola virus, hantavirus pulmonary syndrome, monkey pox and multidrug-resistant mycobacterium tuberculosis (mdr-tb), severe acute respiratory syndrome (sars) associated coronavirus and avian influenza are examples of emerging infections. many of these infections have arisen and become major health issues in the developing world, in part due to selection factors as noted above. additionally, inadequate resource for public health surveillance and treatment of these illnesses plus poor sanitation and nutritional status may contribute to their flourishing. once established, these illnesses then pose concerns for all mankind as we have truly become a global community. for these same reasons even common infections such as rotavirus gastrointestinal infection or respiratory syncytial virus (rsv) exert a greater toll in the developing world. thus, even as molecular biology advances approaches to diagnoses, new challenges to prevention and treatment of infectious diseases continue to emerge. through a discussion of avian influenza and mdr-tb, primarily, we will demonstrate the magnitude of the problems of infectious diseases in the developing world and discuss approaches that can be taken to in an attempt to monitor and contain these new threats as they emerge. throughout history influenza has been a major infectious disease problem for man causing significant morbidity and mortality. influenza viruses are members of the orthomyxoviridae (myxo ¼ mucous, gr.) family and are classified into three types a, b, and c, based on the composition of their viral nuclear proteins. influenza a viruses are further categorized into subtypes based on the antigenic structure of the two-surface membrane glycoproteins, hemagglutinin (h) and neuraminidase (n). both of these proteins play critical roles in the viral life cycle. the hemagglutinin protein is involved in the process of viral attachment and entry to the host epithelial cell to initiate the process of viral infection. the neuraminidase is involved at the other end of the replication cycle as it functions to snip the cell membrane releasing newly made viral particles from the infected cell. there are distinct hemagglutinins (h -h ) and nine neuraminidase subtypes (n -n ) that have been described in nature. until recently, human influenza viruses only contained h , h , h and n or n subtypes. avian influenza viruses may have additional combinations of the hemagglutinin and neuraminidase proteins. the influenza virus genome is composed of segmented pieces of rna with each coding for a separate viral protein. these viral proteins may change slowly (antigenic drift) through point mutations occurring during viral replication or suddenly (antigenic shift). the latter occurs only in influenza a strains and it results from a combination of the segmented genome described above and the ability of influenza a strains of human and avian origin to co-infect the same host (typically, pigs or swines are most receptive), and share their genetic information. during this process of reassortment a new strain acquiring one of the avian surface protein genes (h or n) may emerge. such a novel strain then eludes pre-existing immunity in people previously infected with influenza and may spread rapidly among the entire population. this defines the potential for worldwide spread or an influenza pandemic. a number of influenza pandemics were documented over the th century with the most notable being the - spanish or swine flu epidemic. during this pandemic, a new for the time h ni strain arose leading to an estimated million deaths worldwide. scientists to this date, continue to study this strain attempting to discern virulence factors that contributed to the severity of illness caused by this strain. it has been suggested that the recently observed avian influenza h n strain to be described below may share some of these same virulence properties which may contribute to the % mortality observed in the limited number of human cases reported to date [ ]. specifically, this virus predominately attaches to lower respiratory tract cells (type ii pneumocytes, alveolar macrophages and nonciliated bronchiolar cells) leading to severe lower respiratory tract damage. however, it does not appear to attach well to upper respiratory tract cells, possibly explaining the low human-to-human risk from this strain [ ] . the crowding of people, swine, and bird species in areas of the developing world such as southeast asia creates an environment that favors the origin of such strains. public health vigilance has contributed to prompt recognition of new strains as they emerge and has contributed to control of their spread through destruction of bird and/or swine populations. the h n avian influenza that has attracted so much attention over the last several years does not presently meet the criteria of an antigenically shifted strain. it is presently an avian strain that has not undergone reassortment with a human strain and is not well adapted to humans. only a small number of people (n ¼ , with deaths) have been confirmed as infected to date, and the vast majority of these individuals apparently acquired the virus through close contact with infected birds and/or consuming undercooked infected bird products [ , ] . still the increasing geographic spread of infected birds beyond asia to africa and europe, the transmission to felids (tigers and leopards) through consumption of raw infected chickens in thailand, and the spread of reported human cases from thailand and viet nam in to cambodia, china and indonesia in and azerbaijan, egypt, iraq, and turkey in , raises concerns for a potential pandemic should further adaptation to humans occur. in the event of such a pandemic should arise, where do we stand in treating and preventing the spread of this virus? since human influenza a (h n ) isolates are resistant to the m inhibitors amantadine and rimantadine, these antivirals do not have a role for the treatment or prophylaxis against this strain. the neuraminidase inhibitors oseltamivir, , and zanamivir, , have in vitro activities against the human h n isolates; however, recent data suggest that higher doses for longer periods ( - days vs. the prior recommendation of five days) may be required to be effective [ ] . oseltamivir is an oral agent approved for prophylaxis and treatment of influenza infections. zanamivir is delivered topically to the respiratory tract with similar indications. oseltamivir resistance in human isolates from viet nam has been noted [ , ] . these strains may not be resistant to zanamivir [ ] . stockpiles of drug will be needed for prophylaxis and treatment if they are to be used in the event of a new pandemic. a long-acting neuraminidase inhibitor, peramivir, , has begun phase i clinical trial to begin to determine whether it may be an intravenous option for this disease [ traditionally, vaccination has been the principal approach to protecting individuals against influenza. currently, no influenza a h n vaccine is available although several candidate vaccines are being developed. preliminary data suggest that either higher concentrations of antigens than used in seasonal influenza vaccines and/or addition of adjuvants to these vaccines will be necessary to induce protective responses [ ] . gearing production, to rapidly make necessary quantities, of such a vaccine in the event of pandemic spread will be a great challenge to the vaccine industry. thus, although great progress has been made in understanding the biology of avian influenza h n and although the virus is not presently capable of efficient human-to-human transmission there is reason for concern as noted above. continued surveillance in animal and human populations and infection control measures to limit its spread are critical, while we learn to better combat this virus (or some other future strain) with pandemic potential. in , when selman waksman received the nobel prize for his discovery of streptomycin, the first effective tuberculosis drug, he proclaimed that the conquest of the ''great white plague'', which was previously incurable and associated with up to % mortality, was in sight. five decades later, tuberculosis remains a global epidemic and currently affects billion people, or / of the world's population, killing million of them annually. the world health organization (who) reports that new infections are increasing by % per year. by , who predicts there will be billion new infections, million of who will develop active disease, and million deaths caused by tuberculosis [ ] . tuberculosis is a particularly frustrating disease to cure because of a multitude of confounding factors. in fact, one of the greatest ironies is that the majority of antibiotics used to treat tuberculosis are still very effective. however, successful treatment requires a combination of multiple drugs for at least six months. the majority ( %) of tuberculosis occurs in the developing world, where the high relative cost, poor accessibility of medications, and weak health infrastructure causes poor compliance and subsequent selection of resistant strains of m. tuberculosis to the drugs it was exposed to. two-thirds of people with tb do not have access to effective therapy. but even when therapy is available and properly utilized, it may be incompletely effective against these multi-drug resistant strains of tuberculosis. a beijing/w genotype strain, which has developed resistance to both first and second line tb drugs, is of particular concern. a recent review combining , patients in studies from countries has found it to be epidemic in cuba, the former soviet union, and south africa, and endemic in east asia [ ] . and, estimates place the cost of treating mdr-tb at $ , -$ , per patient, because of the extra isolation, monitoring, medical supervision, and medication costs required. hence, there has been renewed interest in developing new drugs for the treatment of tuberculosis. current goals include compounds which can successfully treat active tuberculosis in a shorter period of time and/or with less frequent, intermittent dosing; compounds to deal with mdr-tb; and compounds to prevent tuberculosis in people at risk of mdr-tb. in fact, a recent cochrane review covering appropriate studies to address the problem of treating latent tuberculosis infection in people exposed to mdr-tb found there were no randomized controlled trials in the database that have assessed the effectiveness of treatment [ ] . there is also a need for relatively inexpensive drugs and medicines that can be given safely with antiretroviral (arv) medications for hiv, because of the growing problem of tuberculosis and hiv co-infection. one-third of the million people infected with hiv, are also infected with tb, mostly in sub-saharan africa. aids patients are particularly vulnerable to tb because of their weakened immune systems, which makes them more susceptible to contracting tb or allows previous infection with tb that has been suppressed and contained by the immune system to become active. tb is a leading cause of death for people living with hiv/aids, but treating both diseases simultaneously is difficult. many of the first-line tb medications like rifampin cause adverse drug-drug reactions if given with arv medications, because both utilize the same hepatic metabolic pathway. although more than years has passed since the last novel tb drug, rifampin, was discovered, there are a large number of new drugs now in the pipeline. many are still in early preclinical stages, but this discussion will focus on those already in clinical human trials and of particular relevance to the developing world. moxifloxacin, , is a methoxyfluoroquinolone which is already available and approved for the treatment of acute respiratory infections such as community-acquired pneumonia, intra-abdominal infections, acute sinusitis, and skin infections. it is an inhibitor of dna gyrase, which is an enzyme important in bacterial growth and replication. it has an excellent safety profile and has already been used by million patients worldwide. however, the drug has not been adequately evaluated in children and pregnant or lactating women. in vitro and in vivo studies have demonstrated good activity against m. tuberculosis [ , ] . moxifloxacin has a minimum inhibitory concentration (mic) against m. tuberculosis fourfold lower than levofloxacin, a second-generation fluoroquinolone already in use for mdr-tb. (older fluoroquinolones like levofloxacin and ofloxacin are only used for mdr-tb treatment because of initial studies showing lack of superiority to existing first-line drugs.) it also has a long half-life and high area under the curve concentration. a murine model study found that substituting moxifloxacin for isoniazid decreased the overall time necessary for tb eradication by two months. moxifloxacin is currently in four different phase ii trials in eight countries -brazil, canada, south africa, spain, tanzania, uganda, the united states, and zambia, with a goal of demonstrating efficacy as a first-line component of a shorter, standardized tb treatment regimen. the hypothesis is that moxifloxacin substitution for isoniazid or ethambutol in the usual four drug standard regimen (isoniazid, rifampin, ethambutol, and pyrazinamide) during the first two months of treatment will significantly increase the proportion of patients with culture-negative sputum at weeks, compared with the standard regimen. phase ii trials, are expected to complete in , with phase iii trials to follow. moxifloxacin metabolism also does not utilize the hepatic cytochrome p- system, and does not lead to adverse reactions when taken alongside arvs. however, one recent study in italy found that / patients treated with long-term moxifloxacin for tb developed adverse reactions. none of the reactions was irreversible or fatal, and moxifloxacin was felt to be safe and result in treatment success in / patients. the success rate was only / in mdr-tb patients [ ] . gatifloxacin, , is another methoxyfluoroquinolone that is actually furthest along in clinical development for tuberculosis treatment. like moxifloxacin, it is used to treat a variety of bacterial infections, has been used globally for many years, and has no interactions with arv drugs. gatifloxacin is a structurally similar to moxifloxacin and likely to have the same mechanism of action against bacterial dna gyrase. this means cross-resistance is possible between these two drugs. gatifloxacin's toxicity and drug-drug interaction profile is similar to moxifloxacin's. early clinical studies in brazil have shown gatifloxacin to possess equivalent antituberculosis activity to moxifloxacin and similar, but slightly less potent, activity to isoniazid [ ] . one murine study suggests that gatifloxacin in combination with ethionamide or pyrazinamide, could be an alternative regimen to cure active tb. this would be important in treating patients with drug-resistant tuberculosis where the standard isoniazid/rifampin-containing regimen would be ineffective [ ] . a large multicenter phase iii clinical trial is currently enrolling a planned patients in south africa, senegal, kenya, benin, and guinea to evaluate whether using gatifloxacin instead of ethambutol can shorten the standard tb treatment in adults to four months. this study began in , and is to be completed in [ ]. an additional quinolone derivative, tmc , , (formerly known as r ) is currently being studied in phase ii trials in patients with tuberculosis. it is a diarylquinolone, with activity against both sensitive and mdr strains of tb. in a murine model it appeared equivalent to both isoniazid and rifampin at lower doses, and superior to the combination of isoniazid, rifampin, and pyrazinamide bactericidal activity by at least log unit when combined at a higher dose with any two of the three. it even appeared to be superior to the combination as monotherapy. in combination with isoniazid or rifampin, tmc decrease tb sterilization time from four to less than two months. its activity appears to be directed against mycobacterial atp synthase, giving it a unique mechanism of action, compared with other tuberculosis drugs, including fluoroquinolones like moxifloxacin. moreover, the half-life was greater than hours, suggesting once weekly dosing is possible [ ] . pa- , , is a nitroimidazopyran discovered in as a potential cancer drug. in , a study revealed its potency against m. tuberculosis [ ] . it has a novel mechanism of action inhibiting protein and lipid synthesis, which is effective against both tb and tested clinical isolates of mdr-tb. it attacks the mycobacteria in both the initial, intensive phase of tb therapy, as well as the later, continuation phase. in the intensive phase, pa- has bactericidal activity comparable to isoniazid, and in the continuation phase, its bactericidal activity is close to the combination of isoniazid and rifampin. pa- also has activity against both actively replicating and the static, slow growing mycobacteria. this has the potential for shortening the course of therapy, since many current drugs are only active against actively replicating mycobacteria, and m. tuberculosis spends weeks to months in its static, slow-growing phase. in a murine model, pa- was more potent than rifampin when used in combination with moxifloxacin and pyrazinamide, and that when all four drugs were used together, the duration of treatment could be reduced to months or less [ ] . pa- successfully completed a phase i clinical trial in the u.s. in august , and a phase ii trial is planned for later in , ending in . pa- has a bioavailability of about % and single oral dose rapidly travel to important target sites like the lung and spleen. pa- also does not inhibit the hepatic cytochrome p- isoenzyme system, suggesting it could be safely used with arvs [ ] . there is no evidence of genotoxicity, by such standard tests as ames. the global alliance for tb drug development, a non-profit, public-private partnership has directed and funded pa- 's development and has obtained exclusive worldwide rights to pa- and its derivatives, so that the technology will be available royalty free in endemic countries. work has also begun on second-generation nitroimidazoles. in japan, a nitroimidazo-oxazole opc- , , has entered phase i clinical trials and a backup nitroimidazo-oxazole is in early development. preclinical data has been encouraging so far [ ] . second-generation nitroimidazopyran analogs with greater in vivo potency in animal models have also been identified. sudoterb belongs to a class of compounds known as pyrroles, which are plant alkaloids. sudoterb has been reported to have potent anti-tb activity in vitro and in vivo. in vitro, sudoterb has bactericidal activity similar to isoniazid and is synergistic with rifampin. the combination of sudoterb with isoniazid, rifampin, and pyrazinamide led to complete sterilization of both sensitive and mdr-tb strains in mice within two months, and in combination with rifampin and pyrazinamide, cured tb in all animals after three months. sudoterb exhibits good oral bioavailability with once daily dosing. phase i studies began in india in [ ] . the structure of sudoterb has not been released. currently, bcg (bacille calmette-gue´rin) is the only licensed tuberculosis vaccine. it has a number of shortcomings, among which is a relatively short period of protection, and poor antibody stimulation response beyond childhood. since there are clearly many problems with treating tb at this point, a lot of effort and money has been focused on creation of a vaccine, as a means of preventing future infections in those at highest risk and putting a cap on the ever increasing tb epidemic. unfortunately, it appears the obstacles in the development of such a vaccine are no less challenging than those hindering drug development. it is well know that bcg varies in effectiveness among those who receive it. this has previously been attributed to different environmental factors or differences in the bcg strains used to create the vaccine. however, a recent gene analysis has found that m. tuberculosis is genetically distinct in different parts of the world and has evolved into different lineages: east-african-indian, east-asian, euro-american, indo-oceanic, and two west-african. these strains appear to have adapted over time to specific human populations, which suggest separate, wholly different vaccines might be required for each region [ ] . those who receive an effective vaccine may still not develop adequate protection. bcg is known to stimulate th immune cells, which leads to the development of protective antibody and drives m. tuberculosis into latency in the body. in many areas of the developing world, parasitic helminthic infections are extremely common. parasites stimulate a th immune cell response, usually associated with an allergic response. this increase in background th response is antagonistic to the th cell response, and, in theory, will blunt or lead to an inadequate immune, as well as cause impaired bactericidal activity [ ] . nevertheless, work continues on a vaccine. a leading candidate, mva a, utilizes a recombinant vaccinia virus ankara (mva) expressing antigen a. mva a completed a phase i clinical trial in the united kingdom in . when given with bcg, th levels were upto -fold higher than groups given mva a or bcg alone [ ] . further clinical trials in the united kingdom and gambia are in progress. another vaccine, mtb f/as a consisting of a recombinant fusion protein (mtb f) formulated in a proprietary adjuvant system (as a) has shown promising results in preclinical studies to induce strong, long lasting cellular and humoral immune responses. in addition, the vaccine has shown a good safety and immunogenicity profile in phase i trials. phase ii trials are planned to being shortly [ ] . even with concerns with the potential efficacy of a vaccine, there may still be a role for its use. combining the use of a vaccine with antituberculosis drugs seems to augment the bactericidal activity of the drugs, particularly with regard to the slowgrowing mycobacteria [ ] . we are still years away from actually seeing any of these developments in tuberculosis treatment actually enter clinical practice. but if their full potential is achieved, then it is likely that none of the current first-line drugs will be part of the new, optimized regimen, which should finally begin to decrease both the incidence of tuberculosis, and the development of mdr-tb. it is clear that although drug and vaccine development for both avian influenza and mdr-tb is well underway, there is so much more that needs to be done. currently, it is the developing world which is most affected by both of these infections; yet, the drugs and vaccines are not being optimized for these areas. drug regimens need to be shorter and simpler, not only to make them affordable to the population at risk but also to prevent the development of drug resistance. vaccines of more widespread efficacy are also needed to protect as well as prevent these infections from becoming global epidemics. hopefully, this review has demonstrated the impact for all mankind of emerging infections in the developing world and will stimulate ongoing development of approaches aimed at addressing these issues for the people affected, and for the entire world. european concerted action on new generation genetic markers and techniques for the epidemiology and control of tuberculosis abstract f- , th icaac abstract f- , th icaac proc. acad key: cord- -berrojmq authors: burri, christian title: sleeping sickness at the crossroads date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: berrojmq human african trypanosomiasis (hat; sleeping sickness) is a disease with truly historic dimensions [...]. these activities marked the start of incredible efforts of the colonial powers to control the disease, but particularly also of the vertical approach towards disease control, with special programs run in parallel with the public health system-which is now one of the challenges in the elimination "end game" with very few patients remaining requiring an integrated public health approach. the detection of the causative agent, the mode of transmission and the first documented major epidemics coincided with the advent of modern pharmacology. it is, therefore, no surprise that there was an interest to find drugs against this disease. in , the colonial powers, in a joint conference, decided to give drug development a high priority and in course, several molecules were developed. the history of the oldest drug still in use against hat, suramin, is described in the context of the history in the article of madeja et al., not only is the drug still manufactured, but also the continued support of bayer since the year is a major contributing factor allowing us to write about the elimination of hat today. the activity of inorganic arsenic-based compounds had already been recognized in the mid- th century and the treatment of "nagana" (animal trypanosomiasis) was described by david livingstone in and david bruce in . this knowledge led to the development of the first organo-arsenic compound atoxyl ® in by paul ehrlich, who used trypanosomes as a model to screen molecules, but was mainly searching for drugs against syphilis. wolferstan thomas in liverpool subsequently showed that atoxyl ® was effective against t. b. gambiense. however, atoxyl ® , meaning 'non-toxic', caused severe adverse drug reactions particularly affecting the optic nerve; it was only active against early stage hat and was followed by tryparsamide in . tryparsamide was developed in the usa and was the first drug to be active against the late stage, although not very active and also prone to extensive resistance development. tryparsamide was also very toxic, with a dose dependent risk damaging the optic nerve. this is documented in a horrifying report from when a lieutenant of the french army in cameroon doubled the prescribed dose of tryparsamide to speed up the recovery of patients. two days later, all these patients were blind. this event prompted the swiss chemist and physician dr. friedheim to investigate alternative drugs. the introduction of a triazine ring lead to the development of melarsen (disodium p-melaminyl-phenyl arsonate) in , which proved to be a very efficient drug against t. b. gambiense, but was still very toxic. in , friedheim first described the trivalent arsenoxide form of melarsan, known as melarsenoxide. he reported its use in treatment of human sleeping sickness in . the main achievement was the reduction of the duration of therapy to six weeks, with two weeks of seven daily injections of . mg/kg each, spaced by an interval of one month. in a further step, capping the arsenic in melarsen oxide with british anti-lewisite, an antidote to the arsenical warfare agent lewisite, reduced the toxicity by a factor of the order of , but the trypanocidal activity only by a factor . . the new drug was called melarsoprol (mel b; arsobal ® ) [ ] . it remained the mainstay of second stage hat treatment until the early s despite the long treatment duration of days, the known related adverse drug reactions, particularly the encephalopathic syndrome that occurs in about % of the patients treated and leads to their death in about % of cases, and the potential for drug resistance. another line of research was the molecules with a diamidine structure discovered in the s. these molecules were detected by serendipity in the search for hypoglycaemic compounds, with the idea in mind that this effect might compromise the very prominent and particular glucose metabolism of trypanosomes. several compounds, however, proved to have a direct trypanocidal effect with the three compounds stilbamidine, pentamidine, and propamidine identified to have the highest activity [ ] . pentamidine does not penetrate the blood brain barrier so its use is limited to first stage hat; despite this drawback it is still is in use today, for treatment of children below six kilograms. the distinctly better safety profile of the drugs only active against first stage disease (pentamidine and suramin) versus melarsoprol was also the advent of the consistent performance of lumbar puncture to determine the disease stage and make a treatment decision. the need for a lumbar puncture was, for over years, a characteristic of hat treatment, a source of patient distress, stigma and technical limitation of treatment. some years after the discovery of pentamidine, the diamidines again became the focus of drug development, although not successful until today. the development of the organo-arsenicals, diamidines, and also drugs developed much later and still in use like eflornithine and nifurtimox, including their drawbacks related to the potential for drug resistance are described in detail in the contribution of de koning. the "scramble for africa" was an investment with very high political and economic stakes, and sleeping sickness was not just a disease; it had become the colonial disease. besides drug development, the responses of the anglophone and francophone colonial powers to trypanosomiasis differed significantly. francophone countries chose to concentrate directly on the medical problems presented by the disease in humans. this included the introduction of "mobile teams" actively searching and screening population for hat cases. this method of systematic case detection and treatment with the aim of elimination of the parasite reservoir was suggested by the french military surgeon eugène jamot and such activities started in in cameroon ("atoxylisation") [ ] . subsequently, the prevalence of hat declined from as high as % in to . - . % in , leading to an expansion of the methodology to other countries. after the second world war, atoxyl ® was replaced by pentamidine and a regular application of the drug every six months to the population at risk was introduced ("pentamidinisation"). in the s in the belgian congo alone, some two million people were subjected to this preventive mass drug administration [ ] . due to the partial presence of t. b. rhodesiense in their territories, the anglophone countries were confronted with the more widespread problem of disease in domestic livestock, which also presented a reservoir for human disease. their approach included vector control (traps, spraying), bush clearing, and game destruction [ , ] , and later the chemopreventive use of veterinary drugs (diminazene, isometamidium, and homidium). the control measures were overall very successful and progressively controlled the disease, reaching a very low, generalized transmission by the mid- s, with a minimum of cases declared in africa in [ ] . however, the measures taken were very costly, but above all very unpopular in the communities. this led to concealment where possible, and made the longterm goal of elimination by chemotherapy difficult, if not impossible [ ] . we could observe similar tendencies when conducting clinical trials in the s; potential patients were mostly hiding away or fleeing the villages at the beginning of mobile team campaigns for reasons of fear of stigmatization, lumbar puncture, pain, and the possibility of being treated with the dangerous drug melarsoprol. the in-depth understanding of the communities' beliefs, needs, and approaches is therefore key in a successful elimination attempt; insight on these topics are presented in the papers by and falisse et al., lee et al., and palmer et al. a factor mentioned by winslow in , which may still be under-researched today, is the relationship between the disease and poverty, particularly inadequate food supply, as the disease leads to unused land, which creates malnutrition [ ] . such a view would require an even more integrated approach towards disease control and elimination. when the colonial powers withdrew from africa between and , a new era began. the young nations created their own institutions with the goal of continuing research towards the elimination of hat (e.g., kenya trypanosomiasis research institute (ketri), nigerian institute for trypanosomiasis research (nitr), uganda trypanosomiasis research organisation (utro), and programme sur la recherche sur la trypanosomiase in côte d'ivoire (prct)). these institutions were reinforced since the s by internationally funded institutes dedicated wholly or partly to trypanosomiasis research (e.g., international laboratory for research on animal diseases (ilrad) in kenya, international centre of insect physiology and ecology (icipe) in kenya, international trypanotolerance centre (itc) in the gambia). however, in the course of monetary adjustments in the s, the decreasing funds available, and the emergence or increase of other health priorities, institutions devoted to a single disease were no longer sustainable and they were continuously integrated or transformed into multilateral institutions. overall, in the years after the independence process, the expenditures for hat were reduced, and awareness and surveillance of the disease decreased. the number of mobile teams was decreased, and it was attempted to transfer activities to the public health system-without having the respective tools, approaches, and knowledge [ ] . this, together with social instability, conflicts, and insecurity constraining disease control interventions led to a significant resurge of gambiense hat in the s and s [ ] , mainly affecting angola, congo, southern sudan, and the west nile district of uganda [ ] . at the end of the s, the situation was comparable to the one in the and ; the number of reported cases was almost , with , cases suspected [ ] . in , we published a special issue in the journal tropical medicine and international health [ ] , asking ourselves whether there were new approaches to roll back hat. not only was this the time of , hat cases suspected in countries of sub-saharan africa, it was also the time when the organo-arsenic drug melarsoprol was still the only treatment available for second stage hat. treatment with melarsoprol required around days of hospitalization with numerous and very painful injections, very severe adverse drug reactions like an encephalopathic syndrome were common and the mortality rate under treatment was as high as - %. in those days, an oncologist tried to comfort us, saying that a % treatment success rate for a disease with an inevitably fatal course was fantastic. we did not share this view, and rather expressed a dream: to make hat "an ordinary" disease, which follows the usual pattern "test, treat, track"-without the need of a lumbar puncture to make treatment decisions, without a high mortality rate under treatment, without the pain, without the stigma. elimination was a very far-fetched goal at this time, but there were some first positive signals that the dimensions of the problem and its impact on society and development were being recognized. the conclusions adopted by the international scientific committee of trypanosomiasis research and control (isctrc) in reflected a new awareness of the disease. the african union member states were urged to give highest priority ranking to african trypanosomiasis in their development programs, and it was recommended that urgent and particular attention should be given to surveillance and intervention in epidemic areas, to drug availability and resistance, and to the implementation of operational research to respond to the needs of control programs. at their meeting in lomé in july the oau heads of state and governments signed a declaration of intent to eradicate tsetse flies on the african continent-something that will likely not happen, but it was the turning point towards manifold activities which make us today work towards elimination of hat as a public health problem of and the interruption of transmission by . at the same time, médecins sans frontières' access to medicines campaign were able to make a compelling case that society needed to rethink drug discovery paradigms for neglected diseases. aventis (now sanofi) was persuaded to repurpose and develop the failed anti-cancer drug eflornithine for use against hat and to donate it at no cost to the who for distribution in africa. millions of dollars were also provided by aventis/sanofi to the who, who could now develop new screening and intervention programmes [ ] . bayer signed a similar contract with the who, a success story and joint effort which has been renewed by both companies until today, and which is one of the strong drivers in control and now elimination. in , the bill and melinda gates foundation selected hat to be one of the first diseases they targeted through the consortium of parasitic drug development (cpdd) and shortly thereafter, the drugs for neglected diseases initiative (dndi) was founded. the beginning of the century was truly an exciting time for neglected diseases and for hat in particular. the changes and significant impact on funding were later summarized in the landmark publication "the new landscape of neglected disease drug development" [ ] . the changed situation immediately led to the initiation of several large scale activities in drug development and the term "elimination" in the context of hat was mentioned for the first time by dr. jannin, leading the anti-hat efforts at the who in [ ] . drug development had been virtually dormant for about years. although the cultivation and test methods for drug screening for anti-trypanosomal drugs had been developed in the s and s [ ] , the money for pursing lead compounds in preclinical work, translational studies and large-scale trials was too scarce in these days. during the s, some initial limited drug activities were carried out on shoestring budgets: eflornithine, which had initially been developed in the s as a potential anti-cancer drug, was found to be active against second stage gambiense hat. this discovery in the s was a scientific breakthrough [ ] and eflornithine was shown to be much safer compared to melarsoprol. the drug received orphan drug status by the us food and drug administration in ; however, production was stopped after a few years and only resumed after significant public and political pressure. eflornithine, however, was only introduced for treatment in a limited number of centres by msf in , but not until by the national sleeping sickness control programs because of its limited availability, the initial high costs, and particularly the logistical challenge to transport the drug and its associated bottles of sterile water per treatment. the turning point was when who launched a kit format and coordinated training of staff from national sleeping sickness control programs [ ] . furthermore, in the s nifurtimox, developed against chagas disease was used in experimental settings mainly to treat melarsoprol refractory cases [ ] . in the mid- s, the pharmacokinetics of melarsoprol was elucidated. the assessment of a subsequently proposed abridged days regimen in a large scale trial with patients in angola (impamel i) allowed the replacement of the empirically derived complex schemes lasting from - days in [ ] [ ] [ ] . whereas the new regimen had major socio-economic advantages, the disappointment was that the frequency of the worst adverse drug reaction, the encephalopathic syndrome, remained at levels of - % of patients treated, still resulting in death in - % of those in whom encephalopathy developed. the metabolism of melarsoprol was elucidated somewhat later [ ] . the finding that the major metabolite melarsenoxide covalently bound to a midsize protein triggered another large-scale clinical trial, which led to the elucidation of the nature of the encephalopathic syndrome. the early s were dominated by the development of the oral prodrug pafuramidine against first stage hat; the program failed at a late point of development, but it contributed much to the understanding of hat chemotherapy and the conduct of clinical trials against hat, which it is described in detail by the paper of dickie et al. in parallel, several trials assessing combinations of eflornithine, melarsoprol, and nifurtimox were conducted. in all trials, the efficacy was better in the combination arms compared to the monotherapies. however, combinations containing melarsoprol resulted in very high frequencies of severe adverse drug reactions and were rapidly abandoned [ ] . a multiple-centre trial, conducted in the republic of congo and the democratic republic of the congo (drc) compared nifurtimox-eflornithine combination therapy (nect) with the standard eflornithine therapy. nect reduces the number of eflornithine infusions from to , the total amount of eflornithine by half and the hospitalization time by one-third [ ] . based on the favourable results of the trials conducted, nect was included for treatment of second stage gambiense hat into the who's essential medicines list in [ ] , and for children in [ ] . nect can be considered a milestone improvement: under optimal conditions, fatality during treatment is . % compared to - % under melarsoprol [ ] . the complexity of its application still restricts the use to the second stage disease, meaning that the lumbar puncture for diagnostic staging is still required [ ] , continuing until today. to identify better alternatives, the drugs for neglected diseases initiative initiated a major compound mining effort in to explore new and old nitroimidazoles as drug leads against human african trypanosomiasis. one of the compounds screened, fexinidazole, proved to be orally active against t. b. gambiense and t. b. rhodesiense in animal studies and had an excellent safety profile. the development of this orally active compound is described in detail in the papers of neau et al., and dickie et al., fexinidazole received a positive scientific opinion from the european medicines agency for treatment of gambiense hat in late , it was approved by the drug regulatory authority of the drc and added to the who list of essential medicines in , and the first official application in the drc happened at the end of january on world ntd day in a public ceremony. this deliberate coincidence of the date depicts the new integrated thinking of hat control and elimination in the framework of ntds clearly. fexinidazole will be an essential component towards hat elimination. however, it has some limitations, which will hamper its widespread use in the field: its absorption is dependent on simultaneous food intake, or else only subtherapeutic drug levels are reached; based on the observation of a lowered efficacy in patients with advanced disease, a lumbar puncture for staging still is necessary in such patients; and the drug has not been tested yet for children below six years [ ] . hence, the search for "the magic bullet" [ ] continues-with an excellent starting position compared to years ago: for the first time in history, we can speak of a modest pipeline of anti-hat drugs. one most promising candidate is in late clinical development, several compounds are well advanced in pre-clinical stages, and medicinal chemistry and lead selection work is continued as currently, the leading novel class of molecules are the boron-containing benzoxaboroles. one candidate, scyx- , acoziborole, entered phase ii/iii assessment in [ ] . the compound is described in the publication of dickie et al. should the development program be successful, acoziborole would further revolutionize the efforts to eliminate and sustain elimination of hat. due to its long half-life of h, it can be potentially used as a single-dose treatment and should it be well tolerated this would provide further options for decentralized use, and maybe even for "ring-treatment" of patient contacts following the example of ring-vaccinations used, e.g., in the control of the ebola virus. with fexinidazole, and potentially even more with acoziborole, the focus will turn away from the discovery and development of better tools, to the understanding of the implementation, optimal use, including the needs and perception of patients. the clinical research programs have contributed to the reduction of cases: new strong partnerships were formed as described by taylor et al. and the conduct of clinical trials in a number of endemic areas per se has had an impact through staff training, attention to disease, and intensified active case search and treatment of a large number of patients as described by mbo et al. besides the improvements of the renewed interest of governments and improved drug treatment, there are several other reasons for the decrease of hat prevalence: the advances in diagnostics are one of the major factors. the serological card agglutination test for trypanosomes (catt) first published in [ ] had a paramount impact on how patients could be screened by mobile teams. the test was adapted and improved several times, and despite its disadvantages (insufficient specificity to confirm diagnosis, only available in larger batches, cold chain necessary), it has kept its place in hat diagnosis. the mini-anion exchange chromatography for trypanosomes (maect) which increases the sensitivity to detect the parasite in the blood significantly was already published in [ , ] , however, only the increased funding available allowed its more consistent use and therefore detection of cases with low parasitemia. the introduction of rapid diagnostic tests is a true advancement, but also lacks the specificity needed to make a final treatment decision [ ] . additional tools were recently developed but so far only introduced to a limited extent into routine use (e.g., loop-mediated isothermal amplification (lamp) [ , ] ; immune trypanolysis test [ ] ), which will both play a role in the "end-game". the question, however, is how such tests will be used in the future, and in what settings. the currently ongoing research program ditect-hat is set up do exactly that: it seeks to validate the performance of diagnostic tools and algorithms for early and rapid diagnosis of gambiense hat for passive case detection, post-elimination monitoring, and for assessing the therapeutic response [ ] . in addition to the optimization of the technical aspects, however, it is of paramount interest to know about local settings, preferences, and the loss of skills in areas with decreasing patient numbers. the paper by palmer et al. reports on such investigations carried out in uganda. benhamou et al., through a case report on a repeatedly misdiagnosed patient, gives us an insight on future challenges for rapid diagnosis if knowledge and interest in the public health system is not maintained and broadened. another unresolved caveat of diagnosis is that a number of patients are determined to be seropositive, but thereafter hat cannot be confirmed. it will be one of the leading discussions when defining future strategies, and what to do in such cases. nkieri et al. investigated the extent of this phenomenon in the still affected regions of drc. on one hand, relapses have always been a major challenge in the treatment of hat and have made follow-up periods of up months after treatment necessary [ ] . on the other hand, until a few years ago, the dogma was "infected, but not treated inevitably leads to the death of the patient". reports on patients surviving for longer periods despite infection with trypanosomes emerged in the past few years [ ] . one of the compartments where trypanosome may survive seems to be the skin [ , ] . this might also explain how hat can re-emerge in so-called silent foci as illustrated by a nine-year-old child, who was diagnosed with gambiense-hat in ghana in , years after the last detected case [ ] . in this light, the findings of mudji et al. also have importance: over ten years after treatment in the framework of clinical trials, a number of patients revisited presented continued signs and symptoms seen in hat (lymphadenopathy, severe headaches, sleep disturbances); since no trypanosomes could be identified by any means, the implication of these findings remain open at this point. in any case, the existence of such long-term cases has a sudden and dramatic impact on the view of hat epidemiology and hat elimination [ ] . besides further epidemiological, parasitological, and molecular research, mathematical modelling may help to improve our epidemiological knowledge and inform about elimination strategies [ ] and their related costs [ ] . this field has significantly developed against all odds in the past years: trypanosomiasis with its extremely focal distribution and the many external factors influencing its transmission has been a true headache over two decades for all modellers and predictive mappers. studies of existing gambiense-hat models in a few foci (i.e., drc, guinea, and chad) suggest that some type of additional infection reservoir is needed to match the observed dynamics of reported hat cases [ , ] . this could arise from another human reservoir (including undiagnosed and latent infections), an animal reservoir, and/or heterogeneities in human risk exposure and surveillance coverage [ ] . the french colonial forces had completely dismissed the value of vector control due the successes of the treatment strategy proposed by laveran. however, vector control may play a larger role in gambiense hat elimination than anticipated. historical investigations, practical intervention studies, and modelling demonstrate the significant role that vector control can play in the control of gambiense hat. recent models suggest vector control will be essential if we are to reach the set target of elimination of the diseases as a public health problem by and beyond [ , ] . the fact that neither modelling nor vector control are represented in this edition does not represent a valuation of these topics. this special issue comes in a very timely moment, because it is now important to secure what has been achieved, to understand missing pieces, and to finish the work. however, several challenges have to be overcome to not to end up, again, in disaster. in , the world health organization, which has played an instrumental role in the control, set the goal for the elimination of human african trypanosomiasis (hat), caused by trypanosoma brucei gambiense (ghat), as a public health problem for and for the total interruption of transmission to humans for . the efforts to maximise output and optimize innovation by the who has intensified since, and several stakeholders and expert groups have been created and convened [ ] . since , the spectacular decrease of the case number has continued: some , cases were reported in , far fewer than the targeted milestone of cases, and in [ , ] . first of all, "donor fatigue" must be avoided. the elimination to "no transmission" in the drc where over % of the cases are nowadays occurring is a herculean task, which will not happen without considerable and continued funding. the conventional measurements of success (e.g., us$ spent per daly prevented) inevitably fail in an elimination scenario. naturally, the amount of money spent per patient identified and treated will soar, so the question to the health economists is, rather how much money do we lose in case efforts would not be continued, factoring in the needed future efforts to re-start and control the disease again. decisions on priorities will be necessary, too: whereas the total number of patients has massively decreased, the area in the drc they are coming from has not. therefore, a vast area still has to be kept under surveillance; this area has to be gradually reduced by safely "closing" focus by focus in order to not jeopardize the efforts. the elimination of hat, malaria, and guinea worm were all believed at a certain point to only be a matter of time-before new reservoirs became known (guinea worm), pestidicide, and drug resistance set in (malaria), and interest was decreased in a premature belief in success (malaria and hat). secondly, the human factor will start to play a key role: in theory, fexinidazole could be applied in , fixed health facilities ( ) an increase up by % from [ ] , and it will be many more, should acoziborole make it to application in a few years. however, hat is a massively stigmatized disease, linked to many beliefs and bad spirits. traditionally, patients after treatment were excluded from working and sexual intercourse for six months [ ] . therefore, the questions are: "will the disease be recognized by the younger physicians and nurses who have never seen a case of hat?" "is the medical staff willing to recognize a suspected case, given this will create a massive workload including trouble with the relatives of the patient and village, paperwork, an invasion of specialists for diagnosis and follow up activities?" "is the medical staff that was told for over years that hat belongs into the specialized hands of the vertical programs willing to assume this task and challenge?" "are the patients willing to accept hat as a diagnosis anymore?" "can we overcome wrong dogma and information?" and "will patients falling into the respective category accept a lumbar puncture?" these questions can only be addressed through the thorough understanding of beliefs, perceptions, preferences, and decision-making processes. therefore, the social and anthropological science, as well as health economics, will start to play a key role in the "end game". the articles presented in this issue by falisse et al., and lee et al. are contributing to this area. thirdly, peace, stability, and a minimal standard of living for the people in the remote regions most affected are necessary to achieve disease elimination-this condition has not yet been met. there is little the scientific community can directly contribute to this-however, knowing that disease and poverty are inextricably linked [ ] , our efforts have to continue. finally, rhodesiense hat (the east african form of the disease) was reduced to as little as cases in [ ] , goals have been reached and it may be seen as a quantité négligable. however, the disease with a zoonotic reservoir has the potential for spectacular returns, and this real danger still exists as described in the contribution of matovu et al. the surveillance and the knowledge of local medical staff has dwindled, innovation was absent for t. b. rhodesiense for decades, and serological instead of microscopic tests are used for diagnosis of other diseases making accidental diagnosis impossible. from january to october , a total of - cases were reported per month from all treatment centres in malawi; in november to january , this number surged to and higher. the cases were reported from populations around the two geographically separate wildlife reserve areas, vwaza and nkhotakota; the reason for this increase is, so far, unknown (personal communication, world health organization, control of neglected tropical diseases, geneva). this outbreak causes major concern and should be a serious warning to everyone who is of the opinion that sleeping sickness has been conquered. similarly, other unexpected priorities, such as the current sars-cov- epidemic, may at all times derail a fragile health system. as soon as the mental and financial attention and priority is on another disease, signals of a hat resurgence may well be overlooked-we should now be well aware about the consequences and impact of late reactions and exponential transmission. compared to when i wrote the conclusion of the hat special issue in [ ] , we are at a completely different point today. we celebrated several marvellous scientific successes in the meantime, tools were improved, patients numbers down-but to reach the set goals and to get completely rid of this horrible disease, the conclusion is the same again: "the goal must now be to maintain the momentum" and "even the biggest efforts of the research scientists, field workers, development agencies, and companies will fail if they are not paralleled by achievements in the political field bringing peace, stability, and a minimal standard of living to the people in the remote regions most affected". the author declares no conflict of interest. the history of african trypanosomiasis. parasites vectors the history of sleeping sickness african trypanosomiasis-centennial review the elusive trypanosome pharmacological aspects of the trypanocidal drug melarsoprol chemotherapy of protozoal infections: amebiasis, giardiasis, trichomoniasis, trypanosomiasis, leishmania and other protozoal infectious the development of drugs for treatment of sleeping sickness: a historical review epidemiology of human african trypanosomiasis control of human african trypanosomiasis: back to square one sleeping sickness-a growing problem? are there new approaches to roll back trypanosomiasis (editorial) the new landscape of neglected disease drug development commentary: sleeping sickness-a growing problem? new developments in human african trypanosomiasis effects of the ornithine decarboxylase inhibitors dl-α-difluoromethylornithine and α-monofluoromethyldehydroornithine methyl ester alone and in combination with suramin against trypanosoma brucei brucei central nervous system models treatment options for second-stage gambiense human african trypanosomiasis. expert rev. anti-infective ther nifurtimox in late-stage arsenical refractory gambiense sleeping sickness efficacy of new, concise schedule for melarsoprol in treatment of sleeping sickness caused by trypanosoma brucei gambiense: a randomised trial efficacy of -day melarsoprol schedule years after treatment for late-stage gambiense sleeping sickness effectiveness of a -day melarsoprol schedule for the treatment of late-stage human african trypanosomiasis: confirmation from a multinational study investigations of the metabolites of the trypanocidal drug melarsoprol nifurtimox-eflornithine combination therapy for second-stage african trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase iii, non-inferiority trial control and surveillance of human african trypanosomiasis: report of a who expert committee; world health organisation update on field use of the available drugs for the chemotherapy of human african trypanosomiasis new who guidelines for treatment of gambiense human african trypanosomiasis including fexinidazole: substantial changes for clinical practice a 'magic bullet' for african sleeping sickness ) for the serological diagnosis of t. b. gambiense trypanosomiasis the separation of trypanosomes from blood by anion exchange chromatography: from sheila lanham's discovery years ago to a gold standard for sleeping sickness diagnosis miniature anion exchange centrifugation for detection of low parasitemias: adaptation for field use sensitivity and specificity of hat sero-k-set, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by trypanosoma brucei gambiense: a case-control study loop-mediated isothermal amplification for detection of african trypanosomes using detergent-enhanced lamp for african trypanosome detection in human cerebrospinal fluid and implications for disease staging immune trypanolysis test as a promising bioassay to monitor the elimination of gambiense human african trypanosomiasis diagnostic tools for human african trypanosomiasis elimination and clinical trials (ditect-hat). . available online untreated human infections by trypanosoma brucei gambiense are not % fatal the skin is a significant but overlooked anatomical reservoir for vector-borne african trypanosomes the dermis as a delivery site of trypanosoma brucei for tsetse flies do cryptic reservoirs threaten gambienses-leeping sickness elimination? mathematical models of human african trypanosomiasis epidemiology seeing beyond : an economic evaluation of contemporary and emerging strategies for elimination of trypanosoma brucei gambiense quantitative evaluation of the strategy to eliminate human african trypanosomiasis in the democratic republic of congo evaluating long-term effectiveness of sleeping sickness control measures in guinea the impact of vector migration on the effectiveness of strategies to control gambiense human african trypanosomiasis human african trypanosomiasis control: achievements and challenges all past events/information related to human african trypanosomiasis monitoring the elimination of human african trypanosomiasis: update to the flipside of eradicating a disease; human african trypanosomiasis in a woman in rural democratic republic of congo: a case report whose elimination? frontline workers' perspectives on the elimination of the human african trypanosomiasis and its anticipated consequences this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -m q rm z authors: mahapatra, sovesh; nath, prathul; chatterjee, manisha; das, neeladrisingha; kalita, deepjyoti; roy, partha; satapathi, soumitra title: repurposing therapeutics for covid- : rapid prediction of commercially available drugs through machine learning and docking date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: m q rm z background the outbreak of the novel coronavirus disease covid , caused by the sars-cov- virus has spread rapidly around the globe during the past months. as the virus infected cases and mortality rate of this disease is increasing exponentially, scientists and researchers all over the world are relentlessly working to understand this new virus along with possible treatment regimens by discovering active therapeutic agents and vaccines. so, there is an urgent requirement of new and effective medications that can treat the disease caused by sars cov . methods and findings we perform the study of drugs that are already available in the market and being used for other diseases to accelerate clinical recovery, in other words repurposing of existing drugs. the vast complexity in drug design and protocols regarding clinical trials often prohibit developing various new drug combinations for this epidemic disease in a limited time. recently, remarkable improvements in computational power coupled with advancements in machine learning (ml) technology have been utilized to revolutionize the drug development process. consequently, a detailed study using ml for the repurposing of therapeutic agents is urgently required. here, we report the ml model based on the naive bayes algorithm, which has an accuracy of around % to predict the drugs that could be used for the treatment of covid- . our study predicts around ten fda approved commercial drugs that can be used for repurposing. among all, we suggest that the antiretroviral drug atazanavir (drugbank id db ) would probably be one of the most effective drugs based on the selected criterions. conclusions our study can help clinical scientists in being more selective in identifying and testing the therapeutic agents for covid treatment. the ml based approach for drug discovery as reported here can be a futuristic smart drug designing strategy for community applications. the recent outbreak of novel coronavirus disease is now considered to be a pandemic threat to the global population - . coronaviruses belong to a family of viruses mainly found in animals but with the recent outbreak, they have transmitted to humans. the new coronavirus, -ncov is termed as severe acute respiratory syndrome-related coronavirus sars-cov- [ ] [ ] [ ] [ ] [ ] [ ] which has now affected more than countries with over , , cases confirmed and , deaths reported all over the world [as on april ]. this could potentially bring major challenges to global healthcare and disastrous effect on the global economy if the virus is not contained within a few months . the common symptoms include cough, fever, shortness of breath, fatigue etc which makes it confusing for the patients to differentiate the symptoms with that of the typical cold and flu [ ] [ ] [ ] [ ] . reports suggest that the virus is transmitted through body fluids of the infected patients, especially when in contact and while sneezing even though exact reasons are not known. unfortunately, no drugs have been approved by regulatory agencies to treat sars-cov- infection until now. efforts are ongoing on war footing to find the effective drug and vaccine to treat this pandemic. coronaviruses are classified into four classes designated as alpha, beta, gamma, and delta sars-cov- spikes also bind to receptors on the human cell surface called angiotensin-converting enzyme (ace ) . like sars-cov and mers-cov, sars-cov- also attacks the lower respiratory system causing viral pneumonia. however, there are also reports that it could affect the gastrointestinal system, heart, kidney, liver, and central nervous system resulting in multiple organ failure . compiling the medical reports and data available from the patients, sarscov- is found to be more transmissible/contagious than sars-cov . rapid development of computer aided technology like ml based on artificial intelligence (ai) can help accelerate the drug development process for different diseases [ ] [ ] [ ] . the advantage of ai approaches like ml is that they can be applied to learn from examples and build predictive models even when our understanding of the underlying biological processes is limited, or when computational simulations based on fundamental physical models are too expensive to be carried away. another advantage of ml is to automatically learn to identify complex patterns that categorize sets from input all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint data and thereby make intelligent decisions based on independent datasets . ml can accurately predict drug-target interactions as an enormous amount of complex information by studying hydrophobic interactions, ionic interactions, hydrogen bonding, van der waals forces, etc. between molecules. bioactivity datasets which are available from the numerous high throughput screens deliver useful means for machine learning classifiers as they contain binary information (active/inactive) as well as numerical values to classify different compounds under consideration , . such a huge number of datasets available on biological activities of molecules, derived from high throughput screens now allows to create predictive computational models. in this study, we have applied a machine learning approach to predict several new potential drugs for the treatment of sars-cov- and validated the predicted drugs. initially, we have trained our model with the inhibitors of the sars coronavirus c-like protease. the fda approved drugs are only taken from the drug bank as a test model to predict the new drugs. these new drugs are again validated using a docking method to ensure that the drugs match with the same active site on the protein. a ranked list of drugs based on energy value is given that can be tested experimentally. our study hypothesizes that the commercial fda approved antiretroviral drug saquinavir may be a potential candidate requiring to limit viral recognition of host cells or disrupt host-virus interactions thus requiring further clinical trial. here, we have at first taken the inhibitors of sars-cov- , which doesn't allow them to replicate in the host. these are screened and collected in the bioassay aid which were used as the main component for the modeling of the training model using ml. the attributes were taken under consideration for more than , compounds. we have not used unsupervised learning to filter out the dataset, because it would have made the dataset much weaker. as mentioned in the method section, we have used a classifying algorithm to train the model and the best among them was further used for the testing and predicting the drugs from the drug bank. the schematic of the process is shown in figure . we have used the naïve bayes against the dataset. it has shown accuracy of approximately . %. with this model, we have used the drugs from the drug bank to get predicted for the identification of the potential drugs which can be used for the treatment of disease caused by sars coronavirus. along with that the model has predicted true positives and true negatives. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . table . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . the pdb structure of sars spike protein receptor binding domain was retrieved from protein data bank (pdb id: ghv). the docking experiment was done with the patchdock server ( https://bioinfo d.cs.tau.ac.il/patchdock/). the results refinement and energy calculation was performed as per the algorithm used in the firedock server. the result having minimum global energy was taken into consideration. out of all drugs predicted, saquinavir (drugbank id -db ) (figure a ) has shown the minimum global energy. figure b shows the ligplot analysis having conventional h-bonding between ligand and tyr-c- . in our study, we have taken those drugs for docking purpose which show protease inhibition activity. the docking of sars protein with the approved drug saquinavir (drugbank id -db ), has a global energy of around - . ev which confirms that it had numerous steric clashes with the adjoining strands and thus highlight its potential to inhibit novel sars coronavirus. saquinavir is a small molecule antiretroviral drug (figure b all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . in addition to the docking of the above drug, we have also docked several other approved drugs available in the drug bank, which are predicted by our ml model with a confidence level of above % and also shows the activity of protease inhibition. with reference to that, we have found that the other drugs predicted by the model with the inclusion of all the parameters taken under consideration can also be quite effective. the rapid identification of active therapeutic agents against sars-cov- is a major challenge. analyzing the available knowledge on their safety profiles, and in some cases, efficacy against other coronaviruses and repurposing existing antiviral drugs is a potentially crucial short-term strategy to tackle covid- . under the current scenario, it takes more than years to bring a drug from the investigational stages to market availability. it is because of the trial and error process or the so-called edisonian approach, where one keeps on analyzing several compounds to find the best possible one. these days with the inclusion of digital medicine, this time span has been reduced to a great extent and people are able to approach in a rational manner for the drug discovery process. here, we have targeted for the repurpssed drugs towards the development of effective treatment of covid- to speed up clinical trial. we have found that ml model created on the basis of the naive bayes algorithm is the most effective one with the accuracy of almost equal to %. the drugs predicted by this model is further verified by the docking process. so, we speculate can expect that our predicted the drugs show immense potential for treatment of the covid- . considering the ongoing efforts to prevent the spread of covid- all over the world, we are optimistic that the outbreak may subside in a few months like sars and mers. however, the outbreak has stressed the urgent need for renewed efforts towards the development of braod-spectrum therapeutic agents to combat coronaviruses which are repeatedly found to be a realistic threat of this century till now. our this findinging will provide a base for further enhanced drug discovery programs. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . the drug bank is an online database which contains detailed data about various medications . today, it is being widely used to facilitate in silico drug target discovery, drug design, drug docking or screening, drug metabolism prediction, drug interaction prediction and for general pharmaceutical education. this database of more than drugs is categorized into many different types as trial stages drugs, approved drugs and withdrawn drugs. in this database, more than % of drugs are approved for various medication purposes . in this research, we have focused only on the fda approved drugs for repurposing purpose which are around with the intention that it will minimize clinical trial in the present situation. these drugs were downloaded in the form of sdfs and after processing, the descriptions generated were taken as the test model for developing the train model which was made on the basis of a database containing the inhibitors of the sars coronavirus. the developed model has predicted few of the potential drugs. the ncbi protein database was used in the process of getting the fasta sequence of the desired protein (sars coronavirus). further, the fasta sequence is used for the modeling of the three-dimensional protein structure and on the basis of this structure the docking of the known and predicted drugs have been carried out . since the datasets are present in the form of sdfs, we have generated the attributes present in the sdfs. first, the information present in the sdfs are generated as csv files which are used as the training dataset and test dataset for preparing the ml models. these csv files containing both the actives and inactive points are split into % as training all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint dataset and % as test dataset. this entire splitting process was random. this process is done by self-written python code to split as per the conditions. we have used machine learning (ml) model to the selected dataset from the pubchem which was considered as inhibitors and tested against the drugs from the drug bank to find more suitable drugs for the coronavirus- . using ml, we have implemented the classification algorithms as described below. the classification is a type of supervised learning in which the computer system can learn from the dataset which contains the detail and practical results. the algorithmic procedure of the classification is to assign an input value according to the description in the datasets . so, for this, it requires a mathematical classifier that can assign specific class (active and inactive) labels to instances defined by the attributes. in this process, the training model is made to learn using dataset where the classification is already assigned and on the basis of which it is able to run on different datasets to classify them according to the present instances. in this study, we have compared the results from the classifier that is naïve bayes classification algorithm. naive bayesian classification algorithm is a simple and elegant approach by assuming that its classification attributes are independent and they don't have any correlation with each other . it is a type of classifier that depends on bayes' hypothesis. naive bayes does work best in two cases: complete independent feature (as expected) and functionally dependent features (as expected) and is a widely tested method for probabilistic induction. this classifier functions well and has advantages over many other induction algorithms. it has no entangled iterative parameter that makes it work for vast data sets. this algorithm is more useful than any other induction algorithms because of its computation speed and reliability. it can be useful for both the binary classification as well as multi-classification . the training model is prepared by % of the original dataset. the dataset is completely classified from where the computer learns and finds the relations among various attributes. the cross-validation is used along with the algorithm to train the model. in this case, the cross-validation is n set with n-folds dataset. then it is supposed to divide the training dataset into n parts, and the n- parts will be used as training data and the other one will be used to validate the rest. this process of iteration goes on for n iteration times. here, we have used -fold and it is chosen as per the size of the dataset , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint generally, the datasets containing binary classification based on several attributes are imbalanced. we observe the similar tred here. these imbalanced datasets are not possible to be handled by the normal classifiers since they give importance to each of the attributes equally which could lead to misclassification errors. this can decrease the accuracy of the dataset for the trained model , . therefore, we have used the misclassification cost where the trained model becomes cost sensitive and able to find the lowest expected cost. this approach is actually much randomized because it neither depends upon the number of attributes nor on the minority class ration; rather it depends on the base classifier , . here, we had two methods to introduce the misclassification cost with the imbalance dataset. the first method is to classify the algorithm into the cost-sensitive one and proceed with the rest settings . the other is the use of a wrapper, which helps in the base classifiers into cost sensitive ones. we have used naïve bayes classifier which uses the cost insensitive algorithm to predict the probability estimations of the test instances and then using this it predicts class labels for the examples of the test dataset. in our report, we have classified our datasets into two classes i.e. active and inactive. so, we used the x matrix which is generally used for the binary classification. in the matrix sections are true positives (active classified as active), false positives (inactive classified as active), false negatives (active classified as inactive) and true negatives (inactive classified as inactive). in this case, the percent of false negatives are more important than the percent of false positives and the upper limit for false positives were set to % , . in this process, we increase the misclassification up to the set percent which also helps in the increasing of the true positives. since, the actives are very less in number, we have replicated them to around - times to match it with the inactives and make the model less biased. there are various methods for the validation of the binary classifiers. the true positive rate is the ratio of the actual actives to the predicted positives and this can be obtained as (tp/tp+fn). the false positive rate is the ratio of the predicted false actives to actual inactives and this can be obtained as (fp/tn+fp). accuracy shows the model's performance relative to the real values and this can be calculated as (tn+tp/tn+tp+fp+fn). the sensitivity shows the model's ability to identify the positive results and this is calculated as (tp/fn+tp) and the specificity shows the model's ability to identify the negative results and this is calculated as (tn/tn+fp). a model with high specificity and sensitivity has a low error rate. the balanced classification rate (bcr) is the mean of the sensitivity and specificity, which provides the accuracy of the model applied on the imbalanced dataset. this bcr can be calculated as . *(specificity+sensitivity). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint apart from the bcr, the mathews correlation coefficient (mcc) is also used whose range varies from - to . the receiver operating characteristic (roc) curve is the visualization of the ratio of fpr to tpr. in this case, the fpr and tpr are placed on the x-and y-axis respectivel. the area under curve shows the probability prediction of the classifier and its ability to classify the randomly chosen instance into the correct class. around drugs were predicted by our ml model which can be effective for the treatment of diseases caused by sars-cov- . there are no available drugs as of now, since the epidemic has just recently accelerated to over , , cases [as of th april, ]. the predicted compounds with above % of confidence were docked using firedock server. another decade, another coronavirus novel coronavirus of pneumonia in wuhan, china: emerging attack and management strategies the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- origin and evolution of pathogenic coronaviruses clinical features of patients infected with novel coronavirus in wuhan discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin coronavirus infections-more than just the common cold viral and bacterial etiology of acute febrile respiratory syndrome among patients in qinghai epidemiological characteristics of pediatric patients with coronavirus disease in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding cryo-em structure of the -ncov spike in the prefusion conformation pathogen genomics in public health severe acute respiratory syndrome-related coronavirus -the species and its viruses, a statement of the coronavirus study group a family cluster of sars-cov- infection involving patients in nanjing a novel coronavirus from patients with pneumonia in china an updated estimation of the risk of transmission of the novel coronavirus ( -ncov) machine learning in virtual screening predicting phospholipidosis using machine learning structure-based virtual screening for drug discovery: a problem-centric review virtual screening of bioassay data pubchem's bioassay database drugbank: a knowledgebase for drugs, drug actions and drug targets protein data bank. rcsb pdb: homepage. rcsb pdb geometric deep learning autonomously learns chemical features that outperform those engineered by domain experts supervised learning an empirical comparison of supervised learning algorithms cross-validation cross-validation methods cost-sensitive boosting for classification of imbalanced data cost-sensitive learning methods for imbalanced data cost-sensitive online classification cost-sensitive learning with conditional markov networks cost-sensitive boosting and technology, government of india. all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- - mhacqa authors: gougis, paul; fenioux, charlotte; funck-brentano, christian; veyri, marianne; gligorov, joseph; solas, caroline; spano, jean-philippe title: anticancer drugs and covid- antiviral treatments in cancer patients: what can we safely use? date: - - journal: eur j cancer doi: . /j.ejca. . . sha: doc_id: cord_uid: mhacqa • more safety data is needed to treat covid- symptomatic patients with anticancer drugs known to increase infections. • we summarized immunosuppressing anticancer drugs; other drugs were studied for drug-drug interactions with antiviral medicines. • a ready-to-use table synthetize these pharmacokinetic and pharmacodynamic interactions between antiviral and anticancer drugs. to the editor, the rapid emergence of covid- pandemics worldwide is of particular concern for fragile populations who are more at risk of acute respiratory distress syndrome and death. patients treated for malignant hemopathy and solid cancers have a four times higher risk of hospitalization due to influenza infection, and a ten times higher risk of death. this fragility could be due to their age, multiple associated comorbidities, lymphopenia, or the immunosuppressive action of a broad spectrum of anticancer drugs. in any of these settings, clinical trials and incoming standard of care could lead to the prescription of antiviral drugs concomitant to non-immunosuppressive anticancer treatments. similar to previous works reporting interactions between hiv antiretrovirals and anticancer drugs, these two classes of medications have a narrow therapeutic index and can have pharmacological interactions. some of them are substrates or interact with hepatic cytochrome p cytochrome isoenzymes (cyp), particularly cyp a , and pharmacokinetic interactions could lead to supra or infratherapeutic concentrations. for example, enzalutamide, a nonsteroidal antiandrogen prescribed for prostate cancer, is both a cyp a substrate and inducer. ritonavir, on the other hand, is a pharmacokinetic booster of lopinavir contained in kaletra, which is explored as a covid- treatment. ritonavir is a substrate and also a potent inhibitor of cyp a . thus, enzalutamide and ritonavir could interfere with each other's metabolism, decrease or increase each other's clearance, and be responsible for severe toxicities or decreased efficacy. favipiravir, an anti-ebov drug, also a candidate for the covid- treatment, is an inhibitor of cyp c , and therefore may increase anticancer drug metabolized through this pathway, such as dabrafenib and enzalutamide. furthermore, cyp a induction could lead to sustained cyp a increased activity for up to week after discontinuation. dabrafenib or enzalutamide, two cyp a inducers, could significantly decrease hydroxychloroquine concentration during the first week of wash-out. among pharmacodynamic interactions, qt interval prolongation could be of particular interest. hydroxychloroquine, which is currently widely prescribed as an anti-coronavirus drug, or azithromycin, are two drugs known to prolong qt interval. concomitant use of qtprolonging anticancer drugs could lead to torsade-de-pointes and be fatal. caution should be observed in this case, and electrocardiographic monitoring should be implemented to monitor qt interval duration during combination therapy. similarly, anticancer drugs could potentiate nephrotoxicity and hepatotoxicity of antiviral treatments. the table (part b) summarizes pharmacokinetic and pharmacodynamics interactions between some currently tested drugs against covid- and anticancer drugs. table: a -class of anticancer drugs with immunosuppressive properties immunosuppressing drugs were defined as drugs associated with significantly more infections or neutropenia compared to the control group or placebo in trials. these drugs were excluded from part b. no interaction driven by other cytochromes were found. red arrows are for interactions relying on clinically significant data. orange arrows are for interactions relying on in vitro data for pharmacokinetic interactions. cytochromes involved in the drug interaction were specified. substrates for which induction but not inhibition could lead to significant interaction are between brackets. when the interaction modifies the pharmacokinetics of the anticancer drug, the arrow was on the bottom-left. antiviral exposition prediction is on the top-right. red boxes are for anticancer drugs with known torsade-de-pointes risk and high risk of renal and liver toxicities. orange boxes are for anticancer drugs prolonging qt without known torsade-de-pointes risk and moderate risk for renal and liver toxicities. data from fda labels were retrieved for drug metabolism, qt prolongation, and nephrotoxicity. livertox database was used for hepatotoxicity . gnrh analogs immunotherapy antipd /pdl cancer patients in sars-cov- infection: a nationwide analysis in china non-aids-related malignancies: expert consensus review and practical applications from the multidisciplinary cancervih working group liverpool covid- interactions drugs@fda: fda-approved drugs national institute of diabetes and digestive and kidney diseases jean-philippe spano: consultant: roche, msd, biogaran ; adboard/symposium: msd, roche, az, leopharma, mylan, pfizer, bms, novartis, pfo, myriads, gilead, lilly. all remaining authors have declared no conflicts of interest. key: cord- -kpg vley authors: ojha, probir kumar; kar, supratik; krishna, jillella gopala; roy, kunal; leszczynski, jerzy title: therapeutics for covid- : from computation to practices—where we are, where we are heading to date: - - journal: mol divers doi: . /s - - -x sha: doc_id: cord_uid: kpg vley abstract: after the spanish flu pandemic caused by the h n virus, the recent coronavirus disease (covid- ) brought us to the time of serious global health catastrophe. although no proven therapies are identified yet which can offer a definitive treatment of the covid- , a series of antiviral, antibacterial, antiparasitic, immunosuppressant drugs have shown clinical benefits based on repurposing theory. however, these studies are made on small number of patients, and, in majority of the cases, have been carried out as nonrandomized trials. as society is running against the time to combat the covid- , we present here a comprehensive review dealing with up-to-date information of therapeutics or drug regimens being utilized by physicians to treat covid- patients along with in-depth discussion of mechanism of action of these drugs and their targets. ongoing vaccine trials, monoclonal antibodies therapy and convalescent plasma treatment are also discussed. keeping in mind that computational approaches can offer a significant insight to repurposing based drug discovery, an exhaustive discussion of computational modeling studies is performed which can assist target-specific drug discovery. graphic abstract: [image: see text] a form of pulmonary disease was first reported in china from a city called wuhan in the hubei province on december , [ ] . the deadly disease was later termed as covid- by the world health organization (who) on february , . the identified causative novel coronavirus ( -ncov) is termed as severe acute respiratory syndrome-related coronavirus sars-cov- as it shares around . % of genome similarity with sars-cov which also previously emerged in china during - [ ] . with the announcement of covid- as 'global pandemic' by who on march , , sars-cov- has eventually affected countries and territories around the world and international conveyances. as of august , , , , cases have been confirmed with , deaths and , , recovery cases, while among the active cases, , , cases are in mild condition and , cases in a serious or critical condition [ ] (fig. a) . the literature reported seven coronaviruses (covs) that are known to cause human disease where the strains e (α-cov), hku (β-cov), oc (β-cov) and nl (α-cov) caused mild infections of the upper respiratory tract in humans [ ] . on the contrary, other two strains sars-cov (occurring in [ ] [ ] and mers-cov (middle east respiratory syndrome occurring in ) and the newly identified sars-cov- belonging to β-cov have caused serious health threat and fatality [ ] . the present scenario and available pathophysiology specify that sars-cov- is highly transmittable and contagious than its progenitor affecting not only the respiratory system but also the gastrointestinal system, central nervous system, kidney, heart and liver leading to multiple organ failure [ ] . the sars-cov- spike s glycoprotein has % identical sequence with human sars with a unique furin-like cleavage site, which is absent in other sars-like covs [ ] . the cryo-em structural evidence has revealed that sars-cov- has - times higher binding affinity to the ace receptor than sars-cov which may lead to higher transmission and contagiousness [ ] . therefore, blocking of the isolated viral s protein at its host receptor region and/or binding within the s protein-ace interface are the two most important strategies to design probable drugs for covid- ( fig. b) [ ] . the sars-cov- virus replicates via multiple processes after entering into the host cell, and the proteins associated with these replication steps are the principal targets to treat the infected patients by blocking the viral replication. the replication-associated proteins are [ ] : a. translation of genomic rna, b. proteolysis of the translated polyprotein with viral c-like proteinase, c. replication of genomic rna with the viral replication complex which comprises ′-to- ′ exonuclease, rnadependent rna polymerase (rdrp), endornase and helicase, ′-o-ribose methyltransferase, d. assembly of viral components. along with the above-mentioned targets, the most commonly employed drug targets and drug discovery strategies employed all over the world right now are illustrated in fig. . presently, there is no specific treatment or approved drugs available to treat covid- . in most of the active cases, physicians are relying on symptom-based treatment for mild cases and primarily oxygen therapy (if required, with ventilator support) for critically ill patients. a set of approved marketed drugs like hydroxychloroquine (hcq) [ , ] , chloroquine (clq) [ ] , combination of hcq and azithromycin [ ] , remdesivir [ ] , lopinavir [ ] and ritonavir [ ] are being evaluated for the infection treatment; their clinical trials are also going on in different pharmaceutical industries. a series of new vaccines are also under clinical trial such as mrna- [ ] , ad -ncov [ ] and chadox ncov [ ] along with existing bacillus calmette-guerin (bcg) vaccine [ , ] to check its efficiency in covid- . due to severity and contagious nature of the sars-cov- , researchers are exploring multiple in silico approaches and artificial intelligence [ ] [ ] [ ] [ ] [ ] [ ] with the aim of identifying target-specific and potent therapeutic agents to speed up the discovery process. the rcsb protein data bank (pdb) (www.rcsb.org) has already deposited around protein crystal structures associated with sars-cov- and covid- to allow understanding important structural binding sites which can be explored in rational designing of small molecules. the current review discusses the most updated and probable drug candidates which are being experimentally used to treat patients in different parts of the world. also, their possible targets and pharmacological mechanisms of action which might not be clear in many cases and their pathophysiology along with the details about the status of convalescent plasma treatment and ongoing vaccine trials are discussed. we have compiled up-to-date in silico studies providing information related to computational tools, employed protein crystal structure used in the study followed by probable future drug candidates evolved from the repurposed virtual screening (vs) study employing docking, molecular dynamics (md) and homology modeling. therefore, the details related to sars-cov- transmission, protein structures, epidemiology, disease spectrum, diagnosis and testing are not discussed here at all as they have already been discussed in multiple literatures and separate reviews [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the present review is significantly different from the other recently published ones on a similar topic in that it covers and gives emphasis on the in silico modeling studies in at the time of writing this article, there are no clinically approved drugs or vaccine available for the treatment of covid- [ ] . however, there are many drugs under trial for the treatment of covid- (chemical structures - in figs. , , ) including angiotensin ii type i receptor (at r) blockers, antiviral drugs, antimalarial drugs, interferon, il- inhibitors, corticosteroids, ascorbic acid, some antibacterial antibiotics, etc. an up-to-date list of drugs under trials against covid- with their targets, mechanisms of action, the developer companies or institutions, uses and recent status are tabulated in table . several industries and research institute are also trying to develop miscellaneous drugs and/ or therapeutics agents, followed by investigation of the effectiveness of combination of drugs listed under box . among these tabulated drugs, most effective ones are discussed here. mechanisms of different categories of drugs used in covid- patients on various stages of sars-cov- life cycle are schematically depicted in fig. . the entry of sars-cov- to the host cell can occur in two ways, i.e., either via plasma membrane fusion or via endosomes (endocytosis blockers: clq and hcq) (fig. ). in both ways, spike proteins (s , s ) of sars-cov- mediate attachment to the membrane of a host cell and engage angiotensin-converting enzyme (ace ) as the entry receptor. inhibitors like convalescent plasma, monoclonal antibodies bind to the spike glycoprotein, thus preventing the viral entry. when virions are taken up into endosomes, the spike protein can be activated by the cellular serine protease tmprss in close proximity to the ace receptor, which initiates fusion of the viral membrane with the plasma membrane. camostat mesylate inhibits the tmprss receptor. the plasma membrane fusion entry is less likely to trigger host cell antiviral immunity and therefore more efficient for viral replication. after the viral rna is released into the host cell, polyproteins are translated. the coronavirus genomic rna encodes non-structural proteins that have a critical role in the synthesis of viral rna and structural proteins which are important for virion assembly. first, polyproteins are translated and cleaved by some of proteases like cl pro , pl pro , etc. (lopinavir, ritonavir and darunavir act as inhibitors of this step) to form rna replicase-transcriptase complex. the non-structural protein rdrp is responsible for replication of structural protein rna. (remdesivir, favilavir and ribavirin act as inhibitors of this enzyme). structural proteins s , s , envelope and membrane are translated by ribosomes that are bound to the endoplasmic reticulum (er) and presented on its surface as preparation of virion assembly. the nucleocapsids (n) remain in cytoplasm and are assembled from genomic rna. they fuse with the virion precursor which is then transported from the er through the golgi apparatus to the cell surface via small vesicles. the mature virions are then released from the infected cell through exocytosis, and then, they search another host cell (fig. ). sars-cov- has transmembrane spikes (s protein). the spikes attached to the lipid membrane of the coronavirus recognize a host cell to attach and infect it with its viral rna. the attachment of the coronavirus s protein to angiotensin converting enzyme (ace ) at its cellular binding site promotes the entry into human cells. the s protein contains two subunits such as an n-terminal s subunit which is responsible for receptor-virus binding and a c-terminal s subunit which is responsible for the fusion of virus with cell membrane [ , ] . the s subunit has two domains such as a receptor-binding domain (rbd) and an n-terminal domain (ntd). at the time of infection, at first coronavirus binds to the human cell through interaction between the cell ace receptor and s -rbd of coronavirus. as a result, conformational changes in the s subunit are triggered followed by virus-cell fusion and entry into the target cell. ace is a natural protein present in the lungs and the intestine (epithelial cells), and in the heart and the kidneys (endothelial cells). ace regulates the blood pressure by converting angiotensin molecules. it was found that the coronavirus which caused the sars outbreak in also binds to the same ace molecule, but in case of sars-cov- , the binding affinity is to times more on human cells than the spike from the sars virus of , which makes it a suitable target for covid- . due to the high affinity of this virus to human cells, it is spread without any difficulty from one person to another than the earlier virus [ ] . the entry of sars-cov- to the host cells via binding with ace enzyme leads to ace down-regulation. as a result, angiotensin ii is produced excessively by the correlated enzyme ace , while a lower amount of ace is not capable of transforming it to the vasodilator heptapeptide angiotensin - . thus, expression of higher ace resulting from frequently medicating covid- patients with at r blockers may resist them against acute lung injury. this can be described by following two complementary mechanisms: ( ) blocking the excessive angiotensin ii-mediated at r activation caused by the viral infection, ( ) upregulation of ace decreasing angiotensin ii production by ace and enhancing the production of the vasodilator angiotensin - [ ] . the role of ace to enter the coronavirus into the host cell and mechanism of action of ace inhibitors to control the covid- are depicted in fig. . table drug candidates under trial against covid- with probable targets, mechanism of action, pathophysiology, application and current status [ , , , , , , , , , drug candidates under trial the sars-cov- uses the s protein to facilitate viral entry into the host cells. the pathogen s protein consists of two subunits s and s , of which "s " allows entry of pathogen and binding of s protein to ace (cellular receptor) (fig. ). in addition, the entry requires s protein priming by cellular proteases, which is responsible for cleavage of s protein. after this, "s " subunit employs fusion of viral and cellular membranes. sars-cov- engages ace as the entry receptor that can be blocked by ace inhibitors and arbidol, and it employs the cellular serine protease tmprss for s protein priming which is inhibited by camostat mesylate (fig. ) . conclusively, sars-cov- / ace interface occurs in the molecular level, and the efficiency of ace usage is a key determinant of sars-cov- transmissibility. although it is widely accepted that coronavirus enters into the host cell through the ace receptor, due to limited number of studies, it is yet to establish how ace , at and at receptors exert their activities in coronavirus-induced diseases [ , ] . thus, ace inhibitors and at receptor antagonists [e.g., l- as a partial antagonist of at receptor and partial agonists of at receptor; losartan, valsartan, irbesartan, candesartan cilexetil, telmisartan, and eprosartan (fda-approved at receptor blockers)] may be used as important drug candidates to control lung injury of covid- patients [ ] . the binding of viral s protein with its receptor ace on host cells followed by viral endocytosis into the cells may also be a possible drug target. for example, the broad-spectrum antiviral drug arbidol recently entered the clinical trial for the treatment of sars-cov- which may act by inhibiting virus-host cell fusion, thus preventing the viral entry into host cells against influenza virus [ ] [ ] [ ] . the serine protease tmprss produced by the host cells plays a key role for cell entry of coronaviruses by s protein priming to the receptor ace binding in human cells (fig. ) . a recent study shows that camostat mesylate, a clinically approved inhibitor of tmprss (responsible for s protein priming), has been able to block sars-cov- infection of lung cells. thus, this drug may be a potential drug candidate for covid- [ ] . remdesivir, a nucleoside analog and a monophosphoramidate prodrug of remdesivir-triphosphate (rdv-tp) developed by gilead sciences inc. (usa), was previously due to the incorporation of additional nucleotides after rdv-tp, it does not cause instant chain termination because these three additional nucleotides may protect the inhibitor from excision by the viral ′- ′ exoribonuclease activity (fig. ) . recent reports showed that the ec value of remdesivir against covid- in veroe cells was . µm, half-cytotoxic concentration (cc ) was greater than µm, and the selective index (si) was greater than . , suggesting that its working concentrations are likely to be achieved in nonhuman primate (nhp) [ , ] . this drug is also able to inhibit virus infection proficiently in human liver cancer huh- cells sensitive to covid- . a recent case study revealed that treatment with remdesivir improved the clinical condition of the first patient infected by sars-cov- in the usa [ ] . a recent in vitro data showed that remdesivir and chloroquine (cq) phosphate are capable of inhibiting sars-cov- infection [ ] . remdesivir is currently being studied in phase-iii clinical trials against sars-cov- in wuhan, china, as on february , , and in the usa. chloroquine (clq) and hydroxychloroquine (hcq) have received deep attention because of positive results from some small studies. an antimalarial drug, clq, has recently been reported to have potential in vitro activity against sars-cov- . clq protects from viral infection by enhancing endosomal ph (making the environment unfavorable) which is required for virus-cell fusion. this drug may also block viral infection by inhibiting viral enzymes or processes like viral dna and rna polymerases, virus assembly, new virus particle transport, immunomodulation of cytokine release and virus release. clq also affects the glycosylation process of ace (as discussed earlier that to enter the host cell, the viral s protein binds with this receptor) [ , , , ] . besides this mechanism, a recent report [ ] showed that this drug also acts by inhibiting the sialic acid containing glycoprotein and gangliosides (act as primary attachment factors along the respiratory tract) mediated attachment to the s protein which is the first step for viral replication. in the ntd of the s protein of sars-cov- , a ganglioside-binding site was recognized. the antimalarial drug clq was found to be a probable blocker of the s-ganglioside interaction. thus, this drug may be used to fight pathogenic coronaviruses especially sars-cov- which is responsible for covid- . a detailed mechanism of action of clq and hcq against sars-cov- is illustrated in fig. . a recent report showed that the ec value of clq against the sars-cov- in veroe cells was . µm, which may be clinically achievable. although specific data are not available, this drug is able to inhibit the exacerbation of pneumonia patients with sars-cov- infection. figure explains two possible mechanisms of clq and hcq against sars-cov- . the mechanism is that clq and its derivative hcq are weak bases, which can raise the ph of acidic intracellular organelles, such as endosomes/ lysosomes, essential for membrane fusion. on the other hand, mechanism- explains the entry of sars-cov- into the host cells also depends upon sialic acid (neu ac) containing glycoproteins and gangliosides that act as the key binding factors along the respiratory tract. a ganglioside-binding site at the n-terminal domain (ntd) of the s glycoprotein of sars-cov- was recognized, and clq was found to be a possible blocker of the s-ganglioside interaction which occurs in the first step of the viral replication cycle (i.e., attachment to the surface of respiratory cells, intermediated by the s protein) [ ] . the interaction was augmented by placing the negative charge of the carboxylate anion of neu ac and one of the two positive charges of clq. sars-cov- especially interacted with -o-acetyl-n-acetylneuraminic acid ( -o-sia). in this case, the carboxylic acid group of the sialic acid interacted with the cationic group of the nitrogen-containing ring of clq. the formed complex of clq and -o-sia was further stabilized by oh-π and van der waals interactions. next, the complex developed from hcq was very close to that obtained from clq, although numerous conformational adjustments happened for the period of the simulations. interestingly, the -oh group of hcq reinforced the binding of clq to neu ac via formation of a hydrogen bond. the formed complex of clq-oh and -o-sia will be stabilized again like clq to form a protective layer against fusion of the sars-cov- . hcq is a hydroxy derivative of clq, which can block the viral infection by a similar mechanism as chloroquine; thus, this drug may also be a potential candidate against sars-cov- . this drug is less toxic (~ %) than fig. the role of ace receptor for the entry of coronavirus into the host cell, and mechanism of action of ace inhibitors and tmprss inhibitors to control covid- chloroquine in animals. it was found that there were seven clinical trials registered as on february , , in the chinese clinical trial registry (http://www.chict r.org.cn), for using hcq to treat covid- . the in vitro results suggested that this drug can efficiently inhibit sars-cov- infection. based on an in vitro report, it was suggested that hcq may be more potent than clq to treat covid- [ , ] . a purine nucleoside presently labeled as avigan, developed by fujifilm toyama chemical of japan, has recently been approved for phase-iii clinical trial (march , ) for the covid- patients. this drug is approved for manufacturing and sale in japan for the treatment of influenza as an antiviral. in case of influenza virus, it selectively inhibits rna polymerase which is essential for viral replication when human cells are infected. it is believed that this drug may be effective for the treatment of covid- as sars-cov- uses same enzyme (rna polymerase) for replication and classification into the same type of single-stranded rna virus like influenza. thus, this drug acts by inhibiting the rdrp leading to inaccurate viral rna synthesis (fig. ). this drug is recommended by the director of the china national center for biotechnology development under the ministry of science and technology to treat covid- . italy has also approved the drug to treat covid- cases. due to the effectiveness of this drug against covid- , it is being mass-produced as generic version in china [ , ] . these drugs are approved hiv- protease inhibitors, used in combination with other anti-retroviral drugs to treat hiv- infection in both adults and pediatric patients who is older than days. coronaviruses encode either two or three protease enzymes like papain-like proteases (pl pro ), a serinetype protease, the main protease, or m pro which cleave the polyproteins into non-structural polyproteins (nsps). these nsps are essential for viral rna synthesis. ritonavir and lopinavir act by inhibiting these protease enzymes. the mechanisms of action of these drugs suppressing coronavirus activity [ ] are depicted in fig. . a combination of these drugs is recommended in italy to treat covid- patients. several trials are going on worldwide using these drugs or in combination of other drugs. a collaborative research from china and the uk conducted a clinical trial to examine the effectiveness of a combination of these two drugs against covid- which was published in the new england journal of medicine (nejm) [ ] . the output of their trial did not provide any significant benefit in the patients with covid- . they suggest that "future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit." among those trials, two trials are investigating against pneumonia caused by covid- . one trial is conducted in the tongji hospital, wuhan, china, using lopinavir-ritonavir against arbidol hydrochloride (influenza drugs) and oseltamivir (nct ). in south korea, a comparative study of lopinavir-ritonavir against hcq in patients with mild cases of covid- (nct ) was made. the two arms of the who solidarity trial are lopinavir-ritonavir alone and in combination with interferon-beta [ ] . ivermectin is an fda-approved broad-spectrum antiparasitic drug, which recently showed in vitro antiviral activity against sars-cov- . it acts by inhibiting the interaction between hiv- integrase protein (in) and the importin (imp) α/β heterodimer which is responsible for in nuclear import [ , ] . therefore, (imp) α/β is unable to bind to the viral protein and preventing it from entering the nucleus, thus inhibiting hiv- replication [ , ] . as a result, inhibition of the antiviral responses is reduced leading to a normal, more efficient antiviral response. the trial of potential monoclonal antibody-based therapy against covid- is going on by using the previous knowledge on the neutralizing monoclonal antibodies (nmab) against similar coronaviruses such as sars-cov and mers-cov. monoclonal antibodies targeting the vulnerable sites of trimeric spike (s) glycoproteins on the viral surface which are responsible for the entry to the host cell are increasingly being recognized as a promising class of drugs against covid- . potential neutralizing monoclonal antibodies mainly targeting the receptor-interaction domain at s subunit ultimately disabled cell-receptor interactions [ ] [ ] [ ] . the detailed mechanism of action of this class of drugs is depicted in fig. . recently, several monoclonal antibodies, namely tocilizumab (atlizumab), mavrilimumab, lenzilumab, leronlimab, gimsilumab, sarilumab, siltuximab (sylvant), camrelizumab (airuika), eculizumab (soliris), etc., are being tried to investigate their potency against covid- disease, and these are tabulated in table . the human monoclonal antibodies (hmabs) are developed by using several strategies against sars-cov- including preparation of hybridomas (using a transgenic mouse), phage display technologies and the immortalization of convalescent b cells. at present, the hmabs production has been carried out with two strains of transgenic mice, i.e., medarex humab-mouse and xeno-mouse [ ] . the difference between these two strains of mice is that mouse l chain genes are still functional in the medarex humab-mouse; thus, these mice can produce chimeric mabs. the xeno-mouse from amgen has all the mouse l chain genes deleted, and b cells produce only human abs. one of these mice monoclonal antibodies [chimeric (medarex humab) (or) hybridomas (xeno-mouse)] was immunized to produce neutralizing antibodies and further evaluated. the developed neutralizing antibodies bind to a specific portion to the rbd (n-terminal (amino acids - , - ), and c-terminal of the rbd (amino acids - , - )) to prevent fusion of sars-cov- with the target cells. due to emergency of covid- patients, recently, fda has recommended to healthcare providers and investigators the use of convalescent plasma collected from individuals who have recovered from covid- that may contain antibodies to sars-cov- . however, till now, this therapy has not yet been shown to be effective and safe against this disease. thus, it is very essential to investigate the safety and effectiveness of covid- convalescent plasma in clinical trials. for this therapy, fda has recommended some guidelines as follows [ ] : ( ) the pathways for use of investigational covid- convalescent plasma; ( ) patient eligibility; ( ) collection of covid- convalescent plasma, including donor eligibility and donor qualifications; ( ) labeling, and ( ) record keeping. the pharmacological safety data including dose, drug-drug interactions and toxicities of selected potential drug candidates are provided in table . due to the worldwide outbreak of the covid- , the general public are keenly watching the progress of development of covid- vaccines [ ] . dr. anthony s. fauci, director of national institute of allergy and infectious diseases (niaid), said that "finding a safe and effective vaccine to prevent infection with sars-cov- is an urgent public health priority." but development of a new vaccine against a new disease is not an easy task. various research institutes and industries are giving their full efforts to develop a vaccine against this pandemic disease with its earliest. thankfully, the progress is rapid due to various reasons such as: ( ) sharing the efforts to sequence the genetic material of sars-cov- by china throughout the world, ( ) coronaviruses were already on the radar of health science researchers, ( ) the knowledge from sars and mers caused by corona viruses and ( ) also learnings from the vaccines against sars and mers which were stopped or postponed when those outbreaks were controlled may still be used to defeat covid- . the progress of most promising vaccines against the pandemic disease covid- being made by various industries and research institutes is tabulated in table . several miscellaneous vaccines under investigation for covid- are listed under box . to combat the covid- pandemic, researchers must fight with the time to save as many lives as possible. to save the time and speed up the drug discovery process, in silico modeling provides one the best possible options. till now, in most of the cases, researchers are trying the computational repurposing theory of existing approved drugs (synthetic as well as from natural origin) for sars-cov- employing docking, homology modeling and molecular dynamics (md) studies to identify probable magic drugs for covid- . huang et al. [ ] computationally designed a short protein fragment or peptide which may block the coronaviruses' ability to enter human cells by binding to the viral protein which is one of the first kinds of peptide treatments routing for experimental efforts. smith and smith [ ] analyzed small drug molecules and natural products (sweetlead library database) employing restrained temperature replica-exchange md simulations combining virtual screening through the ensemble docking to identify the effective drug for covid- which might stop the virus by two ways: (a) disrupting s protein and ace receptor interface stability; or (b) by troubling the capability of the s protein to recognize table pharmacological safety data of selected potential drug candidates [ , , , , , , - , - , , , , ] drug dose drug-drug interaction toxicity chloroquine phosphate (aralen) [ , , , , ] this is a genetically engineered vaccine candidate with the replicationdefective adenovirus type as the vector to express sars-cov- spike protein. preclinical animal studies of this vaccine candidate showed that it can persuade a strong immune response in animal models. preclinical animal safety studies also exhibited a good safety profile a phase-iii trial in saudi arabia is currently underway (fig. a) . ton et al. [ ] identified noncovalent inhibitors for sars-cov- main protease (m pro ) using . billion compounds from the zinc library employing deep docking platform using glide sp module which utilizes qsar models trained on docking scores. the mds protein with . Å resolution bound to a noncovalent inhibitor was used for the docking study, and among the identified noncovalent inhibitors, zinc is the top hit drug candidate (fig. b) . α-ketoamide inhibitors are proposed as new drug candidates by designing, synthesis, followed by a docking study on the main protease (m pro , cl pro ) which has a crucial role in processing the polyproteins that are translated from the viral rna [ ] . three different pdb structures were employed for the study; they are y e, y f and y g. the authors modified previously designed inhibitor ( ) (earlier used for beta-, alpha coronaviruses and c proteases of enteroviruses) by incorporating the p -p amide bond into a pyridone ring to enhance the half-life of the newly designed ( ) compound in plasma followed by replacing the hydrophobic cinnamoyl moiety with less hydrophobic boc group (fig. a) . although the inhibitory concentration decreased from . ± . µm ( ) to . ± . µm ( ), but molecule reported three times higher plasma binding and times better plasma solubility compared to molecule . further, to improve the antiviral activity against betacoronaviruses of clade b, the authors replaced the p cyclohexyl moiety of molecule by the smaller cyclopropyl fragment to produce compound which showed ic value of . ± . μm for the purified recombinant sars-cov- m pro . zhou et al. [ ] reported an integrative antiviral drug repurposing analysis employing pharmacology-based network medicine platform by a two-step process: (a) quantifying the relationship between the human coronaviruses oral bactrl-spike [ ] symvivo corporation oral vaccine for covid- bactrl-spike- will be the firstin-human study of bactrl-spike, and the first-in-human use of orally delivered bactrl. it is a genetically modified, probiotic-based oral vaccine for covid- . the study design is a phase , randomized, observer-blind, placebo-controlled trial in healthy adults [ receiving active vaccine in bacterial medium and receiving placebo (bacterial medium only)] phase-i clinical trial (nct ) (hcov) and host interactome and (b) identifying the drug targets in the human protein-protein interaction network. as per phylogenetic analyses, sars-cov- has the highest nucleotide sequence identity ( . %), followed by the envelope and nucleocapsid protein sequence identities of % and . %, respectively, with sars-cov. employing the network proximity analyses between drug targets and hcov-associated proteins followed by gene set enrichment analysis (gsea), it was possible to identify probable anti-hcov drugs (e.g., melatonin, mercaptopurine, and sirolimus, etc.). later, the drugs were validated through hcov-induced transcriptomics data in human cell lines and enrichment analyses of drug-gene signatures. the authors also reported three effective drug combinations among the probable hits identified through 'complimentary exposure' pattern (fig. b) . grifoni et al. [ ] employed the immune epitope database and analysis resource (iedb) to characterize the sequence similarity between sars-cov and sars-cov- through homology modeling. the epitope prediction identified a priori potential b and t cell epitopes for sars-cov- which are the promising targets for immune recognition, followed by the discovery of diagnostics and future vaccines. within a short period of time, a huge number of in silico results were deposited in the preprint servers from all over the world and at the same time a few are already published. as in most of the cases, multiple in silico drug designing and virtual screening tools were employed for repurposing theory of approved existing drugs [ ] , thus, major information is gathered in table to avoid similar discussion several times . an interesting aspect is that most of the studies are based on repurposing theory of existing approved drug employing docking and md supported vs. majorly the authors relied on approved antiviral drugs along with drugbank database, zinc database, natural compounds' databases along with one of the most discussed molecules of recent time, hcq. thus, the basic ideas of implication of in silico tools and repurposing of approved drugs are same, but the only difference is selection of the target protein in different studies. without any doubt, the crystal structure of covid- main protease (m pro ) in complex with an inhibitor n (pdb: lu ) is the most accessed protein for the drug discovery. but based on the target type, the choice of proteins can be different. thus, to assist researchers, we have classified the target proteins into types covering pdb crystal id from pdb (https :// www.rcsb.org/) available until april , , and enlisted in table . multiple incidents illustrated the outbreak of corona virus in animals, especially in pets [ , ] . animals from dogs, cats, tigers, lions to minks are already tested positive and showed mild to severe symptoms of corona virus, followed by death of few instances. although these events are scattered and not enough to study in the middle of human crisis, we cannot ignore the fact of human to animal transmission. if human to animal transmission is true, then there is a possibility of mutations, insertions and deletions in the genome sequence of this deadly virus in these animals in future with probabilities of zoonotic transfer of a stronger form of present sars-cov- virus from them to human in the near future. thus, these small incidents need to be checked very carefully to avoid any future transmission. the above-mentioned facts help to build an interspecies analysis by kar and leszczynski [ ] for animals to human transmission or vice versa to aid in the drug discovery process of covid- in upcoming days. the in silico interspecies-quantitative structure-activity relationship (i-qsar) modeling correlates and then extrapolates the response (activity/toxicity) data from animal source to human which will be helpful for drug discovery. due to genome sequence similarity of sars-cov- with the pangolins and bat, experimental data of drug candidates to pangolins/bat along with structural and physicochemical properties of drugs can be correlated with human response endpoint which is the first step of modeling. in the next step, extrapolation of animal data to human data can lead to no human testing through developed i-qsar model. once the acceptable predictive i-qsar model is ready, there might be no need of future animal testing. as bat and pangolins are endangered species, thus future introspection can be performed in dogs and cats due to their better accessibility as well as considering them possible carriers of sars-cov- from the recent incidents. researchers from all over the world are trying to find the medicines which will help to stop the transmission of the virulent sars-cov- virus, mitigate the symptoms of the infected patients and help to lower the death toll throughout the world. unfortunately, till now there is no silver gunshot which can solve this pandemic covid- . we have reported here a comprehensive and updated discussion on drug or drug candidates under investigation with their probable targets and mechanisms of action which might not be clear for many cases earlier, along with the effort of ongoing vaccine trials, monoclonal antibodies therapy and convalescent plasma treatment. we have presented here the ongoing computational efforts related to in silico tools to explore the probable drug candidates docking, md (sybyl-x . ) essential oils in garlic including organosulfur compounds ( ) lu inhibitory effect of essential oils from garlic is established and found interactions with the amino acids of the human ace protein and the m pro of sars-cov- (allyl disulfide, allyl trisulfide) [ ] against covid- along with the up-to-date target protein information. the information provided from in silico approaches may work as a magic bullet for the medicinal chemists to accelerate the drug discovery and development process. overall, this review provides a strong intellectual foundation to support progress of ongoing research related to thorough knowledge of drugs or investigational drugs, vaccines and in silico approaches which can be helpful for the development of new drug candidates for the treatment of covid- . sars-cov- main protease (m pro / cl pro ) r , r , r y, r , r , r , r t, r z, rea, rec, reb, ree, red, reg, ref, re , re , re , re , re , re , rfb, rfa, rfd, rfc, rff, rfe, rfh, rfg, rey, rex, rf , rez, rf , rep, rf , res, rf , rer, rf , reu, rf , ret, rf , rew, rev, rf , rei, reh, rek, rej, rem, rel, reo, rf , ren, rfz, rfy, rfr, rfq, rft, rfs, rfv, rfu, rfx, rfw, rfj, rfi, rfl, rfk, rfn, rfm, rfp, rfo, rg , y e, y , w , yb , rf , y g, y f, lu adp ribose phosphatase of nsp from sars-cov- vxs, w . a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin covid- coronavirus pandemic data epidemiology and clinical characteristics of human coronaviruses oc , e, nl , and hku : a study of hospitalized children with acute respiratory tract infection in guangzhou, china a novel coronavirus from patients with pneumonia in china potential of large "first generation" human-to-human transmission of -ncov the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases human coronavirus: host-pathogen interaction in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a sixday follow up: a pilot observational study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro a trial of lopinavir-ritonavir in adults hospitalized with severe covid- a study to evaluate efficacy, safety, and immunogenicity of mrna- vaccine in adults aged years and older to prevent covid- phase i clinical trial of a covid- vaccine in - healthy adults (ctcovid- ) a study of a candidate covid- vaccine (cov ) can a century-old tb vaccine steel the immune system against the new coronavirus? science bcg vaccination to protect healthcare workers against covid- (brace). clinicaltrials.gov website computational design of peptides to block binding of the sars-cov- spike protein to human ace repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors network-based drug repurposing for novel coronavirus -ncov/sars-cov- a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- a review of the novel coronavirus (covid- ) based on current evidence the sars-cov- outbreak: what we know covid- : a new challenge for human beings severe sars-cov- infections: practical considerations and management strategy for intensivists the spike protein of sars-cov-a target for vaccine and therapeutic development mers-cov spike protein: a key target for antivirals novel coronavirus structure reveals targets for vaccines and treatments angiotensin receptor blockers as tentative sars-cov- therapeutics differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl compensation of ace function for possible clinical management of -ncov-induced acute lung injury therapeutic options for the novel coronavirus ( -ncov) the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection (elacoi) structural basis of influenza virus fusion inhibition by the antiviral drug arbidol sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor compounds with therapeutic potential against novel respiratory coronavirus first case of novel coronavirus in the united states breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases more than clinical trials launch to test coronavirus treatments antivirals targeting the polymerase complex of influenza viruses potential inhibitors against -ncov coronavirus m protease from clinically approved medicines all populations monograph) the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro an alphascreen(r)-based assay for high-throughput screening for specific inhibitors of nuclear import structural basis for the neutralization of mers-cov by a human monoclonal antibody mers- towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody mice with a human touch recommendations for investigational covid- convalescent plasma preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov old weapon for new enemy: drug repurposing for treatment of newly emerging viral diseases ct /resul ts?cond=coron aviru s&term=&type=&rslt=&age_v=&gndr=&intr=remde sivir &title s=&outc=&spons =&lead=&id=&cntry =&state =&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_ s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_ s=&lupd_e=&sort= favipiravir combined with tocilizumab in the treatment of corona virus disease ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov surviving sepsis campaign rapid guidelines of the management of critically ill adults with coronavirus disease coronavirus puts drug repurposing on the fast track the covid- epidemic l- , is a partial angiotensin at receptor agonist in the hindquarters vascular bed of the cat baricitinib as potential treatment for -ncov acute respiratory disease effective treatment of severe covid- patients with tocilizumab roche initiates phase iii clinical trial of actemra/roactemra in hospitalised patients with severe covid- pneumonia /antib odies /kinik sa-annou nces-early -evide nce-of-treat ment-respo nse-with-mavri limum ab-in- -patie nts-with-sever e-covid - -pneum onia-and-hyper infla mmati on - therapeutics tracker. regulatory focus humanigen preps lenzilumab for potential battle with deadly covid- symptom /fda-appro ves-emerg ency-ind-use-of-human igens -lenzi lumab -for-compa ssion ate-use-in-covid - -patie nts novant health initiates phase covid- trial with cytodyn's leronlimab. cytodyn. https ://conte nt.equis olve.net/_ bdf ef b daa ec d f da a a/cytod yn/news/ - - _novan t_ healt h_initi ates_phase _ _covid _ _trial _ roivant pushing gimsilumab testing for ards in covid- patients first patient outside u.s. treated in global kevzara ® (sarilumab) clinical trial program for patients with severe covid- file fda ind for aviptadil to treat covid- -induced respiratory distress. p&t community eusa launches trial of siltuximab for covid- complications. fda news immunoregulatory therapy for -ncov. clinicaltrials.gov alexion to start soliris in covid- phase ii trial in next few days bevacizumab in severe or critical patients with covid- cd fc as a non-antiviral immunomodulator in covid- colchicine counteracting inflammation in covid- pneumonia (colcovid- ) covid- drug research: second phase of clinical trial of new drug candidate, sng for sars-cov- coronavirus initiated bidmc launches clinical trial to assess common anti-clotting medication for treatment of covid- -related respiratory failure clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected. world health organization use of ascorbic acid in patients with covid . clinicaltrials pharmacologic treatments for coronavirus disease (covid- ): a review immunity and safety of covid- synthetic minigene vaccine evaluating the safety, tolerability and immunogenicity of bactrl-spike vaccine for prevention of covid- covid % tra cker% v -posti ng.pdf?fbcli d=iwar rx eh bvbmc gf eq nqnim iz u kpjvm aexn hmgm cx ql fbvtr cv cq q. accessed covid- vaccine tracker silico drug design: repurposing techniques and methodologies computational design of ace -based short peptide inhibitors of sars-cov virtual screening based prediction of potential drugs for covid- virtual screening of an fda approved drugs database on two covid- coronavirus proteins simeprevir, potential candidate to repurpose for coronavirus infection: virtual screening and molecular docking study fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study molecular docking study of novel covid- protease with low risk terpenoides compounds of plants repurposing drugs against main protease of sars-cov- : mechanism based insights supported by available laboratory and clinical data identification of potential inhibitors of sars-cov main protease via a rapid in-silico drug repurposing approach in silico screening of chinese herbalmedicines with the potential to directly inhibit novel coronavirus analysis of therapeutic targets for sars-cov and discovery of potential drugs by computational methods predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov ) through a drug-target interaction deep learning model computational search for potential covid- drugs from fda-approved drugs and small molecules of natural origin identifies several anti-virals and plant products identification of sars-cov cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach prediction of the sars-cov ( -ncov) c-like protease ( cl pro ) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates comparative computational study of sars-cov receptors antagonists from already approved drugs investigation into sars-cov- resistance of compounds in garlic essential oil in-silico studies of antimalarialagent artemisinin and derivatives portray more potent binding to lys and lys -binding hotspots of sars-cov spike protein than hydroxychloroquine: potential repurposing of artenimol for covid- structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov infection destabilizing the structural integrity of sars-cov receptor proteins by curcumin along with hydroxychloroquine: an insilco approach for a combination therapy identification of potential molecules against covid- main protease through structure-guided virtual screening approach development of a simple, interpretable and easily transferable qsar model for quick screening antiviral databases in search of novel clike protease ( clpro) enzyme inhibitors against sars-cov diseases computational screening for potential drug candidates against the sars-cov- main protease consensus virtual screening of dark chemical matter and food chemicals uncover potential inhibitors of sars-cov- main protease protein reliability analysis and virtual screening of natural inhibitors for sars-cov- main protease (mpro) through docking, molecular mechanic & dynamic, and admet profiling. struct dyn should pets be tested for coronavirus susceptibility of ferrets, cats, dogs, and different domestic animals to sars coronavirus- from animal to human-interspecies analysis provides novel way of ascertaining and fighting covid- . the innovation acknowledgements pko thanks the ugc, new delhi, for finan- key: cord- -ndcbmgfj authors: takahashi, takuto; luzum, jasmine a.; nicol, melanie r.; jacobson, pamala a. title: pharmacogenomics of covid- therapies date: - - journal: npj genom med doi: . /s - - -y sha: doc_id: cord_uid: ndcbmgfj a new global pandemic of coronavirus disease (covid- ) has resulted in high mortality and morbidity. currently numerous drugs are under expedited investigations without well-established safety or efficacy data. pharmacogenomics may allow individualization of these drugs thereby improving efficacy and safety. in this review, we summarized the pharmacogenomic literature available for covid- drug therapies including hydroxychloroquine, chloroquine, azithromycin, remdesivir, favipiravir, ribavirin, lopinavir/ritonavir, darunavir/cobicistat, interferon beta- b, tocilizumab, ruxolitinib, baricitinib, and corticosteroids. we searched pubmed, reviewed the pharmacogenomics knowledgebase (pharmgkb(®)) website, clinical pharmacogenetics implementation consortium (cpic) guidelines, the u.s. food and drug administration (fda) pharmacogenomics information in the product labeling, and the fda pharmacogenomics association table. we found several drug-gene variant pairs that may alter the pharmacokinetics of hydroxychloroquine/chloroquine (cyp c , cyp d , slco a , and slco b ); azithromycin (abcb ); ribavirin (slc a , slc a , and slc a ); and lopinavir/ritonavir (slco b , abcc , cyp a). we also identified other variants, that are associated with adverse effects, most notable in hydroxychloroquine/chloroquine (g pd; hemolysis), ribavirin (itpa; hemolysis), and interferon β - b (irf ; liver toxicity). we also describe the complexity of the risk for qt prolongation in this setting because of additive effects of combining more than one qt-prolonging drug (i.e., hydroxychloroquine/chloroquine and azithromycin), increased concentrations of the drugs due to genetic variants, along with the risk of also combining therapy with potent inhibitors. in conclusion, although direct evidence in covid- patients is lacking, we identified potential actionable genetic markers in covid- therapies. clinical studies in covid- patients are deemed warranted to assess potential roles of these markers. since the report of the first cluster infections in wuhan, a city in china in december , coronavirus disease (covid- ) has caused an unprecedented global pandemic and healthcare crisis with high mortality and morbidity. in an urgent attempt to mitigate its devastating catastrophe, many drugs without established efficacy have been used in patients either as an offlabel/compassionate use or as a clinical trial. under these extenuating circumstances these agents have been used without good evidence of efficacy and/or extent of toxicities. there is also no or limited data on the pharmacogenomics of these agents, and genomic determinants are important factors in efficacy and/or toxicity of many medications. pharmacogenomics may help clinicians to choose proper first-line agents and initial dosing that would be most likely achieve adequate drug exposure among critically ill patients; those who cannot afford a failure of ineffective therapy. it is also important to minimize the risks of toxicity because covid- particularly affects those with comorbidities on other drug therapies . therefore the purpose of this review was to summarize the pharmacogenomic literature and clinical recommendations available for covid- candidate drug therapies. in selection of drugs of interest, we reviewed the following sources: guidelines by the infectious disease society of america and the national institute of health . we also reviewed interventional clinical trials for covid- in clinicaltrials.gov registered as of june , (searched by a term "covid" and selected the study type "interventional [clinical trials]"). we excluded products derived of human plasma (e.g., convalescent plasma, immunoglobulin). our search was limited to online sources published in english. as a result, we included potential antiviral or immune-based therapy in the following eight categories for the present review: hydroxychloroquine/chloroquine, azithromycin, rna polymerase inhibitors, anti-retrovirus agents, interferon β- b (ifn-β b), il- /il- antagonists, janus kinase inhibitors, and corticosteroids. we then reviewed relevant literature by searching pubmed with the terms including but not limited to "pharmacogenomics", "pharmacogenetics", "polymorphism", and "pharmacokinetics" in combination with each drug name. we also reviewed the pharmacogenomics knowledgebase (pharmgkb ® ) website , clinical pharmacogenetics implementation consortium (cpic) guidelines , the u.s. food and drug administration (fda) pharmacogenomics information in the product labeling , and the fda pharmacogenomics association table . none of the reviewed drugs had applicable cpic guidelines. table summarizes the drugs described in this review and the pharmgkb and fda pharmacogenomic information. hydroxychloroquine and chloroquine the first antiviral drugs officially indicated for covid- in the u.s. were hydroxychloroquine and chloroquine as an fda emergency use authorization issued on march , . this was later revoked on june , based on new data suggesting that the drug's potential benefits may not outweigh its known and potential risks been used for malaria and autoimmune disorders for decades. it is thought to increase ph of phagolysosome and, thereby, interrupts virus fusion, and it also prevents binding of the virus to cell surface receptors . the immunomodulatory effects of these drugs may also play a role in managing the cytokine storm associated with advanced covid- disease. concerns around the heart rhythm problems of these agents for treatment of covid- has been announced from the fda . this is corroborated by a large retrospective study of hospitalized patients with covid- in new york metropolitan region; those treated with hydroxychloroquine and azithromycin had a higher incidence of cardiac arrest and possible higher mortality . the nih guidelines currently do not recommend the use of hydroxychloroquine or chloroquine . although the role of these drugs in covid- may be limited, biomarkers for toxicity may potentially be valuable. hydroxychloroquine and chloroquine are metabolized via cytochrome p (cyp) enzymes including cyp c , cyp a , and cyp d . these drugs are substrates of organic anion transporting polypeptides (oatp), influx cellular membrane transporters encoded by slco . the genes involved in the pharmacokinetics of hydroxychloroquine and chloroquine are considered "very important pharmacogenes (vips)" by the pharmgkb, genes that are particularly important in the field of pharmacogenomics. a study in patients with systemic lupus erythematosus showed that metabolism ratio of the active metabolite of hydroxychloroquine to its parent drug was increased by~ % in those carrying variants in cyp d (rs : cc vs. gg was . vs. . , respectively, p < . ) . in a cohort of malariainfected patients, low-activity alleles of cyp c (i.e., * , * , and * ) were associated with worse reduction of gametocytemia than wild-type alleles day after chloroquine/primaquine treatment (− . vs. − . gametocytes/μl, p = . , respectively) . in the same cohort, a subsequent analysis showed that variant alleles of slco a and slco b had lower gametocytemia clearance than the wild-type alleles after adjusting for cyp c (p = . and . , respectively) . hydroxychloroquine and chloroquine are relatively safe drugs when used for malaria and autoimmune disease, although there are several toxicities of importance. individuals who are glucose- phosphate dehydrogenase (g pd) deficient are considered at higher risk of hemolytic anemia upon exposure to hydroxychloroquine or chloroquine. however this has not been consistently shown. no individuals developed hemolytic anemia among a group of patients with g pd deficiency who received a -day course of chloroquine with methylene blue, nor another group of patients who received a total cumulative hydroxychloroquine exposure of person-months , . another well-recognized adverse effect of hydroxychloroquine and chloroquine is retinopathy, which occurs more frequently with long term use (in years) and higher doses. retinopathy occurred less frequently in those with the minor allele of abca c. a>g (or: . , % ci: . - . , adjusted for clinical factors including treatment duration and dose) . although data on the functional analysis of this variant is limited, mutations in abca are associated with various retinal diseases such as stargardt disease, which is phenotypically similar to chloroquine-induced retinopathy . the average treatment duration in the study was approximately years, thus the effects of abca c. a>g in short-term use of hydroxychloroquine or chloroquine for covid- treatment would likely to be small. of note, hydroxychloroquine is also a cyp d inhibitor, which may reduce cyp d activity. this is particularly of concern when other cyp d substrates that also prolong qt interval, such as ondansetron and haloperidol, are used concomitantly with hydroxychloroquine and chloroquine since these combinations may significantly reduce their metabolism which may further potentiate qt prolongation. azithromycin is a macrolide antibacterial agent with antiinflammatory properties. a recent observational study showed that azithromycin, when used in combination with hydroxychloroquine, may be more effective than hydroxychloroquine alone for covid- . although macrolide agents are associated with numerous drug-drug interactions, azithromycin is not a significant substrate of cyp a , slco b , or slco b , and therefore has fewer interactions than erythromycin or clarithromycin . the pharmacokinetics of azithromycin are, however, influenced by the activity of p-glycoprotein transporter encoded by abcb . genetic variation in abcb showed up to a -fold lower peak azithromycin concentrations in healthy volunteers after a single dose (rs tt/rs tt vs. rs gg/rs cc was . vs. . ng/ml, respectively, p = . ) . higher systemic exposure to azithromycin is of particular concern when it is combined with hydroxychloroquine or chloroquine because of their additive effects on qt prolongation which may result in fatal arrhythmias. rna polymerase inhibitors (remdesivir, ribavirin, and favipiravir) nucleotide analogs, including remdesivir, ribavirin and favipiravir, inhibit viral rna polymerase after metabolism to their active forms by intracellular enzymes. remdesivir became available for severe covid- in the u.s. via fda emergency use authorization on may , , following two randomized, double-blinded, placebo-controlled trials suggesting potential benefits outweighing the known and potential risks , . although no pharmacogenomic data of remdesivir are available, in vitro studies suggest that it is a substrate for drug metabolizing enzymes cyp c , cyp d , and cyp a , and is a substrate for oatp b and pglycoprotein transporters . thus, known variants of these genes could theoretically affect the pharmacokinetics of remdesivir [ ] [ ] [ ] . all of these genes are considered vips by pharmgkb. ribavirin is indicated for hepatitis c virus infection and is also under investigation for covid- . genetic polymorphisms in influx cellular transporters of ribavirin result in up to % variability in the trough concentration; troughs were significantly higher in those with slc a variants (homozygous for the variants ng/ml vs. wild-type ng/ml; p = . ), while significantly lower in those with slc a variants (homozygous for the variants ng/ml vs. heterozygous ng/ml vs. homozygous wildtype ng/ml; p = . ) and slc a variants (homozygous ng/ml vs. heterozygous ng/ml; p = . ) . it is wellrecognized that various itpa (inosine triphosphatase) variants have protective effects against hemolytic anemia, which is the most common and dose-limiting adverse effect of ribavirin . decreased itpa activity in red blood cells leads to accumulation of inosine triphosphate and protects against ribavirin-induced hemolysis. in a meta-analysis of studies, hemoglobin decline was associated with wild-type alleles of itpa; rs cc (or: . , % ci: . - . ), rs aa (or: . , % ci: . - . ), and rs aa (or: . , % ci: . - . ) . a model incorporating itpa genotypes and clinical factors was predictive of the degree of ribavirin-related hemoglobin decrease . of note, hemolytic anemia was also reported from a short-term use of ribavirin for respiratory virus infection . in contrast, the itpa variants in rs were identified to be a risk of thrombocytopenia in a genome-wide association study among patients with hepatitis c who received ribavirin and peg-interferon (or: . , % ci: . - . , p = . × - ) . favipiravir was developed and approved in japan in exclusively for a resistant, novel influenza pandemic and is now under investigation for covid- . although no published studies specifically have addressed its pharmacogenomics, it is metabolized by aldehyde oxidase and partly via xanthine oxidase . notably, variants of aldehyde oxidase are associated with pharmacodynamic outcomes in other drugs which are substrates of aldehyde oxidase such as azathioprine or allopurinol . anti-retrovirus agents (lopinavir/ritonavir, darunavir/cobicistat) lopinavir is a viral protease inhibitor that is primarily used in the treatment of human immunodeficiency virus (hiv), but it has also been used in covid- . hiv-protease inhibitors including lopinavir/ritonavir are currently not recommended for covid- because of lack of efficacy in a small randomized controlled trial and a concern of inadequate drug exposure relative to that required for sars-cov- inhibition . ritonavir inhibits the inactivation of lopinavir via the cyp a pathway and boosts the concentrations of lopinavir and is marketed as a combination product. besides cyp a, several pathways are involved in the pharmacokinetics of lopinavir, including other cyp enzymes and membrane drug transporters. a pharmacogenomic analysis of lopinavir/ritonavir, including variants in hiv-infected caucasians, identified four significant variants. clearance of lopinavir in a population pk model was higher in individuals with slco b * /* and lower in individuals with two or more variant alleles of slco b * , abcc or a cyp a tag compared to the reference group ( . vs. . vs. . l/h, respectively, p < . ) . another genetic association study explored variants for their effects on lopinavir/ritonavir related toxicity among caucasian patients with hiv; variants in the cetp, mcp- , abcc , lep, and slco b genes were associated with dyslipidemia and hyperbilirubinemia, and a variant in il- was associated with diarrhea (all p < . ) . darunavir, also a protease inhibitor used in hiv treatment, is a substrate of cyp a that is used simultaneously with a cyp a inhibitor, cobicistat, in a clinical trial for covid- . several haplotypes in cyp a have been discovered that affect the activity and/or expression of cyp a . those haplotypes could hypothetically affect concentrations of these drugs, but they have yet to be studied specifically with darunavir. although there is no direct evidence that darunavir is a substrate for slco a , a % significantly lower darunavir clearance was observed in carriers of an slco a variant (p < . ) . a family of ifns, particularly ifn-β b, has showed efficacy against sars-and/or mers-coronaviruses and are currently being investigated for covid- either alone or in combination with other therapy (e.g., lopinavir/ritonavir) . as it is commonly seen in other biologic drugs, pharmacogenomics determinants are not well-delineated for ifn-β b. in contrast, reduced efficacy and increased adverse effects secondary to immunogenicity is a specific concern among biologics. in a cohort of swedish patients with multiple sclerosis who received ifn-β b, the risk of biologically relevant neutralizing antibody development was higher in patients with the hla-drb * allele (or: . , % ci: . - . ) and lower with hla-drb * (or: . , % ci: . - . ) . in a two-stage genome-wide association study among cases and controls of ifn-β b-treated patients with multiple sclerosis, a higher risk of drug-induced liver injury was identified (p = . × − , or: . , % ci: . - . ) in patients with variants of irf , which encodes for an interferon regulatory factor and is involved in promotion of liver damage . the results were confirmed in an independent cohort of multiple sclerosis patients for an association with increased peak levels of aspartate aminotransferase (p = . × − ) and alkaline phosphatase (p = . × − ) . il- and il- antagonists (tocilizumab, sarilumab, siltuximab, anakinra) severe covid- is associated with a cytokine-release syndrome with elevated interleukin- (il- ) . tocilizumab, an inhibitor of the il- receptor, is commonly used for rheumatoid arthritis (ra) and cytokine-release syndrome induced by chimeric antigen receptor-t cell therapy, and now it is under investigation for covid- . although several genetic biomarkers have been reported in the efficacy of tocilizumab in ra, including fcgr a, il r, cd , galnt [ ] [ ] [ ] , potential translation of these data to covid- is highly speculative. no studies have addressed pharmacogenomics of tocilizumab in patients with cytokine-release syndrome, which is similar to the physiology in covid- . the only genetic variants potentially involving tocilizumab's pharmacokinetics are in the fcgr a gene, where differences in efficacy is postulated to be due to changes in systemic exposure. in patients with ra treated with tocilizumab, fcgr a rs tt genotype showed higher response at months (vs. gt; or: . ; % ci: . - . ; p = . ). this variant may affect the affinity of the fc fragment of igg receptor to tocilizumab and alter its systemic clearance . polymorphisms of il r are considered to affect intracellular signaling pathway of il- receptor bound to tocilizumab, which may also be applicable to other conditions with upregulated il- pathway . in contrast, variants in cd and galnt are thought to have limited direct effects on tocilizumab. variants in those genes are more likely to affect the downstream signaling pathways of the immune system in ra patients, which may limit generalization to non-ra patients . at this point there is limited evidence that pharmacogenomic biomarkers would be helpful in determining response to tocilizumab therapy in covid- . no relevant pharmacogenomic data is reported in other il- or il- antagonists (i.e., sarilumab, siltuximab, anakinra). a group of janus kinase inhibitors is another potentially effective immunomodulator for covid- that is currently in clinical trials. ruxolitinib is approved by the fda for myeloproliferative diseases and graft-versus-host-disease, and baricitinib for ra. no published studies have addressed the effects of genetic variants on either of the two drugs in any patient population. however, their pharmacokinetics pathways involve a few potentially important pharmacogenes. ruxolitinib is a major and baricitinib is a minor substrate of cyp a , . ruxolitinib is also partly metabolized by cyp c . both of these genes are considered vips in the pharmgkb and subject to notable genetic polymorphisms , . although the main pharmacogene of baricitinib, slc a encoding oat transporter , is not a vip, influence of variants on its activity is previously reported in another substrate drug . the potential role of corticosteroids in the treatment of covid- infected patients is mainly limited to those with acute respiratory distress syndrome (ards). in patients with sars-coronavirus infection, corticosteroid use was associated with delayed viral clearance but also showed possible benefits in those with ards . many variants have been associated with corticosteroids response and toxicities across multiple disease conditions, including genes involved in the receptor binding (e.g., crhr , nr c ), chaperone/ cochaperone protein (e.g., st , stip , fkbp ), metabolizing enzymes (e.g., cyp a , cyp a , cyp a , gstt ), and transporters (e.g., mdr , abcb ) . the mechanistic and metabolic pathways of steroids are complex, and genomic determinants with sufficient evidence for clinical application to covid- were not identified. variants associated with corticosteroids in pharmgkb with level of evidence higher than "low" (level ) were only assessed in combination therapy (e.g., with chemotherapy) or inhaled corticosteroids. no pharmacogenetic information specifically on the effectiveness of corticosteroids for ards was found. summary of clinical implications of pharmacogenomics for covid- we found evidence that several genetic variants may alter the pharmacokinetics of hydroxychloroquine, azithromycin, ribavirin, lopinavir/ritonavir and possibly tocilizumab, which hypothetically may affect clinical response and toxicity in the treatment of covid- . although the level of evidence for most is weak, and has not been directly studied in patients with covid- , some of these potential pharmacogenetic associations are worth further exploration. qt prolongation could hypothetically be exacerbated by a combination of drug-drug, drug-gene, and drug-disease interactions in the treatment of covid- . as previously described in this review, hydroxychloroquine, chloroquine and azithromycin can individually increase risk for qt prolongation, and those drugs have been used in combination in covid- patients. this combination therapy showed higher odds of cardiac arrest in hospitalized patients with covid- in comparison to those on neither therapy (odds ratio . [ % ci: . - . ]) . hydroxychloroquine is a cyp d inhibitor, which calls for a vigilance in drug-drug interactions in patients with covid- , who are at risk of polypharmacy. in particular, some qt-prolonging drugs, that are commonly used in hospitalized patients, are substrates of cyp d which may be inhibited by hydroxychloroquine. this may dramatically increase the risk of qt prolongation. the cyp d * nonfunctional allele is present in nearly % of patients with european ancestry , and thus patients with the cyp d * allele may have even higher risk. patients with the abcb gg/ cc genotype have higher peak concentrations of azithromycin, a qt-prolonging drug . higher concentrations of azithromycin in combination with hydroxychloroquine may be particularly dangerous. baseline qt interval evaluation prior to therapy is important since there is a greater risk of prolongation in individuals with high baseline values. the prevalence of high baseline qt intervals was reported to be . % among those aged ≥ years in a us population study . and finally, the potential combination of these arrhythmogenic risks may be further exacerbated in the setting of covid- , in which the disease is associated with myocardial injury and arrhythmias . the association between ifn-β b and a variant in irf for the adverse outcome of liver damage was one of the strongest pharmacogenetic associations identified in this review . the previous studies of ifn-β b/irf were performed in patients with multiple sclerosis, not covid- . therefore the ifn-β b/irf pharmacogenetic association should be investigated in patients with covid- , especially since a large portion of patients with covid- have abnormal liver function tests and liver injury . individuals homozygous for slco b * showed a greater than fold increase in lopinavir clearance. notably, this variant has a differing allele frequency across racial groups; it is % in european, % in african, and . % in east asian descents. although g pd deficiency is associated with hydroxychloroquine and chloroquine related hemolytic anemia, the evidence suggests that the strength of the association is unclear and possibly because there are varying degrees of g pd deficiency conferred by the genetic variations. the itpa variants, associated with protective effects against ribavirinrelated hemolytic anemia, has pharmgkb level evidence (moderate strength), may have a role in improving ribarivin safety. older age, race, male gender, obesity and comorbid conditions have been established as risk factors for covid- susceptibility and death , . whether they are also precise risk factors for drug treatment failure have yet to be established. it has yet to be determined if host genomics or confounding factors (e.g., access to healthcare, environment) also play a role. several studies though suggest that higher susceptibility to covid- is associated with genetic variants, including those in the ace , ace , tmprss , gstt genes [ ] [ ] [ ] . interestingly, several important ace variants on the x-linked locus were identified in male as hemizygous, which may explain the higher mortality in male . although no direct evidence of pharmacogenomics data in patients with covid- was available at this time, there are plausible mechanisms by which genetic determinants may play a role. studies must be conducted in covid- before pharmacogenomic testing can be recommended. these data support the collection of dna samples for pharmacogenomic studies of the hundreds of currently ongoing clinical trials of covid- therapies. one of the biggest success stories in the field of pharmacogenomics was for a drug used to treat another, highly lethal, infectious disease: abacavir for hiv. the application of a pharmacogenetic test (hla-b* : ) nearly eliminates a potentially fatal hypersensitivity reaction to abacavir . thus, the pharmacogenetic test for abacavir is now standard of care in the treatment of hiv. it took years after the discovery of hiv to develop such a pharmacogenetic success for abacavir. while it is understandable, at these very early stages since the discovery of covid- , the pharmacogenetic data are very limited. the use of modern genomic tools coupled with a proactive assessment of the most likely gene-drug candidates could lead to a quicker understanding of the role of pharmacogenetics for covid- . important in silico efforts are underway to repurpose old drugs for covid- and some of these drugs may have established pharmacogenomic markers in other disease , . it is worth noting potential limitations of the use of pharmacogenetics in the treatment of covid- . in an acute illness such as covid- , pharmacogenetics would only be useful if the genetic test results were already available (i.e., pre-emptive pharmacogenetic testing) or rapidly available (i.e., point-of-care genetic testing). several institutions have already implemented pre-emptive pharmacogenetic testing, and some patients may have results readily available . point-of-care pharmacogenetic tests are available but generally the potential variants of relevance in covid- therapies are not available on this type of testing platform . in the face of unprecedented challenges posed by the covid- pandemic, collaborative efforts among the medical communities are more important than ever to improve the efficacy of these treatments and ensure safety. some large national covid- trials are evaluating pharmacogenomics, which will inform the role of pharmacogenomics markers for future clinical use. no datasets were generated or analyzed during the current study. received: april ; accepted: july ; clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study infectious diseases society of america guidelines on the treatment and management of patients with covid- infection covid- ) treatment guidelines the clinical pharmacogenetics implementation consortium (cpic ® ) table of pharmacogenomic biomarkers in drug labeling request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease review: hydroxychloroquine and chloroquine for treatment of sars-cov- (covid- ) fda drug safety communication: fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state a review of pharmacogenetics of antimalarials and associated clinical implications slco a , slco b and slco b polymorphisms influences chloroquine and primaquine treatment in plasmodium vivax malaria association of polymorphisms of cytochrome p d with blood hydroxychloroquine levels in patients with systemic lupus erythematosus the effect of snps in cyp in chloroquine/primaquine plasmodium vivax malaria treatment examination of hydroxychloroquine use and hemolytic anemia in g pdh-deficient patients safety of the combination of chloroquine and methylene blue in healthy adult men with g pd deficiency from rural burkina faso common synonymous variants in abca are protective for chloroquine induced maculopathy (toxic maculopathy) a photoreceptor cell-specific atp-binding transporter gene (abcr) is mutated in recessive stargardt macular dystrophy hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial pharmgkb summary: macrolide antibiotic pathway, pharmacokinetics/pharmacodynamics influence of abcb gene polymorphisms on the pharmacokinetics of azithromycin among healthy chinese han ethnic subjects remdesivir for the treatment of covid- -preliminary report remdesivir for or days in patients with severe covid- fact sheet for health care providers: emergency use authorization (eua) of remdesivir (gs- ™) cyp c : the pharmacogene variation (pharmvar) consortium cyp d : the pharmacogene variation (pharmvar) consortium cyp a : the pharmacogene variation (pharmvar) consortium role of pharmacogenetic in ribavirin outcome prediction and pharmacokinetics in an italian cohort of hcv- and patients pharmacogenetics of ribavirin-induced anemia in hcv patients relationship between itpa polymorphisms and hemolytic anemia in hcv-infected patients after ribavirin-based therapy: a meta-analysis a formula to estimate the optimal dosage of ribavirin for the treatment of chronic hepatitis c: influence of itpa polymorphisms oral ribavirin for respiratory syncytial virus infection after lung transplantation: efficacy and cost-efficiency genome-wide association study identified itpa/ddrgk variants reflecting thrombocytopenia in pegylated interferon and ribavirin therapy for chronic hepatitis c ebola virus infection: review of the pharmacokinetic and pharmacodynamic properties of drugs considered for testing in human efficacy trials aldehyde oxidase; new approaches to old problems adme pharmacogenetics: investigation of the pharmacokinetics of the antiretroviral agent lopinavir coformulated with ritonavir toxicogenetics of lopinavir/ritonavir in hiv-infected european patients simultaneous pharmacogenetics-based population pharmacokinetic analysis of darunavir and ritonavir in hiv-infected patients human leukocyte antigen genes and interferon beta preparations influence risk of developing neutralizing anti-drug antibodies in multiple sclerosis common variation near irf is associated with ifn-betainduced liver injury in multiple sclerosis clinical features of patients infected with novel coronavirus in wuhan fcgr a/fcgr a gene polymorphisms and clinical variables as predictors of response to tocilizumab and rituximab in patients with rheumatoid arthritis influence of il r gene polymorphisms in the effectiveness to treatment with tocilizumab in rheumatoid arthritis genetic and clinical biomarkers of tocilizumab response in patients with rheumatoid arthritis jakafi (ruxolitinib) olumiant (baricitinib) cyp c : the pharmacogene variation (pharmvar) consortium reduced renal clearance of cefotaxime in asians with a lowfrequency polymorphism of oat (slc a ) sars: systematic review of treatment effects genetic variation in the glucocorticoid pathway involved in interindividual differences in the glucocorticoid treatment cyp d worldwide genetic variation shows high frequency of altered activity variants and no continental structure association of qt interval with mortality by kidney function: results from the national health and nutrition examination survey (nhanes) cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) liver injury in covid- : management and challenges disparities in outcomes among covid- patients in a large health care system in california covid- : the gendered impacts of the outbreak effects of host genetic variations on response to, susceptibility and severity of respiratory infections an evidence for correlation between the glutathione s-transferase t (gstt ) polymorphism and outcome of covid- c and ace polymorphisms are more important confounders in the spread and outcome of covid- in comparison with abo polymorphism the expression and polymorphism of entry machinery for covid- in human: juxtaposing population groups, gender, and different tissues hla-b* screening for hypersensitivity to abacavir network-based drug repurposing for novel coronavirus -ncov/ sars-cov- network-based approach to prediction and population-based validation of in silico drug repurposing the pharmacogenomics research network translational pharmacogenetics program: outcomes and metrics of pharmacogenetic implementations across diverse healthcare systems challenges of development and implementation of point of care pharmacogenetic testing impact of genetic slc transporter and itpa variants on ribavirin serum level, hemoglobin drop and therapeutic response in patients with hcv infection influence of mdr c t polymorphism on lopinavir plasma concentration and virological response in hiv- -infected children individualized protease inhibitormonotherapy: the role of pharmacokinetics and pharmacogenetics in an aged and heavily treated hiv-infected patient single-nucleotidepolymorphisms in hla-and non-hla genes associated with the development of antibodies to interferon-β therapy in multiple sclerosis patients j.a.l. and p.a.j. conceived the project. t.t. conducted initial literature search and drafted the manuscript. t.t. and p.a.j. further refined the manuscript with input from j.a.l. and m.r.n. all authors contributed further literature search, provided critical feedback, and collectively composed the final manuscript. p.a.j was in charge of overall direction and planning. the authors declare no competing interests. correspondence and requests for materials should be addressed to p.a.j.reprints and permission information is available at http://www.nature.com/ reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -mhviub e authors: le, brian l.; andreoletti, gaia; oskotsky, tomiko; vallejo-gracia, albert; rosales, romel; yu, katharine; kosti, idit; leon, kristoffer e.; bunis, daniel g.; li, christine; kumar, g. renuka; white, kris m.; garcía-sastre, adolfo; ott, melanie; sirota, marina title: transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for covid- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: mhviub e the novel sars-cov- virus emerged in december and has few effective treatments. we applied a computational drug repositioning pipeline to sars-cov- differential gene expression signatures derived from publicly available data. we utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and sars-cov- -infected samples. using a rank-based pattern matching strategy based on the kolmogorov-smirnov statistic, the signatures were queried against drug profiles from connectivity map (cmap). we validated sixteen of our top predicted hits in live sars-cov- antiviral assays in either calu- or t-ace cells. validation experiments in human cell lines showed that of the compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against sars-cov- . these initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of covid- . sars-cov- has already claimed at least a million lives, has been detected in at least million people, and has likely infected at least another million. the spectrum of disease caused by the virus can be broad ranging from silent infection to lethal disease, with an estimated infection-fatality ratio around % . sars-cov- infection has been shown to affect many organs of the body in addition to the lungs . three epidemiological factors increase the risk of disease severity: increasing age, decade-by-decade, after the age of years; being male; and various underlying medical conditions . however, even taking these factors into account, there is immense interindividual clinical variability in each demographic category considered . recently, researchers found that more than % of people who develop severe covid- have misguided antibodies-autoantibodies-that attack the innate immune system. another . % or more of people who develop severe covid- carry specific genetic mutations that impact innate immunity. consequently, both groups lack effective innate immune responses that depend on type interferon, demonstrating a crucial role for type interferon in protecting cells and the body from covid- . whether the type interferon has been neutralized by autoantibodies or-because of a faulty gene-is produced in insufficient amounts or induced an inadequate antiviral response, the absence of type ifn-mediated immune response appears to be a commonality among a subgroup of people who suffer from life-threatening covid- pneumonia . while numerous efforts are underway to identify potential therapies targeting various aspects of the disease, there is a paucity of clinically proven treatments for covid- . there have been efforts to therapeutically target the hyperinflammation associated with severe covid- , as well as to utilize previously identified antiviral medications , . one of these antivirals, remdesivir, an intravenously administered rnadependent rna polymerase inhibitor, showed positive preliminary results in patients with severe covid- . in october , the fda approved remdesivir for the treatment of covid- . dexamethasone has also been shown to reduce the mortality rate in cases of severe covid- . nevertheless, the lack of treatments and the severity of the current health pandemic warrant the exploration of rapid identification methods of preventive and therapeutic strategies from every angle. the traditional paradigm of drug discovery is generally regarded as protracted and costly, taking approximately years and over $ billion to develop and bring a novel drug to market . the repositioning of drugs already approved for human use mitigates the costs and risks associated with early stages of drug development, and offers shorter routes to approval for therapeutic indications. successful examples of drug repositioning include the indication of thalidomide for severe erythema nodosum leprosum and retinoic acid for acute promyelocytic leukemia . the development and availability of large-scale genomic, transcriptomic, and other molecular profiling technologies and publicly available databases, in combination with the deployment of the network concept of drug targets and the power of phenotypic screening, provide an unprecedented opportunity to advance rational drug design. drug repositioning is being extensively explored for covid- . high-throughput screening pipelines have been implemented in order to quickly test drug candidates as they are identified [ ] [ ] [ ] [ ] . in the past, our group has successfully applied a transcriptomics- based computational drug repositioning pipeline to identify novel therapeutic uses for existing drugs . this pipeline leverages transcriptomic data to perform a patternmatching search between diseases and drugs. the underlying hypothesis is that for a given disease signature consisting of a set of up and down-regulated genes, if there is a drug profile where those same sets of genes are instead down-regulated and upregulated, respectively, then that drug could be therapeutic for the disease. this method has shown promising results for a variety of different indications, including inflammatory bowel disease , dermatomyositis , cancer [ ] [ ] [ ] , and preterm birth . in existing work from xing et al. , this pipeline has been used to identify potential drug hits from multiple input disease signatures derived from sars-cov or mers-cov data. the results were aggregated to obtain a consensus ranking, with drugs selected for in vitro testing against sars-cov- in vero e cell lines, with four drugs (bortezomib, dactolisib, alvocidib and methotrexate) showing viral inhibition . however, this pipeline has not yet been applied specifically to sars-cov- infection. a variety of different transcriptomic datasets related to sars-cov- were published in the spring of . in may , blanco-melo et al. studied the transcriptomic signature of sars-cov- in a variety of different systems, including human cell lines and a ferret model . by infecting human adenocarcinomic alveolar basal epithelial cells with sars-cov- and comparing to controls, the authors generated a list of differentially expressed genes. they observed two enriched pathways: one composed primarily of type-i interferon-stimulated genes (isgs) involved in the cellular response to viral infection; and a second composed of chemokines, cytokines, and complement proteins involved in the humoral response. after infecting the cell lines, blanco-melo et al. did not detect either ace or tmprss , which are the sars-cov- receptor and sars-cov- protease, respectively . however, supported viral replication was observed, thereby allowing the capture of some of the biological responses to sars-cov- . in may , another study by lamers et al. examined sars-cov- infection in human small intestinal organoids grown from primary gut epithelial stem cells. the organoids were exposed to sars-cov- and grown in various conditions, including wnt-high expansion media. enterocytes were readily infected by the virus, and rna sequencing revealed upregulation of cytokines and genes related to type i and iii interferon responses . a limited amount of transcriptomic data from human samples has also been published. one study detailed the transcriptional signature of bronchoalveolar lavage fluid (of which responding immune cells are often a primary component) of covid- patients compared to controls . despite a limited number of samples, the results were striking enough to reveal inflammatory cytokine profiles in the covid- cases, along with enrichments in the activation of apoptosis and the p signaling pathways. on the drug side, data are available in the form of differential gene expression profiles from testing on human cells. publicly-available versions include the connectivity map (cmap) , which contains genome-wide testing on approximately , drugs, wherein the differential profile for a drug was generated by comparing cultured cells treated with the drug to untreated control cultures. here, we applied our existing computational drug repositioning pipeline to identify drug profiles with significantly reversed differential gene expression compared to predicted inhibitors (including one tested in calu- ) were incubated with sars-cov- infected human embryonic kidney t cells overexpressing ace ( t-ace ) with viral replication determined using an immunofluorescence-based assay. in this study, we applied our drug repositioning pipeline to sars-cov- differential gene expression signatures derived from publicly available rna-seq data ( figure ). the transcriptomic data were generated from distinct types of tissues, so rather than aggregating them together, we predicted therapeutics for each signature and then combined the results. we utilized three independent gene expression signatures (labelled "alv", "exp", and "balf"), each of which consisted of lists of differentially expressed genes between sars-cov- samples and their respective controls. the alv signature was generated from human adenocarcinomic alveolar basal epithelial cells by comparing sars-cov- infection to mock-infection conditions . the exp signature originated from a study where organoids, grown from human intestinal cells expanded in wnt-high expansion media, were infected with sars-cov- and then compared to controls . the balf signature was from a contrast of primary human balf samples from two covid- patients versus three controls . each of these signatures was contrasted with drug profiles of differential gene expression from cmap. for each of the input signatures, we applied a significance threshold false discovery rate (fdr) < . . we further applied minimum fold change thresholds in order to identify the driving genes. the alv signature had only genes, with genes shared with the drug profiles; in order to maintain at least genes for the pattern-matching algorithm to work with, we applied no fold-change threshold. for the exp signature, we applied a |log fc| > cutoff, resulting in genes for the expansion signature ( shared with the drug profiles). for the balf signature, we processed the raw read count data to calculate differential gene expression values. we applied a |log fc| > cutoff, with the balf data yielding , protein-coding genes for the lavage fluid signature ( shared with the drug profiles). the gene lists for each of these signatures can be found in the supplement (tables s , s , s ). we used gsea (gene set enrichment analysis) we used the publicly available single-cell rnaseq dataset gse composed of patients ( healthy, presenting with mild covid- symptoms, and presenting with severe covid- symptoms) to further characterise the expression of dkk ( figure s ). data were re-analyzed following the standard seurat pipeline. from the analyses of the single-cell data, dkk is highly expressed in covid- patients compared to controls, specifically in severe patients and it is expressed by epithelial cells. after analyzing the input sars-cov- signatures, we utilized our repositioning pipeline to identify drugs with reversed profiles from cmap ( figure ). significantly reversed drug profiles were identified for each of the signatures using a permutation approach: hits from the alv signature (table s ), hits from the exp signature (table s ) , and hits from the balf signature (table s ). when visualizing the gene regulation of the input signatures and their respective top drug hits, the overall reversal pattern can be observed ( figure c -e). in total, our analysis identified unique drug hits (table s ) . twenty-five common drug hits were shared by at least two of the signatures (p = . ), with four consensus drug hits (bacampicillin, clofazimine, haloperidol, valproic acid) across all three signatures (p = . ) (table , figure a ). we further characterized the common hits by examining their interactions with proteins in humans. we used known drug targets from drugbank and predicted additional targets using the similarity ensemble approach (sea) . we visualized the known interactions from drugbank in a network ( figure b ). we also aggregated the list of proteins which were found in drugbank for at least of the common hits (table s ) to confirm the validity of our approach, the inhibitory effects of of our drug hits which significantly reversed multiple sars-cov- profiles were assessed in live antiviral assays. the inhibitory effects of haloperidol, clofazimine, valproic acid, and fluticasone were evaluated in sars-cov- infected calu- cells (human lung epithelial cell line), with remdesivir also tested as a positive control. from these five, remdesivir and haloperidol inhibited viral replication ( figure a ), and the inhibitory effect was also observed by microscopy ( figure b ). additionally, drugs (bacampicillin, ciclopirox, ciclosporin, clofazimine, dicycloverine, fludrocortisone, isoxicam, lansoprazole, metixene, myricetin, pentoxifylline, sirolimus, tretinoin) were assessed in a live sars-cov- antiviral assay. remdesivir was again used as a positive control. this testing involved six serial dilutions of each drug to inhibit the replication of sars-cov- in t-ace cells using an immunofluorescence-based antiviral assay . all antiviral assays were paired with cytotoxicity assays using identical drug concentrations in uninfected human t-ace cells. positive control remdesivir and of our predicted drugs (bacampicillin, ciclopirox, ciclosporin, clofazimine, dicycloverine, isoxicam, metixene, pentoxifylline, sirolimus, and tretinoin) showed antiviral efficacy against sars-cov- , reducing viral infection by at least %, that was distinguishable from their cytotoxicity profile when tested in this cell line ( figure ) . several inhibitors showed micromolar to sub-micromolar antiviral efficacy, including clofazimine, ciclosporin, ciclopirox, and metixene. these results not only confirm our predictive methods, but have also identified several clinically-approved drugs with potential for repurposing for the treatment of covid- . here, we used a transcriptomics-based drug repositioning pipeline to predict therapeutic drug hits for three different input sars-cov- signatures, each of which came from distinct human cell or tissue origins. we found significant overlap of the therapeutic predictions for these signatures. twenty-five of our drug hits reversed at least two of the three input signatures (table ) . notably, of the hits from the exp signature were also hits for the balf signature, despite being generated from different types of tissue. the exp signature was generated from intestinal tissue, whereas the balf signature was generated from constituents of the respiratory tract. among the common hits reversing at least two of the signatures were two immunosuppressants (ciclosporin and sirolimus), an antiinflammatory medication (isoxicam), and two steroids (fludrocortisone and fluticasone). our testing of clofazimine demonstrated submicromolar antiviral effects of this drug in sars-co-v- infected t-ace and vero e cells (figures and s ) . clofazimine is an orally administered antimycobacterial drug used in the treatment of leprosy. by preferentially binding to mycobacterial dna, clofazimine disrupts the cell cycle and eventually kills the bacterium . in addition to being an antimycobacterial agent, clofazimine also possesses anti-inflammatory properties primarily by inhibiting t lymphocyte activation and proliferation validation experiments revealed antiviral activity for of drug hits. further clinical investigation into these drug hits as well as potential combination therapies is warranted. we have previously developed and used a transcriptomics based bioinformatics approach for drug repositioning in various contexts including inflammatory bowel disease, dermatomyositis, and spontaneous preterm birth. for a list of differentially expressed genes, the computational pipeline compares the ranked differential expression of a disease signature with that of a profile , , . a reversal score based on the kolmogorov-smirnov statistic is generated for each disease-drug pair, with the idea that if the drug profile significantly reverses the disease signature, then the drug could be potentially therapeutic for the disease. blanco-melo et al. generated a differential gene expression signature using rnaseq on human adenocarcinomic alveolar basal epithelial cells infected with sars-cov- propagated from vero e cells (gse ) . due to the fast-moving nature of the research topic, we opted to use this cell line data in lieu of waiting for substantial patient-level data. this work identified differentially expressed genes (degs) - upregulated and downregulated. we used these genes as the alv signature for our computational pipeline (table s ) (v. . . r package) to call differentially expressed genes (degs). after adjusting for the sequencing platform, the default settings of deseq were used. principal components were generated using the deseq function ( figure s ), and heat maps were generated using the bioconductor package pheatmap (v. . . ) using the rlogtransformed counts ( figure s ). values shown are rlog-transformed and rownormalized. volcano plots were generated using the bioconductor package enhancedvolcano (v. . . ) ( figure s ). retaining only protein-coding genes and applying both a significance threshold and a fold-change cutoff (fdr < . , |log fc| > ), we obtained , genes to be used as the balf signature (table s ). functional enrichment gene-set analysis for gsea (gene set enrichment analysis) was performed using fgsea (v. . . r package) and the input gene lists were ranked by log fold change. the hallmark gene sets used in the gsea analysis were downloaded from msigdb signatures database , . for go (gene ontology) terms, identification of enriched biological themes was performed using the david database . drug gene expression profiles were sourced from connectivity map (cmap), a publicly-available database of drugs tested on cancer cell lines computational gene expression reversal scoring to compute reversal scores, we used a non-parametric rank-based method similar to the kolmogorov-smirnov test statistic. this analysis was originally suggested by the creators of the cmap database and has since been implemented in a variety of different settings [ ] [ ] [ ] [ ] , . similar to past works, we applied a pre-filtering step to the cmap profiles to maintain only drug profiles which were significantly correlated with another profile of the same drug. drugs were assigned reversal scores based on their ranked differential gene expression profile relative to the sars-cov- ranked differential gene expression signature. a negative reversal score indicated that the drug had a profile which reversed the sars-cov- signature; that is, up-regulated genes in the sars-cov- signature were down-regulated in the drug profile and vice versa. single-cell data analysis quantification files were downloaded from geo gse . an individual seurat object for each sample was generated using seurat v. . while the data has been filtered by x's algorithm, we still needed to ensure the remaining cells are clean and devoid of artifacts. we calculated three confounders for the dataset: mitochondrial percentage, ribosomal percentage, and cell cycle state information. for each sample, cells were normalized for genes expressed per cell and per total expression, then multiplied by a scale factor of , and log-transformed. low quality cells were excluded from our analyses-this was achieved by filtering out cells with greater than , and fewer than genes and cells with high percentage of mitochondrial and ribosomal genes (greater than % for mitochondrial genes, and % for ribosomal genes). sctransform is a relatively new technique that uses "pearson residuals" (pr) to normalize the data. pr's are independent of sequencing depththanks . we "regress out" the effects of mitochondrial and ribosomal genes, and the cell cycling state of each cell, so they do not dominate the downstream signal used for clustering and differential expression. we then performed a lineage auto-update disabled r dimensional reduction (runpca function). then, each sample was merged together into one seurat object. data were then re-normalized and dimensionality reduction and significant principal for studies at the gladstone institutes, calu- cells, a human lung epithelial cell line for studies carried out at mount sinai, sars-cov- was propagated in vero e cells and t-ace cells, as previously described in , . two thousand cells were seeded into -well plates in dmem ( % fbs) and incubated for h at °c, % co . then, h before infection, the medium was replaced with predicted covid- fatality rates based on age, sex, comorbidities and health system capacity extrapulmonary manifestations of covid- inborn errors of type i ifn immunity in patients with life-threatening covid- covid- : consider cytokine storm syndromes and immunosuppression a protein interaction map identifies existing drugs targeting sars-cov- silico discovery of candidate drugs against covid- remdesivir for the treatment of covid- -preliminary report food and drug administration. fda approves first treatment for covid- dexamethasone in hospitalized patients with covid- -preliminary report the price of innovation: new estimates of drug development costs old drugs -new uses a sars-cov- protein interaction map reveals targets for drug repurposing network-based drug repurposing for novel coronavirus -ncov/sars-cov- comparative host-coronavirus protein interaction networks reveal panviral disease mechanisms in silico studies on therapeutic agents for covid- : drug repurposing approach discovery and preclinical validation of drug indications using compendia of public gene expression data computational repositioning of the anticonvulsant topiramate for inflammatory bowel disease identification of alphaadrenergic agonists as potential therapeutic agents for dermatomyositis through drugrepurposing using public expression datasets computational discovery of niclosamide ethanolamine, a repurposed drug candidate that reduces growth of hepatocellular carcinoma cells in vitro and in mice by inhibiting cell division cycle signaling reversal of cancer gene expression correlates with drug efficacy and reveals therapeutic targets a drug repositioning approach identifies tricyclic antidepressants as inhibitors of small cell lung cancer and other neuroendocrine tumors computational discovery of therapeutic candidates for preventing preterm birth reversal of infected host gene expression identifies repurposed drug candidates for imbalanced host response to sars-cov- drives development of covid- sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor sars-cov- productively infects human gut enterocytes transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles pgc- α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes single-cell landscape of bronchoalveolar immune cells in patients with covid- drugbank . : a major update to the drugbank database for relating protein pharmacology by ligand chemistry an in vitro microneutralization assay for sars-cov- serology and drug screening schizophrenia: more dopamine, more d receptors clofazimine: current status and future prospects clofazimine is a broad-spectrum coronavirus inhibitor that antagonizes sars-cov- replication in primary human cell culture and hamsters systematic integration of biomedical knowledge prioritizes drugs for repurposing a next generation connectivity map: l platform and the first , , profiles entrez gene: gene-centered information at ncbi circulating levels of gdf- and calprotectin for prediction of in-hospital mortality in covid- patients: a case series who | who model lists of essential medicines molecular signatures database (msigdb) . database for annotation, visualization, and integrated discovery normalization and variance stabilization of single-cell rna-seq data using regularized negative binomial regression interferon_alpha_response interferon_gamma_response e f_targets myc_targets_v g m_checkpoint il _jak_stat _signaling kras_signaling_dn oxidative_phosphorylation estrogen_response_late androgen_response spermatogenesis tnfa_signaling_via_nfkb kras_signaling_up pancreas_beta_cells inflammatory_response myc_targets_v dna_repair epithelial_mesenchymal_transition allograft_rejection unfolded_protein_response complement estrogen_response_early protein_secretion uv_response_up fatty_acid_metabolism hedgehog_signaling apical_surface uv_response_dn notch_signaling reactive_oxigen_species_pathway tgf_beta_signaling il _stat _signaling mitotic_spindle peroxisome pi k_akt_mtor_signaling xenobiotic_metabolism p _pathway glycolysis coagulation apical_junction mtorc _signaling cholesterol_homeostasis adipogenesis key: cord- -lnvc mmf authors: lichtenstein, david; alfa, michelle j. title: cleaning and disinfecting gastrointestinal endoscopy equipment date: - - journal: clinical gastrointestinal endoscopy doi: . /b - - - - . - sha: doc_id: cord_uid: lnvc mmf abstract outbreaks of infection transmission due to contaminated flexible endoscopes have focused the attention of health care personnel, senior management, device manufacturers, and regulators on the need to improve the approach used to offer this valuable service. this chapter presents the principles of flexible endoscope reprocessing along with a pragmatic approach to the judicious selection and proper reprocessing of endoscopic equipment, as well as guidance for prevention and management of infection transmission inclusive of newer sterilization (e.g., hydrogen peroxide vapor) and disinfection (e.g., improved hydrogen peroxide) technologies. it also provides an outline of the quality systems approach that is applicable to flexible endoscope reprocessing and the need for ongoing staff competency and audits of endoscope cleaning, disinfection, and storage practices. furthermore, the most current regulatory, expert organization, and manufacturer's recommendations are reviewed. the field of gastrointestinal (gi) endoscopy has expanded dramatically as new procedures, instruments, and accessories have been introduced into the medical community; more than million gi endoscopies are performed annually in the united states. , although gi endoscopes are used as a diagnostic and therapeutic tool for a broad spectrum of gi disorders, more health care-associated infectious outbreaks and patient exposures have been linked to contaminated endoscopes than to any other reusable medical device. [ ] [ ] [ ] [ ] failure to adhere to established reprocessing guidelines or the use of defective reprocessing equipment accounts for the majority of these cases. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition, complex endoscopes such as the duodenoscope and linear echoendoscope with elevator mechanisms can transmit bacterial infections even when reprocessing protocols are reportedly followed in accordance with manufacturer and societal guidelines. [ ] [ ] [ ] the topic of endoscope reprocessing has largely been taken for granted by many endoscopists; however, standardized cleaning and disinfection protocols have been available for some time, and, with few exceptions, changes have been gradual. this slow evolution with a high safety profile may have engendered some complacency on the part of endoscopists, to the point that many endoscopists are only vaguely aware of what goes on "behind the curtain" of the endoscope reprocessing room. instruments are used on patients, taken away by gi nurses or other health care personnel, reprocessed, and returned ready for patient use. as the complexity of reprocessing and recognition of its importance become a concern to the medical community and our patients, endoscopists must become more educated on these issues and thereby able to participate in informed discussions with their patients. this chapter presents a pragmatic approach to proper reprocessing of endoscopic equipment, with guidance for prevention and management of infection transmission, and includes newer sterilization and disinfection technologies. . semicritical: semicritical devices contact intact mucous membranes and do not ordinarily penetrate sterile tissue. these instruments include endoscopes, bronchoscopes, transesophageal echocardiography probes, and anesthesia equipment. reprocessing of these instruments requires a minimum of hld. . noncritical: noncritical devices contact intact skin (e.g., stethoscopes or blood pressure cuffs). these items should be cleaned by low-level disinfection. endoscopes gi endoscopes are considered semicritical devices, and the resultant minimal standard for reprocessing is hld. this standard is endorsed by governmental agencies including the joint commission (jc), the centers for disease control and prevention (cdc), and the fda. it is also endorsed by gastroenterology societies such as the american society for gastrointestinal endoscopy (asge), american college of gastroenterology (acg), and american gastroenterological association (aga), as well as medical organizations, including the association of perioperative registered nurses (aorn), society of gastroenterology nurses and associates (sgna), association for professionals in infection control and epidemiology (apic), and american society for testing and materials (astm). [ ] [ ] [ ] [ ] hld of endoscopes eliminates all viable microorganisms, but not necessarily all are alike, however. steam is the most extensively utilized process and is routinely monitored by the use of biologic indicators (e.g., spore test strips) to show that sterilization has been achieved. when liquid chemical germicides (lcgs) are used to eradicate all microorganisms, they can be called chemical sterilants; however, the us food and drug administration (fda) and other authorities have stated that these processes do not convey the same level of assurance as other sterilization methods. [ ] [ ] [ ] other commonly used sterilization processes include low-temperature gas such as ethylene oxide (eto), liquid chemicals, and hydrogen peroxide gas plasma. disinfection is defined broadly as the destruction of microorganisms, except bacterial spores, on inanimate objects (e.g., medical devices such as endoscopes). three levels of disinfection are achievable depending on the amount and kind of microbial killing involved. these levels of disinfection are as follows: . high-level disinfection (hld): the destruction of all viruses, vegetative bacteria, fungi, mycobacterium, and some, but not all, bacterial spores. , for lcgs, hld is operationally defined as the ability to kill mycobacteria (a six-log reduction). the efficacy of hld is dependent on several factors and includes the type and level of microbial contamination; effective precleaning of the endoscope; presence of biofilm; physical properties of the object; concentration, temperature, ph, and exposure time to the germicide; and drying after rinsing to avoid diluting the disinfectant. . intermediate-level disinfection: the destruction of all mycobacteria, vegetative bacteria, fungal spores, and some nonlipid viruses, but not bacterial spores. . low-level disinfection: a process that can kill most bacteria (except mycobacteria or bacterial spores), most viruses (except some nonlipid viruses), and some fungi. although this categorization for disinfection levels generally remains valid, there are examples of disinfection issues with prions, viruses, mycobacteria, and protozoa that challenge these definitions. antiseptics are chemicals intended to reduce or destroy microorganisms on living tissue (e.g., skin), as opposed to disinfectants, which are used on inanimate objects (e.g., medical devices such as endoscopes). the difference in the way the same chemical is used to achieve different levels of disinfection and sterilization is important for endoscopy because the contact times for sterilization with any given lcg are generally much longer (hours) than for high-level disinfection (minutes) and may be detrimental to the endoscope. the relative resistance of various microorganisms to lcgs is shown in box . . more than years ago, earle h. spaulding developed a rational approach to disinfection and sterilization of medical equipment based on the risk of infection involved with the use of these instruments. , the classification scheme defined these categories of medical devices and their associated level of disinfection as follows: . critical: critical devices or instruments come into contact with sterile tissue or the vascular system. these devices confer a high risk for infection if they are contaminated. this category includes biopsy forceps, sphincterotomes, surgical instruments, and implants, when used in sterile anatomic locations. reprocessing of these instruments requires sterilization. disinfection at least once daily. the water bottle should be filled with sterile water. [ ] [ ] [ ] [ ] because accessory items often do not have unique identification numbers, it is critical to ensure they are dedicated to and stored with the endoscope that they are used with. this is necessary to ensure that if there is an outbreak, it is possible to identify which accessory components were used. this may require the use of disposable accessory holders or holders such as mesh bags that are also reprocessed along with the accessories. most accessory instruments used during endoscopy either contact the bloodstream (e.g., biopsy forceps, snares, and sphincterotomes) or enter sterile tissue spaces (e.g., biliary tract) and are classified as critical devices. as such, these devices require sterilization. , these accessories may be available as disposable "single-use" or "reusable" instruments. reuse of devices labeled single-use only remains controversial but has been commonly employed in many practices, primarily for economic benefits. , [ ] [ ] [ ] [ ] the fda considers reprocessing a used single-use device into a ready-for-patient-use device as "manufacturing," and as a result, hospitals or third-party reprocessing , companies that reprocess these devices are required to follow the same regulations as the original equipment manufacturers (i.e., obtain [k] and premarket approval application; submit adverse event reports; demonstrate sterility and integrity of the reprocessed devices; and implement detailed quality assurance monitoring protocols). this includes the development of standards and policies to determine the maximum number of uses for the devices and the training of staff in the reprocessing procedures. [ ] [ ] [ ] [ ] the regulatory burden imposed by these requirements essentially eliminated the practice of the reprocessing of single-use devices by most hospitals. aers were developed to replace some of the manual disinfection processes and standardize several important reprocessing steps, thereby eliminating the possibility of human error and minimizing exposure of reprocessing department personnel to chemical bacterial spores. although spores are more resistant to hld than other bacteria and viruses, they are likely to be killed when endoscopes undergo thorough manual cleaning. in addition, survival of small numbers of bacterial spores with hld is considered acceptable because the intact mucosa of the gi tract is resistant to bacterial spore infection. endoscope sterilization, as opposed to hld, is not required for "standard" gi endoscopy, as a reprocessing endpoint of sterilization has not been demonstrated to further reduce the risk of infectious pathogen transmission from endoscopes. sterilization of endoscopes is indicated when they are used as "critical" medical devices, such as intraoperative endoscopy when there is potential for contamination of an open surgical field. , in addition, individual institutional policies may dictate sterilization of duodenoscopes and linear endoscopic ultrasound instruments due to elevator mechanisms that have been difficult to clean and eradicate all bacterial contaminants with hld alone (see the later section on duodenoscope-related infections). despite the complex internal design (fig. . ) of endoscopes, hld is not difficult to achieve with rigorous adherence to currently accepted reprocessing guidelines. endoscope features that challenge the reprocessing procedures include: • complex endoscope design with several long, narrow internal channels and bends that make it difficult to remove all organic debris and microorganisms (e.g., elevator channel and elevator lever cavity of duodenoscopes). • a large variety of endoscope vendors and models require different cleaning procedures and devices and materials. • occult damage (e.g., scratches, crevices) to the endoscope can sequester microorganisms and promote biofilm formation. all valves, caps, connectors, and flushing tubes need to be adequately cleaned, rinsed, and disinfected or sterilized at the same time the patient-used endoscope is being reprocessed. the water bottle used to provide intraprocedural flush solution and its connecting tubing should be sterilized or receive high-level lcgs have inherent limitations; however, they are universally used to reprocess flexible endoscopes and accessories due to their relative convenience, safety, and rapid action. lcgs used as hlds should ideally have the following properties: broad antimicrobial spectrum, rapid onset of action, activity in the presence of organic material, lack of toxicity for patients and endoscopy personnel, long reuse life, low cost, odorless, ability to monitor concentration, and nondamaging to the endoscope or the environment. , hld solutions can act as sterilants if an increased exposure time is used , , ; however, the exposure time required to achieve sterilization with most lcg solutions is far longer than is practical, and therefore these formulations are only used for hld. , the efficacy of chemical disinfectants and sterilants is dependent on their physical properties including concentration and temperature; the length of exposure of the endoscope to the chemical solutions; the type and amount of microbial debris on the endoscope; and the mechanical components of the endoscope such as channels and crevices. because the chemicals are toxic to humans and the environment, proper handling, thorough rinsing, and appropriate disposal are essential for human safety. when selecting a hld product, institutional requirements need to be taken into consideration with important variables including the number of endoscopes processed per day, training requirements, turnaround time, cost information, and regulatory issues regarding safe use of the hld products. health care workers who use hlds need to be familiar with and have readily accessible, product/brand-specific material safety data sheets (msds) and keep current with regulatory changes and new product developments. users should consult with manufacturers of endoscopes and aers for compatibility before selecting an lcg. the most commonly used fda approved lcgs for disinfection of flexible endoscopes include glutaraldehyde, ortho-phthalaldehyde (opa), peracetic acid, and hydrogen peroxide (table . ) , , based chemicals in varying combinations and concentrations. some formulations contain combinations of microbicidal agents, including glutaraldehyde and phenol/phenate, peracetic acid and hydrogen peroxide, and glutaraldehyde and isopropyl alcohol. the fda periodically updates a list of approved hld solutions along with some of their attributes, such as contact time and temperature required for hld. sterilization of endoscopes is indicated on occasions when they are used as critical medical devices during open surgical procedures. the risk for contamination of the operative field exists when a nonsterile endoscope enters the abdomen through sterilants. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] aers continuously bathe the exterior surface of the endoscope and circulate the lcg under pressure through the endoscope channels. the aer manufacturer identifies each endoscope (brand and model) that is compatible with the aer and specifies limitations of reprocessing models of endoscopes and accessories. variations in aers may require customization of the facility design to accommodate requirements for ventilation; water pressure, temperature, and filtration; plumbing; power delivery; and space. all models of aers have disinfection and rinse cycles. in addition, the aers may also have one or more of the following automated capabilities: , , . some aers utilize and discard small quantities of lcg per hld cycle, whereas others have a reservoir of lcg that is reused over multiple cycles. the latter design results in gradual dilution of the lcg and requires intermittent testing to verify maintenance of the minimum effective concentration (mec). product-specific test strips need to be used regularly to monitor these solutions, which should be discarded whenever they fall below the mec or when the use-life expires, whichever comes first. . the temperature and cycle length can be altered to ensure hld or sterilization based on the lcg and type of endoscope. . the aer should ensure circulation of lcgs through all endoscope channels at an equal pressure with flow sensors for automated detection of channel obstruction. . the aer should be self-disinfecting. . vapor recovery systems are available. . low intensity ultrasound waves are an option. . variable number of endoscopes per cycle. . some aers flush the endoscope channels with forced air or with % to % ethyl or isopropyl alcohol followed by forced air to aid in drying the endoscope channels, thereby eliminating residual water, which reduces microbial growth during storage. . the aer should incorporate a self-contained or external water filtration system. lcgs and aers must meet specified performance levels for hld to receive fda clearance. this is defined as a reduction in residual organic loads and a -log killing of resistant indicator organisms (typically mycobacterium bovis). all aers marketed in the united states meet these criteria. the asge has published a summary of vendor-specific aers and their compatible lcgs. the fda has approved labeling some aers as washer-disinfectors due to the introduction of automated, brushless washing of endoscope channels prior to the disinfection cycle. utilization of this aer washing cycle provides an extra margin of safety by providing redundancy of cleaning; however, the existing multisociety guideline and other international standards emphasize that manual cleaning is still necessary when a washer-disinfector is used to assure the overall efficacy of hld. , one aer (steris system e [ss e]; steris corp, mentor, oh) has received fda approval for liquid chemical sterilization, as opposed to hld, for heat-sensitive devices that cannot be sterilized by traditional means. this system uses filtered, ultraviolettreated water that enters the aer and mixes with a peracetic acid-based formulation that is subsequently heated to °c to °c for liquid chemical sterilization. this system is designed for "point of use" sterilization, as sterile storage is not possible. for flexible endoscopes processed through the ss e, there is still a requirement for an alcohol rinse and drying prior to placing the endoscope into a storage cabinet. the fda also requested that aer manufacturers conduct additional validation testing to evaluate aer reprocessing agent/action ever more complex gi flexible endoscopes. the combination of ultrasonic capability with flexible endoscopes has opened up a new tool to use for the diagnosis and staging of cancers. however, along with these improvements that enhance diagnostic capabilities comes the increasing complexity of the endoscope channels. these complexities include double instrument channels with connector bridges, ultrasound probe channels, auxiliary channels, and elevator lever wire channels (sealed and unsealed). these complexities in endoscopes have far-reaching impacts in terms of reprocessing of reusable flexible endoscopes. this has been painfully highlighted by the recent outbreaks of antibiotic resistant bacteria associated with fully reprocessed endoscopes that remain contaminated , , - and act as fomites that transmit bacteria to a high percentage of subsequent patients who are exposed to the contaminated endoscope (see later section on infection control issues for more detailed information on infection transmission). such outbreaks have focused attention on the cleaning and disinfection of flexible endoscopes. there has been a paradigm change in that it is now recognized that reprocessing of gi flexible endoscopes is an extremely complex process that requires a quality systems approach, which includes specific training for reprocessing personnel, adequate monitoring of various stages in the reprocessing cycle, and ongoing documentation of staff competency. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] human factors play a critical role in compliance with reprocessing of gi endoscopes. ofstead et al ( ) demonstrated that compliance with all the reprocessing steps occurred for only an incision, as occurs with selected methods of intraoperative enteroscopy or postsurgical anatomy endoscopic retrograde cholangiopancreatography (ercp). , endoscopes, when sterilized, require low-temperature methods because they are heat labile and therefore, unlike most other medical or surgical devices, they cannot undergo steam sterilization. eto is the most commonly employed low-temperature sterilization process and a valuable method of sterilizing flexible endoscopes. however, a lengthy aeration time is required following eto sterilization to allow desorption of all residual toxic gas from the endoscope. additional steps must be taken, such as the application of a venting valve or the removal of the water-resistant cap to ensure proper perfusion with the gas and to prevent damage to the endoscope due to excessive pressure build-up. in addition, there are potential hazards to staff, patients, and the environment related to eto toxicities (table . ). the international agency for research on cancer has classified eto as a known (group ) human carcinogen. within the past two decades, several new, low-temperature (< °c) sterilization systems have been developed, including hydrogen peroxide gas plasma, vaporized hydrogen peroxide, peracetic acid immersion, and ozone - (see table . ). endoscopes and are not trained on specific cleaning requirements. the use of different sizes and types of channel brushes for the various different channel sizes, the fact that some channels cannot be brushed, and the multitude of different types of cleaning brushes available makes duodenoscope reprocessing a confusing process prone to human error. major changes in gi endoscope reprocessing over the past five years have occurred and include new regulatory requirements, . % of flexible endoscopes reprocessed when cleaning steps were performed manually and disinfection was automated, compared to . % compliance when both cleaning and disinfection were automated. fig. . outlines the basic steps in reprocessing of a gi flexible endoscope. until recently, the only aspect of this process that was monitored was to test the mec of the high-level disinfectant to ensure it contained a sufficient concentration of the active ingredient. it is easy to see from the outline provided in fig. identified. , , , , , [ ] [ ] [ ] [ ] if biofilm forms, the ability of disinfectants to reliably kill microorganisms within biofilm is dramatically reduced. , although most published reports of infectious outbreaks related to flexible endoscopes have involved duodenoscopes, other endoscopes including colonoscopes, gastroscopes, bronchoscopes, cystoscopes, and ureteroscopes (see later sections on reprocessing errors and outbreak management and infection control issues), have been shown to be contaminated and involved in infectious outbreaks. as such, every site offering flexible endoscopy procedures should ensure they have an established quality system for reprocessing these complex devices as recommended by the fda and cdc. table . provides an overview of what components are needed for such a system. as recommended by the cdc, the first step that all health care facilities should undertake is to perform an audit by reviewing their current endoscopy services to ensure they meet all aspects of a quality system approach. this requires input from administration, risk management, endoscopy staff, and infection prevention and control to ensure that all components are adequately assessed and the appropriate policies and procedures documented and implemented. table . provides an overview of the key steps in reprocessing and outlines where mistakes are frequently made, as well as the impact of such mistakes. it is important that audits done for endoscope reprocessing are observational and based on a specific checklist of critical components, as well as data to substantiate processes (e.g., time data to show contact time with detergent during cleaning, as well as transport times to determine how frequently it exceeds hour). table . is a useful aid to review during initial training of staff, as well as during discussion of audit data on a yearly basis. the need to monitor the adequacy of cleaning is a critical step in the total quality system approach (see table . ) to endoscope reprocessing. appropriate benchmarks for cleaning markers have been established. , , , , , [ ] [ ] [ ] there are a number of rapid cleaning tests (rct) available for monitoring organic residuals such as hemoglobin, carbohydrate, and protein, as well as those that monitor atp residuals. there are published data for some of these rapid test methods, , - but when selecting a rapid cleaning monitor, it is important to request that the rapid test manufacturer provide their validation data. review of the pros and cons of the testing method based on the validation data provided by the manufacturer is an important step when selecting the rct. examples of the considerations for selecting the rct are shown in table . . once the rct has been selected, a way to document the rct results is needed (e.g., a record sheet that identifies the scope tested, the person doing the testing, date and time of testing, result of testing, and the result of retesting for those scopes that fail the rct and require recleaning). regardless of the method selected, the site should do initial testing for all endoscopes to determine the current baseline status. this should be performed on the next patient use of each endoscope after completion of manual cleaning and rinsing. if the initial rct fails for the majority of the endoscopes, this device-reprocessing guidelines, and endoscope manufacturers' instructions for use. the key "domino" in this chain of changes was the fda guide to manufacturers of reusable medical devices (fda, march ) that required manufacturers to validate that their cleaning instructions were effective and could achieve predetermined benchmarks. cleaning validation for medical device reprocessing was not previously required; the focus was on validation of the disinfection or sterilization protocols recommended by manufacturers for their medical devices. when transmission of carbapenem-resistant enterobacteriaceae (cre) associated with contaminated duodenoscopes was recognized and first investigated and reported in the united states, it was unclear why reprocessing of duodenoscopes was failing. however, it was clear that transmission rates from contaminated duodenoscopes were high (up to %) and that, in addition to causing infections, patients often became colonized with cre and remained colonized long after the exposure to the cre contaminated duodenoscope. some health care facilities continued to report cre transmission and suggested that the manufacturers' instructions for use (mifus) for endoscope reprocessing were inadequate , , and that was why endoscope contamination was ongoing. although there had been recent design changes by the three main duodenoscope manufacturers whereby the elevator wire channel was sealed, the transmission of cre was reported for duodenoscopes with both sealed and unsealed elevator wire channels. , , however, one thing was clear; it was very difficult to adequately clean the lever cavity in duodenoscopes, and visible patient debris under the elevator lever was detected in one published outbreak. this has prompted new validated cleaning instructions for the lever cavity of duodenoscopes. the cre outbreaks linked to contaminated duodenoscopes prompted the fda to convene an advisory panel meeting in may , and on august , , the fda issued a safety communication with the recommendations from the advisory panel meeting that included: . establishing and implementing a comprehensive quality control program for endoscopy reprocessing to ensure meticulous adherence to mifus for duodenoscope reprocessing, adequate training of reprocessing personnel, and audits to ensure ongoing compliance. . supplemental measures to be considered by sites offering ercp procedures, including: • microbiological culture with quarantine of contaminated endoscopes until culture results become available; • meticulous cleaning and hld followed by one of: • eto sterilization, • liquid chemical sterilization, or • repeat hld. the fda safety alert was followed by a cdc health advisory in september , that indicated an immediate need for sites utilizing duodenoscopes to undertake audits of the reprocessing protocol, as well as staff training and longitudinal audits to document ongoing staff competency. these events and actions have led to a "paradigm shift" regarding the reprocessing of flexible endoscopes, whereby the need for a total quality systems approach has been recognized. it is no longer adequate to accept that endoscope cleaning is being done properly just because the mifus are available to staff; rather, there needs to be evidence of monitoring and ongoing audits of cleaning compliance for all staff who reprocess endoscopes. in addition, the need to ensure the endoscopes are truly dry during storage to prevent biofilm formation has been another stage where monitoring of the endoscope can be done is post-hld. a rapid post-disinfection test (rpdt) that could be completed just prior to the next patient use of the scope to confirm that the endoscope does not contain viable microorganisms would be ideal. however, there is little published data for indicates that there may already be build-up biofilm (bbf) in the scopes being used. remedial action would be needed for the endoscopes, which may include a longer soak time in detergent followed by extended brushing and flushing (as per scope manufacturer's input). if the endoscope fails the rct after the remedial action, then it should be sent to the manufacturer for further remedial action (e.g., change of channels, etc.). there must be documentation of all reprocessing components to ensure there is a way to link which endoscopes (and corresponding accessories) were used on which patient. • document which endoscope was used in the patient's medical record • document in the reprocessing area which personnel cleaned each endoscope, which patient the endoscope was used on, the date/time it was cleaned, and which aer (automated endoscope reprocessor) was used to disinfect the endoscope (or which sterilizer was used). • ensure the mifu for reprocessing is available in the reprocessing area • create a site-specific set of instructions that are based on the mifu but indicate specific detergent, brushes, etc. that are used for reprocessing of each make/model of endoscope used at that site • if pump-assisted flushing is used during manual cleaning, ensure instructions for use are available • for manual cleaning, a succinct visual "aide" consisting of a summary of the process posted above the reprocessing sink, counters, etc., is useful. table . for additional information) • traceability of each endoscope and reusable accessories used • documentation of all monitoring performed for cleaning and minimum effective concentration (mec) testing • timely reprocessing • routine cleaning and decontamination protocol for aer, flushing pump, sinks, connector tubing, endoscope storage cabinets • policy on disposable and reusable ancillary items (e.g., water bottles, connector tubing, etc.) • preventative maintenance (pm) program for endoscopes, aers, flushing pump with repair history records • record keeping of preventative maintenance on all equipment • regular audits to ensure ongoing adequacy of all stages of the program • involvement of infection prevention and control (ipac) and workplace safety in all components of the endoscopy reprocessing is crucial • a structured management scheme with regular review of the endoscopy program that includes reprocessing considerations. • there should be regular review and reporting of monitoring data at appropriate management meetings to identify any potential issues the bedside clean reduces the load of organic material and microbes for the full manual cleaning step. detergent or water solution (as per endoscope manufacturer's instructions) is used to: • wipe exterior using sponge or lint-free cloth • suction or flush all channels (note: this may require use of specific endoscope flushing adaptors as per mifu) • place all accessory components in detergent or water to keep moist during transport. . forget to wipe exterior or forget to suction or flush some channels after patient procedure. . dismantle reusable components such as valves, caps, flushing connectors etc. and process along with endoscope. ensure endoscope is totally immersed in detergent solution. . ideally suction detergent through all channels and discard this material (not in the cleaning basin). brush all openings including suction cylinder, air/water cylinder, instrument port, and outlets on umbilical end. . adequate contact time with detergent (as per detergent manufacturer's instructions) to loosen debris. . rinse away detergent using tap water. . reusable components not properly cleaned. performing brushing while endoscope is not immersed. into the detergent solution. . inadequate brushing of channels due to improper brush size or confusion regarding which brush to use. inadequate contact time with detergent. . many detergents are protein solutions that need to be removed before the disinfection/sterilization step. . increased risk of accumulation of organic material and biofilm forming. increased risk to personnel and environment due to aerosols of infectious debris. contamination of detergent solution with high levels of patient secretions increases the risk of high levels of organic material and microbes on exterior and in channels. inadequate brushing leads to improper cleaning and risk of excessive organic and microbial load for hld. of organic material and microbes. . if detergent is not adequately rinsed away this could lead to accumulation of protein and failure of hld. . once manual cleaning has been completed collect a sample from the channel(s) of the endoscope. minimally a sample from the instrument port to the distal end should be collected and tested. follow the mifu for the rapid cleaning monitor. if the test result is below the manufacturer's cut-off for adequate cleaning, then the scope can be transferred to the aer for hld (or manual hld done) or sent for sterilization. if the test result is above the mifu cut-off, then the endoscope needs to be fully recleaned and tested after the second manual clean. note: for duodenoscopes there should also be a sample from the elevator lever recess. if the endoscope fails the rapid cleaning test after three rounds of cleaning it should be sent to the endoscope manufacturer for evaluation. breach of any of the manual cleaning steps shown in fig. . can lead to patient-derived material remaining in the endoscope that is sent for hld or sterilization. hld or sterilization processes fix the organic residues to the channel surface leading to build-up biofilm formation (bbf). . accumulation of bbf in the endoscope increases the risk of microbes in the bbf matrix surviving hld and possibly being transmitted to subsequent patients. disinfection or sterilization hld and sterilization are intended to kill any remaining microbes left after the cleaning step. hld and sterilization can achieve and log microbial reductions, respectively. all reusable accessory items must also be exposed to hld or sterilization along with the endoscope they are dedicated to. ideally an automated endoscope reprocessor (aer) should be used for hld or sterilization. manual hld instructions are available from manufacturers but the manual process is fraught with concerns including ensuring adequate vapor control to prevent staff exposure to toxic fumes, adequate channel perfusion when using manual syringe to inject hld, dilution of hld from water remaining in channels after manual cleaning, etc. when using an aer, it is necessary to change the sub-micron filter inside the aer as per mifu. . fully reprocessed endoscopes and accessory components need to be handled using clean gloves to ensure skin organisms from bare hands are not transmitted to the endoscope post-hld. . inadequate hld contact due to poor manual perfusion of channels (e.g., bubbles or inadequate immersion). breach in sub-micron filter inside aer leads to contaminated rinse water. handling of endoscope post-hld with un-gloved hands . increased risk of microbes surviving the hld process due to inadequate contact with hld. microbes on the endoscope when put into storage can facilitate biofilm formation. . microbes on hands transferred to disinfected endoscope can survive and subsequently be transmitted to next patient that the endoscope is used for. microbes transferred to endoscope from hands also increase the risk of biofilm formation if there is sufficient moisture. . through drying of endoscope exterior and all channels is critical prior to storage. this can be done using appropriately filtered forced air. if a "channel-purge" storage cabinet is used the manual drying only needs to be done briefly to remove excess water prior to placing the endoscope in the cabinet (ongoing air purging once the endoscope is placed in the cabinet is preferred to ensure dryness is maintained as additional wet endoscopes are sequentially placed into the storage cabinet). there are also small air-flushing pumps that can be used to ensure channel drying prior to placing the endoscope in a regular storage cabinet. ensure adequate time is used for air-flushing to ensure scope is totally dry before being placed in a regular storage cabinet. . ensure fully reprocessed endoscopes are somehow labeled or identified so they can be easily differentiated from patient-used endoscopes that are contaminated. . the volume of alcohol flushed and the time for manual forced air drying is not specified in mifu and is often sub-optimal when performed manually. . contaminated endoscope may be mistaken for a fully reprocessed endoscope and accidentally used on a patient. . inadequate drying during storage is one of the key factors that leads to biofilm formation. once formed, the biofilm protects microbes from being adequately killed by hld or sterilization and increases the risk of organisms being transmitted to patients exposed to this contaminated endoscope. use of a contaminated endoscope can lead to transmission of infectious agents that result in colonization or infection. . contamination of endoscope may lead to microbial replication and biofilm formation if moisture is also present. valves inserted into the valve cylinders can lead to moisture retention that facilitates microbial replication and can lead to biofilm formation. endoscope reprocessing. as outlined by recent guidelines, , , , initial training and ongoing competency assessment are critical to ensuring that staff can effectively reprocess flexible endoscopes. the compliance of reprocessing personnel with endoscope reprocessing protocols should be reviewed at least annually to document ongoing competency. it is clear from some outbreaks that despite having adequate written protocols, staff may create breaches by not following some steps in the protocol. , , as such, observational audits are a useful approach to determining if staff are fully compliant in following the site protocol. if ongoing cleaning monitoring is performed, the results of these tests can be included as part of documentation of ongoing competency. transmission of exogenous pathogens (i.e., not derived from the patient) can be categorized as "nonendoscopic," which is related to care of intravenous lines and administration of medications and anesthesia, or "endoscopic," which is related to transmission by the endoscope, water bottles, and its accessories. outbreaks of infection have been traced to process failures, including endoscopes that are damaged or difficult to clean; aer design problems or failures such as breakdowns in aer water filtration systems; and lack of adherence to reprocessing guidelines for endoscopes and accessories. there are also data that demonstrate that all the steps associated with manual endoscope reprocessing are rarely performed and some essential steps, such as brushing all endoscope channels and adequate drying prior to storage, are frequently deficient. these deficient reprocessing practices can be summarized as follows: , , . inadequate or absent mechanical cleaning of the endoscope and channels before disinfection. the two rpdt tests that are currently available (table . ). in the absence of validated rapid test methods, culture is the only well-studied method for detection of microbial contamination of flexible endoscopes postdisinfection/-sterilization. however, there are a number of considerations when culture is used. there have been a variety of published studies on culture results from endoscopes, but there is little data on the recovery efficiency of the various endoscope extraction methods that have been used. if a patient-ready endoscope is extracted by flushing the channel with bacterial culture media or other harvesting fluids containing various proteins or buffers containing salt, then the endoscope requires recleaning and disinfection prior to being used on a patient. if sterile, high-quality water (i.e., reverse osmosis or deionized water) is used to flush endoscope channels, the endoscope can be dried and then still be safely used on the next patient. personnel who reprocess flexible endoscopes must have thorough initial training regarding the reprocessing of all makes and models of endoscopes that they will be responsible for reprocessing (see tables . and . ). the training process should be documented and new staff not allowed to reprocess endoscopes on their own until they have demonstrated, under supervision, that they are competent to perform reprocessing independently. the use of rapid cleaning monitor (rcm) tests for each endoscope reprocessed during training is an excellent way to document the adequacy of the trainee's ability to perform the cleaning process. reprocessing errors are a common underlying problem for many of the reported outbreaks. , , , the "human factors" study done by showed that inadequate cleaning of channels related to the lack of adequate channel brushing ( % of scopes) and inadequate drying ( % of scopes) prior to storage of endoscopes were the two most common breaches in the cdc guidelines for safe injection practices include the following recommendations: • use aseptic technique when preparing and administering medications and fluids. • a sterile, single-use, disposable needle and syringe should be used for each injection on a single patient. • do not administer medications from single-dose vials or use iv solutions as a common source of supply for multiple patients. • do not keep multidose vials in the immediate patient treatment area. • do not reuse a syringe to access or administer medications from a vial that may be used on multiple patients, even if the needle is changed. • in times of critical need, medications from unopened singledose/single-use vials can be subdivided for multiple patients. however, this should only be performed by qualified health care personnel in accordance with standards in the united states pharmacopeia chapter on pharmaceutical compounding. . an inadequate disinfectant was used or used improperly at an incorrect concentration, temperature, or exposure period. . flawed or malfunctioning aer units or use of incorrect connectors. , , . failure to disinfect or sterilize the irrigation bottle of the endoscope regularly. , . endoscopic accessory instruments were not sterilized. . the endoscope and all channels were not dried adequately before storage. . unrecognized problems with water supply. outbreaks of hepatitis b and c viruses have occurred due to failure to follow fundamental principles of aseptic technique and safe injection practices. , these included improper handling of intravenous sedation tubing, reuse of syringes and needles, and use of single-dose or single-use medical vials on multiple patients. • detects viable organisms of high concern including gram negatives and gram positives. • allows assessment of whether the bacteria detected are multi-antibiotic resistant • for outbreak investigation allows for genetic typing methods (e.g., pfge) to help identify a point-source outbreak • requires to hrs before results of culture are reported. • during outbreak investigation, quarantine of the endoscope pending culture results is necessary. • for routine surveillance (i.e., not an outbreak investigation) the endoscope is often not quarantined and may be used on multiple patients before culture results are available. sites need to have a response plan in place regarding notification if culture shows organisms of concern on an endoscope that has been used on multiple patients (i.e., notify the patient, the doctor or both?) not be detected after hld as these organisms commonly result in a clinically significant infection including gram-negative bacteria (e.g., escherichia coli, klebsiella spp., shigella spp., salmonella spp., pseudomonas aeruginosa, other enterobacteriaceae) as well as staphylococcus aureus, and enterococcus spp. lco are less often associated with disease; these bacteria typically include coagulase-negative staphylococci, micrococci, diptheroids, and bacillus spp. the levels of lco on a surveillance endoscope culture can vary depending on the reprocessing, handling, and culturing practices in a facility. typically, fewer than colony forming units (cfu) of lco does not require intervention as this most likely represents collection process contamination rather than a significant problem with the disinfection or cleaning process. interpretation of culture results with or greater cfu of lco should be considered in the context of typical culture results at the facility. any endoscope found to be contaminated with a hco or unacceptable cfu of lco should cause concern and lead to repeat endoscope reprocessing followed by post-reprocessing cultures. the endoscope should be quarantined until it has been demonstrated to be free of hco and has an acceptable level of lco. positive cultures should also prompt a review of the endoscopy unit reprocessing procedures to ensure adherence to the manufacturer's reprocessing instructions and to ensure proper culture methodology. if a reprocessing breach is identified, appropriate facility personnel should be notified and corrective actions should be immediately implemented. when bacteria are persistently recovered by surveillance cultures, refer to the manufacturer's instructions , for evaluating the endoscope for mechanical defects and consider having the endoscope evaluated by the manufacturer. in addition, when ineffective reprocessing is suspected based on surveillance cultures, it might be helpful to review positive cultures among affected patients to determine whether transmission of relevant pathogens could have occurred. , the vast majority of exogenously acquired endoscope-related infections have been caused by bacterial transmission. the bacteria involved have been true pathogens, which always have the potential to cause infection (e.g., salmonella spp.), or opportunistic pathogens that cause infection if the microbial load is sufficient and/or host-factors are permissive (e.g., pseudomonas aeruginosa). in the hierarchy of relative resistance to hld, vegetative bacteria such as pseudomonas spp. and salmonella spp. are the most susceptible to disinfectants, whereas the mycobacteria are less susceptible and bacterial spores (e.g., bacillus subtilis and clostridium difficile) are the most difficult to eliminate (see box . ). nevertheless, as previously stated, all bacteria with the exception of a few bacterial spores are highly sensitive and eliminated by hld. salmonella is a serious primary pathogen, and pseudomonas is ubiquitous in many water sources, and although both these pathogens have been associated most frequently with endoscopic transmission, they are both sensitive to multiple agents, including glutaraldehyde, and other hlds. transmission of bacterial pathogens from flexible endoscopes has been rare since the adoption of the current multisociety reprocessing guideline, , with the exception of duodenoscope-related infections (discussed later). the most commonly reported infectious agents transmitted during gi endoscopy have been pseudomonas aeruginosa ( cases) [ ] [ ] [ ] and salmonella spp. ( cases). isolated reports of more health care-associated outbreaks and patient exposures have been linked to contaminated endoscopes than to any other reusable medical device. , nevertheless, endoscopy-related transmission of infection is very low and was originally estimated to have an incidence of approximately infection per . million procedures. , this is very likely an underestimate, as many endoscopy-related infections go unrecognized because of inadequate or nonexistent surveillance programs, the absence of clinical symptoms in many patients who are colonized, a long lag time between colonization and clinical infection, and the fact that the pathogens transmitted by endoscopy are often normal enteric flora. endoscope-related transmission of bacterial infection has been rare since the adoption of the current multisociety reprocessing guidelines. , however, recent outbreaks have occurred with duodenoscopes even when the manufacturers and societal guidelines were reportedly followed correctly (see later section on duodenoscope-related infections). the primary concern raised by infectious outbreaks is that current reprocessing guidelines are not adequate to ensure patient safety when undergoing endoscopic procedures. endoscopes can harbor between and enteric organisms at the completion of some patient procedures. this bioburden is reduced by cleaning (i.e., bedside precleaning followed by manual cleaning) by a factor of to log and the hld step is expected to provide a log reduction of any microbes remaining after cleaning. , therefore the margin of safety associated with cleaning and hld of gi endoscopes is low, and any deviation from proper reprocessing could lead to failure to eliminate contamination, with a possibility of subsequent patient-to-patient transmission. biofilms can contribute to reprocessing failure and endoscoperelated infectious outbreaks. biofilms form in endoscope channels, in aers, and within municipal and hospital water supplies as multilayered bacteria within exopolysaccharide. these biofilms protect the bacteria against physical (e.g., brushing, fluid flow) and chemical (e.g., disinfectant) forces, making the microorganisms more difficult to remove or completely kill by hld. , there is evidence that accumulation of fixed material within endoscope channels occurs over repeated usage. biofilms that develop in endoscopes and aers may not be detectable by surveillance culture, as cleaning and disinfection may have destroyed bacteria within the superficial layers but not those within the deeper layers. prompt, meticulous, manual cleaning to remove biologic material and strict adherence to reprocessing protocols is the optimal approach to reduce biofilm formation/accumulation. [ ] [ ] [ ] [ ] better biofilm removal protocols are needed to address this issue. pathogens of concern to the gi endoscopy community and general public include clostridium difficile, helicobacter pylori, escherichia coli, norovirus, human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), and multidrug resistant organisms (mdros) such as m. tuberculosis, vancomycin-resistant enterococcus (vre), methicillin-resistant staphylococcus aureus (mrsa), and cre. all these established pathogens are susceptible to currently available chemical disinfectants and sterilants. , low concern organisms (lco) versus high concern organisms (hco) surveillance cultures of endoscopes are assessed for two general categories of microbial growth, lco and hco. hco should be changed to protect our patients from cjd, citing no reported cases of cjd transmission by endoscopy and the lack of exposure to high-risk cns tissue during endoscopic procedures. , vcjd is a rare but fatal condition caused by the consumption of beef contaminated with a bovine spongiform encephalopathy agent. it differs from cjd in that the mutated prion protein can be found in lymphoid tissue throughout the body, including the gut and tonsils. , only three cases of vcjd have been reported in the united states, and all three patients contracted the disease elsewhere. as vcjd is resistant to conventional disinfectants and sterilants, endoscopy should be avoided, if at all possible, in patients known to harbor this agent. , endoscopes used in individuals with definite, probable, or possible vcjd should be destroyed after use or quarantined to be reused exclusively on that same individual if required. , between and , duodenoscopes resulted in international outbreaks (at least eight in the united states) of antibiotic-resistant infections with cre and other mdros that sickened a reported patients and resulted in deaths. , , , , [ ] [ ] [ ] in addition, transmission resulting in a long-term carrier state has been recognized as a risk of exposure to contaminated duodenoscopes. long-term carriage has important clinical implications due to the development of a delayed infectious complication weeks to months later or patient-topatient transmission of pathogens when these carriers are subsequently admitted to health care or chronic care facilities. investigative cultures identified persistent contamination of duodenoscopes as the cause for patient infections with mdros in most of the outbreaks. , furthermore, these duodenoscopeassociated infections occurred even though the sites reported strict adherence to reprocessing procedures according to manufacturer's instructions and professional guidelines. , , , , it is likely that mdros are acting as a marker for ineffective reprocessing due to the complex design of duodenoscopes that have difficultly reaching crevices and channels involving the elevator mechanism where persistent colonization was identified. , duodenoscopes that persistently yield positive cultures likely harbor biofilms that cannot be eradicated with standard reprocessing. in october the fda and the cdc released an official health advisory alerting health care facilities to review their reprocessing procedures. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in response to the problems with duodenoscope reprocessing, the fda requested all three duodenoscope manufacturers to revise and validate their reprocessing instructions with provisions for additional duodenoscope reprocessing measures. this led to the modification of manufacturers' reprocessing protocols with a larger emphasis on precleaning and manual cleaning before hld. , [ ] [ ] [ ] one duodenoscope manufacturer (olympus; center valley, pa) subsequently modified the design of the closed elevator channel to create a tighter seal. , in addition, the fda has recommended that health care facilities performing ercp consider employing supplemental measures for duodenoscope reprocessing when facilities have the resources to do so. most sites where outbreaks have occurred have chosen per procedure eto sterilization after hld as its primary reprocessing method, and in all reported instances, eto has prevented further mdro transmission. , however, one site reported failure to eliminate mdro contamination of a duodenoscope after hld and eto. alternative reprocessing endoscopic transmission of other enteric bacteria include klebsiella spp., enterobacter spp., serratia spp., and staphylococcus aureus. the few reports of endoscopic transmission of helicobacter pylori were related to inadequate reprocessing of endoscopes and biopsy forceps. [ ] [ ] [ ] current reprocessing guidelines are shown to inactivate clostridium difficile spores, and no cases of endoscopic transmission of this infection or mycobacteria have been reported. in summary, there have not been any observed gi endoscopy-related transmission of bacterial pathogens since introduction of the currently accepted reprocessing standards with the exception of duodenoscope-related outbreaks (discussed later). much greater anxiety is associated with the possibility of transmission of viral infections. this anxiety is surprising because the viruses of greatest concern (hbv, hcv, and hiv) are among the easiest microorganisms destroyed with standard reprocessing. transmission of viral pathogens by gi endoscopy procedures is rare because these microorganisms are obligate intracellular microorganisms that cannot replicate outside living tissue. thus, even when a flexible endoscope is contaminated with viral pathogens, the burden of virus cannot increase, as they are not capable of ex vivo replication. enveloped viruses (e.g., hiv, hbv, hcv) die readily once dried and are more readily killed by hld compared to nonenveloped viruses (e.g., enteroviruses, rotavirus), which can survive in dry conditions. there has been concern about the possibility of hiv transmission by flexible gi endoscopy; however, no cases have been reported to date. [ ] [ ] [ ] there is only one well-documented case of hbv transmission by gi endoscopy that occurred in the setting of inadequate endoscope reprocessing. however, transmission of hbv is very rare or does not occur when accepted reprocessing guidelines are followed. the presence of fungi is associated with prolonged storage of flexible endoscopes. although transmission of trichosporon beigelii and trichosporon asahii occurred in the s, , there are no documented cases of fungal infections by gi endoscopy when updated reprocessing guidelines are followed. a single publication in the s reported circumstantial evidence of strongyloides stercoralis transfer to four patients from a contaminated upper endoscope. no subsequent reports of parasite transmission by gi endoscopes have been identified. creutzfeldt jacob disease (cjd) and variant cjd (vcjd) are degenerative neurologic disorders transmitted by proteinaceous infectious agents called prions. all prions remain infectious for years in a dried state, and resist all routine sterilization and disinfection procedures commonly used by health care facilities. , [ ] [ ] [ ] [ ] cjd is confined to the central nervous system (cns) and is transmitted by exposure to infectious tissues from the brain, pituitary, or eye, whereas tissues and secretions that come into contact with the endoscope during procedures, such as saliva, gingival tissue, intestinal tissue, feces, and blood, are considered noninfectious by the world health organization. , [ ] [ ] [ ] [ ] the cdc and other infection control experts conclude that current guidelines for cleaning and disinfecting medical devices need not will help with the investigation and lead to an expeditious correction of any deficiencies that are identified. • a user facility is not required to report a device malfunction, but it can voluntarily advise the fda of such product problems using the voluntary medwatch form fda under fda's safety information and adverse event reporting program. however, if a device failure leads to a death or serious injury, the fda and the manufacturer must be contacted, as outlined in facility policies, by the designated individual or department at the facility. the fda encourages health care professionals, patients, caregivers, and consumers to submit voluntary reports of significant adverse events or product problems with medical products to medwatch (https://www.accessdata.fda.-gov/scripts/ medwatch/), the fda's safety information and adverse event reporting program • manufacturers are required to report to the fda when they learn that any of their devices may have caused or contributed to a death or serious injury or when they become aware that their device has malfunctioned and would be likely to cause or contribute to a death or serious injury if the malfunction were to recur. , . patient notification and counseling: , in instances where a breach in the reprocessing protocol or damaged equipment poses a risk to patients for adverse events, it becomes the institution's ethical obligation to notify patients in a timely manner. notification may be accomplished by a direct meeting, telephone call, and letter sent by registered mail. the content should include an assessment of the risk, possible adverse events that may occur, symptoms and signs of the adverse event, time range for the adverse event, risk to other contacts, possible prophylactic therapy (including benefits and risks), and recommended medical follow-up. prompt notification allows patients to take precautions to minimize the risk of transmitting infection to others and allows for early serologic testing. this may help distinguish chronic infections from those potentially acquired at the time of endoscopy and to permit earlier initiation of treatment for newly acquired infections. on the other hand, adverse publicity associated with the reporting of a reprocessing error might lead patients to avoid potentially life-saving endoscopic procedures because of an unwarranted fear of infection. personal counseling should be offered to all patients. the risk of infection should be discussed and placed in context to minimize patient anxiety. patients should be advised against donating blood and tissue products and engaging in sexual contact without barrier protection until all serologic testing is complete. a toll-free helpline should be established to provide information to all patients at risk. . develop a long-term follow-up plan (e.g., long-term surveillance, changes in current policies and procedures) and prepare an after action report. there are risks related to infection transmission to personnel who handle patient-used endoscopes as well as to patients. sites offering endoscopy procedures need to ensure the risk to personnel and patients is minimized. flexible gi endoscopes are expensive and easily damaged. unlike surgical instruments where the microbial load is less than methods employed have included double hld after each procedure , or hld with duodenoscope quarantine until negative culture results are obtained. another supplemental option for reprocessing endorsed by the fda includes the use of a liquid chemical sterilant processing system. , surveillance microbiological culturing should be considered in addition to these supplemental reprocessing measures. this involves sampling the duodenoscope channels and the distal end of the scope to identify any bacterial contamination that may be present on the scope after reprocessing. , it must be recognized that the sensitivity of surveillance culturing of the elevator channel, the elevator lever cavity, or other scope channels is unknown. until there are evidence-based guidelines, individual hospitals should choose from these different options based on available information and feasibility for their medical practice. however, at a minimum, there should be an audit of all facilities offering duodenoscope procedures to ensure the site has a quality system in place and is compliant with current mifus and guidelines. breaches of disinfection guidelines and device failures (e.g., endoscopes or aers) are common in health care settings, resulting in potential patient injury or infection transmission. the identification of such a problem may stem from the result of microbiologic surveillance cultures, an infectious outbreak within an institution or isolation of a pathogen from individuals having a recent endoscopic procedure, identification of a break in reprocessing protocol, or a visibly faulty device. endoscopy facilities should have written policies on the roles and responsibilities within the organization to identify, report, and analyze these failures. the investigation of a breach in reprocessing or resultant outbreak should be undertaken using a standardized approach. it should focus on the identification of factor(s) that led to the exposure and protect patients from potential adverse events. the investigation should not be punitive and not attempt to assign blame to any particular individual. rutala et al ( ) described a process for exposure investigation, and the asge has published guidelines for patient assessment and notification when there is a suspected failure in the disinfection or sterilization protocol. these can be summarized as follows: , . confirm that the reprocessing failure occurred and assess the duration of exposure (e.g., review sterilization methods and aer records of biological parameters). . quarantine any endoscopes or associated accessories that malfunctioned or are at risk for inadequate reprocessing. . do not use the devices in question, such as the endoscope or aer, until proper functioning is confirmed. . prepare a list of potentially exposed patients, dates of exposure, and inadequately reprocessed or malfunctioning devices used. . reporting: • inform facility leadership: breaches in patient safety with serious potential infection risks should be reported to facility leadership, including infection control, risk management, public relations, legal department, and selectively to local/state public health agencies, the fda, cdc, and the manufacturers of the involved equipment. , this the workflow should proceed from "dirtiest to cleanest" in the reprocessing area, and there should be physical separation of "dirty" reprocessing areas and "clean" areas. , , this requires appropriate removal of ppe and hand hygiene when leaving the dirty reprocessing area to enter any of the clean areas. staff should take every precaution to reduce the generation of aerosols during reprocessing of gi endoscopes. this includes total immersion of the endoscope during cleaning. , this ensures that any patient material removed from the channels during cleaning is contained within the detergent cleaning solution. care is needed to ensure all brushing steps are done underneath the water surface to reduce aerosols. holding the control head above water to insert the channel brush and then pulling the brush out of the channel while the control head is above the water generates significant aerosols of the contaminated detergent solution. in addition, during the air-flushing process after cleaning is completed, a piece of gauze should be placed over the distal end of the endoscope channel prior to placing it in an aer to prevent creation of aerosols when flushing out residual rinse water. a final, often overlooked step, is rinsing and decontamination of the sink after each endoscope is cleaned. this ensures that the sink does not accumulate microbial contamination over time and act as a reservoir within the reprocessing area to contaminate reprocessing personnel or other endoscopes. if flushing pumps are used as part of the manual cleaning step, they also require routine (usually daily) decontamination as per mifu to ensure they do not become a reservoir of microbes that develop biofilm and subsequently contaminate endoscopes that they are used on. any single-use disposable sharps used in the procedure room should be disposed of in appropriate sharps containers in the procedure room. there should be no single-use disposable sharps transported to the reprocessing area. if there are reusable sharps (e.g., biopsy forceps) used for patient procedures, these should be appropriately transported to the reprocessing area in a labeled, rigid, sealed container that ensures separation from the endoscope. this reduces the risk that the biopsy forceps (or other sharp accessory device) could damage the endoscope during transit. reprocessing of reusable sharps requires specific mifu and adequate staff training to reduce the risk of sharps injuries to reprocessing personnel. single-use, disposable accessories are preferred to eliminate the risks associated with reprocessing of reusable sharps. bacteria for % of instruments, , the load of microorganisms in channels of flexible endoscopes can be as high as bacteria per instrument channel (e.g., for colonoscopes). during transport from the procedure room to the reprocessing area, flexible gi endoscopes require a rigid, sealed container that is appropriately labeled as biohazardous. this protects the endoscope from accidental damage and also ensures that any patient-derived secretions and microorganisms are adequately contained and cannot drip out and contaminate the environment. all reusable accessory items (valves, flushing adaptors, cleaning valves, etc.) should be transported along with the associated endoscope. during transport, the endoscope and all accessory items should be kept moist to prevent drying of patient-derived material. if endoscopes are transported to a central reprocessing facility, evaluation of the time of transport should be conducted to determine the frequency of excessive transit times. there are risks to reprocessing personnel being exposed to patient-derived infectious materials. endoscopes contacting the gi tract can have very high levels of infectious organisms (including bacteria, viruses, fungi, etc.) in channels or on the endoscope surface. to mitigate these risks, reprocessing personnel need to be trained regarding standard precautions, personal protective equipment (ppe), hand hygiene, disposal of sharps, and dealing with chemical and/or infectious material spills. standard precautions are required when reprocessing any patient-used medical device. this means that staff treat all patient-used endoscopes as potentially infectious regardless of the underlying known illnesses that patients might have (e.g. clostridium difficile infection, vre colonization, human papilloma virus infection, candidiasis, etc.). any handling of gi endoscopes should be done with due consideration to the potential to transmit infectious microorganisms to reprocessing personnel. staff must be trained in appropriate ppe and reprocessing considerations aimed at reducing the generation of aerosols. it is critical that appropriate ppe be available , , and include a gown (preferably a water-resistant gown), gloves (appropriate to the task), and a face shield/mask. reprocessing personnel must be adequately trained in the proper donning and doffing of all ppe. gowns, gloves, and a full-face shield (or combined face shield/mask) are required for cleaning of flexible endoscopes. the reprocessing staff needs to be trained in the appropriate use of protective gloves, as well as hand hygiene after removing gloves. utility gloves used for cleaning of endoscopes should never be used at other stages in endoscope reprocessing (i.e., they are dedicated to the cleaning sinks). disposable examination gloves must be available for handling cleaned endoscopes during connection to the aer. fresh disposable gloves are needed for removing and handling fully reprocessed endoscopes from the aer and during manual channel drying and placing the endoscope into the clean storage cabinet. fresh disposable gloves should also be used whenever an endoscope is removed from the clean storage cabinet. the use of gloves helps protect both the reprocessing personnel from contamination with patientderived microorganisms and the fully reprocessed endoscope from contamination with skin-derived microorganisms from reprocessing personnel. staff should always perform hand hygiene immediately after removing any type of glove. handwashing sinks with appropriate soap, as well as waterless hand hygiene agent dispensers, must be available in the reprocessing area. endoscope reprocessing methods: a prospective study on the impact of human factors and automation automated endoscope reprocessors society of gastroenterology nurses and associates: guideline for use of high level disinfectants & sterilants for reprocessing flexible gastrointestinal endoscopes food and drug administration: fda-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices food and drug administration: reprocessing medical devices in health care settings: validation methods and labeling. guidance for industry and food and drug administration staff food and drug administration: supplemental measures to enhance reprocessing: fda safety communication current issues result in a paradigm shift in reprocessing medical and surgical instruments society of gastroenterology nurses and associates: standards of infection prevention in reprocessing flexible gastrointestinal endoscopes impact of ethylene oxide gas sterilization of duodenoscopes after a carbapenem-resistant enterobacteriaceae outbreak the use of channel-purge storage for gastrointestinal endoscopes reduces microbial contamination comparison of clinically relevant benchmarks and channel sampling methods used to assess manual cleaning compliance for flexible gastrointestinal endoscopes the atp test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels the atp test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels control and prevention: interim sampling method for the duodenoscope -distal end and instrument channel control and prevention: interim protocol for healthcare facilities regarding surveillance for bacterial contamination of duodenoscopes after reprocessing united states senate; health, education, labor, and pensions committee: preventable tragedies: superbugs and how ineffective monitoring of medical device safety fails patients. minority staff report risk of transmission of carbapenem-resistant enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy food and drug administration: design of endoscopic retrograde cholangiopancreatography (ercp) duodenoscopes may impede effective cleaning: fda safety communication reprocessing failure antimicrobial efficacy of endoscopic disinfection procedures: a controlled, multifactorial investigation high-level disinfection of gastrointestinal endoscopes: are current guidelines adequate? microbial bioburden in endoscope reprocessing and an in-use evaluation of the high-level disinfection capabilities of cidex pa efficacy of high-level disinfectants for reprocessing gi endoscopes in simulated in-use testing what is disinfection, sterilization? (letter) food and drug administration: guidance for industry and fda reviewers: content and format of premarket notification [ (k)] submissions for liquid chemical sterilants/high level disinfectants are all sterilization processes alike? low-temperature sterilization technologies: do we need to redefine sterilization? disinfection and sterilization of patient-care items centers for disease control and prevention: guideline for disinfection and sterilization in healthcare facilities guideline for disinfection and sterilization of prion contaminated medical instruments disinfection: is it time to reconsider spaulding? chemical disinfection and antisepsis in the hospital disinfection of medical and surgical materials food and drug administration: draft guidance for the content of premarket notifications for endoscopes used in gastroenterology and urology cdc guideline for handwashing and hospital environmental control reprocessing of flexible gastrointestinal endoscopes apic guidelines for infection prevention and control in flexible endoscopy american society for testing and materials: standard practice for cleaning and disinfection of flexible fiberoptic and video endoscopes used in the examination of the hollow viscera guideline implementation: processing flexible endoscopes new developments in reprocessing semicritical items in vitro evaluation of integrity and sterilization of single-use argon beam plasma coagulation probes multisociety guideline on reprocessing flexible gastrointestinal endoscopes department of health and human services burden of gastrointestinal disease in the united states: update overview of infection control problems: principles in gastrointestinal endoscopy infectious disease complications of gi endoscopy: part ii, exogenous infections ercp scopes: what can we do to prevent infections? american society for gastrointestinal endoscopy: guidelines for safety in the gastrointestinal endoscopy unit transmission of infection by gastrointestinal endoscopy and bronchoscopy lessons from outbreaks associated with bronchoscopy jr: the prevention of infection following gastrointestinal endoscopy: the importance of prophylaxis and reprocessing an outbreak of mycobacterium chelonae infection following liposuction mycobacterium chelonae causing otitis media in an ear-nose and-throat practice centers for disease control and prevention: pseudomonas aeruginosa infections associated with transrectal ultrasound-guided prostate biopsies-georgia prevention of flexible bronchoscopy-associated infection new delhi metallo-betalactamase-producing carbapenem-resistant escherichia coli associated with exposure to duodenoscopes a quarantine process for the resolution of duodenoscope-associated transmission of multi drug-resistant escherichia coli withdrawal of a novel design duodenoscope ends outbreak of a vim- -producing pseudomonas aeruginosa disinfection of endoscopes: review of new chemical sterilants used for high level disinfection significant factors in the disinfection and sterilization of flexible endoscopes endoscope reprocessing: where do we go from here? mycobacteria and glutaraldehyde: is high-level disinfection of endoscopes possible? natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning endoscope reprocessing methods: a prospective study on the impact of human factors and automation automated endoscope reprocessors automatic flexible endoscope reprocessors society of gastroenterology nurses and associates: guideline for use of high level disinfectants & sterilants for reprocessing flexible gastrointestinal endoscopes automated washing with the reliance endoscope processing system and its equivalence to optimal manual cleaning food and drug administration: (k) summary for system le liquid chemical sterilant processing system steris corporation: system e tm liquid chemical sterilant processing system food and drug administration: information about automated endoscope reprocessors (aers) and fda's evaluation fda news release: fda orders recall under consent decree for all custom ultrasonics automated endoscope reprocessors food and drug administration: fda recommends health care facilities stop using custom ultrasonics' system plus automated endoscope reprocessors (aers) for reprocessing duodenoscopes; these reprocessors remain available to reprocess other flexible endoscopes: fda safety communication module : cleaning, disinfection and sterilization effectiveness of the system e liquid chemical sterilant processing system for reprocessing duodenoscopes public health agency of canada: infection prevention and control guideline for flexible gastrointestinal endoscopy and flexible bronchoscopy. part iv. issues related to reprocessing flexible endoscopes food and drug administration: fda-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices food and drug administration: fda-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices sterilization, high level disinfection and environmental cleaning public health agency of canada (formerly health canada): infection control guidelines: hand washing, cleaning, disinfection and sterilization in health care apic guideline for selection and use of disinfectants. , , and apic guidelines committee high-level disinfection or "sterilization" of endoscopes? flexible and semi-rigid endoscope processing in health care facilities canadian society of gastroenterology nurses and associations: infection control: recommended guidelines in endoscopy settings multi-society guideline for reprocessing flexible gastrointestinal endoscopes. society for healthcare epidemiology of america apic guideline for infection prevention and control in flexible endoscopy reprocessing endoscopes: united states perspective methodology of reprocessing reusable accessories in vitro evaluation of wire integrity and ability to reprocess single-use sphincterotomes a prospective study of the repeated use of sterilized papillotomes and retrieval baskets for ercp: quality and cost analysis reuse of disposable sphincterotomes for diagnostic and therapeutic ercp: a one-year prospective study food and drug administration: enforcement priorities for single-use devices reprocessed by third parties and hospitals third-party reprocessing of endoscopic accessories methodology of reprocessing one-time use accessories decontaminated single-use devices: an oxymoron that may be placing patients at risk for cross-contamination a performance, safety and cost comparison of reusable and disposable endoscopic biopsy forceps: a prospective, randomized trial risk associated with reprocessed reusable endoscopic instruments society of gastroenterology nurses and associates: standards of infection control in reprocessing of flexible gastrointestinal endoscopes. chicago, sgna advantages and limitations of automatic flexible endoscope reprocessors automated endoscope reprocessors endoscope disinfection a resourcesensitive approach. world gastroenterology organization practice guideline -endoscope disinfection -a resource-sensitive approach british society of gastroenterology: bsg guidelines for decontamination of equipment for gastrointestinal endoscopy: the report of a working party of the british society of gastroenterology endoscopy committee esge-esgena guideline for quality assurance in reprocessing: microbiological surveillance testing in endoscopy canadian standards association (csa): z . - decontamination of reusable medical devices food and drug administration: reprocessing medical devices in health care settings: validation methods and labeling. guidance for industry and food and drug administration staff food and drug administration: supplemental measures to enhance reprocessing: fda safety communication real-time monitoring in managing the decontamination of flexible gastrointestinal endoscopes current issues result in a paradigm shift in reprocessing medical and surgical instruments society of gastroenterology nurses and associates: standards of infection prevention in reprocessing flexible gastrointestinal endoscopes endoscope reprocessing methods: a prospective study on the impact of human factors and automation impact of ethylene oxide gas sterilization of duodenoscopes after a carbapenem-resistant enterobacteriaceae outbreak urgent safety notification: important updated labeling information: new reprocessing instructions for the olympus tjf-q v duodenoscope the role of biofilms in reprocessing of medical devices evaluation of the quality of reprocessing of gastrointestinal endoscopes the use of channel-purge storage for gastrointestinal endoscopes reduces microbial contamination evaluation of endoscope cleanliness after reprocessing: a clinical-use study dynamics of action of biocides in pseudomonas aeruginosa biofilm prevention: health advisory: immediate need for healthcare facilities to review procedures for cleaning, disinfecting, and sterilizing reusable medical devices worse-case soiling levels for patient-used flexible endoscopes before and after cleaning comparison of clinically relevant benchmarks and channel sampling methods used to assess manual cleaning compliance for flexible gastrointestinal endoscopes studies on the chemical sterilization of surgical instruments. i. a bacteriological evaluation ethylene oxide sterilization of medical devices: a review validation of low-temperature steam with formaldehyde sterilization for endoscopes, using validation device comparison of ion plasma, vaporized hydrogen peroxide, and % ethylene oxide sterilizers to the / ethylene oxide gas sterilizer comparison of liquid chemical sterilization with peracetic acid and ethylene oxide sterilization for long narrow lumens comparative evaluation of the sporicidal activity of new low temperature sterilization technologies: ethylene oxide, plasma sterilization systems, and liquid peracetic acid a comparative study of ethylene oxide gas, hydrogen peroxide gas plasma, and low-temperature steam formaldehyde sterilization ethylene oxide sterilization of medical devices: a review current practice of duodenoscope reprocessing carbapenem-resistant enterobacteriaceae and endoscopy: an evolving threat early identification and control of carbapenemase-producing klebsiella pneumoniae, originating from contaminated endoscopic equipment reported gastrointestinal endoscope reprocessing lapses: the tip of the iceberg multidrug-resistant klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography control of a multi-hospital outbreak of kpc-producing klebsiella pneumonia type in france outbreak of ertapenem resistant enterobacter cloacae urinary tract infections due to a contaminated ureteroscope duodenoscope-associated bacterial infections: a review and update infectious diseases linked to cross-contamination of flexible endoscopes transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy endoscopic retrograde cholangiopancreatography-associated ampc escherichia coli outbreak a carbapenem-resistant klebsiella pneumoniae outbreak following bronchoscopy ammi tir water for reprocessing of medical devices tir a compendium of processes, materials, test methods and acceptance . muscarella lf: recommendations for preventing hepatitis c virus infection: analysis of a brooklyn endoscopy clinic's outbreak prevention: transmission of hepatitis b and c viruses in outpatient settings viral hepatitis transmission in ambulatory health care settings guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings evaluation of the risk of transmission of bacterial biofilms and clostridium difficile during gastrointestinal endoscopy ercp scopes: what can we do to prevent infections? disinfection, sterilization and antisepsis: principles and practices in healthcare facilities is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination effectiveness of current disinfection procedures against biofilm on contaminated gi endoscopes modeling microbial survival in buildup biofilm for complex medical devices a study of the efficacy of bacterial biofilm cleanout for gastrointestinal endoscopes evaluation of detergents and contact time on biofilm removal from flexible endoscopes control and prevention: interim protocol for healthcare facilities regarding surveillance for bacterial contamination of duodenoscopes after reprocessing antimicrobial efficacy of endoscopic disinfection procedures: a controlled, multifactorial investigation pseudomonas aeruginosa cross-infection following endoscopic retrograde cholangiopancreatography pseudomonas aeruginosa and enterobacteriaceae bacteremia after biliary endoscopy: an outbreak investigation using dna macrorestriction analysis nosocomial infections from contaminated endoscopes: a flawed automated endoscope washer. an investigation using molecular epidemiology a prospective analysis of fever and bacteremia following ercp polymicrobial sepsis following endoscopic retrograde cholangiopancreatography infection control practices in gastrointestinal endoscopy in the united states: a national survey iatrogenic campylobacter pylori infection is a cause of epidemic achlorhydria development and validation of rapid use scope test strips (rust) to determine the efficacy of manual cleaning for flexible endoscope channels rapid method for the sensitive detection of protein contamination on surgical instruments the atp test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels validation of atp to audit manual cleaning of flexible endoscope channels persistent contamination on colonoscopes and gastroscopes detected by biologic cultures and rapid indicators despite reprocessing performed in accordance with guidelines assessing residual contamination and damage inside flexible endoscopes over time simulated-use validation of a sponge atp method for determining the adequacy of manual cleaning of endoscope channels validation and comparison of three adenosine triphosphate luminometers for monitoring hospital surface sanitization: a rosetta stone for adenosine triphosphate testing the use of rapid indicators for the detection of organic residues on clinically used gastrointestinal endoscopes with and without visually apparent debris the atp test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels hemoglobin assay for validation and quality control of medical device reprocessing control and prevention: interim sampling method for the duodenoscope -distal end and instrument channel detection of persistent vegetative bacteria and amplified viral nucleic acid from in-use testing of gastrointestinal endoscopes natural bioburden levels detected on rigid lumened medical devices before and after cleaning microbiological monitoring of endoscopes: -year review is bacteriologic surveillance in endoscope reprocessing stringent enough? surveillance cultures of samples obtained from biopsy channels and automated endoscope reprocessors after high-level disinfection of gastrointestinal endoscopes pseudomonas aeruginosa sepsis following retrograde cholangiopancreatography (ercp) pseudomonas septicaemia after endoscopic retrograde cholangiopancreatography: an unresolved problem acute hepatitis c virus infections attributed to unsafe injection practices at an endoscopy clinic-nevada nonhospital health care-associated hepatitis b and c virus transmission: united states news release: fda orders duodenoscope manufacturers to conduct postmarket surveillance studies in health care facilities usa: safety communication -fujifilm medical systems validates revised reprocessing instructions food and drug administration: pentax validates reprocessing instructions for ed- tk video duodenoscopes: fda safety communication olympus validates new reprocessing instructions for model tjf-q v duodenoscopes: fda safety communication food and drug administration: fda clears olympus tjf-q v duodenoscope with design modifications intended to reduce infection risk. fda news release urgent medical device removal and corrective action: elevator mechanism replacement, updated operation manual, and new reprocessing instructions for the olympus tjf-q v duodenoscope outbreaks of carbapenem-resistant enterobacteriaceae infections associated with duodenoscopes: what can we do to prevent infections? surveillance of guideline practices for duodenoscope and linear echoendoscope reprocessing in a large healthcare system how to assess risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines reprocessing failure food and drug administration: medical device reporting (mdr) food and drug administration: mandatory reporting requirements: manufacturers, importers and device user facilities assessing the risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines analysis of microbial load on surgical instruments after clinical use and following manual and automated cleaning patient-to-patient transmission of campylobacter pylori infection by fiberoptic gastroduodenoscopy and biopsy conventional cleaning and disinfection techniques eliminate the risk of endoscopic transmission of helicobacter pylori inactivation of clostridium difficile spores by disinfectants inactivation of hepatitis b virus by intermediate-to-high level disinfectant chemicals viral transmission and fibreoptic endoscopy elimination of high titre hiv from fiberoptic endoscopes endoscopic transmission of hepatitis b virus absence of transmission of hepatitis b by fiberoptic upper gastrointestinal endoscopy contamination of an endoscope due to trichosporon beigelli transmission of trichosporon asahii oesophagitis by a contaminated endoscope complications associated with esophagogastroduodenoscopy and with esophageal dilation creutzfeldt-jakob disease: recommendations for disinfection and sterilization managing the risk of nosocomial transmission of prion diseases creutzfeldt-jakob disease: implications for gastroenterology current issues in endoscope reprocessing and infection control during gastrointestinal endoscopy guideline for disinfection and sterilization of prion-contaminated medical instruments variant creutzfeldt-jakob disease (vcjd) and gastrointestinal endoscopy united states senate; health, education, labor, and pensions committee: preventable tragedies: superbugs and how ineffective monitoring of medical device safety fails patients. minority staff report food and drug administration: infections associated with reprocessed duodenoscopes risk of transmission of carbapenem-resistant enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy infection using ercp endoscopes multisociety guideline on reprocessing flexible gi endoscopes: update food and drug administration: design of endoscopic retrograde cholangiopancreatography (ercp) duodenoscopes may impede a complete reference list can be found online at expertconsult .com key: cord- -rgm ffpk authors: senger, mario roberto; evangelista, tereza cristina santos; dantas, rafael ferreira; santana, marcos vinicius da silva; gonçalves, luiz carlos saramago; de souza neto, lauro ribeiro; ferreira, sabrina baptista; silva-junior, floriano paes title: covid- : molecular targets, drug repurposing and new avenues for drug discovery date: - - journal: mem inst oswaldo cruz doi: . / - sha: doc_id: cord_uid: rgm ffpk coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) is a highly contagious infection that may break the healthcare system of several countries. here, we aimed at presenting a critical view of ongoing drug repurposing efforts for covid- as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for covid- treatment. at the moment, the prevention aimed at reducing transmission in the community is the best alternative, indicating that enhanced public health interventions, including social distancing, use of masks and movement restrictions should be implemented to bring the covid- pandemic under control. aggressive isolation measures including travel restriction in china, have successfully led to a progressive reduction of covid- cases. ( ) kissler and colleagues, simulated the relaxation of the protective measures using a post-pandemic mathematical model. ( ) the authors demonstrated that social isolation will be necessary at the best scenario until , and reaffirmed the need and importance of maintaining social isolation. they also suggested that in the absence of such restrictions, the pandemic could last until . thus, in this context, the search for new medicines available to combat this disease is urgent. coronaviruses are members of the family coronaviridae that present crown-like spikes on their surface visualised by electron microscopy. the subfamily coronavirinae contains the four genera alpha, beta, gamma, and deltacoronavirus. coronaviruses infect birds (gamma and deltacoronaviruses) and several mammalian species (mainly alpha and betacoronaviruses), including humans. ( , ) coronaviruses have been isolated from diverse species, including mammals like bats, rodents, bovines, swine, felines, pangolins, horses and others. ( , ) human coronavirus was first identified in by tyrrell and bynoe. ( ) nowadays the seven coronaviruses that can infect humans (hcovs) are classified in alpha coronavirus ( e, nl ) or beta coronavirus [oc , hku , middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and more recently the sars-cov- ]. e, nl , oc , and hku can cause upper and lower respiratory tract infection in adults and children. after the s, two epidemic covs have arisen in humans: the sars-cov and the mers-cov which were reported in and , respectively. ( , ) in , after the first report of novel pneumonia in wuhan, china, the seventh hcov was described. it also causes severe acute respiratory syndrome (therefore called sars-cov- ) and spreads very quickly worldwide . coronavirus are single-stranded positive-sense rna viruses and their genome size is approximately kb, which encodes some important structural proteins. ( ) the spike (s) glycoprotein is a well characterised protein that mediates coronavirus entry into host cells via fusion of the viral and cellular membranes through a pre to post fusion conformation transition. ( ) the s protein s -s subunits bind to cellular receptors that vary according to the coronavirus species: angiotensin-converting enzyme (ace ) in sars-cov, sars-cov- and hcov-nl ; and dipeptidyl peptidase (dpp ) and aminopeptidase n (apn) in mers or others alphacoronaviruses like tgev (porcine transmissible gastroenteritis coronavirus) and porcine respiratory coronavirus (prch). ( , , ) other structural proteins are mandatory to assemble the complete viral particle like nucleocapsid protein (n), membrane protein (m) and the envelope protein (e). furthermore, they can be involved in other processes like morphogenesis, envelope formation, budding or pathogenesis. ( , , ) by genomic sequencing analysis of other coronavirus strains and sars-cov- , andersen and collaborators demonstrated that sars-cov- has mutations resulting in six different amino acids at the receptor-binding domain (rbd) that appears to be optimised for binding to the human receptor ace . ( ) they also showed that the gene encoding the spike protein has an insertion of nucleotides giving it a polybasic (furin) cleavage site at the s -s . in this way, the high-affinity of the sars-cov- spike protein to the human ace is a consequence of natural selection on a human or human-like ace . they suggest some possibilities to explain that: emergence in an animal host before zoonotic transfer; natural selection in humans following zoonotic transfer; or natural selection during the passage. ( ) other researchers did a phylogenetic analysis of genomes of sars-cov- . ( ) they showed important variations in the composition of amino acids that allowed them to classify into different groups. group a has two subclusters that are distinguished by the synonymous mutation t c. while b is derived from a by two mutations t c and c t, type c differs from its parent type b by mutation of g t. the a and c types are found more often outside east asia, in europeans and americans. while the b type is the most common type in east asia. ( ) in december , covid- was initially reported as a new viral pneumonia, due to the clinical characteristics of the large number of cases that emerged in wuhan, china. ( , ) sars-cov- typically causes respiratory sickness, the major clinical characteristics ob-served in infected patients are high fever, dry cough and dyspnea (shortness of breath or difficulty in breathing). minor symptoms include headache, diarrhea, nausea, vomiting, loss of smell and taste. ( ) this clinical condition can progress to moderate or severe pneumonia. ( ) in this case, first there is an accumulation of macrophages in alveoli, followed by release of cytokines and accumulation of fluids. neutrophils can also be recruited by the immune system leading to the destruction of type i and type ii alveolar epithelial cells causing a collapse of the alveoli function and consequently the acute respiratory distress syndrome (ards). in the severe condition, with an increase of the inflammation, the protein rich fluid from lungs enter in the bloodstream causing the systemic inflammatory syndrome (sirs). ( , ) these complicating factors can lead to a multi-organ failure and septic shock, causing patient death. furthermore, some pre-existing conditions can enhance the risk to develop the severe form of the disease, including age over years and a history of chronic diseases like chronic lung disease, asthma, heart diseases, immunosuppressed patients, cancer, diabetes or chronic kidney disease. ( , , , , ) finally, the vast majority of people have mild symptoms or are asymptomatic, which is a big problem because they can also transmit the virus to the non-infected population. ( ) drug repositioning, repurposing, reprofiling or retasking is the evaluation of existing drugs for new therapeutic purposes. ( ) a candidate drug (investigational or approved) for repurposing efforts already has a known safety and toxicity profile, based on at least successful phase i or phase ii clinical trials. ( ) considering the whole process, costs of bringing a repurposed drug to the market have been estimated to be ten times lower and the time is shortened by around a half, compared with a new drug. ( ) even though the clinical phase iii and regulatory aspects remain similar for developing a new drug, drug repurposing possesses many advantages over developing a new drug from scratch: the reduced time and financial investment for development, the lower risk of failure and a consolidated pharmaceutical supply chain for production and distribution to the patients that effectively need treatment. ( ) emerging or reemerging viruses pose major public health concerns globally. ( ) for several pathogenic viruses, considerable efforts have focused on vaccine development and other therapies, like transfusion of convalescent plasma. ( , ) however, during pandemics infected individuals need urgently to be treated on a large scale. a medicine armamentarium for the covid- outbreak is needed immediately and drug repurposing could be one of the best strategies to deal with this pandemic. ( , ) computational and experimental approaches can be used, alone or combined, to achieve a more holistic point of view and increased chance of success in drug repurposing. in the following topic, we will review sars-cov- structure and mechanism of infection in order to discuss molecular targets from the virus or its human host that are being considered for drug repurposing and perhaps future development of new drugs. ongoing drug repurposing efforts will be described in more details later in this article, along with some clinical trials that have been carried out so far for covid- treatment. finally, as treatment availability is of utmost importance when dealing with a pandemic, we bring a discussion on patent protection and ease of large-scale production of some of the drugs that are more advanced in clinical studies. sars-cov- : structure, mechanism of infection and drug targets sars-cov- structure -electron microscopy imaging of sars-cov- virions indicates that they have a spherical or pleomorphic shape, with diameters ranging from to nm, showing prominent spikes of - nm in their surfaces that resemble a solar corona, hence the name "coronavirus". ( , , , ) sars-cov- is an enveloped virus with a single-stranded positive sense ( '- ') rna (+ssrna) (~ kb) containing a 'cap structure and a '-poly-a tail. ( , ) its genomic rna (grna) has a variable number of open reading frames (orfs) that are predicted to encode non-structural (nsp), structural and several accessory proteins ( fig. ). ( , , , , ) orf a and orf b represent more than / of the whole length of grna, and encode two polyproteins: pp a ( - kda) and pp ab ( - kda). ( , ) the polyprotein pp a is translated from orf a while pp ab from orf a/orf b using a - ribosomal frameshift mechanism that occurs near the ' end of orf a which allows continued translation of orf b. ( ) together, pp a and pp ab originate all nsps ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , such as m pro (nsp ) protease and rdrp (nsp ) rna polymerase, which form viral replicase/transcriptase complexes (rtcs), and are encapsulated in double-layered vesicles originated from the endoplasmic reticulum (er). ( , , ) the orfs near the ' end of the grna encode the structural and accessory proteins of sars-covs. ( ) the first ones have a crucial role in the assembly of viral particles and virus invasion. ( , ) the main structural proteins are named: spike (s), envelope (e), nucleocapsid (n) and membrane (m) proteins. most of them reside on the virion surface (s, e, m proteins) while n proteins are found in the core of the particle bound to grna. ( ) s proteins are essential for virus attachment and entry into the host cells, tissue tropism and pathogenesis. ( , ) e proteins exert several roles in virus infection, such as helping in virus assembly and release from infected cells, creating ion channels in cell membranes and suppressing host stress response. ( , , ) n proteins interact with grna to form the ribonucleoprotein. ( , ) m proteins have a role in virion assembly and in determining the shape of the envelope. they also bind to all other structural proteins promoting, for instance, the stabilisation of n protein-rna complexes. ( , ) sars-cov- mechanism of infection -at present, the mechanisms that underlie sars-cov- infection have not been directly described. nonetheless, they seem to be similar to those proposed for other coronaviruses. ( ) in one proposal, virus infection starts with the binding of its s proteins to host receptor ace , a membrane protein largely expressed in the lung and small intestine cells (fig. ). ( , , ) after attachment, s protein is cleaved by host proteases initiating the fusion of virus and cell membranes that culminates in viral grna release into the cytoplasm. this event is proposed to occur through two distinct ways: via plasma membrane (early pathway) or via endosomes (late pathway). in the early pathway, s protein is cleaved by host plasma membrane proteases (e. g., tmprss ) while in the late pathway by endosomal proteases (e. g., cathepsin l). the route taken by the virus to enter the cell appears to be dependent on the availability of these proteases. ( , , ) once in the cytoplasm, grna is readily translated into viral polyproteins (pp a/pp ab), which are cleaved into the individual nsps that compose the rtcs (fig. ) . these complexes recognise transcriptional regulator sequences in grna and begin to transcribe a series of subgenomic rnas that encode structural and accessory proteins, otherwise the whole grna is replicated. ( , , ) upon translation, s, e and m structural proteins are driven to the er-golgi intermediate compartment (ergic) where s proteins go through post-translational modifications (e. g., proteolysis, n-glycosylation). in parallel, a copy of the grna and n proteins bind in the cytoplasm to form the nucleocapsid and move into the ergic. in this compartment, nucleocapsid and the other ( ) virus infection initiates with the binding of virus s proteins to the ace cellular receptors. after attachment, the virus may enter the cell through two distinct mechanisms: early and late pathways. ( ) in the early pathway the genomic ssrna (grna) is liberated into the cytoplasm after the fusion between viral and cell cytoplasmic membranes, an event triggered by membrane proteases (e. g., tmprss ). ( ) the grna is immediately translated into two polyproteins that undergo proteolytic cleavage giving rise to all nonstructural proteins (nsps). ( ) the nsps form the replication-transcription complexes (rtcs) where the grna (blue ribbon) is replicated and the subgenomic rnas (red ribbon) are transcribed. ( ) the subgenomic rnas are translated into viral structural and accessory proteins in the cytosol. ( ) upon translation, e, m and s structural proteins are inserted into er and follow the secretory pathway to the er-golgi intermediate compartment (ergic). ( ) meanwhile, a copy of the grna binds to n proteins in the cytoplasm forming the nucleocapsid, which is transported to the ergic. ( ) virion assembly in the ergic. ( ) the new virion travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. ( ) alternatively, in the late pathway, the virus can undergo endocytosis to initiate the infection. ( ) the virion membrane merges with the endosome membrane after s protein proteolysis by endosomal proteases (e. g., cathepsin l), allowing the grna to be released into the cytoplasm. from this point on, the cycle follows the same pathway described in - steps. the red arrows indicate the general replication pathway and the blue arrows indicate the grna movement through the cycle. some viral and host proteins have been explored as potential targets for drug repurposing. some of these drug candidates are shown with their site of action indicated in the cycle. ( , , , ) viral proteins are assembled into a virion which travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. ( , , ) candidate drug targets -in the search for a treatment for covid- , several viral and host molecular proteins have been explored as potential drug targets. overall, they participate in key events of the virus infection cycle, such as cell entry and replication, as well in host metabolic pathways and immune response. in the following topics we will address in more details some of these targets. drugs under investigation for blocking the main steps of the sars-cov- virus replication cycle are indicated in fig. . virus targets -during sars-cov- grna translation, two proteases, namely m pro and pl pro , act in concert to cleave and release from pp a/pp ab the nsps that compose the rtc. ( ) therefore, these proteases are essential for virus replication and represent useful targets for therapeutic intervention. recently, sars-cov- m pro and pl pro had their d structures published -pdb lu , . Å and pdb w c, . Å, respectivelywhich make them particularly useful for computational structure-based drug design methods. ( ) -chymotrypsin-like protein (synonyms: coronavirus main protease, m pro , cl pro ) of sars-cov- is a . kda homodimeric protein ( aa) that belongs to the cysteine protease class (possibly from c family and pa clan). ( , ) this enzyme catalyses the hydrolysis of peptide bonds of polyproteins (possibly e.c. . . . ) at sites whose amino acid sequences generally follow the pattern leu-gln* (ser, ala, gly) (*marks the cleavage site). ( , ) m pro is part of pp a/pp ab polyproteins (nsp ). ( ) during polyproteins translation, m pro suffers autolyt-ic cleavage and is released from its polyprotein precursor, reaching a mature state that cleaves pp a/pp ab at no less than sites downstream of the nsp coding region. ( , , , ) each protomer of sars-cov- m pro is divided into three domains: chymotrypsin and picornavirus c protease-like i and ii domains, composed by antiparallel β-barrel structures, and domain iii which contains five α-helices arranged into an antiparallel globular cluster responsible for protease dimerisation (fig. a) . domains ii and iii are connected by a long loop, where lies a cleft that serves as a substrate binding site and where catalysis occurs using the cys -his dyad (fig. b ). ( , , ) m pro has been widely explored in drug discovery campaigns using experimental and/or computational approaches. ( , , , , , ) moreover, m pro has no human homologue, which reduces the chances of toxic effects of a given inhibitor. ( ) potential sars-cov- m pro inhibitors include fda-approved antivirals, such as inhibitors of hiv- [e. g., lopinavir ( ) /ritonavir ( ) ] and hcv [e.g., boceprevir ( )] proteases, as well as antineoplastic [e.g. carmofur ( )] and antibacterial [e.g., doxycycline ( )] drugs. ( , , , , , ) chemical structures for compounds - are shown in fig. . nsp is the largest protein encoded by covs (~ kda). in sars-covs it has domains which include a papain-like proteolytic enzyme from the cysteine protease class (c family and ca clan for sars-cov). ( , , ) pl pro is composed of a catalytic domain, an extended right-handed thumb-palm-finger structure with a cys-his-asp catalytic triad, and a ubiquitin-like domain (ubl) (fig. a ). the catalytic cys is located in the thumb subdomain while his and asp in the palm subdomain (fig. b ). pl pro catalyses the hydrolysis reaction of peptide bonds of pp a/pp ab at three sites (nsp /nsp , nsp /nsp and nsp /nsp ) that share the xlxgg* pattern (*represents the cleavage site). ( , , , ) this activity is responsible for nsp release from polyproteins. pl pro also recognises and hydrolyses ubiquitin and isg from cellular proteins. ( , ) the deubiquitination and deisgylation activities are proposed to modulate the post-translational modifications of signaling molecules that trigger innate immune response of the host. ( ) these functions are pivotal for virus infection justifying the search for sars-covs pl pro inhibitors. ( , , , , , ) putative inhibitors of sars-cov- pl pro include fda-approved drugs such as fostamatinib disodium ( ) (a tyrosine kinase inhibitor used in the treatment of chronic immune thrombocytopenia) and natural products [e.g., platycodin d ( )]. ( , ) rna-dependent rna polymerase (rdrp, nsp ) is an essential protein responsible for rna synthesis during viral rna transcription and replication cycles. ( ) as described for sars-cov, the rna polymerase activity of sar-cov- rdrp ( aa) also seems to require the binding of nsp and nsp cofactors to enhance rdrp binding and processivity. ( , ) the overall rdrp structure has a "right hand" rdrp domain, composed by three subdomains (palm, fingers and thumb), and a nidovirus-unique n-terminal extension domain that forms a nidovirus rdrp-associated nucleotidyltransferase (niran) structure. these domains are connected by an interface domain. additionally, this protein also has a n-terminal β-hairpin (fig. a) . the active site of rdrp contains conserved polymerase motifs located in the palm subdomain and its configuration is similar to other rna polymerases. ( ) rdrp substrates, rna template/primer and nucleotide triphosphate (ntps), access the catalytic centre through the template and ntp entry paths, respectively, while the product-template hybrid is released through the rna exit path. ( , ) sars-cov- rdrp shares % identity in amino acid sequence with sars-cov protein. ( ) moreover, the accessory proteins also have a high degree of amino acid sequence identity between these viruses: . % for nsp and . % for nsp -sequences obtained from pdb bv ( . Å) and pdb nur ( . Å) entries. ( , ) therefore, it is reasonable to suggest that sars-cov rdrp inhibitors may also bind to the homologous enzyme from sars-cov- , as demonstrated for remdesivir ( ) (fig. b) , an adenosine triphosphate analog. ( , , ) other potential inhibitors include clinically available drugs, such as favipiravir ( ), a purine nucleic acid analog used in the treatment of influenza, and ribavirin ( ), a synthetic guanosine nucleoside indicated for the treatment of hepatitis c virus (hcv) infection. ( , ) the crucial role of rdrp and the lack of a host homolog turns this enzyme into a valuable target for anti-covs agents. ( ) helicase (nsp ) is a multifunctional protein (predicted to have aa in sars-cov- ) essential for sars-covs rna replication and proliferation. ( , ) sars-cov helicase belongs to helicase superfamily (sf ) and can unwind double-stranded dnas/rnas (helicase activity) in the '- ' direction, an energyconsuming process that is driven by the hydrolysis of nucleosides triphosphate (ntpase activity). ( , , ) its structure contains five domains arranged in a triangular pyramid-shape: the triangular base is composed by two "reca-like" ( a and a) and b domains whereas the zinc binding domain (zbd), in the n-terminal, and the stalk domain are oriented towards the apex. the zbd and b domains are connected by the stalk domain ( fig. ) . ( ) the same domain arrangement is predicted to occur in sars-cov- enzyme. ( ) sars-cov helicase has been explored as a potential target for drugs. ( , , ) recently, some efforts have also been made to predict inhibitors for sars-cov- helicase. ( , , , ) they include drugs used in the clinics to treat acquired immunodeficiency syndrome (aids), such as vapreotide ( ) (somatostatin analog) and atazanavir ( ) (hiv protease inhibitor), as well anti-hcv protease inhibitors [e.g., daclatasvir ( ) ] and bismuth salts [e.g., bismuth potassium citrate ( ) , a compound used in clinical treatment of gastrointestinal diseases]. ( , , ) sars-covs replication also requires other enzymatic activities including (guanine-n )-methyltransferase (n -mtase), '-o-methyltransferase ( '-omtase) and exoribonuclease (exon). ( , , ) the n -mtase domain at the c-terminus of nsp is responsible for adding a methyl group in the n position of rna '-guanosine forming the '-cap structure of viral rnas. ( ) additionally, nsp also has a catalytic domain in its nterminus with '- ' exon activity, which participates in rna proofreading mechanism (fig. ). ( ) '-omtase (nsp ) catalyses the methylation reaction at the ribose '-o position of the first and second nucleotide of the mrnas and it is one of the sars-cov- proteins whose d structure has already been resolved (pdb: w , . Å) (fig. ). ( ) both nsp and use nsp as a cofactor to enhance their '- ' exon and '-omtase activities, respectively. ( ) together, these proteins are responsible for inducing rna modifications that are essential for its stability and translation as well to avoid the activation of host immune response. ( , , ) thus, nsp and have been considered as potential targets for anti-sars agents that may affect their functions in a direct or indi- rect way (e.g., by blocking nsp binding). ( , , , , , , , ) some compounds have been regarded as potential inhibitors of sars-cov- methyl transferases, such as adenine dinucleoside s-adenosylmethionine analogs [e.g., dinucleoside ( ) ], predicted for sars-cov n -mtase activity, and some clinically available drugs [e.g., raltegravir ( ) -a hiv integrase inhibitor], predicted for sars-cov- '-omtase activity. ( , ) chemical structures for compounds - are shown in fig. . spike protein (s) is a viral type i transmembrane glycoprotein (~ - kda) responsible for covs interaction and invasion of host cells. ( , ) this protein is synthesised as a monomer and suffers post-translational modifications in the er before becoming a trimeric glycosylated protein. ( , ) its structure is divided into three main domains: an extracellular, a transmembrane and a short intracellular domain (fig. ). ( ) the former protrudes from the surface of the virus particle creating a crown-like halo and contains two functional subunits: s (bulbous shape), which binds to cellular receptors (e.g., ace ), and s (stalk shape) that promotes the fusion between cell and virus membranes. ( ) in turn, s subunit has two domains: a n-terminal and a c-terminal domain. the latter serves as a rbd for sars-covs being responsible for ace recognition and binding. ( , , ) apart from s and s , sars-cov- s protein also has a ganglioside-binding subdomain at the tip of n-terminal domain, which may allow this protein to interact with gangliosides on cells' surface. in theory, this domain could facilitate virus attachment to the cell and facilitate the contact with ace , being a potential site for drug interference. ( ) fig. : ribbon representation and vdw surface of the severe acute respiratory syndrome coronavirus (sars-cov- ) '-o-methyltransferase d structure (pdb w , . Å, nsp , pink) in complex with its nsp cofactor (light blue). the zinc and chloride ions are represented as gray and green spheres, respectively. image created using maestro, release - (maestro, schrödinger, llc, new york, ny, ). covs s proteins require proteolytic priming or cleavage to become fusion competent. ( ) this is achieved by host proteases (e.g., tmprss , cathepsin l, furins) which cleave the s subunit of s protein at two main positions: s /s interface and s '. the latter is located immediately upstream of the fusion peptide (fp), the fusion functional element of s . contrary to sars-cov, sars-cov- s subunit also has an additional cleavage site for furin proteases in the s /s region, something that also occurs in mers-cov. ( ) it is still unclear its role in sars-cov- infection, but it is speculated that the ubiquitous expression of furins may increase cell and tissue tropism of sars-cov- in comparison to sars-cov, as well altering its transmissibility and pathogenicity. ( , ) taken together, host proteases represent potential targets for anti-sars-cov- drugs, and thus some of them will be discussed in the following topics. ( , , ) covs e proteins are small integral membrane polypeptides ( - aa) encoded by subgenomic rnas. in sars-cov, they are found in virions, but also in large amounts in the ergic where they participate in virus budding and trafficking. their structures are divided into three main domains: n-terminal, transmembrane (tmd) and c-terminal domain. tmd is able to form pentameric α-helical bundles creating ion conductive pores in membranes. ( , , ) the ion channel (ic) activity of e protein has been proposed to alter ion homeostasis, as well induce inflammatory response, which may lead to pulmonary damage. ( , ) thus, the use of inhibitors of ic activity may represent a possible therapeutic strategy for covs-related diseases, including covid- . ( this can be exemplified by the fda-approved drugs gliclazide ( ), a sulfonylurea administered to non-insulin-dependent diabetes mellitus patients, and memantine ( ) , an n-methyl-d-aspartate receptor antagonist used in the management of alzheimer's disease, which have shown ic inhibitory activity in bacteria expressing sars-cov- e proteins. ( ) host cell targets -a key step in sars-covs infection is the attachment of s protein to host angiotensinconverting enzyme (ace ), a membrane-bound carboxypeptidase (family: m , clan: ma). ( , , ) this enzyme catalyses the hydrolysis of angiotensin i and angiotensin ii into angiotensin-( - ) and angiotensin-( - ) peptides, respectively. ( ) ace is expressed in the upper respiratory system, type i and ii alveolar epithelial cells in the lungs, heart, endothelial cells, kidney tubular epithelium and other tissues. ( ) its role as a functional receptor of sars-cov- s protein in host cells makes this protein a potential drug target to treat covid- . there are several therapeutic strategies targeting ace which include: vaccines based on s protein, exogenous administration of a soluble form of ace , and administration of small molecules [e.g., arbidol ( ) , an anti-influenza drug] or antibodies (e.g., sti- , an anti-spike antibody) to block the interaction of ace with s protein. ( , , ) additionally, compounds could also have an anti-sars-covs activity by disturbing ace glycosylation, as proposed for chloroquine ( ) . ( ) renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors [ace-i, e.g., captopril ( ) ] and angiotensin receptor blockers [arb, e.g., losartan ( ) ], are commonly used in clinics to treat hypertension and cardiovascular/renal diseases. ( , ) recently, some researchers have debated over the administration of these drugs to patients with known or suspected covid- . ( , , , ) the main reason for this discussion is that raas inhibitors may induce ace expression, which, in theory, could increase the severity of the infection. ( , ) nonetheless, evidence suggests that these drugs may protect patients from lung injury by suppressing angiotensin ii signaling mediated by angiotensin receptor (at r). ( , , ) further investigation is still required to determine whether ace-i and arb have a beneficial or deleterious role in covid- treatment. ( ) in order to become fusion-competent, sar-covs s proteins must be cleaved by host proteases. in sars-cov- , this process appears to be mediated primarily by tmprss in the plasma membrane. ( ) tmprss (transmembrane protease, serine ) is a transmembrane protein (predicted to have aa) from the serine protease class (family: s clan: pa). ( , ) it is highly expressed in the epithelial cells of the prostate, and relatively less in lungs, colon, liver, kidney, and pancreas. its physiological function is still unclear. ( ) tmprss has a major role in sars-cov- cell entry and replication, and thus represents an interesting therapeutic target since its inhibitors could potentially block virus infection in its initial stages. ( , ) potential tmprss blockers include some serine protease inhibitors [e.g., camostat mesylate ( )], commercially available compounds [e. g., zinc ( ), -[[ -( -fluorophenyl)pyrimidin- -yl]amino~(n)-( -methyl phenyl)benzamide]] and natural products [e.g., withanone ( ) ]. ( , , , ) in the absence of exogenous or membrane-bound proteases (e g. tmprss ) to promote sars-covs infection on cells' surface, the virus can also be internalised via endocytosis, also known as "late pathway". ( ) this process comprises several factors that operate in a sequential and partially overlapping fashion. ( ) endocytosis initiates with the recruitment of endocytic coat proteins (e. g., clathrins) from the cytosol to gather on the inner leaflet of the plasma membrane. then, protein-coated pits bud into vesicles originated from plasma membranes and fuse with each other or with pre-existing early endosomes. ( , ) gradually, early endosomes mature to late endosomes which have an acid environment (approx. ph . ), activating endosomal cathepsin l cysteine proteases (family: c , clan: ca). ( , , ) these enzymes are responsible for triggering the fusion activity of s protein and subsequent insertion of viral sars-cov grna into the cytosol, especially in cells with lower expression of tmprss . ( , ) some key elements of endocytosis have been explored as potential targets for anti-sars-cov drugs, such as agents that increase endosomal ph (avoiding cathepsin l activation) [e.g., chloroquine ( ) and hydroxychloroquine ( ) ] and cathepsin l inhibitors [e.g., teicoplanin ( ) , a glycopeptide antibiotic]. ( , , ) cytokines are short-lived small size proteins ( - kda) that exert an important role in autocrine, paracrine, and endocrine signaling controlling the development and function of several immune and nonimmune cells. ( , ) they are classified in families based on certain properties of their receptor complexes, such as their structure, specificity and composition. ( ) one example is interleukin (il)- family which includes several cytokines, such as il- , that use the common signaling receptor subunit glycoprotein kda (gp ). il- signaling requires the binding of il- to il- receptor (il- r) which consists of a soluble il- binding domain (cd ) and membrane-bound gp . il- acts as the main inducer of fever, inflammation and of hepatic acute phase proteins. therefore, il- /il- r antagonists have a therapeutic effect in inflammatory diseases. ( , ) their use in covid- treatment may be beneficial to avoid the "cytokines storm" syndrome that some patients with the severe form of the disease may develop. ( ) this deleterious event is caused by an amplified immune response and cytokine release that can damage the organs, including the lungs. ( ) tocilizumab and sarilumab are two examples of humanised monoclonal antibodies that act as il- r antagonists which are currently under clinical trials for covid- treatment. ( ) other targets -apart from sars-covs infection, some relevant molecular targets of other viral diseases and host metabolism have also been investigated in covid- drug discovery, such as viral neuraminidases and the dpp cell receptor. neuraminidases (na) or sialidases, are glycoside hydrolases (family: gh ) largely found attached to the envelope of influenza viruses. ( , ) they catalyse the hydrolysis of glycosidic bonds between sialic acid and adjacent sugar residues (ec . . . ) in glycoproteins, glycolipids and oligosaccharides. ( ) na is mainly responsible for cleaving sialic acid acids from cell receptors and on carbohydrate side chains of nascent virion, facilitating its release from infected cells. ( ) its critical role in virus infection and proliferation has been exploited by na inhibitors [e.g., oseltamivir ( ) ] which are administered in clinics to combat influenza infections. apparently, this therapeutic strategy was employed at the beginning of co-vid- outbreak during the peak of influenza season in china when the etiologic agent of this disease was yet unknown. so far, there is no evidence that na inhibitors may have a role in covid- management. ( ) dipeptidyl peptidase- (ddp ), also known as adenosine deaminase complexing protein or t-cell activation antigen cd , is a type ii transmembrane glycoprotein of aa largely expressed in many tissues (e.g. lungs and immune cells). ( , ) ddp belongs to serine protease group (family: s , clan: sc) and catalyses the hydrolysis of n-terminal dipeptide, xaa-yaa-|-zaa-, (preferentially when yaa is a proline, as long as zaa is neither proline or hydroxyproline), from a variety of peptide substrates (ec . . . ), such as chemokines, neuropeptides, vasoactive peptides and growth factors. ( , , ) therefore, it participates in many physiologic and pathological processes, including glucose/insulin metabolism and immune/inflammatory response. ( ) in addition, dpp acts as the functional cell receptor of mers-cov s protein, helping virus entry into the cell. theoretically, this role may also be played in sars-cov- infection, as predicted by computational analysis. ( ) recently, there has been a discussion about the use of ddp inhibitors, such as sitagliptin ( ) (anti-diabetic drug), in the treatment of covid- . ( , , , ) in principle, these inhibitors could be beneficial for type diabetes patients (or even without diabetes) with covid- since it could potentially block virus cell entry/replication, as well suppress inflammatory response. ( , ) however, there is not enough evidence yet to prove this hypothesis. ( , , ) computational approaches -the sars-cov- pandemic is creating a fertile ground for computational approaches to identify viable therapeutic options in the short-term, with the number of published studies growing rapidly. ( , ) classical target-and ligand-based strategies (e.g., molecular docking and similarity analysis), are being applied to fda-approved drugs and compounds in clinical trials to explore possible interactions with viral proteins. ( , , ) artificial intelligence (ai) methods are also being extensively applied to large molecule databases to find existing drugs that could be repurposed based on previously reported antiviral activities. ( , , , ) ai approaches can even explore hidden connections between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. ( , , ) these studies emphasize how important computational methods are in modern drug discovery to analyse the huge amount of biomedical data available. table i summarises a selection of studies that suggested potential drugs for repurposing. below we will describe the main computational methods and results. smith and smith performed a large virtual high-throughput screening to identify drugs, natural products and metabolites that could disrupt the interaction between sars-cov- s protein and human ace receptor. the authors used a supercomputer called summit to conduct the simulations, which included structural modeling of the s protein:ace complex, generation of an ensemble of conformations for the complex and molecular docking. ( ) the ensemble generation was a crucial step in this work, allowing the authors to explore different conformations of the complex and identify drugs that could interact with binding sites not easily identified in static conformations. using the gromacs molecular dynamics simulation suite, six different clusters of conformations were generated by restrained temperature replica-exchange molecular dynamics simulation (t-remd) and submitted to docking with autodock vina using the sweet-lead compound collection. ( , , ) the authors explored two strategies, consisting of disrupting the s protein: ace complex and preventing its formation by docking on the isolated proteins. from this analysis, four compounds [pemirolast ( ) , nitrofurantoin ( ), isoniazid pyruvate ( ) and eriodictyol ( ) ] displayed potential to disrupt the s protein: ace interaction, and three natural compounds [cepharanthine ( ) , ergoloid ( ) and hypericin ( ) ] showed potential to block host recognition. ( ) chemical structures for compounds - are shown in fig. . ge and coworkers constructed a virus-related knowledge graph (or network) by combining seven networks with information about target-drug and protein-protein interactions, molecule similarity and sequence similarity of human and viral sources to predict drugs targeting sars-cov- . ( ) well known biological and interaction databases were employed, such as drugbank, chembl, bindingdb, and uniprot . ( , , , ) the final knowledge graph was assembled by merging the nodes and edges of the seven networks, where each node represent drugs or targets, while the edges between them are the identified relations, such as similarity (i.e., molecular and sequence similarities) and drug-target or target-target interactions. ( ) a graph convolutional network (gcn) was used to learn and extract the hidden information on the nodes and edges of the knowledge-graph, allowing the authors to access novel drug-target and target-target interactions and find molecules that could be repurposed to sars-cov- . ( ) gcn's are powerful neural networks that can access the rich information within the nodes of a graph and return insights about possible relations (e.g., potential drugs and targets interactions), representing a valuable tool in the modern drug discovery scenario, where massive biological data are available in databases such as drugbank and chembl. ( , ) the final knowledge-graph was then mined to gather an initial list of drugs, which was further refined by extracting previous evidence of antiviral activity from pubmed using a deep learning method called biomedical entity relation extraction (bere) and manual inspection. ( ) in a subsequent step, the authors elaborated a final list of drug candidates using transcriptome analysis of ten sars-cov patients. the poly-adp-ribose polymerase (parp ) inhibitor, mefuparib hydrochloride (cvl ) ( ) , currently in phase i clinical trials, was identified as a potential drug for repurposing. in vitro and in vivo validation showed promising inhibition and safety profiles. concretely, compared to arbidol ( ) , one of the standard treatments for covid- in china, cvl showed higher antiviral efficacy and higher concentration in lungs of rats. ( ) the authors also found that cvl significantly inhibited the production of il- induced by the cpg oligodeoxynucleotide (cpg-odn ) in peripheral blood mononuclear cells (pmbc) indicating it might be an alternative to treat the inflammation caused by sars-cov- . furthermore, cvl showed favorable pharmacokinetics and toxicity profiles in rats and monkey models. ( ) this integration of artificial intelligence with wet-lab experiments highlights the importance of in silico methods to mine baricitinib ( ) rheumatoid arthritis human ap -associated protein kinase (aak ) knowledge-graph a pilot trial indicated that baricitinib ( ) improved clinical parameters (e.g., cough and dyspnea) in a small group of patients. c ( ) toremifene ( ) ( ) and mercaptopurine ( ) ] and angiotensin receptor blockers [irbesartan ( ) ]. d ( ) a: cepharanthin ( ) has been shown to decrease the expression levels of s-protein in mrc- cells infected with human coronavirus oc (hcov-oc ). ( ) flavonoids have been described as inhibitors of m pro and ligands of the s-protein of sars-cov, which could give insights on possible antiviral activities of eriodictyol ( ), hypericin ( ) and other flavonoids on sars-cov- . ( , ) b: a recent study showed that atazanavir ( ) inhibited m pro in vitro with greater potency than lopinavir ( ). ( ) c: despite the small size ( patients) and open label, non-randomised format, baricitinib ( ) showed potential safety when used in combination with lopinavir ( ) / ritonavir ( ). ( ) as large amounts of data from databases and accelerate the discovery of potential drugs for the covid- pandemic. beck et al. used a natural language processing (nlp)-based approach to estimate the binding affinity of , fda-approved drugs against potential targets of sars-cov- , including cl pro , s protein, rdrp, helicase, endornase, '-to- '-exonuclease and '-o-ribose methyltransferase. ( ) the premise of their approach is analogous to understanding text in different languages, for instance by learning the semantic relationships of words to execute a task, such as predicting the most probable word in a text or the sentiment expressed by it. ( , , ) their model, called molecular transformerdrug target interaction (mt-dti) was trained on billion smiles strings and the fasta sequence of target proteins, which bypass the need for d structures (e.g., x-ray) of protein-target complexes. ( ) the pre-training approach allows the model to be used for other related tasks without the need to train from scratch, which is especially important when not enough data is available, which is the case for sars-cov- inhibitors. in addition, this approach is able to transfer more general features learned by the model, making it extremely flexible to deal with related tasks. ( , ) therefore, the task consisted of training the model to understand the chemical structure of small compounds and protein targets. the authors found that atazanavir ( ), remdesivir ( ) and efavirenz ( ) are potential inhibitors of m pro , while atazanavir ( ) also yielded nanomolar predicted binding affinity for rdrp, helicase, endornase, '-to- '-exonuclease and '-o-ribose methyltransferase. ( ) it is important to highlight that although these drugs were predicted as nanomolar inhibitors, experimental confirmation is essential to validate the computational analysis. researchers at the uk-based company benevolen-tai (https://www.benevolent.com/) analysed medical data from different sources to create a knowledge graph of biomedical information, showing possible drug-target interactions on its nodes and edges (similar to the previously mentioned approach adopted by ge et al.) to explore new strategies for sars-cov- . ( , ) among the drugs identified by mining the hidden information within the graph, there were inhibitors of the p associated protein kinase (aak ), a regulator of viral endocytosis in at alveolar epithelial cells. ( ) inhibitors of aak could then potentially block viral entry into alveolar cells. however, the authors argued that only one of these drugs, baricitinib ( ), a janus kinase inhibitor, has an acceptable safety profile. in addition, the low therapeutic dosing of baricitinib ( ) ( mg or mg daily) makes it a promising drug for repurposing. ( ) baricitinib ( ) will be further discussed below. zhou et al. developed a network-based approach to identify potential repurposable drugs based on the interactions between their human targets and coronavirusassociated proteins. ( ) the premise is that protein targets that are associated with viral infections are part of a subnetwork of the human protein-protein interaction network. the targets of a repurposable drug should be in or close in proximity to this subnetwork. the authors identified many potential targets, such as poly [adp-ribose] polymerase (parp- ), glycogen synthase kinase (gsk- ), and also biological pathways that could be explored, such as nf-kappa b signaling. ( ) the most interesting drugs selected by the authors are from a diverse set, including toremifene ( ) (selective estrogen receptor modulator), sirolimus ( ) (immunosuppressant), mercaptopurine ( ) (antineoplastic) and irbesartan ( ) (antihypertensive). an interesting finding was that the selected molecules and targets have reported links to viral infection and some have been used for treatment of coronavirus-related disease, such as emodin ( ) in sars-cov, and mercaptopurine ( ) and toremifene ( ) in sars-cov and mers-cov. ( ) in addition to identifying repurposable drugs, possible synergistic combinations between them were also suggested by the network, such as sirolimus ( ) and dactinomycin ( ) , and mercaptopurine ( ) and melatonin ( ) . ( ) in vitro and in vivo studies -genome and replication kinetics of sars-cov, mers-cov and sars-cov- viruses are very similar, suggesting that these diseases could be treated by a single drug that modulates the activity of a common target or mechanism among them. ( , ) so far, no clinically available antiviral drugs have been developed for any of these three viruses. ( ) this fact demonstrates that we did not learn a suitable strategy for treatment from these two prior diseases, and we are not yet prepared to deal with this new coronavirus epidemic, regarding therapeutic options. ( ) experimental target validation (e.g., by genetic and proteomic approaches), cellular in vitro and in vivo animal models of sars-cov- infection, are pivotal for the discovery of molecular pathways involved in pathogenesis, supporting the discovery of new drugs. ( , ) sars-cov- genome encodes less than proteins that can be explored for generating new avenues for further research. heterologous viral protein expression could be used in a myriad of structural and functional targetbased biochemical studies. ( ) these assays do not need to be performed in biosafety level laboratory, democratising the early research on searching for a new drug candidate for covid- treatment. ( ) genome-wide expression studies, nucleotide sequences, protein structures and associated sequences are available online providing a foundation for preclinical drug discovery and development research against sars-cov- (available from: https://www.ncbi.nlm.nih.gov/genbank/sars-cov- -seqs/). it is good to point out that many drugs show in vitro effect (e.g., target and/or cellular assays) that may not last in vivo, in animal models, including humans. an ideal animal model of covid- should reflect the clinical signs, viral replication and pathology reported in humans. ( ) in vivo animal infection experiments with sars-cov- require infrastructure of a biosafety level laboratory, restricting scientific research to well-equipped research groups. animal models are indispensable, because they could identify toxic hits or molecules that could enhance covid- pathogenicity. table ii lists some compounds that have the potential to be clinically tested and that will be discussed in more details below in this topic. remdesivir ( ) , a nucleotide analog pro-drug developed by gilead sciences inc. to fight ebola, acts on viral replication by inhibiting rna polymerase. ( ) this molecule was also active against sars and mers viruses. ( , , ) wang et al. demonstrated reduced viral copy numbers in cell culture supernatant of vero e cells infected with ncov- betacov/wuhan/wiv / , in the presence of varying concentrations of remdesivir ( ) . ( ) this compound blocked virus infection at lowmicromolar concentration (ec = . μm) and showed a high selectivity index (> ). the authors also disclosed that remdesivir ( ) inhibited virus infection efficiently in human liver cancer huh- cells. chloroquine (cq; ) and its less toxic derivative, hydroxychloroquine (hcq; ) , are used to treat malaria and lupus/rheumatoid arthritis, respectively. both drugs have already been described as possible antivirals. ( , , ) cq ( ) and derivatives probably act by decreasing acidity in endosomes and lysosomes, intervening on glycosylation of cellular virus receptors and modulating host immunological activity. ( , ) studies in cultures of vero e cells have suggested that hcq ( ) can affect the virus sars-cov- in both entry and post entry stages at host cells. ( ) this molecule presented an ec ( ) the authors also performed in vitro physiologically based pharmacokinetic models for both drugs, separately. interestingly, when comparing toxicity in five animal models, mcchesney et al. demonstrated that hcq ( ) is two to three times less toxic than cq ( ) itself. ( ) it is also good to point out that chloroquine ( ) has shown in vitro activity against many different viruses, but no significant beneficial effect on animal models. ( ) the association ritonavir ( )/lopinavir ( ) is used together with other antiretrovirals for the treatment of human immunodeficiency virus since the beginning of the century. ( ) ritonavir ( ) is a potent cyp a inhibitor therefore inhibiting the metabolism of lopinavir ( ), increasing its plasma levels. both are reported as peptidomimetic molecules that inhibit hiv- protease activity in a competitive manner. ( ) the m pro plays a major role in homeostasis of viral polyproteins essential for viral function and replication, being considered a validated drug target. ( ) this combination may be useful against ( ) chloroquine ( ) ( , , ) ritonavir ( ) + lopinavir ( ) lopinavir ( ) nd ( ) nitazoxanide ( ) broad-spectrum antiparasitic and antiviral still needs confirmation . μm . ( ) ivermectin ( ) broad-spectrum antiparasitic still needs confirmation μm nd ( ) teicoplanin ( ) antibiotic for gram-positive bacteria still needs confirmation . μm nd ( ) sars-cov- virus by acting on its main protease, an enzyme essential in processing polyproteins translated from viral rna. ( ) choy et al. demonstrated that it inhibits sars-cov- replication in vero e cells with ec value of . μm. ( ) these results corroborate with the study of de wilde et al. that demonstrated antiviral in vitro effect of lopinavir ( ), but not ritonavir ( ), against sars-cov, mers-cov, and hcov- e, with mean ec varying from . to . μm. ( ) other compounds were also tested against sars-cov- in vitro. nitazoxanide ( ), a broad-spectrum antiparasitic and antiviral, inhibits a low-micromolar range with an ec of . μm but has a selective index of only . . ( ) ivermectin ( ), another broad spectrum antiparasitic agent also demonstrated in vitro anti-sars-cov- activity. its ec was determined to be μm when added to vero-hslam cells h post-infection and measuring viral rna at h post-infection. ( ) teicoplanin ( ) , an antibiotic used against gram-positive bacterial infections, was found to be active in vitro against sars-cov- with an ec value of . μm, but in vivo efficacy still needs to be determined. ( ) a robust preclinical drug discovery pipeline comprising in vitro, and in vivo models of sars-cov- infection is particularly important to identify new antivirals for human covid- treatment. this pipeline can munition clinical studies with molecules that have a higher chance to become a drug, decreasing attrition rates. clinical studies -over clinical trials have been unveiled, until the publication of this manuscript, focusing on covid- treatment interventions (available from: https://clinicaltrials.gov/ct /results?cond=covid- ). with a growing number of patients suffering from acute severe respiratory symptoms and hospital capacities reaching its limit, therapeutic options are urgently essential to avoid human mass deaths. drug repurposing approaches of approved (with safe and effectiveness proven) and unapproved molecules, that were promising in pre-clinical and early stages of clinical studies of sars and mers, are in vogue. with this premise, world health organization (who) organised a simplified dynamic platform comparing the effectiveness of treatment strategies around the globe. this clinical trial design, called solidarity, can shrink by % the time of clinical studies, compared with the "gold standard" double-blind, placebo-controlled trials. this strategy could overcome the uncertainty of multiple small trials that do not produce a solid base necessary to establish the relative success of arising probable treatments. on the other hand, this shorter time required reflects the compassionate characteristic of the studies, that cannot rule out placebo effects and patient severe adverse effects, including death. ( ) currently, who is focusing on four most promising therapies: remdesivir ( ); cq ( ) or hcq ( ) ; ritonavir ( )/lopinavir ( ); ritonavir ( ) /lopinavir ( ) plus interferon-beta, an immune response modulator. ( ) a selection of studies with these drugs, alone or in combination, are summarised in table iii. for other studies, the reader can refer to kupferschmidt and cohen, which summarised several clinical studies on drug repurposing for covid- reported so far. ( ) it is worth noting that many clinical studies are beginning to be carried out in different parts of the world and their number is increasing rapidly day by day. remdesivir ( ), a nucleoside analogue that acts by inhibiting rna synthesis, is already in clinical studies for treatment of covid- . ( ) this prodrug was already investigated in clinical trial testing for ebola virus with no effect but showed efficiency against different types of coronaviruses in cell culture and animal models. ( , ) from the drugs in the solidarity trial, remdesivir ( ) has the best potential to be used in clinics, having a low toxicity profile to humans. ( ) this molecule was used compassionately in the first covid- patient diagnosed in the united states by intravenous treatment and improved patient clinical condition. ( ) grein et al. reported a study in a cohort of patients hospitalised for severe covid- who were treated with compassionateuse remdesivir ( ) , clinical improvement was observed in % of the patients ( of ). ( ) there are two clinical trials at phase iii, being designed to evaluate the efficacy and safety of parenteral remdesivir ( ) in mild/moderate (nct ) and severe (nct ) hospitalised adults with covid- . ( ) these trials are evaluating intravenous remdesivir ( ) at a dose of mg on the first day plus mg once daily, for an additional consecutive days. the randomised, double-blind, placebocontrolled, multicentre trials were recently suspended (nct ) or terminated (nct ) because no eligible patients for enrollment were found, due pandemic control in china. ( ) the study nct showed that remdesivir ( ) was not associated with statistically significant clinical benefits in severe forms of covid- . however, the authors observed a numerical reduction in time to clinical improvement in those treated earlier, but still requires confirmation. another study, performed by beigel et al. (with the same dosage and treatment period than the previous works cited above) enrolled patients with lower respiratory tract infection, demonstrated that individuals that were treated with remdesivir ( ) had a shorter time to recovery than placebo group ( days compared with days). ( ) even with a small scientific ballast, due to limited information about safety and effectiveness of using remdesivir ( ), u.s. food and drug administration (fda) approved its emergency use on severe covid- . ( ) the approval was based on review of the topline data on two studies that are recruiting patients, (nct ) and (nct ). other clinical studies are being conducted to address the effect of remdesivir ( ) on co-vid- treatment (available from: https://clinicaltrials. gov/ct /results?cond=covid- &term=remdesivir&cn try=&state=&city=&dist=). other phase iii clinical trials evaluated for co-vid- treatment included cq ( )/hcq ( ) . these studies were endorsed by promising in vitro data that suggested an impairment of viral replication by acting on endosome-mediated viral entry or later phases of viral replication. ( , ) both molecules have a well-studied toxicity profile being used for more than half-century as antimalarials, but in some cases can induce heart side ( ) was not associated with clinical benefits in severe forms. however, an observed numerical reduction in time to clinical improvement in patients treated earlier, but still requires confirmation ( ) ; nct (terminated by epidemic control in china with no eligible patients) remdesivir ( ) ( ) ; nct chloroquine ( ) multicentre > patients mg per day, for days compilation of various clinical studies that are in course, authors conclude that the dosage used could be sufficient ( ) phase iib double-blind, randomised patients mg twice daily for days and mg twice on the first day, once daily for more days higher dosage of chloroquine ( ) has toxic effects and increased lethality, with any clinical benefit ( ) hydroxychloroquine ( ) non-randomised, non-double-blind, non-placebo-controlled patients mg of hydroxychloroquine ( ) daily for days hydroxychloroquine ( ) significantly reduces viral load despite small sample size ( ) ritonavir ( effects. ( ) these drugs have already been examined against at least five other viruses with good initial in vitro results, but without significant effects in animal models and humans. ( ) clinical trials to measure the effectiveness of cq ( ) at mg of chloroquine diphosphate (corresponding to mg of chloroquine ( ) base) per day, for ten consecutive days on the treatment of covid- were performed. ( ) based on results for a very limited number of patients ( ) , it was concluded that cq ( ) presented apparent efficacy and acceptable safety against cov-id- associated pneumonia. borba et al. performed a double-blind, randomised clinical trial designed to assess the safety of cq ( ) in two dosages: mg twice daily for days with an initial dose of mg twice daily on the first day, decreasing to once daily for four more days. ( ) the study suggests that the higher dosage should not be recommended for critically ill patients with covid- because of its potential side effects. it is important to note that both doses studied were above the brazilian recommended dose. a controversial study performed by gautret et al. with a small sample size demonstrated that hcq ( ) treatment daily at mg significantly decreases viral load in covid- patients and its effect is reinforced by azithromycin (az; ) combination. ( ) until this moment there is clinical robustness to support az ( ) therapy in covid- . ( ) mehra et al. performed a registry analysis of , hospitalised patients regarding the use of cq ( ) or hcq ( , combined or not with a macrolide) for treatment of covid- . ( ) the observational study verified that with covid- requiring hospitalisation a regimen containing cq ( ) or hcq ( ) had no evidence of benefit, but instead was associated with an increase in the risk of ventricular arrhythmias and a greater hazard for in-hospital death with covid- . these findings suggest that these drug regimens should not be used outside of clinical trials and urgent confirmation from randomised clinical trials is needed. after the publication, the paper was retracted, which influenced who to return the studies on cq ( ) and hcq ( ) . ( ) recently, on june , who stopped the hcq ( ) arm of the solidarity trials again based on the recovery trials (available from: https://www.recoverytrial.net/). in this study patients were randomised to hcq ( ) and compared with patients randomised to usual care alone. there was no significant difference in any of the parameters evaluated: the primary endpoint of -day mortality, hazard ratio and hospital stay duration or other issues. other study reported that hcq ( ) did not prevent covid- when used as postexposure prophylaxis within days after moderate or high-risk exposure. ( ) robust data from well-designed control randomised clinical trials with large number of patients is still expected for concluding on the efficacy of hydroxychloroquine ( ) . ritonavir ( ) and lopinavir ( ) were developed to target hiv- protease and postulated to inhibit the -chymotrypsin-like protease of sars and mers, being associated with improved clinical outcomes. ( ) the combination has beneficial effects in marmoset monkeys infected with the mers-cov virus. ( ) ( )/( ) may have clinical efficacy against sars-cov- , as seen in the response against sars-cov. ( ) in vitro sensitivity and satisfactory response in a preliminary non-randomised clinical trial of sars-cov to ( )/( ) have already been reported, encouraging its testing in sars-cov- . ( ) a total of patients confirmed severe sars-cov- infection were randomly designated to be given either ( )/( ) ( mg/ mg) twice a day for consecutive days plus standard care or standard care alone. ( ) this first trial was not promising, no benefit was observed on clinical improvement, mortality and viral loads on severe covid- patients. other clinical trials are being carried out and should decide whether these drugs are useful for covid- treatment or not. ritonavir ( )/lopinavir ( ) plus interferon-beta, a cytokine involved in inflammatory modulation, is the last bet of the solidarity megatrial. sheahan et al. demonstrated that this combination improves pulmonary function but does not reduce virus replication or severe lung pathology in mice model of mers-cov. ( ) this combination also showed promising results in mers-cov infection in a nonhuman primate model. ( ) the study (nct ) administered ritonavir ( )/lopinavir ( ) mg + mg/ml twice daily for days and interferon beta- b . mg subcutaneous, every alternate day for days. another study (nct ) will perform a randomised trial to verify the effects of interferon beta a, compared to interferon beta b and the base therapeutic treatment in moderate to severe covid- . currently, there is a lot of research around the world examining drugs that can be used for the treatment of covid- . unfortunately, until this moment no therapeutic options are promptly effectively available. most of the studies have a small number of patients with variable dose and/or duration, fact that hinders a comparison. to successfully undertake the covid- pandemic with new medicines, synchronised clinical trials with randomised, double-blind, placebo-controlled are still needed. infection prevention by social distancing and supportive medical care, are the only strategies to deal with this disease until this moment. the goal of this session is to consider potential obstacles for effective adoption of a repurposed drug to widespread treatment of covid- . both patent protection and scalability of manufacturing processes are key aspects to consider while choosing the best therapeutic option to adopt when dealing with a pandemic. a patent can be defined as a government license that confers the owner the exclusivity over a new invention or medication. with the patent, the inventor is able to exclude others from making and selling the invention for a determined period of time. when it comes to medication, the market exclusivity granted by the patent generates enormous economic profit for the patent holder, once the company will have the monopoly over the product for around years. once the patent term finishes, generic companies can start producing and selling the drug, increasing the market competition over the product. a strategy adopted by some companies is to maximise the term of patent of successful products, extending market exclusivity, for instance, increasing economic rewards with the invention. in order to extend patent terms, the companies can apply for new formulations, new routes of administration, new uses of the drug among other strategies. ( , ) this is very important when considering drug repurposing, once those strategies can make it harder to patent a new method of use for the drug, compromising the legal rights of the new repurposed indication. as a consequence, the expected profit associated with repurposing can be severely affected. ( ) remdesivir ( ) a good example of maximising the term of patent is the drug remdesivir ( ) , which was developed by the american pharmaceutical company gilead sciences, inc. in . nowadays, ( ) has shown promising results as a suitable repurposed drug in the treatment of the covid- disease. indeed, there are many ongoing clinical trials with this drug in cov-id- patients. ( , ) due to the good results concerning remdesivir ( ) , in january , the wuhan institute of virology applied for a patent on gilead's remdesivir ( ) for the treatment of coronavirus, in an attempt to protect china's economic and medical interests. however, remdesivir ( ) has already patents related to the coronavirus filed by gilead science in countries around the globe since . in fact, the company has a robust patent portfolio that includes structures of remdesivir-related compounds (family ), the manufacturing method of the drug (family ), the use of remdesivir ( ) in treating coronaviridae infection (family ), among other patents that claims the use of ( ) for the treatment of a series of viral infections (families and ) (fig. ) . ( ) practically, this means that if the ( ) is proved to be efficient for the treatment of covid- , gilead science is the only pharmaceutical company owing the market exclusivity of the drug, at least until . in fact, the company has already started to produce larger amounts of the drug and they already made improvements to optimise the drug manufacturing timeline. now, the company can produce the drug in months, half the time that they used to need to get the final product. in addition, the company has been donating remdesivir ( ) to ongoing clinical trials, a gesture that will not only help the trial patients, but gilead itself. ( ) baricitinib ( ) -in , a patent assigned to incyte corporation disclosed the preparation of several active compounds as jak inhibitors, including baricitinib ( ). in the same year, lilly and incyte made an agreement allowing lilly co. to manufacture and commercialise the medicine worldwide, making it lilly co. the only supplier around the globe. ( ) there are two patents that protect the drug, one concerning its synthetic pathway and another disclosing the use of ( ) in the treatment of rheumatoid arthritis. both of them will expire in nine years, which could open opportunities for generic drug companies to produce baricitinib ( ) . nowadays, ( ) has been investigated as a drug that could be used in the treatment of covid patients, since its anti-inflammatory activity could minimise inflammatory compli-cations in covid patients. according to the platform clinicaltrials.gov, there are ongoing clinical trials to evaluate the efficacy and safety of baricitinib ( ) in the treatment of covid . ( ) if the drug succeeds, there will be a need for larger and faster production and distribution of the medicine worldwide. there are two patents disclosing a synthetic method for the preparation of ( ). the first one is from and owned by incyte co. and the latest is from , by lilly co. ( , ) the main difference between them is how the central pyrazole ring is installed in the molecule. in the patent from , baricitinib ( ) is obtained by a convergent synthesis. the synthetic pathway starts with the protection in position of -chloro- h-pyrrolo[ , d]pyrimidine ( ) using -(trimethylsilyl)ethoxymethyl chloride ( ) , affording intermediate ( ) . next step is a suzuki-miyaura reaction, coupling the fused ring system to a -pyrazoleboronic acid pinacol ester ( ), giving key intermediate ( ) . parallelly, a couple of steps starting from -(chloromethyl)oxirane ( ) lead to -boc- -azetidinone ( ) that reacts with diethyl cyanomethylphosphonate ( ), affording key intermediate ( ) . next, the key intermediates ( ) and ( ) react in the presence of dbu, giving ( ) . then, steps involving hydrolysis of the boc, sulfonation and pyrrolopyrimidine deprotection afford ( ) with an overall yield of % (fig. ) . ( ) the synthetic pathway described in the patent from has only six steps in a linear approach, as a consequence, the product is obtained in a higher overall yield when compared to the synthetic pathway discussed above. intermediate ( ) is obtained from azetidine- -ol ( ) in a couple of steps, including a sulfonation, an oxidation and the installment of the cyanomethylene moiety (fig. ). furthermore, it is not necessary to protect any position in this sequence. additionally, the oxidation step can be performed under flow conditions. then, intermediate ( ) is reacted with ester ( ), affording ( ) . a suzuki-miyaura reaction involving -boc- -chloro- hpyrrolo [ , -d] pyrimidine ( ) is applied, allowing the formation of the bound between the azetidinylpyrazole group and the pyrrolo[ , -d]pyrimidine system. last step is a hydrolysis of the boc, affording baricitinib ( ) with an overall yield of % (fig. ). ( ) off-patent drugs -fortunately, a number of drugs that have been tested as possible agents in the covid- treatment are off-patent, meaning that generic drug companies already manufacture and commercialise the medicine, making it easier for drug repurposing. ( ) the off-patent drugs that have been tested in covid- clinical trials include cq ( ) , hcq ( ), ritonavir ( ) and lopinavir ( ) . ( , ) a recent research article published by hill and collaborators estimated the minimum cost of production associated with these drugs, showing that all the treatments under evaluation in current clinical trials are cheap to manufacture. however, list prices can be over times higher than the costs to produce the drug. ( ) if any of those drugs become approved for the treatment of covid- , its demand will increase dramatically. therefore, it is important to consider the challenges associated with scaling up the production to meet the demand. for instance, there are few regulatory approved production facilities, and drug manufacturers rely on low-cost suppliers of raw materials in india and china, which might be scarce during a pandemic. consequently, it would be difficult to ensure short term global availability of the treatment, since the production depends on different countries. ( ) those conclusions are very helpful in showing the importance of optimising the way of manufacturing the candidate drugs presented above. for this reason, recent and optimised synthetic pathways found in patents and articles for some ritonavir ( ) and lopinavir ( ) are discussed below, as cq ( ) and hcq ( ) have now been abandoned. finally, we will briefly discuss the ease of manufacturing in large scale each of the drugs we had the synthesis reviewed here. ritonavir ( ) -the first disclosure of ritonavir ( ) is presented in a patent from , assigned to abbott laboratories. even though the patent consists of a range of markush structures, among which is found ( ), its synthesis is not presented. ( ) the synthetic pathway to obtain ( ) was disclosed for the first time in a second-generation patent in , by the same company. ( , ) some drawbacks related to the synthesis presented by abbott include the employment of expensive condensing agents, as -ethyl- -( -dimethylaminopropyl) carbodiimide (edc) and poor reaction yields on the first steps of the synthetic pathway, making this strategy too expensive and not suitable for scale-up production. considering the above deficiencies, a recent chinese patent concerning the synthesis of ritonavir ( ) has been released. the synthetic pathway described in the patent starts with a nucleophilic addition-elimination reaction between the starting material ( -isopropylthiazol- -yl)-nitro-methylamine ( ) and n-[( , , -trichloroethoxy)carbonyl]-l-valine ( ), generating intermediate ( ) . the intermediate is mixed with p-toluenesulfonyl chloride and triethylamine to activate the acid function, followed by the addition of reagent ( ) in a one-pot procedure. the condensation allows the formation of intermediate ( ) , which is submitted to acidic conditions for a boc deprotection, followed by another nucleophilic additionelimination reaction with reagent ( ), thus obtaining the final product ritonavir ( ) (fig. ) . ( ) when comparing both patents, it is possible to highlight important advantages present in the latest one, including the use of cheap and easily available reagents, for instance, the use of ptoluenesulfonyl chloride as an amide condensing agent, the synthetic pathway has a high yield ( % overall), low cost and it is ease to scale-up the production. lopinavir ( ) -the first synthetic methods related to the synthesis of lopinavir ( ) are present in a patent from owned by abbott laboratories. ( ) ( ) has chiral centres, and the synthetic strategies reported by abbott are similar, involving the synthesis of a key intermediate amino alcohol unit, that is then connected to the appropriated side chains. some drawbacks that can be pointed out in the synthesis include the use of -ethyl- -( -dimethylaminopropyl)carbodiimide (edc) as a condensing agent and the weak base -hydroxybenzotriazole, both expensive reagents, making the synthesis not suitable from the viewpoint of cost and industrial ap-plication. in addition, one of the methods described by abbott includes the synthesis of an acid chloride as an intermediate, which is very unstable, and it can be easily decomposed by humidity, making the synthesis not suitable for industrial production. the most recent patent related to the synthesis of lopinavir ( ) is dated and it is from the chinese company shanghai desano pharmaceuticals co. ltd. ( ) the patent is described as technical, with the aim of improving the synthesis of ( ) presented in the patent from , by abbott laboratories. the patent describes the synthesis of lopinavir ( ) by a one-pot procedure starting with the formation of an acid chloride from ( s)-( tetrahydropyramid- -one)- -methylbutanoic acid ( ) followed by addition of a weak base and the reactant ( ) to the reaction system. the amine function of ( ) allows a nucleophilic addition-elimination reaction to occur, affording ( ) after treatment with nahco (fig. ). therefore, it is possible to synthesise lopinavir ( ) in high yield ( %) in an optimised method, which involves a few reagents, mild conditions and is performed in a one-pot procedure. synthetic scalability and cost-effectiveness -the synthetic pathways described above show how much improvement has been done concerning the preparation of these drugs in a more cost-effective way. furthermore, when comparing the drugs discussed above, it is worth pointing out which one could be the easiest one to produce when analysing the factors that influence the final cost of a synthetic pathway, such as chiral centres, cost of starting materials, number of steps and overall yield. regarding chiral centres, ritonavir ( ) and lopinavir ( ) have four chiral centres each, and they are not sold as racemate mixtures. moreover, to synthesise those compounds the chiral centres do not come from natural molecules, but have to be synthetically installed, which makes the chiral starting materials more expensive and enantiomeric excess has to be accessed more carefully at the end of the synthetic pathway. as a consequence, it can take longer and more expensive to obtain the final product. on the other hand, baricitinib ( ) does not have any chiral centres, making the synthetic pathway easier and cheaper to perform. concerning the number of steps and overall yield, baricitinib ( ) has the highest number of steps and lower overall yield when compared to ritonavir ( ) and lopinavir ( ) . however, one of the steps in the synthesis of baricitinib ( ) can be performed under flow conditions, which is very interesting from the industrial point of view. therefore, when taking into account the most recent synthetic pathways proposed for these drugs, the synthesis of baricitinib ( ) is the most cost-effective of the three of them. in conclusion -sars-cov- infection is a lifethreatening disease with such a high transmission rate that can surpass even the most well-structured health systems in developed high-income countries. while vaccines are the ideal solution for preventing the spread of infectious diseases like covid- their development cycle have intrinsic challenges and safety checking steps that require several months or years to complete their development. a similar situation is found for developing new drugs for covid- (as with any other disease), because of the long process involving discovery, validation and safety evaluation of new chemical entities for use in human health. in this scenario, repositioning drugs already in clinical use for treatment of covid- is an important shortcut, as we have discussed. data are accumulating about the molecular pathology of covid- as well as structural information on the viral and host proteins involved in the infection mechanism. this knowledge is essential for allowing researchers to have new insights on approved or investigational drugs that can be repurposed to treat sars-cov- infection. as reviewed here, over targets distributed amongst distinct steps of the sars-cov- replication cycle or host cell mediators are currently being investigated. here, we highlighted several up-to-date examples of potential repurposable drug candidates proposed from either computational approaches or experimental trials (or their combination) at different levels of validation and stages of development. noteworthy, ai methods hold a great promise in finding occult links between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. remdesivir ( ) , cq ( )/hcq ( ) (alone or in association with other drugs) and the association lopinavir ( )/ritonavir ( ) have been the focus of most clinical studies. so far, results have been mostly disappointing with these trials, stressing the importance of carefully performed studies in patients to attest that biological activities observed in vitro actually be translated into clinical efficacy and safety. at the moment, even with limited information about safety and effectiveness, remdesivir is the only drug approved by the fda for emergency use on severe covid- . as discussed here, a major concern with drugs for effectively fighting the pandemic is the drug's industrial production cost and its impact on final treatment cost. gilead, remdesivir's owner company, has recently signed non-exclusive voluntary licensing agreements with five generic pharmaceutical manufacturers to produce remdesivir for distribution in countries, nearly all low-income and lower-middle income countries. hopefully, based on the constantly growing scientific knowledge summarised in this review, other treatment options will be revealed soon enough to help stop the havoc caused by the covid- pandemic. coronavirus disease (covid- ) situation report persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of cov-id- aerodynamic analysis of sars-cov- in two wuhan hospitals sars-cov- in wastewater: potential health risk, but also data source first confirmed detection of sars-cov- in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid- in the community fiocruz divulga estudo sobre a presença do novo coronavírus em esgotos sanitários the effect of human mobility and control measures on the covid- epidemic in china projecting the transmission dynamics of sars-cov- through the postpandemic period genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding coronavirus reverse genetic systems: infectious clones and replicons discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals. emerg microbes infect cultivation of viruses from a high proportion of patients with colds isolation and characterization of viruses related to the sars coronavirus from animals in southern china adaptive evolution of bat dipeptidyl peptidase (dpp ): implications for the origin and emergence of middle east respiratory syndrome coronavirus roles of host gene and non-coding rna expression in virus infection structure, function, and evolution of coronavirus spike proteins the mers-cov receptor dpp as a candidate binding target of the sars-cov- spike. iscience receptor recognition mechanisms of coronaviruses: a decade of structural studies coronavirus envelope protein: current knowledge analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras the proximal origin of sars-cov- phylogenetic network analysis of sars-cov- genomes a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china real-time tracking of self-reported symptoms to predict potential covid- clinical, laboratory and imaging features of covid- : a systematic review and meta-analysis coronavirus infections and immune responses targeting potential drivers of co-vid- : neutrophil extracellular traps similarity in case fatality rates (cfr) of covid- /sars-cov- in italy and china the importance of hypertension as a risk factor for severe illness and mortality in covid- clinical characteristics of fatal and recovered cases of coronavirus disease in wuhan, china: a retrospective study diabetes is a risk factor for the progression and prognosis of covid- older people and covid- : isolation, risk and ageism asymptomatic carriers of covid- as a concern for disease prevention and control: more testing, more follow-up drug repositioning: identifying and developing new uses for existing drugs drug repurposing from the perspective of pharmaceutical companies drug repurposing: progress, challenges and recommendations emerging virus diseases: can we ever expect the unexpected? drug repurposing for new, efficient, broad spectrum antivirals deployment of convalescent plasma for the prevention and treatment of covid- therapeutic options for the novel coronavirus ( -ncov) race to find covid- treatments accelerates coronavirus sars-cov- disease covid- : infection, prevention and clinical advances of the prospective chemical drug therapeutics: a review on coronavirus disease covid- , epidemiology, prevention, and anticipated therapeutic advances a novel coronavirus from patients with pneumonia in china isolation and rapid sharing of the novel coronavirus (sars-cov- ) from the first patient diagnosed with covid- in australia covid- infection: origin, transmission, and characteristics of human coronaviruses targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid- emerging coronaviruses: genome structure, replication, and pathogenesis genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan networkbased drug repurposing for novel coronavirus -ncov/sars-cov- the architecture of sars-cov- transcriptome a sars-cov- protein interaction map reveals targets for drug repurposing genomic characterization of a novel sars-cov- severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response functional studies of the coronavirus nonstructural proteins coronaviruses and the associated potential therapeutics for the viral infections coronavirus membrane fusion mechanism offers a potential target for antiviral development the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade the coronavirus e protein: assembly and beyond from sars and mers covs to sars-cov- : moving toward more biased codon usage in viral structural and nonstructural genes pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid- characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor pharmacologic treatments for coronavirus disease novel coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment structure of mpro from sars-cov- and discovery of its inhibitors merops : the database of proteolytic enzymes, their substrates and inhibitors crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors pharmacophores and biological activities of severe acute respiratory syndrome viral protease inhibitors. expert opin ther pat structural basis for the inhibition of sars-cov- main protease by antineoplastic drug carmofur structural elucidation of sars-cov- vital proteins: computational methods reveal potential drug candidates against main protease, nsp polymerase and nsp helicase rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds insights into the inhibitory potential of selective phytochemicals against mpro of -ncov: a computer-aided study discovery of potential multi-target-directed ligands by targeting host-specific sars-cov- structurally conserved main protease potential inhibitors against -ncov coronavirus m protease from clinically approved medicines gc- , and calpain inhibitors ii, xii inhibit sars-cov- viral replication by targeting the viral main protease glecaprevir and maraviroc are high-affinity inhibitors of sars-cov- main protease: possible implication in covid- therapy fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study computational insights into tetracyclines as inhibitors against sars-cov- mpro via combinatorial molecular simulation calculations nsp of coronaviruses: structures and functions of a large multi-domain protein the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds structural basis for the ubiquitin-linkage specificity and deis-gylating activity of sars-cov papain-like protease evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus disulfiram can inhibit mers and sars coronavirus papainlike proteases via different modes analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods potential treatments for covid- ; a narrative literature review repurposing of fda-approved antivirals, antibiotics, anthelmintics, antioxidants, and cell protectives against sars-cov- papain-like protease structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites structure of the rna-dependent rna polymerase from covid- virus structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus with high potency favipiravir: pharmacokinetics and concerns about clinical trials for -ncov infection anti-hcv, nucleotide inhibitors, repurposing against covid- differential inhibitory activities and stabilisation of dna aptamers against the sars coronavirus helicase development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp upon atp hydrolysis identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov- ) through a drug-target interaction deep learning model state-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in sars-cov- shahzad-ul-hussan s. identification of potential inhibitors of three key enzymes of sars-cov using computational approach sars-coronavirus- nsp possesses ntpase and rna helicase activities that can be inhibited by bismuth salts crystal structure and functional analysis of the sars-coronavirus rna cap ′-o-methyltransferase nsp /nsp complex functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and ′-o-ribose methyltransferase coronavirus nsp , a critical co-factor for activation of multiple replicative enzymes yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n -methyltransferase synthesis of adenine dinucleosides sam analogs as specific inhibitors of sars-cov nsp rna cap guanine-n -methyltransferase singh sk. structure-based virtual screening and molecular dynamics simulation of sars-cov- guanine-n methyltransferase (nsp ) for identifying antiviral inhibitors against covid- virtual screening, adme/t, and binding free energy analysis of anti-viral, anti-protease, and anti-infectious compounds against nsp / nsp methyltransferase and main protease of sars cov- targeting sars-cov- non-structural protein : a virtual drug repurposing study ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence a unique protease cleavage site predicted in the spike protein of the novel pneumonia coronavirus ( -ncov) potentially related to viral transmissibility structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection tmprss and covid- : serendipity or opportunity for intervention? protein-protein interactions of viroporins in coronaviruses and paramyxoviruses: new targets for antivirals? viruses the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel structure and inhibition of the sars coronavirus envelope protein ion channel. baric rs severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus : biology and therapeutic options sars-cov- e protein is a potential ion channel that can be inhibited by gliclazide and memantine angiotensin-( - ) and angiotensin-( - ): function in cardiac and vascular remodelling covid- pandemic, coronaviruses, and diabetes mellitus angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target sti- , a potent anti-sars-cov- antibody, demonstrates ability to completely inhibit in vitro virus infection in preclinical studies new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? recent research advances in renin-angiotensin-aldosterone system receptors covid- and the cardiovascular system reply to: 'interaction between raas inhibitors and ace in the context of covid- ' angiotensin receptor blockers as tentative sars-cov- therapeutics renin-angiotensin-aldosterone system inhibitors in patients with covid- sars-cov : should inhibitors of the renin-angiotensin system be withdrawn in patients with covid- ? tmprss : a potential target for treatment of influenza virus and coronavirus infections nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- computeraided screening for potential tmprss inhibitors: a combination of pharmacophore modeling, molecular docking and molecular dynamics simulation approaches withanone and withaferin-a are predicted to interact with transmembrane protease serine (tmprss ) and block entry of sars-cov- into cells mechanisms of clathrin-mediated endocytosis the endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration cathepsin l-selective inhibitors: a potentially promising treatment for covid- patients interleukin- family cytokines cytokines and cytokine receptors covid- : consider cytokine storm syndromes and immunosuppression covid- : consider il- receptor antagonist for the therapy of cytokine storm syndrome in sars-cov- infected patients the carbohydrate-active enzymes database (cazy) in influenza virus neuraminidase structure and functions coronavirus infections and type diabetesshared pathways with therapeutic implications dpp inhibition: preventing sars-cov- infection and/or progression of covid- ? covid- and diabetes: can dpp inhibition play a role? emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd dipeptidyl peptidase- (dpp ) inhibition in covid- is dpp inhibition a comrade or adversary in cov-id- infection boosting the arsenal against covid- through computational drug repurposing déjà vu: stimulating open drug discovery for sars-cov- repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface peptide-like and small-molecule inhibitors against covid- baricitinib as potential treatment for -ncov acute respiratory disease de novo design of new chemical entities (nces) for sars-cov- using artificial intelligence deeppurpose: a deep learning library for drug-target interaction prediction and applications to repurposing and screening a data-driven drug repositioning framework discovered a potential therapeutic agent targeting covid- natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc infection of mrc- human lung cells inhibition of sars-cov cl protease by flavonoids small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells atazanavir inhibits sars-cov- replication and pro-inflammatory cytokine production baricitinib therapy in covid- : a pilot study on safety and clinical impact gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading sweetlead: an in silico database of approved drugs, regulated chemicals, and herbal isolates for computer-aided drug discovery drugbank . : a major update to the drugbank database for chembl: towards direct deposition of bioassay data bindingdb: a web-accessible database of experimentally determined proteinligand binding affinities uniprot: the universal protein knowledgebase graph convolutional neural networks for predicting drug-target interactions graph convolutional networks: a comprehensive review bere: an accurate distantly supervised biomedical entity relation extraction network recent trends in deep learning based natural language processing deep learning in natural language generation from images deep learning self-attention based molecule representation for predicting drug-target interaction inductive transfer learning for molecular activity prediction: next-gen qsar models with molpmofit a survey on deep transfer learning the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? covid- , sars and mers: are they closely related? overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and -ncov drug target validation: hitting the target coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- multiparametric assays for accelerating early drug discovery laboratory safety aspects of sars at biosafety level animal models for sars and mers coronaviruses remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro to zika and destroy: an antimalarial drug protects fetuses from zika infection in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro teicoplanin potently blocks the cell entry of -ncov broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection potential antivirals and antiviral strategies against sars coronavirus infections chloroquine and hydroxychloroquine in covid- targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro animal toxicity and pharmacokinetics of hydroxychloroquine sulfate of chloroquine and covid- . antiviral res hiv protease inhibitors: a review of molecular selectivity and toxicity anti-hiv drugs: compounds approved within years after the discovery of hiv clpro inhibitors as a potential therapeutic option for covid- : available evidence and ongoing clinical trials computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid- -preliminary report breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a trial of lopinavir-ritonavir in adults hospitalized with severe covid- a randomized, controlled trial of ebola virus disease therapeutics first case of novel coronavirus in the united states compassionate use of remdesivir for patients with severe covid- arguments in favour of remdesivir for treating sars-cov- infections fda -food and drug administration remdesivir eua letter of authorization effects of chloroquine on viral infections: an old drug against today's diseases cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid- retraction: cardiovascular disease, drug therapy, and mortality in covid- retracted: hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- coronaviruses -drug discovery and therapeutic options treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset molecular dynamic simulations analysis of ritronavir and lopinavir as sars-cov clpro inhibitors role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov patent protection strategies data exclusivity, and the development of new drugs. ssrn [internet coronavirus puts drug repurposing on the fast track research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases coronavirus patents: china files for remdesivir patent, but gilead sciences will still come out a winner fierce pharma gilead turbocharges production of covid- hopeful remdesivir lilly and incyte announce collaboration for development and commercialization of oral anti-inflammatory and autoimmune therapies search results: baricitinib | covid- processes and intermediates for the preparation of { (ethylsulfonyl)- inventors; incyte corporation assignee . azetidine and cyclobutane derivatives as jak inhibitors. united states patent us / a minimum costs to manufacture new treatments for covid- dozens of coronavirus drugs are in development -what happens next? inventors; abbott laboratories assignee . process for the preparations of a substituted , -diamino- -hydroxyhexane discovery of ritonavir, a potent inhibitor of hiv protease with high oral bioavailability and clinical efficacy zhou z, inventors; guizhou yongnuo feite bio pharmacy co ltd assignee . preparation method of ritonavir. chinese patent cn a inventors; yancheng desano pharmaceutical co ltd assignee . method used for preparing lopinavir using one-pot method. chinese patent cn a all authors contributed to writing, reviewing and editing the manuscript. mrs, tcse and rfd contributed equally to this manuscript; fps-jr performed the manuscript conceptualisation and final review. the authors declare no conflict of interest concerning this manuscript. key: cord- -npcuo ld authors: gale, robert peter; lazarus, hillard m. title: liaisons dangereuses? new drugs, physicians and the drug industry date: - - journal: bone marrow transplant doi: . /s - - - sha: doc_id: cord_uid: npcuo ld nan us food and drug administration (fda) has approved record numbers of new cancer drugs in recent years. many are relevant to readers of bone marrow transplan-tation who treat acute myeloid leukaemia (aml) including midostarin, gilteritinib, venetoclax, enasideinib, ivosidenib, glasdegib, acute lymphoblastic leukaemia including inotuzumab ozogamicin, blinatumomab, dasatinib, ponatinib and tisagenlecleucel, chronic myeloid leukaemia including ponatinib and bosutinib, plasma cell myeloma including daratumumab, selinexor, elotuzumab, panobinostat and ixazomib, chronic lymphocytic leukaemia including ibrutinib, idelalisib and ofatumumab, lymphoma including vorinostat, mogamulizumab-kpkc, polatuzumab vedotin-piiq, acabrutinib, brentuximab vedotin, zanubrutinib, axicabtagene ciloleucel and tisagenlecleucel, myeloproliferative neoplasm (mpn)-associated myelofibrosis including fedratinib and acute and chronic graftversus-host disease (gvhd) including ibrutinib and ruxolitinib. this is extraordinarily good news but raises questions whether everyone receiving a haematopoietic cell transplant needs and/or benefits from these new drugs and whether physicians were complicit in promoting their approval and subsequent use. these questions are especially important in developing countries where many of these new drugs are not yet or may ever be approved, available and/or affordable. first, do these new drugs really improve survival? for example, many approvals in aml relevant to haematopoietic cell transplantation were based on data from small, sometimes uncontrolled phase- studies. also, many regular approvals were based on surrogate endpoints such as complete or overall response rate, response duration and/or progression-free survival (pfs) rather than survival. for example, between and the us fda-approved cancer drugs, based on surrogate outcomes including based on overall response rate and based on pfs [ ] . all accelerated approvals and one-half of conventional approvals were based on treatment effects with surrogate outcomes. in a recent review fewer than one-half of drugs approved based on a pfs endpoint were shown to improve survival or quality-of-life (qol) [ ] . similarly, between and the european medicines agency (ema) approved cancer drugs only one-half of which were proved to increase survival or improve qol [ ] . another problem with several new drug approvals is the choice of an inappropriate comparator therapy in randomized phase- or phase- trials. finally, many of these approvals were based on analyses of hazard ratios rather than a preferred mean restricted survival time [ ] . another issue is the magnitude of benefit conferred by new drugs like midostaurin and how they should be used in persons with aml with a flt mutation. in the randomized study of midostaurin percent of subjects received an allotransplant with an important imbalance between the midostaurin and placebo cohorts in subjects transplanted in st complete remission ( % versus %; p = . ) [ ] . elsewhere my colleagues and i explain this p value does not mean there is no difference in transplant rates between the arms [ , ] . rather, this p value means it is very likely transplant rates are different and that the null hypothesis is simply not a good statistical model of these (or more extreme) data. more importantly. although the trial reported a survival benefit of pretransplant midostaurin, many physicians fail to appreciate midostaurin was stopped at the time of transplant and that there are not data supporting giving midostaurin after a transplant regardless of whether it was given pretransplant. our informal survey indicates most transplant physicians continue giving midostaurin despite this lack of supporting data. their motto seems: it can't hurt. (sounds like president trump promoting chloroquine, hydroxychloroquine and bleach for covid- .) but who knows? some data suggest one reason many new drugs gain favour with physicians and patients is because they are promoted by nationally or internationally by disease experts often referred to as key opinion leaders (kols) and by drug companies, often in media advertisements. une liaison dangerous? this may be so in some instances [ ] . however, there are several complex considerations here. kols are frequently principle investigators in the clinical trial(s) resulting in a new drug approval. often, they believe, sometimes strongly or even too strongly, the new drug is an important advance. in psychology this process is known as confirmation bias. having invested several years studying a new drug, often with considerable effort and problems working with ethical committees, clinical research organizations (https://www.ashclinicalnews.org/viewpoints/ editors-corner/contract-research-agonizations/); sometimes known as clinical research aggravations), drug company study managers etc. people want to believe their effort worthwhile. otherwise one would seem to have wasted their time, energy and perhaps destroyed your marriage, missed your children growing up, both or perhaps something even worse (a fling in cancun?). but we need to acknowledge other considerations such as the requirement to publish for academic promotion and advancement. it is far easier to publish a study claiming a new, typically more expensive drug is better than an old, typically cheaper drug than the converse. and there are also some less altruistic considerations. everyone likes to address a large, admiring audience, have your headshot (typically of you years ago) projected on a huge screen, travel abroad, be feted at a reception and dine in le bernadine in new york, the ivy in london or la tour d'argent in paris. this behavior is human nature and we cannot unfairly fault them. generic drug makers are likely to take you and the meeting organizers to nathan's famous hot dogs in coney island for dinner (excellent but expensive; currently $ . usd; for $ . ), no fries or, for europeans, traditional pie 'n' mash with eels at cockney's at portobello road, london w ru (£ ). everyone likes to be liked. in contrast, consider a world where drug companies do not develop new drugs for the diseases we need to treat. first, we would be left with methotrexate and corticosteroids to prevent or treat gvhd. almost all new drugs we use in the past years have come from drug companies, not academia. but there are other far-reaching implications. for example, annual educational meetings such as those of the recent transplantation and cellular therapy (tct) meeting in orlando and the european bone marrow transplant group (ebmt) meeting in frankfurt would have rather than attendees. about onethird of the attendees are drug company employees or physicians sponsored to attend the meeting by drug companies. much of the funding for these annual meetings comes from the drug industry, not registration fees. tct and ebmt charge drug companies substantial monies for exhibition space and for the opportunity to present their new drugs in so-called product theatres. imagine an exhibition space at one of these meetings without drug company booths. it would look like grand central terminal devoid of people with only the central lonely information kiosk. a world without free cappuccino or frozen yogurt, video games, giveaways for which physicians earning >$ , per year are willing to que for min. amazing. blood and marrow trans-plantation is filled with advertisements for new drugs. i doubt many journals would be able to achieve widespread distribution at a reasonable cost to society members without advertising income. likewise, their websites are where you are bombarded by unsolicited advertisements for new transplant-related drugs. and think of the exhibit areas at the tct and ebmt meetings. our point is that although its popular to disparage the drug industry for promoting new drugs with sometimes marginal benefits and high costs, the medical community benefits greatly from the investment of these drug companies. much of our medical education in diverse areas is indirectly supported by the drug industry. our lives would be entirely different if drug companies developing and promoting new drugs ceased to exist. and we would be eating hot dogs at nathan's rather than black cod in miso at nobu. remember, the estimated cost of bringing a new cancer drug to approval is about $ million usd [ ] . these monies come from drug company research and development and need to be returned to stockholders with a profit. have we a solution to these potential liaisons dangereuses likely to work? no. it is unreasonable to expect drug companies not to promote drugs they have developed at great cost. professional societies and publishers are addicted to drug company support. individual investigators fight to participate in clinical trials of interesting drugs. coinvestigators fight for lead authourship on publications of successful drug trials which come with designation as a kol and extensive travel on what are labeled educational symposia. regulatory agencies such as us fda and ema have helped curb inappropriate drug labeling and promotional activities but have limited jurisdiction and are subject to political influences. in the us, fda is required by law to approve any drug determined safe and effective whether or not the new drug is safer or better than a current drug. price is dictated by the drug company, not the fda. cost control (if any) lies with the centers for medicare and medicaid services (cms), payor for americans without medical insurance and for persons > years and with pharmacy benefit managers who affect discounts, not price. moreover, us physicians can prescribe any fda-approved drug in settings different from those for which the drug was approved, a practice termed off-label use. again, the only control of off-label use are payors. controls of drug use and pricing are different in some other countries such as the uk where approval based on a determination of safety and efficacy is done by one health authority (medicines and health care products regulatory agency) and appropriateness of use and price are determined by another agency (national institute for health and care excellence (nice). can we correct the threat of liaisons dangereuses? there are some rays of hope. in the us cms and the state children's insurance program are required to enforce the physicians payments sunshine act which requires drug companies to collect and track all financial relationships with physicians and teaching hospitals. the goal is to increase transparency of financial relationships between health care providers and drug companies and uncover potential conflicts of interest. the bill also allows states to enact additional requirements which several states have done. another step is that some universities have limited or completely restricted participation of their faculty in promotional activities. however, data on effectiveness of this ban is far from promising [ ] . for example, in a cohort of academic oncologists at us public medical schools two-thirds received payments from the drug industry in accounting for > % of their annual salary. recently, there has been considerable controversy over participation in china's thousand talents plan under which us academic physicians have established relationships with chinese universities and, in some instances, drug companies receiving received substantial research and personal monies. the fbi is currently investigating these relationships which have led to the firing of several prominent physician scientists. none of these developments is surprising. for example, us medical schools have been selling themselves for a price. for a mere $ million david geffin was able to have the ucla school of medicine, a state institution, named for him. and ucla medical center is named for ex-president ronald reagan after his friends donated $ million. (full disclosure: rpgs wife is in the case family of case western reserve university.) it would seem the california board of regents would be on shaky moral ground trying to control physicians' drug prescribing activities. the truth is physicians and the drug industry have a symbiotic relationship. physicians like to complain but nobody really wants change. but there is hope because what ultimately saves us from these liaisons dangereuses is the moral compass of most physicians who want to do what's best for their patients. our bottom line is some of these new drugs are important advances such as cyclosporine, mycophenolate mofetil and several new antibiotics and anti-fungal drugs. however, the impact of many new approved drugs on transplant outcomes is mostly modest and not everyone needs them. health care resources need to be used to reduce infant mortality, increase childhood vaccination rates, eradicate malaria etc. and most recently to combat the sars-cov- pandemic and treat covid- ; these new drugs are unlikely to be the most effective investment of health care resources, especially in developing countries. despite the above comments, criticisms and admonitions we are standing by to renounce all of these potential liaisons dangereuses if a drug company offers to sponsor us to lecture on a new drug for transplant recipients in milano with dinner at cracco thrown in buon appetito. surrogate endpoints in oncology: when are they acceptable for regulatory and clinical decisions, and are they currently overused cancer drugs approved on the basis of a surrogate end point and subsequent overall survival. an analysis of years of us food and drug administration approvals availability of evidence of benefits on overall survival and quality of life of cancer drugs approved by european medicines agency: retrospective cohort study of drug approvals - uses and limitations of the restricted mean survival time: illustrative examples from cardiovascular outcomes and mortality trials in type diabetes midostaurin plus chemotherapy for acute myeloid leukemia with a flt mutation what is the p-value anyway? what is the (p-) value of the pvalue? following the script: how drug reps make friends and influence doctors research and development spending to bring a single cancer drug to market and revenues after approval comparison of industry payments in with annual salary in a cohort of academic oncologists acknowledgements rpg acknowledges support from the national institute of health research (nihr) biomedical research centre funding scheme. key: cord- - y gou v authors: buckles, thomas c.; ziemba, brian p.; djukovic, danijel; falke, joseph j. title: rapid exposure of macrophages to drugs resolves four classes of effects on the leading edge sensory pseudopod: non-perturbing, adaptive, disruptive, and activating date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: y gou v leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. this study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized raw macrophages. the drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (pdgf, gö , wortmannin). notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. this finding is consistent with the well established clinical safety of these drugs. however, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature pi( , , )p( ) lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (gö , wortmannin); and (iv) activating exposures yielded pseudopod expansion (pdgf). the novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures. leukocytes, including macrophages and neutrophils, possess a sophisticated chemosensory system that controls cellular migration up primary attractant gradients to sites of infection, injury, or tumor formation during the innate immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . at the target site, recruited leukocytes and other cells can release secondary attractants that recruit additional leukocytes, but excessive secondary attractant signaling and recruitment may lead to local inflammation and, in extreme cases, toxic effects (reviewed in [ ] [ ] [ ] [ ] [ ] [ ] ). the chemosensory pathway that directs leukocyte migration up primary and secondary attractant gradients is localized on the broad sensory pseudopod at the leading edge of the polarized cell, where pathway components assemble on the cytoplasmic leaflet of the plasma membrane (reviewed in [ - , , - ] ). the leukocyte chemosensory pathway is highly specialized, conferring unique properties to leukocyte chemotaxis not observed in other cell types. in particular, the leukocyte pathway possesses a positive feedback loop that is able to maintain the stability and ruffling activity of the leading edge pseudopod even in the absence of an attractant gradient, enabling the pseudopod to rapidly detect and direct migration up a newly appearing gradient [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the feedback loop is comprised of multiple essential signaling proteins, cytoskeletal elements, and second messengers, including: phosphoinositide- -kinase (pi k); protein kinase c (pkc); small g-proteins (ras, rac); actin filaments; the signaling lipid phosphatidylinositol- , , -trisphosphate (pip ); and ca + ions [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . activation of any loop component leads to a characteristic co-activation of other loop components, increased activity of the lipid kinase pi k and accumulation of its product pip signaling lipid, and expansion of the leading edge pseudopod. by contrast, inhibition of any loop component yields inactivation of other loop components, decreased pi k activity and pip levels, and contraction of the pseudopod. the leading edge chemosensory pathway also possesses an intrinsic adaptation mechanism that enables the leading edge pseudopod to adapt as needed to changes in its chemical environment. adaptation is a ubiquitous feature of chemosensory pathways in archaea, bacteria, and eukaryotes [ ] [ ] [ ] [ ] [ ] . such intrinsic adaptation adjusts pathway gain and dampens background signals, thereby preventing signal saturation and maintaining sensory function as the cell migrates up an attractant gradient in a complex chemical environment with competing stimuli. in leukocytes, intrinsic pseudopod adaptation is provided by a hardwired adaptation branch of the chemosensory pathway that regulates the net activity of specific pathway components, often by covalent modification [ ] [ ] [ ] [ ] [ ] . different types of leukocytes, such as macrophages and neutrophils, possess different classes of cell surface receptors and other specializations, but appear to share similar mechanisms of chemosensing and intrinsic adaptation [ , , , ] . such intrinsic pseudopod adaptation could in principle counteract perturbations triggered by drug exposure, thereby restoring normal pseudopod morphology and function. alternatively, extrinsic intracellular or extracellular processes besides intrinsic chemosensory adaptation could counteract drug-induced perturbations; such extrinsic mechanisms could extrude, sequester, or chemically modify a drug that perturbs the leading edge pseudopod [ ] [ ] [ ] . the leading edge pseudopod is clearly visualized in live, cultured leukocytes by differential interference contrast microscopy (dicm) or by fluorescence microscopy [ , ] . prior studies have shown that drugs that target key components of the leading edge positive feedback loop yield dramatic changes in the size of the pseudopod and its leading edge pip signal [ , , [ ] [ ] [ ] [ ] [ ] . well characterized inhibitors of the leading edge positive feedback loop that collapse the leading edge pseudopod include wortmannin, a covalent inhibitor of pi k that directly inhibits leading edge pip production, and gö , an inhibitor of pkc protein kinase activity that indirectly inhibits pip production by blocking a key component of the feedback loop [ ] . natural attractants for leukocytes include platelet-derived growth factor (pdgf), a powerful macrophage attractant that activates a receptor tyrosine kinase (rtk, in particular the pdgf receptor) that stimulates pi k and pip production. pdgf has been observed to expand the leading edge pseudopod, increase its pip signal, and drive increased levels of cell polarization [ , ] . the present study begins by developing a novel combination of live cell assays and applying them to cultured, polarized raw macrophages in order to quantify the effects of four therapeutic and three control drugs on leading edge pseudopod morphology. the four selected therapeutic drugs possess well established clinical safety and are investigated within their standard clinical dosages (i.e. total plasma concentrations, [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). three are non-steroidal antiinflammatory drugs (nsaids) that inhibit cyclooxygenase enzymes (cox) and thereby inhibit the production of inflammatory signals [ ] [ ] [ ] : acetylsalicylic acid (aspirin), ibuprofen and diclofenac. the fourth, acetaminophen (paracetamol), is not an nsaid and relieves pain via unknown mechanisms. the three control drugs, wortmannin, gö , and pdgf each target a known pathway component (see above) and are employed at their standard experimental concentrations [ , , [ ] [ ] [ ] [ ] [ ] . the findings demonstrate that the present approach can resolve drug effects on the leukocyte leading edge pseudopod of polarized, cultured macrophages into four operational classes: (i) non-perturbing exposures, (ii) adaptive exposures, (iii) disruptive exposures, and (iv) activating exposures. when any of the four therapeutic drugs are added slowly to mimic the slow rise of drug levels in the plasma of patients following standard oral dosages [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] , these drugs are found to have little or no effect on leading edge morphology, as consistent with their well-established clinical safety. however, rapid addition of the therapeutic and control drugs reveals the four operational classes. acetylsalicylic acid and diclofenac are classified non-perturbing because they have little or no detected effect on the macrophage leading edge pseudopod and its pip signal. for these drugs, either rapid or slow addition retains normal pseudopod morphology in single cell and population studies. in contrast, ibuprofen and acetaminophen are classified as adaptive because rapid addition of either drug to polarized cells yields short-term collapse of the leading edge pseudopod and loss of the pip signal, followed by slow recovery. in contrast to the four therapeutic adaptive drugs, the two non-clinical control inhibitors wortmannin and gö are each known to directly inhibit key components of the leading edge positive feedback loop and rapid addition is observed herein to trigger long term collapse of the pseudopod with no detected recovery as previously observed [ , , [ ] [ ] [ ] [ ] [ ] . at the other extreme, pdgf is a well studied attractant and rapid addition yields leading edge expansion and increased cell polarization over the three hour observation period as predicted [ , ] . overall, the findings provide impetus for future studies probing the inhibitory mechanisms underlying therapeutic drug-triggered pseudopod collapse, as well as the adaptive mechanisms that enable pseudopod recovery following rapid exposure to some perturbing drugs. raw . macrophages were obtained from the american type culture collection (atcc tib- , manassas, va; lots , with cell identity confirmed by atcc via growth properties, morphology, and coi assay). cell media (supplemented dmem) was composed of dmem obtained from thermo fischer scientific (waltham, ma), fetal bovine serum (fbs, contains~ μm bsa as specified by manufacturer) from millipore-sigma (st. louis, mo), penicillin, streptomycin and glutamineplus (l-alanyl-l-glutamine) from atlanta biologicals (flowery branch, ga). dulbelcco's phosphate buffered saline (d-pbs) was from gibco (gaithersburg, md). all cell media was regularly subjected to mycoplasma testing to ensure lack of infection. matriplate well imaging plates were from matrical, inc. (spokane, wa). acetylsalicylic acid (lot slbv ), ibuprofen (lot slbx ), acetaminophen (lot slbr v), diclofenac (lot bcbs v), wortmannin (lot m v), pdgf (lot slb v), and hepes (free acid) were obtained from millipore-sigma (st. louis, mo). gö (lot a/ ) was purchased from tocris biosciences (minneapolis, mn). cellmask deep red and green membrane stains were purchased from thermofischer scientific (waltham, ma). cell-culture grade dmso was purchased from mp biomedicals (santa ana, ca). akt-ph-mrfp mammalian expression plasmid was subcloned by john h. evans [ , ] . cell culture techniques used herein have been previously described [ , ] . briefly, starting from cryogenic stocks, raw macrophages were grown as per atcc recommendation at ˚c under % co in supplemented dmem media containing mm glutamineplus, mm hepes ph . with naoh, u/ml penicillin, μg/ml streptomycin, and supplemented with % fbs (where undiluted fbs contains~ μm bsa as specified by manufacturer), resulting in a final albumin concentration of~ μm. cells were washed in d-pbs ph . with hcl and passaged upon reaching~ % confluency, as recommended by atcc. single cell or population experiments used raw cells following at most or such passages, respectively. for studies of cells transfected with akt-ph-mrfp to monitor pip lipid, the transfection protocol using the neon electroporation system has been previously described [ ] . live-cell microscopy methods have been previously described [ ] . cells were counted by hemocytometer and plated at known density in well imaging plates, then subsequently incubated at ˚c under % co for~ hours to facilitate cell adhesion and generate conditions of steady state cell polarization. in the bottom of each well, cells attach to a flat glass surface and many spontaneously polarize in the absence of an attractant gradient. two microscopes were employed. (i) images for figs a and b and c and d and s and s were captured using a nikon tie microscope equipped with a x, . n.a. objective and a hamamatsu orca-flash . v digital cmos camera. (ii) images for figs c and d and e- l and s were acquired with a nikon tie spinning-disc confocal microscope equipped with a yokogawa csu-x scanning unit, an andor ixon emccd camera, and a x, . n.a. water-immersion objective (fig c and d ) or a x, . n.a. objective (figs e- l and s ). for both microscopes, the imaging stage was enclosed in an environmental chamber maintaining humidity, % co , and ˚c. imaging studies monitored a single cell, or a population of cells, following the addition of drug or its carrier as a control. two types of addition protocols were employed. in rapid addition the drug or carrier was added ( . μl into μl) at t = via a μl micropipettor (gilson) with immediate, gentle pump-mixing with the same micropipettor and tip to yield the final concentration within sec. this pump-mixing procedure slowly drew and expelled μl of media into and out of the tip, and then repeated for a total of pump-mixing cycles during the sec mixing time. in slow addition the drug or carrier was added via a stepwise procedure over a period of min to generate a more gradual rise up to the same final total drug concentration as rapid addition. this slow addition protocol added the drug in four equal increments using a μl pre-calibrated hamilton syringe (each . μl into μl) at t = , , , min with immediate, gentle pump-mixing as above to generate a more gradual, stepwise increase in concentration. final total drug concentrations were pdgf ββ . μm, wortmannin nm, gö . μm, acetylsalicylic acid . mm, diclofenac μm, ibuprofen μm, acetaminophen μm. all drugs were added in carrier dmso with the exception of pdgf ββ, which was added in supplemented dmem. care was taken to ensure that drug effects observed after addition were due to the intended global increase in the total drug concentration, not due to locally high drug concentrations prior to complete mixing. the microscopic field observed following drug addition in single cell and population measurements represented a small fraction (< . % and < %, respectively) of the x mm glass surface area in the observation well. in single cell and population studies, cells appropriate for imaging were selected at random locations in three of the four quadrants of the surface, then drug was added at the outside corner of the remaining quadrant (always the outside corner of the far-left quadrant) followed by pump mixing at that same location. no correlation was observed between drug effects on cells and the distance of the cells from the drug addition. moreover, in single cell measurements each cell observed exhibited approximately the same drug effect, while in population measurements all four quadrants of the large grid showed approximately the same drug effects. such independence of drug effects from the distance to the point of addition indicated the effects were global, not local. a novel cell population analysis was developed to quantify both initial and long-term drug effects, including pseudopod adaptation and recovery following an initial drug-induced perturbation. timecourses of cell populations were measured in a large field containing ± cells (figs , a and b and s ), which was scanned as either x (fig ab) or x ( fig ) unit areas. the unit areas, which served as individual data points, were stitched together into a full field super-image using nikon elements software. for each full field, an initial super-image was captured at t = , then subsequent super-images were captured at the specified timepoints after drug or control addition. analysis of population timecourse data was as follows. at each timepoint, the number of polarized cells with extended leading edge pseudopods was counted in fiji (imagej) yielding an average over the (fig ab) or ( fig fig . imaging highly polarized, actively ruffling cultured raw macrophages and drug-induced collapse of the leading edge pseudopod. raw macrophages were plated on glass at ˚c in the absence of an attractant gradient. individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were imaged as described in methods. these cells migrate slowly on glass and are thus well suited for quantitative imaging of event timecourses at their leading edges. drugs were added at t = to the total concentration indicated in fig . panels a through d images show representative effects of drug addition on the leading edge pseudopod: (a,b) dicm images of a single cell at t = and min after ibuprofen addition, respectively, illustrating the drug-triggered loss of leading edge pseudopod area; (c,d) fluorescence images of the pip sensor aktph-mrfp in a single cell at t = and min after wortmannin addition, respectively [ , ] illustrating the drug-triggered loss of the signaling lipid pip on the leading edge pseudopod. https://doi.org/ . /journal.pone. .g ) unit areas of that super-image. for each super-image, the resulting mean timepoints were normalized to the mean initial value of the t = timepoint. finally, the final global mean timecourse was determined by averaging the corresponding mean normalized timepoints from different super-images (experiments), yielding the global mean timepoints and their sem for n = the number of super-images. the resulting global mean timecourses represent averages over - super-images (experiments) each containing approximately cells and collected from independent wells over - separate days for therapeutic drug treatments, or - separate days for control treatments. single-cell timecourses ( fig ) were measured and analyzed as previously described for individual, polarized cells with extended, actively ruffling leading edge pseudopods [ ] . briefly, for each cell the value of the initial t = timepoint was used to normalize all timepoints. drugs employed in the present study and key parameters. shown are the four representative therapeutic drugs employed (acetylsalicylic acid, acetaminophen, diclofenac, ibuprofen) as well as the three control drugs (gö , wortmannin, pdgf ββ). information provided for each drug includes: chemical structure, molecular weight, pka, therapeutic peak total concentration in human plasma following clinical dosage, total concentration employed in the present cultured cell experiments, target (where known), and percent bound to proteins in human plasma. notes: a recommended therapeutic range of total drug concentration in human plasma; b experimental total drug concentration in cell culture medium (this study); c references [ , ] ; d references [ , ] ; e reference [ ] ; f references [ ] [ ] [ ] [ ] . https://doi.org/ . /journal.pone. .g subsequently, for a group of at least cells in an experiment carried out on the same day, corresponding normalized timepoints were averaged to generate a mean value for each timepoint. finally, the resulting mean timepoints for individual experiments were averaged over all experiments to generate a global mean and its standard error of the mean (sem) for n = the number of experiments. final global averages represented - total cells imaged in at least effects of gradual drug addition on populations of polarized macrophages. raw macrophages were plated on glass at ˚c in the absence of an attractant gradient, then images of cell populations containing large numbers of individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were acquired as described in methods. subsequently, drug or carrier was added in four equal increments at , , , min summing to yield the total drug concentration indicated in fig . images of the cell population were collected min after each incremental addition at the indicated timepoints over a hour observation period (filled symbols). the number of cells in the population displaying extended leading edge pseudopods was counted for each timepoint and normalized to the initial number prior to drug addition. data collected for multiple populations was averaged, yielding the illustrated mean timecourse for each treatment. as an internal control, rapid addition at t = of the full total drug concentration was also carried out for two of the four therapeutics (ibuprofen fast and acetaminophen fast, open symbols; these data can be directly compared with more extensive, independent rapid addition data in fig below) . panel a summarizes the resulting timecourses. panel b compares the dose dependence of drug effects measured min after addition at t = of either x total drug concentration (fig ) or . x total drug concentration. error bars are sem for n = (fast acetaminophen) or (others) experiments carried out on at least three days. error bars smaller than their data point symbol are not visible. cell cultures were prepared for mass spectrometry analysis of drug stability using exactly the same procedures described above for long-term studies of drug effects on cell populations (above). μl of media were taken from the culture immediately after drug addition and mixing, then snap frozen in ln . cultures were allowed to incubate minutes before another μl sample was withdrawn and snap frozen. images of the cell population in each culture were analyzed to ensure the drug effects on the cells were within their characteristic ranges. the number of replicate samples, each prepared from separate cultures, ranged from (acetylsalicylic acid and diclofenac) to (acetaminophen and ibuprofen). means and standard deviations were calculated for each set of replicates. after storage at - ˚c and thawing, samples were diluted into μl meoh containing μm meloxicam as an internal sample standard. samples were then stored at - ˚c for minutes and subsequently spun at , x g for minutes. while not disturbing the precipitated pellet, μl of supernatant was extracted and dried via speedvac. prior to lc-ms analysis, dried study samples containing each drug were reconstituted in μl (ibuprofen, acetaminophen and aspirin) or μl (diclofenac) of mm ammonium acetate in % water/ % acetonitrile/ % methanol + . % formic acid. except for acetaminophen samples, the reconstitution solvent also contained μm -amino-salicylic acid ( -asa) that was used as a post-reconstitution internal standard to monitor final sample preparation and mass spec performance ( -asa overlapped the acetaminophen peak). the lc system was composed of an agilent binary pump, an agilent auto-sampler and an agilent temperature-controlled column compartment (agilent technologies, santa clara, ca). μl of each sample was injected into the lc. chromatography was performed in reverse phase (rp) on agilent eclipse xdb-c column ( x . mm, . μm particle size, agilent technologies, santa clara, ca). the flow rate was . ml/min, autosampler temperature was kept at ˚c, and the column compartment was set at ˚c. chromatography time was min, and all peaks eluted off the column between and min without any overlapping except for acetaminophen and -asa. after the chromatographic separation, ms ionization and data acquisition was performed using ab sciex qtrap mass spectrometer (ab sciex, toronto, on, canada) equipped with methods) , then images of cell populations containing large numbers of spontaneously polarized cells with extended leading edge pseudopods were acquired as described in methods. drugs were added rapidly at t = to the total concentration indicated in fig . shown are timecourses following drug addition illustrating the changing number of cells in the full population that display extended leading edge pseudopods, normalized to the initial number prior to drug addition. panels a and b summarize findings for the four therapeutic drugs (a) and for the three control drugs and two carriers (b), respectively. data collected for multiple population measurements was averaged, yielding the illustrated mean timecourse for each treatment. error bars are sem for n = experiments carried out on at least five days for experimental treatments, or at least two days for control treatments. error bars smaller than their data point symbol are not visible. panels c through l show representative cell images in these studies, as follows. (c,d) small subsection of a dicm super-image used to quantify the number of cells with extended leading edge pseudopods in a population before and min after ibuprofen addition, respectively. dividing cells are occasionally observed as illustrated by the pair at lower left. (e-l) expanded sequential images showing the leading edge pseudopod of a single polarized macrophage within a super-image, illustrating temporary pseudopod collapse after rapid ibuprofen addition at t = , followed by pseudopod adaptation and recovery. panels e-l correspond to timepoints , . , , , , , , min after ibuprofen addition, respectively. scale bars indicate μm. https://doi.org/ . /journal.pone. .g fig . effects of rapid drug addition on polarized macrophages: single cell analysis. raw macrophages were plated on glass at ˚c in the absence of an attractant gradient. individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were imaged for min as previously described ( [ ] and methods). drugs were added rapidly at t = to their standard total concentration (fig ) . panels a and c show the timecourses of leading edge pseudopod area loss following the addition of the four therapeutic drugs (a), and the three control drugs or media controls (c). panels b and d show the timecourses of leading edge pseudopod pip loss following the addition of the four therapeutic drugs (b), and the three control drugs or media controls (d). all timecourses were quantified as previously described [ ] . briefly, a box of uniform dimensions and placement was used to define the leading edge region of each cell, then the leading edge area or fluorescence was quantified at the indicated timepoints. note these parameters do not decay to zero, even for the strongest pseudopod inhibitors wortmannin and gö , because the box contains both the pseudopod and the tip of the cell body at t = . thus, the final timepoints at t = min show the loss of area and fluorescence due to the pseudopod collapse, but retain the signals arising from the cell body remaining within the box, respectively. error bars are sem for n = to cells over at least three experiments on at least two different days. error bars smaller than their data point symbol are not visible. the extracted mrm peaks were integrated using multiquant . software (ab sciex, toronto, on, canada). the two spiked internal standards (meloxicam and -aminosalicylic acid) were used to monitor sample preparation and lc-ms assay performance. a standard mixture of all four study drugs was used as a quality control to measure signal reproducibility. intra-and interday reproducibility for each measured standard was under % without data normalization. replicates suitable for statistical analysis were carried out for all measurements, as described in detail above and reported in the relevant figure legend. no outliers were removed during data analysis and calculation of reported means. data were normalized to the initial t = measurement for individual cells and for populations to facilitate comparisons between different cells and different populations, respectively, given standard biological variability. statistical significance of differences between means was calculated using student's unpaired t-test [ ] a onetailed test was employed for significance of a predicted increase or decrease, while a two-tailed test was used for significance of a possible difference of either sign. the threshold for significance is defined as % (p � . ). to characterize the effects of drugs on the leukocyte leading edge chemosensory pathway, we utilized both cell population and single cell assays to monitor the leading edges of raw macrophages plated at controlled, low density on clean glass in standard media at ˚c. plating on clean glass significantly reduces cell movement and facilitates long-term pseudopod imaging [ , ] . no global attractant was added (excepting experiments with global pdgf addition), and no attractant gradient was imposed. in the absence of added attractant, the leading edge positive feedback loop maintains spontaneous polarization of a sizable subset ( ± %) of the macrophage population. this protocol yields large numbers of relatively immobile, easily imaged polarized cells with well-developed leading edges displaying a ruffling sensory pseudopod (see figs a and c below) [ , ] . activators and inhibitors of the positive feedback loop are known to expand and contract the leading edge pseudopod, respectively, via a signaling mechanism that involves increased or decreased levels of the signaling lipid pip in the cytoplasmic leaflet of its plasma membrane, respectively [ , , , ] . the leading edge pseudopod also possesses an intrinsic adaptation mechanism that damps out fluctuations in the chemical environment as required for movement in an attractant gradient [ , , ] . a novel cell population analysis was developed to quantify both short-and long-term effects of drugs on the leading edge pseudopods of large numbers of polarized raw macrophages over a timescale of - hours. these studies employed dicm to record high resolution images of macrophage populations containing~ total cells, including~ polarized cells with well-defined leading edge pseudopods, captured in subpopulations of~ - total cells per unit area in a grid containing - such areas per superimage (s fig). the number of cells with extended leading edge pseudopods per unit area was quantified before drug addition, and at selected timepoints after drug addition for up to hours. finally, the mean timecourses for - superimages were averaged to generate an overall mean timecourse for effect of each drug on hundreds of polarized cells across all experiments. single cell imaging studies were also carried out to probe the short-term effects of drugs on both the size (area) and pip signal of the leading edge pseudopod in individual raw macrophages. previous studies have shown that leading edge area and pip levels are sensitive indicators of pseudopod perturbation by drugs [ , , , ] . each single cell imaging experiment employed differential interference contrast microscopy (dicm) and z-stacked spinning disk confocal fluorescence microscopy to record high resolution images of the leading edge pseudopod of a polarized, actively ruffling macrophage before addition of each drug, and at selected timepoints for min after drug addition. the resulting dicm images were used to quantify the timecourse of drug effects on pseudopod area, while the fluorescence images quantified levels of a standard fluorescent pip sensor (akt ph domain fused to mrfp [ , ] ) on the pseudopod membrane. for a given drug treatment, timecourses obtained from observations of to single cells were averaged to generate a mean timecourse for drug effects on the leading edge pseudopod area and pip signal. the population and single cell assays are complementary. the population analysis monitors the effects of drugs on the leading edge pseudopods of at least~ polarized cells over a period of hours, making it possible to quantify how drugs impact the frequency of cells with deployed, extended pseudopods, and to ascertain whether the perturbed pseudopods exhibit long term recovery following drug treatment. the single cell analysis provides a higher timeresolution view of the initial effects of drugs on the leading edge pseudopod and its pip signal for at least individual, polarized macrophages over a period of minutes. in both the population and single cell assays, selective observation of polarized, ruffling cells ensures that all studied cells are healthy and possess an active leading edge pseudopod and signaling pathway. representative cell images and drug effects are shown in figs and and s and s for all drugs. fig shows representative dicm and fluorescence images of the leading edge, both before and min after addition of an inhibitory drug, illustrating drug-triggered collapse of both the leading edge area (fig a and b ) and its pip signal (fig c and d) . the drugs employed in the present study, their total concentrations, structures, and other key parameters are summarized in fig . the four therapeutic drugs employed (acetylsalicylic acid, acetaminophen, diclofenac, ibuprofen) are each studied at the same total peak drug concentration reached in human plasma following maximum clinical oral dosage. the three control drugs employed (gö , wortmannin, pdgf ββ) are each studied at their standard total concentrations in cultured cell studies [ , ] . to measure drug effects on populations of polarized raw macrophages, populations of~ well-resolved cells plated on glass in standard media were imaged at ˚c in each experiment (see methods and approach). a target subpopulation of~ cells displayed a readily observed extended, ruffling leading edge pseudopod [ , ] and were counted before drug addition, and at multiple timepoints during a - hour observation period following drug addition at t = . altogether, to experiments were carried out for each treatment on at least days, yielding average timecourses summarizing observations of~ to~ polarized cells, respectively. fig first examines the effects of gradual addition of the four therapeutic drugs on leading edge morphology. in the clinic, these therapeutic drugs are typically administered orally, which yields gradually increasing drug levels in the plasma over a period of - min [ , ] . here, gradual addition of therapeutic drugs was carried out by adding four equal increments over min that sum to yield the maximum total drug concentration achieved at peak plasma levels in patients (fig ) . fig a shows that such gradual, incremental addition of each of the four therapeutics or carrier has no significant effect on the number of polarized cells with extended leading edges per unit observation area ( . > p > . for the full range of treatments and timepoints, respectively). in contrast, however, rapid addition of two therapeutics at t = , yielding the same final, maximum drug concentration achieved by incremental addition above, triggers significant collapse of the leading edge subpopulation within min. specifically, rapid addition of ibuprofen or acetaminophen decreased the number cells with extended leading edges ± % (p < . ) and ± %, (p < . ), respectively, followed by gradual recovery within hrs. as illustrated in fig , gradual and rapid addition of ibuprofen or acetaminophen yields dramatically different effects on the leading edge pseudopod, thus the effects of rapid drug addition were examined more carefully. fig compares timecourses observed following rapid addition of the four therapeutic drugs, as well as three control drugs for which the effects of rapid addition are well documented in the literature. fig a summarizes the effects of the therapeutic drugs, each added rapidly at t = to their standard peak clinical concentration (fig ) , on the relative number of cells possessing extended leading edge pseudopods per unit observation area. rapid acetylsalicylic acid or diclofenac addition had no significant effect on the number of cells with extended pseudopods over a hour observation period, yielding timecourses indistinguishable from that of control vehicle addition (fig a and b ). in contrast, within min of addition, ibuprofen and acetaminophen each triggered significant reduction of the number of cells with extended pseudopods (decreased ± %, p < . and ± %, p = . , respectively; see also figs a and c- l and s . again, as observed in fig a, within hrs of ibuprofen or acetaminophen addition the number of cells with extended pseudopods returned to the initial level, indicating that the macrophage population recovered from each drug perturbation by an unidentified mechanism (fig a; see also figs e- l and s ). figs b and s show the effects of the three control drugs, rapidly added at t = to their standard working concentration (fig ) , on the normalized number of pseudopod-possessing cells in the raw macrophage population. global addition of the pathway activator (pdgf ββ), a known macrophage chemoattractant, triggered a significant increase (by ± %, p = . ) in the number of pseudopod-possessing cells within hours. at the other extreme, addition of a control pathway inhibitor (either wortmannin or gö to inhibit pi k or pkc, respectively) triggered a major decrease (by ± %, p < . or ± %, p < . respectively) in the number of observed pseudopods within min, with little or no recovery during the hour observation period as expected for standard inhibitors of essential components of the leading edge pathway. to further investigate the effects of rapid drug addition on leading edge pseudopod morphology and its signature pip lipid signal, single cell imaging was carried out. such single cell measurements provide enhanced spatial and time resolution of within the min observation period following drug addition, but observations could not be extended over a period of hours for technical reasons (on this timescale, even slowly moving cells plated on glass must be imaged periodically to maintain single cell identity, which bleaches the akt-ph-mrfp pip sensor). for each drug or control treatment, an average timecourse was obtained based on observations of - individual, highly polarized raw macrophages with extended, ruffling leading edge pseudopods, as illustrated above in fig a and c . fig displays average single cell timecourses following rapid addition at t = of the four therapeutic drugs to their standard total concentrations (fig ) . rapid addition at t = of acetylsalicylic acid or diclofenac has little or no detectable effect on the pseudopod area ( fig a) and pip levels (fig c) . by contrast, rapid addition of ibuprofen or acetaminophen triggers significant loss of pseudopod area (decreased ± %, p < . , or ± %, p < . , respectively; fig a) and significant loss of pseudopod pip (decreased ± %, p < . , or ± %, p < . , respectively; fig c) within min. the observation that rapid ibuprofen or acetaminophen addition triggers both collapse of pseudopod area and decreased pip levels is consistent with pseudopod destabilization via inhibition of the leading edge signaling pathway. fig also summarizes the effects of the three control drugs on the leading edge pseudopod. addition of the known pathway activator (pdgf ββ) led to a significant pseudopod area expansion (increased ± %, p < . ) and increased pip levels (by ± %, p < . ) during the min observation period (fig b and d ). by contrast, the two control pathway inhibitors wortmannin and gö each triggered significant losses of both the pseudopod area (decreased ± %, p < . and ± %, p < . , respectively) and the pip signal (decreased ± %, p < . and ± %, p < . , respectively) (fig b and d ). the figure also shows that carriers alone had no detectable effect on the pseudopod. the observation that a subset of the tested compounds significantly perturb pseudopod area and pip levels in single polarized cells raises the possibility these drugs may directly and specifically modulate the enzyme activity of pi k lipid kinase. control studies summarized in supplemental s fig confirm that wortmannin, a known irreversible pi k inhibitor, directly blocks the pip production of purified pi k in vitro as previously observed [ , ] . in contrast, the other drugs have little or no direct effect on the pip production of purified pi k, indicating that only wortmannin directly inhibits pi k activity in vitro. the finding that two therapeutic drugs had no detectable effect on the leading edge pseudopod, while two other therapeutic drugs triggered an initial pseudopod collapse followed by recovery, raised the possibility that drugs might be unstable in the media. quantitative mass spectrometry analysis was carried out to ascertain whether each of the four target drugs was present at the same total concentration during three hours in culture. fig shows that each of the four therapeutic drugs was present in the cell culture media at the intended clinical concentration at the first timepoint after addition, and exhibited relatively little change during the three hour incubation. thus, each of the four drugs remained at an approximately fixed, standard clinical concentration during the timecourses described in this study. the present findings reveal that gradual exposure of cultured raw macrophages to four representative therapeutic drugs (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) has little or no effect on the native morphology of the leading edge sensory pseudopod. thus, when the concentration of each drug is increased in four equal, stepwise increments up to its standard clinical total concentration (fig ) over a period of min, no significant loss of polarized cells with extended pseudopods is detected. these findings are consistent with the well-documented safety of these therapeutics in the clinic, where standard oral dosage also yields a gradually increasing drug level in the plasma up to the total concentration employed herein (fig ) within~ min [ , , [ ] [ ] [ ] . strikingly different results are observed, however, when the same four therapeutic drugs (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) and three control drugs (pdgf, gö , wortmannin) are added rapidly to cultured raw macrophages. such rapid drug addition, in one step at t = that raises the drug up to its standard total concentration (fig ) within sec, yields at least four operational classes of drug effects on the leading edge pseudopod and its signature pip lipid signal. (i) non-perturbing rapid exposures yield little or no effect on pseudopod area, pip levels and stability (as observed for acetylsalicylic acid, diclofenac). (ii) adaptive rapid exposures trigger temporary collapse of the pseudopod followed by recovery (observed for ibuprofen, acetaminophen). (iii) disruptive rapid drug exposures yield long-term pseudopod collapse with little or no adaptation over the hour observation period (gö , wortmannin). (iv) activating exposures yield pseudopod expansion and increased cell polarization (pdgf). to analyze the molecular mechanisms of these four different classes, it is useful to begin by noting the significant drug buffering capacities of human plasma and cell culture media. this buffering capacity is dominated by drug binding to the highly homologous proteins human serum albumin (hsa) and bovine serum albumin (bsa), respectively. both hsa and bsa possess two high affinity drug binding sites and multiple low affinity sites [ , [ ] [ ] [ ] . as a result, in human plasma the four well-studied therapeutic drugs employed herein each show significant binding to hsa at their standard total concentrations, ranging from to % bound (fig ) . drug binding to serum albumins is rapid, often on the msec timescale depending on the drug and albumin concentrations. while the drug binding properties of hsa and bsa are extremely similar [ , ] , their concentrations are quite different in human plasma (hsa, - μm) [ , ] vs cell culture medium (bsa, - μm; provided by % fbs containing - μm bsa (methods)). as a representative example, we can estimate the rate of ibuprofen binding to albumin binding sites under these conditions since its hsa equilibrium association constant (k a = . x m - ) and off-rate constant (k off = . sec - ) are known [ , ] . assuming the standard total clinical concentration of ibuprofen ( μm) and the relevant hsa (~ μm) or bsa (~ μm) concentration, the initial on-rate for drug binding to protein is~ mm per sec or~ mm per sec in human plasma or cell culture media, respectively. it follows that the binding of μm ibuprofen to bsa in cultured cell experiments will occur in less than a second, or during the sec mixing time of our drug addition protocol. two mechanisms are possible for the lack of pseudopod perturbation observed upon rapid addition of the non-perturbing drugs acetylsalicylic acid and diclofenac: either the free drug could be non-perturbing, or serum albumin buffering in the media could ensure that little free drug is available for cell binding. for acetylsalicylic acid, the high total drug concentration ( . mm, fig ) relative to the bsa concentration in cell culture media ( - μm) ensures that high levels of free drug exceeding mm will be present immediately upon addition to cells. moreover, mass spec analysis shows the total drug concentration does not change significantly over the hour observation period (fig ) . it follows that high levels of free acetylsalicylic acid in the millimolar range do not perturb the leading edge pseudopod morphology. for diclofenac, however, the total drug concentration ( μm) is significantly lower than the bsa concentration in the cell media, and the affinity of this drug for serum albumin is very high ( % bound in human plasma). thus the free diclofenac concentration in the cell media is likely sub-micromolar, and the lack of pseudopod perturbation could arise from strong drug buffering by bsa that ensures the free drug concentration remains below its unknown pseudopod perturbation threshold. two mechanisms are also possible for the temporary collapse of the macrophage pseudopod and its signature pip lipid signal upon rapid addition of ibuprofen or acetaminophen. mechanism (i), which is favored by the available evidence, proposes that the pseudopod is strongly perturbed by appearance of a relatively constant level of free drug triggered by rapid drug addition at t = , collapses within min, and then recovers due to cell adaptation to the constant free drug concentration. in contrast, mechanism (ii) proposes that the pseudopod is strongly perturbed by a transient, high local concentration of free drug during addition and mixing that exceeds a drug threshold for pseudopod perturbation. in this model, the transient, high local concentration of drug rapidly decreases as mixing is completed and drug binds to bsa; subsequently, the pseudopod gradually recovers from the initial drug shock simply because the free drug has fallen below its pseudopod perturbation threshold with no contribution from cell adaptation. multiple lines of evidence support mechanism (i) for both drugs as follows. the total concentrations of ibuprofen ( μm) and acetaminophen ( μm) added to cells are significantly higher than the total bsa concentration in the cell media ( - μm) , ensuring that substantial free concentrations of drug are present throughout the observation period. moreover, the affinity of acetaminophen for bsa is low, such that only % of the drug is bound even in human plasma where the serum albumin concentration is~ -fold higher. we cannot formally rule out a version of mechanism (ii) in which rapid addition of the drug stock solution yields a transient spike of high drug concentration; however both mixing and bsa buffering should be complete within~ sec and sec, respectively (see methods and binding rate calculation above). evidence disfavoring this transient high local concentration model is provided by the observed strong reproducibility with which each drug triggers pseudopod collapse. a mechanism requiring high local drug concentrations during mixing would likely yield variable results between replicates, and between cells in different regions of the same coverslip, with cells close to the addition point more strongly perturbed than distal cells. spatial heterogeneity is not observed in the large fields of~ cells scanned for population studies (figs and and s ), and reproducibility is high in studies monitoring - single cells (fig ) . to fully exclude the high local concentration model, future studies will compare the addition of drugs as a x stock solution and as a x stock solution in cell media. (such a comparison was planned but prevented by the covid pandemic and lab shut-down). the cellular mechanism(s) by which rapid addition of ibuprofen or acetaminophen triggers the initial collapse of leading edge pseudopod and pip levels remain unknown, but the findings rule out the possibility that the drugs act as intrinsic pi k inhibitors since neither blocks pip production by purified pi k in a lipid kinase activity assay (s fig). thus, the perturbing drugs must directly or indirectly modulate as yet unidentified component(s) of the leading edge signaling pathway, or trigger a nonspecific intracellular perturbation (likely not a ph change, since the drug perturbations are not correlated with drug pkas (fig ) ). similarly, assuming that mechanism (i) accurately describes ibuprofen and acetaminophen, multiple possibilities exist for the cellular adaptation mechanism(s) that drive pseudopod recovery following the initial drug-triggered collapse. the simplest hypothesis is that the same unknown adaptation mechanism(s) are acting both to drive pseudopod recovery after rapid drug addition, and to prevent pseudopod collapse during slow drug addition. this hypothesis proposes that rapid drug addition overwhelms the adaptation process which is unable to counteract the perturbation in real time, but gradually eliminates the perturbation within hours. in contrast, gradual drug addition allows concurrent gradual adaptation that is able to counteract the drug perturbation in real time. adaptation does not involve extracellular chemical or enzymatic degradation of drugs in the media surrounding the cultured cells, since mass spectrometry analysis indicates that each drug is stable in the media during the three hour cell incubation (fig ) . instead, the adaptation mechanism is hypothesized to be intracellular. such intracellular adaptation could be provided by the intrinsic adaptation mechanisms of the leading edge chemosensory pathway which, like other sensory pathways, must adapt to changing background stimuli and attractant concentrations as the cell moves through different environments [ , , , ] . other possible adaptive mechanisms could include activation of an intracellular sink, pump, degradation or modification reaction that eliminates free drug in the cytoplasm, but has a negligible effect on the bulk extracellular drug concentration. such mechanisms have been reported for other drugs (reviewed in [ , ] ). the mechanisms of the three control drugs are well defined by previous studies. exposures to the non-clinical control inhibitors wortmannin and gö are disruptive, yielding long term collapse of the leading edge pseudopod and its pip lipid signal. these drugs are known to directly inhibit essential components of the leading edge positive feedback loop (pi k and pkc, respectively), and their disruption of the leading edge pseudopod upon rapid addition has been previously described [ , , [ ] [ ] [ ] [ ] [ ] . rapid exposure to pdgf yields pseudopod expansion and increased cell polarization, as previously observed and commensurate with its known importance as a strong macrophage attractant in vitro and in vivo [ , ] . in closing, the present findings suggest a number of directions for future research. further studies are needed to determine the molecular mechanisms by which rapid ibuprofen or acetaminophen addition trigger macrophage leading edge pseudopod collapse, and by which the pseudopod adapts (or recovers) from these drug exposures. given the~ -fold higher concentrations of albumins in plasma compared to cell culture media, the greater drug buffering capacity of plasma will help protect macrophages and other cell types from drug perturbations, (fig ) to ascertain the stability of the total drug concentration in the media surrounding the cells. aliquots of media were removed from cell imaging wells immediately after rapid drug addition at t = to the total concentration indicated in fig , and again at t = hr after completion of imaging, then snap frozen in liquid n . later, the samples were prepared for quantitation with addition of internal standards (methods), then quantitative mass spectrometry was carried out to compare the initial and final total drug concentrations. error bars are sd for n = (acetylsalicylic acid, diclofenac) or (ibuprofen, acetaminophen). https://doi.org/ . /journal.pone. .g but the sensitivity of the cultured macrophage leading edge pseudopod to drugs makes it an excellent model system for detecting and classifying drugs capable of inhibiting macrophages, either transiently or permanently. transient macrophage inhibition could be useful in controlling extreme inflammation events, while long-term inhibition that blocks macrophage participation in the innate immune response would be highly detrimental. it will be important to carry out future studies that directly test whether drug-induced pseudopod collapse causes inhibition of macrophage motility and chemotaxis in vitro and in vivo [ ] . more broadly, we hypothesize that the same four classes of drug effects observed herein for cultured macrophages will also be observed for primary macrophages and other leukocytes such as neutrophils which possess similar chemosensory pathways [ , , , ] , but this prediction also remains to be tested. finally, the effects of other therapeutic and experimental drugs on the leading edge pseudopod remain to be determined. in short, the present study opens several new research paths with major implications for a basic understanding of drug-induced perturbation and adaptation in leukocyte signaling pathways. a single molecule tirfm assay was employed to measure the specific kinase activity of purified pi k molecules on a supported lipid bilayer by counting the number of pi k molecules, as well as the number of product pip molecules they produce as previously described in detail [ ] [ ] [ ] [ ] . this assay utilizes physiological concentrations of class i pi ka and saturating concentrations of fluorescently-labeled grp-ph domain, a high-affinity pip product lipid binder, in order to count each product lipid produced. drug is added to therapeutic dose identified in fig prior to the start of the assay. enzyme rates are calculated as the slope of product lipid created vs. time. bars indicate the lipid kinase activity of pi k in the presence of the indicated drug. error bars are sd where n = for acetylsalicylic acid and diclofenac, or n = for ibuprofen and acetaminophen. (tiff) temporal and spatial regulation of chemotaxis signaling mechanisms for regulation of chemotaxis feedback signaling controls leading-edge formation during chemotaxis moving towards a better understanding of chemotaxis new paradigms in the establishment and maintenance of gradients during directed cell migration self-organization of protrusions and polarity during eukaryotic chemotaxis rac functions in both neutrophils and macrophages to mediate motility and host defense in larval zebrafish neutrophil plasticity in the tumor microenvironment signaling to migration in neutrophils: importance of localized pathways. the international journal of biochemistry & cell biology live imaging of wound inflammation in drosophila embryos reveals key roles for small gtpases during in vivo cell migration calcium flickers steer cell migration role of platelets in leukocyte recruitment and resolution of inflammation triggering the resolution of immune mediated inflammatory diseases: can targeting leukocyte migration be the answer? front pharmacol lipid droplets in leukocytes: organelles linked to inflammatory responses moving in the right direction: how eukaryotic cells migrate along chemical gradients interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells signaling networks that regulate cell migration signaling mechanisms for chemotaxis calcium gradients underlying cell migration molecular players in neutrophil chemotaxis-focus on pi k and small gtpases single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase c-alpha regulation of pi k by pkc and marcks: single-molecule analysis of a reconstituted signaling pathway regulation of a coupled marcks-pi k lipid kinase circuit by calmodulin: single-molecule analysis of a membrane-bound signaling module single-molecule study reveals how receptor and ras synergistically activate pi kalpha and pip signaling neutrophil chemotaxis a pkc-marcks-pi k regulatory module links ca + and pip signals at the leading edge of polarized macrophages activation of phosphatidylinositol -kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation specific translocation of protein kinase calpha to the plasma membrane requires both ca + and pip recognition by its c domain unusual interplay of two types of ras activators, rasgrp and sos, establishes sensitive and robust ras activation in lymphocytes ca + influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages mechanism of specific membrane targeting by c domains: localized pools of target lipids enhance ca + affinity eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity phosphoinositides: tiny lipids with giant impact on cell regulation pi k signalling in inflammation exploring cells with targeted biosensors chemoattractant receptors activate, recruit and capture g proteins for wide range chemotaxis mutually inhibitory ras-pi( , ) p feedback loops mediate cell migration understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids signal transduction: principles, pathways and processes the microtubule-associated protein eb maintains cell polarity through activation of protein kinase c the two-component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes integrating conflicting chemotactic signals. the role of memory in leukocyte navigation a model for direction sensing in dictyostelium discoideum: ras activity and symmetry breaking driven by a gbetagamma-mediated, galpha -ric -dependent signal transduction network bacterial pep-dependent carbohydrate: phosphotransferase systems couple sensing and global control mechanisms cellular memory in eukaryotic chemotaxis moving towards a paradigm: common mechanisms of chemotactic signaling in dictyostelium and mammalian leukocytes the different mechanisms of cancer drug resistance: a brief review mechanisms of antibiotic resistance determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction regulation of chemotaxis by the platelet-derived growth factor receptor-beta the chemotactic response to pdgf-bb: evidence of a role for ras pi- -kinase and mapk regulate mesangial cell proliferation and migration in response to pdgf current clinical recommendations for use of platelet-rich plasma platelet-rich plasma: review of current literature on its use for tendon and ligament pathology platelet-derived growth factor-bb and transforming growth factor beta selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds non-steroidal anti-inflammatory drugs. current status and rational therapeutic use ibuprofen: plasma concentrations in man clinical pharmacokinetics of diclofenac. therapeutic insights and pitfalls drug levels: therapeutic and toxic serum/plasma concentrations of common drugs evolution of nonsteroidal anti-inflammatory drugs (nsaids): cyclooxygenase (cox) inhibition and beyond trends in the use of aspirin and nonsteroidal anti-inflammatory drugs in the general u.s. population use of non-steroidal anti-inflammatory drugs in us adults: changes over time and by demographic mechanism of action of nonsteroidal anti-inflammatory agents mechanism of action of nonsteroidal anti-inflammatory drugs effects of nonsteroidal anti-inflammatory drugs at the molecular level the effect of nonsteroidal anti-inflammatory drugs on tissue healing mutual inhibition between pten and pip generates bistability for polarity in motile cells sensory adaptation of leukocytes to chemotactic peptides adaptation of human neutrophil responsiveness to the chemoattractant n-formylmethionylleucylphenylalanine. heterogeneity and/or negative cooperative interaction of receptors essential role of phosphoinositide -kinase delta in neutrophil directional movement live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues kinetics and metabolism of paracetamol and phenacetin nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation the binding of ibuprofen to plasma proteins in vitro protein binding of diclofenac sodium in plasma and synovial fluid studies on paracetamol binding to serum proteins aspirin binding and the effect of albumin on spontaneous and enzyme-catalysed hydrolysis effects of food on pharmacokinetics of immediate release oral formulations of aspirin, dipyrone, paracetamol and nsaids-a systematic review an overview of clinical pharmacology of ibuprofen structural determinants of phosphoinositide -kinase inhibition by wortmannin, ly , quercetin, myricetin, and staurosporine pharmgkb summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses emerging evidence in nsaid pharmacology: important considerations for product selection nsaids and aspirin: recent advances and implications for clinical management kinetics and mechanism of bilirubin binding to human serum albumin interactive association of drugs binding to human serum albumin species differences of serum albumins: i. drug binding sites a comprehensive spectroscopic and computational investigation to probe the interaction of antineoplastic drug nordihydroguaiaretic acid with serum albumins the correlation of plasma proteins binding capacity and flavopiridol cellular and clinical trial studies on the molecular interaction between albumin and ibuprofen: an afm and qcm-d study mechanisms of gradient detection: a comparison of axon pathfinding with eukaryotic cell migration incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway a high-throughput, multi-cell phenotype assay for the identification of novel inhibitors of the authors gratefully acknowledge helpful conversations with expert colleagues, including: prof. anna huttenlocher from university of wisconsin, madison; and drs. ken hunt, rachel frank, and sourav poddar from the university of colorado sports medicine clinic. furthermore, we gratefully acknowledge resources and experts at university of colorado, boulder integral to the completion of this work: the biofrontiers advanced light microscopy core (dr. joseph dragavon), the biochemistry cell culture facility (dr. theresa stines nahreini, nicole kethley), and the biochemistry mass spectrometry laboratory at the central analytical laboratory (thomas lee). we thank the national institutes of health and the nih/cu molecular biophysics training program for support. finally, we thank the editor and reviewers for helpful comments that enabled us to significantly improve this contribution. conceptualization: thomas c. buckles, brian p. ziemba, joseph j. falke. key: cord- - tapkjb authors: nan title: th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, – october date: - - journal: int j clin pharm doi: . /s - - - sha: doc_id: cord_uid: tapkjb nan pharmacy, sint maartenskliniek, ubbergen, pharmacy, radboud university medical centre, nijmegen, clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from to %. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september , and march , . physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars- and the morisky- questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which ( . %) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from . to . %) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive -day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were /unplanned hospitalizations assessed by phone contact at day after discharge and /the iatrogenic potential exposure scale from to for each patient established by a clinical team. analysis was conducted in intention to treat. results: hospitals corresponding to clusters enrolled patients ( e group v/s c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day ; ( . %) patients in e group had at least one drp v/s ( . %) in c group (or . ; ic % [ . ; . ] p = . ). intervention was especially efficient for patient discharged from surgery unit (or . ic % [ . ; . ]) and aged less than years (or . ic % [ . ; . ] . although intervention decreased patient exposure to drp with high iatrogenic potential (from . to . % p \ . ), un-planned hospitalizations at day weren't different between groups ( . vs. . % p = . ). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc : clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *, , kristine lundereng namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half ( of ) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since , a service where clinical pharmacists participate in multidisciplinary medication reviews in municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during , totally medication reviews were performed in municipalities. in the same period, lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel , arnaud de la blanchardière , vincent cattoir , guillaume saint-lorant *, pharmacy, infectious and tropical diseases, microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since , the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july and november in a -bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: daptomycin prescriptions were analysed. observed indications were: sepsis ( %), infective endocarditis ( %), bone and joint infections ( %) and vascular prosthetic infections ( %). identified pathogens were: mrsa ( %), methicillin-resistant coagulase-negative staphylococci ( %), methicillin-sensitive staphylococcus aureus ( %), enterococci ( %) and methicillinsensitive coagulase-negative staphylococci ( %). daptomycin was prescribed as first-line treatment in % of cases. the mean dose was mg/kg/day [ - mg/kg/day] for a mean duration of days [ ; days] . local guidelines were followed in % of cases. daptomycin use was relevant for % of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in % of cases, respectively, generating an % decrease in consumption and an economy of over € for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. ( ) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. ( ) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. ( ) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc : assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: patients were included in the registry, of which ( %) presented with stemi and ( %) presented with nsteacs. of ( %) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. ( %) had a high or very high crusade score. of these, ( %) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately % of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february and january , hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, had advanced liver disease (fibrosis score: f /f ) and were infected with hcv genotype . prior to starting hcv treatment, patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: / and / , respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, / ( %) patients achieved sustained viral response weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation ( ps) , early mortality (charlson) and drug related problems (drp) . design: clinical and therapeutic variables in patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during months in six units (internal medicine, n = ; nephrology, n = ; geriatrics, n = ; rheumatology, n = ; cardiology, n = and endocrinology, n = ). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization ( ps [ ) , early mortality (charlson [ ) and iatrogenic events (drp c ) . results: in the general population, the average age was . ± . years old and the sex ratio was . . the average treatment used was . ± . charlson scores were higher in geriatric unit ( . ± . ) follow by medical interne unit ( . ± . ). the ps and drp scores were higher in nephrology unit respectively . ± . and . ± . follow by internal medecine unit . ± . and . ± . . on contrary the rheumatology unit presented the lower level for the three scores. patients were considered at high risk for three scores, % (n = ) in nephrology unit (almost % of unit), % (n = ) in geriatric unit, % (n = ) in internal medicine unit, % (n = ) in cardiology unit, % (n = ) in endocrinology unit and % (n = ) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january to may . to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: patients were treated with doacs: with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( %). fifty-nine ( . %) patients experienced at least one bleeding leading to hospitalization: with rivaroxaban ( . %), with dagibatran ( . %) and with apixaban ( . %). thirty-eight severe bleeding were identified ( . %): occurred with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( . %). they included intracranial bleeding ( %) and gastro-intestinal bleeding ( %). seven haemorrhages resulted in hypovolemic shock (dabigatran: , rivaroxaban: , apixaban: ) and of them were fatal (dabigatran: ). rates of bleeding (p = . , v test) and of severe bleeding (p = . , v test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab ( g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc ( - units/kg) is indicated. in case of pcc failure, activated pcc ( - units/kg) is suggested. pcc, activated pcc or idarucizumab ( ) were used in / patients ( %). in rivaroxaban and apixaban-related haemorrhages, patients received activated pcc: two had a ui/kg dose and one had a ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was % ( , p [ . , v test) . pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = ) were more willing to prescribe antihypertensive and antidiabetic medication ( . %) when compared to oral anticoagulants ( . %). these values are higher than those obtained by vella in . the majority of pharmacists ( . %) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may ) and mmf (april ). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the data for the first months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith google searches in march compared to searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first and months after rule implementation (from + to + %) except for the micropakin mg. the months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp : vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc : nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *, , anne gerd granås , ragnhild omli , ingela enmarker , aslak steinsbekk nord university/ntnu, trondheim, hioa, oslo, nord university, namsos, norway, department of nursing, Østersund, sweden, ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april , of invited community pharmacies participated in the study. out of documented observation periods, encounters were analysed (first prescription: /refill prescription: ). counselling was provided to ( . %) clients with an average of . (± . ) themes per encounter. a total of clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake ( / ), dosage ( / ) , and administration ( / ). for the pharmacists (n = ), most important themes to be discussed at first prescription dispensing were indication ( ), administration ( ), and anamnesis ( ); for refill prescription dispensing they were adherence ( ), therapy benefits ( ), and adverse effects ( ). the majority of pharmacists ( ) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients ( ), communication problems (language, ), lack of medical data ( ) , and lack of time ( ) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april , community pharmacists across nhs tayside (n = ) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = / ). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (# ), to confirm a patient's prescription (n = ), secondly (# ), for additional information (n = ). less frequently (# ), portal access was to check repeat medications (n = ). other reasons for access were (# ) to check hospital discharge (n = ) followed by (# ) check on multi-compartment appliance aid status (n = ) or (# ) check the emergency care summary (n = ). there were also instances (# ) when portal access was not found to be helpful (n = ) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis Ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia : chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives point. a maximum score of points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis Ò and stored at accelerated conditions ( °c/ % rh) for weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after , , and days. main outcome measures: score according to eight criteria. results: after weeks, tablets/capsules including of the half tablets showed no visual alteration and were identical to controls. a reduced score ( - points) was given to seven repackaged medicines and one half tablet within weeks: madopar Ò ( ), pravastatin sandoz Ò ( ), carvedilol mepha Ò ( ), plavix Ò ( ), pantoprazol nycomed Ò ( ), adalat Ò cr ( ). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc : drug-related problems and symptom burden in nursing home residents kerstin bitter *, , ulrich jaehde , christina pehe , gabriela heuer , manfred krü ger clinical pharmacy, university of bonn, bonn, aok rheinland/ hamburg, pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions ( %). % of the pharmacists' recommendations were accepted by the gp. % of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around % of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between and via centralised procedure were included in the study based on the orphanet list from january . using the quarterly ims health database, sales data were analysed for slovenia and other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, medicines for rd were approved between and . complete sales data were available for medicines which were included in the comparison. for germany and the united kingdom the continuous use of ( %) and ( %) was observed, respectively. the following italy, france, denmark and sweden use - % of all the medicines. in slovenia, ( %) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time - months after marketing approval). median times below months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from to months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in ( . , . and . euros/inhabitant) while slovenia amounted to . euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec : mapping of the dlqi scores to eq- d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq- d. the dlqi is a specialty-specific measure unlike the eq- d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq- d based utility values for use in economic appraisal of medicines. setting and method: data from patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v for the five eq- d dimensions based on age, sex and all individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq- d domain. using these estimated probabilities, a series of monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq- d domain data results: there are conceptual overlaps between items of the dlqi and eq- d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq- d ' mobility' domain, patients answered 'no' (predicted and patients answered 'some or extreme' (predicted ) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were - . , - . and - . for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were patients in the estimation set and in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from . to . across the mc simulations compared to the actual average utility value of . . conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq- d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *, , tanja kersnik levart hospital pharmacy, the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate Ò ), a consecutive case series study. results: ehrs of patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in with suspected icd- diagnoses from i to i were reviewed. patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining patients (average age . years (range - )) received daily on average . medications (range - ) in . individual doses (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most frequently used drugs were perindopril (n = ), ramipril (n = ), amlodipine (n = ), bisoprolol (n = ) and doxazosin (n = ). patients received ex tempore oral suspensions. dosage regimen was not optimized in % (n = ) of the patients, among those % (n = ) receiving one medication only and % (n = ) receiving more than one medication where at least one was not optimized. furthermore, % (n = ) patients on stabledosage therapy (no dosage change of either medication in last months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average . medication (range - ) (- %) in . individual doses (range - ) (- %). the difference was statistically significant ( % ci, p \ . ) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy ( %) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph : polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *, , christos kampolis , aggelos vontetsianos , jennifer mcintosh , alpana mair , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (& % coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, -rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph : clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra , mara guerreiro *, , , nathany pessoa , maria paula athayde , rodrigo auad , joão josé gomes , josé lamartine soares sobrinho post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from / to / (n = ). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male ( %, n = ), with age raging from to years old ( . %, n = ); most had a low education level ( %, n = ) and low socioeconomic status ( %, n = ). the most common clinical presentation was pulmonary tb. most cases were new ( . %, n = ); recurrence and enrolment after discontinuation were respectively . and . % (n = and n = ). with respect to hiv, . % of patients were seronegative (n = ); about a third ( %; n = ) had not performed hiv test. rates for cure were respectively . % (n = ), . % (n = ) and . % (n = ) in the sites a, b and c. correspondent rates for treatment discontinuation were . %(n = ), . % (n = ), . % (n = ), respectively. tb-related mortality ranged from in site c to . % in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september to june . three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = ) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph : accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *, , pascal paubel , , sylvie burnel oncorif -regional cancer network, health law and health economics deparment, health law institute, inserm, umr s , paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum months, age [ , solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. % were treated with targeted therapy, % with cytotoxic agent, and % with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist ( %). while the delivery of treatment, % of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than - months; % of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; % not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist ( %), general practitioner ( %) and pharmacist ( %). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort ( %); being more actively involved in their cancer care ( %); and state not having any anxiety taking chemotherapy home ( %) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph : general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *, , , victoria throfast the pharmaceutical department, kalmar county council, ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . main outcome measures: bmq-general subscale scores. results: a total of elderly people were included in the study, in the intervention and in the control group. the mean age in the total population was . years and % were women. eleven percent did not use any prescribed drugs while % used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was in the intervention group versus in the control group, the median ''harm'' score was (iqr - ) in both groups and the median ''necessity'' score was in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. . % of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', . % stated ''doctors prescribe too many medicines'', and . % stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life ( . % agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph : maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between st october and st december were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between and , maf-plus applications were reviewed, of which ( . %) were approved. of the rejected applications, ( . %) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac ( . %), respiratory ( . %), and psychiatric ( . %) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from in - to by end- . from onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from . % in - to . % by end- , representing an average year-on-year growth of . %. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between and , this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed validated questionnaires for adolescents aged from to at secondary schools in all regions of slovakia. response rate was . %. questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas . . was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group . % (n = ) of adolescents are treated for chronic disease. mostly they suffer from allergy ( . %, n = ) and skin diseases ( . %, n = ). adolescents with chronic disease use regularly prescription medicines ( . %, n = , p \ . ) and over the counter medicines ( . %, n = , p \ . ). this group of adolescents better accept the pharmacotherapy with parental supervision ( . %, n = , p = . vs. healthy adolescents) and they believe in effectiveness of prescription medicines ( . %, n = , p = . vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that . % (n = ) prefer self-medication without check-ups, . % (n = ) used drugs in the last months without a prescription, . % (n = ) take over the counter medications independently without the supervision of parents, . % (n = ) buy medicines themselves in the pharmacy, . % (n = ) do not take medications as recommended, . % (n = ) believe that they have enough knowledge of medicines which they take, . % (n = ) resp. . %, (n = ) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between - / , through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of patients ( , and in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively . % ( ), . % ( ) and % ( ) in the sites a, b and c, while rates for treatment discontinuation were . % ( ), . % ( ) and % ( ), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ . ). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in . we calculated that a population size of patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \ after presentation with usage of one or more prescription drugs. exclusion criteria: age \ years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity ( = no discrepancy to = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a th year pharmacy student. statistical analysis was carried out with a statistical software package (spss ) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category , , or as defined. results: during the intervention measurement patients were admitted to the er. sixty-five ( ) patients ( . %) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by %, from to discrepancies. the average number of discrepancies per patient after intervention decreased by . %, from average . to . discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc : discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a -point likertscale, d) information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer (n = ). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants ( . ± . years, % female, % employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by pharmacists (response rate . %, . ± . years, . % female). there were . % of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc : patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips study rafal yeisen *, , stein opjordsmoen , , , inge joa , , jan olav johannessen , , jone bjørnestad on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that % of patients suffering from chronic illness are not taking medication as prescribed after months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips study, where twenty-first episode patients ( male, female) participated in semi-structured interviews years after inclusion. they were still using or had used antipsychotics during the last years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: ( ) admission experience as a psychotic's patient; ( ) information from healthcare staff; ( ) limited involvement in decision-making; ( ) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c years old, discharged home with a discharge prescription. patients were called between the nd and th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were . ± . years old, % female, % from internal medicine, and % regularly visit the same pharmacy. of the interviewed patients, have not filled their prescriptions yet and had their prescription filled when they were called. of these, % of them visited the pharmacy on the day of discharge, but it took up to the th day until all of them received their medication. supply problems were encountered by patients ( %), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = . , p = . ). patients experiencing supply problems had statistically significant more medicines on a daily basis ( . ± . vs. . ± . , p = . ). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, % of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p y inhibitors for year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p y inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \ %. results: a total of patients were included. ebm drug prescription at discharge was . % for aspirin (n = ), . % for p y receptor inhibitor (n = ), . % for statin (n = ), . % for acei/arb (n = ) and . % for bb (n = , among patients with lvef \ %). ticagrelor ( . %) was the major p y inhibitor prescribed. overall, ebm drugs were missing at discharge, with of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median . months, n = ), aspirin, statins and acei/ arb prescription rates were . % (n = ), . % (n = ) and . % (n = ) respectively. out of patients ( . %) with lvef \ % received a bb. among patients treated with ticagrelor at discharge, ( . %) were receiving ticagrelor at follow-up, whereas ( . %) were switched to another p y inhibitor. among patients who discontinued ticagrelor (n = , . %), duration of dual antiplatelet therapy was months for % (n = ) and discontinued prematurely (\ year) for % (n = ) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at year remains high with more than % of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in % of patients, whereas ticagrelor was switched for another p y inhibitor in . % of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october -march were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version- toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period visits of patients were evaluated. a total of side effects assessments were recorded. among those assessments ( . %) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = ; ), sensory loss (n = ; ), fatigue (n = ; ), stress (n = ; ), insomnia (n = ; ) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc : implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *, , signe kristensen , lisbeth lund pedersen clinical pharmaceutical services, capital regional pharmacy, head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may , a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of beds. inclusion criteria were patients to which two or more of the following apply: • c years of age • c drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may th and june th , patients were screened at admission to the apw, of which . % met the inclusion criteria ( patients). in this period the pharmacist conducted notes, indicating that . % of the included patients were seen by the pharmacist. in april , patients were in average admitted with . drugs and . inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during months, in a private hospital belonging to a group devoted to healthcare for over years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has inpatient beds, two surgical blocks with rooms and beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: . logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. . furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. . in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. . clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of interventions, relate to revenue optimization and relate to clinical interventions. there was an increase of approximately % in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: ± min) was performed in patients ( . % female; av.(±sd) age: . (± ) years; av.(±sd) medicines at discharge: . (± . )). in % of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support ( . , e.g. proper prescriptions' writing), therapy-related discussions ( . ), untreated indications ( . ), errors in documentation ( . ), and medicines without an indication ( . ). patients ( . %) classified for study inclusion, of whom ( . %) consented to be followed-up and ( %) provided data. roughly % of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient ( . %) reported having received valuable new information. changes in chronic-use medicines occurred in . , . , and . % of patients at -, -, and -month, respectively. at -month, in . % of patients chronic-use medicines were newly prescribed, in . % discontinued. medical specialists initiated these changes in . % of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly % of patients showed moderate to little medication adherence at -month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until , prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since . many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first months of and at the same period of the year in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. . % in versus none in . when prescription analysis was based in the pharmacy unit, % of drp detected by the pharmacist had a potential clinical impact versus % when the pharmacist performed prescription analysis in the care unit (p \ . ). three drp detected in had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc : qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *, , hans van den ameele , jü rgen de fruyt , yves vandekerckhove , frank vancoillie , veerle grootaert pharmacy, psychiatry, cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp and asp ), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october st and march th on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg ) was performed and creatinine and potassium levels were measured. a second ecg (ecg ) was performed at least days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as ms for males and ms for females. clinically relevant qtc-prolongation was defined as c ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: patients (mean age years, %female) were enrolled in the analysis. in patients, an ecg was performed. qtc + were prolonged in . %( / ) of females and . %( / ) of males. no clinical relevant prolongation (c ms) was registered. higher qtc intervals were measured in the geriatric population. . %( / ) of all measured qtc were situated between [ c qtc + b ms] in gpw versus . %( / ) in the other units. significant difference in qtc-changes was associated with sex (p = . ). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ . ). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([ years). hp-pc : implementation of medication reconciliation aase m. raddum *, , anne-lise sagen major sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, avd. volda sjukehus, volda, sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the patients included, a total of discrepancies were revealed. in summary, patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of % of the included patients. at the start of the study, the level of medication reconciliation varied among the wards ( - %), while at the end of the study the levels were increased ( - %). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from / / to / / . we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was . % ( / ). the most consumed herbal products were (prevalence [ % among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: . , . , . and . %. we detected herb-drug interactions or potential for hepatotoxicity in out of patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from / / to / / , in a stable hd population to compare is and iss. the follow-up periods lasted weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p ) and the second (p ) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p and p per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of patients were included. there was no significant difference in mean hb value between p and p ( . ± . mmol/l versus . ± . mmol/l p = . ). anaemia parameters were significantly different between p and p (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ . , except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p and in p : . ± . mg versus . ± . mg (p = . ) and . ± . ui/kg/week versus . ± . ui/kg/ week (p = . ). transfusions occurred less frequently in p than in p (p = . ). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject Ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient h a day, days a week. setting and method: an online and anonymous survey of questions was sent to the residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them % never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: st year (n = ), nd year (n = ), rd year (n = ) and th year (n = ). only % know where a medicine is injected intrathecally on the spinal column, % know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: % answered vincristine, % vinblastine, % bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only % for the cytarabine'one. only residents out of know that ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject Ò mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine Ò (cytarabine) mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is . / compared with . / for those who never did. the respective scores per year of internship are . ( st year), . ( nd year), . ( rd year) and . ( th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of (high) to (low observance) (srap- ) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included patients, % were women and % were diabetic ( % suffering from type diabetes). the mean age was ± years, the average number of medication was . ± . . ( %) patients were not able to estimate their adherence. patients reported srap- scale with an average of . ± . , this estimation was significantly higher than mmas- with an average of . ± . (p \ . ). the proportion of adherence level were identical between srap- and mmas- with respectively and % of high, and % of medium and and % of low adherence. a significand correlation between srap- and mmas- scales (r = . , p \ . ) was found. however no correlation between adherence scale and number of treatment (r = . for mmas- and . for srap- scale) nor number of daily doses (r = . for mmas- and . for srap- scale). on the medications, % presented difficulties with observance. cardiovascular ( . %), diabetes ( . %) and psychiatric ( . %) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap- and mmas- scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc : the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year . in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah . these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc : improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society beers criteria'' and ''stopp-start criteria. design: retrospective observational study that included patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in patients from the original group of , we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start and beers ) . all data were registered, measured and analysed in excel Ò . results: patients and a total of mp were reviewed. discrepancies ( . %) were found between the medical order and the prt, those discrepancies included errors of omission in prt ( . %), absence of discontinuation of medication ( . %), incorrect dosage ( . %). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) ( . %), benzodiazepines (bzds) ( . %), oral hypoglycemiants ( . %), other groups with frequency over %, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. stopp criteria were identified that affected to mp and the distribution was as follows: laxative combinations ( . %), long term ppis ( . %), cns depressants combinations ( . %), long half life bzds combinations ( . %), aspirin incorrect drug strength ( . %) and other groups with lower frequencies, nsaids and prokinetics. start criteria: being all of them by omission of the drug at the time of admission. beers criteria: prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over / , make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant ( transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients ( ) to get them ready for transplant ( ) to give the key messages to support their transplant ( ) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for days of shooting. we defined the key points for the patient and places to film, and fixed the duration ( - min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the days of film shooting, we selected sequences. results: the movie was a succession of six parts. ( ) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at general medicine wards at oslo university hospital for a period of weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of min, were given to doctors and nurses as part of daily ward routines. this was repeated over weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i- analyser, abbott Ò ) and fitted to a two-compartment model by using bayesian analysis (pks Ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss Ò , version . was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration * /daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: adult patients were included (females: %); median age [ - ] years).the first and the second tdm were carried out after . [ . - . ] and [ . - . ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations ( . ( . ) lg/ml) and those predicted ( . ( . ) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs ( . ( . ) mg/l vs. . ( . ) mg/ml, p \ . ). the median cdr in the second tdm was significantly higher than that calculated in the first one ( . [ . - . ] l - vs. . [ . - . ] l- ; p \ . ), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found ( [ - ] ml/min vs. ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a -year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since with a body mass index of . kg m - , pulmonary tuberculosis in with relapse in , and chronic pulmonary aspergillosis since . at admission, a treatment by oral voriconazole at mg/ h was introduced. the trough concentration of voriconazole at steady state was . mg/l (therapeutic range - mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in mg/ h, the trough concentration did not increase significantly ( . mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole mg/ h). according to subtherapeutic voriconazole concentrations (trough concentration: . mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p a * , c * and c * , particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype ( * allelic variant of the c isoenzyme) in our patient should lead to increase drug dosage from to %. finally, the patient was treated by intravenous voriconazole at mg/kg/ h (i.e., increase by %). the maximum concentration performed h after iv route initiation was at . mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q ; q ] = . [ . ; . ] years; % female) were included between june and march . eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension ( . ± . l - vs. . ± . l - , p \ . ). overall, pcz therapy initiation led to an increase in tac c min /d ( . ± . l - before initiation vs. . ± . l - after initiation, p = . ). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension ( . ± . l - vs. . ± . l - , p \ . ). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = . , p = . with pcz enteric-coated tablets and r = . , p = . with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp a inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an years old male, admitted to the neurology unit (d ) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose % infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal ( . g/l). heparin was stopped at d and replaced with apixaban according to following dose regimen . mg twice a day. at d , patient presented with hyperleukocytosis (neutrophils count g/l) and high crp ( mg/l). thus, a cytobacteriological urine test was performed. at d , patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count . g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d . the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a years old male, admitted to the cardiology unit (d ) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d by an oral treatment with cefixime and spiramycine until d . heparin was replaced by apixaban at d ( . mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d (neutrophils count . g/l), followed by agranulocytosis at d (neutrophils count . g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about . g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, ( ) is there a difference in the age of conversion from liquid to solid formulation and ( ) is there a difference in re-prescription rates on day and after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children - years registered in the norwegian prescription database (norpd) - due to dicloxacillin mixture being discontinued from the norwegian market in . the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day and after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was . years, compared to years for other common antibiotics. the average represcription rate for dicloxacillin was . % for children - years and . % % for children - years. the highest re-prescription rate of . % was found in -year olds. corresponding numbers were . , . and . % for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children - years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * million inhabitants of the five nordic countries in the period - . data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from to , both in volume and prevalence of use. denmark had the largest increase in volume, from . to . ddd/ inhabitants/day. sweden had the highest increase in prevalence of use over the period, from . to . users/ inhabitants. iceland had the largest consumption of adhd medications in , . ddd/ inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in : . users/ inhabitants. males aged - years had the largest volume and prevalence of use in , but females' consumption had been increasing faster both in terms of numbers of users (* . faster) and in volume (* faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in and , regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to days while fda concluded that treatment longer than weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production ( days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase Ò for the time period november to may was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and % confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over million adr reports. metoclopramide is a suspected/interacting drug in of the reports, most common ( %) are neurological adrs. the majority ( %) of the metoclopramide adrs occurred within the first days of use. a total of % of the reports was received the last years ( ) ( ) ( ) ( ) ( ) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age ( - years) reported higher proportion of neurological adrs (prr = . , % ci . - . , n = ) than women + years (prr = . , % ci . - . , n = ) but a similar proportion as men - years (prr = . , % ci . - . , n = ). conclusion: there is a . to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service ( %) with around - / inhabitants and represent around % of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under . the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over months, including all icu patients following sidi from june to january . we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing % of icu admissions. the average hospital stay was days for an average cost of . €. this amount is low compared with the average cost of all stays gone through the icu for the period ( , €). ninety percent of patients had a psychiatric history and % a previous sa. the usual treatment was involved in % of sidi. half of the patients arrived conscious with an average of severity score igsii of / , being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period ( / ). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines %, neuroleptics %, antidepressants . % and antiepileptic . %. the main drugs involved are oxazepam %, alprazolam %, cyamemazine %, bromazepam % and quetiapine %. antihypertensives then arrive and represent % of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november were identified. six databases were examined with search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of screened article abstracts, articles were selected for detailed review: trials met the eligibility criteria, describing research on a total of , patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : - = , - = , and - = ( ) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and ( ) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = ) of patients, that underwent sleep studies between the years and were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy ( . %) patients of the patients ( males, females) were diagnosed with osa. forty-five ( . %), ( . %) and ( . %) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of years. angiotensin ii receptor antagonists (arbs) (p-value = . ), sulphonylureas (p-value = . ), insulin therapy (p-value = . ) and non-benzodiazepine sedating agents (p-value = . ) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = . ), angiotensin converting enzyme inhibitors (p-value = . ) and non-benzodiazepine hypnotics (pvalue = . ) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h ) antagonists (p-value = . ), b-adrenergic blocking agents (pvalue = . ) and antiplatelets (p-value = . ). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has , members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: pharmacists participated in the study, . % of the participants were a member of our facebook group and . % of participants were not. . % of the participants have doctoral degree or student, . % have master degree or student, % have bachelor degree from year-pharmacy faculty, . % have bachelor degree from year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while . % of the ams questions were answered correctly in the member group only . % were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce : impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho , , hung-wei lin *, , fang-ju lin , , sheng-ping chang , yen-ming huang graduate institute of clinical pharmacy, school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: ( ) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and ( ) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p ) year. setting and method: a modified chisholm's pharmacy professionalism instrument ( -item, -point likert scale) was administered to p students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity ( . ± . ) and communication ( . ± . ) were recognized by students ( . %) and pharmacists ( . %), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, . ± . ; pharmacists, . ± . ). the -item professionalism scores ranged from . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after year of implementation. design: in january , we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre ( beds). design: a prospective survey was realized over a period of months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from opened up for paid cognitive services, leading to the first such service being implemented in march . the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in pharmacies in oslo, akershus and buskerud in november/december . the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in of the pharmacies, whereof asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march , will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over , deaths in ( % of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at , , and months. results: between january and june , smokers joined the programme, dropouts ( . %). the remaining smokers, ( . %) were male, with mean age of . ± . years. on average, each smoker consumed . ± . cigarettes per day. the mean age of initial tobacco use was . ± . years with . ± . years of consumption. about % reported moderate or high motivation and % medium or high dependence. a total of consultations were held and, on average, each patient received . ± . interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and ( . %) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by patients ( . %). a month after quit date, patients were abstinent ( . %). the number reduced to after months ( . %), to after months ( . %) and to after year ( . %). these data upgrade and are consistent with our previously published results ( ). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc : patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than %'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads ( %) and even more acknowledged that they were uncomfortable with managing patients' side effects ( %). on the other hand only % of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists ( %) have not attended any of ce events related to oncology in last years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in , the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm ; : - .). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july . please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas- ). patients who scored b points, . - . points and points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september and february . the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version . main outcome measures: medication adherence rate (mmas- ), quality of life (sgrq total score) and impact of disease (cat score). results: of patients, majority were men ( %) with mean age years. in average, patients were prescribed . medicines for copd and . medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in . , . and . % of patients, respectively. mean cat scores and sgrq scores were . (range - ) and . (range - ), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; ( ) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) ( ) the type and amount of drug related problems (drp), focusing inappropriate drugs, and ( ) if there are differences between the icmu and stcu in drug use and drps. design: patients c years old admitted and submitted at the imcu or stcu in the study period ( weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was . points out of . by submission from either imcu or stcu, the score was . . systematic drug review identified . drp in the imcu and . in the stcu. imcu patients used . drugs, stcu patients . in the icu, % of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented % of the identified drps. the doctors agreed in % of the suggestions for solution, and started immediate changes in %. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged years or older using or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now patients have been included in the drea-mer study ( % of the target). half of them ( ) were randomized into the intervention group. by now ( %) of these patients have received a patient interview. goal attainment scales were formulated yet. the number of gas ranged from to per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines ( ), reducing pain ( ), increasing mobility ( ), reducing fatigue ( ). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc : oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september , two quizzes were sent to the pharmacists and pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the pharmacists who participate in the survey, % know the all of the oftc specialties, % of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by , , and % of them. only % have dispensed oftc more than times over the past months, % never have. they already have dispensed oftc in noncancer patients ( %) or without opioid maintenance treatment ( %). they consider not know enough about these drugs to be able to provide the necessary advice to patient ( %) and would like specific training on oftc ( %). of the analyzed prescriptions, only % are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient ( %) and/or dosage is higher than four units per day ( %) and/or there is no prescribed opioid maintenance treatment ( %). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = ), dose adjustment (n = ), and clarification of information (n = ). common services were reconstitution of suspension (n = ), provision in advance for continuing supply (n = ), and follow up offers (n = ). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january to april . the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. ) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age ± years old) were included in the study, which represents % of the patients hospitalized in this service. the mean number of drugs per patient was ± . the bpmh were obtained within h to h of admission to hospital. a total of ids were detected, with a mean of . ids per patient. the most frequent type of ids was omission ( %, n = ), error of dose ( %, n = ). the three most common classes involved in ids were hypolipaemic drug (n = ), antidiabetic drugs (n = ) and the drugs for acid related disorders (n = ). the mean of ids per patient ( . ) as well as the percentage of patients affected by a ids ( %) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, % of patients presented with ids considered as minor, % significant and % major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in community pharmacies. questions to assess potential medication problems covered three domains; medication adherence ( ), practical management issues ( ) and medication knowledge ( ) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: patients starting with mdd and patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence . versus . , practical management issues . versus . , medication knowledge . versus . . on the three domains together, patients starting with mdd scored . [ . - . ] potential medication problems compared to . [ . - . ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and % was classified as frail compared to and % respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc : fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round (online survey; importance): the questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round (online survey; relevance): the questions were to be ranked according to decreasing relevance. round (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions ( = most important/relevant) in march/april . results: experts ranked adherence ( . ± . ) as the most important topic, followed by risk-benefit ( . ± . ), disease ( . ± . ), accessing healthcare professionals ( . ± . ), self-care ( . ± . ), lab-monitoring ( . ± . ), medication interactions ( . ± . ), diet/life-style ( . ± . ) and mode of action ( . ± . ). one question was judged as unimportant by all experts. out of the remaining questions, ( . %) were selected as relevant for basic knowledge, ( . %) were combined into four questions and one new question was generated. a total of key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december and an education program was set to assure pharmacists competencies. in june the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v . [ ] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on mur cases, performed by certified pharmacists. in total drps were identified: ( . %) manifested and ( . %) potential. patient had on average two drps, however patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented . % of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in . % of cases; times recommendations were given to physicians. however, in . % of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may the first pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february . guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo . written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc : evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of % and error of margin of %. the study was conducted over a total of pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited . % of them had female pharmacists and . % were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was . ± . . only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two ( . %) pharmacists recommended various medication regimens, including antibiotics. of them, . % referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky -item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: - points = full adherence, points = partial adherence and - = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of min and used the average value in data sheet. results: the research included hypertensive patients ( . % females and . % males). the results showed that almost % of the respondents were non-adherent to the prescribed pharmacotherapy ( . % of those were males and . % were females). the group of partially adherent patients consisted of . % of the respondents ( . % of those were female). only . % respondents were fully adherent according to modified morisky score ( . % of those were women). fully adherent patients reached an average blood pressure . / . mmhg; partially adherent hypertensive patients recorded an average blood pressure . / . mmhg; and in the non-adherent patients has been observed the average blood pressure . / . mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified -item morisky score (mmmas- ). main outcome measures: respondents were patients ( males, females) using at least one antidepressant. the results were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was , which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males ( ) compared to females ( ). the highest level of medication adherence ( ) has been shown in patients - years old, the lowest average adherence level (non-adherence) was observed in patients up to years old ( ). patient living in the city were more adherent to their medication ( ) compared to patients living in countryside ( ). the highest level of the partial medication adherence has been shown in secondary educated patients ( ). partial adherence level was higher in patients with monthly income over € ( ) compared to non-adherent patients with monthly income up to € ( ). in patients using no other medications, only antidepressant, we have observed the highest partial adherence ( ). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. ( ) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) ( ) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects ( . ± . ; male) were included in this study. of them, . % had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in . % of them. the mean of mrci per elderly patient was . ± . . one or more pims use was observed in seventy-four elderly subjects ( . %). of all elderly subjects, . % were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy ( . vs. . %, p \ . ). the total score of mrci was significantly increased with elevated number of pims (r = . , p \ . ). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for long-term patients, on average years old, and % females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, % of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, , and %, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for % of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = ), oxycodone (n = ) and zopiclone (n = ), where , and % had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc : personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as %. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december to march a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included participants, most of whom ( . %) were women. participants average age was . ± . years and the median arterial hypertension duration was ( ; ) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, ( ; ) vs. ( ; ); p = . . group of respondents (n = ) receiving c prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = ) treated with b prescription drugs, respectively . versus . %; p = . . respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = ) compared to the group (n = ), which had to intake fewer tablets every day, respectively, . versus . %; p = . . conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c ) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for in belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in belgian pharmacies to accompany diabetes patients taking dpp- inhibitors and encourage them to be compliant with their treatment. this study concerns of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of pharmacies, patients were included in the program. by the end of april , only of them had completed the program; patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups ( . - . % of patients with mmpr b %). only for the pdc, a statistically significant difference was measured between the intervention and control group ( . vs. . %; p = . ). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm ( ) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: ( ) has the member information, position, telephone and address, ( ) email forum, following a subject line, ( ) a place for meeting requests and then hang up the meeting minutes. ( ) a tool allows you to upload documents to the portal ( ) a search engine. two sessions are schedules and total of mails. we have of members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january and june , in our university hospital, % ( out of ) of the declared adverse drug events have involved insulin: were caused by prescription errors and by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january and december . for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: % in (before the table's diffusion), % in (after its diffusion), % in and % in . prisons units are responsible of % of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: % ganfort Ò (bimatoprost . mg/ml/timolol . %), % xalacom Ò (latanoprost + timolol . %), % azopt Ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost Ò (latanoprost) + ophtim Ò (timolol . %) and for the third: dorzolamide Ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; month prospective study through interviews, at time zero (t ) and after days (t ) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number / . main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of patients ( . % female, mean = . years), . % considered as relevant the leaflet's content, as well . % of them kept it and . % of them read it. suggestions of . % included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times ( . % e . %), however there was a decrease in high adherence to medicine treatment ( . - . %). among patients who read the leaflet, no statistically significant association was found on these two variables at t and t (p = . and p = . , respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = . ). . % of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di : chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *, , anne lefebure , sonia martelli , marion pin , eve maubec , philippe arnaud pharmacy, dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september . the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients ( women, men, mean age [min ; max ]) were treated with chlormethine gel in our hospital. twelve ( %) were treated three times per week, ( %) once a day. before treatment by chlormethine gel, ( %) patients were treated by dermocorticoids, ( %) by dermocorticoids and phototherapy, and ( %) by bexarotene, all of them stopped their treatment on account of inefficacity. one ( %) patient was treated by carmustine and dermocorticoid, and ( %) by only carmustin, all of them stopped it because of supply disruption. one ( %) patient received it in first line therapy. ten ( %) patients showed a response (partial or complete), one ( %) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients ( %) had an involved bsa [ % and four of them ( . %) experienced adverse effects. seven patients ( %) had an involved bsa \ % and three of them had ( . %) side effects. a total of seven patients ( %) presented at least one adverse effect. five patients ( %) stopped the treatment on account of adverse effects; two of them ( %) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity ( ), rash ( ) and telangiectasia ( ) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports % of adverse effects for chlormethine solution versus % in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than % of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *, , capucine devaux , nathalie le guyader diaconesses croix saint-simon hospital, diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin- receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p (cyp) c and moderately inhibits cyp a . drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp a and c were identified. pharmacokinetic literature review was performed using medline Ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp a substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp c catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and hydroxytryptamine type ( ht- ) receptor antagonists are also metabolised by cyp a . aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with ht- receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = ) was obtained from pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = ), muscular pain (n = ), headache (n = ) and arthritic pain (n = ) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by of the patients, of which use the mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = ), heartburn (n = ), dyspepsia (n = ), asthma (n = ) and a history of helicobacter pylori infection (n = ). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = ) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain ( . %), stomach ulcers or gi bleeding ( . %) and elevated blood pressure ( . %) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives ( . %) and ssris ( %) regardless of their significant risks of interacting with nsaids. . % of the pharmacists and doctors believe that nsaids are being over-used and . % state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp c ). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january and april in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: . , mean age: . years [ ; ]. ppi therapy prevalence over the period was %. the ppi were prescribed in the geriatric department in patients (mostly esomeprazole) whereas patients had ppi therapy (mostly esomeprazole) at the admission, for more than years in patients. oral route was the most frequent one (n = ). ppi were administered once a day and only three ppi were administered in the morning. % of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = ), prevention of haemorrhage with corticoid (n = ), prevention of haemorrhage with anti-vitamin k (n = ), dyspeptic disorders (n = ), gastralgy (n = ) and others reasons (n = ). % of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = ) lead to prescribe another dosage (n = ), to stop therapy (n = ) or no change (n = ). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline ( mg followed by mg every h) a tertiary care hospital. design: retrospective observational study. period: january to december . inclusion criteria: episodes use of tigecycline ( mg followed by mg every h. exclusion criteria: time less than days treatment. data source: corporate program stories electronic health. results: we identified episodes in patients ( men, mean age: years ( - )). treatment was directed to multidrug-resistant organism infection in cases (seven klebsiella pneumoniae oxa- , two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was ( - ). tigecycline was administered as monotherapy in three cases, bitherapy and triple combination therapy in . the antibiotics were associated were: beta-lactam ( ), aminoglycosides ( ), quinolones ( ) colistin (three, two inhaled cases), cotrimoxazole ( ) and vancomycin ( ) . in episodes produced clinical and/or microbiological resolution and antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa- infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. patients showed an adverse effect possibly related to therapy tigecycline: diarrhoea after days of treatment and case of liver toxicity after days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm ( ) : - conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di : wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke , kristian svendsen *, department of history, uit -the arctic university of norway, hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in and the ten most recently approved medicines on the european market (until december ). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on january and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june , . data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december ( medicines), information sources on medicines: ( ) vademecum of medicines and ( ) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: patients were included in the professional service of the mds. of them were institutionalized (average age: . years, . % men, . % polymedicated defined as using c prescribed chronic medicines) and the remaining were ambulatory (average age: . years, . % women, . % polymedicated). different medicines prescribed in the institutionalized patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines were eligible for repackaging, medicines could be repackaged according to the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones could not. different medicines prescribed in the ambulatory patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines could be repackaged, medicines would depend on the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones must remain in the original package. discrepancies were observed in the information for ( . %) and ( . %) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills ( . ± . %), public health promotion ( . ± . %) and patient counselling ( . ± . %) than in resolution of drps ( . ± . %) and pharmacotherapy application ( . ± . %), while they significantly enhanced in di manipulation ( . ± . %) compared to baseline assessment ( . ± . %)(p = . ). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *, , lea boissinot , ngauv melodie , stephanie charles weber , isabelle lopez , françois chast pharmacy, hopital cochin, pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: patients completed the questionnaire. the average patients age is . ± . years. all of those interviewed could name their disease. for % of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress ( %), fatigue ( %) or an emotional shock ( %). oedema were located mainly in extremities ( %), abdomen ( %), ent sphere ( %) or face ( %). patients ( %) reported having more than crisis each year (eligible to prophylaxis), among them, patients ( %) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the patients receiving treatment for crisis, were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid ( %) and danazol ( %). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c esterase inhibitor (intravenous) were self-administrated respectively in and % of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september . in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and came out with an oa. one hundred and forty-three answers were collected: officinal pharmacists and nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than % of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss Ò, version . the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', % were considered as the pharmacists' main responsibility, whereas % were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', and % of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of % and supportive that of % of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = . ); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = . ). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april . pi and data were extracted from the software pharma Ò and collected in a summary excel Ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over months, lines of prescriptions from patients aged years old (median [ - ]) were evaluated. sex ratio (m/f) was . . one hundred and two medication problems have been found: overdose ( . %), contraindication (ic) ( . %), under dosage ( . %), wrong rhythm of administration ( . %), forgotten treatment ( . %), dose unit error ( . %), antibiotic indication missing ( . %), drug not listed in the hospital formulary ( . %), potassemie unchecked ( . %), dose unadapted to renal function ( . %) or to inr ( . %), treatment not indicated ( . %), wrong administration route ( . %), antibiotic unreevaluated ( . %), redundancy ( . %). the proposals made to the doctors were: stopping treatment ( . %), posology adaptation ( . %), substitution ( . %), dose unit modification ( . %), adding information about the indication ( . %), treatment renewal ( . %), administration modalities changing ( . %), biological monitoring ( . %), therapeutical monitoring ( . %), antibiotic treatment reevaluation ( . %). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was %. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in , france was the nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level ( : approved by the ma (marketing authorization), : non-valid but certified by international publications or learned society, : nonvalid without scientific proofs, : non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications ( and ) . results: patients have ppis prescriptions ( male, £ years] among patients ( %). % ppis prescriptions began during the hospitalization. ( %) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level ( . %), indication level -gi bleedings-( . %). of the ppis prescription aren't in accordance. details: treatment duration ( %), dosage ( %), indication level -prevention of iatrogenic bleeding risk without nsaids prescription-( %), indication level ( %). regarding level indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level . the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication and ). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce : impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence ( . million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september , individual educational sessions have been set up and since september , collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, ibd outpatients participated to the educational program, patients with crohn's disease and patients with ulcerative colitis ( . % male; median age: ). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce : desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel  . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between and . we included only patients with moderate to severe immediate hypersensitivity reactions (b h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution : , : and : ) in twelve successive steps for about h. results: in the period - were desensitized five patients with platinum group drugs, carboplatin (n = ) and paclitaxel (n = ) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a -month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm ( ) : - the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, patients were included corresponding to prescription lines. reconciliation process required about min per patient. we identified at admission unintentional discrepancies. the most common unintentional discrepancy was the omission of medication ( %). % concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required min per patient. % of healthcare professionals answered to our satisfaction survey to date. % are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment. healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in among which are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs Ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in , healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for - teenagers and adults. every ''malle des savoirs Ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in , we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included - % experienced at least one unintentional medication discrepancy. of the medications used by the patients, - % were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, - % were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april to december . the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were , and % respectively. the proportion of medications involved in undocumented changes for the three subgroups were , and % respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ . ) and at discharge (p \ . ). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than days (p \ . and p: \ . respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ . ). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce : participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *, , erik oie , kirsten k viktil diakonhjemmet hospital pharmacy, department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c years participating at a days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september -december . the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale - ) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age years, % were women, using a mean of . cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas and % had high concern immediately after and months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, % immediately after, and % months after the course. the mean score for bmq-necessity was . (sd . ) prior to course and . ( . ) and . ( . ) immediately after and months after the course, respectively. the corresponding scores for bmq-concern were . ( . ), . ( . ), and . ( . ), respectively. the proportions of patients classified to be 'accepting'were , , and % at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were , , and %, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of narratives were collected ( ups and hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce : impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *, , jacques de grève , veerle foulon , hilde collier , pieter-jan cortoos pharmacy, medical oncology, university hospital brussels, brussels, faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory ( beds) and in-hospital onco-/haematology unit ( beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: patients were included over a period of months (control (n = ); intervention (n = )). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = . ; p = . ). multiple linear regression analysis showed a similar trend (b = . ; p = . ). patients receiving first-line therapy (b = . ; p \ . ) and ambulatory patients (b = . ; p = . ) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file ( vs. . drugs/patient; p \ . ). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high ( %). during the study, interventions shifted from therapy adjustments towards advice on supportive measures ( st contact: %; rd contact: %). improved medication stock control on the ward led to a savings of € , . conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july . hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey Ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a -item likert scale was used to answer to statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of pharmacists responded ( % in quebec vs. % in france). the global response rate is: % ( % quebec vs. % france) (n = / ). % attended at least to one conference on biosimilars ( vs. %). % had already dispensed biosimilars ( vs. %). more than % of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. % considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product ( vs. %). there was no difference between the level of agreement of french and quebec pharmacists for the statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary ( vs. %), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar ( vs. %), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product ( vs. %). pharmacists disagree that a biosimilar can be used for all the indications of the reference product ( vs. %). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = / ) had a computerized register of narcotics and % of care units (n = / ) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = / ) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash . % (p \ . ) with occurrence in males (n = ) and females (n = ), skin erythema . % with equal occurrence in both genders, followed by skin discharge . % with also equal occurrence in both genders. a high occurrence of skin rash . % occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash ( . %), skin discharge ( . %), and skin erythema ( . %). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c years old, treated with c drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first -hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost drp in % of patients, a sample size of patients should allow an estimate of drp rate over % (need of intervention) with a % power and a confidence interval of % (software stata version . ). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/ -february/ , inpatients ( male, . mean age) were included. overall, patients were admitted with drugs used at home and prescribed during the first -hours; pharmacists retrieved discrepancies ( %id, %ind, %ni) and drps, of which % ddis, % off-label, % overdoses, % duplications, % inappropriate drugs, % not notified adrs. the % of drps was known to clinicians and % considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: participants were included, of which ( %) participants had mi-htn. two-thirds were female (p = . ) with a mean age of ± years (p . ), of whom . % had uncontrolled hypertension (p = . ). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [ were statistically associated with a greater likelihood of reporting medicines intolerance (p \ . ). patients who believed that medicines are harmful were [ -times more likely to report mi-htn (p = . ) and -times more likely to have uncontrolled bp ([ / mmhg) (p = . ). patients with high self-perceived sensitivity to medication was -times more prone to mi-htn (p = . ). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between and ( years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of patients met the inclusion criteria and prescriptions were analysed. each prescription contained an average of drugs. a total of drug-drug interactions were identified: . % was classified as moderate; . % as major and . % as contraindicated. almost half of all interactions (n = ) included immunosuppressive agents ( . % was classified as moderate; . % as major and . % as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type diabetes. patients who were diagnosed with type diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on patients who met the inclusion criteria; none of the patients were lost to follow-up. majority ( %) of the patients was female and the mean age was . years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by mmhg, while diastolic blood pressure decreased by . mmhg (p \ . ). hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by . mg/dl (p [ . ). on the other hand, the number of patients reaching the blood pressure goal increased from to ; and those reaching to hba c goal increased from to (p \ . for all). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc : impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type diabetic patients residing at a nursing home nimet saglam *, , sule apikoglu-rabus , betul okuyan , fikret v. izzettin , nuran yildirim clinical pharmacy department, marmara university faculty of pharmacy, darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for months. it consisted of an initial visit, followed by ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority ( %) of the patients was male and the mean age was . years. pharmacist intervention resulted in positive outcomes regarding hba c levels. hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by mg/dl (p [ . ). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january to june ). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose- -phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss . (spss inc, chicago). observation period was months and data were recorded from the clinical records. ( ) . %, sofosbuvir/daclatasvir/ribavirin ( ) . %, sofosbuvir/simeprevir ( ) . %, sofosbuvir/ledipasvir ( ) . %, sofosbuvir/ledipasvir/ribavirin ( ) . %, dasabuvir/ombitasvir/paritaprevir/ritonavir ( ) . %, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin ( ) % ombitasvir/paritaprevir/ritonavir/ ribavirin ( ) . %, sofosbuvir/ribavirin ( ) . %. viral load at week was \ iu/ml in patients and at the end of treatment . conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: -year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with medicines as home treatment. patient was hemodynamically stable during the hospital stay. discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified ( ): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified ( ): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of patients were included and of these % had c discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in % of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february th, -march th, . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about files were examined, contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin ( %) and the most frequent indications were urinary tract infections ( %). among the prescriptions of fq, the establishment of fq was justified in % of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, % did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by - % per year for years. it is estimated that worldwide, more than . million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn . main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total patients for a -months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since and has a leading role for studies conducted in italy (vaccher, ) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, neutral and negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ) please specify your abstract type: research abstract background and objective: the world health organization reports that . million people die each year from cancer, an estimated % of all deaths worldwide and that there is a % increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcomes indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians' satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ), costs ( ) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome int j clin pharm ( ) : - measures. of these outcome indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), side effects management ( ), mortality ( ) and others ( ). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and % of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc : impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between and million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january st until july th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, ( %) neutral and negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost ( ), errors ( ), morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = pill dispensers), . % of pill dispensers had an incorrect cdat status. on average, . drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = ) were . and . respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june in wards. among the patients included, patients ( . %) were treated with antimicrobial agents. patients ( . %) were treated with broad spectrum antibiotics: ( . %) with amx-ac, with pip-tz, with imipenem and with meropenem. the mean age of patients was . ± . and the weight was . ± . kg. their prescriptions were concentrated in three types of wards: ( . %) in icu, ( . %) in medicine, ( . %) in surgery. moreover, we observe that bsa were used to treat ( . %) community acquired infections, ( . %) nosocomial infections, ( . %) used as medical prophylaxis, or surgical prophylaxis (n = , . %). in relation to the type of treatment: were empirical treatment (including prophylaxes) and were targeted treatments ( bacteraemia, joint and bones infections, cardiovascular system infection, urinary tract infections, lower respiratory tract infections, skin and soft tissues infections and others infections). finally, extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a -months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a months follow-up. results: patients were included. among them, % have been taking a bzd for more than years, and % were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. % of the subjects were frail and % pre-frail according to the fried criteria. at the end of the study, % of the patients had their bzd treatments optimized, including % of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a month period. hba c and weight were significantly reduced by . ± . %, p \ . and . ± . kg, p \ . , respectively; systolic bp ( . ± . mmhg, p \ . ), diastolic bp ( . ± . mmhg p \ . ) and triglycerides ( . ± . mg/dl, p . ).genital-and urinary tract infections were reported by . % patients. any diabetic ketoacidosis case was reported. conclusion: sglt- inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt- inhibitors, appears to be an important addition to the therapeutic options for the management of type diabetes, particularly when used as add-on therapy. ( ). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline- to may -with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of titles, and abstracts when necessary, were screened for eligibility. after reviewing process, studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among patients versus , suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a -day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase , patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the -day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the -day multi dose pill box before and after its use. in phase , the chosen patients were given a demonstration on how to use the -day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the patients recruited, were male and were female. the mean age was years (range - ) and the mean number of daily medications (range - ). upon initial scoring using mars, patients were adherent and patients were nonadherent. a higher adherence was observed in patients taking or more medications daily. ten patients accepted to move on to phase of the study and took the device home to use for one week. out of these patients, felt that the way they take their medication improved following use of the device and out of patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ . ). there was no significant association between level of adherence and type of psychiatric condition (p [ . ). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ . ). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. ( ), sofosbuvir/ledipasvir ( ), ombitasvir/ paritaprevir/ritonavir ( ), ombitasvir/paritaprevir/ritonavir + dasabuvir ( ), simeprevir + ifn ( ) . twenty-six patients ( %) were treated for weeks ( week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of %. the interventions included treatment adjustments due to drug interactions ( ), inappropriate treatment according to genotype ( ), duration of treatment ( ) and switch to a more cost-effective therapy ( ). seven pharmacists' interventions concerning treatment switch were applied ( %) resulting in a cost saving of € , . . all assessable patients ( ) have a negative serum hcv rna weeks after the end of treatment (svr = %) while patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc : are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january among pharmaceutical staff in a -bed hospital ( pharmacists, pharmacy residents, pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ °c or t \ °c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: completed questionnaires were collected. collected questionnaires included % from pharmacists (n = ), % from pharmacy residents (n = ) and % from pharmacy technicians (n = ). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively and % of cases. % of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and % of the highest risk situation for a thermosensitive drug (t \ °c). the measures to adopt during a temperature excursion are understood in % of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a -bed university hospital. all adult patients having received at least days of treatment between june and april were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july , temocillin was used in a year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up weeks with therapeutic success. in august , temocillin was used in a year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may among a share of the nursing staff in a int j clin pharm ( ) results: completed questionnaires were collected. % of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at month in % of cases, weeks in % of cases and days in % of cases. % of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively and % of cases. only % of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma Ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv Ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since , a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, patients were included during months. elective surgeries were concerned in % of the cases. at least one umd was identified in patients ( %) (median age: . years old; male/female ratio: . ). of these, ( %) were older than years old. finally, umd were detected, being . by patient. main therapeutic classes concerned cardiovascular system ( %), nervous system ( %) and digestive system ( %). of the umd detected by mr, there were % of omissions, % of inappropriate dosing and % of renewal prescriptions stopped by the patient. finally, % of umd were corrected. of these umd, % were major errors (i.e. causing potential harm), % were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and % were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v. .software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd ) were compared previous and after change to drv/c. results: hiv treatment-experienced patients have received drv/c in dt ( ) or mt ( ). . % were men with a mean age of years. main risk factors were: . % heterosexual, . % msm, . % injection drug users, . % mother-to-child transmission, . % transfusion in haemophiliac patient and . % unknown. cdc category distribution was . % a, . % b, . % c and . % unknown. overall mean nadir cd counts were . ± . cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was . days [range - ]. . % drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were . years. in case of dt, . % were prescribed on patients with prior drv/ r + tc with a mean duration of . years. serum creatinine increases ( . vs. . ; p \ . ) and cd decrease ( . vs. . ; p = . ) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc : developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *, , antoine gantier , florent lapacherie , hélène dewaele , laura foucault , anne-laure raso , emmanuel cirot , said laribi , xavier pourrat pharmacy, emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected mh ( from ltcf), with a sex ratio of . and a medium age of . years old. it represented an average of . mh per day or min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy ( , % of cases), patient ( , %), previous electronic patient file ( , %), prescription sheets ( , %), call to gp ( , . %), gp mail ( , . %), patient's family ( , . %), packs of pills ( , . %), community nurse ( , . %). finally, patients ( %) had been hospitalized, others were discharged. we analysed mh for patients: at least one drp occurred for patients ( %). among patients, ( %) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among patients who did not come from ltcf and who could communicate, were good adherent to treatment ( %). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending min by mh, we identify one drp every min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of months (november -february ) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of . ui cholecalciferol (vitamine d bon Ò ) was administered to vitd-deficient patients and serum -hidroxy-vitamin d (s ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range - ng/ml). main outcome measures: s ohd (\ ng/ml: deficiency; - ng/ml: insufficiency; c ng/ml: sufficiency). results: data from patients with a mean (sd) age of . ( . ) years were collected (males: %). the admission diagnosis was laryngeal squamosis cell carcinoma (n = ), glottis carcinoma (n = ) and nasopharynx, tongue and skull base cancer (n = ). at baseline, , and patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum ohd was . ( . ) ng/ml (deficiency: patients; insufficiency: patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range . ( . ) mg/dl, . ( . ) mg/dl, and . ( . ) mg/dl, respectively. s ohd records were available week after the administration (mean (sd) = . ( . ) ng/ml). and patients still showed deficiency and insufficiency, respectively. primary care's records from patients were available after discharge ( . , . and . ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between st january and st december were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only patients ( . % over all patients receiving antifungal therapy; n = / ) received an antifungal combination therapy during the study period. majority of patients presented risk factors: % of patients had an organ transplant (n = ), % suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), % suffered from solid cancer (one lung cancer and one breast cancer) and % suffered from chronic obstructive bronchopneumopathy (n = ). antifungal combination therapy was used against invasive aspergillosis in % of cases (n = ) among which complications such as brain and cardiac impairment were found in % of patients (n = ). the six remaining patients ( %) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin ( %, n = ) and intravenous liposomal amphotericin b ( %; n = ). combination including liposomal amphotericin b and caspofungin (n = , %) or posaconazole with liposomal amphotericin b (n = ) were found in our study. five patients deceased during the hospitalization of the fungal infection ( %) which shows the gravity of these cases. majority of patients ([ %) was treated less than days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a -months period. setting and method: this is a descriptive retrospective study over a -months period from november to may in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation ( %; n = ), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome ( %; n = ); treatment of invasive aspergillosis ( %; n = ); mycetoma ( %; n = ); zygomycosis or mucormycosis while patient had renal impairment ( %; n = ). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only patients ( %) received a loading dose ( milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: % of patients (n = ) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. patient received a loading dose and was switched to intravenous voriconazole after icu transfer. patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: % of the patients had c crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c ), c admission to hospital last months, admission causes; surgical, malfunction, cancer. the model correctly classified . % of the patients with crmds as high risk. further, . % of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified . % of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc : intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl . % vials reserved for paediatric services and kcl % vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to physicians practicing in departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel Ò . results: % of physicians responded to the survey ( medicine residents, hospital doctors). in paediatric services, % of doctors know that only the kcl . % should be used. % know the unit of prescription to be used (mmol/kg or meq/kg), and % know that the maximum recommended infusion rate is . mmol/kg/hour (or mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion ( g/h) is known to all prescribers, but only % know that the concentration of kcl must be less than g/l. % of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only % of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by % of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: ( ), quetiapine ( ) and ibuprofen ( ) and were psychotropics ( . %). part . the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the sfd in cupboards, were green ( %), yellow ( %) and red ( %); an infovigilance was sent to each manufacturers. part . potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may . conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early , none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of months of prescriptions for patients hospitalized in hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on , patients. ( . %) of them were prescribed nefopam, and ( . %) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that of them were really epileptic. only pi's were effected ( . % of problematic prescriptions), and ( %) of them had an immediate prescription change. . % ( / ) of the patients have a pi in medicine services compared to . ( / ) in surgery services (p \ . ). the results of this study show that % of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for beds for years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a -month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study patients had a daily prescription review. patients ( %) had at least one drug-related problem. lines of prescriptions were mentioned to have at least one rpi. rpi represent % of drug-related problems. ( %) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: years old) and with pi (mean age: years old; p \ . ). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: . ; surgery: . ; p \ . ), the rate of corrected pi (medicine: %, surgery: %, p \ . ), but did not impact the rate of corrected rpi (p = . ). in surgery units the rate of corrected rpi ( / ) is significantly higher than corrected pi ( / ; p \ . ). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than % of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in % of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type dm were included in the study. there were analysed records from patients aged - of whom were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in % of cases, patients aged - , of whom % were men. patients received diuretic treatment, of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the patients, had high serum urea levels ([ mg/dl), had high levels of serum creatinine ([ . mg/dl), and presented risen levels for both, but only were also diagnosed with kidney disease. patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in % of the patients, aged - and % of subjects, aged - , had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc : epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *, , adeline martin-dupray , jacqueline berlioz , pierric giraud pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a -year-old woman suffered from headaches and neck pain since days, she was treated with thiocolchicoside. she took mg in the evening and mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly - min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at mg twice a day. outcome was favourable with no recurrence months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc : acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *, , amélie serra , déborah grosset-janin , jacqueline berlioz , aymeric dopter , jean-henri ruel pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a -year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement ( %) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score / ) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm ( ) : - chewing trouble and dysphagia (osserman score / ). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to / . results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included ( women, men), % had a urinary catheter. . % of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc : off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo , anti-pl , anti-pl . patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a -years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december who received c injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for months, patients (median age (min-max): ( - ), % women) have been treated with rtx for refractory as, the majority with anti-jo antibodies ( ). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies ( ) or cutaneous disorders ( ). cardiac involvement is seldom ( symptomatic patients). the mean age of diagnostic is . years and the mean treatment period is . years. the common treatment is g at day (d ) and d , then g all months. before rtx treatment, seven patients received c other drugs such as cs ( %), azathioprine ( %), methotrexate or mycophenolate mofetil ( %). prednisone and azathioprine are also prescribed with rtx respectively for and %. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is ( - ), d and d included. all patients have presented positive effects on both clinical and biological markers, mainly during the first months after treatment induction. wilcoxon tests show a significant difference in cpk level between d and m , also between d and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since , budget impact due to drug cost amounts to €. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and . % ( / ) of the patients died before discharge. parenteral nutrition was administered an average of . days ( % ci . - . ), of which . ( % ci . - . ) were with ipn. previous spn had been administered in . % ( / ) of the patients. before beginning ipn, the average triglycerides level was . mg/ dl ( % ci . - . ) but at the end of the ipn it was . mg/ dl ( % ci . - . ), which lead to a mean reduction of . mg/dl ( % ci . - . ; p = . ). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of . g ( % ci . - . ; p = . ) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march until june . these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of me registered, of them were wpme ( . %). . % of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a . % of the total amount of wpme and dispensing errors were . %. % of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining . % were system errors after cpoe. the wpme reported took place in the hospitalization wards ( . %), pharmacy ( . %), outpatient services ( . %), intensive care unit ( . %) and day-care hospital unit ( . %). . % occurred at working days and . % at the weekends. wpme were notified by pharmacists ( . %), nurses ( . %) and physicians ( . %). referring to the classification according to nccmerp, . % of wpme didn't reach the patient (category b) whereas . % reached the patient but didn't cause harm (category c) and . % required patient monitoring (category d). the remaining wpme ( . %) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm ( ) : - more than half of wpme ( . %), reporters suggested measures to prevent these errors. conclusion: wpme represents near % of total me reported in our hospital. given that more than % reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: and . both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon Ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc : descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by % compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: patients have been included in the registry between oct and june , of which ( %) were naïve to anticoagulation prior to admission, ( %) were taking warfarin and ( %) were on noacs. atrial fibrillation (af) accounted for ( %) cases, (average chadsvasc score of , hasbled score of ), and ( %) were for lv thrombus. of those naïve to anticoagulation, ( %) were initiated on warfarin and ( %) on a noac (last patients all received noacs). of those on a noac for af, ( %) were dose reduced on triple therapy; apixaban being the most commonly prescribed ( % apixaban, % rivaroxaban, % dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of ddds of common oral urti antibiotics were sold in , distributed as % erythromycin % amoxicillin and % penicillin v. amoxicillin had the highest proportion of liquid with %, followed closely by erythromycin at %. in contrast, only % of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc : proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed - h after the pa (pourrat x and al, ). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of years old ( - ) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between and p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of . ( - ) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was min ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on lines of prescriptions, discrepancies between the patient's information and prescriptions were identified and between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september and april . in july , the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, . % of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc : use and safety of trastuzumab emtansin in her + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her drugs have changed the management of her + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm- ) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm- between january and december . we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version . of the national cancer institute. results: eleven female patients with a median age of . years [ . - . ] and an ecog ( / ) were included. tdm- was prescribed as a third or further line treatment in / patients and as firstline in one patient who develop disease recurrence within months of completing adjuvant therapy. median number of tdm- cycles was . . all treatments discontinuations were due to disease progression ( / ). pfs was . [ . - . months] (patients that received less than three cycles were excluded (n = )). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study ( . vs. . months) could be explained by the later use of tdm- in clinical practice ( / patients received tdm- as third or further line while % in the pivotal study were first or second line). tdm- safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm- in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: . optimization of the detection and reporting of problems related to therapy; . implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: . detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. . information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. . report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. . completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. . monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren Ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren Ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren Ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus Ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since months, this reorganization helped us to propose pharmaceutical notices for prescriptions: • omissions (remove lipids, levocarnyl Ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren Ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus Ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. % ( . % in ) , difficulties in swallowing/psycho-behavioural distress in . % ( . % in ) , and rejection of oral drug in . % ( . % in ) . physicians and nurses indicate the reason in the medical record in . % of case versus % last year. this year, drug were crushed versus drugs in : % concerned nervous system group (vs. % in ), % concerned cardiovascular system group (vs. % in ) , and % concerned alimentary tract and metabolism group (vs. % in ) . nurses use guideline in % of cases versus . % last year. as the previous year, in % of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, % was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid ( ) and the idsa ( ), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm ( ) : - despite recommended dosage adjustment. caspofungin must be prescribed at a dose of mg/day for patients weighing over kg. micafungin must be administered at a dose of mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the Østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for patients, identifying drps and potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = , %) or greatly beneficial (n = , %). most physicians (n = , %) and nurses (n = , %) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = , %) and most nurses (n = , %) wanted more information about the pharmacists work in the emergency department. the majority of ed staff ( % of physicians and % of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of patients were included in the study, median age was years and % were males. they used in average four drugs regularly (range - ). almost three-quarters ( %) of the patients reported high or moderate adherence to all their regularly used drugs (mmas- c (max )). of the patients using oral spasmolytics, % reported high or moderate adherence to these drugs. the majority ( % of the patients) had high perceptions of necessity to their treatment (bmq [ . (max )), and % had a high level of concern (bmq [ . (max )). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, administrations were observed. only % of nurses deliver information about the treatment to their patients before administration and % question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in % of cases. emergency cart is not reachable in % of all cases. % of nurses ask patients to decline their identity. the use-by date on the bottles is checked in % of cases. at the time of preparation of perfusion, labelling does not mention either patient's name ( %) or date and hour of perfusion ( %). int j clin pharm ( ) : - during perfusion, only % of nurses follow diuresis and % watch rate of administration. hydration is not always kept min after the end of perfusion ( %). patient monitoring varies between min and h after perfusion's end. in % of cases, diuresis is monitored after the end of administration. % of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in % of all cases and in the event of adverse side effects, statement was made in % of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments ( vs. and ). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose ( g) for intravenous administration is available on our establishment since . in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving nurses. among the interviewed, ( %) have already used the g dosage. in % of cases, users were very satisfied and % were satisfied. the most positive points noted were: gain of time provided ( . % of satisfaction), less manipulation needed ( . % of satisfaction), and reducing of infectious risk ( . %). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in % of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the % not-user of this new dose, the % showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch g/ g. the capital region pharmacy, clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may nd to june rd were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included patients, of which ( %) were newly-admitted. patients ( %) were transferred between wards. of the transferred patients, ( %) were transferred from the icu to other wards and ( %) were transferred from other wards to the icu. of the newly-admitted patients, ( %) were admitted to the icu and ( %) were admitted to other wards. the pharmacists identified dd; dd ( %) in the transferred and dd ( %) in the newly-admitted patients. in the transferred, dd were all related to the icu. in the newly-admitted, dd ( %) was related to the icu and dd ( %) to other wards. of the dds, ( %) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november and december , . patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since . as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june in hacettepe university hospitals, department of internal diseases intensive care units which consists of beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v. . ) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, patients were admitted to the intensive care units. each patient's medication orders were evaluated and interventions were recommended by postgraduate students. the number of interventions per patient was . . the acceptability rate of interventions by physicians was . %. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems ( . %), no therapeutic drug monitoring ( . %), inappropriate timing of administration and/or dosing intervals ( . %), requiring dose adjustment due to deterioration/improvement of diseases ( . %), inappropriate drug selection ( %) and new indication for drug treatment presented ( %). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed different pathogens in blood culture results of inpatients out of patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of culture results. most frequently resistance were int j clin pharm ( ) results: a total of ( %) questionnaires were completed. of these, approximately % were answered by hospital nurses, the remaining mainly by physicians ( %) and % ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked . and . respectively (scale from (''no benefit'') to (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received / comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june , the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: patients (median age years old) were included from november to march . . % ( / ) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in prescriptions ( . %) and among them . % ( / ) were anxiolytic bzd. among those prescriptions, . % ( / ) already existed before the hospitalization and . % ( / ) were given during the hospitalization ( were prescribed automatically). . % ( / ) of the prescriptions did not respect the legal duration of the amm ( pieces of data were not found). . % ( / ) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, . % ( out of ) could consider stopping their use of bzd. by the end of this study, patients were discharged from hospital, among them . % ( / ) with a prescription of bzd. . % ( / ) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately . days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections ( %) (respiratory disease ( %) and other ( %)), hepatic disease ( %) and neoplasias complications ( . %). patients died during their admission; due to hepatic disorder, due to neoplasia, and due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period . . - . . . pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: patients were included, . % women. median patient age was (range - ) and most of the patients were home-living before admittance ( . %). in total drps were identified at admission, with a median number of (range - ) per patient. drps ( . %) were identified due to medication reconciliation. for patients ( . %) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in of the included patients ( . %), with a median number of (range - ) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc : assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *, , clélia monchablon , anaïs breteau , marie lefebvre-caussin , rémi varin , jean doucet , mikael daouphars , doreya monzat omedit normandie -chu rouen, chu rouen, please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm ( ) : - between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the -months study period, patients were included in six health institutions. the average age was years ( % of patients over years old) and % of the patients were women. % of patients had renal impairment. % of patients were treated with vka, and % with noacs. it was the first prescription of oa for % of patients ( % with vka; % with noacs). the most common indication was the non-valvular atrial fibrillation ( %). in this indication, % of patients had cha ds -vasc score c , and nearly % had a high risk of bleeding (has-bled score c ). drug interactions were observed, and adrs occurred related to oa. % of patients with an adrs had a has-bled score c . . % of prescriptions were considered inappropriate, including % noacs (no monitoring renal function in % of patients over years initiating treatment, inappropriate posology in %, and % of contraindications). the rate of pharmaceutical interventions was %. nearly % of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of %. the study investigated on patients hospitalized in the audited units, including patients ( %) receiving antibiotics. among the patients, % were hospitalized in medical, surgery or obstetrics units we recorded prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' ( % amoxicillin-clavulanate, % ceftriaxone, % fluoroquinolone and % other third-generation cephalosporine) and prescriptions of antibiotics called ''last resort'' ( % carbapenem). the average age of the population was . years (± years). sex ratio was . . % corresponded to curative use and % to prophylactic use. the expertise of infections diseases physician was requested in only % of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections ( %), urinary tract infections ( %) and intraabdominal infections ( %). ninety-two percent of the prescriptions had a proper indication. % of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in % of the cases. only % of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more psychotropic drugs» has been met in . % of assessment. otherwise, or more psychotropic drugs are prescribed in . % of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in . % of assessment. otherwise, more than one benzodiazepine drug is prescribed in . % of assessment from the point of admission. no contra-indication is detected in . % otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more anticholinergic drug is prescribed in . % of assessment. according to the french criteria, one or more inappropriate drug is prescribe in . % of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug ( . %) (the hospital at home team needs these class of drug) followed by nervous system ( . %) (prescribed at the point of admission) and by cardiovascular drugs ( . %) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc : access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a -bed french university hospital. patient blood samples were taken within the university hospital every weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted days after blood sampling. sterile preparations were then stored at a temperature of - °c. studies showed that eye drops were stable days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below °c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: patients benefitted from the preparation. all patients resided more than kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc : computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in , a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may was addressed to head pharmacists of the health facilities in the region, giving a total of head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists ( %) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. ( %) pharmacists planned to develop medication reconciliation in their health facility and ( %) were already carrying out this activity. within these %, medication reconciliation was conducted on paper only for ( %) of them, while ( %) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. ( %) pharmacists had contact with the editor of the software, and had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: ( ) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. ( ) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. ( ) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over weeks in . the mr is performed within days after admission. on average, . information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the patients included, drugs were listed. discrepancies were observed and were studied (status stopped or changed only): one documented intentional discrepancy, undocumented intentional discrepancies and unintentional discrepancies (ud). these uds affected patients ( - medication errors per patient) and corresponded to a non-prescribed drug in % of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in % of cases; cardiovascular drugs in % and antiinfectious in %. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age + , liver transplant, crp [ mg/ l, creatinine [ lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous ( mg one times daily) and liposomal amphotericin b ( mg/kg/day) were initiated. the inflammatory markers remained unchanged days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to mg/ kg/day. after week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within h the crp rose to mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was . mg/l (range [ . - . ) . through serum concentration days later was . mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in . we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on . patients. these patients were admitted to the emergency department of a university mother and child hospital, between october and march . one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = . ; op: k median = . ). the easy use of popi was approved by % evaluators. the median time of use was min s per case (quartiles : . - . ) . as a result, there were % of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a -year-old male patient, weight kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was . mg trimethoprim/kg/day every h as specified for anuric patients on cvvh. patient was initially anuric for days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was ml/min. following diuresis on day , remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal ( ml/min) was added to the extracorporeal clearance, and gave a total clearance of ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged and older) who applied internal service during months ( days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index ( ) and checked drug interactions with micromedex drug interaction program. results: a total of from of patients who applied to the internal service (male . %, female . ; the mean age of patients was . ± . .) were included to this study during months. of them, . % had low education level (\ education years), . % had and more chronic diseases of them, % hospitalized last months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease ( . %), cardiovascular disease ( . %) and cancer ( . %). the mean of patients' home medication number was . ± . and the mean of their mrci scores was . ± . . % in patients hospitalized in the last months. at least one possible drug-drug interactions were found in . % of patients at home medication and in . % of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was . ± . , while the mean number of possible drug-drug interactions at patients' hospital medications was . ± . . of them, . % had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may to october , we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: / , age: ) were included. underlying conditions were lung transplant (n = ), heart transplant (n = ), pulmonary fibrosis (n = ) and acute myeloblastic leukaemia (n = ). the median duration between transplantation and infection was . years ( . - . ). nine patients were exclusively infected by rsv, by hpiv ( hpiv- ; hpiv- ; hpiv- ; hpiv- ; non-identified hpiv), by hmpv and by coronavirus. there were six co-infections: rsv/ hpiv- , rsv/coronavirus, hpiv- /hpiv- and hpiv- or /coronavirus ( patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was mg tid or mg tid if there was renal insufficiency ( patients). the median duration of the treatment was days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv- /coronavirus coinfection had an hpiv- relapse days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv- and hmpv, in opposition to hpiv- twice and hpiv- respectively). virological response rate was % ( / for rsv, / for hpiv, / for coronavirus and / for hmpv). two non-negative viremia patients (rsv and hpiv- /coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week and in (n = ) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every - weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week to (n = ) were pooled together and compared to medicine lists in weeks - (n = ). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c drugs or c risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of beds). data consist of pharmacist notes conducted from august to may . pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified discrepancies between the patients' actual medication at admission and ems at the hospital in patient records ( . discrepancies per patient). in total discrepancies were accepted by the physicians ( . discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november , where out of pharmacist notes were accepted by the physicians resulting in an acceptance rate of . %. from december to may the interventions made by the pharmacist contributed to an increase in acceptance rate to . % ( out of notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was % ( out of notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from . to . %. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc : how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: -prescription by the physician on an electronic prescription software, -pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, -reconstitution at the pharmacy, -administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during months, a total of pi were collected. they concerned patients with an average of . pi per patient. among them, . % ( ) concerned paediatric population. antibiotics were involved in . % ( ) then followed by . % ( ) cases ( . % ( ) biological assessment issues, . % ( ) absence of therapeutic drug monitoring (tdm) and . % ( ) the drug hasn't been adapted to the weight), dosage adjustments in . % ( ), information missing concerning the treatment indication in . % ( ) and miscellaneous pi in . % ( ) such as wrong clinical service on the prescription, etc. approval rate of physicians was . % ( ). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc : the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of months. patients years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: patients, males and females, out of , were included. the mean age was years and the patients received in average prescribed drugs. at admission the average number of stopp criteria were . ± . and . ± . for the start criteria. in average, % of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, % of the suggested start criteria were accepted, which included: calcium and vitamin d supplement, beta- agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january and december , . the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated patients, ( %) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was years and % were male. the male and female ratio was . : . reasons for hospitalization were: digestive system neoplasms ( %), fistula ( %), intestinal obstructions ( %) and other pathologies ( %). of the patients treated with octreotide, ( %) received a lar form. only four patients received doses in accordance with guidelines: one at mg/month lar form and three at mg/month lar form, after having respectively been treated by intravenous octreotide at and mcg/day during - days. medical prescriptions of the remaining patients did not comply: all patients received mg/month after an intravenous treatment of mcg/day, instead of mg/month. from a financial perspective, these misuses have led to an additional cost of . euros for the hospital, excluding tax ( mg: . €/unit and mg: €/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, % of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc : pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until , intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at °c in syringe over h and at °c in pumps over month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *, , xavier deviot , thomas sidibe , zohra mostefaï , michèle minvielle , marta wyrtwal , roselyne gervais pharmacy, geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose ( mg or mg) • to maintain ppi but half dose (from mg to mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: patients were included in the study. % of ppis prescriptions were off-label, % had no indication mentioned in patient's file and % were conform to the marketing authorization. % of patients have been on ppis medication longer than months, which is the recommended treatment' duration in france, % longer than a year and % longer than years. in collaboration with the geriatricians, ppi prescriptions were maintained for % of patients. we reduced the dose in % of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . hp-pc : oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu , marie-françoise hurtaud-roux , ronan bonnefoy , caroline farnoux , philippe sachs , theresa kwon , olivier bourdon , sophie ajzenfisz , sonia prot-labarthe *, pharmacy, hématologie clinique, ap-hp hôpital robert-debré, cardiologie, néonatologie, ap-hp hôpital robert debré, réanimation pédiatrique, ap-hp hôpital robert-debré, néphrologie, pharmacy, ap-hp hôpital robert debré, coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed Ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc : does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *, , vera jordan-von gunten , pierre-auguste petignat , pierre turini , johnny beney division of pharmacy, institut central des hôpitaux, division of internal medicine, medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: °to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. °to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are - km apart. main outcome measures: . for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. . categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: ' prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than h for both formulary and non-stored/non-formulary drugs; and more than % of formulary drugs and around % of non-stored/non-formulary drugs were administered within h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing . or more omitted doses was found for ( . %) prescriptions. among them, only were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than % of formulary drugs and around % of non-stored/non-formulary drugs are administered within h following their prescription. none of the patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *, , roger jeremy , lauriane cornuault , anne lecoeur , franck lemercier , isabelle javerliat , thomas tritz service de pharmacie à usage intérieur, service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february th and may st , a total of patients have been reconciled. patients were excluded due to a lack of reliable sources. mean age was . years old (± . ) and sex ratio m/f was . . % of the reconciliated patients' admissions were scheduled. the mean number of medication was . (± . ). patients ( %) had at least one uid and the mean uids per patient was . (± . ). the most common types of uids were omission ( %), incorrect dose ( %) and incorrect administration frequency ( %). more than % of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; uid were considered to have potential clinically significant impact ( %), a potential major impact ( %) and a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc : delirium in elderly patients: successful use of melatonin gaëlle jouin , aurélie reiter-schatz , pierre bentzinger , fatem-zohra laalou , bénédicte gourieux *, pharmacy-sterilization, orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of patients were included: average age was . years ( - ), sex ratio m/f = . twelve patients ( %) were hospitalized because of an infection (prosthesis or osteoarticular). in % of cases (n = ), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients ( %) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was days ( - ). melatonin was started on an average of days after surgery , and administered at the dose of mg xr in the evening, during an average of days ( - ). cognitive impairments requiring a prescription of melatonin were: confusion ( %, n = ), agitation ( %, n = ), daytime sleepiness ( %, n = ), temporal-spatial disorientation ( %, n = ), nocturnal awakening ( %, n = ), hallucination ( %, n = ), difficult falling asleep ( %, n = ). the average time to recover from confusion was days, agitation days, daytime sleepiness days, temporal-spatial disorientation days, nocturnal awakening days, hallucination days and falling asleep days. melatonin treatment helped stopping benzodiazepines treatment in six patients ( %). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva Ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about % of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection months before, on the th of march). he went to emergency on the th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = . mmol/l) and hypophosphatemia (phosphorus \ . mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses ( g of calcium and . g of phosphorus) and then a week of following daily intakes: phosphorus ( g iv and . g oral); calcium ( g iv and . g oral). however, low-serum corrected calcium and phosphorus levels persist at . mmol/l and . mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose ( g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable ( . g per day); calcium oral supplementation is increased ( . g per day). h between intakes is applied to avoid digestive complexation. h later, corrected calcium levels are normalized at . mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *, , , ida rudberg , lone holst , anne-lise sagen major , central norway hospital pharmacy trust, Å lesund, centre for pharmacy, university of bergen, bergen, møre og romsdal health trust, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of Å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, Å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm ( ) : - usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total patients were admitted. on average, each patient had . dis, and . % of the admitted patients had at least one di. the prevalence of dis was significantly higher among the patients in the pharmacist group compared to the patients in the non-pharmacist group (median@@@ vs. , respectively, p \ . ). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. . % of the dis detected in the pharmacist group were discussed with the physician. the remaining . % were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant ( %) or already adjusted for in clinical practice ( %). for dis the clinical pharmacist suggested a change in prescription, and of these suggestions ( %) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only of dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc : securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *, , linda an , anne fratta , anne auvrignon , , nadia marquis , karine morand pharmacy, risk management committee, hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of physicians, coordinating nurse, hospital pharmacists and pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: failure modes were identified, including critical risk failure modes. critical failures were related to hospital discharge prescriptions and were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of patients were included. overall, mds and mrps were identified. at least one md was revealed in % of the included patients, whereas at least one mrp was identified in % of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, % of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in % of the discussed md-cases and % of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc : prevention and management of drug interactions in oncology day-hospital: results from a months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *, , chloé hugard , céline mongaret , , juliette vella-boucaud , antonin maréchal , olivier bouché , dominique hettler , florian slimano , pharmacy, oncology day hospital, university hospital reims, clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, to april, n = oncologic patients were included with following characteristics: mean age of . , sex ratio : , majority of oncologic thoracic localization ( %). number of oncologic concomitant medications per patient was . ± . (mean ± standard deviation). patients present an average of . ± . comorbidities (excluding cancer) and . ± . linked medications per patient. pharmaceutical analysis revealed potential clinically relevant ddi ( . ± . per patient): % of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and % of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc : loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *, , cécile sanchez , guillaume beraud , nicolas venisse , pauline lazaro , antoine dupuis pharmacy, infectiology, pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during days in units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the patients, were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in % of case, monitoring of steady state concentrations was performed in % of case and dose adjustment after first dosage was required in % of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: g/ h after loading dose of mg/kg • to monitor steady state concentrations in patient with renal failure a total of interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc : assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a -bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october . after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of pims were detected in this population. at discharge, the number of pims decreased to . the median number of pims per patient decreased from (on admission) to (at discharge). this difference was statistically significant (p \ . ; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc : impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on wards, which coupled with one-stop dispensing meant % of discharges require nothing to be supplied at the point of validation, % of discharge prescriptions meeting key performance indicator of being dispensed and ready within h with average turnaround time of min for a discharge prescription and a reduction in missed doses- . % in september to . % in march . this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than % of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june shows across the site % ( ) wards scored below the national . % target and ( %) wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being % ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april . this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of months study period (period i: february-july , and period ii: november-february ), clinical pharmacy postgraduate students followed patients for - times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of recommendations was provided for patients. those recommendations were classified as alteration or discontinuation of drug treatment ( . %), dose adjustment ( . %), change in drug administration time ( . %), inadequate treatment ( . %), healthcare staff training/consulting ( . %), patient education ( . %) and error/deficit in therapeutic drug monitoring ( . %). a majority of recommendations (n = ) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = , geriatrics (n = ), neurology intensive care (n = ) and infectious diseases service (n = ). the following main reason for pharmacist's recommendation was related with dose adjustment (n = ) which were provided in departments of internal intensive care (n = ), infectious diseases service (n = ), neurology (n = ) and internal medicine (n = ). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc : development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *, , eric gautier , astrid bacle , florian charier , christine silvain , pauline lazaro pharmacy, gastroenterology, hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to %, bilirubin superior to micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of patients, patients were included for liver failure ( . %) and for a cholestasis ( . %) mainly in reanimation care units ( . %) and gastro-enterology ( %). among the lines of prescribed medicines, the main pharmacological classes encountered were cardiology, ( %) pain ( %), psychiatry ( %), haemostasis ( %) and antibiotics ( %). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, hl and nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into sections (a-c). the same questionnaire was used for both the first interview (t = ) and after weeks (t = ). an additional section (d) was incorporated at t = to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky -item medication adherence scale (mmas- ) to evaluate adherence to oral chemotherapy. a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = and t = , pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm Ò spss version and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = and t = . main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the patients with hl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. for the patients with nhl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all nhl and hl patients indicated the haematologist as their source of information about the management of lymphoma. of the nhl patients, scored low adherence and scored medium adherence at t = and after weeks (t = ) all nhl patients who participated scored medium adherence. of the hl patients, scored low adherence and scored medium adherence in the first interview (t = ) and after weeks (t = ) all hl patients who participated scored medium adherence. there was a statistically significant increase (p \ . ) in the number of patients who scored medium medication adherence between t = and t = for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may , an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a -month period, patients were reconciled with on average age of . the reconciliation is executed on average . days after the entry in the service. . % of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in . % of the cases an oral interview with the patient and/or the family; in % of the cases the prescriptions, the hometown pharmacist ( . %) and a medical letter ( . %). . drugs are on average on the hospital prescription, and . % ( / ) of the patients are concerned with at least one non intentional divergence (nid). on average there are . nid/patient and . intentional divergences (id)/patient. the main types of nid are omissions ( . %), drug dose errors ( . %) and errors in administration frequency ( . %). after the detection of nid, the proposed modifications to the prescribers are accepted in more than % of the cases ( / ). the average time of a reconciliation is min. exchanges on the id and nid are made with the junior doctors in . % of the cases. conclusion: some nid are occurring for . % of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over years, suffering from chf and on bumetanide therapy. the validated -item morisky medication adherence scale (mmas- ) was administered to patients on admission (t = ), repeated after two weeks hospital stay (t = ) and again one-month post-discharge (t = ). a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = ) and one-month after pharmacist intervention (t = ). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of patients were recruited from the hospital setting, of whom were female and were male with a mean age of years (range - years). on admission (t = ), patients scored high adherence, scored medium adherence and scored low adherence to bumetanide therapy. following weeks at the hospital (t = ), the number of patients scoring high adherence increased from to and the number of patients scoring low adherence decreased from to . one-month post-discharge (t = ), patients scoring high adherence decreased from to and patients scoring low adherence increased from to (p \ . ). a total of patients were recruited from the community pharmacy, of whom were female and were male, with a mean age of years (range - years). after pharmacist intervention (t = ), the number of patients scoring high adherence increased from to , while the patients scoring low adherence decreased from to (p [ . ). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment ( ) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre ( , population). method: a review of the medical records of polymedicated patients (c chronic drugs for c months). the patients' data were collected from january to june from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c years old. treatment alternatives proposed to gp's by pcp were also measured. results: patients were included in the study (average age: . ± . , % women). out of the patients, interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. % of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average . interventions for patient. the prevalence of intervention proposals were: non-indicated drugs ( %), interventions for improve the efficiency of treatments ( %), interactions ( %), inappropriate medication for patients c years old ( %), contraindicated drugs ( %), duplications ( %), medication with low therapeutic effect ( %), inadequate dosages ( %) and omission of a necessary drug ( %). % of these intervention proposals were accepted by the gp: % of the accepted proposals were carried out and from the remaining , . % led to a prescription from a specialist physician. in % of the cases, the patient did not accept the changes. . % were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over million person-years of data from some million active patients. the study encompassed , patients below the age of years with an incident major depression diagnosis between and , and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd- codes (f ), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or . , % ci . - . ) compared to patients with no history of influenza infections. a recent influenza infection recorded within - days prior to the index date yielded an adjusted or of . ( % ci . - . ), and an increasing number of previous influenza infections was associated with increasing odds ratios (c recorded influenza infections, adjusted or . , % ci . - . ). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than years were included and monitored for months in community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp Ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp Ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included elderly patients with an average age of years. in the follow-up period, new drugs were prescribed to patients ( . %). there were prescriptions of new drugs and ( . %) of those were drugs with a clinically significant interaction with warfarin. there were prescriptions of drugs with level c of interaction ( . %), and ( . %) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with prescriptions: amoxicillin/clavulanate %, clindamycin %, ciprofloxacin %, norfloxacin %, azithromycin %, cefuroxime %, clarithromycin %, doxycycline %. the remaining prescriptions included tramadol with paracetamol %, rosuvastatin %, simvastatin %, fluvastatin %, levothyroxine % and torasemide %. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with prescriptions-diclofenac %, ibuprofen %, indomethacin %. among other drugs, prescriptions were antibiotic sulfamethoxazole with trimethoprim %, fenofibrate %, miconazole %, and fluconazole %. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action initiative wg b ( wg b ( - is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of pims in the period - / . items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of pims . % were registered in at least participating country. for the czech republic ( . %), turkey ( . %), spain ( . %) and hungary ( . %) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence- . %) and portugal (high prevalence- . %). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this ( ) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of at three primary care centres ( , population). polymedicated patients were those using c chronic drugs for c months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were and polymedicated patients before and after intervention, respectively. . % (n = , before) and . % (n = , after) of the total patients had at least one pip. the number of pips was reduced from to , while the rate of pip per polymedicated patient decreased from . to . , achieving the limit established by the regional health authority. . % (before) and . % (after) of patients had more than one pip at the same time, up to pips per patient. before and after intervention, more than half of patients with at least one pip were c years old, and approximately out of were c years old. also before and after intervention, out of patients with chronic nsaid and with bzd duplication were women. out of patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd ( . vs. . % of the total pip), medications with low therapeutic effect ( . vs. . %) and inappropriate medication for patients c years old ( . vs. . %). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to % of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of siblings; were prenatally exposed to ssris and were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of g [ % confidence interval (ci) to ] and a decrease in gestational length of . days ( % ci . to . ). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = ). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of . main outcome measures: dbi scores of participants categorised into low ( ), medium ( - ) and high (c ). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of participants, . % ( ) had dbi exposure; . % were exposed to any anticholinergic medication, . % to any sedative medication; mean number of dbi medications . (± . ), mean dbi score: . (± . ). ( . %) participants had dbi score , ( . %) - , and ( . %) c . antiepileptics accounted for the greatest contribution to cumulative score ( . %), antipsychotics ( %) and antidepressants ( %). there was no significant association between higher dbi score and sleep difficulties (p = . ). there was a significant age gradient associated with higher dbi score (p = . ) and significant association between higher scores and increased comorbidity scores; mean fci of . in those with dbi c , . in dbi - and . in those with dbi . conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified -item morisky score and score charts ( ). medication adherence and cvr were evaluated in the whole group (n = , males and females, range - years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the cvr (estimating -year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average ( . ± . ). the average value of cvr in the study group was . %. the highest average medication adherence has been observed in males b years ( . ), with elementary education ( . ), in ex-smokers ( . ), in patients with regular physical activity-at least times a week ( . ), in patients non-adherent to the diabetic diet ( . ), in patients using medications ( . ), and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ). the lowest cvr has been observed in females b years ( . %), in no-smokers ( . %), with elementary education ( . %), in patients with irregular physical activity ( . %), in patients adherent to the diabetic diet ( . ) , in patients using medications ( . %) and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ) . on the other hand, the highest cvr has been observed in males [ years ( . %), smokers ( . %), secondary educated patients ( . %), without any physical activity ( . %), in patients partially adherent to diabetic diet ( . %), using medications ( . %) and, surprisingly, in patients with satisfactory (\ . mmol/l) total cholesterol levels ( . %). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified -item morisky score and questionnaire eq- d- l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = ) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the qol in levels of dimensions results were evaluated as follows: the lowest qol in every dimension = point, the highest = points. the highest vas evaluation has been points and every patient should mark number on the scale - to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average ( . ± . ). the average value of the qol in the study group was . and vas . . the highest medication adherence has been observed in males ( . ± . ), patients \ years old ( . ± . ), with primary education ( . ± . ), with monthly income over € ( . ± . ) and in patients using medications ( . ± . ). the highest qol and vas (qol; vas) has been observed in males ( . ; . ), patients \ years old ( . ; . ), university educated ( . ; . ) , with monthly income over € ( . ; . ) . qol has been highest in patients using medications ( . ), vas has been highest in patients using medication ( . ). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin ( . ), the lowest in patients treated with only insulin therapy ( . ). highest qol was recorded in patients treated with oral antidiabetic agents ( . ), and the lowest qol in patients with insulin therapy ( . ). the highest vas has been observed in patients using only oral antidiabetic agents ( . ), the lowest in patients using combined therapy ( . ). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may . a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december . main outcome measures: appropriateness of antibiotic prescription (type and duration) results: elderly patients were included (women: . % (n = ), mean age: . years). % of antibiotic choices were appropriate and % of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for . % of the cases (n = ). patients received an empirical antibiotherapy ( . %). . % (n = ) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = ). the urine culture results led to a change in antibiotics for . % of the cases. for cystitis, . % of the antibiotics chosen were appropriate (n = ). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was . days (appropriateness: . % (n = )). for pyelonephritis, . % of the antibiotics chosen were appropriate (n = ). the average duration of effective antibiotic treatment was . days (appropriateness: . % (n = )). . % of the patients had a transurethral catheterization (n = ). another infection was diagnosed for . % of the patients (n = ). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising nursing home patients from two geographical different regions in norway; tromsø city (n = ) and lofoten islands (n = ). data was collected from november to january . pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of . ( . ) drugs; . ( . ) regularly and . ( . ) as needed. at least % of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified ( %), followed by the use of antidepressant ( %) and hypnotics ( %). an increasing number of regularly used drugs increased the odds of having pims (or: . ), as well as it lead to . more pims per extra drug used. on average, patients c years had . fewer pims than patients \ years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe : use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july and july , three adherence measures were calculated: ( ) average medication availability (ama); ( ) mean rate of adherent patients with an ama c % (mrap ); ( ) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering transition states: remission, clinical response, relapse) was constructed using the software r . . markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised- . %) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab - %; infliximab - . %; infliximab biosimilar - . %; golimumab - . %; vedolizumab - %). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over years of treatment per patient: adalimumab (n: € , . , r: € , . , - . %), infliximab (n: € , . , r: € , . , - %), infliximab biosimilar (n: € , . , r: € , . , - . %), golimumab (n: € , . , r: € , . , - . %), vedolizumab (n: € , . , r: € , . , - . %) with associated qaly respectively of . , . , . , . , . . from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was € . for years (willingness to pay (wtp) € . /qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was € , . for years (wtp € , . /qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in university hospitals in , one with ads ( beds, ads) and one with cds ( beds, cds ( ) for ads and ( ) for cds (p \ . ). mean number of costly drug per system was for ads and for cds. the global stock value in the wards was , € in ads and , € in cds representing respectively . and . % of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on january . according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june and may . regional and national infliximab consumption between january and february were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to patients, of which ( %) were naive. patients were treated with biosimilar (i.e. . % of all patients), of which were naive. in the end, nearly % of naive patients actually received the biosimilar and . % of patients treated with infliximab switched specialties during treatment. in % of cases, biosimilar prescriptions were consistent with the recommendations (vs. % for infliximab). in % of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. % for infliximab). in february , the share of biosimilars was % in france, % at regional level and % locally. in year, infliximab and its biosimilar's consumption in our hospital have increased by % in quantity and only % in expenditure (+€ m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € , (vs. € , during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € , if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from st january to st july obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec , pre-baseline period; jan-dec , baseline period; jan-sept , transition period between announcement and introduction of trp; oct to jul , period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € , , in pre-baseline period to € , in period after trp introduction although the consumption increased from . to . ddds/ inhabitants/day. cost of ppis decreased the most in baseline period ( %), however trp induced . % cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented % of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for %. still, this resulted in the minor market concentration change; hhi was on average . before and . after trp introduction. further, at least one active substance switch was detected in approx. and % of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from . % in transition period to . % in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec : blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for ahf out of . in % of cases, this stock modification has exceeded %. the overall cost of ls has been reduced by . % ( , €) for the general stock at the central hospital pharmacy (hp) and by . % for offsite-stocks ( , €). the ahf mainly involved in this reduction was fvii mg ( , €), then followed by the strengths of mg and mg ( , € for each). in order to improve the ahf management, several propositions have been implemented: ( ) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, ( ) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, ( ) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip ] and ( ) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of , €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with beds and currently clinical trials, with patients included in clinical trials between january and may . design: retrospective observational study over months. all the clinical trials ongoing between january and may were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were clinical trials ongoing between january and may , but only were selected to be included in this study. the total number of patients included was . the clinical trials selected for this study were conducted in medical specialties: in dermatology, in immunology clinical unit, in hemato oncology, in gastroenterology, in ophtalmology and in neurology. during these months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than million euros. conclusion: during the period of time established, of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between and and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of fix between and have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in , recombinant (rfix) and plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these factors represent almost million ui and million euros, which comprise % of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly % of the fix purchases in . french rfix consumptions are higher than pfix consumptions ( % against %). in the ap-hp hospitals, rfix even account for % of consumptions against % for pfix. both national and ap-hp rfix purchases have steadily increased between and . the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus (hhv- ) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a years old man (creatinine clearance rate (crcl): ml/min), was diagnosed with hiv infection in february (cd at ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: ml/min) and pancytopenia (hemoglobinemia at . g/dl, leucopoenia at . g/l, and thrombopenia at g/l). because of hhv blood pcr at copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day , intravenous etoposide at mg; day , rituximab at mg/ m ; following one week later by rituximab day and oral etoposide at mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in and the national healthcare system (nhs) has since provided the cervarix Ò vaccine free of charge to girls aged . the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately € , while the average estimated cost based on the tender price was approximately € , . this signifies that cost savings based on the tender price compared to wholesale costs were of approximately € , . the cost for the cohort who completed the three dose schedule using the tender price on average was of € , per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of € , per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of participants. participants were pilgrims, aged over , and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, % of whom were female, and the median (iqr) age was ( . - . ) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of (iqr - ) and (iqr: - ) respectively. they were less educated about management ( %), hallmark symptoms ( %), high-risk individuals ( %) and source of coronavirus ( %). almost % of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants ( %) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ . ). the correlation between knowledge, attitude and practices was significant (correlation coefficient: . ; p \ . ). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type diabetes accounts for % of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type diabetes risk assessment program for the kids b years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in community pharmacies. all patients with kids aged b years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type diabetes risk test consisted of questions and identified subjects at risk. the parent of the kid who was identified to have risk for type diabetes was referred to a physician for further examination. also, information regarding type diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type diabetes and the behavioural parameters associated with type diabetes risk. results: the study involved subjects. of the subjects % were identified to be at risk of type diabetes. more girls than the boys had the risk ( vs. . %). those with type diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); . % were spending more than h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in , only ade were reported and analysed by the comedims, including from the paediatric centre ( %), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives ( %), nurses ( %), pharmacists ( %), residential students ( %), doctors ( %) and others ( %). the main impacted steps of the drug circuit are: administration ( %), prescription ( %) and the use or implementation of a sterile medical device ( %). identified causes include related following factors: operational tasks and procedures ( %), health professionals ( %), work environment ( %), organization and management ( %), drugs or associated medical devices ( %). the number of ade reports, taking into account the size of the institution, remains very low. in january , the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed times, and the number of reports increased by % compared to the same period in . conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the , persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type diabetes (t d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t d was investigated. setting and method: three focus group interviews with people with t d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t d describe their experiences of being diagnosed with t d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo . flie questionnaire was administered to patients before chemotherapy, and days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was and a dramatic decline was noticed post chemotherapy (flie score ; p \ . ). the post-chemotherapy score were for nausea and for vomiting ( . , respectively). although the flie score differed significantly between gcgd and gigd (p \ . ), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study ( pre-to post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ . ). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of %. prescription criterion was fulfilled to %: all requirements of pntb were performed using standardized procedure. in what concerns validation, % of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from : to : and on weekends and holidays from : to : . % of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, % were supplemented with micronutrients. pntb of kabiven peripheral administration ml are not supplemented in our centre. of the remaining prescriptions central administration, % were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, % of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in , patients were in opioid substitution treatment, of who were on methadone. in , the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation . . the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of questions divided into sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to community pharmacies. an online format of the questionnaire was also circulated to community pharmacists through the pharmacy council. data was analysed using spss version . main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of responses were obtained and a response rate of . % was achieved. eighteen percent of the pharmacists (n = ) who responded to the questionnaire worked in a community pharmacy located in the north of malta, % in the centre, % in the south, % in the southeast and % in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to % if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while % had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt : evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr , - , - , tie ), the oncogenesis (kit, ret, raf- , braf, brafv e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca . ) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca . only decreased in the . % of the patients after the regorafenib treatment. results: regorafenib was taken by patients ( %men).the average age of these patients was . ± . years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib ( . % patients), intervened colon adenocarcinoma e-iv ( . % patients), sigma adenocarcinoma e-iv ( . % patients) and unresectable and non-intervened rectal adenocarcinoma e-iv ( . % patients).all patients presented metastasis in different locations on the body: liver ( . % patients), diaphragm ( . % patients), intestine ( . % patients) and lung ( . % patients).the % of the patients started the treatment with mg of regorafenib, administrated once a day for weeks followed by one week without this drug; while the . % of the patients started the treatment with mg. however, the . % had to decrease the initial dose and the . % of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the . % of the patients presents native kras. the native kras was presented in the % of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca . ) conclusion: the decrease of cea in the . % of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt : evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november to september . sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca . ) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in % of the patients and ca . no decreased in any patient after nintedanib treatment. results: nintedanib was used in patients ( % men and % smoker).the average age of these patients was years old. the average bmi was kg/m ( - ).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib mg/ h from day to day every weeks; but patients had to reduce the initial dose to mg/ h ( patient) and mg/ h ( patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in % of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen . mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen . mg spray plus a placebo lozenge, or flurbiprofen . mg lozenge plus placebo spray at sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a -point scale, = no relief, = slight relief, = mild relief, = moderate relief, = considerable relief, = almost complete relief, = complete relief) at timed intervals throughout h starting from min post completion of first dosing ( min after administration of the spray, and min after the lozenge had fully dissolved). adverse events (aes) were recorded over h post-dose. results: patients were assessed (n = for spray, n = for lozenge). [ % of patients in either treatment group experienced some relief (a score of [ on the strrs) at min post-dose, which increased to % of patients by h. - % of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at min post-dose, which increased to - % of patients by h. over the h post-dose, a total of drugrelated aes were reported by patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen . mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt : analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may ) and the period before of it (january-may ). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept €, bevacizumab €, ranibizumab €. results: in the period, patients were treated with antiangiogenics. ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. in the period, patients were treated, ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. the consumption of aflibercept decreased a %, bevacizumab consumption increased % an ranibizumab increased a %.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: % . please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was years (range - ), and the average number of drugs per respondent was . (range - ). a total of patients ( . %) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, patients ( . %) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid Ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager . . main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: . ( . , . ) and presents a low heterogeneity (i = %). about % of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and % presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm ( ) all of these patients, except one whose treatment adherence was inadequate, were cirrhotic ( / ), liver transplanted ( / ) and/ or presented hepatocellular carcinoma ( / ). / patients were coinfected with hiv. / patients ( %) were genotype . the total genotype patients treated with daas (svr /relapsed) were , which means that . % ( / ) of all genotype patients has had a relapse. / patients ( %) were treated with ledispavir/sofosbuvir ( . % of a total of patients (svr /relapsed) treated with this option). % of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents . % of the total of patients treated with the older option. conclusion: relapses rate was . %, slightly lower than reported in other studies. according to the references, these results show that genotype is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april , when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from time-points were extracted from medical records and collected in an excel database: t (november -january ) and t (february -april ). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: records were analysed (t -t ), of which contained at least one antibiotic prescription. the most frequent infections were urinary tract ( %), respiratory ( %) and gastro-intestinal ( %). antibiotic therapy was started in . % of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t , an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were clostridium difficile infections ( relapses), confirmed by antibiogram. a total of adrs were identified: of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march and june ( month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos Ò and the electronic medical record software hcis Ò were used. statistical analysis was carried out using microsoft excel Ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of patients ( males) started treatment with pirfenidone, with a median age of . years ( - ). during this period patients were excluded for lack of monitoring. the median fvc, dlco, wt m values prior to pirfenidone therapy, were % ( [ %), . % ( [ %) and m ( - m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt m; however of them didn't meet the dlco criteria (at least %).'' all patients were monitored every months. the median in fvc percentage change at the end of the study was - % (- % to + %). patients ( %) showed an improvement on fvc during treatment with a median change of %. in the other eight patients fvc value decreased with a median of - %. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c % in fvc during first year of treatment). dlco percentage was measured in patients, with a median change of % (- % to + %). dlco decreased in patients. wt m was monitored in patients, with a median change of - . m (- m to + m). adverse effects related to pirfenidone were gastrointestinal disorders ( / ), increase of hepatic ggt ( / ), and dermatologic toxicity ( / ). six patients ( %) required a dose reduction because of gastrointestinal adverse effects. five patients ( %) discontinued treatment with pirfenidone due to hepatotoxicity ( ), gastrointestinal ( ) and dermatologic effects ( ). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in - , based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, patients with mental disabilities, aged - years, consented to have a medication review. they used on int j clin pharm ( ) : - average medicines (range - ). the team identified drp in of the patients (average . , range - ). overall, % of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics ( %), antiepileptics ( %) and anxiolytics ( %). the most frequent drps were unnecessary drug choice ( %), side effects ( %) and too low dose ( %). drps were most common in antipsychotics ( %), antidepressants ( %) and anxiolytics ( %). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt : protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr ) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are ( ) to evaluate the feasibility of a pilot study with a food supplement among pregnant women ( ) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between and weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: ( ) number of included patients as an indicator of feasibility: including a number of patients was taken as a target ( ) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, patients were included with an average age of . years and a median age of ( - ) years. patients used gr : patients were dissatisfied, patients had a neutral opinion and patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost weeks (only tablets of gr were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. patients experienced no adverse events. the remaining patients were not yet evaluated. of the included patients, six patients decided not to use the product: because their gastrointestinal complaints were not serious enough, because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with mg of daily ibrutinib between september and june were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel Ò and spss Ò . results: throughout the period of study a total of patients ( males and female) with a mean age of . ± . years started treatment with ibrutinib. the median of previous treatments were ( ) ( ) ( ) ( ) ( ) including first-line treatment with high dose chemotherapy ( %), steam-cell transplantation ( %), rituximab ( %), bortezomib ( %) and lenalidomide ( %). the median ecog value prior to ibrutinib therapy was (range - ). the mipi status was intermediate risk in patients and high risk in , the disease stage was relapsed in % of the patients. partial response was reported in patients. the mean pfs estimated at the end of the study period was months ( % . - . ). adverse effects related to ibrutinib were: fatigue ( %), diarrhoea ( %) leucocytosis ( %) and infections ( %), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp a and interactions may be frequently present. pharmacy, hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to %) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september to june of patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of patients attended by pharmacy department in our hospital, patients ( males, mean age years (range - )) had started a dtg-based art. out of them, patients were art naive and art-experienced. at the moment of starting dtg, mean cd cells were cell/mm (range - ) and hiv- rna load in plasma was detectable in patients. treatment discontinuation was reported in / patients ( . %) with a median treatment time of days (range - ). / patients ( . %) were naïve and / patients ( . %) pre-treated. most of the patients ( ) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in / patients ( . %). the rest of the patients discontinued due to other motives (clinical trial inclusion ( / ), treated in another hospital ( / ), exitus ( / ) and others ( / ). reasons for the discontinuation were classified in different side effects: / ( . %) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), / ( . %) gastrointestinal effects, / ( . %) headaches, / ( . %) musculoskeletal effects, / ( . %) fatigue, / ( . %) allergy and / ( . %) for other reasons. some patients reported various toxicities at once. conclusion: more than % of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet Ò , and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd count, cd levels end of treatment, sustained viral response (svr) and hcv treatment. results: patients, of whom were men, mean age years were included. patients received daclatasvir and sofosbuvir for hcv, patients had genotype a and b respectively, patients genotype and patient genotype . patients had fibrosis f f , . of the patients they had not received previous treatment (naïve) and had failed to treatment. hiv treatment was modified in patients, patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd levels increased in all patients at the end of treatment for hcv with a median of cells/ul and at the beginning and end respectively. patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype a. these two patients had received previous treatment and had a f and f fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd slightly increased after the treatment. patients received ledipasvir and sofosbuvir, patients had genotype a, patients genotype b and patient genotype . patients had f fibrosis and had f . patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, patients achieved svr. cv increase in patients after the treatment while cd followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to / / , with at least one clinical and analytical control months before the beginning. data were collected from the medical record archinet Ò and outpatient farmatools Ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd % during monotherapy. main outcome measures: inclusion criteria: virological suppression for year prior to the start of monotherapy, no previous ip virological failure, high cd count ([ cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure ( consecutive plasma viral load (vl) [ copies/ml) and without treatment failure (any event causing retirement monotherapy). results: patients with monotherapy, which represent % of patients with antiretroviral therapy (art) at our institution were identified. were excluded ( co-infected with hepatitis virus, with insufficient data and no had more than months included), including patients in the analysis, with a mean age of years and % were men. the median of time monotherapy treatment was . years ( . days), ( . %) patients received darunavir/r and ( . %) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was % with undetectable pvl at follow-up. the median of cd % over the treatment time was cell/ml ( %). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree / ( / ). the objective of the study is to describe the use of cancer drugs through compassionate use in the last years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january until october . the data were obtained from medical records programme diraya Ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded requests of cancer drugs in compassionate use during the years of study. oncology was the service that recorded more authorizations with %, followed with gynaecology with . % and finally endocrinology and haematology with . %. drugs of the requests were approved ( %) and unauthorized ( %) in the years of study. the year in which more applications were received was ( . %) and the least requests were received in ( . %), being the year where all requests were authorized. in fewer applications were authorized, %. in the years , and were authorized . , and . %, respectively. a total of different active drugs were received during the study, the most requested bevacizumab ( %) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus ( %) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel ( %) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab ( %) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib ( %). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of and years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated mu ( ml) in lesions less than cm, mu ( ml) in the larger lesions and repeating each week until complete remission of the lesions. in the nd patient we proceed in the same way but the second week was infiltrated mu ( ml). the following week, infiltrated mml, in metastases and we turn to weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than % reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd / . it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with . ml api ( mu/ml) and then diluted with . ml of a solution of . % albumin, % glucose as stabilizer, to avoid aggregate formation, preparing ml syringes ( mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable days in a refrigerator ( - °c), protected from light. initially patients had approximately a total of injuries. after months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories archinet Ò and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included patients of whom were men. baseline characteristics were: patients with genotype , genotype b, with genotype a and genotype . the degree of fibrosis in the study was patients with f , f and to f . among the patients infected with hcv genotype , had not received prior treatment (naïve) and had failed therapy. the duration of the treatment was weeks to patients and weeks for patients. only patients receiving ribavirin of these had genotype and genotype b. from ribavirin patients it was greater the number of patients in whom the treatment duration was weeks ( patients versus with p-value = . ). the digestive service attended to patients while patients were followed by infectious. the median cv was , , iu/ml. svr was achieved in . % of patients with hcv genotype in . % with genotype b and % with genotype and a. after weeks of treatment % of patients achieved svr and % after weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck Ò . pillcheck Ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: patients were enrolled in the study. average age was . years and patients were taking a mean of . chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy ( . %), to address an adverse reaction ( . %), and to guide initiation of therapy ( . %). an average of . drug therapy problems were identified per patient. pharmacist recommendations included change in therapy ( . %), dose adjustment ( . %), discontinuation of a drug ( . %), and increased monitoring ( . %). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of patients with hip fractures per year ( % surgically treated). data collection was performed from june to march . reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro Ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients ( women and men) were included, median age year-old . in patients, eye drops reconciliation treatment was performed by the pharmacist in of the patients, the geriatrician in cases and the orthopaedic surgeon in . in patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the patients on eye drops treatment during hospital stay, ( . %) of them suffer from acute confusional state. on the other hand, among the patients without medication reconciliation, delirium was registered in cases ( . %). concerning ocular topic treatment, . ± . active principles per patient were observed, being the most frequent timolol ( . %), brinzolamide ( . %) and latanoprost ( . %). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a -year-old-man lung transplant patient for alpha -antitrypsin deficiency in receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in , and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april , which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june led to stop mycophenolate sodium and introduce azathioprine at mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january , a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at . mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites ( -thioguanine and -methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from to %. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june . collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: patients were included in the study ( men- women); median age was [ - years]. a total of . % (n = ) were using a least one cm, most frequently homeopathy ( %, n = ) or phytotherapy ( %, n = ); some patients were using a combination of two cm ( %, n = ). cm are mainly used by women in comparison to men ( . % versus . % and p = . , chi square test). for phytotherapy, at least different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that % (n = ) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to % if we considered only patients taking phytotherapy. interactions included pharmacokinetic ( %, n = ), such as altered hepatic metabolism, and pharmacodynamics ones ( %, n = ). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate ( . % versus . %; p = . ), but not compared to apixaban ( . %; p = . ). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban ( . % for apixaban, . % for dabigatran etexilate and . % for rivaroxaban; p \ . and p \ . , respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials ( . % versus . %; p \ . for dabigatran; . % versus . %; p \ . for rivaroxaban and . % versus . %; p \ . for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm ( ) : - our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including women and men. the age of patients is between and years, with an average of . years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, patients have diabetes mellitus, have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in of the patients: days - mg every h, days - mg every h and a maintenance dose of mg every h. in one patient due to its low imc the dose received was smaller ( - days mg every h, days - mg every h and maintenance dose of mg every h). in relation with the adverse effects, digestive discomfort were observed in of the patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients were reviewed: women and men with a mean age of . years old (range - years). regarding the indication of the prescription, patients have been diagnosed of indolent systemic mastocytosis and the remaining were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment . months, with a range between months and months. the dose received was mg every h in patients, having to be increased to mg times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of patients infected with hcv genotype b treated with the new therapies against hcv from february to december in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of patients with hcv gen. b were reviewed which . % of them were men with a mean age of . years (range - years). of the patients were naive and only of them were hiv co-infected, there were a . % of cirrhotic patients. regarding the degree of hepatic fibrosis, patients had grade f , f grade patients, patients grade f and f grade patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in patients ( without ribavirin and with ribavirin ) using a treatment schedule of weeks in of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in times ( without ribavirin and with ribavirin) choosing only in one of them for a treatment period of weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. patients received simeprevir + sofosbuvir for weeks ( without ribavirin and with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to patients, associating ribavirin in only one case. a treatment duration of weeks was used in patients and weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to patients in -week regimen which was well tolerated achieving svr. digestivo service treated the % of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in . % of patients. regarding the safety, only patients have discontinued the treatment due to adverse effects representing less than % dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april , when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april to april , noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman Ò genotyping assays technology by independent laboratories to confirm the results. results: from april we were asked for pharmacogenetic tests from different healthcare specialty areas: rheumatology ( . %), infectious diseases ( . %), oncology ( . %), cardiology ( . %), vascular surgery ( . %), neurology ( . %), ophthalmology ( . %); this information was asked about different drugs: clopidogrel ( . %), trastuzumab ( . %), ranibizumab ( . %), azathioprine ( . %) and tocilizumab ( . %). from all the genotypes, ( . %) were done after using the drug (study phase) and ( . %) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations affected to the prescription of clopidogrel. conclusion: during the last years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in different healthcare areas affecting drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of medications was identified by consensus ( anticholinergic, sedative). of these, ( %) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics ( medications), antidepressants ( medications) and antiepileptics ( medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between and mg/l to avoid development of resistance or - mg/l to improve penetration in complicated infections. some articles have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december . non obese adult patients with creatinine clearance (crcl) \ ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem Ò (pks Ò ). the statistical analysis was made with medcalc software Ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: patients were included, with a mean age of (± . ) years. % were male and % female. they received a median dose per h of ( - ) mg. the mean of cminr was . mg/l ( % ci . - . ), cminb . mg/l ( % ci . - . ), cminf . ( % ci . - . ) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was . ( % ci . - . ), while r between cminb and cminr was higher: . ( % . - . ). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = - . + . x and y = - . + . x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december to march performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c- Ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range . - ng/ml). results: data from patients, . % women with a mean (sd) age of . ( . ) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was . ( . ) mmol/l; . ( . ) mg/dl; . ( . ) mg/dl; . ( . ) ml/min. . % of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in . % of patients, it was maintained in . %, and it was decreased in . %. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il- receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the - g[c (rs ) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il- genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il- (g[c) (rs ) genetic variant was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. main outcome measures: clinical response was evaluated at , , and months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v. . results: we recruited patients with ra treated with tocilizumab, these were aged . ± . (mean ± sd), ( %) were women. the mean das at baseline was . ± . . of these patients, the il- g[c genetic polymorphism was significantly associated with ''responders'' at months after the baseline (cc vs non-cc p = . , or . , % ci . - . ) but not at (p = . ), (p = . ) and (p = . ) months. conclusion: the il- g[c may be useful as a genetic marker of tocilizumab efficacy at months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp : daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *, , frederic tissot , laurent decosterd , thierry buclin , guy prod'hom , christina orasch , oscar marchetti , farshid sadeghipour , , thierry calandra , véronique erard pharmacy service, infectious diseases service, laboratory and division of clinical pharmacology, service of biomedicine, institute of microbiology, lausanne university hospital, lausanne, school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin mg/ kg/day ( mg/kg/day for enterococci) for c days as a -min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem Ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [ is required for bacteriostatic effect against staphylococci and [ for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of . has been suggested for bacteriostasis and . for a -log bacterial count reduction. results: model-derived mean auc observed in patients was . ± . mg h/l, maximum concentration (cmax) ± mg/ l, minimal concentration (cmin) . ± . mg/l. clearance was . ± . l/h and volume of distribution at steady sate . ± . l, both values found higher than those reported in healthy subjects. all patients ( / ) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and out of values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of ). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients ( u/l and u/l) with cmin of . and . mg/l, respectively. conclusion: daptomycin pk profile in neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in / patients with a staphylococcal infection (two-log drop) and / with e. faecium infection ( -log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines ( mg/kg every weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range - mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor Ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of patients, with a median age of years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to patients: . % showed sitls under the therapeutic range ( . % with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: % of these patients showed sitls below the therapeutic range ( % with ada). it had been necessary to increase the dose or shorten the interval in patients due to inadequate clinical response: . % of these patients with sitls below the therapeutic range ( % with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = - mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study randomly chosen patients with measured plasma valproate concentrations were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other % subtherapeutic), % were drawn at c though , . % were drawn for assessing compliance (nontrough). in the prospective study patients were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other subtherapeutic), % were drawn at c trough , . % were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost % and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: . neovascular membranes with visual acuity higher than . . one eye affection severe cardiovascular pathology (severe episodes in the last months) non-responders to other anti-vegf bevacizumab: . diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c , under treatment with or more drugs and belonging to primary care areas in different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during months number of adverse effects ( . ; p \ . ) and number of clinical problems ( . ; p \ . ).with each year increase in age ) and a significant rise in physician ( . ; p \ . ) and nurse ( . ; p \ . ) home visits. women compared to men resulted in a significant decrease ) but a significant increase in visits to nurses ( . ; p \ . ), hospital admissions ( . ; p \ . ) and hospital visits ( . ; p \ . ). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate ( ng/l) and ls ( ng/l) wherein the concentration of bpa decreases over time to reach ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to . ng/dialyzer in the compartment mimicking the blood and to . ng/dialyzer in the dialysate despite prior rinsing with l of . % nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, genomic unit san cecilio university hospital, genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp c * * polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from to in our hospital. data collected: age, sex, cyp c * (rs ) and cyp c * (rs ) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during months after discharge. polymorphisms were genotyped using taqman Ò genotyping assays technology. main outcome measures: we recruited patients with stroke ( . % men; mean age . ) and patients after pta ( . % men; mean age . ) treated with clopidogrel after discharge %) suffered the primary end-point during months after discharge; of these patients had the cyp c * allele. among patients with pta of the lower limb: % of them had the cyp c * allele and no one a cyp c * allele; ( . %) of these patients suffered the primary end-point during months after the discharge and of these had the * allele of the cyp c isoenzyme * allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr a (a[c) (rs ) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr a (a[c) (rs ) snp was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was . ± . years and % were women. the mean das at baseline was . ± . . we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp : therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b ( mg/kg/ weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus Ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) . % no ifx-b detection (c \ . mcg/ml) and positive ati-b (c [ ua/ml) ( assessments/ patient). atis = , y ua/ml. no clinical response (nr) in . % assessments. (b) . % ifx-b and ati-b (c b ua/ml) no detection ( assessments/ patient). nr %. (c) . % ifx-b detection (c [ . mcg/ml) and negative ati-b ( assessments/ patients) weight: ( - ) kg. twenty assessments, . ( - ) assessments per patient, ( - ) ifx-b doses, % concomitant treatment ( / -azathioprine, / -corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp : serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud , mette i martinsen estimation of t of noacs by t / = ln /kel [kel; elimination constant] using two s-concentration measurements. results: we included patients (median age years, . % women). noac use was detected be serum analysis in patients ( . %; % coherent with mr), while patients ( . %) used warfarin. of the noac users ( . %) had s-concentrations of noacs above the reference range at admission, and five patients ( . %) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users ( vs h, p = . ). blood transfusions were given to . % of noac-users vs . % of warfarin-users (p = . ). mean estimated t of noacs were , . and . h for dabigatran (n = ), apixaban (n = ) and rivaroxaban (n = ), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c ) groups. the mobile phase was composed of . % na po h o (ph . ), acetonitrile, and methanol ( : : , v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was . ml/min at ambient temperature and sample detection was carried out at nm. plasma samples were obtained from patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture h after oral administration of nilotinib. plasma was separated by centrifugation at g for min and stored at - °c until analysis. plasma samples ( ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of - ng/ml at nm, with relative standard deviations (n = ) of . , . , and . % for , , and ng/ml, respectively. the detection limit for nilotinib was ng/ml due to three blank determinations (q = ). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r = . , p \ . ). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract